Design and synthesis of novel pyrrolo[2,3-b]pyridine derivatives targeting V600EBRAF

Article abstract of DOI:10.1016/j.bmc.2020.115493

Several pyrrolo[2,3-b]pyridine-based B-RAF inhibitors are well known and some of them are currently FDA approved as anticancer agents. Based on the structure of these FDA approved ^V600EB-RAF inhibitors, two series of pyrrolo[2,3-b]pyridine scaffold were designed and synthesized in attempt to develop new potent ^V600EB-RAF inhibitors. The 38 synthesized compounds were biologically evaluated for their ^V600EB-RAF inhibitory effect at single dose (10 μM). Compounds with high percent inhibition were tested to determine their IC₅₀ over ^V600EB-RAF. Compounds 34e and 35 showed the highest inhibitory effect with IC₅₀ values of 0.085 μM and 0.080 μM, respectively. Headed for excessive biological evaluation, the synthesized derivatives were tested over sixty diverse human cancer cell lines. Only compound 35 emerged as a potent cytotoxic agent against different panel of human cancer cell lines.

Full text of DOI:10.1016/j.bmc.2020.115493

Journal Pre-proofs
Design and synthesis of novel pyrrolo[2,3-b]pyridine derivatives targeting
V600EBRAF
Mohammed S. Abdel-Maksoud, Eslam M.H. Ali, Usama M. Ammar, Karim I.
Mersal, Kyung Ho Yoo, Chang-Hyun Oh
PII:
S0968-0896(20)30314-X
https://doi.org/10.1016/j.bmc.2020.115493
BMC 115493
DOI:
Reference:
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Bioorganic & Medicinal Chemistry
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5 April 2020
Please cite this article as: M.S. Abdel-Maksoud, E.M.H. Ali, U.M. Ammar, K.I. Mersal, K. Ho Yoo, C-H. Oh,
Design and synthesis of novel pyrrolo[2,3-b]pyridine derivatives targeting V600EBRAF, Bioorganic & Medicinal
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Design and synthesis of novel pyrrolo[2,3-b]pyridine derivatives
targeting V600EBRAF
Mohammed S. Abdel-Maksouda,*, EslamM. H. Alib,c,d,*, Usama M. Ammarb,c,e,, KarimI. Mersalb,c,, Kyung
Ho Yoof , Chang-Hyun Ohb,c,**
aMedicinal &PharmaceuticalChemistry Department,Pharmaceuticaland Drug Industries Research
Division, NationalResearch Centre (NRC), Dokki, Giza, 12622, Egypt.
bCenterfor Biomaterials, Korea Instituteof Science & Technology (KISTSchool), Seoul, Seongbuk-gu,
02792, Republic of Korea.
cUniversity of Science & Technology (UST), Daejeon, Yuseong-gu, 34113, Republicof Korea.
dPharmaceuticalChemistry Department, Faculty of Pharmacy, Modern University of technology and
information (MTI), Cairo, 12055, Egypt.
ePharmaceuticalChemistry Department, Faculty of Pharmacy, Ahram Canadian University, Giza, 12566,
Egypt.
f Chemical Kinomics Research Center, Korea Instituteof Science and Technology, Seoul, Republicof Korea
* Both authorcontributed equally in this work
** Corresponding author(choh@kist.re.kr)

Abstract
Several pyrrolo[2,3-b]pyridine-based B-RAF inhibitors are well known and some of them are
currently FDA approved as anticancer agents. Based on the structure of these FDA approved
V600EB-RAF inhibitors, two series of pyrrolo[2,3-b]pyridine scaffold were designed and
synthesized in attempt to develop new potent V600EB-RAF inhibitors. The 38 synthesized
compounds were biologically evaluated for their V600EB-RAF inhibitory effect at single dose
(10uM). Compounds with high percent inhibition were tested to determine their IC50 over
V600EB-RAF. Compounds 34e and 35 showed the highest inhibitory effect with IC50 values of
0.085 µM and 0.080 µM, respectively. Headed for excessive biological evaluation, the
synthesized derivatives were tested over sixty diverse human cancer cell lines. Only compound
35 emerged as a potent cytotoxic agent against different panel of human cancer cell lines.
Keywords
B-RAF inhibitors; Kinase inhibitor; Anticancer; pyrrolo[2,3-b]pyridine; SAR.
1. Introduction
BRAF is a serine/threonine kinase that plays an important role in MAPK singling pathway. The
activation of MAPK pathway is vital for normal cellular functions such as cell differentiation and
growth[1, 2]. Oncogenic activation of MAPK pathway as a result of BRAF mutation occurs in 50-
70% of melanomas[3, 4], up to 70% of thyroid carcinoma[5-7] and 10% of colorectal cancers[8].
B-RAF kinase can be activated by oncogenic mutations, such as B-RAF V600E mutation (in which
valine amino acid in position 600 is replaced by glutamic acid). V600EBRAF is prevalent in
melanomas 63%[9-13] and papillary thyroid carcinomas up to 50%[3].
Targeting V600EBRAF by small molecules is a good strategy for treatment of MAPK pathway
hyper-activation in case of melanoma[14, 15]. In addition, the combination of V600EBRAF and
MEK inhibitors showed great success in preclinical study for treatment of advanced solid
tumors[16-18].
Due to the widespread of different forms of RAF kinase in different types of cancer, it has been
considered as a very important anticancer drug target. RAF kinase inhibitors have been

extensively developed. Sorafenib (Nexavar®, BAY- 43-9006) is a diarylurea inhibitor for B-RAF
that has been approved for the treatment of advanced renal cell carcinoma (RCC) and
Hepatocellular carcinoma (HCC). Sorafenib is a small molecule inhibitor of several protein
kinases, among which is B-RAF[19, 20]. Vemurafenib (PLX4032, Zelboraf) and dabrafenib
(GSK2118436) were approved by the U.S. Food and Drug Administration (FDA) for treatment of
late-stage melanoma. Both are selective inhibitors of V600E mutated B-RAF kinase and cause
programmed cell death in melanoma cell lines, and hence high potency against melanoma cell
lines. Melanoma cells without this mutation are not inhibited by vemurafenib and
dabrafenib[20-22]. Mono-therapy treatment with V600EBRAF produced significant improve in
both survival rate and patient life style. Despite of the successful of selective V600EBRAF
inhibitors, this success is short-lived and resistance to these inhibitors appear from 5 to 8.8
months.16-19 The acquired resistant to selective V600EBRAF makes the development of new
candidates to be a new therapy for V600EBRAF embedded cancers an essential issue.
Pyrrolo[2,3-b]pyridine scaffold is present in a variety of B-RAF approved and preclinical
investigated inhibitors (Fig. 1)[14, 23-25]. Starting from our previous work on pyrrolopyridine
scaffold and based on our BRAF related researches [26-29] and the high therapeutic
applications of pyrrolo[2,3-b]pyridine scaffold we developed a new pyrrolo[2,3-b]pyridine
candidates in order to check their V600EBRAF inhibitory effect. In the current work, pyrrolo[2,3-
b]pyridine scaffold was used as a building block in attempt to produce potent V600EB-RAF
inhibitors. Two synthetic pathways were used to produce two sets of new pyrrolo[2,3-
b]pyridine-based derivatives 24, 25, 26, 27, 34, and 35 (Fig. 2). The new compounds were
evaluated in vitro against V600EBRAF kinase enzyme. Moreover, all the synthesized derivatives
were subjected to NCI sixty human cancer cell lines assay. The preliminary biological data of the
first group of compounds 24, 25, 26, 27, 34 and 35 showed satisfied enzyme inhibitory activity.
Compounds 34a-g and 35 exhibited high enzymatic and cellular activities upon tested against
V600EBRAF kinase enzyme and NCI sixty human cancer cell lines. The biological evaluation data
indicated that the structural modification in the second group of compounds (34 and 35) has a
tangible effect on enhancing the activity against both the enzyme and human cancer cell lines.

Figure 1: Structures of reported B-RAF inhibitors.

