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1 ISS Том/Volume LIV 2007 Книжка/umber 1-2 СПИСАНИЕ НА БЪЛГАРСКОТО НАУЧНО ДРУЖЕСТВО ПО ФАРМАЦИЯ Главен редактор: Ст. Николов Секретар: Ал. Златков Редакционна колегия: Зл. Димитрова, Св. Богданова, И. Иванов, Г. Китанов, И. Йонкова, Н. Данчев, Г. Петрова, Д. Обрешкова, Ст. Титева, И. Костадинова, Ф. Клерфьой, Е. Х. Хансен, М. Шефер, Р. Грьонинг, Л. Пистели, М. Унзета JURAL F TE BULGARIA PARMACEUTICAL SCIETIFIC SCIETY Editor in Chief: St. ikolov Assistant Editor: Al. Zlatkov Editorial Board: Zl. Dimitrova, Sv. Bogdanova, I. Ivanov, G. Kitanov, I. Jonkova,. Danchev, G. Petrova, D. breshkova, St. Titeva, I. Kostadinova, F. Clerfeuille, E.. ansen, M. Schaefer, R. Gröning, L. Pistelli, M. Unzeta Адрес на редакцията Address of Editorial Board Фармацевтичен факултет Faculty of Pharmacy ул. "Дунав" 2, София , Dunav str., Sofia 1000 Факс (02) Fax (02) Гл. редакпор: (02) Editor in Chief: (+359 2) snikolov@mbox.pharmfac.acad.bg snikolov@mbox.pharmfac.acad.bg

2 СЪДЪРЖАНИЕ Оригинални статии. Дизайн и синтез на нови 1,8-дисубституирани пурин-2,6-диони и 3,6-дисубституирани тиазоло[2,3- f]пурин-2,4-диони като потенциални антиноцицептивни и противовъзпалителни средства...3 К. Йончева, Й. Вандервоорт и А. Лудвиг. Влияние на карбопол върху мукоадхезивните свойства на наночастици за очно приложение, приготвени на базата на съполимер на полимлечна-полигликолова киселина...14 Д. Дренска, М. Варадинова и Н. Бояджиева. Сравняване на антидепресивната активност на антоциани и миансерин при експериментален модел на оксидативен стрес у плъхове...20 Т. Шумкова-Тучева и Н. Бояджиева. Хистоморфологични изследвания върху развитието на хипофизен тумор при третиране с естрогени и с алкохол...25 К. Тодорова, В. Петкова, Зл. Димитрова, С. Захариева и Н. Доганов. Фармакоикономически анализ на профилактичната стратегия за планиране на бременност при жени с инсулинозависим захарен диабет...29 Е. Кръстева, И. Костадинова и Н. Матева. Самолечение с анксиолитици и хипнотици сред студенти и влияние на нервното напрежение...35 От редакционната колегия Инструкции към авторите...39 CTETS riginal Articles. Design and synthesis of new 1,8-disubstituted purine-2,6-diones and 3,6-disubstituted thiazolo[2,3- f]purine-2,4-diones as potential antinociceptive and anti-inflammatory agents...3 K. Yoncheva, J. Vandervoort and A. Ludwig. The influence of carbopol on the mucoadhesive properties of poly(dl-lactide-coglycolide) nanoparticles for ocular purpose...14 D. Drenska, M. Varadinova and. Boyadjieva. Comparison of antidepressant activity of anthocyanins and mianserin in experimental model of oxidative stress in rats...20 T. Shumkova-Tucheva and. Boyadjieva. istomorphological studies on the development of pituitary tumor after treatment with estrogens and alcohol...25 K. Todorova, V. Petkova, Zl. Dimitrova, S. Zaharieva and. Doganov. Pharmacoeconomic analysis of the prophylactic strategy for pregnacy planning in women with insulin-dependent diabetes mellitus...29 E. Krasteva, I. Kostadinova and. Mateva. Self-administration of anxiolytics and hypnotics among students and influence of the stress...35 From the Editorial Board Instructions to authors...42 ФАРМАЦИЯ 1-2/2007 ISS УДК 615 Организационен секретар и стилов редактор Св. Цветанова Корекция Д. Танчева и Св. Цветанова Терминологичен и семантичен контрол д-р Б. Станчева Подписана за печат на г. Печатни коли 5.5, формат 60 x 90/8 Централна медицинска библиотека 1431 София, ул. Св. Г. Софийски 1, тел Fax: nmi@medun.acad.bg; svetlamu@mail.bg

