Evaluation of in vivo wound-healing potential of 2-[4-(2,4-dimethoxy-benzoyl)-phenoxy]-1-[4-(3-piperidin-4-yl-propyl)-piperidin-1-yl]-ethanone derivatives

Article abstract of DOI:10.1016/j.ejmech.2009.03.012

Series of 2-[4-(2,4-dimethoxy-benzoyl)-phenoxy]-1-[4-(3-piperidin-4-yl-propyl)-piperidin-1-yl]-ethanone derivatives 9(a-d) and 10(a-d) were synthesized in good yield. The synthesized compounds were characterized by ¹H NMR, LC-MS, FTIR and eleme

Full text of DOI:10.1016/j.ejmech.2009.03.012

European Journal of Medicinal Chemistry 44 (2009) 3158–3165
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Evaluation of in vivo wound-healing potential of 2-[4-(2,4-dimethoxy-benzoyl)-
phenoxy]-1-[4-(3-piperidin-4-yl-propyl)-piperidin-1-yl]-ethanone derivatives
K. Vinaya a, H. Raja Naika b, C.S. Ananda Kumar a, S.B. Benaka Prasad a, S. Chandrappa a,
,
S.R. Ranganatha a, V. Krishna b, K.S. Rangappa a
*
a Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore 570 006, India
b Phytochemistry and Pharmacology Laboratory, Post Graduate Department of Studies and Research in Biotechnology and Bioinformatics, School of Biological Sciences, Kuvempu
University, Shankaraghatta 577 451, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Series of 2-[4-(2,4-dimethoxy-benzoyl)-phenoxy]-1-[4-(3-piperidin-4-yl-propyl)-piperidin-1-yl]-etha-
none derivatives 9(a–d) and 10(a–d) were synthesized in good yield. The synthesized compounds were
characterized by 1H NMR, LC–MS, FTIR and elemental analysis. All the compounds were screened for in
vivo wound-healing activity by incision and dead space wound models on Swiss albino rats. Significant
wound healing was observed in 10b and 10d treated groups as also the epithelialization of the incision
wound was faster with a high rate of wound contraction in these groups. The tensile strength of the
incision wound was significantly increased in 10b and 10d compared to the Nitrofurazone, the standard
skin ointment. In dead space wound model also the weight of the granulation was higher indicating
increase in collagenation. The SAR correlation studies revealed that the thioamide functional linkage and
electron withdrawing groups influence the wound-healing activity.
Received 14 October 2008
Received in revised form
7 March 2009
Accepted 12 March 2009
Available online 24 March 2009
Keywords:
Piperidine
Wound healing
Granulation
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
process of wound healing: inflammation, proliferation, and
remodeling.
Wound infections are common in developing countries because
of poor hygienic conditions. Staphylococcus aureus, Streptococcus
pyogenes, Escherichia coli, Pseudomonas aeroginosa, Streptococcus
pneumoniae, and Klebsiella pneumoniae are some important
organisms causing wound infection [1]. A wide range of antibiotics
is being used at present for treating wound infections, but they are
now proved to have adverse effects on the human body system
beside, these pathogens develop resistance to the antibiotics tar-
geted against them. In view of this, much attention has been paid to
synthesis of biologically active compounds.
Wound healing is a complex multifaceted process that results in
the contraction and closure of the wound and restoration of
a functional barrier [2]. It is a process of repair that follows injury
caused either to the skin or to other soft tissues of the body.
Following injury, an inflammatory response starts and the cells
below the dermis (the deepest skin layer) begin to increased
collagen (connective tissue) production. Later, the epithelial tissue
(the outer skin) gets regenerated [3,4]. There are three stages in the
Interventions that promote the healing of skin wounds have
been in use around millennia. The phrase ‘to lick ones wounds’
might have been grounded in science to show that mammalian
saliva contains some anti-bacterial agents and growth factors that
aid in wound resolution [5]. Given this historical background, it is
surprising that the modern clinical approach to improving the
healed appearance of skin wounds remains largely devoid of drug-
based therapies. There are no licensed therapeutics in clinical use
that have proven consistency in ameliorating excess deposition of
scar tissue – a frequent undesirable resultant of both surgical and
non-surgical injuries caused to the skin.
Heterocyclic compounds carrying piperidine skeleton are attrac-
tive targets of organic synthesis owing to their pharmacological
activities and their wide occurrence in nature [6–8]. Piperidine
heterocycles play an important role in the field of medicinal chem-
istry. Several derivatives of this class such as herbicidal, insecticidal,
fungicidal, bactericidal, anti-inflammatory, antihistaminic, hypoten-
sive, anticancer, CNS stimulant and depressant and nerve activities
have been found to possess useful biological activities [9–16]. The
great importance of benzophenones is fundamentally due to the
diverse biological [17] and chemical [18] properties that they possess.
The proficiency of benzophenone analogues as chemotherapeutic
agent especially as anti-inflammatory is well documented [19].
* Corresponding author. Tel.: þ91 821 2419661; fax: þ91 821 2412191.
