ATR 101

N-(2,6-bis(1-methylethyl)phenyl)-N'-((1-(4-(dimethylamino)phenyl)cyclopentyl) methyl)urea hydrochloride
N-(2,6-BIS(l-METHYLETHYL)PHENYL)-N'-((l-(4- (DIMETHYLAMINO)PHENYL)CYCLOPENTYL)METHYL)UREA
ATR-101; ATR 101; ATR101; PD132301-2; PD-132301-2; PD 132301-2; PD132301; PD-132301; PD 132301.
IUPAC/Chemical Name: 1-(2,6-diisopropylphenyl)-3-((1-(4-(dimethylamino)phenyl)cyclopentyl)methyl)urea hydrochloride
ATR-101 HCl
CAS#: 133825-81-7 (ATR-101 HCl); 133825-80-6 (ATR-101).

Molecular Formula:	C27H40ClN3O
Molecular Weight:	458.079 g/mol

The Regents Of The University Of Michigan, Atterocor, Inc.

Millendo Therapeutics is developing ATR-101, an ACAT1 inhibitor, for treating adrenal cancers including adrenocortical cancer and congenital adrenal hyperplasia.




ATR-101, also known as PD-132301 (a free base) or PD-132301-2 (a HCl salt), is in clinical development for the treatment of adrenocortical carcinoma (ACC). ATR-101 is a selective inhibitor of ACAT1 (acyl coenzyme A:cholesterol acyltransferase). ACAT1 catalyzes cholesterol ester formation and, in the adrenals, is particularly important in creating a reservoir of substrate for steroid biosynthesis. ATR-101 is uniquely distributed to adrenal tissues and inhibition of adrenal ACAT1 by ATR-101 disrupts steroidogenesis and leads to selective apoptosis of steroid producing adrenocortical-derived cells. Similar effects have been seen in the human ACC cell line, H295R. ATR-101 has shown pre-clinical efficacy in H295R xenograft mouse models. ACC is an ultra-rare malignancy, occurring in about 2 per million population annually.

ATR-101 (Atterocor, Inc., Ann Arbor, MI, USA) is in clinical development for the treatment of adrenocortical carcinoma (ACC). ATR-101 is a selective inhibitor of ACAT1 (acyl coenzyme A:cholesterol acyltransferase). ACAT1 catalyzes cholesterol ester formation and, in the adrenals, is particularly important in creating a reservoir of substrate for steroid biosynthesis. ATR-101 is uniquely distributed to adrenal tissues and inhibition of adrenal ACAT1 by ATR-101 disrupts steroidogenesis and leads to selective apoptosis of steroid producing adrenocortical-derived cells. Similar effects have been seen in the human ACC cell line, H295R. ATR-101 has shown pre-clinical efficacy in H295R xenograft mouse models. ACC is an ultra-rare malignancy, occurring in about 2 per million population annually. ACC is frequently discovered in Stage 4 and the overall disease survival is approximately 17 months. Tumors often overproduce steroids normally produced in the adrenal cortex. Current therapies are toxic, difficult to administer, and poorly effective. Clinical trial information: NCT01898715.
Adrenocortical carcinoma (ACC) generally has poor prognosis. Existing treatments provide limited benefit for most patients with locally advanced or metastatic tumors. We investigated the mechanisms for the cytotoxicity, xenograft suppression and adrenalytic activity of ATR-101 (PD132301-02), a prospective agent for ACC treatment. Oral ATR-101 administration inhibited the establishment and impeded the growth of ACC-derived H295R cell xenografts in mice. ATR-101 induced H295R cell apoptosis in culture and in xenografts. ATR-101 caused mitochondrial hyperpolarization, reactive oxygen release and ATP depletion within hours after exposure, followed by cytochrome c release, caspase-3 activation, and membrane permeabilization. When combined with ATR-101, lipophilic free radical scavengers suppressed the reactive oxygen release, and glycolytic precursors prevented the ATP depletion, abrogating ATR-101 cytotoxicity. ATR-101 directly inhibited F1F0-ATPase activity and suppressed ATP synthesis in mitochondrial fractions. ATR-101 administration to guinea pigs caused oxidized lipofuscin accumulation in the zona fasciculata layer of the adrenal cortex, implicating reactive oxygen release in the adrenalytic effect of ATR-101. These results support the development of ATR-101 and other adrenalytic compounds for the treatment of ACC.

Company	Millendo Therapeutics Inc.
Description	Selective inhibitor of sterol O-acyltransferase 1 (SOAT1; ACAT1)
Molecular Target	Sterol O-acyltransferase 1 (SOAT1) (ACAT1)

PATENT
WO2013142214
https://www.google.co.in/patents/WO2013142214A1?cl=en
PATENT
WO-2016049518
One such promising agent is N-(2,6-bis( 1 -methylethyl)phenyl)-N'-(( 1 -(4-(dimethyl-amino)phenyl)cyclopentyl)methyl)urea hydrochloride ("ATR-101"). The free base form of ATR-101 has the following chemical structure:

The chemical synthesis of ATR-101 has been previously reported by Trivedi et al. (J. Med. Chem. 37: 1652-1659, 1994). This procedure, however, does not provide for ATR-101 in a form suitable for solid-dosing, particularly with regard to capsule or tablet formation, and does not provide for ATR-101 in high purity.
While significant advances have been made in this field, particularly in the context of ATR-101, there remains a substantial need for improved techniques and products for the oral administration of ATR-101 to patients in need thereof, including patients having ACC and/or other disorders or conditions such as Cushing's syndrome and congenital adrenal hyperplasia (CAH).



EXAMPLE 1
SYNTHESIS OF SOLID DRUG FORM OF ATR-101

Step 1 : Preparation of Primary Amine 2 from the Nitrile 1

Tetrahyrofuran (THF) and Compound 1 are charged to a reactor vessel and a lithium aluminum hydride (LAH) solution in THF is added slowly. After the addition, the reaction mixture is warmed to 45°C and stirred until in-process HPLC analysis indicates that the reaction is complete. The reaction mixture is cooled to between 0 and 10°C and aqueous NaOH is added slowly while controlling the temperature to between 0 and 10°C. The mixture is then warmed to between 20 and 25°C and any inorganic salts removed by filtration. The solids are then washed with additional THF.
The filtrate is distilled under vacuum. Acetonitrile (MeCN) is added and the distillation continued to reduce the total volume. H₂0 is added and the solution is cooled to 20°C, and seeded if necessary. Additional water is added to the slurry and cooled to between 0 and 5°C and filtered. The crystallization vessel and filter cake is washed with MeCN and water (1 :2 mixture) and dried under vacuum between 40 to 45°C to produce Compound 2. Typical yield: 85%.
Step 2: Preparation of ATR-101 Free Base

2,6-Diisopropyl aniline hydrochloride (Compound 3) is converted to the corresponding free base by stirring in a mixture of dichloromethane (DCM) and 10% aqueous NaOH. The organic phase is separated and washed with water. The DCM solution containing the aniline free base is concentrated by distillation.
4-dimethylaminopyridine (DMAP) and DCM are charged to a separate reaction vessel. The mixture is cooled and a solution of di-tert-butyl dicarbonate (Boc₂0) in DCM is slowly added while the temperature is maintained between 0 and 5°C. The aniline free base solution is then slowly added to the reaction vessel. A complete conversion of aniline to the isocyanate is verified by in-process HPLC analysis.
Compound 2 and MeCN are charged to a separate vessel and this solution is cooled to between 0 and 5°C. The isocyanate intermediate solution
(prepared above) is slowly added while the temperature is maintained between 0 and 5°C, and stirred until in-process HPLC indicates that the reaction is complete.
The reaction mixture is distilled under vacuum, and isopropyl alcohol
(IP A) is added and the distillation is continued. The resulting solution is cooled and seeded, if necessary. After crystallization occurs, water is added and the mixture is cooled to between 0 and 5°C, and filtered. The crystallization vessel and filter cake is washed with isopropanol: water (1 : 1) and the product cake is dried under vacuum to yield ATR-101 as the free base. Typical yield: 89 %
Step 3 : Preparation of Solid Drug Form of ATR- 101

The ATR-101 free base is dissolved in acetone and filtered to remove particulates. Additional acetone is used to rinse the dissolution vessel and filter. Concentrated hydrochloric acid (HCl) is added while maintaining the reaction at room temperature. The resultant slurry is filtered and the cake is washed with acetone. The resulting solid is dried under vacuum between 40 and 45°C to obtain the solid drug form of ATR-101. Typical yield: 70-80 %.
EXAMPLE 2
CHARACTERIZATION OF THE SOLID DRUG FORM OF ATR-101
The solid drug form of ATR-101 was analyzed to fully characterize the material and provide proof of structure.
Elemental Analysis
An elemental (CHN) analysis was conducted, in duplicate, of the solid drug form of ATR-101. The results are summarized in Table 1 and are in agreement with the theoretical values calculated for the molecular ATR-101 drug substance formula of C₂₇H₃₉N₃O HCl.
Table 1

Chloride Content
The solid drug form of ATR-101 is prepared as its HCl salt. To confirm the chloride content (and the stoichiometry), the hydrochloride salt was analyzed by Ion Chromatography using a validated method. The w/w% result showed 7.8% chloride present. The theoretical value for a mono hydrochloride salt is 7.7%. The experimental result conforms to the theoretical value for the mono-hydrochloride salt.
Mass Spectrometry
Mass spectrometry studies were conducted in accordance with
USP<736> using an AB Sciex API 2000 LC/MS/MS system. The samples were analyzed by electrospray ionization in positive mode. The base peak observed was 422.3 (M+H-HC1), consistent with the parent compound (see Figure 1). Two minor peaks were observed, at 301.3 and 202.3 (uncharacterized fragments). The combined data of the LC/MS and CFIN results support the molecular formula assignment of C27H39N3O and mass of 421.63 g/mol for the free base and C27H39N3O . HCl (mass of 458.09 g/mol) for the mono hydrochloride salt.
Nuclear Magnetic Resonance (NMR) - 1H NMR
The proton NMR spectrum of the solid drug form of ATR-101 was obtained using a Varian Gemini 400 MHz spectrometer and. The sample was dissolved in CD₃OD. The resulting proton NMR spectrum is shown in Figure 2.
Two-Dimensional (2D) NMR
The 2D proton NMR spectrum (COSY) shown in Figure 3 confirmed some of the connectivity expected for the solid drug form of ATR-101. In particular the resonance at 1.2 ppm is strongly correlated to the resonance at 3.1. This correlation together with the splitting pattern observed for the peak at 3.1 strongly suggests an isopropyl moiety. Further, the data from these spectra show a strong correlation between each of the broad peaks at 1.6-2.2 ppm, consistent with a cycloalkyl functionality in which no heteroatoms or other non-alkyl substitution is present.
Carbon 13 NMR (¹³C NMR)
The 100 MHz ¹³C NMR spectrum of the solid drug form of ATR-101 was obtained using a Varian Gemini 400 MHz spectrometer. The sample was dissolved in CD₃OD. The resulting ¹³C NMR spectrum is shown in Figure 4. The numbering of the carbon atoms for the analysis of the spectrum is shown below, and the interpretation is shown in Table 2. The observed signals are consistent with the structure of ATR-101.

