Journal of Scientitic & Industri al Research

Vol. 62 , Jul y 2003 , pp 659-665

Medicinal Significance of Nitroimidazoles - Some Recent Advances

V K Kapoor' , Renu Chadha, Praveen K Venisetty and S Prasanth

. Uni vers ity Institute of Pharmaceuti cal Sciences , Panj ab University , Chandigarh 160014

Received: 29 February 2002; revised: 12 April 2002; accepted: II September 2003

Nitroimidazoles have therapeutic uses as anaerobic anti bacterials and ant i protozoal agents. Recent ly, they ha ve been founcito possess other int eresting biological acti vit ies of therapeutic potenti al such as, radiosensi tizers in treatment of cancer, control of fertili ty, and antitubercul ar therapy. Variolls aspects on the chemistry and modes of acti on of nitroill1idazoles are di scussed.

Keywords : Nit ro imiciazoles, Therapeutic uses of nitroill1idazoles , Biological acti vities

Introduction

Med ic in al chemists have often been skeptica l about the use of nitro group in potential medicinal agents because certa in unt owards observati ons have been made with regard to the nitrocompounds. Compound s like trinitroto luene, employed in expl os ives arsenals , cause methemoglobinaemia following skin absorption , and picric acid useful as antiseptic, turns out to be a systemic poi son. Chl oramphen icol, an antibacterial di scovered in 1948, was found to have a nitrophenyl sysy tem. Since then, other nitroheterocyc les have been di scovered among antimicrobi a l agents and have been used in severe typho id and other sa lmone ll a l infections. The di scovery of azomycin (1) , a 2-nitroimidazole, by Nakamura i and demonstrati on o f its antibacterial antitrichomonal properties2

, revo lutionised the area of nitro imidazo les. Azomycin became the chemical lead fo r extensive synthetic development of over hundered compounds . This resulted in the emergence of a compound now known as metronidazo le (2), which was introduced as a che motherapeutic agent in 1960 in Europe and in 1963 in the US. It became a drug of cho ice in the treatment o f several protozoal infecti ons and infecti ons caused by obligate anaerobes . The present communication rev iews the development of nitro imidazo les as med ic inal agents with their current

* Author for correspondencc e·· mail: renukchadha@rediITmail .coll1

status not only as antimicrobials but hav ing other inte resting biol ogical profiles, discovered recentl y.

Azyomyc in (1) Metronidazo le (2)

Classification of Nitroimidazoles

Depending on the pos itions ava il ab le 111

imidazole hete rocycle, nitroimidazo les have been class ified as 2-nitro, 4-nitro- , and 5-nitro imidazo les. Out of them, 5-nitroimidazoles have establi shed themselves as s ignificant therapeutic agents with anti protozoal and antibacterial properites.

2-Nitroimidazoles

Azomycin (1) di scovered from the extract o f so il Streptomyces cultures was the first 2-nitroimidazole to exhibit activity against several protozoans, specifically trichomonads. It provided a prototype fo r the development of other nitroimidazo les, such as misonidazole (3), which showed activity aga in st intestinal amoebiasis in rats similar to that of metronidazole3.4. Mi sonidazole also find therapeutic use in the treatment of neoplasti c diseases5

Benznidazole (4) is useful in the treatment of South American trypanosomiasis (Chagas' di sease) due to

660 J SCI IND RES VOL 62 JULY 2003

infecti on with Trypanosoma cruzi, especially in early acute stage of the di sease.

Mi sonidazol e (3) Benzindazo le (4 )

4-Nil m imidazole.\"

As such, there appears to be no report on the anti protozoal or antibacterial effects of 4-nitroimidazole derivatives. However, a drug azathioprine (5) which has 4-nitroimidazole-5-·yl moeity attached to purine through sulphur has shown promise as an immunosuppressant.

CH3 I .

s ----<, II )CN"-...

\ N

°2N

N

~ N H

Azathioprine (5)

5 -Ni I milllidm.nies

S-Nitroimidazo les constitute the l,irgest group of nitroimidazoles with anti protozoal and antibacterial actIv ItIes. M etronidazole, 1-(2-hydroxye thyl)-2-methyl-S-nitroimidazo le (2) is one of the most ex tens ively used drug in the treatment of tri chomoniasis, amoeb iasis, ;JI1d g iardi as is and shown to be active against anaerobic bacteria. It is the mos t active agent available against obli gate anaerobes and is of maj or value in the treatment of seri ous infections due to these organisms3

. M etronidazole has been a subjec t of ex tensive chemica l modification s to improve its bi oavail ab ilit / .

