Synthesis, antimicrobial, and mitotic toxicity evaluation of new 6-substituted 2-(benzo[4,5]imidazo[1,2-c]quinazolin-5(6H)-yl)acetic acids

Article abstract of DOI:10.1002/jhet.4161

A series of novel 6-substituted 2-(benzo[4,5]imidazo[1,2-c]quinazolin-5(6H)-yl)acetic acids were synthesized and characterized by ¹H, ¹³C, ¹⁹F NMR, ¹H-¹H-COSY, ¹H-¹³C-HSQC, NOESY, LC-MS, IR, and elemental analysis. The mitotic toxicity of the synthesized compounds was determined according to the Allium test procedure. The 2-(6-(pentafluorophenyl)benzo[4,5]imidazo[1,2-c]quinazolin-5(6H)-yl)acetic acid inhibited mitotic spindle formation, which resulted in significant cytotoxic effect for meristematic cells of Allium cepa l. roots. In a preliminary antimicrobial evaluation, only Streptococcus pyogenes and Candida albicans were slightly susceptible to some of the synthesized compounds.

Full text of DOI:10.1002/jhet.4161

Synthesis, antimicrobial and mitotic toxicity evaluation of new 6-
substituted 2-(benzo[4,5]imidazo[1,2-c]quinazolin-5(6H)-yl)acetic
acids
[
a]
[a]
[a]
Dmytro V. Kravtsov* , Oleksii Yu. Voskoboinik and Serhii I. Kovalenko
Abstract: A series of novel 6-substituted 2-(benzo[4,5]imidazo[1,2-
c]quinazolin-5(6H)-yl)acetic acids was synthesized and
would provide an opportunity to understand the structure-activity
relationship and to identify more advantageous option.
a
1
13
19
1
1
1
13
characterized by H, C, F NMR, H- H-COSY, H- C-HSQC,
NOESY, LC-MS, IR and elemental analysis. Mitotic toxicity of the
synthesized compounds was determined according to the Allium test
Obtained results may be used for purposeful search of
chemotherapeutic agents among compounds with cytotoxic
activities or finding promising objects for studies aimed at
developing of compounds with other types of pharmacological
activity among non-cytotoxic products.
procedure.
The
2-(6-(pentafluorophenyl)benzo[4,5]imidazo[1,2-
c]quinazolin-5(6H)-yl)acetic acid inhibited mitotic spindle formation,
which resulted in significant cytotoxic effect for meristematic cells of
Allium cepa L. roots. In a preliminary antimicrobial evaluation, only
Streptococcus pyogenes and Candida albicans were slightly Results and Discussion
susceptible to some of the synthesized compounds.
Chemistry
Initially, commercially available anthranilic acid, as mentioned
above, was condensed with 1,2-phenylenediamine in a mixture
of concentrated H PO and P 10 at 250°C to afford 1, which
Introduction
3
4
4
O
Benzo[4,5]imidazo[1,2-c]quinazoline scaffold is of significant
focus in the field of medical chemistry due to its broad spectrum
was further treated with carbonyldiimidazole (CDI) to provide
intermediate 2 (this compound had been previously obtained by
[
1]
of
biological
properties,
and bronchodilator
The vast majority
ranging
from
antitumor,
to antimicrobial
of bioactive
[29]
another synthetic method)
(Scheme 1). N-alkylation of this
[
2]
[3,4]
anticonvulsant
activities.
intermediate by ethyl 2-chloroacetate in the presence of
anhydrous K CO gave an almost quantitative yield of 3 after the
[
5-9]
2
3
benzo[4,5]imidazo[1,2-c]quinazolines are represented by 6-aryl
and 6-hetaryl substituted derivatives. The most common
approaches towards their synthesis involve the condensation of
anthranilic acid with 1,2-phenylenediamine in polyphosphoric
refluxing for a few hours in anhydrous dimethylformamide (DMF).
Subsequent hydrolysis of the ester and cyclic urea groups of 3
by treatment with an excess of NaOH in a H
2
O-EtOH solution at
80°C afforded 4. Finally, the target 6-substituted 2-
[8,10]
acid (PPA)
or the cyclodehydrogenation of 2-
(
(
benzo[4,5]imidazo[1,2-c]quinazolin-5(6H)-yl)acetic acids 5a-n
Figure 1) were prepared by refluxing of appropriate aldehyde
nitrobenzaldehyde with 1,2-phenylenediamine in the presence of
KHSO
5
, CeCl
3
∙7H
2
O-CuI-I
2
or air as oxidants, followed by
The cyclization of the resulting 2-
with 4 in AcOH for 6 hours. During this time the color
fluorescence of the solution in UV-A was almost always
gradually changing from blue to green. Potassium salts 6a and
[
11-15]
reduction of nitro group.
(
1H-benzo[d]imidazol-2-yl)aniline with different aldehydes, acyl
chlorides or orthoesters gives 6-substituted 5,6-dihydrobenzo- or
benzo[4,5]imidazo[1,2-c]quinazolines,
Besides, the fusion of 2-substituted 4H-3,1-benzoxazin-4-one
with 1,2-phenylenediamine also yields quite good results.
6b were prepared by treatment of 5k and 5g, respectively, with
[
2,12,16-25]
respectively.
KOH in EtOH.
[
26-28]
Particular attention should be given to the clean-up of the
reaction products between 4 and (R)-2-methylhexanal, which
It should be noted, that despite the large number of publications
on the synthesis of benzo[4,5]imidazo[1,2-c]quinazolines their
was far less straightforward. It was supposed to be
a
diastereomeric mixture, but, according to the TLC data, only one
component was detected as a product of the reaction, which
was unexpected. What is more interesting is that after silica gel
column chromatography of the crude product LC-MS analysis
gave the single molecular ion peak with desired mass.
5,6-disubstituted derivatives are almost unknown. Therefore, this
article reveals the synthetic route for aforementioned class of
compounds. The designed method was used to introduce
carboxymethyl group into the 5-position and various substituents
into the 6-position of benzo[4,5]imidazo[1,2-c]quinazoline
scaffold. The in vitro evaluation of the obtained compounds’
antimicrobial and mitotic toxicity activities was conducted as well.
Moreover, a detailed analysis of the structure of compounds
1
13
Nevertheless, H and C NMR data made it clear that the
sample was a mixture composed of two diastereomers in the 1:1
ratio. It is important to emphasize that the chemical shifts were
almost identical to each other and often came together. The
attempts to separate these diastereomers by column
chromatography were unsuccessful.
[
a]
Dmytro V. Kravtsov*, Oleksii Yu. Voskoboinik, Serhii I. Kovalenko
Department of Organic and Bioorganic Chemistry
Zaporizhzhia State Medical University
Mayakovsky ave., 26, 69035, Zaporizhzhia, Ukraine
E-mail: kravtsovsynthesis@gmail.com
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not been through the copyediting, typesetting, pagination and proofreading process
which may lead to differences between this version and the Version of Record. Please
cite this article as doi: 10.1002/jhet.4161
This article is protected by copyright. All rights reserved.

Scheme 1. Synthesis of 6-substituted 2-(benzo[4,5]imidazo[1,2-c]quinazolin-5(6H)-yl)acetic acids 5a-n. Reaction conditions: (i) PPA, 250°C, 4 h, 25.3%; (ii) CDI,
dioxane, reflux, 2 h, 75.1%; (iii) K CO , DMF, reflux 1 h, then ClCH COOEt, reflux, 3 h, 94.9%; (iv) NaOH, H O-EtOH, 80°C, 4 h, 92.2%; (v) RCHO, AcOH, reflux,
h, 16.8-75.3%.
2
3
2
2
6
Figure 1. Structures of obtained compounds.
In addition, it was found that ketones did not react with 4. Thus,
acetophenone gave only 2% of the desired product according to
the LC-MS data of the residue, while acetone even gave no
tracers. In both cases, about 20% of decarboxylation product of
MeOH with a few drops of conc. HCl as a catalyst for an hour
and a half led to a small amount of methyl ester of 4 after
column chromatography (20-75% EtOAc-hexane). This leads to
the conclusion that the steric hindrance greatly affects reaction
in this case.