Table 2


Fourier Transform Infrared Spectroscopy (IR)
Infrared (IR) spectroscopy was performed using the soid drug form of ATR-101. The resulting spectrum, shown in Figure 5, is consistent with the structure of ATR-101 drug substance. The major peak assignments are presented in Table_3.
Table 3

EXAMPLE 3
COMPARISON WITH PRIOR ART SYNTHESIS OF ATR-101 (BY TRIVEDI ETAL.. J. MED. CHEM. 37: 1652-1659, 1994)

ATR-101
In this experiment, 10.6 g of ATR-101 was synthesized according to the above procedure, which corresponds to the the procedure set forth in Trivedi et al., J. Med. Chem. 137: 1652-1659, 1994 (hereinafter referred to as the "Trivedi procedure"). The purity of ATR-101 as made by the Trivedi procedure was found to be 94.9%, compared to a purity of 98.3% for ATR-101 obtained by the procedure of Example 1 and as evaluated in Example 2.
Step 1 : Alkylation of p-nitrophenylacetonitrile

52
The initial alkylation reaction was run on 15.0 g scale and, according to the Trivedi procedure, should have given 15.7 g (79%) of product 52. However, several problems occurred, and the yield was much lower than expected (6.0 g, 30% yield), although the purity by 1H NMR and melting point (actual: 71-72°C, reported: 76°C) seemed good. Approximately half way through the addition of 1 ,4-bromobutane and p- nitrophenylacetonitrile to NaH, a black solid precipitated out of the purple solution causing the stirbar in the flask to skip and jump. The rate of stirring had to be monitored throughout the remainder of the addition to maintain a sluggish and inefficient mixing of the solution.
After stirring at ambient temperature overnight to ensure reaction completion, the reaction was worked-up as the procedure indicated. First, excess ether was removed using air bubbling, and the black solid was isolated by filtration. Diethyl ether was then added until all of the solids dissolved to give a clear black solution. However, upon washing the ether solution with 2N HC1, a black amorphous solid precipitated from the solution. There was no note of this black solid in the Trivedi procedure, so the work-up was continued without modification. The black solids ended up in the aqueous washes, or stuck to the seperatory funnel. The remainder of the work-up proceeded as expected, and the hot hexanes extraction of the crude solid resulted in light pink planar crystals.
The procedure was repeated with two changes thought to be responsible for the low yield: the anhydrous solvent (from the bottle) was sieve dried to remove trace water, and the stir bar was replaced with a mechanical stirrer to ensure more even mixing of the solution. The procedure was re-run on 10 g scale, which should have yielded 10.5 g of compound 52. However, despite the changes to the procedure, the resulting product and yield was nearly identical to the first run (4.5 g, 34% yield, 71-72°C melting point).
In an attempt to determine where the bulk of material ended up, the aqueous layer from this reaction was re-extracted with diethyl ether, but only resulted in trace amounts of material. The black solids that formed during the work-up were isolated by filtration, and an NMR was taken of the material. The NMR showed peaks corresponding to compound 52. Presumably, this amorphous black solid that resulted after HC1 formation is the main source of lost material, as there appeared to be several grams of it.
Ste 2: Reduction of Nitro Compound

The conversion of nitro compound 52 to the dimethyl amine 53 was done over two steps: palladium catalyzed hydrogenation of the nitro compound to give the free amine 52b, followed by imine formation & reduction to the dimethylamine 53.
An exploratory small scale reaction was run, using 1/10th of the available material (1.0 g compound 52). The reduction of the nitro compound on the 1 gram scale was very rapid, with hydrogen consumption ceasing after 3-4 hours. A crude NMR of an aliquot of the reaction mixture showed very clean amine (52b). The formaldehyde was added, as well as additional Pd/C, and the hydrogenation was continued. The hydrogen was not consumed as quickly for the imine reduction, and the reaction was still progressing when the vessel was pressurized to 55 psi and left shaking overnight (ca. 16h).
After 16 hours, the pressure in the flask had dropped to 30 psi, indicating that the hydrogenation was still progressing overnight. An aliquot NMR confirmed that the reaction had not proceeded to completion.
On large scale, the nitro reduction proceeded very smoothly, consuming hydrogen at a very rapid rate, and going to completion again within 3-4 hours. The reactor was pressurized to 55 psi and shaken overnight, as indicated in the original procedure, before more Pd/C was added, followed by formaldehyde. Hydrogen consumption was again observed to be very sluggish, so the valve to the hydrogen tank was left open to the vessel, and the reaction was shaken for 24 hours.
After 24 hours of shaking, the valve to the vessel was closed, and a drop of 5 psi was observed over 1 hour, indicating that the reaction had not progressed to completion. TLC also showed several polar products, suggesting that the reaction was only ca. 50% complete. The hydrogenation vessel was pressurized to 55 psi with hydrogen, and the valve again left open for an additional 24 hours of hydrogenation.
After 24 hours, the reaction stopped consuming hydrogen, and the vessel was purged and the contents filtered to remove the palladium catalyst. The work-up was performed similarly to the small scale, and the two reactions were combined prior to purification by column chromatography, giving 5.7g (57.5% yield) of the desired dimethylamine product 53.
Step 3 : Reduction of C ano Compound

A small scale RaNi hydrogenation was done and the test reaction went smoothly. Hydrogen consumption was rapid, and the reaction appeared complete after approximately 2 hours. The consumption of hydrogen had ceased, and TLC indicated that there was no compound 53 remaining. After filtration to remove the Raney Nickel, the reaction completion was confirmed by aliquot NMR.
The remaining material was subjected to reduction using the same conditions, and hydrogen consumption and TLC analysis again indicated reaction completion after 2 hours. The material was filtered and combined with the smaller scale reaction material. After concentration to dryness, the crude yield was found to be 5.5 g (96.5% yield), which was very close to the reported yield (99%>).
Step 4: Formation of Urea Com ound

Urea formation is a straightforward procedure, and the small scale test reaction with the amine 54 (500 mg) being combined with 1.0 equivalent of the
isocyanate in 20 parts ethyl acetate. After stirring for 16 hours, the solution was concentrated to dryness to give a white solid. Crude 1H NMR of the solid confirmed that the spectra matched the reported spectra in the Trivedi procedure.
The remaining material was carried forward to ATR-101 freebase without difficulty, and the lots of product were combined. In an effort to remove the residual ethyl acetate, the solids were dissolved in 10 mL of toluene, followed by concentration under reduced pressure. After drying on high- vacuum, ATR-101 freebase was isolated as a sticky white foam (10.6 g, 99% yield). The 1H NMR of the final product showed trace toluene even after extended drying, and the material was moved on to the HC1 salt formation.
The melting point of the solid was later taken and found to be surprisingly low (50-56°C, expected: 132-133°C). The nature of the solid (oily foam) made the determination of the melting point difficult, but it was judged to be completely melted above 60°C.
Step 5: Formation of HC1 Salt

To the ATR-101 freebase in toluene was added 37% HC1, and a gummy white solid precipitated out immediately. The solution was dried by Dean-Stark apparatus over approximately 3 hours with vigorous stirring and heating (bath temp: 160°C). After drying, the solution was cooled and the fine crystalline solid was isolated by filtration and washed with acetone and diethyl ether. The product ATR-101 was dried until a constant weight was achieved (10.6 g, 92% yield) and fully characterized.
Figure 1 is the LC/MS Mass spectrum of the solid drug form of ATR- 101.

Figure 2 is the proton NMR spectrum of the solid drug form of ATR- 101.
Figure 3 is the 2-D ¹H NMR spectrum (COSY) of the solid drug form of ATR-101.
Figure 4 is the ¹³C NMR spectrum of of the solid drug form of ATR- 101.

Figure 5 is the FT-IR spectrum the solid drug form of ATR-101.


Paper
(J. Med. Chem. 37: 1652-1659, 1994

Inhibitors of Acyl-CoA:Cholesterol Acyltransferase (ACAT). 7. Development of a Series of Substituted N-Phenyl-N'-[(1-phenylcyclopentyl)methyl]ureas with Enhanced Hypocholesterolemic Activity

Bharat K. Trivedi, Terri Stoeber Purchase, Ann Holmes, Corinne E. Augelli-Szafran, Arnold D. Essenburg, Katherine L. Hamelehle, Richard L. Stanfield, Richard F. Bousley, Brian R. Krause

J. Med. Chem., 1994, 37 (11), pp 1652–1659

DOI: 10.1021/jm00037a016

http://pubs.acs.org/doi/abs/10.1021/jm00037a016

Patent ID	Date	Patent Title
EP0474733	1994-08-31	ANTIHYPERLIPIDEMIC AND ANTIATHEROSCLEROTIC UREA COMPOUNDS.
WO9015048	1990-12-13	ANTIHYPERLIPIDEMIC AND ANTIATHEROSCLEROTIC UREA COMPOUNDS

Patent ID	Date	Patent Title
US2015087649	2015-03-26	TREATING DISORDERS ASSOCIATED WITH ABERRANT ADRENOCORTICAL CELL BEHAVIOR
US2013267550	2013-10-10	Compounds and Methods for Treating Aberrant Adrenocartical Cell Disorders
EP0858336	2006-12-20	METHOD AND PHARMACEUTICAL COMPOSITION FOR REGULATING LIPID CONCENTRATION
US2005234124	2005-10-20	Carboxyalkylether-ACAT inhibitor combinations
US2004072903	2004-04-15	Carboxyalkylether-acat inhibitors combinations
US6143755	2000-11-07	Pharmaceutical methods of treatment with ACAT inhibitors and HMG-CoA reductase inhibitors
US6124309	2000-09-26	Method and pharmaceutical composition for regulating lipid concentration
US6093719	2000-07-25	Method and pharmaceutical composition for regulating lipid concentration
WO9716184	1997-05-09	METHOD AND PHARMACEUTICAL COMPOSITION FOR REGULATING LIPID CONCENTRATION
EP0474733	1994-08-31	ANTIHYPERLIPIDEMIC AND ANTIATHEROSCLEROTIC UREA COMPOUNDS.

