Design and synthesis of potent N-phenylpyrimidine derivatives for the treatment of skin cancer

Article abstract of DOI:10.1039/c5ra27017k

The development of novel synthetic compounds for the treatment of skin cancer is much needed, as there is a sudden rise in the incidence of skin cancer throughout the world and the available chemotherapy is facing problems of resistance. Hence, present re

Full text of DOI:10.1039/c5ra27017k

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Design and Synthesis of Potent NꢀPhenylpyrimidine Derivatives for the
Treatment of Skin Cancer
Udit J. Chaube, Vivek K. Vyas, Hardik G. Bhatt*
Department of Pharmaceutical Chemistry, Institute of Pharmacy, Nirma University,
Ahmedabad 382 481. India.
*Corresponding author Information:
Department of Pharmaceutical Chemistry, Institute of Pharmacy, Nirma University, S. G.
Highway, Chharodi, Ahmedabad 382 481. India
Tel.:+91 79 30642727; Fax: +91 2717 241916; Eꢀmail: hardikbhatt23@hotmail.com

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Abstract
Development of novel synthetic compounds for the treatment of skin cancer is much needed,
as there are sudden rise in incidences of skin cancer throughout the world and available
chemotherapy is facing problems of resistance. Hence, present research efforts were made to
discover potent molecules against skin cancer. Pharmacophore models were developed by
using GALAHAD module of Sybyl X followed by validation, virtual screening, design and in
silico ADMET studies. Based on features generated in computational studies and structural
importance of pyrimidine moiety in highest QFIT value molecule of virtual hit; it was selected
as a core moiety for further designing of molecules. Fourteen substituted pyrimidine
1
derivatives were designed, synthesized and characterised by H and 13C NMR, mass and
elemental analysis, while purity was checked by HPLC. All these compounds were evaluated
for in vitro studies on five cancer lines; from which, compounds 6a, 6h, 6j and 6k were found
potent, specifically on skin cancer cell line. Based upon results of in vitro studies, they were
selected further, along with 5ꢀFU, for in vivo study by DMBA induced skin cancer model.
Compound 6h showed favourable actions against skin cancer and treated tumours
demonstrating potential of the series. This study could be explored in future to design lead
molecule for the treatment of skin cancer.
Keywords: Skin cancer, Pharmacophore modelling, Virtual screening, NꢀPhenylpyrimidine,
Xꢀray crystallography.

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1. Introduction
Cancer is a disease with uncontrolled proliferation of cells and the potential with which it can
invade to any part of the body. Cancer is one of the leading cause of death in the world and
the second most common disease after cardiovascular disorders.1 It is predicted that by the
end of the year 2020, approximately 16.5 million new cases will be detected, and most of
them from the developing countries.1 Various preventive measures are now available for the
treatment of cancers, but few cancers such as skin cancer have no preventive measures.
Rendering to world cancer report of WHO, skin cancer constitutes nearly 30% of all recently
diagnosed cancers in the world.1ꢀ3 Difficulty associated in the treatment of skin cancer is the
resistance to existing chemotherapy due to the presence of survivin molecule in the cell.2 Few
cytotoxic drugs are available for the treatment of skin cancer, however, they exhibit toxic
effects on kidney, lung, heart, brain, and gastrointestinal tract.4 If skin cancer cannot be
diagnosed at early stage, then it can metastasized to various parts of the body through the
lymph system and blood circulation. UV radiations are mainly responsible for the
development of skin cancer5,6 through the direct and indirect mechanisms like immune
suppression, formation of cyclobutane pyrimidine dimers, p53 gene mutation, oxidative
stress, etc.7ꢀ9 Another factor, which is responsible for the skin cancer, is fair complexion due
to deficiency of UVꢀblocking pigment known as eumelanin.9 Ozone layer depletion, altitude,
latitude and weather conditions also affect the intensity of UV radiations. Rudimentary
mechanism behind the pathogenesis of skin cancer due to UV radiation is the dented DNA,
which results in the formation of assembly of bad cell4,8 and the end result of this is the
formation of tumour. Normally, p53 protein triggers p21 protein and this intrudes the cell
cycle in between G1 and S phase. Due to the mutation in the p53 protein, this normal process
get disturbed, which upshots into the copious growth of defective cells resulted into the skin
cancer. Hence, development of novel chemical compounds for the treatment of skin cancer is

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desirable goal of this work. Here, efforts were made to design novel pyrimidine derivatives
via pharmacophoreꢀbased scaffold hopping method. Designed molecules were synthesised
and evaluated against different cancer cell lines including skin cancer cell line (Aꢀ375).
Potent compounds in in vitro evaluation were further evaluated for in vivo evaluation by
DMBA induced skin cancer animal model.
2. Results and Discussion
2.1 Rational for design of compounds
Involvement of CDK2 in skin cancer via p53 and p21 protein is well known, hence in this
work, eight molecules (1ꢀ8), (Table STꢀ1 under supplementary information) were taken from
the published literature10ꢀ15 for the generation of pharmacophore models. Twenty
pharmacophore models were generated through the GALAHAD module of Sybyl X,16 among
them, modelꢀ15 (Table STꢀ2 under supplementary information) was selected as best model
based on lowest energy value, maximum number of hits, and comparatively higher specificity
value.17ꢀ20 2D representation of the best model 15 is shown in Figure 1 (3Dꢀrepresention is
shown as Figure SFꢀ1 under supplementary information), which consisted of 2 donor atoms,
2 hydrophobic sites, and 1 acceptor atom. Validation of best pharmacophore model was
carried out by GH (Guner Henry) scoring method and ROC (Receiver Operating
Characteristics) curve analysis to check the accuracy and reliability of model.21ꢀ23 GH score
and area under curve (AUC) of ROC curve analysis of modelꢀ15 was 0.703 and 0.7,
respectively (Table STꢀ3 and STꢀ4 under supplementary information). Pharmacophore modelꢀ
15 was used as a 3D search query against NCI database, which consisted of 250000 diverse
anticancer molecules. Compounds having various chemical groups spatially overlap (map)
with corresponding features of pharmacophoric model 15 and were captured as hits with QFIT
value. Retrieved hit NCIIDM81922 with highest QFIT value of 93.18 consisted of pyrimidine

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ring as core moiety in the structure. (Structures of top five hit molecules and their QFIT values
are given in supplementary information).
NꢀPhenylpyrimidine was selected as a core moiety for the design of compounds based on
maximum similarity with the generated pharmacophore. Another advantage associated with
the selection of Nꢀphenylpyrimidine ring is the presence of pyrimidine ring, which is similar
to the most effective marketed drug, 5ꢀfluorouracil (5ꢀFU), available for the treatment of skin
cancer. Based upon these facts and results of pharmacophore modelling, fourteen novel
derivatives were designed and synthesized (6aꢀn) (Scheme 1). In the designed series,
different substitutions were carried out at R1 (Cꢀ5) and R2 (Cꢀ4) positions of Nꢀ
phenylpyrimidine ring. Two different series were prepared by using two different starting
materials; methylcyanoacetate and ethylcyanoacetate, which resulted into methyl and ethyl
substitution at R1 position of Nꢀphenylpyrimidine moiety. Different aliphatic and aromatic
amines were substituted at R2 position to check the effect of additional hydrophobic group on
the skin cancer activity. The designed compounds were predicted for in silico toxicity risk
prediction parameters. Factors of toxicity risk management are mutagenicity, tumorigenicity,
irritant effect and reproductive effect. Results of toxicity prediction study revealed that four
compounds viz. 6f, 6g, 6m, and 6n are at risk of mutagenic toxicity; compounds 6g and 6n
showed tumorigenic effect; compounds 6f and 6m showed irritant effect. All other
compounds are nonꢀtoxic. (Table STꢀ5 under supplementary information).
3. Chemistry
24
Our previously reported synthetic scheme was utilised as a reference for the synthesis of
intermediates and for the target compounds. As shown in scheme 1, methyl or ethyl
substituted S,Sꢀacetal (2a,b) was afforded from methylcyanoacetate or ethylcyanoacetate,

