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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 AFRICURE PHARMA, ROW2TECH, CLEANCHEM LABS as ADVISOR, earlier assignment was with GLENMARK LIFE SCIENCES LTD, as CONSUlTANT, Retired from GLENMARK in Jan2022 Research Centre as Principal Scientist, Process Research (bulk actives) at Mahape, Navi Mumbai, India. Total Industry exp 32 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, 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 32 PLUS year tenure till date Feb 2023, 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 100 million plus hits on Google, 2.5 lakh plus connections on all networking sites, 100 Lakh plus views on dozen plus blogs, 227 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 38 lakh plus views on New Drug Approvals Blog in 227 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 He has total of 32 International and Indian awards

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Efgartigimod alfa-fcab

December 23, 2021 4:30 am / Leave a comment

DKTHTCPPCP APELLGGPSV FLFPPKPKDT LYITREPEVT CVVVDVSHED PEVKFNWYVD
GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK
GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS
DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALKFHYTQKS LSLSPGK
(Disulfide bridge: 6-6′, 9-9′, 41-101, 147-205, 41′-101′, 147′-205′)

Efgartigimod alfa-fcab

Formula	C2310H3554N602O692S14
CAS	1821402-21-4
Mol weight	51279.464

US FDA APPROVED 12/17/2021, To treat generalized myasthenia gravis
Press Release, Vyvgart, BLA 761195

エフガルチギモドアルファ (遺伝子組換え)

PEPTIDE

Treatment of IgG-driven autoimmune diseases

AS ON DEC2021 3,491,869 VIEWS ON BLOG WORLDREACH AVAILABLEFOR YOUR ADVERTISEMENT

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https://www.fda.gov/news-events/press-announcements/fda-approves-new-treatment-myasthenia-gravis

FDA Approves New Treatment for Myasthenia Gravis

Approval is the First of a New Class of Medication for this Rare, Chronic, Autoimmune, Neuromuscular DiseaseFor Immediate Release:December 17, 2021

The U.S. Food and Drug Administration today approved Vyvgart (efgartigimod) for the treatment of generalized myasthenia gravis (gMG) in adults who test positive for the anti-acetylcholine receptor (AChR) antibody.

Myasthenia gravis is a chronic autoimmune, neuromuscular disease that causes weakness in the skeletal muscles (also called voluntary muscles) that worsens after periods of activity and improves after periods of rest. Myasthenia gravis affects voluntary muscles, especially those that are responsible for controlling the eyes, face, mouth, throat, and limbs. In myasthenia gravis, the immune system produces AChR antibodies that interfere with communication between nerves and muscles, resulting in weakness. Severe attacks of weakness can cause breathing and swallowing problems that can be life-threatening.

“There are significant unmet medical needs for people living with myasthenia gravis, as with many other rare diseases,” said Billy Dunn, M.D., director of the Office of Neuroscience in the FDA’s Center for Drug Evaluation and Research. “Today’s approval is an important step in providing a novel therapy option for patients and underscores the agency’s commitment to help make new treatment options available for people living with rare diseases.”

Vyvgart is the first approval of a new class of medication. It is an antibody fragment that binds to the neonatal Fc receptor (FcRn), preventing FcRn from recycling immunoglobulin G (IgG) back into the blood. The medication causes a reduction in overall levels of IgG, including the abnormal AChR antibodies that are present in myasthenia gravis.

The safety and efficacy of Vyvgart were evaluated in a 26-week clinical study of 167 patients with myasthenia gravis who were randomized to receive either Vyvgart or placebo. The study showed that more patients with myasthenia gravis with antibodies responded to treatment during the first cycle of Vyvgart (68%) compared to those who received placebo (30%) on a measure that assesses the impact of myasthenia gravis on daily function. More patients receiving Vyvgart also demonstrated response on a measure of muscle weakness compared to placebo.

The most common side effects associated with the use of Vyvgart include respiratory tract infections, headache, and urinary tract infections. As Vyvgart causes a reduction in IgG levels, the risk of infections may increase. Hypersensitivity reactions such as eyelid swelling, shortness of breath, and rash have occurred. If a hypersensitivity reaction occurs, discontinue the infusion and institute appropriate therapy. Patients using Vyvgart should monitor for signs and symptoms of infections during treatment. Health care professionals should administer appropriate treatment and consider delaying administration of Vyvgart to patients with an active infection until the infection is resolved.

The FDA granted this application Fast Track and Orphan Drug designations. The FDA granted the approval of Vyvgart to argenx BV.

///////////efgartigimod alfa-fcab, Vyvgart, FDA 2021,APPROVALS 2021, myasthenia gravis, argenx BV, Fast Track, Orphan Drug, PEPTIDE,

エフガルチギモドアルファ (遺伝子組換え)

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Pafolacianine

December 2, 2021 4:46 am / Leave a comment

ChemSpider 2D Image | OTL-38 | C61H67N9O17S4

Pafolacianine

OTL-38

Molecular FormulaC₆₁H₆₇N₉O₁₇S₄
Average mass1326.495 Da

FDA APPROVED NOV 2021

2-{(E)-2-[(3E)-2-(4-{2-[(4-{[(2-Amino-4-oxo-3,4-dihydro-6-pteridinyl)methyl]amino}benzoyl)amino]-2-carboxyethyl}phenoxy)-3-{(2E)-2-[3,3-dimethyl-5-sulfo-1-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene ]ethylidene}-1-cyclohexen-1-yl]vinyl}-3,3-dimethyl-1-(4-sulfobutyl)-3H-indolium-5-sulfonate OTL-38Tyrosine, N-[4-[[(2-amino-3,4-dihydro-4-oxo-6-pteridinyl)methyl]amino]benzoyl]-O-[(6E)-6-[(2E)-2-[1,3-dihydro-3,3-dimethyl-5-sulfo-1-(4-sulfobutyl)-2H-indol-2-ylidene]ethylidene]-2-[(E)-2-[3,3-dimethy l-5-sulfo-1-(4-sulfobutyl)-3H-indolium-2-yl]ethenyl]-1-cyclohexen-1-yl]-, inner salt

2-(2-(2-(4-((2S)-2-(4-(((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)amino)benzamido)-2-carboxyethyl)phenoxy)-3-(2-(3,3-dimethyl-5-sulfo-1-(4-sulfobutyl)-1,3-dihydro-2H-indol-2-ylidene)ethylidene)cyclohex-1-en-1-yl)ethenyl)-3,3-dimethyl-5-sulfo-1-(4-sulfobutyl)-3H-indolium inner salt,sodium salt (1:4)

3H-Indolium, 2-(2-(2-(4-((2S)-2-((4-(((2-amino-3,4-dihydro-4-oxo-6-pteridinyl)methyl)amino)benzoyl)amino)-2-carboxyethyl)phenoxy)-3-(2-(1,3-dihydro-3,3-dimethyl-5-sulfo-1-(4-sulfobutyl)-2H-indol-2-ylidene)ethylidene)-1-cyclohexen-1-yl)ethenyl)-3,3-dimethyl-5-sulfo-1 (4-sulfobutyl)-, inner salt,sodium salt (1:4)

1628423-76-6 [RN]

Pafolacianine sodium [USAN]
RN: 1628858-03-6
UNII: 4HUF3V875C

C61H68N9Na4O17S4+5

Intraoperative Imaging and Detection of Folate Receptor Positive Malignant Lesions

Pafolacianine, sold under the brand name Cytalux, is an optical imaging agent.^[1]^[2]

The most common side effects of pafolacianine include infusion-related reactions, including nausea, vomiting, abdominal pain, flushing, dyspepsia, chest discomfort, itching and hypersensitivity.^[2]

It was approved for medical use in the United States in November 2021.^[2]^[3]

Pafolacianine is a fluorescent drug that targets folate receptor (FR).^[1]

Medical uses

Pafolacianine is indicated as an adjunct for intraoperative identification of malignant lesions in people with ovarian cancer.^[1]^[2]

History

The safety and effectiveness of pafolacianine was evaluated in a randomized, multi-center, open-label study of women diagnosed with ovarian cancer or with high clinical suspicion of ovarian cancer who were scheduled to undergo surgery.^[2] Of the 134 women (ages 33 to 81 years) who received a dose of pafolacianine and were evaluated under both normal and fluorescent light during surgery, 26.9% had at least one cancerous lesion detected that was not observed by standard visual or tactile inspection.^[2]

The U.S. Food and Drug Administration (FDA) granted the application for pafolacianine orphan drug, priority review, and fast track designations.^[2]^[4] The FDA granted the approval of Cytalux to On Target Laboratories, LLC.^[2]

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SYN

WO 2014149073

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2014149073

In another aspect of the invention, this disclosure provides a method of synthesizing a compound having the formula

[0029] In a fourth embodiment of the invention, this disclosure provides a method of synthesizing a compound having the formula

[0030]

[0032] wherein C is any carbon isotope. In this embodiment, the amino acid linker is selected from a group consisting of methyl 2-di-tert-butyl dicarbonate-amino-3-(4-phenyl)propanoate, 3-(4-hydroxyphenyl)-2-(di-tert-butyl-dicarbonate methylamino)propanoic acid, 2-amino-4-(4-hydroxyphenyl)butanoic acid, and Tert-butyl (2-di-tert-butyl dicarbonate- amino)-3-(4-hydroxyphenyl)propanoate . In a particular embodiment, the aqueous base is potassium hydroxide (KOH). The method of this embodiment may also further include purifying the compound by preparatory HPLC.

EXAMPLE 1 : General synthesis of Pte – L Tyrosine – S0456 (OTL-0038)

[0088] Scheme:

C3₃H3₇CIF3N

Reactants for Step I:

[0089] A 500 mL round bottom flask was charged with a stirring bar, pteroic acid

(12.0 g, 29.40 mmol, 1 equiv), (L)-Tyr(-O^fBu)-O^fBu- HCI (1 1 .63 g, 35.28 mmol, 1 .2

equiv) and HATU (13.45 g, 35.28 mmol, 1 .2 equiv) then DMF (147 mL) was added to give a brown suspension [suspension A]. DIPEA (20.48 mL, 1 17.62 mmol, 4.0 equiv) was added slowly to suspension A at 23 °C, over 5 minutes. The suspension turned in to a clear brown solution within 10 minutes of addition of DIPEA. The reaction was stirred at 23 °C for 2.5 h. Reaction was essentially complete in 30 minutes as judged by LC/MS but was stirred further for 2.5 h. The formation of Pte_N¹⁰(TFA)_L_Tyr(-O^fBu)-O^fBu HCI (Figure 12) was confirmed by LC/MS showing m/z 409→m/z 684. LC/MS method: 0-50% acetonitrile in 20 mM aqueous NH₄OAc for 5 min using Aquity UPLC-BEH C18, 1 .7μιη 2.1 * 50 mm column . The reaction mixture was cannulated as a steady stream to a stirred solution of aq. HCI (2.0 L, 0.28 M) over the period of 30 minutes to give light yellow precipitate of Pte_N¹⁰(TFA)_L_Tyr(-O^fBu)-O^fBu HCI. The precipitated Pte_N ¹⁰(TFA)_L_Tyr(- O^fBu)-O^fBu HCI was filtered using sintered funnel under aspirator vacuum, washed with water (8 * 300 mL) until the pH of the filtrate is between 3 and 4. The wet solid was allowed to dry under high vacuum for 12 hours on the sintered funnel. In a separate batch, where this wet solid (3) was dried under vacuum for 48 hours and then this solid was stored at -20 ⁰ C for 48 h. However, this brief storage led to partial decomposition of 3. The wet cake (58 g) was transferred to a 500 mL round bottom flask and was submitted to the next step without further drying or purification.

Reactants for Step II:

The wet solid (58 g) was assumed to contain 29.40 mmol of the desired compound (3) (i. e. quantitative yield for the step I ).

[0090] A 500 mL round bottom flask was charged with a stirring bar, Pte_N¹⁰(TFA)_L_Tyr(-O^fBu)-O^fBu HCI as a wet cake (58 g, 29.40 mmol, 1 equiv). A solution of TFA:TIPS:H₂0 (95:2.5:2.5, 200 mL) was added at once to give a light brown suspension. The reaction content was stirred at 23°C for 1 .5 hours and was monitored by LC/MS. The suspension became clear dull brown solution after stirring for 5 minutes. LC/MS method: 0-50% acetonitrile in 20 mM aqueous NH₄OAc for 5 min using Aquity UPLC-BEH C18, 1 .7μιη 2.1 * 50 mm column. The formation of Pte_TFA_L_Tyr (Figure 12) was confirmed by showing m/z 684→m/z 572. Reaction time varies from 30 min to 1 .5 hours depending on the water content of Pte_N¹⁰(TFA)_L_Tyr(-O^fBu)-O^fBu HCI. The reaction mixture was cannulated as a steady stream to a stirred MTBE (1 .8 L) at 23 °C or 100 °C to give light yellow precipitate of Pte_TFA_L_Tyr. The precipitated Pte_TFA_L_Tyr was filtered using sintered funnel under aspirator vacuum, washed with MTBE (6 * 300 mL) and dried under high vacuum for 8 hours to obtain Pte_TFA_L_Tyr (14.98 g, 83.98% over two steps) as a pale yellow solid. The MTBE washing was tested for absence of residual TFA utilizing wet pH paper (pH between 3-4). The yield of the reaction was between 80-85% in different batches. The deacylated side product was detected in 3.6% as judged by LC/MS. For the different batches this impurity was never more than 5%.

Reactants for Step III:

[0091] A 200 mL round bottom flask was charged with a stirring bar and Pte_TFA_L_Tyr (13.85 g, 22.78 mmol, 1 equiv), then water (95 mL) was added to give a yellow suspension [suspension B]. A freshly prepared solution of aqueous 3.75 M NaOH (26.12 mL, 97.96 mmol, 4.30 equiv), or an equivalent base at a corresponding temperature using dimethylsulfoxide (DMSO) as a solvent (as shown in Table 1 ), was added dropwise to suspension B at 23 °C, giving a clear dull yellow solution over 15 minutes [solution B]. The equivalence of NaOH varied from 3.3 to 5.0 depending on the source of 4 (solid or liquid phase synthesis) and the residual TFA. Trianion 5 (Figure 12) formation was confirmed by LC/MS showing m/z 572→m/z 476 while the solution pH was 9-10 utilizing wet pH paper. The pH of the reaction mixture was in the range of 9-10. This pH is crucial for the overall reaction completion. Notably, pH more than 10 leads to hydrolysis of S0456. Excess base will efficiently drive reaction forward with potential hydrolysis of S0456. The presence of hydrolysis by product can be visibly detected by the persistent opaque purple/blue to red/brown color.

TABLE 1 : Separate TFA deprotection via trianion formation; S0456

[0092] The precipitated OTL-0038 product could also be crashed out by adding the reaction solution steady dropwise to acetone, acetonitrile, isopropanol or ethyl acetate/acetone mixture. Acetone yields optimal results. However, viscous reactions could be slower due to partial insolubility and/or crashing out of S0456. In this reaction, the equivalence of the aqueous base is significant. Excess base will efficiently drive reaction forward with potential hydrolysis of S0456. This solution phase synthesis provides Pte_N¹⁰(TFA)_Tyr-OH »HCI salt and desires approximately 4.1 to approximately 4.8 equiv base as a source to hydrolyze the product. Particularly, precipitation of Pte_Tyr_S0456 was best achieved when 1 mL of reaction mixture is added dropwise to the stirred acetone (20 mL). Filtration of the precipitate and washing with acetone (3 x10 mL) gave the highest purity as judged from LC/MS chromatogram.

[0093] During experimentation of this solution-phase synthesis of Pte – L Tyrosine -S0456 (OTL-0038) at different stages, some optimized conditions were observed:

Mode of addition: Separate TFA deprotection via trianion formation; S0456 @ 23 °C; reflux.

Stability data of Pte – L Tyrosine – S0456 (OTL-0038):

Liquid analysis: At 40 °C the liquid lost 8.6% at 270 nm and 1 % at 774 nm. At room temperature the liquid lost about 1 .4% at 270 nm and .5% at 774 nm. At 5 °C the

270 nm seems stable and the 774 nm reasonably stable with a small degradation purity.

Source Purity Linker S0456 Base Solvent Duration % Conversion

4.3-4.6

Solution 0.95

95% 1 equiv equiv H₂0 15 min 100% phase equiv

K₂C0₃

PATENT

US 20140271482

FDA approves pafolacianine for identifying malignant ovarian cancer lesions

https://www.fda.gov/drugs/resources-information-approved-drugs/fda-approves-pafolacianine-identifying-malignant-ovarian-cancer-lesions

On November 29, 2021, the Food and Drug Administration approved pafolacianine (Cytalux, On Target Laboratories, LLC), an optical imaging agent, for adult patients with ovarian cancer as an adjunct for interoperative identification of malignant lesions. Pafolacianine is a fluorescent drug that targets folate receptor which may be overexpressed in ovarian cancer. It is used with a Near-Infrared (NIR) fluorescence imaging system cleared by the FDA for specific use with pafolacianine.

Efficacy was evaluated in a single arm, multicenter, open-label study (NCT03180307) of 178 women diagnosed with ovarian cancer or with high clinical suspicion of ovarian cancer scheduled to undergo primary surgical cytoreduction, interval debulking, or recurrent ovarian cancer surgery. All patients received pafolacianine. One hundred and thirty-four patients received fluorescence imaging evaluation in addition to standard of care evaluation which includes pre-surgical imaging, intraoperative palpation and normal light evaluation of lesions. Among these patients, 36 (26.9%) had at least one evaluable ovarian cancer lesion detected with pafolacianine that was not observed by standard visual or tactile inspection. The patient-level false positive rate of pafolacianine with NIR fluorescent light with respect to the detection of ovarian cancer lesions confirmed by central pathology was 20.2% (95% CI 13.7%, 28.0%).

The most common adverse reactions (≥1%) occurring in patients were nausea, vomiting, abdominal pain, flushing, dyspepsia, chest discomfort, pruritus, and hypersensitivity.

The recommended pafolacianine dose is 0.025 mg/kg administered intravenously over 60 minutes, 1 to 9 hours before surgery. The use of folate, folic acid, or folate-containing supplements should be avoided within 48 hours before administration of pafolacianine.

View full prescribing information for Cytalux.

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

USFDA approves new drug to help identify cancer lesions

This drug is indicated for use in adult patients with ovarian cancer to help identify cancerous lesions during surgery.By The Health Master -December 2, 2021

The U.S. Food and Drug Administration (USFDA) has approved Cytalux (pafolacianine), an imaging drug intended to assist surgeons in identifying ovarian cancer lesions. The drug is designed to improve the ability to locate additional ovarian cancerous tissue that is normally difficult to detect during surgery.

Cytalux is indicated for use in adult patients with ovarian cancer to help identify cancerous lesions during surgery. The drug is a diagnostic agent that is administered in the form of an intravenous injection prior to surgery.

Alex Gorovets, M.D., deputy director of the Office of Specialty Medicine in the FDA’s Center for Drug Evaluation and Research said, “The FDA’s approval of Cytalux can help enhance the ability of surgeons to identify deadly ovarian tumors that may otherwise go undetected.

By supplementing current methods of detecting ovarian cancer during surgery, Cytalux offers health care professionals an additional imaging approach for patients with ovarian cancer.”

The American Cancer Society estimates there will be more than 21,000 new cases of ovarian cancer and more than 13,000 deaths from this disease in 2021, making it the deadliest of all female reproductive system cancers.

Conventional treatment for ovarian cancer includes surgery to remove as many of the tumors as possible, chemotherapy to stop the growth of malignant cells or other targeted therapy to identify and attack specific cancer cells.

Ovarian cancer often causes the body to overproduce a specific protein in cell membranes called a folate receptor. Following administration via injection, Cytalux binds to these proteins and illuminates under fluorescent light, boosting surgeons’ ability to identify the cancerous tissue.

Currently, surgeons rely on preoperative imaging, visual inspection of tumors under normal light or examination by touch to identify cancer lesions. Cytalux is used with a Near-Infrared fluorescence imaging system cleared by the FDA for specific use with pafolacianine.

The safety and effectiveness of Cytalux was evaluated in a randomized, multi-center, open-label study of women diagnosed with ovarian cancer or with high clinical suspicion of ovarian cancer who were scheduled to undergo surgery.

Of the 134 women (ages 33 to 81 years) who received a dose of Cytalux and were evaluated under both normal and fluorescent light during surgery, 26.9% had at least one cancerous lesion detected that was not observed by standard visual or tactile inspection.

The most common side effects of Cytalux were infusion-related reactions, including nausea, vomiting, abdominal pain, flushing, dyspepsia, chest discomfort, itching and hypersensitivity. Cytalux may cause fetal harm when administered to a pregnant woman.

The use of folate, folic acid, or folate-containing supplements should be avoided within 48 hours before administration of Cytalux. There is a risk of image interpretation errors with the use of Cytalux to detect ovarian cancer during surgery, including false negatives and false positives.

References

^ Jump up to:^a ^b ^c ^d https://www.accessdata.fda.gov/drugsatfda_docs/label/2020/214907s000lbl.pdf
^ Jump up to:^a ^b ^c ^d ^e ^f ^g ^h ⁱ “FDA Approves New Imaging Drug to Help Identify Ovarian Cancer Lesions”. U.S. Food and Drug Administration (FDA) (Press release). 29 November 2021. Retrieved 30 November 2021. This article incorporates text from this source, which is in the public domain.
^ “On Target Laboratories Announces FDA Approval of Cytalux (pafolacianine) injection for Identification of Ovarian Cancer During Surgery”. On Target Laboratories. 29 November 2021. Retrieved 30 November 2021 – via PR Newswire.
^ “Pafolacianine Orphan Drug Designations and Approvals”. U.S. Food and Drug Administration (FDA). 23 December 2014. Retrieved 30 November 2021.

External links

“Pafolacianine”. Drug Information Portal. U.S. National Library of Medicine.

Clinical data

Trade names	Cytalux
Other names	OTL-0038
License data	US DailyMed: Pafolacianine
Pregnancy category	Not recommended
Routes of administration	Intravenous
ATC code	None
Legal status
Legal status	US: ℞-only ^[1]^[2]
Identifiers
show IUPAC name
CAS Number	1628423-76-6
PubChem CID	135565623
DrugBank	DB15413
ChemSpider	64880249
UNII	F7BD3Z4X8L
ChEMBL	ChEMBL4297412
Chemical and physical data
Formula	C₆₁H₆₇N₉O₁₇S₄
Molar mass	1326.49 g·mol⁻¹
3D model (JSmol)	Interactive image
show SMILES
show InChI

////////////Pafolacianine, FDA 2021, APPROVALS 2021, Cytalux, OVARIAN CANCER, OTL 38,

[Na+].[Na+].[Na+].[Na+].CC1(C)\C(=C/C=C/2\CCCC(=C2Oc3ccc(C[C@H](NC(=O)c4ccc(NCc5cnc6N=C(N)NC(=O)c6n5)cc4)C(=O)O)cc3)\C=C\C7=[N](CCCCS(=O)(=O)O)c8ccc(cc8C7(C)C)S(=O)(=O)O)\N(CCCCS(=O)(=O)O)c9ccc(cc19)S(=O)(=O)O

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Avalglucosidase alfa

August 14, 2021 2:50 am / Leave a comment

QQGASRPGPR DAQAHPGRPR AVPTQCDVPP NSRFDCAPDK AITQEQCEAR GCCYIPAKQG
LQGAQMGQPW CFFPPSYPSY KLENLSSSEM GYTATLTRTT PTFFPKDILT LRLDVMMETE
NRLHFTIKDP ANRRYEVPLE TPRVHSRAPS PLYSVEFSEE PFGVIVHRQL DGRVLLNTTV
APLFFADQFL QLSTSLPSQY ITGLAEHLSP LMLSTSWTRI TLWNRDLAPT PGANLYGSHP
FYLALEDGGS AHGVFLLNSN AMDVVLQPSP ALSWRSTGGI LDVYIFLGPE PKSVVQQYLD
VVGYPFMPPY WGLGFHLCRW GYSSTAITRQ VVENMTRAHF PLDVQWNDLD YMDSRRDFTF
NKDGFRDFPA MVQELHQGGR RYMMIVDPAI SSSGPAGSYR PYDEGLRRGV FITNETGQPL
IGKVWPGSTA FPDFTNPTAL AWWEDMVAEF HDQVPFDGMW IDMNEPSNFI RGSEDGCPNN
ELENPPYVPG VVGGTLQAAT ICASSHQFLS THYNLHNLYG LTEAIASHRA LVKARGTRPF
VISRSTFAGH GRYAGHWTGD VWSSWEQLAS SVPEILQFNL LGVPLVGADV CGFLGNTSEE
LCVRWTQLGA FYPFMRNHNS LLSLPQEPYS FSEPAQQAMR KALTLRYALL PHLYTLFHQA
HVAGETVARP LFLEFPKDSS TWTVDHQLLW GEALLITPVL QAGKAEVTGY FPLGTWYDLQ
TVPIEALGSL PPPPAAPREP AIHSEGQWVT LPAPLDTINV HLRAGYIIPL QGPGLTTTES
RQQPMALAVA LTKGGEARGE LFWDDGESLE VLERGAYTQV IFLARNNTIV NELVRVTSEG
AGLQLQKVTV LGVATAPQQV LSNGVPVSNF TYSPDTKVLD ICVSLLMGEQ FLVSWC
(Disulfide bridge:26-53, 36-52, 47-71, 477-502, 591-602, 882-896)

Avalglucosidase alfa

アバルグルコシダーゼアルファ (遺伝子組換え)

Avalglucosidase alfa (USAN/INN);
Avalglucosidase alfa (genetical recombination) (JAN);
Avalglucosidase alfa-ngpt

To treat late-onset Pompe disease

Formula	C4490H6818N1197O1299S32
CAS	1802558-87-7
Mol weight	99375.4984

FDA APPROVED Nexviazyme, 2021/8/6, Enzyme replacement therapy product
Treatment of Pompe disease

Biologic License Application (BLA): 761194
Company: GENZYME CORP

https://www.fda.gov/news-events/press-announcements/fda-approves-new-treatment-pompe-diseaseFor Immediate Release:August 06, 2021

Today, the U.S. Food and Drug Administration approved Nexviazyme (avalglucosidase alfa-ngpt) for intravenous infusion to treat patients 1 year of age and older with late-onset Pompe disease.

Patients with Pompe disease have an enzyme deficiency that leads to the accumulation of a complex sugar, called glycogen, in skeletal and heart muscles, which cause muscle weakness and premature death from respiratory or heart failure. Normally, glycogen—the stored form of glucose—breaks down to release glucose into the bloodstream to be used as fuel for the cells.

“Pompe disease is a rare genetic disease that causes premature death and has a debilitating effect on people’s lives,” said Janet Maynard, M.D., deputy director of the Office of Rare Diseases, Pediatrics, Urologic and Reproductive Medicine in the FDA’s Center for Drug Evaluation and Research. “Today’s approval brings patients with Pompe disease another enzyme replacement therapy option for this rare disease. The FDA will continue to work with stakeholders to advance the development of additional new, effective and safe therapies for rare diseases, including Pompe disease.”

Nexviazyme, an enzyme replacement therapy, is an intravenous medication that helps reduce glycogen accumulation. The effectiveness of Nexviazyme for the treatment of Pompe disease was demonstrated in a study of 100 patients who were randomized to take Nexviazyme or another FDA-approved enzyme replacement therapy for Pompe disease. Treatment with Nexviazyme improved lung function similar to the improvement seen with the other therapy.

The most common side effects included headache, fatigue, diarrhea, nausea, joint pain (arthralgia), dizziness, muscle pain (myalgia), itching (pruritus), vomiting, difficulty breathing (dyspnea), skin redness (erythema), feeling of “pins and needles” (paresthesia) and skin welts (urticaria). Serious reactions included hypersensitivity reactions like anaphylaxis and infusion-associated reactions, including respiratory distress, chills and raised body temperature (pyrexia). Patients susceptible to fluid volume overload or with compromised cardiac or respiratory function may be at risk for serious acute cardiorespiratory failure.

The FDA granted this application Fast Track, Priority Review and Breakthrough Therapy designations. Nexviazyme also received an orphan drug designation, which provides incentives to assist and encourage the development of drugs for rare diseases. The FDA granted the approval of Nexviazyme to Genzyme Corporation.

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FDA grants priority review for avalglucosidase alfa, a potential new therapy for Pompe disease

The FDA decision date for avalglucosidase alfa, an investigational enzyme replacement therapy, is set for May 18, 2021
Regulatory submission based on positive data from two trials in patients with late-onset and infantile-onset Pompe disease, respectively
Avalglucosidase alfa received FDA Breakthrough Therapy and Fast Track designations for the treatment of people with Pompe Disease
Pompe disease, a rare degenerative muscle disorder, affects approximately 3,500 people in the U.S.
Milestone reinforces 20+year commitment to Pompe disease community

PARIS – November 18, 2020 – The U.S. Food and Drug Administration (FDA) has accepted for priority review the Biologics License Application (BLA) for avalglucosidase alfa for long-term enzyme replacement therapy for the treatment of patients with Pompe disease (acid α-glucosidase deficiency). The target action date for the FDA decision is May 18, 2021.

Avalglucosidase alfa is an investigational enzyme replacement therapy designed to improve the delivery of acid alpha-glucosidase (GAA) enzyme to muscle cells, and if approved, would offer a potential new standard of care for patients with Pompe disease.

In October, the European Medicines Agency accepted for review the Marketing Authorization Application for avalglucosidase alfa for long-term enzyme replacement therapy for the treatment of patients with Pompe disease. The Medicines and Healthcare Products Regulatory Agency in the UK has granted Promising Innovative Medicine designation for avalglucosidase alfa.

“The hallmarks of Pompe disease are the relentless and debilitating deterioration of the muscles, which causes decreased respiratory function and mobility,” said Karin Knobe, Head of Development for Rare Diseases and Rare Blood Disorders at Sanofi. “Avalglucosidase alfa is specifically designed to deliver more GAA enzyme into the lysosomes of the muscle cells. We have been greatly encouraged by positive clinical trial results in patients with late-onset and infantile-onset Pompe disease.”

Pompe disease is a rare, degenerative muscle disorder that can impact an individual’s ability to move and breathe. It affects an estimated 3,500 people in the U.S. and can manifest at any age from infancy to late adulthood.ⁱ

The BLA is based on positive data from two trials:

Pivotal Phase 3, double-blind, global comparator-controlled trial (COMET), which evaluated the safety and efficacy of avalglucosidase alfa compared to alglucosidase alfa (standard of care) in patients with late-onset Pompe disease. Results from this trial were presented during a Sanofi-hosted virtual scientific session in June 2020 and in October 2020 at World Muscle Society and the American Association of Neuromuscular and Electrodiagnostic Medicine.
The Phase 2 (mini-COMET) trial evaluated the safety and exploratory efficacy of avalglucosidase alfa in patients with infantile-onset Pompe disease previously treated with alglucosidase alfa. Results from this trial were presented at the WORLDSymposium, in February 2020.

Delivery of GAA to Clear Glycogen

Pompe disease is caused by a genetic deficiency or dysfunction of the lysosomal enzyme GAA, which results in build-up of complex sugars (glycogen) in muscle cells throughout the body. The accumulation of glycogen leads to irreversible damage to the muscles, including respiratory muscles and the diaphragm muscle supporting lung function, and other skeletal muscles that affect mobility.

To reduce the glycogen accumulation caused by Pompe disease, the GAA enzyme must be delivered into the lysosomes within muscle cells. Research led by Sanofi has focused on ways to enhance the delivery of GAA into the lysosomes of muscle cells by targeting the mannose-6-phosphate (M6P) receptor that plays a key role in the transport of GAA.

Avalglucosidase alfa is designed with approximately 15-fold increase in M6P content, compared to standard of care alglucosidase alfa, and aims to help improve cellular enzyme uptake and enhance glycogen clearance in target tissues.ⁱⁱ The clinical relevance of this difference has not been confirmed.

