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ORGANIC SPECTROSCOPY

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DR ANTHONY MELVIN CRASTO Ph.D

DR ANTHONY MELVIN CRASTO Ph.D

DR ANTHONY MELVIN CRASTO, Born in Mumbai in 1964 and graduated from Mumbai University, Completed his Ph.D from ICT, 1991,Matunga, Mumbai, India, in Organic Chemistry, The thesis topic was Synthesis of Novel Pyrethroid Analogues, Currently he is working with GLENMARK LIFE SCIENCES LTD, Research Centre as Principal Scientist, Process Research (bulk actives) at Mahape, Navi Mumbai, India. Total Industry exp 30 plus yrs, Prior to joining Glenmark, he has worked with major multinationals like Hoechst Marion Roussel, now Sanofi, Searle India Ltd, now RPG lifesciences, etc. He has worked with notable scientists like Dr K Nagarajan, Dr Ralph Stapel, Prof S Seshadri, Dr T.V. Radhakrishnan and Dr B. K. Kulkarni, etc, He did custom synthesis for major multinationals in his career like BASF, Novartis, Sanofi, etc., He has worked in Discovery, Natural products, Bulk drugs, Generics, Intermediates, Fine chemicals, Neutraceuticals, GMP, Scaleups, etc, he is now helping millions, has 9 million plus hits on Google on all Organic chemistry websites. His friends call him Open superstar worlddrugtracker. His New Drug Approvals, Green Chemistry International, All about drugs, Eurekamoments, Organic spectroscopy international, etc in organic chemistry are some most read blogs He has hands on experience in initiation and developing novel routes for drug molecules and implementation them on commercial scale over a 30 PLUS year tenure till date June 2021, Around 35 plus products in his career. He has good knowledge of IPM, GMP, Regulatory aspects, he has several International patents published worldwide . He has good proficiency in Technology transfer, Spectroscopy, Stereochemistry, Synthesis, Polymorphism etc., He suffered a paralytic stroke/ Acute Transverse mylitis in Dec 2007 and is 90 %Paralysed, He is bound to a wheelchair, this seems to have injected feul in him to help chemists all around the world, he is more active than before and is pushing boundaries, He has 9 million plus hits on Google, 2.5 lakh plus connections on all networking sites, 90 Lakh plus views on dozen plus blogs, 233 countries, 7 continents, He makes himself available to all, contact him on +91 9323115463, email amcrasto@gmail.com, Twitter, @amcrasto , He lives and will die for his family, 90% paralysis cannot kill his soul., Notably he has 33 lakh plus views on New Drug Approvals Blog in 233 countries......https://newdrugapprovals.wordpress.com/ , He appreciates the help he gets from one and all, Friends, Family, Glenmark, Readers, Wellwishers, Doctors, Drug authorities, His Contacts, Physiotherapist, etc

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Rilzabrutinib


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(R)-2-(3-(4-Amino-3-(2-fluoro-4-phenoxyphenyl)-1H-pyrazolo[3,4-d]-pyrimidin-1-yl)piperidine-1-carbonyl)-4-methyl-4-(4-(oxetan-3-yl)piperazin-1-yl)pent-2-enenitrile.png
20200818lnp2-rilza.jpg

PRN 1008, Rilzabrutinib

CAS 1575591-66-0

リルザブルチニブ;

C36H40FN9O3,

MW 665.7597

2-[(3R)-3-[4-amino-3-(2-fluoro-4-phenoxyphenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidine-1-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-1-yl]pent-2-enenitrile

Anti-inflammatory disease, Autoimmune disease treatment

  • OriginatorPrincipia Biopharma
  • Class2 ring heterocyclic compounds; Amines; Anti-inflammatories; Fluorobenzenes; Nitriles; Phenyl ethers; Piperazines; Piperidines; Pyrazoles; Pyrimidines; Skin disorder therapies; Small molecules
  • Mechanism of ActionAgammaglobulinaemia tyrosine kinase inhibitors
  • Orphan Drug StatusYes – Idiopathic thrombocytopenic purpura; Pemphigus vulgaris
  • Phase IIIIdiopathic thrombocytopenic purpura; Pemphigus vulgaris
  • Phase IIAutoimmune disorders
  • 02 Jun 2021Efficacy data from a phase IIa trial in Ankylosing spondylitis presented at the 22nd Annual Congress of the European League Against Rheumatism (EULAR-2021)
  • 07 Apr 2021Sanofi initiates enrollment in a phase I pharmacokinetics trial in healthy volunteers in Australia (PO, Tablet, Capsule) (NCT04748926)
  • 31 Mar 2021Sanofi announces intention to seek regulatory approval for Idiopathic thrombocytopenic purpura in 2023 (Sanofi pipeline, May 2021)

CLIP

https://cen.acs.org/pharmaceuticals/drug-development/Sanofi-acquire-BTK-inhibitor-firm/98/web/2020/08

Sanofi to acquire BTK inhibitor firm Principia for $3.7 billion

Principia is testing its small-molecule compounds in multiple sclerosis and immune system diseases

Sanofi will pay $3.7 billion to acquire Principia Biopharma, a San Francisco-based biotech firm developing small molecules that inhibit Bruton tyrosine kinase (BTK). The price represents about a 75% premium over Principia’s stock market value in early July, before reports surfaced that Sanofi was interested in buying the firm.

BTK is a protein important for both normal B cell development and the proliferation of lymphomas, which are B cell cancers. AbbVie, AstraZeneca, and BeiGene all market BTK inhibitors for treating specific kinds of lymphomas. Sales of AbbVie’s inhibitor, Imbruvica, approached $4.7 billion in 2019.

Other drug firms have been eager to get in on the action as well. In January, Merck & Co. spent $2.7 billion to acquire ArQule, whose experimental noncovalent BTK inhibitor is designed to overcome resistance that some cancers develop after treatment with current covalent BTK inhibitors. Eli Lilly and Company’s $8 billion acquisition of Loxo Oncology in 2019 also included a noncovalent BTK inhibitor.

BTK is also linked to inflammation, and Principia focuses on developing BTK inhibitors for immune system diseases and multiple sclerosis. Its compound rilzabrutinib is currently in clinical trials for pemphigus and immune thrombocytopenia. In 2017, Sanofi struck a deal to develop Principia’s brain-penetrant BTK inhibitor, SAR442168, for multiple sclerosis.

Sanofi announced in April of this year that the inhibitor reduced formation of new lesions—the scarred nervous tissue that gives multiple sclerosis its name—by 85% in a Phase II clinical trial. A Phase III trial of the compound began in June.

Upon announcing its deal to acquire Principia, Sanofi said that both rilzabrutinib and SAR442168 have the potential to become a “pipeline in a product,” indicating they can be used for many immune-related and neurological diseases, respectively.

The anti-inflammatory effects of BTK inhibitors have raised interest in the drugs as treatments for people hospitalized with COVID-19. Notably, the US National Cancer Institute conducted a small study suggesting acalabrutinib may help reduce the respiratory distress and inflammation in people with COVID-19. Based on that preliminary study, AstraZeneca—which markets acalabrutinib as Calquence—is conducting a 60-person randomized trial of the drug for COVID-19.

Sanofi has not indicated interest in investigating Principia’s BTK inhibitors as COVID-19 treatments.Chemical & Engineering NewsISSN 0009-2347 
PATENTWO 2021127231https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2021127231&tab=PCTDESCRIPTION&_cid=P20-KRA0I9-18818-1

SOLID FORMS OF 2-[3-[4-AMTNO-3-(2-FT,TTORO-4-PHENOXY- PHEN¥L)PYRAZOLO[3,4 D]PYRIMIDIN l~YL]PIPERIDINE~l~CARBON¥L] 4~

METHYL-4-[4-(OXETAN-3-YL)PIPERAZIN-l-YLjPENT-2-ENENITRILE

[11 This application claims the benefit of priority to U.S. Provisional Application

No 62/951,958, filed December 20, 2019, and U.S Provisional Application No. 63/122,309, filed December 7, 2020, the contents of each of which are incorporated by reference herein in their entirety.

[2] Disclosed herein are solid forms of 2-[3-[4~amino-3~(2~fluoro-4-phenoxy-plienyl)pyrazolo[3,4-d]pyrimidin-l-yl]piperidine-l Carbonyl]~4-nietliyl-4~[4-(oxetaii~3-yl)piperazin-!~yi]pent-2~enenitriie (Compound (I)), methods of using the same, and processes for making Compound (I), including its solid forms. The solid forms of Compound (I) may be inhibitors of Bruton’s tyrosine kinase (BTK) comprising low residual solvent content.

[3| The enzyme BTK is a member of the Tec family non-receptor tyrosine kinases.

BTK is expressed in most hematopoietic cells, including B cells, mast cells, and macrophages BTK plays a role in the development and activation of B cells. BTK activity has been implicated in the pathogenesis of several disorders and conditions, such as B cell-related hematological cancers (e.g., non-Hodgkin lymphoma and B cell chronic lymphocytic leukemia) and autoimmune diseases (e.g., rheumatoid arthritis, Sjogren’s syndrome, pemphigus, IBD, lupus, and asthma).

[4] Compound (I), pharmaceutically acceptable salts thereof, and solid forms of any of the foregoing may inhibit BTK and be useful in the treatment of disorders and conditions mediated by BTK activity. Compound (I) is disclosed in Example 31 of WO 2014/039899 and has the following structure:

where *C is a stereochemical center. An alternative procedure for producing Compound (!) is described in Example 1 of WO 2015/127310.

[5] Compound (I) obtained by the procedures described in WO 2014/039899 and WO 2015/127310 comprises residual solvent levels well above the limits described in the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (“ICH”) guidelines. In general, manufacturing processes producing residual solvent levels near or above the ICH limits are not desirable for preparing active pharmaceutical ingredients (APIs).

Example 1: Spray Drying Process A

[311] A solution of Compound (I) in dichloromethane (prepared according to Example 31 on pages 86-87 of WO 2014/039899) was washed with pH 3 phosphate buffer to remove basic impurities that are more soluble than Compound (I) in the aqueous layer. The dichloromethane solution was then washed with pH 7 buffer and solvent exchanged into isopropyl acetate. The isopropyl acetate solution was then washed with pH 3 phosphate buffer, bringing Compound (I) into the aqueous layer and removing non-basic impurities. The pH of the aqueous layer was adjusted to pH 9 with 10% sodium hydroxide, and the aqueous layer was extracted with isopropyl acetate. Upon concentration under vacuum, Compound (I) was precipitated from heptane at 0 °C, filtered and dried to give a white amorphous solid as a mixture of the (E) and (Z) isomers, as wet Compound (I). Wet Compound (I) was dissolved in methanol and spray dried at dryer inlet temperature of 125 °C to 155 °C and dryer outlet temperature of 48 to 58 °C to obtain the stable amorphous Compound (I) free base with levels of isopropyl acetate and heptane below 0.5% and 0.05%, respectively.

Example 2: Spray Drying Process B


intermediate A

Compound (!)

[241] A jacketed reactor with overhead stirrer, condenser, nitrogen line, temperature probe, and recirculating fluid chiller/heater was charged with Intermediate A (20.2 kg) and Intermediate B (13.6 kg, 1.5 equiv). DCM (361.3 kg, 14.5 vol) was charged to the reactor. The mixture was agitated, and the batch cooled to 0 °C to 5 °C. The reactor was charged with pyrrolidine (18.3 kg, 6 equiv) and then charged with TMSC1 (18.6 kg, 4 eq). Stirring was continued at 0 °C to 5 °C for 0.5 to 1 hour

[242] At 0 °C to 5 °C, acetic acid (2.0 equiv) was charged to the reactor followed by water (5 equiv). Stirring was continued at 0 °C to 5 °C for 1 to 1.5 hours. Water (10 equiv) was charged to the reactor, and the solution was adjusted to 20 °C to 25 °C. The internal temperature was adjusted to 20 °C to 25 °C and the biphasic mixture was stirred for 15 to 20 mins. Stirring was stopped and phases allowed to separate for at least 0.5 h. The lower aqueous layer was removed.

[243] Water (7 vol) was charged to the reactor. The pH was adjusted to 2.8-3.3 with a 10 wt. % solution of citric acid. Stirring was continued at 0 to 5 °C for 1 to 1.5 hours. Stirring was stopped and phases allowed to separate for at least 0.5 h. The lower aqueous layer was removed.

[244] A jacketed reactor with overhead stirrer, condenser, nitrogen line, temperature probe, and recirculating fluid chiller/heater was charged with an approximately 9% solution of NaHCCri (1 vol) and the organic layer. The internal temperature was adjusted to 20 °C to 25 °C, and the biphasic mixture was stirred for 15 to 20 mins. Stirring was stopped and phases allowed to separate for at least 0.5 h. The lower aqueous layer was removed. The aqueous layer was measured to have a pH greater than 7.

[245] A jacketed reactor with overhead stirrer, condenser, nitrogen line, temperature probe and recirculating fluid chiller/heater was charged with the organic layer. The organic phase ¾s distilled under vacuum at less than 25 °C to 4 total volumes. IP AC (15 vol) was charged to the reactor. The organic phase was distilled under vacuum at less than 25 °C to 10 total volumes. Water (15 vol) followed by pH 2.3 phosphate buffer were charged to the reactor at an internal temperature of 20 °C to 25 °C. The pH adjusted to 3 Stirring was stopped and phases allowed to separate for at least 0.5 h. The organic phase was removed.

[246] The following steps were repeated twice: IP AC (5 vol) was charged to the reactor containing the aqueous layer. Stirring was continued for 0.25 to 0.5 hours. Stirring was stopped and phases allowed to separate for at least 0.5 h. The organic phase was removed. [247] IP AC (15 vol) was charged to the reactor containing the aqueous layer. A pH 10 phosphate buffer was charged to the reactor and the pH adjusted to 10 with 14% NaOH solution. Stirring was continued for 1.5 to 2 hours. Stirring was stopped and phases allowed to separate for at. least 0.5 h. The aqueous layer was discarded. The organic layer was dried over brine.

[248] The organic solution was distilled under vacuum at less than 25 °C to 5 total volumes.

[249] A jacketed reactor with overhead stirrer, condenser, nitrogen line, temperature probe and recirculating fluid chiller/heater was charged with n-heptane (20 vol). The internal temperature was adjusted to 0 to 5 °C, and the IP AC solution was added.

[250] The suspension was filtered. The filter cake was washed with n-heptane and the tray was dried at 35 °C. Compound (I) (24.6 kg) was isolated in 86% yield.

[251] Compound (1) was dissolved in methanol (6 kg) and spray dried to remove residual IP AC and n-heptane.

Example 3: Precipitation Process A

[252] A solution of Compound (I) in dichloromethane (prepared according to Example 31 on pages 86-87 of WO 2014/039899) was quenched with acetic acid and water, followed by washing with pH 3 aqueous solution to remove basic impurities that are more soluble than Compound (1) in the aqueous layer. Washing was repeated as needed to reduce impurities. Methanesulfonic acid was added to the dichloromethane solution, and the dichloromethane solution was concentrated by distillation under reduced pressure, followed by addition of 1% NaCi aqueous solution and isopropyl acetate before adjustment of pH to approximately 3 with potassium hydroxide. The isopropyl acetate layer was removed and discarded. The aqueous layer containing Compound (I) was washed with isopropyl acetate to remove hydrophobic impurities. Washing was repeated as needed to reduce related substance impurities. Residual isopropyl acetate was removed by distillation under reduced pressure. The aqueous solution containing Compound (I) was cooled to 0 to 5°C before adjusting the pH to approximately 9 with potassium hydroxide. The free base of Compound (I) was allowed to precipitate and maturate at 20 °C for 20 hours. The mixture temperature was then adjusted to 20 °C to 25 °C, and the hydrate impurity was verified to be less than 0.3% (< 0.3%). The cake of the free base of Compound (I) was filtered and washed as needed to reduce conductivity. The cake was then allowed to dry on the filter under vacuum and nitrogen swept to reduce water content by Karl-Fischer (KF < 50%) before transferring to the oven for drying. The wet cake of the free base of Compound (1) was dried under vacuum at 25 °C until water content by Karl -Fischer was less than 1.5% (KF < 1.5%), and then dehmiped by milling to yield a uniform white amorphous solid as a mixture of the (E) and (Z) isomers, with no detectible levels of isopropyl acetate or heptane.

Example 4: Precipitation Process 3B

[253] A solution of Compound (I) in dichloromethane (prepared according to Example 31 on pages 86-87 of WO 2014/039899) was quenched with acetic acid and water, followed by washing with pH 3 aqueous solution to remove basic impurities that are more soluble than Compound (I) in the aqueous layer. The washing was repeated as needed to reduce residual solvents and impurities. The dichloromethane solution was then washed with saturated sodium bicarbonate (pH > 7). Dichloromethane was removed by distillation under reduced pressure, followed by addition of water and isopropyl acetate. The pH of the aqueous layer was adjusted to pH to 2.8 – 3.3 with 2 M aqueous sulfuric acid (H2SQ4) at 0 – 5 °C, and the mixture rvas stirred and settled. After phase separation removal of the organic layer, the aqueous layer was washed with isopropyl acetate three times and the residual isopropyl acetate in aqueous layer was distilled out under vacuum at a temperature below 25 °C and the solution was basitied with 5% aqueous KOFI to pH 9 – 10 to a slurry . The resulting suspension was stirred and warmed up to 20 °C to 25 °C and aged for 20 h. The product was filtered and washed with water and dried to give white solid in 86% yield.

Example 5: Precipitation Process C

[254] A solution of Compound (I) in dichloromethane (prepared according to Example 31 on pages 86-87 of WO 2014/039899) was quenched with acetic acid and water, followed by washing to remove basic impurities that are more soluble than Compound (I) in the aqueous layer. Washing was repeated as needed to reduce impurities. Methanesulfonic acid was added to the d chloromethane solution, and the dichloromethane solution was concentrated under reduced pressure to obtain a thin oil. The concentrated oil was cooled to approximately 5°C before washing with an aqueous solution of sodium chloride. The organic phase was discarded. Washing of the aqueous layer was repeated as needed with dichloromethane to remove low level impurities. The pH of the aqueous solution was adjusted to approximately 3 with an aqueous solution of potassium hydroxide. Residual dichloromethane was removed

under reduced pressure. The level of residual acetic acid was determined by, for example, titration. The aqueous solution containing Compound (I) was cooled to a temperature between 0°C and 5°C. Acetic acid was present at 0 wt % to 8 wt. %. Acetic acid level was 0 wt % if the aqueous acid solution was washed with aqueous sodium bicarbonate or another aqueous inorganic base. Optionally, additional acetic acid was added to achieve a 0 wt.% to 8 wt. % acetic acid level. An aqueous solution of potassium hydroxide was constantly charged to the aqueous solution to obtain a pH to approximately 9.5. The free base of Compound (I) was allowed to precipitate and maturate at approximately 20 °C for least 3 hours. The cake (wet solid) of the free base of Compound (I) was filtered and washed with water. The wet cake was then dried under reduced vacuum with slight heat. Alternatively, instead of washing the wet cake with water, the wet cake was reslurried with water at approximately 15 °C for at least 1 hour before filtering. The free base of Compound (I) in the fomi of a wet cake was dried under vacuum with slight heat at 25°C.

[255] FIGs. 12-15 are example SEM images showing the variable morphologies of particles of Compound (I) during the filtration step to isolate Compound (I) based on the amount acetic acid added during the initial step in the precipitation of Compound (Ϊ) (FIG. 12: at 0 wt. % acetic acid; FIG 13: at 3 wt. % acetic acid; FIG. 14: at 5 wt. % acetic acid; FIG 15: at 8 wt. % acetic acid). Filtration speed depended on the morphology and was the fastest for 0 wt. % acetic acid. At 1 wt. % acetic acid, the filtration speed diminished considerably, improving at 2 wt. % to 3 wt. % acetic acid. Morphologies with more open holes (such as, e.g., more porous particles) resulted in improved filtration speeds, whereas more compact particles resulted in decreased filtration speed.

Example 6: Conversion of a Crystalline Form of Compound (Ϊ) to an Amorphous Form

[256] 9.8 grams of a crystalline form of Compound (I) were dissolved in approximately 20 mL of dichloromethane and approximately 120 ml. of brine solution. Then, approximately 1 equivalent of methanesulfonic acid was added. The pH w¾s approximately 2. The layers were separated. The aqueous layer was concentrated at a temperature between 0°C and 5°C to remove residual dichloromethane before slowly adding aqueous KOI I solution (approximately 5%) to adjust the pH to a value between 9 and 10. During aqueous KOH addition, an amorphous form of Compound (I) precipitated out. The slurry was slowly warmed to room temperature and then was stirred for approximately 24 hours before filtering and rinsing the wet cake with water. The wet cake was dried under vacuum with slight heat at approximately 30°C to provide 7 grams of a white to an off-white solid (87% yield and 98 4% purity). XRPD showed that the product was an amorphous solid form of Compound (I).

Example 7: Micronization of Compound (I) Particles Obtained by Precipitation Processes

[257] A fluid jet mill equipment was used during lab scale jet milling trials. The fluid jet mill equipment includes a flat cylindrical chamber with 1.5” diameter, fitted with four symmetric jet nozzles winch are tangentially positioned in the inner wall. Prior to feeding material to the fluid jet mill in each trial, the material was sieved in a 355 iim screen to remove any agglomerates and avoid blocking of the nozzles during the feed of material to the micronization chamber. The material to be processed was drawn into the grinding chamber through a vacuum created by the venturi (P vent ~ 0 5 – 1 0 bar above P grind). The feed flow rate of solids (F_feed) was controlled by a manual valve and an infinite screw volumetric feeder. Compressed nitrogen was used to inject the feed material; compressed nitrogen was also used for the jet nozzles in the walls of the milling chamber. Compressed fluid issuing from the nozzles expands from P grind and imparts very’ high rotational speeds in the chamber. Accordingly, material is accelerated by rotating and expanding gases and subjected to centrifugal forces. Particles move outward and are impacted by high velocity jets, directing the particles radially inward at very high speeds. Rapidly moving particles impact the slower moving path of particles circulating near the periphery of the chamber. Attrition takes place due to the violent impacts of particles against each other. Particles with reduced size resulting from this sequence of impacts are entrained in the circulating stream of gas and swept against the action of centrifugal force toward the outlet at the center. Larger particles in the gas stream are subjected to a centrifugal force and returned to the grinding zone. Fine particles are carried by the exhaust gas to the outlet and pass from the grinding chamber into a collector.

[258] The feeder has continuous feed rate control; however, to more precisely control the feed rate, the full scale of feed rates was arbitrary divided in 10 positions. To calibrate F feed, the feeder was disconnected from milling chamber and 10 g of Compound (I) powder was fed through the feeder operating at various feed rate positions. The mass of powder flowing through the feeder over 6 minutes was marked. The resulting feed rate was directly proportional to feeder position. After processing each of the four trials, the jet mill was stopped, micronized product removed from the container, and the milling chamber checked for any powder accumulation.

Variables/Parameters

F_feed Feed flow rate of solids [kg/h]

P grind Grinding pressure inside the

drying chamber [bar]

P vent Feed pressure in the venturi [bar]

Example 8: Residual Solvent Levels

[251] Retention of process solvents (/.<?., res dual solvents) depends on van der Waal s’ forces that are unique to and an inherent property of each molecule. Additionally, solvent retention depends how the API solid is formed, isolated, washed, and dried (i.e., during the manufacturing process). Because residual solvents may pose safety risks, pharmaceutical processes should be designed to minimize residual solvent levels (e.g , to result in residual solvent levels below the limits established in the ICH guidelines).

[252] Residual solvent analysis was performed using gas chromatography-mass spectrometry. The residual solvent levels in solid forms of Compound (I) prepared by spray drying processes described herein and precipitation processes described herein are provided in Table 2. The residual solvent levels in crude Compound (I) listed in Table 2 are comparable to the residual solvent levels in crude Compound (I) prepared according to the procedures detailed in Example 31 of WO 2014/039899 and Example 1 of WO 2015/127310.

Table 2: Residual solvent levels in solid forms of Compound (I)

PATENTWO 2015127310https://patents.google.com/patent/WO2015127310A1/enExample 1Synthesis of 2-[(3R)-3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-l- yl]-piperidine-l-carbonyl]-4-m iperazin-l-yl]pent-2-enenitrile

Figure imgf000045_0001

Step 1To a solution of 3-[(3R)-3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4- d]pyrimidin-l -yl]-l-piperidyl]-3-oxo-propanenitrile (15 g, 3.12mmol), 2-methyl-2-[4- (oxetan-3-yl)piperazin-l-yl]propanal (794.25mg, 3.74mmol) in DCM (40mL), pyrrolidine (1.54mL,18.71mmol) at 0-5 °C was added, which is followed by TMS-Cl (1.58mL,12.47mmol). The reaction mixture was stirred at 0-5 °C for 3 h and was quenched with 1 M potassium phosphate buffer (pH 3). Layers were separated and the organic layer was washed once more with 1 M potassium phosphate buffer (pH 3). The organic layer was extracted withl M potassium Phosphate buffer at pH 1.5. Layers were separated. The aqueous phase contained the desired product while the impurities stayed in the organic phase. The aqueous phase was neutralized with 1 M potassium phosphate (pH 7) and was extracted with isopropylacetate (10 volumes). Upon concentration 2-[(3R)-3-[4-amino-3-(2-fluoro-4- phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-l-yl]piperidine-l-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-l-yl]pent-2-enenitrile was obtained as a foam having >99% HPLC purity. MS (pos. ion) m/z: 666 (M+l ).The foam containing high levels of residual solvent was dissolved in 2 M HC1 and the resulting solution was placed under vacuum to remove residual organic solvents. pH of the solution was then adjusted to ~ 7 and the resulting paste was filtered and dried in vacuum without heat. This resulted in isolation of 2-[(3R)-3-[4-amino-3-(2-fluoro-4-phenoxy- phenyl)pyrazolo[3,4-d]pyrimidin-l-yl]piperidine-l-carbonyl]-4-methyl-4-[4-(oxetan-3- yl)piperazin- l-yl]pent-2-enenitrile containing residual water up to 10%. Drying under vacuum without heat reduces the water level but lead to generation of impurities.Step 1AAlternatively, the isopropylacetate solution of 2-[(3R)-3-[4-amino-3-(2-fluoro-4- phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin- 1 -yl]piperidine- 1 -carbonyl]-4-methyl-4-[4- (oxetan-3-yl)piperazin-l -yl]pent-2-enenitrile can be concentrated to 4 vol and added to heptane (20 volume) at 0 °C. The resulting suspension was stirred at 0 °C overnight and the product was filtered, washed twice with heptane and dried at 45 °C for 2 days under vacuum to give 2-[(3R)-3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]pyrimidin-l – yl]piperidine-l-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-l-yl]pent-2-enenitrile in 85 – 90 % yield as a free flowing solid. However, the solids obtained by this method contained high residual solvents (3.9 wt% isopropylacetate and 1.7 wt% heptane). In addition, the free base form was not very stable as degradation products were observed during the drying process at less than 45 °C.Salt formationExample 2Preparation of 2-[(3R)-3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)-pyrazolo[3,4-d]pyrimidin- l-yl]-piperidine-l-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)-piperazin-l-yl]pent-2-enenitrile hemisulfate and sulfate saltHemisulfate: To the solution of 2-[(3R)-3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)-pyrazolo[3,4- d]pyrimidin-l-yl]-piperidine -carbonyl]-4-methyl-4-[4-(oxetan-3-yl)-piperazin-l-yl]pent-2- enenitrile (4.2 g) in EtOAc (60 mL, 15 vol) was added sulfuric acid (0.31 g, 0.17 mL, 0.5 eq) in EtOAc (20 mL, 5 vol) at ambient temperature. The suspension was stirred at ambient temperature for ~ 2 hr and then 40 °C for 4 hr and then at ambient temperature for at least 1 hr. After filtration and drying at ambient temperature under vacuum, 1.5 g of white powder was obtained. Solubility of the hemi-sulfate at ambient temperature was > 100 mg/mL in water.Sulfate saltTo the solution of 2-[(3R)-3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)-pyrazolo[3,4- d]pyrimidin-l-yl]-piperidine-l-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)-piperazin-l-yl]pent-2- enenitrile (810 mg) in EtOAc (8 mL, 10 vol) was added sulfuric acid (0.06 mL, 1.0 equiv.) in EtOAc (2.5 mL, 5 vol) at ambient temperature. The resulting suspension was stirred at 40 °C for 2 hr and then cooled to ambient temperature for at least 1 hr. After filtration, solids were dried by suction under Argon for 1 h to give a white powder (0.68 g) in 69% yield.

Figure imgf000047_0001

Example 3Preparation of 2-[(3R)-3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)-pyrazolo[3,4- d]pyrimidin- 1 -yl]-piperidine- 1 -carbonyl] -4-methyl-4-[4-(oxetan-3-yl)-piperazin- 1 -yl]pent-2- enenitrile hydrochlorideTo a solution of 2-[(3R)-3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4- d]pyrimidin- 1 -yl]piperidine- 1 -carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin- 1 -yl]pent-2- enenitrile (100 mg, 0.15 mmol) in CH2CI2 (1ml) at ambient temperature was added 2 equivalent of HC1 (0.3 mmol, 0.15 ml of 2M HC1 in 1 : 1 dioaxane:CH2Cl2). The resulting homogeneous solution was stirred at ambient temperature for 1 h and was added dropwise to 15 volumes of ethylacetate (as compared to CH2C12) resulting in formation of a white solid. The mixtures was aged at ambient temperature for lh and placed at 2-8 C for 19 h. Upon filtration and washing of the filter cake with ethylacetate and drying a white solid was obtained. Analysis by XRPD indicated formation of an amorphous solid. Both Ή-NMR and IC analysis indicated formation of the salt. IC indicated formation mono-HCl salt.

Figure imgf000048_0001

Example 4General procedure for preparation of 2-[(3R)-3-[4-amino-3-(2-fluoro-4-phenoxy- phenyl)pyrazolo[3,4-d]pyrimidin-l-yl]-piperidine-l-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)- piperazin-l-yl]pent-2-enenitrile mono- and di-mesylate saltsTo a solution of 2-[(3R)-3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4- d]pyrimidin-l-yl]piperidine-l-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin-l-yl]pent-2- enenitrile (100 mg, 0.15 mmol) in CH2C12 (1 ml) at ambient temperature was added either 1 equivalent of methanesulfonic acid (0.15 mmol, 0.2 ml of 74 mg/ml solution in CH2C12) or 2 equivalent of methanesulfonic acid (0.3 mmol, 0.4 ml of 74 mg/ml solution in CH2C12). The resulting homogeneous solution was stirred at ambient temperature for 1 h and was added dropwise to 10 volumes of antisolvents (ethylacetate, methyl tert-butylether (MTBE), or cyclohexane) (10 ml as compared to CH2C12) resulting in formation of a white solid. The mixture was aged at ambient temperature for lh and placed at 2-8 °C for 19 h. Upon filtration and washing of the filter cake with the antisolvent and drying, a white solid was obtained. Analysis by XRPD indicated formation of an amorphous solid. Both Ή-NMR and IC analysis indicated formation of the salt as well as counterion ratio.Alternatively 2-[(3R)-3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]- pyrimidin- 1 -yl]piperidine- 1 -carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin- 1 -yl]pent-2- enenitrile can be dissolved in 4 volumes of isopropylacetate and added to 2 equivalent of methanesulfonic acid in 6 volumes of isopropylacetate at 0 °C to generate the dimesylate salt.

Figure imgf000049_0001

1. Theoretical mesylate content, monomesylate=12.6% and dimesylate=22.4%, NO- not determinedExample 5 General procedure for the preparation of carboxylate salt Approximately 20 mg of the compound (I) was dissolved in minimum amount of the allocated solvent system. These were then mixed with the appropriate number of equivalents of counterion dissolved or slurried in the allocated solvent.If compound (I) was insoluble in the selected solvent, slurry of the sample was used after adding 300 μί.If the acid was insoluble in the selected solvent, slurry of the acid was used after adding 300 xL.If the acid was a liquid, the acid was added to the dissolved/slurried compound (I) from a stock solution in the allocated solvent.The suspensions/ precipitates resulting from the mixtures of compound (I) were temperature cycled between ambient (ca. 22°C) and 40°C in 4 hour cycles for ca. 48 hrs (the cooling/heating rate after each 4 hour period was ca. 1 °C/min). The mixtures were visually checked and any solids present were isolated and allowed to dry at ambient conditions prior to analysis. Where no solid was present, samples were allowed to evaporate at ambient. Samples which produced amorphous material, after the treatment outlined above, were re- dissolved and precipitated using anti-solvent (ter/-butylmethylether) addition methods at ambient conditions (ca. 22°C). i.e. the selected anti-solvent was added to each solution, until no further precipitation could be observed visually or until no more anti-solvent could be added. The solvents used in this preparation were acetonitrile, acetone, isopropyl acetate, THF and MTBE. The acid used were oxalic acid, L-aspartic acid, maleic acid, malonic acid, L-tartaric acid, and fumaric acid.Example 6General procedure for preparation of 2-[(3R)-3-[4-amino-3-(2-fluoro-4-phenoxy- phenyl)pyrazolo[3,4-d]pyrimidin-l-yl]-piperidine-l-carbonyl]-4-methyl-4-[4-(oxetan-3-yl)- piperazin-l-yl]pent-2-enenitrile hemicitrate saltTo a solution 2-[(3R)-3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)pyrazolo[3,4-d]- pyrimidin- 1 -yl]piperidine- 1 -carbonyl]-4-methyl-4-[4-(oxetan-3-yl)piperazin- 1 -yl]pent-2- enenitrile (5 g, 7.5 mmol) in ethanol (50 ml) was added citric acid (720.5 mg, 3.76 mmol) dissolved in 2 ml of water. Mixture was stirred at ambient temperature for 15 min, additional 0.5 ml of water was added and the mixture was stirred for 1 h, concentrated in vacuo to a gum. Ethanol was added and the mixture was concentrated. This process was repeated twice more and then CH2CI2 was added to the mixture. Upon concentration a white solid was obtained which was tumble dried under reduced pressure at 40 C for 4 h, then in a vacuum oven for 19h to give 5.4 g of a solid. Analysis by XRD indicated formation of an amorphous solid 

PATENT

WO2014039899, Example 31

Rilzabrutinib (PRN1008) is an oral, reversible covalent inhibitor of Bruton’s tyrosine kinase (BTK) [1].

https://patents.google.com/patent/WO2014039899A1/enExample 31Synthesis of (R)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)- 1 H-pyrazolo[3,4-d]pyrimidin- 1 -yl)piperidine- 1 -carbonyl)-4-methyl-4-(4-(oxetan-3-yl)piperazin- 1 -yl)pent-2-enenitrile

Figure imgf000087_0002

Step 1A solution of 2-bromo-2-methyl-propanal (696.6 mg, 4.61 mmol) in DCM (10 mL) was cooled with an ice bath and l -(oxetan-3-yl)piperazine (328 mg, 2.31 mmol), diluted with 5-10 mL of DCM, was slowly added via addition funnel over a 15 min period. Next, Hunig’s base (0.4 mL, 2.31 mmol) was added and then the cooling bath was removed. The reaction mixture was stirred at room temperature overnight and the DCM layer was washed three times with 0.5N HC1. The combined aqueous layer was neutralized with NaOH to pH 10-11 and extracted with DCM. The combined organic layer was washed with brine and dried over Na?S04. Filtration and removal of solvent afforded 2-methyl-2-[4-(oxetan-3-yl)piperazin-l- yl]propanal as a light yellow liquid, which was used directly in the next step without further purification.Step 2To a cooled (0 °C) solution of 3-[(3R)-3-[4-amino-3-(2-fluoro-4-phenoxy-phenyl)- pyrazolo[3,4-d]pyrimidin-l-yl]-l-piperidyl]-3-oxo-propanenitrile (80 mg, 0.17 mmol), was added 2-methyl-2-[4-(oxetan-3-yl)piperazin-l-yl]propanal (-108 mg, 0.51 mmol) in DCM (10 mL) followed by pyrrolidine (0.08 mL, 1.02 mmol) and TMS-C1 (0.09 raL, 0.68 mmol.) The ice bath was removed, and the reaction stirred 1 hour. Most of the solvent was removed and the residues were purified by chromatography, using 95:5 CH2Cl2:MeOH to obtain 79 mg of (R)-2-(3-(4-amino-3-(2-fluoro-4-phenoxyphenyl)-lH-pyrazolo[3,4-d]-pyrimidin-l- yl)piperidine- 1 -carbonyl)-4-methyl-4-(4-(oxetan-3-yl)piperazin- 1 -yl)pent-2-enenitrile as a white solid. MS (pos. ion) m/z: 666 (M+l).