3 Оригинални статии riginal Articles 3 DESIG AD SYTESIS F EW 1,8-DISUBSTITUTED PURIE-2,6-DIES AD 3,6-DISUBSTITUTED TIAZL[2,3-F]PURIE-2,4-DIES AS PTETIAL ATICICEPTIVE AD ATI-IFLAMMATRY AGETS Department of Pharmaceutical rganic Chemistry, Faculty of Pharmacy, Assiut University, Assiut, Egypt Summary. In this study, the design, synthesis and preliminary pharmacological investigation of novel 8-(2-oxo-2-p-(un)substituted phenyl)-1-substituted-3,7,8,9-tetrahydropurine-2,6-diones (11-20) and 3,6- disubstituted thiazolo[2,3-f]purine-2,4-diones (21-30) are described. 1,8-Disubstituted purine-2,6- diones were prepared by the reaction of 3-substituted-8-thioxo-3,7,8,9-tetrahydropurine-2,6-diones (4-5) with appropriate phenacyl bromides (6-10). Compounds (4-5) were in turn prepared by reaction of 3- substituted-5,6-diaminouracils (3a-b) with carbon disulfide. The derivatives (21-30) were obtained by cyclodehydration of compounds (11-20) in polyphosphoric acid (PPA). The effect of the new prepared derivatives as potential antinociceptive and anti-inflammatory agents was carried out on mice and rats. Aspirin and indomethacin were used as reference drugs. Some of the tested compounds have shown a pronounced analgesic and anti-inflammatory activity. The results here illustrate a promising analgesic and anti-inflammatory activity of the newly synthesized derivatives. Key words: purine-2,6-diones, thiazolo[2,3-f]purinediones, mice, rats, analgesic and anti-inflammatory activities, synthesis ДИЗАЙН И СИНТЕЗ НА НОВИ 1,8-ДИСУБСТИТУИРАНИ ПУРИН-2,6-ДИОНИ И 3,6-ДИСУБСТИТУИРАНИ ТИАЗОЛО[2,3-f]ПУРИН-2,4-ДИОНИ КАТО ПОТЕНЦИАЛНИ АНТИНОЦИЦЕПТИВНИ И ПРОТИВОВЪЗПАЛИТЕЛНИ СРЕДСТВА Катедра по фармацевтична органична химия, Факултет по фармация, Университета в Assiut, Египет Резюме. Описани са дизайнът, синтезът и предварителните фармакологични проучвания на нови 8-(2-оксо-2р-(не)субституирани фенил)-1-субституирани-3,7,8,9-тетрахидропурин-2,6-диони (11-20) и 3,6-дисубституирани тиазоло[2,3-f]пурин-2,4-диони. 1,8-дисубституирани пурин-2,6-диони се приготвят чрез реакцията на 3-субституирани-8-тиоксо-3,7,8,9-тетрахидропурин-2,6-диони (4-5) с подходящи фенацил бромиди (6-10). Съставните компоненти (4-5) се получават чрез реакция на 3-субституиран-5,6-диаминоурацили с въглероден дисулфид. Дериватите са получени чрез циклодехидриране на компоненти (11-20) в полифосфорна киселина. Ефикасността на новите деривати като потенциални антиноцицептивни и противовъзпалителни средства е изследвана върху мишки и плъхове. За сравнение са използвани аспирин и индометацин. Някои от тестваните субстанции показват явна обезболяваща и противовъзпалителна активност. Резултатите от изследването илюстрират обещаваща активност. Ключови думи: пурин-2,6-диони, тиазоло[2,3-f]пуриндиони, мишки, плъхове, аналгетици и противовъзпалителни средства, синтез Introduction The search of new analgesic compounds devoid of the significant side effects of morphine-like opioid agonists (such as physical dependence, respiratory depression, constipation) as well as of the gastrointestinal problems associated with nonsteroidal anti-inflammatory drugs (SAIDs) is one of the challenging tasks of current medicinal chemis-

4 4 ФАРМАЦИЯ, том LIV, кн. 1-2/2007 try. Several purine derivatives and condensed purines have been claimed to possess a multitude of pronounced biological activities. The class of fused purines is considered to be attractive target since its fundamental skeleton is analogous to naturally occurring purine alkaloids. They play an important role in cancer chemotherapy [7, 19, 20] and act as antimicrobial agents [3, 18]. In addition, some purine derivatives such as theophylline and enprofylline are reported to play an important role in the treatment of asthma [6, 17]. Some hydroxypyrimido[2,1-f] purine-2,4,8-triones are reported as interesting potential antiarthritis agents and also exhibit an activity profile different to that of the standard nonsteroidal antiinflammatory agents [4]. Caffeine has intrinsic antinociceptive properties and is used as an adjuvant analgesic drug [13, 20]. Recently, some 1,8-disubstituted purine-2,6-diones (PSB-53 I and PSB-1115, II, chart 1) were reported as potent analgesic and anti-inflammatory agents through adenosine receptor antagonism [1, 8]. In addition, thiazole and fused thiazoloheterocyclic derivatives such as thiazolo[3,2-b]triazoles, thiazolo[4,5-d]pyrimidines and imidazo[3,4-c]thiazoles show antinicecptive and anti-inflammatory action [2, 9, 14, 22]. These facts motivated my interest in the present investigation towards the design and synthesis of new 1,8-disubstituted purine-2,6-diones carrying at 8-position 2-aryl-2-oxoethylsulfanyl moiety and 3,6- disubstituted thiazolo[2,3-f]purine-2,4-dione, in which a third ring (thiazole) was added to the purine skeleton as shown in scheme 1. These derivatives were synthesized and rationalized as potential antinociceptive and anti-inflammatory agents. 