Novel heterocyclic-fused pyrimidine derivatives: Synthesis, molecular modeling and pharmacological screening

Article abstract of DOI:10.1016/j.ejmech.2013.08.042

Novel heterocyclic-fused pyrimidines viz pyrrolo[1,2-c]pyrimidines 4-8, pyrimido[5,4-e]pyrrolo[1,2-c]pyrimidines 9-14, pyrimido[4′,5′:4,5] pyrimido[1,6-a]azepines 16-18, pyrrolo[1′,2′:1,6]pyrimido[4,5-d][1, 3]thiazines 19a,b and 1,3-thiazino[4′,5′:4,5]pyrimido[1,6-a]-azepine 19c were designed and synthesized as potential anticancer agents. In this investigation all the newly synthesized compounds were subjected to cytotoxic screening against MCF-7 breast cancer cell line. Moreover, kinase inhibitory assay was done for compounds 5, 7, 9 and 18 against the non-receptor and receptor tyrosine kinases c-Src and VEGFR, respectively. The tested compounds were more potent against c-Src than VEGFR, and the highest activity was observed for 18 showing 81% c-Src activity inhibition. Finally, molecular docking was performed with c-Src and VEGFR in an attempt to simulate and understand the possible binding interactions underlying the association between these small molecules and the kinase enzyme ATP binding pocket essential amino acids.

Full text of DOI:10.1016/j.ejmech.2013.08.042

Accepted Manuscript
Novel Heterocyclic-Fused Pyrimidine Derivatives: Synthesis, Molecular Modeling and
Pharmacological Screening
Reem K. Arafa, Mai S. Nour, Nehad A. El-Sayed
PII:
S0223-5234(13)00559-X
10.1016/j.ejmech.2013.08.042
EJMECH 6389
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 April 2013
Revised Date: 25 August 2013
Accepted Date: 31 August 2013
Please cite this article as: R.K. Arafa, M.S. Nour, N.A. El-Sayed, Novel Heterocyclic-Fused Pyrimidine
Derivatives: Synthesis, Molecular Modeling and Pharmacological Screening, European Journal of
Medicinal Chemistry (2013), doi: 10.1016/j.ejmech.2013.08.042.
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ACCEPTED MANUSCRIPT
Graphical Abstract