PAPER

https://www.sciencedirect.com/science/article/abs/pii/S0223523421001781?dgcid=rss_sd_all

Therapy based on Bruton’s tyrosine kinase (BTK) inhibitors one of the major treatment options currently recommended for lymphoma patients. The first generation of BTK inhibitor, Ibrutinib, achieved remarkable progress in the treatment of B-cell malignancies, but still has problems with drug-resistance or off-target induced serious side effects. Therefore, numerous new BTK inhibitors were developed to address this unmet medical need. In parallel, the effect of BTK inhibitors against immune-related diseases has been evaluated in clinical trials. This review summarizes recent progress in the research and development of BTK inhibitors, with a focus on structural characteristics and structure-activity relationships. The structure-refinement process of representative pharmacophores as well as their effects on binding affinity, biological activity and pharmacokinetics profiles were analyzed. The advantages and disadvantages of reversible/irreversible BTK inhibitors and their potential implications were discussed to provide a reference for the rational design and development of novel potent BTK inhibitors.

Image 17

///////////////PRN-1008,  PRN 1008, Rilzabrutinib, リルザブルチニブ,
N#CC(=CC(N(C1COC1)C)(C)C)C(=O)N1CCCC1Cn1nc(c2c1ncnc2N)c1ccc(cc1F)Oc1ccccc1

DEUCRAVACITINIB


CID 134821691.png
Deucravacitinib Chemical Structure
2D chemical structure of 1609392-27-9

DEUCRAVACITINIB

BMS-986165

CAS 1609392-27-9, C20H22N8O3, 425.46

6-(cyclopropanecarbonylamino)-4-[2-methoxy-3-(1-methyl-1,2,4-triazol-3-yl)anilino]-N-(trideuteriomethyl)pyridazine-3-carboxamide

6-(cyclopropanecarboxamido)-4-((2-methoxy-3-(1-methyl-1H-1,2,4-triazol-3-yl)phenyl)amino)-N-(methyl-d3)pyridazine-3-carboxamide

Tyk2-IN-4

UNII-N0A21N6RAU

N0A21N6RAU

GTPL10432

EX-A3154

BDBM50507816

NSC825520

s8879

  • OriginatorBristol-Myers Squibb
  • ClassAmides; Aniline compounds; Anti-inflammatories; Antipsoriatics; Antirheumatics; Cyclopropanes; Ethers; Hepatoprotectants; Organic deuterium compounds; Pyridazines; Skin disorder therapies; Small molecules; Triazoles
  • Mechanism of ActionTYK2 kinase inhibitors
  • Phase IIIPlaque psoriasis
  • Phase IICrohn’s disease; Lupus nephritis; Psoriatic arthritis; Systemic lupus erythematosus; Ulcerative colitis
  • Phase IAutoimmune disorders
  • No development reportedInflammatory bowel diseases; Psoriasis
  • 02 Jul 2021Bristol-Myers Squibb plans a phase I pharmacokinetics trial (In volunteers) in USA (PO, Tablet) in July 2021 (NCT04949269)
  • 14 Jun 2021Bristol-Myers Squibb plans a phase III trial for Psoriatic arthritis (Treatment-naïve) in USA, Brazil, Colombia, Czech republic, Hungary, Italy, Mexico, Romania, Spain and Taiwan in July 2021 (NCT04908202) (EudraCT2020-005097-10)
  • 02 Jun 2021Interim efficacy and adverse events data from the phase III POETYK-PSO-1 trial in Psoriatic psoriasis presented at the 22nd Annual Congress of the European League Against Rheumatism (EULAR-2021)

BMS , presumed to be in collaboration with Jinan University and Chinese Academy of Sciences , is developing deucravacitinib, a TYK2 inhibitor, for treating autoimmune diseases, primarily psoriasis. In July 2021, deucravacitinib was reported to be in phase 3 clinical development.

Deucravacitinib (BMS-986165) is a highly selective, orally bioavailable allosteric TYK2 inhibitor for the treatment of autoimmune diseases, which selectively binds to TYK2 pseudokinase (JH2) domain (IC50=1.0 nM) and blocks receptor-mediated Tyk2 activation by stabilizing the regulatory JH2 domain. Deucravacitinib inhibits IL-12/23 and type I IFN pathways.

PAPER

https://pubs.acs.org/doi/abs/10.1021/acs.jmedchem.9b00444

Abstract Image

Small molecule JAK inhibitors have emerged as a major therapeutic advancement in treating autoimmune diseases. The discovery of isoform selective JAK inhibitors that traditionally target the catalytically active site of this kinase family has been a formidable challenge. Our strategy to achieve high selectivity for TYK2 relies on targeting the TYK2 pseudokinase (JH2) domain. Herein we report the late stage optimization efforts including a structure-guided design and water displacement strategy that led to the discovery of BMS-986165 (11) as a high affinity JH2 ligand and potent allosteric inhibitor of TYK2. In addition to unprecedented JAK isoform and kinome selectivity, 11 shows excellent pharmacokinetic properties with minimal profiling liabilities and is efficacious in several murine models of autoimmune disease. On the basis of these findings, 11 appears differentiated from all other reported JAK inhibitors and has been advanced as the first pseudokinase-directed therapeutic in clinical development as an oral treatment for autoimmune diseases.

Bristol Myers Squibb Presents Positive Data from Two Pivotal Phase 3 Psoriasis Studies Demonstrating Superiority of Deucravacitinib Compared to Placebo and Otezla® (apremilast)

04/23/2021.. https://news.bms.com/news/details/2021/Bristol-Myers-Squibb-Presents-Positive-Data-from-Two-Pivotal-Phase-3-Psoriasis-Studies-Demonstrating-Superiority-of-Deucravacitinib-Compared-to-Placebo-and-Otezla-apremilast/default.aspx

Significantly more patients treated with deucravacitinib achieved PASI 75 and sPGA 0/1 compared to patients treated with placebo and Otezla at Week 16, with an increased benefit versus Otezla at Week 24 and maintained through Week 52

Deucravacitinib was well tolerated with a low rate of discontinuation due to adverse events

Deucravacitinib is a first-in-class, oral, selective tyrosine kinase 2 (TYK2) inhibitor with a unique mechanism of action

Results presented as late-breaking research at the 2021 American Academy of Dermatology Virtual Meeting Experience

PRINCETON, N.J.–(BUSINESS WIRE)– Bristol Myers Squibb (NYSE:BMY) today announced positive results from two pivotal Phase 3 trials evaluating deucravacitinib, an oral, selective tyrosine kinase 2 (TYK2) inhibitor, for the treatment of patients with moderate to severe plaque psoriasis. The POETYK PSO-1 and POETYK PSO-2 trials, which evaluated deucravacitinib 6 mg once daily, met both co-primary endpoints versus placebo, with significantly more patients achieving Psoriasis Area and Severity Index (PASI) 75 response and a static Physician’s Global Assessment score of clear or almost clear (sPGA 0/1) after 16 weeks of treatment with deucravacitinib. Deucravacitinib was well tolerated with a low rate of discontinuation due to adverse events (AEs).

This press release features multimedia. View the full release here: https://www.businesswire.com/news/home/20210423005134/en(Graphic: Business Wire)

Deucravacitinib demonstrated superior skin clearance compared with Otezla® (apremilast) for key secondary endpoints in both studies, as measured by PASI 75 and sPGA 0/1 responses at Week 16 and Week 24. Findings include:

PASI 75 Response in POETYK PSO-1 and POETYK PSO-2:

  • At Week 16, 58.7% and 53.6% of patients receiving deucravacitinib achieved PASI 75 response, respectively, versus 12.7% and 9.4% receiving placebo and 35.1% and 40.2% receiving Otezla.
  • At Week 24, 69.0% and 59.3% of patients receiving deucravacitinib achieved PASI 75 response, respectively, versus 38.1% and 37.8% receiving Otezla.
  • Among patients who achieved PASI 75 response at Week 24 with deucravacitinib and continued treatment with deucravacitinib, 82.5% and 81.4%, respectively, maintained PASI 75 response at Week 52.

sPGA 0/1 Response in POETYK PSO-1 and POETYK PSO-2:

  • At Week 16, 53.6% and 50.3% of patients receiving deucravacitinib achieved sPGA 0/1 response, respectively, versus 7.2% and 8.6% receiving placebo and 32.1% and 34.3% receiving Otezla.
  • At Week 24, 58.4% and 50.4% of patients receiving deucravacitinib achieved sPGA 0/1 response, respectively, versus 31.0% and 29.5% receiving Otezla.

“In both pivotal studies, deucravacitinib was superior to Otezla across multiple endpoints, including measures of durability and maintenance of response, suggesting that deucravacitinib has the potential to become a new oral standard of care for patients who require systemic therapy and need a better oral option for their moderate to severe plaque psoriasis,” said April Armstrong, M.D., M.P.H., Associate Dean and Professor of Dermatology at the University of Southern California. “As many patients with moderate to severe plaque psoriasis remain undertreated or even untreated, it is also highly encouraging to see that deucravacitinib improved patient symptoms and outcomes to a greater extent than Otezla.”

Superiority of Deucravacitinib Versus Placebo and Otezla

Deucravacitinib demonstrated a robust efficacy profile, including superiority to placebo for the co-primary endpoints and to Otezla for key secondary endpoints. In addition to PASI 75 and sPGA 0/1 measures, deucravacitinib was superior to Otezla across both studies in multiple other secondary endpoints, demonstrating significant and clinically meaningful efficacy improvements in symptom burden and quality of life measures.

POETYK PSO-1 and POETYK PSO-2 Results at Week 16 and Week 24
Endpoint POETYK PSO-1 (n=666) POETYK PSO-2 (n=1,020)
Deucravacitinib6 mg(n=332) Otezla30 mg(n=168) Placebo(n=166) Deucravacitinib6 mg(n=511) Otezla30 mg(n=254) Placebo(n=255)
PASI 75*a
Week 16 58.7%* 35.1% 12.7% 53.6%* 40.2% 9.4%
Week 24 69.0% 38.1% 59.3% 37.8%
sPGA 0/1*b
Week 16 53.6%* 32.1% 7.2% 50.3%* 34.3% 8.6%
Week 24 58.4% 31.0% 50.4% 29.5%
(Scalp) ss-PGA 0/1c
Week 16 70.8%* 39.1% 17.4% 60.3%* 37.3% 17.3%
Week 24 71.8% 42.7% 59.7% 41.6%
PSSD-Symptoms CFBd
Week 16 -26.7* -17.8 -3.6 -28.3* -21.1 -4.7
Week 24 -31.9 -20.7 -29.1 -21.4
DLQI 0/1e
Week 16 40.7%* 28.6% 10.6% 38.0%* 23.1% 9.8%
Week 24 47.8% 24.2% 41.8% 21.5%
*Co-primary endpoints for POETYK PSO-1 and POETYK PSO-2 were PASI 75 and sPGA 0/1 for deucravacitinib vs placebo at Week 16.
a. PASI 75 is defined as at least a 75% improvement from baseline in Psoriasis Area and Severity Index (PASI) scores. *p<0.0001 vs placebo. †p<0.0001 vs Otezla. ‡p=0.0003 vs Otezla.
b. sPGA 0/1 is defined as a static Physician’s Global Assessment (sPGA) score of clear or almost clear. *p<0.0001 vs placebo. †p<0.0001 vs Otezla.
c. ss-PGA 0/1 is defined as a scalp-specific Physician’s Global Assessment (ss-PGA) score of clear or almost clear in those with ss-PGA of at least 3 (moderate) at baseline. POETYK PSO-1: *p<0.0001 vs placebo. †p<0.0001 vs Otezla. POETYK PSO-2: *p<0.0001 vs placebo. †p<0.0001 vs Otezla. ‡p=0.0002 vs Otezla.
d. Change from baseline (CFB) in Psoriasis Symptoms and Signs Diary (PSSD) captures improvement in symptoms of itch, pain, stinging, burning and skin tightness in patient eDiaries. *p<0.0001 vs placebo. †p<0.0001 vs Otezla.
e. Dermatology Life Quality Index (DLQI) 0/1 scores reflect no effect at all on patient’s life in patients with a baseline DLQI score of ≥2. POETYK PSO-1: *p<0.0001 vs placebo. †p=0.0106 vs Otezla. ‡p<0.0001 vs Otezla. POETYK PSO-2: *p<0.0001 vs placebo. †p<0.0001 vs Otezla.

Safety and Tolerability

Deucravacitinib was well-tolerated and had a similar safety profile in both trials. At Week 16, 2.9% of 419 patients on placebo, 1.8% of 842 patients on deucravacitinib and 1.2% of 422 patients on Otezla experienced serious adverse events (SAEs) across both studies. The most common AEs (≥5%) with deucravacitinib treatment at Week 16 were nasopharyngitis and upper respiratory tract infection with low rates of headache, diarrhea and nausea. At Week 16, 3.8% of patients on placebo, 2.4% of patients on deucravacitinib and 5.2% of patients on Otezla experienced AEs leading to discontinuation. Across POETYK PSO-1 and POETYK PSO-2 over 52 weeks, SAEs when adjusted for exposure (exposure adjusted incidence per 100 patient-years [EAIR]) were 5.7 with placebo, 5.7 with deucravacitinib and 4.0 with Otezla. In the same timeframe across both studies, EAIRs for AEs leading to discontinuation were 9.4 with placebo, 4.4 with deucravacitinib and 11.6 with Otezla. No new safety signals were observed during Weeks 16‒52.

Across both Phase 3 trials, rates of malignancy, major adverse cardiovascular events (MACE), venous thromboembolism (VTE) and serious infections were low and generally consistent across active treatment groups. No clinically meaningful changes were observed in multiple laboratory parameters (including anemia, blood cells, lipids and liver enzymes) over 52 weeks.

“The findings from both studies affirm that deucravacitinib – a first-in-class, oral, selective TYK2 inhibitor with a unique mechanism of action that inhibits the IL-12, IL-23 and Type 1 IFN pathways –may become an oral treatment of choice for people living with psoriasis. We believe deucravacitinib has significant potential across a broad range of immune-mediated diseases, and we are committed to further advancing our expansive clinical program with this agent,” said Mary Beth Harler, M.D., head of Immunology and Fibrosis Development, Bristol Myers Squibb. “We are in discussions with health authorities with the goal of bringing this new therapy to appropriate patients as soon as possible. At Bristol Myers Squibb, we are committed to building an immunology portfolio that addresses pressing unmet needs that exist for those impacted by serious dermatologic conditions and other immune-mediated diseases, to ultimately deliver the promise of living a better life.”

These results are available as a late-breaking research presentation (Session S033 – Late-Breaking Research Abstracts) as part of the 2021 American Academy of Dermatology (AAD) Virtual Meeting Experience (VMX). Full results of both studies will be submitted to a medical journal for peer review. In November 2020 and February 2021, respectively, Bristol Myers Squibb announced positive topline results from POETYK PSO-1 and POETYK PSO-2.

Visit www.bms.com/media/medical-meetings/bms-at-aad-vmx.html for more information on Bristol Myers Squibb’s scientific approach and resources on psoriasis and immune-mediated diseases.

About Deucravacitinib

Deucravacitinib (pronounced doo-krav-a-sih-ti-nib) is a first-in-class, oral, selective tyrosine kinase 2 (TYK2) inhibitor with a unique mechanism of action. Deucravacitinib is the first and only TYK2 inhibitor in clinical studies across multiple immune-mediated diseases. Bristol Myers Squibb scientists designed deucravacitinib to selectively target TYK2, thereby inhibiting signaling of interleukin (IL)-12, IL-23 and Type 1 interferon (IFN), key cytokines involved in psoriasis pathogenesis. Deucravacitinib achieves a high degree of selectivity by uniquely binding to the regulatory, rather than the active, domain of TYK2, which is structurally distinct from the regulatory domains of Janus kinase (JAK) 1, 2 and 3. At therapeutic doses, deucravacitinib does not inhibit JAK1, JAK2 or JAK3. Due to the innovative design of deucravacitinib, Bristol Myers Squibb earned recognition with the 2019 Thomas Alva Edison Patent Award for the science underpinning the clinical development of deucravacitinib.

Deucravacitinib is being studied in multiple immune-mediated diseases, including psoriasis, psoriatic arthritis, lupus and inflammatory bowel disease. In addition to POETYK PSO-1 and POETYK PSO-2, Bristol Myers Squibb is evaluating deucravacitinib in three other Phase 3 studies in psoriasis: POETYK PSO-3 (NCT04167462); POETYK PSO-4 (NCT03924427); POETYK PSO-LTE (NCT04036435). Deucravacitinib is not approved for any use in any country.

About the Phase 3 POETYK PSO-1 and POETYK PSO-2 Studies

PrOgram to Evaluate the efficacy and safety of deucravacitinib, a selective TYK2 inhibitor (POETYK) PSO-1 (NCT03624127) and POETYK PSO-2 (NCT03611751) are global Phase 3 studies designed to evaluate the safety and efficacy of deucravacitinib compared to placebo and Otezla® (apremilast) in patients with moderate to severe plaque psoriasis. Both POETYK PSO-1, which enrolled 666 patients, and POETYK PSO-2, which enrolled 1,020 patients, were multi-center, randomized, double-blind trials that evaluated deucravacitinib (6 mg once daily) compared with placebo and Otezla (30 mg twice daily). POETYK PSO-2 included a randomized withdrawal and retreatment period after Week 24.

The co-primary endpoints of both POETYK PSO-1 and POETYK PSO-2 were the percentage of patients who achieved Psoriasis Area and Severity Index (PASI) 75 response and those who achieved static Physician’s Global Assessment (sPGA) score of 0 or 1 at Week 16 versus placebo. Key secondary endpoints of the trials included the percentage of patients who achieved PASI 75 and sPGA 0/1 compared to Otezla at Week 16 and other measures.

About Psoriasis

Psoriasis is a widely prevalent, chronic, systemic immune-mediated disease that substantially impairs patients’ physical health, quality of life and work productivity. Psoriasis is a serious global problem, with at least 100 million people worldwide impacted by some form of the disease, including around 14 million people in Europe and approximately 7.5 million people in the United States. Up to 90 percent of patients with psoriasis have psoriasis vulgaris, or plaque psoriasis, which is characterized by distinct round or oval plaques typically covered by silvery-white scales. Despite the availability of effective systemic therapy, many patients with moderate to severe psoriasis remain undertreated or even untreated and are dissatisfied with current treatments. People with psoriasis report an impact on their emotional well-being, straining both personal and professional relationships and causing a reduced quality of life. Psoriasis is associated with multiple comorbidities that may impact patients’ well-being, including psoriatic arthritis, cardiovascular disease, metabolic syndrome, obesity, diabetes, inflammatory bowel disease and depression.

About Bristol Myers Squibb

Bristol Myers Squibb is a global biopharmaceutical company whose mission is to discover, develop and deliver innovative medicines that help patients prevail over serious diseases. For more information about Bristol Myers Squibb, visit us at BMS.com or follow us on LinkedInTwitterYouTubeFacebook and Instagram.

Celgene and Juno Therapeutics are wholly owned subsidiaries of Bristol-Myers Squibb Company. In certain countries outside the U.S., due to local laws, Celgene and Juno Therapeutics are referred to as, Celgene, a Bristol Myers Squibb company and Juno Therapeutics, a Bristol Myers Squibb company.

Otezla® (apremilast) is a registered trademark of Amgen Inc.

PATENT

WO-2021129467

Novel crystalline polymorphic forms (CSI and CSII) of deucravacitinib (also known as BMS-986165), useful a tyrosine kinase 2 pseudokinase domain (TYK2) inhibitor for treating psoriasis, systemic lupus erythematosus, and Crohn’s disease.Tyrosine kinase 2 (TYK2) is an intracellular signal transduction kinase that can mediate interleukin-23 (IL-23), interleukin-12 (IL-12) and type I interferon (IFN) These cytokines are involved in inflammation and immune response. 
BMS-986165 is the first and only new oral selective TYK2 inhibitor, clinically used to treat autoimmune and autoinflammatory diseases (such as psoriasis, psoriatic arthritis, lupus and inflammatory bowel disease, Crowe Graciousness, etc.). The results of a phase III clinical study of the drug announced in November 2020 showed that BMS-986165 has shown positive clinical effects in the treatment of moderate to severe plaque psoriasis. In addition, BMS-986165 also shows good therapeutic effects in the treatment of systemic lupus erythematosus and Crohn’s disease. 
The chemical name of BMS-986165 is 6-(cyclopropaneamido)-4-((2-methoxy-3-(1-methyl-1H-1,2,4-triazol-3-yl)benzene (Yl)amino)-N-(methyl-D3)pyridazine-3-carboxamide, the structural formula is shown below, and is hereinafter referred to as “compound I”: 

The crystal form is a solid in which the compound molecules are arranged in a three-dimensional order in the microstructure to form a crystal lattice. The phenomenon of drug polymorphism refers to the existence of two or more different crystal forms of the drug. Because of different physical and chemical properties, different crystal forms of the drug may have different dissolution and absorption in the body, which in turn affects the clinical efficacy and safety of the drug to a certain extent. Especially for poorly soluble solid drugs, the crystal form will have a greater impact. Therefore, drug crystal form must be an important content of drug research and also an important content of drug quality control. 
WO2018183656A1 discloses compound I crystal form A (hereinafter referred to as “crystal form A”) and a preparation method thereof. The crystalline form A disclosed in WO2018183656A1 is the only known free crystalline form of Compound I. The inventor of the present application repeated the preparation method disclosed in WO2018183656A1 to obtain and characterize the crystal form A. The results show that the crystal form A has poor compressibility and high adhesion. Therefore, there is still a need in the art to develop a compound I crystalline form with good stability, good compressibility, and low adhesion for the development of drugs containing compound I. 
The inventor of the present application has paid a lot of creative work and unexpectedly discovered the crystalline form CSI of compound I and the crystalline form CSII of compound I provided by the present invention, which have advantages in physical and chemical properties, preparation processing performance and bioavailability, for example, There are advantages in at least one aspect of melting point, solubility, hygroscopicity, purification, stability, adhesion, compressibility, fluidity, dissolution in vivo and in vitro, and bioavailability, especially good physical and chemical stability and mechanical stability It has good performance, good compressibility, and low adhesion, which solves the problems existing in the prior art, and is of great significance to the development of drugs containing compound I.

PATENT

US9505748 , a family member of WO2014074661 .

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2014074661

Preparation 1

Step l Int1

Step 2 Int2 Step 3 Int3 Step 4 Int4

Example 52

Step 1

[00219] To a solution of 2-methoxy-3-(l-methyl-lH-l ,2,4-triazol-3-yl)aniline (10.26 g, 50.2 mmol) and Int8 (10.5 g, 50.2 mmol) in THF (120 mL) was added lithium bis(trimethylsilyl)amide (LiHMDS, 1M in THF, 151 mL, 151 mmol) in a dropwise manner using a pressure equalized addition funnel. The reaction was run for 10 minutes after the completion of the addition and then quenched with HCl (1M aq., 126 mL, 126 mmol). The reaction was concentrated on a rotary evaporator until the majority of the THF was removed and a precipitate prevailed throughout the vessel. Water (-500 mL) was then added and the slurry sonicated for 5 minutes and stirred for 15 min. The solid was filtered off, rinsing with water and then air dried for 30 minutes. The powder was collected and dissolved in dichloromethane. The organic layer was washed with water and brine and then dried over sodium sulfate, filtered and concentrated to provide the product (12.5 g, 66% yield) (carried on as is). 1H NMR (400MHz, DMSO-d6) δ 11.11 (s, 1H), 9.36 (s, 1H), 8.56 (s, 1H), 7.72 (dd, J=7.8, 1.6 Hz, 1H), 7.60 (dd, J=7.9, 1.5 Hz, 1H), 7.29 (t, J=7.9 Hz, 1H), 7.19 (s, 1H), 3.95 (s, 3H), 3.72 (s, 3H). LC retention time 1.18 [E]. MS(E+) m/z: 377 (MH+).

Step 2

[00220] Intl3 (2.32 g, 6.16 mmol) and cyclopropanecarboxamide (1.048 g, 12.31 mmol) were dissolved in dioxane (62 mL) and Pd2(dba)3 (564 mg, 0.616 mmol), Xantphos (534 mg, 0.924 mmol) and cesium carbonate (4.01 g, 12.3 mmol) were added. The vessel was evacuated three times (backfilling with nitrogen) and then sealed and heated to 130 °C for 140 minutes. The reaction was filtered through CELITE® (eluting with ethyl acetate) and concentrated (on smaller scale this material could then be purified using preparative HPLC). The crude product was adsorbed onto CELITE® using dichloromethane, dried and purified using automated chromatography (100% EtOAc) to provide example 52 (1.22 g, 46% yield). 1H NMR (500MHz, chloroform-d) δ 10.99 (s, 1H), 8.63 (s, 1H), 8.18 (s, 1H), 8.10 (d, J=0.5 Hz, 2H), 7.81 (dd, J=7.9, 1.7 Hz, 1H), 7.51 (dd, J=7.9, 1.4 Hz, 1H), 7.33 – 7.20 (m, 7H), 4.01 (d, J=0.3 Hz, 3H), 3.82 (s, 3H), 1.73 -1.60 (m, 1H), 1.16 – 1.06 (m, 2H), 0.97 – 0.84 (m, 2H). LC retention time 6.84 [N]. MS(E+) m/z: 426 (MH+).

Example 53

[00221] To a homogeneous solution of Example 52 (50 mg, 0.12 mmol) in dichloromethane (3 mL) was added HCI (1M aq., 0.13 mL, 0.13 mmol) resulting in the solution turning yellow. The homogenous solution was concentrated down and then re-concentrated from dichloromethane twice to remove residual water, resulting in a white powder. The powder was suspended in dichloromethane and sonicated for 15 minutes, the powder was then collected via filtration, rinsing with dichloromethane to provide the corresponding HCI salt (38 mg, 70% yield). 1H NMR (500MHz, chloroform-d) δ 12.02 (s, 1H), 8.35 (s, 1H), 8.16 (s, 1H), 8.01 (dd, J=7.9, 1.5 Hz, 1H), 7.57 (br. s., 1H), 7.52 -7.46 (m, 1H), 7.36 (t, J=7.9 Hz, 1H), 4.03 (s, 3H), 3.83 (s, 3H), 2.05 – 1.95 (m, 1H), 1.16 – 1.09 (m, 2H), 1.03 (dd, J=7.4, 3.6 Hz, 2H). LC retention time 0.62 [j]. MS(E+) m/z: 426 (MH+).

[00222] Compare to NMR of parent free base: 1H NMR (500MHz, chloroform-d) δ 10.99 (s, 1H), 8.63 (s, 1H), 8.18 (s, 1H), 8.10 (d, J=0.5 Hz, 2H), 7.81 (dd, J=7.9, 1.7 Hz, 1H), 7.51 (dd, J=7.9, 1.4 Hz, 1H), 7.33 – 7.20 (m, 7H), 4.01 (d, J=0.3 Hz, 3H), 3.82 (s, 3H), 1.73 – 1.60 (m, 1H), 1.16 – 1.06 (m, 2H), 0.97 – 0.84 (m, 2H).

////////////DEUCRAVACITINIB, phase 3, BMS-986165, BMS 986165, psoriasis, systemic lupus erythematosus, Crohn’s disease,

CNC(=O)C1=NN=C(C=C1NC2=CC=CC(=C2OC)C3=NN(C=N3)C)NC(=O)C4CC4

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BELUMOSUDIL


KD025 structure.png
2-(3-(4-((1H-Indazol-5-yl)amino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide.png
2D chemical structure of 911417-87-3

BELUMOSUDIL

C26H24N6O2

MW 452.5

911417-87-3, SLx-2119, KD-025, KD 025, WHO 11343

2-[3-[4-(1H-indazol-5-ylamino)quinazolin-2-yl]phenoxy]-N-propan-2-ylacetamide

2-(3-(4-(lH-indazol-5-ylamino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide

Belumosudil mesylate | C27H28N6O5S - PubChem

Belumosudil mesylate

KD025 mesylate

2109704-99-4

 

UPDATE FDA APPROVED 7/16/2021 To treat chronic graft-versus-host disease after failure of at least two prior lines of systemic therapy, Rezurock

New Drug Application (NDA): 214783
Company: KADMON PHARMA LLC

200 MG TABLET

FDA approves belumosudil for chronic graft-versus-host disease

On July 16, 2021, the Food and Drug Administration approved belumosudil (Rezurock, Kadmon Pharmaceuticals, LLC), a kinase inhibitor, for adult and pediatric patients 12 years and older with chronic graft-versus-host disease (chronic GVHD) after failure of at least two prior lines of systemic therapy.

Efficacy was evaluated in KD025-213 (NCT03640481), a randomized, open-label, multicenter dose-ranging trial that included 65 patients with chronic GVHD who were treated with belumosudil 200 mg taken orally once daily.

The main efficacy outcome measure was overall response rate (ORR) through Cycle 7 Day 1 where overall response included complete response (CR) or partial response (PR) according to the 2014 criteria of the NIH Consensus Development Project on Clinical Trials in Chronic Graft-versus-Host Disease. The ORR was 75% (95% CI: 63, 85); 6% of patients achieved a CR, and 69% achieved a PR. The median time to first response was 1.8 months (95% CI: 1.0, 1.9). The median duration of response, calculated from first response to progression, death, or new systemic therapies for chronic GVHD, was 1.9 months (95% CI: 1.2, 2.9). In patients who achieved response, no death or new systemic therapy initiation occurred in 62% (95% CI: 46, 74) of patients for at least 12 months since response.

The most common adverse reactions (≥ 20%), including laboratory abnormalities, were infections, asthenia, nausea, diarrhea, dyspnea, cough, edema, hemorrhage, abdominal pain, musculoskeletal pain, headache, phosphate decreased, gamma glutamyl transferase increased, lymphocytes decreased, and hypertension.

The recommended dosage of belumosudil is 200 mg taken orally once daily with food.

View full prescribing information for Rezurock.

This review was conducted under Project Orbis, an initiative of the FDA Oncology Center of Excellence. Project Orbis provides a framework for concurrent submission and review of oncology drugs among international partners. For this review, FDA collaborated with Australia’s Therapeutic Goods Administration, Health Canada, Switzerland’s Swissmedic, and the United Kingdom’s Medicines and Healthcare products Regulatory Agency.

This review used the Real-Time Oncology Review (RTOR) pilot program, which streamlined data submission prior to the filing of the entire clinical application, and the Assessment Aid, a voluntary submission from the applicant to facilitate the FDA’s assessment. The FDA approved this application 6 weeks ahead of the FDA goal date.

This application was granted priority review and breakthrough therapy designation. A description of FDA expedited programs is in the Guidance for Industry: Expedited Programs for Serious Conditions-Drugs and Biologics.

Belumosudil mesylate is an orally available rho kinase 2 (ROCK 2) inhibitor being developed at Kadmon. In 2020, the drug candidate was submitted for a new drug application (NDA) in the U.S., under a real-time oncology review pilot program, for the treatment of chronic graft-versus-host disease (cGVHD). The compound is also in phase II clinical development for the treatment of idiopathic pulmonary fibrosis and diffuse cutaneous systemic sclerosis. Formerly, the company had also been conducting clinical research for the treatment of psoriasis and non-alcoholic steatohepatitis (NASH); however, no further development has been reported for these indications. Originally developed by Nano Terra, the product was licensed to Kadmon on an exclusive global basis in 2011. In 2019, Kadmon entered into a strategic partnership with BioNova Pharmaceuticals and established a joint venture, BK Pharmaceuticals, to exclusively develop and commercialize KD-025 for the treatment of graft-versus-host disease in China. The compound has been granted breakthrough therapy designation in the U.S. for the treatment of cGVHD and orphan drug designations for cGVHD and systemic sclerosis. In the E.U. belumosudil was also granted orphan drug status in the E.U. for the treatment of cGVHD.

Kadmon , under license from NT Life Sciences , is developing belumosudil as mesylate salt, a ROCK-2 inhibitor, for treating IPF, chronic graft-versus-host disease, hepatic impairment and scleroderma. In July 2021, belumosudil was reported to be in pre-registration phase.

Belumosudil (formerly KD025 and SLx-2119) is an experimental drug being explored for the treatment of chronic graft versus host disease (cGvHD), idiopathic pulmonary fibrosis (IPF), and moderate to severe psoriasis. It is an inhibitor of Rho-associated coiled-coil kinase 2 (ROCK2; ROCK-II).[1] Belumosudil binds to and inhibits the serine/threonine kinase activity of ROCK2. This inhibits ROCK2-mediated signaling pathways which play major roles in pro- and anti-inflammatory immune cell responses. A genomic study in human primary cells demonstrated that the drug also has effects on oxidative phosphorylation, WNT signaling, angiogenesis, and KRAS signaling.[2] Originally developed by Surface Logix, Inc,[1] Belumosudil was later acquired by Kadmon Corporation. As of July 2020 the drug was in completed or ongoing Phase II clinical studies for cGvHD, IPF and psoriasis.[3]

cGvHD is a complication that can follow stem cell or hematopoietic stem cell transplantation where the transplanted cells (graft) attack healthy cells (host). This causes inflammation and fibrosis in multiple tissues. Two cytokines controlled by the ROCK2 signaling pathway, IL-17 and IL-21, have a major role in the cGvHD response. In a 2016 report using both mouse models and a limited human clinical trial ROCK2 inhibition with belumosudil targeted both the immunologic and fibrotic components of cGvHD and reversed the symptoms of the disease.[4] In October 2017 KD025 was granted orphan drug status in the United States for treatment of patients with cGvHD.[5]

IPF is a progressive fibrotic disease where the lining of the lungs become thickened and scarred.[6] Increased ROCK activity has been found in the lungs of humans and animals with IPF. Treatment with belumosudil reduced lung fibrosis in a bleomycin mouse model study.[7] Belumosudil may have a therapeutic benefit in IPF by targeting the fibrotic processes mediated by the ROCK signaling pathway.

Psoriasis is an inflammatory skin condition where patients experiences eruptions and remissions of thickened, erythematous, and scaly patches of skin. Down-regulation of pro-inflammatory responses was observed with KD025 treatment in Phase 2 clinical studies in patients with moderate to severe psoriasis.[8]
“Substance Name:Substance Name: Belumosudil [USAN]”.

PATENT

WO2012040499  

https://patents.google.com/patent/WO2012040499A2/en

PATENT

CN106916145  

https://patents.google.com/patent/CN106916145A/en

WO 2014055996, WO 2015157556

(7) preparation of SLx-2119:
 
N- isopropyls -2- [3- (4- chloro-quinazolines base)-phenoxy group]-acetamide VI is sequentially added in 25mL tube sealings (1.2mmol), 5- Aminoindazoles (1mmol) and DMF (5mL), load onto condensation reflux unit;Back flow reaction is carried out at 100 DEG C, After 2.5h, raw material N- isopropyls -2- [3- (4- chloro-quinazolines base)-phenoxy group]-acetamide VI is monitored by TLC and reacts complete Afterwards, stop stirring, add water after being quenched, organic layer, saturated common salt water washing, anhydrous Na are extracted with ethyl acetate2SO4Dry, be spin-dried for Obtain SLx-2119, brown solid (yield 87%), as shown in figure 1,1H NMR(500MHz,DMSO)δ(ppm):13.12(br, NH,1H),9.98(br,NH,1H),8.61-8.59(m,1H),8.32(s,1H),8.17(s,1H),8.06-8.03(m,2H), 7.97-7.96(m,1H),7.87-7.84(m,1H),7.66-7.61(m,2H),7.44-7.40(m,1H),7.09-7.08(m, 1H), 4.57 (s, 2H), 4.04-3.96 (m, 1H), 1.11 (d, J=5.0Hz, 6H).
 

Patent

WO-2021129589

Novel crystalline polymorphic forms (N1, N2 and N15) of KD-025 (also known as belumosudil ), useful as a Rho A kinase 2 (ROCK-2) inhibitor for treating multiple sclerosis, psoriasis, rheumatoid arthritis, idiopathic pulmonary fibrosis (IPF), atherosclerosis, non-alcoholic fatty liver and systemic sclerosis. Represents the first filing from Sunshine Lake Pharma or its parent HEC Pharm that focuses on belumosudil.KD-025 is a selective ROCK2 (Rho-associated protein kinase 2, Rho-related protein kinase 2) inhibitor. It has multiple clinical indications such as the treatment of multiple sclerosis, psoriasis, rheumatoid arthritis, and Primary pulmonary fibrosis, atherosclerosis, non-alcoholic fatty liver, etc., among which many indications are in clinical phase I, and psoriasis and systemic sclerosis are in clinical phase II.
The structure of KD-025 is shown in the following formula (1).

