Identification of new pyrrolo[2,3-d]pyrimidines as potent VEGFR-2 tyrosine kinase inhibitors: Design, synthesis, biological evaluation and molecular modeling

Article abstract of DOI:10.1016/j.bioorg.2018.09.001

Vascular endothelial growth factor receptor-2 (VEGFR-2) plays a crucial role in cancer angiogenesis. In the current study, a series of novel pyrrolo[2,3-d]pyrimidine based-compounds was designed and synthesized as VEGFR-2 inhibitors, in accordance to the structure activity relationship (SAR) studies of known type II VEGFR-2 inhibitors. The newly synthesized compounds were evaluated for their ability to inhibit VEGFR-2 kinase enzyme in vitro. All the tested compounds demonstrated highly potent dose-related VEGFR-2 inhibition with IC₅₀ values in nanomolar range. Among these compounds, pyrrolo[2,3-d]pyrimidine derivatives carrying biaryl urea moieties (12d and 15c) exhibited IC₅₀ values of 11.9 and 13.6 nM respectively. Additionally, most of the newly synthesized final compounds were tested on 60 human cancer cell lines. Docking of these compounds into the inactive conformation of VEGFR-2 was performed which showed comparable binding modes to that of the FDA approved VEGFR-2 kinase inhibitors. These newly discovered potent kinase inhibitors could be considered as potential candidates for the development of new targeted anticancer agent.

Full text of DOI:10.1016/j.bioorg.2018.09.001

Accepted Manuscript
Identification of new pyrrolo[2,3-d]pyrimidines as potent VEGFR-2 tyrosine
kinase inhibitors: design, synthesis, biological evaluation and molecular mod-
eling
Mai Adel, Rabah A.T. Serya, Deena S. Lasheen, Khaled A.M. Abouzid
PII:
S0045-2068(18)30709-0
DOI:
https://doi.org/10.1016/j.bioorg.2018.09.001
YBIOO 2498
Reference:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
12 July 2018
31 August 2018
5 September 2018
Please cite this article as: M. Adel, R.A.T. Serya, D.S. Lasheen, K.A.M. Abouzid, Identification of new pyrrolo[2,3-
d]pyrimidines as potent VEGFR-2 tyrosine kinase inhibitors: design, synthesis, biological evaluation and molecular
modeling, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.09.001
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Identification of new pyrrolo[2,3-d]pyrimidines as potent VEGFR-2 tyrosine kinase inhibitors:
design, synthesis, biological evaluation and molecular modeling
Mai Adel*, Rabah A.T. Serya, Deena S. Lasheen and Khaled A.M. Abouzid*
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Ain Shams University, Abbassaia,
Cairo 11566, Egypt
*Corresponding authors: Khaled.abouzid@pharma.asu.edu.eg and mai.adel@pharma.asu.edu.eg
Abstract:
Vascular endothelial growth factor receptor-2 (VEGFR-2) plays a crucial role in cancer
angiogenesis. In the current study, a series of novel pyrrolo[2,3-d]pyrimidine based-compounds was
designed and synthesized as VEGFR-2 inhibitors, in accordance to the structure activity relationship
(SAR) studies of known type II VEGFR-2 inhibitors. The newly synthesized compounds were evaluated
for their ability to inhibit VEGFR-2 kinase enzyme in vitro. All the tested compounds demonstrated
highly potent dose-related VEGFR-2 inhibition with IC50 values in nanomolar range. Among these
compounds, pyrrolo[2,3-d]pyrimidine derivatives carrying biaryl urea moieties (12d and 15c) exhibited
IC50 values of 11.9 and 13.6 nM respectively. Additionally, most of the newly synthesized final
compounds were tested on 60 human cancer cell lines. Docking of these compounds into the inactive
conformation of VEGFR-2 was performed which showed comparable binding modes to that of the FDA
approved VEGFR-2 kinase inhibitors. These newly discovered potent kinase inhibitors could be
considered as potential candidates for the development of new targeted anticancer agent.
1. Introduction
Angiogenesis is a fundamental process for tumor growth as it ensures oxygen and nutrients supply to
proliferating cells through the development of new blood vessels, potentially causing cancer progression
and metastasis.[1]
The vascular endothelial growth factor (VEGF) family, which includes VEGF-A, VEGF-B, VEGF-C,
VEGF-D and placental growth factor (PlGF) represent a group of key proteins involved in the angiogenic
pathway. VEGF proteins and their receptors are highly expressed in many tumor types.[2] Therefore they
represent potential targets for anticancer therapy. The VEGF family members bind three different
tyrosine kinase (TK) receptors: VEGFR1 (Flt-1), VEGFR-2 (Flk-1-KDR), and VEGFR3, which is
expressed on the lymphatic and vascular endothelium.[3]
1

VEGFR-2, a type II transmembrane TK receptor expressed on endothelial cells and on circulating
bone marrow-derived endothelial progenitor cells. It is the principal mediator of the VEGF-induced
angiogenic signaling. This receptor contains three different parts, including an Ig-like domain
extracellular region, a hydrophobic transmembrane region containing the TK domain and the carboxyl
terminal tail. VEGFR-2 binds all VEGF-A isoforms, VEGF-C and VEGF-D. The binding of VEGF-A to
VEGFR-2 induces a cascade of different signaling pathways. Downstream effects of VEGFR-2 activation
in the vascular endothelium include cell proliferation, migration, permeability and survival, resulting in
vasculogenesis and angiogenesis.
Therefore, VEGFR-2 inhibition was considered as an effective approach for the prevention of
angiogenesis [4]. This spurred the researchers focus on the use of angiogenesis inhibitors as an adjunct to
other forms of therapy for preventing progression of malignant neoplasms [5].
Several small molecule tyrosine kinase inhibitors of VEGF receptor have been approved for the
treatment of several types of cancer (Figure 1).
2

Figure 1. Structures of FDA and some clinically approved VEGFR2 inhibitors with their
corresponding IC50.
3

FDA-approved angiogenesis inhibitors in combination with chemotherapy have proven their clinical
worth regarding improved patient survival time and patient tolerability in certain cancers[6].
In the last few years, several pyrrolopyrimidines have been approved by the US FDA and in other
countries for the treatment of different diseases or are currently in phase I/II clinical trials. Due to their
inimitable antioxidant and anti-tumor properties, researchers were inspired to develop novel derivatives
for the treatment of different types of cancer[7].
2. Rationale and Design
The design of the target compounds was based on literature survey and molecular modeling studies of
reported potent VEGFR2 inhibitors. The X-ray co-crystal structures of kinase-bound type-II kinase
inhibitors, as well as analysis of binding modes of sorafenib (PDB code 4ASD)[8] and pyrrolo-[3,2-
d]pyrimidine derivative (1) (Figure 1) (IC50= 6.2nM) (PDB code 3VHE)[9] generally reveal two
conserved hydrogen-bond interactions. The two NH of the urea moiety form H-bonds with the
carboxylate group of Glu885 residue, and the carbonyl group of urea from another H-bond with
backbone-NH of Asp1046 residue. The terminal phenyl moiety occupies the hydrophobic pocket created
by rearrangement of the protein.[10]
Compound (I) forms hydrogen bonds with the backbone-NH of Cys919 through N1 of pyrimidine
ring, however, on exploring the binding mode of sorafenib, an extra hydrogen bond was observed
between its carboxamide NH and Carbonyl oxygen of Cys919. Hence, our interest moved to develop new
pyrrolo[2,3-d]pyrimidine based compounds as VEGFR-2 kinase inhibitors that could retain the essential
hydrogen bonding with the backbone NH of Cys919 but aiming that the rearranged pyrrole- nitrogen
position could attain an extra HB with cys919 mimicking that of sorafenib (Figure 2),
4