1. MDS 2. RX 3. a 2 S 2 3, ac 3 R1 C 3 C, a 2 R a-b 2a-b 12.5%aq. 3, a 2 S 2 4 R % aq. a R1 2 R1 CS 2 /K, ethanol Br 2 R1 reflux S R2 3a-b , R1 = prpyl 5, R1 = butyl S glacial acetic acid 3 days X R2 PPA, 140 C R S 11, 21 R 1 = n-prop. R 2 = 12, 22 R 1 = n-prop. R 2 = Br 13, 23 R 1 = n-prop. R 2 = Cl 14, 24 R 1 = n-prop. R 2 = C 3 15, 25 R 1 = n-prop. R 2 = 2 16, 26 R 1 = n-but. R 2 = 17, 27 R 1 = n-but. R 2 = Br 18, 28 R 1 = n-but. R 2 = Cl 19, 29 R 1 = n-but. R 2 = C 3 20, 30 R 1 = n-but. R 2 = 2 Scheme 1. Synthesis of 1,8-disubstituted purinediones and 3,6-disubstituted thiazolo[2,3-f]purinediones

5 Design and synthesis of new 1,8-disubstituted ФАРМАЦИЯ, том LIV, кн. 1-2/ Results and discussion Chemistry. The general synthetic route to obtain the designed 1,8-disubstituted purine2,6-diones, 3,6-disubstituted thiazolo[2,3-f]purine-2,4-diones and the intermediates used in their preparations is presented in scheme 1. 1-Propyl-8-thioxo-3,7,8,9-tetrahydropurine-2,6-dione 4 and 1-butyl-8-thioxo- 3,7,8,9-tetrahydropurine-2,6-dione 5 were synthesized by reaction of 5,6-diamino-3-propyl- 1 -pyrimidine-2,4-dione 3a and 5,6-diamino-3-propyl- 1 - pyrimidine-2,4-dione 3b, respectively with carbon disulfide in the presence of potassium hydroxide [5]. Their structures were verified by 1-MR (see Exp. part). The compounds 4 and 5 were subjected to the interaction with the prepared phenacyl bromide 6-10 [12, 25, 26] in the presence of potassium hydroxide to give the required 1,8- disubstituted purine-2,6-diones The structure elucidation of these newly synthesized derivatives was confirmed by elemental and spectral data (table 1, 3). The 1 -MR spectra (table 1) of compounds were characterized by appearance of the methylene protons of S-C 2 -C at δ 5.3 to 5.6 ppm and also the introduced aromatic moiety, in addition to the presence of 1- and 7- signals is a strong support for an S-alkylation reaction rather than -alkylation. It is well known that mercaptopurines undergo S-alkylation at lowered temperature while at elevated one 7- is also attacked [11]. The use of glacial acetic acid or ethanolic solution of hydrochloric acid in cyclodehydration of compounds to obtain the required 3,6-disubstituted thiazolo[2,3-f]purine- 2,4-diones did not succeed, after 3 days reflux of compound 11 and 12, the 1-MR indicated the opened not the cyclized one. The new 3,6-disubstituted thiazolo[2,3-f]purine-2,4-diones were synthesized by cyclodehydration of compounds using polyphosphoric acid (PPA). In the 1 -MR data of compounds (table 2), the disappearance of both methylene protons of S-C 2 -C and 7- proton, in addition to the appearance of C7- as downfield proton ( ppm) is a strong evidence for ring cyclization. The structures of the prepared compounds were verified on the basis of elemental analyses and spectroscopic data (table 2, 3). Pharmacology. Analgesic activity. The antinociceptive activity of the newly synthesized compounds was investigated in mice using hot-plate test [24], an acute animal pain model and aspirin was used as standard drug. The results are summarized in table 4. The tested compounds have demonstrated good to potent analgesic effects. Study of analgesic activity at 0.5, 1.0, 2.0, 3.0 and 5.0 h time intervals (table 4, fig. 1-4) revealed the following conclusions: 1. All the tested compounds exhibited analgesic activity comparable to that of aspirin. 2. Some compounds showed more potent activity than aspirin, compounds 13, 14, 23, 25, 27, 29 and 30 were being the most active compounds showing analgesic activity twice that of the reference drugs. 3. Most of the tested compounds had rapid onset of action at 0.5 h time intervals and reached the maximum activity at 2.0 h and 3.0 h time intervals (table 4, fig. 1-4). 4. Some compounds showed the maximum activity at 0.5 h such as compounds 25 and 27, while some compounds showed continuous high activity until 5.0 h such as compounds 18, 25 and Regarding the substitution at the phenyl ring, it seems to be important, that the nonsubstituted derivatives 11, 16, 21 and 26 showed generally less activity if compared with the substituted derivatives. C S I- PSB-53 II- PSB-1115 Chart 1. Structure of PSB-53 and PSB-1115

