Synthesis and analgesic activity of 2-methyl-2-[1-(3-benzoyl-4- substituted-1,4-dihydropyridyl)]acetic acid methyl esters, acetic acids, and acetamides

Article abstract of DOI:10.1002/(SICI)1521-4184(19996)332:6<213::AID-ARDP213>3.0.CO;2-G

A group of 2-methyl-2-[1-(3-benzoyl-4-substituted-1,4- dihydropyridyl)]acetic acid methyl esters (7), weak acetic acids (8), and acetamides (9) were designed for evaluation as less acidic non-ulcerogenic non-steroidal antiinflammatory drugs (NSAIDs). In this respect, the model compound 2-methyl-2-[1-(3-benzoyl-4-phenyl-1,4-dihydropyridyl)]acetic acid (8a), unlike traditional arylacetic acid NSAIDs, was shown to be a weak acid with a pK(a) of 9.17. In contrast to arylacetic acid NSAIDs, the α- methylacetic acid sodium salt of 8a, or the methyl α-methylacetate ester (7a) did not inhibit cyclooxygenase-1 (COX-1) or -2 (COX-2). In vitro stability studies showed that the methyl α-methylacetate ester (7a) acts as a prodrug to the α-methylacetic acid derivative (8a), undergoing rapid (< 10 minutes) and quantitative conversion upon incubation with rat plasma, or incubation with rat liver homogenate (t(1/2) = 25 min). In contrast, the α- methylacetamide (9a) underwent negligible (< 2%) conversion to the α- methylacetic acid derivative (8a) upon incubation with either rat plasma, or rat liver homogenate, for incubation times up to 24 h. The effect of a C-3 para-substituted-benzoyl substituent (R¹ = H, Cl, Me), a C-4 substituent (R² = aryl, benzyl, cyclohexyl, alkyl), and the nature of the N¹-acetic acid moiety [methyl ester (R³ = OMe), acetic acid (R³ = OH), acetamide (R³ = NH₂)] on analgesic activity was determined using the 4% NaCl-induced abdominal constriction (writhing) assay. Compounds 7-9 inhibited writhing 27- 95% relative to the reference drug aspirin (58% inhibition). The analgesic potency with respect to the para-benzoyl substituent was H > Cl or Me. Although the effect of the C-4 R²-substituent on analgesic activity was variable within the ester, acid and amide sub-groups of compounds, compounds having a R²-cyclohexyl substituent generally provided superior analgesic activity relative to those having a lipophilic alkyl substituent. The nature of the R³-substituent (OMe, OH, NH₂) was a determinant of analgesic activity where the potency order was acetic acid methyl ester > acetic acid or acetamide, except when the C-4 R²-substituent was cyclohexyl or benzyl where the potency order was acetamide > acetic acid methyl ester or acetic acid. Reduction of the 5,6-olefinic bond of the 1,4-dihydropyridyl compound (9a, 94% inhibition) to the corresponding 1,2,3,4-tetrahydropyidyl derivative (10, 69% inhibition) reduced analgesic activity.

Full text of DOI:10.1002/(SICI)1521-4184(19996)332:6<213::AID-ARDP213>3.0.CO;2-G

Substituted Acetic Acid Derivatives
213
Synthesis and Analgesic Activity of
2-Methyl-2-[1-(3-benzoyl-4-substituted-1,4-dihydropyridyl)]acetic Acid
Methyl Esters, Acetic Acids, and Acetamides
a)
a)
b)
a),
Sammy A. Agudoawu , Sai-hay Yiu , John L. Wallace , and Edward E. Knaus *
a) Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2N8
b) Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada T2N 4N1
Key Words: 1,4-Dihydropyridines; acetic acid esters; acetic acids; acetamides, analgesic activity
[1]
4S*,2S*; 4R*,2S*; 4S*,2R*) . Aryl- and heteroarylacetic
Summary
acid derivatives that exhibit analgesic and non-steroidal anti-
inflammatory (NSAID) activities possess several common
structural features. These include, (a) a carboxyl group or its
equivalent (ester, amide) separated by one carbon atom from
a flat aromatic nucleus, and (b) one or more large lipophilic
groups attached to the aromatic nucleus that is two, three, or
four carbon or heteratoms removed from the point of attach-
A group of 2-methyl-2-[1-(3-benzoyl-4-substituted-1,4-dihy-
dropyridyl)]acetic acid methyl esters (7), weak acetic acids (8), and
acetamides (9) were designed for evaluation as less acidic non-ul-
cerogenic non-steroidal antiinflammatory drugs (NSAIDs). In this
respect, the model compound 2-methyl-2-[1-(3-benzoyl-4-
phenyl-1,4-dihydropyridyl)]acetic acid (8a), unlike traditional
arylacetic acid NSAIDs, was shown to be a weak acid with a pKa
of 9.17. In contrast to arylacetic acid NSAIDs, the α-methylacetic
acid sodium salt of 8a, or the methyl α-methylacetate ester (7a)
did not inhibit cyclooxygenase-1 (COX-1) or -2 (COX-2). In vitro
stability studies showed that the methyl α-methylacetate ester (7a)
acts as a prodrug to the α-methylacetic acid derivative (8a),
undergoing rapid (< 10 minutes) and quantitative conversion upon
incubation with rat plasma, or incubationwith rat liver homogenate
(t1/2 = 25 min). In contrast, the α-methylacetamide (9a) underwent
negligible (< 2%) conversion to theα-methylacetic acid derivative
(8a) upon incubation with either rat plasma, or rat liver homogen-
ate, for incubation times up to 24 h. The effect of a C-3 para-sub-
stituted-benzoyl substituent (R1 = H, Cl, Me), a C-4 substituent (R2
= aryl, benzyl, cyclohexyl, alkyl), and the nature of the N1-acetic
acid moiety [methyl ester (R3 = OMe), acetic acid (R3 = OH),
acetamide (R3 = NH2)] on analgesic activity was determined using
the 4% NaCl-induced abdominal constriction (writhing) assay.
