The present invention provides compounds useful as inhibitors of PAD4, compositions thereof, and methods of treating PAD4-related disorders.

BACKGROUND OF THE INVENTION

PAD4 is a member of the peptidylarginine deiminase (PAD) family of enzymes capable of catalysing the citrullination of arginine into citrulline within peptide sequences. PAD4 is responsible for the deimination or citrullination of a variety of proteins in vitro and in vivo, with consequences of diverse functional responses in a variety of diseases (Jones J. E. et al,Curr. Opin. Drug Discov. Devel.,12(5), (2009), 616-627). Examples of exemplar diseases include rheumatoid arthritis, diseases with neutrophilic contributions to pathogenesis (for example vasculitis, systemic lupus erythematosus, ulcerative colitis) in addition to oncology indications. PAD4 inhibitors also have wider applicability as tools and therapeutics for human disease through epigenetic mechanisms.

Inhibitors of PAD4 have utility against Rheumatoid Arthritis (RA). RA is an auto-immune disease affecting approximately 1% of the population (Wegner N. et al,Immunol. Rev.,233(1) (2010), 34-54). It is characterised by inflammation of articular joints leading to debilitating destruction of bone and cartilage. A weak genetic association between PAD4 polymorphisms and susceptibility to RA has been suggested, albeit inconsistently, in a number of population studies (Kochi Y. et al,Ann. Rheum. Dis.,70, (2014512-515). PAD4 (along with family member PAD2) has been detected in synovial tissue where it is responsible for the deimination of a variety of joint proteins. This process is presumed to lead to a break of tolerance to, and initiation of immune responses to, citrullinated substrates such as fibrinogen, vimentin and collagen in RA joints. These anti-citrullinated protein antibodies (ACPA) contribute to disease pathogenesis and may also be used as a diagnostic test for RA (e.g. the commercially available CCP2 or cyclic citrullinated protein 2 test). In addition, increased citrullination may also offer additional direct contributions to disease pathogenesis through its ability to affect directly the function of several joint and inflammatory mediators (e.g. fibrinogen, anti-thrombin, multiple chemokines). In a smaller subset of RA patients, anti-PAD4 antibodies can be measured and may correlate with a more erosive form of the disease.

PAD4 inhibitors are also useful for the reduction of pathological neutrophil activity in a variety of diseases. Studies suggest that the process of Neutrophil Extracellular Trap (NET) formation, an innate defence mechanism by which neutrophils are able to immobilise and kill pathogens, is associated with histone citrulllination and is deficient in PAD4 knockout mice (Neeli I. et al,J. Immunol.,180, (2008), 1895-1902 and Li P. et al,J. Exp. Med.,207(9), (2010), 1853-1862). PAD4 inhibitors may therefore have applicability for diseases where NET formation in tissues contributes to local injury and disease pathology. Such diseases include, but are not limited to, small vessel vasculitis (Kessenbrock K. et al,Nat. Med.,15(6), (2009), 623-625), systemic lupus erythematosus (Hakkim A. et al,Proc. Natl. Acad. Sci. USA,107(21), (2010), 9813-9818 and Villanueva E. et al,J. Immunol.,187(1), (2011), 538-52), ulcerative colitis (Savchenko A. et al,Pathol. Int.,61(5), (2011), 290-7), cystic fibrosis, asthma (Dworski R. et al,J. Allergy Clin. Immunol.,127(5), (2011), 1260-6), deep vein thrombosis (Fuchs T et al,Proc. Natl. Acad. Sci. USA,107(36), (2010), 15880-5), periodontitis (Vitkov L. et al,Ultrastructural Pathol.,34(1), (2010), 25-30), sepsis (Clark S. R. et al,Nat. Med.,13(4), (2007), 463-9), appendicitis (Brinkmann V. et al, Science, 303, (2004), 1532-5), and stroke. In addition, there is evidence that NETs may contribute to pathology in diseases affecting the skin, eg in cutaneous lupus erythematosis (Villanueva E. et al,J. Immunol.,187(1), (2011), 538-52) and psoriasis (Lin A. M. et al.,J. Immunol.,187(1), (2011), 490-500), so a PAD4 inhibitor may show benefit to tackle NET skin diseases, when administered by a systemic or cutaneous route. PAD4 inhibitors may affect additional functions within neutrophils and have wider applicability to neutrophilic diseases.

