One-pot multicomponent synthesis and antimicrobial evaluation of novel tricyclic indenopyrimidine-2-amines

Article abstract of DOI:10.1002/jhet.4081

The synthesis of novel tricyclic indenopyrimidine-2-amines from 3,3-dimethyl-/3-methyl-2H-indanones has achieved by base-catalyzed one-pot three-component reaction. The desired products are formed within 10 hours after addition at reflux temperature. This

Full text of DOI:10.1002/jhet.4081

One-pot Multicomponent Synthesis and Antimicrobial Evaluation of Novel Tricyclic
Indenopyrimidine-2-amines
Navjot Kaur1, Pratibha Singh1, Pawandeep Kaur1, Ajar Nath Yadav2 and Karan Singh1*
1Department of Chemistry, Akal College of Basic Sciences, Eternal University, Baru Sahib,
District Sirmaur, H.P.-173101, India
2Department of Biotechnology, Dr. KSG Akal College of Agriculture, Eternal University, Baru
Sahib, District Sirmaur, H.P.-173101, India
*Address correspondence to this author at:
Department of Chemistry,
Akal College of Basic Sciences,
Eternal University, Baru Sahib, Sirmaur- 173101, H.P. (India)
Tel: +919882226019
Fax: +911799276006
E-mails: karansinghji@rediffmail.com; karan.sagwal@yahoo.com
ABSTRACT
The synthesis of novel tricyclic indenopyrimidine-2-amines from 3,3-dimethyl-/3-methyl-2H-
indanones has achieved by base-catalysed one-pot three-component reaction. The desired
products are formed within 10 hours after addition at reflux temperature. This multicomponent
approach offers a viable protocol for the construction of indenopyrimidine-2-amines in single-
step without the isolation of the intermediates 3,3-dimethyl/3-methyl-2-(4-substituted
benzylidene)-2,3-dihydro-1H-inden-1-ones. All the synthesized compounds have been screened
for antimicrobial activity and some of the derivatives show comparable activity against bacterial
and fungal isolates.
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INTRODUCTION
Nitrogen containing heterocyclic compounds constitutes the largest and vital family of organic
compounds due to their enormous synthetic and biological utility. Pyrimidine, a heterocyclic six
membered ring containing two nitrogen atoms possesses a vital place in the pharmaceutical and
medicinal fields due to its antifungal, anti-inflammatory, antipyretic, anti-depressant and
anticancer activities.[1] The fused pyrimidines such as indenopyrimidines have been extensively
studied by various groups of scientists because of their chemical and biological importance. The
indenopyrimidines were found to possess various biologically activities such as antibacterial,[2-4]
antiallergic,[3,5] antitumor,[3,4,6,7] antifolate,[8] tyrosine kinase,[9] calcium channel antagonists,[10]
antiinflammatory, analgesic,[11] antihypertensive,[12] antileishmanial,[13] tuberculostatic,[14]
anticonvulsants,[15] and antiaggressive activities. [16]
The condensation of CCC bis-electrophiles and NCN bis-nucleophiles is the most
classical [3+3] approach to the pyrimidines synthesis using diverse catalytic system. [17] The use
of NaOH, NaOMe, ionic liquids and metal complexes or metal oxides such as silver loaded ZnO2
to the pyrimidine synthesis have been well described in the literature.[18] On the other hand, the
multicomponent reactions are the vital protocols for synthesis of organic compounds due to
advantages such as high atom efficiency, high chemical yields, lesser reaction time, lesser waste
and minimum by-product formation which makes it superior to the classical protocols.[18] The
indenopyrimidines syntheses through multicomponent reactions have been extensively studied
by several research groups.[18-20]
Owing the biological applications of pyrimidines and fused pyrimidines, the preparation
of indenopyrimidines and their functionalized derivatives is of notable interest to reveal novel
derivatives and explore new applications. These indenopyrimidine derivatives can offer strong
opportunity to the organic as well as the medicinal chemists to develop more useful drugs. In
continuation of our work in the synthesis of various heterocyclic compounds [21-29] and owing the
importance of indenopyrimidine amines as the most privileged building blocks in organic and
medicinal chemistry we reported here one-pot three-component synthesis of novel tricyclic
indenopyrimidine derivatives and evaluate their antimicrobial activities.
2
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RESULTS AND DISCUSSION
The literature revealed that 2-benzylideneindan-1-one undergoes dimerization upon heating in
guanidine carbonate using DMF as a solvent. This was possible due to the abstraction of one of
the methylene protons of 5-membered ring of indene system to give a cross-conjugate enolate
followed by intermolecular Michael addition on a second molecule.[30,31] In another report, the
treatment of 2-benzylideneindan-1-one with guanidine-HCl using solid NaOH in refluxing
ethanol afforded indenopyrimidine in excellent yield.[32] Therefore, we thought worthwhile to
investigate the multicomponent strategy for the preparation of 2-aminopyrimidine fused with
five-membered ring of the indene system (Method A) (Scheme 1).
