Novel 5,6-disubstituted pyrrolo[2,3-d]pyrimidine derivatives as broad spectrum antiproliferative agents: Synthesis, cell based assays, kinase profile and molecular docking study

Article abstract of DOI:10.1016/j.bmc.2018.10.004

Two new series of 5-subtituted and 5,6-disubstituted pyrrolo[2,3-d]pyrimidine octamides (4a–o and 6a–g) and their corresponding free amines 5a–m and 7a–g have been synthesized and biologically evaluated for their antiproliferative activity against three h

Full text of DOI:10.1016/j.bmc.2018.10.004

Accepted Manuscript
Novel 5,6-disubstituted pyrrolo[2,3-d]pyrimidine derivatives as broad spectrum
antiproliferative agents: Synthesis, cell based assays, kinase profile and molec-
ular docking study
Ju-Hyeon Lee, Ashraf Kareem El-Damasy, Seon Hee Seo, Changdev G. Gadhe,
Ae NimPae, Nakcheol Jeong, Soon-Sun Hong, Gyochang Keum
PII:
S0968-0896(18)30781-8
https://doi.org/10.1016/j.bmc.2018.10.004
BMC 14563
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
31 May 2018
1 October 2018
7 October 2018
Please cite this article as: Lee, J-H., El-Damasy, A.K., Seo, S.H., Gadhe, C.G., NimPae, A., Jeong, N., Hong, S-S.,
Keum, G., Novel 5,6-disubstituted pyrrolo[2,3-d]pyrimidine derivatives as broad spectrum antiproliferative agents:
Synthesis, cell based assays, kinase profile and molecular docking study, Bioorganic & Medicinal Chemistry (2018),
doi: https://doi.org/10.1016/j.bmc.2018.10.004
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Novel 5,6-disubstituted pyrrolo[2,3-d]pyrimidine derivatives as broad
spectrum antiproliferative agents: Synthesis, cell based assays, kinase
profile and molecular docking study
a ,b, 1
a ,c, 1
a
d
d
Ju-Hyeon Lee
, Ashraf Kareem El-Damasy
,Seon Hee Seo , Changdev G. Gadhe , Ae NimPae ,
b
e,
a,
Nakcheol Jeong , Soon-Sun Hong, *, Gyochang Keum *
a
Center for Neuro-Medicine, Brain Science Institute, Korea Institute of Science and Technology,
Hwarangro 14-gil 5, Seongbuk-gu, Seoul 02792, Korea
b
Department of chemistry, Korea University; Anam-ro 145 Seongbuk-gu, Seoul 136–701, South
Korea
c
Department of Medicinal Chemistry, Faculty of Pharmacy, University of Mansoura, Mansoura 35516,
Egypt
d
Convergence Research Center for Diagnosis, Treatment and Care System of Dementia,Korea
Institute of Science and Technology, Hwarangro 14-gil 5, Seongbuk-gu, Seoul 02792, Korea
e
Department of Drug Development, College of Medicine, Inha University, 3-ga, Sinheung-
dong, Jung-gu, Incheon, 400-712, Republic of Korea
1These authors equally contributed to this work.
*
Corresponding authors.
E-mail addresses: gkeum@kist.re.kr (G. Keum), hongs@inha.ac.kr (S.-S. Hong)
Abstract
Two new series of 5-subtituted and 5,6-disubstituted pyrrolo[2,3-d]pyrimidine octamides (4a–o and
5
a–m) and their corresponding free amines 6a–g and 7a–g have been synthesized and biologically
evaluated for their antiproliferative activity against three human cancer cell lines. The 5,6-
disubstituted octamides 6d–g as well as the amine derivative 7b have shown the best anticancer
activity with single digit micromolar GI values over the tested cancer cells, and low cytotoxic effects
5
0
(
GI > 10.0 M) against HFF-1 normal cell. A structure activity relationship (SAR) study has been
5
0
established and disclosed that terminal octamide moiety at C2 as well as disubstitution with
fluorobenzyl piperazines at C5 and C6 of pyrrolo[2,3-d]pyrimidine are the key structural features
prerequisite for best antiproliferative activity. Moreover, the most active member 6f was tested for its
antiproliferative activity over a panel of 60 cancer cell lines at NCI, and exhibited distinct broad
spectrum anticancer activity with submicromolar GI50 and TGI values over multiple cancer cells.
Kinase profile of compound 6f over 53 oncogenic kinases at 10 M showed its highly selective
inhibitory activity towards FGFR4, Tie2 and TrkA kinases. The observed activity of 6f against TrkA
(
IC = 2.25 M), FGFR4 (IC = 6.71 M) and Tie2 (IC = 6.84 M) was explained by molecular
5
0
50
50
docking study, which also proposed that 6f may be a type III kinase inhibitor, binding to an allosteric
site rather than kinase hinge region. Overall, compound 6f may serve as a promising anticancer lead
compound that could be further optimized for development of potent anticancer agents.
Keywords
Pyrrolo[2,3-d]pyrimidine octamides; Mannich reaction; Antiproliferative activity; FGFR4 kinase;
Tie2; TrKA; Molecular docking

1
. Introduction
Cancer is still one of the most challenging medical problems in the globe, being the second common
cause of mortality after cardiovascular diseases [1]. In spite of the enormous research efforts and
profound improvements in cancer detection and treatment, accomplishment of optimal cancer
therapies is still difficult because of the severe side effects associated with classical cytotoxic
chemotherapeutics, as well as emergence of tumor resistance [2]. Over the last two decades, the
paradigm of anticancer drug development has been switched significantly from conventional
cytotoxic drugs to targeted agents that modulate protein kinases whose activities are more specifically
related to cancerous cells. Nowadays, several kinase inhibitors, derived from diverse scaffolds, are
being developed in attempt to achieve superior anticancer potency as well as minimal side effects [3].
Pyrrolo[2,3-d]pyrimidine, which is most commonly referred to as 7-deazapurine, represents one of the
privileged scaffolds for identification of protein kinase inhibitors. A growing body of literature
emphasized the interesting antiproliferative activities of various pyrrolo[2,3-d]pyrimidine derivatives
through inhibition of multiple receptor tyrosine protein kinases, such as epithelial growth factor
receptor (EGFR) [4], vascular endothelial growth factor receptor-1 (VEGFR-1) [5], platelet-derived
growth factor receptor- (PDGFR-) [4, 6–8], and insulin-like growth factor receptor (IGFR) [9, 10]
(Figure 1). Moreover, certain non-receptor tyrosine protein kinases were potently inhibited by
pyrrolo[2,3-d]pyrimidines (Figure 1). For example, Yoriko et al. discovered RK-20449, a pyrrolo[2,3-
d]pyrimidine member, as a potent hematopoietic cell kinase (HCK) for treatment of leukemia [11].
Recently, Francesca et al. reported the selective Src kinase inhibitory activity of certain pyrrolo[2,3-
d]pyrimidines in vitro active against glioblastoma [12].
O
O
F
HN
Br
H2N
HN
H2N
N
HN
N
N
N
O
N
N
H2N
N
N
N
H3C
N
N
S
N
N
H
N
H
N
O
Cl
VEGFR-2 & PDGFR- inhibitor
F
O
HN
IGF-1R inhibitor
c-Src kinase inhibitor
HCK inhibitor
Figure 1. Representative examples of antiproliferative pyrrolo[2,3-d]pyrimidine derivatives with
protein kinase inhibitory activities.
On the other hand, Mannich bases, the products of classical Mannich reaction, constitute a substantial
class of compounds found in several anticancer chemotherapeutics [13]. Mannich bases or aminoalkyl
derivatives of indoles, bioisostere of pyrrolopyrimidine, and other bicyclic ring systems exhibited
potent cytotoxic activities against various cancer cell lines through different mechanisms of action,
like induction of apoptosis [14], inhibition of isoprenylcysteine carboxyl methyltransferase (Imct)

[15], or inhibition of certain oncogenic kinases (VEGFR and PDGFR) [16] and phosphatidylinositol-
3
-kinase a (PI3K) inhibitors [17] (Figure 2).
N
Cl
CH2OH
N
Cl
R
N
N
H3C
R
N
N
N
NH
O
H
N
N
O
N
O
H
N
H
O
Isoprenylcysteine carboxyl
methyltransferase inhibitor
VEGFR-2 & PDGFR- inhibitor
PI3K inhibitor
Apoptosis inducer
Figure 2. Certain reported Mannich bases with promising antiproliferative activities. The aminoalkyl
moiety is illustrated in orange.
Despite of the extensive investigation of pyrrolopyrimidines as antiproliferative agents, only few
Mannich bases of pyrrolopyrimidine were reported to date, and none of them was examined for its
anticancer activity so far. In view of the aforementioned considerations, and in pursuit of our ongoing
endeavors to identify new potent antiproliferative agents [18–24], four new series of
pyrrolopyrimidine Mannich bases; 4a–o, 5a–m and their corresponding free amines 6a–g and 7a–g
have been synthesized for the first time (Figure 3). The antiproliferative activities of the target
compounds were examined against a panel of three human cancer cell lines; HCT-116 colorectal
carcinoma and MCF-7 and SK-BR3 breast cancer cells. Moreover, the kinase inhibitory activity for
the most potent derivative, compound 6b, was also profiled to investigate the potential mechanism of
action at molecular level.
R2
N
R1
O
N
N
X
O
N
N
HN
HN
R
N
N
N
R
H
H
N
N
H
H
N
R2
4
a-o, R = -CO(CH ) CH
2 6 3
6a-g, R = -CO(CH ) CH
2 6 3
5a-m, R = -H
7
a-g, R = -H
Figure 3. General structure of the target compounds.
2
. Results and discussion
2
.1. Chemistry
As shown in Scheme 1, the main starting material 1 was prepared via treatment of bromoacetaldehyde
diethyl acetal with HCl and sodium acetate, then cyclization with 2,6-diamino-4-pyrimidinone [25].
Since pyrrole ring of compound 1 possesses three active protons (C5-H, C6-H, and NH) with different
reactivity towards the proposed Mannich reaction, we adopted the protection of amino group in
attempt to selectively achieve monosubstitution at C5 of pyrrolo[2,3-d]pyrimidine [26]. Therefore,
compound 1 was reacted with octanoyl chloride in pyridine to furnish the key intermediate

pyrrolo[2,3-d]pyrimidin-2-yl octamide (2) in 75% yield [26]. The corresponding 5-monsubstituted
pyrrolopyrimidines 3, 4a–f, and 4n were generated through Mannich-type reaction of compound 2
with N,N-dibenzylamine or the proper arylpiperazine or piperidine, and 80% aqueous formaldehyde in
acidic conditions [26]. It is noteworthy mentioning that upon completion of Mannich-type reaction,
the reaction mixture was subjected to acidic treatment with 0.5 M aqueous HCl to cleave the
undesired N-alkylated product and afford only C-alkylated derivative. Despite of the octanoyl
protection of amine group in compound 2, the highly electron deficient aryl or benzyl piperazines
provided 5,6-disubstituted pyrrolo[2,3-d]pyrimidin-2-yl octamides 6a–g under the same reaction
conditions. Upon using 1-benzylpiperazine, a mixture of mono and disubstituted pyrrolo[2,3-
d]pyrimidine octamides, 4b and 6a, was formed in 61% and 10% yield, respectively. Also, a mixture
of mono and disubstituted derivatives, 4f and 6c, was obtained in 48% and 52% yield, respectively,
when using 4-nitrophenyl piperazine. In order to solely get the 5-monosubstituted derivatives 4g–m
and 4o, we introduced amine-exchange reaction with compound 3 in MeOH/THF (1/1) in a sealed
tube at 75°C. Amide hydrolysis of compounds 4a–o and 6a–g was performed under basic conditions
[26] with KOH for 72 h to afford the corresponding 2-amino derivatives 5a–m and 7a–g in excellent
yields (Scheme 1).
O
N
O
O
N
Br
i), ii)
HN
H2N
iii)
HN
O
6
HN
O
O
N
N
H
H N
NH2
N
N
2
H
H
1
iv)
2
v)
R1
R1
X
X
O
N
O
N
O
N(CH Ph)2
viii)
2
HN
H N
vii)
O
HN
vi)
O
HN
N
N
H
N
N
N
N
H
2
N
N
H
6
H
6 H
5a-m
4a-o
3
X = N;
N R2
R2
1
N
N
R = Me, Ac, -(CH ) OH, Ph, -
O
N
N
O
N
2
2
CHPh , 4-MeO-C H -, 3-Cl-C H -
2
6
4
6 4
HN
H N
vii)
O
HN
,
4-NO -C H -, 2-CN-C H -, 3-F-
2 6 4 6 4
N
H
N
N
N
N
H
N
C H -CH -, morpholin-(CH ) -,
6
4
2
2 2
2
6 H
furyl-(CO)-
N
1
X = C; R = H, NHBoc
7a-g
6a-g
R2
R2
2
R = Bn, 3,5-Cl -C H -, 4-NO -C H -, 2-F-C H -CH -,
2
6
3
2
6
4
6
4
2
Ph CH-, 3-F-C H -CH -, 4-F-C H -CH -.
2
6
4
2
6
4
2
Scheme 1. Reagents and reaction conditions: i) conc. HCl, NaOAc, 90 °C to rt, ii) 2,6-Diamino-4-
pyrimidinone, NaOAc, 80 °C to rt, quant.; iii) Octanoyl chloride, pyridine, 85 °C, 49–75%; iv) 37%
formalin, aryl piperazine, 80% AcOH, 75 °C, 12%–quant.; v) 37% Formalin, N,N-dibenzylamine,

