Synthesis and evaluation of antimycobacterial activity of new benzimidazole aminoesters

Article abstract of DOI:10.1016/j.ejmech.2013.06.025

Abstract A total of 51 novel benzimidazoles were synthesized by a 4-step reaction starting from basic compound 4-fluoro-3-nitrobenzoic acid under relatively mild reaction conditions. The structure of the novel benzimidazoles was confirmed by mass spectra as well as ¹H NMR spectroscopic data. Out of the 51 novel synthesized compounds, 42 of them were screened for their antimycobacterial activity against Mycobacterium tuberculosis H₃₇Rv strain using BacTiter-Glo Microbial Cell Viability (BTG) method. Results of activity screened using Alamar Blue method was also provided for comparison purposes. Two of the novel benzimidazoles synthesized showed moderately good activity with IC₅₀ of less than 15 μM. Compound 5g, ethyl 2-(4-(trifluoromethyl)phenyl)-1-(2-morpholinoethyl)-1H-benzo[d]imidazole-5-carboxylate, was found to be the most active with IC₅₀ of 11.52 μM.

Full text of DOI:10.1016/j.ejmech.2013.06.025

Accepted Manuscript
Synthesis and evaluation of antimycobacterial activity of new benzimidazole
aminoesters
Yeong Keng Yoon, Mohamed Ashraf Ali, Ang Chee Wei, Tan Soo Choon, Rusli Ismail
PII:
S0223-5234(13)00393-0
10.1016/j.ejmech.2013.06.025
EJMECH 6253
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 September 2012
Revised Date: 22 May 2013
Accepted Date: 4 June 2013
Please cite this article as: Y.K. Yoon, M.A. Ali, A.C. Wei, T.S. Choon, R. Ismail, Synthesis and
evaluation of antimycobacterial activity of new benzimidazole aminoesters, European Journal of
Medicinal Chemistry (2013), doi: 10.1016/j.ejmech.2013.06.025.
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*Graphical Abstract (for review)
Click here to download Graphical Abstract (for review): Graphical_Abstract.doc
ACCEPTED MANUSCRIPT
Graphical Abstract
Synthesis and evaluation of antimycobacterial activity of new
benzimidazole aminoesters
Yeong Keng Yoona, Mohamed Ashraf Alia,b,c*, Ang Chee Weia, Tan Soo Choona ,Rusli
Ismaila
The novel benzimidazoles synthesized showed moderately good activity with IC50 of less than 15
µM.
Compound
5Ig,
ethyl
2-(4-(trifluoromethyl)phenyl)-1-(2-morpholinoethyl)-1H-
benzo[d]imidazole-5-carboxylate, was found to be the most active with IC50 of 11.52 µM.

*Highlights (for review)
ACCEPTED MANUSCRIPT
Research Highlights
1. We synthesised series on novel benzimidazole derivatives.
2. The synthesised compounds were tested invitro against mycobacterial strains.
3. Compound 5Ig identified as a promising inhibitor of M. tuberculosis H37Rv.
4. Compounds serve as potential leads for further drug discovery in tuberculosis area.

*Revised Manuscript
Click here to view linked References
ACCEPTED MANUSCRIPT
Synthesis and evaluation of antimycobacterial activity of new
benzimidazole aminoesters
Yeong Keng Yoona, Mohamed Ashraf Alia,b,c*, Ang Chee Weia, Tan Soo Choona ,Rusli
Ismaile
aInstitute for Research in Molecular Medicine, Universiti Sains Malaysia, Minden 11800,
Penang, Malaysia
cNew Drug Discovery Research, Department of Medicinal Chemistry, Alwar Pharmacy
College, Alwar, Rajasthan-301030, India.
dNew Drug Discovery Research, Department of Medicinal Chemistry, Sunrise University,
Alwar,Rajasthan-301030, India.
eCentre of Excellence for Research in AIDS (CERiA), University Malaya, Minden 59990,
Kuala Lumpur, Malaysia.
Abstract: A total of 51 novel benzimidazoles were synthesized by a 4-step reaction starting
from basic compound 4-fluoro-3-nitrobenzoic acid under relatively mild reaction conditions.
1
The structure of the novel benzimidazoles were confirmed by mass spectra as well as H
NMR spectroscopic data. Out of the 51 novel synthesized compounds, 42 of them were
screened for their antimycobacterial activity against M. tuberculosis H37Rv strain using
BacTiter-Glo™ Microbial Cell Viability (BTG) method. Results of activity screened using
Alamar Blue method was also provided for comparison purposes. Two of the novel
benzimidazoles synthesized showed moderately good activity with IC50 of less than 15 µM.
Compound
5g,
ethyl
2-(4-(trifluoromethyl)phenyl)-1-(2-morpholinoethyl)-1H-
benzo[d]imidazole-5-carboxylate, was found to be the most active with IC50 of 11.52 µM.
Key words: Benzimidazoles, antimycobacterial; azole ; *E-mail: asraf80med@yahoo.com

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Introduction
Tuberculosis (TB) is the oldest documented infectious disease. It is a chronic necrotizing
bacterial infection with wide variety of manifestations caused by Mycobacterium
tuberculosis, which has plagued humans throughout recorded and archeological history [1].
The primary site of infection is the lungs, followed by dissemination via the circulatory and
lymphatic system to secondary sites including the bones, joints, liver and spleen.
In 2010, there were 8.8 million (range, 8.5–9.2 million) incident cases of TB, 1.1 million
(range, 0.9–1.2 million) deaths from TB among HIV-negative people and an additional 0.35
million (range, 0.32–0.39 million) death from HIV-associated TB [2]. The introduction of the
first line drugs like streptomycin, para-aminosalicylic acid and isoniazid for treatment some
50 years ago has witnessed in a remarkable decline in TB cases all over the world. The active
TB is currently treated with a four first-line drug regimen comprising mainly isoniazid,
rifampicin, pyrazinamide and ethambutol for a period of at least 6 months [3,4]. However, it
did not take long for M. tuberculosis to find its way around these compounds, and since the
mid of 1980s, the disease has been undergoing a resurgence driven by variety of changes in
social, medical and economic factors as well as M. tuberculosis’ resistance to the above-
mentioned drugs itself. Despite extensive research in the last 40 years, no new anti-TB drugs
have been introduced into the market by means of passing actual clinical trials [5]. Although
a new TB drug, Bedaquiline, was released in USA [6] in December 2012, the trial was based
on paradoxical surrogate measure to gain “fast track” approval by USFDA. However,
decisions making under such time pressure may lead to unanticipated safety problems as
shown by the higher chances of death by patients taking this drug, indicating treatment failure
[7].