Figure 2: General Structures of the final targeted compounds 24a-h, 25a-h, 26a-g, 27a-g, 34a-g,
and 35.
2. Results & discussion
2.1. Chemistry
The synthesis of the N1 substituted ethyl and propyl- 1H-pyrrolo[2,3-b]pyridin-1-yl)pyrimidin
targeted compounds 24, 25, 26, and 27 (Table 1) was performed as described in Scheme 1,
Scheme 2, and Scheme 3. Starting by protecting 3-methylpyridin-2-amine (1) using di-tert-
butyl dicarbonate to obtain tert-butyl (3-methylpyridin-2-yl)carbamate (2). Compound 2
reacted with butyl lithium to give lithium (E)-((2-((tert-butoxyoxidomethylene)amino)pyridin-3-
yl)methyl)lithium (3). The dilithio compound 3 was condensed with DMF to give tert-butyl (3-(2-
oxoethyl)pyridin-2-yl)carbamate (4) from which 7-azaindole (5) was obtained by acidic
hydrolysis, cyclisation, and dehydration. 7-azaindole (5) was -arylated with 4-chloro-2-
(methylthio)pyrimidine (6) using sodium hydride to give 1-(2-(methylthio)pyrimidin-4-yl)-1H-
pyrrolo[2,3-b]pyridine (7). Oxidation of methylthio group using oxone produced the key
intermediate 1-(2-(methylsulfonyl)pyrimidin-4-yl)-1H-pyrrolo[2,3-b]pyridine (8).

Scheme 1: Synthesis of intermediate 8;Reagentsand conditions:i) Boc anh., DMAP, DIPEA, THF, rt, 12
h.; ii) n-BuLi, THF, 0 °C, 4 h.; iii) DMF, HCl, 0 °C; iv) HCl, reflux, 8h.; v) NaH, DMF, 80 °C, 12 h.; vi) Oxone,
MeOH/H2O, rt, 18 h.
The preparation of N1-sustituted ethane-1,2 diamines 20, and 22 and N1-sustituted propane-
1,3-diamines 21 and 23 was carried out by reaction of 2-aminoethan-1-ol (9a) and 3-
aminopropan-1-ol (9b) with benzyl chloroformate to give benzyl (2-hydroxyethyl)carbamate
(10a) and benzyl (3-hydroxyethyl)carbamate (10b), followed by conversion of OH to mesylate
group with methane sulfonyl chloride to obtain 2-(((benzyloxy)carbonyl)amino)ethyl
methanesulfonate (11a) and 3-(((benzyloxy)carbonyl)amino)propyl methanesulfonate (11b).
The mesylates, 11, were then reacted with sodium azide in dry dimethylformamide to afford
benzyl (2-azidoethyl)carbamate (12a), and benzyl (3-azidopropyl)carbamate (12b) which were
converted to the corresponding amines 13 by reduction with triphenylphosphine in methanol.
Coupling of the produced amines 13 with appropriate sulfonyl chlorides 14 or benzoyl chlorides
15 in dichloromethane gave compounds 16, 17, 18, and 19. The protected amino linkers 16-19
were then subjected to Pd/C in methanol to afford N-(2-aminoethyl)substituted
amides(sulfonamides) and N-(3-aminopropyl)substituted amides(sulfonamides) side chains 20-
23. (Scheme 2).

Scheme 2: Synthesis of side chains 20a-h, 21a-h, 22a-h, and 23 a-h; Reagents and conditions:i) Benzyl
chloroformate, TEA, MC, 0 oC 12 h.; ii) Methane sulfonyl chloride, TEA, MC, 0 oC, 4 h.; iii) NaN3, DMSO,
70 °C, 2 h; iv) Triphenyl phosphine, MeOH, reflux, 2h.; v) Appropriate arylsufonyl chloride oracid
chloride, DIPEA, MC, 0 oC .; vi) H2/Pd-C, MeOH, rt, 2 h .
Nucleophilic substitution reaction of the different aliphatic amines 20-23 on the key
intermediate 8 in presence of N,N-diisopropylethyl amine produced the final target compounds
24, 25, 26, and 27. (Scheme 3).

Scheme 3: Synthesis of the final target compounds 24a-h, 25a-h, 26a-g, and 27a-g.
Table 1: Structure of the final target compounds 24a-h, 25a-h, 26a-g, and 27a-g:
Comp.
24a
n
x
R
Comp.
25h
n
x
R
1
SO2
2
SO2
24b
24c
24d
24e
1
1
1
1
SO2
SO2
SO2
SO2
26a
26b
26c
26d
1
1
1
1
CO
CO
CO
CO
24f
1
SO2
26e
1
CO
24g
24h
1
1
SO2
SO2
26f
1
1
CO
CO
26g
25a
25b
25c
2
2
2
SO2
SO2
SO2
27a
27b
27c
2
2
2
CO
CO
CO

25d
25e
2
2
SO2
SO2
27d
27e
2
2
CO
CO
25f
2
2
SO2
SO2
27f
2
2
CO
CO
25g
27g
For biological evaluation expansion, a new series of the Azaindole derivatives were prepared
with different substitutional pattern owing to enhance the biological inhibitory activity. 1-(3-(5-
subs-1H-pyrrolo[2,3-b]pyridine-3-carbonyl)-2,4-difluorophenyl)-3-(4-substituted-phenyl)urea
34a-g and 35 (Table 2) were synthesized starting from the nitration of 2,6-difluorobenzoic acid
28 using conc. H2SO4 and conc. HNO3 producing the nitrated compound 29. Reaction of
carboxylic acid derivative 29 with thionyl chloride producing the acid chloride form 30. Friedel–
Crafts acylation was carried out between the acid chloride derivative 30 and Azaindole
derivatives 5a,b using aluminium chloride as lewis acid reagent producing the corresponding
(2,6-difluoro-3-nitrophenyl)(1H-pyrrolo[2,3-b]pyridin-3-yl)methanone derivatives 31a,b.
Reduction of the nitro derivatives 31a,b by pd/C under H2 giving the amino derivatives 32a,b.
The targeted urea compounds 34a-g and 35 were prepared by the reaction of the amino
compounds 32a,b with different substituted phenyl isocyanates 33a-g. (Scheme 4).

Scheme 4 : Synthesis of final targetcompounds 34a-gand 35; Reagentsand conditions:i) Conc. HNO3,
Conc. H2SO4, 0 oC, 2 h.; ii) Thionyl chloride, DMF, 70 oC, 5 h.; iii) Aluminiumchloride, DCM, 50°C, 12h; iv)
SnCl2.2H2O, EtOAc, 80 oC, 4 h.; v) THF, 0 oC .
Table 2: Structure of the final target compounds 34a-g and 35:
Comp.
X
R
Comp.
X
R

34a
34b
H
H
34e
34f
H
H
34c
34d
H
H
34g
35
H
Br
2.2. Biological Evaluation
2.2.1. V600E-B-RAF and wild type BRAF Kinases Inhibition
The Azaindole-based compounds were reported as BRAF kinase inhibitors,[30-33] The
synthesized Azaindole derivatives 24a-h, 25a-h, 26a-g, 27a-g, 34a-g and 35 were tested for their
inhibitory effect over V600E-B-RAF using an enzyme-linked immunosorbent assay (ELISA). As
reported in table 3 and table 4, tested compounds exhibited a wide range of inhibition activity
against V600E-B-RAF. The preliminary % inhibition data of first Azaindole-based series 24a-h,
25a-h, 26a-g, 27a-g revealed that most of sulfonamide moiety containing derivatives 24 and 25
have higher inhibition activity than the ones with amide moiety 26 and 27. Moreover, the
propyl linker of the sulfonamide possessing derivatives 25a-h is bearing to have great effect on
the inhibitory activity with % inhibition mean of 97%. Among the highest active inhibitors 25a-
h, the IC50 of seven compounds 25a,b,d,e,f,g,h were determined. The obtained IC50 values are
ranging from 0.35 µM to 1.1 µM. The para-substitution with electron withdrawing groups on
the terminal phenyl sulfonamide in compounds 25b, and 25d enhanced the activity compared
to the electron donating substituted derivative 25g. However, compound 25e with electron
donating methoxy group revealed quite similar inhibitory activity like compounds 25b, and 25d
with electron withdrawing groups. Nevertheless, the meta-substitution with electron
withdrawing groups on the terminal phenyl sulfonamide in compounds 25f and 25h showed
slight drop in inhibitory activity compared to para substituted derivatives 25b, 25d, and 25e.
Compound 25d with para bromo group is the highest active inhibitor (IC50 =0.35 µM) among the
first Azaindole-based set. In our attempts to produce more potent V600E-B-RAF inhibitors, a

