Design, synthesis and anti-inflammatory evaluation of novel pyrrolo[2,3-d]pyrimidin derivatives as potent JAK inhibitors

Article abstract of DOI:10.1016/j.bmc.2019.07.037

Aiming to develop potent JAK inhibitors, two series of 4-(1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidine derivatives (8a–8p and 11a–11i) were designed and synthesized by coalescing various N-acylpiperidine motifs with baricitinib. The pharmacological results based on enzymatic and cellular assays identified the optimized compound 11e, which exerted over 90% inhibition rates against JAK1 and JAK2, and displayed the most compelling anti-inflammatory efficacy superior to baricitinib by inhibiting NO generation from LPS-induced RAW264.7 macrophages. Importantly, low cytotoxity of 11e was revealed by the IC₅₀ value of 88.2 μM against normal RAW264.7 cells. The binding mode of 11e with JAK1 and JAK2 identified the essential structural bases in accord with SARs analysis. Furthermore, cellular morphology observation and western blot analysis disclosed the ability of 11e to relieve cells inflammatory damage by significantly down-regulating LPS-induced high expression of JAK1, JAK2, as well as pro cytokine IL-1β. Together, 11e was verified as a promising lead for JAK inhibitors for the treatment of inflammatory diseases.

Full text of DOI:10.1016/j.bmc.2019.07.037

Accepted Manuscript
Design, synthesis and anti-inflammatory evaluation of novel pyrrolo[2,3-d]pyr-
imidin derivatives as potent JAK inhibitors
Feng Jiang, Linghe Zang, Xiuqi Miao, Fang Jia, Jie Wang, Minglin Zhu, Ping
Gong, Nan Jiang, Xin Zhai
PII:
S0968-0896(19)31018-1
https://doi.org/10.1016/j.bmc.2019.07.037
BMC 15021
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
18 June 2019
17 July 2019
20 July 2019
Please cite this article as: Jiang, F., Zang, L., Miao, X., Jia, F., Wang, J., Zhu, M., Gong, P., Jiang, N., Zhai, X.,
Design, synthesis and anti-inflammatory evaluation of novel pyrrolo[2,3-d]pyrimidin derivatives as potent JAK
inhibitors, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.07.037
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Design, synthesis and anti-inflammatory evaluation of novel pyrrolo[2,3-
d]pyrimidin derivatives as potent JAK inhibitors
Feng Jianga, 1, Linghe Zangb, 1, Xiuqi Miao , Fang Jia , Jie Wang , Minglin Zhu , Ping Gong , Nan
a
a
a
a
a
Jianga, *, Xin Zhaia, *
a
Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education,
Shenyang Pharmaceutical University, Shenyang 110016, China.
b
Department of Pharmacology, Institute of Life Sciences and Biopharmaceuticals, Shenyang
Pharmaceutical University, Shenyang 110016, China.
Abstract
Aiming to develop potent JAK inhibitors, two series of 4-(1H-pyrazol-4-yl)-7H-pyrrolo[2,3-
d]pyrimidine derivatives (8a-8p and 11a-11i) were designed and synthesized by coalescing
various N-acylpiperidine motifs with baricitinib. The pharmacological results based on enzymatic
and cellular assays identified the optimized compound 11e, which exerted over 90% inhibition
rates against JAK1 and JAK2, and displayed the most compelling anti-inflammatory efficacy
superior to baricitinib by inhibiting NO generation from LPS-induced RAW264.7 macrophages.
Importantly, low cytotoxity of 11e was revealed by the IC50 value of 88.2 μM against normal
RAW264.7 cells. The binding mode of 11e with JAK1 and JAK2 identified the essential structural
bases in accord with SARs analysis. Furthermore, cellular morphology observation and western
blot analysis disclosed the ability of 11e to relieve cells inflammatory damage by significantly
down-regulating LPS-induced high expression of JAK1, JAK2, as well as pro cytokine IL-1β.
Together, 11e was verified as a promising lead for JAK inhibitors for the treatment of
inflammatory diseases.
Key words: pyrrolo[2,3-d]pyrimidin, JAK inhibitors, anti-inflammatory, RAW264.7
macrophages.
1
. Introduction
Janus kinases (JAKs), the members of intracellular non-receptor tyrosine protein kinase family,
1
consist of JAK1, JAK2, JAK3 and TYK2 . JAK1, JAK2 and TYK2 are widely existed, whereas
2
JAK3 is astricted to myeloid, hematopoietic, and lymphoid cells . JAKs play a crucial role in
cytokine receptor-mediated signal transduction through phosphorylating signal transducer and
3
activator of transcription (STAT) to regulate the expression of cytokine . Simultaneously, JAK-
4
dependent cytokines are bound up with pathogenesis of inflammatory diseases . Consequently,
JAKs, the gatekeepers in signal transduction pathway, have attracted great attention in recent
years as desirable targets of inflammatory and autoimmune diseases, such as rheumatoid arthritis
5
6
7
(
RA) , psoriasis , Crohn's disease (CD) and so on.
8
Since the first crystal structure of JAK was analyzed in 2005 , multiple JAK inhibitors have

Corresponding author.


Corresponding author.
E-mail address: zhaixin_syphu@126.com (X. Zhai). jiangnan@syphu.edu.cn (N. Jiang);
1
These authors contributed equally to this work.

been approved or entered into clinical trials, which were divide into three classes according to the
selectivity, represented by tofacitinib , baricitinib and filgotinib 11, respectively (Fig. 1). Pan-
JAK inhibitor tofacitinib, the first-in-class JAK inhibitor for RA treatment, exhibited distinguished
therapeutic effect for patients. However, serious side effects including liver toxicity and anemia
were nonnegligible due to its non-selectivity to four isozymes 12,13. Filgotinib, a selective JAK1
inhibitor undergoing phase III clinical trial for RA treatment, was identified as an advanced
candidate with less untoward effects, whereas its maximum dosage in the case of male was strictly
restricted by FDA for adverse effects 14. By contrast, JAK1/2 inhibitor baricitinib revealed
obvious therapeutic advantages for RA due to lower therapeutic dose of 2 mg/day and slight side
9
10
1
5
effects .
Fig. 1. Structures of representative JAK inhibitors.
Aiming to develop potent JAK inhibitors without undesirable side effects, molecular design was
performed based on the docking analysis of baricitinib with JAK1 16 and JAK2 17 (Fig. 2). 4-(1H-
pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidine nucleus and sulfonyl linker were retained for the
pivotal role to provide hydrogen bond interactions with JAK residues. Furthermore, a structural
fragment of piperidine ring was introduced to replace 3-cyanomethylazetidine for better structural
stability. Besides, various alkyl or aryl sidechains were connected with sulfonyl linker to
investigate the affinity to JAK. Hence, a series of N-sulfonylpiperidine derivatives (8a-8p) were
designed successfully.
Fig. 2. Design of target compounds based on the cocrystal structures of baricitinib with JAK1 and JAK2. (A)

Modeled docking of baricitinib with JAK1 (PDB ID 5KHX). (B) Structure of baricitinib. (C) Modeled docking of
baricitinib with JAK2 (PDB ID 3E63). (D) Structures of target compounds.
In initiatory enzymatic assays, 8a-8p were observed excellent effects against JAK1 and JAK2,
whereas the anti-inflammatory effects on inhibiting NO generation from lipopolysaccharide
(LPS)-reduced RAW264.7 macrophages was unsatisfactory, which may be due to the high rigidity
and undesirable solubility of phenylsulfonyl moiety. In this regard, acetyl linker incorporated
various aliphatic amino sidechains was introduced instead of phenylsulfonyl fragements for
improved flexibility and solubility, which resulted in the discovery of N-acetylpiperidine
derivatives 11a-11i.
Herein, twenty-five target compounds were synthesized and examined for in vitro enzymatic
inhibitory activities and anti-inflammatory effects at cellular level. Cytotoxicity of optimized
compound 11e and 11i was screened for preliminary safety assessment. Furthermore, cell
morphology and western blot analysis were carried out to ascertain their anti-inflammatory
potency, thoroughly.
2
. Results and discussion
2
.1 Chemistry
Target pyrrolo[2,3-d]pyrimidin derivatives 8a-8p and 11a-11g were synthesized according to
the pathway described in Scheme 1. Commercially available material tert-butyl-4-
hydroxypiperidine-1-carboxylate was acylated with methanesulfonyl chloride at room temperature
to provide 1, which underwent a nucleophilic substitution with 4-iodo-1H-pyrazole in presence of
C CO to give 2. Intermediate 2 was converted to 3 by reacting with isopropyl magnesium chloride
3
S
and 2-methoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane under the atmosphere of nitrogen in
sequence 18. 4 was obtained by introducing methyl pivalate (POM) group into 4-chloro-7H-
pyrrolo[2,3-d]pyrimidine. Subsequently, Suzuki coupling of 3 and 4 resulted in the formation of
1
9
compound 5 , which was deprotected Boc group under acidic condition to yield key intermediate
6
.
Intermediate 6 was substituted by various sulfonyl chloride or chloroacetyl chloride to afford
a-7p and 9, respectively. Thereinto, various sulfonyl products 7a-7p were further hydrolyzed in
7
condition of potassium carbonate to furnish target compounds 8a-8p. Moreover, amination of
chloroacetylated products 9 with corresponding aliphatic amines in the catalysis of triethylamine
gave rise to target intermediates 10a-10i. Ultimately, target compounds 11a-11i were successfully
acquired through deprotection of POM group and purification by column chromatography.