E-mail addresses: rangappaks@gmail.com, rangappaks@chemistry.uni-mysore.ac.in
(K.S. Rangappa).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.03.012

K. Vinaya et al. / European Journal of Medicinal Chemistry 44 (2009) 3158–3165
3159
Benzophenone analogues synthesized by several scientists have been
reported as effective antiinflammatory agents [20–22].
proton peak in 4 confirm the formation of the product. [4-(2,4-
Dimethoxy-benzoyl)-phenoxy]-acetic acid (4) (1.0 eq) and 4-(3-
(piperidin-4-yl)propyl)piperidine (5) (1.0 eq) in N,N-dimethyl
formamide (DMF) were taken, to which N-methylmorpholine
(NMP) (3.0 eq) and 10% of o-benzotriazol-1-yl-N,N,N1,N1-tetra-
methyl uranium tetrafluoroborate (TBTU) catalyst were added, and
reaction mixture was stirred for 5 h at room temperature, which
gave target key intermediate 6. The absence of –COOH proton peak
and presence of –NH proton peak confirmed the formation of
compound 6. The nucleophilic substitution reaction of 2-[4-
(2,4-dimethoxy-benzoyl)-phenoxy]-1-[4-(3-piperidin-4-yl-propyl)
-piperidin-1-yl]-ethanone (6) with different substituted aromatic
isocyanates (R–N]C]O)/isothiocyanates (R–N]C]S) was carried
out in the presence of triethylamine and dichloromethane as
solvent with a good yield of 74–80%. The absence of –NH and
presence of –CO–NH, –CS–NH proton peak in synthesized deriva-
tives 9(a–d) and 10(a–d) in proton NMR and IR spectra confirmed
the identity of the products. It is also confirmed by IR data, for
carboxamide series 9(a–d) and thioamide series 10(a–d), IR data
showed stretching frequency at 3350–3360 cmꢁ1 for –NH and
1640–1660 cmꢁ1 for –C]O group. The chemical structures and
yield of all the synthesized compounds are given in Table 1.
An essential component of the search for new leads in a drug
designing program is the synthesis of molecules, which are novel
yet resemble known biologically active molecules by virtue of the
presence of some critical structural features. Certain small hetero-
cyclic molecules act as highly functionalized scaffolds and are
known pharmacophores of a number of biologically active and
medicinally useful molecules [23,24]. Anti-inflammatory drugs are
widely used in the treatment of various inflammatory skin disor-
ders [25,26]. The rate of wound healing is invariably brought down
by these agents [27,28]. In view of the above, we planned to
synthesize a system which combines these two biolabile compo-
nents together to give a compact structure like title compounds and
investigate their wound-healing capacity by incision and dead
space wound models.
2. Chemistry
For the synthesis of the target key intermediate compound 6,
the reaction sequences outlined in Scheme 1 were followed.
(4-Hydroxyphenyl)(2,4-dimethoxyphenyl)methanone (3) was
synthesized by Friedel–Crafts reaction with 1,3-dimethoxy benzene
(1) (1.0 eq) and p-hydroxy benzoic acid (2) (1.2 eq) in the presence
of phosphorus oxychloride (7.0 eq) and zinc chloride (2.5 eq) at 60–
70 ꢀC for 2 h. The absence of –COOH and presence of phenolic
proton peak in 1H NMR and IR spectra confirmed the formation of
compound 3. Treatment of (4-hydroxyphenyl)(2,4-dimethox-
yphenyl) methanone (3) with chloroacetic acid (4.5 eq) in potas-
sium carbonate (7.0 eq) solution, and refluxed for 10 h gave the O-
alkylated product. The absence of Ar–OH and presence of –COOH
3. Experimental
Infrared (IR) spectra were recorded using a Jasco FTIR-4100
spectrometer in the wave number range of 4000–400 cmꢁ1
.
Nuclear magnetic resonance (1H NMR) spectra were recorded on
a Bruker AM 400 MHz spectrometer using DMSO-d6 as solvent and
tetramethylsilane as an internal standard. The chemical shifts are
expressed in
d and the following abbreviations are used: s
(singlet), d (doublet), t (triplet), q (quartet) and m (multiplet).
MeO
MeO
O
MeO
MeO
COOH
(ii)
(i)
O
+
O
82%
90%
MeO
OMe
OH
4
2
3
1
HO
O
COOH
(iii)
HN
+
NH
88%
N
NH
MeO
OMe
O
O
5
6
O
O
R1NCO 7(a-d)
(iv)
N
N
N R1
H
MeO
OMe
O
O
O
9(a-d)
6
S
R2NCS 8(a-d)
(v)
N
N
N R2
H
MeO
OMe
O
O
10(a-d)
Scheme 1. Reaction and reagent conditions: (i) ZnCl2, POCl3, 60–70 ꢀC, 2 h. (ii) ClCH2COOH, K2CO3, reflux, 10 h. (iii) N-Methylmorpholine, o-benzotriazol-1-yl-N,N,N1,N1-tetramethyl
uranium tetrafluoroborate (TBTU), N,N-dimethyl formamide, 5 h. (iv) Isocyanates 7(a–d) (R1NCO), triethylamine, dichloromethane, 5–6 h. (v) Isothiocyanates 8(a–d) (R2NCS),
triethylamine, dichloromethane, 5–6 h. 7a: 4-methoxyphenyl isocyanate; 8a: 4-methoxyphenyl isothiocyanate; 7b: 4-fluorophenyl isocyanate; 8b: 2-chlorophenyl isothiocyanate;
7c: 2-chlorophenyl isocyanate; 8c: 2,4-dichlorophenyl isothiocyanate; 7d: 2,4-dichlorophenyl isocyanate; 8d: 4-fluorophenyl isothiocyanate.

3160
K. Vinaya et al. / European Journal of Medicinal Chemistry 44 (2009) 3158–3165
Table 1
3.2. Procedure for the synthesis of [4-(2,4-dimethoxy-benzoyl)-
phenoxy]-acetic acid 4
Chemical structure and yield of the synthesized compounds 9(a–d) and 10(a–d).
Compound
R1 and R2
Yield (%)
Solutions
of
(4-hydroxyphenyl)(2,4-dimethoxyphenyl)-
OCH3
F
methanone (3) (15 g, 5.81 mmol), and potassium carbonate(56.21 g,
40.67 mmol) were taken in water (125 mL). Then chloroacetic acid
(24.7 g, 26.14 mmol) inportion (1 eq/1 h time gap) was slowlyadded
to the reaction mass and adjusted the pH to 9–10 by adding potas-
sium carbonate solution. The reaction mixture was refluxed for 10 h.
