Synthesis and anti-inflammatory activity of acetylsalicylamino acids and peptides

Article abstract of DOI:10.1007/s10600-006-0079-5

Several water-soluble acetylsalicylamino acids and peptides containing neutral and acidic amino acids were synthesized and investigated for anti-inflammatory activity. 2006 Springer Science+Business Media, Inc.

Full text of DOI:10.1007/s10600-006-0079-5

Chemistry of Natural Compounds, Vol. 42, No. 2, 2006
SYNTHESIS AND ANTI-INFLAMMATORY ACTIVITY
OF ACETYLSALICYLAMINO ACIDS AND PEPTIDES
Sh. Kh. Khalikov, M. Kodirov, and S. V. Alieva
UDC 577.126.6+577.152
Several water-soluble acetylsalicylamino acids and peptides containing neutral and acidic amino acids were
synthesized and investigated for anti-inflammatory activity.
Key words: aspirin, acetylsalicylic acid, amino acids, acid chlorides, -hydroxysuccinimide, peptide.
N
Side effects of aspirin, acetylsalicylic acid (Ac-Sal-OH), can be avoided by decreasing the acidity and increasing the
pharmacological activity, which can be achieved by preparing new amino-acid and peptide derivatives of acetylsalicylic
acid [1].
The first attempt to prepare acetylsalicylamino acids was made in 1982 [2].
The goal ofthe present work was tosynthesize water-soluble amino-acid and peptide derivatives of acetylsalicylic acid.
We used amino acids with hydrophobic and hydrophilic groups that affected the conformation of the final product in general
and its solubility in polar and nonpolar solvents. Therefore, we synthesized and investigated the anti-inflammatory activity of
thefollowingcompounds: Ac-Sal-Gly-OH(2), Ac-Sal-Gly-OMe(3), Ac-Sal-Ala-OH(4), Ac-Sal-Ala-OMe(5), Ac-Sal-Val-OH
(6), Ac-Sal-Leu-OH(7), Ac-Sal-Pro-OH(8), Ac-Sal-Phe-OH(9), Ac-Sal-Tyr-OH(10), Ac-Sal-Glu(OH) (11), Ac-Sal-Asp(OH)
2
2
(12), Ac-Sal-Gly-Gly-OH (13), Ac-Sal-Tyr-Pro-OH (14), Ac-Sal-Tyr-Pro-Phe-OH (19).
The first step in the synthesis was activation of the carboxylic acid of acetylsalicylic acid (1) by converting it to
acetylsalicylic acid chloride (Ac-Sal-Cl, 1a) using thionyl chloride and a catalyst of 2-hydroxypyridine (2-HP, 97%) or AlCl
3
(81%) and without a catalyst (75%).
R
COCl
C
O
OCOCH
OCOCH
3
3
2 - 5
1a
2: R = NHCH COOH
4: R = NHCH(CH )COOH
2
3
3: R = NHCH COOCH
5: R = NHCH(CH )COOCH
2
3
3 3
The highest yield (97%) of crystalline acetylsalicylic acid chloride was obtained using 2-HP catalyst.
Glycine, alanine, and their methyl esters were added to the resulting acetylsalicylic acid chloride.
The methyl esters of acetylsalicylamino acids 3 (78%) and 5 (77%) were prepared in higher yields than
acetylsalicylamino acids 2 (48%) and 4 (56%). Obviously this was due to the higher nucleophilicity of the amine group of the
amino-acid esters.
The yields of acetylsalicylamino acids were increased by using activated esters. We found that the most effective was
the -hydroxysuccinimide ester of acetylsalicylic acid (Ac-Sal-ONSu). The sodium salts of valine, leucine, proline,
N
phenylalanine, tyrosine, glutamic, and aspartic acid were condensed with the -hydroxysuccinimide ester ofacetylsalicylic acid
N
by the literature method [3] to give 2 and 4 in increased yields of 84 and 74%, respectively.
