Discovery of diaminopyrimidine-carboxamide derivatives as JAK3 inhibitors

Article abstract of DOI:10.1016/j.bioorg.2020.103851

Selective inhibition of janus kinase (JAK) has been identified as an important strategy for the treatment of autoimmune disorders. Optimization at the C2 and C4-positions of pyrimidine ring of Cerdulatinib led to the discovery of a potent and orally bioavailable 2,4-diaminopyrimidine-5-carboxamide based JAK3 selective inhibitor (11i). A cellular selectivity study further confirmed that 11i preferentially inhibits JAK3 over JAK1, in JAK/STAT signaling pathway. Compound 11i showed good anti-arthritic activity, which could be correlated with its improved oral bioavailability. In the repeat dose acute toxicity study, 11i showed no adverse changes related to gross pathology and clinical signs, indicating that the new class JAK3 selective inhibitor could be viable therapeutic option for the treatment of rheumatoid arthritis.

Full text of DOI:10.1016/j.bioorg.2020.103851

Journal Pre-proofs
Discovery of Diaminopyrimidine-carboxamide Derivatives as JAK3 Inhibi-
tors
Rajesh Bahekar, Nandini Panchal, Shubhangi Soman, Jigar Desai, Dipam
Patel, Anil Argade, Archana Gite, Sanjay Gite, Bhaumin Patel, Jeevan Kumar,
Sachchidanand S, Harilal Patel, Rajesh Sundar, Abhijit Chatterjee, Jogeswar
Mahapatra, Hoshang Patel, Krishnarup Ghoshdastidar, Debdutta
Bandyopadhyay, Ranjit C. Desai
PII:
S0045-2068(19)32066-8
DOI:
https://doi.org/10.1016/j.bioorg.2020.103851
YBIOO 103851
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
4 December 2019
10 April 2020
12 April 2020
Please cite this article as: R. Bahekar, N. Panchal, S. Soman, J. Desai, D. Patel, A. Argade, A. Gite, S. Gite, B.
Patel, J. Kumar, S. S, H. Patel, R. Sundar, A. Chatterjee, J. Mahapatra, H. Patel, K. Ghoshdastidar, D.
Bandyopadhyay, R.C. Desai, Discovery of Diaminopyrimidine-carboxamide Derivatives as JAK3 Inhibitors,
Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.103851
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Discovery of Diaminopyrimidine-carboxamide Derivatives
as JAK3 Inhibitors#
Rajesh Bahekar a,*, Nandini Panchal a,b, , Shubhangi Soman b, *, Jigar Desai a, Dipam Patel a, Anil
a
a
a
a
c
c
Argade , Archana Gite , Sanjay Gite , Bhaumin Patel , Jeevan Kumar , Sachchidanand S ,
d
d
d
d
e
Harilal Patel Rajesh Sundar , Abhijit Chatterjee , Jogeswar Mahapatra , Hoshang Patel ,
,
Krishnarup Ghoshdastidar e, Debdutta Bandyopadhyay e and Ranjit C. Desaia
a Department of Medicinal Chemistry, Zydus Research Centre, Sarkhej-Bavla, N.H. 8A Moraiya,
Ahmedabad 382210, India.
b Department of Chemistry, Faculty of Science, M.S. University of Baroda, Vadodara 390002,
India.
c
Department of Bioinformatics, Zydus Research Centre, Sarkhej-Bavla, N.H. 8A Moraiya,
Ahmedabad 382210, India.
d
Department of Pharmacology, Zydus Research Centre, Sarkhej-Bavla, N.H. 8A Moraiya,
Ahmedabad 382210, India.
e
Department of Cell Biology, Zydus Research Centre, Sarkhej-Bavla, N.H. 8A Moraiya,
Ahmedabad 382210, India.
O
O
O
S
O
S
S
N
NH
4
O
O
NH
4
O
O
NH
N
O
N
O
O
N
NH2
N
NH2
N
NH2
N
N
N
N
N
2
2
H
H
H
5k
11i
Cerdulatinib
ABSTRACT
Selective inhibition of janus kinase (JAK) has been identified as an important strategy for the
treatment of autoimmune disorders. Optimization at the C2 and C4-positions of pyrimidine ring
of Cerdulatinib led to the discovery of a potent and orally bioavailable 2,4-diaminopyrimidine-5-
carboxamide based JAK3 selective inhibitor (11i). A cellular selectivity study further confirmed
that 11i preferentially inhibits JAK3 over JAK1, in JAK/STAT signaling pathway. Compound
11i showed good anti-arthritic activity, which could be correlated with its improved oral
bioavailability. In the repeat dose acute toxicity study, 11i showed no adverse changes related to
gross pathology and clinical signs, indicating that the new class JAK3 selective inhibitor could
be viable therapeutic option for the treatment of rheumatoid arthritis.
_______________________________________________________________________
#ZRC communication No: 596 (Part of Ph.D thesis work of Nandini Panchal).
*Corresponding author: Rajesh Bahekar: Tel.: +91-2717-665555; Fax: +91-2717-665355
E-mail: rajeshbahekar@zyduscadila.com (Rajesh Bahekar)

1. Introduction
Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovitis and joint
destruction. [1-2] The multiple cytokines (tumor necrosis factor-α (TNF-α) and interleukin (IL))
triggers RA pathogenesis by inducing intracellular signal transduction, via JAK/STAT (Janus
kinases and signal transducers and activators of transcription) signaling pathway. [3-6] The JAK
family of enzymes (JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2)) are cytoplasmic protein
tyrosine kinases, associated with various cytokine-mediated signal transduction pathways. [7-9]
Binding of the cytokines to their corresponding receptors induces JAK activation and subsequent
phosphorylation. [10] The activated JAKs phosphorylate STAT proteins, which translocate to the
nucleus and promote cytokine-responsive gene expression. [11] Due to unique role of JAKs in
the immune system, inhibition of JAKs emerged out as one of the most validated and attractive
therapeutic target for the treatment of autoimmune disorders such as RA and other inflammatory
diseases. [12]
JAK3 is expressed in lymphoid cells, drives pro-inflammatory signaling cascades, inducing
cytokine expression in the synovial fibroblasts, activated monocytes and macrophages. [13]
JAK3 pairs with the JAK1 and it is involved in common gamma chain c cytokine (IL-2, IL-4,
IL-7, IL-9, IL-15 and IL-21) signaling pathways, which play important role in the T-cell
differentiation, proliferation and survival. [14]
Clinically, JAK1 inhibition induces undesirable secondary pharmacodynamic effects such as
cholesterol and liver enzyme elevation. [15] JAK2 mediates signaling via hematopoietic
cytokines such as erythropoietin (EPO), thus dose-limiting tolerability and safety issues such as
anemia are being associated with the JAK2 inhibition. [16] Selective JAK3 inhibition only deters
common gamma (γc) chain receptors signaling and spares JAK1 dependent immunoregulatory
cytokines (IL-10, IL-27 and IL-35). [17] Thus, JAK3 selective inhibitors are likely to offer a
better efficacy to the safety ratio in the clinic for the treatment of chronic inflammatory
disorders.
2. Result and discussion
2.1. Design of novel JAK inhibitor:

Over the past decades, structurally diverse JAK inhibitors were identified containing pyrrolo
[2,3-b]pyridine and pyrimidine core as a promising scaffold (Fig. 1). [18-20] Tofacitinib, a low
nanomolar (nM) JAK3 inhibitor but limited selectivity against JAK1 and JAK2 (Fig. 1) and the
Barictinib, a dual JAK1 and JAK2 inhibitor are approved for the treatment of RA. [21, 22]
Momelotinib, a dual JAK1 and JAK2 inhibitor and the Fedratinib, a JAK2 selective inhibitor are
under clinical trials, for the treatment of myelofibrosis. [23, 24] A reversible ATP-competitive
dual SYK/JAK inhibitor, Cerdulatinib is under clinical trial for the treatment of leukemia and
lymphoma. [25] Knowing the potential side effects associated with the PAN JAK inhibitors,
efforts are being directed towards the development of a JAK3 selective inhibitor, for an effective
treatment of autoimmune disorder. [26]
Recently, JAK3 selective inhibitor programs have targeted covalent bond formation with Cys909
as an optimization strategy, including PF-06651600, an irreversible covalent inhibitor of JAK3
(Fig. 1), which is in the clinical trials for the treatment of inflammatory diseases. [27-29] Most of
the irreversible JAK3 inhibitors demonstrated good JAK3 enzymatic selectivity but largely
lacked cellular selectivity or appropriate physicochemical properties to be tested in vivo. [30]
In this article, we present our efforts to develop novel non-covalent inhibitors of the JAK3. It
was hypothesized that the non-covalent JAK3 selective inhibitors could be devoid of systemic
toxicity. An approach undertaken to overcome the JAK isozymes selectivity was to capitalize on
the amino acid sequence differences among the JAKs, more specifically to favor hydrogen
bonding interaction with the JAK3 cysteine (Cys-909) residue in the catalytic domain to develop
the novel, non-covalent JAK3 selective inhibitor. While designing JAK3 inhibitors, attempts
were made to avoid an introduction of the covalent reactive groups (CRGs), in the structural
features to circumvent the possibility of a covalent modification of Cys-909.
Structural modifications were carried out in the Cerdulatinib (having pyrimidine moiety as a key
pharmacophore, Fig. 1). In the docking studies (Fig. 3) Cerdulatinib showed hydrogen bonding
interactions in the hinge region of JAK3 enzyme but no hydrogen bonding was observed in the
catalytic domain. Initial attempts involved maintaining essential hydrogen bonding interactions
in the original scaffold and optimization at the C2-position of pyrimidine ring of Cerdulatinib to
induce additional hydrogen bonding with the Cys-909. Modifications at the C2 position of
pyrimidine ring in the C4 benzylated Cerdulatinib (Set-1, Table 1) led to the single digit nM

