Design, synthesis and docking studies of some novel (R)-2-(4′- chlorophenyl)-3-(4′-nitrophenyl)-1,2,3,5-tetrahydrobenzo[4,5] imidazo [1,2-c]pyrimidin-4-ol derivatives as antitubercular agents

Article abstract of DOI:10.1016/j.ejmech.2014.06.019

Filamenting temperature-sensitive mutant (FtsZ) is a novel target for the treatment of tuberculosis. A series of (R)-2-(4′-chlorophenyl)-3- (4′-nitrophenyl)-1,2,3,5-tetrahydrobenzo[4,5] imidazo[1,2-c]pyrimidin-4-ol derivatives were designed and docked on the FtsZ protein crystal structure (PDB Id: 1RLU, resolution 2.08 ?). Compound 7t showed the highest docking score and H-bond interaction with Arg140 and Gly19. Our strategy for synthesis of (R)-2-(4′-chlorophenyl)-3-(4′-nitrophenyl)-1,2,3,5- tetrahydrobenzo[4,5]imidazo[1,2-c]pyrimidin-4-ol derivatives from o-phenylenediamine as illustrated in scheme. All the synthesized compounds were characterized by FTIR, Mass spectra, ¹H NMR, ¹³C NMR, elemental analysis and purity was confirmed by HPLC and LCMS. Compound 7g was also confirmed by single crystal X-ray analysis. The in silico results are also validated with in vitro antitubercular activity of compound 7t. Compound 7b exhibited in vitro antitubercular activity 3.13 μg/mL and 4.7 μg/mL whereas compound 7t exhibited in vitro antitubercular activity 6.25 μg/mL and 9.4 μg/mL using GAST/Fe medium after week 1 and week 2 respectively against Mycobacterium tuberculosis H₃₇Rv. Medium 7H9/ADC/Tween was found to be very less effective for in vitro antitubercular activity of all the benzimidazole derivatives. Assays for in vitro cytotoxicity against VERO cells of all the synthesized compounds was found to be very less cytotoxic.

Full text of DOI:10.1016/j.ejmech.2014.06.019

Accepted Manuscript
Design, synthesis and docking studies of some novel (R)-2-(4'-chlorophenyl)-3-(4'-
nitrophenyl)-1,2,3,5-tetrahydrobenzo[4,5]imidazo[1,2-c]pyrimidin-4-ol derivatives as
antitubercular agents
Kuldipsinh P. Barot, Shailesh V. Jain, Nirzari Gupta, Laurent Kremer, Shubhra Singh,
Vijay B. Takale, Kruti Joshi, Manjunath D. Ghate, Ph. D, Prof., Post Doc. Director
PII:
S0223-5234(14)00538-8
10.1016/j.ejmech.2014.06.019
EJMECH 7062
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 February 2014
Revised Date: 5 June 2014
Accepted Date: 10 June 2014
Please cite this article as: K.P. Barot, S.V. Jain, N. Gupta, L. Kremer, S. Singh, V.B. Takale, K. Joshi,
M.D. Ghate, Design, synthesis and docking studies of some novel (R)-2-(4'-chlorophenyl)-3-(4'-
nitrophenyl)-1,2,3,5-tetrahydrobenzo[4,5]imidazo[1,2-c]pyrimidin-4-ol derivatives as antitubercular
agents, European Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.06.019.
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ACCEPTED MANUSCRIPT
Highlights
•
•
•
•
•
Novel imidazo [1,2-c] pyrimidin-4-ol derivatives are designed.
Molecular docking study was carried out.
Synthetic methodologies leading to targeted molecules have been reported.
X-ray crystallographic study (ORTEP) of compound 7g was carried out.
Compounds are subjected to screening for antitubercular activity and cytotoxicity assay.

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Graphical Abstract
OH
R
H
H
N
NH2
NH2
N
N
+
CH3COOH
R'
7
1
2
where
R = 4-NO2, 3-NO2, 4-Cl, 2-Cl, 4-OH, 2-OCH3, 3-OH, 3-Br,
4-OH, etc.
R' = 4-Cl, 4-NO2, 4-Br, 4-OCH3, 2-OCH3, 4-COOH, 4-OH,
2-OH, 4-NO2, etc.

ACCEPTED MANUSCRIPT
Design, synthesis and docking studies of some novel (R)-2-(4'-
chlorophenyl)-3-(4'-nitrophenyl)-1,2,3,5-tetrahydrobenzo[4,5]imidazo[1,2-
c]pyrimidin-4-ol derivatives as antitubercular agents
a
a
a
b,e,f
Kuldipsinh P. Barot , Shailesh V. Jain , Nirzari Gupta , Laurent Kremer b,c, Shubhra Singh
,
Vijay B. Takale d, Kruti Joshi d and Manjunath D. Ghate a,*
a Department of Pharmaceutical Chemistry, Institute of Pharmacy, Nirma University, Ahmedabad -
382481, Gujarat, India
b
Laboratoire de Dynamique des Interactions Normales et Pathologiques, CNRS UMR 5235,
Université, Montpellier 2, Eugène Bataillon, 34 095 Montpellier cedex 5, France
c INSERM, DIMNP, Place Eugène Bataillon, 34 095 Montpellier Cedex 05, France
d
Research & Development Department, Piramal Discovery Solution Ltd, Ahmedabad-382213,
Gujarat, India
e IFTM University, Lodhipur Rajput, Delhi Road, Moradabad-244102, Uttar Pradesh, India
f CSIR, Central Drug Research Institute (CDRI), Sector-10, Sitapur Road, Lucknow-226031, India
Corresponding Author
Prof. Manjunath Ghate, Ph. D, Post Doc.
Director
Institute of Pharmacy
Nirma University,
S. G. Highway, Ahemdabad – 382481,
Gujarat, INDIA.
Telephone: +91-2717-241900-04, Fax: -91-2717-241916,
E-mail : barotkuldip@gmail.com
1

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Abstract: Filamenting temperature-sensitive mutant (FtsZ) is a novel target for the treatment of
tuberculosis. A series of (R)-2-(4'-chlorophenyl)-3-(4'-nitrophenyl)-1,2,3,5-tetrahydrobenzo[4,5]
imidazo[1,2-c]pyrimidin-4-ol derivatives were designed and docked on the FtsZ protein crystal
structure (PDB Id: 1RLU, resolution 2.08 Å). Compound 7t showed the highest docking score and
H-bond interaction with Arg140 and Gly19. Our strategy for synthesis of (R)-2-(4'-chlorophenyl)-3-
(4'-nitrophenyl)-1,2,3,5-tetrahydrobenzo[4,5]imidazo[1,2-c]pyrimidin-4-ol derivatives from o-
phenylenediamine as illustrated in scheme. All the synthesized compounds were characterized by
1
13
FTIR, Mass spectra, H NMR, C NMR, elemental analysis and purity was confirmed by HPLC
and LCMS. Compound 7g was also confirmed by single crystal X-ray analysis. The in silico results
are also validated with in vitro antitubercular activity of compound 7t. Compound 7b exhibited in
vitro antitubercular activity 3.13 µg/mL and 4.7 µg/mL whereas compound 7t exhibited in vitro
antitubercular activity 6.25 µg/mL and 9.4 µg/mL using GAST/Fe medium after week 1 and week 2
respectively against M. tuberculosis H37Rv. Medium 7H9/ADC/Tween was found to be very less
effective for in vitro antitubercular activity of all the benzimidazole derivatives. Assays for in vitro
cytotoxicity against VERO cells of all the synthesized compounds was found to be very less
cytotoxic.
Keywords: Tuberculosis, Docking study, Benzimidazole derivatives, In vitro antitubercular activity,
Cytotoxicity assay, Structure-activity relationship (SAR).
2

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1. Introduction
Tuberculosis (TB) is a foremost communicable disease and we have not yet been able to
eradicate the malady. It is primarily caused by Mycobacterium tuberculosis (M. tb) of the
‘‘Tuberculosis Complex’’ [1]. The chemical composition of M. tb cell wall includes peptidoglycans
and a vast array of lipids, including mycolic acids, which are significant determinant of its
virulence. The unique structure of the cell wall of M. tb allow it to lie dormant for many years as a
latent infection, particularly as it can reside inside macrophages, hiding it from the host immune
system [2]. TB is a contagious and deadly disease that has reached pandemic proportions in the
world. According to the World Health Organization (WHO), 10-12 million new cases of TB are
diagnosed each year and making M. tb a leading cause of death in adults (2-3 million/year) due to
an infectious agent [3]. A high proportion of these new cases and deaths occur in HIV-positive
people with a significant number of AIDS deaths in developing countries being attributed to TB
infections. Global population growth is increasing the disease burden, posing a continuing health
and financial burden in various parts of the world, particularly Asia and Africa [4].
The continuous rise in multi-drug resistant strains of M. tb (MDR-TB) has further
contributed to the urgent need for new antitubercular drugs, as no new TB drugs have been
introduced into clinical use in the past 4 decades. Drugs active against resistant forms of TB are less
potent, more toxic and need to be taken for an extended period of time (≥18 months) [5]. Current
emergences of virtually untreatable extensively-drug resistant TB (XDR-TB) posses a new threat to
TB control world-wide. Recent findings by WHO from 2000 to 2004 suggested that 4% of MDR-
TB cases meet the criteria for XDR-TB. Effective treatment of TB in persons co-infected with HIV
is complicated because of drug-drug interactions [6]. Therefore, shorter and simpler regimens are
safe and effective against drug-susceptible and drug-resistant TB, which are appropriate for joint
HIV-TB treatment and amenable to routine clinical settings, are required. New chemotherapeutic
agents to combat the emergence of the resistance and strategies which can effectively shorten the
duration of chemotherapy are urgently needed [7].
Isoniazid (INH), a well-known antitubercular drug, is believed to kill mycobacteria by
inhibiting the biosynthesis of mycolic acids and critical components of the mycobacterial cell wall.
The catalase and peroxidase activities are thought to participate in the drug sensitivity mechanism
by converting INH in vivo into its biologically active form, which then acts on its intracellular target
[8]. Substituted benzimidazole derivatives have been reported to possess antitubercular activity
against M. tb H37Rv. These compounds, after penetration of the mycobacterial cell wall, could be
bio-transformed by esterases or peroxidase-catalases. They are more active than the unmodified
3

