Structure-Based Rationale Design and Synthesis of Aurantiamide Acetate Analogues - Towards a New Class of Potent Analgesic and Anti-inflammatory Agents

Article abstract of DOI:10.1111/j.1747-0285.2012.01331.x

A series of new aurantiamide acetate analogues were synthesized by modifying its N-terminal substitution and the amino acid residue. The structure of all these compounds was established on the basis of analytical and spectral studies. All the new derivatives were evaluated in vivo for their analgesic activity by tail flick method in mice and anti-inflammatory activity against carrageenan-induced oedema in albino rats at different doses (25, 50 and 100mg/kg body weight). All the compounds exhibited significant pharmacological activity with no ulcerogenic liability. In particular, pentapeptides and tricosamers (30 amino acids) containing analogues have demonstrated high potency than the reference standards. These compounds hold promise for further development.

Full text of DOI:10.1111/j.1747-0285.2012.01331.x

ª 2012 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2012.01331.x
Chem Biol Drug Des 2012; 79: 850–862
Research Letter
Structure-Based Rationale Design and Synthesis
of Aurantiamide Acetate Analogues – Towards
a New Class of Potent Analgesic and Anti-
inflammatory Agents
useful in the treatment of inflammatory diseases, but they can also
reduce the risk of Alzheimer's disease (4,5). Although NSAIDs are
the most widely used drugs, their long-term clinical employment is
associated with significant side effects and the steady use deter-
mines the onset of gastrointestinal lesions, bleeding and nephrotox-
icity (6,7). Therefore, the discovery of new safer anti-inflammatory
drugs represents a challenging goal for such a research area.
Ramesh Suhas and Dase Channe Gowda*
Department of Studies in Chemistry, University of Mysore,
Manasagangotri, Mysore 570 006, India
*Corresponding author: D. Channe Gowda,
dchannegowda@yahoo.co.in
A series of new aurantiamide acetate analogues
were synthesized by modifying its N-terminal sub-
stitution and the amino acid residue. The structure
of all these compounds was established on the
basis of analytical and spectral studies. All the
new derivatives were evaluated in vivo for their
analgesic activity by tail flick method in mice and
anti-inflammatory activity against carrageenan-
induced oedema in albino rats at different doses
(25, 50 and 100 mg/kg body weight). All the com-
pounds exhibited significant pharmacological
activity with no ulcerogenic liability. In particular,
pentapeptides and tricosamers (30 amino acids)
containing analogues have demonstrated high
potency than the reference standards. These com-
pounds hold promise for further development.
Aurantiamide acetate (Figure 1), a dipeptide composed of N-ben-
zoylphenylalanine and phenylalaninol acetate, was isolated from
Polygonum chinensis and showed inhibitory effects on Oꢀ2 genera-
tion induced by formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) in
human neutrophils (8). Additionally, its analogue aurantiamide (Fig-
ure 1), a major component of Zanthoxylum dissitum and Aspergillus
penicilloides (9,10), exhibited antibacterial (11), anti-inflammatory
(10), antioxidant (12) and anti-HIV effects.
Several researchers have shown that N-Fmoc amino acids exert
anti-inflammatory activity by recruitment of neutrophils into the
inflammatory site (12–14). In addition, these compounds also
increased intracellular Ca2+ concentrations in Madin–Darby canine
kidney (MDCK) renal tubular cells (15). These agents do not act by
inhibiting lipid metabolic enzymes. They are not steroids, nor do
they increase the circulating levels of endogenous glucocorticoids.
Instead, they blocked recruitment of neutrophils into inflammatory
lesions and, when studied in vitro, inhibited T-cell activation (13).
Key words: analgesics, anti-inflammatory agents, aurantiamide ace-
tate, elastin-based peptides, synthesis
Abbreviations: Ac2O, acetic anhydride; Boc, t-Butoxycarbonyl;
Bpoc, Biphenylisopropyloxycarbonyl; EDCI, 1-(3-Dimethylaminopropyl)-3-
ethyl-carbodiimide.HCl; Fmoc, 9-Fluorenylmethoxycarbonyl; HOBt, 1-Hy-
droxybenzotriazole; Hyp, hydroxyproline; IBCF, isobutyl chloroformate;
NMM, N-methylmorpholine; TFA, trifluoroacetic acid.
Elastin protein-based polymers have their origin in repeating
sequences of the mammalian elastic protein, elastin (16,17). The
most prominent repeating sequence occurs in bovine elastin; it can
be represented as (Val1-Pro2-Gly3-Val4-Gly5)n where n is eleven with-
out a single substitution. Another repeat first found in porcine elas-
tin is (Val1-Pro2-Gly3-Gly4)n, but this repeat has not been found to
Received 2 August 2011, revised 1 December 2011 and accepted for
publication 5 January 2012
Inflammation is a response of the tissue to an infection, irritation
or foreign substance (1) and is a part of the host defence mecha-
nism. Acute and chronic inflammation and different types of arthri-
tis are the inflammatory disorders that are a big blow to humanity,
and continual search for newer non-steroidal anti-inflammatory
agents is the only way to fortify against this awful threat. Non-ste-
roidal anti-inflammatory drugs (NSAIDs) are of huge therapeutic
benefit in the treatment of rheumatoid arthritis, anti-inflammatory,
analgesic and antipyretic activities and are widely used to treat
acute and chronic inflammatory disorders (2,3). NSAIDs are not only
O
NH
NH
CH3
O
OH
HN
HN
O
O
O
O
Aurantiamide
Aurantiamide acetate
Figure 1: Structures of aurantiamide and aurantiamide acetate.
850

Synthesis of Aurantiamide Acetate Analogues
Synthesis
occur with n > 2 without substitution. The next most common recur-
ring sequence in mammalian elastin is a polyhexapeptide (Ala1-
Pro2-Gly3-Val4-Gly5-Val6)n where, with a couple of isomorphous
hydrophobic residue replacements such as Val by Ile or Leu, n is 8
in man (18,19). The monomers, oligomers and high polymers of
these repeats have been synthesized and conformationally charac-
terized (20). These polymers have a number of medical and non-
medical applications (21,22). The peptides used in the present work
are the analogues of elastin sequences.
The peptides were synthesized by classical solution phase method
using t-Butoxycarbonyl (Boc) chemistry. The Boc group used for tem-
porary Na protection was removed with 4 N HCl in dioxane or TFA.
The C terminus carboxyl group was protected by the benzyl ester,
and its removal was effected by hydrogenolysis using HCOONH4 as
hydrogen donor and 10% Pd on carbon as catalyst (28) or hydroly-
sed using 1 N NaOH ⁄ MeOH. All the coupling reactions for peptide
synthesis were achieved with IBCF. The procedure followed for the
synthesis of pentapeptides and tricosamers are outlined in the
Schemes 1 and 2, respectively. The protected peptides were puri-
fied by column chromatography over silica gel and characterized by
physical and analytical techniques. The purity of free peptides was
checked by HPLC and found to have purity >95% in each case. Boc-
Phe-OH was converted to Boc-Phe-CH2OH (9) using IBCF ⁄ NMM ⁄
NaBH4, which was esterified using (acetic anhydride) Ac2O ⁄ pyridine
to obtain 10. Boc group of the ester 10 was deblocked using 4 N
HCl ⁄ dioxane and reacted with various amino acids ⁄ peptides using
EDCI ⁄ HOBt as coupling reagent and NMM as base to obtain the
desired compounds 12–23. The synthetic route is illustrated in
Scheme 3. The Boc group of compound 23 was removed using TFA
to obtain 23a. The benzyl group of compound 16 was hydrogenoly-
sed using HCOONH4 as hydrogen donor and 10% Pd on carbon as
catalyst to obtain 16a.
In view of the aforementioned facts and as part of an ongoing pro-
gramme to develop novel biologically active agents (23–27), herein
we describe the design, synthesis and pharmacological activities of
a series of aurantiamide acetate analogues. As shown in Figure 2,
the points of synthetic modification included (1) the N-terminal sub-
stitution and (2) the amino acid residue.
Experimental Section
Materials and methods
All the amino acids used except glycine were of L-configuration
unless otherwise mentioned. All amino acids, 1-(3-Dimethylamino-
propyl)-3-ethyl-carbodiimide.HCl (EDCI), 1-hydroxybenzotriazole (HOBt)
and trifluoroacetic acid (TFA) were purchased from Advanced Chem.
Tech. (Louisville, KY, USA). Isobutyl chloroformate (IBCF) and N-
methylmorpholine (NMM) were purchased from Sigma Chemical Co.
(St. Louis, MO, USA). All solvents and reagents used for the synthe-
sis were of analytical grade. Silica gel (60–120 mesh) for column
chromatography was purchased from Sisco Research Laboratories
Pvt. Ltd. (Mumbai, India). The progress of the reaction was moni-
tored by TLC using silica gel coated on glass plates with the sol-
vent system comprising chloroform ⁄ methanol ⁄ acetic acid in the
ratio 98:2:3 (Raf ) ⁄ 95:5:3 (Rfb), and the compounds on TLC plates
were detected by iodine vapours. Melting points were determined
on a Thomas Hoover melting point apparatus and are uncorrected.
All the HPLC analyses were performed with Lachrom-2000 Merck-
Hitachi L7100 pump with RP18.250-4 mm column and UV detector
UV-VISL 7400. IR spectra were recorded on Jasco FTIR-4100 Spec-
trometer. 1H NMR spectra were obtained on VARIAN 400 MHz
instrument using CDCl3, and the chemical shifts are reported as
parts per million (d ppm) using TMS as an internal standard. Elec-
trospray ionization mass spectrometry (ESI-MS) was performed on a
Bruker electrospray mass spectrometer.
Synthesis of pentapeptides
The pentapeptides Boc-GZGZŒP-OH [where Z=ZŒ=Val for GVGVP;
Z=ZŒ=Phe for GFGFP; Z=Val and ZŒ=Phe for GVGFP; Z=Glu(OcHx)
and ZŒ=Phe for GE(OcHx)GFP] were synthesized by (3 + 2) coupling
strategy, the tripeptide Boc-Gly-Z-Gly-OH and the dipeptide
HCl.NH2-ZŒ-Pro-OBzl were synthesized separately and coupled to
obtain the pentamer. In this approach, Boc-ZŒP-OBzl was synthe-
sized by the mixed anhydride method in the presence of HOBt,
deblocked to obtain HCl.NH2-ZŒP-OBzl. On the other hand, Boc-Z-
Gly-OBzl was synthesized by the mixed anhydride method,
deblocked and coupled to Boc-Gly-OH to obtain Boc-Gly-Z-Gly-OBzl,
hydrogenolysed using HCOONH4 ⁄ 10% Pd-C or hydrolysed using
1 N NaOH ⁄ MeOH to obtain Boc-Gly-Z-Gly-OH. Now the tripeptide
and dipeptide fragments were coupled by the mixed anhydride
method to obtain Boc-Gly-Z-Gly-ZŒ-Pro-OBzl. These were then trea-
ted with HCOONH4 ⁄ 10% Pd-C or 1 N NaOH ⁄ MeOH to obtain cor-
responding free acid.
Synthesis of tricosamers
Conversion to other
amino acid/peptide
residues
The tricosapeptides Boc-GE(OcHx)GFP GVGVP GVGVP GVGVP GFGFP
GFGFP-OH (3) and Boc-GE(OcHx)GFP GVGVP GVGFP GFGFP GVGVP
GVGFP-OH (4) were synthesized by the [(5 + 5 + 5) + (5 + 5 + 5)]
fragment coupling strategy in solution phase. The pentamers
required for this purpose were synthesized with appropriate side-
chain protection as described earlier. Among the tricosamers 3 and
4, synthesis of 4 has been given below and the synthesis of 3
was followed in a similar way. In this approach, Boc-GVGVP-GVGFP-
OBzl was synthesized using EDCI ⁄ HOBt, deblocked and coupled to
Boc-GFGFP-OH to obtain Boc-GFGFP-GVGVP-GVGFP-OBzl. This frag-
ment was deblocked further to obtain TFA.GFGFP-GVGVP-GVGFP-OBzl.
In an another approach, Boc-GVGVP-GVGFP-OBzl was synthesized
O
NH
CH3
O
Replacement with
Boc, Fmoc, Bpoc or
no substitution
HN
O
O
Figure 2: Design of aurantiamide acetate analogues.
Chem Biol Drug Des 2012; 79: 850–862
851

