Synthesis, Characterization, Stereochemistry and Antimicrobial Evaluation of N-Acyltetrahydrobenzodiazepin-2-ones

Article abstract of DOI:10.14233/ajchem.2020.22331

A few N5-substituted (1) and N1,N5-disubstituted tetrahydro-1,5-benzodiazepin-2-ones (2-8) viz. N5-chloroacetyl-, N5-formyl-, N5-dichloroacetyl-, N1,N5-diethoxycarbonyl-, N1,N5-bischloroacetyl-, N5-piperazinoacetyl- and N5-morpholinoacetyltetrahydro-1,5-benzodiazepin-2-ones have been synthesized. The characterization and conformational analysis of these compounds 2-8 have been carried out using IR and ¹H, ¹³C, DEPT & 2D (COSY & HSQC) NMR spectral techniques. The coupling constants for compounds 3-6 were determined by irradiating the C4-methyl doublet. The appearance of major and minor conformers has been found in the case of benzodiazepin-2-ones (3 and 6) and the spectral data confirm the equilibrium due to ring inversion over the N-C=O rotation. The spectral data and the extracted coupling constant values revealed that the substituted tetrahydro-1,5-benzodiazepin-2-ones (2-8) prefer to adopt boat conformation. The antimicrobial activity of compounds 1-8 has also been evaluated.

Full text of DOI:10.14233/ajchem.2020.22331

Asian Journal of Chemistry; Vol. 32, No. 5 (2020), 1007-1014
A
SIAN
J
OURNAL OF HEMISTRY
C
https://doi.org/10.14233/ajchem.2020.22331
Synthesis, Characterization, Stereochemistry and Antimicrobial
Evaluation of N-Acyltetrahydrobenzodiazepin-2-ones
†,
*
A. AKILA , P. SAKTHIVEL and S. PONNUSWAMY
P.G. & Research Department of Chemistry, Government Arts College (Autonomous), Coimbatore-641018, India
†Present Address: Department of Chemistry, Sri Ramakrishna College of Arts and Science (Autonomous), Coimbatore-641006, India
*Corresponding author: E-mail: kspons2001@gmail.com
Received: 15 July 2019;
Accepted: 13 September 2019;
Published online: 29 April 2020;
AJC-19827
A few N5-substituted (1) and N1,N5-disubstituted tetrahydro-1,5-benzodiazepin-2-ones (2-8) viz. N5-chloroacetyl-, N5-formyl-, N5-dichloro-
acetyl-, N1,N5-diethoxycarbonyl-, N1,N5-bischloroacetyl-, N5-piperazinoacetyl- and N5-morpholinoacetyltetrahydro-1,5-benzodiazepin-
2-ones have been synthesized. The characterization and conformational analysis of these compounds 2-8 have been carried out using IR
and 1H, 13C, DEPT & 2D (COSY & HSQC) NMR spectral techniques. The coupling constants for compounds 3-6 were determined by
irradiating the C4-methyl doublet. The appearance of major and minor conformers has been found in the case of benzodiazepin-2-ones (3
and 6) and the spectral data confirm the equilibrium due to ring inversion over the N-C=O rotation. The spectral data and the extracted
coupling constant values revealed that the substituted tetrahydro-1,5-benzodiazepin-2-ones (2-8) prefer to adopt boat conformation. The
antimicrobial activity of compounds 1-8 has also been evaluated.
Keywords: N-Acyltetrahydrobenzodiazepin-2-ones, Boat conformation, Biological activity.
The introduction of acyl groups at N1 and N5 of tetrahydro-
benzodiazepines results in perihydrogen interaction between
the acyl groups and the ortho-hydrogen of benzene ring, which
could lead to interesting conformational changes [21-31]. The
conformations of diazepines play a key role in deciding their
biological activity [32-36]. Hence, it is of interest to introduce
the conformation directing moieties, such as acyl groups at
the nitrogen site of benzodiazepines and to study the stereo-
chemical consequences on the seven membered rings of benzo-
diazepines. In continuation of the work on N-acyltetrahydro-
1,5-benzodiazepines [21-23,37] and other related systems [38-
43], we report, herein, the synthesis and stereochemistry of
N5-chloroacetyl-, N5-formyl-, N5-dichloroacetyl-, N1,N5-diethoxy-
carbonyl- and N1,N5-bischloroacetyl-, N5-piperazinoacetyl- and
N5-morpholinoacetyltetrahydro-1,5-benzodiazepin-2-ones (2-8),
using NMR spectra.
INTRODUCTION
Benzodiazepines are bicyclic heterocyclic mixes having
benzene nucleus combined to a seven membered ring contain-
ing two nitrogen atom which form a significant class of biolog-
ically and medicinally active compounds [1-3]. Benzodiazepines
are an important class of pharmacologically active compounds
[4,5], which are finding applications as anti-inflammatory,
anticonvulsant, antianxiety, antifungal, antibacterial, analgesic,
antifeedant, seditative, antidepressive and hypnotic agents [6-
10]. Certain derivatives like lofendazam, clobazam and triflu-
bazam are used for the treatment of anxiety and neuroses
including psychomatic disturbances and a few 2,4-diaryl-7,8-
dimethyl-2,3-dihydro-1H-1,5-benzodiazepines have been tested
against breast cancer and have shown moderate activity [11].