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respectively, by reacting with carbon disulphide in the presence of base, potassium hydroxide
(stepꢀI). StepꢀII involved nucleophilic displacement of one ꢀSCH3 group from S,Sꢀacetal
(2a,b) by aniline which resulted in the formation of methyl or ethyl substituted S,Nꢀacetal
(3a,b). StepꢀII product (3a,b) was treated with formamidine acetate in presence of sodium
hydroxide and ethanol to afford product (4a,b), which got cyclised to give pyrimidine moiety
(5a,b) in the presence of HCl saturated dioxane. HCl is known to enhance the reactivity of ꢀ
CN group, which resulted into formation of 5ꢀsubstitutedꢀ6ꢀ(Nꢀphenyl)ꢀ4ꢀchloropyrimidine
(5a,b) as a core moiety. This core moiety was utilised for the synthesis of designed
compounds as shown in synthetic Scheme 1. Chlorine on pyrimidine ring was a point for
nucleophilic substitution by different amines. Seven different aliphatic as well as aromatic
amines were substituted on pyrimidine moiety to afford 14 different final compounds (6aꢀn).
Chemical structure of all compounds were confirmed by 1H and 13C NMR, mass spectral and
elemental analysis data. Purity of compounds were determined from HPLC analysis (Table
1
STꢀ6 under supplementary information). The significant features of H NMR spectra include
appearance of different sets of hydrogen atom resonances corresponding to the protons in the
aromatic rings (on Cꢀ4 and Cꢀ6 of pyrimidine; aromatic protons on Cꢀ6 phenyl ring are
indicated by symbolꢀ ′ ), pyrimidine ring (at Cꢀ2), secondary amines (on Cꢀ4 and Cꢀ6 of
pyrimidine), methyl and ethyl groups (as R1). For synthesized compounds, set of protons
corresponding to the aromatic ring appear in the range of 6.50ꢀ8.00 δ ppm, showing the
expected multiplicity and integration values. The resonance peaks, in the region from 8.01ꢀ
1
8.70 δ ppm, corresponds to the pyrimidine proton. These ArꢀH of H NMR spectrum are
correlated with C atoms from 110ꢀ170 δ ppm in 13C NMR spectra. Signals for the secondary
amine proton (ꢀNHꢀ) appear as singlet in the region of 8ꢀ11 δ ppm. For ꢀCH3 group of methyl
acetate, singlet appears from 3.00 to 3.90 δ ppm. For ꢀCH3 of ethyl acetate, triplet appears in

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the region of 1.20ꢀ1.40 δ ppm and for ꢀCH2 group of ethyl acetate, quartet appears in the
region of 4.13ꢀ4.94 δ ppm (1H NMR, 13C NMR and Mass spectra of few representative
compounds are provided in supplementary information). Crystal structure of compound 6h
was derived by single crystal Xꢀray analysis and is shown in Figure 2 (Detailed description of
results of Xꢀray crystallography studies is provided in supplementary information. Crystal
structure is deposited at the Cambridge Crystallographic Data Centre with deposition number
CCDC 1438291).
4. Biological Evaluation
4.1 Antiproliferative in vitro assay
Synthesized derivatives were screened against five different cancer cell lines, viz. Aꢀ375,
MCFꢀ7, DUꢀ145, HEPꢀ3B and nonꢀcancerous cell line VERO, and their IC50 values are
reported in Table 1. Few compounds showed potent activity on various cell lines.
Compounds 6a (IC50 = 0.0073µM), 6h (IC50 = 0.0047µM), 6j (IC50 = 0.0079µM) and 6k
(IC50 = 0.0876µM) showed potent activity on skin (melanoma) cancer cell line, Aꢀ375.
Compounds 6h (IC50 = 0.0075µM), 6j (IC50 = 0.0042µM) and 6n (IC50 = 0.016µM) showed
potent activity on breast cancer cell line MCFꢀ7. Compounds 6h (IC50 = 0.0222µM), 6k (IC50
= 0.0561µM), 6l (IC50 = 0.018µM) and 6m (IC50 = 0.0302µM) showed potent activity on
Hepatoma cancer cell line HEPꢀ3B. Compounds 6b (IC50 = 0.0073µM) and 6h (IC50
=
0.0075µM) showed potent activity on Prostate cancer cell line DUꢀ145. In case of VERO cell
line, compounds 6c, 6d, 6e, 6i and 6l were found cytotoxic on normal VERO cell line and
rest of the compounds were found to be nonꢀtoxic, showing IC50 value >500 µM. 5ꢀFU
showed IC50 values of 0.0042µM (on Aꢀ375), 1.60µM (on MCFꢀ7), 1.75µM (on HEPꢀ3B)
and 0.07µM (on DUꢀ145). From the results of in vitro activity of synthesized compounds, it
was found that synthesized compounds showed most prominent activity on skin cancer cell

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line (Aꢀ375). Hence, highest active compounds, 6a, 6h, 6j and 6k, were further selected for
in vivo evaluation in swissꢀalbino mice by DMBA induced skin cancer model.
4.2 In vivo assay
In vivo study was carried out in Swissꢀalbino mice by 16 weeks DMBA induced skin cancer
model. Serum biomarkers and oxidative stress parameter levels were checked 12 weeks after
the induction of skin cancer in disease induced mice. A significant increase (p < 0.05) in the
serum level of biomarkers viz. Gamma Glutamyl Transferase (GGT), Lactate Dehydrogenase
(LDH), CꢀReactive Protein (CRP); oxidative stress parameter, Malondialdehyde (MDA) and
significant decrease in serum levels of other oxidative stress parameters viz. Reduced
Glutathione (GSH) and Superoxide Dismutase (SOD) were observed in skin cancer induced
mice as compared to normal control mice group, which confirmed the presence of skin
cancer. After the treatment with synthesized compounds (2%) and standard 5ꢀfluorouracil (5ꢀ
FU) (2%) from 12th to 16th weeks, it was found that compound 6h and 5ꢀFU significantly
reduced (p < 0.05) levels of GGT, LDH, CRP (Figures 3A, 3B and 3C) and MDA (Figure
4A); while, increased levels of GSH and SOD (Figures 4B, 4C) at the end of 16th week which
demonstrated good anticancer activity. Statistical results of serum biomarkers and oxidative
stress parameter levels are shown in Table 2 and 3, respectively. Tumour volume, tumour
burden and survival rate were observed from 6th to 16th weeks after the induction of skin
cancer and data are shown in Table 4. A significant increase (p < 0.05) in tumour volume and
tumour burden were observed at the end of 12th week in disease (skin cancer) induced mice
group. Effect of 5ꢀFU and synthesized compounds 6a, 6h, 6j and 6k on tumour volume and
tumour burden was observed from 12th to 16th week and it was found that at the end of 16th
week, highest reduction in tumour volume and tumour burden was noted in the compound 6h
and 5ꢀFU (Figures 5A and 5B). At the end of the study (16th week), no significant difference
of serum biomarkers levels, oxidative stress parameters, tumour volume and tumour burden