Suhas and Channe Gowda
Gly
Pro
Gly
Z
Z
Boc
Boc
H
OBzl
OH
iii
OBzl
ii
Boc
Boc
OBzl Boc
OBzl Boc
H
OH
OH
H
OBzl
OBzl
iii
iv
i
ii
Boc
OH
H
OBzl
iii
iv
Boc
Boc
OBzl
OH
i. IBCF/HOBt, NMM
ii. HCl/dioxane
iii. IBCF, NMM
iv. HCOONH4/10% Pd-C or 1N NaOH/MeOH
Scheme 1: Schematic representation of the synthesis of pentapeptides Boc-GZGZŒP-OH where Z=ZŒ=Val for GVGVP, Z=ZŒ=Phe for GFGFP,
Z=Val and ZŒ=Phe for GVGFP, Z=Glu(OcHx) and ZŒ=Phe for GE(OcHx)GFP by (3 + 2) fragment coupling method.
using EDCI ⁄ HOBt, deblocked and coupled to Boc-GE(OcHx)GFP-OH to
obtain Boc-GE(OcHx)GFP-GVGVP-GVGFP-OBzl. This fragment was hy-
drolysed using 1 N NaOH ⁄ MeOH to obtain Boc-GE(OcHx)GFP-GVGVP-
GVGFP-OH. Now the 15mer fragments were coupled using EDCI ⁄ HOBt
to obtain Boc-GE(OcHx)GFP GVGVP GVGFP GFGFP GVGVP GVGFP-OBzl
and hydrolysed using 1 N NaOH ⁄ MeOH to obtain Boc-GE(OcHx)GFP
GVGVP GVGFP GFGFP GVGVP GVGFP-OH.
General procedure for the hydrogenolysis of
benzyl esters of pentapeptides
Each of the peptides 1, 2 (0.006 mol) was hydrogenolysed in metha-
nol (10 mL ⁄ g of peptide) using ammonium formate (2.0 equi.) and
10% Pd-C (0.1 g ⁄ g of peptide) for 30 min at room temperature. The
completion of the reaction was monitored by TLC. The catalyst was
filtered and washed with methanol. The combined washings and
GE(OcHx)GFP
GVGVP
Boc
GVGVP
Boc
GVGFP
OBzl
GFGFP
GVGFP
H OBzl
OH
H
OH
i
i
Boc
OBzl
OBzl
OBzl
Boc
H
OBzl
OBzl
OBzl
OcHx
OH
OcHx
ii
ii
Boc
Boc
Boc
H
OH
i
i
Boc
H
OcHx
OcHx
ii
iii
Boc
Boc
OH
OBzl
OBzl
i
OcHx
iii
Boc
OH
i. EDCI/HOBt, NMM
ii. TFA
iii. 1N NaOH/MeOH
Scheme 2: Schematic representation of the synthesis of tricosapeptides by [(5 + 5 + 5) + (5 + 5 + 5)] fragment coupling method.
852 Chem Biol Drug Des 2012; 79: 850–862

Synthesis of Aurantiamide Acetate Analogues
O
O
C
O
NH C
O
NH
O
NH
O
C
O
a
b
O
OH
OH
CH3
C
9
10
O
c
O
C
.HCl
NH2
d
NH
Xaa NH
Y
+
Y-NH-Xaa-COOH
O
C CH3
O
O
C
O
CH3
11
12-23
Y = Fmoc, Boc, Bpoc; Xaa = Amino acids/peptides
Reagents and conditions: a) IBCF, NMM, -15 °C, NaBH4, over night b) Ac2O/pyridine, 1 hr, rt
c) 4N HCl/1,4-dioxane, 30 min, rt d) EDCI/HOBt, NMM, 0 °C, over night at rt
Compound No.
Y
Xaa
His(Trt)
Tyr(Bzl)
Val
Comp. No.
Y
Xaa
Phe
Fmoc
Fmoc
Fmoc
Bpoc
Boc
Boc
Boc
Boc
Boc
Boc
12
13
14
15
16
18
19
20
21
22
Tyr(Cl2-Bzl)
GFGFP
Val
GVGVP
Hyp(Bzl)
GE(OcHx)GFP GVGVP
GVGVP GVGVP GFGFP
GFGFP
Boc
Trp(N-CHO)
Boc
GE(OcHx)GFP GVGVP
GVGFP GFGFP GVGVP
GVGFP
17
23
Scheme 3: Synthesis of aurantiamide acetate analogues.
filtrate were evaporated, and the residue was taken into CHCl3,
washed with water and dried over anhydrous Na2SO4. The solvent was
removed under pressure and triturated with ether, filtered, washed
with ether and dried to obtain debenzylated peptides (5, 6). {(5) Rbf .
0.45; M.P. 125 [Lit. 127, (29)] (6) Rbf 0.46; M.P. 117 [Lit. 118, (29)]}.
TLC, and solvent was evaporated, cooled, neutralized with cold
1 N HCl, extracted with CHCl3, washed with 1 N HCl followed by
water and dried over anhydrous Na2SO4. The solvent was
removed under pressure and triturated with ether, filtered, washed
with ether and dried to obtain debenzylated peptides (7, 8). {(7)
Rbf . 0.48; M.P. 201–202 [Lit. 200–203, (25)] (8) Rbf 0.46; M.P.
164–166 [Lit. 166–169, (25)]}.
General procedure for the hydrolysis of benzyl
esters of tricosapeptides
Each of the tricosamers 3, 4 (0.006 mol) was hydrolysed in meth-
anol (10 mL ⁄ g of peptide) using cold solution of 1 N NaOH
(30 mL) for 2 h. The completion of the reaction was monitored by
Synthesis of Boc-phenylalaninol (9)
To Boc-Phe-OH (1.327 g, 0.005 mol) dissolved in acetonitrile (15 mL)
and cooled to 0 ꢀC was added NMM (0.55 mL, 0.005 mol). The
Chem Biol Drug Des 2012; 79: 850–862
853