Acetylsalicylpeptides were synthesized by a scheme such that the synthesized peptides were easily isolated from the
reaction medium with minimal racemization. We used a synthesis of Ac-Sal-Tyr-Pro-Phe-OH (19) that clearly demonstrated
the possibility in principle of extending the peptide chain stepwise using free terminal amino acids. Part of the Z-Tyr(OBzl)-
Tadzhik State National University, e-mail: shirin41@yandex.ru. Translated from Khimiya Prirodnykh Soedinenii,
No. 2, pp. 169-172, March-April, 2006. Original article submitted October 25, 2005.
0009-3130/06/4202-0204 ©2006 Springer Science+Business Media, Inc.
204

ONp remained unreacted in the synthesis of 15 [4] and was removed using column chromatographyover silica gel (L-100/160)
with elution bybenzene:acetone:acetic acid (100:50:2) and then methanol to isolate the principal product Z-Tyr(OBzl)-Pro-OH
(15).
TheprotectedtripeptideZ-Tyr(OBzl)-Pro-Phe-OH(17)waspreparedbycondensation ofZ-Tyr(OBzl)-Pro-ONSu (15a)
with phenylalanine in DMF. The condensing agent was N,N-dicyclohexylcarbodiimide (DCC). The protected tripeptide 17
was purified by extraction with ethylacetate from an acidified (pH 3-4) aqueous solution of the reaction mixture and subsequent
precipitation from ethylacetate by hexane. The Z- and -Bzl groups were removed from the protected tripeptide 17 by catalytic
hydrogenation over Pd-catalyst. The final product Ac-Sal-Tyr-Pro-Phe-OH (19) was synthesized by condensation of Ac-Sal-
ONSu (15b) with the sodium salt of H-Tyr-Pro-Phe-OH (18) in DMF. Crystalline product 19 was isolated by extraction with
ethylacetate from an acidified aqueous solution of the reaction mixture. The synthetic results led to the conclusion that the
method using the acid chloride and the N-hydroxysuccinimide can be used successfully to synthesize acetylsalicylamino acids
and peptides because the product yield in the final step is rather high.
Yield, %
Ac Sal OH
Tyr
Pro
Phe
ONp
OBzl
Z
H
OH
15
72
Z
OH
OBzl
OBzl
OBzl
15a
Z
Z
ONSu H
OH
OH
62
(17)
Ac
Ac
76
89
ONSu
15b
H
OH
OH
(18)
(19)
The anti-inflammatory activity of the amino-acid and peptide derivatives of acetylsalicylic acid was determined by
screening in rabbits in which inflammation was induced by formalin. The extent of the inflammation was estimated from the
amount of leucocytes in the blood as determined on a Picoscale PS-4 instrument.
The results showed that the synthetic derivatives ofAc-Sal-aminoacids and peptides have anti-inflammatoryactivities
that differ from each other. However, these properties are clearly evident for the peptide derivatives (13 and 19). The anti-
inflammatory activity of the synthesized compounds are given below (dose 5 mg/kg):
Compound
Anti-inflammatory activity, %
1
2
100
65
4
82
8
56
12
13
14
19
20
104
94
111
.
EXPERIMENTAL
Weusedacetylsalicylicacidpreparedbyacetylation ofsalicylic acid and commercial L-aminoacids(Reanal, Hungary).
Chromatography was performed on Silufol plates (Czech Rep.) using CHCl :CH OH (60:13, A), CHCl :CH OH:CH CO H
3
3
3
3
3
2
(90:10:5, B), C H :(CH ) CO:CH CO H (100:50:2, C), and C H OH:CH CO H:H O (3:1:1, D). Elemental analyses were
6
6
3 2
3
2
4
9
3
2
2
performed on a 240B Perkin—Elmer analyzer; amino-acid analysis, on a Durram D 500 analyzer (USA). Chromatograms were
developed using alcoholic ninhydrin (0.5%), I vapor, and Cl -benzidine KI. Hydrogenation was carried out in the presence
2
2
of Pd/C (Merck, 5 and 10 wt.%). Specific optical rotation was determined in a Polamat polarimeter (GDR); melting points, on
a Boetius instrument (GDR). Amino-acid analysis was performed on a Hitachi-635 instrument (Japan) after hydrolysis in HCl
205

(6 N) with phenol (2%) at 105-110°C for 24 h. We used silica gel L-100/160 (Chemapol, Czech Rep.). Solvents were
evaporated in a rotary evaporator at 40-50°C (if the temperature is not given).
