Discovery and biological evaluation of a series of pyrrolo[2,3-b]pyrazines as novel FGFR inhibitors

Article abstract of DOI:10.3390/molecules22040583

Abnormality of fibroblast growth factor receptor (FGFR)-mediated signaling pathways were frequently found in various human malignancies, making FGFRs hot targets for cancer treatment. To address the consistent need for a new chemotype of FGFR inhibitors, here, we started with a hit structure identified from our internal hepatocyte growth factor receptor (also called c-Met) inhibitor project, and conducted a chemical optimization. After exploring three parts of the hit compound, we finally discovered a new series of pyrrolo[2,3-b]pyrazine FGFR inhibitors, which contain a novel scaffold and unique molecular shape. We believe that our findings can help others to further develop selective FGFR inhibitors.

Full text of DOI:10.3390/molecules22040583

molecules
Article
Discovery and Biological Evaluation of a Series of
Pyrrolo[2,3-b]pyrazines as Novel FGFR Inhibitors
Yan Zhang 1,2, Hongchun Liu 2, Zhen Zhang 3,4, Ruifeng Wang 3,4, Tongchao Liu 3,4
,
Chaoyun Wang 1, Yuchi Ma 3,*, Jing Ai 2,*, Dongmei Zhao 4, Jingkang Shen 3 and Bing Xiong 3,
*
1
School of Pharmaceutical Sciences, Binzhou Medical University, Yantai 264003, Shandong, China;
zhangyanzy2014@126.com (Y.Z.); ytwcy@163.com (C.W.)
2
Division of Anti-Tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia
Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China; hchliu@simm.ac.cn
Department of Medicinal Chemistry, State Key Laboratory of Drug Research, Shanghai Institute of Materia
Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China;
18204012193@163.com (Z.Z.); rfwang1992@126.com (R.W.); Tongchao_Liu@163.com (T.L.);
jkshen@simm.ac.cn (J.S.)
Key Laboratory of Structure-Based Drug Design and Discovery of Ministry of Education,
Shenyang Pharmaceutical University, Shenyang 110016, China; dongmeiz-67@163.com
Correspondence: ychma@simm.ac.cn (Y.M.); jai@simm.ac.cn (J.A.); bxiong@simm.ac.cn (B.X.);
Tel.: +86-21-5080-6600 (ext. 5412) (Y.M. & B.X.); +86-21-5080-6600 (ext. 2413) (J.A.);
Fax: +86-21-5080-7088 (Y.M. & B.X.)
3
4
*
Academic Editors: Pierre Koch and Stefan Laufer
Received: 26 January 2017; Accepted: 3 April 2017; Published: 5 April 2017
Abstract: Abnormality of fibroblast growth factor receptor (FGFR)-mediated signaling pathways were
frequently found in various human malignancies, making FGFRs hot targets for cancer treatment.
To address the consistent need for a new chemotype of FGFR inhibitors, here, we started with a hit
structure identified from our internal hepatocyte growth factor receptor (also called c-Met) inhibitor
project, and conducted a chemical optimization. After exploring three parts of the hit compound,
we finally discovered a new series of pyrrolo[2,3-b]pyrazine FGFR inhibitors, which contain a novel
scaffold and unique molecular shape. We believe that our findings can help others to further develop
selective FGFR inhibitors.
Keywords: FGFR1; inhibitor; kinase inhibitor; pyrazine; hinge binder
1. Introduction
In the human genome, a total of 22 fibroblast growth factors (FGFs) have been identified,
and there are four fibroblast growth factor receptors (FGFRs) that respond to and mediate the
signaling from fibroblast growth factors [
FGFR signaling pathways in the regulation of several basic biologic processes, including tissue
development, angiogenesis, and tissue regeneration [ ]. Not surprisingly, abnormal signaling
involving FGFRs has been frequently found in various human malignancies, making FGFRs hot
targets in anticancer drug development [ ]. Aberrant FGFR signaling could be caused by different
1–4]. Increasing evidence highlights the importance of
5,6
7–9
mechanisms, including activating mutations in FGFRs, oncogenic fusion of FGFRs and over-expression
of FGFRs [10]. Currently, several FGFR-targeted agents, mostly small molecules binding to the kinase
domain, are being evaluated in clinical trials for cancer treatment, and the most intriguing and
advanced evaluations are for FGFR-selective inhibitors, such as AZD4547 (
and JNJ-42756493 ( ) (Figure 1) [13]. As investigated by Patani et al. [14], due to the landscape of
activated mutations in FGFR kinases, the above-mentioned FGFR inhibitors ( ) exhibited distinct
1) [11], NVP-BGJ398 (2) [12]
3
1, 2, 3
Molecules 2017, 22, 583; doi:10.3390/molecules22040583
www.mdpi.com/journal/molecules

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effects on different mutants, which indicates that developing FGFR inhibitors with novel scaffolding
are highly in demand, as they may provide unique therapeutic benefits for certain patients.
Figure 1. Representative selective fibroblast growth factor receptor inhibitors.
In the development of tyrosine kinase inhibitors, we previously synthesized a series of
1-sulfonylpyrazolo[4,3-b]pyridines as potent and selective c-Met (hepatocyte growth factor receptor)
inhibitors [15]. Since c-Met and FGFR all belong to the receptor tyrosine kinase subfamily, we also
tested a subset of this series of compounds in a fibroblast growth factor receptor 1 (FGFR1) enzymatic
assay and serendipitously found that compound
4 has weak but definite FGFR1 activity, with an
87.8% inhibition ratio towards FGFR1 at 10 M concentration. Starting from this compound, in the
µ
current study, we present our structure optimization and elaborated the structure–activity relationship
(SAR) of this novel scaffold, in the hope that the investigation can stimulate new ideas for developing
selective FGFR inhibitors as anticancer drugs.
2. Results and Discussion
As reported in c-Met inhibitor development [15], the 1-sulfonylpyrazolo[4,3-b]pyridine acts as
the hinge binder, and the benzene group of compound
Tyr1230 in the c-Met ATP binding site. Since compound
view of chemical ring system and from the shape of the structure, we need to probe its binding
mode. Therefore, we performed a docking study to predict the binding mode of compound in the
4
forms a
π–π interaction with the residue
4
is a novel FGFR inhibitor, both from the
4
FGFR1 ATP binding site prepared from a PDB (Protein Data Bank) structure (PDB code: 3TT0) [16].
As shown in Figure 2, the docking results indicated that the pyrazolo[4,3-b]pyridine ring still acts as
the hinge binder to anchor the molecule in the ATP binding site of FGFR1. Therefore, the benzene
group is located at the middle of the ATP binding site and the linking sulfamide group makes it
perpendicular to the pyrazolo[4,3-b]pyridine. We inspected the predicted complex structure of
bound to FGFR1 kinase and did not find any possible residues could form the interaction with
the benzene group of compound . Then, we designed three other compounds by modifying the
connecting group of sulfamide. As shown in Table 1, three compounds showed moderate inhibitions
towards FGFR1, but the activities are all inferior to the starting compound . This indicated that the
nearly right angle between the benzene group and pyrazolo[4,3-b]pyridine ring is important to the
4
π–π
4
4
binding affinity. Together with the unique shape of compound
group for late optimization.
4, we wanted to retain this connecting

