Chromone-fused cytosine analogues: Synthesis, biological activity, and structure-activity relationship

Article abstract of DOI:10.1080/15257770.2013.873128

The preparation of a series of novel chromone-fused cytosine analogues, i.e., chromeno[2,3-d]pyrimidines has been carried out from substituted 2-amino-4-oxo-4H-chromene-3-carbonitriles with urea, thiourea, and guanidine under different reaction conditions. These chromone-fused cytosine analogues were evaluated for their in vitro activity against Mycobacterium tuberculosis H³⁷Rv strain and different microbial pathogenic strains in cell culture for their structure-activity relationships, respectively. Among the synthesized compounds, 2d, 3a, and 4e showed better results against Mycobacterium tuberculosis H³⁷Rv. The compounds 2a, 2b, and 3a showed potential antibacterial activity against E. coli and P. aeruginosa, while the majority of compounds were found to be active against S. aureus as compared to ampicillin. The synthesized cytosine analogues having an imine (-C&dbnd;NH) have been less sensitive to the bacterial and fungal strains but have a more beneficial effect on Mycobacterium tuberculosis H³⁷Rv. 2014 Copyright Taylor and Francis Group, LLC.

Full text of DOI:10.1080/15257770.2013.873128

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Chromone-Fused Cytosine Analogues:
Synthesis, Biological Activity, and
Structure–Activity Relationship
Dhaval D. Haveliwalaa, Nimesh R. Kamdara, Prashant T. Mistrya &
Saurabh K. Patela
a Department of ChemistryVeer Narmad South Gujarat University,
Surat, Gujarat, India
Published online: 24 Mar 2014.
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To cite this article: Dhaval D. Haveliwala, Nimesh R. Kamdar, Prashant T. Mistry & Saurabh
K. Patel (2014) Chromone-Fused Cytosine Analogues: Synthesis, Biological Activity, and
Structure–Activity Relationship, Nucleosides, Nucleotides and Nucleic Acids, 33:2, 80-91, DOI:
10.1080/15257770.2013.873128
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Nucleosides, Nucleotides and Nucleic Acids, 33:80–91, 2014
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2013.873128
CHROMONE-FUSED CYTOSINE ANALOGUES: SYNTHESIS,
BIOLOGICAL ACTIVITY, AND STRUCTURE–ACTIVITY RELATIONSHIP
Dhaval D. Haveliwala, Nimesh R. Kamdar, Prashant T. Mistry,
and Saurabh K. Patel
Department of Chemistry, Veer Narmad South Gujarat University, Surat, Gujarat, India
The preparation of a series of novel chromone-fused cytosine analogues, i.e., chromeno[2,3-
2
d]pyrimidines has been carried out from substituted 2-amino-4-oxo-4H-chromene-3-carbonitriles
with urea, thiourea, and guanidine under different reaction conditions. These chromone-fused cyto-
sine analogues were evaluated for their in vitro activity against Mycobacterium tuberculosis H37Rv
strain and different microbial pathogenic strains in cell culture for their structure–activity relation-
ships, respectively. Among the synthesized compounds, 2d, 3a, and 4e showed better results against
Mycobacterium tuberculosis H37Rv. The compounds 2a, 2b, and 3a showed potential antibacterial
activity against E. coli and P. aeruginosa, while the majority of compounds were found to be active
against S. aureus as compared to ampicillin. The synthesized cytosine analogues having an imine
(–C NH) have been less sensitive to the bacterial and fungal strains but have a more beneficial
effect on Mycobacterium tuberculosis H37Rv.
Keywords Antibacterial nucleosides; synthetic methodology; nucleoside synthesis; base
modification
INTRODUCTION
The study of the chemistry and biochemistry of nucleoside and nu-
cleotides has been of enormous importance; not only because of the rel-
evance to nucleic acid chemistry and molecular biology but also because
of a number of nucleosides (adenosine, guanosine, uridine, cytidine, and
thymidine) have been found to be a part of useful antibiotic and antitumor
scaffolds. Chemical modifications in the structure of nucleobases are widely
studied in the search of important bioactive molecules. Nevertheless, the
search for new bioactive compounds that lead to the formation of modified
components of nucleobases having useful physicochemical properties still
Received 20 July 2012; accepted 3 December 2013.
The author wish to thank the Head, Department of Chemistry, V.N.S.G.U., Surat for providing
the laboratory facility; Microbiologist, Microcare Laboratory for pharmacological essay and instrumental
facility SAIF, Chandigarh for analytical data.
