INTRODUCTION:

2-Acetoxybenzoic acid, a non-steroidal anti-inﬂammatory drug, could not be used as to its’ potentials because of adverse reactions offered due to presence of free carboxylic acid group. The non-steroidal anti-inﬂammatory drugs (NSAIDs) are widely used for indications extending from inﬂammation and pain to cardiovascular and genitourinary diseases. In the recent years a number of NSAIDs have been introduced into clinical practice. Hunt is on to relieve pain and inﬂammation with freedom of undesirable effects. Gastrointestinal side effects constitute the most frequent of all the adverse reactions of NSAIDs and often these reactions lead to GIT (Gastro Intestinal Tract) ulceration and hemorrhage. GI (Gastro intestine) mucosal injury produced by NSAIDs is generally believed to be causes by two different mechanisms.^1,8,9

The ﬁrst mechanism involves a local action comprised of a direct contact and second on indirect effect on the GI mucosa. The direct contact effect can be attributed to a combination of a local irritation produced by acidic group of NSAIDs and local inhibition of prostaglandin synthesis in the GIT.

The indirect effect can be attributed to combination of an ion trapping mechanism of NSAIDs from the lumen into the mucosa. The second mechanism is based on a generalized systemic action occurring after absorption, which can be demonstrated following intravenous dosing.

Recently, considerable attention has been focused in the development of bio-reversible derivatives, by temporarily masking the acidic group of NSAIDs, as a promising means of reducing or abolishing the GI toxicity.

In the present study well-recognized NSAID, 2-Acetoxybenzoic acid was selected, which suffers with the gastrointestinal side effects. Literature reveals that many efforts had been made to synthesize amino acid ester, glycolamide ester, and amide prodrugs using various amines but few attempts were made to develop amide prodrugs using amino acids.^1,2,5,7

The salient features of the usefulness of conjugation of amino acids with NSAIDs are as follows:

(i) Amino acids are normal dietary constituent and they are non-toxic in moderate doses as compared to other promoities;

(ii)Amino acids have healing effect on gastric lesions produced by NSAIDs;

(iii) A drug with free carboxyl group can be derivatized into corresponding esters or amide of amino acids, so as to alter the physical properties of a parent drug with one or more of the hydrolase enzymes serving as the in-vivo reconversion site(s);

(iv) Being a nutritional substance, the use of amino acids as a derivatizing group might also permit more speciﬁc targeting site for enzymes involved in the terminal phase of digestion;

(v) Many amino acids possess marked anti-inﬂammatory activity against gelatin induced hind paw oedema in rats;

(vi) By using different types of amino acids like non-polar, polar, acidic and basic, the drug molecule can be made more or less polar, or more or less soluble in given solvent.

Thus present work aims to synthesize amide prodrugs of 2-Acetoxybenzoic acid using amino acid ester with the expectation to get non-toxic prodrugs with minimized GIT disturbances while maintaining the useful anti-inﬂammatory and analgesic activities. Various proteolytic enzymes will help in release of 2-Acetoxybenzoic acid by hydrolysis of peptide linkage.^1,2,3,5,6,7

MATERIALS AND METHODS:

Materials:

All the amino acids, namely, glycine, L-phenylalanine, L-glutamic acid, L-aspartic acid were accquired from Raruan Chemicals Co. Ltd., China. Other reagents and solvent used were of Analytical/spectroscopic/HPLC grade as the case desired.

Synthesis of ethyl esters of amino acid hydrochloride salts:^10,11

Amino acid contains both acidic and basic groups in the same molecule and exists in the zwitterionic form. The non-availability of the free amino group in the zwitterionic form of the amino acid restricts its use in the formation of amide. Esteriﬁcation of amino acid in presence of HCl produces amino acid ester hydrochloride in which neutralization of HCl, using aqueous alkali, pyridine or triethylamine, generates free amino group to react as nucleophile in the synthesis of amide.