Sorafenib
Target compounds
Figure 2. Design of pyrrolo[2,3-d]pyrimidine derivatives.
The urea linker lying in the allosteric channel forming hydrogen bonds with N-lobe residue Glu885
and the activation-loop residue Asp 1046 linker is a well-established interaction in many VEGFR-2
inhibitors [11]. Also the amide linker was observed in many potent VEGFR-2 inhibitors as Axitinib,
ponatinib, sunitinib and Apatinib[12,13]. In this study the hydrogen bond donor–acceptor pair, was
maintained as amide or urea moiety aiming interaction with Glu885 and Asp1046 residues. Moreover, the
linker atom between the pyrrolo[2,3-d]pyrimidine core and the central phenyl ring was retained as either
O or NH based on reported SAR to investigate the preferred linker atom oxygen or nitrogen. The
substitution pattern (R) on terminal aromatic moiety was varied, aiming interaction with the terminal
allosteric hydrophobic pocket.
To test our designed hypothesis regarding the extra HB that could be attained by the rearranged
pyrrole-nitrogen, methylation of the N7 position of pyrrolo[2,3-d]pyrimidine scaffold was attempted to
test its influence on activity compared to its desmethyl analogues. In continuation of the optimization
process, we also introduced a methyl group at the 2 position of the pyrrolo[2,3-d]pyrimidine scaffold
aiming that this methyl group, being an electron donating group, may increase the electron density on the
N-1 of pyrimidine which could lead to stronger hydrogen bonding interaction with the Cys919 residue,
hence this may result in increased activity (Figure 3).
5

Figure 3. Schematic representation of the designed pyrrolopyrimidine derivatives
3. Results and discussion
3.1.Chemistry
The synthetic pathways for preparation of the target compounds were depicted in scheme 1, 2 and 3.
Final compounds incorporating substituted amide and urea moieties were obtained utilizing the
corresponding intermediates (3a–h and 5), which were synthesized according to the routes outlined in
schemes 1A,B. p-Nitrobenzoic acid was activated through reaction with thionyl chloride to afford 4-
nitrobenzoyl chloride (1) [14], which was then reacted with different anilines to form the corresponding
amides (2a–h) [15]. Using palladium charcoal as the catalyst, the reduction of the nitro group of 2a–h
6

was performed in ethanol [16] to give corresponding 4-amino-N-substituted benzamides (3a–h) (Scheme
1a).
On the other hand, the urea intermediate (5) was prepared by reacting p-nitroaniline with m-tolyl
isocyanate in dry DCM to afford 4, which was further reduced to the corresponding amino derivative
using 10% Pd-C in ethanol to give 5 [17] (Scheme 2b).
The synthesis of pyrrolo[2,3-d]pyrimidine target compounds was initiated via the preparation of 2-
amino-3-cyano-4,5-dimethylpyrrole (7).
Annulation of this aminopyrrole carbonitrile to the
pyrrolo[2,3]pyrimidine was performed in such a way that no protection was needed. Gewald [18,19]
reported the synthesis of 2-amino-3-cyano-4,5-dimethylpyrrole (7) by the base catalyzed condensation of
3-amino-2-butanone with malononitrile. Since 3-amino-2-butanone (6) is not a stable compound, It was
suggested in literature that it may be used in its stable, N-acetylated form, for pyrrole condensation [20].
However, 6 is not a commercially available compound, while R-acylamino ketones are easily accessible
from the corresponding amino acids and anhydrides through the Dakin-West reaction [21]. This reaction
is usually carried out with primary amino acids [22]. Since (racemic) alanine is cheap, this seemed to be
an attractive approach to synthesize pyrrole 7 [19-21].
The pyrrolo[2,3-d]pyrimidine-4-one (8) was afforded through cyclization of 7 via its reflux with
formic acid [26]. Chlorination of 8 with POCl3 yields the 4-chloropyrrolopyrimidine derivative (9)
[27,28].
The urea based compounds can be classified into two categories: 4-N-pyrrolopyrimidine derivatives
(12a-i) and 4-O-pyrrolopyrimidine derivatives (15a-f). Target compounds (12a-i) were obtained by a
classic nucleophilic substitution reaction between 9 and the 4-amino acetanilide in 1-butanol in the
presence of concentrated HCl in 74−80% yield. Hydrolysis of amide protection can be achieved by
several methods like using strong acids [29] or bases as NaOH [30]. Herein, both methods were adopted
but the reaction worked better by acid hydrolysis of compound 10 using HCl leading to the free amine
derivative (11). Then target compounds 12a-i were produced by the reaction of intermediate 11 with the
corresponding isocyanates under reflux in methylene chloride (Scheme 2).
The 4-O-pyrrolopyrimidine derivatives (15a-f) were obtained through the reaction between 9 and 4-
nitrophenol in DMF at room temperature using K2CO3 as the base to provide the desired intermediate 13
in good yield [31]. Reduction of compound (13) with iron and ammonium chloride in ethanol afforded 14
which was then engaged in a nucleophilic substitution reaction with the corresponding isocyanates under
reflux in methylene chloride (Scheme 2a).
7

