CA2139503A1 - Compounds for cancer imaging and therapy - Google Patents

Abstract

2139503 9426314 PCTABScor01 The present invention relates to compounds having affinity for certain cancer cells, e.g. lung carcinomas, colon carcinomas, renal carcinomas, malignant melanomas, gliomas, neuroblastomas and pheochromocytomas. The compounds also bind with high specificity to cell surface sigma receptors and can therefore be used for diagnostic imaging of any tissue having an abundance of cells with sigma receptors. The compounds are of general formula (I) wherein X
is a radionuclide; R2 is -N(R3)2 or a 5 to 6 member nitrogen containing heterocyclic ring, optionally substituted with at least one alkyl group; the remaining variables are defined on page 5 of the disclosure. Methods are provided for diagnostic imaging and for the detection and treatment of tumors containing the cancer cells described above.

Description

WO 94126314 213 9 ~ 0 3 PCT~IJS93/04309 ~ , AND TH~RAPY
~ ', 5 FIEI.D OF T~IE XNVENTICIN:
The presen~ invention reLa~es ko a ~las~ o~ -compounds ha~ing partlcular a~lnl~y ~or a speci1c ~ell ;~;
sur~ace receptor prs~alen~ on c~r~ain cance~ c311~
lung carcinam s, malignant melan~ma~, gllomas, I.
1~ neu~oblas~omas, pheochxomocy~omas, colan ca~cinom~s, ,~
renal carcinomas an~ the iiXe. X~ pa~ 3~- ~ne ~. .
presen~ inventl~n provides such compoun~s as ag~nts o~ ;~
det~ct~n~ 2nd tra~ting ~umo~s, pa~icula:rly tum~s ;`;~
havlng cancer cells whlch pos~ess a cell sur~ace sigma 15 receptor.

BACKGROUND OF TME INV~NTION: `' Lung carcinomas, malignant melanom~, gliomas, neuroblastomas, pheochromocy~omas, colon carcinomas and 2^ ra.lal ca~ .2 ~ 2~ 5 si~ erm~ o~ canc~r, th~
early detection an~ ~rea~men~ o~ whi~h ~ o~ ~a---.o~
importance. If le~t und~,~ec~ed or u~rsate~ for several .
years or ev~n months th~ media~ r~ ~al t lm~ a~
patients having these kypes of cancers ls dxamatically 25 reduced.
Qf these cancers, lung cancer has lead to ~he :~
highest number of fatalitLes. In 1992 a}one, lung cancer caused about 165,000 deaths within the Uni~e~ ::
States. Two ~ajor types of lung ca.rcinomas are 3O responsible for most of ~hese deaths: small cell lung carcinoma (SCLC) and non~small cell lung carcinoma (NSCLC). ~:~
SCLC is a neuroendocrine tumor that secretes ~everal peptide growth factors including , ~

, SU8STlTUlE SHE~ :

W094/26314 ~ ~3 9 ~ o 3 2 PC'I'~JS93/0430s 1 bombesln/gas~rin releasing pep~ide (BN/GRP). SCLC ls responsive to chemotherapy and radiation therapy, but relapse occuxs frequently, and the median survival kime is only about one year.
NSCLC accounts for ab~ut 75~ of ~11 lung cancex cases and encompasse~ a ~ariet~ carciriomas including adenocarcinoma~, larg~ cell carcinomas and squamous ~ell c~rcLno~i~s. N~C~C kumors secre~e ,;
transforming growth factor-~lpha (~GF-a) to stimulate 1;:
10 canc~r c~ll prolifexatio~, NSC~C ~g g~nexaLly ~reat~id ~.
c~,sme~h~r~ n~ ~urglcaL rssec-,ion. r2owe~rer the m~dl~n sur~iv~l ~lme ~r pa~len~ wl~h NSCLC i~ only .::
about S yea~s.
Mslan~mas axe amon~ the mos~ seriQus manifestations o~ skin cancer and lead to a greater number of fatalities than any oth0r ~orm of skLn cancer. ;
Melanomas can mekastasize th~ough ~hs lymphatic system . , .
~o regional nodes and then vla th~ bl~od to ~ec~ndary si~es on the skin or in khe live~, lungs and braln~ `:
~r,ere~s tne progno~is ror ~uper~icial sp~eading melanoms can he quite good, there is a much poorer prognosis ~o~ :~
nodular melanoma~ in whlch dlstan~ metasta~e~ ~requently ~arm.
Many lives could be saved if lung ca~cinomas, 25 melanomas, gliomas, neuroblas~omas, pheochromocytomas, colon carclnomas and rsnal carclnomas w9~e de~ect~d and treated at an early stage. Moreover many patients are reluctant to undergo radical ~urgical or broad spectrum chemotherapy pxocedures which are requently used to 30 treat such cancers ~ince khese procedures can cause disfiguration or disablement. Accordingly an ~ .
outstanding need exists for highly selective and non- ;
. i '~
: 35 ., SU~ ~ ', WO 9412~314 213 ~ 5 03 PCT~S93/04309 - 3 - :;
'' ';'".
1 ln~asive procedures p~rmi~king early detection and ~:
~rea~ment of cancer. ..
A variety of radiopharmaceuticals have been evaluated ~or d~agnos~ic lmaglng. Fox example, .;
5 Michelot, ~.M. e al. ~1991 J. Nucl. M~d. 32:1573-15~0; :~
Meyniel G. et, al. (1990 ~ 311~ 13~
18; and French Patent ~ubllcation No. 2,642,972 by .~ :
Morean e~ al, na~e disclosed ~Z~I and 1~5~t~
, . .. .
(diethylaminoe~h~ iodob~nzamLd~ B) fo~
~O imaging malignant mel~n~ma in humans. Unfoxtunatel~
the ~yn~he~ i3 probl~a~i~ an~ 3 signi~lcantly, IDA~ i5 k~ken up .in hlgh conc~ntrations by non~melanoma cells i~ th~ liver and lung. ;:;
Accordingly, IDAB doe~ not have optimal sp~clficity or 15 melanoma cells and i~s up~ake b~ n~n-tumor c~lls ~;
undermines its u~ility for ~outin~ ~cr~ning o~ cancer. .:
United St~tes Pa~en~ No. 4,279,887 to ~aldwin et al., United States Patent No. S,1S4j913 ~o ~e Paulls et al. and Murphy et al. ~1990 J. Med.~ 33:171-17 2~ d's~las~ -a 55~io~ 5~ 3 ~ 'g~
imaging ~ne Drain only, e.g. ~ N~3-pnenecnyi-o- :~
iodobenzamide or ~S)-N~ ethyl-2-pyrr~lidln~;l)ms~hyl]- ,:~
2-hydr~xy-3-iodo-6-methoxyb~n-a~ide ~IBZ~). H~WQ~I~r~
the structure and utili~y of the compounds disclosed by ~:~
25 Baldwin et al., De Paul;s e~ al. and Murphy et al. ls distinc~ from those provided herein. `
The present inventlon provides compounds which bind with h~gn speclIicity ana arrini~y ~o ~ne caii surface of cancer cells. These compounds bind, for 30 example, ko receptors on the cancer cell surface. One such receptor is a sigma receptor. Sigma receptors are known to be present on neural tissues and certain .

1:

1.

SUB 8H~

W094/26314 ~39~o3 PCT~S93/04309 ~

1 immortalized neuroblastoma and glioma cell lines (Walker ~:~
e~ al. 1990 Pharmacol. ~eviews 4~: 355~40a; and Villner et al. 1992 in ~
Mechanlsms for Neuromodula~ion and NeuroDrotection? ::
I
5 Kamenka et aL., eds. NPP ~ooks, pp. 341-353). However, , `~
it has been surpri~ingly ound by ~he presont inventors that sigma receptors are prevalen~ ~n som~ type~ ~f. ~;;.
canc~r cells, ~.g. nsu~ stom~, mqlanom~, gll~m~
ph~ochromocytoma, colon, r~nal and lung ca~clnoma c~Lls.
lO There~ore ~he comp~un~ o ~he prasen~ ln~ntlon are use~u; ~o~ d~_c~ing and w~ g ~mGrs, 4.g. t~10~3 can~aining cells with slgma r0c~ptors.
The present campoun~s are also ussul for diagnostic lmaglny any tlssue ha~ing a sigma r~ceptor, 15 e.g. a neural ~issue such as ~he b.~aln or spLnal cord.

S MMA~Y OF T~ INV'~NTION:
~ he present inv~ntion pro~id~s a me~hod ~ar diagnosing a mammal ~or the presenc0 o~ a mammallan :~ tumor wh~ch ~n~Ludes admlni~t~rlng t~ ~ m~mal ~'a-~..o~ ~L~ ~m,~gl.~ dmo~nt of ~ c~mpaund Ol. the pres~n~
invention, ~nd de~ectir1s binding of ~hs c~mpound to A
tumor i~ ~he mammal. Th~ campounds a f th~ p~ss~nt invention are of the general ~ormula I, ~.

Ra --~ (~2 j~--CZ~ Ctl2)y~l~2 Rb~ ~
~R~

'~`' SU8SriTl~E SHl~

4 213 9 ~ 0 3 PCTIUS93/04309 _5_ , whexe i n X is a raclionucLidP;
Z t s =O or two -H;
each ~l is ind~pendently H, halo, lowex alkyl 5 or lower alkaxy; -;.
R~ and ~b are independen~ly H, halo, lower alkyl, lower allcoxy or ~ nd Rb kog~her wlth the carbon ~;
a~oms ~o which ~hey ~re al~tach~d ~arm a c~rcloaïkenv~l or ~;
heterocylLc ~ing; ~: .
R2 is ~N~R,)2 or a 5 to 6 membered nlkrogen ~n~a~ h~ L~ rl~g ~hl~:h i ~s^~ t~
substitu~ed with at l~ast one alkyl substitu~n~;
~ach RJ ~S ind~Dend~ntly hydrog~n or low~r a lkyl;
j and ~r ea~h a~o indep~rlden~ly an ln~eger f~om 0 to ~;
q is an int~ger rom 0 to 2; ~nd i~
~'~h t;~e pr3v:so thaL ~he compoun~ ls noL a~ iod'ns radioisotope o (N-dleth~laminoethyl)-4~iodobenzamide. :.
~r The present invan~ion slso provlde~ a method f~ m.s~ ,m,~ m,r~-~ wh~ rh ~ n~ ~g : administering to a mammal a compo~i~lo~ including a tumor-inhi~l~inq amount o~ a compound o~ ~ormula ~.
The prssent inventlon ~urther pro~ides a 25 method for diagnostic lmaying o a mammali~n tissue which has cell surface sigma recep~ors which lncludes :.
administering to a mammal a dlagnostLG imaging amount o~
a compound o~ ~he pxesen~ invention and detecting an image o a tissua having an abundance o~ cslls with sigma receptorS,~
A further aspect of the present invention provides a method ~or in vitro detec~ion of a canc~r '';
:~ 35 ;~

SllB~ ~ ,","

WO94/26314 PCT~S93/04309 ~39~ 6~ .
, .
l cell in a mamm~lian tissue sample which includes contacting a mammalian tissue sample with an in vitro ,~:
diagnostic imaging amount o~ a compound of formula I for a time and unde~ conditions su~ficlent ~or blnding of , 5 the compound to the cancer cell and detectlng ~uch ~ndlng. ~: Ano~h~r aspect ~ ~he pre~n~ invention pro~dss ~ prs4~ mp~un~ f ~o~rr.u1 a ~, g . ~ . a compoun~ of any one o ~ormula~ X or XV.
Q
P.a ~ ~
~ ~ CZ ~ ~3~ CH2- R4 XI
~ ' .
~i )., ~ - (C~,)2-C~ (C:i2)2- ~, ;;I

(~
y :
Ra~
(CH2)J--C~ NR3--(C~23n~ )2 I~
~,:; Rb~
( R ~
wherein Z, Rn ~ Rb, Rl, ~, ; are as described ~bove;
Q is a radionuclide, halide or an activating ~roup; .~.
R4 is - NtR3)2~or an N-linked 5 to 6 membered nitrogen containing heterocyclic ring which can have at , :
~ ;

:, ;, .....

WO 94/2Ç314 21~ 9 ~ 0 3 PCT~S93/04309 -7- :

1 l~ast one alkyl substituent, wh~rein each R3 is independently lower alkyl or hydrogen; . ::
R5 is a g or 6 memb~red nitrogen containin~
heterocyclLc ring which can have at least one alkyl 5 substituent;
m i~ an integer ~rom 2 to 6;
n i~ an lnt~ger ~rom 3 ko 6. Such pre~er~ed c3~pounds can ~lse ~ ~s~ in th~ me~ho~s o~ ths p~ssant invention~
Compo~ltions ~nd klt~ con~alning ~h~ p~es~nt comp~unds are al~o ~ro-~ld~d harsln.

BRIEF ~ :
Flg, 1 illus~at~e~ ~h~ log molar amoun~ of :~
15 nonradioacti~e IPAB needed ~o comp~ltively inhiblt binding of ~adloactive IP~B to malignan~ melanoma cells. `;
The Kl obtained '~om thess data wa~ 6.8 nM. ,;
~ i~. 2~ p~O'~'J d~ a s~in~is-a~ .a~
obt2ined at 6 hrs. ater a nude mouse beari~g a human ~n malignan~ melanoma tumor rec~ived ~3lI]PA~, ~h0 arrow l ~r~ l r~ h~ ~ n~ t..... ~.~mc~ ;
Fia. 2B provide~s a scin~igraphic lmage obtained at 24 hrs. a~er a nude mouss bearing a human mall~nant melanoma tumor rec~i~ed [l3~I]PAB. The axrow 25 indicates the implanted tumor.
Fig. 3A provides a scintigraphic image obtain2d at 6 hrs. aft~r a nude mouse bearing a human `:
mallgnar,t ~a..oma ~U~..G~ ~a~ d ~ 13~ ,A2 . ~ e ~--e~
lndicates the implanted tumor.
Fig. 3B provides a scintigraphic ima~e obtained at 24 hrs. after a nude mouse bearing a humarl :

", 1 ~' SUE3STITUTE~ StlEEr ~:
. . .

WO 94/26314 PCT/US93/043a9 :,`,, ~9~3 ~8- , ~

1 malignant melanoma tumor received [l3lI]DAB. The arrow ;:
indicates the implanted tumo~.
. .
Fig. 4 provides a scin~igraphic image obtained at 30 hrs. after a nude mouse bearing a human lung 5 adenocarcinoma tumor rec~ived ~ I]P~B, The arrow indicates the implanted tumor.

DETAIL~D D~SC~r~TTON ~ TXE XN~NT~ON:
The present ln~n~lon pro~id~s nov~l compounds 10 and methods ox detec~in~ and t~ating ~rt~ln typss o~
canc~r, ~.g. n~u_o~7~ mas, glioma~, ph~s~hr~moc~tom~t msl~nomas, colon, renal and lung carcinomas. The campounds of the p~e~ent inv~ntl~n bind to ~ cell surface slgma recep~or and exhlbit ~xqulslte cell 15 speciicity and affini~y or ~h~ abo~ cancerous cells .
and for cells having sigma rec~ptors.
In one embadim~n~ tha pres~nt in~ention ls p~
di~2c~ed to a method for de~actlng a ma~alian tumo~ ~
whlch includes admlnis~e~ing to a mammal a diagno~tic .i.
imaging amoun~ of a compound o~ ths pr~sènt lnv~ntion, ~ n.~i ~h c c- ~ ',r ~ r.~ ~ Q ~ cl n~ t ~ h, ~ ~ ~,7 _ ~ g ~3 ~ ~ _ .
the mammal; wherein ~he compourld is of any one o~
; formulae I, II, III or IV: ;~
X .:
Ra I ~ (CH2)~-C~ - NR3-~CH2)~-~R~ I
Rb/~ .
tR,,~ ~

W094/26314 213 9 ~ 0 3 pcTl~ss3ln43os .:

l Q ~:
~a ~ ~.:
1 ~ (C~Z)i CZ ~ NRJ- CH2 -R~

t~

~a ~ ll;
~ ~ ~CH2~ CZ ~ NR~(C~2)m~ R~
F~b~ "~''~' tP.,,)~ :

~ (CH~ C~--N~-(CH~)n- N(~ IV

whe~Qin ~
X is a radionucl ide; : i :
Q is a radlonuclldQ, halide or an act;i~rating arau~:
z i s =o o r ~wo - H;
each R~ is independen~ly H, halo, low~r alkyl or }ower alkoxy; :~
2 R~ and F~b arQ lndependently ~, halo, lower 5 alkyl, lower alkoxy or ~ and Rb together wlth khe carbon atoms to which ~hey are attached ~orm a cya.Loalkenyl hs~e~Qcvlic rin~:
R2 is - N(R,)2 or a 5 to 6 membered nltrogen . ;
containlng he~erocyclic ring which is unsubstltuted or ~:.
3 substituted with at least one alkyl substi~uenk;
I, ~ ~ '.''.
,~,,"""
~; 35 ,. .

, .