potent compound 5k, with moderate isoform selectivity (Table 3). Docking study of 5K reveals
no additional hydrogen bonding in the catalytic domain of the JAK3.
In the second set (Table 2), changes were carried out at the C4-position of the 5k, mainly to
improve JAK3 isoform selectivity, which led to the discovery of a novel 2,4-diaminopyrimidine-
5-carboxamide based JAK3 selective inhibitor 11i. Docking study of 11i showed additional
hydrogen bonding with the Cys-909 of JAK3 (Fig. 3) and no interaction with the Serine residue
of JAK isoforms, in the ATP binding pockets. Based on the in vitro results, highly potent and
selective compound (11i) was assessed in vivo for the pharmacokinetic (PK), efficacy and animal
toxicity studies.
2.2. Chemistry:
Synthesis of 2, 4-diaminopyrimidine-5-carboxamides derivatives (5a-o and 11a-o) was carried
out as depicted in Scheme 1 and 2, following the modified literature procedure. [31] Treatment
of ethyl 4-chloro-2-(methylthio)-pyrimidine-5-carboxylate (1) with benzyl amine gave 4-
benzylamino carboxylate, followed by hydrolysis, to get the acid moiety (2). Compound 2 was
converted into amide (3), using HOBt and EDC.HCl, followed by reaction with aq. ammonia.
Compound 3 converted to reactive methyl sulfinyl derivate (4) using m-CPBA, followed by
treatment with substituted aryl amines furnished compounds 5a-o. For the synthesis of 11a-o,
substituted 4-hydroxy pyrimidine-5-carboxylate (6) was converted to reactive methyl sulfinyl
derivate, using m-CPBA, followed by treatment with 4-aminophenylmethanesulfonate to get
intermediate 7, which was converted to acid (8), using LiOH, as a base. Acid functionality of
compound 8 transformed to amide (9), using aq. ammonia. Phenolic hydroxyl group of
compound 9 was activated into reactive chloro group (10), using POCl3, followed by reaction
with substituted amines, furnished compounds 11a-o.
Compounds 5a-o and 11a-o were prepared in good yield (60 to 80%), under the mild reaction
condition. Spectral data were found to be in conformity with the structures assigned, which
ensure the formation of compounds 5a-o and 11a-o (see experimental section, 4.2).
2.3. JAK3 inhibitory activity (enzyme based biochemical screening):

For in vitro JAK3 inhibitory activity screening, Cerdulatinib and Tofacitinib were used as a
positive control. In Set-1 (Table 1), 2-substituted diaminopyrimidine-5-carboxamides (5a-o)
analogues displayed varying degree of JAK3 inhibitory activities. m-substituted N-phenyl
derivatives, 5a (m-aniline), 5b (oxopropanamide), 5c (oxoaceticacid), 5d (cinnamamide), 5e
(methanesulfonamide), 5f (sulfoximine) and 5g (sulfonylacetamide) showed moderate JAK3
inhibitory activity (IC50: > 120 nM). Compounds 5h and 5i (positional isomer of 5e and 5f)
exhibited enhanced JAK3 inhibitory activity (IC50: 56 and 48 nM). Compound 5j (acetyl
protection of 5h) displayed moderate JAK3 activity (IC50: 102 nM). Replacement of the
sulfonamide group (5h) with methane sulfonate (5k, IC50: 9.5 nM) and iso-propyl sulfonate (5l,
IC50: 22.4 nM) led to higher JAK3 inhibitory activity, whereas o-substitution with the electron
donating groups such as amino (5m, IC50: 56 nM), di-methylamine (5n, IC50: 66 nM) and N-
acetyl derivative (5o: IC50: 89 nM) displayed comparatively weaker JAK3 inhibitory activity.
In Set-1, compound 5k (methane sulfonate) showed potent JAK3 inhibitory activity, which was
found to be comparable to the Cerdulatinib (IC50: 8 nM; Table 1) and 5 fold less potent
compared to Tofacitinib (IC50: 1.6 nM). Thus, in the Set-1, substitution on p-position of phenyl
ring, followed by replacement of N-acetyl to O-sulfonate led to the single digit nM potent
compound (5k), with moderate isoform selectivity (Table 3).
Further, to improve JAK3 isoform selectivity, modifications were carried out in the 5k. In the
second set (Table 2), variations were carried out at the C4-position of pyrimidine, in 5k. As
listed in the Table 2 (compounds 11a-o), compound 11a (debenzylated 5k; IC50: 300 nM) was
found to be less active. Replacement of benzyl group with cycloalkyl groups, compound 11b
(cyclopropyl, IC50: 49 nM), 11c (cyclobutyl, IC50: 37 nM), 11d (cyclopentyl, IC50: 33 nM) and
11e (cyclohexyl, IC50: 79 nM) showed moderate activities. Overall, the cycloalkyl derivatives
were found to be less potent compared to 5k. Compound 11f (pyran, IC50: 12 nM) showed slight
improvement in the activity, while 11g (iso-butane, IC50: 9.8 nM) was found to be equipotent as
the 5k. Racemic compound 11h (iso-pentane, IC50: 20 nM) showed two fold less potency
compared to 5k. Chirally pure compound 11i (R-isomer of 11h) was found to be five-fold more
potent (IC50: 1.7 nM), whereas 11j (S-isomer of 11h) was found to be least potent (IC50: 256
nM). Compound 11k (phenyl ethyl, IC50: 39 nM), fluoro-substituted benzyl analogues, 11l (3-
fluoro), 11m (4-fluoro) and 11n (2-fluoro) showed moderate activities (IC50 >40 nM) compared
to 5k, while 11o (naphthyl, IC50: 189 nM) was found to be less active.

2.4. JAKs Isoform Selectivity (enzyme based biochemical screening):
Most potent compounds (5k and 11i) were evaluated for their selectivity against JAK isoforms
(JAK-  and TYK2). [32] As shown in Table 3, initial hit (5k) showed moderate selectivity (2
to 5X) against JAK isoforms over JAK3. Compound 11i (IC50: 1.7 nM) demonstrated 12, 100,
and 109 fold selectivity over JAK, JAK2 and TYK2 respectively. Selectivity profile of 11i
against all the three isoforms was found to be better than standard compounds. Thus, potency
and selectivity of diaminopyrimidine-5-carboxamides based JAK3 inhibitors can be modulated
using suitable substituents at C2 and C4-position of pyrimidine ring.
2.5. Profiling of 11i (in vitro and ex vivo):
In vitro kinase profiling study of compound 11i was carried out at 1M concentration, against
Millipore panel of 170 purified kinases (n=2) and the % inhibition was found to be < 20% at
1M concentration, including key cysteine containing protein kinases (TEC family (BMX, BTK,
ITK, TXK, and TEC), ErbB family (EGFR, ERBB4, and ERBB2), CLK2, MKK7β, PKG1α and
Aurora kinase), see Table 8.
JAK3 selectivity of 11i was evaluated under physiologic conditions (cellular assay) to
understand role of the associated cytokine receptors and downstream inflammatory pathways
inhibited. To elucidate potency and selectivity profile in a cellular environment, compound 11i
was tested for the inhibition of phosphorylation of downstream signal (STAT proteins), in the
human peripheral blood mononuclear cells (PBMCs) [33]. The different cellular stimuli were
used to induce phosphorylation of STATs (pSTAT), either by dual JAK1/3 (IL-2 stimulus,
pSTAT5), JAK2 (GM-CSF stimulus, pSTAT5), or with the PAN JAK1/JAK2/TYK2 (IL-6
stimulus, pSTAT3) stimuli [34]. As shown in Table 4, 11i showed 27-fold selectivity for
inhibition of the IL-2 (IC50: 22.16 nM) versus the IL-6 readout (IC50: 608 nM) and a 23-fold
selectivity for the inhibition of the GM-CSF (IC50: 511 nM). Tofacitinib displayed similar
potencies but lower selectivity than 11i in the relevant pSTAT assays. Thus, compound 11i
showed preferential inhibition of JAK3 over JAK1, in the JAK/STAT signaling pathway, when
assessed in the PBMC assay.

Ex vivo, compound 11i was evaluated for the plasma protein binding studies (using mice, rat and
human plasma) and liver microsomal stability studies (using immortalized mice, rats and human
liver cell line). Compound 11i showed 75 to 80% plasma protein binding and less than 10%
metabolism at 30 minutes, in microsomal metabolic stability study. Compound 11i was found to
be devoid of CYP (<10% CYP inhibition) at 10 M concentration, for CYP1A2, CYP2C8,
CYP2C9, CYP2D6, CYP2C19 and CYP3A4 and hERG liabilities (IC50: > 10 M).
2.6. Pharmacokinetic study:
In a single dose PK studies (3 mg/kg, po and 1 mg/kg, iv, in male C57BL/6J mice) of
compounds 5k, 11i and Tofacitinib, various PK parameters (Tmax, Cmax, t1/2, Cl, AUC and %F)
were recorded (Table 5). Compound 5k showed moderate AUC, due to its high clearance, which
resulted into overall low bioavailability (15%). Compound 11i showed higher AUC (~10 fold,
compared to std), extended t1/2 (2.56 hr) and good oral bioavailability (%F: ~48 over std, 20%).
Compound 11i showed extended t1/2 and higher AUC, which could be due to its low clearance
compared to standard (11.59 vs 47.56 ml/min/kg, iv).
2.7. In vivo efficacy studies:
2.7.1 Anti-arthritic efficacy of test compounds in Collagen Induced Arthritis (CIA) mice model:
Arthritis was developed in male DBA1j mice, using collagen mixture and mice were recruited
for the study once clinical signs were visible. [35] Considering low bioavailability of 5k, only
11i was evaluated. Eight animals were assigned in each of the three groups [vehicle, positive
control (Tofacitinib, 60 mg/kg) and test compound 11i (30 mg/kg)]. Treatment was continued for
three weeks and percentage inhibition in clinical score was recorded.
As shown in the Fig.2a, standard compound and 11i showed good reduction in the arthritic score,
compared to vehicle control (untreated group). Two fold higher dose of a standard compound
was used, considering >2X difference in the mice oral bioavailability. At 30 mg/kg dose,
compound 11i showed comparable anti-arthritic activity to that of standard compound (dose 60
mg/kg). Body weights of the animals were also recorded 3 times a week as a measure of
treatment related side effect. No changes in the mice body weight was observed in any treatment

