FIILD OJlTHI INVINTION .
This inveotion ~lales to tbe proce.a for the ~isbeazimidazoles 884 its
deri• ·wM ~ U...1 A JA 0 0V :-A. 0~-;:; .
BACKGROUND OPTBE JNVJNTION
The DNA ligands such as-bisbenzimidazolea Hoecbat 33342 .ad Hoecbst 33258, fonn
strong and non-covalftlt linlaJges, with th~ adonine and thymine rich ~gions in !be minor
groove of DNA, sisPifiealy lllterius 1he cbromatin Btructtn (Exp. Cell Res. (1973),
81,474-477; Biochemistry (1990), 29,9029·9039). The dyes Hoechst 33342 sod Hoechlt
33258 are frt-quently used in cytometry to stsin chromosomefl insitn. [J. Hit..-toehf.ra
Cytoch~;,"~m(1985).:B.333.338J. '1bclie two bi.s-b~nriruidarole .::om;x:xmd'-' b~><:<>ill~ brjf.!N,l~
ilnores<:ent \\rhen ilicy hind to DNA For s long time, it hs.s hern kno\VY! th~t Hot';~'hsi
::rn:,g hincitv. Bf.H ciilt'fl!ty to ATrich t;equenct·B in llNl\. [niachim. Bir.•r·hYL ."-ctlf EF81.
~19, J58-J68; J. Mnl Bioi. 1987. 197, 257; B;m:hmni~1ry l~Xn, 3e. le2-}>?')
BiochomiHtry 1991, 30, 10294-HH06; EMlJO J. 1 ~~n, 11, 215 .. 231]. J\ ,,ra;·i~3::y of ·l~?t.l~
:•wl X··r~y 'r/HhJl u~nwtureo of HoedJHI Ti/~JH hmmd to differe.a; .,-,H,i;t'n:u::le~:dd~:·
u.L .., ,,J;j ,.'.)...' .-,._•,·~· h:~· l·.p..; , l~i-'''-""'''•1 £, ,.. .. _.~. _,..~,! · .•L1,\!~".", ,la f~. I'>h.qlr·- ~'.;'J."__". '.. !,'.., •l... . ,•_ :t·rAv (l. .(;;Icf }.... !·,\ ·~·t"f-?' t 'ft.v>;l~iQ ..' j'!". c~-;~,·---h~- J.t .•.~'.':.
holi.x, cuvtwin.g a ntn of fhw- coutiguous AT h~uw pa.UK 'l)K; J:io;;:;. lrei :U 158 })}\.!/
interaction l:l.ppear to be st.abilu;ed by eeveral H-honding and vm ck-r Wtutla cout:Bct!'l
[Structure 1. 177) to ia filet these molecular forces are believed to cootrilde to overall
binding affinity {J. Mol. Bioi (1997) 271, 244-2S7]. The hydrophobic transfer oflipnd
from solution on to its DNA binding sites is more likely to represent the main driving
force for the complex formation [J. Mol. Bioi. (1997) 271. 244-257]. Administratioo of
these compooods prior to irradiation afford protection 888inst the formation of primal)'
lesioDB in the aqueous solutions ofDNA aB well as in the intact cell nucl~us. Theos" DNA
ligands havt'· also be-MJ obsf'J'Ved to rt"duce tht> radiation- induced cytogt"fft>tic d81Jt88t'
••
-2-
and cell death in cell culCures, u well as in whole body irradiated animals [lnd J. Exp.
Bioi (1998) 36, 37.5-384; Br. J. Cancer (1989) 60, 715-721]. However, post-irradiation
treabnent of cells with these ligands bas been obserwd to eobmce cell death in vitro
[IndJ.F.xp.Bioi(1998)36,37S-384]. fue radical scavenging and quenching o DNA
radicals appear to be the mechanisms responsible for protection by Hoechst compounds
administered prior to irradiation [Int. J. Radiation Oncot (1992) 23, .579-.586;
Radioprotection (1997) 32,Cl-89], but its role in eoltan4iing the radiation- induced cell
death when administered after imldiation is not clearly uoderstood
The limitations of these minor groove binding ligands s beiJ18 mutasenic. clstpgffiic ROd
gono I:'X}>il'hHiou alwraimn and rep.Ur inbbititm preve-nt tiF>lH :hom h~iu.~ ur;Hl jn hlc:;:,<'.iJl~.