Some benzodiazepine derivatives are used as valuable synthons
for the preparation of other fused compounds such as triazolo
[12], oxadiazolo [13], oxazino [14] and furobenzodiazepines [15].
Moreover, the derivatives of some benzodiazepines are used
as anticancer [16], antithrombotic [17,18], protein kinase inhi-
bitory and anti-inflammatory agents [19,20].
EXPERIMENTAL
Unless otherwise stated, all the reagents and solvents used
were of high grade and purchased from Aldrich and Merck.
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1008 Akila et al.
Asian J. Chem.
All the solvents were distilled prior to use.All the reported melting
points were taken in open capillaries and are uncorrected. IR
spectra were recorded in Shimadzu FT-IR 8400s and Bruker
α-model spectrometers using KBr pellets. The 1H & 13C NMR
spectra were recorded in CDCl3 solution using TMS as the
internal standard in BrukerAMX 400 & 100 MHz NMR spectro-
meter, respectively, with the chemical shifts referenced to TMS.
0.05 M solutions of the sample prepared in CDCl3 were used
for recording 2D NMR spectra. Electron impact mass spectra
were recorded in JEOL GS mate spectrometer and micro-
analyses were performed on Carlo Erba 1108 CHN analyzer.
The parent benzodiazepin-2-one (1) was prepared accor-
ding to the literature procedure [44,45].
General method for the synthesis of compounds 2-6:
To a solution of tetrahydro-4-methyl-1,5-benzodiazepin-2-one
(1) (0.88 g, 5 mmol) in anhydrous benzene (50 mL) were added
triethylamine (2.8 mL, 20 mmol) and the acylating agents [2:
chloroacetylchloride (0.8 mL, 10 mmol), 3: acetic-formic
anhydride (15 mL), 4: dichloroacetylchloride (0.95 mL, 10
mmol), 5: ethylchloroformate (1.91 mL, 20 mmol), 6: chloro-
acetylchloride (1.6 mL, 20 mmol)] for the synthesis of compounds
2-6, respectively. The reaction mixture was allowed to reflux
on a water bath for 6 h. The course of the reaction was monitored
by TLC (silica, CHCl3 as eluent). The resulting solution was
washed with water (4 × 25 mL). The organic layer was separ-
ated, dried using anhydrous Na2SO4, passed through a short
column of silica (eluent: CHCl3), evaporated and crystallized
from ethanol (for compounds 2, 4, 5 and 6), petroleum ether
(60-80 ºC) and benzene (for compound 3). All the synthesized
compounds were obtained in good yield.
5-(2-Chloroacetyl)-4-methyl-1,3,4,5-tetrahydrobenzo-
[b][1,4]diazepin-2-one (2):Yield: 73%; m.p.: 161-163 ºC; 1H
NMR: δ ppm: 7.24-7.49 (m, 4H, aromatic protons), 9.15 (brs,
1H, NH), 5.32 (m, 1H, H4a), 3.77 (d, 1H, HA of COCH2Cl)
3.66 (d, 1H, HB of COCH2Cl), 2.45 (dd, 1H, H3e), 2.36 (t, 1H,
H3a), 1.34 (d, 3H, CH3); 13C NMR: 18.9 (CH3 at C4), 40.2 (C3),
55.2 (C4), 41.9 (CH2 of NCOCH2Cl), 123.3-136.8 (aromatic
carbon), 165.7 (CO of NCOCH2Cl), 173.0 (C2); Anal. calcd.
(found) % for C12H13N2O2Cl: C 57.04 (56.82); H 5.19 (5.12);
N 11.09 (11.22)
2-Methyl-4-oxo-2,3,4,5-tetrahydro-benz[b][1,4]diaze-
pine-1-carbaldehyde (3): Yield: 75.4 %; m.p.: 244-245 ºC;
1H NMR: δ ppm: 8.40 (s, 1H, CHOMinor), 8.12 (s, 1H, CHOMajor),
7.14-7.48 (m, 4H, aromatic protons), 8.33 (brs, 1H, NH), 5.16
(m, 1H, H4a Major) & 4.65 (m, 1H, H4a Minor), 2.54 (m, 2H, H3),
1.26 (d, 3H, CH3 Major), 1.33 (d, 3H, CH3 Minor); 13C NMR: 19.4
(CH3 at C4), 39.8 (C3), 53.2 (C4), 122.6-129.4 (aromatic carbon),
136.0 & 130.7 (aromatic ipso carbons), 162.0 (CHO), 172.2
(C2);Anal. calcd. (found) % for C11H12N2O2 : C 64.69 (64.38);
H 5.92 (5.85); N 13.72 (13.87).
(found) % for C12H12N2O2Cl2: C 50.19 (49.95); H 4.21 (4.29);
N 9.76 (9.54).
