Synthesis and antiinflammatory and analgesic activity of naproxen amides with amino acid derivatives

Full text of DOI:10.1023/A:1020561127636

Pharmaceutical Chemistry Journal
Vol. 36, No. 5, 2002
SYNTHESIS AND ANTIINFLAMMATORY AND ANALGESIC ACTIVITY
OF NAPROXEN AMIDES WITH AMINO ACID DERIVATIVES
G. L. Levit,1 L. V. Anikina,2,3 Yu. B. Vikharev,2 A. M. Demin,1 V. A. Safin,2,3
T. V. Matveeva,1 and V. P. Krasnov1
Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 36, No. 5, pp. 12 – 15, May, 2002.
Original article submitted January 8, 2002.
COOH
In recent years, there have been attempts to modify the
I, ÒEÀ
well-known nonsteroidal antiinflammatory drugs by reac-
O
N
H
NH2
Dioxane – H2O
tions with natural amino acids, aimed at eliminating unde-
sired side effects of the drug action [1]. In particular, the syn-
thesis of naproxen amide with glycine was reported in [2, 3].
The aim of this study was to obtain naproxen amides
with some amino acid derivatives and characterize the prod-
ucts with respect to the antiinflammatory and analgesic activ-
ity and acute toxicity. The synthesis of naproxen amides
(II – V) with methyl esters of (S )-methionine,
(S )-phenylalanine, (S )-histidine, and (S )-leucine was based
on the condensation of (S )-naproxen chloroanhydride (I)
with the corresponding amino acid esters in DMF in the pres-
ence of triethanolamine (TEA) [4]:
VI
CH3
CO
H
N
COOH
MeO
NH
VII
O
The condensation of naproxen chloroanhydride I with a
copper complex of L-lysine (VIII) in an aqueous dioxane so-
lution in the presence of KOH, followed by decomposition of
the intermediate copper complex IX in a dilute hydrochloric
acid solution, led to (S )-naproxen amide with L-lysine (X):
CH3
H2NCH(R)COOMe, ÒEÀ
DMF
COCl
MeO
COO
I
I, KOH
Cu
CH3
Dioxane – H2O
H
N
H2N
NH2
COOMe
2
VIII
O
R
CH3
MeO
H
N
II – V
CH2
Cu
O
H2N
IV: R =
II: R = CH2CH2SCH3
III: R = CH2Ph
MeO
HN
N
COO
V: R = CH2CH(CH3)2
2
IX
CH3
The condensation of naproxen chloroanhydride with
Ne-formyl-L-lysine (VI) was performed in an aqueous
dioxane solution in the presence of TEA:
H
N
H+
O
H2N
MeO
X
COOH
1
Institute of Organic Synthesis, Ural Division, Russian Academy of Sci-
ences, Yekaterinburg.
Institute of Technical Chemistry, Ural Division, Russian Academy of Sci-
ences, Perm, Russia.
The possibility of selective hydrolysis of an ester group
with the formation of amide (XI) with a free carboxy group
was demonstrated by the alkaline hydrolysis of amide V in
an aqueous acetone solution at 0°C:
2
3
Institute of Natural Sciences, Perm State University, Perm, Russia.
232
0091-150X/02/3605-0232$27.00 © 2002 Plenum Publishing Corporation

Synthesis and Antiinflammatory and Analgesic Activity of Naproxen Amides
233
CH3
temperature overnight. Then the mass was poured into
150 ml of ice-cold water. The precipitated oil was extracted
with ethyl acetate. The extract was sequentially washed with
a 5% NaHCO3 solution, water, 1 N HCl, and water, dried
over Na2SO4, and evaporated to dryness. Finally, the residue
was recrystallized from a hexane – ethyl acetate mixture.
N-[(2S )-2-(6-Methoxy-2-naphthyl)propionyl]-(2S )-me-
H
N
COOMe
CH3
–
OH
Acetone – H2O
O
MeO
CH3
V
CH3
H
N
1
COOH
CH3
CH3
thionine methyl ester (II). Yield, 1.52 g (75%); H NMR
spectrum in CDCl3 (d, ppm): 7.78 – 7.10 (m, 6H, Harom),
6.11 (d, 1H, NH), 4.67 (m, 1H, CaH methionine), 3.91 (s,
3H, OCH3), 3.75 (q, 1H, CH naproxen), 3.65 (s, 3H,
COOCH3), 2.38 (m, 2H, CH2CH), 1.99 (m, 2H, S-CH2), 1.97
(s, 3H, S-CH3), 1.61 (d, 3H, CH–CH3).
