Synthesis and Evaluation of 1H-Pyrrolo[2,3-b]pyridine Derivatives as Novel Immunomodulators Targeting Janus Kinase 3

Abstract

Janus kinases (JAKs) have been known to play crucial roles in modulating a number of inflammatory and immune mediators. Here, we describe a series of 1H-pyrrolo[2,3-b]pyridine derivatives as novel immunomodulators targeting JAK3 for use in treating immune diseases such as organ transplantation. In the chemical modification of compound 6, the introduction of a carbamoyl group to the C5-position and substitution of a cyclohexylamino group at the C4-position of the 1H-pyrrolo[2,3-b]pyridine ring led to a large increase in JAK3 inhibitory activity. Compound 14c was identified as a potent, moderately selective JAK3 inhibitor, and the immunomodulating effect of 14c on interleukin-2-stimulated T cell proliferation was shown. Docking calculations and WaterMap analysis of the 1H-pyrrolo[2,3-b]pyridine-5-carboxamide derivatives were conducted to confirm the substituent effects on JAK3 inhibitory activity.

Janus kinases (JAKs) are cytoplasmic protein tyrosine kinases with four known members (JAK1, JAK2, JAK3, and TYK2) which play important roles in cytokine-mediated signal transduction.^1–5) Cytokines bind to their respective receptors associated with JAKs and induce JAK activation, following phosphorylation of the receptors. Activated JAKs subsequently phosphorylate signal transducers and activators of transcription proteins (STATs) in cytoplasm, which are dimerized to translocate to the nucleus and activate gene transcription to promote cytokine-responsive gene expression.

In the JAK family, JAK3 is specifically associated with the common γc subunit of cytokine receptors of interleukins (ILs), such as IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, all of which are involved in differentiation, proliferation, and survival of T cells.^1–5) The role of JAK3 was studied by analysis of severe combined immunodeficiency patients, showing JAK3 gene mutation and decreased expression of JAK3 protein.^6,7) Further, JAK3 knockout mice exhibited immunodeficiency with remarkably reduced numbers of functional T cells, and no other profound phenotype was observed.^8,9) While other members of JAKs are ubiquitously expressed in whole body, expression of JAK3 is limited to hematopoietic cells.^10,11) Therefore, the effects of JAK3 inhibition are suspected to be limited to immune system. JAK1 is associated with IL-2 receptors and is involved in regulating the function of T cells in concert with JAK3. In addition, JAK1 is also involved in the signaling pathways of IL-6 and interferon (IFN)-γ for inflammatory responses.^1–5) JAK2 participates in differentiation and proliferation of erythrocytes, neutrophils, and thrombocytes by mediating signaling of hematopoietic growth factors such as erythropoietin, colony-stimulating factor, and thrombopoietin.^1–5)

A number of laboratories have attempted to develop JAK inhibitors.^12–14) Pfizer’s group discovered tofacitinib (compound 1, Fig. 1) as a pan-JAK inhibitor^15,16) which proved effective in various animal models^17,18) and was recently approved for use in treating rheumatoid arthritis (RA). Incyte’s group discovered the JAK1 and JAK2 inhibitor baricitinib (compound 2, Fig. 1),¹⁶⁾ which is in phase 3 clinical trials for the treatment of RA. JAK inhibitors presently attract a great deal of attention with regard to their potential therapeutic application for inflammatory and immune diseases such as RA, psoriasis, and organ transplant rejection, and several compounds have been advanced to the clinical stage.^19–21)

Fig. 1. Chemical Structures of JAK Inhibitors

For the treatment of organ transplantation, calcineurin inhibitors (CNIs), such as tacrolimus and cyclosporin A, have been used as a standard immunosuppressive therapy to inhibit IL-2 production and the following T cell activation, and achieved high efficacy to prevent acute transplant rejection.²²⁾ However, the long-term use of CNIs is associated with side effects such as nephrotoxicity and neurotoxicity.^23–25)

In our own research on novel immunomodulators, we mainly focused on JAK3 inhibition, as targeting JAK3 may offer novel and safe immunomodulating regimen due to the effect on IL-2-dependent T cell proliferation and the limited JAK3 expression on lymphoid cells. In compound screening, JAK1 inhibitory activity was evaluated in addition to JAK3 inhibitory activity, as JAK3 and JAK1 are known to regulate the IL-2 signaling pathway in concert. JAK2 inhibitory activity was also evaluated, as JAK2 inhibition may be related to adverse hematopoietic effects such as anemia.²⁶⁾ In our laboratory, we paid attention to hydrogen bond interaction with the hinge region of the ATP-binding site, and a number of heteroaryl compounds were synthesized. Among these compounds, 1H-pyrrolo[2,3-b]pyridine derivative 6 (Table 1) was identified as the initial template compound to show JAK inhibitory activity. We focused on the structure of 6 and attempted to enhance inhibitory activity toward JAK3, as the 1H-pyrrolo[2,3-b]pyridine ring mimicked the pyrrolopyrimidine scaffold of 1 and 2. Here, we report findings from a structure–activity relationship (SAR) study of a series of 1H-pyrrolo[2,3-b]pyridine derivatives on JAK3 inhibitory activity as novel immunomodulators.

Table 1. SARs of C5-Substitutent of 1H-Pyrrolo[2,3-b]pyridine Derivatives

Compd.	R1	R2	h JAK3 IC50a) (nM)	h JAK1 IC50a) (nM)	h JAK2 IC50a) (nM)	Rat T cell IC50b) (nM)
6	H	Me	1100	2900	1800	NTd)
11a	CONH2	Me	1600	10000	5300	2400
14a	CONH2	H	14	55	50	120
15a		H	85	230	57	350
15b		H	3400	5000	2000	3200
15c		H	1200	570	640	NTd)
1c)			0.8	3.7	3.1	23

a) IC₅₀ values are the average of duplicate experiments except for compound 1. b) Inhibitory effect on IL-2-stimulated T cell proliferation using rat spleen cells (n=2). c) IC₅₀ values of JAK assays are the average of four experiments. d) NT=not tested.

Chemistry

As shown in Chart 1, commercially available compound 3 was treated with 2-(trimethylsilyl)ethoxymethyl (SEM) chloride, followed by a Pd-catalyzed coupling reaction with N-methylcyclohexylamine to achieve insertion of an amino group to the C4-position. Deprotection of the SEM group of compound 5 was conducted by treatment with trifluoroacetic acid (TFA), followed by alkalization in the presence of 1,2-diaminoethane to afford the desired compound 6.

Chart 1

Reagents and conditions: (a) SEMCl, NaH, DMF, 0°C; (b) N-Methylcyclohexylamine, Pd(OAc)₂, 2-(di-tert-butylphosphino)biphenyl, Cs₂CO₃, 110°C; (c) 1) TFA, CH₂Cl₂, room temperature, 2) 1 M NaOH, 1,2-diaminoethane, CH₂Cl₂, room temperature.

General synthetic routes of the 1H-pyrrolo[2,3-b]pyridine-5-carboxamide derivatives are shown in Charts 2–4. As shown in Chart 2, protection of compound 3 by treatment of triisopropylsilyl (TIPS) chloride and NaH gave compound 7, which was subjected to ortho-lithiation by sec-BuLi, followed by addition of ethyl chloroformate to introduce an ester group at the C5-position.²⁷⁾ Subsequent deprotection of the TIPS group using tetra-n-butylammonium fluoride (TBAF) afforded 8. Conversion of the ethyl ester group of 8 to a carbamoyl group was carried out by basic hydrolysis and subsequent amidation using carbonyldiimidazole (CDI) and aqueous ammonia, thereby yielding the carboxamide intermediate 10. Nucleophilic substitution at the C4-position with a variety of amines under microwave irradiation afforded the desired compounds 11a–k. The reaction is convenient for conversion of C4-amino group of the 1H-pyrrolo[2,3-b]pyridine-5-carboxamide derivatives.

Chart 2

Reagents and conditions: (a) TIPSCl, NaH, DMF, 5°C; (b) 1) sec-BuLi, ethyl chloroformate, THF, −78°C, 2) TBAF, THF, room temperature; (c) 1 M NaOH, EtOH, 60°C; (d) CDI, DMF, room temperature, then 28% NH₄OH, room temperature; (e) Amines, DIPEA, n-BuOH or NMP, microwave, 150–160°C.

Chart 3

Reagents and conditions: (a) Amines, DIPEA, n-BuOH, microwave, 160°C; (b) 2 M NaOH, EtOH, reflux; (c) HOBt, EDC, DMF, 60°C, then 28% NH₄OH, room temperature.

Chart 4

Reagents and conditions: (a) Amines, HOBt, EDC, DMF, 55°C.