Example 1 Preparation method of crystal form N1 of KD-025[0222]300mg of KD-025 solid was suspended and stirred in 10mL methanol at room temperature. After 22h, it was filtered, suction filtered and placed in a drying oven at 50°C under vacuum overnight to obtain 262mg of powder. The obtained crystal was detected by XPRD and confirmed to be KD-025 crystal form N1; its X-ray powder diffraction pattern was basically the same as that of Fig. 1, its DSC pattern was basically the same as that of Fig. 2, and the TGA pattern was basically the same as that of Fig. 3.

PATENT

WO2006105081 ,

Belumosudil product pat, 

protection in the EU states until March 2026, expires in the US in May 2029 with US154 extension.

Example 82
2-(3-(4-(lH-indazol-5-ylamino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide

[0257] A suspension of 2-(3-(4-(lH-indazol-5-ylamino)qumazolin-2-yl)ρhenoxy)acetic acid (70 mg, 0.14 mmol), PyBOP® (40 mg, 0.077 mmol), DlEA (24 μL, 0.14 mmol) in dry CH2Cl2 : DMF (2 : 0.1 mL) was stirred at RT for 15 minutes. To this solution of activated acid was added propan-2-amine (5.4 mg, 0.091 mmol). After 30 minutes, 1.0 equivalent of DIEA and 0.55 equivalents of PyBOP® were added. After stirring the solution for 15 minutes, 0.65 equivalents of propan-2-aminewere added and the mixture was stirred for an additional 30 minutes. The solvent was removed in vacuo and the crude product was purified using prep HPLC (25-50 90 rnins) to afford 2-(3-(4-(lH-indazol-5-ylamino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide. (40 mg, 0.086 mmol, 61 %).

References

  1. Jump up to:a b Boerma M, Fu Q, Wang J, Loose DS, Bartolozzi A, Ellis JL, et al. (October 2008). “Comparative gene expression profiling in three primary human cell lines after treatment with a novel inhibitor of Rho kinase or atorvastatin”Blood Coagulation & Fibrinolysis19 (7): 709–18. doi:10.1097/MBC.0b013e32830b2891PMC 2713681PMID 18832915.
  2. ^ Park J, Chun KH (5 May 2020). “Identification of novel functions of the ROCK2-specific inhibitor KD025 by bioinformatics analysis”. Gene737: 144474. doi:10.1016/j.gene.2020.144474PMID 32057928.
  3. ^ “KD025 – Clinical Trials”. ClinicalTrials.gov. Retrieved 25 July 2020.
  4. ^ Flynn R, Paz K, Du J, Reichenbach DK, Taylor PA, Panoskaltsis-Mortari A, et al. (April 2016). “Targeted Rho-associated kinase 2 inhibition suppresses murine and human chronic GVHD through a Stat3-dependent mechanism”Blood127 (17): 2144–54. doi:10.1182/blood-2015-10-678706PMC 4850869PMID 26983850.
  5. ^ Shanley M (October 6, 2017). “Therapy to Treat Transplant Complications Gets Orphan Drug Designation”RareDiseaseReport. Retrieved 25 July 2018.
  6. ^ “Pulmonary Fibrosis”. The Mayo Clinic. Retrieved July 25, 2018.
  7. ^ Semedo D (June 5, 2016). “Phase 2 Study of Molecule Inhibitor for Idiopathic Pulmonary Fibrosis Begins”Lung Disease News. BioNews Services, LLC. Retrieved 25 July 2018.
  8. ^ Zanin-Zhorov A, Weiss JM, Trzeciak A, Chen W, Zhang J, Nyuydzefe MS, et al. (May 2017). “Cutting Edge: Selective Oral ROCK2 Inhibitor Reduces Clinical Scores in Patients with Psoriasis Vulgaris and Normalizes Skin Pathology via Concurrent Regulation of IL-17 and IL-10”Journal of Immunology198 (10): 3809–3814. doi:10.4049/jimmunol.1602142PMC 5421306PMID 28389592.
 
Clinical data
Routes of
administration
Oral administration (tablets or capsules)
ATC code None
Identifiers
showIUPAC name
CAS Number 911417-87-3 
PubChem CID 11950170
UNII 834YJF89WO
CompTox Dashboard (EPA) DTXSID80238425 
Chemical and physical data
Formula C26H24N6O2
Molar mass 452.518 g·mol−1
3D model (JSmol) Interactive image
showSMILES
showInChI

////////////BELUMOSUDIL, SLx-2119, KD-025, KD 025, WHO 11343, PHASE 2, cGvHD, IPF,  psoriasis, Breakthrough Therapy, Orphan Drug Designation

CC(C)NC(=O)COC1=CC=CC(=C1)C2=NC3=CC=CC=C3C(=N2)NC4=CC5=C(C=C4)NN=C5

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BPI-7711, Rezivertinib


Rezivertinib.png
img

BPI-7711, Rezivertinib

1835667-12-3

C27H30N6O3, 486.576

N-[2-[2-(dimethylamino)ethoxy]-4-methoxy-5-[[4-(1-methylindol-3-yl)pyrimidin-2-yl]amino]phenyl]prop-2-enamide

Beta Pharma in collaboration Chinese licensee CSPC Pharmaceuticals Group , is developing BPI-7711

In June 2021, this drug was reported to be in phase 3 clinical development.

  • OriginatorBeta Pharma
  • ClassAmides; Amines; Antineoplastics; Indoles; Phenyl ethers; Pyrimidines; Small molecules
  • Mechanism of ActionEpidermal growth factor receptor antagonists
  • Phase IIINon-small cell lung cancer
  • 30 Dec 2020Chemical structure information added
  • 09 Apr 2020Beta Pharma initiates a phase I trial for Non-small cell lung cancer (In volunteers) in China (PO) (NCT04135833)
  • 25 Mar 2020Beta Pharma completes a phase I pharmacokinetic trial for Non-small cell lung cancer (In volunteers) in China (NCT04135820)

GTPL10628

2-Propenamide, N-(2-(2-(dimethylamino)ethoxy)-4-methoxy-5-((4-(1-methyl-1H-indol-3-yl)-2-pyrimidinyl)amino)phenyl)-

N-(2-(2-(Dimethylamino)ethoxy)-4-methoxy-5-((4-(1-methyl-1H-indol-3-yl)-2-pyrimidinyl)amino)phenyl)-2-propenamideThe epidermal growth factor receptor (EGFR, Herl, ErbB l) is a principal member of the ErbB family of four structurally-related cell surface receptors with the other members being Her2 (Neu, ErbB2), Her3 (ErbB3) and Her4 (ErbB4). EGFR exerts its primary cellular functions though its intrinsic catalytic tyrosine protein kinase activity. The receptor is activated by binding with growth factor ligands, such as epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-a), which transform the catalytically inactive EGFR monomer into catalytically active homo- and hetero- dimers. These catalytically active dimers then initiate intracellular tyrosine kinase activity, which leads to the autophosphorylation of specific EGFR tyrosine residues and elicits the downstream activation of signaling proteins. Subsequently, the signaling proteins initiate multiple signal transduction cascades (MAPK, Akt and JNK), which ultimately mediate the essential biological processes of cell growth, proliferation, motility and survival.EGFR is found at abnormally high levels on the surface of many types of cancer cells and increased levels of EGFR have been associated with advanced disease, cancer spread and poor clinical prognosis. Mutations in EGFR can lead to receptor overexpression, perpetual activation or sustained hyperactivity and result in uncontrolled cell growth, i.e. cancer. Consequently, EGFR mutations have been identified in several types of malignant tumors, including metastatic lung, head and neck, colorectal and pancreatic cancers. In lung cancer, mutations mainly occur in exons 18 to 21, which encode the adenosine triphosphate (ATP)-binding pocket of the kinase domain. The most clinically relevant drug- sensitive EGFR mutations are deletions in exon 19 that eliminate a common amino acid motif (LREA) and point mutations in exon 21, which lead to a substitution of arginine for leucine at position 858 (L858R). Together, these two mutations account for nearly 85% of the EGFR mutations observed in lung cancer. Both mutations have perpetual tyrosine kinase activity and as a result they are oncogenic. Biochemical studies have demonstrated that these mutated EGFRs bind preferentially to tyrosine kinase inhibitor drugs such as erlotinib and gefitinib over adenosine triphosphate (ATP).Erlotinib and gefitinib are oral EGFR tyrosine kinase inhibitors that are first line monotherapies for non-small cell lung cancer (NSCLC) patients having activating mutations in EGFR. Around 70% of these patients respond initially, but unfortunately they develop resistance with a median time to progression of 10-16 months. In at least 50% of these initially responsive patients, disease progression is associated with the development of a secondary mutation, T790M in exon 20 of EGFR (referred to as the gatekeeper mutation). The additional T790M mutation increases the affinity of the EGFR kinase domain for ATP, thereby reducing the inhibitory activity of ATP- competitive inhibitors like gefitinib and erlotinib.Recently, irreversible EGFR tyrosine kinase inhibitors have been developed that effectively inhibit the kinase domain of the T790M double mutant and therefore overcome the resistance observed with reversible inhibitors in the clinic. These inhibitors possess reactive electrophilic functional groups that react with the nucleophilic thiol of an active-site cysteine. Highly selective irreversible inhibitors can be achieved by exploiting the inherent non-covalent selectivity of a given scaffold along with the location of a particular cysteine residue within the ATP binding site. The acrylamide moieties of these inhibitors both undergo a Michael reaction with Cys797 in the ATP binding site of EGFRT790M to form a covalent bond. This covalent mechanism is thought to overcome the increase in ATP affinity of the T790M EGRF double mutant and give rise to effective inhibition. However, these inhibitors may cause various undesired toxicities. Therefore, development of new inhibitors for treatment of various EGFR-related cancers is still in high demand. 
PatentCN201580067776) N-(2-(2-(dimethylamino)ethoxy)-4-methoxy-5-((4-(1-methyl-1H- Indol-3-yl)pyrimidin-2-yl)amino)phenyl)acrylamide (compound of formula I) can be prepared by the following synthetic route: 

PATENT

WO2016094821A2

https://patents.google.com/patent/WO2016094821A2/enExample 1N-(2-(2-(Dimethylamino)ethoxy)-4-methoxy-5-((4-(l-methyl-lH-indol-3- yl)pyrimidin-2-yl)amino)phenyl)acrylamide (1) Sche

Figure imgf000022_0001

N-(4-(2-(Dimethylamino)ethoxy)-2-methoxy-5-nitrophenyl)-4-(l-methyl-lH- indol-3-yl)pyrimidin-2-amine (Scheme 1, Intermediate B). To a slurry of NaH (30 mmol, 60% oil dispersion prewashed with hexanes) and 50 mL of 1,4-dioxane was added 2-dimethylaminoethanol (27 mmol, 2.7 mL) dropwise with stirring under N2. After stirring for 1 h, a slurry of A (5.4 mmol) in 50 mL of 1,4-dioxane was added portion-wise over 15 min under a stream of N2. The resulting mixture was stirred overnight, then poured into water and the solid was collected, rinsed with water, and dried under vacuum to yield 2.6 g of product as a yellow solid. A purified sample was obtained from chromatography (silica gel; CH2C12-CH30H gradient). 1H NMR (300 MHz, DMSO) δ 2.26 (s, 6H), 2.70 (t, 2H, J = 6 Hz), 3.87 (s, 3H), 4.01 (s, 3H), 4.32 (t, 2H, J = 6 Hz), 7.00-7.53 (m, 5H), 8.18-8.78 (m, 5H); C24H26N604 m/z MH+ 463.4-(2-(Dimethylamino)ethoxy)-6-methoxy-Nl-(4-(l-methyl-lH-indol-3- yl)pyrimidin-2-yl)benzene-l,3-diamine (Scheme 1, Intermediate C). A suspension of 2.6 g of Intermediate B, 1.6 g of Fe°, 30 mL of ethanol, 15 mL of water, and 20 mL of cone. HC1 was heated to 78 °C for 3 h. The solution was cooled to room temperature, adjusted to pH 10 with 10% NaOH (aq) and diluted with CH2C12. The mixture was filtered through Dicalite, and the filtrate layers were separated. The aqueous phase was extracted with CH2C12 twice, and the combined organic extracts were dried over Na2S04 and concentrated. Column chromatography (silica gel, CH2Cl2-MeOH gradient) afforded 1.2 g of Intermediate C as a solid. C24H28N602 m/z MH+ 433.N-(2-(2-(Dimethylamino)ethoxy)-4-methoxy-5-((4-(l-methyl-lH-indol-3- yl)pyrimidin-2-yl)amino)phenyl)acrylamide (1). To a solution of Intermediate C (2.8 mmol) in 50 mL of THF and 10 mL of water was added 3-chloropropionychloride (2.8 mmol) dropwise with stirring. After 5 h of stirring, NaOH (28 mmol) was added and the mixture was heated at 65°C for 18 h. After cooling to room temperature, THF was partially removed under reduced pressure, and the mixture was extracted with CH2C12, dried over Na2S04, and concentrated. Chromatography of the crude product (silica gel, CH2Cl2-MeOH) afforded 0.583 g of Example 1 as a beige solid. 1H NMR (300 MHz, DMSO) δ 2.28 (s, 6H), 2.50-2.60 (m, 2H), 3.86 (s, 3H), 3.90 (s, 3H), 4.19 (t, 2H, = 5.5 Hz), 5.73-5.77 (m, IH), 6.21-6.27 (m, IH), 6.44-6.50 (m, IH), 6.95 (s, IH), 7.11-7.53 (overlapping m, 3H), 7.90 (s, IH), 8.27-8.30 (overlapping m, 3H), 8.55 (s, IH), 8.84 (s, IH), 9.84 (s, IH) ppm; C27H30N6O3 m/z MH+ 487

PATENT WO2021115425

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2021115425&tab=FULLTEXT&_cid=P20-KQN9F3-73566-1Epidermal growth factor receptors (EGFR, Her1, ErbB1) are the main members of the ErbB family of four structurally related cell surface receptors, and the other members are Her2 (Neu, ErbB2), Her3 (ErbB3) and Her4 (ErbB4). EGFR exerts its main cellular functions through its inherent catalytic tyrosine protein kinase activity. The receptor is activated by binding to growth factor ligands, such as epidermal growth factor (EGF) and transforming growth factor-α (TGF-α). The catalytically inactive EGFR monomer is transformed into a catalytically active homopolymer and Heterodimer. These catalytically active dimers then initiate intracellular tyrosine kinase activity, which leads to autophosphorylation of specific EGFR tyrosine residues and elicits downstream activation of signaling proteins. Subsequently, the signal protein initiates multiple signal transduction cascades (MAPK, Akt, and JNK), which ultimately regulate the basic biological processes of cell growth, proliferation, motility, and survival.

EGFR has been found to have abnormally high levels on the surface of many types of cancer cells, and elevated EGFR levels have been associated with advanced disease, cancer spread, and poor clinical prognosis. Mutations in EGFR can lead to overexpression of the receptor, permanent activation or continuous hyperactivity, leading to uncontrolled cell growth, which is cancer. Therefore, EGFR mutations have been identified in several types of malignant tumors, including metastatic lung cancer, head and neck cancer, colorectal cancer, and pancreatic cancer. In brain cancer, mutations mainly occur in exons 18-21, which encode the adenosine triphosphate (ATP)-binding pocket of the kinase domain. The most clinically relevant drug-sensitive EGFR mutations are deletions in exon 19 and point mutations in exon 21. The former eliminates a common amino acid motif (LREA), and the latter results in position 858 (L858R). The arginine is replaced by leucine. Together, these two mutations account for nearly 85% of the EGFR mutations observed in lung cancer. Both mutations have permanent tyrosine kinase activity, so they are carcinogenic. In at least 50% of patients who initially responded to current therapies, the progression of the disease is related to the development of a secondary mutation, T790M (also known as the goalkeeper mutation) in exon 20 of EGFR.
BPI-7711 is a third-generation EGFR-TKI compound developed by Beida Pharmaceuticals and disclosed in International Patent No. WO2017/218892. It is the N-(2-(2-(dimethylamino) )Ethoxy)-4-methoxy-5-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)phenyl)acrylamide methanesulfonic acid salt:

Need to develop improved properties containing N-(2-(2-(dimethylamino)ethoxy)-4-methoxy-5-((4-(1-methyl-1H-indole-3 -Yl)pyrimidin-2-yl)amino)phenyl)acrylamide pharmaceutically acceptable salt, in particular the pharmaceutical composition of BPI-7711 and its use, and the preparation of said pharmaceutical composition suitable for large-scale production method.

PATENT

WO2021061695 , for another filing, assigned to Beta Pharma, claiming a combination of an EGFR inhibitor (eg BPI-7711) and a CDK4/6 inhibitor, useful for treating cancer.

PATENT

WO-2021121146

Novel crystalline polymorphic form A of rezivertinib – presumed to be BPI-7711 – useful for treating diseases mediated by EGFR mutations eg lung cancer, preferably non-small cell lung cancer (NSCLC).Epidermal growth factor receptor (EGFR) is a type of transmembrane receptor tyrosine kinase in the human body. The activation (ie phosphorylation) of this kinase is of great significance to the inhibition of tumor cell proliferation, angiogenesis, tumor invasion, metastasis and apoptosis. EGFR kinase is involved in the disease process of most cancers, and these receptors are overexpressed in many major human tumors. Overexpression, mutations, or high expression of ligands associated with these family members can lead to some tumor diseases, such as non-small cell lung cancer, colorectal cancer, breast cancer, head and neck cancer, cervical cancer, bladder cancer, and thyroid. Cancer, stomach cancer, kidney cancer, etc. 
In recent years, epidermal growth factor receptor tyrosine kinase has become one of the most attractive targets in current anti-tumor drug research. In 2003, the US FDA approved the first epidermal growth receptor tyrosine kinase inhibitor (EGFR-TKI) drug (gefitinib) for the treatment of advanced non-small cell lung cancer (NSCLC). Development of a generation of EGFR inhibitors. Numerous clinical trials have confirmed that for patients with EGFR-positive non-small cell lung cancer, the therapeutic effect of molecular targeted drugs is significantly better than traditional chemotherapy. 
Although the first-generation EGFR-inhibiting targeted drugs responded well to the initial treatment of many non-small cell lung cancer (NSCLC) patients, most patients will eventually develop disease progression due to drug resistance (such as EGFR secondary T790M mutation). The emergence of drug resistance is caused by various mechanisms based on the mutations in the original EGFR pathway activity. In the drug resistance research on the first generation of EGFR inhibitors, the research frontier is the irreversible third generation EFGR inhibitor. 
But so far, the third-generation EGFR inhibitors worldwide, in addition to AstraZeneca O’Higgins imatinib developed, there is no other effective against T790M resistance mutations in patients with drug approved for clinical use; Several drug candidates for the T790M mutation are in clinical development. The chemical structure of this third-generation EGFR inhibitor is completely different from that of the first-generation. The main difference from the first-generation EGFR inhibitors is that they both use a highly selective core structure to replace the low-selective aminoquinoline core structure of the first and second-generation EGFR-TKIs. Compared with wild-type EGFR, these third-generation compounds are highly specific and selective for the T790M mutation after EGFR positive resistance. 
Chinese Patent Application No. CN201580067776.8 discloses a compound of the following formula I, which also belongs to the third-generation EGFR-TKI class of small molecule targeted drugs. The compound has a high inhibitory effect on non-small cell lung cancer (NSCLC) cells with single-activity mutation and T790M double-mutant EGFR, and its effective inhibitory concentration is significantly lower than the concentration required to inhibit the activity of wild-type EGFR tyrosine kinase. It has good properties, low side effects and good safety.

Chinese Patent Application No. CN201780050034.3 also discloses various salts and corresponding crystal forms of the compound of the above formula I. Example 2 discloses two crystal forms of the methanesulfonate of the compound of formula I, 2A and 2B, respectively.In the following examples, the “room temperature” can be 15-25°C.[0041](1) N-(2-(2-(Dimethylamino)ethoxy)-4-methoxy-5-((4-(1-methyl-1H-indol-3-yl)pyrimidine -2-yl)amino)phenyl)acrylamide (compound of formula I)[0042]

[0043]Known (for example, see CN201580067776.8) N-(2-(2-(dimethylamino)ethoxy)-4-methoxy-5-((4-(1-methyl-1H- Indol-3-yl)pyrimidin-2-yl)amino)phenyl)acrylamide (compound of formula I) can be prepared by the following synthetic route:[0044]

[0045]Step 1-Preparation of Intermediate J:[0046]

[0047]Preparation: In a 10L reaction flask, add 6L of anhydrous tetrahydrofuran solvent, protected by nitrogen, and cool to 0°C. While stirring, slowly add 101 g of sodium hydride (101 g, 2.52 mol), and the internal temperature does not exceed 10° C., and add 234 g of dimethylaminoethanol (234 g, 2.62 mol). After the addition, the temperature is adjusted to room temperature to prepare a sodium alkoxide solution.[0048]In a 30L reaction flask, add N-(4-fluoro-2-methoxy-5-nitrophenyl)-4-(1-methyl-1H-indol-3-yl)-2-pyrimidinamine ( Starting material B) (430g, 1.10mol), then add 9L of tetrahydrofuran, start stirring, dissolve it, control the temperature at 10±10°C, slowly add the prepared sodium alkoxide solution dropwise. Control the temperature at 10±10℃ and keep it for 5.0h. When the raw material content is ≤0.5%, the reaction ends. Control the temperature at 10±10°C, slowly add 3% hydrochloric acid solution dropwise, adjust the pH of the solution to 6-7, stir for 1.5h and then stand for stratification, separate the organic phase, and concentrate to 15-20L. After cooling to 20±5°C, 4.3 kg of water was slowly added dropwise, filtered, and dried to obtain 497 g of yellow powder intermediate J with a yield of 98.0% and an HPLC purity of 99.3%. MS m/z: 463.2 [M+1].[0049]Nuclear magnetic data: 1 HNMR (d 6 -DMSO): δ ppm: 8.78 (s, 1H); 8.42-8.28 (m, 3H); 8.16 (s, 1H); 7.53 (d, 1H, J = 8.28); 7.29- 7.20 (m, 2H); 7.13-7.07 (m, 1H); 7.01 (s, 1H); 4.33 (t, 2H, J = 5.65); 4.02 (s, 3H); 3.88 (s, 3H); 2.71 ( t, 2H, J = 5.77); 2.27 (s, 6H).[0050]Step 2-Preparation of Intermediate K:[0051]

[0052]Preparation: Add 5L of tetrahydrofuran and Intermediate J (350g, 108mmol) to a 10L hydrogenation reactor, add 17.5g of wet palladium charcoal, replace the hydrogenation reactor with hydrogen, adjust the pressure value to 0.2MPa, control the temperature at 25°C, and keep the temperature for reaction. At 9h, HPLC monitors the progress of the reaction, and stops the reaction when the substrate is ≤0.5%. Filter, concentrate the filtrate under reduced pressure until the solvent volume is about 2L, adjust the internal temperature to room temperature, slowly add 4L n-heptane dropwise within 4-7 hours, filter and dry the solid under reduced pressure to obtain 285g of white powder intermediate K The yield was 86%, and the HPLC purity was 99.60%. MS m/z: 433.3 [M+1].

Nuclear magnetic data: 1 HNMR (CDCl 3 ): δ ppm: 8.42 (d, 1H, J = 7.78), 8.28 (s, 1H), 8.26-8.23 (m, 1H), 7.78 (s, 1H), 7.51 (d, 1H,J=8.28),7.41(s,1H),7.26-7.23(m,1H),7.19- 7.11(m,2H),6.72(s,1H), 4.38(br,2H),4.06(t, 2H,J=5.77), 3.88(s,3H), 3.75(s,3H), 2.63(t,2H,J=5.77), 2.26(s,6H).

Step 3-Preparation of compound of formula I:

Add 250 mL of anhydrous tetrahydrofuran solvent and Intermediate K (14 g, 32 mmol) to the reaction flask and stir, cool to 0-5° C., add 10% hydrochloric acid (12 ml), and stir for 20 minutes. At 0-5°C, slowly drop 3-chloropropionyl chloride (5.6 g, 45 mmol) into the reaction flask. Stir for 3 hours, after sampling test (K/(U+K)≤0.5%) is qualified, add 36% potassium hydroxide aqueous solution (75ml, 480mmol), heat to 23-25°C, and stir for 12 hours. Raise the temperature to 50-60°C and stir for 4 hours. After the sampling test (U/(U+L)≤0.1%) is qualified, stand still for liquid separation. Separate the organic phase, wash with 10% brine three times, dry, filter, and concentrate the organic phase to 150 ml. The temperature was raised to 40° C., 150 ml of n-heptane was slowly added dropwise, and the temperature was lowered to room temperature to precipitate crystals. Filtered and dried to obtain 10.71 g of light brown solid (compound of formula I), yield 68%, HPLC purity: 99.8% (all single impurities do not exceed 0.15%). MS m/z: 487.3 [M+1].[0057]Nuclear magnetic data (Figure 1): 1 HNMR (d 6 -DMSO): δppm: 9.84 (s, 1H), 8.90 ~ 8.82 (m, 1H), 8.32-8.25 (m, 2H), 7.89 (s, 1H) ,7.51(d,1H,J=8.25), 7.27~7.10(m,1H), 6.94(s,1H), 6.49(dd,1H,J=16.88,10.13), 6.25(dd,1H,J=16.95 ,1.81),5.80~5.75(m,1H),4.19(t,2H,J=5.57),3.88(d,6H,J=14.63,6H),3.34(s,3H),2.58(d,2H, J=5.5), 2.28 (s, 6H).

(2) N-(2-(2-(Dimethylamino)ethoxy)-4-methoxy-5-((4-(1-methyl-1H-indol-3-yl)pyrimidine -2-yl)amino)phenyl)acrylamide methanesulfonate (Form A) preparation
Example 1

The compound of formula I (3 g, 6.1 mmol) was dissolved in 24 ml of dimethyl sulfoxide DMSO solvent, the temperature was raised to 65° C., and the mixture was stirred and dissolved. Add an equivalent amount of methanesulfonic acid (0.59 g, 6.1 mmol) to the system. The temperature was lowered to 50°C, and 12ml of isopropyl acetate IPAc was slowly added. Stir at 50°C for 1 hour, then lower the temperature to 15°C. 21ml IPAc was added in 4 hours. The solution was stirred and crystallized at 15°C, filtered under reduced pressure, the filter cake was washed with isopropyl acetate, and washed with acetone to reduce the residual DMSO solvent. Blow drying at 50°C (or vacuum drying at 50°C) to obtain 3.16 g of a pale yellow solid (crystal form A). HPLC purity is 100%, yield is 88%, DMSO: <100ppm; IPAc: <100ppm. MS m/z: 487.2 [M+1-MsOH]. Melting point: 242-244°C.
Nuclear magnetic data (figure 2): 1 HNMR(d 6 -DMSO): δppm: 9.57(brs,1H), 9.40(s,1H), 8.71(s,1H), 8.48(s,1H), 8.32(d ,1H,J=7.9),8.29(d,1H,J=5.3),7.96(s,1H),7.51(d,1H,J=8.2),7.23(ddd,1H,J=7.9,7.1,0.8 ), 7.19 (d, 1H, J = 5.4), 7.15 (ddd, 1H, J = 7.8, 7.3, 0.5), 6.94 (s, 1H), 6.67 (dd, 1H, J = 16.9, 10.2), 6.27 ( dd, 1H, J = 16.9, 1.8), 5.57 (dd, 1H, J = 16.9, 1.7), 4.44 (t, 2H, J = 4.6), 3.89 (s, 3H), 3.88 (s, 3H), 3.58 (t, 2H, J=4.6), 2.93 (s, 6H), 2.39 (s, 3H).
After testing, the powder X-ray diffraction pattern of crystal form A obtained in this example has diffraction angle 2θ values of 11.06±0.2°, 12.57±0.2°, 13.74±0.2°, 14.65±0.2°, 15.48±0.2°, 16.58±0.2°, 17.83±0.2°, 19.20±0.2°, 19.79±0.2°, 20.88±0.2°, 22.05±0.2°, 23.06±0.2°, 24.23±0.2°, 25.10±0.2°, 25.71±0.2°, 26.15±0.2°, 27.37±0.2°, 27.42±0.2° has a characteristic peak; its XRPD spectrum is shown in Figure 3 and the attached table, DSC diagram is shown in Figure 4, TGA diagram is shown in Figure 5, and infrared spectrum IR diagram is shown in Figure 6. Show.
Example 2

[0066]The compound of formula I (28.25 g, 58.1 mmol) was dissolved in 224 ml of dimethyl sulfoxide DMSO solvent, the temperature was raised to 15-35° C., and the mixture was stirred to clear. 0.97 equivalents of methanesulfonic acid (5.4 g, 0.97 mmol) were added to the system in batches. Slowly add 448 ml of methyl isobutyl ketone (MIBK). Stir for 1 hour, then lower the temperature to 10-15°C. The solution was reacted with salt formation at 10-15°C, sampled, and HPLC detected the residue of the compound of formula I in the mother liquor (≤0.4%). After the reaction was completed, vacuum filtration was performed to obtain 32 g of the crude methanesulfonate of the compound of formula I.Add 3g of the crude methanesulfonate of the compound of formula I into 24ml of dimethyl sulfoxide DMSO solvent, stir to clear at 65°C, cool down, slowly add 48ml of methyl isobutyl ketone (MIBK) dropwise, stir and crystallize 6-8 After hours, vacuum filtration, drying at 60° C. (or 60° C. vacuum drying) to obtain the target crystal form A. Melting point: 242-244°C. The XRPD pattern of the crystal form is consistent with Figure 3 (Figure 7), and all characteristic peaks are within the error range.

//////////// BPI-7711,  BPI 7711, rezivertinib, phase 3

CN1C=C(C2=CC=CC=C21)C3=NC(=NC=C3)NC4=CC(=C(C=C4OC)OCCN(C)C)NC(=O)C=C

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Uprifosbuvir


Uprifosbuvir structure.svg
Uprifosbuvir.png
ChemSpider 2D Image | Uprifosbuvir | C22H29ClN3O9P

Uprifosbuvir

MK 3682, IDX 21437

ウプリホスブビル;

Formula C22H29ClN3O9P
CAS 1496551-77-9
Mol weight 545.9071

уприфосбувир [Russian] [INN]أوبريفوسبوفير [Arabic] [INN]乌磷布韦 [Chinese] [INN]

propan-2-yl (2R)-2-[[[(2R,3R,4R,5R)-4-chloro-5-(2,4-dioxopyrimidin-1-yl)-3-hydroxy-4-methyloxolan-2-yl]methoxy-phenoxyphosphoryl]amino]propanoate

Isopropyl (2R)-2-{[(R)-{[(2R,3R,4R,5R)-4-chloro-5-(2,4-dioxo-3,4-dihydro-1(2H)-pyrimidinyl)-3-hydroxy-4-methyltetrahydro-2-furanyl]methoxy}(phenoxy)phosphoryl]amino}propanoate

IDX-21437DB15206SB18784D10996Q27281714

Uprifosbuvir (MK-3682) is an antiviral drug developed for the treatment of Hepatitis C. It is a nucleotide analogue which acts as an NS5B RNA polymerase inhibitor. It is currently in Phase III human clinical trials.[1][2][3]

Uprifosbuvir is under investigation in clinical trial NCT02332707 (Efficacy and Safety of Grazoprevir (MK-5172) and Uprifosbuvir (MK-3682) With Elbasvir (MK-8742) or Ruzasvir (MK-8408) for Chronic Hepatitis C Genotype (GT)1 and GT2 Infection (MK-3682-011)).Hepatitis C viruss (HCV) have the newly-increased patients of 3-4 million every year, and World Health Organization (WHO) is estimated in global sense More than 200,000,000, in China more than 10,000,000 patients, HCV belongs to flaviviridae hepatovirus virus to dye person.Long-term hepatitis C virus Gently to inflammation, weight is to liver cirrhosis, hepatocarcinoma for poison infection.And during hepatitis C cirrhosis patients in decompensation, can there are various complication, such as abdomen Water abdominal cavity infection, upper gastrointestinal hemorrhage, hepatic encephalopathy, hepatorenal syndrome, liver failure etc. are showed.The side of HCV infection is treated initially Method is interferon and interferon and ribavirin combination therapy, and only 50% therapist has reaction, and interferon to the method With obvious side effect, such as flu-like symptoms, body weight lower and fatigue and weak, and interferon and ribavirin Conjoint therapy then produces sizable side effect, including haemolysis, anemia and tired etc..U.S. FDA have approved multiple HCV medicines, including the polymerization of protease inhibitor, ucleosides and non-nucleoside in recent years Enzyme inhibitor and NS5A inhibitor etc..The protease inhibitor class medicine of FDA approvals has three:VX‐950 (Telaprevir), SCH-503034 (Boceprevir) and TMC435 (Simeprevir), the shortcoming of protease inhibitor is It is also easy to produce that mutation, toxicity is big, poor bioavailability, it is effective to individual other gene type.Eggs of the Telaprevir as the first generation White enzyme inhibitor has logged out market.The second filial generation and third generation protease inhibitor of high activity and wide spectrum is mainly used as and other One of component of drug combination of hepatitis C medicine.NS5A inhibitor is the highly active anti-HCV medicament of a class.The most representative Daclatasive for having BMS, The Ombitasvir of the Ledipasvir and AbbVie of Gilead, as this kind of medicine independent medication is easy to produce drug resistance, They treat one of drug component of HCV primarily as drug combination.The AG14361 of hepatitis C is generally divided into two kinds of ucleosides and non-nucleoside.At present, clinically only Suo Feibu One ucleosides hepatitis C medicine of Wei is listed by FDA approvals, and other are still in the anti-hepatitis C virus medicine of ucleosides of clinical experimental stage Thing also has the MK-3682 (IDX21437) of Mo Shadong, the AL-335 of the ACH-3422 and Alios of Achillion drugmakers.Third Hepatitis virus have the features such as Multi-genotype and fast variation, and single medicine treatment hepatitis C has generation drug resistance fast, to part Genotype cure rate is low and the various defects such as course for the treatment of length.In order to overcome these defects, the treatment of drug combination is primarily now taken Scheme, in order to overcome these defects, primarily now takes the therapeutic scheme of drug combination, the Sovaldi conducts of FDA approval listings The key component of drug combination, for the patient of 4 type of 1 type of gene and gene be Suo Feibuwei, profit Ba Wei woodss and Polyethylene Glycol-α- The drug combination of interferon three, the course for the treatment of are 12 weeks;For 1 type of gene and the patient of 3 types, the big woods joints of Suo Feibuwei and Li Ba Medication, the course for the treatment of are respectively 12 weeks and 24 weeks.- 2016 years 2013, FDA ratified Suo Feibuwei and NS3 protein inhibitors again in succession Simeprevir shares the patient of 1 type of therapeutic gene;The NS5A inhibitor Daclatavir therapeutic genes 1 of Suo Feibuwei and BMS With the patient of 3 types.Harvoni is the patient that Suo Feibuweijia NS5A inhibitor Ledipasvir is used for 1 type of gene.Even if using Same nucleoside, the NS5A inhibitor and/or NS3 protease inhibitor for sharing varying strength can effectively extend composition of medicine Clinical application range and Shorten the Treatment Process.In June, 2016, FDA have approved Suo Feibuwei and more potent secondary NS5A inhibitor Velpatasvir shares the hepatitis C patient suitable for all gene types, it is not necessary to carry out genetic test.Just in three phases clinic Suo Feibuwei, NS5A inhibitor Velpatasvir and NS3 protease inhibitor Voxilaprevir goes for all of disease People, is try to the course for the treatment of and shortened to 8 weeks from 12 weeks.Suo Feibuwei just in clinical trial target spots different with hepatitis C virus are directed to Drug regimen (such as Suo Feibuweijia new type NS 5A inhibitor Velpatasvir and/or protease inhibitor GS5816), its knot Fruit show than single drug more wide spectrum, effectively, and can be with Shorten the Treatment Process.MSD Corp. is by MK-3682 and NS5A inhibitor Grazoprevir and/or protease inhibitor Elbasvir is used as new drug regimen, effective for all genotype of HCV, And further shorten to the course for the treatment of of 8 weeks.New deuterated nucleoside phosphoric acid ester compound disclosed in patent of the present invention, especially The double deuterated compound such as VI-1b2 in 5 ‘-position, shows than the more preferable bioavailability of former compound MK-3682 and longer partly declines Phase.In addition, this kind of novel nucleoside phosphoramidate is significantly superior to the Suo Feibuwei of clinical practice in terms of anti-hepatitis C activity, On sugared ring, chlorine atom replaces fluorine atom, and cytotoxicity is significantly reduced in surveyed cell line.By to base, sugared ring With the transformation and optimization of prodrug moiety system, the anti-hepatitis C activity of partial synthesis compound is higher than Suo Feibuwei 2-10 times, meanwhile, In the optimization of metabolism key position, synthesis compound shows that in blood plasma the higher metabolic stabilities of peso Fei Buwei and chemistry are steady It is qualitative.Therefore this kind of new deuterated nucleotide phosphate and NS5A inhibitor and/or egg as shown in formula a, a1, a2, b, b1, b2 The newtype drug combination constituted by white enzyme inhibitor is with extremely wide application prospect.Deuterium is the naturally occurring hydrogen isotope of nature, the deuterated isotopic body in common drug all containing trace.Deuterium without It is malicious, “dead”, it is safe to human body, C-D keys are more stable (6-9 times) than c h bond, hydrogen is replaced with after deuterium, can extend medicine Half-life, while pharmacologically active (shape difference of H and D is little, J Med Chem.2011,54,2529-2591) is not affected, in addition Deuterated medicine usually shows more preferable bioavailability and less toxicity, and the active ribonucleoside triphosphote of its metabolism is more stable, So deuterated nucleoside phosphoramidate will be better than corresponding nucleoside medicine in the curative effect of clinical practice.For example, 2013 It is exactly a deuterated compound that the nucleoside anti hepatitis C virus drug ACH-3422 of clinical trial is in the approval of year FDA, with non-deuterium (WO2014169278, WO are 2014169280) than having higher bioavailability and longer half-life for the former compound phase in generation. 
Based on above-mentioned present Research, we design and are prepared for the new deuterated nucleoside that compound VI-1b2 is representative Phosphoramidate.Below we will be described in the architectural feature of deuterated nucleoside phosphoramidate of our inventions, preparation method, Antiviral activity experimental result and it as anti-hepatitis c virus drug combination key component and NS5A inhibitor and/ Or the drug regimen of protease inhibitor is in the application of anti-virus aspect.