second set of Azaindole-based derivatives bearing urea moiety 34a-g and 35 were synthesized
and subjected to V600E-B-RAF enzyme assay evaluation. In similar manner, the inhibition of
wild type BRAF was determined to the final target compounds. Generally, the final compounds
have lower inhibitory effect over wild type BRAF compared to mutated BRAF. Only compounds
26f and 34c produced slightly higher activity over wild type BRAF compared to V600EBRAF. The
% inhibition values are depicted in Table 3.
Table 3: Enzyme % Inhibition Activity of the final target compounds 24a-h, 25a-h, 26a-g, 27a-g,
34a-g and 35 over BRAF (V600E) and wild type BRAF at 10 uM .
BRAF
(V600E) %
Inhibition at
(10 µM)
66.45
BRAF
(V600E) %
Inhibition at (10 µM)
BRAF (WT) %
Inhibition at
BRAF (WT) %
Inhibition at Comp.
(10 µM)
Comp.
(10 µM)
32.90
24a
24b
24c
24d
24e
24f
24g
24h
25a
25b
25c
25d
25e
25f
52.31
55.87
49.21
44.64
39.25
10.21
26.32
55.20
81.41
79.65
66.46
75.21
82.14
86.32
83.29
89.38
86.34
56.77
-1.1
26d
26e
26f
26g
27a
27b
27c
27d
27e
27f
27g
34a
34b
34c
34d
34e
34f
34g
35
12.05
0.1
89.51
-1.90
81.81
73.66
69.37
67.50
80.69
15.33
72.58
61.65
49.63
94.5
92
63.66
53.84
43.44
60.91
58.74
72.14
9.45
67.24
49.99
41.25
86.46
80.11
51.22
79.26
77.57
69.63
68.94
72.54
65.90
56.70
18.15
41.12
65.35
95.54
95.19
96.83
97.73
94.48
96.10
49
92.96
98.7
98.4
81.3
85.6
92.3
25g
25h
26a
26b
26c
96.70
86.27
69.99
0.16
From the revealed inhibitory activity data of the tested compounds, we found that the para-
substitution on the terminal phenyl urea derivatives 34d and 34e has a slight increase in the
activity rather than the unsubstituted derivative 34a. Conversely, the meta- substitution on the

terminal phenyl urea derivative 34c showed a huge drop in the inhibitory activity. 5-Bromo
substitution on the Azaindole ring in compound 35 showed to have no effect in the inhibitory
activity. The IC50 of the most potent inhibitors 34d, 34e, and 35 with para-substitution with
triflouromethyl group and methyl group, respectively were determined Table 4. The three
tested compounds showed Nano molar IC50 and compound 35 showed the most potential
inhibitory effect against V600EBRAF (80 nM).
The data reported in Table 3 and Table 4 revealed that the structural modification on the
second set of the Azaindole-based series 34a-g and 35 has a tangible effect for activity
improvement over the first set of Azaindole-based series 24a-h, 25a-h, 26a-g, and 27a-g.
Table 4: IC50 values of most active V600EBRAF inhibitors compounds against V600EBRAF kinases.
IC50 (µM)
Comp.
BRAF (V600E)
25a
25b
25d
25e
25f
25g
25h
34d
34e
35
0.75 ± 0.012
0.88 ± 0.009
0.35 ± 0.016
0.79 ± 0.010
1.1 ± 0.0210
1 ± 0.0113
0.5 ± 0.002
0.114 ± 0.002
0.085 ± 0.001
0.080 ± 0.003
2.2.2. NCI 60 cell lines one dose test
All the synthesized derivatives were submitted to the NCI, USA for in vitro cytotoxicity assay
against a 60 cell lines panel belonging to nine cancer types (leukemia, non-small cell lung
cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostate cancer and

breast cancer). The first Azaindole-based series 24a-h, 25a-h, 26a-g, and 27a-g exhibited no
remarkable activity against the cell growth according to 60 cell line results ( supporting
information and table 1s ).
100
90
80
70
60
50
40
30
20
10
0
34d 35
Figure 3: % inhibition of 34d, and 35 on most sensitive cancer cell lines at 10 uM.
Among the second set derivatives 34a-g and 35, only compound 35 with Bromo substitution at
C5 of the Azaindole ring showed moderate inhibitory activity against different cell lines (Figure
3) such as Leukemia K-562 (65%), Melanoma M14 (65%), Melanoma SK-MEL-2 (85 %), Ovarian
cancer IGROV1 (75 %), Ovarian cancer SK-OV-3 (87 %), Renal cancer ACHN (70%), and Renal
cancer CAKl-1 (90%). Moreover, compound 34d with CF3 terminal substituted urea, exhibited a
moderate inhibitory activity against Leukemia HL-60(TB) (67 %) and Leukemia K-562 (83 %)
(Fig.3).

Table 5: IC50 of compounds 34d and 35 over most sensitive cell lines
Compound IC50(uM)
Cell line
34d
35
Leukemia
HL-60(TB)
K-562
8.12 ± 0.09
10.91 ± 0.12
7.21 ± 0.14
25.31 ± 0.22
Non-Small Cell Lung cancer
NCI-H-460
NCI-H-522
Colon
26.42 ± 0.31
29.11 ± 0.24
10.53 ± 0.21
9.01 ± 0.15
HCT-116
15.53 ± 0.27
8.71 ± 0.18
Melanoma
M14
24.32 ± 0.21
21.12 ± 0.32
27.02 ± 0.35
12.35 ± 0.62
7.92 ± 0.22
6.02 ± 0.16
26.53 ± 0.13
9.26 ± 0.55
SK-MEL-2
LOX IMVI
MALMA-3M
Ovarian cancer
IGROV1
9.66 ± 0.42
5.06 ± 0.33
4.52 ± 0.27
SK-OV-3
18.78 ± 0.31
Renal cancer
ACHN
21.33 ± 29
14.01 ± 0.37
32.33 ± 0.52
7.62 ± 0.18
3.04 ± 0.16
11.24 ± 0.11
CAKl-1
A498
Prostate cancer
DU-145
27.56 ± 0.26
9.62 ± 0.32
Breast cancer
MDA-MB-231
HS 578T
9.72 ± 0.25
12.24 ± 0.10
9.36 ± 0.42
21.35 ± 0.41

In order to compare the potencies of compounds 34d and 35 over the most sensitive cell lines,
the concentration required to reduce cellular growth to 50% after treatment of the cells with
tested compound compared to untreated cell (IC50) was determined for both tested compounds
(Table 5). For leukemia cell lines, compound 35 exhibited the highest activity with IC 50 7.21 uM
over K-562 while compound 34d was more potent on HL-60(TB) with IC50 8.12 uM. Regarding
non-small cell lung cancer, compound 35 was more potent on both NCI-H-460 and NCI-H-522
with IC50s 10.53 and 9.01 uM, respectively. Compound 35 was also stronger over colon cancer
cell line HCT-116 , melanoma cell lines M14, SK-MEL-2, LOX IMVI and MALMA-3M , ovarian
cancer cell lines IGROV1 and SK-OV-3, renal cancer cell lines ACHN and CAKl-1, prostate cancer
cell line DU-145 and breast cancer cell line HS 578T. Compound 34d showed higher activity
against only MDA-MB-231 breast cancer cell line with IC50 9.72 uM.
2.3. Moleculardocking
Molecular docking study was conducted to demonstrate binding modes prospects of the
second synthetic series 34a-g and 35 into BRAF kinase binding site. In this study, we used the
known crystal structure of PLX4720-BRAF inhibitor complex (PDB: 3C4C). The low root mean
square deviation, RMSD (0.6346) of PLX4720-BRAF kinase domain complex with dock score (-
12.12 kcal/mol, Table 6) proved the validity of the used docking protocol. The resulted docking
poses of the native ligand (PLX4720) showed H-bonding interaction between the N-pryridine
and NH-Pyrole of the Azaindole moiety and Cys 532 and Gln 530 amino acids of the hinge
binder backbone. In addition, the sulphonamide moiety formed three hydrogen bonds between
the SO2 group and Lys 483, Asp 594 and Phe 595 amino acids. The binding affinity of the ligand
was evaluated with energy score (S, Kcal/mol) (Table 6). Applying the same docking protocol
into the synthetic hits 34a-g and 35 showed a similar binding behaviour like the native ligand.
Compounds 34f, 34g, and 35 preserved the H-bonding interaction with Cys 532 in the hinge
binding region (Fig. 4). Moreover, the urea moiety in all compounds played the same role of

sulphonamide moiety in the native ligand PLX4720 by formation of H-bonding interaction with
a variety of amino acids such as Asp 594, Phe 595, and Lys 483. In addition, arene-arene and
arene-H interactions were displayed between Pyridine ring and Pyrrole ring, and Phe 583 and
Gly 464. Unlike the native ligand PLX4720, the terminal substituted phenyl group in compounds
34b, 34d, and 35 showed variable interaction modes with Thr 508, Lys 483, and Leu 505.
A
B