Address correspondence to Saurabh K. Patel, Department of Chemistry, Veer Narmad South Gujarat
University, Udhana-Magdalla Road, Surat 395007, Gujarat, India. E-mail: saurabh@vnsgu.ac.in
80

Chromone-Fused Cytosine Analogues
81
remains as an urgent task. Pyrimidine-based derivatives have a prestigious
position in medicinal chemistry and are important due to their being versa-
tile building blocks for the synthesis of nitrogen-containing heteroaromatic
species of biological importance. Recently, many chemical structures con-
taining a pyrimidine moiety have been reported to exhibit diverse biological
and pharmaceutical activities such as antibacterial,[1] glycogen inhibitory,[2]
antimicrobial,[3,4] kinase Inhibitors,[5] and anti molluscicidal[6] agents. More-
over, pyrimidine-2 ones have been used as anti-inflammatory and analgesic
agents,[7] anti mycobacterium tuberculosis and anti-mycobacterial[8], antivi-
ral[9] and imino pyrimidine were reported to have antiviral[10] activity. Fur-
thermore, imino pyrimidines have been used as antileishmanial agents [11]
,
anti-histaminic activity[12], and antagonists of the human A2A receptor.[13]
In addition, pyrimidine-2-thiones were reported as antimicrobial[14,15] and
antiviral agent.[16]
The natural compounds containing chromone constitute an interesting
class because of their usefulness as biologically active agents.[17,18] Due to
their abundance in plants and their low mammalian toxicity, chromone
derivatives are present in large amounts in the diet of humans.[19]
We recently initiated that the structural modification of nucleobases with
the prospect of yielding new classes of therapeutically active agents. The
synthesis of these types of molecules can be made by several protocols and
different reaction conditions. Although various known methods are reported
concerning the synthesis of some fused pyrimidine derivatives we exploited
the o-amino nitrile functionality in this study to design new routes for the
preparation of biologically active heterocyclic compounds.[20–23] We herein
disclose the synthesis of natural product chromone-containing structurally
related cytosine fused systems and their antitubercular and antimicrobial
profile to provide some structure–activity relationships (SAR).
RESULTS AND DISCUSSION
Eighteen chromone-fused cytosine analogues were synthesized and
screened for their antimicrobial and antitubercular activity. The syn-
thetic procedures adopted to obtain the target compounds are depicted
in Scheme 1. The synthesis of precursor 2-amino-4-oxo-4H-chromene-3-
carbonitriles 1(a–f) derivatives were carried out by the chlorination of 2-
(acetyloxy)benzoic acids with thionyl chloride followed by the treatment of
malononitrile.[24]
The reactive amino and nitrile functionality of the precursor 2-amino-
4-oxo-4H-chromene-3-carbonitriles 1(a–f) was efficiently transformed into
various cytosine analogue 2(a–f), 3(a–f) and 4(a–f) by treatment of urea,
thiourea, and guanidine respectively in an ethanolic solution containing
catalytic amount of sodium ethoxide. The structures of compounds 2(a–f),

82
D. D. Haveliwala et al.
SCHEME 1 Synthetic route to chromeno[2,3-d]pyrimidines.
3(a–f) and 4(a–f) were established through spectroscopic and elemental
analyses data. The IR spectra of the final compounds 2(a–f), 3(a–f), and
4(a–f) revealed that the cyclocondensation occurs at their o-amino nitrile
functionality of the chromone nucleus with the disappearance of –C≡N band
1
in each case. The H-NMR spectra of all the compounds also show charac-
teristic signals for the protons of the cytosine nucleus. Some representative
13C-NMR spectra of compounds further confirm the proposed structures.
Antimycobacterial Assay
Antimycobacterial evaluation was carried out at the Microcare Labo-
ratory, Surat, Gujarat, India. Primary screening of all compounds was per-
formed against the Mycobacterium tuberculosis H37Rv strain. All the compounds
were tested for their antitubercular activity by the L. J. method[21] and com-
pared with standard drug rifampicin (40 μg/mL). The percent inhibition
activity of synthesized compounds is summarized in Fig. 1.
Compounds 2d, 3a, and 4e were the most effective against Mycobacterium
tuberculosis H37Rv with 99% of inhibition, while compounds 2c, 2b, 4d, and
4f showed transitional antitubercular activity between 50% and 85% of inhi-
bition. Among the unsubstituted cytosine analogues, those with a thio group
(>C S) show almost 100% inhibition of mycobacterial tubercular species,
but the introduction of other substituents decreases the activity; typically a
7-chloro substitution at chromone nucleus diminishes activity completely.