ACCEPTED MANUSCRIPT
Novel Heterocyclic-Fused Pyrimidine Derivatives: Synthesis, Molecular
Modeling and Pharmacological Screening
Reem K. Arafa,1,* Mai S. Nour,2 Nehad A. El-Sayed1
1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street,
Cairo 11562, Egypt
2 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, MSA University, 6th October City, Cairo,
Egypt
*To whom correspondences should be addressed, e-mail: rkarafa@cu.edu.eg; tel.: +2 (0100) 207-4028;
fax: +2 (02) 3362-8426
Abstract:
Novel heterocyclic-fused pyrimidines viz pyrrolo[1,2-c]pyrimidines 4-8, pyrimido[5,4-
e]pyrrolo[1,2-c]pyrimidines
9-14,
pyrimido[4',5':4,5]pyrimido[1,6-a]azepines
16-18,
pyrrolo[1',2':1,6]pyrimido[4,5-d][1,3]thiazines 19a,b and 1,3-thiazino[4',5':4,5]pyrimido[1,6-a]-
azepine 19c were designed and synthesized as potential anticancer agents. In this investigation
all the newly synthesized compounds were subjected to cytotoxic screening against MCF-7
breast cancer cell. Moreover, kinase inhibitory assay was done for compounds 5, 7, 9 and 18
against the non-receptor and receptor tyrosine kinases c-Src and VEGFR, respectively. The
tested compounds were more potent against c-Src than VEGFR, and the highest activity was
observed for 18 showing 81% c-Src activity inhibition. Finally, molecular docking was performed
with c-Src and VEGFR in an attempt to simulate and understand the possible binding
interactions underlying the association between these small molecules and the kinase enzyme
ATP binding pocket essential amino acids.
Key words: Fused Pyrimidines; Antiproliferative agents; MCF-7 breast cancer cell line; c-Src and
VEGFR inhibition.
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1. Introduction
A recent WHO report states that by the year 2030 the incidence of cancer worldwide
will grow by about 75%, doubling in some of the developing countries [1]. Cancer can affect any
body organ yet the most common type of cancer that afflicts women worldwide is breast cancer
accounting for 22.9% of all cancers incidences in women. Needless to mention, breast cancer
can also affect men but is 100 times more common in women as compared to men [2,3].
Chemotherapy is one of the medical options for cancer management. Antineoplastic
drugs in medical use can elicit their cytotoxic activity by impairing cellular mitosis via numerous
possible mechanisms of actions being either antimetabolites, alkylating agents, topoisomerase
inhibitors or most recently signal transduction inhibitors [4,5]. Belonging to the latter are
tyrosine kinase inhibitors (TKIs), which play a substantial effect in stopping cellular signaling
leading to halting cell migration, adhesion and proliferation thus eventually causing cell
apoptosis. Amongst the many known TKs, vascular endothelial growth factor receptor (VEGFR)
and cellular-Src (c-Src) are two receptor and cytoplasmic tyrosine kinase, respectively. It is note-
worthy that increased VEGF activity has been implicated with poor prognosis in breast cancer
[6]. Also, an association between high c-Src levels in human breast cancer tissue relative to
adjacent epithelium, as well as disease recurrence has been reported [7-10].
As such, both kinases display important targets for small-molecule TKIs. Many
pyrimidine and fused pyrimidine based VEGFR and c-Src inhibitors were FDA approved as first
and second line cancer therapy agents against breast cancer, bone cancer, prostate cancer,
acute lymphocytic leukemia and other types of cancer [11,12]. Among the numerous known
examples are the pyrimidine (imatinib, dasatinib, nilotinib and pazopanib) and pyrrole
(sunitinib) TKIs which are in clinical use as anticancer agents due to their high activity towards
several families of receptor and non-receptor tyrosine. Albeit the unquestionable clinical
activity of the currently used TKIs, it is generally recognized that the value of these agents is
markedly restricted by the emergence of drug resistance, which ultimately arises in most
treated patients [13-15].
The above mentioned facts and findings call for incessant search for news anticancer
agents. In a contribution to recent scientific endeavors concerned with study of pyrimidines as
cytotoxic agents [16,17], it deemed of interest to initiate a research work directed at designing
a series of heterocyclic-fused (viz pyrrole, azepine, pyrimidine and thiazine) bicyclic and tricyclic
pyrimidines as a backbone scaffold for new potential TKIs. The target compounds design was
based on alterations of number/size/type of the rings fused with the pyrimidine as well as on
variations of the flexibility of the side chains attached to the fused ring system nucleus. We
herein report the synthesis, antiproliferative screening, VEGFR and c-Src inhibitory profiling of
novel pyrrolo[1,2-c]pyrimidine, pyrimido[5,4-e]pyrrolo[1,2-c]pyrimidine, pyrimido[4',5':4,5]-
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pyrimido[1,6-a]azepine, pyrrolo[1',2':1,6]-pyrimido[4,5-d][1,3]thiazine and 1,3-thiazino-
[4',5':4,5]pyrimido[1,6-a]-azepine derivatives.
2.
2.1. Chemistry
The synthetic pathways adopted for the synthesis of the novel fused pyrimidine
Results and Discussion
derivatives are illustrated in schemes 1-4. The known key intermediate enaminonitriles 3a,b
and 15 were employed for the synthesis of the target compounds.
Scheme 1 depicts the synthesis of the bicyclic pyrrolo[1,2-c]pyrimidines with flexible or
semi-flexible side chains at position 3. Thus, treatment of enaminonitrile intermediates 3a and
3b with 2-chloro-N-p-tolylacetamide afforded the derivatives 4a and 4b. On the other hand, the
target compound 5 was obtained via reacting the enaminonitrile 3a with ethyl cyanoacetate.
Moreover, treatment of 3a,b with phenyliso(thio)cyanate yielded the nucleophilic addition
product uriedo derivatives 6a-d. In an attempt to introduce some rigidity in the side chain of
these bicyclic derivatives, the uriedo moiety of 6a was cyclized via reaction with oxalyl chloride
or chloroacetyl chloride affording the imidazoline trione 7 and the imidazoline dione 8,
respectively.
In addition, target pyrimido[5,4-e]pyrrolo[1,2-c]pyrimidine derivatives with various
substituents on the tricyclic backbone were obtained as represented in Scheme 2. Initially,
sodium ethoxide-catalysed intramolecular cyclization of the ureido derivative 6a furnished the
pyrimido[5,4-e]pyrrolo[1,2-c]pyrimidine 9. Furthermore, fusion of the key intermediates 3a,b
with thiourea resulted in nucleophilic substitution followed by cyclization onto the cyano
function yielding the tricyclic compounds 10a,b. Schiff’s base 11 was obtained via the
condensation of the amino group of 10a with the active carbonyl of p-chlorobenzaldehyde.
Also, treatment of the key intermediates 3a,b with formic acid resulted in the formation of
tricyclic derivatives 12a,b. Additionally, treating 12a with phosphorus oxychloride (POCl3) in the
presence of a catalytic amount of phosphorus pentachloride (PCl5) produced the more stable
aromatic chloro derivative 13 which was reacted with hydrazine hydrate affording the tricyclic
hydrazine derivative 14.
On the other hand, the synthesis of the azepine analogs is illustrated in scheme 3
employing the key intermediate enaminonitrile 15. Fusion of 15 with thiourea produced the 1-
aminopyrimido[4',5':4,5]pyrimido[1,6-a]azepine 16 which was then condensed with p-
chlorobenzaldehyde forming schiff’s base 17. On the other hand, reacting 15 with formic acid
afforded the tricyclic pyrimido[4',5':4,5]pyrimido[1,6-a]azepine 18.
Finally, as demonstrated in scheme 4, the reaction of the five membered
enaminonitriles 3a,b or the seven membered analog 15 with carbon disulfide afforded the
target pyrrolopyrimidothiazines 19a,b and the thiazinopyrimidoazepine 19c derivatives,
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respectively. All the postulated structures were supported by spectral and elemental analysis
data.
2.2. In vitro Antitumor Screening and SAR Findings
All the newly synthesized compounds were tested for their antiproliferative activity
against MCF-7 breast cancer cell line in the National Cancer Institute (NCI, Cairo, Egypt). The cell
growth inhibitory activity was measured in vitro using the Sulfo-Rhodamine-B-stain (SRB) assay
method as described by Skehan et al. [18] and the data is shown in Table 1. Also, cLog P values
of the synthesized compounds were calculated using ACD/ChemSketch software version12.01
(listed in Table 1) and were used for the SAR analysis purpose.
Regarding the fused bicyclic pyrrolo[1,2-c]pyrimidines, and referring to the first molecular
manipulation adopted concerning the bioisosteric alteration between the oxo and thioxo
groups, the thioxo derivative 4b showed higher activity than its oxo anolog 4a. The same
behavior was observed for the oxo/thioxo bioisosteric derivatives 6a, 6b and 6c; IC50 values
19.8, 14.2 and 16.6 µg/mL, respectively. This may possibly be explained in terms of the higher
lipophilicity of the thioxo, analogs as compared to their oxo counterparts (cLogP values are
enlisted in table 1).
Moreover, results showed that the cyanoacetamide derivative 5, IC50 12.8 µg/mL and
cLogP 0.61, has better activity than the phenylureido analog 6a which in turn has better activity
than the p-methylacetanilide 4a, IC50 values 19.8 and 21.6 µg/mL; cLogP values 4.42 and 3.16,
respectively. This in part might be attributed to the hydrophilicity of the side chain which when
increases, the cytotoxic activity increases.
Looking at the effect of side chain rigidification of compound 6a through the
introduction of an imidazoline ring, it was found that the imidazoline-2,4,5-trione derivative 7
showed marked increase in the growth inhibitory activity, IC50 10.8 µg/ml, furnishing the most
active derivative in this series. On the other hand, the introduction of the imidazoline-2,5-dione
side chain in compound 8 enhanced the activity only slightly, IC50 18.6 µg/mL as compared with
the flexible analog 6a. Again this increase in activity of the imidazoline derivatives might be
ascribed to either the higher lipophilicity of 7 (cLogP 0.86) compared to 8 (cLogP 1.80) or the
presence of an extra oxygen atom in 7 which might contribute to the molecular target
interaction of these compounds.
Concerning the fused tricyclic pyrimido[5,4-e]pyrrolo[1,2-c]pyrimidine derivatives,
substitution on the N atom in the upper pyrimidine ring with a phenyl group as in compound 9
improved the activity giving the most active derivative in this study, IC50 9.6 µg/mL. This
indicates that bulkiness on the pyrimidine ring is well tolerated and in fact increases the activity
possibly due to offering an extra lipophilicity which either contributes to molecular uptake by
the cells or ligand receptor interaction or both.
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Moreover, in this series, oxo compounds 10a and 12a constantly displayed better
inhibitory activity than their thioxo analogs 10b and 12b which indicates that as the
hydrophilicity increases, the activity increases.
On the other hand, the Schiff's bases 11 and 17 showed no significant alteration in
cytotoxic activity when compared with their amine precursors 10a and 16, respectively.
Furthermore, substituting the carbonyl group of 12a for a hydrazine group in compound
14, improved the cytotoxic activity, IC50 values 11 and 14.8 µg/mL, respectively.
Referring to the fused tricyclic pyrimido[4',5':4,5]pyrimido[1,6-a]azepines derivatives, it
has been found that generally the azepine derivatives (16, 17, 18 and 19c) showed slightly
better growth inhibitory activity when compared to their pyrrolo counterparts (10a, 11, 12a
and 19a). This ring size impact on cytotoxic activity calls for further studies to be properly
understood and justified as to whether activity is solely related to lipophilicity or other
parameters are also involved.
Finally, concerning the bioisosteric switch of the upper pyrimidine ring to a thiazine ring
furnishing the pyrrolo[1',2':1,6]pyrimido[4,5-d][1,3]thiazines 19a,b and the 1,3-
thiazino[4',5':4,5]pyrimido[1,6-a]azepine 19c, it was found that the same SAR findings
pertaining to the tricyclic fused pyrimidines and discussed above are still followed by this series.
Thus, the oxo analog 19a had slightly better cytotoxic activity than the thioxo analog 19b and
the azepine analog 19c was more active than its pyrrole counterpart 19a.
2.3. Kinase Inhibition Assay
Four compounds were selected for profiling against the cytoplasmic and receptor
tyrosine kinases c-Src and VEGFR kinases, respectively and the results are shown in Table 2.
These compounds were chosen to represent one bicyclic derivative with a flexible and another
with rigid side chain (compounds 5 and 7, respectively) as well as one pyrimido[5,4-
e]pyrrolo[1,2-c]pyrimidine derivative (9) and one pyrimido[4',5':4,5]pyrimido[1,6-a]azepine
analog (18).
In general, while derivatives 7 and 18 displayed some inhibitory activity against both
enzymes, the tricyclic derivative 9 only inhibited VEGFR and 5 was found inactive against both
tested enzymes.
The profiling data for two of the four selected compounds against c-Src protein kinase
target showed moderate inhibition where compound 18 showed the highest inhibition of c-Src
activity causing an overall 81% catalytic activity reduction compared to control, while
compound 7 showed lower inhibition of c-Src (45%).
Furthermore, the profiling data against VEGFR protein kinase showed weak enzyme
inhibition by compound 7 (25% reduction in catalytic activity) and very weak inhibition by
compounds 9 and 18 (11 and 3%, respectively).
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It can be inferred from these findings that bicyclic pyrimidines with a pyrrolo[1,2-
c]pyrimidine backbone and a rigid side chain represented by compound 7 and tricyclic
pyrimidines with a pyrimido[4',5':4,5]pyrimido[1,6-a]azepine scaffold represented by 18 display
promising potential building blocks for antiproliferative agents with multi-kinase inhibitory
properties. However, bicyclic derivatives with a flexible side chain and tricyclic analogs with a
pyrrolidine ring in place of the azepine do not seem to be such likely candidates. Further studies
to explore and better understand the essential molecular features required for improving the
kinase inhibitory activity of this class of compounds are underway.
2.4.
Docking Study
In an attempt to understand the molecular basis behind the observed kinase inhibitory
activity, compounds 5, 7, 9 and 18 were docked in the active site of both c-Src and VEGFR
kinases obtained from the protein data bank, PDB ID: 3EL7 and 4AG8, respectively using MOE
software version 2008.10. As depicted in Figure 1, it is clear that while the tricyclic derivative 18
displays two hydrogen bond interactions with c-Src through its active pocket amino acid
Met341, the bicyclic compound 7 exhibited only one such interaction. This might be in part a
reason for the difference in observed kinase inhibitory ability of 7 (45%) and 18 (81%).
active site amino acids (data not shown).
On the other hand, docking with VEGFR showed that the relatively higher activity of 7
(25%) as opposed to the other tested compounds is associated with two hydrogen bonds and a
pi-cationic interaction with the amino acids lys868 and Asp1046. This compared with only two
hydrogen bonds in case of 18 can possibly justify the lower percent enzyme inhibition (3%).
3. Conclusion
A series of fused bicyclic and tricyclic pyrimidine derivatives viz pyrrolo[1,2-
c]pyrimidines 4-8, pyrimido[5,4-e]pyrrolo[1,2-c]pyrimidines 9-14, pyrimido[4',5':4,5]-
pyrimido[1,6-a]azepines 16-18, pyrrolo[1',2':1,6]pyrimido[4,5-d][1,3]thiazines 19a,b and 1,3-
thiazino[4',5':4,5]pyrimido[1,6-a]azepine 19c were synthesized. Antiproliferative screening of
the prepared target compounds was performed against MCF-7 breast cancer cell and many
compounds displayed promising activities. Moreover, Kinase inhibitory ability of compounds 5,
7, 9 and 18 against c-Src and VEGFR tyrosine kinases was assessed and they showed good
results. Finally, molecular docking was performed with the tested kinases to simulate and
potentially understand the possible binding interactions underlying the association between
these small molecules and the kinase enzyme ATP binding pocket essential amino acids. In spite
of the lack of direct correlation between the observed antiproliferative activity and the kinase
inhibitory ability against the tested enzymes, it can be inferred that the cytotoxic effects
displayed by these molecules may be attributed to other yet undisclosed mechanism of action.
Studies in this regards are underway.
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It can be inferred from the results obtained in this study that fused tricyclic derivatives
are more active than fused bicyclic analogs. Besides, azepine derivatives showed higher
cytotoxic activity than their pyrrole counterparts. In conclusion, compounds possessing a fused
pyrimidine skeleton seem to be promising prospective antiproliferative agents.
4. Experimental
4.1.
Chemistry
Melting points were determined on Electrothermal Stuart SMP3 digital melting point
apparatus and were uncorrected Elemental microanalyses were performed at the regional
center for Mycology and Biotechnology, Al-Azhar University. Infrared spectra were determined
(KBr) using Shimadzu Infrared spectrometer (IR-435) and FT-IR 1650 (Perkin Elmer), Faculty of
1
Pharmacy, Cairo University. H-NMR spectra were performed in CDCl3 or DMSO-d6 using Joel,
300 MHz NMR spectrometer, Faculty of Science, Cairo University, and 500 MHz NMR
spectrometer, National Research Institute (NRI, Cairo, Egypt). Mass spectra were carried out
using Finnigan SSQ 7000 Gas chromatograph-Mass, at the micro-analytical unit, Faculty of
Science, Cairo University. Compounds 3a, 3b and 15 were prepared according to reported
procedures [19-22].
4.1.1. General procedure for the preparation of 4a,b
To a mixture of the appropriate enaminonitrile 3a,b (10 mmol) and anhydrous
potassium carbonate (2.07 g, 15 mmol) in dry DMF (5 mL), 2-chloro-N-p-tolylacetamide (2.75 g,
0
15 mmol) was added. The reaction mixture was heated at 100 C for 12 h, cooled to R.T. and
poured onto ice- water. The obtained solid was filtered, dried and crystallized from ethanol.
4.1.1.1. 1-Oxo-2-phenyl-3-(N-p-tolylcarbamoylmethyl)amino-1,2,4a,5,6,7-hexahydro-
pyrrolo[1,2-c]pyrimidine-4-carbonitrile 4a
C23H23N5O2 (401.46), yield % 60; m.p. 140-3 C; IR (KBr, cm-1): 3387, 3336 (2 NH), 3028 (CH
0
arom.), 2954 (CH aliph.), 2187 (CN), 1724, 1678 (2 CO), 1597 (C=N). MS, m/z (%): 401 (M+,
1
66.92), 107 (100). H-NMR (DMSO-d6, 300 MHz): δ ppm 1.73-1.89 (m, 4H, C-5, C-6 pyrrolidine
2×CH2), 2.81 (s, 3H, CH3),4.13-4.20 (m, 2H, C-7 pyrrolidine CH2), 4.34 (s, 2H, NH-CH2-CO), 5.28 (t,
1H, J = 7.8 Hz, C-4a pyrrolidine CH), 6.05 (s, 1H, NH exchanged with D2O),7.82-8.26 (m, 9H,
aromatic protons), 10.92 (s, 1H, NH exchanged with D2O). Anal. Calcd. for C23H23N5O2: C, 68.81;
H, 5.77; N, 17.44. Found: C, 68.63; H, 5.58; N, 17.62.
4.1.1.2. 2-Phenyl-3-(N-p-tolylcarbamoylmethyl)amino-1-thioxo-1,2,4a,5,6,7-hexahydro-
pyrrolo[1,2-c]pyrimidine-4-carbonitrile 4b
C23H23N5OS (417.53), yield % 55; m.p. 135-7 C; IR (KBr, cm-1): 3448, 3313 (2 NH), 3032 (CH
0
arom.), 2958 (CH aliph.), 2183 (CN), 1674 (CO), 1593 (C=N), 1250 (CS). MS, m/z (%): 417 (M+,
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23), 63 (100).1H-NMR (DMSO-d6, 300 MHz): δ ppm 1.30-1.67 (m, 4H, C-5, C-6, pyrrolidine
2×CH2), 2.23 (s, 3H, CH3), 3.33-3.99 (m, 2H, C-7, pyrrolidine CH2), 4.44 (s, 2H, NH-CH2-CO), 5.63
(t, 1H, J = 7.8 Hz, C-4a, pyrrolidine CH), 5.92 (s, 1H, NH exchanged with D2O), 7.06-8.20 (m, 9H,
aromatic protons), 8.50 (s, 1H, NH exchanged with D2O). Anal. Calcd. for C23H23N5OS: C, 66.16;
H, 5.55; N, 16.77. Found: C, 66.19; H, 5.67; N, 17.18.
4.1.1.3. 3-(2-Cyano-1-oxoethyl)amino-1-oxo-2-phenyl-1,2,4a,5,6,7-hexahydropyrrolo[1,2-c]-
pyrimidine-4-carbonitrile 5
A mixture of compound 3a (10 mmol) and ethyl cyanoacetate (10 mmol) in glacial acetic acid
(10 mL) was heated under reflux for 8 h the mixture was cooled to R.T. and poured onto ice
water (30 mL). The separated solid was filtered, washed with water and crystallized from
ethanol.
C17H15N5O2 (321.33), yield % 65; m.p. 253-5 0C; IR (KBr, cm-1): 3390 (NH), 3059 (CH arom.), 2978
(CH aliph.), 2221, 2351 (2 CN), 1724, 1681 (2 CO). MS, m/z (%): 321 (M+, 45), 55 (100). 1H-NMR
(CDCl3, 500 MHz): δ ppm 1.81-1.93 (m, 2H, C-6, pyrrolidine CH2), 2.10-2.03 (m, 2H, C-5
pyrrolidine CH2), 3.44 (s, 2H, CH2-CN), 4.31-4.35 (m, 2H, C-7, pyrrolidine CH2), 4.80 (t, 1H, J = 8.5
Hz, C-4a, pyrrolidine CH), 7.18-7.46 (m, 5H, aromatic protons), 7.80 (s, 1H, NH exchanged with
D2O). Anal. Calcd. for C17H15N5O2: C, 63.54; H, 4.71; N, 21.79. Found: C, 63.71; H, 4.79; N, 21.80.
4.1.2. General procedure for the preparation of 6a-d
To a solution of the appropriate enaminonitrile 3a or 3b (1.97 mmol) in dichloromethane (10
mL) and few drops of triethylamine, phenyliso(thio)cyanate (2.37 mmol) was added and the
mixture was refluxed for 12 h. The solvent was removed under reduced pressure, the formed
residue was triturated with ether and the product was crystallized from ethanol affording the
corresponding derivatives 6a-d, respectively.
4.1.2.1. 1-Oxo-3-(3-phenylureido)-1,2,4a,5,6,7-hexahydropyrrolo-[1,2-c]pyrimidine-4-
carbonitrile 6a
C21H19N5O2 (373.41), yield % 69 m.p. 138-140 C; IR (KBr, cm-1): 3433, 3336 (2 NH), 3035 (CH
0
arom.), 2978 (CH aliph.), 2179 (CN), 1695, 1689(2 CO), 1597 (C=N). MS, m/z (%): 373 (M+, 25.8),
1
64 (100). H-NMR (DMSO-d6, 300 MHz): δ ppm 1.19-1.23 (m, 4H, C-5, C-6, pyrrolidine 2×CH2),
4.50-4.57 (m, 2H, C-7, pyrrolidine CH2), 5.33 (t, 1H, J = 7.8 Hz, C-4a, pyrrolidine CH), 6.03 (s, 1H,
NH exchanged with D2O), 7.00-7.80 (m, 10H, aromatic protons), 8.70 (s, 1H, NH exchanged with
D2O). Anal. Calcd. for C21H19N5O2: C, 67.55; H, 5.13; N, 18.76. Found: C, 67.63; H, 5.18; N, 18.92.
4.1.2.2. 3-(3-Phenylureido)-1-thioxo-1,2,4a,5,6,7-hexahydropyrrolo[1,2-c]pyrimidine-4-
carbonitrile 6b
C21H19N5OS (389.47), yield % 67; m.p. 120-2 C; IR (KBr, cm-1): 3471, 3390 (2 NH), 3035 (CH
0
arom.), 2985 (CH aliph.), 1697 (CO), 1593 (C=N), 1243 (CS). MS, m/z (%): 389 (M+, 25.58), 59
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1
(100). H-NMR (DMSO-d6, 300 MHz): δ ppm 1.31-1.42 (m, 2H,C-6, pyrrolidine CH2), 1.90-
2.13(m, 2H, C-5, pyrrolidine CH2), 3.21-3.44 (m, 2H, C-7, pyrrolidine CH2), 4.55 (t, 1H, J = 7.8 Hz,
C-4a, pyrrolidine CH), 4.94 (s, 1H, NH exchanged with D2O), 7.21-7.49 (m, 10H, aromatic
protons), 7.90 (s, 1H, NH exchanged with D2O). Anal. Calcd. for C21H19N5OS: C, 64.76; H, 4.92; N,
17.98. Found: C, 64.72; H, 4.94; N, 18.12.
4.1.2.3. 1-Oxo-3-(3-phenylthioureido)-1,2,4a,5,6,7-hexahydropyrrolo[1,2-c]pyrimidine-4-
carbonitrile 6c
0
C21H19N5OS: (389.47), yield % 70; m.p. 140-3 C; IR (KBr, cm-1): 3429, 3294 (2 NH), 3055 (CH
arom.), 2974 (CH aliph.), 2179 (CN), 1681 (CO), 1585 (C=N), 1262 (CS). MS, m/z (%): 389 (M+,
0.31), 64 (100).1H-NMR (DMSO-d6, 300 MHz): δ ppm 1.21-1.35 (m, 2H, C-6, pyrrolidine CH2),
1.81-2.00 (m, 2H, C-5, pyrrolidine CH2), 3.44-3.87 (m, 2H, C-7, pyrrolidine CH2), 4.34 (t, 1H, J =
7.8 Hz, C-4a, pyrrolidine CH), 5.14 (s, 1H, NH exchanged with D2O), 7.22-7.49 (m, 10H, aromatic
protons), 8.05 (s, 1H, NH exchanged with D2O). Anal. Calcd.for C21H19N5OS: C, 64.76; H, 4.92; N,
17.98. Found: C, 64.81; H, 4.88; N, 18.13.
4.1.2.4.
3-Phenylthioureido-1-thioxo-1,2,4a,5,6,7-hexahydropyrrolo[1,2-c]pyrimidine-4-
carbonitrile 6d
0
C21H19N5S2 (405.54), yield % 65; m.p. 130-2 C; IR (KBr, cm-1): IR (KBr, cm-1): 3438, 3309 (2 NH),
3046 (CH arom.), 2931 (CH aliph.), 2187 (CN), 1593 (C=N), 1287 (CS). MS, m/z (%): 405 (M+, 25).
1H-NMR (DMSO-d6, 300 MHz): δ ppm 1.24-1.41 (m, 2H, C-6, pyrrolidine CH2), 2.01-2.05 (m, 2H,
C-5, pyrrolidine CH2), 3.07-3.11 (m, 2H, C-7, pyrrolidine CH2), 4.26 (t, 1H, J = 7.8 Hz, C-4a,
pyrrolidine CH), 5.49 (s, 1H, NH exchanged with D2O), 7.25-7.51 (m, 10H, aromatic protons),
8.01 (s, 1H, NH exchanged with D2O). Anal. Calcd. for C21H19N5S2: C, 62.19; H, 4.72; N, 17.27.
Found: C, 62.38; H, 4.96; N, 17.31.
4.1.3. 1-Oxo-2-phenyl-3-(2,4,5-trioxo-3-phenylimidazolidin-1-yl)-1,2,4a,5,6,7-hexahydro-
pyrrolo[1,2-c]pyrimidine-4-carbonitrile 7
To a solution of oxalyl chloride (0.63 g, 5 mmol) in dichloromethane (20 mL), few drops of
triethylamine and compound 6a (1.87 g, 5 mmol) were added and the reaction mixture was
refluxed for 1 h. The solvent was removed under reduced pressure; the obtained precipitate
was washed 3 times with water and the remaining precipitate was filtered, collected and
recrystallized from aqueous ethanol.
C23H17N5O4 (427.41), yield % 75; m.p. 150-3 0C; IR (KBr, cm-1): 3062 (CH arom.), 2981 (CH aliph.),
1
2214 (CN), 1743, 1705, 1647 (4 CO). MS, m/z (%): 427 (M+, 35), 77 (100). H-NMR (CDCl3, 500
MHz): δ ppm 1.21-1.85 (m, 4H, C-5, C-6, pyrrolidine 2×CH2), 3.11-3.36 (m, 2H, C-7, pyrrolidine
CH2), 4.45 (t, 1H, J = 7.8 Hz, C-4a, pyrrolidine CH),7.25-7.54 (m, 10H, aromatic protons). Anal.
Calcd. for C23H17N5O4: C, 64.63; H, 4.01; N, 16.39. Found: C, 64.72; H, 4.08; N, 16.52.
9