2-Methyl-4-oxo-3,4-dihydro-2H-benzo[b]diazepine-
1,5-dicarboxylic acid diethyl ester (5):Yield: 59.3 %; m.p.:
1
117-119 ºC; H NMR: δ ppm: 7.21-7.39 (m, 4H, aromatic
protons), 4.93 (m, 1H, H4a), 2.48 (dd, 1H, H3e), 2.16 (t, 1H, H3a),
1.09-1.32 (m, 9H, CH3 & COOC2H5); 13C NMR: 13.9 &14.4
(C2H5 of N1 & N5), 18.7 (CH3 at C4), 42.6 (C3), 54.5 (C4), 61.7
& 63.5 (CH2 of NCOOCH2CH3), 152.1 & 154.3 (CO of NCOO-
CH2CH3), 127.2-130.8 (aromatic carbon), 136.4 & 132.2
(aromatic ipso carbons), 169.7 (C2); Anal. calcd. (found) %
for C16H20N2O5: C 59.99 (59.73); H 6.29 (6.34); N 8.74 (8.66).
1,5-Bis-(2-chloroacetyl)-4-methyl-1,3,4,5-tetrahydro-
benzo[b][1,4]diazepin-2-one (6): Yield: 75.9 %; m.p.: 125-
127 ºC; 1H NMR: δ ppm: 7.13-7.54 (m, 4H, aromatic protons),
5.16 (m, 1H, H4a Major) & 5.27 (m, 1H, H4a Minor), 3.79 (d, 1H, HA
of COCH2Cl at N1), 4.81 (d, 1H, HA of COCH2Cl at N5), 3.70
(d, 1H, HB of COCH2Cl at N1), 4.76 (d, 1H, HB of COCH2Cl at
N5), 2.50 (m, 1H, H3e Major), 2.41 (m, 1H, H3e Minor), 2.19 (m, 1H,
H3a Major), 2.29 (m, 1H, H3a Minor), 1.12 (d, 3H, CH3 Major), 1.19 (d,
3H, CH3 Minor); 13C NMR: 18.3 (CH3 at C4), 41.5 (CH2 of NCO-
CH2Cl at N1), 47.1 (CH2 of NCOCH2Cl at N5), 42.3 (C3), 53.0
(C4), 129.4-130.0 (aromatic carbon), 128.2-135.9 (aromatic
ipso carbons), 169.1 (CO of NCOCH2Cl at N1), 169.5 (CO of
NCOCH2Cl at N5), 171.0 (C2); Anal. calcd. (found) % for
C14H14N2O3Cl2: C 51.08 (50.87), H 4.29 (4.18), N 8.51 (8.62).
Synthesis of N5-piperazinoacetyltetrahydro-4-methyl-
1,5-benzodiazepin-2-one (7): A mixture of N5-chloroacetyl-
tetrahydro-4-methyl-1,5-benzodiazepin-2-one (2) (2.53 g, 10
mmol), piperazine (0.86 g, 10 mmol) and triethylamine (2.8
ml, 20 mmol) in anhydrous benzene (30 ml) was stirred at
room temperature for 7 h. The precipitated ammonium salt
was washed with water (4 × 10 mL). The resulting benzene
solution was dried over anhydrous Na2SO4, passed through a
short column of silica and concentrated. The pasty mass was
purified by crystallization from benzene and petroleum ether
(60-80 ºC) in the ratio of 95:5.Yield: 63 %; m.p.: 280-282 ºC;
1H NMR: δ ppm: 9.42 (s, 1H, NH), 7.08-7.41 (m, 4H, aromatic
protons), 5.34 (m, 1H, H4a), 3.11 (d, 1H, HA of COCH2), 2.1
(m, 8H, piperazine protons), 2.80 (d, 1H, HB of COCH2), 2.43
(m, 2H, H3), 1.18 (d, 3H, CH3); 13C NMR: 19.2 (CH3 at C4),
52.1 (piperazine carbons), 60.3 (CH2 of NCOCH2), 40.8 (C3),
55.3 (C4), 122.6-130.2 (aromatic carbon), 131.3 & 137.1 (aromatic
ipso carbons), 168.9 (CO of NCOCH2), 174.2 (C2);Anal. calcd.
(found) % for C16H22N4O2: C 63.58 (63.65), H 7.28 (7.33), N
18.54 (18.46).
Synthesis of N5-morpholinoacetyltetrahydro-4-methyl-
1,5-benzodiazepin-2-one (8): A mixture of N5-chloroacetyl-
tetrahydro-4-methyl-1,5-benzodiazepin-2-one (2) (1.25 g, 5
mmol), morpholine (1.8 mL, 20 mmol) and triethylamine (2.8
mL, 20 mmol) in anhydrous benzene (30 mL) was stirred at
room temperature for 7 h. The precipitated ammonium salt
was washed with water (4 × 10 mL). The resulting benzene
solution was dried over anhydrous Na2SO4, passed through a
short column of silica and concentrated. The pasty mass was
purified by crystallization from benzene and petroleum ether
(60-80 ºC) in the ratio of 95:5.Yield: 40.5 %; m.p.: 226-228 ºC;
1H NMR: δ ppm: 8.51 (s, 1H, NH), 7.14-7.41 (m, 4H, aromatic
5-(2,2-Dichloroacetyl)-4-methyl-1,3,4,5-tetrahydro-
benzo[b][1,4]diazepin-2-one (4): Yield: 76.6 %; m.p.: 151-
153 ºC; 1H NMR: δ ppm: 7.25-7.54 (m, 4H, aromatic protons),
8.83 (brs, 1H, NH), 5.30 (m, 1H, H4a), 5.64 (s, 1H, H of COCHCl2),
2.47 (dd, 1H, H3e), 2.35 (t, 1H, H3a), 1.26 (d, 3H, CH3); 13C NMR:
18.7 (CH3 at C4), 40.1 (C3), 56.1 (C4), 63.9 (CH of NCOCHCl2),
123.9-130.9 (aromatic carbon), 137.0 & 129.2 (aromatic ipso
carbons), 172.8 (CO of NCOCHCl2), 163.3 (C2); Anal. calcd.