Studies have demonstrated efficacy of tool PAD inhibitors (for example chloro-amidine) in a number of animal models of disease, including collagen-induced arthritis (Willis V. C. et al,J. Immunol.,186(7), (2011), 4396-4404), dextran sulfate sodium (DSS)-induced experimental colitis (Chumanevich A. A. et al,Am. J. Physiol. Gastrointest. Liver Physiol.,300(6), (2011), G929-G938), spinal cord repair (Lange S. et al,Dev. Biol.,355(2), (2011), 205-14), and experimental autoimmune encephalomyelitis (EAE). The DSS colitis report also demonstrates that chloro-amidine drives apoptosis of inflammatory cells both in vitro and in vivo, suggesting that PAD4 inhibitors may be effective more generally in widespread inflammatory diseases.

PAD4 inhibitors are also useful in the treatment of cancers (Slack. J. L. et al,Cell. Mol. Life Sci.,68(4), (2011), 709-720). Over-expression of PAD4 has been demonstrated in numerous cancers (Chang X. et al,BMC Cancer,9, (2009), 40). An anti-proliferative role has been suggested for PAD4 inhibitors from the observation that PAD4 citrullinates arginine residues in histones at the promoters of p53-target genes such as p21, which are involved in cell cycle arrest and induction of apoptosis (Li P. et al,Mol. Cell Biol.,28(15), (2008), 4745-4758).

The aforementioned role of PAD4 in deiminating arginine residues in histones may be indicative of a role for PAD4 in epigenetic regulation of gene expression. PAD4 is the primary PAD family member observed to be resident in the nucleus as well as the cytoplasm. Early evidence that PAD4 may act as a histone demethyliminase as well as a deiminase is inconsistent and unproven. However, it may reduce histone arginine methylation (and hence epigenetic regulation associated with this mark) indirectly via depletion of available arginine residues by conversion to citrulline. PAD4 inhibitors are useful as epigenetic tools or therapeutics for affecting expression of varied target genes in additional disease settings. Through such mechanisms, PAD4 inhibitors may also be effective in controlling citrullination levels in stem cells and may therefore therapeutically affect the pluripotency status and differentiation potential of diverse stem cells including, but not limited to, embryonic stem cells, neural stem cells, haematopoietic stem cells and cancer stem cells. Accordingly, there remains an unmet need to identify and develop PAD4 inhibitors for the treatment of PAD4-mediated disorders.

SUMMARY OF THE INVENTION

It has now been found that compounds of formula I are useful as inhibitors of PAD4:

or a pharmaceutically acceptable salt thereof, wherein each of R1, R2, R3, n and Ring A is as defined and described herein.

It has also been found that compounds of formula I′ are useful as inhibitors of PAD4:

or a pharmaceutically acceptable salt thereof, wherein each of R1, R2, R3, Ring A, X and n is as defined and described herein.

In some embodiments, a provided compound demonstrates selectivity for PAD4 with respect to PAD2. The present invention also provides pharmaceutically acceptable compositions comprising a provided compound. Provided compounds are useful in treatment of various disorders associated with PAD4. Such disorders are described in detail, herein, and include, for example rheumatoid arthritis, vasculitis, systemic lupus erythematosus, ulcerative colitis, cancer, cystic fibrosis, asthma, cutaneous lupus erythematosis, and psoriasis.

