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ORGANIC SPECTROSCOPY

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DR ANTHONY MELVIN CRASTO Ph.D

DR ANTHONY MELVIN CRASTO Ph.D

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

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Gadopiclenol


STR1
Chemical structure of gadopiclenol [gadolinium chelate of 2,2′,2″-(3,6,9-triaza-1(2,6)-pyridinacyclodecaphane-3,6,9-triyl)tris(5-((2,3-dihydroxypropyl)amino)-5-oxopentanoic acid)]. The PCTA parent structure is shown in red. Two water molecules are included to show the coordination in solution.
Molecules 27 00058 g003 550

Gadopiclenol

ガドピクレノール;

FormulaC35H54N7O15. Gd
CAS933983-75-6
Mol weight970.0912

FDA APPROVED 2022/9/21, Elucirem

Diagnostic agent (MR imaging), WHO 10744, P 03277, UNII: S276568KOY

EluciremTM; G03277; P03277; VUEWAY

(alpha3,alpha6,alpha9-Tris(3-((2,3-dihydroxypropyl)amino)-3-oxopropyl)-3,6,9,15-tetraazabicyclo(9.3.1)pentadeca-1(15),11,13-triene-3,6,9-triacetato(3-)-kappaN3,kappaN6,kappaN9,kappaN15,kappaO3,kappaO6,kappaO9)gadolinium

Molecules 27 00058 g002 550
  • OriginatorGuerbet
  • ClassDiagnostic agents; Gadolinium-containing contrast agents; Macrocyclic compounds; Propylamines; Pyridines
  • Mechanism of ActionMagnetic resonance imaging enhancers
  • RegisteredCNS disorders
  • Phase IIIUnspecified
  • Phase IILiver cancer
  • 21 Sep 2022Registered for CNS disorders (Diagnosis) in USA (IV)
  • 13 Jun 2022Guerbet plans to launch Gadopiclenol in Europe
  • 13 Jun 2022The European Medicines Agency (EMA) accepts brand name EluciremTM for Gadopiclenol

PATENT

https://patents.google.com/patent/WO2020030618A1/en

MRI contrast agents used in daily diagnostic practice typically include gadolinium complex compounds characterized by high stability constants that guarantee against the in vivo release of the free metal ion (that is known to be extremely toxic for living organisms).

Another key parameter in the definition of the tolerability of a gadolinium-based contrast agent is the kinetic inertness (or kinetic stability) of Gd(III)-complex, that is estimated through the half-life (ti/2) of the dissociation (i.e. decomplexation) of the complex.

A high inertness becomes crucial in particular for those complex compounds having lower thermodynamic stability and/or longer retention time before excretion, in order to avoid or minimize possible decomplexation or transmetallation reactions.

EP1931673 (Guerbet) discloses PCTA derivatives of formula

Figure imgf000002_0001

and a synthetic route for their preparation.

EP 2988756 (same Applicant) discloses a pharmaceutical composition comprising the above derivatives together with a calcium complex of 1,4,7, 10-tetraazacyclododecane- 1,4,7, 10-tetraacetic acid. According to the EP 2988756, the calcium complex compensates the weak thermodynamic stability observed for PCTA-based gadolinium complexes, by forming, through transmetallation, a strong complex with free lanthanide ion, thereby increasing the tolerability of the contrast agent.

Both EP1931673 and EP 2988756 further refer to enantiomers or diastereoisomers of the claimed compounds, or mixture thereof, preferentially chosen from the RRS, RSR, and RSS diastereoisomers. Both the above patents disclose, among the specific derivatives, (a3, a6, a9)-tris(3- ((2,3-dihydroxypropyl)amino)-3-oxopropyl)-3,6,9,15-tetraazabicyclo(9.3.1)pentadeca- l(15),l l,13-triene-3,6,9-triacetato(3-)-(KN3,KN6,KN9,KN15,K03,K06,K09)gadolinium, more recently identified as gadolinium chelate of 2,2′,2″-(3,6,9-triaza-l(2,6)- pyridinacyclodecaphane-3,6,9-triyl)tris(5-((2,3-dihydroxypropyl)amino)-5-oxopentanoic acid), (CAS registry number: 933983-75-6), having the following formula

Figure imgf000003_0001

otherwise identified as P03277 or Gadopiclenol.

For Gadopiclenol, EP1931673 reports a relaxivity of 11 mM _1_1Gd 1 (in water, at 0.5 T, 37°C) while EP 2988756 reports a thermodynamic equilibrium constant of 10 14 9 (log Kterm

= 14.9).

Furthermore, for this same compound a relaxivity value of 12.8 mM _11 in human serum (37°C, 1.41 T), stability (log Kterm) of 18.7, and dissociation half-life of about 20 days (at pH 1.2; 37°C) have been reported by the proprietor (Investigative Radiology 2019, Vol 54, (8), 475-484).

The precursor for the preparation of the PCTA derivatives disclosed by EP1931673 (including Gadopiclenol) is the Gd complex of the 3,6,9,15-tetraazabicyclo- [9.3.1]pentadeca-l(15),l l,13-triene-tri(a-glutaric acid) having the following formula

Figure imgf000003_0002

Gd(PCTA-tris-glutaric acid)

herein identified as “Gd(PCTA-tris-glutaric acid)”. In particular, Gadopiclenol is obtained by amidation of the above compound with isoserinol.

As observed by the Applicant, Gd(PCTA-tris-qlutaric acid) has three stereocenters on the glutaric moieties (identified with an asterisk (*) in the above structure) that lead to a 23 = 8 possible stereoisomers. More particularly, the above structure can generate four pairs of enantiomers, schematized in the following Table 1

Table 1

Figure imgf000004_0002

Isomer RRR is the mirror image of isomer SSS and that is the reason why they are called enantiomers (or enantiomer pairs). As known, enantiomers display the same physicochemical properties and are distinguishable only using chiral methodologies, such as chiral chromatography or polarized light.

On the other hand, isomer RRR is neither equal to nor is it the mirror image of any of the other above six isomers; these other isomers are thus identified as diastereoisomers of the RRR (or SSS) isomer. Diastereoisomers may display different physicochemical properties, (e.g., melting point, water solubility, relaxivity, etc.).

Concerning Gadopiclenol, its chemical structure contains a total of six stereocenters, three on the glutaric moieties of the precursor as above discussed and one in each of the three isoserinol moieties attached thereto, identified in the following structure with an asterisk (*) and with an empty circle (°), respectively:

Figure imgf000004_0001

This leads to a total theoretical number of 26 = 64 stereoisomers for this compound. However, neither EP1931673 nor EP 2988756 describe the exact composition of the isomeric mixture obtained by following the reported synthetic route, nor does any of them provide any teaching for the separation and characterization of any of these isomers, or disclose any stereospecific synthesis of Gadopiclenol. Summary of the invention

The applicant has now found that specific isomers of the above precursor Gd(PCTA- tris-glutaric acid) and of its derivatives (in particular Gadopiclenol) possess improved physico-chemical properties, among other in terms of relaxivity and kinetic inertness.

An embodiment of the invention relates to a compound selected from the group consisting of:

the enantiomer [(aR,a’R,a”R)-a,a’,a”-tris(2-carboxyethyl)-3,6,9,15- tetraazabicyclo[9.3.1]pentadeca-l(15),l l,13-triene-3,6,9-triacetato(3-)- Kl\l3,Kl\l6,Kl\l9,Kl\ll5,K03,K06,K09]-gadolinium (RRR enantiomer) having the formula (la):

Figure imgf000005_0001

the enantiomer [(aS,a’S,a”S)-a,a’,a”-tris(2-carboxyethyl)-3,6,9,15-tetraazabicyclo- [9.3.1]pentadeca-l(15),ll,13-triene-3,6,9-triacetato(3-)KN3,KN6,KN9,KN15,K03,K06,K09]- gadolinium (SSS enantiomer) having the formula (lb):

Figure imgf000005_0002

the mixtures of such RRR and SSS enantiomers, and a pharmaceutically acceptable salt thereof.

Another embodiment of the invention relates to an isomeric mixture of Gd(PCTA-tris- glutaric acid) comprising at least 50% of the RRR isomer [(aR,a’R,a”R)-a,a’,a”-tris(2- carboxyethyl)-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-l(15),l l,13-triene-3,6,9- triacetato(3-)-KN3,KN6,KN9,KN15,K03,K06,K09]-gadolinium, of formula (la), or of the SSS isomer [(aS,a’S,a”S)-a,a’,a”-tris(2-carboxyethyl)-3,6,9,15- tetraazabicyclo[9.3.1]pentadeca-l(15),l l,13-triene-3,6,9-triacetato(3-)- Kl\l3,Kl\l6,Kl\l9,Kl\ll5,K03,K06,K09]-gadolinium of formula (lb), or of a mixture thereof, or a pharmaceutically acceptable salt thereof. Another aspect of the invention relates to the amides obtained by conjugation of one of the above compounds or isomeric mixture with an amino group, e.g. preferably, serinol or isoserinol.

An embodiment of the invention relates to an amide derivative of formula (II A)

F( N RI R2)3 (II A)

in which :

F is:

a RRR enantiomer residue of formula Ilia

Figure imgf000006_0001

a SSS enantiomer residue of formula Illb

Figure imgf000006_0002

or a mixture of such RRR and SSS enantiomer residues;

and each of the three -NRIR2 group is bound to an open bond of a respective carboxyl moiety of F, identified with a full circle (·) in the above structures;

Ri is H or a Ci-Ce alkyl, optionally substituted by 1-4 hydroxyl groups;

R2 is a Ci-Ce alkyl optionally substituted by 1-4 hydroxyl groups, and preferably a C1-C3 alkyl substituted by one or two hydroxyl groups.

Another embodiment of the invention relates to an isomeric mixture of an amide derivative of Gd(PCTA-tris-glutaric acid) having the formula (II B)

F'( N RI R2)3 (II B)

in which :

F’ is an isomeric mixture of Gd(PCTA-tris-glutaric acid) residue of formula (III)

Figure imgf000007_0001

said isomeric mixture of the Gd(PCTA-tris-glutaric acid) residue comprising at least 50 % of an enantiomer residue of the above formula (Ilia), of the enantiomer residue of the above formula (Illb), or of a mixture thereof; and each of the -NR1R2 groups is bound to an open bond of a respective carboxyl moiety of F’, identified with a full circle (·) in the above structure, and is as above defined for the compounds of formula (II A).

EXPERIMENTAL PART

HPLC characterization of the obtained compounds.

General procedures

Procedure 1: HPLC Characterization of Gd(PCTA-tris-glutaric acid) (isomeric mixture and individual/enriched isomers).

The HPLC characterization of the Gd(PCTA-tris-glutaric acid) obtained as isomeric mixture from Example 1 was performed with Agilent 1260 Infinity II system. The experimental setup of the HPLC measurements are summarized below.

Analytical conditions

HPLC system HPLC equipped with quaternary pump, degasser, autosampler,

PDA detector ( Agilent 1260 Infinity II system)

Stationary phase: Phenomenex Gemini® 5pm C18 lloA

Mobile phase: H2O/HCOOH 0.1% : Methanol

Elution : Gradient Time (min) H2O/HCOOH 0.1% Methanol

0 95 5

5 95 5

30 50 50

35 50 50

40 95 5

Flow 0.6 mL/min

Temperature 25 °C

Detection PDA scan wavelenght 190-800nm

Injection volume 50 pL

Sample Cone. 0.2 mM Gd(PCTA-tris-glutaric acid) complex

Stop time 40 min

Retention time GdL = 18-21 min.

Obtained HPLC chromatogram is shown in Figure 1

The HPLC chromatogram of the enriched enantiomers pair C is shown in Figure 2.

Procedure 2: HPLC Characterization of Gadopiclenol (isomeric mixture) and compounds obtained by coupling of enantiomers pair C with R, S, or racemic isoserinol.

The HPLC characterization of Gadopiclenol either as isomeric mixture obtained from Example 2, or as the compound obtained by conjugation of enantiomers pair C of the Gd(PCTA-tris-glutaric acid) with R, S, or racemic isoserinol was performed with Thermo Finnigan LCQ DECA XPPIus system. The experimental setup of the HPLC measurements are summarized below.

Analytical conditions

HPLC system HPLC equipped with quaternary pump, degasser, autosampler,

PDA and MS detector (LCQ Deca XP-Plus – Thermo Finnigan )

Stationary phase: Phenomenex Gemini 5u C18 110A

Mobile phase: H2O/TFA 0.1% : Acetonitrile/0.1%TFA

Elution : Gradient Time (min) H2O/TFA 0.1% Acetonitrile/0.1%TFA

0 100 0

5 100 0

22 90 10

26 90 10

Flow 0.5 mL/min

Temperature 25 °C

Detection PDA scan wavelenght 190-800nm

MS positive mode – Mass range 100-2000

Injection volume 50 pL

Sample cone. 0.2 mM Gd complex

Stop time 26 min

Retention time GdL = 20-22min.

Obtained HPLC chromatograms are shown in Figure 6.

Procedure 3: Chiral HPLC method for the separation of enantiomers of the compound C

A specific chiral HPLC method was set up in order to separate the RRR and SSS enantiomers of the enantiomers pair C (compound VI), prepared as described in Example 3. The separation and characterization of the enantiomers were performed with Agilent 1200 system or Waters Alliance 2695 system. The experimental setup of the HPLC measurements are summarized below.

Analytical conditions

HPLC System HPLC equipped with quaternary pump, degasser, autosampler,

PDA detector

Stationary phase SUPELCO Astec CHIROBIOTIC 5 pm 4.6x250mm

Mobile phase H2O/HCOOH 0.025% : Acetonitrile

Elution : isocratic 2% Acetonitrile for 30 minutes

Flow 1 mL/min

Column Temperature 40°C

Detection 210-270 nm. Obtained HPLC chromatogram is shown in Figure 5a) compared to the chromatograms of the pure RRR enantiomer (compound XII of Example 5, Tr. 7.5 min.) and the pure SSS enantiomer (Compound XVII of Example 6, Tr. 8.0 min), shown in figure 5b) and 5c), respectively.

Example 1: Synthesis of Gd(PCTA-tris-glutaric acid) (isomeric mixture)

Gd(PCTA-tris-glutaric acid) as an indiscriminate mixture of stereoisomers has been prepared by using the procedure reported in above mentioned prior-art, according to the following synthetic Scheme 1 :

Scheme 1

Figure imgf000030_0001

a) Preparation of Compound II

Racemic glutamic acid (33.0 g, 0.224 mol) and sodium bromide (79.7 g, 0.782 mol) were suspended in 2M HBr (225 ml_). The suspension was cooled to -5°C and NaN02 (28.0 g, 0.403 mol) was slowly added in small portions over 2.5 hours, maintaining the inner temperature lower than 0 °C. The yellow mixture was stirred for additional 20 minutes at a temperature of -5°C; then concentrated sulfuric acid (29 ml.) was dropped in the mixture. The obtained dark brown mixture was warmed to RT and then extracted with diethyl ether (4×150 ml_). The combined organic phases were washed with brine, dried over Na2S04 and concentrated to a brown oil (21.2 g), used in the following step without further purification. The oil was dissolved in ethanol (240 ml_), the resulting solution was cooled in ice and thionyl chloride (14.5 ml_, 0.199 mol) was slowly added. The slightly yellow solution was stirred at RT for 2 days. Then the solvent was removed in vacuum and the crude oil was dissolved in dichloromethane (200 ml.) and washed with 5% aq. NaHCC>3 (4×50 ml_), water (1×50 ml.) and brine (1×50 ml_). The organic phase was concentrated and purified on silica eluting with petroleum ether-ethyl acetate 3: 1, obtaining 19.5 g of pure product. (Yield 33%).

b) Preparation of Compound IV

A solution of Compound II (17.2 g, 0.0645 mol) in acetonitrile (40 ml.) was added to a suspension of 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-l(15),l l,13-triene (pyclen) Compound (III) (3.80 g, 0.018 mol) and K2CO3 (11.2 g, 0.0808 mol) in acetonitrile (150 ml_). The yellow suspension was heated at 65 °C for 24 h, then the salts were filtered out and the organic solution was concentrated. The orange oil was dissolved in dichloromethane and the product was extracted with 1M HCI (4 x 50 ml_). The aqueous phases were combined, cooled in ice and brought to pH 7-8 with 30% aq. NaOH. The product was then extracted with dichloromethane (4 x 50 ml.) and concentrated to give a brown oil (10.1 g, yield 73%). The compound was used in the following step without further purification.

c) Preparation of compound V

Compound IV (9.99 g, 0.013 mol) was dissolved in Ethanol (40 ml.) and 5M NaOH (40 ml_). The brown solution was heated at 80 °C for 23 h. Ethanol was concentrated; the solution was cooled in ice and brought to pH 2 with cone HCI. The ligand was purified on resin Amberlite XAD 1600, eluting with water-acetonitrile mixture, obtaining after freeze- drying 5.7 g as white solid (yield 73%). The product was characterized in HPLC by several peaks.

d) Preparation of compound VI

Compound V (5.25 g, 0.0088 mol) was dissolved in deionized water (100 ml.) and the solution was brought to pH 7 with 2M NaOH (20 ml_). A GdCh solution (0.0087 mol) was slowly added at RT, adjusting the pH at 7 with 2M NaOH and checking the complexation with xylenol orange. Once the complexation was completed, the solution was concentrated and purified on resin Amberlite XAD 1600 eluting with water-acetonitrile gradient, in order to remove salts and impurities. After freeze-drying the pure compound was obtained as white solid (6.79 g, yield 94%). The product was characterized in HPLC; the obtained HPLC chromatogram, characterized by several peaks, is shown in Figure 1 A compound totally equivalent to compound VI, consisting of an isomeric mixture with a HPLC chromatogram substantially superimposable to that of Figure 1 is obtained even by using (S)-methyl a-bromoglutarate obtained starting from L-glutamic acid.

Example 2: Synthesis of Gadopiclenol (isomeric mixture)

Gadopiclenol as an indiscriminate mixture of stereoisomers has been prepared as disclosed in EP11931673 B1 by coupling the isomeric mixture of Gd(PCTA-tris-glutaric acid) obtained from Example 1 with racemic isoserinol according to the following synthetic Scheme 2:

Scheme 2

Figure imgf000032_0001

Preparation of compound VII

Compound VI (0.90 g, 0.0011 mol) obtained from Example 1 was added to a solution of racemic isoserinol (0.40 g, 0.0044 mol) in water adjusted to pH 6 with cone. HCI. Then N- ethyl-N’-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI-HCI) (1.0 g, 0.0055 mol) and hydroxybenzotriazole (HOBT) (0.12 g, 0.00088 mol) were added and the resulting solution was stirred at pH 6 and RT for 24 h. The product was then purified on preparative HPLC on silica C18, eluting with water/acetonitrile gradient. Fractions containing the pure compound were concentrated and freeze-dried, obtaining a white solid (0.83 g, yield 78%). The product was characterized in HPLC; the obtained HPLC chromatogram is shown in Figure 4a.

Example 3: Isolation of the enantiomers pair related to the peak C.

Compound VI obtained as described in Example 1 (step d) (1.0 g, 0.0013 mol) was dissolved in water (4 ml.) and the solution was acidified to pH 2-3 with cone. HCI. The obtained solution was loaded into a pre-packed column of silica C18 (Biotage® SNAP ULTRA C18 120 g, HP-sphere C18 25 pm) and purified with an automated flash chromatography system eluting with deionized water (4 CV) and then a very slow gradient of acetonitrile. Fractions enriched of the enantiomers pair related to the peak C were combined, concentrated and freeze-dried obtaining a white solid (200 mg).

The HPLC chromatogram of the obtained enriched enantiomers pair C is shown in Figure 2.

Corresponding MS spectrum (Gd(H4L)+:752.14 m/z) is provided in Figure 3

Example 4: Coupling of the enantiomers pair C with isoserinol.

a) Coupling of the enantiomers pair C with R-isoserinol.

Enriched enantiomers pair C collected e.g. as in Example 3 (34 mg, titer 90%, 0.040 mmol) was dissolved in deionized water (5 ml_), and R-isoserinol (16 mg, 0.17 mmol) was added adjusting the pH at 6 with HCI 1M. Then, EDCI-HCI (39 mg, 0.20 mmol) and HOBT (3 mg, 0.02 mmol) were added and the solution was stirred at RT at pH 6 for 48 h. The solution was concentrated and loaded to pre-packed silica C18 column (Biotage® SNAP ULTRA C18 12 g, HP-sphere C18 25 pm), eluting with water/acetonitrile gradient using an automated flash chromatography system. Fractions containing the pure product, or showing a major peak at the HPLC with area greater than 90%, were combined, concentrated and freeze-dried giving a white solid (21 mg, yield 54%).

The HPLC chromatogram of the obtained product is shown in Figure 6b.

b) Coupling of the enantiomers pair C with S-isoserinol

Enriched enantiomers pair C collected e.g. as in Example 3 (55 mg, titer 90%, 0.066 mmol) was dissolved in deionized water (5 mL), and S-isoserinol (34 mg, 0.29 mmol) was added adjusting the pH at 6 with 1M HCI. Then, EDCI-HCI (64 mg, 0.33 mmol) and HOBT (4.5 mg, 0.033 mmol) were added and the solution was stirred at RT at pH 6 for 48 h. The solution was concentrated and loaded to pre-packed silica C18 column (Biotage® SNAP ULTRA C18 12 g, HP-sphere C18 25 pm), eluting with water/acetonitrile gradient using an automated flash chromatography system. Fractions containing the pure product, or showing a major peak at the HPLC with area greater than 90%, were combined, concentrated and freeze-dried giving a white solid (52 mg, yield 81%).

HPLC chromatogram of the obtained product is shown in Figure 6c.

c) Coupling of the enantiomers pair C with racemic isoserinol.

The enriched enantiomers pair C collected e.g. as in Example 3 (54 mg, titer 90%, 0.065 mmol) was dissolved in deionized water (5 mL), and racemic isoserinol (27 mg, 0.29 mmol) was added adjusting the pH at 6 with 1M HCI. Then, EDCI-HCI (62 mg, 0.32 mmol) and HOBT (4.3 mg, 0.032 mmol) were added and the solution was stirred at RT at pH 6 for 24 h. The solution was concentrated and loaded to pre-packed silica C18 column (Biotage® SNAP ULTRA C18 12 g, HP-sphere C18 25 pm), eluting with water/acetonitrile gradient using an automated flash chromatography system. Fractions containing the pure product, or showing a major peak at the HPLC with area greater than 90%, were combined, concentrated and freeze-dried giving a white solid (60 mg, yield 95%).

HPLC chromatogram of the obtained product is shown in Figure 6d. Example 5: Stereoselective synthesis of the RRR Gd(PCTA-tris-glutaric acid) (compound XII).

RRR enriched Gd(PCTA-tris-glutaric acid) acid has been prepared by following the synthetic Scheme 3 below

Scheme 3

Figure imgf000034_0001

comprising :

a) Preparation of Compound VIII

The preparation was carried out as reported in Tetrahedron 2009, 65, 4671-4680.

In particular: 37% aq. HCI (50 pL) was added to a solution of (S)-(+)-5- oxotetrahydrofuran-2-carboxylic acid (2.48 g, 0.019 mol) (commercially available) in anhydrous methanol (20 ml_). The solution was refluxed under N2 atmosphere for 24 h. After cooling in ice, NaHCC>3 was added, the suspension was filtered, concentrated and purified on silica gel with hexanes/ethyl acetate 1 : 1. Fractions containing the pure product were combined and concentrated, giving a colorless oil (2.97 g, yield 89%).

b) Preparation of Compounds IX and X

Compound VIII (445 mg, 2.52 mmol) obtained at step a) was dissolved in anhydrous dichloromethane (6 ml.) and triethylamine (0.87 ml_, 6.31 mmol) was added. The solution was cooled at -40°C and then (triflic) trifluoromethansulfonic anhydride (0.49 ml_,2.91 mmol) was slowly added. The dark solution was stirred at -40°C for 1 h, then a solution of Compound III (104 mg, 0.506 mmol) in anhydrous dichloromethane (3 ml.) and triethylamine (1 ml_, 7.56 mmol) were added and the solution was slowly brought to RT and stirred at RT overnight. The organic solution was then washed with 2M HCI (4x 10 ml_), the aqueous phase was extracted again with dichloromethane (3 x 10 ml_). The organic phases were combined and concentrated in vacuum, obtaining 400 mg of a brown oil that was used in the following step with no further purification.

c) Preparation of Compound XI

Compound X (400 mg, 0.59 mmol) was dissolved in methanol (2.5 ml.) and 5M NaOH (2.5 ml_). The brown solution was heated at 80°C for 22 h to ensure complete hydrolysis. Methanol was concentrated, the solution was brought to pH 1 with concentrated HCI and purified through an automated flash chromatography system with a silica C18 pre-packed column (Biotage® SNAP ULTRA C18 12 g, HP-sphere C18 25 pm), eluting with deionized water/acetonitrile gradient. Fractions containing the pure product were combined, concentrated and freeze-dried (64 mg, yield 18 %). The HPLC showed a major peak.

d) Compound XII

Compound XI (32 mg, 0.054 mmol) was dissolved in deionized water (4 mL) and the pH was adjusted to 7 with 1M NaOH. GdCl3-6H20 (20 mg, 0.054 mmol) was added and the pH was adjusted to 7 with 0.1 M NaOH. The clear solution was stirred at RT overnight and the end of the complexation was checked by xylenol orange and HPLC. The HPLC of the crude showed the desired RRR isomer as major peak: about 80% in area %. The mixture was brought to pH 2 with concentrated HCI and purified through an automated flash chromatography system with a silica C18 pre-packed column (Biotage® SNAP ULTRA C18 12 g, HP-sphere C18 25 pm), eluting with deionized water/acetonitrile gradient. Fractions containing the pure product were combined, concentrated and freeze-dried (36 mg, yield 90%).

By reaction of the collected compound with isoserinol e.g. by using the procedure of the Example 2, the corresponding RRR amide derivative can then be obtained.

Example 6: stereoselective synthesis of the SSS Gd(PCTA-tris-glutaric acid) (compound XVII).

SSS enriched Gd(PCTA-tris-glutaric acid) acid has been similarly prepared by following the synthetic Scheme 4 below Scheme 4

Figure imgf000036_0001

comprising :

a) Preparation of Compound XIII

37% aq. HCI (100 pl_) was added to a solution of (R)-(-)-5-oxotetrahydrofuran-2- carboxylic acid (5.0 g, 0.038 mol) (commercially available) in anhydrous methanol (45 ml_). The solution was refluxed under N2 atmosphere for 24 h. After cooling in ice, NaHC03 was added, the suspension was filtered, concentrated and purified on silica gel with hexanes/ethyl acetate 1 : 1. Fractions containing the pure product were combined and concentrated, giving a colorless oil (6.7 g, yield 99%).

b) Preparation of Compounds XIV and XV

Compound XIII (470 mg, 2.67 mmol) was dissolved in anhydrous dichloromethane (6 ml.) and trimethylamine (0.93 ml_, 6.67 mmol) was added. The solution was cooled down at -40°C and then trifluoromethanesulfonic anhydride (0.50 ml_, 3.07 mmol) was slowly dropped. The dark solution was stirred at -40°C for 1 h, then Compound III (140 mg, 0.679 mmol) and trimethylamine (0.93 ml_, 6.67 mmol) were added and the solution was slowly brought to RT overnight. The organic solution was then washed with water (3 x 5 ml.) and 2M HCI (4 x 5 ml_). The aqueous phase was extracted again with dichloromethane (3 x 10 ml_). the organic phases were combined and concentrated in vacuum, obtaining 350 mg of a brown oil that was used in the following step with no further purification. c) Preparation of Compound XVI

Compound XV (350 mg, 0.514 mmol) was dissolved in methanol (4.5 ml.) and 5M NaOH (4.5 ml_). The obtained brown solution was heated at 80°C for 16 h to ensure complete hydrolysis. Methanol was concentrated, the solution was brought to pH 2 with concentrated HCI and purified through an automated flash chromatography system with a silica C18 pre-packed column (Biotage® SNAP ULTRA C18 12 g, HP-SPHERE C18 25 pm), eluting with a water/acetonitrile gradient. Fractions containing the pure product were combined, concentrated and freeze-dried (52 mg, yield 17%). The HPLC showed a major peak.

d) Preparation of Compound XVII

Compound XVI (34 mg, 0.057 mmol) was dissolved in deionized water (5 mL) and the pH was adjusted to 7 with 1 M HCI. GdCl3-6H20 (20 mg, 0.0538 mmol) was added and the pH was adjusted to 7 with 0.1 M NaOH. The solution was stirred at RT overnight and the end of complexation was checked by xylenol orange and HPLC. The HPLC of the crude showed the desired SSS isomer as major peak: about 85% in area %. The solution was brought to pH 2.5 with concentrated HCI and purified through an automated flash chromatography system with a silica C18 pre-packed column (Biotage® SNAP ULTRA C18 12 g, HP-SPHERE C18 25 pm), eluting with a water/acetonitrile gradient. Fractions containing the pure product SSS were combined, concentrated and freeze-dried (39 mg, yield 87%).

Example 7: Kinetic studies of the dissociation reactions of Gd(PCTA-tris- glutaric acid) (isomeric mixture) in 1.0 M HCI solution (25°C)

The kinetic inertness of a Gd(III)-complex is characterized either by the rate of dissociation measured in 0.1-1.0 M HCI or by the rate of the transmetallation reaction, occurring in solutions with Zn(II) and Cu(II) or Eu(III) ions. However, the dissociation of lanthanide(III)-complexes formed with macrocyclic ligands is very slow and generally proceeds through a proton-assisted pathway without the involvement of endogenous metal ions like Zn2+ and Cu2+.

We characterized the kinetic inertness of the complex Gd(PCTA-tris-glutaric acid) by the rates of the dissociation reactions taking place in 1.0 M HCI solution. The complex (isomeric mixture from Example 1) (0.3 mg) was dissolved in 2.0 mL of 1.0 M HCI solution and the evolution of the solution kept at 25 °C was followed over time by HPLC. The HPLC measurements were performed with an Agilent 1260 Infinity II system by use of the analytical Procedure 1.

The presence of a large excess of H+ ([HCI] = 1.0 M), guarantees the pseudo-first order kinetic conditions.

GdL + yH÷ ^ Gd3+ + HyL y=7 and 8 (Eg. 1) where L is the protonated PCTA-tri-glutaric acid, free ligand, and y is the number of protons attached to the ligand.

The HPLC chromatogram of Gd(PCTA-tris-glutaric acid) is characterized by the presence of four signals (A, B, C and D) having the same m/z ratio (Gd(H4L)+ :752.14 m/z) in the MS spectrum. Each of these peaks is reasonably ascribable to one of the 4 pairs of enantiomers generated by the three stereocenters on the three glutaric arms of the molecule, formerly identified in Table 1. The HPLC chromatogram of this complex in the presence of 1.0 M HCI changes over time: in particular, the areas of peaks A, B, C, and D decrease, although not in the same way for the different peaks, while new signals corresponding to non-complexed diastereoisomers are formed and grow over time. Differences in the decrease of the integral areas of the peaks can be interpreted by a different dissociation rate of the enantiomer pairs associated to the different peaks.

In the presence of [H + ] excess the dissociation reaction of enantiomer pairs of Gd(PCTA-tris-glutaric acid) can be treated as a pseudo-first-order process, and the rate of the reactions can be expressed with the following Eq. 2, where kA, kB, kc and kD are the pseudo-first-order rate constants that are calculated by fitting the area-time data pair, and [A]t, [B]t, [C]t and [D]t are the total concentration of A, B, C and D compounds at time t.

Figure imgf000038_0001

The decrease of the area values of signals of A, B, C, and D has been assessed and plotted over time. Area values of A, B, C and D signals as a function of time are shown in Figure 7.

Area value at time t can be expressed by the following equation:

A. = A + (A0 – A )e kxt

(Eg. 3)

where At, A0 and Ae are the area values at time t, at the beginning and at the end of the reactions, respectively, kx pseudo-first-order rate constants (/fX=/fA, kB, kc and kD) characterizing the dissociation rate of the different enantiomer pairs of Gd(PCTA-tris-glutaric acid) complex were calculated by fitting the area – time data pairs of Figure 7 to the above equation 3. kx rate constants and half-lives (ti/2= In2/ x) are thus obtained, as well as the average the half-life value for the isomeric mixture of Gd(PCTA-tris-glutaric acid), calculated by considering the percentage composition of the mixture. Obtained values are summarized in the following Table 2, and compared with corresponding values referred in the literature for some reference contrast agents. (Gd-DOTA or DOTAREM™). Table 2. Rate constants ( kx ) and half-lives (ti/2= In2/ x) characterizing the acid catalyzed dissociation of the different stereoisomers of Gd(PCTA-tris-glutaric acid), Dotarem® and Eu(PCTA) in 1.0 M HCI (pH 0) ( 25°C)

A B C D

Ms 1) (4.5±0.1) x105 (1.1±0.1)x104 (1.6±0.1)x10-6 (1.2±0.1)x10-5 fi/2 (hour) 4.28 ± 0.03 1.76 ± 0.02 120 ± 3 15.8 ± 0.5

fi/2 (hour)

Figure imgf000039_0001

average

Dotarem a

k, (S‘1) 8.0×10-6

fi/2 (hour) 23 hour

Eu(PCTA) b

*1 (s·1) 5.08X10·4

fi/2 (hour) 0.38 hour

a) Inorg. Chem. 1992, 31 ,1095-1099.

b) Tircso, G. et al. Inorg Chem 2006, 45 (23), 9269-80.

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A gadolinium-based paramagnetic contrast agent, with potential imaging enhancing activity upon magnetic resonance imaging (MRI). Upon administration of gadopiclenol and placement in a magnetic field, this agent produces a large magnetic moment and creates a large local magnetic field, which can enhance the relaxation rate of nearby protons. This change in proton relaxation dynamics, increases the MRI signal intensity of tissues in which this agent has accumulated; therefore, contrast and visualization of those tissues is enhanced compared to unenhanced MRI.

FDA Approves New MRI Contrast Agent Gadopiclenol

September 22, 2022

https://www.diagnosticimaging.com/view/fda-approves-new-mri-contrast-agent-gadopiclenol

Requiring only half of the gadolinium dose of current non-specific gadolinium-based contrast agents (GBCAs), gadopiclenol can be utilized with magnetic resonance imaging (MRI) to help detect lesions with abnormal vascularity in the central nervous system and other areas of the body.

Gadopiclenol, a new magnetic resonance imaging (MRI) contrast agent that offers high relaxivity and reduced dosing of gadolinium, has been approved by the Food and Drug Administration (FDA).1

Approved for use with MRI in adults and pediatric patients two years of age or older, gadopiclenol is a macrocyclic gadolinium-based contrast agent that aids in the diagnosis of lesions with abnormal vascularity in the brain, spine, abdomen, and other areas of the body.

Recently published research demonstrated that gadopiclenol provides contrast enhancement and diagnostic efficacy at half of the gadolinium dosing of other gadolinium-based contrast agents (GBCAs) such as gadobutrol and gadobenate dimeglumine.2

Co-developed by Bracco Diagnostics and Guerbet, gadopiclenol will be manufactured and marketed as Vueway™ (Bracco Diagnostics) and Elucirem™ (Guerbet).1,3

Alberto Spinazzi, M.D., the chief medical and regulatory officer at Bracco Diagnostics, said gadopiclenol is “a first of its kind MRI agent that delivers the highest relaxivity and highest kinetic stability of all GBCAs on the market today.”