N-[(2S )-2-(6-Methoxy-2-naphthyl)propionyl]-(2S )-phe-
nylalanine methyl ester (III). Yield, 1.26 g (60%); 1H NMR
spectrum in CDCl3 (d, ppm): 7.70 – 6.83 (m, 11H, Harom),
5.78 (d, 1H, NH), 4.80 (2t, 1H, CaH phenylalanine), 3.93 (s,
3H, OCH3), 3.69 (q, 1H, CH naproxen), 3.65 (s, 3H,
COOCH3), 3.05, 2.97 (2dd, 2H, CH2–C6H4 phenylalanine),
1.58 (d, 3H, CH–CH3).
O
MeO
XI
The proposed structures of amides II – V, VII, X, and XI
were confirmed by the results of elemental analyses and by
1H NMR data. It should be noted that, according to the H
1
NMR data, the synthesized compounds represent individual
(S ,S )-diastereomers. Amides II – V, VII, X, and XI appear
as colorless crystalline substances. Compounds II – V, VII,
and XI are insoluble in water and well soluble in organic sol-
vents. Compound X is soluble only in diluted acids (hydro-
chloric, trifluoroacetic).
The UV spectra of compounds II – V, VII, X, and XI dis-
play five absorption maxima related to the naphthalene nu-
cleus, including one intense peak at l = 233 – 235 nm and
somewhat less intense, at l = 264, 274, 320, and
333 – 335 nm.
N-[(2S )-2-(6-Methoxy-2-naphthyl)propionyl]-(2S )-
1
histidine methyl ester (IV). Yield, 1.23 g (60%); H NMR
spectrum in CDCl3 (d, ppm): 8.12 (d, 1H, NH), 7.71 – 7.05
(m, 6H, Harom), 7.40 (d, 1H, C2H imidazole), 6.69 (s, 1H,
C4H imidazole), 4.51 (m, 1H, CaH histidine), 3.87 (s, 3H,
OCH3), 3.76 (q, 1H, CH naproxen), 3.52 (s, 3H, COOCH3),
2.90 (m, 2H, CH2 histidine), 1.39 (d, 3H, CH–CH3).
Some physicochemical characteristics of the synthesized
amides are presented in Table 1. The data of elemental analy-
ses agree with the results of analytical calculations using em-
pirical formulas.
N-[(2S )-2-(6-Methoxy-2-naphthyl)propionyl]-(2S )-
1
leucine methyl ester (V). Yield, 1.54 g (80%); H NMR
spectrum in CDCl3 (d, ppm): 7.74 – 7.12 (m, 6H, Harom),
5.73 (d, 1H, NH), 4.60 (2t, 1H, CaH leucine), 3.92 (s, 3H,
OCH3), 3.73 (q, 1H, CH naproxen), 3.63 (3H, COOCH3),
1.65 (m, 2H, CH2CH), 1.60 (d, 3H, CH–CH3 naproxen), 1.40
(m, 1H, CH2CH), 0.89, 0.87 (2d, 6H, (CH3)2).
EXPERIMENTAL CHEMICAL PART
The purity of the products was checked by TLC on
Sorbfil eluted in chloroform – methanol (9 : 1) and devel-
oped by exposure to UV light. The melting points were de-
termined on a Boetius heating table. The UV absorption
Na-[(2S )-2-(6-Methoxy-2-naphthyl)propionyl]-Ne-for-
myl-(2S )-lysine (VII). To a solution of 1.0 g (5.74 mmole)
of He-formyl-L-lysine in 32 ml of a dioxane – water (5 : 3)
mixture was added 1.6 ml (11.48 mmole) TEA. To this sus-
pension, cooled to 0°C on an ice-cold bath, was added
dropwise (with stirring and cooling) a solution of 1.42 g
(5.34 mmole) of naproxen chloroanhydride I in 20 ml
spectra were recorded with
a
Specord UV- VIS
spectrophotometer (Germany) using ethanol solutions. The
1H NMR spectra were measured on a Bruker DRX400 (Ger-
many) with a working frequency of 400 MHz using TMS as
the internal standard. The values of specific rotation of the
polarization plane were determined on an A1-EPO
polarimeter (Russia). The initial naproxen chloroanhydride
(I), or (S )-2-(6-methoxy-2-naphthyl)propionyl chloride, was
synthesized as described in [4].