DETAILED DESCRIPTION OF THE INVENTION

1. General Description of Certain Aspects of the Invention

In some embodiments, such compounds include those of the formulae described herein, or a pharmaceutically acceptable salt thereof, wherein each variable is as defined herein and described in embodiments. In some embodiments, the present invention provides a compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein:R1is hydrogen, —CN, —OR, or C1-6aliphatic optionally substituted with 1-4 groups selected from fluorine, —CN, or OR;R2is hydrogen or C1-10aliphatic optionally substituted with 1-5 groups selected from fluorine, —CN, or —OR;Ring A is

wherein Ring A is optionally substituted with 1-4 groups selected from fluorine, —CN, —OR, or C1-6aliphatic optionally substituted with 1-3 fluorine atoms;each R3is independently halogen, —CN, —R, or —OR;n is 0-3; andeach R is independently hydrogen or C1-6aliphatic optionally substituted with 1-3 fluorine atoms.

or C1-6aliphatic optionally substituted with 1-4 groups selected from fluorine, —CN, or OR;R2is hydrogen or C1-10aliphatic optionally substituted with 1-5 groups selected from fluorine, —CN, or —OR;Ring A is

wherein Ring A is optionally substituted with 1-4 groups selected from fluorine, —CN, —OR, or C1-6aliphatic optionally substituted with 1-3 fluorine atoms;each R3is independently halogen, —CN, —R, or —OR;X is C or N;n is 0-3; andeach R is independently hydrogen or C1-6aliphatic optionally substituted with —OH or 1-3 fluorine atoms.

The terms “measurable affinity” and “measurably inhibit,” as used herein, means a measurable change in PAD4 activity between a sample comprising a compound of the present invention, or composition thereof, and PAD4, and an equivalent sample comprising PAD4 in the absence of said compound, or composition thereof.

3. Description of Exemplary Compounds

According to one aspect, the present invention provides a compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein:R1is hydrogen, —CN, —OR, or C1-6aliphatic optionally substituted with 1-4 groups selected from fluorine, —CN, or OR;R2is hydrogen or C1-10aliphatic optionally substituted with 1-5 groups selected from fluorine, —CN, or —OR;Ring A is

wherein Ring A is optionally substituted with 1-4 groups selected from fluorine, —CN, —OR, or C1-6aliphatic optionally substituted with 1-3 fluorine atoms;each R3is independently halogen, —CN, —R, or —OR;n is 0-3; andeach R is independently hydrogen or C1-6aliphatic optionally substituted with 1-3 fluorine atoms.

According to another aspect, the present invention provides a compound of formula I′:

or C1-6aliphatic optionally substituted with 1-4 groups selected from fluorine, —CN, or OR;R2is hydrogen or C1-10aliphatic optionally substituted with 1-5 groups selected from fluorine, —CN, or —OR;Ring A is

wherein Ring A is optionally substituted with 1-4 groups selected from fluorine, —CN, —OR, or C1-6aliphatic optionally substituted with 1-3 fluorine atoms;each R3is independently halogen, —CN, —R, or —OR;X is C or N;n is 0-3; andeach R is independently hydrogen or C1-6aliphatic optionally substituted with —OH or 1-3 fluorine atoms.

In some embodiments, R1is

In certain embodiments, R1is selected from those depicted in Table 1, below.

As defined above, Ring A is

wherein Ring A is optionally substituted with 1-4 groups selected from fluorine, —CN, —OR, or C1-6aliphatic optionally substituted with 1-3 fluorine atoms.

In some embodiments, Ring A is selected from

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In some embodiments, Ring A is

In certain embodiments, Ring A is selected from those depicted in Table 1, below.

In some embodiments, R1is methyl, R2is 2,2,2-trifluoroethyl, and Ring A is

In some embodiments, R1is ethyl, R2is cyclopropylmethyl, and Ring A is

In some embodiments, R1is methyl, R2is 2,2,2-trifluoroethyl, and Ring A is

In some embodiments, R1is ethyl, R2is ethyl, and Ring A is

In some embodiments, R1is methyl, R2is 2,2,2-trifluoroethyl, and Ring A is

In some embodiments, R1is methyl, R2is 2,2,2-trifluoroethyl, and Ring A is

In some embodiments, R1is methyl, R2is cyclopropylmethyl, and Ring A is

In some embodiments, R1is methyl, R2is cyclopropylmethyl, and Ring A is

As defined above and described herein, n is 0-3. In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In certain embodiments, n is selected from those depicted in Table 1, below.

As described herein, X is C or N. In some embodiments, X is C. In some embodiments, X is N. In certain embodiments, X is selected from those depicted in Table 1, below.