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The benzimidazole nucleus is of significant importance in medicinal chemistry research and
many benzimidazole-containing compounds exhibit important biological properties such as
antiviral [8], anti-inflammatory [9] and anti-HIV [10]. In the light of the affinity they display
towards a variety of enzymes and protein receptors, medicinal chemists thus classify them as
“privileged sub-structures” for drug design [11].
Recently, there have been reported work done on utilizing benzimidazole derivatives to
counter TB with relatively good results [12,13]; thus further reinforcing our belief that
benzimidazole could potentially be a lead compound in our effort to discover new potent anti-
TB agents.
In the present paper, we wish to report the synthesis and antimycobacterial activity of novel
2-substituted benzimidazole derivatives.
2. Results and Discussion
2.1 Chemistry
The procedure to synthesize benzimidazole derivatives was adopted and modified from
literature [14]. Our synthetic study into novel benzimidazoles started with 4-fluoro-3-nitro
benzoic acid which was esterified in the presence of catalytic sulfuric acid in ethanol by
refluxing for 8 hours to afford the ethyl ester 1 in 75% yield. The ethylbenzoate 1 was then
treated with various amines (see Experimental section) and DIPEA in dry dichloromethane at
room temperature yielded amino compound 2, which was reduced to the amine 3 using
ammonium formate and 10% Pd/C for 3 hours to give 60% yield. The structure of 3-amino
ethylbenzoate 3 was confirmed by chomatographic analysis.

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The phenylenediamine 3 was then refluxed with various substituted bisulfite adduct of
aromatic aldehydes [15] in DMF overnight to afford benzimidazole derivatives 5-7 in
moderate to good yields (38-90%). The structure of the novel benzimidazoles were
comfirmed by spectroscopic analysis and further unambiguosly ascertained by single X-ray
crystallographic analysis [16-19]. Among the literature reports available for the synthesis of
benzimidazoles by the reaction of phenylenediamine with acid chloride [20], aldehyde [21]
and acid [22], we found that access into benzimidazole derivatives via this metabisulfite route
is efficient, environmental friendly and afforded good to excellent yield of the
benzimidazoles.
1
The H NMR spectrum of benzimidazole 5g showed a singlet at δ 1.43 ppm due to the
methyl group. The N-methylene protons from the piperazine appeared as a triplet at δ 2.33
1
ppm while the O-methylene showed a triplet at 3.55 ppm. Similar H patterns were obtained
for other substituted benzimidazoles derivatives. Proton NMR assignment for 5g was shown
as representation for the other compounds in the series (Figure 1). The 13C NMR spectrum of
5g which resonated at δ 150.81 and 167.36 ppm are assigned to imine (C=N) and ester
carbonyl carbon respectively.
The mechanism for the formation of the novel benzimidazole derivatives is proposed and
summarized in Scheme 1.
2.2 Pharmacology
A total of 51 novel benzimidazole derivatives were synthesized and 42 of them were then
analyzed for their antimycobacterial activities against Mycobacterium tuberculosis H37RV
(MTB-H37Rv). Another 9 compounds were obtained in gel-like form. As there might be
ambiguity over the trace amount of solvent effect and the consistency of the weight from gel-

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like compounds, we omitted those compounds from further biological activity testing. Results
are shown in Table 3.
In vitro antimycobacterial activity of the compounds were evaluated against Mycobacterium
tuberculosis H37RV (MTB-H37Rv) in a HTS (High Throughput Screening) assay adapted
from the microdilution AlamarBlue (AB) broth method as reported by Collins and Franzblau
[23]. For comparison, an alternative method for end-point detection was assessed using the
Promega reagent BacTiter-Glo™ Microbial Cell Viability (BTG). The BTG assay is a
quantitative ATP assay for bacteria using luciferase production as an end-point detection
point. Data was analyzed using the IDBS Activity Base software and the dose response result
was analyzed using a four parameter logistic fit to the data (Excel Fit equation 205) with the
maximum and minimum locked at 100 and 0. From these curves, EC90 and EC50 values were
calculated. As references, six standard drugs used for TB treatment (Amikacin, Cycloserine,
Ethambutol, Isoniazid, Pyrimethamine and Rifampicin) were also evaluated in the assays.
Comparing the three series of substitution at the 2-position of the benzimidazole core, it can
be concluded that 4-(2-aminoethyl)morpholine gave the best activity. This could be due to the
fact that heterocyclic moiety, such as morpholine group are biologically active. It also
showed that electron donating groups somehow give rise to better antimycobacterial
activities, which is consistent with those reported in literature [24,25]. Thus, a further
modification of the 4-substitution on the sodium bisulfite adduct was carried out to further
improve the activity. We synthesized compounds with a wide range of substitution including
compounds with electron-donating as well as electron-withdrawing groups. Generally, we
found that electron withdrawing group sustituents at 4-position in the phenyl ring is important
for good activities as shown by 5g, 5b, 5e and 5p. Of all 42 compounds which have been
tested, compound 5g, ethyl 2-(4-(trifluoromethyl)phenyl)-1-(2-morpholinoethyl)-1H-