Reference

1. Bracco Diagnostics. Bracco announces FDA approval of gadopiclenol injection, a new macrocyclic high-relaxivity gadolinium-based contrast agent which will be commercialized as VUEWAY™ (gadopiclenol) injection and VUEWAY™ (gadopiclenol) phamarcy bulk package by Bracco. Cision PR Newswire. Available at: https://www.prnewswire.com/news-releases/bracco-announces-fda-approval-of-gadopiclenol-injection-a-new-macrocyclic-high-relaxivity-gadolinium-based-contrast-agent-which-will-be-commercialized-as-vueway-gadopiclenol-injection-and-vueway-gadopiclenol-pharmacy-bulk-p-301630124.html . Published September 21, 2022. Accessed September 21, 2022.

2. Bendszus M, Roberts D, Kolumban B, et al. Dose finding study of gadopiclenol, a new macrocyclic contrast agent, in MRI of central nervous system. Invest Radiol. 2020;55(3):129-137.

3. Guerbet. Guerbet announces U.S. Food and Drug Administration (FDA) approval of Elucirem™ (gadopiclenol) injection for use in contrast-enhanced MRI. Cision PR Newswire. Available at: https://www.prnewswire.com/news-releases/guerbet-announces-us-food-and-drug-administration-fda-approval-of-elucirem-gadopiclenol-injection-for-use-in-contrast-enhanced-mri-301630085.html . Published September 21, 2022. Accessed September 21, 2022.

////Gadopiclenol, FDA 2022, APPROVALS 2022, ガドピクレノール, WHO 10744, P 03277,  EluciremTM, G03277; P03277, VUEWAY, Guerbet

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Eflapegrastim


2D chemical structure of 1384099-30-2
STR1

Eflapegrastim

エフラペグラスチム;

Molecular Formula

  • C15-H28-N2-O6(C2-H4-O)n

Molecular Weight

  • 376.4468
FormulaC3070H4764N806O927S23.(C2H4O)n

UNII: UT99UG9QJX

HM10460A
SPI-2012

  • HNK460

Reducing neutropenia and the incidence of infecton in patients with cancer

(2S)-1-{3-[2-(3-{[(1S,2R)-1-carboxy-2-hydroxypropyl]amino}propoxy)ethoxy]propyl}pyrrolidine-2-carboxylic acid

APPROVED FDA 2022/9/9, Rolvedon

CAS: 1384099-30-2

LAPS-GCSF, ROLONTIS

Antineutropenic, Leukocyte growth factor

Poly(oxy-1,2-ethanediyl), α-hydro-ω-hydroxy-, 1-ether with immunoglobulin G4 [1-[1-(3-hydroxypropyl)proline]] (human Fc fragment), (3→3′)-disulfide with immunoglobulin G4 (human Fc fragment), 1′′-ether with granulocyte colony-stimulating factor [N-(3-hydroxypropyl),17-serine,65-serine] (human) (ACI)

A long-acting, recombinant analog of the endogenous human granulocyte colony-stimulating factor (G-CSF) with hematopoietic activity. Similar to G-CSF, eflapegrastim binds to and activates specific cell surface receptors and stimulates neutrophil progenitor proliferation and differentiation, as well as selected neutrophil functions. Therefore, this agent may decrease the duration and incidence of chemotherapy-induced neutropenia. Eflapegrastim extends the half-life of G-CSF, allowing for administration once every 3 weeks.

  • A long-acting GCSF that consists of 17th serine-G-CSF conjugated to the G4 fragment HMC001 via a PEG linker.

PATENT

 WO2021113597

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2021113597

Neutropenia is a relatively common disorder most often due to chemotherapy treatments, adverse drug reactions, or autoimmune disorders. Chemotherapy-induced neutropenia is a common toxicity caused by the administration of anticancer drugs. It is associated with life-threatening infections and may alter the chemotherapy schedule, thus impacting on early and long term outcome. Febrile Neutropenia (FN) is a major dose-limiting toxicity of myelosuppressive chemotherapy regimens such as docetaxel, doxorubicin, cyclophosphamide (TAC); dose-dense doxorubicin plus cyclophosphamide (AC), with or without subsequent weekly or semiweekly paclitaxel; and docetaxel plus cyclophosphamide (TC). It usually leads to prolonged hospitalization, intravenous administration of broad-spectrum antibiotics, and is often associated with significant morbidity and mortality.

Current therapeutic modalities employ granulocyte colony-stimulating factor (G-CSF) and/or antibiotic agents to combat this condition. G-CSF or its other polypeptide derivatives are easy to denature or easily de-composed by proteolytic enzymes in blood to be readily removed through the kidney or liver. Therefore, to maintain the blood concentration and titer of the G-CSF containing drugs, it is necessary to frequently administer the protein drug to patients, which causes excessive suffering in patients. To solve such problems, G-CSF was chemically attached to polymers having a high solubility such as polyethylene glycol (“PEG”), thereby increasing its blood stability and maintaining suitable blood concentration for a longer time.

Filgrastim, tbo-filgrastim, and pegfilgrastim are G-CSFs currently approved by the US Food and Drug Administration (FDA) for the prevention of chemotherapy-induced neutropenia, While the European guidelines also include lenograstim as a recommended G-CSF in solid tumors and non-myeloid malignancies, it is not approved for use in the US. Binding of PEG to G-CSF, even though may increase blood stability, does dramatically reduce the titer needed for optimal physiologic effect. Thus there is a need to address this shortcoming in the art.

PATENT

WO2021112654

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2021112654

Eflapegrastim

[54]

Eflapegrastim, as known as Rolontis ®, SPI-2012, HM10460A, and 17,65S-G-CSF, is a long-acting granulocyte-colony stimulating factor (G-CSF) that has been developed to reduce the severity and duration of severe neutropenia, as well as complications of neutropenia, associated with the use of myelosuppressive anti-cancer drugs or radiotherapy. Eflapegrastim consists of a recombinant human G-CSF analog (ef-G-CSF) and a recombinant fragment of the Fc region of human immunoglobulin G4 (IgG4), linked by a Bifunctional polyethylene glycol linker. In certain embodiments, the recombinant human G-CSF analog (ef-G-CSF) varies from human G-CSF (SED ID NO: 1) at positions 17 and 65 which are substituted with serine (SED ID NO: 2). Without wishing to be bound by theory, it is believed that the Fc region of human IgG4 increases the serum half-life of ef-G-CSF.

[55]

ef-G-CSF is produced by transformed E. coli in soluble form in the periplasmic space. Separately, the Fc fragment is produced in transformed E. coli as an inclusion body. The ef-G-CSF and the Fc fragment are independently isolated and purified through successive purification steps. The purified ef-G-CSF (SEQ ID NO: 2) and Fc fragment (SEQ ID NOs: 3 and 4) are then linked via a 3.4 kDa PEG molecule that was designed with reactive groups at both ends. Eflapegrastim itself is the molecule resulting from the PEG linker binding at each of the N-termini of ef-G-CSF and the Fc fragment. The G-CSF analog is conjugated to the 3.4 kDa polyethylene glycol analogue with propyl aldehyde end groups at both ends, (OHCCH 2CH 2(OCH 2CH 2nOCH 2CH 2CHO) at the nitrogen atom of its N-terminal Thr residue via reductive amination to form a covalent bond. The resulting G-CSF-PEG complex is then linked to the N-terminal Pro at the nitrogen of the recombinant Fc fragment variant produced in E. coli via reductive amination to yield the final conjugate of Eflapegrastim.

[56]

Example 1: Preparation of Eflapegrastim ( 17,65S-G-CSF-PEG-Fc)

[120]

Step 1: Preparation of Immunoglobulin Fc Fragment Using Immunoglobulin

[121]

Preparation of an immunoglobulin Fc fragment was prepared as follows.

[122]

200 mg of 150-kDa immunoglobulin G (IgG) (Green Cross, Korea) dissolved in 10 mM phosphate buffer was treated with 2 mg of a proteolytic enzyme, papain (Sigma) at 37℃ for 2 hrs with gentle agitation.

[123]

After the enzyme reaction, the immunoglobulin Fc fragment regenerated thus was subjected to chromatography for purification using sequentially a Superdex column, a protein A column and a cation exchange column. In detail, the reaction solution was loaded onto a Superdex 200 column (Pharmacia) equilibrated with 10 mM sodium phosphate buffer (PBS, pH 7.3), and the column was eluted with the same buffer at a flow rate of 1 ml/min. Unreacted immunoglobulin molecules (IgG) and F(ab’)2, which had a relatively high molecular weight compared to the immunoglobulin Fc fragment, were removed using their property of being eluted earlier than the Ig Fc fragment. Fab fragments having a molecular weight similar to the Ig Fc fragment were eliminated by protein A column chromatography (FIGURE 1). The resulting fractions containing the Ig Fc fragment eluted from the Superdex 200 column were loaded at a flow rate of 5 ml/min onto a protein A column (Pharmacia) equilibrated with 20 mM phosphate buffer (pH 7.0), and the column was washed with the same buffer to remove proteins unbound to the column. Then, the protein A column was eluted with 100 mM sodium citrate buffer (pH 3.0) to obtain highly pure immunoglobulin Fc fragment. The Fc fractions collected from the protein A column were finally purified using a cation exchange column (polyCAT, PolyLC Company), wherein this column loaded with the Fc fractions was eluted with a linear gradient of 0.15-0.4 M NaCl in 10 mM acetate buffer (pH 4.5), thus providing highly pure Fc fractions. The highly pure Fc fractions were analyzed by 12% SDS-PAGE (lane 2 in FIGURE 2).

[124]

Step 2: Preparation of 17,65S-G-CSF-PEG Complex

[125]

3.4-kDa polyethylene glycol having an aldehyde reactive group at both ends, ALD-PEG-ALD (Shearwater), was mixed with human granulocyte colony stimulating factor ( 17,65S-G-CSF, MW: 18.6 kDa) dissolved in 100 mM phosphate buffer in an amount of 5 mg/ml at a 17,65S-G-CSF: PEG molar ratio of 1:5. To this mixture, a reducing agent, sodium cyanoborohydride (NaCNBH 3, Sigma), was added at a final concentration of 20 mM and was allowed to react at 4℃ for 3 hrs with gentle agitation to allow PEG to link to the amino terminal end of 17,65S-G-CSF. To obtain a 1:1 complex of PEG and 17,65S-G-CSF, the reaction mixture was subjected to size exclusion chromatography using a Superdex R column (Pharmacia). The 17,65S-G-CSF-PEG complex was eluted from the column using 10 mM potassium phosphate buffer (pH 6.0) as an elution buffer, and 17,65S-G-CSF not linked to PEG, unreacted PEG and dimer byproducts where PEG was linked to 17,65S-G-CSF molecules were removed. The purified 17,65S-G-CSF-PEG complex was concentrated to 5 mg/ml. Through this experiment, the optimal reaction molar ratio for 17,65S-G-CSF to PEG, providing the highest reactivity and generating the smallest amount of byproducts such as dimers, was found to be 1:5.

[126]

Step 3: Preparation of the 17,65S-G-CSF-PEG-Fc Conjugate

[127]

To link the 17,65S-G-CSF-PEG complex purified in the above step 2 to the N-terminus of an immunoglobulin Fc fragment, the immunoglobulin Fc fragment (about 53 kDa) prepared in Step 1 was dissolved in 10 mM phosphate buffer and mixed with the 17,65S-G-CSF-PEG complex at an 17,65S-G-CSF-PEG complex:Fc molar ratio of 1:1, 1:2, 1:4 and 1:8. After the phosphate buffer concentration of the reaction solution was adjusted to 100 mM, a reducing agent, NaCNBH 3, was added to the reaction solution at a final concentration of 20 mM and was allowed to react at 4℃ for 20 hrs with gentle agitation. Through this experiment, the optimal reaction molar ratio for 17,65S-G-CSF-PEG complex to Fc, providing the highest reactivity and generating the fewest byproducts such as dimers, was found to be 1:2.

[128]

Step 4: Isolation and Purification of the G-CSF-PEG-Fc Conjugate

[129]

After the reaction of the above step 3, the reaction mixture was subjected to Superdex size exclusion chromatography so as to eliminate unreacted substances and byproducts and purify the 17,65S-G-CSF-PEG-Fc protein conjugate produced. After the reaction mixture was concentrated and loaded onto a Superdex column, 10 mM phosphate buffer (pH 7.3) was passed through the column at a flow rate of 2.5 ml/min to remove unbound Fc and unreacted substances, followed by column elution to collect 17,65S-G-CSF-PEG-Fc protein conjugate fractions. Since the collected 17,65S-G-CSF-PEG-Fc protein conjugate fractions contained a small amount of impurities, unreacted Fc and interferon alpha dimers, cation exchange chromatography was carried out to remove the impurities. The 17,65S-G-CSF-PEG-Fc protein conjugate fractions were loaded onto a PolyCAT LP column (PolyLC) equilibrated with 10 mM sodium acetate (pH 4.5), and the column was eluted with a linear gradient of 0-0.5 M NaCl in 10 mM sodium acetate buffer (pH 4.5) using 1 M NaCl. Finally, the 17,65S-G-CSF-PEG-Fc protein conjugate was purified using an anion exchange column. The 17,65S-G-CSF-PEG-Fc protein conjugate fractions were loaded onto a PolyWAX LP column (PolyLC) equilibrated with 10 mM Tris-HCl (pH 7.5), and the column was then eluted with a linear gradient of 0-0.3 M NaCl in 10 mM Tris-HCl (pH 7.5) using 1 M NaCl, thus isolating the 17,65S-G-CSF-PEG-Fc protein conjugate in a highly pure form.

[130]

[131]

Example 2: Efficacy Study of Eflapegrastim by Different Dosing Regimens in Rats with Docetaxel/Cyclophosphamide induced Neutropenia

[132]

The efficacy of Eflapegrastim (HM10460A), a long acting G-CSF analogue, was compared with Pegfilgrastim by different dosing regimens in a chemotherapy-induced neutropenic rat model.

[133]

In the following study, the Eflapegrastim was created essentially as described in Example 1.

[134]

(i) Materials for Study

[135]

[Table 1] Test Articles

NameBatch/Lot No.Storage ConditionPurity (%)Expiration DateSupplier
HM10460A9066170012~8 ℃RP-HPLC: 98.6% IE-HPLC: 97.4%
SE-HPLC: 98.6%
01/31/2019
Pegfilgrastim10703342~8 ℃Amgen

[136]

[Table 2] Vehicles

NameCompositionStorage ConditionSupplier
Dulbecco’s phosphate buffered saline (DPBS)2~8 ℃Sigma-Aldrich

[137]

[Table 3] Neutropenia-Inducing Agents

NameBatch/Lot No.Storage ConditionPurity (%)Expiration DateSupplier
Cyclo-phosphamideC32500002~8 ℃Sigma-Aldrich
Docetaxel17006RT (20 – 25 ℃)10/31/2020Hanmi Pharmaceutical Co.

[138]

Preparing HM10460A Solutions for Subcutaneous Administration

[139]

Preparation of a 61.8 ㎍/kg HM10460A solution for subcutaneous administration: a stock solution of HM10460A (6.0 mg/mL) 92.7 μL was diluted with DPBS 17907.3 μL.

[140]

Preparation of a 372.0 ㎍/kg HM10460A solution for subcutaneous administration: a stock solution of HM10460A (6.0 mg/mL) 558.0 μL was diluted with DPBS 17442.0 μL.

[141]

Preparation of a 496.0 ㎍/kg HM10460A solution for subcutaneous administration: a stock solution of HM10460A (6.0 mg/mL) 744.0μL was diluted with DPBS 17256.0 μL.

[142]

The test article was prepared based on G-CSF protein dosage on drug label(HM10460A.)

[143]

The HM10460A solution for subcutaneous administration was then diluted with DPBS to a final dose concentration of 2 mL/kg.

[144]

Preparing Pegfilgrastim Solutions for Subcutaneous Administration

[145]

Preparation of a 103.3 ㎍/kg Pegfilgrastim solution for subcutaneous administration: a stock solution of Pegfilgrastim (10 mg/mL) 93.0 μL was diluted with DPBS 17907.0 μL.

[146]

Preparation of a 620.0 ㎍/k Pegfilgrastim solution for subcutaneous administration: a stock solution of Pegfilgrastim (10 mg/mL) 558.0 μL was diluted with DPBS 17442.0 μL.

[147]

The Pegfilgrastim solution for subcutaneous administration was then diluted with DPBS to a final dose concentration of 2 mL/kg.

[148]

Preparing Solutions of Neutropenia-Inducing Agents

[149]

To induce neutropenia in rats, Docetaxel/cyclophosphamide was administered using a 1/3 human equivalent dose (Docetaxel 4 mg/kg and CPA 32 mg/kg) (“TC”).

[150]

Preparation of a 32 mg/kg cyclophosphamide solution for subcutaneous administration: cyclophosphamide powder (CPA, Sigma, USA) 2560.0 g was diluted with distilled water (DW, Daihan, Korea) 80000.0 μL.

[151]

Preparation of a 4 mg/kg docetaxel solution for subcutaneous administration: Docel inj. (Hanmi Pharmaceutical, Korea) (42.68 mg/mL) 29070.0 μL was diluted with a commercial formulation buffer (FB, Etahnol 127.4mg/mL in DW) 30930.0 μL.

[152]

The docetaxel and cyclophosphamide solutions for subcutaneous administration were then diluted with FB to a final dose concentration of 1 mL/kg. HM10460A and Pegfilgrastim were diluted with DPBS to a final dose concentration of 2 mL/kg.

[153]

(ii) Methods

[154]

Test System

[155]

[Table 4]

Species and StrainRats
Crl: CD Sprague Dawley (SD)
Justification for SpeciesSD rats were chosen due to their extensive characterization collected from various preclinical studies, especially with the study done to test G-CSF analogue1), 2).
SupplierOrient Bio corp. Korea 143-1, Sangdaewondong, Jungwon-gu, Seongnam-si, Gyeonggi-do, Korea
Number of animalsMale 125 (at group allocation)
Age8 weeks (at group allocation)
Body weight range239.54 ~ 316.46 g (at start of dosing)
Neutropenia induction with chemotherapyNormal SD rats were administered with Docetaxel 4 mg/kg and CPA 32 mg/kg once intraperitoneally to induce neutropenia. Docetaxel and CPA were injected to induce neutropenia in a rat model according to 4 different regimens: Concomitant (G2-G7), 2 hour (G8-G13), 5 hour (G14-G19), and 24 hour (G20-G25) prior to test article administration.

[156]

Animal Care and Identification

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Eflapegrastim

25/10/2019by Christian Hilscher

Neutropenia in Breast Cancer: Spectrum Pharmaceuticals has submitted an updated regulatory submission to the US FDA for its biologic Rolontis

10/25/2019 Spectrum Pharmaceutical announced that it has filed an updated Biologics License Application (BLA) with the US Food and Drug Administration (FDA) for Rolontis (eflapegrastim).

The BLA for Rolontis is supported by data from two identically designed Phase 3 clinical trials – ADVANCE and RECOVER – that evaluated the safety and efficacy of eflapegrastim in 643 patients with early breast cancer for the treatment of neutropenia with myelosuppressive chemotherapy.

In both studies, eflapegrastim demonstrated the pre-specified hypothesis of non-inferiority (NI) in Duration of Severe Neutropenia (DSN) and a similar safety profile to pegfilgrastim .

Eflapegrastim also demonstrated non-inferiority to pegfilgrastim in DSN across all 4 cycles in both studies (all NI p<0.0001), the company writes.
© arznei-news.de – Source: Spectrum Pharmaceuticals

Eflapegrastim, sold under the brand names Rolvedon among others, is a long-acting G-CSF analog developed by Hanmi Pharmaceutical and licensed to Spectrum Pharmaceuticals.[2] Eflapegrastim is a leukocyte growth factor.[1] It is used to reduce the risk of febrile neutropenia in people with non-myeloid malignancies receiving myelosuppressive anti-cancer agents.[1]

Eflapegrastim was approved for medical use in the United States in September 2022.[1][3][4]

Medical uses

Eflapegrastim is indicated to decrease the incidence of infection, as manifested by febrile neutropenia, in adults with non-myeloid malignancies receiving myelosuppressive anti-cancer drugs associated with clinically significant incidence of febrile neutropenia.[1]

Its efficacy has been shown to be non-inferior to pegfilgrastim.[1]

References

  1. Jump up to:a b c d e f “Archived copy” (PDF). Archived (PDF) from the original on 19 September 2022. Retrieved 19 September 2022.
  2. ^ pharmaceutical, hanmi. “Pipeline – R&D”Hanmi PharmaceuticalArchived from the original on 2 February 2017. Retrieved 23 January 2017.
  3. ^ “Rolvedon: FDA-Approved Drugs”U.S. Food and Drug Administration (FDA)Archived from the original on 19 September 2022. Retrieved 18 September 2022.
  4. ^ “Spectrum Pharmaceuticals Receives FDA Approval for Rolvedon (eflapegrastim-xnst) Injection”Business Wire (Press release). 9 September 2022. Archived from the original on 9 September 2022. Retrieved 18 September 2022.

External links

  • “Eflapegrastim”Drug Information Portal. U.S. National Library of Medicine.
  • Clinical trial number NCT02643420 for “SPI-2012 vs Pegfilgrastim in the Management of Neutropenia in Participants With Breast Cancer With Docetaxel and Cyclophosphamide (ADVANCE) (ADVANCE)” at ClinicalTrials.gov
  • Clinical trial number NCT02953340 for “SPI-2012 vs Pegfilgrastim in Management of Neutropenia in Breast Cancer Participants With Docetaxel and Cyclophosphamide” at ClinicalTrials.gov
Clinical data
Trade namesRolvedon
Other namesEflapegrastim-xnst, HM-10460A, SPI-2012
Routes of
administration
Subcutaneous
ATC codeNone
Legal status
Legal statusUS: ℞-only [1]
Identifiers
CAS Number1384099-30-2
ChemSpiderNone
UNIIUT99UG9QJX
KEGGD11188

////////////Eflapegrastim, Rolvedon, APPROVALS 2022, FDA 2022, エフラペグラスチム , HM10460A, SPI-2012, HNK460, ROLONTIS

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Terlipressin acetate


Terlipressin.png
Terlipressin acetate.png
2D chemical structure of 1884420-36-3

Terlipressin acetate

テルリプレシン酢酸塩

C52H74N16O15S2. (C2H4O2)x

CAS: 914453-96-6 ACETATEFREE  FORM 14636-12-5

Terlipressin acetate (JAN);
Heamopressin (TN);
Terlivaz (TN)

Cardiovascular agent

Antidiuretic, Vasoconstrictor, Arginine vasopressin receptor agonist

USFDA APPROVED 2022/9/14

An inactive peptide prodrug that is slowly converted in the body to lypressin. It is used to control bleeding of ESOPHAGEAL VARICES and for the treatment of HEPATORENAL SYNDROME.

SVG Image
IUPAC CondensedH-Gly-Gly-Gly-Cys(1)-Tyr-Phe-Gln-Asn-Cys(1)-Pro-Lys-Gly-NH2.CH3CO2H
SequenceGGGCYFQNCPKG
IUPACglycyl-glycyl-glycyl-L-cysteinyl-L-tyrosyl-L-phenylalanyl-L-glutaminyl-L-asparagyl-L-cysteinyl-L-prolyl-L-lysyl-glycinamide (4->9)-disulfide acetic acid
  • EINECS 238-680-8
  • Terlipressin
  • Terlipressina
  • Terlipressina [INN-Spanish]
  • Terlipressine
  • Terlipressine [INN-French]
  • Terlipressinum
  • Terlipressinum [INN-Latin]
  • UNII-7Z5X49W53P

acetic acid;(2S)-1-[(4R,7S,10S,13S,16S,19R)-19-[[2-[[2-[(2-aminoacetyl)amino]acetyl]amino]acetyl]amino]-7-(2-amino-2-oxoethyl)-10-(3-amino-3-oxopropyl)-13-benzyl-16-[(4-hydroxyphenyl)methyl]-6,9,12,15,18-pentaoxo-1,2-dithia-5,8,11,14,17-pentazacycloicosane-4-carbonyl]-N-[(2S)-6-amino-1-[(2-amino-2-oxoethyl)amino]-1-oxohexan-2-yl]pyrrolidine-2-carboxamide

FREE FORM

Molecular Structure of 14636-12-5 (Terlipressin)
Formula:C52H74N16O15S2
Molecular Weight:1227.39

14636-12-5

(2S)-1-[(4R,7S,10S,13S,16S,19R)-19-[[2-[[2-[(2-aminoacetyl)amino]acetyl]amino]acetyl]amino]-13-benzyl-10-(2-carbamoylethyl)-7-(carbamoylmethyl)-16-[(4-hydroxyphenyl)methyl]-6,9,12,15,18-pentaoxo-1,2-dithia-5,8,11,14,17-pentazacycloicosane-4-carbonyl]-N-[(1S)-5-amino-1-(carbamoylmethylcarbamoyl)pentyl]pyrrolidine-2-carboxamide;N-(N-(N-Glycylglycyl)glycyl)-8-L-lysinevasopressin;Glypressin;Terlipressin Acetate;Remestyp;Thymosin α1 Acetate;Gly-Gly-Gly-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Lys-Gly-NH2 (disulfide bridge 4:9);Glycylpressin;

/////////

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Terlipressin, sold under the brand name Terlivaz among others, is an analogue of vasopressin used as a vasoactive drug in the management of low blood pressure. It has been found to be effective when norepinephrine does not help. Terlipressin is a vasopressin receptor agonist.[1]

Medical uses

Terlipressin is indicated to improve kidney function in adults with hepatorenal syndrome with rapid reduction in kidney function.[1]

Indications for use include norepinephrine-resistant septic shock[2] and hepatorenal syndrome.[3] In addition, it is used to treat bleeding esophageal varices.[4]

Contraindications

Terlipressin is contraindicated in people experiencing hypoxia or worsening respiratory symptoms and in people with ongoing coronary, peripheral or mesenteric ischemia.[1] Terlipressin may cause fetal harm when used during pregnancy.[1]

Society and culture

Terlipressin is available in New Zealand,[5] Australia, the European Union,[6] India, Pakistan & UAE. It is sold under various brand names including Glypressin.

Clinical data
Trade namesTerlivaz
AHFS/Drugs.comInternational Drug Names
Routes of
administration
Intravenous
ATC codeH01BA04 (WHO)
Legal status
Legal statusUS: ℞-only [1]
Pharmacokinetic data
Protein binding~30%
Identifiers
showIUPAC name
CAS Number14636-12-5 
PubChem CID72081
DrugBankDB02638 
ChemSpider65067 
UNII7Z5X49W53P
KEGGD06672 
CompTox Dashboard (EPA)DTXSID7048952 
ECHA InfoCard100.035.149 
Chemical and physical data
FormulaC52H74N16O15S2
Molar mass1227.38 g·mol−1
3D model (JSmol)Interactive image
showSMILES
showInChI
  (verify)

References

  1. Jump up to:a b c d e “Archived copy” (PDF). Archived (PDF) from the original on 2022-09-19. Retrieved 2022-09-19.
  2. ^ O’Brien A, Clapp L, Singer M (2002). “Terlipressin for norepinephrine-resistant septic shock”. Lancet359 (9313): 1209–10. doi:10.1016/S0140-6736(02)08225-9PMID 11955542S2CID 38463837.
  3. ^ Uriz J, Ginès P, Cárdenas A, Sort P, Jiménez W, Salmerón J, Bataller R, Mas A, Navasa M, Arroyo V, Rodés J (2000). “Terlipressin plus albumin infusion: an effective and safe therapy of hepatorenal syndrome”. J Hepatol33 (1): 43–8. doi:10.1016/S0168-8278(00)80158-0PMID 10905585.
  4. ^ Ioannou G, Doust J, Rockey D (2003). Ioannou GN (ed.). “Terlipressin for acute esophageal variceal hemorrhage”Cochrane Database Syst Rev (1): CD002147. doi:10.1002/14651858.CD002147PMC 7017851PMID 12535432.
  5. ^ http://www.medsafe.govt.nz/profs/datasheet/g/Glypressin01mgmlFerringinj.pdf Archived 2021-12-20 at the Wayback Machine[bare URL PDF]
  6. ^ “Terlipressin”Archived from the original on 2019-06-26. Retrieved 2018-01-23.

External links

////Terlipressin acetate, テルリプレシン酢酸塩 , FDA 2022, APPROVALS

2022, CC(=O)O.C1CC(N(C1)C(=O)C2CSSCC(C(=O)NC(C(=O)NC(C(=O)NC(C(=O)NC(C(=O)N2)CC(=O)N)CCC(=O)N)CC3=CC=CC=C3)CC4=CC=C(C=C4)O)NC(=O)CNC(=O)CNC(=O)CN)C(=O)NC(CCCCN)C(=O)NCC(=O)N

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Spesolimab


(Heavy chain)
QVQLVQSGAE VKKPGASVKV SCKASGYSFT SSWIHWVKQA PGQGLEWMGE INPGNVRTNY
NENFRNKVTM TVDTSISTAY MELSRLRSDD TAVYYCTVVF YGEPYFPYWG QGTLVTVSSA
STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW NSGALTSGVH TFPAVLQSSG
LYSLSSVVTV PSSSLGTQTY ICNVNHKPSN TKVDKRVEPK SCDKTHTCPP CPAPEAAGGP
SVFLFPPKPK DTLMISRTPE VTCVVVDVSH EDPEVKFNWY VDGVEVHNAK TKPREEQYNS
TYRVVSVLTV LHQDWLNGKE YKCKVSNKAL PAPIEKTISK AKGQPREPQV YTLPPSREEM
TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL DSDGSFFLYS KLTVDKSRWQ
QGNVFSCSVM HEALHNHYTQ KSLSLSPGK
(Light chain)
QIVLTQSPGT LSLSPGERAT MTCTASSSVS SSYFHWYQQK PGQAPRLWIY RTSRLASGVP
DRFSGSGSGT DFTLTISRLE PEDAATYYCH QFHRSPLTFG AGTKLEIKRT VAAPSVFIFP
PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS QESVTEQDSK DSTYSLSSTL
TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRGEC
(Disulfide bridge: H22-H96, H146-H202, H222-L215, H228-H’228, H231-H’231, H263-H323, H369-H427, H’22-H’96, H’146-H’202, H’222-L’215, H’263-H’323, H’369-H’427, L23-L89, L135-L195, L’23-L’89, L’135-L’195)

Spesolimab

スペソリマブ (遺伝子組換え)

FormulaC6480H9988N1736O2012S46
cas2097104-58-8
Mol weight145878.0547
Antipsoriatic, Anti-IL-36 receptor antagonist

fda approved 2022/9/1, spevigo

BI 655130; Spesolimab-sbzo

  • OriginatorBoehringer Ingelheim
  • ClassAnti-inflammatories; Antipsoriatics; Monoclonal antibodies; Skin disorder therapies
  • Mechanism of ActionInterleukin 36 receptor antagonists
  • Orphan Drug StatusYes – Generalised pustular psoriasis
  • RegisteredGeneralised pustular psoriasis
  • Phase II/IIIUlcerative colitis
  • Phase IICrohn’s disease; Hidradenitis suppurativa; Palmoplantar pustulosis
  • DiscontinuedAtopic dermatitis
  • 01 Sep 2022First global approval – Registered for Generalised pustular psoriasis in USA (IV)
  • 01 Sep 2022Adverse events data from the Effisayil 1 phase II trial in Generalised pustular psoriasis released by Boehringer Ingelheim
  • 03 Aug 2022Boehringer Ingelheim anticipates regulatory approval in Generalised pustular psoriasis by 2022

Spesolimab (BI 655130) is a humanised monoclonal antibody, being developed by Boehringer Ingelheim, for the treatment of generalised pustular psoriasis, Crohn’s disease, palmoplantar pustulosis, ulcerative colitis and hidradenitis suppurativa.

What causes Palmoplantar Pustulosis?

Researchers have found some possible causes including smoking, infections, certain medications and genetics. Smoking: Many patients who have PPP are smokers or have smoked in the past. Smoking may cause sweat glands to become inflamed, especially on the hands and feet, which causes pustules to form.

FDA approves the first treatment option for generalized pustular psoriasis flares in adults

  • More than half of patients treated with SPEVIGO® (spesolimab-sbzo) injection, for intravenous use showed no visible pustules one week after receiving treatment
  • Spesolimab is a monoclonal antibody that inhibits interleukin-36 (IL-36) signaling

https://www.boehringer-ingelheim.us/press-release/fda-approves-first-treatment-option-generalized-pustular-psoriasis-flares-adults

Ridgefield, Conn., September 1, 2022 – Boehringer Ingelheim announced today the U.S. Food and Drug Administration has approved SPEVIGO, the first approved treatment option for generalized pustular psoriasis (GPP) flares in adults. SPEVIGO is a novel, selective antibody that blocks the activation of the interleukin-36 receptor (IL-36R), a key part of a signaling pathway within the immune system shown to be involved in the cause of GPP.

“GPP flares can greatly impact a patient’s life and lead to serious, life-threatening complications,” said Mark Lebwohl, M.D., lead investigator and publication author, and Dean for Clinical Therapeutics, Icahn School of Medicine at Mount Sinai, Kimberly and Eric J. Waldman Department of Dermatology, New York. “The approval of SPEVIGO is a turning point for dermatologists and clinicians. We now have an FDA-approved treatment that may help make a difference for our patients who, until now, have not had any approved options to help manage GPP flares.”

Distinct from plaque psoriasis, GPP is a rare and potentially life-threatening neutrophilic skin disease, which is characterized by flares (episodes of widespread eruptions of painful, sterile pustules). In the United States, it is estimated that 1 out of every 10,000 people has GPP. Given that it is so rare, recognizing the signs and symptoms can be challenging and consequently lead to delays in diagnosis.

“This important approval reflects our successful efforts to accelerate our research with the aim to bring innovative treatments faster to the people most in need,” said Carinne Brouillon, Member of the Board of Managing Directors, responsible for Human Pharma, Boehringer Ingelheim. “We recognize how devastating this rare skin disease can be for patients, their families and caregivers. GPP can be life-threatening and until today there have been no specific approved therapies for treating the devastating GPP flares. It makes me proud that with the approval of SPEVIGO we can now offer the first U.S. approved treatment option for those in need.” 

In the 12-week pivotal Effisayil 1 clinical trial, patients experiencing a GPP flare (N=53) were treated with SPEVIGO or placebo. After one week, patients treated with SPEVIGO showed no visible pustules (54%) compared to placebo (6%). 

In Effisayil 1, the most common adverse reactions (≥5%) in patients that received SPEVIGO were asthenia and fatigue, nausea and vomiting, headache, pruritus and prurigo, infusion site hematoma and bruising, and urinary tract infection.

“GPP can have an enormous impact on patients’ physical and emotional wellbeing. With the FDA approval of this new treatment, people living with GPP now have hope in knowing that there is an option to help treat their flares,” said Thomas Seck, M.D., Senior Vice President, Medicine and Regulatory Affairs, Boehringer Ingelheim. “SPEVIGO represents Boehringer Ingelheim’s commitment to delivering meaningful change for patients living with serious diseases with limited treatment options.”