Acetylsalicylic Acid Chloride (1a). Ac-Sal-OH (1, 0.9 g, 5 mmol) prepared as before [5] was dissolved in CHCl
3
(6 mL) and treated with SOCl (3 mL, 5 mmol) and a catalytic amount of AlCl . The reaction mixture was heated for 4 h at
2
3
80-85°C, cooled, and filtered to remove unreacted Ac-Sal-OH. Solvent was evaporated. The oily product was dissolved in
absolute ether, washed with water, dried over Na SO , and filtered. The filtrate was evaporated to afford 1a (0.8 g, 81%),
2
4
mp 58-59°C, R 0.86 (B) and 0.76 (D).
f
The reaction was performed analogously using 2-HP catalyst and without catalyst to give yields of 97 and 75%,
respectively.
Ac-Sal-Gly-OH (2). A. A solution of freshlyprepared acetylsalicylic acid chloride (1a, 2.5 g, 12.5 mmol) in dioxane
(20 mL) was stirred, cooled to -5°C to produce a suspension, and treated dropwise over 30 min with glycine (1.22 g, 12.5 mmol)
in NaOH (10.2 mL, 12.5 mmol, 1.22 N). The reaction mixture was left overnight at room temperature. Solvent was evaporated
to produce an oil that was dissolved in water and acidified with HCl (3 N) until the pH was 3-4. The product was extracted with
ethylacetate (3 × 20 mL) and washed with HCl (3 N) and water. Solvent was evaporated to produce an oil that was dissolvd
in ether and precipitated with hexane. Repeated reprecipitation from ethylacetate by ether produced 2 (1.5 g, 51%),
mp 120-121°C, R 0.86 (A) and 0.81 (B).
f
B. Glycine (0.375 g, 5 mmol) was dissolved in NaOH (4 mL, 5 mmol, 1.25 N). Water was evaporated. The solid was
dissolved in DMF, cooled, stirred, and treated with acetylsalicylic acid N-hydroxysuccinimide (1.355 g, 5 mmol) [3] in DMF.
The reaction mixture was stirred at 0°C for 2 h and left for 1 d. Solvent was evaporated. The solid was dissolved in water and
washed with ether (3 × 20 mL). The aqueous layer was acidified with HCl (3 N) until the pH was 3, extracted with ethylacetate
(3 × 20 mL), and washed with water until neutral. Solvent was removed. The solid was recrystallized from ethylacetate to
afford 2 (0.99 g, 84%), C H NO (237.11), mp 121-122°C, R 0.86 (A) and 0.81 (B). Amino-acid analysis: Gly 1.01(1).
f
11 11
5
Ac-Sal-Gly-OMe (3). Glycine methyl ester hydrochloride (1.25 g, 10 mmol) prepared as before [6] was dissolved with
stirring in CHCl (20 mL), cooled to -5°C, and treated with Et N (2 g-eq) and dropwise with cooled (-5°C) Ac-Sal-Cl (1.98 g,
3
3
10 mmol) in CHCl (20 mL). The mixture was stirred for another 1 h and left for 1 d, washed several times with NaHCO
3
3
solution, H O, and HCl (0.01 N), washed again with H O, dried over Na SO , and evaporated to afford 3 (oil, 1.9 g, 77%),
2
2
2
4
R 0.75 (B) and 0.69 (C).
f
Ac-Sal-Ala-OH (4). Alanine (0.64 g, 7.2 mmol) was dissolved in NaOH (5.9 mL, 7.2 mmol, 1.22 N) and evaporated
to dryness. The solid was dissolved in DMF, cooled, stirred, and treated with the N-hydroxysuccinimide ester of acetylsalicylic
acid (2.0 g, 7.2 mmol) that was dissolved earlier in DMF. The reaction mixture was stirred at 0°C for 2 h and left for 1 d.