Avalglucosidase alfa is currently under clinical investigation and its safety and efficacy have not been evaluated by any regulatory authority worldwide.

About Sanofi

Sanofi is dedicated to supporting people through their health challenges. We are a global biopharmaceutical company focused on human health. We prevent illness with vaccines, provide innovative treatments to fight pain and ease suffering. We stand by the few who suffer from rare diseases and the millions with long-term chronic conditions.

With more than 100,000 people in 100 countries, Sanofi is transforming scientific innovation into healthcare solutions around the globe.

Sanofi, Empowering Life

/////////Avalglucosidase alfa, FDA 2021, Nexviazyme, APPROVALS 2021, PEPTIDE, Enzyme replacement therapy , Pompe disease, アバルグルコシダーゼアルファ (遺伝子組換え), Fast Track, Priority Review, Breakthrough Therapy, orphan drug designation, genzyme, sanofi

Asparaginase erwinia chrysanthemi (recombinant)-rywn

July 6, 2021 10:57 am / Leave a comment

Sequence:

1ADKLPNIVIL ATGGTIAGSA ATGTQTTGYK AGALGVDTLI NAVPEVKKLA51NVKGEQFSNM ASENMTGDVV LKLSQRVNEL LARDDVDGVV ITHGTDTVEE101SAYFLHLTVK SDKPVVFVAA MRPATAISAD GPMNLLEAVR VAGDKQSRGR151GVMVVLNDRI GSARYITKTN ASTLDTFKAN EEGYLGVIIG NRIYYQNRID201KLHTTRSVFD VRGLTSLPKV DILYGYQDDP EYLYDAAIQH GVKGIVYAGM251GAGSVSVRGI AGMRKAMEKG VVVIRSTRTG NGIVPPDEEL PGLVSDSLNP301AHARILLMLA LTRTSDPKVI QEYFHTY

>Protein sequence for asparaginase (Erwinia chrysanthemi) monomer
ADKLPNIVILATGGTIAGSAATGTQTTGYKAGALGVDTLINAVPEVKKLANVKGEQFSNM
ASENMTGDVVLKLSQRVNELLARDDVDGVVITHGTDTVEESAYFLHLTVKSDKPVVFVAA
MRPATAISADGPMNLLEAVRVAGDKQSRGRGVMVVLNDRIGSARYITKTNASTLDTFKAN
EEGYLGVIIGNRIYYQNRIDKLHTTRSVFDVRGLTSLPKVDILYGYQDDPEYLYDAAIQH
GVKGIVYAGMGAGSVSVRGIAGMRKAMEKGVVVIRSTRTGNGIVPPDEELPGLVSDSLNP
AHARILLMLALTRTSDPKVIQEYFHTY

References:

Therapeutic Targets Database: TTD Biologic drug sequences in fasta format [Link]

Asparaginase erwinia chrysanthemi (recombinant)-rywn

JZP458-201

JZP458

CAS Registry Number 1349719-22-7

Protein Chemical FormulaC₁₅₄₆H₂₅₁₀N₄₃₂O₄₇₆S₉

Protein Average Weight 140000.0 Da

Rylaze, FDA APPROVED 6/30/2021, BLA 761179

L-Asparaginase (ec 3.5.1.1, L-asparagine amidohydrolase) erwinia chrysanthemi tetramer alpha4Asparaginase (Dickeya chrysanthemi subunit)

Other Names

Asparaginase Erwinia chrysanthemi
Crisantaspase
Cristantaspase
Erwinase
Erwinaze
L-Asparagine amidohydrolase (Erwinia chrysanthemi subunit)

D733ET3F9O

1349719-22-7

Asparaginase erwinia chrysanthemi [USAN]

UNII-D733ET3F9O

L-Asparaginase (erwinia)

Erwinia asparaginase

L-Asparaginase, erwinia chrysanthemi

Asparaginase (erwinia chrysanthemi)

Erwinase

Asparaginase erwinia chrysanthemi

Erwinaze

Crisantaspase

Crisantaspase [INN]

L-Asparaginase (ec 3.5.1.1, L-asparagine amidohydrolase) erwinia chrysanthemi tetramer alpha4

Asparaginase erwinia sp. [MI]

Asparaginase erwinia chrysanthemi (recombinant) [USAN]

Asparaginase erwinia chrysanthemi (recombinant)

JZP-458

A hydrolase enzyme that converts L-asparagine and water to L-aspartate and NH3.

NCI: Asparaginase Erwinia chrysanthemi. An enzyme isolated from the bacterium Erwinia chrysanthemi (E. carotovora). Asparagine is critical to protein synthesis in leukemic cells, which cannot synthesize this amino acid due to the absence of the enzyme asparagine synthase. Asparaginase hydrolyzes L-asparagine to L-aspartic acid and ammonia, thereby depleting leukemic cells of asparagine and blocking protein synthesis and tumor cell proliferation, especially in the G1 phase of the cell cycle. This agent also induces apoptosis in tumor cells. The Erwinia-derived product is often used for those patients who have experienced a hypersensitivity reaction to the E. Coli formulation. (NCI Thesaurus)

Treatment of Acute Lymphoblastic Leukemia (ALL)
Antineoplastic Agents

10MG/0.5ML

INJECTABLE;INTRAMUSCULAR

Label (PDF)
Letter (PDF)

Label (PDF)

PATENT

WO 2011003633

https://patents.google.com/patent/WO2011003633A1/en

The present invention concerns a conjugate of a protein having substantial L-asparagine aminohydrolase activity and polyethylene glycol, particularly wherein the polyethylene glycol has a molecular weight less than or equal to about 5000 Da, particularly a conjugate wherein the protein is a L-asparaginase from Erwinia, and its use in therapy.Proteins with L-asparagine aminohydrolase activity, commonly known as L- asparaginases, have successfully been used for the treatment of Acute Lymphoblastic Leukemia(ALL) in children for many years. ALL is the most common childhood malignancy (Avramis and Panosyan, Clin. Pharmacokinet. (2005) 44:367-393).[0003] L-asparaginase has also been used to treat Hodgkin’s disease, acute myelocytic leukemia, acute myelomonocytic leukemia, chronic lymphocytic leukemia, lymphosarcoma, reticulosarcoma, and melanosarcoma (Kotzia and Labrou, J. Biotechnol. 127 (2007) 657-669).The anti-tumor activity of L-asparaginase is believed to be due to the inability or reduced ability of certain malignant cells to synthesize L-asparagine (Kotzia and Labrou, J. Biotechnol. 127 (2007) 657-669). These malignant cells rely on an extracellular supply of L-asparagine. However, the L-asparaginase enzyme catalyzes the hydrolysis of L-asparagine to aspartic acid and ammonia, thereby depleting circulating pools of L-asparagine and killing tumor cells which cannot perform protein synthesis without L-asparagine (Kotzia and Labrou, J. Biotechnol. 127 (2007) 657-669).[0004] L-asparaginase from E. coli was the first enzyme drug used in ALL therapy and has been marketed as Elspar® in the USA or as Kidrolase® and L-asparaginase Medac® in Europe. L- asparaginases have also been isolated from other microorganisms, e.g., an L-asparaginase protein from Erwinia chrysanthemi, named crisantaspase, that has been marketed as Erwinase® (Wriston Jr., J.C. (1985) “L-asparaginase” Meth. Enzymol. 113, 608-618; Goward, CR. et al. (1992) “Rapid large scale preparation of recombinant Erwinia chrysanthemi L-asparaginase”, Bioseparation 2, 335-341). L-asparaginases from other species of Erwinia have also been identified, including, for example, Erwinia chrysanthemi 3937 (Genbank Accession#AAS67028), Erwinia chrysanthemi NCPPB 1125 (Genbank Accession #CAA31239), Erwinia carotovora (Genbank Accession #AAP92666), and Erwinia carotovora subsp. Astroseptica (Genbank Accession #AAS67027). These Erwinia chrysanthemi L-asparaginases have about 91-98% amino acid sequence identity with each other, while the Erwinia carotovora L- asparaginases have approximately 75-77% amino acid sequence identity with the Erwinia chrysanthemi L-asparaginases (Kotzia and Labrou, J. Biotechnol. 127 (2007) 657-669).[0005] L-asparaginases of bacterial origin have a high immunogenic and antigenic potential and frequently provoke adverse reactions ranging from mild allergic reaction to anaphylactic shock in sensitized patients (Wang, B. et al. (2003) “Evaluation of immunologic cross reaction of anti- asparaginase antibodies in acute lymphoblastic leukemia (ALL and lymphoma patients),Leukemia 17, 1583-1588). E. coli L-asparaginase is particularly immunogenic, with reports of the presence of anti-asparaginase antibodies to E. coli L-asparaginase following i.v. or i.m. administration reaching as high as 78% in adults and 70% in children (Wang, B. et al. (2003) Leukemia 17, 1583-1588).[0006] L-asparaginases from Escherichia coli and Erwinia chrysanthemi differ in their pharmacokinetic properties and have distinct immunogenic profiles, respectively (Klug Albertsen, B. et al. (2001) “Comparison of intramuscular therapy with Erwinia asparaginase and asparaginase Medac: pharmacokinetics, pharmacodynamics, formation of antibodies and influence on the coagulation system” Brit. J. Haematol. 115, 983-990). Furthermore, it has been shown that antibodies that developed after a treatment with L-asparaginase from E. coli do not cross react with L-Asparaginase from Erwinia (Wang, B. et al., Leukemia 17 (2003) 1583-1588). Thus, L-asparaginase from Erwinia (crisantaspase) has been used as a second line treatment of ALL in patients that react to E. coli L-asparaginase (Duval, M. et al. (2002) “Comparison of Escherichia co/z-asparaginase with £Vwzm^‘α-asparaginase in the treatment of childhood lymphoid malignancies: results of a randomized European Organisation for Research and Treatment ofCancer, Children’s Leukemia Group phase 3 trial” Blood 15, 2734-2739; Avramis and Panosyan,Clin. Pharmacokinet. (2005) 44:367-393).[0007] In another attempt to reduce immunogenicity associated with administration of microbial L-asparaginases, an E. coli L-asparaginase has been developed that is modified with methoxy- polyethyleneglycol (mPEG). This method is commonly known as “PEGylation” and has been shown to alter the immunological properties of proteins (Abuchowski, A. et al. (1977) “Alteration of Immunological Properties of Bovine Serum Albumin by Covalent Attachment of Polyethylene Glycol,” J.Biol.Chem. 252 (11), 3578-3581). This so-called mPEG-L- asparaginase, or pegaspargase, marketed as Oncaspar® (Enzon Inc., USA), was first approved in the U.S. for second line treatment of ALL in 1994, and has been approved for first- line therapy of ALL in children and adults since 2006. Oncaspar® has a prolonged in vivo half-life and a reduced immunogenicity/antigenicity.[0008] Oncaspar® is E. coli L-asparaginase that has been modified at multiple lysine residues using 5 kDa mPEG-succinimidyl succinate (SS-PEG) (U.S. Patent No. 4,179,337). SS-PEG is aPEG reagent of the first generation that contains an instable ester linkage that is sensitive to hydro lysis by enzymes or at slightly alkaline pH values (U.S. Patent No. 4,670,417; Makromol. Chem. 1986, 187, 1131-1144). These properties decrease both in vitro and in vivo stability and can impair drug safety.[0009] Furthermore, it has been demonstrated that antibodies developed against L-asparaginase from E. coli will cross react with Oncaspar® (Wang, B. et al. (2003) “Evaluation of immunologic cross-reaction of anti-asparaginase antibodies in acute lymphoblastic leukemia (ALL and lymphoma patients),” Leukemia 17, 1583-1588). Even though these antibodies were not neutralizing, this finding clearly demonstrated the high potential for cross-hypersensitivity or cross-inactivation in vivo. Indeed, in one report 30-41% of children who received pegaspargase had an allergic reaction (Wang, B. et al. (2003) Leukemia 17, 1583-1588).[0010] In addition to outward allergic reactions, the problem of “silent hypersensitivity” was recently reported, whereby patients develop anti-asparaginase antibodies without showing any clinical evidence of a hypersensitivity reaction (Wang, B. et al. (2003) Leukemia 17, 1583-1588). This reaction can result in the formation of neutralizing antibodies to E. coli L-asparaginase and pegaspargase; however, these patients are not switched to Erwinia L-asparaginase because there are not outward signs of hypersensitivity, and therefore they receive a shorter duration of effective treatment (Holcenberg, J., J. Pediatr. Hematol. Oncol. 26 (2004) 273-274).[0011] Erwinia chrysanthemi L-asparaginase treatment is often used in the event of hypersensitivity to E. co/z-derived L-asparaginases. However, it has been observed that as many as 30-50% of patients receiving Erwinia L-asparaginase are antibody-positive (Avramis andPanosyan, Clin. Pharmacokinet. (2005) 44:367-393). Moreover, because Erwinia chrysanthemi L-asparaginase has a significantly shorter elimination half-life than the E. coli L-asparaginases, it must be administered more frequently (Avramis and Panosyan, Clin. Pharmacokinet. (2005) 44:367-393). In a study by Avramis et al., Erwinia asparaginase was associated with inferior pharmacokinetic profiles (Avramis et al., J. Pediatr. Hematol. Oncol. 29 (2007) 239-247). E. coli L-asparaginase and pegaspargase therefore have been the preferred first-line therapies for ALL over Erwinia L-asparaginase.[0012] Numerous biopharmaceuticals have successfully been PEGylated and marketed for many years. In order to couple PEG to a protein, the PEG has to be activated at its OH terminus. The activation group is chosen based on the available reactive group on the protein that will bePEGylated. In the case of proteins, the most important amino acids are lysine, cysteine, glutamic acid, aspartic acid, C-terminal carboxylic acid and the N-terminal amino group. In view of the wide range of reactive groups in a protein nearly the entire peptide chemistry has been applied to activate the PEG moiety. Examples for this activated PEG-reagents are activated carbonates, e.g., p-nitrophenyl carbonate, succinimidyl carbonate; active esters, e.g., succinimidyl ester; and for site specific coupling aldehydes and maleimides have been developed (Harris, M., Adv. Drug – A -DeI. Rev. 54 (2002), 459-476). The availability of various chemical methods for PEG modification shows that each new development of a PEGylated protein will be a case by case study. In addition to the chemistry the molecular weight of the PEG that is attached to the protein has a strong impact on the pharmaceutical properties of the PEGylated protein. In most cases it is expected that, the higher the molecular weight of the PEG, the better the improvement of the pharmaceutical properties (Sherman, M. R., Adv. Drug Del. Rev. 60 (2008), 59-68; Holtsberg, F. W., Journal of Controlled Release 80 (2002), 259-271). For example, Holtsberg et al. found that, when PEG was conjugated to arginine deaminase, another amino acid degrading enzyme isolated from a microbial source, pharmacokinetic and pharmacodynamic function of the enzyme increased as the size of the PEG attachment increased from a molecular weight of 5000Da to 20,000 Da (Holtsberg, F.W., Journal of Controlled Release 80 (2002), 259-271).[0013] However, in many cases, PEGylated biopharmaceuticals show significantly reduced activity compared to the unmodified biopharmaceutical (Fishburn, CS. (2008) Review “The Pharmacology of PEGylation: Balancing PD with PK to Generate Novel Therapeutics” J. Pharm. Sd., 1-17). In the case of L-asparaginase from Erwinia carotovora, it has been observed that PEGylation reduced its in vitro activity to approximately 57% (Kuchumova, A.V. et al. (2007) “Modification of Recombinant asparaginase from Erwinia carotovora with Polyethylene Glycol 5000” Biochemistry (Moscow) Supplement Series B: Biomedical Chemistry, 1, 230-232). The L-asparaginase from Erwinia carotovora has only about 75% homology to the Erwinia chrysanthemi L-asparaginase (crisantaspase). For Oncaspar® it is also known that its in vitro activity is approximately 50% compared to the unmodified E. coli L-asparaginase.[0014] The currently available L-asparaginase preparations do not provide alternative or complementary therapies— particularly therapies to treat ALL— that are characterized by high catalytic activity and significantly improved pharmacological and pharmacokinetic properties, as well as reduced immunogenicity. L-asparaginase protein has at least about 80% homology or identity with the protein comprising the sequence of SEQ ID NO:1, more specifically at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homology or identity with the protein comprising the sequence of SEQ ID NO:1. SEQ ID NO:1 is as follows:ADKLPNIVILATGGTIAGSAATGTQTTGYKAGALGVDTLINAVPEVKKLANVKGE QFSNMASENMTGDVVLKLSQRVNELLARDDVDGVVITHGTDTVEESAYFLHLTV KSDKPVVFVAAMRPATAISADGPMNLLEAVRVAGDKQSRGRGVMVVLNDRIGSA RYITKTNASTLDTFKANEEGYLGVIIGNRIYYQNRIDKLHTTRSVFDVRGLTSLPKV DILYGYQDDPEYLYDAAIQHGVKGIVYAGMGAGSVSVRGIAGMRKAMEKGVVVIRSTRTGNGIVPPDEELPGLVSDSLNPAHARILLMLALTRTSDPKVIQEYFHTY (SEQ ID NO:1) [0048] The term “comprising the sequence of SEQ ID NO:1” means that the amino-acid sequence of the protein may not be strictly limited to SEQ ID NO:1 but may contain additional amino-acids.ExamplesExample 1 : Preparation of Recombinant Crisantaspase [0100] The recombinant bacterial strain used to manufacture the naked recombinant Erwinia chrysanthemi L-asparaginase protein (also referred to herein as “r-crisantaspase”) was an E. coli BL21 strain with a deleted ansB gene (the gene encoding the endogenous E. coli type II L- asparaginase) to avoid potential contamination of the recombinant Erwinia chrysanthemi L- asparaginase with this enzyme. The deletion of the ansB gene relies on homologous recombination methods and phage transduction performed according to the three following steps:1) a bacterial strain (NMI lOO) expressing a defective lambda phage which supplies functions that protect and recombine electroporated linear DNA substrate in the bacterial cell was transformed with a linear plasmid (kanamycin cassette) containing the kanamycin gene flanked by an FLP recognition target sequence (FRT). Recombination occurs to replace the ansB gene by the kanamycin cassette in the bacterial genome, resulting in a ΛansB strain; 2) phage transduction was used to integrate the integrated kanamycin cassette region from the ΛansB NMI lOO strain to the ansB locus in BL21 strain. This results in an E. coli BL21 strain with a deleted ansB gene and resistant to kanamycin; 3) this strain was transformed with a FLP -helper plasmid to remove the kanamycin gene by homologous recombination at the FRT sequence. The genome of the final strain (BL21 ΛansB strain) was sequenced, confirming full deletion of the endogenous ansB gene.[0101] The E. co/z^‘-optimized DNA sequence encoding for the mature Erwinia chrysanthemi L- asparaginase fused with the ENX signal peptide from Bacillus subtilis was inserted into an expression vector. This vector allows expression of recombinant Erwinia chrysanthemi L- asparaginase under the control of hybrid T5/lac promoter induced by the addition of Isopropyl β- D-1-thiogalactopyranoside (IPTG) and confers resistance to kanamycin.[0102] BL21 ΛansB strain was transformed with this expression vector. The transformed cells were used for production of the r-crisantaspase by feed batch glucose fermentation in Reisenberg medium. The induction of the cell was done 16h at 23°C with IPTG as inducer. After cell harvest and lysis by homogenization in 1OmM sodium phosphate buffer pH6 5mM EDTA (Buffer A), the protein solution was clarified by centrifugation twice at 1500Og, followed by 0.45μm and 0.22μm filtration steps. The recombinant Erwinia chrysanthemi L-asparaginase was next purified using a sequence of chromatography and concentration steps. Briefly, the theoretical isoelectric point of the Erwinia chrysanthemi L-asparaginase (7.23) permits the recombinant enzyme to adsorb to cation exchange resins at pH6. Thus, the recombinant enzyme was captured on a Capto S column (cation exchange chromatography) and eluted with salt gradient in Buffer A. Fractions containing the recombinant enzyme were pooled. The pooled solution was next purified on Capto MMC column (cation exchange chromatography) in Buffer A with salt gradient. . The eluted fractions containing Erwinia chrysanthemi L-asparaginase were pooled and concentrated before protein separation on Superdex 200pg size exclusion chromatography as polishing step. Fractions containing recombinant enzymes were pooled, concentrated, and diafiltered against 10OmM sodium phosphate buffer pH8. The purity of the final Erwinia chrysanthemi L-asparaginase preparation was evaluated by SDS-PAGE (Figure 1) and RP-HPLC and was at least 90%. The integrity of the recombinant enzyme was verified byN-terminal sequencing and LC-MS. Enzyme activity was measured at 37°C using Nessler’s reagent. The specific activity of the purified recombinant Erwinia chrysanthemi L-asparaginase was around 600 U/mg. One unit of enzyme activity is defined as the amount of enzyme that liberates lμmol of ammonia from L-asparagine per minute at 37°C. Example 2: Preparation of 10 kDa mPEG-L- Asparaginase Conjugates[0103] A solution of L-asparaginase from Erwinia chrysanthemi was stirred in a 100 mM sodium phosphate buffer at pH 8.0, at a protein concentration between 2.5 and 4 mg/mL, in the presence of 150 mg/mL or 36 mg/mL 10 kDa mPEG-NHS, for 2 hours at 22°C. The resulting crude 10 kDa mPEG-L-asparaginase was purified by size exclusion chromatography using a Superdex 200 pg column on an Akta purifier UPC 100 system. Protein-containing fractions were pooled and concentrated to result in a protein concentration between 2 and 8 mg/mL. Two 10 kDa mPEG-L-asparaginase conjugates were prepared in this way, differing in their degree of PEGylation as determined by TNBS assay with unmodified L-asparaginase as a reference, one corresponding to full PEGylation (100% of accessible amino groups (e.g., lysine residues and/or the N-terminus) residues being conjugated corresponding to PEGylation of 78% of total amino groups (e.g., lysine residues and/or the N-terminus)); the second one corresponding to partial PEGylation (39% of total amino groups (e.g., lysine residues and/or the N-terminus) or about 50% of accessible amino groups (e.g., lysine residues and/or the N-terminus)) . SDS-PAGE analysis of the conjugates is shown in Figure 2. The resulting conjugates appeared as an essentially homogeneous band and contained no detectable unmodified r-crisantaspase.Example 3: Preparation of 5 kDa mPEG-L-Asparaginase Conjugates[0104] A solution of L-asparaginase from Erwinia chrysanthemi was stirred in a 100 mM sodium phosphate buffer at pH 8.0, at a protein concentration of 4 mg/mL, in the presence of 150 mg/mL or 22.5 mg/mL 5 kDa mPEG-NHS, for 2 hours at 22°C. The resulting crude 5 kDa mPEG-L-asparaginase was purified by size exclusion chromatography using a Superdex 200 pg column on an Akta purifier UPC 100 system. Protein-containing fractions were pooled and concentrated to result in a protein concentration between 2 and 8 mg/mL. Two 5 kDa mPEG-L- asparaginase conjugates were prepared in this way, differing in their degree of PEGylation as determined by TNBS assay with unmodified L-asparaginase as a reference, one corresponding to full PEGylation (100% of accessible amino groups (e.g., lysine residues and/or the N-terminus) being conjugated corresponding to PEGylation of 84% of total amino groups (e.g., lysine residues and/or the N-terminus)); the second one corresponding to partial PEGylation (36% of total amino groups (e.g., lysine residues and/or the N-terminus) or about 43% of accessible amino groups (e.g., lysine residues and/or the N-terminus)). SDS-PAGE analysis of the conjugates is shown in Figure 2. The resulting conjugates appeared as an essentially homogeneous band and contained no detectable unmodified r-crisantaspase.Example 4: Preparation of 2 kDa mPEG-L-Asparaginase Conjugates[0105] A solution of L-asparaginase from Erwinia chrysanthemi was stirred in a 100 mM sodium phosphate buffer pH 8.0 at a protein concentration of 4 mg/mL in the presence of150 mg/mL or 22.5 mg/mL 2 kDa mPEG-NHS for 2 hours at 22°C. The resulting crude 2 kDa mPEG-L-asparaginase was purified by size exclusion chromatography using a Superdex 200 pg column on an Akta purifier UPC 100 system. Protein containing fractions were pooled and concentrated to result in a protein concentration between 2 and 8 mg/mL. Two 2 kDa mPEG-L- asparaginase conjugates were prepared in this way, differing in their degree of PEGylation as determined by TNBS assay with unmodified L-asparaginase as reference, one corresponding to maximum PEGylation (100% of accessible amino groups (e.g., lysine residues and/or the N- terminus) being conjugated corresponding to PEGylation of 86% of total amino groups (e.g., lysine residues and/or the N-terminus)); the second one corresponding to partial PEGylation (47% of total amino groups (e.g., lysine residues and/or the N-terminus) or about 55% of accessible amino groups {e.g., lysine residues and/or the N-terminus)). SDS-PAGE analysis of the conjugates is shown in Figure 2. The resulting conjugates appeared as an essentially homogeneous band and contained no detectable unmodified r-crisantaspase.Example 5: Activity of mPEG-r-Crisantaspase Conjugates[0106] L-asparaginase aminohydrolase activity of each conjugate described in the proceeding examples was determined by Nesslerization of ammonia that is liberated from L-asparagine by enzymatic activity. Briefly, 50μL of enzyme solution were mixed with 2OmM of L-asparagine in a 50 mM Sodium borate buffer pH 8.6 and incubated for 10 min at 37°C. The reaction was stopped by addition of 200μL of Nessler reagent. Absorbance of this solution was measured at 450 nm. The activity was calculated from a calibration curve that was obtained from Ammonia sulfate as reference. The results are summarized in Table 2, below:Table 2: Activity of mPEG-r-crisantaspase conjugates

* the numbers “40%” and “100%” indicate an approximate degree of PEGylation of respectively 40-55% and 100% of accessible amino groups (see Examples 2-4, supra).** the ratio mol PEG / mol monomer was extrapolated from data using TNBS assay, that makes the assumption that all amino groups from the protein (e.g., lysine residues and the N-terminus) are accessible.[0107] Residual activity of mPEG-r-crisantaspase conjugates ranged between 483 and 543 Units/mg. This corresponds to 78-87% of L-asparagine aminohydrolase activity of the unmodified enzyme. Example 6: L-Asparagine-Depleting Effect of Unmodified Crisantaspase

PAPER

Biotechnology and Applied Biochemistry (2019), 66(3), 281-289. |

https://iubmb.onlinelibrary.wiley.com/doi/10.1002/bab.1723

Crisantaspase is an asparaginase enzyme produced by Erwinia chrysanthemi and used to treat acute lymphoblastic leukemia (ALL) in case of hypersensitivity to Escherichia coli l-asparaginase (ASNase). The main disadvantages of crisantaspase are the short half-life (10 H) and immunogenicity. In this sense, its PEGylated form (PEG-crisantaspase) could not only reduce immunogenicity but also improve plasma half-life. In this work, we developed a process to obtain a site-specific N-terminal PEGylated crisantaspase (PEG-crisantaspase). Crisantaspase was recombinantly expressed in E. coli BL21(DE3) strain cultivated in a shaker and in a 2-L bioreactor. Volumetric productivity in bioreactor increased 37% compared to shaker conditions (460 and 335 U L⁻¹ H⁻¹, respectively). Crisantaspase was extracted by osmotic shock and purified by cation exchange chromatography, presenting specific activity of 694 U mg⁻¹, 21.7 purification fold, and yield of 69%. Purified crisantaspase was PEGylated with 10 kDa methoxy polyethylene glycol-N-hydroxysuccinimidyl (mPEG-NHS) at different pH values (6.5–9.0). The highest N-terminal pegylation yield (50%) was at pH 7.5 with the lowest poly-PEGylation ratio (7%). PEG-crisantaspase was purified by size exclusion chromatography and presented a K_M value three times higher than crisantaspase (150 and 48.5 µM, respectively). Nonetheless, PEG-crisantaspase was found to be more stable at high temperatures and over longer periods of time. In 2 weeks, crisantaspase lost 93% of its specific activity, whereas PEG-crisantaspase was stable for 20 days. Therefore, the novel PEG-crisantaspase enzyme represents a promising biobetter alternative for the treatment of ALL.

ADKLPNIVILATGGTIAGSAATGTQTTGYKAGALGVDTLINAVPEVKKLANVKGEQFSN

MASENMTGDVVLKLSQRVNELLARDDVDGVVITHGTDTVEESAYFLHLTVKSDKPVV

FVAAMRPATAISADGPMNLLEAVRVAGDKQSRGRGVMVVLNDRIGSARYITKTNAST

LDTFKANEEGYLGVIIGNRIYYQNRIDKLHTTRSVFDVRGLTSLPKVDILYGYQDDPEY

LYDAAIQHGVKGIVYAGMGAGSVSVRGIAGMRKAMEKGVVVIRSTRTGNGIVPPDEE

LPGLVSDSLNPAHARILLMLALTRTSDPKVIQEYFHTY

Figure S1 – Amino acid sequence of the enzyme crisantaspase without the signal peptide and with the lysines highlighted in red (Swiss-Prot/TrEMBL accession number: P06608|22-348 AA).

……………………………………………………………………………………………………………………………..

As a component of a chemotherapy regimen to treat acute lymphoblastic leukemia and lymphoblastic lymphoma in patients who are allergic to E. coli-derived asparaginase products
Press ReleaseFor Immediate Release:June 30, 2021

FDA Approves Component of Treatment Regimen for Most Common Childhood Cancer

Alternative Has Been in Global Shortage Since 2016

Today, the U.S. Food and Drug Administration approved Rylaze (asparaginase erwinia chrysanthemi (recombinant)-rywn) as a component of a chemotherapy regimen to treat acute lymphoblastic leukemia and lymphoblastic lymphoma in adult and pediatric patients who are allergic to the E. coli-derived asparaginase products used most commonly for treatment. The only other FDA-approved drug for such patients with allergic reactions has been in global shortage for years.

“It is extremely disconcerting to patients, families and providers when there is a lack of access to critical drugs for treatment of a life-threatening, but often curable cancer, due to supply issues,” said Gregory Reaman, M.D., associate director for pediatric oncology in the FDA’s Oncology Center of Excellence. “Today’s approval may provide a consistently sourced alternative to a pivotal component of potentially curative therapy for children and adults with this type of leukemia.”

Acute lymphoblastic leukemia occurs in approximately 5,700 patients annually, about half of whom are children. It is the most common type of childhood cancer. One component of the chemotherapy regimen is an enzyme called asparaginase that kills cancer cells by depriving them of substances needed to survive. An estimated 20% of patients are allergic to the standard E. coli-derived asparaginase and need an alternative their bodies can tolerate.

Rylaze’s efficacy was evaluated in a study of 102 patients who either had a hypersensitivity to E. coli-derived asparaginases or experienced silent inactivation. The main measurement was whether patients achieved and maintained a certain level of asparaginase activity. The study found that the recommended dosage would provide the target level of asparaginase activity in 94% of patients.

The most common side effects of Rylaze include hypersensitivity reactions, pancreatic toxicity, blood clots, hemorrhage and liver toxicity.

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 Health Canada, where the application review is pending.

Rylaze received Fast Track and Orphan Drug designations for this indication. Fast Track is a process designed to facilitate the development and expedite the review of drugs to treat serious conditions and fulfill an unmet medical need. Orphan Drug designation provides incentives to assist and encourage drug development for rare diseases.

The FDA granted approval of Rylaze to Jazz Pharmaceuticals.

REF

Click to access 761179Orig1s000ltr.pdf

https://www.prnewswire.com/news-releases/jazz-pharmaceuticals-announces-us-fda-approval-of-rylaze-asparaginase-erwinia-chrysanthemi-recombinant-rywn-for-the-treatment-of-acute-lymphoblastic-leukemia-or-lymphoblastic-lymphoma-301323782.html#:~:text=Jazz%20Pharmaceuticals%20Announces,details%20to%20follow

DUBLIN, June 30, 2021 /PRNewswire/ — Jazz Pharmaceuticals plc (Nasdaq: JAZZ) today announced the U.S. Food and Drug Administration (FDA) approval of Rylaze^™ (asparaginase erwinia chrysanthemi (recombinant)-rywn) for use as a component of a multi-agent chemotherapeutic regimen for the treatment of acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma (LBL) in pediatric and adult patients one month and older who have developed hypersensitivity to E. coli-derived asparaginase.¹ Rylaze is the only recombinant erwinia asparaginase manufactured product that maintains a clinically meaningful level of asparaginase activity throughout the entire duration of treatment, and it was developed by Jazz to address the needs of patients and healthcare providers with an innovative, high-quality erwinia-derived asparaginase with reliable supply.