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Figure 2. The docking study of compound 4 in FGFR1 binding site. The protein structure was extracted
from the crystal structure (PDB code: 3TT0) and is shown in green, while compound
with a stick model and in cyan.
4 was depicted
Table 1. Structures and FGFR1 enzymatic activities of compounds 4–7 a.
FGFR1 Inhibition (%) b
Compound No.
4
Structure
10 µM
87.8
5
6
46.1
53
7
55.9
a
In our enzymatic assay, the IC50 value of AZD4547 on FGFR1 is 1.8
Inhibition values are given as the mean (%) from two separate experiments. FGFR1: fibroblast growth factor
±
0.1 nM (mean
±
standard deviation).
b
receptor 1.
Most of the tyrosine kinase inhibitors comprise a hydrogen bonding acceptor to interact with
the hinge part of the kinase domain, which is to mimic the binding pattern of the adenine group
of the ATP molecule [17]. Since the hinge binder forms hydrogen bonding and Van der Waals
interactions with the binding site residues, it makes a great contribution to anchoring the whole
inhibitor in the kinase domain. Therefore, we modified it with the aim of improving the binding
activity. We retained the important nitrogen atom as a hydrogen bond receptor to the backbone of
the residue ALA564 and synthesized three compounds listed in Table 2. Changing the scaffold into
the 1H-pyrrolo[3,2-b]pyridine ring (
5H-pyrrolo[2,3-b]pyrazine ( ) dramatically increase the binding activity. Even at a concentration of
M, it still showed more than 90% inhibition in the FGFR1 enzymatic assay [18 19]. We also changed
8) slightly decreased the binding activity, while changing into
9
1
µ
,
the bicyclic scaffold into monocycle ring by opening the pyrrole (10), and the activity dropped by
about 10-fold, as indicated by the inhibition ratio in Table 2.

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Table 2. Structures and FGFR1 enzymatic activities of compounds 8–10.
FGFR1 Inhibition (%)
Compound No.
4
Structure
10 µM
1 µM
87.8
47.1
8
9
80.4
93.3
31.6
92.5
10
50.0
15.4
Although we did not observe any possible residues could make direct interaction with the benzene
group of compound by looking at the cocrystal structure (PDB code: 3TT0), we are aware that the
protein kinase domain has a complex conformational landscape, which has been extensively studied
via crystal structure analyses and computer simulations [20 22]. Then, by analogy to developing
4
–
c-Met inhibitors, we optimized the benzene group and synthesized over a dozen compounds listed
in Table 3, and indeed found that this part is critical to the binding affinity. Substitution of the
benzene group with saturated cyclopentane (11) or cyclohexane (12) dramatically decreased the
binding activity. Generally, the electronic properties of substituents on benzene group are not essential
to the binding, if comparing compounds 17 and 19 to compound 9. These compounds are showing
similar sub-micromolar enzymatic activities. By scrutiny of the structure–activity relationship, we
found that the steric characteristics may be more important, and the meta position on the benzene
sulfamide is favorable for the binding, which is evident from the comparison of compounds 17 and 18
or 19 and 20. The slightly large substitution such as acetyl group (21) on meta position of benzene is
also tolerated, while larger propionyl group (22) decreased the activity. These reinforce that the steric
characteristics of the inhibitors are vital to the binding interactions. From the exploration of this part,
we found that the compound containing nitrobenzene group (17) stood out as a potent inhibitor with
enzymatic activity about 85 nM. Then, this substructure pattern was retained and the methyl pyrazole
group was subjected to further optimization.
,
To probe the SAR around the methyl pyrazole part, we synthesized analogs 23–25 by substituting
the methyl group with different acyl groups. When R group is 1-(1H-pyrazol-1-yl)ethan-1-one (23) or
1-(1H-pyrazol-1-yl)propan-1-one (24), it showed reduced activity, giving less than a 40% inhibition ratio
at the concentration of 0.1
µ
M (Table 4). While the compound with 1-(1H-pyrazol-1-yl)propan-2-one
25) has the similar activity to compound 17. Modifying the methyl pyrazole to benzene groups
26 27) follows a similar trend, in that the phenylmethanol substitution (27) is more active than the
(
(
,
acetophenone substitution (26). In general, the modification at the methyl pyrazole part did not
improve the binding activity, and only 25 and 27 were found to have a similar potency to compound
17, with IC50 values of 45 nM and 113 nM, respectively.

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Table 3. Structures and FGFR1 enzymatic activities of compounds 11–22.
FGFR1 Inhibition (%)
Compound No.
11
R
10 µM
1 µM
0.1 µM
34.7
20.8
26.8
12
9
53.3
90.5
24.1
63.4
29.4
46
13
14
15
59.7
45.2
55.6
32.6
27.2
31.2
40.3
22
23.6
16
17
18
19
74.5
92.1
15.5
82.2
34.8
76.4
12.8
57.5
13.6
61.1
17.4
34.7
20
21
22
42.4
79
24.2
50.9
17.3
31.8
44.7
27.7
38.7
Since this series of compounds started from the project of developing c-Met inhibitors, we wanted
to assess the selectivity of this series. Therefore, three potent compounds were picked to test in
c-Met enzymatic assay. As shown in Table 5, three compounds demonstrated nearly no inhibition
towards c-Met even at concentration of 1
FGFR inhibitors.
µM, indicating compounds of this series may be selective

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Table 4. Structures and FGFR1 enzymatic activities of compounds 23–28.
FGFR1 Inhibition (%)
Compound No.
17
R
1 µM
0.1 µM
0.01 µM
76.4
61.1
ND
23
24
25
58.8
60.9
63.4
37.8
30.2
55.7
36.5
29.3
32.8
26
27
57.9
75.5
35.7
52.2
ND
ND
28
62.4
34
27.2
Note: ND = not determined.
Table 5. FGFR1 and c-Met enzymatic activities of compounds 17, 25 and 27.
c-Met Inhibition (%)
FGFR1 IC50 (nM) a
Compound No.
1 µM
0.1 µM
0.01 µM
17
25
27
85.9 ± 21.2
45.9 ± 14.5
113.8 ± 5.8
30.6
14.1
30.1
−1.5
−5.4
5.0
5.5
8.4
1.1
a
IC50 values are given as the mean
±
SD (nM) from two separate experiments. c-Met: hepatocyte growth
factor receptor.
3. Materials and Methods
3.1. Elisa Kinase Assay
The effects of compounds on the activities of indicated (FGFR1 and c-Met) kinases were
determined using enzyme-linked immunosorbent assays (ELISAs) with purified recombinant