Htffefonc. the- de·velop!ltf"1J1 (){ DNA bittding hgB-nds (Minm .. Gn1ovr. Hindin.g Lw&it.h.
p~i:rticuhu ly) 1lurt rJlot d )i ~diopH>im.trvt' e:HJ:d v:ithout 1-'iguit'k~.• n t ~mrt.a.gu~~.c]l)· and
;;yiotu);ir dJ£•rtf' c~:m p!ny ~~ flignihnml rdr· m t®lc~i~:ul rn.di&ion pwh•r:tio.>t
~~ctivity.
Yet another object of tbiFJ iuvcu1ion ill to t;yuthesiu, ben1.imidn.zolefl DNA hgnnds \vhicl!
can afford radioprotective effect without signifiQIDt JDUtagwncity and cytotoxic effectB.
••
-3-
DETAILED DESCRIPTION OJi' THE INV.H~IION
~j_
According to this inveotio.n there is provided a process fot the ~esir of
b ..... __ ..... deri .gg ..
eozuntwu.u es . vatfiNI compnstng:
(i) chloroaoiJin~ with zinc dust and acetic anhydride to produc~ 5
(ii) reacting with HN~ to produce 2-nitro-.5-
(iii) adding sodimn me oxide to 2-nitro-5-chloroaniline;
(iv) heatins 2-nitro-5-c oaniline. methyl pipt:t1l.zine, Hnhydrouot< K;t(-:0~ 1md
Dimethyl fonnamide l00-120°C p1oduc1:~ ~. :tui,JiiFt} vdti.c:b \F t ;y,\~·5 bv
polrins ice snd is ' .,
~ / ..
nih onniline;
(v) fn~gtiug 5 ( 4 · mt~tilylpijJet?Ll 1 'y})· L · uih o:~JiJim< \Vitti ?dJC t,:· ;H
.1.~''(4'' ,, .... ,. Ft.· 1
• • -. ·_ ····n,.,'-~t'~j!·r'~r:A,-~~ 1·•!i-·'. : 'ii •'" ,_,.,L.J!.)Io ... i'"'.cc~ :·.
(viii) heatin,cj 2~amino-4-[ .S '-{4"-Methylpip -I" -yl)beozimidszol-2 '-yl]aniliue
and 3-4-dimetboxy bmzaldehyde using 'trobeuzene as a solv~nt at 110-
1.500C to produce (DMA) I.e .5-(4 methylpiperazin-1-yl)-2-[2'-(3.4-
dimethoxypbenyl )· 5 '-benzimidazolyl] beozimi
(ix) beating 2-amino-4-[5'-(4''-Metbylpiperazin-1"- ) benzimidazol-2'-yl] aniline
and 5-Formyl-[3-methoxy-4-hydroxy beorimi le' ] usmg nitroben.r.ene
ot llOOC to 1500(' in Pff.SNlCf' of argon to produce (TBZ) i.t" 'li-{4-
••
-4-
. -1-yl)-2-{2' {2 .. -(4-hydroxyOXVI[)
I)e.tii\'))Y'benzimidazolyl} -Y- benzimidazolyl) bf'tlZimidazoJe.