Vol. 32, No. 5 (2020)
Synthesis, Stereochemistry and Antimicrobial Evaluation of N-Acyltetrahydrobenzodiazepin-2-ones 1009
protons), 5.31 (m, 3H, H3 & H4a), 2.85 (d, 1H, HA of COCH2),
2.29 & 3.58 (m, 8H, morpholine protons), 2.66 (d, 1H, HB of
COCH2), 2.06 (d, 3H, CH3); 13C NMR: 19.2 (CH3 at C4), 60.3
& 55.3 (morpholine carbons), 60.0 (CH2 of NCOCH2), 40.3
(C3), 53.1 (C4), 122.6-130.2 (aromatic carbon), 131.2 & 137.1
(aromatic ipso carbons), 168.9 (CO of NCOCH2), 174.2 (C2);
Anal. calcd. (found) % for C16H21N3O3: C 63.36 (63.21); H
6.93 (6.98); N 13.86 (13.75).
In IR spectra of compounds 2-4, a stretching band for
amine NH was absent (3296 cm-1) and for benzodiazepin-2-
ones 5 & 6, the stretching bands for both amine NH and amide
NH (3254 cm-1) were absent, which confirms the formation of
products 2-6. The presence of amide NH peak at 3230 & 3382
cm-1 for compounds 7 & 8, respectively, confirms the formation
of compounds 7 & 8. The ring C=O stretching values of comp-
ounds 2-4 were increased to 1694, 1686 & 1695 cm-1, respec-
tively, when compared to the parent 1 (1664 cm-1) due to the -I
effect of acyl groups attached at N5. The C=O stretching vibrations
of N-acyl moiety in compounds 2-4 were observed at 1658,
1642 & 1643 cm-1, respectively. In the case of N1,N5-disubsti-
tuted compounds 5 & 6, there is a remarkable increase in the
ring C=O stretching vibrations due to the substitution at N1
site (5: 1705 & 6: 1754 cm-1) and the C=O stretching vibrations
at N1 & N5 positions were observed below 1680 cm-1 (5: 1667
& 6: 1678, 1628 cm-1). The C=O stretching vibrations of ring
and N-acyl moiety of compounds 7 & 8 were observed around
1677 & 1655 cm-1 as broad peaks.
RESULTS AND DISCUSSION
Tetrahydrobenzodiazepin-2-ones (2-6) were synthesized
by the action of chloroacetyl chloride, acetic-formic anhydride,
dichloroacetyl chloride, ethylchloroformate and chloroacetyl
chloride, respectively, on tetrahydrobenzodiazepin-2-one (1) in
dry benzene and using triethylamine as catalyst (Scheme-I),
while compounds 7 and 8 were synthesized by the action of
piperazine and morpholine, respectively, on N5-chloroacetyl-
tetrahydro-1,5-benzodiazepin-2-ones (2) in dry benzene and
using triethylamine again as catalyst (Scheme-I). Onlymonoacyl
derivatives 3 & 4 were obtained by the reaction of acetic-formic
anhydride and dichloroacetyl chloride on compound 1. How-
ever, a controlled reaction of chloroacetyl chloride with comp-
ound 1 gave monoacyl derivative 2 and the addition of excess
reagent yielded diacyl product 6. However, there was a difficulty
in isolating monoethoxycarbonyl derivative and only diethoxy-
carbonyl compound 5 was isolated.
In the 1H NMR spectra of compounds 2-4, an absence of
amine NH signals (δ 3.50 ppm) confirms the monoacylation.