In some embodiments, the compound of formula I or formula I′ is selected from those depicted below in Table 1.

In certain embodiments, the present invention provides any compound described above and herein, or a pharmaceutically acceptable salt thereof. In some embodiments, the present invention provides a compound as depicted in Table 1, above, or a pharmaceutically acceptable salt thereof.

In some embodiments, the present invention provides any compound described above and herein in isolated form.

4. Uses, Formulation and Administration

Pharmaceutically Acceptable Compositions

According to another embodiment, the invention provides a composition comprising a compound of this invention or a pharmaceutically acceptable derivative thereof and a pharmaceutically acceptable carrier, adjuvant, or vehicle. The amount of compound in compositions of this invention is such that is effective to measurably inhibit PAD4, in a biological sample or in a patient. In certain embodiments, the amount of compound in compositions of this invention is such that is effective to measurably inhibit PAD4, in a biological sample or in a patient. In certain embodiments, a composition of this invention is formulated for administration to a patient in need of such composition. In some embodiments, a composition of this invention is formulated for oral administration to a patient.

The term “subject,” as used herein, is used interchangeably with the term “patient” and means an animal, preferably a mammal. In some embodiments, a subject or patient is a human. In other embodiments, a subject (or patient) is a veterinary subject (or patient). In some embodiments, a veterinary subject (or patient) is a canine, a feline, or an equine subject.

Most preferably, pharmaceutically acceptable compositions of this invention are formulated for oral administration. Such formulations may be administered with or without food. In some embodiments, pharmaceutically acceptable compositions of this invention are administered without food. In other embodiments, pharmaceutically acceptable compositions of this invention are administered with food.

A compound of the current invention can be administered alone or in combination with one or more other therapeutic compounds, possible combination therapy taking the form of fixed combinations or the administration of a compound of the invention and one or more other therapeutic compounds being staggered or given independently of one another, or the combined administration of fixed combinations and one or more other therapeutic compounds. A compound of the current invention can besides or in addition be administered especially for tumor therapy in combination with chemotherapy, radiotherapy, immunotherapy, phototherapy, surgical intervention, or a combination of these. Long-term therapy is equally possible as is adjuvant therapy in the context of other treatment strategies, as described above. Other possible treatments are therapy to maintain the patient's status after tumor regression, or even chemopreventive therapy, for example in patients at risk.

As used herein, the term “combination,” “combined,” and related terms refers to the simultaneous or sequential administration of therapeutic agents in accordance with this invention. For example, a compound of the present invention may be administered with another therapeutic agent simultaneously or sequentially in separate unit dosage forms or together in a single unit dosage form. Accordingly, the present invention provides a single unit dosage form comprising a compound of the current invention, an additional therapeutic agent, and a pharmaceutically acceptable carrier, adjuvant, or vehicle.

The amount of both an inventive compound and additional therapeutic agent (in those compositions which comprise an additional therapeutic agent as described above) that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. Preferably, compositions of this invention should be formulated so that a dosage of between 0.01-100 mg/kg body weight/day of an inventive compound can be administered.

In those compositions which comprise an additional therapeutic agent, that additional therapeutic agent and the compound of this invention may act synergistically. Therefore, the amount of additional therapeutic agent in such compositions will be less than that required in a monotherapy utilizing only that therapeutic agent.

Compounds and compositions described herein are generally useful for the inhibition of PAD4.

The activity of a compound utilized in this invention as an inhibitor of PAD4, may be assayed in vitro, in vivo or in a cell line. In vitro assays include assays that determine the inhibition of PAD4. Detailed conditions for assaying a compound utilized in this invention as an inhibitor of PAD4 are set forth in the Examples below. In some embodiments, a provided compound inhibits PAD4 selectively as compared to PAD2.

Provided compounds are inhibitors of PAD4 and are therefore useful for treating one or more disorders associated with activity of PAD4. Thus, in certain embodiments, the present invention provides a method for treating a PAD4-mediated disorder comprising the step of administering to a patient in need thereof a compound of the present invention, or pharmaceutically acceptable composition thereof.