Chart 3 shows alternative synthetic routes of the 1H-pyrrolo[2,3-b]pyridine-5-carboxamide derivatives. The intermediate 8 was reacted with a variety of amines under microwave irradiation to give corresponding C4-substituted compounds 12a–c, after which the ester groups were hydrolyzed to give carboxylic acids 13a–c. Condensation of 13a–c with aqueous ammonia using 1-hydroxybenzotriazole (HOBt) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) prepared the desired compounds 14a–c.

As shown in Chart 4, carboxylic acid in Chart 3 is available for the conversion of the amide moiety at the C5-position. Compound 13a was condensed with several amines using HOBt and EDC to afford the N-substituted-1H-pyrrolo[2,3-b]pyridine-5-carboxamide derivatives 15a–c.

Results and Discussion

Our synthesized compounds were evaluated for inhibitory activity toward human JAK3, and IC₅₀ values were calculated. JAK1 and JAK2 inhibitory activity were also investigated for JAK selectivity. To evaluate the cellular immunomodulating effect, the inhibitory effect on IL-2-stimulated T cell proliferation using rat spleen cells was tested.

As shown in Table 1, our initial compound, 1H-pyrrolo[2,3-b]pyridine analogue 6 showed potential inhibitory activity toward JAKs (JAK3, JAK1, and JAK2 IC₅₀=1100, 2900, and 1800 nM, respectively). The ligand binding pocket of JAK3 is known to be comprised of a hinge region in the ATP-binding site and a spatial cavity surrounded by hydrophobic amino acid residues.²⁸⁾ In order to improve JAK3 inhibitory activity, interaction with the JAK3 binding pocket is important. We therefore performed docking calculations of our compounds to human JAK3 (PDB code: 3LXK²⁸⁾), indicating that the 1H-pyrrolo[2,3-b]pyridine scaffold was located near the hinge region and the C4-substituent was directed to the hydrophobic cavity (Fig. 2). Given that 6 had a space allowing a substituent in C5-position, introduction of a carbamoyl group was investigated. Results showed that 1H-pyrrolo[2,3-b]pyridine-5-carboxamide derivative 11a maintained moderate JAK3 inhibitory activity (IC₅₀=1600 nM), equipotent to 6. On the other hand, when an N-methyl group at the C4-position of 6 was deleted to solve the steric hindrance with C5-carbamoyl group, compound 14a achieved an over 100-fold increase in JAK3 inhibitory activity (IC₅₀=14 nM) compared to 11a. Although JAK1 and JAK2 inhibitory activity of 14a were also increased, several-fold selectivity for JAK3 was maintained. We then investigated the acceptability of modifying the carbamoyl group. Given that N-methyl carboxamide analogue 15a maintained moderate JAK3 inhibitory activity (IC₅₀=85 nM), introduction of other substituted groups was examined. However, 15b and 15c showed substantial decreases in JAK3 inhibitory activity (IC₅₀=3400 and 1200 nM, respectively), indicating intolerance to introduction of bulky aliphatic or aromatic substituted carbamoyl groups. Therefore, 1H-pyrrolo[2,3-b]pyridine-5-carboxamide was deemed a potential scaffold for development of a JAK inhibitor. Of note, 14a showed moderate cellular inhibition of IL-2-stimulated rat T cell proliferation (IC₅₀=120 nM).

Fig. 2. Predicted Binding Mode of Compound 6 to Human JAK3 (PDB Code: 3LXK)

Given the above findings, we investigated conversion of the C4-substituent of 1H-pyrrolo[2,3-b]pyridine-5-carboxamide derivatives, as shown in Table 2. Similarly to N-methylcyclohexylamine (11a), conversion to piperidine (11b) dramatically decreased JAK inhibitory activity (JAK3, IC₅₀=3200 nM), indicating that the NH moiety at the C4-position was critical for JAK inhibition. Therefore, a variety of secondary amine derivatives were investigated. The size of the cyclohexyl ring of 14a was altered, and incorporation of 3-, 5-, and 7-membered cycloalkyl rings was investigated. The JAK inhibitory activity of these compounds increased with ring size, in order of cyclopropane (14b), cyclopentane (11c), and cycloheptane (11d). Compared to 14a, the cycloheptylamine analogue 11d showed potent JAK inhibitory activity, particularly for JAK3 (JAK3, JAK1, and JAK2 IC₅₀=3.5, 25, and 13 nM, respectively), suggesting the effect of hydrophobic interaction for JAK inhibition.

Table 2. SARs of C4-Substitutent of 1H-Pyrrolo[2,3-b]pyridine-5-carboxamide Derivatives

Compd.	R	h JAK3 IC50a) (nM)	h JAK1 IC50a) (nM)	h JAK2 IC50a) (nM)	Rat T cell IC50b) (nM)
14a		14	55	50	120
11b		3200	1900	2400	NTc)
14b		110	NTc)	470	2600
11c		15	45	44	100
11d		3.5	25	13	63
11e		7.7	59	60	230
11f		25	290	71	300
14c		5.1	47	30	86

a) IC₅₀ values are the average of duplicate experiments. b) Inhibitory effect on IL-2-stimulated T cell proliferation using rat spleen cells (n=2). c) NT=not tested.

We next examined cleavage of the cyclohexyl ring of 14a, and 3-pentylamine analogue 11e showed increased JAK3 inhibitory activity over 14a (IC₅₀=7.7 nM). In contrast, replacement of the cyclohexylamine with cyclohexylmethylamine (11f) led to a slight decrease in JAK3 inhibitory activity (IC₅₀=25 nM) compared to 14a, indicating that a branched structure in the α-position of the C4-amino group was also important for achieving good inhibitory activity. We also investigated methylation of the cyclohexyl ring of 14a, and 2-methyl-cyclohexylamine analogue 14c had approximately 3-fold increased JAK3 inhibitory activity with equipotent JAK1 and JAK2 inhibitory activity (JAK3, JAK1, and JAK2 IC₅₀=5.1, 47, and 30 nM, respectively) compared to 14a. In the cellular assay, 11d and 14c had increased inhibitory activity (IC₅₀=63 and 86 nM, respectively) compared to 14a, likely due to an increase in JAK3 and JAK1 inhibitory activity. As for JAK2 inhibitory activity, 14c had less potent than 11d. Given its potential profile with regard to JAK inhibitory activity and cellular potency, 14c was selected for further chemical modification and biological evaluation.

As shown in Table 3, the effect of substituent on the cyclohexyl ring moiety of 14c was investigated. At the 2-position of the ring, conversion to an ethyl (11g) or di-methyl group (11h) maintained potent JAK3 inhibitory activity (IC₅₀=5.2 and 3.7 nM, respectively) and increased cellular inhibitory activity of IL-2-stimulated T cell proliferation (IC₅₀=30 and 37 nM, respectively). We next assessed conversion of the substituted position of the methyl group. The 3-methylcyclohexylamine analogue 11i showed potent JAK3 inhibitory activity (IC₅₀=3.0 nM), while the 4-methylcyclohexylamine analogue 11j had slightly decreased inhibitory activity (IC₅₀=14 nM) compared to 14c. It was revealed that introduction of a substituted group to the cyclohexyl ring moiety was effective in increasing JAK3 inhibition. Because a number of compounds showed good in vitro pharmacological activity, their metabolic stability was tested using rat liver microsomes prior to in vivo evaluation. Results showed that 14c, 11g, 11h, and 11i had high intrinsic clearance (CL_int) values of more than 1000 mL/min/kg.

Table 3. SARs of Modification of Cyclohexyl Ring Moiety in C4-Substituent

Compd.	R	h JAK3 IC50a) (nM)	h JAK1 IC50a) (nM)	h JAK2 IC50a) (nM)	Rat T cell IC50b) (nM)	Rat CLintc) (mL/min/kg)	c Log Pd)
14c		5.1	47	30	86	>1000	2.8
11g		5.2	55	48	30	>1000	3.3
11h		3.7	54	26	37	>1000	3.1
11i		3.0	29	23	25	>1000	2.8
11j		14	50	38	97	NTe)	2.8
11k		9.7	280	190	530	267	1.0

a) IC₅₀ values are the average of duplicate experiments. b) Inhibitory effect on IL-2-stimulated T cell proliferation using rat spleen cells (n=2). c) In vitro metabolism with rat liver microsomes in presence of NADPH-generating system (n=2). d) c Log P values are calculated using ACD/Labs Software, version 12.01. e) NT=not tested.

