Anti-inflammatory and Anti-ulcer Activities of New Fused Thiazole Derivatives Derived from 2-(2-Oxo-2H-chromen-3-yl)thiazol-4(5H)-one

The reaction of the 2-(4-oxo-4,5-dihydrothiazol-2-yl)acetonitrile (1) with salicylaldehyde (2) in 1,4-dioxane containing a catalytic amount of piperidine gave the coumarin derivative 3. The latter reacted with different reagents to give pyrano[4,5-b]thiazole, pyrido[4,5-b]thiazole and thieno[5,4-b]thiazole dereivatives. The anti-inflammatory and anti-ulcer activities of the newly synthesized products were evaluated and the results showed that compounds 7a, 8a, 10b, 13b, 15b, 18a, 19b, 19c, and 19d showed higher activity compared to the rest of the compounds. In addition to this, toxicity of such active compounds was studied against shrimp larvae where compounds 10b, 18a, 19c and 19d showed to be non-toxic against the tested organisms.


Introduction
Thiazole is a core structural motif present in a variety of natural products, such as vitamin B1 (thiamine) and penicillin.Thiazole derivatives also exhibit a broad spectrum of medicinal and biological properties, such as antibacterial, antifungal, 1 anti-inflammatory, 2 antiviral, 3 antimalarial, 4 and anti-HIV activities. 5Thiazole analogs have also been reported as ligands at estrogen receptors, 6 neuropeptides, 7 Y5 adenosine receptors, 8 and act as inhibitors of human platelet aggregation factor, 9 urokinase, 10 and poly(ADP-ribose) polymerase-1. 11Selenazoles have been reported to possess antibacterial properties, 12 superoxide-anion-scavenging activity, 13 and exhibit cytotoxicity and DNA fragmentation effects in human HT-1080 fibrosarcoma cells. 14The structures of sulfathiazole, meloxicam, and selenazofurin and their pharmacological activities are given in Fig. 1.In many report structural modification of the heterocyclic rings through the construction of new heterocyclic nuclei enhances the pharmaceutical applications of the resulting molecules. 15,16This encouraged our efforts in this work to modify 2-(4-oxo-4,5-dihydrothiazol-2yl)acetonitrile through its reaction with salicyladehyde to produce a chromen-3-yl)thiazol which was used as the key starting compound for many further heterocyclic transformations.The anti-inflammatory and anti-ulcer evaluations of the newly synthesized compounds were studied.
Compound 3 was ready for thiophene formation through the well known Gewald's thiophene synthesis. 17,18hus, the reaction of 3 with elemental sulfur and either malononitrile (4) or ethyl cyanoacetate (4') gave the thieno [2,3-d]thiazole derivatives 8a and 8b, respectively.The analytical and spectral data of 8a,b were the tools of their structural elucidation.Thus, the 1  The 2-amino group present in compounds 8a and 8b is capable for the amide group formation.Thus, the reaction of either 8a or 8b with ethyl cyanoacetate in refluxing dimethylformamide gave the amide derivatives 12a and 12b, respectively.The latter compounds readily underwent cyclization when heated in sodium ethoxide solution to give the 7-phenylthiazolo[4',5':4,5]thieno [2,3-d]pyridine derivatives 13a and 13b, respectively.On the other hand, the reaction of either compound 12a or 12b with acetophenone (14) gave the Knoevenagel condensation products 15a and 15b, respectively (Scheme 3).
The presence of the but-2-enenitrile moiety in compounds 15a and 15b is suitable for thiophene synthesis.Thus, the reaction of either compound 15a or 15b with elemental sulfur gave the thiophene derivatives 16a and 16b, respectively.On the other hand, the reaction of eit-her 15a or 15b with any of benzenediazonium chloride (17a), 4-chlorobenzenediazonium chloride (17b), or 4-methoxybenzenediazonium chloride (17c) gave the arylhydrazone derivatives 18a-c, respectively (Scheme 4).The spectral and analytical data of compounds 18a-c were in agreement with their respective structures (see experimental section).The latter compounds underwent cyclization when heated in sodium ethoxide solution to give corresponding pyridazine derivatives 19a-c, respectively.

1. Anti-inflammatory Evaluation Method
Carrageenin-induced rat hind paw oedema model was used.The method adopted resembles essentially that described by Winter. 19The animals were studied for toxicity of DMSO up to 10% v/v in saline, and 5% DMSO was selected as a vehicle to suspend the standard drugs and the test compounds.
Albino rats weighing between 150 and 250 g of either sex were starved for 18 h prior to the experiment.The animals were weighed, marked for identification and divided into groups of six.The standard drugs, ibuprofen (20 mg/kg body weight), mefenamic acid (100 mg/kg body weight) and three graded doses (10, 20 and 40 mg/kg body weight) of the test compounds were given orally as a suspension using 5% DMSO as a vehicle.
One hour later foot paw oedema was induced by injecting 0.1 mL of 1% carrageenin subcutaneously into the planter portion of the right hind paw of each rat.Initial foot paw volume was measured immediately by mercury plethysmometer.Oedema was measured 3 h after carrageenin administration.The swelling in test group animals was used to calculate the % inhibition +/-SEM of oedema achieved by the compound at the test dose compared with the vehicle control group.The % protection of oedema was calculated according to the formula: (1)   where V t and V c are the volume of oedema in test compounds and control groups, respectively.