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benzo[d]imidazole-5-carboxylate was found to be the most active with IC50 of 11.52 µM,
IC90 of 16.53 µM and MIC of 50 µM respectively. It was followed closely by 5b, ethyl 2-(4-
bromophenyl)-1-(2-morpholinoethyl)-1H-benzo[d]imidazole-5-carboxylate, with IC50 of
12.54 µM, IC90 of 14.48 µM and MIC of 50 µM respectively.
Both these compounds are more active than the standard drugs cycloserine and
pyrimethamine. However none of the compounds screened in the present derivatives are
found to be more potent than other used standard drugs. This clearly showed that the presence
of electron donating group substitution at 4-position of the phenyl ring, caused marked
improvement in antimycobacterial activity.
All the compounds were also tested for cytotoxicity (IC50) in VERO cells at concentrations of
62.5 μg/mL. After 72 h of exposure, viability was assessed on the basis of cellular conversion
of MTT into a formazan product using the Promega CellTiter 96 nonradioactive cell
proliferation assay according to the manufacturer’s protocol. All of the active compounds
were found to be non-toxic till 62.5 μg/mL.
Encourage by the positive results we have reported here, further modification on the 2-
susbstituted position on the benzimidazole core as well as 4-position on the bisulfite adducts
as currently in progress in our laboratory.
3. Experimental
3.1. Chemistry
All chemicals were supplied by Sigma-Aldrich (U.S.A) and Merck Chemicals (Germany).
Purity of the compounds was checked on thin layer chromatography (TLC) plates (silica gel
G) in the solvent system chloroform-methanol (9:1). The spots were located under short

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(254nm)/long (365nm) UV light. Elemental analyses were performed on Perkin Elmer 2400
1
Series II CHN Elemental Analyzer and were within ± 0.3% of the calculated values. H and
13
13C NMR were performed on Bruker Avance 300 (1H: 300 MHz, C: 75MHz) spectrometer
in CDCl3 for (5 and 6 series) and CD3OD (7 series) using TMS as internal standard. Mass
spectra were recorded on Varian 320-MS TQ LC/MS using ESI. Crystal structure analysis
was carried out using Bruker SMART APEXII CCD area-detector diffractometer. Melting
point was performed on Gallenkamp MFB595.010M Melting Point apparatus. Column
chromatography purification was done in solvent system chloroform-methanol (9:1) using
Silica Gel 60 (0.063 – 0.200 mm).
3.1.1. Procedure for the preparation of Ethyl-4-fluoro-3-nitrobenzoate (1)
4-Fluoro-3-nitrobenzoic acid (5 g, 27 mmol) was refluxed in ethanol (50 mL) and
concentrated H2SO4 (2 mL) for 8 hours. After completion of reaction (as evident from TLC),
the solvent was evaporated under reduced pressure. The aqueous layer was extracted with
ethyl acetate (25 mL x 3). The organic layer was dried over Na2SO4 and concentrated under
reduced pressure to yield 1 as cream-coloured powder (75%).
3.1.2. General procedure for the preparation of 4-(2-substituted amino)-3-nitro-
ethylbenzoate (2)
Ethyl-4-fluoro-3-nitrobenzoate,
1
(0.5 g, 2.34 mmol), amine [for 5: 4-(2-
Aminoethyl)morpholine; 6: 1-(3-Aminopropyl)-2-pyrrolidinone); 7: Aqueous ammonia
solution, 38%] (2.58 mmol) and N,N-Diisopropylethylamine, DIPEA (0.49 mL, 2.78 mmol)
were mixed in dichloromethane (10 mL). The reaction mixture was stirred overnight at room
temperature. After completion of reaction (as evident from TLC), the reaction mixture was
washed with water (10 mL x 2) followed by 10% Na2CO3 solution (10 mL). The organic

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layer was dried over Na2SO4 and concentrated under reduced pressure to afford 2 as brown
oil/yellow solid.
3.1.3. General procedure for the preparation of Ethyl-3-amino-4-(2-substituted
amino)benzoate (3)
4-(2-substituted amino)-3-nitro-ethylbenzoate, 2 (1 mmol), ammonium formate (3 mmol) and
Pd/C (50 mg) were mixed in ethanol (10 mL). The reaction mixture was refluxed until
completion (solution turned colourless). The reaction mixture was then filtered through Celite
545. The filtrate was evaporated under reduced pressure. It was resuspended in ethyl acetate
and washed with water, dried over Na2SO4 and evaporated to dryness to yield 3 (60%).
3.1.4. General procedure for the preparation of sodium bisulfite addcuts of 4-
substituted benzaldehyde (4)
Appropriate benzaldehyde (10 mmol) was dissolved in ethanol (20 mL). Sodium
metabisulfite (15 mmol) in 5 mL water was added in portion over 5 minutes. The reaction
mixture was stirred at room temparature for 1 hour and subsequently stirred at 4oC overnight.
The precipitate formed was filtered and dried to afford sodium bisulfite adducts (55%-90%).
3.1.5. General procedure for the preparation of 2-substituted benzimidazole derivatives
(5-7)
Ethyl-3-amino-4-(2-substituted amino)benzoate, 3 (1 mmol) and various sodium bisulfite
adducts, 4 (1.5 mmol) were dissolved in DMF (5 mL). The reaction mixture was stirred at
90oC under N2 atmosphere for 24-48 hours. After completion of reaction (evident by TLC),
the reaction mixture was diluted in ethyl acetate (25 mL) and washed with water (10 mL x 3).
The organic layer was collected, dried over Na2SO4 and evaporated under reduced pressure to

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afford crude products. Final compounds 5-7 were obtained in 38-90% yields after
recrystallisation from ethanol or column purification.
3.2 Biology
The test samples were analyzed in vitro against Mycobacterium tuberculosis H37Rv (Mtb
H37Rv) in a high throughput screen using an assay adapted from the microdilution
AlamarBlue (AB) broth assay as reported by Collins and Franzblau [20]. All manipulations of
Mtb H37Rv were conducted in accordance with the Biosafety in Microbiological and
Biomedical Laboratories (BMBL) 5th Edition in Biosafety Level 3 containment laboratories.
In brief, our assay uses Mtb H37Rv in a 384-well plate format with in-plate DMSO carrier
controls, 3.198 µM amikacin, 0.17 µM amikacin controls and 320 compounds. The Mtb
H37Rv + DMSO carrier control provides a 100% growth control for each plate. The 3.198 µM
amikacin completely inhibits growth of the bacteria and is used in place of uninoculated
media as the background control; whereas, the 0.17 µM amikacin control approximates the
MIC of amikacin ranging from 30-80% inhibition indicative of a positive growth inhibition
and proper assay performance. The media used for both compound preparation and Mtb
H37Rv plating was assessed for contamination by plating two 384 well plates with media
alone. The plates were checked for contamination by visual inspection and end-point
detection. The compounds were evaluated in 10-point stacked plate dose response method.
Compounds were serially diluted 1:2 from 100 µM thru 0.195 µM. The plates were read
fluorometrically after incubation with the compounds and addition of AB. Tests were
performed in triplicate.
An alternative method for end-point detection was assessed using the Promega reagent
BacTiter-Glo™ Microbial Cell Viability (BTG). The BTG plates were briefly incubated for