W094/26314 ~39~ 3 PCT~S93/04309 --10- ;

1 each R3 is ind~penderltly hydrogen or lower alkyl; ~ .
R4 ls ~ N~R,)2 ox an N~linked 5 to 6 membered nitrogen containlng he~erocyclic rin~ whlch can have at 5 least one alkyl subs~ituent, wherein ~ch ~J iS
independently lawex alkyl or hydrogen~
~ a 5 or 6 memberod ni~rogen containing hoterocycllc ring which ~an hav~ a~ Qns ~lk~l sub~tituent; ;~
~O ~ and y ar~ independ~ntl~ an lnteger Erom 0 ~o ~; '.' q is an int3ger ~rorn O to 2, m ls an intog~ ~rom ~ to ~
n is an in~egor ~rom 3 to 6; and 15 with the proviso tha~ the~compound is not an iodine ~adioisotope o~ (N-diethylamino~thyl)-4-lod~b2nzamide. ~
The presen~ lnvention also pro~id~ a msthod ~; :
: fo_ 4rea~'r.g a mam~2'ian t~ wh~ch ir~ ud3~ ;.
: : administerîng to a mammal a compQ~ltlon includlng a .`~:
2~ tumor-inhiblting amount of a comp~und o~ ~ormula I, II, ;~
III or I~
The present ~n~ention urth~ pro~ides a method for diaanostlc imaging of a mammalian tissu~
which has cell surface sigma receptors which includ~s 25 administering to a ma~mal a diagnosti~ imaging amount of a compound of the present inventLon and det~cting an image of a tlssue having an abundance of cells with : ~igma receptors.
The pres~nt In~ention furthex provides a ~::
method for in vitro detection of a cancer cell in a mammalian tissue sample whlch includes contaating a ~:
: mammallan tlssue sample with an in vLtro diag~ostic j";',~

~ , ~u~

.... : . . .. . .. ~ . . , -WO ~4/26314 213 9 ~ 0 3 PCT/US93/04309 1 imaging amoun~ of a compound of ~ormu}a I for a tlm~ and ~ .
under c~nditlons su~lclen~ for blnding o~ the compound to the cancer cell and de'cecting such bindlng.
When used :eor diagnos~ic imaging X or Q as a ~;
5 radionuclide is us~d. Moreove~ X o~ Q radlonuclide groups which are preerably us~d ~or dlagno~tic lmaging ar~ y-emittlng radionuclides whl~h can be d~tected by radloimag i ng~ prorsdurss, ~ ~ g . by ~cintl~~a~ m~gi;~g .
Such ~-em~ting radionuclide~ eml~ radial:ion which ls 10 gu~icentl~ penetrating to be cletec~sd ~hrollgh tl~sue~. ;
M3re~er, for dl~Gne~ic irnaging p--s~ r~ nu~
do not emi~ a particl~, ~.g. a~ cx or J3 partic:le.
P~eferred X and ~ groups for dla~n~sk~c imagins lnclude lZ3I 124~ 125~ F 7~r and 77~r. ~ore preerr~d 15 and Q groups for diagnos~ic im~ging include lZ~ 2sI and 9F. l23I is especlally preferr~d ~or di~gn~stlc im~g~ng. `~
When used ~or therapeukic purpo~s X or Q as a radlonucl~de is used. Pre~erably X and ~ radionuclides employed for therap~ are ~-em3~t3Ang or an a~mitting i 20 radionuclides. HOW9~Q~. as contemplatsd h~r~in, ~n~

activity can be used in plac~ of a X o~ Q radienl~cl~d~
The pref~rred X and Q grsup~ ~o~ tre?ting c-~nc~_s ;:
i cl~lde l31I 2ll~t al2pb, 2lzBi, 76~r, "Br and ~he like~
25 How0ver, compounds for trea~ing cancer more preferably ~;~
have X or Q as ~
: As provided herein Q is a radionuclide, a ~a;~de o~ an activ~ing g~oup. Compound~ ha~ing Q as a halide or an activating group are provided as non~
3O radioactive compounds o~ the pres~nt inven~ion which can be readily converted in~o the correspond.~ng radioactive : compound. Since the utility of a radioactive compound , ~

":
8UE35TlTlJTE SHEET

W~94/26314 ~39~03 -12~ PCT~S93/04309 1 relates to the speciflc activity of such a radioactive compound, it is often pxeferred to add ~he radionuclide just before use. Accord.~ngly compounds having Q as halide or as an actlva~ing group are provlded, for 5 example, in a orm useful for storage or t~ansport.
When Q is a halide~ such a halide is ~ .
preferably Br or I.
A~ pr~ldQ~ h~aln ~ h~n~ u~ ~3 a group which is easlly dlsplaced by a radionucllde ~ia :~:
el~ctrophillc ar~matic ~ub~ti~u~lon. Pr~err~d a~ki~atlng g~oup3 lnclude ~ri~ tln, ~Jm~h~ls~
t-butyldime~hylsilyl, lodld~ and ~he llke.
According ~ ~he pres~n~ ln~n~lon Z ~s =O or two h~drogen a~om subskituents. Slnce the -C~ moiety 15 ls ad~acent ~o an amine, wh~n Z is ~O an ~mlde (-CO-MH~
is ~ormed, When Z ~s two hydrogen atoms a meth~len0 t-CH2-) is formed. There~ore ~ompound~ o th~ present lnvention can be amide or alkylamlno compounds, e.g.
compounds o ~oxmula I can have one o~ the following side ch2ins~ -Crl2 ~ y R2 ~ -~ ( CH2 ) ~ CH2 -~R ~-- ( C~2 ) y F~2 ' In a preferr~d embodlment ~ ~9 ~, i.e. th~ ~CZ- group forms a ca~bonyl. When -CZ-NR~- is -CH2-NR~-, the R, is , .
25 preferably an alkyl.
The term lower alkyl, when used sinyly ar in combinatiQn, refers to al~yl groups con~aining one to s~x ~ .s. ~o-~2r ~kyls may De straign~ cnain or branched and include such groups 2S methyl, ethyl, .~ 1 30 propyl, isopropyl, butyl, s~c-hutyl ~ isobutyl, k-butyl, pentyl, isopentyl, neopen~yl, hexyl and the like. Tho preferred alkyl groups contain one to four carbon akoms.

: 35 , SUBSTITUTE S~ ET

213~3~03 W0~4t26314 PCT~S93l04309 -~3-.
1 ~s used herein, a lower alkylene, singly or in combination with o~her groups, contains up to slx carbon ~:
atoms in the maln chain and a total of 10 carbon atoms if the alkylene is br~nched. Lower alkylene groups 5 include methylene, ethylene, propylene, i~opropylene, butylene, t~butyl0ne, ~ec~butylene, isobutyl0n0, ~: .
am~lene, isoamylene, pen~ylen~, isap~ntylene, hexyl~ne I ;
and th~ lik~. ~h~ ~r~Q~red low~r ~lkylsn~ aroups ~::
contain one to four carbon ~toms.
lQ The ~r~ cycloalk~nyl r~fers ko ~ pa~tially j.
sa~uraLed c~;~lic s~ruç~ur~ . a ~ng, ha~l~g 3~7 ~ing carbon atoms which can have one or ~wo unsaturations.
Since ths cycloalk~nyl group~ o khe pr~nt lnvention are fused to a phenyl mo.lety such cyclo~lkenyls a~e 15 partially unsaturatQd. ~he sub~ect cycl~alk~nyls groups l~clude such groups ~s cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexeny~ ~nd cyclohept~nyl rings.
A3 ~ h~3~ r a~ t~ a~g ~
alkyl group attached to the main chaln vla an oxygcn atom.
~al~ ref~r~ t~ ~ haloa~n, ~eclally bromin~, iodine, chlorine and 1uo~ine. A~ used hereln a halo group is a commonly available, non-radioactive halogen :~
i~otope. Preferred h~lo group.s include io~ide, ~:~
25 chlorlde, bromide and the lik~
As employed hereln, a h~terocylic ring means a sa~urated, partiaLly saturated or aromatic heterocyclic ~ h~ r.~ ~4 l~ac~ c~.e nltregen or oxvu~n ring atom. ~: .
As is known to the skilled artisan a saturat~d heterocyclic ring has no double bonds. As used herein a :
partially saturated heterocyclic .ring can has at least one double bond.

:
:,, ;, ,~

wo g4~2~l4 9 Q3 -14- PCT/US93l0430g l The present heterocyclic rings can have tlp to three heteroa~oms and up to a total of six ring atoms.
Accordingly heterocyclic rlngs o the present invention .:
can havQw abou~ ~ to abou~, S ring carbon a~Qms, . .. :.
5 Pre~0rably a heterocycllc rln~ has only one nikrog~n or one oxygen he~eroa~omt or one nl.~rogen atom ~nd one oxygen he~ero~tom. He~eroc~cliG r$ng~ can also hav~ a ~:
m~xtu-e cf r.~ro~n or oxygen hetexoa~oms, e.g. .~`
morpholine wl~h one oxygen ~nd on~ ni~rogen. It is lO pre~erred that the hekerocylic rlng ~nt~ln one or two r- ng hetQ-oa_oms t m~s~ preLgrred iS ene ~ing ni~rogen or oxygen he teroatom .
H~terocyclic rings o~ the p~esent ln~ntlon are monocyc~ic; such mono~ycllG rings can ~ fused to a 15 phenyl ring to form a bic~clic ring.
Representativ~ partlally saturated and heteraaromaklc heterocyciic rlngs include ~u~an, pyran, ox24ine ~ i soxazine ~ pyrr~le ~ pyrazole ~ pyrid~n~, pyrazine, triazole, tetrazole, tri~zine, pyrlmidlnQ, 20 pyridazine, furazan and th~ llk~. ~r~s~
he~rQ~ "r~ J~ *~ L~i~a an~ tn~
Representative saturats~ hsts~cycli~ rin~
include tetrahydro~uran, pyrazol~dine, ~m~d2zolidine, pyrrol~dine, aæetidine, piperidine, piperazlne and 25 mO~pholine, As used herein ~ and R~ are independently H, halo, lower alkyl, lower alkoxy or Ra and Rb to~ether n ~ ~h ~he carbon a~oms tO wnich they axe attached form a cycloalkenyl or heterocylic ring. When R~ and Rb ! ` ~ 3o together form a cycloalkenyl or heterocylic ring, such a ring is fused to the phenyl.

.;';
' ' .

,;.

WOg4/26314 213 9 ~ ~ 3 PCT~S93/04309 j -lS-1 Such a cycloalkenyl rlng ~ormed from R~ and Rb ;.
has only one unsaturation in ~he cycloalkenyl ring and that unsa~uration is centlibut,ed by the phenyl ring to ~ :
which t,he cycloalkenyl is fused. While a cycloalkenyl 5 formed by R~ and ~b can be a 5 o~ 6 membared rlng, such ring~ are pre~erably 5 memb~r~d r~ng~, e.g.
cyclopentenyl. Examplss o~ ~he ~u~ed cycloalkenyl-ph~ny1 rin~ inclu~e ind~n~;l and t~trahïdr~n~hthll, s.g~, 5,6,7,~-~etrahydronaph~h~l, and the like. ~;
. lO Wh~n a,het0roc~cllc ring i8 ~orm~d by R~ and : R~, the he~Qr~s~cll~ ring ~re~rablv h~ ~ne nltroaan o~
oxygen hetexoa~om and 5 o~ 6 ~ing atom~. A~ u~ed herein, he~erocyclic is as ds~insd hereinabo~. The heterocyclic ring cont,ains at l~as~ ~wo rlng carbon atoms when the h~kerocyclic ring is a 5 membered xing, and the num~er o~ ring carbon a~om~ p~es~n~ can range from 2-4 carbon xing atoms. ~h~n ~he heterocycllc ring ;~
~s a 6-m2."~ere~ ~iny/ the numD~r of ring carbon atoms ,,;
can range from 2-5 ring carbon a~oms. ~hu~, ~h~ to~al number of ring carbon atoms will r~nae rom 6-~ xlng memberQd heterocyclic rlny and ~-~ ring carbon atoms I :
when ths phenyl ring is fuss~ to a 5-mem~srad ~`
heterocyclic ring~ The heterocyclic ring can corltain up ;~-25 to 3 ring heteroatoms. The preferred ring hetaroatoms : are oxygen and nitrogen, especl~lly oxygen. Proferred heterocyclic rings forrned by R~ and Rb include :~
dihydrofuranyl, dihydropyrrolyl, tetrahydropyridinyl and the like.
As provided herein each R, is indep~ndent~y H, ~:.
halo, lower alk~l or lower alkoxy, In a preferred embodiment Rl is H, halo or lower alkoxy. More .,; .
. .
1 : .

8UE~ST~TUTE~ 5HEE~T

WO 94/26314 ~,~39~ PCT/US93/04309 1 pre~err~d R~ groups include H and halo. ~lowever, ln one ; .
ernboclimen~ R1 is preferably alko~
The variable q is defined hereln as an iAt~gsr ranging from O to 2 whlch descr.ibe~ khe number of R~
5 groups on the phenyl m~ie~y. Sinc~ ~he phenyl is aiso subs~itu~ed with R~, Rb, X ~or Q) and a ~i~e ch~in amiAe or a~ine moi~y, the maxlmal number a~ R, groups ls ~
(i,~. q ca~ maxima~ly ~e ~). Wh~n ~ i~ less ~h~n 2 some positions on ~he phsnyl group a.re unsubs~l~utsd; in thls 10 case a hydrog~n ls presant ~t ~h~ p~ ions h~ving na R, ~U~ P~ u~3 ~r ~ a ~0 1. An espec~ally preferr~d ~alue or q ~s 0, i~e. kh~ phonyl has hydrogen at all poslti.ons ~xc~pt khoss o~cup~.ed by R~, R~r X (or Q) and th~ amide ~ amirle slde chain 15 moiety .
In a preferx~d ernbodiment the Q(o~
Ra ~b~ .

g~oup i5 selected fxom the i~ollowlng: I :

y~ Q/~>~

~` 1 3O

35 Q ~l or E~r , SU13STITUTE SHE~ET
,. ..
,::

WO94/26314 21 3 9~ ~ PCT~S93/04309 '' "'~

;;

Q ~ s ,;

~:~

:, ' wherein Rl is as descrlbe~ hereinabove and Q is a radionuclide ~e~ ), a h~lide or an actl~at.Lna reup.
_ A~ J-~a~ c~ J~ ) vr membered ni.~rogen containing h~t~roc~ic ring whi~h is .:~
unsubstitu~ed or substitu~e~ with ~t l Q~S~ cnQ ~1 k~
substituQn~; wherein each R3 i5 independenkly hydrsgen ~-25 or lower alkyl. Prefexably ~3 is lower al~yl in the -N(R~)2 ~roups of the pr~sent invention. Pre~erred R2 heterocyclic rings include N-piperidinyl r N- ~ .
pyrroliàinyi, ~-pyridinyl, N-morpholinyl, N-pyrrolyl, piperidinyl, pyrrolidinyl, pyrldlnyl, morpholinyl or 3~ pyrrolyl, which can be substituted with an R6 lower alkyl. R6 i5 preferably attached to the ni~rogen o ~he piperidinyl, pyrrolidinyl or morpholinyl rings.

: `
'`.
. .

$UE3STITUTE SHlE~ i~
:.

wo 94~2~14 9~ PCT~S93/~4309 l In one embodiment R2 can be R4 as defined herein. In another embodiment ~ can be ~5 as de1ned herein. In stlll another embodim2nt R2 can be ~ N(R3)2 a~ deflned herein.
As provided herein, R4 is ~ N(R3) 2 or an N-llnked 5 to 6 membered ni~rogen containing he~erocycllc rlng whlch can have ~t le~st one alkyl subskltuent. ~g da~in~d h~r~in N~l~n~ m3ar.~ ~ha~ th~ nitrogen containing h~terocycllc ring 19 attached to th~ maln lO chaln ~hrough a nitrogen ~om. R4 is us~d in ~ormula II
to lndlcate ~ ~re~er~n~e ~ h~a~ ec ~hs ~J ~-v~
pr~ent wi~hln ~he heterocyclic rlng ~o the main chain.
Preferred R4 h~t~rocycli~ rlngs include rings o~ the ~ormulae:

R

or ~5 R~
X~ ~
-N~

3 .
".''''' ..

:~
'.':".,'.

SOE3STITUTE SHE~T
.. .

WO94/26314 213 9 ~ 0 3 PCT~S93/04309 1 wherein R6 ls hydrogen or lower alkyl and each i is ,~;
independently an .integer from 0 to 1. Preferred R
heterocyclic rings inclu~e N~pipe~ldinyl, N~
pyrrolidinyl, N py.ridine and ~he like. ' In another embodiment pxeerred compounds have .
he~erocyclic xings tha~ ~r9 not at~achQd ~ia ~he rLng ni~rogen, ~5 is used in formula ~ o d~scrlb~ such l~
com~ounds, wher~in ~5 is a ~ o~ 6 membered nit~ogen ~ ;;
containing heterocyclic rlng which c~n hav~ a~ s~ one lO alkyl substltuont. In a p~erred ~mbodlm~nk R5 i~ ~ny ~`
c~ h~ ~oll~

R-~ , R~$(~N

R.~ , R,~ r t CH2 ) ~ NR

2~ R6 wherein each i is ~ndependently an integer from 0 to 1 and R6 iS nydrogen or lower alkyL. More preferred Rs heterocyclic rings include piperidinyl, pyrrolidinyl or :
, 30 which are N-substituted wIth an Rs lower al~yl, or ..
pyridinyl which can have an R5 lower alkyl. Such an R6 ~`
1"'.,;.

` . . .
il ., ..:;", &UBSTITUTE SHEET
',~

wn 94/26314 ~39~3 -20- PCT~S93/04309 l lower alkyl is preferably methyl, ekhyl, propyl or butyl. . l;~
The compounds ~f ormula IV have an -N(R3)2 group which is hydrayen or lower allcyl. In a prefexred 5 embodimen~ for ~N~R3), R~ is lower alkyl, e.g. methyl, ethrl, propyl or butyl.
The variable ~, as u~d har~in, re~ers ~o an integer r~n~ing ~r~m 0 te 5 whi~h deLLneS tha l~gth the alkylene chain sepa~atlng ~h0 phenyl and -CZ-lO mo~e~ies o~ the ~esent compo~nd~. Pref~rably, ~ i~ an ~ntege- L ro~ C t~ 3 . ~re ~ 3~1y, ~ 13 an intager ::
rom 0 to 2. Far compound~ where CZ- is ~CO , ~ ig pref~rably 0.
As de~ined herein y is 0 to 6. ~he variable y 15 defines the ~ength of th~ alkylene chaln separatlng the -CZ-NR3- and ~2 grQups ln the -CZ-N~3 ~(CH2)y~ Rz moiety of formula I. Preferably y is l ~o 3; more preferably y is 1 to 2.
Llke y, the vaxlable m de~ines th~ 1~ngth of ::
ths a~kyl Q~ ~h,_~ " ~g~3_3~y ~h~ J~ a~d t~ Rs y-r~p in ~ne -~Z~R~-~CH2 )m~ mol~ty of f~rmula III. ::
The v~riable m is an ln~eger ranging ~om ~ to 6.
However, m ~s pr~era~ly 2 to 4 and m~re preferab1y 2 ~ : ko 3.
: 25 The leng~h of ~he alkylene chaln separating ';
the -CZ-NH- and the -N(R~)z moietles in formula IV ~s described herein by n. The vaxiable n is an lntegex ranging ~rom 3 ~o 6. In a preferred embodlment n is 3.
Preferred compounds o~ the present lnventlon .:
include the followlng.

.~
` ::
""
i~`

SUBSTITUTE SHE T
, ,:

; .- ~ .. - ~; .

2139~ 03 .;
WO 94/26314 PCT/US93/04309 ~ ~

. .

o ~ow~x low~
a~ o ~ \~ a1k~

, '.

