[Title of the invention]
New a-Acyl P-Phenylpropanoic Acid Derivative as PPAR-a Based Hypolipidemic
Agent.
[Field of the invention and use of invention]
The present invention relates to new a-acyl P-phenylpropanoic acid derivative as
PPAR-a based hypolipidemic agent.
[Prior art and problem to be solved/background of the invention]
Peroxisome Proliferator-Activated Receptors (PPARs) are ligand-activated
transcription factors which are members of the nuclear hormone receptor super
family. Generally, PPARs act as intracellular sensors for fatty acids, allowing cells to
adjust lipid metabolism (at the level of gene expression) after lipid availability
[Wilson, T.M. et. al. J. Med. Chem., 2000, 43(4), 527-550 and Berger, J. et. al.,
Annu. Rev. Med., 2002, 53, 409-435.].Three types of PPARs have been identified -
PPAR a, 8, and y. PPAR a is supposed to control mainly genes involved in fatty acid
P-oxidation and to play a pivotal role in energy homeostasis [Guerre-Millo, M. et. al.,
J. Biol. Chem., 2000, 275(22), 16638-16642.]. Activation of PPAR y leads to
modulation of genes involved in lipid metabolism and hormones that affects whole
body energy metabolism [Gage, J.R. et. al., Organic Synth., 1989, 68,83-91.]. PPAR 5
function has been recently linked to basal lipid metabolism, embryo implantation,
colon cancer, and inflammation [Escher, P. at. al., Endocrinology, 2001, 142(10),
4195—4202.]. PPAR a is an important modulator of metabolic syndrome which
include insulin resistance (hyperinsulinemia), abnormal glucose metabolism,
hypertension, dyslipidemia, obesity, hyperuricemia and hypercoagulation state. PPAR
a was recently been linked as possible therapeutic target for treating cardiovascular
complications. PPAR a occurs in mitochondria, peroxisomes, and microsomes in the
liver. PPAR a acts on transcription and protein levels of critical enzymes in 0-
oxidation and GO- oxidation pathways. Such enzymes include acyl CoA oxidase,
carnitine palmitoyl transferase I, mitochondrial hydroxymethylglutaryl-CoA synthase,
and cytochrome P450 4A enzymes (CYP4A). PPAR a on activation increases
expression of these genes and mediates hepatic fatty acid oxidation, whereas genetic
inactivation of PPAR a genes leads massive accumulation of lipids in the liver, severe
hypoketonemia, hypoglycemia, hypothermia, and elevated plasma free fatty acid
levels. Thus, it can be concluded that PPAR a is a key factor governing metabolic
adaptation to increased fatty acids level [Guan, Y. et al., J. Am. Soc. Nephrol., 2004,
15, 2801-2815.]. The structure of phenylpropanoic acid derivatives as PPAR a agonist
comprises of four key regions- these are a carboxylic acid head group, a central
phenyl ring, a linker and a lipophilic tail [Fievet, C. et. al., Curr. Opin. Pharm., 2006,
6(6), 606-614.]. On the basis of previous comparative QSAR studies [Tripathi, R.B.
et. al., Der. Pharma. Chemica, 2010, 2(2), 303-311.], molar refractivity value of 143
was found to be optimal for maximum agonist activity. New substitutions were
proposed in oxime ethers of a-acyl-p-phenylpropanoic acid in this range and (1) RMKT-
01 was proposed which also passes the Lipinski's rule of 5 for drug likeness.
Therefore, the present work involves the synthesis of new a-acyl-P-phenylpropanoic
acid and its characterization with the objectives of discovering novel and potent
hypolipidemic agent with minimum side effects. The synthesized compound was
evaluated for its hypolipidimic potency by in vitro transactivation assay to establish
importance of new derivative as hypolipidemic agent
1
(1)
[Objects of the invention]
The principal object of this invention is to provide a new a-acyl P-phenylpropanoic
acid derivative.
Another object of this invention is to provide a new PPAR-a based hypolipidemic
agent.
A further object is to provide a new hypoglycemic agent.
The present invention also relates to provide histopathological evaluation on
hepatotocytes.
Another object of this invention is to provide the process of preparation of new a-acyl
P-phenylpropanoic acid derivative and characterization of synthesized compound by
spectroscopic and chromatographic techniques for the authentication of structure and
purity.
Another object is to ascertain the bioactive conformation of the new a-acyl Pphenylpropanoic
acid derivative in the binding site of PPAR-a receptor by molecular
docking simulation studies.
A further object of this invention is to predict the toxicity profile of new a-acyl pphenylpropanoic
acid derivative by Computer Aided Drug Discovery Program PASS
(Prediction of Biological Activity Spectra for Substances).
[Summary of invention]
Clofibrate the most widely used hypolipidemic drug and other related agents are
relatively ineffective for Type Ila hyperlipidemia and they are not entirely devoid of
untoward side effects (Meinhertz, H et. al., Side effects of Drugs, Excerpta Medica,
Amsterdam and Oxford,1978,p358).
PPAR a is a key factor governing metabolic adaptation to increased fatty acids level
[Guan, Y. et. al., J. Am. Soc. Nephrol., 2004, 15, 2801-2815.]. Currently no marketed
hypolipidemic drug is available whose mechanism of action is based on activation of
PPAR a.
Hence it is a need of time to develop drugs, which are safe hypolipidemics.
The present invention relates to provide new a-acyl P-phenylpropanoic acid
derivative.
The present invention also relates to provide the process of preparation and
characterization via spectroscopy and chromatography, of new a-acyl Pphenylpropanoic
acid derivative.
Further, the present invention also relates to new a-acyl P-phenylpropanoic acid
derivative as hypolipidemic agent evaluated in-vitro.
The present invention also relates to provide a new hypoglycemic agent evaluated invivo.
The present invention also relates to provide histopathological evaluation on
hepatotocytes.
Also, the present invention relates to predict the toxicity profile of new a-acyl pphenylpropanoic
acid derivative by Computer Aided Drug Discovery Program PASS
(Prediction of Biological Activity Spectra for Substances).
[Detailed description]
Accordingly, the present invention provides a new a-acyl p-phenylpropanoic acid
derivative with structural formula (1) RM-KT-01.