6 6 ФАРМАЦИЯ, том LIV, кн. 1-2/2007 Table MR data of 1,8-disubstituted-3,7-dihydropurine-2,6-diones (11-20) R' R S Compd. R R 1-R C 2 C8-aryl 3-7- thers 11 n-propyl 0.95 (t, 3), 1.70 (m, 2), 4.10 (t, 2) 12 n-propyl Br 0.95 (t, 3), 1.70 (m, 2), 4.15 (t, 2) 13 n-propyl Cl 0.90 (t, 3), 1.70 (m, 2), 4.20 (t, 2) 14 n-propyl C (t, 3), 1.65 (m, 2), 4.20 (t, 2) 15 n-propyl (t, 3), 1.65 (m, 2), 4.10 (t, 2) 16 n-butyl 0.95 (t, 3), 1.60 (m, 4), 4.15 (t, 2) 17 n-butyl Br 0.95 (t, 3), 1.55 (m, 4), 4.15 (t, 2) 18 n-butyl Cl 0.90 (t, 3), 1.60 (m, 4), 4.15 (t, 2) 19 n-butyl C (t, 3), 1.60 (m, 4), 4.15 (t, 2) 20 n-butyl (t, 3), 1.60 (m, 2), 4.15 (t, 2) (m, 3), 8.85 (m, 2) (d, 2), 8.70 (d, 2) (d, 2), 8.85 (d, 2) (d, 2), 8.55 (d, 2) (d, 2), 9.10 (d, 2) (m, 3), 8.85 (m, 2) (d, 2), 8.70 (d, 2) (d, 2), 8.75 (d, 2) (d, 2), 8. (d, 2) (d, 2), 9.10 (d, 2) (s, 3) (s, 3) Regarding the effect of 1-substitution, it is difficult to detect which is more favorable propyl or butyl substituent, but in some cases the butyl substituent seems to be more favorable than propyl. It is clear if the propyl substituted activities, compounds 24 and 25 are compared to that of the butyl derivatives 29 and 30, respectively. 7. The interesting thing was that the analgesic activity of the tested compounds increased after cyclization (addition of the thiazole ring to the purinedione skeleton), for example the analgesic profile of the compounds 11, 15, 16, 19 and 20 was enhanced after cyclization compared to 21, 25, 26, 29 and 30, respectively. Anti-inflammatory activity. Compounds 13, 17, 23, 25, 29 and 30 were selected among the most active analgesic compounds and were tested in vivo for their anti-inflammatory effect. They were tested using carragennan induced paw edema in rats using indomethacin as a refrence drug [23]. The results of tested compounds and indomethacin were presented as the time course dependent size of edema and percentage of edema inhibition at a dose (75 mg/kg) and time intervals 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 h

7 Design and synthesis of new 1,8-disubstituted ФАРМАЦИЯ, том LIV, кн. 1-2/ (table 5, 6 and fig. 5). All the tested compounds showed moderate to potent anti-inflammatory effect. The compounds 23 and 25 showed more activity than that of indomethacin. The enhancement of the activity after cyclization and formation of fused thiazolopurinediones was again here clear if the activity of compound 23 was compared with that of 13. Although the derivatives 29 and 30 were more potent analgesic than 23, it showed higher activity as anti-inflammatory agent. Table MR data of 3,6-disubstituted-1-thiazolo[2,3-f]purine-2,4-diones (21-30) R' R S Compd. R R 3-R C7- C6-aryl 1- thers 21 n-propyl 0.90 (t, 3), (br s, 5) (m, 2), 4.10 (t, 2) 22 n-propyl Br 0.95 (t, 3), (br s, 5) (m, 2), 4.10 (t, 2) 23 n-propyl Cl 0.95 (t, 3), (br s, 5) (m, 2), 4.00 (t, 2) 24 n-propyl C (t, 3), 1.60 (m, 2), 4.10 (t, 2) (d, 2), 8.15 (d, 2) (s, 3) 25 n-propyl (t, 3), 1.65 (m, 2), 4.10 (t, 2) 26 n-butyl 1.00 (t, 3), 1.65 (m, 4), 4.25 (t, 2) 27 n-butyl Br 1.00 (t, 3), 1.60 (m, 4), 4.20 (t, 2) 28 n-butyl Cl 0.97 (t, 3), 1.60 (m, 4), 4.15 (t, 2) 29 n-butyl C (t, 3), 1.60 (m, 4), 4.25 (t, 2) 30 n-butyl (t, 3), 1.60 (m, 2), 4.20 (t, 2) (d, 2), 9.15 (d, 2) (br s, 5) (d, 2), 7.95 (d, 2) (br s, 5) (d, 2), 8.00 (d, 2) (d, 2), 8.95 (d, 2) (s, 3) 11.90