group, compared to vehicle control group. Thus improved PK of 11i justifies its potent in vivo
efficacy in the CIA mice model.
2.7.2 Anti-arthritic efficacy of test compounds in AIA (Adjuvant induced Arthritis) rat model:
Anti-arthritic efficacy of the compound 11i was evaluated in a rat AIA model.[36] As shown in
the Fig.2b, standard and 11i showed good reduction in the paw volume, compared to vehicle
control (untreated group). Compound 11i suppressed paw swelling in a dose-dependent manner
(ED50: 10 mg/kg) and at 30 mg/kg dose, efficacy of 11i was found to be comparable to that of
standard (Tofacitinib, 60 mg/kg). Body weight was not significantly affected in rats, in any
treatment group compared to the vehicle control group.
2.8 Safety pharmacology:
To assess the safety profile of compound 11i, repeat dose acute toxicity studies (14 days) was
carried out in male Wistar rats (100 mg/kg, po, once daily) and various parameters such as gross
pathology, clinical signs, body weight, organ weight and serum chemistry/hematological
changes were recorded. Daily oral administration of compounds 11i (10X of ED50 dose), over a
period of 2 weeks did not affect the survival of Wistar rats and also no adverse changes related to
gross pathology, clinical signs, body weight and feed consumption were noticed, compared to
control group. As shown in Table 6 and 7, the hematological parameters (WBC and RBC) of
compounds 11i were found to be comparable to that of control animals. Similarly, compound 11i
showed no significant changes in serum hepatotoxicity assessment parameters as compared to
the control group. Also compound 11i treated groups showed no changes in the key organs
(heart, kidney, spleen and brain) weights.
2.9. Docking study:
In the docking studies, Cerdulatinib binds in a similar orientation to that of 4,6-
diaminonicotinamide of the co-crystallized ligand (9YV; 4-(benzylamino)-6-({4-[(1-
methylpiperidin-4-yl)carbamoyl]phenyl}amino)pyridine-3-carboxamide), maintaining hydrogen
bonding interactions with the hinge region of JAK3 (a ‘classical’ triad hinge binding interaction),
Fig.3. The docked poses of the core of Cerdulatinib and the co-crystallized ligand superimposed
well. Compound 11i showed similar interactions as that of Cerdulatinib, in the hinge region and

superimposed very well with the core of Cerdulatinib. However, 11i showed additional
interaction with the Cys909 (hydrogen bond between NH of Cys909 with oxygen of methyl
sulfonate group), which was not observed with the Cerdulatinib. An additional interactions of 11i
with the Cys909, in the catalytic domain of JAK3 enzyme likely to contribute towards its potent
and selective JAK3 inhibitory activity. The docking score for Cerdulatinib and 11i was found to
be -8.6 and -9.9 kcal mol-1 respectively.
3. Conclusion
In summary, we have described discovery and characterization of a novel JAK3 inhibitor,
compound 11i. Two series of 2,4-diaminopyrimidine-5-carboxamide derivatives were evaluated
as a JAK3 inhibitors. Modifications at the C2-position of pyrimidine ring led to an identification
of a single digit nM potent JAK3 inhibitor (5k), with moderate isoform selectivity. Further
structure-activity relationship (SAR) studies on the C4-position of 5k resulted in to the discovery
of (R)-4-((5-carbamoyl-4-((3-methylbutan-2-yl)amino)pyrimidin-2-yl)amino)phenyl methane
sulfonate (11i) that showed improved isoform selectivity in the biochemical assay. Compound
11i selectively inhibits JAK3 cytokine signaling in the primary cells, which translated to the
promising efficacy in animal models of RA. In the repeat dose acute toxicity study, 11i showed
no adverse changes related to gross pathology, clinical signs and liver toxicity, indicating that the
new class JAK3 selective inhibitor could be viable therapeutic option for the treatment of
rheumatoid arthritis.
4. Experimental section
4.1 Materials and methods:
NMR spectra were measured on a Varian Unity 400 (1H at 400 MHz, 13C at 100 MHz), magnetic
resonance spectrometer. Spectra were taken in the indicated solvent at ambient temperature.
Chemical shifts (δ) are given in parts per million (ppm) with tetramethylsilane as an internal
standard. Multiplicities are recorded as follows: s = singlet, d = doublet, t = triplet, q = quartet, br
= broad, m = multiplet. Coupling constants (J values) are given in Hz. Mass spectra are recorded
on Perkin-Elmer Sciex API 3000. Reactions were monitored using thin layer silica gel
chromatography (TLC) using 0.25 mm silica gel 60F plates from Merck. Plates were visualized
by treatment with UV, acidic p-anisaldehyde stain, KMnO4 stain with gentle heating. Products

were purified by column chromatography using silica gel 100-200 mesh and the solvent systems
indicated.
4.2 General synthesis:
4.2.1. 4-(Benzylamino)-2-(methylthio)pyrimidine-5-carboxylic acid (2)
Ethyl 4-chloro-2-(methylthio)pyrimidine 5-carboxylate (1) 13.3 g (57.2 mmol) dissolved in
dioxane 100 mL and DIPEA 10.99 mL (62.9 mmol) was added to the reaction mixture at 0°C,
followed by addition of benzyl amine 6.13 g (57.2 mmol) and the reaction mixture was stirred at
0°C for 30 min, and then 6 h. at RT. LiOH 6.85 g (286 mmol) was dissolved in 50 mL water and
added in to reaction mixture and stirred overnight at 28°C. After completion of reaction, the
reaction mixture was concentrated by rotary evaporation to remove a majority of the dioxane,
and then acidified with 10 M HC1 to pH ~ 5, resulting in the formation of a white precipitate.
The solid compound was filtered, washed with water and dried under vacuum to afford the 4-
(benzylamino)-2 (methylthio) pyrimidine-5-carboxylic acid (2) as white solid. (13.23 g, 84 %). 1H
NMR (DMSO-d6, 400 MHz)  ppm: 2.59 (s, 3H), 4.69 (d, J = 6.0 Hz, 3H), 7.28 - 7.22 (m ,1H),
7.30 - 7.33 (m, 4H), 8.52.(s, 1H), 9.25 (s, 1H). ESI-MS: m/z Calcd for C13H14N3O2S [M+1]+
276.33, found 276.05.
4.2.2. 4-(Benzylamino)-2-(methylthio)pyrimidine-5-carboxamide (3).
4-(Benzylamino)-2-(methylthio)pyrimidine-5-carboxylic acid (2) 12 g (43.6 mmol) was
dissolved in DMF 100 mL, under nitrogen atmosphere and treated with HOBT 6.68 g (43.6
mmol) and EDC.HCI 16.68 g (87.0 mmol) at RT. After stirring 1 hr., aq. Ammonia (40 mL) was
added at 0° C, and it was stirred further for 1 h., at RT. Reaction mixture was quenched by ice -
water (200 mL), resulted in the formation of a white precipitate. The solid compound was
filtered, washed with water and dried under to afford 4-(benzylamino)-2-(methylthio)pyrimidine-
5-carboxamide (3) (11.36 g, 95 %). 1H NMR (DMSO-d6, 400 MHz)  ppm: 2.40 (s, 3H), 4.67 (d,
J = 6.0 Hz, 2H), 7.23 - 7.28 (m, 1H), 7.31- 7.35 (m, 4H), 7.45 (s, 1H), 8.04 (s, 1H), 8.53 (s, 1H),
9.48 (t, J = 6.0 Hz,1H). ESI-MS : m/z Calcd for C13H15N4OS [M+1]+ 275.34, found 275.08.
4.2.3. 4-(Benzylamino)-2-(methylsulfinyl)pyrimidine-5-carboxamide (4).
4-(Benzylamino)-2-(methylthio)pyrimidine-5-carboxamide (3; 11 g (40.1 mmol)) was dissolved
in a mixture of dioxane and chloroform (1:1; 290 mL). Reaction mixture was cooled up to -2°C