“We are excited to bring this important new treatment to patients who are in critical need, and we are grateful to FDA for the approval of Rylaze based on its established safety and efficacy profile. We are pleased Rylaze was approved before the trial is complete and are diligently working to advance additional clinical trial data. We are committed to quickly engaging with FDA to evolve the Rylaze product profile with additional dosing options and an IV route of administration,” said Bruce Cozadd, chairman and CEO of Jazz Pharmaceuticals. “Thank you to our collaborators within the Children’s Oncology Group, the clinical trial investigators, patients and their families, and all of the other stakeholders who helped us achieve this significant milestone.”

Rylaze was granted orphan drug designation for the treatment of ALL/LBL by FDA in June 2021. The Biologics Licensing Application (BLA) approval followed review under the Real-Time Oncology Review (RTOR) program, an initiative of FDA’s Oncology Center of Excellence designed for efficient delivery of safe and effective cancer treatments to patients.

The company expects Rylaze will be commercially available in mid-July.

“The accelerated development and approval of Rylaze marks an important step in bringing a meaningful new treatment option for many ALL patients – most of whom are children – who cannot tolerate E. coli-derived asparaginase medicine,” said Dr. Luke Maese, assistant professor at the University of Utah, Primary Children’s Hospital and Huntsman Cancer Institute. “Before the approval of Rylaze, there was a significant need for an effective asparaginase medicine that would allow patients to start and complete their prescribed treatment program with confidence in supply.”

Recent data from a Children’s Oncology Group retrospective analysis of over 8,000 patients found that patients who did not receive a full course of asparaginase treatment due to associated toxicity had significantly lower survival outcomes – regardless of whether those patients were high risk or standard risk, slow early responders.²

About Study JZP458-201
The FDA approval of Rylaze, also known as JZP458, is based on clinical data from an ongoing pivotal Phase 2/3 single-arm, open-label, multicenter, dose confirmation study evaluating pediatric and adult patients with ALL or LBL who have had an allergic reaction to E. coli-derived asparaginases and have not previously received asparaginase erwinia chrysanthemi. The study was designed to assess the safety, tolerability and efficacy of JZP458. The determination of efficacy was measured by serum asparaginase activity (SAA) levels. The Phase 2/3 study is being conducted in two parts. The first part is investigating the intramuscular (IM) route of administration, including a Monday-Wednesday-Friday dosing schedule. The second part remains active to further confirm the dose and schedule for the intravenous (IV) route of administration.

The FDA approval of Rylaze was based on data from the first of three IM cohorts, which demonstrated the achievement and maintenance of nadir serum asparaginase activity (NSAA) greater than or equal to the level of 0.1 U/mL at 48 hours using IM doses of Rylaze 25 mg/m². The results of modeling and simulations showed that for a dosage of 25 mg/m² administered intramuscularly every 48 hours, the proportion of patients maintaining NSAA ≥ 0.1 U/mL at 48 hours after a dose of Rylaze was 93.6% (95% CI: 92.6%, 94.6%).¹

The most common adverse reactions (incidence >15%) were abnormal liver test, nausea, musculoskeletal pain, fatigue, infection, headache, pyrexia, drug hypersensitivity, febrile neutropenia, decreased appetite, stomatitis, bleeding and hyperglycemia. In patients treated with the Rylaze, a fatal adverse reaction (infection) occurred in one patient and serious adverse reactions occurred in 55% of patients. The most frequent serious adverse reactions (in ≥5% of patients) were febrile neutropenia, dehydration, pyrexia, stomatitis, diarrhea, drug hypersensitivity, infection, nausea and viral infection. Permanent discontinuation due to an adverse reaction occurred in 9% of patients who received Rylaze. Adverse reactions resulting in permanent discontinuation included hypersensitivity (6%) and infection (3%).¹

The company will continue to work with FDA and plans to submit additional data from a completed cohort of patients evaluating 25mg/m² IM given on Monday and Wednesday, and 50 mg/m² given on Friday in support of a M/W/F dosing schedule. Part 2 of the study is evaluating IV administration and is ongoing. The company also plans to submit these data for presentation at a future medical meeting.

Investor Webcast
The company will host an investor webcast on the Rylaze approval in July. Details will be announced separately.

About Rylaze^™ (asparaginase erwinia chrysanthemi (recombinant)-rywn)
Rylaze, also known as JZP458, is approved in the U.S. for use as a component of a multi-agent chemotherapeutic regimen for the treatment of acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma (LBL) in pediatric and adult patients one month and older who have developed hypersensitivity to E. coli-derived asparaginase. Rylaze has orphan drug designation for the treatment of ALL/LBL in the United States. Rylaze is a recombinant erwinia asparaginase that uses a novel Pseudomonas fluorescens expression platform. JZP458 was granted Fast Track designation by the U.S. Food and Drug Administration (FDA) in October 2019 for the treatment of this patient population. Rylaze was approved as part of the Real-Time Oncology Review program, an initiative of the FDA’s Oncology Center of Excellence designed for efficient delivery of safe and effective cancer treatments to patients.

The full U.S. Prescribing Information for Rylaze is available at: <http://pp.jazzpharma.com/pi/rylaze.en.USPI.pdf>

Important Safety Information

RYLAZE should not be given to people who have had:

Serious allergic reactions to RYLAZE
Serious swelling of the pancreas (stomach pain), serious blood clots, or serious bleeding during previous asparaginase treatment

RYLAZE may cause serious side effects, including:

Allergic reactions (a feeling of tightness in your throat, unusual swelling/redness in your throat and/or tongue, or trouble breathing), some of which may be life-threatening
Swelling of the pancreas (stomach pain)
Blood clots (may have a headache or pain in leg, arm, or chest)
Bleeding
Liver problems

Contact your doctor immediately if any of these side effects occur.

Some of the most common side effects with RYLAZE include: liver problems, nausea, bone and muscle pain, tiredness, infection, headache, fever, allergic reactions, fever with low white blood cell count, decreased appetite, mouth swelling (sometimes with sores), bleeding, and too much sugar in the blood.

RYLAZE can harm your unborn baby. Inform your doctor if you are pregnant, planning to become pregnant, or nursing. Females of reproductive potential should use effective contraception (other than oral contraceptives) during treatment and for 3 months following the final dose. Do not breastfeed while receiving RYLAZE and for 1 week after the final dose.

Tell your healthcare provider if there are any side effects that are bothersome or that do not go away.

These are not all the possible side effects of RYLAZE. For more information, ask your healthcare provider.

You are encouraged to report negative side effects of prescription drugs to the FDA. Visit www.fda.gov/medwatch, or call 1-800-FDA-1088 (1-800-332-1088).

About ALL
ALL is a cancer of the blood and bone marrow that can progress quickly if not treated.³ Leukemia is the most common cancer in children, and about three out of four of these cases are ALL.⁴ Although it is one of the most common cancers in children, ALL is among the most curable of the pediatric malignancies due to recent advancements in treatment.^5,6 Adults can also develop ALL, and about four of every 10 cases of ALL diagnosed are in adults.⁷ The American Cancer Society estimates that almost 6,000 new cases of ALL will be diagnosed in the United States in 2021.⁷ Asparaginase is a core component of multi-agent chemotherapeutic regimens in ALL.⁸ However, asparaginase treatments derived from E. coli are associated with the potential for development of hypersensitivity reactions.⁹

About Lymphoblastic Lymphoma
LBL is a rare, fast-growing, aggressive subtype of Non-Hodgkin’s lymphoma, most often seen in teenagers and young adults.⁸ LBL is a very aggressive lymphoma – also called high-grade lymphoma – which means the lymphoma grows quickly with early spread to different parts of the body.^10,11

About Jazz Pharmaceuticals plc
Jazz Pharmaceuticals plc (NASDAQ: JAZZ) is a global biopharmaceutical company whose purpose is to innovate to transform the lives of patients and their families. We are dedicated to developing life-changing medicines for people with serious diseases – often with limited or no therapeutic options. We have a diverse portfolio of marketed medicines and novel product candidates, from early- to late-stage development, in neuroscience and oncology. We actively explore new options for patients including novel compounds, small molecules and biologics, and through cannabinoid science and innovative delivery technologies. Jazz is headquartered in Dublin, Ireland and has employees around the globe, serving patients in nearly 75 countries. For more information, please visit www.jazzpharmaceuticals.com and follow @JazzPharma on Twitter.

About The Children’s Oncology Group (COG)
COG (childrensoncologygroup.org), a member of the NCI National Clinical Trials Network (NCTN), is the world’s largest organization devoted exclusively to childhood and adolescent cancer research. COG unites over 10,000 experts in childhood cancer at more than 200 leading children’s hospitals, universities, and cancer centers across North America, Australia, and New Zealand in the fight against childhood cancer. Today, more than 90% of the 14,000 children and adolescents diagnosed with cancer each year in the United States are cared for at COG member institutions. Research performed by COG institutions over the past 50 years has transformed childhood cancer from a virtually incurable disease to one with a combined 5-year survival rate of 80%. COG’s mission is to improve the cure rate and outcomes for all children with cancer.

Caution Concerning Forward-Looking Statements
This press release contains forward-looking statements, including, but not limited to, statements related to Jazz Pharmaceuticals’ belief in the potential of Rylaze to provide a reliable therapeutic option for adult and pediatric patients to maximize their chance for a cure, plans for a mid-July 2021 launch of Rylaze, the availability of a reliable supply of Rylaze and other statements that are not historical facts. These forward-looking statements are based on Jazz Pharmaceuticals’ current plans, objectives, estimates, expectations and intentions and inherently involve significant risks and uncertainties. Actual results and the timing of events could differ materially from those anticipated in such forward-looking statements as a result of these risks and uncertainties, which include, without limitation, effectively launching and commercializing new products; obtaining and maintaining adequate coverage and reimbursement for the company’s products; delays or problems in the supply or manufacture of the company’s products and other risks and uncertainties affecting the company, including those described from time to time under the caption “Risk Factors” and elsewhere in Jazz Pharmaceuticals’ Securities and Exchange Commission filings and reports (Commission File No. 001-33500), including Jazz Pharmaceuticals’ Annual Report on Form 10-K for the year ended December 31, 2020 and future filings and reports by Jazz Pharmaceuticals. Other risks and uncertainties of which Jazz Pharmaceuticals is not currently aware may also affect Jazz Pharmaceuticals’ forward-looking statements and may cause actual results and the timing of events to differ materially from those anticipated. The forward-looking statements herein are made only as of the date hereof or as of the dates indicated in the forward-looking statements, even if they are subsequently made available by Jazz Pharmaceuticals on its website or otherwise. Jazz Pharmaceuticals undertakes no obligation to update or supplement any forward-looking statements to reflect actual results, new information, future events, changes in its expectations or other circumstances that exist after the date as of which the forward-looking statements were made.

Jazz Media Contact:
Jacqueline Kirby
Vice President, Corporate Affairs
Jazz Pharmaceuticals plc
CorporateAffairsMediaInfo@jazzpharma.com
Ireland, +353 1 697 2141
U.S. +1 215 867 4910

Jazz Investor Contact:
Andrea N. Flynn, Ph.D.
Vice President, Head, Investor Relations
Jazz Pharmaceuticals plc
investorinfo@jazzpharma.com
Ireland, +353 1 634 3211

References

Rylaze (asparaginase erwinia chrysanthemi (recombinant)-rywn) injection, for intramuscular use Prescribing Information. Palo Alto, CA: Jazz Pharmaceuticals, Inc.
Gupta S, Wang C, Raetz EA et al. Impact of Asparaginase Discontinuation on Outcome in Childhood Acute Lymphoblastic Leukemia: A Report From the Children’s Oncology Group. J Clin Oncol. 2020 Jun 10;38(17):1897-1905. doi: 10.1200/JCO.19.03024
National Cancer Institute. Adult Acute Lymphoblastic Leukemia Treatment (PDQ^®)–Patient Version. Available at www.cancer.gov/types/leukemia/patient/adult-all-treatment-pdq. Accessed June 29, 2021
American Cancer Society. Key Statistics for Childhood Leukemia. Available at https://www.cancer.org/cancer/leukemia-in-children/about/key-statistics.html. Accessed June 29, 2021.
American Cancer Society. Cancer Facts & Figures 2019. www.cancer.org/research/cancer-facts-statistics/all-cancer-facts-figures/cancer-facts-figures-2019.html. Accessed June 29, 2021.
Pui C, Evans W. A 50-Year Journey to Cure Childhood Acute Lymphoblastic Leukemia. Seminars in Hematology. 2013;50(3), 185-196.
American Cancer Society. Key Statistics for Acute Lymphocytic Leukemia (ALL). Available at https://cancerstatisticscenter.cancer.org/?_ga=2.8163506.1018157754.1621008457-1989786785.1621008457#!/data-analysis/NewCaseEstimates. Accessed June 29, 2021.
Salzer W, Bostrom B, Messinger Y et al. 2018. Asparaginase activity levels and monitoring in patients with acute lymphoblastic leukemia. Leukemia & Lymphoma. 59:8, 1797-1806, DOI: 10.1080/10428194.2017.1386305.
Hijiya N, van der Sluis IM. Asparaginase-associated toxicity in children with acute lymphoblastic leukemia. Leuk Lymphoma. 2016;57(4):748–757. DOI: 10.3109/10428194.2015.1101098.
Leukemia Foundation. Lymphoblastic Lymphoma. Available at https://www.leukaemia.org.au/disease-information/lymphomas/non-hodgkin-lymphoma/other-non-hodgkin-lymphomas/lymphoblastic-lymphoma/. Accessed June 29, 2021.
Mayo Clinic. Acute Lymphocytic Leukemia Diagnosis. Available at https://www.mayoclinic.org/diseases-conditions/acute-lymphocytic-leukemia/diagnosis-treatment/drc-20369083. Accessed June 29, 2021.

SOURCE Jazz Pharmaceuticals plc

Pegcetacoplan

June 9, 2021 8:56 am / Leave a comment

Sequence:

1ICVWQDWGAH RCTXK

Sequence:

1ICVWQDWGAH RCTXK

Sequence Modifications

Type	Location	Description
terminal mod.	Lys-15	C-terminal amide
terminal mod.	Lys-15′	C-terminal amide
bridge	Cys-2 – Cys-12	disulfide bridge, dimer
bridge	Lys-15 – Lys-15′	covalent bridge, dimer
bridge	Cys-2′ – Cys-12′	disulfide bridge, dimer
uncommon	Oaa-14	–
uncommon	Oaa-14′	–

Pegcetacoplan

ペグセタコプラン;

FDA APPROVED Empaveli, 2021/5/14

Protein Sequence

Sequence Length: 30, 15, 15multichain; modifiedPoly(oxy-1,2-ethanediyl), α-hydro-ω-hydroxy-, 15,15′-diester with N-acetyl-L-isoleucyl-L-cysteinyl-L-valyl-1-methyl-L-tryptophyl-L-glutaminyl-L-α-aspartyl-L-tryptophylglycyl-L-alanyl-L-histidyl-L-arginyl-L-cysteinyl-L-threonyl-2-[2-(2-aminoethoxy)ethoxy]acetyl-N⁶-carboxy-L-lysinamide cyclic (2→12)-(disulfide)Polymer

Poly(oxy-1,2-ethanediyl), alpha-hydro-omega-hydroxy-, 15,15′-diester with N-acetyl-Lisoleucyl-L-cysteinyl-L-valyl-1-methyl-L-tryptophyl-L-glutaminyl-L-alpha-aspartyl-L-tryptophylglycyl-L-alanyl-L-histidyl-L-arginyl-L-cysteinyl-L-threonyl-2-(2-(2-aminoethoxy)ethoxy)acetyl-N6-carboxy-L-lysinamide cyclic (2�->12)-(disulfide)

O,O’-bis((S2,S12-cyclo(N-acetyl-L-isoleucyl-L-cysteinyl-L-valyl-1-methyl-Ltryptophyl-L-glutaminyl-L-alpha-aspartyl-L-tryptophylglycyl-L-alanyl-L-histidyl-L-arginyl-L-cysteinyl-L-threonyl-2-(2-(2-aminoethoxy)ethoxy)acetyl-L-lysinamide))-N6.15-carbonyl)polyethylene glycol(n = 800-1100)

APL-2
WHO 10743

Formula	C170H248N50O47S4. (C2H4O)n3872.40 g·mol⁻¹
EfficacyDisease	Complement inhibitorParoxysmal nocturnal hemoglobinuria
CAS	2019171-69-6
Comment	Treatment of paroxysmal nocturnal hemoglobinuria (PNH), complement-mediated nephropathies, and age-related macular degeneration (AMD)

OriginatorApellis Pharmaceuticals
ClassAnti-inflammatories; Anti-ischaemics; Antianaemics; Cyclic peptides; Eye disorder therapies; Polyethylene glycols; Urologics
Mechanism of ActionComplement C3 inhibitors
Orphan Drug StatusYes – Paroxysmal nocturnal haemoglobinuria; Autoimmune haemolytic anaemia; Glomerulonephritis

RegisteredParoxysmal nocturnal haemoglobinuria
Phase IIIAge-related macular degeneration
Phase IIAmyotrophic lateral sclerosis; Autoimmune haemolytic anaemia; Glomerulonephritis; IgA nephropathy; Lupus nephritis; Membranous glomerulonephritis
Phase I/IIWet age-related macular degeneration
DiscontinuedIschaemia

02 Jun 2021Apellis Pharmaceuticals plans a phase III trial for Glomerulonephritis in the second half of 2021
25 May 2021Top-line efficacy and safety results from the phase III PRINCE trial for Paroxysmal nocturnal haemoglobinuria released by Apellis Pharmaceuticals
18 May 2021Registered for Paroxysmal nocturnal haemoglobinuria in USA (SC) – First global approval

Pegcetacoplan, sold under the brand name Empaveli, is a medication used to treat paroxysmal nocturnal hemoglobinuria (PNH).^[1]^[2]

The most common side effects include injection-site reactions, infections, diarrhea, abdominal pain, respiratory tract infection, viral infection, and fatigue.^[2]

Paroxysmal nocturnal hemoglobinuria is characterized by red blood cell destruction, anemia (red blood cells unable to carry enough oxygen to tissues), blood clots, and impaired bone marrow function (not making enough blood cells).^[1]

Pegcetacoplan is the first treatment for paroxysmal nocturnal hemoglobinuria that binds to complement protein C3.^[1] Pegcetacoplan was approved for medical use in the United States in May 2021.^[1]^[3]

Pegcetacoplan is a complement inhibitor indicated in the treatment of paroxysmal nocturnal hemoglobinuria (PNH).^5,7 Prior to its FDA approval, patients with PNH were typically treated with the C5 inhibiting monoclonal antibody eculizumab.⁵ Patients given eculizumab experienced less hemolysis caused by the membrane attack complex, but were still somewhat susceptible to hemolysis caused by C3b opsonization.^5,6 Pegcetacoplan was developed out of a need for an inhibitor of complement mediated hemolysis further upstream of C5.^5,6 Pegcetacoplan is a pegylated C3 inhibitor that can disrupt the processes leading to both forms of hemolysis that threaten patients with PNH.⁵

Pegcetacoplan was granted FDA approval on 14 May 2021.⁷

Medical uses

Pegcetacoplan is indicated to treat adults with paroxysmal nocturnal hemoglobinuria (PNH).^[1]^[2]

EMPAVELI contains pegcetacoplan, a complement inhibitor. Pegcetacoplan is a symmetrical molecule comprised of two identical pentadecapeptides covalently bound to the ends of a linear 40-kiloDalton (kDa) PEG molecule. The peptide portions of pegcetacoplan contain 1-methyl-L-tryptophan (Trp(Me)) in position 4 and amino(ethoxyethoxy)acetic acid (AEEA) in position 14.

The molecular weight of pegcetacoplan is approximately 43.5 kDa. The molecular formula is C₁₉₇₀H₃₈₄₈N₅₀O₉₄₇S₄. The structure of pegcetacoplan is shown below.

EMPAVELI™ (pegcetacoplan) Structural Formula - Illustration

EMPAVELI injection is a sterile, clear, colorless to slightly yellowish aqueous solution for subcutaneous use and is supplied in a 20-mL single-dose vial. Each 1 mL of solution contains 54 mg of pegcetacoplan, 41 mg of sorbitol, 0.384 mg of glacial acetic acid, 0.490 mg of sodium acetate trihydrate, and Water for Injection USP. EMPAVELI may also contain sodium hydroxide and/or additional glacial acetic acid for adjustment to a target pH of 5.0.

FDA approves new treatment for adults with serious rare blood disease..

https://www.fda.gov/drugs/drug-safety-and-availability/fda-approves-new-treatment-adults-serious-rare-blood-disease

FDA has approved Empaveli (pegcetacoplan) injection to treat adults with paroxysmal nocturnal hemoglobinuria (PNH), a rare, life-threatening blood disease. Empaveli is the first PNH treatment that binds to compliment protein C3.

PNH is characterized by red blood cell destruction, anemia (red blood cells unable to carry enough oxygen to tissues), blood clots, and impaired bone marrow function (not making enough blood cells). The disease affects 1-1.5 people per million. Individuals are typically diagnosed around ages 35 to 40. PNH can be serious, with median survival of 10 years after diagnosis. However, some patients live for decades with only minor symptoms.

PNH is caused by gene mutations that affect red blood cells. Red blood cells in people with these mutations are defective and can be destroyed by the immune system, which causes anemia.

The effectiveness of Empaveli was evaluated in a study enrolling 80 patients with PNH and anemia who had been taking eculizumab, a treatment previously approved for PNH. Patients first completed a four-week period during which they received Empaveli 1,080 mg twice weekly in addition to eculizumab at their previous dose. After the first four weeks, patients were randomly assigned to receive either Empaveli or their current dose of eculizumab for 16 weeks.

After 16 weeks, the severity of anemia was compared in the two treatment groups on the basis of hemoglobin concentration (a laboratory measure of anemia). In both treatment groups, the average hemoglobin was 8.7 g/dL at baseline, indicating severe anemia. (Normal hemoglobin values in adult men are 14 g/dL or above; normal values in adult women are 12 g/dL or above.) During the 16 weeks of treatment, patients in the Empaveli group had an average increase in their hemoglobin of 2.4 g/dL. Meanwhile, patients in the eculizumab group had an average decrease in their hemoglobin of 1.5 g/dL.

Empaveli is available only through a restricted program under a risk evaluation and mitigation strategy. Meningococcal (a type of bacteria) infections can occur in patients taking Empaveli and can become life-threatening or fatal if not treated early. Empaveli may also predispose individuals to serious infections, especially infections caused by encapsulated bacteria. Patients should be monitored for infusion-related reactions. Empaveli can interfere with certain laboratory tests. The most common side effects are injection site reactions, infections, diarrhea, abdominal pain, respiratory tract infection, viral infection, and fatigue.

Empaveli received priority review, fast track and orphan drug designations for this indication.

FDA granted the approval of Empaveli to Apellis Pharmaceuticals.

Adverse effects

Meningococcal (a type of bacteria) infections can occur in people taking pegcetacoplan and can become life-threatening or fatal if not treated early.^[1] Pegcetacoplan may also predispose individuals to serious infections, especially infections caused by encapsulated bacteria.^[1]

History

The effectiveness of pegcetacoplan was evaluated in a study enrolling 80 participants with paroxysmal nocturnal hemoglobinuria and anemia who had been taking eculizumab, a treatment previously approved for paroxysmal nocturnal hemoglobinuria.^[1]

References

^ Jump up to:^a ^b ^c ^d ^e ^f ^g ^h ⁱ “FDA approves new treatment for adults with serious rare blood disease”. U.S. Food and Drug Administration (FDA). 14 May 2021. Retrieved 14 May 2021. This article incorporates text from this source, which is in the public domain.
^ Jump up to:^a ^b ^c ^d https://pi.apellis.com/files/PI_Empaveli.pdf
^ “Apellis Announces U.S. Food and Drug Administration (FDA) Approval of Empaveli (pegcetacoplan) for Adults with Paroxysmal Nocturnal Hemoglobinuria (PNH)” (Press release). Apellis Pharmaceuticals. 14 May 2021. Retrieved 14 May 2021 – via GlobeNewswire.

External links

“Pegcetacoplan”. Drug Information Portal. U.S. National Library of Medicine.
Clinical trial number NCT03500549 for “Study to Evaluate the Efficacy and Safety of APL-2 in Patients With Paroxysmal Nocturnal Hemoglobinuria (PNH)” at ClinicalTrials.gov

Clinical data
Trade names	Empaveli
Other names	APL-2
License data	US DailyMed: Pegcetacoplan
Routes of administration	Subcutaneous infusion
Drug class	Complement inhibitor
ATC code	None
Legal status
Legal status	US: ℞-only ^[1]^[2]
Identifiers
CAS Number	2019171-69-6
UNII	TO3JYR3BOU
KEGG	D11613
ChEMBL	ChEMBL4298211
Chemical and physical data
Formula	C₁₇₀H₂₄₈N₅₀O₄₇S₄
Molar mass	3872.40 g·mol⁻¹

/////////Pegcetacoplan, ペグセタコプラン , FDA 2021, APPROVALS 2021, APL-2, WHO 10743, Apellis Pharmaceuticals, Empaveli, priority review, fast track, orphan drug

https://www.sec.gov/Archives/edgar/data/1492422/000156459020007350/apls-10k_20191231.htm

NEW DRUG APPROVALS

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RIDINILAZOLE

January 27, 2021 10:56 am / 1 Comment on RIDINILAZOLE

RIDINILAZOLE

SMT19969

Molecular FormulaC₂₄H₁₆N₆
Average mass388.424 Da
ридинилазол [Russian] [INN]ريدينيلازول [Arabic] [INN]利地利唑 [Chinese] [INN]
リジニラゾール;

10075
2,2′-Di(4-pyridinyl)-3H,3’H-5,5′-bibenzimidazole
308362-25-6[RN]6,6′-Bi-1H-benzimidazole, 2,2′-di-4-pyridinyl-

Summit Therapeutics (formerly Summit Corp ) is developing ridinilazole the lead compound from oral narrow-spectrum, GI-restricted antibiotics, which also include SMT-21829, for the treatment of Clostridium difficile infection and prevention of recurrent disease.

Ridinilazole (previously known as SMT19969) is an investigational small molecule antibiotic being evaluated for oral administration to treat Clostridioides difficile infection (CDI). In vitro, it is bactericidal against C. difficile and suppresses bacterial toxin production; the mechanism of action is thought to involve inhibition of cell division.^[1] It has properties which are desirable for the treatment of CDI, namely that it is a narrow-spectrum antibiotic which exhibits activity against C. difficile while having little impact on other normal intestinal flora and that it is only minimally absorbed systemically after oral administration.^[2] At the time ridinilazole was developed, there were only three antibiotics in use for treating CDI: vancomycin, fidaxomicin, and metronidazole.^[1]^[2] The recurrence rate of CDI is high, which has spurred research into other treatment options with the aim to reduce the rate of recurrence.^[3]^[4]

As of 2019, two phase II trials have been completed and two phase III trials comparing ridinilazole to vancomycin for CDI are expected to be completed in September 2021.^[2][5][6] Ridinilazole was designated as a Qualified Infectious Disease Product (QIDP) and was granted Fast Track status by the U.S. FDA.^[2] Fast Track status is reserved for drugs designed to treat diseases where there is currently a gap in the treatment, or a complete lack thereof.^[7] The QIDP designation adds five more years of exclusivity for ridinazole upon approval.^[8]

PATENT

WO-2021009514

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2021009514&tab=PCTDESCRIPTION&_cid=P22-KKEXJ9-06647-1

Process for preparing ridinilazole useful for treating Clostridium difficile infection. Also claimed is the crystalline form of a compound.

The present invention relates to processes for the preparation of 2,2′-di(pyridin-4-yl)-1/-/,T/-/-5,5′-bibenzo[d]imidazole (which may also be known as 5,5’-bis[2-(4-pyridinyl)-1/-/-benzimidazole], 2,2′-bis(4-pyridyl)-3/-/,3’/-/-5,5′-bibenzimidazole or 2-pyridin-4-yl-6-(2-pyridin-4-yl-3/-/-benzimidazol-5-yl)-1/-/-benzimidazole), referenced herein by the INN name ridinilazole, and pharmaceutically acceptable derivatives, salts, hydrates, solvates, complexes, bioisosteres, metabolites or prodrugs thereof. The invention also relates to various crystalline forms of ridinilazole, to processes for their preparation and to related pharmaceutical preparations and uses thereof (including their medical use and their use in the efficient large-scale synthesis of ridinilazole).

WO2010/063996 describes various benzimidazoles, including ridinilazole, and their use as antibacterials (including in the treatment of CDAD).

WO 2011/151621 describes various benzimidazoles and their use as antibacterials

(including in the treatment of CDAD).

W02007056330, W02003105846 and W02002060879 disclose various 2-amino benzimidazoles as antibacterial agents.

W02007148093 discloses various 2-amino benzothiazoles as antibacterial agents.

W02006076009, W02004041209 and Bowser et at. (Bioorg. Med. Chem. Lett., 2007, 17, 5652-5655) disclose various substituted benzimidazole compounds useful as anti-infectives that decrease resistance, virulence, or growth of microbes. The compounds are said not to exhibit intrinsic antimicrobial activity in vitro.

US 5,824,698 discloses various dibenzimidazoles as broad-spectrum antibiotics, disclosing activity against both Gram-negative and Gram-positive bacteria, including Staphylococcus spp.and Enterococcus spp. However, this document does not disclose activity against anaerobic spore-forming bacteria and in particular does not disclose activity against any Clostridioides spp. (including C. difficile).

US 2007/0112048 A1 discloses various bi- and triarylimidazolidines and bi- and

triarylamidines as broad-spectrum antibiotics, disclosing activity against both Gram negative and Gram-positive bacteria, including Staphylococcus spp., Enterococcus spp. and Clostridioides spp. However, this document does not disclose compounds of formula (I) as described herein.

Chaudhuri et al. (2007) J.Org. Chem. 72, 1912-1923 describe various bis-2-(pyridyl)-1 H-benzimidazoles (including compounds of formula I as described herein) as DNA binding agents. This document is silent as to potential antibacterial activity.

Singh et al. (2000) Synthesis 10: 1380-1390 describe a condensation reaction for producing 2,2′-di(pyridin-4-yl)-1/-/,T/-/-5,5′-bibenzo[d]imidazole using 4-pyridine

carboxaldehyde, FeCI₃, 0₂, in DMF at 120°C.

Bhattacharya and Chaudhuri (2007) Chemistry – An Asian Journal 2: 648-655 describe a condensation reaction for producing 2,2′-di(pyridin-4-yl)-1/-/,T/-/-5,5′-bibenzo[d]imidazole using 4-pyridine carboxaldehyde and nitrobenzene at 120°C.

WO2019/068383 describes the synthesis of ridinilazole by metal-ion catalyzed coupling of 3,4,3’,4’-tetraaminobiphenyl with 4-pyridinecarboxaldehyde in the presence of oxygen, followed by the addition of a complexing agent.

PATENT

WO2010063996

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2010063996&_cid=P22-KKEXNB-07493-1

claiming antibacterial compounds. Bicyclic heteroaromatic compounds, particularly bi-benzimidazole derivatives.

WO2007056330, WO2003105846 and WO2002060879 disclose various 2-amino benzimidazoles as antibacterial agents.

WO2007148093 discloses various 2-amino benzothiazoles as antibacterial agents.