'', ".

o~N1;~

lo~r al~g~

iower r I~' ~ ~O- ai~cyi ,~

o M~

"~3 ` ' ~ ~ ~35 1`~

:

~31lJElSTITUTE SHEET ~:~

..... ...... ..... ... .

wo 94/~6314 ~ ~3 -22- PCT/US931û4309 k~ }~ t~
:1 ower al~yl-O~ ~ ~o~ lowe~ alkyl J~o lower alkyl ~- lower ~lkyl :: 2~

C) NH t C~ R~
o~ ~t 3 ~

"~.

''~
SUBSTITUTE SHEET
`

2139~0.~

--~3--~ ~(\N~

~ R
~X ~'''`'' C~ or ~r o~ C~II~VN(ll,), I 13 >

or SU13STITUTE SHE~ET 1 ~

WO94/26314 PCT~S93/04309 ~9~3 -24-1The various combinations and permuta~ions of the Markush groups o~ X, ~, Z, R~, Rb, Rl, R2, R3~ R4 and R~ described her~in are contempla~ed ko be wlthin the scope of the presen~ invention. Moreover, the present ~.
5 in~en~lon al~o encompas~es compounds and composltions :.
which contain les~ than all o~ the elements in the ; ;
Mar~ush grouplng. Thus, the p~es0n~ compounds and composltions con~ain one or more elem~nt~ o~ sa~h o~ tha Markush grouping~ in X, Q~ r ~b~ R2, RJ~ R~ and 5 lO and ~he ~arlous combina~lons ther~o~. Thus, ~or examDl~, ~he pres~n~ ~n~nt~on ~n~ m~ s ~ha~ ~. ma~
be one or more ~ th~ subs~ltuen~ lls~sd h~reinabov~ o~
any and all o~ the subs~ituonts o ~(X~)2, R4 an~ R~
The pr~sent compoun~ can blnd ~o a speciflc 15 cell recep~or preva~ent on certaln types o cancer cells. Such canc~r c~lls include ~ung car~inoma, colon carcinoma, renal carcinoma, melanoma, glloma, ` .
pheochromoc~.~tema, neu-~blas~e~ and r~la~ed cell~. An .~;
example of ~he cell recep~or to whlch the pres~nt 2Q compounds bind is a cell surace ~igma r~c~ptor. .:
The binding cha~c~ri~ic~ ~ 4h~
compounds were de~ermined by observing whether bindlng :~.
was inhib~ted by known sigma rece~tor antagonists. Many antagonis~s are known which ha~e demonstrated binding .~
25 specificikies for a given cell s~rface receptor. Such .--antagonists can be tested as comp~titive lnhlbi~ors ~or cellular binding by compounds o~ the pres~nt invention. ,:
If a c~iven anta~oni st 1~ ~ Cc!!'!2~Qt $ t~ ~:s ' -.hi~ l ~ ,r ~'.e recepto.r to which the antagonisk binds must also bind 3O the subject compounds.
For example, as demonstrated by the present inventors, a malignan~ melanoma cell llne blnds the.
:

' ';:
'',~

SUBSTITUTE SHEFr `
i ~

WV94/26314 213 9 ~ 0^3 PCT~S93/04309 1 present compounds wi~h s~rong specifîcity and affinity.
Only antagoni.st~ which bind ~o the same site as the ~;~
presen~ compounds can inhibit binding ~f the sub~ect compounds. ~n~agonists which can be tested include 5 antagani~ts speci~ic or cell recepto~s such as sigma (e.g. uslng SE2466-2), sigma-l ~e~g. ~uphana~lne ~t low concentrations~, sigm~ a~ luph~nazln~ at hlgh ~;
co~cgn~a~i~n~ Cp3~ n~ .g. ~ 3~ am~
(e~g. raclop~ide), melan~cyt~ ~cre~ing ho~mone raceptor lO ~e g m~lanocyte ~ec~a~lnc1 ho~m~r~s peptide), S~ ~:~
h~dro~rypt~mln~ . mians~rin3, ~-hydroxJ- ~:
~ryptamine- la ( e . g . N~N~l90), 5-hy~roxytryp~amlne~lc (e.g. ketanserins), S-hydroxykryptamin~ e.g.
k~tanserine and mianse~in ) and 5~hyd~oxytryp~amin~-3 15 ( e . g . 3-tropanyl-dichlorob~n ) c~ ecep~or~ and the like. ~.
As provlded hereln, antagonlsts wlth d~ n~_a~P~ ~S~d~ 2~ci~'~'L, ~O~ ~1! sur~ s'~a receptoxs (e.g. ~luphenazine) can act as c~mpetltlve '!'''~,'"
"~ binding inhlbitors or compoun;ls oE the present ~n~ nt~ n,, `Tn, ,~~ c~ n,L~ h~t d~ ind ~
cell sur~ace sigma recep~ors cannot lnhibit bindlng of l the present compounds to meLanoma cells. Thereore, ~he present compounds can bind ~o cell sux~ace sigma 25 recep~ors. :;
Cell types which have sigma receptors lnclude normal neural ~issues (e.g., brain, ~pinal cord and the ~ a~ -w~ a~ l~n~ ~ar~ ~! r.~ vlv~. ~a~ 2, carcinoma, melanoma, pheochromocy~oma, glioma, neuro~lastoma and the like. For example, several lung carcinoma cell types have demonstrated binding afinity for the present compounds including an adenocarcinoma, a SUBSTITOTE SIHEET

W094/2~14 ~9~303 -26- PCT~593/04309 1 squamous carcinoma and large cell lung carcinoma cells. .:::
In a ~ur~her example metas~atic malignant melanoma cells ~.
ha~e demonstra~ed high a~finlty and speci~icity ~or the present compounds. In a preferred embodiment the l .;
5 present compounds are used to detec~ and treat melanomas l:
and non-small cell ~ung carclnQma ~N~C~C). Such N5CLC .:~
cancers lnclude lung adenoc~rcinom~, lung ~quamous cell .
carclnoma, large cell lung c~rclnoma and th~ like. ~:~
Accordlng to the pre~ent invention a metho~
10 for de~ecting a mammali~n ~umor ar a ~issu~ containing cell surf3ce sigm~ ra_~pL~ inslu~ admini~ring to a mammal a compositlon lncluding a diagnostlc imaglng amount of at 10~st one of L~he pr~s~n~ compound~. Such a diagnos~ic lmaging ~mount is a dosage of a~ leask one of ;~
15 the sub~ect compounds which pe~mlts~sufflci~n~ ~umor or tissue localizatlon of the compoun~ ~o aLlow detection of the tumor or tissue. Thls dosage can ~ange from ~:
about ahou~ 1 yg ~o about l g of the compound per li~r which can be administered in do~es o~ abou~ 1 ng/kg body ~:~
weight to about 10 ua/~ bod~ wsiaht. ~r~s~r~d do~ag~s __ ~ ~ ' r.. .
:: ^$ ~ r`~5~ J a-a J~ yc UL ~ iL iU ~g ~' to about 2 ug~kg ~or diagnos~ic im~ing. Mor~o~sr, ~or diagnostlc imaging the amount Q~ radioact~lty administerod should be conside~ed. Preerably about 0.1 25 mlllicuries (mCi) to about 20 mCi of radio~cti~e compound is administered.
As described herein a tumor or tissue labeLed w~_h ~.e o. ...c~a 0c th~ s~n' c~ urlds can ~e detected using a radiation det~c~or, e.g. a y-radiation detector. One such procedure utllizes scintlgraphy.
Tomographic imaging procedures such as single photon . ::
emission computed tomography ~SPECT) or positron ~:
....
. !

SUBSTITUTE 5HE~ET

213 9 ~ 0 3 PCTIUS93104309 WO94/~14 -~7-., ,'.~.
l e~is~ion tomo~raphy (PET) can also be used to improve vlsualizat~on.
In ano~her ~mbodiment the presen~ invent~ion ls dlrected to a me~hod for treating a mammalian tumor ;.~
5 which includes administQring to a mammal a composition :~;
including a ~umo~-inhibiking amoun~ Q~ a~ l~ast one `~;~
compound of the present ~nventlon. Su~h ~ tumor~
inhlbit~ng amount is 2n amoun~ o~ ~ lsa3t on~ o~ ~h~
subjec~ compounds which p~mits ~u~lcl~n~ tumor l~ loc~lization of the compound to diminish tumo~ g~owth or s~zs. As ~ s~ aw~h Q~ ar.
monltored by any known diagno~ic imaglng procedure, e.g. by using the pres~nt me~hod~. This dosage can range from about O.l mmol~/kg body w~ight to about S00 15 mmoletkg body weight. A p~e~er~d ~o~ag~ ls ~bout 5 to about 50 mmole/kg body weigh~
The amount of radioacti~lty adminlst~rad can ;a-i d_p2nding on the typ8 of radionuclide. Howe~er, with this in mind the amount of radioactivity which ls ;~
2~ administered can lJar~ f~om ab~u~ 1 ~rl ~o a~ mrl.
~:~f~ 'i, abv~-~ lv m~i tu ~bouc ouu m~
administ~r~d.
Moreov~r wh~n consid~r~ng a des~ for diagnostic imaging or therapy, the specific activity of 25 the radioactiv~ compound should be taken into consideration. Such a speciflc activity l~ preera~1y `:
very high, e.g. ~or l23I~labeled compounds the spsclic ~ s~.eu~ at l~as~ about l,uOu Ci~'mM to a~out 50,000 Ci~mM. More preferably the specl~lc activity 30 for ~Z~I-labeled ~ompounds ~5, e.g. about lO,OOû Ci/mM to about 22,0ûû Ci/mM.

;

SUBSTITUTE SHEr "` ,.

wo 94/26314 ~ ~3 -2~- PCT~593104309 l In another embodimen~ ~he present invention :;
provides a method for in vitro d~tection o~ a cancer ~:
cell in a mammalian tissue sample which includes contacting a mammallan tlssue sample with an in vitro 5 diagnostic imaging amou~t o.~ a compound o~ any one of ~ormulae I, II, III ox ~ ~or a ~ime an~ und~r conditions su~iclen~ ~or bindlng o the compound to a ~.
cell sur~ace sigma r~c~ptor on ~hs c~n~s~ ~ll and detecbing such binding.
Samples ~n be ~oL~e~ed by procedure~ known .
to the sk~l~sd a-~san, ~ y ~o ~ J ~ a ~'~3us biopsy or a body fluid, by aspira~ing ~or tracheal or ~:
pulmona~y samples and the like.
As used her~in any mammallan tlssue can bs :~
15 tested in vitro. Preerred tlssues ~or in vitro testing i~clude lung, bronchial,:lymph, ~kin, bra~n, li~er, any :~
tiS9119 o ner~ou~ orlgin and ~he li~. Sample~ can be se_~L^ne~, e.g. with a m.icro~ome, to ~acllita~e microscopic examina~ion and observ~tion oE b~und compound. Samples can also be ~x~ appr~p~labe ~;
~a~7'~ P~v~c u~ ~L~r incll~atl~n ~lth one ~f the pr~sent compounds ts impro~e ~he hi3~0lagical .~.:
quali~y o~ sample ti~sues.
Conditions suff iGient ~or binding of the 25 compound ~o a cell surface sigma receptor on the cancer ~`
cell include standard tissue culture ~onditlons, i.~.
~amples can be cultured in ~itro and incubat~d with one Or 'h~ ~rcs~nt compounds in physiological media. Such conditions are well known to the sXilled artisan. ...
30 Alternatively, samples can be fixed and then incubated with a ~ompound of the present invention in an isotonic or physiologlcal buffer.
;:

:``,;

SUBSTITIJITE SHE~T ,:

WO 94/26314 213 9 ~ O 3 PCT/IJS~3/04309 , ~
_~9_ ' ., ~n amount of a~ least one of the present ~;:
compounds f or in vitro detec~ion o~ a cancer cell can ~ ~:
xang~ f~om about 1 ng/l to about 1000 ~g/l. P. preferred ,;
amoun~ is abou~ 1 y~/l 'co ahout 100 ~g/l.
When the pr~s~nt compounds ar~ u~ed f or in vitro diagnosls o~ cancer :X or Q a~ a radionuclide is l:
~ ~ 1, .
u~ed, Preferable X and Q radionu~lldes o~ in vitro diagnosls o~ S~anc~ r lncLIld3 lZsI, l~F/ -~tS-al.kyl, -~sSoJt -3~so4~ -14C~ CH~ arld ~h~
For detection o~ cellula~ bindlrlg of on~ a th~ p~ga~n~ cornpounds r s~ s ~3r~ h~ J ~ b~3f' ' ;~
presence o a ~elect~d compound, th~n washed and counted in a standard scin~illa~on c~unter. Alternatively samples can b~ dipped ln pheto~muLsion and ~he sign~l 15 detected under light m~croscopy ~er several days, as exposed silver grains.
Co~pounds of ~he presen~ inv~ntion can be pr~pared by any p~oceduxe availabl0 to the skilled artisan using protec~ing groupsj lea~Lng group~
2~ activatin~ grou~s ~n~ ~h~ l$ke ~3 ..3e~e~. ~t rting ~ v~ c~n ve cnosen wnlcn nave ~he desir~d ~1~ P~a~ :
R3, R4, R5 and P~6 g~ou2s at the re~ulsite pa~itions.
Alternativgl y, 2 le~ sreup may be u~d in plac0 o the des $ red ~1~ R2, R~, R4 ~ R, or R6 group, and the 25 appropriate group may r~place the leaving group ln a later synthetic step. Another altexnat~ve ls to employ a protecting group on a reactive group whLch may be pr~san~ ~n scar~ing ma~erials, , an amine or slmilar reactive group on the chasen starting material. The use of leaving ar protecting groups prevents undesirable ~:
side reactions from occurriny, while permittlng desired reac~ions to take place.
:;
`~

' .

SUBSTITUTE S~IEET
,:

WO~4/26314 ~39 ~ PCT~S93/04309 l As is goner~lly known in ~he art, and for the purposes of the present invention, a leaving group ~s :~.
defined as a group which is r~adily broken away ~rom its union wi~h a carbon atom. ~These groups are readlly !~
5 L^ecognizable by one skilled in the art. Suitable leaving groups are generally el2ctron at~raatin~ groups, ,~:
either becau~ o~ ~helr el~ctronega~i~ity or b0cause they have an inducti~e e~ 4, ~n~ m~ lnclud3 ~oups such as halides, N3, ~IO-~ryl, or HSO3 Aryl group~, and lO the like. For ex~mple, a lea~lng group can be present a~ t~s p~s~t~n ~. X ~ n ~ ~t~ ~ln~ '3~
presenk compounds; such a l~aving group ls pre~arably a halide, e.g. Br or I.
A protecting group l~ co~al~ntly boun~ to a 15 reactive group ~o ~ender the r~a~tlv~ group un~eac~ive while a}lowing desi~ed rkactions to take~ place. To be useful, a protec~lng group must in ~ddi~i~n be easily rsmo;sd ~iLhGut cnemicaily al~ering the r~ainder o~ the ~:
molecule, and must regenerate the correct structure o 2a the reactive aroup. Exampl~ og p~ts~ sr~ups ~4e~s~ e ~t~ e~ ~,~mpi~, primary an~ secondary amino : groups include acetyl, carbobQnz~xy ~cle~ed by a~ld hydrolysis~, benzyl (clea~sd by catal~tic hydrogenation), tert-~utoxycarbonyl ~cleaved by mild 25 acid treatment) and 9-fluorenylmethoxycarbonyl (cleaved by secondary amines). ~ comprehensive revlew o useful protecting group~ is prov~ded in Greene, l9~l Protective ~-_?~ gLanic ~y~nesls (John W~ley & Sons, New York). :
As provided herein an activat~ng group is a ~;
: group whlch is easily d~splaced by a radionuclide via ~:
electrophilic aromatic substitution~ The activating ' ~ , ` :~

SUE~STITUTE SHEET
`~:

WO94/2~314 213~ i~3 . PCT~593/04309 j ~

l group is used ~o facilltate substitution of a '~
radionuclide onto ~he present compounds. Ac~ivating ¦;:
groups contemplated by the present lnvention include ~'~
tr~butyl-tln, ~rimethylsllyl, t~butyldlmethylsilyl, 5 iodide and the llke.
The present compounds can be prepared rom readily available starting materlal~, ~or exampl~, hy ~:
amldati~n of a ~ubstltuts~ pheni~ alX~lca~b~xilat~ or substltu~ed benzolc acid wikh ~n appropri~e ~mlne. ';
lO Such a reaction yleld~ a compound o any en~ o~ ormulaQ
I to I~.
In an ~xemplary procedùr0 or synthe~ls o a benzamide compoun~ of ~ormula ~, a substituted phenylcarboxyalkyl can b~ used ~s a skar~lng ma~erlal, 15 For example, a phenylcarboxy~lkyl ~) ha~ing a lea~ing group (Y) a~ the desired X ~or Q) posi~l~n can be amidated in the pre~ence of a halagenating reagent with an amine of formula VI, as depic~ed below.
~y ~ .
~ ~CH2)~ CO-- OH ~ NH~-~CH2)y R2 ~~--~

( R ~

v ~ ~ ' 3 ;~
~.

SILJBSTITUTE SH~
;, Wo 94/263l4 g~3 -32- PCT~S93/04309 ~(CHz)~--CO--NH--(CH2 )y--11~

~.
wherein Y 1~ a lea~i~g group ~n~ t, q t ~, ~ and 0 F~2 ~rl3 as de~cri~ed herein~bo~r~ . Pre~erably Y ls a halo ~ :
~roup, . g . Cl, 3~ o~ c~ea p~e~re~ x~ gr~ups ~re ~r in a meta p~ition arld I ln a pa.ra pa~ltion relatlv~ to the carboxyl group, wh~ th~ X c~r Q ~ to be placed in ~ ~;
suGh a rQspect~ve meta or pa~ po~l tion .
Halogenatlng reag~nt~ ~o~ the ab~v0 dasc~l~ed reaction includ~ t,hos~ which can con~re.r~ ~h~ ca~boxylate to an acid haIlde, e ~ g . thionyl halide such as SOCl2, ~ 15~ ~Cl3 and tne lik~. A pre~erred halog~nating reagent is SOC12 in ~h~ p~esence o~ c~imethylormamide.
~? To facilitate '~r:n~tJ~n Oc su~h an acid :
h.~ 2, ~ L~tion can ~e ne~ted to re~lu~
temperatusss. ~ ~refQrred s~l~ent for thls reaction is a nonpolar vol~t ~1~ sol~rerlt, ~, g . chloroIorm . The acid chloride 90 ormed is suf ~iciently stable to be ;
25 isolated, for example, by evaporation of solvent. After : conversion of V to the acid halide, the amine (e.g. ~
can be condensed with the acid halide in the presence of a ~2 ~uch as ~riethylamine. Ths solvent ~or this r~action is also pre~erably a nonpolar solvent, e . g . .:
3~ chlorof orm .
The skllled artisan can readily madify the reactions describ~d above to g~nerate a compound of any , ,~ .
. ..