4
was observed. The refluxing of 4 and 4-bromoacetophenone in
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The chemical structures of all synthesized compounds were
verified by spectroscopic methods. Chemical ionization mass
spectra of the compounds 2-4 and 5a-n displayed the correct
molecular ions in accordance with the suggested structures. The
diastereotopic methylene protons in acetic acid moiety of
compounds 5b-n have a different chemical environment due to
the presence of chiral carbon atom at C(6). Hence, signals of
above-mentioned protons were registered as a doublet of
2
doublets at the range of 4.15-5.04 ppm with J coupling constant
1
3
of 18.2-19.0 Hz. The C NMR-spectra additionally proved the
structure of synthesized 6-substituted 2-(benzo[4,5]imidazo[1,2-
c]quinazolin-5(6H)-yl)acetic acids 5a-n. Thus, the signal of the
carbon atom at the 6-position at the range 60.6-78.8 ppm was
characteristic and confirmed the formation of partially saturated
1
3
pyrimidine-containing polycyclic system. In C NMR spectrum of
compound 5l the signals of carbon atoms in pentafluorophenyl
group were registered as a series of hardly distinct low-intensive
multiplets, which melted into the background noise due to the C-
Figure 2. Selected NOE correlations for diastereomeric mixture 5c.
6 5
F couplings. However, the presence of the C F group in 5l was
clearly confirmed by F NMR (see Supporting Information File).
1
9
Biology
IR spectra of compounds 5g and 5k were characterized by
-1
The 6-substituted 2-(benzo[4,5]imidazo[1,2-c]quinazolin-5(6H)-
yl)acetic acids 5a-n and potassium salts 6a and 6b were tested
in vitro for their antibacterial activity against 7 strains of Gram-
medium-weak absorption bands at 1704-1709 cm (C=O str.)
-1
-
and medium-strong ones at 1612-1616 cm (COO str. as.) and
-1
-
1
395-1392 cm (COO str. s.). The aforementioned fact makes it
negative bacteria and
according to the Kirby–Bauer disc diffusion method.
2 strains of Gram-positive bacteria
possible to assume that compounds 5g and 5k might exist in
zwitterion form. In IR spectra of potassium salts 6a and 6b there
are no absorption bands of C=O group, while the COO bands
[
31]
From
-
screening result, it was observed that 5c, 5e, 5h, 5l and 6a were
slightly (12 mm) active against Streptococcus pyogenes
compared to ciprofloxacin (20 mm) as a reference broad-
spectrum antibiotic (Table 1). The antifungal activity was
similarly evaluated in vitro against Candida albicans. This strain
showed very small inhibition zones of 9 mm around a 5g disk
and 8 mm around a 5k disk.
became more intense. All of them show strong absorption band
-1
at 742 cm (C-Haryl bend.), which indicates that they are 1,2-
[
30]
substituted benzenoid compounds. The chemical structure of
1
1
1
5
c was fully characterized based on the H- H-COSY and H-
1
3
C-HSQC data. The stereochemistry was unambiguously
determined by NOESY experiment (see Figure 2 and Supporting
Information File).
Table 1. Antimicrobial screening results of the tested compounds 5a-n, 6a and 6b.
Compounds
Diameter of the inhibition zone in mm
5
5
5
5
5
a
b
c
d
e
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
9
6
12
6
12
6
5
f
5
g
h
6
5
12
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5
i
j
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
5
5k
6
6
6
6
6
6
6
6
6
8
5
l
6
12
6
6
6
6
6
6
6
6
6
5m
6
6
6
6
6
6
6
6
6
5n
6a
6b
6
6
6
6
6
6
6
6
6
6
6
12
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
Ciprofloxacin
Ketoconazole
18
-
20
-
32
-
37
-
30
-
35
-
28
-
30
-
27
-
-
17
A few interesting features of the structure-activity relationship
were discovered. Thus, according to the reported study 6-
decreased the activity against above-mentioned strain. The
replacement of the carboxy and hydroxyl groups in 5k by their
potassium salts (6a) decreased antifungal activity and
significantly increased activity against S. pyogenes. It should be
noted that compound 5k as well as jatrorrhizine comprised (4-
hydroxy-3-methoxybenzyl)(methyl)amino fragment and both
(
pyridine-3-yl)benzo[4,5]imidazo[1,2-c]quinazoline was active
[
8]
against a number of bacteria and fungi, while 2-(6-(pyridin-3-
yl)benzo[4,5]imidazo[1,2-c]quinazolin-5(6H)-yl)acetic acid (5m)
was not (Figure 3A). Apparently, the partial saturation
benzo[4,5]imidazo[1,2-c]quinazoline fragment as well as
introduction of acetic acid moiety into the 5-position led to the
loss of antimicrobial activity. Additional introduction of aryl
fragment with methoxy group at the para position (5g) caused
the occurrence of growth inhibition activity towards to C.
albicans strain. The replacement of methoxy group by hydroxyl
group (5f) resulted in the loss of antifungal activity. At the same
time, compound 5k that contained both as methoxy and hydroxyl
groups in aryl fragment turned out to have caused a slightly
higher antifungal activity than 5g. The introduction of long
aliphatic fragment (5c) and benzene ring (5e) into the 6-position
of heterocyclic system resulted in the appearance of activity
against S. pyogenes. The introduction of oxygen-containing
groups into the aryl fragment at the 6-position (5f, 5g, 5k) and
substitution of aryl fragment by heterocyclic moiety (5m, 5n)
[
32]
showed a weak antifungal activity,
which implies that this
fragment is a key structural component for further studies to
develop new antifungal agents (Figure 3B).
Subsequently the mitotic toxicity of synthesized 6-substituted 2-
(benzo[4,5]imidazo[1,2-c]quinazolin-5(6H)-yl)acetic acids 5a-n
[
33,34]
were examined according to the Allium test procedure.
The
above-mentioned method was chosen due to its conventional
application for screening of mutagenic and cytotoxic effects of
chemical factors, availability and reproducibility. Moreover, this
test was described as an effective drug safety evaluation
[
35]
[36]
method. Methotrexate (m) was used as a reference agent,
while tap water (w) and KOH (k) – as intact ones. Figure 4
shows images of the bulbs and root length taken at the
beginning of the experiment and at the end of days 1, 2 and 3.
Figure 3. The structure-activity relationship between the new synthesized compounds and previously reported ones.
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inhibitory activity of obtained compounds. On the one hand,
mitotic index of 5c, 5i and 5n was the same as for methotrexate,
namely equal to 0.0% (Table 3). On the other hand, the severe
degenerative changes were induced by 5i, 5n (karyolysis) and
especially 5c (karyolysis and extensive cell fragmentation),
which indicated a highly toxic effect (Figure 5G).
Figure 4. Growth retardation effects of compounds 5a-n. w tap water, k 0.001
N KOH in tap water, m 1 mmol/l methotrexate in tap water with 0.002 N KOH,
5a-n 1 mmol/l 5a-n in tap water with 0.001 N (0.002 N for 5f and 5k) KOH.
First of all, it is noteworthy that m led to a complete inhibition of
root growth of Allium cepa L., thus, the total increase of root
length in 3 days was 0 mm, while it was 25 mm in w and 47 mm
in k (Table 2). The microscopic study showed that under the
action of methotrexate meristematic root tip cells undergo some
degenerative changes, such as karyopyknosis and cell
fragmentation (Figure 5F). Compounds 5c, 5e, 5h, 5i, 5l and 5n
exhibited a significant growth inhibition effect. The analysis of
the structure-roots growth inhibition activity relationship revealed
that the introduction of halogen or halogen-containing group into
aryl (hetaryl) fragment at the 6-position had promoted the growth
Figure 5. Cellular alterations observed by analysing meristematic cells of
Allium cepa L. roots. Treatment in parentheses. A normal interphase (tap
water). B normal prophase (tap water), C normal methaphase (tap water), D
normal anaphase (tap water), E normal telophase (tap water), F karyopyknosis
(methotrexate), G karyolysis (5c), H c-mitosis (5l) (600×).