The absence of both amine NH (δ 3.50 ppm) and amide NH
(δ 8.2 ppm) signals in compounds 5 & 6 revealed the acylation
of both sites. In mass spectra of compounds 2-4, the molecular
ion peaks were observed at m/z 252, 204 and 287 and their
fragmentation patterns correspond to chloroacetyl, formyl and
dichloroacetyl derivatives, respectively. The preferred confor-
H
R1
O
O
9
6
9
NH2
N
1
N
1
10
11
10
11
2
4
2
4
8
8
7
HCl
Z
Y
CH3CH=CHCOOH
+
3
3
∆
7
5
5
NH2
N
N
6
CH3
CH3
H
R2
1
2-6
H
H
O
O
9
9
N
1
N
1
10
10
11
2
4
2
4
8
7
8
7
Z
Y
3
3
5
5
11
N
N
6
6
CH3
COCH2Cl
CH3
COCH2R
7-8
2
Z
Y
R1
H
R2
R
–
–
–
–
–
Compound
ClCOCH2Cl
HCOOCOCH3
ClCOCHCl2
ClCOOCH2CH3
ClCOCH2Cl
Piperazine
Et3N/benzene
Benzene
COCH2Cl
CHO
2
3
4
5
6
7
8
H
Et3N/benzene
Et3N/benzene
Et3N/benzene
Et3N/benzene
Et3N/benzene
H
COCHCl2
COOEt
ClCOCH2Cl
–
COOEt
ClCOCH2Cl
–
–
C4H9N2
Morpholine
–
C4H8NO
Scheme-I: Synthesis of N5-acyl and N1,N5-diacyltetrahydro-1,5-benzodiazepin-2-ones (2-8)

1010 Akila et al.
Asian J. Chem.
mation of benzodiazepin-2-ones (2-6, 7 & 8) were derived from
the 1H & 13C NMR spectral data in comparison with those of
the parent amines 1 and 2. The DEPT and 2D (1H-1H COSY
& HSQC) NMR spectra were also used for the assignments.
The coupling constants J3a,4a and J3e,4a were determined by
irradiating the C4-methyl doublet and the corresponding dihedral
angles were estimated using dihedral angle estimation by ratio
method (DAERM) [46] and are presented in Table-1. The parent
tetrahydrobenzodiazepin-2-one (1) has been reported to exist
in a boat conformation on the basis of the coupling constant
values 7.5 (J3a,4a) and 4.2 Hz (J3e,4a) observed between H3a/H3e
and H4 protons and also by X-ray crystallography [45].
result in an eclipsing interaction between N5-C4/N5-C11 and
C=O bonds and the α-carbon is expected to be shielded [47].
Even if there was a fast N-C=O rotation, α-carbons would
encounter shielding effect [47]. However, C4-carbon signal for
compounds 2 and 4 does not show a significant shielding/
deshielding effect compared to that of parent diazepine 1. Hence,
X-ray crystal structures of compounds 2 and 4 were used to
predict the orientation of acyl groups at N5. The X-ray crystal
structures [48,49] showed that acyl groups in compounds 2
and 4 at N5 prefer to adopt an exo orientation (Fig. 1, exo).
Ring conformations of compounds 2 and 4: The N5-acyl
derivatives 2 and 4 may prefer to adopt the chair conformations
CE & CA or any of the boat conformations BE & BA (Fig. 2).
In chair CA and boat BA forms, the coupling constants J3a,4a
and J3e,4a were expected to be around 2-5 Hz. But one of the
observed coupling constant of compounds 2 and 4 was larger
(Table-1). In addition, analysis using Dreiding models indicates
that chair CA and boat conformations BA require an approxi-
mate cis (φ3a,4a) and trans (φ3e,4a) angle of 60º. However, the cis
and trans angles calculated using DAERM [46] obtained from
the coupling constant values were 48º and 168º for compounds
2 and 4, respectively. Hence, on the basis of the observed
coupling constants and calculated dihedral angles, the possi-
bility of chair CA and boat BA conformations was ruled out.
TABLE 1
VICINAL COUPLING CONSTANT DATA (Hz) AND
THE CORRESPONDING DIHEDRAL ANGLES (°) ESTIMATED
USING DAERM OF BENZODIAZEPIN-2-ONES (2-8)
AND PARENT AMINE (1 and 2)
Compounds
3J3e,4a
3J3a, 4a
φ3e,4a
48
46
48
44
49
45
45
41
φ3a,4a
168
166
168
164
169
165
165
161
5.2
5.5
5.0
6.0
5.0
6.0
5.6
4.2
12.8
12.0
12.5
12.0
13.0
12.8
12.0
7.5
2
3
4
5
6
7
8
1
Major & minor
Major & minor
N5-Chloroacetyl and N5-dichloroacetyltetrahydro-1,5-
benzodiazepin-2-ones (2 and 4): The 1H and 13C NMR spectra
of N5-acyltetrahydrobenzodiazepin-2-ones (2 and 4) show only
isochronous nature of proton and carbon signals at room temp-
erature. This observation might be explained due to either a
fast rotation about N-C=O bond (or) the N-C=O moiety due
to the arrest in one of the possible orientations viz. exo or endo.
Between these two possible planar orientations of N-C=O group,
the one in which oxygen is directed towards the benzene ring
is designated as endo and the other in which oxygen is away
from the benzene ring is exo (Fig. 1).
Fig. 2. Possible conformations of N5-acyltetrahydro-1,5-benzodiazepin-2-
ones 2-4 and 7-8
The coupling constants cannot be used to differentiate
between the conformations CE and BE since the C2-C3-C4
parts of them were similar. Thus, a choice between the confor-
mations CE and BE can be decided by using Dreiding models,
which indicate that the dihedral angle between the planes C10-
N1-C2 and N1-C2-C3 would be around 60º for chair confor-
mation CE and around 0º for boat conformation BE. The X-ray
crystal structures [48,49] of compounds 2 and 4 were used to
decide the conformation and the preferred conformation was
found to be BE for both compounds. Hence, it is concluded
that compounds 2 and 4 prefer to adopt a boat conformation
BE with exo orientation of -COCH2Cl, -COCHCl2 groups at
N5 position, respectively (Fig. 3).