8UB~IT LJTE SHEET
~ .
.

WO94/263l4 213 95 0 3 PCT~US93/0~309 ~;
~33-l one af formulae I, II, III or IV. For example, toproduce a compound o~ formula II, an am~ne o~ the formula NH2- CHz- R~ can be used in place of the compound of ~ormula VI. Sim~-larly, to pro~uce a 5 c~mpuund of formula III or IV, an amine of the ~ormula NH2~ ( CH2 ~ m - ~5 or NHz~ ~C~2)n~ N~J)2~ ~espectlvely, can ~ .
be u~ed in place o ~I.
W~sn Z i~ he lea~ing group ~') can be directly replaced to produce a compoun~ ~ any one af lO formulae I, I}, III or IV. When Z is two -~, the ~.
carhorlJ~l c~ ~ ~h~ ~^m~ ~e "~i~ti or~ed ~y .,h~ a~ov2 conden~ation must be conver~ed in~o a methylene. To convert the -CO-NH- to a -C~ ~NH- a reducing ~gsnt can be used, e.g. ~oron hydxide, so~um borohydrate, lithium 15 aluminium hydride and the lik~, ~ pre~erred reducing agent i9 boron hydride (~H3) in the presence of : tetrahydro~uran (THF). ~or exa~ple, the carb~nyL o a ;:
compound of ~ormula VI~ can be converted to a msthylsne by the following xeaction.
2~ y Ra ~
~ ( CHz ) ~--CO--N~ CH2 ) y--F~2 ~ ;

: ~R~
: : 25 -~:
VII
, ~
, .

:
,~

S~J~sTl~lJTE S

WO 941263fl4 PCTliflLJS93/04309 .,:
~39~Q3 34 tetrahydro~furan ~ / , , ;''~

~fa~,~
J~ ( cHa ) ffff~f~f f~f~fffN~ J~ ~ ~2 Jf y -R2 ~)ff~ ~:
10' ,,, :,.
When the Rf afff -CZ-NRf- is lnw~r alkyl, ~fuch a :;
lower alkyl is addeaff, o . g, b~ ff-fff l ksrl ation, af tfff-a ~,f 5 condensation orf the acid hff~fffll~le f~nd thfa amine and afl:f ff~
conver~fion of ~he amidf~ (-CO-~H-) tfO the ~lkylaminf~ffff l:
(-CH2-NH~ ) . Pffflkylation can be d;,n~ by any ff~rallabl2 procfff.?dure, e.g. ufffing an alkyl halicle with a 30dium salt in dimethylfformamlde ox ethanol ff Fo~ exampleff, fff~n alkyl :::
2f~ halLde ~f e . g~ ' f~fd~fms~han-f ) can bfe rfzac~fffad with a corrffffpounff I
ULf~ ~v~ in ff nff~ prfffff3qnc~ o~ sff3fff~um bicarff~fonate sodium carbfona~cff~ff using dimetn~.lf~ffrmamide a~ff s~fflvefffn'c.
T i~ ff~f compound of ar,ffy Of ne of fforrnffful fffffff~
or IV is de~ ~ red, a C? group can replace the Y leaving ..
25 group, e . g . on ~rII or VIII . ~s pro~lded herein Q is a radionuclide, a halid~ or an act~vating group. When Q
1.9 a halide a starting material having the cl~sired h~ e a~ tha posi~ion o~ Q an be utilized, ~.g. V can be bromophenyl carboxyalkyl, lodophenyl carboxyalkyl 30 iodobenzoic acid, and the l~ke. An activating group ::~
~an he placed at the position of Y by available pocedures to qenera~e a compound o any one of formulae ; 35 , ~;

W~94/2~14 21 3 9 ~ ~ 3 PCT~US93/04309 ~35~

1 II, III or IV, wherein ~ is the ~ctivating group. The acti~ating group ~Q) can in turn be readily replaced by a radionuclide (i.e. X) to gen0rat~ compounds of formulae I, II, ~II or ~, wherein X i~ th~ desired 5 rad~nuclide.
For example, act.ivation can b~ achi~ved using palladium cat~lyzed s~arlnyl~tion with bls(trlbutyltin), as d~ict~d bslow. .;:~
~r :
lV ~ ~
2 ) ~C~ 3 - ~ C~2)y- ~2 V~ Ia ~ ~ a ) ¦ (triphenylpho~phlne) 4 palladlum ~tribu~yltln) 2 ~ / ~ tri~thylamine Q ~ :
Ra ~
I ~ (CH ~ O -~3~ !C~!y- ~Z ~X

~R~)~
In this case Q i5 tributyl~in (~u3$n). This reac~ion i~ ;~
25 effective whether Z i5 -O or two -H. .:~
When using t-butyldimethylsilyl chloride (TBDMSCl) or trimethylsi1yL chloride with N hutyL ~::
~ ~ I hJu.. ~ C, ~-~u~ ithium, a protec~ing group (R1) is ~::
first placed on the -CZ-NR~- ~mine, i ~3 .iS hydrogen.
3O When the R3 of the -CZ-NR~- is lower alk~l, no such protecting group is nQeded~ Protecting groups used for a -C~-NR3- amine can be any protecting group for a :~
: secondary amine~ e.g. carbobenzoxy (i.e. CBz, cleaved by .
,~

~....

.
SUBSTITlJTE SHE~Er WO94/~14 ~ 9 ~ ~ 3 PCT~S93/04309 l acid hydrolysis~, benzyl (cleaved by ca~alytic hydrog~nation)~ tert-butoxycarbonyl (l.e. t~BOC, cleaved by mild acid treatment) and the like. The silylation reaction ~n then be performed as depic~ed below, e.g~
5 using an amine protected .~ompoun~ of formula VIIX.

Ra ~ 1~

~C~2)~ lz~ N~ (~) ~ R

¦ t-J.~ ~h~il3ilyl chïo~id~
~ ~ N-butyl lithiu~

~ ¦7 ( CH2 ) ~ H2~N--( C~Z ) y~--~2 t h~

2 In this case Q is trim~thy1silanQ ~Me Si). Th~
condltions u~d for this reac~ien ~ n,~ v'~
temperature (~.g. -78C) and a polar sol~an~ (e.g. ,1 tetrahydrouran ) . .;
The R7 group can be removed by standaxd techni~ues, e.g. when R;, is C~ or t-~OC acid hyd~olysls ;-can remove R~ and restore the secondary amine ~NH~
Silylation is preferred ~or compound~ whereln Z is two - :
H.
The radioactively labeLed compounds of the present invention can be produced with high speci~ic ~:
activity and high yi~ld by reacting a r~dioisotope ~e.g. ;::
3I, l25I or 13 I) with an activated intermediate ~e.g. a l~

, ~

,.:
$lJ13STITlJTE~ SHEE~T

.. . .......... . . . , . . .. ...... , . .. .. .. - . . .. ,. ~ . -WO94/~14 21 3 9 S O 3 PCT~S93/04309 .
l compound of formula IX or X~ in the presence of an oxidizing agent. ~ny oxidizing reagent whlch can :~
conver~ the nega~ively charged rad~onuclide ko a positively charged radionuclide can be used. Preferred 5 oxidizing reagen~s lnclude lodogen boads, peroxides such ~s peracetic.acld, hydrogen perox~d~ and ~he lik~, as well as ~~chlo~o-4-toluene-sulonami.de (i.e. chloramine- . .
T). A mor~ pr~err~d oxi~i~lng ~agent l~ chloramine~
: An acid, e.g. ~Cl, can al~o be added. :.`
An ex~mple of a ~eac~lon wher~ the r~ a r_~ a~ ing ~u~ i~ d3pi~d ~ ;
below ~sing, e.y. a compound of ~ormula I~. ,`;
`::

1 ~ (CH2)~ ~O - N~ G~12)y~ ~ IX ;~
Rb~ .
~ R ~

r~dioi~o~ope oxldizin~ agent acid x Ba ~
~ _ ( CH2 ) y--CO--NR~ ( CH2 ~ y ~2 2~ Rb~X . ~.:
~R~
.:
: When R and R togekher with the carbon atoms a ~ .
to whlch they are attached form a cycloalkenyl or 3 heterocyclic ring the deslred cycloalkenyl or heterocyclic ring can be in place on the ~tart~ng materlal. For example, the R~ and R~ of formula V

SUBSTIT~ E SHEET
.:
.~

w~94e~l4 ~ 38- PCT~S93104309 1 togethex wlth the carbon atoms to which they are attached can form the desired cycloalkenyl or heterocyclic ring. .;
As is recognized by the skllled ar~isan, th~ ~ , 5 above procedures can be modi~ied for making ~he presen-t 1:
compounds ~o include other known and commonly available procedures. The procedu~es provid~d hereln are inten~ed to ~e ill~t-a~ive ~nd are no~ exilaus~ive; there~or~ the illustrated procedures should no~ b~ vlewed as llmlting j;
lO the inventlon in any wa~.
A~4hor ~ n~ ;,g p~ n~ ln~n~i~n provides a compar~m~ntalize~ ki~ ~or detectl~n o~ a mammalian ~umor which lncludes a ~i~s~ con~ainer adapted to contain at least on0 e~ ~he compounds o the present l5 invention, A further ~mbodimen~ o~ th~ present in~entl~n provides a compar~mentalized ki~ ~or ~eating a ~a~,~al'an ~"o~ wh~ch inciudes a llrs~ conta~ner a~ap~ed ~ ;
to contain at least ane o~ th~ compounds o~ ~he present 2~ inv~ntion, :~
r~.m.pe~ s _~ ~.h~ praJe~ .v-~ ion wnicn a~e -;
provided in a kit for d~tecting e- t_~atir.g a ma~mallan , :~
tumor can have any one of formula~ I, TI, ~, IV, ~II, ~III, IX or X. However more preferred compounds for the 25 present kits are of any one of fa-muale II, III, IV, VII, ~III or IX. Especially pxeferred compounds of the ~:
present ~nvention which placed ln klts include compounds L X " ~"
~ l u a ~. .
Compounds provided in the present kits preferably have a Q rather than an X group and such a Q
group is preferably an activating group. Activating groups presnt on compounds provided in the subject kits ,: ' ,, ~.:

'~

wos4/26314 ~ 9 ~ 0 3 PCT~S93/04309 -39- ! .

1 include tribu~yl-tin, trimethylsilyl or t-butyldimethylsilyl. Tributyl-tin is an especlally pre~erred ackivating group ~or compounds provlded in the present kits. .. .
The klts of the pr~sen~ inven~lon can be adapked ko con~ain anokher c~nkainer havlng ~ reagQn~
~or replacing a ackiva~ing group with a radlonucllde.
~r ~x2mple su~h a ~ag~n4 c3n ~9 an oxldl~in~ reagen~, :
e.g. chloramlne-T.
~n a ~u~ther embodim~n~, the kl~ a~ the pressnt inv~n~ion can ~ a~p~3~ to cont~Ln ~na~hQ_ con~ain~r having a material ~r ~parating unattached radionuclid~ ~rom ~h~i radiolabeled ~omp~unds o~ the present inven~lon having an a~ached X group. Such a 15 materi~l can ba any chr~ma~ogr~phic material including a thin layer chroma~og~aphy pla~0, a molecular exc}usl~n resLn, a sillca gel, a reverse phase r~sln and the like.
ror con~nience, suc;~ re~ins ~an al50 b~ pro~lded in ~ns form of a prepacked column.
2~ The presen~ compounds can h~ admlnl~t~r~ to a m~,m, m,3 t ~ g ~ rh ~ ~m~ 4 ~ --f.~ J ~
pharmaceutical composi~lons con~aln a dLagno~lc lmaginy :~
or an anti~tumor amount o~ at least ane of the DrQssnt compounds tog~ther with a pharmaceu~ically acceptable 25 carr~r.
The composltions can be administered by well-known routes lncluding oral, intravenous, intramuscular, in~ranasai, intradermai, subcutaneous, parentexal, enteral, topical and the like. Dependlng on the route of administratlon, the pharmaceutical composition may require protective coatings.

.. ..
':',...
SUBSTITUTE SHEET

wo 94/~6314 ~39~3 PCT~S93/04309 , , -40- :

1 The subject compounds may be lncorpora~ed into ;.
a cream, solution or suspension ~ox topical admlnistration~ ' ' The pharmaceutical fo.rms suitable for ~ . , 5 injectlon lnclude sterll~,a~ueous solutlons or disperslons and sterile powder~ ~Qr th~ extemporaneous prepara~ion of sterile in~ectable ~olutions or ', dlsp~s~ons. In all case~ th~ u1tlmate ~olutlon Xorm must be sterl~s an~ ~luid~ I~ypic~l c~rlers include 10 solvent or disper~ion medium conkalnin~, ~or oxamplo, wa~er~ buf~e~ aqugous sel~;4~o~s (l.~, ~ blocompatibie bu~ers), ethanol, polyo1 ~glycerol, propylene gly~ol, polyethylens glycol and the llke), suitable mlxtur~s ~
thsreof, sur~ac~ant~ or v~g~tabl~ oils. Steriliza~ion :':
15 can be accompli,shed by any ar~ racQgnized technlquo, including but not limited ~o, ad~l~ion o antl~acterial ;~
or antifungal agents, for exampl~, paraben, c~lorobutanol, phenol, so~bic acid, thimero~al, and the like. Fur~her, isot~nic agents, such as sugar~ or sodium chlorids mav bQ in~orpe--te~ e s~bJ~c~

ProductLon of sterLie Lnjectable solutlons ~:
contain~ng 2~ 1 east on~ o~ tha pressnt campounds is accomplished by incorporating ~hese compounds ln the 25 required amount in the approp~iate solvent with various ingredients enumerated a~ove, as r~quired, ~ollowed by sterilization, pre~erably ~ilter steriLiæation. To ~btain a s~erile powder, the above ~olutions are vacuum-dried or freeze-dried as necessary.
When the present compounds are administered orally, the pharmaceutical compositLons containing an ;
effectlve dosage of the compound, can also contain an .;.
.

. ~.;

SUBSTITUTE SHEET ~

:
WO94/26314 21 3 9 5 0 3 PCT~S93/04309 1 inert diluen~, an assimilable edible carrier and the lilce. O~ally adminis~ered composi~ions can be provided in hard or soft shell gelatin capsules, tablets, elixirs, suspensions, s~rups and the like.
The subjec~ compounds are ~hu~ prepared for convenien~ and effective administration in pha~maceutically eE~ective amoun~s with ~ ~uitabla ph?rmacs~-t~ca~1y ccop~ble c2rrLsr in a d~ags which permits diagno~tic lmaglng ar cance~ cell death. The~e lO amounts are pr~ferably abou~ 1 ~g ~o abou~ 1 g o the compound ~er li~er an~ re ad~}nls~sred in dos~5 Or abou~ 1 ng/kg body weight ~o ~bout 10 ugt~g body weigh-t, or from about 0.1 mmole/kg body wsight to about 500 mmol0/kg body weight. Preferred composltions pro~lde 15 eEfective dosages of the p~esent compounds in the range of about 10 ng to about 2 ug/kg for diagnostias and preferably about 5 to about 50 mmole/kg body weight for thera2'i Moreover when cansldering a dosage ~o~ ~
dia~nostic imaain~ of therapv, ~.he s~e~ acti~Ltv of Q _a~ "~,v'~ C ~,~k~,. ' ..-'~
consideration. Such a SpQcifi~ activity is pr~f~akly very high, e.g. ~or l23I-labeled compounds the spsclf~c activity should be at leas~ ab~ut 1,0~0 Ci/mM ~o about 25 50,000 Ci/m~. More preferably the specific actlvity for ~2~I-labeled compounds is, e.g. about 10,000 Ci/mM to about 30,000 Cl/mM.
As used h~rein, a pharmaceu~ically acceptable carr.ier includes solvents, dispersion media, coatings, ant~bacterial and antifungal agents, isotonlc agents, and the like which are physiologically acceptable. The use of such media and agents are well-known in the art.

.
~.
~UBSTlTUTE SHET ~

W054/26314 9~3 -42- rCT/lJS93/04309 The ~ollow.ing Examples ~ur~her illustrate the lnventlon.

'''' :

~, ~, ,~

SUBSTl rUTE SHEET

wos4l263l4 213 9 5 0 3 PCT~S93/0~309 -~3- ;

Synthesis o~ ~5I- ~
(2-Pipexi ~ .

Materials and Me~hods Melting points were d~term~ned wl~h a Flsher-Johns apparatus. lH and ~lC ~ gp~c~ra were recor~ed on a Brucker 300 ~M spectrometex. Vnles~ na~od, chemical :.
shi~ts were expresged as ppm usin~ te~ramethyl~llan~ a~ ~ :
an ln~ernal standard. Th~ ~hin lay~r ~hromatography (TLC) system consist~d o~ Analtech uniplate ~illca gel ~ -GF plate~ ~2$0 microns, 10 x ~0 cm), ~sind CHCl3/MeOH:80/~0 as ~olv~nt. ~a~ioac~lve ~po~ wer~
scanned and reco~ded by a ~loscan 300 ~magin~ scann~r e~uippe~ wlt~l automatic plate reader. Mass spectra (chemiczl ioni2a~ion) wsr~ recordsd on Finnlgan 1015 mass spec~rometer. Mal3lI was ob~ained rom duPont NEN
~nd ~a~Z5I was obtained rom aristol Meyo~s ~quibb.
Elemental analyses were pexformed by Galbraith : Labora~ory of Knoxville, TN.

Preparatiorl o~
(2-piper~ y~ino~th~l}4-b A round bottom ~la~k was charged witA 4-bromobenzoic acid (2.0 g, 9.95 mmol) in chloro~arm `.
25 ( 150 mL) . To th~ solution was added thionyl chlorLde ( 3 mL) in chloroorm ( 10 mL), 2-3 dxops o~ . :
dimethylformamide ~DMF). The slurry was heated at ~e1ux for 3 hr while monit~rin~ the rea~tion through a bubbler. A t::lear solution of 4-bromobenzoyl chloride ~.
3 was obtained, the volatiles were removed and a light yellow oil was obtained which solidified upon coollng.

,.

, ,.

