Green synthesis of tetrahydropyrimidine analogues and evaluation of their antimicrobial activity

Article abstract of DOI:10.1016/j.bmcl.2013.02.099

It is the first report of 1,3,4,5-tetrasubstituted 1,2,3,6- tetrahydropyrimidines derivatives, catalyzed by ZrOCl₂. The mild reaction conditions, excellent yields in shorter reaction time and evasion of cumbersome workup procedures make this pr

Full text of DOI:10.1016/j.bmcl.2013.02.099

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2632–2635
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Green synthesis of tetrahydropyrimidine analogues and evaluation
of their antimicrobial activity
⇑
Sunil N. Darandale, Dattatraya N. Pansare, Nayeem A. Mulla, Devanand B. Shinde
Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad 431004, MS, India
a r t i c l e i n f o
a b s t r a c t
Article history:
It is the first report of 1,3,4,5-tetrasubstituted 1,2,3,6-tetrahydropyrimidines derivatives, catalyzed by
ZrOCl2. The mild reaction conditions, excellent yields in shorter reaction time and evasion of cumbersome
workup procedures make this process economically lucrative for industrial application. The novelty and
highlight of the present method is the promising antibacterial and antifungal activity shown by com-
pounds (4a, 4b, 4e, 4f, 4h and 4l) compared to standard.
Received 3 January 2013
Revised 8 February 2013
Accepted 22 February 2013
Available online 4 March 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
One-pot
Green synthesis
1,2,3,6-Tetrahydropyrimidines derivatives
Antimicrobial activity
Pyrimidines and their analogues are considered as important
bioactive heterocycles. Tetrahydropyrimidine is an important het-
erocyclic ring which is widely explored in the past decade for
exhibiting interesting biological activities. In particular they are
screened for antiviral, anti-inflammatory and muscarinic agonist
activities.1–3 Tetrahydropyrimidine is also responsible for salt and
heat sensitivity of protein–DNA interactions.4 However, literature
reveals very few synthetic methods for synthesis of polysubstitut-
ed 1,2,3,6-tetrahydropyrimidines, which were also associated with
drawbacks like low yields, high temperature, long reaction time
and complicated procedures.5 Thus, one of the major challenge be-
fore synthetic chemist is the establishment of practical synthetic
method for the synthesis of tetrahydropyrimidine analogues. These
challenges can mainly be overcome by the employment of multi-
component reactions in synthesis, which would give higher yields
of complex product in shorter reaction time.6
nitric acid, acetic acid, sulphamic acid, sulphanilic acid and Zirconyl
oxychloride (ZrOCl2) at reflux temperature in water (Table 1).The
ZrOCl2 (Table 1, entry 8) was found to be superior catalyst for this
reaction by considering the reaction time (20 min) and yield (90%)
whereas the rest of the catalysts required longer reaction time
(45–140 min) and yielded (57–78%) less product. In addition the
ZrOCl2 is easily available having good activity at low cost and is easy
to handle due to its low toxicity when compared to the other used
catalyst (Table 1).
To evaluate the exact concentration of ZrOCl2 required for the
reaction, we investigated the model reaction (4a) using different
concentrations (Table 2) such as 2, 4, 6, 8, 10, 12 mol % the product
was formed in 55, 67, 72, 85, 90, 90% respectively. The result re-
vealed that, when the reaction was carried out in presence of 2, 4,
6 mol % of catalyst, it gave lower yield of product even after pro-
longed duration. While 8, 10 and 12 mol % of catalyst gave excellent
yields of product in shorter time. The optimal results were obtained
by 10 mol % of catalyst and this concentration was ideal to carry out
reaction smoothly (Table 2, entry 5). In order to evaluate the effect of
solvent, reactions were carried out in various solvent as enlisted in
(Table 3). Acetonitrile and isopropyl alcohol afforded moderate yield
72% and 65%, respectively. Methanol, ethanol and aqueous ethanol
(50%) resulted in good yields of 82%, 90% and 90%, respectively.
Whereas,waterfurnishedtheproductinexcellent90%yield(Table3,
entry 6), making it the most favorable solvent.