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20 minutes at room temperature, sealed with Perkin Elmer clear TopSeal A and read from the
top using luminescence on an Perkin Elmer Envision. Tests were performed in triplicate.
Results from both type of assays were presented in this paper for comparison purposes.
3.2.1. Autofluorescence
Compounds in media were pre-read from the high concentration plate with no AlamarBlue or
bacteria added. Fold increase was calculated using the median of the positive control wells
from the AlamarBlue Mtb H37Rv assay for the Mtb H37Rv control wells. The criteria for a
compound being considered autofluorescent was defined as having >50% fluorescence of the
Mtb H37Rv control wells. None of the analyzed compounds were found to be autofluorescent.
Acknowledgments
The authors wish to express their gratitude and appreciation to Pharmacogenetics and Novel
Therapeutics Research Cluster, Institute for Research in Molecular Medicine, University
Science Malaysia, Penang for supporting this work. This work was funded through Research
Grant No.RUC (1001/PSK/8620012) and HiCoE research Grant No (311.CIPPM.4401005).
This work was also supported by the National Institutes of Health and the National Institute
of Allergy and Infectious Diseases, Contract No. HHSN272201100012I for
antimycobacterial screening.
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Table captions, Figures and Schemes
Table captions, Figures and Schemes
Table 1: Physical properties and analytical results of compounds 5a-q, 6a-q and 7a-q
Table 2: 1H NMR and 13C NMR results for compounds 5a-q, 6a-q and 7a-q.
Table 3: Antimycobacterial activity of 2-substituted benzimidazole derivatives against M.
tuberculosis H37Rv.
Scheme 1: Protocol for synthesis of titled compounds.

Table(s)
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Table 1
Compound
R1
R2
Formula
C22H25N3O3
C22H24N3O3Br
C22H24N4O5
C23H27N3O5
C/H/N Calculated;
[M+H]
380.3
458.2
425.2
427.2
Yield (%)
M.p (oC)
(C/H/N Found)
69.56% / 6.56% / 11.11%;
(69.42% / 6.79% / 11.25%)
57.67% / 5.33% / 9.21%;
(57.80% / 5.23% / 9.40%)
62.22% / 5.67% / 13.20%;
(62.40% / 5.58% / 13.22%)
64.89% / 6.41% / 9.90%;
(65.01% / 6.29% / 10.02%)
50
65
78
70
-
5a
5b
5c
5d
O
O
O
O
N
205-206
210-211
201-202
Br
N
N
N
NO2
OCH3
OH
OCF3
N
C23H24N3O4F3
C24H30N4O3
59.60% / 5.20% / 9.08%;
(59.62% / 5.20% / 9.03%)
68.22% / 7.21% / 13.30%;
(68.29% / 7.27% / 13.23%)
464.2
423.3
86
39
187-188
203-204
5e
5f
O
O
N
N
1

ACCEPTED MANUSCRIPT
C23H24N3O3F3
C27H34N4O3
C26H32N4O4
C27H27N4O3F
61.67% / 5.42% / 9.37%;
448.2
463.3
465.3
476.2
85
73
77
76
183-184
-
5g
5h
5i
O
O
O
O
CF3
N
N
N
N
(61.66% / 5.40% / 9.37%)
70.12% / 7.40% / 12.11%;
(70.25% / 7.45% / 12.82%)
67.20% / 6.90% / 12.21%;
(67.20% / 6.87% / 12.24%)
68.33% / 5.67% / 11.80%;
(68.48% / 6.83% / 12.60%)
N
186-187
210-211
N
O
F
5j
N
C23H27N3O4
C23H25N3O5
67.50% / 6.74% / 10.32%;
(67.60% / 6.67% / 10.36%)
65.19% / 6.03% / 9.94%;
(65.17% / 6.08% / 10.10%)
410.3
424.3
68
87
189-190
201-202
5k
5l
O
O
OCH3
N
N
O
O
2

ACCEPTED MANUSCRIPT
OCH3
C32H34N4O5
69.31% / 6.20% / 10.11%;
555.3
38
-
5m
O
N
(69.49% / 6.39% / 10.01%)
N
O
C23H25N3O5
C22H25N3O4
C22H24N3O3Cl
C22H25N3O5
65.19% / 6.03% / 9.94%;
(65.24% / 6.13% / 10.00%)
66.78% / 6.42% / 10.61%;
(66.51% / 6.59% / 10.53%)
63.80% / 5.81% / 10.21%;
(63.84% / 5.65% / 10.28%)
64.24% / 6.13% / 10.22%;
(64.26% / 6.15% / 10.03%)
424.2
397.2
415.2
412.2
89
55
63
42
215-216
193-194
190-191
-
5n
5o
5p
5q
O
O
O
O
COOH
OH
N
N
N
N
Cl
HO
OH
O
C23H25N3O3
70.56% / 6.46% / 10.71%;
(70.52% / 6.41% / 11.72%)
392.3
79
92-93
6a
N
3