o
Scheme 1. Reagents and conditions: (a) TEA, methanesulfonyl chloride, DCM , 0 C then r.t. 1 h; (b) 4-iodo-1H -
o
o
o
pyrazole CsCO
dioxaborolane, N
Pd(Pph Cl , CsCO
3
, NMS, 80 C, 8 h; (c) iPr-MgCl, THF, -10 C then 20 C, 2 h; 2-methoxy-4,4,5,5-tetramethyl-1,3,2
o
2
, THF, r.t. 12 h; (d) NaH, THF, 0 C, 30 min then chloromethyl pivalate, r.t. 1.5 h; (e)
o
3
)
2
2
3
, dioxane, 80 C, 8 h; (f) HCl, CH
3
OH, r.t. 8 h; (g) various sulfonyl chloride, TEA, DMA, 60
oC, 4 h; (h) K
CO
, DMA, 80 C, 4 h; (i) 2-chloroacetyl chloride, DCM, 0 C, 30 min; (j) various amines, DMA,
o
o
2
3
o
1
10 C, 3 h; (k) NaOH, CH
3
OH, r.t. 1.5 h.
2
2
.2 Biological activity and discussion
.2.1 In vitro enzymatic assays and anti-inflammatory activities evaluation
The in vitro inhibition rates of phosphorylation levels of the kinase-specific ligand peptides
were applied to evaluate the inhibitory activities of target compounds 8a-8p and 11a-11i against
JAK1 and JAK2. All results were summarized in Table 1 and Table 2, taking baricitinib as a
positive control.
As shown in Table 1, most N-sulfonyl substituted compounds exhibited excellent activities
against two kinases with the inhibition rates over 90%. Among them, the originally designed
compound 8a (R = ethyl), which possessed the most structural similarity to baricitinib, performed
a great inhibition rates of 95.3% and 93.0% to JAK1 and JAK2. While replacing ethyl with methyl,

cyclopropyl or phenyl group led to a conspicuous decrease in potency (8b-8d). However,
introduction of 2-methylphenyl and 4-methylphenyl groups as R moiety discovered the
compounds 8e and 8f, which showed surprising potency comparable with baricitinib. The results
disclosed that methyl group may increase the binding interaction of molecule with JAK within the
active site. In order to clarify the influence of the substituents on phenyl ring on biological activity,
compounds 8g-8p were designed and synthesized. Based on the enzyme activities screening,
compounds 8i-8k bearing nitrophenyl motif gained promising potency superior to baricitinib.
Confusingly, introducing chlorine and bromine groups caused a mild decline in activity (8l-8n),
but obvious improved potency was displayed by fluorine substituted compounds 8o and 8p. The
differences might be attribute to the strong polarity and hydrogen-bonding acceptor action of
fluorine and nitro groups.
Table 1: In vitro inhibition rates of 8a-8p against JAK1 and JAK2.
N
N
N
N
HN
O
N
S
O
R
(
%) Inhibition at 10 μM a
(%) Inhibition at 10 μM a
Compd.
R
Compd.
R
JAK1
95.2
JAK2
93.0
JAK1
96.6
JAK2
95.1
O2N
8
a
Et
8i
8j
NO2
8
b
Me
22.1
59.4
2.7
56.0
63.2
16.7
94.7
98.7
87.1
69.6
92.4
99.6
63.7
72.8
60.3
93.4
98.3
98.1
84.3
91.4
95.5
99.1
NO2
8
c
8k
8l
Cl
8
d
Br
8
e
94.7
92.4
72.5
53.2
8m
8n
8o
Br
F
8
f
CN
8
g
F
O
8
h
8p
93.6
94.3
98.6
96.0
F
Baricitinib
a
Values are the average of two independent experiments.
Macrophages are crucial immune cells which are involved in the pathogenesis of inflammatory
disorders through secreting inflammatory mediators, such as NO, pro-inflammatory cytokines

interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) 20. Encouraged by enzymatic assays
results, the inhibition on NO generation from LPS-induced RAW264.7 macrophages were used to
estimate the anti-inflammatory activities of 8a-8p at cellular level. All compounds were detected
using Griess method at the concentrations of 2.5, 5, and 10 μg/mL, respectively, and values are
summarized in Fig. 3 as the average of three independent experiments.
Fig. 3. The effects of 8a-8p on NO generation from LPS-reduced RAW264.7 macrophages. RAW264.7 cells were
pre-treated with or without target compounds (2.5, 5, 10 μg/mL) for 2 h. Then, the cells except control group were
incubated with LPS (5 μg/mL) for 18 h. Subsequently, the supernatant was collected for NO detection by the
Griess method. Each compound was detected in three independent experiments. Data are expressed as the means ±
SEM, *p<0.01, **p<0.05 vs. LPS group; # p<0.01 vs. control group.
As depicted in Fig. 3, most compounds exhibited moderate anti-inflammatory activities in a
dose-dependent manner, and the NO generation was markedly inhibited by target compounds at 5
and 10 μg/mL. Further analysis indicated that anti-inflammatory activities in cellular level of
target compounds are basically parallel to enzymatic inhibition results. Compounds accompanied
with weaker enzymatic inhibition showed unexpected cellular potency, as can be seen from the
activities of 8b-8d, 8g and 8h. The similar regularity was also demonstrated by the residual
compounds both with good enzymatic and cellular potency except for 8f and 8o. Unfortunately,
even if the most prominent compound 8i at 2.5, 5 and 10 μg/mL reduced the NO concentration to
2
2.9, 16.5 and 6.2 μM, respectively, it was less active than baricitinib (16.3, 13.0 and 5.9 μM).
The unsatisfactory anti-inflammatory activities may be due to the strong rigidity of molecular
conformation and poor solubility.
Inspirited by the preliminary SARs, structural optimization based on the predicted binding
mode was performed by replacing sulfonyl with acetyl as a linker for better flexibility, meanwhile
various aliphatic amino sidechains were introduced into the acetyl linker to improve solubility.
Accordingly, analogues 11a-11i were synthesized and evaluated by the enzymatic and cellular
assays (Table 2 and Fig. 4).
As illustrated in Table 2, most compounds showed moderate to good efficacy against JAK1 and
JAK2. The pharmacological results demonstrated that the bulk of hydrophilic aliphatic amino tails

exerted a dramatically effect on inhibition. Compounds 11a and 11b bearing dimethylamine and
diethylamine sidechains were less active, while introduction of bulky amines groups, such as 4-
methylpiperidinyl in 11e and 4-carboxypiperidinyl in 11i, were envisioned for the most excellent
potency with inhibition rate exceeding 90%. Notably, all compounds except for 11h presented a
better selectivity to JAK2 superior to JAK1, which contributed to further development of selective
JAK2 inhibitor.
Table 2: In vitro inhibition rates of 11a-11i against JAK1 and JAK2.
N
N
N
N
HN
N
O
R
(
%) Inhibition at 10 μM a
(%) Inhibition at 10 μM a
Compd.
R
Compd.
R
JAK1
46.9
JAK2
79.6
JAK1
69.6
JAK2
88.4
N
N
O
1
1a
11f
11g
N
N
N
1
1b
38.6
55.1
65.6
90.3
77.8
82.3
87.5
94.7
72.8
83.0
92.5
94.3
83.6
61.8
98.4
96.0
OH
1
1c
N
11h
N
N
N
N
COOH
1
1d
11i
N
1
1e
Baricitinib
a
Values are the average of two independent experiments.
Fig. 4. The effects of 11a-11i on NO generation from LPS-reduced RAW264.7 macrophages. RAW264.7 cells

were pre-treated with or without target compounds (2.5, 5, 10 μg/mL) for 2 h. Then, the cells except control group
were incubated with LPS (5 μg/mL) for 18 h. Subsequently, the supernatant was collected for NO detection by the
Griess method. Each compound was detected in three independent experiments. Data are expressed as the means ±
SEM, *p<0.01, **p<0.05 vs. LPS group; # p<0.01 vs. control group.
As a continuation, the effects of nine compounds (11a-11i) on the NO generation from LPS-
reduced RAW264.7 macrophages were identified in a dose-dependent manner (Fig. 4).
Surprisingly, all compounds displayed more remarkable activities than baricitinib. Especially, the
most promising compound 11e at 2.5, 5 and 10 μg/mL reduced the NO concentrations
significantly to 13.8, 5.1 and 3.9 μM, respectively, superior to positive drug baricitinib (16.3, 13.0
and 5.9 μM). The results proved that our previous optimization by coupling aliphatic aminoacetyl
moiety was reasonable for the improved anti-inflammatory activities of pyrrolo[2,3-d]pyrimidin
analogues at cellular level.
2
.2.3 In vitro growth inhibitory effects of 11e and 11i on RAW264.7 macrophages
To ascertain the cytotoxity of target compounds, the in vitro growth inhibitory effects of the
high-efficiency compounds 11e and 11i on normal RAW264.7 cells were tested by MTT assay. A
concentration range from 10 to 200 μM was evaluated for IC50 values. After a 24 h incubation,
significant growth inhibitory effects were noted to the 11e and 11i in a dose dependent manage
(Fig. 5). To our delight, both of them reflected secure inhibitions against RAW264.7 cells with the
IC values of 88.2 μM and 80.9 μM, respectively.
5
0
Fig. 5. The effects of 11e and 11i on RAW264.7 cells.
2
.2.4 Effect of 11e and 11i on cellular morphology of LPS-induced RAW264.7 macrophages
To visually assess anti-inflammation efficacy of target compounds, we observed the changes of
cellular morphology of RAW264.7 macrophages pre-treated with LPS, LPS+11e (10 μg/mL),
LPS+11i (10 μg/mL), and LPS+baricitinib (10 μg/mL), respectively (Fig. 6). Compared with
control group cells, the cells treated with LPS was not membrane integrity and displayed apoptotic
or necrosis morphology such as cell shrinkage, rounded and release of apoptotic bodies. By
contrast, pre-treated with compounds 11e and 11i protected the cells from LPS-induced
inflammatory damage partly.