In contrast, a 7-chloro substitution containing a keto group (>C O) shows
the maximum inhibition of the tubercular pathogen. The introduction of
electron withdrawing groups shows more pronounced activity than electron

Chromone-Fused Cytosine Analogues
83
FIGURE 1 Antitubercular activity of chromeno[2,3-d]pyrimidines.
donating groups. In imino cytosine analogues, the introduction of methyl
group (–CH3) at eighth position shows maximum inhibition, while a change
in the position of the methyl group decreases the activity. The 7-chloro
substitution also shows appreciable inhibition, while the other substitutions
reduce the inhibition of tubercular species.
Antimicrobial Assay
In vitro antibacterial activity of synthesized compounds 2,3,4(a–f) was
determined against isolated species of bacterial strains, i.e., Staphylococcus
aureus [MTCC-96] and Streptococcus pyogenes [MTCC – 442] as gram posi-
tive, Escherichia coli [MTCC-443] and Pseudomonas aeruginosa [MTCC-1688]
as gram negative and antifungal activity against two fungal strain Candida
albicans [MTCC-227] and Aspergillus niger [MTCC-282]. The minimum in-
hibitory concentration (MIC), i.e., the lowest concentration of compound
that completely inhibited microbial growth was determined by the broth
dilution technique.[25] Ampicillin and greseofulvin were used as standard
drugs for bacterial and fungal strains, respectively. The procedure was per-
formed with two-fold dilution of the compounds in RPMI 1640 medium
buffered to pH 7.0. The MICs were determined after 24 hours and 48 hours
of static incubation at 35◦C. The results of all compounds in vitro tested
against bacterial as well as fungal strains are summarized in Table 1.
The MIC values (Table 1) show that the chromone ring containing
different substituents on the aromatic ring elicited an interesting activity

84
D. D. Haveliwala et al.
TABLE 1 Antimicrobial assay of chromeno[2,3-d]pyrimidines
Minimal Fungicidal
Minimal Bactericidal Concentrations (μg/mL)
Concentrations (μg/mL)
Code No.
E. coli
P. aeruginosa
S. aureus
S. pyogenus
C. albicans
A. niger
2a
2b
2c
2d
2e
2f
3a
3b
3c
3d
3e
3f
4a
4b
4c
4d
4e
4f
A
62.5
62.5
250
200
250
200
62.5
200
250
150
250
100
500
150
250
100
250
250
100
—
100
100
250
100
250
200
100
250
250
150
250
200
500
200
250
200
250
250
100
—
150
150
250
200
200
200
150
250
100
250
100
200
1000
200
500
500
500
500
250
—
250
250
500
250
200
250
250
500
250
250
200
250
1000
500
500
500
500
500
100
—
1000
500
500
250
250
>1000
1000
500
500
250
250
>1000
500
>1000
>1000
500
1000
>1000
—
500
1000
>1000
1000
1000
>1000
500
1000
>1000
1000
500
>1000
500
>1000
>1000
>1000
>1000
>1000
—
B
500
100
Ampicillin (A) was used as standard drugs for bacterial strains, while greseofulvin (B) was used as
standard drugs for fungal strains.
against gram negative bacteria. The observed activities are particularly good
against E. coli. Compounds 2a, 2b, and 3a show highest activity against E. coli
and the values are even better than the standard drug ampicillin. Only the
strain S. pyogenus is resistant to most of the synthesized compounds. Among
the unsubstituted derivatives, 2a and 3a are found as be more sensitive
than ampicillin against E. coli, P. aeruginosa, and S. aureus. In fact –NO2
derivatives are totally inactive on both gram negative bacteria, while dibromo
and monochloro compounds possess similar MIC value as ampicillin against
P. aeruginosa. More interestingly, the compounds 2a, 2b, 2d, 2e, 2f, 3a, 3c,
3e, 3f, and 4b show MIC value in range of 100–200 μg/mL against S. aureus;
in particular, the chloro and methyl derivatives (3c and 3e) having sulfur-
containing cytosine nucleus show remarkable (100 μg/mL) MIC value than
ampicillin.