General method for the synthesis of amides II – V. To
a solution of 5.4 mmole of an amino acid methyl ester hydro-
chloride in 10 ml DMF was added 0.75 ml (5.4 mmole)
TEA. To this suspension, cooled to 0°C on an ice-cold bath,
was added dropwise (with stirring and cooling) a solution of
1.47 g (5.4 mmole) of naproxen chloroanhydride I in 5 ml
DMF and (simultaneously, in two portions) 0.75 ml
(5.4 mmole) of TEA. Still on the ice-cold bath, the reaction
mass was stirred for 2 h and then allowed to stand at room
TABLE 1. Melting Points and Specific Rotation of Compounds
II – V, VII, X, and XI
20
[a] , deg
D
Com-
pound
Empirical
formula
M.p., °C
II
III
IV
V
106 – 107 + 15.7 (c, 1.84; chloroform)
106 – 107 + 8.6 (c, 2.0; acetone)
211 – 213 – 8.4 (c, 1.0; methanol)
99 – 101 + 27.5 (c, 2.0; chloroform)
129 – 131 –9.5 (c, 1.2; acetone)
C20H25NO4S
C24H25NO4
C21H23N3O4
C21H27NO4
C21H26N2O5
VII
X
225 – 227 + 34.2 (c, 2.0; 1 N CF3COOH) C20H26N2O4
135 – 137 –15.0 (c, 1.0; methanol) C20H25NO4
XI

234
G. L. Levit et al.
dioxane. Still on the ice-cold bath, the reaction mass was
stirred for 2 h and then allowed to stand at room temperature
overnight. Then the mass was poured into 100 ml of ice-cold
water and acidified with 1 N HCl to pH 1. The precipitated
oil was extracted with ethyl acetate. The extract was washed
with a saturated NaCl solution, dried over Na2SO4, and evap-
orated to dryness. Finally, the residue was recrystallized from
a hexane – acetone mixture.
tion of 2.0 g (8.08 mmole) of naproxen chloroanhydride I in
30 ml dioxane. Still on the ice-cold bath, the reaction mass
was stirred for 2 h and then allowed to stand at room temper-
ature overnight. The precipitated copper complex IX was
separated by filtration, washed on the filter sequentially with
water, 5% NaHCO3 solution, and water (to pH 7.0), and eth-
anol, and dried in air to obtain 1.98 g (79%) of copper com-
plex IX. The blue-violet residue of IX was stirred in 20 ml of
1 N HCl for 2 h at room temperature. The resulting white
precipitate was separated by filtration, washed on the filter
with (to pH 7.0) and ethanol, and dried over P2O5. The re-
sulting amide X was reprecipitated from 2 N trifluoroacetic
acid.
1
Yield of compound VII, 1.88 g (85%); H NMR spec-
trum in acetone-d6 (d, ppm): 8.11 (s, 1H, HCO), 7.78 – 7.04
(m, 6H, Harom), 7.2 (d, 1H, NH), 4.41 (m, 1H, CaH lysine),
3.90 (s, 3H, OCH3), 3.9 (q, 1H, CH naproxen), 3.21 (q, 2H,
NHCH2CH2), 1.90 – 1.55 (m, 6H, CH2CH2CH2CH), 1.50 (d,
3H, CH–CH3 naproxen).
Yield of compound X, 1.39 g (60%); 1H NMR spectrum
Ne[-[(2S )-2-(6-Methoxy-2-naphthyl)propionyl]-(2S )-
lysine (X). To a solution of 1.18 g (6.46 mmole) of L-lysine
hydrochloride and 0.96 g (14.53 mmole) KOH in 20 ml of
water was added a solution of 0.81 g (3.23 mmole)
CuSO4 × 5H2O in 10 ml water. To the resulting solution of an
L-lysine copper complex (VIII), cooled to 0°C on an ice-cold
bath, was added dropwise (with stirring and cooling) a solu-
+
in DMSO-d6/CF3COOD (d, ppm): 7.54 (s, 3H, NH3 ),
7.51 – 6.89 (m, 6H, Harom), 7.32 (bs, 1H, NH), 3.74 (s, 1H,
CaH lysine), 3.68 (q, 1H, CH naproxen), 3.63 (s, 3H, OCH3),
3.05 (bs, 2H, NHCH2CH2), 1.72 (m, 2H, CH2CH),
1.35 – 1.15 (m, 4H, CH2CH2), 1.35 (d, 3H, CH–CH3
naproxen).