The EPA awarded the greener reaction conditions to the pharmaceutical company Merck & Co. for building a prodrug synthesis that eliminated the use of toxic reagents. Prodrugs are molecules that get metabolized by our bodies into an active pharmaceutical. Some hepatitis C and HIV medications are prodrugs and get synthesized through a method call pronucleotide (ProTide) synthesis. The method uses toxic and corrosive thionyl chloride, plus an excess of expensive pentafluorophenol that generates a lot of waste. Merck’s new method creates their target compounds in 90 to 92% yields without these reagents and eliminates the need for halogenated solvents entirely through strategic catalyst loading and the use of different starting materials from the traditional route.

20200616lnp3-structure.jpg

The design of greener chemicals award went to the development of more environmentally friendly versions of chemicals called thermoset binders, which can serve as carpet adhesives and are involved in the manufacture of mineral and fiberglass products. Generally, these chemicals are based on formaldehyde or polycarboxylic acids, and they can give off toxic formaldehyde and often use small amounts of sulfuric and hypophosphorous acid as catalysts to activate them. The insulation and commercial roofing company Johns Manville created a new binder based on the reaction between renewable dextrose, fructose, and other simple sugars, bound together by the α-carbon-containing cross-linking agent glyoxal. The reaction also uses a biodegradable acid in water as a catalyst. The binder can be made in just one step instead of the traditional multistep synthesis. Also, the synthesis can be done directly at the manufacturing site, instead of beforehand like with the traditional approach, meaning this new binder creates fewer of the health and environmental hazards that come from storage and transportation.

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US 20170226146,

Paper

Organic Process Research & Development (2021), 25(3), 661-667.

https://pubs.acs.org/doi/10.1021/acs.oprd.0c00487

Abstract Image

A novel application of the synthesis of pronucleotide (ProTide) 5′-phosphoramidate monoesters promoted by aluminum-based Lewis acids is described. In the multikilogram synthesis of uprifosbuvir (MK-3682, 1), a clinical candidate for the treatment of hepatitis C, this methodology provided >100:1 diastereoselectivity at the phosphorus stereocenter and >100:1 selectivity for the 5′-mono phosphorylation over undesired bisphosphorylation side products. The high diastereoselectivity and mono/bis ratio achieved enabled elimination of the tedious workup associated with the tert-butyl magnesium chloride protocol commonly used to install this functionality in similar nucleotide prodrugs, achieving a near doubling of the isolated yield from 45% to 81%. The process development and purity control strategy of MK-3682, as well as handling of the pyrophoric reagent on scale, will also be discussed.

PAPER

Science (Washington, DC, United States) (2020), 369(6504), 725-730.

Science (Washington, DC, United States) (2017), 356(6336), 426-430.

Chemical Science (2017), 8(4), 2804-2810.

PATENT

CN 106543253

https://patents.google.com/patent/CN106543253A/zh

PATENT

WO 2014058801

https://patents.google.com/patent/WO2014058801A1/enExample 1Preparation of 2′-Chloro Nucleoside Analogs

Scheme 1

Figure imgf000136_0001
Figure imgf000136_0002

Ethyl (3R)-2-chloro-3-[(4R)-2,2-dimethyl-l,3-dioxolan-4-yl]-3-hydroxy-2- methylpropanoate (A2):

Figure imgf000137_0001

[00273] A 5 L flange flask was fitted with a thermometer, nitrogen inlet, pressure equalizing dropping funnel, bubbler, and a suba»seal. Methyl lithium solution (1.06 L, 1.6 M in diethylether, 1.7 equiv.) was added, and the solution was cooled to about -25 °C.Diisopropyl amine (238 ml, 1.7 equiv.) was added using the dropping funnel over about 40 minutes. The reaction was left stirring, allowing to warm to ambient temperature overnight. C02(s)/acetone cooling was applied to the LDA solution, cooling to about -70 °C.[00274] i?-Glyceraldehyde dimethylacetal solution (50% in DCM) was evaporated down to -100 mbar at a bath temp of 35 °C, to remove the DCM, then azeotroped with anhydrous hexane (200 ml), under the same Buchi conditions. 1H NMR was used to confirm that all but a trace of DCM remained.[00275] The fresh aldehyde (130 g, 1 mol) and ethyl 2-chloropropionionate (191 ml, 1.5 equiv.) were placed in a 1 L round bottom flask, which was filled with toluene (800 ml). This solution was cooled in a C02(s)/acetone bath, and added via cannula to the LDA solution over about 50 minutes, keeping the internal temperature of the reaction mixture cooler than -60 °C. The mixture was stirred with cooling (internal temp, slowly fell to ~ -72 °C) for 90 min, then warmed to room temperature over 30 minutes using a water bath. This solution was added to a sodium dihydrogen phosphate solution equivalent to 360 g of NaH2P04 in 1.5 L of ice/water, over about 10 minutes, with ice-bath cooling. The mixture was stirred for 20 minutes, then transferred to a sep. funnel, and partitioned. The aqueous layer was further extracted with EtOAc (2 x 1 L), and the combined organic extracts were dried over sodium sulfate. The volatiles were removed in vacuo (down to 20 mbar). The resultant oil was hydrolyzed crude.

(3R,4R,5R)-3-chIoro-4-hydroxy-5-(hydroxymethyI)-3-methyIoxoIan-2-one (A4):

Figure imgf000137_0002

H O CI[00276] The crude oil A2 was taken up in acetic acid (1.5 L, 66% in water) and heated to 90 °C over one hour, then at held at that temperature for one hour. Once the mixture had cooled to room temperature, the volatiles were removed in vacuo, and azeotroped with toluene (500 ml). The resultant oil was combined with some mixed material from an earlier synthesis and columned in two portions (each -1.25 L of silica, 38→ 75% EtOAc in DCM). The lower of the two main spots is the desired material; fractions containing this material as the major component were combined and the solvent removed in vacuo to give 82 g of orange solid whose 1 H NMR showed the material to be of about 57% purity (of the remainder 29% was the indicated epimer). This material was recrystallized fromtoluene/butanone (600 ml / -185 ml), the butanone being the ‘good’ solvent. The resultant solid was filtered washing with toluene and hexane, and dried in vacuo to give product of about 92% purity (30 g).(2R,3R,4R)-2-[(benzoyIoxy)methyI]-4-chIoro-4-methyI-5-oxooxoIan-3-yI benzoate(A5):

Figure imgf000138_0001

[00277] A 2 L 3 -neck round bottom flask was fitted with an overhead stirrer, thermometer and pressure equalizing dropping funnel (→N2). The intermediate A4 (160 mmol) in acetonitrile (1 L) was added, followed by 4-dimethylaminopyridine (3.2 mmol) and benzoyl chloride (352 mmol). Finally triethylamine (384 mmol) was added over 10 minutes using the dropping funnel. The addition of the triethylamine is accompanied by a mild exotherm, which obviated the addition of a cold water bath to keep the internal temperature below 25 °C. The reaction was stirred at ambient temperature for 2.5 hours. The reaction mixture was transferred to a sep. funnel with EtOAc (2 L) and half saturated brine (2 L), and partitioned. The aqueous layer was re-extracted with EtOAc (1 L). The combined organic layers were washed with 50%> sodium bicarbonate/25%) brine (1.5 L) and dried over sodium sulfate, to give 62 g of solid. This was recrystallized from 1.8 L of 1 : 1 toluene/trimethylpentane (95 °C), to give 52.4 g of product.[00278] 1H NMR (CDCls, 400 MHz): δ (ppm) 1.91 (s, 3H), 4.57 (dd, J= 5.12Hz and J = 12.57Hz, 1H), 4.77 (dd, J= 3.29Hz and J= 12.68Hz, 1H), 4.92-4.96 (m, 1H), 5.60 (d, J = 8.36Hz, 1H), 7.38-7.66 (m, 6H), 7.97-7.99 (m, 2H), 8.08-8.10 (m, 2H); MS (ESI) m/z= 411.1(MNa ).

3,5-Di-0-benzoyl-2-C-chloro-2-C-methyl-D-ribofuranose (A6):

Figure imgf000139_0001

[00279] To a solution of A5 (14.48 mmol) in anhydrous tetrahydrofurane (70 ml) was added under inert atmosphere at -35°C, LiAlH(OtBu)3 (1M in tetrahydrofurane, 21.7 mmol) over a 30 min period. The reaction mixture was stirred for 1 hour at -20 °C and quenched by addition of a saturated NH4C1 solution, keeping the temperature bellow 0 °C. Ethyl acetate was added and the white suspension was filtered through a pad of celite and washed with ethyl acetate. The filtrate was extracted with ethyl acetate twice. The combined organic layers were dried over anhydrous sodium sulfate, filtered and evaporated under reduced pressure. The residue was purified by chromatography on silica gel (eluent: petroleum ether/ethyl acetate 0 to 20%). The product was dried in vacuum (50 °C) overnight to afford expected intermediate as a colorless oil in 96% yield (mixture α/β: 45/55).[00280] 1H NMR (CDC13, 400 MHz): δ (ppm) 1.74 (s, 1.75HP), 1.76 (s, 1.25Ha), 4.42-4.69 (m, 3H), 5.30 (d, J= 12.8Hz, 0.55HP), 5.43-5.47 (m, 0.45Ha), 5.60 (d, J= 7.0Hz, 0.55HP), 5.78 (d, J= 7.0Hz , 0.45Ha), 7.35-7.41 (m, 2H), 7.45-7.56 (m, 3H), 7.59-7.65 (m, 1H), 7.96- 8.04 (m, 2H), 8.06-8.14 (m, 2H); MS (ESI) m/z= 413 (MNa+).3,5-Di-0-benzoyl-2-C-chloro-2-C-methyl-D-arabinofuranosyl bromide (A7):

Figure imgf000139_0002

[00281] To a solution of A6 (12.80 mmol) in anhydrous dichloromethane (80 ml) was added under inert atmosphere at -20 °C, triphenylphosphine (18.0 mmol). The reaction mixture was stirred for 15 minutes at -20 °C and CBr4 (19.20 mmol) was added. The reaction mixture was then stirred for 1 hour at -20 °C. The crude was partially concentrated under reduced pressure (bath temperature bellow 30 °C) and directly purified by chromatography on silica gel (eluent: petroleum ether/ethyl acetate 0 to 30%) to afford a mixture of β sugar A7a (1.67 g) and a sugar A7b (2.15 g) as a colorless gum in 66%> global yield.[00282] 1H NMR (CDC13, 400 MHz): β sugar δ (ppm) 1.93 (s, 3H), 4.60-4.88 (m, 3H), 6.08 (d, J= 7.9 Hz, 1H), 6.62 (s, 1H), 7.31-7.38 (m, 2H), 7.41-7.55 (m, 3H), 7.59-7.65 (m, 1H), 8.00-8.05 (m, 2H), 8.06-8.12 (m, 2H); a sugar δ (ppm) 1.88 (s, 3H), 4.66-4.89 (m, 3H), 5.37 (d, J= 4.88Hz, 1H), 6.44 (s, 1H), 7.41-7.55 (m, 4H), 7.54-7.65 (m, 2H), 8.00-8.05 (m, 2H), 8.14-8.20 (m, 2H); MS (ESI) m/z= 476/478 (MNa+).3 ,5′-Di-0-benzoyl-2′-C-chloro-2′-C-methyl-4-benzoyl-cytidine (A8):

Figure imgf000140_0001

[00283] To a suspension of N-benzoyl cytosine (9.48 mmol), and a catalytic amount of ammonium sulfate in 4-chlorobenzene (24 ml) was added HMDS (28.44 mmol). The reaction mixture was heated during 2 hours at 140 °C. The solvent was removed under inert atmosphere and the residue was taken in 4-chlorobenzene (15 ml). Then, A7b (4.74 mmol) in chlorobenzene (10 ml) was added dropwise to the reaction mixture followed by SnCl4 (14.22 mmol) dropwise. The reaction mixture was stirred at 70 °C overnight, cooled to room temperature and diluted with dichloromethane and a saturated NaHC03 solution. The white suspension was filtered through a pad of celite and washed with dichloromethane. The filtrate was extracted with dichloromethane twice. The combined organic layers were dried over anhydrous Na2S04, filtered and evaporated under reduced pressure to afford expected intermediate as a white solid in 89% yield.[00284] 1H NMR (DMSO, 400 MHz): δ (ppm) 1.58 (s, 3H), 4.68-4.81 (m, 3H), 5.68 (brs, 1H), 6.55 (brs, 1H), 7.36 (d, J= 7.84 Hz, 1H), 7.39-7.76 (m, 9H), 7.88-8.07 (m, 6H), 8.30 (d, J= 7.84 Hz, 1H); MS (ESI) m/z= 588 (MH+).3′,5′-Di-0-benzoyl-2,-C-chloro-2,-C-methyluridine (A9):

Figure imgf000140_0002

[00285] A suspension of A8 (4.19 mmol) in an acetic acid/water mixture (67 ml/17 ml, v/v), was heated at 110 °C for 3 hours. The reaction mixture was evaporated to dryness and co-evaporated with toluene (three times) to afford expected intermediate in quantitative yield as an oil which was directly used for the next step; MS (ESI) m/z= 485 (MH+). 2 -C-Chloro-2 -C-methyluridine (301):

Figure imgf000141_0001

H O CI[00286] Intermediate A9 (4.19 mmol) in 7 N methanolic ammonia (80 ml) was stirred at room temperature for 24 hours. The mixture was evaporated to dryness, diluted with water and transferred into a separatory funnel. The aqueous layer was extracted withdichloromethane and water was removed under reduced pressure. The residue was purified by flash RP18 gel chromatography (eluent: water/acetonitrile 0 to 40%) to afford pure expected compound as a white foam in 79% yield.[00287] 1H NMR (DMSO, 400 MHz): δ (ppm) 1.44 (s, 3H), 3.60-3.68 (m, 1H), 3.80-3.94 (m, 3H), 5.39 (t, J= 4.45 Hz, 1H), 5.63 (d, J= 8.26 Hz, 1H), 5.93 (d, J= 5.72 Hz, 1H), 6.21 (s, 1H), 8.16 (d, J= 8.90 Hz, 1H), 11.44 (m, 1H); MS (ESI) m/z= 277 (MH+).2′-C-Chloro-2′-C-methyl-3-benzyloxymethyluridine (Al 1):

Figure imgf000141_0002

H O CI[00288] To a solution of 301 (0.361 mmol) in anhydrous DMF (4 ml) was added at -5 °C, DBU (0.723 mmol) followed by benzyloxymethylchloride (0.542 mmol). The reaction mixture was stirred for 45 minutes between -5 °C and 5 °C. The solvent was evaporated under reduced pressure and the residue was purified by chromatography on silica gel (eluent: dichloromethane/methanol 0 to 10%) to afford pure expected intermediate as a white solid in 80% yield.[00289] 1H NMR (DMSO, 400 MHz): δ (ppm) 1.41 (s, 3H), 3.61-3.69 (m, 1H), 3.82-3.95 (m, 3H), 4.57 (s, 2H), 5.32 (s, 2H), 5.43 (t, J= 4.46Hz, 1H), 5.80 (d, J= 8.08Hz, 1H), 5.96 (d, J= 4.46 Hz, 1H), 6.23 (s, 1H), 7.22-7.36 (m, 5H), 8.25 (d, J= 8.22Hz, 1H); MS (ESI) m/z= 397 (MH+). Isopropyl (2S)-2-[[chloro(phenoxy)phosphoryl]amino]propanoate (A12a):

Figure imgf000142_0001

2,2-Dimethylpropyl (2S)-2-[[chloro(phenoxy)phosphoryl]amino]propanoate (A12b):

Figure imgf000142_0002

[00290] To a solution of aminoester, HC1 salt (0.434 mmol) in anhydrous dichloromethane (or acetonitrile) (4 ml) (3 times vacuo/nitrogen) under nitrogen was added at -30°C phenyldichlorophosphate (0.434 mmol) followed by N-methylimidazole (2.90 mmol)(or only 1.45 mmol for A12b). The reaction mixture was stirred at -30°C during 1 hour. The reaction was monitored by LC/MS (the sample was quenched by methanol or water) to check the complete formation of expected intermediate A12a [MS (ESI) m/z= 302 (MH+)(-OMe compounder A12b [MS (ESI) m/z= 314 (MH~)].Compound (A13a), (A13b) or (83ii):[00291] To the previous reaction mixture containing A12 was added All (or 302) (0.29 mmol) at -25°C under nitrogen. The reaction mixture was allowed to warm up slowly to room temperature overnight, and then diluted with dichloromethane and water (or with NaHCC”3 and EtOAc). The organic layer was extracted, dried, filtered and evaporated under reduced pressure. The crude residue was purified by chromatography on silica gel (eluent: dichloromethane/methanol 0 to 10%) (followed by preparative HPLC for A29).Compound (A13a):

Figure imgf000142_0003

[00292] Mixture of diastereoisomers; MS (ESI) m/z= 666 (MH+). Compound (A13b):

Figure imgf000143_0001

[00293] Mixture of diastereoisomers; MS (ESI) m/z= 692.3 (MH ).Compound (83ii):

Figure imgf000143_0002

[00294] Glassy solid; 1H NMR (CDCI3, 400MHz): δ (ppm) 1.19-1.24 (m, 9H), 1.35 (d, J = 7.1Hz, 3H), 3.95-4.05 (m, 1H), 4.31 (d, J= 8.1Hz, 2H), 4.41 (d, J= 9.0Hz, 1H), 4.59 (d, J = 7.1Hz, 2H), 4.98 (heptuplet, J= 6.28Hz, 1H), 6.38 (brs, 1H), 6.52 (s, 1H), 7.08-7.15 (m, 1H), 7.23-7.30 (m, 4H), 8.07 (s, 1H), 8.31 (s, 1H); 31P NMR (CDC13, 161.98 MHz): δ (ppm) 3.96 (s, IP); MS (ESI) m/z= 569.20 (MH+).Compounds (40iia) and (40iib):

Figure imgf000143_0003

[00295] To a solution of A13 (0.29 mmol) in anhydrous ethanol (6 ml) was added trifluoroacetic acid (2.9 mmol) dropwise (then 3 times vacuo/nitrogen purges), followed by Palladium hydroxide (20% on Carbon). The reaction mixture was purged 3 timesvacuo/nitrogen, and 3 times vacuo/hydrogen and then stirred under hydrogen for 5 hours. The reaction mixture was diluted with ethyl acetate and filtered through a pad of celite. The filtrate was evaporated under reduced pressure, and the crude compound was purified by preparative MS/HP LC to afford two pure compounds in 48% global yield.[00296] Compound 40ii (diastereoisomer 1): white solid; 1H NMR (CDC13, 400 MHz): δ (ppm) 1.22-1.26 (m, 6H), 1.37 (d, J= 7.08 Hz, 3H), 1.51 (s, 3H), 3.71-3.88 (m, 2H), 3.97- 4.06 (m, 1H), 4.16-4.18 (m, 1H), 4.45-4.57 (m, 2H), 4.97-5.07 (m, 1H), 5.57 (d, J= 8.20 Hz, 1H), 6.39 (s, 1H), 7.18-7.37 (m, 5H), 7.44 (d, J= 8.20 Hz, 1H), 8.40 (s, 1H); 31P NMR (CDC13, 161.98 MHz): δ (ppm) 4.20 (s, IP); MS (ESI, El+) m/z= 546 (MH+).[00297] Compound 40ii (diastereoisomer 2): white solid; 1H NMR (CDC13, 400 MHz): δ (ppm) 1.24-1.26 (m, 6H), 1.36 (d, J= 7.04 Hz, 3H), 1.59 (s, 3H), 3.69-3.77 (m, 1H), 3.91- 3.99 (m, 2H), 4.17-4.19 (m, 1H), 4.43-4.59 (m, 2H), 5.01-5.06 (m, 1H), 5.68 (d, J= 8.20 Hz, 1H), 6.42 (s, 1H), 7.21-7.39 (m, 5H), 7.60 (d, J=8.20 Hz, 1H), 8.14 (s, 1H); 31P NMR (CDC13, 161.98 MHz): δ (ppm) 3.47 (s, IP); MS (ESI) m/z= 546 (MH+).Compound 42ii:

Figure imgf000144_0001

[00298] Compound 42ii was synthesized from compound A13b (0.144 mmol) as described for compound 40ii.[00299] White solid; 1H NMR (MeOD, 400 MHz) δ (ppm) 0.94 (s, 9H), 1.40 (d, J= 7.10 Hz, 3H), 1.53 (s, 3H), 3.76 (d, J= 10.43 H, 1H), 3.86 (d, J= 10.44 H, 1H), 3.98-4.06 (m, 2H), 4.18-4.22 (m, 1H), 4.39-4.44 (m, 1H), 4.52-4.57 (m, 1H), 5.62 (d, J= 8.18 Hz, 1H), 6.40 (s, 1H), 7.20-7.29 (m, 3H), 7.36-7.41 (m, 2H), 7.74 (d, J= 8.18 Hz, 1H); 31P NMR (MeOD, 161.98 MHz) δ (ppm) 3.68 (s, IP); MS (ESI) m/z = 574.08 (MH+).

PAPER

US 20170226146

https://patents.google.com/patent/US20170226146A1/en

  • [0250]
  • [0251]
    A 3-neck 100 mL jacketed round bottom flask with nitrogen inlet and mechanical stirrer was charged with compound 4 (3.0 g, 10.8 mmol), compound 13 (0.484 g, 2.17 mmol, 0.20 equiv), 2-butanone (21 mL), and 2,6-lutidine (2.53 mL, 21.7 mmol, 2.0 equiv). The resulting slurry was cooled to −15° C., then a solution of compound 12 (7.96 g, 13.0 mmol) in 2-butanone (3 mL) was added over 14 hours. The reaction mixture was allowed to stir at −15° C. for an additional 25 hours and then warmed to 20° C. n-Heptane (16 mL) was added with stirring over a 1 hour period then the mixture was allowed to stir at 25° C. for 3 hours, then filtered through a fitted funnel. The filter cake was slurry-washed with a 3:2 mixture of 2-butanone and n-heptane (10 mL and then 15 mL), then dried by pulling nitrogen stream through the fritted funnel. The filter cake was slurried in a 10:1 mixture of water and 2-butanone (21 mL) and then filtered. This slurrying and filtration sequence was repeated two more times. The resulting filter cake was dried with nitrogen stream through the fritted funnel to provide compound 6.

Example 21Alternate Preparation of Compound A

  • [0252]
  • [0253]
    Compound 6 (0.072 mmol, 1 equiv), K2HPO(63.0 mg, 0.361 mmol) and compound 14 (5.45 mg, 0.018 mmol) were added to a 1 dram vial with 4 A mol sieves (40 mg). To the resulting mixture was added DCM (800 μl), then the resulting reaction was allowed to stir for 5 minutes. To the reaction mixture was then added compound 14 (28.7 mg, 0.094 mmol, 1.3 equiv) and the resulting reaction was allowed to stir for about 15 hours at room temperature to provide Compound A.
  • [0256]
  • [0257]
    A 100 mL reactor with nitrogen inlet and mechanical stirrer was charged with compound 4 (7.00 g, 25.3 mmol), compound 15 (0.225 g, 0.506 mmol, 0.020 equiv), 1,3-dioxolane (42 mL), and 2,6-lutidine (4.42 mL, 38.0 mmol, 1.5 equiv). The mixture was cooled to −10° C. and a 33 wt % solution of compound 12 in isopropyl acetate (29 mL, 30 mmol) was added over 1 hour. The reaction mixture was allowed to stir at −10° C. for additional 40 hours, then isopropyl acetate (28 mL) was added, and the resulting mixture was warmed to 0° C. A 10 wt % aqueous NaHSOsolution was added (14 mL), and the mixture was allowed to stir at 30° C. for 30 minutes, then the layers were separated. To the organic layer was added an aqueous solution containing 5 wt % NaHCOand 5 wt % Na2SO(21 mL). The mixture was allowed to stir at 50° C. for 6 h. The layers were separated. To the organic layer was added 10 wt % aqueous NaCl solution (21 mL). The mixture was allowed to stir at 50° C. for 30 min. The organic layer was separated, combined with isopropyl acetate (5 mL) and concentrated in vacuo to half volume at 20000 pa in a 50° C. bath. The resulting solution was solvent-switched with isopropanol (4×35 mL) to 60 g weight. The mixture was seeded with 100 mg of compound A at 60° C. The resulting slurry was allowed to stir at 55° C. for 30 minutes, then n-Heptane (35 mL) was added over 1 hour at 55° C. The resulting slurry was allowed to stir for an additional 1 hour at 55° C., then cooled to room temperature and filtered. The filter cake was washed with a 1:1 mixture of isopropanol and n-heptane (3×14 mL), followed by n-heptane (14 mL), then dried under nitrogen to provide Compound A.

PAPER

https://pubs.rsc.org/en/content/articlelanding/2021/sc/d1sc01978c#!divAbstract

Uprifosbuvir is an antiviral agent developed for treatment of chronic hepatitis C infections. Its original synthesis route requires twelve steps with an overall yield of only 1 %. Such a difficult and time-consuming synthesis approach is acceptable for the early trial phase of a new drug, but impractical for broad application as hepatitis C treatment or for repurposing against novel viral diseases.

Artis Klapars, John Y. L. Chung, and colleagues, Merck & Co., Inc., Rahway, NJ, USA, and WuXi STA, Shanghai, China, have developed a synthesis route for uprifosbuvir requiring only five steps and starting from readily available uridine. Initially, uridine is selectively oxidized after OH-acylation with pivaloyl chloride in an acyl migration/oxidation process driven by complexation with the Lewis acid BF3*OEt2 in toluene. In the second step, methylation is achieved by MeMgBr/MgCl2 in a toluene/anisole mixture where a more reactive methyl-manganese species is formed in-situ from the Grignard reagent, providing high yield and a good diastereomeric ratio (dr). Subsequently, the tertiary chloride group is introduced. Due to the high functional-group density, a cyclodehydration step is required before chlorination to avoid side reactions. The chlorination is carried out using dichlorodimethylsilane with FeCl3*6H2O and tetramethyldisiloxane as additives which avoids the hazardous use of HCl gas under pressure required in the initial synthesis. In the final step, the regioselective phosphoramidation is achieved using a chlorophosphoramidate precursor and a dimeric chiral imidazole carbamate catalyst which led to a dr of 97:3 starting from a 1:1 diastereomeric mixture of the chlorophosphoramidate reagent.

Uprifosbuvir was synthesized with an overall yield of 50 %, a vast improvement compared to the 1 % of the original synthesis route. Additionally, the newly developed synthesis steps have the potential to provide easier access to other nucleoside-based antiviral agents.


Efficient synthesis of antiviral agent uprifosbuvir enabled by new synthetic methods

Artis Klapars,  *a

This article is Open Access

Creative Commons BY license

All publication charges for this article have been paid for by the Royal Society of Chemistry

Abstract

An efficient route to the HCV antiviral agent uprifosbuvir was developed in 5 steps from readily available uridine in 50% overall yield. This concise synthesis was achieved by development of several synthetic methods: (1) complexation-driven selective acyl migration/oxidation; (2) BSA-mediated cyclization to anhydrouridine; (3) hydrochlorination using FeCl3/TMDSO; (4) dynamic stereoselective phosphoramidation using a chiral nucleophilic catalyst. The new route improves the yield of uprifosbuvir 50-fold over the previous manufacturing process and expands the tool set available for synthesis of antiviral nucleotides.

Graphical abstract: Efficient synthesis of antiviral agent uprifosbuvir enabled by new synthetic methods

Scheme 1 Synthetic approaches to uprifosbuvir 1 with the two main challenges highlighted. (a) Me2NH, AcOH, EtOH/MeOH, 80 °C, 1.5 h; (b) Ca(OH)2, water, 70 °C, 24 h, 19% over 2 steps.9

Scheme 3 Complexation-driven selective acyl migration/oxidation to access 12. (a) PivCl, pyridine, 0 °C, 16 h; (b) BF3·OEt2, PhMe, 40 °C, 10 h; (c) TEMPO, Bu4NBr, AcOOH, dioctyl sulphide, PhMe, −10 °C to 20 °C, 24 h, 83% from 5.

Scheme 6 Completion of uprifosbuvir synthesis. (a) TMS-Cl, iPrOH, 70 °C, 12 h; (b) NEt3, iPrOAc, wiped film evaporation, 80%; (c) PhOP(O)Cl2, NEt3, iPrOAc, −20 °C, 2 h, 90%; (d) C6F5OH, NEt3, iPrOAc, −5 °C to 10 °C, 18 h, 76%;26 (e) 4, 3 mol% 24, 2,6-lutidine, 1,3-dioxolane, −10 °C, 24 h, 88%; (f) 4, tBuMgCl, THF, −5 °C to 5 °C, 15 h, 50%;27 (g) 4, Me2AlCl, 2,6-lutidine, THF, 35 °C, 16 h, 81%.27

Scheme 7 Summary of uprifosbuvir synthesis. AY = assay yield; IY = isolated yield. 

https://www.rsc.org/suppdata/d1/sc/d1sc01978c/d1sc01978c1.pdf

PAPERhttps://www.sciencedirect.com/science/article/abs/pii/S0960894X17308314

References

  1. ^ Soriano V, Fernandez-Montero JV, de Mendoza C, Benitez-Gutierrez L, Peña JM, Arias A, Barreiro P (August 2017). “Treatment of hepatitis C with new fixed dose combinations”. Expert Opinion on Pharmacotherapy18 (12): 1235–1242. doi:10.1080/14656566.2017.1346609PMID 28644739S2CID 205819421.
  2. ^ Borgia G, Maraolo AE, Nappa S, Gentile I, Buonomo AR (March 2018). “NS5B polymerase inhibitors in phase II clinical trials for HCV infection”. Expert Opinion on Investigational Drugs27 (3): 243–250. doi:10.1080/13543784.2018.1420780PMID 29271672S2CID 3672885.
  3. ^ Lawitz E, Gane E, Feld JJ, Buti M, Foster GR, Rabinovitz M, et al. (September 2019). “Efficacy and safety of a two-drug direct-acting antiviral agent regimen ruzasvir 180 mg and uprifosbuvir 450 mg for 12 weeks in adults with chronic hepatitis C virus genotype 1, 2, 3, 4, 5 or 6”. Journal of Viral Hepatitis26 (9): 1127–1138. doi:10.1111/jvh.13132PMID 31108015S2CID 160014275.
 
Clinical data
Trade names Uprifosbuvir
Legal status
Legal status US: Investigational New Drug
Identifiers
showIUPAC name
CAS Number 1496551-77-9
PubChem CID 90055716
DrugBank DB15206
ChemSpider 57427403
UNII JW31KPS26S
KEGG D10996
ChEMBL ChEMBL3833371
Chemical and physical data
Formula C22H29ClN3O9P
Molar mass 545.9 g·mol−1
3D model (JSmol) Interactive image
showSMILES
showInChI

Uprifosbuvir (MK-3682) is an antiviral drug developed for the treatment of Hepatitis C. It is a nucleotide analogue which acts as an NS5B RNA polymerase inhibitor. It is currently in Phase III human clinical trials.[1][2][3]

References

  1. ^ Soriano V, Fernandez-Montero JV, de Mendoza C, Benitez-Gutierrez L, Peña JM, Arias A, Barreiro P (August 2017). “Treatment of hepatitis C with new fixed dose combinations”. Expert Opinion on Pharmacotherapy18 (12): 1235–1242. doi:10.1080/14656566.2017.1346609PMID 28644739S2CID 205819421.
  2. ^ Borgia G, Maraolo AE, Nappa S, Gentile I, Buonomo AR (March 2018). “NS5B polymerase inhibitors in phase II clinical trials for HCV infection”. Expert Opinion on Investigational Drugs27 (3): 243–250. doi:10.1080/13543784.2018.1420780PMID 29271672S2CID 3672885.
  3. ^ Lawitz E, Gane E, Feld JJ, Buti M, Foster GR, Rabinovitz M, et al. (September 2019). “Efficacy and safety of a two-drug direct-acting antiviral agent regimen ruzasvir 180 mg and uprifosbuvir 450 mg for 12 weeks in adults with chronic hepatitis C virus genotype 1, 2, 3, 4, 5 or 6”. Journal of Viral Hepatitis26 (9): 1127–1138. doi:10.1111/jvh.13132PMID 31108015S2CID 160014275.

//////////uprifosbuvir, MK 3682, ウプリホスブビル, уприфосбувирأوبريفوسبوفير , 乌磷布韦 , IDX-21437DB15206SB18784D10996Q27281714, IDX 21437, PHASE 3 
CC(C)OC(=O)C(C)NP(=O)(OCC1C(C(C(O1)N2C=CC(=O)NC2=O)(C)Cl)O)OC3=CC=CC=C3

wdt-24

NEW DRUG APPROVALS

ONE TIME

$10.00

Sitravatinib


Sitravatinib.png
File:Sitravatinib.svg - Wikipedia

Sitravatinib

1-N‘-[3-fluoro-4-[2-[5-[(2-methoxyethylamino)methyl]pyridin-2-yl]thieno[3,2-b]pyridin-7-yl]oxyphenyl]-1-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide

1-N’-[3-fluoro-4-[2-[5-[(2-methoxyethylamino)methyl]pyridin-2-yl]thieno[3,2-b]pyridin-7-yl]oxyphenyl]-1-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide

MG-91516

1,1-Cyclopropanedicarboxamide, N-[3-fluoro-4-[[2-[5-[[(2-methoxyethyl)amino]methyl]-2-pyridinyl]thieno[3,2-b]pyridin-7-yl]oxy]phenyl]-N’-(4- fluorophenyl)-

N-(3-fluoro-4-((2-(5-(((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide

シトラバチニブ; ситраватиниб , سيترافاتينيب , 司曲替尼 , 
FormulaC33H29F2N5O4S
Cas1123837-84-2
Mol weight629.6763

MG-516

Sitravatinib (MGCD516)

UNII-CWG62Q1VTB

CWG62Q1VTB

MGCD-516

MGCD516

Antineoplastic, Receptor tyrosine kinase inhibitor

Sitravatinib (MGCD516) is an experimental drug for the treatment of cancer. It is a small molecule inhibitor of multiple tyrosine kinases.

Sitravatinib is being developed by Mirati Therapeutics.[1]

Ongoing phase II trials include a trial for liposcarcoma,[2] a combination trial for non-small cell lung cancer,[3] and a combination trial with nivolumab for renal cell carcinoma.[4]

Mirati Therapeutics and licensee BeiGene are developing sitravatinib, an oral multitargeted kinase inhibitor which inhibits Eph, Ret, c-Met and VEGF-1, -2 and -3, DDR, Trk, Axl kinases, CHR4q12, TYRO3 and Casitas B-lineage, in combination with immune checkpoint inhibitors, for treating advanced solid tumors.

In March 2021, sitravatinib was reported to be in phase 3 clinical development.

PDT PATENT

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2009026717

WO2009026717 , in which sitravatinib was first disclosed, claiming heterocyclic compounds as multi kinase inhibitors.