ACCEPTED MANUSCRIPT
4.1.4. 1-Oxo-2-phenyl-3-(2,5-dioxo-3-phenylimidazolidin-1-yl)-1,2,4a,5,6,7-hexahydro-
pyrrolo[1,2-c]pyrimidine-4-carbonitrile 8
To a solution of compound 6a (1.87 g, 5 mmol) in dichloromethane (20 mL), chloroacetyl
chloride (0.85 g, 7.5 mmol) was added dropwise and the reaction mixture was refluxed for 3 h.
The solvent was removed under reduced pressure and the obtained crystals were recrystallized
from aqueous ethanol.
C23H19N5O3 (413.43), yield % 65; m.p. 253-5 0C; IR (KBr, cm-1): 3015 (CH arom.), 2974 (CH aliph.),
2214 (CN), 1724, 1678 (3 CO). MS, m/z (%): 413 (M+, 29), 63 (100). 1H-NMR (CDCl3, 500 MHz): δ
ppm 1.20-1.61 (m, 4H, C-5, C-6, pyrrolidine 2×CH2), 3.01-3.11 (m, 2H, C-7, pyrrolidine CH2), 4.24
(t, 1H, J = 9.00 Hz, C-4a, pyrrolidine CH), 4.25 (s, 2H,N-CH2-C=O),7.25-8.00 (m, 10H, aromatic
protons). Anal. Calcd. for C23H19N5O3: C, 66.82; H, 4.63; N, 16.94. Found: C, 66.91; H, 4.67; N,
17.08.
4.1.5. 1-Imino-2,5-diphenyl-1,5,8,9,10,10a-hexahydropyrimido[5,4-e]pyrrolo[1,2-c]pyrimidine-
3,6(2H,4H)-dione 9
Compound 6a (3.73 g, 10 mmol) was refluxed in freshly prepared sodium ethoxide solution (0.5
g sodium metal in 15 mL absolute ethanol) for 2 h. The mixture was cooled and poured onto
ice-water; the obtained precipitate was filtered and recrystallized from aqueous ethanol.
C21H19N5O2 (373.41), yield % 60; m.p. 220-2 0C; IR (KBr, cm-1): 3313 (NH), 3035 (CH arom.), 2920
(CH aliph.), 1681 (2 CO). MS, m/z (%): 373 (M+, 20), 119 (100). 1H-NMR (CDCl3, 500 MHz): δ ppm
1.24-1.91 (m, 4H, C-9, C-10, pyrrolidine 2×CH2), 4.55-4.87 (m, 2H, C-8, pyrrolidine CH2), 5.35 (t,
1H, J = 10 Hz, C-10a, pyrrolidine CH), 7.00-7.5 (m, 10H, aromatic protons), 7.08 (s, 1H, NH
exchanged with D2O), 7.71 (s, 1H, NH exchanged with D2O). Anal. Calcd. for C21H19N5O2: C,
64.55; H, 5.13; N, 18.76. Found: C, 64.52; H, 5.18; N, 18.94.
4.1.6. General procedure for preparation of 10a,b and 16
Compound 3a or 3b (5 mmol) and thiourea (0.76 g, 10 mmol) were mixed in a mortar and fused
0
in oil bath at 180 C for 30 min. The molten product was boiled for 10 minutes with hot water,
the mixture was allowed to cool to R.T. and the product was solidified by cooling and addition
of methanol (15 mL). The crystals were collected by filtration and recrystallized from aqueous
ethanol.
4.1.6.1. 1-Amino-5-phenyl-3-thioxo-3,5,8,9,10,10a-hexahydropyrimido[5,4-e]pyrrolo[1,2-c]-
pyrimidin-6(4H)-one 10a
C15H15N5OS (313.38), yield % 70; m.p. 230-2 C; IR (KBr, cm-1): 3321-3190 (NH, NH2), 3035 (CH
0
arom.), 2939 (CH aliph.), 1687 (CO), 1243 (CS). MS, m/z (%): 313 (M+, 11.45), 54 (100). 1H-NMR
(DMSO-d6, 300 MHz): δ ppm 1.02-1.07 (m, 2H, C-9, pyrrolidine CH2), 1.41-1.72 (m, 2H, C-10,
pyrrolidine CH2), 4.11-4.31 (m, 2H, C-8, pyrrolidine CH2), 4.05 (t, 1H, J = 7.8 Hz, C-10a,
10