In continuation of our work,7 on the development of synthetic
methodologies and screening of the analogues, we have discovered
efficient method for the synthesis of 1,3,4,5-tetrasubstituted 1,2,3,6-
tetrahydropyrimidine analogues. The 1,3,4,5-tetrasubstituted
1,2,3,6-tetrahydropyrimidine analogues were synthesized effi-
ciently through the three component reactions of substituted
amines, substituted but-2-ynedioate and formaldehyde using
ZrOCl2 in water (Scheme 1). Initially, for the model reaction (4a), ani-
line (2 mmol) was treated with diethyl acetylene dicarboxylate
(1 mmol) and formaldehyde (4 mmol) without use of catalyst and
by using different catalysts like hydrochloric acid, sulphuric acid,
The possible mechanism of the reaction is shown in (Scheme 2)
wherein, the reactions involving substituted amines, formalde-
hyde, dimethyl and diethyl acetylene dicarboxylates (substituted
but-2-ynedioate) occur in the presence of 10 mol % ZrOCl2 in
aqueous medium. In the first step the amine reacts with the
⇑
Corresponding author. Tel.: +91 0240 2403308; fax: +91 0240 2400413.
E-mail address: dbssunil09@rediffmail.com (D.B. Shinde).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.02.099

S. N. Darandale et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2632–2635
2633
R
NH2
NH2
COOR
ROOC
ROOC
ZrOCl2
H2O
/ 20 Min
N
+
R
+
HCHO
+
R
N
COOR
R
4a-l
1
2
3
1
Scheme 1. Synthesis of 1,3,4,5-tetrasubstituted 1,2,3,6-tetrahydropyrimidines catalyzed by ZrOCl2 in water.
reaction indicating the flexibility and sturdiness of the method.
The practical applicability of this method was evaluated by using
substituted amines and both dimethyl and diethyl acetylene dicar-
boxylates to afford the desired products in good yields in shorter
reaction time (Table 4).
All the synthesized compounds were screened for in vitro anti-
microbial activity. The antibacterial activity was evaluated
against different bacterial strains such as Staphylococcus aureus
(NCIM-2901), Bacillus subtilus (NCIM-2063) and Escherichia coli
Table 1
The effect of catalyst on model reaction (4a)a
Entry
Catalyst
Time (min)
Yieldb (%)
1
2
3
4
5
6
7
8
No catalyst
180
140
125
90
75
55
32
57
62
65
72
76
78
90
Hydrochloric acid
Sulphuric acid
Nitric acid
Acetic acid
Sulphamic Acid
Sulphanilic Acid
Zirconyl oxychloride
45
20
(NCIM-2256). Minimum inhibitory concentration (MIC,
lg/mL) of
antibacterial activity was determined using broth dilution method
as per CLSI guidelines.8–11 Ciprofloxacin and ampicillin were used
as a standard drug for the comparison of antibacterial activity (Ta-
ble 5). The antifungal activity was evaluated against different fun-
gal strains such as Candida albicans (NCIM3471), Aspergillus flavus
(NCIM539) and Aspergillus niger (NCIM1196). MIC values of anti-
fungal activity were determined using standard agar dilution
method as per CLSI guidelines.8–11 Fluconazole and miconazole
were used as standard drugs for the comparison of antifungal
activity (Table 5). Dimethyl sulfoxide was used as solvent control.
From the antimicrobial data, it is observed that all the newly
synthesized compounds shows good to moderate level of antibac-
terial and antifungal activity (Table 5). The antimicrobial activity
data (Table 5), reveals that compounds (4a, 4b, 4e and 4f), all of
which having diethyl but-2-ynedioate, are found to be most active
and potent as antimicrobial agents among the series. These com-
pounds were found to be active against the tested fungal and bac-
a
Reaction condition: Aniline (2 mmol), diethyl acetylene dicarboxylate
(1 mmol), formaldehyde (4 mmol) in water (5 mL) at reflux temperature using
various catalyst (10 mol %).
b
Isolated yields.