8
0% AcOH, 75 °C, 24–74%; vi) Appropriate amines, MeOH/THF (1:1), 75 °C, 11–97%; vii) 5 M aq.
KOH, MeOH/THF, rt, 60%–quant.; viii) 37% Formalin, aryl or benzyl piperazine, 80% acetic acid, 75
C, 9%–quant.
°
2
2
.2. In vitro screening of the antiproliferative activities.
.2.1. Preliminary evaluation against three human cancer cell lines
All final compounds 4a–o, 5a–m, 6a–g, and 7a–g were preliminary evaluated for their
antiproliferative activity at 10 μM concentration against three human cancer cell lines, HCT-116
(human colorectal carcinoma) and MCF-7 and SK-BR3 (breast cancer cells), by MTT assay method
and BIIB021 was used as a reference compound. The percentages of growth inhibition (GI) as well as
GI50 of compounds 4a–o against the three cell lines are listed in Table 1. The compound was
considered active if it achieves GI > 65% and/or GI <8.0 M.
5
0
Table 1. In vitro growth inhibitory data of the 5-substituted pyrrolopyrimidinyl octamides 4a–o
against MCF-7, SK-BR-3 and HCT116 cancer cell lines.
R1
X
O
N
N
O
6
HN
N
H
N
H
4a-o
MCF-7
SKBR3
HCT116
Compound
No.
X R1
a
b
a
b
a
b
%
GI GI50
% GI GI50
% GI GI50
4
a
N C
6
H
5
-
50.26 -c
67.56 0.63 ± 0.03
73.90 3.99 ± 0.82
c
4
4
4
b
c
d
N (C
N 4-CH
N 3-Cl-C
6
H
5
)
2
-CH-
58.43 -
67.41 0.41 ± 0.12
71.36 1.23 ± 0.11
69.69 0.95 ± 0.06
75.47 1.01 ± 0.23
71.44 8.94 ± 2.63
72.73 7.17 ± 3.00
c
3
O-C
6
H
4
-
49.19 -
c
6
H
4
-
43.40 -
4
e
N 2-CN-C
N 4-NO
N 3-F-C
N CH
imidazole-
N CH (C=O)-
N 2-furyl-(C=O)-
N HOCH CH
6
H
4
-
59.21 -c
40.37 -c
85.74 7.84 ± 0.65
59.56 -c
68.17 -c
35.78 -c
68.54 -c
41.99 -c
88.56 -c
84.61 0.97 ± 0.16
79.52 1.46 ± 0.13
4
4
4
f
g
2
-C
6
H
4
-
61.61 8.04 ± 1.45
91.18 5.58 ± 0.72
50.36 -c
86.09 -c
40.25 -c
71.16 -c
62.16 -c
90.36 -c
68.47 -c
92.83 5.34 ± 0.59
51.02 -c
80.48 -c
43.83 -c
60.20 -c
67.29 -c
93.05 -c
6
H
4.-CH
2
-
h
3
-
4
4
4
4
4
4
4
i
-
j
3
k
l
2
2
-
m
n
o
N Morpholin-CH
2
CH
2
-
C H
70.21 13.43 ± 2.64 77.13 15.04 ± 5.44 51.06 14.90 ± 4.22
36.45 -c
C (CH
3
)
3
CO(C=O)NH-
59.32 -c
50.28 -c
BIIB021
-
-
84.81 0.392±0.03
71.94 0.347±0.023 86.20 0.227±0.015
a
%
Growth inhibition values were obtained using MTT assay at 10 M concentration after incubation
b
for 72 hours, and the bold figures refer to %GI > 65. IC values (±SD) were obtained from the dose-
response curve. Not determined.
5
0
c

Close examination of the results revealed that the piperazine derivatives (4a–m) generally possess
superior antiproliferative activities to piperidines 4n and 4o. On the terminal nitrogen of piperazine,
various structural fragments were attached for exploration of their anticancer activities. Direct
installation of phenyl (4a) or substituted phenyl (4c–f) was found to be favorable for activity. The
phenyl piperazine derivative 4a exerted remarkable antiproliferative activity (GI = 0.63 ± 0.03 M)
5
0
against SKBR3 cell line and moderate activity (GI = 3.99 ± 0.82 M) against HCT116 cell line.
5
0
Substitution of phenyl piperazine with electron withdrawing chlorine or cyano group is tolerable for
retaining the activity (4d, GI = 0.95 ± 0.06 M; 4e, GI = 0.97 ± 0.16 M) against SKBR3, while
5
0
50
introduction of p-nitro group greatly reduced the antiproliferative activity (4f, GI = 8.04 ± 1.45 M)
5
0
against SKBR3. Furthermore, installation of p-methoxy moiety as electron donating group led to two
folds drop in activity (4c, GI = 1.23 ± 0.11 M) against SKBR3. Replacement of phenylpiperazine
5
0
with 1-benzylpiperazine improved the activity (4b, SKBR3, GI = 0.41 ± 0.12 M; HCT116, GI =
5
0
50
1
.01 ± 0.23 M). Interestingly, introduction of m-fluorobenzyl moiety broaden the spectrum of
antiproliferative activity to include MCF7 cell line with moderate potency (4g, GI = 5.34–7.84 M).
5
0
Replacing the bulky phenyl group with small lipophilic methyl (4h) or acetyl (4j) decreased the
activity, while insertion of hydrophilic imidazole ring (4i) or 2-morpholinoethyl (4m) was favorable
for the anticancer activity (4i, %GI = 68.17–86.09; 4m, %GI = 88.65–93.05). In contrast to the
promising activity of several octamide derivatives, their corresponding free amines 5a–m showed
modest antiproliferative activity (Table 2). This superiority of octamides than amines may be
attributed to the increase in compound lipophilicity, permeability and penetration into cancer cells and
thus the cellular potency. Relatively, the m-chlorophenyl piperazine derivative 5d is the best in this
series with compounds with GI values of 8.28 ± 1.37 M and 7.66 ± 1.05 M against SKBR3 and
5
0
HCT116 cell lines, respectively.
Table 2. In vitro growth inhibitory data of the 2-amino-5-substituted pyrrolopyrimidinones 5a–m
against MCF-7, SK-BR-3 and HCT116 cancer cell lines.
R1
X
O
N
N
HN
H N
N
2
H
5
a-m
MCF-7
GIa
SKBR3
HCT116
% GIa
Compound
No.
X R1
%
GI50b
% GIa
GI50b
GI50b
5
a
N C
6
H
5
-
49.73
-c
30.71
-c
-c
48.97
-c
5
5
5
5
b
c
d
N (C
6
H
5
)
2
-CH-
79.55
51.73
54.23
51.08
33.89 ± 1.59 4.22
80.45
20.27 ± 2.26
c
c
N 4-CH
N 3-Cl-C
N 2-CN-C
3
O-C
6
H
4
-
-
57.48
67.46
73.79
11.17 ± 1.55 69.22
8.28 ± 1.37 79.29
23.7 ± 1.26 71.97
-
c
6
H
4
-
-
7.66 ± 1.05
e
6
H
4
-
-c
8.37 ± 1.22

5
f
N 4-NO
N 3-F-C
N CH
imidazole-
N CH (C=O)-
N HOCH CH
morpholin-
2
-C
6
H
4
-
26.81
-c
27.50
-c
28.54
-c
5
5
5
5
5
g
h
i
6
H
4
-CH
2
-
20.62
20.21
38.76
16.21
18.80
-c
-c
-c
-c
-c
16.80
28.96
38.84
24.21
32.84
-c
-c
-c
-c
-c
31.12
16.48
50.64
13.81
17.82
-c
-c
-c
-c
-c
3
-
-
j
3
k
2
2
-
5
5
l
N
19.46
-c
-c
27.11
17.50
-c
-c
15.40
24.13
-c
CH
2 2
CH -
m
C
H-
23.76
-c
BIIB021
N
-
84.81
0.392±0.03 71.94
0.347±0.023 86.20
0.227±0.015
a
%
Growth inhibition values were obtained using MTT assay at 10 M concentration after incubation
b
for 72 hours, and the bold figures refer to %GI > 65. IC values (±SD) were obtained from the dose-
response curve. Not determined.
5
0
c
Regarding the 5,6-disubstituted pyrrolopyrimidinyl octamides 6a–g and their amino derivatives 7a–g,
their antiproliferative activities are shown in Table 3 and Table 4, respectively.
Table 3. In vitro growth inhibitory data of the 5,6-Bis-substituted pyrrolopyrimidinyl octamides 6a–g
against MCF-7, SK-BR-3 and HCT116 cancer cell lines.
R2
N
O
N
N
O
6
HN
N
N
N
H
H
N
R2
6a-g
MCF-7
GIa
SKBR3
HCT116
Compound
No.
R2
(C
%
GI50b
% GIa
GI50b
% GIa
GI50b
6
a
6
H
5
)
2
-CH- 15.16
-C - 10.70
-C
-CH
-c
67.33
6.43 ± 0.22
41.53
18.38 ± 4.80
c
c
c
6
6
6
b
c
d
3,5-Cl
4-NO
2
6
H
3
-
42.54
64.59
93.40
-
6.88
27.04
97.01
-
c
c
c
2
6
H
4
- 12.56
-
-
-
C
6
H
5
2
-
96.57
2.78 ± 0.01
3.64 ± 0.03
4.09 ±0 .03
2
-F-C
CH
-F-C
CH
-F-C
6
6
6
H
H
H
4
4
4
-
-
-
6
6
6
e
f
96.13
94.76
4.20 ± 0.03
93.39
93.06
93.52
3.73 ± 0.06
97.28
95.54
97.14
1.89 ± 0.30
2
-
3
2.15 ± 0.09
1.75 ± 0.15
3.91 ± 0.34
3.60 ± 0.06
2.25 ± 0.12
2
-
4
g
97.23
84.81
4.13 ± 0.15
2
CH -
BIIB021
-
0.392 ± 0.03 71.94
0.347 ± 0.023 86.20
0.227 ± 0.015
a
%
Growth inhibition values were obtained using MTT assay at 10 M concentration after incubation
b
for 72 hours, and the bold figures refer to %GI > 65. IC values (±SD) were obtained from the dose-
response curve. Not determined.
5
0
c
Table 4. In vitro growth inhibitory data of the 2-Amino-5,6-bis-substituted pyrrolopyrimidinones 7a–
g against MCF-7, SK-BR-3 and HCT116 cancer cell lines.

R2
N
O
N
N
HN
H N
N
N
2
H
N
7
a-g
R2
MCF-7
GIa
SKBR3
HCT116
Compound
No.
R2
(C
%
GI50b
% GIa
GI50b
% GIa
GI50b
7
a
6
H
5
)
2
-CH- 13.77
-C - 82.46
-C
-CH
-c
11.08
-c
10.69
-c
7
7
7
b
c
d
3,5-Cl
4-NO
2
6
H
3
4.02 ± 0.53
90.03
6.79
15.69
3.22 ± 0.14
91.73
1.94
21.36
2.89 ± 0.13
c
c
c
2
6
H
4
- 1.26
21.19
-
-
-
c
c
c
C
6
H
5
2
-
-
-
-
2
-F-C
CH
-F-C
CH
-F-C
6
6
6
H
H
H
4
4
4
-
-
-
7
7
7
e
f
8.91
-c
-c
-c
58.96
10.50
23.21
-c
12.80
13.59
-c
2
-
3
16.08
-c
-c
2
-
4
g
18.36
-c
22.97
-c
2
CH -
BIIB021
-
84.81
0.392 ± 0.03 71.94
0.347 ± 0.023
86.20
0.227 ± 0.015
a
%
Growth inhibition values were obtained using MTT assay at 10 M concentration after incubation
b
for 72 hours, and the bold figures refer to %GI > 65. IC values (±SD) were obtained from the dose-
response curve. Not determined.
5
0
c
As revealed from the results listed in Table 3, the benzylpiperazine derivatives 6d–g exerted superior
antiproliferative activities to phenyl piperazines 6a–c. In this set of compounds, compound 6f was
found to be the most potent member with GI values of 2.15 ± 0.09 M, 3.91 ± 0.34 M and 2.25 ±
5
0
0
.12 M against MCF7, SKBR3 and HCT116 cell lines, respectively. Upon comparing 6f with its
relevant monosubstituted derivative 4g, it could be observed that disubstitution is more auspicious for
the antineoplastic activity. Compounds 6e and 6g, the positional isomers of 6f, exhibited comparable
activity (marginally superior or inferior) to the unsubstituted benzyl piperazine 6d. As expected, 7a–g,
the corresponding amino derivatives of 6a–g, showed weak growth inhibitory activities against the
three tested cancer cell lines. The only exception was the 3,5-dichlorophenyl piperazine derivative 7b,
which displayed reasonable activity with GI values of 4.02 ± 0.53 M, 3.22 ± 0.14 M and 2.89 ±
5
0
0
.13 M against MCF7, SKBR3 and HCT116 cell lines, respectively.
2
.2.2. In vitro antiproliferative activities against NCI-60 cell line panel
2
.2.2.1. Single dose testing
Prompted by the promising preliminary antiproliferative activity and in order to interrogate the
antiproliferative activity of the target compounds on broad extent, compounds 6f and 7b as
representative examples were submitted to National Cancer Institute (NCI, Bethesda, Maryland,
USA) [27] for appraisal of their antiproliferative activities against clinically important cancer cell
lines. According to the NCI protocol, compounds 6f and 7b were tested at a single dose concentration
of 10 μM over a panel of approximately 60 human cancer cell lines comprising nine human cancer

