Synthesis and in vitro antimicrobial activities of 2-hydroxy-6-methyl-7- (arylamino)-1,7-dihydropurin-8-ones

Article abstract of DOI:10.1016/j.bmc.2004.04.007

A number of 2-hydroxy-6-methyl-7-(arylamino)-1,7-dihydropurin-8-ones have been synthesized. 3-Oxo-2-(arylhydrazono)butyric acid ethyl ester were acetylated and treated with triethyl amine and formamide in presence of 1,4-dioxane to yield N-(5-acetyl-4-eth

Full text of DOI:10.1016/j.bmc.2004.04.007

Bioorganic & Medicinal Chemistry 12 (2004) 3135–3139
Synthesis and in vitro antimicrobial activities of 2-hydroxy-6-
methyl-7-(arylamino)-1,7-dihydropurin-8-ones
Pratibha Sharma,* Shikha Sharma and Nilesh Rane
School of Chemical Sciences, D.A. University, Indore 452 017, India
Received 25 February 2004; revised 7 April 2004; accepted 8 April 2004
Abstract—A number of 2-hydroxy-6-methyl-7-(arylamino)-1,7-dihydropurin-8-ones have been synthesized. 3-Oxo-2-(arylhydrazo-
no)butyric acid ethyl ester were acetylated and treated with triethyl amine and formamide in presence of 1,4-dioxane to yield N-(5-
acetyl-4-ethoxy-2-oxo-2,5-dihydro-imidazol-1-yl)-N-arylacetamide, which on refluxation with urea and freshly prepared sodium
ethoxide yielded the title compound. All the newly synthesized compounds have been characterized by spectroscopic and elemental
analysis data. The synthesized compounds were screened against a representative panel of susceptible and resistant Gram-positive
and Gram-negative bacteria using a standard antibiotic drug purinthol as control. Quantitative structure–activity relationship has
also been interpreted in terms of correlation of biological activity with molecular refractive index parameters (MR) and Hammett
substituent constant (r).
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
report the synthesis and in vitro antibacterial behaviour
2-hydroxy-6-methyl-7-(arylamino)-1,7-dihydropurin-8-
ones. 3-Oxo-2-(arylhydrazono)butyric acid ethyl ester22
1a–k was acetylated23 to yield 2-(acetyl arylhydrazono)-
3-oxo butyric acid ethyl ester 2a–k, which in turn was
treated with triethyl amine and formamide in presence
of 1,4-dioxane for 4 h to give N-(5-acetyl-4-ethoxy-2-
oxo-2,5-dihydro-imidazol-1-yl)-N-arylacetamide 3a–k.
In order to extend the synthesis to obtain the desired
purine, it was refluxed with urea in freshly prepared
sodium ethoxide for 3 h and then subjected to acid
hydrolysis23 to yield 2-hydroxy-6-methyl-7-(arylamino)-
1,7-dihydropurin-8-ones 4a–k. Structures of these syn-
thesized compounds have been elucidated on the basis of
IR, NMR and elemental analysis data and purity was
ascertained by TLC (methanol and toluene, 4:6 v/v).
Purines are reported to have a wide range of biological
activities.1–4 The application of purine and its deriva-
tives is found in the research areas as cyclin-dependent
kinase inhibitors,5 antitumour,6 antimycobacterial,7;8
corticotropin releasing hormone receptor antagonist,9
anti-rhinovirus,10 xanthine oxidase inhibitor11 and
anticonvulsant agents.12 They appear in nucleic acids
and cofactors that play an essential role in the modu-
lation of protein function and signal transduction.13
These and other findings have resulted in large number
of synthetic investigations.14–17
Resistance of various bacteria against commercially
available antibiotics is increasing day-by-day.18 As a
result need for new antibiotic agents to combat this
problem is also increasing. These threats have rekindled
our interest to search for new types of lead compounds.
In the present context and in continuation of our pre-
vious work on heterocyclic compounds19–21 we herein
2. Chemistry
The synthesis of novel purine derivatives having different
substituents is shown in Scheme 1. The key intermediate
3-oxo-2-(arylhydrazono) butyric acid ethyl ester 1a–k
was prepared according to the developed method.22
This requisite starting material was acetylated23 in
order to protect –NH moiety. This acetylated compound
2a–k was refluxed with formamide in presence of
Keywords: 2-Hydroxy-6-methyl-7-(arylamino)-1,7-dihydropurin-8-ones;
Molecular refractive index parameters (MR); Hammett substituent
constant (r).