About SPEVIGO
SPEVIGO is indicated for the treatment of GPP flares in adults. SPEVIGO is contraindicated in patients with severe or life-threatening hypersensitivity to spesolimab-sbzo or to any of the excipients in SPEVIGO. Reactions have included drug reaction with eosinophilia and systemic symptoms (DRESS).

What is SPEVIGO?
SPEVIGO is a prescription medicine used to treat generalized pustular psoriasis (GPP) flares in adults. It is not known if SPEVIGO is safe and effective in children.

U.S. FDA grants Priority Review for spesolimab for the treatment of flares in patients with generalized pustular psoriasis (GPP), a rare, life-threatening skin disease

https://www.boehringer-ingelheim.us/press-release/us-fda-grants-priority-review-spesolimab-treatment-flares-patients-generalized

December 15, 2021 – Boehringer Ingelheim today announced that the U.S. Food and Drug Administration (FDA) has accepted a Biologics License Application (BLA) and granted Priority Review for spesolimab for the treatment of generalized pustular psoriasis (GPP) flares. 

FDA grants Priority Review to applications for medicines that, if approved, would offer significant improvement over available options in the safety or effectiveness of the treatment, diagnosis, or prevention of serious conditions. The FDA has granted spesolimab Orphan Drug Designation for the treatment of GPP, and Breakthrough Therapy Designation for spesolimab for the treatment of GPP flares in adults.

“The FDA acceptance of our filing for spesolimab is a critical step in our efforts to bring this first-in-class treatment to people living with GPP,” said Matt Frankel, M.D., Vice President, Clinical Development and Medical Affairs, Specialty Care, Boehringer Ingelheim. “There is an urgent unmet need for an approved treatment option that can rapidly clear painful GPP flares.”

GPP is a rare, life-threatening neutrophilic skin disease, which is distinct from plaque psoriasis. It is characterized by episodes of widespread eruptions of painful, sterile pustules (blisters of non-infectious pus). There is a high unmet need for treatments that can rapidly and completely resolve the signs and symptoms of GPP flares. Flares greatly affect a person’s quality of life and can lead to hospitalization with serious complications, including heart failure, renal failure, sepsis, and death.

About spesolimab
Spesolimab is a novel, humanized, selective antibody that blocks the activation of the interleukin-36 receptor (IL-36R), a signaling pathway within the immune system shown to be involved in the pathogeneses of several autoimmune diseases, including GPP. Spesolimab is also under investigation for the prevention of GPP flares and for the treatment of other neutrophilic skin diseases, such as palmoplantar pustulosis (PPP) and hidradenitis suppurativa (HS).

About generalized pustular psoriasis (GPP)
GPP is a rare, heterogenous and potentially life-threatening neutrophilic skin disease, which is clinically distinct from plaque psoriasis. GPP is caused by neutrophils (a type of white blood cell) accumulating in the skin, resulting in painful, sterile pustules all over the body. The clinical course varies, with some patients having a relapsing disease with recurrent flares, and others having a persistent disease with intermittent flares. While the severity of GPP flares can vary, if left untreated they can be life-threatening due to complications such as sepsis and multisystem organ failure. This chronic, systemic disease has a substantial quality of life impact for patients and healthcare burden. GPP has a varied prevalence across different geographical regions and more women are affected than men.

Boehringer Ingelheim Immunology: Pioneering Science, Inspired By Patients
Living with fibrotic and inflammatory diseases greatly impacts patients’ lives emotionally and physically. These patients are our guides, partners and inspiration as we redefine treatment paradigms. As a family-owned company, we can plan long-term. Our goal is to discover and develop first-of-their-kind therapies. With a deep understanding of molecular pathways, we are pioneering scientific breakthroughs that target, repair and prevent many fibrotic and inflammatory diseases. By building on long-term external collaborations, we strive to bring treatment breakthroughs to patients in the shortest time. We won’t rest until we can give people the chance to live the lives they want.

Boehringer Ingelheim
Boehringer Ingelheim is working on breakthrough therapies that improve the lives of humans and animals. As a leading research-driven biopharmaceutical company, the company creates value through innovation in areas of high unmet medical need. Founded in 1885 and family-owned ever since, Boehringer Ingelheim takes a long-term perspective. Around 52,000 employees serve more than 130 markets in the three business areas, Human Pharma, Animal Health, and Biopharmaceutical Contract Manufacturing. Learn more at www.boehringer-ingelheim.com.

MPR-US-101971

////////Spesolimab, monoclonal antibody, fda 2022, approvals 2022, Orphan Drug Status, Generalised pustular psoriasis, BI 655130, Spesolimab-sbzo, peptide, monoclonal antibody

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Lenacapavir sodium


Lenacapavir.svg

Lenacapavir.pngChemSpider 2D Image | N-[(1S)-1-(3-{4-Chloro-3-[(methylsulfonyl)amino]-1-(2,2,2-trifluoroethyl)-1H-indazol-7-yl}-6-[3-methyl-3-(methylsulfonyl)-1-butyn-1-yl]-2-pyridinyl)-2-(3,5-difluorophenyl)ethyl]-2-[(3bS,4aR)-5,5-diflu oro-3-(trifluoromethyl)-3b,4,4a,5-tetrahydro-1H-cyclopropa[3,4]cyclopenta[1,2-c]pyrazol-1-yl]acetamide | C39H32ClF10N7O5S2

Lenacapavir sodium

レナカパビルナトリウム

Formula
C39H31ClF10N7O5S2. Na
C39H32ClF10N7O5S2 FREE FORM
CAS
2283356-12-5
2189684-44-2 FEE FORM
Mol weight
990.2641
 968.28 FREE FORM

2022/8/17 EMA APPROVED, SUNLECA

N-[(1S)-1-[3-[4-chloro-3-(methanesulfonamido)-1-(2,2,2-trifluoroethyl)indazol-7-yl]-6-(3-methyl-3-methylsulfonylbut-1-ynyl)pyridin-2-yl]-2-(3,5-difluorophenyl)ethyl]-2-[(2S,4R)-5,5-difluoro-9-(trifluoromethyl)-7,8-diazatricyclo[4.3.0.02,4]nona-1(6),8-dien-7-yl]acetamide

Treatment of HIV-1 infection

PF-3540074, to GS-CA1,

GS-6207

GS-HIV

GS-CA1

GS-CA2

Lenacapavir, sold under the brand name Sunlenca, is a medication used to treat HIV/AIDS.[1] It is taken by mouth or by subcutaneous injection.[1]

The most common side effects include reactions at the injection site and nausea.[1]

Lenacapavir was approved for medical use in the European Union in August 2022.[1]

HIV/AIDS remains an area of concern despite the introduction of numerous successful therapies, mainly due to the emergence of multidrug resistance and patient difficulty in adhering to treatment regimens.1,2 Lenacapavir is a first-in-class capsid inhibitor that demonstrates picomolar HIV-1 inhibition as a monotherapy in vitro, little to no cross-resistance with existing antiretroviral agents, and extended pharmacokinetics with subcutaneous dosing.1,2,3,5

Lenacapavir was first globally approved by the European Commission to treat adults with multi-drug resistant HIV infection.7 It is currently being investigated in clinical trials in the US.

U.S. Patent Application No. 15/680,041 discloses novel compounds useful for treating a Retroviridae viral infection, including an infection caused by the HIV virus. One specific compound identified therein is a compound of formula I:

PATENTS

  1.  WO 2018/035359 A1
  2. Different formulations and salts: WO 2019/035904 A1; WO 2019/035973 A1

PATENT

WO 2019/161280 A1

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2019161280

I. Synthesis of Starting Materials and Intermediates

Example la: Preparation of (S)-1-(3,6-dibromopyridin-2-yl)-2-(3,5-difluorophenyl)ethan- 1-amine (VIII-02), or a co-crystal, solvate, salt, or combination thereof, and starting materials and/or intermediates therein

wherein R4 and R5 are each independently hydrogen, methyl, phenyl, benzyl, 4-nitrobenzyl, 4-chlorobenzyl, 4-brornobenzylamine, or 4-methoxybenzyl

Synthesis of 3,6-dibromopicolinaldehyde (1a)

[00553] A dry reaction flask with magnetic stir-bar was charged with 2,5-dibromopyridine (1.0 g). The flask was inerted under nitrogen, THF (4.2 mL) was added, and the thin slurry agitated. Separately, a dry glass reactor was charged with 2,2,6,6-tetramethylpiperidinylmagnesium chloride, lithium chloride complex (TMPMgCl●LiCl) (5.8 mL, 6.3 mmol). The TMPMgCl●LiCl solution was agitated and cooled to about -20 °C. The 2,5-dibromopyridine solution was added to the TMPMgCl●LiCl solution over about 30 min, maintaining a temperature below about -18 °C. Upon completing the addition, the flask was rinsed forward to the reactor with three additional portions of THF (1 mL x 2), and aged at about -20 for about 1 hour. A solution of N,N-dimethylformamide (1.6 mL, 20 mmol) in THF (1.6 mL) was added to the reactor over about 15 min. The reaction mixture was aged for a further 15 min. and quenched by the addition of a solution of acetic acid (1.9 mL, 34 mmol) in water (10 mL) over about 20 minutes, maintaining a temperature of no more than about 0 °C. To the reactor was added isopropyl acetate (10 mL) and the reaction mixture was warmed to about 20 °C. After aging for 30 min, the mixture was filtered through diatomaceous earth and the reactor rinsed with a mixture of isopropyl acetate (10 mL), saturated aqueous ammonium chloride (10 mL) and 0.2 M aqueous hydrochloric acid (10 mL). The reactor rinse was filtered and the pH of the combined reaction mixture was adjusted to about 8-9 by the addition of a 10% aqueous sodium hydroxide solution (about 6 mL). The mixture was filtered a second time to remove magnesium salts and transferred to a separatory funnel. The phases were separated and the aqueous phase was extracted with isopropyl acetate (3 x 10 mL). The combined organic extracts were washed with 50% saturated aqueous sodium chloride (20 mL), dried over anhydrous sodium sulfate, and filtered. The solution was concentrated to dryness by rotary evaporation and purified by chromatography (eluting with 0-100% ethyl acetate in heptane) to afford 3,6-dibromopicolinaldehyde (1a) as a solid. 1H NMR (400 MHz, DMSO-d6) δ 9.94 (q, J = 0.6 Hz, 1H), 8.19 (dq, J = 8.4, 0.6 Hz, 1H), 7.82 (dt, J = 8.4, 0.7 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 189.33, 148.59, 145.66, 140.17, 133.19, 120.27.

Synthesis of 3,6-dibromopicolinaldehyde (1a)

[00554] A solution of 2,5-dibromo-6-methylpyridine (8.03 g) in THF (81 mL) was cooled to about 0 °C. To this solution was charged tert-butyl nitrite (4.33 g), followed by a dropwise addition of potassium tert-butoxide (28 mL, 1.5 equiv, 20 wt% solution in THF). The reaction mixture was agitated at about 0 °C until the reaction was complete. The reaction mixture was diluted with THF (24 mL), and quenched with ammonium chloride (6.38 g, 119 mmol) in water (43 mL). The reaction mixture was distilled under vacuum to approximately 55 mL to afford a slurry, which was filtered and washed twice with water (2x 24 mL) to afford 1h. 1H NMR (400 MHz, DMSO-d6) δ 11.69 (s, 1H), 8.08 (d, J = 8.4 Hz, 1H), 7.67 (s, 1H), 7.61 (d, J = 8.5 Hz, 1H).

[00555] A solution of glyoxylic acid (407 L, 50 wt% in water) was heated to about 80 °C and in portions was charged with 1h (40.69 kg, 145.4 mol) . Reaction mixture was held at this temperature until the reaction was complete. The reaction mixture was cooled to about 20 °C, filtered, and the filter cake was washed with water until the filtrate had a pH ≥ 5, to afford 1a. 1H NMR (400 MHz, DMSO-d6) δ 9.95 (s, 1H), 8.22 (d, J = 8.4 Hz, 2H), 7.85 (d, J = 8.4 Hz, 1H).

Synthesis of (E)-N-benzhydryl-1-(3,6-dibromopyridin-2-yl)methanimine (1b-02)

[00556] Compound 1a (5.0 g, 18.0 mmol) in toluene (20 mL) was heated to about 50 °C and benzhydrylamine (3.47 g, 18.9 mmol) was charged in one portion and agitated at this temperature until the reaction was deemed complete. Methanol (61 mL) was charged and the reaction mixture was distilled to a volume of approximately 25 mL. Methanol (40 mL) was charged and the reaction mixture was distilled to a volume of approximately 30 mL. The resulting slurry was filtered and rinsed with two portions of methanol (15 mL each) and dried under vacuum to afford 1b-02.

[00557] Alternatively, compound 1a (10.0 g, 37.8 mmol) in 2-methyltetrahydrofuran (50 mL) was heated to about 50 °C and benzhydrylamine (7.28 g, 39.7 mmol) was charged dropwise. The reaction was agitated at this temperature until it was deemed complete. The reaction mixture was distilled to a volume of approximately 30 mL. To the reaction mixture was charged heptane (100 mL) and 1b-02 seed (59.3 mg, 0.138 mmol). The resulting slurry was filtered, rinsed with two portions of heptane (2x 20 mL), and dried under vacuum to afford 1b-02. 1H NMR (400 MHz, DMSO-d6) δ 8.73 (s, 1H), 8.12 (d, J = 8.4 Hz, 1H), 7.67 (d, J = 8.4 Hz, 1H), 7.44 – 7.40 (m,

4H), 7.38 – 7.32 (m, 4H), 7.28 – 7.22 (m, 2H), 5.88 (s, 1H).

Synthesis of (E)-N-benzhydryl-1-(3,6-dibromopyridin-2-yl)methanimine (1b-02)

[00558] 1a (2.00 g) was combined with isopropanol (7.6 mL) and agitated at ambient temperature. To this mixture was added potassium metabisulfite (0.96 g) in water (3.8 mL), dropwise. This mixture was agitated for at least 90 minutes and the resulting slurry was filtered. The filter cake was rinsed twice with isopropanol (6 mL then 12 mL) to afford 1i-1. 1H NMR (400 MHz, DMSO-d6) δ 7.92 (d, J = 8.3 Hz, 1H), 7.47 (d, J = 8.3 Hz, 1H), 5.48 – 5.38 (m, 2H).

[00559] li-1 (1.00 g) was combined with 2-methyltetrahydrofuran (3.5 mL) and agitated at ambient temperature. To this slurry was charged potassium hydroxide (443.8 mg, 7.91 mmol) in water (4 mL) and the biphasic mixture was agitated for 2 hours. The layers were separated and the aqueous layer was extracted with an additional portion of 2-methyltetrahydrofuran (3.5 mL). To the combined organics was charged benzhydrylamine (0.47 mL, 2.7 mmol). The reaction mixture was concentrated in vacuo (-300 mbar, 45 °C bath) to a volume of approximately 3 mL. Heptane (7 mL) was charged and the mixture was agitated. The resulting slurry was filtered to afford 1b-02 1H NMR (400 MHz, DMSO-d6) δ 8.73 (s, 1H), 8.12 (d, J = 8.4 Hz, 1H), 7.67 (d, J = 8.4 Hz, 1H), 7.44 – 7.40 (m, 4H), 7.38 – 7.32 (m, 4H), 7.28 – 7.22 (m, 2H), 5.88 (s, 1H).

Synthesis of (E)-N-benzhydryl-1-(3,6-dibromopyridin-2-yl)methanimine (1b-02)

[00560] Compound 1a (1.0 g) was added to a reactor, and toluene (6.0 mL) was added to the reactor. The mixture was agitated. Aminodiphenylmethane (0.73 g, 1.05 equiv.) was added to the reaction mixture. The jacket was heated to about 60 °C, and the mixture was allowed to age for about 1 hour. After about one hour, the mixture was carried forward to the next step. 1H NMR (400 MHz, DMSO-d6) δ 8.68 (s, 1H), 8.05 (d, J = 8.4 Hz, 1H), 7.60 (d, J = 8.4 Hz, 4H), 7.40 – 7.34 (m, 7H), 7.29 (td, J = 6.9, 6.5, 1.7 Hz, 5H), 7.22 – 7.16 (m, 3H), 5.81 (s, 1H).

Synthesis of N-(1-(3,6-dibromopyridin-2-yl)-2-(3,5-difluorophenyl)ethyl)-1,1-diphenylmethanimine (1d-02)

[00561] A solution of1b-02 in toluene (1.0 g in 3.8 mL) was stirred in a reactor at about 60 °C. Tetrabutylammonium bromide (0. 08 g, 0.10 equiv.) was added, 3,5-difluorobenzylbromide (0.60 g, 1.20 equiv.) was added, and potassium hydroxide (50% in water, 1.3 g, 5 equiv.) was added. The mixture was aged for about 4 hours and sampled for conversion. When the reaction was complete, the aqueous phase was removed, and water (3.1 mL) was added to the reactor. Contents were agitated and phases were allowed to settle. The aqueous phase was removed, and the toluene solution of1d-02 was carried forward to the next step. 1H NMR (400 MHz, Chloroform-d) δ 7.78 (dd, J = 8.6, 1.0 Hz, 1H), 7.64 – 7.60 (m, 2H), 7.59 – 7.53 (m, 1H), 7.49 (d, J = 8.3 Hz, 1H), 7.47 (s, 0H), 7.45 (s, 0H), 7.43 (d, J = 0.7 Hz, 0H), 7.41 – 7.34 (m, 3H), 7.33 (t, J = 1.4 Hz, 1H), 7.28 (t, J = 7.3 Hz, 2H), 7.22 (s, 0H), 7.18 (d, J = 8.3 Hz, 1H), 6.87 (dd, J = 7.7, 1.7 Hz, 2H), 6.55 (dt, J = 9.0, 2.3 Hz, 1H), 6.50 (dd, J = 7.0, 4.9 Hz, 3H), 5.26 (s, 0H), 5.16 (t, J = 6.9 Hz, 1H), 3.32 (dd, J = 13.2, 6.6 Hz, 1H), 3.16 (dd, J = 13.1, 7.2 Hz, 1H).

Synthesis of 1-(3,6-dibromopyridin-2-yl)-2-(3,5-difluorophenyl)ethan-1-amine (X) from N-(1-(3,6-dibromopyridin-2-yl)-2-(3,5-difluorophenyl)ethyl)-1,1-diphenylmethanimine (1d-02)

[00562] A solution of 1d-02 in toluene (1.0 g in 3.0 mL) was stirred in a reactor at about 60 °C. Sulfuric acid (0.93 g, 5 equiv.) was diluted into water (3.5 mL), and added to the reactor. The mixture was aged for about 4 hours. When the reaction was complete, the aqueous phase was removed. The aqueous phase was recharged to the reactor, and heptane (2.5 mL) was added. The mixture was agitated and agitation stopped and layers allowed to settle. The aqueous phase was removed, and heptane was discharged to waste. Toluene (5.0 mL) and potassium hydroxide (50% in water, 2.1 g, 10 equiv.) was added to the reactor. The aqueous acidic solution was added to the reactor. The mixture was agitated for about 10 minutes, and agitation stopped and phases allowed to settle. The aqueous phase was discharged to waste. Water (2.5 mL) was added to the reactor, and the mixture was agitated for about 5 minutes, and agitation was stopped and the phases were allowed to settle. The aqueous phase was discharged to waste. The toluene solution of 1-(3,6-dibromopyridin-2-yl)-2-(3,5-difluorophenyl)ethan-1-amine (X) was carried forward to the next step. 1H NMR (400 MHz, Chloroform-d) δ 7.60 (d, J = 8.3 Hz, 1H), 7.21 (d, J = 8.3 Hz, 1H), 6.74 – 6.67 (m, 2H), 6.66 – 6.58 (m, 1H), 4.57 – 4.45 (m, 1H), 3.02 (dd, J = 13.5, 5.2 Hz, 1H), 2.72 (dd, J = 13.5, 8.6 Hz, 1H), 1.77 (s, 3H).

Synthesis of (S)-1-(3.6-dibromopyridin-2-yl)-2-(3,5-difluorophenyl)ethan-1-amine (R)-2-hydroxy-2-phenyl acetate (VIII-03)

[00563] A solution of X in toluene (1.0 g in 7.1 mL) was stirred in a reactor at about 60 °C. The mixture was distilled to minimum volumes (2.9 mL), and methyl tert-butyl ether was added (7.1 mL). (R)-(-)-Mandelic acid (0.41 g, 1 equiv.) was added, and the mixture was cooled to about 0 °C. The newly formed slurry was filtered, providing (S)-1-(3,6-dibromopyridin-2-yl)-2-(3,5-difluorophenyl)ethan-1-amine (R)-2-hydroxy-2-phenylacetate (VIII-03). 1H NMR (400 MHz, DMSO-d6) δ 7.93 (d, J = 8.4 Hz, 1H), 7.49 (d, J = 8.4 Hz, 1H), 7.34 (d, J = 7.3 Hz, 2H), 7.28 – 7.14 (m, 4H), 7.01 (tt, J = 9.4, 2.3 Hz, 1H), 6.79 (d, J = 7.4 Hz, 3H), 4.77 (s, 1H), 4.55 (d, J = 6.6 Hz, 1H), 3.02 (s, 1H), 2.92 (d, J = 6.7 Hz, 2H), 1.05 (s, 2H).

Synthesis of (S)-1-(3,6-dibromopyridin-2-yl)-2-(3,5-difluorophenyl)ethan-1-amine N-acetyl-D- Leucine (VIII-04)

[00564] A reactor was charged with X (15.0 g), N-acetyl-D-leucine (8.28 g) and zinc oxide (0.311 g). Toluene (375 mL) was charged to the reactor followed by 2-pyridinecarboxaldehyde (183 μL). The mixture was aged at about 55 °C for about 6 hrs. and then held at about 35 °C for about 4 days. The mixture was cooled to about 0 °C and held for about 17 hrs. The product was isolated by filtration and the filter cake was washed with cold toluene (2 x 75 mL). The filter cake was re-charged to the reactor. Ethanol (150 mL) was added and the mixture distilled to remove residual toluene. Once the toluene was removed, the reactor volume was adjusted with ethanol to about 90 mL and the mixture was cooled to about 25 °C. Water (210 mL) was added over approximately 10 min. and the mixture aged for approximately 12 hrs. The slurry was filtered and the solids were dried to afford VIII-04. 1H NMR (400 MHz, DMSO-d6) δ 8.03 (d, J = 8.0 Hz, 1H). 7.95 (d, J = 8.3 Hz, 1H), 7.49 (d, 7 8.3 Hz, 1H), 7.03 (tt, J = 9.5, 2.4 Hz, 1H),

6.87 (dtd, J = 8.4, 6.2, 2.2 Hz, 2H), 5.49 (s, 3H), 4.42 (dd, J = 7.9, 5.9 Hz, 1H), 4.18 (q, J = 7.8 Hz, 1H), 2.93 (dd, J = 13.3, 5.9 Hz, 1H), 2.85 (dd, J = 13.2, 8.0 Hz, 1H), 1.83 (s, 3H), 1.71 -1.54 (m, 1H), 1.47 (dd, J = 8.4, 6.2 Hz, 2H), 0.88 (d, J = 6.6 Hz, 3H), 0.83 (d, J = 6.5 Hz, 3H).

13C NMR (101 MHz, DMSO-d6) δ 174.72, 169.03, 162.07 (dd, J = 245.5, 13.3 Hz), 161.79, 143.51, 142.82 (t, J = 9.4 Hz), 139.72, 128.39, 119.30, 113.36 – 111.39 (m), 101.73 (t, J = 25.7 Hz), 55.19, 50.69, 41.74 (d, J = 2.3 Hz), 40.51, 24.36, 22.91, 22.44, 21.46.

Example 1b: Preparation of alternative starting materials and intermediates for use in the formation of (S)-1-(3,6-dibromopyridin-2-yl)-2-(3,5-difliiorophenyl)ethan-1-amine (VIII), or a co-crystal, solvate, salt, or combination thereof

Synthesis of (R)-1-(3,6-dibromopyridin-2-yl)-2-(3,5-difluorophenyl)ethan-1-ol (XII)

[00565] A stainless steel autoclave equipped with a glass inner tube was charged with compound XI (1.00 g) and (A)-RuCY-XylBINAP (16 mg, 0.05 equiv.). EtOH (1.0 mL) and IPA (1.0 mL) followed by tert-BuOK (1.0 M solution in THE, 0.51 mL, 0.2 equiv.) were added to the autoclave. After being purged by H2, the autoclave was charged with 3 MPa 
of H2. The mixture was stirred at about 20 °C for about 10 h. To the mixture, cone. HCl aqueous solution was added and pH was adjusted to 2. 1H NMR (400 MHz, CDCl3): δ 7.72 ( d, J = 8.2 Hz, 1H), 7.33 (d, J = 8.2 Hz, 1H), 6.80 -6.72 (m, 2H), 6.68 (tt, J = 9.2, 2.4 Hz, 1H), 5.16 (dd, J = 8.2, 3.4 Hz, 1H), 3.60 (br, 1H), 3.12 (dd, J = 13.8, 3.4 Hz, 1H), 2.81 (dd, J = 13.8, 8.2 Hz,

1H). 13C NMR (100 MHz, CDC13): d 162.8 (dd, J= 246.4, 12.9 Hz), 160.1, 143.0, 141.3 (t, j = 9.1 Hz), 139.8, 128.7 (t, J= 35.7 Hz), 117.9, 112.3 (m), 102.1 (t, J= 25.0 Hz), 72.0, 43.0. 19F NMR (376 MHz, CDCl3): δ -112.1 (m).

Synthesis of N-(1-(3,6-dibromopyridin-2-yl)-2-(3,5-difluorophenyl)ethyl)-15-chloranimine (X-02)

[00566] Compound XIII (.0 g) was dissolved in THF (4.2 mL) and was cooled over an ice bath. Diphenylphosphoryl azide (0.66 mL, 1.2 equiv.) was added followed by DBU (0.46 mL, 1.2 equiv.) over about 25 min at below about 4 °C. The dark mixture was aged about 1 hour, and the cooling bath was removed. After about 2.5 hours age at RT, some starting material was still present so more diphenylphosphoryl azide (0.15 equiv.) and DBU (0.15 equiv.) were added after cooling over an ice bath. After about 2 hours, more diphenylphosphoryl azide (0.08 equiv.) and DBU (0.08 equiv.) were added. The reaction mixture was allowed to age overnight for about 16 h to allow the conversion to azide intermediate complete. The reaction mixture was cooled over an ice bath and triphenylphosphine (1.0 g, 1.5 equiv.) was added over about 15 min at about 6 °C). The cooling bath was removed after about 10 min and the reaction mixture was agitated for additional about 2.5 hours. To this reaction mixture was added water (0.18 mL, 4 equivalents) and the resulting mixture was aged for about 15 hours at room temperature. The mixture was diluted with EtOAc (5.0 mL) and was washed with water (4.2 mL + 2.0 mL). The aqueous layer was back extracted with EtOAc (4.0 mL) and the EtOAc layer was washed with water (1.0 mL). The organic layers were combined, concentrated via rotary evaporation and evaporated with EtOAc (4 x 4.0 mL) to dry. The residue was dissolved to a 50 ml solution in EtOAc, and cooled over an ice bath to become slurry. To the cold slurry 4N HCl/dioxane (0.76 mL, 1.2 equiv.) was added and the slurry was aged about 2 hours at room temperature. The solid product was filtered and the filter cake was rinsed with EtOAc and dried at about 35 to 50 °C under vacuum to give X-02.

[00567] Recrystallization: A portion of the above obtained X-02 (1.0 g) was mixed with EtOAc (10 mL) and was heated to 65 °C to afford thick slurry. The slurry was aged at about 65 °C for about 2 hours, and overnight at room temperature. The solids were filtered with recycling the mother liquor to help transfer the solids. The filter cake was rinsed with EtOAc, and dried overnight at about 50 °C vacuum to afford X-02. 1H NMR (300 MHz, DMSO-d) δ 8.78 (br s, 3 H), 8.06-8.02 (m, 1 H), 7.64-7.61 (m, 1 H), 7.15-7.08 (m, 1 H), 6.83-6.78 (m, 2 H), 4.87-4.82 (m, 1 H), 3.35-3.25 (m, 1 H), 3.17-3.05 (m, 1 H). 19F NMR (282.2 MHz, Chloroform-d) δ – 109.9-110.1 (m).

Synthesis of 1-(3,6-dibromopyridin-2-yl)-2-(3,5-difluorophenyl)ethyl methanesulfonate (XIII-A)

[00568] Compound XIII (1.0 g) and DMAP (0.1 equiv.) were dissolved in THF (4.5 mL) and cooled over an ice bath. Triethylamine (Et3N) (0.39 mL, 1.1 equiv.) was added followed by methanesulfonyl chloride (218 μL, 1.1 equiv.). The cooling bath was removed, and the mixture was aged about 1.5 hours at room temperature. The reaction mixture was cooled over an ice bath and quenched with water (10 mL). The mixture was diluted with EtOAc and the phases were separated. The aqueous phase was extracted with EtOAc, and the combined organic phase was dried (Na2SO4) and was passed through silica gel with EtOAc. The filtrate was concentrated to afford the mesylate (XIII-A). 1H NMR (300 MHz, Chloroform-d) δ 7.72-7.66 (m, 1 H), 7.38-7.32 (m, 1 H), 6.78-6.63 (m, 3 H), 6.17-6.13 (m, 1 H), 3.40-3.25 (m, 2 H), 2.87 (s, 3 H). 19F NMR (282.2 MHz, Chloroform-d) δ -109.3—109.5 (m).

Synthesis of 1-(3,6-dibromopyridin-2-yl)-2-(3,5-difluorophenyl)ethan-1-amine (X) from 1-(3,6- dibromopyridin-2-yl)-2-(3,5-difluorophenyl)ethyl methanesulfonate (XIII-A)

[00569] A glass pressure bottle was charged with the mesylate (XIII-A) (1.0 g), 28-30% ammonium hydroxide (19 mL) and MeOH (4.7 mL). The mixture was sealed and heated at about 70 °C for about 16 hours, and extracted with 2-MeTHF/ EtOAc. The organic layer was dried (Na2SO4) and purified by silica gel chromatography (10-60% EtOAc/hexanes) to afford racemic amine X. 1H NMR (300 MHz, Chloroform-d) δ 7.70-7.60 (m, 1 H), 7.30-7.20 (m, 1 H), 6.78-6.60 (m, 3 H), 4.46-4.58 (m, 1 H), 3.00-3.16 (m, 1 H), 2.70-2.80 (m, 1 H). 19F NMR (282.2 MHz, Chloroform-d) δ -110.3 – 110.4 (m).

Synthesis of (Z)-N-(1-(3,6-dibrornopyridin-2-yl)-2-(3,5-difluorophenyl)vinyl)acetamide (1f)

[00570] A glass reactor was charged with XI (1.0 g). Ethanol (5.0 mL) was added, and the slurry was agitated while hydroxylamine hydrochloride (0.88 g) was charged. Pyridine (1.0 mL) was added and the mixture heated at about 55-65 °C for about two hours. The mixture was cooled to about 20 °C, transferred to a flask, and concentrated to approximately 75 mL by rotary evaporation. The concentrate was returned to the reactor, rinsing through with isopropyl acetate (5.0 mL). Residue remaining in the flask was carefully (gas evolution) rinsed into the reactor with saturated aqueous sodium bicarbonate (5.0 mL). The bi-phasic mixture was agitated, the phases separated, and the organic extract washed with water (3.2 mL) and saturated sodium chloride (3.2 mL). The organic extract was dried over anhydrous sodium sulfate, filtered, and concentrated to dryness by rotary evaporation to yield 1e which was used without further purification.

[00571] A glass reactor was charged with iron powder (<10 micron, 0.30 g mmol) followed by acetic acid (1.6 mL) and acetic anhydride (0.72 mL). The slurry was de-gassed by holding the reactor contents under vacuum until bubbling was observed, and back-filled with nitrogen (3 cycles). The mixture was heated at 115-120 °C for 2 hours and cooled to 40 °C. Compound le from the previous step in isopropyl acetate (2.0 mL) was added over 30 min. Upon completing the addition, the temperature was raised to 45-65 °C and the mixture aged for about 2 hours. A slurry of diatomaceous earth (1.0 g) in isopropyl acetate (2.0 mL) was added, followed by toluene (2.0 mL). The slurry was filtered, hot, through a Buchner funnel and the reactor and filter cake were washed with warm isopropyl acetate (3 x 1.8 mL). The filtrate was transferred to a reactor and the solution washed with 0.5% aqueous sodium chloride (4.2 mL). Water (3.1 mL) was added to the reactor and the mixture was cooled to about 5 °C. The pH was adjusted to 7-9 with the addition of 50 wt% aqueous sodium hydroxide; following separation, the organic extract was warmed to room temperature and washed with aqueous 1% (w/w) sodium chloride NaCl (3.6 mL). The organic extract was discharged to a flask and dried over anhydrous sodium sulfate (ca. 0.8 g), filtered through diatomaceous earth, and concentrated to approximately 4 mL at 100 mmHg and 45 °C water bath. The warm solution was returned to the reactor, rinsing forward with isopropyl acetate to a produce a total volume of approximately 5.2 mL. This solution was heated further to 50 °C with agitation, cooled to about 35 °C, and seeded with pure 1f (0.006 g). Heptane (9.6 mL) was added over a period of about 4 hours, the solution was cooled to about 10 °C, and the product was isolated by filtration. The filter cake was washed with 33.3% iPAc in heptane (4.0 mL) and dried in a vacuum oven at 40 °C with nitrogen sweep for approximately 24 hours. Compound 1f, a mixture of geometric isomers (approximately 94:6 ratio) was isolated. Major isomer: 1H NMR (400 MHz, DMSO-d6) δ 9.96 (s, 1H), 8.04 (d, J = 8.4 Hz, 1H), 7.66 (d, J= 8.4 Hz, 1H), 7.05 (s, 1H), 6.97 (tt, J = 9.2, 2.2 Hz, 1H), 6.40 – 6.31 (m,

2H), 1.97 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 168.37, 162.04 (dd, J = 245.1, 13.9 Hz), 154.47, 143.63, 139.45, 139.40 – 139.18 (m), 135.99, 129.44, 120.66, 113.80, 111.23 – 109.68 (m), 101.77 (t, J = 26.0 Hz), 23.49.

Synthesis of (S)-N-(1-(3,6-dibromopyridin-2-yl)-2-(3,5-difluorophenyl)ethyl)acetamide (1g)

[00572] Preparation of catalyst solution: A flask was charged with [IrCl(cod)((S)-segphos)] (110 mg) and the internal atmosphere was replaced with N2. EtOAc (200 mL) was added to the flask and the mixture was stirred until the catalyst solid was dissolved.

[00573] A stainless steel autoclave was charged with compound 1f (1.0 mg). EtOAc (16 mL) and followed by the catalyst solution prepared above (4.0 mL, 0.001 equiv.) were added to the autoclave. After being purged by H2, the autoclave was charged with 3 MPa of H2.