Scheme 10



Example 52
N-(3-Fluoro-4-(2-(5-((2-methoxyethylamino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7- yloxy)phenyl)-N-(4-fluorophenyl)cyclopropane- 1 , 1 -dicarboxamide

Step 1 : tert-Butyl (6-(7-(2-Fluoro-4-(1-(4-fluorophenylcarbamoyl)-cyclopropanecarboxamido)phenoxy)thieno [3 ,2-b]pyridin-2-yl)pyridin-3 -y l)methyl(2-methoxyethyl)carbamate (146)
To aniline 126 (0.58 g, 1.1 mmol) and DIPEA (0.58 mL, 0.43 g, 3.3 mmol) in dry DMF

(20 mL) was added 1-(4-fluorophenylcarbamoyl)cyclopropanecarbpxylic acid (0.35 g, 1.5 mmol) and HATU (0.72 g, 1.9 mmol) and the mixture was stirred at r.t. for 18 h. It was then partitioned between ethyl acetate and water, the organic phase was washed with water, IM NaOH, brine, dried (MgSO4), filtered, and concentrated. Silica gel chromatography (ethyl acetate) afforded title compound Ϊ46 (0.60 g, 74 % yield). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 10.40 (s, 1H), 10.01 (s, 1H), 8.52-8.49 (m, 2H), 8.33 (s, 1H), 8.27-8.24 (m, 1H), 7.92-7.88 (m, 1H), 7.78 (dd, J = 8.2, 2.1 Hz, 1H) 7.65-7.60 (m, 2H), 7.52-7.42 (m, 2H), 7.14 (t, J = 8.8 Hz, 2H), 6.65 (d, J = 5.1 Hz 1H), 4.47 (s, 2H), 3.42-3.30 (m, 4H), 3.22 (s, 3H), 1.46-1.30 (m, 13H). MS (m/z): 730.1 (M+H).
Step 2. N-(3-Fluoro-4-(2-(5-((2-methoxyethylamino)methyl)pyridin-2-yl)thieno[3,2-blpyridin-7-yloxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide (147)
To the compound 146 (0.59 g, 0.81 mmol) in dichloromethane (50 mL) was added TFA (3 mL). The solution was stirred for 18 h then concentrated. The residue was partitioned between dichloromethane and 1 M NaOH, and filtered to remove insolubles. The organic phase was collected, washed with IM NaOH, brine, dried (MgSO4), filtered, and concentrated to afford title compound 147 (0.35 g, 69 % yield).

1H NMR (400 MHz, DMSO-d6) δ (ppm): 10.40 (s, 1H), 10.01 (s, 1H), 8.55 (d, J = 1.6 Hz, 1H), 8.51 (d, J = 5.3 Hz, 1H), 8.31 (s, 1H), 8.22 (d, J = 8.0 Hz, 1H), 7.92-7.87 (m, 2H), 7.65-7.61 (m, 2H), 7.52-7.43 (m, 2H), 7.17-7.12 (m, 2H), 6.64 (d, J = 5.5 Hz, 1H), 3.77 (s, 2H), 3.40 (t, J = 5.7 Hz, 2H), 3.23 (s, 3H), 2.64 (t, J = 5.7 Hz, 2H), 1.46 (br s, 4H). MS (m/z): 630.1 (M+H).

PATENT

WO 2009026720 

https://patents.google.com/patent/WO2009026720A1

PATENT

WO-2021050580

Novel, stable crystalline polymorphic forms (form D) of sitravatinib , useful for treating a multi tyrosine kinase-associated cancer eg sarcoma, glioma, non-small cell lung, bladder, kidney, ovarian, gastric, breast or liver cancer. 

 International publication No. W02009/026717A disclosed compounds with the inhibition activities of multiple protein tyrosine kinases, for example, the inhibition activities of VEGF receptor kinase and HGF receptor kinase. In particular, disclosed N-(3-fluoro-4-((2-(5-(((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane- 1,1 -di carboxamide (Compound 1) is a multi-tyrosine kinase inhibitor with demonstrated potent inhibition of a closely related spectrum of tyrosine kinases, including RET, CBL, CHR4ql2, DDR and Trk, which are key regulators of signaling pathways that lead to cell growth, survival and tumor progression.

[003]

Compound 1

[004] Compound 1 shows tumor regression in multiple human xenograft tumor models in mice, and is presently in human clinical trials as a monotherapy as well as in combination for

treating a wide range of solid tumors. Compound 1 is presently in Phase 1 clinical trial for patients with advanced cancer, in Phase 2 studies for patients with advanced liposarcoma and non-small cell lung cancer (NSCLC).

[005] The small scale chemical synthesis of the amorphous Compound 1 had been disclosed in the Example 52 (compound 147) of W02009/026717A, however, in order to prepare the API of Compound 1 with high quality and in large quantity, crystalline forms of Compound 1 would be normally needed so the process impurities could be purged out by recrystallization.

Practically, it is difficult to predict with confidence which crystalline form of a particular compound will be stable, reproducible, and suitable for phamaceutical processing. It is even more difficult to predict whether or not a particular crystalline solid state form will be produced with the desired physical properties for pharmaceutical formulations.

[006] For all the foregoing reasons, there is a great need to produce crystalline forms of Compound 1 that provide manufacturing improvements of the pharmaceutical composition.

The present invention advantageously addresses one or more of these needs.

EXAMPLE 1

Preparation of N-(3-fluoro-4-((2-(5-(((2-methoxyethyl)amino)methyl)pyridin-2- yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane-l,l- dicarboxamide (Compound 1)

[0085] This Example illustrates the preparation ofN-(3-fluoro-4-((2-(5-(((2-methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N-(4-fluorophenyl)cyclopropane- 1,1 -di carboxamide (Compound 1).

[0086] Step 1: N-(Y6-bromopyridin-3-vDmethvD-2-methoxyethan-l-amine (Compound 1A)

Compound 1A

[0087] To a stirred solution of 2-Methoxyethylamine (3.0 eq) in dichloromethane (DCM) (12 vol) was added Molecular sieves (0.3 w/w) and stirred for 2 hours at 25±5°C under nitrogen atmosphere. The reaction mass water content was monitored by Karl Fischer analysis until the water content limit reached 0.5 % w/w. Once the water content limit was reached, the reaction mass cooled to 5±5°C and 6-bromonicotinaldehyde (1.0 eq) was added lot wise over period of 30 minutes to the above reaction mass at 5±5°C. The reaction mass was stirred for 30±5 minutes at 5±5°C and acetic acid (1.05 eq) was added drop wise at 5±5°C. After completion of the addition, the mass was slowly warmed to 25±5°C and stirred for 8 h to afford Compound 1 A. The imine formation was monitored by HPLC.

[0088] Step 2: tert-butyl (Y6-brom opyri din-3 -vQmethvO(2-m ethoxy ethvDcarbamate (Compound

IB)

Compound 1B

[0089] Charged Compoud 1A (1.0 eq) in THF (5.0 vol) was added and the reaction mass was stirred for 30 minutes at 25±5°C under nitrogen atmosphere. The reaction mass was cooled to temperature of about 10±5°C. Di-tert- butyl dicarbonate (1.2 eq) was added to the reaction mass at 10±5°C under nitrogen atmosphere and the reaction mass temperature was raised to 25±5°C and the reaction mass for about 2 hours. The progress of the reaction was monitored by HPLC. After IPC completion, a prepared solution of Taurine (1.5 eq) in 2M aq NaOH (3.1 vol) was charged and stirred at 10±5°C for 16 h to 18 h. The reaction mass was further diluted with 1M aq.NaOH solution (3.7 vol) and the layers were separated. The aqueous layer was extracted with DCM (2 x 4.7vol) and the extract combined with the organic layer. The combined organic layers were washed with 1M aq.NaOH solution (3.94 vol), followed by water (2×4.4 vol), and dried over sodium sulfate (2.0 w/w) . The filtrate was concentrated under reduced pressure below 40° C until no distillate was observed. Tetrahydrofuran (THF) was sequentially added (1×4 vol and lx 6vol) and concentrated under reduced pressure below 40°C until no distillate was observed to obtained Compound IB as light yellow colored syrup liquid.

[0090] Step 3: tert-butyl 7-chlorothieno[3.2-b1pyridin-2-yl)pyridin-3-yl )methyl)(2- 

methoxyethvDcarbamate (Compound 1C)

Compound 1C

[0091] To a stirred solution of 7-chlorothieno[3,2-b]pyridine (1.05 eq) in tetrahydrofuran (7 vol) was added n-butyl lithium (2.5 M in hexane) drop wise at -15±10°C and stirred for 90 minutes at same temperature under nitrogen atmosphere. Zinc chloride (1.05 eq) was added to the reaction mass at -15±10°C. The reaction mass was slowly warmed to 25±5°C and stirred for 45 minutes under nitrogen atmosphere to afford Compound 1C. The progress of the reaction was monitored by HPLC.

[0092] Step 4: tert-butyl (Y6-(7-(4-amino-2-fluorophenoxy)thieno[3.2-b1pyridin-2-v0pyridin-3-vDmethvD(2-methoxyethvDcarbamate (Compound ID)

Compound 1D

[0093] 3-fluoro-4-hydroxybenzenaminium chloride (1.2 eq) in DMSO (3.9 vol) at 25±5°C was charged under nitrogen atmosphere and the reaction mass was stirred until observance of a clear solution at 25±5°C. t-BuOK was added lot wise under nitrogen atmosphere at 25±10°C. The reaction mass temperature was raised to 45±5°C and maintained for 30 minutes under nitrogen atmosphere. Compound 1C was charged lot-wise under nitrogen atmosphere at 45±5°C and stirred for 10 minutes at 45± 5°C.The reaction mixture was heated to 100± 5°C and stirred for 2 hrs. The reaction mass is monitored by HPLC.

[0094] After reaction completion, the reaction mass was cooled to 10± 5°C and quenched with chilled water (20 vol) at 10±5°C. The mass temperature was raised to 25± 5°C and stirred for 7-8 h. The resulting Compound ID crude was collected by filtration and washed with 2 vol of water. Crude Compound ID material taken in water (10 vol) and stirred for up to 20 minutes at 25±5°C. The reaction mass was heated to 45±5°C and stirred for 2-3 h at 45±5°C, filtered and vacuum-dried.

[0095] Crude Compound ID was taken in MTBE (5 vol) at 25±5°C and stirred for about 20 minutes at 25±5°C. The reaction mass temperature was raised to 45±5°C, stirred for 3-4 h at 45±5°C and then cooled to 20±5°C. The reaction mass was stirred for about 20 minutes at 20±5°C, filtered, followed by bed wash with water (0. 5 vol) and vacuum-dried.

[0096] The crude material was dissolved in acetone (10 vol) at 25±5°C and stirred for about 2h at 25±5°C. The reaction mass was filtered through a celite bed and washed with acetone (2.5 vol). The filtrate was slowly diluted with water (15 vol) at 25±5°C. The reaction mass was stirred for 2-3 h at 25±5°C, filtered and bed washed with water (2 vol) & vacuum-dried to afford Compound ID as brown solid.

[0097] Step 5 : 1 -((4-((2-(5-(((tert-butoxycarbonv0(2-methoxy ethvOaminolmethvOpyri din-2 -yl )thieno[3.2-b]pyridin-7-yl )oxy)-3 -fluorophenyl icarbamoyl level opropane-1 -carboxylic acid (Compound IE)

Compound 1E

[0098] To a solution of Compound ID (1.0 eq.) in tetrahydrofuran (7 vol.), aqueous potassium carbonate (1.0 eq.) in water (8 vol.) was added. The solution was cooled to 5±5°C, and stirred for about 60 min. While stirring, separately triethylamine (2.0 eq.) was added to a solution of 1,1-cyclopropanedicarboxylic acid (2.0 eq.) in tetrahydrofuran (8 vol.), at 5±5°C, followed by thionyl chloride (2.0 eq.) and stirred for about 60 min. The acid chloride mass was slowly added to the Compound ID solution at 5±5°C. The temperature was raised to 25±5°C and stirred for 3.0 h. The reaction was monitored by HPLC analysis.

[0099] After reaction completion, the mass was diluted with ethyl acetate (5.8 vol.), water (5.1 vol.), 10% (w/w) aqueous hydrochloric acid solution (0.8 vol.) and 25% (w/w) aqueous sodium chloride solution (2 vol.). The aqueous layer was separated and extracted with ethyl acetate (2 x 5 vol.). The combined organic layers were washed with a 0.5M aqueous sodium bicarbonate solution (7.5 vol.). The organic layer was treated with Darco activated charcoal (0.5 w/w) and sodium sulfate (0.3 w/w) at 25±5°C for 1.0 h. The organic layer was filtered through celite and washed with tetrahydofuran (5.0 vol.). The filtrate was concentrated under vacuum below 50°C to about 3 vol and co-distilled with ethyl acetate (2 x 5 vol.) under vacuum below 50°C up to ~ 3.0 vol. The organic layer was cooled to 15±5°C, stirred for about 60 min., filtered, and the solid was washed with ethyl acetate (2.0 vol.). The material was dried under vacuum at 40±5°C until water content was less than 1% to afford Compound IE as brown solid.

[00100] Step 6: tert-butyl (Y6-(7-(2-fluoro-4-(T-(Y4-fluorophenvDcarbamovDcvclopropane-l-carboxamido)phenoxy)thieno[3.2-b]pyridin-2-v0pyri din-3 – (2- 
methoxyethvDcarbamate (Compound IF)

[00101] Pyridine (1.1 eq.) was added to a suspension of Compound IE (1.0 eq.) in tetrahydrofuran (10 vol.) and cooled to 5±5°C. Thionyl chloride (2.0 eq.) was added and stirred for about 60 min. The resulting acid chloride formation was confirmed by HPLC analysis after quenching the sample in methanol. Separately, aqueous potassium carbonate (2.5 eq.) solution (7.0 vol. of water) was added to a solution of 4-fluoroaniline (3.5 eq.) in tetrahydrofuran (10 vol.), cooled to 5±5°C, and stirred for about 60 min. The temperature of the acid chloride mass at 5±5°C was raised to a temperature of about 25±5°C and stirred for 3 h. The reaction monitored by HPLC analysis.

[00102] After completion of the reaction, the solution was diluted with ethyl acetate (25 vol.), the organic layer was separated and washed with a 1M aqueous sodium hydroxide solution (7.5 vol.), a 1M aqueous hydrochloric acid solution (7.5 vol.), and a 25% (w/w) aqueous sodium chloride solution (7.5 vol.). The organic layer was dried and and filtered with sodium sulfate (1.0 w/w). The filtrate was concentrated ~ 3 vol under vacuum below 50°C and co-distilled with ethyl acetate (3 x 5 vol.) under vacuum below 50°C to ~ 3.0 vol. Ethyl acetate (5 vol.) and MTBE (10 vol.) were charged, heated up to 50±5°C and stirred for 30-60 min. The mixture was cooled to 15±5°C, stirred for about 30 min., filtered, and the solid was washed with ethyl acetate (2.0 vol.). MGB3 content was analyzed by HPLC analysis. The material was dried under vacuum at 40±5°C until the water content reached about 3.0% to afford Compound IF as brown solid.

[00103] Step 7 : N-(3-fluoro-4-((2-(5-(((2-methoxyethv0amino)methv0pyridin-2-yl )thieno[3.2-b]pyridin-7-yl )oxy)phenyl)-N-(4-fluorophenyl level opropane-1. 1 -dicarboxamide (Compound 1)

Compound 1

[0100] To a mixture of Compound IF in glacial acetic acid (3.5 vol.) concentrated hydrochloric acid (0.5 vol.) was added and stirred at 25±5°C for 1.0 h. The reaction was monitored by HPLC analysis.

[0101] After reaction completion, the mass was added to water (11 vol.) and stirred for 20±5°C for 30 min. The pH was adjusted to 3.0 ± 0.5 using 10% (w/w) aqueous sodium bicarbonate solution and stirred for 20±5°C for approximately 3.0 h.. The mass was filtered, washed with water (4 x 5.0 vol.) and the pH of filtrate was checked after every wash. The material was dried under vacuum at 50±5°C until water content was about 10%.

[0102] Crude Compound 1 was taken in ethyl acetate (30 vol.), heated to 70±10°C, stirred for 1.0 h., cooled to 25±5°C, filtered, and washed with ethyl acetate (2 vol.). The material was dries under vacuum at 45±5°C for 6.0 h.

[0103] Crude Compound 1 was taken in polish filtered tetrahydrofuran (30 vol.) and pre washed Amberlyst A-21 Ion exchange resin and stirred at 25±5°C until the solution became clear. After getting the clear solution, the resin was filtered and washed with polish filtered tetrahydrofuran (15 vol.). The filtrate was concentrated by -50% under vacuum below 50°C and co-distilled with polish filtered IPA (3 x 15.0 vol.) and concentrated up to -50% under vacuum below 50°C. Charged polish filtered IPA (15 vol.) was added and the solution concentrated under vacuum below 50°C to – 20 vol. The reaction mass was heated to 80±5°C, stirred for 60 min. and cooled to 25±5°C. The resultant reaction mass was stirred for about 20 hours at 25±5°C. The reaction mass was cooled to 0±5°C, stirred for 4-5 hours, filtered, and washed with polish filtered IPA (2 vol.). The material was dried under vacuum at 45±5°C, until the water content was about 2%, to obtain the desired product Compound 1. ¾-NMR (400 MHz, DMSO- d): 510.40 (s, 1H), 10.01 (s, 1H), 8.59 – 8.55 (m, 1H), 8.53 (d, J= 5.6 Hz, 1H), 8.32 (s, 1H), 8.23 (d, J= 8.0 Hz, 1H), 7.96 – 7.86 (m, 2H), 7.70 – 7.60 (m, 2H), 7.56 – 7.43 (m, 2H), 7.20 – 7.11 (m, 2H), 6.66 (d, J= 5.6 Hz, 1H), 3.78 (s, 2H), 3.41 (t, J= 5.6 Hz, 2H), 3.25 (s, 3H), 2.66 (t, J= 5.6 Hz, 2H), 1.48 (s, 4H)ppm. MS: M/e 630 (M+l)+.

EXAMPLE 2

Preparation of Crystalline Form D of N-(3-fluoro-4-((2-(5-(((2- methoxyethyl)amino)methyl)pyridin-2-yl)thieno[3,2-b]pyridin-7-yl)oxy)phenyl)-N-(4- fluorophenyl)cyclopropane-l, 1-dicarboxamide

EXAMPLE 2A: Preparation of Compound 1 Crystalline Form D

[0104] To a 50 L reactor, 7.15 Kg of Compound 1, 40 g of Form D as crystal seed and 21 L acetone (>99%) were added. The mixture was heated to reflux ( ~56 °C) for 1~2 h. The mixture was agitated with an internal temperature of 20±5 °C for at least 24 h. Then, the suspension was filtered and washed the filter cake with 7 L acetone. The wet cake was dried under vacuum at <45 °C, to obtain 5.33 kg of Compound 1 of desired Form D

[0105] X-Ray Powder Diffraction (XRPD)

The XRPD patterns were collected with a PAN alytical X’ Pert PRO MPD diffractometer using auincident beam of Cu radiation produced using au Optix long, fine-focus source. An elliptically graded multilayer mirror was used to focus Cu Ka X -rays through the specimens and onto the detector. Prior to the analysis, a silicon specimen (NIST SRM 640e) was analyzed to verify the observed position of the Si Ill peak is consistent with the NIST-certified position. A specimen of each sample was sandwiched between 3 -pm -thick films and analyzed in transmission geometly. A beam-stop, short autiscatter extension, and an autiscatter knife edge were used to minimize the background generated by air. Sober slits for the incident aud diffracted beauls were used to minimize broadening from axial divergence. The diffraction patterns were collected using a scanning position-sensitive detector (X’Celerator) located 240 mm from the specimens and Data Collector software v. 2.2b. Pattern Match v2.3.6 was used to create XRPD patterns.

[0106] The X-ray powder diffraction (XRPD) pattern was used to characterize the Compound 1 obtained, which showed that the Compound 1 was in Crystalline Form D of Compound 1 (Compound 1 Form D), see Figure 1A. The XRPD pattern yielded is substantially the same as that shown in Figure 3C.

[0107] Differential Scanning Calorimetry (DSC)

[0108] DSC was performed using a Mettler-Toledo DSC3+ differential scanning calorimeter. Temperature calibration was performed using octane, phenyl salicylate, indium, tin, and zinc. The TAWN sensitivity was 11.9. The samples were placed into aluminum DSC pans, covered with lids, and the weights were accurately recorded. A weighed aluminum pan configured as the sample pan was placed on the reference side of the cell. The pan lids were pierced prior to sample analyses. The method name on the thermograms is an abbreviation for the start and end temperature as well as the heating rate; e.g., -30-250-10 means “from ambient to 250°C, at 10°C/min.” The nitrogen flow rate was 50.0 mL/min. This instrument does not provide gas pressure value as required by USP because it is the same as atmospheric pressure.

[0109] A broad small endotherm with a peak maximum at approximately 57°C to 62°C (onset ~20°C to 22°C) followed by a sharp endotherm with a peak maximum at approximately 180°C (onset ~178°C) were observed. These events could be due to the loss of volatiles and a melt, respectively (see Figure IB).

[0110] In an alternative embodiment Form D was prepared as follows. Designated Material O was suspended in 600 pL of acetone. Initial dissolution was observed followed by re precipitation. The amount of suspended solids was not measured because the target of the experiment was to get a suspension with enough solids to slurry isolate and collect XRPD data. Based on the solubility of Form D in acetone a very rough estimate for the scale of the experiment is about 80-100mg. The suspension was stirred at ambient temperature for approximately 2 5 weeks after which the solids were isolated by centrifugation with filtration. XRPD data appeared to be consistent with Form D The sample was then dried in vacuum oven at ~40 °C for ~2 5 hours. The XRPD pattern of the final solids was consistent with Form D EXAMPLE 2B: Preparation of Compound 1 Form D

[0111] 427.0 mg of Compound 1 was dissolved in 5 mL of THF to obtain a clear brown solution. The resulting solution was filtered, and the filtrate evaporated under flow of nitrogen. A sticky solid was obtained, which was dried under vacuum in room temperature for ~5 min, still a sticky brown solid obtained. It was dissolved in 0.2 mL of EtOAc and sonicated to dissolve. The solution obtained was stirred at room temperature for 15 min and a solid precipitated. The resulting solid was added 0.4 mL of EtOAc and stirred in room temperature for 21 h 40 min to ontian a suspension. The solid was spparated from mother liquor by centrifugation, then the resulting solid was resuspended the in 0.6 mL of EtOAc and stirred in room temperature for 2 days. The solid was isolated by centrifugation, to obtain Compound 1 of desired Form D.

[0112] The X-ray powder diffraction (XRPD) pattern was used to characterize the Compound 1 obtained, which showed that the Compound 1 was in Crystalline Form D of Compound 1 (Compound 1 Form D).

EXAMPLE 2C: Preparation of Compound 1 Form D

[0113] Single crystal X-ray diffraction data of Compound 1 was collected at 180 K on a Rigaku XtaLAB PRO 007HF(Mo) diffractometer, with Mo Ka radiation (l = 0.71073 A). Data reduction and empirical absorption correction were performed using the CrysAlisPro program. The structure was solved by a dual-space algorithm using SHELXT program. All non-hydrogen atoms could be located directly from the difference Fourier maps. Framework hydrogen atoms were placed geometrically and constrained using the riding model to the parent atoms. Final structure refinement was done using the SHELXL program by minimizing the sum of squared deviations of F2 using a full-matrix technique.

Preparation of Compound 1 Form D ( a Single Crystal )

[0114] Compound 1 Form D was dissolved in a mixture of acetone/ ACN (1/2) with the concentration of Compound 1 at ~7 mg/mL. A block single crystal was obtained, which was a single crystal.

[0115] The XRPD pattern was used to characterize the single crystal of Compound 1 Form D obtained, see Figure 2A. The crystal structural data are summarized in Table IB. The refined single crystal structure were shown in Figure 2B. The single crystal structure of Compound 1 Form D is in the P-1 space group and the triclinic crystal system. The terminal long alkyl chain is found to have large ellipsoids, indicating high mobility with disordered atoms.

[0116] The theoretical XRPD calculated from the single crystal structure and experimental XRPD are essentially similar (Figure 2A). A few small peaks are absent or shift because of orientation preference, disorder and tested temperature (180 K for single crystal data and 293 K for experimental one).

[0117] Table IB. Crystal Data and Structure Refinement for Compound 1 Form D (a Single Crystal)

References

  1. ^ http://www.mirati.com/go/mgcd516/
  2. ^ “MGCD516 in Advanced Liposarcoma and Other Soft Tissue Sarcomas – Full Text View – ClinicalTrials.gov”.
  3. ^ “Phase 2 Study of Glesatinib, Sitravatinib or Mocetinostat in Combination With Nivolumab in Non-Small Cell Lung Cancer – Full Text View – ClinicalTrials.gov”.
  4. ^ “MGCD516 Combined With Nivolumab in Renal Cell Cancer (RCC) – Full Text View – ClinicalTrials.gov”.
Identifiers
showIUPAC name
CAS Number1123837-84-2
ChemSpider52083477
UNIICWG62Q1VTB
KEGGD11140
Chemical and physical data
FormulaC33H29F2N5O4S
Molar mass629.68 g·mol−1
3D model (JSmol)Interactive image
hideSMILESCOCCNCc1ccc(nc1)c2cc3c(s2)c(ccn3)Oc4ccc(cc4F)NC(=O)C5(CC5)C(=O)Nc6ccc(cc6)F
hideInChIInChI=1S/C33H29F2N5O4S/c1-43-15-14-36-18-20-2-8-25(38-19-20)29-17-26-30(45-29)28(10-13-37-26)44-27-9-7-23(16-24(27)35)40-32(42)33(11-12-33)31(41)39-22-5-3-21(34)4-6-22/h2-10,13,16-17,19,36H,11-12,14-15,18H2,1H3,(H,39,41)(H,40,42)Key:WLAVZAAODLTUSW-UHFFFAOYSA-N

///////////// sitravatinib, phase 3, シトラバチニブ , MGCD516, MG-516Sitravatinib (MGCD516)UNII-CWG62Q1VTBCWG62Q1VTBMGCD-516ситраватиниб , سيترافاتينيب , 司曲替尼 , Antineoplastic, MGCD 516

#sitravatinib, #phase 3, #シトラバチニブ , #MGCD516, #MG-516#Sitravatinib (MGCD516), #UNII-#CWG62Q1VTB, #CWG62Q1VTB, #MGCD-516ситраватиниб , سيترافاتينيب , 司曲替尼 , #Antineoplastic, #MGCD516

COCCNCC1=CN=C(C=C1)C2=CC3=NC=CC(=C3S2)OC4=C(C=C(C=C4)NC(=O)C5(CC5)C(=O)NC6=CC=C(C=C6)F)F

NIROGACESTAT


Nirogacestat.png
img
Structure of NIROGACESTAT

NIROGACESTAT

(2S)-2-[[(2S)-6,8-difluoro-1,2,3,4-tetrahydronaphthalen-2-yl]amino]-N-[1-[1-(2,2-dimethylpropylamino)-2-methylpropan-2-yl]imidazol-4-yl]pentanamide

489.6 g/mol, C27H41F2N5O

CAS 1290543-63-3

PF-03084014, 1290543-63-3, PF-3084014, 865773-15-5QZ62892OFJUNII:QZ62892OFJUNII-QZ62892OFJнирогацестат [Russian] [INN]نيروغاسيستات [Arabic] [INN]尼罗司他 [Chinese] [INN]ニロガセスタット;

orphan drug designation in June 2018 for the treatment of desmoid tumors, and with a fast track designation

 Nirogacestat, also known as PF-03084014, is a potent and selective gamma secretase (GS) inhibitor with potential antitumor activity. PF-03084014 binds to GS, blocking proteolytic activation of Notch receptors. Nirogacestat enhances the Antitumor Effect of Docetaxel in Prostate Cancer. Nirogacestat enhances docetaxel-mediated tumor response and provides a rationale to explore GSIs as adjunct therapy in conjunction with docetaxel for men with CRPC (castration-resistant prostate cancer).

Nirogacestat was disclosed to be a gamma-secretase inhibitor, which can inhibit Aβ-peptide production. SpringWorks Therapeutics (a spin-out of Pfizer ) is developing nirogacestat, as hydrobromide salt, a gamma-secretase inhibitor, for treating aggressive fibromatosis. In February 2021, nirogacestat was reported to be in phase 3 clinical development.

Nirogacestat is a selective gamma secretase (GS) inhibitor with potential antitumor activity. Nirogacestat binds to GS, blocking proteolytic activation of Notch receptors; Notch signaling pathway inhibition may follow, which may result in the induction of apoptosis in tumor cells that overexpress Notch. The integral membrane protein GS is a multi-subunit protease complex that cleaves single-pass transmembrane proteins, such as Notch receptors, at residues within their transmembrane domains. Overexpression of the Notch signaling pathway has been correlated with increased tumor cell growth and survival.

Nirogacestat has been used in trials studying the treatment of Breast Cancer, HIV Infection, Desmoid Tumors, Advanced Solid Tumors, and Aggressive Fibromatosis, among others.

SpringWorks Therapeutics

Nirogacestat (Gamma Secretase Inhibitor)

Nirogacestat is an oral, selective, small molecule, gamma secretase inhibitor (GSI) in Phase 3 clinical development for patients with desmoid tumors. Gamma secretase is a protease complex that cleaves, or divides, multiple transmembrane protein complexes, including Notch, which, when dysregulated, can play a role in activating pathways that contribute to desmoid tumor growth.

Gamma secretase has also been shown to directly cleave BCMA, a therapeutic target that is highly expressed on multiple myeloma cells. By inhibiting gamma secretase with nirogacestat, membrane-bound BCMA can be preserved, thereby increasing target density while simultaneously reducing levels of soluble BCMA, which may serve as decoy receptors for BCMA-directed therapies. Together, these mechanisms combine to potentially enhance the activity of BCMA therapies and improve outcomes for multiple myeloma patients. SpringWorks is seeking to advance nirogacestat as a cornerstone of multiple myeloma combination therapy in collaboration with industry leaders who are advancing BCMA therapies.

SpringWorks Therapeutics Announces Clinical Collaboration with Pfizer

By Satish  October 05, 2020 

SpringWorks Therapeutics today announced that the company has entered into a clinical trial collaboration agreement with Pfizer to evaluate SpringWorks Therapeutics’ investigational gamma secretase inhibitor (GSI), nirogacestat, in combination with Pfizer’s anti-B-cell maturation antigen (BCMA) CD3 bispecific antibody, PF‐06863135, in patients with relapsed or refractory multiple myeloma.

Gamma secretase inhibition prevents the cleavage and shedding of BCMA from the surface of myeloma cells. In preclinical models, nirogacestat has been shown to increase the cell surface density of BCMA and reduce levels of soluble BCMA, thereby enhancing the activity of BCMA-targeted therapies, including CD3 bispecific antibodies.

Saqib Islam, Chief Executive Officer of SpringWorks Therapeutics Said: This collaboration is another important step in continuing to advance our goal of developing nirogacestat as a best-in-class BCMA potentiator, and we are pleased to work with Pfizer to study nirogacestat in combination with PF‐06863135, which has recently demonstrated promising monotherapy clinical data, We now have five collaborations with industry-leading BCMA developers to evaluate nirogacestat in combinations across modalities. We look forward to generating clinical data with our collaborators to further evaluate the ability of nirogacestat to improve outcomes for patients with multiple myeloma.

Under the terms of the agreement, Pfizer will sponsor and conduct the Phase 1b/2 study to evaluate the safety, tolerability and preliminary efficacy of the combination, and will assume all costs associated with the study, other than expenses related to the manufacturing of nirogacestat and certain expenses related to intellectual property rights. Pfizer and SpringWorks Therapeutics will also form a joint development committee to manage the clinical study, which is expected to commence in the first half of 2021.

Chris Boshoff, MD, PhD, Chief Development Officer for Pfizer Oncology at Pfizer Said: Entering into this clinical collaboration is a proud milestone in our strong relationship with SpringWorks,We believe that studying nirogacestat in combination with PF-06863135 could hold significant therapeutic promise for patients with relapsed or refractory multiple myeloma, and we look forward to working together to advance this important area of research.

In addition to its ongoing clinical collaborations with BCMA-directed therapies, SpringWorks is also currently conducting a global Phase 3, double-blind, randomized, placebo-controlled clinical trial (the DeFi Trial) to evaluate nirogacestat in adults with progressing desmoid tumors.

About Nirogacestat

Nirogacestat is an investigational, oral, selective, small molecule gamma secretase inhibitor in Phase 3 clinical development for desmoid tumors, which are rare and often debilitating and disfiguring soft-tissue tumors. Gamma secretase cleaves multiple transmembrane protein complexes, including Notch, which is believed to play a role in activating pathways that contribute to desmoid tumor growth.

In addition, gamma secretase has been shown to directly cleave membrane-bound BCMA, resulting in the release of the BCMA extracellular domain, or ECD, from the cell surface. By inhibiting gamma secretase, membrane-bound BCMA can be preserved, increasing target density while reducing levels of soluble BCMA ECD, which may serve as decoy receptors for BCMA-directed therapies. Nirogacestat’s ability to enhance the activity of BCMA-directed therapies has been observed in preclinical models of multiple myeloma. SpringWorks is evaluating nirogacestat as a BCMA potentiator and has five collaborations with industry-leading BCMA developers to evaluate nirogacestat in combinations across modalities, including with an antibody-drug conjugate, two CAR T cell therapies and two bispecific antibodies. In addition, SpringWorks and Fred Hutchinson Cancer Research Center have entered into a sponsored research agreement to further characterize the ability of nirogacestat to modulate BCMA and potentiate BCMA directed therapies using a variety of preclinical and patient-derived multiple myeloma models developed by researchers at Fred Hutch.

Nirogacestat has received Orphan Drug Designation from the U.S. Food and Drug Administration (FDA) for the treatment of desmoid tumors (June 2018) and from the European Commission for the treatment of soft tissue sarcoma (September 2019). The FDA also granted Fast Track and Breakthrough Therapy Designations for the treatment of adult patients with progressive, unresectable, recurrent or refractory desmoid tumors or deep fibromatosis (November 2018 and August 2019).

About PF‐06863135

PF‐06863135 is an anti-B-cell maturation antigen (BCMA) CD3 bispecific antibody being investigated in a Phase 1 clinical study to treat relapsed or refractory multiple myeloma. This bispecific antibody can be administered subcutaneously and has been optimized for binding affinity to both BCMA and CD3, enabling more potent T-cell-mediated tumor cell toxicity.

Source: SpringWorks Therapeutics

FDA Grants Breakthrough Designation to Nirogacestat for Desmoid Tumors

The FDA has granted nirogacestat, an investigational gamma-secretase inhibitor, with a breakthrough therapy designation for the treatment of adult patients with progressive, unresectable, recurrent or refractory desmoid tumors or deep fibromatosis.

The FDA has granted nirogacestat (PF-03084014), an investigational gamma-secretase inhibitor, with a breakthrough therapy designation for the treatment of adult patients with progressive, unresectable, recurrent or refractory desmoid tumors or deep fibromatosis.1

The breakthrough designation was granted as a result of positive findings seen in phase I and II trials of nirogacestat monotherapy in patients with desmoid tumors. A phase III trial has also been initiated investigating nirogacestat in patients with desmoid tumors or aggressive fibromatosis (NCT03785964).

“We are committed to pursuing the rapid development of nirogacestat given the important need for new therapies for patients with desmoid tumors and are pleased to receive this breakthrough therapy designation,” Saqib Islam, CEO of SpringWorks, the company developing the small molecule inhibitor, said in a statement. “We are currently enrolling adult patients in our phase III DeFi trial and will continue to work closely with the FDA with the goal of bringing nirogacestat to patients as quickly as possible.”

The open-label, single-center phase II trial of nirogacestat enrolled 17 patients with desmoid tumors who were not eligible for surgical resection or definitive radiation therapy and who had experienced disease progression after at least 1 prior treatment regimen. Patients received 150 mg twice per day of continuous, oral nirogacestat in 21-day cycles.2

The median age of patients was 34 years (range, 19-69), 82% of the patients were female, and 53% of patients had aCTNNB1T41A somatic missense mutation. The median number of prior therapies was 4 (range, 1-9), which included cytotoxic chemotherapy in 71% and a tyrosine kinase inhibitor in 59%.

Sixteen patients were evaluable for response. After a median follow-up of more than 25 months, 5 patients (29%) achieved a partial response and 11 (65%) had stable disease, for a disease control rate of 100%. Ten patients (59%) remained on treatment with nirogacestat for more than 2 years.

Grade 1/2 adverse events were observed in all patients, with diarrhea (76%) and skin disorders (71%) being the most common toxicities. The only treatment-related grade 3 event was reversible hypophosphatemia, which was reported in 8 patients (47%) and was considered to be a class effect of gamma-secretase inhibitors. Four patients met the criteria for dose reduction.