Table 2
Effect of concentration of ZrOCl2 on model reaction (4a)a
Entry
Catalyst (Mol %)
Time (min)
Yieldb (%)
1
2
3
4
5
6
2
4
6
8
10
12
60
45
25
25
10
20
55
67
72
85
90
90
a
Reaction condition: Aniline (2 mmol), diethyl acetylene dicarboxylate
(1 mmol), Formaldehyde (4 mmol) in water (5 mL) at reflux temperature using
various concentration of ZrOCl2.
b
Isolated yields.
terial strains, having MIC values (15–60
12.5–50 g/mL for fungus) compatible with standard drugs, except
for bacterium S. aureus which shows MIC values between (60 and
100 g/mL). The compounds (4h, 4k and 4l) containing dimethyl
lg/mL for bacteria and
l
Table 3
Screening of solvent for model reaction (4a)a
l
Entry
Solvent
Time (min)
Yieldb (%)
but-2-ynedioate, shows reduced antibacterial properties compared
to (4b, 4e and 4f), while the antifungal activity of compounds (4h,
4k and 4l) remained unchanged for the tested fungal strains. The
compounds (4d and 4j), both formed by m-nitro aniline, are least
1
2
3
4
5
6
Acetonitrile
Isopropyl alcohol
Methanol
Ethanol
Ethanol–water (50%)
Water
25
30
15
13
10
10
72
65
82
90
90
90
active against tested bacterial strains (MIC 60–100
lg/mL) and
fungal strains (MIC 100 g/mL). The remaining compounds (4c,
l
4g and 4i) have intermediate antimicrobial activity compared to
standard. In short the compounds (4a, 4b, 4e, 4f, 4h, 4k, and 4l)
show promising antifungal activity indicating the future scope
for optimization. The structure activity relationship of the series
can be explained as,
a
Reaction condition: Aniline (2 mmol), diethyl acetylene dicarboxylate
(1 mmol), (4 mmol) formaldehyde and ZrOCl2 (10 mol %) in various solvents (5 mL)
at reflux temperature.
b
Isolated yields.
formaldehyde to form an N-methyleneaniline derivative with the
loss of water molecule. In the next step of hydroamination, the
amine, formaldehyde and the dicarboxylates reacts with each
other to form phenyl amino maleate derivatives. The final step is
the cycloaddition of N-methyleneaniline and phenyl amino male-
ate derivatives to form 1,3,4,5-tetrasubstituted 1,2,3,6-tetrahydro-
pyrimidine derivatives in good to excellent yields. The 1,3,4,
5-tetrasubstituted 1,2,3,6-tetrahydropyrimidines were obtained
within 15–20 min (Table 4) in high yields (85–91%). The reaction
proceeded smoothly to furnish the desired products in good yields
without substantial difference in time required for completion of
(1) Effect of alkyne substituent: The diethyl but-2-ynedioate (4a–
f) gave more potent molecules for the antibacterial activity.
The replacement of diethyl but-2-ynedioate by dimethyl
but-2-ynedioate (4g–l), resulted in diminished antibacterial
activity, however their antifungal properties remained
unchanged. In short the diethyl but-2-ynedioate was more
specific towards bacterial strains compared with dimethyl
but-2-ynedioate.
(2) Effect of Amine: Introduction of phenyl ring on both nitrogen of
pyrimidine ring (4a, 4g) shows promising antibacterial and
antifungal activity compared to standard drugs. Introduction

2634
S. N. Darandale et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2632–2635
CH2
H
H
ZrOCl2
-H2O
R
NH2
+
R
N
O
H
COOR
COOR
COOR
COOR
R
NH2
Hydro
+
R
N
+
Amination
H
H
HO
O
H
ROOC
ROOC
R
ROOC
ROOC
ROOC
R
R
N
H2C
N
N
-H2O
OH
ROOC
N
R
N
R
N
R
H
1,3,4,5-tetrasubstituted
1,2,3,6-tetrahydropyrimidines
Scheme 2. Plausible reaction mechanism for the synthesis of 1,3,4,5-tetrasubstituted 1,2,3,6-tetrahydropyrimidines catalyzed by ZrOCl2 in water.
