CA2247822A1 - Methods of treating or preventing interstitial cystitis - Google Patents

Abstract

This invention provides methods for the treatment or prevention of interstitial cystitis or urethral syndrome in a mammal which comprise administering to a mammal in need thereof an effective amount of a compound of formula (I), where R1 and R2 are independently selected from the group consisting of hydrogen, methyl, methoxy, chloro, and trifluoromethyl, with the proviso that no more than one of R1 and R2 can be hydrogen; and Y is (1), (2), (3), (4), (5), (6), N-Ra, or CH-NRbRc, where Ra, Rb, and Rc are independently selected from the group consisting of hydrogen and C1-C6 alkyl; or a pharmaceutically acceptable salt or solvate thereof.

Description

W O 97t33583 PCTrUS97/03555 o METHODS OF TREATING OR PREVENTING
INTERSTITIAL ~Y~ 1 l~l~lS
~ 5 R~rk~round o~~e Invf~n~ n Tachykinins are a f~mily of peptides which share a common slmi~l~ted carboxy t~rTnin~l sequence. Sllhst~nre P was the ~irst peptide of 10 this family to be i.col~ted, although its pllrific~1;on and the determinz 1;on of its primary sequence did not occur until the early 1970's.
Between 1983 and 1984 several groups reported the i.cnl~t;~n of two novel m~mm~ n tachykinins, now termed neurokinin A (also known as sllhst~nce K, neuromedin L, and neurokinin ~), and neurokinin 15 B (also known as neuromedin K and neurokinin O- &~, J.E. Maggio, Pep~ c~ 6 (Suppl~mPnt 3):237-243 (1985) for a review of these discovt,l;es.
Tachykinins are widely distributed in both the central and peripheral nervous ~y~ ..s, are released from nerves, and exert a variety 2 o of biologir~l ~c1;on.c, which, in most cases, depend upon activation of specific receptors e~pressed on the membrane of target cells. Tachykinins are also produced by a number of non-neural tissues.
The m~mm~ n tachykinins substance P, neurokinin A, and neurokinin B act through three major receptor subtypes, denoted as 2 5 NK-1, NK-2, and NK-3, respectively. These receptors are present in a variety of organs.
Sl~hstsnce P is believed int~r alia to be invo1ved in the neulo~..cmic.cir)Il of pain sçnC~t;nn.q~ includi~g the p~in ~c.cori~ted with migraine he~ rh~.q and with ar1~hritis. These peptides have also been 3 o i~nplir~ted in gastrointostin~l disorders and diseases of the gastr~intes{ in~l tract such as infl~mm~tory bowel disease. Tachykinins have also been implic~ted as playing a role in numerous other m~ ie~
as ~liqcll.qsed i~
Tachykinins play a mnajor role in me~ ng the sf~n~q~1~on 3 5 and tr~n-qmiq~4ion of pain or nociception, especially _igraine he?~ rhes.

W O 97/33S83 PCT~U~97/03555 see. e ~.. S.~. Shepheard, et aL? Bri~;.ch Jollrn:~l of Ph~rn~r.~ 108 20 (1993); S.M. Moussaoui, ~L, ~,llro~ n Jol~rns~l of Ph~rrn~roloFy 238:421-424 (1993); and W.S. Lee, et al., Rri*.ch Jon~l of Ph~ r-~lo~y, 112:920-924 (1994).
In view of the wide number of r.linir~l ms~ i.q.s associated with an excess of tachykini~Ls, the development of tachykinin receptor antagonists will serve to cont~ol these rlinir~l con-li*~ns The e~r1i~xt tachykinin receptor antagonists were peptide del;vaLiv~s. These antagonists proved to be of limited ph~rm~l~.eutical u1ility because of their metabolic inQ~hility Recent pllhlir.~*on.q have ~l~srri~hed novel r.lf~c.cç~ of non-peptidyl tachykinin lec~tor antagonists wlbich generally have greater oral bioav~ hility and metabolic st~hility than the earlier classes of tachykinin lec~toL antagonists. ~.Y~mrles of such newer non-peptidyl tachykinin receptor antagonists are found in United States Patent 5,328,927, issued July 12, 1994; United States Patent 5,360,820, issued November 1, 1994; United States Patent 5,344,830, issued September 6, 1994; United States Patent 5,331,089, issued July 19, 1994; European Patent Pllhlir n 591,040 A1, pl~hliched April 6, 1994; Patent Cooperation Treaty pllhl;cs.*nn WO 94/01402, p~ he-l Janu. ry 20, 1994; Patent Cooperation Treaty pllhlir~tion WO 94/04494, published March 3, 1994; and Patent Coopers.~on Treaty pllhlif~gtis)n WO 93/011609, pnhli.che~ January 21, 1993.
T..~ l cystitis is a chronic (l~ stinginflsmn~st~ry 25 disorder of t he hl s(l~l~r T~e disease is most common in women r~n~in~ in age from about 1~hirty to sixty with onset of the con~ition typically ~~;~ F at about forty years of age. It is ch~;s~- LY- ;,ed by a number of urinary ~lifficlllt;~.s, such as suprapubic pressure a~d pain, with bladder fil ling, urinary frequency, noctu~ia, dysuria, urgency adn irrit~tive 30 voiding s~.q.~or~isted with mo~hnlogi~sl and hist~-logi-~l changes in 1 he hlz~ r The con~ on is rh~r~ctQri7ed as "inlQ. ~ 1 cystitis" because it is believed the conrlition does not affect the surface of the blflrl-ler, but instead involves the spaces between the cells, namely the int~r.s*~s, in the lining of the bl~ ?r.