The mixture was stirred at about 130 °C for about 6 hours and cooled to room temperature and H2 was vented out. The reaction mixture was purified by silica gel column chromatography (EtOAc/Hexane = 1/4 to 1/1) to afford 1g. 1H NMR (400 MHz, CD2Cl2): d 7.70 ( d, J = 8.0 Hz, 1H), 7.30 (d, J = 8.0 Hz, 1H), 6.68 (tt, J = 9.2, 2.4 Hz, 1H), 6.64 -6.58 (m, 2H), 6.49 (brd, j = 8.0 Hz, 1H), 5.74 (ddt, J = 8.0, 7.2, 6.4 Hz, 1H), 3.10 (dd, J = 13.6, 6.4 Hz, 1H), 2.99 (dd, J = 13.6, 7.2 Hz), 1.95 (s, 3H). 13C NMR (100 MHz, CD2Cl2): δ 169.5, 163.3 (dd, J = 246.0, 12.9 Hz), 159.1, 143.6, 141.4 (t, J = 9.1 Hz), 140.7, 129.1, 119.9, 112.9 (m), 102.6 (t, J= 25.1 Hz), 53.0, 41.3, 23.6. 19F NMR (376 MHz, CD2Cl2): δ -111.3 (m).

Synthesis of (S)-1-(3,6-dibromopyridin-2-yl)-2-(3,5-difluorophenyl)ethan-1-amine (VIII) from 1-(3,6-dibromopyridin-2-yl)-2-(3,5-difluorophenyl)ethan-1-one (XI), Method 1

[00574] A glass-lined reactor was charged with isopropylamine (about 18 g) and triethanolamine (3.8 g). Water (231 mL) was added and the pH was adjusted to about 7.5 by the addition of concentrated hydrochloric acid. A portion of the buffer solution (23 mL) was removed. The transaminase enzyme (2.5 g) was added to the reactor as a suspension in buffer solution (12 mL), followed by addition of pyridoxal phosphate monohydrate (50 mg) as a solution in buffer solution (12 mL). A solution of XI (1.0 g) in dim ethyl sulfoxide (23 mL) was added to the reactor and the mixture was heated at about 35 °C for about 48 hours with constant nitrogen sparging of the solution. The reaction mixture was cooled to about 20 °C the unpurified amine was removed by filtration. The filter cake was washed with water (3 x 7.7 mL) and the product was dried at about 60 °C under vacuum with nitrogen sweep to afford VIII.

Synthesis of (S)-1-(3.6-dibromopyridin-2-yl)-2-(3.5-difluorophenyl)ethan-1-amine (VIII) from 1-(3,6-dibromopyridin-2-yl)-2-(3,5-difluorophenyl)ethan-1-one (XI), Method 2

[00575] A stainless steel reactor was charged with XI (1.0 g) and p-toluenesulfonic acid (0.49 g). Ammonia (7 M in methanol, 3.7 mL) was added and the vessel was sealed and heated at about 60 °C for about 18 hours. The mixture was cooled to about 20 °C and sparged for about 30 min to remove excess ammonia. A solution of diacetato[(R)-5,5′-bis(diphenylphosphino)-4,4′-bi-1,3-benzodioxole]ruthenium(II) (0.10 g) in methanol (0.5 mL) was added to the reactor, which was sealed and heated at about 60 °C under a hydrogen atmosphere (400 psi) for a further about 6-10 hours. Upon cooling to about 20 °C the mixture was filtered through a plug of silica, rinsing with additional methanol (5.0 mL). Concentration of the filtrate by rotary evaporation affords VIII.

Example 1c: Preparation of 1-(3,6-dibromopyridin-2-yl)-2-(3,5-difluorophenyI)ethan-1-amine (X) by racemization of (S)-1-(3,6-dibromopyridin-2-yl)-2-(3,5-difluorophenyl)ethan-1-amine (VIII)

[00576] A vial was charged with zinc acetate (25 mol %), enantioenriched VIII (1.0 g, 92:8 enantiomer ratio), toluene (10 mL), and 2-formylpyridine (5 mol %). The vial was wanned to about 60 °C and stirred for about 4 h.

Example 2: Preparation of (S)-1-(3-bromo-6-(3-methyl-3-(methylsulfonyl)but-1-yn-1-yl)pyridin-2-yl)-2-(3,5-difluorophenyl)ethan-1-amine (VI)

[00577] A glass-lined reactor was charged with (S)-1-(3,6-dibromopyridin-2-yl)-2-(3,5-difluorophenyl)ethan-1-amine (R)-mandelic acid salt (VIII-03) (1.0 g), 3-methyl-3-(methylsulfonyl)but-1-yne (IX) (about 0.3 g), and dichlorobis(triphenylphosphine)palladium(II) (about 0.39 g). The reactor was evacuated and purged with nitrogen to inert. To this reactor was added 2-methyltetrahydrofuran (6.4 kg) and triethylamine (0.92 kg 5.0 equiv.). The reaction mixture was agitated at about 65-75 °C until the reaction was deemed complete by HPLC analysis. Upon cooling to about 30-40 °C the reaction mixture was discharged to another reactor and the parent reactor was rinsed with 2-methyltetrahydrofuran (4.6 g) and the resulting solution transferred to the receiving reactor. To the reactor was added water (5.0 g) and the biphasic mixture agitated at about 30-40 °C for about 30 min. Agitation was ceased and the mixture was allowed to layer for 30 min. The lower aqueous layer was discharged and the remaining organic solution held for about 15 hours. A solution of A-acetyl-L-cysteine (196 g) and sodium hydroxide (0.80 g) in water (11.8 g) was prepared. To the reactor was added approximately half of the N-acetyl-L-cysteine solution (6.7 g). The mixture was agitated at about 55-65 °C for about 30 min. The temperature was adjusted to about 30-40 °C and agitation was ceased. After about 30 min had elapsed, the lower aqueous phase was discharged. The remaining alkaline N-acetyl-L-cysteine solution (5.4 kg) was added and the mixture was heated, with agitation, to about 55-65 °C and held for about 30 min. The temperature was adjusted to about 30-40 °C and agitation was ceased. After about 30 min had elapsed, the lower aqueous phase was discharged. To the reactor was added a solution of sodium chloride (0.26 g) in water (4.9 g) and the mixture agitated at about 30-40 °C for about 30 min. Agitation was ceased and the biphasic mixture allowed to layer for about 30 min. The lower aqueous layer was discharged and the contents cooled to about 15-25 °C and held for about 16 hours. The mixture was concentrated at about 55-65 °C. The concentrated solution was cooled to about 30-40 °C and heptane (3.4 kg) was added over about 2 hours. The resulting slurry was cooled to about 20 °C and aged for about 20 h, and filtered. The filter cake was washed with 2-methyltetrahydrofuran/heptane (1:1 v/v,2 mL) and the solids dried in a vacuum oven at about 40 °C to yield (S)-1-(3-bromo-6-(3-methyl-3-(methylsulfonyl)but-1-yn-1-yl)pyridin-2-yl)-2-(3,5-difluorophenyl)ethan-1-amine (VI)). 1H NMR (400 MHz, DMSO-d6) δ 8.05 (d, J = 8.2 Hz, 1H), 7.42 (d, J = 8.2 Hz, 1H), 7.01 (tt, J = 9.5, 2.4 Hz, 1H), 6.97 – 6.84 (m, 2H), 4.41 (dd, J = 8.5, 5.2 Hz, 1H), 3.20 (s, 3H), 2.93 (dd, J = 13.3, 5.2 Hz, 1H), 2.79 (dd, J = 13.3, 8.5 Hz, 1H), 1.99 (s, 2H), 1.68 (s, 6H). 13C NMR (101 MHz, DMSO-d6) δ 162.25, 162.00 (dd, J = 245.2, 13.4 Hz), 143.88 (t, J= 9.4 Hz), 141.09, 139.72, 127.51, 120.08, 112.58 – 112.12 (m), 101.45 (t, J= 25.7 Hz), 87.94, 84.25, 57.24, 55.90, 42.57, 34.99, 22.19.

Example 2a: Preparation of 3-methyl-3-(methylsulfonyl)but-1-yne (IX)

[00578] Sodium methansulfmate (418.1 g), copper (II) acetate (26.6 g), N,N,N’,N’- Tetramethylethylenediamine (TMEDA, 34.0 g), and isopropyl acetate (2100 mL) were added to a reactor and the suspension was agitated at 20 – 25 °C. 3-Chloro-3-methylbut-1-yne (3-CMB,

300 g) was added slowly to maintain a constant temperature of about 20 – 25 °C. The reaction mixture was then heated to about 30 °C until the reaction was complete. The mixture was cooled to about 20 °C and washed twice with 5% aqueous sulfuric acid (600 mL). The combined

aqueous layers were then extracted with isopropyl acetate (600 mL). The combined organic layers were then washed with water (600 mL). The product was then isolated by crystallization from isopropyl acetate (900 mL) and n-heptane (1.8 kg) at about 0 °C. The wet cake was then washed with cold n-heptane to afford IX. 1H NMR (400 MHz, DMSO-d6) δ 3.61 (s, 1H), 3.07 (s, 3H), 1.55 (s, 6H); 13C NMR (10Q MHz, DMSO) d 82.59, 77.76, 56.95, 34.95, 22.77.

Example 3a: Preparation of (3bS,4aR)-3-(trifluoromethyI)-1,3b,4,4a-tetrahydro-5H-cyclopropa[3,4]cyclopenta[1,2-c]pyrazol-5-one (XV) from lithium (Z)-2,2,2-trifluoro-1-(3-oxobicyclo[3.1.0]hexan-2-ylidene)ethan-1-olate (3a)

Synthesis of 3-(trifluoromethyl)-3b,4,4a,5-tetrahydro-1H-cyclopropa[3,4]cyclopenta[1,2-c]pyrazole (3b)

[00579] A reactor was charged with 3a (1.0 g) and AcOH (4.2 ml) and the resulting solution was adjusted to about 20 °C. Hydrazine hydrate (0.29 g, 1.4 equiv.) was added over about 60 min at about 17-25 °C and the reaction mixture was stirred for about 2 hours at about 20-25 °C, warmed up to about 45 to 50 °C over about 30 min, and aged at about 50 °C overnight. Water was slowly (5 mL) added at about 50 °C and product started to crystallize after addition of 5 mL of water. Another 5 mL of water was added at about 50 °C, and the slurry was cooled down to about 20 °C in about one hour and held overnight at about 20 °C. The solids were filtered, washed with water (4X 3 mL), and dried under vacuum at about 30 °C to yield 3b. 1H NMR (400 MHz, Chloroform-d) δ 2.99 (dd, J = 17.0, 6.1 Hz, 1H), 2.89 – 2.78 (m, 1H), 2.14 (dddd, J = 9.1, 7.9, 3.6, 2.5 Hz, 2H), 1.13 (td, J = 7.8, 5.1 Hz, 1H), 0.36 – 0.26 (m, 1H).

Isolation of (3bS,4aS)-3-(trifluoromethyl)-3b,4,4a,5-tetrahydro-1H-cyclopropa[3,4]cyclopenta[1,2-c]pyrazole (3c)

[00580] Chiral purification of 3b (1.0 g) was achieved using a 8×50 mm simulated moving bed (SMB) chromatography system and Chiralpak IG (20 μ particle size) stationary phase using acetonitrile as a mobile phase to afford 3c. 1H NMR (400 MHz, Chloroform-d) δ 3.00 (dd, J = 17.0, 5.7 Hz, 1H), 2.90 – 2.77 (m, 1H), 2.21 – 2.05 (m, 2H), 1.13 (td, J = 7.8, 5.1 Hz, 1H), 0.35 – 0.27 (m, 1H).

Synthesis of (3bS,4aR)-3-(trifluoromethyl)-1,3b,4,4a-tetrahydro-5H-cyclopropa[3,4]cyclopenta[1,2-c]pyrazol-5-one (XV)

[00581] A reactor was charged with water (7 mL) and CuCl2 ● 2H2O (0.09 g, 0.1 equiv). To the reactor was added pyridine (0.42 g, 1 equiv.) and 3c. tert-Butylhydroperoxide (70% in water, 5.5 g, 8 equiv.) was added over about 0.5 hour. The reaction mixture was stirred at about 20 °C for about 2.5 days and quenched with aqueous sodium metabisulfite solution (0.73 g in 2.5 mL water). The quenched reaction mixture was extracted with isopropyl acetate (20 mL), and the aqueous layer was back extracted with isopropyl acetate (2.0 ml). The organic layers were combined and washed with aqueous ethylenediaminetetraacetic acid (EDTA) solution 0.16 g EDTA 10 ml in water), the aqueous layer was dropped, and the organic layer was further washed with aqueous EDTA solution (0.015 g EDTA in 20 ml water). The washed organic layer was concentrated to dryness. To the residue was added isopropyl acetate (2.0 ml) and heptane (2.0 mL). The solution was seeded and stirred overnight at about 20 °C, further diluted with heptane (2.0 mL), and the mixture was concentrated to dryness. The residue was suspended in heptane (4.0 mL) at about 40 °C. The solid was filtered and the filter cake was washed with heptane (1.0 mL) and dried at about 40 °C to yield XV. 1H NMR (400 MHz, Chloroform-d) δ 2.84 (dt, J = 6.8, 4.2 Hz, 1H), 2.71 – 2.64 (m, 1H), 1.79 – 1.67 (m, 2H).

Example 3b: Preparation of (3bS,4aR)-3-(trifluoromethyl)-1,3b,4,4a-tetrahydro-5H- cyclopropa[3,4]cyclopenta[1,2-c]pyrazol-5-one (XV) from lithium (Z)-1-((1S,5R)-4,4- dimethoxy-3-oxobicyclo[3.1.0]hexan-2-ylidene)-2,2,2-trifluoroethan-1-olate (3d-02)

[00582] Hydrazine sulfate (0.45 g, 0.95 equiv.) and ketal lithium salt 3d-02 (1.0 g) were dissolved in ethylene glycol (9.5 mL), and the solution was heated to about 40 °C for about 16 hours. Reaction was cooled to room temperature and water (9.0 mL) was added. Reaction was polish filtered andThe filtrate was collected and to this receiving flask was added water (10 mL, 2x). Slurry was cooled in ice water bath for about five hours, and filtered. Solids were washed with ice water (10 mL, 2x), deliquored, and dried to afford XV. 1H NMR (400 MHz, CDCl3) δ 11.83 (bs, 1H), 2.93 – 2.77 (m, 1H), 2.77 – 2.58 (m, 1H), 1.86 – 1.57 (m, 2H). 19F NMR (376 MHz, CDCl3) δ -61.69. 13C NMR (101 MHz, CDCl3) δ 188.56, 144.08, 142.92, 121.82, 119.15, 36.28, 31.87, 14.15.

Example 3c: Preparation of (3bS,4aR)-3-(trifiuoromethyl)-1,3b,4,4a-tetrahydro-5H- cyclopropa[3,4]cyclopenta[1,2-c]pyrazol-5-one (XV) from (1S,2S)-2-iodo-N-methoxy-N- methylcyclopropane-1-carboxamide (3f) and 1-(4-methoxybenzyl)-4-(4,4,5,5-tetramethyl- 1,3,2-dioxaborolan-2-yl)-3-(trifluoromethyl)-1H-pyrazole (3i) and preparation of starting materials and/or intermediates therein

Synthesis of (1S,2S)-2-iodo-N-methoxy-N-methylcyclopropane-1-carboxamide (3f)

[00583] Starting material iodoacid 3e is a mixture of 3e and cyclopropane carboxylic acid (des-iodo 3e) with mole ratio of 3e to des-iodo 3e of 2:1 by NMR. A mixture of 3e (1.0 g),

N,O-dimethyl hydroxyl amine-HCl (0.46 g) and carbonyl diimidazole (1.72 g) in THF was stirred overnight at room temperature. The reaction mixture was diluted with water, extracted with CH2Cl2, and concentrated to afford unpurified 3f (1.8 g). The unpurified 3f was purified by column chromatography to afford 3f which was a mixture of Wei nr eb amide 3f and des-iodo-3f (about 80:20 by HPLC).

Synthesis of 1-(4-methoxybenzyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3- (trifluoromethyl)-1H-pyrazole (3i)

[00584] To a suspension of NaH (60%, 0.31 g, 1.1 equiv.) in DMF (7.5 mL), a solution of 3g (1.0 g) in DMF (7.5 mL) was added dropwise over about 15 min at about 3 to 7 °C. The reaction mixture was stirred at room temperature for about 1 h and a solution of PMBCl (1.2 g, 1.05 equiv.) in DMF (4.2 mL) was added dropwise in about 25 min at room temperature. The reaction mixture was stirred at room temperature overnight, poured into water (17 mL), and extracted with diethyl ether (3×17 mL). The ether layers were combined and washed with water (2 x 17 mL) and brine (17 mL), dried over Na2SO4, and concentrated in vacuo to give unpurified 3h. Unpurified 3h was absorbed in silica gel (4.3 g) and purified by silica gel chromatography (eluting with 5-25% EtOAc in hexanes) to give 3h (1.5 g).

[00585] To solution of iodopyrazole 3h (1.0 g) in THF (8 mL) i-PrMgCl (2M in ether, 1.8 mL, 1.1 equiv.) was added dropwise over about 10 min at below about 5 °C. The resulting solution was stirred at about 0 °C for about 70 min and 2-methoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (970 mg, 1.81 equiv.) was added at below about 6 °C. The reaction mixture was warmed up to room temperature, quenched by addition of saturated NH4Cl (20 mL), and

extracted with EtOAc (2 x 20 mL). The combined organic layer was washed with saturated NH4Cl (10 mL) and concentrated to unpurified oil, which was combined with the unpurified oil from a previous batch (prepared using 1.1 g of 3h), absorbed on silica gel (6 g), and purified via silica gel chromatography (eluting with 5-40% EtOAc/Hexanes,). Boronate 3i was obtained. 1H NMR (300 MHz, Chloroform-d) δ 7.60 (s, 1 H), 7.23-7.19 (m, 2 H), 6.90-6.85 (m, 2 H), 5.25

(s, 2 H), 3.81 (m, 3 H), 1.29 (s, 12 H).

Synthesis of (1R,2S)-N-methoxy-2-(1-(4-methoxybenzyl)-3-(trifluoromethyl)-1H-pyrazol-4-yl)-N-methylcyclopropane-1-carboxamide (3j)

[00586] A mixture of unpurified iodide 3f (1.0 g), boronate 3i (about 2.2 g), CsF (4.5 equiv.), Pd(OAc)2 (0.1 equiv.), and PPh3 (0.5 equiv.) in DMF (58 mL) was degassed by bubbling N2 and heated at about 87 °C for about 15 hours. The reaction mixture was diluted with water,

extracted with MTBE, concentrated and the unpurified product was purified by column chromatography to give 3j. 1H NMR (300 MHz, Chloroform-d) δ 7.18-7. 14 (m, 3 H), 6.86-6.82 (m, 2 H), 5.24-5.08 (m, 2 H), 3.77 (s, 3 H), 3.63 (s, 3 H), 3.05 (s, 3 H), 2.37-2.32 (m, 1 H), 1.50-1.42 (m, 1 H), 1.32-1.21 (m, 2 H).

Synthesis of (3bS,4aR)-1-(4-methoxybenzyl)-3-ftrifluoromethyl)-1,3b,4,4a-tetrahydro-5H-cyclopropa[3,4]cyclopenta91,2-c]pyrazol-5-one (3k)

[00587] Compound 3j (1.0 g) was treated with freshly prepared LDA (3.3 eq then 0.7 equiv.) at about -67 °C for about 2.5 hours. The reaction mixture was quenched with saturated NH4Cl (12.5 mL) and diluted with MTBE (63 mL). The organic layer was washed with brine, concentrated, and purified by column chromatography to give 3k. 1H NMR (300 MHz, Chloroform-d) δ 7.36-7.33 (m, 2 H), 6.86-6.83 (m, 2 H), 5.28 (s, 2 H), 3.78 (s, 3 H), 2.73-2.65

(m, 1 H), 2.60-2.53 (1 H), 1.70-1.61 (m, 2 H).

Synthesis of (3bS,4aR)-3-(trifluoromethyl)-1,3b,4,4a-tetrahydro-5H-cyclopropa[3,4]cyclopenta[1.2-c]pyrazol-5-one (XV)

[00588] A mixture of 3k (1.0 g) and TFA (5 mL) was heated at about 75 °C for about 3 hours and concentrated. The residue was dissolved in DCM (50 mL), washed with saturated NaHCO3 and brine, concentrated, and purified by column chromatography to give XV. 1H NMR (300 MHz, Chloroform-d) δ 2.86-2.80 (m, 1 H), 2.68-2.63 (m, 1 H), 1.77-1.65 (m, 2 H).

Example 3d: Resolution of 2-(2,2,2-trifluoroacetyl)bicyclo[3.1.0]hexan-3-one (3I) with quinine

[00589] A flask was charged with 3I (1.0 g), acetone (2.5 ml), and quinine (1.7 g, 0.65 equiv). The mixture was stirred at about 15 to 25 °C for about 18 hours and the solids were isolated by filtration and washed with acetone to provide the quinine salt 3n.

Example 4a: Preparation of ethyl 2-((3bS,4aR)-5-oxo-3-(trifluoromethyl)-3b,4,4a,5-tetrahydro-1H-cyclopropa[3,4]cyclopenta[1,2-c]pyrazol-1-yl)acetate (XIV) from (3bS,4aR)-3-(trifluoromethyl)-1,3b,4,4a-tetrahydro-5H-cyclopropa[3,4]cyclopenta[1,2-c]pyrazol-5-one (XV)


[00590] Acetonitrile (5 vol.) was added to a reactor containing XV (1.0 g). N,N-Diisopropylethylamine (0.80 g, 1.25equiv.) was added at about 0 °C. Ethyl bromoacetate (0.91 g, 1.1 equiv.) was added over about 1 hour at about 0 °C. The reaction was stirred at about 5 °C for about 30 minutes and warmed to about 10 °C. The reaction was stirred until complete as determined by HPLC, warmed to about 20 °C, and extracted with MTBE (2 vol.) and saturated NaCl (6 vol.). The aqueous layer was removed and the organic phase was concentrated and diluted with EtOH (3 vol.). The reaction was crystallized by the addition of H2O (7.8 vol.) at about 20 °C. The mixture was cooled to about 5 °C over about 2 hours and maintained at about 5 °C for about 0.5 hour. The mixture was filtered at about 5 °C and washed with cold water (4 vol). The product was dried at about 40 °C under vacuum to give XIV. 1H NMR (400 MHz, Chloroform-d) δ 4.97 (s, 2H), 4.31 – 4.17 (m, 2H), 2.77 (dddd, J= 6.4, 5.2, 2.9, 2.3Hz, 1H), 2.65 – 2.55 (m, 1H), 1.74 – 1.64 (m, 2H), 1.34 – 1.19 (m, 5H), 0.94 – 0.84 (m, 1H).

Example 4b: Preparation of ethyl 2-((3bS,4aR)-5-oxo-3-(trifluoromethyl)-3b,4,4a,5-tetrahydro-1H-cyclopropa[3,4]cyclopenta[1,2-c]pyrazol-1-yl)acetate (XIV) from (1R,5S)-bicyclo[3.1.0]hexan-2-one (4a)

Synthesis of (1R,5R)-2,2-dimethoxybicyclo[3.1.0]hexan-3-ol (4b-02)

[00591] Potassium hydroxide (KOH) (2.2 g, 3.50 equiv.) and anhydrous methanol (13 mL) were added to a reactor and the reaction mixture was warmed to about 55 °C and agitated until

KOH solids were dissolved completely. The mixture was adjusted to about 0 to 6 °C and compound 4a (1.0 g) was slowly added while maintaining the internal temperature at NMT 6 °C. The reaction mixture was agitated for about 45 min at about 0 to 6 °C. Diacetoxy iodobenzene (PhI(OAc)2, 5.0 g, 1.5 equiv.) was added over about 2 hours while maintaining the internal temperature at NMT 6 °C. The reaction mixture was agitated for NLT 1 hour at about 0 to 6 °C. Water (10 g) and heptane (10 mL) were added to the reaction mixture and the biphasic was agitated for NLT 30 min at about 19 to 25 °C The aqueous layer was separated and washed with heptane (10 mL). The combined organic layer was extracted twice with aqueous solution of methanol (MeOH, 10 mL) and water (5 g). The combined aqueous layer was concentrated under vacuum. The aqueous layer was extracted twice with DCM (15 mL and 5 mL). The combined organic layer was concentrated and dried under vacuum. The unpurified compound 4b-02 was obtained. 1H NMR (600 MHz, CDCl3): d 3.98 (d, 1H), 3.45 (s, 3H), 3.25 (s, 3H),

2.40 (s, 1H), 2.21 (m, 1H), 1.78 (d, 1H), 1.48 (m, 1H), 1.38 (m, 1H), 0.83 (q, 1H), 0.58 (m, 1H).

13C NMR (150 MHz, CDCl3): δ 110.91, 72.19, 51.18, 49.02, 34.08, 21.66, 14.75, 8.37.

Synthesis of (1R,5R)-2,2-dimethoxybicyclo[3.1.0]hexan-3-one (4c-02)

[00592] Oxalyl chloride (0.96 g, 1.20 equiv.) and dichloromethane (10 mL) were added to a reactor and the mixture was cooled to about -78 °C. Dimethyl sulfoxide (DMSO, 1.2 g, 2.4 equiv.) was added over about 30 min while maintaining the internal temperature below about -60 °C. After agitation for about 5 min, the solution of compound 4b-02 (1.0 g) in dichloromethane (6 mL) was added over about 30 min while maintaining the internal temperature below about -60 °C and the reaction mixture was agitated for about 20 min at about -60 °C. Triethylamine (TEA, 3.1 g, 4.8 equiv.) was added over about 40 min at about -60 °C, and the reaction mixture was warmed to about 10 to 20 °C. Water (15 g) was added and the biphasic was agitated about 30 min at about 10 to 20 °C. After phase separation, the aqueous layer was back-extracted with dichloromethane (10 mL). Combined organic layer was concentrated until no distillate was observed. To the residue was added MTBE (1 mL), filtered and evaporated to afford unpurified compound 4c-02. 1H NMR (600 MHz, CDCl3): d 3.45 (s,

3H), 3.27 (s, 3H), 2.79 (ddd, 1H), 2.30 (d, 1H), 1.73 (td, 1H), 1.63 (m, 1H), 0.96 (m, 1H), 0.25 (td, 1H). 13C NMR (150 MHz, CDCl3): δ 207.75, 102.13, 50.93, 50.50, 38.87, 19.15, 9.30, 8.56.

Synthesis of lithium (Z)-1-((1S,5R)-4,4-dimethoxy-3-oxobicyclo[3.1.0]hexan-2-ylidene)-2,2,2-trifluoroethan-1-olate (3d-02)

[00593] A reactor was charged with compound 4c-02 (1.0 g), ethyl trifluoroacetate (CF3COOEt, 0.91 g, 1.0 equiv.) and tetrahydrofuran (THF, 0.5 mL) and the reaction mixture was cooled to about -10 to 0 °C. The 1M solution of lithium bis(trimethylsilyl)amide (LiHMDS, 7.0 mL, 1.10 equiv.) was added over about 40 min while maintaining the internal temperature below about 0 °C. The reaction mixture was agitated for about 2 hours at about -10 to 0 °C until the reaction was complete. After then, the reaction mixture was wanned to about 20 °C followed by charging tert-butyl methyl ether (MTBE, 10 mL) and water (10 g). After agitating for about 30 min, the organic layer was separated and the aqueous layer was back-extracted twice with mixture of MTBE (6 mL) and THF (4 mL). The combi ned organic layer was concentrated until no distillate was observed. To the unpurified solids, THF (3 mL) and heptane (15 mL) were added at about 20 °C, and the reaction mixture was cooled to about 0 °C and agitated about 1 hour. The resulting slurry was filtered and wet cake was washed with heptane (7 g) and dried under vacuum at about 40 °C to afford compound 3d-02. 1H NMR (600

MHz, DMSO-d6): d 3.31 (s, 3H), 3.27 (s, 3H) 2.01 (m, 1H), 1.42 (td, 1H), 0.96 (m, 1H), 0.08 (q, 1H). (600 MHz, CDCl3 with THF) δ 3.44 (s, 3H), 3.24 (s, 3H), 2.26 (m, 1H), 1.48 (m, 1H), 1.04 (q, 1H), 0.25 (m, 1H). 13C NMR (150 MHz, DMSO-d6): 193.20, 120.78, 118.86, 105.53,

104.04, 50.66, 49.86, 17.34, 16.20, 13.78.

Synthesis of ethyl 2-((3bS.4aR)-5-oxo-3-(trifluoromethyl)-3b,4,4a,5-tetrahydro-1H-cyclopropa[3,4]cyclopenta[1,2-c]pyrazol-1-yl)acetate (XIV)


[00594] Compound 3d-02 (1.0 g), ethyl hydrazinoacetate hydrochloride (EHA-HCl, 0.60 g,

1.0 equiv.) and absolute ethanol (EtOH, 15 mL) were added to a reactor and the reaction mixture was cooled to about 0 – 5 °C. Sulfuric acid (H2SO4, 0.19 g, 0.50 equiv.) was added while maintaining the internal temperature below about 5 °C. Triethyl orthoformate (0.86 g, 1.50 equiv.) was added and the reaction mixture was agitated at about 0 to 5 °C for about 15 hours. The reaction mixture was warmed to about 20 to 25 °C and water (30 g) was added over about 15 minutes. The content was cooled to about 0 to 5 °C and agitated for about 1 hour. The slurry was filtered and wet cake was washed with water (5 g) and dried under vacuum at about 45 °C to afford XIV 1H NMR (600 MHz, CDCl3): d 4.97 (s, 1H), 4.23 (qd, 2H), 2.77 (quint. 1H), 2.60 (quint, 1H), 1.69 (m, 2H), 1.28 (t, 3H). 13C NMR (150 MHz, CDCl3): d 187.14, 165.98, 143.35, 143.12, 121.37, 119.59, 62.34, 51.83, 35.35, 31.72, 14.00, 13.73.

Example 4c: Kinetic resolution of ethyl 2-(5-oxo-3-(trifluoromethyl)-3b,4,4a,5-tetrahydro- 1H-cyclopropa[3,4]cyclopenta[1,2-c]pyrazol-1-yl)acetate (XVII) to form ethyl 2- ((3bS,4aR)-5-oxo-3-(trifluoromethyl)-3b,4,4a,5-tetrahydro-1H- cyclopropa[3,4]cyclopenta[1,2-c]pyrazol-1-yl)acetate (XIV)

[00595] Compound XVII (1.0 g), (R)-2-methyl-CBS-oxazaborolidine (0.0.05 g, 0.05 equiv.), and tetrahydrofuran (11.9 g) were combined and cooled to about 0 to 5 °C. A solution of borane dimethyl sulfide complex (0.14 g, 0.55 equiv.) in tetrahydrofuran (0.67 g) was added to the mixture, and the mixture was agitated at about 0 to 5 °C until the reaction was deemed complete. Methanol (1 mL) was added to the mixture at about 0 to 5 °C over about 1 h, and the mixture was adjusted to about 15 to 25 °C. The mixture was concentrated under vacuum and combined with tetrahydrofuran (2.7 g). The mixture was combined with 4-dimethylaminopyridine (0.18, 0.44 equiv.) and succinic anhydride (0.30 g, 0.87 equiv.) and agitated at about 15 to 25 °C until the reaction was deemed complete. The mixture was combined with tert-butyl methyl ether (5.2 g) and washed with 1 M aqueous HCl (6.7 g), twice with 5 wt % aqueous potassium carbonate (6.7 g each), and 5 wt % aq. sodium chloride (6.7 g). The organics were concentrated under reduced pressure to an oil which was dissolved in dichloromethane (0.1 g) and purified by flash column chromatography (2.0 g silica gel, 20:80 to 80:20 gradient of ethyl acetate:hexanes). The combined fractions were concentrated under vacuum to give XIV.

Example 4d: Preparation of (1R,5S)-bicyclo[3.1.0]hexan-2-one (4a)

[00596] 4-Tosyloxycyclohexanone (50 mg), (8α,9S)-6′-methoxycinchonan-9-amine trihydrochloride (16 mg), trifluoroacetic acid (28 μL), lithium acetate (49 mg), water (3.4 μL), and 2-methyltetrahydrofuran (0.75 mL) were combined in a vial. The mixture was agitated at about 20 °C until the reaction was complete. 4a was isolated by vacuum distillation. 1H NMR (400 MHz, CDCl3) δ2.05 (m, 5H), 1.74 (m, 1H), 1.18 (m, 1H), 0.91 (m, 1H).

Example 5: Synthesis of ethyl 2-((3bS,4aR)-3-(trifluoromethyl)-4,4a- dihydrospiro[cyclopropa[3,4]cyclopenta[1,2-c]pyrazole-5,2′-[1,3]dithiolane]-1(3bH)- yl)acetate (5h) from (1R,5R)-2,2-dimethoxybicyclo[3.1.0]hexan-3-ol (4b-02)

Synthesis of (1R,5R)-spiro[bicyclo[3.1.0]hexane-2,2′-[1,3]dithiolan1-3-ol (5d)

[00597] A mixture of ketal alcohol 4b-02 (1.0 g), ethanedi thiol (0.91 g), MeCN (7.5 ml) and BiCl3 (0.30 g) was agitated at r.t. overnight. The solids were removed by filtration and the filtrate was concentrated and the residue was further purified by flash column on silica gel to obtain the two isomers. Major product: 1H NMR (400 MHz, Chloroform-d) δ 3.82 (ddt, J = 6.1, 1.3, 0.6 Hz, 1H), 3.41 – 3.32 (m, 2H), 3.31 -3.23 (m, 1H), 3.14 – 3.06 (m, 1H), 2.71 (s, 1H),

2.33 (dddd, J = 14.0, 6.2, 4.8, 1.4 Hz, 1H), 2.00 (d, J = 13.9 Hz, 1H), 1.79 – 1.72 (m, 1H), 1.54 -1.46 (m, 1H), 1.04 (dt, J = 5.1, 3.9 Hz, 1H), 0.63 – 0.54 (m, 1H). Minor product: 1H NMR (400 MHz, Chloroform-d) δ 3.83 (q, J = 9.1 Hz, 1H), 3.43 – 3.34 (m, 2H), 3.33 – 3.25 (m, 2H), 2.35 (d, J= 11.2 Hz, 1H), 2.18 (ddd, J = 12.7, 6.7, 0.4 Hz, 1H), 1.84 (ddd, J= 8.1, 6.3, 3.7 Hz, 1H),

1.60 – 1.51 (m, 1H), 1.43 – 1.35 (m, 1H), 0.65 (tdt, J= 8.1, 5.9, 0.8 Hz, 1H), 0.57 (dddd, J= 5.9, 4.2, 3.7, 0.6 Hz, 1H).

Synthesis of (1R,5R)-spiro[bicyclo[3.1.0]hexane-2,2′-[1,3]dithiolan1-3-one (5e)

[00598] To a dried flask was sequentially added dithiolane alcohol 5d (1.0 g), CH2Cl2 (25 ml), anhydrous DMSO (8.5 ml), and tri ethylamine (3.5 ml) and the resulting mixture was aged at room temperature for about 21 hours. The reaction mixture was transferred to a separatory funnel, diluted with CH2Cl2 (30 ml), washed with 1 M HCl (25 ml), and water (25 ml). The CH2Cl2 layer was concentrated to a solid and further purify by flash column chromatography on silica gel eluted with gradient EtOAc/n-heptane (0-20%) to obtain 5e. 1H NMR (400 MHz, Chloroform-d) δ 3.57 (dddd, J = 10.5, 5.6, 4.3, 0.5 Hz, 1H), 3.49 – 3.41 (m, 1H), 3.39 – 3.28 (m, 2H), 3.10 (ddd, J = 18.3, 5.6, 2.2 Hz, 1H), 2.29 (d, J = 18.3 Hz, 1H), 1.89 (ddd, J = 8.0, 7.0, 3.9

Hz, 1H), 1.63 (tdd, J= 7.3, 5.6, 4.1 Hz, 1H), 1.05 (tdd, J = 8.0, 6.3, 2.2 Hz, 1H), 0.21 (dt J = 6.4, 4.0 Hz, 1H).