In order to clarify the metabolic pathway of this series of compounds, 14c was investigated to determine the profiles of its metabolites in rat liver microsomes (Fig. 3). Findings revealed that the scaffold moiety, 1H-pyrrolo[2,3-b]pyridine-5-carboxamide, was hardly metabolized, with the methylcyclohexyl ring mainly oxidized to afford hydroxylated compound 11k. The major metabolite 11k was synthesized and investigated for its biological profile, showing good JAK3 inhibitory activity (IC₅₀=9.7 nM) and significantly reduced values for microsomal clearance (CL_int=267 mL/min/kg) compared to the parent compound 14c. The result indicated that decrease in molecular lipophilicity by introducing polar functional group was effective in improving metabolic stability (c Log P=1.0 and 2.8 for 11k and 14c, respectively).

Fig. 3. Metabolic Pathway of 14c in Rat Liver Microsomes

Pharmacokinetic (PK) study of 14c and 11k was investigated in rats, and the parameters are shown in Table 4. In intravenous dosing, 14c showed high total clearance (CL_tot=62.4 mL/min/kg), comparable to hepatic blood flow in rats, while, in oral dosing, 14c resulted in low exposure of plasma concentration (C_max=126 ng/mL and area under curve from 0 to 24 h (AUC)_0–24=303 ng·h/mL) and low oral bioavailability (F=11%). In a parallel artificial membrane permeability assay (PAMPA),²⁹⁾ 14c showed acceptable membrane permeability (P_e=39×10⁻⁶ cm/s). Findings suggested that the poor in vitro metabolic stability of 14c was reflected in the in vivo PK profiles. In contrast, 11k showed over 5-fold improved oral absorption (C_max=1129 ng/mL and AUC_0–24=1523 ng·h/mL), possibly due to reduction of microsomal CYP-mediated drug metabolism. Chemical modification of 1H-pyrrolo[2,3-b]pyridine-5-carboxamide derivatives revealed that the hydrophobicity of the C4-substituent was critical for PK profile as well as pharmacological activity.

Table 4. Pharmacokinetic Parameters of 14c and 11k in Rats

Compd.	Intravenouslya)				per osb)
	AUC0–24 (ng·h/mL)	t1/2 (h)	Vdss (L/kg)	CLtot (mL/min/kg)	Cmax (ng/mL)	tmax (h)	AUC0–24 (ng·h/mL)	Fc) (%)
14c	810	0.4	1.8	62.4	126	1.3	303	11
11k	NTd)	NTd)	NTd)	NTd)	1129	1.5	1523	—

a) Dosed at 3 mg/kg (n=2). b) Dosed at 10 mg/kg (n=3). c) F=bioavailability. d) NT=not tested.

Our optimization study on C4- and C5-substitutents of the 1H-pyrrolo[2,3-b]pyridine scaffold led to a large increase in JAK3 inhibitory activity, and 14c achieved more than 200-fold increase in activity over the initial compound 6. To validate the effect of structural modification for JAK3 inhibitory activity, we analyzed docking calculations of 1H-pyrrolo[2,3-b]pyridine-5-carboxamide derivatives to human JAK3. Figure 4A shows the predicted binding mode of 14c to human JAK3. This result shows that the 1H-pyrrolo[2,3-b]pyridine moiety closely interacted with Glu903 and Leu905 in the hinge region, and the N1-hydrogen atom and N7-nitrogen atom served as hydrogen donor and acceptor, respectively. In addition to the hydrogen bond interaction, the proton of the C2-position interacted with gate-keeper amino acid Met902, and the aromaticity of the pyrrolopyridine ring contributed to CH–π interaction with Val836 and Leu828. The 1H-pyrrolo[2,3-b]pyridine-5-carboxamide moiety was overlapped with the pyrrolopyrimidine of 1, while the C4-substituent occupied the space in the hydrophobic cavity, corresponding to the aminopiperidine moiety of 1.

Fig. 4. (A) Predicted Binding Mode of Compound 14c to Human JAK3 (PDB Code: 3LXK, Orange: 14c, Green: 1). (B) 14c with WaterMap Unfavorable Water Molecules (ΔG=>2.0 kcal/mol)

With respect to C4- and C5-positions of 14c, an intramolecular hydrogen bond was observed between the NH proton of the C4-amine and the carbonyl group of the C5-carboxamide. The intramolecular interaction allowed the cycloalkyl group at C4-position to direct efficiently to the hydrophobic cavity of JAK3, which had a large space capable of accommodating bulky substituted groups. It was suggested that conformational restriction by the intramolecular hydrogen bond contributed to enhance affinity to the hydrophobic cavity, resulting in potent JAK inhibitory activity of 14c compared to 6 and 11a. Further, the carbamoyl group was located around the polar and narrow hinge region, where a small functional group might be tolerable. Substitution of the carbamoyl group seemed to hinder access to the hinge region, which was disadvantageous for binding to JAK3—findings consistent with SARs showing that N-substituted carboxamide analogues (15a–c) had lower JAK3 inhibitory activity than 14a. Given that both hydrogen bond interaction with the hinge region and hydrophobic interaction with the cavity are essential for binding to JAK3, the 4-cycloalkylamino-1H-pyrrolo[2,3-b]pyridine-5-carboxamide derivatives were favorably located in the binding pocket of JAK3.

In addition to docking calculations, to analyze SARs on the C4-substitutent, we examined predicted binding energy using the WaterMap program, a recently developed protocol that combines molecular dynamics, solvent clustering, and statistical thermodynamics to assess the enthalpy, entropy, and free energy (ΔG) of water “hydration sites.”^30,31) The ΔG was computed for the human JAK3 structure (PDB code: 3LXK), and 11 unfavorable waters (ΔG=>2.0 kcal/mol) were detected in the ligand binding pocket of human JAK3 (Fig. 4B).

Based on the docking calculations of our series of compounds, the 1H-pyrrolo[2,3-b]pyridine-5-carboxamide scaffold displaced 5 unfavorable water molecules (W1 to W5) in the hinge region. In contrast, the aliphatic ring moiety of the C4-substituent contributed to displacement of 3 unfavorable water molecules (W6 to W8) located in the hydrophobic site. As shown in Fig. 5, weak correlation (R²=0.45) was observed between the experimental inhibitory activity (pIC₅₀) and the WaterMap free energy liberation (ΔG_pred). The ΔG_pred estimates the desolvation energy generated when a compound displaces the waters in the binding pocket. In the hydrophobic site, 3 unfavorable water molecules (W6 to W8) were displaced to enhance the inhibitory activity, associated with an increase in spatial volume of the C4-substituent. This observation was consistent with SAR findings that enhancement of the bulkiness of the C4-substituent led to an increase in JAK3 inhibitory activity, and that substituted cyclohexylamine analogues (14c and 11g) were more potent than unsubstituted cyclohexylamine analogue 14a. The results of computational analyses are useful for further optimization of the 1H-pyrrolo[2,3-b]pyridine-5-carboxamide derivatives.

Fig. 5. Correlation between the Experimental Activity and the WaterMap Free Energy Liberation of Binding Site Waters for 1H-Pyrrolo[2,3-b]pyridine-5-carboxamide Derivatives in Tables 2 and 3

Conclusion

We synthesized 1H-pyrrolo[2,3-b]pyridine derivatives as novel immunomodulators targeting JAK3 and evaluated the biological profiles with respect to JAK inhibitory activity and immunomodulating effects on T cell proliferation. The results of a SAR study revealed that a carbamoyl group at the C5-position and a substituted cycloalkylamino group at the C4-position of the 1H-pyrrolo[2,3-b]pyridine scaffold played an important role in increasing JAK3 inhibitory activity, and 14c was identified as a potent and moderately selective JAK3 inhibitor. Modification of the C4-substituent of 14c led to increase in JAK3 inhibitory activity and cellular inhibitory activity on IL-2-stimulated T cell proliferation (11g, 11h, and 11i). However, these compounds had high molecular lipophilicity and showed poor metabolic stability in liver microsomes. We clarified metabolic pathway of 14c to convert to the hydroxylated compound 11k. Introduction of a hydroxy group to the hydrophobic C4-substituent decreased molecular lipophilicity, and thereby 11k showed a reduction in metabolic clearance and subsequent improvement of oral absorption. Docking calculations to JAK3 supported putative docking modes of 1H-pyrrolo[2,3-b]pyridine-5-carboxamide derivatives, indicating effective occupancy of the JAK3 binding site to interact with the hinge region and hydrophobic cavity. In addition, using the WaterMap program, the displacement effect against unfavorable water molecules in the binding pocket was correlated with JAK3 inhibitory activity. Compared with the pyrrolopyrimidine derivatives such as 1 and 2, C5-carbamoyl group of the 1H-pyrrolo[2,3-b]pyridine scaffold allows for intramolecular hydrogen bond to maintain active conformation in the JAK3 binding site. Given the findings, the 1H-pyrrolo[2,3-b]pyridine-5-carboxamide derivatives are promising as novel immunomodulators targeting JAK3 for the treatment of immune diseases.