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was observed between compound 6h and 5ꢀFU treated groups, which confirmed the
anticancer activity of 6h. 100% survival rate was observed in the normal control group, skin
cancer control group treated with 5ꢀFU, 6h and 6j from 6th to 16th week.
Figure 7(A to D) represented photographs of caudal region skin of the mice and
histopathological results of skin samples of normal control group, skin cancer control group,
skin cancer treated with 5ꢀFU and skin cancer treated with compound 6h, respectively. Figure
7A, normal control group, represented normal caudal region skin of the mice and
histopathological section having various layers of skin. In skin cancer control mice,
photograph (Fig 7B) showed that generated tumour mass is thoroughly spread over the caudal
region skin of the mice. Histopathological study also showed disruption of fatty layer with
presence of keratin pearls. The presence of rete ridges extending through the connective
tissue was also seen, along with the mark presence of dysplastic and hyperplastic epithelial
cells. Photographs shown in Figure 7C and 7D showed that tumour mass was completely
removed from the caudal region skin of the mice which were treated with compound 6h and
5ꢀFU. Histopathological sections of skin from mice treated with compound 6h and 5ꢀFU
showed the complete absence of keratin pearls, dysplastic epithelial cells and rete ridges,
which proved the potency of the 6h for the treatment. (Colour representation is given as
Figure SFꢀ2)
5. Structure Activity Relationship
Structure activity relationship study of synthesized compounds for their effectiveness on all
four cell lines, viz. Skin cancer, Breast cancer, Hepatoma cancer and Prostate cancer is
discussed in this section.

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R1 substitution of the pyrimidine ring was substituted with methyl as well as ethyl groups to
check the effect of bulkiness on the activity against the skin cancer cell line. Compounds with
methyl substitution at R1 position resulted into the lower activity as compared to ethyl
substituted compounds, which clearly indicated that groups with higher carbon number like
ethyl or higher at R1 position is required to increase the potency of synthesized compounds.
Apart from highest active compound 6h, compounds with ethyl substitution at R1 position
(6j, 6k, 6l and 6n) were found more potent than compounds 6c, 6d, 6e and 6g, which were
substituted with methyl group at R1 position. R2 position on pyrimidine ring was substituted
with aliphatic and aromatic amines. Results of in vitro anticancer activity revealed that
compounds with aromatic amine exhibited more potent anticancer activity as compared to
aliphatic amines. In the first series, compounds 6a, 6b, 6c and 6d exhibited more potent
activity than compounds 6f and 6g; while in the second series, compounds 6h, 6j, 6k and 6l
exhibited more potent activity than compounds 6m and 6n. The compounds which showed
good in vitro anticancer activity were evaluated for in vivo anticancer assay. Four best
compounds 6a, 6h, 6j and 6k were further screened for in vivo anticancer activity. Amongst
them, compound 6h showed most promising activity as compared to 6a, 6j and 6k and
emerged as the most potent compound of the series. Structural features like presence of ethyl
group at R1 position and sulphanilamide at R2 position in compound 6h are found crucial and
required for anticancer activity.
In MCFꢀ7 cell line, compounds 6h, 6j and 6k were found more potent as compared to
compounds 6a, 6c and 6d proving the importance of ethyl substitution at R1 position.
Further, comparison between 6h, 6j and 6k revealed that 6h and 6j were found more potent
than compound 6k, as compound 6k consisted of electron withdrawing nitro group on
aromatic amine at R2 position, while compounds 6h and 6j consisted of the ꢀSO2NH2 and ꢀ
OCH3, which are electron donating groups.

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In HEPꢀ3B cell line, compounds 6h, 6i and 6m were found more potent as compared to 6a,
6b and 6e. This result again proved the importance of the ethyl group at R1 substitution which
is present in case of 6h, 6i and 6m. Further comparison between 6h, 6i and 6m indicated that
substitution at R2 with substituted aromatic amine, specifically sulphanilamide, is more
favourable in context with potency as compared to the alicyclic or aliphatic amine. This can
be proved by the compounds 6h, 6i and 6m, where 6i and 6m were found to be less potent as
compared to the compound 6h, which consisted of the aromatic amine like sulphanilamide at
R2 position, while in contrast, compound 6i consisted of piperazine and compound 6m
consisted of urea at R2 position.
On the other hand, methyl substitution at R1 position was proved to be active against DUꢀ145
cell line. This results were in contrast with other three cell line study results, where ethyl
group at R1 position was found more effective. This could be proved by compounds 6b and
6d, where compounds 6b and 6d were found more potent as compared to 6i and 6k. Further
comparison between compounds 6b and 6d showed that compound 6b was found more
potent as compared to compound 6d; as compound 6b consisted of piperazine at R2
substitution, while in case of compound 6d, electron withdrawing group nitro group was
present on aromatic amine at R2 position.
6. Conclusion
Based upon the pharmacophoreꢀbased virtual screening fourteen novel NꢀPhenyl pyrimidine
derivatives were designed, synthesised and evaluated against different cancer lines viz. skin
cancer (Aꢀ375), breast cancer (MCFꢀ7), hepatocellular carcinoma (HEPꢀ3B) and prostate
cancer (DUꢀ14). Structure activity relationship study proved that ethyl group at R1 and
aromatic amines at R2 positions of NꢀPhenyl pyrimidine were required for activity against the
skin cancer (Aꢀ375). Similarly in case of breast cancer (MCFꢀ7) ethyl group was proved

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effective at R1 position with electron donating substituted aromatic amine at R2 position. In
case of hepatocellular carcinoma (HEPꢀ3B), R2 position was found to favourable for aromatic
amines compared to alicyclic or aliphatic amines in combination with ethyl group at R1
position. In contrast, methyl substitution at R1 position was found more effective against
prostate cancer (DUꢀ145) in combination with electron donor amines at R2 position. As
compounds 6a, 6h, 6j and 6k revealed good anticancer activity against the skin cancer cell
line (Aꢀ375), they were further evaluated in DMBA induced skin cancer animal model.
Among these four derivatives, compound 6h was found to be best as it treated the tumours
completely. Results of biological screening was quite promising which indicated that
compound 6h is equipotent to 5ꢀFU and it is considered as a good lead compound in future
derivatization with bulky group at R1 and aromatic amines at R2 positions of NꢀPhenyl
pyrimidine to design novel compound which can give good anticancer activity against the
skin cancer.
7. Experimental Section
All reactions were performed using oven dried borosilicate glassware and dry solvents.
Precoated silica gel plates (MERCK) were used for TLC to monitor progress of the reaction.
UV chamber and iodine fume chamber were used for detection of spots on TLC plates. REMI
Rotaꢀmantle was used for refluxing and stirring the reactions. Rotary vacuum evaporator
(BUCHI type) was used for the recovery of the solvents. IR lamp was used for drying
products. Melting point was recorded on VEEGO corporation melting point apparatus. All
the compounds were purified by column chromatography and purity of all the compounds
were checked through the JASCO HPLC instrument. Purity of all the compounds were found
to be more than 95%. Mass spectra were recorded on WATERS mass spectrometer using
1
Electron Spray Ionization (ESI) as ion source. The H and 13C NMR were recorded using