Table 4
Synthesis of 1,3,4,5-tetrasubstituted 1,2,3,6-tetrahydropyrimidines derivatives (4a–l) using ZrOCl2 as catalyst in watera
Entry
Amine
Alkynes
Time (min)
Melting point (°C)
Yieldb (%)
4a
4b
4c
4d
4e
4f
4g
4h
4i
Aniline
p-Chloro aniline
Diethyl but-2-ynedioate
Diethyl but-2-ynedioate
Diethyl but-2-ynedioate
Diethyl but-2-ynedioate
Diethyl but-2-ynedioate
Diethyl but-2-ynedioate
Dimethyl but-2-ynedioate
Dimethyl but-2-ynedioate
Dimethyl but-2-ynedioate
Dimethyl but-2-ynedioate
Dimethyl but-2-ynedioate
Dimethyl but-2-ynedioate
15
17
15
18
20
20
16
18
15
20
19
20
234–236
263–265
219–221
237–239
258–260
272–274
197–199
259–261
241–243
250-252
235–237
243–245
90
88
90
89
86
85
91
87
88
91
89
90
p-Hydroxy aniline
m-Nitro aniline
2 Amino pyridine
2 Amino 6-methoxybenzothiazole
Aniline
p-Chloro aniline
p-Hydroxy aniline
m-Nitro aniline
4j
4k
4l
2 Amino pyridine
2 Amino 6-methoxybenzothiazole
a
Reaction condition: Substituted amines (2 mmol), substituted but-2-ynedioate (1 mmol), formaldehyde (4 mmol) and ZrOCl2 (10 mol %) in water (5 mL) at reflux
temperature.
b
Isolated yields.
Table 5
Antimicrobial activity of the synthesized compounds (4a–l)
Compound
MIC valuesa
E. coli
(
l
g/mL)
B. subtilus
S. aureus
C. Albicans
A. Flavus
A. Niger
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
25
25
40
60
20
20
30
40
80
100
40
25
25
50
—
60
80
15
15
50
100
60
15
60
60
80
80
60
40
15
50
—
50
25
75
100
25
25
50
25
75
100
25
25
—
25
25
50
100
12.5
12.5
25
25
50
100
12.5
12.5
—
—
25
25
25
50
100
25
12.5
25
25
50
100
25
12.5
—
—
25
12.5
100
100
100
100
90
100
90
80
80
100
25
50
—
Ciprofloxacin
Ampicillin
Fluconazole
Miconazole
—
40
12.5
—
—
—
12.5
a
Values are the average of three readings.
of p-chloro (4b, 4h) on phenyl ring decreases the antibacterial
activity against bacterium S. aureus (MIC from 60 to 80 g/
mL for (4b) and for (4h) MIC from 90 to 100 g/mL)
whereas it increases antifungal activity against the fungus
C. albicans (MIC from 50 to 25 g/mL for both 4b and 4h),
when compared to (4a, 4g) respectively. The introduction
of p-hydroxy on phenyl ring (4c, 4i) reduces the overall
antimicrobial activity of molecule on the tested strains
compared to (4a, 4g) respectively. The introduction of
m-nitro on phenyl ring (4d, 4j) further reduces the activity
compared to (4a, 4g) respectively and were the least
active molecule of the series.
l
l
l

S. N. Darandale et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2632–2635
2635
(3) Effect of heterocyclic amine: The 2 amino pyridine (4e, 4k)
Supplementary data
and 2 amino 6-methoxybenzothiazole (4f, 4l) makes the
molecule more potent compared to the respective unsubsti-
tuted analogues (4a, 4g). This clearly highlights that the het-
erocycles help in increasing the antimicrobial activity.
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
02.099.
(4) Strain specific effects of heterocycles: In compounds (4f, 4l),
the replacement of pyridine ring by 6-methoxybenzothiaz-
ole ring, increased the activity against bacterium E. coli
References and notes
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Res. 2000, 33, 879; (c) Kappe, C. O. Tetrahedron 1993, 49, 6937.
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(b) Lagoja, I. M. Chem. Biodivers. 2005, 2, 1; (c) Kappe, C. O. Eur. J. Med. Chem.
2000, 35, 1043.
3. (a) Wisen, S.; Androsavich, J.; Evans, C. G.; Chang, L.; Gestwicki, J. E. Bioorg. Med.
Chem. Lett. 2008, 18, 60; (b) Evans, C. G.; Wisen, S.; Gestwicki, J. E. J. Biol. Chem.
2006, 281, 33182.
(MIC from 60 to 15
lg/mL) and fungus A. niger (MIC from
25 to 12.5 g/mL), while the MIC values of this compounds
l
for other bacterial and fungus strains remained unchanged.