Compounds 7–9 inhibited writhing 27–95% relative to the refer-
ence drug aspirin (58% inhibition). The analgesic potency with
respect to the para-benzoyl substituent was H > Cl or Me. Al-
though the effect of the C-4 R2-substituent on analgesic activity
was variable within the ester, acid and amide sub-groups of com-
pounds, compounds having a R2-cyclohexyl substituent generally
provided superior analgesic activity relative to those having a
lipophilic alkyl substituent. The nature of the R3-substituent (OMe,
OH, NH2) was a determinant of analgesic activity where the
potency order was acetic acid methyl ester > acetic acid or
acetamide, except when the C-4 R2-substituent was cyclohexyl or
benzyl where the potency order was acetamide > acetic acid methyl
ester or acetic acid. Reduction of the 5,6-olefinic bond of the
1,4-dihydropyridyl compound (9a, 94% inhibition) to the corre-
sponding 1,2,3,4-tetrahydropyidyl derivative (10, 69% inhibition)
reduced analgesic activity.
[2]
ment of the acetic acid side chain . The observation that the
acetic acid derivatives (1) bear some structural similarity to
[3]
the NSAID ketoprofen (2) , and that the non-ulcerogenic
antiinflammatory indan-1-acetic acid (3) induces minimal
[4]
gastric irritation , prompted us to investigate the pharma-
cological action(s) exhibited by this novel class of com-
pounds 1. We now describe the synthesis and pharmaco-
logical evaluation of 2-methyl-2-[1-(3-benzoyl-4-substi-
tuted-1,4-dihydropyridyl)]acetic acidsesters, acetic acids and
1
acetamides of general formula 4 wherein the R -substituent
2
is H, Cl or Me, the R -substituent is aryl, arylalkyl, alkyl or
3
cycloalkyl, and the R -substituent is OMe, OH or NH (see
2
structures in Figure 1).
Figure 1. Structures of 2-methyl-2-[1-(3-benzoyl-4-phenyl-1,4-dihy-
dropyridyl)]acetic acid methyl ester (1a), acetic acid sodium salt (1b) and
acetamide (1c), ketoprofen (2), 5,6-dimethoxyindan-1-acetic acid (3), and
2-methyl-2-[1-(3-benzoyl-4-substituted-1,4-dihydropyridyl)]acetic acid es-
ters (4, R3 = OMe), acetic acids (4, R3 = OH) and acetamides (4, R3 = NH2).
Chemistry
The methyl 2-methyl-2-[1-(3-benzoyl-4-substituted-1,4-
dihydropyridyl)]acetates (7a–o) were synthesized using the
procedures illustrated in Scheme 1. Thus, reaction of the
1
3-benzoylpyridine compound (5, R = H, Cl, Me) with
Introduction
racemic methyl 2-bromopropionate in acetone at reflux tem-
In an earlier study, we described the synthesis and charac- perature afforded the corresponding pyridinium bromide salt
terization of 2-methyl-2-[1-(3-benzoyl-4-phenyl-1,4-dihy- (6). The subsequent regiospecific copper-catalyzed reac-
[5]
2
2
dropyridyl)]acetic acid methyl ester (1a), acetic acid sodium tion of a Grignard reagent (R MgCl; R = Ph, 4-Cl-C H -,
6
4
salt (1b), and acetamide (1c) diastereomers (4R*,2R*; 4-Me-C H -, cyclohexyl, PhCH , n-Bu, i-Bu, n-hexyl,
6
4
2
Arch. Pharm. Pharm. Med. Chem.
© WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999
0365-6233/99/0606/0213 $17.50 +.50/0

214
Agudoawu, Yiu, Wallace, and Knaus
n-tetradecyl) with the pyridinium salt (6) yielded the corre-
sponding methyl acetate ester (7a–o) in 14–91% yields. No
product arising from reaction of the benzoyl substituent of the
pyridinium salt (6) with the Grignard reagent was observed.
This is attributed to the fact that pyridinium salts are so
reactive to Grignard reagents in the presence of cuprous
iodide that selective reaction at C-4 of the pyridinium salt
takes place even when a reactive substituent, such as a ketone,
Results and Discussion
The 1,4-dihydropyridyl ring system present in 2-methyl-2-
[1-(3-benzoyl-4-aryl (cycloalkyl, arylalkyl or alkyl)-1,4-di-
hydropyridyl)]acetic acid analogs (7–9) possesses
conformational and steric properties that are significantly
different from those present in traditional benzoylarylacetic
acids (NSAIDs) such as ketoprofen (2). Accordingly, the
1,4-dihydropyridyl ring system is more puckered than the
planar aryl(heteroaryl) ring system due to distortion at the
1,4-dihydropyridyl N-1 and C-4 positions (see Figure 2).
Theoretical ab initio STO-3G calculations for 4-phenyl-3,5-
dicarboxy-1,4-dihydropyridine showed that a 1,4-dihydro-
pyridine boat-conformation is more favored relative to a
planar ring conformation, that ring distortion is greater at C-4
[6]
is present at C-3 . The presence of an electron-withdrawing
3-benzoyl group, that is conjugated with the enamine moiety,
stabilizes the 1,4-dihydropyridyl ring system which prevents
[7]
aromatization .
than at N-1, and that a pseudo-axial orientation of the C-4
[8,9]
phenyl moiety is unequivocally favored
. These confor-
mational differences, together with steric and physicochemi-
cal effects due to the 1,4-dihydropyridyl N-1, C-3 and C-4
substituents, are expected to change the overall size of the
molecule, tissue biodistribution and potential mechanism of
action of compounds 7–9.
Scheme 1. Reagentsandconditions: (a) racemicBrCH(Me)CO2Me, acetone,
reflux, 8 h; (b) 0.1 equiv. CuI, THF, R2MgCl, –23 °C, 0.5 h; (c) aqueous
NaOH, EtOH-H2O, 25 °C, 2 h; (d) NH3, MeOH, 25 °C, 48 h.
The methyl acetate esters (7a–o), which contain two asym-
metric carbons at the dihydropyridyl C-4 and α-methyl ace-
tate methine, positions exist as a mixture of four dia-
Figure 2. Boat-shaped structure for 2-methyl-2-[1-(3-benzoyl-4-substituted-
1,4-dihydropyridyl)]acetic acid methyl esters (7), acetic acids (8) and
acetamides (9).
1
stereomers (SS, SR, RS, RR). The H NMR spectra of the
methyl acetate derivatives (7a–o) exhibit dual resonances
There are two contributing factors responsible for the gas-
trointestinal ulcerogenicity and hemorrhage resulting from
NSAID therapy. These include local irritation of the gastric
(approximate ratio of about 1:1) for some of the dihydro-
[1]
pyridyl ring and ester OMe protons, as reported previously .