Findings from the phase I study also showed a disease control rate of 100% with nirogacestat. However, the median progression-free survival was not reached in either study due to a lack of patients progressing on treatment. Only 1 patient discontinued treatment due to an adverse event between the 2 studies.1

The FDA had previously granted nirogacestat with an orphan drug designation in June 2018 for the treatment of desmoid tumors, and with a fast track designation in November 2018 for the treatment of adult patients with progressive, unresectable, recurrent or refractory desmoid tumors or deep fibromatosis.

References

  1. SpringWorks Therapeutics Receives Breakthrough Therapy Designation for Nirogacestat for the Treatment of Adult Patients with Progressive, Unresectable, Recurrent or Refractory Desmoid Tumors [press release]. Stamford, CT: SpringWorks Therapeutics, Inc; August 29, 2019. https://bit.ly/30IV0Eb. Accessed September 3, 2019.
  2. Kummar S, O&rsquo;Sullivan Coyne G, Do KT, et al. Clinical Activity of the &gamma;-Secretase Inhibitor PF-03084014 in Adults With Desmoid Tumors (Aggressive Fibromatosis).J Clin Oncol.2017;35(14):1561-1569. doi: 10.1200/JCO.2016.71.1994.

PAPER

str1-png

Bioorganic & medicinal chemistry letters (2011), 21(9), 2637-40.

https://www.sciencedirect.com/science/article/abs/pii/S0960894X10018822

Design, synthesis, and in vivo characterization of a novel series of tetralin amino imidazoles as γ-secretase inhibitors: Discovery of PF-3084014 - ScienceDirect
Design, synthesis, and in vivo characterization of a novel series of tetralin amino imidazoles as γ-secretase inhibitors: Discovery of PF-3084014 - ScienceDirect
Design, synthesis, and in vivo characterization of a novel series of tetralin amino imidazoles as γ-secretase inhibitors: Discovery of PF-3084014 - ScienceDirect

PATENT

WO 2016089208

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2016089208

PATENT

WO-2021029854

Novel, stable crystalline polymorphic (A to N) and amorphous forms of nirogacestat hydrobromide , useful for treating desmoid tumors such as multiple myeloma, a cancer having a mutation in a Notch pathway gene, adenoid cystic carcinoma and T-cell acute lymphoblastic leukemia.

(S)-2-(((S)-6,8-difluoro-l,2,3,4-tetrahydronaphthalen-2-yl)amino)-N-(l-(2- methyl- l-(neopentylamino) propan-2-yl)-lH-imidazol-4-yl)pentanamide (“Compound 1”) is a gamma-secretase inhibitor which can inhibit Ab-peptide production.

[0003] Not all compounds that are gamma-secretase inhibitors have characteristics affording the best potential to become useful therapeutics. Some of these characteristics include high affinity at the gamma-secretase, duration of gamma-secretase deactivation, oral bioavailability, tissue distribution, and stability (e.g., ability to formulate or crystallize, shelf life). Favorable characteristics can lead to improved safety, tolerability, efficacy, therapeutic index, patient compliance, cost efficiency, manufacturing ease, etc.

[0004] In addition, the isolation and commercial -scale preparation of a solid state form of hydrobromide salts of Compound 1 and corresponding pharmaceutical formulations having acceptable solid state properties (including chemical stability, thermal stability, solubility, hygroscopicity, and/or particle size), compound manufacturability (including yield, impurity rejection during crystallization, filtration properties, drying properties, and milling properties), and formulation feasibility (including stability with respect to pressure or compression forces during tableting) present a number of challenges.

[0005] Accordingly, there is a current need for one or more solid state forms of hydrobromide salts of Compound 1 that have an acceptable balance of these properties and can be used in the preparation of pharmaceutically acceptable solid dosage forms.

Crystalline Form A

[0147] In one aspect, the present disclosure relates to crystalline Form A of a hydrobromide salt of (S)-2-(((S)-6,8-difluoro-l,2,3,4-tetrahydronaphthalen-2-yl)amino)- N-(l -(2 -methyl- l-(neopentylamino) propan-2-yl)-lH-imidazol-4-yl)pentanamide having Formula (I),

[0148] In one embodiment, crystalline Form A is anhydrous.

[0149] In another embodiment, the melting point of crystalline Form A is about 254 °C.

[0150] In another embodiment, Form A is characterized by an XRPD pattern having peaks at 8.8 ± 0.2, 9.8 ± 0.2, and 23.3 ± 0.2 degrees two theta when measured by Cu Ka radiation. In another embodiment, Form A is characterized by an XRPD pattern having peaks at 8.8 ± 0.2, 9.8 ± 0.2, 23.3 ± 0.2, 25.4 ± 0.2, 28.0 ± 0.2, and 29.3 ± 0.2 degrees two theta when measured by Cu Ka radiation. In another embodiment, Form A is characterized by an XRPD pattern having peaks at 8.8 ± 0.2, 9.8 ± 0.2, 20.0 ± 0.2, 23.3 ± 0.2, 25.4 ± 0.2, 28.0 ± 0.2, 29.3 ± 0.2, and 32.5 ± 0.2 degrees two theta when measured by Cu Ka radiation.

Patent

Product case, WO2005092864 ,

hold protection in the EU states until March 2025, and expire in the US in February 2026 with US154 extension.

PATENT

WO2020208572 , co-assigned to GSK and SpringWorks, claiming a combination of nirogacestat with anti-BCMA antibody (eg belantamab mafodotin ), for treating cancer.

PATENT

US10590087 , for a prior filing from Pfizer, claiming crystalline forms of nirogacestat hydrobromide.

////////////NIROGACESTAT, orphan drug designation, esmoid tumors,  fast track designation, PF-03084014, PF 03084014, QZ62892OFJ , UNII:QZ62892OFJ ,UNII-QZ62892OFJ, ,нирогацестат , نيروغاسيستات , 尼罗司他 , ニロガセスタット, phase 3

CCCC(C(=O)NC1=CN(C=N1)C(C)(C)CNCC(C)(C)C)NC2CCC3=C(C2)C(=CC(=C3)F)F

TROFINETIDE


ChemSpider 2D Image | Trofinetide | C13H21N3O6
Trofinetide structure.png

Trofinetide

  • Molecular FormulaC13H21N3O6
  • Average mass315.322 Da

Tofinetide , NNZ-256610076853400-76-7[RN]
glycyl-2-methyl-L-prolyl-L-glutamic acid
H-Gly-PMe-Glu-OHL-Glutamic acid, glycyl-2-methyl-L-prolyl-UNII-Z2ME8F52QLZ2ME8F52QLтрофинетид [Russian] [INN]تروفينيتيد [Arabic] [INN]曲非奈肽 [Chinese] [INN]

IUPAC CondensedH-Gly-aMePro-Glu-OH
SequenceGXE
HELMPEPTIDE1{G.[*C(=O)[C@@]1(CCCN1*)C |$_R2;;;;;;;;_R1;$|].E}$$$$
IUPACglycyl-alpha-methyl-L-prolyl-L-glutamic acid

An (1-3) IGF-1 analog with neuroprotective activity.

OPTICAL ROT; -52.4 °   Conc: 0.19 g/100mL;  water ;  589.3 nm; Temp: 20 °C; Len: 1.0 dm…Tetrahedron 2005, V61(42), P10018-10035 

EU Customs Code CN, 29339980

Harmonized Tariff Code, 293399

  • L-Glutamic acid, glycyl-2-methyl-L-prolyl-
  • glycyl-2-methyl-L-prolyl-L-glutamic acid
  • Glycyl-L-2-methylprolyl-L-glutamic acid
2D chemical structure of 853400-76-7

Trofinetide (NNZ-2566) is a drug developed by Neuren Pharmaceuticals that acts as an analogue of the neuropeptide (1-3) IGF-1, which is a simple tripeptide with sequence GlyProGlu formed by enzymatic cleavage of the growth factor IGF-1 within the brain. Trofinetide has anti-inflammatory properties and was originally developed as a potential treatment for stroke,[1][2] but has subsequently been developed for other applications and is now in Phase II clinical trials against Fragile X syndrome and Rett syndrome.[3][4][5]

Trofinetide (NNZ-2566), a neuroprotective analogue of glypromate, is a novel molecule that has a profile suitable for both intravenous infusion and chronic oral delivery. It is currently in development to treat traumatic brain injury.

In February 2021, Neuren is developing trofinetide (NNZ-2566, phase 2 clinical ), a small-molecule analog of the naturally occurring neuroprotectant and N-terminus IGF-1 tripeptide Glypromate (glycine-proline-glutamate), for intravenous infusion treatment of various neurological conditions, including moderate to severe traumatic brain injury (TBI), stroke, chronic neurodegenerative disorders and peripheral neuropathies. At the same time, Neuren is also investigating an oral formulation of trofinetide (phase 3 clinical) for similar neurological indications, including mild TBI.

Autism Spectrum Disorders and neurodevelopment disorders (NDDs) are becoming increasingly diagnosed. According to the fourth edition of the American Psychiatric Association’s (APA) Diagnostic and Statistical Manual oƒ Mental Disorders (DSM-4), Autism spectrum disorders (ASD) are a collection of linked developmental disorders, characterized by abnormalities in social interaction and communication, restricted interests and repetitive behaviours. Current classification of ASD according to the DSM-4 recognises five distinct forms: classical autism or Autistic Disorder, Asperger syndrome, Rett syndrome, childhood disintegrative disorder and pervasive developmental disorder not otherwise specified (PDD-NOS). A sixth syndrome, pathological demand avoidance (PDA), is a further specific pervasive developmental disorder.

More recently, the fifth edition of the American Psychiatric Association’s (APA) Diagnostic and Statistical Manual oƒ Mental Disorders (DSM-5) recognizes recognises Asperger syndrome, childhood disintegrative disorder, and pervasive developmental disorder not otherwise specified (PDD-NOS) as ASDs.

This invention applies to treatment of disorders, regardless of their classification as either DSM-4 or DSM-5.

Neurodevelopment Disorders (NDDs) include Fragile X Syndrome (FXS), Angelman Syndrome, Tuberous Sclerosis Complex, Phelan McDermid Syndrome, Rett Syndrome, CDKL5 mutations (which also are associated with Rett Syndrome and X-Linked Infantile Spasm Disorder) and others. Many but not all NDDs are caused by genetic mutations and, as such, are sometimes referred to as monogenic disorders. Some patients with NDDs exhibit behaviors and symptoms of autism.

As an example of a NDD, Fragile X Syndrome is an X-linked genetic disorder in which affected individuals are intellectually handicapped to varying degrees and display a variety of associated psychiatric symptoms. Clinically, Fragile X Syndrome is characterized by intellectual handicap, hyperactivity and attentional problems, autism spectrum symptoms, emotional lability and epilepsy (Hagerman, 1997a). The epilepsy seen in Fragile X Syndrome is most commonly present in childhood, but then gradually remits towards adulthood. Hyperactivity is present in approximately 80 percent of affected males (Hagerman, 1997b). Physical features such as prominent ears and jaw and hyper-extensibility of joints are frequently present but are not diagnostic. Intellectual handicap is the most common feature defining the phenotype. Generally, males are more severely affected than females. Early impressions that females are unaffected have been replaced by an understanding of the presence of specific learning difficulties and other neuropsychiatric features in females. The learning disability present in males becomes more defined with age, although this longitudinal effect is more likely a reflection of a flattening of developmental trajectories rather than an explicit neurodegenerative process.

The compromise of brain function seen in Fragile X Syndrome is paralleled by changes in brain structure in humans. MRI scanning studies reveal that Fragile X Syndrome is associated with larger brain volumes than would be expected in matched controls and that this change correlates with trinucleotide expansion in the FMRP promoter region (Jakala et al, 1997). At the microscopic level, humans with Fragile X Syndrome show abnormalities of neuronal dendritic structure, in particular, an abnormally high number of immature dendritic spines (Irwin et al, , 2000).

Currently available treatments for NDDs are symptomatic – focusing on the management of symptoms – and supportive, requiring a multidisciplinary approach. Educational and social skills training and therapies are implemented early to address core issues of learning delay and social impairments. Special academic, social, vocational, and support services are often required. Medication, psychotherapy or behavioral therapy may be used for management of co-occurring anxiety, ADHD, depression, maladaptive behaviors (such as aggression) and sleep issues, Antiepileptic drugs may be used to control seizures.

Patent

WO 2014085480,

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2014085480

str1-1

EP 0 366 638 discloses GPE (a tri-peptide consisting of the amino acids Gly-Pro-Glu) and its di-peptide derivatives Gly-Pro and Pro-Glu. EP 0 366 638 discloses that GPE is effective as a neuromodulator and is able to affect the electrical properties of neurons.

WO95/172904 discloses that GPE has neuroprotective properties and that administration of GPE can reduce damage to the central nervous system (CNS) by the prevention or inhibition of neuronal and glial cell death.

WO 98/14202 discloses that administration of GPE can increase the effective amount of choline acetyltransferase (ChAT), glutamic acid decarboxylase (GAD), and nitric oxide synthase (NOS) in the central nervous system (CNS).

WO99/65509 discloses that increasing the effective amount of GPE in the CNS, such as by administration of GPE, can increase the effective amount of tyrosine hydroxylase (TH) in the CNS to increase TH-mediated dopamine production in the treatment of diseases such as Parkinson’s disease.

WO02/16408 discloses certain GPE analogs having amino acid substitutions and certain other modification that are capable of inducing a physiological effect equivalent to GPE within a patient. The applications of the GPE analogs include the treatment of acute brain injury and neurodegenerative diseases, including injury or disease in the CNS.

EXAMPLES

The following examples are intended to illustrate embodiments of this invention, and are not intended to limit the scope to these specific examples. Persons of ordinary skill in the art can apply the disclosures and teachings presented herein to develop other embodiments without undue experimentation and with a likelihood of success. All such embodiments are considered part of this invention.

Example 1: Synthesis of N,N-Dimethylglycyl-L-prolyl)-L-glutamic acid

The following non-limiting example illustrates the synthesis of a compound of the invention, N,N-Dimethylglycyl-L-prolyl-L-glutamic acid

All starting materials and other reagents were purchased from Aldrich; BOC=tert-butoxycarbonyl; Bn=benzyl.

BOC-L-proline-(P-benzyl)-L-glutamic acid benzyl ester

To a solution of BOC-proline [Anderson GW and McGregor AC: J. Amer. Chem. Soc: 79, 6810, 1994] (10 mmol) in dichloromethane (50 mi), cooled to 0°C, was added triethylamine (1 .39 ml, 10 mmol) and ethyl chloroformate (0.96 ml, 10 mmol). The resultant mixture was stirred at 0 °C for 30 minutes. A solution of dibenzyl-L-glutamate (10 mmol) was then added and the mixture stirred at 0° C for 2 hours then warmed to room temperature and stirred overnight. The reaction mixture was washed with aqueous sodium bicarbonate and citric acid (2 mol 1-1) then dried (MgSO4) and concentrated at reduced pressure to give BOC-L-proline-L-glutamic acid dibenzyl ester (5.0 g, 95%).

L-proline-L-glutamic acid dibenzyl ester

A solution of BOC-L-glutamyl-L-proline dibenzyl ester (3.4 g, 10 mmol), cooled to 0 °C, was treated with trifluoroacetic acid (25 ml) for 2 h. at room temperature. After removal of the volatiles at reduced pressure the residue was triturated with ether to give L-proline-L-glutamic acid dibenzyl ester.

N,N-Dimethylglycyl-L-prolyl-L-glutamic acid

A solution of dicyclohexylcarbodiimide (10.3 mmol) in dichloromethane (10 ml) was added to a stirred and cooled (0 °C) solution of L-proline-L-glutamic acid dibenzyl ester (10 mmol), N,N-dimethylglycine (10 mmol) and triethylamine ( 10.3 mmol) in dichloromethane (30 ml). The mixture was stirred at 0°C overnight and then at room temperature for 3 h. After filtration, the filtrate was evaporated at reduced pressure. The resulting crude dibenzyl ester was dissolved in a mixture of ethyl acetate (30 ml) and methanol (30 ml) containing 10% palladium on charcoal (0.5 g) then hydrogenated at room temperature and pressure until the uptake of hydrogen ceased. The filtered solution was evaporated and the residue recrystallised from ethyl acetate to yield the tripeptide derivative.

It can be appreciated that following the method of the Examples, and using alternative amino acids or their amides or esters, will yield other compounds of Formula 1.

Eample 2: Synthesis of Glycyl-L-2-Methyl-L-Prolyl-L-Glutamate

L-2-Methylproline and L-glutamic acid dibenzyl ester p-toluenesulphonate were purchased from Bachem, N-benzyloxycarbonyl-glycine from Acros Organics and bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BoPCl, 97%) from Aldrich Chem. Co.

Methyl L-2-methylprolinate hydrochloride 2

Thionyl chloride (5.84 cm3, 80.1 mmol) was cautiously added dropwise to a stirred solution of (L)-2-methylproline 1 (0.43 g, 3.33 mmol) in anhydrous methanol (30 cm3) at -5 °C under an atmosphere of nitrogen. The reaction mixture was heated under reflux for 24 h, and the resultant pale yellow-coloured solution was. concentrated to dryness in vacuo. The residue was dissolved in a 1 : 1 mixture of methanol and toluene (30 cm3) then concentrated to dryness to remove residual thionyl chloride. This procedure was repeated twice more, yielding hydrochloride 2 (0.62 g, 104%) as an hygroscopic, spectroscopically pure, off-white solid: mp 127- 131 °C; [α]D -59.8 (c 0.24 in CH2Cl2); vmax (film)/cm-1 3579, 3398 br, 2885, 2717, 2681 , 2623, 2507, 1743, 1584, 1447, 1432, 1374, 1317, 1294, 1237, 1212, 1172, 1123, 981 , 894, 861 and 764; δH (300 MHz; CDCl3; Me4Si) 1.88 (3H, s, Proα-CH3), 1 .70-2.30 (3H, br m, Proβ-HAΗΒ and Proγ-H2), 2.30-2.60 (1H, br m, Proβ-HAΗΒ), 3.40-3.84 (2H, br m, Proδ-H2), 3.87 (3H, s, CO2CH3), 9.43 (1H, br s, NH) and 10.49 ( 1H, br s, HCl); δC (75 MHz; CDCl3) 21.1 (CH3, Proα-CH3), 22.4 (CH2, Proγ-C), 35.6 (CH2, Proβ-C), 45.2 (CH2, Proδ-C), 53.7 (CH3, CO2CH3), 68.4 (quat., Proα-C) and 170.7 (quat, CO); m/z (FAB+) 323.1745 [M2.H35Cl.H+: (C7H13NO2)2. H35Cl.H requires 323.1738] and 325.1718 [M2.H37Cl.H+: (C7H13NOz)2. H37Cl.H requires 325.1708],

N-Benxyloxycarbonyl-glycyl-L-2-methylproline 5

Anhydrous triethylamine (0.45 cm3, 3.23 mmol) was added dropwise to a mixture of methyl L-2-methylprolinate hydrochloride 2 (0.42 g, 2.34 mmol) and N-benzyloxycarbonyl-glycine (98.5%) 3 (0.52 g, 2.45 mmol) in methylene chloride (16 cm3), at 0 °C, under an atmosphere of nitrogen. The resultant solution was stirred for 20 min and a solution of 1 ,3-dicyclohexylcarbodiimide (0.56 g, 2.71 mmol) in methylene chloride (8 cm3) at 0 °C was added dropwise and the reaction mixture was warmed to room temperature and stirred for a further 20 h. The resultant white mixture was filtered through a Celite™ pad to partially remove 1 ,3-dicyclohexylurea, and the pad was washed with methylene chloride (50 cm3). The filtrate was washed successively with 10% aqueous hydrochloric acid (50 cm3) and saturated aqueous sodium hydrogen carbonate (50 cm3), dried (MgSO4), filtered, and concentrated to dryness in vacuo. Further purification of the residue by flash column chromatography (35 g SiO2; 30-70% ethyl acetate – hexane; gradient elution) afforded tentatively methyl N-benzyloxycarbonyl-glycyl-L-2-methylprolinate 4 (0.56 g), containing 1 ,3-dicyclohexylurea, as a white semi-solid: Rf 0.65 (EtOAc); m/z (ΕI+) 334.1534 (M+. C17H22N2O5 requires 334.1529) and 224 ( 1 ,3-dicyclohexylurea).

To a solution of impure prolinate 4 (0.56 g, ca. 1.67 mmol) in 1,4-dioxane (33 cm3) was added dropwise 1 M aqueous sodium hydroxide (10 cm3, 10 mmol) and the mixture was stirred for 19 h at room temperature. Methylene chloride ( 100 cm3) was then added and the organic layer extracted with saturated aqueous sodium hydrogen carbonate (2 x 100 cm3). The combined aqueous layers were carefully acidified with hydrochloric acid (32%), extracted with methylene chloride (2 x 100 cm3), and the combined organic layers dried (MgSO4), filtered, and

concentrated to dryness in vacuo. Purification of the ensuing residue (0.47 g) by flash column chromatography ( 17 g SiO2; 50% ethyl acetate – hexane to 30% methanol – dichloromethane; gradient elution) gave N-protected dipeptide 5 (0.45 g, 60%) as a white foam in two steps from hydrochloride 2. Dipeptide 5 was shown to be exclusively the frafw-orientated conformer by NMR analysis: Rf 0.50 (20% MeOH – CH2Cl2); [α]D -62.3 (c 0.20 in CH2Cl2); vmax (film)/cm-1 3583, 3324 br, 2980, 2942, 1722, 1649, 1529, 1454, 1432, 1373, 1337, 1251 , 1219, 1179, 1053, 1027, 965, 912, 735 and 698; δH (300 MHz; CDCl3; Me4Si) 1.59 (3H, s, Proα-CH3), 1 .89 (1H, 6 lines, J 18.8, 6.2 and 6.2, Proβ-HAHB), 2.01 (2H, dtt, J 18.7, 6.2 and 6.2, Proγ-H2), 2.25-2.40 (1H, m, Proβ-HAΗΒ), 3.54 (2H, t, J 6.6, Proδ-H2), 3.89 (1H, dd, J 17.1 and 3.9, Glyα-HAHB), 4.04 (1H, dd, J 17.2 and 5.3, Glyα-HAΗΒ), 5.11 (2H, s, OCH2Ph), 5.84 (I H, br t, J 4.2, N-H), 7.22-7.43 (5H, m, Ph) and 7.89 (1 H, br s, -COOH); δC (75 MHz; CDCl3) 21.3 (CH3, Proα-CH3), 23.8 (CH2, Proγ-C), 38.2 (CH2, Proβ-C), 43.6 (CH2, Glyα-C), 47.2 (CH2, Proδ-C), 66.7 (quat, Proα-C), 66.8 (CH2, OCH2Ph), 127.9 (CH, Ph), 127.9 (CH, Ph), 128.4, (CH, Ph), 136.4 (quat., Ph), 156.4 (quat., NCO2), 167.5 (quat., Gly-CON) and 176.7 (quat., CO); m/z (EI+) 320.1368 (M+. C16Η20Ν2Ο5 requires 320.1372).

Dibenzyl N-benzyloxycarbonyl-glycyl-L-2-methylprolyl-L-glutamate 7

Triethylamine (0.50 cm3, 3.59 mmol) was added dropwise to a solution of dipeptide 5 (0.36 g, 1.12 mmol) and L-glutamic acid dibenzyl ester /Moluenesulphonate 6 (0.73 g, 1.46 mmol) in methylene chloride (60 cm3) under nitrogen at room temperature, and the reaction mixture stirred for 10 min. Bis(2-oxo-3-oxazoIidinyl)phosphinic chloride (BoPCl, 97%) (0.37 g, 1.41 mmol) was added and the colourless solution stirred for 17 h. The methylene chloride solution was washed successively with 10% aqueous hydrochloric acid (50 cm3) and saturated aqueous sodium hydrogen carbonate (50 cm3), dried (MgSO4), filtered, and evaporated to dryness in vacuo. Purification of the resultant residue by repeated (2x) flash column chromatography (24 g SiO2; 30-70% ethyl acetate – hexane; gradient elution) yielded ƒully protected tripeptide 7 (0.63 g, 89%) as a colourless oil. Tripeptide 7 was shown to be exclusively the trans-orientated conformer by NMR analysis: Rf 0.55 (EtOAc); [α]D -41.9 (c 0.29 in CH2Cl2); vmax (film)/cm-1 3583, 3353 br, 2950, 1734, 1660, 1521, 1499, 1454, 1429, 1257, 1214, 1188, 1166, 1051, 911, 737 and 697; δH (400 MHz; CDCl3; Me4Si) 1.64 (3H, s, Proot-CH3), 1.72 (1H, dt, J 12.8, 7.6 and 7.6, Proβ-HAHB), 1.92 (2H, 5 lines, J 6.7, Proγ-H2), 2.04 (1H, 6 lines, J 7.3 Gluβ-HAHB), 2.17-2.27 (1H, m, Gluβ-HAΗΒ), 2.35-2.51 (3H, m, Proβ-HAΗΒ and Gluγ-H2), 3.37-3.57 (2H, m, Proδ-H2), 3.90 (1 H, dd, J 17.0 and 3.6, Glyα-HAHB), 4.00 (1H, dd, J 17.1 and 5.1, Glyα-HAΗΒ), 4.56 (1H, td, J 7.7 and 4.9, Glyα-H), 5.05-5.20 (6H, m, 3 x OCH2Ph), 5.66-5.72 (1H, br m, Gly-NH), 7.26-7.37 (15H, m, 3 x Ph) and 7.44 (1H, d, J 7.2, Glu-NH); δC (100 MHz; CDCl3) 21.9 (CH3, Proα-CH3), 23.4 (CH2, Proγ-C), 26.6 (CH2, Gluβ-C), 30.1 (CH2, Gluγ-C), 38.3 (CH2, Proβ-C),

43.9 (CH2, Glyα-C), 47.6 (CH2, Proδ-C), 52.2 (CH, Glua-C), 66.4 (CH2, OCH2Ph), 66.8 (CH2, OCH2Ph), 67.1 (CH2, OCH2Ph), 68.2 (quat, Proα-C), 127.9 (CH, Ph), 128.0 (CH, Ph), 128.1, (CH, Ph), 128.2, (CH, Ph), 128.2, (CH, Ph), 128.3, (CH, Ph), 128.4, (CH, Ph), 128.5, (CH, Ph), 128.5, (CH, Ph), 135.2 (quat., Ph), 135.7 (quat., Ph), 136.4 (quat, Ph), 156.1 (quat, NCO2), 167.3 (quat., Gly-CO), 171.4 (quat., CO), 172.9 (quat., CO) and 173.4 (quat., CO); m/z (FAB+) 630.2809 (MH+. C35H40N3O8 requires 630.2815).

Glycyl-L-2-methylprolyl-L-glutamic acid (G-2-MePE)

A mixture of the protected tripeptide 7 (0.63 g, 1.00 mmol) and 10 wt % palladium on activated carbon (0.32 g, 0.30 mmol) in 91 :9 methanol – water (22 cm3) was stirred under an atmosphere of hydrogen at room temperature, protected from light, for 23 h. The reaction mixture was filtered through a Celite™ pad and the pad washed with 75 :25 methanol – water (200 cm3). The filtrate was concentrated to dryness under reduced pressure and the residue triturated with anhydrous diethyl ether to afford a 38: 1 mixture of G-2-MePE and tentatively methylamine 8 (0.27 g, 86%) as an extremely hygroscopic white solid. Analytical reverse-phase HPLC studies on the mixture [Altech Econosphere C 18 Si column, 150 x 4.6 mm, 5 ☐m; 5 min flush with H2O (0.05% TFA) then steady gradient over 25 min to MeCN as eluent at flow rate of 1 ml/min; detection using diode array] indicated it was a 38: 1 mixture of two eluting peaks with retention times of 13.64 and 14.44 min at 207 and 197 nm, respectively. G-2-MePE was shown to be a 73 :27 trans:cis mixture of conformers by 1H NMR analysis (the ratio was estimated from the relative intensities of the double doublet and triplet at δ 4.18 and 3.71 , assigned to the Gluα-H protons of the major and minor conformers, respectively):

mp 144 °Cɸ;

[ α]D -52.4 (c 0.19 in H2O);

δα (300 MHz; D2O; internal MeOH) 1.52 (3H, s, Proα-CH3), 1.81-2.21 (6H, m, Proβ-H2, Proγ-H, and Gluβ-H2), 2.34 (1.46H, t, J 7.2, Gluy-H2), 2.42* (0.54H, t, 77.3, Gluγ-H2), 3.50-3.66 (2H, m, Pro6-H2), 3.71 * (0.27H, t, J 6.2, Gluoc-H), 3.85 (1H, d, J 16.6, Glyα-HAHB), 3.92 (1H, d, J 16.6, Glyα-HAΗΒ) and 4.18 (0.73H, dd, J 8.4 and 4.7, Glua-H);

δC (75 MHz; D2O; internal MeOH) 21.8 (CH3, Proα-CH3), 25.0 (CH2, Proγ-C), 27.8* (CH2: Gluβ-C), 28.8 (CH2, Gluβ-C), 32.9 (CH2, Gluγ-C), 40.8 (CH2, Proβ-C), 42.7 (CH2, Glyα-C), 49.5 (CH2, Proδ-C), 56.0* (CH, Gluα-C), 56.4 (CH, Gluα-C), 69.8 (quat, Proα-C), 166.5 (quat., Gly-CO), 177.3 (quat., Pro-CON), 179.2 (quat., Gluα-CO), 180.2* (quat., Gluγ-CO) and 180.6 (quat., Gluγ-CO);

m/z (FAB+) 3 16.1508 (MH+. C13H22N3O6 requires 316.1509).

PATENT

WO02094856

Example

The following non-limiting example illustrates the synthesis of a compound of the invention, NN-dimethylglycyl-L-prolyl-L-glutamic acid.

All starting materials and other reagents were purchased from Aldrich;
BOC = tert-butoxycarbonyl; Bn = benzyl.

BOC-(γ-benzyl)-L-prolyl-L-glutamic acid benzyl ester
To a solution of BOC-proline [Anderson GW and McGregor AC: J. Amer. Chem.

Soc: 79, 6180, 1957] (10 mmol) in dichloromethane (50 ml), cooled to 0 °C, was added triethylamine (1.39 ml, 10 mmol) and ethyl chloroformate (0.96 ml, 10 mmol). The resultant mixture was stirred at 0 °C for 30 minutes. A solution of dibenzyl L-glutamate (10 mmol) was then added and the mixture stirred at 0 °C for 2 hours then warmed to room temperature and stirred overnight. The reaction mixture was washed with aqueous sodium bicarbonate and citric acid (2 mol l“1) then dried (MgS04) and concentrated at reduced pressure to give BOC-(γ-benzyl)-L-prolyl-L-glutamic acid dibenzyl ester (5.0 g, 95%).

(7-Benzyl)-L-prolyl-L-glutamic acid dibenzyl ester
A solution of BOC-(γ-benzyl)-L-prolyl-L-glutamic acid dibenzyl ester (3.4 g, 10 mmol), cooled to 0 °C, was treated with trifluoroacetic acid (25 ml) for 2 hr at room temperature. After removal of the volatiles at reduced pressure the residue was triturated with ether to give (γ-benzyl)-L-prolyl-L-glutamic acid dibenzyl ester (I).

N,N-Dimethylglycyl-L-prolyl-L-glutamic acid
A solution of dicyclohexylcarbodiimide (10.3 mmol) in dichloromethane (10 ml) was added to a stirred and cooled (0 °C) solution of (7-benzyl)-L-prolyl-L-glutamic acid dibenzyl ester (10 mmol), TVN-dimethylglycine (10 mmol) and triethylamine
(10.3 mmol) in dichloromethane (30 ml). The mixture was stirred at 0 °C overnight and then at room temperature for 3 h. After filtration, the filtrate was evaporated at reduced pressure. The resulting crude dibenzyl ester was dissolved in a mixture of ethyl acetate (30 ml) and methanol (30 ml) containing 10% palladium on charcoal (0.5 g) then hydrogenated at room temperature and pressure until the uptake of hydrogen ceased. The filtered solution was evaporated and the residue recrystallized from ethyl acetate to yield the tri-peptide derivative.

It will be evident that following the method of the Example, and using alternative amino acids or their amides or esters, will yield other compounds of Formula 1.

Testing; Material and Methods
The following experimental protocol followed guidelines approved by the

University of Auckland animal ethics committee.
Preparation of cortical astrocyte cultures for harvest of metabolised cell culture supernatant

One cortical hemisphere from a postnatal day 1 rat was used and collected into

4ml of DMEM. Trituration was done with a 5ml glass pipette and subsequently through an 18 gauge needle. Afterwards, the cell suspension was sieved through a lOOμm cell strainer and washed in 50ml DMEM (centrifugation for 5min at 250g). The sediment was resuspended into 20ml DMEM+10% fetal calf serum. 10 Milliliters of suspension was added into each of two 25cm3 flasks and cultivated at 37°C in the presence of 10% C02, with a medium change twice weekly. After cells reached confluence, they were washed three times with PBS and adjusted to Neurobasal/B27 and incubated for another 3 days. This supernatant was frozen for transient storage until usage at -80°C.

Preparation of striatal and cortical tissue from rat E18/E19 embryos
A dam was sacrificed by C02-treatment in a chamber for up to 4 minutes and was prepared then for cesarean section. After surgery, the embryos were removed from their amniotic sacs, decapitated and the heads put on ice in DMEM/F12 medium for striatum and PBS + 0.65% D(+)-glucose for cortex.

Striatal tissue extraction procedure and preparation of cells
Whole brain was removed from the skull with the ventral side facing upside in DMEM/F12 medium. The striatum was dissected out from both hemispheres under a stereomicroscope and the striatal tissue was placed into the Falcon tube on ice.

The collected striatal tissue was triturated by using a PI 000 pipettor in 1ml of volume. The tissue was triturated by gently pipetting the solution up and down into the pipette tip about 15 times, using shearing force on alternate outflows. The tissue pieces settled to the bottom of the Falcon tube within 30 seconds, subsequently the supernatant was transferred to a new sterile Falcon tube on ice. The supernatant contained a suspension of dissociated single cells. The tissue pieces underwent a second trituration to avoid excessively damaging cells already dissociated by over triturating them. 1 Milliliter of ice-cold DMEM/F12 medium was added to the tissue pieces in the first tube and triturated as before. The tissue pieces were allowed to settle and the supernatant was removed to a new sterile Falcon tube on ice. The cells were centrifuged at 250g for 5 minutes at 4°C. The resuspended cell pellet was ready for cell counting.

Plating and cultivation of striatal cells
Striatal cells were plated into Poly-L-Lysine (O.lmg/ml) coated 96-well plates (the inner 60 wells only) at a density of 200,000 cells /cm2 in Neurobasal/B27 medium (Invitrogen). The cells were cultivated in the presence of 5% C02 at 37°C under 100% humidity. Complete medium was changed on days 1, 3 and 6.

Cortical tissue extraction procedure and preparation of cells
The two cortical hemispheres were carefully removed by a spatula from the whole brain with the ventral side facing upside into a PBS +0.65% D(+)-glucose containing petri dish. Forcips were put into the rostral part (near B. olfactorius) of the cortex for fixing the tissue and two lateral – sagittal oriented cuttings were done to remove the paraform and entorhinal cortices. The next cut involved a frontal oriented cut at the posterior end to remove the hippocampal formation. A final frontal cut was done a few millimeters away from the last cut in order to get hold of area 17/18 of the visual cortex.

The collected cortices on ice in PBS+0.65% D(+)-glucose were centrifuged at 350g for 5min. The supernatant was removed and trypsin/EDTA (0.05%/0.53mM) was added for 8min at 37°C. The reaction was stopped by adding an equal amount of DMEM+10%) fetal calf serum. The supernatant was removed by centrifugation followed by two subsequent washes in Neurobasal/B27 medium.

The cells were triturated once with a glass Pasteur pipette in 1 ml of
Neurobasal/B27 medium and subsequently twice by using a 1ml insulin syringe with a 22 gauge needle. The cell suspension was passed through a lOOμm cell strainer and subsequently rinsed by 1ml of Neurobasal B27 medium. Cells were counted and adjusted to 50,000 cells per 60μl.

Plating and cultivation of cortical cells

96-well plates were coated with 0.2mg/ml Poly-L-Lysine and subsequently coated with 2μg/ml laminin in PBS, after which 60μl of cortical astrocyte-conditioned medium was added to each well. Subsequently, 60μl of cortical cell suspension was added. The cells were cultivated in the presence of 10% C02 at 37°C under 100%) humidity. At day 1, there was a complete medium change (1:1- Neurobasal/B27 and astrocyte-conditioned medium) with addition of lμM cytosine-β-D-arabino-furanoside (mitosis inhibitor). On the second day, 2/3 of medium was changed. On day 5, 2/3 of the medium was changed again.