* Corresponding author. Tel.: +917312460208; fax: +917312470352;
e-mail: drpratibhasharma@yahoo.com
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.04.007

3136
P. Sharma et al. / Bioorg. Med. Chem. 12 (2004) 3135–3139
R
O
C
O
O
C
O
R
NH
C
C
CH
O
N
C
C
CH
O
H3C
3
3
(i)
N
N
O
1a-k
2a-k
CH
3
CH
3
(ii)
R
O
H3C
N
CH
3
C
N
O
O
O
N
Compd
R
CH
3
3a-k
4a
4b
4c
4d
4e
4f
H
2'-OCH3
2'-Cl
3'-Cl
4'-Cl
(iii),(iv)
2'-NO2
3'-NO2
4'-NO2
H C
3
H
N
4g
4h
4i
R
NH
OH
N
2'-OH
3'-OH
N
N
4j
O
4k
2'-CH3
4a-k
Scheme 1. Reagents and conditions: (i) (CH3CO)2O; (ii) (C2H5)3N, HCONH2, 1,4-dioxane, reflux 4 h; (iii) (NH2)2CO, C2H5ONa, reflux 3 h; (iv)
concd H2SO4.
Table 1. Analytical data for compound 3a,c,f
Compd
Yield (%)
MP (ꢁC)
Mol. formula
15H17O4N3
Micro analytical data (%) found (calcd)
3a
3c
3f
74
76
71
171–173
165–167
160–161
C
C, 59.35 (59.40); H, 5.62 (5.65); N, 13.80 (13.85)
C, 53.30 (53.34); H, 4.73 (4.77); N, 12.40 (12.44)
C, 51.65 (51.72); H, 4.58 (4.63); N, 16.05 (16.09)
C15H16O4N3Cl
C15H16O6N4
triethylamine and 1,4-dioxane for 4 h to afford the
corresponding N-(5-acetyl-4-ethoxy-2-oxo-2,5-dihydro-
imidazol-1-yl)-N-arylacetamide 3a–k in 70–80% yield as
depicted in Table 1.
synthesized by condensation–cyclization of N–C–N
fragment with a C–C–C fragment. The N–C–N frag-
ment employed in the course of study was generated
from urea and the reaction has been carried out in
the presence of freshly prepared sodium ethoxide
solution.
The reaction of 3a–k with urea in freshly prepared
sodium ethoxide solution followed by acidic hydrolysis23
yielded 2-hydroxy-6-methyl-7-(arylamino)-1,7-dihydro-
purin-8-ones 4a–k. Hence, pyrimidine ring, which was
fused with imidazole ring to yield purine skeleton was
All the synthesized compounds 4a–k exhibited satisfac-
tory elemental analysis and spectral data consistent with
the structures.

P. Sharma et al. / Bioorg. Med. Chem. 12 (2004) 3135–3139
Table 2. In vitro antibacterial activity of 4a–k (zone of inhibition, %)
3137
Compd
R
MR
Bacteria testeda
Ec
Ag
40
At
)9
)34
)26
)20
3
Pd
Bs
26
4a
4b
4c
4d
4e
4f
H
48.878
55.313
53.745
53.745
53.745
55.078
55.078
55.078
50.403
50.403
53.670
12
)24
)12
)8
20
3
20-OCH3
20-Cl
)20
7
)30
)17
)13
13
)21
)5
5
30-Cl
40-Cl
13
53
)13
)7
47
27
33
20
37
20-NO2
30-NO2
40-NO2
20-OH
30-OH
20-CH3
)32
)29
)6
)14
)11
)17
)20
)16
16
)27
)20
7
)16
)11
32
4g
4h
4i
4
)7
)3
16
21
4j
4k
8
)4
)10
10
a Ag A. globiformis; At A. tumefaciens; Ec E. coli; Pd P. diminuata; Bs B. subtilis.
