Development of selective inhibitors for the treatment of rheumatoid arthritis: (R)-3-(3-(Methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)-3-oxopropanenitrile as a JAK1-selective inhibitor

Article abstract of DOI:10.1016/j.bmc.2018.01.021

A series of 3(R)-aminopyrrolidine derivatives were designed and synthesized for JAK1-selective inhibitors through the modification of tofacitinib's core structure, (3R,4R)-3-amino-4-methylpiperidine. From the new core structures, we selected (R)-N-methyl-N-(pyrrolidin-3-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine as a scaffold for further SAR studies. From biochemical enzyme assays and liver microsomal stability tests, (R)-3-(3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)-3-oxopropanenitrile (6) was chosen for further in vivo test through oral administration. Compound 6 showed improved selectivity for JAK1 compared to that of tofacitinib (IC₅₀ 11, 2.4 × 10², 2.8 × 10³, and 1.1 × 10² nM for JAK1, JAK2, JAK3, and TYK2, respectively). In CIA and AIA model tests, compound 6 exhibited similar efficacy to tofacitinib citrate.

Full text of DOI:10.1016/j.bmc.2018.01.021

Accepted Manuscript
Development of selective inhibitors for the treatment of rheumatoid arthritis:
(R)-3-(3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)-3-
oxopropanenitrile as a JAK1-selective inhibitor
Chieyeon Chough, Misuk Joung, Sunmin Lee, Jaemin Lee, Jong Hoon Kim, B.
Moon Kim
PII:
S0968-0896(17)32304-0
https://doi.org/10.1016/j.bmc.2018.01.021
BMC 14175
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 November 2017
22 January 2018
24 January 2018
Please cite this article as: Chough, C., Joung, M., Lee, S., Lee, J., Kim, J.H., Moon Kim, B., Development of selective
inhibitors for the treatment of rheumatoid arthritis: (R)-3-(3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-
yl)amino)pyrrolidin-1-yl)-3-oxopropanenitrile as a JAK1-selective inhibitor, Bioorganic & Medicinal Chemistry
(2018), doi: https://doi.org/10.1016/j.bmc.2018.01.021
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Fonts or abstract dimensions should not be changed or altered.
Development of selective inhibitors for the
treatment of rheumatoid arthritis: (R)-3-(3-
(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-
yl)amino)pyrrolidin-1-yl)-3-
Leave this area blank for abstract info.
oxopropanenitrile as a JAK1-selective
inhibitor
Chieyeon Chougha,+, Misuk Joungb,+, Sunmin Leeb, Jaemin Leeb, Jong Hoon Kimc and B. Moon Kima,*
a Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 08876, South Korea
b Yang Ji Chemical Co., Ltd., Gyeonggi Bio-Center, Suwon, Gyeonggi-do 16229, South Korea
c Han Wha Pharma Co., Ltd., 109, Yagam-gil, Nam-myeon, Chuncheon, Gangwon-do 24468, South Korea
+ These two individuals contributed equally to this work.

Bioorganic & Medicinal Chemistry
Development of selective inhibitors for the treatment of rheumatoid arthritis: (R)-3-
(3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)-3-
oxopropanenitrile as a JAK1-selective inhibitor
Chieyeon Chougha, + , Misuk Joungb, + , Sunmin Leeb , Jaemin Leeb , Jong Hoon Kimc and B. Moon Kima, 
a Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 08876, South Korea
b Yang Ji Chemical Co., Ltd., Gyeonggi Bio-Center, Suwon, Gyeonggi-do 16229, South Korea
c Han Wha Pharma Co., Ltd., 109, Yagam-gil, Nam-myeon, Chuncheon, Gangwon-do 24468, South Korea
+ These two individuals contributed equally to this work.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A series of 3(R)-aminopyrrolidine derivatives were designed and synthesized for JAK1-selective
inhibitors through the modification of tofacitinib’s core structure, (3R,4R)-3-amino-4-
methylpiperidine. From the new core structures, we selected (R)-N-methyl-N-(pyrrolidin-3-yl)-
7H-pyrrolo[2,3-d]pyrimidin-4-amine as a scaffold for further SAR studies. From biochemical
enzyme assays and liver microsomal stability tests, (R)-3-(3-(methyl(7H-pyrrolo[2,3-
d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)-3-oxopropanenitrile (6) was chosen for further in vivo
test through oral administration. Compound 6 showed improved selectivity for JAK1 compared
to that of tofacitinib (IC50 11, 2.4x102, 2.8x103, and 1.1x102 nM for JAK1, JAK2, JAK3, and
TYK2, respectively). In CIA and AIA model tests, compound 6 exhibited similar efficacy to
tofacitinib citrate.
Available online
Keywords:
JAK inhibitor
rheumatoid arthritis
JAK1-selective
collagen-induced arthritis mouse model
adjuvant-induced arthritis rat model
2018 Elsevier Ltd. All rights reserved.
1. Introduction
shown distinguished therapeutic efficacy for patients who have
not responded to the treatment of biologics such as adalimumab
Rheumatoid arthritis (RA) is a chronic autoimmune disease
related to inflammatory disorder that damages not only joints but
also a wide variety of body systems.1 Much effort has been
concentrated in search of the RA’s therapeutic targets and
recently several targets2,3 such as cytokines,4,5 G-protein coupled
receptors,6 and kinases7,8 have been identified. Several disease-
modifying antirheumatic drugs (DMARDS)9 have been used,
however, they have been found to be inappropriate for long term
use due to low therapeutic response and some side effects. Since
then a few biologics such as etanercept, infliximab, and
adalimumab have been introduced.10 Though these biologics
exhibit better efficacies than the synthetic ones, their use has
been limited because of high cost, limited i.v. administration,
etc.11,12 Recently new therapeutic targets such as Janus kinase
(JAK)/signal transducer and activator of transcription (STAT)
signal pathway13 have been identified for RA treatment. Isolation
of JAK kinases was first made in 198914 and their roles were
discovered in 1994.15
and etanercept, it has been known to exhibit some serious
adverse effects.19,20 They include anemia, liver toxicity, lipid
level increase, etc., some of which may result from nonselective
inhibition of JAK family enzymes. For example, anemia is
believed to result from the inhibition of JAK2 isozyme.21,22 Since
tofacitinib is a pan-JAK inhibitor suppressing all JAK isozymes
including JAK1, JAK2, JAK3 and TYK2, the need for a new
selective inhibitor against a single JAK isozyme has surfaced
recently.23-32 Of the four isozymes, JAK1 has been focused as a
selective target for treating rheumatoid arthritis because it can
more effectively control the levels of the cytokines involved in
the disease symptoms than the others.33 The representative JAK1-
selective inhibitors include filgotinib (GLPG0634),26,34-41
upadacitinib (ABT-494),42-46 solcitinib (GSK2586184),35,47-50
itacitinib (INCB039110),51-54 PF-04965842.55-56 The most
advanced drug candidate as a JAK1-selective inhibitor is
Galapagos’s filgotinib, and its phase II result provided the proof-
of-concept in selective JAK1 inhibition.24 Its phase III clinical
trial started in 2016.57-59
Pfizer’s tofacitinib, the first US FDA-approved oral
rheumatoid arthritis drug, is believed to exhibit its efficacy
However, from filgotinib’s toxicological tests in rat and dog
through the inhibition of Janus kinases (JAKs).16-18 Though it has
models, adverse effects in testes were reported.60 Due to this
———
 Corresponding author. Tel.: +82-2-880-6644; fax: +82-2-872-7505; e-mail: kimbm@snu.ac.kr

Figure 1. Interactions of tofacitinib with JAK1 or JAK2.
Scheme 1. Synthesis of substituted (R)-N-alkyl-N-(pyrrolidin-3-yl)-7H-
pyrrolo[2,3-d]pyrimidin-4-amines.
we hypothesized that changing the piperidine moiety of
tofacitinib can alter the binding affinity with JAK2 more than
that with JAK1.
Figure 2. Docking simulation of a) tofacitinib and b) compound 6 at JAK2
(PDB ID: 3FUP) and c) an overlay of the lowest conformations of tofactinib
(red color) and compound 6 at JAK2.
Based upon our hypothesis, we selected a pyrrolidine moiety
in place of the piperidine of tofacitinib. A docking simulation
using AutoDock 4.2 program63-65 was performed to assess the
effect of the pyrrolidine substitution at the piperidine site of the
inhibitors (Figure 2). The estimated binding energies of
tofacitinib and our representative compound 6 at JAK1 (PDB ID:
3EYG) were -8.10 and -7.50 kcal/mol, respectively. Besides,
estimated binding energies of -8.98 and -7.93 kcal/mol,
respectively, for tofacitinib and compound 6 were obtained in the
case of JAK2 binding (PDB ID: 3FUP). Increased intermolecular
energy of compound 6 with JAK2 appears to result from the
absence of its interactions with Ser963 and Leu983 of JAK2.
From the above result, we expected that compound 6 would
exhibit lower binding affinity for JAK2 through the substitution
into pyrrolidine moiety. In addition, since the methyl group of C9
at tofactinib appears to interact with Leu855 at JAK2, replacing
the methyl group by another alkyl group may also influence the
binding affinity at JAK2. According to the docking results, we
designed inhibitors possessing several substituted pyrrolidine
moieties equipped with various alkyl groups at the bridging
amino group of compound 6.
problem, the US FDA set lower maximum dosage in the case of
male at the phase III clinical trials. In relation with these results,
we would like to report our new JAK1 selective inhibitors aimed
at resolving the toxicological issue. Our design principle was
centered on searching for a JAK1-selective inhibitor possessing a
substituted 3(R)-aminopyrrolidine moiety in place of the (3R,4R)-
3-amino-4-methylpiperidine of tofacitinib. Here we describe our
medicinal chemistry effort in the discovery of 3(R)-(3-
(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-
yl)-3-oxopropanenitrile as a more selective inhibitor against
JAK1 over JAK2 than tofacitinib (IC50 3.1 nM for JAK1 vs 4.2
nM for JAK2).61
2. Strategy
According to the tofacitinib’s X-ray crystal structure reported
by N. K. Williams et al.,62 the interactions between the piperidine
moiety of tofacitinib and each isozyme including JAK1 and
JAK2 appear to be the basis for binding affinity differentiation
(Figure 1). Especially, the carbon atoms C4, C5, and C7 of the
piperidine ring may play an important role: notable interactions
are those of C4 and C5 with Arg1007, Asn1008, Gly1020, and
Asp1021 at JAK1 (Asp981, Gly993, and Asp994 at JAK2) and
C7 with Ser963, Arg1007, and Leu1010 at JAK1 (Ser936,
Arg980, and Leu983 at JAK2). However, the C2 and N3 atoms
appear to be involved in binding JAK2, but not JAK1. Therefore,
3. Synthesis
Three 3-aminopyrrolidine derivatives with varying R1 and R2
substituents at the 4-position were chosen for the studies, namely
(R)-1-benzylpyrrolidin-3-amine (1a), (R)-4,4-dimethyl-1-((R)-1-
phenylethyl)pyrrolidin-3-amine
(1b),
and
(R)-6-((R)-1-

Table 2. The IC50 values against JAK1 and JAK2 and the
selectivity indices of substituted (R)-N-(pyrrolidin-3-yl)-7H-
pyrrolo[2,3-d]pyrimidin-4-amines with varying R1 and R2
groups.
Table 1. The IC50 values of compounds 6 – 9 and tofacitinib
against JAK1 and JAK2 and the selectivity indices of
substituted (R)-N-methyl-N-(pyrrolidin-3-yl)-7H-pyrrolo[2,3-
d]pyrimidin-4-amines according to the substitution at the 4-
position of the pyrrolidine ring.
SIa
23
23
Cmpd
R1
R2
IC50 (nM)
SIa
Cmpd
A
IC50 (nM)
JAK2
JAK1
JAK2
JAK1
11
Me
Et
11
62
2.4x102
1.3x103
1.7x103
23
21
11
6
9
10
2.4x102
6
7
1.6x102
H
Me
Et
5.1x102
4.1
19
17
1.9x103
57
3.7
14
5.8
82
11
12
13
14
2.6x102
5.9x103
1.1x102
1.4x103
16
4.5x102
9.9
28
8
a SI: Selectivity Index = JAK2 IC50 / JAK1 IC50
commercially available 3(R)-amino-1-benzylpyrrolidine (1a) for
our structure-activity-relationship (SAR) studies involving many
substituents at the pyrrolidine nitrogen because of the simple
synthetic steps.
2.0
5.0
Tofacitinib
a SI: Selectivity Index = JAK2 IC50 / JAK1 IC50
phenylethyl)-6-azaspiro[3.4]octan-8-amine (1c). Except for the
commercially available (R)-3-amino-1-benzylpyrrolidine (1a),
compounds 1b and 1c were synthesized according to published
methods.66,67 Scheme 1 shows a synthetic sequence leading to the
pyrrrolidines 5aa – 5c, from which a variety of derivatives (6 –
42) were prepared as potential JAK1 inhibitors: 1) the primary
amino group of 1a-c was protected from the reaction with di-tert-
butyl dicarbonate, acetic anhydride, or cyclopropanecarbonyl
chloride, 2) the N-tert-butoxycarbonyl-, N-acetyl- or N-
cyclopropanecarbonyl- protected compounds 2aa – 2c were
treated with LiAlH4 to yield alkylated amines 3aa – 3c, 3) the
alkylamines 3aa – 3c and the unprotected amine 1a were allowed
to react with 6-chloro-7-deazapurine to produce compounds 4aa
– 4c, 4) hydrogenolysis using palladium on carbon and
ammonium formate removed the benzyl group of 4aa – 4ad or 1-
phenylethyl moiety of 4b and 4c. The desired inhibitors 6 – 42
were obtained from 5aa – 5c through amide coupling,
sulfonylation, alkylation, carbonylation, etc.
With the N-alkylated compounds in hand, we fixed the
pyrrolidine nitrogen with either cyanoacetate or 3-
cyanobenzenesulfonyl group as R1 at 1-position and probed the
inhibitory activities by changing the R2 at 6-position from
hydrogen to cyclopropylmethyl group. In both cyanoacetyl- and
3-cyanophenylsulfonyl-substituted
pyrrolidine
derivatives,
increasing from methyl to ethyl and to cyclopropylmethyl
decreased the inhibitory activities against JAK1, although JAK2
inhibitions were not as much affected. In the case of compound
11, where there is no alkyl substitution on the 3-amino group,
quite low level of inhibition against JAK1 was observed. It turns
out that the 3-cyanophenylsulfonyl substitution resulted in better
inhibition on JAK1 than the cyanoacetyl one in all the cases
examined, although mixed results were obtained in selectivity
indices. After the results of Table 2, we chose methyl group as R2
and
(R)-N-methyl-N-(pyrrolidin-3-yl)-7H-pyrrolo[2,3-
d]pyrimidin-4-amine as a scaffold for further SAR studies.
To find a new lead compound, we screened the inhibitory
activities for JAK1 and JAK2 of compounds possessing a variety
of substituents at the 1-nitrogen of pyrrolidine moiety (Table 3).
First, a comparison between amide and alkylamine groups of
similar size (6 vs 15) was attempted and the amide group
appeared to increase the affinity for JAK1 isozyme. This
hypothesis also appears to apply to the urea functionality with
compound 20 exhibiting 22 nM IC50 value for JAK1. If the
inhibitors contain an amide or urea side chain bulkier than the
cyanomethyl group as in 6, their inhibitions for JAK1 isozyme
were less effective (16, 18, and 19). However, in the case of 20,
its inhibitory activity was similar to that of compound 6 although
it has an N-phenyl side chain, which is larger than that of
compound 6. With compounds 6 and 17, similar inhibitory
activities were observed, which suggests that the planar or linear
group at the side chain of amide offsets the ill effect the side
chain length. The introduction of the sulfonamide on the 1-
nitrogen of the pyrrolidine core improved the inhibitory activities
for JAK1 (16 vs 25). Moreover, the arenesulfonamides (12 and
27 – 40) exhibited higher inhibitory activities than the
sulfonamides having alkyl or heterocyclic groups (21 – 26). As
for the substitutions at the benzene ring, inhibitors with
substituents at ortho-position (31 and 33) showed lower
4. Results and discussions
4.1. Enzyme assay
The 7-deazapurine moiety of tofacitinib was considered to be
critical in securing the ATP-binding site of JAK isozymes,
therefore it was kept in our scaffold structure. First, to evaluate
the effect of the substituents at the 4-position of the pyrrolidine
ring, we prepared cyanoacetyl derivatives 6 – 8 from the three
parent pyrrolidine precursors, 5aa, 5b, and 5c. We then screened
the inhibitory efficiencies of the derivatives substituted with
dimethyl and spirocyclic moieties at the 4-position of the
pyrrolidine core, which is believed to correspond to the 4-
position of the piperidine of tofacitinib (Table 1). The
unsubstituted inhibitor 6 exhibited an IC50 value of 11 nM for
JAK1 and its selectivity index was 23, which was higher than
that of tofacitinib (IC50 2.0 nM and 9.9 nM for JAK1 and JAK2,
repectively, n = 3). The dimethyl-substituted 7 was 23-fold less
potent against JAK1 than that of compound 6, however, the
spirocyclic derivative 8 had similar levels of IC50’s to compound
6. After identifying the fact that the derivative 8 having (R)-6-
azaspiro[3.4]octan-8-amine moiety showed the best selectivity of
JAK1 over JAK2, we selected the scaffold derived from