S-Cyaoo-2 [3-methoxy-4-
Diamino beozonitrile
xy beuzimidazole] Y -yl i1 produced by reading 3,4-
J-metboxy-4-hydroxy benzaldehyde. 5-Cyano-2 [Jtreated
with Raney nickel w
presence offonnic acid to produce ~-Formyl-[3-metboxy-4-hydroxy benzimidazole]
~IBRWU:~WJ~al.-~~~~~~IZIIIudlllZOies hiiVin,g bi1nlbtrtituted pl:wnyl
pharmacoJogic~d ;,;c:tivity of
5. (4~m~th)-'\pipt>,.rat:m 1-:yl) · 2~ p·. ~.(3,.4· ·dimllttmJ:yptu~n.rl)-5' ··h~nrimhia;.~~"bi}
(UMA)
(flJ1... . ) wa<; cornpared tn thm of Hoechm 33.142. "lhe 1wo COIDfH.mndwrv::~l;c;n ~h~~t Ho!'.ch£i 3B4) in more cyioto~ic than Hocchnt ·r~:':>: h"1 th:· ');~"'11
phmJOhr !~} oup ,If Hoochr;t .B258 II> b£~1n.~ repkrcd by nthoJ:y group i;1 ~)ocd~J;l :~.~ ;·
K.''"'Pillg uhovt' obr'l'fV?!liom~ in mwd, wt~ JuJ>vo intnrdnc~d ~wo nJt'4ho;;y ;~;nmp "'· ('+
hif1ht':n:,jmichwk : :td o:w: mc:ff•mty nud r.Ht~ hyd!·nxyl Ji;JC!Up in i:dHhrwi~:·:,J~;;Inlt; 'i i<
:ynllwr.in ofbi:,b,:m.Jmith~zo}C" ..,..,~:; n:;>rir.d out b~:;iu:.Jly a.r;: dt\f;nibod by J:.elh' ,< rd 0 9><)
Ufllng 2 · Arnino·A · f ~'/ ·( 4 (( ~J\{eiliylpipcr1i.zin .. J c• ·Yl }bc.nzimidnwJ~2' .. yJ)anilinc nnd :l/ ·
dim~thoxy Vt.11J.Wld..•hyoo in 30% yield. This nwthodology is diJf6rent from the weiliod
described by Lown et aJ. where they have condensed 2-arylbenzimidazo)e with oorylenediamine
. lbe terbeomnidazole was prepwed usilt8 a methodology desa-ibed by
Edmond J. La Voie (1995) using an equimolar mixture of 2-Amino-4-{5'-(4"MetbyJpiperazin-
1"-yJ)benzimidazol-2'-yl)aniJine and 5-Fonnyl-(3-methoxy-4-hydroxy
benzimidazole] m nitrobenzene at 140-150°C for 36 h in 25% vield. In this reaction the
initially formed schiffbase lUldergoes oxidative cyclization by nitTobenzene to give
I
•
-5-
~rbeozimidazole. Howewr. we inlroduced two i.mpommt modificatiODB m
bisbem.imidazole and tetbenzimidazole. This is the finrt report of synthesis of
terbeozimidazole having bisubltitutioo oo the pbeoyl fins. In the biabeazimidazole the
two methoxy JUoupB are introduced wbidJ are eledron donating whereas in the
terbenzimidazole one metboiy 8fOUP was replaced by hydroxyl BfOUP to observe the
stability ofDNA-drug complex using hydroxyl group capable ofhydrogen hooding.
Both of the above JDelltiontd compounds ~ non-cytotoxic evtn at 1 OOJIM
concentration even upto 72 hmn after treabneot in bonum glioma cell line BMG-1. Cell
survival assay showed that 5-(4-methylpiperazin-I-yJ)-2-[2'-(J,4-dimethoxyphenyJ)-Y~
benz.imida.zolyl] betzzimid111..ok (DMJ..) lut(j 73% edJ ffiJtvivnl ru lOOJ.tM concentrntion
\Vhen~a.s5--{ 4-methylpipernzin··l-yl )-· 2 ··12' { 2" ··( 4 ··hydroxy-3m~thox.ypheuyl)
)"hewin.ud~7nlyl.}-5'·henzitnidazoiyl] hen1jmidezole (IRZ) n1 lower coocerdtlliio.n 1 e.,
l Oj.LM hw; sbawu increHBe in growth whereBB at l OOJlM showed 92% cell survival. These
results an tBrther supported by IJl)wth kinetics.