Scheme 1. One-pot three-component synthesis of indenopyrimidine-2-amines 4a-j
The Friedel-Craft reaction of 3-substituted but-2-enoic acid in refluxing dry benzene
using anhydrous AlCl3 afforded the corresponding 3,3-disubstituted-2H-indanones (Scheme
2).[33]
3
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Scheme 2. Synthesis of 3,3-disubstituted-2H-indanone 1a-b
Having 3,3-disubstituted-2H-indanone in hand, the optimization study for a one-pot
three-component reaction involving 3-methyl-2H-indanone (1.0 equiv.), benzaldehyde (1.0
equiv.) and guanidinium hydrochloride (2.0 equiv.) was carried out by performing a series of
experiments using various reaction conditions. The results are summarized in Table 1. When the
reaction was performed in the absence of base and solvent then no product formation was
observed at room temperature or heating at 120 °C for 24 h (Table 1, entries 1 and 2). The
reaction was attempted using various bases such as TEA, pyridine, K2CO3, Cs2CO3, KOH and
NaOH. The most encouraging results were obtained in KOH and NaOH (Table 1, entries 7 and
8, respectively). By increasing the quantity of NaOH from 2 to 5 equivalents in refluxing ethanol
afforded the indenopyrimidine product 4a in 73% yield (Table 1, entry 10).
Table 1. Optimization of reaction condition
Temp
°C
Time
h
Yielda
%
Entry
Base
Solvent
1
2
3
4
5
6
7
8
9
10
11
12
13
14
-
-
-
-
rt
120
24
24
10
10
10
10
10
10
10
10
12
10
10
10
NR
NR
8
11
15
17
39
45
59
73
71
67
61
TEA (3.0 eq)
Pyridine (3.0 eq)
K2CO3 (3.0 eq)
Cs2CO3 (3.0 eq)
KOH (2.0 eq)
NaOH (2.0 eq)
NaOH (3.0 eq)
NaOH (5.0 eq)
NaOH (5.0 eq)
NaOH (6.0 eq)
NaOH (5.0 eq)
NaOH (5.0 eq)
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
MeOH
DMF
Reflux
Reflux
Reflux
Reflux
Reflux
Reflux
Reflux
Reflux
Reflux
Reflux
Reflux
90
53
4
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15
NaOH (5.0 eq)
MeCN
Reflux
10
51
Reaction conditions: 3-Methyl-2H-indanone (1.0 equiv.), benzaldehyde (1.0 equiv.) and
guanidinium hydrochloride (2.0 equiv.), Base, Solvent (10 V), temperature.
aYields of isolated products.
Repeating the experiment for 12 h resulted in a significant decrease of yield (71%, Table
1, entry 11). Whereas the reaction in other solvents such as MeOH, DMF and acetonitrile gave
the desired product 4a in 61%, 53% and 51% yield, respectively (Table 1, entries 13-15). The
best condition for one-pot three-component reaction appeared thus to be the use of 5.0 equivalent
of NaOH in refluxing ethanol (Table 1, entry 10) and consequently, used for further exploration
of the preparing compound 4, having a number of substituents. The results are summarized in
Table 2. To our delight all the substituted indenopyrimidine-2-amines resulted into good yields
(66-83%) and were not particularly substituents dependent.
In order to compare the effect of multicomponent strategy on the reaction yield, it was
considered worthwhile to attempt the synthesis of 4 in stepwise manner (Method B) (Scheme 3).
Scheme 3. Synthesis of indenopyrimidine-2-amine 4a-j in stepwise manner
Thus, 3-methyl-2H-indanone 1a was converted to 2-benzylidene-3-methyl-2,3-dihydro-
1H-inden-1-one 5a which was refluxed with guanidinium hydrochloride in ethanol using NaOH
as base catalyst afforded indenopyrimidine-2-amine 4a in 65% yield as compared to 73% yield
obtained in one-pot multicomponent strategy. Other derivatives 4b-j were synthesized in a
similar manner in 49-71% yield (Table 2).
Table 2. Yields of a series of indenopyrimidine derivatives 3a-j
% Yield
Entry
R
R′
R″
Product
4a
Method Aa Method Bb
1
CH3
H
H
73
65
5
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2
3
4
5
6
7
8
9
10
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
H
H
CH3
Cl
OMe
F
H
CH3
Cl
76
70
68
83
79
66
74
78
81
63
59
53
71
61
49
58
67
69
4b
4c
4d
4e
4f
4g
4h
4i
H
CH3
CH3
CH3
CH3
CH3
OMe
F
4j
aYields of isolated products 4 with respect to 1
bYields of isolated products 4 with respect to 5
The IR spectrum of compound 4a revealed three medium intensity bands at 3301, 3150
cm-1 and 1485 cm-1 which corresponds to –NH and –C=N stretching, respectively. The 400 MHz
1H NMR spectrum of 4a was quite informative. It displayed a three-proton doublet (J= 7.2 Hz) at
δ 1.05 which can undoubtedly be ascribed to methyl group at C5. A one-proton quartet (J= 7.2
Hz) appeared at 4.53 ascribed to C5-H of the 5-membered ring of indene system. The two-proton
singlet appeared at δ 6.69 belongs to –NH2 group. In the aromatic region one-proton doublet
located at δ 7.67 (J = 7.2 Hz) which can be ascribed to aromatic ring. The other eight protons of
the aromatic rings appeared as one three-proton and the other five-proton multiplets in the region
13
δ 7.94-7.85 & 7.57-7.45, respectively. In case of C NMR spectral analysis of 4a, the methyl
carbon of 5-membered ring of indene and amino carbon resonated at 19.2 ppm and 159.6 ppm,
respectively. The other signals of magnetically non-equivalent carbon atoms appeared at their
respective chemical shifts. The mass spectral analysis of 4a has shown the molecular ion peak at
m/z 288 [M+H]+.
The structures of all novel synthesized products 4a-j were determined by spectroscopic
1
(IR, H NMR, 13C NMR and MS) methods (Supporting Information File-1) and purity was
further determined by its elemental analysis.