Table 2. Roots lengths of AIIium cepa L. after 3 days of cultivation in 1 mmol/l 5a-n aqueous solution with 0.001 N (0.002 N for 5f and 5k) KOH.
Compounds
mmol/l
Root length in mm
Total increase of root
length in mm
1
Day 0
Day 1
22
Day 2
Day 3
5a
10
31
36
26
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5
b
5
9
13
10
18
7
18
10
30
10
25
18
11
12
37
19
8
24
10
37
12
31
23
12
13
40
26
10
30
11
33
57
9
19
1
5
c
5d
6
31
7
5
e
5
5
f
10
5
22
11
8
21
18
7
5
g
h
5
5
5
i
10
8
11
29
12
6
3
5
j
32
21
4
5
k
5
5
l
6
5m
8
16
7
21
11
31
46
9
22
5
5n
6
[
a]
w
8
23
32
9
25
47
0
[
b]
k
10
9
[
c]
m
[
a] Tap water. [b] 0.001 N KOH in tap water. [c] 1 mmol/l methotrexate in tap water with 0.002 N KOH.
[
37]
Since c-mitosis have been observed in root tips that had been
exposed to phenyl (5e) and pentafluorophenyl (5l) derivatives,
these compounds are of particular interest as potential
chemotherapeutic agents (Figure 5H). Moreover, any other
abnormalities of root apex cells were not detected. C-mitosis is
characterized by well separated metaphase chromosomes
dispersed in the cell, not oriented along the equatorial plate. The
occurrence of c-mitosis indicates that 5e and 5l have caused the
inhibition of spindle formation, similar to the effect of
colchicine. Compared to tap water, 5e and 5l reduced the
mitotic index by 57.14% and 81.63%, respectively. The
percentage of cells in c-mitosis for 5e and 5l was closely
correlated to their mitotic index values and was 14.3% and
33.3%, respectively. Thus, it can be concluded that the
replacement of all hydrogen atoms of phenyl group at the 6-
position by fluorine atoms considerably increased the inhibition
of spindle formation.
Table 3. Mitotic toxicity of compounds 5a-n.
Compounds
mmol/l
Total cells
counted
Cells in mitotic
stage
Mitotic index (%)
C-mitosis (%)
1
5
5
5
5
5
a
b
c
d
e
632
691
617
637
660
624
673
22
26
0
3.5
3.8
0.0
3.9
2.1
3.7
3.6
0.0
0.0
0.0
0.0
14.3
0.0
0.0
25
14
23
24
5
f
5g
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5
h
679
602
618
651
645
645
611
658
610
624
13
0
1.9
0.0
4.7
3.8
0.9
3.6
0.0
4.9
6.4
0.0
0.0
0.0
0.0
0.0
33.3
0.0
0.0
0.0
0.0
0.0
5
i
5
j
29
25
6
5
k
5
l
5m
23
0
5n
[
a]
w
32
39
0
[
b]
k
[
c]
m
[
a] Tap water. [b] 0.001 N KOH in tap water. [c] 1 mmol/l methotrexate in tap water with 0.002 N KOH.
Any chromosome aberrations, abnormalities in the mitotic cycle
or degenerative changes to the other synthesized compounds
were not detected. As we consider, compounds that have not Experimental Section
revealed anti-mitotic activity are interesting objects for screening
of types of biological activities, which are not associated with
inhibition of cell growth.
Chemistry
The substance
1
was synthesized according to the reported
[
10]
procedure.
All commercially available chemicals were used without
further purification. Melting points were uncorrected. IR spectra were
recorded on a Shimadzu IR Prestige-21 Fourier spectrometer fitted with
an attenuated total reflectance sampling accessory. The elemental
analyses (C, H, and N) were performed using the Elementar vario EL
cube analyzer. NMR spectra were measured on a Bruker DRX 500 (400
Conclusions
The convenient method for synthesis of 5,6-disubstituted
benzo[4,5]imidazo[1,2-c]quinazolines based on sequential
1
13
19
alkylation
of
benzo[4,5]imidazo[1,2-c]quinazolin-6(5H)-one,
or 500 MHz, H; 100, 125 or 150 MHz,
C; 470 MHz,
F).
1
13
19
hydrolytic cleavage of pyrimidone fragment and two component
heterocyclocondensation with carbonyl-containing reagents has
been elaborated. The structures of the obtained 6-substituted 2-
Tetramethylsilane ( H, C) or CFCl ( F) were used as an internal
3
reference for the spectra. The chemical shift values (δ) and coupling
constants (J) are expressed in parts per million (ppm) and hertz (Hz),
respectively. Chemical shifts were referenced to residual non deuterated
(
benzo[4,5]imidazo[1,2-c]quinazolin-5(6H)-yl)acetic acids have
1
13
solvent (dimethyl sulfoxide (DMSO) H: δ = 2.50 ppm, C: δ = 40.0 ppm
been confirmed by a modern physicochemical method. The
preliminary screening for antimicrobial activity and mitotic toxicity
of the obtained compounds has been conducted. It has been
1
13
and trifluoroacetic acid (TFA) H: δ = 11.18 ppm, C: δ = 153.0, 105.7
ppm). Column chromatography was carried out using Merck silica gel 60
(0.040-0.063 mm) as the stationary phase and analytical TLC was
established,
that
6-substituted
2-(benzo[4,5]imidazo[1,2-
performed using Merck silica gel 60 F254 aluminium sheets. Mass spectra
were recorded with an LC-MS instrument using chemical ionization (CI).
LC-MS data were acquired with an Agilent 1200 HPLC system equipped
with a DAD/ELSD/LCMS-6120 diode matrix and mass-selective detector,
c]quinazolin-5(6H)-yl)acetic acids are not active against most of
the studied strains, but some of the synthesized compounds
have been slightly active against S. pyogenes (5c, 5e, 5h, 5l,
column: Poroshell 120 SBC18, 4.6×30 mm; eluent A: MeCN-H
2
O 99:1
6a) and C. albicans (5g, 5k). According to the Allium test result
with 0.1% of HCO H; eluent B: H O with 0.1% of HCO H.
2
2
2
the substance 5k or its dipotassium salt 6a showed no
significant mitotic toxicity effect. The pentafluorophenyl
derivative 5l displayed potent in vitro cytotoxic effect (inhibition
of mitotic spindle formation) for root meristematic cells of Allium
cepa L. and it showed an activity close to that of the positive
control methotrexate. The analysis of structure-biological activity
correlations made it possible to detect the fragments that are
essential for occurrence of the proper type of biological activity.
The absence of mitotic toxicity of some of the obtained
compounds showed high prospects of their further screening for
other types of biological activity.
Benzo[4,5]imidazo[1,2-c]quinazolin-6(5H)-one (2)
1
3.0 g (62.1 mmol) of 1 and 11.5 g (70.9 mmol) of CDI in 75 ml of
dioxane were refluxed for 2 h. The reaction mixture was poured into 200
ml of H O and neutralized with conc. HCl, making solution slightly acid.