TABLE 2. Acute Toxicity, Antiinflammatory, and Analgesic Activity of Naproxen Amides with L-Amino Acid Derivatives
Antiinflammatory activity (carrageenan edema)*
Analgesic activity (vinegar convulsions)*
Percentage edema
Edema growth
LD50,
mg/kg
Convulsions
inhibited relative
to control, %
Compound
Dose,
mg/kg
growth relative
to initial
Dose,
mg/kg
Number
of convulsions
inhibited relative
to control,%
background, %
II
> 3000
25
25
25
23
25
23
22
15
–
19.6
26.4
29.8
30.3
26.4
25.3
19.0
47.6
–
103
106
105
98
39.1
59.76 ± 4.93
n = 10
P > 0.05
13.50 ± 2.00
n = 6
P > 0.05
III
...
3.0
54.65 ± 2.96
n = 10
P < 0.05
21.50 ± 3.15
n = 6
P > 0.5
IV
V
> 3000
55.7
63.9
37.6
76.7
83.5
75.2
–
52.12 ± 4.45
n = 10
P < 0.01
9.83 ± 4.34
n = 6
P < 0.05
> 3000
51.76 ± 13.13
n = 6
8.00 ± 3.47
n = 6
P < 0.02
P > 0.05
VII
...
105
98
54.68 ± 8.77
n = 10
P > 0.05
13.83 ± 2.02
n = 6
P > 0.5
X
> 3000
55.52 ± 3.31
n = 10
P < 0.02
5.17 ± 2.32
n = 6
P < 0.002
XI
...
94
60.17 ± 9.28
n = 6
3.67 ± 1.52
n = 6
P < 0.001
P > 0.1
Naproxen
Control
547 (400 – 750)
–
63
38.95 ± 1.52
n = 10
P < 0.001
5.50 ± 2.66
n = 6
P < 0.01
–
74.28 ± 6.00
n = 10
22.17 ± 3.36
n = 6
* n, number of test animals; P is the confidence level of differences relative to control.

Synthesis and Antiinflammatory and Analgesic Activity of Naproxen Amides
235
TABLE 3. Dynamics of the Antiinflammatory Activity of Compound IV upon Peroral Administration
Antiinflammatory activity upon drug introduction,* tested after
Com- Dose,
pound mg/kg
1 h
2 h
3 h
4 h
8 h
24 h
A
B
A
B
A
B
A
B
A
B
A
B
IV
25
15
–
23.1
43.7
30.6
7.1
–2.9
–129.2
27.07 ± 2.67
n = 6
P > 0.1
34.05 ± 4.20
n = 6
P < 0.02
50.11 ± 5.01
n = 6
P < 0.01
70.23 ± 9.28
n = 6
57.09 ± 6.47
n = 6
24.94 ± 3.51
n = 6
P < 0.001
P > 0.5
P > 0.5
Nap-
roxen
10.1
–
30.5
–
19.6
–
28.3
–
34.4
–
26.0
–
31.65 ± 6.69
n = 6
42.03 ± 6.53
n = 6
58.10 ± 4.90
n = 6
P > 0.05
54.20 ± 3.01
n = 6
P < 0.002
36.40 ± 2.55
n = 6
P < 0.01
8.05 ± 1.27
n = 6
P > 0.1
P > 0.5
P = 0.1
Control
35.22 ± 5.24
n = 10
60.44 ± 8.17
n = 10
12.24 ± 4.65
n = 10
75.57 ± 4.44
n = 10
55.48 ± 4.68
n = 10
10.88 ± 1.50
n = 10
* n is the number of test animals; P is the confidence level of differences relative to control; A, percentage edema growth relative to initial
background; B, percentage edema-growth inhibition relative to control.
N-[(2S )-2-(6-Methoxy-2-naphthyl)propionyl]-(2S )-
leucine (XI). To a solution of 0.36 g (1 mmole) of ester V in
3 ml acetone, cooled to 0°C on an ice-cold bath, was added
dropwise (with stirring and cooling) 2.4 ml (2.4 mmole) of
1 N NaOH solution, and the reaction mixture was kept on the
cool bath for 18 h. To this mixture was added 3 ml of diethyl
ether, after which the aqueous layer was separated, washed
again with diethyl ether, and neutralized with 1 N HCl to
pH 6. The precipitate was separated by filtration, washed on
the filter with water, and recrystallized from a hexane – ace-
tone (4 : 3) mixture.
also studied upon peroral administration, with the
antiinflammatory effect determined 1, 2, 3, 4, 8, and 24 h af-
ter model edema induction [7].