Antimicrobial Activities
The Minimum inhibitory concentrations (MICs) of all compounds 4a-j were evaluated
against Gram-positive (Bacillus cereus MTCC1272 and Staphylococcus aureus MTCC737) and
Gram-negative (Escherichia coli MTCC118 and Yersinia enterocolitica MTCC861) bacteria,
and the fungal species Candida albicans MTCC227 by the broth macrodilution method[34] using
standard drugs ampicillin and miconazole. As shown in Table 3, the compounds 4e and 4j
6
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(containing p-fluoro substituents) exhibited comparable activity to the reference standard
ampicillin against the Gram (-ve) bacterial strain E. coli evidenced by minimum inhibitory
concentration of 78.1 µg/ml. While compounds 4b (containing p-methyl substituents), 4e
(containing p-fluoro substituents), 4f and 4j exhibited MIC of 39.06 µg/ml against the Gram (-
ve) bacterial strain Y. enterocolitica. The MIC value of these compounds was found comparable
to the reference standard ampicillin. The compounds 4a, 4c, 4d, 4e, 4f and 4g were having MIC
of 78.01 µg/ml against the Gram (+ve) bacterial strains S. aureus. The compound 4j was found
an excellent compound to be active against all Gram (-ve) and Gram (+ve) bacterial strains E.
coli, Y. enterocolitica, and S. aureus. Regarding the antifungal evaluation, the compounds 4c, 4d,
4e, 4f, 4i and 4j showed comparable antifungal activity against C. albicans with MIC 78.1 µg/ml
as compared to reference standard miconazole. All other compounds displayed lower activity
against all bacterial and fungal isolates.
Table 3. Minimum inhibitory concentrations (MICs) of test compounds 4 against fungal and
bacterial isolates.
Test compounds
MIC (µg/mL)
Gram-negative bacteria
Gram-positive bacteria
Fungi
Escherichia
coli
Yersinia
enterocolitica
78.1
Bacillus
cereus
78.1
Staphylococcus
aureus
Candida
albicans
156.25
156.25
78.1
4a
156.25
156.25
156.25
78.1
156.25
156.25
156.25
156.25
78.1
39.06
78.1
78.1
39.06
39.06
78.1
156.25
156.25
39.06
39.06
78.1
78.1
78.1
78.1
78.1
78.1
78.1
78.1
39.06
39.06
156.25
78.1
78.1
78.1
78.1
156.25
78.1
78.1
78.1
78.1
156.25
156.25
78.1
78.1
-
4b
4c
4d
4e
4f
4g
4h
4i
4j
78.1
156.25
156.25
156.25
78.1
Ampicillin#
Miconazole##
78.1
-
-
-
-
78.1
#Standard antibacterial agent. ##Standard antifungal agent.
But there is still a need to carry out further modifications in indenopyrimidines structure
in order to detect more efficacious antimicrobial lead molecules. Moreover, cytotoxicity
evaluation on test microorganisms and mammalian cell lines is also necessary to establish
7
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correlation between antimicrobial potential and therapeutic safety of the indenopyrimidines
under investigation. Follow-up studies with most promising and non-cytotoxic derivatives are
important to elucidate their possible molecular mechanism of action.
CONCLUSIONS
In summary, a one-pot multicomponent synthesis of novel tricyclic indenopyrimidine-2-amines
from 3,3-disubstituted-2H-indanone has been reported. This multicomponent approach offers a
viable protocol for the construction of indenopyrimidine-2-amines in single-step without the
isolation of the intermediates 3,3-dimethyl/3-methyl-2-(4-substituted benzylidene)-2,3-dihydro-
1H-inden-1-one. All the synthesized compounds have been screened for antimicrobial activity
and some of the derivatives show comparable activity against bacterial and fungal isolates.
Notably, tricyclic indenopyrimidine-2-amines can be further used as versatile building blocks for
the synthesis of various useful derivatives that may be of great interest in developing the novel
pyrazole-based drugs. The investigation about further derivatization of indenopyrimidine-2-
amines is still ongoing in our laboratory.
MATERIALS AND METHODS
Melting points were determined in open glass capillaries on a Fisher-Johns melting point
apparatus and are uncorrected. Nuclear magnetic resonance (NMR) spectra were recorded at
room temperature on FT-NMR Cryo-magnet Spectrometer 400 MHz (Bruker) in DMSO-
d6/CDCl3 as solvent and tetramethylsilane (TMS) as an internal standard (δ = 0 ppm), and the
values were reported in the following order: chemical shift (δ = 0 ppm), multiplicity (s = singlet,
d = doublet, t = triplet, q = quartet, m = multiplet), coupling constant (J in Hz), and integration.
IR spectra were taken as KBr pellets on a Perkin Elmer RXI FT-IR spectrometer. The mass
spectra were recorded on a JOEL JMS 600 mass spectrometer at an ionising potential of 70eV
(EI) electron impact ionisation mode using the direct inlet system. All the reactions were
monitored by thin-layer chromatography (TLC) on pre-coated silica gel 60 F254 (mesh); spots
were visualised under UV light at 254 nm. Elemental analyses were obtained using a Carlo Erba
EA1108 microanalyser.
Synthesis of 3,3-dimethyl-/3-methyl-2H-indanone 1a-b[33]
8
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A solution of crotonic acid (25 g, 29.07 mmol) in benzene (75 mL) was added dropwise in
suspension of AlCl3 (115.95 g, 87.21 mmol) in dry benzene (112 mL). The mixture was refluxed
for 5 hours. The reaction progress was monitored by thin layer chromatography at regular time
intervals. After completion of reaction, the mixture was poured into ice cooled 10% HCl and
extracted with diethyl ether. The organic layer was washed with aqueous saturated solution of
NaHCO3 and water. Then the combined organic layer was dried over anhydrous Na2SO4 and
evaporated under reduced pressure to afford 34.2 g of 3-methyl-2H-indanone 1a.