3. Biological activities
0
-5
4'-Cl
We had conducted antibacterial screening studies versus
several clinically relevant Gram-positive and Gram-
negative bacteria in an effort to unravel interesting leads
from synthesized compounds. Nutrient agar media was
prepared for bacterial growth. Stock solutions of tested
compound were prepared in dimethylformamide
(DMF). Inocula containing approximately 107 CFU/mL
of bacteria were prepared from broth culture in log
phase growth. Filter paper disc method by Rapper24 was
employed to determine the antibacterial activity of
synthesized compounds. The inhibition zone of each test
solution was measured in mm and reported in terms of
4'-NO2
H
3'-OH
-10
-15
-20
-25
-30
-35
2'-OH
2'-CH3
3'-Cl
2'-Cl
3'-NO2
2'-NO2
2'-OCH3
55 56
48
49
50
51
52
MR
53
54
%
inhibition
using
the
formula
inhibition
ð%Þ ¼ ½ða ꢀ bÞ=aꢁ ꢂ 10025 and presented in Table 2,
where a and b stands for zone of inhibition of control
drug and synthesized compounds, respectively.
Figure 1. Plot of biological activity versus molecular refractive index
(MR) screened against A. tumefaciens.
All the newly synthesized compounds were assayed for
their antimicrobial activity (zone of inhibition %) against
Arthobacter globiformis, Bacillus subtilis as Gram-posi-
tive bacteria and Agrobacterium tumefaciens, Escherichia
coli, Pseudomonas diminuata as Gram-negative bacteria.
An overview of the antibacterial data in Table 2 shows
that synthesized compounds gives promising results
when tested against these bacteria in vitro compared to
the reference drug purinthol. The screening data revealed
that compound exhibit promising activity against A.
tumefaciens, while least against A. globiformis. Accord-
ing to systemic perusal of the data presented in the Table
2, compound 4b exhibited the best in vitro profile in our
abbreviated testing panel. The data pertaining to Table 2
reveals that the activity shown by synthesized purine
follows the pattern A. tumefaciens > P. diminuata > E.
coli > B. subtilis > A. globiformis.
60
4'-Cl
50
4'-NO2
H
40
3'-OH
2'-OH
30
2'-CH3
3'-Cl
20
10
0
2'-Cl
3'-NO2
-10
-20
-30
2'-NO2
2'-OCH3
55 56
48
49
50
51
52
53
54
MR
Interestingly in a view to establish a quantitative struc-
ture–activity relationship (QSAR), molecular refractive
index (MR) values, determined by the method of Dreis-
bach,26 has been correlated with the biological activity
and reported in Table 2.
Figure 2. Plot of biological activity versus molecular refractive index
(MR) screened against A. globiformis.
activity of 2-hydroxy-6-methyl-7-(arylamino)-1,7-di-
hydropurin-8-ones has been observed to be highest
against A. tumefaciens and least against A. globiformis as
has been justified on the basis of increase in molecular
refractive index (MR) values.
A perusal of graphical representation (Figs. 1 and 2)
reveals the linearity of drug activity with molecular
refractive index (MR) data. It is interpreted that the

3138
P. Sharma et al. / Bioorg. Med. Chem. 12 (2004) 3135–3139
silica gel G 0.50 mm plate Merck no 5700. Melting
0
-5
4'-Cl
points were determined in open capillary tubes using an
electric melting point apparatus. All the melting points
were reported are uncorrected.
4'-NO2
H
-10
-15
-20
-25
-30
3'-OH
4.1. Synthesis of N-(5-acetyl-4-ethoxy-2-oxo-2,5-dihydro-
imidazol-1-yl)-N-arylacetamide 3a–k
3'-Cl
In a 250 round bottom flask 3-oxo-2- (arylhydrazono)
butyric acid ethyl ester22 1a–k (0.02 M), was taken and
subjected to acetylation using literature procedure.23 To
this triethylamine (2.4 mL, 0.02 M), formamide (1 mL,
0.02 M) and measured quantity of 1,4-dioxane (50 mL)
were added and all the content were refluxed for 4 h to
give 3a–k.
3'-NO2
0.0
0.2
0.4
σ
0.6
0.8
Figure 3. Plot of biological activity versus Hammett substituent con-
stant (r) screened against A. tumefaciens.
