Ir aq i J.Pharm.Sc i., Vol.15 (1 ) ,2006 74 Conjugation of Steroidal and Non – Steroidal Anti- Inflammatory Drugs as Possible Mutual Prodrug Muthanna D. Saud* Receive d 30-12-2002 Ac cepted 15-5-2005 ABSTRACT Prednis olone (S AID) was c onjugated with ibup ro fe n (NS AID) thro ugh an a mino ac id (glycine) as a s pac er arm to s ynthe size the fo llowing c omp ound: Pre dniso lo ne – g ly cine – ibupro fe n. The me thod e mploye d co nsists o f co nve rting the carbo xylic ac id functio n of (R,S) – ib up rofen – glycine to the highly reac tiv e acid c hloride and s ub se que nt rea ction with the C21 hydroxyl group of prednis olone . This re active intermed ia te was found to re act as we ll with the C17 te rtia ry hyd ro xyl group of the s te ro id to fo rm thre e c omp ounds and e ight d ia stereo mers. Thes e res ults w ere confirmed by T.L.C, and the des ired c omp ound was s epa ra te d by co lumn chro matogra phy. The id entity of the p re pared c omp ound was e stab lished using U.V sp ec tros cop y, IR sp ec tros cop y and eleme ntal mic roa na lysis.The partition co effic ie nt (PC) fo r this co mpound wa s es tima te d and fo und to b e more so luble in the organic p has e (n – octanol). Prelimina ry kinetic stud y indica te d that the comp ound ne eds more than 1 5 hours fo r significa nt hydrolys is in p hos phate buffer pH 7.8. خالصة ال عقار الستيرويدي مضاد (مع عقار االيبوبروفين ) مضاد لاللتهابعقار ستيرويدي (تضمنت هذه الدراسة ربط عقار البردنزولون  –كاليسـين   –باستخدام حامض أميني هو الكاليسين كذراع مباعدة فراغية٬ لتحضير المركب النهائي وهو بردنزولون ) لاللتهاب ب  . ايبوبروفرين ي مرـك وبروفين   إن الطريقة المستخدمة للربط قد اعتمدت على تحويل مجموعة الكاربوكسيل ـف كاليسـين   –االيـب ولقد تبين أن هـذه المجموعـة   . إلى مجموعة كلورايد الكاربوكسيل الفعالة جدا كمجموعة باحثة عن االلكترونات) المحضر سابقا( 2باإلضافة إلى تفاعلها مع مجموعة الهيدروكسيل على ذرة الكاربون  في جزيئة البردنزولون لتكوين آصرة االستر٬ فأنها كذلك  1 .في جزيئة البردنزولون 17اعل مع مجموعة الهيدروكسيل على ذرة الكاربون تتف ذي    إن النتائج المستحصلة قد تم التأكد منها باستخدام تقنية كروماتوغرافيا الطبقة الرقيقة٬ وكذلك فلقد تم فصل المركب النهـائي اـل 2ة الكاربون ترتبط فيه مجموعة الكاربوكسيل الفعالة مع مجموعة الهايدروكسيل على ذر بشكل كمي ونقي٬ وباستخدام تقنية  1 .كروماتوغرافيا العمود لقد تم التأكد من صحة التركيب الكيمياوي لهذا المركب باستخدام طيف األشعة فوق البنفسجية٬ وطيف األشعة تحت الحمراء٬ وكذلك  مركب المحضر حيث تبين أن هذا المركب أكثر ذوبانا لقد تم حساب مقدار معامل التجزئة لل. بالتحليل الكمي الدقيق لعناصر المركب ول الفوسـفات          . في المذيب العضوي منه في المذيب المائي ي محـل ذا المركـب ـف ل ـه ة لسـرعة تحـل كما تبين من خالل دراسـة ابتدائـي 1إن تحلل هذا المركب يتطلب أكثر من , 7,8الدوارئي ذي األس الهيدروجيني  .ساعة 5 INTRODUCTION Drug ta rgeting to spe cific rec eptors or spe cific orga ns ha s bee n one of the main objec tive s o f the med ic inal and pha rma ce utic al c he mis ts from the be ginning of the p as t ce ntury. Howev er, only in the pas t 30 years or s o hav e there be en a ny p ro mis ing de ve lo pments in a chiev ing this goa l(1). The s ite – s pec ific delivery of d rug is ind ee d a v ery attrac tiv e goa l be cause this prov id es one of the mo st significant potential wa ys to improve the therape utic ind ex of the drugs(2). When a drug is delive red p re fe re ntially to the s ite of the ac tion by v irtue of this