ACCEPTED MANUSCRIPT
pyrrolidine CH), 6.44 (s, 1H, NH exchanged with D2O), 7.11-7.52 (m, 5H, aromatic protons), 8.23
(s, 2H, NH2 exchanged with D2O). Anal. Calcd. for C15H15N5OS: C, 57.49; H, 4.82; N, 22.35. Found:
C, 57.58; H, 4.91; N, 22.52.
4.1.6.2. 1-Amino-5-phenyl-8,9,10,10a-tetrahydropyrimido[5,4-e]-pyrrolo[1,2-c]pyrimidine-
3,6(4H,5H)-dithione 10b
C15H15N5S2 (329.08), yield % 52; m.p. 222-4 C; IR (KBr, cm-1): 3313-3186 (NH, NH2), 2935 (CH
aliph), 1543 (C=N), 1234 (CS). MS, m/z (%): 329 (M+, 14.3), 54 (100). H-NMR (DMSO-d6, 300
0
1
MHz): δ ppm 1.12-1.81 (m, 4H, C-9, C-10, pyrrolidine 2×CH2), 3.90-4.11 (m, 2H, C-8, pyrrolidine
CH2), 5.06 (t, 1H, J = 7.8 Hz, C-11a, pyrrolidine CH), 5.30 (s, 1H, NH exchanged with D2O), 7.28-
7.60 (m, 5H, aromatic protons), 7.93 (s, 2H, NH2 exchanged with D2O). Anal. Calcd. for
C15H15N5S2: C, 54.69; H, 4.59; N, 21.26. Found: C, 54.74; H, 4.62; N, 21.39.
4.1.6.3. 1-Amino-5-phenyl-3-thioxo-3,5,8,9,10,11,12,12a-octahydropyrimido[4',5':4,5]-
pyrimido[1,6-a]azepin-6(4H)-one 16
C17H19N5OS (341.43), yield % 56; m.p. 240-3 C; IR (KBr, cm-1): 3178-3305 (NH, NH2), 3062 (CH
0
arom.), 2854 (CH aliph.), 1670 (CO), 1593 (C=N), 1238 (CS). MS, m/z (%): 341 (M+, 15), 79 (100).
1H-NMR (DMSO-d6, 300 MHz): δ ppm 1.21-1.53 (m, 4H, C-10, C-11, azepine 2×CH2), 1.78-1.88
(m, 2H, C-9, azepine CH2), 2.07-2.26 (m, 2H, C-12, azepine CH2), 3.22-3.41 (m, 2H, C-8, azepine
CH2), 4.43 (t, 1H, J = 7.8 Hz, C-12a, azepine CH), 5.51 (s, 1H, NH exchanged with D2O), 7.07-7.36
(m, 5H, aromatic protons), 7.60 (s, 2H, NH2 exchanged with D2O). Anal. Calcd. for C17H19N5OS: C,
59.80; H, 5.61; N, 20.51. Found: C, 59.64; H, 5.24; N, 20.22.
4.1.7. General procedure for the preparation of 11 and 17
To a solution of compound 10a or 16 (10 mmol) and p-chlorobenzaldehyde (1.41 g, 10 mmol) in
absolute ethanol, drops of glacial acetic acid were added. The reaction mixture was refluxed for
18 h, a precipitate started forming that was filtered out while hot. The obtained solid was then
crystallized from aqueous ethanol.
4.1.7.1. 1-[(E)-(4-Chlorobenzylidene)amino]-5-phenyl-3-thioxo-3,5,8,9,10,10a-hexahydro-
pyrimido[5,4-e]pyrrolo[1,2-c]pyrimidin-6(4H)-one 11
0
C22H18ClN5OS (435.93), yield % 48; m.p. 225-7 C; IR (KBr, cm-1): 3325 (NH), 3066 (CH arom.),
2958 (CH aliph.), 1543 (C=N), 1296 (CS). MS, m/z (%):437 (M++2, 11), 435 (M+, 35), 412 (100).
1H-NMR (DMSO-d6, 300 MHz): δ ppm 1.24-1.43 (m, 2H, C-9, pyrrolidine CH2), 1.91-2.10 (m, 2H,
C-10, pyrrolidine CH2), 3.75-3.79 (m, 2H, C-8, pyrrolidine CH2), 4.04 (t, 1H, J = 7.8 Hz, C-10a,
pyrrolidine CH), 5.07 (s, 1H, NH exchanged with D2O), 6.30 (s, 1H, CH=N), 7.13-8.22 (m, 9H,
aromatic protons). Anal. Calcd. for C22H18ClN5OS: C, 60.61; H, 4.16; N, 16.07. Found: C, 60.74; H,
4.23; N, 16.36.
11