C
D

Figure 4. 2D and 3D interaction diagram of docking study output (PDB ID: 3C4C) A) 2D and 3D
interaction of PLX4720 (native ligand) with BRAF kinase enzyme domain; B) 2D and 3D
interaction of 34b with BRAF kinase enzyme domain C) 2D and 3D interaction of 34f with BRAF
kinase enzyme domain; D) 2D and 3D interaction of 35 with BRAF kinase enzyme domain.
Table 6: Dockingresults of target compounds 34a-g and 35 with BRAF kinase enzyme domain (PDBID:
3C4C).
Energy score
(Kcal/mol)
Hydrogen bond
length (Å)
2.96
Compound
PLX4720
Amino acid Binding group
Interaction
Cys 532
Gln 530
Asp 594
Phe 595
Lys 483
Gly 464
Phe 583
Asp 594
Phe 595
Lys 483
Ser 535
Asp 594
Asp 594
Asp 594
Thr 508
N pyridine
NH pyrrole
SO2
SO2
SO2
Pyridine ring
Pyrrole ring
CO
CO
CO
N pyridine
NH
NH
Phenyl
Br
H-bond
H-bond
H-bond
H-bond
H-bond
Arene-H
Arene-H
H-bond
H-bond
H-bond
H-bond
H-bond
H-bond
Arene-H
H-bond
2.96
2.65
2.72
2.56
-12.12
-12.24
-12.38
34a
34b
3.19
3.45
3.35
3.27
3.3
3.62
3.85

Phe 583
Asp 594
Phe 595
Gly 464
Lys 483
Asp 594
Phe 583
Phe 583
Cys 532
Cys 532
Cys 532
Asp 594
Lys 483
Leu 505
Thr 508
Pyrrole ring
CO
Arene-Arene
H-bond
H-bond
Arene-H
H-bond
H-bond
Arene-H
Arene-H
H-bond
H-bond
H-bond
H-bond
H-bond
H-bond
H-bond
34c
34d
34e
-13.20
-13.81
-12.51
3.23
2.84
CO
Pyridine ring
CF3
2.82
2.99
CO
NH
NH
34f
34g
-12.48
-12.40
NH pyrrole
N pyridine
Br-Azaindole
NH
CO
Br-Phenyl
Br-Phenyl
3.46
2.88
3.55
3.27
2.63
3.66
3.08
35
-12.09
3. Conclusion
Two series of Azaindole-based derivatives were synthesized and tested against V600E B-RAF
kinase to investigate their potency. The biological evaluation data revealed that the substitution
arrangement modification in the second set of azaindole-based derivatives has a remarkable
effect in enhancing the activity. In the first set of compound, SAR studies revealed that most of
sulfonamide moiety containing derivatives 24 and 25 have higher inhibition activity over the
amide bearing derivatives 26 and 27. Additionally, the terminal propyl linker containing
derivatives 25a-h showed higher potency than the one with ethyl linker 24a-h. Compound 25d
with para bromo group is the highest active V600EBRAF inhibitor (IC50 = 0.35 µM) among the first
Azaindole-based set. The substitution diversity on the urea moiety containing second set
showed variable inhibitory activities. Compound 35 showed that most potential inhibitory
effect against V600EBRAF (IC50 = 0.080 µM). Among the synthesised compounds of the two series,
compound 35 exhibited cytotoxicity activity against different panel of human cancer cell lines.
Overall, the second set of the Azaindole derivatives represent a therapeutically promising
scaffold for future structural optimization to develop a new anticancer agent.
4. Experimental
4.1. Chemistry

General
The intermediate compounds 8 and 20-23 as well as the target compounds 24a–h, 25a–h, 26a–
g and 27a–g were purified by flash column chromatography using silica gel 60 (0.040–0.063
1
mm, 230–400 mesh ASTM) and technical grade solvents. H NMR and 13C NMR analyses were
carried out on a Bruker Avance 400 spectrometer using tetramethylsilane (TMS) as an internal
standard. Melting points were measured on a Walden Precision Apparatus Electrothermal 9300
apparatus and were uncorrected. LC-MS analysis was conducted using the following system:
Waters 2998 photodiode array detector, Waters 3100 mass detector, Waters SFO system
fluidics organizer, Waters 2545 binary gradient module, Waters reagent manager, Waters 2767
sample manager, Sunfire™ C18 column (4.6×50 mm, 5 μm particle size); Solvent gradient=95%
A at 0 min, 1% A at 5 min; solvent A: 0.035% TFA in water; solvent B: 0.035% TFA in MeOH; flow
rate=3.0 ml/min; the AUC was calculated using Waters MassLynx 4.1 software. The solvents and
liquid reagents were transferred using hypodermic syringes. All the solvents and reagents were
purchased from commercial companies, and used as such.
Synthesis of 1H-pyrrolo[2,3-b]pyridine (5)
It was carried out utilizing the 4-step procedure previously reported in the literature[34].
Synthesis of 1-(2-(methylthio)pyrimidin-4-yl)-1H-pyrrolo[2,3-b]pyridine (7)
To a solution of compound 5 (10 g, 84.74 mmol) in DMF (40 ml) under N2 is added sodium
hydride (3.55 g of a 60% suspension in mineral oil, 88.8 mmol) and stirred for 1 h. 4-chloro-2-
(methylthio)pyrimidine 6 (15 g, 93.21 mmol, 1.1 eq) in DMF is slowly added and the reaction
stirred for 12 h at 80 °C. The reaction is quenched with a NaHCO3 solution before extracting
with DCM. The combined extracts were washed with water and brine then dried over
anhydrous Na2SO4. The solvent is removed in vacuo to give the title intermediate 7.
Yield: 85%. m.p.: 137 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.74 (d, J = 5.6 Hz, 1H, Ar-H), 8.7 (d, J =
5.6 Hz, 1H, Ar-H), 8.43 (m, 2H, Ar-H), 8.14 (dd, J = 1.6 Hz, J = 8 Hz, 1H, Ar-H), 7.34 (q, J = 3.2 Hz,
1H, Ar-H), 6.86 (d, J = 4 Hz, 1H, Ar-H), 2.6 (s, 3H, SCH3). 13C NMR (100 MHz, DMSO-d6) δ 172.74

(Ar-C), 159.61 (Ar-C), 156.74 (Ar-C), 148.12 (Ar-C), 144.36 (Ar-C), 130.66 (Ar-C), 125.54 (Ar-C),
124.14 (Ar-C), 119.95 (Ar-C), 104.27 (Ar-C), 105.63 (Ar-C), 14.1 (SCH3).
Synthesis of 1-(2-(methylsulfonyl)pyrimidin-4-yl)-1H-pyrrolo[2,3-b]pyridine (8)
To a solution of compound 7 (10 g, 41.32 mmol) in methanol (100 ml) is added a solution of
oxone (84.5 g, 124 mmol, 3 eq.) in water (100 ml) dropwise at rt and stirred at rt for 48 h. The
reaction mixture was concentrated under vacuo. The reaction mass was extracted with
dichloromethane (20 ml) and the organic layer was separated. The aqueous layer was extracted
with dichloromehane. The combined extracts were dried over anhydrous Na2SO4. The solvent
was evaporated under vacuo. The crude residue was purified by column chromatography
(hexane:ethyl acetate; 2:1) to give the titled product 8.
Yield: 80%. m.p.: 197 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.51 (t, J = 4.4 Hz, 2H, Ar-H), 8.34 (d, J =
4 Hz, 1H, Ar-H), 8.26 (d, J = 6.8 Hz, 1H, Ar-H), 8.14 (q, J = 1.6 Hz, 1H, Ar-H), 7.34 (t, J = 3.2 Hz, 1H,
Ar-H), 6.85 (d, J = 4 Hz, 1H, Ar-H), 3.53 (s, 3H, SO2CH3). 13C NMR (100 MHz, DMSO-d6) δ 164.79
(Ar-C), 160.84 (Ar-C), 151.97 (Ar-C), 144.08 (Ar-C), 142.65 (Ar-C), 130.56 (Ar-C), 125.86 (Ar-C),
119.17 (Ar-C), 115.99 (Ar-C), 104.09 (Ar-C), 103.98 (Ar-C), 100.68 (Ar-C), 95.64 (Ar-C), 46.07
(SO2CH3).
Synthesis of N1-sustituted ethane(propane)-1,2(3)diamine (20a-h, 21a-h, 22a-g, and 23a-g)
Synthesis of side chains (20a-h, 21a-h, 22a-g, and 23a-g) was carried out through a previously
reported pathway[35, 36].
Synthesis of N-(2-((4-(1H-pyrrolo[2,3-b]pyridin-1-yl)pyrimidin-2-
yl)amino)ethyl(propyl))substituted sulfonamide(amide) (24a-h, 25a-h, 26a-g, and 27a-g)
To a solution of compound 8 (0.36 mmol) in DMSO (4 ml), compounds 20a-h, 21a-h, 22a-g, and
23a-g (0.54 mmol) and DIPEA (500 mg, 3 mmol) were added. The reaction mixture was stirred
at 90 °C for 8 h. The reaction mixture was cooled and extracted between Ethyl acetate and
water. The organic extract was dried over anhydrous Na2SO4. The solvent was evaporated
under vacuo. The crude residue was purified by column chromatography (hexane:ethyl acetate;
2:1) to give the titled products 24a-h, 25a-h, 26a-g, and 27a-g.