Under the 88!Tle conditions, the cells were imldiated witb 20y. 5Gy and lOGy of
:n.lfliniion r.:ad phy~i(.·o-chemical stnclies like UV-Vis r.pectroscopy, floon~cence
Objective of t.he lnV<':ntion
The cytotoxic effect of DMA and lBZ on exponentially growing tumor cells BM0-1 was
studi&d 81 a fimction of time. Cells were treated foc 1 b with the ligands and allowed to
grow for 24, 48 and 72 hours. l1te MTT assay was perfom1ed at the specified time points.
It was observed that Hoechst 33342 was highly cytotoxic at l O,..M conc&ntration whereas
DMA and TBZ do not show any cytotoxicity t-ven at 1 oo~tM concentration. There
iieemed to be no effect on dte metabolic tlt.atlli! of the treated cellt~ However. strikingly
•
-6-
the uwtabolic activity of the Cmlted cells see~d to be eobmced as coOJpan}d to the
Ulltrested coolrol cells. To fur1her support our results we have pedOI"'Ded celliUIVivaJ
8118.)' at O.luM, IOuM amd IOOuM cooceotndioo of drus having oootrolas uolreated cell.
These ~ werv done dlrice in six setB • ~ally growing cells were usod in
1bese experimeots and cell survival W88 studied uams Macrocolony assay. The platins
efficiency was nearly 7~'- The parent CGIDpOl8ld Hoechst 11142 showed A significant
effect on cell survival At )ow coocentnltiODB, Hoechst 33342 do not seem to havl'
conaiderable e1fect on cell survival whereas at 1 OJ!M cooceotratioo the IDI'Viving ftadioo
reached remained 4.5% and at IOOJ!M only 10% of cells survived DMA at low
cotlct-.ntmtion (O.l,lpM) did not show n.oy dfef'l on m11vivn.l hut &t JOpM there i~ R dir);t
( 3%) deen;nr;c in survivio,g fr'f¥:iion H.nd Hr J OOp.M, Tio/e of the t..•ells m.JJvived TB? J:wfi "H
~tign.ifieMHy differt::xlf hduwiar fflHl the two lip;R:H.i£. Ai low concentrntionH (upto H!Jlivfl
htdc (9'%) t.bntmr:.:l\ in mxrv)vju,g J.iachon. 'ftw ,;.iii•d ofDMA and TBZ on p.rolrlhahnn .. ,~·
o:ponentis~lly f10'tNing cdh; nt l OGJ 1M -:·onrentra!ion we;s Hlso Hturlied The r;r c·.nltbyn:
n.m DNA V\-'U8 t;hoslm us the· DNA rmrleritd ruH.I liglmds wru·f! inculmtE, U!)
l~umple 7:
l-AmiDo-4-15'-(4 "-Methylpiperadu-1 "-yl)bell&imida&ol-l' -yl]aDitiue :
A solution of 4-[5'-(4"-metbylpiperazio-1"-yl)benzimidazol-2'-yl]-2-nitroaniline {1.04 g
,30nunol) in ethyl acetate/methanol (80 ml of 2:1 mixture) was treated with 5%
palladiwn on carbon (250 rug) and hydrogenated at room temperature and atmospheric
••
-10-
preBSUI"e. When hydrogen uptake bas ceased • the solution was filtered (celite) and
c~ed without delay to afford tbe orange-brown colored diamine .