Fo ll ow ing the sllccess with metronidazole, a hect ic ac ti vity of synthes is ing and tes ting other 5-nitroimidazo le was initiated . As the 1-(2-hydroxyeth yl) side chain at posi tion I of metron idazo le is read il y ox idi sed metabolically , repl aceme nt of this side c hain resulted in the

development of other clinically useful antibacterial and antiprotozoal drugs, like ti n iclazo le (6) , panidazole (7), ornidazole (8), secnidazo le (9), carnidazole (10), and ternidazole (11)5.

Tinidazole (6)

OH I

CH2CHCH2CI

I O,N'(rCH, Ornidazole (8)

s II

CH2CH2NHCOCH3

I O,N--(,(CH,

Carnidazole (10)

fH 2CH2 < ~N O,N,(,(CH,

Panidazo le (7)

OH I

CH 2CHCH 3

I O,N'(rCH, Secnidazo le (9)

CH2CH2CH20H

I O,N'('(CH,

Ternidazo le (11)

Nimorozole (12) which lacks meth y l at postion 2, and other 5-nitroimidazoles having varied substituents at pos ition 2 are: dime trid azo le (13) , ipronidazole (14), ronidazole OS) , 'MP' nitroimidazole (16), 'MCA' nitroimidazole (17), and azanidazole (18). Ronidazole is used in vete rinary practice also for the treatment and contro l of swine dysentry.

r-\ CH CH -N 0 12 2 ~

o,N-<'-fj

Nimorazole (12) Dimetridazole (13)

KAPOOR el al.: MEDICINAL SIGNIFICANCE OF NITROIMIDAZOLES 66 1

CH I 3 CH

/. 3

02N\CN-Y-CH ~ II " CH

N 3

Ipronidazo le (14) Ronidazo le (15)

'MF' Nitroimidazo le (16) 'MeA' Nitroimida7.0 le (17)

Azan idazole (18)

A new 5-nitroimidazole fex inidazole (HOE 239) (19) with a 4-methylthi o- phenoxymethyl group at postion 2 was fou nd to be hi ghl y ac ti ve against cure

experimental murine eNS-trypanosomiasis with a I d treatment, when given in combination with melarsopro l6. Sulphimidazo\e (20) is the most recent compound in wh ich a p-aminobenzene­sulphonamide' moeity has been attached at position 2 of the 5-nitroimidazo le rin£ . It possesses a useful spectrum of activity .

Fexi nidazol e (19)

CH3 I 0 ON N II._~

2 'i IrNHS~NH2 \-N

Sulphimidazole (20)

Mechanism of Antimicrobial Actions of 5-Nitroimidazoles

The mechani sm of acti on of nitro imidazo les has been studied in detaiI R

. " • T he nitroimidazoles are se lectively toxic to anaerobic or microaerop hilic microorgani sms and for anox ic or hypoxic cell s. The S-nitroimjdazoles, as represented by metronidazole, require metabolic activati on by sens iti ve organi sms for their antimicrobial effec t. Afte r the drug enters into the cell the nitro group of the imidazole heterocyc le accepts e lec trons from the e lectron transport prote ins with suffic ie ntl y low negati ve redox potential such as, flavoproti ens inc luding xanthine ox idase, NADPH cyt P450 reductase and NADPH cyt c reductase in mammalian cell s and ferredox in or the ir equivalent , such as pyru vate dehydrogenase fe rredoxin oxidoreductase in protozoan or bacte ri a. The rate of intrace llul a r reduc ti on is important because it dete rmines the rate of drug uptake into the cell via a concentration gradient. The complex reduction is cata lysed by nitroreductase in mammalian ce ll and by iron-su lphur co mpl exes in other case. The e lectrons are supplied by e ither reduced nicotinamide adenine dinuc leotide phospahtt' (NADPH) or sulphi de. The reduction of nitroimidazo les is a complex process in volv ing sequentia l add ition of an e lectron , protonat ion and disprotonati on, as shown in F igure I . The che micall y reac tive intermediates, formed during the e lectron reduction of the nitro group to the corresponding hyd roxy lamine, account for the antimicrobial activity

e . -R-N02 ---( 1)

R-NO; + f-+ - R-N02H ---(2)

2R-N02H - R-N02 + R-N(OH)2 ---(3)

R-N(OH)2 --__ R-NO + Hp ---(4)

- -e . R-NO? - R-N02

---(.5)

+ . R-N02 + H - R-NOH

---(6)

R-NOH + R-NO?H - R-NHOH + R-N02 ---(7)

e . R-NHOH - R-N HOH --- (8)

R-NHOH + 2H - R-NH2 + H20 ---(9)