Synthesis of lithium (Z)-2,2,2-trifluoro-1-((1R,5S)-3-oxospiro[bicyclo[3.1.0]hexane-2,2′-[1,3]dithiolan]-4-ylidene)ethan-1-olate (5f)

[00599] To a flask with dithiolane ketone 5e (1.0 g) under N2 was added anhydrous THF (8.8 ml), and the mixture was cooled to about -78 °C and followed by addition of LiHMDS (1 M in THF, 7.4 ml) over about 5 min. The resulting mixture was agitated at about -78 °C for about 0.5 hours, and ethyl trifluoroacetate (0.88 ml) was added. The resulting mixture was agitated at about -78 °C for about 10 minutes, at about 0 °C for about 1 hour, and at room temperature overnight. THF was removed under reduced pressure and the residue was crystallized in n-heptane (about 18 ml). The solid product was isolated by filtration, and the filter cake was rinsed with n-heptane (4.1 ml), and dried at about 50 °C under vacuum to provide 5f. 1H NMR (400 MHz, Acetonitrile-d3) δ 6.98 (s, 0H), 5.20 (s, 0H), 3.60 – 3.50 (m, 2H), 3.46 – 3.36 (m, 2H), 2.28 – 2.20 (m, 1H), 1.80 (ddd, J = 8.3, 7.2, 4.1 Hz, 1H), 1.39 (s, 1H), 1.03 (ddd, J = 8.3, 6.7, 4.8 Hz, 1H), 0.17 (ddd, J = 4.7, 4.2, 3.6 Hz, 1H).

Synthesis of (3bS,4aR)-3-(trifluoromethyl)-1,3b,4,4a-tetrahydrospiro[cvciopropa[3.4]cyclopenta[1,2-c]pyrazole-5,2′-[1,3]dithiolane] (5g)

[00600] To flask containing the dithiolane lithium salt 5f (1.0 g) was added water (10 ml), hydrazine hydrate (0.88 ml) and acetic acid (10 ml). The reaction mixture was heated at about 35 °C for about 2 hours, and at about 55 °C for about 2 hours. Water was removed under reduced pressure and the residue was diluted with acetic acid (20 ml) and heated at about 55 °C for about 0.5 hour and held at room temperature overnight. The reaction mixture was further heated at about 65 °C for about 20 hours, and cooled down and concentrated to remove volatile components by rotavap. The residue was triturated with water (50 ml) at about 0 °C and the solid residue was isolated and further washed with ice-cold water (2×10 ml). The solids were further dried to afford unpurified 5g. 1H NMR (400 MHz, Chloroform-d) δ 3.65 – 3.46 (m, 4H), 2.60 (dddd, J = 8.3, 5.6, 4.2, 0.7 Hz, 1H), 2.47 – 2.38 (m, 1H), 1.33 (dddd, J= 8.2, 7.4, 5.7, 0.7 Hz, 1H), 0.66 (dddd, J = 5.7, 4.3, 3.6, 0.7 Hz, 1H)

Synthesis of ethyl 2-((3bS,4aR)-3-(trifluoromethyl)-4,4a-dihydrospiro[cyclopropa[3,4]cyclopenta[1,2-c]pyrazole-5.2′-[1,3]dithiolane]-1(3bH)-yl)acetate

(5h) from (3bS,4aR)-3-(trifluoromethyl)-1,3b,4,4a-tetrahydrospiro[cyclopropa[3,4]cyclopenta[1,2-c]pyrazole-5,2′-[1,3]dithiolane] (5g)

[00601] A reactor was charged with dithiolane pyrazole 5g (1.0 g) and THF (15 ml). The contents were adjusted to about 0 to -5 °C and followed by addition of ethyl bromoacetate (0.44 ml, 1.1 equiv.). To the resulting mixture NaHMDS (2 M, 2.0 ml, 1.1 equiv.) was added over about 10 min via syringe pump at about -2.5 to 0 °C and the mixture was held for about 3 hours, a second portion of ethyl bromoacetate (0.050 ml, 0.12 equiv.) was added, and the mixture was aged for about 1 hour. The reaction mixture was quenched by excess water (2 ml) to form 5h.

Synthesis of ethyl 2-((3bS,4aR)-3-(trifluoromethyl)-4,4a-dihydrospiro[cyclopropa[3,4]cyclopenta[1,2-c]pyrazole-5,2′-[1,3]dithiolanel-1(3bH)-yl)acetate

(5h) from lithium (Z)-2,2,2-trifluoro-1-((1R,5S)-3-oxospiro[bicyclo[3.1.0]hexane-2.2′- [1,3]dithiolanl-4-ylidene)ethan-1-olate (5f)

[00602] A 100 ml flask was charged with ethanol (5 ml). The contents were cooled to about 0 °C and acetyl chloride (1.1 g, 4.0 equiv.) was added over about 10 min. The mixture was agitated at about 0 °C for about 20 minutes and at room temperature for about 20 minutes. To the freshly prepared HCl ethanol solution was added EHA.HCl (0.68 g, 1.2 equiv.) and dithiolane lithium salt 5f (1.0 g). The reaction mixture was heated at about 40 °C for about 22 hours. Ethanol was removed under reduced pressure, and the residue was partitioned between ethyl acetate (5 ml) and water (5 ml). The aqueous layer was discarded, and the organic layer was sequentially washed with aqueous NaHCO3 (5%, 5 ml) and brine (5%, 5 ml) and 5h was

obtained in the EtOAc layer. 1H NMR (400 MHz, DMSO-d6) d 5.14 – 4.97 (m, 2H), 4.14 (qd, J = 7.1, 1.0 Hz, 2H), 3.67 – 3.35 (m, 4H), 2.69 (ddd, J= 8.2, 5.6, 4.2 Hz, 1H), 2.44 (ddd, J= 7.2,

5.5, 3.5 Hz, 1H), 1.37 – 1.29 (m, 1H), 1.21 – 1.14 (m, 3H), 0.44 (ddd, J = 5.3, 4.2, 3.6 Hz, 1H).

Synthesis of ethyl 2-((3bS,4aR)-3-(trifluoromethyl)-4,4a-dihydrospiro[cyclopropa[3,4]cyclopenta[1,2-c]pyrazole-5,2′-[1,3]dithiolanel-1 (3bH)-yl)acetate (5h) from (1R,5R)-spiro[bicyclo[3.1.0]hexane-2.2′-[1,3]dithiolanl-3-one (5e)

[00603] 5e (756 mg) was charged to a vessel and dissolved in 2-methyltetrahydrofuran (7.6 mL). To this solution was charged ethyl trifluoroacetate (0.57 g) and the resulting solution was cooled to about 0 °C. Lithium hexamethyldisilazide (1.0 M solution in THF, 4.5 g) was charged over about 60 minutes and reaction was agitated until complete. A solution of sulfuric acid (2.0 g) in water (5.6 mL) was charged, then the reaction was warmed to about 20 °C and agitated for about 20 minutes. Layers were separated and aqueous layer was extracted twice with 2-methyltetrahydrofuran (5.3 mL). Combined organic layer was concentrated to about 0.4 mL and N,N-diisopropylamine (0.5 g) was charged. The product was crystallized by the addition of heptane (11 ml). The slurry was filtered and the filter cake was washed with heptane, then deliquored thoroughly, and dried to afford 5f-01. 1H NMR (400 MHz, Acetonitrile-d3) δ 7.84 (m, 2H), 3.58 (d, J = 8.7 Hz, 2H), 3.47 – 3.27 (m, 4H), 2.20 (s, 1H), 1.81 – 1.68 (m, 1H), 1.24 (dd, J = 6.5, 0.6 Hz, 12H), 0.99 (q, J = 6.5 Hz, 1H), 0.13 (s, 1H).

[00604] Acetyl chloride (1.02 g) was charged to a cooled reaction vessel containing ethanol (5.0 mL) at about 0 °C, then warmed to about 20 °C and agitated for about 30 minutes. In a separate vessel, 5f-01 (1.00 g), ethyl hydrazinoacetate hydrochloride (0.48 g), and lithium chloride (0.39 g) were combined, and the acetyl chloride/ethanol solution was charged to this mixture, followed by tri ethyl orthoformate (1.16 g). The mixture was heated to about 45 °C and agitated until reaction was complete. The reaction was then concentrated to 2 volumes and dichlorom ethane (5.0 mL) was added followed by water (5.0 mL). Layers were separated and organic layer was washed with 5 wt % aqueous sodium bicarbonate followed by 10 wt % aqueous sodium chloride to afford a solution of 5h in dichloromethane that was carried forward into the subsequent step. 1H NMR (400 MHz, DMSO-d6) δ 5.27 – 4.79 (m, 2H), 4.14 (qd, J =

7.1, 1.1 Hz, 2H), 3.70 – 3.42 (m, 4H), 2.68 (dtd, J = 8.0, 6.4, 5.9, 4.4 Hz, 1H), 2.44 (ddd, J = 7.2, 5.5, 3.6 Hz, 1H), 1.32 (ddd, J = 8.2, 7.2, 5.4 Hz, 1H), 1.18 (t, J = 7.1 Hz, 3H), 0.44 (dt, J = 5.4, 3.9 Hz, 1H); 13C NMR (101 MHz, DMSO-d6) δ 167.14, 148.36, 133.80 (q, J = 38.3 Hz), 128.77 (m), 121.54 (q, J = 268.4 Hz), 65.33, 61.79, 51.14, 41.30, 40.98, 40.49, 23.57, 15.52, 14.33; 19F NMR (376 MHz, DMSO-d6) δ -60.31.

Synthesis of (1R,5R)-spiro[bicyclo[3.1.0]hexane-2,2′-[1,3]dithiolan]-3-one (5e) from (1R,5R)-spiro[bicyclo[3.1.0]hexane-2,2′-[1,3]dithiolan]-3-one (5e) from (1R,5S)-bicyclo[3.1.0]hexan-2-one (4a)

[00605] Tert-butyl nitrite (1.31 g) was charged to a vessel containing 4a (1.00 g, 1.0 equiv) and tetrahydrofuran (5.0 mL) at about 20 °C. Potassium tert-butoxide (6.1 g, 1.7M in tetrahydrofuran) was charged over not less than 30 minutes. The mixture was then agitated until the reaction was complete. The reaction was quenched with aqueous citric acid (2.00 g in 10.00 g water) and extracted with dichloromethane (10.0 mL, 3x). This solution was partially concentrated and the product was isolated by the addition of heptane (6.0 mL). The slurry was filtered and the filter cake was washed with heptane (2.0 mL), then deliquored thoroughly to afford 4d 1H NMR (400 MHz, DMSO-d6) δ 12.26 (s, 1H), 2.73 (d, J = 18.5 Hz, 1H), 2.63 (ddd, J = 18.6, 5.3, 2.0 Hz, 1H), 2.17 – 2.01 (m, 2H), 1.34 (dddd, J= 9.2, 7.1, 4.9, 2.0 Hz, 1H), 0.77 (td, J= 4.6, 3.4 Hz, 1H).

[00606] 1,2-Ethanedithiol (0.41 g) was charged to a vessel containing a solution of 4d (0.50 g, 4.0 mmol) in glacial acetic acid (2.5 mL) at about 20 °C. para-toluenesulfonic acid monohydrate (0.15 g) was added and the mixture was agitated until the reaction was complete. The product was isolated by the addition of water (2 mL). The slurry was filtered and the filter cake was washed with water, then deliquored thoroughly to afford 5i. 1H NMR (400 MHz,

DMSO-d6) δ 10.93 (s, 1H), 3.63 – 3.51 (m, 2H), 3.51 – 3.42 (m, 1H), 3.39 – 3.31 (m, 1H), 2.83 (d, J= 17.4 Hz, 1 H), 2.59 – 2.52 (m, 1H), 1.87 (ddd, J = 8.0, 6.2, 3.7 Hz, 1H), 1.65 (dddd, J=

7.7, 6.2, 5.2, 3.9 Hz, 1H), 0.93 (tdd, J = 7.6, 5.5, 1.7 Hz, 1H), 0.02 (dt, J= 5.5, 3.8 Hz, 1H).

[00607] Para-toluenesulfonic acid (0.90 g) was charged to a vessel containing a suspension of 5i (0.50 g, 2.5 mmol) in methyl ethyl ketone (2.5 mL) and water (2.5 mL). The mixture was agitated at about 85 °C until the reaction was complete. The product was isolated from the reaction mixture by cooling to about 20 °C, adding water (2.50 mL), and cooling to about 0 °C. The slurry was filtered and the filter cake was washed with water, then deliquored thoroughly to afford 5e. 1H NMR (400 MHz, DMSO-d6) δ 3.55 – 3.37 (m, 3H), 3.28 – 3.13 (m, 1H), 3.03 (ddd, J = 18.5, 5.6, 2.2 Hz, 1H), 2.20 (d, J = 18.5 Hz, 1H), 1.84 (ddd, J = 8.0, 7.0, 3.8 Hz, 1H), 1.66 (tdd, J = 7.2, 5.6, 4.1 Hz, 1H), 1.03 (tdd, J = 7.9, 5.9, 2.1 Hz, 1H), 0.06 (dt, J = 6.0, 4.0 Hz, 1H).

Example 6: Preparation of 2-((3bS,4aR)-5,5-difluoro-3-(trifluoromethyl)-3b,4,4a,5-tetrahydro-1H-cyclopropa[3,4]cyclopenta[1,2-c]pyrazol-1-yl)acetic acid (VII) from ethyl 2-((3bS,4aR)-5-oxo-3-(trifluoromethyl)-3b,4,4a,5-tetrahydro-1H-cyclopropa[3,4]cyclopenta[1,2-c]pyrazol-1-yl)acetate (XIV)

Synthesis of ethyl 2-((3bS,4aR)-3-(trifluoromethyl)-4,4a-dihydrospiro[cyclopropa[3,4]cyclopenta[1,2-c]pyrazole-5,2′-[1,3]dithiolane]-1(3bH)-yl)acetate (5h) from ethyl 2-((3bS,4aR)-5-oxo-3-(trifluoromethyl)-3b,4,4a,5-tetrahydro-1H-cyclopropa[3,4]cyclopenta[1,2-c]pyrazol-1-yl)acetate (XIV)


[00608] Dichloromethane (27 g) was added to a reactor containing XIV (1.0 g) and cooled to about 10 °C. To this was added 1,2-ethanedithiol (0.18 g, 1.2 equiv.). To this was added boron trifluoride acetic acid complex (3.3 g, 2.5 equivalents) over about 25 minutes, and the reaction mixture was agitated at about 20 °C until complete. A solution of calcium chloride dihydrate (0.80g, 0.78 equiv) in 0.10 N hydrochloric acid (16 g) was added over about 1 hour at about 10 °C, and the mixture was agitated for about 90 minutes at about 20 °C. The organic layer was washed successively with water (8 g) and sodium bicarbonate solution (5 wt/wt%). The organic layer was concentrated to afford 5h. 1H NMR (400 MHz, DMSO-d6) δ 5.27 – 4.79 (m, 2H),

4.14 (qd, J = 7.1, 1.1 Hz, 2H), 3.70 – 3.42 (m, 4H), 2.68 (dtd, J = 8.0, 6.4, 5.9, 4.4 Hz, 1H), 2.44 (ddd, J = 7.2, 5.5, 3.6 Hz, 1H), 1.32 (ddd, J = 8.2, 7.2, 5.4 Hz, 1H), 1.18 (t, J= 7.1 Hz, 3H), 0.44 (dt, J = 5.4, 3.9 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 167. 14, 148.36, 133.80 (q, J= 38.3 Hz), 128.77 (m), 121.54 (q, J= 268.4 Hz), 65.33, 61.79, 51.14, 41.30, 40.98, 40.49, 23.57,

15.52, 14.33. 19F NMR (376 MHz, DMSO-d6) δ -60.31.

Synthesis of ethyl 2-((3bS,4aR)-5,5-difluoro-3-(trifluoromethyl)-3b,4,4a,5-tetrahydro-1H-cyclopropa[3,4]cyclopenta[1,2-c]pyrazol-1-yl)acetate (VII-A)

[00609] Dichloromethane (26 g) was added to a reactor containing 1,3-dibromo-5,5-dimethylhydantoin (DBDMH, 2.4 g, 3.1 equiv.) and cooled to about -10 °C. To this was added 70% hydrofluoric acid/pyridine complex (1.3 g, 17 equiv.), followed by a solution of 5h (1.0 g) in dichloromethane (3 g). The reaction was agitated at about 0 °C until complete. A solution of potassium hydroxide (3.7 g, 25 equivalents) and potassium sulfite (1 .9 g, 4 equiv.) in water (24 g) was added, maintaining an internal temperature of about 5 °C, and agitated for about 30 minutes at about 20 °C. Layers were separated and organic layer was washed with hydrochloric acid (1.1 g, 4 equiv.) in water (9.6 g). The organic layer was concentrated to afford VII-A. 1H NMR (400 MHz, DMSC-d6) δ 5.31 – 5.04 (m, 2H), 4.17 (q, J = 7.1 Hz, 2H), 2.78 – 2.57 (m,

2H), 1.47 (dddd, J = 8.5, 7.1, 5.5, 1.4 Hz, 1H), 1.19 (t, J = 7.1 Hz, 3H), 1.04 (tdt, J= 5.3, 4.0,

1.8 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 166.79, 143.15 (t, J= 29.4 Hz), 134.65 (q, J=

39.0 Hz), 132.99, 121.05 (q, J= 268.4 Hz), 120.52 (t, J= 243.3 Hz), 62.09, 52.49, 27.95 (dd, J = 34.7, 29.0 Hz), 23.82 (d, J = 2.6 Hz), 14.25, 12.14 (t, J = 3.1 Hz). 19F NMR (376 MHz, DMSO-d6) δ -60.47, -79.68 (dd, J= 253.5, 13.2 Hz), -103.09 (dd, J = 253.3, 9.8 Hz).

Synthesis of 2-((3bS,4aR)-5,5-difluoro-3-(trifluoromethyl)-3b,4,4a,5-tetrahydro-1H- cyclopropa[3,4]cyclopenta[1,2-c]pyrazol-1-yl)acetic acid (VII)

[00610] A reactor was charged with a solution of VII-A (1.0 g) in dichloromethane (18 g) and cooled to about 5 °C. To this was added ethanol (1.5 g), followed by potassium hydroxide (45 wt/wt%, 0.74 g, 2.0 equiv.). The reaction mixture was agitated at about 20 °C until complete. Water (3.7 g) was added and the reaction mixture was agitated for about 30 minutes. Organic layer was removed and reaction was cooled to about 10 °C. Dichloromethane (18 g) was added, followed by 2N hydrochloric acid (3.3 g, 2,2 equiv.). Reaction was warmed to about 20 °C and agitated for 10 minutes. Layers were separated and aqueous phase was washed with dichloromethane (18 g). Organic layers were combined and concentrated on rotary evaporator to afford VII. 1H NMR (400 MHz, DMSO-d6) δ 13.50 (s, 1H), 5.14 – 4.81 (m, 2H), 2.82 – 2.56 (m, 2H), 1.46 (dddd, J = 8.5, 7.1, 5.5, 1.4 Hz, 1H), 1.08 – 1.00 (m, 1H). 13C NMR (101 MHz, DMSO-d6) δ 168.16, 143.05 (t, J = 29.4 Hz), 134.40 (q, J = 38.9 Hz), 132.80, 121.11 (q, J = 268.4 Hz), 120.55 (t, J = 243.3 Hz), 52.54, 27.97 (dd, J = 34.7, 29.0 Hz), 23.81 (d, J = 2.5 Hz), 12.13 (t, J = 3.1 Hz). 19F NMR (376 MHz, DMSO-d6) δ -60.39 (d, J = 1.4 Hz), -79.83 (dd, J = 253.2, 13.1 Hz), -102.97 (dd, J= 253.2, 9.8 Hz).

Example 7: Preparation of 4-chloro-7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1- (2,2,2-trifluoroethyl)-1H-indazol-3-amine (V-02) and its mesylated derivatives

Synthesis of 4-chloro-7-bromo-1-(2,2,2-trifluoroethyl)-1H-indazol-3-amine (V-A)

[00611] To a reactor was added tetrahydrofuran (THF, 275 kg) and diisopropyl amine (DIPA, 30 kg) and the mixture was cooled to about -35 °C. nButyl lithium (2.5 mol/L in hexanes, 74 kg) was charged slowly keeping the reaction temperature less than -30 °C. The mixture was agitated at-35 °C until the reaction was complete. 1-bromo-4-chloro-2-fluorobenzene (52 kg) was charged keeping reaction temperature less than 30 °C and the mixture was agitated at -35°C until reaction was complete. N,N-dimethylformamide (DMF, 36 kg) was charged keeping reaction temperature less than -30 °C and the mixture was agitated at about -35 °C until reaction was complete. Hydrochloric acid (HCl, 18 mass% in water, 147 kg) was charged keeping reaction temperature less than -5 °C. The reaction was warmed to about 0 °C, water (312 kg) was added, and the reaction was extracted with methyl tert-butyl ether (MTBE, 770 kg). The organic was warmed to about 20 °C and washed with brine (NaCl, 23.5 mass% in water, 1404 kg). The mixture was distilled to about 3-4 volumes and heptane was charged (354 kg). The product was isolated by distillation to 3-4 volumes. The slurry was filtered and washed with heptane (141 kg) and dried to afford 6a. 1H NMR (400 MHz, DMSO-d6) δ 10.23 (d, J = 1.2 Hz, 1H), 8.00 (dd, J = 8.7, 1.4 Hz, 1H), 7.44 (dd, J = 8.7, 1.4 Hz, 1H).

[00612] 6a (98.5 kg) was charged to a reactor containing acetic anhydride (105 kg) and acetic acid (621 kg) at 20 °C. The mixture was heated to about 45 °C and hydroxyl amine hydrochloride (31.5 kg) was charged. The reaction was heated to about 75 °C and agitated until the reaction was complete. The product was isolated from the reaction mixture by adding water (788 kg) at about 45 °C. The mixture was cooled to about 25 °C and then the slurry was filtered. The filtered cake was washed with water (197 kg,). The cake was dried to afford 6b. 1H NMR (400 MHz, DMSO-d6) δ 8.11 (dd, J= 8.8, 1.4 Hz, 1H), 7.58 (dd, J = 8.8, 1.4 Hz, 1H).

[00613] To a reactor was charged 6b (84 kg), isopropanol (318 kg), and water (285 kg).

Hydrazine hydrate (20 wt% in water, 178 kg) was charged and the mixture was heated to about 80 °C until the reaction was complete. The product was isolated by cooling the reaction to about 25 °C. The slurry was filtered and the filtered cake was washed with a mixture of isopropanol (127 kg) and water (168 kg). The wet solids were recharged to the reactor and water (838 g) was added. The mixture was agitated at about 25 °C and then filtered and washed with water

(168 g, 2 rel). The cake was dried to afford 6c 1H NMR (400 MHz, DMSO-d6) δ 12.20 (s, 1H), 7.41 (d, J= 7.9 Hz, 1H), 6.84 (d, J= 7.9 Hz, 1H), 5.31 (s, 2H).

[00614] 6c (75 kg) was charged to a reactor containing N,N-dimethylformamide (75 kg). Potassium phosphate (99.8 kg) was charged to the reactor at about 25 °C and the mixture was agitated. 2,2,2-trifluoroethyl trifluoromethanesulfonate (74.3 kg) was charged at about 25 °C and the mixture was agitated until the reaction was complete. Water (375 kg) was charged and the mixture was agitated at about 20 °C. The slurry was filtered and washed with water (150 kg). N,N-dimethylformamide (424 kg) and the wet solid were charged to a reactor at about 20 °C.

The mixture was agitated at about 45 °C. 5 % hydrochloric acid (450 kg) was charged drop-wise to the mixture at about 45 °C. The mixture was cooled to about 25 °C. The slurry was filtered and washed with water (375 g). Water (375 kg) and the filtered solid were charged to a reactor at about 20 °C. The mixture was agitated for about 1 hour at about 20 °C. The slurry was filtered and washed with water (375 kg). The cake was dried to afford V-A. 1H NMR (400 MHz, DMSO-d6) δ 7.57 (d, J= 8.1 Hz, 1H), 6.98 (d, J = 8.1 Hz, 1H), 5.70 (s, 2H), 5.32 (q, J = 8.6 Hz,

2H).

Synthesis of 4-chloro-7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-(2,2,2-trifluoroethyl)- 1 H-indazol-3-amine (V-02)

[00615] A reactor containing tetrahydrofuran (27 g) and V-A (1.0 g) was cooled to about 0 °C. Chlorotrimethylsilane (7.6 g, 2.3 equiv) was added, followed by the slow addition of lithium bis(trimethylsilyl)amide (5.7 g, 1 M in THF, 2.1 equiv.). The mixture was stirred at about 0 °C until bistrimethylsilane protection was complete. The solution was washed with ammonium chloride in water (10 wt%, 52 g), toluene (44 g) was added, and the biphasic mixture was filtered through celite. The organic and aqueous phases were separated and the aqueous phase was washed with toluene (44 g). The organics were combined, washed with brine (58 g), and azeotropically distilled . The solution was cooled to about 0 °C, isopropylmagnesium chloride lithium chloride complex (2.7 g, 1.3 M in THF, 1.2 equiv.) was added and the reaction was stirred at about 0 °C until lithium halogen exchange was complete. Isopropoxyboronic acid pinacol ester (6.8 g, 1.2 equiv.) was added and the reaction was stirred at about 0°C until botylation was complete. At about 0 °C, The reaction was quenched with aqueous hydrochloric acid (52 g, 1 M), acetonitrile (16 g) was added, and the mixture was stirred until trimethylsilane deprotection was complete. The solution was extracted with ethyl acetate (45 g) and the organic was washed twice with brine (2 x 58 g). The solution was concentrated to low volumes (26 g), dim ethylformami de (47 g) was added, and the solution was concentrated again (51 g). The product was crystallized by the addition of water (50 g). The slurry was filtered and filter cake was washed with heptane (14 g). The solids were dried to afford V-02. 1H NMR (400 MHz, DMSO-d6) δ 7.70 (dd, J = 7.6, 1.0 Hz, 1H), 7.07 (dd, J = 7.6, 1.0 Hz, 1H), 5.58 (s, 2H), 5.46 (q, J = 9.1Hz, 2H), 1.32 (s, 12H).

Synthesis of 4-chloro-7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-(2,2,2-trifiuoroethyl)- 1 H-indazol-3-amine (V-02)

[00616] To a reactor was charged V-A (30 kg), bis(pinacolato)diboron (27.9 kg), bis(triphenylphosphine)palladium (II) dichloride (0.9 kg, 1.5 mol%), N,N-dimethylformamide (56 kg, 2 rel. vol.) and toluene (157 kg, 6 rel vol.). The mixture was heated to about 105 °C until the reaction was complete. The mixture was cooled to about 25 °C, filtered through celite (15 kg, 0.5 rel. wt.) and rinsed forward with ethyl acetate (270 kg, 10 rel vol.). PSA-17 palladium scavenger (3 kg, 10 wt%) was added and the mixture was stirred at about 45 °C. The mixture was filtered and the cake was washed with ethyl acetate (54 kg, 2 rel. vol.). The mixture was washed twice with lithium chloride (180 kg, 6 rel. vol.) and once with brine (NaCl, 23.5 mass% in water, 180 kg, 6 rel. vol.). The mixture was then concentrated to about 5-6 rel. vol. under vacuum, heated to about 45 °C then cooled to about 25 °C. Heptane (102 kg, 5 rel. vol.) was charged and the mixture was concentrated to about 4-5 rel. vol. The product was isolated by charging heptane (41 kg, 2 rel. vol.) and cooling the mixture to about 0 °C. The slurry was filtered and washed with heptane (41 kg, 2 rel. vol.). The wet solids were recharged to the reactor with ethyl acetate (27 kg, 1 rel. vol.) and heptane (82 kg, 4 rel. vol.), heated to about 65 °C, and then cooled to about 5 °C. The slurry was filtered and washed with heptane (41 kg, 2 rel. vol.). The cake was dried to afford V-02. 1H NMR (400 MHz, DMSO-d6) δ 7.70 (dd, J =

7.6, 1.0 Hz, 1H), 7.07 (dd, J = 7.6, 1.0 Hz, 1H), 5.58 (s, 2H), 5.46 (q, J = 9.1Hz, 2H), 1.32 (s, 12h).

Synthesis of N-(4-chloro-7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-(2,2,2- trifluoroethyl)-1H-indazol-3-yl)-N-(methylsulfonyl)methanesulfonamide (V-04)

[00617] To a 100 mL reactor was added V-02 (5.00 g), 2-methyltetrahydrofuran (50 mL), and triethylamine (11.1 mL). The mixture was cooled to about 10 °C and methanesulfonyl chloride (2.58 mL, 33.3 mmol) was added to the mixture. The mixture was agitated at about 10 °C until reaction was complete. The mixture was concentrated to dryness and the residue was purified by column chromatography to afford V-04. 1H NMR (400 MHz, DMSO-d6) δ 7.96 (d, J = 7.7 Hz, 1H), 7.50 (d, J = 7.6 Hz, 1H), 5.95 (q, J = 8.8 Hz, 2H), 3.66 (s, 6H), 1.37 (s, 12H).

Synthesis of N-(4-chloro-7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2)-1-(2,2,2,- trifluoroethyl)-1H-indazol-3-yl)methanesulfonamide (V-03)

[00618] To a 100 mL reactor was added V-02 (5.00 g), 2-methyltetrahydrofuran (50 mL), and triethylamine (11.1 mL, 79.6 mmol). The mixture was cooled to about 10 °C and methanesulfonyl chloride (2.58 mL) was added to the mixture. The mixture was agitated at about 10 °C until reaction was complete. To the mixture was added 2-methyltetrahydrofuran (21.5 g) and sodium hydroxide (0.43 g) and the mixture was agitated at about 25 °C until the reaction was complete. To the resulting solution was added 2-methyltetrahydrofuran (21.5 g), water (25 g) and acetic acid to achieve a pH of less than 7. The lower aqueous layer was then removed and the organic layer was washed with brine (5 wt%, 7.8g). The organic layer was then concentrated to dryness and the residue was purified by column chromatography to afford V-03. 1H NMR (400 MHz, DMSO-d6) δ 9.96 (s, 1H), 7.86 (d, J = 7.6 Hz, 1H), 7.34 (d, J = 7.6 Hz, 1H), 5.80 (q, J = 8.9 Hz, 2H), 3.22 (s, 3H), 1.36 (s, 12H).

Synthesis of N-(7-bromo-4-chloro-1-(2,2,2-trifluoroethyl)-1H-indazol-3-yl)-N- (methylsulfonyl)methanesulfonamide (V-06)

[00619] To a reactor was added V-A (3 g), 2-methyltetrahydrofuran (25.8 g), and triethylamine (7.6 mL). The mixture was cooled to about 10 °C, methanesulfonyl chloride (1.8 mL) was added, and the mixture was stirred until reaction was complete. The reaction mixture was washed with aqueous sodium chloride (30 mL) and the organic layer was evaporated to dryness. The residue was purified by column chromatography to afford V-06. 1H NMR (400 MHz, DMSO-d6) δ 7.83 (d, J = 8.0 Hz, 1H), 7.35 (d, J = 8.1 Hz, 1H), 5.79 (q, J = 8.5 Hz, 2H), 3.62 (s, 6H).

Synthesis of N-(7-bromo-4-chloro-1-(2,2,2-trifluoroethyl)-1H-indazol-3-yl)methanesulfonamide (V-05)

[00620] To a reactor was added V-02 (3 g), 2-methyltetrahydrofuran (30 mL), and triethylamine (7.6 mL). The mixture was cooled to about 10 °C, methanesulfonyl chloride (1.8 mL) was added, and the mixture was stirred until reaction was complete. The reaction mixture was washed with aqueous sodium chloride (30 mL) and the organic portion was concentrated to dryness.

[00621] To the resulting mixture (2.7g) was added 2-methyltetrahydrofuran (15 mL) and sodium hydroxide (1M in water, 15 mL). The mixture was stirred at about 20 °C until the reaction was complete. The aqueous layer was removed and the organic was washed with acetic acid (0.7M in water, 10 mL) and sodium chloride (5 wt% in water, 10 mL).The organic layer was then concentrated to dryness and the residue was purified by column chromatography to afford V-05. 1H NMR (400 MHz, DMSO-D6) δ 10.03 (s, 1H), 7.71 (dd, J = 8.0, 1.6 Hz, 1H), 7.20 (dd, J = 8.1, 1.6 Hz, 1H), 5.64 (q, J = 8.7 Hz, 3H), 3.19 (2, 3H).

Synthesis of N-(4-chloro-7-(4,4,5,5-tetramethyl-1,3,2,-dioxaborolan-2-yl)-1-(2,2,2- trifluoroethyl)-1H-indazol-3-yl)-N-(methylsulfonyl)methanesulfonamide (V-04)

[00622] To a reactor was charged V-06 (148 mg), bis(pinacolato)diboron (93 mg), potassium acetate (90 mg) and bis(triphenylphosphine)palladium (II) chloride (4.3 mg, 1.5 mol%). N,N- dimethylformamide (0.2 mL) and toluene (0.6 mL) were added and the reaction was heated to about 105 °C until completion. V-04 was formed. 1H NMR (400 MHz, DMSO-D6) δ 7.96 (d, J = 7.7 Hz, 1H), 7.50 (d, J= 7.6 Hz, 1H), 5.95 (q, J= 8.8 Hz, 2H), 3.66 (s, 6H), 1.37 (s, 12H).

Synthesis of N-(4-chloro-7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-(2,2,2- trifluoroethyl)-1H-indazol-3-yl)methanesulfonamide (V-03)

[00623] To a reactor was charged V-05 (124 mg), bis(pinacolato)diboron (93 mg), potassium acetate (90 mg) and bis(triphenylphosphine)palladium (II) chloride (4.3 mg, 1.5 mol%). N,N- dimethylform amide (0.2 mL.) and toluene (0.6 mL, 6 rel. vol.) were added and the reaction was heated to about 105 °C until completion. V-03 was formed. 1H NMR (400 MHz, DMSO-d6) δ

9.96 (s, 1 H), 7.86 (d, J= 7.6 Hz, 1H), 7.34 (d, J= 7.6 Hz, 1H), 5.80 (q, J = 8.9 Hz, 2H), 3.22 (s,

3H), 1.36 (s, 12H).