Table 3. The IC50 values against JAK1 and JAK2 and the selectivity indices of substituted (R)-N-methyl-N-(pyrrolidin-3-yl)-
7H-pyrrolo[2,3-d]pyrimidin-4-amines.
IC50 (nM)
IC50 (nM)
R
Cmpd
R
SIa
23
Cmpd
SIa
3.8
JAK1
11
JAK2
JAK1
14
JAK2
53
2.4x102
1.9x103
6
29
53
36
11
1.1x102
10
15
30
7.4x102
10
2.7x104
1.7x102
3.9x103
36
17
56
52
4.1
11
3.7x102
57
7.1
14
16
17
18
31
12
32
70
1.2x103
1.1x102
1.1x102
22
4.3x103
5.5x102
39
25
1.1x102
1.9
1.1x103
18
10
19
20
33
34
9.5
70
4.7x103
67
3.6
89
25
21
35
1.4x102
79
4.5x103
2.4x103
32
30
29
19
7.3x102
1.8x103
25
95
22
23
36
37
1.7x102
4.8x103
4.6x102
1.9x104
28
14
29
26
9.5
13
1.5x103
3.4x103
6.9x102
58
3.6x102
53
24
25
26
38
39
40
34
6.5x102
28
25
4.3x102
1.3x102
15
66
3.9x103
4.4x103
59
16
27
28
41
42
5.2
2.7x102
a SI: Selectivity Index = JAK2 IC50 / JAK1 IC50

Table 4. Liver microsomal stabilities of 6, 12, 34 and 39.
Cmpd
% Remaining during 30 min
Human (%)
97.6
Dog (%)
92.8
Rat (%)
92.8
3.7
Mouse (%)
> 100
10.0
6
26.3
31.6
12
34
39
13.4
10.4
2.5
3.1
21.0
14.2
11.4
8.4
Table 5. In vitro ADME profiles of 6.
Plasma protein binding (% bound)
Human
14.7
Rat
17.7
Plasma stability (% remaining)
Human
Rat
30 min
96.3
120 min
99.8
30 min
> 100
120 min
> 100
Figure 3. The kinome tree of 6 against 345 kinases at the 10 M concentration
drawn by the web accessible KinMap program.
Caco-2 permeability (×10-5 cm/sec)
4.3. Kinase profiling and human ether-a-go-go related gene
(hERG) potassium channel assay
Papp, A to B
0.38
Papp, B to A
0.77
Efflux ratio
2.02
Inhibitors targeting ATP-binding site of a kinase can inhibit
other kinases because it generally resembles the structure of
dephosphorylated ATP. Therefore, we carried out kinase
profiling for compound 6 against 345 kinases. At 10 M
concentration, the kinases inhibited to over 90% were only three
kinases, JAK1, JAK2, and TYK2. And the kinases with 80-90%
inhibition included 6 kinases: JAK3, ROCK-II (human), ROCK-
II (rat), DCAMKL3, CLK1, and Flt4. This result indicates that
the selectivity of compound 6 may be superior to tofacitinib’s
one, which inhibited 26 kinases over 90% at the same
concentration, as reported by D. C. Borie and colleages.17
Additionally, we identified that compound 6 exhibited IC50
values of 2.8x103 and 1.1x102 nM’s for JAK3 and TYK2,
respectively, which are two other important targets for pan-JAK
inhibitors.
CYP450 inhibition (activity % at 10 µM)
1A2
89.3
2C9
> 100
2C19
91.8
2D6
> 100
3A4
98.2
inhibition than the meta- or para-counterparts, presumably due to
steric interaction with JAK1 except for the fluorine substitution
cases (28 – 30). In the case of the selectivity for JAK1 over
JAK2, the inhibitors with substitution at para-position (30, 32,
and 35) showed 2.5 to 7.9-fold improved JAK1 selectivity
compared to those having meta-substitutions. Consequently,
compounds 32 and 39 were the most selective for JAK1 over
JAK2.
4.2. In vitro ADME
Since we aimed to develop a drug candidate that can be
administered orally for rheumatoid arthritis, we tested some
selected compounds (6, 12, 34 and 39) for liver microsomal
stabilities in several species to predict the liver first-pass effect.
Remaining percentages of the three compounds were measured
after 30 min incubation with human, dog, rat and mouse liver
microsomes (LM). As shown in Table 4, the amide 6 showed
good stabilities (over 90% remaining) against LM of all species,
but the benzenesulfonamides 12, 34 and 39 showed low
stabilities (below 30% remaining) in almost all the species so that
the LM stability screening for the other benzenesulfonamide
derivatives. Therefore, compound 6, of which the
cyanoacetamide group at the nitrogen of pyrrolidine moiety
composes the structure of tofacitinib, was selected for in vivo
efficacy tests.
For the prediction for cardiotoxicity of compound 6, hERG
assay was performed at HEK293 cell with the automated patch
clamp method. Compound 6 showed IC50 value of 93 M. When
filgotinib, competitive JAK1-selective inhibitor, was carried out
under the same condition, it showed the IC50 value of 85 M.
4.4. Pharmacokinetics of 6
To address oral bioavailability of compound 6, we then
carried out the pharmacokinetic tests in dogs, rats, and mice. The
vehicles for oral administration and intravenous injection were
corn oil and the solution of 10% ethanol and 90% PEG400,
respectively, because of low solubility of compound 6 in water.
In the case of pharmacokinetics through intravenous injection,
the drug exposure generally tended to be decreased so that the
bioavailability at all species became over 100%, which is similar
to the results reported by K. W. Ward et al.68 and R. Weaver et
al.69
In addition, we performed in vitro ADME tests such as plasm
protein binding, plasma stability, Caco-2 permeability, and
CYP450 inhibition for the selected compound 6. It showed low
human plasma protein bound percentage of 14.7%, which comes
from the low lipophilicity of compound 6. In human and rat
plasma stability tests, compound 6 displayed high plasma
In the case of oral administration at 10 mg/kg dosage in male
Sprague Dawley rats, compound 6 showed 2.1 hours of half-life
(t1/2), 4.3x103 ng·h/mL of area under curve from 0 to infinite
stability in both tests. Compound
6 showed moderate
(AUC0 inf), 1.9x103 ng/mL of maximum concetration (Cmax), and

permeability in Caco-2 permeability test like filgotinib
(filgotinib’s Papp, A to B = 0.37x10-5 cm/sec).26 However, unlike
filgotinib (filgotinib’s efflux ratio = 16.5), compound 6 did not
seem to be heavily influenced by efflux mechanism.
Investigation of compound 6 against five major CYP450 isozymes
at 10 M concentration exhibited minimal degrees of inhibition.
0.30 hour of the time to reach the maximum concentration (Tmax).
Though the profiles of t1/2, Cmax, and Tmax were similar to the
reported tofacitinib’s ones (t1/2 = 2.0 h, Cmax = 2.4x103 ng/mL,
Tmax = 0.31 h), compound 6 surpassed tofacitinib with AUC0
inf

value of 2.8x103 ng·h/mL on drug exposure.70 In comparison

Table 6. Pharmacokinetic profiles of 6.
Species
Beagle dog
S. D. rat
ICR mouse
Route
N
Dose (mg/kg)
Cmax (ng/mL)
Tmax (h)
P.O.
4M
I.V.
4M
3
P.O.
4M
I.V.
4M
5
P.O.
4M
I.V.
4M
5
5
10
10
1.9x103
1.1
1.9x103
0.30
1.0x103
0.30
t1/2 (h)
1.7
1.6
2.1
0.70
9.5x102
9.3x102
0.9
2.1
0.9
AUC0 inf (ng·h/mL)
6.5x103
6.4x103
3.1
2.2x103
2.1x103
2.4
4.3x103
4.1x103
2.9
2.1x103
1.9x103
3.1
6.8x102
5.1x102
1.4
AUC0 t (ng·h/mL)

MRT (h)
F (%)
1.8x102
2.2x102
1.9x102
Figure 4. Plasma concentrations after a) oral administration and b) intravenous injection of 6 in Beagle dogs, Sprague-Dawley rats, and ICR mice.
with the reported profiles of filgotinib through oral treatment at 5
mg/kg dosage, filgotinib has a longer half-life (t1/2 = 3.9 h), but a
reduced paw swelling to a similar extent as tofacitinib citrate (10
mg/kg/day) treatment (Figure 6b).
slightly lower drug exposure (AUC0 = 1.7x103 ng·h/mL) than
t
compound 6,26 although direct comparison with filgotinib and 6
is impossible because of their different oral administration
dosages. Compound 6 showed a superior drug exposure to
5. Conclusions
From our SAR studies based on substituted pyrrolidine-
containing JAK1 selective inhibitors, we have identified our lead
tofacitinib (AUC0
= 2.3x103 ng·h/mL)70 in the PK study in
inf
male beagle dogsat 5 mg/kg dosage. However, PK profiles of
compound,
(R)-3-(3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-
yl)amino)pyrrolidin-1-yl)-3-oxopropanenitrile (6) as a potential
rheumatoid arthritis drug candidate. Its inhibitory activity and
selectivity for JAK1 over other JAK’s was based on the
replacement of the piperidine moiety of tofacitinib by a
pyrrolidine moiety. Compound 6 showed desirable selectivity
index for JAK1 over JAK2, JAK3 and TYK2, which may lead to
decreased side effects. Its human liver microsomal stability was
identified to withstand the liver first-pass. Also, we have shown
that compound 6 has improved efficacies compared to tofacitinib
for treating rheumatoid arthritis in CIA and AIA models, albeit in
a somewhat increased dose because of decreased inhibition of
JAK isozymes, especially JAK2 and JAK3 by compound 6 than
tofacitinib at the same serum concentration. Furthermore,
compound 6 surpassed filgotinib, a JAK1-selective inhibitor in
phase III, on many in vivo efficacy factors, although compound 6
has similar drug exposure through oral administration to
compound 6 in dogs are inferior to the reported values of
filgotinib, which features 5.2 hours of half-life (t1/2) and 1.4x104
26
ng·h/mL of AUC0
.
t

4.5. In vivo efficacy of 6
To identify in vivo efficacies in rheumatoid arthritis models,
we employed collagen-induced arthritis (CIA) in DBA/1J
mouse71 and adjuvant-induced arthritis (AIA) in Lewis rat.72 In
mouse CIA model, following indexes were used to evaluate the
efficacy of compound 6: clinical arthritis score, paw volume,
serum concentration of IL-6 and TNF-, bone surface/volume
ratio, histopathological semiquantitative score of ankle joint,
thickness of ankle joint, thickness of articular surface cartilage
and inflammatory cell infiltration in ankle joint (Figure 5). In
clinical arthritis score, treatment with compound 6 (100
mg/kg/day) showed more potent inhibition of arthritis symptom
than a JAK1-selective inhibitor filgotinib treatment (100
mg/kg/day) and showed similar arthritic score of tofacitinib
citrate treatment (50 mg/kg/day) at day 8 to 11 (Figure 5a).
However, its symptom relief level was nearly identical to those
of other drugs at day 18. Treatment with compound 6 resulted in
significant improvements of all other evaluation criteria
compared to vehicle treatment. Furthermore, the ameliorating
effects of compound 6 treatment were even better than filgotinib
or tofacitinib citrate in serum cytokine concentration, bone
filgotinib. In summary, compound
6
has desirable
physicochemical properties and efficacy as an oral JAK1-
selective inhibitor and these findings suggest that compound 6
can be a good candidate for the treatment of rheumatoid arthritis.
6. Experimental Section
6.1. Docking simulation
The initial structures of ligands, tofacitinib and compound 6,
were obtained through the optimization with Gaussian 03
package73 with Hatree-Fock method at 6-31G basis set level.
JAK1 (PDB ID: 3EYG) and JAK2 (PDB ID: 3FUP) were
prepared with removing waters and other ligands from only chain
A. Rigid docking simulations were performed with AutoDock 4.2
and AutoDockTools 1.5.6.63 The grid box composed of 60x60x60
points with spacing of 0.375 angstrom. The Lamarckian genetic
algorithm was used as search method for best docking
surface/volume
ratio,
histopathological
features
and
inflammatory cell infiltration. In a rat AIA model, clinical
arthritis score and paw thickness were assessed to evaluate
efficacy (Figure 6). Treatment with compound 6 (20 mg/kg/day)
significantly attenuated arthritis symptoms to a similar extent as
filgotinib (20 mg/kg/day) treatment (Figure 6a) and significantly

Figure 5. Effects of 6 treatment on collagen-induced arthritis in DBA/1J mice: a) the clinical arthritis scores for 18 days, b) the volumes of right hind paws on
days 1 and 15, c-d) the concentrations of IL-6 and TNF-, respectively, at the serums sampled after autopsy, e) the bone surface/volume ratios of right hind
ankle joints measured by micro-CT, f) the histopathological semiquantitative scores of right hind ankle joints, g) the right hind ankle joint thicknesses, h-i) the
articular surface cartilage thicknesses (tibia and talus) in right hind ankle joints, and j) the numbers of inflammatory cells infiltrated in the right hind ankle joints.
The significance symbols are ** = significantly different between G1 and G2 (P <0.01), + = significantly different from G2 (P <0.05), and ++ = significantly
different from G2 (P <0.01).
Figure 6. Effects of 6 treatment on adjuvant-induced arthritis in Lewis rats: a) the clinical arthritis scores and b) the volumes of right hind paws. The data were
measured twice per week for 14 days.
system 400 MHz DD2MR400, Bruker Biospin AVANCE II 400,
and Varian NMR System 500 MHz. Bruker Compact Ultra High
Resolution ESI Q-TOF mass spectrometer was used for the MS
data. The purities of synthesized compounds were analyzed
through the use of 256 nm-wavelength absorption spectra on
Agilent HPLC 1100 and 1260 infinity with 6120 Quadrupole
LC/MS detector. Additionally, their optical rotation data were
obtained from JASCO’s P-1030 Polarimeter.
conformations. The other options were default settings at
AutoDockTools 1.5.6.
6.2. Chemistry
All reagents for the syntheses were obtained from
commercially available sources and used without any further
purification. Except for the commercially available (R)-3-amino-
1-benzylpyrrolidine (1a) and (R)-6-((R)-1-phenylethyl)-6-
azaspiro[3.4]octan-8-amine (1c)66,67 were synthesized according
to published methods. The detailed synthetic procedures of (R)-
4,4-dimethyl-1-((R)-1-phenylethyl)pyrrolidin-3-amine (1b) was
provided in Supporting information. All final products were
purified by flash column chromatography and Merck silica gel 60
(0.040-0.063 mm) was used for flash column chromatography.
6.2.1. Synthesis of tert-butyl (R)-(1-
benzylpyrrolidin-3-yl)carbamate, 2aa
Sodium bicarbonate (5.92 g, 70.5 mmol) in 118 mL of
deionized water was added to (3R)-(+)-benzylaminopyrrolidine
1a (5.00 g, 28.4 mmol) solution in 118 mL of acetonitirile and
the mixture was stirred at room temperature for 10 minutes. Di-
tert-butyl dicarbamate (6.22 g, 28.5 mmol) was then added and
the mixture was stirred at room temperature overnight. After the
1
The structures of the compounds were identified through H and
13C NMR spectroscopy and high resolution mass spectrometry
(MS) analyses. NMR spectra were taken from Agilent NMR