The amide based derivatives (16a-h) were synthesized via the coupling of 9 with the key
intermediates (3a-h). (see Scheme 1)
The N-methyl pyrrolopyrimidine derivative (18), was prepared from the methylated intermediate 17.
Methyl iodide was used as an alkylating agent as reported for N-methylation of pyrrolopyrimidines
[32,33] using NaH as a base and DMF as a solvent. Nucleophilic substitution reaction between 17 and the
synthesized urea intermediate (5) afforded the target compound. (Scheme 2b)
For the synthesis of the 2-methyl substituted pyrrolopyrimidine derivatives (24a-d and 25a-c,h), It
started through the formation of substituted 1H-Pyrrolo[2,3-d]pyrimidin-4(7H)-one (20) from 2-
acylamino-3-cyanopyrrole derivative (7) through an intermediate amide (19) resulting from hydrolysis of
the cyano function followed by intramolecular cyclohydration of 19 through the heating of the compound
in a mixture of phosphorous pentaoxide, N,N-dimethylcyclohexylamine (DMCA) and water in 1:1:1.5
ratio[34]. The reaction proceeded smoothly at temperature ranging from 180°C to 200°C.
For compounds (24a-d) and (25a-c,h), synthesis was carried out using the same procedure used for
the preparation of 12a-i and 18a-f, respectively. (Scheme 3).
1A
O
O
O
R
O
R
OH
N
Cl
c
b
N
a
H
H
O2N
O2N
O2N
H2N
2a-h
3a-h
1
2a, 3a; R = 3-F
2b, 3b; R = 4-F
2e, 3e; R = 4-OCH3
2f, 3f; R = 3-Cl
2c, 3c; R = 3-CF3,4-Cl
2d, 3d; R= 3-OCH3
2g, 3g; R = 4-Cl
2h, 3h; R = 3,4 diCl
1B
H
H
H
H
N
NH2
N
N
N
N
C
b
a
O
+
O
O
O2N
O2N
H2N
4
5
8

Scheme 1. Reagents and conditions: [A] (a) SOCl2, reflux, 7 hrs, 60% (b) Aniline
derivatives, DCM, rt, 2-24 hrs, 40-86% (c) H2, Pd/C, EtOH, rt, 1 hr, 80-85%. [B] (a) DCM, rt,
24 hrs, 50% (b) H2, Pd/C, MeOH, rt, 1 hr, 85%.
9

10

Scheme 2a. Reagents and conditions: (a) Acetic anhydride, AcOH, TEA, DMAP, 50°C, 8
hrs (b) 30% NaOH, <60°C then 0°C, 66% (c) Formic acid (85%), 110 °C, 5 h, 42% (d) POCl3,
reflux, 3 hrs, 85% (e) HCl, 1-BuOH, reflux, 24 hr, 60% (f) 2N HCl, 20 hr, reflux, 80% (g)
Substituted phenylisocyanate, DCM, reflux, 24 hrs, 30-80% (h) ) p-nitrophenol, DMF, K2CO3,
140°C, 72 hrs, 65% (i) NH4Cl, Fe, EtOH, 1.5 h, reflux, 35% (j) Substituted phenylisocyanate,
Acetonitrile/toluene or DCM, reflux, 1 hr, 25-55% (k) HCl, 1-BuOH, reflux, 18-48 hrs, 20-
40%
Scheme 2b. Reagents and conditions: (a) DMF, NaH, MeI, 0°C, 2 hrs, 50% (b) 5, HCl, 1-
BuOH, reflux, 24 hr.
11

Scheme 3. Reagents and conditions: (a) Acetic anhydride, gl. AcOH, reflux, 20 min, 50%
(b) P2O5, H2O, DMCA, 180-200°C, 3 hrs, 75% (c) POCl3, reflux, 3 hrs, 90% (d) HCl, 1-BuOH,
reflux, 24 hrs, 85% (e) 2N HCl, 20 hr, reflux, 70% (f) Substituted phenylisocyanates, DCM, rt,
24 hrs, 50-75% (g) HCl, 1-BuOH, reflux, 24 hr, 40-50% .
3.2. Biological evaluation
In vitro VEGFR-2 tyrosine kinase activity
12

3.2.1. Initial screening at a single dose of 10 µM concentration.
The assay was performed to evaluate the VEGFR-2 inhibitory activity of the designed compounds
using Kinase-Glo Plus luminescence kinase assay kit (Promega). It measures kinase activity by
quantitating the amount of ATP remaining in solution following a kinase reaction. The luminescent signal
from the assay is correlated with the amount of ATP present and is inversely correlated with the amount
of kinase activity. In an initial screening; all synthesized final compounds were evaluated for their
inhibitory activity against VEGFR-2 kinase at a single dose concentration of 10 µM. (Sorafenib as a lead
compound showed 95% VEGFR-2 inhibition)[35]. The compounds showed either very poor or excellent
inhibitory activity against VEGFR-2 kinases as shown in Tables 1,2.
Table 1: Percent inhibition of VEGFR-2 enzymatic activity achieved by the amide based
derivatived at 10 µM:
O
R
N
H
HN
N
N
R1
N
H
(16a-f, 25a-c,h)
R1
Compound
ID
R
VEGFR-2
inhibition%
3-F
4-F
H
H
10
3
6
4
2
4
3
3
0
0
16a
16b
3-CF3,4-Cl
3-OCH3
4-OCH3
3-Cl
H
16c
H
16d
H
16e
H
16f
4-Cl
H
16g
3.4 diCl
3-F
H
16h
CH3
CH3
25a
4-F
25b
13

3-CF3,4-Cl
3.4 diCl
CH3
CH3
0
0
25c
25h
Table 2: Percent inhibition of VEGFR-2 enzymatic activity achieved by the urea based
derivatives at 10 µM and The IC50 values for compounds (12a-i, 15a-f):
H
H
N
N
R
O
X
N
N
N
R2
R1
14

Compound ID
X
R
R1
R2
VEGFR-2
IC50
inhibition%
NH
NH
NH
NH
NH
NH
NH
NH
NH
O
3-Cl
4-Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
H
H
H
H
99
87
97
100
93
96
87
94
99
89
44
95
100
84
100
20
2
1.8 µM
52.2 nM
22.9 nM
11.9 nM
77.7 nM
19.6 nM
21.2 nM
26.9 nM
50.6 nM
49.2 nM
3.0 µM
13.7 nM
43.7 nM
1.4 µM
32.8 nM
----
12a
12b
12c
H
3.4 diCl
3-CH3
H
H
12d
12e
3-OCH3
4-OCH3
4-Br
H
H
12f
H
12g
12h
12i
3-Cl, 4-CH3
3-CF3,4-Cl
4-Cl
H
H
H
15a
15b
15c
O
3,4 diCl
3-CH3
H
O
H
O
3-OCH3
3-Cl, 4-CH3
3-CF3,4-Cl
3-CH3
H
15d
15e
O
H
O
H
15f
NH
NH
NH
NH
NH
H
18
3, 4-di Cl
3-OCH3
3-Cl, 4-CH3
3-CF3,4-Cl
CH3
CH3
CH3
CH3
----
24a
24b
24c
3
----
2
----
0
----
90 nM [36]
24d
Sorafenib
95
At this concentration, the 4,5-dimethylpyrrolopyrimidine derivatives (12a, 12d, 12i, 15d, 15f)
incorporating a substituted biarylurea motif linked via an ether or NH linker to the parent scaffold,
demonstrated a potent inhibition of 99-100% of the VEGFR-2 kinase activity. Moreover, compounds
(12c, 12e, 12f, 12h, 15c) showed inhibitory activity above 93%, Nevertheless, significant inhibition
ranging between 84-90 % was also exhibited by compounds (12b, 12g, 15a, 15e).
Structure activity relationship among the newly synthesized pyrrolo[2,3-d]pyrimidines have been
closely investigated. Compared to the very weak inhibitory activity (2-10%) exhibited by the amide
derivatives (16a-h) at 10 µM concentration, it was revealed that the incorporation of the biarylurea
15