cell types; leukemia, non-small cell lung, colon, central nervous system, melanoma, ovarian, renal,
prostate, and breast tumor cell lines. The growth percentages of all tested cell lines after treatment
with the tested compounds are illustrated in Figure 4.
Figure 4. % Growth of NCI 60 cell line panel upon treatment with compounds 6f (upper panel) and
7
b (lower panel) at 10 M concentration.
Close inspection of the results presented in Figure 4, demonstrated the broad spectrum
antiproliferative activity of compound 6f over all the nine examined cancer types. Compound 6f
exerted distinct cytotoxic effects (minus % growth) against 52 cell lines out of 56. Of particular
importance, compound 6f displayed lethal activity over HCT-116 (colon cancer), SF-539 (CNS
cancer), SK-MEL-28 (melanoma), and the renal carcinoma cells ACHN and UO-31 with percentages
growth values of −96.68%, −96.93%, −96.98%, −96.57% and −96.54%, respectively. In contrast to
the broad spectrum cytotoxic activity of 6f, the pyrrolopyrimidine 2-amine derivative 7b manifested
good cytostatic effect (% growth < 40) with limited spectrum of activity against 14 cancer cell lines
derived from leukemia, melanoma, colon and breast cancer types. Such finding further emphasizes the
essential role of octamide moiety for achieving the favorable antiproliferative activity.
2
.2.2.2. Five-dose testing
Compounds 6f and 7b satisfied the criteria set by NCI for activity in the preliminary assay and
consequently they were further tested in a five-dose testing mode at 10-fold dilution (100–0.01 M)
over the full panel. Three response parameters were determined for each compound; GI (the
5
0

concentration producing 50% growth inhibition, a measure of compound's potency), TGI (the
concentration producing 100% growth inhibition, a measure of compound's efficacy) and LC (the
5
0
concentration causing 50% lethality, a measure of compound's cytotoxicity and efficacy). The
GI values of compounds 6f and 7b are showed in Table 5.
5
0
a,b
Table 5. GI values (M) of compounds 6f and 7b over NCI-60 cell line panel.
5
0
Cancer type/
Cell line
Cancer type/
Cell line
6
f
7b
6f
7b
CCRF-CEM
0.25
2.07
MDA-MB-435
0.32
1.62
b
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
A549/ATCC
HOP-62
HOP-92
1.74
0.21
0.26
0.36
0.21
0.34
1.42
1.14
1.84
1.86
-b
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
-
2.21
1.79
1.63
2.06
1.8
1.95
1.81
1.58
2.64
2.05
b
2.33
2.58
2.41
1.46
1.82
1.67
1.35
15.8
2.03
-
Leukemia
Melanoma
1.57
0.20
1.68
1.43
0.78
1.21
b
NCI-H226
NCI-H23
-
Non-Small Cell
Lung Cancer
Ovarian Cancer
0.21
NCI-H322M
1.46
6.33
NCI/ADR-RES
1.5
>100
NCI-H460
NCI-H522
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
0.38
1.91
1.4
0.25
0.17
0.30
0.26
0.72
0.19
1.96
1.74
1.69
1.7
1.63
1.93
1.74
1.83
1.72
SK-OV-3
786-0
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
1.9
4.62
1.57
1.69
1.94
5.1
1.58
1.91
1.8
1.54
1.25
b
-
1.32
0.51
1.48
1.74
0.38
Renal Cancer
Colon Cancer
CNS Cancer
KM12
SW-620
UO-31
11.7
SF-268
SF-295
SF-539
1.26
1.41
-b
1.77
1.82
1.72
PC-3
1.3
1.76
1.92
1.58
Prostate Cancer
DU-145
MCF7
1.1
0.19
MDA-MB-
SNB-19
1.56
1.77
0.59
2.01
2
31/ATCC
SNB-75
U251
LOX IMVI
1.34
0.17
0.20
1.42
1.67
1.94
HS 578T
BT-549
T-47D
1.72
1.53
1.6
1.78
1.72
1.78
Breast Cancer
Melanoma
MALME-3M
M14
0.67
0.21
2.00
1.83
MDA-MB-468
0.48
1.72
a
Bold figures refer to sub-micromolar GI50 values.
b
c
NT: not tested, ND: not determined
As illustrated in Table 5, the fluoro substituted pyrrolopyrimidine octamide 6f showed high potency
with sub-micromolar or one-digit micromolar (less than 2.0 M) GI values over 26 and 24 cell lines,
5
0
respectively. Leukemia, colon cancer and melanoma were found to be the most sensitive types of
cancer responsive to compound 6f. It showed equipotent activity towards CCRF-CEM and PRMI-
8
226 leukemia cells and HCC-2998 and HT29 colon cancer cell lines with GI value of 260 nM.
50
Moreover, compound 6f strongly suppressed the proliferation of LOX IMVI, M14 and UACC-257
melanoma cell lines with GI value of 200 nM. Besides the previously mentioned targeted cellular
5
0

potencies of compound 6f, it exhibited significant broad spectrum antiproliferative activity over other
multiple cell lines such as U251CNS cancer, OVCAR-8 ovarian cancer, and MCF7 breast cancer cell
lines with GI values of 0.17, 0.21 and 0.19 M, respectively. On the other hand, the
5
0
pyrrolopyrimidine 2-amine derivative 7b exerted remarkable antiproliferative activity (mean
GI value = 1.8 M) against most of the tested cell lines, yet with inferior potency when compared
5
0
with compound 6f. It was evident that the octamide derivative 6f is more potent than the free amine
compound 7b regarding most of the cell lines, particularly leukemia, colon cancer and leukemia. Such
conclusion implies the significance of octamide side chain for antiproliferative activity of this new
series of compounds.
In view of the efficacy related parameters (TGI and LC values) of compounds 6f and 7b (Table 6), it
5
0
is obvious that compound 6f is also more efficacious than 7b towards most of the examined cell lines.
Compound 6f completely inhibited the proliferation of several cancer cells at submicromolar
concentrations, with about 10 times superiority than compound 7b. For instance, 6f exerted TGI
values of 0.32, 0.42, and 0.55 M against HCT-116 colon cancer cell, LOX IMVI melanoma cell, and
K-562 leukemia cell line, respectively. In terms of LC values, both compounds 6f and 7b showed
5
0
limited efficacy against leukemia cell lines with LC more than 100 M. Upon considering both TGI
5
0
and LC values, it was observed that compound 6f exerted remarkable efficacies against HCT-116
5
0
colon cancer cell, U251 CNS cancer, LOX IMVI melanoma, OVCAR-8 ovarian, and MCF-7 breast
cancer cell line, being able to induce total growth inhibition (TGI) and 50% lethality (LC ) at
5
0
submicromolar concentrations.
Table 6. TGI and LC values (M) of compounds 6f and 7b over the most sensitive cell lines.
5
0
Cancer type
6f
TGI
7b
TGI
>100 -b
Cell line
K-562
a
LC50
LC50
Bold figures refer to sub-micromolar TGI
and LC50 values.
0.55
>100
b
ND: not determined
Leukemia
MOLT-4
SR
HCC-2998
HCT-116
SW-620
0.74
0.55
0.57
0.32
0.38
>100 8.56
>100 4.46
>100
>100
2
.2.3. In vitro cytotoxic activities
b
2.62
0.6
0.74
3.16
3.17
3.5
-
against HFF-1 cell line panel
Colon
CNS
6.18
7.12
To get insights about the differential
cytotoxicity of this novel series of
compounds, representative examples
of the potent compounds 6f and 7b
have been tested against the human
foreskin fibroblast (HFF-1) normal
cell utilizing MTT assay (Table 7).
U251
0.33
0.61
3.14
5.9
-b
LOX IMVI
M14
UACC-257
OVCAR-8
0.42
0.47
0.46
0.41
0.89
1.24
1.09
0.8
3.57
3.63
4.76
4.48
Melanoma
Ovarian
Breast
7.2
28.9
b
-
MCF7
0.43
0.99
3.43
-b
MDA-MB-
1
.96
5.38
3.98
-b
2
31/ATCC
Interestingly, both compounds 6f and 7b showed high GI values (> 10.0 μM), which reveal their
5
0
selective cytotoxic activities towards human cancer cells rather normal cell lines.
Table 7. Cytotoxicity evaluation of compounds 6f and 7b against the HFF-1 normal cell line.

Compound No. % growth inhibition at 10 M GI50 (μM)
2
.3. In vitro kinase screening
6
7
f
b
14.12
15.77
> 10.0
> 10.0
Aiming at investigation of the mechanism of
action of this new series of compounds and
its kinase inhibitory profile, compound 6f, the most active member, was tested at 10 μM concentration
over a panel of 53 oncogenic kinases at Reaction Biology Corporation (RBC, Malvern, PA, USA)
[28]. As shown in Figure 5, compound 6f displayed highly potent inhibitory activity (above 95%)
against three kinases; Tie-2 (97.4%), TrkA (95.3%) and FGFR4 (95.3%). Moreover, it exerted good
inhibitory activity (70.3%–85.2%) towards FMS, IGFIR and LYN kinases, and moderate activity
against FGFR1 (57.4%), FGFR3 (39.2%) and Yes-1 (45.5%). Meanwhile, it showed modest activity
against the other kinases with inhibition percentage less than 35.
Figure 5. Inhibition percentages of compound 6f over a panel of 53 oncogenic protein kinases at 10
M concentration.
Since compound 6f displayed distinct activity against FGFR4, it was interesting to further examine
the kinase inhibitory activity of the relevant derivatives 6d, 6e, 6g and 7b over the four subtypes of
FGFR (Table 8). As revealed from the results, all the tested pyrrolopyrimidine octamides 6d, 6e, 6g
exerted moderate activity (about 50%) towards FGFR1 which is comparable to that of 6f. In contrast,
the 2-amino pyrrolopyrimidine 7b did not possess any inhibitory activity against FGFR1.
Furthermore, all compounds showed relatively weak inhibition over FGFR2, FGFR3 and FGFR4
except 6f and 7b which exhibited particular selectivity towards FGFR4 kinase.
Table 8. In vitro enzymatic inhibitory activity of 6d–g and 7b over the four isozymes of FGFR
kinase.a
Compound /
Kinases
% Enzyme inhibition (relative to DMSO controls) (10 μM)
FGFR1
47.1
FGFR2
24.04
FGFR3
34.62
FGFR4
27.47 b
6
d
6
6
6
7
e
f
g
59.83
54.45
50.52
-8.65
22.99
25.93
14.67
-0.49
39.95
39.22
31.68
6.1
31.49 b
95.38
30.74b
53.17
b

a
IC50 (M)
3.70E-09
1.63E-09
1.41E-08
9.73E-08
Staurosporine
Compound
s were
tested in single dose duplicate mode at a concentration of 10 μM.
b
Compound was tested in in single dose duplicate mode at a concentration of 11.1 μM.
Encouraged by the promising results of single dose biochemical screening, we further tested the most
active compounds in cell based assay (6d–g and 7b) in a 10-dose testing mode to determine their IC50
values over FGFR4, Tie2 and TrkA kinases (Table 9). While the four tested octamides 6d–6g showed
reasonable and comparable activity (average IC values = 7.0 M) against FGFR4 and Tie2 kinases,
5
0
they exerted three times remarkable activity against TrkA (average IC values = 2.5 M). It seems
5
0
that the position of fluorine group has marginal contribution on the kinase activity against TrkA.
Among the three fluoro substituted derivatives, compound 6f was the most potent member with IC50
value of 2.25 M against TrkA. Interestingly, the free amine 7b was found to possess weak activity
towards all three tested kinases with IC values more than 12 M, which points out the significance
5
0
of the lipophilic octamide moiety for retaining the kinase inhibitory activity.
Table 9. IC50 (M) values of 6d–g and 7b against FGFR4, Tie2/Tek and TrkA kinases.
a
Compound /
Kinases
IC50 (M)a
FGFR4
Compounds were
tested in a 10-dose IC50
mode with 3-fold serial
dilution using 10 μM
ATP.
Tie2/Tek
TrkA
6
d
7.05
7.67
6.71
7.57
26.3
8.69
5.80
6.84
8.21
24.8
2.29
3.15
2.25
2.76
12.4
6
6
e
f
In agreement with
the
above
6
7
g
b
mentioned
IC50 (M)
Staurosporine
0
.0973
0.0595
< 0.001
biochemical assay
findings, compound 6f exerted high potencies over numerous cell lines belonging to TrkA correlated
cancer types, like colon [29], melanoma [30] ovarian [31] and breast cancers [32]. However, the
moderate potency 6f against TrkA (IC = 2.25 μM) cannot justify its broad spectrum antiproliferative
5
0
activity over multiple cancer cell lines. Therefore, TrkA inhibition can be one of the mechanisms by
which compound 6f displays its antiproliferative activity, with the possibility of presence of additional
underlying mechanisms that contribute to this strong anticancer effect.
2
.4. Molecular docking study
In order to rationalize the observed FGFR4, TIE2, and TrkA kinase inhibitory activity from a three
dimensional (3D) structural perspective, 6f, 7b, and staurosporine were docked in the kinase domain.
Figure 6 illustrates the 3D-interaction model of 6f with FGFR4, TIE2, and TrkA, while the
corresponding 2D interaction model is shown in Supplementary Figure 1. The docking model
demonstrates that 6f was located deep inside the extended hydrophobic pocket formed by the DFG out
conformation and αC-helix region in FGFR4, TIE2, and TrkA. Glide docking score of 6f inside
FGFR4, TIE2, and TrkA is −7.01, −6.77, and −7.08 kcal/mol, respectively. In FGFR4 (Figure 6A),
piperazine, pyrrole, and pyrimidine rings of 6f were involved in salt-bridge and π-anion interactions