reaction, the solution was concentrated under educed pressure
and the residue was extracted with dichloromethane three times.
The combined organic layers were dried over anhydrous sodium
sulfate, filtered and concentrated. The residue was purified with
flash column chromatography (methanol:dichloromethane =
2:98). Removing the solvent in vacuo provided 4.24 g of tert-
sodium hydroxide solution, and 8.10 mL of deionized water.
Then, celite 545 was added and the mixture was stirred for 30
minutes before being filtered through a celite 545 pad. The
filtered solution was concentrated under reduced pressure and the
residue was extracted with dichloromethane three times.
Combined organic layers were dried over anhydrous sodium
sulfate, filtered and concentrated. The residue was purified with
flash column chromatography (methanol:dichloromethane
:ammonium hydroxide = 5:90:5). Removing the solvent in vacuo
provided 2.17 g of (R)-1-benzyl-N-methylpyrrolidin-3-amine
1
butyl (R)-(1-benzylpyrrolidin-3-yl)carbamate (65.2% yield). H
NMR (400 MHz, CDCl3) δ 7.36 – 7.26 (m, 5H), 4.86 (bs, 1H),
4.18 (bs, 1H), 3.61 (s, 2H), 2.79 (bs, 1H), 2.65 – 2.61 (m, 1H),
2.54 (d, J = 8.0 Hz, 1H), 2.34 – 2.25 (m, 2H), 1.61 – 1.51 (m,
1H), 1.46 (s, 9H). []D +2.5o (c 0.620, CHCl3).
1
(98.6% yield). H NMR (400 MHz, CDCl3) δ 7.34 – 7.24 (m,
5H), 3.62 (s, 2H), 3.25 – 3.19 (m, 1H), 2.74 (dd, J = 9.4, 6.8 Hz,
1H), 2.64 (dt, J = 8.6, 6.0 Hz, 1H), 2.52 (dt, J = 8.4, 6.0 Hz, 1H),
2.41 – 2.37 (m, 1H), 2.38 (s, 3H), 2.19 – 2.09 (m, 1H), 2.02 (bs,
1H), 1.63 – 1.56 (m, 1H).
In the cases of 2b and 2c, the desired products were
synthesized
phenylethyl)pyrrolidin-3-amine
from
(R)-4,4-dimethyl-1-((R)-1-
(1b) and (R)-6-((R)-1-
phenylethyl)-6-azaspiro[3.4]octan-8-amine (1c), respectively,
instead of (3R)-(+)-benzylaminopyrrolidine 1a according to the
aforementioned process (vide supra).
In the cases from 3ab to 3c, the desired products were
synthesized from 2ab – 2c, respectively, instead of (R)-(1-
benzylpyrrolidin-3-yl)carbamate 2aa according to the
aforementioned process (vide supra).
6.2.1.1. tert-Butyl ((R)-4,4-dimethyl-1-((R)-1-
phenylethyl)pyrrolidin-3-yl)carbamate, 2b
6.2.2.1. (R)-1-Benzyl-N-ethylpyrrolidin-3-amine,
3ab
1
Yield: 335 mg (95.7%). H NMR (400 MHz, CDCl3) δ 7.29 –
7.20 (m, 5H), 4.61 (d, J = 10.4 Hz, 1H), 3.79 – 3.74 (m, 1H),
3.23 (m, 1H), 2.89 (q, J = 9.6, 7.2 Hz, 1H), 2.51 (d, J = 9.2 Hz,
1H), 2.31 – 2.19 (m, 2H), 1.43 (s, 9H), 1.30 (d, J = 6.4 Hz, 3H),
1.10 (s, 3H), 0.98 (s, 3H). []D +7.4o (c 0.153, CHCl3).
1
Yield: 1.61 g (94.0%). H NMR (400 MHz, CDCl3) δ 7.31 –
7.27 (m, 5H), 3.64 (s, 2H), 3.51 (d, J = 12.8 Hz, 1H), 2.67 – 2.58
(m, 2H), 2.53 – 2.50 (m, 1H), 2.29 – 2.25 (m, 2H), 2.06 – 1.95
(m, 2H), 1.69 – 1.60 (m, 2H), 1.13 – 1.04 (m, 3H).
6.2.1.2. tert-Butyl ((R)-6-((R)-1-phenylethyl)-6-
azaspiro[3.4]octan-8-yl)carbamate, 2c
6.2.2.2. (R)-1-Benzyl-N-
(cyclopropylmethyl)pyrrolidin-3-amine, 3ac
Yield: 563 mg (quantitative yield). 1H NMR (400 MHz,
CDCl3) δ 7.32 – 7.22 (m, 5H), 4.71 (d, J = 8.8 Hz, 1H), 3.95 –
3.90 (m, 1H), 3.21 (q, J = 6.4 Hz, 1H), 2.77 – 2.70 (m, 2H), 2.51
(d, J = 9.6 Hz, 1H), 2.21 (dd, J = 10.0, 3.6 Hz, 1H), 2.07 – 2.02
(m, 2H), 1.89 – 1.73 (m, 4H), 1.45 (s, 9H), 1.31 (d, J = 6.4 Hz,
3H). []D +8.4o (c 0.387, CHCl3).
1
Yield: 1.68 g (64.0%). H NMR (400 MHz, CDCl3) δ 7.34 –
7.29 (m, 5H), 3.62 (d, J = 7.2 Hz, 2H), 3.66 – 3.33 (m, 1H), 2.80
– 2.76 (m, 1H), 2.65 – 2.55 (m, 2H), 2.44 – 2.38 (m, 2H), 2.36 –
2.32 (m, 2H), 1.61 – 1.55 (m, 2H), 0.97 – 0.93 (m, 1H), 0.51 –
0.47 (m, 2H), 0.12 – 0.09 (m, 2H).
6.2.2.3. (R)-N,4,4-Trimethyl-1-((R)-1-
phenylethyl)pyrrolidin-3-amine, 3b
In the cases of 2ab and 2ac, the desired products were
synthesized through substitution reactions with acetic anhydride
and cyclopropanecarbonyl chloride instead of di-tert-butyl
dicarbamate according to the aforementioned process (vide
supra).
1
Yield: 238 mg (97.9%). H NMR (400 MHz, CDCl3) δ 7.38 –
7.07 (m, 5H), 3.26 (q, J = 13.2, 6.4 Hz, 1H), 3.08 (q, J = 9.2, 7.2
Hz, 1H), 2.73 (t, J = 7.2 Hz, 1H), 2.37 (s, 3H), 2.36 (d, J = 4.4
Hz, 1H), 2.31 – 2.26 (m, 1H), 2.24 – 2.17 (m, 1H), 1.30 (d, J =
6.4 Hz, 3H), 1.06 (s, 3H), 0.96 (s, 3H).
6.2.1.3. (R)-N-(1-Benzylpyrrolidin-3-yl)acetamide,
2ab
1
6.2.2.4. (R)-N-Methyl-6-((R)-1-phenylethyl)-6-
Yield: 2.12 g (85.0%). H NMR (400 MHz, CDCl3) δ 7.34 –
azaspiro[3.4]octan-8-amine, 3c
7.24 (m, 5H), 5.93 (s, 1H), 4.46 – 4.42 (m, 1H), 3.60 (s, 2H),
2.90 – 2.86 (m, 1H), 2.62 – 2.51 (m, 2H), 2.30 – 2.22 (m, 2H),
1.93 (s, 3H), 1.64 – 1.60 (m, 1H). []D +19.7o (c 0.410, CHCl3).
1
Yield: 308 mg (75.0%). H NMR (400 MHz, CDCl3) δ 7.33 –
7.23 (m, 5H), 3.24 (q, J = 6.4 Hz, 1H), 3.03 (dd, J = 9.6, 6.4 Hz,
1H), 2.81 (t, J = 6.4 Hz, 1H), 2.66 (d, J = 9.2 Hz, 1H), 2.55 (d, J
= 9.2 Hz, 1H), 2.41 (s, 3H), 2.21 (s, 1H), 2.17 (dd, J = 9.6, 5.6
Hz, 1H), 1.99 – 1.95 (m, 1H), 1.90 – 1.63 (m, 4H), 1.34 (d, J =
6.4 Hz, 4H).
6.2.1.4. (R)-N-(1-Benzylpyrrolidin-3-
yl)cyclopropanecarboxamide, 2ac
Yield: 3.02 g (quantitative yield). 1H NMR (400 MHz, CDCl3)
δ 8.39 (d, J = 5.2 Hz, 1H), 7.62 – 7.48 (m, 5H), 4.96 (s, 1H), 4.28
– 4.21 (bs, 2H), 3.81 (s, 1H), 3.52 (d, J = 11.2 Hz, 1H), 3.05 –
2.88 (m, 2H), 2.54 – 2.47 (m, 1H), 2.30 – 2.23 (m, 1H), 0.94 –
0.91 (m, 2H), 0.89 – 0.84 (m, 1H), 0.78 – 0.75 (m, 2H). []D
+16.3o (c 0.397, CHCl3).
6.2.3. Synthesis of (R)-N-(1-benzylpyrrolidin-3-yl)-
N-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine, 4aa
A solution of (R)-1-benzyl-N-methylpyrrolidin-3-amine 3aa
(420 mg, 2.21 mmol) in 11.0 mL of deionized water was placed
in a 50 mL round bottom flask. Consequently, 6-chloro-7-
deazapurine (372 mg, 2.42 mmol) and potassium carbonate (609
mg, 4.41 mmol) were added and the mixture was refluxed for 18
hours. After the reaction, it was cooled at room temperature and
the aqueous mixture was extracted with 20 mL of
dichloromethane three times. The combined organic layers were
dried over anhydrous sodium sulfate, filtered and concentrated.
The residue was purified with flash column chromatography
(methanol:dichloromethane = 2:98). Removing the solvent in
6.2.2. Synthesis of (R)-1-benzyl-N-
methylpyrrolidin-3-amine, 3aa
A tert-butyl (R)-(1-benzylpyrrolidin-3-yl)carbamate 2aa (3.20
g, 11.6 mmol) solution in 58.0 mL of tetrahydrofuran was placed
o
in a 100 mL round bottom flask. After it was cooled at -40 C,
lithium aluminum hydride (2.64 g, 69.6 mmol) was slowly added
to the stirred mixture. The reaction mixture was refluxed for 4
hours and then cooled down to -40 oC. The reaction was
quenched with 2.70 mL of deionized water, 2.70 mL of 15%

vacuo provided 507 mg of (R)-N-(1-benzylpyrrolidin-3-yl)-N-
methyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine (74.8% yield). 1H
NMR (400 MHz, CDCl3) δ 10.40 (s, 1H), 8.29 (s, 1H), 7.51 –
7.20 (m, 5H), 7.03 (s, 1H), 6.59 (d, J = 2.2 Hz, 1H), 5.66 (s, 1H),
3.65 (dd, J = 62.5, 12.9 Hz, 2H), 3.42 (s, 3H), 2.98 (dd, J = 13.5,
7.8 Hz, 1H), 2.83 (dd, J = 10.3, 3.4 Hz, 1H), 2.69 – 2.53 (m, 1H),
2.44 – 2.21 (m, 2H), 1.96 – 1.83 (m, 1H).
Then, 10w/w% palladium on charcoal (638 mg, 5 wt%) and 10.1
g of ammonium formate (262 mg, 4.15 mmol) were added and
the reaction mixture was stirred at 60 – 70 oC overnight. After the
reaction, it was filtered through a celite 545 pad before the
solution was concentrated under reduced pressure. The residue
was
purified
with
flash
column
chromatography
(methanol:dichloromethane:ammonium hydroxide = 10:88:2).
Removing the solvent in vacuo provided 325 mg of (R)-N-
methyl-N-(pyrrolidin-3-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine
In the cases of 4ab, 4ac, 4ad, 4b, and 4c, the desired products
were synthesized from 3ab, 3ac, 1a, 3b, and 3c, respectively,
instead of (R)-1-benzyl-N-methylpyrrolidin-3-amine 3aa
according to the aforementioned process (vide supra).
1
(72.0% yield). H NMR (400 MHz, CDCl3) δ 12.16 (bs, 1H),
8.33 (s, 1H), 7.09 (d, J = 3.5 Hz, 1H), 6.58 (d, J = 3.4 Hz, 1H),
5.62 – 5.42 (m, 1H), 3.42 – 3.32 (m, 3H), 3.29 (dd, J = 11.5, 8.4
Hz, 1H), 3.24 – 3.12 (m, 1H), 3.10 – 3.01 (m, 1H), 2.98 (dd, J =
11.5, 6.2 Hz, 1H), 2.66 (bs, 1H), 2.26 – 2.10 (m, 1H), 1.91 (td, J
= 14.9, 7.6 Hz, 1H).
6.2.3.1. (R)-N-(1-Benzylpyrrolidin-3-yl)-N-ethyl-
7H-pyrrolo[2,3-d]pyrimidin-4-amine, 4ab
1
Yield: 296 mg (10.0%). H NMR (400 MHz, CDCl3) δ 10.84
In the cases from 5ab to 5c, the desired products were
synthesized from 4ab – 4c, respectively, instead of (R)-N-(1-
benzylpyrrolidin-3-yl)-N-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-
amine 4aa according to the aforementioned process (vide supra).
(s, 1H), 8.31 (s, 1H), 7.39 – 7.33 (m, 5H), 7.06 (d, J = 3.6 Hz,
1H), 6.51 (d, J = 3.6 Hz, 1H), 5.57 (bs, 1H), 3.93 – 3.85 (m, 2H),
3.78 – 3.75 (m, 1H), 3.65 – 3.58 (m, 1H), 2.98 (bs, 1H), 2.84 (bs,
1H), 2.70 (bs, 1H), 2.49 – 2.33 (m, 2H), 2.01 – 1.93 (m, 1H),
1.36 (t, J = 7.2 Hz, 3H).
6.2.4.1. (R)-N-Ethyl-N-(pyrrolidin-3-yl)-7H-
pyrrolo[2,3-d]pyrimidin-4-amine, 5ab
6.2.3.2. (R)-N-(1-Benzylpyrrolidin-3-yl)-N-
(cyclopropylmethyl)-7H-pyrrolo[2,3-d]pyrimidin-
4-amine, 4ac
1
Yield: 189 mg (88.8%). H NMR (400 MHz, CDCl3) δ 10.30
– 10.01 (bs, 1H), 8.31 (s, 1H), 7.09 (d, J = 3.6 Hz, 1H), 6.51 (d, J
= 3.6 Hz, 1H), 5.10 – 5.04 (m, 1H), 3.81 (q, J = 7.2 Hz, 2H), 3.51
(s, 1H), 3.35 – 3.29 (m, 2H), 3.14 – 3.04 (m, 2H), 2.26 – 2.20 (m,
1H), 2.10 – 2.01 (m, 1H), 1.40 (t, J = 7.2 Hz, 3H).
1
Yield: 313 mg (12.4%). H NMR (400 MHz, CDCl3) δ 9.59 –
9.54 (bs, 1H), 8.30 (s, 1H), 7.36 – 7.30 (bs, 5H), 7.03 (bs, 1H),
6.69 (bs, 1H), 5.54 (bs, 1H), 3.78 – 3.68 (m, 3H), 3.63 (bs, 1H),
3.00 (bs, 1H), 2.62 (bs, 1H), 2.39 (bs, 2H), 2.01 (bs, 1H), 1.64
(bs, 1H), 0.62 – 0.54 (m, 1H), 0.44 – 0.41 (m, 1H), 0.39 – 0.36
(m, 1H).
6.2.4.2. (R)-N-(Cyclopropylmethyl)-N-(pyrrolidin-
3-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine, 5ac
1
Yield: 162 mg (70.7%). H NMR (400 MHz, CDCl3) δ 10.40
6.2.3.3. (R)-N-(1-Benzylpyrrolidin-3-yl)-7H-
pyrrolo[2,3-d]pyrimidin-4-amine, 4ad
– 10.10 (bs, 1H), 8.32 (d, J = 4.4 Hz, 1H), 7.10 (d, J = 3.6 Hz,
1H), 6.67 (d, J = 3.6 Hz, 1H), 4.95 – 4.91 (m, 1H), 3.76 – 3.60
(m, 2H), 3.39 – 3.34 (m, 1H), 3.26 – 3.24 (m, 2H), 3.04 – 2.97
(m, 1H), 2.24 – 2.12 (m, 2H), 1.25 – 1.13 (m, 2H), 0.69 – 0.62
(m, 2H), 0.45 – 0.39 (m, 2H).
1
Yield: 292 mg (58.5%). H NMR (400 MHz, CDCl3) δ 11.20
(s, 1H), 8.36 (s, 1H), 7.37 – 7.26 (m, 5H), 7.05 (d, J = 3.6 Hz,
1H), 6.40 (d, J = 3.2 Hz, 1H), 5.80 (d, J = 8.0 Hz, 1H), 4.89 (s,
1H), 3.71 (s, 2H), 3.02 – 3.00 (m, 1H), 2.88 (d, J = 8.4 Hz, 1H),
2.81 – 2.76 (m, 1H), 2.51 – 2.39 (m, 2H), 1.92 – 1.83 (m, 1H).
6.2.4.3. (R)-N-(Pyrrolidin-3-yl)-7H-pyrrolo[2,3-
d]pyrimidin-4-amine, 5ad
6.2.3.4. N-((R)-4,4-Dimethyl-1-((R)-1-
phenylethyl)pyrrolidin-3-yl)-N-methyl-7H-
pyrrolo[2,3-d]pyrimidin-4-amine, 4b
1
Yield: 191 mg (94.8%). H NMR (400 MHz, DMSO-d6) δ
11.47 (s, 1H), 8.08 (s, 1H), 7.31 (d, J = 6.8 Hz, 1H), 7.05 (d, J =
3.2 Hz, 1H), 5.73 (d, J = 3.6 Hz, 1H), 4.63 – 4.50 (m, 1H), 3.12 –
3.07 (m, 1H), 3.02 – 2.95 (m, 1H), 2.87 – 2.81 (m, 1H), 2.75 (dd,
J = 11.2, 3.2 Hz, 1H), 2.10 – 2.01 (m, 1H), 1.75 – 1.67 (m, 1H).
1
Yield: 106 mg (30.7%). H NMR (400 MHz, CDCl3) δ 9.03
(s, 1H), 8.21 (s, 1H), 7.38 – 7.19 (m, 5H), 6.96 (q, J = 3.6, 2.0
Hz, 1H), 6.61 (q, J = 3.2, 1.6 Hz, 1H), 5.14 (d, J = 8.4 Hz, 1H),
3.48 (s, 3H), 3.18 (q, J = 13.2, 6.4 Hz, 1H), 2.98 (d, J = 9.2 Hz,
1H), 2.68 (d, J = 12.0 Hz, 1H), 2.48 (q, J = 11.2, 8.4 Hz, 1H),
2.10 (d, J = 8.8 Hz, 1H), 1.39 (d, J = 6.4 Hz, 3H), 1.35 (s, 3H),
0.98 (s, 3H).
6.2.4.4. (R)-N-(4,4-Dimethylpyrrolidin-3-yl)-N-
methyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine, 5b
1
Yield: 58.2 mg (79.1%). H NMR (400 MHz, CDCl3) δ 9.57
(d, J = 24.8 Hz, 1H), 8.24 (d, J = 14.8 Hz, 1H), 7.01 – 6.98 (m,
1H), 6.67 – 6.61 (m, 1H), 5.34 – 5.23 (m, 1H), 3.56 – 3.40 (m,
3H), 3.19 – 3.16 (m, 1H), 2.97 – 2.55 (m, 2H), 2.32 (s, 1H), 1.33
(d, J = 25.2 Hz, 3H), 0.94 (d, J = 12.0 Hz, 3H).
6.2.3.5. N-Methyl-N-((R)-6-((R)-1-phenylethyl)-6-
azaspiro[3.4]octan-8-yl)-7H-pyrrolo[2,3-
d]pyrimidin-4-amine, 4c
6.2.4.5. (R)-N-Methyl-N-(6-azaspiro[3.4]octan-8-
yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine, 5c
1
Yield: 272 mg (60.0%). H NMR (400 MHz, CDCl3) δ 9.06
(s, 1H), 8.25 (s, 1H), 7.37 – 7.22 (m, 5H), 6.98 (dd, J = 3.6, 2.4
Hz, 1H), 6.61 (dd, J = 3.6, 2.0 Hz, 1H), 5.48 (s, 1H), 3.29 (s, 3H),
3.18 (d, J = 6.8 Hz, 2H), 2.65 – 2.46 (m, 4H), 1.98 – 1.91 (m,
2H), 1.87 – 1.80 (m, 2H), 1.73 – 1.69 (m, 1H), 1.40 (d, J = 6.4
Hz, 3H).
1
Yield: 163 mg (84.5%). H NMR (400 MHz, CDCl3) δ 10.62
(s, 1H), 8.29 (s, 1H), 7.07 (d, J = 3.6 Hz, 1H), 6.62 (d, J = 4.0
Hz, 1H), 5.40 (s, 1H), 3.43 (dd, J = 12.4, 8.4 Hz, 1H), 3.30 (s,
3H), 3.26 – 3.19 (m, 2H), 3.09 (dd, J = 12.4, 5.2 Hz, 1H), 2.37 –
2.32 (m, 2H), 2.03 (d, J = 7.2 Hz, 1H), 1.95 – 1.88 (m, 2H), 1.86
– 1.81 (m, 2H).
6.2.4. Synthesis of (R)-N-methyl-N-(pyrrolidin-3-
yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine, 5aa
A (R)-N-(1-benzylpyrrolidin-3-yl)-N-methyl-7H-pyrrolo[2,3-
d]pyrimidin-4-amine 4aa (638 mg, 2.08 mmol) solution in 20.8
mL of methanol was placed in a 100 mL round bottom flask.
6.2.5. Syntheses of (R)-3-(3-(methyl(7H-
pyrrolo[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-
yl)-3-oxopropanenitrile, 6