R", Imidazole heterocycle

Figure i- Biorecluction of nilruimiclazo\es

662 J SCI INO RES VOL 62 JULY 2003

of nitroimidazoles. The exact process by which these intermedi ates destroy the cell s has not been elucidated but it has been suggested that these · intermedi ates react with cellul ar mac romolecules, such as deoxy ribonucleic ac id (DNA), proteins, and membranes. The entiti es which are cytotox ic and cause DNA damage are nitro- free radical and the superox ide ani on, fo rmed by its reacti on with oxygen, as well as the products of further reducti on of nitro group [i.e. nitroso (R-NO), nitroso-free radical (R-NO)] or hydroxy lam ine (R-NHOH).

Pharmacokinetics

The pharmacok inetics of nitroimidazo les, including th at of metronidazo le, has been studied in tensive l/ ' 2 They are readily absorbed foll owing ad min istrati on by mouth with excellent degrees of bioava il ability. Peak pl asma concentrati on of approx imately 5 and 10 /-lg/mL are ac hieved usually wi thin I to 2 h of single dose ad ministrati on of 250 i.lnd 500 mg, respecti vely, of metronidazo le. Nitroimidazo les get widely di stributed in the body. Metronidazo le is reported to appear in most of body ti ssues and flu ids , li ke bil e, bone, breast mil k, cereberospinal flui d, seminal fluids, and vaginal secreti ons.

Metro nidazole gets metaboli zed by the li ver th rough side chain ox idation and glucuronide format ion. The principa l ox ida ti on metabolites are 1-(2-hydrox yethy 1)-2-hydrox ymethyl-5-n i tro i midazole which also have anti bacteri al act ivity . It is detectable in plas ma and uri ne. The other major metabolite wh ich has no anti bac teri al act ivity is 2-methyl-5-nitroil1li dazo le- I-aceti c ac id . Small amounts of reduced acetamide and N-(2-hyd roxyethyl)oxamic acid (HOA) have also been detected in ur ine.

The half- li ves of 5- nit ro imidazoles vary from 8-20 h. The va lues repo rted1 are metronidazo le (8 h), orinidazole ( 12 - 14 h), benznidazo le (1 0.5 - 13.6 h), and sec nidazo le (20 h).

Recent Advances in Othe." Biological Profiles

Besides having anti protozoal and antibacteri al activity nitroimidazo les have ex hibited other in terestin a bioloaical ac ti vities, which are useful in

b b

therapeuti cs. These are nitroimidazo les as radi o-sensiti zers, antifertility potenti al of nitroimidazo les, and antitubercu lar profile of certain nitri ­midazopyrans.

Nitroimidazoles as Radiosensitizers in the Treatment of Cancer

The hypox ic cell s present in most of the so lid tumours, limit the successful local control of these tumours by radi otherapy of growth , essenti all y outstripping the tumour's vascul ar sys tem, hence reducing the suppl y of essenti al nutrients, parti cul arly oxygen. However, ti ssue oxygen tension decreases with di stance from the capill ary and graduall y fall s to a level in sufficient for cell di vision. Eventuall y the oxygen deprived cell s die and thi s causes the loca l necros is observed in most solid tu mours. A viab le hypoxic cell , which occurs in the in terface regions between the well-oxygenated ti ssue and the necrot ic regions, are radi ati on res istant relati ve to ox ic ce ll s. So the radi ation res istance is the largest fac tor influencing loca l tumour control by radiati on.

In the earl y I 970s, researchers in Canada and England , di scovered that ni tro furans and nitroimidazoles are radi osensiti zers of hypox ic (oxygen deficient) ce ll s. Cellul ar response to ioni zing radi ation IS strongly influenced by oxygen concentrati on; hypox ic ce ll s are much more resistant to radiati on damage than are normal ly oxygenated cell s. The sensiti zing effect of molecu lar oxygen is probably a consequence of the rap id chemica l reac tions between oxygen and short-li ved free radicals generated by radi ati on absorption. It has been suggested that electron affinic (or eas il y reduced) chemical s mi ght mimic the sensitizin g ac ti on of oxygen and thereby overcome the radio res istance of hypox ic cell s. The di scovery of the radio sensiti zing properties of nitro-heterocyc les led to the in tense interest in the medica l applicati on of radiosensiti zers. The correlation between one electron reduction potenti al and ability of nitro compounds to act as radi osensitiziers of hypox ic cell s i, ce ntral to the deve lopment of agents useful as adjunc ts in radi o the rapy. The f irst e lectro n affi ni c sensitizer based on the nitro fun cti o nal group to be tested