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polar isosteres of isonicotinic acid, which may be due to better penetration of these agents into the
cell wall of the mycobacteria [9,10].
In the last 40 years, few drugs have been approved by the Food and Drug Administration
(FDA) to treat TB, reflecting the inherent difficulties in drug discovery and clinical testing of new
agents and the lack of pharmaceutical industry research in the area [11]. In addition to the current
drugs approved by the FDA for the treatment of TB and to the drugs that commonly are
recommended for the treatment of TB but are not FDA approved, many compounds are under
investigation as potential antitubercular drugs [12]. Among them, benzimidazole derivatives were
first reported in 1998 as antimycobacterial agents. The most potent derivative showed minimum
inhibitory concentration (MIC) values ranging from 0.7 to 1.5 µg/mL against M. tb. Activity toward
drug-resistant strains of M. tb was similar to that found toward sensitive strains. Finally, it was also
active toward non-tuberculosis mycobacteria with MICs higher that against M. tb [13].
Filamenting temperature-sensitive mutant Z (FtsZ), a tubulin homologue, is a highly
conserved and ubiquitous bacterial cell division protein. Similar to the process of microtubule
formation by tubulin, FtsZ polymerizes in a GTP-dependent manner, forming a highly dynamic
cytokinetic structure, designated as the Z-ring, at the mid-point of the cell. The recruitment of the
other cell division proteins leads to Z-ring contraction and results in septum formation [14].
Because of the requirement of FtsZ in mycobacterial cytokinesis, inhibition of FtsZ is a promising
target for antitubercular drug discovery. While tubulin and FtsZ share structural and functional
homology and because tubulin inhibitors are known to affect FtsZ assembly, the limited sequence
homology at the protein level affords an opportunity to discover FtsZ-specific compounds with
limited cytotoxicity to eukaryotic cells. Since FtsZ is a proposed novel drug target, compounds
targeting FtsZ are expected to be active against drug resistant M. tb strains. Furthermore, the
validation of FtsZ as a novel antitubercular drug target has been confirmed by the work of various
groups [15].
Southern Research Institute researchers have screened known tubulin inhibitors against M.
tb and identified several benzimidazole, pyridopyrazine and pteridine based FtsZ inhibitors with
potent antituberular activity [16,17]. They have concluded that substituted benzimidazole
derivatives interfered and delayed the M. tb cell division processes. Benzimidazole derivatives are
also reported as potent antitubercular agents. They are polar and ionisable aromatic compounds and
used as a remedy to optimize solubility and bio-availability parameters of proposed molecules.
Clubbed-[1,2,3] triazole by fluorine benzimidazoles are also very potent antitubercular agents
4

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against M. tb H37Rv. It has broadened the scope in remedying various dispositions in clinical
medicines and play vital role in the field of medicinal chemistry which is an important
pharmacophore and exhibits potent in vivo and in vitro antitubercular activities [18,19]. Extensive
research works are also reported on benzimidazole but relatively very little is known so far about
substituted benzimidazoles. Ordinary organic synthesis has been widely employed for the synthesis
of diverse heterocycles. In bacteria, the main cytoskeletal element involved in cell division is the Z-
ring, a membrane associated structure consisting of polymers of the essential tubulin-related protein
FtsZ. The Z-ring then reduces in diameter during cell division, drawing the cytoplasmic membrane
inwards. A number of accessory proteins that are essential for cell division require being located to
the cell centre during cell division. The localization of these proteins is achieved through
recruitment to Z-ring by either direct or indirect interaction with FtsZ [20,21]. Threrefore, docking
studies were performed on FtsZ target to understand the basis of the mechanism of antitubercular
activity of the benzimidazole derivatives synthesized in this study. The binding domain identified to
accommodate GTP (Guanosine diphosphate monothiophosphate) appears large enough to hold the
benzimidazole derivatives synthesized in this study.
Taking into account the structural similarity of the pyridopyrazine moiety, pteridine moiety,
albendazole, and thiabendazole, they envisioned that the benzimidazole scaffold would be a good
starting point for the development of novel FtsZ inhibitors, which have activity against both drug-
sensitive and drug-resistant M. tb. Therefore, substituted benzimidazoles were investigated for
potent antitubercular activity [17]. Specifically, we report the activity of 1,2,3,4-tetrahydrobenzo
[4,5] imidazo [1,2-c]pyrimidin-4-ol derivatives against M. tb H37Rv. Importantly, it substantiates
disubstituted benzimidazoles for the development of next generation mycobacterial inhibitors with
activity against difficult to treat clinical strains.
2. Results and discussion
2.1. Chemistry
Since benzimidazoles are well known for their in vivo and in vitro antitubercular activities
and their mode of action has been characterized [22]. Our work has focused on the synthesis of
novel molecules based on the benzimidazole skeleton and the synthesized compounds were tested
for their in vitro antitubercular activity. The synthetic pathways are illustrated in scheme. Molecular
modeling of their binding to ENR was also performed to study whether this target was the ideal site
to exhibit their mechanism of antitubercular activity or not. Our strategy for synthesis of (R)-2-(4'-
chlorophenyl)-3-(4'-nitrophenyl)-1,2,3,5-tetrahydrobenzo[4,5]imidazo[1,2-c]pyrimidin-4-ol
7a
5