References

1: Wolfgang GH, MacDonald JR, Vernetti LA, Pegg DG, Robertson DG. Biochemical alterations in guinea pig adrenal cortex following administration of PD 132301-2, an inhibitor of acyl-CoA:cholesterol acyltransferase. Life Sci. 1995 Feb 17;56(13):1089-93. PubMed PMID: 9001442.
2: Saxena U, Ferguson E, Newton RS. Acyl-coenzyme A:cholesterol-acyltransferase (ACAT) inhibitors modulate monocyte adhesion to aortic endothelial cells. Atherosclerosis. 1995 Jan 6;112(1):7-17. PubMed PMID: 7772069.
3: Reindel JF, Dominick MA, Bocan TM, Gough AW, McGuire EJ. Toxicologic effects of a novel acyl-CoA:cholesterol acyltransferase inhibitor in cynomolgus monkeys. Toxicol Pathol. 1994 Sep-Oct;22(5):510-8. PubMed PMID: 7899779.
4: Krause BR, Black A, Bousley R, Essenburg A, Cornicelli J, Holmes A, Homan R, Kieft K, Sekerke C, Shaw-Hes MK, et al. Divergent pharmacologic activities of PD 132301-2 and CL 277,082, urea inhibitors of acyl-CoA:cholesterol acyltransferase. J Pharmacol Exp Ther. 1993 Nov;267(2):734-43. PubMed PMID: 8246149.
5: Dominick MA, McGuire EJ, Reindel JF, Bobrowski WF, Bocan TM, Gough AW. Subacute toxicity of a novel inhibitor of acyl-CoA: cholesterol acyltransferase in beagle dogs. Fundam Appl Toxicol. 1993 Feb;20(2):217-24. PubMed PMID: 8383621.
6: Dominick MA, Bobrowski WA, MacDonald JR, Gough AW. Morphogenesis of a zone-specific adrenocortical cytotoxicity in guinea pigs administered PD 132301-2, an inhibitor of acyl-CoA:cholesterol acyltransferase. Toxicol Pathol. 1993;21(1):54-62. PubMed PMID: 8397438.
///////ATR 101, 133825-81-7, ATR-101 HCl,  133825-80-6,  Millendo Therapeutics,  ACAT1 inhibitor, treating adrenal cancers,  adrenocortical cancer,  congenital adrenal hyperplasia, Atterocor, Inc., Ann Arbor, MI, USA
O=C(NCC1(C2=CC=C(N(C)C)C=C2)CCCC1)NC3=C(C(C)C)C=CC=C3C(C)C.[H]Cl

INCB24360 (epacadostat)

Epacadostat

(Z)-N-(3-bromo-4-fluorophenyl)-N'-hydroxy-4-[2-(sulfamoylamino)ethylamino]-1,2,5-oxadiazole-3-carboxamidine

1,2,5-Oxadiazole-3-carboximidamide, 4-[[2-[(aminosulfonyl)amino]ethyl]amino]-N-(3-bromo-4-fluorophenyl)-N'-hydroxy-

1204669-58-8

INCB024360

N-(3-Brom-4-fluorphenyl)-N'-hydroxy-4-{[2-(sulfamoylamino)ethyl]amino}-1,2,5-oxadiazol-3-carboximidamid

UNII 71596A9R13

(Z)-N-(3-bromo-4-fluorophenyl)-N'-hydroxy-4-(2-(sulfamoylamino)ethylamino)-1,2,5-oxadiazole-3-carboximidamide

1,2,5-Oxadiazole-3-carboximidamide, 4-[[2-[(aminosulfonyl)amino]ethyl]amino]-N'-(3-bromo-4-fluorophenyl)-N-hydroxy-

Molecular Formula, C₁₁H₁₃BrFN₇O₄S
Average mass438.233 Da
cas 1204669-58-8 (or 1204669-37-3)

Synonym:	IDO1 inhibitor INCB024360 indoleamine-2,3-dioxygenase inhibitor INCB024360
Code name:	INCB 024360 INCB024360
Chemical structure:	1,2,5-Oxadiazole-3-carboximidamide, 4-((2-((Aminosulfonyl)amino)ethyl)amino)-N-(3-bromo-4-fluorophenyl)-N'-hydroxy-, (C(Z))-

Company	Incyte Corp.
Description	Indoleamine 2,3-dioxygenase 1 (IDO1) inhibitor
Molecular Target	Indoleamine 2,3-dioxygenase 1 (IDO1)
Mechanism of Action	Indoleamine 2,3-dioxygenase (INDO) inhibitor
Therapeutic Modality	Small molecule

 

• OriginatorIncyte Corporation
• DeveloperFred Hutchinson Cancer Research Center; Incyte Corporation; Merck AG
• ClassAmides; Antineoplastics; Imides; Oxadiazoles; Small molecules
• • Phase IIFallopian tube cancer; Malignant melanoma; Non-small cell lung cancer; Ovarian cancer; Peritoneal cancer; Solid tumours

  Most Recent Events

  • 15 Jan 2016Phase-II clinical trials in Solid tumours (Combination therapy, Late-stage disease, Second-line therapy or greater) in USA (PO)
  • 11 Jan 2016Phase-II clinical trials in Non-small cell lung cancer (Combination therapy, Late-stage disease, Second-line therapy or greater) in USA (PO)
  • 11 Jan 2016The US FDA and Health Canada approve IND application and Clinical Trial Application, respectively, for a phase Ib trial in Ovarian cancer (Combination therapy, Recurrent, Second-line therapy or greater)

In 2016, orphan drug designation was assigned to the compound in the US. for the treatment of stage IIB-IV melanoma

EpacadostatAn orally available hydroxyamidine and inhibitor of indoleamine 2,3-dioxygenase (IDO1), with potential immunomodulating and antineoplastic activities. epacadostat targets and binds to IDO1, an enzyme responsible for the oxidation of tryptophan into kynurenine. By inhibiting IDO1 and decreasing kynurenine in tumor cells, epacadostat increases and restores the proliferation and activation of various immune cells, including dendritic cells (DCs), NK cells, and T-lymphocytes, as well as interferon (IFN) production, and a reduction in tumor-associated regulatory T cells (Tregs). Activation of the immune system, which is suppressed in many cancers, may inhibit the growth of IDO1-expressing tumor cells. IDO1 is overexpressed by a variety of tumor cell types and DCsINCB24360 (epacadostat), An Agent For Cancer Immunotherapy

Incyte and Merck Expand Clinical Collaboration to Include Phase 3 Study Investigating the Combination of Epacadostat with Keytruda® (pembrolizumab) as First-line Treatment for Advanced Melanoma

Pivotal study to evaluate Incyte’s IDO1 inhibitor in combination with Merck’s anti-PD-1 therapy in patients with advanced or metastatic melanoma
WILMINGTON, Del. and KENILWORTH, N.J. -- October 13, 2015 -- Incyte Corporation (Nasdaq: INCY) and Merck (NYSE:MRK), known as MSD outside the United States and Canada, today announced the expansion of the companies’ ongoing clinical collaboration to include a Phase 3 study evaluating the combination of epacadostat, Incyte’s investigational selective IDO1 inhibitor, with Keytruda® (pembrolizumab), Merck’s anti-PD-1 therapy, as first-line treatment for patients with advanced or metastatic melanoma. The Phase 3 study, which is expected to begin in the first half of 2016, will be co-funded by Incyte and Merck.
“We are very pleased to expand our collaboration with Merck and to move the clinical development program for epacadostat in combination with Keytruda into Phase 3,” said Hervé Hoppenot, President and Chief Executive Officer of Incyte. “We believe the combination of these two immunotherapies shows promise and, if successfully developed, may help to improve clinical outcomes for patients with metastatic melanoma.”
“The initiation of this large Phase 3 study with Incyte in the first-line advanced melanoma treatment setting is an important addition to our robust immunotherapy clinical development program for Keytruda,” said Dr. Roger Dansey, senior vice president and therapeutic area head, oncology late-stage development, Merck Research Laboratories. “We continue to explore the benefit that Keytruda brings to patients suffering from advanced melanoma when used alone, and we are pleased to be able to add this important combination study with epacadostat to our Keytruda development program.”
Under the terms of the agreement Incyte and Merck have also agreed, for a period of two years, not to initiate new pivotal studies of an IDO1 inhibitor in combination with a PD-1/PD-L1 antagonist as first-line therapy in advanced or metastatic melanoma with any third party. During this time, the companies will each offer the other the opportunity to collaborate on any new pivotal study involving an IDO1 inhibitor in combination with a PD-1/PD-L1 antagonist for types of melanoma and lines of therapy outside of the current collaboration agreement.
The agreement is between Incyte and certain subsidiaries and Merck through its subsidiaries.
Epacadostat and Keytruda are part of a class of cancer treatments known as immunotherapies that are designed to enhance the body’s own defenses in fighting cancer; the two therapies target distinct regulatory components of the immune system. IDO1 is an immunosuppressive enzyme that has been shown to induce regulatory T cell generation and activation, and allow tumors to escape immune surveillance. Keytruda is a humanized monoclonal antibody that blocks the interaction between PD-1 and its ligands, PD-L1 and PD-L2. Preclinical evidence suggests that the combination of these two agents may lead to an enhanced anti-tumor immune response compared with either agent alone.
Safety and efficacy data from the ongoing Phase 1/2 study evaluating the combination of epacadostat with Keytruda in patients with advanced malignancies is scheduled to be highlighted as a late-breaking oral presentation (Abstract #142) at the upcoming Society for Immunotherapy of Cancer 30th Anniversary Annual Meeting & Associated Programs, November 4–8, 2015 at the Gaylord National Resort & Convention Center in National Harbor, MD.

Metastatic Melanoma

Melanoma, the most serious form of skin cancer, strikes adults of all ages and accounts for approximately five percent of all new cases of cancer in the United States each year. The number of new cases of melanoma continues to rise by almost three percent each year which translates to 76,000 new cases yearly in the U.S. alone.[i] The 5-year survival rate for late-stage or metastatic disease is 15 percent.[ii] 

About Epacadostat (INCB024360)

Indoleamine 2,3-dioxygenase 1 (IDO1) is an immunosuppressive enzyme that has been shown to induce regulatory T cell generation and activation, and allow tumors to escape immune surveillance. Epacadostat is an orally bioavailable small molecule inhibitor of IDO1 that has nanomolar potency in both biochemical and cellular assays and has demonstrated potent activity in enhancing T lymphocyte, dendritic cell and natural killer cell responses in vitro, with a high degree of selectivity. Epacadostat has shown proof-of-concept clinical data in patients with unresectable or metastatic melanoma in combination with the CTLA-4 inhibitor ipilimumab, and is currently in four proof-of-concept clinical trials with PD-1 and PD-L1 immune checkpoint inhibitors in a variety of cancer histologies.