`' '. .
SUE~STlTlJTE SHE~E~T
` .''.'' ~39 _44_ PCT~S93/0430 1 The 4-bromobenzoyl chloride was dissolved in .~
chloroform (30 mL) and added ~o a flask cont,aining 1 (2- :
aminoethyl)-piperidine (1.~9 g, 10 mmol) in chl~roform ;~
(20 mL). Triethylamine ~10 mL) wa~ added dropwise. The 5 mixture was stirxed a~ room temperatur~ ~or 1 hr and the ~olatiles were remov~ in vacuo. ~he resul~ing ~lurry ~ ;
was w~shed with 2~ sodium bicarbona~0 (2 x 50 m~). The `~
org~nlcs war3 d~s~olved in C~;Cl~ (100 mL), ~par~ted fram aqu00us layer and dried o~e~ anh~drous ~a2$O~. Th9 10 sol~ent was remo~ed to give a colorless s~lld ~A, 2.7 y~Qld~ 8~ 2 ~r~3/;~ 4~ pm~
1.~6 (t, 2H, CH2); 1.54 (broad m, 4~, CH2); 2.43 (broad s, 4H, NC~2); ~.52~2.56 (t, 2H, NCH2); 2.6~ (m, 2H, NCH2); 3.49-3.53 (d~, ZH, NCH~); 7.~1 (bs, lH, ~H); 7.S2-15 7~55 (m, 2H, arom); 7.65-7.6~ (m, 2H, arom).

Prepartion ~f This was prepar~d using a proc~du~e llke that 2~ described above for A ~u~ 5~ 4 ~ o~An~ acld ~3 ~ al. ~ w~i~e ~Oil~ (B) w2s obtalned in 89~ yield. lH R (? ppm~: 1. 43-1.45 (broad m, 2H, NC~2);
1.53-1.60 (broAd mr 4H, ~CH2~; 2.41 ~broad m, 4~, ~C~2); `~
2.50-2.54 (t, 2H, J=7.RH2, NCHz); 3.44-3.4R (dt, 2H, 25 NCH2); 7.02 (bs, lH, ~H); 7.47-7.4g (m, 2H, arom. ); ,, 7.73-7.76 (m, 2H, arom.). m.p. }14-115 C. Anal Cl4HlgN2Ol calcd. C, 46~91; H, 5.31; N, 7.R2, ~ound C, 46.91; H, `:;
5.~ 8.
'"'~

1'`"

,, ",...

,`.

~: '.', SlJE~STlTUTE SHE~ET `::

WO94/2~l4 21 3 9 ~ 0 3 PCT~S93/0430g l Preparation of A flame dried flask was charged with 4 ~romobenzamide (1.0 y, 3.Zl mmol) in triethylamine ~4~ mL). Tetrakls(triphenylphosphine)palladlum (370 mg, 0.321 mmol), and bistributyltin ~Z.4 g, 3.80 mmol) were add~d, and ~h~ mixture wa~ re~luxed un~e~ ni~gan ~or 1~ hr. The mixtur~ was co~d, so~vent3 d9~aZ~ LrOm the black resldue, and ~he ~olatlles were rem~ad in }O vacuo. The xesuLting blacX oil wa~ pa~ed through a silic~ 5_1 c~1umn w~th ~lution wlth. CH~73 ¦100 m~), ~ollowed by ~lut~on with C~tCll/MeOH: 90/10. The de~ired frac~ions, as chara~teri~d by thln lay~r chroma~ography, were pooled and ~ol~ent wa~ evaporat0d lS to gi~e an oil ~C, 0.4 g, 56%). m/e ~ 5~3 (M ~H) ~100%), 233 (M -Sn~u3) (40~ H R (d ppm): 0.82-O.g3 (m, 16H, Bu~ and CH2); 1.01-1.05 (m, 4H, ~U3); 1.22-1.37 (m, ~H, ~ .A5-1.57 (r" 8~ lperidi~yl xLng); 2.45-2.5~ (t, 2H, NCH2 plperdiny.L ring); 2.60-2.63 ~t, 2H, 20 J-6Hz, NCH2); 3.53-3.58 (dt, 2~, J 5.34Hz, NC~); 7.30 7.41 ~bs, lH. ~H): 7.4~-7.7P tm 4w ~r.~. 13r p la, :~
ppm): 9.60, 13.58, 25.7~3, 27.30, 29.00, 36.0g, 54.2g, ;~.
57.lS, 126.~3, 128.39, 132.00, 136.5:4~ 167.6g.

Radiolabeling of n-tribut~ltin PA~ ~C) with I-125 to Yield l25I(2 pi .
To 100 uL of an ethanolic solu~ion o~ (2- : :
piperidinylaminoethyl)4-tributyl~lnbenzamidQ (1 mg~ml), was added a solution of ~l25I~sodlum iodlde (1.5 mCi, `~-3 uL) ln 0.1 N NaOH, followed by the addit~on of 0.05 N :-! 30 HCl (50 uL) to adjust the pH of the solution to pH 4.5- ::

6. Fifty uL of a freshly prepared solution of N-chloro- -.
~' i ' SUBSTITUTE~ SHEE~T
..

WO94/26314 ~,~39 ~ PCT/US93/04309 ,~

l 4-toluenesulfonamide sodium monohydra~e chloramine-T ::
(1 mg/ml) was added to t~ above mixture. The contents ~.
were s~irred ~or 10-15 minutes a~ room ~emperature and 100 uL of a solukion of sodlum metabisul~ite (200 mg/ml) ~ .
5 were added. The reaction mix~ure was neutrallzed with.a saturated solution o Na~CO3 ~0.~ rnL). 0.4 mL o~ normal sallne was added and ~he organlc~ wer~ exkracted ln CHC13 ~ m~ ater vor~exing 30 secsnd3. The chloro~orm layer was evapora~ed in a str~am o~ ni~rogen.
10 The radioactivi~y o~ the aqueous lay~r an~ the organic residue was countsd, Ths tot~1 ~s~o~er3d ~dl'o~t~
in the residue ranged ~rom 74 to 89% ~n~6). ~h~ r~siduo ~D) was dissol~ed in 90~ e~hanol, and 10~ 0.01 M
phosph~t,e bufLer ~00 UL). ~ por~ion o~ D was spot~ed on 15 a TLC-SG plats alony with a sample o~ nonradioac~i~e (2-piperidinylamino~thyl)4-icdoben~.amide (~, as abo~e).
The TLC-SG plates were ~eveloped wi~h CHCl~/MeOH: 90/10 (Rf=0.4S). Another por~i~n ~ s ln~ecb2d into a Gllson HPLC fitted with a Wa~ers Z-modul~ xadi~l ;
20 compression separation system containing a mlcro ~ondaPak C-18 rev~rse ~hase oL~n. ~ Jr~ b~L ',~' Rheodyne 4125 in~ector (0.5 mL loop~. The r~t~nti~n time for D (l25I-P~B) using isocratic elution with g9/10 E~OH~0.1M phosphake bu~fer (pH~6.7) at a ~low ra~e of ,~;
1 mL/min, was 8.5 min., a valu~,id~ntlcal t~ tha~ of non-radioactive (2-piperidinylaminoethyl)4- ~.
iodobenzamide. ~;

Radi,lolabeling of n-tributyltin PAB ~C) with l3lI
Yield _ I(2-pl~eridinylaminoeth 1)4-i ~ l ~0 . . :
The same protocol as described above far i .
5I(2-piperidinylaminoethyl)4-iodobenzamide ~D) was used .
' '.' SlJE3STlTtJTE SHEET

W094/26314 ~ 0 3 PCT~Sg3/04309 1 except that the amount o~ 0.05 N HCl added to adjust pHbetween 4.5-6 was dif~eren~ due ~o diffexent concentra~ion o~ aqueous sodlum hyd~oxide solution in which Na~31I was commerclally suppl~e~. The worXup of 5 and the purlfication o~ 13lI(2-piperidlnylaminoethyl)4 iodobenxamide ~E) was identlcal ~o ~g~2-piperidinylaminoethyl)4-iodobenzamld~ (D) 3bove.
Ths reactlons dsscr~bsd here~nabo~e ar~
depicted in Reactian Scheme X.

~EAC~ON SC~

~ OH SOC~ ~ A~N~ ;
X~ ~N~> X~bJJ ';

NE!3 la: X .. ~r ~:
Ib: X .. I

NE'3 ~u35n ~;

1) NaI ;~:
Z) ~.05 N HCI. pH ~
3) Chloramine-T ,:
- :' ~ ~ ~N3 79 94%
, :

SUBSTITUTE SHEEI' wo 94,26~ 39 ~3 PCT/US93/04309 EXAMPLE
5ynthe~is o~ ~;
5-ioclo~ ( N, N~ e~hylaminoethyl ) ~
~ " .
. .
5Materials and Metllod~
~ _ ,. ..
Mel~lng polnts were d~termlnQd wlth a Flsher~
Johns apparatus. l~t ~nd 13C t~ spec~,r~ ~re~ recorded ~n ~ ~ .
Brucker 30Cl A~ spectrome~er~ Unless n~t~d, chemlc~
shi~ts were ~3xpres~d as ppm Usin~ t~ramethylsil~ne ~s 10 an internal standard. Th~ t:hln lay~x chr~ma~ogr~phy .:
(~I3 C) S~s~-gl~l C~ns~3J''e'~ 0~ ~nal~e~h un~plats s~ a gel GF plates ~250 microns, 10 x 20 cm), u~ing .;~;
CHCl~/MeO~:80/20 ~s sol~rent adioactive spot~ w~re scanned ~nd recorded by a ~iosc~an 3ao i~aging s~anner 15 e~u~pped with automa~ic plate r~ad~r. Mass spectra (chemical ioni~ation) were recorded on ~innigan 1015 mass spec~rometer, Na~ was obtained from duP~nt NEN ,~:
and Na~~I was obtained rom Bristol Mey~rs Squlbb. l.. `
Elemental analyses were per~ormed by ~albraith 20 ~bo~ U......... cx~ , TL~. 1 1.~; ,'.

To a solution ~,3-dihydrobenzouran (25 g~
0.21 mol) in chloroorm ~100 mL) was ~dded dropwise at , .
25 0C, a solution of bromine (67 g, 0.42 mol) with `~:
stirring. The reaction ~ix~uxe was stirred overnight at .
xoom temperature. The excess bromine was dastroyed by ::
addition of a saturated sollltion of sodium thiosul~ate .:.
(30 ml). The organic layer was separated r~m the 3O ~norganic layer and washed with 2~ sodium bicarbonate (2 .
X 50 ml), then dried over anhydrous sodium sul~ate. `
The volatiles were removed in vacuo to provide a light ;

:. .
:
SU~STITWTE SHEET

WO94t26314 2 I 3 9 ~ 0 3 PCT~S93/04309 , -49-1 yellow oil ~51 g, 87~ H NMR ~CDCL~) ~ ppm: 3.~1-3.27 (~, J - 9 Hz, 2~, CH2): 4.57-4.63 ~t, J ~ 9 Hz, 2 H, OCH2): 7.14-7.15 ~t, l H, arom.): 7.32-7.33 (t/ 1 ~, arom.).
~;
Synth~ a~ 5-bromo-7- ~;
~ 1'."""';
To the a~ove dibxomo compound ~ lS g, S3, 9 mmol) was added anhydrous t,e~rahydrouran ~50 ml). The .
solut,ion was co~le~ a~ -78 C unde~ nlt~ogen atmosphere. 1.~
A solution of n-bu~hyl~ hlum (~.0 M. ~7 ml) ~as ~ddsd .. ;:
to the mixture dropwise. The mix~u~ tul^n~d lighk `-yellow brown. Aft0~ 5 mi~ute of stir~lng at -7~ C, carbon dioxide was ~ubblèd th~ough ~he mixture, gi~ing a ~.
5 s~raw yellow color to the m~x~ure. Tho mix~ure Wa5 then warmed up to room ~empe-atu~e an~ s~ir~sd for 30 minutes. A dirty white color solid was ob~aln~d upon :;
filteration (7.0 g, 53~ H NMR ~d~MS~) 2.~5-2.95 ~.
(t, J - 9 H~, 2H, C~2: 4.3S-4.4S (~, J ~ 9 Hz, 2~, OCH2)~

7.1 (m, lH, arom): 7.4 (m, 1~, arom). 13C NMR ( d6-DMS0 ~ and CDCll): 27.~8, 71.78, lL0.51, 114.17, 131~2, 131.25, 153.09, 164.87. ~nal., C9H7BrO3 calcd. C, 44.44i H, 2.88; found C, 44.52; H, 2.97.
. .
Synthesis of 5-bromo~~N, N'-diethylamino~ , ;;

A round bottom flask was chargQd wi~h .
bromocarboxylic acid (1.79 g, 7.36 ~nol) and chloro~orm ::;
(50 ml). The slurry was skirred and thionyl chloride (~.0 ml) in chloroform (~ ml) was added to the slurry ~`:
3 along with 2 drops of DMF. The mixture was refluxed for 90 min to give a clear solution. The volatiles were removed in vacuo to give yellow solid. This acid ~::

";

~ ~, S~E~STITUTE SHE~
` ~.

wo 94/2~14 z~3g ~ 3 PCT~S93/04309 . -50-1 chloride was used wi~hout fur~her purification ~or the ".
condensation with amine. To another flask containing N,N-dlethylethylenediamine ~0.82 g, 6.99 mmol) and :~
triethylamine (lS ml) ~nd CHClJ (30 ml) was added a 5 solution of th~ abo~e acid chlorlde in CHC13 ~1$ ml~.
The mix~ure was stlrred or 3 hourg. The~vola~iles were removed, the residue was wash~d wlth water ~50 ml) and the org~n~s wor~ dissol~ed ~n CHCl~ (,S ~). Th~ ~ ;
organic layer was sep~rated, drl~d o~er anhyd. N~2$O4, lO and the volatilss remov0d agaln to glve a ligh~ yellow ,;~
color oil. Th~ oLi Wa5 ou~ d ~ 3s~g~ ~h~ h a silica gel column a~d elution with CHCl~/MeOH:90/10.
The ~rackions con~ainin~ ~he dssirsd compound w~re ~:~
pooled together, ~nd th0 ~olatiles were remo~ed to gi~
15 the c~rboxamlde ~l.g y, 80~ 'LC silic~ gel R (0.7) CHCl3/~leOH: 90/10~ The hyd~ochloride salt was made with an ethanolic solu~ion of hydrogen chloride gas upon t~it~xa~ion ~h anhydrous ~,h~ N;~R ~DCl~) c ppm~
0.9~-1.00 ~t, J Y 7 Hx, 6 H, NCH2C~3): 2.46-2.53 ~q, ~ = !
2Q 7 Hz~ 4 H, ~CH;CH~: 2.55-2.59 (t, J ~ 7 ~z, 4 H NCH ): :;
3.17-3.~2 ~, J ~ Q U 7 ~ 7 u, r~z ~ ~, 4~_ ~, A ~ ~ ""
NCH2): 4.63-4.69 (t, J = 9 Hz, 2 H, OCH ~: 7.3~ (m 1 H. I:~
arom~: 7.961-7.968 (m, 1 ~, arom):. Anal.
Cl5H2~BrN2O2.2HCl, Calcd. C,47.68; H,5.82; N,7.41; E~und 25 C,47.38; H,5.~0; N,7.35. -~
,':' Synthesis of 5-tributyltin-(N,~'-di~thylamino-ethvl~-2.3-dihvdroben7.ofuran-7 _ b~x~m~.dQ ! J`
A round bo~tom flask was charged wlth 5-bromo~
carboxamide (1.0 g, 2.93 mrnol), bis(tributyltin) (~.4 g, 3 4.1 mmol), palladium tetrakis (triphenylphosphine) ~0.35 g, 0.29 mmol) and triethylamine ~55 ml). The mixture ~-:

, ~.
SUE3STITUTE SHEET ~

WO9~/26314 21~ 9 ~ fJ 3 PCT~S93/04309 -51~
. .: ., ;
l was refluxed for 3 hours. The volatiles wexe removecl in ~.
vacuo and the resi~ue was dissolved in CHCl~. Thls ;:
solu~ion was loaded onto a sillca gel colurnn ~nd eluted ~.
first wlth C~C13 ( 100 ml) and then wl~h CHCl3/M~aOH: 90/10 5 wh~.reby a llght brown band was collec~ed. Th~ volatil~s ~., were r~moved ln vacuo to giv~ ~n oil ~1.3 g). The TL ;
show~d a sllghtly impure cQmpound. ~h~ oil w~s pa~sed through a shor si~Lc~ gQl column agal~ and gl~t~ with : :~
CHCl3/MeOH: 90/10 ~o gl~e O.g g puro tln comp~ound. TLC ..
lv (sillca gel) Rf~0.,4S (CHCl3/MeOH: 90/10). lH NM~ ;
~CDc13) 0.33-1.60 (~ 3 3 ~, nBUJ and r~Cr~ 3~ r 2.il~
~q, J ~ 7 Hz, 4 H, ~C~2CH~); 2.60-2.66 ~t, 2 H, C~l2);
3.18-3.23 (t, J = 8 Hz, ~2); 3 44~3~50 ~, J ~ 6 Hz, H, CH2); 4.64-4.70 (~, J 9 Hz, 2 H, OCH2~; 7.32 (m, 1 15 H, arom); 7.~7 (m, 1 H, arom). lJC ~CDClJ) (~ ppm):
9.67, 12.01, 13.60, lfi.45, 2~ 7, 27.30, ~7.~2, 2Q.03, 37.58, 47.06, 51.72, 71.74, 115.~7, 127.26, 132.65, 135.53, 136.76, 158.0~, 164.92.
~ ~;
V S y ~ ~ ~h ~ ~ g--~ N ~ ~ .~. J
e ~ h v l ~ - 2 ~ h ~ ~ h~ =
Tributyltincarbox~mlde (300 mg) and lodln~
~0.8 g) were stirred t~g~ther in CHC13 a~ room temperature for 48 hours. The mix~ure was qu~nched with a saturated solution of sodium thiosul~ate. The organic 5 layer was ~eparated, dried and the volatiles were removed in vacuo to give a colorless oil. The oil was ~s~d ~ h th~ c~ ~n ~n~ t~ w~ th CHCl3~MeOH: 95/5. The first few fractians contained tributyltin ~odide and were discarded. The later 3 fractions provided the deslred iodo (0.2 g, 9s~) compound. TLC slli.ca gel Rf = 0.3 ~CHCl3/MeOH:90/10).