W O 97133583 PCT~US97/03555 Urethral syndrome is a related painful voiding disorder of unknown etiology affecting wo_en exhihitin~ many of the con.litinnc set forth above.
As noted in United States Patent 5,145,859, issued ~ 5 September 8, 1992, the entire cont~-ntc of which are herein incorporatedby l~r~e..ce, there are a number of compounds proposed to treat these con-litionc, based on ~liffering theories as to the etiology of intel~LiLial cystitis and urethral syndrome. None of these tre~t~Pnt regimens has V~ll cf mplet~ly successful to date Because of the current ~li.cs~t;cf~l~fion of the currently marketed tre~hn~nts for in~ 1 cystitis within the affected pop~ tion, there exists a need for a more {~.ffic~fious and safe tre~t~nPnt ~llmms-ry of ~e ~n T'nis iIlvention provides methods for the tre~t~nent or ~v~-ltion of intel;iLiLial cystitis or urethral syndrome in a msnnmsil which comrri.ce ~lminictering to a m~mm~l in need thereof an ~eclive amount of a compound of Formula T
~N ' I

where R1 and R2 are indep~n-lP.nt~y selected from the group 2 5 consisting of hy~o~ , methyl, methoxy, chloro, and tri~luoromethyl, with the proviso that no more than one of Rl and R2 can be hydrogen; and Yis W O 97/33583 PCTrUS97/03555 N~ N - N ~ , N~, ..

CH - N 3 CH ~ , CH ~

N-Ra, or CH-NRbRC

where Ra, Rb, and Rc are indepP.n~ntly sPlecte~l from the group con.QiQ~ing of hydrogen and Cl-C6 alkyl;
or a ph~r n~rellt~ lly acceptable salt or solvate thereof.
I~et~ilrd 1 )esr.r nti-~n an-l rlef~ d P'.mhollimP.n~c;

The terms and abbrevi~*on.~ used in the in~t~nt e.7r~m}~les have their no~m~l me~ning.~ unless o1~herwise ~l~s;gn~te~l For f~ mrlP
15 "~C" refers to degrees Celsius; "N" refers to norm~l or norm~lity; "mol"
refers to mole or Imoles; "mmol" refers to millimol~ or n~illimnlerc; ~g~ refersto gram or grams; "kg" refers to kilogram or kilograms; "L" refers to liter or liters; "ml" means millilit,er or millili~rs; "M" refers to molar or mnl~ri1y; ''MS'' refers to mass spectrometry; and "NMR" refers to nuclear 2o ms~gnetiC resonSmre spe.;~losc~,~y.
As used herein, the term "Cl-C6 alkyl" refers to straight or br~n~.h~ monovalent, sa~ ted ~ h~t~e chains of 1 to 6 carbon atoms and includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, a~ld hexyl. The term "C l-C6 alkyl"
25 inl-ln(l~s within its (lP.finit~( n the term "Cl-C3 alkyl".
"Halo" represents chloro, fluoro, bromo or iodo.
The term "h~lofnrm~te" as used herein refers to an ester o~ a h~lnformic acid, this compound having the formula W O 97133583 PCT~US97/03555 X--C~
o--Rd wherein X is halo, and Rd is Cl-C6 alkyl. Pl~elled hPl~rmqtss are bromoformqtqs and chloloro. ~ teC Especially pl~erled are 5 chl~ orO. .,~qts.s Those hPlnform~tes wherein Rd is C3-C6 alkyl are especially ple~ ~ ed. Most ~l~elled is isobutylchloloro. ~e~te The compounds prepared in the processes of the present invention have an asymme~rir- center. As a consequence of this c~iral center, the compounds produced in the present invention may occur as 10 r~qrPmqtqs, mixtures of çnPntiomers and as individual enqn~inmer.c, as well as diastereomers and ...ix~ s of diastereomers. Processes for prepPring such asymmetric forms, individual icomers and comhinqtion.5 thereof, are within the scope of the present inven*nn The terms "R" and "S" are used herein as commonly used in 15 organic rhPmictry to denote specific configuration of a l~hiral center. The term "R" (rectus) refers to that configuration of a chiral center with a clockwise rQlq.*onchir of group p~ori*es (hi~hest to second lowest) when viewed along the bond ~ow~1 the lowest priority group. The term "S"
(sinister) refers to that configuration of a chiral center with a 20 counterclockwise rPl~*~n.~hir of group priori*~s (high~.qt to second lowest) when viewed, long the bond tow~d the lowest priority group.
The priority of groups is based upon their qtomic number (in order of decre~,ing qfomir number). A par1ial list of priori*~s and a ~ c~ ion of stereorh~mi-~try is contqined in NOMENCLATURE OF ORGANIC
2 5 COMPOUNDS: PRINCIPLES AND PRACTICE, (J.H. Fletr~her, et al., eds., 1974) atpages 103-120.
In e.~l~i*on to the (R)-(S) ~y:j~e~.~, the older D-L system is also used in this ~lnc-lment to denote absolute configuration, especially with la~e~ ce to amino acids. In this :iy:j~em a Fischer projection formula is 3 0 oriented so that the number 1 carbon of the mq-in chqin is at the top. The prefix "D" iS used to represent the absolute configuration of the isomer in which the ~ln~ on~l (determining) group is on the right side of the carbon atom at the chiral center and "L", that of the isomer in which it is on the left.