and treated with m-CPBA (60%; 17.3 g, 60.1 mmol) and it was stirred for 50 min at 0°C.
Reaction mixture was quenched with 10% aq. sodium metabisulfite (290mL) and extracted with
chloroform. Organic layer was washed with aq. 10 % sodium bicarbonate. The organic layer was
dried over sodium sulfate (42 g) and the solvent was removed under reduced pressure to afford
4-(benzylamino)-2-(methylsulfinyl)pyrimidine-5-carboxamide (4) as white solid. (9.31 g, 80 %).
1H NMR (DMSO-d6, 400 MHz)  ppm 2.75 (s, 3H), 4.70 (d, J = 6.0 Hz, 2H), 7.24 - 7.31 (m,
1H), 7.33 - 7.38 (s, 4H), 7.74 (s, 1H), 8.28 (s, 1H), 8.76 (s, 1H), 9.60 (t, J = 5.6 Hz, 2H). ESI-MS
: m/z Calcd for C13H15N4O2S [M+1]+ 291.34, found 291.00.
4.2.4. General procedure for the synthesis of compound 5a-o.
To a solution of 4-(benzylamino)-2-(methylsulfinyl)pyrimidine-5-carboxamide (4; 1.0 eq) and
PTSA (1.1 eq)), in NMP was added different substituted aryl amines (1.0 eq), at RT. The
reaction mixture was heated at 100-110°C for 1-2 h. After completion of reaction, the mixture
was diluted with water and the compound was extracted in EtOAc. Organic layer was dried over
sodium sulfate, filtered and concentrated under vacuum to afford the crude product. Crude
product was purified by flash chromatography over silica gel (100-200 mesh) with 2 %
MeOH/CHCl3 to provide the desired title product 5a-o.
4.2.4.1. 2-((3-Aminophenyl)amino)-4-(benzylamino)pyrimidine-5-carboxamide (5a). Refer to
general procedure for the synthesis of 5a, as described in section 4.1.4. 51.2% isolated yield;
white solid. 1H NMR (DMSO-d6, 400 MHz)  ppm 4.67 (d, J = 5.6 Hz, 2H), 6.18 (d, J = 6.8 Hz,
1H), 6.82-6.86 (m, 2H), 6.97 (s, 1H), 7.22 - 7.35 (m, 5H), 8.51 (s, 1H), 9.20 (s, 1H), 9.47 (t, J =
13
6 Hz, 1H). C NMR (DMSO-d6, 100 MHz) δ ppm 43.9, 100.5, 100.9, 103.2, 107.0, 126.6,
127.0, 128.7, 129.5, 139.9, 142.9, 148.0, 161.8, 165.1, 166.2, 169.6. ESI-MS : m/z Calcd for
C18H19N6O [M+1] + 335.38, found 334.90.
4.2.4.2.
Methyl-2-((3-((4-(Benzylamino)-5-carbamoylpyrimidin-2-yl)amino)phenyl)amino)-2-
oxoacetate (5b). Refer to general procedure for the synthesis of 5b, as described in section 4.1.4.
49.5% isolated yield; white solid. 1H NMR (DMSO-d6, 400 MHz)  ppm 3.84 (s, 3H), 4.72 (d, J
13
= 6.0 Hz, 2H), 7.18 - 7.31 (m, 8H), 8.25 (s, 1H), 8.55 (s, 1H), 9.71 (s, 2H), 10.68 (s, 1H). C
NMR (DMSO-d6, 100 MHz) δ ppm 43.8, 51.1, 100.4, 107.8, 111.0, 113.6, 126.5, 127.1, 128.8,
129.6, 137.2, 140.0, 142.7, 155.0, 162.2, 164.5, 165.9, 166.8, 169.7. ESI-MS: m/z Calcd for
C21H21N6O4 [M] + 420.43, found 420.80.

4.2.4.3. 2-((3-((4-(Benzylamino)-5-carbamoylpyrimidin-2-yl)amino)phenyl)amino)-2-oxoacetic
acid (5c). Refer to general procedure for the synthesis of 5c, as described in section 4.1.4. 51.9%
isolated yield; white solid. 1H NMR (DMSO-d6, 400 MHz)  ppm 4.71 (d, J = 6.0 Hz, 2H), 6.98
13
- 7.70 (m, 7H), 7.86 - 7.94 (m, 1H), 8.17 (s, 1H), 8.53 (s, 1H), 9.62 (s, 2H), 10.58 (s, 1H). C
NMR (DMSO-d6, 100 MHz) δ ppm 43.8, 100.5, 107.9, 110.9, 113.8, 126.5, 127.1, 128.8, 129.9,
137.8, 140.1, 143.0, 156.9, 162.0, 164.1, 166.0, 166.9, 169.5. ESI-MS: m/z Calcd for
C20H18N6O4 [M] + 406.40, found 406.02.
4.2.4.4. 4-(Benzylamino)-2-((3-cinnamamidophenyl)amino)pyrimidine-5-carboxamide (5d).
Refer to general procedure for the synthesis of 5d, as described in section 4.1.4. 48.8% isolated
yield; white solid. 1H NMR (DMSO-d6, 400 MHz)  ppm 4.74 (d, J = 6.0 Hz, 2H), 6.84 (s, 1H),
6.88 (s, 1H), 7.21 - 7.32 (m, 5H), 7.38 - 7.45 (m, 5H), 7.47 - 7.61 (m, 3H), 8.14 (s, 1H), 8.56 (s,
13
1H), 9.53 (s, 2H), 10.11 (s, 1H). C NMR (DMSO-d6, 100 MHz) δ ppm 43.5, 100.4, 108.0,
111.2, 113.9, 119.0, 126.7, 127.4, 128.0, 128.4, 128.7, 128.9, 129.6, 135.6, 137.1, 139.8, 142.0,
143.2, 162.2, 165.8, 166.7, 167.2, 169.8. ESI-MS: m/z Calcd for C27H25N6O2 [M]+ 464.53, found
464.90.
4.2.4.5.
4-(benzylamino)-2-((3-(methylsulfonamido)phenyl)amino)pyrimidine-5carboxamide
(5e). Refer to general procedure for the synthesis of 5e, as described in section 4.1.4. 50.8%
isolated yield; white solid.1H NMR (DMSO-d6, 400 MHz)  ppm 3.33 (s, 3H), 4.70 (d, J = 5.6
Hz, 2H), 7.10 - 7.05 (m ,2H), 7.31 - 7.35 (m, 4H), 7.80 - 7.88 (m, 2H), 7.94 (s, 1H), 8.59 (s, 1H),
13
9.55 (s, 1H), 9.69 (s, 1H). C NMR (DMSO-d6, 100 MHz) δ ppm 40.0, 44.5, 101.0, 101.5,
107.4, 109.0, 126.5, 127.1, 128.6, 130.0, 138.3, 139.5, 143.0, 161.9, 165.5, 167.0, 169.1. ESI-
MS: m/z Calcd for C19H20N6O3S [M] + 412.47, found 412.22.
4.2.4.5. 4-(Benzylamino)-2-((3-(S-methylsulfonimidoyl)phenyl)amino)pyrimidine-5-carboxamide
(5f). Refer to general procedure for the synthesis of 5f, as described in section 4.1.4. 45.9%
isolated yield; white solid. 1H NMR (DMSO-d6, 400 MHz)  ppm 2.51 (s, 3H), 4.65 (d, J = 6.0
Hz, 2H), 6.80 - 6.87 (m ,2H), 7.26 - 7.27 (m, 5H), 7.33 - 7.34 (m, 2H), 8.37 (s, 1H), 9.61 (s, 1H),
13
9.78 (s, 1H). C NMR (DMSO-d6, 100 MHz) δ ppm 43.8, 44.2, 100.9, 112.2, 114.5, 120.3,
126.8, 127.0, 128.7, 128.9, 140.1, 142.3, 146.8, 162.0, 165.9, 168.2, 169.8. ESI-MS: m/z Calcd
for C19H20N6O2S [M] + 396.47, found 396.20

4.2.4.6.
4-(Benzylamino)-2-((3-(N-(methylsulfonyl)acetamido)phenyl)amino)pyrimidine-5
carboxamide (5g). Refer to general procedure for the synthesis of 5g, as described in section
1
4.1.4. 49.6% isolated yield; white solid. H NMR (DMSO-d6, 400 MHz)  ppm 1.83 (s, 3H),
3.46 (s, 3H), 4.70 (d, J = 6.0 Hz, 2H), 7.03 (d, J = 7.2 Hz, 1H), 7.23 - 7.26 (m, 1H), 7.31 - 7.35
(m, 5H), 7.66 (d, J = 7.2 Hz, 1H), 7.93 (S, 1H), 8.59 (s, 1H), 9.64 (s, 1H), 9.75 (s, 1H). 13C NMR
(DMSO-d6, 100 MHz) δ ppm 20.0, 43.7, 44.0, 101.2, 108.4, 113.9, 119.9, 126.9, 127.2, 128.6,
129.1, 131.5, 140.0, 142.5, 162.3, 166.2, 166.5, 168.5, 169.7. ESI-MS: m/z Calcd for
C21H23N6O4S [M+1]+ 455.50, found 455.00.
4.2.4.7.
4-(Benzylamino)-2-((4-(methylsulfonamido)phenyl)amino)pyrimidine-5-carboxamide
(5h). Refer to general procedure for the synthesis of 5h, as described in section 4.1.4. 47.8%
isolated yield; white solid. 1H NMR (DMSO-d6, 400 MHz)  ppm 2.90 (s, 3H), 4.67 (d, J = 6.0
Hz, 2H), 7.05 (d, J = 9.2 Hz, 2H), 7.32 - 7.33 (m, 5H), 7.58 (d, J = 8.8 Hz, 2H), 8.54 (s, 1H),
13
9.39 (s, 1H), 9.45 (s, 1H), 9.47 (s, 1H). C NMR (DMSO-d6, 100 MHz) δ ppm 43.9, 43.6,
100.8, 117.9, 118.6, 126.6, 126.9, 127.8, 128.2, 128.9, 139.8, 162.5, 166.2, 168.5, 169.8. ESI-
MS: m/z Calcd for C19H20N6O3S [M]+ 412.47, found 412.80.
4.2.4.8. 4-(Benzylamino)-2-((4-(S-methylsulfonimidoyl)phenyl)amino)pyrimidine-5-carboxamide
(5i). Refer to general procedure for the synthesis of 5i, as described in section 4.1.4. 45.2%
1
isolated yield; white solid. H NMR (DMSO-d6, 400 MHz)  ppm 2.50 (s, 3H) 4.41 (d, J = 6.0
Hz, 2H), 6.64 (d, J = 8.8 Hz, 2H), 7.22 - 7.32 (m, 5H), 7.54 (d, J = 8.0 Hz, 2H), 8.45 (s, 1H),
13
9.42 (s, 1H), 9.65 (s, 1H). C NMR (DMSO-d6, 100 MHz) δ ppm 43.8, 45.2, 101.7, 115.0,
126.2, 126.5, 126.7, 128.0, 136.2, 140.0, 141.3, 162.4, 166.1, 168.8, 169.5. ESI-MS: m/z Calcd
for C19H20N6O2S [M]+ 396.47, found 396.80.
4.2.4.9.
4-(Benzylamino)-2-((4-(N-(methylsulfonyl)acetamido)phenyl)amino)pyrimidine-5-
carboxamide (5j). Refer to general procedure for the synthesis of 5j, as described in section
4.1.4. 49.7% isolated yield; white solid.1H NMR (DMSO-d6, 400 MHz)  ppm 1.88 (s, 3H),
3.49 (s, 3H), 4.70 (d, J = 6.0 Hz, 2H), 7.22 - 7.31 (m, 5H), 7.33 - 7.36 (m, 2H), 7.78 (d, J = 8.8
Hz, 2H), 8.58 (s, 1H), 9.57 (t, J = 6.0 Hz, 1H), 9.74 (s, 1H). ). 13C NMR (DMSO-d6, 100 MHz) δ
ppm 19.5, 42.9, 43.7, 100.5, 117.6, 119.6, 121.8, 126.4, 126.9, 127.9, 139.7, 140.1, 162.8, 166.9,
167.2, 168.8, 169.2. ESI-MS m/z Calcd for C21H23N6O4S [M]+ 454.50, found 454.90.

