Synthesis and Pharmacological Activities of Some New Triazolo- and Tetrazolopyrimidine Derivatives

A new series of fused triazolo- and tetrazolopyrimidine derivatives 2–14 were synthesized and their anti-inflammatory and ulcerogenic activities were evaluated. The pharmacological screening showed that many of these obtained compounds have good anti-inflammatory activities, comparable to the reference drug. The toxicity of the compounds was also assayed via the determination of their LD50 values. The structures of newly synthesized compounds were confirmed by IR, 1H-NMR, MS spectral data and elemental analysis.


Introduction
In our previous work, some polycyclic heterocyclic derivatives were studied as 5α-reductase inhibitors, antiviral and anti-tumor agents [1]. Some of these compounds also showed aromatase and quinone reductase-2 inhibitors [2], anti-inflammatory, analgesic and antipyretic [3,4], and anti-arthritic and immunosuppressive activities [5]. Pyrimidines and fused pyrimidines, being integral parts of DNA and RNA, play an essential role in several biological processes and also have considerable chemical and pharmacological importance as antibiotics, antibacterials, cardiovascular as well as agrochemical and veterinary products [6][7][8][9][10][11][12]. Heterocyclic compounds play an important role in designing new classes of structural entities of medicinal importance with potentially new mechanisms of action. These heterocyclic compounds are well known to possess diverse pharmacological properties, viz. antimicrobial, analgesic, anti-inflammatory, anticancer, anticonvulsant and antimalarial activities [13]. On the other hand, we have reported that some of our new substituted heterocyclic compounds exhibited antiparkinsonian [14], antitumor [15,16], and anti-inflammatory [17] activities. In addition, during the last few years, condensed thienopyrimidine derivatives have received considerable attention. Many of these derivatives were found to possess a variety of pronounced activities such as anti-inflammatory and analgesic [18][19][20][21], antimicrobial [22][23][24][25], anti-avian influenza virus (H5N1) [26], anti-herpes simplex virus type-1 (HSV-1) and hepatitis-A virus (HAV), serotonin 5-HT 6 receptor antagonist [27], antiarrhythmic [28] agent properties. Pyrimidine derivatives have been previously reported as platelet aggregation inhibitors, antagonists, anti-conceptive and anti-parkinsonism [29][30][31][32] agents. Heterocyclic compounds have also exhibited anthelmintic, anti HIV activity and hypoglycemic activities [33]. In view of these observations and as continuation of our previous works on heterocyclic chemistry, we report herein the synthesis of some new heterocyclic containing pyrazolothienopyrimidine moieties and the study of their anti-inflammatory activities in comparison to indomethacin used as positive control.

Chemistry
2-(4-Methoxypheyl)-4-chlorocycloocteno [4,5]thieno [2,3-e]pyrimidine (1) [34] was treated with hydrazine hydrate to afford the corresponding hydrazinopyrimidine 2. Reaction of compound 1 with cyanoacetylhydrazine in boiling butanol afforded the corresponding 3-(cyanomethyl)triazolopyrimidine 3. Compound 1 was also reacted with amino acids to give imidazo derivatives 4 and 5, respectively. Treatment of 1 with sodium azide in boiling acetic acid gave the corresponding tetrazolopyrimidine 6 (Scheme 1). On the other hand, when compound 2 was refluxed with diethyl oxalate or chloroacetylchloride it afforded triazino derivatives 7 and 8, respectively. The IR and 1 H-NMR spectra of 7 and 8 revealed the absence of the NH 2 group. Also, reaction of compound 2 with ethyl chloroformate or acetic anhydride gave triazolopyrimidine derivatives 9 and 10, respectively. The IR spectrum of compound 9 showed absorption bands due to C=O and NH; its 1 H-NMR showed a singlet at 6.24 ppm due to the NH (amide) group, while the 1 H-NMR of compound 10 showed a singlet at 2.16 ppm due to the CH 3 group (Scheme 2). Reaction of compound 2 with phenyl isothiocyanate in boiling ethanol yielded compound 11, which was treated with methanolic sodium hydroxide to give the cyclized compound 12. The 1 H-NMR spectrum of compound 12 showed a singlet at δ = 10.23 ppm due to the NH group. Finally, compound 2 was reacted with benzaldehyde to give arylidenehydrazine derivative 13, which was cyclized with bromine and sodium carbonate to give triazolopyrimldine 14 (Scheme 3).

Pharmacological Screening
Two pharmacological activities namely; anti-inflammatory and ulcerogenic activities were tested despite their different biological receptors. Eleven representative compounds-2-10, 12 and 14-were evaluated as anti-inflammatory agents. Compounds 2 and 10 were also evaluated for ulcerogenic activity. The activities of these compounds are different according to the structure and function groups (Tables 1 and 2).

Ulcerogenicity
Compounds 2 and 10 showed higher anti-inflammatory activities than the other compounds so they were also screened for their ulcerogenic activity at dose levels of 10, 50 and 100 mg/kg b.m. ( Table 2). The tested compound 2 and 10 showed no ulcerogenic activity of 1.4 to 2.1 mm. Table 2. Ulcerogenic activities of compounds 2 and 10 (Gastric Ulceration in mice a ).

Acute Toxicity
To determine the acute toxicity of the compounds, the LD 50 values for compounds 2, 3, 6, 9 and 10 were determined by injecting different gradual increased doses of the tested compounds into male adult mice, then calculating the dose corresponding to 50% animal death (Table 3).