PATENT
WO 2014066834
https://www.google.com/patents/WO2014066834A1?cl=en
EXAMPLE 1
4-({2-[(Aminosulfonyl)amino]ethyl}amino)- V-(3-bromo-4-fluorophenyl)- V -hydroxy- l,2,5-oxadiazole-3-carboximidamide

Step 1: 4-Amino-N'-hydroxy-l,2,5-oxadiazole-3-carboximidamide
[00184] Malononitrile (320.5 g, 5 mol) was added to water (7 L) preheated to 45 °C and stirred for 5 min. The resulting solution was cooled in an ice bath and sodium nitrite (380 g, 5.5 mol) was added. When the temperature reached 10 °C, 6 N hydrochloric acid (55 mL) was added. A mild exothermic reaction ensued with the temperature reaching 16 °C. After 15 min the cold bath was removed and the reaction mixture was stirred for 1.5 hrs at 16-18 °C. The reaction mixture was cooled to 13 °C and 50% aqueous hydroxylamine (990 g, 15 mol) was added all at once. The temperature rose to 26 °C. When the exothermic reaction subsided the cold bath was removed and stirring was continued for 1 hr at 26-27 °C, then it was slowly brought to reflux. Reflux was maintained for 2 hrs and then the reaction mixture was allowed to cool overnight. The reaction mixture was stirred in an ice bath and 6 N hydrochloric acid (800 mL) was added in portions over 40 min to pH 7.0. Stirring was continued in the ice bath at 5 °C. The precipitate was collected by filtration, washed well with water and dried in a vacuum oven (50 °C) to give the desired product (644 g, 90%). LCMS for C3H6N5O2
(M+H)+: m/z = 144.0. ¹³C MR (75 MHz, CD3OD): δ 156.0, 145.9, 141.3. Step 2: 4-Amino-N-hydroxy-l,2,5-oxadiazole-3-carboximidoyl chloride [00185] 4-Amino-N^,-hydroxy-l ,2,5-oxadiazole-3-carboximidamide (422 g, 2.95 mol) was added to a mixture of water (5.9 L), acetic acid (3 L) and 6 Ν hydrochloric acid (1.475 L, 3 eq.) and this suspension was stirred at 42 - 45 °C until complete solution was achieved. Sodium chloride (518 g, 3 eq.) was added and this solution was stirred in an ice/water/methanol bath. A solution of sodium nitrite (199.5 g, 0.98 eq.) in water (700 mL) was added over 3.5 hrs while maintaining the temperature below 0 °C. After complete addition stirring was continued in the ice bath for 1.5 hrs and then the reaction mixture was allowed to warm to 15 °C. The precipitate was collected by filtration, washed well with water, taken in ethyl acetate (3.4 L), treated with anhydrous sodium sulfate (500 g) and stirred for 1 hr. This suspension was filtered through sodium sulfate (200 g) and the filtrate was concentrated on a rotary evaporator. The residue was dissolved in methyl i-butyl ether (5.5 L), treated with charcoal (40 g), stirred for 40 min and filtered through Celite. The solvent was removed in a rotary evaporator and the resulting product was dried in a vacuum oven (45 °C) to give the desired product (256 g, 53.4%). LCMS for C3H4CIN4O2 (M+H)+: m/z = 162.9. ¹³C NMR (100 MHz, CD3OD): 5 155.8, 143.4, 129.7.
Step 3: 4-Amino-N'-hydroxy-N-(2-methoxyethyl)-l,2,5-oxadiazole-3-carboximidamide [00186] 4-Amino-N-hydroxy-l ,2,5-oxadiazole-3-carboximidoyl chloride (200.0 g, 1.23 mol) was mixed with ethyl acetate (1.2 L). At 0-5 °C 2-methoxyethylamine [Aldrich, product # 143693] (119.0 mL, 1.35 mol) was added in one portion while stirring. The reaction temperature rose to 41 °C. The reaction was cooled to 0 - 5 °C. Triethylamine (258 mL, 1.84 mol) was added. After stirring 5 min, LCMS indicated reaction completion. The reaction solution was washed with water (500 mL) and brine (500 mL), dried over sodium sulfate, and concentrated to give the desired product (294 g, 1 19%) as a crude dark oil.
LCMS for C₆Hi_{2 5}0₃ (M+H)⁺: m/z = 202.3. 1H NMR (400 MHz, DMSO- ): δ 10.65 (s, 1 H), 6.27 (s, 2 H), 6.10 (t, J = 6.5 Hz, 1 H), 3.50 (m, 2 H), 3.35 (d, J = 5.8 Hz, 2 H), 3.08 (s, 3 H).
Step 4: N'-Hydroxy-4-[(2-methoxyethyl)amino]-l,2,5-oxadiazole-3-carboximidamide
[00187] 4-Amino-N-hydroxy-N-(2-methoxyethyl)-l,2,5-oxadiazole-3- carboximidamide (248.0 g, 1.23 mol) was mixed with water (1 L). Potassium hydroxide (210 g, 3.7 mol) was added. The reaction was refluxed at 100 °C overnight (15 hours). TLC with 50% ethyl acetate (containing 1% ammonium hydroxide) in hexane indicated reaction completed (product Rf = 0.6, starting material Rf = 0.5). LCMS also indicated reaction completion. The reaction was cooled to room temperature and extracted with ethyl acetate (3 x 1 L). The combined ethyl acetate solution was dried over sodium sulfate and concentrated to give the desired product (201 g, 81%) as a crude off-white solid. LCMS for C6H12N5O3 (M+H)⁺: m/z = 202.3 ^LH NMR (400 MHz, OMSO-d₆): δ 10.54 (s, 1 H), 6.22 (s, 2 H), 6.15 (t, J = 5.8 Hz, 1 H), 3.45 (t, J= 5.3 Hz, 2 H), 3.35 (m, 2 H), 3.22 (s, 3 H). Step 5: N-Hydroxy-4-[(2-methoxyethyl)amino]-l,2,5-oxadiazole-3-carboximidoyl chloride
[00188] At room temperature N'-hydroxy-4-[(2-methoxyethyl)amino]- 1 ,2,5- oxadiazole-3-carboximidamide (50.0 g, 0.226 mol) was dissolved in 6.0 M hydrochloric acid aqueous solution (250 mL, 1.5 mol). Sodium chloride (39.5 g, 0.676 mol) was added followed by water (250 mL) and ethyl acetate (250 mL). At 3-5 °C a previously prepared aqueous solution (100 mL) of sodium nitrite (15.0 g, 0.217 mol) was added slowly over 1 hr. The reaction was stirred at 3 - 8 °C for 2 hours and then room temperature over the weekend. LCMS indicated reaction completed. The reaction solution was extracted with ethyl acetate (2 x 200 mL). The combined ethyl acetate solution was dried over sodium sulfate and concentrated to give the desired product (49.9 g, 126%) as a crude white solid. LCMS for
C₆HioClN₄03 (M+H)⁺: m/z = 221.0. ^!H NMR (400 MHz, DMSO-d₆): δ 13.43 (s, 1 H), 5.85 (t, J= 5.6 Hz, 1 H), 3.50 (t, J= 5.6 Hz, 2 H), 3.37(dd, J= 10.8, 5.6 Hz, 2 H), 3.25 (s, 3 H).
Step 6 : N-(3-Bromo-4-fluorophenyl)-N'-hydroxy-4- [(2-methoxyethyl)amino] - 1 ,2,5- oxadiazole-3-carboximidamide [00189] N-Hydroxy-4-[(2-methoxyethyl)amino]- 1 ,2,5-oxadiazole-3-carboximidoyl chloride (46.0 g, 0.208 mol) was mixed with water (300 mL). The mixture was heated to 60 °C. 3-Bromo-4-fluoroaniline [Oakwood products, product # 013091] (43.6 g, 0.229 mol) was added and stirred for 10 min. A warm sodium bicarbonate (26.3 g, 0.313 mol) solution (300 mL water) was added over 15 min. The reaction was stirred at 60 °C for 20 min. LCMS indicated reaction completion. The reaction solution was cooled to room temperature and extracted with ethyl acetate (2 x 300 mL). The combined ethyl acetate solution was dried over sodium sulfate and concentrated to give the desired product (76.7 g, 98%) as a crude brown solid. LCMS for Ci₂Hi₄BrF₅0₃ (M+H)⁺: m/z = 374.0, 376.0. 1H NMR (400 MHz, DMSO- t_f): δ 11.55 (s, 1 H), 8.85 (s, 1 H), 7.16 (t, J= 8.8 Hz, 1 H), 7.08 (dd, J= 6.1, 2.7 Hz, 1 H), 6.75 (m, 1 H), 6.14 (t, J= 5.8 Hz, 1 H), 3.48 (t, J = 5.2 Hz, 2 H), 3.35 (dd, J= 10.8, 5.6 Hz, 2 H), 3.22 (s, 3 H).
Step 7: 4-(3-Bromo-4-fluorophenyl)-3-{4- [(2-methoxyethyl)amino]-l,2,5-oxadiazol-3- yl}-l,2,4-oxadiazol-5(4H)-one
[00190] A mixture of N-(3-bromo-4-fluorophenyl)-N'-hydroxy-4-[(2- methoxyethyl)amino]-l,2,5-oxadiazole-3-carboximidamide (76.5 g, 0.204 mol), 1,1 '- carbonyldiimidazole (49.7 g, 0.307 mol), and ethyl acetate (720 mL) was heated to 60 °C and stirred for 20 min. LCMS indicated reaction completed. The reaction was cooled to room temperature, washed with 1 N HC1 (2 x 750 mL), dried over sodium sulfate, and concentrated to give the desired product (80.4 g, 98%) as a crude brown solid. LCMS for