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proteins [19]. Briefly, 20
µg/mL poly (Glu, Tyr)4:1 (Sigma, St. Louis, MO, USA) was pre-coated
in 96-well plates as a substrate. A 50-
buffer (50 mmol/L HEPES (pH 7.4), 50 mmol/L MgCl2, 0.5 mmol/L MnCl2, 0.2 mmol/L Na3VO4, and
1 mmol/L DTT) was added to each well; 1 L of various concentrations of compounds diluted in 1%
µL aliquot of 10 µmol/L ATP solution diluted in kinase reaction
µ
DMSO (v/v) (Sigma) were then added to each reaction well. DMSO (1%, v/v) was used as the negative
control. The kinase reaction was initiated by the addition of purified tyrosine kinase proteins FGFR1
(Millipore, Darmstadt, Germany) or c-Met (Millipore) diluted in 49
incubation for 60 min at 37 C, the plate was washed three times with phosphate-buffered saline (PBS)
containing 0.1% Tween 20 (T-PBS). Anti-phosphotyrosine (PY99) antibody (Sanra Cruz, Dallas, USA)
µ
L of kinase reaction buffer. After
◦
(100
µ
L; 1:500, diluted in 5 mg/mL bovine serum albumin (BSA) T-PBS) was then added. After a 30-min
◦
incubation at 37 C, the plate was washed three times, and 100
µL horseradish peroxidase-conjugated
goat anti-mouse IgG (Calbiochem, Shanghai, China) (1:2000, diluted in 5 mg/mL BSA T-PBS) was
added. The plate was then incubated at 37◦C for 30 min and washed 3 times. A 100-
L aliquot of a
solution containing 0.03% H2O2 and 2 mg/mL o-phenylenediamine in 0.1 mol/L citrate buffer (pH
5.5) was added. The reaction was terminated by the addition of 50 L of 2 mol/L H2SO4 as the
µ
µ
color changed, and the plate was analyzed using a multi-well spectrophotometer (SpectraMAX 190,
Molecular Devices, Palo Alto, CA, USA) at 490 nm. The inhibition rate (%) was calculated using the
following equation:
IR = [1 − (A490/A490control)] × 100%.
(1)
where IR, A490 and A490control are the inhibition rate, the absorbance value of the tested compound at
490 nm and the absorbance value of the negative control compound at 490 nm, respectively. The IC50
values were calculated from the inhibition curves in two separate experiments.
3.2. Docking Study
The BGJ398-bound FGFR1 complex structure (PDB code: 3TT0) was downloaded from the
PDB database, and prepared with protein preparation module in Schrödinger software package
(Schrödinger, New York City, NY, USA). Then the Glide software (Schrödinger) was used to build the
grid file within FGFR1 ATP binding site. Compound 4 was minimized with an OPLS2015 force field,
and then it was docked into the FGFR1 ATP site with default XP precision parameters implemented in
Glide software. Finally, the best predicted binding conformation of compound 4 was illustrated with
the Pymol program (Schrödinger).
3.3. Chemistry
Compounds
coupling of commercially available 29 with 1-methylpyrazole-4-boronic acid pinacol ester (30) provided
31. Compounds were prepared by deprotonation of compound 31 followed by addition of the
4–7 were synthesized according to the procedures outlined in Scheme 1. Suzuki
4–7
corresponding electrophile reagents.
Scheme 1. The synthesis route of compounds 4–7. Reagents and conditions: (
Dioxane:H2O (v/v = 4:1), 80 C, 2 h, 86% yield; (b) NaH, Dimethylformamide (DMF), R-SO2Cl, room
a
) Pd(dppf)Cl2, K2CO3,
◦
temperature (r.t.), 1–4 h, 76–83% yield.

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Compounds
8
and
9
were prepared according to the procedure in Scheme 2. Treatment of
compounds 32 and 33 with 30 via Suzuki coupling afforded compounds 34 and 35 which were
sulfonylated to afford 8 and 9, respectively.
Scheme 2. The synthesis route of compounds
8
and
9
. Reagents and conditions: (
a
) Pd(dppf)Cl2,
K2CO3, Dioxane:H2O (v/v = 4:1), 80 ◦C, 3 h, 89% yield (34), 82% yield (35); (
benzenesulfonyl chloride, r.t., 2 h, 82% yield (8), 79% yield (9).
b) NaH, DMF,
Compound 10 was prepared according to Scheme 3. Commercially available 5-bromo-2-
methylpyridin-3-amine (36) were sulfonylated to afford 37. Conventional Suzuki coupling of 37
and 1-methylpyrazole-4-boronic acid pinacol ester (30) afforded compound 10.
Scheme 3. The synthesis route of compound 10. Reagents and conditions: (
a) benzenesulfonyl chloride,
NaH, DMF, r.t., 85% yield; (◦
b) 1-Methylpyrazole-4-boronic acid pinacol ester, Pd(dppf)Cl2, K2CO3,
Dioxane:H2O (v/v = 4:1), 80 C, 3 h, 80% yield.
Compounds 11
33 was first treated via Suzuki coupling then sulfonylated with corresponding sulfonyl chlorides
to afford compounds 11 22 Correspondingly, compound 33 firstly was sulfonylated with
–28 were prepared according to the procedures outlined in Scheme 4. Compound
–
.
3-nitrobenzenesulfonyl chloride (38) then treated via Suzuki coupling with corresponding boric acid
or boric acid ester to afford compounds 23–28.
Scheme 4. The synthesis route of compounds 11
Dioxane:H2O (v/v = 4:1), 80 C, 3 h, 82% yield; (
–
28. Reagents and conditions: (
a) Pd(dppf)Cl2, K2CO3,
◦
b) NaH, DMF, R-SO2Cl, r.t., 1–4 h, 72–87% yield;
◦
(
c) NaH, DMF, r.t., 2 h, 80% yield; (
d) Pd(dppf)Cl2, K2CO3, Dioxane:H2O (v/v = 4:1), 80 C, 2–4 h,
81–92% yield.