de sire d differential distribution, it will sp are the re st of the bod y; thus it will b e s ignifica ntly re duc e the ov erall toxic ity while maintaining its the ra peutic be ne fits(3). One o f the a pproa ches for site – spe cific d rug de live ry is the che mical ap pro ac h or s o ca lled site – s pe cific c he mic al de live ry systems (CDS s) which prov id e a wid e v arie ty of pos sibilitie s fo r s ite – e nhanc ed or s ite – spe cific d eliv ery(1,4,5). re actions by whic h the p arent drug is cov alently co upled with o ne or mo re c arrier moietie s. By d es ign, a fter d eliv ery the CDS will unde rgo a v arie ty o f enzymatic convers ions, which prod uce inte rme diates a ll hav ing diffe re nt physica l prop erties and varying rates of formatio n a nd eliminatio n, thus ultima te ly allowing a p re fe re ntia l and fa vorab le d is tribution o f the prec urso r prodrug at the s ite of the a ctio n where ultima te ly the drug is re le as ed(6,7). Co lo n – spe cific de live ry of bioa ctiv e c omp ounds re ce iv ed exte nsive inves tigatio ns , utilizing the s ignifica ntly variable bioe nvironments of the diffe re nt p arts of the G.I.T.(8,9). * Dep artmen t of Phar mace utic al Chemistry, Co lleg e of Ph arma cy,Un iver sity o f Bagh dad , Bagh dad - Ir aq . Ir aq i J.Pharm.Sc i., Vol.15 (1 ) ,2006 75 Cortico steroids we re c urre ntly use d for the trea tme nt o f infla mmatory bowe l dise ase s. The y are us ed eithe r alone or in c ombina tio n with other d rugs(10,11). In a rece nt inv estiga tion in this lab oratory dexame thas one (SAID) was c onjugated to metro nidazole through a phosp hod ie ster linkage . This p oss ible p ro drug, which wa s fo und to be insolub le in the a queous med ium at low pH, was sugge sted to be a ble to re ac h the lo wer p art of the G.I.T. in which it will gain e nough aqueous s olubility to b e hyd ro lyze d through e nzymatic and /o r no n – enzyma tic proc ess es to libe ra te its ac tiv e moietie s(12). In this inv es tiga tion we wo uld like to re po rt the s ynthes is o f the follow ing conjuga te : Prednisolone – glycine – ibupr ofen Ib uprofen – glyc ine conjuga te , which wa s previously synthe sized (13), was c onverted to the a cid chloride through rea ctio n with thio nyl chlo ride . This re ac tive intermed ia te wa s allo wed to re act with the C21 hydroxyl group of prednis olone to form the final c onjugate. The rea ctiv e intermed ia te wa s found to re ac t, though to a muc h le ss er exte nt, with C1 7 hyd ro xyl group o f the cortis one a s will b e des crib ed in the follo wing se ctions . EXPERIMENTAL SECTION Materials:- The amino a cid glycine wa s purchas ed from HOP KINS a nd WILLIAMS LTD. Engla nd. Pre diso lo ne and ib uprofen we re a gift from the Jo rd anian P ha rma ceutic al Ma nufac turing Comp any LTD. The ide ntity and purity o f thes e co mpound s were c hec ke d a cco rd ing to the B.P and Merc k Inde x. N,N' – dic yclohexylcarbo diimide (DCC) was from ACROS USA. The remaining c he mic als were o f re age nt grade , a nd were us ed a s such without further purifica tion, sinc e they we re of the highe st commercially a vailable p urity. Ge neral Me thods:- All re ac tions, througho ut this work that ne ed a co ns ta nt tempe ra ture , were carrie d out in a thermos ta ted do uble jac ke te d flas k connec ted to a co