N-(2-((4-(1H-pyrrolo[2,3-b]pyridin-1-yl)pyrimidin-2-yl)amino)ethyl)benzenesulfonamide (24a)
Yield: 70%. m.p.: 221 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.43 (dd, J = 1.6 Hz, J = 8.2 Hz, 2H, Ar-
H), 8.38 (d, J = 5.6 Hz, 1H, Ar-H), 8.21 (d, J = 5.6 Hz, 1H, Ar-H), 8.13 (dd, J = 1.6 Hz, J = 8 Hz, 1H,
Ar-H), 7.83 (d, J = 6.8 Hz, 2H, Ar-H), 7.6 (m, 3H, Ar-H), 7.31 (t, J = 4.8 Hz, 2H, Ar-H), 6.83 (d, J =
3.6 Hz, 1H, Ar-H), 3.42 (t, J = 6.8 Hz, 2H, NHCH2-), 3 (d, J = 6.4 Hz, 2H, -CH2-NHSO2). 13C NMR
(100 MHz, DMSO-d6) δ 162.28 (Ar-C), 160.54 (Ar-C), 156.43 (Ar-C), 146.09 (Ar-C), 143.94 (Ar-C),
140.96 (Ar-C), 132.82 (Ar-C), 130.17 (Ar-C), 129.66 (Ar-C), 126.86 (Ar-C), 123.44 (Ar-C), 118.58
(Ar-C), 104.51 (Ar-C), 40.6 (NHCH2-), 39.35 (CH2-NHSO2). LC/MS calculated for C19H18N6O2S is
394.12, Found: 394.45 (M+1)+.
N-(2-((4-(1H-pyrrolo[2,3-b]pyridin-1-yl)pyrimidin-2-yl)amino)ethyl)-4-
fluorobenzenesulfonamide (24b)
Yield: 65%. m.p.: 185-187 °C. 1H NMR (400 MHz, CDCl3) δ 8.42 (m, 4H, Ar-H), 8.27 (d, J = 4 Hz,
1H, Ar-H), 7.9 (q, J = 4 Hz, 2H, Ar-H), 7.52 (q, J = 7.2 Hz, 1H, Ar-H), 7.21 (q, J = 12.2 Hz, 1H, Ar-H),
7.12 (t, J = 8.4 Hz, 1H, Ar-H), 6.65 (d, J = 4 Hz, 1H, Ar-H), 6.5 (brs, 1H, Ar-H), 6.14 (brs, 1H,
NHCH2), 5.50 (s, 1H, NHSO2), 3.64 (d, J = 5.2 Hz, 2H, CH2-CH2-NHSO2), 3.31 (s, 2H, CH2-CH2-
NHSO2). 13C NMR (100 MHz, CDCl3) δ 161.97 (Ar-C), 159.32 (Ar-C), 156.95 (Ar-C), 148.27 (Ar-C),
142.5 (Ar-C), 129.69 (Ar-C), 129.6 (Ar-C), 129.28 (Ar-C), 129.04 (Ar-C), 126.92 (Ar-C), 125.14 (Ar-
C), 122.96 (Ar-C), 117.68 (Ar-C), 116.11 (Ar-C), 104.26 (Ar-C), 41.24 (-CH2-NHSO2). LC/MS
calculated for C19H17FN6O2S is 412.11, Found: 413.1 (M+1)+.
N-(2-((4-(1H-pyrrolo[2,3-b]pyridin-1-yl)pyrimidin-2-yl)amino)ethyl)-4-
chlorobenzenesulfonamide (24c)
Yield: 67%. m.p.: 181-183 °C. 1H NMR (400 MHz, CDCl3) δ 8.41 (d, J = 9.6 Hz, 2H, Ar-H), 8.29 (s,
1H, Ar-H), 7.96 (d, J = 6.4 Hz, 1H, Ar-H), 7.85 (d, J = 5.2 Hz, 2H, Ar-H), 7.48 (t, J = 10 Hz, 3H, Ar-H),
7.29 (s, 1H, Ar-H), 7.22 (s, 1H, Ar-H), 6.66 (s, 1H, Ar-H), 5.85 (s, 1H, NHCH2), 5.38 (s, 1H, NHSO2),
3.64 (s, 2H, NHCH2-), 3.32 (s, 2H, CH2-NHSO2). 13C NMR (100 MHz, CDCl3) δ 162.37 (Ar-C),
159.21(Ar-C), 156.83(Ar-C), 148.04 (Ar-C), 143.94 (Ar-C), 140.99 (Ar-C), 132.02 (Ar-C), 130.16
(Ar-C), 129.64 (Ar-C), 126.87 (Ar-C), 123.94 (Ar-C), 118.52 (Ar-C), 104.5 (Ar-C), 42.18 (NHCH2-),
39.36 (CH2-NHSO2). LC/MS calculated for C19H17 ClN6O2S is 428.08, Found: 429.1 (M+1)+.

N-(2-((4-(1H-pyrrolo[2,3-b]pyridin-1-yl)pyrimidin-2-yl)amino)ethyl)-4-
bromobenzenesulfonamide (24d)
Yield: 72%. m.p.: 189-191 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.42 (dd, J = 1.44 Hz, J = 7.4 Hz,
1H, Ar-H), 8.38 (d, J = 5.48 Hz, 1H, Ar-H), 8.22 (d, J = 5.48 Hz, 1H, Ar-H), 8.13 (dd, J = 5.48 Hz, J =
8.4 Hz, 1H, Ar-H), 7.93 (s, 1H, Ar-H), 7.75 (q, J = 5.6 Hz, 3H, Ar-H), 7.31 (q, J = 3.04 Hz, 2H, Ar-H),
6.82 (d, J = 3.92 Hz, 1H, Ar-H), 3.4 (d, J = 6.44 Hz, 2H, NHCH2), 3.02 (t, J = 6.44 Hz, 2H,-CH2-
NHSO2). 13C NMR (100 MHz, DMSO-d6) δ 162.12 (Ar-C), 148.06 (Ar-C), 143.93 (Ar-C), 140.29 (Ar-
C), 132.71 (Ar-C), 130.15 (Ar-C), 128.93 (Ar-C), 126.58 (Ar-C), 123.94 (Ar-C), 118.51 (Ar-C),
104.51 (Ar-C), 42.05 (NHCH2-), 39.36 (-CH2-NHSO2). LC/MS calculated for C19H17 BrN6O2S is
473.3, Found: 474.6 (M+1)+.
.
N-(2-((4-(1H-pyrrolo[2,3-b]pyridin-1-yl)pyrimidin-2-yl)amino)ethyl)-4-
methoxybenzenesulfonamide (24e)
Yield: 55%. m.p.: 195-197 °C. 1H NMR (400 MHz, CDCl3) δ 8.42 (t, J = 1.64 Hz, 2H, Ar-H), 8.36 (d,
J = 5.64 Hz, 1H, Ar-H), 8.3 (d, J = 3.96 Hz, 1H, Ar-H), 7.96 (q, J = 1.56 Hz, 1H, Ar-H), 7.78 (s, 1H,
Ar-H), 7.76 (s, 1H, Ar-H), 7.29 (s, 2H, Ar-H), 7.22 (d, J = 4.8 Hz, 1H, Ar-H), 6.92 (s, 1H, Ar-H), 6.89
(s, 1H, Ar-H), 6.66 (d, J = 3.96 Hz, 1H, Ar-H), 3.78 (s, 3H, OCH3), 3.63 (q, J = 5.8 Hz, 2H, NHCH2-),
3.29 (q, J = 5.6 Hz, 2H, CH2-CH2-NHSO2). 13C NMR (100 MHz, CDCl3) δ 162.49 (Ar-C), 162.14 (Ar-
C), 156.38 (Ar-C), 148.04 (Ar-C), 143.91 (Ar-C), 132.56 (Ar-C), 130.13 (Ar-C), 129.05 (Ar-C),
123.093 (Ar-C), 118.5 (Ar-C), 114.72 (Ar-C), 104.46 (Ar-C), 55.96 (OCH3), 42.09 (NHCH2-), 39.35
(CH2-NHSO2). LC/MS calculated for C20H20N6O3S is 424.4, Found: 425.1 (M+1)+.
N-(2-((4-(1H-pyrrolo[2,3-b]pyridin-1-yl)pyrimidin-2-yl)amino)ethyl)-3-
(trifluoromethyl)benzenesulfonamide (24f)
Yield: 63%. m.p.: 199-201 °C. 1H NMR (400 MHz, CDCl3) δ 8.44 (s, 1H, Ar-H), 8.36 (d, J = 5.6 Hz,
2H, Ar-H), 8.26 (d, J = 3.96 Hz, 1H, Ar-H), 8.11 (s, 1H, Ar-H), 7.97 (d, J = 1.6 Hz, 1H, Ar-H), 7.76 (d,
J = 7.84 Hz, 1H, Ar-H), 7.58 (t, J = 7.84 Hz, 1H, Ar-H), 7.29 (s, 1H, Ar-H), 7.23 (q, J = 3.04 Hz, 1H,
Ar-H), 6.67 (d, J = 3.96 Hz, 1H, Ar-H), 6.45 (brs, 1H, NHCH2), 5.34 (brs, 1H, NHSO2), 3.65 (q, J =