The antifungal activity data support the specific observation that com-
pounds 2d (7-Cl) and 2e (8-CH3) possess better antifungal activity than grese-
ofulvin (B) against C. albicans. Similar activity is also observed in compounds
3d (7-Cl) and 3e (8-CH3) which contain a sulfur-based cytosine nucleus. The
replacement of a keto (–C O) or thio (–C S) group on cytosine with an

Chromone-Fused Cytosine Analogues
85
imine (–C NH) group results in the complete loss of antifungal activity.
Most of the compounds were insensitive toward A. niger.
Among the synthesized cytosine analogues, imine (–C NH) group com-
pounds in general reduce the potency against all the tested micro-organisms
as compared to keto (–C O) and thio (–C S) group. The unsubstituted
chromeno[2,3-d]pyrimidines containing keto (–C O) and thio (–C S)
groups show very potent activity against E. coli. bacteria.
CONCLUSIONS
Novel chromone fused cytosine analogues have been successfully syn-
thesized by simple condensation with urea, thiourea, and guanidine, respec-
tively. A majority of the compounds were found to be active against S. aureus.
Compounds 2a, 2b, and 3a show higher antimicrobial potency against E.
coli, while compounds 2d, 2e, 3d, and 3e were found to be more potent
against C. albicans as compared to the standard drug. The antimicrobial
study shows that the synthesized compounds have been found to be more
active toward bacteria than fungi. The SAR of the title compounds showed
that the presence of an imino functionality on cytosine and groups like 8-
methyl, 7-chloro, and 9-methyl on the chromone ring are responsible for
the good antitubercular activity.
EXPERIMENTAL
Chemistry
All organic solvents used for the synthesis were of analytical grade.
The solvents were dried and freshly distilled under moisture free atmo-
sphere. TLC (Merck, 0.25 mm thick) was performed on coated silica gel
plates in the toluene–ethyl acetate (3:1) solvents system. The plates were
detected in UV (254 nm). Melting points were taken in open capillaries
and are uncorrected. IR spectra were recorded using KBr pellets on Nicolet
400 IR-spectrophotometer (Thermo Fisher). 1H and 13C-NMR spectra were
recorded on a Varian 400 MHz spectrometer using DMSO-d6 or CDCl3 as
the solvent and TMS as the internal standard.
General Procedure for Synthesis of Compounds 2(a–f)
A mixture of 1(a–f) (0.05 mol) and urea (0.05 mol) in absolute ethanol
(20 mL) containing sodium ethoxide (0.05 mol) was refluxed for 8 hours.
The reaction mixture was left to cool to room temperature, then poured
into ice cold water (50 mL) and neutralized with dilute hydrochloric acid;
the separated precipitates were filtered and recrystallized from methanol to
give compounds 2(a–f).

86
D. D. Haveliwala et al.
4-amino-2H-chromeno[2,3-d]pyrimidine-2,5(1H)-dione (2a)
Solid brown; Yield 68%; m. p. 246–249◦C; IR (KBr, ν, cm−1): 3299
(–NH2), 3126 (>NH), 1738 (>C O pyrimidine ring), 1665 (chromone ring
>C O), 1615 (–C N–); 1H-NMR: (400 MHz, CDCl3:DMSO-d6): δ 6.66–6.73
(2H, multiplet, Ar–H9/H7), 7.17 (1H, ddd, Ar–H8, J = 8.28, 2.14, 1.80 Hz),
7.67 (1H, dd, Ar–H6, J = 8.28, 1.80 Hz), 8.09 (2H, broad s, –NH2, D2O ex-
changeable), 8.35 (1H, s, >NH); 13C-NMR (400 MHz, CDCl3) δ (ppm): 116,
123, 125, 129 135, 157 (aromatic carbons), 172.9 (C–NH2), 177.5 (chromone
>C O), 148.5 (pyrimidone >C O), 76 and 173 (C C at pyrimidine ring);
Anal. Calcd. for C11H7N3O3: C, 57.65, H, 3.08, N, 18.33, Found: C, 57.64, H,
3.10, N, 18.34.
4-amino-7,9-dibromo-2H-chromeno[2,3-d]pyrimidine-2,5(1H)-dione (2b)
Solid brown; Yield 74%; m. p. 272–276◦C; IR (KBr, ν, cm−1): 3281
(–NH2), 3112 (>NH), 1728 (>C O pyrimidine ring), 1668 (chromone ring
>C O), 1624 (–C N–), 726 (C–Br); 1H-NMR: (400 MHz, CDCl3:DMSO-d6):
7.85 (1H, d, Ar–H8, J = 2.0 Hz), 8.26 (1H d, Ar–H6, J = 2.0 Hz), 8.36 (2H,
broad s, –NH2, D2O exchangeable), 8.44 (1H, s, >NH); Anal. Calcd. for
C11H5N3O3Br2: C, 34.14, H, 1.28, N, 10.86, Found: C, 34.16, H, 1.30, N,
10.84.