nsta nt temp era ture circ ulator a nd re frigerator o f Ultra – te mp 200 0 Jullab lo VC. Chiller, Germany. Melting po ints were meas ured us ing a n electrothe rma l melting point a ppa ra tus and were uncorre cted . Thin layer chro matogra phy (TLC) us ing silica gel coa te d glas s plates was p erfo rme d to follow up chemica l re actions. The purity o f the p re pared c omp ounds wa s checked by thin layer c hroma to graphy plates of (20X20) s ilica ge l (60 F254) with 0 .2 5mm layer thic kne ss o btaine d from me rc k, Ge rmany. Chromatogra ms were eluted by o ne of the following s olvent s ys te ms: A: Me ntho l: Ammonia (100 :1 .5 V:V) B: Benzene: Ether: Ac etic a cid: Me ntho l (12 0:60:18 :1 V:V) C: Chloroform D: Acetone: n – Hexane (33 :6 7 V:V) E: Benzene: Ether: Menthol (6 0:35:5 V:V) The chro matogra phic s pots we re rev ea le d b y e ithe r re activity with iodine v ap or or by ob se rving them under UV light. IR sp ec tra were reco rd ed on Perkin – Elmer s pe ctro sc opy, Engla nd. UV sp ec tra we re c arried out a t the Na tional Cente r for P ha rma ceutic al Re se arch and Quality Control, Baghda d, us ing Cec il L – 41 1, F ra nc e. Column Chroma tograp hy were carrie d o ut using glas s c olumn (75 cmX2 0mm) p re pa cka ge d with 5 0gm of s ilica gel (Kies elgel 6 0) s us pe nde d in 10 0ml of c hloroform. Ele menta l Micro Analysis (CHN) was p erfo rme d a t the Univ ersity of Mouse l, Colle ge of S cienc e using (CHN) a nalyzer type 11 06 Ca rlo Erba. The pH va lues we re me as ured using Pye Unica m p H meter (Philips), Holla nd. Chemical Synthesis:- Synthe sis of ibupr ofe n – glycine acid c hlor ide , (N – [2(4 – isobutyl phenyl) propionyl] – glycyl c hloride), compound I(14 )., (scheme 1):- Ibuprofen – glucine (1.5gm, 5.7m mo l) w hich was prev io us ly synthe size d(13), was d is so lv ed in 1 0ml chlo ro fo rm a nd the s olution was coo le d to 5ºC. An e xc ess thio nyl c hlorid e (2.5ml) was a dde d d ro p w is e with co ntinuo us stirring, during which the te mperature of the re ac tion mixture was kep t below 10ºC. The mixture was then refluxed for more tha n 2hrs . until the e vo lution of gas eous S O2 and HCl w ere c ea se d. The so lv ent wa s e va porated to d ryne ss in va cuo and the re sidue was red is so lv ed in c hloroform a nd ev apo ra te d. This proce ss wa s repe ated s eve ra l time s in o rd er to remo ve exce ss thionyl c hlorid e. Ibuprofen – glyc ine a cid c hlorid e was o btaine d as a faint yello w o ily residue and w as used as s uc h for rea ctio n with p re dnis olone . Ir aq i J.Pharm.Sc i., Vol.15 (1 ) ,2006 76 Synthesis of ibupr ofen – glycine – pre dnisolone (N –[ - 2 –(4 – isobutyl phe nyl) propionyl] – glycyl – (11,17α – dihydroxy pre gna – 1,4 – diene – 3,20 – dione – 21 – yl) – e ste r) (compound II):- Ib up rofen – glycine ac id chlo ride (1 .4 gm, 5mmo l) was diss olved in 1 0ml of dry dichlo romethane, and the solution wa s coo le d to 0 ºC. Pre dnis olone (2.52 gm, 7 mmole) wa s diss olved in 2 00ml a cetone a nd 1.5ml of triethyla mine was a dd ed. The cortis one s olution wa s add ed d ro p wis e to the co oled ac id chloride so lution o ve r p erio d of 3 – 4hrs with co ntinuo us stirring. The re ac tion mixture was the n stirred at 25ºC for 48hrs . Afte r that it was filtered to remov e trie thyl ammo nium c hlorid e and the filtrate wa s