The analgesic activity was studied on a group of male
and female white mongrel mice weighing 16 – 18 g on the
model of vinegar convulsions [8]. The synthesized com-
pounds were introduced perorally with a 2% starch jelly; An-
imals in the control group received pure starch jelly. The
model convulsions were induced, 1 h after treatment with the
synthesized compounds, by intraperitoneal injections of
0.75% aqueous acetic acid solution in a dose of 0.25 ml per
10 g animal body weight. The test results were evaluated by
counting the number of convulsions over a time period of
10 min and by calculating the percentage inhibition of con-
vulsions relative to control.
Yield of compound X, 0.24 g (70%); 1H NMR spectrum
in CDCl3 (d, ppm): 7.73 – 7.12 (m, 6H, Harom), 5.76 (d, 1H,
NH), 4.54 (m, 1H, CaH leucine), 3.91 (s, 3H, OCH3), 3.76
(q, 1H, CH naproxen), 1.60 (d, 3H, CH–CH3 naproxen), 1.57
(m, 2H, CH2CH), 1.42 (m, 1H, CH2CH), 0.87, 0.86 (2d, 6H,
(CH3)2).
The acute toxicity for intraperitoneal injections was de-
termined in a group of male and female white mice weighing
16 – 20 g and evaluated as LD50 [9].
EXPERIMENTAL PHARMACOLOGICAL PART
The experimental data were statistically processed in
terms of the Student criterion [10]. The effects were consid-
ered as reliable for p < 0.05. It was established that, of the to-
tal of seven derivatives studied, only two naproxen
amides – with L-histidine methylate (IV) and phenylalanine
methylate (III) – exhibit reliable antiinflammatory activity
(Table 2). Investigation of the dynamics of the
antiinflammatory action for the most active compound IV
upon peroral administration showed a reliable effect 2 and
3 h after introduction (Table 3), while the maximum effect of
naproxen was observed 4 and 8 h after treatment.
Four compounds (IV, V, X, and XI) of the total of seven
studied exhibited a reliable analgesic effect (Table 2). Most
of the synthesized compounds are inferior to naproxen with
respect to the acute toxicity (Table 2).
Thus, the most promising results were observed for
naproxen amide with methyl ester, which, while possessing
less pronounced antiinflammatory and analgesic properties,
exhibits a somewhat lower acute toxicity.
The antiinflammatory activity was studied on a group of
both male and female rats weighing 180 – 220 g. The model
edema was induced by subplantar 0.1-ml injections of a 1%
aqueous carrageenan solution [5]. The synthesized com-
pounds suspended in a 2% starch jelly were intraperitoneally
injected 1 h before model edema induction. The drug dose
corresponded to ED50 for naproxen (15 mg/kg [6]), with a
coefficient taking into account the molecular mass of each
amide. Animals in the control group received pure 2% starch
jelly. The results, evaluated as an increase in the foot volume
relative to the initial value (measured before test), were de-
termined oncometrically 4 h after carrageenan injection.
From these data, the percentage edema growth relative to the
initial volume and a percentage inhibition of the model
edema growth relative to that in the control group were cal-
culated.
For the most active compound, the antiinflammatory ac-
tion with respect to the carrageenan edema development was

236
G. L. Levit et al.
5. C. A. Winter, E. A. Risley, and G. W. Nuss, Proc. Soc. Exp.
ACKNOWLEDGMENTS
Biol. (N. J.), 111(3), 544 – 547 (1962).
6. R. D. Syubaev, M. D. Mashkovskii, G. Ya. Shvarts, and V. I.
This study was performed within the framework of the
Regional Program of “Support for Interdisciplinary Projects
between the Ural and Siberian Divisions of the Russian
Academy of Sciences.”
Pokryshkin, Khim.-Farm. Zh., 19(1), 33 – 39 (1985).
7. F. P. Trinus, B. M. Klebanov, V. I. Kondratenko, et al. (eds.),
Methodological Recommendations on the Experimental (Pre-
clinical) Investigation of Nonsteroidal Antiinflammatory Phar-
maceuticals [in Russian], Pharmaceutical Committee of the
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