W O 97/33583 PCT~US97/03SS5 The term "treating" (or "treat") as used herein includes its generally accepted mes2nin~ which ~n~on-rasses prohihi*n~, preventing, re~LLA i ..; .-g, and slowing, ~ g, or ~ ~g progre.~.eion, ~v~l~y, or a resultant sy~ptom. As such, the methods o~ this invention ~nComrass 5 both therapeutic and prophylactic ~mini.~tration.
Patent Cooperation Treaty Puhlic~ion WO 9~/1401~, pllhli.ched May 26, 1995, te~rhee, inter ~ a series of tachykinin receptor antagonists of Formula II

Rl r/--~ R2 cH2 ) m z~

HN (cH2 ) n-N Y

II

wherein:
m and n are independently 0-6;
Z is ~(C~IR4)p~(CHR63q~~ where, p is O or l;

qisOorl;and R4 and R6 are indepPn-len~ly selected ~om the group consisting of hydrogen and Cl-C3 alkyl;

Y~s N ~ , N - N 3 , N ~ , CH - N ~ ,CH ~ , CH

N-Ra, or CH-NRbRC, where Ra, Rb, and Rc are independently selected from the group consisting of hydrogen and Cl-C6 alkyl; and Rl and R2 are independently hydrogen, halo, Cl-C6 alkoxy, Cl-C6 alkylthio, nitro, trifluoromethyl, or Cl-C6 ~lkyl;

or a ph~rm~celltic~lly acceptable sa~t or solvate thereof.
These compounds have been shown to be very active, specific tachykinin receptor antagonists. Par1~ rly pl~rellad compounds are those of Formula II in which m and n are both 1; Rl and R2 are indepçn(l~ntly hydrogen, methoxy, ethoxy, chloro, fluoro, trifluoromethyl, methyl, and ethyl; Z is methylene; and Y, when c- mhined with the heterocyclic group to which it is ~tt~ -hed, forms 4-~piperidin-1-yVpiperidin-l-yl~ 4-(cyclohexyVpiperazin-1-yl, 4-(phenyl)piperazin-1-yl, or 4-2 ~ (phenyl)piperidin- 1-yl.
Especially ~laLell~d is the compound ~R)-3-(lH-indol-3-yl)-1-tN-(2-me~o~ybenzyl)acetyl~mino]-2-[N-(2-(4-(piperidin-1-yl)piperidin-1-yl)acetyVam~o]propane and the ph~rmaceutically acceptable salts and solvates thereo~ Most especially ~l~erlad is the compound ~R)-3-(lE-2 5 indol-3-yV- l-lN-(2-methoxybenzyl)acetyl ~mino]-2-[N-(2-(4-(piperidin- 1-yVpiperidin-l-yVacetyVamino]propane dihydrorl~ ri-le trihydrate.
The most pl~rellad method of synth~ci~in~ this compound is depicted in Scheme I, in~. Many of the steps of this syn~ç.~i.c are described in Patent Cooperation Treaty p~lhlic~tion WO 95/14017, W O 97/33583 PCTtUS97tO3S5S

pnhli.c:hed May 26, 1995, and European Patent Applis~s-1;on Plll)lioz~1~f)n 693,489, published January 24, 1996.