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1H-NMR (400 MHz) and 13C-NMR (125 MHz or 151 MHz) spectra were recorded by using a Varian
Mercury-400, a Mercury-500 and a Mercury-600 High Performance Digital FT-NMR spectrometer
(Varian, Palo Alto, CA, USA) with tetramethylsilane (TMS) as an internal standard. Abbreviations for
peak patterns in NMR spectra: br = broad, s = singlet, d = doublet, and m = multiplet. Low-resolution
mass spectra were obtained with a Finnigan LCQ Deca XP mass spectrometer using a CAPCELL PAK
C18 (50mm
C18 (50 mm
mode. Purity of all compounds was determined by analytical Gilson high-performance liquid
chromatography (HPLC) using an YMC ODS3 column (50 mm 4.6 mm, 5 ZM). Conditions were
as follows: CH3CN/H2O eluent at 2.5 mL
min−1 flow (containing 0.1% trifluoroacetic acid (TFA)) at
×
2.0 mm, 5 ZM) (Finnigan, Palo Alto, CA, USA) or an Agilent ZORBAX Eclipse XDB
×
2.1 m, 5 ZM) (Agilent, Santa Clara, CA, USA) in positive or negative electrospray
×
·
35 ◦C, 8 min, gradient 5% CH3CN to 95% CH3CN, monitored by UV absorption at 214 nm and 254
nm. TLC analysis was carried out with glass precoated silica gel GF254 plates (Jiangyou, Qingdao,
Shandong, China). TLC spots were visualized under UV light. Flash column chromatography was
performed with a Teledyne ISCO CombiFlash Rf system (Teledyne, Huntsville, AL, USA). All solvents
and reagents were used directly as obtained commercially unless otherwise noted. Anhydrous
dimethylformamide was purchased from Acros (Shanghai, China) and was used without further
drying. All air and moisture sensitive reactions were carried out under an atmosphere of dry Argon
with heat-dried glassware and standard syringe techniques.
6-(1-Methyl-1H-pyrazol-4-yl)-1H-pyrazolo[4,3-b]pyridine (31). A solution of 6-bromo-1H-pyrazolo
[4,3-b]pyridine (29) (330mg, 1.52 mmol), 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
1H-pyrazole (30) (378 mg, 1.82 mmol), PdCl2(dppf)-CH2Cl2 (61.9 mg, 80 µmol) and K2CO3 (628
mg, 4.54 mmol) in 1,4-dioxane:water (15 mL, 2:1, v) in a microwave tube was flushed with N2 for 5
min then sealed. The tube was heated at 80 ◦C for 2 h. Then the reaction mixture was evaporated to
dryness. The residue was purified by flash chromatography to give 31 (260 mg, 86% yield); LC–MS
m/z (ESI) found (M + H)+ 200.0 (M + H)+; 1H-NMR (400 MHz, DMSO-d6)
δ13.29 (s, 1H), 8.80 (s, 1H),
8.36 (s, 1H), 8.24 (s, 1H), 8.07 (s, 2H), 3.90 (s, 3 H).
6-(1-Methyl-1H-pyrazol-4-yl)-1-(phenylsulfonyl)-1H-pyrazolo[4,3-b]pyridine (4). Sodium hydride (29 mg,
1.2 mmol) was suspended in 3 mL of anhydrous DMF. 6-(1-methyl-1H-pyrazol-4-yl)-1H-pyrazolo
[4,3-b]pyridine (31) (160 mg, 0.8 mmol) was dissolved in 5 mL of anhydrous DMF was slowly added
dropwise, and the mixture was stirred for 30 min benzenesulfonyl chloride (214 mg, 0.94 mmol)
dissolved in 5 mL of anhydrous DMF was slowly added in drops, and the mixture was stirred for
4 h at room temperature. The reaction solution was poured to 0.1 N hydrochloric acid, which was
then brought to basic using aqueous sodium bicarbonate solution, and extracted with ethyl acetate.
The organic layer was collected, and distilled under reduced pressure. The remaining substance was
purified by flash column chromatography to give
H)+ 340.0 (M + H)+; 1H-NMR (400 MHz, CDCl3)
4
δ
(224 mg, 82% yield); LC-MS m/z (ESI) found (M +
8.84 (d, J = 1.7 Hz, 1 H), 8.49 (s, 1 H), 8.38 (s, 1 H),
8.02 (d, J = 7.4 Hz, 2 H), 7.94 (s, 1H), 7.86 (s, 1H), 7.62 (t, J = 7.4 Hz, 1 H), 7.51 (t, J = 7.4 Hz, 2 H), 4.03 (s,
3 H). 13C-NMR (126 MHz, CDCl3)
128.85, 128.03, 127.72 (C × 2), 119.32, 116.03, 39.38. Retention time 2.95 min, >98% purity.
Compounds 5–7 were prepared with a similar procedure as that used for 4.
δ
146.75, 142.02, 141.60, 137.29, 137.20, 134.61, 134.41, 129.42 (C 2),
×
1-(Benzylsulfonyl)-6-(1-methyl-1H-pyrazol-4-yl)-1H-pyrazolo[4,3-b]pyridine (
(M + H)+ 354.1 (M + H)+; 1H-NMR (400 MHz, CDCl3)
8.72 (d, J = 1.9 Hz, 1H), 8.50 (d, J = 0.8 Hz,
1H), 7.74 (d, J = 0.8 Hz, 1H), 7.69 (dd, J = 1.9, 0.8 Hz, 1H), 7.67 (s, 1H), 7.11–7.03 (m, 3H), 6.97 (dd,
J = 7.9, 1.6 Hz, 2H), 4.70 (s, 2H), 4.00 (s, 3H). 13C-NMR (126 MHz, CDCl3)
146.47, 141.71, 140.45,
137.16, 136.00, 130.55 (C 2), 129.49, 128.56 (C 2), 128.37, 127.73, 126.14, 119.12, 115.27, 60.31, 39.32.
Retention time 2.97 min, >98% purity.
5). LC–MS m/z (ESI) found
δ
δ
×
×
1-Benzyl-6-(1-methyl-1H-pyrazol-4-yl)-1H-pyrazolo[4,3-b]pyridine (
290.1 (M + H)+; 1H-NMR (400 MHz, CDCl3)
8.73 (d, J = 1.8 Hz, 1H), 8.28 (d, J = 0.9 Hz, 1H), 7.81
6
). LC–MS m/z (ESI) found (M + H)+
δ