The precipitate was filtered, washed with H O and dried at 60°C to
provide a white-gray solid. Yield: 10.93 g (75.1%); m.p. >300°C; H NMR
2
2
1
3
6
(500 MHz, DMSO-d ): δ 11.95 (s, 1H), 8.36 (d, J = 7.9 Hz, 1H), 8.31 (d,
3
3
3
J = 8.0 Hz, 1H), 7.86 (d, J = 7.9 Hz, 1H), 7.65 (t, J = 7.7 Hz, 1H), 7.55
13
–
6
7.29 (m, 4H); C NMR (125 MHz, DMSO-d ): δ 148.1, 146.8, 144.0,
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1
1
37.6, 132.7, 131.1, 125.5, 124.9, 124.1, 123.8, 119.6, 116.4, 115.2,
Yield: 0.38 g (50.0%) as a brown solid; m.p. 231°C; H NMR (500 MHz,
+
3
3
112.3; MS (CI): m/z 236.1 [M + H] ; Anal. Calcd. for C14
9 3
H N O: C, 71.48;
DMSO-d
6
): δ 12.89 (s, 1H), 8.02 (d, J = 7.6 Hz, 1H), 7.67 (d, J = 7.0 Hz,
1H), 7.52 (d, J = 7.4 Hz, 1H), 7.37 (t, J = 7.9 Hz, 1H), 7.30 – 7.19 (m,
H), 6.93 (t, J = 7.5 Hz, 1H), 6.77 (d, J = 8.3 Hz, 1H), 6.31 (q, J = 5.9
Hz, 1H), 4.35 (d, J = 18.2 Hz, 1H), 4.26 (d, J = 18.3 Hz, 1H), 1.35 (d, J
5.9 Hz, 3H); C NMR (125 MHz, DMSO-d ): δ 171.7, 146.3, 144.2,
6
42.4, 132.4, 132.2, 125.3, 122.7, 122.7, 119.2, 119.2, 114.5, 114.0,
10.1, 68.3, 51.2, 19.6; MS (CI): m/z 294.0 [M + H] ; Anal. Calcd. for
C H N O : C, 69.61; H, 5.15; N, 14.33; Found: C, 69.57; H, 5.10; N,
17 15 3 2
3
3
H, 3.86; N, 17.86; Found: C, 71.41; H, 3.89; N, 17.91.
3
3
3
2
2
2
3
Ethyl 2-(6-oxobenzo[4,5]imidazo[1,2-c]quinazolin-5(6H)-yl)acetate (3)
13
=
1
1
10.93 g (46.5 mmol) of 2 and 3.17 g (22.9 mmol) of anhydrous K
2 3
CO in
+
75 ml of dry DMF were refluxed for 1 h. 5.47 g (44.6 mmol) of ethyl 2-
chloroacetate in 10 ml of dry DMF was added dropwise to the stirring
mixture. The reaction mixture was refluxed for 3 h and poured into 500 ml
14.37.
of H
2 2
O. The precipitate was filtered, washed with H O and dried at 60°C
1
2-((R)-6-((R)-Hexan-2-yl)benzo[4,5]imidazo[1,2-c]quinazolin-5(6H)-
yl)acetic acid with 2-((S)-6-((R)-hexan-2-yl)benzo[4,5]imidazo[1,2-
c]quinazolin-5(6H)-yl)acetic acid (1:1) (5c)
to afford a white solid. Yield: 14.17 g (94.9%); m.p. 207°C; H NMR (400
MHz, DMSO-d ): δ 8.49 (d, J = 7.7 Hz, 1H), 8.38 (d, J = 8.0 Hz, 1H),
6
3
3
3
3
7.81 (d, J = 7.7 Hz, 1H), 7.69 (t, J = 8.3 Hz, 1H), 7.52 – 7.37 (m, 4H),
3
3
13
5
.15 (s, 2H), 4.26 (q, J = 7.1 Hz, 2H), 1.32 (t, J = 7.0 Hz, 3H); C NMR
): δ 168.5, 147.0, 147.0, 143.9, 137.7, 133.2, 131.1,
25.9, 125.5, 124.6, 124.5, 119.8, 115.8, 115.2, 113.0, 61.9, 45.1, 14.5;
(125 MHz, DMSO-d
6
Yield: 0.16 g (16.8%) as a light brown solid; R
f
= 0.80 (DCM-EtOH 6:4);
m.p. 70°C (softens), 81-87°C (melts); H NMR (500 MHz, DMSO-d ): δ
7.98 (d, J = 7.7 Hz, 1H, H-C(1)), 7.65 (d, J = 7.5 Hz, 1H, H-C(11)), 7.55
1
1
6
+
3
3
MS (CI): m/z 322.2 [M + H] ; Anal. Calcd. for C18
15 3 3
H N O : C, 67.28; H,
3
3
3
4.71; N, 13.08; Found: C, 67.24; H, 4.68; N, 13.12.
(d, J = 7.5 Hz, 1H, H-C(8)), 7.53 (d, J = 8.3 Hz, 1H, H-C(8)), 7.33 (t, J
3
=
8
1
CH
7.9 Hz, 1H, H-C(3)), 7.25 – 7.17 (m, 2H, H-C(9), H-C(10)), 6.97 (t, J =
.5 Hz, 1H, H-C(4)), 6.91 (t, J = 6.1 Hz, 1H, H-C(2)), 6.06 (d, J = 5.1 Hz,
H, H-C(6)), 6.04 (d, J = 5.6 Hz, 1H, H-C(6)), 4.58 (d, J = 14.6 Hz, 1H,
3
3
(
2-(1H-Benzo[d]imidazol-2-yl)phenyl)glycine (4)
3
2
2
2
2
COOH), 4.53 (d, J = 14.6 Hz, 1H, CH
2
COOH), 4.18 (d, J = 18.2 Hz,
COOH), 2.06 – 1.94 (m,
14.17 g (44.1 mmol) of 3 and 26.5 g (0.66 mol) of NaOH in 310 ml of
2
1
1
1
H, CH
H, CHCH
.04 – 1.00 (m, 4H, CH
2
COOH), 4.15 (d, J = 18.2 Hz, 1H, CH
2
2
H O-EtOH 85:15 were heated at 80 °C for 4 h with stirring. The resulting
3
), 1.44 – 1.30 (m, 1H, CHCH
CH CH ), 0.77 – 0.65 (m, 6H, CHCH
C NMR (150 MHz, DMSO-d
47.2 (N(7)-C-N(12)), 144.2 (C-N(7)), 144.1 (C-N(7)), 144.0 (C-N(12)),
2
), 1.25 – 1.15 (m, 1H, CHCH
, CH CH
2
),
solution was filtered hot and 53 ml (determined by titration of starting
NaOH solution, using phenolphthalein as an internal indicator) of conc.
HCl was added. The precipitate was filtered, washed with H O and dried
2
2
2
3
3
2
3
);
13
6
): δ 171.9 (COOH), 147.3 (N(7)-C-N(12)),
1
at 60°C to give a white-yellow solid. Yield: 10.87 g (92.2%); m.p. 145°C;
1
3
143.9 (C-N(12))), 133.8 (C-N(5)), 133.6 (C-N(5)), 131.8 (C(3)), 131.8
(C(3)), 125.0 (C(1)), 122.3 (C(9)), 122.2 (C(9)), 122.1 (C(10)), 122.0
(C(10)), 119.5 (C(2)), 119.4 (C(2)), 119.0 (C(11)), 118.9 (C(11)), 116.4
H NMR (400 MHz, DMSO-d
6
): δ 12.68 (s, 1H), 9.42 (s, 1H), 7.90 (d, J =
3
7.0 Hz, 1H), 7.62 – 7.51 (m, 2H), 7.24 (t, J = 7.1 Hz, 1H), 7.21 – 7.14 (m,
3
3
13
2H), 6.71 (t, J = 7.5 Hz, 1H), 6.63 (d, J = 8.4 Hz, 1H), 4.08 (s, 2H);
C
(
C-C(1)), 116.3 (C-C(1)), 115.7 (C(4)), 111.0 (C(8)), 110.8 (C(8)), 75.6
(C(6)), 75.4 (C(6)), 54.8 (CH COOH), 54.6 (CH COOH), 41.0 (CHCH ),
0.9 (CHCH ), 31.5 (CH CH ), 31.4 (CHCH ), 28.9 (CH CH ), 28.8
), 22.6 (CH CH CH ), 22.5 (CH CH CH ), 15.3 (CHCH ), 15.0
), 14.1 (CH CH ), 14.0 (CH CH ); H- H-COSY cross-peaks:
NMR (100 MHz, DMSO-d
15.7, 111.6, 111.4, 45.0; MS (CI): m/z 268.0 [M + H] ; Anal. Calcd. for
: C, 67.40; H, 4.90; N, 15.72; Found: C, 67.46; H, 4.86; N,
6
): δ 172.6, 152.6, 147.5, 131.4, 128.0, 122.5,
+
2
2
3
1
4
3
2
3
2
2
3
15 13 3 2
C H N O
(
(
CHCH
CHCH
2
2
2
3
2
2
3
1
3
1
5.76.