II. Synthesis of the Compound of Formula I

Example 8: Preparation of N-((S)-1-(3-bromo-6-(3-methyl-3-(methylsulfonyl)but-1-yn-1- yl)pyridin-2-yl)-2-(3,5-difluorophenyl)ethyl)-2-((3bS,4aR)-5,5-difluoro-3-(trifluoromethyl)- 3b,4,4a,5-tetrahydro-1H-cyclopropa[3,4]cyclopenta[1,2-c]pyrazol-1-yl)acetamide (IV)

Synthesis of N-((S)-1-(3-bromo-6-(3-methyl-3-(methylsulfonyl)but-1-yn-1-yl)pyridin-2-yl)-2- (3,5-difluorophenyl)ethyl)-2-((3bS.4aR)-5,5-difluoro-3-(trifluoromethyl)-3b,4,4a,5-tetrahydro- 1H-cyclopropa[3,4]cyclopenta[1,2-c]pyrazol-1-yl)acetamide (IV) from (S)-1-(3-bromo-6-(3- methyl-3-(methylsulfbnyl)but-1-yn-1-yl)pyridin-2-yl)-2-(3.5-difluorophenyl)ethan-1-amine (VI) Method 1

[00624] n-Propyl phosphonic anhydride (T3P, 3.1 g, 1.5 equiv.) was slowly added to a reactor containing amine VI (1.5 g), acid VII (1.0 g, 1.1 equiv.), triethylamine (Et3N, 0.5 g, 1.5 equiv.), and acetonitrile (MeCN, 8.0 g). The mixture was agitated at about 20 °C until the reaction was complete. The product was crystallized from the reaction mixture with DMF (0.63 g), and water (15 g). The slurry was filtered and the filter cake was washed with a mixture of acetonitrile and water (2 x 2.5 g). The cake was dried to afford IV. 1H NMR (400 MHz, DMSO-d6) δ9.19 (d, J = 8.3 Hz, 1H), 8.12 (d, J = 8.3 Hz, 1H), 7.50 (d, J = 8.3 Hz, 1H), 7.07 (tt, J = 9.4, 2.4 Hz, 1H),

6.96 – 6.87 (m, 2H), 5.52 (td), J = 8.8, 5.3 Hz, 1 H), 4.93 – 4.73 (m, 2H), 3.22 (s, 3H), 3.11 -2.90 (m, 2H), 2.66 – 2.52 (m, 2H), 1.69 (s, 6H), 1.45 – 1.36 (m, 1H), 1.02 – 0.93 (m, 1H). 13C NMR (100 MHz, DMSO-d6): δ 164.42, 163.62, 163.49, 161.17, 161.04, 158.19, 142.92, 142.20, 142.10, 142.01, 141.63, 140.23, 134.11, 133.73, 132.14, 128.66, 122.23, 120.49, 119.56, 112.49, 112.25, 104.75, 102.25, 88.62, 84.20, 57.44, 53.85, 53.03, 35.21, 23.41, 22.46, 22.40, 11.79.

Synthesis of N-((S)-1-(3-bromo-6-(3-methyl-3-(methylsulfonyl)but-1-yn-1-yl)pyridin-2-yl)-2-(3,5-difluorophenyl)ethyl)-2-((3bS,4aR)-5,5-difluoro-3-(trifluoromethyl)-3b,4,4a,5-tetrahydro-1H-cyclopropa[3,4]cyclopenta[1,2-c]pyrazol-1-yl)acetamide (IV) from (S)-1-(3-bromo-6-(3-methyl-3-(methylsulfonyl)but-1-yn-1-yl)pyridin-2-yl)-2-(3,5-difluorophenyl)ethan-1-amine (VI) Method 2


[00625] N-methylmorpholine (NMM, 0.51 g, 2.3 equiv.) was added to a vessel containing amine VI (1.0 g), acid VII (1.0 g), 1-hydroxybenzotriazole hydrate (HOBt ● H2O, 0.17 g, 0.5 equiv.), N-(3-dimethylaminopropyi)-N’-ethylcarbodiimide (EDCI ● HCl, 0.52 g, 1.25 equiv.), and acetonitrile (MeCN, 7.8 g). The mixture was agitated at about 20 °C until the reaction was complete. The product was crystallized from the reaction mixture with DMF (2.8 g), and water (10 g). The slurry was filtered and the filter cake was washed with a mixture of acetonitrile and water. The cake was dried to afford IV. 1H NMR (400 MHz, DMSO-d6) δ9.19 (d, J = 8.3 Hz, 1H), 8.12 (d, J = 8.3 Hz, 1H), 7.50 (d, J = 8.3 Hz, 1H), 7.07 (tt, J = 9.4, 2.4 Hz, 1H), 6.96 – 6.87 (m, 2H), 5.52 (td), J = 8.8, 5.3 Hz, 1 H), 4.93 – 4.73 (m, 2H), 3.22 (s, 3H), 3.11 – 2.90 (m, 2H), 2.66 – 2.52 (m, 2H), 1.69 (s, 6H), 1.45 – 1.36 (m, 1H), 1.02 – 0.93 (m, 1H). 13C NMR (100 MHz, DMSO-d6): δ 164.42, 163.62, 163.49, 161.17, 161.04, 158.19, 142.92, 142.20, 142.10, 142.01, 141.63, 140.23, 134.11, 133.73, 132.14, 128.66, 122.23, 120.49, 119.56, 112.49, 112.25, 104.75, 102.25, 88.62, 84.20, 57.44, 53.85, 53.03, 35.21, 23.41, 22.46, 22.40, 11.79.

Example 9: Preparation of N-((S)-1-(3-(3-amino-4-chloro-1-(2,2,2-trifluoroethyl)-1H- indazol-7-yl)-6-(3-methyl-3-(methylsulfonyl)but-1-yn-1-yl)pyridin-2-yl)-2-(3,5- difluorophenyl)ethyl)-2-((3bS,4aR)-5,5-difluoro-3-(trifluoromethyl)-3b,4,4a,5-tetrahydro- 1H-cyclopropa[3,4]cyclopenta[1,2-c]pyrazol-1-yl)acetamide (III)

Synthesis of compound III-03

[00626] To a reactor was added IV (1 .0 g), potassium bicarbonate (0.43 g, 1.3 equiv), dichlorobis(tricyclohexylphosphine)palladium(II) (28 mg, 2.5mol%), V-02 (0.67 g), butyl acetate (7.3 g) and water (2.1 g). The reactor was inerted and the mixture was agitated at about 85 °C (75-90 °C) until the reaction was complete. The mixture was cooled to about 40 °C and passed through celite (0.52 g). The celite cake was rinsed with butyl acetate (1.8 g). The filtrate and rinse were combined and this solution was washed twice with a mixture of N-acetyl-L-

cysteine (0.31 g) dissolved in water (5.2 g) and sodium hydroxide in water (5 wt%, 5.4 g). The organics were washed twice with sodium chloride in water (5 wt%, 11 g). The solution was azeotropically distilled into 1-propanol (3.3 g). To the propanol solution at about 50 °C was added methanesulfonic acid (0.31 g, 2.25 equiv.) and the product was crystallized using dibutyl ether (5.1 g). The slurry was cooled to about 10 °C, filtered, and the filter cake was washed with a 5:1 mixture of propanol in dibutyl ether (1.6 g). The solids were dried to afford III-03 1H NMR (400 MHz, DMSO-d6) δ 9.19 (d, J = 8.3 Hz, 2H), 7.84 – 7.69 (m, 4H), 7.11 (d, J = 7.7 Hz, 2H), 7.07 – 6.95 (m, 3H), 6.82 (d, J = 7.7 Hz, 2H), 6.54 – 6.40 (m, 4H), 4.90 (d, J = 16.4 Hz, 2H), 4.76 – 4.60 (m, 4H), 4.15 (dq, J = 16.6, 8.4 Hz, 2H), 3.75 (dt, J = 16.3, 8.7 Hz, 2H), 3.25 (s, 7H), 2.99 – 2.86 (m, 4H), 2.63 – 2.50 (m, 3H), 2.41 (s, 14H), 1.73 (d, J = 2.1 Hz, 13H), 0.93 (dd, J = 6.1, 3.9 Hz, 2H).

Synthesis of N-((S)-1-(3-(3-amino-4-chloro-1-(2,2,2-trifluoroethyl)-1H-indazol-7-yl)-6-(3-methyl-3-(methylsulfonyl)but-1-yn-1-yl)pyridin-2-yl)-2-(3,5-difluorophenyl)ethyl)-2- ((3bS,4aR)-5,5-difluoro-3-(trifluoromethyl)-3b,4,4a,5-tetrahydro-1H-cyclopropa[3,4]cyclopenta[1,2-c]pyrazol-1-yl)acetamide (III)

[00627] Aqueous sodium hydroxide (0.2 M; 2.2 equivalents; 9.2 g) was added to a reactor containing III-03 (1.0 g) in MeTHF (8.3 g) at about 20 °C. The biphasic mixture was agitated for about 15 min, and the aqueous layer was removed. The organic layer was washed four times with 2.0 wt% aqueous sodium chloride (9.8 g) and was distilled. The solution containing III was used directly in the II process below. A sample was concentrated to dryness for analysis. 1H NMR (400 MHz, CDCl3): δ 7.44 ( m, 1H), 7.39 (br, 1H), 7.18 (m, 1H), 6.90 (m, 1H), 6.65 (m 1H), 4.10 (m, 2H), 3.72 (m, 4H), 2.78 (m 2H), 2.56 (br, 4H), 1.31 (s, 9H). 13C NMR (100 MHz, DMSO-d6): δ 176.88, 158.95, 141,06, 129.55, 112.79, 109.56, 106.83, 66.66, 65.73, 57.45,

54.12, 39.53, 27.63.

Example 10: Preparation of N-((S)-1-(3-(4-chloro-3-(N- (methylsulfonyl)methylsulfonamido)-1-(2,2,2-trifluoroethyl)-1H-indazol-7-yl)-6-(3-methyl-3-(methylsulfonyl)but-1-yn-1-yl)pyridin-2-yl)-2-(3,5-difluorophenyl)ethyl)-2-((3bS,4aR)-5,5-difluoro-3-(trifluoromethyl)-3b,4,4a,5-tetrahydro-1H-cyclopropa[3,4]cyclopenta[1,2-c]pyrazol-1-yl)acetamide (II)

[00628] Methanesulfonyl chloride (0.32 g, 2.5 equivalents) was added to a reactor containing III (1.0 g), triethylamine (0.69 g, 6.0 equivalents), and MeTHF (11 g) at about 10 °C. The mixture was agitated at about 10 °C until the reaction was complete. The reaction mixture was washed with water (6.4 g) for about 15 minutes, and warmed to about 20 °C. The layers were separated and the organic layer was washed for about 15 minutes with 10 wt% aqueous sodium chloride (6.9 g). The layers were separated and the organic layer was used directly in the next step. An aliquot was concentrated to dryness for analysis. 1H NMR (400 MHz, δ6-DMSO; 9: 1 mixture of atropi somers): δ 9.20 (d, J = 7.9 Hz 1 H), 8.99* (d, J = 8.6 Hz, 1 H), 7.96* (d, J = 7.9 Hz, 1 H), 7.83 (d, J = 8.0 Hz, 1 H), 7.80* (d, J = 7,9 Hz, 1 H), 7.76 (d, J – 8.0 Hz, 1 H), 7.45 (d, J = 7.7 Hz, 1 H), 7.41* (d, J = 7.8 Hz, 1 H), 7.31* (d, J = 7.8 Hz, 1 H), 7.02 (tt, J = 9.4, 2.1 Hz,

1 H), 6.92* (s, 1 H), 6.91 (d, J = 7.7 Hz, 1 H), 6.48 (m, 2 H), 4.92* (s, 1 H), 4.88 (d, J = 16.4 Hz, 1 H), 4.79* (d, J = 16.8 Hz, 1 H), 4.73* (d, J = 16.4 Hz, 1 H), 4.71* (m, 1 H), 4.69 (m, 1 H), 4.62* (s, 1 H), 4.60 (m, 1 H), 4.38* (dq, J = 16.4, 8.2 Hz, 1 H), 4.12 (dq, J = 16.7, 8.4 Hz, 1 H), 3.68* (s, 3 H), 3.66* (s, 3 H), 3.63 (s, 3 H), 3.58 (s, 3 H), 3.26 (s, 3 H), 3.12* (dd, 7 = 13.8, 10.5 Hz, 1 H), 3.05 (dd, J = 13.5, 5.8 Hz, 1 H), 2.97 (dd, J = 13.5, 8.5 Hz, 1 H), 2.78* (dd, J = 13.7, 3.9 Hz, 1 H), 2.59 (m, 1 H), 2.53 (m, 1 H), 1.75 (s), 1.75 (s, 6 H), 1 .39 (m, 1 H), 0.98 (m, 1 H).

13C NMR (100 MHz, DMSO-d6, 9:1 mixture of atropi somers): δ 164.5, 163.6*, 162.1 (dd, ,7 = 246.3, 13.4 Hz), 162.0* (dd, J = 246.1, 13.3 Hz), 158.7, 158.4*, 142.7 (t, J = 29.3 Hz), 142.3, 142.0*, 141.8 (t, J= 9.4 Hz), 140.6*, 139.9, 139.7*, 139.3, 135.8*, 135.0, 133.8 (q, J = 39.0 Hz), 132.2*, 132.1 (m), 131.6, 129.6, 129.4*, 126.7, 125.3, 125.2*, 124.1*, 123.4, 122.8*, 122.7 (q, J= 280.9 Hz), 120.7 (q, J = 268.3 Hz), 119.9 (t, J = 243.7 Hz), 119.8, 119.5*, 119.0*, 118.9, 112.0, 102.2 (t, J= 225.7 Hz), 101.8*, 88.4, 84.5, 57.3, 52.93, 52.86, 52.7, 52.5*, 50.7 (q, J = 33.8 Hz), 50.3*, 42.6*, 42.4, 42.3*, 42.2, 39.51, 39.5, 38.9*, 35.1, 27.5 (dd, J = 35.0, 28.6 Hz), 23.1, 22.4, 22.3, 11.5. (* signals arising from minor atropisomer)

Example 11: Preparation of N-((S)-1-(3-(4-chIoro-3-(methylsuIfonamido)-1-(2,2,2- trifluoroethyl)-1H-indazol-7-yl)-6-(3-methyl-3-(methylsulfonyl)but-1-yn-1-yl)pyridin-2-yl)- 2-(3,5-difluorophenyl)ethyl)-2-((3bS,4aR)-5,5-difluoro-3-(trifluoromethyl)-3b,4,4a,5- tetrahydro-1H-cyclopropa[3,4]cyclopenta[1,2-c]pyrazol-1-yl)acetamide (I)

Synthesis of sodium (4-chloro-7-(2-((S)-1-(2-((3bS.4aR)-5,5-difluoro-3-(trifluoromethyl)- 3b,4,4a,5-tetrahydro-1H-cyclopropa[3,4]cyclopenta[1,2-c]pyrazol-1-yl)acetamido)-2-(3,5- difluorophenyl)ethyl)-6-(3-methyl-3-(methylsulfonyl)but-1-yn-1-yl)pyridin-3-yl)-1-(2,2,2- trifluoroethyl)-1H-indazol-3-yl)(methylsulfonyl)amide (1-02)

[00629] Sodium hydroxide (1 M, 2.9 g, 3.0 equiv.) was added to a reactor containing II (1.0 g) and 2-methyltetrahydrofuran (8.4 g) at about 35 °C. The mixture was agitated until the reaction was deemed complete. The reaction mixture was adjusted to between about 20 and 40 °C and the bottom layer was removed. The organic layer was washed with water (2.9 g) for about 15 minutes, and the bottom layer was removed. The organic solvent was swapped for ethanol and the solution was concentrated to about 5 volumes and the temperature was adjusted to about 35 °C. n-Heptane (3.4 g) was slowly added, and the mixture was aged for about 12 hours. The solids were collected by filtration, and the filter cake was washed with ethanol/n- heptane (1:1). The resultant wet cake was dried under vacuum to afford 1-02. 1H NMR (400 MHz, DMSO-d6) δ 9.09 (d, J = 8.0 Hz, 1H), 8.93* (d, J = 8.5 Hz), 7.80 – 7.72* (m), 7.71 (s, 2H), 6.99 (tt, J = 9.5, 2.4 Hz, 1H), 6.94 (d, J = 7.6 Hz, 1H), 6.90* (d, J = 6.3 Hz), 6.69 (d, J = 7.6 Hz, 1H), 6.57 – 6.51* (m), 6.48 – 6.40 (m, 2H), 4.90 (d, J = 16.5 Hz, 1H), 4.77 (d, J = 16.4

Hz, 1H), 4.70 (td, J = 8.3, 5.2 Hz, 1H), 4.63* (d, J = 16.5 Hz), 4.22 (dq, J= 16.7, 8.4 Hz, 1H), 3.90 – 3.75 (m, 1H), 3.26 (s, 3H), 2.92 (td, J = 13.8, 8.5 Hz, 2H), 2.83* (s), 2.80 (s, 3H), 2.64 – 2.51 (m, 2H), 1.74 (d, J = 2,2 Hz, 6H), 1.44 – 1.34 (m, 1H), 0.94 (dq, J = 6.0, 3.7 Hz, 1H); 13C NMR (100 MHz, dmso) δ 164.39, 163.43, 163.39, 163.25, 160.94, 160.91, 160.81, 158.93,

158.22, 152.64, 151.94, 142.92, 142.72, 142.63, 142.43, 142.34, 142.19, 142.10, 142.00, 141.43,

141.14, 139.55, 139.36, 133.95, 133.56, 133.17, 132.12, 131.93, 131.68, 129.66, 129.56, 128.17,

127.91, 126.86, 126.76, 125.02, 122.35, 122.21, 122.08, 122.05, 119.93, 119.88, 119.38, 118.88,

118.18, 117.54, 117.21, 117.04, 112.18, 112.02, 111.95, 111.84, 111.78, 102.28, 102.03, 101.81,

88.14, 88.00, 84.69, 84.65, 57.33, 53.22, 52.96, 52.76, 52.44, 40.15, 39.94, 39.73, 39.52, 39.31, 39.10, 38.97, 38.89, 38.65, 35.10, 35.08, 27.86, 27.56, 27.52, 27.23, 23.19, 22.42, 22.41, 22.30, 22.28, 11.63. * Signals arising from minor atropisomer. 13C NMR data is reported for the mixture of atropisomers.

Synthesis of N-((S)-(3-(4-chloro-3-(methylsulfonamido)-1-(2,2,2-trifluoroethyl)-1H-indazol-7-yl)-6-(3-methyl-3-(methylsulfonyl)but-1-yn-1-yl)pyridin-2-yl)-2-(3,5-difluorophenyl)ethyl)-2-((3bS,4aR)-5,5-difluoro-3-(trifluoromethyl)-3b,4,4a,5-tetrahydro-1H-cyclopropa[3,4]cyclopenta[1,2-c]pyrazol-1-yl)acetamide (I) from sodium (4-chioro-7-(2-((S)-1-(2-((3bS.4aR)-5,5-difluoro-3-(trifluoromethyl)-3b,4,4a,5-tetrahydro-1H-cyclopropa[3,4]cyclopenta[1,2-c]pyrazol-l-yl)acetamido)-2-(3.5-difluorophenyl)ethyl)-6-(3-methyl-3-(methylsulfonyl)but-1-yn-1-yl)pyridin-3-yl)-1-(2,2,2-trifluoroethyl)-1H-indazol-3-yl)(methylsulfonyl)amide (I-02)

[00630] Compound I-02 (1.0 g) and glacial acetic acid (2.1 g) were combined at about 20 °C and were agitated until dissolved. The resultant solution was transferred to a reactor containing water (15 g) over about 1 hour. The resultant slurry was further agitated for about one hour, and was filtered. The wet cake was washed with water (2 x 5 g), deliquored, and dried at about 60 °C under vacuum to provide I. 1H NMR (400 MHz, δ6-DMSO; 5:1 mixture of atropi somers) δ 10.11* (s), 10.00 (s, 1 H), 9.25 (d, J= 8.0 Hz, 1 H), 8.92* (d, J = 8.4 Hz), 7.90* (d, J = 7.6 Hz), 7.81 (d, J = 8.0 Hz, 1 H), 7.76 (d, J= 8.0 Hz, 1 H), 7.32 (d, J = 7.6 Hz, 1 H), 7.23* (d, J = 8.0 Hz), 7.19* (d, J = 8.0 Hz), 7.02 (tt, J = 9.4, 2,4 Hz, 1 H), 6.94* (m), 6.86 (d, J = 7.6 Hz, 1 H), 6.54* (m), 6.48 (m, 2 H), 4.92 (d, J = 16.4 Hz, 1 H), 4.77* (d, J = 16.4 Hz), 4.71 (d, J = 16.4 Hz, 1 H), 4.68* (m), 4.51 (dq, J = 16.4, 8.3 Hz, 1 H), 4.19* (dq, J = 16.4, 8.2 Hz), 3.96 (dq, J = 16.8,

8.4 Hz, 1 H), 3.27 (s, 3 H), 3.24* (s), 3.17 (s, 3 H), 3.11* (dd, J = 13.0, 3.4 Hz), 3.02 (dd, J = 13.6, 5.6 Hz, 1 H), 2.95 (dd, J = 13.8, 8.6 Hz, 1 H), 2.92* (m), 2.60 (m, 1 H), 2.55 (m, 1 H), 1.74 (s, 6 H), 1.40 (m, 1 H), 0.96 (m, 1 H); 13C NMR (100 MHz, δ6-DMSO; 5:1 mixture of atropisomers) δ 164.5, 163.4*, 162.1 (dd, 7 = 246.0, 13.4 Hz), 162.0* (dd, 7 = 246.1, 13.4 Hz), 158.8, 158.1 *, 142.7 (t, 7 = 29.3 Hz), 142.3, 142.1* (m), 141.9 (t, J= 9.5 Hz), 141.7*, 140.2*, 140.0*, 139.8*, 139.5, 139.3, 139.2, 133.8 (q, J= 38.7 Hz), 132.0 (m), 131.7*, 131.1, 130.3*, 130.0, 126.8, 126.4, 126.2*, 123.0* (m), 122.9 (q, J = 281.7 Hz), 122.7*, 122.1, 120.7 (q, J = 268.3 Hz), 119.9 (t, J= 243.4 Hz), 119.0, 118.7*, 117.5*, 117.4, H2.0 (m), 102.1 (t, J= 25.6 Hz), 101.9* (m), 88.5*, 88.4, 84.5, 57.3, 52.8, 52.7, 52.4*, 50.2 (q, J= 33.3 Hz), 50.0 (m),

41.4*, 41.2, 39.8, 38.7, 35.1, 27.5 (dd, J= 35.1, 29.0 Hz), 23.2, 22.4, 22.3, 22.2*, 11.6. * Signals arising from the minor atropisomer.

[00631] Alternatively, a premixed solution of acetic acid (1.5 g), ethanol (12 g), and water (0.3 g) were combined with Compound I-02 at 20 °C and were agitated until dissolved. The resultant solution was transferred to a reactor containing water (100 g) over about 30 minutes. The resultant slurry was further agitated for about one hour, and was filtered. The wet cake was washed with water (2 x 25 g), deliquored, and dried at about 60 °C under vacuum to provide I.

Synthesis of N-((S)-1-(3-(4-chloro-3-(methylsulfonamido)-1-(2,2,2-trifluoroethyl)-1H-indazol- 7-yl)-6-(3-methyl-3-(methylsulfonyl)but-1-yn-1-yl)pyridin-2-yl)-2-(3,5-difluorophenyl)ethyl)-2- ((3bS,4aR)-5,5-difluoro-3-(trifluoromethyl)-3b,44a,5-tetrahydro-1H-cyclopropa[3,4]cyclopenta[1,2-c]pyrazol-1-yl)acetamide(I) from N-((S)-1-(3-(3-amino-4-chloro-1-(2,2,2-trifluoroethyl)-1H-indazol-7-yl)-6-(3-methyl-3-(methylsulfonyl)but-1-yn-1-yl)pyridin-2-yl)-2-(3,5-difluorophenyl)ethyl)-2-((3bS,4aR)-5,5-difluoro-3-(trifluoromethyl)- 3b,4,4a,5-tetrahydro-1H-cyclopropa[3,4]cyclopenta[1,2-c]pyrazol-1-yl)acetamide (III)

[00632] A reactor was charged with III (1.0 g) followed by cyclopentyl methyl ether (2.0 mL). The contents were adjusted to about 80 °C. In a separate reactor, methanesulfonic acid anhydride (0.3g, 1.5 equiv.) was dissolved in cyclopentyl methyl ether (6 mL). The solution was added to the first reactor via a syringe pump over 5 h. Following addition, the reaction mixture was aged for 16 h. The reaction mixture was quenched with water (10 mL). UPLC analysis of the organic phase showed I with 94.8% purity.

Synthesis of N-((S)-1-(3-(4-chloro-3-(methylsulfonamido)-1-(2,2,2-trifluoroethyl)-1H-indazol- 7-yl)-6-(3-methyl-3-(methylsulfonyl)but-1-yn-1-yl)pyridin-2-yl)-2-(3,5-difluorophenyl)ethyl)-2- ((3bS,4aR)-5,5-difluoro-3-(trifluoromethyl)-3b,4,4a,5-tetrahydro-1H- cyclopropa[3,4]cyclopenta[1,2-c]pyrazol-1-yl)acetamide (I) from N-((S)-1-(3-bromo-6-(3- methyl-3-(methylsulfonyl)but-1-yn-1-yl)pyridin-2-yl)-2-(3,5-difluorophenyl)ethyl)-2- ((3bS,4aR)-5,5-difluoro-3-(trifluoromethyl)-3b,4,4a,5-tetrahydro-1H- cyclopropa[3,4]cyclopenta[1,2-c]pyrazol-1-yl)acetamide (IV)

[00633] To a 40 mL vial was added IV (1 .00 g), potassium bicarbonate (420 mg), palladium(II) chloride (4.9 mg, 2.0 mol%), cyclohexyl diphenylphosphine (13.4 mg, 3.6 mol%), V-03 (849 mg), 2-methyltetrahydrofuran (8.0 mL) and water (2.0 mL). The vial was inerted and the mixture was agitated at about 68 °C (65-73 °C) until the reaction was complete. The mixture was cooled to about 40 °C and the aqueous layer was removed. The organic layer was washed with aqueous acetic acid (5% w/v, 5.1 g). The organic was then concentrated to dryness and the residue was purified by column chromatography to afford I. 1H NMR (400 MHz, DMSO-d6) δ 10.12 (s, 0.2H), 10.00 (s, 1H), 9.25 (d, J = 8.2 Hz, 1H), 8.92 (d, J = 8.6 Hz, 0H),

7.90 (d, J = 7.9 Hz, 0.1H), 7.85 – 7.71 (m, 2H), 7.52-7.50 (m, 0.1H), 7.32 (d, J = 7.7 Hz, 1H),

7.21 (q, J= 9.6 Hz, 0.4H), 7.11 – 6.97 (m, 1H), 6.94-6.89 (m, 0.2H), 6.86 (d, J = 7.7 Hz, 1H),

6.55 (d, J = 7.4 Hz, 0.4H), 6.52 – 6.43 (m, 2H), 4.92 (d, J = 16.4 Hz, 1H), 4.81-4.66 (m, 1.5H),

4.64-4.45 (m, 2.4H), 4.28-4.13 (m, 0.2H), 4.08-3.92 (m, 1.6H), 3.32 (s, 0.7H), 3.30-3.22 (m, 4.4H), 3.17 (s, 3H), 3.08-2.89 (m, 2.2H), 2.69 – 2.53 (m, 2.2H), 2.12 (s, 0.2H), 1.99 (s, 1H), 1.91 (s, 0.3H), 1.80 – 1.70 (m, 6H), 1.48-1.36 (m, 1.2H), 1.23 – 1.12 (m, 1.3H), 0.96 (s, 1.2H).

Synthesis of N-((S)-1-(3-(4-chloro-3-(methylsulfonamido)-1-(2,2,2-trifluoroethyl)-1H-indazol- 7-yl)-6-(3-methyl-3-(methylsulfonyl)but-1-yn-1-yl)pyridin-2-yl)-2-(3.5-difluorophenyl)ethyl)-2- ((3bS,4aR)-5,5-difluoro-3-(trifluoromethyl)-3b,4,4a,5-tetrahydro-1H- cyclopropa[3,4]cvclopenta[1,2-c]pyrazol-1-yl)acetamide(I) from N-((S)-1-(3-bromo-6-(3- methyl-3-(methylsulfonyl)but-1-yn-1-yl)pyridin-2-yl)-2-(3,5-difluorophenyl)ethyl)-2- ((3bS,4aR)-5,5-difluoro-3-(trifluoromethyl)-3b,4,4a,5-tetrahydro-1H- cyclopropa[3,4]cyclopenta[1,2-c]pyrazol-1-yl)acetamide (IV)

[00634] To a 40 mL vial was added IV (1.00 g), potassium bicarbonate (420 mg), palladium(II) chloride (4.9 mg, 2.0 mol%), cyclohexyl diphenylphosphine (13.4 mg, 3.6 mol%), V-04 (923 mg), 2-methyltetrahydrofuran (8.0 mL) and water (2.0 mL). The vial was inerted and the mixture was agitated at about 68 °C (65-73 °C) until the reaction was complete. The mixture was cooled to about 40 °C and the aqueous layer was removed. The organic was stirred with aqueous sodium hydroxide (5 % w/w, 6.3 g) at 40 °C until reaction was complete. The organic was washed with aqueous acetic acid (5% w/v, 5.1 g). The organic was then concentrated to dryness and the residue was purified by column chromatography to afford I. 1H NMR (400 MHz, DMSO-d6) δ 10.12 (s, 0.2H), 10.00 (s, 1H), 9.25 (d, J = 8.2 Hz, 1H), 8.92 (d, J = 8.6 Hz, 0H), 7.90 (d, J = 7.9 Hz, 0.1H), 7.85 – 7.71 (m, 2H), 7.52-7.50 (m, 0.1H), 7.32 (d, J = 7.7 Hz, 1H), 7.21 (q, J = 9.6 Hz, 0.4H), 7.11 – 6.97 (m, 1H), 6.94-6.89 (m, 0.2H), 6.86 (d, J =

7.7 Hz, 1H), 6.55 (d, J = 7.4 Hz, 0.4H), 6.52 – 6.43 (m, 2H), 4.92 (d, J = 16.4 Hz, 1H), 4.81- 4.66 (m, 1.5H), 4.64-4.45 (m, 2.4H), 4.28-4.13 (m, 0.2H), 4.08-3.92 (m, 1.6H), 3.32 (s, 0.7H), 3.30-3.22 (m, 4.4H), 3.17 (s, 3H), 3.08-2.89 (m, 2.2H), 2.69 – 2.53 (m, 2.2H), 2.12 (s, 0.2H), 1.99 (s, 1H), 1.91 (s, 0.3H), 1.80 – 1.70 (m, 6H), 1.48-1.36 (m, 1.2H), 1.23 – 1.12 (m, 1.3H), 0.96 (s, 1.2H).

SYN

Luíza Cruz

https://drughunter.com/lenacapavir-synthesis-highlights/

L 1 L 2

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Lenacapavir
Lenacapavir.svg
Clinical data
Trade names Sunlenca
Other names GS-CA1, GS-6207
Routes of
administration
By mouthsubcutaneous
ATC code
Legal status
Legal status
  • EU: Rx-only [1]
Identifiers
CAS Number
DrugBank
ChemSpider
UNII
KEGG
ChEMBL
PDB ligand
Chemical and physical data
Formula C39H32ClF10N7O5S2
Molar mass 968.28 g·mol−1
3D model (JSmol)

History

Lenacapavir is being developed by Gilead Sciences.[2]

As of 2021, it is in phase II/III clinical trials.[3] It is being investigated as a treatment for HIV patients infected with multidrug-resistant virus and as a twice-yearly injectable for pre-exposure prophylaxis (PrEP).[3][4]

Society and culture

Legal status

On 23 June 2022, the Committee for Medicinal Products for Human Use (CHMP) of the European Medicines Agency (EMA) adopted a positive opinion, recommending the granting of a marketing authorization for the medicinal product Sunlenca, intended for the treatment of adults with multidrug‑resistant human immunodeficiency virus type 1 (HIV‑1) infection.[5] The applicant for this medicinal product is Gilead Sciences Ireland UC.[5] Lenacapavir was approved for medical use in the European Union in August 2022.[1]

References

  1. Jump up to:a b c d e f “Sunlenca EPAR”European Medicines Agency (EMA). 22 June 2022. Archived from the original on 26 August 2022. Retrieved 25 August 2022. Text was copied from this source which is copyright European Medicines Agency. Reproduction is authorized provided the source is acknowledged.
  2. ^ Link JO, Rhee MS, Tse WC, Zheng J, Somoza JR, Rowe W, et al. (August 2020). “Clinical targeting of HIV capsid protein with a long-acting small molecule”Nature584 (7822): 614–618. Bibcode:2020Natur.584..614Ldoi:10.1038/s41586-020-2443-1PMC 8188729PMID 32612233S2CID 220293679.
  3. Jump up to:a b Boerner H (11 March 2021). “Lenacapavir Effective in Multidrug Resistant HIV”MedscapeArchived from the original on 16 March 2021. Retrieved 15 March 2021.
  4. ^ Highleyman L (15 March 2021). “Lenacapavir Shows Promise for Long-Acting HIV Treatment and Prevention”POZArchived from the original on 19 July 2021. Retrieved 15 March 2021.
  5. Jump up to:a b “Sunlenca: Pending EC decision”European Medicines Agency. 23 June 2022. Archived from the original on 26 June 2022. Retrieved 26 June 2022. Text was copied from this source which is copyright European Medicines Agency. Reproduction is authorized provided the source is acknowledged.

External links

////////////Lenacapavir sodium, approvals 2022, ema 2022, レナカパビルナトリウム , HIV, SUNLECA, GS-6207GS-HIVGS-CA1GS-CA2,  PF-3540074,  GS-CA1, eu 2022

[H][C@]12C[C@@]1([H])C(F)(F)C1=C2C(=NN1CC(=O)N[C@@H](CC1=CC(F)=CC(F)=C1)C1=NC(=CC=C1C1=CC=C(Cl)C2=C1N(CC(F)(F)F)N=C2NS(C)(=O)=O)C#CC(C)(C)S(C)(=O)=O)C(F)(F)F

OTERACIL POTTASIUM


ChemSpider 2D Image | RR4580000 | C4H2KN3O4

OTERACIL

UNII4R7FFA00RX, CAS Number2207-75-2,  WeightAverage: 195.175, Monoisotopic: 194.96823705, Chemical FormulaC4H2KN3O4

[K+].OC1=NC(=NC(=O)N1)C([O-])=O

1,3,5-Triazine-2-carboxylic acid, 1,4,5,6-tetrahydro-4,6-dioxo-, potassium salt (1:1)

218-627-5[EINECS]

2207-75-2[RN]

4,6-Dihydroxy-1,3,5-triazine-2-carboxylic acid potassium salt

  • KOX
  • NSC 28841
  • Oxonate
  • Oxonate, potassium

CDSCO APPROVED,01.02.2022

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Gimeracil bulk & Oteracil potassium bulk and Tegafur 15mg/20mg, Gimeracil 4.35mg/5.8mg and Oteracil 11.8mg/15.8mg capsules

indicated in adults for the treatment of advanced gastric cancer when given in combination with cisplatin.