WO2006076009, WO2004041209 and Bowser et al. (Bioorg. Med. Chem. Lett., 2007, 17, 5652-5655) disclose various substituted benzimidazole compounds useful as anti-infectives that decrease resistance, virulence, or growth of microbes. The compounds are said not to exhibit intrinsic antimicrobial activity in vitro.

US 2007/0112048 A1 discloses various bi- and triarylimidazolidines and bi- and triarylamidines as broad-spectrum antibiotics, disclosing activity against both Gram-negative and Gram-positive bacteria, including Staphylococcus spp., Enterococcus spp.

and Clostridium spp. However, this document does not disclose compounds of general formula (I) as described herein.

Chaudhuri et al. (J.Org. Chem., 2007, 72, 1912-1923) describe various bis-2-(pyridyl)-1 H-benzimidazoles (including compounds of formula I as described herein) as DNA binding agents. This document is silent as to potential antibacterial activity.

PATENT

Product PATENT, WO2010063996 ,

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2010063996&_cid=P22-KKEXS7-08574-1

protection in the EP until 2029 and expire in the US in December 2029.

PAPER

https://www.frontiersin.org/articles/10.3389/fmicb.2018.01206/full

PAPER

Synthesis (2000), (10), 1380-1390.

https://www.thieme-connect.de/products/ejournals/abstract/10.1055/s-2000-7111

PAPERT

Chemistry – An Asian Journal (2007), 2(5), 648-655.

https://onlinelibrary.wiley.com/doi/abs/10.1002/asia.200700014

Studies of double‐stranded‐DNA binding have been performed with three isomeric bis(2‐(n‐pyridyl)‐1H‐benzimidazole)s (n=2, 3, 4). Like the well‐known Hoechst 33258, which is a bisbenzimidazole compound, these three isomers bind to the minor groove of duplex DNA. DNA binding by the three isomers was investigated in the presence of the divalent metal ions Mg²⁺, Co²⁺, Ni²⁺, Cu²⁺, and Zn²⁺. Ligand–DNA interactions were probed with fluorescence and circular dichroism spectroscopy. These studies revealed that the binding of the 2‐pyridyl derivative to DNA is dramatically reduced in the presence of Co²⁺, Ni²⁺, and Cu²⁺ ions and is abolished completely at a ligand/metal‐cation ratio of 1:1. Control experiments done with the isomeric 3‐ and 4‐pyridyl derivatives showed that their binding to DNA is unaffected by the aforementioned transition‐metal ions. The ability of 2‐(2‐pyridyl)benzimidazole to chelate metal ions and the conformational changes of the ligand associated with ion chelation probably led to such unusual binding results for the ortho isomer. The addition of ethylenediaminetetraacetic acid (EDTA) reversed the effects completely.

PAPER

Journal of Organic Chemistry (2007), 72(6), 1912-1923.

https://pubs.acs.org/doi/10.1021/jo0619433

Three symmetrical positional isomers of bis-2-(n-pyridyl)-1H-benzimidazoles (n = 2, 3, 4) were synthesized and DNA binding studies were performed with these isomeric derivatives. Like bisbenzimidazole compound Hoechst 33258, these molecules also demonstrate AT-specific DNA binding. The binding affinities of 3-pyridine (m-pyben) and 4-pyridine (p-pyben) derivatized bisbenzimidazoles to double-stranded DNA were significantly higher compared to 2–pyridine derivatized benzimidazole o-pyben. This has been established by combined experimental results of isothermal fluorescence titration, circular dichroism, and thermal denaturation of DNA. To rationalize the origin of their differential binding characteristics with double-stranded DNA, computational structural analyses of the uncomplexed ligands were performed using ab initio/Density Functional Theory. The molecular conformations of the symmetric head-to-head bisbenzimidazoles have been computed. The existence of intramolecular hydrogen bonding was established in o-pyben, which confers a conformational rigidity to the molecule about the bond connecting the pyridine and benzimidazole units. This might cause reduction in its binding affinity to double-stranded DNA compared to its para and meta counterparts. Additionally, the predicted stable conformations for p-, m-, and o-pyben at the B3LYP/6-31G* and RHF/6-31G* levels were further supported by experimental pK_a determination. The results provide important information on the molecular recognition process of such symmetric head to head bisbenzimidazoles toward duplex DNA.

Patent

US 8975416

PATENT

WO 2019068383

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2019068383

Clostridium difficile infection (CDI) is the leading cause of infectious healthcare-associated diarrhoea. CDI remains a challenge to treat clinically, because of a limited number of antibiotics available and unacceptably high recurrence rates. Because of this, there has been significant demand for creating innovative therapeutics, which has resulted in the development of several novel antibiotics.

Ridinilazole (SMT19969) is the INN name of 5,5’bis[2-(4-pyridinyl)-lH-benzimidazole], which is a promising non-absorbable small molecule antibiotic intended for oral use in the treatment of CDI. It has been shown to exhibit a prolonged post-antibiotic effect and treatment with ridinilazole has resulted in decreased toxin production. A phase 1 trial demonstrated that oral ridinilazole is well tolerated and specifically targets Clostridia whilst sparing other faecal bacteria.

Ridinilazole has the following chemical structure:

Bhattacharya & Chaudhuri (Chem. Asian J., 2007, No. 2, 648-655) report performing double-stranded DNA binding with three benzimidazole derivatives, including ridinilazole. The compounds have been prepared by dissolving the reactants in nitrobenzene, heating at 120°C for 8- 1 Oh and purifying the products by column chromatography over silica gel. The compounds were obtained in 65-70% yield. Singh et al., (Synthesis, 2000, No. 10, 1380-1390) describe a catalytic redox cycling approach based on Fe(III) and molecular oxygen as co-oxidant for providing access to benzimidazole and

imidazopyridine derivatives, such as ridinilazole. The reaction is performed at high temperatures of 120°C and the product is isolated in 91% yield by using silica flash chromatography.

Both processes are not optimal, for example in terms of yield, ease of handling and scalability. Thus, there is a need in the art for an efficient and scalable preparation of ridinilazole, which overcomes the problems of the prior art processes.

Example 1 : Preparation of crude ridinilazole free base

A solution of 3,4,3′,4′-tetraaminobiphenyl (3.28 g, 15.3 mmol) and isonicotinaldehyde (3.21 g, 30.0 mmol) in DMF (40 mL) was stirred at 23 °C for one hour. Then anhydrous ferric chloride (146 mg, 0.90 mmol), water (0.10 mL, 5.4 mmol) and additional DMF (2 mL) were added and fresh air was bubbled into the solution during vigorous stirring for 5 hours at room temperature. Next, water (80 mL) and EDTA (0.29 g) were added resulting in a brownish suspension, which was stirred overnight. The product was isolated by filtration, washed with water, and dried in a desiccator in vacuo as a brown powder (5.56 g; 95%). The addition of EDTA had held iron in solution and the crude ridinilazole contained significantly lower amounts of iron than comparative example 1.

Example 12: Formation of essentially pure ridinilazole free base

To a suspension von ridinilazole tritosylate (1 10 mg, 0.12 mmol) in water (35 mL) featuring a pH value of about 4.5 stirring at 70 °C sodium bicarbonate (580 mg, 6.9 mmol) were added and caused a change of color from orange to slightly tan. The mixture, now at a pH of about 8.5, was cooled down to room temperature and the solids were separated by filtration, washed with water (1 ML) and dried in vacuo providing 40 mg (85%) essentially pure ridinilazole as a brownish powder.

Spectroscopic analysis:

¾ NMR (DMSO-de, 300 MHz): δ 7.55 (d, J = 8.4 Hz, 2H), 7.70 (d, J = 8.4 Hz, 2H), 7.88 (s, 2H), 8.13 (d, J = 5.8 Hz, 4H), 8.72 (d, J = 5.8 Hz, 4H) ppm.

¹³C NMR (DMSO-d₆, 75 MHz): δ 1 13.4 (2C), 1 16.4 (2C), 120.4 (4C), 121.8 (2C), 135.7 (2C), 138.7 (2C), 140.7 (2C), 141.4 (2C), 150.3 (4C), 151.1 (2C) ppm.

IR (neat): v 3033 (w), 1604 (s), 1429 (m), 1309 (m), 1217 (m), 1 1 15 (w), 998 (m), 964 (m), 824 (m), 791 (s), 690 (s), 502 (s) cm .

UV-Vis (MeOH): 257, 341 nm.

The sharp peaks in the ¾ NMR indicated that iron had been efficiently removed.

Comparative example 1 : Preparation of ridinilazole

A solution of 3,4,3′,4′-tetraaminobiphenyl (0.69 g, 3.2 mmol) and isonicotinaldehyde (0.64 g, 6.0 mmol) in DMF (20 mL) was stirred at 80°C for one hour. Then ferric chloride hexahydrate (49 mg, 0.18 mmol), water (0.10 mL, 5.4 mmol) and additional DMF (2 mL) were added and fresh air was bubbled into the solution during vigorous stirring for 10 hours at 120 °C. After cooling to room temperature water (50 mL) and the mixture was stirred for one hour. A black crude product was isolated by filtration and comprised ridinilazole and iron.

References

^ Jump up to:^a ^b Cho JC, Crotty MP, Pardo J (March 2019). “Clostridium difficile infection”. Annals of Gastroenterology. 32 (2): 134–140. doi:10.20524/aog.2018.0336. PMC 6394264. PMID 30837785.
^ Jump up to:^a ^b ^c ^d Carlson TJ, Endres BT, Bassères E, Gonzales-Luna AJ, Garey KW (April 2019). “Ridinilazole for the treatment of Clostridioides difficile infection”. Expert Opinion on Investigational Drugs. 28 (4): 303–310. doi:10.1080/13543784.2019.1582640. PMID 30767587.
^ Bassères E, Endres BT, Dotson KM, Alam MJ, Garey KW (January 2017). “Novel antibiotics in development to treat Clostridium difficile infection”. Current Opinion in Gastroenterology. 33 (1): 1–7. doi:10.1097/MOG.0000000000000332. PMID 28134686. These tables highlight the increased drug development directed towards CDI due to the rise in prevalence of infections and to attempt to reduce the number of recurrent infections.
^ Vickers RJ, Tillotson G, Goldstein EJ, Citron DM, Garey KW, Wilcox MH (August 2016). “Ridinilazole: a novel therapy for Clostridium difficile infection”. International Journal of Antimicrobial Agents. 48 (2): 137–43. doi:10.1016/j.ijantimicag.2016.04.026. PMID 27283730. there exists a significant unmet and increasing medical need for new therapies to treat CDI, specifically those that can reduce the rate of disease recurrence.
^ Clinical trial number NCT03595553 for “Ri-CoDIFy 1: Comparison of Ridinilazole Versus Vancomycin Treatment for Clostridium Difficile Infection” at ClinicalTrials.gov
^ Clinical trial number NCT03595566 for “Ri-CoDIFy 2: To Compare Ridinilazole Versus Vancomycin Treatment for Clostridium Difficile Infection” at ClinicalTrials.gov
^ “Fast Track”. U.S. Food and Drug Administration. 2018-11-03.
^ “”HHS spurs new antibiotic development for biodefense and common infections””. Public Health Emergency. U.S. Department of Health and Human Services. Retrieved 2020-12-04.

Clinical data

Other names	SMT19969
ATC code	None
Identifiers
IUPAC name [show]
CAS Number	308362-25-6
PubChem CID	16659285
ChemSpider	17592423
UNII	06DX01190R
KEGG	D11958
Chemical and physical data
Formula	C₂₄H₁₆N₆
Molar mass	388.42 g/mol
3D model (JSmol)	Interactive image
SMILES [hide]c6cc(c5nc4ccc(c3ccc2nc(c1ccncc1)[nH]c2c3)cc4[nH]5)ccn6

/////////RIDINILAZOLE, SMT19969, SMT 19969, ридинилазол , ريدينيلازول , 利地利唑 , リジニラゾール , Qualified Infectious Disease Product, QIDP, Fast Track , PHASE 3, Clostridioides difficile infection ,

LYS 228

February 24, 2020 10:24 am / Leave a comment

2D chemical structure of 1810051-96-7

LYS228

BOS-228
LYS-228

Molecular Formula, C16-H18-N6-O10-S2

Molecular Weight, 518.4783

(3S,4R)-3-((Z)-2-(2-Ammoniothiazol-4-yl)-2-((1-carboxycyclopropoxy)imino)acetamido)-2-oxo-4-((2-oxooxazolidin-3-yl)methyl)azetidine-1-sulfonate

RN: 1810051-96-7
UNII: 29H7N9XI1B

UNII-005B24W9YP

005B24W9YP

Lys-228 trihydrate

2091840-43-4

Yclopropanecarboxylic acid, 1-(((Z)-(1-(2-amino-4-thiazolyl)-2-oxo-2-(((3S,4R)-2-oxo-4-((2-oxo-3-oxazolidinyl)methyl)-1-sulfo-3-azetidinyl)amino)ethylidene)amino)oxy)-, hydrate (1:3)

1-[(Z)-[1-(2-amino-1,3-thiazol-4-yl)-2-oxo-2-[[(3S,4R)-2-oxo-4-[(2-oxo-1,3-oxazolidin-3-yl)methyl]-1-sulfoazetidin-3-yl]amino]ethylidene]amino]oxycyclopropane-1-carboxylic acid;trihydrate

BOS-228 (LYS-228) is a monobactam discovered at Novartis and currently in phase II clinical development at Boston Pharmaceuticals for the treatment of complicated urinary tract infection and complicated intraabdominal infections in adult patients.

The compound has been granted fast track and Qualified Infectious Disease Product (QIDP) designation from the FDA.

In October 2018, Novartis licensed to Boston Pharmaceuticals worldwide rights to the product.

Paper

https://pubs.acs.org/doi/10.1021/acs.oprd.9b00330

Patent

US 20150266867

PATENT

WO 2017050218

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2017050218&tab=FULLTEXT

Compound X: 1- ( ( (Z) – (1- (2-aminothiazol-4-yl) -2-oxo-2- ( ( (3S, 4R) -2-oxo-4- ( (2-oxooxazolidin-3-yl) methyl) -1-sulfoazetidin-3-yl) amino) ethylidene) amino) oxy) cyclopropanecarboxylic acid.

[0126]

Step 1: Benzhydryl 1- ( ( (Z) – (1- (2- ( (tert-butoxycarbonyl) amino) thiazol-4-yl) -2-oxo-2- ( ( (3S, 4R) -2-oxo-4- ( (2-oxooxazolidin-3-yl) methyl) azetidin-3-yl) amino) ethylidene) amino) oxy) cyclopropanecarboxylate. To a solution of (Z) -2- ( (1- ( (benzhydryloxy) carbonyl) cyclopropoxy) imino) -2- (2- ( (tert-butoxycarbonyl) amino) thiazol-4-yl) acetic acid (854 mg, 1.59 mmol) prepared according to published patent application US2011/0190254, Intermediate B (324 mg, 1.75 mmol) and HATU (785 mg, 2.07 mmol) in DMF (7.9 mL) , DIPEA was added (832 μL, 4.77 mmol) . After 1 h of stirring, it was poured into water and extracted with EtOAc. Brine was added to the aqueous layer, and it was further extracted with ethyl acetate (EtOAc) (3x) . The combined organic layers were dried over Na ₂SO ₄ and concentrated in vacuo. The crude residue was purified via silica gel chromatography (0-10％MeOH-DCM) to afford the title compound (1.09 g, 97％) as a beige foam. LCMS: R _t ＝ 0.97 min, m/z ＝705.3 (M+1) Method 2m_acidic.

[0127]

Instead of HATU, a variety of other coupling reagents can be used, such as any of the typical carbodiimides, or CDMT (2-chloro-4, 6-dimethoxy-1, 3, 5-triazine) and N-methylmorpholine to form the amide bond generated in Step 1.

[0128]

Step 2: (3S, 4R) -3- ( (Z) -2- ( (1- ( (benzhydryloxy) carbonyl) cyclopropoxy) imino) -2- (2- ( (tert-butoxycarbonyl) amino) thiazol-4-yl) acetamido) -2-oxo-4- ( (2-oxooxazolidin-3-yl) methyl) azetidine-1-sulfonic acid. Benzhydryl 1- ( ( (Z) – (1- (2- ( (tert-butoxycarbonyl) amino) thiazol-4-yl) -2-oxo-2- ( ( (3S, 4R) -2-oxo-4- ( (2-oxooxazolidin-3-yl) methyl) azetidin-3-yl) amino) ethylidene) amino) oxy) cyclopropanecarboxylate (1.00 g, 1.42 mmol) in DMF (7.0 mL) at 0 ℃ was treated with SO ₃·DMF (448 mg, 2.84 mmol) . After 2 h of stirring at rt, the solution was poured into ice-cold brine and extracted with EtOAc (3x) . The combined organic layers were dried over Na ₂SO ₄ and concentrated in vacuo, affording the title compound (assumed quantitative) as a white solid. LCMS: Rt ＝0.90 min, m/z ＝ 785.2 (M+1) Method 2m_acidic.

[0129]

Step 3: 1- ( ( (Z) – (1- (2-aminothiazol-4-yl) -2-oxo-2- ( ( (3S, 4R) -2-oxo-4- ( (2-oxooxazolidin-3-yl) methyl) -1-sulfoazetidin-3-yl) amino) ethylidene) amino) oxy) cyclopropanecarboxylic acid.

[0130]

[0131]

To a solution of (3S, 4R) -3- ( (Z) -2- ( (1- ( (benzhydryloxy) carbonyl) cyclopropoxy) imino) -2- (2- ( (tert-butoxycarbonyl) amino) thiazol-4-yl) acetamido) -2-oxo-4- ( (2-oxooxazolidin-3-yl) methyl) azetidine-1-sulfonic acid (1.10 g, 1.40 mmol) in DCM (1.5 mL) at 0℃, TFA (5.39 mL, 70.0 mmol) was added, and after 10 minutes, the ice bath was removed. Additional TFA (3.24 mL, 42.0 mmol) was added after 1 hr at rt and the solution was diluted with DCM and concentrated in vacuo after an additional 30 min. Optionally, anisole may be added to the TFA reaction to help reduce by-product formation, which may increase the yield of desired product in this step. The crude residue was purified by reverse phase prep HPLC (XSelect CSH, 30 x 100 mm, 5 μm, C18 column； ACN-water with 0.1％formic acid modifier, 60 mL/min) , affording the title compound (178 mg, 23％) as a white powder. LCMS: R _t ＝ 0.30 min, m/z ＝ 518.9 (M+1) Method 2m_acidic； ¹H NMR (400 MHz, DMSO-d ₆) δ 9.27 (d, J ＝ 9.0 Hz, 1H) 6.92 (s, 1H) 5.23 (dd, J ＝ 9.1, 5.7 Hz, 1H) 4.12-4.23 (m, 3H) 3.72-3.62 (m, 2H assumed； obscured by water) 3.61-3.52 (m, 1H assumed； obscured by water) 3.26 (dd, J ＝ 14.5, 5.9 Hz, 1H) 1.36 (s, 4H) . ¹H NMR (400 MHz, D ₂O) δ 7.23 (s, 1H) , 5.48 (d, J ＝ 5.8 Hz, 1H) , 4.71-4.65 (m, 1H) , 4.44 (t, J ＝ 8.2 Hz, 2H) , 3.89-3.73 (m, 3H) , 3.54 (dd, J ＝ 14.9, 4.9 Hz, 1H) , 1.65-1.56 (m, 2H) , 1.56-1.46 (m, 2H) . The product of this process is amorphous. Compound X can be crystallized from acetone, ethanol, citrate buffer at pH 3 (50 mM) , or acetate buffer at pH 4.5 (50 mM) , in addition to solvents discussed below.

PAPER

Bioorganic & Medicinal Chemistry Letters (2018), 28(4), 748-755.

https://www.sciencedirect.com/science/article/pii/S0960894X18300064

PATENT

WO 2019026004

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2019026004&tab=PCTDESCRIPTION

Over the past several decades, the frequency of antimicrobial resistance and its association with serious infectious diseases have increased at alarming rates. The increasing prevalence of resistance among nosocomial pathogens is particularly disconcerting. Of the over 2 million (hospital-acquired) infections occurring each year in the United States, 50 to 60% are caused by antimicrobial-resistant strains of bacteria. The high rate of resistance to commonly used antibacterial agents increases the morbidity, mortality, and costs associated with nosocomial infections. In the United States, nosocomial infections are thought to contribute to or cause more than 77,000 deaths per year and cost approximately $5 to $10 billion annually.

Important causes of Gram-negative resistance include extended-spectrum 13- lactamases (ESBLs), serine carbapenemases (KPCs) and metallo-13-lactamases (for example NDM-1 ) in Klebsiella pneumoniae, Escherichia coli, and Proteus mirabilis, high-level third-generation cephalosporin (AmpC) 13-lactamase resistance among Enterobacter species and Citrobacter freundii, and multidrug-resistance genes observed in Pseudomonas, Acinetobacter, and Stenotrophomonas. The problem of antibacterial resistance is compounded by the existence of bacterial strains resistant to multiple antibacterials. For example, Klebsiella pneumonia harboring NDM-1 metallo-13- lactamase carries frequently additional serine-13-lactamases on the same plasmid that carries the NDM-1 .

Thus there is a need for new antibacterials, particularly antibacterial compounds that are effective against existing drug-resistant microbes, or are less susceptible to development of new bacterial resistance. Monobactam antibiotic, which is referred to herein as Compound X, is primarily effective against Gram-negative bacteria, including strains that show resistance to other monobactams.

The present invention relates to a process for the preparation of monobactam antibiotic Compound X and intermediates thereof.

More particularly, the present invention relates to a process for the preparation of Compound X

Compound X

also referred to as 1 -(((Z)-(1 -(2-aminothiazol-4-yl)-2-oxo-2-(((3S,4R)-2-oxo-4-((2-oxooxazolidin-3-yl)methyl)-1 -sulfoazetidin-3-yl)amino)ethylidene)amino)oxy)cyclopropanecarboxylic acid, or a salt thereof, or a solvate including hydrate thereof.

Patent application number PCT/US2015/02201 1 describes certain monobactam antibiotics. Compound X may be prepared using the method disclosed in PCT/US2015/02201 1 , in particular example 22, and in PCT/CN2016/099482.

A drawback from these processes is that they exhibit a large number of process steps and intermediate nitrogen protection/deprotection steps, reducing the overall yield and efficiency. Furthermore, these processes require several chromatographic purification steps to be carried out in course of the processes. We have found that the preparation of Compound X, as previously prepared on a manufacturing scale, possesses a number of disadvantages, in particular poor handling characteristics.

It would thus be beneficial to develop alternative or improved processes for the production of Compound X that do not suffer from some or all of these disadvantages.

Compound ^x Compound ^x

Scheme 1

Preparation of Compound X from Intermediates 22 and 2A

Scheme 3

Examples

The Following examples are merely illustrative of the present disclosure and they should not be considered as limiting the scope of the disclosure in any way, as these examples and other equivalents thereof will become apparent to those skilled in the art in the light of the present disclosure, and the accompanying claims.

Synthesis of Compound 8 (R = benzyl)

1 .50kg oxazolidin-2-one (7b) was charged into the reactor. 7.50kg THF was charged and the stirring started. The mixture was cooled to 10~20°C. 2.18kg potassium fert-butoxide was charged intol 2.00kg THF and stirred to dissolve.

The potassium fert-butoxide solution was added dropwise into the reactor while maintaining the temperature at 10-20 °C. The reaction was stirred for 1 ~2hrs at 10-20 °C after the addition. The solution of 2.36kg methyl-2-chloroacetate (7a) in 3.00kg of THF was added to the reactor while maintaining the temperature at 10-20 °C. The reaction mixture was stirred for 16-18 h at 20-25 °C. The IPC (in process control) showed completion of the reaction. The mixture was centrifuged and the wet cake was washed with 7.50kg THF. The filtrate was concentrated and the crude 7 was provided as reddish brown liquid, which was used for the next step without further purification,

¹H NMR (400 MHz, CHLOROFORM- /) δ ppm 3.65 – 3.71 (m, 2 H) 3.74 (s, 3 H) 4.02 (s, 2 H) 4.34 – 4.45 (m, 2 H).

The dried reactor was exchanged with N2 three times. 3.71 kg LiHMDS solution in THF/Hep (1 M) and 1 .30kg THF were charged under nitrogen protection. The stirring was started and the solution was cooled to -70—60 °C. The solution of 0.71 kg benzyl acetate (6) in 5.20 kg THF was added dropwisely at -70— 60 °C, and the resulted mixture was stirred for 1 -1 .5 h after the addition. The solution of 0.65kg 7 in 3.90kg THF was added dropwise while maintaining the temperature at -70—60 °C, then stirred for 30-40 minutes. The reaction mixture was warmed to 20-25 °C and stirring was continued for 0.5-1 .0 h. IPC showed 6 was less than1 .0% (Otherwise, continue the reaction till IPC passes). The reaction mixture was poured into 13.65 kg aqueous citric acid below 10 °C. The mixture was stirred for 15-20 minutes after the addition. Phases were separated and the organic layer was collected. The aqueous layer was extracted with EA (6.50kg ^* 2). The organic layer was combined, washed by 6.50 kg 28% NaCI solution and dried with 0.65

kg anhydrous MgSC . The mixture was filtered and the wet cake was washed with 1 .30kg EA. The filtrate was concentrated under vacuum to provide crude 8. The crude 8 was stirred in 2.60 kg MTBE at 20-25 °C for 1 -1 .5 h. The mixture was cooled to 0-10 °C and stirred for 1 .5-2.0 h and filtered. The filter cake was washed with 0.65kg pre-cooled MTBE and dried under vacuum (<-0.096Mpa) at 20-25 °C for 12~16hrs till a constant weight to give 513 g of 8 as a white solid, Yield: 45%, HPLC purity 96.4%,¹ H NMR (400 MHz, CHLOROFORM-c δ ppm 3.48 – 3.55 (m, 1 H) 3.56 – 3.63 (m, 2 H) 3.66 – 3.74 (m, 1 H) 4.17 – 4.26 (m, 2 H) 4.31 – 4.44 (m, 2H) 5.12 – 5.24 (m, 2 H) 7.30 – 7.44 (m, 5 H).

Synthesis of Compound 9 (R = benzyl)

The dried reactor was charged with 3.75kg HOAc and 1 .50 kg 8. The stirring was started and the reaction mixture was cooled to 0-5 °C. 3.53kg aqueous NaN02 was added dropwise at 0-10 °C, and the reaction mixture was stirred for 15-30 minutes after the addition. IPC showed 8 was less than 0.2%. The reaction mixture was treated with 7.50kg EA and 7.50 kg water. Phases were separated and the organic layer was collected. The aqueous layer was extracted with EA (7.50kg ^* 2). The organic layers were combined, washed with 7.50 kg 28% NaCI solution, and concentrated under vacuum to provide crude 9. The crude 9 was slurried with 5.25 kg water at 10-20 °C for 3~4hrs, and filtered. The wet cake was washed with 1 .50kg water. The solid was dried under vacuum (<-0.096 Mpa) at 45-50 °C for 5-6 h till a constant weight to give 1 .44 Kg of 9, yield: 86.9%, HPLC purity 92.9%,¹H NMR (400 MHz, CHLOROFORM- /) δ ppm 3.60 – 3.76 (m, 2 H) 4.44 (t, J=8.07 Hz, 2 H) 4.60 (s, 2 H) 5.25 – 5.41 (m, 2 H) 7.30 – 7.43 (m, 5 H) 1 1 .62 (br s, 1 H).

Synthesis of Compound 9a (R = benzyl)

The dried reactor was charged with 0.58 kg Zn, 4.72kg (Βο Ο, 6.00 kg water, 1 .20 kg NH₄CI and 6.00kg THF. The reaction mixture was stirred and heated to 50-55 °C. The solution of 0.60 kg 9 in 4.20kg THF

was added dropwisely while maintaining the temperature at 50-55 °C. The reaction mixture was stirred for 0.5-1 .Ohrs after the addition. IPC showed 9 was less than 0.1 %. The reaction mixture was treated withl .50 kg ethyl acetate and stirred for 15-20 minutes. Phase was separated and the water layer was extracted by1 .50 kg ethyl acetate. The organic layers were combined, washed with 6.00 kg 28% NaCI solution and concentrated under vacuum to provide crude 9a. The crude 9a was stirred with 3.60kg^*2 n-heptane to remove excess (Βο Ο. The residue was purified by silica gel chromatography column eluted with ethyl acetate: Heptane= 1 :1 to provide crude 9a solution. The solution was concentrated under reduced pressure to obtain crude 9a. The crude 9a was slurried with 1 .80 kg MTBE for 2.0-3. Ohrs, filtered, and the wet cake was washed with MTBE. The solid was dried under vacuum (<-0.096 Mpa) at 50-55 °C for 16-18 h till a constant weight to give 392 g of 9a as a white solid, Yield: 51 %, HPLC purity 98.1 %,¹H NMR (400 MHz, DMSO-cfe) δ ppm 1.17 – 1 .57 (m, 9 H) 3.39 – 3.61 (m, 2 H) 4.20 – 4.45 (m, 3 H) 5.10 – 5.32 (m, 3 H) 5.75 (s, 1 H) 7.38 (br s, 5 H) 7.75 – 7.99 (m, 1 H).

Synthesis of compound (VII) (R = benzyl, X = CI)

9a VII

The dried reactor was charged with 13.0kg HCI in IPA and the stirring was started. 1 .33 kg 9a was charged in portions at 20-25 °C. The mixture was stirred at 20-25 °C for 3-4 h. IPC showed 9a was less than 0.1 %. The reaction solution was concentrated under vacuum 40-45 °C. The residue was treated with 21 .58kg MTBE at 20-25 °C for 3-4 h. The mixture was filtered and the wet cake was washed with 2.60kg MTBE. The solid was dried under vacuum (<-0.096 Mpa) at 45-50 °C for 5-6 h till a constant weight to give 1 .045 Kg of compound VII (R = benzyl, X = CI) as a yellow solid, Yield: 93.7%, HPLC purity 99.2%,¹ H NMR (400 MHz, DMSO-cfe) δ ppm 3.16 – 3.74 (m, 3 H) 4.10 – 4.35 (m, 4 H) 5.09 – 5.39 (m, 2 H) 7.27 – 7.60 (m, 5 H) 8.72 (br s, 2 H).

Synthesis of compound (Vile) (R = benzyl)

VII Vile

To an autoclave (3L) were added VII (R = benzyl, X = CI) (100 g, 304.2 mmol, 1 .0 equiv.), DCM (2650 g, 26.5 equiv., w/w) and (S-BINAP)RuCl2 (2.4 g, 3.04 mmol, 0.01 equiv.), successively. Air in the autoclave was replaced with N2 5 times. N2 in the autoclave was was replaced with H2 5 times. The solution was stirred with 250-260 r/min and H2 (2.1 ±0.1 MPa) at 40±5°C for 24 h. The reaction mixture was filtered, and the filter cake was washed with DCM (400 g, 4.0 equiv., w/w). The filter cake was slurried with IPA (785 g, 7.85 equiv., w/w) and H2O (40 g, 0.4 equiv., w/w) overnight (18-20 h). The mixture was filtered. The filter cake was washed with IPA (200 g, 2.0 equiv., w/w) and dried at 45±5°C overnight (18-20 h). Vile (R = benzyl) was obtained as off-white solid, 80.4 g, 79.9% yield, 95.5% purity, 97.6% de, >99.5% ee. ¹H NMR (400 MHz, DMSO-cfe) δ ppm 3.34-3.38 (m, 2 H) 3.50-3.52 (m, 1 H) 3.60-3.62 (m, 1 H) 4.18-4.24 (m, 4 H) 5.23 (s, 2H) 6.16 (s, 1 H) 7.32 (m, 5H) 8.74 (s, 1 H).