8 8 ФАРМАЦИЯ, том LIV, кн. 1-2/2007 Table 3. Yields, melting points and analytical data of the final products Compd. Formula MW Elemental Analyses, Calc./Found Yield [%] Mp [ C] C S 11 C S C Br 4 3 S C Cl 4 3 S C S C S C S C Br 4 3 S C Cl 4 3 S C S C S C S C Br 4 2 S C Cl 4 3 S C S C S C S C Br 4 2 S C Cl 4 2 S C S C S dec dec >

9 Design and synthesis of new 1,8-disubstituted ФАРМАЦИЯ, том LIV, кн. 1-2/ Table 4. Analgesic effects of the tested compounds and aspirin in the hot plate test Compd. The average reaction time (second) at different times after compound administration ± SEM 0.5 h 1 h 2 h 3 h 5 h ± 0.25* 28 ± 0.10** 16 ± ± 1.55* 21 ± 1.20* ± 1.** 15 ± ± 1.40* 17 ± 1.42** 20 ± 1.60* ± ± 1.80* 34 ± 0.90* 40 ± 1.70** 24 ± 2.11* ± 1.00** 29 ± 0.40** 36 ± 0.35** 46 ± 0.*** 26 ± 1.32** ± 0.33** 22 ± 0.90* 20 ± ± 1.* 19 ± 2.20** ± ± 0.53** 19 ± ± ± 1.56** ± 0.40* 26 ± 0.17** 37 ± 1.70** 31 ± 1.05** 17 ± 1.08* ± ± 2.6* 29 ± 1.94* 30 ± 1.35** 21 ± 1.46* ± 2.55* 30 ± 0.30** 17 ± 0.40* 16 ± 0.15** 17 ± 0.83* ± 1.00** 30 ± 1.75* 25 ± 0.60* 26 ± 2.17* 25 ± 1.92* ± 2.25* 37 ± 0.55** 22 ± ± ± 1.89* ± 0.85** 22 ± 0.53** 18 ± 1.48** 17 ± 1.00* 18 ± 1.57** ± 0.75* 23 ± 1.00* 38 ± 0.30** 28 ± 0.28** 27 ± 1.72* ± 0.85** 37 ± 0.44** 22 ± ± ± 2.10* ± 0.76*** 33 ± 1.44** 34 ± 2.70* 32 ± 2.20** 30 ± 1.82* ± ± ± 0.80** 16 ± 0.89* 16 ± 0.92* ± 1.87** 26 ± 0.64** 26 ± 0.37** 23 ± ± 1.18** ± 0.** 23 ± 0.10*** 17 ± 0.31** 18 ± 0.44** 18 ± 2.30* ± 1.20** 39 ± 1.10** 25 ± ± ± 1.81* ± 2.75** 46 ± 1.00* 41 ± 1.65** 32 ± 2.45** 28 ± 2.12** Control 17 ± 0.3* 16 ± 0.15** 19 ± 0.93* 17 ± ± 1.17** Aspirin 26 ± 0.1** 20 ± 2.00* 20 ± 0.74** 19 ± ± 2.10* SEM standard error mean, *p < 0.05, **p < 0.01, ***p < Table 5. Anti-inflammatory effects of compounds 13, 17, 23, 25, 29 and 30 and indomethacin on carrageenan induced paw edema in rats Compd. V L (mm) Thickness of right paws (mm, mean ± SEM) 0 h 0.5 h 1 h 2 h 3 h 4 h 5 h ± ± ± 0.88 ** 6.90 ± 1.22 * 6.10 ± 1.15 * 5.80 ± 0.57 ** 5.10 ± 1.18 * 5.10 ± 0.96 ** ± ± 1.23 * 7.30 ± 0.68 ** 6.94 ± 1.02 * 6.30 ± 1.15 * 5.77 ± 0.77 ** 5.40 ± 1.38 * 5.20 ± 0.56 ** ± ± 0.93 * 6.67 ± 1.23 ** 5.34 ± 1.48 * 5.02 ± 0.40 ** 4.75 ± 1.19 * 3.90 ± 0.87 * 3.94 ± 1.12 * ± ± 1.43* 6.60 ± 0.33 * 5.14 ± 1.48 * 4.80 ± 1.40 * 4.65 ± 1.65 ** 3.85 ± 0.87 ** 3.84 ± 0.16 * ± ± 1.57 * 7.12 ± 0.87 * 6.80 ± 1.22 * 6.05 ± 1.35 * 5.68 ± 0.87 ** 5.00 ± 1.28 * 4.88 ± 1.96 * ± ± ± 1.80 * 6.66 ± 1.52 * 6.15 ± 1.65 * 5.40 ± 0.37 ** 5.20 ± 1.58 * 4.38 ± 1.16 * Control 3.07 ± ± 0.04 * 7.74 ± 0.5 * 7.81 ± 0.3 * 7.69 ± 0.6 ** 7.82 ± 0.6 * 7.84 ± 0. ** 7.88 ± 0.3 ** IM 3.06 ± ± ± 0.98 ** 5.84 ± 1.12 * 5.10±0.90 ** 4.68 ± 1.02 * 4.16 ± 1.20 * 3.98 ± 0.28 ** SEM standard error mean, *p < 0.05, **p < 0.01, ***p <

10 10 ФАРМАЦИЯ, том LIV, кн. 1-2/2007 Table 6. Inhibitory effect of compound 14, 17, 23, 25, 29 and 30 and indomethacin on carrageenan induced paw edema in rats Compd. Percentage of paw edema inhibition 0 h 0.5 h 1 h 2 h 3 h 4 h 5 h Control IM Table 7. Ulcerogenic effects of compounds 13, 17, 23, 25, 29 and Indomethacin in rats Compd. Dose mg/kg IM Ulcer score* 4/6 3/6 0/6 2/6 2/6 0/6 0/6 0/6 2/6 0/6 2/6 3/6 5/6 ot tested Ulcer index ± SEM 1.15 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.64 * umber of rats with lesions that were more than 0.5 mm in length per total numbers Average 30 reaction 25 time h 1h 2h 3h 5h Time Aspirin Fig. 1. Mean licking time (average reaction time) in the hotplate test of compounds and aspirin Average reaction time h 1h 2h 3h 5h Time Aspirin Fig. 2. Mean licking time (average reaction time) in the hotplate test of compounds and aspirin. Average reaction time h 1h 2h 3h 5h time Aspirin Fig. 3. Mean licking time (average reaction time) in the hotplate test of compounds and aspirin Average 30 reaction 25 time h 1h 2h 3h 5h Time Aspirin Fig. 4. Mean licking time (average reaction time) in the hotplate test of compounds and aspirin