Fig. 1. Relative (endo/exo) orientations of acyl groups in benzodiazepin-
2-ones 2-4 and 7-8 at N5
The shielding of α-carbon signals in 13C NMR spectra of
compounds in 2 and 4 compared to that of parent compound 1
is used to fix the orientation of acyl group at N5. If C=O group
is oriented towards the α-carbon (syn orientation), it would

Vol. 32, No. 5 (2020)
Synthesis, Stereochemistry and Antimicrobial Evaluation of N-Acyltetrahydrobenzodiazepin-2-ones 1011
4. The possible orientations of C=O groups at N1 and N5 are
shown in Fig. 5. One of the ipso carbons was deshielded by
4.2 ppm and the other is shielded by 1.9 ppm when compared
to the parent 1. Hence, similar to compounds 2and 4, the ethoxy-
carbonyl group at N5 prefers exo orientation (syn to C4, C11 is
deshielded), while ethoxycarbonyl group at N1 position prefers
to adopt an endo orientation (syn to C10, C10 is shielded). Thus,
it is concluded that N1,N5-diethoxycarbonyltetrahydro-1,5-
benzodiazepin-2-one (5) also prefers to adopt a boat confor-
mation BE with endo and exo orientation of the ethoxycarbonyl
Fig. 3. Preferred conformation of N5-acyltetrahydro-1,5-benzodiazepin-2-
ones 2-4 and 7-8
groups at N1 and N5 positions, respectively (Fig. 6).
N5-Formyltetrahydro-1,5-benzodiazepin-2-one (3): The
1H NMR spectrum of N5-formyltetrahydro-1,5-benzodiazepin-
2-one (3) shows two sets of proton signals for CH3 & Ha at C4
and CHO due to major and minor conformers in equilibrium.
However, the intensity of minor conformer is as low as 8 %.
Hence in 13C NMR spectrum, the chemical shifts were observed
only for major conformer. The comparable coupling constant
and dihedral angle values of the major conformer of compound
3 (Table-1) with those of compounds 2 and 4 indicate that the
major conformer of compound 3 also prefers to adopt BE
conformation with exo orientation of C=O group. The 3J3e,4a
&
3J3a,4a values for the minor & major conformers were found to
be the same (Table-1). Hence, the C2-C3-C4 parts of them
should be similar. However, a perusal of 1H NMR data indicates
that C4-Ha of major is more deshielded (5.16 ppm) compared
to that of minor (4.65 ppm). In the case of N1,N5-diformyl-
2,2,4-trimethyl-1H-tetrahydro-1,5-benzodiazepine, the major
conformer was found to be boat in which C4-Ha was more
deshielded compared to that of the minor conformer which
prefers to exist in a chair conformation [22]. Hence, based on
the above discussion, similar conformational equilibrium due
to ring inversion over the N-C=O rotation was expected for
compound 3 also and the minor conformer may prefer to adopt
the chair conformation CE. Hence, the conformational equili-
brium between boat BE and chair CE conformation with exo
orientation of N-C=O is preferred by compound 3 (Fig. 4). The
X-ray crystal structure of compound 3 [50] also confirmed
that acyl group at N5 prefers exo orientation.
Fig. 5. Relative (exo/endo) orientations of diacyl groups in benzodiazepin-
2-ones 5 & 6 at N1 and N5
Fig. 6. Preferred conformation of N1,N5-diacyltetrahydro-1,5-benzodia-
zepin-2-ones 5 & 6
N1,N5-Bischloroacetyltetrahydro-1,5-benzodiazepin-2-
one (6): The NMR spectral data of N1,N5-bischloroacetyltetra-
hydro-1,5-benzodiazepin-2-one (6) were also similar to that
of compound (5) for the major conformer. Hence, the major
conformer of compound (6) prefers a boat conformation with
endo and exo orientation of C=O groups at N1 & N5 positions.
In this case also, an intensity of minor conformer is 20 %.
Furthermore, the coupling constants and estimated dihedral
angles were similar to compound 3. Hence, similar to compound
3, compound 6 also expected to exist in a conformational equil-
ibrium between the boat BE (major) & chair CE (minor) with
endo & exo orientation of C=O of N1 & N5, respectively (Fig. 7).
Fig. 4. Major and minor isomers of N5-formyltetrahydro-1,5-benzodiaze-
pin-2-one 3
N1,N5-Diethoxycarbonyltetrahydro-1,5-benzodiazepin-
2-one (5): The NMR spectra of N1,N5-diethoxycarbonyltetra-
hydro-1,5-benzodiazepin-2-one (5) also showed a isochronous
nature of proton and carbon signals at room temperature. The
CH2 protons of ethoxycarbonyl groups at N1 & N5 showed an
unsymmetrical quartet at δ 4.27 & 4.10 ppm, respectively.