Alternative synthesis of compound 9a (R = benzyl)

Mg(OtBu)2

To a flask was added 5a (1 .88 g, 12.93 mmol), THF (40 mL), and CDI (2.20 g, 13.58 mmol) at 25 °C. The mixture was stirred for 3 h. To the reaction mixture was added 5b (2.00 g, 6.47 mmol), and Mg(OfBu)2 (2.21 g, 12.93 mmol). The reaction mixture was stirred at 25 °C for 24 h. The reaction mixture was concentrated under vacuum to remove most of the THF solvent. To the concentrated solution was added MTBE (40 mL), followed by addition of an aqueous solution of HCI (1 M, 60mL) to adjust to pH = 2-3. Two phases were separated, and the water phase was extracted with MTBE (20 mL). The combined organic phase was washed with aqueous NaHCC (5%, 50 mL) and brine (20%, 40 mL). The organic phase was concentrated to a weight of -19 g, and a lot of white solid was obtained in the concentration process. The suspension was cooled to 0 °C, and filtered. The filter cake was washed with cold MTBE (5 mL) and dried under vacuum to obtain product 9a (1 .6g, 63% yield).

Synthesis of compound (Vile) (R = benzyl, PG = Cbz)

Vile Vile

To a flask (5 L) were added Vile (R = benzyl) (140 g, 423.2 mmol, LOequiv.), H₂0 (1273 g, 9.09 equiv., w/w) and toluene (2206 g, 15.76 equiv., w/w). The solution was stirred and cooled to 0-5 °C with ice bath. Then NaHCOa (78.4 g, 933 mmol, 2.22 equiv.) was added and CbzCI (89.6 g, 527 mmol, 1 .24 equiv.) was dropped into the stirring solution, respectively. The solution was stirred at 30±5 °C overnight (18-20 h). Heptane (3612 g, 25.8 equiv., w/w) was added dropwise to the stirring solution over 1 h at 20-30 °C. The mixture was filtered. The filter cake was washed with heptane (280 g, 2.00 equiv., w/w) and MTBE (377 g, 2.69 equiv., w/w), respectively. The filter cake was dried at 45±5°C overnight (18-20 h). Vile (R = benzyl, PG = Cbz) was obtained as an off-white solid, 169.4 g, 93% yield, 96.7% purity, 98% de, >99.5% ee, ¹ H NMR (400 MHz, DMSO-cfe) δ ppm 3.23-3.24 (m, 1 H) 3.30 (m, 1 H) 3.51 -3.55 (m, 2 H) 3.99 (s, 1 H) 4.17-4.21 (m, 3 H) 5.02-5.03 (m, 2H) 5.12 (s, 2H) 5.46-5.48 (d, 1 H) 7.33-7.36 (m, 10H) 7.75-7.73 (d, 1 H).

Synthesis of compound (IV) (PG = Cbz)

Vile IV

Vile (R = benzyl) (220 g, 513.5 mmol, 1 .0 equiv.) was dissolved in THF (1464g, 6.65 equiv., w/w). The solution was filtered. The filter cake was washed with THF (488g, 2.22 equiv., w/w). The filtrate (Vile) was collected. To an autoclave (3L) were added the filtrate (Vile). The reactor was cooled down to -75 – -65 °C with dry-ice/EtOH bath, and bubbled with NH3 for not less than 4 h. Then the solution was stirred at 25±5 °C with NH3 (0.5-0.6 MPa) for 24 h. The autoclave was deflated to release NH3. The reaction solution was concentrated with a rotary evaporator to remove THF until the residue was around 440 g. The residue was slurried with EA (2200 g, 10 equiv., w/w) at 70±2 °C, then cooled to 25±5 °C and stirred for 16-18 h. The mixture was filtered. The filter cake was washed with EA (440 g). The filter cake was slurried with EA (1320 g, 6.00 equiv. w/w), and the temperature was raised to 70±2 °C, then cooled to 25±5 °C and stirred for 16-20 h. The mixture was filtered. The filter cake was washed with EA, and dried at 50±5 °C overnight (18-20 h). IV (PG = Cbz) was obtained as off-white solid, 141 g, 81 .5% yield, 99.1 % purity, >99.5% assay, ¹H NMR (400 MHz, DMSO-cfe) δ ppm 3.12 – 3.23 (m, 2 H) 3.31 (br s, 1 H) 3.56 (t, J=8.01 Hz, 2 H) 3.88 (quin, J=6.02 Hz, 1 H) 3.93 – 4.03 (m, 1 H) 4.20 (t, J=8.01 Hz, 2 H) 5.02 (s, 2 H) 5.27 (d, J=5.87 Hz, 1 H) 7.12 (s, 1 H) 7.22 – 7.45 (m, 5 H).

Synthesis of compound (III) (PG = Cbz, LG = S0₂CH₃)

IV III

To a flask was added IV (PG = Cbz) (14.00 g, 41 .50 mmol, 1 .00 equiv), and dry 1 , 2-dimethoxyethane (300 mL) under N2. The mixture was stirred at -5°C ~ 0°C for 1 h to obtain a good suspension. MsCI (7.89 g, 68.89 mmol, 5.33 mL, 1 .66 eq) in 1 , 2-dimethoxyethane (20.00 mL) was added dropwise during 30 min, and Et3N (12.60 g, 124.50 mmol, 17.26 mL, 3.00 eq) in 1 , 2-dimethoxyethane (20.00 mL) was added dropwise during 30 min side to side. The reaction mixture was stirred for additional 5 min at -5°C ~ 0°C, and was quenched with water (6 mL). The reaction mixture was concentrated to remove DME. The solid was slurried in water (250 mL) and MTBE (125 mL) for 1 h. The solid was collected by filtration, and then slurried in water (250 mL) for 1 hr. The solid was collected by filtration, and washed with water (25 mL) to give white solid. The solid was slurried in EA (150 mL) and dried in vacuum at 60°C for 24 h to give III (PG = Cbz, LG = SO2CH3) (15.00 g, 36.1 1 mmol, 87.01 % yield), ¹H NMR (400 MHz, DMSO-cfe) δ ppm 3.17 (s, 3 H) 3.26 (br d, J=15.04 Hz, 1 H) 3.47 – 3.57 (m, 1 H) 3.64 (br d, J=6.36 Hz, 2 H) 4.22 (br dd, J=17.79, 8.50 Hz, 2 H) 4.50 (br s, 1 H) 4.95 – 5.17 (m, 3 H) 7.21 – 7.56 (m, 5H) 7.43 (s, 1 H) 7.63 – 7.89 (m, 2 H).

Synthesis of compound II (PG = Cbz, LG = SO2CH3, M⁺ = NBu₄⁺)

O OMs o CISO3H, 2-picoline – ° O ?yO

HN Bu₄NHS0₄^< NHCbz

“Cbz

III II

To a flask was added 2-picoline (1 1 .50 g, 12.23 mL) and DMF (10 mL). The solution was cooled to 5 ^SC, followed by slow addition of chlorosulfonic acid (7.20 g, 4.14 mL). The temperature was increased to 20 ^SC. Ill (PG = Cbz, LG = SO2CH3) (5.13 g, 12.35 mmol) was added to the reaction mixture. The reaction mixture was heated to 42 ^SC for 18h. IPC (in process control) showed complete conversion of starting material. The reaction was cooled to 20 ^SC and dropwise added to a solution of tetrabutylammonium hydrogen sulfate (4.6 g, 13.6 mmol) in the mixed solvents of dichloromethane (100 mL) and water (100 mL) at 5^SC. The phases were separated and the water phase was extracted with dichloromethane (2^*50mL). The combined organic phase was washed with water (5^*100mL). The organic phase was concentrated to dryness and purified by column chromatography (dichloromethane/methanol = 15/1 v/v) to afford II (PG = Cbz, LG = SO2CH3, M⁺ = NBii4⁺) (8.4 g, 92.30%), ¹ H NMR (400 MHz, CHLOROFORM-c/) δ ppm 0.99 (t, J=7.34 Hz, 12 H) 1 .36 – 1 .50 (m, 8 H) 1 .54 – 1 .76 (m, 8 H) 3.15 (br d, J=8.31 Hz, 2 H) 3.21 – 3.35 (m, 8 H) 3.47 (br dd, J=14.73, 7.27 Hz, 1 H) 3.54 – 3.65 (m, 1 H) 3.67 – 3.81 (m, 2 H) 4.17 – 4.32 (m, 1 H) 4.39 – 4.62 (m, 1 H) 4.74 (br s, 1 H) 5.1 1 (s, 3 H) 5.32 – 5.50 (m, 1 H) 6.47 (br s, 1 H) 7.29 – 7.47 (m, 5 H) 8.69 – 8.94 (m, 1 H).

Synthesis of compound (IA)

A solution of II (PG = Cbz, LG = SO2CH3, M⁺ = NBu₄⁺) (4.0 g) in dichloromethane (38 mL) was pumped to tube A at rate of 2.0844 mL/min, and a solution of KHCO3 (3.0 g) in water (100 mL) was pumped to tube B at a rate of 1 .4156 mL/min side to side. These two streams were mixed in a cross-mixer then flowed to a tube coil that was placed in an oil bath at 100 °C. The residence time of the mixed stream in the coil was 2 min. The reaction mixture flowed through a back-pressure regulator that was set at ~ 7 bars, and was collected to a beaker. After completion of the collection, two phases was separated. The organic phase was concentrated to dryness. The residue was slurried in ethyl acetate (5 mL). The solid was filtered and the filter cake was dried to give IA (2.6 g, 75%),

¹H NMR (400 MHz, CHLOROFORM-c/) δ ppm 1.00 (t, J=7.27 Hz, 12 H) 1 .42 (sxt, J=7.31 Hz, 8 H) 1 .62 (quin, J=7.83 Hz, 8 H) 3.13 – 3.39 (m, 8 H) 3.54 – 3.69 (m, 2 H) 3.81 (dd, J=14.98, 2.51 Hz, 1 H) 3.96 – 4.13 (m, 1 H) 4.22 – 4.47 (m, 3 H) 4.99 – 5.23 (m, 3 H) 6.42 (br d, J=9.29 Hz, 1 H) 7.26 – 7.44 (m, 5 H).

Synthesis of compound 2A

Step 1

To a stirring solution of compound 16b (2 g, 10.14mmol, 1 .0 eq) in DMF (20 ml_) was added CS2CO3 (5.29g, 16.22 mmol, 1 .6 eq), then the resulting solution was stirred at room temperature for 10mins, then compound 16a (5.27g, 20.28mmol, 2eq) was added dropwise to the mixture for 2 minutes, then the resulting solution was stirred for another 2 hours. TLC showed the starting material was consumed completely. The mixture was added with water (60mL) and extracted with MTBE (20mL^*3). The combined organic layers were dried over anhydrous sodium sulfate and concentrated. The crude was slurried in heptane to give 1 .65 g 16 as a white solid (Yield: 57%), ¹H NMR (400 MHz, DMSO-cfe) δ ppm 7.48-7.28 (m, 10 H), 5.00-4.96 (t, J=6.0 Hz, 1 H), 3.81 (s, 3H), 3.44-3.42 (m, 2H), 2.40-2.37 (m, 2H).

Compound 16 (1 g, 2.66mmol, 1 eq) was dissolved in THF (20mL) under Nitrogen, and cooled to -40 °C. NaHMDS (1 .6mL, 2.0M THF solution, 1 .2 eq) was added dropwise. The reaction was stirred for 1 h at -40 °C. HPLC indicated the reaction was finished. The reaction was quenched with 10% Citric acid, extracted with MTBE (25 ml_ x 2). The combined organic layers were washed with brine (30 ml_), dried with Na2S04, filtered and concentrated to give 17 as a yellow solid, which was used for the next step without purification (assay yield: 65%); ¹H NMR (400 MHz, DMSO-cfe) δ ppm 7.27-7.13 (m, 10 H), 3.46 (s, 3H), 1 .21 -1 .17(dd, J=7.2, 10.4 Hz, 2H ); 1 .14-1 .1 1 (dd, J=7.2, 10.4 Hz, 2H).

Step 3

Compound 17 (100 mg) was dissolved in methanol (5 mL) and 2.0 M HCI IPAC solution (5 mL). The solution was heated at 45 °C for 3 days. HPLC indicated the reaction was finished. The reaction was cooled to room temperature and was diluted with 10 mL water. The reaction mixture was washed with MTBE (10 mL x 2), organic layer was discarded and the aqueous layer was concentrated to give compound 2A HCI (32 mg, 62% yield), ¹ H NMR (400 MHz, DMSO-cfe) δ ppm 3.80-3.44 (br, 4H), 1 .56 (s, 2H), 1 .38 (s, 2H).

Step 4

To a solution of 2A HCI (0.70 g, 4.57 mmol) in methanol (5 mL) was added triethylamine (1 .26 mL, 9.14 mmol) at room temperature. The solution was stirred for 20 min, and the solvent was removed under vacuum. To the residue was added IPAC (10 mL) leading to precipitation. The solid was filtered, and the filtrate was concentrated to provide 2A (0.50g, 94% yield) containing ca. 6 wt% Et3N-HCI.

Synthesis of Compound X from compound of formula (I), (IA)

Compound ^x

To a flask was charged 21 (1 .00 g, 68.43 wt%, 2.50 mmol) and DMF (10 mL). The suspension was cooled to -20 °C, to which was added diphenylphosphinic chloride (0.52 mL, 2.75 mmol). The solution was stirred at -20 °C for 30 min, followed by addition of a mixed solution of (IA) (1 .52g, 3.00 mmol) and triethylamine (0.52 mL, 3.76 mmol) in DMF (2mL). The reaction mixture was stirred at 20 °C for 20 h, followed by addition of MTBE (20 mL). The reaction mixture was adjusted to pH = 2-3 using aqueous HCI solution (37%). To the mixture was added isopropanol (100 mL). The resulting mixture was stirred for 4 h to obtain a suspension. The suspension was filtered and the filter cake was dried under vacuum to afford crude 22 (1 .17 g). The crude 22 was slurried in a combined solvent of THF/H2O (= 12 mL / 3mL), and filtered to afford 22 (0.744 g, 75 wt% by Q-NMR, 53.3% yield). ¹H NMR (400 MHz, DMSO-cfe) δ ppm 3.47 – 3.55 (m, 2 H) 3.59 – 3.63 (m, 2 H) 4.13 – 4.21 (m, 3 H ) 5.05 (dd, J=8.8, 5.6 Hz, 1 H) 8.22 (s, 1 H) 9.73 (d, J=8.7 Hz, 1 H).

To a suspension of 22 (580 mg, 75 wt%, 1 .037 mmol) in DMAC (1 .5 mL) was added 2A (214.3 mg, 85 wt%, 1 .556 mmol). The reaction was stirred at 25 °C for 3 days, and in process control showed 22, Compound X = 4/96, and Z/E = 91 /9. the mixture was slowly added into 15ml acetone to precipitate yellowish solid. The reaction mixture was filtered to afford Compound X (0.7 g, 34 wt% by QNMR, 44% yield).

Synthesis of compound 3 (R² = CH(Ph)₂)

^R2 = CH(Ph)₂

2-(2-aminothiazol-4-yl)-2-oxoacetic acid (Y) (10.00 g, 47.93 mmol) and compound W (R² = CH(Ph)₂) (13.31 g, 46.98 mmol) were suspended in DMAC (40 mL), followed by addition of triethylamine (5.01 mL, 35.95 mmol). The reaction mixture was stirred at 20 °C for 5 h. HPLC showed completion of the reaction, and Z/E

= 97/3. To the reaction mixture was added water (120 mL) with stirring. The mixture was stirred for 20 min to obtain a suspension. The suspension was filtered and the filter cake was washed with water (50 mL).

The filter cake was slurried in a combined solvent of THF/ethyl acetate (50 mL / 50 mL) at 60 °C and cooled to 20 °C. The solid was filtered and dried at 50 °C for 3 h to get 3 (R² = CH(Ph)₂) (19.5 g, 88% yield). ¹H

NMR (400 MHz, DMSO-cfe) δ ppm 1.37 -1 .42 (m, 2 H) 1 .44 – 1 .49 (m, 2 H) 6.87 (s, 1 H) 6.94 (s, 1 H) 7.22

– 7.30 (m, 6 H) 7.45 – 7.49 (m, 4 H).

Alternative Synthesis of Compound X from compound of formula (I), (IA)

Compound ^x

IA (40.14 g, 62.63 mmol) was dissolved in methanol (200 ml_), followed by addition of Pd/C (10%, 1 .1 g). The reaction mixture was maintained under hydrogen atmosphere (1 -2 bar) at 20 °C for 24 h. In process control showed completion of the reaction. The reaction mixture was filtered. The filtrate was concentrated to give an oil of IB (M⁺ = NBu₄⁺) (58.20 g, 55 wt% by Q-NMR, 100% yield). ¹ H NMR (400 MHz, DMSO-cfe) δ ppm 0.93 (t, J=7.3 Hz, 12 H) 1 .23 – 1 .36 (m, 8 H) 1 .57 (m, 8 H) 2.99 – 3.28 (m, 8 H) 3.37 (dd, J=14.3, 7.5 Hz, 1 H) 3.65 – 3.70 (m, 3 H) 3.84 – 3.88 (m, 1 H) 4.08 (d, J=5.6 Hz, 1 H) 4.18 – 4.22 (m, 2 H).

3 (R² = CH(Ph)₂) (0.95 g, 2.17 mmol) was dissolved in THF (20 ml_). To the solution was added /V-methyl morpholine (0.77 g, 7.60 mmol) and 2-chloro-4,6-dimethoxy-1 ,3,5-triazine (0.57 g, 3.26 mmol). The reaction mixture was stirred at 20 °C for 1 h followed by addition of IB (M⁺ = NBu ⁺) (2.70 g, 48.98 wt%, 2.61 mmol). The reaction was stirred at 20 °C for 5 h. In process control showed completion of the reaction. To the reaction mixture was added ethyl acetate (20 ml_). The organic phase was washed with brine (10 ml_). Solvent was removed. Acetone (40ml) was added to dissolve residue. TFA (1 .24 g, 10.86 mmol) dissolved in acetone (3 ml) was added slowly. The white solid was filtered and washed by acetone (10 ml) two times. Dried at 40 °C for 5h to get compound 4 (R² = CH(Ph)₂). ¹ H NMR (400 MHz, DMSO-cfe) δ ppm 1 .49 – 1 .55 (m, 4 H) 3.27 (dd, J=14.4, 6.2 Hz, 1 H) 3.49 – 3.65 (m, 2 H) 3.71 (dd, J=14.4, 6.2 Hz, 1 H) 4.04 – 4.10 (m, 1 H) 4.07 (dd, J=16.0, 8.6 Hz, 1 H) 4.17 (dd, J=1 1 .8, 6.0 Hz, 1 H) 5.28 (dd, J=9.0, 5.7 Hz, 1 H) 6.88 (s, 1 H) 7.03 (s, 1 H) 7.18 – 7.32 (m, 6 H) 7.43 (m, 4 H) 9.45 (d, J=9.0 Hz, 1 H).

Crude 4 (R² = CH(Ph)₂) (2.13 g) was dissolved in dichloromethane (20 ml_). The solution was cooled to 0 °C. To the solution was added anisole (0.68 ml_, 6.24 mmol) and trifluoroacetic acid (2.16 ml_, 28.08 mmol). The reaction was warmed to 20 °C, and stirred for 15 h. In process control showed completion of the

reaction. The aqueous phase was separated and added to acetone (40 mL) to obtain a suspension. The suspension was filtered to afford Compound X (0.98 g, 54.5% yield over two steps). ¹ H NMR (400 MHz, DMSO-c/e) δ ppm 1.40 (m, 4 H) 3.26 (dd, J=14.4, 6.0 Hz, 1 H) 3.54 – 3.69 (m, 3 H) 4.14 – 4.21 (m, 3 H) 5.25 (dd, J= 8.9, 5.7 Hz, 1 H) 7.02 (s, 1 H) 9.38 (d, J=9.0 Hz, 1 H).

REF

Synthesis and optimization of novel monobactams with activity against carbapenem-resistant Enterobacteriaceae – Identification of LYS228
57th Intersci Conf Antimicrob Agents Chemother (ICAAC) (June 1-5, New Orleans) 2017, Abst SATURDAY-297

//////////////LYS228, LYS 228, BOS-228, LYS-228, monobactam, Novartis, phase II, Boston Pharmaceuticals, complicated urinary tract infection, complicated intraabdominal infections, fast track, Qualified Infectious Disease Product, QIDP,

Nc1nc(cs1)\C(=N\OC2(CC2)C(=O)O)\C(=O)N[C@H]3[C@@H](CN4CCOC4=O)N(C3=O)S(=O)(=O)O

Selinexor

July 16, 2019 9:07 am / Leave a comment

Selinexor

セリネクソル

KPT-330

UNII-31TZ62FO8F

(Z)-3-[3-[3,5-bis(trifluoromethyl)phenyl]-1,2,4-triazol-1-yl]-N‘-pyrazin-2-ylprop-2-enehydrazide

Formula	C17H11F6N7O
CAS	1393477-72-9
Mol weight	443.306

FDA, APPROVED 2019/7/3, Xpovio

CAS : 1393477-72-9 (free base) 1421923-86-5 (E-isomer) 1621865-82-4 (E-isomer) Unknown (HCl)

Treatment of cancer, Antineoplastic, Nuclear export inhibitor

Selinexor (INN, trade name Xpovio; codenamed KPT-330) is a selective inhibitor of nuclear export used as an anti-cancer drug. It works by quasi-irreversibly binding to exportin 1 and thus blocking the transport of several proteins involved in cancer-cell growth from the cell nucleus to the cytoplasm, which ultimately arrests the cell cycle and leads to apoptosis.^[1] It is the first drug with this mechanism of action.^[2]^[3]

Selinexor was granted accelerated approval by the U.S. Food and Drug Administration in July 2019, for use as a drug of last resort in people with multiple myeloma. In clinical trials, it was associated with a high incidence of severe side effects, including low platelet counts and low blood sodium levels.^[3]^[4]

Selinexor is an orally available, small molecule inhibitor of CRM1 (chromosome region maintenance 1 protein, exportin 1 or XPO1), with potential antineoplastic activity. Selinexor modifies the essential CRM1-cargo binding residue cysteine-528, thereby irreversibly inactivates CRM1-mediated nuclear export of cargo proteins such as tumor suppressor proteins (TSPs), including p53, p21, BRCA1/2, pRB, FOXO, and other growth regulatory proteins. As a result, this agent, via the approach of selective inhibition of nuclear export (SINE), restores endogenous tumor suppressing processes to selectively eliminate tumor cells while sparing normal cells. CRM1, the major export factor for proteins from the nucleus to the cytoplasm, is overexpressed in a variety of cancer cell types.

Selinexor has been used in trials studying the treatment of AML, Glioma, Sarcoma, Leukemia, and Advanced, among others.

Selinexor, also known as KPT-330, is an orally bioavailable, potent and selective XPO1/CRM1 Inhibitor. Selinexor is effective in acquired resistance to ibrutinib and synergizes with ibrutinib in chronic lymphocytic leukemia. Selinexor potentiates the antitumor activity of gemcitabine in human pancreatic cancer through inhibition of tumor growth, depletion of the antiapoptotic proteins, and induction of apoptosis. Selinexor has strong activity against primary AML cells while sparing normal stem and progenitor cells.

SYN

Medical uses

Selinexor is restricted for use in combination with the steroid dexamethasone in people with relapsed or refractory multiple myelomawhich has failed to respond to at least four or five other therapies (so-called “quad-refractory” or “penta-refractory” myeloma),^[5] for whom no other treatment options are available.^[3]^[4] It is the first drug to be approved for this indication.^[6]

Adverse effects

In the clinical study used to support FDA approval, selinexor was associated with high rates of pancytopenia, including leukopenia(28%), neutropenia (34%, severe in 21%), thrombocytopenia (74%, severe in 61% of patients), and anemia (59%).^[4]^[7] The most common non-hematological side effects were gastrointestinal reactions (nausea, anorexia, vomiting, and diarrhea), hyponatremia (low blood sodium levels, occurring in up to 40% of patients), and fatigue.^[7]^[8] More than half of all patients who received the drug developed infections, including fatal cases of sepsis.^[7] However, these data are from an open-label trial, and thus cannot be compared to placebo or directly attributed to treatment.

Mechanism of action

Schematic illustration of the Ran cycle of nuclear transport. Selinexor inhibits this process at the nuclear export receptor (upper right).

Like other so-called selective inhibitors of nuclear export (SINEs), selinexor works by binding to exportin 1 (also known as CRM1). CRM1 is a karyopherin which performs nuclear transport of several proteins, including tumor suppressors, oncogenes, and proteins involved in governing cell growth, from the cell nucleus to the cytoplasm; it is often overexpressed and its function misregulated in several types of cancer.^[1] By restoring nuclear transport of these proteins to normal, SINEs lead to a buildup of tumor suppressors in the nucleus of malignant cells and reduce levels of oncogene products which drive cell proliferation. This ultimately leads to cell cycle arrest and death of cancer cells by apoptosis.^[1]^[2]^[7] In vitro, this effect appeared to spare normal (non-malignant) cells.^[1]^[8]

Because CRM1 is a pleiotropic gene, inhibiting it affects many different systems in the body, which explains the high incidence of adverse reactions to selinexor.^[2] Thrombocytopenia, for example, is a mechanistic and dose-dependent effect, occurring because selinexor causes a buildup of the transcription factor STAT3 in the nucleus of hematopoietic stem cells, preventing their differentiation into mature megakaryocytes (platelet-producing cells) and thus slowing production of new platelets.^[2]

Chemistry

Selinexor is a fully synthetic small-molecule compound, developed by means of a structure-based drug design process known as induced-fit docking. It binds to a cysteine residue in the nuclear export signal groove of exportin 1. Although this bond is covalent, it is not irreversible.^[1]

History

Selinexor was developed by Karyopharm Therapeutics of Newton, Massachusetts, a pharmaceutical company devoted entirely to the development of drugs that target nuclear transport. It was approved by the FDA on July 3, 2019, on the basis of a single uncontrolled clinical trial. The decision was controversial, and overruled the previous recommendation of an FDA Advisory Panel which had voted 8–5 against approving the drug, due to concerns about efficacy and toxicity.^[3]

Research

Under the codename KPT-330, selinexor was tested in several preclinical animal models of cancer, including pancreatic cancer, breast cancer, non-small-cell lung cancer, lymphomas, and acute and chronic leukemias.^[9] In humans, early clinical trials (phase I) have been conducted in non-Hodgkin lymphoma, blast crisis, and a wide range of advanced or refractory solid tumors, including colon cancer, head and neck cancer, melanoma, ovarian cancer, and prostate cancer.^[9] Compassionate use in patients with acute myeloid leukemia has also been reported.^[9]

The pivotal clinical trial which served to support approval of selinexor for people with relapsed/refractory multiple myeloma was an open-label study of 122 patients known as the STORM trial.^[7] In all of the enrolled patients, selinexor was used as fifth-line or sixth-line therapy after conventional chemotherapy, targeted therapy with bortezomib, carfilzomib, lenalidomide, pomalidomide, and a monoclonal antibody (daratumumab or isatuximab)^[5]; nearly all had also undergone hematopoietic stem cell transplantation to no effect.^[7] The overall response rate was 25%, and no patients had a complete response.^[7] However, the response rate was higher in patients with high-risk myeloma (cytogenetic abnormalities associated with a worse prognosis).^[5] The median time to progression was 2.3 months overall and 5 months in patients who responded to the drug.^[2]

As of 2019, phase I/II and III trials are ongoing,^[3]^[9] including the use of selinexor in other cancers and in combinations with other drugs used for multiple myeloma.^[2]

PATENT

WO 2013019561

WO 2013019548

US 9079865

PATENT

WO 2016025904 A

https://patents.google.com/patent/WO2016025904A1/tr

International Publication No. WO 2013/019548 describes a series of compounds that are indicated to have inhibitory activity against chromosomal region maintenance 1 (CRM1, also referred to as exportin 1 or XPO1) and to be useful in the treatment of disorders associated with CRM1 activity, such as cancer. (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1H-1,2,4-triazol-1-yl)-N’-(pyrazin-2-yl)acrylohydrazide (also referred to as selinexor) is one of the compounds disclosed in International Publication No. WO 2013/019548. Selinexor has the chemical structure shown in Structural Formula I:

Example 1. Preparation of Selinexor Lot No.1305365 (Form A).

[00274] Selinexor for Lot No. 1305365 was made in accordance with the following reaction scheme:

[00275] A solution of propane phosphonic acid anhydride (T3P^®, 50% in ethyl acetate, 35Kg) in THF (24.6Kg) was cooled to about -40 °C. To this solution was added a solution of KG1 (13.8Kg) and diisopropylethylamine (12.4Kg) in tetrahydrofuran (THF, 24.6Kg). The resulting mixture was stirred at about -40°C for approximately 2.5 hours.

[00276] In a separate vessel, KJ8 (4.80Kg) was mixed with THF (122.7Kg), and the resulting mixture cooled to about -20°C. The cold activated ester solution was then added to the KJ8 mixture with stirring, and the reaction was maintained at about -20°C. The mixture was warmed to about 5°C, water (138.1Kg) was added and the temperature adjusted to about 20°C. After agitating for about an hour, the lower phase was allowed to separate from the mixture and discarded. The upper layer was diluted with ethyl acetate (EtOAc). The organic phase was then washed three times with potassium phosphate dibasic solution (~150Kg), then with water (138.6Kg).

[00277] The resulting organic solution was concentrated under reduced pressure to 95L, EtOAc (186.6Kg) was added and the distillation repeated to a volume of 90L. Additional EtOAc (186.8Kg) was added and the distillation repeated a third time to a volume of 90L. The batch was filtered to clarify, further distilled to 70L, then heated to about 75°C, and slowly cooled to 0 to 5°C. The resulting slurry was filtered and the filter cake washed with a mixture of EtOAc (6.3Kg) and toluene (17.9Kg) before being dried in a vacuum oven to provide selinexor designated Lot No. 1305365 (Form A).

Example 2. Preparation of Selinexor Lot No.1341-AK-109-2 (Form A).

[00278] The acetonitrile solvate of selinexor was prepared in accordance with Example 6.

[00279] The acetonitrile solvate of selinexor (2.7g) was suspended in a mixture of isopropanol (IPA, 8mL) and water (8mL), and the resulting mixture heated to 65 to 70 °C to effect dissolution. The solution was cooled to 45 °C, and water (28mL) was added over 15 minutes, maintaining the temperature between 40 and 45 °C. The slurry was cooled to 20 to 25 °C over an hour, then further cooled to 0 to 5 °C and held at that temperature for 30 minutes before being filtered. The filter cake was washed with 20% v/v IPA in water and the product dried under suction overnight, then in vacuo (40°C).

Example 3. Preparation of SelinexorSelinexorSelinexor Lot No. PC-14-005 (Form A).

[00280] The acetonitrile solvate of selinexor (Form D) was prepared in accordance with the procedure described in Example 6.

[00281] The acetonitrile solvate of selinexor (1.07Kg) was suspended in a mixture of IPA (2.52Kg) and water (3.2Kg) and the mixture heated to 70 to 75 °C to dissolve. The temperature was then adjusted to 40 to 45 °C and held at that temperature for 30 minutes. Water (10.7Kg) was added while maintaining the temperature at 40 to 45 °C, then the batch was cooled to 20 to 25 °C and agitated at that temperature for 4 hours before being further cooled to 0 to 5 °C. After a further hour of agitation, the slurry was filtered and the filter cake washed with a cold mixture of IPA (0.84Kg) and water (4.28Kg) before being dried.

Example 4. Preparation of SelinexorSelinexorSelinexor Lot No. PC-14-009 (Form A).

[00282] The acetonitrile solvate of selinexor (Form D) was prepared in accordance with the procedure described in Example 6.

[00283] The acetonitrile solvate of selinexor (1.5Kg) was suspended in IPA (3.6Kg) and water (4.5Kg) and warmed to 37 to 42 °C with gentle agitation. The suspension was agitated at that temperature for 4 hours, and was then cooled to 15 to 20 °C over 1 hour. Water (15.1Kg) was added, maintaining the temperature, then the agitation was continued for 1 hour and the batch was filtered. The filter cake was washed with a mixture of IPA (1.2Kg) and water (6Kg), then dried under a flow of nitrogen.