11 Design and synthesis of new 1,8-disubstituted ФАРМАЦИЯ, том LIV, кн. 1-2/ % Edema Inhibition 90,00 80,00 70,00 60,00,00 40,00 30,00 20,00 10,00 0,00 0 h 0.5 h 1 h 2 h 3 h 4 h 5 h Time IM Fig. 5. Inhibitory effect of compounds 13, 17, 23, 25, 29, 30 and indomethacin on carrageenan induced paw edema in rats Gastric ulceration. The test was carried out in rats according to a reported method [10]. Compounds 13, 17, 23, 25, 29 and 30 were selected for this test. The compounds 23, 25, 29 and 30 showed no ulcerogenic effect at dose 30 mg/kg. All compounds showed less gastric ulcer than indomethacin at higher doses (25-75 mg/kg). The results are shown in table 7. Experimental Section Chemical Synthesis: 1 MR spectra were performed on Varian EM Mz spectrometer at the Faculty of Pharmacy, Assiut University, Egypt. DMS-d 6 was used as solvent and the chemical shifts are given in δ (ppm) values downfield from TMS as internal standard. Elemental microanalyses were performed at the Department of Chemistry, Faculty of Science, Assiut university, Egypt. Melting points were determined on electrothermal melting point apparatus SMPi (U.K.) and were uncorrected. Purity of the prepared compounds was checked by TLC on silica gel 60 F 254 (Merck) aluminum plates, using dichloromethane:methanol (9:1) as the mobile phase. 3-substituted 5,6-diaminouracils (3a-b) were prepared from 6-aminouracil via regioselective alkylation followed by nitrosation and reduction as described [15, 16]. Phenacyl bromides (6-10) were prepared from p-(un)substituted acetophenone derivatives by bromination in ether-dioxan (2:1) in presence of aluminum chloride as catalyst [12, 25, 26]. Synthesis of 3-Alkyl-8-thioxo-3,7,8,9-tetrahydropurine-2,6-diones (4-5). General procedure. Potassium hydroxide (1.5 g, 27 mmol) was dissolved in 40 ml ethanol, then carbon disulfide (2.05 g, 27 mmol) was added followed by addition of 3-substituted 5,6- diaminouracil (27 mmol) 3a or 3b respectively. The reaction mixture was refluxed for 4 h, diluted with warm water (30 ml) and stirred well, then acetic acid (3 ml) in water (5 ml) was added portionwise. The reaction mixture was allowed to cool in refrigerator for 3 h, the product was collected by filtration and crystallized from ethanol. 4: Yield 80%, mp > 330º, 1 -MR: δ (s, 7-), (s, 3), (s, 9), 4.00 (t, 2), 1.65 (m, 2), 0.90 (t, 3). 5: Yield 82%, mp º 1 -MR: δ (s, 7-), (s, 3), (s, 9), 4.10 (t, 2), 1.60 (m, 4), 0.95 (t, 3). Synthesis of 1,8-disubstituted-3,7-dihydropurine- 2,6-diones (11-20).General procedure. To a solution of 3-substituted 8-thioxo-3,7,8,9-tetrahydropurine- 2,6-diones (2.89 mmol), 4-5 dissolved in aqueous sodium hydroxide (1%, 20 ml) was added portionwise with stirring a solution of the appropriately p(un)substituted phenacyl bromide (2.89 mmol) 6-10 in ethanol (5 ml). The reaction mixture was stirred at ambient temperature for 4-5 h. The reaction mixture was kept at room temperature overnight and the product was collected by filtration, washed with water and crystallized from ethanol/water to afford the compounds Synthesis of 3,6-disubstituted-1-thiazolo[2,3-f]purine-2,4-diones (21-30). General procedure. To a stirred freshly prepared polyphosphoric acid from phosphorus pentaoxide (8 g) and phosphoric acid (6 ml), it was added the appropriate 8-(2-xo-2- (un)substituted phenyl-ethylsulfanyl)-1-propyl(butyl)- 3,7,8,9-tetrahydropurine-2,6-dione (compounds 11-20, 5.3 mmol) and the reaction mixture was heated at C for 5-6 h. The reaction mixture was cooled, poured into ice-water and neutralized with sodium carbonate solution. The precipitate solid was filtered off, washed with water and crystallized from DMF/water to afford compounds

12 12 ФАРМАЦИЯ, том LIV, кн. 1-2/2007 Pharmacology. The pharmacological screening was carried out at the Department of Pharmacology, Faculty of Medicine, Assiut University. Materials and methods Chemicals: Indomethacin (Indacin ampoule, Memphis Company), Carrageenan (Sigma-Aldrich, USA). Acetylsalicylic acid and sodium carboxymethyl cellulose were purchased from the local market. Animals: Male mice (30-35 g) and rats (120-1 g) were used for screening analgesic and anti-inflammatory activity respectively. The animals were bred and housed in group of five mice or rats under standardized environmental conditions