ACCEPTED MANUSCRIPT
O
O
O
C23H24N3O3Br
C23H24N4O5
C24H27N3O5
58.67% / 5.13% / 8.91%;
470.1
437.2
438.3
77
69
80
113-114
121-122
109-110
6b
6c
6d
Br
N
N
N
(58.90% / 5.25% / 8.79%)
63.32% / 5.54% / 12.80%;
(63.42% / 5.57% / 12.83%)
NO2
OCH3
65.89% / 6.21% / 9.62%;
(66.01% / 6.27% / 9.67%)
OH
O
O
O
O
C24H24N3O4F3
C25H30N4O3
C24H24N3O3F3
C28H34N4O3
60.67% / 5.10% / 8.78%;
(60.69% / 5.11% / 8.74%)
476.2
435.3
460.2
475.3
90
72
90
86
100-101
112-113
99-100
-
6e
6f
OCF3
N
N
N
N
69.12% / 7.01% / 12.90%;
(69.16% / 7.13% / 12.92%)
N
62.72% / 5.33% / 9.24%;
(62.75% / 5.36% / 9.25%)
6g
6h
CF3
70.89% / 7.20% / 11.80%;
(70.91% / 7.08% / 11.73%)
N
4

ACCEPTED MANUSCRIPT
O
O
C27H32N4O4
68.11% / 6.82% / 11.83%;
477.3
487.2
88
90
133-134
140-141
6i
6j
N
O
N
N
(68.11% / 6.80% / 11.79%)
F
C28H27N4O3F
69.11% / 5.56% / 11.20%;
(69.08% / 5.80% / 11.14%)
N
O
O
C24H27N3O4
C24H25N3O5
68.40% / 6.50% / 10.00%;
(68.35% / 6.50% / 10.10%)
422.3
436.2
81
89
100-101
-
6k
6l
OCH3
N
N
O
66.22% / 5.82% / 9.71%;
(66.20% / 5.80% / 9.81%)
O
O
OCH3
C33H34N4O5
70.03% / 6.13% / 9.89%;
(70.28% / 6.29% / 9.65%)
567.4
40
-
6m
N
N
O
5

ACCEPTED MANUSCRIPT
O
O
O
O
C24H25N3O5
C23H25N3O4
C23H24N3O3Cl
C23H25N3O5
66.22% / 5.82% / 9.71%;
434.2
408.2
426.2
424.2
82
66
76
57
-
6n
6o
6p
6q
COOH
OH
N
N
N
N
(66.21% / 5.80% / 9.76%)
67.81% / 6.21% / 10.34%;
(67.72% / 6.37% / 10.33%)
128-129
112-113
146-147
64.89% / 5.72% / 9.91%;
(64.85% / 5.82% / 9.96%)
Cl
HO
65.12% / 6.03% / 9.91%;
(65.07% / 6.15% / 9.20%)
OH
-H
-H
-H
C16H14N2O2
C16H13N2O2Br
C16H13N3O4
72.20% / 5.30% / 10.50%;
(72.16% / 5.25% / 10.58%)
55.71% / 3.80% / 8.10%;
(55.70% / 3.79% / 8.07%)
61.70% / 4.22% / 13.53%;
(61.70% / 4.24% / 13.50%)
267.1
345.0
312.0
79
73
75
177-178
190-191
204-205
7a
7b
7c
Br
NO2
6

ACCEPTED MANUSCRIPT
OCH3
OH
-H
C17H16N2O4
65.40% / 5.21% / 9.02%;
313.1
80
228-229
7d
(65.44% / 5.23% / 9.00%)
-H
-H
-H
-H
-H
C17H13N2O3F3
C18H19N3O2
C17H13N2O2F3
C21H23N3O2
C20H21N3O3
58.31% / 3.71% / 8.00%;
(58.29% / 3.72% / 8.07%)
69.90% / 6.21% / 13.62%;
(69.95% / 6.17% / 13.59%)
61.11% / 3.91% / 8.43%;
(61.16% / 3.85% / 8.38%)
72.20% / 6.61% / 11.99%;
(72.30% / 6.69% / 11.91%)
68.42% / 6.01% / 11.98%;
(68.41% / 6.02% / 11.96%)
351.0
310.1
335.1
350.2
352.2
87
84
90
76
77
186-187
220-221
183-184
190-191
204-205
7e
7f
OCF3
N
7g
7h
7i
CF3
N
N
O
7

ACCEPTED MANUSCRIPT
F
-H
C21H16N3O2F
69.81% / 4.51% / 11.61%;
362.2
66
>230
7j
(69.79% / 4.51% / 11.60%)
N
-H
-H
C17H16N2O3
C17H14N2O4
68.90% / 5.41% / 4.50%;
(68.88% / 5.50% / 4.47%)
65.81% / 4.60% / 9.02%;
(65.85% / 4.64% / 8.91%)
297.1
311.0
89
89
188-189
210-211
7k
7l
OCH3
O
O
OCH3
-H
C26H23N3O4
70.72% / 5.30% / 9.51%;
(70.62% / 5.53% / 9.22%)
442.3
42
-
7m
N
O
-H
C17H14N2O4
65.81% / 4.60% / 9.02%;
(65.84% / 5.63% / 9.08%)
311.0
71
>230
7n
COOH
8

ACCEPTED MANUSCRIPT
-H
-H
-H
C16H14N2O3
C16H13N2O2Cl
C16H14N2O4
68.11% / 5.00% / 9.91%;
283.1
301.1
299.1
82
85
75
209-210
182-183
>230
7o
7p
7q
OH
Cl
(68.19% / 5.05% / 9.82%)
63.89% / 4.41% / 9.32%;
(63.90% / 4.40% / 9.30%)
64.41% / 4.30% / 9.42%;
(64.29% / 4.39% / 9.49%)
HO
OH
9