Experimental

¹H-NMR spectra were recorded on a Brucker Biospin Avance400 or AV400M spectrometer. Chemical shifts are expressed in δ units using tetramethylsilane as an internal standard (NMR peak description: s=singlet; d=doublet; t=triplet; q=quartet; m=multiplet; br=broad peak). Mass spectra (MS) were recorded on a Hitachi LC/3DQMS M8000 or an Agilent HP1100 LC/MCD spectrometer. High-resolution mass spectroscopy (HR-MS) spectra were recorded on a Waters LCT Premier XE. Column chromatography was carried out using silica gel 60N (Kanto Chemical, 63–210 µm) or HI-FLASH™ Column (Yamazen). The purity of all compounds screened in biological assays was >95%, as judged by either or both HPLC or elemental analyses. HPLC analysis was conducted using a Hitachi LaChrom Elite system with a TOSOH TSK-gel ODS-80TM column (150 mm×4.6 mm, 5 µm) at 40°C and a 1.0 mL/min flow rate using acetonitrile and 0.01 M HClO₄ aqueous solution as the eluent. Elemental analysis values were recorded on an Elementar Vario EL III or YANACO MT-6 and were within 0.4% of the theoretical values Calcd for C, H, and N. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of AstellasPharma Inc. Further, the AstellasPharma Inc. Tsukuba Research Center was awarded Accreditation Status by the AAALAC International. All efforts were made to minimize the number of animals used and to avoid suffering and distress.

4-Chloro-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridine (4)

To a solution of 4-chloro-1H-pyrrolo[2,3-b]pyridine (3) (4.6 g, 30 mmol) in N,N-dimethylformamide (DMF) (46 mL), 60% NaH (1.4 g, 36 mmol) was added portionwise at 0°C. The mixture was stirred at the same temperature for 1 h, and then SEMCl (6.8 mL, 39 mmol) was added. After stirring at the same temperature for additional 4 h, the mixture was poured into H₂O (150 mL) and extracted with ether (three times). The organic layer was then washed with H₂O, dried over MgSO₄, and concentrated under reduced pressure. The residue was purified by column chromatography (n-hexane/EtOAc=97/3 to 88/12) to give the title compound (7.16 g, 84%). ¹H-NMR (DMSO-d₆) δ: −0.12 (9H, s), 0.80 (2H, t, J=1.6 Hz), 3.50 (2H, t, J=1.6 Hz), 5.64 (2H, s), 6.60 (1H, d, J=3.7 Hz), 7.28 (1H, d, J=5.1 Hz), 7.77 (1H, d, J=3.7 Hz), 8.24 (1H, d, J=5.1 Hz). MS electrospray ionization (ESI) m/z: 283 (M+H)⁺.

N-Cyclohexyl-N-methyl-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-pyrrolo[2,3-b]pyridin-4-amine (5)

To a mixture of 4 (350 mg, 1.24 mmol) and N-methylcyclohexylamine (1.6 mL, 12.4 mmol) were added 2-(di-tert-butylphosphino)biphenyl (74 mg, 0.25 mmol), Cs₂CO₃ (403 mg, 1.24 mmol), and Pd(OAc)₂ (28 mg, 0.12 mmol). The mixture was stirred under N₂ gas atmosphere at 110°C for 2 h. After cooling to room temperature, CHCl₃ and MeOH were added for dilution. After stirring at room temperature for 10 min, the mixture was filtrated through a pad of Celite®. The filtrate was concentrated under reduced pressure, and the residue was purified by column chromatography (n-hexane/EtOAc=9/1 to 4/1) to give the title compound (145 mg, 33%). ¹H-NMR (DMSO-d₆) δ: −0.08 (9H, s), 0.81 (2H, t, J=8.0 Hz), 1.09–1.23 (1H, m), 1.02–1.05 (1H, m), 1.31–1.45 (2H, m), 1.56–1.68 (3H, m), 1.71–1.87 (4H, m), 2.95 (3H, s), 3.49 (2H, t, J=8.0 Hz), 3.90–3.99 (1H, m), 5.52 (1H, s), 6.28 (1H, d, J=5.6 Hz), 6.49 (1H, d, J=4.0 Hz), 7.31 (1H, d, J=4.0 Hz), 7.88 (1H, d, J=5.6 Hz). MS (ESI) m/z: 360 (M+H)⁺.

N-Cyclohexyl-N-methyl-1H-pyrrolo[2,3-b]pyridin-4-amine (6)

To a solution of 5 (140 mg, 0.39 mmol) in CH₂Cl₂ (1.4 mL) was added TFA (1.5 mL, 19.6 mmol). The mixture was stirred at room temperature for 2 h. After concentration under reduced pressure, the residue was dissolved with CH₂Cl₂ (1.4 mL). To the solution were added 1 M NaOH aqueous solution (1.6 mL, 1.56 mmol) and 1,2-diaminoethane (78 µL, 1.17 mmol), and then the mixture was stirred at room temperature for 16 h, extracted with CHCl₃, and washed with H₂O. The organic layer was then dried over MgSO₄ and concentrated under reduced pressure. The residue was purified by column chromatography (CHCl₃/MeOH=100/0 to 95/5) to give the title compound (58 mg, 65%). ¹H-NMR (DMSO-d₆) δ: 1.06–1.22 (1H, m), 1.29–1.44 (2H, m), 1.54–1.69 (3H, m), 1.69–1.87 (4H, m), 2.92 (3H, s), 3.91–4.02 (1H, m), 6.19 (1H, d, J=5.8 Hz), 6.4 (1H, d, J=3.7 Hz), 7.13 (1H, d, J=3.6 Hz), 7.82 (1H, d, J=5.5 Hz), 11.25 (1H, s). MS (ESI) m/z: 230 (M+H)⁺. Anal. Calcd for C₁₄H₁₉N₃·0.1H₂O: C, 72.75; H, 8.37; N, 18.18. Found: C, 72.96; H, 8.36; N, 18.2.

4-Chloro-1-(triisopropylsilyl)-1H-pyrrolo[2,3-b]pyridine (7)

To a solution of 3 (25 g, 164 mmol) in DMF (250 mL), 60% NaH (7.9 g, 197 mmol) was added portionwise at 5°C. The mixture was stirred at the same temperature for 1 h, and then TIPSCl (36 mL, 172 mmol) was added. After stirring for additional 1 h, EtOAc and H₂O were added to the mixture. The organic layer was separated, washed with saturated NaHCO₃ aqueous solution and brine, dried over MgSO₄, and concentrated under reduced pressure. The residue was purified by column chromatography with n-hexane to give the title compound (40 g, 79%). ¹H-NMR (DMSO-d₆) δ: 1.06 (18H, d, J=7.5 Hz), 1.79–1.91 (3H, m), 6.68 (1H, d, J=3.5 Hz), 7.24 (1H, d, J=5.2 Hz), 7.60 (1H, d, J=3.5 Hz), 8.20 (1H, d, J=5.2 Hz). MS (ESI) m/z: 309 (M+H)⁺.

Ethyl 4-Chloro-1H-pyrrolo[2,3-b]pyridine-5-carboxylate (8)

To a solution of 7 (15 g, 48.6 mmol) in tetrahydrofuran (THF) (150 mL), 1 M sec-BuLi in cyclohexane and n-hexane (97.1 mL, 97.1 mmol) was added dropwise at −78°C under Ar gas atmosphere. The mixture was stirred at the same temperature for 1 h, and then ethyl chloroformate (9.29 mL, 97.1 mmol) was added at −78°C. After stirring at the same temperature for additional 30 min, the mixture was quenched with saturated NH₄Cl aqueous solution and extracted with EtOAc. The extract was washed with H₂O and brine, dried over MgSO₄, and concentrated under reduced pressure. The residue was dissolved in THF (120 mL), and 1 M TBAF in THF (56 mL, 56 mmol) was added. The mixture was stirred at room temperature for 1 h and then diluted with EtOAc, washed with H₂O, dried over MgSO₄, and concentrated under reduced pressure. The residue was triturated with IPE, and the precipitate was filtrated to give the title compound (9.6 g, 88%). ¹H-NMR (DMSO-d₆) δ: 1.36 (3H, t, J=7.1 Hz), 4.36 (2H, q, J=7.1 Hz), 6.64–6.67 (1H, m), 7.70–7.73 (1H, m), 8.71 (1H, s), 12.41 (1H, br). MS (ESI) m/z: 223 (M−H)⁻.