Suhas and Channe Gowda
solution was cooled to )15 € 1 ꢀC, and IBCF (0.65 mL, 0.005 mol)
was added under stirring while maintaining the temperature at
)15 ꢀC. After stirring the reaction mixture for 10 min at this tem-
perature, NaBH4 (0.400 g, 0.01 mol) in DMF (5 mL) was added
slowly. The reaction mixture was stirred over night at room temper-
ature. Acetonitrile and DMF were removed under reduced pressure,
and the residue was poured into water. The compound was taken
into CHCl3 and washed with 5% NaHCO3 solution (2 · 20 mL),
water (2 · 20 mL), 0.1 N cold HCl solution (2 · 20 mL) and finally
brine solution (2 · 20 mL). The CHCl3 layer was dried over anhy-
drous Na2SO4, and the solvent was removed under reduced pres-
sure. The product obtained was white shiny solid that was kept
under vacuum overnight.
Deblocking of Boc group of 10
Compound 10 (0.293 g, 0.001 mol) was deblocked with 4 N
HCl ⁄ 1,4-dioxane (3 mL) for 1.5 h. Excess HCl and dioxane were
removed under reduced pressure, triturated with ether, filtered,
washed with ether and dried under vacuum (yield 100%) to obtain
2-amino-3-phenylpropyl acetate.HCl 11.
General procedure for the coupling of 2-amino-
3-phenylpropyl acetate.HCl 11 with Y-NH-Xaa-
OH where Y = Fmoc, Bpoc, Boc; Xaa = His(trt),
Tyr(Bzl), Val, Hyp, N-formyl Trp, Phe and
peptides 5–8
To Y-NH-Xaa-OH (0.001 mol) and HOBt (0.153 g, 0.001 mol) dis-
solved in DMF (10 mL ⁄ g of compound) and cooled to 0 ꢀC was
added NMM (0.11 mL, 0.001 mol). EDCI (0.191 g, 0.001 mol) was
added under stirring while maintaining the temperature at 0 ꢀC.
The reaction mixture was stirred for 10 min, and a precooled solu-
tion of 2-amino-3-phenylpropyl acetate.HCl (0.293 g, 0.001 mol) and
NMM (0.11 mL, 0.001 mol) in DMF (3 mL) was added slowly. After
20 min, pH of the solution was adjusted to 8 by the addition of
NMM and the reaction mixture was stirred over night at room tem-
perature. DMF was removed under reduced pressure, and the resi-
due was poured into about 100 mL ice-cold 90% saturated KHCO3
solution and stirred for 30 min. The precipitated product was taken
into CHCl3 and washed with 5% NaHCO3 solution (2 · 30 mL),
water (2 · 30 mL), 0.1 N cold HCl solution (2 · 30 mL) and finally
brine solution (2 · 30 mL). The CHCl3 layer was dried over anhy-
drous Na2SO4, and the solvent was removed under reduced pres-
sure. The products (12–23) so obtained were recrystallized from
ether ⁄ petroleum ether.
Yield: 90%; M.P.: 94 ꢀC [Lit. 94–96 ꢀC (30)]; IR (m, cm)1): 1707
1
(C=O), 3541 (-OH); H NMR (DMSO d6, d ppm): Boc = 1.31 (9H, s);
Phe = 2.79–2.84 (2H, d, -bCH2), 3.26–3.60 (2H, t, -CH2OH), 3.65 (1H,
t, -OH), 4.72 (1H, m, -aCH), 7.15–7.28 (5H, m, ArH), 8.60 (1H, d, -
NH); Mass: 251 (M+).
Synthesis of 2-(tert-butoxycarbonylamino)-3-
phenylpropyl acetate (10)
Compound
9 (1.257 g, 0.005 mol) was esterified with Ac2O
(0.75 mL, 0.005 mol) in pyridine (10 mL) and stirred for 1 h at
room temperature. After completion of the reaction monitored by
TLC, solvent was removed under reduced pressure. The residue
was taken into ether and washed with 0.1 N HCl solution
(3 · 20 mL) followed by brine solution (3 · 20 mL). The combined
extracts were dried over anhydrous Na2SO4, and the solvent was
removed under reduced pressure to obtain white solid.
Yield: 92.3%; M.P.: 104 ꢀC; IR (m, cm)1): 1685 (C=O); 1H NMR
(DMSO d6, d ppm): Boc = 1.31 (9H, s); Phe = 2.79–2.84 (2H, d,
-bCH2), 4.74 (1H, m, -aCH), 7.15–7.28 (5H, m, ArH), 8.60 (1H, d,
-NH); Ester = 2.58 (1H, s, -CH3), 4.28 (2H, d, -CH2); Mass: 293
(M+).
Procedure for the deblocking of Boc group of
compound 23
Compound 23 (0.307g, 0.0001 mol) was deblocked with TFA
(3 mL) for 40 min at room temperature. The excess solvent was
Table 1: 1H NMR data of the final protected peptides (1–4)
Boc-G1V2G3V4P5-OBzl (1)
Boc = 1.43 (9H, s); -NH = 8.34–8.42 (4H, m); Gly1 = 3.83 (2H, s, -aCH); Val2 = 0.98 (6H, d, -(CH3)2), 2.70 (1H, m, -bCH),
4.75 (1H, s, -aCH); Gly3 = 4.13 (2H, s, -aCH); Val4 = 0.98 (6H, d, -(CH3)2), 2.69 (1H, m, -bCH), 4.78 (1H, s, -aCH);
Pro5 = 2.09–3.64 (6H, m, -CH2), 4.59 (1H, m, -aCH); OBzl = 5.05 (2H, m, CH2), 7.17–7.36 (5H, m, Ar-H)
Boc-G1F2G3F4P5-OBzl (2)
Boc = 1.41 (9H, s); -NH = 8.04–8.12 (4H, m); Gly1 = 3.87 (2H, s, -aCH); Phe2 = 3.53, 3.57 (2H, m, -bCH2), 4.79 (1H, s,
-aCH), 7.21–7.32 (5H, m, ArH); Gly3 = 4.12 (2H, s, -aCH); Phe4 = 3.47, 3.61 (2H, m, -bCH2), 4.81 (1H, s, -aCH), 7.21–7.32
(5H, m, ArH); Pro5 = 1.80–3.64 (6H, m, -CH2), 4.55 (1H, m, -aCH); OBzl = 5.08 (2H, m, CH2), 7.21–7.32 (5H, m, Ar-H)
Boc-GE(OcHx)GFP GVGVP GVGVP
GVGVP GFGFP GFGFP-OBzl (3)
Boc = 1.42 (9H, s); -NH = 8.29–9.30 (24H, m); Gly = 4.09, 4.94 (24H, s, -aCH); Val = 0.98, 1.00 (36H, m, (-CH3)2), 2.46,
2.63 (6H, m, -bCH), 4.44 (6H, m, -aCH); Glu = 1.11–1.24 (10H, m, -CH2 of cyclohexyl ring), 2.05, 2.19 (4H, m, -b,cCH2),
3.69 (1H, m, -CH of cyclohexyl ring), 4.75 (1H, m, -aCH); Phe = 3.54, 3.70 (10H, m, -bCH2), 4.20, 4.65 (5H, m, -aCH),
7.00–7.63 (25H, m, ArH); Pro = 2.00, 3.52, 3.66 (36H, m, -CH2), 4.76 (6H, m, a-CH); OBzl = 5.10 (2H, m, CH2), 7.00–7.63
(5H, m, Ar-H)
Boc-GE(OcHx)GFP GVGVP GVGFP
GFGFP GVGVP GVGFP-OBzl (4)
Boc = 1.40 (9H, s); -NH = 8.38–9.34 (24H, m); Gly = 4.08, 4.90 (24H, s, -aCH); Val = 0.96, 1.02 (36H, m, (-CH3)2), 2.42,
2.60 (6H, m, -bCH), 4.39 (6H, m, -aCH); Glu = 1.10–1.22 (10H, m, -CH2 of cyclohexyl ring), 2.07, 2.14 (4H, m, -b,cCH2),
3.65 (1H, m, -CH of cyclohexyl ring), 4.69 (1H, m, -aCH); Phe = 3.55, 3.74 (10H, m, -bCH2), 4.19, 4.57 (5H, m, -aCH),
7.19–7.69 (25H, m, ArH); Pro = 2.03, 3.54, 3.70 (36H, m, -CH2), 4.72 (6H, m, a-CH); OBzl = 5.13 (2H, m, CH2), 7.19–7.69
(5H, m, Ar-H)
854
Chem Biol Drug Des 2012; 79: 850–862