OC4H9
(1)
In embodiment of the invention, a new a-acyl P-phenylpropanoic acid derivative with
structural formula (1), wherein
a-acyl P-phenylpropanoic acid derivative is:
(Z)-2-(3-(2-(2-(4-carbamoylphenyl)-5-methyloxazol-4-yl) ethoxy) benzyl)-3-
(butoxyimino) butanoic acid IUPAC name of (1) (RM-KT-01)
In embodiment of the invention, a novel use of the compound wherein compound
having structural formula (1), shows hypolipidemic activity.
In yet another embodiment of the invention, compound having structural formula (1)
produced an EC5o of 78 nM on PPAR a receptor studied in vitro transactivation assay.
In embodiment of the invention, a novel use of the compound wherein compound
having structural formula (1), shows hypoglycemic activity.
In vitro PPAR-a transactivation assay
The in vitro transactivation assay was determined spectrophotometrically in a 24 well
plate in ELISA reader. [Makadia, P. et. al., Bioorg. Med. Chem., 2011, 19, 771-782.]
Cell culture: HepG2 cells (ATCC, USA) were maintained in growth medium
composed of MEM (Sigma) supplemented with 10% FBS (Hyclone), 1 * MEM non
essential amino acid (Sigma) and 1 mM sodium pyruvate and 1%
penicillin/streptomycin (Sigma).
Transient transfection: HepG2 cells were seeded in 24 well plates at a density of
400,000 cells/well in 1 mL of medium per well. Cells were transfected using the
3
transfection reagent superfect (Qiagen). Cells were transfected with 0.08ug of the
pSG5 expression vector containing the cDNA of PPARa or PPARy or PPAR8 cotransfected
with PPRE3-TK-luc. Cells were incubated at 37 °C, 5% C02 for 3 h. After
this, 1.0 mL of the medium containing the ligands to the respective wells were added.
The cells were then incubated at 37 °C, 5% C02 for 20-22 h. After the incubation
period, cells were first washed with PBS, lysed and supernatant was collected.
Supernatant was then assayed for luciferase and P-galactosidase activity. The
luciferase activity was determined using commercial fire-fly luciferase assay
according to the supplier's [Promega] instructions in white 96-well plate [Nunc]. PGalactosidase
activity was determined in ELISA reader at 415 nm. The ratio of
luciferase versus P-galactosidase was calculated and fold induction was calculated
with respect to DMSO. EC50 values for the test compound were calculated by
nonlinear regression analysis using Graph Pad Prism .
The synthesized compound (1) RM-KT-01 was screened for human PPAR (hPPAR) a
agonistic activity on full length PPAR a receptor transfected in HepG2 cells as
described in the experimental section. The in vitro PPAR a agonistic activity of
synthesized compound was reported in terms of nM.
As shown in Fig. 1, EC50 of synthetic compound is 78 nM. The in vitro activity
suggests relevancy of presence of phenyl carboxamide group at one end and «-butyl
group attached with phenylpropanoic acid chain of oxime ether of a-acyl-P
phenylpropanoic acid. The result of in vitro transactivation assay also suggests that
(1) RM-KT-01 is PPAR a selective and shows no activity on PPAR y and PPAR 5.
c
o
o
<3
14<
12'
~ 6.
o 4«
llPPARc trajuartivation
EC50:
78 nM
> i mm i i MWI i i mm 1 » m i l 1
10-2.J 101-5 10-0.5 100.5 1015
Concentration (uM)
Fig. 1 EC50 value of synthetic compound when tested by in vitro transactivation assay.
EC50 value is the concentration of the test compound that affords half-maximum
transactivation.
Chemistry
The synthetic route for the synthesis of new a-acyl P-phenylpropanoic acid derivative
is depicted in Scheme 1.
! ^
Scheme 1 Procedure for the synthesis of new a-acyl P-phenylpropanoic acid
derivative.
CHO
V
+
4M HCI in dioxane
OH
CONH,
(I) (II)
H2NOC
H2NOC (VI)
H,NOC
N'
(IX)
N
I
OC4H,
H2NOC
80 °C
NaCN.DMF
H,NOC
KOH.aq.THF.reflux
BH3.SMe2,THF
H,NOC
(VII)
MeS02CI, Et,N,
HO N-OC4H,
Cs2C03
CH,CN
H2NOC
(VIII)
THF/MeOH/NaOH
°AH2NOC
(X)
OH
N
I
OC4H9
Synthesis of [(E)-{2E)-But-2-en-2-yl](4-carbamoylphenyl) methylidene} amino]olate
(III) - 4-Formyl benzamide (lg, 0.0067 mol) was dissolved in acetic acid (20ml) and
treated with diacetyl monooxime (0.670 gm, 0.0067 mol). A stream of dry HC1 was
bubbled for 2 h at 0 °C and for additional 2 h at ambient temperature through the
solution (slightly exothermic). The reaction mixture was poured onto ice water and
extracted two times with dichloromethane. The combined extracts were washed with
water, saturated aqueous sodium bicarbonate solution (until pH 8 was adjusted) and
brine. The organic layer was dried over sodium sulfate and the solution was
concentrated under reduced pressure. Chloroform was added and the solution was
brought to a volume of approximately 100 ml under reduced pressure. Chloroform
was added and the solution of crude dimethyl 2 (4-benzamide) oxazole 3-oxides was
cooled to 0 °C. %yield=28M%, TLC Solvent system; «Hexane:Chloroform:
Ethylmethylketone (6:2:2), Rf 4-formyl benzamide = 0.68, Rf butane 2,3 dione
monooxime = 0.77, Rf oxazolenoxide = 0.70.
Synthesis of 4-[4-Chloromethyl) 5-methyl-l, 3 oxazole -2-ylJ benzamide (IV) - In
oxazole N-oxide a solution of phosphorous oxychloride in chloroform was added
within 10 min. The reaction mixture was heated under reflux for 12 h, cooled to 0 °C
and made basic (pH 10) by carefully adding concentrated aqueous NH3 solution. The
suspension was poured onto ice water and extracted two times with dichloromethane.