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(s, 1H), 7.72 (s, 1H), 7.64–7.59 (m, 1H), 7.38–7.30 (m, 3H), 7.25–7.21 (m, 2H), 5.64 (s, 2H), 4.00 (s, 3H).
13C-NMR (126 MHz, CDCl3)
140.67, 140.55, 137.55, 137.52, 137.10, 135.76, 131.18, 128.82 (C 2),
128.54, 127.92, 127.73 (C × 2), 121.94, 101.51, 47.90, 39.22. Retention time 3.05 min, 98.25% purity.
δ
×
6-(1-Methyl-1H-pyrazol-4-yl)-1-phenethyl-1H-pyrazolo[4,3-b]pyridine ( ). LC–MS m/z (ESI) found (M +
H)+ 304.1 (M + H)+; 1H-NMR (400 MHz, CDCl3)
8.74 (d, J = 1.9 Hz, 1H), 8.06 (d, J = 1.0 Hz, 1H), 7.96
(s, 1H), 7.88 (s, 1H), 7.75 (s, 1H), 7.32–7.24 (m, 3H), 7.14–7.10 (m, 2H), 4.68 (t, J = 7.3 Hz, 2H), 4.01 (s,
3H), 3.35 (t, J = 7.3 Hz, 2H). 13C-NMR (126 MHz, CDCl3)
153.72, 147.61, 145.87, 142.01, 140.34, 139.20,
7
δ
δ
137.77, 137.60, 136.48, 129.91, 129.02, 120.95, 110.86, 107.81, 105.68, 64.3, 39.36, 34.2. Retention time
3.08 min, >98% purity.
6-(1-Methyl-1H-pyrazol-4-yl)-1H-pyrrolo[3,2-b]pyridine (34). A solution of 6-bromo-1H-pyrrolo[3,2-b]
pyridine (32) (100 mg, 0.51 mmol), 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-
pyrazole (30) (116 mg, 0.56 mmol), PdCl2(dppf)-CH2Cl2 (37 mg, 50 µmol) and K2CO3 (140 mg, 1.01
mmol) in 1,4-dioxane:water(6 mL, 2:1, v) in a microwave tube was flushed with N2 for 5 mins then
sealed. The tube was heated at 80 ◦C for 3 h. Then the reaction mixture was evaporated to dryness.
The residue was purified by flash chromatography to give 34 (90 mg, 89% yield); LC–MS m/z (ESI)
found (M + H)+ 199.1 (M + H)+; 1H-NMR (400 MHz, DMSO-d6)
δ
11.31 (brs, 1H), 8.59 (s, 1H), 8.21 (s,
1H), 7.93 (s, 1H), 7.87 (s, 1H), 7.60 (t, J = 2.8 Hz, 1H), 6.53 (t, J = 2.8 Hz, 1H), 3.88 (s, 3H).
6-(1-Methyl-1H-pyrazol-4-yl)-1-(phenylsulfonyl)-1H-pyrrolo[3,2-b]pyridine ( ). Sodium hydride (7 mg,
8
0.28 mmol) was suspended in 3 mL of anhydrous DMF. 6-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo
[3,2-b]pyridine (34) (50 mg, 0.25 mmol) was dissolved in 3 mL of anhydrous DMF was slowly added
dropwise, and the mixture was stirred for 30 min benzenesulfonyl chloride (38 µL, 0.28 mmol) dissolved
in 3 mL of anhydrous DMF was slowly added in drops, and the mixture was stirred for 2 h at room
temperature. The reaction solution was poured to 0.1 N hydrochloric acid, which was then brought
to basic using aqueous sodium bicarbonate solution, and extracted with ethyl acetate. The organic
layer was collected, and distilled under reduced pressure. The remaining substance was purified by
flash column chromatography to give
(M + H)+; 1H-NMR (400 MHz, CDCl3)
7.89 (t, J = 1.7 Hz, 1H), 7.87–7.85 (m, 1H), 7.77 (d, J = 3.8 Hz, 2H), 7.63–7.56 (m, 1H), 7.49 (t, J = 7.7 Hz,
2H), 6.88 (dd, J = 3.8, 0.7 Hz, 1H), 4.02 (s, 3H). 13C-NMR (126 MHz, CDCl3)
147.08, 144.47, 138.00,
136.93, 134.28, 129.53 (C 2), 129.25, 128.86, 127.36, 126.70 (C 2), 124.81, 120.18, 117.08, 110.45, 39.25.
Retention time 2.92 min, >98% purity.
8
δ
(70 mg, 82% yield); LC–MS m/z (ESI) found (M + H)+ 339.1
8.70 (d, J = 1.7 Hz, 1H), 8.32 (d, J = 1.2 Hz, 1H), 7.91 (s, 1H),
δ
×
×
Compounds 9 were prepared with a similar procedure as that used for 8.
3-(1-Methyl-1H-pyrazol-4-yl)-5-(phenylsulfonyl)-5H-pyrrolo[2,3-b]pyrazine ( ). LC–MS m/z (ESI) found (M
+ H)+ 340.0 (M + H)+; 1H-NMR (400 MHz, CDCl3)
8.71 (s, 1H), 8.28–8.19 (m, 2H), 8.09 (s, 1H), 8.03 (s,
1H), 7.94 (d, J = 4.1 Hz, 1H), 7.62 (d, J = 7.4 Hz, 1H), 7.54 (t, J = 7.7 Hz, 2H), 6.80 (d, J = 4.1 Hz, 1H),
4.04 (s, 3H). 13C-NMR (126 MHz, CDCl3)
142.22, 140.54, 139.05, 138.04, 137.96, 137.77, 134.47, 129.14
9
δ
δ
(C × 2), 129.10, 128.96, 128.24 (C × 2), 120.98, 106.58, 39.37. Retention time 2.99 min, >99% purity.
N-(5-Bromo-2-methylpyridin-3-yl)benzenesulfonamide (37). To a stirred solution of the 5-bromo-2-
methylpyridin-3-amine (36) (200 mg, 1.07 mmol) in anhydrous dichloromethane (15 mL) was added
benzenesulfonyl chloride (152
in vacuo, diluted with EtOAc (40 mL) and saturated NaHCO3 solution (20 mL) and partitioned.
The aqueous layer was extracted with EtOAc (2 20 mL). The combined organic layers were dried
(Na2SO4), filtered and concentrated to afford 37 (300 mg, 85% yield); LC–MS m/z (ESI) found (M + H)+
328.1 (M + H)+; 1H-NMR (400 MHz, CDCl3)
8.37 (d, J = 2.1 Hz, 1H), 7.92 (d, J = 2.1 Hz, 1H), 7.82–7.76
µL, 1.12 mmol). After 1 h, The mixture was then partially concentrated
×
δ
(m, 2H), 7.67–7.61 (m, 1H), 7.56–7.49 (m, 2H), 2.17 (s, 3H).
N-(2-Methyl-5-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)benzenesulfonamide (10). A solution of N-(5-bromo-
2-methylpyridin-3-yl)benzenesulfonamide (37) (100 mg, 0.31 mmol), 1-methyl-4-(4,4,5,5-tetramethyl-