ev apo ra te d to d rynes s under v ac uo to a v ery thick brown p as t. The pa sty re sidue was dis solve d in e thyl ac etate and washed with 0.1N HCl, then with distilled wa ter, the n with 5 % NaHCO3 so lution, twic e with dis tille d w ater. The e thyl ac etate laye r wa s drie d ov er a nhydrous ca lcium chlo ride a nd filtered. Comp ound II was the n sep arated a s dias te re ome ric mixture b y column chro matograp hy using s olvent s ystem E as the mob ile pha se (15). Many a tte mpts were pe rformed to crys ta llize the pas t p ro duc t but a ll were faile d. The purity a nd identity of this c ompo und were co nfirmed us ing T.L.C., U.V. sp ectros cop y (figure 1 ) I.R s pe ctro sco py a nd C.H.N analysis . (Nujol) : 3 600 – 3 300 , broa d (N – H, O – H s tre tc h. hyd rogen b ond ing). 307 0 (C – H aromatic stre tch), 2 950 (C – H) alip ha tic stretch), 1 750 (C=O, ester), 17 20 – 171 0 (C=O), ke to ne), 1 660 – 1 650 (N – C=O amide ), 1 380 , 13 50 (gem – d imethyl C – H be nd.), 16 20(C=C s tretch.), 1 220 – 1 190 (C – C – O s tre tc h.), 142 0 (C – N stretch), 700 (C – H a ro matic o ut o f plane b end .), 90 0 N – H wagging. Eleme ntal a nalys is, c alculate d for C36 H 4 7 NO 7 – H2O: C; 69 .3 4, H; 0 7.86, N; 2.24, fo und: C;70.12 ,H;0 7,52,N;2.55 .T.L.C; Rf va lues; 0.83(A); 0.35 and 0.4 (B); 0 ,1 8(C); 0.75 (D); 0 .5 2 and 0.61(E). Fig(1) UV Spe ctrum of Co mpou nd II De te rmina tion o f Pa rtition Co e fficie nt:- Partition c oeffic ie nt (PC) fo r a s olute could be de te rmined us ing the follo wing relatio n: P C = Where Co = the co nce ntratio n of the s olute in organic p ha se, and Cw = the c onc entration of the so lute in the a queous phase . Partition co efficient for c ompo und II has b een performed by a dding 2 5mg of the s olute to a sep aratory funnel c onta ining 25 ml of water pre – saturate d with octa nol and 2 5ml of octanol pre satura te d with wa te r. The sep aratory funnel was inv erte d se veral times during 30 min., a fter that it wa s le ft fo r complete sep aration of the two p ha ses . The aq ueo us p has e was a na lyzed fo r the s olute. A sta nda rd curv e had be en constructed by meas uring the a bso rb anc e of diffe re nt conce ntra tions of c omp ound II, (figure 2). The Cw Co Ir aq i J.Pharm.Sc i., Vol.15 (1 ) ,2006 77 pa rtition co efficient fo r our c ompo und wa s fo und to b e 82.3 . Fig .2 Abso rba nce of c ompo und II at λ 3 70 ve rs us c once ntratio n (solve nt e tha nol) On the othe r ha nd the s ta bility of c ompo und II in phosp ha te b uffe r (0 .1 M, pH 7.8) wa s determine d ov er a p erio d of 24 hrs b y incub ation of 10 0mg of c omp ound II in 10ml of a queous phosp ha te b uffe r at 37ºC. An aliq uot (2 ml) of e ach s amp le wa s ta ke n at c erta in inte rv als (3 0min, 10hrs , 2 0hrs, and 25hrs ) and was me asure d fo r the remaining amount o f compound II b y converting the abs orbance to the c orre spo nd ing conce ntra tion. A p lo t was constructed , c onc entratio n of the re maining amo unt of co mpound II v ersus time (figure 3 ). Fig.3 Showing the hydrolys is of compo und II in pho sphate buffe r (0.1 M , pH 7.89 ) at diffe re nt time inte rvals RESULT and DISCUSSION Synthesis of Ibupr ofe n – Glycine acid chloride:- The goal for this inv es tiga tion was to co njuga te the c arbo xyl moiety of ibup ro fe n – glycine with the