Fig. 6. The effects of 11e and 11i on morphology of LPS-induced RAW264.7 macrophages. RAW264.7 cells were
treated with LPS, LPS+11e (10 μg/mL) , LPS+11i (10 μg/mL), and LPS+baricitinib (10 μg/mL), respectively.
2
.2.5 Regulating effects of 11e and 11i on expression of JAK and IL-1β in RAW264.7
macrophages
In intensive anti-inflammatory activities screening, we investigate the regulating effects of
compounds 11e and 11i on the expression of JAK1, JAK2 and pro IL-1β in RAW264.7 cells by
western blot analysis. As shown in Fig. 7, compounds 11e and 11i could significantly down-
regulate LPS-induced high expression of JAK1 and JAK2 in a dose dependent manage.
Meanwhile, 11e and 11i also reversed LPS-induced pro IL-1β up-regulation. Remarkably, the
suppression effects of 11e at concentration of 10 μg/mL on JAK1 and JAK2 superior to positive
medicine baricitinib, which uncovered excellent potential of 11e on the prevention of
inflammatory diseases.
Fig. 7. The effects of 11e and 11i on the phosphorylation of JAK1 and JAK2.
2
.3 Molecular docking
To shed light on the binding mode, molecular docking analysis of 11e with JAK1 and JAK2
was performed using Acclrys Discovery Studio 3.0, respectively, based on the co-crystal
structures of baricitinib with JAK1(PDB ID code: 5KHX) and JAK2 (PDB ID code: 3E63).

Fig. 8. The binding poses of baricitinib and 11e with JAK1 and JAK2. (A) Modeled docking of baricitinib with
JAK1 (PDB ID 5KHX). (B) Predicted binding conformation for 11e in the binding site of JAK1. (C) 2D diagram
of the interaction between 11e and JAK1. (D) Modeled docking of baricitinib with JAK2 (PDB ID 3E63). (E)
Predicted binding conformation for 11e in the binding site of JAK2. (F) 2D diagram of the interaction between 11e
and JAK2.
As indicated in Fig. 8A-8B, 11e bound to JAK1 through hydrogen bond interactions with
Glu957, Leu959 and Asn1008 in a similar manner as baricitinib. Noteworthy, oxygen atom of
acetyl fragment of 11e formed an additional hydrogen bond of 2.9 Å with Arg1007, validating the
rationality and importance of acyl linker. Meanwhile, pyrrolo[2,3-d] pyrimidin skeleton of 11e
combined with Leu1010, Ala906, Val938, Leu881, Val889 and Lys911 through hydrophobic
interaction (Fig. 8C). Especially, the methyl on 4-methylpiperidinyl motif formed an unique alkyl
interaction with Lys911, which may interpret the excellent anti-inflammatory activity of 11e. Fig.
8
D-8F expounded the same binding sites of 11e as baricitinib with JAK2 protein. Pyrrolo[2,3-d]
pyrimidin skeletons of both compounds formed two hydrogen bonds interactions with Glu930 and
Leu932. Simultaneously, acetyl linker of 11e attached to Lys857 through hydrogen bonds
interaction, as the same role as sulfonyl segment in baricitinib. The hydrophobic interactions were
formed between 11e and Ala880, Leu983, Leu855 and Val863. In any case, the docking analysis
indicated the reasonability of structure optimization in accordance with the SARs analysis.
3
. Conclusion
In an attempt to develop promising JAK inhibitors as anti-inflammatory agents, two series of 4-
(1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d] pyrimidine derivatives (8a-8p and 11a-11i) were designed
and synthesized by coalescing various N-acylpiperidine successively. Most compounds exhibited
excellent inhibitions against JAK1 and JAK2 with inhibition rates over 90%. Structure
optimization by introducing aliphatic aminoacetyl resulted in eight compounds 11a-11i, which
displayed preferable cellular anti-inflammatory activities by inhibiting LPS-induced NO

generation from RAW264.7 macrophages superior to rigid sulfonyl derivatives (8a-8p) and
reference baricitinib. The most compelling 11e at 2.5, 5 and 10 μg/mL significantly reduced NO
concentrations to 13.8, 5.1 and 3.9 μM, respectively, compared with baricitinib (16.3, 13.0 and 5.9
μM). Importantly, low cytotoxity of 11e was revealed by the IC value of 88.2 μM against normal
5
0
RAW264.7 cells. Furthermore, cellular morphology and western blot analysis disclosed the ability
of 11e to relieve cells inflammatory damage by down-regulating LPS-induced high expression of
JAK1, JAK2, as well as pro cytokine IL-1β, significantly. Molecular docking modes were
consistent with observed SARs. Overall, 11e was identified as a promising lead for JAK inhibitors
for the treatments of inflammatory diseases.
4
. Experiment
4
.1 Chemistry
All melting points were obtained on a Büchi Melting Point B-540 apparatus (Büchi Lab
ortechnik, Flawil, Switzerland) and were uncorrected. Mass spectra (MS) were taken in ES
I mode on Agilent 1100 LC–MS (Agilent, Palo Alto, CA, USA). H NMR and 13C NMR
spectra were recorded on Bruker spectrometers (Bruker Bioscience, Billerica, MA, USA) w
ith TMS as an internal standard. Column chromatography was run on silica gel (200-300
mesh) from Qingdao Ocean Chemicals (Qingdao, Shandong, China). Unless otherwise note
d, all materials were obtained from commercially available sources and were used without
further purification.
1
4
.1.1Preparation of tert-butyl-4-((methylsulfonyl)oxy)cyclohexane-1-carboxylate (1)
Methanesulfonyl chloride was added dropwise into a mixture of tert-butyl-4-hydroxypiper
idine-1-carboxylate (20.0 g, 0.100 mol), and triethylamine (14.1 g, 0.100 mol) in dichloro
methane (200 mL) under ice bath. The reaction mixture was stirred at room temperature f
or 1 h. The mixture was poured in water (150 ml) and stirred for 10 min. The organic la
yer was washed with brine, dried over anhydrous Na SO , and then filtered and concentrat
2
4
ed to yield the compound 1 as a white solid (27.8 g, 97.3%). MS (ESI) m/z: 302.1 [M+
Na]+.
4
.1.2 Preparation of tert-butyl-4-(4-iodo-1H-pyrazol-1-yl)piperidine-1-carboxylate (2)
4
-Iodo-1H-pyrazole (7.0 g, 0.036 mol) and barium carbonate (15.3 g, 0.047 mol) were d
o
issolved in nitromethylpyrrolidone (100 mL) under an atmosphere of nitrogen at 80 C. A
solution of tert-butyl-4-((methylsulfonyl)oxy)cyclohexane-1-carboxylate 1 (15.3 g, 0.047 mol)
in nitromethylpyrrolidone (100 mL) was added dropwise and the mixture was stirred for 8
h. Then, the mixture was cooled to room temperature and poured into a mixed solvent of
methyl tert-butyl ether (100 ml) and water (100 ml). After stirred for 10 min, the organic
layer was poured into N-heptane (300 ml). The precipitates was collected by filtration to
give a white solid 2 (7.88 g, 57.9%). MS (ESI) m/z: 400.3 [M+Na]+.
4
.1.3 Preparation of tert-butyl-4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazol-1-
yl)piperidine-1-carboxylate (3)
Tert-butyl-4-(4-iodo-1H-pyrazol-1-yl)piperidine-1-carboxylate 2 (25.0 g, 0.036 mol) was di
ssolved in tetrahydrofuran (50 ml) at -10 °C and a solution of isopropyl magnesium chlori

