Synthesis and biological evaluation of some new tricyclic pyrrolo[3,2-e]tetrazolo[1,5-c]pyrimidine derivatives as potential antitubercular agents

Article abstract of DOI:10.1002/ardp.201800040

A series of new tricyclic pyrrolo[3,2-e]tetrazolo[1,5-c]pyrimidines 8a–l were synthesized and characterized by IR, NMR (¹H and ¹³C), and mass spectral analysis. The newly synthesized compounds 8a–l were inspected for their in vitro antitubercular activity against Mycobacterium tuberculosis (MTB) H₃₇Ra using an established XTT reduction menadione assay (XRMA). The title compounds exhibited minimum inhibitory concentrations (MIC₉₀) ranging from 0.09 to >30 μg/mL. Five compounds (8c, 8i–l) were further confirmed for their dose-dependent effect against MTB. These compounds were evaluated in the THP-1 infection model, where 8i (MIC₉₀ = 0.35 μg/mL), 8j (MIC₉₀ = 1.17 μg/mL), 8k (MIC₉₀ = 2.38 μg/mL), and 8l (MIC₉₀ = 1.17 μg/mL) demonstrated significant antitubercular activity. All the ex vivo active compounds showed insignificant cytotoxicity against the human cancer cell lines, HeLa, MCF-7, and THP-1. Inactivity of all these compounds against Gram positive and Gram negative bacteria indicates their specificity. Molecular docking studies in the active site of the sterol 14alpha-demethylase (CYP51) enzyme revealed a similar binding mode to the native ligand in the crystal structure, thereby helping to understand the ligand–protein interactions and to establish a structural basis for inhibition of MTB. The results suggest novel pharmacophores as selective and specific inhibitors against MTB that can be explored further to synthesize lead compounds against tuberculosis. In summary, the results clearly indicate the identification of some novel, selective, and specific inhibitors against MTB that can be explored further for potential antitubercular drugs.

Full text of DOI:10.1002/ardp.201800040

Received: 14 February 2018
Revised: 1 June 2018
Accepted: 8 June 2018
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DOI: 10.1002/ardp.201800040
FULL PAPER
Synthesis and biological evaluation of some new tricyclic
pyrrolo[3,2-e]tetrazolo[1,5-c]pyrimidine derivatives as
potential antitubercular agents
1
1
2
2
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|
|
|
Yogesh Patil
Ramesh Shingare
Amit Choudhari
Rachana Borkute
2
1
|
Dhiman Sarkar
Balaji R. Madje
1
Department of Chemistry, Vasantrao Naik
Mahavidyalaya, Aurangabad, India
Abstract
2
Combi Chem Bio Resource Centre, National
A series of new tricyclic pyrrolo[3,2-e]tetrazolo[1,5-c]pyrimidines 8a–l were synthe-
Chemical Laboratory, Pune, India
1
13
sized and characterized by IR, NMR ( H and C), and mass spectral analysis. The newly
synthesized compounds 8a–l were inspected for their in vitro antitubercular activity
against Mycobacterium tuberculosis (MTB) H37Ra using an established XTT reduction
menadione assay (XRMA). The title compounds exhibited minimum inhibitory
concentrations (MIC90) ranging from 0.09 to >30 μg/mL. Five compounds (8c, 8i–l)
were further confirmed for their dose-dependent effect against MTB. These
compounds were evaluated in the THP-1 infection model, where 8i
(MIC90 = 0.35 μg/mL), 8j (MIC90 = 1.17 μg/mL), 8k (MIC90 = 2.38 μg/mL), and 8l
Correspondence
Dr. Balaji R. Madje, Department of Chemistry,
Vasantrao Naik Mahavidyalaya, Aurangabad
431003, Maharashtra, India.
Email: drmadjebr@gmail.com
Funding information
Department of Science and Technology, New
Delhi; Science and Engineering Research
Board
(
MIC90 = 1.17 μg/mL) demonstrated significant antitubercular activity. All the ex vivo
active compounds showed insignificant cytotoxicity against the human cancer cell
lines, HeLa, MCF-7, and THP-1. Inactivity of all these compounds against Gram positive
and Gram negative bacteria indicates their specificity. Molecular docking studies in the
active site of the sterol 14alpha-demethylase (CYP51) enzyme revealed a similar
binding mode to the native ligand in the crystal structure, thereby helping to
understand the ligand–protein interactions and to establish a structural basis for
inhibition of MTB. The results suggest novel pharmacophores as selective and specific
inhibitors against MTB that can be explored further to synthesize lead compounds
against tuberculosis. In summary, the results clearly indicate the identification of some
novel, selective, and specific inhibitors against MTB that can be explored further for
potential antitubercular drugs.