3.64 Hz, 2H, NH-CH2-), 3.35 (q, J = 5.28 Hz, 2H, CH2-NHSO2). 13C NMR (100 MHz, CDCl3) δ 162.16
(Ar-C), 148.04 (Ar-C), 143.94 (Ar-C), 142.28 (Ar-C), 131.31 (Ar-C), 130.97 (Ar-C), 130.44 (Ar-C),
130.16 (Ar-C), 129.58 (Ar-C), 123.93 (Ar-C), 123.4 (Ar-C), 118.52 (Ar-C), 104.48 (Ar-C), 41.05
(NHCH2-), 39.36 (-CH2-NHSO2). LC/MS calculated for C20H17F3N6O2S is 462.45, Found: 463.1
(M+1)+.
N-(2-((4-(1H-pyrrolo[2,3-b]pyridin-1-yl)pyrimidin-2-yl)amino)ethyl)-4-
methylbenzenesulfonamide (24g)
Yield: 67%. m.p.: 221-223 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.42 (q, J = 3.24 Hz, 2H, Ar-H), 8.37
(d, J = 5.48 Hz, 1H, Ar-H), 8.21 (d, J = 5.52 Hz, 1H, Ar-H), 8.13 (d, J = 1.44 Hz, 1H, Ar-H), 7.69 (d, J
= 8.12 Hz, 3H, Ar-H), 7.35 (s, 1H, Ar-H), 7.32 (s, 1H, Ar-H), 7.3 (t, J = 4.76 Hz, 1H, Ar-H), 6.82 (d, J
= 3.96 Hz, 1H, Ar-H), 3.4 (t, J = 6.52 Hz, 2H, NHCH2-), 2.97 (s, 2H, CH2-NHSO2), 2.3 (s, 3H, CH3).
13C NMR (100 MHz, DMSO-d6) δ 148.04 (Ar-C), 143.93 (Ar-C), 142.99 (Ar-C), 130.16 (Ar-C),
130.03 (Ar-C), 126.93 (Ar-C), 123.93 (Ar-C), 118.51 (Ar-C), 104.48 (Ar-C), 41.01 (CH2-CH2-NHSO2),
39.35 (NHCH2-), 21.33 (CH3). LC/MS calculated for C20H20N6O2S is 408.48, Found: 409.1 (M+1)+.
N-(2-((4-(1H-pyrrolo[2,3-b]pyridin-1-yl)pyrimidin-2-yl)amino)ethyl)-3-
bromobenzenesulfonamide (24h)
Yield: 59%. m.p.: 185-187 °C. 1H NMR (400 MHz, CDCl3) δ 8.41 (q, J = 4 Hz, 2H, Ar-H), 8.34 (d, J =
5.4 Hz, 1H, Ar-H), 8.29 (d, J = 3.48 Hz, 1H, Ar-H), 7.96 (d, J = 7.56 Hz, 1H, Ar-H), 7.85 (d, J = 7.44
Hz, 2H, Ar-H), 7.46 (t, J = 7.44 Hz, 2H, Ar-H), 7.29 (s, 1H, Ar-H), 7.22 (d, J = 4.88 Hz, 1H, Ar-H),
6.66 (d, J = 3.6 Hz, 1H, Ar-H), 5.88 (s, 1H, NHCH2), 5.41 (s, 1H, NHSO2), 3.64 (d, J = 5.36 Hz, 2H,
NHCH2-), 3.32 (d, J = 5.08 Hz, 2H, CH2-NHSO2). 13C NMR (100 MHz, CDCl3) δ 161.95 (Ar-C),
159.32 (Ar-C), 156.93 (Ar-C), 148.29 (Ar-C), 143.47 (Ar-C), 139.94 (Ar-C), 132.52 (Ar-C), 129.25
(Ar-C), 129.03 (Ar-C), 126.93 (Ar-C), 125.22 (Ar-C), 123.97 (Ar-C), 117.95 (Ar-C), 104.2 (Ar-C),
101.56 (Ar-C), 41.28 (NH-CH2-), 29.7 (-CH2-NHSO2). LC/MS calculated for C19H17 BrN6O2S is 473.3,
Found: 474.3 (M+1)+.
N-(3-((4-(1H-pyrrolo[2,3-b]pyridin-1-yl)pyrimidin-2-yl)amino)propyl)benzenesulfonamide (25a)