with Asp630 of DFG motif. The Cys608 forms conventional hydrogen bond with piperazine rings
protonated NH and π-alkyl interactions with m-fluorobenzyl moiety. Another m-fluorobenzyl moiety
forms alkyl interactions with Arg635 and Ile640, and π-anion interaction with Asp516 of αC-helix.
Furthermore, octamide docked nearby the hinge region and interacted with the Val550, Glu551, and
Ala553. Referring to the docking model of 6f inside TIE2 (Figure 6B), Lys855 was involved in
hydrogen-bond and π-cationic interaction with octamide and central pyrrole and pyrimidine moiety.
Asp982 in DFG motif forms salt-bridge with one of the protonated nitrogen in piperazine ring.
Moreover, Glu872 and Asp868 of αC-helix forms salt-bridge and hydrogen-bond interactions with
both the protonated nitrogens in piperazine ring, respectively. Val875 and Arg963 form π-alkyl and
hydrogen-bond interaction with m-fluorobenzyl moiety. In addition, the octamide moiety was found
to be penetrated into the pocket formed by the hinge region (Tyr904, and Ala905). Docked model of
6
f inside TrkA is shown in Figure 6C. The nitrogen of pyrrole ring was engaged in a hydrogen bond
interaction with Glu560 of αC-helix. Moreover, Leu563 and Leu564 of αC-helix formed hydrophobic
interactions with the pyrimidine moiety. Furthermore, the terminal part of octamide appeared to make
close contacts via π-alkyl and alkyl interactions with Leu657 and Phe669, respectively. Also, 6f
appeared to form close van der Waals contact with various amino acid residues (like Val524, Lys544,
Leu546, Leu563, Val573, Phe589), and gatekeeper Phe589. Besides, salt bridge (ionic) interactions
were formed between the protonated nitrogen of both piperazinyl moieties and Asp668 of DFG motif,
and Glu560 of αC-helix. Moreover, the fluorine of m-fluorobenzyl was involved in halogen bond with
the Phe521 residue of glycine-rich loops in the DFG-out conformation.
Additional docking experiments were carried out for 7b, which has glide docking score of −5.08,
−
5.52, and −5.73 kcal/mol against FGFR4, TIE2, and TrkA, respectively. From the docking mode of
b inside FGFR4, TIE2, and TrkA (Supplementary Figures 2 and 3), it is evident that the octamide
chain is necessary to improve the binding affinity. The docking model of 7b inside FGFR4
Supplementary Figure 2A) shows that in the absence of octamide chain, 7b is devoid of any
7
(
interaction with the hinge region, but interacts with the gatekeeper Val550. Asp630 from DFG motif
is engaged with the central pyrrole and pyrimidine ring through π-anion interaction. Met524, Ile534,
Leu619, Ala629, and Phe631 interact with m-dichlorobenzene via π-alkyl interactions. The chlorine of
another m-dichlorobenzene is involved in alkyl interaction with Leu526. Glu520 and Met524 of αC-
helix form salt-bridge and hydrogen bond interactions with protonated nitrogen of piperazine,
respectively. Regarding the binding mode of 7b inside TIE2 (Supplementary Figure 2B), Asp982
from DFG motif makes strong attractive charge interaction with the protonated nitrogen of piperazine
ring. Also, Asp868 and Glu872 from αC-helix engage via salt bridge interaction with the single and
double piperazine of 7b, respectively. Moreover, Ile886 from the activation loop, Leu876, Leu879
from αC-helix and Phe960, His962, and Arg963 form π-alkyl, hydrogen bond, and π-sigma
interactions with m-dichlorobenzene and the pyrimidine ring. Upon investigation of the docking
model of 7b into TrkA kinase (Supplementary Figure 2C), it was observed that Asp668 from DFG

motif makes a salt bridge and π-anion interactions with the protonated nitrogen and pyrrole ring.
Asp556 and Glu560 residues, from αC-helix, were engaged with protonated nitrogen of piperazine
moieties through single and double salt bridge interactions, respectively. Furthermore, αC-helix
Leu564 and Leu567, Ile572, Val573, Leu641, Phe646 and gatekeeper Phe589 were involved in alkyl
and π-alkyl interactions with m-dichlorobenzene. Moreover, another m-dichlorobenzene shared π-
alkyl interactions with Ser552 and Ala553 from αC-helix, and halogen and alkyl interactions with the
Phe521 and Leu546 from lysine-rich loop.
On the other hand, staurosporine was docked inside FGFR4, TIE2 and TrkA with the glide docking
score of −3.45, −5.88, and −11.48 kcal/mol, respectively. The docking model of staurosporine is
depicted in the Supplementary Figure 4. The putative binding mode of staurosporine inside FGFR4
showed hydrogen bond and π-anion interactions between DFG (Asp630) and αC-helix (Glu520) with
pyrrolidone moiety. Val523 and Cys608 were engaged with staurosporine via π-sigma and π-sulfur
interactions. Staurosporine exhibited van der Waals interactions with Phe478, Lys503, Ile527,
Phe631, Gly632, Leu633, and Arg635. The docked model of staurosporine with TIE2 (Supplementary
Figure 4B) exhibited van der Waals and hydrogen bond interactions with the gatekeeper (Ile902), and
hinge region (Glu903, Tyr904, and Ala905), respectively. The pyrrolidone rings oxygen formed a
hydrogen bond with the main chain of Ala905. In addition, the methoxy group of staurosporine
interacted through π-sigma interactions with DFG motif (Phe983). Moreover, Asp912 from αC-helix
participated via salt bridge interaction with the protonated nitrogen of staurosporine. Ala853 and
Val838 shared π-alkyl and π-sigma interactions with staurosporine. The docking mode of
staurosporine inside TrkA (Supplementary Figure 4C) exhibited strong hydrogen bonds, π-sigma, and
salt bridge interaction network, which resulted in higher glide docking score for staurosporine. Two
hydrogen bonds are formed between the hinge region residues (Glu590 and Met592) and pyrrolidone
rings NH and C=O. Additionally, gatekeeper residue (Phe589) and DFG motif (Phe669) shared π-π T-
shaped and stacked interactions. Asp596 from αD-helix participated in salt bridge interactions with
the protonated N-Met. Moreover, Leu516, Val524, and Leu657 from β1, β2 sheet and catalytic loop
formed π-alkyl and π-sigma interactions. Also, Ala542 and Met671 forms π-alkyl and alkyl
interactions and Gly517, Lys544, Val573, Tyr591, Gly595, Ser672, Ile675, and Tyr676 interacted
through van der Waals interactions.
Surprisingly, 6f did not show any binding interaction with the hinge region residues in TrkA but it
showed alkyl interactions in FGFR4 and TIE2. Additional alkyl interaction of 6f from hinge region
residues in FGFR4 and TIE2 did not benefit concerning docking score. Our observation is following
the previous report suggesting that specific kinase inhibitors exert their activities through binding to
the juxtamembrane (JM) region, a critical activity-controlling segment of receptor tyrosine kinases
(RTKs), rather than hinge region [33]. Compound 7b without octamide resulted in barren of any
interactions with the hinge region and directly affects the docking score against FGFR4, TIE2, and
TrkA. This observation implies that octamide is necessary to maintain the activity. Furthermore,

reference compound staurosporine showed dramatic increase in the glide docking score against TrkA
compared with TIE2, and FGFR4. A putative binding model showed that staurosporine could form
two hydrogen bond between pyrrolidone ring and hinge region residues (Glu590 and Met592) inside
TrkA, and one hydrogen bond with Ala905 inside TIE2, and no hydrogen bond in FGFR4. Such
observation suggests that for a ligand to become highly potent kinase inhibitors, it is essential to form
the critical hydrogen bonds with the hinge region of kinases. Though, 6f could participate in alkyl
interactions with the hinge regions of FGFR4 and TIE2, still it is less active because it does not fulfill
the criterion of a hydrogen bond with hinge region. Taken together, we can conclude that the
structural features of 6f allow profitable binding with TrkA kinase. Also, it could be suggested that 6f
might be a type III kinase inhibitor which lacks any interaction with the hinge region but binds to an
allosteric pocket formed by DFG-out conformation and αC-helix region.
Beside the aforementioned docking studies for the active compounds 6f and 7b, further docking
investigations were performed for compounds 6b, 7e, and 7f to get some structural insights about their
lack of antiproliferative effects. Compound 6b was found to be unable to dock into the binding
pockets of FGFR4, TIE2 and TrkA, suggesting that combining both 3,5-dichloro-phenyl with
octamide moiety in single molecule is detrimental for the activity, because the 3D-shape obtained by
6
b could not be complementary to fit into the binding pocket. Docking results of compound 7e, and 7f
suggests that the octamide moiety is critical for the activity as it extend into the ATP binding site near
hinge region and forms van der Waals contact with the surrounding residues.
The docking models of 7e and 7f inside FGFR4 (Supplementary Figure 5A, B) showed docking
scores of −4.71 kcal/mol and −4.80 kcal/mol, respectively. Piperazinyl moieties of the 7e retain the
salt bridge interactions with the Asp516 and Glu520 of αC-helix, respectively. In absence of octamide
moiety, 7e interacts with the allosteric pocket including Asp630 from DFG motif. In case of
compound 7f, different orientation in the pocket was observed. Piperazinyl moieties retain the salt-
bridge interaction with the Glu520 of αC-helix and Asp630 of DFG motif. Asp516 shows the pi-
cation interactions with the fused ring structure. This compound is devoid of any interaction with the
hinge region and this could be the probable reason for its inactivity against FGFR4.
Docking models of 7e and 7f inside TIE2 are shown in Supplementary Figure 5C, D. Unlike FGFR4,
neither 7e nor 7f interacts near hinge region of TIE2. Docking score for 7e and 7f are −4.71 kcal/mol
and −4.55 kcal/mol, repsectively. They bind into the allosteric pocket formed by DFG motif, αC-
helix, and β2- β3 strands. One of the piperazine moiety of 7e could form attractive charge interaction
with Asp868, and Glu872 from αC-helix. The other piperazine moiety interacts with DFG motif’s
Asp982. Substitution on the benzyl ring (2-F vs 3-F) leads to change in the orientation of respective
functional groups inside the allosteric pocket, while keeping conserved salt bridge interactions
maintained. 7f docking model also shows the salt bridge interactions with Asp868, and Glu872 of αC-
helix and piperazinyl moiety. Asp982 from DFG motif shows salt-bridge interaction with the

piperazine and pi-anion interactions with central pyrrolopyrimidinone. 3F-phenyl shows halogen bond
interaction with the Ile886, amide-pi stacked interaction with Ala981.
In the absence of octamide moiety in compounds 7e and 7f, docking modes inside TrkA
(Supplementary Figure 5E, F) suggest that 7e and 7f docked nearby allosteric pocket but missed the
interactions with hinge region. It can be seen from the 7e docked model that both the piperazinyl
moiety makes the salt-bridge interaction with the Glu556 and Asp560 of αC-helix. Overall, the ligand
was reoriented in the binding pocket and became devoid of any interactions with the hinge region.
The docked model of 7f inside TrkA shows the similar interaction patterns as that of the 7e, and
piperazinyl moieties show salt-bridge interactions with the Asp556 and Glu560 of αC-helix. 3-F-
phenyl moieties showed π–π stacking interactions with Phe521 from P-loop and Phe589 from
gatekeeper. Docking score for the 7e and 7f are −4.43 kcal/mol and −4.51 kcal/mol, respectively. 2D-
interaction images were shown in Supplementary Figure 6.
To become potent ATP competitive inhibitors for the kinases, small molecules need to form
hydrogen-bond as well as other interactions with the hinge region. In the current study, absence of
octamide substituent in compounds 7e and 7f makes them inactive because they miss the essential
interactions with the surrounding environment of hinge region. Despite absence of octamide moiety,
3
,5-dichlorophenyl piperazinyl compound 7b showed considerable growth inhibition, but kinase
inhibitory activity 7b was low as deduced from 3D-binding mode in docking study devoid of
interaction with hinge region and lower glide docking score than 6f. Also, 3,5-dichlorophenyl
piperazinyl compound 6b having octamide moiety could not be complementary to fit in the binding
pocket of FGFR4, TIE2 and TrkA, as revealed from the docking study, and displayed no enzyme
activity as well.