Example 8:
!§-.Cyo&-2 (3-metllaxy-•-lryboxy lten,..,..uole) ~'-yl : A solution of 1.224 8 (9.2
DDDOI) ofpnwious)y obtained 3.4-Dimnino benzonitrile • 1.4 g (9.2mmol) ofl-methoxy-
4-hydroxy beozaldehyde in oitrobeozene are taken in a three necked ro1md bottom flask
under nitrogen and healed at 140° C. The reaction mixture is heated for 18 hours with
Btirrin& nitrob~e is 1hen remov~ mder reduced preSBUre to obtain 1he brown
colored crude product (14). The final product is then obtained through silica gel (60-120
mesh Ei7..e) column chromatogrnphy nsing EtOAc I Mt-OH as eluent mp. 226-230° C; m
:1:425.3 (O·.Jl), :l2<>3.3 (-NH). 2221.8 (nCN), l/.7&.7 (C>O-C) cm-1
;
1H NMR o H . .'5 {H
lH, ~'H), 9.:'52. ( E, lH, ·OH),7.85 (cl, HI, C7). 7}J2 (cl, 1H, C6), 7.72 (fi, Hl CY),7.ri3
(dJH,C'.'S'). 6.93 (1!, jH, C6'), 3.90 (s,3H, OCH:s)
Kutmp!e ~:
by the d.ropwisf~ uddition of 2 N NrtOH. Th~· pntduct Wflfl ohbiined hy tx1nK1jfln •,v;th
ethyl acetate. 1be ethyl a.cetai.e eJdrnct was dried (N~SO~) ood conce:ntrnted in vacuo to
give the yellow color-ed compound in 30% yield. m p. 258 -260°C ~ IR 3435.6 ( -OH)
,3194.55 (NH} , 1675.6 (CHO), 1594, 1506, 1441.6, 1280.7cm .t ; 1H NMR ~ 13.7 (b,
lH. -NH), 10.2 (S,lH. -CHO) ,9.5 (s.1H. -OH), 8.25 (d.lH. C6), 7.78 (s,lH. C4), 7.75
(d.1H. C7), 7.7 (s, lH. CJ'), 7.65 (d,1H. C5'), 6.92 (d.lH. C6'), 3.92 (s,3H. -OCIL)
Hxampl~ 10:
~-<•-mtt.hylplperadn-1-yl)-1-(1' -(3,-'-cUmethosyphtnyl)-~· -btulmltluolyl)
bendmidnole : A solution offre~>hly prepared 2-Amino-4-(5'-(4"-Methylpiperazin-1 "-
•
-11-
yJ)benz.imidazoJ-2'-yl]aniline (1.18g.3.67mmol) and 3,4-dimethoxy benzaldehyde ( 0.6lg
,3.67uunol) in ni~ (110 ml) is heated at 140-1Scf C for 24 h. The solv• is then
removed uoder reduced pre88UI'e to give the final crude product as a brown colored eolid.
The product is purified by column chromatography on BUCID 688 Liquid (MPLC)
Pump using silica 3\'l (70-230 mnb size) and EtOAc/ M\'OH as eluent to Jliv\' a yellow
colored compoUDd aod characterized by spectroscopic techniques .m. p. 220°C Yield :
30%; m 3556, 2922, 1629. 1508, 1417, 1371, 1296, 1022, 810cm"1
;
1H NMR
(DMSO~) ~ 2.24 (a,3~CH3), 2.46 (t,4H,~)=4Hz). 3.5 (t,4H,~, J=4Hz), 3.86
(s,3}L0Clh), 6. 77 (d,lll,Ar-h)=8Hz), 7.13 (d,2ILAr-IL J= 9Hz), 7.64 (d,liLArH)=-
·K5Hz ), 7.78 (d,1H,.Ar·H,J~4l Hz), 8.03 (rnJH,Ar·-H),8.17(d,2H.Ar-b, J::-9Jh:), 8.3
(sJH,.Ar·H), U.O (hs,2H,NU) .D10 excllP.... nge reF-ulted in the di~appeenmct>: oftl~,, p~a.¥ 0
S 13.0.; EIMS ; 46'7 (.1\f), 42.\411. 261, 23.\ 220. !94, 118, 91, 5~. 44
Y:u.mpk 11 :
5-(<-mdh-yq~.tptra.a:i.n-1- yt)Z· '1' {2"- (4 -b,·dr(];J.)'· 3mdbfl.'T:yf.ht:n)·l)5' 'henrlmidur.l)I;
~'- ht mlru.ldu:nlJl] b~rti!"lm.tc:hu~olt : J-t roluiion nf fn,ghJy prepHred 2-Amino-4-f ~· •>+' c ·•
Melhylpipt1'RZin·,J .. -.Yl)h(~m.imidH.:·o1-2'-yJ]enilh: (}JH m.g OH/.rumol) m~:J "~I'c1rmyj..JJ ..