Progressof the reactionwasmonitored byTLC. Uponcompletion, the
reaction mass cooled to the room temperature, diluted with water,
acidifiedusing 3 M HCl solution (pH ¼ 4–5) and extractedtwicewith
ethyl acetate. The organic layer was washed with water and brine
solution until the bottom impurities were removed. The compound
was extracted into the sodium carbonate solution from the organic
layer and the aqueous layer was washed by using ethyl acetate to
remove remaining initial compound. The aqueous layer was acidi-
fied using conc. HCl (pH ¼ 1). The pure compound got precipitated in
aqueous solution. The compound was filter and dried. 1H NMR
9a
78
9b
9c
77
75
Cl
Cl
Cl
9d
80
80
74
(DMSO-d6, 400 MHz)d: 9.75 (s,1H, –COOH)7.92 (s,1H, Ar-H), 7.83 (d,
OCH3
1H, Ar-H), 7.35 (d,1H, Ar-H), 7.00 (d,1H, Ar-H), 6.73 (d,1H, Ar-H), 6.60
(dd, 2H, Ar-H), 5.18 (s, 2H, –OCH2), 3.81(s, 3H, ꢁ OCH3), 3.72(s, 3H, ꢁ
OCH3). IR (KBr, cmꢁ1): 1674, 1045, 1248. Anal. Calcd for C17H16O6 (in
%): C-64.55, H-5.10. Found C-64.50, H-5.04.
10a
10b
Cl
3.3. Procedure for the synthesis of 2-[4-(2,4-dimethoxy-benzoyl)-
phenoxy]-1-[4-(3-piperidin-4-yl-propyl)-piperidin-1-yl]-
ethanone 6
Cl
Cl
F
10c
10d
78
78
A solution of [4-(2,4-dimethoxy-benzoyl)-phenoxy]-acetic acid
(4) (5 g, 1.57 mmol) in dry N,N-dimethyl formamide was taken,
4-(3-(piperidin-4-yl)propyl)piperidine 5 (3.32 g, 1.57 mmol) was
added to the solution, and then N-methylmorpholine (4.78 g,
4.73 mmol) and 10% of the TBTU catalyst were added. The reaction
mixture was stirred at room temperature for 5 h and progress of the
reaction was monitored by TLC. Upon completion of the reaction,
water was added and the reaction mixture was filtered, washed
with ether and dried under vacuum. A pink amorphous solid
compound with 88% yield was obtained. 1H NMR (DMSO-d6,
Mass and purity were recorded on a LC-MSD-Trap-XCT. Elemental
(CHNS) analyses were obtained on Vario EL III Elementar. The
purity of the compounds was checked by thin layer chromatog-
raphy (TLC). Silica gel column chromatography was performed
using Merck 7734 silica gel (60–120 mesh) and Merck made TLC
plates. All the reagents and chemicals were from Sigma Aldrich
Chemicals Pvt Ltd.
400 MHz) d: 8.02 (s, 1H, Ar-H), 7.78 (d, 1H, Ar-H), 7.34 (d, 1H, Ar-H),
6.97 (d, 1H, Ar-H), 6.78 (s, 1H, Ar-H), 6.60 (dd, 2H, Ar-H), 5.31 (s, 2H,
–OCH2), 3.84 (s, 3H, –OCH3), 3.72 (s, 3H, –OCH3), 2.87–3.03 (m, 8H,
–N–CH2–), 2.05 (s, 1H, –NH), 2.02 (br s, 2H, –CH2–), 1.22–1.32 (m,
14H, –CH2–). IR (KBr, cmꢁ1): 1687, 1040, 1249. Anal. Calcd for
C30H40N2O5 (in %): C-70.84, H-7.93, N-5.51. Found C-70.79, H-7.89,
N-5.46.
3.1. Procedure for the synthesis of (4-hydroxyphenyl)
(2,4-dimethoxyphenyl)methanone 3
A solution of p-hydroxy benzoic acid (2) (23.99 g, 17.39 mmol)
was taken, phosphorus oxychloride (92.9 mL, 101.43 mmol) was
added slowly under stirring condition. Then dimethoxybenzene (1)
(20 g, 14.49 mmol) was added, and finally zinc chloride was added
(48.96 g, 36.22 mmol). The reaction mixture was heated at 60–
70 ꢀC for 2 h. Progress of the reaction was monitored by TLC. Upon
completion, the reaction mass was poured slowly into the ice cold
water with stirring. The compound was extracted with ethyl
acetate, the organic layer was washed with water and brine solu-
tion. Evaporation of the organic layer was carried out under
reduced pressure followed by recrystallisation by using methanol
and water; pure compound with 90% yield was obtained. 1H NMR
3.4. General procedure for synthesis of 2-[4-(2,4-dimethoxy-
benzoyl)-phenoxy]-1-[4-(3-piperidin-4-yl-propyl)-piperidin-1-yl]-
ethanone derivatives 9(a–d) and 10(a–d)
A solution of 2-[4-(2,4-dimethoxy-benzoyl)-phenoxy]-1-[4-(3-
piperidin-4-yl-propyl)-piperidin-1-yl]-ethanone (6) (1.0 eq) in dry
dichloromethane was taken and cooled to 0–5 ꢀC in an ice bath.
Triethylamine (3.0 eq) was added to this cold reaction mixture and
stirred for 10 min, and then different isocyanates (1.0 eq) or iso-
thiocyanates (1.0 eq) were added, and allowed to stir at room
temperature for 5–6 h. Progress of the reaction mixture was
monitored by TLC. Upon completion, the solvent was removed
under reduced pressure and residue was taken in water and
extracted with ethyl acetate. The organic layer was washed with
10% ammonium chloride solution and finally water wash was given
to organic layer and dried with anhydrous sodium sulphate, the
solvent was evaporated to get crude product which was purified by
(DMSO-d6, 400 MHz) d: 8.18 (s, 1H, –OH) 7.97 (s, 1H, Ar-H), 7.80 (d,
1H, Ar-H), 7.36 (d, 1H, Ar-H), 7.02 (d, 1H, Ar-H), 6.78 (d, 1H, Ar-H),
3.81(s,3H, ꢁ OCH3), 3.70(s, 3H, ꢁ OCH3) 6.62 (dd, 2H, Ar-H). IR (KBr,
cmꢁ1): 1669, 1042, 1258. Anal. Calcd for C15H14O4 (in %): C-69.76, H-
5.46. Found C-69.69, H-5.41.

K. Vinaya et al. / European Journal of Medicinal Chemistry 44 (2009) 3158–3165
3161
column chromatography over silica gel (60–120 mesh) using hex-
ane:ethyl acetate (8:2) as eluent.