General procedure for the synthesis of compounds 5a-n
.6 mmol of 4 and 3.1 mmol of appropriate carbonyl compound in 10 ml
1
3
2
3
2
3
6
.91/7.98 (H-C(2)/H-C(1)), 7.21/7.65 (H-C(10)/H-C(11)), 7.21/7.54 (H-
C(9)/H-C(8)), 6.97/7.33 (H-C(4)/H-C(3)), 4.16/4.55
CHH`COOH/CHH`COOH), 2.00/6.05 (CHCH /H-C(6)), 1.19/1.38
CHCHH`/CHCHH`),1.02/1.38 (CH CH CH /CHCHH`),
CH CH CH /CHCHH`), 0.74/2.00 (CHCH /CHCH ),
CHCH /CHCH ), 0.70/1.02 (CH CH /CH CH
(
(
(
(
3
2
2
2
3
1.02/1.19
0.68/2.00
of AcOH were refluxed for 6 h (5a and 5f fell out of AcOH during the
reaction and after cooling the precipitate was collected by suction
filtration). After evaporation in vacuo, the residue was treated with MeOH.
The precipitate was filtered, washed with MeOH and dried at 60°C to
afford the desired product at >90% purity (5c was very soluble in MeOH,
so the residue after evaporation of solvent was purified by column
2
2
3
3
3
1
13
3
3
2
3
2
3
); H- C-HSQC cross-
peaks:7.33/131.9 (H-C(3)/C(3)), 7.98/124.9 (H-C(1)/C(1)), 7.21/122.3 (H-
C(9), H-C(10)/C(9), C(10)), 7.65/119.0 (H-C(11)/C(11)), 6.91/119.3 (H-
C(2)/C(2)), 6.97/115.6 (H-C(4)/C(4)), 7.54/110.8 (H-C(8)/C(8)), 6.05/75.5
(
(
(
(
H-C(6)/C(6)), 4.55/54.8 (CHH`COOH/ CHH`COOH), 4.17/54.7
chromatography (SiO
diastereomeric mixture in a 1:1 ratio).
2
,
DCM-EtOH 6:4) to give inseparable
CHH`COOH/CHH`COOH), 2.00/41.1 (CHCH
3
/CHCH
3
), 1.02/31.6
1.02/29.0
CH
CH
2
2
2
CH
CH
CH
3
3
2
/CH
/CH
2
CH
CH
3
3
2
),
),
1.38/31.5
1.19/28.9
(CHCH
(CHCH
2
/CHCH
/CHCH
2
),
),
2
2
2
1.06/22.7
2-(Benzo[4,5]imidazo[1,2-c]quinazolin-5(6H)-yl)acetic acid (5a)
(CH
3
CH /CH
CH
2
CH
3
), 0.68/15.2 (CHCH
CH /CH CH ); NOESY cross-peaks:
6.91/7.98 (H-C(2)/ H-C(1)), 7.21/7.65 (H-C(10)/ H-C(11)), 7.21/7.53-7.55
H-C(9)/H-C(8)), 6.04-6.06/7.53-7.55(H-C(6)/H-C(8)), 6.97/7.33 (H-C(4)/
3 3
/CHCH ), 0.72/14.9
(CHCH
3
/CHCH
3
), 0.71/12.7 (CH
2
3
2
3
1
Yield: 0.39 g (53.4%) as a light yellow solid; m.p. 265°C; H NMR (500
MHz, DMSO-d ): δ 8.03 (d, J = 7.6 Hz, 1H), 7.68 (d, J = 7.4 Hz, 1H),
6
3
3
(
3
3
H-C(3)), 6.91/7.33 (H-C(2)/H-C(3)), 4.53/6.97 (CHH`COOH/H-C(4)),
4.15-4.18/ 6.04-6.06 (CHH`COOH/ H-C(6)), 4.15-4.18/4.53-4.58
7
6
2
1
.53 (d, J = 7.5 Hz, 1H), 7.38 (t, J = 7.9 Hz, 1H), 7.30 – 7.17 (m, 2H),
.97 (t, J = 7.4 Hz, 1H), 6.81 (d, J = 8.3 Hz, 1H), 5.68 (s, 2H), 4.33 (s,
H); C NMR (125 MHz, DMSO-d
32.1, 125.5, 122.8, 122.7, 119.7, 119.1, 114.4, 113.4, 110.3, 60.6, 50.6;
3
3
13
(
6
CHH`COOH/ CHH`COOH), 2.00/6.04-6.06 (CHCH
3
/H-C(6)), 1.38/6.04-
), 1.02/7.55
6
): δ 171.6, 147.2, 144.7, 144.0, 133.1,
.06 (CHCH /H-C(6)), 1.38/2.00 (CHCH /CHCH
2
2
3
+
(CH CH CH / H-C(8)), 1.02/6.04-6.06 (CH CH CH / H-C(6)), 1.02/2.00
MS (CI): m/z 280.2 [M + H] ; Anal. Calcd. for C16
13 3 2
H N O : C, 68.81; H,
2
2
3
2
2
3
(
(
0
(
(
(
CH
2
CH
2
CH
3
/CHCH
3
), 1.02/1.38 (CH
2
CH
2
CH
3
/CHCH
2
), 0.74/7.53
CH /H-C(6)),
4
.69; N, 15.05; Found: C, 68.87; H, 4.64; N, 15.09.
CHCH
.68/6.04 (CH
CH CH CH /CHCH
CHCH /CHCH ),
CH CH /CH CH
3
/H-C(8)), 0.68/7.55 (CHCH
3
/H-C(8)), 0.74/6.06 (CH
/CHCH
/CHCH ),
/CHCH ),
); MS (CI): m/z 364.2 [M + H] ; Anal. Calcd. for
2
3
2
CH /H-C(6)), 1.38/2.00 (CHCH
3
2
3
), 1.02/2.00
0.68/2.00
2-(6-Methylbenzo[4,5]imidazo[1,2-c]quinazolin-5(6H)-yl)acetic acid
2
2
3
3
),
0.74/2.00
(CH
(CHCH
CH
3
3
(5b)
3
3
0.70/1.19
2
3
2
0.70/1.08-1.02
+
2
3
2
2
CH
3
For internal use, please do not delete. Submitted_Manuscript
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22 25 3 2
C H N O : C, 72.70; H, 6.93; N, 11.56; Found: C, 72.65; H, 6.97; N,
2-(6-(4-Bromophenyl)benzo[4,5]imidazo[1,2-c]quinazolin-5(6H)-
11.62.
yl)acetic acid (5h)
1
(
5
E)-2-(6-(1-Phenylprop-1-en-2-yl)benzo[4,5]imidazo[1,2-c]quinazolin-
(6H)-yl)acetic acid (5d)
Yield: 0.57 g (50.4%) as a brown solid; m.p. 274°C; H NMR (400 MHz,
3
3
3
TFA-d): δ 8.52 (d, J = 8.0 Hz, 1H), 8.27 (d, J = 8.3 Hz, 1H), 8.20 (t, J =
.9 Hz, 1H), 8.12 (t, J = 7.8 Hz, 1H), 8.08 – 7.97 (m, 3H), 7.86 (d, J =
.3 Hz, 1H), 7.79 (d, J = 7.9 Hz, 2H), 7.73 (t, J = 7.8 Hz, 1H), 7.66 (s,
1H), 7.55 (d, J = 8.3 Hz, 1H), 5.04 (d, J = 19.0 Hz, 1H), 4.92 (d, J =
9.0 Hz, 1H); C NMR (100 MHz, TFA): δ 166.4, 135.0, 134.5, 128.8,
24.2, 122.0, 120.6, 119.2, 119.0, 118.4, 117.1, 117.0, 113.6, 106.7,
104.8, 103.2, 98.0, 66.0, 41.6; MS (CI): m/z 434.0 [M + H] ; Anal. Calcd.