in the preclinical animal house, Department of Pharmacology, Faculty of Medicine, Assiut University. They were fed ad libitum with rodents s chow and allowed free access to drink water and were used only once. The experiments were performed during the light phase between 7.00 AM and 4.00 PM. 1. Analgesic activity. ot plate test: The analgesic activity of the newly synthesized compounds was determined in the hot plate test [24] in comparison to aspirin as reference drug. Male mice (5/group) were injected intraperitoneally (i.p.) with the tested compounds (suspension or solution in 1% acmc) at dose level (75 mg/kg). The control group was similarly treated with 1% acmc while the reference group received aspirin (200 mg/kg). At the time indicated, they were placed individually on a hot plate apparatus (Model-DS 30, Socrel [comerio (Va) Italy) maintained at 52 ± 1 C. The reaction time in sec. was taken as the time period from the instant the animal reached the hot plate until the moment the animal licked its feet (licking latencies) or jumped out [24]. Each mouse was placed individually on the hot plate after 0.5, 1.0, 2.0 and 5.0 h, respectively after administration and the average reaction time ( AVR ) for each animal was calculated as a measure of the analgesic activity. The results are presented in table 4, fig Anti-inflammatory activity. The anti-inflammatory activity of the newly synthesized compounds (14, 17, 23, 25, 29 and 30) was determined according to paw induced edema method [23] in comparison to indomethacin as a reference drug. The test is based on the pedal inflammation in rat paws induced by subplantar injection of carrageenan suspension (0.2 ml of 1% solution in normal saline) in the right hind paw of the rats. Male albino rats were divided into groups (5/group). The thickness of rat paw was measured by Varnier Caliper (SMIEC, China) before and after 1 h of carrageenan injection to determine the induced inflammation. The tested compounds of a dose (75 mg/kg) were injected i.p. to the animals. The control group received a vehicle (1% acmc) while the reference group received indomethacin (10 mg /kg). Results of anti-inflammatory activity of the tested compounds and the reference drug were listed in tables 5, 6 and fig. 5. The percentage of edema and percentage of edema inhibition were calculated [24] where: % Variation (edema) = % Edema inhibition = (V R V L ) 100 V R : Average right paw thickness, V L : Average left paw thickness. Gastric ulceration. Male albino rats were divided into groups (6/group), the rats were fasted for 24 h [25].The tested compounds 13, 17, 23, 25, 29, 30 and indomethacin were administered orally as a suspension in 1% acmc. After 6 h, the rats were killed, the stomachs were removed for macroscopic and microscopic investigation. Ulcer was defined as at least one lesion that was 0.5 mm or more in length [10]. All lesions of more than 0.1 mm in length were summed to obtain ulcer index. The results are shown in table 7. Statistical analysis. All data were represented as mean ± standard error mean (SEM). The significance of the results from control was calculated using the student t-test (InState 2.03, Macintosh). The results were regarded as significant (*) when p < 0.05, highly significant (**) when p < 0.01 and extremely significant (***) when p < Acknowledgment. I am grateful to Dr. Moustafa Mahmoud amdy, Department of Pharmacology, Faculty of Medicine, Assiut University, Egypt for his help and valuable comments during the study of the analgesic and anti-inflammatory activity. I am also grateful to Dr. Rasha Ibrahim Anwar, Department of Anatomy, Faculty of Medicine, Assiut University, Egypt for her kind help and valuable comments for ulcerogeneic effect study. V R (V R V L ) control (V R V L ) treated x 100 (V R V L ) control