moiety in compounds (12a-i and 15a-f) resulted in a significant increase in enzymatic activity in the 4,5-
dimethylpyrrolopyrimidine series, which is mostly attributed to the extra H-bond with Glu885 which
proved to be essential for inhibitory activity and also it seems that the urea linker allows more free
movement of the terminal aromatic group that extends to occupy the back hydrophobic pocket.
Comparison of the VEGFR-2 inhibitory activity among the biarylurea derivatives revealed that the
replacement of NH linker by an ether linker at 4-position of the pyrrolopyrimidine core didn’t influence
the enzymatic inhibition. Incorporation of different substituents at both the 3 and 4 position of the
terminal phenyl ring was generally well tolerated. Best inhibitory activity that was consistent in both the
O- and NH-urea series was observed with the 3-CH3 derivatives (12d, 15c), the 3-OCH3 (12e, 15d), as
well as the disubstituted 3-CF3, 4-Cl derivatives (12i, 15f). As expected from the initial design,
methylation of pyrrolopyrimidine nitrogen in compound (18) resulted in significant loss of VEGFR-2
inhibitory activity (18 compared to 12d). Regarding the effect of methylation at position 2 of
pyrrolopyrimidine scaffold, it resulted in a complete loss of VEGFR-2 inhibitory activity as revealed by
the 2,4,5-trimethyl-substituted pyrrolopyrimidines bearing biarylureas (24a-d) which showed VEGFR-2
inhibition (0-3%) compared to the dimethyl analogues (12a-i) (84-100%). As expected, the 2,4,5-
trimethylpyrrolopyrimidine derivatives bearing amide tail (25a-c,h) revealed no inhibitory activity
mimicking the amide derivatives (16a-h).
3.2.2. Measurement of potential enzyme inhibitory activity (IC50)
Promising candidates, which exhibited VEGFR-2 inhibition percent above 80% at 10 µM
concentration were further investigated for their dose-related VEGFR-2 enzymatic inhibition at 5
different concentrations (1 nM - 10 nM - 100 nM - 1 µM - 10 µM) to subsequently calculate their IC50
values. (Table 2)
Twelve of the investigated compounds exhibited potent VEGFR-2 inhibitory activity with IC50
values in nanomolar range. The pyrrolo[2,3-d]pyrimidine-based derivatives (12d and 15c), both having a
m-tolyl substituted urea tail showed the most potent nanomolar VEGFR-2 inhibition (IC50 of 11.9 and
13.7 nM, respectively) suggesting that the m-tolyl substitution is optimum for activity.
3.2.3. In-vitro antiproliferative activity assay against NCI 60-cell line panel
Thirteen of the final compounds were selected by the Developmental Therapeutics Program (DTP)
of the National Cancer Institute (NCI), division of cancer treatment and diagnosis, NIH, Bethesda,
Maryland, USA (www.dtp.nci.nih.gov), namely (16c, 16e, 16f , 16g, 12a, 12b, 12e, 15a, 15c, 15d, 15f,
24b, 24d). Compounds selection for screening is based on drug-likeliness, and the ability of the submitted
16

compounds to add diversity to the NCI small molecule compound collection. The operation of this screen
utilizes 60 different human tumor cell lines, representing leukemia, melanoma, lung, colon, brain, ovary,
kidney, prostate and breast cancers [37]. All compounds selected were tested at initial single dose 10 µM
inhibition percent assay on the full NCI 60 cell panel. Upon analysis of the NCI-60 results, almost all of
the Compounds except for 15f showed weak activity against most of the investigated cell lines. The poor
antiproliferative activity displayed by these derivatives may be attributed to the lack of activity against
other kinases as VEGFR is not highly expressed in these cell lines, thus it can be suggested that
compounds (12a, 12b, 12e, 15a, 15c, 15d, 15f) that have been selected by NCI may enjoy good
selectivity profiles. Compound (15f), showed the highest cell growth inhibition with mean growth
inhibition percent of 42.36%. It exhibited broad spectrum and good anti-proliferative activity against
several NCI cell panel, namely; the leukemic CCRF-CEM, K-562, MOLT-4, RPMI-8226 and SR cancer
cell line with growth inhibition 75.93, 73.38, 72.57, 55.30 and 90.99%, respectively, the non-small cell
lung cancer A549/ATCC, EKVX, NCI-H460 and NCI-H522 cell line with cell growth inhibition 54.66,
56.90, 63.88 and 74.71%, respectively, the colon cancer HCT-116, HCT-15, HT29, KM12 cell line with
cell growth inhibition 57.63, 51.70, 50, 72.75%, respectively, the melanoma LOX IMVI and MDA-MB-
435 cell line with 65.60% and 54.56% inhibition, respectively, the prostate cancer PC-3 cell line with
65.04% inhibition, the breast cancer MCF7, MDA-MB-231/ATCC and T-47D cell line with 68.37, 55.39
and 63.08% inhibition, respectively. It should be noted that this compound bears similar substitution
pattern to the multikinase inhibitor (Sorafenib). Also, compounds (15c and 15d) belonging to the same
pyrrolo[2,3-d]pyrimidine linked to biarylureas via an ether linker showed selective cell growth inhibition
against the leukemic cancer SR cell line (60.78 and 89.5%, respectively).
3.2.4. In vitro HUVEC Anti-proliferative assay
Most of endothelial cell assays utilize human umbilical vein endothelial cells (HUVECs) as good
representatives of vascular endothelial cells.[38]
The most active two compounds (12d and 15c) were selected to be tested for their ability to inhibit
HUVEC cell line proliferation in vitro, using sorafenib as control. The results are illustrated in (Figure 4)
Compounds (12d and 15c) manifested potent ability to inhibit VEGF-induced HUVEC cell line
proliferation with IC50 values of 0.31 and 3.74 µm, respectively compared to sorafenib 0.16. These
findings substantiate our initial findings that these compounds are selective on HUVEC cells
overexpressing VEGFR with no appreciable cytotoxicity on the rest of cancer cell line panels.
17