4.2.4.10. 4-((4-(Benzylamino)-5-carbamoylpyrimidin-2-yl)amino)phenyl methanesulfonate (5k).
Refer to general procedure for the synthesis of 5k, as described in section 4.1.4. 51.5% isolated
yield; white solid. 1H NMR (DMSO-d6, 400 MHz)  ppm 3.33 (s, 3H) 4.68 (d, J = 5.6 Hz, 2H),
7.18 (d, J = 8.4 Hz, 2H), 7.22 - 7.35 (m, 5H), 7.73 (d, J = 8.4 Hz, 2H), 8.56 (s, 1H), 9.57 (s, 1H),
13
9.66 (s, 1H). C NMR (DMSO-d6, 100 MHz) δ ppm 37.5, 44.0, 100.5, 120.5, 122.6, 127.4,
127.8, 128.9, 139.8, 143.5, 157.7, 160.2, 161.9, 169.4. ESI-MS: m/z Calcd for C19H20N5O4S
[M]+ 413.45, found 413.90.
4.2.4.11. 4-((4-(Benzylamino)-5-carbamoylpyrimidin-2-yl)amino)phenyl propane-2-sulfonate
(5l). Refer to general procedure for the synthesis of 5l, as described in section 4.1.4. 50.5%
isolated yield; white solid. 1H NMR (DMSO-d6, 400 MHz)  ppm 1.41 (d, J = 6.8 Hz, 6H), 3.64
- 3.67 (m, 1H), 4.68 (d, J = 6.0 Hz, 2H), 7.13 (d, J = 9.2 Hz, 2H), 7.23 - 7.34 (m, 5H), 7.71 (d, J
= 9.2 Hz, 2H), 8.56 (s, 1H), 9.55 (s, 1H), 9.65 (s, 1H). 13C NMR (DMSO-d6, 100 MHz) δ ppm
16.0, 43.8, 49.8, 100.7, 121.0, 122.4, 127.3, 127.7, 128.6, 138.9, 142.9, 158.0, 160.9, 162.2,
169.8. ESI-MS: m/z Calcd for C21H24N5O4S [M+1]+ 442.51, found 442.10.
4.2.4.12. 2-Amino-4-((4-(benzylamino)-5-carbamoylpyrimidiyl) amino) phenyl methane
sulfonate (5m). Refer to general procedure for the synthesis of 5m, as described in section 4.1.4.
49.8% isolated yield; white solid. 1H NMR (DMSO-d6, 400 MHz)  ppm 3.33 (s, 3H) 4.68 (d, J
= 6.0 Hz, 2H), 5.18 (s, 2H), 6.88 - 6.95 (m, 2H), 7.19 - 7.26 (m, 2H), 7.30 - 7.36 (m, 4H), 8.53
(s, 1H), 9.37 (s, 1H), 9.49 (t, J = 5.8 Hz, 2H). 13C NMR (DMSO-d6, 100 MHz) δ ppm 37.9, 44.0,
100.1, 107.1, 108.1, 123.2, 127.3, 127.7, 128.8, 130.7, 139.9, 140.0, 141.1, 157.7, 160.4, 161.9,
169.5. ESI-MS: m/z Calcd for C19H21N6O4S [M+1]+ 429.47, found 429.00.
4.2.4.13.
4-((4-(Benzylamino)-5-carbamoylpyrimidin-2-yl)amino)-2-(dimethylamino)phenyl
methane sulfonate (5n). Refer to general procedure for the synthesis of 5n, as described in
1
section 4.1.4. 46.8% isolated yield; white solid. H NMR (DMSO-d6, 400 MHz)  ppm 2.67 (s,
6H), 3.32 (s, 3H), 4.72 (d, J = 5.6 Hz, 2H), 7.05 (d, J = 8.8 Hz, 1H), 7.23 - 7.29 (m, 2H), 7.33 -
7.36 (m, 4H), 7.57 (s, 1H), 8.57 (s, 1H), 9.55 (s, 2H). 13C NMR (DMSO-d6, 100 MHz) δ ppm
37.4, 42.9, 44.2, 100.9, 107.7, 108.4, 127.1, 127.9, 128.5, 130.4, 140.0, 140.2, 143.8, 157.3,
160.7, 161.4, 169.2. ESI-MS: m/z Calcd for C21H25N6O4S 456.52 [M]+, found 456.90.
4.2.4.14. 4-((4-(Benzylamino)-5-carbamoylpyrimidin-2-yl)amino)-2-carbamoylphenyl methane
sulfonate (5o). Refer to general procedure for the synthesis of 5o, as described in section 4.1.4.

1
48.5% isolated yield; white solid. H NMR (DMSO-d6, 400 MHz)  ppm 2.05 (s, 3H), 3.33 (s,
3H), 4.71 (d, J = 6.0 Hz, 2H), 7.26 - 7.19 (m, 6H), 7.47 (d, J = 7.6Hz, 1H), 8.31 (s, 1H), 8.57 (s,
1H), 9.50 (s, 1H), 9.54 (s, 1H), 9.67(s, 1H).13C NMR (DMSO-d6, 100 MHz) δ ppm 23.8, 38.1,
43.9, 100.7, 109.1, 116.2, 123.1, 127.2, 127.5, 128.8, 131.2, 139.6, 139.8, 142.0, 157.7, 160.3,
161.9, 163.2, 169.4. ESI-MS: m/z Calcd for C21H23N6O5S [M+1]+ 471.50, found 471.19.
4.2.5. Preparation of Ethyl 4-hydroxy-2-((4-((methylsulfonyl)oxy) phenyl)amino) pyrimidine -5-
carboxylate (7)
Ethyl 4-hydroxy-2-(methylthio)pyrimidine-5-carboxylate (6; 10 g; 46.7 mmol) was dissolved in
a mixture of dioxan and chloroform (1:1; 262 mL). Reaction mixture was cooled up to -2°C,
treated with m-CPBA (60%; 20.14 g, 70.0 mmol) and stirred for 50 min at 0°C. Reaction
mixture was quenched by 10% aq. sodium metabisulfite (262 mL), extracted with chloroform
and washed with aq. 10 % sodium bicarbonate. The organic layer was dried over sodium sulfate
(40 g) and the solvent was evaporated under reduced pressure to obtain ethyl 4-hydroxy-2-
(methylsulfinyl)pyrimidine-5-carboxylate as a white solid. (8.6 g, 80 %), which was further used
in the next step without purification.
To the mixture of ethyl 4-hydroxy-2-(methylsulfinyl)pyrimidine-5-carboxylate (8.6 g; 37.4
mmol) and PTSA (7.82 g; 41.1 mmol), dissolved in NMP (80ml) was added 4-
(methylsulfonyl)aniline (6.99 g; 37.4 mmol), at RT. The reaction mixture was heated at 100-
110°C for 1-2 h. After completion of reaction, the mixture was diluted with water and the
compound was extracted in EtOAc. Organic layer was dried over sodium sulfate, filtered and
concentrated under vacuum to afford the crude product. Crude product was purified by flash
chromatography over silica gel (100-200 mesh) with 2 % MeOH/CHCl3 to get the ethyl 4-
hydroxy-2-((4-((methylsulfonyl) oxy)phenyl)amino) pyrimidine-5-carboxylate (7) (9.9 g, 75%).
1H NMR (DMSO-d6, 400 MHz)  ppm 1.26 (t, J = 7.0 Hz, 3H), 3.48 (s, 3H), 4.20 (q, J = 6.8 Hz,
2H), 6.57 (d, J = 9.2 Hz, 2H), 7.34 (d, J = 8.8 Hz, 2H), 8.49 (s, 1H), 9.75 (s, 1H), 11.25 (s, 1H):
ESI-MS: m/z Calcd for C14H16N3O6S [M+1]+ 354.35, found 354.58.
4.2.6. 4-hydroxy-2-((4-((methylsulfonyl)oxy)phenyl)amino)pyrimidine-5-carboxylic acid (8).
Ethyl 4-hydroxy-2-((4-((methylsulfonyl)oxy)phenyl)amino)pyrimidine-5-carboxylate (7; 9.0 g,
25.5 mmol) was dissolved in THF (90mL). LiOH (3.05 g, 127 mmol) dissolved in water (5mL)
was added in to the reaction mixture and stirred overnight at 25°C. After completion of reaction,