The combined extracts were washed with ice water/brine and dried over sodium
sulfate. Removal of the solvent under reduced pressure gave yellow oil which was
purified by column chromatography (silica gel, cyclohexane/AcOEt) to obtain
compound as yellow oil, which solidified upon standing. % yield = 10%, TLC Solvent
system; «Hexane:Chloroform:ethyl methyl ketone (7:1.5:1.5), Rf 4-chloromethyl 5-
methyl oxazole benzamide = 0.75, Rf oxazole N-oxide = 0.71.
Synthesis of 4-[4-(Cyano methyl)- 5-methyl-l,3-oxazole2-ylJ benzamide (V) - To a
solution of 4(4-(chloro methyl)5-methyl oxazole-2yl)benzamide in DMF powdered
potassium cyanide and potassium iodide were added. Resultant mixture was heated to
80 °C for 3.5h. The resultant mixture was cooled to room temperature, potassium
carbonate was dissolve in water and added drop wise to the reaction mixture to
precipitate the product which was isolated by filtration and washed with water. %
yield = 57.25%. TLC Solvent system; n Hexane: Chloroform:Methanol (4:3:3), Rf
for4-(4-(Chloromethyl)-5 methyl oxazole-2-yl) benzamide = 0.67, Rf 4-(4-
(Cyanomethyl)-5 methyl oxazole-2-yl) benzamide = 0.64, m.p.= 317-320 °C.
Synthesis of 2-[2-(4-Carbamoylphenyl)-5-methyll,3-oxazole-4yl] aceticacid (VI) -
The above prepared 4(4-(Cyanomethyl)5methyloxazole2yl)benzamide (lgm, 0.0041
mol) was dissolved in 100 ml EtOH/water=l/l and treated with 10 eq. of NaOHpellets
. Hydrolysis was allowed to proceed over night at 85 °C. Pouring onto crashed
ice/HCI, twofold extraction with AcOEt, washing with water, drying over magnesium
sulfate, evaporation of the solvents, leads to the compound. % yield = 44.85%, TLC
Solvent system; «-hexane:chloroform:methanol (3:4:4), Rf for 4 (4(Cyanomethyl)-5-
methyloxazole-2-yl) benzamide = 0.66, Rf for 2(-2(4-carbamoyl phenyl) 5-
methyloxazole-4-yl) acetic acid= 0.58, m.p.= 355-358 °C.
Synthesis of 4-[4-(2-Hydroxyethyl)-5- methyl-1,3-oxazole -2-yl) benzamide (Vll) -
The above prepared 2(-2(4-Carbamoyl phenyl)5-methyloxazole-4-yl) acetic acid
(lgm, 0.0038 mol) was dissolved in 12 ml of abs. THF and treated at 0 °C with 1M
6
BH3 THF (2.5 eq.). The reaction mixture was then kept overnight at ambient
temperature. Careful quenching with MeOH and ice, twofold extraction with AcOEt,
washing with water and brine, drying over magnesium sulfate, and evaporation of the
solvents left a crude product which was refluxed for 30 min. in MeOH to liberate
quantitatively the free alcohol. % yield = 61%, TLC solvent system; nhexane:
chloroform:methanol (4:2:2), Rf for 2(-2(4-Carbamoyl phenyl)5-
methyloxazole-4-yl)acetic acid = 0.55, Rf for 4-(4-(2-Hydroxyethyl)-5- methyloxazole
-2-yl) benzamide = 0.58, m.p.= 328-332 °C.
Synthesis of {2-[2-(4-Carboyl phenyl)-5- methyl 1,3-oxazole-4-yl] ethoxyj methane
sulfonic acid (VIII) - A solution of crude 4-(4-(2-Hydroxyethyl)-5- methyloxazole -2-
yl) benzamide (lgm, 0.0040 mol) in CH2C12 was treated with methanesulfonyl
chloride, and triethylamine. The reaction mixture was stirred at ambient temperature
overnight and was diluted with CH2CI2 (20 ml). The mixture was washed with water,
and the organic layer was dried (MgSCu), filtered, and concentrated. The crude
product was purified by silica gel chromatography (hexanes/EtOAc 10/1 to 2/1) to
afford the title compound. % yield = 45%, TLC Solvent system; nhexane:
chloroform:ethyl methyl ketone (4: 3: 4), Rf for 4-(4-(2-Hydroxyethyl)-5-
methyloxazole -2-yl) benzamide = 0.61, Rf for 2(2-(4-Carboyl phenyl)-5-
methyloxazole-4-yl) ethoxy) methane sulfonic acid = 0.57, m.p.= 372-374 °C.
Synthesis of Methyl 2(3-(2-(2-(4-carbamoylphenyl)5-methyloxazole4-ylethoxy)
benzyl)3-(butoxyimino) butanoate (IX) - A round bottomed flask was initially
charged with Methyl2-(4-hydroxybenzyl)-3(butoxyimino)butanoate (lgm, 0.0034
mol), 2-(2-(4-Carbamoylphenyl)-5methyloxazole-4-yl)ethylmethanesulfonate
(1.15gm, 0.0034 mol), anhydrous DMF, and then cesium carbonate. The mixture was
stirred at ambient temperature for 16h. The reaction mixture was poured into ether.
The organic layer washed with brine, and dried with sodium sulphate. The crude
residue was diluted with dichloromethane and the mixture was stirred at ambient
temperature for 2h and concentrate under a stream N2 the crude product was purified
by chromatography to obtain the product. % yield = 40.6%, TLC Solvent system; nhexane:
chloroform:methanol (4:3:3), Rf for methyl 2-(4-Hydroxybenzyl)-3-
(butoxyimino) butanoate = 0.71, Rf for 2(2-(4-Carboyl phenyl)-5- methyloxazole-4-
yl) ethoxy) methane sulfonic acid = 0.60, Rf for Methyl 2-(3-(2-(2-(4-
carbamoylphenyl)-5-methyloxazole-4-yl)ethoxy)benzyl)3-(butoxyimino) butanoate =
0.66, m.p.= 280-285 °C.