de in tetrahydrofuran (60% w/w, 60 ml) was added under an atmosphere of nitrogen whil
e keeping the temperature below 0 °C. After the addition was completed, the mixture was
heated up to 20 °C and stirred for 2 h. Subsequently, 2-methoxy-4,4,5,5-tetramethyl-1,3,2-d
ioxaborolane (13.8 g, 0.087 mol) was dissolved in tetrahydrofuran (10 ml), and slowly add
ed dropwise to the above reaction system. Then the reaction was stirred at room temperat
ure for 12 h. After completion of reaction indicated by TLC, the mixture was pooled into
saturated ammonium chloride solution (200 ml) and stirred for 20 min, and then extracted
with ethyl acetate (200 mL × 2), The organic layer was washed with water and brine, dri
ed with anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting
residue was purified by column chromatography (eluent = dichloromethane:methanol = 97:
3
) to afford 3 as a white solid (15.38 g, 61.5%). MS (ESI) m/z: 378.2 [M+H]+.
.1.4 Preparation of (4-chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)methyl pivalate (4)
To a solution of 4-chloro-7H-pyrrolo[2,3-d] pyrimidine (15.4 g, 0.100 mol) in tetrahydro
4
furan, 60% sodium hydride (8.0 g, 0.200 mol) was added in batches under ice bath. The
reaction mixture was stirred under ice bath for 0.5 h, and then chloromethyl pivalate (30.1
g, 0.200 mol) was added to the mixture. Subsequently, the reaction was stirred at room te
mperature for 1.5 h. After completed, the mixture was poured into saturated ammonium c
hloride solution. The mixture was extracted with ethyl acetate (50 mL × 5), the organic la
yer was washed with brine, dried over anhydrous Na SO , and then filtered and concentrat
2
4
ed to yield 4 as a yellow solid (27.5 g, 85.0%). MS (ESI) m/z: 268.4 [M+H]+
4
1
.1.5. Preparation of tert-butyl-4-(4-(7-((pivaloyloxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-
H-pyrazol-1-yl)piperidine-1-carboxylate (5)
A mixture of 3 (11.93 g, 0.032 mol), bistriphenylphosphine palladium dichlorideand (0.6
g, 0. 95 mmol) and barium carbonate (9.3 g, 0.095 mol) in anhydrous dioxane (200 m
7
L) was heated at 80 °C under an atmosphere of nitrogen for 8 h, After the reaction finis
hed, the mixture was poured in water and then extracted with DCM (200 mL × 2). The
organic layer was washed with water and brine, dried with anhydrous sodium sulfate, and
concentrated under reduced pressure. The resulting residue was purified by column chroma
tography (eluent = petroleum ether:ethyl acetate = 5:1) to afford 5 as a gray red solid (10.4
7
g, 62.4%). MS (ESI) m/z: 483.3 [M+H]+.
4
.1.6. Preparation of (4-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)
methyl pivalate (6)
Tert-butyl-4-(4-(7-((pivaloyloxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl) pi
peridine-1-carboxylate 5 (10.47 g, 0.022 mol) was treated with HCl in methanol (4 mol/L,
4
0 mL) at room temperature for 8 h. Then the mixture was filtered to give compound 6
as a white solid. (7.07 g, 85.2%). MS (ESI) m/z: 381.3 [M+H]+.
4
.1.7. General procedure for the preparation of compounds (7a-7p)
A mixture of (4-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo [2,3-d] pyrimidin-7-yl) me
thyl pivalate 6 (1.00 g, 0.0037 mol) and triethylaminein (1.12 g, 0.011 mol) were dissolve
d in N,N-dimethylacetamide (10 ml), followed by addition of various sulfonyl chloride (0.0
o
0
44 mol) and was stirred at 60 C for 4 h. The reaction mixture was poured into ice wat

er (50 ml). The residue was filtered and washed with water, and dried to give compounds
7
a-7p.
4
.1.7.1. (4-(1-(1-(ethylsulfonyl) piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-y
l)methyl pivalate (7a). Yield: 68%; MS (ESI) m/z: 474 [M+H]+.
4
.1.7.2. (4-(1-(1-(methylsulfonyl)piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-
yl)methyl pivalate (7b). Yield: 83%; MS (ESI) m/z: 461 [M+H]+.
4
.1.7.3. (4-(1-(1-(cyclopropylsulfonyl) piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyrimi
din-7-yl)methyl pivalate (7c). Yield: 68%; MS (ESI) m/z: 487 [M+H]+.
4
.1.7.4. (4-(1-(1-(phenylsulfonyl)piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-
yl)methyl pivalate (7d). Yield: 72%; MS (ESI) m/z: 523 [M+H]+.
4
.1.7.5. (4-(1-(1-(o-tolylsulfonyl)piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-
yl) methyl pivalate (7e). Yield: 76%; MS (ESI) m/z: 537 [M+H]+.
4
.1.7.6. (4-(1-(1-tosylpiperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)methyl p
ivalate (7f). Yield: 68%; MS (ESI) m/z: 537 [M+H]+.
4
.1.7.7. (4-(1-(1-((4-cyanophenyl)sulfonyl)piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyr
imidin-7-yl)methyl pivalate (7g). Yield: 58%; MS (ESI) m/z: 546 [M-H]-.
4
.1.7.8. (4-(1-(1-((4-methoxyphenyl)sulfonyl)piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]
pyrimidin-7-yl)methyl pivalate (7h). Yield: 58%; MS (ESI) m/z: 553 [M+H]+.
4
.1.7.9. (4-(1-(1-((2-nitrophenyl)sulfonyl)piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d pyri
midin-7-yl)methyl pivalate (7i). Yield: 78%; MS (ESI) m/z: 568 [M+H]+.
4
.1.7.10. (4-(1-(1-((3-nitrophenyl)sulfonyl)piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyr
imidin-7-yl)methyl pivalate (7j). Yield: 74%; MS (ESI) m/z: 568 [M+H]+.
4
.1.7.11. (4-(1-(1-((4-nitrophenyl)sulfonyl)piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyr
imidin-7-yl)methyl pivalate (7k). Yield: 80%; MS (ESI) m/z: 568 [M+H]+.
4
.1.7.12. (4-(1-(1-((4-chlorophenyl)sulfonyl)piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]p
yrimidin-7-yl)methyl pivalate (7l). Yield: 68%; MS (ESI) m/z: 557 [M+H]+.
4
.1.7.13. (4-(1-(1-((3-bromophenyl)sulfonyl)piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]p
yrimidin-7-yl)methyl pivalate (7m). Yield: 70%; MS (ESI) m/z: 601 [M+H]+.
4
.1.7.14. (4-(1-(1-((4-bromophenyl)sulfonyl)piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]p
yrimidin-7-yl)methyl pivalate (7n). Yield: 71%; MS (ESI) m/z: 601 [M+H]+.
4
.1.7.15. (4-(1-(1-((4-fluorophenyl)sulfonyl)piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]p
yrimidin-7-yl)methyl pivalate (7o). Yield: 68%; MS (ESI) m/z: 541 [M+H]+.
4
.1.7.16. (4-(1-(1-((3,5-difluorophenyl)sulfonyl)piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-
d]pyrimidin-7-yl)methylpivalate (7p). Yield: 76%; MS (ESI) m/z: 559 [M+H]+.
4
.1.8 General procedure for the preparation of compounds (8a-8p)