ACCEPTED MANUSCRIPT
Table 2
Compound
5a
NMR (δ ppm)
1H NMR : 1.43 (3H, t, J = 7.2 Hz), 2.38 (4H, t, J = 4.8 Hz), 2.74 (2H, t, J = 6.9 Hz), 3.62 (4H, t, J = 4.8 Hz), 4.30-4.50 (4H, m),
6.90-7.10 (5H, m), 7.48 (1H, d, J = 8.4 Hz), 8.10 (1H, dd, J1 = 1.5 Hz, J2 = 8.4 Hz), 8.55 (1H, s)
13C NMR : 14.81, 43.78, 54.33, 57.79, 61.26, 67.12, 110.09, 122.64, 124.70, 125.43, 128.70, 129.2, 129.18, 130.48, 139.12,
143.08, 156.18, 167.48
5b
1H NMR : 1.44 (3H, t, J = 6.9 Hz), 2.25 (4H, t, J = 4.8 Hz), 2.66 (2H, t, J = 6.6 Hz), 3.45 (4H, t, J = 4.8 Hz), 4.44 (2H, q, J = 6.9
Hz), 4.54 (2H, t, J = 6.6), 7.66 (2H, d, J = 8.4 Hz), 7.75 (1H, d, J = 8.4 Hz), 7.90 (2H, d, J = 8.4 Hz), 8.08 (1H, dd, J1 = 1.5 Hz, J2
= 8.4 Hz), 8.38 (1H, s)
13C NMR : 14.38, 42.79, 53.90, 57.49, 60.93, 66.69, 109.72, 122.44, 124.57, 124.73, 125.36, 129.15, 131.00, 132.08, 138.77,
142.73, 143.08, 154.62, 167.00
5c
1H NMR : 1.45 (3H, t, J = 6.9 Hz), 2.34 (4H, t, J = 4.8 Hz), 2.78 (2H, t, J = 6.9 Hz), 3.55 (4H, t, J = 4.8 Hz), 4.40-4.50 (4H, m),
7.52 (1H, d, J = 9 Hz), 8.10-8.20 (3H, m), 8.44 (2H, d, J = 9 Hz), 8.59 (1H, s)
13C NMR : 14.38, 42.77, 53.86, 57.49, 60.51, 66.61, 109.96, 122.50, 125.56, 125.88, 126.18, 129.72, 129.99, 138.68, 142.51,
154.25, 167.03
5d
1H NMR : 1.44 (3H, t, J = 7.2 Hz), 2.38 (4H, t, J = 4.8 Hz), 2.75 (2H, t, J = 6.9 Hz), 3.61 (4H, t, J = 4.8 Hz), 3.97 (3H, s), 4.40-
4.50 (4H, m), 7.04 (1H, d, J = 8.1 Hz), 7.25 (1H, dd, J1= 1.8 Hz, J2 = 8.1 Hz), 7.36 (1H, s), 7.46 (1H, d, J = 8.1 Hz), 8.06 (1H, dd,
J1 = 1.8 Hz, J2 = 8.1 Hz), 8.55 (1H, s)
13C NMR : 14.21, 41.96, 54.30, 57.73, 59.20, 61.68, 67.27, 111.29, 116.37, 122.30, 125.73, 126.50, 131.76, 148.97, 150.05,
10

ACCEPTED MANUSCRIPT
152.10, 167.52
5e
5f
1H NMR : 1.43 (3H, t, J = 7.2 Hz), 2.33 (4H, t, J = 4.8 Hz), 2.75 (2H, t, J = 6.9 Hz), 3.55 (4H, t, J = 4.8 Hz), 4.40-4.50 (4H, m),
7.60 (1H, d, J = 9 Hz), 7.82 (2H, d, J = 9 Hz), 7.99 (2H, d, J = 9 Hz), 8.12 (1H, dd, J1 = 1.5 Hz, J2 = 9 Hz), 8.57 (1H, s)
13C NMR : 14.38, 42.79, 53.90, 57.49, 60.93, 66.69, 109.72, 122.44, 124.57, 124.73, 125.36, 129.15, 131.00, 132.08, 138.77,
142.73, 143.08, 154.62, 167.00
1H NMR : 1.44 (3H, t, J = 7.2 Hz), 2.36 (4H, t, J = 4.8 Hz), 2.80 (2H, t, J = 6.9 Hz), 2.90 (6H, s), 3.54 (4H, t, J = 4.8 Hz), 4.40-
4.50 (4H, m), 6.65 (2H, d, J = 8.4 Hz), 7.25 (1H, d, J = 8.4 Hz), 7.50 (2H, d, J = 8.4 Hz), 8.00 (1H, dd, J1 = 1.5 Hz, J2 = 8.4 Hz),
8.56 (1H, s)
13C NMR : 14.45, 40.20, 42.30, 54.11, 57.66, 60.79, 66.82, 109.09, 120.58, 122.70, 122.93, 123.19, 129.50, 130.75, 131.80,
135.74, 142.03, 153.68, 167.67
5g
5h
1H NMR : 1.43 (3H, t, J = 7.2 Hz), 2.33 (4H, t, J = 4.8 Hz), 2.75 (2H, t, J = 6.9 Hz), 3.55 (4H, t, J = 4.8 Hz), 4.40-4.50 (4H, m),
7.50 (1H, d, J = 9 Hz), 7.83 (2H, d, J = 9 Hz), 8.00 (2H, d, J = 9 Hz), 8.10 (1H, dd, J1 = 1.5 Hz, J2 = 9 Hz), 8.58 (1H, s)
13C NMR : 14.80, 43.71, 54.22, 57.89, 61.28, 67.01, 110.17, 121.54, 122.80, 125.00, 125.73, 127.89, 129.33, 131.64, 139.05,
143.02, 150.81, 167.36
1H NMR : 1.40-1.50 (9H, m), 2.36 (4H, t, J = 4.8 Hz), 2.70 (2H, t, J = 6.6 Hz), 2.80 (4H, t, J = 6.6 Hz), 3.55 (4H, t, J = 4.8 Hz),
4.40-4.50 (4H, m), 6.65 (2H, d, J = 8.4 Hz), 7.25 (1H, d, J = 8.4 Hz), 7.50 (2H, d, J = 8.4 Hz), 8.02 (1H, dd, J1 = 1.5 Hz, J2 = 8.4
Hz), 8.58 (1H, s)
13C NMR : 14.63, 25.35, 26.59, 42.75, 51.24, 53.40, 56.78, 60.89, 66.80, 109.18, 117.50, 121.69, 122.09, 129.48, 130.33, 131.72,
135.84, 142.56, 152.98, 167.50
5i
1H NMR : 1.45 (3H, t, J = 6.9 Hz), 2.47 (4H, t, J = 4.8 Hz), 2.82 (2H, t, J = 6.6 Hz), 3.29 (4H, t, J = 4.5 Hz), 3.70 (4H, t, J = 4.8
Hz), 3.92 (4H, t, J = 4.5 Hz), 4.40-4.60 (4H, m), 7.04 (2H, d, J = 9 Hz), 7.49 (1H, d, J = 9 Hz), 7.75 (2H, d, J = 9 Hz), 8.07 (1H,
11