3S :
,;

SUBSTITUTE SHIE~ET

~ 5~_ rCT~ss3/o43~g l lH NMR ~C~Cl3): 1.34-1.39 (k, J = 8 Hz, 6 H, NCH2CH~
3.14-3.33 (overlapping multiplet and triplet, 8 H); ::
3.85-3.91 (q, J = 6 Hz, 2 H, NC~I2); 4.71-~.76 (t, J ~ 9 ,:~
Hz, 2 H, OCHz); 7.54 (m, l H, arom); 8.02 (m, 1 H, 5 ~rom).
.
Synthesi~ ~ 5-b~omo~ 2~aminoethyl- ~ .
pipexidinyl)-2~3~dih~ uran~7-c~rboxami~
A r~und bottom ~lask wa~ charged w.L~h bromocarboxyl.ic acid (2.0 g, 8~ mmol) and chlo~oorm (50 ml). The slurrv was s~irr~d and ~hl~nyl chlorids (4,0 ml) in chloro~orm ~10 ml) w~s add~d to the ~lu~ry along with 2-3 drops ~f dimethyl~ormamide. The mlx~ur~
w~s ~efluxed ~or 6C mLn ~o glve a cle~r solu~ion. The .
1 volatiles wer~ remov~d in vacuo ~o give a yellow ~olid. ~:~
Th~ acid ~hloridQ was us~ withou~ ~rth~ puxiflcation ~.
for the condensation with amlne. To ano~her fl~sk containing 1-(2-aminoethyl)piper~e~e ~ , 8.98 mmol), trlethylamine (15 ml) and C~c13 ~4~ ml) was added 20 a solution of ~he abovo acid chlorlde irl CHCl~ ~0 ml).
The mix~ure was stirr0d ~or 3 hours ~ r~n~ ~Qm,r~
The volatiles were ~emoved an~ ~he ~esidu~ was takan up in CHC13 ~100 ml) and washed with water (2 x 50 ml).
The organic layer was ~ep2rated, dried ~ve~ anhyd Na2S04, and the volatiles removed in vacuo to give a light yellow oil. The oil was purified by passage through a silica gel column when elution with CHCl~MeOH: ~0/10.
The desir~d ~r~ .s we-e -_...~'..~d an~ th~ volatiles were evapora~ed to give light yellow oll (2.4 g, 83~).
Rf (TLC silica yel CHCl3/MeO~:90/10) - 0.7. lH NMR (~
ppm): 1.42-1.60 (m, 6 H, piperidinyl cHz's); 2.42 (bs, 4 H, piperidinyl NCH2); 2.48-2.S2 tt, J - 6 Hz, 2 H, CH2);
.

SUE3STITUTE~ SHE~
'~.

l:
W094/2~14 213 9 ~ 0 3 PCT~S93/04309 j~
-53- 1:

l 3.20-3.26 (t/ J - g Hz, 2 H, NCH2); 3.48-3.54 (m, 2 H, NHCH2); 4.68-4.73 ~t, J ~ 9 Hæ, 2 H, OCH2); 7.33-7.35 (m, t 1 ~1, ar~m); 7.98-7.9g ~m, 1 ~1, arom); B.OS (bt, 1 H, 1 ;
~) ~':,:' Synthesis of S-n-txibutyltln-1-(2~aminoothyl~
ni~eridinv~ 3-dihvdrob~nzo~uran ~7 ggL3~ab~n~C~
5-bromo-caxbox~ml~ 0 g, S,63 mmol), I :
bis~tributyltln) ~3.~ g, 5.7 mmol), and palladlu~
t~trakis (triphenylphosphlne) ~.33 g, 0.~8 mmol) wexe ~ -re1uxed overnlaht ( 15 hr5 ) in trle~hyl~min~ ~100 ml~
The black r~sidue was separ~t~ ~rom khe sol~nt. The volaSlles were removed and the y~llow ~esidue was passed through a siiica gel column and elu~0d ~irst with CHC1J ~:
tl50 ml) and then wi~h C~C1J/M~OH: 90/10. The ~actions conta~ning ~h~ dssired compoun~ ~er~ comblnsd tog0thQr -~
and the volatiles were removed to give a light ~ellow viscous oil (~.4 g~. ~ NMQ ( ~ pp~ O ~ Q2 ~ ~7 ( 4 ~ J -7 Hz, 9 H, nBu3); 0.98-1.5g (m, 25 ~, nBu~; anc~
piperidinyl CH2); 2 . 48 (bm, 4 H, pipericlinyl NC~"); 2 . 49-2.54 ~ = 7 ~z. 2 ~, C~ ~: 3~21-3.2.~ t = 9 ~. 7. ~
H, CH2); 3.5i-3.55 (m, 2 H, NCH2); 4.63-4.69 (t~ J - 9 ,;
Hz, ~ H, CCH2); 7.34 (m, 1 H, arom); 7.95 ~m, 1 H, arom ) .
The reactions described hereinabove are 5 depict~d in Reaction Scheme II.

:

.
"

$UBSTITILJTE~ SHEET
~'.

WO 94/26314 PCTIUS93/04309 -- ~:
39~3 ; -54- ;
'"'",~ :~
,~, 1'~' ,'','' ','~

R}:ACTIO~ SC~ Iï , ~:
,~

--~\. SOC~
11 / ~ 1 1 \ :., Br~~
~N~ C~ x~

C ~ ~ R

~\ ~ ~ 0 5n ~, '25 ;,.

'~
''~

"~

su~3~;T~T~J~E ~3H :

WO94t26314 ~l 3 9 ~ ~ 3 PCT~S93/04309 1, .

In Vikr~ Compatitive ~inding of Radioactive and Nonradiaactive ~2-Piveridinvl~minoeth~lS-4-Iodo~enzamide ~ .
Competitive binding s~udies indicate ~hat compounds of the p~es~nt lnv~ntion bi:nd malign~n~
melanoma oells wlth ver~ hlgh a~~nit~.

A205~ cell~, d~rlv~ rom a b~in m tasta~
of human ma llgn~n-~ m~l~rl~ma (Todaro e~ al. lg80 ~x~c.
N2stl ~ Acad. Sci. USA 77: sas~ ) were ob~ainqd ~om tha National Insti~u~es o Health. The~e c~lls w0~e g~own inS DMEM2 medium ~Dulbecco's mo~sification o~ Eagle's medium, E~lEi~s) supplemensted wl~h lO~ fe~al ~oviri~S se.~um :~;
and 0 . 03~ L-glutamine .
! 2~ inJ~lai"' no~hy' ) ~-iodso~sn~.amide ( i . e .
IPAB, B) and ~2sI~2-pip~ridlny.lamlnoekhyl)4-iodob4nzamide ~) was synthesized as describecl in Example 1.

In VltroIa~L~_~L~IL~be~9~ ,~
A2058 cells, grown as descxibed above, were harves~ed with calclum and magnesium xee phosphate buffer (0.1 M) containlng 0.~2% EDTA. C011s wsre washed twice with ice-cold RPMI 1640 medium (Gibco) without glutamine and ~esuspended i~ the sam~ medium. Carrier-free rI25IlPAB (0.1 ml! was adde~ to Q~h~ etc ^~
0.1 ml test A2058 cells (1.5 X ~o6 cel1s in suspension).
To observe competitive binding hy non-~adiQ~ctive IPAB, , I ~,~ :
J varying concentrations of non~radioactlve IPAB were add~d in a volume of 0.1 ml. Cells were incubated at ~:~
.

~ 35 STITUTE SIH EET
'`:

WO94/2~14 ~ ~ PCT~S93/0430 ~ $6-l 37C ~or 5 hr af ter add.ition o~ radioactive and nonradioactive IPAB.
After incubation, c~115 w~re collected by ~:
centr~fugation for ~ min and washed twice with RPMI 1640 5 medium. The xadioac~ivl~y bound ~ cells was counted using a Packard Autogamma 5650 ~cln~llla~ion counter. ;~
Data w~re analy~ed wikh an ~NP~O~ er~tive, non-l~near lea~ s~u~r~ cur~ ~iL ~ing program.

0 ~ _3ul t3 Fi~ a~ h~ ;P~ blnds to human malignant melan~ma cells wi~h hiyh a~lnlty. In particular, Fig. l shows ~he amoun~ o~ nQnradioac~iv~
IPAB n~eded to compstL~,iv~ly ~nh.~bit blndLng of 15 radioactive IPAB. ~indlng o~ 50~ of the radioac~ive IPA8 was compe~i~ively inhi~ d by as llttle as 6.8 nM
(i.e. Kl is 6.~ nM). These ~ata indic~e that IPA~ ;
binding is so highly selec~ive an~ stable that khe : interactlorl o~ IPAB wit~l human mallgnant mel~noma cells ~ V 1 ~ k ~ 1 y !) C ~ 1~ _ S ~ T ~ ,~ ~ S ~1 ~, S ~
r~ap~r.

, :, ~ :

.; ', ..~,.
` 30 ;~

SlJB~3TITlJTE SHEET

~ ::

WO 94/26314 PCT/TJS93/04309 ::
-57- ;;

1 EXAMPLE 4 .:
~_ .
In Vi~ro Binding Competition Between :
Pharmaco~ogical Antagonists and ( 2-Piperidin~lAmirloethyl ) -4-Iodo~ nzamide Competitive bindlng studies ln~lcaJce that compounds of the p~es~n~ lnvention blnd cell sllr~a~e sigma receptors on m~llgnant m~lanom~ cell~.
!

klat~rial~ ~nd Metho~
A2058 c~lls, ~erived from a ~rain me~astasis o~ hum~n m~lignasl~ melanom~ (~odara ~ Proc.
Natl . ~c~d . Sci . US.~ 77: 5258 ) are ob~ained ~rom the National ~nstituk~s o~ ~eal~h. Th~ celLs ~re grown in DMEM2 medium (Dulbecco's mc:dl~lcation ~ Eagle's medlum, 15 EMEM) supplem~nted wl~h 10~ e~al bovin~ semm and 0 . 03%
I.-glutamine .
~ 2-Piperidinylaminoe~hyl ) 4 ~ iodabenzamidQ ( i . e .
IPAB, s) and I(2-pipe~idinylamlnoetnyi)~ iodoben~,ainide ~:
~ D) is synthesized as cie~cribed ln Exampls 1.
;~O Ph.~ are~ ir~l anta~enists and the correspo~lng recep~ors wnic'n ara ~ "~ 4C~_ I
2 (i.e. si~ma recQptor an~aganist), ~luph~nazin~ (slama- ~:
l at low concentra~iens 2nd sl~mP-2 at high : concentrations), SC~23390 (dopamine~ aclopride 2r (dopamine-2), melanocyte secrekin~ hormane peptidQ
~melanocyte secreting hormone receptor), mianserln (S- ~:
hydroxytryptamlne-l receptor), NAN-l90 ~S-hydroxy-tryptamine-la recep~or), kat~nserina ~-hydroxy~ryptamine-lc receptor), ketanserine and ;~:
mianserin (S-hydroxytryptamine-2 receptor) and 3-tropanyl dichlaroben (S-hydroxytryp~amine-3 receptor~. ~

~:
,;
S~ BSTI~JTE S~EF~

;,, WO94/2~14 ~ ~ 3 - 58- PCT~S93/04309 ,_ :~

In V~tro Cell Bindinq Assay A20g8 cells, ~rown as described above, are harve~ed with calc.ium and magn~sium ~xee phosphate buf~er ~0.~ M) con~alning a.o2% EDTA. Cells are washed 5 twice with ice-cold ~PMI ~1640 medium (Gibco) wlthout glutamine and resuspended in ~he ~am~ medium~ Carrier-~ree ~l2gI~AB (0.1 ml) i~ added to elgh~ allqu~ks o~ 0.1 ml test A205~ ccSlls (l.S X 106 c~ n sUspc~ns~n)~ To observe competltive blnding by ph~rmacological lO antagonlsts, ~arying concentrakion~s of the ant~gonlst9 2r~ th~n add~d in a tolum~3 o, 0~1 ml. Csl~ e -' incubated a~ 37C for 5 hr a~ter a~ lon o~ an antagonist and th~ radloactiv~ IPAB. ;:~
A~ter incuba~ion, c~ ar~ collect~d by I5 centriuga~ion or S mln and washed twice wlth RPM~ 1640 medium. The radloacti~ity bound ~o cells is count~d ~:
using a Packard Autogamma 5650 scin~illa~ion count~r. : ;
Data can be analyzed with an INPLOT~
lterative, non-linear least squara curve El~ing ~^ ~rogram.
-F~PS U 1 ~S , ' An~agonists with d~monstrated binding ~ :
specificity for cell ~urace ~igma recept~rs (~.g.
25 fluphenazine) can act as competiti~e binding inhibitors of IPAB binding to maligna~ melanoma c911s. In contrastr antagonists that do not bind to c~ll surface sigma rec~p~ors canno~ inhibit binding of radioact~e IPAB to melanoma cells. Such data in~icate that th~
3O present compounds bind to cell surface sigma receptors.

SUBSTIITUTIE S~EET

.

wos4l2~l4 21 3 .9 ~ V ~ PCT~S93/04309 -59- .
.

1 ~XAMPLE 5 Binding C~mpe~ikion Be~ween Pharmacological ~n~agonisks ~nd Materials and M~th~ds ~:
_ _ . __ ~ 2-Piperidinylamin~h~l)4~lo~obenzamldo ~i.e.
IPAB, B) was synth~siz~d as c1esc~ib~d in Exampl~ l.
A sigma-l bindlng assay w~s pQr~med in lO guinoa pig brain memb~an~s and ~a~ C6 glioma cQll3 (purchased '~om Am~rican Tis~u~ and Cell Collectlon, Rockville, M~) ln ~he presence o~ ~ ~igma~l selective lig~n~, t 3H~ pentaz~ine, ~ sigma-2 binding assay wa~ pe~Iorm~d ln ra~
15 liv~r membranes in ~he pre~0nce ~ ~ slgma-~ selecti~0 llgand, ~3nJ~TG, in ~h3 px~s~nce 0~ dex~ra}lorphall to mask sigma~l sites.
: ;.
ion:
A plasma membran~-mltochandrial (P2) m~mbrane ~r~ctiQn w~s orQDar~d f~om rozen ~uinea pig b~ains (Pel-Freez~, Rog~rs~ AK), minus cerebellum. The b~ain tissue was thawed slowly be~re homogenlza~lon. A crude P2 m~mbrane ~raction w~s also prepar~d from ~he li~ers :~
25 of rat Spsagu~-Dawley ra~s (150-220 g, Taconic Farms) liver. The anlmals were decapi~a~ed and theLr li~ers ~;:
were minced and homogenized. The tissue homogenization was carried out at ~ C in m.~ ~'s~ue WQJ~ht ~f 10 mM
Tris-HCl/0.32 M sucrose, p~=7.4 using 10 mo~or-driv0n -~
3o strokes ln a Potter-Elvehj~m Te1On glass homogenizer.
he crude homugenate was centrifuged for 10 min at lOOOg and the crude nuclear (Pl) pellet was discard~d.

~,:

$UBSTI~UTE SHE~ET ; `
... ..
'~' ' .. , .. . .. , . ~ . . , ~, WO94/2~14 2 ~ 9 ~ e PCT/US9310430 1 Supernatants were centrifuged at 31000 g fox 15 min to yield a plasma membrane~mi~ochondrial pellet (P2). This pellet was resuspended in 3 ml/g in lO mM kri~ HCl, pH
7~4 and used for binding studies. Pxotein 5 concenkrations were dekermlned, by the method, of Lowry.
~arious concentratLons of khe ~PAB ranging ~rom 0.5 - 1000 nM wex~ lncubatad with guin~a plg brain membranes (300-500 microgram p~oteln) in the ~resence of 3 nm [~ penkazocine ~p~ci~lc acki~ity 52 Cl/mmol) lO in 0.5 ml o 50 mM Tri~-HCl ~or 60 min ~t 37C. The ~mcun~ ef non speciJ~ h~n~ing was ds~xmir.ed by the additlan of 10 mM Tris-HCl, p~l 8.0 ~ollowed by rapld, ~ilteratlon through gla~s ~ rs u~Lng a ~randel Cell ~;
harvester tGal~h~rsburg, MD)~ Fll~er~ we~e washed ~wi,e ~i 15 with ice-cold buffer. Prior -to u~e, filters wera s~aked, ~;
ln 0.5% polyathyleneimine ~or abeut 30 min ,t 25~ C. .;
Similarly rats Iiv,9x mem~ranes (sl,~ma~2) o~ C6 glloma cell homo~sn~te~ o in~u~ wLth 3 nM rJH~D~G ~3q.4 Ci~mmol) in the presence of 1 mlcrom~lar cold 20 dextrallorphan and var.ious concen~ra~ion o the ::
unlabeled XPA~. The amoun~ of non spsciic bl~din~ was ~.
determined by incuba~ion uf membran0s in the pre~ence of 5 micromolar haloperidol. ~:
When the assay was terminated, the membranes 25 were filtered and the iltra~e washed twice as above.
The radioactivity was counted in Ecoscint ~National Diagnostics, Manville, NJ) after an overnigh~ extxaction ,U,.~g .
The amount of IPAB required to inhibit binding o~ sigma-1 and sigma-2 selective ligands by 50~ (i.e.
the ICso values) was derived using the computerized iterative curve~fitting program, GraphPAD. K1 values '~

8lJB~3TlT~JTE SHE~ET

WO 94/26314 21 ~ 9 .~ ~ 3 PCT~S93/û4309 were calculated from the IC50 values using Cheng-Prusof f equation~

Resultg The K1 value~ for IPAB are shown in Table 1.

Sigm~- 1 Slgma-~ Sigma -~Gu i nea_P l g Br~ i n ~ C~_I D_ a . 89 nM 24 . O nM 130 nM

Thes~ da~a demonst~ate ~hat ~PAB blnds to cell sur~ace sigma receptors with ~re~y high aeinity.

` ~.