(M+H)⁺: m/z = 400.0, 402.0. 1H NMR (400 MHz, DMSO-c½): δ 7.94 (t, J = 8.2 Hz, 1 H), 7.72 (dd, J = 9.1, 2.3 Hz, 1 H), 7.42 (m, 1 H), 6.42 (t, J= 5.7 Hz, 1 H), 3.46 (t, J = 5.4 Hz, 2 H), 3.36 (t, J= 5.8 Hz, 2 H), 3.26 (s, 3 H).
Step 8: 4-(3-Bromo-4-fluorophenyl)-3-{4-[(2-hydroxyethyl)amino]-l,2,5-oxadiazol-3- yl}-l,2,4-oxadiazol-5(4H)-one
[00191] 4-(3-Bromo-4-fluoroplienyl)-3-{4-[(2-metlioxyethyl)amino]-l,2,5-oxadiazol- 3-yl}-l,2,4-oxadiazol-5(4H)-one (78.4 g, 0.196 mol) was dissolved in dichloromethane (600 mL). At -67 °C boron tribromide (37 mL, 0.392 mol) was added over 15 min. The reaction was warmed up to -10 °C in 30 min. LCMS indicated reaction completed. The reaction was stirred at room temperature for 1 hour. At 0 - 5 °C the reaction was slowly quenched with saturated sodium bicarbonate solution (1.5 L) over 30 min. The reaction temperature rose to 25 °C. The reaction was extracted with ethyl acetate (2 x 500 mL, first extraction organic layer is on the bottom and second extraction organic lager is on the top). The combined organic layers were dried over sodium sulfate and concentrated to give the desired product (75 g, 99%) as a crude brown solid. LCMS for Ci₂HioBrFN₅0₄ (M+H)⁺: m/z = 386.0, 388.0.
1H NMR (400 MHz, DMSO-^): δ 8.08 (dd, J = 6.2, 2.5 Hz, 1 H), 7.70 (m, 1 H), 7.68 (t, J = 8.7 Hz, 1 H), 6.33 (t, J = 5.6 Hz, 1 H), 4.85 (t, J= 5.0 Hz, 1 H), 3.56 (dd, J= 10.6, 5.6 Hz, 2 H), 3.29 (dd, J= 11.5, 5.9 Hz, 2 H).
Step 9 : 2-({4- [4-(3-Bromo-4-fluorophenyl)-5-oxo-4,5-dihydro- 1 ,2,4-oxadiazol-3-yl] - l,2,5-oxadiazol-3-yl}amino)ethyl methanesulfonate
[00192] To a solution of 4-(3-bromo-4-fluorophenyl)-3-{4-[(2-hydroxyethyl)amino]- l,2,5-oxadiazol-3-yl}-l,2,4-oxadiazol-5(4H)-one (1.5 kg, 3.9 mol, containing also some of the corresponding bromo-compound) in ethyl acetate (12 L) was added methanesulfonyl chloride (185 mL, 2.4 mol) dropwise over 1 h at room temperature. Triethylamine (325 mL, 2.3 mol) was added dropwise over 45 min, during which time the reaction temperature increased to 35 °C. After 2 h, the reaction mixture was washed with water (5 L), brine (1 L), dried over sodium sulfate, combined with 3 more reactions of the same size, and the solvents removed in vacuo to afford the desired product (7600 g, quantitative yield) as a tan solid. LCMS for C HnBrFNsOeS a (M+Na)⁺: m/z = 485.9, 487.9. ^!H NMR (400 MHz, DMSO- d₆): δ 8.08 (dd, J = 6.2, 2.5 Hz, 1 H), 7.72 (m, 1 H), 7.58 (t, J = 8.7 Hz, 1 H), 6.75 (t, J = 5.9 Hz, 1 H), 4.36 (t, J = 5.3 Hz, 2 H), 3.58 (dd, J = 11.2, 5.6 Hz, 2 H), 3.18 (s, 3 H).
Step 10: 3-{4-[(2-Azidoethyl)amino]-l,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)- l,2,4-oxadiazol-5(4H)-one
To a solution of 2-({4-[4-(3-bromo-4-f uorophenyl)-5-oxo-4,5-dihydro-l ,2,4- oxadiazol-3-yl]-l ,2,5-oxadiazol-3-yl}amino)ethyl methanesulfonate (2.13 kg, 4.6 mol, containing also some of the corresponding bromo-compound) in dimethylformamide (4 L) stirring in a 22 L flask was added sodium azide (380 g, 5.84 mol). The reaction was heated at 50 °C for 6 h, poured into ice/water (8 L), and extracted with 1 : 1 ethyl acetate:heptane (20 L). The organic layer was washed with water (5 L) and brine (5 L), and the solvents removed in vacuo to afford the desired product (1464 g, 77%) as a tan solid. LCMS for CnHgBrFNsOs a
(M+Na)⁺: m/z = 433.0, 435.0. ^!H NMR (400 MHz, DMSO-J₆): δ 8.08 (dd, J = 6.2, 2.5 Hz, 1 H), 7.72 (m, 1 H), 7.58 (t, J= 8.7 Hz, 1 H), 6.75 (t, J = 5.7 Hz, 1 H), 3.54 (t, J = 5.3 Hz, 2 H), 3.45 (dd, J= 1 1.1 , 5.2 Hz, 2 H).
Step 11: 3-{4-[(2-Aminoethyl)amino]-l,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)-
1.2.4- oxadiazol-5(4H)-one hydrochloride
[00194] Sodium iodide (1080 g, 7.2 mol) was added to 3-{4-[(2-azidoethyl)amino]-
1.2.5- oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)-l ,2,4-oxadiazol-5(4H)-one (500 g, 1.22 mol) in methanol (6 L). The mixture was allowed to stir for 30 min during which time a mild exotherm was observed. Chlorotrimethylsilane (930 mL, 7.33 mol) was added as a solution in methanol (1 L) dropwise at a rate so that the temperature did not exceed 35 °C, and the reaction was allowed to stir for 3.5 h at ambient temperature. The reaction was neutralized with 33 wt% solution of sodium thiosulfate pentahydrate in water (-1.5 L), diluted with water (4 L), and the pH adjusted to 9 carefully with solid potassium carbonate (250 g - added in small portions: watch foaming). Di-ieri-butyl dicarbonate (318 g, 1.45 mol) was added and the reaction was allowed to stir at room temperature. Additional potassium carbonate (200 g) was added in 50 g portions over 4 h to ensure that the pH was still at or above 9. After stirring at room temperature overnight, the solid was filtered, triturated with water (2 L), and then MTBE (1.5 L). A total of 11 runs were performed (5.5 kg, 13.38 mol). The combined solids were triturated with 1 : 1 THF:dichloromethane (24 L, 4 runs in a 20 L rotary evaporator flask, 50 °C, 1 h), filtered, and washed with dichloromethane (3 L each run) to afford an off- white solid. The crude material was dissolved at 55 °C tetrahydrofuran (5 mL/g), treated with decolorizing carbon (2 wt%) and silica gel (2 wt%), and filtered hot through celite to afford the product as an off-white solid (5122 g). The combined MTBE, THF, and dichloromethane filtrates were concentrated in vacuo and chromatographed (2 kg silica gel, heptane with a 0-100% ethyl acetate gradient, 30 L) to afford more product (262 g). The combined solids were dried to a constant weight in a convection oven (5385 g, 83%).
In a 22 L flask was charged hydrogen chloride (4 N solution in 1 ,4-dioxane, 4 L, 16 mol). tert-Butyl [2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-l ,2,4- oxadiazol-3-yl]-l ,2,5-oxadiazol-3-yl}amino)ethyl]carbamate (2315 g, 4.77 mol) was added as a solid in portions over 10 min. The slurry was stirred at room temperature and gradually became a thick paste that could not be stirred. After sitting overnight at room temperature, the paste was slurried in ethyl acetate (10 L), filtered, re-slurried in ethyl acetate (5 L), filtered, and dried to a constant weight to afford the desired product as a white solid (combined with other runs, 5 kg starting material charged, 41 13 g, 95%). LCMS for
Ci₂H_nBrFN₆0₃ (M+H)⁺: m/z = 384.9, 386.9. 1H NMR (400 MHz, DMSO-^): δ 8.12 (m, 4 H), 7.76 (m, 1 H), 7.58 (t, J = 8.7 Hz, 1 H), 6.78 (t, J = 6.1 Hz, 1 H), 3.51 (dd, J = 1 1.8, 6.1 Hz, 2 H), 3.02 (m, 2 H).
Step 12: tert-Butyl ({[2-({4-[4-(3-bromo-4-nuorophenyl)-5-oxo-4,5-dihydro-l,2,4- oxadiazol-3-yl]-l,2,5-oxadiazol-3-yl}amino)ethyl]amino}sulfonyl)carbamate
A 5 L round bottom flask was charged with chlorosulfonyl isocyanate [Aldrich, product # 142662] (149 mL, 1.72 mol) and dichloromethane (1.5 L) and cooled using an ice bath to 2 °C. teri-Butanol (162 mL, 1.73 mol) in dichloromethane (200 mL) was added dropwise at a rate so that the temperature did not exceed 10 °C. The resulting solution was stirred at room temperature for 30-60 min to provide tert-bvAy\ [chlorosulfonyl]carbamate.
A 22 L flask was charged with 3- {4-[(2-aminoethyl)amino]- 1 ,2,5-oxadiazol-3- yl}-4-(3-bromo-4-fluorophenyl)-l,2,4-oxadiazol-5(4H)-one hydrochloride (661 g, 1.57 mol) and 8.5 L dichloromethane. After cooling to -15 °C with an ice/salt bath, the solution oi tert- Vmtvl i Vi 1 r>rosulfonyl]carbamate (prepared as above) was added at a rate so that the temperature did not exceed -10 °C (addition time 7 min). After stirring for 10 min, triethylamine (1085 mL, 7.78 mol) was added at a rate so that the temperature did not exceed -5 °C (addition time 10 min). The cold bath was removed, the reaction was allowed to warm to 10 °C, split into two portions, and neutralized with 10% cone HC1 (4.5 L each portion). Each portion was transferred to a 50 L separatory funnel and diluted with ethyl acetate to completely dissolve the white solid (-25 L). The layers were separated, and the organic layer was washed with water (5 L), brine (5 L), and the solvents removed in vacuo to afford an off- white solid. The solid was triturated with MTBE (2 x 1.5 L) and dried to a constant weight to afford a white solid. A total of 4113 g starting material was processed in this manner (5409 g, 98%). 1H NMR (400 MHz, DMSO-^): δ 10.90 (s, 1 H), 8.08 (dd, J = 6.2, 2.5 Hz, 1 H), 7.72 (m, 1 H), 7.59 (t, J = 8.6 Hz, 1 H), 6.58 (t, J = 5.7 Hz, 1 H), 3.38 (dd, J= 12.7, 6.2 Hz, 2 H), 3.10 (dd, J= 12.1 , 5.9 Hz, 2 H), 1.41 (s, 9 H).
Step 13: N-[2-({4-[4-(3-Bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-l,2,4-oxadiazol-3-yl]- l,2,5-oxadiazol-3-yl}amino)ethyl]sulfamide
[00198] To a 22 L flask containing 98:2 trifluoroacetic acid:water (8.9 L) was added tert-bvXyl ({[2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-l,2,4-oxadiazol-3-yl]- l,2,5-oxadiazol-3-yl}amino)ethyl]amino}sulfonyl)carbamate (1931 g, 3.42 mol) in portions over 10 minutes. The resulting mixture was stirred at room temperature for 1.5 h, the solvents removed in vacuo, and chased with dichloromethane (2 L). The resulting solid was treated a second time with fresh 98:2 trifluoroacetic acid:water (8.9 L), heated for 1 h at 40- 50 °C, the solvents removed in vacuo, and chased with dichloromethane (3 x 2 L). The resulting white solid was dried in a vacuum drying oven at 50 °C overnight. A total of 5409 g was processed in this manner (4990 g, quant, yield). LCMS for C₁₂H₁₂BrFN₇0₅S (M+H)⁺: m/z = 463.9, 465.9. 1H NMR (400 MHz, DMSO- ): δ 8.08 (dd, J = 6.2, 2.5 Hz, 1 H), 7.72 (m, 1 H), 7.59 (t, J= 8.7 Hz, 1 H), 6.67 (t, J = 5.9 Hz, 1H), 6.52 (t, J= 6.0 Hz, 1 H), 3.38 (dd, J = 12.7, 6.3 Hz, 2 H), 3.11 (dd, J = 12.3, 6.3 Hz). Step 14: 4-({2-[(Aminosulfonyl)amino]ethyl}amino)-N-(3-bromo-4-fluorophenyl)-N'- hydroxy-l,2,5-oxadiazole-3-carboximidamide