K E Y W O R D S
antituberculosis, cytotoxicity, molecular docking, tricyclic pyrrolo[3,2-e]tetrazolo[1,5-
c]pyrimidine
1
|
INTRODUCTION
One-third of the world's population is infected with latent TB, which is
at a risk of developing active TB disease. Despite the availability of
Among widespread infectious diseases, tuberculosis (TB) caused by
several first and second line drugs, TB remains the top 10 death causes
worldwide with leading in death counter among the infectious diseases
[1]
Mycobacterium tuberculosis (MTB) is the primary health problem.
|
Arch Pharm Chem Life Sci. 2018;1–8.
wileyonlinelibrary.com/journal/ardp
© 2018 Deutsche Pharmazeutische Gesellschaft
1

2
PATIL ET AL.
|
[2]
well above HIV/AIDS. The current preferred treatment strategy for
active drug-sensitive TB disease is a minimum of 6 months of therapy
with isoniazid, rifampicin, pyrazinamide, ethambutol during the first
protons. 2.49 (t) and 4.53 (dd) belong to pyrimidine ring attached CH
and CH, respectively.
2
Compound 4 was treated with aqueous hydrochloric acid solution
1
2
months (the intensive phase of treatment), followed by isoniazid
(5 N) to get 4-hydroxypyrrolo[2,3-d]pyrimidine (5). The H NMR
(
INH) and rifampicin (RIF) for 4 months (the continuation phase).
spectrum showed δ 6.47 (d), 7.03 (d), and 7.83 (s) aromatic ring proton.
Besides, two singlets were observed at δ 11.78 and 11.86 with
However, use of these drugs over longer duration results in the
emergence of resistant strains. The treatment for resistant strains has
to be switched to second or third line drugs; further exploding the cost
of the treatment. Thus, their arises the need for the development of
potent and cost-effective drug scaffolds for antitubercular.
2
D O exchangeable due to ─OH and ─NH. Chlorination of compound 5
with phosphorus oxychloride produced 4-chloropyrrolo[2,3-d]pyrimi-
dine (6). This was further treated with sodium azide in DMF solvent to
get our core moiety (7). This tricyclic compound was confirmed and
characterized by IR, mass, and NMR. All experimental procedure and
spectral details of compounds were in full agreement with the
proposed structure.
[3]
[4]
In the literature, tetrazole and pyrrolo[2,3-d]pyrimidine are
well-explored scaffold and also crucial constituent for various drug
molecules. Moreover, shows widespread activity such as antimicro-
[5,6]
[7,8]
[9,10]
[11,12]
bial,
tensive,
antifungal,
13]
antimalarial,
anticancer,
antihyper-
Here, while compound 6 reacts with sodium azide two possibilities
will be occurred. Either tetrazole ring formation or nucleophilic
[
[14–18]
and antituberculosis.
Some tetrazole and pyrimidine
[
25]
contain a moiety which shows antituberculosis activity as depicted in
Figure 1.
displacement to afford 4-azido derivative.
Literature evidence
suggests tetrazole ring formation can be confirmed by IR spectrum of
−
1
Diversity in the scaffold is a key ongoing exercise for the drug
design and development to achieve better affinity and efficacy to the
molecules. The present investigation deals with the combination of
both scaffolds tetrazole and pyrrolo[2,3-d]pyrimidine to get the
synergetic effect. Tricyclic pyrrolo[3,2-e]tetrazolo[1,5-c]pyrimidine
core moiety also shows activity such as anticancer, antimalarial, and
compound 7. It showed no absorbance band in the 2100 cm region
[26]
which exclude the possibility 4-azido derivative
further alkyl, benzyl
halide, and sulfonyl substitution on tricyclic compound (7) produced
derivatives (8a–l) with moderate to better yield (Table 1). Spectral data
are incorporated in the Supporting Information.
[19,20]
antihypertensive.
All the newly synthesized tricyclic 7H-pyrrolo
2.2
|
Biological evaluation
[3,2-e]tetrazolo[1,5-c]pyrimidine (7) and its derivatives were screened
for antituberculosis and antibacterial activities along with docking
study.
All the newly synthesized compounds (8a–l) were tested against
M. tuberculosis H37Ra (ATCC 25177) at two time points, i.e., days 8 and
1
2 using an established XTT reduction menadione assay (XRMA).
Among the synthesized compounds, 8c, 8i–8l displayed excellent
activity toward M. tuberculosis H37Ra with MIC90 value less than
2
2
|
RESULTS AND DISCUSSION
Chemistry
6
μg/mL. According to the structure–activity relationships of antitu-
.1
|
berculosis compounds 8j (MIC: 5.63), 8k (MIC: 2.13), and 8l (MIC: 2.51)
greatly influence on introduction of sulfonyl amide group contain
methyl, phenyl, and 4-methyl phenyl, respectively. Other attempts to
introduce aliphatic and aromatic compounds without sulfonyl group
failed to produce active compound except 8c and 8i, which contain
aliphatic propyl and aromatic biphenyl ring with MIC values 28.11 and
0.33 μg/mL, respectively. The results of the screening are tabulated in
Table 2.
Synthesis of pyrrolo[2,3-d]pyrimidines plenty permutation and combi-
[21]
nation have been published depending upon the target molecules.
We chalked out a synthetic pathway for our target molecules which is
illustrated in Scheme 1.
Synthesis of 4-chloropyrrolo[2,3-d]pyrimidine was reported in several
[22–24]
literatures.