ACCEPTED MANUSCRIPT
dd, J1 = 1.5 Hz, J2 = 9 Hz), 8.56 (1H, s)
13C NMR : 14.44, 45.46, 53.77, 56.75, 61.06, 63.40, 66.99, 110.20, 118.30, 122.04, 122.28, 123.05, 129.38, 130.56, 132.03,
135.97, 142.57, 153.03, 167.65
5j
5k
5l
1H NMR : 1.45 (3H, t, J = 7.2 Hz), 2.36 (4H, t, J = 4.8 Hz), 2.77 (2H, t, J = 6.9 Hz), 3.57 (4H, t, J = 4.8 Hz), 4.40-4.50 (4H, m),
7.03 (2H, d, J = 9.6 Hz), 7.40-7.50 (3H, m), 8.10-8.20 (2H, m), 8.60 (1H, s), 8.77 (2H, s)
13C NMR : 14.50, 42.71, 54.08, 57.66, 61.05, 67.09, 111.13, 120.52, 122.24, 124.57, 125.63, 129.10, 132.05, 132.28, 133.40,
149.86, 153.86, 162.97, 167.85
1H NMR : 1.44 (3H, t, J = 6.9 Hz), 2.36 (4H, t, J = 4.8 Hz), 2.74 (2H, t, J = 6.6 Hz), 3.59 (4H, t, J = 4.8 Hz), 3.91 (3H, s), 4.30-
4.50 (4H, m), 7.06 (2H, d, J = 9 Hz), 7.45 (1H, d, J = 9 Hz), 7.76 (2H, d, J = 9 Hz), 8.04 (1H, dd, J1 = 1.5 Hz, J2 = 9 Hz), 8.54
(1H, s)
13C NMR : 14.59, 42.61, 53.68, 57.47, 59.20, 61.67, 66.89, 108.15, 109.29, 120.12, 121.80, 150.04, 151.61, 167.68
1H NMR : 1.45 (3H, t, J = 7.2 Hz), 2.37 (4H, t, J = 4.8 Hz), 2.76 (2H, t, J = 6.9 Hz), 3.60 (4H, t, J = 4.8 Hz), 4.40-4.50 (4H, m),
6.10 (2H, s), 7.30 (1H, d, J = 9 Hz), 7.45-7.55 (3H, m), 8.06 (1H, d, J = 9 Hz), 8.53 (1H, s)
13C NMR : 14.60, 41.43, 54.30, 57.71, 61.05, 66.73, 102.83, 108.10, 108.64, 109.57, 109.85, 121.10, 122.80, 124.42, 125.86,
149.97, 151.45, 162.97, 167.67
5m
1H NMR : 1.44 (3H, t, J = 7.2 Hz), 2.33 (4H, t, J = 4.8 Hz), 2.75 (2H, t, J = 6.9 Hz), 3.55 (4H, t, J = 4.8 Hz), 3.73 (3H, s), 4.40-
4.50 (4H, m), 4.74 (1H, d, J = 6.6 Hz), 5.23 (1H, d, J = 6.6 Hz), 6.82 (2H, d, J = 9 Hz), 6.99 (2H, d, J = 9 Hz), 7.00-7.30 (5H, m),
7.50 (1H, d, J = 9 Hz), 8.10 (1H, dd, J1 = 1.5 Hz, J2 = 9 Hz), 8.58 (1H, s)
13C NMR : 14.53, 42.92, 52.10, 53.89, 56.09, 57.88, 61.00, 61.74, 67.09, 112.35, 114.44, 125.30, 125.70, 128.81, 129.86, 143.95,
153.80, 167.79, 168.93
5n
1H NMR : 1.44 (3H, t, J = 7.2 Hz), 2.68 (4H, t, J = 4.8 Hz), 3.01 (2H, t, J = 4.8 Hz), 3.79 (4H, t, J = 4.8 Hz), 4.46 (2H, q, J = 7.2
12