Figure 6. Docking model of 6f inside (A) FGFR4, (B) TIE2, and (C) TrkA. For clarity purpose, only residues
having interactions were shown. 6f is shown in stick model and surrounding residues were shown in line model.
Various interactions are shown in dashes. (For more clarity see the supplementary figure 1). (D) Superposed
model of 6f shown by green, blue, and red inside FGFR4, TIE2, and TrkA, respectively.
2
.5. In vivo pharmacokinetic properties of 6f.
The in vivo pharmacokinetic (PK) properties of compound 6f were evaluated (Table 10). The AUC∞
values of compound 6f are 29.9±5.8 gmin/mL and 3.7±0.7 gmin/mL in intravenous and oral
administration, respectively. 6f was moderately distributed (Tmax = 2.25 h) to reach the blood after
oral administration. Moreover, it showed oral bioavailability (F) value of 12.3%.
Table 10. Pharmacokinetic parameters after intravenous (n=3) and oral (n = 4) administration (10
mg/kg) of 6f to male rats.
Intravenous
29.9 ± 5.8
19.0 ± 2.9
700.1 ± 306.1
-a
Oral
The values are
presented as
mean ± SD.
SD, standard
deviation;
AUC0–∞(μg min/ml)
AUClast(μg min/ml)
Terminal half-life (min)
3.7 ± 0.7
3.4 ± 0.5
99.4 ± 21.5
0.01 ± 0.00
135 (60~240)b
-a
C
max (μg/ml)
AUC
under
plasma
(area
the
T
max (min)
-a
CL (ml/mim/kg)
342.4 ± 60.6
12.3
F (%)
concentration

versus time curve) = dose/clearance; Cmax, peak plasma concentration; Tmax, time to reach Cmax; CL, time-
a
b
averaged body clearance; F, bioavailability; Not determined; Median (range) for Tmax
.
3
. Conclusion
In this study, four new series of 5-substituted and 5,6-disubstituted pyrrolo[2,3-d]pyrimidine
octamides and their corresponding amines have been synthesized through Mannich-type reaction and
evaluated for their antiproliferative activities over three human cancer cells. Among them, compounds
6
d–g and 7b have exerted strong growth inhibitory activities with one-digit micromolar GI values
50
over the tested cell lines. A structure activity relationship (SAR) study has been made and disclosed
that terminal octamide moiety at C2 as well as disubstitution with fluorobenzyl piperazines at C5 and
C6 of pyrrolo[2,3-d]pyrimidine are the key structural features prerequisite for best anticancer activity.
The most active member 6f was tested for its antiproliferative activity over a panel of 60 cancer cell
lines at NCI, and exhibited distinct broad spectrum anticancer activity. Kinase screening of compound
6
f over 53 cancer relevant kinases at 10 M showed its highly selective inhibitory effects against
FGFR4, Tie2 and TrkA kinases. Further determination of IC50 values of 6f showed its promising
activity against TrkA kinase. Such finding was justified by of molecular docking, which also revealed
that 6f did not bind with kinase hinge region, and therefore it may be a type III kinase inhibitor. The
distinguished antiproliferative activity and low toxicity profile of 6f afford a new promising
chemotherapeutic candidate that could be optimized for further development of potent anticancer
agents.
4
. Experimental
4
.1. General
1
13
H and C NMR spectra were recorded on a Bruker Avance 400 or 300 MHz spectrometer, using
CDCl , DMSO-d , or D O as a NMR solvent. TMS was used as the internal standard and chemical
3
6
2
shifts (δ) are reported in parts per million (ppm) and s, d, t, and m are designated as singlet, doublet,
triplet and multiplet respectively. Coupling constants (J) are reported in hertz (Hz). The reaction
progress was monitored on TLC plate (Merck, silica gel 60 F254), and flash column chromatography
was performed on silica gel (Merck, 230e 400 mesh) with the indicated solvent system. Solvents and
liquid reagents were transferred using hypodermic syringes. All solvents and reagents were
commercially available and used without further purification.
4
.2. 2-Amino-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one (1) [25]
To a suspension of bromoacetaldehyde diethyl acetal (10.4 mL, 68.98 mmol) in water (35 mL) was
added concentrated hydrochloric acid (1.5 mL). The reaction mixture was stirred at 90 °C. After 30
min, the solution was cooled to room temperature, and sodium acetate (6.8 g, 82.75 mmol) was added.
The mixture solution was added to a suspension of 2,6-diamino-4-pyrimidinone (10.0 g, 79.29 mmol)
and sodium acetate (3.5g, 42.82 mmol) in water (75 mL). The reaction mixture was stirred at 80 °C.
After 2 h, the mixture was stirred at 0 °C for 90 min. The precipitate was filtered off, and washed with
1
a little amount of cold water and acetone to afford the title product (11.9 g, quantitative yield); H

NMR (300 MHz, DMSO-d ) δ 10.97 (s, 1H), 10.23 (s, 1H), 6.61−6.60 (m, 1H), 6.18−6.17 (m, 1H),
6
6
.05 (s, 2H).
4
.3. N-(4-Oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-2-yl)octanamide (2) [26]
To a suspension of compound 1 (20.0 g, 133.21 mmol) in pyridine (133 mL) was added octanoyl
chloride (68.2 mL, 399.60 mmol) at 0 °C. The reaction mixture was heated at 85 °C. After 30 min, the
mixture was cooled to room temperature, and neutralized with 6.5 % NH in EtOH (400 mL) at 0 °C.
3
The dark red solution was left at room temperature for overnight. A crystalline precipitate was
1
formed, filtered off, and washed with ethanol and ether to give the title product (17.4g, 75% yield); H
NMR (400 MHz, DMSO-d ) δ 11.75 (s, 1H), 11.66 (s, 1H), 11.36 (s, 1H), 6.95 (s, 1H), 6.41 (s, 1H),
6
1
3
2
.44(t, J = 7.4 Hz, 2H), 1.59 (t, J = 6.5 Hz, 2H), 1.28 (s, 8H), 0.87 (t, J = 6.3 Hz, 3H); C NMR (75
MHz, DMSO-d ) δ 176.4, 157.3, 148.3, 146.6, 120.0, 104.2, 102.7, 36.4, 31.6, 28.9, 24.9, 22.5, 14.4.
6
4
.4. N-(5-((Dibenzylamino)methyl)-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-2-yl)octanamide
3)
To a suspension of compound 2 (4.00 g, 14.48 mmol) in 80% aqueous acetic acid (58 mL) were added
7% formalin solution (2.17 mL, 28.95 mmol) and N,N-dibenzylamine (5.57 mL, 28.95 mmol). The
reaction mixture was stirred at 75 °C for 24 h, and cooled to room temperature. Aqueous solution of
.5 M hydrochloric acid (72 mL) was added to the reaction mixture at room temperature. After 30
(
3
0
min, this mixture was neutralized with aqueous ammonia, and the mixture was then extracted with
dichloromethane. The organic layer was washed with brine, dried (Na SO ), and filtered. The solvent
2
4
was evaporated under reduced pressure, and the residue was purified by flash column chromatography
1
with dichloromethane/methanol (10/1, 5/1, v/v) to obtain compound 3 (5.23 g, 74% yield); H NMR
(
300 MHz, DMSO-d ) δ 11.55 (s, 1H), 11.36 (br. s, 1H), 7.40 (d, J = 7.0 Hz, 4H), 7.31 (t, J = 7.3 Hz,
6
4
6
1
2
4
H), 7.23−7.19 (m, 2H), 6.87 (s, 1H), 3.76 (s, 2H), 3.57 (s, 4H), 2.42 (t, J = 7.3 Hz, 2H), 1.57 (t, J =
1
3
.2 Hz, 2H), 1.26 (s, 8H), 0.85 (t, J = 6.4 Hz, 3H); C NMR (75 MHz, DMSO-d ) δ 176.4, 157.8,
6
48.2, 146.6, 140.2, 128.8, 128.6, 127.1, 118.0, 116.2, 103.3, 57.3, 48.8, 36.4, 31.5, 28.83, 28.81,
4.9, 22.5, 14.4.
.5. General procedure for synthesis of compounds 4a–o
Method A: (4a–f and 4n)
To a suspension of compound 2 (1 eq.) in 80% aqueous acetic acid (4 mL) were added 37% formalin
solution (2 equiv.) and the appropriate piperazine or piperidine derivative (2 equiv.). The reaction
mixture was stirred at 75 °C for 24 h, and then cooled to room temperature. Aqueous solution of 0.5 N
hydrochloric acidwas added at room temperature. After 30 min, this mixture was neutralized with
aqueous ammonia, and the mixture was then extracted with dichloromethane. The organic layer was
washed with brine, dried over Na SO , and filtered. The solvent was evaporated under reduced
2
4
pressure, and the residue was purified by flash column chromatography using the appropriate elution
system to afford the target compounds 4a–f and 4n in pure form.
Method B: (4g–m and 4o)

To a suspension of compound 3 (1.0 equiv.) in MeOH/THF (1/1) was added the proper substituted
piperazine or piperidine derivative (5.0 equiv.) in sealed tube. The reaction mixture was stirred at 75
°
C for 24 h, and cooled to room temperature. The mixture solvent was evaporated under reduced
pressure, and the residue was purified by flash column chromatography using the appropriate elution
system to produce the target compounds 4g–m and 4o in pure form.
4
.5.1.
N-(4-Oxo-5-((4-phenylpiperazin-1-yl)methyl)-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-2-
yl)octanamide (4a)
Flash column chromatography was performed using dichloromethane/methanol (15/1, 10/1, v/v) to
1
obtain compound 4a (12% yield); H NMR (300 MHz, DMSO-d ) δ 11.74 (s, 1H), 11.70 (s, 1H),
6
1
3
0
1
4
1.39 (s, 1H), 7.19 (t, J = 7.9 Hz, 2H), 6.91 (d, J = 8.3 Hz, 2H), 6.75 (t, J = 7.1 Hz, 1H), 6.28 (s, 1H),
.51 (s, 2H), 3.27 (s, 2H), 3.13 (s, 4H), 2.42 (t, J = 7.3 Hz, 2H), 1.57 (t, J = 6.3 Hz, 2H), 1.26 (s, 8H),
1
3
.85 (t, J = 6.3 Hz, 3H); C NMR (75 MHz, DMSO-d ) δ 176.5, 157.0, 151.5, 148.6, 146.5, 130.4,
6
29.3, 119.2, 115.8, 104.1, 102.4, 55.0, 52.9, 48.5, 36.3, 31.6, 28.9, 25.0, 22.5, 14.4.
.5.2. N-(5-((4-Benzhydrylpiperazin-1-yl)methyl)-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-2-
yl)octanamide (4b)
Flash column chromatography was performed using dichloromethane/methanol (40/1, v/v) to obtain
1
compound 4b (61% yield); H NMR (300 MHz, DMSO-d ) δ 11.72 (s, 1H), 11.61 (s, 1H), 11.37 (s,
6
1
1
0
1
4
H), 7.40 (d, J = 7.2 Hz, 5H), 7.27 (t, J = 7.5 Hz, 3H), 7.16 (t, J = 7.3 Hz, 2H), 6.21 (s, 1H), 4.25 (s,
H), 3.45 (s, 2H), 2.42 (t, J = 7.2 Hz, 6H), 2.32 (br. s, 2H+2H), 1.57 (t, J = 6.6 Hz, 2H), 1.26 (s, 8H),
1
3
.85 (t, J = 6.4 Hz, 3H); C NMR (75 MHz, DMSO-d ) δ 176.5, 157.1, 148.5, 146.4, 143.3, 130.4,
6
30.1, 129.0, 128.0, 127.3, 104.1, 102.3, 75.5, 54.8, 52.9, 51.8, 36.3, 31.5, 28.8, 25.0, 22.5, 14.4.
.5.3.
N-(5-((4-(4-Methoxyphenyl)piperazin-1-yl)methyl)-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-
d]pyrimidin-2-yl)octanamide(4c)
Flash column chromatography was performed using dichloromethane/methanol (40/1, v/v) to obtain
1
compound 4c (quantitative yield); H NMR (300 MHz, DMSO-d ) δ 11.74 (s, 1H), 11.69 (s, 1H),
6
1
2
3
1
4
1.39 (s, 1H), 6.86 (d, J = 6.8 Hz, 2H), 6.79 (d, J = 9.2 Hz, 2H), 6.28 (s, 1H),3.67 (s, 3H), 3.51 (s,
H), 3.01 (s, 4H), 2.43 (t, J = 7.4 Hz, 2H), 1.58 (t, J = 6.7 Hz, 2H), 1.27 (s, 8H), 0.86 (t, J = 6.7 Hz,
1
3
H); C NMR (75 MHz, DMSO-d ) δ 176.5, 157.0, 153.3, 148.6, 146.5, 145.9, 130.4, 117.8, 114.7,
6
04.1, 102.4, 55.6, 55.0, 53.0, 49.9, 36.3, 31.6, 28.9, 28.8, 25.0, 22.5, 14.4.
.5.4.
N-(5-((4-(3-Chlorophenyl)piperazin-1-yl)methyl)-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-
d]pyrimidin-2-yl)octanamide (4d)
Flash column chromatography was performed using dichloromethane/methanol (40/1, v/v) to obtain
1
compound 4d (30% yield); H NMR (300 MHz, DMSO-d ) δ 11.74 (s, 1H), 11.68 (s, 1H), 11.38 (s,
6
1
1
H), 7.19 (t, J = 8.1 Hz, 1H), 6.91 (s, 1H), 6.87 (d, J = 8.4 Hz, 2H), 6.76 (d, J = 7.6 Hz, 1H), 6.28 (s,
H), 3.51 (s, 2H), 3.17 (s, 4H), 2.43 (t, J = 7.3 Hz, 2H), 1.57 (t, J = 6.3 Hz,2H), 1.26 (s, 8H), 0.85 (t, J
=
6.4 Hz, 3H); 13C NMR (75 MHz, DMSO-d ) δ 176.5, 157.1, 152.7, 148.6, 146.5, 134.3, 130.8,
6
1
30.3, 118.4, 114.9, 114.1, 104.2, 102.5, 55.0, 52.7, 48.0, 36.3, 31.6, 28.9, 28.8, 25.0, 22.5, 14.4.