13 Design and synthesis of new 1,8-disubstituted ФАРМАЦИЯ, том LIV, кн. 1-2/ References 1. A b o S a l e m,. M. et al. Antinociceptive effects of novel A2B adenosine receptor antagonists. J. Pharmacol. Exp. Ther., 308, 2004, B a l k a n, A. et al. Evaluation of the analgesic and antiinflammatory activities of some thiazolo[4,5-d]pyrimidines. Arzneimittelforschung, 52, 2002, B a s y o u n i, W. M.,. M. osni et S. M. elmy. Synthesis and antimicrobial activity of some new 6-substituted 9-arylpurine derivatives. Egypt. J. Chem., 42, 1999, B l y t h i n, D. J. et al. Antiinflammatory activity of substituted 6-hydroxypyrimido[2,1-f]purine-2,4,8(1,3,9)- triones. Atypical nonsteroidal anti-inflammatory agents. J. Med. Chem., 29, 1986, D i e t z, A. J. et R.. Burgison. Synthesis of some 8-alkylthio-2-thiophyllines and 8-alkylthio-6-thiothyophyllines. J. Med. Chem., 9, 1966, F e o k t i s t o v, I. et I. Biaggioni. Role of adenosine in asthma. Drug Dev. Res., 39, 1996, a n, Y. et al. Preparation of purine derivatives and analogs as antitumor agents. W , a y a l l a h, A. M. Et al. E.1,8-Disusbstituted xanthine derivatives: synthesis of potent A2B-selective adenosine receptor antagonists. J. Med. Chem., 45, 2002, u g o n, B. et al. Synthesis of -substituted 4,6-dioxo-imidazo[3,4-c]thiazols and their analgesic activity in mice. J. Parm. Pharmacol., 53, 2001, I k u t a,. et al. Synthesis and anti-inflammatory activities of 3-(3,5-di-trt-butyl-4-hydroxybenzylidene)pyrolidin-2-ones. J. Med. Chem., 30, 1987, L i s t e r, J.. In: Fused Pyrimidines, Ed. D. J. Brown, Wiley-interscience Purines, part 2, ew York, 1971, M a c h i n k a y a, L. V. et A. S. Prodberezina. Bromination of cyclic ketones with dioxane dibromide. Zh. bshch. Khim., 28, 1958, 11-13, CA, 53, 1959, M a l e c, D. et E. Michalskaл The effect of methylxanthines on morphine analgesia in mice and rats. Pol. J. Pharmacol., 40, 1988, M g o n z o, R., A. Geronikaki et P.. Kourounakis. Snthesis and antiinflammatory activity of some new thiazole derivatives. Pharmazie,, 1995, M u e l l e r, C. E. et J. Sandoval-Ramirez. A new versatile synthesis of xanthines with variable substituents in the 1-, 3-, 7- and 8-positions. Synthesis, 1995, M u e l l e r, C. E. Synthesis of 3-substituted 6-aminouracils. Tetrahedron Lett. 32, 1991, P e r s s o n, C. G., K. E. Anderson et G. Kjellin. Effects of enprofylline and theophylline may show the role of adenosine. Life Sci., 38, 1986, R a j, S. C. V. et S. Dhala. Effect of naturally occurring xanthines on bacteria. I. Antimicrobial action and potentiating effect on antibiotic spectra. Appl. Microbiol., 13, 1965, S a d z u k a, Y. et al. Increased effects of MPDAX, a novel xanthine derivative, on antitumor activity of doxorubicin. Toxicology Lett., 1, 2004, S a w y n o k, J. et T. L. Ayksh. Caffeine as an analgesic adjuvant; a review of pharmacology and mechanism of action. Pharmacol. Rev., 45, 1993, T a i s e i, U. et al. Synthesis, antitumor activity and vascular relaxing effect of purino[7,8-g]-6-azapteridines and [1,2,4]triazino[3,2-f]purines. Chem. Pharm. Bull., 35, 987, T o z k o p a r a n, B. et al. Synthesis, characterization and anti-inflammatory-analgesic properties of 6-(α-amino-4-chlorobenzyl)thiazol[3,2-b]triazol-5-ols. Arzneimittelforschung, 54, 2004, V a l e n c i a, E. et al. Antinociceptive, anti-inflammatory and antipyretic effects of lapidin, a bicyclic sesquiterpene. Planta Med., 60, 1994, V á z q u e z, M. M. et al. Analgesic and antipyretic activities of an aqueous extract and the flavone linarin of Buddleia cordata. Planta Med., 62, 1996, Y a n o v s k a y a, L. A. et A. P. Terentev. Bromination with dioxane-dibromide. II. Bromination of aldehydes and ketones. Zh. bshch. Khim., 22, 1952, CA, 47, 1953, Y a n o v s k a y a, L. A. Use of dioxane dibromide in bromination of organic compounds. Dokl. Akad. auk SSSR, 71, 19, , CA, 44, 19, Постъпила 05 декември 2006 г. Address for correspondence: Alaa A. K. M. ayallah Department of Pharmaceutical rganic Chemistry Faculty of Pharmacy, Assiut University Assiut 71526, Egypt alaa_hayalah@yahoo.com Адрес за кореспонденция Alaa A. K. M. ayallah Катедра по фармацевтична органична химия Факултет по фармация, Университета в Assiut Assiut 71526, Egypt alaa_hayalah@yahoo.com