Synthesis of Aurantiamide Acetate Analogues
removed under reduced pressure, triturated with ether, filtered,
washed with ether and dried under vacuum (yield 100%) to obtain
23a.
Deprotection of benzyl ester of compound 16
Compound 16 (0.497 g, 0.001 mol) was hydrogenolysed using the
procedure as described for compound 5.
Table 2: Physical and analytical data of the synthesized compounds
Entry Rfa Rfb Yield (%) M.P. (ꢀC) Mol.wt. Exact mass 1H NMR (DMSO d6, d ppm)
12
0.42 0.50 85.8
110–112 794.93 794.35
Fmoc = 4.14–4.17 (2H, m, -CH2), 4.69 (1H, m, -CH), 7.10–7.48 (8H, m, ArH); -NH = 8.22–8.23
(2H, m); His(trt) = 3.06, 3.24 (2H, m, -bCH2), 4.81 (1H, t, -aCH), 6.97 (1H, s, -CH), 7.10–7.48
(15H, m, ArH), 8.02 (1H, s, -CH), Ester = 2.50 (1H, s, -CH3), 2.77–2.81 (2H, d, -CH2), 4.29 (2H,
d, -CH2), 4.73 (1H, m, -CH), 7.10–7.48 (5H, m, ArH)
13
0.41 0.52 80.7
121
668.78 668.29
Fmoc = 4.10–4.14 (2H, m, -CH2), 4.53 (1H, m, -CH), 7.11–7.39 (8H, m, ArH); -NH = 8.15–8.17
(2H, m); Tyr(Bzl) = 2.66–2.79 (2H, m, -bCH2), 4.79 (1H, t, -aCH), 5.11 (2H, s, -CH2 of benzyl),
7.11–7.39 (9H, m, ArH); Ester = 2.48 (1H, s, -CH3), 2.76–2.80 (2H, d, -CH2), 4.30 (2H, d, -CH2),
4.71 (1H, m, -CH), 7.11–7.39 (5H, m, ArH)
14
15
16
0.41 0.51 83.0
0.51 0.64 81.0
0.52 0.65 82.5
132
88
515
441
497
514
440
496
Fmoc = 4.12–1.14 (2H, m, -CH2), 4.53 (1H, m, -CH), 7.22–7.44 (8H, m, ArH); -NH = 8.19–8.21
(2H, m); Val = 0.86 (6H, d, -(CH3)2), 2.68 (1H, m, -bCH), 4.77 (1H, d, -aCH); Ester = 2.49 (1H, s,
-CH3), 2.80–2.84 (2H, d, -CH2), 4.27 (2H, d, -CH2), 4.72 (1H, m, -CH), 7.22–7.44 (5H, m, ArH)
Bpoc = 1.71–1.72 (6H, m, -CH3), 7.21–7.70 (9H, m, ArH); -NH = 8.10 (2H, m); Val = 0.71–0.77
(6H, d, -(CH3)2), 2.66 (1H, m, -bCH), 4.75 (1H, d, -aCH); Ester = 2.57 (1H, s, -CH3), 2.79–2.84
(2H, d, -CH2), 4.28 (2H, d, -CH2), 4.73 (1H, m, -CH), 7.21–7.70 (5H, m, ArH)
Gum
Boc = 1.33 (9H, s); -NH = 7.96 (1H, s); Hyp(Bzl) = 1.91–1.99 (2H, m, -bCH2), 2.89 (1H, m, -cCH),
3.39–3.57 (2H, t, -dCH2), 4.44 (1H, m, -aCH), 4.49 (2H, s, -CH2 of benzyl), 7.16–7.37 (5H, m,
ArH); Ester = 2.57 (1H, s, -CH3), 2.79–2.84 (2H, d, -CH2), 4.28 (2H, d, -CH2), 4.74 (1H, m, -CH),
7.16–7.37 (5H, m, ArH)
17
18
19
20
0.51 0.64 86.9
0.52 0.63 89.8
0.50 0.61 91.3
0.50 0.57 93.8
117
112
127
98
508
440
616
798
507
441
615
799
Boc = 1.32 (9H, s); -NH = 8.00–8.24 (2H, s); Trp(N-CHO) = 4.22 (2H, d, -bCH2), 4.86 (1H, t, -aCH),
9.63 (1H, s, -CHO), 7.16–7.84 (4H, m, ArH); Ester = 2.58 (1H, s, -CH3), 2.79–2.84 (2H, d, -CH2),
4.28 (2H, d, -CH2), 4.74 (1H, m, -CH), 7.16–7.37 (5H, m, ArH)
Boc = 1.32 (9H, s); -NH = 8.20–8.22 (2H, s); Phe = 2.80–2.85 (2H, m, -bCH2), 4.82 (1H, t, -aCH),
7.11–7.30 (5H, m, ArH), Ester = 2.55 (1H, s, -CH3), 2.77–2.83 (2H, d, -CH2), 4.26 (2H, d, -CH2),
4.72 (1H, m, -CH), 7.11–7.30 (5H, m, ArH)
Boc = 1.31 (9H, s); -NH = 8.22–8.24 (2H, s); Tyr(Cl2-Bzl) = 2.79–2.85 (2H, m, -bCH2), 4.80 (1H, t,
-aCH), 5.09 (2H, s, -CH2 of benzyl), 7.14–7.32 (9H, m, ArH); Ester = 2.53 (1H, s, -CH3), 2.79–
2.85 (2H, d, -CH2), 4.24 (2H, d, -CH2), 4.70 (1H, m, -CH), 7.14–7.32 (5H, m, ArH)
Boc = 1.38 (9H, s); -NH = exchanged; Gly1 = 3.79 (2H, s, -aCH); Phe2 = 3.51, 3.53 (2H, m,
-bCH2), 4.62 (1H, s, -aCH), 6.97–7.31 (5H, m, ArH); Gly3 = 4.13 (2H, s, -aCH); Phe4 = 3.54, 3.58
(2H, m, -bCH2), 4.62 (1H, s, -aCH), 6.97–7.31 (5H, m, ArH); Pro5 = 2.10–2.16, 3.65–3.67 (6H, m,
-CH2), 4.49 (1H, m, -aCH); Ester = 2.60 (1H, s, -CH3), 2.21 (2H, d, -CH2), 4.50–4.51 (2H, d, -
CH2), 4.61 (1H, m, -CH), 6.97–7.31 (5H, m, ArH)
21
22
0.49 0.56 92.8
0.55 0.75 81.4
108
180
702
703
Boc = 1.35 (9H, s); -NH = 7.91–8.23 (5H, m); Gly1 = 3.80 (2H, s, -aCH); Val2 = 0.82–0.86 (6H, d,
-(CH3)2), 2.74 (1H, m, -bCH), 4.31 (1H, s, -aCH); Gly3 = 4.16 (2H, s, -aCH); Val4 = 0.82–0.86 (6H,
d, -(CH3)2), 2.74 (1H, m, -bCH), 4.33 (1H, s, -aCH); Pro5 = 1.81–2.49, 3.39–3.55 (6H, m, -CH2),
4.31 (1H, m, -aCH); Ester = 2.49 (1H, s, -CH3), 2.75–2.77 (2H, d, -CH2), 4.50–4.51 (2H, d, -CH2),
4.71 (1H, m, -CH), 6.97–7.65 (5H, m, ArH)
3072.2 3071
Boc = 1.26 (9H, s); -NH = 7.95 (25H, m); Gly = 4.11, 4.83 (24H, s, -aCH); Val = 0.86, 0.89 (36H,
m, (-CH3)2), 2.44, 2.59 (6H, m, -bCH), 4.43 (6H, m, -aCH); Glu = 1.27 (10H, m, -CH2 of cyclo-
hexyl ring), 2.06, 2.17 (4H, m, -b,cCH2), 3.53 (1H, m, -CH of cyclohexyl ring), 4.54 (1H, m,
-aCH); Phe = 3.55, 3.76 (10H, m, -bCH2), 4.29, 4.54 (5H, m, -aCH), 6.52–7.50 (25H, m, ArH);
Pro = 2.05, 3.55, 3.73 (36H, m, -CH2), 4.81 (6H, m, a-CH); Ester = 2.51 (1H, s, -CH3), 2.76–2.79
(2H, d, -CH2), 4.53–4.54 (2H, d, -CH2), 4.75 (1H, m, -CH), 6.52–7.50 (5H, m, ArH)
23
0.54 0.73 85.4
179–181 3072.2 3071
Boc = 1.38 (9H, s); -NH = 8.11–8.25 (24H, m); Gly = 4.05, 4.91 (24H, s, -aCH); Val = 0.88, 0.97
(36H, m, (-CH3)2), 2.44, 2.64 (6H, m, -bCH), 4.46 (6H, m, -aCH); Glu = 1.10–1.22 (10H, m, -CH2
of cyclohexyl ring), 2.04, 2.14 (4H, m, -b,cCH2), 3.70 (1H, m, -CH of cyclohexyl ring), 4.74 (1H,
m, -aCH); Phe = 3.54, 3.75 (10H, m, -bCH2), 4.18, 4.59 (5H, m, -aCH), 6.69–7.58 (25H, m, ArH);
Pro = 2.04, 3.50, 3.64 (36H, m, -CH2), 4.71 (6H, m, a-CH); Ester = 2.53 (1H, s, -CH3), 2.74–2.77
(2H, d, -CH2), 4.51–4.55 (2H, d, -CH2), 4.77 (1H, m, -CH), 6.69–7.58 (5H, m, ArH)
Chem Biol Drug Des 2012; 79: 850–862
855