3435,3195,1632,1560, 1413,12.tH; 1HNMR & 13.4-·13.55(b,3H), 9.5 (s, HI), 8.4) (c,
IH ), 8.39 (s, IH), 8.03 - 8.08 ( m,4H), 7.9 (d,lH), 7.68 (d,lH), 7.38 (d,lH), 6.91-6.98
(m,JH), 3.92 (s, JH), 3.23 (t,4H), 2.68 (t, 4H), 2.60 (s,JH) The mus of TBZ was
observt'd on MAI.DI : 570.3
Cytotoxicity Assay
The cytotoxicity was detennined using the M1T microtiter plate tetrazolium
cytotoxicity assay (MTA). The ln.mao brain malignant glioma c_,lJiine BMG-1 was us~d.
lbt> cytotoxicity asBay wu performed using 96-wt>JI microliter plates. 3000 ceJJs/ well
•
-12-
were plated and ~ of die lipods was done 24 hourB post plating. For
dete:nninatioa of IClO • ceJJs weft exposed continuously wi1h varying concentration of
drug and M1T 8lllllJI were performed at the end of fourth day. To the oootrol and treated
cells • 20 f1l of 5mglml MIT in PBS was added. inalbated for 2 hours at l'fC and then
1he medimn was removed 150 Jl1 ofDMSO was added and 1he plate wu read st 540 nm
wi1h refer-once at 630 on .
Maao~oiODJ Assay
Cells were washed wi1h HBSS and harvested usin,g 0.0.5% trypain. I>ependins on
the treatment&, 200-1200 cells were plated in 90-nun Petri dishes and incubated at 3"fC
in a 5% C(h Jnmidified atmo~ for 8-10 days. Colonies \Wre fixed in methanol and
f!tBined with 1% crystal violet Colonies contruning more than 50 <~dJs were co'IIDtt:>d ru;
liliown in Tnble 1&.2.
their prolifemt.io-n kinetics wnH mee.s-r1red af 1.4-h in.terv~d~ by trypsiruzinig end co1mting
tots1 cells pt"S flnfk nsins R bemnt:ytomrfet·
Table 1
Thermal Denaturation atudiel of ligand-DNA complex in pre~ence and
absence of radiation
r-· ---··· ----- ----- .. --~-----------· -·- ----------- ------------ ---------- --- Rru.io Dose (Gy) --:1);(llc) -- --
Ligand [hgand]J{DNAJ
----------- ----- 1----------- --- -- -------------------- 1------·----·--
0 74.3
--· --
0.01 100 75
TBZ 0 77.7
0.1 100 81.5
0 71.5
0.01 100 67.5
Hoechst 33258 I 0 81.7
_l2l__ 100 79.3
1
•
-13-
TabJel
CeO Sarrival Assay ill BMG-1 cells wida aad without irraatiea
lliabnmt % SlnivaJ fuwtion
Coatrol 67 1
DMA 1 6.5 0.97
D 1 61 1
64 0.9.5
73 1.07
71 l.OS
69 1.02
line. T}mur)l lllt: hvo f~dl !int·s, Hd~ t.md BMO·l 1:dJ Jint rue no! nrernonable to dcmmmtnue the invitro cfie~,..i of th>.>:.w IIgu.ndu on
different ce II I ines.
The results with the hlw) cell lines are not significantly different. but it can be
concluded that both DMA and mz are better radioprotectors in comparison to Hoechst
33342
•
-14-
Below presented are the result& of tho growth kinetics and Macrocolony assay
performed with the two cell lines.