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tetramethylsilane (TMS) as an internal standard on BRUKER AVANCEꢀII 400 MHz
spectrometer. Chemical shifts (δ) were reported in ppm, downfield of TMS and coupling
constants (J) are given in Hz. Single crystal XRD of most potent compound 6h was recorded
using RIGAKU SCX mini diffractometer.
Following procedures were used for synthesis of products 2a to 5a:
7.1 Synthesis of methylꢀ2ꢀcyanoꢀ3,3ꢀbis(methylthio)acrylate (2a): Potassium hydroxide
(11.2g, 0.2mol) was weighed and dissolved in 5mL of water, 15mL of DMF was added with
cooling and stirring was continued for 10min. In the stirring mixture, methylcyanoacetate
(1a) (8.8mL, 0.1mol) was added, followed by carbon disulphide (6mL, 0.1mol). Above
mixture was stirred for 1hr at 25ºC and was cooled. DMS (18.9mL, 0.2mol) was added drop
wise, maintaining the temperature at 0°C. Above mixture was added to the crushed ice.
Yellow crystalline product of methylꢀ2ꢀcyanoꢀ3,3ꢀbis(methylthio)acrylate (2a) was
precipitated out, washed with water and dried. The product was recrystallized with nꢀhexane
yielding pure white colour needle shaped crystals (yield 65.88%, m.p. 54ꢀ56ºC).
7.2 Synthesis of methylꢀ2ꢀcyanoꢀ3ꢀ(methylthio)ꢀ3ꢀ(phenylamino)acrylate (3a):
Compound 2a (4.06g, 0.02mol) and aniline (1.82mL, 0.02mol) were refluxed in ethyl alcohol
for 2hr. Resulted reaction mixture was cooled on the ice bath. White colour needle shaped
crystals of methylꢀ2ꢀcyanoꢀ3ꢀ(methylthio)ꢀ3ꢀ(phenylamino)acrylate (3a) were obtained,
filtered and dried (yield 76.19%, m.p. 62ꢀ63ºC).
7.3 Synthesis of methylꢀ3ꢀ(aminomethyleneamino)ꢀ2ꢀcyanoꢀ3ꢀ(phenylamino)acrylate
(4a): Formamidine acetate (2.08g, 0.02mol) was added to 1M ethanolic sodium hydroxide
solution. Reaction mixture was allowed to stir for 45min. Compound 3a (2.5g, 0.01mol) in
ethanol was added drop wise by dropping funnel to the reaction mixture. The reaction

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mixture was stirred for 6hr by maintaining the iceꢀcold condition and added to the crushed
ice. White colour solid product of methylꢀ3ꢀ(aminomethyleneamino)ꢀ2ꢀcyanoꢀ3ꢀ
(phenylamino)acrylate (4a) was precipitated out, filtered and dried (yield 75.62 %, m.p. 69ꢀ
71ºC)
7.4 Synthesis of methylꢀ4ꢀchloroꢀ6ꢀ(phenylamino)pyrimidineꢀ5ꢀcarboxylate (5a):
Compound 4a (4.8g, 0.02mol) was added, with stirring, to 25mL of saturated solution of 1,4ꢀ
dioxane with dry HCl gas. The stream of dry HCl gas was passed to the same reaction
mixture for 24hr. Reaction mixture was added to the crushed ice. White precipitates were
obtained
and
were
filtered.
Obtained
compound
methylꢀ4ꢀchloroꢀ6ꢀ
(phenylamino)pyrimidineꢀ5ꢀcarboxylate (5a) was again dissolved in the saturated bicarbonate
solution to neutralize the excessive acid. The compound was filtered and dried (yield 66.37%,
m.p. 57ꢀ59ºC).
Following procedures were used for synthesis of products 2b to 5b:
7.5 Synthesis of ethylꢀ2ꢀcyanoꢀ3,3ꢀbis(methylthio)acrylate (2b): Same as compound 2a;
instead of methylcyanoacetate, ethylcyanoacetate (1b) (10.6mL, 0.1mol) was used and
resulted into yellow crystalline product 2b. The product was recrystallized from nꢀhexane
yielded pure white colour needle shaped crystals (yield 63.14%, m.p. 56ꢀ58ºC).
7.6 Synthesis of ethylꢀ2ꢀcyanoꢀ3ꢀ(methylthio)ꢀ3ꢀ(phenylamino)acrylate (3b): Same as
compound 3a; instead of methylꢀ2ꢀcyanoꢀ3,3ꢀbis(methylthio)acrylate, ethylꢀ2ꢀcyanoꢀ3,3ꢀ
bis(methylthio)acrylate (2b) (2.17g, 0.01mol) was used and resulted into white colour needle
shaped crystalline product 3b (yield 51.52%, m.p. 65ꢀ67ºC).

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7.7 Synthesis of ethylꢀ3ꢀ(ꢀaminomethyleneamino)ꢀ2ꢀcyanoꢀ3ꢀ(phenylamino)acrylate
(4b): Same as compound 4a; instead of methylꢀ2ꢀcyanoꢀ3ꢀ(methylthio)ꢀ3ꢀ
(phenylamino)acrylate, ethylꢀ2ꢀcyanoꢀ3ꢀ(methylthio)ꢀ3ꢀ(phenylamino)acrylate (3b) (2.6g,
0.01mol) was used and resulted into white colour solid product 4b (yield 76.92%, m.p. 70ꢀ
73ºC).
7.8 Synthesis of ethylꢀ4ꢀchloroꢀ6ꢀ(phenylamino)pyrimidineꢀ5ꢀcarboxylate (5b): Same as
compound 5a; instead of methylꢀ3ꢀ(ꢀaminomethyleneamino)ꢀ2ꢀcyanoꢀ3ꢀ(phenylamino)
acrylate, ethylꢀ3ꢀ(aminomethyleneamino)ꢀ2ꢀcyanoꢀ3ꢀ(phenylamino)acrylate (4b) (5.16g,
0.02mol) was used which resulted into white colour solid product 5b (yield 63.29%, m.p. 90ꢀ
92ºC).
Following procedures were used for synthesis of final step products 6a to 6n:
7.9
Synthesis
of
methylꢀ4ꢀbenezenesulfonamideꢀ6ꢀ(phenylamino)pyrimidineꢀ5ꢀ
carboxylate (6a). Methylꢀ4ꢀchloroꢀ6ꢀ(phenylamino)pyrimidineꢀ5ꢀcarboxylate (5a) (2.6g,
0.01mol) was mixed with the sulphanilamide (1.7g, 0.01mol) in 15mL of isopropyl alcohol.
Catalytic amount of the concentrated HCl was added to the reaction mixture and was refluxed
for 6hr and transferred to the crushed ice. White solids of 6a got precipitated out, filtered and
1
dried (yield 55%, m.p. 210ꢀ212ºC). H NMR (400 MHz, DMSOꢀd6) δ PPM: 3.96 (s, 3H, ꢀ
COOCH3), 4.50 (s, 2H, 4ꢀSO2NH2ꢀC6H4ꢀNHꢀ), 7.13 (d, 2H, J = 8 Hz, Arꢀ2′,6′ꢀH), 7.35 (t,
2H, J = 8 Hz, Arꢀ3′,5′ꢀH), 7.50 (t, 1H, J = 7.6 Hz, Arꢀ4′ꢀH), 7.58 (d, 2H, J = 8 Hz, Arꢀ2,6ꢀ
H), 7.83 (d, 2H, J = 8 Hz, Arꢀ3,5ꢀH), 8.26 (s, 1H, Cꢀ2 proton of pyrimidine), 10.05 (s, 1H, ꢀ
NHꢀC6H5), 10.31 (s, 1H, 4ꢀSO2NH2ꢀC6H4ꢀNHꢀ). 13C NMR (400 MHz, DMSOꢀd6) δ PPM:
87.16, 121.65, 122.70, 124.05, 126.36, 128.53, 130.43 (2C), 138.48 (2C), 140.90. 141.73,
149.20 (2C), 158.80 (2C), 159, 167.49. Elemental analysis: Anal. Calcd for C18H17N5O4S: C,