Suhas and Channe Gowda
30 min
1 h
2 h
3 h
4 h
80
70
60
50
40
30
20
10
0
12 13 14 15 16 16a 17 18 19 20 21 22 23 23a Std.
Compounds
Figure 3: Diagrammatic representation of the analgesic activity of the compounds.
Pharmacology
for the purpose. The significant difference between groups was sta-
For analgesic activity, healthy adult Swiss albino mice of either sex
weighing 25–30 g were used. For anti-inflammatory activity, healthy
adult Wistar rats of either sex weighing between 75 and 125 g
were used. Ibuprofen and phenyl butazone served as reference
standards for analgesic and anti-inflammatory activities, respec-
tively. All the animals were maintained as per the norms of the
Committee for the Purpose of Control and Supervision of Experi-
ments on Animal (CPCSEA), and the study protocols were approved
by CPCSEA and Institutional Animal Ethics Committee constituted
tistically analysed by one-way ANOVA followed by Dunnett's t-test.
Analgesic activity
The analgesic activity of the synthesized compounds was deter-
mined by the standard tail immersion method in mouse (31,32).
Albino mice of either sex weighing 25–30 g were divided into
groups of five animals each. At intervals, the mouse was held, so
that its tail was totally immersed in a bath at the temperature of
Table 3: Analgesic and anti-inflammatory activities of the synthesized compounds
Entry
Time
Mean tail withdrawal latencies (s) € SE*
Mean values (€SE) of oedema volume at different intervals*
30 min 1 h 2 h 3 h 4 h
30 min
1 h
4.34 € 0.38
2 h
4.98 € 0.52
3 h
5.12 € 0.12
4 h
Control
12
13
14
15
1.96 € 0.45
3.08 € 0.48 0.55 € 0.006 0.54 € 0.006 0.54 € 0.006 0.53 € 0.006 0.52 € 0.006
0.32 € 0.01 0.29 € 0.006 0.23 € 0.006 0.23 € 0.006
0.36 € 0.006 0.32 € 0.006 0.26 € 0.006 0.27 € 0.00
33.78 € 0.51 36.37 € 0.32 41.42 € 0.32 44.46 € 0.42 28.43 € 0.32 0.36 € 0.02
25.22 € 0.66 31.32 € 0.30 36.33 € 0.41 42.58 € 0.37 26.44 € 0.43 0.39 € 0.02
23.38 € 0.59 28.42 € 0.20 34.72 € 0.16 39.38 € 0.30 21.47 € 0.30 0.37 € 0.01
25.11 € 0.49 30.49 € 0.39 35.48 € 0.34 40.63 € 0.25 22.46 € 0.35 0.39 € 0.01
27.45 € 0.59 33.65 € 0.23 38.22 € 0.19 45.39 € 0.37 27.34 € 0.30 0.35 € 0.01
15.57 € 0.34 17.98 € 0.23 19.38 € 0.22 23.51 € 0.33 17.28 € 0.29 0.46 € 0.02
12.17 € 0.25 15.32 € 0.12 17.54 € 0.32 20.33 € 0.16 14.35 € 0.19 0.48 € 0.02
16.64 € 0.28 21.31 € 0.27 26.53 € 0.39 33.61 € 0.35 18.48 € 0.35 0.44 € 0.01
20.66 € 0.57 26.27 € 0.26 32.58 € 0.14 37.42 € 0.29 21.45 € 0.26 0.40 € 0.02
16.63 € 0.43 22.60 € 0.32 27.42 € 0.25 34.34 € 0.29 19.48 € 0.26 0.43 € 0.02
19.40 € 0.30 25.46 € 0.24 30.50 € 0.29 35.44 € 0.40 22.51 € 0.36 0.42 € 0.02
31.57 € 0.22 38.37 € 0.24 42.49 € 0.29 47.32 € 0.32 29.54 € 0.32 0.35 € 0.02
23.53 € 0.33 25.53 € 0.40 27.52 € 0.39 30.29 € 0.25 25.24 € 0.20 0.41 € 0.02
19.67 € 0.75 21.50 € 0.24 23.34 € 0.24 26.41 € 0.31 22.70 € 0.17 0.44 € 0.02
34.60 € 0.35 39.28 € 0.33 43.57 € 0.32 48.43 € 0.39 31.31 € 0.31 0.32 € 0.01
24.21 € 0.35 26.99 € 0.11 28.42 € 0.20 33.45 € 0.28 23.20 € 0.18 0.40 € 0.01
20.59 € 0.33 22.85 € 0.61 25.40 € 0.19 29.19 € 0.14 20.38 € 0.20 0.42 € 0.02
37.51 € 0.24 42.54 € 0.32 52.56 € 0.18 60.45 € 0.22 35.44 € 0.34 0.30 € 0.01
28.12 € 0.40 30.37 € 0.21 32.67 € 0.37 40.53 € 0.25 30.32 € 0.27 0.35 € 0.02
26.43 € 0.24 28.12 € 0.40 30.32 € 0.37 34.41 € 0.26 22.51 € 0.19 0.40 € 0.02
35.52 € 0.29 38.40 € 0.28 44.49 € 0.28 51.61 € 0.30 33.41 € 0.24 0.31 € 0.01
26.46 € 0.51 28.34 € 0.21 30.39 € 0.21 38.38 € 0.20 22.53 € 0.27 0.38 € 0.01
22.65 € 0.37 24.32 € 0.16 26.34 € 0.16 31.56 € 0.27 20.39 € 0.17 0.42 € 0.02
0.36 € 0.01
0.33 € 0.02
0.33 € 0.01
0.30 € 0.006 0.25 € 0.006 0.26 € 0.00
0.27 € 0.006 0.26 € 0.00
16
0.30 € 0.006 0.28 € 0.006 0.21 € 0.006 0.21 € 0.006
16a
16b
16a
17
0.43 € 0.006 0.40 € 0.01
0.37 € 0.01
0.37 € 0.006
0.39 € 0.006
0.45 € 0.01
0.41 € 0.01
0.38 € 0.01
0.41 € 0.01
0.38 € 0.01
0.42 € 0.006 0.40 € 0.01
0.36 € 0.01
0.35 € 0.01
0.38 € 0.01
0.35 € 0.01
0.30 € 0.006 0.34 € 0.006
0.29 € 0.006 0.31 € 0.006
0.32 € 0.01
18
19
0.33 € 0.006
0.31 € 0.006 0.32 € 0.006
20
0.29 € 0.006 0.26 € 0.006 0.19 € 0.006 0.19 € 0.006
20a
20b
21
0.38 € 0.01
0.41 € 0.02
0.35 € 0.01
0.37 € 0.01
0.34 € 0.006 0.34 € 0.006
0.34 € 0.01 0.35 € 0.006
0.28 € 0.006 0.26 € 0.006 0.18 € 0.006 0.18 € 0.00
21a
21b
22
0.37 € 0.01
0.40 € 0.01
0.26 € 0.01
0.32 € 0.01
0.34 € 0.006 0.31 € 0.006 0.32 € 0.00
0.37 € 0.006 0.34 € 0.006 0.34 € 0.00
0.21 € 0.006 0.13 € 0.006 0.13 € 0.00
22a
22b
23
0.30 € 0.01
0.27 € 0.006 0.29 € 0.006
0.30 € 0.006 0.32 € 0.00
0.36 € 0.006 0.33 € 0.01
0.27 € 0.006 0.24 € 0.006 0.14 € 0.006 0.14 € 0.00
23a
23b
23a
Std.
Std.a
Std.b
0.34 € 0.01
0.39 € 0.01
0.32 € 0.01
0.36 € 0.006 0.33 € 0.006 0.34 € 0.006
0.28 € 0.02 0.25 € 0.006 0.26 € 0.006
0.29 € 0.006 0.27 € 0.006 0.26 € 0.006 0.25 € 0.006
0.29 € 0.006 0.30 € 0.006
22.39 € 0.23 30.43 € 0.20 35.42 € 0.31 40.51 € 0.28 29.55 € 0.26 0.36 € 0.006 0.32 € 0.01
35.33 € 0.21 40.72 € 0.22 43.24 € 0.30 49.57 € 0.43 32.47 € 0.38 0.34 € 0.01
18.28 € 0.23 21.31 € 0.14 23.39 € 0.25 28.34 € 0.21 20.38 € 0.27 0.41 € 0.02
14.27 € 0.27 19.30 € 0.12 21.30 € 0.12 25.47 € 0.18 14.79 € 0.13 0.44 € 0.02
0.39 € 0.01
0.41 € 0.01
0.37 € 0.006 0.32 € 0.006 0.35 € 0.006
0.38 € 0.02 0.37 € 0.006 0.38 € 0.006
a50 mg ⁄ kg body weight.
b25 mg ⁄ kg body weight.
*Significantly different from the control value at p < 0.05.
856
Chem Biol Drug Des 2012; 79: 850–862