W O 97133583 PCT~US97103555 ~I~.h~me I

OH N-Me-morpholine~ Q ~ OH
( a ) ~ ~ NH2 tr~tyl chloride ~H ~N~IM
Tr (b) NMM Q_ ~HN~H3 ~OCH3 ~ Tr Ic~ Q~ ~ Q~ ~

(d) Q~ ~ Ac~o ~ ru ~b CH3 ~I NC)H--~

wherein "Tr" refers to a trityl group, and "NMM" refers to N-methylmorph ..l i n ~?

W O 97/33583 PCT~US97/03SS5 ~r.h~me I (con~nued) CH3 ~ ~ N~ ~ H3 oxalic acid NH ~ ~ CH3 ( :E ) C ~ . oxalate V

H3 ~ H3 ~ ~ O CH3 ~~ O CH3 (g) ~ HCl, H2O ~ ~ 2 HCl . oxalate V V

W O 97/33583 PCT~US97/03555 Synt~e.ci~ of (~ 2~ (2-((4-cyclohexyl)piperazin-l-yl)acetyl)~mino]-3-(lH-indol-3-yl)- 1-1N-(2-methoxybenzyl)acetylamino]propane CH

~ N

(a) Preparation of (:R)-3-(lH-indol-3-yV-2~
triphenylmethyl~mino)propanoic acid [N-~ yl~l~yptophan]

Tritylation ~OH ~OH

H H trityl Chlo ~uLr~ethy]silane (70.01, 0.527 moV was added at a moderate rate to a stirred slurry of D-tryptophan (100.0 g, 0.490 moV in anhydrous methylene rhl-7ri-1a (800 ml) under a nitrogen ~t~no.sphere.
This ll~ ,Ul-2 was continuously stirred for 4.25 hours. Triethylamine (147.0 ml, 1.055 moV was added, followed by the ~d~ on of a sol~ on of triphenylmethyl rhlorill~ (147.0 g, 0.552 mol) in methylene rhlori~e (400 ml) using an ~ ion filnn~l The miX~lre was stirred at room - 2 0 temp~la~ule, under a nitrogen ~f~nosphere for at least 20 hours. The re~r~inn was qnenrhe~l by the ~ nn of me~n- l (500 ml), The solution was concentrated on a rotary evaporator to near dryness and the mixture was redissolved in methylene rhl- ri~e and ethyl acetate. An aqueous work-up involving a 5% citric acid solution (2X) and CA 02247822 l998-08-3l W O 97/33~83 PCT~US97/03SSS

brine (2X~ was then p~rform~d The organic layer was dried over zlnhydrous sodium sulfate, filtered, and conc-sntrated to dryness on a rotary evaporator. The solid was dissolved in hot diethyl ether ~ollowed by the s~ on of hex~nf~.c to promote cryst~ m By this process 173.6 g (0.389 moV of analy~cally pure ( R)-3-(lH-indol-3-yl)-2-(N-triphenylmethyl~n~ino)propanoic acid was ~ ted as a white solid in two crops giving a total of 7g% yield.
FDMS 446 ~).
lH NMR ~DMSO-d~ ~ 2.70 (m, 1~1), 2.83 (m, 2H), 3.35 (m, lH), 6.92-7.20 10 (m, 12H), 7.30-7.41 (m, 8H), 10.83 (s, lE~, 11 73 (br s, 1El).
Analysis for C~(iH26N2o2:
Theory: C, 80.69; H, 5.87; N, 6.27.
Found: C, 80.47; H, 5.92; N, 6.10.

(b) Preparation of (R)-3-(lH-}ndol-3-yl)-N-(2-met~oxybenzyl)-2-(N-triphenylmethylamino)propf~n ~mill~