To
an
(R)-N-methyl-N-(pyrrolidin-3-yl)-7H-pyrrolo[2,3-
6.2.5.4. (R)-3-(3-((Cyclopropylmethyl)(7H-
pyrrolo[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-
yl)-3-oxopropanenitrile, 10
d]pyrimidin-4-amine 5aa (103 mg, 0.474 mmol) solution in 4.70
mL of n-butanol in a 10 mL round bottom flask, ethyl
cyanoacetate
(0.505
mL,
4.75
mmol)
and
1,8-
1
Yield: 38.4 mg (54.0%). 95.3% purity by HPLC. H NMR
diazabicyclo[5.4.0]undec-7-ene (0.0355 mL, 0.237 mmol) were
added and the mixture was heated at 80 C for 24 hours. The
o
(400 MHz, CDCl3) δ 12.03 (d, J = 14.0 Hz, 1H), 8.35 (d, J = 5.1
Hz, 1H), 7.17 (d, J = 5.0 Hz, 1H), 6.67 (m, 1H), 5.30 (ddt, J =
25.0, 16.8, 8.3 Hz, 1H), 3.93 (m, 2H), 3.63 (m, 3H), 3.49 (m,
3H), 2.33 (ddt, J = 17.5, 12.0, 9.6 Hz, 2H), 1.18 (m, 1H), 0.69 (t,
J = 8.9 Hz, 2H), 0.36 (m, 2H). 13C NMR (100 MHz, CDCl3) δ
160.3, 157.1, 152.3, 150.4, 121.4, 114.0, 103.2, 101.8, 56.6, 50.4,
48.9, 45.1, 29.6, 26.0, 11.9, 5.1, 4.9. HRMS (ESI) calcd for
C17H21N6O: 325.1777. Obsd: 325.1775. []D +24.5o (c 1.08,
CHCl3).
reaction solution was concentrated under reduced pressure and
the residue was purified with flash column chromatography
(methanol:dichloromethane = 2:98). Removing the solvent in
vacuo provided 101 mg of (R)-3-(3-(methyl(7H-pyrrolo[2,3-
d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)-3-oxopropanenitrile
1
(74.8% yield). 98.7% purity by HPLC. H NMR (400 MHz,
CDCl3) δ 10.98 (s, 1H), 8.33 (s, 1H), 7.13 (d, J = 3.6 Hz, 1H),
6.60 (d, J = 3.6 Hz, 1H), 5.76 (m, 1H), 3.90 (dt, J = 14.9, 8.0 Hz,
1H), 3.70 (ddd, J = 26.4, 16.1, 8.3 Hz, 1H), 3.51 (m, 4H), 3.35
(d, J = 14.8 Hz, 3H), 2.27 (m, 2H). 13C NMR (100 MHz, CDCl3)
δ 160.3, 157.8, 152.3, 150.9, 120.9, 113.8, 103.6, 102.1, 55.0,
48.0, 45.2, 32.5, 26.9, 26.0. HRMS (ESI) calcd for C14H17N6O:
285.1464. Obsd: 285.1452. []D +42.6o (c 1.00, CHCl3).
In the cases of 7, 8, 9, and 10, the desired products were
synthesized from 5b, 5c, 5ab, and 5ac, respectively, instead of
(R)-N-methyl-N-(pyrrolidin-3-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-
amine 5aa according to the aforementioned process (vide supra).
6.2.6. Synthesis of (R)-3-((3-(methyl(7H-
pyrrolo[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-
yl)sulfonyl)benzonitrile, 12
To an (R)-N-methyl-N-(pyrrolidin-3-yl)-7H-pyrrolo[2,3-
d]pyrimidin-4-amine 5aa (70.0 mg, 0.322 mmol) solution in 1.50
mL of dichloromethane in a 5 mL round bottom flask, 3-
cyanobenzenesulfonyl chloride (68.6 mg, 0.340 mmol) and N,N-
diisopropylethylamine (0.0590 mL, 0.339 mmol) were added.
Then, the reaction solution was stirred at room temperature
overnight before being concentrated under reduced pressure. The
residue was purified by flash column chromatography
(methanol:dichloromethane = 2:98). Removing the solvent in
vacuo provided 88.6 mg of (R)-3-((3-(methyl(7H-pyrrolo[2,3-
d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)sulfonyl)benzonitrile
6.2.5.1. (R)-3-(3,3-Dimethyl-4-(methyl(7H-
pyrrolo[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-
yl)-3-oxopropanenitrile, 7
1
Yield: 41.4 mg (57.1%). 97.7% purity by HPLC. H NMR
1
(400 MHz, CDCl3) δ 12.19 (s, 1H), 8.31 (s, 1H), 7.14 (s, 1H),
6.61 (s, 1H), 5.69 (dd, J = 39.2, 6.0 Hz, 1H), 4.03 (m, 1H), 3.82
(dd, J = 34.5, 12.6 Hz, 1H), 3.54 (m, 3H), 3.41 (m, 1H), 3.34 (d,
J = 10.4 Hz, 3H), 1.25 (d, J = 2.8 Hz, 3H), 1.04 (m, 3H). 13C
NMR (100 MHz, CDCl3) δ 160.4, 158.0, 152.3, 150.3, 120.8,
113.9, 103.1, 102.2, 62.2, 59.9, 49.1, 44.6, 33.9, 28.1, 26.0, 21.6.
HRMS (ESI) calcd for C16H21N6O: 313.1777. Obsd: 313.1772.
[]D -8.93o (c 0.864, CHCl3).
(72.4% yield). 97.0% purity by HPLC. H NMR (400 MHz,
DMSO-d6) δ 11.68 (s, 1H), 8.32 (d, J = 7.8 Hz, 1H), 8.23 (d, J =
7.7 Hz, 1H), 8.17 (d, J = 8.0 Hz, 1H), 8.06 (t, J = 4.6 Hz, 1H),
7.88 (td, J = 7.8, 2.2 Hz, 1H), 7.14 (d, J = 2.1 Hz, 1H), 6.48 (s,
1H), 5.27 (m, 1H), 3.51 (dd, J = 7.6, 4.4 Hz, 1H), 3.44 (m, 1H),
3.36 (s, 3H), 3.23 (m, 1H), 3.17 (s, 1H), 2.03 (dd, J = 15.0, 7.6
Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ 156.8, 151.7, 150.4,
137.1, 136.8, 131.9, 131.0, 130.9, 121.2, 117.6, 112.9, 102.5,
101.3, 54.0, 48.7, 46.8, 31.7, 27.5. HRMS (ESI) calcd for
C18H19N6O2S: 383.1290. Obsd: 383.1285. []D -45.7o (c 0.530,
CHCl3).
6.2.5.2. (R)-3-(8-(Methyl(7H-pyrrolo[2,3-
d]pyrimidin-4-yl)amino)-6-azaspiro[3.4]octan-6-
yl)-3-oxopropanenitrile, 8
In the cases of 11, 13, and 14, the desired products were
synthesized from 5ad, 5ab, and 5ac, respectively, instead of (R)-
N-methyl-N-(pyrrolidin-3-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-
amine 5aa according to the aforementioned process (vide supra).
1
Yield: 23.7 mg (19.0%). 95.0% purity by HPLC. H NMR
(400 MHz, CDCl3) δ 11.84 (s, 1H), 8.36 (s, 1H), 7.15 (s, 1H),
6.62 (s, 1H), 5.96 (dd, J = 19.0, 6.3 Hz, 1H), 3.94 (ddd, J = 39.2,
19.0, 12.0 Hz, 2H), 3.73 (m, 2H), 3.50 (d, J = 5.8 Hz, 2H), 3.28
(d, J = 4.9 Hz, 3H), 2.23 (m, 1H), 1.99 (m, 5H). 13C NMR (100
MHz, CDCl3) δ 160.0, 158.2, 152.4, 150.6, 120.8, 113.7, 103.1,
102.3, 62.0, 58.9, 49.9, 47.7, 35.7, 33.6, 26.4, 26.0, 16.3. HRMS
(ESI) calcd for C17H21N6O: 325.1777. Obsd: 325.1770. []D
+7.04o (c 0.557, CHCl3).
6.2.6.1. (R)-3-((3-((7H-Pyrrolo[2,3-d]pyrimidin-4-
yl)amino)pyrrolidin-1-yl)sulfonyl)benzonitrile, 11
1
Yield: 42.0 mg (38.2%). 97.6% purity by HPLC. H NMR
(400 MHz, DMSO-d6) δ 11.49 (s, 1H), 8.14 (s, 1H), 8.06 (s, 1H),
7.98 (d, J = 7.7 Hz, 1H), 7.92 (d, J = 7.5 Hz, 1H), 7.58 (t, J = 7.7
Hz, 1H), 7.13 (d, J = 4.1 Hz, 1H), 7.03 (s, 1H), 6.36 (s, 1H), 4.38
(d, J = 4.3 Hz, 1H), 3.49 (m, 2H), 3.31 (m, 1H), 3.25 (m, 1H),
2.06 (dd, J = 12.3, 6.2 Hz, 1H), 1.88 (m, 1H). 13C NMR (100
MHz, DMSO-d6) δ 155.2, 151.1, 150.2, 137.2, 136.4, 131.6,
130.7, 130.4, 121.0, 117.5, 112.7, 102.6, 98.7, 53.3, 50.0, 46.7,
30.3. HRMS (ESI) calcd for C17H17N6O2S: 369.1134. Obsd:
369.1128. []D -27.4o (c 1.09, DMSO).
6.2.5.3. (R)-3-(3-(Ethyl(7H-pyrrolo[2,3-
d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)-3-
oxopropanenitrile, 9
1
Yield: 50.9 mg (55.0%). 96.1% purity by HPLC. H NMR
(400 MHz, DMSO-d6) δ 11.66 (s, 1H), 8.11 (d, J = 2.5 Hz, 1H),
7.16 (s, 1H), 6.48 (m, 1H), 5.35 (ddd, J = 54.1, 16.5, 8.3 Hz, 1H),
3.95 (dd, J = 19.0, 8.5 Hz, 1H), 3.87 (dd, J = 18.9, 3.9 Hz, 1H),
3.70 (m, 1H), 3.62 (dd, J = 9.2, 6.9 Hz, 3H), 3.45 (dd, J = 17.5,
8.8 Hz, 1H), 3.24 (m, 1H), 2.16 (m, 1H), 2.07 (m, 1H), 1.19 (dd,
J = 11.2, 6.1 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 161.3,
156.1, 151.8, 150.5, 121.5, 116.0, 101.7, 101.1, 54.4, 47.4, 44.2,
28.5, 26.8, 25.5, 15.7. HRMS (ESI) calcd for C15H19N6O:
299.1620. Obsd: 299.1617. []D +78.5o (c 1.09, DMSO).
6.2.6.2. (R)-3-((3-(Ethyl(7H-pyrrolo[2,3-
d]pyrimidin-4-yl)amino)pyrrolidin-1-
yl)sulfonyl)benzonitrile, 13
1
Yield: 84.3 mg (73.3%). 95.1% purity by HPLC. H NMR
(400 MHz, CDCl3) δ 11.64 (s, 1H), 8.17 (d, J = 14.8 Hz, 2H),
8.11 (d, J = 7.9 Hz, 1H), 7.92 (d, J = 7.6 Hz, 1H), 7.72 (t, J = 7.8
Hz, 1H), 7.13 (d, J = 3.0 Hz, 1H), 6.42 (d, J = 3.0 Hz, 1H), 5.23
(m, 1H), 3.71 (m, 4H), 3.33 (m, 1H), 3.26 (m, 1H), 2.20 (m, 2H),