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54.13; H, 4.28; N, 17.67; O, 16.09; S, 8.05. Found: C, 54.17; H, 4.24; N, 17.70; O, 16.13; S,
8.01. MS (EI) m/z for C18H17N5O4S, found 400.1 MH+.
7.10 Synthesis of methylꢀ4ꢀ(piperazinꢀ1ꢀyl)ꢀ6ꢀ(phenylamino)pyrimidineꢀ5ꢀcarboxylate
(6b): Same as compound 6a; instead of sulphanilamide, piperazine (0.86g, 0.01mol) was
used and refluxed for 2hr yielding white colour solid product of 6b. (yield 55%, m.p. 164ꢀ
1
167ºC). H NMR (400 MHz, DMSOꢀd6) δ PPM: 2.14 (s, 1H, ꢀNHꢀ of piperazine), 2.45 (t,
2H, 2,6ꢀposition protons of piperazine), 2.58 (t, 2H, 3,5ꢀ position protons of piperazine), 3.68
(s, 3H, ꢀCOOCH3), 6.93 (d, 2H, J = 8 Hz, Arꢀ2′,6′ꢀH), 7.09 (t, 2H, J = 8 Hz, Arꢀ3′,5′ꢀH), 7.42
(t, 1H, J = 7.6 Hz, Arꢀ4′ꢀH), 8.15 (s, 1H, Cꢀ2 proton of pyrimidine), 10.16 (s, 1H, ꢀNHꢀC6H5).
13C NMR (400 MHz, DMSOꢀd6) δ PPM: 51.68 (2C), 55.17 (2C), 87.16, 113.83, 122.68,
124.51, 128.15 (2C), 129.94 (2C), 131.40, 156.08, 160.17, 167.78. Elemental analysis: Anal.
Calcd for C16H19N5O2: C, 61.34; H, 6.19; N, 22.27; O, 10.22. Found: C, 61.37; H, 6.14; N,
22.30; O, 10.25. MS (EI) m/z for C16H19N5O2, found 314.1 MH+.
7.11 Synthesis of methylꢀ4ꢀ(4ꢀmethoxyphenylamino)ꢀ6ꢀ(phenylamino)pyrimidineꢀ5ꢀ
carboxylate (6c):
Same as compound 6a; instead of sulphanilamide, 4ꢀmethoxyaniline (1.2g, 0.01mol) was
used and refluxed for 6hr yielding white colour solid product of 6c. (yield 95%, m.p. 145ꢀ
1
147ºC). H NMR (400 MHz, DMSOꢀd6) δ PPM: 3.86 (s, 3H, 4ꢀOCH3ꢀC6H4ꢀNHꢀ), 3.96 (s,
3H, ꢀCOOCH3), 6.91 (d, 2H, J = 8 Hz, Arꢀ2′,6′ꢀH), 7.28 (t, 2H, J = 8 Hz Arꢀ3′,5′ꢀH), 7.35 (t,
1H, J = 8 Hz, Arꢀ4′ꢀH), 7.42 (d, 2H, J = 8 Hz, Arꢀ3,5ꢀH), 7.60 (d, 2H, J = 8 Hz, Arꢀ2,6ꢀH),
8.15 (s, 1H, Cꢀ2 proton of pyrimidine), 9.92 (s, 1H, ꢀNHꢀC6H5), 10.16 (s, 1H, 4ꢀOCH3ꢀC6H4ꢀ
NHꢀ). 13C NMR (400 MHz, DMSOꢀd6) δ PPM: 16.47, 76.28, 85.80, 113.63, 117.63, 122.66,
123.78, 124.96 (2C), 126.51 (2C), 128.15, 131.40 (2C), 138.36 (2C), 156.08, 158.94, 160.17.
Elemental analysis: Anal. Calcd for C19H18N4O3: C, 65.12; H, 5.19; N, 15.90; O, 13.2. Found
C, 65.17; H, 5.15; N, 15.93; O, 13.7. MS (EI) m/z for C19H18N4O3, found 351.1 MH+.

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7.12
Synthesis of methylꢀ4ꢀ(4ꢀnitrophenylamino)ꢀ6ꢀ(phenylamino)pyrimidineꢀ5ꢀ
carboxylate (6d): Same as compound (6a); instead of sulphanilamide, 4ꢀnitroaniline (1.4g,
0.01mol) was used and refluxed for 6hr yielding yellow colour solid product of 6d. (yield
95%, m.p. 160ꢀ162ºC). 1H NMR (400 MHz, DMSOꢀd6) δ PPM: 3.67 (s, 3H, ꢀCOOCH3), 6.14
(d, 2H, J = 8 Hz, Arꢀ2′,6′ꢀH), 6.95 (t, 2H, J = 8 Hz, Arꢀ3′,5′ꢀH), 6.96 (t, 1H, J = 8 Hz, Arꢀ
4′ꢀH), 7.51 (d, 2H, J = 8 Hz, Arꢀ2,6ꢀH), 7.65 (d, 2H, J = 8 Hz, Arꢀ3,5ꢀH), 8.19 (s, 1H, Cꢀ2
proton of pyrimidine), 10.03 (s, 1H, ꢀNHꢀC6H5), 10.09 (s, 1H, 4ꢀNO2ꢀC6H4ꢀNHꢀ). 13C NMR
(400 MHz, DMSOꢀd6) δ PPM: 14.10, 62.35, 76.23, 125.36, 128.52 (2C), 130.11 (2C),
130.81, 134.22 (2C), 136.31 (2C), 136.35, 137.10, 149.10, 155.85, 169.85. Elemental
analysis: Anal. Calcd for C18H15N5O4: C, 59.19; H, 4.17, N; 19.16; O, 17.2. Found: C, 59.16;
H, 4.15; N, 19.20; O, 17.4. MS (EI) m/z for C18H15N5O4, found 366.3 MH+.
7.13 Synthesis of methylꢀ4ꢀ(4ꢀtoludino)ꢀ6ꢀ(phenylamino)pyrimidineꢀ5ꢀcarboxylate (6e):
Same as compound (6a); instead of sulphanilamide, 4ꢀmethylaniline (1.07g, 0.01mol) was
used and refluxed for 6hr yielding white colour solid product of 6e. (yield 78%, m.p. 195ꢀ
197ºC). 1H NMR (400 MHz, DMSOꢀd6) δ PPM: 3.41 (s, 3H, 4ꢀCH3ꢀC6H4ꢀNHꢀ), 3.67 (s, 3H,
ꢀCOOCH3), 7.10 (d, 2H, J = 8 Hz, Arꢀ2′,6′ꢀH), 7.14 (t, 2H, J = 8 Hz, Arꢀ3′,5′ꢀH), 7.27 (t, 1H,
J = 8 Hz, Arꢀ4′ꢀH), 7.36 (d, 2H, J = 6.6 Hz, Arꢀ2,6ꢀH), 7.43 (d, 2H, J = 8 Hz, Arꢀ3,5ꢀH),
8.19 (s, 1H, Cꢀ2 proton of pyrimidine), 9.06 (s, 1H, ꢀNHꢀC6H5), 9.14 (s, 1H, 4ꢀCH3ꢀC6H4ꢀ
NHꢀ). 13C NMR (400 MHz, DMSOꢀd6) δ PPM: 20.37, 51.69, 85.69, 117.66, 120.27, 121.70,
122.80 (2C), 123.88 (2C), 126.51, 128.67, 130.92 (2C), 133.02 (2C), 138.40, 160.14, 169.91.
Elemental analysis: Anal. Calcd for C19H18N4O2: C, 68.19; H, 5.45; N, 16.49; O, 9.71. Found:
C, 68.22; H, 5.41; N, 16.46; O, 9.67. MS (EI) m/z for C19H18N4O2, found 334 MH+.
7.14 Synthesis of methylꢀ4ꢀureidoꢀ6ꢀ(phenylamino)pyrimidineꢀ5ꢀcarboxylate (6f): Same
as compound (6a); instead of sulphanilamide, urea (0.6g, 0.01mol) was used and refluxed for
1
2hr yielding white colour solid product of 6f. (yield 98%, m.p. 200ꢀ202ºC). H NMR (400