4-Chloro-1H-pyrrolo[2,3-b]pyridine-5-carboxylic Acid (9)

To a solution of 8 (10.5 g, 46.7 mmol) in EtOH (84 mL) was added 1 M NaOH aqueous solution (140 mL, 140 mmol), and the mixture was stirred at 60°C for 1.5 h. The mixture was cooled to 4°C and acidified with 1 M HCl aqueous solution. The precipitate was filtrated and washed with H₂O to give the title compound (9.0 g, 98%). ¹H-NMR (DMSO-d₆) δ: 6.62–6.64 (1H, m), 7.67–7.70 (1H, m), 8.71 (1H, s), 12.32 (1H, br s), 13.22 (1H, br s). MS (ESI) m/z: 195 (M−H)⁻.

4-Chloro-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (10)

To a solution of 9 (9.0 g, 45.8 mmol) in DMF (72 mL) was added CDI (8.2 g, 50.4 mmol), and the mixture was stirred at room temperature for 1 h. To the mixture was added 28% NH₄OH aqueous solution (10.0 mL, 164 mmol) dropwise at 4°C, and the mixture was stirred at room temperature for 1 h. To the mixture was added EtOAc, and the precipitate was filtrated to give the title compound (7.5 g, 84%). ¹H-NMR (DMSO-d₆) δ: 6.57 (1H, d, J=3.6 Hz), 7.63 (1H, br s), 7.65 (1H, d, J=3.6 Hz), 7.90 (1H, br s), 8.29 (1H, s), 12.12 (1H, br). MS (ESI) m/z: 218 (M+Na)⁺.

4-[Cyclohexyl(methyl)amino]-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (11a)

In the vessel of a microwave reactor, 10 (25 mg, 0.128 mmol) and N-methylcyclohexylamine (0.085 mL, 0.639 mmol) were suspended in n-BuOH (0.2 mL). The vessel was then sealed, and the mixture was reacted at 150°C for 30 min under microwave irradiation. The mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (CHCl₃/MeOH=100/0 to 95/5) to give the title compound (9 mg, 26%). ¹H-NMR (DMSO-d₆) δ: 0.97–1.37 (3H, m), 1.42–1.88 (7H, m), 2.91 (3H, s), 3.49–3.66 (1H, m), 6.46–6.52 (1H, m), 7.24–7.33 (2H, m), 8.07 (1H, br), 8.21 (1H, s), 11.56 (1H, br s). MS (ESI) m/z: 273 (M+H)⁺. Anal. Calcd for C₁₅H₂₀N₄O·0.5H₂O: C, 64.03; H, 7.52; N, 19.91. Found: C, 63.98; H, 7.52; N, 20.15.

4-(Piperidin-1-yl)-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (11b)

The title compound was prepared from 10 and piperidine in accordance with the procedure for preparing 11a in 38% yield. ¹H-NMR (DMSO-d₆) δ: 1.57–1.73 (6H, m), 3.27–3.42 (4H, m), 6.56–6.59 (1H, m), 7.27–7.36 (2H, m), 7.85–7.93 (1H, m), 8.18 (1H, s), 11.56 (1H, br). MS (ESI) m/z: 245 (M+H)⁺. Anal. Calcd for C₁₃H₁₆N₄O: C, 63.91; H, 6.60; N, 22.93. Found: C, 63.81; H, 6.62; N, 22.81.

4-(Cyclopentylamino)-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (11c)

The title compound was prepared from 10 and cyclopentylamine in accordance with the procedure for preparing 11a in 29% yield. ¹H-NMR (DMSO-d₆) δ: 1.43–1.78 (6H, m), 1.90–2.10 (2H, m), 4.35–4.50 (1H, m), 6.57–6.63 (1H, m), 7.00 (1H, br), 7.08–7.14 (1H, m), 7.64 (1H, br), 8.34 (1H, s), 9.61–9.70 (1H, m), 11.43 (1H, br s). MS (ESI) m/z: 245 (M+H)⁺. Anal. Calcd for C₁₃H₁₆N₄O·0.1H₂O: C, 63.45; H, 6.64; N, 22.77. Found: C, 63.64; H, 6.64; N, 22.62.

4-[(Cyclohexylmethyl)amino]-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (11f)

The title compound was prepared from 10 and cyclohexanemethylamine in accordance with the procedure for preparing 11a in 39% yield. ¹H-NMR (DMSO-d₆) δ: 0.92–1.34 (5H, m), 1.50–1.88 (6H, m), 3.45 (2H, dd, J=6.0, 12.0 Hz), 6.54–6.60 (1H, m), 6.98 (1H, br), 7.08–7.10 (1H, m), 7.65 (1H, br), 8.34 (1H, s), 9.61–9.66 (1H, m), 11.43 (1H, br s). MS (ESI) m/z: 273 (M+H)⁺. Anal. Calcd for C₁₅H₂₀N₄O·0.1H₂O: C, 65.72; H, 7.43; N, 20.44. Found: C, 65.83; H, 7.38; N, 20.29.

4-{[(1S,2R)-2-(Hydroxymethyl)cyclohexyl]amino}-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (11k)

The title compound was prepared from 10 and [(1R,2S)-2-aminocyclohexyl]methanol hydrochloride in accordance with the procedure for preparing 11a in 41% yield. ¹H-NMR (DMSO-d₆) δ: 1.13–2.00 (9H, m), 3.19–3.56 (2H, m), 4.51–4.64 (1H, m), 6.78–6.84 (1H, m), 7.29–7.36 (1H, m), 7.69 (1H, br), 8.38 (1H, br), 8.53 (1H, s), 10.96–11.05 (1H, m), 12.51 (1H, br s). MS (ESI) m/z: 289 (M+H)⁺. HR-MS m/z: 289.1666 (M+H)⁺ (Calcd for C₁₅H₁₈N₄O: 288.1586).

4-{[(1S,2R)-2-Ethylcyclohexyl]amino}-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (11g)

In the vessel of a microwave reactor, to a solution of 10 (100 mg, 0.511 mmol) in N-methylpyrrolidone (NMP) (1 mL) were added (1S,2R)-2-ethylcyclohexanamine hydrochloride (167 mg, 1.02 mmol) and N,N-diisopropylethylamine (DIPEA) (0.27 mL, 1.53 mmol). The vessel was sealed, and the mixture was reacted at 160°C for 1.5 h under microwave irradiation. After cooling to room temperature, H₂O was added, and the mixture was extracted with CHCl₃. The organic layer was separated, washed with H₂O, dried over MgSO₄, and concentrated under reduced pressure. The residue was purified by column chromatography (CHCl₃/MeOH=100/0 to 90/10) to give the title compound (58 mg, 40%). ¹H-NMR (DMSO-d₆) δ: 0.78 (3H, t, J=7.2 Hz), 1.21–1.68 (10H, m), 1.82–1.89 (1H, m), 4.29–4.32 (1H, m), 6.51–6.53 (1H, m), 7.00 (1H, br), 7.08–7.11 (1H, m), 7.67 (1H, br), 8.35 (1H, s), 9.87–9.92 (1H, m), 11.43 (1H, br s). MS (ESI) m/z: 287 (M+H)⁺. Anal. Calcd for C₁₆H₂₂N₄O·0.3H₂O: C, 65.86; H, 7.81; N, 19.20. Found: C, 65.65; H, 7.43; N, 19.25.

4-(Cycloheptylamino)-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (11d)

The title compound was prepared from 10 and cycloheptylamine in accordance with the procedure for preparing 11g in 57% yield. ¹H-NMR (DMSO-d₆) δ: 1.46–1.71 (10H, m), 1.89–2.10 (2H, m), 4.07–4.23 (1H, m), 6.50–6.54 (1H, m), 7.00 (1H, br), 7.08–7.13 (1H, m), 7.62 (1H, br), 8.31 (1H, s), 9.67 (1H, d, J=8.1 Hz), 11.43 (1H, br s). MS (ESI) m/z: 273 (M+H)⁺. Anal. Calcd for C₁₅H₂₀N₄O·0.3H₂O: C, 64.86; H, 7.48; N, 20.17. Found: C, 64.82; H, 7.38; N, 20.10.

4-(Pentan-3-ylamino)-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (11e)

The title compound was prepared from 10 and 3-aminopentane in accordance with the procedure for preparing 11g in 24% yield. ¹H-NMR (DMSO-d₆) δ: 0.92 (6H, t, J=7.6 Hz), 1.48–1.69 (4H, m), 3.87–3.97 (1H, m), 6.50–6.53 (1H, m), 6.93 (1H, br), 7.08–7.12 (1H, m), 7.69 (1H, br), 8.35 (1H, s), 9.55 (1H, d, J=8.8 Hz), 11.42 (1H, br s). MS (ESI) m/z: 247 (M+H)⁺. Anal. Calcd for C₁₃H₁₈N₄O: C, 63.39; H, 7.37; N, 22.75. Found: C, 63.22; H, 7.24; N, 22.65.