[00199] To a crude mixture of N-[2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5- dihydro-l,2,4-oxadiazol-3-yl]-l,2,5-oxadiazol-3-yl}amino)ethyl]sulfamide (2.4 mol) containing residual amounts of trifluoroacetic acid stirring in a 22 L flask was added THF (5 L). The resulting solution was cooled to 0 °C using an ice bath and 2 N NaOH (4 L) was added at a rate so that the temperature did not exceed 10 °C. After stirring at ambient temperature for 3 h (LCMS indicated no starting material remained), the pH was adjusted to 3-4 with concentrated HC1 (-500 mL). The THF was removed in vacuo, and the resulting mixture was extracted with ethyl acetate (15 L). The organic layer was washed with water (5 L), brine (5 L), and the solvents removed in vacuo to afford a solid. The solid was triturated with MTBE (2 x 2 L), combined with three other reactions of the same size, and dried overnight in a convection oven to afford a white solid (3535 g). The solid was recrystallized (3 x 22 L flasks, 2:1 watenethanol, 14.1 L each flask) and dried in a 50 °C convection oven to a constant weight to furnish the title compound as an off-white solid (3290 g, 78%). LCMS for CnHnBrF yC S (M+H)⁺: m/z = 437.9, 439.9. i NMR (400 MHz, DMSO-J^): δ 11.51 (s, 1 H), 8.90 (s, 1 H), 7.17 (t, J= 8.8 Hz, 1 H), 7.11 (dd, J= 6.1, 2.7 Hz, 1 H), 6.76 (m, 1 H), 6.71 (t, J = 6.0 Hz, 1 H), 6.59 (s, 2 H), 6.23 (t, J= 6.1 Hz, 1 H), 3.35 (dd, J= 10.9, 7.0 Hz, 2 H), 3.10 (dd, J= 12.1, 6.2 Hz, 2 H).
PATENT
WO 2010005958
https://www.google.com/patents/WO2010005958A2?cl=en
EXAMPLES Example 1
4-({2-[(Aminosulfonyl)amino]ethyl}amino)-7V-(3-bromo-4-fluorophenyl)-iV'-hydroxy- l,2,5-oxadiazole-3-carboximidamide

Step A: 4-Amino-N'-hydroxy-l,2,5-oxadiazole-3-carboximidamide

Malononitrile [Aldrich, product # M1407] (320.5 g, 5 mol) was added to water (7 L) preheated to 45 ⁰C and stirred for 5 min. The resulting solution was cooled in an ice bath and sodium nitrite (380 g, 5.5 mol) was added. When the temperature reached 10 ⁰C, 6 N hydrochloric acid (55 mL) was added. A mild exothermic reaction ensued with the temperature reaching 16 ⁰C. After 15 min the cold bath was removed and the reaction mixture was stirred for 1.5 hrs at 16-18 ⁰C. The reaction mixture was cooled to 13 ⁰C and 50% aqueous hydroxylamine (990 g, 15 mol) was added all at once. The temperature rose to 26 ⁰C. When the exothermic reaction subsided the cold bath was removed and stirring was continued for 1 hr at 26-27⁰C, then it was slowly brought to reflux. Reflux was maintained for 2 hrs and then the reaction mixture was allowed to cool overnight. The reaction mixture was stirred in an ice bath and 6 N hydrochloric acid (800 mL) was added in portions over 40 min to pH 7.0. Stirring was continued in the ice bath at 5 ⁰C. The precipitate was collected by filtration, washed well with water and dried in a vacuum oven (50 ⁰C) to give the desired product (644 g, 90%). LCMS for C₃H₆N₅O₂ (M+H)+: m/z = 144.0. ¹³C NMR (75 MHz, CD₃OD): δ 156.0, 145.9, 141.3. Step B: 4-Amino-N-hydroxy-l,2,5-oxadiazole-3-carboximidoyl chloride

4-Amino-N'-hydroxy-l,2,5-oxadiazole-3-carboximidamide (422 g, 2.95 mol) was added to a mixture of water (5.9 L), acetic acid (3 L) and 6 Ν hydrochloric acid (1.475 L, 3 eq.) and this suspension was stirred at 42 - 45 ⁰C until complete solution was achieved. Sodium chloride (518 g, 3 eq.) was added and this solution was stirred in an ice/water/methanol bath. A solution of sodium nitrite (199.5 g, 0.98 eq.) in water (700 mL) was added over 3.5 hrs while maintaining the temperature below 0 ⁰C. After complete addition stirring was continued in the ice bath for 1.5 hrs and then the reaction mixture was allowed to warm to 15 ⁰C. The precipitate was collected by filtration, washed well with water, taken in ethyl acetate (3.4 L), treated with anhydrous sodium sulfate (500 g) and stirred for 1 hr. This suspension was filtered through sodium sulfate (200 g) and the filtrate was concentrated on a rotary evaporator. The residue was dissolved in methyl f-butyl ether (5.5 L), treated with charcoal (40 g), stirred for 40 min and filtered through Celite. The solvent was removed in a rotary evaporator and the resulting product was dried in a vacuum oven (45 ⁰C) to give the desired product (256 g, 53.4%). LCMS for C₃H₄ClN₄O₂(M+H)+: m/z = 162.9. 13c NMR (100 MHz, CD₃OD): δ 155.8, 143.4, 129.7.
Step C: 4-Amino-N'-hydroxy-N-(2-methoxyethyl)- 1 ,2,5-oxadiazole-3-carboximidamide

4-Amino-N-hydroxy-l,2,5-oxadiazole-3-carboximidoyl chloride (200.0 g, 1.23 mol) was mixed with ethyl acetate (1.2 L). At 0-5⁰C 2-methoxyethylamine [Aldrich, product # 143693] (119.0 mL, 1.35 mol) was added in one portion while stirring. The reaction temperature rose to 41 ⁰C. The reaction was cooled to 0 - 5 °C. Triethylamine (258 mL, 1.84 mol) was added. After stirring 5 min, LCMS indicated reaction completion. The reaction solution was washed with water (500 mL) and brine (500 mL), dried over sodium sulfate, and concentrated to give the desired product (294 g, 119%) as a crude dark oil. LCMS for C₆Hi₂N₅O₃ (M+H)⁺: m/z = 202.3. ¹H NMR (400 MHz, DMSO-J₆): δ 10.65 (s, 1 H), 6.27 (s, 2 H), 6.10 (t, J= 6.5 Hz, 1 H), 3.50 (m, 2 H), 3.35 (d, J= 5.8 Hz, 2 H), 3.08 (s, 3 H).
Step D: N'-Hydroxy-4-[(2-methoxyethyl)amino]-l ,2,5-oxadiazole-3-carboximidamide

4-Amino-N'-hydroxy-N-(2-methoxyethyl)-l,2,5-oxadiazole-3-carboximidaniide (248.0 g, 1.23 mol) was mixed with water (1 L). Potassium hydroxide (210 g, 3.7 mol) was added. The reaction was refluxed at 100 ⁰C overnight (15 hours). TLC with 50% ethyl acetate (containing 1% ammonium hydroxide) in hexane indicated reaction completed (product Rf= 0.6, starting material Rf = 0.5). LCMS also indicated reaction completion. The reaction was cooled to room temperature and extracted with ethyl acetate (3 x 1 L). The combined ethyl acetate solution was dried over sodium sulfate and concentrated to give the desired product (201 g, 81%) as a crude off-white solid. LCMS for C₆H₁₂N₅O₃ (M+H)⁺: m/z = 202.3 ¹H NMR (400 MHz, DMSO-Gk): δ 10.54 (s, 1 H), 6.22 (s, 2 H), 6.15 (t, J= 5.8 Hz, 1 H), 3.45 (t, J= 5.3 Hz, 2 H), 3.35 (m, 2 H), 3.22 (s, 3 H).
Step E: N-Hydroxy-4-[(2-methoxyethyl)amino]-l,2,5-oxadiazole-3-carboximidoyl chloride

Ν. ,Ν O
At room temperature N'-hydroxy-4-[(2-methoxyethyl)amino]-l,2,5-oxadiazole-3- carboximidamide (50.0 g, 0.226 mol) was dissolved in 6.0 M hydrochloric acid aqueous solution (250 mL, 1.5 mol). Sodium chloride (39.5 g, 0.676 mol) was added followed by water (250 mL) and ethyl acetate (250 mL). At 3-5 ⁰C a previously prepared aqueous solution (100 mL) of sodium nitrite (15.0 g, 0.217 mol) was added slowly over 1 hr. The reaction was stirred at 3 - 8 ⁰C for 2 hours and then room temperature over the weekend. LCMS indicated reaction completed. The reaction solution was extracted with ethyl acetate (2 x 200 mL). The combined ethyl acetate solution was dried over sodium sulfate and concentrated to give the desired product (49.9 g, 126%) as a crude white solid. LCMS for C₆Hi₀ClN₄O₃ (M+H)⁺: m/z = 221.0. ¹H NMR (400 MHz, DMSO-J₆): δ 13.43 (s, 1 H), 5.85 (t, J= 5.6 Hz, 1 H), 3.50 (t, J= 5.6 Hz, 2 H), 3.37(dd, J= 10.8, 5.6 Hz, 2 H), 3.25 (s, 3 H).
Step F: N-(3-Bromo-4-fluorophenyl)-N'-hydroxy-4-[(2-methoxyethyl)amino]- 1 ,2,5- oxadiazole-3 -carboximidamide