Method 1:

Amino acid (0.01 mol/l) in ethanolic hydrogen chloride (100 mL) was taken in a round bottom ﬂask ﬁxed with reﬂux condenser placed over a water bath and reﬂuxed for 2 h. After reﬂuxing, evaporation of the solvent at reduced pressure gave crude ethyl ester hydrochloride. Crude product was dried in vacuum desiccator to get the solid product which was recrystallized by dissolving the product in minimum volume of absolute alcohol by slow addition of ether followed by cooling at 0^oC. Recrystallized product was washed with anhydrous ether and dried under vacuum. This method was utilized for syntheses of ethyl ester hydrochloride salts of glycine, L-phenylalanine.

Method 2:

Amino acid (0.025 mol/l), absolute alcohol (400mL) and concentrated HCl were taken in a round bottom ﬂask. The solvent was distilled off, at a very slow rate, at 70^oC. Dry toluene was added time to time in the reaction mixture to remove the water produced during the esteriﬁcation reaction. After the complete removal of the alcohol a little more quantity of alcohol was further added along with dry toluene. After 2 h, the remaining solvent was evaporated under high vacuum to get the crude ethyl ester hydrochloride of amino acid. Product was collected and dried. Recrystallisation was performed by dissolving the product in minimum quantity of warm alcohol followed by slow addition of anhydrous ether at 4^oC. Product was dried under vacuum and collected in airtight container. This method was utilized for synthesis of ethyl ester hydrochloride salts of L-aspartic acid, and L-glutamic acid.

General procedure for acylation of amino acid ethyl ester hydrochloride:^10,11

Amino acid ester hydrochloride (0.01 mol/l) was taken in 5% aqueous solution of potassium carbonate at 2-5^oC, followed by dropwise addition of solution of salt of drug in ethanol. Reaction mixture was stirred for 22 hrs. at the same temperature. White precipitate was obtained and it was removed, and then washed with distilled water. Product was dried in vacuum and recrystallised by selective precipitation using alcohol-water mixture. It was dried in air and stored in tightly closed container.

Characterization of the synthesized prodrugs:⁴

The synthesized compounds were subjected to thin layer chromatography in order to check their purity. The prepared plates of silica gel G adsorbents were dried and activated. The solvent system C₃H₇OH:H₂O:NH₃ (6:4:0.06) was used. Iodine vapour was used as detecting agent. All compounds gave single spot. The melting points of the synthesized prodrugs were determined by open capillary tube using VEEGO melting point apparatus and are uncorrected. UV spectroscopy was performed on Shimadzo 1700 (CH₃OH). The IR spectra of the compounds were obtained on IR spectrophotometer (Prkin Elmer). The ¹HNMR spectral analysis of the synthesized prodrugs was done on NMR spectrophotometer (BRUKER AVANCE II 400) (CDCl₃)as solvent. MASS spectra were recorded on Jeol SX102-FAB mass spectrometer. All these data are summarized in Table-1

Table-1

Pr.	M. formula	M. Wt.	Melting Point	% Yield	Rf value	Partition Coeff.
AGE	C₁₂O₅H₁₅N	253	128-132^oC	94.66	0.58	59.81
APAE	C₁₉O₅H₂₁N	403	194-198^oC	92.66	0.72	58.71
AGLE	C₁₇O₇H₂₃N	389	175-178^oC	93.66	0.65	43.79
AAPE	C₁₆O₇H₂₁N	365	120-122 ^oC	90.66	0.76	50.80

P=Product; Solvent system: C₃H₇OH:H₂O:NH₃ (6:4:0.06), melting points are uncorrected.

Chemistry:

AGE (2-Acetoxybenzoglycine-ethylester), yield 94.66%, white crystalls, λ_max 276 nm., IR {3438.4(-NH), 3029(Ar.=CH), 2698.2(Ali.-CH), 1752.9(-C=O ester), 1306.8(-C=O carbox.), 1486.3-1458.2(Ali.-CH bend.)}, H¹ NMR (CDCl₃), 1.30-1.27 {(t) CH₃, -OC(=O)-C}, 2.28 {(t) CH₃, -OC(=O)-C}, 3.17-3.65 {(d) CH₃, -NC(=O)}, 4.25-4.23 {(s), CH₃, -N-C=O}, 7.10-7.08 {(d) Ar. –OC(=O)C}, 7.33-7.30 {(t) Ar. –OC(=O)C}, 8.00 {(d) -NH}, (m/e) M+ 253,238,210,208,166,151.