Coupling ~ OH ~ ~ N ~R~2 H trityl H trityl To a stirred solution of ~R)-3-(lH-indol-3-yV-2-(N-~ h~nylme~hyls~minQ)propanoic a~id (179.8 g, 0.403 mol), 2-methoxybenzyl~e (56.0 ml, 0.429 moV, and hyJ~o~yl~enzot~sle 25 Ly~ate (57.97 g, 0.429 mol) in anhydrous tetrahy~vLu~ (1.7 1) a~d anhydrous N,N-dime1~hylforrn~mi(1~ ~500 ml) under a nitrogen atmosphere at 0~C, were added triethyl~mine (60.0 ml, 0.430 moV and 1-(3-dimethylaminopropyl)-3-ethoxycarbor~ hydrochlori-le (82.2~ g, 0.429 mol). The mixture was allowed to warm to room temperature under 30 a niLlv~ no,sphere for at least 20 hours. The ; xl ~ . ~ was concf~ntrated on a rotary evaporator and then redisso~ved in methylene ~hlr~ and an aqueous work-up of 5% citric acid solution (2X~, saturated sodium bicarbonate solution (2X~, and brLne (2X~ was p~ d I~e W O 97/33583 PCT~US97/0355 organic layer was dried over anhydrous sodium sulfate and concentrated to dryness on a rotary evaporator. The desired product was then - recrystallized from hot ethyl acetate to yield 215.8 g (0.381 mol, 95%) of analytica~ly pure m ~te~ri s~l 5 FDMS 565 ~M~.
lH NMR (CDC13) ~ 2.19 (dd, J=6.4 Hz, ~=14.4 Hz, lH), 2.64 (d, J=6.5 Hz, lH), 3.19 (dd, J=4.3 Hz, ~1~=14.4 Hz, lH), 3.49 (m, lH), 3.63 (s, 3H), 3.99 (dd, J=5.4 Hz, ~o=14.2 Hz, lH), 4.25 (dd, J=7.1 Hz, ~o=14.2 Hz, lH), 6.64 (d, ~=2.1 Hz, lH), 6.80 (d, J=8.2 Hz, lH), 6.91 (t, J=7.4 Hz, lH), 7.06-7.38 lo (m, 21 H), 7.49 (d, J=7.9 Hz, lH), 7.75 (s, lH~.
Analysis for C88H3sN3o2:
Theory: C, 80.68; H, 6.24; N, 7.43.
Found: C, 80.65; H, 6.46; N, 7.50.

(c) Preparation of ~R)-3-(lH-indol-3-yV-l-~N-(2-methoxybenzyl)amino]-2-(N-triphenylmethyl~mino)propane Reduction of Carbonyl ~ ~ ~ ~ NH ~ ~Rl H trityl R2 H trityl R2 RED-AL~ [a 3.4 M, solution of sodium bis(2-methoxyethoxy)aluminum hydride in toluenel (535 ml, 1.819 mol), dissolved in anhydrous tetrahy~,ful~ (400 ml) was slowly added using an s~ on filnn~l to a ~ x; ..~ solution of the acylation product, ~R)-3-(lH-indol-3-yl)-N-(2 -methoxybenzyV-2-(N-triphenylmethyl~mino)prop~n~m~ (228.6 g,0.404 mols) produced ~a~, in anhydrous tetrahydl~.rul~ (1.() L) under a nitrogen atmosphere. The re?c~ n mixture became a purple solution. The re~ction was qll~n~hed 30 after at least 20 hours by the slow ~ on of excess saturated 3~ochelle's salt solll~on (potassium sodium tartrate tetrahydrate). The organic layer was isolated, washed with brine (2X), dried over anhydrous sodium sulfate, filtered, and concentrated to an oil on a rotary evaporator. No CA 02247822 l998-08-3l W O 97l33583 PCT~US97/03555 further pllrificP1ion was done and the product was used directly in the next step.

(d) Preparation of ~R)-3-(1H-indol-3-yl)-1-[N-(2-5 methoxybenzyl)-acetyl~mino]-2-~N-t;riphenylme1~hyl~mino)propane Acylation of Secondary Amine trityl To a s~irring s~ ;on of (R)-3-(lH-indol-3-yV-l-[N-(2-methoxybenzyl)amino]-2-~-triphenylmet;hylPmin- )propane (0.404 mol) in anhydrous tetrahy.L~Lu~an (1.2 L) under a ~itroge~ ~t~nosphere at 1)~C
was added triethylPmine (66.5 ml, 0.477 mol) and acetic ~nhydride (45.0 ml, 0.477 mol). After 4 hours, the .~ 5 .. e was concent~ated on a rotary evaporator, re.li~,s..lved in methylene r.hloritle and ethyl Pcet~te, washed with water (2X) and brine (2X), dried over aIlhydrous sodium s~llfP~e, filtered, and cQncf~ntrPted to a solid on a rotary evaporator. The resulting solid was dissolved in chlo~ ~ and lo~-le~l onto silica gel 60 (230-400 2 o mesh)andelutedwitha1:1...ix~ of ethyl ~etDte andh~Y~n~s The product was then cryst~lli7e~ from an ethyl acetate/h~x~ne.s ~ - - . a. The resulting product of ~ )-3-(lH-indol-3-yl)-1-1N-(2-m ç~ oxybenzyvacetyl ~min ol -2-~N-triphenylmethyl s~min o)propane was c~yst~ ed and i~ol~d over three crops giving 208.97 grams (87% yield) 2 5 of analytically pure ms~
Analysis for C40H3sN3O2:
Theory: C, 80.91; H, 6.62; N, 7.08.
Found: C, 81.00; H, 6.69; N, 6.94.

