Indian Journal of Chemistry
Vol. 56B, November 2017, pp. 1207-1211
Urea/thiourea derivatives of Gly/Pro conjugated 2,3-dichlorophenyl piperazine as
potent anti-inflammatory agents: SAR studies
D M Suyoga Vardhana, H K Kumaraa, M B Sridharab & D Channe Gowda*a
a
Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore 570 006, India
b
Department of Chemistry, Ranichannamma University, Vidya Sangama, Belagavi 591 156, India
E-mail: dchannegowda@yahoo.co.in
Received 8 August 2016; accepted (revised) 4 September 2017
Two series of urea/thiourea derivatives of Gly/Pro conjugated 2,3-dichlorophenyl piperazine have been synthesized and
characterized as novel anti-inflammatory agents. The results demonstrate that the derivatives 1f, 1g, 1j, 1k, 2g, 2j and 2k
show good anti-inflammatory properties with IC50 values of 42 ± 0.32 µM, 30 ± 0.51 µM, 65 ± 0.61 µM, 60 ± 0.52 µM, 60
± 0.22 µM, 57 ± 0.37 µM and 35 ± 0.15 µM respectively. These results reveal that compounds with electron withdrawing
moieties (F and Cl) particularly at para position exhibit excellent anti-inflammatory activity compared to the standards
indomethacin (IC50 = 58 ± 0.37 µM) and ibuprofin (IC50 = 67 ± 0.43 µM).
Keywords: Amino acids, piperazine, conjugation, urea/thiourea, anti-inflammatory, SAR
Pain and inflammation are among the main problems
that significantly influence the lifestyle of millions of
people worldwide. There are various therapies
available for the treatment of pain and inflammation,
but these therapies are not always effective and can
cause adverse effects that, in many cases, limit the
treatment. Therefore, a major challenge remains for
biomedical research: the identification of new
compounds for the treatment of pain and
inflammation able to induce low or no side effects1,2.
Traditional non-steroidal anti-inflammatory drugs,
such as aspirin, operate via inhibition of the COX-1
isoenzyme or via inhibition of both COX-1 and COX2 isoenzymes in drugs such as indomethacin,
naproxen, ibuprofen, and flurbiprofen3. Inhibition of
the COX-2 isoenzyme leads to a reduction in the
production of prostanoids, such as prostaglandins and
thromboxanes,
which
are
responsible
for
inflammatory effects4. On the other hand, the
inhibition of the COX-1 isoenzyme is mainly
responsible for gastrointestinal side effects, including
ulcerations and bleeding5. Therefore, the discovery of
new safer anti-inflammatory drugs represents a
challenging goal for such a research area.
—————
List of Abbreviations: Boc: t-Butoxycarbonyl; EDCI: 1-(3Dimethylaminopropyl)-3-ethyl-carbodiimide.HCl; HOBt: 1Hydroxybenzotriazole; NMM: N-Methyl morpholine; PBS:
Phosphate buffer saline; PZN: 1-(2,3-Dichlorophenyl)piperazine;
TFA: Trifluoroacetic acid.
Indeed, amino acids and their metabolic and
physiological ramifications are among the most
investigated topics in biomedical science. The
synthesis of compounds containing amino acids has
attracted the attention of chemists due to their
interesting biological activities with low toxicity and
ample bioavailability6. Furthermore, it is well known
that a number of nitrogen containing heterocyclic
compounds exhibited a wide variety of biological
activities. Among them, piperazine derivatives have
owed their importance in many medicinal molecules.
Piperazine derivatives were originally used in
veterinary medicines which combat parasitic infections
in poultry, stimulants at low doses, and cause
hallucinations at higher level doses7. In addition to the
nitrogen functionalities such as urea and thiourea
which have been found in broad spectrum of biological
8
9
activities like antidiabetic , anti-inflammatory ,
10
11,12
antiviral , antibacterial, antifungal, herbicidal
,
antituberculosis13-16, and antidepressant activity on
central nervous system17 as well as purification agents
for the effluent of organic and inorganic molecules in
18
industrial, agricultural, and mining industries .