1.34 (t, J = 6.9 Hz, 3H). 13C NMR (100 MHz, CDCl3) δ 156.5,
152.1, 150.4, 138.6, 136.1, 131.7, 131.2, 130.4, 121.2, 117.4,
114.0, 102.7, 101.5, 55.6, 49.5, 47.0, 40.6, 28.9, 16.0. HRMS
(ESI) calcd for C19H21N6O2S: 397.1447. Obsd: 397.1442. []D -
63.5o (c 0.568, CHCl3).
To a 2-azidoacetic acid (247 mg, 2.44 mmol) solution in 8.0
mL of N,N-dimethylformamide in a 25 mL round-bottom flask,
N,N’-dicyclohexylcarbodiimide (503 mg, 2.44 mmol) and N,N-
diisopropylethylamine (0.850 mL, 4.88 mmol) were added and
the reaction mixture was stirred for 15 minutes. In a second 25
mL round-bottom flask, (R)-N-methyl-N-(pyrrolidin-3-yl)-7H-
pyrrolo[2,3-d]pyrimidin-4-amine 5aa (265 mg, 1.22 mmol) was
placed and the reaction mixture of 2-azidoacetic acid was
transferred to this second flask. The reaction mixture was
refluxed overnight and then cooled at room temperature. The
mixture was filtered through a celite 545 pad and the solution
was concentrated under reduced pressure. The residue was
6.2.6.3. (R)-3-((3-((Cyclopropylmethyl)(7H-
pyrrolo[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-
yl)sulfonyl)benzonitrile, 14
1
Yield: 62.2 mg (61.0%). 95.0% purity by HPLC. H NMR
(400 MHz, CDCl3) δ 11.91 (s, 1H), 8.19 (s, 1H), 8.11 (d, J = 7.7
Hz, 1H), 8.04 (s, 1H), 7.93 (d, J = 7.4 Hz, 1H), 7.73 (t, J = 7.3
Hz, 1H), 7.15 (s, 1H), 6.60 (s, 1H), 5.05 (m, 1H), 3.66 (m, 2H),
3.60 (d, J = 4.9 Hz, 2H), 3.48 (m, 1H), 3.31 (dd, J = 15.4, 7.6 Hz,
1H), 2.26 (d, J = 7.2 Hz, 2H), 1.12 (s, 1H), 0.65 (d, J = 2.7 Hz,
2H), 0.33 (d, J = 2.1 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ
156.6, 152.0, 149.9, 138.3, 136.0, 131.8, 131.3, 130.3, 121.2,
117.4, 113.9, 103.2, 101.8, 56.9, 51.3, 49.7, 47.4, 29.2, 11.7, 4.8.
HRMS (ESI) calcd for C21H23N6O2S: 423.1603. Obsd: 423.1598.
[]D -31.5o (c 1.49, CHCl3).
purified
with
column
chromatography
(methanol:dichloromethane = 2:98). Removing the solvent in
vacuo provided 41.0 mg of (R)-2-azido-1-(3-(methyl(7H-
pyrrolo[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-yl)ethan-1-one
1
(5.27% yield). 96.2% purity by HPLC. H NMR (400 MHz,
CDCl3) δ 11.97 (d, J = 32.0 Hz, 1H), 8.34 (d, J = 1.9 Hz, 1H),
7.15 (dd, J = 6.5, 3.6 Hz, 1H), 6.59 (s, 1H), 5.75 (m, 1H), 3.92
(m, 3H), 3.79 (dd, J = 19.2, 10.6 Hz, 1H), 3.57 (tt, J = 11.9, 8.2
Hz, 2H), 3.34 (m, 3H), 2.21 (m, 2H). 13C NMR (100 MHz,
CDCl3) δ 166.2, 157.8, 152.3, 150.7, 121.1, 103.6, 101.8, 55.0,
51.3, 46.7, 44.6, 32.3, 26.7. HRMS (ESI) calcd for C13H17N8O:
301.1525. Obsd: 301.1522. []D +33.2o (c 0.753, CHCl3).
6.2.7. Synthesis of (R)-3-(3-(methyl(7H-
pyrrolo[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-
yl)propanenitrile, 15
To an
(R)-N-methyl-N-(pyrrolidin-3-yl)-7H-pyrrolo[2,3-
6.2.9. Synthesis of (R)-3-methyl-1-(3-(methyl(7H-
pyrrolo[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-
yl)butan-1-one, 18
d]pyrimidin-4-amine 5aa (60.0 mg, 0.276 mmol) solution in 1.00
mL of dichloromethane in a 5 mL round-bottom flask, 3-
bromopropionitrile (0.0240 mL, 0.289 mmol) and N,N-
diisopropylethylamine (0.0720 mL, 0.413 mmol) were added.
The reaction mixture was stirred at room temperature overnight
and then concentrated under reduced pressure. The residue was
purified by column chromatography (methanol:dichloromethane
= 2:98). Removing the solvent in vacuo provided 55.3 mg of (R)-
3-(3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-
To an (R)-N-methyl-N-(pyrrolidin-3-yl)-7H-pyrrolo[2,3-
d]pyrimidin-4-amine 5aa (70.0 mg, 0.322 mmol) solution in 1.00
mL of dichloromethane in a 5 mL round-bottom flask, isovaleryl
chloride (38.8 mg, 0.322 mmol) and N,N-diisopropylethylamine
(0.0590 mL, 0.339 mmol) were added. The reaction mixture was
stirred at room temperature overnight and then concentrated
under reduced pressure. The residue was purified by column
chromatography (methanol:dichloromethane = 2:98). Removing
the solvent in vacuo provided 66.7 mg of (R)-3-methyl-1-(3-
(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-
1
1-yl)propanenitrile (74.7% yield). 100% purity by HPLC. H
NMR (400 MHz, CDCl3) δ 12.32 (s, 1H), 8.32 (s, 1H), 7.10 (d, J
= 3.4 Hz, 1H), 6.58 (d, J = 3.3 Hz, 1H), 5.73 (s, 1H), 3.42 (s,
3H), 3.06 (t, J = 7.1 Hz, 1H), 2.94 (dd, J = 9.9, 3.1 Hz, 1H), 2.83
(m, 1H), 2.72 (m, 1H), 2.64 (t, J = 9.2 Hz, 1H), 2.56 (t, J = 6.8
Hz, 2H), 2.34 (m, 2H), 1.93 (dt, J = 13.0, 10.0 Hz, 1H). 13C NMR
(100 MHz, CDCl3) δ 157.6, 151.8, 150.6, 120.4, 118.8, 103.1,
102.1, 57.2, 54.4, 53.7, 50.8, 32.4, 29.3, 17.7. HRMS (ESI) calcd
for C14H19N6: 271.1671. Obsd: 271.1665. []D +35.3o (c 1.07,
CHCl3).
1
yl)butan-1-one (68.7% yield). 98.7% purity by HPLC. H NMR
(400 MHz, CDCl3) δ 11.67 (s, 1H), 8.38 (s, 1H), 7.14 (s, 1H),
6.60 (s, 1H), 5.72 (m, 1H), 3.81 (m, 2H), 3.49 (m, 2H), 3.34 (d, J
= 11.5 Hz, 3H), 2.18 (m, 4H), 1.50 (d, J = 35.8 Hz, 1H), 0.94 (d,
J = 44.9 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 171.6, 157.7,
151.9, 150.2, 121.1, 103.5, 101.7, 54.9, 47.8, 45.4, 43.8, 32.1,
29.7, 25.5, 22.8. HRMS (ESI) calcd for C16H24N5O: 302.1981.
Obsd: 302.1977. []D +29.6o (c 1.47, CHCl3).
In the cases of compound 16, the desired products were
synthesized through substitution reactions with n-butyl bromide
instead of 3-bromopropionitrile according to the aforementioned
process (vide supra).
6.2.10. Synthesis of isobutyl (R)-3-(methyl(7H-
pyrrolo[2,3-d]pyrimidin-4-yl)amino)pyrrolidine-1-
carboxylate, 19
6.2.7.1. (R)-N-(1-Butylpyrrolidin-3-yl)-N-methyl-
7H-pyrrolo[2,3-d]pyrimidin-4-amine, 16
To an (R)-N-methyl-N-(pyrrolidin-3-yl)-7H-pyrrolo[2,3-
d]pyrimidin-4-amine 5aa (70.0 mg, 0.322 mmol) solution in 1.00
mL of dichloromethane in a 5 mL round-bottom flask, isobutyl
chloroformate (44.0 mg, 0.322 mmol) and N,N-
diisopropylethylamine (0.0560 mL, 0.321 mmol) were added.
The reaction solution was stirred at room temperature overnight
and then concentrated under reduced pressure. The residue was
purified by column chromatography (methanol:dichloromethane
= 2:98). Removing the solvent in vacuo provided 41.0 mg of
1
Yield: 90.0 mg (83.3%). 100% purity by HPLC. H NMR
(400 MHz, DMSO-d6) δ 11.69 (s, 1H), 8.11 (s, 1H), 7.11 (d, J =
3.1 Hz, 1H), 6.58 (d, J = 3.2 Hz, 1H), 5.53 (dt, J = 15.1, 7.7 Hz,
1H), 3.25 (s, 4H), 3.08 (m, 2H), 2.81 (m, 3H), 2.16 (m, 1H), 1.95
(m, 1H), 1.50 (dt, J = 15.2, 7.4 Hz, 2H), 1.25 (dq, J = 14.5, 7.3
Hz, 2H), 0.82 (t, J = 7.3 Hz, 3H). 13C NMR (100 MHz, DMSO-
d6) δ 156.7, 151.6, 150.4, 121.1, 102.5, 101.5, 54.5, 54.3, 53.7,
52.9, 32.7, 28.2, 27.1, 19.7, 13.6. HRMS (ESI) calcd for
C15H24N5: 274.2032. Obsd: 274.2027. []D +10.6o (c 3.42,
DMSO).
isobutyl
(R)-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-
yl)amino)pyrrolidine-1-carboxylate (40.2% yield). 97.7% purity
1
by HPLC. H NMR (400 MHz, CDCl3) δ 11.83 (s, 1H), 8.33 (s,
1H), 7.11 (d, J = 2.8 Hz, 1H), 6.58 (d, J = 3.3 Hz, 1H), 5.72 (d, J
= 6.3 Hz, 1H), 3.90 (d, J = 6.5 Hz, 2H), 3.76 (d, J = 9.2 Hz, 1H),
3.69 (m, 1H), 3.45 (m, 2H), 3.33 (s, 3H), 2.15 (dd, J = 21.9, 12.3
Hz, 2H), 1.95 (d, J = 6.1 Hz, 1H), 0.94 (d, J = 6.2 Hz, 6H). 13C
NMR (100 MHz, CDCl3) δ 157.9, 155.5, 152.0, 150.6, 120.8,
6.2.8. Synthesis of (R)-2-azido-1-(3-(methyl(7H-
pyrrolo[2,3-d]pyrimidin-4-yl)amino)pyrrolidin-1-
yl)ethan-1-one, 17

103.4, 102.0, 71.5, 54.7, 46.8, 44.8, 32.0, 28.2, 19.2, 9.5. HRMS
(ESI) calcd for C16H24N5O2: 318.1930. Obsd: 318.1924. []D
+23.7o (c 0.550, CHCl3).
sulfonyl chloride, respectively, instead of methanesulfonyl
chloride according to the aforementioned process (vide supra).
6.2.12.1. (R)-N-Methyl-N-(1-
((trifluoromethyl)sulfonyl)pyrrolidin-3-yl)-7H-
pyrrolo[2,3-d]pyrimidin-4-amine, 22
6.2.11. Synthesis of (R)-3-(methyl(7H-pyrrolo[2,3-
d]pyrimidin-4-yl)amino)-N-phenylpyrrolidine-1-
carboxamide, 20
1
Yield: 72.0 mg (64.3%). 97.4% purity by HPLC. H NMR
To an (R)-N-methyl-N-(pyrrolidin-3-yl)-7H-pyrrolo[2,3-
d]pyrimidin-4-amine 5aa (70.0 mg, 0.322 mmol) solution in 1.00
mL of dichloromethane in a 5 mL round-bottom flask, N,N-
diisopropylethylamine (0.0590 mL, 0.339 mmol) was added and
the mixture was treated with phenyl isocyanate (0.0350 mL,
0.322 mmol). The reaction solution was stirred for 2 hours before
being concentrated under reduced pressure. The residue was
purified by column chromatography (methanol:dichloromethane
= 2:98). Removing the solvent in vacuo provided 81.5 mg of (R)-
3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-
(400 MHz, CDCl3) δ 12.77 (s, 1H), 8.37 (d, J = 8.8 Hz, 1H), 7.18
(m, 1H), 6.59 (m, 1H), 5.84 (m, 1H), 3.89 (m, 2H), 3.62 (m, 1H),
3.53 (m, 1H), 3.36 (d, J = 8.7 Hz, 3H), 2.29 (dd, J = 17.0, 8.6 Hz,
2H). 13C NMR (100 MHz, CDCl3) δ 157.6, 152.2, 150.3, 121.3,
120.4 (q, J = 323.8 Hz), 103.7, 101.6, 54.7, 48.9, 47.7, 32.4, 28.5.
HRMS (ESI) calcd for C12H15F3N5O2S: 350.0899. Obsd:
350.0893. []D +19.4o (c 2.79, CHCl3).
6.2.12.2. (R)-N-(1-(Ethylsulfonyl)pyrrolidin-3-yl)-
N-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine, 23
phenylpyrrolidine-1-carboxamide (75.4% yield). 99.8% purity by
HPLC. H NMR (400 MHz, DMSO-d6) δ 11.72 (s, 1H), 8.22 (s,
1
1
Yield: 53.4 mg (53.6%). 98.2% purity by HPLC. H NMR
(400 MHz, CDCl3) δ 11.30 (s, 1H), 8.34 (s, 1H), 7.13 (s, 1H),
6.61 (s, 1H), 5.80 (m, 1H), 3.70 (dd, J = 19.4, 10.1 Hz, 2H), 3.43
(d, J = 10.3 Hz, 2H), 3.38 (s, 3H), 3.08 (q, J = 7.3 Hz, 2H), 2.28
(s, 1H), 2.19 (m, 1H), 1.43 (t, J = 7.3 Hz, 3H). 13C NMR (100
MHz, CDCl3) δ 157.6, 151.6, 149.9, 121.1, 103.8, 102.0, 54.9,
48.6, 46.6, 44.6, 29.8, 28.8, 8.1. HRMS (ESI) calcd for
C13H20N5O2S: 310.1338. Obsd: 310.1335. []D +13.1o (c 1.24,
CHCl3).
1H), 8.18 (s, 1H), 7.54 (d, J = 8.1 Hz, 2H), 7.23 (t, J = 7.8 Hz,
2H), 7.18 (d, J = 2.3 Hz, 1H), 6.92 (t, J = 7.2 Hz, 1H), 6.64 (d, J
= 3.1 Hz, 1H), 5.56 (m, 1H), 3.75 (m, 1H), 3.67 (m, 1H), 3.45
(m, 2H), 3.25 (s, 3H), 2.16 (m, 2H). 13C NMR (100 MHz,
DMSO-d6) δ 157.1, 154.1, 151.8, 150.6, 140.5, 128.3, 121.7,
121.1, 119.5, 102.6, 101.6, 54.2, 46.8, 44.4, 31.6, 27.6. HRMS
(ESI) calcd for C18H21N6O: 337.1777. Obsd: 337.1772. []D
+43.6o (c 2.44, DMSO).
6.2.12.3. (R)-N-(1-(Isopropylsulfonyl)pyrrolidin-3-
yl)-N-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine,
24
6.2.12. Synthesis of (R)-N-methyl-N-(1-
(methylsulfonyl)pyrrolidin-3-yl)-7H-pyrrolo[2,3-
d]pyrimidin-4-amine, 21
1
To an (R)-N-methyl-N-(pyrrolidin-3-yl)-7H-pyrrolo[2,3-
d]pyrimidin-4-amine 5aa (70.0 mg, 0.322 mmol) solution in 1.00
mL of dichloromethane in a 5 mL round bottom flask,
methanesulfonyl chloride (36.9 mg, 0.322 mmol) and N,N-
diisopropylethylamine (0.0590 mL, 0.339 mmol) were added.
Then, the reaction solution was stirred at room temperature
overnight before being concentrated under reduced pressure. The
residue was purified by flash column chromatography
(methanol:dichloromethane = 2:98). Removing the solvent in
Yield: 36.0 mg (34.6%). 99.1% purity by HPLC. H NMR
(400 MHz, CDCl3) δ 12.23 (s, 1H), 8.35 (s, 1H), 7.14 (d, J = 3.2
Hz, 1H), 6.59 (d, J = 2.6 Hz, 1H), 5.78 (m, 1H), 3.75 (m, 2H),
3.46 (m, 2H), 3.36 (s, 3H), 3.28 (dt, J = 13.6, 6.8 Hz, 1H), 2.26
(m, 1H), 2.16 (m, 1H), 1.41 (d, J = 6.8 Hz, 6H). 13C NMR (100
MHz, CDCl3) δ 157.8, 152.2, 150.6, 120.9, 103.5, 101.9, 55.0,
53.6, 49.0, 47.2, 32.3, 28.9, 16.8. HRMS (ESI) calcd for
C14H22N5O2S: 324.1494. Obsd: 324.1487. []D +18.2o (c 0.950,
CHCl3).
vacuo
provided
40.0
mg
of
(R)-N-methyl-N-(1-
6.2.12.4. (R)-N-Methyl-N-(1-
(methylsulfonyl)pyrrolidin-3-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-
1
(propylsulfonyl)pyrrolidin-3-yl)-7H-pyrrolo[2,3-
amine (42.1% yield). 96.9% purity by HPLC. H NMR (400
d]pyrimidin-4-amine, 25
MHz, DMSO-d6) δ 11.70 (s, 1H), 8.15 (d, J = 1.3 Hz, 1H), 7.17
(d, J = 2.9 Hz, 1H), 6.62 (d, J = 2.7 Hz, 1H), 5.58 (m, 1H), 3.52
(t, J = 9.0 Hz, 1H), 3.46 (m, 1H), 3.31 (dd, J = 17.4, 8.7 Hz, 1H),
3.23 (dd, J = 10.0, 4.4 Hz, 4H), 2.98 (d, J = 1.3 Hz, 3H), 2.14 (m,
2H). 13C NMR (100 MHz, DMSO-d6) δ 157.0, 151.8, 150.5,
121.1, 102.6, 101.5, 54.2, 48.4, 46.3, 33.4, 31.8, 27.9. HRMS
(ESI) calcd for C12H18N5O2S: 296.1181. Obsd: 296.1175. []D
+23.0o (c 1.20, DMSO).
1
Yield: 49.0 mg (54.9%). 97.1% purity by HPLC. H NMR
(400 MHz, CDCl3) δ 12.36 (s, 1H), 8.34 (s, 1H), 7.15 (s, 1H),
6.58 (s, 1H), 5.78 (m, 1H), 3.67 (m, 2H), 3.41 (d, J = 9.3 Hz,
2H), 3.36 (s, 3H), 3.01 (m, 2H), 2.27 (s, 1H), 2.19 (m, 1H), 1.90
(dd, J = 14.5, 7.2 Hz, 2H), 1.09 (t, J = 7.1 Hz, 3H). 13C NMR
(100 MHz, CDCl3) δ 157.7, 152.1, 150.5, 121.0, 103.5, 101.8,
54.8, 51.5, 48.5, 46.5, 32.3, 28.7, 17.1, 13.3. HRMS (ESI) calcd
for C14H22N5O2S: 324.1494. Obsd: 324.1488. []D +11.3o (c 1.52,
CHCl3).
In the cases from 22 to 42, the desired products were
synthesized
through
substitution
reactions
with
trifluoromethanesulfonyl chloride, ethanesulfonyl chloride, 2-
propanesulfonyl chloride, 1-propanesulfonyl chloride, 1-methyl-
1H-imidazole-4- sulfonyl chloride, benzenesulfonyl chloride, 2-
fluorobenzene-1-sulfonyl chloride, 3-fluorobenzene-1-sulfonyl
6.2.12.5. (R)-N-Methyl-N-(1-((1-methyl-1H-
imidazol-4-yl)sulfonyl)pyrrolidin-3-yl)-7H-
pyrrolo[2,3-d]pyrimidin-4-amine, 26
1
chloride,
cyanobenzenesulfonyl
chloride, 4-cyanobenzenesulfonyl
4-fluorobenzenesulfonyl
chloride,
3-cyanobenzenesulfonyl
chloride, 2-
2-
Yield: 17.0 mg (22.4%). 98.4% purity by HPLC. H NMR
chloride,
(400 MHz, DMSO-d6) δ 11.70 (s, 1H), 8.10 (s, 1H), 7.89 (s, 2H),
7.16 (s, 1H), 6.52 (s, 1H), 5.31 (dd, J = 15.6, 7.8 Hz, 1H), 3.74 (s,
3H), 3.50 (m, 2H), 3.27 (m, 2H), 3.12 (s, 3H), 1.99 (dd, J = 15.6,
8.0 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ 156.8, 151.6,
150.3, 140.2, 135.8, 126.1, 121.2, 102.4, 101.4, 54.2, 48.7, 46.9,
33.6, 31.4, 27.7. HRMS (ESI) calcd for C15H20N7O2S: 362.1399.
Obsd: 362.1393. []D +12.5o (c 0.477, DMSO).
nitrobenzenesulfonyl chloride, 3-nitrobenzenesulfonyl chloride,
4-nitrobenzenesulfonyl chloride, 3-toluenesulfonyl chloride, 4-
methoxybenzenesulfonyl chloride, 4-toluenesulfonyl chloride, 4-
(trifluoromethyl)benzenesulfonyl chloride, 2-naphthalenesulfonyl
chloride, piperidine-1-sulfonyl chloride, and morpholine-4-