Molecules 2017, 22, 583
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1,3,2-dioxaborolan-2-yl)-1H-pyrazole (30) (70 mg, 0.34 mmol), PdCl2(dppf)-CH2Cl2 (20 mg, 24 µmol)
and K2CO3 (84 mg, 0.61 mmol) in 1,4-dioxane:water(15 mL, 2:1, v) in a microwave tube was flushed
with N2 for 5 mins then sealed. The tube was heated at 80 ◦C for 3 h. Then the reaction mixture was
evaporated to dryness. The residue was purified by flash chromatography to give 10 (80 mg, 80%
yield); LC–MS m/z (ESI) found (M + H)+ 329.1 (M + H)+; 1H-NMR (400 MHz, DMSO-d6)
δ
8.45 (d,
J = 2.0 Hz, 1H), 7.78–7.75 (m, 2H), 7.73 (d, J = 1.4 Hz, 1H), 7.71 (s, 1H), 7.67 (s, 1H), 7.59 (t, J = 7.5 Hz,
1H), 7.47 (t, J = 7.5 Hz, 2H), 5.30 (s, 1H), 3.97 (s, 3H), 2.16 (s, 3H). 13C-NMR (126 MHz, CDCl3)
149.37,
2), 118.85, 39.23,
δ
143.60, 139.24, 136.71, 133.43, 131.00, 129.32 (C
20.03. Retention time 2.54 min, >98% purity.
×
2), 128.46, 127.44, 127.31, 127.03 (C
×
Compounds 11–28 were prepared with a similar procedure as that used for 8.
3-(1-Methyl-1H-pyrazol-4-yl)-5H-pyrrolo[2,3-b]pyrazine (35). LC–MS m/z (ESI) found (M + H)+ 199.1
(M + H)+; 1H-NMR (400 MHz, DMSO-d6)
9.30 (s, 1H), 8.72 (s, 1H), 8.11 (s, 1H), 7.97 (s, 1H), 7.28 (s,
1H), 6.74 (dd, J = 3.7, 1.9 Hz, 1H), 4.02 (s, 3H).
5-(Cyclopentylsulfonyl)-3-(1-methyl-1H-pyrazol-4-yl)-5H-pyrrolo[2,3-b]pyrazine (11). LC–MS m/z (ESI)
found (M + H)+ 311.2 (M + H)+; 1H-NMR (400 MHz, CDCl3)
8.80 (s, 1H), 8.09 (d, J = 0.8 Hz, 1H), 8.07
(d, J = 0.8 Hz, 1H), 7.83 (d, J = 4.1 Hz, 1H), 6.83 (d, J = 4.1 Hz, 1H), 4.03 (s, 3H), 3.16–3.01 (m, 1H), 2.20
(m, 2H), 2.01 (m, 2H), 1.96–1.62 (m, 4H). 13C-NMR (126 MHz, CDCl3)
142.25, 140.70, 139.08, 138.03,
2). Retention time 2.57 min,
δ
δ
δ
137.65, 129.80, 129.30, 120.83, 105.69, 64.00, 40.57, 27.62 (C
>99% purity.
×
2), 25.86 (C
×
5-(Cyclohexylsulfonyl)-3-(1-methyl-1H-pyrazol-4-yl)-5H-pyrrolo[2,3-b]pyrazine (12). LC–MS m/z (ESI)
found (M + H)+ 345.1 (M + H)+; 1H-NMR (400 MHz, CDCl3)
8.81 (s, 1H), 8.07 (d, J = 0.7 Hz, 1H), 8.05
δ
(d, J = 0.7 Hz, 1H), 7.84 (d, J = 4.0 Hz, 1H), 6.85 (d, J = 4.0 Hz, 1H), 4.01 (s, 3H), 3.82 (m, 1H), 2.01–1.85
(m, 4H), 1.69 (dd, J = 10.9, 4.8 Hz, 2H), 1.32–1.17 (m, 4H). Retention time 2.59 min, 98.54% purity.
2-((3-(1-Methyl-1H-pyrazol-4-yl)-5H-pyrrolo[2,3-b]pyrazin-5-yl)sulfonyl)benzonitrile (13). LC–MS m/z (ESI)
found (M + H)+ 364.0 (M + H)+; 1H-NMR (400 MHz, CDCl3)
δ
8.71 (s, 1H), 8.66–8.62 (m, 1H), 8.16 (d,
J = 4.2 Hz, 1H), 8.01–7.96 (m, 2H), 7.89–7.83 (m, 2H), 7.77 (dd, J = 7.6, 1.3 Hz, 1H), 6.89 (d, J = 4.2 Hz,
1H), 4.02 (s, 3H). 13C-NMR (126 MHz, CDCl3)
142.29, 140.40, 139.90, 139.20, 138.37, 137.78, 135.52,
δ
134.39, 132.82, 131.76, 130.15, 129.00, 120.65, 115.00, 111.37, 106.99, 39.36. Retention time 3.04 min,
>99% purity.
5-((4-Chlorophenyl)sulfonyl)-3-(1-methyl-1H-pyrazol-4-yl)-5H-pyrrolo[2,3-b]pyrazine (14). HPLC 96.66%;
1
LC–MS m/z (ESI) found (M + H)+ 373.0 (M + H)+; H-NMR (400 MHz, CDCl3)
δ 8.73 (s, 1H), 8.19
(d, J = 8.5 Hz, 2H), 8.09 (s, 1H), 8.03 (s, 1H), 7.91 (d, J = 4.1 Hz, 1H), 7.51 (d, J = 8.5 Hz, 2H), 6.82 (d,
J = 4.1 Hz, 1H), 4.05 (s, 3H). 13C-NMR (126 MHz, CDCl3)
142.31, 141.36, 140.46, 139.06, 138.17, 137.77,
2), 128.92, 128.89, 120.87, 106.91, 39.39. Retention time 2.98 min,
δ
136.36, 129.69 (C
96.66% purity.
×
2), 129.53 (C
×
5-((2,4-Difluorophenyl)sulfonyl)-3-(1-methyl-1H-pyrazol-4-yl)-5H-pyrrolo[2,3-b]pyrazine (15). LC–MS m/z
(ESI) found (M + H)+ 375.1 (M + H)+; 1H-NMR (400 MHz, CDCl3)
8.71 (s, 1H), 8.44 (td, J = 8.5, 6.0
Hz, 1H), 8.01 (dd, J = 4.1, 1.3 Hz, 1H), 7.99 (d, J = 0.7 Hz, 1H), 7.91 (s, 1H), 7.16–7.10 (m, 1H), 6.90 (ddd,
J = 10.4, 8.5, 2.4 Hz, 1H), 6.85 (d, J = 4.1 Hz, 1H), 4.02 (s, 3H). 13C-NMR (126 MHz, CDCl3)
168.08,
δ
δ
166.01, 161.34, 159.25, 142.19, 140.38, 139.07, 138.16, 137.76, 133.92, 129.52, 128.76, 120.74, 112.10, 106.53,
39.34. Retention time 2.97 min, 94.53% purity.
5-((3,4-Dichlorophenyl)sulfonyl)-3-(1-methyl-1H-pyrazol-4-yl)-5H-pyrrolo[2,3-b]pyrazine (16). LC–MS m/z
(ESI) found (M + H)+ 407.0 (M + H)+; 1H-NMR (400 MHz, CDCl3)
δ
8.75 (s, 1H), 8.50 (d, J = 2.2 Hz,
1H), 8.10 (d, J = 0.7 Hz, 1H), 8.06 (s, 1H), 8.03 (dd, J = 8.5, 2.2 Hz, 1H), 7.90 (d, J = 4.1 Hz, 1H), 7.62
(d, J = 8.5 Hz, 1H), 6.84 (d, J = 4.1 Hz, 1H), 4.06 (s, 3H). 13C-NMR (126 MHz, CDCl3)
142.44, 140.33,
δ
139.75, 139.07, 138.37, 137.69, 137.34, 133.79, 131.27, 130.76, 129.06, 128.62, 127.09, 120.71, 107.13, 39.38.
Retention time 3.02 min, >99% purity.