In certain embodiments, a PAD4-mediated disorder is a disease, condition, or disorder mediated by inappropriate PAD4 activity. In some embodiments, a PAD4-mediated disorder is selected from the group consisting of rheumatoid arthritis, vasculitis, systemic lupus erythematosus, ulcerative colitis, cancer, cystic fibrosis, asthma, cutaneous lupus erythematosis, and psoriasis. In a further embodiment, the disorder mediated by inappropriate PAD4 activity is rheumatoid arthritis. In a further embodiment, the disorder mediated by inappropriate PAD4 activity is systemic lupus. In a further embodiment, the disorder mediated by inappropriate PAD4 activity is vasculitis. In a further embodiment, the disorder mediated by inappropriate PAD4 activity is cutaneous lupus erythematosis. In a further embodiment, the disorder mediated by inappropriate PAD4 activity is psoriasis.

In one embodiment there is provided a method of treatment of rheumatoid arthritis, vasculitis, systemic lupus erythematosus, ulcerative colitis, cancer, cystic fibrosis, asthma, cutaneous lupus erythematosis, or psoriasis, which method comprises administering to a human subject in need thereof, a therapeutically effective amount of a provided compound or a pharmaceutically acceptable salt thereof.

In one embodiment there is provided a method of treatment of rheumatoid arthritis, which method comprises administering to a human subject in need thereof, a therapeutically effective amount of a provided compound, or a pharmaceutically acceptable salt thereof. In one embodiment there is provided a method of treatment of systemic lupus, which method comprises administering to a human subject in need thereof, a therapeutically effective amount of a provided compound, or a pharmaceutically acceptable salt thereof. In one embodiment there is provided a method of treatment of vasculitis, which method comprises administering to a human subject in need thereof, a therapeutically effective amount of a provided compound, or a pharmaceutically acceptable salt thereof. In one embodiment there is provided a method of treatment of cutaneous lupus erythematosis, which method comprises administering to a human subject in need thereof, a therapeutically effective amount of a provided compound, or a pharmaceutically acceptable salt thereof. In one embodiment there is provided a method of treatment of psoriasis, which method comprises administering to a human subject in need thereof, a therapeutically effective amount of a provided compound, or a pharmaceutically acceptable salt thereof.

All features of each of the aspects of the invention apply to all other aspects mutatis mutandis.

Method A

Gradient Time (minutes)—% B

Method B

A: 2 mM ammonium bicarbonate in HPLC grade water pH10

Method C

Gradient Time (minutes)—% B

Method D

Gradient Time (minutes)—% B

Method E

Chiral HPLC preparative method

Method F

Chiral purity analysis method

Compounds of the present invention were prepared according to Scheme 1, below.

To a stirred solution of ethyl 1H-pyrrolo[2,3-b]pyridine-2-carboxylate (CAS 221675-35-0, 4.0 g, 21.03 mmol) in DMF (40 ml) at 0° C. was added NaH (60%, 0.95 g, 23.87 mmol) portion wise over 5 min under a nitrogen atmosphere. The resulting mixture was stirred at room temperature for 45 min. (Bromomethyl)cyclopropane (2.45 ml, 25.24 mmol) was added and the reaction mixture was stirred at room temperature for 18 h. The solvent was removed in vacuo and the residue suspended in THF (40 ml). To this mixture was added 5M aqueous sodium hydroxide (20 ml, 100 mmol) and the mixture stirred at 50° C. for 18 h. The solvent was removed in vacuo and the remaining material was acidified with 5M aqueous hydrochloric acid (20 ml) whilst stirring in an ice-bath. The suspension was stirred for 10 min then the precipitate collected by vacuum filtration. The solid was washed with water (2×100 ml) and dried to obtain 3.48 g (76.5%) of 1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid EV-AQ1977-001 as a white powder. LCMS (method D): retention time 1.02 min, M/z=217 (M+1).