Proudare:
Macrocolo.ll)' Anay (CeD SurviYal Anay)
Both cell lines in triplicate were washed with HBSS, plated and grown for four
days before harvestiu& using 0.05% trypsin. Inpmding on the treatmentB, 200 tol200
cells were plated in 90 DDil petri dishes BOd incubated at Jfc in 5% C~ humidified
atmosphere for 8 to10 days. Colonies were fixed with methanol and stained with 1 %
crystal violet. Colonies containing more than 50 ceJls were counted
Proli:fteration Kinetin
Both the ceJJ lines in triplicstes were Heeded at 7000 to 8000 cel1sl n:Ji, snd their
proliferation kinetics was studied at 24 b intervals foHowins trypsiniz.ation and cmmting
total cells per flask .mas abaemocytometer.
Irradiatiea Proce.t.H:
Exponentially growing cells, 24 h after plating were imldialed at room
tt'mpe ro ! ,'). -gHPUO- ··_',·' -~'. ~r , -;. .. ; . -
T\~dllylpij•~'S"i:'ij!l. 1"-yl)ben?imidi~:r.ol· :1 ' y1]H.uilinc;
lttFltnr., 2· NuinoM1·f 5 '-(t~" .. Mcillylpipern..-r.in··l "-yl )l>cn7jwid.1i:dli· ? ' .. yJ]H:t.i lin:
u.nd .3--4-dimotJJOX.J {)l-'m:aloobyde using uitrobcozone ~lS a ~>olvont at ll0-
l500C to produce (DMA) 1.e 5-(4-metbylpiperazin-l-yl)-2-[2'-(3,4-
.dimethoxypheoyl )· 5 • -beozimidazolyl] benzimidazole;
(ix) beating 2-.uiuo-4-(5,-(4••-MethytpipeniZin-l .. -,1) bemimidazol-2'-yl] aniline
and 5-Formyl-[3-medwxy-4-hydroxy btmrimidazole] usin3 nitrobenzene at
llOOC to 1500C in presence of argon to produce (1BZ) i.e 5-(4-
metblypiperazine-1-yl)-2-[2' {2"-(4-hydroxy-
3methoxypheoyl)5 .. benzimidazolyl} --5 ·- beozimidazolyl] benzimidazole.
L-K 5-(4- methylpipCI'BZin-1-yl)-2-(2'-2"·(4-hydroxy-3-methoxyphenyl)-Y,
'
benzimidazolyl) -5 • -beazimidazolyl] benzimidazole, 113Z as produced by the
process 88 claimed in chum 1.
3 ¥· 2-Nitro-5-chloroaniline as produced by the process as claimed in claim I
-20-
t..t l'A. 5-(4'- methylpiperazin-1-yl)-2-nitroaoiline as produced by tho procoss as claim~d
in claim I.
0 Jt1: 2-Amino-4-(4"-methylpiperazin-1'-yl) ooiline 88 produced by the process 88
claimed in claim 1.
6 J.8:' 4-Amino-J-nitrobenzonitrile 88 produced by the process 88 claimed in claim 8.
{ ~- Ethyi-4-Amino-3-nitrobenzenecarboximidate hydrochloride 88 produced by the
process as claimed in claim 7.
f!2 1:0. 4-[5'-(4''-metbylpiperazin-r'-yl) benzimidazol-2'-yl]-2-nitroaoiline u produced
by the process as claimed in claim 1.
Cf ~ 2-·Ami.rJo·-4--[5' -(4,-Mf!thylpiperozin-l.,··yl)benzimidaY...o!-2 '·yl]muline ~-if' produc~:Jd
by d1E1 prucegs as daimf.'d in chum 1.
l o 'f#. 5··Cys:no·2(3-metboxy·4-hydroxy be.m..imidaz.ole]5'"yl as ptoch.wed by the proe:ettH
as claimed in claim 3.
t 1 ;,6. 5-Fonnyl13 mAaoxy-4-hydroxy benzimidazole] 88 produced by ile p1ocess 88
claimed in claim 2.