Yield: 72%. m.p.: 165-167 °C. 1H NMR (400 MHz, CDCl3) δ 8.42 (dd, J = 1.28 Hz, J = 7.2 Hz,1H, Ar-
H), 8.34 (s, 2H, Ar-H), 8.31 (d, J = 3.92 Hz, 1H, Ar-H), 7.95 (dd, J = 1.28 Hz, J = 7.2 Hz, 1H, Ar-H),
7.79 (d, J = 6.92 Hz, 2H, Ar-H), 7.49 (t, J = 7.24 Hz, 1H, Ar-H), 7.42 (t, J = 7.44 Hz, 2H, Ar-H), 7.21
(q, J = 2.96 Hz, 1H, Ar-H), 6.66 (d, J = 3.88 Hz, 1H, Ar-H), 5.36 (brs, 1H, NHSO2), 3.58 (q, J = 6.04
Hz, 2H, NH-CH2-CH2-), 3.08 (q, J = 6.12 Hz, 2H, CH2-NHSO2), 1.82 (t, J = 5.92 Hz, 2H, CH2-CH2-CH2-
). 13C NMR (100 MHz, CDCl3) δ 162.14 (Ar-C), 159.25 (Ar-C), 156.93 (Ar-C), 148.26 (Ar-C), 143.45
(Ar-C), 140.19 (Ar-C), 132.4 (Ar-C), 129.6 (Ar-C), 129.01 (Ar-C), 126.81 (Ar-C), 125.21 (Ar-C),
123.95 (Ar-C), 117.91 (Ar-C), 101.19 (Ar-C), 101.11 (Ar-C), 41.07 (NH-CH2-), 37.86 (CH2-NHSO2),
29.7 (CH2-CH2-CH2-). LC/MS calculated for C20H20N6O2S is 408.48, Found: 409.1(M+1)+.
N-(3-((4-(1H-pyrrolo[2,3-b]pyridin-1-yl)pyrimidin-2-yl)amino)propyl)-4-
fluorobenzenesulfonamide (25b)
Yield: 70%. m.p.: 141- 143°C. 1H NMR (400 MHz, CDCl3) δ 8.42 (d, J = 4.04 Hz, 1H, Ar-H), 8.38 (d,
J = 5.36 Hz, 1H, Ar-H), 8.33 (d, J = 5.56 Hz, 1H, Ar-H), 8.27 (t, J = 4.08 Hz, 1H, Ar-H), 7.95 (d, J =
7.6 Hz, 1H, Ar-H), 7.85 (d, J = 3.96 Hz, 2H, Ar-H), 7.49 (m, 1H, Ar-H), 7.21 (d, J = 2.52 Hz, 1H, Ar-
H), 7.12 (t, J = 8.48 Hz, 1H, Ar-H), 6.65 (d, J = 3.72 Hz, 1H, Ar-H), 5.5 (brs, 1H, NHSO2), 3.63 (d, J =
5.16 Hz, 2H,NH- CH2-CH2- 3.31 (s, 2H, -CH2-NHSO2), 1.82 (t, J = 6 Hz, 2H, CH2-CH2-CH2-). 13C NMR
(100 MHz, CDCl3) δ 166.11 (Ar-C), 163.59 (Ar-C), 162.11 (Ar-C), 159.16 (Ar-C), 156.96 (Ar-C),
148.24 (Ar-C), 143.49 (Ar-C), 129.73 (Ar-C), 129.64 (Ar-C), 129.57 (Ar-C), 129.29 (Ar-C), 125.15
(Ar-C), 123.93 (Ar-C), 117.96 (Ar-C), 116.53 (Ar-C), 116.11 (Ar-C), 104.27 (Ar-C), 40.1 (NH-CH2-
CH2), 37.89 (-CH2-NHSO2), 29.69 (CH2-CH2-CH2-). LC/MS calculated for C20H19 FN6O2S is 426.47,
Found: .426.1(M)+.
N-(3-((4-(1H-pyrrolo[2,3-b]pyridin-1-yl)pyrimidin-2-yl)amino)propyl)-4-
chlorobenzenesulfonamide (25c)
Yield: 72%. m.p.: 161-163 °C. 1H NMR (400 MHz, CDCl3) δ 8.42 (dd, J = 1.4 Hz, J = 7.6 Hz,1H, Ar-
H), 8.33 (s, 2H, Ar-H), 8.31 (d, J = 3.92 Hz, 1H, Ar-H), 7.94 (dd, J = 1.44 Hz, J = 7.8 Hz, 1H, Ar-H),
7.79 (d, J = 7.08 Hz, 2H, Ar-H), 7.49 (t, J = 7.28 Hz, 1H, Ar-H), 7.42 (t, J = 7.48 Hz, 2H, Ar-H), 7.2
(q, J = 3.04 Hz, 1H, Ar-H), 6.66 (d, J = 3.92 Hz, 1H, Ar-H), 5.41 (brs, 1H, NHSO2), 3.48 (q, J = 6.32

Hz, 2H, NH-CH2-CH2-), 3.08 (q, J = 6.28 Hz, 2H, CH2-CH2-NHSO2), 1.81 (t, J = 5.92 Hz, 2H, CH2-CH2-
CH2-). 13C NMR (100 MHz, CDCl3) δ 162.11 (Ar-C), 159.21 (Ar-C), 156.93 (Ar-C), 148.26 (Ar-C),
143.45 (Ar-C), 140.2 (Ar-C), 132.39 (Ar-C), 129.25 (Ar-C), 129.01 (Ar-C), 126.81 (Ar-C), 125.21
(Ar-C), 123.95 (Ar-C), 117.91 (Ar-C), 104.19 (Ar-C), 101.06 (Ar-C), 40.09 (NH-CH2-CH2-), 37.88 (-
CH2-CH2-NHSO2), 29.7 (CH2-CH2-CH2-). LC/MS calculated for C20H19 ClN6O2S is 442.97, Found:
.426.1(M)+.
N-(3-((4-(1H-pyrrolo[2,3-b]pyridin-1-yl)pyrimidin-2-yl)amino)propyl)-4-
bromobenzenesulfonamide (25d)
Yield: 63%. m.p.: 160-162 °C. 1H NMR (400 MHz, CDCl3) δ 8.4 (dd, J = 1.53 Hz, J = 8.2 Hz,1H, Ar-
H), 8.3 (t, J = 3.96 Hz, 2H, Ar-H), 7.93 (dd, J = 1.56 Hz, J = 8.4 Hz, 1H, Ar-H), 7.79 (d, J = 7.25 Hz,
2H, Ar-H), 7.48 (t, J = 7.28 Hz, 1H, Ar-H), 7.41 (t, J = 7.56 Hz, 2H, Ar-H), 7.19 (q, J = 3.04 Hz, 1H,
Ar-H), 6.64 (d, J = 3.96 Hz, 1H, Ar-H), 5.49 (brs, 1H, NHSO2), 3.57 (q, J = 6.32 Hz, 2H,NH-CH2-CH2),
3.07 (q, J = 6.28 Hz, 2H, CH2-CH2-NHSO2), 1.81 (t, J = 5.96 Hz, 2H, CH2-CH2-CH2). 13C NMR (100
MHz, CDCl3) δ 162.07 (Ar-C), 159.16 (Ar-C), 156.92 (Ar-C), 148.24 (Ar-C), 143.43 (Ar-C), 140.16
(Ar-C), 132.4 (Ar-C), 129.24 (Ar-C), 129.02 (Ar-C), 126.82 (Ar-C), 125.22 (Ar-C), 123.94 (Ar-C),
117.9 (Ar-C), 101.17 (Ar-C), 100.99 (Ar-C), 40.15 (NH-CH2-CH2-), 37.93 (-CH2-NHSO2), 29.7 (CH2-
CH2-CH2-). LC/MS calculated for C20H19 BrN6O2S is 487.38, Found: 488.1(M+1)+.
N-(3-((4-(1H-pyrrolo[2,3-b]pyridin-1-yl)pyrimidin-2-yl)amino)propyl)-4-
methoxybenzenesulfonamide (25e)
Yield: 65%. m.p.: 157-159 °C. 1H NMR (400 MHz, CDCl3) δ 8.42 (dd, J = 1.52 Hz, J = 8.4 Hz, 1H, Ar-
H), 8.34 (s, 2H, Ar-H), 8.32 (d, J = 3.92 Hz, 1H, Ar-H), 7.95 (dd, J = 1.56 Hz, J = 8.4 Hz, 1H, Ar-H),
7.72 (d, J = 8.32 Hz, 2H, Ar-H), 7.21 (q, J = 3 Hz, 1H, Ar-H), 6.87 (d, J = 8.64 Hz, 2H, Ar-H), 6.66 (d,
J = 3.92 Hz, 1H, Ar-H), 5.4 (s, 1H, Ar-H), 3.79 (s, 3H, OCH3), 3.58 (q, J = 6.08 Hz, 2H, NH-CH2-CH2-
CH2), 3.05 (q, J = 6.2 Hz, 2H, -CH2-NHSO2), 1.81 (t, J = 6.04 Hz, 2H, CH2-CH2-CH2). 13C NMR (100
MHz, CDCl3) δ 162.65 (Ar-C), 162.11 (Ar-C), 159.25 (Ar-C), 156.92 (Ar-C), 148.26 (Ar-C), 143.43
(Ar-C), 131.75 (Ar-C), 129.25 (Ar-C), 129.11 (Ar-C), 128.97 (Ar-C), 125.24 (Ar-C), 123.95 (Ar-C),
117.9 (Ar-C), 114.15 (Ar-C), 104.15 (Ar-C), 101.05 (Ar-C), 55.49 (OCH3), 40.07 (NH-CH2-), 37.92