Anti-ulcer Evaluation Method
Animals Wistar albino rats weighing 150-200 g of either sex maintained under standard husbandary conditions (temperature 23 ± 2 °C, relative humidity 55 ± 10% and 12 h light/dark cycle) were used for the screening.Animals were fed with standard laboratory food ad libitum during the study period.
Albino rats of either sex were divided into four groups of six animals each.Animals were fasted for 24 h before the study, but had free access to water.Animals in the control group received only distilled water.Each of the given compounds at 250 and 500 mg/kg, (p.o.) were given to the animals in the treatment group.Ranitidine (50 mg/kg) was used as a standard.After 1 h of drugs treatment, they were anaesthetized with the help of anesthetic ether; the abdomen was opened by a small midline incision below the xiphoid process.Pyloric portion of the stomach was slightly lifted out and ligated according to the method of Shay 20 avoiding traction to the pylorus or damage to its blood supply.The stomach was replaced carefully and the abdominal wall was closed by interrupted sutures.Rats were sacrificed by an over dose of anaesthetic ether after 4 h of pyloric ligation.The abdomen was opened, cardiac end of the stomach was dissected out and the contents were drained into a glass tube.The volume of the gastric juice was measured and centrifuged at 2000 rpm for 10 min.From the supernatant, aliquots (1 mL each) were taken for the determination of pH, total and free acidity.Each stomach was examined for lesions in the fore stomach portion and indexed according to severity.
Determination of pH: an aliquot of 1 mL gastric juice was diluted with 1 mL of distilled water and pH of the solution was measured using pH meter.

3. Toxicity Method on Shrimp Larvae
All toxicity tests were 96-h static renewal tests and water quality measurements (dissolved oxygen, pH, temperature, salinity) were taken in the control containers each day.Tests were run in a Revcos Environmental Chamber at 25 °C, 20% salinity, and a 16 h light/8 h dark cycle.A media change was made every 24 h.Larvae used for all tests were one to two days old and exposed in 600 mL glass beakers containing 400 mL of media with 10 larvae/beaker and three replicates/concentration.Larvae were fed newly hatched Artemia after daily media exchange.The concentration of each compound was taken in terms 10, 100 and 100 mg/mL.Adult shrimp toxicity tests were also run to complete the grass shrimp toxicity profile.Adult shrimp (acclimated for two weeks before testing) were exposed in 4 L wide mouth glass jars containing 2 L of media and 10 shrimp/jar with two replicates/concentration, according to the modified method published by Delorenzo 21 and were run under conditions as described above for larvae.
The reaction of either compound 8a or 8b with ethyl cyanoacetate gave the N-cyanoacetamido derivatives 12a or 12b, respectively.Such changed showed a remarkable decrease of anti-inflammatory activity in the case of 12a.On the other hand, it showed an increase of anti-inflammatory activity of 12b.For the thiazolo[4',5':4,5]thieno[2,3-b]pyridine derivatives 13a,b the presence of the OH group in compound 13b is responsible for its higher anti-inflammatory activity than compound 13a.The reaction of either compound 12a or 12b with acetophenone gave the condensation products 15a and 15b, respectively, where compound 15b with the COOEt had higher activity than compound 15a with the CN group.However, the thiophene derivatives 16a,b obtained from 15a,b showed the reverse: compound 16a with the CN group showed higher anti-inflammatory activity than compound 16b with the COOEt moiety.Considering the arylhydrazone derivatives 18a-c, it is clear from Table 1 that compound 18a with the unsubstituted aryl moiety has the maximum anti-inflammatory activity among the three compounds.However, cyclization of compounds 18a-c in sodium ethoxide solution gave the pyridazine derivatives 19a-c where compound 19b with 4-chloroaryl group showed the highest anti-inflammatory activity among the three compounds, followed by the compound 19c with the 4-methoxyaryl group having the intermediate activity of the three compounds.

5. Anti-ulcer Activity
From Table 2 it is clear that compounds 6a-c, 7a-c, 8b, 10b, 13a, 16b, 19b and 19c showed the maximum antiulcer activity.Moreover, these twelve compounds showed inhibition effect higher than the reference drug ranitidine.On the other hand, most of the newly synthesized products showed a moderate anti-ulcer activity at concentrations 250 and 500 mg/kg.Moreover, compounds 19b and 19c showed the maximum antiulcer activity among the tested compounds.