To a solution of compounds 8a-8p (0.001 mol) in N, N-dimethylacetamide (1 0ml) was
o
added potassium carbonate (0.42 g, 0.003 mol), the reaction mixture was stirred at 80 C
for 4 h. After completion of the reaction, the mixture was pooled into ice water (50 ml)
and filtered, the residue was washed with water and purified by column chromatography (
eluen = dichloromethane:methanol = 50:1) to afford compounds 8a-8p.
4
.1.8.1 4-(1-(1-(ethylsulfonyl)piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidine (8a).
1
White solid; yield: 82%; H NMR (400 MHz, DMSO) δ 12.07 (s, 1H), 8.68 (s, 1H), 8.66 (s, 1H),
8
.31 (s, 1H), 7.59-7.54 (m, 1H), 7.01 (dd, J = 3.5, 1.6 Hz, 1H), 4.47 (td, J = 11.1, 5.5 Hz, 1H),
3
.75 (d, J = 12.6 Hz, 2H), 3.05 (dd, J = 11.9, 10.0 Hz, 2H), 2.23-2.02 (m, 4H), 1.25 (t, J = 7.3 Hz,
+
o
3
H); MS (ESI) m/z: 361 [M+H] . M.p.: 177~180 C. Anal. Calcd for C H N O S (%): C, 53.32;
16 20 6 2
H, 5.59; N, 23.32. Found (%): C, 53.33; H, 5.61; N, 23.25.
4
.1.8.2 4-(1-(1-(methylsulfonyl)piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidine (8
1
b). Grayish white solid; yield: 66%; H NMR (400 MHz, DMSO) δ 12.07 (s, 1H), 8.68 (
s, 1H), 8.66 (s, 1H), 8.31 (s, 1H), 7.59-7.54 (m, 1H), 7.01 (dd, J = 3.5, 1.6 Hz, 1H), 4.
4
5
1
0
7 (td, J = 11.1, 5.5 Hz, 1H), 3.75 (d, J = 12.6 Hz, 2H), 3.12 (q, J = 7.4 Hz, 2H), 3.0
(dd, J = 11.9, 10.0 Hz, 2H), 2.23-2.02 (m, 4H), 1.25 (t, J = 7.3 Hz, 3H); 13C NMR (
01 MHz, DMSO-d ) δ 152.56(1C), 151.39(1C), 150.51(1C), 138.97(1C), 129.54(1C), 127.
6
2(1C), 121.28(1C), 113.26(1C), 100.37(1C), 58.27(1C), 44.99(2C), 35.18(1C), 31.75(2C).
+
o
MS (ESI) m/z: 347 [M+H] . M.p.: 172~175 C. Anal. Calcd for C H N O S (%): C, 52.
1
5
18
6
2
0
1; H, 5.24; N, 24.26. Found (%): C, 52.03; H, 5.27; N, 24.20.
4
.1.8.3 4-(1-(1-(cyclopropylsulfonyl)piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidi
1
ne (8c). White solid; yield: 75%; H NMR (400 MHz, DMSO) δ 12.07 (s, 1H), 8.68 (s,
1
H), 8.66 (s, 1H), 8.31 (s, 1H), 7.59-7.54 (m, 1H), 7.01 (dd, J = 3.5, 1.6 Hz, 1H), 4.47
(td, J = 11.1, 5.5 Hz, 1H), 3.75 (d, J = 12.6 Hz, 2H), ,3.05 (dd, J = 11.9, 10.0 Hz, 2H),
+
2
.23-2.02 (m, 4H), 1.25 (t, J = 7.3 Hz, 2H); MS (ESI) m/z: 373 [M+H] . M.p.: 192~196
o
C. Anal. Calcd for C H N O S (%): C, 54.82; H, 5.41; N, 22.56. Found (%): C, 54.80;
1
7
20
6
2
H, 5.43; N, 22.57.
4
.1.8.4 4-(1-(1-(cyclopropylsulfonyl)piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidi
1
ne (8d). Yellow solid; yield: 70%; H NMR (400 MHz, DMSO) δ 8.74 (s, 1H), 8.64 (s,
1
H), 7.81 (d, J = 7.3 Hz, 2H), 7.70 (d, J = 7.7 Hz, 2H), 7.12 (d, J = 3.6 Hz, 1H), 7.12
(d, J = 3.6 Hz, 1H), 5.59 (s, 2H), 4.46-4.24 (m, 1H), 3.79 (d, J = 11.9 Hz, 2H), 3.23 (s,
+
o
2
H), 2.13 (dd, J = 16.4, 5.9 Hz, 4H); MS (ESI) m/z: 409 [M+H] . M.p.: 182~185 C. A
nal. Calcd for C H N O S (%): C, 58.81; H, 4.94; N, 20.57. Found (%): C, 58.80; H, 4.9
2
0
20
6
2
7
; N, 20.60.
4
.1.8.5 4-(1-(1-(o-tolylsulfonyl)piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidine (8
1
e). Yellow solid; yield: 80%; H NMR (400 MHz, DMSO) δ 12.07 (s, 1H), 8.65 (s, 1H),
8
.64 (s, 1H), 8.29 (s, 1H), 7.88-7.82 (m, 1H), 7.61 (dd, J = 7.5, 1.1 Hz, 1H), 7.59-7.54 (
m, 1H), 7.46 (dd, J = 15.8, 7.7 Hz, 2H), 6.99 (dd, J = 3.5, 1.7 Hz, 1H), 4.51-4.38 (m,
1
H), 3.75 (d, J = 12.4 Hz, 2H), 2.85 (dt, J = 12.2, 6.2 Hz, 2H), 2.61 (s, 3H), 2.16 (d, J
+
=
9.9 Hz, 2H), 2.06 (ddd, J = 24.3, 12.1, 4.1 Hz, 2H); MS (ESI) m/z: 423 [M+H] . M.p.:
o
1
87~190 C. Anal. Calcd for C H N O S (%): C, 59.70; H, 5.25; N, 19.89. Found (%):
2
1
22
6
2
C, 59.73; H, 5.28; N, 19.85.

4
.1.8.6 4-(1-(1-tosylpiperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidine (8f). Yellow
1
solid; yield: 80%; H NMR (400 MHz, DMSO) δ 12.07 (s, 1H), 8.65 (s, 1H), 8.59 (s, 1
H), 8.27 (s, 1H), 7.68 (d, J = 8.2 Hz, 2H), 7.56 (dd, J = 3.4, 2.5 Hz, 1H), 7.48 (d, J =
8
1
4
1
.0 Hz, 2H), 6.98 (dd, J = 3.5, 1.7 Hz, 1H), 4.33 (tt, J = 9.8, 4.7 Hz, 1H), 3.75 (d, J =
2.0 Hz, 2H), 3.17 (d, J = 5.2 Hz, 2H), 2.43 (s, 3H), 2.20 -2.06 (m, 4H); MS (ESI) m/z:
+
o
23 [M+H] . M.p.: 183~186 C. Anal. Calcd for C H N O S (%): C, 59.70; H, 5.25; N,
2
1
22
6
2
9.89. Found (%): C, 59.73; H, 5.27; N, 19.84.
4
.1.8.7 4-((4-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)piperidin-1-yl)sulfonyl)benzo
1
nitrile (8g). White solid; yield: 69%; H NMR (400 MHz, DMSO) δ 12.07 (s, 1H), 8.65
(s, 1H), 8.61 (s, 1H), 8.27 (s, 1H), 8.17 (d, J = 8.3 Hz, 2H), 7.99 (d, J = 8.3 Hz, 2H),
7
.57-7.55 (m, 1H), 6.99 (d, J = 2.0 Hz, 1H), 4.42-4.29 (m, 1H), 3.81 (d, J = 12.0 Hz, 2
+
H), 2.60 (t, J = 11.9 Hz, 2H), 2.20-2.08 (m, 4H); MS (ESI) m/z: 434 [M+H] . M.p.: 246
o
~
248 C. Anal. Calcd for C H N O S (%): C, 58.19; H, 4.42; N, 22.62. Found (%): C,
2
1
19
7
2
5
8.16; H, 4.47; N, 22.63.
4
.1.8.8 4-(1-(1-((4-methoxyphenyl)sulfonyl)piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]py
1
rimidine (8h). White solid; yield: 83%; H NMR (400 MHz, DMSO) δ 12.07 (s, 1H), 8.6
2
(d, J = 21.2 Hz, 2H), 8.27 (s, 1H), 7.73 (d, J = 7.1 Hz, 2H), 7.56 (s, 1H), 7.18 (d, J
7.0 Hz, 2H), 6.98 (s, 1H), 4.32 (s, 1H), 3.87 (s, 3H), 3.74 (d, J = 9.0 Hz, 2H), 2.45 (
=
+
o
s,2H), 2.14 (s, 4H); MS (ESI) m/z: 439 [M+H] . M.p.: 242~246 C. Anal. Calcd for C H
2
1
N O S (%): C, 57.52; H, 5.06; N, 19.17. Found (%): C, 57.55; H, 5.01; N, 19.20.
2
2
6
3
4
.1.8.9 4-(1-(1-((2-nitrophenyl)sulfonyl)piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyrim
1
idine (8i). Yellow solid; yield: 68%; H NMR (400 MHz, DMSO) δ 12.10 (s, 1H), 8.67 (
d, J = 5.9 Hz, 2H), 8.31 (s, 1H), 8.09 (dd, J = 7.6, 1.4 Hz, 1H), 8.04 (dd, J = 7.8, 1.3
Hz, 1H), 7.96 (dd, J = 7.5, 1.4 Hz, 1H), 7.93 (dd, J = 3.5, 1.6 Hz, 1H), 7.90 (dd, J =
7
.5, 1.3 Hz, 1H), 4.58-4.41 (m, 1H), 3.88 (d, J = 12.6 Hz, 2H), 3.01 (t, J = 11.2 Hz, 2
H), 2.22 (d, J = 10.1 Hz, 2H), 2.11 (qd, J = 12.2, 4.0 Hz, 2H); MS (ESI) m/z: 452 [M-
-
o
H] . M.p.: 231~232 C. Anal. Calcd for C H N O S (%): C, 52.97; H, 4.22; N, 21.62. F
2
0
19
7
4
ound (%): C, 53.00; H, 4.20; N, 21.62.
4
.1.8.10 4-(1-(1-((3-nitrophenyl)sulfonyl)piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyri
1
midine (8j). Yellow solid; yield: 73%; H NMR (400 MHz, DMSO) δ 12.07 (s, 1H), 8.65
(
s, 1H), 8.62 (s, 1H), 8.58 (dd, J = 8.2, 1.4 Hz, 1H), 8.44 (t, J = 1.8 Hz, 1H), 8.26 (d,
J = 9.6 Hz, 2H), 7.99 (t, J = 8.0 Hz, 1H), 7.58-7.55 (m, 1H), 6.99 (dd, J = 3.5, 1.6 Hz,
H), 4.33 (dt, J = 10.5, 5.5 Hz, 1H), 3.85 (d, J = 12.0 Hz, 2H), 2.63 (dt, J = 11.7, 6.0
1
+
o
Hz,2H), 2.13 (tt, J = 12.2, 6.6 Hz, 4H); MS (ESI) m/z: 454 [M+H] . M.p.: 236~239 C.
Anal. Calcd for C H N O S (%): C, 52.97; H, 4.22; N, 21.62. Found (%): C, 53.00; H,
2
0
19
7
4
4
.21; N, 21.63.
4
.1.8.11 4-(1-(1-((4-nitrophenyl)sulfonyl)piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyri
1
midine (8k). Yellow solid; yield: 79%; H NMR (400 MHz, DMSO) δ 12.07 (s, 1H), 8.6
5
(s, 1H), 8.60 (s, 1H), 8.28 (d, J = 6.1 Hz, 1H), 7.90 (d, J = 8.4 Hz, 2H), 7.73 (d, J
8.4 Hz, 2H), 7.56 (s, 1H), 6.99 (s, 1H), 4.36 (t, J = 10.6 Hz, 1H), 3.77 (d, J = 11.6
=
Hz, 2H), 3.17 (d, J = 5.2 Hz, 2H), 2.11 (dd, J = 13.2, 9.8 Hz, 4H); MS (ESI) m/z: 454
+
o
[
M+H] . M.p.: 225~226 C. Anal. Calcd for C H N O S (%): C, 52.97; H, 4.22; N, 21.6
20 19 7 4