723. MS (ESI) m/z: 697.25 (M þ Hþ). Anal. Calcd for C37H43Cl2N3O6
(in %): C-63.79, H-6.22, N-6.03. Found C-63.84, H-6.18, N-6.09.
3.4.1. Synthesis of 4-[3-[1-[2-[4-(2,4-dimethoxy-
benzoyl)phenoxy]acetyl]-4-piperidyl] propyl]-N-(4-
methoxyphenyl)piperidine-1-carboxamide 9a
3.4.5. Synthesis of 4-[3-[1-[2-[4-(2,4-dimethoxy-
benzoyl)phenoxy]acetyl]-4-piperidyl] propyl]-N-(4-
methoxyphenyl)piperidine-1-carbothioamide 10a
The product obtained was pale yellow oily from 2-[4-
(2,4-dimethoxy-benzoyl)-phenoxy]-1-[4-(3-piperidin-4-yl-propyl)
-piperidin-1-yl]-ethanone (6) (0.25 g, 0.491 mmol) and 4-
methoxyphenyl isocyanate (7a) (0.328 g, 2.27 mmol) and triethyl-
The product obtained was pale yellow oily from 2-[4-
(2,4-dimethoxy-benzoyl)-phenoxy]-1-[4-(3-piperidin-4-yl-propyl)
-piperidin-1-yl]-ethanone (6) (0.25 g, 0.491 mmol), 4-methox-
yphenyl isothiocyanate (8a) (0.292 g, 1.77 mmol) and triethylamine
amine (0.667 g, 6.81 mmol). 1H NMR (DMSO-d6, 400 MHz)
d: 9.23
(0.667 g, 6.81 mmol). 1H NMR (DMSO-d6, 400 MHz)
d: 9.25 (s, 1H,
(s, 1H, –NH), 7.81 (d, 2H, Ar-H), 7.33 (m, 2H, Ar-H), 6.95 (m, 4H, Ar-
H), 6.84 (d, 1H, Ar-H), 6.59 (d, 2H, Ar-H), 5.29 (s, 2H, –OCH2), 3.89 (s,
3H, –OCH3), 3.83 (s, 3H, –OCH3), 3.73 (s, 3H, –OCH3), 2.85–2.98 (m,
8H), 2.05 (br s, 2H), 1.09–1.28 (m, 14H). IR (KBr, cmꢁ1): 3356, 2889,
1716, 1648, 1293, 1250, 1124, 1105. MS (ESI) m/z: 658.34 (M þ Hþ).
Anal. Calcd for C38H47N3O7 (in %): C-69.38, H-7.20, N-6.39. Found C-
69.44, H-7.26, N-6.36.
–NH), 7.79 (d, 2H, Ar-H), 7.35 (m, 2H, Ar-H), 6.96 (m, 4H, Ar-H), 6.82
(d, 1H, Ar-H), 6.56 (d, 2H, Ar-H), 5.25 (s, 2H, –OCH2), 3.85 (s, 3H,
–OCH3), 3.80 (s, 3H, –OCH3), 3.72 (s, 3H, –OCH3), 2.02 (br s, 8H),
1.58–1.75 (br s, 4H), 1.09–1.22 (m, 12H), 1.0 (m, 4H). IR (KBr, cmꢁ1):
2939, 2881, 1730, 1643, 1274, 1245, 1128, 1117, 1041. MS (ESI) m/z:
674.32 (M þ Hþ). Anal. Calcd for C38H47N3O6S (in %): C-67.73, H-
7.03, N-6.24. Found C-67.80, H-6.98, N-6.28.
3.4.2. Synthesis of 4-[3-[1-[2-[4-(2,4-dimethoxy-
benzoyl)phenoxy]acetyl]-4-piperidyl] propyl]-N-(4-
fluorophenyl)piperidine-1-carboxamide 9b
3.4.6. Synthesis of 4-[3-[1-[2-[4-(2,4-dimethoxy-
benzoyl)phenoxy]acetyl]-4-piperidyl] propyl]-N-(2-
chlorophenyl)piperidine-1-carbothioamide 10b
The product obtained was pale yellow oily from 2-[4-
(2,4-dimethoxy-benzoyl)-phenoxy]-1-[4-(3-piperidin-4-yl-propyl)
-piperidin-1-yl]-ethanone (6) (0.25 g, 0.491 mmol) and 4-fluo-
rophenyl isocyanate (7b) (0.301 g, 2.27 mmol) and triethylamine
The product obtained was pale yellow oily from 2-[4-
(2,4-dimethoxy-benzoyl)-phenoxy]-1-[4-(3-piperidin-4-yl-propyl)
-piperidin-1-yl]-ethanone (6) (0.25 g, 0.491 mmol), 2-chlorophenyl
isothiocyanate (8b) (0.30 g, 1.77 mmol) and triethylamine (0.667 g,
(0.667 g, 6.81 mmol). 1H NMR (DMSO-d6, 400 MHz)
d: 9.19 (s, 1H,
6.81 mmol). 1H NMR (DMSO-d6, 400 MHz)
d: 9.19 (s, 1H, –NH), 7.81
–CONH), 7.79 (d, 2H, Ar-H), 7.27–7.33 (m, 2H, Ar-H), 6.94 (m, 4H, Ar-
H), 6.86 (d, 1H, Ar-H), 6.54 (d, 2H, Ar-H), 5.25 (s, 2H, –OCH2), 3.87 (s,
3H, –OCH3), 3.72 (s, 3H, –OCH3), 2.89–2.98 (m, 8H), 2.81 (br s, 2H),
1.74 (m, 2H), 1.09–1.26 (m, 12H). IR (KBr, cmꢁ1): 3346, 2885, 1710,
1639, 1287, 1252, 1121, 1039. MS (ESI) m/z: 646.32 (M þ Hþ). Anal.