Synthesis of 2-(3-(2-(4-Carbamoylphenyl)-5-methyloxazole-4-yl)ethoxy)benzyl)-3-
(butoxyimino) butanoic acid(X)- A round bottomed flask was charged with Methyl 2-
(3-(2-(2-(4-carbamoylphenyl)-5-methyloxazole-4yl)ethoxy)benzyl)3-
(butoxyimino)butanoate (lgm, 0.0019 mol), ethanol, and then aqueous 2N NaOH
.The solution was heated at 55 °C. for 1 h. The mixture was concentrated, acidified
using 5% H2SO4 (1.5 mL), and partitioned between CH2CI2 (15 mL) and brine (15
mL). The organic layer was dried (Na2S04), filtered, and concentrated and to gave
product. % yield = 36%, TLC Solvent system; n hexane:ethyl acetate: methanol
(4:2:4), Rf for Methyl2(3-(2-(2-(4-carbamoylphenyl)5methyloxazole-4-
yl)ethoxy)benzyl)3-(butoxyimino) butanoate = 0.58, Rf for Methyl 2-(3-(2-(2-(4-
carbamoylphenyl)-5methyloxazole-4-yl)ethoxy)benzyl)3-(butoxyimino) butanic acid
= 0.51, m.p. 310-315 °C. MS (ESI) m/z 507.31 (M4), m/z 508.8 (M+l), m/z 509.12
(M+2).calculated for C28H33N3O6: C, 66.26; H, 6.56; N, 8.28; O, 18.91. Found: C,
66.21; H, 6.48; N, 8.18; O, 18.13.
Spectral analysis of synthesized compound is shown in Table 1 and Table 2
Table 1 Infra Red spectral data of synthesized compounds (III-X).
Compound
III
IV
V
VI
VII
VIII
IX
X
Spectra] data (cm1)
1660 (C=0 str. of amide), 3284 (N-H str. of primary amine), 1151 (C-O-C str.), 1697
(C=N str. of oxime), 1461 & 1377 (asymmetric and symmetric peaks of N=0),
1585&1461 (aromatic C=C ring str.), 2921 (C-H str.)
1647 (C=0 str. of amide), 3286 (N-H str. of pri. amine), 1614, 1485 (aromatic C=C
ring), 1130 (C-O-C Str.), 1693 (C=N Str. of oxime), 750 (C-Cl Str.), 2928 (aromatic CH
str.)
3387, 3280 (N-H str. of pri. amine), 1652 (C=0 str. of amide), 1660 (C=N str. of
oxime), 1143 (C-O-C str.) 1298 (C-N str.), 2240 (C=N str. of nitrile) 1577-1507 &
1450-1402 (aromatic C=C ring str.), 3140 (aromatic C-H str.) 2953 (aliphatic C-H str.)
1640 (C=0 str. of amide), 3350-3261 (N-H str. of primary amine), 1128 (C-O-C str.),
1680 (C=N str. of oxime), 1280 (C-N str.), 1710 (C=0 str. of carboxylic acid), 1260 (CO
str. of carboxylic acid), 1591-1517 & 1481-1414 (aromatic C=C ring str.), 3110
(aromatic C-H str.)
3337-3290 (N-H str. of primary amine), 1625 (C=0 str. of amide), 1143(C-0-C str.),
1660 (C=N str. of oxime), 1298 (C-N str. Of amide), 1577-1500, 1450-1402 (aromatic
C=C ring str.), 2963 (aliphatic C-H str.), 3068 (aromatic C-H str.) & a broad peak of
OH.
3360, 3261 (N-H str. of amine), 1660 (C=0 str. of amide), 1112 (C-O-C str.), 1242(C-N
str.), 1689 (C=N str. of oxime), 1531-1448 (aromatic C=C ring str.), 1340, 1151(asym.
& symmetric S=0 str.), 654 (S-0 str. of sulfonic acid), 2941 (aliphatic C-H str.),
3050 (aromatic C-H str.)
1647 (C=0 str. of amide), 3343 & 3270 (NH str. of pri. amine), 1172 (C-O-C str.), 1676
(C=N str. of oxime), 1257, 1022 (asym. & symm. str. of Ar-O- R), 1720 (C=0 str. of
ester), 1600-1442 (aromatic C=C ring str.), 3072 (aromatic C-H str.), 2948 (aliphatic CH
str.)
1652 (C=0 str. of amide), 3351, 3278 (NH str. of pri. amine), 1108(C-O-C str.),
1667(C=N str.), 1253, 1170 (asym. &sym. str. of Ar-O-R), 1690 (C=0 str. of acid),
1218 (C-0 str. of acid) 1614-1438 (C=C str. of aromatic ring), 2952 (aliphatic C-H str.),
3031 (aromatic C-H str.)
s
Table 2 *HNMR spectral data of synthesized compounds (V-X).
Compound Spectral data.
5 7.98 (dd, 2H, J„= 8.2 Hz, Jm = 2.36 Hz) 5 7.97 (dd, 2H, J0= 8.12 Hz, Jm = 2.34 Hz) 5
6.17 (s,2H), 8 3.67 (s,2H), 8 2.43 (s,3H).
VI 8 11.59 (s,lH), 8 7.99, (dd, 2H, J0 = 7.2 Hz, Jm = 1.96 Hz), 8 7.96 (dd, 2H, J0 = 6.23 Hz,
Jm= 2.2 Hz), 8 6.17 (s, 2H), 8 4.14 (s, 2H), 8 2.13 ( s, 3H).
K// 8 11.80 (s, 1H), S 7.96 (dd, 2H, J0 = 8.4 Hz, Jm = 2.04 Hz), 5 7.93 (dd, 2H, J0 =7.8 Hz, J„
= 2.24 Hz), 8 2.95, (t, 2H, J= 7.4 Hz), 8 3.65 (t, 2H, J= 7.2 Hz), 8 6.17 (s, 2H), 5 2.42 (
s, 3H).
VIII 8 7.96 (dd, 2H, J0 = 8.4 Hz, Jm = 2.04 Hz), 8 7.93 (dd, 2H, J0 = 7.8 Hz, Jm = 2.24 Hz), 8
2.95 (t, 2H, 7= 7.4 Hz), 8 3.65 (t, 2H, J= 7.2 Hz), 5 6.17 (s, 2H), 8 2.42 (s,3H), 8 1.80 (
s,lH).