Solvent was evaporated. The solid was dissolved in water, washed with ether (3 × 15 mL), acidified with HCl (3 N) until the
pH was 3-4, and extracted with ethylacetate (3 × 20 mL). The extract was washed with HCl (3 N) and water until neutral and
evaporated to afford an oil that was dissolved in ether, precipitated by hexane, and reprecipitated from ethylacetate by ether.
The resulting solid was separated to afford 4 (1.35 g, 74%), C H O N (251.11), mp 117-119°C, R 0.74 (A), 0.81 (B), and
f
12 13
5
0.69 (C). Amino-acid analysis: Ala 1.0(1).
Ac-Sal-Ala-OMe (5) was prepared analogously to 3 starting with alanine methyl ester hydrochloride (1 g, 7.1 mmol)
[6], DMF (20 mL), Et N (1 mL), and Ac-Sal-Cl (1.4 g, 7.1 mmol) to afford 5 (1.48 g, 78%), oil, R 0.88 (A) and 0.83 (B).
f
2
Ac-Sal-Val-OH (6) was prepared analogously to 4 starting with valine (0.84 g, 7.2 mmol) and Ac-Sal-ONSu (2 g,
7.2 mmol). The oily product was reprecipitated from ethylacetate with ether and purified over a column of silica gel (2.6 ×
60 cm) with elution by system C to afford 6 (1.36 g, 68%), C H O N (280.02), mp 184-186°C, R 0.70 (A), 0.78 (B), and
f
14 18
5
0.54 (C). Amino-acid analysis: Val 1.02(1).
Ac-Sal-Leu-OH (7) was prepared analogously to 4 from leucine (0.655 g, 5 mmol) and Ac-Sal-ONSu (1.385 g,
5 mmol) to afford 7 (1.2 g, 73%), C H O N (295.15), mp 192-194°C, R 0.61 (A), 0.72 (B), and 0.65 (C). Amino-acid
f
15 18
5
analysis: Leu 1.0(1).
Ac-Sal-Pro-OH (8) was prepared analogously to 4 from proline (0.632 g, 5 mmol) and Ac-Sal-ONSu (1.385 g,
5 mmol) to afford 8 (1.63 g, 80%), C H O N (230.14), mp 149-150°C, R 0.56 (A), 0.80 (B), and 0.64 (C). Amino-acid
f
14 15
5
analysis: Pro 1.0(1).
Ac-Sal-Phe-OH (9) was prepared analogously to 4 from phenylalanine (0.825 g, 5 mmol) and Ac-Sal-ONSu
(1.385 g, 5 mmol) to afford 9 (1.2 g, 69%), C H O N (279.0), mp 181-183°C, R 0.58 (A), 0.71 (B), and 0.65 (C). Amino-
f
18 16
5
acid analysis: Phe 1.01(1).
206

Ac-Sal-Tyr-OH (10) was prepared analogously to 4 from tyrosine (0.98 g, 5.41 mmol) and Ac-Sal-ONSu (1.5 g,
5.41 mmol) to afford 10 (1.41 g, 76%), C H O N (295.04), mp 167-169°C, R 0.67 (A), 0.63 (B), and 0.69 (C). Amino-acid
f
18
6 6
analysis: Tyr 1.0(1).
Ac-Sal-Glu(OH) (11) was prepared analogously to 4 from glutamic acid (1.470 g, 10 mmol) and Ac-Sal-ONSu
2
(2.77 g, 10 mmol) toafford 11 (2.68 g, 68%), C H O N (309.14), mp 185-187°C, R 0.69 (A), 0.57 (B), and 0.61 (C). Amino-
f
14 15
7
acid analysis: Glu 0.94(1).
Ac-Sal-Asp(OH) (12) was prepared analogously to 4 from aspartic acid (0.665 g, 5 mmol) and Ac-Sal-ONSu
2
(1.385 g, 5 mmol) to afford 12 (1.15 g, 78%), C H O N (295.18), mp 171-172°C, R 0.61 (A), 0.25 (B), and 0.20 (C). Amino-
f
13 13
7
acid analysis: Asp 1.03(1).