Compound ID
IC50 µM
HUVEC
Sorafenib
12d
0.16 ±0.01
0.31 ±0.01
3.74 ±0.18
15c
Figure 4: The IC50 values of target compounds (Sorafenib, 12d, 15c)) on HUVEC cell line
3.3. Molecular modeling study
3.3.1. Docking study
This docking study was performed using CDOCKER protocol in Accelrys Discovery Studio® 2.5
software to gain an insight into the SAR of the target compounds, and to interpret the biological
evaluation results on the basis of the ligand-protein interactions. The X-ray crystal structure of VEGFR-2
complexed with its pyrimidine-based inhibitor (I) was obtained (PDB: 3VHE). In order to validate the
CDOCKER protocol's predictability of the correct poses, the co-crystallized ligand (I) was redocked, and
the docked pose showed RMSD value of 0.3082 Å when aligned to the X-ray geometry (the co-
crystallized bioactive conformation) [Figure 5] indicating the validation of the used protocol in
predicting the pose of the inhibitors in the X-ray structure of VEGFR-2.
Figure 5: The alignment between the co-crystallized bioactive conformer (green) and the pose of the
same compound retrieved from docking at VEGFR-2 binding site using CDOCKER.
18

Docking of the target compounds revealed that the urea based 4,5-dimethylpyrrolopyrimidine
derivatives (12a-i) and (15a-f) fulfilled the basic key interactions of VEGFR-2 inhibitors, where the
hydrogen bonds with Cys919, Glu 885, Asp1046 residues were established, as well as the Pi-cation
interactions with Lys868 residue and the Pi-sigma interaction with Leu 840. The Pi- Pi interaction with
Phe 918 was also observed. Additionally a hydrogen bonding interaction was established between the NH
of pyrrole and the cys 919 amino acid residue as predicted from the initial design. These findings further
interpreted their potent VEGFR-2 inhibitory activity observed for the urea-based derivatives. This was
also consistent with the previously reported SAR studies on various VEGFR-2 inhibitors, which revealed
that the substituted biarylurea moiety is favorable for optimal interaction with the hydrophobic back
pocket of VEGFR-2 kinase[17,39]. Compounds with amide linkage (16a-h) missed the key interaction
with Glu885 residue which seemed to be an essential feature for type-II inhibitors, resulting in lower
docking scores. These results may also interpret their VEGFR-2 kinase inhibitory inactivity in the kinase
assay. Compound (18) was synthesized to test the effect of the methylation of the NH of pyrrole on
activity. Based on biological data the compound showed great drop of activity and upon investigation of
the docking results, the presence of three adjacent methyl groups led to reorientation of the compound in
a manner that it was unable to form the key interaction with cys 919, this may explain the activity loss.
Although 2,4,5-trimethylpyrrolopyrimidines (24a-d) comprising urea linkage were expected to show
potent inhibitory activity, yet the docking study revealed that the 2-methyl group led to repositioning of
the pyrrolopyrimidine core. Thus, these derivatives missed also the key hydrogen bonding with cys 919
and all the hydrophobic interactions expected including the Pi-sigma interaction with Leu 840, and the Pi-
Pi interaction with Phe 918 in almost all compounds. This may explain the loss of inhibitory activity of
these compounds. The 2,4,5-trimethylpyrrolopyrimidine derivatives (25a-c,h) with amide linkage lacks
many interactions due to both the absence of urea key interactions and the presence of the extra methyl
group.
19

B
A
C
D
F
E
Figure 6. [A] Docking pose of compound (I) (PDB code 3VHE) showing the same key interactions as
reported. [B,C] Docking poses of the urea based target compounds (12d and 15c) showing the extra HB
with Cys919 [D] Docking of the amide based derivative (16a) missing the key interaction with Glu885
residue [E] Docking of 24c showing the loss of the two HB interactions with Cys919 [F] Docking of 18
showing the loss of HB interactions with Cys919
20

3.3.2. Field alignment study
Molecules interact through their electronic properties (electrostatic and Van der Waals forces),
proteins perceive the ligands as molecular fields rather than mere 3D arrangements of individual atoms.
Therefore, molecules with similar potential fields (similar set of field points) are expected to exert similar
biological activities through establishing the same interactions within the same binding site, even if they
have diverse structures, since field patterns are not directly affected by 2D connectivity of the molecule
but rather by its 3D properties [40].
Based on this theory, was applied the field alignment technique provided by Cresset's
FieldAlign® module, version 2.0.1 to align our designed compounds to the bioactive conformation of (I)
(PDB: 3VHE). In FieldAlign, each molecule is described as a Field Point Pattern (FPP), using the local
extrema of four molecular fields: positive electrostatic, negative electrostatic, surface (van der Waals
interactions), and hydrophobic. The resulting FPPs are aligned and scored to give a measure of similarity
between a query molecule and a database molecule in specific conformations.
Alignment of the target compound (12d) against sorafenib cocrystallized under PDB code: 4ASD,
showed great similarity, where all the backbone of the bioactive conformer of sorafenib was preserved
[Figure 7].
Alignment with the pyrrolopyrimidine based compound (I) cocrystallized under PDB code:
3VHE also showed matching of their molecular fields that are manifested by the high alignment score
(0.880-0.709). Besides, the alignment agrees with our predicted binding mode from both literature survey
and docking study. The pyrrolo[2,3-d]pyrimidine nucleus of the target compounds is aligned with
pyrrolo[3,2-d]pyrimidine nucleus of the reference compound (I) and the 5-methyl group of the
pyrrolopyrimidine nucleus of the designed compounds aligned with the N-methyl of the reference
compound (I).
In addition, the hydrophobic substituted phenyl ring, The O or NH linkers and the biphenyl urea
in the designed compounds were aligned with the terminal phenyl ring, the O linker and the biphenyl urea
moieties in both sorafenib and compound (I), respectively. It should be mentioned that compound (12d)
with the highest inhibitory potency (IC50 =11.9) represented by pink colour showed the best alignment
with highest score; 0.880 [Figure 7].
21

Figure 7: [A] Field alignment of compound 12d and Sorafenib showing similar molecular fields
suggesting a similar binding mode to VEGFR-2. [B] Compound 12d (pink) aligned to compound (I)
(grey). Spherical field points: designed compounds, icosahedral field points: Reference compound. Field
points are condensed field pattern that make fields computationally viable. Larger field points represent
stronger points of potential interaction. Cyan: Negative field points, Red: Positive field points, Yellow:
van der Waals surface field points, Gold: Hydrophobic field points.
An important aspect about the SAR of the designed pyrrolopyrimidine series with the 2-methyl
substitution (24a-d) and also compound 18 with 7-methyl substitution was not justified enough by the
molecular docking. Field alignment study was performed in an attempt to understand the main reason for
activity loss upon incorporation of these methyl groups. These compounds displayed unexpected loss of
the inhibitory activity in contrast with other urea derivatives. Upon comparing their molecular fields to
other active compounds, an extra hydrophobic field around the methyl group was observed. This finding
may suggest the presence of unflavored interaction at this point due to the incorporation of a hydrophobic
group which may account for loss of activity [Figure 8].
22