4-[(2,2-Dimethylcyclohexyl)amino]-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (11h)

The title compound was prepared from 10 and 2,2-dimethylcyclohexan-1-amine hydrochloride in accordance with the procedure for preparing 11g in 66% yield. ¹H-NMR (DMSO-d₆) δ: 0.94 (3H, s), 1.00 (3H, s), 1.30–1.54 (6H, m), 1.63–1.71 (1H, m), 1.83–1.91 (1H, m), 3.69–3.77 (1H, m), 6.47–6.50 (1H, m), 6.95 (1H, br), 7.08–7.12 (1H, m), 7.69 (1H, br), 8.34 (1H, s), 9.83 (1H, d, J=8.8 Hz), 11.42 (1H, br s). MS (ESI) m/z: 287 (M+H)⁺. HR-MS m/z: 287.1867 (M+H)⁺ (Calcd for C₁₆H₂₂N₄O, 286.1794).

4-[(3-Methylcyclohexyl)amino]-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (11i)

The title compound was prepared from 10 and 3-methylcyclohexylamine in accordance with the procedure for preparing 11g in 77% yield. ¹H-NMR (DMSO-d₆) δ: 0.87–0.92 (3H, m), 0.97–1.81 (8H, m), 2.02–2.12 (1H, m), 3.82–3.92 (0.4H, m), 4.33–4.39 (0.6H, m), 6.47–6.55 (1H, m), 6.98 (1H, br), 7.10–7.16 (1H, m), 7.76 (1H, br), 8.35 (0.4H, s), 8.36 (0.6H, s), 9.60 (0.4H, d, J=7.6 Hz), 10.01 (0.6H, d, J=8.4 Hz), 11.49 (1H, br s). MS (ESI) m/z: 273 (M+H)⁺. HR-MS m/z: 273.1719 (M+H)⁺ (Calcd for C₁₅H₂₀N₄O, 272.1637).

4-[(4-Methylcyclohexyl)amino]-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (11j)

The title compound was prepared from 10 and 4-methylcyclohexylamine in accordance with the procedure for preparing 11g in 95% yield. ¹H-NMR (DMSO-d₆) δ: 0.88–0.94 (3H, m), 1.09–1.31 (3H, m), 1.37–1.58 (2H, m), 1.62–1.81 (3H, m), 2.03–2.11 (1H, m), 3.76–3.86 (0.4H, m), 4.21–4.29 (0.6H, m), 6.46–6.53 (1H, m), 6.97 (1H, br), 7.09–7.14 (1H, m), 7.74 (1H, br), 8.34 (0.4H, s), 8.36 (0.6H, s), 9.55 (0.4H, d, J=8.0 Hz), 9.97 (0.6H, d, J=8.0 Hz), 11.43 (1H, br s). MS (ESI) m/z: 273 (M+H)⁺. HR-MS m/z: 273.1714 (M+H)⁺ (Calcd for C₁₅H₂₀N₄O, 272.1637).

Ethyl 4-{[(1S,2R)-2-Methylcyclohexyl]amino}-1H-pyrrolo[2,3-b]pyridine-5-carboxylate (12c)

In the vessel of a microwave reactor, 8 (258 mg, 1.15 mmol) and (1S,2R)-2-methylcyclohexanamine hydrochloride (292 mg, 1.95 mmol) were suspended in n-BuOH (1.03 mL), and DIPEA (0.70 mL, 4.02 mmol) was added. The vessel was then sealed, and the mixture was reacted at 160°C for 2 h under microwave irradiation. The mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (n-hexane/EtOAc=3/1 to 1/1) to give the title compound (185 mg, 53%). ¹H-NMR (DMSO-d₆) δ: 0.91 (3H, d, J=6.9 Hz), 1.32 (3H, t, J=7.1 Hz), 1.35–2.16 (9H, m), 4.23–4.34 (3H, m), 6.59 (1H, d, J=3.5 Hz), 7.17 (1H, d, J=3.5 Hz), 8.68 (1H, s), 9.02–9.06 (1H, m), 11.66 (1H, br). MS (ESI) m/z: 302 (M+H)⁺.

Ethyl 4-(Cyclohexylamino)-1H-pyrrolo[2,3-b]pyridine-5-carboxylate (12a)

The title compound was prepared from 8 and cyclohexylamine in accordance with the procedure for preparing 12c in 56% yield. ¹H-NMR (DMSO-d₆) δ: 1.32 (3H, t, J=7.1 Hz), 1.33–1.77 (8H, m), 1.99–2.08 (2H, m), 3.95–4.08 (1H, m), 4.26 (2H, q, J=7.1 Hz), 6.55 (1H, d, J=3.5 Hz), 7.18 (1H, d, J=3.5 Hz), 8.54 (1H, s), 8.84–8.88 (1H, m), 11.67 (1H, br s). MS (ESI) m/z: 288 (M+H)⁺.

Ethyl 4-(Cyclopropylamino)-1H-pyrrolo[2,3-b]pyridine-5-carboxylate (12b)

The title compound was prepared from 8 and cyclopropylamine in accordance with the procedure for preparing 12c in 100% yield. ¹H-NMR (DMSO-d₆) δ: 0.57–0.72 (2H, m), 0.87–1.04 (2H, m), 1.31 (3H, t, J=7.1 Hz), 3.00–3.15 (1H, m), 4.25 (2H, q, J=7.1 Hz), 6.97–7.07 (1H, m), 7.12–7.23 (1H, m), 8.52 (1H, s), 8.76 (1H, d, J=2.1 Hz), 11.68 (1H, s). MS (ESI) m/z: 246 (M+H)⁺.

4-{[(1S,2R)-2-Methylcyclohexyl]amino}-1H-pyrrolo[2,3-b]pyridine-5-carboxylic Acid (13c)

To a solution of 12c (150 mg, 0.50 mmol) in EtOH (1.5 mL) was added 2 M NaOH aqueous solution (1.12 mL, 2.24 mmol), and the mixture was stirred under reflux for 20 h. After cooling to room temperature, the mixture was acidified with 1 M HCl aqueous solution (pH=4–5) and extracted with a mixture solution of CHCl₃ and MeOH (4 : 1). The organic layer was separated, dried over MgSO₄, and concentrated under reduced pressure to give the title compound (175 mg, >100%), which was used in the next reaction without further purification. ¹H-NMR (DMSO-d₆) δ: 0.93 (3H, d, J=6.9 Hz), 1.23–2.01 (9H, m), 4.38–4.40 (1H, m), 6.88–6.89 (1H, m), 7.37–7.40 (1H, m), 8.64 (1H, s), 10.20–10.24 (1H, m), 12.76 (1H, br s), 13.80 (1H, br). MS (ESI) m/z: 274 (M+H)⁺.

4-(Cyclohexylamino)-1H-pyrrolo[2,3-b]pyridine-5-carboxylic Acid (13a)

The title compound was prepared from 12a in accordance with the procedure for preparing 13c in 73% yield. ¹H-NMR (DMSO-d₆) δ: 1.14–2.16 (10H, m), 4.03–4.22 (1H, m), 6.77 (1H, d, J=6.8 Hz), 7.33–7.39 (1H, m), 8.59 (1H, s), 9.80–9.90 (1H, m), 12.48 (1H, br s), 13.83 (1H, br). MS (ESI) m/z: 260 (M+H)⁺

4-(Cyclopropylamino)-1H-pyrrolo[2,3-b]pyridine-5-carboxylic Acid (13b)

The title compound was prepared from 12b in accordance with the procedure for preparing 13c in 88% yield. ¹H-NMR (DMSO-d₆) δ: 0.56–0.71 (2H, m), 0.86–1.02 (2H, m), 2.96–3.14 (1H, m), 6.96–7.05 (1H, m), 7.10–7.19 (1H, m), 8.49 (1H, s), 8.89–9.06 (1H, m), 11.61 (1H, s), 12.38 (1H, br). MS (ESI) m/z: 218 (M+H)⁺.

4-{[(1S,2R)-2-Methylcyclohexyl]amino}-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (14c)

To a solution of 13c (170 mg, 0.622 mmol) in DMF (1.7 mL) were added HOBt (126 mg, 0.933 mmol) and EDC (145 mg, 0.933 mmol) at room temperature, and the mixture was stirred at 60°C for 1 h. After cooling to room temperature, 28% NH₄OH aqueous solution (0.17 mL, 1.24 mmol) was added, and the mixture was stirred at the same temperature for 1 h. After adding H₂O, the mixture was extracted with a solution of CHCl₃ and MeOH (4 : 1). The organic layer was separated and washed with H₂O, and the extract was dried over MgSO₄ and concentrated under reduced pressure. The residue was purified by column chromatography (CHCl₃/MeOH=100/0 to 90/10) to give the title compound (102 mg, 60%). ¹H-NMR (DMSO-d₆) δ: 0.90 (3H, d, J=6.8 Hz), 1.20–1.98 (9H, m), 4.11–4.22 (1H, m), 6.46–6.55 (1H, m), 6.99 (1H, br), 7.07–7.13 (1H, m), 7.64 (1H, br), 8.36 (1H, s), 9.85–9.90 (1H, m), 11.43 (1H, br). MS (ESI) m/z: 273 (M+H)⁺. Anal. Calcd for C₁₅H₂₀N₄O: C, 66.15; H, 7.40; N, 20.57. Found: C, 65.81; H, 7.42; N, 20.31.