4
.5.5.
N-(5-((4-(2-Cyanophenyl)piperazin-1-yl)methyl)-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-
d]pyrimidin-2-yl)octanamide (4e)
Flash column chromatography was performed using dichloromethane/methanol (40/1, v/v) to obtain
1
compound 4e (18% yield); H NMR (300 MHz, DMSO-d ) δ11.75 (br. s, 1H), 11.70 (s, 1H), 11.39
6
(br. s, 1H), 7.68 (d, J = 7.6 Hz, 1H), 7.58 (t, J = 7.8 Hz, 1H), 7.14 (d, J = 8.2 Hz, 1H), 7.08 (t, J = 7.5
Hz, 1H), 6.29 (s, 1H), 3.55 (s, 2H), 3.15 (s, 5H), 2.50 (s, 3H), 2.42 (t, J = 7.2 Hz, 2H), 1.57 (t, J = 6.3
1
3
Hz, 2H), 1.26 (s, 8H), 0.85 (t, J = 6.1 Hz, 3H); C NMR (75 MHz, DMSO-d ) δ 176.5, 157.1, 155.7,
6
1
3
4
48.6, 146.5, 134.8, 134.7, 130.2, 122.4, 119.5, 118.7, 105.2, 104.2, 102.5, 54.8, 52.8, 51.6, 36.3,
1.6, 28.8, 25.0, 22.5, 14.4.
.5.6.
N-(5-((4-(4-Nitrophenyl)piperazin-1-yl)methyl)-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-
d]pyrimidin-2-yl)octanamide (4f)
Flash column chromatography was performed using dichloromethane/methanol (40/1, 20/1, v/v) to
1
obtain compound 4f (48% yield); H NMR (300 MHz, DMSO-d ) δ 8.04 (d, J = 9.4 Hz, 2H), 7.01 (d,
6
J = 9.5 Hz, 2H), 6.28 (s, 1H), 3.53 (s, 2H), 3.46 (s, 4H), 2.43 (t, J = 7.4 Hz, 2H), 1.58 (t, J = 6.3 Hz,
1
3
2
1
2
4
H), 1.27 (s, 8H), 0.86 (t, J = 6.4 Hz, 3H); C NMR (75 MHz, DMSO-d ) δ 176.5, 157.1, 155.2,
6
48.6, 146.5, 137.3, 130.1, 126.2, 113.1, 104.2, 102.5, 54.8, 52.4, 46.7, 36.4, 31.5, 28.84, 28.80, 25.0,
2.5, 14.4.
.5.7.
N-(5-((4-(3-Fluorobenzyl)piperazin-1-yl)methyl)-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-
d]pyrimidin-2-yl)octanamide (4g)
Flash column chromatography was performed using dichloromethane/methanol(10/1, v/v) to obtain
1
compound 4g (16% yield); H NMR (300 MHz, DMSO-d ) δ 11.62 (br. s, 1H), 11.56 (s, 1H), 11.33
6
(s, 1H), 7.33 (t, J = 6.9 Hz, 1H), 7.12−7.02 (m, 3H), 6.79 (s, 1H), 3.72 (br. s, 2H), 3.46 (s, 2H), 2.42
13
(
t, J = 7.4 Hz, 2H), 2.40 (br. s, 5H), 1.57 (t, J = 6.8 Hz, 2H), 1.26 (s, 8H), 0.85 (t, J = 6.3 Hz, 3H); C
NMR (150 MHz, DMSO-d ) δ 175.9, 163.0, 161.4, 157.3, 147.6, 146.1, 141.4, 130.02, 129.96, 124.6,
6
1
15.1, 115.0, 113.7, 113.5, 103.0, 61.2, 52.2, 51.8, 35.9, 31.1, 29.8, 28.3, 24.4, 22.0, 13.9.
.5.8. N-(5-((4-Methylpiperazin-1-yl)methyl)-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-2-
4
yl)octanamide (4h)
Flash column chromatography was performed using dichloromethane/methanol (10/1, 5/1, v/v) to
1
obtain compound 4h (31% yield); H NMR (400 MHz, DMSO-d ) δ 11.65 (br. s, 1H), 11.52 (s, 1H),
6
1
1
1
4
1.33 (br. s, 1H), 6.77 (s, 1H), 3.65 (s, 2H), 2.43 (t, J = 6.2 Hz, 6H), 2.29 (br. s, 4H), 2.13 (s, 3H),
1
3
.59 (br. s, 2H), 1.28 (s, 8H), 0.87 (t, J = 6.4 Hz, 3H); C NMR (75 MHz, DMSO-d ) δ 176.3, 157.7,
6
48.0, 146.5, 118.7, 115.3, 103.5, 55.3, 52.9, 52.4, 46.3, 36.4, 31.6, 28.8, 24.9, 22.5, 14.4.
.5.9.
N-(5-((1H-Imidazol-1-yl)methyl)-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-2-
yl)octanamide (4i)
Flash column chromatography was performed using dichloromethane/methanol (20/1, 10/1, v/v) to
1
obtain compound 4i (40% yield); H NMR (300 MHz, DMSO-d ) δ 7.82 (s, 1H), 7.28 (s, 1H), 6.98 (s,
6
1
H), 6.88 (s, 1H), 5.22 (s, 2H), 3.32 (s, 2H), 2.43 (t, J = 7.5 Hz, 2H), 1.58 (t, J = 6.2 Hz, 2H), 1.27 (s,

1
3
8
1
4
H), 0.86 (t, J = 6.4 Hz, 3H); C NMR (75 MHz, DMSO-d ) δ 176.5, 157.6, 148.7, 147.1, 137.2,
6
27.8, 119.9, 119.5, 115.0, 102.3, 42.1, 36.4, 31.5, 28.8, 24.9, 22.5, 14.4.
.5.10.N-(5-((4-Acetylpiperazin-1-yl)methyl)-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-2-
yl)octanamide (4j)
Flash column chromatography was performed using dichloromethane/methanol/water/aq. NH3
1
(
80/16/1/1, v/v/v/v) to obtain compound 4j (97% yield); H NMR (300 MHz, MeOD-d ) δ 7.91 (s,
4
1
H), 6.77 (s, 1H), 4.07 (s, 2H), 3.69−3.66 (m, 4H), 2.93 (br. s, 2H), 2.87 (br. s, 2H), 2.23 (t, J = 7.5
1
3
Hz, 2H), 2.09 (s, 3H), 1.61 (t, J = 6.9 Hz, 2H), 1.29 (s, 8H), 0.88 (t, J = 6.4 Hz, 3H); C NMR (75
MHz, MeOD-d ) δ 170.2, 161.0, 153.0, 151.8, 118.5, 109.9, 99.1, 78.2, 52.1, 51.2, 51.0, 44.6, 39.9,
4
3
6.4, 31.6, 29.2, 28.9, 25.8, 22.3, 19.8, 13.1.
4
.5.11.N-(5-((4-(Furan-2-carbonyl)piperazin-1-yl)methyl)-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-
d]pyrimidin-2-yl)octanamide (4k)
Flash column chromatography was performed using dichloromethane/methanol (10/1, 5/1, v/v) to
1
obtain compound 4k (5.4% yield); H NMR (300 MHz, MeOD-d ) δ 7.69 (s, 1H), 7.58 (s, 1H),
4
6
.99−6.97 (m, 1H), 6.83 (s, 1H), 6.52−6.51 (m, 1H), 6.44 (s, 1H), 3.90 (s, 1H), 3.81 (br. s, 4H), 3.61
(
(
s, 1H), 2.66 (s, 3H), 2.56 (s, 2H), 2.44 (t, J = 7.3 Hz, 2H), 1.69 (t, J = 6.3 Hz, 2H), 1.29 (s, 8H), 0.88
1
3
t, J = 6.3 Hz, 3H); C NMR (75 MHz, MeOD-d ) δ 176.1, 159.6, 147.0, 144.4, 130.0, 119.8, 116.6,
4
1
16.5, 113.3, 111.1, 104.0, 102.3, 54.5, 52.5, 52.0, 36.5, 31.5, 28.84, 28.78, 24.9, 22.4, 13.4.
4
.5.12.N-(5-((4-(2-Hydroxyethyl)piperazin-1-yl)methyl)-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-
d]pyrimidin-2-yl)octanamide (4l)
Flash column chromatography was performed using dichloromethane/methanol/aqueous NH (40/8/1,
3
1
v/v/v) to obtain he compound 4l (13% yield); H NMR (300 MHz, DMSO-d ) δ 11.64 (br. s, 1H),
6
1
1
1
4
1.52 (s, 1H), 11.35 (br. s, 1H), 6.75 (s, 1H), 4.33 (br. s, 1H), 3.63 (s, 2H), 3.44 (br. s, 4H), 2.41 (s,
0H), 1.56 (br. s, 2H), 1.26 (s, 8H), 0.85 (s, 3H); 13C NMR (75 MHz, DMSO-d ) δ 176.3, 157.7,
6
48.0, 146.4, 118.6, 115.2, 103.5, 60.8, 59.0, 53.8, 52.9, 52.5, 36.3, 31.6, 28.8, 24.9, 22.5, 14.4.
.5.13.N-(5-((4-(2-Morpholinoethyl)piperazin-1-yl)methyl)-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-
d]pyrimidin-2-yl)octanamide (4m)
Flash column chromatography was performed using dichloromethane/methanol/aqueous NH (40/8/1,
3
1
v/v/v) to obtain compound 4m (14% yield); H NMR (300 MHz, CD OD) δ 7.35 (br. s, 2H), 6.90 (s,
3
1
H),3.91 (s, 2H), 3.70−3.68 (m, 5H), 2.54−2.44 (m, 15H), 1.71 (t, J = 7.5 Hz, 2H), 1.34 (s, 8H), 0.92
t, J = 6.3 Hz, 3H).
.5.14.N-(4-Oxo-5-(piperidin-1-ylmethyl)-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-2-yl)octanamide
4n)
(
4
(
Flash column chromatography was performed using dichloromethane/methanol (25/1, 15/1, v/v) to
1
obtain compound 4n (30% yield); H NMR (300 MHz, DMSO-d ) δ 11.73 (s, 1H), 11.61 (s,1H),
6
1
2
1.38 (s, 1H), 6.21 (s, 1H), 3.40 (s, 2H), 2.43 (t, J = 7.4 Hz, 2H), 2.33 (s, 4H), 1.56 (t, J = 6.3 Hz,
1
3
H), 1.49 (s, 5H), 1.38 (br. s, 3H), 1.27 (s, 8H), 0.86 (t, J = 6.4 Hz, 3H); C NMR (75 MHz, DMSO-

d6) δ 176.4, 157.0, 148.5, 146.4, 130.9, 104.1, 102.1, 55.8, 54.1, 36.3, 31.5, 28.8, 25.8, 25.0, 24.4,
2
2.5, 14.4.
4
.5.15.tert-Butyl(1-((2-octanamido-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-
yl)methyl)piperidin-4-yl)carbamate(4o)
Flash column chromatography was performed using dichloromethane/methanol/aqueous NH (40/8/1,
3
1
v/v/v) to obtain compound 4o(11% yield); H NMR (300 MHz, DMSO-d ) δ 11.62 (br. s, 1H), 11.50
6
(s, 1H), 11.30 (br. s, 1H), 6.76 (s, 1H), 3.63 (s, 2H), 3.32 (s, 2H), 3.14 (br. s, 1H), 2.84 (d, J = 9.7 Hz,
2
0
4
H), 2.42 (t, J = 7.0 Hz, 2H), 1.98 (t, J = 10.1 Hz, 2H), 1.62−1.57 (m, 4H), 1.36 (s, 9H), 1.27 (s, 8H),
.86 (t, J = 6.4 Hz, 3H).
.6. General procedure for synthesis of compounds 5a–m
To a solution of the appropriate octamide derivative 4 (1.0 equiv.) in MeOH/THF (1/1) was added
aqueous 5 N KOH (0.25 equiv.). The reaction mixture was stirred at room temperature for 3 days. The
solvent was evaporated under reduced pressure, and the residue was washed with CH CN and filtered,
3
or the residue was purified with flash column chromatography using the proper eluent to afford
compounds 5a–m in pure form.
4
.6.1.
2-Amino-5-((4-phenylpiperazin-1-yl)methyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one
(5a)
1
8
6
3
1
4
1% yield; H NMR (300 MHz, DMSO-d ) δ 10.98 (s, 1H), 10.17 (s, 1H), 7.19 (t, J = 7.4 Hz, 2H),
6
.91 (d, J = 8.6 Hz, 2H), 6.76 (t, J = 6.7 Hz, 1H), 6.05 (s, 1H), 6.03 (s, 2H), 3.42 (s, 2H), 3.32 (s, 4H),
.12 (s, 4H); 13C NMR (100 MHz, DMSO-d ) δ 159.0, 152.6, 151.9, 151.5, 129.4, 127.2, 119.2,
6
15.8, 101.7, 100.2, 55.2, 52.8, 48.6.
.6.2. 2-Amino-5-((4-benzhydrylpiperazin-1-yl)methyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-
one (5b)
1
6
7
6% yield; H NMR (300 MHz, DMSO-d ) δ 10.93 (s, 1H), 10.21 (s, 1H), 7.39 (d, J = 7.0 Hz, 5H),
6
.26 (t, J = 7.2 Hz, 3H), 7.16 (d, J = 7.1 Hz, 2H), 6.08 (s, 1H), 6.03 (s, 1H), 4.15 (s, 1H), 2.33 (s, 4H);
1
3
C NMR (75 MHz, DMSO-d ) δ 159.0, 152.7, 151.8, 143.3, 129.0, 128.0, 127.3, 100.3, 75.4, 54.7,
6
5
2.6, 51.6.
4
.6.3.2-Amino-5-((4-(4-methoxyphenyl)piperazin-1-yl)methyl)-3,7-dihydro-4H-pyrrolo[2,3-
d]pyrimidin-4-one (5c)
1
6
6
0% yield; H NMR (300 MHz, DMSO-d ) δ 11.22 (s, 1H), 7.00 (br. s., 2H), 6.88 (d, J = 8.9 Hz, 2H),
6
1
3
.79 (d, J = 8.8 Hz, 2H), 6.00 (s, 1H), 3.66 (s, 3H), 3.44 (s, 3H), 3.02 (s, 5H); C NMR (100 MHz,
DMSO-d ) δ 162.4, 153.3, 146.0, 126.1, 117.8, 114.7, 101.6, 100.3, 55.6, 52.9, 50.0.
6
4
.6.4.2-Amino-5-((4-(3-chlorophenyl)piperazin-1-yl)methyl)-3,7-dihydro-4H-pyrrolo[2,3-
d]pyrimidin-4-one(5d)
1
8
9% yield; H NMR (300 MHz, DMSO-d ) δ 11.10 (s, 1H), 7.19 (t, J = 7.9 Hz, 1H), 6.93 (s, 1H), 6.89
6
1
3
(
d, J = 9.1 Hz, 1H), 6.76 (d, J = 7.6 Hz, 2H), 5.95 (s, 1H), 3.43 (s, 4H), 3.18 (s, 4H); C NMR (75