Unfavourable interactio
with the binding site
Figure 8: A comparison of the hydrophobic fields of different compounds: [A] Hydrophobic field of
Sorafenib, [B] Hydrophobic field of compound (I), [C] Hydrophobic field of compound (12e) as a
representative example of the active urea derivatives, [D] Hydrophobic field of compound (24b) as an
example of the inactive urea derivatives with 2-methyl substitution of the pyrrolopyrimidine ring. [E]
Hydrophobic field of compound (18) with 7-methyl substitution of the pyrrolopyrimidine ring.
23

4.
Conclusion
Novel pyrrolo [2,3-d]pyrimidine derivatives were designed, synthesized and evaluated for their in
vitro VEGFR-2 inhibitory activity as well as their anti-proliferative activity against NCI 60 cell line
panel. Twelve of the biaryl urea based derivatives exhibited potent dose-related VEGFR-2 inhibitory
activity with IC50 values in the nanomolar range. The pyrrolo[2,3-d]pyrimidine derivatives (12d and 15c)
both bearing a m-tolyl urea tail via an NH or ether linker showed highest potent nanomolar VEGFR-2
inhibition (IC50 of 11.9 nM and 13.7 nM respectively). The amide-based derivatives showed weak
VEGFR-2 inhibition (0-10%) at 10 µM concentration. The 2,4,5-trimethylpyrrolopyrimidine derivatives
showed no inhibitory activity (0-3%); which indicates that the incorporation of the methyl group at the 2-
position of the pyrrolopyrimidine core leads to complete loss of activity. Compound (18) with N-methyl
substitution showed decreased activity owing to pyrrole protection. In addition, compounds (12d and
15c) manifested in vitro good ability to inhibit VEGF-induced HUVEC cell line proliferation with IC50
values of 0.31 and 3.74 µm respectively. These results were further explained using molecular docking
studies which revealed the ability of the urea-based derivatives to form a network of key interactions,
known to be essential for type II VEGFR-2 inhibitors. However, their amide-based analogues missed a
key interaction with Glu 885 residue. Moreover, the presence of the 2 or 7-methyl substitution of the
pyrrolopyrimidine core led to repositioning of the pyrrolopyrimidine nucleus; hence the compounds
missed the key interactions characterizing type II VEGFR-2 inhibitors including hydrogen bonding with
cys 919 and the hydrophobic interactions expected including the Pi-sigma interaction with Leu 840 and
the Pi- Pi interaction with Phe 918 in almost all compounds.
5.
Future perspective
The high VEGFR-2 inhibitory activity of the designed compounds with IC50 values in the nanomolar
range, and the concluded structure activity relationship from the biological evaluation and molecular
modelling studies, will be taken in consideration in the design of more potent and highly selective
VEGFR-2 inhibitors. Further optimization will be performed by inserting different substitutions at the
central phenyl ring. These modifications are predicted to elaborate new VEGFR-2 inhibitors with very
high potency and selectivity.
6.
Experimental
24

6.1. Chemistry and analysis.
Starting materials and reagents were purchased from Sigma-Aldrich (USA), Alfa- Aesar and Loba chemie
Organics and were used without further purification. Solvents were purchased from Fisher scientific or
Sigma-Aldrich and used without further purification. Anhydrous methylene chloride was dried by
distillation over calcium hydride. Chemical reactions were monitored by analytical thin layer
chromatography (TLC), on silica gel 60 F254 packed on Aluminum sheets, purchased from Merck, and
were visualized under U.V. light (254 nm). Column chromatography was conducted on silica 60 (35-70
microns). Intermediates monitoring was carried out using melting points, or 1H-NMR which matched the
reference data. Melting points were determined by Stuart Scientific apparatus. 1H-NMR spectra were
recorded on a Bruker 400 MHz spectrometer in δ scale (ppm), using DMSO as solvent and TMS as
internal standard signal at the faculty of pharmacy, Ain-shams university. While 13C-NMR spectra were
recorded at 100 MHz. EI-MS spectra were recorded byTriple Quadrupole LC/Ms/Ms mass spectrometer
API 200 (AB Sciex Instrument) at the regional center of mycology and biotechnology at Al-Azhar
University. Elemental analyses were performed at the regional center of mycology and biotechnology at
Al-Azhar University. The hydrogenation was carried out using (Parr Shaker) hydrogenator apparatus at
the Faculty of Pharmacy, Ain Shams University.
Compounds 1[14], 2a-h [15,41–45], 3a-g [46–49] 4 and 5 [17], were prepared according to the
reported procedures.
4-Amino-N-(3,4-dichlorophenyl)benzamide (3h)
The titled compound was obtained as buff solid (85%); m.p. 220°C; 1H NMR (400 MHz, DMSO-d6)
δ 10.04 (s, 1H, NH D2O exchangeable), 8.15 (s, 1H, ArH), 7.75 (d, J = 9.0 Hz, 1H, ArH), 7.71 (d, J = 8.7
Hz, 2H, ArH), 7.56 (d, J = 9.0 Hz, 1H, ArH), 6.63 (d, J = 9.0 Hz, 2H, ArH), 3.38 (s, 2H, NH D2O
exchangeable).
2-Amino-3-cyano-4,5-dimethylpyrrole (7): [24]
A mixture of acetic anhydride (16.9 g, 165 mmol), acetic acid (2.26 g, 37.6 mmol), triethylamine (19
g, 188 mmol) and 4-(dimethylamino)-pyridine (0.1 g, 0.75 mmol) were charged into a reaction flask and
heated to 50 °C. Racemic D,L alanine (6.79 g, 76.2 mmol)was added in small portions over 4 hrs with
stirring maintaining reaction temperature at 45 to 55 °C. After completion of D,L alanine addition, the red
coloured mixture was continuously stirred for an additional 8 hrs at 50 °C.
25