~e)Prep~r~*on of(R)-2-~ no-3-(1H-indol-3-yV-1-[N-(2-methoxybenzyl)acetylamino]propane Deprotection CA 02247822 l998-08-3l W O 97/33583 PCTrUS97/03555 ~ ~ C~ ~
. trityl Formic acid (9.0 1, 238.540 mmoV was added to a ~irring soll~tion of ~R)-3-(lH-indol-3-yl)- 1-[N-(2-methoxybenzyl)acetyl~mino3-2-~N-triphenylmethyl~mino)propane (14.11 g, 23.763 mmoV in anhydrous methylene ~hlorirle under a nitrogen ~tTnosphçle at 0~C. After 4 hours, the re~ct;Qn .--i x~ ~ ~ e was c~nc~ntrated to an oil on a rotary evaporator and re.licc~lved in diethyl ether and 1.0 N hydrorhlnrir~ acid. The aqueous layer was washed twice with diethyl ether and basified with sodium hydroxide to a pH greater than 12. The product was extracted out with methylene nhlori~la (4~). The organic axtr~ctC were comhined, dried over anhydrous sodium slllf~te, filtered, and cnnc~ntrated on a rotary evaporator to a white foam. The compound (R)-2-amino-3-(lH-indol-3-yl)-1-[N-(2-me~oxybenzyl)acetyl~mino]propane (7.52 g, 21.397 mmnlci) was ;CQk~te~1 giving a 90% yield. No further pllrifi~ *nn was necessary.
(f) Preparation of (;R)-2-amino-3-(lE-indol-3-yl)-1-rN-(2-metl~oxybenzyl)acetyls~mino]propane dihy-Lo~ lnrilla ~ C R2 trityl ~2 HCl A stirrinE solution of ~R)-3-(lH-indol-3-yl)-1-1N-(2-methoxybenzyVacetyl~mino]-2-~N-tripheny~ethyl~mino)propane in two 2 5 volumes of methylene ~hlori fl~? was cooled to between -40~C and -50~C.
- Anhydrous hydrogen r.hlori(le gas was added at such a rate that the temperature of the re~.~tion mixtnre did not exceed 0~C. The re~tion ule was stirred for 30 minutes to one hour at 0-10~C.
To this re~tion ~ule was added two volumes of methyl t-3 G butyl ether and the resulting mixtl~re was allowed to stir for 30 minutes to one hour at 0-10~C. The resulting cryst~lline solid was removed by filtration and then washed with methyl t-butyl ether. The re~r~inn product was dried under vacuum at 50~C. (Yield >98%) Analysis for C2lH2sN3O2 ~ 2 ~ICl:
Theory: C, 59.44; H, 6.41; N, 9.90.
Found: C, 60.40; H, 6.60; N, 9.99.

(g) Preparation of 2-((4-cyclohexyl)piperazin-1-yl)acetic acid potassium salt hydrate Cyclohe~yl~i~eraz;ine (10.0 g, 0.059 mol) was added to ten volumes of methylene rhl~ at room temp~ ,Ule. To this ...;x l ~~. e was added sodium hydroxide (36 ml of a 2N solution, 0.072 moV and tetrabutyl~nmoniwn bromide (1.3 g, 0.004 mol). After the ~rlllit;~n of the 15 sodium hydroxide and tetrabutyl~qmmf nium bromide, methyl bromo~etate (7.0 ml, 0.073 mol) was added and the re~r~;- n ...; ,~I -., e was stirred for four to six hours. The progress of the re~rtion was m~ . ed by gas chrom~tc~ .hy.
The organic fraction was separated and the aqueous phase 2 0 was back-~xtr~rtQ~l with me1~hylene rhl~ri~l~ The organic phases were comhined and washed twice with ~inni7:ed water, once with satulaled sodium bicarbonate solution, and then with brine. The Or{~Zmio phase was dried over m~nesiwn sulfate and the solvents were removed ill vacuo to yield methyl 2-((4-cyclohexyl)piper~in-l-yv~cet~te as a yellowish oil.
The title compound was prepared by dissolving the methyl 2-((4-cyclohexyl)piperazin-1-yVacetate (10.0 g, 0.042 mol) in te~ volumes of diethyl ether. This solution was cooled to 15~C and then potassium trime~ylsilanoate (5.9 g, 0.044) was added. This "';~I,Ule was then stirred for four to six hours. The re~ct;on product was removed by 3 0 filtration, washed twice with five volumes of diethyl ether, then washed twice with iive volumes of he~r~ne.~, and then dried in a vacuum oven for 12-24 hours at 50~C.
Analysis for Cl2H2lKN202 ~ 1.5 H20:
l~eory: C, 49.63; H, 7.98; N, 9.6~.
Found: C, 49.54, H, 7.72; N, 9.11.