1
Oil, 81% yield; TLC solvent system: 10% EtOAc: pet-ether, Rf: 0.7; H NMR (400 MHz,
CDCl3): δ 7.73 (d, J = 8.0 Hz, 1H, ArH), 7.61 (t, J = 7.6 Hz, 1H, ArH), 7.51 (dd, J = 7.6 Hz, 0.8
Hz, 1H, ArH), 7.37 (t, J = 6.4 Hz, 1H, ArH), 3.47-3.42 (m, 1H, CH3CH-), 2.95 (dd, J = 19.2 Hz,
7.6 Hz, 1H, -COCH2), 2.29 (dd, J = 19.2 Hz, 3.6 Hz, 1H, -COCH2), 1.41 (d, J = 6.8 Hz, 3H,
CH3). ESI-MS (m/z): 147 [M+H]+. Anal. Calcd for C20H19N3O: C, 82.16; H, 6.89%. Found: C,
82.09; H, 6.93%.
The 3,3-dimethyl-2H-indanone 1b is also prepared from 3-methyl crotonic acid by following the
same procedure as for 1a.
3,3-Dimethyl-2H-indanone 1b
1
Oil, yield 89%; TLC solvent system: 10% EtOAc: pet-ether, Rf: 0.6; H NMR (CDCl3, 400
mHz): δ 7.71 (d, J= 8.0 Hz, 1H, ArH), 7.71-7.35 (m, 3H, ArH), 2.59 (s, 2H, CH2CO), 1.43 (s,
6H, 2 x CH3). ESI-MS (m/z): 161 [M+H]+. Anal. Calcd for C20H19N3O: C, 82.46; H, 7.55%.
Found: C, 82.39; H, 7.51%.
Method A
Synthesis of 4-(4-substituted phenyl)-5-methyl-5H-indeno[1,2-d]pyrimidin-2-amines 4
An equimolar mixture of aldehydes (1 mmol) and 3,3-disubstituted-2H-indanones (1 mmol) was
dissolved in ethanol (10 mL) at room temperature, followed by the addition of solid NaOH (5
mmol) and guanidinium hydrochloride (2 mmol) and refluxed for 10 hours. The reaction
progress was monitored by thin layer chromatography at regular time intervals. When the
reaction was complete, ethanol was removed under high vacuum. The crude compound was
9
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purified by column chromatography using 100-200 mesh silica. The solvent was evaporated and
dried under high vacuum to afford compounds 4a-j.
5-Methyl-4-phenyl-5H-indeno[1,2-d]pyrimidin-2-amine 4a
Off-white solid, TLC solvent system: 50% EtOAc in pet-ether, Rf: 0.5; m.p. 140-143 °C. IR (υ
1
cm-1, KBr): 3301, 3150 (NH str), 1485 (C=N str). H NMR (400 MHz, DMSO-d6): δ 7.94-7.88
(m, 3H, ArH), 7.67 (d, J = 7.2 Hz, 1H, ArH), 7.57-7.46 (m, 5H, ArH), 6.69 (bs, 2H, NH2), 4.53
13
(q, J = 7.2 Hz, 1H, CH3CH-), 1.05 (d, J = 7.2 Hz, 3H, CH3). C NMR (100 MHz, CDCl3): δ
166.1 (C=N), 163.2 (C=N), 159.7, 138.8, 135.5, 132.2, 128.7, 127.9, 126.6, 125.3, 124.2, 122.6,
121.7, 119.1, 36.5, 19.1 (CH3). ESI-MS (m/z): 274.26 [M+H]+. Anal. Calcd for C18H15N3: C,
79.10; H, 5.53; N, 15.37%. Found: C, 78.95; H, 5.47; N, 15.31%.
5-Methyl-4-(4-methylphenyl)-5H-indeno[1,2-d]pyrimidin-2-amine 4b
Off-white solid, TLC solvent system: 50% EtOAc in pet-ether, Rf: 0.6; m.p. 210-211 °C. IR (υ
cm-1, KBr): 3295, 3165 (NH str), 1489 (C=N str). 1H NMR (400 MHz, DMSO-d6): δ 7.88 (d, J =
7.6 Hz, 1H, ArH), 7.85 (d, J = 7.6 Hz, 2H, ArH), 7.67 (d, J = 7.2 Hz, 1H, ArH), 7.55 (t, J = 7.2
Hz, 1H, ArH), 7.47 (t, J = 7.2 Hz, 1H, ArH), 7.34 (d, J = 7.6 Hz, 2H, ArH), 6.65 (s, 2H, NH2),
4.52 (q, J = 7.2 Hz, 1H, CH3CH-), 2.40 (s, 3H, ArCH3), 1.07 (d, J = 7.2 Hz, 3H, CH3). 13C NMR
(100 MHz, CDCl3): δ 165.9 (C=N), 163.4 (C=N), 159.9, 138.6, 135.4, 130.9, 129.7, 128.3,
126.1, 125.3, 125.2, 122.3, 121.5, 119.6, 35.3, 19.1 (CH3), 18.9 (CH3). ESI-MS (m/z): 288.28
[M+H]+. Anal. Calcd for C19H17N3: C, 79.41; H, 5.96; N, 14.62%. Found: C, 79.35; H, 5.89; N,
14.66%.