Acetic anhydride, acetic acid and triethylamine, were distilled off by vacuum distillation (15-20
mbar), gradually increasing the path temperature to a maximum of 100 °C. The residue of the produced
acylamino ketone intermediate (6) was cooled to room temperature and water was added (40 mL).
Malonodinitrile (4.71 g, 71.3 mmol) was then added, and the reaction mixture was slowly poured into
30% aqueous sodium hydroxide solution (25 mL), keeping the reaction temperature below 60 °C. The
resulting mixture was cooled to 0°C, filtered, washed with water (45 mL), and dried under vacuo to yield
6.65 g of the pyrrole derivative (7) as beige solid in 66% yield; m.p. 163-165°C (as reported) [24].
5,6-Dimethyl-1H-pyrrolo[2,3-d]pyrimidin-4(7H)-one (8):[26]
Compound (7) (3 g, 22.19 mmol) was boiled in 85% formic acid (30 mL) for 5 hrs at 110 °C.
After cooling, 200 mL of ice water was added to the reaction mixture. The separated crystals were
filtered and washed with water and diethyl ether to give the titled compound (8) as white solid in 42%
yield; m.p. 362°C (as reported) [27].
4-Chloro-5,6-dimethyl-7H-Pyrrolo[2,3-d]pyrimidine (9):[28]
A mixture of 8 (3.147 g, 10 mmol, 1 equiv.) and POCl3 (15 mL, 160mmol,16 equiv.) was heated
under reflux with stirring for 3 hrs, after which excess POCl3 was distilled off, then ethyl acetate (200
mL) and iced water (100 mL) were added. The mixture was stirred until the black residue was dissolved,
the organic layer was separated, dried over MgSO4 and evaporated under vacuo, to yield a pale yellow
solid which was recrystallized from ethanol to afford the target compound (9) in 85% yield, m.p. 247°C
(as reported) [27].
N-(4-(5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)phenyl)acetamide (10):
To a solution of the 4-chloro pyrrolo[2,3-d]pyrimidine derivative (9) (1 mmol: 1 equiv.) in
butanol (15 mL), 2-3 drops of conc. HCl were added, followed by the addition of 4-amino-acetanilide
(1.5 mmol; 1.5 equiv.). The mixture was heated under reflux for 24 hrs, after which, the resulting mixture
was concentrated under vacuo then diethyl ether was added dropwise, the resultant solid was collected by
filtration, washed with diethyl ether, allowed to dry. The residue was purified by silica gel column
chromatography (eluent gradient CH2Cl2: MeOH= 98:2 to 95:5) to give 10 as buff solid in 60% yield; 1H
NMR (400 MHz, DMSO-d6) δ 11.62 (s, 1H, NH D2O exchangeable), 10.02(s, 1H, NH D2O
exchangeable), 8.66 (s, 1H, NH D2O exchangeable), 8.08 (s, 1H,pyrimidine), 7.60 (d, 2H, ArH), 7.43 (d,
2H, ArH), 2.38 (s, 3H, CH3 Aliphatic), 2.28 (s, 3H, CH3), 2.06 (s, 3H, CH3).
26

N1-(5,6-Dimethyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl)benzene-1,4-diamine (11):
To 2.95 g (10 mmol) of compound (10), 2N HCl (20 mL) was added and the mixture was refluxed
for 20 hrs. The resulting mixture was poured on ice cooled water, then solid Na2CO3 was added portion
wise till effervescence ceases. The reaction mixture was stirred at room temperature for half an hour and
the resulting precipitate was filtered, washed with water and dried to give compound (11) as grey powder
1
in 80% yield; H NMR (400 MHz, DMSO-d6) δ 11.24 (s, 1H, NH D2O exchangeable), 8.02 (s,
1H,pyrimidine), 7.82 (d, 2H, ArH), 7.63 (s, 1H, NH D2O exchangeable), 6.80 (d, 2H, ArH), 4.84 (s, 2H,
NH2 D2O exchangeable), 2.24 (s, 3H, CH3), 2.01 (s, 3H, CH3).
1-(Substituted-phenyl)-3-(4-(5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)phenyl)urea
(12a-i):
General procedure:
Compound (11) (0.2g, 0.79mmol: 1 equiv.) was dissolved in DCM (20 mL) and the appropriate
isocyanate (0.79mmol: 1 equiv.) was added to the solution. The mixture was stirred under reflux for 24
hrs and then the precipitate formed was filtered and washed with diethyl ether. The residue was purified
by silica gel column chromatography (eluent gradient CH2Cl2: MeOH= 98:2 to 95:5) to give the titled
compounds (12a-i) in yields (30-80%).
1-(3-Chlorophenyl)-3-(4-(5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)phenyl)urea
(12a):
1
The titled compound was separated as off-white powder (30%); m.p. ˃320°C; H NMR (400
MHz, DMSO-d6) δ 11.42 (s, 1H, NH D2O exchangeable), 9.82 (s, 1H, NH D2O exchangeable of urea),
9.80 (s, 1H, NH D2O exchangeable of urea), 8.13 (s, 1H, pyrimidine), 7.99 (s, 1H, NH D2O
exchangeable, ), 7.73 (d, 1H, ArH), 7.69 (d, 2H, ArH), 7.42 (d, 1H, ArH), 7.39 – 7.31 (m, 3H, ArH), 7.17
(d, 1H, ArH), 2.41 (s, 3H, CH3), 2.27 (s, 3H, CH3); 13C NMR (101 MHz, DMSO) δ 160.23, 154.02,
153.24, 150.89, 148.07, 140.22, 135.09, 134.92, 129.07, 128.88, 122.90, 119.44, 119.10, 118.92, 115.67,
104.42, 103.97, 11.15, 10.91; MS: (Mwt.: 406.86) : m/z (% rel. Int.), 408.08 (M++2, 2.61%), 406.02 (M+,
10.63%), 160.99(100%); Anal. Calcd for C21H19ClN6O: C, 61.99; H, 4.71; N, 20.66. Found: C, 62.25; H,
4.80; N, 20.94.
1-(4-Chlorophenyl)-3-(4-(5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)phenyl)urea
(12b):
The titled compound was separated as buff powder (54%); m.p. ˃320°C;1H NMR (400 MHz,
DMSO-d6) δ 11.36 (s, 1H, NH D2O exchangeable), 8.79 (s, 1H, NH D2O exchangeable of urea), 8.63 (s,
27