the reaction mixture was concentrated by rotary evaporation to remove excess THF. Mixture was
acidified (10 M HC1 to pH ~ 5), which led to the formation of a white precipitate. The solid
compound filtered, washed with water and dried under vacuum to afford 4-hydroxy-2-((4-
((methylsulfonyl)oxy)phenyl)amino)pyrimidine-5-carboxylic acid (8), as a white solid. (6.63 g,
80%). 1H NMR (DMSO-d6, 400 MHz)  ppm 3.56 (s, 3H), 7.37 (d, J = 8.8 Hz, 2H), 7.67 (d, J =
8.8 Hz, 2H), 8.51 (s, 1H), 9.85 (s, 1H). ESI-MS: m/z Calcd for C12H12N3O6S [M+1]+ 326.30,
found 326.01.
4.2.7. 4-((5-carbamoyl-4-hydroxypyrimidin-2-yl)amino)phenyl methanesulfonate (9).
4-Hydroxy-2-((4-((methylsulfonyl)oxy)phenyl)amino)pyrimidine-5-carboxylic acid (8; 6.0 g,
18.44 mmol) was dissolved in DMF (50 mL) and treated with HOBT (2.82 g; 18.44 mmol) and
EDC.HCI (7.07 g; 36.9 mmol), at RT. After stirring 1 hr., aq. Ammonia (20 mL) was added at
0°C and stirred for 1 hr., at RT. The reaction mixture was quenched with water (200 mL) which
resulted in the formation of a white precipitate. The solid compound was filtered, washed with
water and dried under reduced pressure to afford 4-((5-carbamoyl-4-hydroxypyrimidin-2-
yl)amino)phenyl methanesulfonate (9; 4.19 g, 70%). 1H NMR (DMSO-d6, 400 MHz)  ppm 3.33
(s, 3H), 7.21 (d, J = 8.8 Hz, 2H), 7.87 (d, J = 8.8 Hz, 2H), 8.37 (s, 1H), 9.33 (s, 1H), 17.6 (s,
1H). ESI-MS: m/z Calcd for C12H13N4O5S [M+2]+ 324.31, found 326.05.
4.2.8. 4-((5-carbamoyl-4-chloropyrimidin-2-yl)amino)phenyl methanesulfonate (10).
To a solution of 4-((5-carbamoyl-4-hydroxypyrimidin-2-yl)amino)phenyl methanesulfonate (9;
4.0 g,12.33 mmol), in toluene (50 mL) was added DIPEA (2.6 mL, 14.8 mmol) and POCl3 (2.3
mL, 24.67 mmol) drop wise, at 0-5oC. The reaction mixture was refluxed for 2 hr. After
completion of reaction, mixture was cooled to RT, quenched with ice - water (180 mL) to afford
white precipitate. The precipitated compound was filtered, washed with cold water to get 4-((5-
1
carbamoyl-4-chloropyrimidin-2-yl)amino)phenyl methanesulfonate (10; 2.96 g, 70%). H NMR
(DMSO-d6, 400 MHz)  ppm 3.37 (s, 3H), 7.34 (d, J = 9.2 Hz, 2H), 7.80 (d, J = 9.2 Hz, 2H),
8.92 (s, 1H), 10.70 (s, 1H). ESI-MS: m/z Calcd for C12H12ClN4O4S [M+1]+ 343.75, found
343.80.
4.2.9. General procedure for the synthesis of compound 11a-o.

4-((5-carbamoyl-4-chloropyrimidin-2-yl)amino)phenyl methanesulfonate (10; 1.0 eq) was
dissolved in dioxane (40 mL) and DIPEA (1.1 eq) was added to the reaction mixture at 0°C
followed by the addition of different amines (1.0 eq). The reaction mixture was stirred at 0°C for
6 hr. After completion of reaction, the mixture was diluted with water and the compound was
extracted in EtOAc. Organic layer was dried over sodium sulfate, filtered and concentrated under
vacuum to afford the crude product. Crude product was purified by flash chromatography over
silica gel (100-200 mesh), with 2 % MeOH/CHCl3 to provide the desired title product (11a-o).
4.2.9.1. 4-((4-Amino-5-carbamoylpyrimidin-2-yl)amino)phenyl methanesulfonate (11a). Refer to
the general procedure for the synthesis of 11a, as described in section 4.1.9. 49.4% isolated
yield; white solid. 1H NMR (DMSO-d6, 400 MHz)  ppm 3.33 (s, 3H), 7.22 (d, J = 9.2 Hz, 2H),
7.89 (d, J = 9.2 Hz, 2H), 8.54 (s, 1H), 9.58 (s, 1H). 13C NMR (DMSO-d6, 100 MHz) δ ppm 37.4,
100.1, 120.8, 122.3, 140.2, 142.9, 155.1, 158.2, 160.5, 161.8. ESI-MS: m/z Calcd for
C12H13N5O4S [M]+ 323.33, found 323.80.
4.2.9.2. 4-((5-Carbamoyl-4-(cyclopropylamino)pyrimidin-2-yl)amino)phenyl methanesulfonate
(11b). Refer to the general procedure for the synthesis of 11b, as described in section 4.1.9.
1
48.9% isolated yield; white solid. H NMR (DMSO-d6, 400 MHz)  ppm 0.54 (t, J = 3.0 Hz,
2H), 0.84 (t, J = 3.4 Hz, 2H), 2.66 - 2.67 (m, 1H), 3.32 (s, 3H), 7.26 (d, J = 9.2 Hz, 2H), 8.02 (d,
J = 8.8 Hz, 2H), 8.53 (s, 1H), 9.22 (s, 1H), 9.75 (s, 1H). 13C NMR (DMSO-d6, 100 MHz) δ ppm
7.5, 25.0, 37.1, 100.5, 121.0, 122.5, 140.5, 144.0, 158.2, 160.4, 161.8, 169.4. ESI-MS: m/z Calcd
for C15H18N5O4S [M+1]+ 364.39, found 364.00.
4.2.9.3. 4-((5-Carbamoyl-4-(cyclobutylamino)pyrimidin-2-yl)amino)phenyl methane sulfonate
(11c). Refer to the general procedure for the synthesis of 11c, as described in section 4.1.9.
49.7% isolated yield; white solid. 1H NMR (DMSO-d6, 400 MHz)  ppm 1.25 (m, 2H), 1.78 (m,
2H), 2.37 (m, 2H), 3.33 (s, 3H), 4.51- 4.55 (m, 1H), 7.28 (d, J = 9.2 Hz, 2H), 7.87 (d, J = 9.2 Hz,
2H), 8.53 (s, 1H), 9.30 (d, J = 7.2 Hz, 1H), 9.67 (s, 1H). 13C NMR (DMSO-d6, 100 MHz) δ ppm
12.5, 30.2, 37.3, 51.2, 100.8, 121.5, 122.3, 140.1, 144.5, 158.8, 160.9, 161.7, 169.3. ESI-MS:
m/z Calcd for C16H20N5O4S [M+1]+ 378.42, found 378.00.
4.2.9.4. 4-((5-Carbamoyl-4-(cyclopentylamino)pyrimidin-2yl)amino) phenylmethane sulfonate
(11d). Refer to the general procedure for the synthesis of 11d, as described in section 4.1.9.
1
48.2% isolated yield; white solid. H NMR (DMSO-d6, 400 MHz)  ppm 1.46 - 149 (m, 2H),

1.50 - 173 (m, 4H), 1.98 - 2.06 (m, 2H), 3.32 (s, 3H), 4.30 - 4.38 (m, 1H), 7.25 (d, J = 9.2 Hz,
13
2H), 7.88 (d, J = 9.2 Hz, 2H), 8.52 (s, 1H), 9.20 (d, J = 7.2 Hz, 1H), 9.65 (s, 1H). C NMR
(DMSO-d6, 100 MHz) δ ppm 23.8, 33.2, 37.5, 52.0, 100.2, 120.4, 122.6, 140.1, 143.5, 157.5,
160.2, 161.4, 169.5. ESI-MS: m/z Calcd for C17H22N5O4S [M+1]+ 392.45, found 392.00.
4.2.9.5. 4-((5-Carbamoyl-4-(cyclohexylamino)pyrimidin-2-yl)amino) phenylmethanes sulfonate
(11e). Refer to the general procedure for the synthesis of 11e, as described in section 4.1.9.
1
47.5% isolated yield; white solid. H NMR (DMSO-d6, 400 MHz)  ppm 1.24 - 1.31 (m, 3H ),
1.35 - 1.44 (m, 2H), 1.62 - 1.59 (m, 1H), 1.70 - 1.73 (m, 2H), 1.95 - 1.98 (m, 2H), 3.33 (s, 3H),
3.93 - 3.94 (m, 1H), 7.25 (d, J = 9.2 Hz, 2H), 7.86 (d, J = 8.8 Hz, 2H), 8.52 (s, 1H), 9.17 (d, J =
7.2 Hz, 1H ), 9.66 (s, 1H). 13C NMR (DMSO-d6, 100 MHz) δ ppm 24.8, 25.7, 32.6, 37.5, 49.0,
100.0, 120.3, 122.6, 140.1, 143.5, 157.7, 160.3, 161.0, 169.6. ESI-MS: m/z Calcd for
C18H24N5O4S [M+1]+ 406.47, found 406.00.
4.2.9.6.
4-((5-Carbamoyl-4-((cyclopentylmethyl)amino)pyrimidin-2-yl)amino)phenylmethane
sulfonate (11f). Refer to the general procedure for the synthesis of 11f, as described in section
1
4.1.9. 48.2% isolated yield; white solid. H NMR (DMSO-d6, 400 MHz)  ppm 1.18 - 1.22 (m,
2H), 1.53 - 155 (m, 2H), 1.56 - 160 (m, 2H), 1.62 - 1.75 (m, 2H), 1.98 - 2.00 (m, 1H), 3.51 (s,
5H), 7.28 (d, J = 8.8 Hz, 2H), 7.85 (d, J = 8.0 Hz, 2H), 8.51 (s, 1H), 9.40 (s, 1H), 9.85 (s, 1H).
13C NMR (DMSO-d6, 100 MHz) δ ppm 25.2, 30.3, 37.5, 45.3, 100.5, 120.9, 122.8, 139.4, 143.9,
158.9, 159.4, 161.8, 169.1. ESI-MS: m/z Calcd for C18H24N5O4S [M+1]+ 406.47, found 406.30.
4.2.9.7.
4-((5-Carbamoyl-4-(isobutylamino)pyrimidin-2-yl)amino)phenyl
methanesulfonate
(11g). Refer to the general procedure for the synthesis of 11g, as described in section 4.1.9.
1
46.3% isolated yield; white solid. H NMR (DMSO-d6, 400 MHz)  ppm 0. 93 (d, J = 6.8 Hz,
6H), 3.10 (s, 2H), 3.33 (s, 3H), 4.10 - 4.15 (m, 1H), 7.25 (d, J = 9.2 Hz, 2H), 7.87 (d, J = 9.2 Hz,
2H), 8.53 (s, 1H), 9.38 (s, 1H), 9.66 (s, 1H). 13C NMR (DMSO-d6, 100 MHz) δ ppm 21.3, 29.9,
37.4, 49.2, 100.1, 121.2, 122.0, 139.6, 144.0, 159.0, 159.9, 161.9, 169.4. ESI-MS: m/z Calcd for
C16H21N5O4S [M]+ 379.44, found 379.50.
4.2.9.8. 4-((5-Carbamoyl-4-((3-methylbutan-2-yl)amino)pyrimidin-2-yl)amino)phenyl methane
sulfonate (11h). Refer to the general procedure for the synthesis of 11h, as described in section
4.1.9. 48.1% isolated yield; white solid. 1H NMR (DMSO-d6, 400 MHz)  ppm 0.90 (d, J = 6.8
Hz, 3H), 0.94 (d, J = 6.8 Hz, 3H), 1.13 (d, J = 6.8 Hz, 3H), 1.85 - 1.90 (m, 1H), 3.34 (s, 3H),