Based on literature condensation of ethyl cyanoacetate
with2-bromo-1,1-diethoxyethanegavedesiredethyl2-cyano-4,4-diethoxy
butanoate (3). Cyclization of compound 3 by using thiourea produced
The synthesized compounds 8c and 8i–l were further tested
against mycobacteria within THP-1 host macrophages (Table 3).
Compounds exhibiting MIC90 less than 5 μg/mL was considered as a
potent compound. Ex vivo studies against M. tuberculosis revealed the
strong anti-tubercular activity of 8i–l. Moreover, all the compounds
showed time-dependent activity when measured at two time points,
i.e., days 8 and 12 (Table 3). Among derivatives, 8i showed significant
activity with MIC90 0.76 and 0.35 μg/mL, respectively, at days 8 and
12. However, the activity is not profound as that of rifampicin.
Experimental details have been presented in the Supporting
Information.
6-amino-5-(2,2-diethoxyethyl)-2-mercaptopyrimidine-4-ol were obtained.
Further desulfurization by using Raney nickel produced 6-amino-5-(2,2-
diethoxyethyl)pyrimidin-4-ol (4).
To avoid some lacuna of reported synthetic route, i.e., desulfuri-
zation stage and get directly pyrimidine ring compound (4) from
compound 3, cyclization has been performed with formamidine
acetate instead of thiourea. 6-Amino-5-(2,2-diethoxyethyl)pyrimidin-
4
-ol (4) formation was confirmed by spectral data. IR spectrum shows
−1
two absorption bands at 3387 and 3337 cm due to primary amine
−
1
1
(
─NH
of compound 4 showed δ 7.69 (s) is due to pyrimidine ring aromatic
proton, 6.06 (s) and 11.47(s) D O exchangeable proton of ─NH and
OH, respectively. 1.05 (m), 3.37 (q), and 3.56 (q) show 2 O-CH -CH
2
) and 3148 cm due to phenolic ─OH. The H NMR spectrum
The active compounds (8i–l) from ex vivo infection model were
evaluated for cytotoxicity on three human cancer cell lines THP-1
(acute monocytic leukemia), HeLa (human epithelial cervical cancer),
2
2
─
2
3

PATIL ET AL.
| 3
FIGURE 1 Antituberculosis active compounds contain tetrazole and pyrimidine skeleton
SCHEME 1 Synthetic route for the preparation of title compounds 8a–l. Reagents and conditions: (a) K
2
CO
3
, DMF, 145–150°C, 78%; (b)
, DMF, 25–30°C,
3
, DMF, 25–30°C, 82–92%; (g) alkyl/benzyl sulfonyl chloride, aq. NaOH, acetone, 25–30°C, 70–75%
formamidine acetate, NaOEt, EtOH, 76–78°C, 85%; (c) 5 N HCl, H
5%; (f) alkyl/benzyl halide, K CO
2 3 3
O, 25–30°C, 90%; (d) POCl , 100–105°C, 95%; (e) NaN
8
2

4
PATIL ET AL.
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TABLE 1 Physical data of tricyclic pyrrolo[3,2-e]tetrazolo[1,5-c]pyrimidine derivatives (8a–l)
Entry
Compound
R
Yields (%)
88
m.p. (°C)
154–156
146–148
134–136
138–141
140–142
175–177
182–184
188–190
179–181
156–158
169–172
150–152
1
2
3
4
5
6
7
8
9
8a
8b
8c
8d
8e
8f
8g
8h
8i
Methyl
Ethyl
90
Propyl
92
Ethyl-4-butanoate
3-Butene
85
82
Benzyl
86
4-Methylbenzyl
4-Nitrobenzyl
2′-Nitrile-1,1′-biiphenyl benzyl
Methyl sulfonyl
Phenyl sulfonyl
4-Methylphenyl sulfonyl
80
82
92
10
11
12
8j
75
8k
8l
72
70
MCF-7 (human mammary epithelial) using MTT assay and IC50 values
were determined. The assay measures the reduction of yellow
Compounds 8a–l were further screened against (Gram +ve
Bacillus subtilis and Staphylococcus aureus, Gram −ve Escherichia coli
and Pseudomonas aeruginosa) at 3 µg/mL concentration, to assess
their selectivity toward MTB. The antimicrobial activity is summa-
rized in Supporting Information Table S1. None of the compound
showed significant activity toward any of the screened bacterial
strain.
3
-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide by
mitochondrial succinate dehydrogenase. The cytotoxic effect of these
compounds was checked to determine the growth inhibition (GI50 and
GI90) (Table 4). It has been noticed that all the active derivatives (8i–l)
showed GI90 values >100 µg/mL in all the cancer cell lines.
Further, the selectivity of the compounds 8i–l toward human cell
lines against MTB was determined via their selectivity index (SI) (Table
2
.3
|
Molecular docking study
5
). SI is a significant parameter used to calculate the efficiency of
antimicrobial agents. It is a ratio between IC50 (cytotoxicity) and MIC90
anti-tubercular activity) value. According to drug susceptibility study
Besides, theoretical predictions of molecular docking studies were
found to be in harmony with the in vitro antitubercular data. To gain an
insight into binding mode and the thermodynamic interactions
that govern the binding of the most active pyrrolo[3,2-e]tetrazolo-
[1,5-c]pyrimidine derivatives can be utilized for further screening
(Supporting Information).