Calcd for C37H44FN3O6 (in %): C-68.82, H-6.87, N-6.71. Found C-
68.88, H-6.82, N-6.73.
(d, 2H, Ar-H), 7.33 (m, 2H, Ar-H), 7.21 (m, 4H, Ar-H), 6.91 (d, 1H, Ar-
H), 6.60 (d, 2H, Ar-H), 5.29 (s, 2H, –OCH2), 3.83 (s, 3H, –OCH3), 3.70
(s, 3H, –OCH3), 2.85–2.98 (m, 8H), 2.05 (br s, 2H), 1.12–1.2 (m, 14H).
IR (KBr, cmꢁ1): 2952, 2876, 1741, 1648, 1270, 1241, 1129, 1097, 722.
MS (ESI) m/z: 678.27 (M þ Hþ). Anal. Calcd for C37H44ClN3O5S (in
%): C-65.52, H-6.54, N-6.20. Found C-65.58, H-6.59, N-6.26.
3.4.7. Synthesis of 4-[3-[1-[2-[4-(2,4-dimethoxy-
benzoyl)phenoxy]acetyl]-4-piperidyl] propyl]-N-(2,4-
dichlorophenyl)piperidine-1-carbothioamide 10c
3.4.3. Synthesis of 4-[3-[1-[2-[4-(2,4-dimethoxy-
benzoyl)phenoxy]acetyl]-4-piperidyl] propyl]-N-(2-
chlorophenyl)piperidine-1-carboxamide 9c
The product obtained was pale yellow oily from 2-[4-
(2,4-dimethoxy-benzoyl)-phenoxy]-1-[4-(3-piperidin-4-yl-propyl)
-piperidin-1-yl]-ethanone (6) (0.25 g, 0.491 mmol), 2,4-dichlor-
ophenyl isothiocyanate (8c) (0.361 g, 1.77 mmol) and triethylamine
The product obtained was pale yellow oily from 2-[4-
(2,4-dimethoxy-benzoyl)-phenoxy]-1-[4-(3-piperidin-4-yl-propyl)
-piperidin-1-yl]-ethanone (6) (0.25 g, 0.491 mmol) and 2-chlor-
ophenyl isocyanate (7c) (0.337 g, 2.27 mmol) and triethylamine
(0.667 g, 6.81 mmol). 1H NMR (DMSO-d6, 400 MHz)
d: 9.17 (s, 1H,
(0.667 g, 6.81 mmol). 1H NMR (DMSO-d6, 400 MHz)
d: 9.20 (s, 1H,
–NH), 7.75 (d, 1H, Ar-H), 7.35 (d, 1H, Ar-H), 7.25 (d, 2H, Ar-H), 7.05
(m, 2H, Ar-H), 6.95 (d, 1H, Ar-H), 6.70 (d, 2H, Ar-H), 6.54 (br s, 1H,
Ar-H), 5.34 (s, 2H, –OCH2), 3.82 (s, 3H, –OCH3), 3.73 (s, 3H, –OCH3),
2.85–2.98 (m, 8H), 2.05 (br s, 2H), 1.09–1.22 (m, 14H), 1.0 (m, 4H). IR
(KBr, cmꢁ1): 2959, 2880, 1735, 1653, 1276, 1240, 1124, 1058, 727. MS
(ESI) m/z: 713.23 (M þ Hþ). Anal. Calcd for C37H43Cl2N3O5S (in %): C-
62.35, H-6.08, N-5.90. Found C-62.41, H-6.13, N-5.96.
–NH), 7.80 (d, 2H, Ar-H), 7.35 (m, 2H, Ar-H), 7.12 (m, 4H, Ar-H), 6.89
(d, 1H, Ar-H), 6.62 (d, 2H, Ar-H), 5.27 (s, 2H, –OCH2), 3.84 (s, 3H,
–OCH3), 3.72 (s, 3H, –OCH3), 2.83–3.0 (m, 8H), 2.03 (br s, 2H), 1.12–
1.28 (m, 14H). IR (KBr, cmꢁ1): 3361, 2920, 2862, 1728, 1674, 1278,
1256, 1123, 1045, 725. MS (ESI) m/z: 662.30 (M þ Hþ). Anal. Calcd
for C37H44ClN3O6 (in %): C-67.11, H-6.70, N-6.35. Found C-67.16, H-
6.77, N-6.41.