Hydrolysis of the methyl acetates (7, mixture of four dias-
tereomers) with aqueous sodium hydroxide at 25 °C for two
hours afforded the corresponding acetic acid analogs (8a–g,
mixture of four diastereomers) in 21–78% yields. Am-
monolysis of the methyl acetate esters (7, mixture of four
diastereomers) yielded the corresponding acetamide analogs
(9a–g, mixture of four diastereomers) in 48–95% yields. A
complete list of the compounds prepared (7–9) is provided in
Table 1.
Hydrogenation of methyl 2-methyl-2-[1-(3-benzoyl-4-
phenyl-1,4-dihydropyridyl)]acetate (7a, mixture of four dias-
tereomers) using 10% Pd/C and hydrogen gas afforded
methyl 2-methyl-2-[1-(4-phenyl-5-benzoyl-1,2,3,4-tetrahy-
ropyridyl)]acetate (10, 47%, mixture of four diastereomers)
as illustrated in Scheme 2.
mucosa due to drug acidity, and inhibition of Prostaglandin
[11]
E (PGE ) biosynthesis in the gastric mucosal barrier
.
2
2
Attachment of the α-methylacetic acid moiety to the 1,4-di-
3
hydropyridyl N-1 sp hybridized nitrogen atom is expected
to reduce acidity significantly since the N-substituted-α-
methylacetic acid moiety (8) may be viewed as an alanine
[NCH(Me)CO H] moiety. It was therefore expected that
2
α-methylacetic acids (8) would be weak acids like alanine
(pK = 9.87) or phenylalanine (pK = 9.24), compared to
a
a
stronger acids such as acetic acid (pK = 4.74) or phenylacetic
a
acid (pK = 4.25). The pK of 9.17 for 2-methyl-2-[1-(3-ben-
a
a
zoyl-4-phenyl-1,4-dihydropyridyl)]acetic acid (8a), deter-
mined using a potentiometric method {Henderson-
–
Hasselbach equation; pK = pH + log [HA] / log [A ]}
a
[10]
reported by Albert and Searjent , was in good agreement
with the postulated pK value. The weak α-methylacetic acids
a
(8) would therefore be expected to cause less local gastric
irritation than commonly used acidic NSAIDs, since many
acidic compounds have a tendency to accumulate in the
[12]
stomach wall soon after oral administration
. In contrast,
non-acidic compounds generally have less tendency to accu-
mulate in stomach tissue and show better gastrointestinal
[13]
tolerance
. The ulcerogenic liability of 8a was determined
Scheme 2. Reagents and conditions: (a) H2 gas at 30 psi, 10% Pd/C, EtOAc,
using a modified method based on the procedure of Nagai et
Arch. Pharm. Pharm. Med. Chem. 332, 213–218 (1999)

Substituted Acetic Acid Derivatives
215
Table 1. Physical data and analgesic activities for methyl 2-methyl-2-[1-(3-para-substituted-benzoyl-4-substituted-1,4-dihydropyridyl)]acetates (7a–o),
2-methyl-2-[1-(3-benzoyl-4-substituted-1,4-dihydropyridyl)]acetic acids (8a–g), 2-methyl-2-[1-(3-benzoyl-4-substituted-1,4-dihydropyridyl)]acetamides
(9a–g), and methyl 2-methyl-2-[1-(4-phenyl-5-benzoyl-1,2,3,4-tetrahydropyridyl)]acetate (10).
—————————————————————————————————————————————————————————————–
Cmpd
R1
R2
R3
Mp
°C
Yield
%
Exact Mass
Calcd. Found
Analgesic activity
% inhibition[a]
—————————————————————————————————————————————————————————————–
7a
7b
7c
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Ph
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OH
–
69[b]
51
28
91
28
21
23
14
72
80
22
27
21
39
50
30
52
54
21
65
37
78
67[b]
62
95
73
60
48
46
47
–
–
94.5 ± 1.5
57.5 ± 2.0
65.4 ± 2.1
62.8 ± 1.8
57.5 ± 3.7
61.1 ± 16.4
66.5 ± 1.4
72.8 ± 3.1
72.2 ± 7.1
39.7 ± 2.6
80.0 ± 2.5
47.6 ± 3.5
53.0 ± 2.6
51.0 ± 5.3
62.7 ± 1.7
30.1 ± 4.7
43.4 ± 2.1
62.8 ± 1.8
NT[k]
4-Cl-C6H4-
4-Me-C6H4-
cyclohexyl
PhCH2
Me
oil
381.1106
381.1104
361.1685
270.1129[c]
361.1678
ND[d]
oil
361.1678
7d
7e
oil
353.1991
oil
361.1678
7f
oil
–
7g
7h
7i
n-Bu
oil
327.1834
326.1716[e]
327.1833
ND[f]
i-Bu
oil
327.1834
Me(CH2)5
Me(CH2)13
Ph
oil
–
7j
oil
–
ND[g]
7k
7l
oil
381.1132
381.1131
415.0731
304.0744[h]
304.0739[i]
361.1674
ND[j]
4-Cl-C6H4-
PhCH2
cyclohexyl
Ph
oil
415.0739
7m
7n
7o
8a
8b
8c
8d
8e
8f
8g
9a
9b
9c
oil
395.1288
oil
387.1601
oil
361.1678
Ph
93–95
98–100
84–86
83–85
105–115
68–70
89–90
–
–
4-Cl-C6H4-
cyclohexyl
PhCH2
Me
n-Bu
i-Bu
OH
–
ND[j]
OH
OH
–
–
ND[j]
ND[j]
ONa
OH
OH
–
–
ND[l]
30.6 ± 2.3
NT
NT
ND[j]
318.1368
–
318.1362
–
Ph
NH2
NH2
NH2
NH2
NH2
NH2
NH2
75.9 ± 2.3
62.5 ± 5.7
95.6 ± 3.6
75.5 ± 2.9
NT
4-Me-C6H4-
cyclohexyl
PhCH2
n-Bu
95–96
90–92
85–87
70–73
78–80
121–123
oil
346.1681
338.1994
344.1525
312.1837
312.1837
–
346.1678
255.1126[m]
344.1521
312.1837
312.1837
ND[n]
9d
9e
9f
i-Bu
NT
9g
10
Aspirin
Me(CH2)13
27.6 ± 11.3
68.6 ± 5.0
57.8 ± 2.8
–
ND[o]
—————————————————————————————————————————————————————————————–
[a]Inhibitory activity in the rat 4% NaCl-induced abdominal constriction (writhing) assay. The result is the mean value ± SEM using five animals follow-
ing a 50 mg/kg intraperitoneal dose of the test compound.