(
of TB, compounds that exhibited SI values >10 were considered to be
nontoxic and effective. It was found that 8i–l have a SI of ≥10 in MTB
when compared to all the three cell lines. Importantly, 8i possessed a
more favorable selectivity index (SI > 100) against all three cell lines.
TABLE 2 In vitro antitubercular activity of compounds against tuberculosis H37Ra
Day 8
Day 12
Entry
IC50 (μg/mL)
6.93
MIC90 (μg/mL)
>30
IC50 (μg/mL)
2.12
MIC90 (μg/mL)
>30
8
8
8
8
8
8
8
8
8
8
8
8
a
b
c
d
e
f
12.87
7.11
>30
20.09
0.15
>30
28.11
>30
9.12
>30
17.29
14.3
26.06
7.07
>30
>30
>30
>30
>30
>30
g
h
i
1.73
>30
6.17
>30
>30
>30
>30
>30
0.09
0.33
0.12
0.72
4.74
2.07
1.2
j
0.83
5.63
0.71
k
l
0.37
2.13
0.74
1.08
2.51
0.43
Rifampicin
0.020
0.42
0.0019
0.80

PATIL ET AL.
| 5
TABLE 3 Ex vivo antitubercular activity of selected compounds at days 8 and 12
Day 8
Day 12
Entry
IC50 (µg/mL)
6.30
MIC90 (µg/mL)
26.58
0.76
IC50 (µg/mL)
0.59
MIC90 (µg/mL)
25.51
0.35
8
8
8
8
8
c
i
0.03
0.10
j
0.40
3.50
0.21
1.17
k
l
0.27
3.84
0.77
2.38
0.12
4.68
0.40
1.17
Rifampicin
0.020
0.53
0.0019
0.80
3
|
CONCLUSION
All the chemicals such as sodium salt XTT and MTT, DMSO,
ampicillin, and rifampicin were purchased from Sigma–Aldrich, USA.
Dubos medium was purchased from DIFCO, USA. Synthesized
compounds were dissolved in DMSO and it was used as a stock
solution (10 mg/mL) for further biological testing.
In summary, a series of new pyrrolo[3,2-e]tetrazolo[1,5-c]pyrimidine
and its derivatives contain alkyl, benzyl, and sulfonyl moieties. These
compounds were characterized and screened for antituberculosis
activity. Among all tested compounds 8i–l demonstrated significant
inhibition against MTB H37Ra. SI values for these compounds were
The InChI codes of some of the investigated compounds together
with some biological activity data are provided as Supporting
Information.
≥
10 against all the cell lines. These results are encouraging to better
define and optimize for further screening.
4
.1.2 Synthesis of ethyl 2-cyano-4,4-
|
4
4
4
|
EXPERIMENTAL
Chemistry
diethoxybutanoate (3)
A solution of ethyl 2-cyanoacetate (10 g, 88 mmol), 2-bromo-1,1-
diethoxyethane (17.5 g, 88 mmol) were dissolved into DMF (50 mL)
.1
|
2 3
and anhydrous K CO (24.4 g, 132 mmol) under nitrogen atmosphere.
.1.1 General
|
After addition, the reaction mixture was maintained at 145–150°C for
5–6 h. The reaction mixture was monitored by TLC. After completion
of the reaction, mixture was cooled at ambient temperature and
diluted with water (100 mL). The mixture was then extracted with
heptane (3 × 40 mL), washed with water (50 mL) and brine solution
All reagents, solvents, and raw materials are commercially available and
were used without further purification. Melting points were deter-
mined by the open capillary method and are uncorrected. IR spectra
−1
(
neat in cm ) were recorded using Perkin Elmer Spectrum-100
1
13
analyzer. NMR ( H and C) spectra were recorded in CDCl
3
and
4
(50 mL). The combined organic layer was dried over anhydrous MgSO ,
DMSO using a JEOL 400 MHz FT NMR spectrometer; the chemical
shifts are reported in ppm relative to TMS. The following abbreviations
were used for spin multiplicity: s = singlet, d = doublet, t = triplet,
dd = double doublet, m = multiplet, brs = broad singlet. Chemical shifts
filtered, and concentrated in vacuo. The organic residue was purified by
column chromatography on silica gel (using 10% ethyl acetate in
hexane as eluting solvents) to afford pale yellow oil of 3 (15.8 g, 78%).