Example 5. Preparation of Selinexor Lot Nos.1339-BS-142-1, 1339-BS-142-2 and PC-14-008 (Form A).

[00284] A reactor, under nitrogen, was charged with KG1 (1Kg, 1.0 Eq), KJ8 (0.439 Kg, 1.4 Eq) and MeTHF (7L, 7 parts with respect to KG1). Diisopropylethylamine (0.902Kg, 2.45 Eq with respect to KG1) was added to the reaction mixture at -20 °C to -25 °C with a MeTHF rinse. To the reaction mixture, 50% T3P® in ethyl acetate (2.174Kg, 1.2 Eq with respect to KG1) was then charged, maintaining the temperature at -20 °C to -25 °C with a MeTHF rinse. After the completion of the addition, the reaction mixture was stirred briefly

and then warmed to 20 °C to 25 °C. Upon completion, the reaction mixture was washed first with water (5L, 5 parts with respect to KG1) and then with dilute brine (5L, 5 parts with respect to KG1). The organic layer was concentrated by vacuum distillation to a volume of 5 L (5 parts with respect to KG1), diluted with acetonitrile (15L, 15 parts with respect to KG1) at approximately 40 °C and concentrated again (5L, 5 parts with respect to KG1). After solvent exchange to acetonitrile, the reaction mixture was then heated to approximately 60 °C to obtain a clear solution. The reaction mixture was then cooled slowly to 0-5 °C, held briefly and filtered. The filter cake was washed with cold acetonitrile (2L, 5 parts with respect to KG1) and the filter cake was then dried under a stream of nitrogen to provide the acetonitrile solvate of selinexor (Form D) as a slightly off-white solid.

[00285] Form D of selinexor (0.9Kg) was suspended in IPA (2.1Kg, 2.7L, 3 parts with respect to Form D) and water (2.7Kg, 2.7L, 3 parts with respect to Form D) and warmed to approximately 40 °C. The resulting suspension was agitated for about 4 hours, selinexor, cooled to approximately 20 °C, and diluted with additional water (9Kg, 10 parts with respect to Form D). The mixture was stirred for a further 4-6 hours, then filtered, and the cake washed with a mixture of 20% IPA and water (4.5L, 5 parts with respect to Form D). The filter cake was then dried under vacuum to provide selinexor designated Lot No. PC-14-008 as a white crystalline powder with a >99.5% a/a UPLC purity (a/a=area to area of all peaks; UPLC-ultra performance HPLC).

Example 6. Preparation of Selinexor Lot No.1405463 (Form A).

[00286] Selinexor Lot No. 1405463 was prepared in accordance with the following reaction scheme:

[00287] A reactor was charged with KG1 (15.8Kg), KJ8 (6.9Kg) and MeTHF (90Kg). Diisopropylethylamine (14.2Kg) was added to the reaction mixture over approximately 35 minutes at about -20 °C. Following the addition of the diisopropylethylamine, T3P® (50%

solution in EtAOc, 34.4Kg) was added maintaining the temperature at -20 °C. The mixture stirred to complete the reaction first at -20 °C, then at ambient temperature.

[00288] Upon completion of the reaction, water (79Kg) was added over about 1 hour. The layers were separated and the organic layer was washed with a mixture of water (55Kg) and brine (18Kg), The mixture was filtered, and the methyl-THF/ethyl acetate in the mixture distillatively replaced with acetonitrile (volume of approximately 220L). The mixture was warmed to dissolve the solids, then slowly cooled to 0 to 5 °C before being filtered. The filter cake was washed with acetonitrile to provide the acetonitrile solvate of

selinexorSelinexorSelinexor (Form D).

[00289] The acetonitrile solvate of selinexorSelinexorSelinexor was dried, then mixed with isopropanol (23Kg) and water (55Kg). The slurry was warmed to about 38 °C and held at that temperature for approximately 4 hours before being cooled to 15 to 20 °C. Water (182Kg) was added. After a further 5 hours of agitation, the mixture was filtered and the filter cake washed with a mixture of isopropanol (14Kg) and water (73Kg), before being dried under vacuum (45 °C). The dried product was packaged to provide

selinexorSelinexorSelinexor Lot No. 1405463 (Form A).

Example 7. Polymorphism Studies of Selinexor.

[00290] A comprehensive polymorphism assessment of selinexor was performed in a range of different solvents, solvent mixtures and under a number of experimental conditions based on the solubility of selinexor. Three anhydrous polymorphs of

selinexorSelinexorSelinexor were observed by XRPD investigation, designated Form A, Form B and Form C. Form A is a highly crystalline, high-melting form, having a melting point of 177 °C, and was observed to be stable from a physico-chemical point of view when exposed for 4 weeks to 25 °C/97% relative humidity (RH) and to 40 °C/75% RH. A solvated form of selinexor was also observed in acetonitrile, designated Form D. A competitive slurry experiment confirmed Form A as the stable anhydrous form under the conditions investigated, except in acetonitrile, in which solvate formation was observed. It was further found that in acetonitrile, below 50 °C, only Form D is observed, at 50 °C both Form A and Form D are observed, and at 55 °C, Form A is observed .

PATENT

CN 106831731

https://patents.google.com/patent/CN106831731A/en

Selinexor is an orally bioavailable selective nuclear export inhibitors, 2012 for the first time in clinical, so far carried out a total of 21 trials, indications include chronic myelogenous leukemia, acute myelogenous leukemia, acute lymphatic leukemia, prostate cancer, melanoma, non-small cell lung cancer, glioma, neuroblastoma into, gynecological cancer, diffuse large B-cell lymphoma, squamous cell carcinoma, colorectal cancer and the like. May 2014, FDA granted orphan drug designation Selinexor treatment of acute myeloid leukemia and diffuse large B-cell lymphoma, in June 2014, EMA is also granted orphan drug designation Selinexor treatment of both diseases. January 2015, received FDA orphan drug to treat multiple myeloma identified.

[0003] Currently, the synthesis process has been disclosed, the following reaction equation:

[0006] wherein the compound is 5 Selinexor drug.

[0007] In this method, however, easy to produce Intermediate 1-2 double bond is easily reversed when synthetically produced from trans impurities, in addition to more difficult to impact yield; Intermediate 3 Intermediate 4 Synthesis APIs 5 when required ultra-low temperature, and the product was purified by column required, only a yield of 20%.

SUMMARY

[0008] The object of the present invention to provide a novel compound Selinexor drug synthesis of 5, in order to solve technical problems.

[0009] – novel synthetic method of Se species I inexor drug, comprising the steps of:

Synthesis [0010] A, Compound 7

[0011] Compound 6, dichloromethane and ethyl acetate mixture, stirred and dissolved, compound 4, T3P (n-propyl phosphoric anhydride) and DIPEA (N, N- diisopropylethylamine) at a low temperature; the reaction was stirred for 25-35min at a low temperature, dichloromethane and water were added after the completion of the reaction, liquid separation, the organic phase was evaporated to dryness to give crude compound 7, crude without purification cast down;

[0012] B, Synthesis of Compound 8

[0013] the compound obtained in Step 7, and mixed sodium iodide acetic acid, warmed to 110-120 ° C, the reaction 2.5-3.5h; After completion of the reaction, the system cooled to room temperature, water and dichloromethane were added, stirred for 8 after -15min, standing layered organic phase was washed with saturated sodium bicarbonate and saturated sodium chloride, dried over anhydrous sodium sulfate and distilled to give crude compound 8, was dissolved in DMF (dimethyl fumarate) to give compound in DMF 8;

Synthesis [0014] C, of Compound 5

[0015] Compound 1, DBAC0 (triethylenediamine), the DMF mixed and dissolved with stirring, dropwise adding to the reaction system of the compound obtained in DMF step 8, after the addition was complete, stirring was continued for 3-4 hours; the reaction after completion, water and ethyl acetate were added to the system, the organic phase is evaporated to dryness and petroleum ether and recrystallized from ethyl acetate to give compound 5.

[0016] Preferably, said step A, the low temperature is 0-2 ° C.

[0017] Preferably, said step B in DMF, the crude compound 8 concentration of less than 1%.

[0018] The novel synthetic methods of the present invention Selinexor drug, the chemical equation is as follows:

〇

[0020] The present invention has the following advantages: novel synthetic method Selinexor drug of the present invention to overcome the conventional synthesis process, is easy to produce trans impurities, more difficult in addition, the influence the yield and the need for ultra-low temperature, and the product requires problems purified by column, the yield is very low, reducing the synthetic steps, increased yield, there is provided a new process for the synthesis of the drug Selinexor.

[0021] In addition to the above-described objects, features and advantages of the present invention as well as other objects, features and advantages. Below the invention will be described in further detail present.

Example 1

[0024] – novel synthetic method of Se species I inexor drug, comprising the steps of:

Synthesis [0025] A, Compound 7

[0026] 50ml three □ flask, 15ml of dichloromethane and 0.2g compound 6,15ml ethyl acetate, stirred and dissolved, was added 0.3g of compound 4 and 3gT3P, 0.75gDIPEA at 0 ° C; the system at 0 ° C the reaction was stirred for 30min, 50ml of dichloromethane and 30ml of water were added after the completion of the reaction, liquid separation, the organic phase was evaporated to dryness to give crude compound 7, crude without purification cast down;

[0027] B, Synthesis of Compound 8

[0028] 50ml three-necked flask, added the compound obtained in Step 7,40ml of glacial acetic acid and 1.38g of sodium iodide was heated to 115. (:, The reaction 3H; After completion of the reaction, cooled to room temperature system, the system will be transferred to 500ml flask, 50ml of water was added and IOOml dichloromethane, after stirring IOmin, standing separation, the organic phase was washed with saturated sodium bicarbonate and saturated washed with sodium chloride, dried over anhydrous sodium sulfate and distilled to give crude compound 8, was dissolved in IOmL DMF to give DMF solution of compound 8;

Synthesis [0029] C, of Compound 5

[0030] After 50ml 3-necked flask was added 0.2g compound 1,0.24gDBAC0,20mlDMF, dissolved with stirring, dropwise adding to the reaction system in DMF compound obtained in Step 8, after the addition was complete, stirring continued for 3.5 hours; after completion of the reaction, 20ml water was added to the system and 50ml ethyl acetate, the organic phase is evaporated to dryness and petroleum ether to ethyl acetate to give 0.158g of compound 5, yield 50.9%.

[0031] Example 2

[0032] – new type Se Iinexor drug synthesis, comprising the steps of:

Synthesis [0033] A, Compound 7

[0034] 50ml three □ flask, 15ml of dichloromethane and 0.2g compound 6,15ml ethyl acetate, stirred and dissolved, was added 0.3g of compound 4 and 3gT3P, 0.75gDIPEA at 1 ° C; system at 1 ° C the reaction was stirred for 35min, 50ml of dichloromethane and 30ml of water were added after the completion of the reaction, liquid separation, the organic phase was evaporated to dryness to give crude compound 7, crude without purification cast down;

[0035] B, Synthesis of Compound 8

Three-neck flask [0036] 50ml of addition of the compound obtained in Step 7,40ml glacial acetic acid and 1.38g of sodium iodide was heated to 120. (:, The reaction for 2.5 h; After completion of the reaction, cooled to room temperature system, the system will be transferred to 500ml flask, 60ml water and 120ml dichloromethane was added, after stirring for 15min, allowed to stand for separation, the organic phase was washed with saturated sodium bicarbonate and washed with saturated sodium chloride, dried over anhydrous sodium sulfate and distilled to give crude compound 8, 12mLDMF was dissolved in DMF to give a solution of compound 8;

Synthesis [0037] C, of Compound 5

[0038] After 50ml 3-necked flask was added 0.2g compound 1,0.24gDBAC0,20mlDMF, dissolved with stirring, dropwise adding to the reaction system of the compound obtained in DMF step 8, after the addition was complete, stirring continued for 3 hours; after completion of the reaction, 25ml of water and 50ml of ethyl acetate was added to the system, the organic phase is evaporated to dryness and petroleum ether to ethyl acetate to give 0.152g of compound 5, yield 49.0% billion

[0039] Example 3

[0040] – novel synthetic method of Se species I inexor drug, comprising the steps of:

Synthesis [0041] A, Compound 7

Three [0042] 50ml of flask, 15ml of dichloromethane and 0.2g compound 6,15ml ethyl acetate, stirred and dissolved, was added 0.3g of compound 4 and 3gT3P, 0.75gDIPEA at 2 ° C; system from 0 ° C the reaction was stirred for 25min, 40ml of dichloromethane and 35ml of water were added after the completion of the reaction, liquid separation, the organic phase was evaporated to dryness to give crude compound 7, crude without purification cast down;

[0043] B, Synthesis of Compound 8

Three-neck flask [0044] 50ml of addition of the compound obtained in Step 7,35ml glacial acetic acid and 1.38g of sodium iodide was heated to 110. (:, The reaction for 3.5 h; After completion of the reaction, cooled to room temperature system, the system will be transferred to 500ml flask, 50ml of water was added and dichloromethane IOOml After Smin of stirring, standing separation, the organic phase was washed with saturated sodium bicarbonate and washed with saturated sodium chloride, dried over anhydrous sodium sulfate and distilled to give crude compound 8, was dissolved in IOmL DMF to give DMF solution of compound 8;

Synthesis [0045] C, of Compound 5

[0046] 50ml three-neck flask was added 0.2g compound 1,0.24gDBA⑶, 20mlDMF, and dissolved with stirring, dropwise adding to the reaction system of the compound obtained in DMF step 8, after the addition was complete, stirring was continued for 4 hours; after completion of the reaction, 20ml of water and 40ml ethyl acetate were added to the system, the organic phase is evaporated to dryness and petroleum ether to ethyl acetate to give 0.155g of compound 5, yield 49.9% billion

PATENT

WO 2017118940

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2017118940&tab=PCTDESCRIPTION

The drug compound having the adopted name “Selinexor” has chemical name:(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-IH-l,2,4-triazol-1 -yl)-N’-(pyrazin-2yl) acrylohydrazide as below.

Selinexor (KPT-330) is a first-in-class, oral Selective Inhibitor of Nuclear Export / SINE™ compound. Selinexor functions by binding with and inhibiting the nuclear export protein XP01 (also called CRM1 ), leading to the accumulation of tumor suppressor proteins in the cell nucleus. This reinitiates and amplifies their tumor suppressor function and is believed to lead to the selective induction of apoptosis in cancer cells, while largely sparing normal cells. Over 1 ,200 patients have been treated with Selinexor in company and investigator-sponsored Phase 1 and Phase 2 clinical trials in advanced hematologic malignancies and solid tumors. Karyopharm has initiated four later-phase clinical trials of Selinexor, including one in older patients with acute myeloid leukemia (SOPRA), one in patients with Richter’s transformation (SIRRT), one in patients with diffuse large B-cell lymphoma (SADAL) and a single-arm trial of Selinexor and lose-dose dexamethasone in patients with multiple myeloma (STORM). Patients may receive a twice-weekly combination of Selinexor in combination with low dose dexamethasone. Randomized 1 :1 , Selinexor will be dosed either at 60mg + dexamethasone or at 100 mg + dexamethasone.

US 8999996 B2 discloses Selinexor and a pharmaceutically acceptable salt thereof, pharmaceutical compositions and use for treating disorders associated with CRM1 activity. Further, it discloses preparative methods for the preparation of compounds disclosed therein including Selinexor by reacting (Z)-3-(3- (3,5-

bis(trifluoromethyl)phenyl)-IH-l,2,4-triazol-l-yl)acrylic acid in 1 :1 CH₂CI₂: AcOEt with 2-Hydrazinopyrazine at -40 °C followed by addition of T₃P[Propylphosphonic anhydride] (50%) and DIPEA. After 30 minutes, the reaction mixture was concentrated and the crude oil was purified by preparative TLC using 5% MeOH in CH2CI2 as mobile phase (under ammonia atmosphere) to afford 40 mg of Selinexor with purity: 95.78%. However, it is not disclosed about the nature of the compound obtained therein.

WO 2016025904 A1 discloses various crystalline forms of Selinexor namely Form A, Form B, Form C, Form D, compositions and MoU thereof for the treatment of disorder associated with CRM1 activity and their preparative processes.

Prior art process for the preparation of Selinexor suffers from disadvantages interms of process such as the use of lengthy procedures to practice and resulting in low yields, which may not be viable at industrial scale. Synthetic product obtained therein has very low purity and contains significant amounts of unreacted starting materials and trans-isomer of Selinexor, which are further purified by time consuming and expensive chromatographic separations leading to loss of yield. Hence, there remains a need for improved process for the preparation of Selinexor which is industrially viable and reproducible. Particularly, it is desirable to have a process avoiding purification steps still meeting desired pharmaceutical quality.

EXAMPLES

Example-1 : Preparation of isopropyl (Z)-3-(3-(3,5-bis(trifluoromethyl) phenyl)-1 H- -triazol-1 -yl)acrylate

3-(3,5-bis(trifluoromethyl)phenyl)-1 H-1 ,2,4-triazole (250 g) was dissolved in tetrahydrofuran (2 I) under nitrogen atmosphere at 27°C and cooled to -5°C. 1 ,4- diazabicyclo[2.2.2]octane (DABCO, 1 99.5 g) was added to the reaction mixture at -5°C and stirred at the same temperature for 40 minutes. Isopropyl (Z)-3- iodoacrylate (234.8 g in 500 mL of tetrahydrofuran) was added drop wise to the reaction mixture in 1 hour 1 0 minutes at -5°C and stirred at the same temperature for 2 hours. After the completion of the reaction, the reaction mixture was added to ice cold water (2 I) and separated the organic layer. The aqueous layer was extracted with ethyl acetate (2 x 1 I). The combined organic layer was washed with brine solution (1 I) and dried over sodium sulphate. The dried solution was evaporated completely under vacuum at 40°C to obtain crude product with HPLC purity of 93.53% The crude product was triturated with hexane (700 mL) and stirred for 20 minutes at -30°C and filtered the solid. Trituration of crude product with hexane was repeated for three times and dried under vacuum to obtain the title compound with HPLC purity of 97.46% and trans-isomer content of 0.66%. Yield: 297 g Example-2: Preparation of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1 H-1 ,2,4- triazol-1 -yl)acr lic acid.

To a mixture of tetrahydrofuran (300 mL) and water (300 mL), Isopropyl (Z)-3-(3- (3,5-bis(trifluoromethyl)phenyl)-1 H-1 ,2,4-triazol-1 -yl)acrylate (30 g) was added and cooled to 0°C. Lithium hydroxide monohydrate (16.03 g) under cooling condition at 0°C was added to the reaction mixture and stirred the reaction mixture at same temperature for 7 hours. After completion of the reaction, 2 N HCI (180 mL) was added to adjust the pH of the reaction mixture to 2 and extracted it with ethyl acetate (300 mL). Organic layer was dried over sodium sulphate and evaporated under vacuum at 40°C. The crude compound was stirred with hexane (150 mL) and filtered the solid. Dried the compound under vacuum at 40°C for 0.5 hour to obtain the title compound with HPLC purity of 97.25% with trans-isomer content of 3 %. Yield: 24 g

Example-3: Purification of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1 H-1 ,2,4- tria

A mixture of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1 H-1 ,2,4-triazol-1 -yl)acrylic acid (24 g) and acetone (240 mL) was stirred for complete dissolution at 30°C. Dicyclohexyl amine (1 5 mL) was added drop wise for 20 minutes under stirring at the same temperature. Acetone (50 mL) was added to the reaction mixture and stirred for 2 hours at 27°C. Filtered the solid and washed with hot acetone (150 mL) and dried in vacuum drier at 30°C for 1 hour to obtain the Dicyclohexyl amine salt of (Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1 H-1 ,2,4-triazol-1 -yl)acrylic acid. To the above salt, dichloromethane (150 mL) and water (1 00 mL) was added and stirred for complete dissolution at 30and adjusted the pH of the solution with 2 N sulphuric acid (100 mL) to 2. Filtered the reaction mixture and washed the product with water (1 00 mL) and then with hexane (150 mL). The solid was dried under vacuum at 40°C for 0.5 hour to obtain title compound with HPLC purity 99.98% with no detectable content of trans-isomer. Yield: 17 g

Example-4: Preparation of Selinexor

(Z)-3-(3-(3,5-bis(trifluoromethyl)phenyl)-1 H-1 ,2,4-triazol-1 -yl)acrylic acid (10 g) was combined with a mixture of acetonitrile (1 00 mL) and ethyl acetate (50 mL) then added the 2-hydrazinylpyrazine (3.76 g) and stirred for 5 min. Reaction mixture was cooled to 0°C and diisopropyl ethyl amine (16.63 ml) and then Propylphosphonic anhydride (T₃P, 33.31 mL) was added at 0°C and stirred the reaction mixture for 2.5 hours at the same temperature. After completion of the reaction, the reaction mixture was quenched with cold water (100 mL) and extracted the product with ethyl acetate (2 x 150 mL). The combined organic layer was dried over sodium sulphate and evaporated the solvent under vacuum at 40°C to obtain the crude product as yellow syrup. The obtained crude product was combined with dichloromethane (1 00 mL) and filtered the solid and washed with dichloromethane (2 x 50 mL). The solid was dried under vacuum at 40°C to obtain the title compound with purity by HPLC of 99.86%. Yield : 7 g

PATENT
WO 2018129227

References

^ Jump up to:^a ^b ^c ^d ^e Fung HY, Chook YM (2014). “Atomic basis of CRM1-cargo recognition, release and inhibition”. Semin Cancer Biol. 27: 52–61. doi:10.1016/j.semcancer.2014.03.002. PMC 4108548. PMID 24631835.
^ Jump up to:^a ^b ^c ^d ^e ^f Gandhi UH, Senapedis W, Baloglu E, Unger TJ, Chari A, Vogl D; et al. (2018). “Clinical implications of targeting XPO1-mediated nuclear export in multiple myeloma”. Clin Lymphoma Myeloma Leuk. 18 (5): 335–345. doi:10.1016/j.clml.2018.03.003. PMID 29610030.
^ Jump up to:^a ^b ^c ^d ^e Feuerstein, Adam (2019-07-03). “FDA approves new multiple myeloma drug despite toxicity concerns”. STAT. Retrieved 2019-07-06.
^ Jump up to:^a ^b ^c Mulcahy, Nick (2019-07-03). “FDA Approves Selinexor for Refractory Multiple Myeloma”. Medscape. Retrieved 2019-07-06.
^ Jump up to:^a ^b ^c Chim CS, Kumar SK, Orlowski RZ, Cook G, Richardson PG, Gertz MA; et al. (2018). “Management of relapsed and refractory multiple myeloma: novel agents, antibodies, immunotherapies and beyond”. Leukemia. 32 (2): 252–262. doi:10.1038/leu.2017.329. PMC 5808071. PMID 29257139.
^ Barrett, Jennifer (2019-07-03). “New Treatment for Refractory Multiple Myeloma Granted FDA Approval”. Pharmacy Times. Retrieved 2019-07-07.
^ Jump up to:^a ^b ^c ^d ^e ^f ^g “XPOVIO Prescribing Information” (PDF). Newton, MA: Karyopharm Therapeutics. 2019-07-03. Retrieved 2019-07-06.
^ Jump up to:^a ^b Chen C, Siegel D, Gutierrez M, Jacoby M, Hofmeister CC, Gabrail N (2018). “Safety and efficacy of selinexor in relapsed or refractory multiple myeloma and Waldenstrom macroglobulinemia”. Blood. 131 (8): 855–863. doi:10.1182/blood-2017-08-797886. PMID 29203585.
^ Jump up to:^a ^b ^c ^d Parikh K, Cang S, Sekhri A, Liu D; et al. (2014). “Selective inhibitors of nuclear export (SINE)—a novel class of anti-cancer agents”. J Hematol Oncol. 7: 78. doi:10.1186/s13045-014-0078-0. PMC 4200201. PMID 25316614.

REFERENCES

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2: Crochiere ML, Hannus S, Hansen K, Becker F, Baloglu E, Lee M, Kauffman M, Shacham S, Landesman Y. XPO1 target occupancy measurements confirm the selinexor recommended phase 2 dose. Oncotarget. 2017 Nov 30;8(66):110503-110516. doi: 10.18632/oncotarget.22801. eCollection 2017 Dec 15. PubMed PMID: 29299164; PubMed Central PMCID: PMC5746399.

3: Chim CS, Kumar SK, Orlowski RZ, Cook G, Richardson PG, Gertz MA, Giralt S, Mateos MV, Leleu X, Anderson KC. Management of relapsed and refractory multiple myeloma: novel agents, antibodies, immunotherapies and beyond. Leukemia. 2017 Jan 16. doi: 10.1038/leu.2017.329. [Epub ahead of print] Review. PubMed PMID: 29257139.

4: Bobillo S, Abrisqueta P, Carpio C, Raheja P, Castellví J, Crespo M, Bosch F. Promising activity of selinexor in the treatment of a patient with refractory diffuse large B-cell lymphoma and central nervous system involvement. Haematologica. 2017 Dec 14. pii: haematol.2017.181636. doi: 10.3324/haematol.2017.181636. [Epub ahead of print] PubMed PMID: 29242296.

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6: Corno C, Stucchi S, De Cesare M, Carenini N, Stamatakos S, Ciusani E, Minoli L, Scanziani E, Argueta C, Landesman Y, Zaffaroni N, Gatti L, Perego P. FoxO-1 contributes to the efficacy of the combination of the XPO1 inhibitor selinexor and cisplatin in ovarian carcinoma preclinical models. Biochem Pharmacol. 2018 Jan;147:93-103. doi: 10.1016/j.bcp.2017.11.009. Epub 2017 Nov 16. PubMed PMID: 29155058.

7: Azmi AS, Li Y, Muqbil I, Aboukameel A, Senapedis W, Baloglu E, Landesman Y, Shacham S, Kauffman MG, Philip PA, Mohammad RM. Exportin 1 (XPO1) inhibition leads to restoration of tumor suppressor miR-145 and consequent suppression of pancreatic cancer cell proliferation and migration. Oncotarget. 2017 Jul 17;8(47):82144-82155. doi: 10.18632/oncotarget.19285. eCollection 2017 Oct 10. PubMed PMID: 29137251; PubMed Central PMCID: PMC5669877.

8: Chen Y, Zhang L, Huang J, Hong X, Zhao J, Wang Z, Zhang K. Dasatinib and chemotherapy in a patient with early T-cell precursor acute lymphoblastic leukemia and NUP214-ABL1 fusion: A case report. Exp Ther Med. 2017 Nov;14(5):3979-3984. doi: 10.3892/etm.2017.5046. Epub 2017 Aug 28. PubMed PMID: 29067094; PubMed Central PMCID: PMC5647690.

9: Body S, Esteve-Arenys A, Miloudi H, Recasens-Zorzo C, Tchakarska G, Moros A, Bustany S, Vidal-Crespo A, Rodriguez V, Lavigne R, Com E, Casanova I, Mangues R, Weigert O, Sanjuan-Pla A, Menéndez P, Marcq B, Picquenot JM, Pérez-Galán P, Jardin F, Roué G, Sola B. Cytoplasmic cyclin D1 controls the migration and invasiveness of mantle lymphoma cells. Sci Rep. 2017 Oct 24;7(1):13946. doi: 10.1038/s41598-017-14222-1. PubMed PMID: 29066743; PubMed Central PMCID: PMC5654982.

10: Broccoli A, Argnani L, Zinzani PL. Peripheral T-cell lymphomas: Focusing on novel agents in relapsed and refractory disease. Cancer Treat Rev. 2017 Nov;60:120-129. doi: 10.1016/j.ctrv.2017.09.002. Epub 2017 Sep 18. Review. PubMed PMID: 28946015.

11: Soung YH, Kashyap T, Nguyen T, Yadav G, Chang H, Landesman Y, Chung J. Selective Inhibitors of Nuclear Export (SINE) compounds block proliferation and migration of triple negative breast cancer cells by restoring expression of ARRDC3. Oncotarget. 2017 May 18;8(32):52935-52947. doi: 10.18632/oncotarget.17987. eCollection 2017 Aug 8. PubMed PMID: 28881784; PubMed Central PMCID: PMC5581083.

12: Garg M, Kanojia D, Mayakonda A, Ganesan TS, Sadhanandhan B, Suresh S, S S, Nagare RP, Said JW, Doan NB, Ding LW, Baloglu E, Shacham S, Kauffman M, Koeffler HP. Selinexor (KPT-330) has antitumor activity against anaplastic thyroid carcinoma in vitro and in vivo and enhances sensitivity to doxorubicin. Sci Rep. 2017 Aug 29;7(1):9749. doi: 10.1038/s41598-017-10325-x. PubMed PMID: 28852098; PubMed Central PMCID: PMC5575339.

13: Conforti F, Zhang X, Rao G, De Pas T, Yonemori Y, Rodriguez JA, McCutcheon JN, Rahhal R, Alberobello AT, Wang Y, Zhang YW, Guha U, Giaccone G. Therapeutic Effects of XPO1 Inhibition in Thymic Epithelial Tumors. Cancer Res. 2017 Oct 15;77(20):5614-5627. doi: 10.1158/0008-5472.CAN-17-1323. Epub 2017 Aug 17. PubMed PMID: 28819023.

14: Arango NP, Yuca E, Zhao M, Evans KW, Scott S, Kim C, Gonzalez-Angulo AM, Janku F, Ueno NT, Tripathy D, Akcakanat A, Naing A, Meric-Bernstam F. Selinexor (KPT-330) demonstrates anti-tumor efficacy in preclinical models of triple-negative breast cancer. Breast Cancer Res. 2017 Aug 15;19(1):93. doi: 10.1186/s13058-017-0878-6. PubMed PMID: 28810913; PubMed Central PMCID: PMC5557476.

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16: Muz B, Azab F, de la Puente P, Landesman Y, Azab AK. Selinexor Overcomes Hypoxia-Induced Drug Resistance in Multiple Myeloma. Transl Oncol. 2017 Aug;10(4):632-640. doi: 10.1016/j.tranon.2017.04.010. Epub 2017 Jun 29. PubMed PMID: 28668761; PubMed Central PMCID: PMC5496204.

17: Gupta A, Saltarski JM, White MA, Scaglioni PP, Gerber DE. Therapeutic Targeting of Nuclear Export Inhibition in Lung Cancer. J Thorac Oncol. 2017 Sep;12(9):1446-1450. doi: 10.1016/j.jtho.2017.06.013. Epub 2017 Jun 21. PubMed PMID: 28647672; PubMed Central PMCID: PMC5572747.

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Selinexor
Clinical data

Trade names	Xpovio
Pregnancy category	Known to cause fetal harm
Routes of administration	Oral
Legal status
Legal status	US: ℞-only
Pharmacokinetic data
Protein binding	95%
Metabolism	Hepatic oxidation, glucuronidation, and conjugation, by CYP3A4, UGTand GST
Elimination half-life	6–8 h
Identifiers
IUPAC name [show]
CAS Number	1393477-72-9
PubChem CID	71481097
DrugBank	DB11942
UNII	31TZ62FO8F
Chemical and physical data
Formula	C₁₇H₁₁F₆N₇O
Molar mass	443.313 g·mol⁻¹
3D model (JSmol)	Interactive image
SMILES [hide] C1=CN=C(C=N1)NNC(=O)/C=C\N2C=NC(=N2)C3=CC(=CC(=C3)C(F)(F)F)C(F)(F)F

Karyopharm’s Selinexor Receives Fast Track Designation from FDA for the Treatment of Patients with Penta-Refractory Multiple Myeloma

NEWTON, Mass., April 10, 2018 (GLOBE NEWSWIRE) — Karyopharm Therapeutics Inc. (Nasdaq:KPTI), a clinical-stage pharmaceutical company, today announced that the U.S. Food and Drug Administration (FDA) has granted Fast Track designation to the Company’s lead, oral Selective Inhibitor of Nuclear Export (SINE) compound selinexor for the treatment of patients with multiple myeloma who have received at least three prior lines of therapy. The FDA’s statement, consistent with the design of Karyopharm’s Phase 2b STORM study, noted that the three prior lines of therapy include regimens comprised of an alkylating agent, a glucocorticoid, Velcade® (bortezomib), Kyprolis® (carfilzomib), Revlimid® (lenalidomide), Pomalyst® (pomalidomide) and Darzalex® (daratumumab). In addition, the patient’s disease must be refractory to at least one proteasome inhibitor (Velcade or Kyprolis), one immunomodulatory agent (Revlimid or Pomalyst), glucocorticoids and to Darzalex, as well as to the most recent therapy. The Company expects to report top-line data from the STORM study at the end of April 2018.