Ac-Sal-Gly-Gly-OH (13). H-Gly-Gly-OH (0.66 g, 7.2 mmol) [7] was dissolved in NaOH (4.62 mL, 7.2 mmol,
1.08 N), cooled to 0°C, stirred, and treated with Ac-Sal-ONSu (1.38 g, 7.2 mmol) that was dissolved earlier in DMF. The
reaction was carried out at 0-1 C for 3 h and left for 1 d. Solvent was evaporated. The solid was dissolved in distilled water
(40 mL), washed with ether (3 × 15 mL), acidified with HCl (3 N) until the pH was 3, and extracted with ethylacetate (3 ×
20 mL). The ethylacetate extract was washed with HCl (3 N) and water until the washings were neutral. Solvent was
evaporated. The solid was reprecipitated from ethylacetate with ether to afford 13 (1.2 g, 82%), C H O N (294.13),
13 14
6 2
mp 210-212°C, R 0.59 (A), 0.41 (B), and 0.50 (C). Amino-acid analysis: Gly 2.04(2).
f
Ac-Sal-Tyr-Pro-OH (14) was prepared analogously to 13 from H-Tyr-Pro-OH (0.08 g, 0.29 mmol) [7] and Ac-Sal-
ONSu (0.08 g, 0.29 mmol) to produce a thick mass that was purified over a silica-gel column (3 × 25 cm). Impurities were
eluted from the column using system C. The principal product eluted with CH OH to afford 14 (0.12 g, 81%), mp 113-115°C,
3
24
R 0.55 (A), 0.37 (B), and 0.41 (C), [α]
-16.18° (c 1, CH OH). Amino-acid analysis: Tyr 1.0(1), Pro 1.02(1).
3
f
D
Z-Tyr(OBzl)-Pro-OH (15) was prepared analogously to 13 from proline (1.00 g, 8.76 mmol) and Z-Tyr(OBzl)-ONp
(4.62 g, 8.76 mmol) [7] to afford 15 (3.18 g, 72%), C H O N (398.386), mp 68-69°C, R 0.86 (B), 0.50 (C), and 0.90 (D),
f
21 22
6 2
24
[α]
-10.24° (c 1, ethanol).
H-Tyr-Pro-OH (16). Compound 15 (0.2 g) was hydrogenated in CH OH to afford amorphous 16 (0.08 g, 72%), R
D
f
3
0.1 (A) and 0.21 (B).
Z-Tyr(OBzl)-Pro-Phe-OH (17) was prepared analogously to 13 from 15 (2 g, 3.9 mmol) in ethylacetate,
N-hydroxysuccinimide (0.5 g, 4.34 mmol), DCC (0.89 g, 4.34 mmol), and phenylalanine (0.66 g, 4 mmol) to afford 17
(1.23 g, 62%), mp 76-78°C, R 0.96 (A), 0.92 (B), 0.49 (C).
f
H-Tyr-Pro-Phe-OH (18). Compound 17 (0.2 g, 0.308 mmol) was hydrogenated in CH OH to afford 18 (0.1 g, 76%)
3
(amorphous), R 0.1 (A) and 0.16 (B).
f
Ac-Sal-Tyr-Pro-Phe-OH (19) was prepared analogously to 13 from 18 (0.1 g, 0.23 mmol) and Ac-Sal-ONSu
(0.063 g, 0.23 mmol). The product was isolated as an oil and treated with water. The emulsion was washed with ether (3 ×
20 mL). The aqueous layer was acidified with HCl (3 N) until the pH was 3, extracted with ethylacetate (3 × 20 mL), and
washed with HCl (3 N) and water. The ethylacetate was evaporated. The solid was worked up from ethylacetate with ether to
24
afford 19 (0.12 g, 89%), mp 118-120°C, R 0.50 (A), 0.33 (B), and 0.38 (C), [α]
-28° (c 1, CH OH). Amino-acid analysis:
3
f
D
Tyr 1.0(1), Pro 1.02(1), Phe 1.02.
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207