W O 97/33S83 PCTAUS97/03~55 (h) Preparation of ~ 2-[N-(2-((4-cyclohexyl)piperazin-1-- yl)acetyl)amino]-3-(lH-indol-3-yl)- 1-1N-(2-methoxybenzyl)acetylsmino]propane The title compound was prepared by first cooling 2-((4-cyclohexyVpiperazin-l-yVacetic acid potassium salt to a tempeld~ule between -8~C and -15~C in 5 volumes of 9nhydrous methylene rhlori~le To this mixt~lre was added isobutylchlol..r... ~ te at a rate such that the 10 tempela~ule did not exceed -8~C. The resulting resr~; on .. .; xl ~ . a was stirred for about 1 hour, the tempela~ule bPing nlsintsinQd between -8~C
and - 15~C.
To this mixt~lre was then added ~R)-2-amino-3-(lH-indol-3-yl)-l-lN-(2-methoxybenzyVacetyl~mino]propane dihydrorhlnrifle at such a 15 rate that the tempelatula did not exceed 0~C. Next added to this ..~ i x~ ~- . e was N-methyl morpholine at a rate such that the te~np~a~ula did not exceed 0~C. This ~--i xl ~- ~ e was then stirred for about 1 hour at a tempeld~ule between -15~C and -8~C.
The re~r*on was quenched by the ~s~ i*on of 5 vnlllmQ.s of 2 0 water. The org~nic layer was washed once with a ssl ~ te~l sodium bicarbonate solution. The orgsnir phase was then dried over anhydrous pots4.qillm carbonate and filtered to remove the drying agent. To the filtrate was then added 2 equivalents of concPn~ated hydrorhlorir, acid, followed by 1 volume of isopropyl alcohol. The methylene rhlori .lP was 2 5 then PYch~n~ed with isc.y~ yl slr,nhol under vacuum by dis~;llst;on The final volume of isopropyl slrchnl was then cnncentrated to three vnlllme.q by vacuum. The re~r*nn ...ix~ was cooled to 20~C to 25~C and the product was allowed to crystallize for at least one hour. The de~qired product was then recovered by filtration and washed with 3 o snffif~ient isopropyl ~lrohol to give a colorl~.q iiltrate. The crystal cake was then dried under vacuum at 50~C. MS 560 (M+1+).
lH NMR (CDCl3) ~ 1.09-1.28 (m, 5H), 1.64 (d, J=10 Hz, 1H), 1.80-1.89 (m, 4H), 2.10 (s, 3H), 2.24-2.52 (m, 9H), 2.90 (s, 2H), 2.95 (d, J=7 Hz, 1H), 3.02 (d, J=7 Hz, lEl), 3.12 (dd, J=5, 14 Hz, lH), 3.77 (s, 3H), 4.01 (dd, 3 5 J=10, 14 Hz, lH), 4.49 (ABq, J=17 Hz, 43 Hz, 2H), 4.56 (m, 1H), 6.79-6.87 W O 97/33583 PCT~US97/03555 (m, 3~, 7.05-7.24 (m, 4H~, 7.34-7.41 (m, 2H~, 7.67 (d, J=8 Hz, 1H), 8.22 (s, lH~.
Analysis for C33H4sNsO3:
Theory: C, 70.81; H, 8.10; N, 12.51.
Found: C, 70.71; H, 8.21; N, 12.42.

Synthesis of ~ 3-(lH-indol-3-yv-l-[N-(2-met~l~xybenzyl)acetyl~minol-2 LN-(2-(4-(piperidin- l-yl)piperidin- l-yvacetyl)amino]propane ~ ~CH3 - 'X~
H O ~ CH3 ~ N