2u ;:

; ;~

~;:

i: ~
1:
~.
~, SUBSTITUTE Sl~ ET
'";

WO g4/26314 PCT/~JS93/0430g ,,.~ ' ~,~39 3C~ -62-Biodls~rlbuti~n a~
5I- ~ 2-Piperidin~Aminc)ethy 1 L~4-IodoBenzamide ,. :
Biodistribution éxperlm0n~s were pex~o~med to a~ses~ th~ tumor-spe~ficiky u;~ the pr~sen~ c:ompound~.

l~tsrials and U-eho~ls ~:
A2058 ~umor a~lls, d~rlve~ ~.rom a b~ain ;~
metastasis OL human rnallgnan~ m~l~noma ~Toclaro et al.
1980 Proc . N~tl . Acad . Sci . US~ 77: 5~5~ ~ w~re oktairl~d ~xom ~he Naklonal l~nstl~ute~ o ~ealth. ;~:~
N~n-small c~ll lung carcln~ma cell lines NC~-157, Nc;-a3a an~ NCl l~g~ were ob~in~d ~x~om th~
National Cancer Instl~ute. ~he NC~ ~157 cell line is ~ ~
squamous carclnoma cell line, while NCI-~38 is an aden~carcinoma cell line and ~C~-12g~ i9 a large c~
lung c~;nom~ c~
Tumor ~ells wer0 gro~n in DMEM2 medlum (Dulbecco's modiication of Eagle's medi~m, E~EM~ ;~
suPpl emented wit:h Ln ~; f ~ r ~ r~g ~.g~ ,.."
glutamine.
l25I-N-(dlethylaminoe~hyl~4-iodobenzamide ~i.e.
['2sI]DAB) was p~èpared A5 described ln John et ~1. (1993 Nucl. Med~ ~lol. 20: 75-79).
l29I(2-piperidinylaminoe~hyl)4-lodobenæamlde (i.e. ~l25IJPAB) (D~ was synthesized as de~cribed ln :~
S; X~r l ~ 1 .
.

3o For ln vivo studies, tumor cells were harvested using calcium and magnes~um fr~e PBS

:

SUBSTtTlJTE SHE~
.

.

WO94/26314 213 ~ ~ 0 3 PCT~S93/04~09 ~ -63- :

l containing ~,02% EDTA. Suspension o~ 5 x 106 cells (vlab~llty gre~er than 95~) in 0.~ mL o medium were innoculated subcutaneously in ~emale Balb/c nu/nu mice. `;
Afker about two weeks, ~solid tumors of about 1 cm in ";
5 diameter appeared in ~pproximately 85~ o all innoculated mice. Mice with soli~ ~umor~ havlng a : .
diameter of abou~ 1 cm were used ~or biodistxibution ~:
s'udles.
Balb/c nu/nu mlce ~17-22 g~ ware ln~ected ,~
lO int~avenously wl~h 0.2 ml of a sallne ~olution ~o~alning ~ ~'T ~p~3 ~ J-6 ~-Ci ) . At 1~ 5 ar~ 2~ h~
in~ection, blood ~amples w~re ~ollec~ed by cardiaa ~.
puncture and ~he mica were sacri1~ed immedlately thereater by cardiectomy while under halothane 15 anesthesia. The organs o in~er~s~ were subse~uently excissd, blot~sd wlth ~ssu~ pap~r, w~lghsd, and the ~;
radioactivity was coun~ed using a Packard au~omatic counter ~autogamma 5650), The ~ in~ect~d do5e/g ID~g~ values were datermined by comparison oP ti59U~
2G _ ~s~ Qg '~ h S~_s~Zb1y ~ 5~"- t~ h~ -ln~ec~e~ 9IJPAB aose a~ e~ Dy cne welgn~ oi ~n~
organ. Tha ~alues obtained were normaliz~d to a mouae :.
w~ighing 20 g. The di~'3~ence~ ~etween t~29I]PAB and ~`
t~25I~DAB were ~xamined by Stud~nt'~ unpaired t tests.
~5 R~ul~s Tables 1-3 illustr2te the biodistrlbution of [lZ5I]PAB and ~ I]DA~ in nude mice bearing human A2058 melanoma xenogxafts in the ~lank at one, six, and ~wenty-four hours, respectively, after administration o the imaglng agent.

;

~ ., SU13STITI )TE SHEET

WO94/26~14 2~39 3~ PCT~S93/04309 1 At one hr. posk-injection ~Table 1), the ...
concentration of ~lZgIJDAB ~% injected dose/gm) was higher than the tumor concentration of ~ZsI]PAB ln several ~issues lncluding non-tumorous liver, muscle, 5 lung and heart tissues. ~here~re, while ~l2sI~DAB
collected in ~he tumor at a marglnally higher level ~han ~25~PAB, ~125I]DAB w~s signllGantly 1ess speci~ic or .
the ~umo~ site than E ~ l~Aa By 6 hrs. ater admlnl~e~l~g th~ di~gnostic 10 agents, mice recei~ing ~la~I] P~ h~d more o~ thl~
diagnostic ag~n4 ~' n ~h~ o~ ~han ~n~ o4h~_ t~ gSUg ~ Tn contragt ~ 125~ ]DAB was found a~ higher concen~ra~ions ln the liver than in the kumor. ~oreo~e~, the COTlCOrltratiOn o~ t 125~ ~A3 was signlicant1y high~r than tha~ o [l2sI}PA~ in non-canc0rous blood, li~er and intestinal ti~5U9S.
By 24 hrs. mlce ~ecelving ~l2sI~PAB had about four-fold more ~g;]P~B ~n ~heir ~umors than in thoir livers. In contrast mice recei~ing ~ ~]DA~ had only ..;
,~ a~out half ~s ~.uch rl25I~A~i in ~h~ tumors ~g ~he~Y
iivars. lnese data indicate tha~ nîgn ie~eis or : [l25I~D.~B are non~-speci ~cally local~d in ~h~ e~.
These d~ta also indicats ~hat ~l~5I~DAB has 1~ss tum~r specificity than [l25I]PAB.
Moreover, ~he ~umor concentxation of [lZsI]PAB
: was almost twice as high as that of ~ 12~I ]DA~ indicating that IPAB ~indq to tumor cells with gxeater affinity and stability than IDAB. These data lndlcate that [~--I]PAB
is highly specific for malignant tumors which contain cells having sigm`a receptors.

:

~UBSTITlJTE 9HEET
j,.
,,, .

21 3 9 ~ D 3 PCT~S93l04309 . WO9~/263t~

1 Table 1 ~ .' Biodistribution o~ N-(piperidinylaminaeth~
4-iodo ~l25I~benzamide, ~lZ~I~PAB, and `~
N2s(diethylaminoeth~1)4-iodo ~lZI]benzamide, ~ IlD~B, in nude mice x~nogxa~ted with human melanotic mel ~ 9i_ ~ `
, ' .
.1 . .
~ Value for ' ;
lO ~ Hour ~ l2~]PAB ~l2~ AB Dif~rence .'~ .
Blood 0.~67(.168) 1.03(.318)NS
Liver 6.35~,770) 1~.7~1.6~)~.001 Spleen 3.11t.789) 3.46~.206)NS
15 K~dney 3.82(.56l~ 4.63~.905)NS
Bone 0.750(.0663) 1.04(.476)NS
Muscle 0.55Z(.0711) 0.988(.125)<.001 Stomach 3.23(.697) 3.~4(1.98) NS
Intestine 10.64(.541) 5.04(1.47)~.001 2~ T~-y-~Oid 4.2~(.594) 5.6~(1.0~).013 Lung 2.34(.~77) 6.32(1.55)~.001 Heart 1.14(.~07) 1.67(.21~).001 Brain 0.895(.08~7) 1.04(.0855).015 Tumor 3.~7t.470) S.18(1.31).044 RATIQ
Tumor/Blood q.l6(1.09) 5.68~2.75) Tumor/Muscle 7.16(1.S0) S.33(1.65) .

, .
, .
;:

SUIE~STIT~JTE SHEET

~:

WO94/26314 PCT~S93/04309 ~39~ 66~

l Table ~ ~:

Biodistributilon o~ N-(piperidinylamin~ethyl)-4-iodo ~ g}]ben~amide, ~l2~I~P~B, and N5(di~thylamlnoethyl)4-iodo ~ 12 I~benzamide, S~ I3D~B, ln nude mice ~enograted with human melanotic melanoma f% ID/a~ mean ~s~d. dev.), ~

P Valu~ ~or 6 Hour ~ l~5I~PAB ~lZ~D~B Di~er~nce Blood 0.208(.0542) 1.03(Ø1g7) .001 I.iver 1.16(.212) 3,74~.427) ~.001 Spleen 0 . 330 ( . 105 ) 0 . 2~0 ( . 0990 ) NS
Kidne~r 0.483(.131) 0.435(.0909) r~s Bone 0 . 115 ( . 0236 ) 0 . 100 ( . 02~7 ) NS
Muscle ~ . 0983 ( . 03~6 ) 0 . Og67 ( . 0356 ) NS
S'oma~n 0 . 757 ~ . 298 ) 0 . 475 ( . 164 ) NS
In~estlne 2 . 46 ( 1.18 ) 0 . 423 ( . 0963). 00 Thyroid 0 ~ 5 ~ 0 3 ~ ~ ~ 4 ~ 2 4 ) ~S
,.3O/;.u5~ 9g3J ~S
Heart 0, t 67 (, 0372 ) 0 . 150 ~ . 0329 ) NS
Braln 0 . 122 ( . 0331 ) 0 .132 ~, C254 ) iJ~
Tumor 2.91(.463) ~ 2.83(.388) NS

RATIO
Tumor/Blood 14.9(5.07) ~8.1(5.84) .002 TumGr~Muscle 32.S(12.0) 33.3(14.0) NS
:~ .
: _ _ TlJTE SHET
.:

WO94/2~14 213 9 ~ 0 3 PCT~S93/04309 ; ;
j ~67-1 Table 3 ~ ' ,' ;.

Blodistribution o N-~plperi~inylamlnoethyl)- :
4-iodo [~2gI]benzamider ~Z5lI~PAB, and N-~dle~hylaminoethyl)4-~odo t I]benzamid~, 5~l25I]~AB, in nude mlce x~nog~ated with human melan~tic me_ noma_L~ g , ,',,.'~

P Value ~o~
24 Hou~ ~ lZsI~P~B ~l19I]~B Dl~~r~nc~

~lood 0.0617~.018) 0.0350~.0084).00~ ~;
L~ver 0.263(.02~6) 1.12(.232) c,001 Spleen 0.0383~.015) 0~o35o~n23) NS
15 ~idney 0.0850~.016) 0.06~.01g7) NS
Bone 0.0133~.0052) 0.0133~005~) N~ ~:
Muscle 0.0117~.0041) 0.0150~.0084)NS
Stomach 0.130(0.881) 0,445~.386) NS
Intestine 0.132~.0852) 0.123~.0717) NS ;~
: ~ Th.y~ n,l~ n / , 14 3 ) ~ ' ~ ~ ~ 7 )~JS `
~ung 0.0717(.0075) 0.0633(.0273JNS
Heart 0~0283(.0075) 0.0233~.017~)NS
Brain 0.0067(.0052) ~.0~33(.00~
Tumor 1.028(.239) 0.553~.241~ ,006 ;-i~;
RATIO ,, Tumor/Blood 17.8~6.10) I5.5~4.69) NS
; .
: Tumor/Muscle 94.S(32.5) 39.7~9.61~ .003 -:

,, .
, ' ', ',.".'' 1,:
i'''''.' SUBSTITUTE SHEET' . .

WO~q/2~14~ PC~'~S93/04309 ~9~ 68-Biodistriblltlon of 2~PiperldinylAminoeth~l~-4-Iodo~enzamide Biodistrlbution.experimen~,s were performad to 5 as~ess the ~umor-speci~lciky o~ ~he present compounds.

v~r~ nd K~ ~ h~
~lon-small ce.~l lung carclnoma eell lines NC
157, NCI-~38 and ~CI-129g wer~ ob~aln~ rom ~h~
lO National Cance~ ~ns~i~ute. The NC~-1$7 c~ in~ i~ a squ~mous ra-~in~m~ 1 line, ~ le NCL~83~ i3 an adenocarclnoma cell line and NCI~l~gg is a large c~ll lung carcinoma c~
Tumor cells wer~ grown in ~MEM2 m~dium (Dulbecco's modi~icati~n of Eagle's medium, ~MSM) supplemen~ed with 10~ al bo~inQ s~rum and 0.03% L-glutamine.
(dietnylaminoQ~hyl)4-iodo~enzamide (l.e.
~lZ5I]DAB) was prepared as de.scribed in J~hn et al. (1993 20 NUC1 . MQd ~ Biol. 2~ 7S-7a), l45L;2-pip~ri~inyiaminoe~nyi~ o~o~enzamLae (~.e. El25I]PAB~ (D) was synth~siz~d as des~ ed ln Example 1. .;~

....
Animal Blod ~ ~
, For in vivo studies, tumor cells were ;:
harvested using calcium and magnesium free P~S
containing 0,02~ EDTA. Suspension of 5 x 106 cells (viability ~reaker than 95%) in 0.2 mL of medium were ; I ` 3 lnnoculated subcutaneously in femal~ Balb/c nu/nu mice. .. ;:
Ater about two weeks, solid tu~ors of about 1 cm in : diam~ter appeared in approximately 85~ of all :
':

$UE3STlTl.)TE~ SHEET
.....

W094/26314 ~I 3 9 ~ ~ 3 PCT/USg3/04309 l innoculated mice. Mice with solid ~umors ha~ing a dlameter of abou~ l cm were used for biod.is~ribution ::
studi~s. ~.
Balb/c nu/nu mice ~l7-22 g) were lnjected 5 intravenously with 0.2 ml of a sallne solutlon `~ `
containing ~12'~]PAB (5~6 ~Ci). ~ and 24 hr. after injectlon, blood samples w~x~ coll~cted by c~dlaa puncture and ~he mlce were ~ac~liced lmme~i~taly thereaftex by c~rdie~t~my whil~ und~r h~loth~ne lO anesthesia. Th9 organs o~ interost were ~ubse~u~nkly excised, blotts~ with tlgs~e pap~r, w~ighed, and khe radl~activi~,y was counted uslng a Packa~d aut~m~ic counter (au~ogamma 5650). The ~ inject~d dosc/g (~
ID/g) vaiues were dete~m~ned by ~omparison o~ tissu~
15 radioacti~ ies wi~h su.i~ably diluted aliquo~s o~ thQ
injscted [1~s~]PAB dos~ dl~Ld~d by ~h~ weLght o the organ. The v~lues ob~aine~ wer~ normalLzed to a mouse weiahing 20 g.
.

Tables 4-5 .illus~xa~e the biodis~ibution of ,:
[l25I]~AB and [125I]PAB, respec~ively, in nude mics , ;
bearing human squamous cell carcinoma xenografts in the :~
flank a~ one, six, and ~wen~-four hours after 25 administration of the lmaging agent.
By 24 hrs. mice recei~ing ~ I~PAB had more [125I]PAB in their tumors than any other tissue. In ;:
contrast mice receiving [125I!DAB had about six-fold more 5I]DAB in thelr livers as ~heir tumo~s. These data 30 indicate that hlgh levels of ~125I]DAB are non- ;
specifîcally localixed in the liver. These data also I ';
'`..
: 35 .~
~,~

SUg3STlTUTE Sl~ ET
: '' W094/26314 ~9~3Q3 P~T~US93/04309 l indicate tha~ ~l25I]DAB has le5s ~umor speci~icik~ than I]PAB.
Moreover, the ~umor concen~ration o ~l~5I]P~B
was more ~han three-~Ql~ higher than that o~ ~l25I~DAB at 5 24 hrs. post-injection lndica~ing that IP~B binds to tumor cells with greater ~f~nit~ and stabilit~ than I~AB. These data indicate that ~5I~P~ ls highl~
sp~cific f~r lun~ car~ln~mas whl~h ~on~ain c~lls h~ving sigma receptors. ~:
..

~5 `" '' ' '. '~' .

, ,~
, .,'' SU113STITUTE 3HE~ET ;;
~.

WO94/26314 21 3 9 5 o 3 PCT~Sg3/04309 , . .
l Table 4 ::

, Biodistribution Q~ N-(diethylaminoethyl)- :
4-iodo ~l2~X~benzamlde~ El25I}D~B, in nude mice xenograft~d with human squamous cell caxcinoma r"~,, ID/gi mea~ d~, d~y~

Tlssue 1 hr ~ hr 24 h~
;' ~ , ~loo~ 1.3~ ~.4~) 0.3~ 4) O.~Z7 ~ 0~ ~
I,L~er13.77 (0.72) g.~4 ~0.5~) 1.2S (0.10) :~;
Spl~en 3.S8 (0.28) ~.Sl ~0.0~) 0.01 ~0.00) ;:
Kidne~ 7.64 (0.35) 1.~7 (0.16) 0.05 (0.00) 15 B4ne 1.85 (O.Z~) 0,21 ~0.01) Stomach 5.41 ~0.53) 2.7 ~0~93) 0.15 ~0.02) Intestine5~64 (O.S8) 1.34 ~0~Z7) 0.05 ~0.01) Thyr~d ~.2~ (a.72) 1.43 (O.SJ) 0.03 ~O.Oi) Lung 6.3~ ~0.8~) 1.07 ~0.14) 0.03 ~0.00) 20 Heart 2.~1 (0.16) 0.38 (0.11) 0.00 t0.00) :
B~ain 1.57 ~n.n~ n ~3 (~,05) ~n~ tV-~vi Tumor 5.13 (0.74) 2.17 ~0.093 0.18 (0.0~
. . - - - l.;

~

.

... ..
' ` 3 :..

.~ .

~SUIBE3TITlJTE Sg~lEET
.:

~ ~3 9 - 72- PC1~593/04309 1 T~b Biodistrlibution of (piperidin~laminooethyl)-4-i~do ~2~I~benzarnide, ~l2gI~P~, ln nude mice xenogra~ted wi~h human ~quamou~ cell carcinoma _________L ~ -, ~

Tissue 1 hr 6 hr 24 hr .
" , ~lood1.9 (0 33) 0~51 ~C.C3) 0,1~ ~.37) Liver10.48 ~ 6) 3.47 (0.23) 0-39 ~0~0~
Spleen3.97 ~O.lB)0.8~ ~0.2a) 0.05 (0.00) Kidney6.56 (0.04)1.~1 ~0.28) 0.13 ~0.~0) BonQ2.03 (0~28) 0.5G ~0.18) 0.02 (0.00) ~.
S'omach6.57 (1.31)3.46 ¢0~18) 0.20 (0.03) Intestine12.63 ~0.43)11.41 ~0.52) 0.38 (0.09) :~:
Thyr~d7.8~ (0.3;) 2.aO (0.49) 0.09 (0.01) Lung 4.91 ~0.25) 1.10 ~0.03) 0,08 ~0.00) -.:
Heart2.25 ~0.07) 0.49 ¢0.03~ 0~03 !~~
Brain t.. ~n !n 17) ~'~9 (V-VLj U.Ui ~O.U~
Tumor4.27 (0-40) 3.Z7 (0~33) 0.55 (a~

`
'',",''' , '.