Recently, some of urea-based compounds act as kinase
inhibitors and as novel therapeutics in cancer
19
treatment due to their unique binding mode and
kinase inhibition profile. Having more importance in
pharmacological applications of urea and thiourea
derivatives, there is a possibility to design more
1208
INDIAN J. CHEM., SEC B, NOVEMBER 2017
efficient urea and thiourea derivatives as antiinflammatory agents.
Led by the previous facts and coupled with our
ongoing project aimed at investigating new bioactive
molecules derived from amino acids-heterocyclic
conjugates20-25, the present work involves the synthesis
of two series of Gly/Pro conjugated 2,3-dichlorophenyl
piperazine derived urea and thiourea compounds and
screening for their in vitro anti-inflammatory activity.
Results and Discussion
The synthetic route for the proposed compounds
viz., urea and thiourea derivatives of the conjugates of
Gly and Pro is outlined in Scheme I. The physical and
spectroscopic data of all the synthesized compounds
are in close agreement with our earlier work20. In our
previous investigations22, it was noticed that the
presence of electron withdrawing substituents enhances
the activity compared to electron donating groups22.
This formed the basis for the present work wherein, we
have chosen only electron withdrawing groups.
All the synthesized compounds were evaluated for
their in vitro anti-inflammatory activity on human
erythrocyte and the results are presented in Table I.
Compounds 1f, 1g, 2j and 2k with an IC50 values of
42 ± 1.11 µg/mL, 30 ± 0.85 µg/mL, 57 ± 1.05 µg/mL
and 35 ± 0.92 µg/mL respectively have shown
excellent anti-inflammatory activity as compared to
standards indomethacin 58 ± 0.58 µg/mL and
ibuprofen 67 ± 0.62 µg/mL. Compounds 1j, 1k, 1o
and 2g with an IC50 value 65 ± 0.98, 60 ± 0.88, 65 ±
0.68 and 60 ± 1.25 respectively are equipotent with
standards. Compounds 1a, 1b, 2a and 2b without any
substitution on phenyl ring have shown poor or
moderate activity. On this basis, the SAR study
revealed that, compounds with electron withdrawing
groups (F, Cl and Br) on aromatic phenyl ring of
ureas and thioureas show higher activity than the
compounds with no substitution. Another interesting
feature observed based on the activity profile is that
halogen substituents particularly at para position are
found to be most active than the compounds with
ortho and meta substituents. In general, the SAR
study divulged that halogen containing urea and
thiourea compounds preferably halogens at para
substitution show higher anti-inflammatory activity
than the unsubstituted compounds.
Experimental Section
Amino acids used were of L-configuration unless
otherwise mentioned. TFA was purchased from
Advanced Chem. Tech. (Louisville, KY, USA).
Reagents and Conditions: (a) EDCI, HOBt, NMM, 0°C, overnight at RT; (b) TFA, 45 min; (c) R-C6H4-N=C=Z, NMM.