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MHz, DMSOꢀd6) δ PPM: 3.67 (s, 3H, ꢀCOOCH3), 7.11 (s, 2H, ꢀNHꢀCOꢀNH2), 7.27 (t, 2H, J
= 8 Hz, Arꢀ3′,5′ꢀH), 7.36 (d, 2H, J = 8 Hz, Arꢀ2′,6′ꢀH), 7.41 (t, 1H, J = 8 Hz, Arꢀ4′ꢀH), 7.61
(s, 1H, ꢀNHꢀC6H5), 8.09 (s, 1H, Cꢀ2 proton of pyrimidine), 8.22 (s, 1H, ꢀNHꢀCOꢀNH2). 13C
NMR (400 MHz, DMSOꢀd6) δ PPM: 51.43, 76.19, 117.68, 123.30, 126.49 (2C), 128.68 (2C),
129.25, 138.46, 152.19, 165.84, 169.12. Elemental analysis: Anal. Calcd for C13H13N5O3: C,
54.17; H, 4.32; N, 24.27; O, 16.28. Found: C, 54.20; H, 4.34; N, 24.30; O, 16.25. MS (EI)
m/z for C13H13N5O3, found 288.2 MH+.
7.15 Synthesis of methylꢀ4ꢀthioureidoꢀ6ꢀ(phenylamino)pyrimidineꢀ5ꢀcarboxylate (6g):
Same as compound (6a); instead of sulphanilamide, thiourea (0.76g, 0.01mol) was used and
refluxed for 2hr yielding white colour solid product of 6g. (yield 75.34%, m.p. 214ꢀ216ºC).
1H NMR (400 MHz, DMSOꢀ d6) δ PPM: 3.26 (s, 3H, ꢀCOOCH3), 6.46 (s, 2H, ꢀNHꢀCSꢀNH2),
7.10 (d, 2H, J = 8 Hz, Arꢀ2′,6′ꢀH), 7.88 (t, 2H, J = 8 Hz, Arꢀ3′,5′ꢀH), 8.18 (t, 1H, J = 8 Hz,
Arꢀ4′ꢀH), 8.31 (s, 1H, ꢀNHꢀC6H5), 8.37 (s, 1H, Cꢀ2 proton of pyrimidine), 9.05 (s, 1H, ꢀNHꢀ
CSꢀNH2). 13C NMR (400 MHz, DMSOꢀd6) δ PPM: 75.30, 113.10, 115.56, 119.10, 120.81,
125.10 (2C), 129.15 (2C), 133.16, 161.10, 163.14, 170.07. Elemental analysis: Anal. Calcd
for C13H13N5O2S: C, 51.35; H, 4.33; N, 23.03; O, 10.49; S, 10.59. Found: C, 51.32 H, 4.30,
N, 23.08, O, 10.45 S, 10.56 MS (EI) m/z for C13H13N5O2S, found 304.4 MH+.
7.16
Synthesis
of
ethylꢀ4ꢀbenezenesulfonamideꢀ6ꢀ(phenylamino)pyrimidineꢀ5ꢀ
carboxylate (6h): Ethylꢀ4ꢀchloroꢀ6ꢀ(phenylamino)pyrimidineꢀ5ꢀcarboxylate (5b) (2.7g,
0.01mol) was mixed with the sulphanilamide (1.72g, 0.01mol) in 15mL of isopropyl alcohol.
Catalytic amount of the concentrated HCl was added to the reaction mixture and was refluxed
for 6hr and transferred to the crushed ice. White solid of 6h got precipitated out, filtered and
1
dried. (yield 49%, m.p. 214ꢀ218ºC). H NMR (400 MHz, DMSOꢀd6) δ PPM: 1.20 (t, 3H, J =
6.79 Hz, ꢀCOOCH2CH3), 3.40 (s, 2H, 4ꢀSO2NH2ꢀC6H4ꢀNHꢀ), 4.13 (q, 2H, J = 6.8 Hz, ꢀ
COOCH2CH3), 7.12 (d, 2H, J = 6.99 Hz, Arꢀ2′,6′ꢀH), 7.33 (t, 2H, J = 6.99 Hz, Arꢀ3′,5′ꢀH),

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7.61 (t, 1H, J = 8 Hz Arꢀ4′ꢀH), 7.81 (d, 2H, J = 9.33 Hz, Arꢀ2,6ꢀH), 7.84 (d, 2H, J = 8 Hz
Arꢀ3,5ꢀH), 8.31 (S, 1H, Cꢀ2 proton of pyrimidine), 10.07 (s, 1H, ꢀNHꢀC6H5), 10.32 (s, 1H, 4ꢀ
SO2NH2ꢀC6H4ꢀNHꢀ). 13C NMR (400 MHz, DMSOꢀd6) δ PPM: 13.88, 62.11, 87.36, 121.20,
122.20, 123.91, 126.46 (2C), 128.62 (2C), 129.23, 138.37, 138.43 (2C), 141.71 (2C), 158.81,
159.72, 166.98. Elemental analysis: Anal. Calcd for C19H19N5O4S: C, 55.29; H, 4.47; N,
16.39; O, 15.54; S, 7.59. Found: C, 55.25; H, 4.43; N, 16.35; O, 15.57; S, 7.56. MS (EI) m/z
for C19H19N5O4S, found 414.2 MH+. Xꢀcrystallographic structure of this compound is shown
in (Figure 2).
7.17 Synthesis of ethylꢀ4ꢀ(piperazinꢀ1ꢀyl)ꢀ6ꢀ(phenylamino)pyrimidineꢀ5ꢀcarboxylate (6i):
Same as compound 6h; instead of sulphanilamide, piperazine (0.86g, 0.01mol) was used and
refluxed for 2hr yielding white colour solid product of 6i. (yield 49.2%, m.p. 179ꢀ182ºC). 1H
NMR (400 MHz, DMSOꢀd6) δ PPM: 1.30 (t, J = 6.8 Hz, 3H, ꢀCOOCH2CH3), 2.60 (t, 2H, J =
8 Hz, 2,6ꢀposition protons of piperazine), 2.65 (t, 2H, J = 8 Hz, 3,5ꢀposition protons of
piperazine), 3.35 (s, 1H, ꢀNHꢀ of piperazine), 4.25 (q, 2H, J = 6.8 Hz, ꢀCOOCH2CH3), 6.40
(d, 2H, J = 8 Hz, Arꢀ2′,6′ꢀH), 7.29 (t, 2H, J = 8 Hz, Arꢀ3′,5′ꢀH), 7.35 (t, 1H, J = 8 Hz, Arꢀ4′ꢀ
H), 8.27 (S, 1H, Cꢀ2 proton of pyrimidine), 8.32 (s, 1H, ꢀNHꢀC6H5). 13C NMR (400 MHz,
DMSOꢀd6) δ PPM: 14.11, 45.91 (2C), 52.85 (2C), 60.78, 115.25, 117.37, 118.25 119.33
(2C), 120.34 (2C), 129.33, 154.22, 166.46, 169.30. Elemental analysis: Anal. Calcd for
C17H21N5O2: C, 62.16 H, 6.34, N, 21.39, O, 9.47. Found: C, 62.20; H, 6.37; N, 21.36; O,
9.50. MS (EI) m/z for C17H21N5O2 found 328.12 MH+.
7.18 Synthesis of ethylꢀ4ꢀ(4ꢀmethoxyphenylamino)ꢀ6ꢀ(phenylamino)pyrimidineꢀ5ꢀ
carboxylate (6j): Same as compound 6h; instead of sulphanilamide, 4ꢀmethoxyaniline (1.2g,
0.01mol) was used and refluxed for 6hr yielding white colour solid product of 6j. (yield 78%,
1
m.p. 185ꢀ187ºC). H NMR (400 MHz, DMSOꢀd6) δ PPM: 1.41 (t, 3H, J = 6.8 Hz, ꢀ