1H, NH D2O exchangeable of urea), 8.09 (s, 1H, pyrimidine), 7.85 (s, 1H, NH D2O exchangeable), 7.60
(d, J = 8.4 Hz, 2H, ArH), 7.49 (d, J = 8.3 Hz, 2H, ArH), 7.39 (d, J = 8.5 Hz, 2H, ArH), 7.33 (d, J = 8.5
Hz, 2H, ArH), 2.39 (s, 3H, CH3), 2.26 (s, 3H, CH3).13C NMR (101 MHz, DMSO) δ 161.73, 153.97,
153.00, 150.06, 147.15, 139.36, 134.68, 129.25, 129.05, 128.87, 125.60, 122.53, 120.09, 119.24, 119.10,
104.443, 103.96, 11.16, 10.91; MS: (Mwt.: 406.86) : m/z (% rel. Int.), 408.08 (M++2, 0.54%), 406.02
(M+, 1.78%), 278.99 (100%); Anal. Calcd for C21H19ClN6O: C, 61.99; H, 4.71; N, 20.66. Found: C,
62.75; H, 4.82; N, 20.74.
1-(3,4-Dichlorophenyl)-3-(4-(5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidin-4ylamino)phenyl)urea
(12c):
1
The titled compound was separated as white powder (62%); m.p. ˃320°C; H NMR (400 MHz,
DMSO-d6) δ 11.36 (s, 1H, NH D2O exchangeable), 9.56 (s, 1H, NH D2O exchangeable of urea), 9.23 (s,
1H, NH of urea D2O exchangeable), 8.09 (s, 1H, pyrimidine), 7.97 – 7.80 (m, 3H, 2ArH+ 1 NH D2O
exchangeable), 7.60 (d, J = 8.4 Hz, 2H, ArH), 7.50 (dd, J = 8.8, 3.2 Hz, 1H, ArH), 7.47 – 7.33 (m, 2H,
ArH), 2.39 (s, 3H, , CH3), 2.26 (s, 3H, CH3);13C NMR (101 MHz, DMSO) δ 160.24, 153.97, 153.08,
150.81, 150.08, 140.97, 135.29, 134.74, 131.42, 130.95, 128.85, 123.02, 122.50, 119.51, 119.39, 119.06,
118.50, 104.43, 103.97, 11.16, 10.92;MS: (Mwt.: 441.31) : m/z (% rel. Int.), 443.14 (M++2,
4.75%),442.04 (M++1, 5.75%), 441.01 (M+, 22.64%), 368.23 (100%); Anal. Calcd for C21H18Cl2N6O: C,
57.15; H, 4.11; N, 19.04; Found: C, 57.03; H, 4.23; N, 19.38.
1-(4-(5,6-Dimethyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)phenyl)-3-m-tolylurea (12d):
1
The titled compound was separated as white powder (63%); m.p. ˃320°C; H NMR (400 MHz,
DMSO-d6) δ 11.36 (s, 1H, NH D2O exchangeable), 8.58 (s, 1H, NH D2O exchangeable of urea), 8.60 (s,
1H, NH of urea D2O exchangeable), 8.09 (s, 1H, pyrimidine), 7.84 (s, 1H, NH D2O exchangeable), 7.60
(d, J = 8.4 Hz, 2H, ArH), 7.40 (d, J = 8.5 Hz, 2H, ArH), 7.31 (d, J = 6.1 Hz, 1H), 7.23 (d, J = 7.7 Hz, 1H,
ArH), 7.15 (dd, J = 9.2, 6.5 Hz, 1H, ArH), 6.78 (d, J = 7.3 Hz, 1H, ArH), 2.40 (s, 3H, CH3), 2.27 (d, 6H,
CH3), 2.26; 13C NMR (101 MHz, DMSO) δ160.17, 154.00, 153.09, 150.79, 150.07, 140.22, 138.39,
135.09, 134.92, 129.07, 128.88, 122.90, 122.58, 119.44, 119.10, 118.92, 115.77, 104.42, 103.97, 21.70,
11.15, 10.91; MS: (Mwt.: 386.44) : m/z (% rel. Int.), 386.08 (M+, 49.90%), 279.01 (100%); Anal. Calcd
for C22H22N6O: C, 68.53; H, 5.79; N, 21.75. Found: C, 57.03; H, 4.23; N, 21.97.
1-(4-(5,6-Dimethyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)phenyl)-3-(3-methoxyphenyl)urea
(12e):
28

1
1
The titled compound was separated as white powder (60%); m.p. ˃320°C; H NMR (400 H
NMR (400 MHz, DMSO-d6) δ 11.36 (s, 1H, NH D2O exchangeable), 8.66 (s, 1H, NH D2O
exchangeable of urea), 8.57 (s, 1H, NH of urea D2O exchangeable), 8.09 (s, 1H, pyrimidine), 7.85 (s, 1H,
NH D2O exchangeable), 7.60 (d, J = 8.5 Hz, 2H, ArH), 7.46 – 7.31 (m, 2H, ArH), 7.19 (d, J = 9.7 Hz,
2H, ArH), 7.04 – 6.82 (m, 1H, ArH), 6.55 (d, J = 7.9 Hz, 1H, ArH), 3.74 (s, 3H, OCH3), 2.40 (s, 3H,
CH3), 2.26 (s, 3H, CH3);13C NMR (101 MHz, DMSO) δ 160.15, 154.00, 153.05, 150.79, 150.07, 141.53,
135.16, 134.83, 130.08, 129.99, 128.88, 122.59, 119.51, 118.99, 110.91, 107.56, 104.43, 104.32, 103.98,
55.38, 11.15, 10.91, MS: (Mwt.: 402.44) : m/z (% rel. Int.), 402.21 (M+, 8.26%), 64.89 (100%); Anal.
Calcd for C22H22N6O2: C, 65.66; H, 5.51; N, 20.88. Found: C, 56.89; H, 5.72; N, 21.19.
1-(4-(5,6-Dimethyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)phenyl)-3-(4-
methoxyphenyl)urea (12f):
1
1
The titled compound was separated as off-white powder (75%); m.p. ˃320°C; H NMR (400 H
NMR (400 MHz, DMSO-d6) δ 11.35 (s, 1H, NH D2O exchangeable), 8.52 (s, 1H, NH of urea), 8.45 (s,
1H, NH D2O exchangeable of urea), 8.09 (s, 1H, pyrimidine), 7.83 (s, 1H, NH D2O exchangeable), 7.58
(d, J = 8.0 Hz, 2H, ArH), 7.42 – 7.31 (m, 4H, ArH), 6.87(d, 2H, J = 8.0 Hz, ArH), 3.72 (s, 3H, OCH3),
13
2.39 (s, 3H, CH3), 2.26 (s, 3H, CH3); C NMR (101 MHz, DMSO) δ 154.84, 153.35, 151.69, 150.06,
135.13, 133.32, 130.86, 125.87, 122.64, 120.44, 118.91, 118.87, 114.44, 111.59, 107.31, 104.39, 103.99,
55.63, 11.14, 10.9; MS: (Mwt.: 402.44) : m/z (% rel. Int.), 402.21 (M+, 6.25%); Anal. Calcd for
C22H22N6O2: C, 65.66; H, 5.51; N, 20.88. Found: C, 65.76; H, 5.68; N, 21.18.
1-(4-Bromophenyl)-3-(4-(5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)phenyl)urea
(12g):
1
The titled compound was separated as off-white powder (80%); m.p. ˃320°C ;1H NMR (400 H
NMR (400 MHz, DMSO-d6) δ 11.36 (s, 1H, NH D2O exchangeable), 8.85 (d, 1H, NH of urea D2O
exchangeable), 8.67 (d, 1H, NH of urea D2O exchangeable), 8.09 (s, 1H, pyrimidine), 7.85 (s, 1H, NH
D2O exchangeable), 7.60 (d, J = 8.3 Hz, 2H, ArH), 7.51-7.33 (m, 6H, ArH), 2.39 (s, 3H, CH3), 2.26 (s,
3H, CH3); 13C NMR (101 MHz, DMSO) δ 152.98, 150.83, 150.07, 140.33, 139.81, 135.28, 134.69,
131.97, 131.94, 122.53, 120.66, 120.46, 119.56, 119.07, 113.42, 104.44, 103.97, 11.17, 10.92; MS:
(Mwt.: 451.1) : m/z (% rel. Int.), 451.14 (M+, 0.84%); Anal. Calcd for C21H19BrN6O: C, 55.89; H, 4.24;
N, 18.62. Found: C, 65.12; H, 4.37; N, 18.89.
29