[b]The synthesis and characterization was reported previously[1]
[c]270.1129 [M – cyclohexyl]+
.
[d]ND = not determined. Anal. calcd. for C17H19NO3: C 71.54, H 6.72, N 4.91. Found: C 71.39, H 6.66, N 4.93.
[e]326.1716 [M – 1]+
[f]Anal. calcd. for C22H29NO3: C 74.33, H 8.22, N 3.94. Found: C 74.22, H 8.38, N 4.14.
[g]Characterized as the acetamide derivative 9g.
[h]304.0744 [M – benzyl]+
[i]304.0739 [M – cyclohexyl]+
[j]The compound was characterized as the methyl ester (7) prepared by adding a solution of diazomethane in MeOH to a solution of the acid (8). The 1H NMR
of the methyl ester prepared in this way was identical to the corresponding methyl ester (7).
[k]NT = not tested.
[l]Anal. calcd. for C16H16NO3Na.1/2H2O: C 63.57, H 5.67, N 4.63. Found: C 63.97, H 5.36, N 4.34.
[m]255.1126 [M – cyclohexyl]+; Anal. calcd. for C21H26N2O2.1/4H2O: C 73.55, H 7.79, N 8.17. Found: C 73.96, H 7.71, N 8.07.
[n]Anal. calcd. for C29H44N2O2: C 76.95, H 9.80, N 6.19. Found: C 76.82, H 9.84, N 6.22.
[o]Anal. calcd. for C23H23NO2.1/2H2O: C 73.65, H 6.42, N 3.91. Found: C 73.30, H 6.36, N 3.94.
Arch. Pharm. Pharm. Med. Chem. 332, 213–218 (1999)

216
Agudoawu, Yiu, Wallace, and Knaus
[14]
2
al.
. Although Ibuprofen was ulcerogenic in rats (UD
=
the C-4 R -subsituent was cyclohexyl or benzyl where the
50
250 mg/kg po), 8a was completely devoid of any ulcerogenic potency order was acetamide > acetic acid methyl ester or
effect for a single 1200 mg/kg po dose at eight hours post drug acetic acid. These comparative data suggest there may be a
2
3
administration. A subsequent chronic ulcerogenicity study cooperative effect between the R - and R -substituents with
showed that 8a, administered at 600 mg/kg po twice a day for respect to analgesic activity. Reduction of the 5,6-olefinic
six days, was completely devoid of any gastric irritation or bond of the 1,4-dihydropyridyl compound (9a, 94% inhibi-
ulcerogenicity.
tion) to the corresponding 1,2,3,4-tetrahydropyridyl deriva-
The methyl α-methyl acetates (7), and α-methyl acetamides tive (10, 69% inhibition) reduced analgesic activity. Methyl
2-methyl-2-[1-(3-benzoyl-4-phenyl-1,4-dihydropyridyl)]ac
etate (7a) and 2-methyl-2-[1-(3-benzoyl-4-cyclohexyl-1,4-
dihydropyridyl)]acetamide (9c) exhibited the most potent
analgesic activity (95% inhibition) relative to the reference
drug aspirin (58% inhibition) for a 50 mg/kg intraperitoneal
dose.
(9), could serve as prodrugs to α-methyl acetic acids (8) upon
bioconversion by esterase(s), and amidase(s), respectively.
The in vitro stability of methyl 2-methyl-2-[1-(3-benzoyl-4-
phenyl-1,4-dihydropyridyl)]acetate (7a) and the correspond-
ing acetamide derivative (9a) were therefore investigated.
Incubation of 7a with rat plasma at 37 °C resulted in its rapid,
and quantitative, conversion to the α-methyl acetic acid (8a)
in less than 10 min. A similar incubation of 7a with rat liver
Acknowledgments
homogenate resulted in a slower rate of ester cleavage (t
=
1/2
25 min). In contrast, the acetamide (9a) underwent negligible
(< 2%) conversion to 8a upon incubation with either rat
plasma, or rat liver homogenate, for incubation times up to
24 h.
The ability of the α-methylacetic acid derivative (1b, so-
diumsaltof8a)and the methylα-methylacetate(7a) to inhibit
cyclooxygenase-1 (COX-1) and -2 (COX-2) was investigated
in view of their structural similarity to NSAIDs such as
ketoprofen (2). These studies showed that neither 1b or 8a
inhibited unstimulated COX-1 activity as measured by the
We are grateful to the Medical Research Council of Canada (Grant No.
MA-13262) for financial support of this research.
Experimental
Melting points were determined using a Thomas-Hoover capillary appa-
ratus and are uncorrected. 1H NMR spectra were recorded on a Bruker
AM-300 spectrometer. Chemical shifts are reported in ppm (δ) values
relative to TMS as internal reference. The assignment of exchangeable
protons (OH, NH2) was confirmed by the addition of [D2]H2O. Infrared
spectra were acquired using a Nicolet 5DX-FT spectrometer. Exact Mass
measurements (HRMS) were obtained using an AEI MS-50 mass spectrome-
ter (electron impact). Measurements of pH were performed using an Orion
Model SA520 digital pH meter. Column chromatography was performed
using Merck 7734 (100–200 mesh) silica gel. Preparative thin-layer chroma-
tography (PTLC) was carried out using Camag Kieselgel DF-5 plates, 1 mm
in thickness, activated at 120 °C overnight prior to use. Tetrahydrofuran
(THF) was dried over sodium-benzophenone and distilled just prior to use.
All chemical reagents, organometallic reagents in ”sure-sealed” containers,
were purchased from the Aldrich Chemical Co. Lipopolysaccaride from E.
coli and indomethacin were obtained from the Sigma Chemical Co.
production of thromboxane B (TXB ), or COX-2 activity as
2
2
measured by lipopolysaccaride stimulated PGE produc-
2
[15]
tion , at a 100 mg/kg oral dose in rats. Thus, this class of
α-methylacetic acid derivatives (7–8), unlike traditional
NSAIDs, do not act as inhibitors of the COX-1 and COX-2
isozymes.