4-(4-Chlorophenyl)-5-methyl-5H-indeno[1,2-d]pyrimidin-2-amine 4c
Off-white solid, TLC solvent system: 30% EtOAc in pet-ether, Rf: 0.6; m.p. 191-194 °C. IR (υ
cm-1, KBr): 3308, 3205 (NH str), 1491 (C=N str). 1H NMR (400 MHz, DMSO-d6): δ 7.97 (d, J =
9.0 Hz, 2H, ArH), 7.88 (d, J = 7.5 Hz, 1H, ArH), 7.67 (t, J = 7.2 Hz, 1H, ArH), 7.62-7.54 (m,
3H, ArH), 7.48 (t, J = 7.2 Hz, 1H, ArH), 6.73 (s, 2H, NH2), 4.53 (q, J = 7.2 Hz, 1H, CH3CH-),
1.08 (d, J = 7.2 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3): δ 165.7 (C=N), 162.3 (C=N),
159.7, 139.1, 135.1, 133.9, 130.7, 128.3, 127.9, 126.1, 125.2, 122.3, 121.5, 119.6, 35.3, 19.1
10
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35
37
(CH3). ESI-MS (m/z): 308.17 [M+H; Cl]+, 310.19 [M+H, Cl]+. Anal. Calcd for C18H14ClN3:
C, 70.24; H, 4.58; N, 13.65%. Found: C, 70.16; H, 4.54; N, 13.69%.
4-(4-Methoxyphenyl)-5-methyl-5H-indeno[1,2-d]pyrimidin-2-amine 4d
Off-white solid, TLC solvent system: 50% EtOAc in pet-ether, Rf: 0.3; m.p. 192-195 °C. IR (υ
cm-1, KBr): 3316, 3175 (NH str), 1490 (C=N str). 1H NMR (400 MHz, DMSO-d6): δ 7.93 (d, J =
8.7 Hz, 2H, ArH), 7.88 (d, J = 7.2 Hz, 1H, ArH), 7.67 (d, J = 7.5 Hz, 1H, ArH), 7.55 (t, 7.2 Hz,
1H, ArH), 7.47 (t, 7.2 Hz, 1H, ArH), 7.08 (d, J = 8.7 Hz, 2H, ArH), 6.62 (s, 2H, NH2), 4.52 (q, J
= 7.2 Hz, 1H, CH3CH-), 3.85 (s, 3H, OCH3), 1.10 (d, J = 7.2 Hz, 3H, CH3). 13C NMR (100 MHz,
CDCl3): δ 165.9 (C=N), 163.1 (C=N), 159.9, 159.3 (C-O), 138.6, 135.4, 128.3, 126.1, 125.3,
125.1, 122.3, 121.5, 119.1, 112.3, 48.3 (OCH3), 37.3, 19.2 (CH3). ESI-MS (m/z): 304.24
[M+H]+. Anal. Calcd for C19H17N3O: C, 75.23; H, 5.65; N, 13.85%. Found: C, 75.15; H, 5.60; N,
13.91%.
4-(4-Fluorophenyl)-5-methyl-5H-indeno[1,2-d]pyrimidin-2-amine 4e
Light yellow solid, TLC solvent system: 50% EtOAc in pet-ether, Rf: 0.4; m.p. 216-218 °C. IR
(υ cm-1, KBr): 3310, 3185 (NH str), 1487 (C=N str). 1H NMR (400 MHz, CDCl3): δ 8.02 (d, J =
7.2 Hz, 1H, ArH), 7.90-7.86 (m, 2H, ArH), 7.54-7.44 (m, 3H, ArH), 7.22 (t, J = 7.6 Hz, 2H,
13
ArH), 5.17 (s, 2H, NH2), 4.35 (q, J = 7.2 Hz, 1H, CH3CH-), 1.16 (d, J= 7.2 Hz, 3H, CH3). C
NMR (100 MHz, CDCl3): δ 166.1 (C=N), 162.3 (C=N), 161.5 (C-F), 159.4, 139.5, 135.7, 130.1,
128.3, 126.1, 125.2, 122.3, 121.5, 119.6, 115.1, 35.3, 19.1 (CH3). ESI-MS (m/z): 292.24
[M+H]+. Anal. Calcd for C18H14FN3: C, 74.21; H, 4.84; N, 14.42%. Found: C, 74.15; H, 4.79; N,
14.37%.
5,5-Dimethyl-4-phenyl-5H-indeno[1,2-d]pyrimidin-2-amine 4f
Yellow solid, TLC solvent system: 50% EtOAc in pet-ether, Rf: 0.5; m.p. 145-147 °C. IR (υ cm-
1, KBr): 3326, 3155 (NH str), 1487 (C=N str). 1H NMR (400 MHz, DMSO-d6): δ 7.84 (d, J = 7.6
Hz, 1H, ArH), 7.59-7.38 (m, 8H, ArH), 6.69 (s, 2H, NH2), 1.22 (s, 6H, 2 x CH3). 13C NMR (100
MHz, CDCl3): δ 165.8 (C=N), 163.2 (C=N), 159.1, 151.8, 133.5, 132.1, 128.9, 127.9, 126.6,
125.3, 122.6, 122.1, 121.7, 119.1, 41.1, 25.3 (2 x CH3). ESI-MS (m/z): 288.28 [M+H]+. Anal.
Calcd for C19H17N3: C, 79.41; H, 5.96; N, 14.62%. Found: C, 79.35; H, 5.91; N, 14.67%.