3
3
7
8
3
3
1
Yield: 0.50 g (48.5%) as a green solid; m.p. 255°C; H NMR (500 MHz,
DMSO-d
1
3
2
2
3
3
6
): δ 12.81 (s, 1H), 8.04 (d, J = 7.8 Hz, 1H), 7.69 (d, J = 6.2 Hz,
13
1
H), 7.52 – 7.44 (m, 1H), 7.36 – 7.31 (m, 3H), 7.30 – 7.19 (m, 5H), 7.04
3
3
1
(s, 1H), 6.88 (t, J = 7.3 Hz, 1H), 6.78 (d, J = 8.3 Hz, 1H), 6.64 (s, 1H),
+
2
2
13
4
.39 (d, J = 18.5 Hz, 1H), 4.28 (d, J = 18.2 Hz, 1H), 1.38 (s, 3H);
): δ 171.3, 146.5, 144.3, 143.0, 136.4, 134.5,
32.8, 132.3, 129.5, 129.4, 128.8, 127.7, 125.1, 123.0, 122.7, 119.2,
C
for C22
3 2
H16BrN O : C, 60.84; H, 3.71; N, 9.68; Found: C, 60.92; H, 3.67; N,
NMR (125 MHz, DMSO-d
1
1
Anal. Calcd. for C25
7
6
9.72.
+
18.4, 112.8, 111.9, 111.0, 78.8, 49.9, 12.0; MS (CI): m/z 396.2 [M + H] ;
: C, 75.93; H, 5.35; N, 10.63; Found: C,
H N O
21 3 2
2-(6-(4-(Trifluoromethyl)phenyl)benzo[4,5]imidazo[1,2-c]quinazolin-
5(6H)-yl)acetic acid (5i)
5.98; H, 5.41; N, 10.69.
1
2
-(6-Phenylbenzo[4,5]imidazo[1,2-c]quinazolin-5(6H)-yl)acetic acid
Yield: 0.49 g (44.5%) as a brown solid; m.p. 262°C; H NMR (500 MHz,
3
3
(
5e)
6
DMSO-d ): δ 12.81 (s, 1H), 8.12 (d, J = 7.7 Hz, 1H), 7.70 (d, J = 7.9 Hz,
H), 7.65 (d, J = 8.1 Hz, 2H), 7.44 (d, J = 8.1 Hz, 2H), 7.41 – 7.34 (m,
H), 7.22 (t, J = 7.4 Hz, 1H), 7.19 (t, J = 7.3 Hz, 1H), 6.99 (t, J = 7.5 Hz,
1H), 6.85 (d, J = 8.3 Hz, 1H), 4.40 (d, J = 18.3 Hz, 1H), 4.33 (d, J =
18.3 Hz, 1H); C NMR (125 MHz, DMSO-d ): δ 171.0, 146.4, 144.3,
CF = 32.7 Hz), 127.1, 126.3 (d,
JCF = 3.6 Hz), 125.4, 123.1, 123.0, 119.8, 119.4, 114.8, 114.0, 110.5,
3
3
1
3
3
3
3
1
Yield: 0.57 g (61.2%) as a brown solid; m.p. 259°C; H NMR (500 MHz,
DMSO-d ): δ 12.81 (s, 1H), 8.10 (d, J = 7.7 Hz, 1H), 7.68 (d, J = 8.0 Hz,
6
3
2
2
3
3
13
3
3
1
5
1
1
1
1
H), 7.36 (t, J = 7.3 Hz, 1H), 7.32 (d, J = 7.7 Hz, 1H), 7.29 – 7.23 (m,
H), 7.22 – 7.18 (m, 2H), 7.15 (t, J = 7.6 Hz, 1H), 6.96 (t, J = 7.5 Hz,
H), 6.82 (d, J = 8.4 Hz, 1H), 4.30 (d, J = 18.2 Hz, 1H), 4.22 (d, J =
8.2 Hz, 1H); C NMR (150 MHz, DMSO-d
42.6, 138.9, 132.9, 132.3, 129.6, 129.3, 126.3, 125.4, 122.9, 122.7,
19.5, 119.2, 114.2, 114.0, 110.6, 73.0, 50.7; MS (CI): m/z 356.0 [M +
6
2
3
3
1
43.3, 142.3, 132.8, 132.4, 129.9 (d,
J
3
3
2
2
19
13
72.2, 51.3; F NMR (470 MHz, DMSO-d ): δ -61.24 (s); MS (CI): m/z
6
): δ 171.0, 146.5, 144.2,
6
+
4
24.0 [M + H] ; Anal. Calcd. for C23
16 3 3 2
H F N O : C, 65.25; H, 3.81; N, 9.92;
Found: C, 65.20; H, 3.77; N, 9.97.
+
H] ; Anal. Calcd. for C22
17 3 2
H N O : C, 74.35; H, 4.82; N, 11.82; Found: C,
74.29; H, 4.78; N, 11.86.
2-(6-(4-Nitrophenyl)benzo[4,5]imidazo[1,2-c]quinazolin-5(6H)-
yl)acetic acid (5j)
2
-(6-(4-Hydroxyphenyl)benzo[4,5]imidazo[1,2-c]quinazolin-5(6H)-
1
yl)acetic acid (5f)
Yield: 0.66 g (63.5%) as a yellow solid; m.p. 246°C; H NMR (400 MHz,
3
DMSO-d
7
6
3
): δ 12.74 (s, 1H), 8.18 – 8.00 (m, 3H), 7.67 (d, J = 7.0 Hz, 1H),
.45 (d, J = 8.9 Hz, 2H), 7.40 (s, 1H), 7.39 – 7.32 (m, 2H), 7.25 – 7.12
(m, 2H), 6.96 (t, J = 7.5 Hz, 1H), 6.83 (d, J = 8.3 Hz, 1H), 4.39 (d, J =
18.3 Hz, 1H), 4.32 (d, J = 18.3 Hz, 1H); C NMR (100 MHz, DMSO-d6):
δ 171.0, 148.2, 146.3, 145.7, 144.3, 142.1, 132.7, 132.5, 127.6, 125.4,
124.6, 123.2, 123.0, 120.0, 119.4, 115.0, 114.0, 110.5, 71.9, 51.4; MS
1
Yield: 0.66 g (68.0%) as a light yellow solid; m.p. 281°C; H NMR (500
MHz, DMSO-d ): δ 12.79 (s, 1H), 9.64 (s, 1H), 8.12 (d, J = 7.7 Hz, 1H),
6
3
3
2
3
2
13
3
3
7
7
8
.68 (d, J = 8.0 Hz, 1H), 7.37 (t, J = 7.8 Hz, 1H), 7.25 – 7.18 (m, 2H),
.17 – 7.11 (m, 3H), 7.06 (s, 1H), 6.96 (t, J = 7.5 Hz, 1H), 6.81 (d, J =
.4 Hz, 1H), 6.68 (d, J = 8.2 Hz, 2H), 4.25 (d, J = 18.2 Hz, 1H), 4.16 (d,
3
3
3
2
+
2
13
(CI): m/z 401.2 [M + H] ; Anal. Calcd. for C22H16N O : C, 66.00; H, 4.03;
J = 18.2 Hz, 1H); C NMR (125 MHz, DMSO-d
6
): δ 171.0, 158.7, 146.5,
44.0, 142.9, 132.9, 132.4, 129.1, 128.1, 125.5, 122.9, 122.7, 119.3,
19.1, 116.0, 113.9, 113.6, 110.9, 73.1, 50.1; MS (CI): m/z 372.2 [M +
4
4
N, 13.99; Found: C, 66.07; H, 4.08; N, 14.04.