4.2. Synthesis of 2-hydroxy-6-methyl-7-(arylamino)-1,7-
dihydropurin-8-ones 4a–k
60
4'-Cl
4'-NO2
50
Compound 3a–k (0.02 M) was refluxed with urea (1.2 g,
0.02 M) in freshly prepared sodium ethoxide solution (6 g
of sodium metal and 50 mL of absolute ethanol) for 3 h.
On cooling the product so formed was hydrolyzed23 using
sulfuric acid and the compound obtained was recrystal-
lized from the solution of ethanol and DMF (1:1 v/v).
The purity of synthesized compound was ascertained by
TLC (methanol + toluene, 4:6 v/v). Structures of the
synthesized compounds have been ascertained on the
basis of spectroanalytical data.28–31 Synthetic pathway of
all the sequential steps is depicted in Scheme 1.
H
40
3'-OH
30
20
3'-Cl
10
0
3'-NO2
-10
0.0
0.2
0.4
0.6
0.8
σ
Figure 4. Plot of biological activity versus Hammett substituent con-
stant (r) screened against A. globiformis.
References and notes
1. Kim, H. O.; Park, Y. H.; Moon, H. R.; Jeong, L. S.
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8. Gundersen, L. L.; Nissen-Meyer, J.; Spilsberg, B. J. Med.
Chem. 2002, 45, 1383.
Likewise, drug activity has also been correlated with
Hammett substituent constant (r)27 (Figs. 3 and 4).
Again, it can inferred that drug activity follows the
pattern of linearity with (r) values. All the synthesized
compounds bearing substituents viz. H, 30-OH, 30-Cl
and 30-NO2 have been observed to fit in regression line
whereas, 40-Cl, 40-NO2 deviate from it indicating that
compounds having aromatic ring incorporating electron
attracting groups at para position decreases the activity
but if they are present on meta/ortho position they tend
to increase the activity to a greater extent. Furthermore,
insertion of hydroxyl group also increases the activity of
the synthesized compounds. In general it has been
concluded that the antibacterial results follow the pat-
tern
9. Cocuzza, A. J.; Chidester, D. R.; Culp, S.; Fitzgerald, L.;
Gilligan, P. Bioorg. Med. Chem. Lett. 1999, 9, 1063.
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1991, 34, 157.
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Pharmacol. 1994, 25, 1027.
13. Jacobso, K. A.; Daly, J. W.; Manganiello, V. Purines in
Cellular Signaling: Targets for New Drugs; Springer: New
York, 1990.
4b > 4f > 4g > 4c > 4d > 4k > 4i > 4j > 4a > 4h > 4e.
4. Experimental
1H NMR spectra were recorded at 300 MHz Bruker
advance DRX 300 instrument using TMS as an internal
standard. IR spectra were run on Perkin Elmer model
377 spectrophotometer in KBr pellets. Analytical thin
layer chromatography was performed using E. Merck

P. Sharma et al. / Bioorg. Med. Chem. 12 (2004) 3135–3139
3139
14. Brun, V.; Legraverend, M.; Grierson, D. S. Tetrahedron
2002, 58, 7911.
15. Austin, R. E.; Okonya, J. F.; Bond, D. R.; Al-Obedi, F.
Tetrahedron Lett. 2002, 43, 6169.
16. Shawali, A. S.; Mosselhi, M. A. N.; Tawik, N. M. J. Org.
Chem. 2001, 66, 4055.
17. Hirota, K.; Monguchi, Y.; Sajiki, H.; Kiatde, Y. Synlett
1997, 6, 697.
18. Bush, K.; Macielag, M. Curr. Opin. Chem. Biol. 2000, 4, 433.
19. Sharma, P.; Kumar, A.; Pandey, P. Phosphorus, Sulphur
and Silicon 2003, 178, 583.
20. Sharma, P.; Kumar, A.; Sharma, S. Ind. J. Chem. 2004,
43B, 385.
CH3), 7.40 (s, 5H, C6H5), 8.21, 9.50 (s, 2H, 2 · NH). Anal.
Calcd for C12H11O2N5: C, 56.03; H, 4.28; N, 27.24.
Found: C, 55.95; H, 4.20; N, 27.21.
