Ir aq i J.Pharm.Sc i., Vol.15 (1 ) ,2006 80 Phosphodiester Conjugation of Metronidazole and Dexamethasone as Possible Mutual Prodrug Muthana D. Saud* and Suhair M. Ghani Received 30-12-2002 Accepted 15-5-2005 ABSTRACT As p os sible mutua l prod rug had b een s ynthes ized that contain metro nidazole and dexame thazone co njuga te d through phosp hod ie ster linkage . The ratio nale for this typ e of c onjugate is to get a prodrug with p oss ible site – s pe cific de live ry of its ac tive c ons titue nts into the lower parts of the G.I.T. This c ompo und was s ynthe size d by the rea ction of de xame thzo ne – 2 1 – phos pha te with me tronid azo le to fo rm:(1 – (de xame thaz o ne – 21 – pho sphory l) – me tro nidaz ole ) This c onjugate was p erfo rme d us ing dicyc lo he xylca rb odiimid e (DCC) as a c ondensing a gent. The id entity o f the prep ared compound had be en confirmed us ing T.L.C., U.V. sp ectros cop y, IR sp ectrosc op y and e lemental a nalysis. The p artitio n coe fficient fo r it had als o b ee n d etermined through n – Octano l / wate r partitioning system. الخالصة وعـة مجم ول مـع داز ي الميترونـي كسيل ف رو وعة الهيد مجم عن طريق تكثيف رونيدازول ميت مع ال ن لقد تم ربط فوسفات الدكساميثازو ون رقم ى ذرة الكارب عل وسفات 2الف د 1 ودايئماـي رب كا سـيل وهك سايكل ون باسـتخدام الداي ميثاز كسـا ف ) DCC(في جزيئة الد كـث عامـل م ك مــذي عد وباسـتخدام البريــدين ك سـا كعامــل م وكــذلك دراســة . ب و حمــراء ة فــوق البنفســجية وتحـت ال الشــع اف ا راســة أطـي ولقــد أثبتــت د ي للعناصر كم ة الى التحليل ال ة باالضاف طبقة الرقيق وغرافيا ال مات وي, كرو كيميا حة تركيبه ال كب الجديد وص وة المر مل .نقا عا تم حساب م جزئة ال) PC(الت هما ا ن مذيبي خدام ست محضر با ركب ال والماءللم كتانول . و INTRODUCTION Metro nidazole (2 – Me thyl – 5 – nitroimidazole – 1 – e thanol). (Flagyl, others ), is a synthe tic antimicrobial a gent which was found to have pa rticula rly high activity in vitro and in vivo a gainst a wide variety of a nae ro bic protozo al p aras ites a nd ana erob ic ba cteria (1,2). Re ce nt studies indicate d that a comb ination of metro nida zole and a n anti – inflamma to ry s te ro id is one o f the mos t effe ctiv e re gimen for the tre atment of inflammatory b owel dise as e inc luding ulcerative c olitis and cohn's dis eas e(3). In o rd er to optimize drug ac tio n new drug fo rmula tion have b een d eve lo ped b ase d on adv anced tec hnologica l d eliv ery sys tem(4,5) or the p ro – drugs a pproa ch(6,7). Pro – d rugs should be s ee n in the light of the still growing nee d fo r de live ry s ystem whic h may enable one to trans port a ctive agent se lectively to the ta rget and c onseque ntly re le ase the drug over de sired pe riod of time . S eve ra l type s o f e ster pro – drugs for me tronid azo le ha d b een synthe size d utilizing pro – moieties that impart diffe re nt p hysicoc he mic al p ro pertie s fo r this drug through the hyd ro xyl functional gro up of it. Mos t of thes e pro – drugs were synthe sized in an attemp t to inc re as e water s olubility of me tronid azo le to be use d in prepa ring pa re ntal dos age fo rms (8,9,10,11). On the other ha nd a wide v arie ty of co rticos teroids p ro – drugs ha ve b een s ynthe size d and are in clinic al use . Thes e pro – drugs were synthe size d utilizing the C – 21 hydroxyl group to make e sters of diffe re nt phys ic ochemica l prop erties (12). Cortico steroids were us ed in the se pro – drugs either a s p ro moiety for ta rge ting the ac tiv e s pe cies as in the cas e of a ntic anc er agents (13,14), or a s the a ctiv e spe cies which we re co njuga ted to promo ie ties to c ha nge *De par tme nt o f Ph arma ce utic al Ch emistry Colle ge o f Pha rmac y, Unive rs ity of Baghdad ,Baghdad- Ir aq. Ir aq i J.Pharm.Sc i., Vol.15 (1 ) ,2006 81 the p hys ic oc hemic al p ro pertie s(12), or for ta rgeting thes e cortic os te ro id s to the de sire d tis sue (15,16). Mutua l pro – d rugs fo r co rticos te ro id s were also s ynthesized through co njuga tion w ith no n – steroida l anti – inflammatory drugs using a n amino a cid as s pa cer arm(17). The pre se nt rep ort de sc ribe s the synthe sis of a co mpound c onta ining de xamethas one and metronida zole linke d thro ugh a phosp ho die ster linkage. This p oss ib le mutua l pro – drug wa s de signed, thro ugh it's inhe re nt physico che mical prope rties to be cleav ed in the lowe r pa rt of the GIT e sp ecially the co lo n. EXPERIMENTAL SECTION Mater ials: Metro nidazole, wa s a gift fro m S amarra drug ind ustry, d exa metha so ne so dium phosp hate wa s a gift from J ord ania n pharmace utical manufa cturing co mpany LTD. The purity of the se two co mpound s wa s checked ac cording to the B.P and Me rc k Inde x. N,N – dicyclohexylc arbod iimide (DCC) wa s purc has ed fro m ACROS, USA. The remaining chemica ls were of rea gent grad e, a nd were us ed as such without further p urification, sinc e they w ere of the highest co mmercially av aila ble purity. Gener al Me thods All rea ctio ns , thro ughout this wo rk that ne ed a co ns ta nt temperature, were carrie d out in a the rmo stated double jacketed flas k co nne cted to a co ns ta nt temperature circulator and refrigerator o f ultratemp – 2 000 , Jullab o VC. Melting po ints were me asure d us ing a n elec trothe rma l me lting po int a ppa ra tus and are unconne cted. Thin – la yer c hromatogra phy (T.L.C.) using silic a gel c oate d gla ss plate s was p erfo rme d to fo llow up c he mic al rea ction. Purity o f the p re pared c omp ound was c hec ke d by thin – layer chroma to graphy plate s 20x20 cm o f silic a gel 6 0 F 254 with 0.25 mm la ye r thicknes s, Me rc k, Germany. Chroma to grams we re e lute d by the follo wing so lv ent systems : A. Iso propyl alco hol – wa te r – c onc entrated ammo nium hydroxid e s olutio n (7 :2 :1 ). B. Chlo ro fo rm – me thanol – wa te r – a cetic ac id (25 :1 5:4:2). The chromatographic spots were re vealed by e ither rea ctivity with iodine va por or by observing them unde r UV light. UV sp ectra were re cod ed on P ye Unic am UV sp ectrophotome te r, SP 8 – 010 0 Germany. IR spe ctra we re rec orded on P ye Unic am S P – 300 s pe ctro pho to meter, Ge rma ny. C – H – N a na lysis was pe rformed at the Unive rs ity o f Mo sul, Co llege of Sc ienc e, Using (C.H.N) analyzer, typ e 110 6 Carlo Erb a. Che mis try: Synthesis of 1 – (Dexamethasone 21 – phosphoryl) – Me tronidazole, Comound I, Figure (1). Fig.1 chimica l structure of c ompo und I Ge ne ra l Proce dure (18) : De xame thas one p hos phate, s odium sa lt, 6.169 g (12mmo l) was diss olv ed in the minimum v olume of d is tille d water. To this solution was a dd ed dilute HCl so lution d rop wis e with s tirring until the comp le te precipitatio n of the free phos pho ric ac id e ster of d examethaso ne . The suspe ns io n was filte re d, a nd the white p re cipita te o f the free acid wa s co llec te d, washed with distilled water and d ried . The fre e p hos phoric ac id es ter of dexame thas one was dis solve d in 150 m of anhyd rous pyridine . To this solution wa s ad ded 4.9g (2 4 mmole) of (DCC) a nd the mixture was stirred at 2 5ºC for 2hrs , during which dicyc lo he xylurea (DCU) was p rec ipita te d and filte re d. To the filtrate the re wa s ad ded 1 .0 26g (6 mmole) of metro nidazole and the reac tion mixture wa s stirred for two d ays a t 25 ºC. The mixture wa s eva po ra te d to drynes s, a nd then the fina l tra ces o f pyridine we re remov ed by coe vap oratio n with tolue ne (50 ml). The res id ue was treated with 200 ml of 5 0% aqueous etha no l, a nd the ins oluble DCU was re move d by filtratio n. The filtrate was eva porated to d rynes s, re disolve d in 1 00ml of 50% aq ueo us ethano l, and a pplied to a silica gel column (3.5x26c m) prep acked with 5 0% aqueous ethano l. The ma teria l was then eluted Ir aq i J.Pharm.Sc i., Vol.15 (1 ) ,2006 82 us ing is op rop yl alcohol – water – concentra te d ammo nium hyd ro xide (7 :2 :1 ) as the mobile phase . The fractions b etwee n 65 and 1 25ml were po oled, eva po ra te d to d ryne