Scheme I — Synthesis of uriedo/thiouriedo derivatives of Gly/Pro conjugated 2,3-dichlorophenyl piperazine
VARDHAN et al.: POTENT ANTI-INFLAMMATORY AGENTS
1209
Table I — Anti-inflammatory activities of synthesized urea and thiourea derivatives
Z
NH
Cl
Xaa
N
Cl
N
R
Where Xaa = Gly (1a-o) and Pro (2a-o)
Entry
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
1m
1n
1o
a
R
H
H
2F
2F
3F
4F
4F
3Cl
3Cl
4Cl
4Cl
2Br
3Br
3Br
4Br
Indomethacin
Z
O
S
O
S
O
O
S
O
S
O
S
S
O
S
O
Anti-inflammatory activity IC50 (µg/mL)a
R
> 300
H
2a
225 ± 1.85
H
2b
125 ±1.25
2F
2c
75 ± 0.98
2F
2d
215 ± 1.61
3F
2e
42 ± 1.11
4F
2f
30 ± 0.85
4F
2g
220 ± 1.10
3Cl
2h
215 ± 1.29
3Cl
2i
65 ± 0.98
4Cl
2j
60 ± 0.88
4Cl
2k
225 ± 1.32
2Br
2l
285 ± 1.41
3Br
2m
185 ± 1.15
3Br
2n
65 ± 0.68
4Br
2o
58 ± 0.58
Ibuprofen
Z
O
S
O
S
O
O
S
O
S
O
S
S
O
S
O
> 300
300 ± 2.00
255 ± 1.72
145 ± 1.21
140 ± 1.01
100 ± 0.98
60 ± 1.25
146 ± 1.54
120 ± 0.93
57 ± 1.05
35 ± 0.92
200 ± 1.12
225 ± 1.34
160 ± 0.85
80 ± 0.93
67 ± 0.62
Values are mean of three determinations, the range of which are < 5% of mean in all cases
NMM and phenyl isocyanates/isothiocyanates were
purchased from Sigma Chemical Co. (St. Louis, MO).
Melting points were determined on a Superfit melting
point apparatus (India) and are uncorrected. TLC was
performed on pre-coated silica gel plates (Kieselgel 60
F254, E. Merck, Germany) with the solvent system
comprising chloroform/methanol/acetic acid in the ratio
98:2:3 (Rfa) and 95:5:3 (Rfb) and the compounds on TLC
were detected by iodine vapors. Solvent and other
chemicals used were of analytical grade. IR spectra of
the compounds were recorded on Jasco spectrometer
(USA). 1H NMR spectra were obtained on VARIAN
400 MHz instrument (USA) using DMSO-d6 as solvent
and the chemical shifts are reported as parts per million
(δ, ppm) using TMS as an internal standard. Mass
spectra were obtained on Bruker (model HP-1100)
(USA) electrospray mass spectrometer.
Conjugation of Gly/Pro to 1-(2,3-dichlorophenyl)piperazine20
1-(2,3-Dichlorophenyl)piperazine.HCl was synthesized as previously reported26. To Boc-Gly/Pro-OH
(0.002 mol) dissolved in acetonitrile (10 mL/g of
compound) and cooled to 0°C was added NMM (0.21
mL, 0.002 mol). To this EDCI (0.383 g, 0.002 mol)
dissolved in acetonitrile (4 mL) was added and stirred
while maintaining the temperature at 0°C. After
stirring the reaction mixture for 10 min at this
temperature, HOBt (0.306 g, 0.002 mol) in DMF
(3 mL) was added slowly. The reaction mixture was
stirred for an additional 10 min and a pre-cooled
solution of 2,3-dichlorophenyl piperazine. HCl (0.536
g, 0.002 mol) and NMM (0.21 mL, 0.002 mol) in
DMF (5 mL) was added slowly. After 20 min the pH
of the solution was adjusted to 8 by addition of NMM
and the reaction mixture was stirred over night at RT.
Acetonitrile was removed under reduced pressure and
the residual DMF was poured into about 100 mL icecold 90% saturated KHCO3 solution and stirred for 30
min. The precipitate was extracted into chloroform
and washed sequentially with 5% NaHCO3 solution
(3 × 20 mL), water (3 × 20 mL), 0.1N cold HCl
(3 × 20 mL) followed by brine solution. The organic
layer was dried over anhydrous Na2SO4, the solvent
was removed under reduced pressure, triturated with
ether and dried.
1210
INDIAN J. CHEM., SEC B, NOVEMBER 2017
General procedure for the synthesis of ureido and
thioureido derivatives (1a-1o/2a-2o)
Boc-Xaa-PZN (0.150 g) [where Xaa = Gly or Pro]
was stirred with 1.5 mL of TFA for 45 min at RT.