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derivatives of benzimidazole is very simple as illustrated in scheme. As per prescribed procedure,
compound 3 was synthesized by the condensation of o-phenylenediamine with acetic acid and water
by refluxing at 80-82oC for 4-4.5 h to obtain the intermediate derivative 3 in good yields [23].
Compounds 3 was refluxed with aromatic aldehyde derivatives at 100-110oC for 1.0 h and then
160-170oC for 30 min to obtain the corresponding alkene derivatives 4 [24]. Compound 4 was
refluxed with aniline derivatives with formaldehyde and triethylamine in ethanol at 70-72oC for 3.5-
4.0 h to obtain corresponding aniline substituted benzimidazole derivative 5 [25]. Epoxide was
formed by the condensation of compound 5 with sodium hypochlorite and (R,R)-Jacobsen’s catalyst
with dichloromethane as solvent at 50-52oC for 2.5-3.0 h [26]. Cyclization of the compound 6 was
take place by refluxing in aluminum trichloride with methanol as solvent at 75-77oC to afford the
target molecules 7a-7t [27]. Synthesized compounds were purified by column chromatography and
1
structures of the compounds were confirmed by FTIR, Mass spectra, H NMR, 13C NMR and
elemental analysis. Structure of compound 7g was also confirmed by single crystal X-ray analysis.
Purity of all the final synthesized compounds was confirmed by HPLC and purity of compound 7g
and 7j was also confirmed by LCMS. A series of cyclic substituted benzimidazole derivatives were
synthesized using o-, m- or p-substituted benzaldehydes and substituted aniline derivatives with
different reagents to form the desired heterocyclic benzimidazole derivatives. Percentage yield of
the intermediate and final compounds were in the range of 68-87% and 60-67% respectively.
NH2
NH2
N
N
(a)
(b)
CH3COOH
+
CH3
R
N
H
N
H
4
1
2
3
(c)
N
N
O
N
N
OH
R
H
R
H
R
N
(d)
NH
(e)
N
N
NH
R'
R'
R'
7
6
5
Scheme. Synthesis of (R)-2-(4'-chlorophenyl)-3-(4'-nitrophenyl)-1,2,3,5-tetrahydrobenzo[4,5]
imidazo[1,2-c]pyrimidin-4-ol derivatives 7a−7t. Reagents and conditions: (a) H2O, 80-82oC, 4 h;
(b) 100-110oC for 1.0 h and then 160-170oC for 30 min, Ar-CHO; (c) C2H5OH, HCHO, TEA, Ar-
NH2, 70-72oC, 3.5-4.0 h; (d) NaOCl, DCM, (R,R)-Jacobsen’s catalyst, 50-52oC, 2.5-3.0 h; (e) AlCl3,
CH3OH, 75-77oC.
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The IR spectrum of compound 7a illustrates broad stretching band around 2930.31 cm-1 and
3598.52 cm-1 due to aromatic CH and OH respectively and strong stretching band at 1090.55 cm-1
accounting for C-N stretch. Strong stretching band of NO2 and Cl was observed at 1381.75 cm-1
1
and 746.31cm-1 respectively. H NMR of compound 7a shows sharp singlets at δ 2.1 accountable
for OH and which was absent on D2O exchange. The two protons of benzimidazole ring was
appeared as doublet at δ 7.49-7.51 with J value 8.6 Hz and other doublet at δ 7.31-7.31 with J value
2.0 Hz for four aromatic hydrogen. Proton of CH2 and CH was appeared as doublet at δ 4.48 and
3.90 respectively. 13C NMR shows a peak at δ 145.54 because of Ar-C of benzimidazole ring
system. Peak of CH and CH2 was appeared at δ 76.74 and 49.29 respectively. Further, molecular ion
peak (M+, 96%), (M+2, 30%) and (M-1, 94%) were found at m/z 420.9, 422.8 and 419.0
respectively which confirm its molecular fragmentation patterns. Elemental analysis of C, H and N
were within ±0.4% of the predicted values. HPLC purity at retention time 9.747 min was found as
98.39% where 0.1% TFA in HPLC grade water and 0.1% TFA in HPLC grade ACN selected as
mobile phase.
Broad stretching band around 2932.23 cm-1 and 3611.05 cm-1 due to aromatic CH and OH
respectively were found of the IR spectrum of compound 7b. Strong stretching band of NO2 was
1
observed at 1383.68 cm-1. H NMR of compound 7b shows sharp singlets at δ 2.1 accountable for
OH and which was absent on D2O exchange. The two protons of benzimidazole ring was appeared
as singlet at δ 7.87 and multiplet at δ 7.06-7.11 with four aromatic hydrogen. Proton of CH2 and CH
was appeared as doublet at δ 4.68 and 3.49 respectively. 13C NMR shows a peak at δ 175.32 due to
Ar-C of aromatic ring system. Peak of CH and CH2 was appeared at δ 76.74 and δ 50.53
respectively. Further, molecular ion peak (M+1, 96%) and (M-1, 97%) were found at m/z 432.1 and
430.0 respectively confirms its molecular fragments. Elemental analysis of C, H and N are within
±0.4% of the predicted values.
Strong stretching band of NH was observed at 3492.45 cm-1 and broad stretching band
around 3076.87, 2948.63 cm-1 and 3775.94 cm-1 due to aromatic CH and OH respectively was found
1
of the IR spectrum of compound 7g. H NMR of compound 7g shows sharp singlets at δ 2.02
accountable for OH and which was absent on D2O exchange. Two protons of aromatic ring were
appeared as doublet at δ 7.66 and multiplet at δ 6.68-6.75 with two aromatic hydrogens. Proton of
CH2 and CH was appeared as doublet at δ 5.68 and 4.52 respectively. 13C NMR of the compound 7g
confirms resonance of CH3 and C-N at δ 21.7 and δ 170.04 respectively. It shows a peak at δ 148.47
because of Ar-C of aromatic ring system. Peak of CH and CH2 was appeared at δ 77.37 and 62.54
respectively. Further, molecular ion peak (M+1, 98%) and (M-1, 97%) was found at m/z 412.8 and
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411.0 respectively which confirms its molecular fragmentation. Elemental analysis of C, H and N
are within ±0.4% of the predicted values. HPLC purity at retention time 5.598 min was found as
97.67% where 0.1% TFA in HPLC grade water and 0.1% TFA in HPLC grade ACN selected as
mobile phase. LCMS purity was found as 100% at retention time 3.015 min which was acquired by
UPLC-02 using the method of Formic_Normal at 230.0 nm.
The IR spectrum of compound 7j illustrates strong stretching band of NH at 3329.50 cm-1
and broad stretching band around 2781.81, 2978.52 cm-1 and 3550.31 cm-1 due to aromatic CH and
1
OH respectively. H NMR of compound 7j shows sharp singlets at δ 1.87 and δ 3.91 accountable
for OH and disappeared on D2O exchange. The two protons of aromatic ring was appeared as
multiplet at δ 7.47 and singlet at δ 7.51 for one aromatic hydrogen. Proton of CH2 and CH was
appeared as doublet at δ 6.86, 5.12 and δ 3.93 respectively. 13C NMR shows a peak at δ 156.62
because of Ar-C of aromatic ring system. Peak of CH and CH2 was appeared at δ 108.51 and 76.29
respectively. Further, molecular ion peak (M+, 97%), (M+2, 30%), (M+4, 10%) and (M-1, 74%)
were found at m/z 426.8, 428.5, 430.7 and 425.0 respectively confirms its fragments. Elemental
analysis of C, H and N were within ±0.4% of the predicted values. HPLC purity at retention time
4.216 min found as 98.25% where 0.1% TFA in HPLC grade water and 0.1% TFA in HPLC grade
ACN selected as mobile phase. LCMS purity was found as 99.5% at retention time 1.903 min
which was acquired by UPLC-01 using the method of Formic_Normal at 309.0 nm. Single isomer
identification of compound 7g was also confirmed by chiral HPLC methods of analysis.
2.2. Analogue design
Our impetus to investigate the benzimidazole subclasses, exemplified by analogues 7a-7t,
was driven by its ease of synthesis and the likelihood that compounds with lowered or absent
enzymatic induction effects would be generated. This latter expectation was based on the known
diminution of FtsZ induction with the progression from isoniazid. Within this same order of
structural subclasses, there is also a trend of isoniazid possessing increased potency against drug
susceptible isolates of slow-growing mycobacteria and better in vivo efficacy in mice. Our analysis
of the recently published structures has revealed an approach for the chemical elaboration of the
benzimidazole core in a novel way that should lead to enhanced binding affinity to both wild-type
and resistant strains [28].
2.3. Molecular docking study
The X-ray crystal co-ordinates of M. tb FtsZ in complex with GTP- gamma-S at a resolution
of 2.08 Å were obtained from the Protein Data Bank under the accession code 1RLU.pdb. M. tb
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FtsZ crystallized as a tight, laterally oriented dimer distinct from the longitudinal polymer observed
for αβ-tubulin. FtsZ and tubulin are known to pass through cycles of polymerization and
depolymerisation, but the structural mechanisms underlying this cycle remain to be determined.
This protein play an essential role in bacterial cell division and interruption of this process is a
bactericidal event [29]. Ramachandran plot of the backbone torsion angles PHI and PSI for local
geometry and the location of buried polar residues or exposed non-polar residues were examined.
Energy refinement procedure was performed using these structures. Kollman charges were used for
the protein while Gasteigere Huckel charges were calculated for the ligand. Then, this model was
subjected to energy minimization following the gradient termination of the Powell method for 3000
iterations using Kollman united force field with non bonding cut off set at 109.0 and the dielectric
constant set at 4.5. While FtsZ retains only limited sequence similarity to tubulin, the three-
dimensional X-ray structure shows a high degree of similarity to the structure of α and β tubulin. 3-
D structure of GTP was obtained from its X-ray crystal co-ordinate which was found in 1RLU.pdb
file [30].
The binding site of the benzimidazole derivatives was estimated using a variety of scoring
functions that have been compiled into the single consensus score (Total Score). The Surflex-Dock
scoring function (originally used within Hammerhead9) was tuned to predict the binding affinities
of 34 protein-ligand complexes, with its output being represented in units of -log(Kd). Prior to start
of the docking operation, essential hydrogens and Gasteiger charges were added to the
macromolecule [31]. Molecular docking of inhibitors into the active site of protein identifies the
binding orientations and the protein–inhibitor interactions responsible for the observed activity. The
benzimidazole derivatives were docked to FtsZ using Surflex Dock in Sybyl X1.2 [32]. Molecular
docking is used to determine the binding orientation of inhibitors to their protein targets to predict
their affinity and activity when bound to the protein. Surflex is a fully automatic flexible molecular
docking algorithm that combines the scoring function from the Hammerhead docking system with a
search engine that relies on a surface based molecular similarity method as a mean to rapidly
generate suitable putative poses for molecular fragments [33]. The dominant terms are the
hydrophobic contact term and a polar contact term that has a directional component and is scaled by
formal charges on the protein and ligand atoms. These functional terms are characterized on the
basis of distances between Vander Waals surfaces, with negative values indicating interpenetration
[34]. The docking study reveals compound 7t as highest active compound having docking score
7.1272.
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Twenty poses were obtained from all compounds. Out of them all, second pose of compound
7t obtains highest occupancy and highest docking score of 7.1272 (Table A of supplementary file).
Compound 7t shows various hydrogen bonding and hydrophobic interaction with the aminoacids.
Hydrogen bonding of compound 7t with Arg140, Gly19, Gly101 and Glu102 takes within the
distance of less than 2.5 Å take place (Fig. 1). The terminal -NH of the Gly19 make a hydrogen
bond with oxygen of the hydroxyl group of the linker heterocyclic ring of the 7t (N-H---O-H, 2.48
Å). Another hydrogen bonding is observed between hydrogen of the same hydroxyl group and
carboxyl oxygen of the amide linkage of the Gly101 (C-O---H-O, 2.05 Å). Two more hydrogen
bonding is observed with the hydroxyl group of the terminal aryl substitution of compound 7t.
Carboxyl oxygen of the Glu102 make bonding with the hydrogen of the hydroxyl group (C-O---H-
O, 2.14 Å) and oxygen of the same hydroxyl group make another hydrogen bonding with guanidino
amine hydrogen of Arg140 (N-H---OH, 2.20 Å). The occupancy of the molecule in the binding
pocket of the enzyme is the other reason of the potent activity. Compound 7t occupies the whole
cavity in the receptor site for the inhibition of the FtsZ (Fig. 2a). The terminal aryl substitution is
oriented near positively charged Arg139 and Arg140, because the negatively charged benzene ring
cloud is attracted towards the positively charged amino acid residues (Fig. 2b). Most of the amino
acids are oriented in the same manner as per the GTP interaction with the receptor. In the receptor,
the active site is in the V-shaped cavity and the most active compounds perfectly fit into the pocket,
hence validate the in silico results (Fig. 3). The proposed docking hypothesis is proposed to become
the reason of inhibitory action on Mtb H37Rv strain.
Fig. 1. Hydrogen bond interaction of compound 7t and compound 7t is in the orange coloured ball
and stick model.
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Fig. 2. (a) The docking pose of compound 7t in the receptor site and cyan coloured area indicated
the excluded volume of the compound 7t. (b) Interacting amino acids of binding site and compound
7t in the field as orange coloured ball and stick model.
Fig. 3. The occupancy of most potent compounds in the binding pocket and the orange colour cloud
indicates the excluded volume of the compounds in the lines format. The colour of binding domain
is as per cavity depth.
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2.3. Pharmacology
2.3.1. In vitro antitubercular activity
Benzimidazoles exhibits various types of pharmacological activities including antimicrobial,
antitubercular, anticancer etc [22,35]. All the final synthesized substituted benzimidazole
derivatives 7a-7t were evaluated for in vitro antitubercular activity against M. tb H37Rv. MICs were
determined in quadruplicate in iron-supplemented GAST or in Middlebrook 7H9 broth base
supplemented with 0.2% glycerol, 0.4% glucose, 0.5% BSA fraction V, 0.08% NaCl and 0.05%
Tween 80 in 96w plates according to the broth microdilution method using drugs from DMSO stock
solutions or with control wells treated with an equivalent amount of DMSO. Briefly, two-fold serial
dilutions of compounds in DMSO were added to 50 ꢀL of medium in round-bottom 96-well plates
(Nunc). An equal volume of M. tb H37Rv ATCC27294 diluted to 10,000 cells/mL in the respective
medium was added to each well. Positive controls included isoniazid. Plates were incubated for 1
and 2 weeks at 37 °C after which growth was determined using an inverted enlarging mirror. The
MIC was determined as the lowest concentration of compound that completely inhibited growth of
the bacterium. The MIC for isoniazid was 0.02 ꢀg/mL [36]. The result of MICs of all the final
targeted synthesized compounds and standard drug are reported in Table 1. Thus, all the final
synthesized substituted benzimidazole derivatives were evaluated for in vitro antitubercular activity
against M. tb H37Rv. Compounds 7b, 7j and 7t are active against H37Rv strain of M. tb. MIC value
of compound 7b, 7j and 7t was found to be 3.13 µg/mL, 6.25 µg/mL and 6.25 µg/mL respectively.
Table 1
In vitro antitubercular activity of benzimidazole derivatives against Mycobacterium tuberculosis
H37RV.
OH
R
H
H
N
N
N
R'
Compd.
R
R'
1-week MICa 2-week MICa
1-week MICa
in µg/mL
2-week MICa
in µg/mL
in µg/mL
(GAST/Fe)
100
in µg/mL
(GAST/Fe) (7H9/ADC/Tween) (7H9/ADC/Tween)
4-NO2
3-NO2
4-Cl
4-Cl
>100
4.7
>100
25
>100
37
7a
7b
7c
4-NO2
3,5-Cl
3.13
37
50
100
>=100
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2-Cl
3,4-OCH3
4-OH
3,5-Cl
3,5-Cl
4-Br
>100
100
75
>100
>100
75
>=100
>100
>=100
100
>100
>100
>100
>100
7d
7e
7f
2-NH2, 3,4-
CH3
2,6-CH3
37
75
7g
2-OCH3
3,5-Cl
2,6-CH3
2,6-Cl
50
100
6.25
37
100
100
9.4
100
50
100
>100
50
7h
7i
2-OCH3
3-OH
37
7j
2-NO2
3-Br
4-NO2
37
37
50
7k
7l
4-OCH3
4-OCH3
4-OCH3
2-OCH3
4-COOH
>100
12.5
50
>100
19
>100
>=100
100
50
>100
>100
>=100
75
Pyridine
4-OCH3
2-OH
7p
7q
7r
7s
75
19
25
2-OH, 5-
OCH3
>100
>100
>100
>100
4-OH
2,6-CH3
6.25
0.02
9.4
12.5
0.02
19
7t
0.02
0.02
Isoniazid
aMinimum Inhibitory Concentration (MICs) against Mycobacterium tuberculosis H37Rv
2.3.2. Assays for in vitro cytotoxicity against VERO cells
In vitro cytotoxicity of the active compounds was measured using Vero cells grown in
DMEM medium containing 10% fetal bovine serum. The cell suspensions were plated in 96-well
tissue culture plates (20,000 cells/well) and incubated overnight at 37 °C in the presence of 5% CO2
to allow cells to adhere. The medium was removed and replaced by 180 µl of fresh medium
containing the test compounds at two different concentrations (25 µg/ml and 50 µg/ml). Cells were
then incubated for a further 48 h. 20 µl of Cell-Titer Blue Reagent (Promega) were then added to
each well and incubated for a further 2 h at 37 °C with 5% CO2. Optical density was recorded at
560 nm using a microplate reader. Percentage of cell survival was calculated considering the control
wells (cells incubated in DMSO-containing medium). Table 2 shows that most compounds are non
toxic for the Vero cells, as a compound is considered toxic if it causes over 50% inhibition at
concentration 10 fold higher than its MIC. The most active compounds 7j and 7t only showed
moderate cytotoxicity (% Survival = Optical density of the test compound/Optical density of the
control *100).
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Table 2
In vitro cytotoxicity assay against VERO cells.
% Survival
% Survival
of Vero cells
of Vero cells
(50 µg/ml)
ND
69
(25 µg/ml)
ND
66
Compound
7a
7b
69
63
7c
ND
ND
ND
73
ND
ND
ND
64
7d
7e
7f
7g
ND
ND
61
ND
ND
63
7h
7i
7j
61
53
7k
ND
56
ND
66
7l
7p
ND
69
ND
63
7q
7r
ND
63
ND
66
7s
7t
100
100
Isoniazid
ND, not determined
2.4. Crystallography
Suitable needle shape crystals of compound 7g was formed by evaporation of solution of
compound in dichloromethane and the crystal structure was measured by "Single crystal X-ray
analysis". Bruker Smart Apex CCD area detector was used for the collection of diffraction data
o
using a graphite monochromated Mo Kα radiation (λ = 0.71073 Å) at 20 C. The structure of
compound was solved using the program SHELXL and Fourier difference techniques and was
refined by full-matrix least-squares method on F2. All hydrogen atoms were added theoretically and
crystal structure of compound 7g is shown in Fig. 4.
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Fig. 4. X-ray crystal structure (ORTEP) of compound 7g.
2.5. Structure-activity relationship (SAR) study
The structure-activity relationship study demonstrated that (R)-2-(4'-nitrophenyl)-3-(3'-
nitrophenyl)-1,2,3,5-tetrahydrobenzo [4,5] imidazo [1,2-c]pyrimidin-4-ol 7b, (R)-2-(2',6'-
dicholrophenyl)-3-(3'-hydroxyphenyl)-1,2,3,5-tetrahydrobenzo [4,5]imidazo[1,2-c]pyrimidin-4-ol
7j and (R)-2-(2',6'-dimethylphenyl)-3-(4'-hydroxyphenyl)-1,2,3,5-tetrahydrobenzo[4,5]imidazo[1,2-
c]pyrimidin-4-ol 7t were exhibited good in vitro antitubercular activity against M. tb H37Rv. But, 3-
nitrophenyl and 4-nitrophenyl containing compound 7b is cytotoxic up to certain level due to the
presence of nitro group. Therefore, compound 7b is not favorable for therapeutic potency against
M. tb H37Rv. The cyclic substituted benzimidazole ring is more favorable for the potent
antitubercular activity. Optimum in vitro antitubercular activity was found by these compounds may
be due to formation of free substituted benzimidazole-NAD+ complex. It may be responsible for the
inhibition of Mycobacterium cell wall bio-synthesis. 3-Hydroxyphenyl; 2,6-dichlorophenyl; 4-
hydroxyphenyl and 2,6-dimethylphenyl are more favorable for the formation of substituted
benzimidazole-NAD+ complex. Benzimidazole containing 3,4-dimethylaminophenyl and 2,6-
dimethylphenyl or 3-hydroxyphenyl and 3,4-dichlorophenyl are highly favorable moieties for
potent in vitro antitubercular activity against M. tb H37Rv.
3. Conclusion
The present study describes the docking, synthesis, structure ellucidations, in vitro
antitubercular activity and cytotoxicity assay against VERO cells of benzimidazole derivatives. We
have synthesized and evaluated a series of cyclic substituted benzimidazole derivatives in good
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yields. All the synthesized compounds were evaluated for in vitro antitubercular activity against M.
tb H37Rv. Compound 7b, 7j and 7t showed good in vitro antitubercular activity and other
compounds showed moderate to significant in vitro antitubercular activity against M. tb H37Rv. All
the synthesized compounds were found to be very less cytotoxic using in vitro cytotoxicity assay
against VERO cells. The potency, selectivity and low cytotoxicity of these compounds make them
valid leads for improved antitubercular developments.
4. Experimental protocols
4.1. Technical details
All the reagents were purchased from Sigma-Aldrich Chemicals and were used without
further purification. All solvents were distilled and dried using dry sieves as the usual manner. TLC
analysis was carried out on aluminum foil pre-coated with silica gel 60 F254 (Sigma-Aldrich,
Bangalore dealer). Melting points were determined on a Thomas micro hot stage apparatus and are
uncorrected. FTIR spectra were determined as KBr solid disc method on a Shimadzu model 470
1
13
spectrophotometer. H NMR and C NMR were recorded using Bruker Avance II 400 MHz and
100 MHz spectrometer using DMSO as NMR solvent and are reported in ppm down field from the
residual DMSO. Elemental analysis was performed on a Elementar Vario Micro Cube analyzer and
results were within ±0.4% of the predicted values for all compounds. FTIR (KBr) spectrum of all
the synthesized compounds has strong O-H absorptions at about 3500-3700 cm-1. The medium
strong C=C band in IR spectra of all the compounds appeared at 1450-1575 cm-1, which is similar
as that of the ordinary C=C absorption (1400-1600 cm-1). The formation of H-bond leads to an
increase of their polarity, so the strength of their double bond decreased and absorption moved to
lower wave number. The medium strong C-H band and C-N band in IR spectra of all the
compounds appeared at 3000-3050 cm-1 and 1160-1350 cm-1 respectively which is similar as that of
the ordinary C-H absorption (3000-3100 cm-1) and C-N absorption (1080-1360 cm-1). The 1H NMR
spectrum exhibited different signals at different ppm which were assigned to the different types of
protons. 13C NMR showed various δ values at different ppm for all the target synthesized
compounds. Compound 7g is also confirmed by single crystal X-ray analysis (ORTEP). Purity of
the final compounds were determined by HPLC (HP-03 Offline) instrument using 0.1% TFA in
HPLC Grade water and 0.1% TFA in HPLC Grade ACN. LCMS purity was acquired using UPLC-
02 and UPLC-01 by Formic_Normal at 230.0 nm and 309.0 nm. Single isomer identification was
observed by chiral SFC and chiral HPLC methods of analysis.
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4.1.1. General procedure for synthesis of 2-methyl-1H-benzo[d]imidazole (3)
5.43g (0.03 mmol) o-Phenylenediamine 1 was mixed with 20 mL water and 5.4 g (0.09
c
mmol) acetic acid 2. Resulting reaction mixture was allowed to reflux at 80-85 C for 3.5-4.0 h.
After completion of the reaction, it was cooled and basified by the gradual addition of concentrated
ammonia solution. Precipitated crude product was filtered by vacuum filtration and recrystalized
from 10% aqueous ethanol. Mobile phase: Tolune: Acetonitrile = 2 : 1. Yield 87.15%. mp 130-132
oC. IR (KBr) cm-1: 1465.63 (C=C, Ar), 3496.31 (N-H), 1087.66 (C-N), 1457.36 (C=N), 2950.55 (-
1
CH3). H NMR (400 MHz, DMSO) δ ppm 7.52-7.58 (m, 2H), 7.22-7.27 (m, 2H), 5.0 (s, 1H),
2.51(s, 3H). 13C NMR (DMSO, 100 MHz) δ: 153.25, 138.37, 124.51, 115.20, 18.16. Anal. Calcd for
C8H8N2: C, 72.70; H, 6.10; N, 21.20; Found: C, 71.16; H, 5.37; N, 20.78. MS (ESI, m/z): 133.2
(M+1, 98%), 131.0 (M-1, 63%).
4.1.2. General procedure for the synthesis of (E)-2-(4-nitrostyryl)-1H-benzo[d]imidazole (4)
2-Methyl-1H-benzo[d]imidazole 3 (0.05 mmol) was mixed with 4-nitro benzaldehyde (0.8
mmol). Resulting reaction mixture was allowed to reflux on silicon bath. When temperature reached
at 100 °C, mixture melted, and at 110 °C, water elimination was observed by water drops appearing
on the neck of flask. Reaction mixture became viscous and water elimination ceased after 1 h.
Temperature increased to 160-170 °C and held for 0.5 h. After completion of the reaction, it was
purified by dissolving in pyridine, precipitation with water and resolving by heating. Methanol
treatment was done to remove any soluble impurities. Further purification was accomplished by
column chromatography. Mobile phase: Ethyl acetate: Hexane = 6 : 4. Yield 81.20%. mp 151-153
oC. IR (KBr) cm-1: 1602.56 (C=C, Ar), 3320.82 (N-H), 1307.50 (C=N), 1698.98 (CH=CH, alkene),
1446.35 (N-O, NO2). 1H NMR (400 MHz, DMSO) δ ppm 8.21-8.24 (d, J = 7.6 Hz, 2H), 8.01-8.03
(d, J = 7.6 Hz, 2H), 7.52-7.58 (m, 2H), 7.22-7.28 (m, 3H), 7.07-7.09 (d, J = 15.6 Hz, 1H), 5.0 (s,
1H). 13C NMR (DMSO, 100 MHz) δ: 157.31, 142.04, 137.62, 133.27, 126.38, 124.09, 121.82,
114.30, 111.56. Anal. Calcd for C15H11N3O2: C, 67.92; H, 4.18; N, 15.84; O, 12.06; Found: C,
66.52; H, 3.72; N, 16.26; O, 11.63. MS (ESI, m/z): 266.1 (M+1, 97%), 264.0 (M-1, 45%).
4.1.3. General procedure for synthesis of (E)-4-chloro-N-((2-(4-nitrostyryl)-1H-benzo [d] imidazol-
1-yl)methyl)aniline (5).
(E)-2-(4-Nitrostyryl)-1H-benzo[d]imidazole 4 (0.03 mmol) was dissolved in ethanol (10
vol). It was allowed to stirred at room temperature for 20 min. Formaldehyde (0.02 mmol) was
added drop wise to the above reaction mixture. Then, 4-nitro aniline (0.04 mmol) was added to
above reaction mixture. After 15 min, triethylamine (0.01 mmol) was added drop wise to above
reaction mixture. Resulting reaction mixture was allowed to reflux at 70-72oC for 3.5-4.0 h. After
17