11
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5,5-Dimethyl-4-(4-methylphenyl)-5H-indeno[1,2-d]pyrimidin-2-amine 4g
Brown solid, TLC solvent system: 50% EtOAc in pet-ether, Rf: 0.6; m.p. 215-217 °C. IR (υ cm-1,
1
KBr): 3336, 3167 (NH str), 1491 (C=N str). H NMR (400 MHz, DMSO-d6): δ 7.84 (d, J = 7.2
Hz, 1H, ArH), 7.58-7.23 (m, 7H, ArH), 6.66 (s, 2H, NH2), 2.39 (s, 3H, CH3), 1.23 (s, 6H, 2 x
13
CH3). C NMR (100 MHz, CDCl3): δ 165.7 (C=N), 163.1 (C=N), 159.1, 151.2, 132.4, 130.9,
129.7, 128.3, 126.1, 125.3, 125.1, 122.3, 121.5, 119.6, 41.3, 28.1 (2 x CH3), 19.2 (CH3). ESI-MS
(m/z): 302.29 [M+H]+. Anal. Calcd for C20H19N3: C, 79.70; H, 6.35; N, 13.94%. Found: C,
79.59; H, 6.31; N, 13.99%.
4-(4-Chlorophenyl)-5,5-dimethyl-5H-indeno[1,2-d]pyrimidin-2-amine 4h
Brown solid, TLC solvent system: 30% EtOAc in pet-ether, Rf: 0.6; m.p. 195-197 °C. IR (υ cm-1,
KBr): 3396, 3208 (NH str), 1495 (C=N str). 1H NMR (400 MHz, CDCl3): δ 7.99 (d, J = 8.4 Hz,
1H, ArH), 7.53-7.37 (m, 5H, ArH), 7.36 (d, J = 8.8 Hz, 2H, ArH), 5.12, (s, 2H, NH2), 1.30 (s,
13
6H, 2 x CH3). C NMR (100 MHz, CDCl3): δ 165.7 (C=N), 162.3 (C=N), 159.7, 151.1, 134.1,
133.9, 130.7, 128.3, 127.9, 126.1, 122.3, 122.1, 121.5, 119.6, 41.3, 28.1 (2 x CH3). ESI-MS
(m/z): 322.26 [M+H; 35Cl]+, 324.27 [M+H, 37Cl]+. Anal. Calcd for C19H16ClN3: C, 70.91; H,
5.01; N, 13.06%. Found: C, 70.86; H, 4.95; N, 13.01%.
4-(4-Methoxyphenyl)-5,5-dimethyl-5H-indeno[1,2-d]pyrimidin-2-amine 4i
Yellow solid, TLC solvent system: 50% EtOAc in pet-ether, Rf: 0.3; m.p. 202-204 °C. IR (υ cm-
1, KBr): 3316, 3185 (NH str), 1488 (C=N str). 1H NMR (400 MHz, DMSO-d6): δ 7.83 (d, J = 7.2
Hz, 1H, ArH), 7.58 (d, J = 7.6 Hz, 1H, ArH), 7.52 (dt, J = 7.2, 1.2 Hz, 1H, ArH), 7.42 (dt, J =
7.2, 1.2 Hz, 1H, ArH), 7.35 (d, J = 8.8 Hz, 2H, ArH), 7.05 (d, J = 8.8 Hz, 2H, ArH), 6.65 (s, 2H,
NH2), 3.82 (s, 3H, OCH3), 1.24 (s, 6H, 2 x CH3). 13C NMR (100 MHz, CDCl3): δ 165.7 (C=N),
163.1 (C=N), 159.6, 159.1 (C-O), 151.6, 133.4, 127.3, 125.9, 125.3, 122.3, 121.9, 121.5, 119.1,
112.3, 49.3 (OCH3), 41.3, 28.2 (2 x CH3). ESI-MS (m/z): 318.35 [M+H]+. Anal. Calcd for
C20H19N3O: C, 75.69; H, 6.03; N, 13.24%. Found: C, 75.60; H, 5.97; N, 13.30%.
4-(4-Fluorophenyl)-5,5-dimethyl-5H-indeno[1,2-d]pyrimidin-2-amine 4j
12
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Yellow solid, TLC solvent system: 50% EtOAc in pet-ether, Rf: 0.4; m.p. 212-214 °C. IR (υ cm-
1, KBr): 3329, 3197 (NH str), 1497 (C=N str). 1H NMR (CDCl3, 400 mHz): δ 7.98 (d, 1H, J= 7.2
13
Hz, ArH), 7.51-7.05 (m, 7H, NH2, ArH), 5.35 (s, 2H, NH2), 1.43 (s, 6H, 2 x CH3). C NMR
(100 MHz, CDCl3): δ 166.2 (C=N), 162.1 (C=N), 161.7 (C-F), 159.4, 151.5, 133.7, 130.1, 128.3,
126.1, 122.9, 122.3, 121.5, 119.1, 115.2, 41.3, 28.1 (2 x CH3). ESI-MS (m/z): 306.29 [M+H]+.
Anal. Calcd for C19H16FN3: C, 74.74; H, 5.28; N, 13.76%. Found: C, 74.69; H, 5.22; N, 13.71%.