After completion of the reaction (monitored by TLC),
TFA was removed under vacuum, triturated with dry
ether, filtered, washed with ether and dried to obtain
TFA.H-Xaa-PZN.
Further, TFA.H-Xaa-PZN (0.001 mol) was dissolved
in DMF (10 mL/g of compound), cooled to 0°C and
NMM (0.10 mL, 0.001 mol) was added. To this
solution respective substituted phenyl isocyanates and
isothiocyanates (0.0012 mol) was added dropwise
while maintaining the temperature at 0°C. The reaction
mixture was stirred for 8 h slowly warming to RT.
DMF was evaporated under high vacuum and the
residue was poured into about 50 mL ice-cold 90%
saturated KHCO3 solution and stirred for 30 min. The
precipitate was extracted into chloroform and washed
sequentially with 5% NaHCO3 solution (2 × 10 mL),
water (2 × 10 mL), 0.1N citric acid (2 × 10 mL)
followed by brine solution. The organic layer was dried
over anhydrous Na2SO4. The solvent was removed
under reduced pressure, triturated with hexane and
dried under vacuum. The analytic and spectroscopic
data of all the synthesized compounds are in close
agreement with our earlier data published20.
Anti-inflammatory activity
Human erythrocyte suspension
The whole blood was collected from a healthy
volunteer who had not taken any NSAIDS for
2 weeks prior to the experiment and collected in
heparinzed vacutainer. The blood was washed three
times with 0.9% saline and centrifuged
simultaneously for 10 min at 3000 rpm. The packed
cells were washed with 0.9% saline and 40% v/v
suspension made using isotonic phosphate buffer
which was composed of 154 mM NaCl in 10 mM
sodium phosphate buffer at pH 7.4 used as stock
erythrocyte or RBC suspension.
Hypotonic solution-induced haemolysis
The membrane stabilizing activity of the compounds
was assessed according to the reported method27 with
slight modification. The test sample consisted of stock
erythrocyte (RBC) suspension 0.5 mL mixed with 5
mL of hypotonic solution (50 mM NaCl in 10 mM
sodium phosphate buffered saline at pH 7.4)
containing different concentrations of sample (25, 50,
100, 200 and 300 µM/mL). The control consists of 0.5
mL RBC suspension mixed with 5 mL of hypotonic
buffered solution alone. The standard drugs
indomethacin and ibuprofen were treated similar to
test concentration. The experiment was carried out in
triplicate. The mixtures were incubated for 10 min at
RT, centrifuged for 10 min at 3000 rpm and
absorbance of the supernatant was measured
spectrophotometrically at 540 nm. The percentage
inhibition of haemolysis or membrane stabilization
was calculated from the following equation.
 A -A 
% Inhibition of haemolysis =  1 2  × 100
 A1 
Where:
A1 = Absorbance of hypotonic buffered solution alone
A2 = Absorbance of test /standard sample in hypotonic
solution
Conclusion
In summary, novel anti-inflammatory agents were
synthesized by conjugating 2,3-dichlorophenyl
piperazine with glycine and proline and converting
them into ureido and thioureido derivatives. The
results indicated that halogen substituents on urea and
thiourea moieties are essential to exhibit good antiinflammatory properties. Further, it was noticed that
compounds containing halogen at para substitutions
on the phenyl ring showed excellent activity
compared to compounds with ortho and meta
substituted compounds. This confirms that variation
of the position of the substituents also affected the
anti-inflammatory activity profile.
Supplementary Information
Supplementary information is available in the website
http://nopr.niscair.res.in/handle/123456789/60.
Acknowledgements
The authors gratefully acknowledge University
Grants Commission (UGC), New Delhi for awarding
UGC-Post Doctoral Fellowship (PDFSS), BSR
faculty fellowship and DST Inspire fellowship.
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