4-amino-7-nitro-2H-chromeno[2,3-d]pyrimidine-2,5(1H)-dione (2c)
Solid brown; Yield 69%; m. p. 285–287◦C; IR (KBr, ν, cm−1): 3295
(–NH2), 3120 (>NH), 1736 (>C O pyrimidine ring), 1666 (chromone ring
>C O), 1618 (–C N–), 1520 (C–NO2); 1H-NMR: (400 MHz, CDCl3:DMSO-
d6): 7.31 (1H, d, Ar–H9, J = 9.16 Hz),), 8.32 (1H dd, Ar–H8, J = 9.10,
2.56 Hz), 8.64 (1H, dd, Ar–H6, J = 2.56 Hz), 8.23 (2H, broad s, –NH2, D2O
exchangeable), 8.48 (1H, s, >NH); Anal. Calcd. for C11H6N4O5: C, 48.18,
H, 2.21, N, 20.43, Found: C, 49.19, H, 2.22, N, 20.44.
4-amino-7-chloro-2H-chromeno[2,3-d]pyrimidine-2,5(1H)-dione (2d)
Pale yellow; Yield 72%; m. p. 258–261◦C; IR (KBr, ν, cm−1): 3272 (–NH2),
3100 (>NH), 1724 (>C O pyrimidine ring), 1669 (chromone ring >
C O), 1621 (–C N–), 812 (C–Cl); 1H-NMR: (400 MHz, CDCl3:DMSO-d6):
7.23 (1H, d, Ar–H9, J = 8.8 Hz), 7.55 (1H, dd, Ar–H8, J = 8.8, 2.6 Hz), 7.84
(1H, d, Ar–H6, J = 2.6 Hz), 8.26 (1H, s, >NH), 8.56 (2H, broad s–NH2, D2O
exchangeable); Anal. Calcd. for C11H6N3O3Cl: C, 50.11, H, 2.29, N, 15.94,
Found: C, 50.09, H, 2.27, N, 15.96.
4-amino-8-methyl-2H-chromeno[2,3-d]pyrimidine-2,5(1H)-dione (2e)
Brown; Yield 71%; m. p. 275–278◦C; IR (KBr, ν, cm−1): 3301 (–NH2),
3127 (>NH), 1742 (>C O pyrimidine ring), 1665 (chromone ring >C O),
1614 (–C N–), 1360 (C–CH3); 1H-NMR: (400 MHz, CDCl3:DMSO-d6): 2.45

Chromone-Fused Cytosine Analogues
87
(3H, s, Ar–CH3), 7.00 (1H, d, Ar–H9, J = 1.64), 7.16 (1H dd, Ar–H7, J =
1.64, 7.88 Hz), 7.94 (1H, d, Ar–H6, J = 7.88 Hz), 8.17 (2H, broad s, –NH2,
D2O exchangeable), 8.08 (1H, s, >NH); Anal. Calcd. for C12H9N3O3: C,
59.26, H, 3.73, N, 17.28, Found: C, 59.28, H, 3.71, N, 17.27.
4-amino-9-methyl-2H-chromeno[2,3-d]pyrimidine-2,5(1H)-dione (2f)
Solid brown; Yield 74%; m. p. 262–264◦C; IR (KBr, ν, cm−1): 3302
(–NH2), 3135 (>NH), 1738 (>C O pyrimidine ring), 1667 (chromone ring
>C O), 1625 (–C N–), 1356 (C–CH3); 1H-NMR: (400 MHz, CDCl3:DMSO-
d6): 2.38 (3H, s, Ar–CH3), 6.87 (1H, t, Ar–H7, J = 7.28 Hz), 7.26 (1H dd,
Ar–H8, J = 7.24, 2.82 Hz), 7.75 (1H, dd, Ar–H6, J = 7.28, 2.82 Hz), 8.33
(2H, broad s, –NH2, D2O exchangeable), 8.17 (1H, s, >NH); Anal. Calcd.
for C12H9N3O3: C 59.26, H, 3.73, N, 17.28, Found: C, 59.25, H, 3.73, N,
17.28.