−
1 1
IR (film): υ 2190 (─CN), 1775 (CO) cm ; H NMR (400 MHz, CDCl
δ 1.21 (m, J = 7.1 Hz, 2CH ), 1.31 (t, J = 7.2 Hz, CH ), 2.24 (m, CH
3.51 (m, ─OCH ─CH ), 3.70 (m, CH, and ─OCH ─CH ), 4.26 (q,
J = 7.1 Hz, ─OCH ─CH ), 4.69 (t, J = 5.6 Hz, 1H); C NMR (100 MHz,
CDCl ): δ 14.2, 15.3, 33.7, 62.8, 62.9, 100.1, 116.5, 166; MS (EI): m/z
calcd. for [C11
3
):
1
3
for C NMR are reported in ppm relative to the solvent peak. Mass
spectrometry was carried out using an Agilent LC/MSD Trap 1100
series. The reaction monitoring and purity of the compounds were
checked by thin layer chromatography (TLC) by using silica gel coated
aluminum sheets (Silica Gel 60 F254).
3
3
2
),
2
3
2
3
1
3
2
3
3
H19NO
4
+H]: 230.13. Found: 230.13.
TABLE 4 Growth inhibition of concentration of selective actives on of cell line
THP-1
HeLa
MCF-7
Code
GI50 (μg/mL)
>100
GI90 (μg/mL)
>100
GI50 (μg/mL)
>100
GI90 (μg/mL)
>100
GI50 (μg/mL)
>100
GI90 (μg/mL)
>100
8
8
8
8
i
j
>100
>100
>100
>100
>100
>100
k
l
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
Rifampicin
>100
>100
>100
>100
>100
>100

6
PATIL ET AL.
|
TABLE 5 Selectivity index of active analogs against Mycobacterium
tuberculosis H37Ra at day 12
gave compound 6 white solid (10.2 g, 90%), m.p. 154–158°C. IR (film)
−1 1
υ: 3120, 2966 (NH), 1600, 1557 (CC) cm
DMSO-d ): δ 6.59 (d, J = 3.4 Hz, 1H), 7.69 (d, J = 3.4 Hz, 1H), 8.58 (s,
1H), 12.57 (s, NH); C NMR (100 MHz, DMSO-d
; H NMR (400 MHz,
Code
THP-1
3.92
HeLa
3.92
MCF-7
3.92
6
1
3
8
8
8
8
8
c
i
6
): δ 99.35, 117.13,
1
1
28.63, 150.83, 152.16, 152.31; MS (EI): m/z calcd. for C ClN +H]:
6
H
4
3
285.71
85.47
42.01
85.47
285.71
85.47
42.01
85.47
285.71
85.47
42.01
85.47
+
+
54.0. Found: 154 (M+H) , 156 (M+2+H) .
j
k
l
4.1.6 Synthesis of 7H-pyrrolo[3,2-e]tetrazolo[1,5-c]-
|
pyrimidine (7)
4
.1.3
|
Synthesis of 6-amino-5-(2,2-diethoxy-ethyl)
A mixture of 4-chloropyrrolo[2,3-d]pyrimidine (6) (15 g, 100 mmol), and
sodium azide (8 g, 125 mmol) in DMF (75 mL) was stirred at ambient
temperaturefor12 h. The reactionmixturewasmonitored by TLC. After
completion of the reaction, mixture was filtered, washed with water
pyrimidin-4-ol (4)
A solution of ethyl 2-cyano-4, 4-diethoxy butanoate (10 g, 44 mmol) in
5
0 mL of ethanol and sodium ethoxide (2.3 g, 44 mmol) was added in
(
(
(
75 mL), anddried under vacuum to afford compound 7 pale yellow solid
14.2 g, 90%), m.p. 200–203°C. IR (film) υ: 3135, 3071 (NH), 1628
portionwiseunderaninertatmosphereatambienttemperature.Freshly
prepared solution of formamidine acetate in ethanol (5.7 g, 54 mmol in
−1 1
CC) cm ; H NMR (400 MHz, DMSO-d
.67 (d, J = 3.3 Hz, Ar-H), 9.83 (s, Ar-H), 12.95 (s, NH); C NMR
100 MHz, DMSO-d ): δ 101.34, 101.80, 125.80, 132.81, 142.05,
46.35; MS (EI): m/z calcd. for [C +H]: 161.05. Found: 161.
6
): δ 7.05 (d, J = 3.3 Hz, Ar-H),
5
0 mL) was added in dropwise manner into the reaction mass and the
13
7
mixturewasrefluxed at 78°C for 5 h. The reactionmixture was gradually
cooled at 25–30°C, then further cooled at 5–10°C and the pH was
adjustedat7using5N HCl(5 mL). Then, thereactionmixturewasstirred
at 5–10°C for anhour, filteredand washed with water (50 mL), and dried
in vacuo to afford compound 4 brown solid (8.4 g, 85%), m.p.
(
6
1
6 4 6
H N
4.1.7
|
General procedure for the synthesis of
−1
1
1
93–195°C. IR (film) υ: 3387 (NH
2
), 3148 (OH), 1662 (CC) cm
): δ 1.05 (t, 2CH ), 2.49 (t, ─CH ), 3.37 (q,
), 3.56 (q, ─OCH ─CH ), 4.53 (m, CH), 6.06 (s, NH ), 7.69
s, Ar-H), 11.47 (s, OH); C NMR (100 MHz, DMSO-d ): δ 15.31, 28.74,
1.36, 93.16, 101.85, 146.97, 161.44, 161.73; MS (EI): m/z calcd. for
−H]: 226.13. Found: 226.