IX 8 8.14 (dd, 2H, J„= 8.36 Hz, J„ = 2.76 Hz), 8 7.95 (dd, 2H, J„= 7.6 Hz, Jm= 2.86 Hz), 8
6.95 (dd, 2H), 8 6.77 (dd, 2H), 8 4.56 (s, 2H), 8 4.26 (dd, 1H, J= 7.36 Hz), 8 3.65 ( s,
3H), 8 3.49 (t, 2H, J= 7.2 Hz), 8 3.16 ( q ,2, J„= 7.2 Hz ), 8 3.14 (t, 3H, J= 7.36 Hz), 8
3.04 (t, 2H, J= 7.36 Hz), 8 2.73 (dd, 2H, J„ = 7.36 Hz), 8 1.60(q, 2H, J„ = 7.36 Hz), 8
1.49 (q, 2H, J0= 7.36Hz), 8 0.93(s, 3H, 7= 7.2Hz).
X 8 11.10 (s, 2H), 8 8.12 (dd, 2H, J0= 8.36 Hz, Jm= 2.96 Hz), 8 7.94 (dd, 2H, J„= 7.6 Hz,
Jm = 2.86 Hz), 8 6.97 (dd, 2H, Ju = 7.4 Hz, Jm = 2.86 Hz), 8 6.74 (dd, 2H, J„ = 7.2 Hz,
ym= 2 Hz), 8 5.89 (s, 2H), 8 5.31 (t, 2H, J = 7.36), 8 4.22 (t, 2H, J= 7.36 Hz), 8 3.49 (t,
1H, J„= 7.2 Hz), 8 3.22 (t, 2H, J= 7.36 Hz), 8 3.17 (s, 3H), 8 2.62 (dd, 2H, J„ = 7.36
Hz), 8 2.42 (s, 3H), 8 1.50 (q, 2H, Ju= 7.36 Hz), S 1.50 (q, 2H, J„= 7.36 Hz), 8 1.39 (q,
2H,J0=7.36Hz).
In vivo effects of (1) RM-KT-01 on i.p. Glucose Tolerance Test (i.p. GTT)
Encouraged with the in vitro PPAR a agonistic activity, (1) RM-KT-01 was then
tested for hypoglycemic activity. An i.p. Glucose Tolerance Test (i.p. GTT) was
conducted on three groups of Albino wistar rats, each group having six animals each,
i.p. GTT was performed as described in experimental section and the results were
reported as in Table 3.
As shown in Table 3, glibenclamide, a known hypoglycemic drug, reduces blood
glucose level to normal in 60 minutes and subsequently reduces glucose level from
normal, whereas (1) RM-KT-01 lowers blood glucose level after 60 minutes but
maintains after 180 minutes. In other words standard drug causes hypoglycemia and
maintains the condition for long duration whereas test drug lowers blood glucose level
in normal pace and maintains blood glucose level afterwards. For more satisfactory
conclusion, test compound was evaluated on in vivo hypoglycemic animal model.
M^„
Table 3 Hypoglycemic activity of synthetic compound on BGL (mg/dl) of glucose
fed rats (LP. Glucose Tolerance Test).
Groups
I-
(Glucose
control)
II-
(Standard)
III-
(Treated)
Dose
Vehicle
Glibenclamide
(5 mg/Kg)
RM-KT-01
(5mg/Kg)
Time (minutes)
0
86.50±2.89
85.50±1.76
86.00±5.41
30
145.83±3.87
141.7±3.45*'*
135.25±4.43*
60
120.17±2.41
94.33±2.81
105.17±3.17
90
110.00±2.67
81.50±6.29*"*
80.83±4.94*"
180
80.67±3.36
65.17±4.64-"
69.50±5.14*
Results were expressed in Mean±SEM, N= 6 and analysed by one-way ANOVA followed by
Dunnett Test. * significant difference( P < 0.05) compared to group 2, " significant difference ( P <
0.01) and significant difference (P < 0.001) vs group 1 at respective time in minutes.
In vivo effects on alloxan induced diabetic animals
Encouraging in vitro results and satisfactory i.p. GTT test suggests potency of (1)
RM-KT-01. Thus for more conclusive results, test compound was investigated on
alloxan induced diabetic animal model.
As shown in Table 4, compared with Group I on day 4 , blood glucose level from
Group II induced by alloxan showed a significant increase (P < 0.05) and it indicated
that alloxan induced diabetic animal model was established. After treatments in 7
days diabetic model, the blood glucose level of Group III and Group V decreased
significantly (P < 0.05) when compared with Group II. Thus above results depict that
the synthetic test drug act in same pace as standard i.e. glibenclamide to normalize
blood glucose level.
U^Table 4 In vivo effect of synthetic compound on BGL (mg/dl) of alloxan treated rats
Groups
I- (Normal
Control)
II- (Diabetic
Control)
III-
(Standard)
IV- (Treated)
V - (Treated)
Dose
Vehicle
Alloxan
(160mg/Kg)
GHbenclamide
(5mg/Kg)
RM-KT-01
(5mg/Kg)
RM-KT-01
(lOmg/Kg)
Time (days)
0
80.60±2.29
82.60±2.60
75.60±3.70*
68.60±4.62***
74.60±4.11*
4
73.60*3.41***
453.20±5.38
402.80±6.29**
427.00±6.99
472.44.26*"
7
84.20±2.25***
263.20*3.02
120.20±4.76***
200.60±4.45*
150.00±5.12***
Results were expressed in MeaniSEM, N= 5 and analysed by one-way ANOVA followed by
Dunnett Test. * significant difference( P < 0.05) compared to group 2, * significant difference
(P < 0.01) and '** significant difference (P < 0.001) vs group 2 on respective day.
Histopathological evaluation:
Fig. 2, indicates a clear demarcation between control (A) and alloxan treated liver (B)
as later shows marked inflammation and vacuolization. This inflammation [Cindoruk
M., et. al. BMC Gastroenterology 2007; 7(44)] and vacuolization are signs of cell
injury. They occur due to increased permeability of cell membranes to intercellular
water leading to water accumulation within the cell. This produces cytoplasmic
vacuolization [Hussein W. F., et. al. Life Science Journal 2011; 8(3): 373-383].