The acetic acid methyl esters (7), acetic acids (8) and
acetamides (9) were investigated to determine the effect of a
1
para-benzoyl substitutent (R = H. Cl, Me), the C-4 substi-
2
tuent (R = phenyl, benzyl, cyclohexyl, alkyl), and the nature
3
of the R -substituent (OMe, OH, NH ) upon analgesic activ-
2
General Method for the Preparation of Methyl 2-methyl-2-[1-(3-para-sub-
ity. Analgesic activity was determined using the 4% NaCl-in-
duced writhing assay and the results are summarized in
Table 1. Compounds 7–9 inhibited writhing 27–95% relative
to the reference drug aspirin (58% inhibition) for a 50 mg/kg
intraperitoneal dose. In the acetic acid methyl ester series (7),
the relative potency order with respect to the R -para-ben-
zoyl substituent was H > Cl and H > Me [H (7a) > Cl (7k);
stituted-benzoyl-4-substituted-1,4-dihydropyridyl)]acetates 7a–o
A solution of the respective 3-benzoylpyridine (5, 10.9 mmol) and rac-
methyl 2-bromopropionate (2.6 g, 16.4 mmol) in dry acetone (20 ml) was
heated at reflux for 8 h. After cooling to 25 °C, the solid was filtered and
dried to give the corresponding pyridinium bromide salt (6, 70–75% yield),
which was used immediately in the subsequent reaction. A mixture of 6
(4.3 mmol) in dry THF (50 ml) was stirred under a nitrogen atmosphere until
the solution became homogeneous. The reaction mixture was cooled to
–23 °C in a dry ice-CCl4 bath, a solution of the Grignard reagent R2MgCl
(R2 = Ph, 4-Cl-C6H4-, PhCH2, 4-Me-C6H4- cyclohexyl, Me, n-Bu, i-Bu,
Me(CH2)5, Me(CH2)13; 6.4 mmol of a 2 M solution in THF) was added
dropwise, and the reaction mixture was maintained at –23 °C for 30 min prior
to warming to 25 °C. The reaction mixture was stirred for a further 1.5 h
before quenching by addition of saturated aqueous NH4Cl (5 ml). Diethyl
ether (30 ml) was added, the organic layer was separated and washed
successively with solutions of 30% NH4OH:saturated NH4Cl (3:1, v/v;
30 ml), water (2 × 10 ml), and then brine (10 ml). The organic layer was dried
(MgSO4), and the solvent was removed in vacuo to yield a brown oil. This
oil was purified by elution from a silica gel column using an EtOAc-hexane
gradient going from 5:95 to 15:85 v/v as eluent to afford the respective
product 7a–o as an oil (mixture of four diastereomers) [see Table 1 for R1-,
R2- and R3-substituents, % yield and Exact Mass (high resolution mass
spectral) data]. Infrared (IR) and 1H NMR spectral data for 7a–o are quali-
tatively similar except for differences with respect to the R1- and R2-substi-
tuents. Representative IR and 1H NMR spectral data for 7b, 7d, 7g, and 7k
are listed below.
1
H (7b) > Cl (7l); H (7e) ≥ Cl (7m); H (7d) > Cl (7n); H (7a)
2
> Me (7o)] The effect of the C-4 R -substituent on analgesic
activity was variable within the acetic acid methyl ester [Ph
(7a) > i-Bu (7h) = Me(CH ) (7i) > n-Bu (7g) > 4-Me-C H -
2 5
6 4
(7c) ≈ cyclohexyl (7d) = Me (7f) benzyl (7e) and 4-Cl-C H -
6
4
(7b) > Me(CH ) (7j)], acetic acid [cyclohexyl (8c) > 4-Cl-
2 13
C H - (8b) > Ph (8a) = Me (8e)], and acetamide [cyclohexyl
6
4
(9c) > Ph (9a) = benzyl (9d) > 4-Me-C H - (9b) > Me(CH )
6
4
2 13
2
(9g)] series of compounds. In general, the C-4 R -cyclohexyl
compounds show superior analgesic activity, whereas those
2
possessing a more lipophilic R = Me(CH ) substituent
2 13
were the least active analgesic agents. Overall the nature of
3
the R -substituent (OMe, OH, NH ) was a determinant of
2
activity where the potency order profile was acetic acid
2
methyl ester [7, R = Ph, 4-Cl-C H -, 4-Me-C H -, Me,
6
4
6 4
Me(CH ) ] > acetic acid (8) or acetamide (9), except when
2 13
Arch. Pharm. Pharm. Med. Chem. 332, 213–218 (1999)

Substituted Acetic Acid Derivatives
217
Methyl 2-Methyl-2-[1-(3-benzoyl-4-para-chlorophenyl-1,4-dihydro-
2-Methyl-2-[1-(3-benzoyl-4-para-chlorophenyl-1,4-dihydropyridyl)]acetic
pyridyl)]acetate 7b
Acid 8b
IR (film): ν = 1745 cm–1 (CO2), 1679 (C=O); 1H NMR (CHCl3-d1): δ 1.44
(d, JMe,CH = 7.5Hz, 3H, CHMe), 3.74 [3.72] (s, 3H, OMe), 4.0 [4.1] (q, JMe,CH
= 7.5 Hz, 1H, CHMe), 4.84 [4.82] (d, J4,5 = 4.9 Hz, 1H, H-4), 5.06 (dd, J5,6
= 8.2, J4,5 = 4.9 Hz, 1H, H-5), 6.0 [6.04] (dd, J5,6 = 8.2, J2,6 = 1.5 Hz, 1H,
H-6), 6.98 [6.96] (d, J2,6 = 1.5 Hz, 1H, H-2), 7.20–7.50 (m, 9H, phenyl
hydrogens). The ratio of the major:minor set of resonances was about 3:2
with the δ values for the minor set of resonances when present shown in
square brackets.
IR (KBr): ν = 3057 cm–1 (OH), 1802 (CO2), 1679 (C=O); 1H NMR
(CHCl3-d1): δ 1.48 [1.56] (d, JMe,CH = 7.2 Hz, 3H, CHMe), 3.95 (q, JCH,Me
= 7.2 Hz, 1H, CHMe), 4.80 (d, J4,5 = 4.9 Hz, 1H, H-4), 5.05 (5.08) (dd, J5,6
= 7.1, J4,5 = 4.9 Hz, 1H, H-5), 6.00 [6.02] (d, J5,6 = 7.1 Hz, 1H, H-6), 6.70
(br s, 1H, OH), 6.99 [6.96] (s, 1H, H-2), 7.10–7.70 (m, 9H, phenyl hydro-
gens). The ratio of the two sets of resonances was about 1:1 with the δ values
for the second set of resonances when present shown in square brackets.