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COOCH2CH3), 3.75 (s, 3H, 4ꢀOCH3ꢀC6H4ꢀNHꢀ), 4.42 (q, 2H, J = 6.8 Hz, ꢀCOOCH2CH3),
6.92 (d, 2H, J = 8 Hz, Arꢀ2′,6′ꢀH), 7.09 (t, 2H, J = 8 Hz, Arꢀ3′,5′ꢀH), 7.33 (t, 1H, J = 7.6 Hz,
Arꢀ4′ꢀH), 7.46 (d, 2H, J = 8 Hz, Arꢀ3,5ꢀH), 7.62 (d, 2H, J = 8 Hz, Arꢀ2,6ꢀH), 8.19 (S, 1H, Cꢀ
2 proton of pyrimidine), 9.95 (s, 1H, ꢀNHꢀC6H5), 10.13 (s, 1H, 4ꢀOCH3ꢀC6H4ꢀNHꢀ). 13C
NMR (400 MHz, DMSOꢀd6) δ PPM: 13.97, 55.18, 60.41, 85.92, 113.72, 122.17, 123.67,
124.00, 124.41 (2C), 128.60 (2C), 129.25, 131.39 (2C), 138.65 (2C), 155.95, 160.02, 167.22.
Elemental analysis: Anal. Calcd for C20H20N4O3: C, 65.85; H; 5.39; N, 15.47; O, 13.2. Found,
C, 65.82, H; 5.37, N 15.43; O, 13.5. MS (EI) m/z for C20H20N4O3, found 365.2 MH+.
7.19 Synthesis of ethylꢀ4ꢀ(4ꢀnitrophenylamino)ꢀ6ꢀ(phenylamino) pyrimidineꢀ5ꢀ
carboxylate (6k): Same as compound 6h; instead of sulphanilamide, 4ꢀnitroaniline (1.38g,
0.01mol) was used and refluxed for 6hr yielding yellow colour solid product of 6k. (yield
1
74%, m.p. 150ꢀ153ºC). H NMR (400 MHz, DMSOꢀd6) δ PPM: 1.21 (t, 3H, J = 6.8Hz, ꢀ
COOCH2CH3), 4.14 (q, J = 6.8 Hz, 2H, ꢀCOOCH2CH3), 6.62 (d, 2H, J = 8 Hz, Arꢀ2′,6′ꢀH),
6.74 (t, 2H, J = 8 Hz, Arꢀ3′,5′ꢀH), 7.27 (t, 1H, J = 7.6 Hz, Arꢀ4′ꢀH), 7.35 (d, 2H, J = 8 Hz,
Arꢀ2,6ꢀH), 7.87 (d, 2H, J = 8 Hz, Arꢀ3,5ꢀH), 8.01 (S, 1H, Cꢀ2 proton of pyrimidine), 10.01 (s,
1H, ꢀNHꢀC6H5), 11.07 (s, 1H, 4ꢀNO2ꢀC6H4ꢀNHꢀ). 13C NMR (400 MHz, DMSOꢀd6) δ PPM:
13.18, 121.46, 122.91 (2C), 124.20, 125.30 (2C), 128.62, 130.37 (2C), 136.62, 136.33 (2C),
139.23, 140.70, 149.72, 158.85, 159.34, 169.95. Elemental analysis: Anal. Calcd for
C19H17N5O4: C, 60.3; H, 4.64; N, 18.4; O, 16.89. Found: C, 60.8; H, 4.67; N, 18.36; O, 16.85.
MS (EI) m/z for C19H17N5O4, found 380.3 MH+.
7.20 Synthesis of ethylꢀ4ꢀ(4ꢀtoluidino)ꢀ6ꢀ(phenylamino) pyrimidineꢀ5ꢀcarboxylate (6l):
Same as compound 6h; instead of sulphanilamide, 4ꢀtoluidine (1.07g, 0.01mol) was used and
1
refluxed for 6hr yielding white colour solid product of 6l. (yield 60%, m.p. 205ꢀ208 ºC). H
NMR (400 MHz, DMSOꢀd6) δ PPM: 1.29 (t, 3H, J = 6.8 Hz, ꢀCOOCH2CH3), 2.29 (s, 3H, 4ꢀ

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CH3ꢀC6H4ꢀNHꢀ), 3.45 (q, J = 6.8 Hz, 2H, ꢀCOOCH2CH3), 6.14 (d, 2H, J = 8 Hz, Arꢀ2′,6′ꢀH),
6.95 (t, 2H, J = 8 Hz, Arꢀ3′,5′ꢀH), 7.10 (t, J = 7.6 Hz, 1H, Arꢀ4′ꢀH), 7.14 (d, J = 8 Hz, 2H,
Arꢀ2,6ꢀH), 7.29 (d, 2H, J = 8 Hz, Arꢀ3,5ꢀH), 9.15 (S, 1H, Cꢀ2 proton of pyrimidine), 10.03 (s,
1H, ꢀNHꢀC6H5), 10.10 (s, 1H, 4ꢀCH3ꢀC6H4ꢀNHꢀ). 13C NMR (400 MHz, DMSOꢀd6) δ PPM:
12.17, 64.12, 78.31, 120.36, 126.52, 129.81 (2C), 132.11, 134.35 (2C), 135.10 (2C), 137.22,
139.31 (2C), 141.61, 144.10, 146.62, 156.85, 170.85. Elemental analysis: Anal. Calcd for
C20H20N4O2: C, 68.6; H, 5.67; N, 16.03; O, 9.12. Found: C, 68.57; H, 5.70; N, 16.05; O, 9.15.
MS (EI) m/z for C20H20N4O2, found 349.2 MH+.
7.21 Synthesis of ethylꢀ4ꢀureidoꢀ6ꢀ(phenylamino)pyrimidineꢀ5ꢀcarboxylate (6m):
Same as compound 6h; instead of sulphanilamide, urea (0.67g, 0.01mol) was used and
refluxed for 2hr yielding white colour solid product of 6m. (yield 60.72%, m.p. 215ꢀ217ºC).
1H NMR (400 MHz, DMSOꢀd6) δ PPM: 1.21 (t, 3H, J = 6.8 Hz, ꢀCOOCH2CH3), 4.10 (q, 2H,
J = 6.8 Hz, ꢀCOOCH2CH3), 6.38 (s, 2H, ꢀNHꢀCOꢀNH2), 7.07 (t, 2H, J = 8 Hz, Arꢀ3′,5′ꢀH),
7.15 (d, 2H, J = 8 Hz, Arꢀ2′,6′ꢀH), 7.50 (s, 1H, ꢀNHꢀC6H5), 7.27 (t, 1H, J = 7.6 Hz, Arꢀ4′ꢀH),
8.24 (s, 1H, Cꢀ2 proton of pyrimidine), 8.41 (s, 1H, ꢀNHꢀCOꢀNH2). 13C NMR (400 MHz,
DMSOꢀd6) δ PPM: 14.10, 60.10, 93.20, 113.10, 115.56, 116.41, 118.10 (2C), 129.15 (2C),
145.46, 156.21, 159.23, 168.12. Elemental analysis: Anal. Calcd for: C14H15N5O3, C, 55.57;
H, 5.30; N, 23.20; O, 15.47. Found: C, 55.60; H, 5.27; N, 23.23; O, 15.50. MS (EI) m/z for
C14H15N5O3, found 302.2 MH+.
7.22 Synthesis of ethylꢀ4ꢀthioureidoꢀ6ꢀ(phenylamino)pyrimidineꢀ5ꢀcarboxylate (6n):
Same as compound 6h; instead of sulphanilamide, thiourea (0.76g, 0.01mol) was used and
refluxed for 2hr yielding white colour solid product of 6n. (yield 80.14%, m.p. 219ꢀ221 ºC).