ss. A yello wis h to white crys ta lline po wer wa s co llec te d as the ammonium s alt of c omp ound 1; M.P 12 8 – 1 30ºC, the yield was 45 %. The UV s pec trum of the c omp ound (0.1mg/ml distilled wa te r) show λmax at 3 20 nm. λ max for de xame thas one p ho sphate and me tronid azole were found to be 243nm a nd 264nm re spec tively (Figure (2)). Fig.2 U.V spectru m D = Dexa me thason sodium phosphate M = Me tronida z ol I = Co mpoun d I , So lv e nt : Dis tille d wa te r IR s pe ctrum (Figure (3)) rev ealed the fo llowing ab so rp tion freq ue ncies , cm – 1, (Nujol): 3 232 (O – H) Hydroge n bo nde d; 293 9 (C – H a liphatic ); 172 0 (C=O); 1 662 , 161 2 (C=O, C=C, C=N); 14 90, 124 2 (P=O); 153 5 (- NO2 group); 10 72 (P – O – C); 887 (C – N) stretching fo r he te ro a ro matic comp ound. Fig.3 IR Spe ctrum o f Compound I T.L.C for compound [I], Rf v alue , solve nt sys tem, A (0.88 ), solve nt B (0.53 ). Tab le (1). Ele menta l analys is c alcula te d fo r C28H37O10N3 PF., NH4H2O C, 50 .8 3; H,6.50 ; N, 8 .4 7 fo und C, 51 ,26; H, 6.93 ; N, 8.13. Table 1: The Rf va lues for the reactants and compoun d (I) us ing two diffe re nt so lve nt syste ms , whe re:Solve nt A: isopropyl alcohol – wate r conc. Ammonium hydroxide (7:2:1). Solvent B: chloroform – methanol – wate r – acetic acid (25:15:4:2). De te rminatio n of Pa rtition Coe fficie nt The pa rtition c oeffic ie nt (PC) o f a s olute is defined a s the ratio of the co nce ntratio ns of solute distribute d b etwee n two immisc ib le solve nts at eq uilibrium, and it is usual to prese nt the ratio s that in fav our o f organic pha se : C PC = -- (I) Cw Where Co = the co nce ntratio n of the s olute in the orga nic pha se , and Cw = the co nce ntratio n of the s olute in the aq ueo us p has e. Partition co efficient for comp ound [I] had b een performed by a dding 5 0mg of the s olute to a sep aratory funnel c onta ining 50 ml of water per – saturate d with octa nol and 5 0ml of octanol per – sa turated with water. The s epa ra tory funne l was inverted s ev eral time s during 30min after tha t s epa ra to ry funne l was left for comp le te sep aration of the two pha se s. The a queous phase was a nalyzed for the s olute. A sta nda rd curv e had be en constructed by meas uring the ab so rba nc e of diffe re nt conce ntra tions o f co mpound [I], Figure (4 ). The p artitio n coe fficient for o ur comp ound was calcula ted ac cording to eq ua tion (I), a nd was fo und to be 0.42. The substance S olve nt S ys te m Rf value Comp ound (I) A 0.88 Metro nidazole A 0.96 Dexa metha so ne phosp hate A 0.72 Comp ound (I) B 0.53 Metro nidazole B 0.7 Dexa inetha so ne phosp hate B 0.35 Ir aq i J.Pharm.Sc i., Vol.15 (1 ) ,2006 83 Fig .4 U.V a bso rbance at λ 320 o f diffe re nt c oncentra tions of compound I RESULTS and DISCUSSION Co mpound [I] had be en s ynthesized thro ugh the follo wing step s: In the first step de xamethas one p hos phate diso dium sa lt was c onverted to de xamethas one phosp hate ac id es te r by acidific atio n with dilute hyd ro cho ric ac id equatio n (I) (S che me I). In the se cond step , de xa mehta sone phosp horic cid e ster wa s c onv erte d to the p hos phate anhyd ride using d ic yc lo hexylca rb odiimid e (DCC) as a d ehydrative co upling rea gent, Eq. (2 ). The re ac tive anhyd ride thus fo rme d wa s allo wed to rea ct with the primary hyd ro xyl group of metronida zo le which ac ts as a nucleop hile that a tta ck the p hos phate. The metho d us ed for the synthe sis of co mpound [I] is a mo difica tion o f proce dure s well es ta blis he d in the a re a o f nuc lc otid e synthe sis(19,20), as we ll a s fo r conjuga tion of nuclcos id es with c ortico sterio des thro ugh phosp hate d ie ster linkage (21). The co upling ge nt DCC, was introduced b y Kho ra na d uring nucleo side polyp hos phate synthe sis to promo te s ynthetic re actions invo lving dehyd ration(22). This rea gent was then us ed b y She eha n et al., as a co nde ns ing agent for amide bond fo rma tio n during pe ptid e