2-Methyl-2-[1-(3-benzoyl-4-cyclohexyl-1,4-dihydropyridyl)]acetic Acid 8c
IR (KBr): ν = 3057 cm–1 (OH), 1802 (CO2), 1671 (C=O); 1H NMR
(CHCl3-d1): δ 0.98-2.00 (m, 13H, cyclohexyl H-2, H-3, H-4, H-5, H-6,
CHMe), 2.62–2.68 (m, 1H, cyclohexyl H-1), 3.45–3.60 (m, 1H, H-4), 4.16
[4.00] (q, JCH,Me = 7.1 Hz, 1H, CHMe), 5.62 [5.56] (dd, J5,6 = 8.7, J4,5 = 4.5
Hz, 1H, H-5), 6.72 (br s, 1H, OH), 6.96 (d, J5,6 = 8.7 Hz, 1H, H-6), 7.30–7.65
(m, 6H, phenyl hydrogens, H-2). The ratio of the two sets of resonances was
about 1:1. The δ values for the second set of resonances when present are
shown in square brackets.
Methyl 2-Methyl-2-[1-(3-benzoyl-4-cyclohexyl-1,4-dihydropyridyl)]acetate
7d
IR (film): ν = 1753 cm–1 (CO2), 1671 (C=O); 1H NMR (CHCl3-d1):
δ 1.0–1.3 (m, 6H, cyclohexyl H-3, H-4, H-5), 1.46 [1.44] (d, JMe,CH = 7.4
Hz, 3H, CHMe), 1.60–1.82 (m, 5H, cyclohexyl H-1, H-2, H-6), 3.66–3.74
(m, 1H, H-4), 3.76 [3.75] (s, 3H, OMe), 3.92 (q, JCH,Me = 7.4 Hz, 1H, CHMe),
4.92–5.04 (m, 1H, H-5), 6.02 [6.00] (d, J5,6 = 7.6 Hz, 1H, H-6), 6.88 (s, 1H,
H-2), 7.35–7.65 (m, 5H, phenyl hydrogens). The ratio of the two sets of
resonances was about 1:1 with the δ values for the second set of resonances
when present shown in square brackets.
2-Methyl-2-[1-(3-benzoyl-4-isobutyl-1,4-dihydropyridyl)]acetic Acid 8g
IR (KBr): ν = 3080 cm–1 (OH), 1802 (CO2), 1679 (C=O); 1H NMR
(Me2SO-d6): δ 0.80–1.00 (m, 6H, CHMe2), 1.18–1.44 (m, 5H, CHCH2,
CHMe), 1.68–1.82 (m, 1H, CH2CHMe2), 3.54–3.60 (m, 1H, H-4), 4.24 (q,
JCH,Me = 6.9 Hz, 1H, CHMe), 4.98 [5.02] (dd, J5,6 = 7.1, J4,5 = 4.8 Hz, 1H,
H-5), 6.08 (d, J5,6 = 7.1 Hz, 1H, H-6), 6.90 [6.92] (s, 1H, H-2), 7.36–7.52
(m, 5H, phenyl hydrogens). The ratio of the two sets of resonances was about
1:1. The δ values for the second set of resonances when present are shown
in square brackets.
Methyl 2-Methyl-2-[1-(3-benzoyl-4-n-butyl-1,4-dihydropyridyl)]acetate 7g
IR (film): ν = 1745 cm–1 (CO2), 1679 (C=O); 1H NMR (CHCl3-d1): δ 0.90
(t, JCH2,Me = 7.2 Hz, 1H, CH2Me), 1.20–1.34 (m, 6H, CH2CH2CH2CH3),
1.38 [1.40] (d, JMe,CH = 6.8 Hz, 3H, CHMe), 3.68–3.80 (m, 1H, H-4), 3.76
[3.78] (s, 3H, OMe), 3.92 (q, JCH,Me = 6.8 Hz, 1H, CHMe), 4.98 (5.02] (dd,
J
5,6 = 7.2, J4,5 = 5.3 Hz, 1H, H-5), 5.94 (d, J5,6 = 7.2 Hz, 1H, H-6), 6.86 (s,
1H, H-2), 7.30–7.56 (m, 5H, phenyl hydrogens). The ratio of the two sets of
resonances was about 1:1 with the δ values for the second set of resonances
when present shown in square brackets.
General Method for the Preparation of 2-Methyl-2-[1-(3-benzoyl-4-substi-
tuted-1,4-dihydropyridyl)]acetamides 9a–g
A saturated solution of ammonia in MeOH (10 ml) was added to a solution
of the methyl acetate ester (7, 2.7 mmol) in MeOH (20 ml), the reaction flask
was sealed with a rubber septum, and the reaction was allowed to proceed
for 48 h at 25 °C with stirring. The solvent was removed in vacuo and the
respective product was purified by silica gel TLC using EtOAc-hexane (3:1,
v/v) as development solvent. Extraction of the band containing the product
using EtOAc (2 × 100 ml) and removal of the solvent in vacuo afforded the
respective product (9a–g) as a mixture of four diastereomers [see Table 1 for
R1-, R2- and R3-substituents, mp, % yield and Exact Mass (high resolution
Methyl 2-Methyl-2-[1-(3-para-chlorobenzoyl-4-benzyl-1,4-dihydro-
pyridyl)]acetate 7k
IR (film): ν = 1753 cm–1 (CO2), 1679 (C=O); 1H NMR (CHCl3-d1): δ 1.24
(d, JMe,CH = 7.2 Hz, 3H, CHMe), 2.76–2.90 (m, 2H, CH2Ph), 3.68 [3.70] (s,
3H, OMe), 3.76 (q, JCH,Me = 7.2 Hz, 1H, CHMe), 3.96–4.12 (m, 1H, H-4),
4.94 (dd, J5,6 = 8.6, J4,5 = 5.2 Hz, 1H, H-5), 5.84 (dd, J5,6 = 8.6, J2,6 = 1.5
Hz, 1H, H-6), 6.72 [6.74] (d, J2,6 = 1.5 Hz, 1H, H-2), 7.14–7.56 (m, 9H,
phenyl hydrogens). The ratio of the two sets of resonances was about 3:2 with
the δ values for the minor set of resonances when present shown in square
brackets.
1
mass spectral) data]. Infrared (IR) and H NMR spectral data for 9a–g are
qualitatively similar except for differences with respect to the R1- and
R2-substituents. Representative IR and 1H NMR spectral data for 9b and 9d
are listed below.