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completion of reaction, it was distilled off to remove ethanol from reaction mixture. Then, it was
quinched with ice cold water and extracted with dichloromethane. Dichloromethane layer was
distilled off to get crude product and further purification was accomplished by column
o
chromatography. Mobile phase: Ethyl acetate: Hexane = 8 : 2. Yield 70.26%. mp 180-182 C. IR
(KBr) cm-1: 1440.56 (C=C, Ar), 2952.48 (-CH2), 3361.32, (N-H), 1052.94 (C-N), 1401.28 (C=N),
1691.27 (CH=CH, alkene), 1238.08 (-NO2), 763.67 (-Cl). 1H NMR (400 MHz, DMSO) δ ppm 8.21-
8.24 (d, J = 7.6 Hz, 2H), 8.01-8.03(d, J = 7.6 Hz, 2H), 7.52-7.58 (m, 2H), 7.22-7.28 (m, 5H), 7.07-
7.09 (d, J = 15.6 Hz, 1H), 6.51-6.53 (d, J = 7.4 Hz, 2H), 5.68 (s, 2H), 4.0 (s, 1H). 13C NMR
(DMSO, 100 MHz) δ: 158.63, 147.82, 143.54, 138.37, 136.52, 134.76, 129.73, 123.64, 121.86,
112.08, 111.94, 65.30. Anal. Calcd for C22H17ClN4O2: C, 65.27; H, 4.23; Cl, 8.76; N, 13.84; O,
7.90; Found: C, 64.86; H, 3.81; Cl, 8.34; N, 14.25; O, 7.49. MS (ESI, m/z): 404.9 (M+, 94%), 406.7
(M+2, 33%), 403.0 (M-1, 61%).
4.1.4. General procedure for synthesis of 4-chloro-N-((2-((2S,3S)-3-(4-nitrophenyl)oxiran-2-yl)-1H-
benzo[d]imidazol-1-yl)methyl)aniline (6).
(E)-4-Chloro-N-((2-(4-nitrostyryl)-1H-benzo [d] imidazol-1-yl)methyl)aniline 5 (0.05
mmol) was dissolved in dichloromethane (10 vol). Sodium hypochlorite (1.01 mmol) was added to
above reaction mixture at rt. After 30 min, R,R-Jacobsen’s catalyst (0.01 mmol) was added portion
wise to above reaction mixture. It was allowed to reflux at 50-52oC for 2.5-3.0 h. After completion
of reaction, it was allowed to cool at rt and quinched in water followed by extracting with
dichloromethane. Dichloromethane layer was distilled off to get crude product. Further purification
was accomplished by Combiflash chromatography to get pure product. Mobile phase: Methanol:
o
Dichloromethane = 1 : 9. Yield 68.26%. mp 214-216 C. IR (KBr) cm-1: 1585.20 (C=C, Ar),
1
2991.05 (-CH2), 3333.36 (N-H), 1151.29 (C-N), 1417.42 (C=N), 1374.03 (-NO2), 765.01 (-Cl). H
NMR (400 MHz, DMSO) δ ppm 8.19-8.21 (d, J = 7.6 Hz, 2H), 7.59-7.62 (m, 4H), 7.22-7.27 (m,
13
4H), 6.52-6.54 (d, J = 7.6 Hz, 2H), 5.5 (s, 2H), 4.2 (s, 2H), 4.0 (s, 1H). C NMR (DMSO, 100
MHz) δ: 167.31, 158.76, 145.84, 141.33, 139.09, 134.78, 131.67, 125.39, 121.66, 115.38, 66.37,
63.86, 55.03. Anal. Calcd for C22HI7ClN4O3: C, 62.79; H, 4.07; Cl, 8.42; N, 13.31; O, 11.41; Found:
C, 62.38; H, 3.65; Cl, 8.02; N, 13.73; O, 10.99. MS (ESI, m/z): 420.8 (M+, 96%), 422.5 (M+2,
30%), 419.4 (M-1, 54%).
4.1.5. General procedure for synthesis of (R)-2-(4'-chlorophenyl)-3-(4'-nitrophenyl)-1,2,3,5-
tetrahydrobenzo[4,5]imidazo[1,2-c]pyrimidin-4-ol (7a).
4-Chloro-N-((2-((2S,3S)-3-(4-nitrophenyl)oxiran-2-yl)-1H-benzo[d] imidazol-1-yl) methyl)
aniline 6 (0.05 mmol) was dissolved in methanol (10 vol). It was allowed to stir for 15 min at rt.
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Then, Aluminum trichloride (1.05 mmol) was added to it. Resulting reaction mixture was allowed
to reflux at 75-77oC for 2.0-2.5 h. After completion of reaction, it was distilled off to remove
methanol from the reaction mixture. Then, it was neutralized by sodium bicarbonate solution and
extracted with dichloromethane and dichloromethane layer was distilled off to get crude product.
Further purification was accomplished by column chromatography to get pure product. Mobile
phase: Methanol: Dichloromethane = 0.5 : 9.5. Yield 65.25%. mp 285-287 °C. IR (KBr) cm-1:
1583.27, 1664.27 (C=C, Ar), 3439.42 (N-H), 2930.31 (-CH2), 1090.55 (C-N), 3598.52 (-OH),
1
746.31 (-Cl), 1381.75 (-NO2). H NMR (400 MHz, DMSO) δ ppm 7.49-7.51 (d, J = 8.6 Hz, 1H),
7.31-7.32 (d, J = 4.0 Hz, 1H), 6.98-7.0 (d, J = 5.0 Hz, 1H), 6.84 (s, 3H), 6.76-6.78 (dd, J = 8.60,
2.28 Hz, 2H), 6.65 (s, 4H), 4.48 (s, 1H), 3.90 (s, 1H), 3.79 (s, 1H), 3.53 (s, 1H), 2.1 (s, 1H, D2O
exchangeable, OH). 13C NMR (DMSO, 100 MHz) δ: 176.75, 145.54, 139.92, 139.31, 138.21,
128.92, 127.62, 127.56, 127.15, 127.12, 126.33, 121.54, 119.73, 77.37, 77.26, 77.05, 76.74, 63.52,
55.84, 49.29, 43.63, 39.14. Anal. Calcd for C22H17ClN4O3 : C, 62.79; H, 4.07; Cl, 8.42; N, 13.31; O,
11.41; Found: C, 62.39; H, 3.72 ; Cl, 8.13; N, 12.94; O, 10.95. MS (ESI, m/z): 420.9 (M+, 96%),
422.8 (M+2, 30%), 419.0 (M-1, 94%), 299.7 (C16H14ClN3O, 11%), 310.0 (C16H14N4O3, 15%),
332.7 (C17H17ClN2O3, 14%). HPLC purity (RT 9.747 min, 98.39%) where mobile phase 0.1% TFA
in HPLC grade water and 0.1% TFA in HPLC grade acetonitrile.
4.1.6. (R)-2-(4'-Nitrophenyl)-3-(3'-nitrophenyl)-1,2,3,5-tetrahydrobenzo [4,5] imidazo[1,2-c]
pyrimidin-4-ol (7b).
Yield 67.23%. mp 290-292 °C. IR (KBr) cm-1: 1667.16, 1583.27 (C=C, Ar), 2932.23 (-CH2),
1
3213.79 (-NH), 1084.76 (C-N), 3611.05 (-OH), 1383.68 (-NO2). H NMR (400 MHz, DMSO) δ
ppm 7.87 (s, 2H), 7.62-7.66 (m, 3H), 7.50-7.56 (m, 3H), 7.06-7.11 (m, 4H), 4.68 (s, 2H), 3.49 (s,
13
1H), 2.27 (s, 1H), 2.1 (s, 1H, D2O exchangeable, OH). C NMR (DMSO, 100 MHz) δ: 176.75,
175.32, 148.54, 145.06, 128.78, 127.44, 126.57, 77.37, 77.26, 77.05, 76.74, 58.66, 50.53, 48.71,
43.90, 40.57, 40.36, 39.94, 39.32. Anal. Calcd for C22H17N5O5: C, 61.25; H, 3.97; N, 16.23; O,
18.54; Found: C, 60.80; H, 3.57; N, 15.86; O, 18.23. MS (ESI, m/z): 432.1 (M+1, 96%), 430.0 (M-
1, 94%), 309.3 (C16H14N4O3, 11%), 341.2 (C17H15N3O5, 20%), 343.5 (C17H17N3O5, 7%), 379.3
(C17H13N5O5, 23%), 415.8 (C22H17N5O4, 34%).
4.1.7. (R)-2-(3',5'-Dichlorophenyl)-3-(4'-chlorophenyl)-1,2,3,5-tetrahydrobenzo [4,5]imidazo [1,2-
c]pyrimidin-4-ol (7c).
Yield 63.28%. mp 295-297 °C. IR (KBr) cm-1: 1582.31 (C=C, Ar), 2951.52 (-CH2), 3202.22
1
(-NH), 1156.12 (C-N), 3597.56 (-OH), 749.20, 677.85 (-Cl). H NMR (400 MHz, DMSO) δ ppm
8.10-8.12 (m, 1H), 7.65 (s, 1H), 7.46-7.50 (m, 2H), 7.41-7.43 (m, 1H), 7.34-7.38 (m, 1H), 7.28-7.30
19