Furthermore, except at N1 site, most of the NMR spectral data
of compound 5 were comparable to that of compounds 2 and

1012 Akila et al.
Asian J. Chem.
8 also preferred to adopt boat conformation BE with exo
orientation of the acyl groups at N5 position.
Antibacterial activity: The synthesized compounds 1-8
were screened for their in vitro growth inhibitory action against
different strains of bacteria viz., Staphylococcus aureus,
Staphylococcus albus, Escherichia coli, Salmonella paratyphi
and Klebsiella pneumonia using Muller-Hint agar medium by
disc diffusion technique [51]. Sterile Muller-Hinton agar plates
were prepared and the agar surface was inoculated with the
bacteria. Compounds 1-8 were dissolved in 1mL of DMSO in
various concentrations in separate tubes. Commercially avail-
able sterile discs were soaked in the preparation for 0.5 h. It
was then placed in empty petri plates for air-drying. Using
sterile forceps, the discs were placed on the surface of agar
plates and gently pressed onto the agar surface. The culture
plates were inverted and incubated for 24-48 h at 37 ºC. After
incubation, zone of clearance was observed and its diameter
measured using microscope. Zone of inhibition of compounds
was compared with standard ciprofloxacin for antibacterial
activity. The results showed that the synthesized compounds
possess a broad spectrum of activity against the tested micro-
organisms (Table-2).
Among benzodiazepinones, N-chloroacetyl benzodiaze-
pinone (2) showed a better antibacterial activity against E. coli
and S. aureus. The introduction of piperazine moiety at N5
position retains the activity and morpholine moiety at N5
position of compound 2 increases the activity against S. aureus
and decreases the activity of other organisms.
All the compounds, in general, dictate superior antibacterial
activity against S. aureus and in particular the compounds 4
& 8 are more active than the standard.All of them show better
activity against E. coli, moderate activity against K. pneumonia
and less activity against S. albus & S. paratyphi.
Antifungal activity: Compounds 1-8 were also screened
for their antifungal strains viz. Aspergillus niger, Aspergillus
fumigates, Candida albicans, Monascus ruber and Aspergillus
parasites. Assays measuring inhibition of mycelia growth on
agar media were used. Compounds 1-8 were dissolved in 1 mL
of sterile DMSO serving as a stock solution. Then, it was
transferred to 4 mL Sabouraud dextrose agar (SDA) growth
media in separate tubes and autoclaved at 121 ºC for 15 min.
These tubes were allowed to cool to 50 ºC and non-solidified
SDA of each tube was loaded with various concentrations of
drug solution. Then, the tube was inoculated with 4 mm dia-
meter piece of inoculums removed from 7 days old culture of
fungus [52,53]. All these tubes were incubated at 28 1 ºC for
10 days. A relative humidity was maintained at 40-50 % in the
incubation room. Growth in the media was determined [54]
Fig. 7. Major and minor isomers of N1,N5-bischloroacetyltetrahydro-1,5-
benzodiazepin-2-one 6
N5-Piperazinoacetyl- and N5-morpholinoacetyltetra-
hydro-1,5-benzodiazepin-2-one (7 & 8): The C4-carbon
signal for compound 7 was shielded by 1.7 ppm which confirms
the exo orientation of C=O. However, compound 8 did not
show significant shielding/deshielding effect compared to that
of parent diazepine 2. Therefore, the X-ray crystal structure
of compound 2 was used to predict the orientation of acyl
groups at N5 and it shows that chloroacetyl group at N5 prefers
to adopt an exo orientation. Hence, it was decided that N-CO
groups at N5 position of compounds 7 and 8 also adopt exo
orientation.
The N-acyl derivatives of benzodiazepinones 7 and 8 may
prefer any one of the possible conformations (Fig. 2).As discu-
ssed earlier, N5-chloroacetyltetrahydro-1,5-benzodiazepin-2-
one (2) prefers to adopt boat conformation BE with exo orien-
tation of the -COCH2Cl group at N5-position (Fig. 3). In the
chair CA and boat BA conformations, the coupling constants
J3a,4a and J3e,4a are expected to be around 2-5 Hz. However, one
of the observed coupling constants was larger for compounds
7 and 8 (7: 6.0 & 12.8 Hz and 8: 5.6 & 12.0 Hz). In addition,
analysis using Dreiding models indicates that chair CA and
boat BA forms require an approximate cis and trans dihedral
angle of 60º. But observed dihedral angles (45º and 165º
calculated using DAERM) eliminate the possibility of CA and
BA forms.
In CE and BE conformations, the C2-C3-C4 part is almost
similar, and the coupling constant cannot be used to decide the
possibility of conformations between CE and BE. Thus, a
choice between the conformations CE and BE can be decided
using Dreiding models, which indicate that expected dihedral
angles would be around 60º for CE and around 0º for boat
conformation BE. The preferred conformation of parent comp-
ound 2 , observed coupling constants and dihedral angles were
used to decide the conformation of compounds 7 & 8 and the
preferred conformation was assigned to be BE for compounds
7 & 8 (Fig. 3).