ACCEPTED MANUSCRIPT
4.1.7.2. 1-[(E)-(4-Chlorobenzylidene)amino]-5-phenyl-3-thioxo-3,5,8,9,10,11,12,12a-
octahydropyrimido[4',5':4,5]pyrimido[1,6-a]azepin-6(4H)-one 17
0
C24H22ClN5OS (463.98), yield % 42; m.p. 165-7 C; IR (KBr, cm-1): 3313 (NH), 3059 (CH arom.),
2964 (CH aliph.), 1701 (CO), 1573 (C=N), 1242 (CS). MS, m/z (%): 465 (M++2, 46), 463 (M+, 15).
1H-NMR (DMSO-d6, 300 MHz): δ ppm 1.21-1.33 (m, 4H, C-10, C-11, azepine 2×CH2), 1.72-1.81
(m, 2H, C-9, azepine CH2), 2.00-2.13 (m, 2H, C-12, azepine CH2), 3.75-3.79 (m, 2H, C-8, azepine
CH2), 4.32 (t, 1H, J = 7.8 Hz, C-12a, azepine CH), 5.53 (s, 1H, NH exchanged with D2O), 5.89 (s,
1H, CH=N), 7.07-7.46 (m, 9H, aromatic protons). Anal. Calcd. for C24H22ClN5OS: C, 62.13; H, 4.78;
N, 15.09. Found: C, 62.41; H, 4.58; N, 14.87.
4.1.8. General procedure for preparation of 12a,b and 18
A solution of compound 3a or 3b (5 mmol) in formic acid (15 mL, 85 %) was refluxed for 5 h; the
reaction mixture was cooled and then poured onto ice-water. The obtained solid was washed
with water and crystallized from aqueous ethanol to afford compounds 12a,b.
4.1.8.1. 5-Phenyl-8,9,10,10a-tetrahydropyrimido[5,4-e]pyrrolo[1,2-c]pyrimidine-1,6(2H,5H)-
dione 12a
C15H14N4O2 (282.3), yield % 85; m.p. 255-6 C; IR (KBr, cm-1): 3390 (NH), 3059 (CH arom.), 2978
0
(CH aliph.), 1724, 1678 (2 CO), 1593 (C=N). MS, m/z (%): 282 (M+, 13.89), 254 (100). H-NMR
1
(DMSO-d6, 300 MHz): δ ppm 1.83-1.89 (m, 2H, C-9, pyrrolidine CH2), 1.94-2.05 (m, 2H, C-10,
pyrrolidine CH2), 4.27-4.31 (m, 2H, C-8, pyrrolidine CH2), 4.87 (t, 1H, J = 7.8 Hz, C-10a,
pyrrolidine CH), 5.69 (s, 1H, CH=N), 6.90-7.45 (m, 5H, aromatic protons), 7.86-8.32 (s, 1H, NH
exchanged with D2O). Anal. Calcd. for C15H14N4O2: C, 63.82; H, 5.00; N, 19.85. Found: C, 63.93;
H, 5.12; N, 20.08.
4.1.8.2. 5-Phenyl-6-thioxo-5,6,8,9,10,10a-hexahydropyrimido[5,4-e]pyrrolo[1,2-c]pyrimidin-
1(2H)-one 12b
C15H14N4OS (298.36), yield % 74; m.p. 165-7 0C; IR (KBr, cm-1): 3383 (NH), 3059 (CH arom.), 2974
1
(CH aliph.), 1712 (CO), 1593 (C=N), 1254 (CS). MS, m/z (%): 298 (M+, 1.53), 63 (100). H-NMR
(CDCl3, 500 MHz): δ ppm 1.57-1.88 (m, 2H, C-9, pyrrolidine CH2), 1.91-1.99 (m, 2H, C-10,
pyrrolidine CH2), 4.36 (t, 2H, C-8, pyrrolidine CH2), 5.03 (t, 1H, J = 10 Hz, C-10a, pyrrolidine CH),
6.32 (s, 1H, CH=N), 7.08-7.41 (m, 5H, aromatic protons), 7.63 (s, 1H, NH exchanged with D2O).
Anal. Calcd. for C15H14N4OS: C, 60.38; H, 4.73; N, 18.78. Found: C, 60.42; H, 4.81; N, 18.97.
4.1.8.3. 5-Phenyl-8,9,10,11,12,12a-hexahydropyrimido[4',5':4,5]pyrimido[1,6-a]azepine-
1,6(2H,5H)-dione 18
C17H18N4O2 (310.35), yield % 70; m.p. 220-2 0C; IR (KBr, cm-1): 3394 (NH), 3075 (CH arom.), 2935
(CH aliph.), 1693, 1681 (2 CO), 1597 (C=N). MS, m/z (%): 310 (M+, 20), 79 (100). 1H-NMR (CDCl3,
12