(CH2-NHSO2), 30.03 (CH2-CH2-CH2). LC/MS calculated for C21H22N6O3S is 438.51, Found:
439.1(M+1)+.
N-(3-((4-(1H-pyrrolo[2,3-b]pyridin-1-yl)pyrimidin-2-yl)amino)propyl)-3-
(trifluoromethyl)benzenesulfonamide (25f)
Yield: 75%. m.p.: 139-141 °C. 1H NMR (400 MHz, CDCl3) δ 8.37 (t, J = 0.94 Hz, 1H, Ar-H), 8.29 (s,
2H, Ar-H), 8.27 (d, J = 3.96 Hz, 1H, Ar-H), 8.11 (s, 1H, Ar-H), 7.97 (d, J = 7.28 Hz, 1H, Ar-H), 7.9
(dd, J = 1.4 Hz, J = 7.8 Hz,1H, Ar-H), 7.74 (d, J = 7.36 Hz, 1H, Ar-H), 7.54 (t, J = 6.4 Hz, 1H, Ar-H),
7.16 (q, J = 2.96 Hz, 1H, Ar-H), 6.61 (d, J = 3.92 Hz, 1H, Ar-H), 5.62 (brs, 1H, NH-SO2), 3.57 (q, J =
6.12 Hz, 2H, NH-CH2-CH2), 3.1 (d, J = 5.72 Hz, 2H, CH2-CH2-NHSO2), 1.83 (t, J = 5.44 Hz, 2H, CH2-
CH2-CH2-). 13C NMR (100 MHz, CDCl3) δ 167.79 (Ar-C), 165.04 (Ar-C), 158.97 (Ar-C), 156.94 (Ar-
C), 148.2 (Ar-C), 143.42 (Ar-C), 141.65 (Ar-C), 132.11 (Ar-C), 130.94 (Ar-C), 129.85 (Ar-C), 128.99
(Ar-C), 127.27 (Ar-C), 124.55 (Ar-C), 117.9 (Ar-C), 104.21 (Ar-C), 101.01 (Ar-C), 40.22 (NH-CH2-
CH2), 37.97 (-CH2-NHSO2), 29.69 (CH2-CH2-CH2-). LC/MS calculated for C21H19 F3N6O2S is 476.48,
Found: 477.1(M+1)+.
N-(3-((4-(1H-pyrrolo[2,3-b]pyridin-1-yl)pyrimidin-2-yl)amino)propyl)-4-
methylbenzenesulfonamide (25g)
Yield: 65%. m.p.: 181-183 °C. 1H NMR (400 MHz, CDCl3) δ 8.44 (dd, J = 1.48 Hz, J = 7.4 Hz, 1H, Ar-
H), 8.36 (s, 2H, Ar-H), 8.32 (d, J = 4 Hz, 1H, Ar-H), 7.96 (dd, J = 1.52 Hz, J = 8.6 Hz,1H, Ar-H), 7.66
(d, J = 7.48 Hz, 2H, Ar-H), 7.29 (s, 1H, Ar-H), 7.21 (m, 3H, Ar-H), 6.67 (d, J = 3.96 Hz, 1H, Ar-H),
5.24 (s, 1H, NHSO2), 3.6 (q, J = 6.44 Hz, 2H, NH-CH2-CH2-), 3.06 (q, J = 6.36 Hz, 2H,-CH2-SO2),
2.35 (s, 3H, CH3), 1.81 (t, J = 6.04 Hz, 2H, CH2-CH2-CH2). 13C NMR (100 MHz, CDCl3) δ 162.19 (Ar-
C), 148.29 (Ar-C), 143.46 (Ar-C), 143.13 (Ar-C), 129.61 (Ar-C), 129.26 (Ar-C), 126.87 (Ar-C),
125.21 (Ar-C), 123.95 (Ar-C), 117.91 (Ar-C), 101.17 (Ar-C), 40.02 (NH-CH2-), 37.83 (-CH2-NHSO2),
30.22 (CH2-CH2-CH2-), 21.41 (CH3). LC/MS calculated for C21H22 N6O2S is 422.51, Found:
423.0(M+1)+.
N-(3-((4-(1H-pyrrolo[2,3-b]pyridin-1-yl)pyrimidin-2-yl)amino)propyl)-3-
bromobenzenesulfonamide (25h)

Yield: 75%. m.p.: 165-167 °C. 1H NMR (400 MHz, CDCl3) δ 8.42 (t, J = 0.96 Hz, 1H, Ar-H), 8.33 (s,
2H, Ar-H), 8.31 (d, J = 3.92 Hz, 1H, Ar-H), 7.94 (dd, J = 1.12 Hz, J = 7.2 Hz, 1H, Ar-H), 7.79 (d, J =
6.92 Hz, 2H, Ar-H), 7.49 (t, J = 7.2 Hz, 1H, Ar-H), 7.42 (t, J = 7.36 Hz, 2H, Ar-H), 7.2 (q, J = 2.96 Hz,
1H, Ar-H), 6.65 (d, J = 3.84 Hz, 1H, Ar-H), 5.42 (brs, 1H, NHSO2), 3.58 (q, J = 6.24 Hz, 2H, NH-
CH2), 3.07 (q, J = 6.2 Hz, 2H, CH2-NHSO2), 1.81 (t, J = 5.84 Hz, 2H, CH2-CH2-CH2-NHSO2). 13C NMR
(100 MHz, CDCl3) δ 172.61 (Ar-C), 162.1 (Ar-C), 159.18 (Ar-C), 156.93 (Ar-C), 148.25 (Ar-C),
143.44 (Ar-C), 140.18 (Ar-C), 132.39 (Ar-C), 129.25 (Ar-C), 129.01 (Ar-C), 126.81 (Ar-C), 125.22
(Ar-C), 123.94 (Ar-C), 117.91 (Ar-C), 104.18 (Ar-C), 101.05 (Ar-C), 40.1 (NH-CH2-CH2), 37.89 (CH2-
NHSO2), 30.1 (CH2-CH2-CH2-NHSO2). LC/MS calculated for C20H19 BrN6O2S is 487.38, Found:
488.1(M+1)+.
N-(2-((4-(1H-pyrrolo[2,3-b]pyridin-1-yl)pyrimidin-2-yl)amino)ethyl)benzamide (26a)
Yield: 74%. m.p.: 199-201 °C. 1H NMR (400 MHz, CDCl3) δ 8.43 (t, J = 5.6 Hz, 2H, Ar-H), 8.34 (d, J
= 3.48 Hz, 2H, Ar-H), 7.94 (d, J = 7.64 Hz, 1H, Ar-H), 7.76 (d, J = 6.84 Hz, 2H, Ar-H), 7.4 (s, 1H, Ar-
H), 7.29 (s, 2H, Ar-H), 7.25 (q, J = 4.84 Hz, 1H, Ar-H), 6.63 (d, J = 3.76 Hz, 1H, Ar-H), 3.79 (d, J =
14.2 Hz, 4H, CH2-CH2-NHCO). 13C NMR (100 MHz, CDCl3) δ 167.95 (C=O), 157.19 (Ar-C), 148.3
(Ar-C), 143.47 (Ar-C), 131.32 (Ar-C), 129.32 (Ar-C), 128.36 (Ar-C), 126.94 (Ar-C), 125.3 (Ar-C),
124.05 (Ar-C), 118.09 (Ar-C), 104.51 (Ar-C), 101.28 (Ar-C), 41.12 (NHCH2-), 29.7 (CH2-NHCO).
LC/MS calculated for C20H18N6O is 358.41, Found: 459.1(M+1)+.
N-(2-((4-(1H-pyrrolo[2,3-b]pyridin-1-yl)pyrimidin-2-yl)amino)ethyl)-4-fluorobenzamide (26b)
Yield: 78%. m.p.: 205-207 °C. 1H NMR (400 MHz, CDCl3) δ 8.44 (t, J = 4.84 Hz, 2H, Ar-H), 8.32 (t, J
= 4 Hz, 2H, Ar-H), 7.95 (q, J = 1.36 Hz, 1H, Ar-H), 7.76 (s, 2H, Ar-H), 7.23 (q, J = 4.8 Hz, 1H, Ar-H),
6.94 (s, 2H, Ar-H), 6.64 (d, J = 4 Hz, 1H, Ar-H), 3.81 (s, 2H, NHCH2-), 3.75 (s, 2H, CH2-NHCO). 13C
NMR (100 MHz, CDCl3) δ 166.86 (C=O), 157.21 (Ar-C), 148.27 (Ar-C), 143.54 (Ar-C), 129.38 (Ar-
C), 129.3 (Ar-C), 129.21 (Ar-C), 125.2 (Ar-C), 124.04 (Ar-C), 118.18 (Ar-C), 115.43 (Ar-C), 115.21
(Ar-C), 104.62 (Ar-C), 41.04 (2xCH2-). LC/MS calculated for C20H17FN6O is 376.4., Found:
376.9(M)+.
N-(2-((4-(1H-pyrrolo[2,3-b]pyridin-1-yl)pyrimidin-2-yl)amino)ethyl)-4-chlorobenzamide (26c)