1
6.2.12.6. (R)-N-Methyl-N-(1-
Yield: 68.8 mg (56.0%). 98.3% purity by HPLC. H NMR
(phenylsulfonyl)pyrrolidin-3-yl)-7H-pyrrolo[2,3-
d]pyrimidin-4-amine, 27
(400 MHz, CDCl3) δ 12.29 (s, 1H), 8.28 (d, J = 3.7 Hz, 1H), 8.11
(dd, J = 7.4, 2.8 Hz, 1H), 7.91 (m, 1H), 7.74 (m, 2H), 7.12 (s,
1H), 6.55 (s, 1H), 5.73 (d, J = 6.8 Hz, 1H), 3.80 (t, J = 8.8 Hz,
1H), 3.67 (td, J = 10.1, 3.5 Hz, 1H), 3.43 (m, 2H), 3.32 (d, J =
3.5 Hz, 3H), 2.22 (ddd, J = 18.3, 11.1, 4.5 Hz, 2H). 13C NMR
(100 MHz, CDCl3) δ 157.6, 152.1, 150.4, 140.7, 135.7, 133.2,
133.0, 130.4, 121.0, 116.5, 110.8, 103.5, 101.8, 54.6, 48.7, 47.2,
32.3, 28.5. HRMS (ESI) calcd for C18H19N6O2S: 383.1290. Obsd:
383.1287. []D -12.6o (c 2.34, CHCl3).
1
Yield: 101 mg (84.2%). 99.3% purity by HPLC. H NMR
(400 MHz, CDCl3) δ 12.18 (s, 1H), 8.26 (s, 1H), 7.87 (dd, J =
7.0, 1.4 Hz, 2H), 7.65 (m, 1H), 7.58 (m, 2H), 7.10 (d, J = 3.0 Hz,
1H), 6.50 (d, J = 2.9 Hz, 1H), 5.60 (m, 1H), 3.64 (dd, J = 12.1,
5.7 Hz, 1H), 3.45 (dd, J = 13.6, 5.3 Hz, 1H), 3.35 (m, 1H), 3.27
(d, J = 2.1 Hz, 3H), 3.12 (dt, J = 16.6, 4.7 Hz, 1H), 2.17 (ddd, J =
12.3, 10.0, 7.7 Hz, 1H), 2.05 (m, 1H). 13C NMR (100 MHz,
CDCl3) δ 157.6, 152.1, 150.5, 135.8, 133.1, 129.3, 127.9, 120.9,
103.4, 101.8, 54.4, 49.3, 47.2, 32.2, 28.5. HRMS (ESI) calcd for
C17H20N5O2S: 358.1338. Obsd: 358.1333. []D -29.2o (c 1.21,
CHCl3).
6.2.12.11. (R)-4-((3-(Methyl(7H-pyrrolo[2,3-
d]pyrimidin-4-yl)amino)pyrrolidin-1-
yl)sulfonyl)benzonitrile, 32
1
Yield: 80.5 mg (65.8%). 100% purity by HPLC. H NMR
6.2.12.7. (R)-N-(1-((2-
Fluorophenyl)sulfonyl)pyrrolidin-3-yl)-N-methyl-
7H-pyrrolo[2,3-d]pyrimidin-4-amine, 28
(400 MHz, DMSO-d6) δ 11.66 (s, 1H), 8.09 (m, 2H), 8.03 (d, J =
3.0 Hz, 1H), 7.98 (dd, J = 4.7, 3.8 Hz, 2H), 7.09 (d, J = 1.3 Hz,
1H), 6.43 (s, 1H), 5.24 (m, 1H), 3.44 (m, 2H), 3.17 (ddd, J =
18.1, 10.9, 5.2 Hz, 2H), 3.06 (d, J = 2.8 Hz, 3H), 1.96 (m, 2H).
13C NMR (100 MHz, DMSO-d6) δ 156.8, 151.7, 150.4, 139.8,
133.6, 128.2, 121.1, 117.7, 115.6, 102.6, 101.4, 54.1, 48.7, 46.8,
31.7, 27.6. HRMS (ESI) calcd for C18H19N6O2S: 383.1290. Obsd:
383.1284. []D -10.7o (c 2.72, DMSO).
1
Yield: 96.8 mg (80.6%). 99.4% purity by HPLC. H NMR
(400 MHz, CDCl3) δ 12.58 (s, 1H), 8.29 (s, 1H), 7.91 (t, J = 6.8
Hz, 1H), 7.60 (d, J = 5.0 Hz, 1H), 7.27 (m, 2H), 7.11 (s, 1H),
6.51 (s, 1H), 5.68 (m, 1H), 3.73 (d, J = 8.2 Hz, 1H), 3.63 (t, J =
9.1 Hz, 1H), 3.40 (m, 1H), 3.34 (d, J = 8.5 Hz, 1H), 3.28 (s, 3H),
2.20 (s, 1H), 2.10 (dd, J = 19.9, 9.1 Hz, 1H). 13C NMR (100
MHz, CDCl3) δ 160.3, 157.7, 157.5, 152.0, 150.3, 135.2 (d, J =
8.4 Hz), 131.4, 125.4 (d, J = 14.9 Hz), 124.6 (d, J = 3.6 Hz),
121.0, 117.4 (d, J = 22.0 Hz), 102.5 (d, J = 172.3 Hz), 54.5, 48.4,
46.6, 32.1, 28.5. HRMS (ESI) calcd for C17H19FN5O2S:
376.1243. Obsd: 376.1236. []D -18.5o (c 3.38, CHCl3).
6.2.12.12. (R)-N-Methyl-N-(1-((2-
nitrophenyl)sulfonyl)pyrrolidin-3-yl)-7H-
pyrrolo[2,3-d]pyrimidin-4-amine, 33
1
Yield: 101 mg (78.1%). 97.5% purity by HPLC. H NMR
(400 MHz, CDCl3) δ 11.92 (s, 1H), 8.30 (s, 1H), 8.05 (dd, J =
7.3, 1.7 Hz, 1H), 7.72 (m, 2H), 7.64 (dd, J = 7.5, 1.6 Hz, 1H),
7.12 (d, J = 3.2 Hz, 1H), 6.56 (d, J = 3.3 Hz, 1H), 5.75 (dd, J =
15.9, 7.8 Hz, 1H), 3.76 (m, 2H), 3.46 (ddd, J = 17.0, 9.8, 7.2 Hz,
2H), 3.32 (s, 3H), 2.26 (m, 1H), 2.18 (m, 1H). 13C NMR (100
MHz, CDCl3) δ 157.7, 152.2, 150.6, 148.5, 133.9, 131.8, 131.6,
131.1, 124.2, 120.9, 103.5, 101.9, 54.7, 48.6, 47.0, 32.3, 28.7.
HRMS (ESI) calcd for C17H19N6O4S: 403.1188. Obsd: 403.1182.
[]D +12.6o (c 2.17, CHCl3).
6.2.12.8. (R)-N-(1-((3-
Fluorophenyl)sulfonyl)pyrrolidin-3-yl)-N-methyl-
7H-pyrrolo[2,3-d]pyrimidin-4-amine, 29
Yield: 101 mg (84.0%). 100% purity by HPLC. 1H NMR (400
MHz, CDCl3) δ 12.64 (s, 1H), 8.27 (s, 1H), 7.65 (d, J = 7.5 Hz,
1H), 7.55 (dd, J = 14.7, 6.6 Hz, 2H), 7.33 (m, 1H), 7.11 (d, J =
2.8 Hz, 1H), 6.49 (d, J = 2.9 Hz, 1H), 5.59 (dd, J = 14.9, 7.4 Hz,
1H), 3.63 (t, J = 7.3 Hz, 1H), 3.47 (t, J = 9.3 Hz, 1H), 3.33 (dd, J
= 10.2, 6.6 Hz, 1H), 3.26 (s, 3H), 3.13 (dd, J = 16.7, 9.1 Hz, 1H),
2.15 (m, 1H), 2.06 (m, 1H). 13C NMR (100 MHz, CDCl3) δ
163.8, 161.3, 157.4, 152.0, 150.2, 137.9 (d, J = 6.5 Hz), 131.1 (d,
J = 7.7 Hz), 123.5 (d, J = 3.2 Hz), 121.0 (s), 120.2 (d, J = 21.2
Hz), 115.0 (d, J = 24.1 Hz), 102.5 (d, J = 172.4 Hz), 54.3, 49.1,
47.0, 32.1, 28.3. HRMS (ESI) calcd for C17H19FN5O2S:
376.1243. Obsd: 376.1239. []D -39.8o (c 3.38, CHCl3).
6.2.12.13. (R)-N-Methyl-N-(1-((3-
nitrophenyl)sulfonyl)pyrrolidin-3-yl)-7H-
pyrrolo[2,3-d]pyrimidin-4-amine, 34
1
Yield: 104 mg (81.0%). 99.6% purity by HPLC. H NMR
(400 MHz, CDCl3) δ 10.63 (s, 1H), 8.70 (s, 1H), 8.50 (d, J = 7.9
Hz, 1H), 8.22 (s, 1H), 8.19 (d, J = 8.1 Hz, 1H), 7.80 (t, J = 7.9
Hz, 1H), 7.08 (s, 1H), 6.54 (s, 1H), 5.60 (m, 1H), 3.73 (t, J = 7.5
Hz, 1H), 3.50 (dd, J = 11.6, 7.1 Hz, 1H), 3.40 (m, 1H), 3.31 (s,
3H), 3.18 (dd, J = 16.8, 9.2 Hz, 1H), 2.15 (m, 2H). 13C NMR
(100 MHz, CDCl3) δ 157.6, 152.2, 150.5, 148.6, 138.7, 133.2,
130.7, 127.5, 122.8, 121.0, 103.5, 101.9, 54.4, 49.1, 47.2, 32.5,
28.4. HRMS (ESI) calcd for C17H19N6O4S: 403.1188. Obsd:
403.1184. []D -41.1o (c 1.24, CHCl3).
6.2.12.9. (R)-N-(1-((4-
Fluorophenyl)sulfonyl)pyrrolidin-3-yl)-N-methyl-
7H-pyrrolo[2,3-d]pyrimidin-4-amine, 30
1
Yield: 93.5 mg (77.8%). 98.0% purity by HPLC. H NMR
(400 MHz, CDCl3) δ 11.08 (s, 1H), 8.24 (s, 1H), 7.89 (dd, J =
8.4, 5.1 Hz, 2H), 7.26 (dd, J = 9.2, 7.5 Hz, 2H), 7.09 (d, J = 2.7
Hz, 1H), 6.52 (d, J = 2.9 Hz, 1H), 5.61 (m, 1H), 3.65 (t, J = 7.5
Hz, 1H), 3.38 (dt, J = 10.1, 6.9 Hz, 2H), 3.31 (d, J = 8.7 Hz, 3H),
3.11 (m, 1H), 2.18 (d, J = 4.7 Hz, 1H), 2.08 (dd, J = 14.9, 6.4 Hz,
1H). 13C NMR (100 MHz, CDCl3) δ 166.8, 164.2, 157.6, 152.1,
150.5, 132.1, 130.6 (d, J = 9.6 Hz), 120.9, 116.6 (d, J = 22.5 Hz),
90.4, 54.4, 49.2, 47.2, 32.3, 28.5. HRMS (ESI) calcd for
C17H19FN5O2S: 376.1243. Obsd: 376.1237. []D -35.9o (c 0.670,
CHCl3).
6.2.12.14. (R)-N-Methyl-N-(1-((4-
nitrophenyl)sulfonyl)pyrrolidin-3-yl)-7H-
pyrrolo[2,3-d]pyrimidin-4-amine, 35
1
Yield: 95.3 mg (74.0%). 99.1% purity by HPLC. H NMR
(400 MHz, CDCl3) δ 10.99 (s, 1H), 8.42 (d, J = 8.6 Hz, 2H), 8.22
(s, 1H), 8.05 (d, J = 8.6 Hz, 2H), 7.09 (d, J = 2.1 Hz, 1H), 6.52
(d, J = 2.6 Hz, 1H), 5.59 (dt, J = 15.6, 7.9 Hz, 1H), 3.70 (m, 1H),
3.51 (m, 1H), 3.38 (dd, J = 10.2, 7.0 Hz, 1H), 3.30 (s, 3H), 3.18
(dd, J = 16.9, 9.2 Hz, 1H), 2.15 (m, 2H). 13C NMR (100 MHz,
CDCl3) δ 157.6, 152.2, 150.9, 150.5, 142.3, 129.0, 124.6, 120.7,
103.5, 102.1, 54.5, 49.1, 47.2, 32.5, 28.4. HRMS (ESI) calcd for
C17H19N6O4S: 403.1188. Obsd: 403.1185. []D -63.5o (c 0.568,
CHCl3).
6.2.12.10. (R)-2-((3-(Methyl(7H-pyrrolo[2,3-
d]pyrimidin-4-yl)amino)pyrrolidin-1-
yl)sulfonyl)benzonitrile, 31