2
. Found (%): C, 53.02; H, 4.21; N, 21.60.
4
.1.8.12 4-(1-(1-((4-chlorophenyl)sulfonyl)piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]py
1
rimidine (8l). White solid; yield: 73% ; H NMR (400 MHz, DMSO) δ 12.09 (s, 1H), 8.
6
6 (s, 1H), 8.61 (s, 1H), 8.28 (s, 1H), 7.84-7.80 (m, 2H), 7.78-7.74 (m, 2H), 7.57 (dd, J
=
3.4, 2.5 Hz, 1H), 6.99 (dd, J = 3.5, 1.7 Hz, 1H), 4.46-4.29 (m, 1H), 3.78 (d, J = 12.0
+
Hz, 2H), 2.60-2.51 (m, 2H), 2.23-2.03 (m, 4H); MS (ESI) m/z: 442 [M+H] . M.p.: 204~2
o
0
4
6 C. Anal. Calcd for C H ClN O S (%): C, 54.24 H, 4.32; N, 18.97. Found (%): C, 5
2
0
19
6
2
.26; H, 4.29; N, 18.98.
4
.1.8.13 4-(1-(1-((3-bromophenyl)sulfonyl) piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]p
yrimidine (8m). White solid; yield: 80%; 1H NMR (400 MHz, DMSO) δ 12.24 (s, 1H), 8.7
1
(s, 1H), 8.68 (s, 1H), 8.34 (s, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.95 (s, 1H), 7.83 (d, J
7.9 Hz, 1H), 7.66 (t, J = 7.9 Hz, 1H), 7.64-7.57 (m, 1H), 7.05 (d, J = 1.9 Hz, 1H), 4.4
=
9
-4.31 (m, 1H), 3.82 (d, J = 11.8 Hz, 2H), 2.57 (dt, J = 19.7, 10.0 Hz, 2H), 2.13 (ddd,
+
o
J = 18.2, 15.4, 8.1 Hz, 4H); MS (ESI) m/z: 487 [M+H] . M.p.: 212~215 C. Anal. Calcd
for C H BrN O S (%): C, 49.29 H, 3.93; N, 16.39. Found (%): C, 49.31; H, 3.90; N, 1
2
0
19
6
2
6
.41.
4
.1.8.14 4-(1-(1-((4-bromophenyl)sulfonyl)piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]py
1
rimidine (8n). White solid; yield: 63%; H NMR (400 MHz, DMSO) δ 12.07 (s, 1H), 8.6
5
(s, 1H), 8.60 (s, 1H), 8.28 (d, J = 6.1 Hz, 1H), 7.90 (d, J = 8.4 Hz, 2H), 7.73 (d, J
8.4 Hz, 2H), 7.56 (s, 1H), 6.99 (s, 1H), 4.36 (t, J = 10.6 Hz, 1H), 3.77 (d, J = 11.6
=
Hz, 2H), 3.17 (d, J = 5.2 Hz, 2H), 2.11 (dd, J = 13.2, 9.8 Hz, 4H); MS (ESI) m/z: 487
+
o
[
M+H] . M.p.: 218~221 C. Anal. Calcd for C H BrN O S (%): C, 49.29 H, 3.93; N, 16
20 19 6 2
.
39. Found (%): C, 49.27; H, 3.90; N, 16.40.
4
.1.8.15 4-(1-(1-((4-fluorophenyl)sulfonyl) piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]py
1
rimidine (8o). White solid; yield: 76%; H NMR (400 MHz, DMSO) δ 12.09 (s, 1H), 8.6
6
(s, 1H), 8.61 (s, 1H), 8.28 (s, 1H), 7.84-7.80 (m, 2H), 7.78-7.74 (m, 2H), 7.57 (dd, J
3.4, 2.5 Hz, 1H), 6.99 (dd, J = 3.5, 1.7 Hz, 1H), 4.46-4.29 (m, 1H), 3.78 (d, J = 12.0
=
Hz, 2H), 2.60-2.51 (m, 2H), 2.23-2.03 (m, 4H); 13C NMR (101 MHz, DMSO) δ 166.40,
1
0
9
6
63.22, 151.35, 138.94, 132.48(2C), 130.05, 129.28, 128.24, 121.28, 117.26, 115.05(2C), 1
-
5.39, 100.30, 57.80, 56.20, 45.44, 31.77, 31.53. MS (ESI) m/z: 425 [M-H] . M.p.: 190~1
o
3 C. Anal. Calcd for C H FN O S (%): C, 56.33 H, 4.49; N, 19.71. Found (%): C, 5
2
0
19
6
2
.31; H, 4.51; N, 19.70.
4
.1.8.16 4-(1-(1-((3,5-difluorophenyl)sulfonyl)piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]p
1
yrimidine (8p). White solid; yield: 63%; H NMR (400 MHz, DMSO) δ 12.06 (s, 1H), 8.
6
5 (s, 1H), 8.62 (s, 1H), 8.28 (s, 1H), 7.74 (t, J = 9.2 Hz, 1H), 7.56 (s, 3H), 6.99 (d, J
2.1 Hz, 1H), 4.38 (t, J = 10.7 Hz, 1H), 3.82 (d, J = 12.0 Hz, 2H), 2.67 (dd, J = 27.1,
=
+
o
1
7.3 Hz,2H), 2.22-2.03 (m, 4H); MS (ESI) m/z: 445 [M+H] . M.p.: 197~200 C. Anal. Ca
lcd for C H F N O S (%): C, 54.05 H, 4.08; N, 18.91. Found: C, 54.07; H, 4.10; N, 18.8
2
0
18
2
6
2
9
.
4
.1.9. Preparation of (4-(1-(1-(2-chloroacetyl)piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-
d]pyrimidin-7-yl)methyl pivalate (9)

To a solvent of (4-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)met
hyl pivalate 6 (2.00 g, 0.0058 mol) in chloroform (20 ml), a solution of 2-chloroacetyl ch
loride (0.70 g, 0.006 mol) in chloroform (10 ml) was added in ice bath. The reaction mix
ture was stirred in ice bath for 30 min, and then filtered to give compound 9 as a white
solid (2,21 g, 82.7%). MS (ESI) m/z: 459 [M+H]+.
4
.1.10. General procedure for the preparation of compounds (10a-10i)
Arious secondary amines (0.0096 mol) were reacted with 9 (1.1 g, 0.0024 mol) in dime
o
thylacetamide (10 ml) at 110 C for 3 h. Subsequently, the mixture was pooled into water
and extracted with DCM (20 mL × 2). The organic layer was washed with water and bri
ne. After dried with anhydrous sodium sulfate, the compound 10a-10i were obtained by co
ncentration
4
.1.10.1. (4-(1-(1-(dimethylglycyl)piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-7
-
yl)methyl pivalate (10a). Yield: 87%; MS (ESI) m/z: 468 [M+H]+.
4
.1.10.2. (4-(1-(1-(diethylglycyl)piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-y
l)methyl pivalate (10b). Yield: 81%; MS (ESI) m/z: 496 [M+H]+.
4
.1.10.3. (4-(1-(1-(2-(pyrrolidin-1-yl)acetyl)piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]p
yrimidin-7-yl)methyl pivalate (10c). Yield: 84%; MS (ESI) m/z: 494 [M+H]+.
4
.1.10.4. (4-(1-(1-(2-(piperidin-1-yl) acetyl) piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]
pyrimidin-7-yl) methyl pivalate (10d). Yield: 83%; MS (ESI) m/z: 508 [M+H]+.
4
2
.1.10.5. (4-(1-(1-(2-(4-methylpiperidin-1-yl)acetyl)piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[
,3-d]pyrimidin-7-yl)methyl pivalate (10e). Yield: 80%; MS (ESI) m/z: 522 [M+H]+.
4
.1.10.6. (4-(1-(1-(2-morpholinoacetyl)piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyrrolo[2,3-d]pyrimi
din-7-yl)methyl pivalate (10f). Yield: 69%; MS (ESI) m/z: 510 [M+H]+.
4
.1.10.7. 2-(4-(1-(1-(2-(4-methylpiperazin-1-yl) acetyl) piperidin-4-yl)-1H-pyrazol-4-yl)-7H-pyr
rolo[2,3-d] pyrimidin-7-yl) ethyl pivalate (10g) Yield: 71%; MS (ESI) m/z: 537 [M+H]+.
4
7
.1.10.8. 2-(4-(1-(1-(2-(4-(2-hydroxyethyl)piperazin-1-yl)acetyl piperidin-4-yl)-1H-pyrazol-4-yl)-
H-pyrrolo[2,3-d]pyrimidin-7-yl)ethyl pivalate (10h). Yield: 70%; MS (ESI) m/z: 567 [M+
H]+.
4
.1.10.9. 1-(2-oxo-2-(4-(4-(7-((pivaloyloxy)methyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol
1-yl)piperidin-1-yl)ethyl)piperidine-4-carboxylic acid (10i). Yield: 67%; MS (ESI) m/z: 552
M+H]+.
-
[
4
.1.11. General procedure for the preparation of compounds (11a-11g)
At ambient temperature, compounds 10a-10i (0.002 mol) were dissolved in methanol (10
ml). A aqueous solution of sodium hydroxide (1.5 g, 0.0096 mol) was added into the reac
tion. After stirred for 1.5 h, the mixture was poured into water and then extracted with D
CM (20 mL × 2). The organic layer was washed with water and brine, dried with anhydr
ous sodium sulfate, and concentrated under reduced pressure. The resulting residue was pu
rified by column chromatography (eluent = dichloromethane:methanol = 50:1) to afford tar