APAE (2-Acetoxybenzophenylalanine-ethylester), yield 92.66%, white crystalls, λ_max 276 nm., IR {3417.6(-NH), 3064.9(Ar.=CH), 2587-2698.2(Ali.-CH), 1753.6(-C=O ester), 1306.3(-C=O carbox.), 1480-1458(Ali.-CH bend.)}, H¹ NMR (CDCl₃), 1.59-1.58 {(d) CH₃, -OC(=O)-C}, 2.29 {(s) CH₃, -C(=O)O}, 7.09-7.07 {(d) Ar. –C=C-}, 7.33-7.31 {(t), Ar. -C=C-}, 7.57-7.53 {(t) Ar. –OC(=O)C, -C(=O)N}, 8.00 {(d) -NH}, (m/e) M+ 403,388,360,358,326,226,211.

AGLE (2-Acetoxybenzoglutamate-ethylester), yield 93.66%, white crystalls, λ_max 276 nm., IR {3446.1(-NH), 3070.9(Ar.=CH), 2698.8(Ali.-CH), 1752.8(-C=O ester), 1306.2(-C=O carbox.), 1457.2(Ali.-CH bend.)}, H¹ NMR (CDCl₃), 2.29 {(t) CH₃, -NC, -C-}, 7.09-7.07 {(d) Ar. –OC(=O)C, -C(=O)N}, 7.30-7.28 {(t) Ar. –OC(=O)N, -C(=O)N}, 7.56-7.52 {(t), Ar. –OC(=O)C, -C(=O)N}, 8.00 {(d) -NH}, (m/e) M+ 389,374,346,344,202,187.

AAPE (2-Acetoxybenzoaspartate-ethylester), yield 90.66%, white crystalls, λ_max 276 nm., IR {3428.7(-NH), 3079.9(Ar.=CH), 2697.4(Ali.-CH), 1753.7(-C=O ester), 1306.5(-C=O carbox.), 1480.3-1458.2(Ali.-CH bend.)}, H¹ NMR (CDCl₃), 1.54-1.53 {(d) CH₃, -OC(=O)C}, 2.28 {(s) CH₃, -C(=O)O}, 2.58 {(s) CH₃, -C(=O)O}, 3.84-3.82 {(d) CH₃, -OC(=O)C}, 7.10-7.08 {(d) Ar. –OC(=O)C, -C(=O)N}, 7.34-7.30 {(t) Ar. –OC(=O)N, -C(=O)N}, 7.59-7.55 {(t), Ar. –OC(=O)C, -C(=O)N}, 8.00 {(d) -NH}, (m/e) M+ 365,350,322,277,237,222,194.

Partition coefﬁcient:⁴

20 mg of prodrug was weighed and dissolved in 20 mL chloroform. The contents were thoroughly shaken for 24 hrs. at room temperature followed by transferring in separating funnel. Absorbance at 276 nm. was observed for both the solvents.

P_O/W = (C_CHCL3/C_H2O)

Where, C = Concentration in respective solvents,

Hydrolysis studies:⁴

Hydrolysis studies were studied using SGF and SIF (1.2 and 7.4 pH respectively), 20 mg of drug and prodrug were hydrolysed in both of the medium. Hydrolysis rate was determined using the concentration curve and absorbance of the samples withdrawn at 10-10 min interval.Temperature was maintained at 37⁰±1, on dissolution apparatus with 900 ml of each of the media solution.

Pharmacological evaluations:

All the synthesized prodrugs along with 2-Aceoxybenzoic acid were evaluated for analgesic, anti-inﬂammatory activities, antipyretic activity and ulcerogenic index. The prodrugs were compared with standard drug for these activities. The methods employed for this purpose were as following:

Anti-inﬂammatory activity:¹²

Anti-inﬂammatory activity of synthesized prodrugs was determined by hind paw oedema method utilizing carrageenan (0.1 mL, 1%) as phlogistic agent. Carra-geenan produces inﬂammation and oedema by causing the release of histamine, 5-HT and bradykynin. The animals used were Wistar rats (albino rats). Rats (100-200 g) were divided into 6 groups, each comprising of 6 rats, including a control and standard group. The initial volume of right hind paw of albino rats was measured by plethysomometer, without administration of the drug/ prodrug. The drug/ prodrugs (dose of each prodrug was calculated equivalent to 7.3 mg/kg body weight) were administered orally in 1% suspension of sodium carboxymethylcellulose (containing 0.1% m/v of drug or prodrug). After 30 min of administration of drug/ prodrugs, carrageenan (0.1 mL, 1% m/v solution in normal saline) was injected into the planter surface of right hind paw of each animal as phlogistic agent. The volume of right hind paw of albino rats was measured by plethysmometer after 2, 4 and 6 hrs. The mean difference in the volume of the right hind paw of rats was compared with standard and control. Percent anti-inﬂammatory activity was calculated using the following formula:

Anti-inﬂammatory % = [1 – (Vt – Vc)] × 100

where Vc = Mean relative change of the volume of the right hind paw of rats in the control group; Vt= Mean relative change of the volume of the right hind paw of rats in the test group.

Analgesic activity:¹²

Analgesic activity of the synthesized prodrugs was determined by hot plate method using thermal stimulus. A hot plate analgesiometer was used for the determination of pain threshold of Wistar rats (albino rats). Rats (100-200 g) were divided into 6 groups, each comprising of 6 rats. The normal reaction time, i.e., the time taken to response to pain was noted. The drug/ prodrug (dose of each prodrug was calculated to equivalent to 7.3 mg/kg body weight) was administered orally in 1% suspension of sodium carboxymethylcellulose. Percent analgesia was calculated using the following formula

Analgesia % = [1 – (T2/T1)] × 100

Where, T1 = reaction time in sec. before administration of drug/ prodrug; T2 = reaction time in sec. after administration of drug/ prodrug.

Antipyretic activity:¹²

A 15% suspension of Brewer’s yeast in 0.9% saline is prepared. Groups of 6 male or female Wistar rats with a body weight of 100 g are used. By insertion of a thermocouple to a depth of 2 cm into the rectum the initial rectal temperatures are recorded. 18 hrs. post challenge, the rise in rectal temperature is recorded. The measurement is repeated after 30 min. The animals receive the test compound or the standard drug by oral administration. Percent Antipyretic activity was calculated using the following formula

Antipyretic % = [1-(T2/T1)]*100

Where, T1 = initial temp. after introduction of Pyresis;

T2 = temp. at each interval.

Ulcerogenic index:¹³

Gastro-intestinal toxicity of the synthesized prodrugs was measured and compared with the drug by measuring ulcerogenic index. For this purpose male albino rats (Wistar rats) were selected, weighing between 130-150 g. The rats were divided into 6 groups each comprising of 6 rats, including a control and standard group. The prodrug/ drug (100 mg) was suspended in 100 mL of 2% m/v suspension of acacia. Measured volume of the suspension containing dose equivalent to 7.3 mg/kg of body weight of drug/prodrug was administered orally to the test group daily for ﬁve days. The rats were fasted after the administration of last dose, thereafter they were sacriﬁced by decapitation and the stomach was removed, opened and washed with distilled water. The lesions on the gastric mucosa were counted by visual examination using a 2x2 binocular magniﬁer. Ulcers greater than 0.5 mm were recorded.

An ulcer index UI is calculated:

UI = [U_N + U_S + U_P]/10

Where, U_N= average of number of ulcers per animal,

U_S = average of severity score

U_P = percentage of animals with ulcers

RESULTS AND CONCLUSION:

Acylation of amino acid ester was performed using various conditions like in the presence of 5% K₂CO₃ at 0-2^oC. All these methods suffer with the drawback of hydrolysis of acid chloride, slow rate of reaction and difﬁculty in the collection of ﬁnal product; due to lump formation. Recrystallisation of the compounds was performed by selective precipitation with water-alcohol mixture. In some cases the oily products were obtained which were crystallized by induced crystallization using acetone/petroleum ether in the ethanolic solution of the prodrug.

The synthesized prodrugs of 2-Acetoxybenzoic acid were subjected to solubility, partition coefﬁcient, and hydrolytic studies. Solubility studies showed that the drug and all prodrugs were insoluble in water, but they showed moderate to high solubility in various solvents such as methanol, ethanol, chloroform, dichloromethane and benzene, which indicates lipophilic behaviour of the compounds.