Thus it indicates that such a modifications are important
in making the molecule more potent against the bacterium
E. coli and fungus A. niger.
4. Rodina, M.; Vilenchik, K.; Moulick, J.; Aguirre, J.; Kim, A.; Chiang, J.; Litz, C. C.;
Clement, Y.; Kang, Y.; She, N.; Wu, S.; Felts, P.; Wipf, J.; Massague, X.; Jiang, J. L.;
Brodsky, G. W.; Krystal, G. Nat. Chem. Biol. 2007, 3, 498.
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(c) Biswnath, D.; Kanth, B. S.; Shinde, D. B.; Kamble, V. T. Helv. Chim. Acta 2011,
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6. Hulme, C. In Multicomponent Reactions; Zhu, J., Bienayme , H., Eds.; Wiley-VCH:
Weinheim, 2005, pp. 311–341.
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8. Medicinal Microbiology; Cruickshank, R., Duguid, J. P., Marmion, B. P., Swain, R.
H. A., Eds., 2nd ed.; Churchill Livingstone: London, 1975; p 2.
9. Collins, A. H. Microbiological Methods, 2nd ed.; Butterworth: London, 1976.
10. Khan, Z. K. In vitro and vivo screening techniques for bioactivity screening and
evaluation, Proc. Int. Workshop UNIDO-CDRI, 1997, 210.
11. (a) Duraiswamy, B.; Mishra, S. K.; Subhashini, V.; Dhanraj, S. A.; Suresh, B.
Indian J. Pharm. Sci. 2006, 68, 389; (b) Saundane, A. R.; Rudresh, K.;
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Highlight of synthesis of 1,3,4,5-tetrasubstituted 1,2,3,6-tetrahy-
dropyrimidines derivatives:12
The novelty and highlight in synthesis of 1,3,4,5-tetrasubstitut-
ed 1,2,3,6-tetrahydropyrimidines derivatives is (i) the first report
of 1,3,4,5-tetrasubstituted 1,2,3,6-tetrahydropyrimidines deriva-
tives, catalyzed by ZrOCl2; (ii) evasion of cumbersome workup pro-
cedures; (iii) development of eco-friendly process by omission of
organic solvents; (iv) Establishment of ZrOCl2 as a versatile catalyst
which is non-toxic and economically feasible to use in multicom-
ponent reactions and also for hydroamination reactions; (v) Excel-
lent yields in shorter reaction time making the process
economically lucrative for industrial application; (vi) opening the
horizon for the synthesis of series of 1,3,4,5-tetrasubstituted
1,2,3,6-tetrahydropyrimidines with promising antibacterial and
antifungal activity which are yet to be explored and exploring
them for other biological applications.
In conclusion, we report a green, practical and facile approach
for the synthesis of some new tetrahydropyrimidine analogues
by multicomponent reaction of substituted amines, dialkyl acety-
lene dicarboxylates, and formaldehyde using ZrOCl2 in water. The
mild reaction conditions, shorter reaction time and promising anti-
bacterial activity of (4a, 4b, and 4f) and antifungal activity (4e, 4f,
4k and 4l) of the compounds compared to standard are the advan-
tages of the present method. Lastly to develop potent antibacterial
agent, diethyl but-2-ynedioate is better choice than dimethyl but-
2-ynedioate, while for the development of antifungal agent both
alkynes were equally pertinent.
12. General procedure for the synthesis of 1,3,4,5-tetrasubstituted 1,2,3,6-
tetrahydropyrimidines derivatives (4a–l):
In a 50 ml round bottom flask, substituted amine (2 mmol), substituted but-2-
ynedioate (1 mmol), formaldehyde (4 mmol) and ZrOCl2 (10 mol %) in (5 mL)
water. The reaction mixture was refluxed. The progress of reaction was
monitored on TLC (30% ethyl acetate/n-hexane). The reaction got to completion
within 15–20 min (Table 5), the product was obtained by filtration and dried
in-vaccuo. The product was recrystallized using ethanol as solvent, with 85–
91% yield (Table 5).
Acknowledgments
The authors are thankful to the Head, Department of Chemical
Technology, Dr. Babasaheb Ambedkar Marathwada University,
Aurangabad 431004, MS, India for providing the laboratory facility.