Synthesis of Aurantiamide Acetate Analogues
ꢀ
ꢁ
55 € 5 ꢀC. The time until the typical reaction, a violent jerk of the
tail, was recorded and noted as the basal reaction time. The ani-
mals were administered orally with control (DMSO), test compounds
(12–23, 16a, 23a) and ibuprofen (standard) at a dose of
100 mg ⁄ kg body weight (the compounds that showed potent activ-
ity were treated with 25 and 50 mg ⁄ kg body weight). Time taken
for tail withdrawal response was recorded at 30 min, 1 h, 2 h, 3 h,
4 h and 24 h after administering the compounds. Increase in reac-
tion time (interval for withdrawal of tail by the animal) was consid-
ered as an analgesic activity. The percentage analgesic activity was
calculated using the formula,
% alalgesic activity of test compound treated group
% alalgesic activity of ibuprofen treated group
Potency ¼
Anti-inflammatory activity
The anti-inflammatory activity was evaluated using carrageenan-
induced rat hind paw oedema method (33). The animals fasted for
24 h were divided into control, standard and test groups each con-
sisting of five rats. The first group of rats was treated with DMSO
(control), second group was administered orally with a dose of
100 mg ⁄ kg of phenyl butazone (standard), and third group was trea-
ted with 100 mg ⁄ kg of test compounds (the compounds that
showed potent activity were treated with 25 and 50 mg ⁄ kg body
weight). After 30 min, the animals were injected subcutaneously
with 0.1 mL of freshly prepared suspension of carrageenan solution
(1% in 0.9% saline) into the subplantar region of the right hind
paw of rats. The volume of hind paw was measured using vernier
calipers both in control and in animals treated with standard and
test compounds at 30 min, 1 h, 2 h, 3 h, 4 h and 24 h after carra-
geenan challenge. The initial paw volume was measured within
30 seconds of the injection. The anti-inflammatory activity was
ꢀ
ꢁ
T2 ꢀ T1
80 ꢀ T1
% Analgesic activity ¼
ꢁ 100
where T1 is reaction time before treatment, T2 is reaction time after
treatment, and 80 is the cut-off time.
Potency of the tested compounds was calculated relative to ibupro-
fen 'reference standard' treated group according to the following
equation.
Table 4: Analgesic activity of the synthesized compounds
Entry
Time
% analgesic activity € SE*
30 min
1 h
2 h
3 h
4 h
24 h
Potency
12
13
14
15
40.77 € 0.66
29.80 € 0.85
27.45 € 0.76
29.66 € 0.63
32.66 € 0.75
17.44 € 0.44
13.09 € 0.33
18.56 € 0.36
23.95 € 0.73
18.79 € 0.55
22.35 € 0.39
37.94 € 0.28
27.64 € 0.42
19.67 € 0.75
41.82 € 0.44
28.51 € 0.44
23.87 € 0.42
45.55 € 0.31
33.80 € 0.22
29.20 € 0.31
43.00 € 0.37
31.40 € 0.65
22.65 € 0.29
26.18 € 0.30
42.75 € 0.27
20.91 € 0.30
15.77 € 0.34
42.32 € 0.43
35.66 € 0.40
31.82 € 0.27
34.56 € 0.52
37.73 € 2.41
18.03 € 0.31
14.51 € 0.16
23.76 € 0.35
28.98 € 0.34
24.13 € 0.43
27.91 € 0.32
44.98 € 0.32
28.01 € 0.53
22.68 € 0.32
46.18 € 0.43
29.94 € 0.15
24.47 € 0.81
50.49 € 0.43
36.40 € 0.27
33.52 € 0.51
45.01 € 0.37
31.72 € 0.27
26.51 € 0.37
34.48 € 0.26
48.24 € 0.46
22.43 € 0.19
19.76 € 0.16
48.57 € 0.43
42.05 € 0.51
39.63 € 0.21
40.66 € 0.45
44.30 € 0.25
19.19 € 0.29
16.74 € 0.43
30.88 € 0.50
36.67 € 0.37
29.90 € 0.33
34.02 € 0.39
49.99 € 0.39
30.04 € 0.39
24.48 € 0.31
51.43 € 0.42
31.24 € 0.26
27.21 € 0.26
63.42 € 0.24
37.44 € 0.49
33.78 € 0.50
52.66 € 0.38
33.87 € 0.27
28.48 € 0.21
40.57 € 0.41
51.00 € 0.41
24.54 € 0.33
21.76 € 0.16
52.54 € 0.56
50.02 € 0.50
45.76 € 0.41
47.42 € 0.34
53.73 € 0.52
24.55 € 0.45
20.32 € 0.21
40.76 € 0.45
43.13 € 0.38
39.03 € 0.39
40.49 € 0.53
56.36 € 0.42
33.62 € 0.34
28.43 € 0.42
57.84 € 0.52
37.83 € 0.37
32.15 € 0.18
73.88 € 0.30
47.28 € 0.33
39.12 € 0.35
62.08 € 0.40
44.42 € 0.27
35.32 € 0.36
47.26 € 0.38
59.36 € 0.58
31.01 € 0.28
27.18 € 0.24
32.96 € 0.42
30.36 € 0.56
23.90 € 0.39
25.19 € 0.46
31.73 € 0.47
17.28 € 0.29
14.65 € 0.24
21.65 € 0.44
23.88 € 0.34
21.32 € 0.34
25.25 € 0.47
34.40 € 0.42
28.80 € 0.26
25.51 € 0.22
36.70 € 0.41
26.16 € 0.23
22.49 € 0.26
42.06 € 0.43
35.41 € 0.35
25.26 € 0.25
39.43 € 0.32
25.28 € 0.34
22.50 € 0.21
34.40 € 0.34
38.20 € 0.50
22.49 € 0.35
15.23 € 0.17
39.32 € 0.24
0.89
0.84
0.77
0.80
0.91
–
–
–
–
16
29.82 € 0.18
16a
16b
16a
17
–
–
–
–
–
–
–
0.69
0.73
0.66
0.68
0.95
–
–
0.97
–
–
1.24
–
–
1.05
–
18
19
20
40.40 € 1.64
20a
20b
21
–
–
42.43 € 1.50
21a
21b
22
–
–
48.40 € 0.53
22a
22b
23
–
–
46.63 € 0.78
23a
23b
23a
Std.
Std.a
Std.b
–
–
–
–
0.80
1.00
–
35.57 € 0.51
–
–
–
'–' = not observed.
Potency was calculated as regards the percentage change of the ibuprofen.
a50 mg ⁄ kg body weight.
b25 mg ⁄ kg body weight.
*Significantly different from the control value at p < 0.05.
Chem Biol Drug Des 2012; 79: 850–862
857

Suhas and Channe Gowda
Table 5: Anti-inflammatory activity of the synthesized compounds
Entry
Time
% anti-inflammatory activity*
30 min
1 h
2 h
3 h
4 h
24 h
Potency
12
13
14
15
32.10 € 0.60
27.17 € 0.55
30.77 € 0.35
29.00 € 0.53
35.17 € 0.55
14.30 € 0.50
10.47 € 0.78
18.20 € 1.31
24.70 € 0.52
19.10 € 0.56
21.00 € 0.53
37.17 € 0.58
23.47 € 0.55
17.90 € 0.60
40.73 € 0.35
25.60 € 0.52
21.00 € 0.53
44.41 € 0.22
33.93 € 0.65
26.43 € 0.72
42.57 € 0.25
20.97 € 0.67
28.87 € 0.70
32.10 € 0.35
37.01 € 0.50
22.50 € 0.61
17.33 € 0.49
40.00 € 0.40
33.73 € 0.93
32.50 € 0.35
37.70 € 0.40
42.73 € 0.51
19.37 € 0.57
16.17 € 0.68
23.10 € 0.50
28.73 € 0.40
23.10 € 0.50
28.73 € 0.40
45.00 € 0.70
27.13 € 0.21
23.10 € 0.50
46.90 € 0.61
30.50 € 0.61
25.00 € 0.50
51.23 € 0.58
40.00 € 0.40
32.50 € 0.35
48.77 € 0.58
26.70 € 0.26
36.23 € 0.38
39.57 € 0.85
44.97 € 0.70
26.87 € 0.45
22.40 € 0.26
45.70 € 0.53
40.97 € 1.19
38.90 € 0.70
45.07 € 0.59
47.53 € 0.49
25.93 € 0.45
21.30 € 0.50
33.30 € 0.60
35.17 € 0.65
29.63 € 0.55
35.17 € 0.65
51.23 € 0.58
35.17 € 0.65
31.10 € 0.53
52.47 € 0.49
37.13 € 0.93
31.97 € 0.81
61.70 € 0.56
44.07 € 0.42
38.90 € 0.70
56.20 € 0.53
34.07 € 0.40
40.43 € 0.38
45.70 € 0.53
49.39 € 0.53
31.73 € 0.25
29.83 € 0.87
57.07 € 0.29
51.57 € 0.58
50.00 € 0.40
53.43 € 0.55
59.63 € 0.49
29.67 € 0.12
25.60 € 0.52
42.37 € 0.21
46.03 € 0.38
40.23 € 0.57
42.27 € 0.35
63.40 € 0.36
36.73 € 0.25
35.77 € 0.81
64.63 € 1.06
42.30 € 0.30
36.73 € 0.25
76.23 € 0.58
49.87 € 0.12
43.00 € 0.36
73.47 € 0.47
38.93 € 0.45
46.27 € 0.06
53.50 € 0.44
51.78 € 0.21
40.60 € 0.17
31.20 € 0.36
53.30 € 0.63
46.10 € 0.17
47.93 € 0.12
47.80 € 0.35
56.77 € 0.15
25.83 € 0.29
21.87 € 0.23
31.53 € 0.42
38.13 € 0.23
32.67 € 0.23
35.90 € 0.17
62.13 € 0.23
30.87 € 0.23
30.67 € 0.70
63.30 € 0.30
36.03 € 0.45
32.27 € 0.23
74.17 € 0.29
42.40 € 0.53
36.43 € 0.75
71.70 € 0.44
31.60 € 0.53
40.40 € 0.53
49.33 € 0.61
49.67 € 0.58
30.53 € 0.50
23.97 € 0.35
26.32 € 0.24
–
–
–
29.82 € 0.18
–
–
–
1.10
1.00
0.97
1.03
1.15
–
16
16a
16b
16a
17
–
0.82
0.89
0.78
0.82
1.22
–
–
1.25
–
–
1.47
–
–
1.42
–
–
–
–
18
19
20
33.82 € 0.23
–
–
38.86 € 0.37
–
–
44.11 € 0.25
–
–
41.75 € 0.40
–
–
–
20a
20b
21
21a
21b
22
22a
22b
23
23a
23b
23a
Std.
Std.a
Std.b
–
1.03
1.00
–
36.23 € 0.18
–
–
–
Potency was calculated as regards the percentage change of the phenyl butazone.
'–' = not observed.
a50 mg ⁄ kg body weight.
b25 mg ⁄ kg body weight.
*Significantly different from the control value at p < 0.05.
expressed as a percentage inhibition of the inflammation in treated
animals in comparison with the control group (34).
Potency of the tested compounds was calculated relative to phenyl
butazone 'reference standard' treated group according to the follow-
ing equation.
ꢀ
ꢁ
Vt
Vc
% inhibition of oedema ¼ 1 ꢀ
ꢁ 100
ꢀ
ꢁ
% oedema inhibition of test compound treated group
% oedema inhibition of phenyl butazone treated group
Potency ¼
where Vc and Vt are the mean relative changes in the volume of
paw oedema in the control and test treated groups, respectively.
30 min
1 h
2 h
3 h
4 h
80
70
60
50
40
30
20
10
0
12 13 14 15 16 16a 17 18 19 20 21 22 23 23a Std.
Compounds
Figure 4: Diagrammatic representation of the anti-inflammatory activity of the compounds.
858
Chem Biol Drug Des 2012; 79: 850–862