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(d, J = 7.5 Hz, 1H), 7.15-7.19 (m, 2H), 7.08-7.10 (d, J = 7.8 Hz, 1H), 6.83 (s, 1H), 5.12 (s, 1H), 3.97
(s, 2H), 3.34 (s, 1H), 1.70 (s, 1H, D2O exchangeable, OH). 13C NMR (DMSO, 100 MHz) δ: 170.15,
148.61, 139.29, 130.15, 129.56, 128.52, 127.63, 126.71, 119.96, 117.97, 77.37, 63.35, 52.33, 50.12,
46.52, 44.12, 40.51, 36.17, 34.58, 29.28, 22.04, 21.70. Anal. Calcd for C22H16Cl3N3O: C, 59.41; H,
3.63; Cl, 23.91; N, 9.45; O, 3.60; Found: C, 58.96; H, 3.35; Cl, 22.12; N, 9.07; O, 3.17. MS (ESI,
m/z): 444.9 (M+, 97%), 446.7 (M+2, 31%), 448.4 (M+4, 15%), 443.0 (M-1, 48%), 300.0
(C16H14ClN3O, 10%), 334.0 (C16H13Cl2N3O, 29%), 356.8 (C17H16Cl3NO, 41%), 397.0
(C18H16Cl3N3O, 26%), 341.5 (C22H19N3O, 18%).
4.1.8. (R)-2-(3',5'-Dichlorophenyl)-3-(2'-chlorophenyl)-1,2,3,5-tetrahydrobenzo[4,5]imidazo [1,2-
c] pyrimidin-4-ol (7d).
Yield 61.36%. mp 290-292 °C. IR (KBr) cm-1: 1509.03 (C=C, Ar), 2969.84 (-CH2), 3338.18
1
(-NH), 1036.55 (C-N), 3624.55 (-OH), 665.32, 770.42 (-Cl). H NMR (400 MHz, DMSO) δ ppm
7.56-7.63 (m, 4H), 7.37-7.52 (m, 2H), 7.12-7.18 (m, 1H), 6.96-7.0 (m, 4H), 5.67 (s, 2H), 4.57-4.62
13
(q, 1H), 3.50 (s, 1H), 2.52-2.54 (m, 1H, D2O exchangeable, OH). C NMR (DMSO, 100 MHz) δ:
170.15, 145.61, 139.20, 138.98, 130.15, 129.95, 128.52, 127.63, 127.40, 126.71, 124.01, 119.74,
117.97, 117.56, 58.66, 50.53, 48.71, 43.50, 40.57, 40.38, 33.73, 33.32. Anal. Calcd for
C22H16Cl3N3O: C, 59.41; H, 3.63; Cl, 23.91; N, 9.45; O, 3.60; Found: C, 58.43; H, 3.30; Cl, 22.48;
N, 9.17; O, 3.21. 443. MS (ESI, m/z): 444.8 (M+, 95%), 446.7 (M+2, 32%), 448.4 (M+4, 11%),
443.3 (M-1, 60%), 299.0 (C16H14ClN3O, 14%), 334.2 (C16H13Cl2N3O, 32%), 428.8 (C22H16Cl3N3,
5%), 341.4 (C22H19N3O, 23%), 325.4 (C22H19N3, 20%).
4.1.9. (R)-2-(3',5'-Dichlorophenyl)-3-(3',4'-dimethoxyphenyl)-1,2,3,5-tetrahydrobenzo [4,5] imidazo
[1,2-c]pyrimidin-4-ol (7e).
Yield 63.41%. mp 287-289 °C. IR (KBr) cm-1: 1576.52 (C=C, Ar), 3037.34, 2928.38,
2852.20 (C-H), 3325.64 (-NH), 1088.62 (C-N), 3578.27 (-OH), 648.92 (-Cl), 1001.25 (C-O),
1440.56 (C-H, OCH3). 1H NMR (400 MHz, DMSO) δ ppm 7.49-7.51 (m, 3H), 7.31-7.32 (d, J = 4.0
Hz, 2H), 6.98-7.0 (d, J = 5.0 Hz, 1H), 6.84 (s, 2H), 6.76-6.78 (dd, J = 8.6, 4.2 Hz, 2H), 4.57-4.60 (d,
J = 12.8 Hz, 1H), 4.45-4.48 (d, J = 12.8 Hz, 1H), 3.90 (s, 1H), 3.79 (s, 6H), 3.53 (s, 1H), 2.52 (s,
1H, D2O exchangeable, OH). 13C NMR (DMSO, 100 MHz) δ: 163.42, 158.25, 146.40, 145.64,
144.46, 144.18, 119.73, 117.19, 114.64, 111.47, 107.58, 107.51, 60.81, 57.43, 55.63, 40.16, 39.74,
39.11. Anal. Calcd for C24H21Cl2N3O3: C, 61.29; H, 4.50; Cl, 15.08; N, 8.93; O, 10.20; Found: C,
60.84; H, 4.09; Cl, 14.71; N, 8.52; O, 9.82. MS (ESI, m/z): 470.6 (M+, 96%), 472.8 (M+2, 31%),
474.5 (M+4, 14%), 469.1 (M-1, 82%), 440.2 (C23H19Cl2N3O2, 20%), 407.1 (C23H20ClN3O2, 4%).
20