Group III (C) shows ground glass appearance of hepatocytes with the appearance of
some binucleated cells. In Group (C), alloxan induced cell inflammation along with
binucleated nuclei indicates partial improvement when treated with standard drug
glibenclamide. Ground glass appearance reflects adaptation of cells upon recovery
[Hussein W. F., et. al. Life Science Journal 2011; 8(3): 373-383]. Group IV (D) and
Group V (E) shows ground glass appearance with binucleated cells indicating partial
regeneration, but there is still presence of some cytoplasmic vacuolization. Thus,
histopathological evaluation shows satisfactory results which supports
pharmacological efficacy of (1) RM-KT-01.
All together, in vivo results of animal diabetic show that (1) RM-KT-01 exhibits good
hypoglycemic activity in Albino wistar rats.
$
Fig. 2 Histopathological evaluation of rat liver after alloxan administration for
diabetes and treatment with respective drugs. Group I (A) shows a normal liver from
control group; Group II (B) shows inflammation and cytoplamic vacuolization
(arrows) caused by alloxan; Group III (C) treated with alloxan and standard drug
glibenclamide (5 mg/Kg) shows ground glass appearance (arrow) indicating signs of
recovery; Group IV (D) treated with test drug (5 mg/Kg) and Group V (E) treated
with test drug (10 mg/Kg) shows binucleated cells and ground glass appearance
indicating signs of recovery.
Molecular Docking Simulations
Following steps were undertaken to carry out molecular docking simulations on
propanoic acid derivative (3-[5-methoxy-l-(4-methoxy-benzenesulfonyl)-lH-indol-3-
yl]-propanoic acid) as PPAR-a through softwares AutoGrid 4 and AutoDock 4.
Ligand Preparation: All 2-D chemical structures were sketched in ChemDraw™
Ultra 8.0 (ChembridgeSoft Corporation, MA, USA). These 2D structures were
converted into 3D by Chem3D™ Ultra 8.0 (ChembridgeSoft Corporation, MA, USA).
These chemical structures were geometrically minimized through MM2 method by
taking default dynamics parameters (step interval 2 fs, frame interval 10 fs) on a PC
with operating system Windows XP. Electrostatic charges were assigned by
Gastgeiger-Hiickel method. These conformations were then utilized as starting
conformations to perform molecular docking.
Receptor Preparation: The X-ray crystal structure of PPAR a (pdb id: 3etl) was
obtained from Brookhaven Protein Data Bank (http://www.rcsb.org/pdb).
Grid Box Formation: The binding pocket in the receptor was defined by a grid box
(no. of grid points: 42x, 48y, 40z; spacing: 0.375; grid center: 6.86x, 32.614y, -
7.929z). All extended conformations of ligands fit in the box. This was ensured by
placing the grid box in the centre of the ligand.
Docking Method Validation: To ensure that the ligand orientations and positions
obtained from the docking studies represent valid and reasonable potential binding
modes of the inhibitors, the docking methods and parameters used were validated by
redocking the crystallized propanoic acid derivative and overlaying the docked and
crystallized propanoic acid derivative, chemical structures and calculating the rms
value.
Grid Maps Preparation Autogrid: utility of the AutoDock suite was run to prepare
map files for different atom types in ligands and receptor viz. A, C, OA, N, NA, SA.
These map files are in turn taken up by AutoDock for carrying docking simulations.
Docking Parameters: Primary approach for conformational searching in AutoDock is
a Lamarckian genetic algorithm (LGA). A population of trial conformations is
created, and then in successive generations these conformations mutate, exchange
conformational parameters, and compete in a manner analogous to biological
evolution, ultimately selecting individuals with lowest binding energy. The
"Lamarckian" aspect is an added feature that allows individual conformations to
search their local conformational space, finding local minima, and then pass this
information to later generations. It uses a semi empirical free energy force field to
predict binding free energies of small molecules to macromolecular targets. The force
field is based on a comprehensive thermodynamic model that allows incorporation of
intramolecular energies into the predicted free energy of binding. This is performed
by evaluating energies for both the bound and unbound states.
Result Analysis of Docking Simulation: The results of all dockings were evaluated
based on hydrophobic and polar interactions between ligand and receptor active site
residues and the calculation of binding energy which must come in the empirical
range -5 to -15 kcal/mol.
Validation of molecular docking simulation.
The reference ligand forms hydrogen bond interactions with CYS276, SER280,
TYR314 and TYR464. Binding energy of proposed molecules was within the
empirical range of-5 to -15 kcal/mol. Figs. 3-4 illustrate the docking mode of
^Wr 'Jgfr
propanoic acid derivative with PPAR a and superimposed picture of docked and
crystallized propanoic acid derivative with a low rms value of 0.51. A good
retrospective validation with a training set consisting of AZ242, GW735,
Gemfibrozil, clofibrate and benzafibrate [Cronet, P. et. al., Structure, 2001, 9, 699-
706; Sierra, M.L. et. al., J. Med. Chem., 2007, 50(4), 685-695 and Scatena, R. et. al.,
Eur. J. Pharmacol., 2007, 567(1-2), 50-58.] paved the way for docking molecules
from different reported series.
Tyr314A
Fig. 3 (a) 2-D picture of binding interactions of propanoic acid derivative with
residues of PPAR a receptor. (Courtesy RCSB Protein Data Bank) (b) Overlay of
crystal and docked bioactive conformations of propanoic acid derivative with rms
value 0.51. (Black dashed lines - hydrogen bonds, salt bridges, metal interactions;
green solid lines - hydrophobic interactions.)
K>^k>\*
#rh
14
Fig. 4 Bioactive conformation of (1) RM-KT-01 with interactions (dotted lines) to
residues of PPAR a site. (Atom colour codes: Red (O), Blue (N), White (C) and
Yellow (S)). Residues involved are indicated with sequence number.
Table 3 Molecular docking results for some PPAR a receptor agonists.
C.N.