MHz, DMSO-d ) δ 166.2, 158.4, 153.7, 152.8, 134.2, 130.8, 124.8, 118.4, 115.0, 114.1, 101.7, 100.8,
6
5
5.8, 52.6, 48.0.
.6.5. 2-(4-((2-Amino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-5-yl)methyl)piperazin-1-
4
yl)benzonitrile (5e)
1
7
3% yield; H NMR (300 MHz, DMSO-d ) δ 11.00 (s, 1H), 10.22 (s, 1H), 7.70−7.68 (m, 1H),
6
1
3
7
.61−7.57 (m, 1H), 7.17−7.06 (m, 2H), 6.07 (s, 2H), 3.46 (s, 2H), 3.13 (s, 4H), 2.50 (s, 3H); C NMR
(
75 MHz, DMSO-d ) δ 159.1, 155.7, 152.7, 151.8, 134.8, 134.7, 127.0, 122.4, 119.5, 118.7, 105.2,
6
1
4
4
01.8, 100.2, 54.9, 52.6, 51.6.
.6.6. 2-Amino-5-((4-(4-nitrophenyl)piperazin-1-yl)methyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-
-one (5f)
1
Quantitative yield; H NMR (400 MHz, DMSO-d ) δ 11.19 (br. s, 1H), 8.03 (d, J = 8.7 Hz, 2H), 7.01
6
1
3
(
d, J = 9.1 Hz, 3H), 5.98 (s, 1H), 3.75–3.45 (m, 10H); C NMR (100 MHz, DMSO-d ) δ 164.69,
6
1
4
4
57.10, 154.81, 153.04, 136.81, 125.71, 124.59, 113.07, 112.65, 101.39, 100.07, 67.03, 55.05, 51.89,
6.19, 25.13.
.6.7.
2-Amino-5-((4-(3-fluorobenzyl)piperazin-1-yl)methyl)-3,7-dihydro-4H-pyrrolo[2,3-
d]pyrimidin-4-one (5g)
Flash column chromatography was performed using dichloromethane/methanol/aqueous NH (40/8/1,
3
1
v/v/v) to afford compound 5g (51% yield); H NMR (300 MHz, MeOD-d ) δ 7.33−7.25 (m, 1H),
4
7
2
1
5
4
.10−7.04 (m, 2H), 6.97 (t, J = 8.4H, 1H), 6.67 (s, 1H), 3.88 (s, 2H), 3.49 (s, 2H), 2.73 (br. s, 4H),
1
3
.52 (br. s, 4H); C NMR (75 MHz, MeOD-d ) δ 164.5, 161.3, 161.1, 152.9, 151.3, 140.2, 140.1,
4
29.6, 129.5, 124.8, 124.82, 124.78, 117.7, 115.7, 115.4, 113.8, 113.5, 111.9, 99.5, 78.1, 61.6, 52.8,
2.1, 51.9, 51.3.
.6.8.
5h)
2-Amino-5-((4-methylpiperazin-1-yl)methyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one
(
Flash column chromatography was performed using dichloromethane/methanol/aqueous NH (40/8/ 1,
3
1
v/v/v) to afford compound 5h (73% yield); H NMR (300 MHz, CD OD) δ 7.91 (s, 1H), 6.66 (s, 1H),
3
1
3
3
1
4
.85 (s, 2H), 2.70 (br. s, 4H), 2.51 (br. s, 4H), 2.24 (s, 3H); C NMR (75 MHz, CD OD) δ 161.0,
3
52.9, 151.4, 117.7, 112.1, 99.5, 78.2, 53.9, 52.1, 51.1, 44.5.
.6.9. 5-((1H-Imidazol-1-yl)methyl)-2-amino-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one (5i)
A solution of compound 14o (105 mg, 0.30 mmol) in MeOH/THF (1/1) (1.5 mL) was added aqueous
M KOH (0.015 mL). This reaction mixture was stirred at room temperature for 3 days. The mixture
5
was concentrated under reduced pressure, and the residue was purified by column chromatography
1
with dichloromethane/methanol/aqueous NH (40/8/1, v/v/v) to afford compound 15o (94% yield); H
3
NMR (300 MHz, DMSO-d ) δ 10.97 (s, 1H), 10.35 (br. s, 1H), 7.70 (s, 1H), 7.24 (s, 1H), 6.80 (s, 1H),
6
1
3
6
1
.63 (s, 1H), 6.12 (s, 2H), 5.11 (s, 2H); C NMR (75 MHz, DMSO-d ) δ 159.6, 153.0, 152.0, 137.4,
6
28.4, 119.7, 116.5, 114.6, 98.5, 42.2.

4
.6.10.
5-((4-Acetylpiperazin-1-yl)methyl)-2-amino-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one
(5j)
Flash column chromatography was performed using dichloromethane/methanol/aqueous NH (40/8/1,
3
1
v/v/v) to afford compound 5j (138 mg, quantitative yield); H NMR (300 MHz, CD OD) δ6.70 (s,
3
1
3
1
H), 3.91 (s, 2H), 3.59 (br. s, 2H), 2.73 (br. s, 2H), 2.67 (br. s, 4H), 2.09 (s, 3H); C NMR (75 MHz,
CD OD) δ 170.2, 160.8, 152.7, 151.5, 117.9, 111.8, 99.4, 52.1, 51.7, 51.3, 19.7.
3
4
.6.11.2-Amino-5-((4-(2-hydroxyethyl)piperazin-1-yl)methyl)-3,7-dihydro-4H-pyrrolo[2,3-
d]pyrimidin-4-one (5k)
Flash column chromatography was performed using dichloromethane/methanol/aqueous NH (40/8/1,
3
1
v/v/v) to afford the compound 5k (58% yield); H NMR (300 MHz, CD OD) δ 6.66 (s, 1H), 3.85 (s,
3
2
H), 3.66 (t, J = 6.0 Hz, 2H), 3.36 (s, 1H), 2.70 (br. s, 4H), 2.60 (br. s, 4H), 2.52 (t, J = 6.0 Hz, 2H);
1
3
C NMR (75 MHz, CD OD) δ 161.0, 152.9, 151.2, 117.7, 112.2, 99.6, 59.8, 58.4, 52.5, 52.0, 51.2.
3
4
.6.12.2-Amino-5-((4-(2-morpholinoethyl)piperazin-1-yl)methyl)-3,7-dihydro-4H-pyrrolo[2,3-
d]pyrimidin-4-one (5l)
Flash column chromatography was performed using dichloromethane/methanol/ aqueous NH (40/8/1,
3
1
v/v/v) to afford compound 5l (43% yield); H NMR (300 MHz, CD OD) δ 6.70 (s, 1H), 3.94 (s, 2H),
3
1
3
3
1
4
1
1
1
4
.68 (t, J = 4.6 Hz, 5H), 2.80 (br. s, 4H), 2.62−2.50 (m, 11H); C NMR (75 MHz, CD OD) δ 161.0,
3
52.9, 151.5, 117.9, 111.3, 99.4, 66.2, 55.4, 54.4, 53.7, 52.2, 52.0, 51.1.
.6.13. 2-Amino-5-(piperidin-1-ylmethyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one (5m)
1
4% yield; H NMR (300 MHz, DMSO-d ) δ 10.90 (s, 1H), 10.28 (br. s, 1H), 6.08 (s, 2H), 5.97 (s,
6
1
3
H), 3.30 (s, 2H), 2.29 (s, 4H), 1.47 (s, 5H), 1.36 (s, 2H); C NMR (75 MHz, DMSO-d ) δ 159.3,
6
52.8, 151.8, 127.6, 101.4, 100.1, 56.0, 54.0, 25.9, 24.5.
.7. General procedure for synthesis of compounds 6a–g
Compounds 6a–g were synthesized from compound 2 following the same procedures described for
compounds 4a–o.
4
.7.1.
N-(5,6-Bis((4-benzhydrylpiperazin-1-yl)methyl)-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-
d]pyrimidin-2-yl)octanamide (6a)
Flash column chromatography was carried out using dichloromethane/methanol (40/1, 25/1, v/v) to
1
obtain compound 6a (10% yield); H NMR (300 MHz, DMSO-d ) δ 11.62 (br. s, 1H), 11.50 (s, 1H),
6
1
1.33 (br. s, 1H), 7.38 (t, J = 7.5 Hz, 8H), 7.30−7.23 (m, 8H), 7.20−7.12 (m, 4H), 4.23 (s, 1H), 4.15
(s, 1H), 3.66 (s, 2H), 3.47 (s, 2H), 2.41 (t, J = 6.7 Hz, 10H), 2.27 (br. s, 7H), 1.55 (t, J = 6.3 Hz, 2H),
1
3
1
1
3
4
.24 (s, 8H), 0.84 (t, J = 6.1 Hz, 3H); C NMR (75 MHz, DMSO-d ) δ 176.4, 157.5, 147.4, 146.3,
6
43.5, 143.4, 128.9, 128.8, 128.0, 127.3, 127.2, 113.2, 104.0, 75.8, 75.5, 53.2, 52.5, 52.1, 51.8, 50.9,
6.3, 31.5, 28.8, 25.0, 22.5, 14.4.
.7.2.N-(5,6-Bis((4-(3,5-dichlorophenyl)piperazin-1-yl)methyl)-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-
d]pyrimidin-2-yl)octanamide (6b)