General Procedure for Synthesis of Compounds 3(a–f)
A mixture of 1(a–f) (0.05 mol) and thiourea (0.05 mol) in absolute
ethanol (20 mL) containing sodium ethoxide (0.05 mol) was refluxed for
8 hours. The reaction mixture was left to cool to room temperature, then
poured into ice cold water (50 mL) and neutralized with dilute hydrochlo-
ric acid; the separated precipitates were filtered and recrystallized from
methanol to give compounds 3(a–f).
4-amino-2-thioxo-1,2-dihydro-5H-chromeno[2,3-d]pyrimidin-5-one (3a)
Yellow, Yield 66%, m.p. > 300 ◦C; IR (KBr, ν, cm−1): 3308 (–NH2), 3144
(>NH), 1668 (>C O), 1622 (–C N–), 1338 (>C S); 1H-NMR: (400 MHz,
CDCl3:DMSO-d6): δ 6.78–6.93 (2H, multiplet, Ar–H9/H7), 7.22 (1H, ddd,
Ar–H8, J = 8.20, 2.12, 1.78 Hz), 7.73 (1H, dd, Ar–H6, J = 8.20, 1.78 Hz),
8.35 (2H, broad s, –NH2, D2O exchangeable), 8.56 (1H, s, >NH); 13C-
NMR (400 MHz, CDCl3) δ (ppm): 115, 121, 125, 129 140, 158 (aromatic
carbons), 175.1 (>C O), 180.4 (>C S), 167.5 (C–NH2), 77 and 177
(C C at pyrimidine ring); Anal. Calcd. for C11H7N3O2S: C, 53.87, H, 2.88,
N, 17.13, S, 13.07; Found: C, 53.88, H, 2.89, N, 17.15, S, 13.09.
4-amino-7,9-dibromo-2-thioxo-1,2-dihydro-5H-chromeno[2,3-d]pyrimidin-
5-one (3b)
Colorless, Yield 67%, m.p. 295- 297 ◦C; IR (KBr, ν, cm−1): 3359 (–NH2),
3100 (>NH), 1684 (>C O), 1601 (–C N–), 1325 (>C S), 731 (C–Br);
1H-NMR: (400 MHz, CDCl3:DMSO-d6): 7.97 (1H, d, Ar–H8, J = 2.2 Hz),
8.31 (1H d, Ar–H6, J = 2.2 Hz), 8.56 (2H, br s, NH2, D2O exchangeable),
8.74 (1H, s, >NH); Anal. Calcd. for C11H5N3O2SBr2: C, 32.38, H, 1.25, N,
10.43, S, 7.96; Found: C, 32.37, H, 1.24, N, 10.42, S, 8.00.

88
D. D. Haveliwala et al.
4-amino-7-nitro-2-thioxo-1,2-dihydro-5H-chromeno[2,3-d]pyrimidin-5-one (3c)
Brown, Yield 73%, m.p. > 300 ◦C; IR (KBr, ν, cm−1): 3294 (–NH2), 3138
(>NH), 1673 (>C O), 1617 (–C N–), 1345 (>C S), 1525 (C–NO2); 1H-
NMR: (400 MHz, CDCl3:DMSO-d6): 7.54 (1H, d, Ar–H9, J = 9.04 Hz), 8.19
(1H, dd, Ar–H8, J = 9.04, 2.84 Hz), 8.70 (1H, dd, Ar–H6, J = 2.84 Hz), 9.01
(2H, broad s–NH2, D2O exchangeable), 8.61 (1H, s, >NH); Anal. Calcd. for
C11H6N4O4S: C, 45.48, H, 2.12, N, 19.28, S, 11.00, Found: C, 45.46, H, 2.15,
N, 19.25, S, 11.00.
4-amino-7-chloro-2-thioxo-1,2-dihydro-5H-chromeno[2,3-d]pyrimidin-5-one
(3d)
Brown, Yield 69%, m.p. 290–292◦C; IR (KBr, ν, cm−1): 3312 (–NH2),
3153 (–NH), 1675 (>C O), 1632 (–C N–), 1329 (>C S), 816 (C–Cl); 1H-
NMR: (400 MHz, CDCl3:DMSO-d6): 7.29 (1H, d, Ar–H9, J = 8.8 Hz), 7.42
(1H, d d, Ar–H8, J = 8.8, 2.68 Hz), 7.88 (1H, d, Ar–H6, J = 2.68 Hz), 8.28
(2H, broad s–NH2, D2O exchangeable), 8.17 (1H, s, >NH); Anal. Calcd. for
C11H6N3O2SCl: C, 47.24, H, 2.16, N, 15.02, S, 11.46; Found: C, 47.26, H,
2.15, N, 15.05, S, 11.48.