Synthesis of Aurantiamide Acetate Analogues
25 mg
50 mg
100 mg
Results and Discussion
80
60
40
20
0
Chemistry
Peptides were synthesized by classical solution phase method using
IBCF ⁄ HOBt as coupling agent and NMM as base. H NMR of the
1
final protected peptides (1–4) is provided in Table 1. Boc-Phe-OH
was subjected to reduction using IBCF ⁄ NMM ⁄ NaBH4. IR spectra of
this compound showed a band at stretching frequency of 3541
cm)1 for –OH functional group. In H NMR spectrum, disappearance
1
of COOH peak and appearance of a peak at d 3.60 (1H, t) for –OH
and d 3.65 (2H, t, -CH2OH) confirmed its synthesis. Further, this
compound was esterified with Ac2O ⁄ pyridine, and its formation was
confirmed by the absence of –OH stretching frequency in IR and
the presence of a peak at d 2.58 (3H, s) for –OCOCH3 in the PMR
spectrum. Boc group of the ester was deblocked using TFA and
reacted with respective N-protected amino acids ⁄ peptides to obtain
the desired compounds. The synthesis of final compounds was con-
firmed by the absence of COOH peak and presence of –NH peak at
16
20
21
22
23
Std.
Compounds
Figure 5: Diagrammatic representation of the analgesic activity
of the potent compounds at different doses at 3-h interval.
25 mg
50 mg
100 mg
80
60
40
20
0
1
d ꢂ8.00 (1H, s) in the H NMR spectra. Further, all the synthesized
compounds were confirmed by mass spectra that are in accordance
with their molecular formulae. The analytical and spectroscopic data
of the synthesized compounds are given in Table 2.
Pharmacology
16
20
21
22
23
Std.
Analgesic and anti-inflammatory activities
Analgesic and anti-inflammatory activities of the synthesized com-
pounds were determined by tail flick method in mice and acute
carrageenan-induced paw oedema method in rats, respectively, at
a dose of 100 mg ⁄ kg body weight (Figures 3 and 4). The analge-
sic and anti-inflammatory properties were recorded at successive
time intervals of 30 min, 1 h, 2 h, 3 h, 4 h and 24 h and com-
pared with that of ibuprofen and phenyl butazone, respectively,
which were used as reference standards. From the results
(Tables 3–5), it is observed that all the synthesized compounds
have exhibited significant analgesic and anti-inflammatory proper-
ties. When N-protected amino acids ⁄ peptides alone were sub-
jected to analgesic and anti-inflammatory activities, they showed
ꢂ10% and 20–35% activity at 3-h interval respectively. Encour-
aged by these results, different N-protected amino acids and pep-
tides were used for the modification of amino acid residue in
aurantiamide acetate.
Compounds
Figure 6: Diagrammatic representation of the anti-inflammatory
activity of the potent compounds at different doses at 3-h interval.
Ulcerogenic activity
Adult albino rats of either sex were fasted 24 h prior to the admin-
istration of compounds. Water was allowed ad libitum to the ani-
mals. The compounds (which have shown the promising anti-
inflammatory activity) and phenyl butazone were given orally and
the animals killed 8 h after treatment. The stomach, duodenum and
jejunum were removed and examined with a hand lens for any evi-
dence of (i) shedding of epithelium, (ii) red spots below skin and
bleeding and (iii) erosion or discrete ulceration with or without per-
foration. The presence of any one of these was considered to be
an evidence of ulcerogenic activity (35).
30 min
1 h
2 h
3 h
4 h
24 h
80
70
60
50
40
30
20
10
0
12
16
20
21
Compounds
22
23
Std.
Figure 7: Comparative diagrammatic representation of analgesic activity of the compounds at different time intervals.
Chem Biol Drug Des 2012; 79: 850–862
859

Suhas and Channe Gowda
30 min
1 h
2 h
3 h
4 h
24 h
80
70
60
50
40
30
20
10
0
12
16
20
21
Compounds
22
23
Std.
Figure 8: Comparative diagrammatic representation of anti-inflammatory activity of the compounds at different time intervals.
Previous report (12) suggested that there is no preference for either
the S- or R-configuration on the activity in aurantiamide acetate.
Hence, in the present study, we included only the L-amino acids.
Earlier investigations (12–14) have shown that 9-fluorenylmethoxy-
carbonyl (Fmoc) group is essential for the compounds to exhibit
potent activity. Accordingly, we included three Fmoc amino acids,
viz. Fmoc-His(Trt), Fmoc-Tyr(Bzl) and Fmoc-Val (12–14). Among
these, compound 12 containing Fmoc-His(Trt) has shown enhanced
results which is presumably because of the presence of imidazole
ring, whereas the other two have shown moderate activity. This
was followed by the replacement of Fmoc group by Boc. Eventually,
compounds containing Boc amino acids (16–19) have revealed
slightly lower activity compared with Fmoc-containing compounds.
This indicated that aromatic group at the N-terminus has an impact
on the activity. One exception to this is compound 16 that has
exhibited more potency than the standard used which is attributed
to the presence of benzylated hydroxyl group in proline. Further,
when benzyl group of compound 16 was removed (16a), there was
a dramatic decrease in the activity. This observation showed that
side-chain functionality is necessary to exert potent activity (12,13).
Subsequently, dependence of activity on the N-terminal Boc group
was verified by subjecting compound 23 to TFA treatment, which
resulted in decreased activity (23a). This revealed the importance
of Boc group or N-substitution in exerting better pharmacological
properties. On the other hand, when Fmoc of Val (14) was replaced
by Bpoc (15), there was an increase in the activity which may be
due to the presence of biphenyl system in the latter.
reduced dose, that is, 25 and 50 mg ⁄ kg body weight. It was
found that 100 mg ⁄ kg body weight of the compound is required
to exhibit maximum activity (Figures 5 and 6). It was noticed that
activity was maximum at 3-h interval and gradually decreased
(Figures 7 and 8). These observations were in correlation with
the standards used. Several compounds of this series showed
better results than the reference drugs at 24 h after the treat-
ment which may be due to the biocompatibility nature of the
amino acids ⁄ peptides (20,21).
Ulcerogenic activity
Considering the potent compounds of the series, they were studied
for ulcerogenic activity. The result clearly showed that the com-
pounds were less ulcerogenic and devoid of any evidences of shed-
ding of epithelium ⁄ red spots below skin ⁄ bleeding ⁄ erosion, etc.
Conclusion
In summary, the current investigation reports the synthesis and
pharmacological evaluation of novel aurantiamide acetate analogues
with different N-terminal as well as side-chain substitutions. Sev-
eral representatives of this series were identified as highly potent
analgesic and anti-inflammatory agents with no ulcerogenic liability.
It was evident that activity is dose as well as time dependent. This
report also demonstrates for the first time that Boc group is essen-
tial for the pharmacological properties. Further, presence of bulky
substituents on the side chain of the amino acids is necessary to
exhibit enhanced activity. It was also noticed that compounds con-
taining peptides (20–23) have shown highly potent activity than
the reference standards. Hence, these compounds could be devel-
oped as new lead analgesic and anti-inflammatory agents. The
work thus provides the platform for future reports, which involves
varying amino acid composition as well as the length of the peptide
chain.
Prompted by the aforementioned results, we next investigated the
effect of replacement of amino acids by elastin-based peptides.
Among the pentapeptide analogues 20 and 21, compound contain-
ing GVGVP (21) has shown slightly increased activity over GFGFP
analogue (20) though the latter contains more hydrophobic Phe
units. When the pentapeptides were replaced by tricosamers, we
obtained highly potent results than the reference standards. Further,
among the tricosamer containing analogues 22 and 23, compound
22 has revealed slightly increased activity over 23 although the
amino acid composition in both remains the same. This could be
due to the altered position of amino acids as well as the charge
separation in them.
Acknowledgment
One of the authors RS gratefully acknowledges Lady Tata Memorial
Trust, Mumbai for the award of Research Scholarship.
Furthermore, compounds (12, 16, 20–23) that showed highly
potent activity at 100 mg ⁄ kg body weight were subjected to
860
Chem Biol Drug Des 2012; 79: 850–862