1
1
+
H] ; Anal. Calcd. for C22
17 3 3
H N O : C, 71.15; H, 4.61; N, 11.31; Found: C,
2-(6-(4-Hydroxy-3-methoxyphenyl)benzo[4,5]imidazo[1,2-
71.19; H, 4.53; N, 11.39.
c]quinazolin-5(6H)-yl)acetic acid (5k)
2-(6-(4-Methoxyphenyl)benzo[4,5]imidazo[1,2-c]quinazolin-5(6H)-
Yield: 0.45 g (42.8%) as a light yellow solid; m.p. 209°C; IR: ṽ = 3408 (O-
yl)acetic acid (5g)
H, str., br.), 3069 (C-Haryl, str., m.), 2926 (C-Halkyl, str., m.), 1898
(
1
aromatic overtone), 1709 (C=O, str., w.), 1632 – 1513 (C=Caryl, str., v.),
616 (COO , str., as., s.), 1460 (C-Halkyl, bend., m.), 1392 (COO , str., s.,
-
-
Yield: 0.47 g (47.0%) as a light yellow solid; m.p. 258°C; IR: ṽ = 3406 (O-
s.), 1288 – 1257 (C-O, str., s.), 1174 – 1028 (C-N, str., v.), 863 (1,2,4-
subst. C-Haryl, bend., m.), 835 (1,2,4-subst. C-Haryl, bend., m.), 742 cm
H, str., br.), 3005 (C-Haryl, str., m.), 2929 – 2835 (C-Halkyl, str., m.), 1900
-1
-
(
1
aromatic overtone), 1704 (C=O, str., m.), 1612 (COO , str., as., m.),
1
-
(
1
1,2-subst. C-Haryl, bend., s.); H NMR (500 MHz, DMSO-d
6
): δ 12.80 (s,
571 – 1499 (C=Caryl, str., v.), 1460 (C-Halkyl, bend., m.), 1395 (COO ,
3
3
H), 9.20 (s, 1H), 8.09 (d, J = 7.4 Hz, 1H), 7.66 (d, J = 7.9 Hz, 1H), 7.36
(t, J = 7.6 Hz, 1H), 7.23 – 7.17 (m, 2H), 7.14 (t, J = 7.5 Hz, 1H), 7.01 –
.97 (m, 2H), 6.95 (t, J = 7.4 Hz, 1H), 6.80 (d, J = 8.3 Hz, 1H), 6.64 (d,
J = 8.2 Hz, 1H), 6.58 (d, J = 8.1 Hz, 1H), 4.22 (d, J = 18.3 Hz, 1H),
str., s., m.), 1242 (C-O, str., s.), 1174 – 1029 (C-N, str., v.), 828 (1,4-
3
3
-
1
1
subst. C-Haryl, bend., m.), 742 cm (1,2-subst. C-Haryl, bend., s.); H NMR
3
3
3
6
(
6
500 MHz, DMSO-d ): δ 12.81 (s, 1H), 8.10 (d, J = 7.6 Hz, 1H), 7.67 (d,
J = 7.8 Hz, 1H), 7.36 (t, J = 7.8 Hz, 1H), 7.27 (d, J = 7.8 Hz, 1H), 7.22
7.18 (m, 3H), 7.15 (t, J = 7.5 Hz, 1H), 7.12 (s, 1H), 6.96 (t, J = 7.4 Hz,
H), 6.83 (d, 2H), 6.80 (d, J = 8.6 Hz, 1H), 4.25 (d, J = 18.2 Hz, 1H),
3
3
2
3
3
3
2
13
3
3
4.15 (d, J = 18.2 Hz, 1H), 3.62 (s, 3H); C NMR (125 MHz, DMSO-d6):
–
1
4
3
2
δ 171.1, 148.0, 147.9, 146.6, 144.2, 142.9, 133.1, 132.3, 129.5, 125.3,
2
13
122.8, 122.6, 119.3, 119.2, 116.1, 113.9, 113.8, 111.2, 111.0, 73.4, 56.0,
6
.19 (d, J = 18.3 Hz, 1H), 3.66 (s, 3H); C NMR (125 MHz, DMSO-d ):
+
5
0.1; MS (CI): m/z 402.2 [M + H] ; Anal. Calcd. for C23
19 3 4
H N O : C, 68.82;
δ 171.0, 160.2, 146.5, 144.3, 142.7, 132.9, 132.3, 130.9, 127.9, 125.4,
22.9, 122.7, 119.3, 119.2, 114.6, 114.0, 113.8, 110.8, 72.8, 55.5, 50.3;
H, 4.77; N, 10.47; Found: C, 68.78; H, 4.71; N, 10.52.
1
+
MS (CI): m/z 386.2 [M + H] ; Anal. Calcd. for C23
19 3 3
H N O : C, 71.68; H,
4.97; N, 10.90; Found: C, 71.61; H, 4.93; N, 10.95.
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2
-(6-(Pentafluorophenyl)benzo[4,5]imidazo[1,2-c]quinazolin-5(6H)-
0.1000 g (0.26 mmol) of 5g and 0.0145 g (0.26 mmol) of KOH in 3 ml of
96% EtOH were stirred for 10 min and worked up as described for 6a to
provide the product as a light green solid. Yield: 0.10 g (91.7%); m.p.
yl)acetic acid (5l)
1
121°C; IR: ṽ = 3368 (H O), 3063 (C-Haryl, str., m.), 2930 (C-Halkyl, str., m.),
Yield: 0.34 g (29.3%) as a brown solid; m.p. 259°C; H NMR (500 MHz,
2
-
3
1610 (COO , str., as., s.), 1533 – 1487 (C=Caryl, str., v.), 1450 (C-Halkyl
,
6
DMSO-d ): δ 12.93 (s, 1H), 8.12 (d, J = 7.4 Hz, 1H), 7.80 (s, 1H), 7.70
d, J = 7.4 Hz, 1H), 7.38 (t, J = 7.6 Hz, 1H), 7.31 – 7.14 (m, 3H), 6.99 (t,
J = 7.3 Hz, 1H), 6.79 (d, J = 8.1 Hz, 1H), 4.44 (d, J = 18.8 Hz, 1H),
.33 (d, J = 18.7 Hz, 1H); C NMR (125 MHz, DMSO-d
46.4, 144.0, 141.9, 132.3, 132.1, 124.9, 123.4, 123.2, 119.6, 119.4,
13.5, 113.2, 109.7, 65.2, 51.1; F NMR (470 MHz, DMSO-d
-
3
3
bend., m.), 1384 (COO , str., s., m.), 1303 – 1245 (C-O, str., s.), 1173 –
(
-1
3
3
2
1028 (C-N, str., v.), 827 (1,4-subst. C-Haryl, bend., m.), 742 cm (1,2-
subst. C-Haryl, bend., s.); Anal. Calcd. for C23H18KN O : C, 65.23; H, 4.28;
2
13
4
1
1
1
2
6
): δ 171.1,
3
3
N, 9.92; Found: C, 65.29; H, 4.32; N, 9.97.
19
6
): δ -
3
3
42.40 (d,
3.7, 7.6 Hz); MS (CI): m/z 446.0 [M
: C, 59.33; H, 2.72; N, 9.44; Found: C, 59.29; H, 2.77; N,
J
FF = 24.8 Hz), -152.86 (t, JFF = 22.3 Hz), -161.52 (td, JFF
=
Biology
+
+
H] ; Anal. Calcd. for
22 12 5 3 2
C H F N O
Antimicrobial screening
9.48.
2
Sterilized filter paper disks (200 g/m , 6 mm diameter) impregnated with
a solution of the test compound (5a-n, 6a, 6b) in DMF (1 mg/ml) and
dried at 60°C for 1 h were placed on a Mueller-Hinton agar, Mueller-
Hinton agar with 5% sheep blood or Sabouraud agar plate seeded with
the appropriate test organism. The plates containing bacteria were
incubated for 24 h at 37°C, while the one with C. albicans for 24 h at
28°C. The utilized test organisms were: S. aureus (ATCC 25923) and S.
pyogenes (the bacterial strain was isolated from the patient’s mucous
membrane of the pharynx, who was diagnosed with nasopharyngitis) as
examples of Gram-positive bacteria, P. vulgaris (HX19 №222), S.
typhimurium (79), E. coli (ATCC 25922), P. aeruginosae (ATCC 27853),
S. sonnei (S-form), K. pneumonia (K-56 №3534/51) and K. aerogenes
(NCTC 10006) as examples of Gram-negative bacteria. The test
compounds were also evaluated for their in vitro antifungal potential
against C. albicans (ATCC 885-653). Ciprofloxacin was used as a
standard for antibacterial assay and ketoconazole – for the antifungal
one in the same concentration as for the test compounds. Inhibition zone
diameters were measured with a ruler on the undersurface of the Petri
dish or with calipers near the agar surface in mm.