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3-(1-Methyl-1H-pyrazol-4-yl)-5-((3-nitrophenyl)sulfonyl)-5H-pyrrolo[2,3-b]pyrazine (17). LC–MS m/z (ESI)
found (M + H)+ 384.2 (M + H)+; 1H-NMR (400 MHz, CDCl3)
9.40 (t, J = 2.1 Hz, 1H), 8.77 (s, 1H),
8.49 (dd, J = 10.8, 8.0 Hz, 2H), 8.24 (s, 1H), 8.06 (s, 1H), 7.92 (d, J = 4.2 Hz, 1H), 7.78 (t, J = 8.0 Hz, 1H),
6.86 (dd, J = 4.2, 0.7 Hz, 1H), 4.08 (s, 3H). 13C-NMR (126 MHz, CDCl3)
148.09, 142.68, 140.32, 139.87,
δ
δ
139.00, 138.45, 137.37, 133.39, 130.67, 129.68, 128.93, 128.39, 124.61, 120.43, 107.41, 39.37. Retention time
3.01 min, >99% purity.
3-(1-Methyl-1H-pyrazol-4-yl)-5-((4-nitrophenyl)sulfonyl)-5H-pyrrolo[2,3-b]pyrazine (18). LC–MS m/z (ESI)
found (M + H)+ 384.0 (M + H)+; 1H-NMR (400 MHz, CDCl3)
δ 8.75 (s, 1H), 8.48–8.44 (m, 2H), 8.37 (d,
J = 8.8 Hz, 2H), 8.10 (s, 1H), 8.03 (s, 1H), 7.91 (d, J = 4.1 Hz, 1H), 6.87 (d, J = 4.1 Hz, 1H), 4.06 (s, 3H).
Retention time 3.02 min, 98.86% purity.
3-((3-(1-Methyl-1H-pyrazol-4-yl)-5H-pyrrolo[2,3-b]pyrazin-5-yl)sulfonyl)aniline (19). LC–MS m/z (ESI)
found (M + H)+ 354.1 (M + H)+; 1H-NMR (600 MHz, CDCl3)
δ 9.05 (d, J = 1.9 Hz, 1H), 8.67 (s, 1H),
8.17–8.14 (m, 2H), 8.06 (dd, J = 8.4, 0.8 Hz, 1H), 8.04 (d, J = 0.8 Hz, 1H), 7.98 (d, J = 4.1 Hz, 1H), 7.42 (d,
J = 8.4 Hz, 1H), 6.80 (d, J = 4.1 Hz, 1H), 4.05 (s, 3H). Retention time 2.79 min, 94.56% purity.
4-((3-(1-Methyl-1H-pyrazol-4-yl)-5H-pyrrolo[2,3-b]pyrazin-5-yl)sulfonyl)aniline (20). LC–MS m/z (ESI)
found (M + H)+ 355.07 (M + H)+; 1H-NMR (400 MHz, CDCl3)
δ 8.69 (s, 1H), 8.09 (d, J = 0.8 Hz, 1H),
8.03 (s, 1H), 8.01 (d, J = 2.1 Hz, 1H), 7.99 (d, J = 2.1 Hz, 1H), 7.93 (d, J = 4.1 Hz, 1H), 6.76 (d, J = 4.1 Hz,
1H), 6.66 (d, J = 2.0 Hz, 1H), 6.64 (d, J = 2.0 Hz, 1H), 4.04 (s, 3H). Retention time 2.75 min, 98.68% purity.
N-(3-((3-(1-Methyl-1H-pyrazol-4-yl)-5H-pyrrolo[2,3-b]pyrazin-5-yl)sulfonyl)phenyl)acetamide (21). HPLC
94.55%; LC–MS m/z (ESI) found (M + H)+ 396.1 (M + H)+; 1H-NMR (400 MHz, CDCl3)
δ 8.72 (s, 1H),
8.61 (s, 1H), 8.20 (s, 1H), 8.06 (s, 1H), 7.91 (t, J = 6.0, 6.3 Hz, 2H), 7.66 (d, J = 8.2 Hz, 1H), 7.47 (d,
J = 8.2 Hz, 1H), 7.44 (d, J = 12.3 Hz, 1H), 6.80 (d, J = 4.1 Hz, 1H), 4.01 (s, 3H), 2.04 (s, 3H). Retention
time 3.12 min, 94.55% purity.
N-(3-((3-(1-Methyl-1H-pyrazol-4-yl)-5H-pyrrolo[2,3-b]pyrazin-5-yl)sulfonyl)phenyl)propionamide
(22).
LC–MS m/z (ESI) found (M + H)+ 410.1 (M + H)+; 1H-NMR (400 MHz, CDCl3)
δ
8.71 (s, 1H), 8.61 (s,
1H), 8.22 (s, 1H), 8.07 (s, 1H), 7.92 (t, J = 6.1 Hz, 2H), 7.67 (d, J = 8.1 Hz, 1H), 7.47 (d, J = 8.1 Hz, 1H),
7.42 (d, J = 12.5 Hz, 1H), 6.81 (d, J = 4.1 Hz, 1H), 4.04 (s, 3H), 2.41 (q, J = 7.6 Hz, 2H), 1.24 (t, J = 7.6 Hz,
3H). Retention time 3.13 min, 97.72% purity.
1-(4-(5-((3-Nitrophenyl)sulfonyl)-5H-pyrrolo[2,3-b]pyrazin-3-yl)-1H-pyrazol-1-yl)ethan-1-one (23). LC–MS
1
m/z (ESI) found (M + H)+ 412.0 (M + H)+; H-NMR (400 MHz, CDCl3)
δ 9.28 (s, 1H), 8.91 (s, 1H),
8.86 (s, 1H), 8.58 (d, J = 8.2 Hz, 1H), 8.51 (d, J = 8.2 Hz, 1H), 8.40 (s, 1H), 8.02 (d, J = 4.2 Hz, 1H), 7.81
(t, J = 8.2 Hz, 1H), 6.91 (d, J = 4.2 Hz, 1H), 2.83 (s, 3H). 13C-NMR (126 MHz, CDCl3)
δ
169.38, 148.24,
142.03, 140.64, 140.34, 140.19, 139.73, 139.00, 133.57, 130.68, 129.81, 129.04, 126.08, 124.04, 123.66, 107.39,
21.67. Retention time 2.64 min, 90.95% purity.
1-(4-(5-((3-Nitrophenyl)sulfonyl)-5H-pyrrolo[2,3-b]pyrazin-3-yl)-1H-pyrazol-1-yl)propan-1-one (24). LC–MS
m/z (ESI) found (M + H)+ 426.3 (M + H)+; 1H-NMR (400 MHz, CDCl3)
δ 9.27 (t, J = 2.0, 2.0 Hz, 1H), 8.91
(d, J = 0.8 Hz, 1H), 8.86 (s, 1H), 8.61–8.56 (m, 1H), 8.53–8.48 (m, 2H), 8.38 (d, J = 0.8 Hz, 1H), 8.32 (s, 1H),
8.02 (d, J = 4.2 Hz, 1H), 6.91 (d, J = 4.2 Hz, 1H), 3.27 (q, J = 7.4 Hz, 2H), 1.38 (t, J = 7.4 Hz, 3H). 13C-NMR
(126 MHz, CDCl3)
δ 172.88, 148.25, 141.86, 140.77, 140.28, 139.75, 139.00, 138.69, 133.60, 130.66, 129.74,
129.04, 126.18, 124.36, 124.02, 123.35, 107.39, 29.70. Retention time 2.65 min, 95.32% purity.
1-(4-(5-((3-Nitrophenyl)sulfonyl)-5H-pyrrolo[2,3-b]pyrazin-3-yl)-1H-pyrazol-1-yl)propan-2-one (25). LC–MS
m/z (ESI) found (M + H)+ 426.2 (M + H)+; 1H-NMR (400 MHz, CDCl3)
δ
9.40 (t, J = 2.0 Hz, 1H), 8.79
(s, 1H), 8.48 (m, 2H), 8.30 (d, J = 0.8 Hz, 1H), 8.14 (d, J = 0.8 Hz, 1H), 7.95 (d, J = 4.1 Hz, 1H), 7.79 (t,
J = 8.0 Hz, 1H), 6.87 (d, J = 4.1 Hz, 1H), 5.11 (s, 2H), 2.29 (s, 3H). 13C-NMR (151 MHz, CDCl3)
201.10,
δ
147.99, 142.15, 140.19, 139.74, 139.23, 138.40, 138.03, 133.42, 130.72, 130.44, 128.98, 128.59, 124.75, 121.22,
107.31, 61.19, 27.02. Retention time 2.66 min, 94.67% purity.