3.4.8. Synthesis of 4-[3-[1-[2-[4-(2,4-dimethoxy-
benzoyl)phenoxy]acetyl]-4-piperidyl] propyl]-N-(4-
3.4.4. Synthesis of 4-[3-[1-[2-[4-(2,4-dimethoxy-
fluorophenyl)piperidine-1-carbothioamide 10d
benzoyl)phenoxy]acetyl]-4-piperidyl] propyl]-N-(2,4-
The product obtained was pale yellow oily from 2-[4-
(2,4-dimethoxy-benzoyl)-phenoxy]-1-[4-(3-piperidin-4-yl-propyl)
-piperidin-1-yl]-ethanone (6) (0.25 g, 0.491 mmol), 4-fluorophenyl
isothiocyanate (8d) (0.271 g, 1.77 mmol) and triethylamine
dichlorophenyl)piperidine-1-carboxamide 9d
The product obtained was pale yellow oily from 2-[4-
(2,4-dimethoxy-benzoyl)-phenoxy]-1-[4-(3-piperidin-4-yl-propyl)
-piperidin-1-yl]-ethanone (6) (0.25 g, 0.491 mmol) and 2,4-
dichlorophenyl isocyanate (7d) (0.413 g, 2.27 mmol) and triethyl-
(0.667 g, 6.81 mmol). 1H NMR (DMSO-d6, 400 MHz)
d: 9.23 (s, 1H,
amine (0.667 g, 6.81 mmol). 1H NMR (DMSO-d6, 400 MHz)
d: 9.22
–NH), 7.80 (d, 2H, Ar-H), 7.28 (m, 2H, Ar-H), 6.96 (m, 4H, Ar-H), 6.53
(d, 2H, Ar-H), 5.27 (s, 2H, –OCH2), 3.92 (s, 3H, –OCH3), 3.83 (s, 3H,
–OCH3), 2.85–2.92 (m, 8H), 2.08 (br s, 2H), 1.09–1.19 (m, 14H), 1.0
(m, 4H). IR (KBr, cmꢁ1): 2892, 1717, 1632, 1280, 1252, 1125, 1041. MS
(ESI) m/z: 662.30 (M þ Hþ). Anal. Calcd for C37H44FN3O5S (in %): C-
67.15, H-6.70, N-6.35. Found C-67.21, H-6.65, N-6.36.
(s, 1H, –NH), 7.76 (d, 1H, Ar-H), 7.33 (d, 1H, Ar-H), 7.25 (d, 2H, Ar-H),
7.02 (m, 2H, Ar-H), 6.94 (d, 1H, Ar-H), 6.68 (d, 2H, Ar-H), 6.51 (br s,
1H, Ar-H), 5.31 (s, 2H, –OCH2), 3.84 (s, 3H, –OCH3), 3.73 (s, 3H,
–OCH3), 2.87–3.02 (m, 8H), 2.02 (br s, 2H), 1.12–1.22 (m, 14H). IR
(KBr, cmꢁ1): 3356, 2935, 2874, 1731, 1640, 1270, 1248, 1120, 1041,

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K. Vinaya et al. / European Journal of Medicinal Chemistry 44 (2009) 3158–3165
3.5. Pharmacological activity
with interrupted sutures of 1 cm apart using a surgical thread (No.
000) and curved needle (No. 11). The wounds were left undressed
and drugs were topically applied to the wound once a day, till the
completion of healing. The skin breaking strength of the 10-day-old
wound was measured by continuous constant water technique of
Lee and Tong [32]. The skin breaking strength is expressed as the
minimum weight (in g) of water necessary to bring about the
gapping of the wound.
3.5.1. Drug formulations
Two types of drug formulations were prepared from synthe-
sized compounds. For topical application, 1% w/w ointment with
cream base from all test samples was prepared by fusion method
(melting ingredients method) as described by Bharath [29]. For
each 1000 g of ointment cream base, 20 g white bees wax, 30 g
hard paraffin, 50 g cetyl alcohol were added and placed in an
evaporating dish. White soft paraffin (900 g) on a butter paper
transferred into the dish. The dish was placed on water bath, by
lowering the volume in dish as far as possible on the water bath,
stirred well for melting the ingredients. The dish was removed from
the water bath, the content was decanted and strained into another
hot dish to remove foreign matter and stirred until the ointment
cooled and it was allowed to settle. For oral administration,
suspensions were prepared by dissolving 4 mg/mL of the synthe-
sized compound incorporate 1% (w/v) of Tween-80, which served
as a suspension medium for the compounds. The drugs were
administered orally through a feeding tube. All formulations were
prepared fresh as and when required.
For dead space wound model the animals were anaesthetized
with light ether anesthesia and the dead space wounds were
created by subcutaneous implantation of sterilized cylindrical grass
piths (2.5 cm ꢂ 0.3 cm), one on either side on the dorsal para-
vertebral surface of the rats. The granulation tissues formed on the
grass piths were removed on the 10th post wounding day and
subjected to breaking strength test and histological study. Ten
percentage of neutral formalin solution was used to fix the gran-
ulation tissues for 24 h and dehydrated with a sequence of ethanol–
xylene series of solutions [33]. The inflicted materials were
embedded with paraffin at 40–60 ꢀC. Microtome sections of 10
mm
thicknesses were taken. The processed sections were stained with
hematoxylin–eosin and observed under microscope.
3.5.4. Statistical analysis
3.5.2. Animals
The results are expressed as mean ꢃ S.E. of four animals in each
group. The data were evaluated by one-way ANOVA followed by
Tukey’s pair-wise comparison test using statistical software SPSS
Inc. (Chicago, USA). The significance at *P < 0.05 and **P < 0.01
levels were compared with that of control.
Male Wistar strain rats of either sex weighing 150–200 g were
procured from the Central animal house, National College of
Pharmacy, Shimoga, Karnataka. They were maintained at standard
housing conditions and fed with commercial diet (Hindustan Lever
Ltd., Bangalore) and watered ad libitum during the experiment. The
Institutional Animal Ethical Committee (Reg. No.144/1999/CPCSEA/
SMG) permitted the study. The staircase method [30] was adopted
for the determination of the acute toxicity. Healthy albino mice of
either sex weighing 20–25 g were used to determine the safer dose.
The drugs were administered orally.
4. Results
The promotion of wound-healing activity is also well gauged by
the tensile strength of the incision and dead space wound model.
Generally wound-healing agents have the property to enhance the
deposition of collagen content, which provides strength to the
tissues and forms cross-linkages between collagen fibers [34].
Significant wound-healing activity was observed in animals treated
with the compounds 9(a–d) and 10(a–d) compared with those
which received the reference standard and control treatments. In
the dead space wound model, the compounds 10b, 10d showed
high skin-breaking strength up to 623.25 ꢃ 3.48 g and
645.69 ꢃ 4.09 g as compared with the control group of animals. A
significant (P < 0.01) increase was observed in the weight of the
granulation tissue in animals treated with the synthesized
compounds. Compounds 9c, 10c showed less skin-breaking
strength of up to 541.82 ꢃ 4.25 g and 619.15 ꢃ 7.78 g respectively.