4.06 - 4.11 (m, 1H), 7.26 (d, J = 9.2 Hz, 2H), 7.85 (d, J = 9.2 Hz, 2H), 8.53 (s, 1H), 9.27 (d, J =
13
8.4 Hz, 3H), 9.65(s,1H). C NMR (DMSO-d6, 100 MHz) δ ppm 16.6, 18.0, 18.4, 31.9, 37.1,
50.1, 99.5, 119.9, 122.2, 139.6, 143.1, 157.1, 159.8, 161.0, 169.2. ESI-MS: m/z Calcd for
C17H24N5O4S [M+1]+ 394.46, found 394.20.
4.2.9.9.
(R)-4-((5-Carbamoyl-4-((3-methylbutan-2-yl)amino)pyrimidin-2-yl)amino)
phenyl
methane sulfonate (11i). Refer to the general procedure for the synthesis of 11i, as described in
section 4.1.9. 47.4% isolated yield; white solid. 1H NMR (DMSO-d6, 400 MHz)  ppm 0.90 (d, J
= 6.8 Hz, 3H), 0.94 (d, J = 6.8 Hz, 3H), 1.13 (d, J = 6.4 Hz, 3H,), 1.84 - 1.92 (m ,1H), 3.34 (s,
3H), 4.05 - 4.12 (m ,1H), 7.26 (d, J = 9.2 Hz, 2H), 7.87 (d, J = 9.2 Hz, 1H), 8.54 (s, 1H), 9.28 (d,
J = 8.4 Hz, 2H), 9.64 (s, 1H). 13C NMR (DMSO-d6, 100 MHz) δ ppm 17.1, 18.4, 18.8, 32.4,
37.5, 50.6, 100.0, 120.3, 122.6, 140.0, 143.5, 157.7, 160.3, 161.5, 169.7. ESI-MS: m/z Calcd for
C17H24N5O4S [M+1]+ 394.46, found 394.14.
4.2.9.10. (S)-4-((5-Carbamoyl-4-((3-methylbutan-2-yl)amino)pyrimidin-2-yl)amino) phenyl
methane sulfonate (11j). Refer to the general procedure for the synthesis of 11j, as described in
section 4.1.9. 47.4% isolated yield; white solid. 1H NMR (DMSO-d6, 400 MHz)  ppm 0.88 (d, J
= 6.8 Hz, 3H), 0.94 (d, J = 6.8 Hz, 3H), 1.12 (d, J = 6.8 Hz, 3H), 1.85 - 1.89 (m, 1H), 3.33(s,
1H), 4.06 - 4.11 (m, 1H), 7.26 (d, J = 9.2 Hz, 2H) 7.85 (d, J = 9.2 Hz, 2H), 8.52 (s, 1H), 9.27 (d,
J = 8.4 Hz, 1H), 9.64 (s, 1H). 13C NMR (DMSO-d6, 100 MHz) δ ppm 16.9, 18.1, 18.6, 32.1,
37.3, 50.2, 100.1, 120.1, 122.3, 140.1, 143.4, 157.5, 160.0, 161.2, 169.5. ESI-MS: m/z Calcd for
C17H24N5O4S [M+1]+ 394.46, found 394.20.
4.29.11. (R)-4-((5-Carbamoyl-4-((1-phenylethyl)amino)pyrimidin-2-yl)amino)phenyl methane
sulfonate (11k). Refer to the general procedure for the synthesis of 11k, as described in section
4.1.9. 47.8% isolated yield; white solid. 1H NMR (DMSO-d6, 400 MHz)  ppm 1.48 (d, J = 8.2
Hz, 3H), 3.32 (s, 3H), 5.20 - 5.23 (m, 1H), 7.21-7.16 (m, 3H), 7.23 - 7.42 (m, 4H), 7.60 - 7.66
(m, 2H), 8.55 (s, 1H), 9.63 (m, 2H). 13C NMR (DMSO-d6, 100 MHz) δ ppm 20.9, 37.4, 55.2,
100.4, 121.1, 122.7, 127.1, 127.9, 128.7, 139.7, 140.3, 143.9, 158.5, 160.8, 162.2, 169.8. ESI-
MS: m/z Calcd for C20H21N5O4S [M]+ 427.48, found 427.80.
4.2.9.12. 4-((5-Carbamoyl-4-((3-fluorobenzyl)amino)pyrimidin-2-yl)amino)phenyl methane
sulfonate (11l). Refer to the general procedure for the synthesis of 11l, as described in section
1
4.1.9. 46.2% isolated yield; white solid. H NMR (DMSO-d6, 400 MHz)  ppm 3.33 (s, 3H),

4.67 (d, J = 6.0 Hz, 2H), 7.04 - 7.19 (m. 5H), 7.35 - 7.41 (m, 1H), 7.68 (d, J = 9.2 Hz, 2H), 8.57
(s, 1H), 9.59 (t, J = 5.6 Hz, 2H), 9.68 (s, 1H). 13C NMR (DMSO-d6, 100 MHz) δ ppm 37.6, 43.7,
100.3, 112.5, 117.9, 121.1, 122.8, 123.1, 127.0, 139.5, 142.1, 143.8, 158.2, 159.9, 160.9, 162.0,
169.6. ESI-MS: m/z Calcd for C19H18FN5O4S [M]+ 431.44, found 431.80.
4.2.9.13. 4-((5-Carbamoyl-4-((4-fluorobenzyl)amino)pyrimidin-2-yl)amino)phenyl methane
sulfonate (11m). Refer to the general procedure for the synthesis of 11m, as described in section
1
4.1.9. 47.3% isolated yield; white solid. H NMR (DMSO-d6, 400 MHz)  ppm 3.32 (s, 3H),
4.67 (d, J = 6.0 Hz, 2H), 7.15 - 7.20 (m, 4H), 7.35 - 7.39 (m, 2H), 7.72 (d, J = 8.8 Hz, 2H), 8.56
13
(s, 1H), 9.55 (s, 1H), 9.67 (s, 1H). C NMR (DMSO-d6, 100 MHz) δ ppm 37.5, 43.4, 100.5,
115.2, 121.0, 122.5, 127.7, 139.2, 140.3, 143.2, 159.4, 160.2, 160.8, 162.3, 169.1. ESI-MS: m/z
Calcd for C19H18FN5O4S [M]+ 431.44, found 431.90.
4.2.9.14. 4-((5-Carbamoyl-4-((2-fluorobenzyl)amino)pyrimidin-2-yl)amino)phenyl methane
sulfonate (11n) Refer to the general procedure for the synthesis of 11n, as described in section
1
4.1.9. 47.9% isolated yield; white solid. H NMR (DMSO-d6, 400 MHz)  ppm 3.32 (s, 3H),
4.67 (d, J = 6 Hz, 2H), 7.13 - 7.17 (m, 3H), 7.21 - 7.33 (m, 3H), 7.69 (d, J = 8.8 Hz, 2H), 8.57 (s,
1H), 9.55 (s, 1H), 9.67 (s, 1H). 13C NMR (DMSO-d6, 100 MHz) δ ppm 37.2, 39.3, 100.6, 114.7,
121.4, 122.8, 124.0, 128.1, 129.5, 139.0, 139.2, 143.5, 159.2, 160.5, 161.2, 162.9, 169.7. ESI-
MS: m/z Calcd for C19H18FN5O4S [M]+ 431.44, found 431.90.
4.2.9.15.
4-((5-Carbamoyl-4-((naphthalen-1-ylmethyl)amino)pyrimidin-2-yl)amino)
phenyl
methanes ulfonate (11o). Refer to the general procedure for the synthesis of 11o, as described in
section 4.1.9. 50.5% isolated yield; white solid.1H NMR (DMSO-d6, 400 MHz)  ppm 1.67 (d, J
= 6.8 Hz, 3H), 3.28 (s, 3H), 6.05 (t, J = 6.4 Hz, 1H), 6.52 - 6.58 (m, 2H), 7.36 - 7.44 (m, 2H),
7.45 - 7.48 (m, 2H), 7.64 - 7.85 (m, 2H), 7.89 -7.91 (m, 1H), 8.01 (d, J = 8.0 Hz, 1H), 8.27 (d, J
= 8.4 Hz, 1H), 8.59 (s, 1H), 8.97 (s, 1H), 9.83 (s, 1H), 12.90 (d, J = 9.2 Hz, 1H). 13C NMR
(DMSO-d6, 100 MHz) δ ppm 21.5, 37.6, 57.9, 100.2, 121.8, 122.4, 122.9, 125.8, 126.9, 128.1,
131.0, 139.4, 140.9, 143.5, 159.7, 161.0, 162.7, 169.2. ESI-MS: Calcd for C24H25N5O4S [M+2]+
477.54, found 479.21.
4.3.
In vitro and ex vivo assay:
4.3.1: Enzymatic potency of JAK inhibitors (biochemical assay):