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1-(4-(5-((3-Nitrophenyl)sulfonyl)-5H-pyrrolo[2,3-b]pyrazin-3-yl)phenyl)ethan-1-one (26). LC–MS m/z (ESI)
found (M + H)+ 422.3 (M + H)+; 1H-NMR (400 MHz, CDCl3)
9.32 (t, J = 2.0 Hz, 1H), 9.12 (s, 1H),
δ
8.60–8.55 (m, 1H), 8.53–8.48 (m, 1H), 8.25 (d, J = 8.4 Hz, 2H), 8.18 (d, J = 8.4 Hz, 2H), 8.09 (d, J = 4.2 Hz,
1H), 7.79 (t, J = 8.1 Hz, 1H), 6.96 (d, J = 4.2 Hz, 1H), 3.73 (d, J = 0.8 Hz, 3H). Retention time 2.65 min,
91.84% purity.
(4-(5-((3-Nitrophenyl)sulfonyl)-5H-pyrrolo[2,3-b]pyrazin-3-yl)phenyl)methanol (27). LC–MS m/z (ESI)
1
found (M + H)+ 410.0 (M + H)+; H-NMR (400 MHz, CDCl3)
δ 9.30 (t, J = 2.0 Hz, 1H), 9.02 (s,
1H), 8.57 (dt, J = 7.9, 1.5 Hz, 1H), 8.48 (ddd, J = 8.2, 2.0, 1.1 Hz, 1H), 8.13 (d, J = 1.5 Hz, 1H), 8.11 (s,
1H), 8.02 (d, J = 4.2 Hz, 1H), 7.77 (t, J = 8.1 Hz, 1H), 7.57 (d, J = 8.1 Hz, 2H), 6.91 (d, J = 4.2 Hz, 1H),
4.83 (s, 2H). 13C-NMR (126 MHz, CDCl3)
δ
148.17, 147.07, 142.88, 140.39, 140.11, 139.84, 139.22, 135.31,
133.66, 130.68, 129.64, 128.97, 127.64 (C
min, 94.62% purity.
×
2), 127.20 (C 2), 124.29, 107.25, 64.85. Retention time 2.71
×
3-(7-Methoxy-1H-indol-2-yl)-5-((3-nitrophenyl)sulfonyl)-5H-pyrrolo[2,3-b]pyrazine (28). LC–MS m/z (ESI)
found (M + H)+ 449.1 (M + H)+; 1H-NMR (400 MHz, CDCl3)
9.43 (s, 1H), 9.19 (s, 1H), 9.07 (s, 1H), 8.58
δ
(s, 1H), 8.50 (s, 1H), 7.99 (s, 1H), 7.81 (s, 1H), 7.23–7.03 (m, 2H), 6.97–6.87 (m, 1H), 6.78 (d, J = 8.4 Hz,
1H), 4.12 (s, 3H). Retention time 2.94 min, 96.21% purity.
4. Conclusions
Starting with the scaffold of 1-sulfonylpyrazolo[4,3-b]pyridine discovered in c-Met inhibitor
development, we conducted a hit optimization towards the inhibitory activity of FGFR1. We explored
the SAR of this new chemotype on three parts, namely the scaffold, π–π stacking part and methyl
pyrazolepart. We found that the scaffold is essential to the binding activity, and substituting the
pyrazolo[4,3-b]pyridine with pyrrolo[2,3-b]pyrazine increases about of the inhibitory activity on
FGFR1 about 10-fold. The π–π stacking part is also vital to the biological activity, showing greater
importance of the steric effect than the electronic effect. In summary, we elaborated a series of FGFR
inhibitors containing the novel scaffold of pyrrolo[2,3-b]pyrazine. This scaffold, together with the
interesting shape of this phenotype, could help others to further develop selective FGFR inhibitors for
cancer treatment.
Acknowledgments: We are grateful for financial support from the National Natural Science Foundation of China
(Grant No. 81473243); NSFC-Shandong Joint Fund for Marine Science Research Centers (Grant No. U1406402);
Youth Innovation Promotion Association and the “Personalized Medicines—Molecular Signature-based Drug
Discovery and Development”, Strategic Priority Research Program of the Chinese Academy of Sciences, Grant No.
XDA12020317).
Author Contributions: Bing Xiong, Yuchi Ma and Jing Ai designed the research; Yan Zhang, Yuchi Ma,
Zhen Zhang, Ruifeng Wang, Tongchao Liu and Chaoyun Wang conducted the research; Hongchun Liu, Jing Ai,
Jingkang Shen, Dongmei Zhao and Bing Xiong analyzed the data; Bing Xiong, Yuchi Ma, and Jing Ai wrote
the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Not available.
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