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4.1.10. (R)-2-(4'-Bromophenyl)-3-(4'-hydroxyphenyl)-1,2,3,5-tetrahydrobenzo[4,5]imidazo[1,2-c]
pyrimidin-4-ol (7f).
Yield 60.28%. mp 293-295 °C. IR (KBr) cm-1: 1605.45, 1507.10 (C=C, Ar), 2880.17 (-CH2),
1
3428.81 (-NH), 1171.54, 1252.54 (C-N), 3677.59 (-OH), 1056.78 (C-O), 537.07 (-Br). H NMR
(400 MHz, DMSO) δ ppm 8.11 (s, 4H), 7.62-7.68 (m, 2H), 7.50-7.55 (m, 2H), 7.06-7.14 (m, 4H),
4.66 (s, 2H), 3.44 (s, 1H), 2.52 (s, 1H), 2.27-2.31 (t, J = 7.5 Hz, 1H), 1.86-1.91 (m, 1H), 1.70-1.72
(m, 1H, D2O exchangeable, OH). 13C NMR (DMSO, 100 MHz) δ: 185.61, 160.99, 154.95, 140.93,
138.40, 135.97, 130.39, 129.14, 127.50, 126.06, 123.4, 75.72, 42.31, 39.38, 27.69, 26.68, 25.47,
21.56, 13.50. Anal. Calcd for C22H18BrN3O2: C, 60.56; H, 4.16; Br, 18.31; N, 9.63; O, 7.33; Found:
C, 60.15; H, 3.76; Br 17.90; N, 9.23; O, 6.92. MS (ESI, m/z): 436.8 (M+, 99%), 438.9 (M+2, 94%),
435.0 (M-1, 65%), 281.0 (C16H15N3O2, 20%), 343.8 (C16H14BrN3O, 43%), 421.2 (C22H18BrN3O,
17%), 357.3 (C22H19N3O2, 14%), 349.0 (C17H18BrO2, 30%). HPLC purity (RT 11.68 min, 95.14%)
where mobile phase 0.1% TFA in HPLC grade water and 0.1% TFA in HPLC grade acetonitrile.
4.1.11. (R)-2-(2',6'-Dimethylphenyl)-3-(2'-amino,3,4-dimethylphenyl)-1,2,3,5-tetrahydrobenzo [4,5]
imidazo[1,2-c]pyrimidin-4-ol (7g).
Yield 61.29%. mp 289-291 °C. IR (KBr) cm-1: 1509.99, 1588.09, 1650.77 (C=C, Ar),
3076.87 (-CH3), 2948.63 (C-H), 1148.4, 1226.5, 1274.72 (C-N), 3775.94 (-OH), 3492.45 (-NH). 1H
NMR (400 MHz, DMSO) δ ppm 7.66 (s, 2H), 7.42 (s, 1H), 7.29 (s, 1H), 7.0 (s, 1H), 6.86 (s, 2H),
6.68-6.75 (m, 2H), 5.69 (m, 1H), 5.68 (s, 1H), 5.48 (s, 1H), 4.52 (s, 1H), 2.99 (s, 6H), 2.31 (s, 6H),
2.02 (s, 1H, D2O exchangeable, OH). 13C NMR (DMSO, 100 MHz) δ: 170.4, 148.47, 139.06,
138.98, 130.15, 129.88, 128.65, 127.61, 126.71, 120.09, 119.74, 117.58, 62.54, 52.02, 50.14, 46.62,
46.12, 42.05, 40.81, 36.17, 34.95, 22.04, 21.7. Anal. Calcd for C26H28N4O: C, 75.70; H, 6.84; N,
13.58; O, 3.88; Found: C, 75.24; H, 6.47; N, 13.11; O, 3.48. MS (ESI, m/z): 412.8 (M+, 99%), 411.0
(M-1, 97%), 307.3 (C18H20N4O, 34%), 294.3 (C18H19N3O, 36%), 396.5 (C26H28N4, 32%), 324.4
(C22H19N3, 49%). HPLC purity (RT 5.598 min, 97.67%) where mobile phase 0.1% TFA in HPLC
grade water and 0.1% TFA in HPLC grade acetonitrile. LCMS (RT 3.015 min, 99%) acquired using
UPLC-02 by Formic_Normal at 230.0 nm.
4.1.12. (R)-2-(3',5'-Dichlorophenyl)-3-(2'-methoxyphenyl)-1,2,3,5-tetrahydrobenzo [4,5] imidazo
[1,2-c]pyrimidin-4-ol (7h).
Yield 63.78%. mp 286-288 °C. IR (KBr) cm-1: 1469.49 (-OCH3), 1586.16 (C=C, Ar),
2970.80 (-CH2), 2780.85 (C-H), 1088.62 (C-N), 3475.1 (-NH), 3623.59 (-OH), 777.17 (-Cl),
1
2970.8, 2780.85 (-CH2). H NMR (400 MHz, DMSO) δ ppm 7.74 (m, 1H), 7.46-7.52 (m, 1H),
7.38-7.42 (m, 2H), 7.22-7.33 (m, 5H), 7.15-7.17 (d, J = 8.1 Hz, 2H), 5.55 (s, 2H), 3.8 (s, 1H), 3.35
21