Propanoic
acid
derivative
AZ242
GW735
Gemfibrozil
Clofibrate
Benzafibrate
(1) RM-KT-
01
Obs. Ki
(uM)
0.5-1.0
1
0.004-
0.02
230
700
1000
0.08
Pred. Ki
OiM)
1.17
16.57
0.693
44.28
46.08
99.26
0.20
Binding
energy
(kcal/mole)
-8.09
-6.52
-8.4
-5.94
-5.92
-5.46
-9.15
Residues
involved in
molecular
interactions
CYS276,
SER280,
TYR314,
TYR464
ILE317,
PHE273
HIS440,
TYR464,
THR279,
SER280
SER280
PHE273,
GLN277
PHE351,
SER280,
CYS276
GLU269,
LEU331,
VAL 332,
THR279,
SER 280
Hydrophobic
Pocket
PHE273,ILE
354, MET355
PHE273
PHE273,
PHE351
PHE273,
PHE351
PHE273
PHE273
PHE 273
^
o^
iH
The docking area consists of a lipophilic pocket lined by aromatic amino acid residues
PHE273, ILE 354, and MET 355 as depicted in Table 3.
Docking simulation on proposed molecule
In order to ascertain the interaction of the proposed molecule with the residues of the
target receptor and predict their binding affinity, a series of docking and scoring
functions were performed. On the basis of such studies it was found that the (1) RMKT-
01 molecule should show more potency compared to other molecules which were
proposed on the basis of QSAR equation.
Prediction of toxicity profile
In order to accelerate search for potent New Chemical Entities (NCEs), the assistance
of computer-aided drug discovery program PASS (Prediction of Biological Activity
spectra) was used to predict the activity of potent (1) RM-KT-01 molecule. PASS
predicts not only for the desirable pharmacological effect but also for molecular
mechanisms of action and different unwanted side effects like mutagenicity,
carcinogenicity, teratogenicity and embryotoxicity. Such analysis of heterogeneous
sets increases considerably the chance of discovering NCEs. The (1) RM-KT-01
molecule did not show any undesirable effects when passed through PASS.
Acknowledgements: The authors are thankful to Dr. Rajesh Bahekar, Kamlesh
Thakur,
Ritesh Mathure, Monika Shringi and Saumya Gupta who have helped compiling this
work.
,uA
©*t
16

U »'*-
IGIHW-'
We claim,
1. A new a-acyl p-phenylpropanoic acid derivative of formula ((11)).. \ 3 ^ ^
H,NOC
(1)
2. A new a-acyl p-phenylpropanoic acid derivative of formula (1) as claimed in
claim 1 wherein a-acyl P-phenylpropanoic acid derivative is:
(Z)-2-(3-(2-(2-(4-carbamoylphenyl)-5-methyloxazol-4-yl)ethoxy)benzyl)-3
(butoxyimino) butanoic acid (1) (RM-KT-01)
3. A process for the preparation of new a-acyl p-phenylpropanoic acid derivative
having general formula (1) as claimed in claim 1 and depicted in scheme 1.
CONH
^ OH
(II)
4M HCI in dioxanc
?^
HjNOC
N
(111)
CHCIj.POCI,
N'
N
HjNOC ^ (,v)
O ^ ,COOH
(VI)
KOH.aqTHFrcnux
BH,.SMe2.THF
H2NOC
„..f- McSOjCL EtjN.
(IX)
N
OCjH,
N-OCjH,
CsjCOj
CH,CN
H2NOC
O - ^ /—0
(VIll) °ir
THF/MoOH/NaOH
(X)
OC,H, J J
0 A i ^ ^
^^'•^ I ^ r^ '^m r>
Synthesis of [(E)-{2E)-BUt-'2^7^-P2--2y-ly]l(]4{-4c-acrahrabmamoyolyplhpehneynly) l)m etmheytlhidvelindeejn ef
amino]olate (III) - 4-Formyl benzamide (Ig, 0.0067 mol) was dissolved in
acetic acid (20ml) and treated with diacetyl monooxime (0.670 gm, 0.0067
mol). A stream of dry HCl was bubbled for 2 h at 0 °C and for additional 2 h
at ambient temperature through the solution (slightly exothermic). The
reaction mixture was poured onto ice water and extracted two times with
dichloromethane. The combined extracts were washed with water, saturated
aqueous sodium bicarbonate solution (until pH 8 was adjusted) and brine. The
organic layer was dried over sodium sulfate and the solution was concentrated
under reduced pressure. Chloroform was added and the solution was brought
to a volume of approximately 100 ml under reduced pressure. Chloroform was
added and the solution of crude dimethyl 2 (4-benzamide) oxazole 3-oxides
was cooled to 0 °C. %yield=28M%, TLC Solvent system;
nHexanerChloroform: Ethylmethylketone (6:2:2).
Synthesis of 4-[4-Chloromethyl) 5-methyl-l, 3 oxazole -2-yl] benzamide (IV) -
In oxazole N-oxide a solution of phosphorous oxychloride in chloroform was
added within 10 min. The reaction mixture was heated under reflux for 12 h,
cooled to 0 °C and made basic (pH 10) by carefully adding concentrated
aqueous NH3 solution. The suspension was poured onto ice water and
extracted two times with dichloromethane. The combined extracts were
washed with ice water/brine and dried over sodium sulfate. Removal of the
solvent under reduced pressure gave yellow oil which was purified by column
chromatography (silica gel, cyclohexane/AcOEt) to obtain compound as
yellow oil, which solidified upon standing. % yield = 10%, TLC Solvent
system; «Hexane:Chloroform:ethyl methyl ketone (7:1.5:1.5).
Synthesis of 4-f4-(Cyano methyl)- 5-methyl-l,3-oxazole2-yl] benzamide (V) -
To a solution of 4(4-(chloro methyl)5-methyl oxazole-2yl)benzamide in DMF
powdered potassium cyanide and potassium iodide were added. Resultant
mixture was heated to 80 °C for 3.5h. The resultant mixture was cooled to
room temperature, potassium carbonate was dissolve in water and added drop
wise to the reaction mixture to precipitate the product which was isolated by
filtration and washed with water. % yield = 57.25%. TLC Solvent system; n
Hexane: Chloroform:Methanol (4:3:3), m.p.= 317-320 °C.