4-amino-8-methyl-2-thioxo-1,2-dihydro-5H-chromeno[2,3-d]pyrimidin-5-one
(3e)
Light Brown, Yield 71%, m.p. > 300◦C; IR (KBr, ν, cm−1): 3297 (–NH2),
3115 (–NH), 1670 (>C O), 1627 (–C N–), 1331 (>C S), 1367 (C–CH3);
1H-NMR: (400 MHz, CDCl3:DMSO-d6): 2.37 (3H, s, Ar–CH3), 7.17 (1H, d,
Ar–H9, J = 1.62 Hz) 7.29 (1H dd, Ar–H7, J = 1.62 Hz, 7.88 Hz), 8.04 (1H,
d, Ar–H6, J = 7.88 Hz), 8.35 (2H, broad s, –NH2, D2O exchangeable), 8.21
(1H, s, >NH); Anal. Calcd. for C12H9N3O2S: C, 55.59, H, 3.50, N, 16.21,
S, 12.37; Found: C, 55.50, H, 3.51, N, 16.24, S, 12.38.
4-amino-9-methyl-2-thioxo-1,2-dihydro-5H-chromeno[2,3-d]pyrimidin-5-one
(3f)
Light Brown, Yield 70%, m.p. 291–293◦C; IR (KBr, ν, cm−1): 3307
(–NH2), 3132 (–NH), 1668 (>C O), 1621 (–C N–), 1324 (>C S), 1361
(C–CH3); 1H-NMR: (400 MHz, CDCl3:DMSO-d6): 2.39 (3H, s, Ar–CH3), 6.72
(1H, t, Ar–H7, J = 7.28 Hz), 7.27 (1H, dd, Ar–H8, J = 7.24, 2.81 Hz), 7.57
(1H, dd, Ar–H6, J = 7.28, 2.81 Hz), 8.38 (2H, broad s, –NH2, D2O exchange-
able), 8.25 (1H, s, >NH); Anal. Calcd. for C12H9N3O2S: C, 55.59, H 3.50, N,
16.21, S, 12.37; Found: C, 55.60, H 3.51, N, 16.24, S, 12.38.
General Procedure for Synthesis of Compounds 4(a–f)
A mixture of 1(a–f) (0.05 mol) and guanidine nitrate (0.05 mol) in
absolute ethanol (20 mL) containing sodium ethoxide (0.05 mol) was re-
fluxed for 8 hr. The reaction mixture was left to cool to room temperature,

Chromone-Fused Cytosine Analogues
89
then poured into ice cold water (50 mL) and neutralized with dilute hy-
drochloric acid; the separated precipitates were filtered and recrystallized
from methanol 4(a–f).
4-amino-2-imino-1,2-dihydro-5H-chromeno[2,3-d]pyrimidin-5-one (4a)
Solid beige; Yield 73%; m. p. 234–236◦C; IR (KBr, ν, cm−1): 3483, 3303
1
(–NH2), 1665 (>C O), 1609 (–C N–); H-NMR: (400 MHz, DMSO-d6):
δ 6.62–6.87 (2H, multiplet, Ar–H9/H7), 7.07 (1H, ddd, Ar–H8, J = 8.18,
2.14, 1.80 Hz), 7.48 (1H, dd, Ar–H6, J = 8.18, 1.80 Hz), 8.47 (2H, broad s,
–NH2, D2O exchangeable), 8.66 (1H, s, >NH); 13C-NMR (400 MHz, CDCl3)
δ (ppm): 118, 121, 124, 130, 137, 156 (aromatic carbons), 153.6 (>C NH),
167.2 (C–NH2), 176.5 (>C O), 74 and 183 (C C at pyrimidine ring); Anal.
Calcd. for C11H8N4O2: C, 57.89, H, 3.53, N, 24.55; Found: C, 57.91, H, 3.51,
N, 24.55.