ACCEPTED MANUSCRIPT
(s, 1H), 2.95-2.97 (d, J = 7.8 Hz, 3H), 2.53-2.55 (m, 1H), 1.85-1.91 (m, 1H, D2O exchangeable,
OH). 13C NMR (DMSO, 100 MHz) δ: 169.53, 156.03, 153.98, 142.73, 141.99, 140.54, 136.19,
135.04, 134.48, 132.18, 129.92, 128.60, 126.05, 123.12, 121.8, 118.58, 109.22, 46.58, 31.58, 29.02,
21.03, 16.38. Anal. Calcd for C23H19Cl2N3O2: C, 62.74; H, 4.35; Cl, 16.10; N, 9.54; O, 7.27; Found:
C, 62.33; H, 3.93; Cl, 15.68; N, 9.95; O, 6.85. MS (ESI, m/z): 440.8 (M+, 96%), 442.9 (M+2, 31%),
444.6 (M+4, 12%), 439.1 (M-1, 71%), 295.0 (C17H17N3O2, 25%), 334.3 (C16H13Cl2N3O, 32%),
371.4 (C23H21N3O2, 15%), 425.0 (C23H19Cl2N3O, 14%), 406.0 (C23H20ClN3O2, 9%).
4.1.13. (R)-2-(2',6'-Dimethylphenyl)-3-(2'-methoxyphenyl)-1,2,3,5-tetrahydrobenzo [4,5] imidazo
[1,2-c]pyrimidin-4-ol (7i).
Yield 65.28%. mp 293-295 °C. IR (KBr) cm-1: 1587.13 (C=C), 3322.75 (-NH), 1229.4,
1
1267.97 (C-N), 1469.49 (-OCH3), 2965.02, 2924.52 (-CH3), 2782.78 (C-H), 3500.17 (-OH). H
NMR (400 MHz, DMSO) δ ppm 7.60-7.66 (m, 2H), 7.49-7.50 (m, 2H), 7.06-7.11 (m, 6H), 6.96-
7.02 (m, 1H), 5.67 (s, 1H), 4.68 (s, 2H), 4.10 (s, 1H), 2.54 (s, 3H), 2.27-2.31 (t, J = 7.5 Hz, 6H),
13
1.85-1.92 (m, 1H, D2O exchangeable, OH). C NMR (DMSO, 100 MHz) δ: 185.65, 161.0, 140.9,
138.44, 135.87, 130.29, 128.80, 127.44, 126.02, 123.47, 78.83, 78.17, 75.73, 42.35, 36.78, 27.75,
26.73, 25.48, 21.58, 13.47. Anal. Calcd for C25H25N3O2: C, 75.16; H, 6.31; N, 10.52; O, 8.01;
Found: C, 74.73; H, 5.91; N, 10.11; O, 8.41. MS (ESI, m/z): 400.0 (M+1, 95%), 398.2 (M-1, 71%),
294.0 (C18H19N3O, 14%), 295.0 (C17H17N3O2, 21%), 372.0 (C23H21N3O2, 12%), 384.1 (C25H25N3O,
20%), 341.2 (C22H19N3O, 14%), 386.0 (C24H23N3O2, 27%).
4.1.14. (R)-2-(2',6'-Dicholrophenyl)-3-(3'-hydroxyphenyl)-1,2,3,5-tetrahydrobenzo [4,5] imidazo
[1,2-c]pyrimidin-4-ol (7j).
Yield 61.27%. mp 287-289 °C. IR (KBr) cm-1: 1586.16, 1468.53 (C=C), 2978.52, 2781.81 (-
1
CH2), 3329.50 (-NH), 1088.62, 1228.43 (C-N), 3550.31 (-OH), 775.24 (-Cl). H NMR (400 MHz,
DMSO) δ ppm 8.10-8.12 (m, 1H), 7.51 (s, 1H), 7.47 (m, 2H), 7.41-7.43 (m, 1H), 7.34-7.38 (m,
1H), 7.28-7.30 (m, 1H), 7.13-7.19 (m, 2H), 7.00-7.10 (d, J = 7.0 Hz, 1H), 6.86 (s, 1H), 5.12 (s, 1H),
13
3.93 (s, 1H), 3.91 (s, 1H), 3.65 (s, 1H), 3.34 (s, 1H), 1.87 (s, 1H, D2O exchangeable, OH). C
NMR (DMSO, 100 MHz) δ: 164.87, 156.62, 153.83, 137.47, 135.28, 133.22, 131.98, 129.95,
127.80, 125.68, 120.53, 114.65, 109.71, 76.29, 55.33, 39.34, 32.24, 4.70, 23.25, 19.72. Anal. Calcd
for C22H17Cl2N3O2: C, 61.98; H, 4.02; Cl, 16.63; N, 9.86; O, 7.51; Found: C, 61.57; H, 3.61; Cl,
16.21; N, 10.28; O, 7.92. MS (ESI, m/z): 426.8 (M+, 98%), 428.5 (M+2, 30%), 430.7 (M+4, 10%),
425.0 (M-1, 74%), 281.0 (C16H15N3O2, 7%), 335.0 (C16H13Cl2N3O, 21%), 358.1 (C22H19N3O2,
14%), 410.3 (C22H17Cl2N3O, 35%). HPLC purity (RT 4.216 min, 98.25%) where mobile phase
0.1% TFA in HPLC grade water and 0.1% TFA in HPLC grade acetonitrile. LCMS (RT 1.903 min,
22

ACCEPTED MANUSCRIPT
99.5%) acquired using UPLC-01 by Formic_Normal at 309.0 nm.
4.1.15. (R)-2-(4'-Nitrophenyl)-3-(2'-nitrophenyl)-1,2,3,5-tetrahydrobenzo [4,5] imidazo[1,2-c]
pyrimidin-4-ol (7k).
Yield 64.92%. mp 293-295 °C. IR (KBr) cm-1: 1595.81, 1436.71 (C=C), 2944.77 (-CH2),
1
1087.66, 1161.90 (C-N), 3273.57 (-NH), 3696.87 (-OH), 1349.93 (-NO2). H NMR (400 MHz,
DMSO) δ ppm 7.45 (m, 5H), 7.12-7.14 (m, 2H), 6.94-7.02 (m, 5H), 4.07-4.15 (m, 1H), 3.86-3.89 (t,
J = 6.66 Hz, 2H), 3.41 (s, 1H), 2.20-2.29 (m, 1H, D2O exchangeable, OH). 13C NMR (DMSO, 100
MHz) δ: 154.31153.66, 130.42, 129.50, 127.95, 124.82, 120.25, 119.88, 109.63, 108.56, 107.51,
79.17, 78.51, 54.76, 52.62, 50.28, 40.14, 39.51, 38.89, 38.28, 28.58, 25.24. Anal. Calcd for
C22H17N5O5: C, 61.25; H, 3.97; N, 16.23; O, 18.54; Found: C, 60.84; H, 3.55; N, 16.65; O, 18.13.
MS (ESI, m/z): 432.1 (M+1, 98%), 430.2 (M-1, 95%), 309.7 (C16H14N4O3, 31%), 343.5
(C17H17N3O5, 33%).
4.1.16. (R)-2-(4'-Methoxyphenyl)-3-(3'-bromophenyl)-1,2,3,5-tetrahydrobenzo[4,5]imidazo [1,2-c]
pyrimidin-4-ol (7l).
Yield 60.28%. mp 288-290 °C. IR (KBr) cm-1: 568.89 (-Br), 1494.56 (-OCH3), 3500.06 (-
1
OH), 1585.20 (C=C), 2982.37, 2775.06 (-CH2), 3256.22 (-NH), 1194.69, 1375.96 (C-N). H NMR
(400 MHz, DMSO) δ ppm 7.88-7.91 (m, 2H), 7.32-734 (m, 4H), 7.25-7.29 (m, 4H), 7.17-7.22 (m,
2H), 5.50 (s, 2H), 3.39 (s, 1H), 2.92-2.96 (t, J = 7.16 Hz, 1H), 2.71 (s, 1H, D2O exchangeable, OH),
13
2.33-2.36 (t, J = 7.16 Hz, 3H). C NMR (DMSO, 100 MHz) δ: 199.86, 156.53, 142.99, 134.70,
128.37, 128.14, 127.35, 127.17, 125.52, 80.07, 77.57, 77.25, 76.93, 72.75, 57.78, 51.19, 36.31,
35.12, 31.53, 31.21, 22.13. Anal. Calcd for C23H20BrN3O2: C, 61.34; H, 4.48; Br, 17.74; N, 9.33; O,
7.11; Found: C, 61.52; H, 3.64; Br, 16.85; N, 8.18; O, 2.85. MS (ESI, m/z): 450.7 (M+, 99%), 452.5
(M+2, 97%), 449.1 (M-1, 73%).
4.1.17. (R)-2-(2',6'-Dimethylphenyl)-3-(3'-hydroxyphenyl)-1,2,3,5-tetrahydrobenzo [4,5] imidazo
[1,2-c] pyrimidin-4-ol (7n).
Yield 61.27%. mp 289-291 °C. IR (KBr) cm-1: 1491.67, 1528.31, 1651.73 (C=C), 1110.8,
1
1216.86 (C-N), 3102.9 (-CH2), 3322.75 (-NH), 3788.47 (-OH), 2965.02 (-CH3). H NMR (400
MHz, DMSO) δ ppm 7.67-7.71 (t, J = 9.3 Hz, 3H), 7.50-7.57 (m, 1H), 7.40-7.46 (m, 1H), 7.22-7.33
(m, 4H), 7.16-7.20 (m, 2H), 5.61 (s, 2H), 3.81 (s, 1H), 3.35 (s, 1H), 2.91 (s, 1H, D2O exchangeable,
OH), 2.63 (s, 1H, D2O exchangeable, OH), 2.50 (d, J = 6.4 Hz, 6H). 13C NMR (DMSO, 100 MHz)
δ: 172.11, 169.50, 156.11, 154.0, 14.67, 140.49, 136.5, 134.65, 130.25, 126.31, 123.06, 121.77,
118.60, 110.29, 99.49, 79.23, 78.57, 46.18, 39.51, 37.71, 28.77, 20.76, 16.43, 13.82. Anal. Calcd for
23