To a stirred solution of 1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid (EV-AQ1977-001, 700 mg, 3.24 mmol) in dry DMF (15 ml) were added HATU (1.57 g, 4.05 mmol) and DIPEA (712 μl, 4.05 mmol). The mixture was stirred at room temperature for 2 h before methyl 5-amino-6-(methylamino)pyridine-3-carboxylate (CAS 211915-53-6, 91%, 709 mg, 3.56 mmol) was added and the mixture stirred at room temperature for 16 h and at 50° C. for 4 h. Further methyl 5-amino-6-(methylamino)pyridine-3-carboxylate (91%, 129 mg, 0.65 mmol) was added and stirring at 50° C. was continued for 16 h. The solvent was removed in vacuo the resulting brown oil dissolved in acetic acid (15 ml) and heated at 80° C. for 10 h and at 70° C. for 16 h. The reaction mixture was concentrated under reduced pressure to obtain a brown oil which was purified by flash column chromatography (17-85% EtOAc/heptane) to obtain 650 mg (55.6%) of methyl 2-[1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl]-3-methyl-3H-imidazo[4,5-b]pyridine-6-carboxylate EV-AT1616-001 as a yellow powder. LCMS (method D): retention time 1.22 min, M/z=362 (M+1).

To methyl 2-[1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl]-3-methyl-3H-imidazo[4,5-b]pyridine-6-carboxylate (EV-AT1616-001, 650 mg, 1.80 mmol) in MeOH (20 ml) was added 2M NaOH (10 ml). The mixture was stirred at 50° C. for 3 h. The solvent was removed in vacuo and the resulting cloudy aqueous solution was acidified with 5M HCl (5 ml) whilst stirring. Stirring was continued for 5 min then the precipitate was collected by vacuum filtration, washed with water (2×10 ml) and dried to obtain 610 mg (97.6%) of 2-[1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl]-3-methyl-3H-imidazo[4,5-b]pyridine-6-carboxylic acid EV-AT1617-001 as a pale yellow powder. LCMS (method D): retention time 1.11 min, M/z=348 (M+1).

A stirred solution of 2-[1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl]-3-methyl-3H-imidazo[4,5-b]pyridine-6-carboxylic acid (EV-AT1617-001, 100 mg, 0.29 mmol) and HATU (126 mg, 0.33 mmol) in DMSO (2.4 ml) and MeCN (1.5 ml) was treated with DIPEA (61 μl, 0.35 mmol) at room temperature. The mixture was stirred for 2 h then tert-butyl octahydro-1H-pyrrolo[2,3-c]pyridine-1-carboxylate (949559-11-9, 72 mg, 0.32 mmol) was added and the reaction mixture stirred for 1.5 h at room temperature. The reaction mixture was diluted with water (0.3 ml) and purified by preparative HPLC (basic method) to obtain 137 mg (84.8%) of tert-butyl 6-{2-[1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl]-3-methyl-3H-imidazo[4,5-b]pyridine-6-carbonyl}-octahydro-1H-pyrrolo[2,3-c]pyridine-1-carboxylate EV-AT1622-001 as a white powder. LCMS (method D): retention time 1.34 min, M/z=556 (M+1).

1.25M HCl in EtOH (0.5 ml) was added to a solution of tert-butyl 6-{2-[1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl]-3-methyl-3H-imidazo[4,5-b]pyridine-6-carbonyl}-octahydro-1H-pyrrolo[2,3-c]pyridine-1-carboxylate (EV-AT1622-001, 99%, 20 mg, 0.04 mmol) in EtOH (1 ml). The reaction mixture was stirred at 50° C. for 4 h then concentrated to a white residue under reduced pressure. This material was freeze-dried from water (3 ml) to obtain 20.8 mg (quantitative) of 2-[1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl]-3-methyl-6-{octahydro-1H-pyrrolo[2,3-c]pyridine-6-carbonyl}-3H-imidazo[4,5-b]pyridine hydrochloride EV-AT1623-001 as a yellow powder. LCMS (method A): retention time 1.79 min, M/z=456 (M+1).