6.2.12.15. (R)-N-Methyl-N-(1-(m-
tolylsulfonyl)pyrrolidin-3-yl)-7H-pyrrolo[2,3-
d]pyrimidin-4-amine, 36
129.4, 129.2, 129.1, 128.9, 127.9, 127.6, 123.0, 120.8, 103.2,
101.6, 54.3, 49.2, 47.1, 32.0, 28.3. HRMS (ESI) calcd for
C21H22N5O2S: 408.1494. Obsd: 408.1495. []D -40.3o (c 3.97,
CHCl3).
1
Yield: 101 mg (84.9%). 95.0% purity by HPLC. H NMR
6.2.12.20. (R)-N-Methyl-N-(1-(piperidin-1-
ylsulfonyl)pyrrolidin-3-yl)-7H-pyrrolo[2,3-
d]pyrimidin-4-amine, 41
(400 MHz, CDCl3) δ 12.57 (s, 1H), 8.27 (s, 1H), 7.66 (d, J = 9.2
Hz, 2H), 7.44 (m, 2H), 7.10 (d, J = 3.0 Hz, 1H), 6.48 (d, J = 2.7
Hz, 1H), 5.58 (dt, J = 14.9, 7.4 Hz, 1H), 3.62 (t, J = 7.5 Hz, 1H),
3.44 (m, 1H), 3.33 (dd, J = 10.2, 6.4 Hz, 1H), 3.25 (s, 3H), 3.10
(dd, J = 16.9, 9.1 Hz, 1H), 2.44 (s, 3H), 2.14 (dd, J = 9.7, 5.3 Hz,
1H), 2.03 (m, 1H). 13C NMR (100 MHz, CDCl3) δ 157.4, 152.0,
150.2, 139.4, 135.5, 133.8, 129.0, 128.1, 124.9, 120.9, 103.3,
101.6, 54.3, 49.2, 47.1, 32.0, 28.4, 21.4. HRMS (ESI) calcd for
C18H22N5O2S: 372.1494. Obsd: 372.1495. []D -39.6o (c 3.34,
CHCl3).
1
Yield: 91.0 mg (77.8%). 98.7% purity by HPLC. H NMR
(400 MHz, CDCl3) δ 12.65 (s, 1H), 8.36 (s, 1H), 7.15 (s, 1H),
6.58 (s, 1H), 5.78 (m, 1H), 3.60 (m, 2H), 3.35 (s, 3H), 3.34 (m,
2H), 3.26 (d, J = 4.7 Hz, 4H), 2.28 (dd, J = 9.6, 7.4 Hz, 1H), 2.13
(m, 1H), 1.64 (d, J = 4.2 Hz, 4H), 1.56 (d, J = 3.4 Hz, 2H). 13C
NMR (100 MHz, CDCl3) δ 157.6, 152.0, 150.4, 120.9, 103.3,
101.7, 77.5, 77.2, 76.9, 54.6, 49.4, 47.3, 47.1, 32.0, 28.5, 25.5,
23.8. HRMS (ESI) calcd for C16H25N6O2S: 365.1760. Obsd:
365.1755. []D +13.7o (c 3.06, CHCl3).
6.2.12.16. (R)-N-Methyl-N-(1-tosylpyrrolidin-3-
yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine, 37
1
6.2.12.21. (R)-N-Methyl-N-(1-
(morpholinosulfonyl)pyrrolidin-3-yl)-7H-
pyrrolo[2,3-d]pyrimidin-4-amine, 42
Yield: 87.4 mg (73.5%). 99.4% purity by HPLC. H NMR
(400 MHz, CDCl3) δ 12.57 (s, 1H), 8.26 (s, 1H), 7.73 (d, J = 6.8
Hz, 2H), 7.35 (d, J = 7.1 Hz, 2H), 7.10 (s, 1H), 6.48 (s, 1H), 5.58
(m, 1H), 3.61 (d, J = 7.6 Hz, 1H), 3.41 (t, J = 9.0 Hz, 1H), 3.31
(dd, J = 8.7, 6.2 Hz, 1H), 3.25 (s, 3H), 3.08 (dd, J = 16.3, 7.7 Hz,
1H), 2.44 (s, 3H), 2.13 (s, 1H), 2.04 (dd, J = 18.2, 9.9 Hz, 1H).
13C NMR (100 MHz, CDCl3) δ 157.4, 151.9, 150.2, 143.8, 132.5,
129.8, 127.8, 120.8, 103.2, 101.6, 54.2, 49.2, 47.0, 32.0, 28.3,
21.5. HRMS (ESI) calcd for C18H22N5O2S: 372.1494. Obsd:
372.1490. []D -41.5o (c 3.24, CHCl3).
1
Yield: 47.0 mg (40.2%). 96.8% purity by HPLC. H NMR
(400 MHz, CDCl3) δ 12.37 (s, 1H), 8.35 (s, 1H), 7.15 (s, 1H),
6.58 (s, 1H), 5.79 (m, 1H), 3.76 (m, 4H), 3.65 (m, 2H), 3.38 (m,
2H), 3.36 (s, 3H), 3.28 (m, 4H), 2.27 (dt, J = 10.4, 8.5 Hz, 1H),
2.16 (m, 1H). 13C NMR (100 MHz, CDCl3) δ 157.7, 152.2, 150.5,
121.0, 103.5, 101.8, 66.5, 54.7, 49.4, 47.6, 46.5, 32.2, 28.6.
HRMS (ESI) calcd for C15H23N6O3S: 367.1552. Obsd: 367.1547.
[]D +12.9o (c 1.53, CHCl3).
6.2.12.17. (R)-N-(1-((4-
Methoxyphenyl)sulfonyl)pyrrolidin-3-yl)-N-methyl-
7H-pyrrolo[2,3-d]pyrimidin-4-amine, 38
6.3. In vitro enzyme assays and in vitro ADME tests
Yield: 107 mg (86.2%). 100% purity by HPLC. 1H NMR (400
MHz, CDCl3) δ 12.56 (s, 1H), 8.27 (s, 1H), 7.79 (d, J = 6.6 Hz,
2H), 7.10 (s, 1H), 7.02 (d, J = 6.6 Hz, 2H), 6.48 (s, 1H), 5.58 (s,
1H), 3.87 (s, 3H), 3.59 (t, J = 8.5 Hz, 1H), 3.40 (t, J = 9.0 Hz,
1H), 3.31 (d, J = 6.3 Hz, 1H), 3.25 (s, 3H), 3.08 (m, 1H), 2.14 (s,
1H), 2.03 (m, 1H). 13C NMR (100 MHz, CDCl3) δ 163.1, 157.4,
151.9, 150.2, 129.9, 127.1, 120.8, 114.3, 103.2, 101.6, 55.6, 54.2,
49.2, 47.0, 32.0, 28.3. HRMS (ESI) calcd for C18H22N5O3S:
388.1443. Obsd: 388.1441. []D -37.5o (c 4.03, CHCl3).
All enzyme inhibition assay results were obtained by
commercially available KinaseProfilerTM services (Eurofins
Scientific, UK) at Km values for ATP. The 50% inhibitory
concentration (IC50) of each compound was determined with
GraphPad Prism software. The kinome tree of the inhibition
percentages of 345 kinases at the 10 M concentration for 6 was
drawn by KinMap web accessible tool.74 All in vitro ADME and
hERG assays were performed by commercially available services
at the New Drug Development Center, Daegu-Gyeongbuk
Medical Innovation Foundation, South Korea and Drug
Discovery Platform Technology Group, Korea Research Institute
of Chemical Technology, South Korea.
6.2.12.18. (R)-N-Methyl-N-(1-((4-
(trifluoromethyl)phenyl)sulfonyl)pyrrolidin-3-yl)-
7H-pyrrolo[2,3-d]pyrimidin-4-amine, 39
6.4. Pharmacokinetic study
1
Yield: 99.2 mg (72.9%). 99.8% purity by HPLC. H NMR
Beagle dogs (10 – 12 kg), Sprague Dawley rats (7 – 8 weeks
(400 MHz, DMSO-d6) δ 11.71 (s, 1H), 8.05 (m, 5H), 7.12 (s,
1H), 6.48 (s, 1H), 5.28 (dd, J = 14.4, 7.2 Hz, 1H), 3.51 (s, 1H),
3.43 (dd, J = 18.2, 8.2 Hz, 3H), 3.22 (m, 2H), 3.12 (m, 3H), 2.04
(m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 157.2, 152.1, 150.8,
140.0, 133.2 (q, J = 32.4 Hz), 128.9, 127.0 (d, J = 3.1 Hz), 123.9
(q, J = 272.7 Hz), 121.5, 102.9, 101.7, 54.5, 49.1, 47.2, 32.1,
28.0. HRMS (ESI) calcd for C18H19F3N5O2S: 426.1212. Obsd:
426.1204. []D -29.6o (c 3.29, CHCl3).
old) and ICR mice (7 – 8 weeks old) were kept in an
o
environmentally controlled breeding room (25  2 C, 60  5%
humidity, 12h dark/light cycle) with free access to food and
water. The free base form of compound 6 clearly dissolved in
10% ethanol and 90% PEG400 with 1 mL/kg dose volume for
intravenous administration. Blood samplings were performed at
0.08, 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 hours after intravenous
administration. For oral administration, compound 6 was
suspended in corn oil with 5 mL/kg dose volume. Animals were
fasted for 16 hours before oral administration but had free access
to water. Blood samplings were performed at 0.25, 0.5, 1, 2, 4, 6,
8, 12 and 24 hours after oral administration. 20 L of the
sampled plasma was diluted with 180 L of acetonitrile
containing an internal standard. It was then vortexed and
centrifuged under 15000 rpm at 4 oC. After the centrifugation, the
supernatant was analyzed by LC-MS/MS, Nexera XR system
(Shimadzu, Japan) with TSQ vantage triple quadruple (Thermo,
USA). The column was Kinetex XB-C18 column (2.1 x 100 mm,
2.6 m particle size; Phenomenex, USA) and pharmacokinetic
6.2.12.19. (R)-N-Methyl-N-(1-(naphthalen-2-
ylsulfonyl)pyrrolidin-3-yl)-7H-pyrrolo[2,3-
d]pyrimidin-4-amine, 40
1
Yield: 112 mg (84.7%). 96.8% purity by HPLC. H NMR
(400 MHz, CDCl3) δ 12.57 (s, 1H), 8.43 (s, 1H), 8.21 (s, 1H),
7.97 (m, 2H), 7.91 (d, J = 7.9 Hz, 1H), 7.85 (d, J = 8.6 Hz, 1H),
7.61 (td, J = 15.0, 7.0 Hz, 2H), 7.02 (d, J = 2.5 Hz, 1H), 6.39 (d,
J = 2.6 Hz, 1H), 5.52 (m, 1H), 3.68 (m, 1H), 3.50 (t, J = 9.4 Hz,
1H), 3.37 (dd, J = 9.9, 6.7 Hz, 1H), 3.20 (s, 3H), 3.16 (m, 1H),
2.10 (ddd, J = 18.9, 8.3, 6.1 Hz, 1H), 2.00 (m, 1H). 13C NMR
(100 MHz, CDCl3) δ 157.4, 151.9, 150.2, 134.9, 132.8, 132.2,

parameters were obtained by the non-compartmental analysis
model in Phoenix WinNonlin 6.4 version (Pharsight, USA).
Kruskal-Wallis test or one-way analysis of variance test was
performed to determine statistically significant differences,
which were defined as P < 0.05.
6.5. Mouse collagen-induced arthritis
Acknowledgments
Male DBA1/J mice (6 weeks old) were purchased from Japan
SLC, Inc and all mice were housed in specific pathogen-free
(SPF) conditions with free access to food and water. After 7 days
of acclimation, mice were immunized with 0.1 mL of emulsion
of 1:1 mixture of type II collagen (2 mg/mL) and complete
Freund’s adjuvant by subcutaneous injection at 1.5 cm distal
from the tail base. After 21 days, immunized mice were boosted
by another injection with 0.1 mL of emulsion of type II collagen
and incomplete Freund’s adjuvant. The emulsions were prepared
according to manufacturer’s instruction.75 When all mice
indicated signs of arthritis, treatment with test articles and
assessment of arthritis were initiated (day 1). The immunized and
boosted mice were randomized into 4 treatment groups (n = 10
each) and same-aged naïve mice were assigned to a normal group
(n = 6). All test articles or vehicle were orally administered once
daily and the clinical arthritis scores were assessed twice weekly
for 18 days. Corn oil was used as a vehicle and all test articles
were suspended in vehicle. Paw volumes were measured by
LE7500 plethysmometer (Panlab, Spain) on days 1 and 15. The
severity of each paw was evaluated and scored according to the
following criteria where 0 = normal; 0.5 = redness of the toe, but
not swollen; 1 = one toe inflamed and swollen; 2 = more than one
toe, but not entire paw, inflamed and swollen, or mild swelling of
entire paw; 3 = entire paw inflamed and swollen; and 4 = very
inflamed and swollen paw or ankylosed paw.76 The clinical
arthritis score was represented by the total scores of each paw.
On day 19, all individuals were sacrificed and autopsies were
performed. Serum cytokines including IL-6 and TNF- were
measured by ELISA kits (ProcartaPlex Mix and Match
customized, Mouse 5 plex, BMS). For the histopathological
studies, the right hind paws of each mouse were fixed by 10%
formalin solution and the hematoxylin-eosin staining was
performed on the ankle and third digit of the paw. The
histopathological score was semiquantitatively measured
according to the following criteria where 0 = normal; 1 =
infiltration of inflammatory cells; 2 = synovial hyperplasia and
pannus formation; and 3 = bone erosion and destruction.77 The
obtained images were analyzed by iSolution EL ver 9.1 (IMT i-
solution Inc., Canada) and the micro-CT analyses of all
individuals were performed by viviCT 80 micro-CT (SCANCO
Medical, Switzerland) to measure bone surface/volume ratio.
Student’s t-test or one-way analysis of variance test was
performed to determine statistically significant differences. The
data for clinical arthritis scores were statistically analyzed by the
Kruskal-Wallis test or Mann-Whitney test where a significant
difference was defined as P < 0.05.
This research was performed in collaboration with DGMIF
Drug Development Center, and supported by Bio & Medical
Technology Development Program through the National
Research Foundation of Korea (NRF) funded by the Ministry of
Science, ICT & Future Planning (2014M3A9D9033717).
References and notes
1.
2.
Deane, K. D.; El-Gabalawy, H. Nat. Rev. Rheumatol. 2014, 10,
212.
Jacques, P; Van den Bosch, F. Expert Opin. Emerg. Drugs 2013,
18, 231.
3.
4.
Emery, P. Nat. Rev. Rheumatol. 2015, 11, 69.
Meier, F. M. P.; Frerix, M.; Hermann, W.; Müller-Ladner, U.
Immunotherapy 2013, 5, 955.
5.
6.
Burmester, G. R.; Feist, E.; Dörner, T. Nat. Rev. Rheumatol. 2014,
10, 77.
Neumann, E.; Khawaja, K.; Muller-Ladner, U. Nat. Rev.
Rheumatol. 2014, 10, 429.
7.
8.
9.
Müller, S.; Knapp, S. Expert Opin. Drug Discov. 2010, 5, 867.
Kelly, V; Genovese, M. Rheumatology 2013, 52, 1155.
Buer, J. K. Inflammopharmacology 2015, 23, 163.
10. O'Dell, J. R. N. Engl. J. Med. 2004, 350, 2591.
11. Detert, J.; Klaus, P. Biologics 2015, 9, 35.
12. Mócsai, A.; Kovács, L.; Gergely, P. BMC Med. 2014, 12, 43.
13. Shuai, K.; Liu, B. Nat. Rev. Immunol. 2003, 3, 900.
14. Wilks, A. F. Proc. Natl. Acad. Sci. U. S. A. 1989, 86, 1603.
15. Darnell Jr., J. E.; Kerr, I. M.; Stark, G. R. Science 1994, 264,
1415.
16. US Food and Drug Administration. Center for drug evaluation and
research, Application number: 203214Orig1s000, Approval letter.
https://www.accessdata.fda.gov/drugsatfda_docs/nda/2012/20321
4Orig1s000MedR.pdf; 2012 Accessed 17.08.30.
17. 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, 2003, 302, 875.
18. Dowty, M. E.; Jesson, M. I.; Ghosh, S.; Lee, J.; Meyer, D. M.;
Krishnaswami, S.; Kishore, N. J. Pharmacol. Exp. Ther. 2014,
348, 165.
19. Kremer, J. M; Bloom, B. J.; Breedveld, F. C.; Coombs, J. H.;
Fletcher, M. P.; Gruben, D.; Krishnaswami, S.; Burgos-Vargas,
R.; Wilkinson, B.; Zerbini, C. A. F.; Zwillich, S. H. Arthritis
Rheum. 2009, 60, 1895.
6.6. Rat adjuvant-induced arthritis
AIA was induced in SPF Lewis LEW/SsNSlc rats (Japan SLC
Inc., Japan). After 2 weeks of acclimation, 10 weeks old rats
were immunized by the subcutaneous injection of 0.1 mL of
complete Freund’s adjuvant containing 10 mg/mL of heat-killed
mycobacterium (Chondrex, Inc., USA) at a 2.0 cm distal from the
rat tail base. After 12 days of immunization (day 1), the rats were
randomized into 4 treatment groups (n = 10 each) and received
test articles or vehicles alone once daily for 14 days. Same-aged
naïve mice were assigned to a normal group (n = 5). The clinical
arthritis score and paw thicknesses were evaluated twice weekly
for 14 days. The criteria for the clinical arthritis score are 0 =
normal; 1 = mild edema or erythema; 2 = moderate edema; 3 =
severe edema; and 4 = ankylosis. The paw thicknesses were
measured by electric caliper CD-15CPX (Mitutoyo Corp., Japan).
20. Feist, E.; Burmester, G. R. Rheumatology. 2013, 52, 1352-1357.
21. Tefferi, A. Blood. 2012, 119, 2721.
22. O'Shea, J. J.; Holland, S. M.; Staudt, L. M. N. Engl. J. Med. 2013,
368, 161.
23. Dymock, B. W.; Yang, E. G.; Chu-Farseeva, Y.; Yao, L. Future
Med. Chem. 2014, 6, 1439.
24. Norman, P. Expert Opin. Investig. Drugs. 2014, 23, 1067.
25. Gras, J. Drug. Future. 2014, 39, 547.
26. Menet, C. J.; Fletcher, S. R.; Van Lommen, G.; Geney, R.; Blanc,
J.; Smits, K.; Jouannigot, N.; Deprez, P.; van der Aar, E. M.;
Clement-Lacroix, P.; Lepescheux, L.; Galien, R.; Vayssiere, B.;
Nelles, L.; Christophe, T.; Brys, R.; Uhring, M.; Ciesielski, F.;
Van Rompaey, L. J. Med. Chem. 2014, 57, 9323.
27. Pardanani, A.; Gotlib, J. R.; Jamieson, C.; Cortes, J. E.; Talpaz,
M.; Stone, R. M.; Silverman, M. H.; Gilliland, D. G.; Shorr, J.;
Tefferi, A. J. Clin. Oncol. 2011, 29, 789.