Cerebellar microexplants from P8 animals: preparation, cultivation and fixation

The laminated cerebellar cortices of the two hemispheres were explanted from a P8 rat, cut into small pieces in PBS + 0.65% D(+)glucose solution and triturated by a 23gauge needle and subsequently pressed through a 125 μm pore size sieve. The microexplants that were obtained were centrifuged (60 g) twice (media exchange) into serum-free BSA-supplemented START V-medium (Biochrom). Finally, the
microexplants were reconstituted in 1500 μl STARTV-medium (Biochrom). For cultivation, 40μl of cell suspension was adhered for 3 hours on a Poly-D-Lysine
(O.lmg/ml) coated cover slip placed in 35mm sized 6-well plates in the presence of 5% C02 under 100% humidity at 34°C. Subsequently, 1ml of STARTV-medium was added together with the toxins and drugs. The cultures were monitored (evaluated) after 2-3 days of cultivation in the presence of 5% C02 under 100% humidity. For cell counting analysis, the cultures were fixed in rising concentrations of paraformaldehyde (0.4%, 1.2%, 3% and 4% for 3min each) followed by a wash in PBS.
Toxin and drug administration for cerebellar, cortical and striatal cells: analysis

All toxin and drug administration experiments were designed that 1/100 parts of okadaic acid (30nM and lOOnM concentration and 0.5mM 3-nitropropionic acid for cerebellar microexplants only), GPE (InM -ImM) and G-2Methyl-PE (InM-lmM) were used respectively at 8DIV for cortical cultures and 9DIV for striatal cultures. The incubation time was 24hrs. The survival rate was determined by a colorimetric end-point MTT-assay at 595nm in a multi-well plate reader. For the cerebellar microexplants four windows (field of 0.65 mm2) with highest cell density were chosen and cells displaying neurite outgrowth were counted.

Results
The GPE analogue G-2Methyl-PE exhibited comparable neuroprotective capabilities within all three tested in vitro systems (Figures 12-15).

The cortical cultures responded to higher concentrations of GPE (Figure 12) /or

G-2Methyl-PE (lOμM, Figure 13) with 64% and 59% neuroprotection, respectively.

Whereas the other 2 types of cultures demonstrated neuroprotection at lower doses of G-2Methyl-PE (Figures 14 and 15). The striatal cells demonstrated
neuroprotection within the range of InM to ImM of G-2Methyl-PE (Figure 15) while the postnatal cerebellar microexplants demonstrated neuroprotection with G-2Methyl-PE in the dose range between InM and lOOnM (Figure 14).

While this invention has been described in terms of certain preferred embodiments, it will be apparent to a person of ordinary skill in the art having regard to that knowledge and this disclosure that equivalents of the compounds of this invention may be prepared and administered for the conditions described in this application, and all such equivalents are intended to be included within the claims of this application.

PATENT

WO-2021026066

Composition and kits comprising trofinetide and other related substances. Also claims a process for preparing trofinetide and the dosage form comprising the same. Disclosed to be useful in treating neurodegenerative conditions, autism spectrum disorders and neurodevelopmental disorders.

Trofinetide is a synthetic compound, having a similar core structure to Glycyl-Prolyl-Glutamic acid (or “GPE”). Trofinetide has been found to be useful in treating neurodegenerative conditions and recently has been found to be effective in treating Autism Spectrum disorders and Neurodevelopmental disorders.

Formula (Ila),

Example 1: Trofinetide Manufacturing Process

In general, trofinetide and related compounds can be manufactured from a precursor peptide or amino acid reacted with a silylating or persilylating agent at one or more steps. In the present invention, one can use silylating agents, such as N-trialkylsilyl amines or N-trialkylsilyl amides, not containing a cyano group.

Examples of such silylating reagents include N,O-bis(trimethylsilyl)acetamide (BSA), N,O-bis(trimethylsilyl)trifluoroacetamide, hexamethyldisilazane, N-methyl-N-(trimethylsilyl)acetamide (TMA), N-methyl-N-(trimethylsilyl)trifluoroacetamide, N-(trimethylsilyl)acetamide, N-(trimethylsilyl)diethylamine, N-(trimethylsilyl)dimethylamine, 1-(trimethylsilyl)imidazole, 3-(trimethylsilyl)-2-oxazolidone.

Step 1: Preparation of Z-Gly-OSu

Several alternative procedures can be used for this step.

Procedure 1A

One (1) eq of Z-Gly-OH and 1.1 eq of Suc-OH were solubilized in 27 eq of iPrOH and 4 eq of CH2Cl2 at 21 °C. The mixture was cooled and when the temperature reached -4 °C, 1.1 eq of EDC.HCl was added gradually, keeping the temperature below 10 °C. During the reaction a dense solid appeared. After addition of EDC.HCl, the mixture was allowed to warm to 20 °C. The suspension was cooled to 11 °C and filtered. The cake was washed with 4.9 eq of cold iPrOH and 11 eq of IPE before drying at 34 °C (Z-Gly-OSu dried product -Purity: 99.5%; NMR assay: 96%; Yield: 84%).

Procedure 1B

This Procedure is for a variant of Procedure 1A, and differs by replacing iPrOH with ACN. One (1) eq of Z-Gly-OH and 1.1 eq of Suc-OH were solubilized in 22 eq of ACN at 35 °C. The mixture was cooled in an ice bath. When the temperature reached 1 °C, 0.9 eq of DCC in 5.5 eq of ACN was added gradually to keep the temperature below 5 °C. The coupling reaction took about 20 hrs. During the reaction, DCU precipitated and was removed by filtration at the end of the coupling. After filtration, DCU was washed with ACN to recover the product. The mixture of Z-Gly-OSu was then concentrated to reach 60% by weight. iPrOH (17 eq) was added to initiate the crystallization. Quickly after iPrOH addition a dense solid appeared. An additional 17 eq of iPrOH was needed to liquify the suspension. The suspension was cooled in an ice bath and filtered. The solid was washed with 9 eq of iPrOH before drying at 45 °C (Z-Gly-OSu dried product – Purity: 99.2%; HPLC assay: 99.6%; Yield: 71%).

Step 2: Preparation of Z-Gly-MePro-OH

Several alternative procedures can be used for this step.

Procedure 2A

 One (1) eq of MePro.HCl was partially solubilized in 29 eq of CH2Cl2 at 35 °C with 1.04 eq of TEA and 1.6 eq of TMA. The mixture was heated at 35 °C for 2 hrs to perform the silylation. Then 1.02 eq of Z-Gly-OSu was added to the mixture. The mixture was kept at 35 °C for 3 hrs and then 0.075 eq of butylamine was added to quench the reaction. The mixture was allowed to return to room temperature and mixed for at least 15 min. The Z-Gly-MePro-OH was extracted once with 5% w/w NaHCO3 in 186 eq of water, then three times successively with 5% w/w NaHCO3 in 62 eq of water. The aqueous layers were pooled and the pH was brought to 2.2 by addition of 34 eq of HCl as 12N HCl at room temperature. At this pH, Z-Gly-MePro-OH formed a sticky solid that was solubilized at 45 °C with approximately 33 eq of EtOAc and 2.3 eq of iButOH. Z-Gly-MePro-OH was extracted into the organic layer and washed with 62 eq of demineralized water. The organic layer was then dried by azeotropic distillation with 11.5 eq of EtOAc until the peptide began to precipitate. Cyclohexane (12 eq) was added to the mixture to complete the precipitation. The suspension was cooled at 5 °C for 2 hrs and filtered. The solid was washed with 10 eq of cyclohexane before drying at 45 °C (Z-Gly-MePro-OH dried product – Purity: 100%; HPLC assay: 100%; Yield 79%).

Procedure 2B

This Procedure is for a variant of Procedure 2A. One (1) eq of MePro.HCl was partially solubilized in 36.6 eq of CH2Cl2 at 34 °C with 1.01 eq of TEA and 0.1 eq of TMA. Then 1.05 eq of Z-Gly-OSu was added to the mixture, followed by 1.0 eq of TEA. The mixture was maintained at 35 °C for approximately 1 hr, cooled to 25 to 30 °C and 0.075 eq of DMAPA was added to stop the reaction. One hundred (100) eq of water, 8.6 eq of HCl as 12N HCl and 0.3 eq of KHSO4 were added to the mixture (no precipitation was observed, pH=1.7). Z-Gly-MePro-OH was extracted into the organic layer and washed twice with 97 eq of demineralized water with 0.3 eq of KHSO4, then 100 eq of demineralized water, respectively. EtOAc (23 eq) was added to the mixture and CH2Cl2 was removed by distillation until the peptide began to precipitate. Cyclohexane (25 eq) was added to the mixture to complete the precipitation. The suspension was cooled at -2 °C overnight and filtered. The solid was washed with 21 eq of cyclohexane before drying at 39 °C (Z-Gly-MePro-OH dried product – Purity: 98.7%; NMR assay: 98%; Yield 86%).

Procedure 2C


In reactor 1, MePro.HCl (1 eq) was suspended in EtOAc (about 7 eq). DIPEA (1 eq) and TMA (2 eq) were added, and the mixture heated to dissolve solids. After dissolution, the solution was cooled to 0 °C. In reactor 2, Z-Gly-OH (1 eq) was suspended in EtOAc (about 15 eq). DIPEA (1 eq), and pyridine (1 eq) were added. After mixing, a solution was obtained, and cooled to -5 °C. Piv-Cl (1 eq) was added to reactor 2, and the contents of reactor 1 added to reactor 2. Upon completed addition, the contents of reactor 2 were taken to room temperature. The conversion from Z-Gly-OH to Z-Gly-MePro-OH was monitored by HPLC. When the reaction was complete, the reaction mixture was quenched with DMAPA (0.1 eq), and washed with an aqueous solution comprised of KHSO4, (about 2.5 wt%), NaCl (about 4 wt%), and conc. HCl (about 6 wt%) in 100 eq H2O. The aqueous layer was re-extracted with EtOAc, and the combined organic layers washed with an aqueous solution comprised of KHSO4 (about 2.5 wt%) and NaCl (about 2.5 wt%) in 100 eq H2O, and then with water (100 eq). Residual water was removed from the organic solution of Z-Gly-MePro-OH by vacuum distillation with EtOAc. The resulting suspension was diluted with heptane (about 15 eq) and cooled to 0 °C. The product was isolated by filtration, washed with cold heptane (about 7 eq), and dried under vacuum at 45 °C. Z-Gly-MePro-OH (85% yield) was obtained.

Step 3: Preparation of Z-Gly-MePro-Glu-OH

Several alternative procedures can be used in this step.

Procedure 3A

 H-Glu-OH (1.05 eq) was silylated in 2 eq of CH2Cl2 with 3.5 eq of TMA at 65 °C. Silylation was completed after 2 hrs. While the silylation was ongoing, 1.0 eq of Z-Gly-MePro-OH and 1.0 eq of Oxyma Pure were solubilized in 24 eq of CH2Cl2 and 1.0 eq of DMA at room temperature in another reactor. EDC.HCl (1.0 eq.) was added. The activation rate reached 97% after 15 min. The activated Oxyma Pure solution, was then added to silylated H-Glu-OH at 40 °C and cooled at room temperature. Coupling duration was approximately 15 min, with a coupling rate of 97%. Addition of 8.2% w/w NaHCO3 in 156 eq of water to the mixture at room temperature (with the emission of CO2) was performed to reach pH 8. Z-Gly-MePro-Glu-OH was extracted in water. The aqueous layer was washed twice with 29 eq of CH2Cl2. Residual CH2Cl2 was removed by concentration. The pH was brought to 2.5 with 2.5N HCl, followed by 1.4 eq of solid KHSO4 to precipitate Z-Gly-MePro-Glu-OH. The mixture was filtered and the solid was washed with 3 x 52 eq of water. The filtered solid was added to 311 eq of demineralized water and heated to 55-60 °C. iPrOH (29 eq) was added gradually until total solubilization of the product. The mixture was slowly cooled to 10 °C under moderate mixing during 40 min to initiate the crystallization. The peptide was filtered and washed with 2 x 52 eq of water before drying at 45 °C (Z-Gly-MePro-Glu-OH dried product – Purity: 99.5%; NMR assay: 96%; Yield 74%).

Procedure 3B

One (1) eq of Z-Gly-MePro-OH and 1.05 eq of Suc-OH were solubilized in 40 eq of ACN and 30 eq of CH2Cl2 at room temperature. The mixture was cooled in an ice bath, and when the temperature was near 0 °C, 1.05 eq of DCC dissolved in 8 eq of ACN was added gradually, keeping the temperature below 5 °C. After addition of DCC, the mixture was progressively heated from 0 °C to 5 °C over 1 hr, then to 20 °C between 1 to 2 hrs and then to 45 °C between 2 to 5 hrs. After 5 hrs, the mixture was cooled to 5 °C and maintained overnight. The activation rate reached 98% after approximately 24 hrs. DCU was removed by filtration and washed with 13.5 eq of ACN. During the activation step, 1.1 eq of H-Glu-OH was silylated in 30 eq of ACN with 2.64 eq of TMA at 65 °C. Silylation was completed after 2 hrs. Z-Gly-MePro-OSu was then added gradually to the silylated H-Glu-OH at room temperature, with 0.4 eq of TMA added to maintain the solubility of the H-Glu-OH. The mixture was heated to 45 °C and 0.7 eq of TMA was added if precipitation occurred. The coupling duration was about 24 hrs to achieve a coupling rate of approximately 91%. The reaction was quenched by addition of 0.15 eq of butylamine and 2.0 eq of TEA. Water (233 eq) was added and the mixture concentrated until gelation occurred. Z-Gly-MePro-Glu-OH was extracted in water by addition of 5% w/w NaHCO3 in 233 eq of water and 132 eq of CH2Cl2. The aqueous layer was washed twice with 44 eq of CH2Cl2. Residual CH2Cl2 was removed by distillation. The pH was brought to 2.0 with 24 eq of HCl as 12N HCl followed by 75 eq of HCl as 4N HCl. At this pH, Z-Gly-MePro-Glu-OH precipitated. The mixture was cooled in an ice bath over 1 hr and filtered. The solid was washed with 186 eq of cold water before drying at 45 °C (Z-Gly-MePro-Glu-OH dried product – HPLC Purity: 98.4%; NMR assay: 100%; Yield 55%).

Procedure 3C

This Procedure is for a variant of Procedure 3A. H-Glu-OH (1.05 eq) was silylated in 3.7 eq of CH2Cl2 with 3.5 eq of TMA at 62 °C. Silylation was completed after approximately 1.5 to 2 hrs, as evidenced by solubilization. During the silylation step, 1.0 eq of Z-Gly-MePro-OH and 1.0 eq of Oxyma Pure were solubilized in 31.5 eq of CH2Cl2 at 22 °C. One (1.06) eq of EDC.HCl was added to complete the activation. The silylated H-Glu-OH was then added to the activated Oxyma Pure solution. The temperature was controlled during the addition to stay below 45 °C. Desilylation was performed by addition of a mixture of 2.5% w/w KHSO4 in 153 eq of water and 9 eq of iPrOH to reach a pH of 1.65. Residual CH2Cl2 was removed by concentration. The mixture was cooled to 12 °C to precipitate the Z-Gly-MePro-Glu-OH. The mixture was filtered and the solid was washed with 90 eq of water before drying at 36 °C.

Procedure 3D

This Procedure is for a variant of Procedure 3A. H-Glu-OH (1.05 eq.) was silylated in 3.9 eq of CH2Cl2 with 3.5 eq of TMA at 62 °C. Silylation was completed after 2 hrs, as evidenced by Solubilization. During the silylation step, 1 eq of Z-Gly-MePro-OH and 1 eq of Oxyma Pure were solubilized in 25 eq of CH2Cl2 at 23 °C. One (1) eq of EDC.HCl was added. To complete the activation, an additional 0.07 eq of EDC. HCl was added. Silylated H-Glu-OH was then added to the activated Oxyma Pure solution. Temperature was controlled during the addition to stay below 45 °C. Desilylation was performed by addition of a mixture of 2.5% w/w KHSO4 in 160 eq of water and 9.6 eq of iPrOH to reach pH 1.63.

Residual CH2Cl2 was removed by concentration. The mixture was cooled to 20 °C to precipitate the Z-Gly-MePro-Glu-OH. The mixture was filtered and the solid was washed with 192 eq of water before drying at about 25 °C for 2.5 days. The solid was then solubilized at 64 °C by addition of 55 eq of water and 31 eq of iPrOH. After solubilization, the mixture was diluted with 275 eq of water and cooled to 10 °C for crystallization. The mixture was filtered and the solid was washed with 60 eq of water before drying at 27 °C (Z-Gly-MePro-Glu-OH dried product – Purity: 99.6%; NMR assay: 98%; Yield 74%).

Procedure 3E

 In reactor 1, H-Glu-OH (1.05 eq) was suspended in ACN (about 2.2 eq). TMA (about 3.5 eq) added, and the mixture was heated to dissolve solids. After dissolution, the solution was cooled to room temperature. In reactor 2, Z-Gly-MePro-OH (1 eq) was suspended in ACN (14 eq). Oxyma Pure (1 eq) and EDC.HCl (1 eq) were added. The mixture was stirred at room temperature until the solids dissolved. The contents of reactor 2 were added to reactor 1. The conversion from Z-Gly-MePro-OH to Z-Gly-MePro-Glu-OH was monitored by HPLC. Upon completion the reaction mixture was added to an aqueous solution comprised of KHSO4 (about 2.5 wt%) dissolved in about 100 eq H2O. ACN was removed from the aqueous suspension of Z-Gly-MePro-Glu-OH by vacuum distillation with H2O. After stirring at room temperature, the product in the resulting suspension was isolated by filtration and washed with water. The solid obtained was dissolved in an aqueous solution comprised of NaHCO3 (about 5 wt%) in 110 eq H2O, and recrystallized by addition of an aqueous solution comprised of KHSO4 (about 10 wt%) in 90 eq H2O. The product was isolated by filtration, washed with water, and dried under vacuum at 45 °C. Z-Gly-MePro-Glu-OH (75% yield) was obtained.

Step 4: Deprotection and Isolation of Trofinetide

Several alternative procedures can be used in this step.

Procedure 4A

 Z-Gly-MePro-Glu-OH (1 eq) was suspended in water (about 25 eq) and EtOAc (about 15 eq). Pd/C (0.025 eq by weight and containing 10% Pd by weight) was added, and the reaction mixture hydrogenated by bubbling hydrogen through the reaction mixture at room temperature. The conversion from Z-Gly-MePro-Glu-OH to trofinetide was monitored by HPLC, and upon reaction completion the catalyst was removed by filtration, and the layers separated. Residual EtOAc was removed from the aqueous solution containing trofinetide by sparging with nitrogen or washing with heptane. The aqueous solution was spray-dried to isolate the product. Trofinetide (90% yield) was obtained. Alternatively, deprotection can be accomplished using MeOH only, or a combination of iPrOH and MeOH, or by use of ethyl acetate in water.

Procedure 4B

This Procedure is for a variant of Procedure 4A, excluding EtOAc. Z-Gly-MePro-Glu-OH (1 eq) was suspended in water (about 50 eq). Pd/C (0.05 eq, 5% Pd by weight) was added, and the reaction mixture hydrogenated at room temperature with a pressure of 5 bar. The conversion from Z-Gly-MePro-Glu-OH to trofinetide was monitored by HPLC. Upon

reaction completion the catalyst was removed by filtration, and the aqueous layer washed with EtOAc (about 5 eq). Residual EtOAc was removed from the aqueous solution containing trofinetide by sparging with nitrogen or washing with heptane. The aqueous solution was spray-dried to isolate the product. Trofinetide (90% yield) was obtained.

Procedure 4C

This Procedure is for a variant of Procedure 4A, replacing EtOAc with MeOH. Z-Gly-MePro-Glu-OH (1 eq) was suspended in MeOH (100 eq) and water (12 eq). Pd/Si (0.02 eq by weight) was added and the mixture was heated at 23 °C for the hydrogenolysis. Solubilization of the peptide occurred during the deprotection. The conversion from Z-Gly-MePro-Glu-OH to trofinetide was monitored by HPLC, and upon reaction completion the catalyst was removed by filtration and the layers were washed with MeOH and iPrOH. The solvents were concentrated under vacuum at 45 °C, and trofinetide precipitated. The precipitate was filtered and dried at 45 °C to provide trofinetide.

Procedure 4D

This Procedure is for a variant of Procedure 4A, replacing Pd/C with Pd/Si. One (1.0) eq of Z-Gly-MePro-Glu-OH was partially solubilized in 105 eq of MeOH and 12 eq of water. Pd/Si (0.02 eq by weight) was added and the mixture was heated at 23 °C for the hydrogenolysis. Solubilization of the peptide occurred during the deprotection. At the end of the deprotection (conversion rate approximately 99% after 1 hr), the catalyst was filtered off and washed with 20-30 eq of MeOH. iPrOH (93 eq) was added and MeOH was replaced by iPrOH by concentration at 45 °C under vacuum. The peptide was concentrated until it began to precipitate. The peptide was filtered and dried at 45 °C (H-Gly-MePro-Glu-OH dried product: Purity: 98.1%; NMR assay: 90%; Yield 81%).

Procedure 4E

This Procedure is for a variant of Procedure 4A, removing H2O and replacing Pd/C with Pd/Si. One (1.0) eq of Z-Gly-MePro-Glu-OH was partially solubilized in 44 eq of MeOH. Pd/Si type 340 (0.02 eq by weight) was added and the mixture was kept at 20 °C for the hydrogenolysis. Solubilization of the peptide occurred during the deprotection. At the end of the deprotection (conversion rate about 99.9%, after 3-3.5 hrs), the catalyst was filtered off and washed with 8 eq of MeOH. Deprotected peptide was then precipitated in 56 eq of iPrOH. After 30 min at 5 °C, the peptide was filtered and washed with three times with 11 eq of iPrOH before drying at 25 °C (H-Gly-MePro-Glu-OH dried product: Purity: 99.4%; HPLC assay: ~98%; Yield: 81%).

Procedure 4F

This Procedure is for a variant of Procedure 4A. One (1) eq of Z-Gly-MePro-Glu-OH was partially solubilized in 14 eq of EtOAc and 25 eq of water. Pd/C (0.01 eq by weight) was added and the mixture was kept at 20 °C for the hydrogenolysis. Solubilization of the peptide occurred during the deprotection. At the end of the deprotection (conversion rate about 100%, after about 3.5 hrs), the catalyst was filtered off and washed with a mixture of 3.5 eq of EtOAc and 6 eq of water. The aqueous layer was then ready for spray-drying (Aqueous H-Gly-MePro-Glu-OH peptide solution: Purity: 98.6%; Yield: ~95%).

Procedure 4G

This Procedure is for a variant of Procedure 4A, replacing Pd/C with Pd/Si, EtOAc with MeOH, and removing H2O. Pd/Si type 340 (0.02 eq by weight) was added to 2.9 vols of MeOH for pre-reduction during 30 min. One (1.0) eq of Z-Gly-MePro-Glu-OH was partially solubilized in 34 eq of MeOH. The reduced palladium was then transferred to the peptide mixture. The mixture was kept at 20 °C for the hydrogenolysis. Solubilization of the peptide occurred during the deprotection. Pd/C type 39 (0.007 eq by weight) was added to the mixture to increase reaction kinetics. At the end of the deprotection, the catalyst was filtered off and washed with 13.6 eq of MeOH. The deprotected peptide was then precipitated in 71 eq of iPrOH. After about 40 min, the peptide was filtered and washed with 35 eq of iPrOH. The peptide was dried below 20 °C and was then ready for solubilization in water and spray-drying.

Procedure 4H

This Procedure is for a variant of Procedure 4A. One (1.0) eq of Z-Gly-MePro-Glu-OH was partially solubilized in 24.8 eq of water and 13.6 eq of EtOAc. Pd/C type 39 (0.025 eq by weight) was added to the peptide mixture. The mixture was kept at 20 °C for the hydrogenolysis. Solubilization of the peptide occurred during the deprotection. At the end of the deprotection (19 hrs), the catalyst was removed by filtration and washed with 5.3 eq of water and 2.9 eq of EtOAc. The biphasic mixture was then decanted to remove the upper organic layer. The aqueous layer was diluted with water to reach an H-Gly-MePro-Glu-OH concentration suitable for spray-drying the solution.

Example 2: Alternative Trofinetide Manufacturing Process

An alternative method for synthesis of Trofinetide is based on U.S. Patent No.

8,546,530 adapted for a tripeptide as follows.

The persilylated compounds used to synthesis Formula (Ia) (trofinetide) are obtained by silylating a corresponding peptide or amino acid by reaction with a silylating agent, optionally in an organic solvent. The persilylated peptide or amino acid can be isolated and purified if desired. One can use the persilylated peptide or amino acid in situ, e.g. by combining a solution containing persilylated peptide or amino acid with a solution containing, optionally activated, peptide or amino acid.

In step 2, the persilylated compound of an amino acid is obtained by silylating a corresponding amino acid (for example, H-MePro-OH) by reaction with a silylating agent, optionally in an organic solvent. The persilylated amino acid can be isolated and purified if desired. One can use the persilylated amino acid in situ, e.g. by combining a solution containing the persilylated amino acid with a solution containing, optionally activated, amino acid (for example, Z-Gly-OH).

In step 3, the persilylated compound of an amino acid is obtained by silylating a corresponding amino acid (for example, H-Glu-OH) by reaction with a silylating agent, optionally in an organic solvent. The persilylated amino acid or peptide can be isolated and purified if desired. It is however useful to use the persilylated amino acid or peptide in situ, e.g. by combining a solution containing the persilylated amino acid with a solution containing, optionally activated (for example, by using EDC.HCl and Oxyma Pure), peptide (for example, Z-Gly-MePro-OH).

In the present invention, it is useful to use silylating agents, such as N-trialkylsilyl amines or N-trialkylsilyl amides, not containing a cyano group. Examples of such silylating reagents include N,O-bis(trimethylsilyl)acetamide (BSA), N,O-bis(trimethylsilyl)trifluoroacetamide, hexamethyldisilazane, N-methyl-N-(trimethylsilyl)acetamide (TMA), N-methyl-N-(trimethylsilyl)trifluoroacetamide, N-(trimethylsilyl)acetamide, N-(trimethylsilyl)diethylamine, N-(trimethylsilyl)dimethylamine, 1-(trimethylsilyl)imidazole, 3-(trimethylsilyl)-2-oxazolidone.

The reaction of step 2 is generally carried out at a temperature from 0 °C to 100 °C, optionally from 10 °C to 40 °C, and optionally from 15 °C to 30 °C.

The reaction of step 3 is generally carried out at a temperature from 0 °C to 100 °C, optionally from 10 °C to 60 °C, optionally from 15 °C to 50 °C.

In the reaction of step 2, generally 0.5 to 5 equivalents, optionally 1 to 3 equivalents, optionally about 1.5 to 2.5 equivalents of silylating agent are used relative to the molar amount of functional groups to be silylated. Use of 2 to 4 equivalents of silylating agent relative to the molar amount of functional groups to be silylated is also possible. “Functional groups to be silylated” means particular groups having an active hydrogen atom that can react with the silylating agent such as amino, hydroxyl, mercapto or carboxyl groups.

In the reaction of step 3, generally 0.5 to 5 equivalents, optionally 2 to 4.5 equivalents, optionally about 3 to 4 equivalents of silylating agent are used relative to the molar amount of functional groups to be silylated. Use of 2.5 to 4.5 equivalents of silylating agent relative to the molar amount of functional groups to be silylated is also possible.

It is understood that “persilylated” means an amino acid or peptide or amino acid analogue or peptide analogue in which the groups having an active hydrogen atom that can react with the silylating agent are sufficiently silylated to ensure that a homogeneous reaction medium for a coupling step is obtained.

In the process according to the invention, the reaction between the amino acid or peptide and the persilylated amino acid or peptide is often carried out in the presence of a carboxyl group activating agent. In that case the carboxylic activating reagent is suitably selected from carbodiimides, acyl halides, phosphonium salts and uronium or guanidinium salts. More optionally, the carboxylic activating agent is an acyl halide, such as isobutyl chloroformate or pivaloyl chloride or a carbodiimide, such as EDC.HC1 or DCC.

Good results are often obtained when using additional carboxylic activating reagents which reduce side reactions and/or increase reaction efficiency. For example, phosphonium and uronium salts can, in the presence of a tertiary base, for example, N,N-diisopropylethylamine (DIPEA) and triethylamine (TEA), convert protected amino acids into activated species. Other reagents help prevent racemization by providing a protecting reagent. These reagents include carbodiimides (for example, DCC) with an added auxiliary nucleophile (for example, 1-hydroxy-benzo triazole (HOBt), 1-hydroxy-azabenzotriazole (HOAt), or Suc-OH) or derivatives thereof. Another reagent that can be utilized is TBTU. The mixed anhydride method, using isobutyl chloroformate, with or without an added auxiliary nucleophile, is also used, as is the azide method, due to the low racemization associated with it. These types of compounds can also increase the rate of carbodiimide-mediated couplings. Typical additional reagents include also bases such as N,N-diisopropylethylamine (DIPEA), triethylamine (TEA) or N-methylmorpholine (NMM).

When the silylation is carried out in the presence of a solvent, said solvent is optionally a polar organic solvent, more optionally a polar aprotic organic solvent. An amide type solvent such as N,N-dimethylformamide (DMF) or N,N-dimethylacetamide (DMAC)

can be used. In the present invention for step 2, one can use an alkyl acetate solvent, in particular ethyl acetate is more particularly optional.

In the present invention for step 3, one can use a chlorinated hydrocarbon solvent or alkyl cyanide solvent, in particular dichloromethane or acetonitrile are more particularly optional.

In another embodiment, silylation is carried out in a liquid silylation medium consisting essentially of silylating agent and amino acid or peptide.

In the present invention, amino acid or peptide is understood to denote in particular an amino acid or peptide or amino acid analogue or peptide analogue which is bonded at its N-terminus or optionally another position, to a carboxylic group of an amino protected amino acid or peptide.

Example 3: Specifications for Compositions Containing Compounds of Formula (I)

1 ICH guideline Q3C on impurities: guideline for residual solvents

Example 4: Alternative Manufacturing of Trofinetide Example 1, Step 4, Procedure 4B

This Procedure is for a variant of Step 4, Procedure 4B. Z-Gly-MePro-Glu-OH (1 eq) was added in portions to Pd/C (0.027 eq by weight and containing 5% Pd by weight) in about 50 eq of water. The reaction mixture was hydrogenated at 20 °C at a pressure of 5 bar for at least 4 cycles of 4 hrs each. Pd/C (0.0027 eq by weight) was charged between cycles, as needed, to speed up the reaction. The conversion from Z-Gly-MePro-Glu-OH to trofinetide was monitored by HPLC. Upon reaction completion the catalyst was removed by filtration, washed with water (12.5 eq) and the aqueous layer washed with EtOAc (about 14 eq). After phase separation, residual EtOAc was removed from the aqueous solution containing

trofinetide by sparging with nitrogen under vacuum at 20 °C for about 3 hrs. The aqueous solution was filtered. The final concentration of trofinetide was about 25 wt% and the solution was then ready for spray-drying to isolate the product.

Example 5: Alternative Composition of Trofinetide

A composition comprising a compound of Formula (I)

or a stereoisomer, hydrate, or pharmaceutically acceptable salt thereof, and a compound of Formula (II):

or a stereoisomer, hydrate, or pharmaceutically acceptable salt thereof, and/or a compound of Formula (III):

or a stereoisomer, hydrate, or pharmaceutically acceptable salt thereof, wherein R1, R2, R3 and R4 independently are selected from the group consisting of hydrogen and C1-4 alkyl, provided that least one of R1, R2, R3 and R4 is C1-4 alkyl, and wherein the composition comprises at least 90 wt%, such as 91 wt%, 92 wt%, 93 wt%, 94 wt%, 95 wt%, 96 wt%, or 97 wt% of the compound of Formula (I) on an anhydrous basis.

Example 6: Alternative Composition of Trofinetide

A composition comprising a compound of Formula (Ia)

or a hydrate, or pharmaceutically acceptable salt thereof, and a compound of Formula (II):

or a stereoisomer, hydrate, or pharmaceutically acceptable salt thereof, and/or a compound of Formula (III):

or a stereoisomer, hydrate, or pharmaceutically acceptable salt thereof, wherein R1, R2, R3 and R4 independently are selected from the group consisting of hydrogen and C1-4 alkyl, provided that least one of R1, R2, R3 and R4 is C1-4 alkyl, and wherein the composition comprises at least 90 wt%, such as 91 wt%, 92 wt%, 93 wt%, 94 wt%, 95 wt%, 96 wt%, or 97 wt% of the compound of Formula (Ia) on an anhydrous basis.

Example 7: A Product of Trofinetide

A product, including a kit containing a dosage form with instructions for use, comprising a compound of Formula (Ia)

or a hydrate, or pharmaceutically acceptable salt thereof, and a compound of Formula (IIa)


or a hydrate, or pharmaceutically acceptable salt thereof, wherein the product comprises between 95 wt% and 105 wt%, such as 96 wt%, 97 wt%, 98 wt%, 99 wt%, 100 wt%, 101

wt%, 102 wt%, 103 wt%, or 104 wt% of the specified amount of the compound of Formula (Ia) in the product.

Example 8: A Product of Trofinetide

A product, including a kit containing a dosage form with instructions for use, comprising a compound of Formula (Ia)

or a hydrate, or pharmaceutically acceptable salt thereof, and a compound of Formula (IIa)

 or a hydrate, or pharmaceutically acceptable salt thereof, and additionally comprising one or more compounds selected from the group consisting of Formula (III), Formula (IIIa), Formula (IV), Formula (V), Formula (VI), Formula (VII), Formula (VIII), and Formula (IX), wherein the composition comprises between 95 wt% and 105 wt%, such as 96 wt%, 97 wt%, 98 wt%, 99 wt%, 100 wt%, 101 wt%, 102 wt%, 103 wt%, or 104 wt% of the specified amount of the compound of Formula (Ia) in the product.

Example 9: Analysis of Products and Compositions

The products and compositions disclosed herein may be analyzed by liquid chromatography, a suitable chromatographic method using UPLC, e.g. using materials and conditions such as Waters Acquity CSH C18, 1.7 µm, 150 x 2.1 mm column, water with 0.1 % TFA (mobile phase A), and water/ACN 70/30 + 0.1 % TFA (mobile phase B), ranging from (4% phase A/6% phase B to 100% phase B and flushed with 4% phase A/6% phase B).

Flow rate: 0.35 ml/min, Column temperature: 40 °C, autosampler temperature: 4 °C, injection volume: 4 ml (e.g. prepared by weighing about 10 mg of powder in a 10 ml volumetric flask and diluted to volume with water). Examples of detectors are UV (ultraviolet, UV 220 nm) and MS (mass spectrometry).

INDUSTRIAL APPLICABILITY

This invention finds use in the pharmaceutical, medical, and other health care fields.

PATENT

WO2014085480 ,

claiming use of trofinetide for treating autism spectrum disorders including autism, Fragile X Syndrome or Rett Syndrome.

EP 0 366 638 discloses GPE (a tri-peptide consisting of the amino acids Gly-Pro- Glu) and its di-peptide derivatives Gly-Pro and Pro-Glu. EP 0 366 638 discloses that GPE is effective as a neuromodulator and is able to affect the electrical properties of neurons.

W095/172904 discloses that GPE has neuroprotective properties and that administration of GPE can reduce damage to the central nervous system (CNS) by the prevention or inhibition of neuronal and glial cell death.

WO 98/14202 discloses that administration of GPE can increase the effective amount of choline acetyltransferase (ChAT), glutamic acid decarboxylase (GAD), and nitric oxide synthase (NOS) in the central nervous system (CNS).

WO99/65509 discloses that increasing the effective amount of GPE in the CNS, such as by administration of GPE, can increase the effective amount of tyrosine hydroxylase (TH) in the CNS for increasing TH-mediated dopamine production in the treatment of diseases such as Parkinson’s disease.

WO02/16408 discloses GPE analogs capable of inducing a physiological effect equivalent to GPE within a patient. The applications of the GPE analogs include the treatment of acute brain injury and neurodegenerative diseases, including but not limited to, injury or disease in the CNS.

Example

The following non-limiting example illustrates the synthesis of a compound of the invention, NN-dimethylglycyl-L-prolyl-L-glutamic acid.

All starting materials and other reagents were purchased from Aldrich;
BOC = tert-butoxycarbonyl; Bn = benzyl.

BOC-(γ-benzyl)-L-prolyl-L-glutamic acid benzyl ester
To a solution of BOC-proline [Anderson GW and McGregor AC: J. Amer. Chem.