The other compounds showed poor epithelialization. In the incision
3.5.3. Wound-healing activity
Ten groups of animals containing four each were used for each
of the incision and dead space wound models. The animals of group
I were considered as the control, the animals of group II served as
the reference standard and were treated with 0.2% (w/w) Nitro-
furazone ointment (Aventis Pharma Limited, Pune, India). The
animals of groups III, IV, V, and VI were treated with compounds
9(a–d), and the groups VII, VIII, IX, and X were treated with
compounds 10(a–d), respectively.
In incision wound model [31], 6 cm long paravertebral incisions
were made through full thickness of the skin on either side of the
vertebral column of the rat. Care was taken to see that incision was
at least 1 cm lateral to vertebral column. The wounds were closed
Table 2
Effect of oral administration of compounds 9(a–d) and 10(a–d) on dead space wound model.
Compound
Granulation tissue
Breaking strength (g)
Wet weight (mg/100 g)
Dry weight (mg/100 g)
Control
Nitrofurazone
9a
9b
9c
9d
10a
10b
10c
10d
80.54 ꢃ 2.26
134.79 ꢃ 1.89**
112.48 ꢃ 5.31**
91.31 ꢃ 4.09
12.36 ꢃ 0.43
26.04 ꢃ 2.06**
21.59 ꢃ 3.16*
17.80 ꢃ 1.59*
21.01 ꢃ 3.78
355.42 ꢃ 6.44
623.25 ꢃ 3.48**
414.19 ꢃ 2.33**
451.60 ꢃ 2.87**
541.82 ꢃ 4.25**
361.60 ꢃ 4.32
104.85 ꢃ 2.08**
79.00 ꢃ 1.28
17.81 ꢃ 0.61**
12.36 ꢃ 0.43
26.04 ꢃ 2.06**
21.02 ꢃ 0.70**
26.27 ꢃ 0.43**
80.54 ꢃ 2.26
355.42 ꢃ 6.44
623.25 ꢃ 3.48**
619.15 ꢃ 7.78**
645.69 ꢃ 4.09**
134.79 ꢃ 1.89**
88.02 ꢃ 0.72*
122.94 ꢃ 0.67**
Each value represents mean ꢃ SE of 4 animals.
*P < 0.05 compared to control.
**P < 0.01 compared to control.

K. Vinaya et al. / European Journal of Medicinal Chemistry 44 (2009) 3158–3165
3163
Table 3
Effect of oral administration of compounds 9(a–d) and 10(a–d) on incision wound
model.
Compound
Breaking strength (g)
Control
Nitrofurazone
9a
9b
9c
9d
10a
10b
10c
10d
371.66 ꢃ 10.45
875.00 ꢃ 85.39**
503.89 ꢃ 52.09*
552.06 ꢃ 60.16*
605.49 ꢃ 65.05**
445.12 ꢃ 54.16*
675.00 ꢃ 85.39**
748.38 ꢃ 79.31**
612.50 ꢃ 119.68*
760.88 ꢃ 82.92**
Each value represents mean ꢃ SE of 4 animals.
*P < 0.05 compared to control.
**P < 0.01 compared to control.
wound model, 10b and 10d treated animals demonstrated high
skin-breaking strength up to 748.38 ꢃ 79.31 g and 760.88 ꢃ 82.92 g,
respectively. A significant (P < 0.01) increase was observed in the
weight of the granulation tissue in the animals treated with the
synthesized compounds. Compounds 9c and 10c showed less skin-
breaking strength up to 605.49 ꢃ 65.05 g and 612.50 ꢃ 119.68 g
respectively.
The data presented in Tables 2 and 3 show that the tensile
strength of the incision wound was significantly increased in the
animals treated with compounds 10(a–d) and it was similar to that
observed with the reference drug Nitrofurazone. A moderate gain
in tensile strength was observed in compounds 9(a–d) treated
animals and it was not significant when compared with control
groups. The effect of oral administration of suspensions of
compounds 9(a–d) and compounds 10(a–d) on dead space wound
model was assessed by the weight of granulation tissue and its
tensile strength. Enhanced collagen maturation is indicated by
increased cross-linking of collagen fibers. The increase in weight of
the granulation tissue is indication of the presence of higher
protein content. Among the treated animals the response was best
in compounds 10(a–d) treated animals.
Histological section of the granuloma tissue of control animal
showed incomplete healing with less epithelialization, macro-
phages and lesser collagen formation indicating incomplete healing
of the wound (Fig. 1A). Nitrofurazone applied section of granuloma
tissue showed complete epithelialization and development depo-
sition of collagen fibers (Fig. 1B). The section of the granuloma
tissue treated with compound 9c showed moderate epithelializa-
tion and collagenation and also retention of macrophages with
moderate epithelialization and fibrosis. But the compounds 10b
and 10d treated animals showed complete epithelialization and
increased collagen formation and complete healing with more of
fibroblasts (Figs. 2 and 3).
Fig. 1. (A) Histological section of the granuloma tissue of control animal showing
incomplete healing with less epithelialization arrow showing macrophage and lesser
collagen formation indicated incomplete healing of the wound (H&E, 10ꢂ). (B) Histo-
logical section of granuloma tissue of standard skin ointment Nitrofurazone applied
animal showing complete epithelialization. Arrowhead indicates the deposition of
collagen fibers (H&E, 10ꢂ).
other. The standard drug Nitrofurazone was used as a reference to
assess the healing potency of the proposed drug. The results of the
present study clearly indicated that 10b and 10d enhanced healing
of wounds, while other synthesized compounds failed to show any
effect. The activity may be attributed to the anti-inflammatory
activity of the piperidine [13] and benzophenone [20–22] moieties
and also the presence of electron withdrawing chloro and fluoro
groups on the substituted thioamide moiety.