N-Hydroxy-4-[(2-methoxyethyl)amino]-l,2,5-oxadiazole-3-carboximidoyl chloride (46.0 g, 0.208 mol) was mixed with water (300 mL). The mixture was heated to 60 °C. 3-Bromo-4- fluoroaniline [Oakwood products, product # 013091] (43.6 g, 0.229 mol) was added and stirred for 10 nrn^'n. A warm sodium bicarbonate (26.3 g, 0.313 mol) solution (300 mL water) was added over 15 min. The reaction was stirred at 60 ⁰C for 20 min. LCMS indicated reaction completion. The reaction solution was cooled to room temperature and extracted with ethyl acetate (2 x 300 mL). The combined ethyl acetate solution was dried over sodium sulfate and concentrated to give the desired product (76.7 g, 98%) as a crude brown solid. LCMS for Ci₂Hi₄BrFN₅O₃ (M+H)⁺: m/z = 374.0, 376.0. ¹H NMR (400 MHz, DMSO-J₆): δ 11.55 (s, 1 H), 8.85 (s, 1 H), 7.16 (t, J= 8.8 Hz, 1 H), 7.08 (dd, J= 6.1, 2.7 Hz, 1 H), 6.75 (m, 1 H), 6.14 (t, J= 5.8 Hz, 1 H), 3.48 (t, J= 5.2 Hz, 2 H), 3.35 (dd, J= 10.8, 5.6 Hz, 2 H), 3.22 (s, 3 H).
Step G: 4-(3-Bromo-4-fluorophenyl)-3-{4-[(2-methoxyethyl)amino]-l,2,5-oxadiazol-3-yl}- 1 ,2,4-oxadiazol-5(4H)-one

A mixture of N-(3-bromo-4-fluorophenyl)-N'-hydroxy-4-[(2-methoxyethyl)amino]-l,2,5- oxadiazole-3-carboximidamide (76.5 g, 0.204 mol), l,r-carbonyldiimidazole (49.7 g, 0.307 mol), and ethyl acetate (720 mL) was heated to 60 ⁰C and stirred for 20 min. LCMS indicated reaction completed. The reaction was cooled to room temperature, washed with 1 Ν HCl (2 x 750 mL), dried over sodium sulfate, and concentrated to give the desired product (80.4 g, 98%) as a crude brown solid. LCMS for C₁₃H₁₂BrFN₅O₄ (M+H)⁺: m/z = 400.0, 402.0. ¹H NMR (400 MHz, OMSO-d₆): δ 7.94 (t, J= 8.2 Hz, 1 H), 7.72 (dd, J= 9.1, 2.3 Hz, 1 H), 7.42 (m, 1 H), 6.42 (t, J= 5.7 Hz, 1 H), 3.46 (t, J= 5.4 Hz, 2 H), 3.36 (t, J= 5.8 Hz, 2 H), 3.26 (s, 3 H).
Step H: 4-(3-Bromo-4-fluorophenyl)-3-{4-[(2-liydroxyethyl)amino]-l,2,5-oxadiazol-3-yl}- 1 ,2,4-oxadiazol-5(4H)-one

4-(3-Bromo-4-fluorophenyl)-3-{4-[(2-methoxyetliyl)amino]-l,2,5-oxadiazol-3-yl}-l,2,4- oxadiazol-5(4H)-one (78.4 g, 0.196 mol) was dissolved in dichloromethane (600 mL). At -67 ⁰C boron tribromide (37 mL, 0.392 mol) was added over 15 min. The reaction was warmed up to -10 ⁰C in 30 min. LCMS indicated reaction completed. The reaction was stirred at room temperature for 1 hour. At 0 - 5 ⁰C the reaction was slowly quenched with saturated sodium bicarbonate solution (1.5 L) over 30 min. The reaction temperature rose to 25 ⁰C. The reaction was extracted with ethyl acetate (2 x 500 mL, first extraction organic layer is on the bottom and second extraction organic lager is on the top). The combined organic layers were dried over sodium sulfate and concentrated to give the desired product (75 g, 99%) as a crude brown solid. LCMS for C₁₂H₁₀BrFN₅O₄ (M+H)⁺: m/z = 386.0, 388.0. ¹H NMR (400 MHz, DMSO-^₆): δ 8.08 (dd, J= 6.2, 2.5 Hz, 1 H), 7.70 (m, 1 H), 7.68 (t, J= 8.7 Hz, 1 H), 6.33 (t, J= 5.6 Hz, 1 H), 4.85 (t, J= 5.0 Hz, 1 H), 3.56 (dd, J= 10.6, 5.6 Hz, 2 H), 3.29 (dd, J= 11.5, 5.9 Hz, 2 H).
Step I: 2-({4-[4-(3-Bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-l,2,4-oxadiazol-3-yl]-l,2,5- oxadiazol-3-yl}amino)ethyl methanesulfonate

To a solution of 4-(3-bromo-4-fluorophenyl)-3-{4-[(2-hydroxyethyl)amino]-l,2,5-oxadiazol- 3-yl}-l,2,4-oxadiazol-5(4H)-one (1.5 kg, 3.9 mol, containing also some of the corresponding bromo-compound) in ethyl acetate (12 L) was added methanesulfonyl chloride (185 mL, 2.4 mol) dropwise over 1 h at room temperature. Triethylamine (325 mL, 2.3 mol) was added dropwise over 45 min, during which time the reaction temperature increased to 35 ⁰C. After 2 h, the reaction mixture was washed with water (5 L), brine (I L), dried over sodium sulfate, combined with 3 more reactions of the same size, and the solvents removed in vacuo to afford the desired product (7600 g, quantitative yield) as a tan solid. LCMS for
Ci₃HnBrFN₅O₆SNa (M+Na)⁺: m/z = 485.9, 487.9. ¹H NMR (400 MHz, DMSCW₆): δ 8.08 (dd, J= 6.2, 2.5 Hz, 1 H), 7.72 (m, 1 H), 7.58 (t, J= 8.7 Hz, 1 H), 6.75 (t, J- 5.9 Hz, 1 H), 4.36 (t, J= 5.3 Hz, 2 H), 3.58 (dd, J= 11.2, 5.6 Hz, 2 H), 3.18 (s, 3 H).
Step J: 3-{4-[(2-Azidoethyl)amino]-l,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)- 1 ,2,4-oxadiazol-5(4H)-one

To a solution of 2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-l,2,4-oxadiazol-3-yl]- l,2,5-oxadiazol-3-yl}amino)ethyl methanesulfonate (2.13 kg, 4.6 mol, containing also some of the corresponding bromo-compound) in dimethylformamide (4 L) stirring in a 22 L flask was added sodium azide (380 g, 5.84 mol). The reaction was heated at 50⁰C for 6 h, poured into ice/water (8 L), and extracted with 1 : 1 ethyl acetate:heptane (20 L). The organic layer was washed with water (5 L) and brine (5 L), and the solvents removed in vacuo to afford the desired product (1464 g, 77%) as a tan solid. LCMS for C₁₂H₈BrFN₈O₃Na (M+Na)⁺: m/z =
433.0, 435.0. ¹H NMR (400 MHz, DMSO-*/*): δ 8.08 (dd, J= 6.2, 2.5 Hz, 1 H), 7.72 (m, 1 H), 7.58 (t, J= 8.7 Hz, 1 H), 6.75 (t, J= 5.7 Hz, 1 H), 3.54 (t, J= 5.3 Hz, 2 H), 3.45 (dd, J= 11.1, 5.2 Hz, 2 H).
Step K: 3-{4-[(2-Aminoethyl)amino]-l,2,5-oxadiazol-3-yl}-4-(3-bromo-4-fluorophenyl)- 1 ,2,4-oxadiazol-5(4H)-one hydrochloride

Sodium iodide (1080 g, 7.2 mol) was added to 3-{4-[(2-azidoethyl)amino]-l,2,5-oxadiazol-3- yl}-4-(3-bromo-4-fluorophenyl)-l,2,4-oxadiazol-5(4H)-one (500 g, 1.22 mol) in methanol (6 L). The mixture was allowed to stir for 30 min during which time a mild exotherm was observed. Chlorotrimethylsilane (930 mL, 7.33 mol) was added as a solution in methanol (1 L) dropwise at a rate so that the temperature did not exceed 35 ⁰C, and the reaction was allowed to stir for 3.5 h at ambient temperature. The reaction was neutralized with 33 wt% solution of sodium thiosulfate pentahydrate in water (~1.5 L), diluted with water (4 L), and the pΗ adjusted to 9 carefully with solid potassium carbonate (250 g - added in small portions: watch foaming). Di-fe/t-butyl dicarbonate (318 g, 1.45 mol) was added and the reaction was allowed to stir at room temperature. Additional potassium carbonate (200 g) was added in 50 g portions over 4 h to ensure that the pΗ was still at or above 9. After stirring at room temperature overnight, the solid was filtered, triturated with water (2 L), and then MTBE (1.5 L). A total of 11 runs were performed (5.5 kg, 13.38 mol). The combined solids were triturated with 1 : 1 TΗF:dichloromethane (24 L, 4 runs in a 20 L rotary evaporator flask, 50 ⁰C, 1 h), filtered, and washed with dichloromethane (3 L each run) to afford an off- white solid. The crude material was dissolved at 55 ⁰C tetrahydrofuran (5 mL/g), treated with decolorizing carbon (2 wt%) and silica gel (2 wt%), and filtered hot through celite to afford the product as an off-white solid (5122 g). The combined MTBE, THF, and dichloromethane filtrates were concentrated in vacuo and chromatographed (2 kg silica gel, heptane with a 0-100% ethyl acetate gradient, 30 L) to afford more product (262 g). The combined solids were dried to a constant weight in a convection oven (5385 g, 83%).
In a 22 L flask was charged hydrogen chloride (4 N solution in 1,4-dioxane, 4 L, 16 mol). fert-Butyl [2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-l,2,4-oxadiazol-3-yl]- l,2,5-oxadiazol-3-yl}amino)ethyl]carbamate (2315 g, 4.77 mol) was added as a solid in portions over 10 min. The slurry was stirred at room temperature and gradually became a thick paste that could not be stirred. After sitting overnight at room temperature, the paste was slurried in ethyl acetate (10 L), filtered, re-slurried in ethyl acetate (5 L), filtered, and dried to a constant weight to afford the desired product as a white solid (combined with other runs, 5 kg starting material charged, 4113 g, 95%). LCMS for C₁₂H_nBrFN₆O₃ (M+H)⁺: m/z
= 384.9, 386.9. ¹H NMR (400 MHz, DMSO-J₆): δ 8.12 (m, 4 H), 7.76 (m, 1 H), 7.58 (t, J= 8.7 Hz, 1 H), 6.78 (t, J= 6.1 Hz, 1 H), 3.51 (dd, J= 11.8, 6.1 Hz, 2 H), 3.02 (m, 2 H).
Step L: tert-Butyl ({[2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-diliydro-l,2,4-oxadiazol- 3-yl]-l,2,5-oxadiazol-3-yl}amino)ethyl]amino}sulfonyl)carbamate