, ,~ .
:

$UBSTITUTE 51HE~T

WO94n63l421 3 9 ~ D 3 PCT~593/04~0~ ¦1 ~73-1 ~XAMPL~ 8 Diagnostic Imaging Uslng sI-(2-PiperidinylAminoethyl)-4-IodOBenZamid.e f I ~
5Thess experlments illustr~e the presen~
diagnostic imaging pr~cedures and ~he ben~fit o~ :
utllizing the presen~ compaunds in such procedures. .:
~i ;
M ~J~I~LJ~
~2058 c~lls, derl~e~ ~om ~ brain meta~asis of human mali~nan~ melanoma ~o~axa e~ al. 1~80 P~oc.
Natl. ~cad. Sci. U5A 77: 5~58) were ohkalned ~rom the ~;:
Natlonal Ins~tutes o~ H~alth. ~ human lung ~ ;~
adenocarcino~ cell line, NCX~ a3~ was ob~alned ~om the -;m~
15 Natlonal Cancer Ins~i~ute. These cell~ wer~ grown in DMEM2 medium (Dulbecco's modi~ication of ~agle's medium, :~ :
EMEM) supplemented with 10~ fetal bo~ln~ serum and 0.03~
L-alutam~ns . ,' .',' l3lI-N-~diethylaminoethyl)4-ifodobenzamld~ ~i.e.
20 [lJlI~DAB) was pxepared as described in John et al, (1993 '` "
Nucl. Med. BioL. 2~ 7~-79! .-;
I~I(2-piperidinylaminoethyl)4-iodobenzamide ~ -(D) was syntheslzed as described in Example 1.

~Y.:_~L~L_b~2L1~5 Balb/c nulnu mice (17-22 g) bearlng human melanoma or non-small cell lung carcinoma xenograft t~,.o-~ ~r~ ~"~a~ad intraven~usiy wîth O . 2 m; or saiine solution containing [l3lI~PAB or [l3lI]DAB (150-200 ~Ci).
; 30 The anlmals were anesthetized with kètamine containing .:;:
rompun before the imaging studies. The images were .
':' ~. `
1;
~, SUBSl~ITUTE~ S~EET

' :

~ 74_ PCT~593/04309 1 obtained using a scintigraphic camera with a pln-hole collimator at Ç and 24 hr. post injection.
Figs. 2 and 3 provid~ scin~igrams of nude rnice implanted wi~h human m~lanoma x~nografts and treated 5 with 1 l3ll ~ PAB and ~ l3lI ~ DA8, respec~ively .
~t 6 and 24 hrs, postln~ectlon, ~I~lIJPA~ wa5 ,~ ' detected only within ~he ~umor (Flgs. 2A and 2B). In conkra~t, no ~ l3lI ~DAB wa~ d~te~d i;l th~ tumor at .
ei~her 6 or ~ hrs. ~e~ ~dministra~lon ~Figs. 3~ and 3B). Moreover, considerabl~ upt~ke o~ ~JiI~A~ had occurr~d in -k~s li~sr~ o~ mic~ rs~ivlng ~h~
and 24 hrs. post-adminlst~atlon ( Flg~ . 3A ~nd 3 L~ttle or no ~l3lI~PAB was obse~v~d in th~ 9r at .~
either 6 or 24 hrs. post-admlnistr~tion, Th~se data ~:
15 indica~e IPAB is a signiflcan~ly be~ter diagno~tic agent -~
for ~umo~ imaging than IDAB.
Fig. 4 provides a scintigram e~ a nude m~u~e lmDlan~ed w~h ~ mas. ad~noca~nom~ xen~gra~ 30 hxs.
after in~ection of t~ PAB. The~e sclntegr~phlc 20 imaging studies easll~ vi~ualized the implanted tum~_ ~L . ~"
bo~h 24 and 30 hrs. ~o~-i.n~ec~

~ `;
;, ~5 . ``
: , ~
.~:

SuB!~TlTllJTE S~EET
... .

Claims (95)

WHAT IS CLAIMED:

1. A method for diagnosing a mammal for the presence of a mammalian tumor which comprises administering to a mamma} a diagnostic imaging amount of a compound of the formula:
I

wherein:
X is a radionucleide Z is =O or two -H;
each R1 is independently H, halo, lower alkyl, lower alkoxy;
Ra and Rb are independently H, halo, lower alkyl, lower alkoxy or Ra and Rb together with the carbon atoms to which they are attached form a cycloalkenyl or heterocyclic ring;
R2 is -N(R3)2 or a 5 to 6 membered nitrogen containing heterocyclic ring which is unsubstituted or substituted with at least one alkyl substituent;
each R3 is independently hydrogen or lower alkyl;
j and y are independently an integer from 0 to 6;
q is an integer from 0 to 2;
with the proviso that said compound is not an iodine radioisotope of (N-diethylaminoethyl)-4-iodobenzamide;

and detecting binding of said compound to a tumor in said mammal.

2. A method for treating a mammalian tumor which comprises administering to a mammal a composition comprising a tumor-inhibiting amount of a compound of the formula:

I

wherein:
X is a radionuclide;
Z is =O or two -H;
each R1 is independently H, halo, lower alkyl, lower alkoxy;
Ra and Rb are independently H, halo, lower alkyl, lower alkoxy or Ra and Rb together with the carbon atoms to which they are attached form a cycloalkenyl or heterocyclic ring;
R2 is -N(R3)2 or a 5 to 6 membered nitrogen containing heterocyclic ring which is unsubstituted or substituted with at least one alkyl substituent;
each R3 is independently hydrogen or lower alkyl;
j and y are independently an integer from 0 to 6;
q is an integer from 0 to 2; and with the proviso that said compound is not an iodine radioisotope of (N-diethylaminoethyl)-4-iodobenzamide.

3. The method of Claim 1 wherein X is a .gamma.-emitting radionuclide.

4. The method of Claim 1 wherein X is 123I, 124I, 125I, 131I, 10F, 76Br or 77Br.

5. The method of Claim 1 wherein X is 123I.

6. The method of Claim 2 wherein X is a .beta.-emitting or an .alpha.-emitting radionuclide.

7. The method of Claim 2 wherein X is 131I, 211At, 76Br, 212Pb, 212Bi or 77Br.

8. The method of Claim 2 wherein X is 131I.

9. The method according to Claim 1 or 2 wherein Z is =O.

10. The method according to Claim 1 or 2 wherein Z is two -H.

11. The method according to Claim 1 or 2 wherein each R1 is independently H, halo or lower alkyl

12. The method according to Claim 1 or 2 wherein each R1 is H.

13. The method according to Claim 1 or 2 wherein q is 2.

14. The method of Claim 13 wherein R1 is alkoxy.

15. The method according to Claim 1 or 2 wherein q is 1.

16. The method of Claim 15 wherein R1 is alkoxy.

17. The method according to Claim 1 or 2 wherein q is 0.

18. The method according to Claim 1 or 2 wherein Ra and Rb are independently H, halo, lower alkyl or Ra and Rb together with the carbon atoms to which they are attached form a cycloalkenyl or heterocylic ring.

19. The method according to Claim 1 or 2 wherein Ra and Rb are independently H, halo, lower alkoxy or lower alkyl.

20. The method according to Claim 1 or 2 wherein Ra and Rb are independently H, halo, or lower alkyl.

21. The method according to Claim 1 or 2 wherein Ra and Rb are independently H or halo.

22. The method according to Claim 1 or 2 wherein Ra and Rb together with the carbon atoms to which they are attached form a cycloalkenyl or heterocylic ring.

23. The method according to Claim 1 or 2 wherein is:
, , , , , , , or .

24. The method of Claim 1 or 2 wherein j is 0 to 2.

25. The method of Claim 1 or 2 wherein j is 0.

26. The method of Claim 1 or 2 wherein y is 1 or 2.

27. The method of Claim 1 or 2 wherein R2 is -N(R3)2.

28. The method of Claim 27 wherein R3 is hydrogen.

29. The method of Claim 27 wherein R3 is lower alkyl.

30. The method of Claim 1 or 2 wherein R2 is a 5 to 6 membered nitrogen containing heterocyclic ring which is unsubstituted or substituted with at least one alkyl substituent.

31. The method of Claim 30 wherein said heterocyclic ring is:

, , , , , , , or .

32. The method of Claim 30 wherein said heterocyclic ring is N-piperidinyl, N-pyrrolidinyl, N-pyridinyl, N-morpholinyl, piperidinyl, pyrrolidinyl, pyridinyl or morpholinyl which can be substituted with least one lower alkyl.

33. The method of Claim 30 wherein said heterocyclic ring is or piperidinyl or pyrrolidinyl which is N-substituted with lower alkyl.

34. The method of Claim 33 wherein said lower alkyl is methyl, ethyl, propyl or butyl,

35. The method of Claim 1 or 2 wherein said compound is:

, , , , , , , or .

36. The method of Claim 1 or 2 wherein said tumor is a lung carcinoma, a colon carcinoma, a renal carcinoma, a melanoma, a glioma, a pheochromocytoma or a neuroblastoma.

37. The method of Claim 1 or 2 wherein said lung carcinoma is an adenocarcinoma, a squamous carcinoma or a large cell lung carcinoma.

38. The method of Claim 1 or 2 wherein said tumor comprises cancer cells which have a cell surface sigma receptor.

39. The method of Claim 1 wherein the dectection of the binding of said compound to a tumor is observed after about 6 to about 30 hours.

40. A method for in vitro detection of a cancer cell in a mammalian tissue sample which includes contacting a mammalian tissue sample with an in vitro diagnostic amount of a compound of the formula:
I
wherein:
X is a radionuclide;
Z is =O or two -H;
each R1 is independently H, halo, lower alkyl, lower alkoxy;
Ra and Rb are independently H, halo, lower alkyl, lower alkoxy or Ra and Rb together with the carbon atoms to which they are attached form a cycloalkenyl or heterocylic ring;

R2 is -N(R3)2 or a 5 to 6 membered nitrogen containing heterocyclic ring which is unsubstituted or substituted with at least one alkyl substituent;
each R3 is independently hydrogen or lower alkyl;
j and y are independently an integer from 0 to 6;
q is an integer from 0 to 2;
with the proviso that the compound is not a radioisotape of (N-diethylaminoethyl)-4-iodobenzamide;
for a time and under conditions sufficient for binding of said compound to a cancer cell and detecting said binding.

41. The method of Claim 40 wherein X is 125I, 16F, 35S-alkyl, 35SO3, 35SO4 or 3H.

42. The method of Claim 40 wherein said compound is:

, , , , , , , , , or .

43. The method of Claim 40 wherein said cancer cell has a cell surface sigma receptor.

44. A compound of the formula:

II

wherein:
Q is a radionuclide, halide or an activating group;
Z is =O or two -H;
each R1 is independently H, halo, lower alkyl, lower alkoxy;
Ra and Rb are independently H, halo, lower alkyl, lower alkoxy or Ra and Rb together with the carbon atoms to which they are attached form a cycloalkenyl or heterocylic ring;
R4 is -N(R3)2 or an N-linked 5 to 6 membered nitrogen containing heterocyclic ring which can have at least one alkyl substituent, wherein each R3 is independently lower alkyl or hydrogen;
j is an integer from 0 to 6; and q is an integer from 0 to 2.

45. A compound of the formula:

III

wherein:

Q is a radionuclide, halide or an activating group;
Z is =O or two -H;
each R1 is independently H, halo, lower alkyl, lower alkoxy;
Ra and Rb are independently H, halo, lower alkyl, lower alkoxy or Ra and Rb together with the carbon atoms to which they are attached form a cycloalkenyl or heterocyclic ring;
R5 is a 5 or 6 membered nitrogen containing heterocyclic ring which can have at least one alkyl substituent;
m is an integer from 2 to 6;
j is an integer from 0 to 6; and q is an integer from 0 to 2.

46. A compound of the formula:

IV

wherein:
Q is a radionuclide, halide or an activating group;
Z is =O or two -H;
each R1 is independently H, halo, lower alkyl, lower alkoxy;
Ra and Rb are independently H, halo, lower alkyl, lower alkoxy ox Ra and Rb together with the carbon atoms to which they are attached form a cycloalkenyl or heterocylic ring;

each R3 is independently lower alkyl or hydrogen;
n is an integer from 3 to 6;
j is an integer from 0 to 6; and q is an integer from 0 to 4.

47. The compound of any one of Claims 44-46 wherein Q is a radionuclide.

48. The compound of Claim 47 wherein said radionuclide is 123I, 124I, 125I, 131I, 18F, 211At, 76Br or 77Br.

49. The compound of Claim 47 wherein said radionuclide is 123I or 131I.

50. The compound of any one of Claims 44-46 wherein Q is an activating group.

51. The compound of Claim 50 wherein said activating group is iodide, tributyl-tin, trimethylsilyl or t-butyldimethylsilyl.

52. The compound of any one of Claims 44-46 wherein Z is =O.

53. The compound of any one of Claims 44-46 wherein Z is two -H.

54. The compound of any one of Claims 44-46 wherein each R1 is independently H, halo, lower alkyl.

55. The compound of any one of Claims 44-46 wherein each R1 is alkoxy.

56. The compound of any one of Claims 44-46 wherein each R1 is H.

57. The compound of any one of Claims 44-46 wherein q is 2.

58. The compound of Claim 57 wherein each R1 is alkoxy.

59. The compound of any one of Claims 44-46 wherein q is 0.

60. The compound of Claim 44 wherein R4 is -N(R3)2 and each R3 is independently lower alkyl.

61. The compound of claim 44 wherein R4 is an N-linked 5 to 6 membered nitrogen containing heterocyclic ring which can have at least one alkyl substituent.

62, The compound of Claim 61 wherein said heterocyclic ring is , , or , .

63. The compound of Claim 51 wherein said heterocyclic ring is N-piperidinyl, N-pyrrolidinyl or N-pyridinyl.

64. The compound of Claim 45 wherein R5 is a 5 or 6 membered nitrogen containing heterocyclic ring which can have at least one alkyl substituent.

65. The compound of Claim 64 wherein said heterocyclic ring is piperidinyl, pyrrolidinyl or pyridinyl which can have at least one alkyl substituent.

66. The compound of Claim 64 wherein said heterocyclic ring is:
, , , or .

67. The compound of Claim 45 wherein m is 2.

68. The compound of Claim 46 wherein each R3 is lower alkyl.

69. The compound of Claim 46 wherein each R3 is hydrogen.

70. The compound of Claim 46 wherein n is 3.

71. A compound having any one of the formulae:

, , , , , , , , or .

72. A pharmaceutical composition comprising a diagnostic imaging amount of the compound of any one of Claims 44-46 and 71, and a pharmaceutically acceptable carrier therefor.

73. A pharmaceutical composition comprising an anti-tumor amount of the compound of any one of Claims 44-46 and 71 and a pharmaceutically acceptable carrier therefor.

74. A compartmentalized kit for detection or treatment of a mammalian tumor comprising a container having at least one compound of the formula:

IX

wherein:
Q is a radionuclide, halide or an activating group;
Z is =O or two -H;
each R1 is independently H, halo, lower alkyl, lower alkoxy;
Ra and Rb are independently H, halo, lower alkyl, lower alkoxy or Ra and Rb together with the carbon atoms to which they are attached form a cycloalkenyl ox heterocylic ring;
R2 is -N(R3)2 or a 5 to 6 membered nitrogen containing heterocyclic ring which is unsubstituted or substituted with at least one alkyl substituent;
each R3 is independently hydrogen or lower alkyl;

j and y are independently an integer from 0 to 6;
q is an integer from 0 to 2;
with the proviso that said compound is not an iodine radioisotope of (N-diethylaminoethyl)-4-iodobenzamide.

75. The kit of Claim 74 wherein Q is an activating group.

76. The kit of Claim 75 wherein said activating group is iodide, tributyl-tin, trimethylsilyl or t-butyldimethylsilyl.

77. The kit of Claim 74 which further comprises another container having a reagent for replacing said activating group with a radionuclide.

78. The kit of Claim 71 wherein said reagent is an oxidizing reagent.

79. The kit of Claim 78 wherein said oxidizing agent is chloramine-T.

80. The kit of Claim 74 which further comprises a material for for separating unattached radionuclide from said compound.

81. The kit of Claim 80 wherein said material is a chromatographic material.

82. The kit of Claim 81 wherein said chromatographic material is a thin layer chromatography plate, a molecular exclusion resin or a reverse phase resin.

83. A kit for detection or treatment of a mammalian tumor which comprises a container having at least one of the compounds of any one of Claims 44-46 and 71.

84. The kit of Claim 83 wherein said compound has Q as an activating group.

85. The kit of Claim 84 wherein said activating group is iodide, tributyl-tin, trimethylsilyl or t-butyldimethylsilyl.

86. The kit of Claim 83 which further comprises another container having a reagent for replacing said activating group with a radionuclide.

87. The kit of Claim 86 wherein said reagent is an oxidizing reagent.

88. The kit of Claim 87 wherein said oxidizing agent is chloramine-T.

89. A method for diagnostic imaging of a mammalian tissue which has cell surface sigma receptors which comprises administering to a mammal a diagnostic imaging amount of a compound of the formula:

I

wherein:
X is a radionuclide;
Z is =O or two -H;
each R1 is independently H, halo, lower alkyl, Lower alkoxy;
Ra and Rb are independently H, halo, lower alkyl, lower alkoxy or Ra and Rb together with the carbon atoms to which they are attached form a cycloalkenyl or heterocylic ring;

R2 is -N(R3)2 or a 5 to 6 membered nitrogen containing heterocyclic ring which is unsubstituted or substituted with at least one alkyl substituent;
each R3 is independently hydrogen or lower alkyl;
j and y are independently an integer from 0 to 6;
q is an integer from 0 to 2;
with the proviso that said compound is not an iodine radioisotope of (N-diethylaminoethyl)-4-iodobenzamide;
and detecting an image of a tissue having an abundance of cells with sigma receptors.

90. The method of Claim 89 wherein said tissue is a neural tissue.

91. The method of Claim 90 wherein said tissue is brain tissue.

92. The method of Claim 90 wherein X is a .gamma.-emitting radionuclide.

93. The method of Claim 90 wherein X is 122I, 124I, 125I, 131I, 18F, 76Br or 77Br.

94. The method of Claim 90 wherein X is 123I.

95. The method of Claim 90 wherein said compound is:

, , , , , , , , , or .