28. Schenkel, L. B.; Huang, X.; Cheng, A.; Deak, H. L.; Doherty, E.;
Emkey, R.; Gu, Y.; Gunaydin, H.; Kim, J. L.; Lee, J.; Loberg, R.;
Olivieri, P.; Pistillo, J.; Tang, J.; Wan, Q.; Wang, H.-L.; Wang, S.-
W.; Wells, M. C.; Wu, B.; Yu, V.; Liu, L.; Geuns-Meyer, S. J.
Med. Chem. 2011, 54, 8440.
29. Thoma G, Nuninger F, Falchetto R, et al. Thoma, G.; Nuninger,
F.; Falchetto, R.; Hermes, E.; Tavares, G. A.; Vangrevelinghe, E.;
Zerwes, H.-G. J. Med. Chem. 2011, 54, 284.
51. Huang, T.; Xue, C.-B.; Wang, A.; Kong, L.; Ye, H. F.; Yao, W.;
Rodgers, J. D.; Shepard, S.; Wang, H.; Shao, L.; Li, H.-Y.; Li, Q.
PCT Int. Appl. WO 2011112662 A1, 2011.
52. Zhang, Y.; Warren, M. S.; Zhang, X.; Diamond, S.; Williams, B.;
Punwani, N.; Huang, J.; Huang, Y.; Yeleswaram, S. Drug Metab.
Dispos. 2015, 43, 485.
53. Bissonnette, R.; Luchi, M.; Fidelus-Gort, R.; Jackson, S.; Zhang,
H.; Flores, R.; Newton, R.; Scherle, P.; Yeleswaram, S.; Chen, X.;
Menter, A. J. Dermatol. Treat. 2016, 27, 332.
54. Mascarenhas, J. O.; Talpaz, M.; Gupta, V.; Foltz, L. M.; Savona,
M. R.; Paquette, R.; Turner, A. R.; Coughlin, P.; Winton, E.;
Burn, T. C.; O’Neill, P.; Clark, J.; Hunter, D.; Assad, A.;
Hoffman, R.; Verstovsek, S. Haematologica 2017, 102, 327.
55. Brown, M. F.; Fenwick, A. E.; Flanagan, M. E.; Gonzales, A.;
Johnson, T. A.; Kaila, N.; Mitton-Fry, M. J.; Strohbach, J. W.;
Tenbrink, R. E.; Trzupek, J. D.; Unwalla, R. J.; Vazquez, M. L.
PCT Int. Appl. WO 2014128591 A1, 2014.
56. Coffman, K. J.; Duerr, J. M.; Kaila, N.; Parikh, M. D.; Reese, M.
R.; Samad, T.; Sciabola, S.; Tuttle, J. B.; Vazquez, M. L.;
Verhoest, P. R. Can. Pat. Appl. CA 2899888 A1, 2016.
57. Filgotinib Alone and in Combination With Methotrexate (MTX)
in Adults With Moderately to Severely Active Rheumatoid
Arthritis Who Are Naive to MTX Therapy. (2016). Retrieved
30. Takeuchi, T.; Tanaka, Y.; Iwasaki, M.; Ishikura. H.; Saeki, S.;
Kaneko, Y. Ann. Rheum. Dis. 2016, 75, 1057.
31. Liang J, Tsui V, Van Abbema A, et al. Liang, J.; Tsui, V.; Van
Abbema, A.; Bao, L.; Barrett, K.; Beresini, M.; Berezhkovskiy,
L.; Blair, W. S.; Chang, C.; Driscoll, J.; Eigenbrot, C.; Ghilardi,
N.; Gibbons, P.; Halladay, J.; Johnson, A.; Kohli, P. B.; Lai, Y.;
Liimatta, M.; Mantik, P.; Menghrajani, K.; Murray, J.; Sambrone,
A.; Xiao, Y.; Shia, S.; Shin, Y.; Smith, J.; Sohn, S.; Stanley, M.;
Ultsch, M.; Zhang, B.; Wu, L. C.; Magnuson, S. Eur. J. Med.
Chem. 2013, 67, 175.
32. Siu, T.; Brubaker, J.; Fuller, P.; Torres, L.; Zeng, H.; Close, J.;
Mampreian, D. M.; Shi, F.; Liu, D.; Fradera, X.; Johnson, K.;
Bays, N.; Kadic, E.; He, F.; Goldenblatt, P. ; Shaffer, L.; Patel, S.
B.; Lesburg, C. A.; Alpert, C.; Dorosh, L.; Deshmukh, S. V.; Yu,
H.; Klappenbach, J.; Elwood, F.; Dinsmore, C. J.; Fernandez, R.;
Moy, L.; Young, J. R. J. Med. Chem. 2017, 60, 9676.
from
http://clinicaltrials.gov/ct2
(Identification
No.
33. Menet, C. J.; Mammoliti, O.; López-Ramos, M. Future Med.
Chem. 2015, 7, 203.
NCT02886728).
58. Filgotinib in Combination With Methotrexate in Adults With
Moderately to Severely Active Rheumatoid Arthritis Who Have
an Inadequate Response to Methotrexate. (2016). Retrieved from
http://clinicaltrials.gov/ct2 (Identification No. NCT02889796).
59. Filgotinib Versus Placebo in Adults With Active Rheumatoid
Arthritis (RA) Who Have an Inadequate Response to Biologic
34. Van't Klooster, G. A. E.; Brys, R. C. X.; Van Rompaey, L. J. C.;
Namour, F. S. PCT Int. Appl. WO 2013189771 A1, 2013.
35. Menet, C. J. M.; Van Rompaey, L. J. C.; Fletcher, S. R.; Blanc, J.;
Jouannigot, N.; Hodges, A. J.; Smits, K. K. PCT Int. Appl. WO
2010010190 A1, 2010.
36. Menet, C. J. M.; Smits, K. K. PCT Int. Appl. WO 2010149769
A1, 2010.
37. Namour, F.; Diderichsen, P. M.; Cox, E.; Vayssière, B.; Van der
Aa, A.; Tasset, C.; Van‘t Klooster, G. Clin. Pharmacokinet. 2015,
54, 859.
Disease-modifying
Treatment. (2016). Retrieved from http://clinicaltrials.gov/ct2
(Identification No. NCT02873936).
Anti-rheumatic
Drug(s)
(DMARDs)
60. Galapagos NV. Risks related to product development, regulatory
approval and commercialization. http://reports.glpg.com/annual-
report-2016/en/risk-factors/product-development-regulatory-
approval-and-commercialization.html; 2016 Accessed 17.08.30.
61. Meyer, D. M.; Jesson, M. I.; Li, X.; Elrick, M. M.; Funckes-
Shippy, C. L.; Warner, J. D.; Gross, C. J.; Dowty, M. E.; Ramaiah,
S. K.; Hirsch, J. L.; Saabye, M. J.; Barks, J. L.; Kishore, N.;
Morris, D. L. J. Inflamm. 2010, 7, 41.
62. Williams, N. K.; Bamert, R. S.; Patel, O.; Wang, C.; Walden, P.
M.; Wilks, A. F.; Fantino, E.; Rossjohn, J.; Lucet, I. S. J. Mol.
Biol. 2009, 387, 219.
63. Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R.
K.; Goodsell, D. S.; Olson, A. J. J. Comput. Chem. 2009, 30,
2785.
64. Lee, K. H.; Lee, D. H.; Hwang, S.; Lee, O. S.; Chung, D. S.;
Hong, J.-I. Org. Lett. 2003, 5, 1431.
65. Ulmann, P. A.; Braunschweig, A. B.; Lee, O.-S.; Wiester, M. J.;
Schatz, G. C.; Mirkin, C. A. Chem. Commun. 2009, 5121.
66. Kimura, Y.; Atarashi, S.; Kawakami, K.; Sato, K.; Hayakawa, I. J.
Med. Chem. 1994, 37, 3344.
67. Kawakami, K.; Atarashi, S.; Kimura, Y.; Takemura, M.;
Hayakawa, I. Chem. Pharm. Bull. 1998, 46, 1710.
68. Ward, K. W.; Azzarano, L. M.; Evans, C. A.; Smith, B. R.
Xenobiotica 2004, 34, 353.
38. Namour, F.; Desrivot, J.; Van der Aa, A.; Harrison, P.; Tasset, C.;
Van’t Klooster, G. Drug Metab. Lett. 2016, 10, 38.
39. Westhovens, R.; Taylor, P. C.; Alten, R.; Pavlova, D.; Enríquez-
Sosa, F.; Mazur, M.; Greenwald, M.; Van der Aa, A.; Vanhoutte,
F.; Tasset, C.; Harrison, P. Ann. Rheum. Dis. 2017, 76, 998.
40. Kavanaugh, A.; Kremer, J.; Ponce, L.; Cseuz, R.; Reshetko, O. V.;
Stanislavchuk, M.; Greenwald, M.; Van der Aa, A.; Vanhoutte, F.;
Tasset, C.; Harrison, P. Ann. Rheum. Dis. 2017, 76, 1009.
41. Vanhoutte, F.; Mazur, M.; Voloshyn, O.; Stanislavchuk, M.; Van
der Aa, A.; Namour, F.; Galien, R.; Meuleners, L.; Van't Klooster,
G. Arthritis Rheumatol. 2017, 69, 1949.
42. Wishart, N.; Argiriadi, M. A.; Calderwood, D. J.; Ericsson, A. M.;
Fiamengo, B. A.; Frank, K. E.; Friedman, M.; George, D. M.;
Goedken, E. R.; Josephsohn, N. S.; Li, B. C.; Morytko, M. J.;
Stewart, K. D.; Voss, J. W.; Wallace, G. A.; Wang, L.; Woller, K.
R. PCT Int. Appl. WO 2011068881 A1, 2011.
43. Mohamed, M.-E. F.; Camp, H. S.; Jiang, P.; Padley, R. J.;
Asatryan, A.; Othman, A. A. Clin. Pharmacokinet. 2016, 55,
1547.
44. Genovese, M. C.; Smolen, J. S.; Weinblatt, M. E.; Burmester, G.
R.; Meerwein, S.; Camp, H. S.; Wang, L.; Othman, A. A.; Khan,
N.; Pangan, A. L.; Jungerwirth, S. Arthritis Rheumatol. 2016, 68,
2857.
45. Kremer, J. M.; Emery, P.; Camp, H. S.; Friedman, A.; Wang, L.;
Othman, A. A.; Khan, N.; Pangan, A. L.; Jungerwirth, S.;
Keystone, E. C. Arthritis Rheumatol. 2016, 68, 2867.
46. Mohamed, M.-E. F.; Jungerwirth, S.; Asatryan, A.; Jiang, P.;
Othman, A. A. Br. J. Clin. Pharmacol. 2017, 83, 2242.
47. van Vollenhoven, R. F.; Layton, M.; Kahl, L.; Schifano, L.;
Hachulla, E.; Machado, D.; Staumont-Sallé, D.; Patel, J. Lupus
2015, 24, 648.
48. Kahl, L.; Patel, J.; Layton, M.; Binks, M.; Hicks, K.; Leon, G.;
Hachulla, E.; Machado, D.; Staumont-Sallé, D.; Dickson, M.;
Condreay, L.; Schifano, L.; Zamuner, S.; van Vollenhoven, R. F.;
on behalf of the, JAK115919 Study Team. Lupus 2016, 25, 1420.
49. Ludbrook, V. J.; Hicks, K. J.; Hanrott, K. E.; Patel, J. S.; Binks,
M. H.; Wyres, M. R.; Watson, J.; Wilson, P.; Simeoni, M.;
Schifano, L. A.; Reich, K.; Griffiths, C. E. M. Br. J. Dermatol.
2016, 174, 985.
69. Weaver, R.; Riley, R. J. Rapid Commun. Mass Spectrom. 2006,
20, 2559.
70. European Medicines Agency. Assessment report
International non-proprietary name: tofacitinib, Procedure No.
EMEA/H/C/002542/0000; 25 July 2013.
- Xeljanz,
http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_
-_Public_assessment_report/human/002542/WC500154697.pdf;
2013 Accessed 17.08.30.
71. Brand, D. D.; Latham, K. A.; Rosloniec, E. F. Nat. Protoc. 2007,
2, 1269.
72. Whiteley, P.E.; Dalrymple, S. A. Models of Inflammation:
Adjuvant-Induced Arthritis in the Rat. In: Enna SJ, ed. Current
Protocols in Pharmacology. Vol. 2. New York, NY: John Wiley
& Sons, Inc; 2001:5.5.1–5.5.5.
73. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;
Robb, M. A.; Cheeseman, J. R.; Montgomery, Jr., J. A.; Vreven,
T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.;
Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.;
Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.;
Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.;
Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.;
50. De Vries, L. C. S.; Ludbrook, V. J.; Hicks, K. J.; D'Haens, G. R.
BMJ Case Rep. 2017, 2017.

Hratchian, H. P.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo,
J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.;
Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.;
Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.;
Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.;
Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.;
Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.;
Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz,
P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham,
M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P.
M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.;
Pople, J. A. Gaussian 03. Revision C.02 ed. Wallingford CT:
Gaussian, Inc.; 2004.
74. Eid, S.; Turk, S.; Volkamer, A.; Rippmann, F.; Fulle, S. BMC
Bioinformatics, 2017, 18, 16.
75. Chondrex, Inc. Protocol for the Successful Induction of Collagen-
Induced
Arthritis
(CIA)
in
Mice;
2017.
https://www.chondrex.com/documents/Mouse%20CIA.pdf; 2017
Accessed 17.09.30.
76. Hooke Laboratories. CIA Induction in DBA/1 Mice; 2013.
https://hookelabs.com/protocols/pdf/CIA%20Induction%20in%20
DBA1%20Mice.pdf; 2017 Accessed 17.09.30.
77. Sohn, K. C.; Kang, S. J.; Kim, J. W.; Kim, K. Y.; Ku, S. K.; Lee,
Y. J. Biomol. Ther. 2013, 21, 290.