OH

I CH2 CHCH

20H

I

(rNO,

Misonidazole Desmethylmisonidazo le

KAPOOR el al.: MEDICINAL SIGNIFICANCE OF NITROIMIDAZOLES 663

SR - 2508 R003 - 8799

clinica ll y was metronidazo le that was a lready in c linica l use as an anti tri chomonal agent. The lead compound, however, in this series was 2-nitro­imidazo le , mi sonidazo le, ex tensive ly in vestigated clinica ll y as radi osenti zie r. The neuro logical toxic ity of mi sonidazo le is the majo r limitati on in its applicati on !} . It has been shown that the neurotox ic property is due to lipophilic characte r. In an effort to reduce thi s side effec t, less lipophili c 2-nitro imidazo les , such as desmethylmi sonidazole and SR-2508 were systhes ized which showed less neurotox ic ity than mi sonidazole in keeping with the ir lower lipophilic ity. A Roche compound R003- 8799 which has a bas ic side chain has been found to be superi or to mi sonidazole with improved tumour uptake. The improved uptake has been attributed to the ac id-base properti es o f the piperidine group in the s ide chain structure. During the development of the chemosensiti zers, numerou s compounds have been synthes ized which were substanti a lly more effic ient in vitro than would be predicted on the bas is of the e lectron-affinity re lati o nship . Thi s is parti cul arl y true of some compounds that react effic iently with intrace lluar g lutathi one". Most of these compounds show little , if any, acti vity in vivo. M ore recentl y, derivatives of mjsonidazole which contain in a side chain a mo nofuncti onal a lkylating groups, such as az iridine, have been prepared . A lead compound in this series is RSU 1069, chemicall y 1-(2-nitro-l­imidazo lyl )-3-( l-aziridinyl )-2-propanoI

14. It has been

found to be more efficie nt radi osensitizer than

OH I

iH2CHCH2-N:j

()(NO, N

RSU 1069 RB 6 145

mj sonidazole, both in vitro and in vivo. However, gastrointestinal toxic ity limits the dose than can be admini stered clinica ll y to levels that woul d be expected to provide a benefit in cancer therapy. RB 6 145 is a prod rug o f RS U I 069 that has lower tox ic ily with simil ar potency .

Binding of Nitroimidazoles to Hypoxic Cells - Use in Detection of Cancer

Nitro imidazoles are reduced under hypoxic conditions to intermedi ates that bind ce llul ar macromolecules . Thi s has been ex plo ited in the detection of tumour hypox ia ls . Earl y studies ut ili zed 14C-labe lled mi sonidazo le and autoradi ography, and served to demonstrate that hypox ia does occur in patient 's tumours. Another approach has been to use nitroimidazole derivati ves that can be detected immunohi stochemicall / 6

. F luorinated nitro imdazo les have been synthes ized for use in non-in vas ive de tection of hypox ia.

Chemosensitization

Nitroimidazo les potentiate some chemo-therapeutic agents, parti cul arly a lky lating agents . Hypox ia is a prerequi s ite fo r chemosensit ization ; which g ives it some spec ific ity for tumours. T hree mechani sms, which have been sugges ted to account for the chemosensitizati on and observed experimenta ll y are deple ti on of intrace llula r thio ls: modification of pharmacokinetics, and increased cross-linking of DNA .

Antifertility Potential of Nitroimidazoles

Ornidazo le, 1-(3 -c hl o ro-2-h ydroxy) propy l-2-methyl-5-nitroimidazole has been found to be an effective antifertility agent in male rats onl yl 7 It ac ts rapidl y and revers ibl y to decrease the motil ity of distal epididymal spermatozoa rather than on maj or epididymal secretions. IS It does not prevent access of spermatozoa to eggs in the oviduct while inhibiting fertilization l9, and reduces the acti vities of two glycolytic enzymes of epididymal spermatozoa without reducing the ability of sperms to undergo capactation and acrosomal loss ill vitro.2o Ornidazo le is used clinically for the treatment of genita l tract infections in both women and men and thus ha some potentia l as a bas is for human contracepti ve agents. In contrast, metronidazole is reported to take 6wk to reduce spermatogenes is and render male rats infert ile,

I f . ? I

as a resu t 0 azoosperml a- .