ACCEPTED MANUSCRIPT
Hz), 4.64 (2H, t, J = 4.8 Hz), 7.50 (1H, d, J = 9 Hz), 7.82 (2H, d, J = 9 Hz), 8.10-8.20 (3H, m), 8.60 (1H, s)
13C NMR : 14.46, 42.78, 53.84, 57.49, 60.48, 66.60, 110.05, 112.51, 124.13, 125.09, 128.73, 129.13, 129.96, 130.17, 134.95,
143.85, 167.81, 169.04
5o
5p
1H NMR : 1.45 (3H, t, J = 7.2 Hz), 2.37 (4H, t, J = 4.8 Hz), 2.73 (2H, t, J = 6.9 Hz), 3.60 (4H, t, J = 4.8 Hz), 4.30-4.50 (4H, m),
6.88 (1H, d, J = 8.4 Hz), 7.47 (2H, d, J = 8.4 Hz), 7.59 (2H, d, J = 8.4 Hz), 8.08 (1H, dd, J1 = 1.5 Hz, J2 = 8.4 Hz), 8.54 (1H, s)
13C NMR : 14.60, 42.62, 53.74, 57.50, 62.00, 67.03, 107.83, 109.87, 110.10, 111.04, 123.05, 126.59, 153.40, 157.70, 167.98
1H NMR : 1.44 (3H, t, J = 6.9 Hz), 2.26 (4H, t, J = 4.8 Hz), 2.68 (2H, t, J = 6.6 Hz), 3.46 (4H, t, J = 4.8 Hz), 4.42 (2H, q, J = 6.9
Hz), 4.54 (2H, t, J = 6.6), 7.66 (2H, d, J = 8.4 Hz), 7.73 (1H, d, J = 8.4 Hz), 7.84 (2H, d, J = 8.4 Hz), 8.07 (1H, dd, J1 = 1.5 Hz, J2
= 8.4 Hz), 8.40 (1H, s)
13C NMR : 14.39, 42.70, 53.80, 57.11, 60.90, 65.69, 109.95, 122.54, 124.67, 125.92, 126.13, 127.45, 137.97, 142.72, 154.53,
167.50
5q
1H NMR : 1.43 (3H, t, J = 7.2 Hz), 2.38 (4H, t, J = 4.8 Hz), 2.75 (2H, t, J = 6.9 Hz), 3.60 (4H, t, J = 4.8 Hz), 4.40-4.50 (4H, m),
6.66 (1H, s), 6.85 (1H, dd, J1= 1.8 Hz, J2 = 8.1 Hz), 7.44 (1H, d, J = 8.1 Hz), 7.50 (1H, d, J = 8.1 Hz), 8.16 (1H, dd, J1 = 1.8 Hz,
J2 = 8.1 Hz), 8.57 (1H, s)
13C NMR : 14.57, 42.60, 53.74, 57.45, 61.78, 67.62, 106.85, 108.95, 111.10, 112.83, 118.92, 120.89, 122.40, 125.33, 130.37,
153.60, 158.74, 168.00
6a
6b
1H NMR : 1.43 (3H, t, J = 7.2 Hz), 2.10-2.30 (4H, m), 2.55 (2H, t, J = 5.7 Hz), 3.40 (2H, t, J = 4.8 Hz), 3.50 (2H, t, J = 5.7 Hz),
4.30-4.50 (4H, m), 6.90-7.10 (5H, m), 7.48 (1H, d, J = 8.4 Hz), 8.10 (1H, dd, J1 = 1.5 Hz, J2 = 8.4 Hz), 8.55 (1H, s)
13C NMR : 14.78, 18.24, 28.07, 31.15, 40.22, 42.93, 47.28, 61.28, 109.80, 122.78, 124.86, 125.53, 129.29, 129.63, 130.43,
130.59, 138.97, 143.13, 155.51, 167.44, 175.71
1H NMR : 1.45 (3H, t, J = 6.9 Hz), 2.10-2.30 (4H, m), 2.55 (2H, t, J = 5.7 Hz), 2.66 (2H, t, J = 6.6 Hz), 3.40 (2H, t, J = 4.8 Hz),
13

ACCEPTED MANUSCRIPT
3.50 (2H, t, J = 5.7 Hz), 4.46 (2H, q, J = 6.9 Hz), 4.54 (2H, t, J = 6.6), 7.66 (2H, d, J = 8.4 Hz), 7.75 (1H, d, J = 8.4 Hz), 7.90 (2H,
d, J = 8.4 Hz), 8.08 (1H, dd, J1 = 1.5 Hz, J2 = 8.4 Hz), 8.40 (1H, s)
13C NMR : 14.58, 18.17, 28.04, 30.16, 40.23, 41.79, 46.4, 61.03, 108.10, 122.65, 124.78, 125.40, 126.19, 128.77, 129.21, 129.82,
130.56, 139.11, 143.13, 156.25, 167.57, 174.99
6c
1H NMR : 1.45 (3H, t, J = 6.9 Hz), 2.10-2.30 (4H, m), 2.52 (2H, t, J = 5.7 Hz), 3.35 (2H, t, J = 4.8 Hz), 3.46 (2H, t, 5.7 Hz), 4.30-
4.50 (4H, m), 7.52 (1H, d, J = 9 Hz), 8.10-8.20 (3H, m), 8.44 (2H, d, J = 9 Hz), 8.59 (1H, s)
13C NMR : 14.58, 18.23, 28.56, 28.68, 31.01, 38.55, 41.37, 46.79, 61.50, 109.96, 122.50, 125.56, 125.88, 126.18, 129.72, 129.99,
138.68, 142.51, 154.25, 167.03, 175.01
6d
1H NMR : 1.44 (3H, t, J = 7.2 Hz), 2.10-2.30 (4H, m), 2.67 (2H, t, J = 5.7 Hz), 3.30 (2H, t, J = 4.8 Hz), 3.68 (2H, t, 5.7 Hz), 3.97
(3H, s), 4.30-4.50 (4H, m), 7.04 (1H, d, J = 8.1 Hz), 7.25 (1H, dd, J1= 1.8 Hz, J2 = 8.1 Hz), 7.36 (1H, s), 7.46 (1H, d, J = 8.1 Hz),
8.06 (1H, dd, J1 = 1.8 Hz, J2 = 8.1 Hz), 8.60 (1H, s)
13C NMR : 14.70, 18.14, 28.17, 30.93, 40.05, 41.93, 47.22, 59.20, 61.68, 111.29, 116.37, 122.30, 125.73, 126.50, 131.76, 148.97,
150.05, 152.10, 167.50, 175.03
6e
6f
1H NMR : 1.44 (3H, t, J = 7.2 Hz), 2.10-2.30 (4H, m), 2.66 (2H, t, J = 5.7 Hz), 3.33 (2H, t, J = 4.8 Hz), 3.56 (2H, t, 5.7 Hz), 4.40-
4.50 (4H, m), 7.60 (1H, d, J = 9 Hz), 7.82 (2H, d, J = 9 Hz), 7.99 (2H, d, J = 9 Hz), 8.12 (1H, dd, J1 = 1.5 Hz, J2 = 9 Hz), 8.57
(1H, s)
13C NMR : 14.71, 18.03, 28.15, 31.00, 40.02, 42.80, 46.17, 61.74, 110.15, 121.18, 122.80, 124.99, 127.88, 129.26, 131.59,
139.06, 143.04, 150.82, 154.74, 167.37, 174.93
1H NMR : 1.44 (3H, t, J = 6.9 Hz), 2.10-2.30 (4H, m), 2.59 (2H, t, J = 5.4 Hz), 2.85 (6H, s), 3.40 (2H, t, J = 4.8 Hz), 3.55 (2H, t,
5.4 Hz), 4.40-4.50 (4H, m), 6.65 (2H, d, J = 8.4 Hz), 7.25 (1H, d, J = 8.4 Hz), 7.50 (2H, d, J = 8.4 Hz), 8.00 (1H, dd, J1 = 1.5 Hz,
J2 = 8.4 Hz), 8.60 (1H, s)
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