ChemSpider 2D Image | selinexor | C17H11F6N7O

The FDA’s Fast Track program facilitates the development of drugs intended to treat serious conditions and that have the potential to address unmet medical needs. A drug program with Fast Track status is afforded greater access to the FDA for the purpose of expediting the drug’s development, review and potential approval. In addition, the Fast Track program allows for eligibility for Accelerated Approval and Priority Review, if relevant criteria are met, as well as for Rolling Review, which means that a drug company can submit completed sections of its New Drug Application (NDA) for review by FDA, rather than waiting until every section of the NDA is completed before the entire application can be submitted for review.

“The designation of Fast Track for selinexor represents important recognition by the FDA of the potential of this anti-cancer agent to address the significant unmet need in the treatment of patients with penta-refractory myeloma that has continued to progress despite available therapies,” said Sharon Shacham, PhD, MBA, Founder, President and Chief Scientific Officer of Karyopharm. “We are fully committed to working closely with the FDA as we continue development of this potential new, orally-administered treatment for patients who currently have no other treatment options of proven benefit.”

About the Phase 2b STORM Study

In the multi-center, single-arm Phase 2b STORM (Selinexor Treatment of Refractory Myeloma) study, approximately 122 patients with heavily pretreated, penta-refractory myeloma receive 80mg oral selinexor twice weekly in combination with 20mg low-dose dexamethasone, also dosed orally twice weekly. Patients with penta-refractory disease are those who have previously received an alkylating agent, a glucocorticoid, two immunomodulatory drugs (IMiDs) (Revlimid® (lenalidomide) and Pomalyst® (pomalidomide)), two proteasome inhibitors (PIs) (Velcade® (bortezomib) and Kyprolis® (carfilzomib)), and the anti-CD38 monoclonal antibody Darzalex® (daratumumab), and their disease is refractory to at least one PI, at least one IMiD, Darzalex, glucocorticoids and their most recent anti-myeloma therapy. Overall response rate is the primary endpoint of the study, with duration of response and clinical benefit rate being secondary endpoints. All responses will be adjudicated by an Independent Review Committee (IRC).

About Selinexor

Selinexor (KPT-330) is a first-in-class, oral Selective Inhibitor of Nuclear Export (SINE) compound. Selinexor functions by binding with and inhibiting the nuclear export protein XPO1 (also called CRM1), leading to the accumulation of tumor suppressor proteins in the cell nucleus. This reinitiates and amplifies their tumor suppressor function and is believed to lead to the selective induction of apoptosis in cancer cells, while largely sparing normal cells. To date, over 2,300 patients have been treated with selinexor, and it is currently being evaluated in several mid- and later-phase clinical trials across multiple cancer indications, including in multiple myeloma in a pivotal, randomized Phase 3 study in combination with Velcade® (bortezomib) and low-dose dexamethasone (BOSTON), in combination with low-dose dexamethasone (STORM) and as a potential backbone therapy in combination with approved therapies (STOMP), and in diffuse large B-cell lymphoma (SADAL), and liposarcoma (SEAL), among others. Additional Phase 1, Phase 2 and Phase 3 studies are ongoing or currently planned, including multiple studies in combination with one or more approved therapies in a variety of tumor types to further inform Karyopharm’s clinical development priorities for selinexor. Additional clinical trial information for selinexor is available at www.clinicaltrials.gov.

About Karyopharm Therapeutics

Karyopharm Therapeutics Inc. (Nasdaq:KPTI) is a clinical-stage pharmaceutical company focused on the discovery, development and subsequent commercialization of novel first-in-class drugs directed against nuclear transport and related targets for the treatment of cancer and other major diseases. Karyopharm’s SINE compounds function by binding with and inhibiting the nuclear export protein XPO1 (or CRM1). In addition to single-agent and combination activity against a variety of human cancers, SINE compounds have also shown biological activity in models of neurodegeneration, inflammation, autoimmune disease, certain viruses and wound-healing. Karyopharm, which was founded by Dr. Sharon Shacham, currently has several investigational programs in clinical or preclinical development.

/////////Selinexor, FDA 2019, セリネクソル ,KPT-330, KPT 330 , KPT330, AML, Glioma, Sarcoma, Leukemia, Fast Track, CANCER

Cavosonstat (N-91115)

May 4, 2019 10:03 am / Leave a comment

Cavosonstat (N-91115)

CAS 1371587-51-7

C₁₆H₁₀ClNO₃, 299.71 g/mol

UNII-O2Z8Q22ZE4, O2Z8Q22ZE4, NCT02589236; N91115-2CF-05; SNO-6

3-chloro-4-(6-hydroxyquinolin-2-yl)benzoic acid

Treatment of Chronic Obstructive Pulmonary Diseases (COPD), AND Cystic fibrosis, Nivalis Therapeutics, phase 2

The product was originated at Nivalis Therapeutics, which was acquired by Alpine Immune Sciences in 2017. In 2018, Alpine announced the sale and transfer of global rights to Laurel Venture Capital for further product development.

In 2016, orphan drug and fast track designations were granted to the compound in the U.S. for the treatment of cystic fibrosis.

Originator N30 Pharma
Developer Nivalis Therapeutics
Class Small molecules
Mechanism of Action Cystic fibrosis transmembrane conductance regulator modulators; Glutathione-independent formaldehyde dehydrogenase inhibitors; Nitric oxide stimulants

Orphan Drug Status Yes – Cystic fibrosis

20 Jul 2018 Laurel Venture Capital acquires global rights for cavosonstat from Alpine Immune Sciences
20 Jul 2018 Laurel Venture Capital plans a phase II trial for Asthma
24 Jun 2018 Biomarkers information updated

Cavosonstat, alos known as N91115) an orally bioavailable inhibitor of S-nitrosoglutathione reductase, promotes cystic fibrosis transmembrane conductance regulator (CFTR) maturation and plasma membrane stability, with a mechanism of action complementary to CFTR correctors and potentiators.

Cavosonstat (N91115) was an experimental therapy being developed by Nivalis Therapeutics. Its primary mechanism of action was to inhibit the S-nitrosoglutathione reductase (GSNOR) enzyme and to stabilize cystic fibrosis transmembrane regulator (CFTR) protein activity. A press release published in February announced the end of research for this therapy in cystic fibrosis (CF) patients with F508del mutations. The drug, which did not meet primary endpoints in a Phase 2 trial, had been referred to as the first of a new class of compounds that stabilizes the CFTR activity.

History of cavosonstat

During preclinical studies, N91115 (later named cavosonstat) demonstrated an improvement in cystic fibrosis transmembrane regulator (CFTR) stability.

A Phase 1 study was initiated in 2014 to evaluate the safety, tolerability, and pharmacokinetics (how a drug is processed in the body) of the drug in healthy volunteers. Later that year, the pharmacokinetics of the drug were assessed in another Phase 1 trial involving CF patients with F508del mutation suffering from pancreatic insufficiency. Results were presented a year later by Nivalis, revealing good tolerance and safety in study participants.

A second, much smaller Phase 2 study (NCT02724527) assessed cavosonstat as an add-on therapy to ivacaftor (Kalydeco). This double-blind, randomized, placebo-controlled study included 19 participants who received treatment with cavosonstat (400 mg) added to Kalydeco or with placebo added to Kalydeco. The primary objective was change in lung function from the study’s start to week 8. However, the treatment did not demonstrate a benefit in lung function measures or in sweat chloride reduction at eight weeks (primary objective). As a result, Nivalis decided not to continue development of cavosonstat for CF treatment.

The U.S. Food and Drug Administration (FDA) had granted cavosonstat both fast track and orphan drug designations in 2016.

How cavosonstat works

The S-nitrosoglutathione (GSNO) is a signaling molecule that is present in high concentrations in the fluids of the lungs or muscle tissues, playing an important role in the dilatation of the airways. GSNO levels are regulated by the GSNO reductase (GSNOR) enzyme, altering CFTR activity in the membrane. In CF patients, GSNO levels are low, causing a loss of the airway function.

Cavosonstat’s mechanism of action is achieved through GSNOR inhibition, which was presumed to control the deficient CFTR protein. Preclinical studies showed that cavosonstat restored GSNO levels.

PATENT

WO 2012083165

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2012083165

The chemical compound nitric oxide is a gas with chemical formula NO. NO is one of the few gaseous signaling molecules known in biological systems, and plays an important role in controlling various biological events. For example, the endothelium uses NO to signal surrounding smooth muscle in the walls of arterioles to relax, resulting in vasodilation and increased blood flow to hypoxic tissues. NO is also involved in regulating smooth muscle proliferation, platelet function, and neurotransmission, and plays a role in host defense. Although NO is highly reactive and has a lifetime of a few seconds, it can both diffuse freely across membranes and bind to many molecular targets. These attributes make NO an ideal signaling molecule capable of controlling biological events between adjacent cells and within cells.

[0003] NO is a free radical gas, which makes it reactive and unstable, thus NO is short lived in vivo, having a half life of 3-5 seconds under physiologic conditions. In the presence of oxygen, NO can combine with thiols to generate a biologically important class of stable NO adducts called S-nitrosothiols (SNO’s). This stable pool of NO has been postulated to act as a source of bioactive NO and as such appears to be critically important in health and disease, given the centrality of NO in cellular homeostasis (Stamler et al., Proc. Natl. Acad. Sci. USA, 89:7674-7677 (1992)). Protein SNO’s play broad roles in the function of cardiovascular, respiratory, metabolic, gastrointestinal, immune, and central nervous system (Foster et al., Trends in Molecular Medicine, 9 (4): 160-168, (2003)). One of the most studied SNO’s in biological systems is S-nitrosoglutathione (GSNO) (Gaston et al., Proc. Natl. Acad. Sci. USA 90: 10957-10961 (1993)), an emerging key regulator in NO signaling since it is an efficient trans-nitrosating agent and appears to maintain an equilibrium with other S-nitrosated proteins (Liu et al., Nature, 410:490-494 (2001)) within cells. Given this pivotal position in the NO-SNO continuum, GSNO provides a therapeutically promising target to consider when NO modulation is pharmacologically warranted.

[0004] In light of this understanding of GSNO as a key regulator of NO homeostasis and cellular SNO levels, studies have focused on examining endogenous production of GSNO and SNO proteins, which occurs downstream from the production of the NO radical by the nitric oxide synthetase (NOS) enzymes. More recently there has been an increasing understanding of enzymatic catabolism of GSNO which has an important role in governing available concentrations of GSNO and consequently available NO and SNO’s.

[0005] Central to this understanding of GSNO catabolism, researchers have recently identified a highly conserved S-nitrosoglutathione reductase (GSNOR) (Jensen et al., Biochem J., 331 :659-668 (1998); Liu et al., (2001)). GSNOR is also known as glutathione-dependent formaldehyde dehydrogenase (GSH-FDH), alcohol dehydrogenase 3 (ADH-3) (Uotila and Koivusalo, Coenzymes and Coƒactors., D. Dolphin, ed. pp. 517-551 (New York, John Wiley & Sons, (1989)), and alcohol dehydrogenase 5 (ADH-5). Importantly GSNOR shows greater activity toward GSNO than other substrates (Jensen et al., (1998); Liu et al., (2001)) and appears to mediate important protein and peptide denitrosating activity in bacteria, plants, and animals. GSNOR appears to be the major GSNO-metabolizing enzyme in eukaryotes (Liu et al., (2001)). Thus, GSNO can accumulate in biological compartments where GSNOR activity is low or absent (e.g. , airway lining fluid) (Gaston et al., (1993)).

[0006] Yeast deficient in GSNOR accumulate S-nitrosylated proteins which are not substrates of the enzyme, which is strongly suggestive that GSNO exists in equilibrium with SNO-proteins (Liu et al., (2001)). Precise enzymatic control over ambient levels of GSNO and thus SNO-proteins raises the possibility that GSNO/GSNOR may play roles across a host of physiological and pathological functions including protection against nitrosative stress wherein NO is produced in excess of physiologic needs. Indeed, GSNO specifically has been implicated in physiologic processes ranging from the drive to breathe (Lipton et al., Nature, 413: 171-174 (2001)) to regulation of the cystic fibrosis transmembrane regulator (Zaman et al., Biochem Biophys Res Commun, 284:65-70 (2001)), to regulation of vascular tone, thrombosis, and platelet function (de Belder et al., Cardiovasc Res.; 28(5):691-4 (1994)), Z. Kaposzta, et al., Circulation; 106(24): 3057 – 3062, (2002)) as well as host defense (de Jesus-Berrios et al., Curr. Biol., 13: 1963-1968 (2003)). Other studies have found that GSNOR protects yeast cells against nitrosative stress both in vitro (Liu et al., (2001)) and in vivo (de Jesus-Berrios et al., (2003)).

[0007] Collectively, data suggest GSNO as a primary physiological ligand for the enzyme S-nitrosoglutathione reductase (GSNOR), which catabolizes GSNO and

consequently reduces available SNO’s and NO in biological systems (Liu et al., (2001)), (Liu et al., Cell, 116(4), 617-628 (2004)), and (Que et al., Science, 308, (5728): 1618-1621 (2005)). As such, this enzyme plays a central role in regulating local and systemic bioactive NO. Since perturbations in NO bioavailability has been linked to the pathogenesis of numerous disease states, including hypertension, atherosclerosis, thrombosis, asthma, gastrointestinal disorders, inflammation, and cancer, agents that regulate GSNOR activity are candidate therapeutic agents for treating diseases associated with NO imbalance.

[0008] Nitric oxide (NO), S-nitrosoglutathione (GSNO), and S-nitrosoglutathione reductase (GSNOR) regulate normal lung physiology and contribute to lung pathophysiology. Under normal conditions, NO and GSNO maintain normal lung physiology and function via their anti-inflammatory and bronchodilatory actions. Lowered levels of these mediators in pulmonary diseases such as asthma, chronic obstructive pulmonary disease (COPD) may occur via up-regulation of GSNOR enzyme activity. These lowered levels of NO and GSNO, and thus lowered anti-inflammatory capabilities, are key events that contribute to pulmonary diseases and which can potentially be reversed via GSNOR inhibition.

[0009] S-nitrosoglutathione (GSNO) has been shown to promote repair and/or regeneration of mammalian organs, such as the heart (Lima et al., 2010), blood vessels (Lima et al., 2010) skin (Georgii et al., 2010), eye or ocular structures (Haq et al., 2007) and liver (Prince et al., 2010). S-nitrosoglutathione reductase (GSNOR) is the major catabolic enzyme of GSNO. Inhibition of GSNOR is thought to increase endogenous GSNO.

[0010] Inflammatory bowel diseases (IBD’s), including Crohn’s and ulcerative colitis, are chronic inflammatory disorders of the gastrointestinal (GI) tract, in which NO, GSNO, and GSNOR can exert influences. Under normal conditions, NO and GSNO function to maintain normal intestinal physiology via anti-inflammatory actions and maintenance of the intestinal epithelial cell barrier. In IBD, reduced levels of GSNO and NO are evident and likely occur via up-regulation of GSNOR activity. The lowered levels of these mediators contribute to the pathophysiology of IBD via disruption of the epithelial barrier via dysregulation of proteins involved in maintaining epithelial tight junctions. This epithelial barrier dysfunction, with the ensuing entry of micro-organisms from the lumen, and the overall lowered anti-inflammatory capabilities in the presence of lowered NO and GSNO, are key events in IBD progression that can be potentially influenced by targeting GSNOR.

[0011] Cell death is the crucial event leading to clinical manifestation of

hepatotoxicity from drugs, viruses and alcohol. Glutathione (GSH) is the most abundant redox molecule in cells and thus the most important determinant of cellular redox status. Thiols in proteins undergo a wide range of reversible redox modifications during times of exposure to reactive oxygen and reactive nitrogen species, which can affect protein activity. The maintenance of hepatic GSH is a dynamic process achieved by a balance between rates of GSH synthesis, GSH and GSSG efflux, GSH reactions with reactive oxygen species and reactive nitrogen species and utilization by GSH peroxidase. Both GSNO and GSNOR play roles in the regulation of protein redox status by GSH.

[0012] Acetaminophen overdoses are the leading cause of acute liver failure (ALF) in the United States, Great Britain and most of Europe. More than 100,000 calls to the U.S. Poison Control Centers, 56,000 emergency room visits, 2600 hospitalizations, nearly 500 deaths are attributed to acetaminophen in this country annually. Approximately, 60% recover without needing a liver transplant, 9% are transplanted and 30% of patients succumb to the illness. The acetaminophen-related death rate exceeds by at least three-fold the number of deaths due to all other idiosyncratic drug reactions combined (Lee, Hepatol Res 2008; 38 (Suppl. 1):S3-S8).

[0013] Liver transplantation has become the primary treatment for patients with fulminant hepatic failure and end-stage chronic liver disease, as well as certain metabolic liver diseases. Thus, the demand for transplantation now greatly exceeds the availability of donor organs, it has been estimated that more than 18 000 patients are currently registered with the United Network for Organ Sharing (UNOS) and that an additional 9000 patients are added to the liver transplant waiting list each year, yet less than 5000 cadaveric donors are available for transplantation.

[0014] Currently, there is a great need in the art for diagnostics, prophylaxis, ameliorations, and treatments for medical conditions relating to increased NO synthesis and/or increased NO bioactivity. In addition, there is a significant need for novel compounds, compositions, and methods for preventing, ameliorating, or reversing other NO-associated disorders. The present invention satisfies these needs.

Schemes 1-6 below illustrate general methods for preparing analogs.

[00174] For a detailed example of General Scheme 1 see Compound IV-1 in Example 1.

[00175] For a detailed example of Scheme 2, A conditions, see Compound IV-2 in Example 2.

[00176] For a detailed example of Scheme 2, B conditions, see Compound IV-8 in Example 8.

[00177] For a detailed example of Scheme 3, see Compound IV-9 in Example 9.

[00178] For a detailed example of Scheme 4, Route A, see Compound IV-11 in Example 11.

[00179] For a detailed example of Scheme 4, Route B, see Compound IV-12 in Example 12.

[00180] For a detailed example of Scheme 5, Compound A, see Compound IV-33 in Example 33.

[00181] For a detailed example of Scheme 5, Compound B, see Compound IV-24 in Example 24.

[00182] For a detailed example of Scheme 5, Compound C, see Compound IV-23 in Example 23.

Example 8: Compound IV-8: 3-chloro-4-(6-hydroxyquinolin-2-yl)benzoic acid

[00209] Followed Scheme 2, B conditions:

[00210] Step 1: Synthesis of 3-chloro-4-(6-methoxyquinolin-2-yl)benzoic acid:

[00211] A mixture of 2-chloro-6-methoxyquinoline (Intermediate 1) (200 mg, 1.04 mmol), 4-carboxy-2-chlorophenylboronic acid (247 mg, 1.24 mmol) and K₂CO₃(369 mg, 2.70 mmol) in DEGME / H₂O (7.0 mL / 2.0 mL) was degassed three times under N₂ atmosphere. Then PdCl₂(dppf) (75 mg, 0.104 mmol) was added and the mixture was heated to 110 °C for 3 hours under N₂ atmosphere. The reaction mixture was diluted with EtOAc (100 mL) and filtered. The filtrate was washed with brine (20 mL), dried over Na₂SO₄, filtered and concentrated to give 3-chloro-4-(6-methoxyquinolin-2-yl)benzoic acid (150 mg, yield 46%) as a yellow solid, which was used for the next step without further purification.

[00212] Step 2: Synthesis of Compound IV-8: To a suspension of 3-chloro-4-(6-methoxyquinolin-2-yl)benzoic acid (150 mg, 0.479 mmol) in anhydrous CH₂Cl₂ (5 mL) was added AlCl₃ (320 mg, 2.40 mmol). The reaction mixture was refluxed overnight. The mixture was quenched with saturated NH₄Cl (10 mL) and the aqueous layer was extracted with CH₂Cl₂ / MeOH (v/v=10: l, 30 mL x3). The combined organic layer was washed with brine, dried over Na₂SO₄, filtered, and concentrated to give the crude product, which was purified by prep-HPLC (0.1% TFA as additive) to give 3-chloro-4-(6-hydroxyquinolin-2-yl)benzoic acid (25 mg, yield 18%). ¹H NMR (DMSO, 400 MHz): δ 10.20 (brs, 1H), 8.30 (d, J = 8.4 Hz, 1H), 8.10-8.00 (m, 2H), 7.95 (d, J = 9.2 Hz, 1H), 7.80 (d, J = 8.0 Hz, 1H), 7.72 (d, J = 8.8 Hz, 1H), 7.38 (dd, J = 6.4, 2.8 Hz, 1H), 7.22 (d, J = 2.4 Hz, 1H), MS (ESI): m/z 299.9 [M+H]⁺.

PATENT

WO 2012048181

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2012048181

PATENT

WO 2012170371

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2012170371&recNum=133&docAn=US2012040821&queryString=(%20&

REFERENCES

1: Donaldson SH, Solomon GM, Zeitlin PL, Flume PA, Casey A, McCoy K, Zemanick ET,
Mandagere A, Troha JM, Shoemaker SA, Chmiel JF, Taylor-Cousar JL.
Pharmacokinetics and safety of cavosonstat (N91115) in healthy and cystic
fibrosis adults homozygous for F508DEL-CFTR. J Cyst Fibros. 2017 Feb 13. pii:
S1569-1993(17)30016-4. doi: 10.1016/j.jcf.2017.01.009. [Epub ahead of print]
PubMed PMID: 28209466.

//////////Cavosonstat, N-91115, Orphan Drug Status, NCT02589236, N91115-2CF-05, SNO-6, PHASE 2, N30 Pharma, Nivalis Therapeutics, CYSTIC FIBROSIS, FAST TRACK

O=C(O)C1=CC=C(C2=NC3=CC=C(O)C=C3C=C2)C(Cl)=C1

IMETELSTAT

January 28, 2019 10:44 am / Leave a comment

Image result for IMETELSTAT

2D chemical structure of 868169-64-6

IMETELSTAT

CAS 868169-64-6, N163L

Molecular Formula, C148-H211-N68-O53-P13-S13, Molecular Weight, 4610.2379,

Nucleic Acid Sequence

Sequence Length: 135 a 1 c 4 g 3 tmodified

DNA d(3′-amino-3′-deoxy-P-thio)(T-A-G-G-G-T-T-A-G-A-C-A-A) 5′-[O-[2-hydroxy-3-[(1-oxohexadecyl)amino]propyl] hydrogen phosphorothioate]

PHASE 3, GERON, Myelodysplasia

Image result for IMETELSTAT

ChemSpider 2D Image | Imetelstat sodium | C148H197N68Na13O53P13S13

IMETELSTAT SODIUM

CAS 1007380-31-5, GRN163L, GRN 163L Sodium Salt

Molecular Formula:	C₁₄₈H₁₉₈N₆₈Na₁₃O₅₃P₁₃S₁₃
Molecular Weight:	4895.941 g/mol

5′-(O-(2-hydroxy-3-((1-oxohexadecyl)amino)propyl)phosphorothioate)-d(3′-amino-3′-deoxy-p-thio)(t-a-g-g-g-t-t-a-g-a-c-a-a), sodium salt (13)

DNA, d(3′-amino-3′-deoxy-p-thio)(T-A-G-G-G-T-T-A-G-A-C-A-A), 5′-(o-(2-hydroxy-3-((1-oxohexadecyl)amino)propyl) hydrogen phosphorothioate), sodium salt (1:13)

UNII-2AW48LAZ4I, Antineoplastic

In 2014, Geron entered into an exclusive worldwide license and collaboration agreement with Janssen Biotech for the treatment of hematologic cancers. However, in 2018, the agreement was terminated and Geron regained global rights to the product.

In 2015, imetelstat was granted orphan drug status in the U.S. for the treatment of myelodysplastic syndrome, as well as in both the U.S. and the E.U. for the treatment of myelofibrosis. In 2017, fast track designation was received in the U.S. for the treatment of adult patients with transfusion-dependent anemia due to low or intermediate-1 risk myelodysplastic syndromes (MDS) who are non-del(5q) and who are refractory or resistant to treatment with an erythropoiesis stimulating agent (ESA).

Imetelstat Sodium is the sodium salt of imetelstat, a synthetic lipid-conjugated, 13-mer oligonucleotide N3′ P5′-thio-phosphoramidate with potential antineoplastic activity. Complementary to the template region of telomerase RNA (hTR), imetelstat acts as a competitive enzyme inhibitor that binds and blocks the active site of the enzyme (a telomerase template antagonist), a mechanism of action which differs from that for the antisense oligonucleotide-mediated inhibition of telomerase activity through telomerase mRNA binding. Inhibition of telomerase activity in tumor cells by imetelstat results in telomere shortening, which leads to cell cycle arrest or apoptosis.

Imetelstat sodium, a lipid-based conjugate of Geron’s first-generation anticancer drug, GRN-163, is in phase III clinical trials at Geron for the treatment of myelodysplastic syndrome, as well as in phase II for the treatment of myelofibrosis.

Geron is developing imetelstat, a lipid-conjugated 13-mer thiophosphoramidate oligonucleotide and the lead in a series of telomerase inhibitors, for treating hematological malignancies, primarily myelofibrosis.

Imetelstat, a first-in-class telomerase inhibitor and our sole product candidate, is being developed for the potential treatment of hematologic myeloid malignancies. Imetelstat is currently in two clinical trials being conducted by Janssen under the terms of an exclusive worldwide collaboration and license agreement.

Originally known as GRN163L, imetelstat sodium (imetelstat) is a 13-mer N3’—P5’ thio-phosphoramidate (NPS) oligonucleotide that has a covalently bound 5’ palmitoyl (C16) lipid group. The proprietary nucleic acid backbone provides resistance to the effect of cellular nucleases, thus conferring improved stability in plasma and tissues, as well as significantly improved binding affinity to its target. The lipid group enhances cell permeability to increase potency and improve pharmacokinetic and pharmacodynamic properties. The compound has a long residence time in bone marrow, spleen and liver. Imetelstat binds with high affinity to the template region of the RNA component of telomerase, resulting in direct, competitive inhibition of telomerase enzymatic activity, rather than elicit its effect through an antisense inhibition of protein translation. Imetelstat is administered by intravenous infusion.

Preclinical Studies with Imetelstat

A series of preclinical efficacy studies of imetelstat have been conducted by Geron scientists and academic collaborators. These data showed that imetelstat:

Inhibits telomerase activity, and can shorten telomeres.
Inhibits the proliferation of a wide variety of tumor types, including solid and hematologic, in cell culture systems and rodent xenograft models of human cancers, impacting the growth of primary tumors and reducing metastases.
Inhibits the proliferation of malignant progenitor cells from hematologic cancers, such as multiple myeloma, myeloproliferative neoplasms and acute myelogenous leukemia.
Has additive or synergistic anti-tumor effect in a variety of cell culture systems and xenograft models when administered in combination with approved anti-cancer therapies, including radiation, conventional chemotherapies and targeted agents.

Clinical Experience with Imetelstat

Over 500 patients have been enrolled and treated in imetelstat clinical trials.

PHASE 1

Six clinical trials evaluated the safety, tolerability, pharmacokinetics and pharmacodynamics both as a single agent and in combination with standard therapies in patients with solid tumors and hematologic malignancies:

Single agent studies of imetelstat were in patients with advanced solid tumors, multiple myeloma and chronic lymphoproliferative diseases. Combination studies with imetelstat were with bortezomib in patients with relapsed or refractory multiple myeloma, with paclitaxel and bevacizumab in patients with metastatic breast cancer, and with carboplatin and paclitaxel in patients with advanced non-small cell lung cancer (NSCLC).
Doses ranging from 0.5 mg/kg to 11.7 mg/kg were tested in a variety of dosing schedules ranging from weekly to once every 28 days.
The human pharmacokinetic profile was characterized in clinical trials of patients with solid tumors and chronic lymphoproliferative diseases. Single-dose kinetics showed dose-dependent increases in exposure with a plasma half-life (t1/2) ranging from 4-5 hours. Residence time in bone marrow is long (0.19-0.51 µM observed at 41-45 hours post 7.5 mg/kg dose).
Telomerase inhibition was observed in various tissues where the enzymes’s activity was measurable.

PHASE 2

Imetelstat was studied in two randomized clinical trials, two single arm proof-of-concept studies and an investigator sponsored pilot study:

Randomized trials were in combination with paclitaxel in patients with metastatic breast cancer and as maintenance treatment following a platinum-containing chemotherapy regimen in patients with NSCLC.
Single arm studies were as a single agent or in combination with lenalidomide in patients with multiple myeloma and as a single agent in essential thrombocythemia (ET) or polycythemia vera (PV).
An investigator sponsored pilot study was as a single agent in patients with myelofibrosis (MF) or myelodysplastic syndromes (MDS).

SAFETY AND TOLERABILITY

The safety profile of imetelstat across the Phase 1 and 2 trials has been generally consistent. Reported adverse events (AEs) and laboratory investigations associated with imetelstat administration included cytopenias, transient prolonged activated partial thromboplastin time (aPTT; assessed only in Phase 1 trials), gastrointestinal symptoms, constitutional symptoms, hepatic biochemistry abnormalities, and infusion reactions. Dose limiting toxicities include thrombocytopenia and neutropenia.

A Focus on Hematologic Myeloid Malignancies

Early clinical data from the Phase 2 clinical trial in ET and the investigator sponsored pilot study in MF suggest imetelstat may have disease-modifying activity by suppressing the proliferation of malignant progenitor cell clones for the underlying diseases, and potentially allowing recovery of normal hematopoiesis in patients with hematologic myeloid malignancies.

Results from these trials were published in the New England Journal of Medicine:

Current Clinical Trials

Imetelstat is currently being tested in two clinical trials: IMbark, a Phase 2 trial in myelofibrosis (MF), and IMerge, a Phase 2/3 trial in myelodysplastic syndromes (MDS).

IMbark

IMbark is the ongoing Phase 2 clinical trial to evaluate two doses of imetelstat in intermediate-2 or high-risk MF patients who are refractory to or have relapsed after treatment with a JAK inhibitor.

Internal data reviews were completed in September 2016, April 2017 and March 2018. The safety profile was consistent with prior clinical trials of imetelstat in hematologic malignancies, and no new safety signals were identified. The data supported 9.4 mg/kg as an appropriate starting dose in the trial, but an insufficient number of patients met the protocol defined interim efficacy criteria and new patient enrollment was suspended in October 2016. As of January 2018, median follow up was approximately 19 months, and median overall survival had not been reached in either dosing arm. In March 2018, the trial was closed to new patient enrollment. Patients who remain in the treatment phase of the trial may continue to receive imetelstat, and until the protocol-specified primary analysis, all safety and efficacy assessments are being conducted as planned in the protocol, including following patients, to the extent possible, until death, to enable an assessment of overall survival.

IMerge

IMerge is the ongoing two-part Phase 2/3 clinical trial of imetelstat in red blood cell (RBC) transfusion-dependent patients with lower risk MDS who are refractory or resistant to treatment with an erythropoiesis stimulating agent (ESA). Part 1 is a Phase 2, open-label, single-arm trial of imetelstat administered as a single agent by intravenous infusion, and is ongoing. Part 2 is designed to be a Phase 3, randomized, controlled trial, and has not been initiated.

Preliminary data as of October 2017 from the first 32 patients enrolled in the Part 1 (Phase 2) of IMerge were presented as a poster at the American Society of Hematology Annual Meeting in December 2017.