;
compounds 8a–i
H NMR (400 MHz, DMSO-d
6
3
2
To
a solution of 7H-pyrrolo[3,2-e]tetrazolo[1,5-c]pyrimidine (7)
─
OCH ─CH
2
3
2
3
2
(
12 mmol), potassium carbonate (18 mmol) and appropriate alkyl and
1
3
(
6
aryl halide (14 mmol) were added DMF (10 mL) at ambient tempera-
ture. The reaction mixture was monitored by TLC. After completion of
the reaction, mixture was quenched into water (50 mL) and precipita-
tion obtained. The residue was filtered, washed with water, and dried
under vacuum to afford compounds 8a–i (82–92%).
6
[
10 17 3 3
C H N O
4
.1.4 Synthesis of 4-hydroxypyrrolo[2,3-d]-pyrimidine
|
(5)
7
-Methyl pyrrolo[3,2-e]tetrazolo[1,5-c]pyrimidine (8a)
A mixture of 6-amino-5-(2, 2-diethoxy-ethyl)pyrimidin-4-ol (4) (10 g,
4 mmol) was added 50 mL of 5 N HCl, then reaction mass stirred at
Off white solid, yield: 88%, m.p. 154–156°C. IR (film) υ: 3115, 2979,
4
−
1 1
1
635, 1487, 1352 cm
;
H NMR (400 MHz, DMSO-d
), 7.05 (d, J = 3.4 Hz, Ar-H), 7.73 (d, J = 3.4 Hz, Ar-H), 9.88 (s, Ar-H);
): δ 32.19, 100.28, 101.80, 129.68,
32.81, 141.28, 146.22; MS (EI): m/z calcd. for [C +H]: 175.07.
Found: 175.0.
6
): δ 3.98 (s,
ambient temperature for 4 h. The reaction mixture was monitored by
TLC. After completion of the reaction, mixture was filtered, washed
with water (50 mL), and dried under vacuum to obtain compound 5 off
white solid (5.3 g, 90%), m.p. 242–245°C. IR (film) υ: 3094, 3059 (OH),
CH
3
1
3
C NMR (100 MHz, DMSO-d
6
1
7 6 6
H N
−1 1
1
663, 1574 (CC) cm
6
; H NMR (400 MHz, DMSO-d ): δ 11.86 (s,
NH), 11.78 (s, OH), 7.83 (s, 1H), 7.03 (d, J = 3.4 Hz, 1H), 6.47 (d,
13
7-Ethyl pyrrolo[3,2-e]tetrazolo[1,5-c]pyrimidine (8b)
Pale yellow solid, yield: 90%, m.p. 146–148°C. IR (film) υ: 3103, 3057,
J = 3.4 Hz, 1H); C NMR (100 MHz, DMSO-d
6
): δ 101.98, 107.66,
20.36, 143.23, 148.08, 158.49; MS (EI): m/z calcd. for [C O+H]:
36.12. Found: 136.1.
1
1
6 5 3
H N
−
1 1
1
633, 1487 cm ; H NMR (400 MHz, DMSO-d
6
): δ 1.45 (t, J = 7.2 Hz,
CH
3
), 4.42 (q, J = 7.2 Hz, N-CH
2
-CH
3
3
), 7.07 (d, J = 3.4 Hz, Ar-H), 7.80 (d,
1
J = 3.4 Hz, Ar-H), 9.88 (s, Ar-H); C NMR (100 MHz, DMSO-d
6
): δ
4
.1.5
|
Synthesis of 4-chloropyrrolo[2,3-d]pyrimidine
1
6.51, 41.08, 100.18, 102.62, 128.97, 133.50, 141.40, 146.97; MS
(
6)
(
EI): m/z calcd. for [C +H]: 189.08. Found: 189.
8 8 6
H N
A mixture of 4-hydroxy pyrrolo[2,3-d]pyrimidine (5) (10 g, 74 mmol) in
POCl (30 mL) was refluxed for an hour. The reaction mixture was
3
7-Propyl pyrrolo[3,2-e]tetrazolo[1,5-c]pyrimidine (8c)
Pale yellow solid, yield: 90%, m.p. 134–136°C. IR (film) υ: 3078, 1717,
monitored by TLC. After completion of the reaction, mixture was
quenched into ice water then precipitation observed. The obtained
precipitation was filtered, washed with excess of water, and dried
under vacuum. The residue purified by recrystallization using toluene
−
1 1
1630, 1461 cm ; H NMR (400 MHz, DMSO-d ): δ 0.85 (t, J = 7.1 Hz,
6
CH ), 1.86 (m, ─CH ─CH ), 4.35 (t, J = 7.1 Hz, N─CH ─CH ), 7.07 (d,
3
2
3
2
2
13
J = 3.4 Hz, Ar-H), 7.78 (d, J = 3.4 Hz, Ar-H), 9.87 (s, Ar-H); C NMR

PATIL ET AL.
| 7
(
100 MHz, DMSO-d
32.69, 140.97, 146.19; MS (EI): m/z calcd. for [C
Found: 203.