ACCEPTED MANUSCRIPT
500 MHz): δ ppm1.24-1.35 (m, 4H, C-10, C-11, azepine 2×CH2), 1.63-1.78 (m, 2H, C-9, azepine
CH2), 2.11-2.20 (m, 2H, C-12, azepine CH2), 3.21-3.33 (m, 2H, C-8,azepine CH2), 4.55 (t, 1H, J =
10.8 Hz, C-12a, azepine CH), 5.65 (s, 1H, CH=N), 7.35-7.62 (m, 5H, aromatic protons), 7.70 (s,
1H, NH exchanged with D2O). Anal. Calcd. for C17H18N4O2: C, 65.79; H, 5.85; N,18.05. Found: C,
65.83; H, 5.92; N, 18.23.
4.1.9. 1-Chloro-5-phenyl-8,9,10,10a-tetrahydropyrimido[5,4-e]pyrrolo[1,2-c]pyrimidine-6(5H)-
one 13
A mixture of 3a (2.8 mmol) and phosphorus pentachloride (0.3 g) in phosphorus oxychloride (5
mL) was refluxed for 4 h. The mixture was cooled to R.T. and poured onto crushed ice portion
wise with continuous stirring. After that the mixture was treated with 10% NaOH solution (0.5 g
NaOH in 50 mL H2O) with stirring and scratching till a precipitate started forming. The obtained
solid was filtered and crystallized from ethanol.
0
C15H13N4OCl (300.74), yield % 65; m.p. above 300 C; IR (KBr, cm-1): 3059 (CH arom.), 2877 (CH
1
aliph.), 1681 (CO). MS, m/z (%): 302 (M++2, 36), 300 (M+, 12), 54 (100). H-NMR (CDCl3, 500
MHz): δ ppm1.83-1.99 (m, 2H, C-9, pyrrolidine CH2), 2.05-2.3 (m, 2H, C-10, pyrrolidine CH2),
4.29-4.34 (m, 2H, C-8, pyrrolidine CH2), 4.86 (t, 1H, J = 10.8 Hz, C-10a, pyrrolidine CH), 5.77 (s,
1H, CH=N), 7.21-7.45 (m, 5H, aromatic protons). Anal. Calcd. for C15H13N4OCl: C, 59.91; H, 4.36;
N, 18.63. Found: C, 60.02; H, 4.39; N, 18.87.
4.1.10. 1-Hydrazino-5-phenyl-8,9,10,10a-tetrahydropyrimido[5,4-e]pyrrolo[1,2-c]pyrimidine-
6(5H)-one 14
A mixture of 13 (5 mmol) and excess hydrazine hydrate (30 mL, 98 %) was refluxed in absolute
ethanol for 4 h. The mixture was cooled to R.T., poured onto ice-water, and then the compound
was extracted with CHCl3 (3 × 10 mL). The solvent was removed under reduced pressure and
the separated crystals were recrystallized from ethanol to give 14.
0
C15H16N4O (296.33), yield % 30; m.p. above 300 C; IR (KBr, cm-1): 3201- 3332 (NH2, NH), 3059
1
(CH arom.), 2927 (CH aliph.), 1681 (CO). MS, m/z (%): 296 (M+, 28), 54 (100). H-NMR (CDCl3,
500 MHz): δ ppm 1.24-1.44 (m, 2H, C-9, pyrrolidine CH2), 1.91-2.17 (m, 2H, C-10, pyrrolidine
CH2), 3.75-3.79 (m, 2H, C-8, pyrrolidine CH2), 4.11 (t, 1H, J = 10.8 Hz, C-10a, pyrrolidine CH), 5.75
(s, 1H, CH=N), 6.77 (s, 2H, NH2 exchanged with D2O), 7.22-7.47 (m, 5H, aromatic protons), 8.09
(s, 1H, NH exchanged with D2O). Anal. Calcd. for C15H16N4O: C, 60.80; H, 5.44; N, 28.36. Found:
C, 60.86; H, 5.48; N, 28.42.
4.1.11. General procedure for the preparation of 19a-c
To a mixture of 3a,b and 15 (10 mmol) and ethanolic solution of 1M KOH (15 mL), carbon
disulphide (15 mmol) was added. The mixture was refluxed for 16h, cooled to R.T., poured onto
13