Structure Activity Relationships
Structure activity relationships based on the obtained results indicated that substitution of pyrimidine derivatives modulated the anti-inflammatory activity. Compounds 2, 3, 6, 9 and 10 have high anti-inflammatory activity due to the presence of electron-donating moieties which increase the pharmacological activity. The sequence of anti-inflammatory properties regarding substitution of electron-donating group in pyrimidine derivatives is as follows: hydrazine > methyl > cyanomethyl > tetrazine > amide as exhibited in compounds 2 > 10 > 3 > 6 > 9, respectively. Compounds 7, 8 and 12 have moderate anti-inflammatory activity due to the presence of electron-withdrawing moieties which decrease the pharmacological activity. The sequence of anti-inflammatory properties regarding substitution of electron-withdrawing groups in pyrimidine derivatives is: amide > carbonyl adjacent to amide > phenyl amine as exhibited in compounds 8 > 7 > 12, respectively. Also, compounds 4, 5 and 14 have weak anti-inflammatory activity due to the presence of electron-withdrawing moieties which strongly decrease the pharmacological activity. The sequence of anti-inflammatory properties regarding electron-withdrawing group substitution in pyrimidine derivatives is in this case: carbonyl of imidazole > carbonyl of pyrimidine > phenyl as exhibited in compounds 4 > 5 > 14, respectively.

General
Melting points were determined on open glass capillaries using an Electrothermal IA 9000 SERIES digital melting point apparatus (Electrothermal, Essex, UK) and are uncorrected. Elemental analyses were performed with all final compounds on an Elementar Vario EL, located at the Microanalytical Unit, National Research Centre, Cairo, Egypt and were found within ± 0.4% of the theoretical values. The IR spectra (KBr) were recorded on a FT IR-8201 PC spectrophotometer. The 1 H-NMR spectra were measured with a Jeol FTGNM-EX 270, 270 MHz instrument in DMSO-d 6 and the chemical shifts were recorded in (δ, ppm) relative to TMS. The mass spectra were run at 70 eV with a Finnigan SSQ 7000 spectrometer using EI and the m/z values are indicated in Dalton. TLC (silica gel, aluminum sheets 60F 254 , Merck, Darmstadt, Germany) was used to follow the reactions.

Animals
Female albino mice (16-18 g) and Sprague Dawley mice (100 g) obtained from the Theodor Bilharz Research Institute (TBRI, Giza city, Egypt) were used. Approval of the institutional animal ethical committee for the animal studies was obtained from the Office of Environmental Health and Radiation Safety, ACUC Protocol 1096-5. The animals were maintained according to accepted standards of animal care.

Anti-inflammatory Activity
Newly synthesized thienopyrimidine derivatives were dissolved in 0.5% carboxymethyl cellulose (CMC) as a homogeneous solution and administered intraperitonneally (i.p.). One hundred and eight rats were divided into eighteen groups, each group consisting of six animals. Anti-inflammatory activity of the compounds was studied in mice using carrageenan induced edema. A suspension of the tested compound and the reference drug, indomethacin in aqueous solution was administered orally at a dose 5 mg/kg. Control animals were treated with 0.5% CMC only. After 30 min, 0.1 mL of freshly prepared 1.0% carrageenan solution (in formol saline) was injected into the sub-plantar region of the right hind paw according to Hernandez-Perez [35]. The right paw volume was measured using a digital plethysmometer (Model 7150, Ugo Basile, Varese, Italy), directly before and after 1, 2, 3 h, intervals after administration of the tested compounds.

Ulcerogenic Activity
Seventy-two mice were divided into twelve groups. Ulcerogenic activity was evaluated after oral administration of the tested compounds or indomethacin at doses of 10, 50, and 100 mg/kg. Control mice received 0.5% CMC. Food but not water was removed 24 h before administration of the tested compounds. After 6 h, the mice were sacrificed; the stomach was removed and opened along the greater curvature, washed with distilled water and cleaned gently by dipping in saline. The mucosa damage for each stomach was examined using a stereoscopic microscope and compared with the reference drug indomethacin according to the reported procedure [36].

Acute Toxicity
The median lethal doses (LD 50 ) of the most active compounds 2, 3, 6, 9 and 10 were determined in mice [37]. Groups of male adult mice, each of six animals, were injected i.p. with graded doses of each of the test compounds. The percentage of mortality in each group of animals was determined 24 h, after injection. Computation of LD 50 was processed by a graphical method.

Statistical Analysis
Assay results are shown as mean ± SE. Statistical analyses were carried out with Sigma Plot software (SPSS Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) followed by Tukey's post test was used to assess the presence of significant differences. Differences were considered statistically significant at p ≤ 0.05.

Conclusions
The objective of the present study was to synthesize and investigate the anti-inflammatory activities of some new thienopyrimidine derivatives. The starting material 1 was used to synthesize the following compounds: hydrazine thinopyrimidine 2, triazolothinopyrimidine 3, benzopyrimidinothieno pyrimidine 5 and tetrazolothienopyrimidine 6. Compound 2 was used to synthesize the triazino-and triazolothinopyrimidine derivatives 7, 8, 9 and 10, respectively. Also, compound 2 was carried out to synthesize of triazolothinopyrimidine derivatives 12 and 14. The newly synthesized compounds 2-10, 12 and 14 were screened for their anti-inflammatory activity compared to indomethacin which was used as reference drug. Compounds 3, 6, 9 and 10 possess strong anti-inflammatory activity, and compounds 8, 7 and 12 possess moderate anti-inflammatory activity, while compounds 4, 5 and 14 possess weak anti-inflammatory activity.