ACCEPTED MANUSCRIPT
C24H23N3O2: C, 74.78; H, 6.01; N, 10.90; O, 8.30; Found: C, 74.37; H, 5.61; N, 11.32; O, 7.89. MS
(ESI, m/z): 386.2 (M+1, 97%), 384.0 (M-1, 94%).
4.1.18. (R)-2-(4'-Methoxyphenyl)-3-(3'-pyridinylphenyl)-1,2,3,5-tetrahydrobenzo [4,5] imidazo
[1,2-c]pyrimidin-4-ol (7p).
Yield 52.37%. mp 281-283 °C. IR (KBr) cm-1: 3657.34 (-OH), 1490.70 (-OCH3), 1652.70,
1
1529.27 (C=C), 2960.20 (-CH2), 3322.75, (-NH), 1216.66, 1346.07 (C-N). H NMR (400 MHz,
DMSO) δ ppm 7.81-7.82 (d, J = 4.0 Hz, 1H), 7.75-7.77 (d, J = 8.6 Hz, 1H), 7.38-7.45 (m, 2H),
7.29-7.32 (d, J = 8.6 Hz, 1H), 7.22-7.24 (d, J = 7.2 Hz, 1H), 7.04-7.19 (m, 4H), 6.81-6.90 (m, 2H),
4.57 (s, 2H), 3.81 (s, 1H), 3.10 (s, 1H), 2.55 (s, 1H, D2O exchangeable, OH), 2.06-2.11 (m, 3 H).
13C NMR (DMSO, 100 MHz) δ: 180.55, 156.82, 148.47, 145.34, 143.87, 140.53, 136.42, 131.56,
128.04, 125.98, 119.48, 77.18, 73.49, 49.98, 43.83, 40.30, 39.77, 32.37, 31.84, 17.42, 13.37, 12.18.
Anal. Calcd for C22H20N4O2: C, 70.95; H, 5.41; N, 15.04; O, 8.59; Found: C, 69.88; H, 4.55; N,
16.60; O, 7.37. MS (ESI, m/z): 373.0 (M+1, 98%), 371.1 (M-1, 88%).
4.1.19. (R)-2-(4'-Methoxyphenyl)-3-(4'-methoxyphenyl)-1,2,3,5-tetrahydrobenzo [4,5] imidazo [1,2-
c]pyrimidin-4-ol (7q).
Yield 58.39%. mp 274-27 °C. IR (KBr) cm-1: 3548.38 (-OH), 1428.99 (-OCH3), 1649.80,
1
1509.03 (C=C), 2955.38 (-CH2), 3291.87 (-NH), 1230.36, 1326.79 (C-N). H NMR (400 MHz,
DMSO) δ ppm 7.61-7.67 (m, 3H), 7.50-7.55 (m, 3H), 7.07-7.12 (s, 6H), 4.68 (s, 2H), 4.10 (s, 1H),
3.52-3.56 (m, 3H), 3.33-3.35 (m, 3H), 1.92 (s, 1H), 1.72 (s, 1H, D2O exchangeable, OH). 13C NMR
(DMSO, 100 MHz) δ: 185.61, 161.06, 154.99, 140.92, 138.43, 135.9, 130.67, 130.33, 129.15,
127.49, 126.06, 123.47, 78.92, 78.27, 75.73, 42.33, 40.17, 39.34, 27.72, 25.48, 21.58, 13.49. Anal.
Calcd for C24H23N3O3: C, 71.80; H, 5.77; N, 10.47; O, 11.96; Found: C, 70.37; H, 5.67; N, 11.31;
O, 10.83. MS (ESI, m/z): 402.0 (M+1, 98%), 400.0 (M-1, 96%).
4.1.20. (R)-2-(2'-Methoxyphenyl)-3-(2'-hydroxyphenyl)-1,2,3,5-tetrahydrobenzo [4,5] imidazo [1,2-
c]pyrimidin-4-ol (7r).
Yield 58.37%. mp 277-279 °C. IR (KBr) cm-1: 3759.48 (-OH), 1439.6 (-OCH3), 1509.03,
1
1561.09, 1649.8 (C=C), 2795.31 (-CH2), 3195.31 (-NH), 1113.69, 1235.18 (C-N). H NMR (400
MHz, DMSO) δ ppm 7.72-7.75 (m, 2H), 7.43-7.46 (m, 2H), 7.06-7.10 (m, 2H), 6.99-7.03 (m, 4H),
6.84-6.88 (m, 2H), 4.48-4.61 (m, 2H), 3.84 (s, 1H), 3.39-3.47 (m, 1H), 3.11 (s, 3H), 2.88-2.89 (d, J
13
= 4.4 Hz, 2H, D2O exchangeable, OH). C NMR (DMSO, 100 MHz) δ: 189.22, 147.09, 141.68,
134.13, 132.03, 130.08, 129.45, 129.41, 126.60, 123.11, 119.62, 77.05, 76.73, 53.38, 53.7, 46.91
41.01, 32.03, 22.68. Anal. Calcd for C23H21N3O3: C, 71.30; H, 5.46; N, 10.85; O, 12.39; Found: C,
24

ACCEPTED MANUSCRIPT
70.52; H, 4.68; N, 10.22; O, 11.96. MS (ESI, m/z): 388.0 (M+1, 96%), 386.2 (M-1, 85%).
4.1.21. (R)-2-(4'-Carboxylphenyl)-3-(2'-hydroxy,5-methoxyphenyl)-1,2,3,5-tetrahydrobenzo [4,5]
imidazo [1,2-c]pyrimidin-4-ol (7s).
Yield 60.25%. mp 279-281 °C. IR (KBr) cm-1: 3657.34 (-OH), 1420.32 (-OCH3), 1509.03
(C=C), 2953.45 (-CH2), 3356.22 (-NH), 1177.33, 1326.79 (C-N), 1714.41 (C=O of COOH),
1227.47 (C-O of COOH). 1H NMR (400 MHz, DMSO) δ ppm 12.19 (s, 1H), 7.50-7.51 (d, J = 7.64
Hz, 2H), 7.24-7.34 (m, 7H), 7.11-7.15 (t, J = 7.3 Hz, 2H), 5.54 (s, 2H), 5.32 (s, 1H), 4.55 (s, 1H),
3.39 (s, 2H, D2O exchangeable, OH), 2.77 (s, 3H). 13C NMR (DMSO, 100 MHz) δ: 177.63, 146.15,
143.58, 143.48, 127.71, 125.48, 125.13, 78.95, 78.62, 78.30, 77.97, 71.34, 56.30, 51.87, 45.39,
41.0, 40.18, 38.92, 35.79, 26.78, 23.81. Anal. Calcd for C24H21N3O5: C, 66.81; H, 4.91; N, 9.74; O,
18.54; Found: C, 65.48; H, 4.16; N, 8.56; O, 17.28. MS (ESI, m/z): 432.1 (M+1, 99%), 430.0 (M-1,
83%).
4.1.22. (R)-2-(2',6'-Dimethylphenyl)-3-(4'-hydroxyphenyl)-1,2,3,5-tetrahydrobenzo [4,5] imidazo
[1,2-c]pyrimidin-4-ol (7t).
Yield 52.81%. mp 274-276 °C. IR (KBr) cm-1: 3696.87 (-OH), 2959.23 (-CH3), 1469.49
(C=C), 3349.34 (-NH), 1166.76, 1267.34 (C-N). 1H NMR (400 MHz, DMSO) δ ppm 7.56-7.63 (m,
4H), 7.37-7.52 (m, 2H), 7.12-7.18 (m, 1H), 6.96-7.03 (m, 4H), 5.67 (s, 2H), 5.44 (s, 1H), 4.57-4.62
(q, J = 7.0 Hz, 1H), 4.20 (s, 1H), 2.52-2.54 (m, 6H), 2.10 (s, 1H, D2O exchangeable, OH). 13C NMR
(DMSO, 100 MHz) δ: 167.49, 158.10, 141.51, 140.86, 138.05, 136.68, 131.23, 130.44, 130.35,
128.82, 127.40, 126.46, 123.48, 121.21, 120.34, 116.34, 78.91, 78.58, 78.25, 66.23, 46.27, 39.53,
38.90, 14.30. Anal. Calcd for C24H23N3O2: C, 74.78; H, 6.01; N, 10.90; O, 8.30; Found: C, 73.20;
H, 5.45; N, 11.16; O, 7.25. MS (ESI, m/z): 386.0 (M+1, 99%), 384.2 (M-1, 94%).
Acknowledgements
Authors would like to thank Institute of Pharmacy, Nirma University for providing facilities
for experimental work. We are also thankful to Piramal Discovery Solutions for providing spectral
analysis of compounds. Authors would also like to thank Dr. Helena Boshoff, Tuberculosis
Research Section, LCID, NIAID, NIH, USA for in vitro antitubercular activity. Shubhra Singh is
supported by a Raman-Charpak Fellowship and is also thankful to IFTM University, Moradabad for
registering her in their university. Authors are also thankful to Laboratoire de Dynamique des
Interactions Normales et Pathologiques, France for in vitro cytotoxicity assay against vero cells. We
are also thankful to Department of Science & Technology (DST), Govt. of India for providing
INSPIRE fellowship which helps as financial support for research work.
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