ACCEPTED MANUSCRIPT
ice-water, acidified with hydrochloric acid (35%) and left overnight. The obtained precipitate
was filtered off, left to dry and crystallized from aqueous ethanol.
4.1.11.1.
5-Phenyl-3-thioxo-3,4,8,9,10,10a-hexahydro-1H-pyrrolo[1',2':1,6]pyrimido[4,5-
d][1,3]thiazine-1,6(5H)-dione 19a
0
C15H13N3O2S2 (331.41), yield % 80; m.p. 235-6 C; IR (KBr, cm-1): 3390 (NH), 3059 (CH arom.),
2978 (CH aliph.), 1724, 1681 (2 CO), 1199 (CS). MS, m/z (%): 331 (M+, 24.39), 330 (100). 1H-NMR
(DMSO-d6, 300 MHz): δ ppm 1.19-1.21 (m, 2H, C-9, pyrrolidine CH2), 1.65-1.73 (m, 2H, C-10,
pyrrolidine CH2), 4.10-4.32 (m, 2H, C-8, pyrrolidine CH2), 5.32 (t, 1H, J = 7.8 Hz, C-10a,
pyrrolidine CH), 5.61 (s, 1H, NH exchanged with D2O), 6.91-7.42 (m, 5H, aromatic protons).
Anal. Calcd. for C15H13N3O2S2: C, 54.36; H, 3.95; N, 12.68. Found: C, 54.43; H, 3.89; N, 12.87.
4.1.11.2.
5-Phenyl-3,6-dithioxo-3,4,5,6,8,9,10,10a-octahydro-1H-pyrrolo[1',2':1,6]-
pyrimido[4,5-d][1,3]thiazin-1-one 19b
0
C15H13N3OS3 (347.48), yield % 62; m.p. 140-2 C; IR (KBr, cm-1): 3363 (NH), 3051 (CH arom.),
2966 (CH aliph.), 1701 (CO), 1239 (CS). MS, m/z (%): 347 (M+, 32), 76 (100): 1H-NMR (DMSO-d6,
300 MHz): δ ppm 1.40-1.91 (m, 4H, C-9, C-10, pyrrolidine 2×CH2), 4.32 (s, 1H, NH exchanged
with D2O),4.55-4.67 (m, 2H, C-8, pyrrolidine CH2), 5.43 (t, 1H, J = 7.8 Hz, C-10a, pyrrolidine CH),
7.12-7.44 (m, 5H, aromatic protons). Anal. Calcd. for C15H13N3OS3: C, 51.85; H, 3.77; N, 12.09.
Found: C, 51.92; H, 3.83; N, 12.24.
4.1.11.3.
5-Phenyl-3-thioxo-3,4,8,9,10,11,12,12a-octahydro-1H-[1,3]-thiazino[4',5':4,5]-
pyrimido[1,6-a]azepine-1,6(5H)-dione 19c
C17H17N3O2S2 (359.47), yield % 54; m.p. 210-2 C; IR (KBr, cm-1): 3331 (NH), 3035 (CH arom.),
2956 (CH aliph.), 1720, 1670 (2 CO), 1239 (CS). MS, m/z (%): 359 (M+, 15), 93 (100): H-NMR
0
1
(DMSO-d6, 300 MHz): δ ppm 1.21-1.42 (m, 4H, C-10, C-11, azepine 2×CH2), 1.70-1.89 (m, 2H, C-
9, azepine CH2), 2.11-2.41 (m, 2H, C-12, azepine CH2), 3.07-3.47 (m, 2H, C-8, azepine CH2), 4.34
(t, 1H, J = 7.8 Hz, C-12a, azepine CH), 4.41 (s, 1H, NH exchanged with D2O),7.36-8.26 (m, 5H,
aromatic protons). Anal. Calcd. for C17H17N3O2S2: C, 56.80; H, 4.77; N, 11.69. Found: C, 56.89; H,
4.78; N, 11.87.
4.2.
In vitro Anticancer Screening
The antiproliferative activity of all the synthesized compounds was assessed in vitro
using the Sulfo-Rhodamine-B-stain (SRB) assay method as described by SKehan et al. [18] The
SRB assay, which was developed in 1990, is one of the most widely used methods where it
relies on the ability of SRB to bind to protein components of the cells that have been fixed to
14

ACCEPTED MANUSCRIPT
tissue-culture plates by trichloroacetic acid (TCA). As the binding of SRB is stoichiometric, the
amount of dye extracted from stained cells is directly proportional to the cell mass.
Cells were plated in 96-microwell microtiter plate (104 cells/well) for 24 h before
treatment with the test compounds to allow attachment of cells to the wall of the plate. Test
compounds were dissolved in DMSO and diluted with saline to the appropriate volume.
Different concentrations of the compound under test (0, 5, 12.5, 25, 50 µg/ml) were added to
the cell monolayer. Triplicate wells were prepared for each individual dose. Monolayer cells
were incubated with the tested compounds for 48 h at 37oC and in atmosphere of 5% CO2.
After 48 h, cells were fixed, washed, and stained for 30 min with 0.4 % (wt/vol) SRB dissolved in
1% acetic acid. Excess unbound dye was removed by four washes with 1 % acetic acid and
attached stain was recovered with Tris-EDTA buffer. Color intensity was measured in an ELISA
reader at a wavelength of 570 nm. The relation between surviving fraction and drug
concentration is plotted to get the survival curve for breast tumor cell line after the specified
time from which the molar concentration required for 50 % inhibition of cell viability (IC50) was
calculated.
4.3.
Kinase Assay
To determine whether any of the selected compounds can affect the activity of the
selected protein kinase targets, profiling of compounds 5, 7, 9 and 18 against c-Src kinase and
VEGFR receptor tyrosine kinase targets was performed. The kinase assay was carried out at a
single concentration (10 μM) in duplicate and the data enlisted in Table 2 is the average of both
experiments.
c-Src and VEGFR were cloned, expressed and purified. The selected compounds 5, 7, 9
and 18 were used to make stock solutions which were then diluted to form the assay solutions
used for profiling against the selected kinases.
A radioisotope assay format was used for profiling evaluation of the kinase targets and
all assays were performed in a designated radioactive working area. Protein kinase assays were
performed at ambient temperature for 20-30 minutes in a final volume of 25 μl according to
the following assay reaction recipe:
Component 1: 5 μl of diluted active protein kinase target (~10-50 nM final protein
concentration in the assay)
Component 2: 5 μl of stock solution of substrate (1-5 μg of peptide or protein substrate)
Component 3: 5μl of kinase assay buffer or protein kinase activator in kinase assay buffer
Component 4: 5 μl of compound (250 μM) or 10% DMSO
Component 5: 5 μl of 33P-ATP (250 M stock solution, 0.8 Ci)
The assay was initiated by the addition of 33P-ATP and the reaction mixture incubated at
ambient temperature for 30 minutes. After the incubation period, the assay was terminated by
spotting 10 μl of the reaction mixture onto Multiscreen phosphocellulose P81 plate. The plate
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was washed 3 times for approximately 15 minutes each in a 1% phosphoric acid solution. The
radioactivity on the P81 plate was counted in the presence of scintillation fluid in a Trilux
scintillation counter.
Blank control was set up for each protein kinase target which included all the assay
components except the addition of the appropriate substrate (replaced with equal volume of
assay dilution buffer). The corrected activity for each kinase target was determined by
removing the blank control value.
The results for profiling 5, 7, 9 and 18 against c-Src and VEGFR protein kinase targets are
displayed as % inhibition compared to control and presented in Table 2.
4.4. Docking study
Docking was performed using MOE version 2008.10. Compounds 7 and 18 were docked
in the active site of c-Src obtained from the protein databank as a co-crystal structure with a
pyrazolopyrimidine native ligand (PDB ID: 3EL7). On the other hand, 7 and 9 were docked in the
active site of VEGFR obtained from the protein databank as a co-crystal structure with the
VEGFR inhibitor axitinib (PDB ID: 4AG8).
Prior to performing the docking, protein-ligand complexes obtained from the protein
databank were subjected 3D protonated; partial charges were calculated using Amber99
forcefield; water of crystallization was deleted; the active sites were isolated; the amino acids
involved were recognized; and ligand interactions with the active site amino acids were studied.
Validation was performed by re-docking of the native inhibitors and rmsd values below one
were obtained for both enzymes (data not shown).
The 3D structures of the fused pyrimidines 7, 9 and 18 were built using MOE builder and
structures were 3D protonated; subjected to conformation analysis using systematic search and
the least energetic conformers were selected and used for molecular docking.
Docking was performed using Alpha triangle placement method and default parameters
were not changed. Poses were prioritized based on affinity London dG scoring method and
those with the best affinity were used for study of ligand-receptor interactions.
References
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441-455.
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List of Captions
1- Scheme 1: Synthesis of pyrrolo[1,2-c]pyrimidines with flexible and semi-flexible side
chains 4-8.
2- Scheme 2: Synthesis of pyrimido[5,4-e]pyrrolo[1,2-c]pyrimidines 9-14.
3- Scheme 3: Synthesis of pyrimido[4',5':4,5]pyrimido[1,6-a]azepines 16-18.
4- Scheme 4: Synthesis of heterocyclic-fused pyrimidothiazines 19.
5- Table 1. Antiproliferative activity of the synthesized fused pyrimidine derivatives against
MCF-7 cell line
6- Table 2. Enzyme inhibition of c-Src and VEGFR by selected fused pyrimidine derivatives
at 10 μM.
7- Figure 1: 2D (left panel) and 3D (right panel) overlay of derivatives 7 (magenta) and 18
(green) in the active pocket of c-Src kinase.
8- Figure 2: 2D overlay of derivatives 7 (magenta) and 18 (green) in the active pocket of
VEGFR kinase.
18