C21 hyd roxyl group of prednis olone thro ugh e ster linkage. To a chie ve this go al the c arboxylic ac id functionality should be a ctiv ated . The activation ma y either b e thro ugh the fo rma tion of a cid a nhydrid e us ing (DCC) as the dehydrating agent(16,17), o r though conversion to the ac id chlo ride whic h is ve ry reac tive inte rme diate. In this inve stigation ib up rofen – glycine was converted to the ac id chlo ride (scheme 1) thro ugh rea ction with thio nyl chloride. The a dva ntages of thio nyl chlo ride o ver o ther chlorinating a ge nts lie s in the fac t the byp ro duc ts o f the reac tio n (SO 2 and HCl) are gas es a nd ca n be ea sily remo ved througho ut the c ourse of the re ac tion. More ov er any exc es s of the low – b oiling thionyl c hloride (7 9ºC) is eas ily remo ved by distillation. The ac id chloride thus formed is highly re active electop hilic spe cies , whic h we nt add ition at the ca rb onyl group by an a mine or hyd ro xyl groups follo wed by elimina tion of the chloride re sidue, to form an amide or e ster linkage s. Synthesis of Ibuprofen – Glycine – Prednisolone (Compound II) (sc heme 1):- This w as p erfo rme d b y reac tion of ib uprofen – glycine a cid chloride with the C 21 hyd ro xyl group of p re dnis olone in the p re se nce of trie thylamine to ab stract the lib erated HCl. Pre dnis olone (sc he me 1 ) ha s thre e hydroxyl group s, a prima ry hydroxyl a t C21, a s eco nda ry hyd ro xyl group at C 11 a nd a te rtia ry one a t C17. Bec aus e of the high rea ctiv ity o f ac id c hlorid e, re action with C17 hydroxyl group could also be occ urre d. On the o ther hand re action with the C11hydroxyl gro up ha d be en exclud ed be ca use C11 hyd ro xyl group is sterica lly hinde re d due to the pres ence of two methyl groups at C 10 and C 13 (18). Acco rd ingly, comp ound II is not the o nly one that is fo rme d through es te rific atio n, comp ound III is a ls o formed, though to a less er extend, by e sterification with C17 hydroxyl group . In add ition to that esterifica tion o f bo th C21, a nd C17 wa s fo und to be occ urre d to form comp ound IV (sc he me 1). Rea rrangeme nt of the C17 e ster (c ompo und III) to the mo re s ta ble C21 es te r might ta ke s place in the p re sence o f aqueo us or non – aq ue ous medium. This rea rrange ment is fa ster than hyd ro lysis to the pa re nt s teroid(19). Ste re oche mistry :- Ibuprofen use d in this inv es tiga tion conta ins one chiral c enter a nd exist a s a ra cemic mixture compos ed of e qual amounts of two enantiome rs hav ing R and S configura tions. Acc ordingly, ib uprofen – Ir aq i J.Pharm.Sc i., Vol.15 (1 ) ,2006 78 glyc ine ac id c hlorid e (c ompo und I, scheme 1) will exist in thes e tw o configura tions. Prednis olone, o n the othe r hand , is an optically ac tive molec ule w hich has mo re than one chiral cente r. Bec ause the rea ctio n we are de aling with d oe s no t affe ct a ny chiral ce nter a t prednis olone molec ule, the co nfiguratio ns of the se chiral ce nters rema in co nsta nt. As a re sult, the sp ecific rotation of p re dniso lo ne will not b e changed and for s implific ation we a rb itrary co nside re d it to has an S co nfiguration. Bas ed o n thes e co ns id era tio ns, it was exp ec te d that the major product (co mpound II, sc he me I) will e xist in two d is te re ome ric fo rms , (S ,S ), and (S,R). On the othe r ha nd, whe n este rifica tio n o cc urs at C17 hydroxyl group of prednis olone (c omp ound III), it will a ls o gene ra te two dias te re ome rs . Fina lly, whe n b oth C17 and C21 hyd ro xyl groups of p re dniso lo ne ha ve be en es te rifie d (c omp ound IV), this s itua tio n w ill res ult in the fo rma tio n of fo ur d ia stereo mers (S,S ,S ), (S ,S ,R), (S,R,S), a nd (S,R,R). Acc ordingly, there will be eight d ia stereomers re sulted from this re action. In a previous work, whe n dicyclohe xylc arbod iimide had be en used fo r co njugatio n of hydroc ortiso ne with ib up rofen, es terific ation was found to oc cur exclusive ly at C21 hydroxyl gro up of hydroco rtisone(13,18). Se paratio n of Dias te re o me rs:- Of a number of T.L.C. s olve nt systems experienc ed in this inve stigation, system E wa s fo und to giv e the be st se paration of dias te re ome rs . This s olv ent system ha d b ee n us ed s ucc es sfully b y othe r inve stigators for the se paration of different d ia stereo mers(15). Using this s ys te m, the fina l p ro duc t, a fter purifica tio n, showed eight spo ts of significantly diffe re nt Rf v alues : 0.37 , 0.44, 0.52 , 0.61 , 0.71 , 0.7 7, 0 .8 5 and 0.91. Beca us e of the low po la rity of this so lv ent system, the re la tive ly non – pola r distereo mers of co mpound IV will e xpe cted to mo ve fas te r and be in the uppe r p art o f the plate. The dias te re ome rs of c omp ound III be ing hav ing the highe st p olarity in c ompa riso n w ith the othe r two c ompo unds, will mov e the sho rter distance s and a ppe ar a t the lower p art of the plate . Comp ound II dia stereomers which hav e re la tive ly me dium polarity will e xpe cted to oc cup y the middle situation on the T.L.C. plate. The se rationa l expe ctatio ns had b een the ba sis fo r sep aratio n of the dias te re ome rs of co mpound II thro ugh column c hromato gra phy which we re further confirmed by IR sp ectrosc op y and C.H.N analys is . Comp ound II tha t wa s synthe size d and id entified througho ut this wo rk, was found to has relative ly high partition co efficient which gav e a n indication of low s olub ility in the aqueous ga stric fluid(20). More ov er it has re la tive ly high molec ular weight a rea so n that will de crea se the pos sibility of its ab so rp tion through G.I.T. A preliminary inve stigatio n of its s tability at aqueous phosp hate buffer, p H 7.8, indicated that its significant hydrolys is too k abo ut 1 5 – 25 hours , during which it will e xpe cted to re ach the lowe r parts of G.I.T whe re it may libe ra te its ac tive s pe cies the re in. Ir aq i J.Pharm.Sc i., Vol.15 (1 ) ,2006 79 REFERENCES 1. Bodo r, N., Ann.N.Y. Aca d. Sc i., 1 987 , 5 07, 285 . 2. Bodo r, N., and Forag, H., J . Med . Ch em. 1 983 , 26 , 313 . 3. Gregoria dis, G., Na tu re, 197 7 , 2 65, 4 07 . 4. S inkula , AA, An n. N.Y Acad . S ci., 1 987 , 50 7 , 281 . 5. S te lla V.J . Med . Ch em. 1 980 , 23, 127 5 6. S te lla V. J . and Oliyai R., Ann. Rev. Phar mac ol. To xicol. 1 993 , 33 , 52 1. 7. Bund ga ard , H., Des ig n of Prod ru gs, N.Y. Elsev ie r, 198 5. 8. Redd y, S.M., Shina, V.R., a nd Red dy, D.S., Drug s of Tod ay 19 99 , 3 5, 5 37 . 9. Mo oter, V., Symyn, C., and Kinge t, R., Int. J . Ph ar m. 19 92 , 8 7, 37 . 10. 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