Synthesis of Aurantiamide Acetate Analogues
anti-inflammatory agent, increased intracellular Ca2+ concentra-
tions in MDCK renal tubular cells. Int J Immunopharma-
col;22:915–921.
References
1. Turner R.A. (1965) Screening Methods in Pharmacology. New
York: Academic Press; p. 152.
16. Sandberg L.B., Leslie J.G., Leach C.T., Torres V.L., Smith A.R.,
Smith D.W. (1985) Calculations of electrostatic interactions in
biological systems and in solutions. Pathol Biol;33:266–275.
17. Yeh H., Ornstein-Goldstein N., Indik Z., Sheppard P., Anderson
N., Rosenbloom J.C., Cicila G., Yoon K., Rosenbloom J. (1987)
Sequence variation of bovine elastin mRNA due to alternative
splicing. Coll Relat Res;7:235–247.
2. Paul A.I. (1996) Analgesic-antipyretic and anti-inflammatory
agents and drugs employed in the treatment of gout. In: Peery
B.M., Raymond W.R., editors. Goodman and Gilmans, The Pharma-
cological Basis of Therapeutic. New York: McGraw Hill; p. 6178.
3. Ashutoshkar (2003) Medicinal Chemistry, 2nd edn. New Delhi:
New Age International Publishers; p. 329.
18. Sandberg L.B., Soskel N.T., Leslie J.B. (1981) Elastin structure,
4. Andersen K., Launer L.J., Ott A., Hoes A.W., Breteler M.M.B.,
Hofman A. (1995) Do nonsteroidal anti-inflammatory drugs
decrease the risk for Alzheimer's Disease? The Rotterdam study.
Neurology;45:1441–1445.
5. Breitner J.C.S. (1996) The role of anti-inflammatory drugs in the
prevention and treatment of Alzheimer's Disease. Annu Rev
Med;47:401–411.
6. Sveigard H.H., Dalgaard L. (2000) Evaluation of blood-brain bar-
rier passage of a Muscarine M1 agonist and a series of analo-
gous tetrahydropyridines measured by in vivo microdialysis.
Pharm Res;17:70–76.
7. Ma Y., Loesewetter D.O., Kagpda E.M., Huang B.X., Eckelman
W.C. (2002) Identification of metabolites of fluorine-18-labelled
M2 Muscarinic receptor agonist, 3-(3[(3-fluoropropyl)thio]-1,2,5-
thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine, produced by
human and rat hepatocytes. J Chromatogr B;766:319–329.
8. Tsai P.L., Wang J.P., Chang C.W., Kuo S.C., Lee Chao P.D. (1998)
Constituents and bioactive principles of Polygonum chinensis.
Phytochemistry;49:1663–1666.
9. Isshiki K., Asai Y., Tanaka S., Nishio M., Uchida T., Okuda T.,
Kamatsubara S., Sakurai N. (2001) Aurantiamide acetate, a
selective cathepsin inhibitor, produced by Aspergillus penicillo-
ides. Biosci Biotechnol Biochem;65:1195–1197.
10. Ferreira E.O., Salvador M.J., Pral E.M., Alfieri S.C., Ito I.Y., Dias
D.A. (2004) A new heptasubstituted (E)-aurone glucoside and
other aromatic compounds of Gomphrena agrestis with biologi-
cal activity. Z Naturforsch C;59:499–505.
11. Ng T.B., Liu Y., Cheng C.H.K., Wang Z. (2003) Antioxidant activity
of compounds from the medicinal herb Aster tataricus. Comp
Biochem Physiol C: Toxicol Pharmacol;136:109–115.
12. Yen C.T., Hwang T.L., Wu Y.C., Hsieh P.W. (2009) Design and
synthesis of new N-(Fluorenyl-9-methoxycarbonyl) (Fmoc)-dipep-
tides as anti-inflammatory agents. Eur J Med Chem;44:1933–
1940.
biosynthesis, and relation to disease states. Engl
Med;304:566–579.
J
19. Indik Z., Yeh H., Ornstein-Goldstein N., Sheppard P., Anderson
N., Rosenbloom J., Peltnen L., Rosenbloom J. (1987) Alternative
splicing of human elastin mRNA indicated by sequence analysis
of cloned genomic and complementary DNA. Proc Natl Acad Sci
USA;84:5680–5684.
20. Gowda D.C., Parker T.M., Harris R.D., Urry D.W. (1994) Synthe-
sis, characterizations, and medical applications of bioelastic
materials. In: Basava C., Ananthramaiah G.M., editors. Peptides.
Boston: Birkhauser; p. 81–111.
21. Urry D.W., Nicol A., Gowda D.C., Hoban L.D., Mckee A., Wil-
liams T., Olsen D.B., Cox B.A. (1993) Medical application of bio-
elastic materials. In: Gebelein C.G., editor. Biotechnological
Polymers: Medical, Pharmaceutical and Industrial Applications.
Atlanta, GA: Technomic Publishing; p. 82–103.
22. Urry D.W., McPherson D.T., Xu J., Daniell H., Guda C., Gowda
D.C., Jing N., Parker T.M. (1996) Protein-based polymeric materi-
als (Synthesis and properties). In: Salamone J.C., editor. Polymeric
Materials Encyclopedia. Boca Raton, FL: CRC Press; p. 7263–7279.
23. Shivakumara K.N., Prakasha K.C., Suresha G.P., Suhas R., Gowda
D.C. (2007) Synthesis and antimicrobial study of amino acids con-
jugated benzylpiperazine derivatives. mySCIENCE;II:100–106.
24. Suresha G.P., Prakasha K.C., Shivakumara K.N., Kapfo W., Gow-
da D.C. (2009) Design and synthesis of heterocyclic conjugated
peptides as novel antimicrobial agents. Int
Ther;15:25–30.
J Pept Res
25. Suhas R., Chandrashekar S., Gowda D.C. (2011) Synthesis of
elastin based peptides conjugated to benzisoxazole as a new
class of potent antimicrobials – a novel approach to enhance
biocompatibility. Eur J Med Chem;46:704–711.
26. Suresha G.P., Suhas R., Kapfo W., Gowda D.C. (2011) Urea ⁄ thio-
urea derivatives of quinazolinone-lysine conjugates: synthesis
and structure-activity relationships of a new series of antimicro-
bials. Eur J Med Chem;46:2530–2540.
27. Suhas R., Chandrashekar S., Gowda D.C. (2011) A new family of
highly potent inhibitors of microbes: synthesis and conjugation
of elastin based peptides to piperazine derivative. Int J Pept
Res Ther; DOI: 10.1007/s10989-011-9282-8.
13. Burch R.M., Weitzberg M., Blok N., Muhlhauser R., Martin D.,
Farmer S.G., Bator J.M., Connor J.R., Green M., Ko C., Kuhn W.,
McMillan B.A., Raynor M., Shearer B.G., Tiffany C. (1991) N-(Flu-
orenyl-9-methoxycarbonyl) amino acids, a class of antiinflamma-
tory agents with a different mechanism of action. Proc Natl
Acad Sci USA;88:355–359.
28. Anwer M.K., Spatola A.F. (1980) An advantageous method for
the rapid removal of hydrogenolysable protecting groups under
ambient conditions: synthesis of leucine-enkephalin. Synthe-
sis;11:929–932.
29. Gowda D.C., Gowda B.K.K., Rangappa K.S. (2001) Sequence
dependence of oxidation of some repeating pentapeptide
sequences of elastin with electrolytically generated Mn(III): syn-
14. Hamilton G.S., Mewshaw R.E., Bryant C.M., Feng Y., Endemann
G., Madden K.S., Janczak J.E., Perumattam J., Stanton L.W.,
Yang X. (1995) Fluorenylalkanoic and benzoic acids as novel
inhibitors of cell adhesion processes in leukocytes. J Med
Chem;38:1650–1656.
15. Jan C.R., Wang J.L., Chou K.J., Cheng J.S., Lee K.C., Tseng L.L.,
Wang S.P., Tang K.Y., Huang J.K. (2000) NPC-14686, a novel
Chem Biol Drug Des 2012; 79: 850–862
861

Suhas and Channe Gowda
thesis, kinetics and mechanistic study. J Phys Org Chem;14:716–
724.
33. Winter C.A., Risley E.A., Nuss G.W. (1962) Carrageenan-induced
oedema in hind paw of the rat as an assay for anti-inflamma-
tory drugs. Proc Soc Exp Biol Med;111:544–547.
34. Kulkarni S.K. (1999) Hand Book of Experimental Pharmacology,
3rd edn. New Delhi: Vallabh Prakashan, p. 149.
35. Djahanjuiri B. (1969) Effect of a single dose of phentolamine
and mj 1999 on aspirin-induced gastric ulceration in rats. J
Pharma Pharmacol;21:341–345.
30. Quagliato D.A., Andrae P.M., Matelan E.M. (2000) Efficient pro-
cedure for the reduction of a-amino acids to enantiomerically
pure a-methylamines. J Org Chem;65:5037–5042.
31. Vogel G.H., Vogel W.H. (1998) Drug Discovery and Evaluation.
Berlin: Springer Verlag, p. 413.
32. Ghosh M.N. (1984) Fundamentals of Experimental Pharmacology,
2nd edn. Calcutta: Scientific Book Agency, p. 145.
862
Chem Biol Drug Des 2012; 79: 850–862