The data showed that among the subset of 13 patients who had not received prior treatment with either lenalidomide or a hypomethylating agent (HMA) and did not have a deletion 5q chromosomal abnormality (non-del(5q)), 54% achieved RBC transfusion-independence (TI) lasting at least 8 weeks, including 31% who achieved a 24-week RBC-TI. In the overall trial population, the rates of 8- and 24-week RBC-TI were 38% and 16%, respectively. Cytopenias, particularly neutropenia and thrombocytopenia, were the most frequently reported adverse events, which were predictable, manageable and reversible.

Based on the preliminary data from the 13-patient subset, Janssen expanded Part 1 of IMerge to enroll approximately 20 additional patients who were naïve to lenalidomide and HMA treatment and non-del(5q) to increase the experience and confirm the benefit-risk profile of imetelstat in this refined target patient population

PATENT

WO 2005023994

WO 2006113426
WO 2006113470

WO 2006124904

WO 2008054711

WO 2008112129

US 2014155465

WO 2014088785

PATENT

WO 2016172346

http://appft1.uspto.gov/netacgi/nph-Parser?Sect1=PTO1&Sect2=HITOFF&d=PG01&p=1&u=/netahtml/PTO/srchnum.html&r=1&f=G&l=50&s1=20160312227.PGNR.

PATENT

WO2018026646

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2018026646

Patients of acute myeloid leukemia (AML) have limited treatment options at diagnosis; treatment typically takes the form of chemotherapy to quickly reduce the leukemic cell burden. Invasive leukapheresis procedures to remove large numbers of leukocytes (normal and diseased) may be applied in parallel to chemotherapy to temporarily lower tumor cell burden. Induction phase chemotherapy can be successful but, most healthy cells residing in patient bone marrow are also killed, causing illness and requiring additional palliative therapy to ward off infection and raise leukocyte counts. Additional rounds of chemotherapy can be used in an attempt to keep patients in remission; but relapse is common.

[0005] Telomerase is present in over 90% of tumors across all cancer types; and is lacking in normal, healthy tissues. Imetelstat sodium is a novel, first-in-class telomerase inhibitor that is a covalently-lipidated 13-mer oligonucleotide (shown below) complimentary to the human telomerase RNA (hTR) template region. Imetelstat sodium does not function through an anti-sense mechanism and therefore lacks the side effects commonly observed with such therapies. Imetelstat sodium is the sodium salt of imetelstat (shown below):

Imetelstat sodium

Unless otherwise indicated or clear from the context, references below to imetelstat also include salts thereof. As mentioned above, imetelstat sodium in particular is the sodium salt of imetelstat.

[0006] ABT-199/venetoclax (trade name Venclexta) is an FDA approved Bcl-2 inhibitor for use in chronic lymphocytic leukemia (CLL) patients with dell7p who are relapsed/refractory. ABT-199 is also known as ABT 199, GDC0199, GDC-0199 or RG7601. The chemical name for ABT-199 is 4-[4-[[2-(4-chlorophenyl)-4,4-dimethylcyclohexen-l-yl]methyl]piperazin-l-yl]-N-[3-nitro-4-(oxan-4-ylmethylamino)phenyl]sulfonyl-2-(lH-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide (Cas No. 1257044-40-8). Unless otherwise indicated or clear from the context, references below to ABT-199 also include pharmaceutically acceptable salts thereof. Specifically in the Examples however, ABT-199 was used in the free base form.

[0007] ABT-199, shown below in the free base form, is highly specific to Bcl-2, unlike other first generation inhibitors which show affinity for related Bel family members and induce greater side effects. Inhibition of Bcl-2 blocks the pro-apoptotic signals caused by damage to or abnormalities within cellular DNA and ultimately leads to programmed cell death in treated cells via the caspase cascade and apoptosis through the intrinsic pathway.

ABT-199 (shown in the free base form)

PATENT

WO-2019011829

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2019011829&tab=PCTDESCRIPTION&maxRec=1000

Improved process for preparing imetelstat . claiming use of a combination comprising a telomerase inhibitor, specifically imetelstat sodium and a Bcl-2 inhibitor, specifically ABT-199 for treating hematological cancer such as acute myeloid leukemia, essential thrombocythemia and polycythemia vera, specifically acute myeloid leukemia.

Imetelstat (SEQ ID NO: 1 ) is a N3′- P5′ thiophosphoramidate oligonucleotide covalently linked to a palmitoyl lipid moiety and has been described in WO-2005/023994 as compound (1 F). The sodium salt of imetelstat acts as a potent and specific telomerase inhibitor and can be used to treat telomerase-mediated disorders, e.g. cancer, including disorders such as myelofibrosis (MF), myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML).

The structure of imetelstat sodium is shown below :

The structure of imetelstat can also be represented as shown below

imetelstat

The LPT group represents the palmitoyi lipid that is covalently linked to the N3′- P5′ thiophosphor-amidate oligonucleotide. The base sequence of the thirteen nucleotides is as follows :

TAGGGTTAGACAA and is represented by the bases B₁ to B₁₃. The -NH-P(=S)(OH)-and -0-P(=S)(OH)- groups of the structure can occur in a salt form. It is understood that salt forms of a subject compound are encompassed by the structures depicted herein, even if not specifically indicated.

Imetelstat sodium can also be represented as follows

o H

LPT = CH3-(CH₂)i4-C-N-CH2-(CHOH)-CH2-

The -NH-P(=S)(OH)- group and the thymine, adenine, guanine and cytosine bases can occur in other tautomeric arrangements then used in the figures of the description. It is understood that all tautomeric forms of a subject compound are encompassed by a structure where one possible tautomeric form of the compound is described, even if not specifically indicated.

Prior art

The synthetic scheme used in WO-2005/023994 to prepare imetelstat as compound (1 F) is described in Scheme 1 and Scheme 2. The synthesis of this oligonucleotide is achieved using the solid-phase phosphoramidite methodology with all reactions taking place on solid-phase support. The synthesis of imetelstat is carried out on controlled pore glass (LCAA-CPG) loaded with

3-palmitoylamido-1-0-(4, 4′-dimethoxytrityl)-2-0-succinyl propanediol. The oligonucleotide is assembled from the 5′ to the 3′ terminus by the addition of protected nucleoside 5′-phosphor-amidites with the assistance of an activator. Each elongation cycle consists of 4 distinct, highly controlled steps : deprotection, amidite coupling, sulfurization and a capping step.

Scheme 1 : imetelstat synthetic scheme cycle 1

3. Sulfurization

In Scheme 1 the solid-phase supported synthesis starts with removal of the acid-labile 4,4-dimethoxy-trityl (DMT) protecting group from the palmitoylamidopropanediol linked to the solid-phase support. The first phosphoramidite nucleotide is coupled to the support followed by sulfurization of the phosphor using a 0.1 M solution of phenylacetyl disulfide (PADS) in a mixture of acetonitrile and 2,6-lutidine (1 : 1 ratio). Then a capping step is applied to prevent any unreacted solid-phase support starting material from coupling with a phosphoramidite nucleotide in the following reaction cycles. Capping is done using an 18:1 :1 mixture of THF / isobutyric anhydride / 2,6-lutidine.

After the first cycle on the solid-phase support, chain elongation is achieved by reaction of the 3′-amino group of the support-bound oligonucleotide with an excess of a solution of the protected nucleotide phosphoramidite monomer corresponding to the next required nucleotide in the sequence as depicted in Scheme 2.

Scheme 2 : imetelstat synthetic scheme cycle 2-13

In Scheme 2 the first cycle is depicted of the chain elongation process which is achieved by deprotection of the 3′-amino group of the support-bound oligonucleotide (a), followed by a coupling reaction of the 3′-amino group of the support-bound oligonucleotide (b) with an excess of a solution of a 5′-phosphoramidite monomer corresponding to the next required nucleotide in the sequence of imetelstat. The coupling reaction is followed by sulfurization of the phosphor of the support-bound oligonucleotide (c) and a capping step (see Scheme 3) to prevent any unreacted solid-phase support starting material (b) from coupling with a 5′-phosphoramidite nucleotide in the following reaction cycles. The reaction cycle of Scheme 2 is repeated 12 times before the solid-phase support-bound oligonucleotide is treated with a 1 :1 mixture of ethanol and concentrated ammonia, followed by HPLC purification to obtain imetelstat.

Scheme 3

The capping step using an 18:1 : 1 mixture of THF / isobutyric anhydride / 2,6-lutidine is done to convert after the coupling step any remaining solid-phase support bound oligonucleotide (b) with a primary 3′-amino group into oligonucleotide (e) with a protected (or ‘capped’) 3′-amino group in order to prevent the primary 3′-amino group from coupling with a phosphoramidite nucleotide in the next reaction cycles.

WO-01/18015 discloses in Example 3 with SEQ ID No. 2 a N3’^P5′ thiophosphoramidate oligonucleotide and a process for preparing this oligonucleotide encompassing a capping step.

Herbert B-S et al. discusses the lipid modification of GRN163 (Oncogene (2005) 24, 5262-5268).

Makiko Horie et al. discusses the synthesis and properties of 2′-0,4′-C-ethylene-bridged nucleic acid oligonucleotides targeted to human telomerase RNA subunit (Nucleic Acids Symposium Series (2005) 49, 171-172).

Description of the invention

The coupling reaction in the solid-phase support bound process disclosed in WO-01/18015 and WO-2005/023994 include a capping step to prevent any unreacted primary 3′ amino groups on the support-bound oligonucleotide from reacting during subsequent cycles.

It has now surprisingly been found that the use of a capping step as described in the prior art is superfluous and that imetelstat can be prepared using a 3-step cycle without an additional capping step with nearly identical yield and purity compared to the prior art 4-step cycle that uses a specific capping step. Eliminating the capping step from each cycle benefits the overall process by reducing the number of cycle steps by 22% (from 54 to 42 steps) and consequent reduction of process time. Also, the solvent consumption is reduced due to the reduction of cycle steps which makes for a greener process.

Wherever the term “capping step” is used throughout this text, it is intended to define an additional chemical process step wherein the primary free 3′-amino group on the solid-phase support bound oligonucleotide is converted into a substituted secondary or tertiary 3′-amino group that is not capable of participating in the coupling reaction with a protected 3′-aminonucleoside-5′-0-cyanoethyl-N,N-diisopropylamino-phosphoramidite monomer in the ensuing coupling step.

In one embodiment, the present invention relates to a method of synthesizing an oligonucleotide N3′ – P5′ thiophosphoramidate of formula

imetelstat

the method comprises of

a) providing a first 3′-amino protected nucleotide attached to a solid-phase support of formula (A) wherein PG is an acid-labile protecting group;

b) deprotecting the protected 3′-amino group to form a free 3′-amino group;

c) reacting the free 3′-amino group with a protected 3′-aminonucleoside-5′-0-cyanoethyl-N,N- diisopropylaminophosphoramidite monomer of formula (B _n) wherein n = 2 to form an internucleoside N3′- P5′-phosphoramidite linkage;

mer (B’n)

d) sulfurization of the internucleoside phosphoramidite group using an acyl disulfide to form a N3′- P5′ thiophosphoramidate;

e) repeating 1 1 times in successive order the deprotection step b), the coupling step c) with a protected 3′-aminonucleoside-5′-0-cyanoethyl-N,N-diisopropylamino-phosphoramidite monomer of formula (B _n) wherein the protected nucleoside base B’ in monomer (B _n) is successively the protected nucleobase B₃ to B₁₃ in the respective 1 1 coupling steps, and the sulfurization step d);

f) removing the acid-labile protecting group PG; and

g) cleaving and deprotecting imetelstat from the solid-phase support;

characterized in that no additional capping step is performed in any of the reaction steps a) to e).

In one embodiment, the present invention relates to a method of synthesizing the N3′ – P5′

thiophosphoramidate oligonucleotide imetelstat of formula

imetelstat

the method comprises of

a) providing a first 3′-amino protected nucleotide attached to a solid-phase support of formula (A) wherein PG is an acid-labile protecting group;

b) deprotecting the protected 3′-amino group to form a free 3′-amino group;

c) reacting the free 3′-amino group with a protected 3′-aminonucleoside-5′-0-cyanoethyl- Ν,Ν-diisopropylaminophosphoramidite monomer of formula (B _n), wherein B _n with n = 2 is protected A, to form an internucleoside N3′- P5′-phosphoramidite linkage;

mer

d) sulfurization of the internucleoside phosphoramidite group using an acyl disulfide to form a N3′- P5′ thiophosphoramidate;

e) repeating 1 1 times in successive order the deprotection step b), the coupling step c) with a protected 3′-aminonucleoside-5′-0-cyanoethyl-N,N-diisopropylamino-phosphoramidite monomer of formula (B _n) wherein the nucleoside base B’ of monomer (B _n) is protected B except when B is thymine, and wherein B_n is successively nucleobase B₃ to B₁₃ in the respective 1 1 coupling steps, and the sulfurization step d);

f) removing the acid-labile protecting group PG; and

g) deprotecting and cleaving imetelstat from the solid-phase support;

characterized in that no additional capping step is performed in any of the reaction steps a) to e).

In one embodiment, the present invention relates to a method of synthesizing the N3′ – P5′

thiophosphoramidate oligonucleotide imetelstat of formula

imetelstat

thymine

adenine

guanine

cytosine

9 H

LPT =CH₃-(CH₂)i4-C-N-CH2-(CHOH)-CH2-

the method comprises of

a) providing a first protected 3′-amino nucleotide attached to a solid-phase support of formula (A) wherein PG is an acid-labile protecting group;

b) deprotecting the PG-protected 3′-amino nucleotide to form a free 3′-amino nucleotide of formula (A’);

c) coupling the free 3′-amino nucleotide with a protected 3′-aminonucleoside-5′-0- cyanoethyl-N,N-diisopropylaminophosphoramidite monomer (B _n), wherein B _nwith n = 2 is protected A, to form an internucleoside N3′- P5′-phosphoramidite linkage;

monomer (B’_n)

d) sulfurizing the N3′- P5′-phosphoramidite linkage using an acyl disulfide to form an internucleoside N3′- P5′ thiophosphoramidate linkage;

e) repeating 1 1 times in successive order:

the deprotecting step b);

the coupling step c) with a protected 3′-aminonucleoside-5′-0-cyanoethyl-N,N- diisopropylamino-phosphoramidite monomer (B _n) wherein the nucleoside base B’ of monomer (B _n) is protected B except when B is thymine, and wherein B_n is successively nucleobase B₃ to B₁₃ in the respective 1 1 coupling steps; and

the sulfurizing step d);

to produce a protected N3′ – P5′ thiophosphoramidate oligonucleotide imetelstat attached to the solid-phase support;

f) removing the 3′-terminal acid-labile protecting group PG from the protected N3′ – P5′ thiophosphoramidate oligonucleotide imetelstat; and

g) deprotecting and cleaving the protected N3′ – P5′ thiophosphoramidate oligonucleotide imetelstat from the solid-phase support to produce imetelstat;

characterized in that no additional capping step is performed in any of the reaction steps a) to e).

A wide variety of solid-phase supports may be used with the invention, including but not limited to, such as microparticles made of controlled pore glass (CPG), highly cross-linked polystyrene, hybrid controlled pore glass loaded with cross-linked polystyrene supports, acrylic copolymers, cellulose, nylon, dextran, latex, polyacrolein, and the like.

The 3′-amino protected nucleotide attached to a solid-phase support of formula (A)

can be prepared as disclosed in WO-2005/023994 wherein a controlled pore glass support loaded with 3-palmitoylamido-1-0-(4, 4′-dimethoxytrityl)-2-0-succinyl propanediol has been coupled with a protected 3′-aminonucleoside-5′-0-cyanoethyl-N,N-diisopropylaminophosphoramidite monomer of formula (B^ )

monomer (B’-| ) wherein B’-| = T

wherein PG is an acid-labile protecting group. Suitable acid-labile 3′-amino protecting groups PG are, but not limited to, e.g. triphenylmethyl (i.e. trityl or Tr), p-anisyldiphenylmethyl (i.e. mono-methoxytrityl or MMT), and di-p-anisylphenylmethyl (i.e. dimethoxytrityl or DMT).

The protected 3′-aminonucleoside-5′-0-cyanoethyl-N,N-diisopropylaminophosphoramidite monomers of formula (B _n) have a 3′-amino protecting group PG which is an acid-labile group, such as triphenylmethyl (i.e. trityl or Tr), p-anisyldiphenylmethyl (i.e. monomethoxytrityl or MMT), or di-p-anisylphenylmethyl (i.e. dimethoxytrityl or DMT). Furthermore the nucleoside base B’ is protected with a base-labile protecting group (except for thymine).

ed A ed C ed A ed A

B’s = protected A G = guanine

B’g = protected G C = cytosine

The nucleotide monomers and B’₂ to B’₁₃ are used successively in the 13 coupling steps starting from the provision of a solid-phase support loaded with 3-palmitoylamido-1-0-(4, 4′-dimethoxytrityl)-2-0-succinyl propanediol and coupled to nucleotide monomer and the following cycle of 12 deprotection, coupling, and sulfurization reactions wherein the nucleotide monomers B’₂ to B -I 3 are used.

The 3′-amino protecting group PG can be removed by treatment with an acidic solution such as e.g. dichloroacetic acid in dichloromethane or toluene.

The nucleoside base B’ in the protected 3′-aminonucleoside-5′-0-cyanoethyl-N,N-diisopropyl-aminophosphoramidite monomers of formula (B _n) is protected with a base-labile protecting group which is removed in step g). Suitable base-labile protecting groups for the nucleoside base adenine, cytosine or guanine are e.g. acyl groups such as acetyl, benzoyl, isobutyryl, dimethyl-formamidinyl, or dibenzylformamidinyl. Under the reaction conditions used in oligonucleotide synthesis the thymine nucleoside base does not require protection. Such protected 3′- amino-nucleoside-5′-0-cyanoethyl-N,N-diisopropylaminophosphoramidite monomers of formula (B _N) having a 3′-amino protected with an acid-labile group protecting group PG and a nucleoside base B’ protected with a base-labile protecting group are commercially available or can be prepared as described in WO-2006/014387.

The coupling step c) is performed by adding a solution of protected 3′-aminonucleoside-5′-0-cyanoethyl-N,N-diisopropylaminophosphoramidite monomer of formula (B_N) and a solution of an activator (or a solution containing the phosphoramidite monomer (B_N) and the activator) to the reaction vessel containing the free amino group of an (oligo)nucleotide covalently attached to a solid support. The mixture is then mixed by such methods as mechanically vortexing, sparging with an inert gas, etc. Alternately, the solution(s) of monomer and activator can be made to flow through a reaction vessel (or column) containing the solid-phase supported (oligo)nucleotide with a free 3′-amino group. The monomer and the activator either can be premixed, mixed in the valve-block of a suitable synthesizer, mixed in a pre-activation vessel and preequilibrated if desired, or they can be added separately to the reaction vessel.

Examples of activators for use in the invention are, but not limited to, tetrazole, 5-(ethylthio)-1 H-tetrazole, 5-(4-nitro-phenyl)tetrazole, 5-(2-thienyl)-1 H-tetrazole, triazole, pyridinium chloride, and the like. Suitable solvents are acetonitrile, tetrahydrofuran, dichloromethane, and the like. In practice acetonitrile is a commonly used solvent for oligonucleotide synthesis.

The sulfurization agent for use in step d) is an acyl disulfide dissolved in a solvent. Art know acyl disulfides are e.g. dibenzoyl disulphide, bis(phenylacetyl) disulfide (PADS), bis(4-methoxybenzoyl) disulphide, bis(4-methylbenzoyl) disulphide, bis(4-nitrobenzoyl) disulphide and bis(4-chlorobenzoyl) disulfide.

Phenylacetyl disulfide (PADS) is a commonly used agent for sulfurization reactions that it is best ‘aged’ in a basic solution to obtain optimal sulfurization activity (Scotson J.L. et al., Org. Biomol. Chem., vol. 14, 10840 – 10847, 2016). A suitable solvent for PADS is e.g. a mixture of a basic solvent such as e.g. 3-picoline or 2,6-lutidine with a co-solvent such as acetonitrile, toluene, 1-methyl-pyrrolidinone or tetrahydrofuran. The amount of the basic solvent to the amount of the co-solvent can be any ratio including a 1 :1 ratio. Depending upon the phosphite ester to be converted into its corresponding thiophospate, both ‘fresh’ and ‘aged’ PADS can be used however ‘aged’ PADS has been shown to improve the rate and efficiency of sulfurization. ‘Aged’ PADS solutions are freshly prepared PADS solutions that were maintained some time before usage in the sulfurization reaction. Aging times can vary from a few hours to 48 hours and the skilled person can determine the optimal aging time by analysing the sulfurization reaction for yield and purity.

For the preparation of imetelstat in accordance with the present invention, a PADS solution in a mixture of acetonitrile and 2,6-lutidine, preferably in a 1 :1 ratio, with an aging time of 4 to 14 hours is used. It has been found that when 2,6-lutidine is used, limiting the amount of 2,3,5-collidine (which is often found as an impurity in 2,6-lutidine) below 0.1 % improves the efficiency of sulfurization and less undesirable phosphor oxidation is observed.

In step g) imetelstat is deprotected and cleaved from the solid-phase support. Deprotection includes the removal of the β-cyanoethyl groups and the base-labile protecting groups on the nucleotide bases. This can be done by treatment with a basic solution such as a diethylamine (DEA) solution in acetonitrile, followed by treatment with aqueous ammonia dissolved in an alcohol such as ethanol.

The reaction steps a) to f) of the present invention are carried out in the temperature range of 10°C to 40°C. More preferably, these reactions are carried out at a controlled temperature ranging from 15°C to 30°C. In particular reaction step b) of the present invention is carried out in the temperature range of 15°C to 30°C; more in particular 17°C to 27°C. In particular reaction step d) of the present invention is carried out in the temperature range of 17°C to 25°C; more in particular 18°C to 22°C; even more in particular 19°C. The step g) wherein imetelstat is deprotected and cleaved from the solid-phase support is carried out at a temperature ranging from 30°C to 60°C. Depending upon the equipment and the specific reaction conditions used, the optimal reaction temperature for each step a) to g) within the above stated ranges can be determined by the skilled person.

After each step in the elongation cycle, the solid-phase support is rinsed with a solvent, for instance acetonitrile, in preparation for the next reaction.

After step g), crude imetelstat is obtained in its ammonium salt form which is then purified by a preparative reversed phase high performance liquid chromatography (RP-HPLC) by using either polymeric or silica based resins to get purified imetelstat in triethyl amine form. An excess of a sodium salt is added, and then the solution is desalted by diafiltration thereby yielding imetelstat sodium which is then lyophilized to remove water.

Experimental part

‘Room temperature’ or ‘ambient temperature’ typically is between 21-25 °C.

Experiment 1 (no capping step)

All the reagents and starting material solutions were prepared including 3% dichloroacetic acid (DCA) in toluene, 0.5 M 5-(ethylthio)-1 H-tetrazole in acetonitrile, 0.15 M of all 4 nucleotide monomers of formula (B _n) in acetonitrile, 0.2 M phenyl acetyl disulfide (PADS) in a 1 :1 mixture of acetonitrile and 2,6-lutidine and 20% DEA (diethylamine) in acetonitrile.

The oligonucleotide synthesis was performed in the direction of 5′ to 3′ utilizing a repetitive synthesis cycle consisting of detritylation followed by coupling, and sulfurization performed at ambient temperature.

A column (diameter : 3.5 cm) was packed with a solid-support loaded with 3-palmitoylamido-1-0- (4, 4′-dimethoxytrityl)-2-0-succinyl propanediol (3.5 mmol based on a capacity of 400 μιηοΙ/g) that was coupled with the nucleotide monomer B Detritylation was achieved using 3% dichloroacetic acid (DCA) in toluene (amount is between 6.5 and 13.4 column volumes in each detritylation step) and the solid-support bound nucleotide was washed with acetonitrile (amount: 5 column volumes). Coupling with the next nucleotide monomer of formula (B _n) was achieved by pumping a solution of 0.5 M 5-(ethylthio)-1 H-tetrazole in acetonitrile and 0.15 M of the next nucleotide monomer of formula (B _n) in the sequence, dissolved in acetonitrile, through the column. The column was washed with acetonitrile (amount : 2 column volumes). Then sulfurization was performed by

pumping a solution of 0.2 M phenyl acetyl disulfide (PADS) in a 1 :1 mixture of acetonitrile and 2,6-lutidine mixture through the column followed by washing the column with acetonitrile (amount : 5 column volumes).

The synthesis cycle of detritylation, coupling with the next nucleotide monomer of formula (B _n) and sulfurization was repeated 12 times, followed by detritylation using 3% dichloroacetic acid (DCA) in toluene (amount is between 6.5 and 13.4 column volumes).

4 to 24 hours at 55°C, cooled to room temperature, and slurry was filtered to remove the polymeric support. The solution comprising imetelstat in its ammonium form was subjected to the HPLC analysis procedure of Experiment 3.

Experiment 2 (with capping step)

A column (diameter : 3.5 cm) was packed with a solid-support loaded with 3-palmitoylamido-1-0-(4, 4′-dimethoxytrityl)-2-0-succinyl propanediol (3.5 mmol based on a capacity of 400 μιηοΙ/g) that was coupled with the nucleotide monomer B Detritylation was achieved using 3% dichloroacetic acid (DCA) in toluene (amount is between 6.5 and 13.4 column volumes in each detritylation step) and the solid-support bound nucleotide was washed with acetonitrile (amount : 5 column volumes). Coupling with the next nucleotide monomer of formula (B _n) was achieved by pumping a solution of 0.5 M 5-(ethylthio)-1 H-tetrazole in acetonitrile and 0.15 M of the next nucleotide monomer of formula (B _n) in the sequence, dissolved in acetonitrile, through the column. The column was washed with acetonitrile (amount : 2 column volumes). Then sulfurization was performed by pumping a solution of 0.2 M phenyl acetyl disulfide (PADS) in a 1 :1 mixture of acetonitrile and 2,6-lutidine mixture through the column followed by washing the column with acetonitrile (amount :

5 column volumes).

The sulfurization was followed by a capping step. Each capping in a given cycle used 37-47 equivalents (eq.) of the capping agent NMI, and 9-1 1 equivalents of the capping agent B isobutryic anhydride (IBA), and 1 .4-1.8 equivalents of 2,6 lutidine. Capping agents A and B were pumped through the column with separate pumps at different ratios such as 50:50, 35:65, 65:35.

The synthesis cycle of detritylation, coupling with the next nucleotide monomer of formula (B _n) and sulfurization, and capping step was repeated 12 times, followed by detritylation using 3% dichloroacetic acid (DCA) in toluene (amount is between 6.5 and 13.4 column volumes).

Upon completion of the synthesis cycle, the crude oligonucleotide on the solid-support support was treated with a diethylamine (DEA) solution followed by treatment with ammonium hydroxide solution: ethanol (3: 1 volume ratio) at a temperature of 55°C. The reaction mixture was aged for 4 to 24 hours at 55°C, cooled to room temperature, and slurry was filtered to remove the polymeric support. The solution comprising imetelstat in its ammonium form was subjected to the HPLC analysis procedure of Experiment 3.

Experiment 3 : comparision of no-capping vs. capping

Imetelstat obtained in Experiment 1 and Experiment 2 was analysed by HPLC. The amount of the desired full length oligonucleotide having 13 nucleotides was determined and listed in the Table below for Experiment 1 and Experiment 2. Also, the total amount of shortmer, specifically the 12mer, was determined and listed in the Table below for Experiment 1 and Experiment 2.

HPLC analysis method :

column type: Kromasil C18, 3.5 μιτι particle size, 4.6 X 150 mm

eluent:

A: 14.4 mM TEA/386 mM HFIP (hexafluoroisopropanol) /100 ppm(w/v) Na₂EDTA in water B: 50% MeOH, 50% EtOH containing 5% IPA

Gradient :

Step Run time (minutes) %B

1 0 10

2 5 10

3 12 26 (linear)

4 35 45 (linear)

5 40 50 (linear)

6 42 50

7 44 10 (linear)

8 50 10

Table : capping vs. no-capping experiments (Experiment 1 was run twice and results are listed as Experiment 1a and 1 b).

The HPLC analysis of Experiment 1 and Experiment 2 demonstrates that yield and purity are comparable for the no-capping experiment vs. the capping experiment.

Main peak % includes Full length oligonucleotide + PO impurities + depurinated impurities.

PO impurities are impurities including one or more oxophosphoramidate internucleoside linkages instead of thiophosphoramidate internucleoside linkages.

Solvent use and reaction time

0.45 L of acetonitrile/mmol is used to prepare capping agent A and capping agent B reagents which corresponds to approximately 25 % of the overall acetonitrile use during the preparation of the reagents. Since each chemical reaction step is followed by a solvent wash, after each capping step too, a solvent wash takes place which is equivalent to about 40 column volumes of the solvent. Considering that about 212 column volumes of the solvent wash is done for a given synthesis run, about 19 % of the wash solvent is used for the capping steps. Each capping step takes between 3 – 6 minutes. This corresponds to about 8 % of the overall synthesis time including the 13 cycles and DEA treatment.

Experiment 4 (detritylation temperature)

The detritylation temperature has an impact in terms of controlling n-1 and depurinated impurities. The temperature of the deblocking solution at the entrance of the synthesizer was chosen between 17.5 and 27 °C (at 3.5 mmol scale) and the selected temperature was kept the same for all detritylation steps. The acetonitrile washing was also kept at the same temperature of the deblocking solution. The % depurinated impurities increased linearly with temperature while n-1 was higher at lower temperatures.

Temperature n-1 % Depurinated Impurity %

17.5 10.7 5.3

19 7.6 6.4

22 5.4 8.7

25 6.1 10.8

27 5.3 12.3

Experiment 5 (sulfurization step temperature)

In the experiments below, the temperature (RT means room temperature) of the PADS solution used in the sulfurization reactions was tested for the % of less favourable PO impurities (these are impurities where phosphor oxidation occurred instead of sulfurization). Lower temperature results in lower PO %.

SEQ ID NO:1 – imetelstat and imetelstat sodium

5′-R-TAGGGTTAGACAA-NH₂-3′

wherein R represents palmitoyl [(CH₂)₁ CH₃] amide is conjugated through an aminoglycerol linker to the 5′-thiophosphate group of an N3′ – P5′ thiophosphoramidate (NPS) -linked oligonucleotide.

///////////IMETELSTAT, GRN163L, PHASE 3, orphan drug, FAST TRACK

CCCCCCCCCCCCCCCC(=O)NCC(COP(=S)([O-])OCC1C(CC(O1)N2C=C(C(=O)NC2=O)C)NP(=S)([O-])OCC3C(CC(O3)N4C=NC5=C4N=CN=C5N)NP(=S)([O-])OCC6C(CC(O6)N7C=NC8=C7N=C(NC8=O)N)NP(=S)([O-])OCC9C(CC(O9)N1C=NC2=C1N=C(NC2=O)N)NP(=S)([O-])OCC1C(CC(O1)N1C=NC2=C1N=C(NC2=O)N)NP(=S)([O-])OCC1C(CC(O1)N1C=C(C(=O)NC1=O)C)NP(=S)([O-])OCC1C(CC(O1)N1C=C(C(=O)NC1=O)C)NP(=S)([O-])OCC1C(CC(O1)N1C=NC2=C1N=CN=C2N)NP(=S)([O-])OCC1C(CC(O1)N1C=NC2=C1N=C(NC2=O)N)NP(=S)([O-])OCC1C(CC(O1)N1C=NC2=C1N=CN=C2N)NP(=S)([O-])OCC1C(CC(O1)N1C=CC(=NC1=O)N)NP(=S)([O-])OCC1C(CC(O1)N1C=NC2=C1N=CN=C2N)NP(=O)(OCC1C(CC(O1)N1C=NC2=C1N=CN=C2N)N)[S-])O.[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+]

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OTL-38

Pafolacianine sodium [USAN]RN: 1628858-03-6UNII: 4HUF3V875C

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Asparaginase erwinia chrysanthemi (recombinant)-rywn

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Pafolacianine sodium [USAN]
RN: 1628858-03-6
UNII: 4HUF3V875C