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1H NMR (400 MHz, DMSOꢀd6) δ PPM: 1.24 (t, 3H, J = 6.8 Hz, ꢀCOOCH2CH3), 4.20 (q, J =
6.8 Hz, 2H, ꢀCOOCH2CH3), 6.94 (d, 2H, J = 8 Hz, Arꢀ2′,6′ꢀH), 7.07 (t, 2H, J = 8 Hz, Arꢀ
3′,5′ꢀH), 7.84 (t, 1H, J = 7.6 Hz, Arꢀ4′ꢀH), 8.38 (s, 2H, ꢀNHꢀCSꢀNH2), 8.47 (s, 1H, Cꢀ2
proton of pyrimidine), 8.61 (s, 1H, ꢀNHꢀC6H5), 9.01 (s, 1H, ꢀNHꢀCSꢀNH2). 13C NMR (400
MHz, DMSOꢀd6) δ PPM: 10.10, 75.10, 115.66, 120.30, 121.81, 125.10, 129.25 (2C), 130.41
(2C), 134.46, 162.10, 164.84, 170.12. Elemental analysis: Anal. Calcd for C14H15N5O2S: C,
52.4; H, 4.73; N, 22.09; O, 10.08; S, 10.3. Found: C, 52.43; H, 4.77; N, 22.04; O, 10.05; S,
10.6 MS (EI) m/z for C14H15N5O2S, found 318.4 MH+.
7.23 Antiproliferative in vitro assay
All synthesized compounds were evaluated for their anticancer activity against Aꢀ375
(Melanoma), MCFꢀ7 (Breast), HEPꢀ3B (Hepatoma) and DUꢀ145 (Prostate) cancer cell lines.
To evaluate toxic effects of these compounds, they were also screened against nonꢀcancerous
cell line, VERO. For culturing of all these cell lines, DMEM supplemented with 5% HBSS,
penicillin, streptomycin and amphotericin B were utilised.25 These cell lines were kept in CO2
incubator which consisted of humidified atmosphere of 5% CO2 at 37ºC with 90% humidity.
Stock solutions of all compounds were prepared in 2% DMSO. Different concentrations of
these compounds ranging from 100µM to 1nM were prepared from the stock solution. Cells
were seeded into 96ꢀwell plates and incubated for 24hr. After 24hr, different concentrations
of compounds were added into 96ꢀwell plate and incubated for 24hr. Effect of compounds
was determined after 24hr by performing MTT assay. This assay was performed by using (3ꢀ
(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium bromide) solution (20ml of 5mg/ml). This
solution was added to each well and incubation was continued for additional 3hr resulted into
formation of dark blue formazan crystals within the cells. These crystals were solubilised by

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DMSO and absorbance was estimated by ELISA plate reader. IC50 values of all these
compounds were determined by nonꢀlinear regression method by using Graph pad prism 6.
7.24 Induction of skin cancer 26ꢀ28
All experiments were performed in compliance with the relevant laws and institutional
guidelines, and experimental protocols carried out in present study were permitted by the
Institutional Animal Ethics Committee (IAEC) of Institute of Pharmacy, Nirma University,
Ahmedabad, India (vide protocol number: IP/PCHEM/FAC/15ꢀ1/022). All experimental
methods are in harmony with CPCSEA guidelines, Ministry of Environment and Forests,
Government of India.
Male Swissꢀalbino mice weighing 25ꢀ30g were chosen for the study and were maintained
under controlled conditions of temperature (25° ± 2°C), humidity (55 ± 5%) and 12hr lightꢀ
dark cycle. Male Swissꢀalbino mice were kept for 1 week acclimatization period.
Subsequently the mice were treated with depilatory cream to remove the hairs from the skin,
from lumbar region to caudal region of their back. After removing the hairs from the skin,
mice were kept for 2 days under standard conditions to check whether the mice were in hair
cycle or not. Mice with no hair growth were chosen for the study and were grouped
accordingly. Skin cancer was induced by Dimethylbenz(a)anthracene (DMBA) and croton
oil. 1mg/ml concentration of DMBA solution was prepared by dissolving 50mg of DMBA in
50ml of acetone. 50µl of the DMBA solution was sprayed directly at caudal region skin of
the mice. Application was made two times in a week, while in 1st week, with period of 2
days gap between the successive doses. Total of 100µl of DMBA solution was sprayed within
3mm3 area on the caudal skin region of the mice. Two weeks from the initiation of DMBA
application, 1% croton oil solution was applied twice a week from 3rd week to 16th week. 1%
croton oil was prepared by dissolving 1ml of croton oil in 100ml of acetone. During this, the

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treatment with synthesised compounds was started from 7th week and continued till 16th
week. Compounds were applied twice a week, followed by application of croton oil in
disease induced groups with gap of 1 hour between the two applications.
7.25 Measurement of various parameters 26ꢀ28
At the end of 16th week, blood was collected by retroꢀorbital puncture from each mice and
various parameters were evaluated as per procedure provided in supplementary information.
After collection of blood samples, one animal each from all groups were killed and caudal
region of skin was excised from the mice, stored in 10% v/v neutral buffered formalin for
further histopathological study as detailed in supplementary information.

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Electronic Supplementary Information:
1
Detailed information regarding results of computational approaches; Chromatogram and H
NMR, 13C NMR and Mass spectra of few representative compounds; Colour figure and
detailed description of results of Xꢀray crystallography studies; Colour figure with
photographs of caudal region skin of the mice and histopathological results of skin samples
and pharmacological evaluation methods are available in supplementary information as pdf
file. ChemꢀDraw file of Schemeꢀ1 and original “.cif” files of crystal structure and reflection
list are also provided in supplementary information. Crystal structure is deposited at the
Cambridge Crystallographic Data Centre with deposition number CCDC 1438291.
Acknowledgement:
The authors are thankful to Nirma University, Ahmedabad, India for providing necessary
facilities to carry out the research work.
Abbreviations Used:
5ꢀFU, 5ꢀfluorouracil; DMBA, dimethylbenz(a)anthracene; DMEM, dulbecco’s modified
eagle’s medium; HBSS, hank’s balanced salt solution; GGT, gamma glutamyl transferase;
LDH, lactate dehydrogenase; CRP, Cꢀreactive protein; MDA, malondialdehyde; GSH,
reduced glutathione; SOD, superoxide dismutase.

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