29. Analytical data for compound 4c: mp (ꢁC) 176–178; IR
(KBr) (cmꢀ1) 3780 (OH, enolized), 3470 (NH, bonded),
3210 (N–H ring), 1675 (C@O), 1590 (C@C/C@N), 1520,
1473, 1410 (C'C ring str.), 1300 (C–N), 1230 (C@O),
1012 (C–H in plane bending), 840, 740 (C–H out of plane
bending, 1,2-disubstituted benzene ring), 660 (C–Cl); 1H
NMR (d ppm) 1.30 (s, 1H, OH), 2.50 (s, 3H, CH3), 7.42 (d,
Ar–H6), 8.25, 9.20 (s, 2H, 2 · NH). Anal. Calcd for
C12H10O2N5Cl: C, 49.40; H, 3.43; N, 24.01. Found: C,
49.31; H, 3.33; N, 23.90.
21. Sharma, P.; Kumar, A.; Mandloi, A. Synth. Commun.
2003, 373, 33.
22. Jain, R.; Pandey, P. Bull. Electrochem. 1987, 2, 177.
23. Furniss, B. S.; Hannaford, A. J.; Roger, V.; Smith, P. W.
G.; Tatchell, A. K. In Vogel’s Textbook of Organic
Chemistry (Longmann) 1984.
24. Rapper, R. P. The Actinomycates 1901, 88, 12.
25. Kumar, A.; Sharma, P.; Nigam, A.; Indapurkar, P. Ind. J.
Chem. 1997, 36B, 445.
26. Dreisbach, R. R. Physical Properties of Chemical Com-
pound; Am. Chem. Soc: Washington, DC, 1961.
27. Skagerberg, B.; Bonelli, D.; Clementi, S.; Cruciani, G.;
Ebert, C. Quant. Struct. Act. Relat. 1989, 8, 32.
28. Analytical data for compound 4a: mp (ꢁC) 177–179; IR
(KBr) (cmꢀ1) 3761 (OH, enolized), 3473 (NH, bonded),
3217 (N–H ring), 1672 (C@O), 1595 (C@C/C@N), 1522,
1475, 1400 (C'C ring str.), 1302 (C–N), 1234 (C@O),
1017, 947 (C–H in plane bending, phenyl ring), 845, 771
(C–H out of plane bending, monosubstituted benzene
ring); 1H NMR (d ppm) 1.21 (s, 1H, OH), 2.50 (s, 3H,
30. Analytical data for compound 4g: mp (ꢁC) 172–174; IR
(KBr) (cmꢀ1) 3760 (OH, enolized), 3453 (NH, bonded),
3200 (N–H ring), 1680 (C@O), 1595 (C@C/C@N), 1500,
1472, 1400 (C'C ring str.), 1361 (NO2), 775 (C–H out of
1
plane bending, 1,3-disubstituted benzene ring); H NMR
(d ppm) 1.57 (s, 1H, OH), 2.50 (s, 3H, CH3), 7.08–7.17 (dd,
Ar–H4), 7.22 (t, Ar–H5), 7.28 (d, Ar–H6) 7.93 (s, 1H, Ar–
H2), 8.23, 9.52 (s, 2H, 2 · NH). Anal. Calcd for
C12H10O4N6: C, 47.68; H, 3.31; N, 27.81. Found: C,
47.60; H, 3.25; N, 27.73.
31. Analytical data for compound 4k: mp (ꢁC) 170–172; IR
(KBr) (cmꢀ1) 3771 (OH, enolized), 3460 (NH, bonded),
3215 (N–H ring), 1675 (C@O), 1595 (C@C/C@N), 1530,
1480, 1430 (C'C ring str.), 745 (C–H out of plane
bending, 1,2-disubstituted benzene ring); 1H NMR (d
ppm) 0.09 (s, 3H, CH3), 1.40 (s, 1H, OH), 2.51 (s, 3H,
CH3), 7.05 (d, Ar–H3), 7.57 (d, Ar–H6), 7.73–7.80 (t, Ar–
H4, Ar–H5), 8.20, 9.52 (s, 2H, 2 · NH). Anal. Calcd for
C13H13O2N5: C, 57.56; H, 4.80; N, 25.83. Found: C, 57.48;
H, 4.75; N, 25.70.