4-(Cyclohexylamino)-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (14a)

The title compound was prepared from 13a in accordance with the procedure for preparing 14c in 60% yield. ¹H-NMR (DMSO-d₆) δ: 1.14–2.01 (10H, m), 3.91–4.01 (1H, m), 6.48–6.54 (1H, m), 7.03 (1H, br), 7.10–7.13 (1H, m), 7.70 (1H, br), 8.34 (1H, s), 9.64–9.68 (1H, m), 11.43 (1H, br s). MS (ESI) m/z: 259 (M+H)⁺. Anal. Calcd for C₁₄H₁₈N₄O: C, 65.09; H, 7.02; N, 21.69. Found: C, 65.08; H, 7.12; N, 21.40.

4-(Cyclopropylamino)-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (14b)

The title compound was prepared from 13b in accordance with the procedure for preparing 14c in 74% yield. ¹H-NMR (DMSO-d₆) δ: 0.49–0.66 (2H, m), 0.80–1.00 (2H, m), 2.90–3.09 (1H, m), 6.90–7.02 (1H, m), 7.03 (1H, br), 7.04–7.18 (1H, m), 7.73 (1H, br), 8.35 (1H, s), 9.58 (1H, d, J=2.1 Hz), 11.45 (1H, s). MS (ESI) m/z: 217 (M+H)⁺. Anal. Calcd for C₁₁H₁₂N₄O·0.05H₂O·0.1CH₃OH: C, 60.75; H, 5.70; N, 25.53. Found: C, 60.97; H, 5.62; N, 25.11.

4-(Cyclohexylamino)-N-methyl-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (15a)

To a solution of 13a (25 mg, 0.096 mmol) in DMF (0.375 mL) were added HOBt (19.5 mg, 0.145 mmol), EDC (22.5 mg, 0.145 mmol), and MeNH₂·HCl (9.8 mg, 0.145 mmol), and the mixture was stirred at 55°C for 1 h. To the solution were added H₂O and EtOAc, and the mixture was extracted with EtOAc. The extract was then washed with H₂O, dried over MgSO₄, and concentrated under reduced pressure. The residue was purified by column chromatography (CHCl₃/MeOH=100/0 to 90/10) to give the title compound (5 mg, 19%). ¹H-NMR (DMSO-d₆) δ: 1.18–1.81 (8H, m), 1.91–2.05 (2H, m) 2.73 (3H, d, J=4.4 Hz), 3.84–4.01 (1H, m), 6.47–6.50 (1H, m), 7.11–7.14 (1H, m), 8.14–8.24 (1H, m), 8.27 (1H, s), 9.36–9.45 (1H, m), 11.42 (1H, br s). MS (ESI) m/z: 273 (M+H)⁺. Anal. Calcd for C₁₅H₂₀N₄O·0.5H₂O: C, 64.03; H, 7.52; N, 19.91. Found: C, 63.91; H, 7.57; N, 20.07.

N-Cyclohexyl-4-(cyclohexylamino)-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (15b)

The title compound was prepared from 13a and cyclohexylamine in accordance with the procedure for preparing 15a in 34% yield. ¹H-NMR (DMSO-d₆) δ: 1.05–2.01 (20H, m), 3.63–3.82 (1H, m), 3.84–4.01 (1H, m), 6.48–6.49 (1H, m), 7.11–7.13 (1H, m), 7.96 (1H, d, J=7.7 Hz), 8.31 (1H, s), 9.32 (1H, d, J=8.0 Hz), 11.44 (1H, br s). MS (ESI) m/z: 341 (M+H)⁺. Anal. Calcd for C₂₀H₂₈N₄O·0.2H₂O: C, 69.82; H, 8.32; N, 16.28. Found: C, 69.82; H, 8.35; N, 16.06.

4-(Cyclohexylamino)-N-phenyl-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (15c)

The title compound was prepared from 13a and aniline in accordance with the procedure for preparing 15a in 37% yield. ¹H-NMR (DMSO-d₆) δ: 1.15–1.81 (8H, m), 1.92–2.09 (2H, m), 3.91–4.11 (1H, m), 6.59–6.60 (1H, m), 7.04–7.44 (4H, m), 7.68 (2H, d, J=7.9 Hz), 8.50 (1H, s), 9.10–9.21 (1H, m), 10.13 (1H, br s), 11.77 (1H, br s). MS (ESI) m/z: 335 (M+H)⁺. Anal. Calcd for C₂₀H₂₂N₄O·0.2H₂O: C, 71.07; H, 6.68; N, 16.58. Found: C, 70.98; H, 6.62; N, 16.60.

Docking Calculation

Docking calculations were performed on the crystal structure of tofacitinib (1) bound to JAK3 (PDB code: 3LXK²⁸⁾). The protein-ligand complex was prepared with the Protein Preparation Wizard in Maestro (version 9.3; Schrödinger, LLC, New York, NY, 2012), with impref applying the appropriate side-chain protonation states, refinement, and structure minimization. Docking grids were generated and defined based on the centroid of tofacinitib in the ATP binding site incorporating hydrogen bond constraints to the hinge region and hydrophobic region constraints. Ligands were prepared using LigPrep (version 2.5; Schrödinger, LLC, New York, NY, 2012) and Confgen (version 2.3; Schrödinger, LLC, New York, NY, 2012), and the energy-minimized conformation of each ligand was used to input molecules into docking calculations. Ligand receptor docking was carried out using XP mode in Glide (version 5.8; Schrödinger, LLC, New York, NY, 2012). The top-scoring pose, as assessed by GlideScore, was employed for discussions.

WaterMap

A WaterMap (version 1.4; Schrödinger, LLC, New York, NY, 2012) calculation was conducted on the crystal structure of tofacitinib bound to JAK3 (PDB code: 3LXK) via the structure preparation method described above. WaterMap was run in the default mode using the tofacitinib structure to define the binding site but removing the structure in the MD simulation. ΔG_pred of binding and ligand strain energies were calculated using the ab initio form of the displaced-solvent functional as described by Abel et al.³⁰⁾

Human JAK Assay

The human JAK1, JAK2, and JAK3 kinase-domains were purchased from Carna Biosciences, Inc. (Kobe, Japan), and the assay for kinase inhibitory activity³²⁾ was performed using a streptavidin-coated 96-well plate. A final 50-µL reaction mixture contained 15 mM Tris–HCl (pH 7.5), 0.01% Tween 20, 2 mM dithiothreitol (DTT), 10 mM MgCl₂, 250 nM Biotin-Lyn-Substrate-2 (Biotin-XEQED EPEGD YFEWL EPE, X=ε-Acp; Peptide Institute, Inc., Osaka, Japan) and ATP. The final concentrations of ATP were 200, 10, and 8 µM for JAK1, JAK2, and JAK3, respectively. Test compounds were dissolved in dimethyl sulfoxide (DMSO). The reaction was initiated by adding the kinase domain (JAK1: 60 ng/mL, JAK2: 20 ng/mL, JAK3: 16 ng/mL), followed by incubation at room temperature for 1 h. Kinase activity was measured as the rate of phosphorylation of Biotin-Lyn-Substrate-2 using horseradish peroxidase (HRP)-conjugated anti-phosphotyrosine antibody (HRP-PY-20; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, U.S.A.) using a phosphotyrosine-specific enzyme-linked immunosorbent assay (ELISA). The experiments were performed in duplicate for test compounds, except for 1, and the IC₅₀ value of each experiment was calculated using linear regression analysis. Assays for 1 were performed in four experiments, and the IC₅₀ values were calculated using Sigmoid-Emax non-linear regression analysis.