2-Methyl-2-[1-(3-benzoyl-4-para-tolyl-1,4-dihydropyridyl)]acetamide 9b
General Method for the Preparation of 2-Methyl-2-[1-(3-benzoyl-4-substi-
tuted-1,4-dihydropyridyl)]acetic Acids 8a–g
IR (KBr): ν = 3312 cm–1 (NH2), 3180 (NH2), 1720 (CONH), 1679 (C=O);
1H NMR (CHCl3-d1): δ 1.36 (d, JMe,CH = 6.6 Hz, 3H, CHMe), 2.28 (s, 3H,
-C6H4-Me), 3.82 (q, JCH,Me = 6.6 Hz, 1H, CHMe), 4.78 (d, J4,5 = 4.8 Hz, 1H,
H-4), 5.10 (dd, J5,6 = 7.8, J4,5 = 4.8 Hz, 1H, H-5), 5.92 (d, J5,6 = 7.8 Hz, 1H,
H-6), 5.96 and 6.36 (two s, 1H each, NH2), 7.00 (s, 1H, H-2), 7.06–7.50 (m,
9H, phenyl hydrogens).
Aqueous NaOH (10 ml of a 1% w/v solution, 0.75 mmol) was added
dropwise to a solution of the respective methyl acetate ester (7, 0.75 mmol)
in EtOH-H2O (4:1, v/v, 12.5 ml) at 25 °C with stirring. The reaction was
allowed to proceed with stirring until TLC (0.25 mm thickness silica gel G
microplate) indicated that the reaction was completed (about 2 h). Removal
of the solvent in vacuo, addition of water (10 ml), and acidification with 5N
HCl gave a yellow solid which was filtered off and dried in a drying pistol
to afford the respective acetic acid products 8a–g as a mixture of four
diastereomers (see Table 1 for R1-, R2- and R3-substituents, mp and % yield
data). The acetic acid products 8a–d, 8f were characterized by reconversion
to their respective methyl ester derivatives (7) in quantitative yield by
addition of a solution of excess diazomethane in MeOH at 25 °C with stirring.
2-Methyl-2-[1-(3-benzoyl-4-benzyl-1,4-dihydropyridyl)]acetamide 9d
IR (KBr): ν = 3328 cm–1 (NH2), 3180 (NH2), 1720 (CONH), 1687 (C=O);
1H NMR (CHCl3-d1): δ 1.24 [1.28] (d, JMe,CH = 7.5 Hz, 3H, CHMe), 2.66
(dd, Jgem = 14.2, Jvic = 2.9 Hz, 1H, CHH’Ph), 3.16 (dd, Jgem = 14.2, Jvic
=
5.5Hz, 1H, CHH’Ph), 3.52 [3.60] (q, JCH,Me = 7.5 Hz, 1H, CHMe), 4.20–4.26
(m, 1H, H-4), 4.66 and 4.70 (two s, 1H total, NH), 5.00–5.10 (m, 1H, H-5),
5.34 (s, 1H, NH), 5.80 [5.83] (dd, J5,6 = 7.2, J2,6 = 1.5 Hz, 1H, H-6), 6.62
[6.72] (d, J2,6 = 1.5 Hz, 1H, H-2), 7.10–7.62 (m, 10H, phenyl hydrogens).
The ratio of the two sets of resonances was about 1:1. The δ values for the
second set of resonances when present are shown in square brackets.
1
Infrared (IR) and H NMR spectral data for 8a–g are qualitatively similar
except for differences with respect to the R1-, R2- and R3-substituents.
1
Representative IR and H NMR spectral data for 8b, 8c and 8g are listed
below.
Arch. Pharm. Pharm. Med. Chem. 332, 213–218 (1999)

218
Agudoawu, Yiu, Wallace, and Knaus
Methyl 2-Methyl-2-[1-(4-phenyl-5-benzoyl-1,2,3,4-tetrahydropyridyl)]-
acetate 10
Animals were anesthetized with pentobarbital 3 h after administration of
the test compound. Blood samples (2 ml) were taken from the descending
aorta and divided into two samples. The first aliquot (1 ml) was transferred
into a glass tube and placed in a water bath at 37 °C. After 45 min, 10 µg of
indomethacin, prepared as a 0.1 mg/ml solution in NaHCO3 (1.25% w/v),
was added (100 µl) and gently mixed. When aggregating, platelets release
thromboxane A2, which is synthesized via the enzyme cyclooxygenase
(COX-1). The production of this enzyme is measured by the release of the
stable hydration product, thromboxane B2. This assay can therefore be used
to measure systemic inhibition of COX by an NSAID.
The remaining blood was aliquoted (1 ml) and incubated for 15 min with
gentle shaking in a water bath at 37 °C. Lipopolysaccaride (5 µg/ml) was
added and the incubation was allowed to proceed for an additional 5 h. The
incubation was terminated by addition of 100 µl of 0.1 mg/ml indomethacin
in 1.25% NaHCO3 solution. Lipopolysaccaride-stimulated COX-2 reactivity
represents predominately a COX-2 response primarily from mononuclear
cells [15]. Samples were centrifuged at 4 °C for 10 min at 1,000g. Serum was
transferred to Eppendorf tubes and stored tat –20 °C for subsequent meas-
urement of prostaglandin E2 by ELISA.
10% Palladium-on-charcoal (20 mg) was added cautiously to a solution of
7a (0.5 g, 1.4 mmol) in EtOAc (10 ml) in a pressure bottle, and the reaction
was allowed to proceed in the presence of hydrogen gas at 30 psi with shaking
for 24 h at 25 °C. Filtration of the mixture, and removal of the solvent in
vacuo gave a yellow oil that was purified by preparative TLC using EtOAc-
hexane (1:1, v/v) as development solvent to afford 10 (mixture of four
diastereomers) as an oil (Rf = 0.65, 200 mg, 47%); IR (film): IR (KBr): ν =
1745 cm–1 (CO2), 1679 (C=O); 1H NMR (CHCl3-d1): δ 1.44 [1.42] (d, JMe,CH
= 7.2 Hz, 3H, CHMe), 1.90–2.15 (m, 2H, H-3), 2.90–3.18 (m, 2H, H-2), 3.74
[3.76] s, 3H, OMe), 3.88–4.02 (two overlapping q, JCH,Me = 7.2 Hz, 1H,
CHMe), 4.32–4.40 (m, 1H, H-4), 7.18–7.65 (m, 11H, phenyl hydrogens,
H-6). The ratio of the two sets of resonances was about 1:1. The δ values for
the second set of resonances when present are shown in square brackets.