Soc: 79, 6180, 1957] (10 mmol) in dichloromethane (50 ml), cooled to 0 °C, was added triethylamine (1.39 ml, 10 mmol) and ethyl chloroformate (0.96 ml, 10 mmol). The resultant mixture was stirred at 0 °C for 30 minutes. A solution of dibenzyl L-glutamate (10 mmol) was then added and the mixture stirred at 0 °C for 2 hours then warmed to room temperature and stirred overnight. The reaction mixture was washed with aqueous sodium bicarbonate and citric acid (2 mol l“1) then dried (MgS04) and concentrated at reduced pressure to give BOC-(γ-benzyl)-L-prolyl-L-glutamic acid dibenzyl ester (5.0 g, 95%).

(7-Benzyl)-L-prolyl-L-glutamic acid dibenzyl ester
A solution of BOC-(γ-benzyl)-L-prolyl-L-glutamic acid dibenzyl ester (3.4 g, 10 mmol), cooled to 0 °C, was treated with trifluoroacetic acid (25 ml) for 2 hr at room temperature. After removal of the volatiles at reduced pressure the residue was triturated with ether to give (γ-benzyl)-L-prolyl-L-glutamic acid dibenzyl ester (I).

N,N-Dimethylglycyl-L-prolyl-L-glutamic acid
A solution of dicyclohexylcarbodiimide (10.3 mmol) in dichloromethane (10 ml) was added to a stirred and cooled (0 °C) solution of (7-benzyl)-L-prolyl-L-glutamic acid dibenzyl ester (10 mmol), TVN-dimethylglycine (10 mmol) and triethylamine
(10.3 mmol) in dichloromethane (30 ml). The mixture was stirred at 0 °C overnight and then at room temperature for 3 h. After filtration, the filtrate was evaporated at reduced pressure. The resulting crude dibenzyl ester was dissolved in a mixture of ethyl acetate (30 ml) and methanol (30 ml) containing 10% palladium on charcoal (0.5 g) then hydrogenated at room temperature and pressure until the uptake of hydrogen ceased. The filtered solution was evaporated and the residue recrystallized from ethyl acetate to yield the tri-peptide derivative.

It will be evident that following the method of the Example, and using alternative amino acids or their amides or esters, will yield other compounds of Formula 1.

PAPER

Tetrahedron (2005), 61(42), 10018-10035.  (CLICK HERE)

The synthesis of ten proline-modified analogues of the neuroprotective tripeptide GPE is described. Five of the analogues incorporate a proline residue with a hydrophobic group at C-2 and two further analogues have this side chain locked into a spirolactam ring system. The pyrrolidine ring was also modified by replacing the γ-CH2 group with sulfur and/or incorporation of two methyl groups at C-5.

Graphical Abstract

PAPER

Bioorganic & Medicinal Chemistry Letters (2005), 15(9), 2279-2283

A series of GPE analogues, including modifications at the Pro and/or Glu residues, was prepared and evaluated for their NMDA binding and neuroprotective effects. Main results suggest that the pyrrolidine ring puckering of the Pro residue plays a key role in the biological responses, while the preference for cis or trans rotamers around the Gly-Pro peptide bond is not important.

Graphical abstract

A series of Pro and/or Glu modified GPE analogues is described. Compounds incorporating PMe and dmP showed higher affinity for glutamate receptors than GPE and neuroprotective effects similar to those of this endogenous tripeptide in culture hippocampal neurons exposed to NMDA.

PATENT

US 20060251649

WO 2006127702

US 20070004641

US 20080145335

WO 2012102832

WO 2014085480

US 20140147491

References

  1. ^ Bickerdike MJ, Thomas GB, Batchelor DC, Sirimanne ES, Leong W, Lin H, et al. (March 2009). “NNZ-2566: a Gly-Pro-Glu analogue with neuroprotective efficacy in a rat model of acute focal stroke”. Journal of the Neurological Sciences278 (1–2): 85–90. doi:10.1016/j.jns.2008.12.003PMID 19157421S2CID 7789415.
  2. ^ Cartagena CM, Phillips KL, Williams GL, Konopko M, Tortella FC, Dave JR, Schmid KE (September 2013). “Mechanism of action for NNZ-2566 anti-inflammatory effects following PBBI involves upregulation of immunomodulator ATF3”Neuromolecular Medicine15 (3): 504–14. doi:10.1007/s12017-013-8236-zPMID 23765588S2CID 12522580.
  3. ^ Deacon RM, Glass L, Snape M, Hurley MJ, Altimiras FJ, Biekofsky RR, Cogram P (March 2015). “NNZ-2566, a novel analog of (1-3) IGF-1, as a potential therapeutic agent for fragile X syndrome”. Neuromolecular Medicine17 (1): 71–82. doi:10.1007/s12017-015-8341-2PMID 25613838S2CID 11964380.
  4. ^ Study Details – Rett Syndrome Study
  5. ^ Neuren’s trofinetide successful in Phase 2 clinical trial in Fragile X
PHASESTATUSPURPOSECONDITIONSCOUNT
3Enrolling by InvitationTreatmentRett’s Syndrome1
3RecruitingTreatmentRett’s Syndrome1
2CompletedSupportive CareInjuries, Brain1
2CompletedTreatmentFragile X Syndrome (FXS)1
2CompletedTreatmentInjuries, Brain1
2CompletedTreatmentRett’s Syndrome2
2TerminatedTreatmentConcussions1
1CompletedTreatmentBrain Injuries,Traumatic2
Legal status
Legal statusUS: Investigational New Drug
Identifiers
IUPAC name[show]
CAS Number853400-76-7 
PubChem CID11318905
ChemSpider9493869
UNIIZ2ME8F52QL
Chemical and physical data
FormulaC13H21N3O6
Molar mass315.322 g·mol−1
3D model (JSmol)Interactive image
SMILES[hide]C[C@]1(CCCN1C(=O)CN)C(=O)N[C@@H](CCC(=O)O)C(=O)O
InChI[hide]InChI=1S/C13H21N3O6/c1-13(5-2-6-16(13)9(17)7-14)12(22)15-8(11(20)21)3-4-10(18)19/h8H,2-7,14H2,1H3,(H,15,22)(H,18,19)(H,20,21)/t8-,13-/m0/s1Key:BUSXWGRAOZQTEY-SDBXPKJASA-N

////////////Tofinetide , NNZ 2566, PHASE 2, PHASE 3. NEUREN, Amino Acids, Peptides, Proteins,

CC1(CCCN1C(=O)CN)C(=O)NC(CCC(=O)O)C(=O)O

Devimistat


Devimistat Chemical Structure
DEVIMISTAT
6,8-Bis(benzylthio)octanoic acid.png

Devimistat

CPI-613

Molecular Weight388.59
FormulaC₂₂H₂₈O₂S₂
CAS No.95809-78-2
SMILESO=C(O)CCCCC(SCC1=CC=CC=C1)CCSCC2=CC=CC=C2

phase III, hematological cancer

6,8-Bis(benzylsulfanyl)octanoic acid

Octanoic acid, 6,8-bis[(phenylMethyl)thio]-

Octanoic acid, 6,8-bis((phenylmethyl)thio)-

Rafael Pharmaceuticals (formerly Cornerstone Pharmaceuticals), a subsidiary of Rafael Holdings, is developing devimistat, the lead candidate from a program of thioctans and their derivatives that act as pyruvate dehydrogenase and alpha-ketoglutarate inhibitors and stimulators of pyruvate dehydrogenase kinase (PDK), using the company’s proprietary Altered Energy Metabolism Directed (AEMD) platform, for the iv treatment of hematological cancer [phase III, January 2021].

Devimistat (INN; development code CPI-613) is an experimental anti-mitochondrial drug being developed by Rafael Pharmaceuticals.[1] It is being studied for the treatment of patients with metastatic pancreatic cancer and relapsed or refractory acute myeloid leukemia (AML).

Devimistat’s mechanism of action differs from other drugs, operating on the tricarboxylic acid cycle and inhibiting enzymes involved with cancer cell energy metabolism. A lipoic acid derivative different from standard cytotoxic chemotherapy, devimistat is currently being studied in combination with modified FOLFIRINOX to treat various solid tumors and heme malignancies.

Regulation

The U.S. Food and Drug Administration (FDA) has designated devimistat as an orphan drug for the treatment of pancreatic cancer, AML, myelodysplastic syndromes (MDS), peripheral T-cell lymphoma, and Burkitt’s lymphoma, and given approval to initiate clinical trials in pancreatic cancer and AML.

Clinical trials

Clinical trials of the drug are underway including a Phase III open-label clinical trial[2] to evaluate efficacy and safety of devimistat plus modified FOLFIRINOX (mFFX) versus FOLFIRINOX (FFX) in patients with metastatic adenocarcinoma of the pancreas.

Developed as part of Rafael’s proprietary Altered Metabolism Directed (AMD) drug platform, CPI-613® was discovered at Stony Brook University. CPI-613® is designed to target the mitochondrial tricarboxylic acid (TCA) cycle, an indispensable process essential to tumor cell multiplication and survival, selectively in cancer cells.

The attacks of CPI-613® on the TCA cycle also substantially increases the sensitivity of cancer cells to a diverse range of chemotherapeutic agents. This synergy allows for combinations of CPI-613® with lower doses of these generally toxic drugs to be highly effective with lower patient side effects. Combinations with CPI-613® represent a diverse range of potential opportunities to substantially improve patient benefit in many different cancers.

The U.S. Food and Drug Administration (FDA) has given Rafael approval to initiate pivotal clinical trials in pancreatic cancer and acute myeloid leukemia (AML), and has designated CPI-613® as an orphan drug for the treatment of pancreatic cancer, AML, Myelodysplastic syndromes (MDS), peripheral T-cell lymphoma and Burkitt’s lymphoma. The EMA has granted orphan drug designation to CPI-613® for pancreatic cancer and AML.


Learn more about recent developments involving CPI-613®CPI-613® (devimistat) Fact Sheet

he FDA granted a Fast Track designation to devimistat for the treatment of patients with acute myeloid leukemia.

The FDA has granted a Fast Track designation to devimistat (CPI-613) for the treatment of patients with acute myeloid leukemia (AML), Rafael Pharmaceuticals, announced in a press release.1

“This designation underscores the pressing need to find new ways to combat this aggressive disease,” said Jorge Cortes, MD, director of the Georgia Cancer Center at Augusta University, and principal investigator on the phase 3 clinical trial, in a statement. “It brings hope not only to clinicians, but to patients who hear that they have been diagnosed.”

The first-in-class agent devimistat targets enzymes that are involved in cancer cell energy metabolism. This therapy substantially increases the sensitivity of cancer cells to a diverse range of chemotherapies, and this synergy allows for potential combinations that could be more effective with devimistat and lower doses of drugs that are generally toxic.

“Receiving Fast Track designation, especially during a pandemic that has created significant challenges for many trials across the globe, is a testament to the dedicated work of the Rafael team,” stated Sanjeev Luther, president and CEO of Rafael Pharmaceuticals, Inc.

Devimistat combinations appear promising with a diverse range of potential opportunities to improve benefit in patients with various cancer types. Two pivotal phase 3 clinical trials, including the AVENGER 500 study in pancreatic cancer (NCT03504423) and ARMADA 2000 for AML (NCT03504410), have been approved for initiation by the FDA.

The primary end point of the multicenter, open-label, randomized ARMADA 2000 study is complete response (CR), and secondary end points include overall survival and CR plus CR with partial hematologic recovery rate. To be eligible to enroll to the study, patients must be aged ≥50 years with a documented AML diagnosis that has relapsed from or became refractory to previous standard therapy. Patients must have an ECOG performance status of 0 to 2 and an expected survival longer than 3 months.

Five hundred patients are expected to be enrolled and randomized in the study. To enroll, patients could not have received prior radiotherapy or cytotoxic chemotherapy for their current AML. Those with active central nervous system involvement, active uncontrolled bleeding, history of other malignancy, or known hypersensitivity to study drugs are ineligible to enroll to the trial as well.

This study aims to determine the safety and efficacy of devimistat in combination with high-dose cytarabine and mitoxantrone in older patients with relapsed/refractory AML compared with high-dose cytarabine and mitoxantrone therapy alone. Other control groups include patients treated with mitoxantrone, etoposide, and cytarabine and the combination of fludarabine, cytarabine, and filgrastim. The addition of devimistat is expected to improve the CR rate in patients who are aged 50 years or older with relapsed/refractory AML.

In a prior phase 1 study of devimistat plus high-dose cytarabine and mitoxantrone in patients with relapsed/refractory AML, the addition of devimistat sensitized AML cells to chemotherapy treatment.2

The objective response rate was 50% including CRs in 26 of 62 evaluable patients. Median overall survival was 6.7 months. In patients above age 60, the CR or CR with incomplete hematologic recovery rate was 47% and the median survival was 6.9 months.

This designation for this experimental anti-mitochondrial agent follows news of another Fast Track designation granted to devimistat for the treatment of patients with metastatic pancreatic cancer in November 2020, as well as an Orphan Drug designation granted in October 2020 for the treatment of patients with soft tissue sarcoma.

References

1. Rafael Pharmaceuticals Receives FDA Fast Track Designation for CPI-613® (devimistat) for the treatment of acute myeloid leukemia (AML). News Release. Rafael Pharmaceuticals, Inc. December 15, 2020. Accessed December 15, 2020. https://bit.ly/34g6YsR

2. Pardee TS, Anderson RG, Pladna KM, et al. A Phase I Study of CPI-613 in Combination with High-Dose Cytarabine and Mitoxantrone for Relapsed or Refractory Acute Myeloid Leukemia. Clin Cancer Res. 2018;24(9):2060-2073. doi:10.1158/1078-0432.CCR-17-2282 P[APERJournal of the American Chemical Society (1954), 76, 4109-12.https://pubs.acs.org/doi/abs/10.1021/ja01645a016
PAPERJournal of the American Chemical Society (1955), 77, 416-19.https://pubs.acs.org/doi/abs/10.1021/ja01607a057PAPERJustus Liebigs Annalen der Chemie (1958), 614, 66-83.https://chemistry-europe.onlinelibrary.wiley.com/doi/abs/10.1002/jlac.19586140108PATENTWO 2009123597WO 2009110859WO 2010110771PATENTCN 111362848

PATENT

WO-2021011334

Deuterated derivatives of 6,8-bis(benzylsulfanyl)octanoic acid (CPI-613 or devimistat ) or its salts for treating cancer.

CPI-613 (6,8-bis(benzylsulfanyl)octanoic acid) is a first-in-class investigational small-molecule (lipoate analog), which targets the altered energy metabolism unique to many cancer cells. CPI-613 is currently being evaluated in two phase III clinical trials, and has been granted orphan drug designation for the treatment of pancreatic cancer, acute myeloid leukemia (AML), peripheral T-cell lymphoma (PTCL), Burkitt lymphoma and myelodysplastic syndromes (MDS).

[0004] One limitation to the clinical utility of CPI-613 is its very rapid metabolism. After IV dosing the half-life of 6,8-bis(benzylsulfanyl)octanoic acid is only about 1-2 hours (Pardee,

T.S. et al, Clin Cancer Res. 2014, 20, 5255-64). The short half-life limits the patient’s overall exposure to the drug and necessitates administration of relatively high doses. For safety reasons, CPI-613 is administered via a central venous catheter as an IV infusion over 30-120 minutes, with higher doses requiring longer infusion times.

The terms“6,8-bis(benzylsulfanyl)octanoic acid” and“ 6,8-bis-benzylthio-octanoic acid” refer to the compound known as CPI-613 or devimistat, having the chemical structure

PATENT

WO2020132397

claiming the use of CPI-613 in combination with an autophagy inhibitor eg chloroquine for treating eg cancers.

CPI-613 (6,8-bis-benzylthio-octanoic acid) is a first-in-class investigational small-molecule (lipoate analog), which targets the altered energy metabolism that is common to many cancer cells. CPI-613 has been evaluated in multiple phase I, I/II, and II clinical studies, and has been granted orphan drug designation for the treatment of pancreatic cancer, acute myeloid leukemia (AML), peripheral T-cell lymphoma (PTCL), Burkitt lymphoma and myelodysplastic syndromes (MDS).

PAPER

https://pubs.acs.org/doi/10.1021/op200091t

An Efficient, Economical Synthesis of the Novel Anti-tumor Agent CPI-613

Cite this: Org. Process Res. Dev. 2011, 15, 4, 855–857

Publication Date:May 2, 2011
https://doi.org/10.1021/op200091t

An efficient and practical synthesis of the novel anti-tumor compound 6,8-dithiobenzyl octanoic acid, CPI-613 (2), was developed and executed on a practical scale. CPI-613 can be made in a single vessel from (±)-lipoic acid (1) via reductive opening of the disulfide ring followed by benzylation of the sulfhydryls with benzyl bromide. CPI-613 was isolated by simple crystallization in high yield and purity. The process is scaleable and has been demonstrated at up to 100 kg.CPI-613 (2) was isolated [4.7 kg (90%)] with an HPLC purity of 99.8 area %. Mp 66–67 °C. IR: 3050, 1710, 1400, 668 cm–11H NMR (400 MHz, CDCl3) δ 7.40–7.20 (m, 10 H), 3.80–3.60 (m, 4 H), 2.60–2.50 (m, 2 H), 2.44 (t, J = 8.7, 2 H), 2.23 (t, J = 8.1, 2 H) 2.03–1.30 (m, 8 H). Anal. Calc for C22H28O2S2: C, 68.00; H, 7.26; S, 16.50. Found: C, 67.99; H, 7.31; S, 16.37. 

References

  1. ^ “CPI-613”. Rafael Pharmaceuticals.
  2. ^ Philip PA, Buyse ME, Alistar AT, Rocha Lima CM, Luther S, Pardee TS, Van Cutsem E (October 2019). “A Phase III open-label trial to evaluate efficacy and safety of CPI-613 plus modified FOLFIRINOX (mFFX) versus FOLFIRINOX (FFX) in patients with metastatic adenocarcinoma of the pancreas”Future Oncology15 (28): 3189–3196. doi:10.2217/fon-2019-0209PMC 6854438PMID 31512497.
Clinical data
Other namesCPI-613
Legal status
Legal statusInvestigational
Identifiers
IUPAC name[show]
CAS Number95809-78-2
PubChem CID24770514
DrugBank12109
ChemSpider28189062
UNIIE76113IR49
ChEMBLChEMBL3186849
CompTox Dashboard(EPA)DTXSID70914807
ECHA InfoCard100.231.125 
Chemical and physical data
FormulaC22H28O2S2
Molar mass388.58 g·mol−1
3D model (JSmol)Interactive image
SMILES[hide]C1=CC=C(C=C1)CSCCC(CCCCC(=O)O)SCC2=CC=CC=C2

//////////devimistat, CPI-613, CPI 613, phase 3, hematological cancer , Fast Track designation, ORPHAN DRUG, 

BINDARIT


Bindarit.png
ChemSpider 2D Image | bindarit | C19H20N2O3
Bindarit Chemical Structure

BINDARIT

  • Molecular FormulaC19H20N2O3
  • Average mass324.374 Da

CAS 130641-38-2

2-[(1-benzylindazol-3-yl)methoxy]-2-methylpropanoic acid

2-[(1 -benzyl-1 H-indazol-3-yl)methoxy]-2-methylpropanoic acid

2-[(1-benzyl-1H-indazol-3-yl)methoxy]-2-methylpropanoic acidJQ11LH711MPropanoic acid, 2-methyl-2-[[1-(phenylmethyl)-1H-indazol-3-yl]methoxy]- [ACD/Index Name]биндарит [Russian] [INN]بينداريت [Arabic] [INN]宾达利 [Chinese] [INN]PHASE 2Bindarit has been used in trials studying the prevention and treatment of Coronary Restenosis and Diabetic Nephropathy.

Bindarit, an inhibitor of monocyte chemotactic protein synthesis, protects against bone loss induced by chikungunya virus infection

Bindarit (AF2838) is a selective inhibitor of the monocyte chemotactic proteins MCP-1/CCL2MCP-3/CCL7, and MCP-2/CCL8, and no effect on other CC and CXC chemokines such as MIP-1α/CCL3, MIP-1β/CCL4, MIP-3/CCL23. Bindarit also has anti-inflammatory activity.

As is known, MCP-1 (Monocyte Chemotactic Protein-1 ) is a protein belonging to the β subfamily of chemokines. MCP-1 has powerful chemotactic action on monocytes and exerts its action also on T lymphocytes, mastocytes and basophils (Rollins BJ. , Chemokines, Blood 1997; 90: 909-928; M.

Baggiolini, Chemokines and leukocyte traffic, Nature 1998; 392: 565-568).

Other chemokines belonging to the β subfamily are, for example, MCP-2 (Monocyte Chemotactic Protein-2), MCP-3, MCP-4, MIP-1 α and MIP-1 β, RANTES.

The β subfamily differs from the α subfamily in that, in the structure, the first two cysteines are adjacent for the β subfamily, whereas they are separated by an intervening amino acid for the α subfamily. MCP-1 is produced by various types of cells (leukocytes, platelets, fibroblasts, endothelial cells and smooth muscle cells).

Among all the known chemokines, MCP-1 shows the highest specificity for monocytes and macrophages, for which it constitutes not only a chemotactic factor but also an activation stimulus, consequently inducing processes for producing numerous inflammatory factors (superoxides, arachidonic acid and derivatives, cytokines/chemokines) and amplifying the phagocytic activity.

The secretion of chemokines in general, and of MCP-1 in particular, is typically induced by various pro-inflammatory factors, for instance interleukin-1 (IL-1 ), interleukin-2 (IL-2), TNFα (Tumour Necrosis Factor α), interferon-γ and bacterial lipopolysaccharide (LPS).

Prevention of the inflammatory response by blocking the chemokine/chemokine receptor system represents one of the main targets of pharmacological intervention (Gerard C. and Rollins B. J., Chemokines and disease. Nature Immunol. 2001 ; 2:108-1 15).

There is much evidence to suggest that MCP-1 plays a key role during inflammatory processes and has been indicated as a new and validated target in various pathologies.

Evidence of a considerable physiopathological contribution of MCP-1 has been obtained in the case of patients with articular and renal inflammatory diseases (rheumatoid arthritis, lupus nephritis, diabetic nephropathy and rejection following transplant).

However, more recently, MCP-1 has been indicated among the factors involved in inflammatory pathologies of the CNS (multiple sclerosis, Alzheimer’s disease, HIV-associated dementia) and other pathologies and conditions, with and without an obvious inflammatory component, including atopic dermatitis, colitis, interstitial lung pathologies, restenosis, atherosclerosis, complications following a surgical intervention (for instance angioplasty, arterectomy, transplant, organ and/or tissue replacement, prosthesis implant), cancer (adenomas, carcinomas and metastases) and even metabolic diseases such as insulin resistance and obesity.

In addition, despite the fact that the chemokine system is involved in controlling and overcoming viral infections, recent studies have demonstrated that the response of certain chemokines, and in particular of MCP-1 , may have a harmful role in the case of host-pathogen interactions. In particular, MCP-1 has been indicated among the chemokines that contribute towards organ and tissue damage in pathologies mediated by alpha viruses characterized by monocyte/macrophage infiltration in the joints and muscles (Mahalingam S. et al. Chemokines and viruses: friend or foes? Trends in Microbiology 2003; 1 1 : 383-391 ; RuIIi N. et al. Ross River Virus: molecular and cellular aspects of disease pathogenesis. 2005; 107: 329-342).

Monocytes are the main precursors of macrophages and dendritic cells, and play a critical role as mediators of inflammatory processes. CX3CR1 , with its ligand CX3CL1 (fractalkine), represents a key factor in regulating the migration and adhesiveness of monocytes. CX3CR1 is expressed in monocytes, whereas CX3CL1 is a transmembrane chemokine in endothelial cells. Genetic studies in man and in animal models have demonstrated an important role in the physiopathology of inflammatory diseases of CX3CR1 and CX3CL1. There is in fact much evidence to suggest a key contribution of CX3CR1 and of its ligand in the pathogenesis and progression of articular, renal, gastrointestinal and vascular inflammatory diseases (e.g. rheumatoid arthritis, lupus nephritis, diabetic nephropathy, Crohn’s disease, ulcerative colitis, restenosis and atherosclerosis). The expression of CX3CR1 is over-regulated in T cells, which are believed to accumulate in the synovium of patients suffering from rheumatoid arthritis. In addition, the expression of CX3CL1 is over-regulated in endothelial cells and fibroblasts present in the synovium of these patients. Consequently, the CX3CR1/CX3CL1 system plays an important role in controlling the type of cell and the mode of infiltration of the synovium and contributes towards the pathogenesis of rheumatoid arthritis (Nanki T. et al., “Migration of CX3CR1-positive T cells producing type 1 cytokines and cytotoxic molecules into the synovium of patients with rheumatoid arthritis”, Arthritis & Rheumatism (2002), vol. 46, No. 1 1 , pp. 2878-2883). In patients suffering form renal damage, the majority of the inflammatory leukocytes that infiltrate the kidneys express CX3CR1 , and in particular it is expressed on two of the main cell types involved in the most common inflammatory renal pathologies and in kidney transplant rejection, T cells and monocytes (Segerer S. et al., Expression of the fractalkine receptor (CX3CR1 ) in human kidney diseases, Kidney International (2002) 62, pp. 488-495).

Participation of the CX3CR1/CX3CL1 system has been suggested also in inflammatory bowel diseases (IBD). In point of fact, in the case of patients suffering from IBD (e.g. Crohn’s disease, ulcerative colitis), a significant increase in the production of CX3CL1 by the intestinal capillary system and a – A – significant increase in CX3CR1 -positive cells have been demonstrated, both at the circulatory level and in the mucosa (Sans M. et al., “Enhanced recruitment of CX3CR1 + T cells by mucosal endothelial cell-derived fractalkine in inflammatory bowel diseases”, Gastroenterology 2007, vol. 132, No. 1 , pp. 139-153).

Even more interesting is the demonstration of the key role played by the CX3CR1/CX3CL1 system in vascular damage and in particular under pathological conditions, for instance atherosclerosis and restenosis. CX3CR1 is indicated as a critical factor in the process of infiltration and accumulation of monocytes in the vascular wall, and CX3CR1 polymorphism in man is associated with a reduced prevalence of atherosclerosis, coronary disorders and restenosis (Liu P. et al., “Cross-talk among Smad, MAPK and integrin signalling pathways enhances adventitial fibroblast functions activated by transforming growth factor-1 and inhibited by Gax” Arterioscler. Thromb. Vase. Biol. 2008; McDermott D. H. et al., “Chemokine receptor mutant CX3CR1 -M280 has impaired adhesive function and correlates with protection from cardiovascular diseases in humans”, J. Clin. Invest. 2003; Niessner A. et al., Thrombosis and Haemostasis 2005).

IL-12 and IL-23 are members of a small family of proinflammatory heterodimeric cytokines. Both cytokines share a common subunit, p40, which is covalently bonded either to the p35 subunit to produce the mature form of IL-12, or to the p19 subunit to produce the mature form of IL-23. The receptor for IL-12 is constituted by the subunits IL-12Rβ1 and IL-12Rβ2, while the receptor for IL-23 is constituted by the subunits IL-12Rβ1 and IL-23R. IL-12 and IL-23 are mainly expressed by activated dendritic cells and by phagocytes. The receptors for the two cytokines are expressed on the T and NK cells, and NK T cells, but low levels of complexes of the receptor for IL-23 are also present in monocytes, macrophages and dendritic cells.

Despite these similarities, there is much evidence to suggest that IL-12 and IL-23 control different immunological circuits. In point of fact, whereas IL-12 controls the development of Th1 cells, which are capable of producing gamma-interferon (IFN-γ), and increases the cytotoxic, antimicrobial and antitumoral response, IL-23 regulates a circuit that leads to the generation of CD4+ cells, which are capable of producing IL-17. The induction of IL-23- dependent processes leads to the mobilization of various types of inflammatory cell, for instance TH-17, and it has been demonstrated as being crucial for the pathogenesis of numerous inflammatory pathologies mediated by immonological responses. Typical examples of pathologies associated with the expression of p40 are chronic inflammatory diseases of the articular apparatus (e.g. rheumatoid arthritis), of the dermatological apparatus (e.g. psoriasis) and of the gastrointestinal apparatus (e.g. Crohn’s disease). However, IL-23 also exerts a role in promoting tumour incidence and growth. In point of fact, IL-23 regulates a series of circuits in the tumoral microenvironment, stimulating angiogenesis and the production of inflammation mediators.

Psoriasis is a chronic inflammatory skin disease that affects 3% of the world’s population (Koo J. Dermatol. Clin. 1996; 14:485-96; Schon M. P. et al., N. Engl. J. Med. 2005; 352: 1899-912). A type-1 aberrant immune response has been correlated with the pathogenesis of psoriasis, and the cytokines that induce this response, such as IL-12 and IL-23, may represent suitable therapeutic objects. The expression of IL-12 and IL-23, which share the subunit p40, is significantly increased in psoriasis plaques, and preclinical studies have demonstrated a role of these cytokines in the pathogenesis of psoriasis. More recently, the treatment of anti- IL-12 and IL-23 monoclonal antibodies of patients suffering from psoriasis proved to be effective in improving the signs of progression and seriousness of the disease and has subsequently reinforced the role of IL-12 and IL-23 in the physiopathology of psoriasis. Crohn’s disease is a chronic inflammatory pathology of the digestive apparatus and may affect any region thereof – from the mouth to the anus. It typically afflicts the terminal tract of the ileum and well-defined areas of the large intestine. It is often associated with systemic autoimmune disorders, such as mouth ulcers and rheumatic arthritis. Crohn’s disease affects over 500 000 people in Europe and 600 000 people in the United States.

Crohn’s disease is a pathology associated with a Th1 cell-mediated excessive activity of cytokines. IL-12 is a key cytokine in the initiation of the inflammatory response mediated by Th1 cells. Crohn’s disease is characterized by increased production of IL-12 by cells presenting the antigen in intestinal tissue, and of gamma-interferon (IFN-γ) and TNFα by lymphocytes and intestinal macrophages. These cytokines induce and support the inflammatory process and thickening of the intestinal wall, which are characteristic signs of the pathology. Preclinical and clinical evidence has demonstrated that inhibition of IL-12 is effective in controlling the inflammatory response in models of intestinal inflammation and/or in patients suffering from Crohn’s disease.

The relationship between cancer and inflammation is now an established fact. Many forms of tumours originate from sites of inflammation, and inflammation mediators are often produced in tumours.

IL-23 has been identified as a cytokine associated with cancer and, in particular, the expression of IL-23 is significantly high in samples of human carcinomas when compared with normal adjacent tissues. In addition, the absence of a significant expression of IL-23 in the normal adjacent tissues suggests an over-regulation of IL-23 in tumours, reinforcing its role in tumour genesis.

European patent EP-B-O 382 276 describes a number of 1-benzyl-3-hydroxymethylindazole derivatives endowed with analgesic activity. In turn, European patent EP-B-O 510 748 describes, on the other hand, the use of these derivatives for preparing a pharmaceutical composition that is active in the treatment of autoimmune diseases. Finally, European patent EP-B-1 005 332 describes the use of these derivatives for preparing a pharmaceutical composition that is active in treating diseases derived from the production of MCP-1. 2-Methyl-2-{[1-(phenylmethyl)-1 H-indazol-3-yl]methoxy}propanoic acid is thought to be capable of inhibiting, in a dose-dependent manner, the production of MCP-1 and TNF-α induced in vitro in monocytes from LPS and Candida albicans, whereas the same compound showed no effects in the production of cytokines IL-1 and IL-6, and of chemokines IL-8, MIP-1 α, and RANTES (Sironi M. et al., “A small synthetic molecule capable of preferentially inhibiting the production of the CC chemokine monocyte chemotactic protein-1 “, European Cytokine Network. Vol. 10, No. 3, 437-41 , September 1999).

European patent application EP-A-1 185 528 relates to the use of triazine derivatives for inhibiting the production of IL-12. European patent application EP-A-1 188 438 and EP-A-1 199 074 relate to the use of inhibitors of the enzyme PDE4, for instance Rolipram, Ariflo and diazepine-indole derivatives, in the treatment and prevention of diseases associated with excessive production of IL-12. European patent application EP-A-1 369 1 19 relates to the use of hyaluronane with a molecular weight of between 600 000 and 3 000 000 daltons for controlling and inhibiting the expression of IL-12. European patent application EP-A-1 458 687 relates to the use of pyrimidine derivatives for treating diseases related to an overproduction of IL-12. European patent application EP-A-1 819 341 relates to the use of nitrogenous heterocyclic compounds, for instance pyridine, pyrimidine and triazine derivatives, for inhibiting the production of IL-12 (or of other cytokines, such as IL-23 and IL-27 which stimulate the production of IL-12). European patent application EP-A-1 827 447 relates to the use of pyrimidine derivatives for treating diseases related to an overproduction of IL-12, IL-23 and IL-27.

European patent applications EP-A-1 869 055, EP-A-1 869 056 and EP-A-1 675 862 describe 1 ,3-thiazolo-4,5-pyrimidine derivatives that are capable of acting as CX3CR1 receptor antagonists.

Despite the activity developed thus far, there is still felt to be a need for novel pharmaceutical compositions and compounds that are effective in the treatment of diseases based on the expression of MCP-1 , CX3CR1 and p40. The Applicant has found, surprisingly, novel 1-benzyl-3-hydroxymethylindazole derivatives with pharmacological activity.

The Applicant has found, surprisingly, that the novel 1-benzyl-3-hydroxymethylindazole derivatives according to formula (I) of the present invention are capable of reducing the production of the chemokine MCP-1. More surprisingly, the Applicant has found that the novel 1-benzyl-3-hydroxymethylindazole derivatives according to formula (I) of the present invention are capable of reducing the expression of the chemokine MCP-1.

Even more surprisingly, the Applicant has found that the 1-benzyl-3-hydroxymethylindazole derivatives according to formula (I) of the present invention are capable of reducing the expression of the subunit p40 involved in the production of the cytokines IL-12 and IL-23, and the expression of the receptor CX3CR1.

SYN

PATENTS

EP 0382276

https://patents.google.com/patent/EP0382276A2/en

PATENT

WO 2009109613

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2009109613

Preparation of compound 29

2-[(1 -benzyl-1 H-indazol-3-yl)methoxy]-2-methylpropanoic acid The preparation of product 29 was performed as described in patent application EP 382 276.

PATENT

WO 2011015502

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2011015502

Example 5

Preparation of 2-[(1-benzyl-1H-indazol-3-yl)methoxy]-2-methylpropanoic acid

Ethyl-2-hydroxyisobutyrate (18.5 g, 140 mmol, 1.2 eq.), toluene (100 ml_) and DMF (20 ml_) were placed in a three-necked flask fitted with a mechanical stirrer and a reflux condenser under an inert atmosphere. A dispersion of 60% NaH (5.6 g, 140 mmol, 1.2 eq.) was added to the mixture in portions over a period of approximately 1.5 hours. A solution of i -benzyl-3-chloromethyl-I H-indazole (30 g,

117 mmol, 1 eq.) in toluene (90 ml_) and DMF (60 ml_) was then added dropwise. The reaction mixture was heated to approximately 90°C and kept at that temperature until the reaction was complete (checked by TLC, approximately 10 hours). After cooling to room temperature the mixture was washed with acidified water and water. The organic phase was concentrated under reduced pressure and the oily residue obtained was treated with 10 M NaOH (36 ml_) at reflux temperature for at least 3 hours. The product, which was precipitated out by the addition of concentrated HCI, was filtered and dried. Yield: 32.3 g of white solid (85%).

mp: 133-134°C.

Elemental analysis:Calculated: C (70.35), H (6.21 ), N (8.64), Found: C (70.15), H (6.17), N (8.63).

1H NMR (300 MHz, DMSO-d6) δ (ppm) 1.44 (s, 6H), 4.76 (s, 2H), 5.60 (s, 2H), 7.14 (t, 1 H, J = 7.6 Hz), 7.20-7.34 (m, 5H), 7.37 (ddd, 1 H, J = 8.3 Hz, 7.0 Hz, 1.1 Hz), 7.66 (d, 1 H, J = 8.4 Hz), 7.94 (d, 1 H, J = 8.1 Hz), 12.77 (s, 1 H).

13C NMR (300 MHz, DMSO-d6) δ (ppm) 24.48, 24.48, 51.63, 59.65,76.93, 109.69, 120.22, 121.06, 122.62, 126.28, 127.36, 127.36, 127.44, 128.46, 128.46, 137.49, 140.31 , 141.97, 175.46.

PATENT

WO 2011015501

https://patents.google.com/patent/WO2011015501A1

PATENT

US 8350052

US 8354544

US 8835481

//////////////BINDARIT, JQ11LH711M, биндарит , بينداريت , 宾达利 , AF2838, AF 2838, PHASE 2

CC(C)(C(=O)O)OCC1=NN(C2=CC=CC=C21)CC3=CC=CC=C3

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