Synthesis of 2-[2-(4-Carbamoylphenyl)-5-methyll,3-oxazole-4yl] aceticacid
(VI) - The above prepared 4(4-(Cyanomethyl)5methyloxazole2yl)benzamide
(Igm, 0.0041 mol) was dissolved in 100 ml EtOH/water=l/l and treated with
10 eq. of NaOH-pellets . Hydrolysis was allowed to proceed over night at 85
°C. Pouring onto crashed ice/HCI, twofold extraction with AcOEt, washing
with water, drying over magnesium sulfate, evaporation of the solvents, leads
to the compound. % yield = 44.85%, TLC Solvent system; nhexane:
chlorofonn:methanol (3:4:4), m.p.= 355-358 °C.
Synthesis of 4-[4-(2-Hydroxyethyl)-5- methyl-1,3-oxazole -2-yl) benzamide
(VII) - The above prepared 2(-2(4-Carbamoyl phenyl)5-methyloxazole-4-yl)
acetic acid (Igm, 0.0038 mol) was dissolved in 12 ml of abs. THF and treated
at 0 °C with IM BH3 THF (2.5 eq.). The reaction mixture was then kept
overnight at ambient temperature. Careful quenching with MeOH and ice.
18
t> vxA
>r''^
' — •?• K"
i ti.-"-X^'3MAY 20»
twofold extraction with AcOEt, washing with water and brine, drying over
magnesium sulfate, and evaporation of the solvents left a crude product which
was refluxed for 30 min. in MeOH to liberate quantitatively the free alcohol.
% yield = 61%, TLC solvent system; n-hexane:chlorofonn:methanol (4:2:2),
m.p.= 328-332 °C.
Synthesis of {2-[2-(4-Carboyl phenyl)-5- methyll,3-oxazole-4-yl] ethoxy}
methane sulfonic acid (VIII) - A solution of crude 4-(4-(2-Hydroxyethyl)-5-
methyloxazole -2-yl) benzamide (Igm, 0.0040 mol) in CH2CI2 was treated
with methanesulfonyl chloride, and triethylamine. The reaction mixture was
stirred at ambient temperature overnight and was diluted with CH2CI2 (20 ml).
The mixture was washed with water, and the organic layer was dried
(MgS04), filtered, and concentrated. The crude product was purified by silica
gel chromatography (hexanes/EtOAc 10/1 to 2/1) to afford the title compound.
% yield = 45%, TLC Solvent system; n-hexane:chloroform:ethyl methyl
ketone (4: 3: 4), m.p.= 372-374 °C.
Synthesis of Methyl 2(3-(2-(2-(4-carbamoylphenyl)5-methyloxazole4-ylethoxy)
benzyl)3-(butoxyimino) butanoate (IX) - A round bottomed flask was
initially charged with Methyl2-(4-hydroxybenzyl)-3(butoxyimino)butanoate
(Igm, 0.0034 mol), 2-(2-(4-Carbamoylphenyl)-5methyloxazole-4-
yl)ethylmethanesulfonate (1.15gm, 0.0034 mol), anhydrous DMF, and then
cesium carbonate. The mixture was stirred at ambient temperature for 16h.
The reaction mixture was poured into ether. The organic layer washed with
brine, and dried with sodium sulphate. The crude residue was diluted with
dichloromethane and the mixture was stirred at ambient temperature for 2h
and concentrate under a stream Nj the crude product was purified by
chromatography to obtain the product. % yield = 40.6%, TLC Solvent system;
«-hexane:chloroform:methanol (4:3:3), m.p.= 280-285 °C.
Synthesis . of 2-(3-(2-(4-Carbamoylphenyl)-5-methyloxazole-4-
yl)ethoxy)benzyl)-3-(butoxyimino) butanoic acid(X)- A round bottomed flask
was charged with Methyl 2-(3-(2-(2-(4-carbamoylphenyl)-5-methyloxazole-
4yl)ethoxy)benzyl)3-(butoxyimino)butanoate (Igm, 0.0019 mol), ethanol, and
then aqueous 2N NaOH .The solution was heated at 55 °C. for 1 h. The
mixture was concentrated, acidified using 5% H2SO4 (1.5 mL), and partitioned
between CH2CI2 (15 mL) and brine (15 mL). The organic layer was dried
(Na2S04), filtered, and concentrated and to gave product. % yield = 36%, TLC
Solvent system; n hexane:ethyl acetate: methanol (4:2:4), m.p. 310-315 °C.
4. Use of compound as claimed in claim 1, wherein the compound having
general structural formula (1), shows PPAR a receptor agonistic activity and
the ECso value is 78 nM.
5. The new compound a-acyl P-phenylpropanoic acid derivative as claimed in
(1) possesses in vivo hypoglycemic activity. It showed dose dependency in
alloxan induced diabetic rats with BGL (mg/dl) of 200.60±4.45 at 5mg/Kg and
150.00±5.12 at lOmg/Kg dose after 7 days of treatment which is equivalent to
standard drug glibenclamide at 5mg/kg.
19
\ ^
IHWI
O 3 m 20H
6. The new compound a-acyl p-phenylpropanoic acid derivative as claimed in
(1) possesses in vivo hypoglycemic activity. In Glucose Tolerance Test
conducted on glucose fed rats, glibenclamide, a known hypoglycemic drug,
reduces blood glucose level to normal in 60 minutes and subsequently reduces
glucose level from normal, whereas (1) lowers blood glucose level after 60
minutes but maintains after 180 minutes. In other words standard drug causes
hypoglycemia and maintains the condition for long duration whereas test drug
lowers blood glucose level in norma! pace and maintains blood glucose level
afterwards. It showed BGL (mg/dl) of 69.50±5.14 after 180 minutes of
treatment at 5mg/Kg dose.
7. The new compound a-acyl (3-phenylpropanoic acid derivative as claimed in
(1) on histopathological evaluation shows ground glass appearance with
binucleated cells indicating partial regeneration of hepatocytes (doses 5
mg/Kg and 10 mg/Kg) fi-om inflammation and cytoplamic vacuolization
caused by exposure to alloxan in comparison to standard drug glibenclamide
(dose 5 mg/Kg).