The partition coefficient studies at pH 7.4/1.2 of the drug and prodrugs were determined, Partition coefﬁcient study of prodrugs showed that the major fraction of the prodrugs was partitioned towards the organic phase. It indicates enhancement in the lipophilic property, which might be favorable to biological absorption. Po/w of drug and prodrugs were found to be b/w 50-60.

Hydrolytic studies in SIF and SGF of the compounds, showed that the drug is mostly hydrolized in SGF (47.24% remained) where as the prodrugs were less hydrolized in the SGF (91-78% remained). There is a marked hydrolysis in the SIF media (59-49% remained).

Pharmacological screening of the synthesized prodrugs was done for anti-inﬂammatory, analgesic, antipyretic and ulcerogenic activity. In each study albino rats of either sex weighing (100 gm.) were used. The dose of drug or prodrug was given in form of 1% CMC suspension through oral route. In case of prodrugs of the dose administered was equivalent to asprin (7.3 mg/Kg of body weight).

The analgesic activity of synthesized prodrugs 2-Acetoxybenzophenylalanine-ethylester and 2-Acetoxybenzoglycine-ethylester was found highest of all the compounds. Of all them 2-Acetoxybenzophenylalanine-ethylester was most active its onset of action was 30 min. others remained as comparable with 2-Acetoxybenzoic acid.

The anti-inﬂammatory activities obtained, 120-150 min. after the administration of standard drug was found highest of the other time intervals. All compounds showed same activity as of standard drug up till 60 min. at 120-150 min. 2-Acetoxybenzoglutamate-ethylester was the most active compound to show the anti-inflamatory activity.

The anti-pyretic activity was showed only by the 2-Acetoxybenzoaspartate-ethylester. Uptill 60 min. there was no response of any compound. At 90 min. the response was noted, till 180 min. all the compounds showed the same values of response. Only 2-Acetoxybenzoaspartate-ethylester gave highest response at 120 mn.

Higher percentage of the pharmacological effect observed for the amide derivatives might be the result of either better absorption of the amides from the GI tract resulting in higher bioavailability of the compounds or due to higher selectivity of these derivatives towards the COX-2 enzyme than the parent drug.

Ulcerogenic Index of the synthesized prodrugs was recorded to observe the extent of gastrointestinal side effects. The ulcerogenic index of standard drug was found 22.33. The ulcerogenic index of prodrugs was found better as compared to the STD group.

The stability of the prodrugs in buffer of pH 1.2 indicated that the prodrugs would be absorbed intact without exposing the GIT to the acidic carboxy group. Hence, the observed ulcerogenicity of the prodrugs has been attributed to the systemic inhibition of prostaglandins by the parent drug, which was released upon hydrolysis of amides in circulaton.

All the prodrugs showed good activity in comparison to standard drug in the initial phase, after the administration, but a gradual increase was observed in the activity. This might be due to various factors including hydrolysis rate, dissolution rate and protein binding etc. Ulcerogenic index of the prodrug was found much lesser in comparison to standard drug. The minimized side effects obtained in the prodrug might be due to inhibition of direct contact of carboxyl group of the drug to the gastric mucosa, which is mainly responsible for the damage. It is also due to negligible dissolution as well as hydrolysis in acidic buffer (pH 1.2).

On the basis of the results, it is concluded that prodrug approach can be successfully applied in attaining the goal of minimized gastrointestinal toxicity without loss of desired pharmacological activity of the drug. The good pharmacological response indicates that the absorption of the prodrugs might be regulated by some other means like presence of amino acid transport system besides dissolution. All the synthesized prodrug showed the better results as compared to the STD drug, they showed excellent pharmacological response and encouraging hydrolysis rate in SIF. On contrary prodrugs with increased aliphatic side chain length or introduction of aromatic substitution showed enhanced partition coefﬁcient but diminished hydrolysis rate. Such prodrugs can be considered for sustained release purpose.

ACKNOWLEDGEMENT:

We are thankful to the management of VNS. Institute of Pharmacy, for financial as well as infrastructural support to carry out above research work. Our team is also thankful to Raruan Chemicals Co. Ltd., China for providing us free samples of amino-acids.

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Received on 13.07.2011 Modified on 14.08.2011

Asian J. Research Chem. 4(11): Nov., 2011; Page 1695-1700