get compounds 11a-11i.
4
.1.11.1. 1-(4-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)piperidin-1-yl)-2-(dimethyla
1
mino)ethan-1-one (11a). Yield: 61%; H NMR (400 MHz, DMSO) δ 12.12 (s, 1H), 8.69 (
s, 2H), 8.33 (s, 1H), 7.59 (dd, J = 3.3, 2.4 Hz, 1H), 7.03 (dd, J = 3.5, 1.5 Hz, 1H), 4.5
9
(dd, J = 9.8, 5.7 Hz, 1H), 4.51 (d, J = 13.1 Hz, 1H), 4.22 (d, J = 13.1 Hz, 1H), 3.42
(s, 4H), 3.22 (d, J = 13.2 Hz, 2H), 3.14 (t, J = 12.6 Hz, 2H), 2.24 (s, 6H), 2.08-1.83 (
+
o
m, 2H); MS (ESI) m/z: 354 [M+H] . M.p.: 197~200 C. Anal. Calcd for C H N O: C, 6
1
8
23
7
1
.17 H,6.56; N, 27.74. Found (%): C, 61.20; H, 6.51; N, 27.76.
4
.1.11.2. 1-(4-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)piperidin-1-yl)-2-(diethylam
1
ino)ethan-1-one (11b). Yield: 50%; H NMR (400 MHz, DMSO) δ 8.78-8.63 (m, 2H), 8.3
4
(d, J = 9.5 Hz, 1H), 7.58 (t, J = 3.6 Hz, 1H), 7.12-6.95 (m, 1H), 4.62 (d, J = 30.0 H
z, 1H), 4.52-4.37 (m, 1H), 4.25 (dd, J = 26.0, 14.1 Hz, 1H), 3.51 (s, 1H), 3.18 (d, J =
3.5 Hz, 1H), 2.88 (t, J = 11.9 Hz, 1H), 2.76 (s, 1H), 2.17 (d, J = 37.0 Hz, 2H), 2.06-1
92 (m, 2H), 1.23 (s, 6H), 1.12-0.78 (m, 4H); 13C NMR (101 MHz, DMSO-d ) δ 167.65(
1
.
6
1
1
3
C), 152.55(1C), 151.39(1C), 150.56(1C), 138.94(1C), 129.51(1C), 127.01(1C), 121.16(1C),
13.25(1C), 100.34(1C), 66.68(2C), 61.48(1C), 58.97(1C), 53.47(2C), 44.53(1C), 33.08(1C),
+
o
2.21(1C). MS (ESI) m/z: 382 [M+H] . M.p.: 149~152 C. Anal. Calcd for C H N O (%
2
0
27
7
): C, 62.97 H, 7.13; N, 25.70. Found (%): C, 63.00; H, 7.15; N, 25.68.
4
.1.11.3. 1-(4-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)piperidin-1-yl)-2-(pyrrolidi
1
n-1-yl ethan-1-one (11c). Yield: 48%; H NMR (400 MHz, DMSO) δ 12.07 (s, 1H), 8.64
(s, 2H), 8.28 (s, 1H), 7.55 (d, J = 2.0 Hz, 1H), 6.98 (d, J = 3.3 Hz, 1H), 4.63-4.51 (m,
1
H), 4.52-4.39 (m, 1H), 4.17 (d, J = 12.8 Hz, 1H), 3.27 (d, J = 13.4 Hz, 4H), 3.16 (t, J
=
=
12.1 Hz, 1H), 2.75 (t, J = 11.8 Hz, 1H), 2.07 (s, 2H), 2.03-1.91 (m, 1H), 1.87 (dt, J
+
11.9, 8.2 Hz, 1H), 1.69 (s, 4H), 1.21 (s, 2H); MS (ESI) m/z: 380 [M+H] . M.p.: 155~
o
1
8
56 C. Anal. Calcd for C H N O (%): C, 63.30 H, 6.64; N, 25.84. Found (%): C, 63.2
2
0
25
7
; H, 6.66; N, 25.86.
4
1
,
.1.11.4. 1-(4-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)piperidin-1-yl)-2-(piperidin-
1
-yl)ethan-1-one (11d). Yield: 52%; H NMR (400 MHz, DMSO) δ 12.09 (s, 1H), 8.66 (s
2H), 8.31 (s, 1H), 7.61-7.54 (m, 1H), 7.03-6.98 (m, 1H), 4.63-4.53 (m, 1H), 4.48 (d, J
=
13.3 Hz, 1H), 4.25 (d, J = 13.3 Hz, 1H), 3.24 (d, J = 13.2 Hz, 1H), 3.17 (t, J = 11.9
Hz, 1H), 3.02 (d, J = 13.1 Hz, 1H), 2.76 (t, J = 11.7 Hz, 1H), 2.36 (s, 4H), 2.11 (t, J
13.2 Hz, 2H), 2.04-1.80 (m, 2H), 1.58-1.46 (m, 4H), 1.38 (d, J = 4.2 Hz, 2H); MS (E
=
+
o
SI) m/z: 393 [M+H ] . M.p.: 153~155 C. Anal. Calcd for C H N O (%): C, 64.10 H, 6
2
1
27
7
.
92; N, 24.92. Found (%): C, 64.09; H, 6.99; N, 25.00.
4
.1.11.5. 1-(4-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)piperidin-1-yl)-2-(4-methylp
1
iperidin-1-yl)ethan-1-one (11e). Yield: 53%; H NMR (400 MHz, DMSO) δ 8.71 (s, 1H),
8
.67 (d, J = 4.3 Hz, 1H), 8.31 (d, J = 4.9 Hz, 1H), 7.62 (d, J = 3.6 Hz, 1H), 7.03 (t, J
3.1 Hz, 1H), 4.64-4.53 (m, 1H), 4.48 (d, J = 13.0 Hz, 1H), 4.19 (dd, J = 18.7, 8.9 H
z, 1H), 3.17 (t, J = 11.8 Hz, 2H), 2.84 (s, 1H), 2.82-2.72 (m, 1H), 2.22-1.95 (m, 5H), 1.
4-1.79 (m, 1H), 1.60 (s, 2H), 1.34 (d, J = 8.4 Hz, 1H), 1.27-1.07 (m, 4H), 0.86 (dd, J
16.4, 6.7 Hz, 3H); 13C NMR (101 MHz, DMSO) δ 167.99, 151.39, 150.49, 147.88, 139.0
, 127.03, 121.08, 113.32, 100.23, 61.99, 58.96, 53.88, 53.50, 46.47, 46.61, 34.33, 33.33,
=
9
=
6