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Scheme 1: Synthesis of pyrrolo[1,2-c]pyrimidines with flexible and semi-flexible side chains 4-8.
CH3
CN
CN
CN
HN
H
5
4
3
NH2
NH
N
c
b
CN
CN
a
6
O
2
N
N
N
N
N
N
N
H
7
1
X
3a,b
3a X=O
3b X=S
X
X
4a,b
4a X=O
4b X=S
1
2a,b
2a X=O
2b X=S
d
O
CN
CN
e
NH
N
N
O
5
O
HN
O
N
N
O
CN
CN
CN
Y
f
O
g
N
N
NH
O
N
N
N
N
N
N
O
X
O
6a-d
6a X=Y=O
8
7
6b X=S, Y=O
6c X=O, Y=S
6d X=S, Y=S
Reagents and solvents: a C6H5-NCX, Et3N, CH2Cl2; b NaBH4, absolute ethanol; c 2-Chloro-N-p-
tolylacetamide, DMF, K2CO3; d ethyl cyanoacetate, acetic acid; e C6H5-NCY, Et3N, CH2Cl2; f (CO)2Cl2,
Et3N, CH2Cl2; g ClCH2COCl, CH2Cl2.
19

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Scheme 2: Synthesis of pyrimido[5,4-e]pyrrolo[1,2-c]pyrimidines 9-14.
2
3
HN
O
N
N
a
b
6a
1
3a,b
10
NH
4
5
9
N
7
8
6
O
9
HC N
N
N
N
S
S
N
H2N
N
c
d
NH
NH
Cl
10a
N
O
X
10a,b
11
10a X=O
10b X=S
H
N
O
N
Cl
N
N
H2NHN
N
N
N
e
g
N
f
N
N
N
12a
N
X
O
O
12a,b
12a X=O
12b X=S
13
14
Reagents and solvents: a C6H5-NCO, Et3N, CH2Cl2; b NaOC2H5; c NH2CSNH2; d p-chloro
benzaldehyde, ethanol; e HCOOH; f POCl3, PCl5; g NH2NH2.H2O, absolute ethanol
20

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Scheme 3: Synthesis of pyrimido[4',5':4,5]pyrimido[1,6-a]azepines 16-18.
S
H2N
N
N
N
HC N
N
N
S
NH
b
NH
a
Cl
N
CN
O
O
NH2
16
17
N
N
H
O
N
O
15
N
c
N
N
O
18
Reagents and solvents: a NH2CSNH2; b p-chloro benzaldehyde, ethanol; c HCOOH
Scheme 4: Synthesis of heterocyclic-fused pyrimidothiazines 19.
S
N
S
O
NH
CS2, KOH, ethanol
3a,b or 15
N
n
X
19a-c
19a n=1, X=O
19b n-1, X=S
19c n=2, X=O
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Table 1. Antiproliferative activity of the synthesized fused pyrimidine derivatives against
MCF-7 cell line.
Compound
cLog P
IC50*
Compound
no.
cLog P
IC50*
no.
4a
4b
5
µg/mL
µg/mL
3.16
3.76
0.61
4.42
4.51
5.03
5.11
0.86
1.80
1.62
0.57
----
21.6±0.8
12.8±0.2
12.8±0.2
19.8±0.3
14.2±0.3
16.6±0.4
20.8±0.6
10.8±0.3
18.6±0.2
9.6±0.2
10b
11
1.17
3.93
-0.13
0.48
0.92
1.70
5.06
1.00
1.63
2.23
2.75
22.8±0.2
12.4±0.2
14.8±0.3
20±0.5
12a
12b
14
6a
6b
6c
6d
7
11±0.4
16
10.8±0.2
10.6±0.2
11.2±0.3
17±0.4
17
18
8
19a
19b
19c
9
18.8±0.3
14±0.2
10a
Dox
13.6±0.4
14.1±0.3
* Values represent the mean of three experiments ± SD.
Table 2. Enzyme inhibition of c-Src and VEGFR by selected fused pyrimidine derivatives at 10
μM.
% Activity Inhibition*
Compound no
c-Src
0
VEGFR
5
7
0
25
11
3
45
0
9
18
81
* Values represent the mean of two experiments.
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Figure 1: 2D (left panel) and 3D (right panel) overlay of derivatives 7 (magenta) and 18 (green) in the
active pocket of c-Src kinase.
Figure 2: 2D overlay of derivatives 7 (magenta) and 18 (green) in the active pocket of VEGFR kinase.
23

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Highlights
ꢀ Design and synthesis of novel heterocyclic-fused pyrimidines.
ꢀ Antiproliferative activity of novel heterocyclic-pyrimidines against MCF7.
ꢀ Evaluation of VEGFR and c-Src enzyme inhibition by heterocyclic-fused pyrimidines.
ꢀ Docking of the new compounds in the active site of VEGFR and c-Src.

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Supplementary Material
Novel Heterocyclic-Fused Pyrimidine Derivatives: Synthesis, Molecular
Modeling and Pharmacological Screening
Reem K. Arafa,1,* Mai S. Nour,2 Nehad A. El-Sayed1
1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street,
Cairo 11562, Egypt
2 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, MSA University, 6th October City, Cairo,
Egypt
Fig. 1: I.R. spectrum of 4a.
Fig. 2: 1HNMR spectrum of 4a.
Fig. 3: I.R. spectrum of 12a.
Fig. 4: 1HNMR spectrum of 12a.
Fig. 5: Mass spectrum of 12a.
Fig. 6: I.R. spectrum of 13.
Fig. 7: 1HNMR spectrum of 13.
Fig. 8: Mass spectrum of 13.
Fig. 9: I.R. spectrum of 14.
Fig. 10: 1HNMR spectrum of 14.
*To whom correspondences should be addressed, e-mail: rkarafa@cu.edu.eg; tel.: +2 (0100) 207-4028;
fax: +2 (02) 3362-8426
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Fig. 1: I.R. spectrum of 4a.
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Fig. 2: 1HNMR spectrum of 4a.
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Fig. 3: I.R. spectrum of 12a.
Fig. 4: 1HNMR spectrum of 12a.
4