Flash column chromatography was carried out using dichloromethane/methanol (40/1, v/v) to obtain
1
compound 6b (34% yield); H NMR (300 MHz, DMSO-d ) δ 11.67 (s, 1H), 11.63 (s, 1H), 11.36 (s,
6
1
4
H), 6.90 (d, J = 8.5 Hz, 4H), 6.82 (d, J = 6.2 Hz, 2H), 3.73 (s, 2H), 3.57 (s, 2H), 3.22 (s, 4H), 3.16 (s,
1
3
H), 2.43 (t, J = 7.3 Hz, 2H), 1.58 (t,J = 6.6 Hz, 2H), 1.26 (s, 8H), 0.86 (t, J = 6.3 Hz, 3H); C NMR
(
75 MHz, DMSO-d ) δ 176.4, 157.5, 153.14, 153.10, 147.6, 146.5, 135.1, 135.0, 128.8, 117.3, 117.2,
6
1
13.42, 113.40, 104.0, 52.6, 52.1, 51.0, 47.7, 47.6, 36.3, 31.6, 28.8, 25.0, 22.5, 14.4.
.7.3. N-(5,6-Bis((4-(4-nitrophenyl)piperazin-1-yl)methyl)-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-
4
d]pyrimidin-2-yl)octanamide (6c)
Flash column chromatography was carried out using dichloromethane/methanol (40/1, 20/1, v/v) to
1
obtain compound 6c (52% yield); H NMR (300 MHz, DMSO-d ) δ 8.03 (dd, J = 9.4 Hz, 2.1 Hz, 4H),
6
7
1
1
4
4
.00 (t, J = 9.0 Hz, 4H), 3.75 (s, 2H), 3.59 (s, 2H), 3.46 (s, 5H), 3.40 (s, 6H), 2.42 (t, J = 7.4 Hz, 2H),
1
3
.57 (t, J = 6.3 Hz, 2H), 1.27 (s, 8H), 0.86 (t, J = 6.4 Hz, 3H); C NMR (75 MHz, DMSO-d ) δ 176.4,
6
57.5, 155.2, 147.6, 146.5, 137.3, 137.1, 128.8, 126.2, 113.1, 113.0, 104.0, 52.6, 52.0, 50.8, 46.9,
6.8, 36.3, 31.5, 28.8, 25.0, 22.5, 14.4.
.7.4. N-(5,6-Bis((4-benzylpiperazin-1-yl)methyl)-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-2-
yl)octanamide (6d)
Flash column chromatography was carried out using dichloromethane/methanol (40/1, 25/1, v/v) to
1
obtain compound 6d (9% yield); H NMR (300 MHz, DMSO-d ) δ 11.62 (br. s, 1H), 11.33 (br. s,
6
1
H), 11.28 (s, 1H), 7.24−7.17 (m, 10H), 3.67 (s, 2H), 3.48 (s, 2H), 3.42 (s, 2H), 3.38 (s, 2H),
1
3
2
.41−2.37 (m, 18H), 1.57 (t, J = 6.7 Hz, 2H), 1.25 (s, 8H), 0.84 (t, J = 6.4 Hz, 3H); C NMR (75
MHz, DMSO-d ) δ 176.1, 157.9, 157.6, 148.1, 147.5, 146.5, 146.3, 138.71, 138.66, 138.5, 129.1,
6
1
5
29.0, 128.7, 128.31, 128.28, 127.1, 127.0, 118.4, 115.1, 113.3, 104.0, 103.6, 62.73, 62.67, 53.2, 53.1,
3.0, 52.8, 52.4, 51.1, 36.4, 31.6, 28.91, 28.89, 25.1, 22.5, 14.3; HRMS (ESI-TOF) m/z calcd for
+
C H N O [M+H] : 653.4291, found: 653.4291.
3
8
53
8
2
4
.7.5.
N-(5,6-Bis((4-(2-fluorobenzyl)piperazin-1-yl)methyl)-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-
d]pyrimidin-2-yl)octanamide (6e)
Flash column chromatography was carried out using dichloromethane/methanol (10/1, 5/1, v/v) to
1
obtain compound 6e (15% yield); H NMR (300 MHz, DMSO-d ) δ 11.48 (s, 1H), 11.36 (br. s, 1H),
6
7
1
1
1
2
6
4
.39−7.24 (m, 4H), 7.18−7.08 (m, 4H), 3.64 (s, 2H), 3.49 (s, 2H), 3.46 (s, 4H), 2.43−2.31 (m, 18H),
1
3
.56 (t, J = 6.8 Hz, 2H), 1.25 (s, 8H), 0.84 (t, J = 6.3 Hz, 3H); C NMR (75 MHz, DMSO-d ) δ 176.3,
6
62.8, 159.6, 157.5, 147.5, 146.4, 131.92, 131.86, 129.5, 129.4, 129.3, 128.8, 125.2, 125.1, 125.0,
24.9, 124.5, 115.7, 115.6, 115.4, 113.3, 103.9, 54.9, 54.2, 53.1, 52.7, 52.4, 51.0, 45.9, 36.3, 31.6,
+
8.8, 25.0, 22.5, 14.4; HRMS (ESI-TOF) m/z calcd for C H FN O [M+H] : 689.4103, found:
3
8
51
8
2
89.4110.
.7.6.
N-(5,6-Bis((4-(3-fluorobenzyl)piperazin-1-yl)methyl)-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-
d]pyrimidin-2-yl)octanamide (6f)

Flash column chromatography was carried out using dichloromethane/methanol (40/1, 20/1, 10/1, v/v)
1
to obtain compound 6f (70% yield); H NMR (300 MHz, DMSO-d ) δ 11.59 (s, 1H), 11.52 (s,1H),
6
1
1
1
1
1.35 (s, 1H), 7.37−7.28 (m, 2H), 7.12−7.00 (m, 6H), 3.65 (s, 2H), 3.48−3.41 (m, 6H), 2.43−2.38 (m,
1
3
7H), 1.55 (t, J = 5.9 Hz, 2H), 1.24 (s, 8H), 0.84 (t, J = 6.4 Hz, 3H); C NMR (75 MHz, DMSO-d ) δ
6
76.3, 164.3, 161.1, 147.5, 146.5, 142.1, 142.0, 141.9, 130.5, 130.4, 128.9, 125.1, 115.6, 115.4, 114.2,
13.9, 103.9, 61.7, 53.1, 52.9, 52.4, 51.1, 36.3, 31.6, 28.8, 25.0, 22.5, 14.4; HRMS (ESI-TOF) m/z
+
calcd for C H FN O [M+H] : 689.4103, found: 689.4106.
3
8
51
8
2
4
.7.7.
N-(5,6-Bis((4-(4-fluorobenzyl)piperazin-1-yl)methyl)-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-
d]pyrimidin-2-yl)octanamide (6g)
Flash column chromatography was carried out using dichloromethane/methanol (40/1, 25/1, v/v) to
1
obtain compound 6g (18% yield); H NMR (300 MHz, DMSO-d ) δ 11.46 (s, 1H), 7.32−7.25 (m,
6
4
H), 7.14−7.06 (m, 4H), 3.65 (s, 2H), 3.48 (s, 2H), 3.40 (d, J = 9.2 Hz, 4H), 2.67 (t, J = 4.8 Hz, 1H),
1
3
2
.44−2.27 (m, 16H), 1.57 (t, J = 6.3 Hz, 2H), 1.25 (s, 8H), 0.83 (t, J = 6.4 Hz, 3H); C NMR (75
MHz, DMSO-d ) δ 176.1, 163.2, 160.0, 157.6, 147.5, 146.3, 134.8, 134.7, 134.6, 130.8, 130.7, 128.7,
6
1
1
4
15.2, 114.9, 113.3, 104.0, 62.5, 61.7, 54.3, 53.1, 52.9, 52.4, 51.1, 46.0, 36.4, 31.6, 28.9, 25.1, 22.5,
+
4.3; HRMS (ESI-TOF) m/z calcd for C H FN O [M+H] : 689.4103, found: 689.4107.
3
8
51
8
2
.8. General procedure for synthesis of compounds 7a–g
Compounds 7a–gwere synthesized from their corresponding octamides 6a–g following the same
procedures described for compounds 5a–m.
4
4
.8.1. 2-Amino-5,6-bis((4-benzhydrylpiperazin-1-yl)methyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-
-one (7a)
Flash column chromatography was carried out using dichloromethane/methanol/aqueous NH (40/8/1,
3
1
v/v/v) to afford compound 7a (92% yield); H NMR (400 MHz, THF-d ) δ 7.39 (d, J = 7.5 Hz, 8H),
8
7
3
1
3
4
.19 (q, J = 7.9 Hz, 8H), 7.12–7.08 (m,, 4H), 5.89 (br. s, 2H), 4.21 (s, 1H), 4.15 (s, 1H), 3.75 (s, 2H),
1
3
.49 (s, 2H), 2.53–2.35 (m, 16H), 1.39 (s, 2H); C NMR (100 MHz, THF-d ) δ 144.38, 144.10,
8
29.17, 129.13, 128.77, 127.58, 127.58, 127.47, 77.44, 77.13, 54.25, 53.78, 53.37, 53.03, 52.66,
0.77.
.8.2.2-Amino-5,6-bis((4-(3,5-dichlorophenyl)piperazin-1-yl)methyl)-3,7-dihydro-4H-pyrrolo[2,3-
d]pyrimidin-4-one (7b)
1
6
3% yield; H NMR (300 MHz, DMSO-d ) δ 10.99 (br. s, 1H), 10.95 (s, 1H), 6.91-6.89 (m, 4H), 6.82
6
(
s, 2H), 6.06 (s, 2H), 3.70 (s, 2H), 3.48 (s, 2H), 3.19 (s, 8H), 2.50 (s, 8H); 13C NMR (75 MHz,
DMSO-d ) δ 159.6, 153.1, 152.7, 150.9, 135.1, 125.8, 117.3, 117.2, 113.4, 111.9, 100.1, 52.5, 52.0,
6
+
5
1.2, 47.6; HRMS (ESI-TOF) m/z calcd for C H Cl N O [M+H] : 635.1375, found: 635.1378.
2
8
31
4
8
4
.8.3.
2-Amino-5,6-bis((4-(4-nitrophenyl)piperazin-1-yl)methyl)-3,7-dihydro-4H-pyrrolo[2,3-
d]pyrimidin-4-one (7c)
Flash column chromatography was carried out using dichloromethane/methanol/aqueous NH (40/8/1,
3
1
v/v/v) to afford compound 7c (60 mg, 47% yield); H NMR (300 MHz, D O) δ 5.54−5.49 (m, 4H),
2

4
3
4
.75 (d, J = 8.4 Hz, 2H), 4.61 (d, J = 9.2 Hz, 2H), 1.51−1.48 (m, 4H), 1.36−1.28 (m, 3H), 1.14 (br. s,
H), 1.07−0.99 (m, 5H), 0.95 (br. s, 4H).
.8.4. 2-Amino-5,6-bis((4-benzylpiperazin-1-yl)methyl)-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-
one (7d)
Flash column chromatography was carried out using dichloromethane/methanol/aqueous NH (40/8/1,
3
1
v/v/v) to afford compound 7d (54% yield); H NMR (300 MHz,DMSO-d ) δ 10.71 (s, 1H), 10.02 (s,
6
1
H), 7.28 (s, 10H), 6.00 (s, 2H), 3.57 (br. s, 8H), 2.35 (br. s, 12H).
.8.5. 2-Amino-5,6-bis((4-(2-fluorobenzyl)piperazin-1-yl)methyl)-3,7-dihydro-4H-pyrrolo[2,3-
4
d]pyrimidin-4-one (7e)
Flash column chromatography was carried out using dichloromethane/methanol/aqueous NH (40/8/
3
1
1
, v/v/v) to afford compound 7e (94% yield); H NMR (300 MHz, CDCl ) δ 7.35−7.24 (m, 4H),
3
1
3
7
.14−6.99 (m, 4H), 3.83 (s, 2H), 3.60−3.55 (m, 6H), 2.69 (br. s, 4H), 2.52 (s, 12H); C NMR (75
MHz, CDCl ) δ 167.0, 165.1, 163.8, 157.3, 155.2, 135.9, 135.8, 133.3, 133.2, 133.0, 129.9, 127.8,
3
1
5
4
27.5, 127.4, 127.3, 127.2, 119.22, 119.18, 118.92, 118.88, 115.2, 103.8, 59.4, 58.8, 58.7, 57.1, 56.3,
6.1, 55.9, 55.5, 54.8, 48.8.
.8.6.
2-Amino-5,6-bis((4-(3-fluorobenzyl)piperazin-1-yl)methyl)-3,7-dihydro-4H-pyrrolo[2,3-
d]pyrimidin-4-one (7f)
Flash column chromatography was carried out using dichloromethane/methanol/aqueous NH (40/8/
3
1
1
, v/v/v) to afford the compound 7f (73% yield); H NMR (300 MHz, DMSO-d ) δ 8.24 (s, 2H),
6
7
.35−7.28 (m, 2H), 7.12−7.00 (m, 6H), 6.42 (s, 1H), 4.24 (s, 2H), 3.12 (br. s, 4H), 2.50 (s, 2H), 2.42
(
br. s, 4H), 2.38 (br. s, 4H); 13C NMR (75 MHz, DMSO-d ) δ 164.3, 161.1, 160.6, 153.1, 152.0,
6
1
1
4
41.6, 141.5, 140.9, 140.8, 131.8, 130.6, 130.5, 130.3, 127.6, 126.4, 125.1, 125.0, 115.8, 115.7, 115.6,
15.4, 114.5, 114.2, 113.9, 106.7, 99.0, 61.7, 60.9, 52.7, 52.6, 52.3, 51.2, 51.1, 50.5, 49.5, 43.4.
.8.7.
2-Amino-5,6-bis((4-(4-fluorobenzyl)piperazin-1-yl)methyl)-3,7-dihydro-4H-pyrrolo[2,3-
d]pyrimidin-4-one (7g)
Flash column chromatography was carried out using dichloromethane/methanol/aqueous NH (40/8/
3
1
1
7
1
1
1
4
, v/v/v) to afford compound 7g (90% yield); H NMR (300 MHz, CD OD) δ 7.29−7.22 (m, 4H),
3
.01−6.93 (m, 4H), 3.79 (s, 2H), 3.54 (s, 2H), 3.48 (s, 2H), 3.44 (s, 2H), 2.63 (br. s, 4H), 2.47 (s,
1
3
2H); C NMR (75 MHz, CD OD) δ 163.78, 163.74, 161.0, 160.53, 160.49, 153.3, 151.0, 132.9,
3
32.8, 132.75, 132.71, 131.01, 130.99, 130.91, 130.88, 125.9, 115.0, 114.9, 114.72, 114.65, 111.5,
00.0, 61.9, 52.4, 52.2, 51.6, 50.8.
.9. Cancer cell line screening
The Antiproliferative activities of all target compounds were investigated against the human cancer
cell lines using the MTT assay. HCT-116 (Human colorectal carcinoma), MCF-7 and SK-BR3 (breast
cancer cells) were supplied from the Korea Cell Line Bank (KCLB). All cell lines were grown in
RPMI 1640/DMEM (Gibco BRL) supplemented with 10% (V/V) heat inactivated Fetal Bovine Serum
4
(
FBS) and maintained at 37 °C in a humidified atmosphere with 5% CO . The cells (5 x 10 cells/mL)
2