4-amino-7,9-dibromo-2-imino-1,2-dihydro-5H-chromeno[2,3-d]pyrimidin-5-one
(4b)
Solid beige (methanol); Yield 70%; m. p. 223–225◦C; IR (KBr, ν, cm−1):
1
3467, 3315 (–NH2), 1669 (>C O), 1621 (–C N–), 728 (C–Br); H-NMR:
(400 MHz, CDCl3:DMSO-d6): 7.87 (1H, d, Ar–H8, J = 2.0 Hz), 8.23 (1H d,
Ar–H6, J = 2.0 Hz), 8.47 (2H, br s, NH2, D2O exchangeable), 8.68 (1H, s,
>NH); Anal. Calcd. for C11H6N4O2Br2: C, 34.23, H, 1.57, N, 14.51; Found:
C, 34.25, H, 1.59, N, 14.51.
4-amino-7-nitro-2- imino-1,2-dihydro-5H-chromeno[2,3-d]pyrimidin-5-one (4c)
Solid beige (methanol); Yield 66%; m. p. 256–258◦C; IR (KBr, ν, cm−1):
1
3473, 3308, (–NH2), 1673 (>C O), 1616 (–C N–), 1528 (C–NO2); H-
NMR: (400 MHz, CDCl3:DMSO-d6): 7.66 (1H, d, Ar–H9, J 9.2 Hz), 8.36
(1H, dd, Ar–H8, J = 9.2, 2.84 Hz), 8.69 (1H, dd, Ar–H6, J = 2.84 Hz), 8.89
(2H, broad s–NH2, D2O exchangeable), 8.79 (2H, s–NH); Anal. Calcd. for
C11H7N5O4: C, 48.36, H, 2.58, N, 25.63; Found: C, 48.38, H, 2.56, N, 25.64.
4-amino-7-chloro-2-imino-1,2-dihydro-5H-chromeno[2,3-d]pyrimidin-5-one
(4d)
Solid yellow (methanol); yield 71%; m. p. 201–203◦C; IR (KBr, ν, cm−1):
1
3453, 3313 (–NH2), 1667 (>C O), 1613 (–C N–), 810 (C–Cl); H-NMR:
(400 MHz, CDCl3:DMSO-d6): 7.19 (1H, d, Ar–H9, J = 8.8 Hz), 7.39 (1H,
dd, Ar–H8, J = 8.8, 2.68 Hz), 7.82 (1H, d, Ar–H6, J = 2.68 Hz), 8.34 (2H,
broad s–NH2, D2O exchangeable), 8.21 (1H, s, >NH); Anal. Calcd. for
C11H7N2O4Cl: C, 50.30, H 2.59, N, 21.33; Found: C, 50.32, H 2.62, N, 21.33.

90
D. D. Haveliwala et al.
4-amino-2-imino-8-methyl-1,2-dihydro-5H-chromeno[2,3-d]pyrimidin-5-one
(4e)
Solid light brown (methanol); yield 66%; m. p. 218–220◦C; IR (KBr, ν,
cm−1): 3462, 3303 (–NH2), 1669 (>C O), 1623 (–C N–), 1355 (C–CH3);
1H-NMR: (400 MHz, CDCl3:DMSO-d6): 2.36 (3H, s, Ar–CH3), 7.01(1H, d,
Ar–H9, J = 1.63 Hz) 7.24(1H dd, Ar–H7, J = 1.63, 7.88 Hz), 7.86 (1H, d,
Ar–H6, J = 7.88 Hz), 8.26 (2H, broad s, –NH2, D2O exchangeable), 8.14
(1H, s, >NH); Anal. Calcd. for C12H10N4O2: C, 59.50, H, 4.16, N, 23.13;
Found: C, 59.53, H, 4.13, N, 23.14.
4-amino-2-imino-9-methyl-1,2-dihydro-5H-chromeno[2,3-d]pyrimidin-5-one
(4f)
Solid light brown (methanol); yield 68%; m. p. 207–209◦C; IR (KBr, ν,
cm−1): 3482, 3299 (–NH2), 1666 (>C O), 1614 (–C N–), 1358 (C–CH3);
1H-NMR: (400 MHz, CDCl3:DMSO-d6): 2.45 (3H, s, Ar–CH3), 6.66 (1H, t,
Ar–H7, J = 7.28 Hz), 7.26 (1H, dd, Ar–H8, J = 7.24, 2.80 Hz), 7.84 (1H, dd,
Ar–H6, J = 7.28, 2.80 Hz), 8.10 (2H, broad s, –NH2, D2O exchangeable),
8.08 (1H, s, >NH); Anal. Calcd. for C12H10N4O2: C, 59.50, H, 4.16, N, 23.13;
Found: C, 59.51, H, 4.15, N, 23.15.
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