111 mg of tert-butyl 6-{2-[1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl]-3-methyl-3H-imidazo[4,5-b]pyridine-6-carbonyl}-octahydro-1H-pyrrolo[2,3-c]pyridine-1-carboxylate EV-AT1622-001 were dissolved to 14 mg/mL in methanol and then purified by chiral HPLC (method E) to obtain 48.4 mg (87.2%) of tert-butyl (3aS,7aR)-6-{2-[1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl]-3-methyl-3H-imidazo[4,5-b]pyridine-6-carbonyl}-octahydro-1H-pyrrolo[2,3-c]pyridine-1-carboxylate EV-AT1622-002 (absolute stereochemistry arbitrarily assigned) as a white powder and 48.4 mg (87.2%) of tert-butyl (3 aR,7aS)-6-{2-[1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl]-3-methyl-3H-imidazo[4,5-b]pyridine-6-carbonyl}-octahydro-1H-pyrrolo[2,3-c]pyridine-1-carboxylate EV-AT1622-003 (absolute stereochemistry arbitrarily assigned) as a white powder.

Synthesised according to the procedures described in Scheme 1 via synthesis of methyl 1-(cyclopropylmethyl)-6-(difluoromethoxy)-1H-pyrrolo[2,3-b]pyridine-2-carboxylate EV-AY2833-002 as described in Scheme 1.1:

Ethyl 6-chloro-1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridine-2-carboxylate (EV-AY7167-001 synthesised according to Scheme 1, 90%, 1.45 g, 4.68 mmol), Pd2(dba)3(214 mg, 0.23 mmol),tBu-BippyPhos (237 mg, 0.47 mmol) and potassium hydroxide (788 mg, 14.0 mmol) were combined in dioxane (7.0 ml) and water (7.0 ml) in a pressure tube. The reaction mixture was purged with nitrogen for 5 minutes then the vessel was sealed and heated at 70° C. for 1.5 h. The reaction mixture was allowed to cool to room temperature then filtered through glass fibre filter paper. The filtrate was diluted with water (10 ml) and extracted with EtOAc (30 ml). The aqueous layer was acidified to pH 5 with 2M HCl and the resulting precipitate filtered and dried to obtain 0.80 g (62.2%) of 1-(cyclopropylmethyl)-6-hydroxy-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid EV-AX5573-002 as an off-white powder. LCMS (method D): retention time 0.95 min, M/z=233 (M+1).

2M (Diazomethyl)(trimethyl)silane (1729 μl in diethylether) was added to a stirred suspension of 1-(cyclopropylmethyl)-6-hydroxy-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid (EV-AX5573-002, 73%, 550 mg, 1.73 mmol) in anhydrous toluene (6.0 ml) and methanol (anhydrous, 2.0 ml, 49.44 mmol) under an atmosphere of nitrogen. The resulting mixture was stirred at room temperature for 1.5 h. Acetic acid (0.70 ml) was added until the bright yellow colour disappeared. The reaction mixture was concentrated in vacuo and triturated with DCM (5 ml). The solid was filtered off under vacuum and dried to afford 131 mg (28.1%) of 1-(cyclopropylmethyl)-6-hydroxy-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid EV-AY4950-001 as a pale beige solid. LCMS (method D): retention time 1.13 min, M/z=247 (M+1).

To a stirred suspension of methyl 1-(cyclopropylmethyl)-6-hydroxy-1H-pyrrolo[2,3-b]pyridine-2-carboxylate (EV-AY2828-001 synthesised according to steps 1 and 2, 672 mg, 2.73 mmol) in acetonitrile (15.0 ml) was added 2,2-difluoro-2-(fluorosulfonyl)acetic acid (0.71 ml, 6.82 mmol) and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was filtered and the filtrate was concentrated in vacuo. The crude residue was purified by flash column chromatography (0-100% EtOAc/heptane) to obtain 450 mg (55.7%) of methyl 1-(cyclopropylmethyl)-6-(difluoromethoxy)-1H-pyrrolo[2,3-b]pyridine-2-carboxylate EV-AY2833-002 as an off-white solid. LCMS (method D): retention time 1.39 min, M/z=297 (M+1).