Rat T Cell Proliferation

Spleen cells from male Lewis rats (Charles River Japan, Inc., Kanagawa, Japan) were suspended in RPMI1640 (Sigma, St. Louis, MO, U.S.A.) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin, and 50 µM 2-mercaptoethanol at a density of 1.5×10⁶ cells/mL. Rat splenocytes were cultured with 1 µg/mL concanavalin A (Sigma) for 24 h at 37°C in 5% CO₂. After being washed, 4×10⁴ splenocytes were incubated with 3 ng/mL IL-2 (BD Biosciences, San Diego, CA, U.S.A.) and test compounds at designated concentrations in 96-well tissue culture plates. After incubation for 3 d, alamarBlue® (Life Technologies, Carlsbad, CA, U.S.A.) was added to each of the test wells, followed by incubation for 6 h. The fluorescence intensity was measured at an excitation wavelength of 545 nm and an emission wavelength of 590 nm. The experiments were performed in duplicate for test compounds, and the IC₅₀ value of each experiment was calculated using linear regression analysis.

In Vitro Liver Microsomal Stability

To estimate stability against rat hepatic CYPs, test compound (0.2 or 1.0 µM) was incubated with male SD rat liver microsomes (0.2 mg protein/mL) in the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH) (1 mM) and ethylenediaminetetraacetic acid (EDTA) (0.1 mM) in phosphate buffer (100 mM) at 37°C. The percentage compound remaining was determined via LC/MS/MS.

Pharmacokinetic Study

The pharmacokinetic characterization was conducted in male SD rats. Compound 14c was administrated at 10 mg/kg orally and 3 mg/kg intravenously in 50% polyethylene glycol. Compound 11k was administrated at 10 mg/kg orally in 50% propylene glycol. Blood samples were taken at multiple points up to 24 h after a single administration of 14c and 11k. Concentrations of the unchanged compound in plasma were determined using LC/MS/MS.

Acknowledgment

We deeply thank Dr. Mitsuru Ohkubo for his useful advice in this study, and Ms. Misato Ito, Ms. Masako Kuno, Dr. Noboru Chida, Dr. Masamichi Inami, Dr. Hidetsugu Murai, and Mr. Keitaro Kadono for performing the biological experiments. We also thank Dr. Toru Asano for his helpful support in preparation of this manuscript. The authors are grateful to the staff of the Division of Analytical Science Laboratories for elemental analysis and spectral measurements.

Conflict of Interest

All authors were employees of Astellas Pharma Inc. when this study was conducted and have no further conflicts of interest to declare.

Supplementary Materials

The online version of this article contains supplementary materials.

References

• 1) Imada K., Leonard W. J., Mol. Immunol., 37, 1–11 (2000).
• 2) Leonard W. J., Lin J. X., J. Allergy Clin. Immunol., 105, 877–888 (2000).
• 3) O’Shea J. J., Pesu M., Borie D. C., Changelian P. S., Nat. Rev. Drug Discov., 3, 555–564 (2004).
• 4) Ghoreschi K., Laurence A., O’Shea J. J., Immunol. Rev., 228, 273–287 (2009).
• 5) O’Shea J. J., Plenge R., Immunity, 36, 542–550 (2012).
• 6) Macchi P., Villa A., Giliani S., Sacco M. G., Frattini A., Porta F., Ugazio A. G., Johnston J. A., Candotti F., O’Shea J. J., Vezzoni P., Notarangelo L. D., Nature (London), 377, 65–68 (1995).
• 7) Russell S. M., Tayebi N., Nakajima H., Riedy M. C., Roberts J. L., Aman M. J., Migone T. S., Noguchi M., Markert M. L., Buckley R. H., O’Shea J. J., Leonard W. J., Science, 270, 797–800 (1995).
• 8) Nosaka T., van Deursen J. M., Tripp R. A., Thierfelder W. E., Witthuhn B. A., McMickle A. P., Doherty P. C., Grosveld G. C., Ihle J. N., Science, 270, 800–802 (1995).
• 9) Park S. Y., Saijo K., Takahashi T., Osawa M., Arase H., Hirayama N., Miyake K., Nakauchi H., Shirasawa T., Saito T., Immunity, 3, 771–782 (1995).
• 10) Johnston J. A., Kawamura M., Kirken R. A., Chen Y. Q., Blake T. B., Shibuya K., Ortaldo J. R., McVicar D. W., O’Shea J. J., Nature (London), 370, 151–153 (1994).
• 11) Witthuhn B. A., Silvennoinen O., Miura O., Lai K. S., Cwik C., Liu E. T., Ihle J. N., Nature (London), 370, 153–157 (1994).
• 12) Wilson L. J., Expert Opin. Ther. Pat., 20, 609–623 (2010).
• 13) Dymock B. W., See C. S., Expert Opin. Ther. Pat., 23, 449–501 (2013).
• 14) Norman P., Exper. Opin. Ther. Pat., 22, 1233–1249 (2012).
• 15) Flanagan M. E., Blumenkopf T. A., Brissette W. H., Brown M. F., Casavant J. M., Shang-Poa C., Doty J. L., Elliott E. A., Fisher M. B., Hines M., Kent C., Kudlacz E. M., Lillie B. M., Magnuson K. S., McCurdy S. P., Munchhof M. J., Perry B. D., Sawyer P. S., Strelevitz T. J., Subramanyam C., Sun J. M., Whipple D. A., Changelian P. S., J. Med. Chem., 53, 8468–8484 (2010).
• 16) Gomez-Puerta J. A., Mocsai A., Curr. Top. Med. Chem., 13, 760–773 (2013).
• 17) Milici A. J., Kudlacz E. M., Audoly L., Zwillich S., Changelian P., Arthritis Res. Ther., 10, R14 (2008).
• 18) Changelian P. S., Flanagan M. E., Ball D. J., Kent C. R., Magnuson K. S., Martin W. H., Rizzuti B. J., Sawyer P. S., Perry B. D., Brissette W. H., McCurdy S. P., Kudlacz E. M., Conklyn M. J., Elliott E. A., Koslov E. R., Fisher M. B., Strelevitz T. J., Yoon K., Whipple D. A., Sun J., Munchhof M. J., Doty J. L., Casavant J. M., Blumenkopf T. A., Hines M., Brown M. F., Lillie B. M., Subramanyam C., Shang-Poa C., Milici A. J., Beckius G. E., Moyer J. D., Su C., Woodworth T. G., Gaweco A. S., Beals C. R., Littman B. H., Fisher D. A., Smith J. F., Zagouras P., Magna H. A., Saltarelli M. J., Johnson K. S., Nelms L. F., Des Etages S. G., Hayes L. S., Kawabata T. T., Finco-Kent D., Baker D. L., Larson M., Si M. S., Paniagua R., Higgins J., Holm B., Reitz B., Zhou Y. J., Morris R. E., O’Shea J. J., Borie D. C., Science, 302, 875–878 (2003).
• 19) Pesu M., Laurence A., Kishore N., Zwillich S. H., Chan G., O’Shea J. J., Immunol. Rev., 223, 132–142 (2008).
• 20) Clark J. D., Flanagan M. E., Telliez J. B., J. Med. Chem., 57, 5023–5038 (2014).
• 21) West K., Curr. Opin. Investig. Drugs, 10, 491–504 (2009).
• 22) Denton M. D., Magee C. C., Sayegh M. H., Lancet, 353, 1083–1091 (1999).
• 23) Safa K., Riella L. V., Chandraker A., Curr. Opin. Nephrol. Hypertens., 22, 689–697 (2013).
• 24) Liptak P., Ivanyi B., Nat. Clin. Pract. Nephrol., 2, 398–404 (2006).
• 25) Bechstein W. O., Transpl. Int., 13, 313–326 (2000).
• 26) Neubauer H., Cumano A., Muller M., Wu H., Huffstadt U., Pfeffer K., Cell, 93, 397–409 (1998).
• 27) L’Heureux A., Thibault C., Ruel R., Tetrahedron Lett., 45, 2317–2319 (2004).
• 28) Chrencik J. E., Patny A., Leung I. K., Korniski B., Emmons T. L., Hall T., Weinberg R. A., Gormley J. A., Williams J. M., Day J. E., Hirsch J. L., Kiefer J. R., Leone J. W., Fischer H. D., Sommers C. D., Huang H. C., Jacobsen E. J., Tenbrink R. E., Tomasselli A. G., Benson T. E., J. Mol. Biol., 400, 413–433 (2010).
• 29) Hirayama F., Koshio H., Ishihara T., Hachiya S., Sugasawa K., Koga Y., Seki N., Shiraki R., Shigenaga T., Iwatsuki Y., Moritani Y., Mori K., Kadokura T., Kawasaki T., Matsumoto Y., Sakamoto S., Tsukamoto S., J. Med. Chem., 54, 8051–8065 (2011).
• 30) Abel R., Young T., Farid R., Berne B. J., Friesner R. A., J. Am. Chem. Soc., 130, 2817–2831 (2008).
• 31) Yang Y., Lightstone F. C., Wong S. E., Expert Opin. Drug Discov., 8, 277–287 (2013).
• 32) http://www.carnabio.com/english/support/thesis.html, and references cited therein.