Thus, it is concluded that N-piperazinoacetyl- and N-mor-
pholinoacetyltetrahydro-4-methylbenzodiazepin-2-ones 7 and
TABLE-2
ANTIBACTERIAL ACTIVITY OF THE COMPOUNDS 1-8
Zone of inhibition (mm)
Sample (100 µg/L)
Organisms
Ciprofloxacin
(10 µg/L)
1
8
8
8
6
8
2
8
9
10
10
8
3
8
8
9
6
9
4
9
7
8
8
9
5
7
8
7
7
6
8
9
9
9
9
7
8
7
6
8
8
8
9
7
6
8
8
Staphylococcus aureus
Staphylococcus albus
Escherichia coli
Salmonella paratyphi
Klebsiella pneumonia
8
25
11
26
20
10

Vol. 32, No. 5 (2020)
Organisms
Synthesis, Stereochemistry and Antimicrobial Evaluation of N-Acyltetrahydrobenzodiazepin-2-ones 1013
TABLE-3
ANTIFUNGAL ACTIVITY OF THE COMPOUNDS 1-8
Zone of inhibition (mm)
Sample (100 µg/L)
Clotrimazole
(10 µg/L)
1
8
8
8
11
9
2
9
9
7
10
11
3
8
10
7
12
9
4
8
5
8
6
20
8
13
9
7
8
9
8
15
9
8
7
13
9
13
8
Aspergillus niger
Aspergillus fumigates
Candida albicans
Monascus ruber
12
11
15
13
15
8
7
10
8
9
10
10
7
Aspergillus parasites
9
by measuring linear growth (mm) of the compounds 1-8, and
compared with clotrimazole which was used as a standard
reference (Table-3).
ound 2 against Aspergillus fumigates, compound 7 possessed
a comparable activity and compound 8 was more active.
Conclusion
The parent benzodiazepinone 1 exhibits superior anti-
fungal activity against Monascus ruber, better activity against
Aspergillus fumigates, good activity against Aspergillus niger
and Aspergillus parasites and moderate activity against Candida
albicans. The chloroacetyl benzodiazepinone 2 shows superior
activity against Aspergillus fumigates, better activity against
Monascus ruber, Aspergillus niger and Aspergillus parasites
and moderate activity against Candida albicans.
The diformyl benzodiazepinone 3 shows superior activity
against Monascus ruber and Aspergillus parasites, better activity
against Aspergillus niger, good activity against Aspergillus
parasites and moderate activity against Candida albicans. The
compound containing dichloroacetyl group at N5 position 4
shows better activity against Aspergillus fumigates, Monascus
ruber, Aspergillus niger and Aspergillus parasites and moderate
activity against Candida albicans. The diethoxy carbonyl benzo-
diazepinone 5 exhibits superior antifungal activity against
Aspergillus fumigates and better activity against Monascus
ruber and Aspergillus niger, good activity against Candida
albicans and moderate activity against Aspergillus parasites.
The introduction of two chloroacetyl groups at N1 & N5 positions
of benzodiazepinone 1, results in the formation of compound
6 and it shows marked improvement in activity against all the
organisms. The bischloroacetyl benzodiazepinone 6 has superior
activity against Aspergillus niger and Candida albicans, better
activity against Monascus ruber and Aspergillus fumigates and
good activity against Aspergillus parasites.
Compound 7, obtained by the substitution of piperazine
moiety in compound 2 exhibits superior antifungal activity
against Monascus ruber andAspergillus fumigates, better activity
against Aspergillus niger, good activity against Candida albicans
and Aspergillus parasites. The replacement of morpholine in
the place of piperazine of compound 7 results in the formation
of compound 8 and it shows superior activity against Monascus
ruber and Aspergillus fumigates and good activity against
Aspergillus niger, Candida albicans and Aspergillus parasites.
All the compounds have superior activity against
Aspergillus fumigates and Monascus ruber. Compounds 6, 7
& 8 were more active than standard and among these the bis-
chloroacetyl benzodiazepinone 6 showed a superior antifungal
activity against Aspergillus niger. Introduction of piperazine
and morpholine moieties at N5 position of compound 2 incre-
ased the activity against Monascus ruber and Candida albicans
and decreases the activity against Aspergillus niger &Aspergillus
parasites. However, when compared to the activity of comp-
On the basis of the above observations, it is concluded
that N5-chloroacetyl, N5-formyl and N5-dichloroacetyl-, N5-
piperazinoacetyl- and N5-morpholinoacetyltetrahydro-1,5-
benzodiazepin-2-ones 2-4 and 7-8, respectively, prefer a boat
conformation (BE) with exo orientation (syn to C4) of acyl
groups at N5 position and N1,N5-diethoxycarbonyl and N1,N5-
bischloroacetyltetrahydro-1,5-benzodiazepin-2-ones 5 & 6,
prefer a boat conformation (BE) with endo orientation (syn to
C10) of acyl groups at N1 position and exo orientation (syn to
C4) of acyl groups at N5 position. The synthesized compounds
1-8 display a superior and better antimicrobial activity against
selected the antibacterial and fungal strains when compared
to the standard.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interests
regarding the publication of this article.
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