Synthesised according to the procedures described in Scheme 1 via synthesis of methyl 1-(cyclopropylmethyl)-6-(2-hydroxypropan-2-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxylate EV-AW6273-002 as described in Scheme 1.2:

To a solution of methyl 6-chloro-1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridine-2-carboxylate (EV-AW6269-001, 90%, 1.16 g, 3.93 mmol) in anhydrous dioxane (3 ml) were added tributyl(1-ethoxyethenyl)stannane (1.59 ml, 4.71 mmol), Xantphos (0.17 g, 0.29 mmol) and Pd2dba3(0.09 g, 0.10 mmol). The reaction mixture was stirred at 90° C. for 17 h. The solvent was removed in vacuo and 1M HCl (50 ml) and DCM (50 ml) were added to the residue. The biphasic mixture was stirred for 20 minutes then the organic layer was separated and concentrated in vacuo. The crude residue was purified by flash column chromatography (0-10% EtOAc/heptane) to obtain 0.512 g (47%) of methyl 6-acetyl-1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridine-2-carboxylate EV-AW6271-002 as an off-white solid. LCMS (method D): retention time 1.40 min, M/z=273 (M+1).

Methylmagnesium chloride (3M in THF, 642 μl) was added dropwise to a stirred solution of methyl 6-acetyl-1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridine-2-carboxylate (EV-AW6271-002, 510 mg, 1.84 mmol) in dry THF (5 mL) at −78° C. The reaction was stirred at −78° C. for 2.5 h. Further methylmagnesium chloride (3M in THF, 61 μl) was added at −78° C. and stirring was continued for 30 minutes. The reaction was quenched with water (20 ml) and THF was removed in vacuo. 1M HCl was added to the aqueous layer until pH 3. The aqueous layer was extracted with EtOAc (2×30 ml). The combined extracts dried over sodium sulfate, filtered and concentrated in vacuo. The crude residue was purified by flash column chromatography (0-35% EtOAc/heptane) to obtain 410 mg (76%) of methyl 1-(cyclopropylmethyl)-6-(2-hydroxypropan-2-yl)-1H-pyrrolo[2,3-b]pyridine-2-carboxylate EV-AW6273-002 as an off-white solid. LCMS (method D): retention time 1.23 min, M/z=289 (M+1).

The following compounds were synthesised according to Scheme 1, Scheme 1.1, Scheme 1.2 and procedures described above:

Compounds of the present invention were assayed as inhibitors of PAD4 using the assay protocol described below.

Compounds were solubilised in 100% DMSO to achieve 100 mM final compound concentration. Compound stock solutions were stored at RT. A series of dilutions were prepared in DMSO and mixed 8 times with 20 μL mixing volume. Final assay conditions were as follows:

0.25 μL of compound solution was added to 10 μL of 200 nM PAD4 in assay buffer (100 mM Tris-HCl pH 7.6, 2 mM DTT). After 5 mins, 10 μL of 100 μM of substrate in buffer (100 mM Tris-HCl pH 7.6, 2 mM DTT, 2 mM CaCl2) was added and the reaction incubated for 60 mins at 37° C. The enzymatic reaction was quenched by addition of 40 μl of 5% TCA in ACN (1.7% TCA final concentration) stop solution. Arginine containing substrate and citrulline containing product (+1 Da mass shift) were subjected to solid phase extraction on Agilent RapidFire (RF) 300 system and detected on a coupled, triple quadrupole Agilent 6460 QQQ mass spectrometry (MS) device under application of multiple reaction monitoring (MRM) for quantitation.

Table 2, below, shows the activity of selected compounds of this invention in the PAD4 assays described above. The compound numbers correspond to the compound numbers in Table 1. Compounds having an activity designated as “A” provided an IC50≦1 μM; compounds having an activity designated as “B” provided an IC50of 1.0-5.0 μM; compounds having an activity designated as “C” provided an IC50of 5.0-10.0 μM; and compounds having an activity designated as “D” provided an IC50of ≧10.0 μM. The term pIC50=−log(IC50). Compounds having an activity designated as “E” provided a pIC50≦4; compounds having an activity designated as “F” provided a pIC50of 4.0-5.0; compounds having an activity designated as “G” provided a pIC50of 5.0-6.0; and compounds having an activity designated as “H” provided a pIC50of ≧6. “NA” stands for “not assayed.”