6
): δ 10.88, 23.39, 46.80, 100.30, 101.76, 128.73,
Ar-H), 7.43 (d, Ar-2H), 7.56 (m, Ar-5H), 7.77 (d, J = 7.8 Hz, Ar-H), 7.92
1
3
1
9
H
10
N
6
+H]: 203.1.
(d, J = 5.1 Hz, Ar-H), 7.94 (s, Ar-H), 9.92 (s, Ar-H); C NMR (100 MHz,
DMSO-d ): δ 48.23, 101.06, 102.21, 110.07, 118.47, 127.58, 128.25,
29.03, 130.05, 133.35, 133.51, 133.81, 137.22, 137.93, 141.12,
6
1
7
-(Ethyl 4-butanoate)pyrrolo[3,2-e]tetrazolo[1,5-c]pyrimidine
8d)
White solid, yield: 85%, m.p. 138–141°C. IR (film) υ: 1713, 1630, 1486,
13 7
143.96, 146.30; MS (EI): m/z calcd. for [C20H N +H]: 352.1. Found:
(
351.9.
−1 1
1
410 cm ; H NMR (400 MHz, DMSO-d
.15–2.08 (m, ─CH ─CH ), 2.30 (t, J = 7.3 Hz, ─CO─CH
─CH ), 4.43 (t, J = 6.8 Hz, N─CH ─CH
J = 3.4 Hz, Ar-H), 7.77 (d, J = 3.4 Hz, Ar-H), 9.89 (s, Ar-H); C NMR
100 MHz, DMSO-d ): δ 13.95, 25.45, 30.53, 44.51, 59.86, 100.54,
01.89, 128.67, 132.77, 141.03, 146.18, 172.01; m/z calcd. for
+H]: 275.12. Found: 275.
6
): δ 1.12 (t, J = 7.1 Hz, CH
), 3.99 (q,
), 7.09 (d,
3
),
4.1.8
|
General procedure for the preparation of
2
2
2
2
compounds 8j–l
J = 7.1 Hz, ─OCH
2
3
2
2
13
To a mixture of 4-chloropyrrolo[2,3-d]pyrimidine (12 mmol) and
appropriate sulfonyl chloride (15 mmol) in acetone (10 mL) was added
a solution of aqueous sodium hydroxide (50%) (12 mmol). The reaction
mixture was monitored by TLC. After completion of the reaction,
mixture was precipitated then filtered, washed with mixture of
acetone/water 1:1 (5 mL), and dried under vacuum to afford
compounds 8j–l (70–75%).
(
6
1
12 14 6 2
[C H N O
7
-(3-Butene)pyrrolo[3,2-e]tetrazolo[1,5-c]pyrimidine (8e)
White solid, yield: 82%, m.p. 140–142°C. IR (film) υ: 3107, 3054, 1637,
−
1 1
1
534, 1489 cm ; H NMR (400 MHz, DMSO-d
CH ), 4.96 (m, N─CH
─CHCH ), 4.45 (t, J = 7.0 Hz, CHCH
.82–5.72 (m, ─CH ─CHCH ), 7.04 (d, J = 3.4 Hz, Ar-H), 7.74 (d,
J = 3.4 Hz, Ar-H), 9.83(s, Ar-H); CNMR(100 MHz, DMSO-d
6
): δ 2.60 (q, J = 6.8 Hz,
2
2
2
2
─CH ),
2
7-(Methylsulfonyl)pyrrolo[3,2-e]tetrazolo[1,5-c]-pyrimidine (8j)
Brown color solid, yield: 75%, m.p. 156–158°C. IR (film) υ: 3120, 2966,
−1 1
5
2
2
1
3
6
):δ34.45,
4.79, 100.54, 101.97, 117.70, 129.01, 132.84, 134.76, 141.15,
46.38; MS (EI): m/z calcd. for [C10 +H]: 215.0. Found: 214.7.
1353, 1262 cm ; H NMR (400 MHz, DMSO-d
6 3
): δ 3.98 (s, N─CH ),
4
1
7.05 (d, J = 3.4 Hz, Ar-H), 7.73 (d, J = 3.4 Hz, Ar-H), 9.88 (s, Ar-H) ppm,
1
3
H N
10 6
C NMR (100 MHz, DMSO-d
32.81, 141.28, 146.22; MS (EI): m/z calcd. for [C
239.0. Found: 238.9.
6
): δ 38.19, 100.28, 101.80, 129.68,
1
7 6 6 2
H N O S+H]:
7
-(Benzyl)pyrrolo[3,2-e]tetrazolo[1,5-c]pyrimidine (8f)
Brown solid, yield: 86%, m.p. 175–177°C. IR (film) υ: 3127, 3054, 1633,
−
1 1
1
534, 1513 cm ; H NMR (400 MHz, DMSO-d
6
): δ 5.63 (s, N─CH
2
),
7-(Phenylsulfonyl)pyrrolo[3,2-e]tetrazolo[1,5-c]pyrimidine (8k)
Brown color solid, yield: 72%, m.p. 169–172°C. IR (film) υ: 3120, 2966,
−1 1
7
.12 (d, J = 3.2 Hz, Ar-H), 7.25–7.35 (m, Ar-5H), 7.85 (d, J = 3.4 Hz, Ar-
1
3
H), 9.90 (s, Ar-H); C NMR (100 MHz, DMSO-d
6
): δ 48.55, 100.85,
02.04, 127.30, 127.66, 128.57, 128.82, 133.10, 137.21, 140.97,
46.20; MS (EI): m/z calcd. for [C13 +H]: 251.1. Found: 251.