Determination of the pKa Value for 2-Methyl-2-[1-(3-benzoyl-4-phenyl-
1,4-dihydropyridyl)]acetic Acid 8a
The pKa, calculated using the Henderson-Hasselebach equation {pKa =
pH + log [HA] / log [A–]} using the method of Albert and Searjent[10] for 8a
was 9.17 ± 0.01 (n = 5).
Analgesic Assay
Analgesic activity was determined using the 4% sodium chloride-induced
writhing (abdominal constriction) assay reported by Fukawa et al.[16] as
Ulcerogenicity Assay
described previously[17]
.
Six male Sprague-Dawley rats weighing 100–120 g, fasted for 24 h, were
sacrificed 8 h after oral administration of the test compound (100–
1200 mg/kg po dose). The stomach, sternum, and duodenum were removed
for macroscopic and microscopic assessment to determine the presence, or
absence, of lesions which were used to calculate the UD50 value which is
defined as the dose of test compound causing lesions in 50% of animals. A
chronic ulcerogenesis assay was performed for 8a by administering a
600 mg/kg po dose twice daily for six days.
References
[1] S. A. Agudoawu, S.-H. Yiu, E. E. Knaus, Can. J. Chem. 1997, 75,
1106–1109.
[2] G. H. Hamor, Nonsteroidal anti-inflammatory agents in Principles of
Medicinal Chemistry (Ed.: W. O. Foye, 2nd edition), Lea and Febiger,
Philadelphia, 1981, chapter 22.
In Vitro Stability in Rat Plasma, or Rat Liver Homgenate
[3] A. Allais, G. Roussecui, R. Deract, J. Benzoni, L. Chifflot, Eur. J. Med.
Chem. 1974, 9, 381–389.
Rat liverhomogenate (1:3, w/v) was prepared by homogenizingwhole liver
from a Sprague-Dawley rat with phosphate buffer (pH = 7.4). Rat plasma
was obtained by centrifugation of whole rat blood, obtained from Sprague-
Dawley rats, at 3,000 rpm for 15 min, and then removal of the plasma using
a pipet. Standard solutions of methyl 2-methyl-2-[1-(3-benzoyl-4-phenyl-
1,4-dihydropyridyl)]acetate (7a), and 2-methyl-2-[1-(3-benzoyl-4-phenyl-
1,4-dihydropyridyl)]acetamide (9a), were prepared by dissolving the test
compound (8.5–13.7 mg) in EtOH (5 ml). An aliquot (100 µl) of the standard
solution (7a or 9a) was added to either rat plasma (2 ml), or rat liver
homogenate (3 ml) and the mixture was incubated at 37 °C with stirring. At
selected time intervals (2, 5, 10, 30 min, 1, 1.5, 2, 3, 4.5, 6, 8, 23 h), an aliquot
of the incubation mixture (100 µl) was withdrawn, mixed with ice-cold
MeCN (400 µl), and stored at –20 °C prior to HPLC analysis. Quantitative
HPLC analysis (concentrations were determined using Water’s Millennium
2010 software) was performed using a Millipore 3.9 × 150 mm reverse phase
C-18 column with MeCN:0.005 M aqueous tetrabutyl ammonium hydrogen
phosphate buffer (0.45:0.55, v/v) as the mobile phase at a flow rate of
1 ml/min, and UV detection at 205 nm. The retention times for 7a, 9a, the
acetic acid hydrolysis product 8a, and the internal standard methyl 2-[1-(3-
benzoyl-4-phenyl-1,4-dihydropyridyl)]acetic acid were 9.6, 2.7, 4.0, and 6.6
min, respectively.
[4] A. Mukherjee, S. C. Lahiri, Synthesis and pharmacological evaluation
of some potential non-steroidal anti-inflammatory agents with low
gastric irritancy, Medicinal Chemistry Division, 193rd ACS National
Meeting, Abstract No. 43, 1987, Denver, USA, April 5–10.
[5] D. L. Comins, A. H. Abdullah, J. Org. Chem. 1982, 47, 4315–4319.
[6] D. L. Comins, E. D. Stroud, J. J. Herrick, Heterocycles 1984, 22,
151–157.
[7] U. Eisner, J. Kuthan, Chem. Rev. 1972, 42, 1–42.
[8] H. J. Hofmann, R. Cimiraglia, FEBS Lett. 1988, 241, 38–40.
[9] H. J. Hofmann, R. Cimiraglia, J. Mol. Struct. (Theochem.), 1990, 205,
1–11.
[10] A. Albert, E. P. Serjeant, In The Determination of Ionization Constants,
A Laboratory Manual, Chapman and Hall, 1984, p. 14–17.
[11] D. Robinson, Am. J. Med. 1983, 75, 26–31.
[12] F. Luzzani, G. Colombo, P. Shiatti, D. Selva, A. Glasser, Pharmacol.
Res. 1984, 16, 755–763.
[13] T. Shen, J. Med. Chem. 1981, 24, 1–5.
In Vivo Inhibition of Cyclooxygenase-1 (COX-1) and Cyclooxygenase-2
(COX-2)
[14] Y. Nagai, K. Kirio, H. Nakamura, H. Uno, H. Nishimura, J. Med. Chem.
1987, 26, 222–226.
Male Sprague-Dawley rats, 100–150 g in weight, were fed standard labo-
ratory chow and tap water ad libitum. All experimental protocols were
approved by the Animal Care Committee of the University of Calgary. In
vivo assessment of COX-1/COX-2 inhibitory activity was determined based
on a modification of a previously reported method[15]. Rats were divided into
three groups (3 animals in each group). The rats received via oral gavage
either vehicle (10% w/v acacia), 1a or 7a (100 mg/kg po). Test compounds
(1a, 7a) were prepared by grinding the appropriate weight with acacia first,
and then adding distilled water to form a suspension. The ratio of the acacia
to distilled water used was 10% w/v.
[15] K. Glaser, M.-L. Sung, K. O’Neill, M. Belfast, D. Hartman, R. Carlson,
A. Kreft, D. Kubrak, C. L. Weichman, Eur. J. Pharmacol. 1995, 281,
107–111.
[16] K. Fukawa, O. Kawano, M. Hibi, N. Misaka, S. Ohba, Y. Hatanaka, J.
Pharmacol. Methods 1980, 4, 251–259.
[17] J. K. Buolamwini, E. E. Knaus, Drug Design Delivery 1990, 7, 19–31.
Received: March 30, 1999 [FP381]
Arch. Pharm. Pharm. Med. Chem. 332, 213–218 (1999)