2
-(6-(Pyridin-3-yl)benzo[4,5]imidazo[1,2-c]quinazolin-5(6H)-yl)acetic
acid (5m)
1
Yield: 0.70 g (75.3%) as a light yellow solid; m.p. 274°C; H NMR (500
MHz, DMSO-d ): δ 12.55 (s, 1H), 8.55 (s, 1H), 8.49 – 8.43 (m, 1H), 8.11
6
3
3
3
(d, J = 7.6 Hz, 1H), 7.70 (d, J = 7.7 Hz, 1H), 7.49 (d, J = 8.1 Hz, 1H),
3
3
7.44 (d, J = 7.6 Hz, 1H), 7.39 (t, J = 7.8 Hz, 1H), 7.34 (s, 1H), 7.29 –
3
3
7
.16 (m, 3H), 7.00 (t, J = 7.5 Hz, 1H), 6.87 (d, J = 8.3 Hz, 1H), 4.41 (d,
2
2
13
J = 18.3 Hz, 1H), 4.34 (d, J = 18.2 Hz, 1H); C NMR (125 MHz,
): δ 171.0, 150.8, 147.5, 146.4, 144.3, 142.4, 134.6, 133.9,
32.7, 132.5, 125.4, 124.5, 123.2, 123.0, 120.0, 119.4, 114.9, 114.1,
DMSO-d
6
1
1
+
10.5, 71.0, 51.4; MS (CI): m/z 357.2 [M + H] ; Anal. Calcd. for
: C, 70.77; H, 4.53; N, 15.72; Found: C, 70.82; H, 4.49; N,
21 16 4 2
C H N O
15.78.
2-(6-(5-(Trifluoromethyl)thiophen-2-yl)benzo[4,5]imidazo[1,2-
c]quinazolin-5(6H)-yl)acetic acid (5n)
1
Yield: 0.74 g (66.1%) as a brown solid; m.p. 252°C; H NMR (500 MHz,
DMSO-d ): δ 12.87 (s, 1H), 8.09 (dd, J = 7.7, 1.3 Hz, 1H), 7.73 (s, 1H),
Allium test
6
3
7
=
.72 – 7.70 (m, 1H), 7.61 – 7.57 (m, 1H), 7.51 – 7.48 (m, 1H), 7.43 (t, J
3
7.0 Hz, 1H), 7.28 – 7.24 (m, 2H), 7.23 – 7.20 (m, 1H), 7.05 (t, J = 7.3
Equal-sized bulbs of Allium cepa L. (4.1-5.9 g) were chosen and placed
initially in tap water and incubated at an average temperature of 20°C for
2-3 days. When the roots have reached the length of 0.5-1 cm, the bulbs
were transferred to 20 ml test tubes with the test solutions and exposed
for 3 days at room temperature. A small amount of each of the above
solutions was added to each respective test tube each day in order to
replace that lost through evaporation. Roots length was measured to the
nearest millimetre using a ruler every day at the same time. Then the
rootlets were collected and fixed immediately in Clarke’s fluid (EtOH-
AcOH 3:1) for 24 h. Afterwards, the rootlets were removed from the fixing
solution and transferred to 70% EtOH and stored at 4°C for further
experimental work. In the next stage, the root tips were stained with 1%
acetocarmine (1.0 g of carmine in 100 ml of 45% AcOH had been
refluxed for 3 hours and filtered) with heating on the water bath for 12
min. The root tip was removed from the stain and rinsed in 45% AcOH.
The root tip was placed on a microscope slide and 3 mm of the root tip
was removed with a scalpel. Two drops of 45% AcOH were added and
the root tip was squashed beneath a 40×40 mm cover glass. Observation
was done at 600× through the light microscope (MICMED-1 LOMO,
objective 40×0.65, eyepiece K15×). The mitotic index was calculated by
examining about 600 cells of each root tip. The mitotic index is the
percentage of cells in various stages of mitosis relative to the total
number of cells examined. A negative control group was treated with tap
water and 0.001 N KOH in tap water. Methotrexate hydrate (Sigma-
Aldrich) was used as a positive control. The test solutions and a positive
control were prepared by the following procedure. In a 50 ml beaker were
placed 24 ml of tap water, 1 ml of 0.025 N KOH in tap water (0.05 N KOH
for methotrexate hydrate, 5f and 5k) and 0.025 mmol of the test
3
2
2
Hz, 1H), 6.96 (d, J = 8.3 Hz, 1H), 4.44 (d, J = 18.2 Hz, 1H), 4.33 (d, J =
13
6
18.2 Hz, 1H); C NMR (125 MHz, DMSO-d ) δ 170.9, 146.5, 146.0,
3
2
144.2, 141.7, 132.6, 132.5, 130.1 (q, JCF = 3.5 Hz), 129.4 (q, JCF = 38.5
1
Hz), 127.0, 125.4, 123.3, 123.2, 122.4 (d, JCF = 268.7 Hz), 120.7, 119.5,
1
9
115.6, 114.6, 110.5, 68.4, 51.3; F NMR (470 MHz, DMSO-d
6
): δ -54.19
+
(
s); MS (CI): m/z 430.0 [M + H] ; Anal. Calcd. for C21H F N O S: C,
14 3 3 2
5
8.74; H, 3.29; N, 9.79; Found: C, 58.68; H, 3.24; N, 9.83.
Potassium
c]quinazolin-5(6H)-yl)acetate (6a)
2-(6-(3-methoxy-4-oxidophenyl)benzo[4,5]imidazo[1,2-
0.1000 g (0.25 mmol) of 5k and 0.0279 g (0.50 mmol) of KOH in 3 ml of
96% EtOH were stirred for 10 min. The resulting solution was
concentrated under reduced pressure at room temperature. The solid
residue was thoroughly dried in vacuo to give the product as a beige
solid. Yield: 0.11 g (92.5%); m.p. 242-247°C; IR: ṽ = 3367 (H
2
O), 3066
-
(
(
–
C-Haryl, str., m.), 2925 (C-Halkyl, str., m.), 1613 (COO , str., as., s.), 1488
C=Caryl, str., s.), 1450 (C-Halkyl, bend., m.), 1385 (COO , str., s., s.), 1301
1228 (C-O, str., s.), 1169 – 1028 (C-N, str., v.), 840 (1,2,4-subst. C-Haryl
-
,
-1
bend., m.), 820 (1,2,4-subst. C-Haryl, bend., m.), 742 cm (1,2-subst. C-
aryl, bend., s.); Anal. Calcd. for C23 : C, 57.84; H, 3.59; N,
.80; Found: C, 57.80; H, 3.53; N, 8.86.
H
8
17 2 3 4
H K N O
Potassium
c]quinazolin-5(6H)-yl)acetate (6b)
2-(6-(4-methoxyphenyl)benzo[4,5]imidazo[1,2-
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compound (5a-n) or methotrexate hydrate with stirring at room
temperature. The test compounds (5a-n) and methotrexate hydrate were
not completely soluble in the alkaline solution, so the slight precipitate
was observed at the bottom of the beaker. The resulting liquid was
suspended and transferred to the test tube without decantation or
filtration.
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The authors express their gratitude to Smyk T.I., Oleksandriia
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[
Keywords: antimicrobial • benzimidazoles • c-mitosis • mitotic
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Entry for the Table of Contents
FULL PAPER
Text for Table of Contents
Dmytro V. Kravtsov*, Oleksii Yu.
Voskoboinik and Serhii I. Kovalenko
Page No. – Page No.
Synthesis, antimicrobial and mitotic
toxicity evaluation of new 6-
substituted 2-(benzo[4,5]imidazo[1,2-
c]quinazolin-5(6H)-yl)acetic acids
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