5. Discussion
The breaking strength is the strength of a healing wound and is
measured experimentally by the amount of force required to
disrupt it. In the beginning, a wound will be having little breaking
strength because the clot will be holding the edges together.
Thereafter, breaking strength increases rapidly as collagen depo-
sition increases and cross linkages are formed between the collagen
fibers. Shirwaikar et al. [36] and Singh et al. [37] in their studies also
showed significant increase in the breaking strength of wounds in
animals treated with synthesized compounds.
Control of inflammation is an important feature of a wound-
healing process, since excessive inflammation impedes wound
healing [35]. Such prolonged, inflammation and its consequent
effect is a major challenge in recalcitrant wounds. The synthesis of
2-[4-(2,4-dimethoxy-benzoyl)-phenoxy]-1-[4-(3-piperidin-4-yl-
propyl)-piperidin-1-yl]-ethanone amide and thioamide derivatives
with different isocyanates/isothiocyanates containing substituted
aromatic groups led to the production of novel derivatives 9(a–d)
and 10(a–d). These set of compounds were tried in our study to
assess the extent of wound-healing capacity on various phases of
wound healing, which run concurrently, but independent of each
The presence of any foreign body in the subcutaneous area
initiates the formation of granulation tissue around the wound,
having the appearance of pink granules protruding from the floor of

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K. Vinaya et al. / European Journal of Medicinal Chemistry 44 (2009) 3158–3165
In the maturation phase, which is the final phase of wound healing
the wound undergoes contraction resulting in a smaller amount of
apparent scar tissue. Granulation tissue formed in the final part of
the proliferative phase is primarily composed of fibroblasts,
collagen, edema, and new small blood vessels. The increase in dry
granulation tissue weight in the test treated animals suggests
higher protein content.
From the results obtained, structure–activity relationship (SAR)
can be drawn for compounds 9(a–d) and 10(a–d). In this connec-
tion, several different electron donating or electron withdrawing
groups attached to phenyl ring as substituents have been studied
for wound-healing efficacy. Upon introduction of electron donating
methoxy group in 9a, 10a of phenyl ring showed poor wound-
healing activity, on the other hand remaining compounds showed
superior wound-healing activity with electron withdrawing chloro
and fluoro groups. Similarly the SAR study for carboxamide deriv-
atives 9(a–d) revealed that, electron withdrawing groups 9b, 9c and
9d attached to phenyl ring as substituents showed superior healing
activity compared to the electron donating group 9a. On the other
hand, compounds 9c and 10b with one electron withdrawing
chloro group at position -2 showed superior activities compared to
9d and 10c having two chloro groups at the -2nd and -4th position.
Finally N-aryl thioamides 10(a–d) substituted derivatives showed
potent wound-healing activity compared to those having N-aryl
amides 9(a–d). The above four SAR correlation studies revealed that
the nature of the functional linkage (amide/thioamide) influences
the wound-healing activity. Recent studies with 2-[4-(2,4-dime-
thoxy-benzoyl)-phenoxy]-1-[4-(3-piperidin-4-yl-propyl)-piper-
Fig. 2. Histological section of the granuloma tissue from 10b treated animal removed
after 10th day of treatment showing complete epithelialization and increased collagen
deposition. Arrows showing increase in collagen formation and complete healing with
more of fibroblasts (H&E, 10ꢂ).
the wound. When microscopically observed these granules show
newly formed capillaries, fibroblasts and leucocytes. As more and
more collagen fibers are formed, vascularizatison tissue decreases.
The breaking strength of the granulation tissue increases pro-
portionately with the collagen deposition. Due to oral administra-
tion of compounds 9(a–d) and 10(a–d), the breaking strength of the
granulation tissue increased. Earlier reports indicate that the
increase in weight of the granulation tissue is due to the higher
content of protein [38].
Wound healing is a complex and dynamic process of restoring
cellular structures and tissue layers in damaged tissue as close as
possible to its normal state. Wound contracture is a process that
occurs throughout the healing process, commencing in the fibro-
blastic stage whereby the area of the wound undergoes shrinkage.
idin-1-yl]-ethanone
6
have shown that substituents like
sulfonamides and carboxamides have potent antimicrobial prop-
erties [39], may also have a role in wound contraction and increased
rate of epithelization.
6. Conclusion
The size and lipophilicity of the substitution on the phenyl
moiety are considered to be key factors in determining the wound-
healing activity. Halogens like chlorine or fluorine are very useful to
modulate the electronic effects on phenyl rings of the synthesized
compounds. Moreover, these atoms may also influence the steric
characteristics and the hydrophilic–hydrophobic balance of the
molecule. On the other hand, ketones, amide and thioamide func-
tional groups have different steric, electronic and lipophilic char-
acteristics. These structural requirements are present in the
synthesized compounds and they are expected to get strong
wound-healing property of the benzophenones containing
a substituted N-trimethylene dipiperidine moiety. Therefore, this
work presents a novel class of potent, wound-healing activities of
the compounds. Among the compounds tested 10b and 10d proved
to be the most effective.
Acknowledgments
One of the authors (K. Vinaya) is grateful to Council of Scientific
and Industrial Research (CSIR), New Delhi for financial support
under CSIR-SRF order No. 09/119(0172)2K8 EMR-I. The CHNS, IR
and other data obtained from the instruments granted under DST-
FIST and UGC-SAP (phase I) No.F.540/10/DRS/2004-05 (SAP-I) pro-
grammes are greatly acknowledged.
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Fig. 3. Histological section of the granuloma tissue from 10d treated animal removed
after 10th day of treatment showing complete epithelialization and increased collagen
deposition. Arrows showing increase in collagen formation and complete healing with
more of fibroblasts (H&E, 10ꢂ).
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