Oteracil Potassium is the potassium salt of oxonate, an enzyme inhibitor that modulates 5- fluorouracil (5-FU) toxicity. Potassium oxonate inhibits orotate phosphoribosyltransferase, which catalyzes the conversion of 5-FU to its active or phosphorylated form, FUMP. Upon oral administration, Oxonate is selectively distributed to the intracellular sites of tissues lining the small intestines, producing localized inhibitory effects within the gastrointestinal tract. As a result, 5-FU associated gastrointestinal toxic effects are reduced and the incidence of diarrhea or mucositis is decreased in 5-FU related therapy.

Oteracil is an adjunct to antineoplastic therapy, used to reduce the toxic side effects associated with chemotherapy. Approved by the European Medicines Agency (EMA) in March 2011, Oteracil is available in combination with Gimeracil and Tegafur within the commercially available product “Teysuno”. The main active ingredient in Teysuno is Tegafur, a pro-drug of Fluorouracil (5-FU), which is a cytotoxic anti-metabolite drug that acts on rapidly dividing cancer cells. By mimicking a class of compounds called “pyrimidines” that are essential components of RNA and DNA, 5-FU is able to insert itself into strands of DNA and RNA, thereby halting the replication process necessary for continued cancer growth.

Oteracil’s main role within Teysuno is to reduce the activity of 5-FU within normal gastrointestinal mucosa, and therefore reduce’s gastrointestinal toxicity 1. It functions by blocking the enzyme orotate phosphoribosyltransferase (OPRT), which is involved in the production of 5-FU.

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SYNTHESIS

https://patents.google.com/patent/CN103435566A/zh

str1
STR2
STR3

SYN

https://europepmc.org/article/pmc/pmc7717319

Poje et al. reported a two-step, gram-scale preparation of the TS-1 additive oteracil 21 (Scheme 16).226 Iodine-mediated-oxidation of uric acid 116 produced dehydroallantoin 117 as the major product, and subsequent treatment with potassium hydroxide resulted in the rearranged product oteracil 21.227

An external file that holds a picture, illustration, etc.
Object name is nihms-1649941-f0037.jpg

Synthesis of Oteracil 21a

aReagents and conditions: (a) LiOH, I2, H2O, 5 °C, 5 min, then AcOH, 75%; (b) aq KOH, 20 min, rt, 82%.

(226) Poje M; Sokolić-Maravić L The mechanism for the conversion of uric acid into allantoin and dehydro-allantoin: A new look at an old problem. Tetrahedron 1986, 42 (2), 747–751. [Google Scholar]

(227) Sugi M; Igi M EP Patent 0957096, 1999.

EP0957096A1 *1998-05-111999-11-17SUMIKA FINE CHEMICALS Co., Ltd.Method for producing potassium oxonate

CN101475539A *2009-02-112009-07-08鲁南制药集团股份有限公司Refining method for preparing high-purity oteracil potassium

CN102250025A *2011-05-182011-11-23深圳万乐药业有限公司Preparation method suitable for industrially producing oteracil potassium

CN102746244A *2012-07-272012-10-24南京正大天晴制药有限公司Refining method of oteracil potassium

//////////OTERACIL POTTASIUM, KOX, NSC 28841, Oxonate, Oxonate potassium, INDIA 2022, APPROVALS 2022, CANCER

[K+].OC1=NC(=NC(=O)N1)C([O-])=O

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NEW DRUG APPROVALS

ONE TIME

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GIMERACIL


Gimeracil.png

GIMERACIL

C5H4ClNO2, 145.54

103766-25-2

5-chloro-4-hydroxy-1H-pyridin-2-one

5-Chloro-2,4-dihydroxypyridine

5-chloropyridine-2,4-diol

5-Chloro-4-hydroxy-2(1H)-pyridone

Ts-1 (TN)

CDSCO APPROVED,01.02.2022

File:Animated-Flag-India.gif - Wikimedia Commons

Gimeracil bulk & Oteracil potassium bulk and Tegafur 15mg/20mg, Gimeracil 4.35mg/5.8mg and Oteracil 11.8mg/15.8mg capsules

indicated in adults for the treatment of advanced gastric cancer when given in combination with cisplatin.

Combination of
TegafurAntineoplastic drug
GimeracilEnzyme inhibitor
OteracilEnzyme inhibitor
Clinical data
Trade namesTeysuno, TS-1
Other namesS-1[1]
AHFS/Drugs.comUK Drug Information
License dataEU EMAby Tegafur
Pregnancy
category
Contraindicated
Routes of
administration
By mouth
ATC codeL01BC53 (WHO)
Legal status
Legal statusUK: POM (Prescription only) [2]EU: Rx-only [3]In general: ℞ (Prescription only)
Identifiers
CAS Number150863-82-4
PubChem CID54715158

Tegafur/gimeracil/oteracil, sold under the brand names Teysuno and TS-1,[3][4] is a fixed-dose combination medication used for the treatment of advanced gastric cancer when used in combination with cisplatin,[3] and also for the treatment of head and neck cancer, colorectal cancer, non–small-cell lung, breast, pancreatic, and biliary tract cancers.[5]: 213 

The most common severe side effects when used in combination with cisplatin include neutropenia (low levels of neutrophils, a type of white blood cell), anaemia (low red blood cell counts) and fatigue (tiredness).[3]

Tegafur/gimeracil/oteracil (Teysuno) was approved for medical use in the European Union in March 2011.[3] It has not been approved by the U.S. Food and Drug Administration (FDA).[5]: 213 

Medical uses

In the European Union tegafur/gimeracil/oteracil is indicated in adults for the treatment of advanced gastric cancer when given in combination with cisplatin.[3]

Contraindications

In the European Union, tegafur/gimeracil/oteracil must not be used in the following groups:

  • people receiving another fluoropyrimidine (a group of anticancer medicines that includes tegafur/gimeracil/oteracil) or who have had severe and unexpected reactions to fluoropyrimidine therapy;[3]
  • people known to have no DPD enzyme activity, as well as people who, within the previous four weeks, have been treated with a medicine that blocks this enzyme;[3]
  • pregnant or breastfeeding women;[3]
  • people with severe leucopenia, neutropenia, or thrombocytopenia (low levels of white cells or platelets in the blood);[3]
  • people with severe kidney problems requiring dialysis;[3]
  • people who should not be receiving cisplatin.[3]

Mechanism of action

Tegafur is the actual chemotherapeutic agent. It is a prodrug of the active substance fluorouracil (5-FU).[3] Tegafur, is a cytotoxic medicine (a medicine that kills rapidly dividing cells, such as cancer cells) that belongs to the ‘anti-metabolites’ group. Tegafur is converted to the medicine fluorouracil in the body, but more is converted in tumor cells than in normal tissues.[3] Fluorouracil is very similar to pyrimidine.[3] Pyrimidine is part of the genetic material of cells (DNA and RNA).[3] In the body, fluorouracil takes the place of pyrimidine and interferes with the enzymes involved in making new DNA.[3] As a result, it prevents the growth of tumor cells and eventually kills them.[3]

Gimeracil inhibits the degradation of fluorouracil by reversibly blocking the dehydrogenase enzyme dihydropyrimidine dehydrogenase (DPD). This results in higher 5-FU levels and a prolonged half-life of the substance.[6]

Oteracil mainly stays in the gut because of its low permeability, where it reduces the production of 5-FU by blocking the enzyme orotate phosphoribosyltransferase. Lower 5-FU levels in the gut result in a lower gastrointestinal toxicity.[6]

Within the medication, the molar ratio of the three components (tegafur:gimeracil:oteracil) is 1:1:0.4.[7]

The maximum tolerated dose differed between Asian and Caucasian populations (80 mg/m2 and 25 mg/m2 respectively), perhaps due to differences in CYP2A6 genotype.[5]: 213 

Research

It is being developed for the treatment of hepatocellular carcinoma.[8] and has activity in esophageal,(Perry Chapter 33) breast,[citation needed] cervical,[citation needed] and colorectal cancer.[9]

  • Tegafur
  • Gimeracil
  • Oteracil potassium

References

  1. ^ Liu TW, Chen LT (201). “S-1 with leucovorin for gastric cancer: how far can it go?”. Lancet Oncol17 (1): 12–4. doi:10.1016/S1470-2045(15)00478-7PMID 26640038.
  2. ^ “Teysuno 20mg/5.8mg/15.8mg hard capsules – Summary of Product Characteristics (SmPC)”(emc). Retrieved 30 July 2020.
  3. Jump up to:a b c d e f g h i j k l m n o p q r “Teysuno EPAR”European Medicines Agency (EMA). Retrieved 30 July 2020. Text was copied from this source which is © European Medicines Agency. Reproduction is authorized provided the source is acknowledged.
  4. ^ “ティーエスワン 患者さん・ご家族向け総合情報サイト | 大鵬薬品工業株式会社”.
  5. Jump up to:a b c DeVita, DeVita; Lawrence, TS; Rosenberg, SA (2015). DeVita, Hellman, and Rosenberg’s Cancer: Principles and Practice of Oncology (10th ed.). LWW. ISBN 978-1451192940.
  6. Jump up to:a b A. Klement (22 July 2013). “Dreier-Kombination gegen Magenkrebs: Teysuno”. Österreichische Apothekerzeitung (in German) (15/2013): 23.
  7. ^ Peters GJ, Noordhuis P, Van Kuilenburg AB et al. (2003). “Pharmacokinetics of S-1, an oral formulation of ftorafur, oxonic acid and 5-chloro-2,4-dihydroxypyridine (molar ratio 1:0.4:1) in patients with solid tumors”. Cancer Chemother. Pharmacol52 (1): 1–12. doi:10.1007/s00280-003-0617-9PMID 12739060S2CID 10858817.
  8. ^ “BCIQ”.
  9. ^ Miyamoto Y, Sakamoto Y, Yoshida N, Baba H (2014). “Efficacy of S-1 in colorectal cancer”. Expert Opin Pharmacother15 (12): 1761–70. doi:10.1517/14656566.2014.937706PMID 25032886S2CID 23637808.

External links

  • “Tegafur”Drug Information Portal. U.S. National Library of Medicine.
  • “Gimeracil”Drug Information Portal. U.S. National Library of Medicine.
  • “Oteracil”Drug Information Portal. U.S. National Library of Medicine.

Gimeracil is an adjunct to antineoplastic therapy, used to increase the concentration and effect of the main active componets within chemotherapy regimens. Approved by the European Medicines Agency (EMA) in March 2011, Gimeracil is available in combination with Oteracil and Tegafur within the commercially available product “Teysuno”. The main active ingredient in Teysuno is Tegafur, a pro-drug of Fluorouracil (5-FU), which is a cytotoxic anti-metabolite drug that acts on rapidly dividing cancer cells. By mimicking a class of compounds called “pyrimidines” that are essential components of RNA and DNA, 5-FU is able to insert itself into strands of DNA and RNA, thereby halting the replication process necessary for continued cancer growth.

Gimeracil’s main role within Teysuno is to prevent the breakdown of Fluorouracil (5-FU), which helps to maintin high enough concentrations for sustained effect against cancer cells 2. It functions by reversibly and selectively blocking the enzyme dihydropyrimidine dehydrogenase (DPD), which is involved in the degradation of 5-FU 1. This allows higher concentrations of 5-FU to be achieved with a lower dose of tegafur, thereby also reducing toxic side effects.

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Metabolism, Biochemical Actions, and Chemical Synthesis of Anticancer Nucleosides, Nucleotides, and Base Analogs. - Abstract - Europe PMC
Metabolism, Biochemical Actions, and Chemical Synthesis of Anticancer Nucleosides, Nucleotides, and Base Analogs. - Abstract - Europe PMC
An external file that holds a picture, illustration, etc. Object name is nihms-1649941-f0002.jpg

SYNTHESIS

https://www.semanticscholar.org/paper/A-Convenient-Synthesis-of-Gimeracil-Li-Zhu/8c04bd3d12699b5c7b9f55cf4723cc0aaf7e3d70

A Convenient Synthesis of Gimeracil | Semantic Scholar

SYN

https://europepmc.org/article/pmc/pmc7717319

Synthesis of Gimeracil 20a

aReagents and conditions: (a) CH3C(OCH3)3, MeOH, then (CH3)2NHCH(OCH3)2, reflux, 92%; (b) aq AcOH, 130 °C, 2 h, 95%; (c) SO2Cl2, HOAc, 50 °C, 0.5 h, 91%; (d) 40% H2SO4, 130 °C, 4 h, 91%; (e) SO2Cl2, HOAc, 50 °C, 45 min, 86%; (f) 75% H2 SO4, 140 °C, 3 h, then NaOH, then pH 4–4.5, 89%

str1

In 1953, Kolder and Hertog reported a synthesis of the TS-1 additive gimeracil 20, which was completed in seven steps using 4-nitropyridine N-oxide as starting material.222 Later, Yano et al. reported an alternative gram-scale synthesis (Scheme 15).223 The one-pot, three component condensation of malononitrile 111, 1,1,1-trimethoxyethane, and 1,1-dimethyoxytrimethylamine generated the dicyano intermediate 112, which was into 2(1H)-pyridinone 113.224 Selective chlorination of 113 was followed by acid-mediated demethylation, hydrolysis, and decarboxylation, to afford gimeracil 20. Interestingly, Xu et al. found that treatment of intermediate 113 with sulfuryl chloride resulted in dichloro 115 formation, which could still be converted to gimeracil 20 by treatment with sulfuric acid.225

(222) Kolder CR; den Hertog HJ Synthesis and reactivity of 5-chloro-2,4-dihydroxypyridine. Rec. Trav. Chim 1953, 72, 285–295. [Google Scholar]

(223) Yano S; Ohno T; Ogawa K Convenient and practical synthesis of 5-chloro-4-hydroxy-2(1H)-pyridinone. Heterocycles 1993, 36, 145–148. [Google Scholar]

(224) Mittelbach M; Kastner G; Junek H Synthesen mit Nitrilen, 71. Mitt. Zur Synthese von 4-Hydroxynicotinsaure aus Butadiendicarbonitrilen. Arch. Pharm 1985, 318 (6), 481–486. [Google Scholar]

(225) Xu Y; Mao D; Zhang F CN Patent 1915976, 2007.

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Darinaparsin


69819-86-9.png
img
2D chemical structure of 69819-86-9
SVG Image
IUPAC CondensedH-gGlu-Cys(Unk)-Gly-OH
SequenceXXG

Darinaparsin

ダリナパルシン , Darvias

JAPAN 2022 APPROVED, PMDA 2022/6/20

(2S)-2-amino-5-[[(2R)-1-(carboxymethylamino)-3-dimethylarsanylsulfanyl-1-oxopropan-2-yl]amino]-5-oxopentanoic acid

(S)-2-amino-5-(((R)-1-((carboxymethyl)amino)-3-((dimethylarsino)thio)-1-oxopropan-2-yl)amino)-5-oxopentanoic acid

Glycine, L-gamma-glutaMyl-S-(diMethylarsino)-L-cysteinyl-

FormulaC12H22AsN3O6S
CAS69819-86-9
Mol weight411.3062
EfficacyAntineoplastic
Commentorganic arsenical

Zinapar, ZIO-101, DMAs(III)G, clarinaparsinUNII-9XX54M675GSP-02L

  • OriginatorTexas A&M University; University of Texas M. D. Anderson Cancer Center
  • DeveloperSolasia Pharma; ZIOPHARM Oncology
  • ClassAmines; Antineoplastics; Arsenicals; Oligopeptides; Pentanoic acids; Small molecules; Sulfides
  • Mechanism of ActionApoptosis stimulants; Cell cycle inhibitors; Reactive oxygen species stimulants
  • Orphan Drug StatusYes – Peripheral T-cell lymphoma
  • PreregistrationPeripheral T-cell lymphoma
  • DiscontinuedLiver cancer; Lymphoma; Multiple myeloma; Non-Hodgkin’s lymphoma; Solid tumours
  • 28 Mar 2022No recent reports of development identified for phase-I development in Peripheral-T-cell-lymphoma in China (IV, Injection)
  • 26 Jan 2022ZIOPHARM Oncology is now called Alaunos Therapeutics
  • 11 Dec 2021Safety and efficacy data from a phase II trial in Peripheral T-cell lymphoma presented at the 63rd American Society of Hematology Annual Meeting and Exposition (ASH-2021)

Darinaparsin is a small-molecule organic arsenical with potential antineoplastic activity. Although the exact mechanism of action is unclear, darinaparsin, a highly toxic metabolic intermediate of inorganic arsenicals (iAs) that occurs in vivo, appears to generate volatile cytotoxic arsenic compounds when glutathione (GSH) concentrations are low. The arsenic compounds generated from darinaparsin disrupt mitochondrial bioenergetics, producing reactive oxygen species (ROS) and inducing ROS-mediated tumor cell apoptosis; in addition, this agent or its byproducts may initiate cell death by interrupting the G2/M phase of the cell cycle and may exhibit antiangiogenic effects. Compared to inorganic arsenic compounds such as arsenic trioxide (As2O3), darinaparsin appears to exhibit a wide therapeutic window.

Darinaparsin, also know as ZIO-101 and SP-02, is a small-molecule organic arsenical with potential antineoplastic activity. Although the exact mechanism of action is unclear, darinaparsin, a highly toxic metabolic intermediate of inorganic arsenicals (iAs) that occurs in vivo, appears to generate volatile cytotoxic arsenic compounds when glutathione (GSH) concentrations are low. The arsenic compounds generated from darinaparsin disrupt mitochondrial bioenergetics, producing reactive oxygen species (ROS) and inducing ROS-mediated tumor cell apoptosis; in addition, this agent or its byproducts may initiate cell death by interrupting the G2/M phase of the cell cycle and may exhibit antiangiogenic effects.

Darinaparsin is an organic arsenical composed of dimethylated arsenic linked to glutathione, and is being investigated for antitumor properties in vitro and in vivo. While other arsenicals, including arsenic trioxide, have been used clinically, none have shown significant activity in malignancies outside of acute promyelocytic leukemia. Darinaparsin has significant activity in a broad spectrum of hematologic and solid tumors in preclinical models. Here, we review the literature describing the signaling pathways and mechanisms of action of darinaparsin and compare them to mechanisms of cell death induced by arsenic trioxide. Darinaparsin has overlapping, but distinct, signaling mechanisms. We also review the current results of clinical trials with darinaparsin (both intravenous and oral formulations) that demonstrate significant antitumor activity.

PAPER

 Biochemical Pharmacology (Amsterdam, Netherlands), 126, 79-86; 2017

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PATENT

WO 2015085208

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2015085208

Preparation of Darinaparsin

[0071] Sterile water (15.5 L) and ethyl alcohol (200 proof, 15.5 L) were charged in a reaction flask prior to the addition of L-glutathione (3.10 kg). While being stirred, the reaction mixture was cooled to 0-5 °C prior to the addition of triethylamine (1.71 L). Stirring was continued until most of the solids were dissolved and the solution was filtered. After filtration, the reaction mixture was cooled to 0-5 °C prior to the addition of chlorodimethylarsine (1.89 kg) over 115 minutes while maintaining the temperature at 0-5 °C. Stirring continued at 0-5 °C for 4 hours before acetone (30.6 L) was added over 54 minutes while maintaining the temperature at 0-5 °C. The suspension was stored at 0-5°C overnight prior to filtration. The solid was collected in a filter funnel, washed successively with ethyl alcohol (200 proof, 13.5 L) and acetone (13.5 L) and dried in suction for 23 minutes. A second similar run was performed and the collected solids from both runs were combined. Ethyl alcohol (200 proof, 124 L) and the combined solids (11.08 kg) were charged in a vessel. The slurry was stirred at ambient temperature for 2 hours before filtration, washing successively with ethyl alcohol (200 proof, 27 L) and acetone (27 L) and dried in suction for 60 minutes. The resulting solid was transferred to drying trays and dried in a vacuum oven at ambient temperature for 66 hours to provide darinaparsin as a solid with the differential scanning calorimetry (DSC) thermogram of Figure 1, with an extrapolated onset temperature at about 191.36° C and a peak temperature at about 195.65° C.

PATENT

WO 2010021928

Step 1

Dimethylchloroarsine. Dimethylarsinic acid, (CH3)2As(O)OH was supplied by the Luxembourg Chemical Co., Tel Aviv, Israel. The product was accompanied by a statement of its purity and was supplied as 99.7% pure. The dimethylarsinic acid was dissolved in water-hydrochloric acid to pH 3. A stream of sulfur dioxide was passed through this solution for about one hour. Dimethylchloroarsine separated as a heavy, colorless oil. The two liquid phases, water/(CH3)2AsCl were separated using a separatory funnel. The chlorodimethylarsine was extracted into diethylether and the ether solution was dried over anhydrous sodium sulfate. The dried solution was transferred to a distillation flask which was heated slowly to evaporate the ether. The remaining liquid, dimethylchloroarsine was purified by distillation. The fraction boiling at 106-109°C was collected. The product, a colorless oil. 1H NMR resonance at 1.65 ppm.

Step 2

SGLU-1: Glutathione (14.0 g, 45.6 mmol) was stirred rapidly in glyme while dimethylchoroarsine (6.5 g, 45.6 mmol) was added dropwise. Pyridine (6.9 g, 91.2 mmol) was then added to the slurry and the mixture was subsequently heated to reflux. The heat was removed immediately and the mixture stirred at room temperature for 4 h. Isolation of the resultant insoluble solid and recrystallization from ethanol afforded 4 as the pyridine hydrochloride complex (75% yield). mp 115-118°C; NMR (D20) δ1.35 (s, 6H), 1.9-4.1 (m’s, 10H), 7.8-9.0 (m, 5H); mass spectrum (m/e) 140, 125, 110, 105, 79, 52, 45, 36.

PATENT

WO 2009075870

Step 1

Example 1. Preparation of Dimethylchloroarsine (DMCA). A 3-neck round-bottom flask (500 mL) equipped with mechanical stirrer, inlet for nitrogen, thermometer, and an ice bath was charged with cacodylic acid (33 g, 0.23 mol) and cone. hydrochloric acid (67 mL). In a separate flask, a solution of SnCl2·2H2O (54 g, 0.239 mol) in cone. hydrochloric acid (10 mL) was prepared. The SnCl2·2 H2O solution was added to the cacodylic acid in HCl solution under nitrogen while maintaining the temperature between 5 °C and 10 °C. After the addition was complete, the ice bath was removed and the reaction mixture was stirred at ambient temperature for 1 h. The reaction mixture was transferred to a separatory funnel and the upper layer (organic) collected. The bottom layer was extracted with dichloromethane (DCM) (2 × 25 mL). The combined organic extract was washed with 1 N HCl (2 × 10 mL) and water (2 × 20 mL). The organic extract was dried over MgSO4 and DCM was removed by rotary evaporation (bath temperature 80 °C, under nitrogen, atmospheric pressure). The residue was further distilled under nitrogen. Two tractions of DMCA were collected. The first fraction contained some DCM and the second fraction was of suitable quality (8.5 g, 26% yield). The GC analysis confirmed the identity and purity of the product.

Step 2

Example 3. Preparation of S-Dimethylarsinoglutathione (SGLU-1). In a 3 L three-neck flask equipped with a mechanic stirrer, dropping funnel and thermometer under an inert atmosphere was prepared a suspension of glutathione (114.5 g, 0.37 mol) in a 1:1 (v/v) mixture of water/ethanol (1140 mL) and cooled to below 5 °C. The mixture was treated slowly (over 15 min) with triethylamine (63.6 mL, 0.46 mol) while maintaining the temperature below 20 °C. The mixture was cooled to 4 °C and stirred for 15 min and then the traces of undissolved material removed by filtration. The filtrate was transferred in a clean 3 L three-neck flask equipped with a mechanic stirrer, dropping funnel, nitrogen inlet, and thermometer and DMCA (70 g, 0.49 mol) (lot # 543-07-01-44) was added slowly while maintaining the temperature at 3-4°C. The reaction mixture was stirred at 1-4°C for 4 h, and acetone (1.2 L) was added over a period of 1 h. The mixture was stirred for 90 min between 2 and 3°C and the resulting solid was isolated by filtration. The product was washed with ethanol (2 × 250 mL) and acetone (2 × 250 mL) and the wet solids were suspended in ethanol 200 Proof (2000 mL). The product was isolated by filtration, washed with ethanol (2 × 250 mL) and acetone (2 × 250 mL) and dried in vacuum for 2 days at RT to give 115 g (75%) of SGLU-1, HPLC purity > 99.5% (in process testing).

PATENT

WO 2007027344

Example 2 Preparation of S-Dimethylarsinoglutathione A 5 L, three necked round bottom flask was equipped with a mechanical stirrer assembly, thermometer, addition funnel, nitrogen inlet, and a drying tube was placed in a cooling bath. A polyethylene crock was charged with glutathione-reduced (200 g) and deionized water (2 L) and stirred under a nitrogen atmosphere to dissolve all solids. The mixture was filtered to remove any insoluble material and the filtrate was transferred to the 5 L flask. While stirring, ethanol, 200 proof (2 L) was added and the clear solution was cooled to 0-5° C. using an ice/methanol bath. Pyridine (120 g) was added followed by a dropwise addition of Me2AsCl (120 g) over a minimum of 1 hour. The reaction mixture was stirred at 0-5° C. for a minimum of 2 hours prior to removal of the cooling bath and allowing the mixture to warm to room temperature under a nitrogen atmosphere with stirring. The reaction mixture was stirred overnight (>15 hrs) at room temperature under a nitrogen atmosphere at which time a white solid may precipitate. The reaction mixture was concentrated to a slurry (liquid and solid) at 35-45° C. using oil pump vacuum to provide a white solid residue. As much water as possible is removed, followed by two coevaporations with ethanol to azeotrope the last traces of water. The white solid residue was slurried in ethanol, 200 pf. (5 L) under a nitrogen atmosphere at room temperature overnight. The white solid was filtered and washed with ethanol, 200 pf. (2×500 mL) followed by acetone, ACS (2×500 mL). The resulting solid was transferred to drying trays and vacuum oven dried overnight at 25-35° C. using oil pump vacuum to provide pyridinium hydrochloride-free S-dimethylarsinoglutathione as a white solid. melting point of 189-190° C.

PATENT

WO 20060128682

Step 1

Dimethylchloroarsine. Dimethylarsinic acid, (CH3)2As(O)OH was supplied by the Luxembourg Chemical Co., Tel Aviv, Israel. The product was accompanied by a statement of its purity and was supplied as 99.7% pure. The dimethylarsinic acid was dissolved in water-hydrochloric acid to pH 3. A stream of sulfur dioxide was passed through this solution for about one hour. Dimethylchloroarsine separated as a heavy, colorless oil. The two liquid phases, water/(CH3)2AsCl were separated using a separatory funnel. The chlorodimethylarsine was extracted into diethylether and the ether solution was dried over anhydrous sodium sulfate. The dried solution was transferred to a distillation flask which was heated slowly to evaporate the ether. The remaining liquid, dimethylchloroarsine was purified by distillation. The fraction boiling at 106-109° C. was collected. The product, a colorless oil. 1H NMR resonance at 1.65 ppm.

Step 2

Pyridine Hydrochloride Free Synthesis of S-Dimethylarsinoglutathione (GLU) Dimethylarsinoglutathione is made using an adapted of Chen (Chen, G. C., et al. Carbohydrate Res. (1976) 50: 53-62) the contents of which are hereby incorporated by reference in their entirety. Briefly, dithiobis(dimethylarsinoglutamine) is dissolved in dichloromethane under nitrogen. Tetramethyldiarsine is added dropwise to the solution and the reaction is stirred overnight at room temperature under nitrogen and then exposed to air for 1 h. The mixture is then evaporated to dryness and the residue is washed with water and dried to give a crude solid that is recrystallized from methanol to give S-dimethylarsinoglutathione.

//////////

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Solasia Announces Submission of New Drug Application for Anti-cancer Drug DARINAPARSIN for Peripheral T-Cell Lymphoma in Japan

Solasia Pharma K.K. (TSE: 4597, Headquarters: Tokyo, Japan, President & CEO: Yoshihiro Arai, hereinafter “Solasia”) today announced submission of a New Drug Application (NDA) for its new anti-cancer drug darinaparsin (generic name, development code: SP-02) as a treatment for relapsed or refractory peripheral T-cell lymphoma to the Ministry of Health, Labour and Welfare (MHLW). Based on positive results of R&D on darinaparsin, centered primarily on the results of the Asian Multinational Phase 2 Study (study results released in June 2020), Solasia filed an NDA for the drug with the regulatory authority in Japan ahead of anywhere else in the world.

Solasia expects to obtain regulatory approval in 2022 and to also launch in the same year. If approved and launched, darinaparsin would be the third drug Solasia successfully developed and brought to market since its founding and is expected to contribute to the treatment of PTCL.

Mr. Yoshihiro Arai, President and CEO of Solasia, commented as follows:
“No standard treatment has been established for relapsed or refractory PTCL as of yet. I firmly believe that darinaparsin, with its novel mechanism of action that differs from those of already approved drugs, will contribute to patients and healthcare providers at clinical sites as a new treatment option for relapsed or refractory PTCL. Since founding, Solasia has conducted R&D on five pipeline drugs. Of the five, we have successfully developed and brought to market two drugs, i.e., began providing them to patients, and today, we submitted an NDA for our first anti-cancer drug. Under our mission to provide patients with ‘Better Medicine for a Brighter Tomorrow’, we will continue aiming to contribute to patients’ treatment and enhanced quality of life. ”

About darinaparsin (SP-02)
Darinaparsin, an organoarsenic compound with anticancer activity, is a novel mitochondrial-targeted agent being developed for the treatment of various hematologic and solid tumors. The proposed mechanism of action of the drug involves the disruption of mitochondrial function, increased production of reactive oxygen species, and modulation of intracellular signal transduction pathways. Darinaparsin is believed to exert anticancer effect by inducing cell cycle arrest and apoptosis. Darinaparsin has been granted orphan drug designation in the US and EU.
For more information, please visit at https://solasia.co.jp/en/pipeline/sp-02.html

About Asian Multinational Phase 2 Study
The Asian Multinational Phase 2 Study was a multinational, multicenter, single-arm, open-label, non-randomized study to evaluate the efficacy and safety of darinaparsin monotherapy in patients with relapsed or refractory PTCL conducted in Japan, Korea, Taiwan, and Hong Kong. (CT.gov Identifier: NCT02653976).
Solasia plans to present the results of the study at an international academic conference to be held in the near future.

About peripheral T-cell lymphoma (PTCL)
Please visit at https://solasia.co.jp/en/pipeline/sp-02.html

About Solasia
Please visit at https://solasia.co.jp/en/

/////////////Darinaparsin, Darvias, JAPAN 2022,  APPROVALS 2022, PMDA, ダリナパルシン  , Zinapar, ZIO-101, DMAs(III)G, clarinaparsinUNII-9XX54M675GSP-02LOrphan Drug

C[As](C)SCC(C(=O)NCC(=O)O)NC(=O)CCC(C(=O)O)N

Pimitespib


Pimitespib Chemical Structure
Benzamide, 3-ethyl-4-[3-(1-methylethyl)-4-[4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl]-1H-pyrazolo[3,4-b]pyridin-1-yl]-.png

Pimitespib

TAS 116

CAS 1260533-36-5

Antineoplastic, Hsp 90 inhibitor

3-ethyl-4-[4-[4-(1-methylpyrazol-4-yl)imidazol-1-yl]-3-propan-2-ylpyrazolo[3,4-b]pyridin-1-yl]benzamide

Pimitespib (TAS-116) is an oral bioavailable, ATP-competitive, highly specific HSP90α/HSP90β inhibitor (Kis of 34.7 nM and 21.3 nM, respectively) without inhibiting other HSP90 family proteins such as GRP94. Pimitespib demonstrates less ocular toxicity.

FormulaC25H26N8O
CAS1260533-36-5
Mol weight454.5269

JAPAN APPROVED 2022/6/20, ピミテスピブ

Jeselhy

Taiho. originator

日本医薬品一般的名称(JAN)データベース

Pimitespib is a specific inhibitor of heat shock protein 90 (Hsp90) subtypes alpha and beta, with potential antineoplastic and chemo/radiosensitizing activities. Upon oral administration, pimitespib specifically binds to and inhibits the activity of Hsp90 alpha and beta; this results in the proteasomal degradation of oncogenic client proteins, which inhibits client protein dependent-signaling, induces apoptosis, and inhibits the proliferation of cells overexpressing HSP90alpha/beta. Hsp90, a family of molecular chaperone proteins that are upregulated in a variety of tumor cells, plays a key role in the conformational maturation, stability, and function of “client” proteins within the cell,; many of which are involved in signal transduction, cell cycle regulation and apoptosis, including kinases, cell-cycle regulators, transcription factors and hormone receptors. As TAS-116 selectively inhibits cytosolic HSP90alpha and beta only and does not inhibit HSP90 paralogs, such as endoplasmic reticulum GRP94 or mitochondrial TRAP1, this agent may have less off-target toxicity as compared to non-selective HSP90 inhibitors.

Patent

WO2011004610

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2011004610

PATENT

CN108623496

3-Ethyl-4-fluorobenzonitrile is an important intermediate for the preparation of a variety of new drugs under development, such as TAS-116, a Phase II clinical drug of Taiho Pharmaceuticals for the treatment of gastrointestinal stromal tumors.
         
        Patent WO2005105760 discloses its preparation method. In the method, tetrakis(triphenylphosphine) palladium is used as a catalyst, and 3-bromo-4-fluorobenzonitrile is coupled with tetraethyl tin in a solvent hexamethylphosphoramide for a heating reaction for 15 hours to obtain 3 -Ethyl-4-fluorobenzonitrile. The method uses highly toxic tetraethyl tin, which brings great harm to operators and the environment, and is difficult to carry out industrial production. Meanwhile, the product 3-ethyl-4-fluorobenzonitrile obtained by the preparation method is an oily substance, which is purified by column chromatography with complicated operation, which is unfavorable for industrial production, and the specific purity of the product is not described.
         