synthe sis(23). Pyridine , d uring this c ours e of rea ctio ns, ha s tw o role s. It a cts a s a po werful s olvent a nd as a ca ta list acc ording to the following (Sc heme II)(2 4). DCC was used s uc ces sfully in this labo ra tory as a co nd ens ing agent fo r many re ac tions that involve s ynthes is of c arboxylic acid e sters, and fo r amide b ond fo rma tion a s well(25). In a p re vious work(12), a ttemp ts at condensa tion of p re dniso lo ne 2 1 – pho sphate or pre dnis one 21 – p ho sphate with 5 – hyd ro xyl gro up of nuc le os id es in the p re se nce of DCC and pyridine at room te mpe ra ture and a t re flux were not s ucc es sful. This fa ilure could be attributed in pa rt as d ue to the metho d app lie d in whic h the pred niso lo ne 21 – pho sphate was Ir aq i J.Pharm.Sc i., Vol.15 (1 ) ,2006 84 no t conve rted to the free ac id e ster prior to re ac tion with DCC and pyridine . In add ition to that, the pro ce dure employe d did no t allo wed s ufficient time for the p hos phate anhyd ride formation prior to the re ac tion with the alcohol functio nality, as we did in our proce dure . Co nfirmation of the molec ular s truc ture of co mpound [I] was provide d by eleme ntal analysis , UV, IR and TLC. Lipo philic ity is a te rm c ommonly use d to d es cribe the te nde nc y fo r a chemica l a gent to pa rtition itself b etwee n aq ueo us and organic biop has es . P artitio n co efficients p ro vide a co nve nient mea sure of lipo philic ity a nd are often used in e stablishing the relative rates with which che mical substa nce s penetra te lipoida l memb ranes or pa rtic ip ate in the formation of a hyd ro pho bic bond(26). Partition c oeffic ie nt a s was p re viously de fine d , is an imp orta nt parame ter for mea suring the relativ e affinitie s of the s olute fo r an aq ue ous a nd non – aq ueo us or lipid phase . The greater the value o f PC (e quatio n (1 )), the highe r the lip id so lubility o f the so lute . On the o ther hand , the lowe r the va lue of P C the highe r the a que ous solub ility for the so lute . n – Oc ta no le is the s olve nt for whic h mo st pa rtition co effic ie nt v alues hav e b ee n publis hed . It is claime d that the octa nol / water system is sa tisfac to ry mode l for the b io lo gical system b ec aus e the orga nic p has e is not co mpletely no n polar and conta ins a s ignific ant amo unt of wa ter in a stab le , hydroge n bo nde d co mplex(27). In contras t, partitioning systems such a s hexane / wate r and chloroform / water co ntain so little water in the o rganic p has e that they are po or mod es fo r the lipid bila yer / water fo und in the b ody. Lipid s oluble drugs us ua lly cross ce llular bound arie s by d is solving in o r interacting with the lip id me mbrane s and diffus ing ac ros s into the intra ce llular aqueous phas e. Mo st drugs are wea k o rganic elec trolyte s p re se nt in eq uilibrium betwee n two fo rms , of which o nly the no n – io nize d from po ss ess lipid s olubility. Since our comp ound was fo und to hav e re la tive ly lo w p artitio n c oe fficient, it has a s a re sult re la tive ly low lipid s olubility, and a s a co nse quenc e it will p oorly abs orbed from the G.I.T after o ra l ab sorption. In a dd ition to that, it has relatively la rge molec ular s ize, whic h is a n add itiona l factor fo r poo r abs orptio n. On the other ha nd our co mpound is a phosp hate d ie ster, a nd this typ e of conjuga tion is res is ta nt to che mic al cleav age by the G.I fluid. So we expec t that our comp ound will p ersist fo r a lo ng pe riod o f time d uring which it will pas s the G.I tract and reac h the colon in a signific ant qua ntity to e xert its loca l effe ct afte r co mplete hydrolysis a nd lib eratio n of the active mo ie ties . The U.V. spe ctrum of the c ompo und (0 .1 mg/ml distilled wa ter) s how λ max at 320 nm. λmax fo r dexame thas one phos pha te and metro nidazole was found to be 2 43nm and 264 nm re sp ectively. 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