+
o
3
2.37, 30.35, 22.19. MS (ESI) m/z: 408[M+H] . M.p.: 157~160 C. Anal. Calcd for C H
22 2
N O (%): C, 64.84 H, 7.17; N, 24.06. Found (%): C, 64.86; H, 7.20; N, 24.08.
7
9
4
.1.11.6. 1-(4-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)piperidin-1-yl)-2-morpholin
1
oethan-1-one (11f). Yield: 49%; H NMR (400 MHz, DMSO) δ 12.10 (s, 1H), 8.66 (d, J
=
2.0 Hz, 2H), 8.30 (s, 1H), 7.57 (s, 1H), 7.00 (s, 1H), 4.58 (t, J = 11.4 Hz, 1H), 4.48
d, J = 12.7 Hz, 1H), 4.21 (d, J = 12.6 Hz, 1H), 3.59 (s, 4H), 3.29 (d, J = 13.5 Hz, 1
H), 3.26-3.05 (m, 2H), 2.77 (t, J = 12.1 Hz, 1H), 2.43 (s, 4H), 2.10 (d, J = 14.6 Hz, 2
(
+
o
H), 2.05-1.80 (m, 2H); MS (ESI) m/z: 396 [M+H ] . M.p.: 164~166 C. Anal. Calcd for
C H N O (%): C, 60.74 H, 6.37; N, 24.79. Found (%): C, 60.79; H, 6.40; N, 24.81.
2
0
25
7
2
4
.1.11.7. 1-(4-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)piperidin-1-yl)-2-(4-methylp
1
iperazin-1-yl)ethan-1-one (11g). Yield: 47%; H NMR (400 MHz, DMSO) δ 12.13 (s, 1H),
8
.68 (d, J = 3.7 Hz, 2H), 8.33 (d, J = 5.1 Hz, 1H), 7.58 (d, J = 3.3 Hz, 1H), 7.01 (d,
J = 3.5 Hz, 1H), 4.58 (td, J = 11.3, 5.6 Hz, 1H), 4.49 (d, J = 13.1 Hz, 1H), 4.23 (d, J
13.3 Hz, 1H), 3.50 (t, J = 6.2 Hz, 3H), 3.36-3.25 (m, 1H), 3.18 (dd, J = 13.5, 10.4 H
z, 1H), 3.08 (t, J = 8.2 Hz, 1H), 2.78 (t, J = 11.9 Hz, 1H), 2.44 (s, 4H), 2.38 (t, J = 6
3 Hz, 4H), 2.12 (t, J = 12.1 Hz, 2H), 2.07-1.97 (m, 1H), 1.89 (qd, J = 12.1, 4.0 Hz, 1
=
.
+
o
H); MS (ESI) m/z: 409 [M+H] . M.p.: 159~161 C. Anal. Calcd for C H N O (%): C, 6
2
1
28
8
1
.74 H, 6.91; N, 27.43. Found (%): C, 61.79; H, 6.90; N, 27.41.
4
.1.11.8. 1-(4-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)piperidin-1-yl)-2-(4-(2-hydr
1
oxyethyl)piperazin-1-yl)ethan-1-one (11h). Yield: 40%; H NMR (400 MHz, DMSO) δ 12.1
0
(s, 1H), 8.66 (d, J = 2.9 Hz, 2H), 8.31 (s, 1H), 7.57 (s, 1H), 7.00 (d, J = 2.1 Hz, 1H
)
, 4.58 (t, J = 11.3 Hz, 1H), 4.48 (d, J = 12.7 Hz, 1H), 4.23 (d, J = 13.1 Hz, 1H), 3.50
3.41 (m, 2H), 3.27 (d, J = 13.3 Hz, 1H), 3.17 (t, J = 12.1 Hz, 1H), 3.07 (d, J = 13.2
-
Hz, 1H), 2.81 (s, 2H), 2.75 (d, J = 13.8 Hz, 1H), 2.12 (t, J = 20.2 Hz, 2H), 2.00 (d, J
11.0 Hz, 2H), 1.97-1.79 (m, 2H), 1.57 (d, J = 10.4 Hz, 2H), 1.30 (s, 2H), 1.15-1.01 (
=
+
o
m, 2H); MS (ESI) m/z: 439 [M+H] . M.p.: 182~187 C. Anal. Calcd for C H N O (%):
2
2
30
8
2
C, 60.26 H, 6.90; N, 25.55. Found (%): C, 60.26; H, 6.93; N, 25.51.
4
.1.11.9. 1-(2-(4-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)piperidin-1-yl)-2-oxoethy
1
l)piperidine-4-carboxylic acid (11i). Yield: 42%; H NMR (400 MHz, DMSO) δ 12.07 (s,
1
H), 8.63 (d, J = 3.6 Hz, 2H), 8.26 (s, 1H), 7.53 (d, J = 3.5 Hz, 1H), 6.96 (d, J = 3.6
Hz, 1H), 4.64-4.51 (m, 2H), 4.45 (d, J = 12.7 Hz, 1H), 4.12 (s, 2H), 3.81 (d, J = 13.3
Hz, 1H), 3.13 (t, J = 12.0 Hz, 1H), 2.80 (t, J = 12.1 Hz, 1H), 2.07 (d, J = 10.5 Hz, 2
+
H), 1.93 (ddd, J = 30.8, 19.9, 9.6 Hz, 3H), 1.19 (s, 4H); MS (ESI) m/z: 438 [M+H] . M.p
o
.
: 179~183 C. Anal. Calcd for C H N O (%): C, 57.52 H, 5.98; N, 25.55. Found (%):
2
1
26
8
3
C, 57.55; H, 6.00; N, 25.51.
4
4
.2 Biological assay
.2.1. In vitro protein kinase inhibition against JAKs
The inhibitory effects of target compounds against human JAK1 were evaluated by surveying
the phosphorylation of Ulight-CAGAGAIETDKEYYTVKD (JAK1) substrate, employing a
human recombinant enzyme and the LANCE detection assay. The target compound, reference
compound or water (control) were blended with the enzyme (4.4 ng) in a buffer (40 mM

Hepes/Tris pH 7.4, 8 mM MgCl , 0.8 mM EGTA/Tris, 0.008% Tween 16 and 3.6 mM DTT). In
2
sequence, 25 μM ATP and 100 nM of the substrate Ulight-CAGAGAIETDKEYYTVKD (JAK1)
were added, and the mixture was incubated at ambient temperature for 60 min. Then the enzyme
was filtered from the reaction mixture. After incubation, 13 mM EDTA was added to stop the
reaction. In 5 min, the anti-phopho-PT66 antibody labeled by europium chelate was mixed. 60
more min later, the fluorescence transfer was tested at ex = 337 nm, em = 616 nm and em = 665
nm using a microplate reader (Envision, Perkin Elmer). The enzyme activities were detected by
dividing the signal at 665 nm by that at 616 nm (ratio). The results were summerized as a percent
inhibition, taking staurosporine as the standard inhibitory reference. The inhibitory effects of
target compounds against human JAK2 were evaluated by surveying the phosphorylation of
Ulight-CAGAGAIETDKEYYTVKD (JAK2) substrate, employing a human recombinant enzyme
and the LANCE detection assay. The process was similar to the determination of inhibition of
JAK1. In the assay, 10 μM ATP and enzyme (0.96 ng) were improved.
4
.2.2 Nitric oxide determination
5
RAW264.7 cells (5 × 10 /well) were seeded in 96-well culture plates and were pre-treated with
or without target compounds (2.5, 5, 10 μg/ml) for 2 h. Then, the cells except control group were
incubated with LPS (5 μg/ml) for 18 h. The supernatant was collected for nitric oxide detection by
Griess reagent (Nan Jing Jian Cheng biochemical company). Following the instruction, Griess
reagentⅠ(50 μl) and Ⅱ (50 μl) were reacted with equal volume of cell culture media (50 μl)
successively. The absorbance was measured at 540 nm.
4
.2.3 MTT analysis
4
RAW264.7 cells (5 × 10 ) were plated into 96-well plates and incubated overnight at 37 °C in
5
% CO . The cells were treated with or without serial concentration of corresponding compounds
2
(15, 25, 50, 75, 100, 150, 200 μM) for 24 h and then collected for MTT analysis. After washed
three times by PBS, cells were incubated in 100 µL of 0.5 mg/mL MTT solution at 37 °C for 3 h.
The residual cell layer was dissolved in 150 µL of DMSO, and the absorbency for formazan
crystals was measured at 490 nm wavelength using a microplate reader (Thermo Scientific,
Shanghai, China). Inhibition ratio was calculated. The results which expressed as IC50 was
statistical computing by computer software.
Inhibition ratio (%) = (A492control - A492sample)/ (A492control - A492blank)  100%
4
.2.4 Cellular morphology Observation
5
RAW264.7 cells (5 × 10 /well) were seeded in 96-well culture plates and were pre-treated with
or without corresponding target compounds (10 μg/ml) for 2 h. Then, the cells except control
group were incubated with LPS (5 μg/ml) for 18 h. Then the cells were examined under a light
microscope for cellular morphology evaluation.
4
.2.5 Western blot analysis
6
The RAW264.7 cells (5 × 10 ) were harvested and then lysed in lysis buffer (50 mM Hepes pH
.4, 1% Triton X-100, 2 mM sodium orthovanadate, 100 mM sodium fluoride, 1 mM EDTA, 1
7
mM EGTA, 1 mM PMSF, 10 µg/ml aprotinin and 10 µg/ml leupeptin) at 4 °C for 60 min. The
lysate was centrifuged at 15,000 × g for 10 min and the supernatants were used for Western blot

analysis. Equal amounts of total protein were separated by 12% SDS PAGE and electro-
transferred to a PVDF membrane (Bio-Rad). Antibodies against JAK1 (1:1000), JAK2 (1:1000),
Pro IL-1β (1:500), and β-actin (1:2000) overnight at 4°C, followed by horseradish peroxidase
(
HRP)-conjugated secondary antibody and visualization was performed with chemiluminescence
ECL) detection kit followed by autoradiography.
(
Acknowledgements
This work was supported by Provincial Natural Science Foundation of Liaoning
(No.1552626507244).
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Research highlights
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Novel pyrrolo[2,3-d]pyrimidin derivatives were designed and synthesized.
Most compounds exerted over 90% inhibition rates against JAK1 and JAK2.
11e showed compelling anti-inflammatory efficacy on LPS-induced RAW264.7 cells.
11e revealed low cytotoxity by IC value of 88.2 μM against normal RAW264.7 cells.
5
0
11e significantly down-regulating high expression of JAK1, JAK2 and pro IL-1β.