Method B
Synthesis of 3-methyl-2-(4-substituted benzylidene)-2,3-dihydro-1H-inden-1-one 5
Aromatic aldehyde (1.0 equivalent) was added to a solution of 3-methylindanone (1.0
equivalent) in 10 V of EtOH followed by dropwise addition of an aqueous solution of NaOH (3.0
equivalents). The solution was refluxed for 2 hours. The reaction progress was monitored by thin
layer chromatography at regular time intervals. After completion of reaction, the mixture was
extracted with EtOAc and washed with water and brine solution. The organic layer was dried
over anhydrous Na2SO4 and evaporated under reduced pressure. Then, recrystallized with EtOH
and dried under reduced pressure to get pure product 5a-j.
2-Benzylidene-3-methyl-2,3-dihydro-1H-inden-1-one 5a35
Brown solid, 76% yield; TLC solvent system: 10% EtOAc in pet-ether, Rf: 0.6; m.p. 78-80 °C.
1H NMR (400 MHz, CDCl3): δ 7.91 (d, J = 7.6 Hz, 1H, ArH), 7.68-7.63 (m, 4H, ArH), 7.56 (dd,
J = 8.0 Hz, 1.2 Hz, 1H, ArH), 7.48-7.40 (m, 4H, =CH, ArH), 4.44 (q, J = 6.8 Hz, 1H, CH3CH-),
1.42 (d, J = 6.8 Hz, 3H, CH3). ESI-MS (m/z): 235 [M+H]+.
3-Methyl-2-(4-methylbenzylidene)-2,3-dihydro-1H-inden-1-one 5b
Off-white solid, 69% yield; TLC solvent system: 10% EtOAc in pet-ether, Rf: 0.8; m.p. 75- 77
°C. 1H NMR (400 MHz, CDCl3): δ 7.90 (d, J = 7.6 Hz, 1H, ArH), 7.66-7.62 (m, 2H, ArH), 7.57-
7.54 (m, 3H, =CH, ArH), 7.43 (t, J = 8.0 Hz, 1H, ArH), 7.27 (d, J = 8.0 Hz, 2H, ArH), 4.41 (q, J
= 6.8 Hz, 1H, CH3CH-), 2.41 (s, 3H, ArCH3), 1.42 (d, J = 6.8 Hz, 3H, CH3). ESI-MS (m/z): 249
[M+H]+.
2-(4-Chlorobenzylidene)-3-methyl-2,3-dihydro-1H-inden-1-one 5c
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Light brown solid, 72% yield; TLC solvent system: 30% EtOAc in pet-ether, Rf: 0.8; m.p. 84-86
°C. 1H NMR (400 MHz, CDCl3): δ 7.90 (d, J = 7.2 Hz, 1H, ArH), 7.66 (dt, J = 7.6 & 1.2Hz, 1H,
ArH), 7.61-7.55 (m, 4H, ArH), 7.46-7.42 (m, 3H, =CH, ArH), 4.39 (q, J = 6.8 Hz, 1H, CH3CH-),
1.40 (d, J = 6.8 Hz, 3H, CH3). ESI-MS (m/z): 269 [M+H; 35Cl]+, 271 [M+H, 37Cl]+.
2-(4-Methoxybenzylidene)-3-methyl-2,3-dihydro-1H-inden-1-one 5d
Brown solid, 82% yield; TLC solvent system: 30% EtOAc in pet-ether, Rf: 0.7; m.p. 69-71 °C.
1H NMR (400 MHz, CDCl3): δ 7.89 (d, J = 7.2 Hz, 1H, ArH), 7.65-7.61 (m, 4H, =CH, ArH),
7.55 (d, J = 7.6 Hz, 1H, ArH), 7.43 (t, J = 7.6 Hz, 1H, ArH), 6.98 (d, J = 8.8 Hz, 2H, ArH), 4.37
(q, J = 6.8 Hz, 1H, CH3CH-), 3.87 (s, 3H, OCH3), 1.43 (d, J = 6.8 Hz, 3H, CH3). ESI-MS (m/z):
265 [M+H]+.
2-(4-Fluorobenzylidene)-3-methyl-2,3-dihydro-1H-inden-1-one 5e
Yellow solid, 83% yield; TLC solvent system: 20% EtOAc in pet-ether, Rf: 0.5; m.p. 75-77 °C.
1H NMR (400 MHz, CDCl3): δ 7.90 (d, J = 7.6 Hz, 1H, ArH), 7.67-7.63 (m, 4H, =CH, ArH),
7.56 (dd, J = 7.6 & 0.8 Hz, 1H, ArH), 7.44 (t, J = 7.6 Hz, 1H, ArH), 7.15 (t, J = 8.8 Hz, 2H,
ArH), 4.39 (q, J = 6.8 Hz, 1H, CH3CH-), 1.41 (d, J = 6.8 Hz, 3H, CH3). ESI-MS (m/z): 253
[M+H]+.
2-Benzylidene-3,3-dimethyl-2,3-dihydro-1H-inden-1-one 5f
1
Oil, 80% yield, TLC solvent system: 10% EtOAc in pet-ether, Rf: 0.6; H NMR (400 MHz,
CDCl3,): δ 8.00 (d, J = 6.8 Hz, 1H, ArH), 7.87 (d, J = 7.6 Hz, 1H, ArH), 7.78 (s, 1H, =CH), 7.66-
7.63 (m, 3H, ArH), 7.61 (d, J = 1.2 Hz, 4H, ArH), 1.52 (s, 6H, 2 x CH3). ESI-MS (m/z): 248
[M+H]+.