        Therefore, looking for a new method for preparing 3-ethyl-4-fluorobenzonitrile with cheap and easy-to-obtain raw materials, safe and simple operation, high product purity and low cost suitable for industrial production, which will speed up the research process of related new drugs under development. , it is of great significance to reduce the production cost of related new drugs.
Example 1 3-Bromo-4-fluorobenzonitrile
         
        3-Bromo-4-fluorobenzaldehyde (250g, 1.23mol) was dissolved in acetonitrile (1.5L), then hydroxylamine sulfonic acid (67g, 1.48mol) was added, and the reaction was refluxed for 4h. TLC showed that the conversion of the raw materials was complete, and the reaction solution was concentrated. To a small volume, add water (2L) and stir for 30min, cool to 5-10°C and continue stirring for 10min, filter, dissolve the filter cake with methyl tert-butyl ether (1.2L), wash twice with 500ml of water, saturated with 200ml Washed with sodium bicarbonate solution, dried over anhydrous sodium sulfate, filtered, the filtrate was adsorbed with activated carbon (10g), filtered, concentrated under reduced pressure to remove the solvent, added n-heptane (250ml), cooled and stirred in an ice-salt bath for 1h, filtered, reduced Press drying to give 3-bromo-4-fluorobenzonitrile (217 g, 88% yield). 1 H NMR (CDCl 3 ,400MHz):δ7.91(m,1H),7.63(m,1H),7.24(m,1H)。
        Example 2 3-Bromo-4-fluorobenzonitrile
         
        Add tetrahydrofuran (100ml) to a 250ml reaction flask, add 3-bromo-4-fluorobenzaldehyde (10g, 49.2mmol) and ammonia (40ml) under stirring, add elemental iodine (25g, 98.5mmol) in batches under cooling to 5°C ), then raised to ambient temperature and reacted for 2 to 3 hours, the reaction was completed, the reaction solution was poured into a 10% aqueous solution of sodium sulfite (200g), extracted twice with methyl tert-butyl ether (100ml), dried over anhydrous sodium sulfate , concentrated under reduced pressure to remove the solvent, added n-heptane (20 ml), cooled to 0-10 °C and stirred for 1 h, filtered, and dried under reduced pressure to obtain 3-bromo-4-fluorobenzonitrile (9.6 g, yield: 97.5 %). The NMR spectrum of this compound was determined and was identical to the product of Example 1.
        Example 3 3-ethyl-4-fluorobenzonitrile
         
        3-Bromo-4-fluorobenzonitrile (200 g, 1 mol) and [1,1-bis(diphenylphosphino)ferrocene]palladium(II) chloride dichloromethane complex (4.08 g, 5mmol) was dissolved in THF (1.2L), 1.0M/L diethylzinc n-hexane solution (600mL, 0.6mol) was added at 40-50°C, and the temperature was raised to 50-60°C for 4-5h. TLC showed The raw materials reacted completely. After the reaction solution was cooled to room temperature, it was added to 5% dilute hydrochloric acid (1 L), the layers were separated, the organic layer was washed twice with 500 ml of water, and then concentrated under reduced pressure to remove the solvent. Then n-hexane (600mL) and activated carbon (20g) were added, refluxed for 0.5h, cooled to room temperature, filtered, then added activated carbon (10g) to the filtrate, refluxed for 0.5h, cooled to room temperature, filtered, and cooled to -50°C to -60°C and filtered, and the filter cake was dried under reduced pressure at 10-20°C to obtain 3-ethyl-4-fluorobenzonitrile (112 g, yield: 75%) as an off-white solid, melting point 23.1-27.4°C. 1 H NMR (CDCl 3 , 400MHz): δ 7.50 (m, 2H), 7.09 (m, 1H), 2.69 (q, J=7.6Hz, 2H), 1.24 (t, 3H, J=7.6Hz), HPLC purity 99.6%.
        HPLC assay conditions:
        Chromatographic UV detector: DAD
        Chromatography pump: 1100 quaternary pump
        Chromatographic column: Agilent (USA) ZORBAX SB-C184.6×150mm, 5μm PN883975-902 Chromatographic conditions:
        Mobile Phase A: Water
        Mobile Phase B: Acetonitrile
         
        Injection volume: 5 μL, flow rate: 1.0 mL/min, column temperature: room temperature, detection wavelength: 210 nm.

Acylation of 2-fluoro-4-iodopyridine with isobutyric anhydride in presence of BuLi and DIEA in THF at -78 °C gives 1-(2-fluoro-4-iodo-3-pyridinyl)-2-methylpropan-1-one ,

This upon cyclization using hydrazine hydrate  at 65 °C gives 4-iodo-3-isopropylpyrazolo[3,4-b]pyridine.

N-Protection of intermediate  with PMB-Cl in the presence of base NaH in solvent DMF at 0 °C affords 4-iodo-3-isopropyl-1-(4-methoxybenzyl)pyrazolo[3,4-b]pyridine,

This is  coupled with 4-(4-imidazolyl)-1-methylpyrazole in the presence of Cu2O, 4,7-dimethoxy-1,10-phenanthroline, Cs2CO3 and PEG-diamine in solvent  NMP or DMSO at 130 °C to furnish 4-[4-(4-pyrazolyl)-imidazol-1-yl]pyrazolo[3,4-b]pyridine derivative .

N-Deprotection of PMB-protected pyrazolo[3,4-b]pyridine derivative by using TFA and anisole gives free pyrazolo[3,4-b]pyridine ,

This on condensation with 3-ethyl-4-fluorobenzonitrile  in the presence of Cs2CO3 in DMF at 95 °C yields 4-(pyrazolo[3,4-b]pyridin-1-yl)benzonitrile .

Finally, partial hydrolysis of nitrile  by means of aqueous NaOH and H2O2 in DMSO/EtOH gives the Pimitespib TAS-116 .

CLIP

https://pubs.acs.org/doi/abs/10.1021/acs.jmedchem.8b01085

J. Med. Chem.2019, 62, 2, 531–551

Publication Date:December 7, 2018

https://doi.org/10.1021/acs.jmedchem.8b0108

Abstract Image

The molecular chaperone heat shock protein 90 (HSP90) is a promising target for cancer therapy, as it assists in the stabilization of cancer-related proteins, promoting cancer cell growth, and survival. A novel series of HSP90 inhibitors were discovered by structure–activity relationship (SAR)-based optimization of an initial hit compound 11a having a 4-(4-(quinolin-3-yl)-1H-indol-1-yl)benzamide structure. The pyrazolo[3,4-b]pyridine derivative, 16e (TAS-116), is a selective inhibitor of HSP90α and HSP90β among the HSP90 family proteins and exhibits oral availability in mice. The X-ray cocrystal structure of the 16e analogue 16d demonstrated a unique binding mode at the N-terminal ATP binding site. Oral administration of 16e demonstrated potent antitumor effects in an NCI-H1975 xenograft mouse model without significant body weight loss.

3-Ethyl-4-(3-Isopropyl-4-(4-(1-methyl-1H-Pyrazol-4-yl)-1H-Imidazol-1-yl)-1H-Pyrazolo[3,4-b]pyridin-1-yl)benzamide (16e). Yield 64% (2 steps), white powder. UPLC−MS (ESI) m/z: 454.8 [M + H]+ , tR = 1.19 min. UPLC purity 99.65%. 1 H NMR (400 MHz, CDCl3): δ 1.14 (t, J = 7.5 Hz, 3H), 1.25 (d, J = 7.0 Hz, 6H), 2.62 (q, J = 7.3 Hz, 2H), 3.18 (spt, J = 6.8 Hz, 1H), 3.98 (s, 3H), 5.88 (br s,1H), 6.22 (br s, 1H), 7.13 (d, J = 5.1 Hz, 1H), 7.39 (d, J = 1.1 Hz, 1H), 7.58 (d, J = 8.1 Hz, 1H), 7.78−7.81 (m, 3H), 7.86 (d, J = 1.5 Hz, 1H), 7.96 (d, J = 1.8 Hz, 1H), 8.59 (d, J = 4.7 Hz, 1H). HRMS: calcd for C25H26N8O, 455.2308 [M + H]+ ; found, 455.2311.

PAPER

Journal of Medicinal Chemistry (2021), 64(5), 2669-2677.

PATENT

WO 2016181990

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2016181990

Compound 1 in the present invention is 3-ethyl-4- {3-isopropyl-4- (4- (1-methyl-1H-pyrazol-4-yl) -1H-imidazole-1-yl) -1H-. Pyrazolo [3,4-b] pyridin-1-yl} benzamide (formula below). Compound 1 is known to have HSP90 inhibitory activity and exhibit excellent antitumor activity. Compound 1 can be synthesized based on the production methods described in Patent Documents 1 and 2.

[0013]

[hua 1]

Patent Document 1: International Publication No. 2012/093708
Patent Document 2: International Publication No. 2011/004610

Comparative Example 1 3-Ethyl-4- {3-isopropyl-4- (4- (1-methyl-1H-pyrazole-4-yl) -1H-imidazole-1-yl) -1H-pyrazolo [3, 4-b] Pyridine-1-yl} Synthesis of type I crystals of benzamide
3-Ethyl-4 obtained according to the production method described in International Publication No. 2012/093708 and International Publication No. 2011/004610. -{3-Isopropyl-4- (4- (1-methyl-1H-pyrazole-4-yl) -1H-imidazole-1-yl) -1H-pyrazolo [3,4-b] pyridin-1- A white solid (3.58 g) of yl} benzamide was added to ethanol (7.84 mL) and stirred at room temperature for 2 hours. After sampling, it was washed with ethanol (7.84 mL) and dried under reduced pressure at 70 to 80 ° C. for 20 hours to obtain type I crystals (yield: 2.40 g, yield: 61.2%, purity: 98.21%). rice field.
Further, as shown in FIG. 1, the type I crystal has a diffraction angle (2θ) of 8.1 °, 10.9 °, 12.1 °, 14.0 °, and 14.9 in the powder X-ray diffraction spectrum. °, 16.2 °, 17.7 °, 20.2 °, 21.0 °, 21.5 °, 22.6 °, 24.3 °, 25.4 ° 26.4 °, 27.0 ° , 28.3 °, 30.2 °, 30.9 °, 31.5 °, 32.7 °, 34.7 °, 35.4 ° and 36.6 ° showed characteristic peaks.

[0032]

1H-NMR (DMSO-d 6):δppm 9.35 (1H,d,J=4.88Hz), 8.93 (1H,d,J=1.22Hz), 8.84 (1H,brs), 8.72 (1H,d,J=1.95Hz), 8.70 (1H,s) ,8.63 (1H,d,J=1.22Hz), 8.60 (1H,dd,J=8.29,1.95Hz), 8.46 (1H,s) ,8.25 (1H,d,J=8.29Hz), 8.22 (1H,brs), 8.12 (1H,d,J=4.88Hz), 4.59 (3H,s) ,3.95 (1H,tt,J=6.83,6.83Hz), 3.21 (2H,q,J=7.56Hz), 1.83(6H,d,J=6.83Hz), 1.75 (3H,t,J=7.56Hz):LRMS(ESI)m/z 455[M+H]

PATENT

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2015050235

Synthesis of Test Compound The
following synthesis example compounds (Synthesis Examples 1 to 3) were synthesized according to the method described in WO2011 / 004610.

[0361]

Synthesis Example 1: 4- {3-Isopropyl-4- (4- (1-methyl-1H-pyrazole-4-yl) -1H-imidazol-1-yl) -1H-pyrazolo [3,4-b] pyridine -1-yl} -3-methylbenzamide

[0362]

[Changing 22]

PATENT

WO 2011004610

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2011004610

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Pimitespib

3-Ethyl-4-{4-[4-(1-methyl-1H-pyrazol-4-yl)-1H-imidazol-1-yl]-3-(propan-2-yl)-1H-pyrazolo[3,4-b]pyridin-1-yl}benzamide

C25H26N8O : 454.53
[1260533-36-5]

//////////Pimitespib, ピミテスピブ,  JAPAN 2022, APPROVALS 2022, TAS 116, Jeselhy

O=C(N)C1=CC=C(N2N=C(C(C)C)C3=C(N4C=C(C5=CN(C)N=C5)N=C4)C=CN=C32)C(CC)=C1

Vutrisiran sodium, ALN 65492, Votrisiran


RNA, (Um-​sp-​(2′-​deoxy-​2′-​fluoro)​C-​sp-​Um-​Um-​Gm-​(2′-​deoxy-​2′-​fluoro)​G-​Um-​Um-​(2′-​deoxy-​2′-​fluoro)​A-​Cm-​Am-​Um-​Gm-​(2′-​deoxy-​2′-​fluoro)​A-​Am-​(2′-​deoxy-​2′-​fluoro)​A-​Um-​Cm-​Cm-​Cm-​Am-​sp-​Um-​sp-​Cm)​, complex with RNA (Um-​sp-​Gm-​sp-​Gm-​Gm-​Am-​Um-​(2′-​deoxy-​2′-​fluoro)​U-​Um-​(2′-​deoxy-​2′-​fluoro)​C-​(2′-​deoxy-​2′-​fluoro)​A-​(2′-​deoxy-​2′-​fluoro)​U-​Gm-​Um-​Am-​Am-​Cm-​Cm-​Am-​Am-​Gm-​Am) 3′-​[[(2S,​4R)​-​1-​[29-​[[2-​(acetylamino)​-​2-​deoxy-​β-​D-​galactopyranosyl]​oxy]​-​14,​14-​bis[[3-​[[3-​[[5-​[[2-​(acetylamino)​-​2-​deoxy-​β-​D-​galactopyranosyl]​oxy]​-​1-​oxopentyl]​amino]​propyl]​amino]​-​3-​oxopropoxy]​methyl]​-​1,​12,​19,​25-​tetraoxo-​16-​oxa-​13,​20,​24-​triazanonacos-​1-​yl]​-​4-​hydroxy-​2-​pyrrolidinyl]​methyl hydrogen phosphate] (1:1)

Vutrisiran Sodium

Nucleic Acid Sequence

Sequence Length: 44, 23, 2113 a 9 c 8 g 14 umultistranded (2); modified

Vutrisiran sodium

  • ALN 65492
  • Votrisiran

C530H672F9N171Na43O323P43S6 : 17289.77
[1867157-35-4 , Vutrisiran]

FormulaC530H672F9N171O323P43S6.43Na  ORC530H672F9N171Na43O323P43S6
CAS1867157-35-4 , VURISIRAN
Mol weight17289.7661

FDA APPROVED, AMVUTTRA, 2022/6/13

ブトリシランナトリウム
EfficacyGene expression regulator
  DiseasePolyneuropathy of hereditary transthyretin-mediated amyloidosis [D
CommentRNA interference (RNAi) drug
Treatment of transthyretin (TTR)-mediated amyloidosis (ATTR amyloidosis)

UNII28O0WP6Z1P UNII

Vutrisiran
Vutrisiran Sodium is a sodium salt of an siRNA derivative targeting transthyretin (TTR) covalently linked to a triantennary GalNAc3 complex at the 3’ end of the sense strand. The siRNA moiety is composed of a duplex oligonucleotide of sense strand consisting of chemically modified 21 nucleotide residues and antisense strand consisting of chemically modified 23 nucleotide residues each.

Vutrisiran is a double-stranded small interfering ribonucleic acid (siRNA) that targets wild-type and mutant transthyretin (TTR) messenger RNA (mRNA).7 This siRNA therapeutic is indicated for the treatment of neuropathies associated with hereditary transthyretin-mediated amyloidosis (ATTR), a condition caused by mutations in the TTR gene.2 More than 130 TTR mutations have been identified so far,3 but the most common one is the replacement of valine with methionine at position 30 (Val30Met).2 The Val30Met variant is the most prevalent among hereditary ATTR patients with polyneuropathy, especially in Portugal, France, Sweden, and Japan.2

TTR mutations lead to the formation of misfolded TTR proteins, which form amyloid fibrils that deposit in different types of tissues. By targeting TTR mRNA, vutrisiran reduces the serum levels of TTR.6,7 Vutrisiran is commercially available as a conjugate of N-acetylgalactosamine (GalNAc), a residue that enables the delivery of siRNA to hepatocytes.5,7 This delivery platform gives vutrisiran high potency and metabolic stability, and allows for subcutaneous injections to take place once every three months.8 Another siRNA indicated for the treatment of polyneuropathy associated with hereditary ATTR is patisiran.2 Vutrisiran was approved by the FDA in June 2022.

CLIP

https://www.nature.com/articles/s41392-020-0207-x

figure 1

Schematic illustrations of the working mechanisms of miRNA (a) and siRNA (b)

figure 2

Structures of chemical modifications and analogs used for siRNA and ASO decoration. According to the modification site in the nucleotide acid, these structures can be divided into three classes: phosphonate modification, ribose modification and base modification, which are marked in red, purple and blue, respectively. R = H or OH, for RNA or DNA, respectively. (S)-cEt-BNA (S)-constrained ethyl bicyclic nucleic acid, PMO phosphorodiamidate morpholino oligomer

figure 3

Representative designs for the chemical modification of siRNA. The sequences and modification details for ONPATTRO®, QPI-1007, GIVLAARI™ and inclisiran are included. The representative siRNA modification patterns developed by Alnylam (STC, ESC, advanced ESC and ESC+) and arrowhead (AD1-3 and AD5) are shown. Dicerna developed four GalNAc moieties that can be positioned at the unpaired G–A–A–A nucleotides of the DsiRNA structure. 2′-OMe 2′-methoxy, 2′-F 2′-fluoro, GNA glycol nucleic acid, UNA unlocked nucleic acid, SS sense strand, AS antisense strand

figure 6

siRNA delivery platforms that have been evaluated preclinically and clinically. Varieties of lipids or lipidoids, siRNA conjugates, peptides, polymers, exosomes, dendrimers, etc. have been explored and employed for siRNA therapeutic development by biotech companies or institutes. The chemical structures of the key component(s) of the discussed delivery platforms, including Dlin-DMA, Dlin-MC3-DMA, C12-200, cKK-E12, GalNAc–siRNA conjugates, MLP-based DPC2.0 (EX-1), PNP, PEI, PLGA-based LODER, PTMS, GDDC4, PAsp(DET), cyclodextrin-based RONDEL™ and dendrimer generation 3 are shown. DLin-DMA (1,2-dilinoleyloxy-3-dimethylaminopropane), DLin-MC3-DMA (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino) butanoate, DPC Dynamic PolyConjugates, MLP membrane-lytic peptide, CDM carboxylated dimethyl maleic acid, PEG polyethylene glycol, NAG N-acetylgalactosamine, PNP polypeptide nanoparticle, PEI poly(ethyleneimine), LODER LOcal Drug EluteR, PLGA poly(lactic-co-glycolic) acid, PTMS PEG-PTTMA-P(GMA-S-DMA) poly(ethylene glycol)-co-poly[(2,4,6-trimethoxybenzylidene-1,1,1-tris(hydroxymethyl))] ethane methacrylate-co-poly(dimethylamino glycidyl methacrylate), GDDC4 PG-P(DPAx-co-DMAEMAy)-PCB, where PG is guanidinated poly(aminoethyl methacrylate) PCB is poly(carboxybetaine) and P(DPAx-co-DMAEMAy) is poly(dimethylaminoethyl methacrylate-co-diisopropylethyl methacrylate), PEG-PAsp(DET) polyethylene glycol-b-poly(N′-(N-(2-aminoethyl)-2-aminoethyl) aspartamide), PBAVE polymer composed of butyl and amino vinyl ether, RONDEL™ RNAi/oligonucleotide nanoparticle delivery

Vutrisiran SodiumVutrisiran Sodium is a sodium salt of an siRNA derivative targeting transthyretin (TTR) covalently linked to a triantennary GalNAc3 complex at the 3’ end of the sense strand. The siRNA moiety is composed of a duplex oligonucleotide of sense strand consisting of chemically modified 21 nucleotide residues and antisense strand consisting of chemically modified 23 nucleotide residues each.C530H672F9N171Na43O323P43S6 : 17289.77
[1867157-35-4 , Vutrisiran]

REF

Nucleic Acids Research (2019), 47(7), 3306-3320. 

Drug Metabolism & Disposition (2019), 47(10), 1183-1201.  

PATENT

WO 2020128816

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2020128816

The present invention relates to pharmaceutical compositions and methods of treatment comprising administering to a patient in need thereof a combination of a benzoxazole derivative transthyretin stabilizer or a pharmaceutically acceptable salt or prodrug thereof and an additional therapeutic agent for the treatment of transthyretin amyloidosis. Particularly, the present invention relates to pharmaceutical compositions and methods of treatment comprising administering to a patient in need thereof 2-(3,5-dichlorophenyl)-1,3-benzoxazole-6-carboxylic acid or a pharmaceutically acceptable salt or prodrug thereof and one or more additional therapeutic agent for the treatment of transthyretin amyloidosis.

The present invention relates to pharmaceutical compositions and methods of treatment comprising administering to a patient in need thereof a combination of a benzoxazole derivative transthyretin stabilizer or a pharmaceutically acceptable salt or prodrug thereof and one or more additional therapeutic agent. Particularly, the present invention relates to pharmaceutical compositions and methods of treatment comprising administering to a patient in need thereof 2-(3,5-dichlorophenyl)-1,3-benzoxazole-6-carboxylic acid or a pharmaceutically acceptable salt or prodrug thereof and one or more additional therapeutic agent. The compositions and methods of the invention are useful in stabilizing transthyretin, inhibiting transthyretin misfolding, proteolysis, and treating amyloid diseases associated thereto.

Transthyretin (TTR) is a 55 kDa homotetrameric protein present in serum and cerebral spinal fluid and which functions as a transporter of L-thyroxine (T4) and holo-retinol binding protein (RBP). TTR has been found to be an amyloidogenic protein that, under certain conditions, can be transformed into fibrils and other aggregates which can lead to disease pathology such as polyneuropathy or cardiomyopathy in humans.

US Patent Nos. 7,214,695; 7,214,696; 7,560,488; 8, 168.683; and 8,653,119 each of which is incorporated herein by reference, discloses benzoxazole derivatives which act as transthyretin stabilizers and are of the formula

or a pharmaceutically acceptable salt thereof; wherein Ar is 3,5-difluorophenyl, 2,6-difluorophenyl, 3,5-dichlorophenyl, 2,6-dichlorophenyl, 2-(trifluoromethyl)phenyl or 3-(trifluoromethyl)phenyl. Particularly, 2-(3,5-dichlorophenyl)-1,3-benzoxazole-6-carboxylic acid (tafamidis) of the formula

is disclosed therein. Tafamidis is an orally active transthyretin stabilizer that inhibits tetramer dissociation and proteolysis that has been approved in certain jurisdictions for the treatment of transthyretin polyneuropathy (TTR-PN) and is currently in development for the treatment of transthyretin cardiomyopathy (TTR-CM). US Patent No. 9,249, 112, also incorporated herein by reference, discloses polymorphic forms of the meglumine salt of 2-(3,5-dichlorophenyl)-1,3-benzoxazole-6-carboxylic acid (tafamidis meglumine). US Patent No. 9,770,441 discloses polymorphic forms of the free acid of 2-(3,5-dichlorophenyl)-1,3-benzoxazole-6-carboxylic acid (tafamidis), and is also incorporated by reference herein.

Summary of the Invention

The present invention provides pharmaceutical compositions and methods comprising the compound 2-(3,5-dichlorophenyl)-1,3-benzoxazole-6-carboxylic acid or a pharmaceutically acceptable salt or prodrug thereof, and one or more additional therapeutic agent. Particular embodiments of this invention are pharmaceutical compositions and methods comprising 2-(3,5-dichlorophenyl)-1,3-benzoxazole-6-carboxylic acid or a pharmaceutically acceptable salt or prodrug thereof, and one or more additional therapeutic agents selected from the group consisting of agents that lower plasma levels of TTR such as an antisense therapy, TTR gene editing therapy, transcriptional modulators, translational modulators, TTR protein degraders and antibodies that bind and reduce TTR levels; amyloid reduction therapies such as anti amyloid antibodies (either TTR selective or general), stimulators of amyloid clearance, fibril disruptors and therapies that inhibit amyloid nucleation; other TTR stabilizers; and TTR modulators such as therapeutics which inhibit TTR cleavage. Particularly, the present invention provides pharmaceutical compositions and methods comprising tafamidis or tafamidis meglumine salt with one or more additional therapeutic agents. More particularly, the present invention provides pharmaceutical compositions and the present invention provides pharmaceutical compositions and methods comprising tafamidis or tafamidis meglumine salt with one or more additional therapeutic agents. More particularly, the present invention provides pharmaceutical compositions and the present invention provides pharmaceutical compositions and methods comprising tafamidis or tafamidis meglumine salt with one or more additional therapeutic agents. More particularly, the present invention provides pharmaceutical compositions and

methods comprising a polymorphic form of tafamidis free acid or a polymorphic form of tafamidis meglumine salt with one or more additional therapeutic agents.

The present invention also provides a method of treating or preventing transthyretin amyloidosis in a patient, the method comprising administering to a patient in need thereof a therapeutically or prophylactically effective amount of 2-(3,5-dichlorophenyl)-1,3-benzoxazole- 6-carboxylic acid or a pharmaceutically acceptable salt or prodrug thereof, and one or more additional therapeutic agents.

A particular embodiment of the present method of treatment is the method comprising a pharmaceutical composition comprising 2-(3,5-dichlorophenyl)-1,3-benzoxazole-6-carboxylic acid or a pharmaceutically acceptable salt or prodrug thereof, and one or more additional therapeutic agent are administered orally. Additional embodiments of this invention are methods of treatment as described above wherein the 2-(3,5-dichlorophenyl)-1,3-benzoxazole-6-carboxylic acid or a pharmaceutically acceptable salt or prodrug thereof, and one or more additional therapeutic agent are administered parenterally (intravenously or subcutaneously). Further embodiments of this invention are methods of treatment wherein the 2-(3,5-dichlorophenyl)-1, 3-benzoxazole-6-carboxylic acid or a pharmaceutically acceptable salt or prodrug thereof is administered orally and the one or more additional therapeutic agent is administered either orally or parenterally. Another embodiment of the present invention is wherein a pharmaceutical composition comprising 2-(3,5-dichlorophenyl)-1,3-benzoxazole-6-carboxylic acid or a pharmaceutically acceptable salt or prodrug thereof in combination with one or more additional therapeutic agent is administered parenterally and then 2-(3,5-dichlorophenyl)-1,3-benzoxazole-6-carboxylic acid or a pharmaceutically acceptable salt or prodrug thereof is administered orally. A particular method of treatment is a method of treating TTR amyloidosis such as TTR polyneuropathy or TTR Another embodiment of the present invention is wherein a pharmaceutical composition comprising 2-(3,5-dichlorophenyl)-1,3-benzoxazole-6-carboxylic acid or a pharmaceutically acceptable salt or prodrug thereof in combination with one or more additional therapeutic agent is administered parenterally and then 2-(3,5-dichlorophenyl)-1,3-benzoxazole-6-carboxylic acid or a pharmaceutically acceptable salt or prodrug thereof is administered orally. A particular method of treatment is a method of treating TTR amyloidosis such as TTR polyneuropathy or TTR Another embodiment of the present invention is wherein a pharmaceutical composition comprising 2-(3,5-dichlorophenyl)-1,3-benzoxazole-6-carboxylic acid or a pharmaceutically acceptable salt or prodrug thereof in combination with one or more additional therapeutic agent is administered parenterally and then 2-(3,5-dichlorophenyl)-1,3-benzoxazole-6-carboxylic acid or a pharmaceutically acceptable salt or prodrug thereof is administered orally. A particular method of treatment is a method of treating TTR amyloidosis such as TTR polyneuropathy or TTR 5-dichlorophenyl)-1,3-benzoxazole-6-carboxylic acid or a pharmaceutically acceptable salt or prodrug thereof is administered orally. A particular method of treatment is a method of treating TTR amyloidosis such as TTR polyneuropathy or TTR 5-dichlorophenyl)-1,3-benzoxazole-6-carboxylic acid or a pharmaceutically acceptable salt or prodrug thereof is administered orally. A particular method of treatment is a method of treating TTR amyloidosis such as TTR polyneuropathy or TTR

cardiomyopathy, the method comprising administering to a patient in need thereof a therapeutically effective amount of 2-(3,5-dichlorophenyl)-1,3-benzoxazole-6-carboxylic acid or a pharmaceutically acceptable salt or prodrug thereof in combination with one or more additional therapeutic agents.

Brief Description of the Drawings

REF

Biochemical Pharmacology (Amsterdam, Netherlands) (2021), 189, 114432.

PATENT

WO 2021041884 

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2021041884

Exemplary RNAi agents that reduce the expression of TTR include patisiran and vutrisiran.

The ter s “antisense polynucleotide agent”, “antisense oligonucleotide”, “antisense compound”, and “antisense agent” as used interchangeably herein, refer to an agent comprising a single-stranded oligonucleotide that specifically binds to the target nucleic acid molecules via hydrogen bonding (e.g., Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding) and inhibits the expression of the targeted nucleic acid by an antisense mechanism of action, e.g., by RNase H. In some embodiments, an antisense agent is a nucleic acid therapeutic that acts by reducing the expression of a target gene, thereby reducing the expression of the polypeptide encoded by the target gene. Exemplary antisense agents that reduce the expression of TTR include inotersen and Ionis 682884/ ION-TTR-LRx (see, e.g., WO2014179627 which is incorporated by reference in its entirety). Further antisense agents that reduce the expression of TTR are provided, for example in WO2011139917 and WO2014179627, each of which is incorporated by reference in its entirety.

REF

Clinical Pharmacology & Therapeutics (Hoboken, NJ, United States) (2021), 109(2), 372-382

Annals of Plastic Surgery (2021), 86(2S_Suppl_1), S23-S29.

Journal of Cardiovascular Pharmacology (2021), 77(5), 544-548. 

Annals of Pharmacotherapy (2021), 55(12), 1502-1514.

Kidney International (2022), 101(2), 208-211

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figure 7

Tissues targeted by siRNA and miRNA therapeutics currently being investigated at the clinical stage. The corresponding therapeutic names are shown beside the tissues

CLIP

Vutrisiran An Investigational RNAi Therapeutic for ATTR Amyloidosis Vutrisiran has not been approved by the U.S. Food and Drug Administration, European Medicines Agency, or any other regulatory authority and no conclusions can or should be drawn regarding the safety or effectiveness of this investigational therapeutic. Overview • Vutrisiran is an investigational RNAi therapeutic in development for the treatment of transthyretin-mediated (ATTR) amyloidosis, which encompasses both hereditary ATTR (hATTR) amyloidosis and wild-type ATTR (wtATTR) amyloidosis.1, 2 • Vutrisiran inhibits the production of disease-causing transthyretin (TTR) protein by the liver, leading to a reduction in the level of TTR in the blood.1, 2 • Vutrisiran is administered subcutaneously (under the skin) and utilizes one of Alnylam’s delivery platforms known as the Enhanced Stabilization Chemistry (ESC)-GalNAc-conjugate delivery platform.1, 2 • Vutrisiran is administered every three months.2 • Vutrisiran is under review by the U.S. Food and Drug Administration (FDA), the European Medicines Agency (EMA), and the Brazilian Health Regulatory Agency (ANVISA). Vutrisiran has been granted Orphan Drug Designation in the U.S. and the European Union (EU) for the treatment of ATTR amyloidosis. Vutrisiran has also been granted a Fast Track designation in the U.S. for the treatment of the polyneuropathy of hATTR amyloidosis in adults. In the U.S. vutrisiran has received an action date under the Prescription Drug User Fee Act (PDUFA) of April 14, 2022. The Company received orphan drug designation in Japan. Alnylam has global commercial rights to vutrisiran, assuming regulatory approvals. Clinical Development • A Phase 1 clinical study of vutrisiran was conducted in 80 healthy volunteers (60 received vutrisiran and 20 received placebo). Vutrisiran demonstrated an acceptable safety profile and a single dose reduced serum TTR for a period of at least 90 days.2 • The safety and efficacy of vutrisiran are being evaluated in the HELIOS Phase 3 clinical program, currently consisting of two clinical trials: HELIOS-A and HELIOS-B. • HELIOS-A is a randomized, open-label, global multi-center Phase 3 study of 164 adult patients with hATTR amyloidosis with polyneuropathy.1 • The primary endpoint of HELIOS-A is change from baseline in the modified Neuropathy Impairment Score +7 (mNIS+7) at 9 months. • Secondary endpoints at 9 months include the Norfolk Quality of Life-Diabetic Neuropathy (Norfolk QoL-DN) Total Score and the 10-Meter Walk Test (10-MWT). • The 9-month endpoints will be analyzed at 18 months with the addition of other secondary endpoints. • HELIOS-B is a randomized, double-blind, placebo-controlled Phase 3 study of 655 adult patients with ATTR amyloidosis with cardiomyopathy (including both hATTR and wtATTR amyloidosis).3 • The primary endpoint will evaluate the efficacy of vutrisiran versus placebo for the composite outcome of all-cause mortality and recurrent cardiovascular (CV) events (CV hospitalizations and urgent heart failure (HF) visits) at 30-36 months. • Secondary endpoints include the change from baseline in the 6-minute walk test (6-MWT), health status measured using the Kansas City Cardiomyopathy Questionnaire Overall Summary (KCCQ-OS), echocardiographic assessments of mean left ventricular wall thickness and global longitudinal strain, the N-terminal prohormone B-type natriuretic peptide (NT-proBNP) as a cardiac biomarker, and all-cause mortality, rate of recurrent CV events, and composite of all-cause mortality and recurrent all-cause hospitalizations and urgent HF visits at month 30 or 30-36 months. Page 2 © 2021 Alnylam Pharmaceuticals, Inc. All rights reserved. TTRsc02-USA-00012 v4 About ATTR Amyloidosis • ATTR amyloidosis is a rare, underdiagnosed, rapidly progressive, debilitating, and fatal disease caused by misfolded TTR that accumulates as amyloid fibrils in multiple tissues including the nerves, heart, and GI tract. There are two types of ATTR amyloidosis: hATTR amyloidosis and wtATTR amyloidosis.4,5,6 • hATTR amyloidosis is an inherited condition that is caused by variants (i.e., mutations) in the transthyretin (TTR) gene.5,7,8 TTR protein is produced primarily in the liver and is normally a carrier of vitamin A.9 The variant results in misfolded TTR proteins that accumulate as amyloid deposits in multiple tissues, including the nerves, heart and gastrointestinal (GI) tract.5, 6, 7 It is a multisystem disease that can include sensory and motor, autonomic, and cardiac symptoms. The condition can have a debilitating impact on a patient’s life and may lead to premature death with a median survival of 4.7 years following diagnosis.8,10 It is estimated that there are approximately 50,000 patients with hATTR amyloidosis worldwide.11 • wtATTR amyloidosis is a non-hereditary condition that occurs when misfolded wild-type TTR accumulates as amyloid deposits in multiple organs. It predominantly manifests as cardiac symptoms, but other systems are also involved, and commonly leads to heart failure and mortality within 2.5 to 5.5 years.12,13,14,15,16,17,18,19 wtATTR amyloidosis affects an estimated 200,000-300,000 people worldwide.20 • Alnylam is committed to developing multiple treatment options for people who are living with ATTR amyloidosis to help manage the debilitating and progressive nature of the disease. For more information about vutrisiran, please contact media@alnylam.com. For more information on HELIOS-A (NCT03759379) and HELIOS-B (NCT04153149) please visit http://www.clinicaltrials.gov or contact media@alnylam.com. Current information as of November 2021

CLIP

Alnylam announces extension of review period for new drug vutrisiran to treat ATTR amyloidosis

https://www.medthority.com/news/2022/4/alnylam-announces-3-month-extension-of-review-period-for-new-drug-application-for-vutrisiran-to-treat-attr-amyloidosis/

Alnylam announces 3-month extension of review period for new drug application for vutrisiran to treat ATTR amyloidosis.

Alnylam Pharmaceuticals, Inc., a RNAi therapeutics company, announced that the FDA has extended the review timeline of the New Drug Application (NDA) for vutrisiran, an investigational RNAi therapeutic in development for the treatment of transthyretin-mediated (ATTR) amyloidosis, to allow for the review of newly added information related to the new secondary packaging and labelling facility.

Alnylam recently learned that the original third-party secondary packaging and labelling facility the Company planned to use for the vutrisiran launch was recently inspected and the inspection requires classification for the FDA to take action on the vutrisiran NDA. The inspection observations were not directly related to vutrisiran. In order to minimize delays to approval, Alnylam has identified a new facility to pack and label vutrisiran and submitted an amendment to the NDA for review by the FDA. The updated Prescription Drug User Fee Act (PDUFA) goal date to allow for this review is July 14, 2022. No additional clinical data have been requested by the FDA.

////////////Vutrisiran sodium,  APPROVALS 2022, FDA 2022, FDA APPROVED, AMVUTTRA, 2022/6/13, ブトリシランナトリウム , ALN 65492, Votrisiran, siRNA

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