664 1 SCII ND RES VOL 62 .I UL Y 2003

So me obsevati ons with regard to structure acti vity re lati onship fo r anti fe rtil ity potenti a l have been drawn n Of the th ree funct ional groups attached to the imidazo le ring of ornidazo le, viz.. methyl, nitro , and (chl orohydroxy) propy l, the three-carbon side cha in at pos il ion I is essent ial fo r anti fe rtility acti on. T he compounds that lac k thi s s ide cha in , such as 2-methy l-5 -ni iroimidazoie or those hav ing side chains of one carbon, suc h as dimetridazo le or of two carbons with either a hyd roxy l group, such as metroni dazo le or chl orine, such as 1-(2-chloroethyl )-2- lllethy l-S-nitro il1lidazo le are all in acti ve. Even when the side c hain has three-carbon, suc h as ornidazo le, but lacks chl oro and hydroxy functi ons, such as 1-(2,3-epox y )pro py 1-2-methyl-5-n i tro i l1lidazole or whi ch has a longer s ide c hain, such as tinidazo le has no effect on fe rtili ty .

Antitubercular Profile of Certain Nitmimidazopyrans

A se ri es o r hi cyc lic ni troimdazofurans, o ri gina lly in vestigated as radi osensiti zers fo r ll se in cancer chemotherap/.1, were found to possess ac ti vity aga inst cultured repli cating Mycobacterium flIben:lI/osis (MTB) and had s ignificant ill vivo ac ti vi ty in a mu rine in fec ti on mode l"4-26. T he lead compollnd in thi s se ri es, CGI-· I 734 ) was mutagenic, d iscourag ing furthe r in vesti ga ti on of the antibacte ri a l acti vity of the compound se ri es26 T hese studies however, suggested, that the bi cyc ii c nitro imidazo les might be po tenti al ant itubercular agents. A se ri es of over three hundred ~-su bs t i tuted ni tro imi dazopyrans (NAPs) were synthes ized on the bas is o f the structure of CGI- ) 734 I 27 Over 100 o f these compound s possessed subs tantial anti tubercul ar act ivity, matching or exceed ing th at ofCGI- I 734 I.

T he ac ti ve compounds lacked the mutagenic ity. Structure act ivi ty re lationship studi es foc using on an titube rcul a r activ ity revea led substantia l va ri ety in the tol e rated substi tue nt s at C3, but optimal activity was ach ieved with lipophilic groups . The stereoche mi stry at C3 was important fo r activi ty, as

CGI- ! 734 1 PA- 824

the S-enantiomers we re generall y at least la-fo ld more acti ve than the R-e nanti omers, NAP acti vity was found to be highly specifi c for the MTB complex, and showed only modest or no act IvIty against mycobacte ria outs ide the MTB complex (M. avium, M. megmatis, M.che lonae alld M . jortll itum)27

Over 50 NAP compounds with demonstrated acti vity aga inst cultured MTB were :'creened for ill vivo antitubercular activity, using a short-te rm murine mode l of infection with an MTB reporte r stra in expressing firefl y luc ife rase (rMTB-Lux). A lthough PA-824 was not the most potent N AP against cultured MTB c linica l iso lates, it was found to be mos t acti ve in infected mice when ora ll y ad min iste red at 25 mg/kg . Thi s indicated that PA-824 mig ht possess more des irable pharmacokine ti c properties than the other more potent NAP compounds that were tes ted.

The acti vity o f PA-824 aga inst both mult id rug­res is tant and non-repli cating mi croaerophi I ic MTB sugges ted that the NAPs ac t by a new mechani sm. S tudies on the macromo lecul a r e ffec ts of PA ·824 on MTB revealed that both prote in and li p id sy nthes is were substanti a lly inhibi ted at drug concentrati o ns and time inte rva ls when nuc le ic ac id sy nthes is was unaffected . Lipid synthes is was not e ffected by treatment with amikac in , an aminoglycoside pro te in synthes is inhibitor, ind icating that the e ffec t on li p id synthes is is spec ific and not a conseq ue nce prote in synthes is inhibition in MTB . As de mo nstrated by PA-824, culture and mode ls of anima l in fe t ion the NAPs possess bac te ri c ida l ac tivity against MTB comparabl e to that o f isoni azid at doses tha t are ,afe in mice and guinea pi g. PA-824 and the NA Ps are a lso small , ora lly acti ve mo lecul es that shouid be amenab le to iarge-sca le produc tion, making them prac ti cal a nd att rac tive candidates fo r the c linical deve lopment of a new antitubercul ar the rapy. In add iti on, activi ty aga inst muti drug-res isitant tubercul os is and demonstrab le acti vity by PA-824 against sta ti c MTB could offe r signifi cant potenti a! for reduc in g the durat ion of the rapy over current antitubercul ar

I . · "7 the rapies that target on y grow Ing organ ls lll s- .

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KAPOOR el ai.: MEDICINAL SIGNIFICANCE OF NITROIMIDAZOLES 665

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