3,3-Dimethyl-2-(4-methylbenzylidene)-2,3-dihydro-1H-inden-1-one 5g
1
Oil, 77% yield; TLC solvent system: 10% EtOAc in pet-ether, Rf: 0.8; H NMR (400 MHz,
CDCl3): δ 8.00 (d, J = 7.6 Hz, 1H, ArH), 7.66-7.27 (m, 8H, =CH, ArH), 2.41 (s, 3H, ArCH3),
1.52 (s, 6H, 2 x CH3). ESI-MS (m/z): 263 [M+H]+.
2-(4-Chlorobenzylidene)-3,3-dimethyl-2,3-dihydro-1H-inden-1-one 5h
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Oil, 96% yield, TLC solvent system: 30% EtOAc in pet-ether, Rf: 0.8; 1H-NMR(CDCl3, 400 Hz)
δ 8.00 (d, J = 8.8 Hz, 2H, ArH), 7.86 (d, J = 8.0 Hz, 1H, ArH), 7.78 (d, J = 8.0 Hz, 1H, ArH),
7.70-7.61 (m, 3H, =CH, ArH), 7.51 (d, J = 8.8 Hz, 2H, ArH), 1.51 (s, 6H, 2 x CH3). ESI-MS
(m/z): 283 [M+H; 35Cl]+, 285 [M+H, 37Cl]+.
2-(4-Methoxybenzylidene)-3,3-dimethyl-2,3-dihydro-1H-inden-1-one 5i
1
Oil, 80% yield; TLC solvent system: 30% EtOAc in pet-ether, Rf: 0.7; H NMR (400 MHz,
CDCl3): δ 8.01 (d, J = 7.2 Hz, 1H, ArH), 7.65-7.61 (m, 4H, =CH, ArH), 7.55 (d, J = 7.6 Hz, 1H,
ArH), 7.43 (t, J = 7.6 Hz, 1H, ArH), 6.98 (d, J = 8.8 Hz, 2H, ArH), 3.87 (s, 3H, OCH3), 1.51 (s,
6H, 2 x CH3). ESI-MS (m/z): 279 [M+H]+.
2-(4-Fluorobenzylidene)-3,3-dimethyl-2,3-dihydro-1H-inden-1-one 5j
1
Oil, 84% yield; TLC solvent system: 20% EtOAc in pet-ether, Rf: 0.5; H NMR (400 MHz,
CDCl3): δ 8.00 (d, J = 7.6 Hz, 1H, ArH), 7.67-7.44 (m, 8H, =CH, ArH), 1.51 (s, 6H, 2 x CH3).
ESI-MS (m/z): 267 [M+H]+.
Synthesis of 5H-indeno[1,2-d]pyrimidin-2-amines 4
Solid NaOH (5.0 eq.) was added to a solution containing 2.0 eq. of guanidine-HCl in 10
mL of EtOH and stirred for an hour. The mixture was filtered to remove salt. The filtrate was
added into a solution of corresponding sample of compound 5 (1.0 eq.) in another 10 mL of
ethanol and reflux for 10 h. The reaction was monitored by thin layer chromatography. After
completion of the reaction, ethanol was removed under high vacuum. The crude compound was
purified by column chromatography. The solvent was evaporated and dried under high vacuum
to afford compound 4a-j. The isolated yield for each compound is given in Table-2.
Biological Assay
Test Microorganisms
Bacterial cultures (Bacillus cereus MTCC1272, Staphylococcus aureus MTCC737,
Escherichia coli MTCC118, Yersinia enterocolitica MTCC861) and fungus Candida albicans
MTCC227 were obtained from Microbial Type Culture Collection, IMTECH, Chandigarh
15
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(India). Cultures were regularly maintained in the laboratory using standard media and
incubation conditions.
Antimicrobial Activity
Indenopyrimidine-2-amines 4 were evaluated for their antimicrobial activities by broth
macrodilution method as described previously by Sood et al.[34] Briefly, to prepare the stock
solution, 10 mg of the test compounds were dissolved in DMSO (4 mL) and similarly, the
standard drugs ampicillin and miconazole were dissolved in appropriate quantity of sterile
distilled water (2.5 mg/mL). The bacterial and fungal inocula were made in saline solution
(0.85% NaCl) and cell counts were adjusted to 5 x 107cfu/mL by using 0.5 McFarland standard.
MICs of test compounds and test drugs were determined in sterile test tubes containing 2.0 mL
of nutrient broth or Mueller-Hinton broth (for bacteria) and Sabouraud dextrose broth (for
Candida). Two mL stock solution of test compounds and standard antimicrobial agents were
added to first tube of their respective series and diluted serially to obtain different concentrations.
The resultant diluted concentrations of test compounds and standard antimicrobial agents were
2500, 1250, 625, 312.5, 156.25, 78.1, 39.06, 19.53, 9.76 μg/mL. Broth tubes were inoculated
with 10 μL of microbial suspension so that final counts were 5 x 105cfu/mL. Tubes containing
medium or medium plus DMSO, and tubes without microbial inoculum served as growth and
sterility controls, respectively. Broth tubes were incubated at 35-37 °C for 24 h and examined for
the presence or absence of growth. The minimum concentration of test compounds and standard
antimicrobials that resulted in complete growth inhibition was recorded as MIC.
ACKNOWLEDGEMENTS
The authors are grateful to Prof. H.S. Dhaliwal, Vice-Chancellor, Eternal University,
Baru Sahib for providing facilities and financial assistance. The authors would like to
acknowledge GVK Biosciences Pvt. Ltd. Hyderabad and Regional Sophisticated Instrumentation
Centre, Punjab University, Chandigarh, for recording the analytical data during the study.
SUPPORTING INFORMATION
The supporting information is available in the file “Supporting Information File-1”
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