5. 1. Macroscopic Evaluation of Stomach
The stomachs were opened along the greater curvature, rinsed with saline to remove gastric contents and blood clots and examined by a 10× magnifier lens to assess the formation of ulcers.The numbers of ulcers were counted.Scoring of ulcer was made as follows: (0 Mean ulcer score for each animal was expressed as ulcer index (UI).The percentage of ulcer protection was determined as follows.UI was measured by using the following formula: (2 where UI is ulcer index; UN average number of ulcers per animal; US average number of severity score; UP percen-tage of animals with ulcers.Percentage inhibition of ulceration was calculated as below: (3)

6. Toxicity
Bioactive compounds are often toxic to shrimp larvae.Thus, in order to monitor these chemicals in vivo lethality to shrimp larvae (Artemia salina), brine shrimp lethality assay was used.Results were analyzed with LC 50 program to determine LC 50 values and 95% confidence intervals. 22Results are given in Table 4 for the compounds which exhibited optimal anti-inflammatory and anti-ulcer activity, that is the eleven compounds 7b, 8a, 10a, 10b, 13a, 13b, 15b, 18a, 19b, 19c and 19d.The shrimp lethality assay is considered as a useful tool for preliminary assessment of toxicity, and it has been used for the detection of fungal toxins, plant extract toxicity, heavy metals, cyanobacteria toxins, pesticides, and cytotoxicity testing of dental materials, 23 natural and synthetic organic compounds. 24It has also been shown that A. salina toxicity test results have a correlation with rodent and human acute oral toxicity data.Generally, a good correlation was obtained between A. salina toxicity test and the rodent data.Likewise, the predictive screening potential of the aquatic invertebrate tests for acute oral toxicity in humans, including A. salina toxicity test, was slightly better than the rat test for test compounds. 25n order to prevent the toxicity results from possible false effects originating from solubility of compounds and possible toxicity effect of DMSO, compounds were prepared by dissolving in DMSO in the suggested DMSO volume ranges.It is clear from Table 3 that compounds 10b, 18a, 19c and 19d showed no toxicity against the tested organisms.On the other hand, compound 7b, 13b and 15b are very toxic compounds, while the rest of compounds are harmful.

1. General
All melting points were determined on an Electrothermal digital melting point apparatus and are uncorrected.IR spectra (KBr discs) were recorded on a FTIR plus 460 or Pye Unicam SP-1000 spectrophotometer (Pye Unicam, UK, Cambridge). 1

Conclusions
In summary, we have shown herein that our strategy is applicable for the synthesis of a wide range of thiazole derivatives and particularly of such which are incorporated into heterocyclic cores and for fused derivatives.Compounds 10b, 13b, 18a, 19b and 19c showed the maximum anti-inflammatory activities while compounds 6-c, 7a-c, 8b, 10b, 13a, 16b, 19b and 19c showed high anti-ulcer activities among the synthesized compounds.The toxicity of selected compounds was studied against shrimp larvae where compounds 10b, 18a, 19c and 19d showed to be non toxic against the tested organisms.

Sheme 1 .
C NMR spectrum sho-Synthesis of compounds 3, 6a-c and 7a-c wed beside the expected signals, δ 58.6 ppm indicating the presence of the thiazole CH 2 , two signals at δ 164.3, 168.6 ppm equivalent to the two CO groups and a signal at δ 170.3 ppm for the C=N.
H NMR and 13 C NMR spectra were recorded with Varian Gemini-200 (200 MHz, Varian UK) and JEOL AS 500 MHz (JEOL, Japan) instruments in DMSO-d 6 as the solvent, using TMS as the internal standard, chemical shifts are expressed as δ ppm.The Mohareb et al.: Anti-inflammatory and Anti-ulcer Activities ... mass spectra were recorded with Hewlett Packard 5988 A GC/MS system (Hewlett Packard, Agilent, USA) and GCMS-QP 1000Ex Shimadzu (EI, 70 eV) (Shimadzu, Japan) instruments.Analytical data were obtained from Vario EL III Elemental CHNS analyzer (Germany).
R. M. Mohareb would like to thank the Alexander von Humboldt Foundation in Bonn, Germany for affor-ding him regular fellowships in Germany for finance and completing his research work.

Table 1 .
Anti-inflammatory evaluation of the newly synthesis products

Table 3 .
Toxicity of the most potent compounds

Table 2 .
Effect of DMSO solution of the given compounds on gastric ulcer induced by pylorus ligation in rats, pH, total and free acidity in pyloric ligation induced ulceration in rats Mohareb et al.: Anti-inflammatory and Anti-ulcer Activities ...