1557, 1353, 1262 cm
6
; H NMR (400 MHz, DMSO-d ): δ 7.40 (d,
1
1
J = 3.7 Hz, Ar-H), 7.69 (t, J = 7.8 Hz, Ar-2H), 7.80 (t, J = 7.6 Hz, Ar-H),
1
3
H
10
N
6
8.20 (m, Ar-H), 10.05 (s, Ar-H) ppm; C NMR (100 MHz, DMSO-d
6
): δ
1
04.64, 106.64, 127.07, 128.80, 129.96, 135.48, 135.64, 136.80,
7
-(4-Methylbenzyl)pyrrolo[3,2-e]tetrazolo[1,5-c]pyrimidine (8g)
8 6 2
140.64, 145.88; MS (EI): m/z calcd. for [C12H N O S+H]: 301.04.
Yellow solid, yield: 86%, m.p. 182–184°C. IR (film) υ: 3108, 3055, 1637,
Found: 301.
−
1 1
1
486, 1260 cm ; H NMR (400 MHz, DMSO-d
N─CH ), 7.12 (m, Ar-3H), 7.20 (d, J = 8.0 Hz, Ar-2H), 7.83 (d, J = 3.4 Hz,
Ar-H), 9.90 (s, Ar-H); C NMR (100 MHz, DMSO-d
6 3
): δ 2.24 (s, CH ), 5.57 (s,
2
7-[(4-Methylphenyl)sulfonyl]pyrrolo[3,2-e]tetrazolo[1,5-c]-
pyrimidine (8l)
13
6
): δ 20.57, 48.34,
00.80, 102.02, 127.38, 128.78, 129.11, 133.09, 134.23, 136.98, 140.94,
46.21; MS (EI): m/z calcd. for [C14 +H]: 265.0. Found: 264.8.
1
1
Dark brown color solid, yield: 70%, m.p. 150–152°C. IR (film) υ: 3120,
−1 1
H N
12 6
2966, 1557 cm ; H NMR (400 MHz, DMSO-d
6 3
): δ 2.24 (s, CH ), 7.12
(
m, Ar-3H), 7.20 (d, J = 8.0 Hz, Ar-2H), 7.83 (d, J = 3.4 Hz, Ar-H), 9.90 (s,
1
3
7
-(4-Nitrobenzyl)pyrrolo[3,2-e]tetrazolo[1,5-c]pyrimidine (8h)
Ar-H) ppm; C NMR (100 MHz, DMSO-d
127.38, 128.78, 129.11, 133.09, 134.23, 136.98, 140.94, 146.21; MS
(EI): m/z calcd. for [C13 S+H]: 315.06. Found: 315.
6
): δ 20.57, 100.80, 102.02,
Yellow solid, yield: 86%, m.p. 188–190°C. IR (film) υ: 3108, 3055,
−
1 1
1
637, 1513, 1251, 1074 cm ; H NMR (400 MHz, DMSO-d
s, N─CH ), 7.12 (m, Ar-3H), 7.71 (d, J = 8.0 Hz, Ar-2H), 7.83 (d,
J = 3.4 Hz, Ar-H), 9.90 (s, Ar-H); C NMR (100 MHz, DMSO-d
6
): δ 5.57
10 6 2
H N O
(
2
1
3
6
): δ
8.34, 100.80, 102.02, 121.38, 128.78, 129.11, 133.09, 134.23,
40.94, 146.21, 162.1; MS (EI): m/z calcd. for [C13 +H]: 296.1.
ACKNOWLEDGMENTS
4
1
H
10
N
7
O
2
Authors are thankful to the Department of Science and Technology,
New Delhi, India for financial support under Fast Track Young Scientist
Schemes and are grateful to the Principal and Head Department of
Chemistry for providing research facilities. Amit Choudhari (File No:
PDF/2016/003615) is grateful to the Science and Engineering
Research Board (SERB), Government of India, New Delhi for the
award of a National Post-Doctoral Fellowship.
Found: 296.
7
-(2′-Nitrile-1,1′-biphenylbenzyl)pyrrolo[3,2-e]tetrazolo[1,5-c]-
pyrimidine (8i)
−1
White solid, yield: 86%, m.p. 179–181°C. IR (film) υ: 3108, 3055 cm
;
1
6 2
H NMR (400 MHz, DMSO-d ): δ 5.73 (s, N─CH ), 7.16 (d, J = 3.4 Hz,

8
PATIL ET AL.
|
CONFLICT OF INTEREST
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https://doi.org/10.1002/ardp.201800040