A 5 L round bottom flask was charged with chlorosulfonyl isocyanate [Aldrich, product #
142662] (149 mL, 1.72 mol) and dichloromethane (1.5 L) and cooled using an ice bath to 2 ⁰C. tert-Butanol (162 mL, 1.73 mol) in dichloromethane (200 mL) was added dropwise at a rate so that the temperature did not exceed 10 ⁰C. The resulting solution was stirred at room temperature for 30-60 min to provide tert-butyl [chlorosulfonyljcarbamate.
A 22 L flask was charged with 3-{4-[(2-aminoethyl)amino]-l,2,5-oxadiazol-3-yl}-4-(3- bromo-4-fluorophenyl)-l,2,4-oxadiazol-5(4H)-one hydrochloride (661 g, 1.57 mol) and 8.5 L dichloromethane. After cooling to -15 ⁰C with an ice/salt bath, the solution of tert-butyl [chlorosulfonyl]carbamate (prepared as above) was added at a rate so that the temperature did not exceed -10 ⁰C (addition time 7 min). After stirring for 10 min, triethylamine (1085 mL, 7.78 mol) was added at a rate so that the temperature did not exceed -5 ⁰C (addition time 10 min). The cold bath was removed, the reaction was allowed to warm to 10 ⁰C, split into two portions, and neutralized with 10% cone HCl (4.5 L each portion). Each portion was transferred to a 50 L separatory funnel and diluted with ethyl acetate to completely dissolve the white solid (~25 L). The layers were separated, and the organic layer was washed with water (5 L), brine (5 L), and the solvents removed in vacuo to afford an off-white solid. The solid was triturated with MTBE (2 x 1.5 L) and dried to a constant weight to afford a white solid. A total of 4113 g starting material was processed in this manner (5409 g, 98%). *Η NMR (400 MHz, OMSO-d₆): δ 10.90 (s, 1 H), 8.08 (dd, J= 6.2, 2.5 Hz, 1 H), 7.72 (m, 1 H), 7.59 (t, J= 8.6 Hz, 1 H), 6.58 (t, J= 5.7 Hz, 1 H), 3.38 (dd, J= 12.7, 6.2 Hz, 2 H), 3.10 (dd, J = 12.1, 5.9 Hz, 2 H), 1.41 (s, 9 H). Step M: N-[2-({4-[4-(3-Bromo-4-fluorophenyl)-5-oxo-4,5-dmydro-l ,2,4-oxadiazol-3-yl]- l,2,5-oxadiazol-3-yl}amino)ethyl]sulfamide

To a 22 L flask containing 98:2 trifluoroacetic acid:water (8.9 L) was added tert-butyl ({[2- ({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-diliydro-l,2,4-oxadiazol-3-yl]-l,2,5-oxadiazol-3- yl}amino)ethyl]amino}sulfonyl)carbamate (1931 g, 3.42 mol) in portions over 10 minutes. The resulting mixture was stirred at room temperature for 1.5 h, the solvents removed in vacuo, and chased with dichloromethane (2 L). The resulting solid was treated a second time with fresh 98:2 trifluoroacetic acid:water (8.9 L), heated for 1 h at 40-50 ⁰C, the solvents removed in vacuo, and chased with dichloromethane (3 x 2 L). The resulting white solid was dried in a vacuum drying oven at 50 ⁰C overnight. A total of 5409 g was processed in this manner (4990 g, quant, yield). LCMS for C]₂H₁₂BrFN₇O₅S (M+H)⁺: m/z = 463.9, 465.9.
¹H NMR (400 MHz, OM$>O-d₆): δ 8.08 (dd, J= 6.2, 2.5 Hz, 1 H), 7.72 (m, 1 H), 7.59 (t, J= 8.7 Hz, 1 H), 6.67 (t, J= 5.9 Hz, IH), 6.52 (t, J= 6.0 Hz, 1 H), 3.38 (dd, J= 12.7, 6.3 Hz, 2 H), 3.11 (dd, J= 12.3, 6.3 Hz).
Step N: 4-( {2-[(Aminosulfonyl)amino]ethyl} amino)-N-(3-bromo-4-fluorophenyl)-N- hydroxy-l,2,5-oxadiazole-3-carboximidamide
To a crude mixture of N-[2-({4-[4-(3-bromo-4-fluorophenyl)-5-oxo-4,5-dihydro-l,2,4- oxadiazol-3-yl]-l,2,5-oxadiazol-3-yl}amino)ethyl]sulfamide (2.4 mol) containing residual amounts of trifluoroacetic acid stirring in a 22 L flask was added THF (5 L). The resulting solution was cooled to 0 °C using an ice bath and 2 Ν NaOH (4 L) was added at a rate so that the temperature did not exceed 10 ⁰C. After stirring at ambient temperature for 3 h (LCMS indicated no starting material remained), the pH was adjusted to 3-4 with concentrated HCl (-500 mL). The THF was removed in vacuo, and the resulting mixture was extracted with ethyl acetate (15 L). The organic layer was washed with water (5 L), brine (5 L), and the solvents removed in vacuo to afford a solid. The solid was triturated with MTBE (2 x 2 L), combined with three other reactions of the same size, and dried overnight in a convection oven to afford a white solid (3535 g). The solid was recrystallized (3 x 22 L flasks, 2: 1 water: ethanol, 14.1 L each flask) and dried in a 50 ⁰C convection oven to a constant weight to furnish the title compound as an off-white solid (3290 g, 78%). LCMS for C_nH₁₄BrFN₇O₄S (M+H)⁺: m/z = 437.9, 439.9. ¹H NMR (400 MHz, DMSO-J₆): δ 11.51 (s, 1 H), 8.90 (s, 1 H), 7.17 (t, J= 8.8 Hz, 1 H), 7.11 (dd, J= 6.1, 2.7 Hz, 1 H), 6.76 (m, 1 H), 6.71 (t, J= 6.0 Hz, 1 H), 6.59 (s, 2 H), 6.23 (t, J= 6.1 Hz, 1 H), 3.35 (dd, J= 10.9, 7.0 Hz, 2 H), 3.10 (dd, J= 12.1, 6.2 Hz, 2 H).
The final product was an anhydrous crystalline solid. The water content was determined to be less than 0.1% by Karl Fischer titration.


CLIP

INCB24360

Company:Incyte Corp.
Target: IDO1
Disease: Cancer

Incyte’s Andrew P. Combs presented the company’s clinical candidate for cancer immunotherapy. The basic tenet of this burgeoning field is that the human body’s immune system is a tremendous resource for fighting disease; scientists just need to figure out how to unleash it. One target that’s proven to be particularly attractive for this purpose in recent years is indoleamine-2,3-dioxygenase-1, or IDO1 (C&EN, April 6, page 10).

IDO1 plays a role in signaling the immune system to stand down from attacking foreign bodies it might otherwise go after, such as fetuses. Tumors also produce IDO1 to evade the immune system, so molecules that can inhibit this enzyme could bring the full force of the body’s defenses to bear on these deadly invaders.

Incyte’s search for an IDO1 inhibitor began with a high-throughput screen, which led to a proof-of-concept compound. But the compound had poor oral bioavailability. What’s more, the molecule and its analogs underwent glucuronidation during its metabolism: Enzymes tacked on a glucuronic acid group to the structure’s amidoxime, which was key to its activity.

The chemists reasoned they could block this metabolism by sterically hindering that position. Making such molecules proved to be more difficult than they expected. But then they unearthed a Latvian paper from 1993 that gave them the synthetic method they needed to make the series of compounds that would lead to their clinical candidate INCB24360 (epacadostat).

With its furazan core, as well as its amidoxime, bromide, and sulfuric diamide functional groups, INCB24360 is something of an odd duck, Combs acknowledged. “Some of you in the audience may be looking at this and saying, ‘That molecule does not look like something I would bring forward or maybe even make,’ ” he said, noting that the structure breaks many medicinal chemistry rules. “We’re a data-centric company, and we followed the data, not the rules,” Combs told C&EN.

The compound has completed Phase I clinical trials and is now being used in collaborative studies with several other pharmaceutical companies that combine INCB24360 with other cancer immunotherapy agents.

TEAMWORK

Incyte’s team (from left): Andrew Combs, Dilip Modi, Joe Glenn, Brent Douty, Padmaja Polam, Brian Wayland, Rick Sparks, Wenyu Zhu, and Eddy Yue.

Credit: Incyte

WO2007113648A2 *	Mar 26, 2007	Oct 11, 2007	Pfizer Products Inc.	Ctla4 antibody combination therapy
US20070185165 *	Dec 19, 2006	Aug 9, 2007	Combs Andrew P	N-hydroxyamidinoheterocycles as modulators of indoleamine 2,3-dioxygenase
US20100055111 *	Feb 14, 2008	Mar 4, 2010	Med. College Of Georgia Research Institute, Inc.	Indoleamine 2,3-dioxygenase, pd-1/pd-l pathways, and ctla4 pathways in the activation of regulatory t cells
US20120058079 *	Nov 11, 2011	Mar 8, 2012	Incyte Corporation, A Delaware Corporation	1,2,5-Oxadiazoles as Inhibitors of Indoleamine 2,3-Dioxygenase

REFERENCES

1: Vacchelli E, Aranda F, Eggermont A, Sautès-Fridman C, Tartour E, Kennedy EP, Platten M, Zitvogel L, Kroemer G, Galluzzi L. Trial watch: IDO inhibitors in cancer therapy. Oncoimmunology. 2014 Dec 15;3(10):e957994. eCollection 2014 Nov. Review. PubMed PMID: 25941578; PubMed Central PMCID: PMC4292223.
2: Liu X, Shin N, Koblish HK, Yang G, Wang Q, Wang K, Leffet L, Hansbury MJ, Thomas B, Rupar M, Waeltz P, Bowman KJ, Polam P, Sparks RB, Yue EW, Li Y, Wynn R, Fridman JS, Burn TC, Combs AP, Newton RC, Scherle PA. Selective inhibition of IDO1 effectively regulates mediators of antitumor immunity. Blood. 2010 Apr 29;115(17):3520-30. doi: 10.1182/blood-2009-09-246124. Epub 2010 Mar 2. PubMed PMID: 20197554.
3: Koblish HK, Hansbury MJ, Bowman KJ, Yang G, Neilan CL, Haley PJ, Burn TC, Waeltz P, Sparks RB, Yue EW, Combs AP, Scherle PA, Vaddi K, Fridman JS. Hydroxyamidine inhibitors of indoleamine-2,3-dioxygenase potently suppress systemic tryptophan catabolism and the growth of IDO-expressing tumors. Mol Cancer Ther. 2010 Feb;9(2):489-98. doi: 10.1158/1535-7163.MCT-09-0628. Epub 2010 Feb 2. PubMed PMID: 20124451.

//////////1204669-58-8 , INCB024360, INCB24360, epacadostat, PHASE 2, CANCER, orphan drug designation

Fc1ccc(cc1Br)N/C(=N\O)c2nonc2NCCNS(N)(=O)=O