Human JAK1-3 and TYK2 kinase domains were purchased from Carna Biosciences (Japan), and
the assay was performed using a streptavidin-coated 96-well plate. The reaction mixture
contained 15 mM Tris-HCl (pH 7.5), 0.01% Tween 20, 2 mM DTT, 10 mM MgCl2, 250 nM
Biotin-Lyn-Substrate-2 (Peptide Institute, Inc., Osaka, Japan) and ATP. The final concentrations
of ATP were 10 μM for JAK1-3 and TYK2. Test compounds were dissolved in DMSO and the
reaction was initiated by adding the kinase domain, followed by incubation at room temperature
for 1 hr. Kinase activity was measured as the rate of phosphorylation of Biotin-Lyn-Substrate-2,
using HRP-conjugated anti-phosphotyrosine antibody (HRP-PY-20; Santa Cruz Biotechnology,
CA, USA) with a phosphotyrosine-specific ELISA. All the experiments were performed in
duplicate. The IC50 values were calculated using linear regression analysis [32].
4.3.2. JAK cellular assays using human peripheral blood mononuclear cells (PBMCs):
PBMCs were collected from healthy volunteers (as per Zydus Research Centre, ethical
committee protocol), into sodium heparin vacutainer tubes. After incubation with compound 11i
and Tofacitinib at 37°C for 30 min, blood was triggered with either recombinant human IL-6
(400 ng/mL; R&D Systems), IL-2 (20 ng/mL; R&D Systems), GM-CSF (100 ng/mL; Pepro-
Tech), or vehicle (PBS plus 0.1% [w/v] BSA), at 37°C for 20 min and treated with pre-warmed
13 lysis/fix buffer (BD Biosciences) to lyse RBCs and fix leukocytes. Cells were permeabilized
with 100% MeOH and incubated with anti-pSTAT3 and anti-CD4 (IL- 6-triggered samples),
anti-pSTAT5 and anti-CD4 Abs (IL-2-triggered samples), or anti-pSTAT5 Abs (GM-CSF-
triggered samples; all Abs were from BD Biosciences) at 4°C, for 30min, washed once with PBS
and analyzed on a FACS Canto II flow cytometer.[33, 34] IC50 values were determined, using
Prism software (Version 7, GraphPad), for Tofacitinib and 11i, Table 4.
4.3.3. Plasma protein binding:
An equilibrium dialysis method was used to determine the plasma fraction unbound (fu) values.
Briefly, dialysis membranes (MWCO 12‐14K) and 96‐well dialysis devices were assembled
following the manufacturer's instructions (HT Dialysis, LLC, USA). Human plasma samples
containing 1 μmol/L test compounds with 1% DMSO were dialyzed against PBS for 6 hours in a
humidified incubator (75% relative humidity; 5% CO2/95% air) at 37°C at 450 RPM.
Quadruplicates of binding were measured for each compound. Samples were matrix‐matched

and quenched with cold acetonitrile containing internal standard(s). The solutions were
centrifuged and the supernatant was analyzed using LC‐MS/MS.
4.4.
In vivo efficacy studies:
The animal experiments were carried out in rats and mice, bred in-house. Animals were housed
in groups of 6 animals per cage, for a week, in order to habituate them to vivarium conditions (25
 4 oC, 60-65 % relative humidity, 12 : 12 h light dark cycle, with lights on, at 7.30 am). All the
animal experiments were carried out according to the internationally valid guidelines following
approval by the ‘Zydus Research Center, animal ethical committee’.
4.4.1. Protocol for Collagen Induced Arthritis (CIA) study in mice:
CIA study is a representative animal model of human rheumatoid arthritis [35]. Following 7 days
acclimation, male DBA1j (8 to 12-weeks old) mice were randomly assigned to groups according
body weight. Mice were immunized subcutaneously in the tail using bovine type II collagen mix
in complete Freund’s adjuvant (CFA). Twenty-one days after the first immunization, mice were
given booster dose of collagen in incomplete Freund’s adjuvant (IFA). Mice were monitored
every other day after the booster dose for the development of arthritis. Mice were recruited for
the study once clinical signs were visible. Eight animals were assigned each of three groups
[vehicle, Tofacitinib and test compound, 11i], treatment was continued for three weeks and
percentage inhibition in clinical score is recorded as per graded score. Body weights of the
animals were also recorded 3 times a week as a measure of treatment related side effect and paw
thickness measured twice a week (Fig. 2a). The mice were scored in a blinded manner, for signs
of arthritis in each paw according to the following scale: 0 - no swelling or redness/normal paw;
1- swelling and/or redness in one digit; 2- swelling and/or redness in two or more digits; and 3-
entire paw is swollen or red. The severity score was reported as the sum of all four paws for each
mouse and expressed as the average severity score for each group.
4.4.2. Protocol for Adjuvant Induced Arthritis (AIA) model in rats:
Arthritis was induced in female Lewis rats by inoculation with Freund's complete adjuvant
(CFA). [36] Briefly, on day 0, rats were anesthetized (mixture of ketamine and xylazine
(80:ꢀ10ꢀmg/kg, intraperitoneally)) and then injected with 0.1ꢀmL CFA (1ꢀmg/mL of heat-
inactivated Mycobacterium tuberculosis) intra-dermally, at the base of the tail. Eight animals

were assigned in each of the three groups [vehicle (2% sodium carboxymethylcellulose (CMC),
positive control (Tofacitinib, 60 mg/kg) and test compound 11i (3, 10 and 30 mg/kg)]. Treatment
was continued for 20 days (once daily, by oral gavage) and paw edema determined by measuring
changes in the paw volume, using a plethysmometer, on days 10, 12, 14, 16, 18 and 20, post-
CFA injection. Statistical analysis was performed by one-way ANOVA (Dunnett's method, data
represent the mean ± S.E.M., n = 8/group).
4.5. Pharmacokinetic studies:
A comparative single dose (3 mg/kg, po and 1 mg/kg, iv) PK study of compounds 5k, 11i and
Tofacitinib was evaluated in overnight fasted male C57BL/6J mice (n=6). Serial blood samples
were collected in the micro-centrifuge tubes containing EDTA, at pre-dose, 0.15, 0.3, 0.5, 0.75,
1, 2, 4, 6, 8, 24 and 30 h post-dose, after the compounds administration. Approximately 0.3 ml of
blood was collected at each time points and centrifuged at 4oC. The obtained plasma was frozen,
stored at-70oC and the concentrations of compounds in plasma were determined by the LC-
MS/MS (Shimadzu LC10AD, USA), using YMC hydrosphere C18 (2.0 x 50 mm, 3 m) column
(YMC Inc., USA). The pharmacokinetic parameters, such as Tmax, t1/2, Cmax, AUC and %F
were calculated using a non-compartmental model of WinNonlin software version 5.2.1.
4.6.
Docking studies:
Multiple structures of JAK3, co-crystallized with various ligands, were analyzed, and the
structure with PDB ID 5W86 (solved at 2.6Å) was selected due to core similarity of the co-
crystallized ligand and Cerdulatinib. [37] The protein was prepared using protein preparation
wizard of Schrodinger Suite 2018-4, at pH 7.4. Ligands were prepared using Ligprep module at
pH 7.0 ± 0.5, with default settings. Glide grid was used with default options for grid generation
and Glide SP docking was used for docking simulation with default settings. The docked pose of
Cerdulatinib, 5k and 11i was analyzed with respect to docking score, docking pose and hydrogen
bonding interactions with the key region residues of the JAK3 enzyme.
Acknowledgments
Authors thank the management of Zydus Research Centre, Cadila Healthcare Ltd. for support
and encouragement and the analytical department for providing analytical data.

Corresponding Author: Rajesh Bahekar
E-mail: rajeshbahekar@zyduscadila.com
Notes
The authors declare no competing financial interest.
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37. Schrödinger Release, 2018-3: Glide, Schrödinger, LLC, New York, NY, 2018.

CN
O
S
N
O
O
O
N
N
N N
CN
N
NH
N
N
N
N
N
N
N
H
H
N
N
H
PF-00651600
Tofacitinib
Baricitinib
JAK1 : IC50 =>10000 nM
JAK2 : IC50 = >10000 nM
JAK3 : IC50 = 33.1 nM
JAK1 : IC50 =3.2 nM
JAK2 : IC50 = 4.2nM
JAK3 : IC50 = 1.6 nM
JAK1 : IC50 =5.9 nM
JAK2 : IC50 = 5.7 nM
JAK3 : IC50 =>400 nM
H
HN
O S O
O
N
CN
O
O
S
N
NH
N
O
NH
N
O
N
O
N
NH2
N
N
N
N
N
N
N
H
H
H
N
Momelotinib
Cerdulatinib
JAK1 : IC50 = 12 nM
Fedratinib
JAK1 : IC50 = 105 nM
JAK2 : IC50 = 3 nM
JAK1 : IC50 = 2.7 nM
JAK2 : IC50 = 4.5 nM
JAK3 : IC50 = 332 nM
JAK2 : IC50
JAK3 : IC50
=
=
6 nM
8 nM
JAK3 : IC50 = 1000 nM
Fig. 1. Structures of 1H-pyrrolo[2,3-b]pyridine and Pyrimidine based JAK inhibitors