(a) Preparation of 2-(4-(piperidill-l-yl)piperi~lin-l-yvacetic a~d, potassium salt 4-~Pipe~din-l-yl)pip~ri~ine (1.20 kg, 7.13 mol) was added to methylene rhlori~e (12.0 L) under a nitroge~ ~t~no,sphere Tetrabutyl~mmonium bromide (0.150 kg, 0.47 mol) and sodium hydroxide (1.7 L of a 5 N solution, 8.5 moV were then added. The re~r~;r,n rni~ture 2 o was cooled to 10-15~C and ~nethyl brr,mo~oetate (1.17 kg, 7.65 moV was added and the resultiIIg ~-- i x ~ e was s~rred for a rninimum of 16 hours.
Deionized water (1.2 L~ was then added to the ~ u~e and the layers separated. The aqueous layer was back-extracted with methylene rhlo~(l~ (2.4 L). The or~ni(. fr~t;on~ were crmhined and 2 5 washed with ~le~ioni7:ed water (3 x 1.2 L), a sa~ulated sodium bicarbonate solution (1.1 L) and a saturated sodium rhlori~le solution (1.1 L). The organic fraction was then dried over anhydrous m~ne~sium sulfate and W O 97/33583 PCTrUS97/035S5 cnnc.qntrated to an oil on a rotary evaporator to yield 1.613 kg (93.5%) of methyl 2-(4-(piperidin- 1-yl)piperidin- l-yl)s--~etst~
A snl~ on of methyl 2-[4-(piperidin-1-yl)piperidin-1-yllacetate (2.395 kg, 9.96 mol) in met~nnl (2.4 L) was added to a solution ~ 5 of potsqcillm hydroxide (0.662 kg, 10.0 mol @ 85% purity) in me~n~l (10.5 L) under a nitrogen ~t~osphere. The re~c1ion mixt lre was heated to 45-50~C for a minimum of 16 hours.
A solvent e~ h~nge from methanol to acetone (15.0 L) was pçrfnrmed on the solution on a rotary evaporator. This snl-lt;on was slowly cooled to room temperature over 16 hours. l~ne resulting solids were filtered, rinsed with ~etone (5.0 L) and then dried to yield 2.4713~g (93.8%) of 2-(4-~piperidin-1-yl)piperidin-1-yl)acetic acid, potassium salt.
MS 265 ~+l) (b) Preparation of ~R)-3-(lH-indol-3-yV-1-[N-(2-methoxybenzyl)acetyl~mino]-2-[N-(2-(4-(piperidin- 1-yl)piperidin- 1-Yvacetyvamino]propane The title compound was prepared by first ~.1...i ,.; ..g ~R)-2-20 amino-3-(lH-indol-3-yl)-l-~N-(2-me~xybenzyvacetyl~mino~propane dihy~o~ lori-le (50.0 g, 0.118 mol) with 100 1 of methylene rhlori~3 under a nitrogen s~t~nosph~re~
In a ~econ~ flask, under a nitrogen ~hnosphere~ 2-(4-(piperidin-l-yVpiperidin-l-yl)acetic acidpotassium salt (62.3 g, 0.236 moV
25 was added to 600 ml of methylene r.~lori(l~ This mixt-lre was cooled to about -10~C and .s1;rring was continued. To this mixt-lre isobutylchlol. r(J. ..~te (23 ml, 0.177 moV was added dropwise such that the tempera~ule of the 2-(4-(piperidin-l-yvpiperidin-l-yvacetic acid potassium salt ...; ~ . e never rose appreciably.
This re~ ~on .. ixl.. e was stirred at about -10~C for about 1.5 hours at which time the (R)-2-~mino-3-(lH-indol-3-yV-l-[N-(2-methoxybenzyVacetylamino]propane dihydro~hlori-l~?/methylene l hlori~
mixt~lre prepared supra was slowly added to the 2-(4-(piperidin-1-Yvpiperidin-l-yvacetic acid potassium 35 salt/isobutylchloloro~ te/methylene r.hlori~e solution. The resulting W O ~7/33583 PCT~US97/03SSS

.Ule was then st~rred for about 1 hour at a tennpe~alule between -1~~C
and -8~C.
The re~r1;on ..~i xl . . e was removed from the ice bath and allowed to warm to 15-20~(~ and the re~rPon was ~uenched by the 5 ~ nn of 200 ml of water. The pH of the solution was adjusted to 2.3-2.7 by the ~rl~liton of lN sulfuric acid. The layers were separated and the aqueous layer was washed with 100 ml of methylene rhloride The organic fr~ctionq were comhined and washed wit_ water (100 ml). The water wash was back extracted with methylene Ghk~ritlQ (50 o ml) and comhined with the aqueous fraction from above Methylene rhlorid~ (500 ml) was added to the cnmbined aqueous layers and the mixture was stirred at room temperature for 15 minutes as bS~qificS~l;nn with 2N sodium hydroxide to a ~;nal pH of 9.8 to 10.2 was achieved.
The organic and aqueous fr~ ;nnq were separated. The aqueous 15 fraction was washed with methylene rhlo~l~ and the methylene rhlorifl was added to the organic frac~ion The organic fraction was then washed with a mil~l~U12 of sa~ulated sodiulm bicarbonate ~qol~ on (100 ml) and water (~0 ml). The bicarbonate wash was separated from t~he organic fraction and back extracted with met hylene chln~ (50 ml). The back 2 0 extraction was comhined with the metllylene rhls)? ifl~ fraction and the comhined frnc1;onq were dried over magnesium sul~te. The magnesium sulfate was removed by fil~5l1~0n alld the v~ ;les were removed by vacuum di~ hon to yield the ti1~e product as a foam. (72.5 g, ~98%
yield). MS ~59~M+l) NMR (DMSO-d6 3:2 .... ~ixl.. c, of amide rctqnler.q) ~ 1.25-1.70 (m, lOH), 1.77-2.00 (m, 2~, 1.95 (s, 3/5 3H), 2.04 (s, 2/5 3~, 2.10-2.97 (m, 9E~, 3.10-3.65 (m, 3H), 3.72 (s, 2l5 3H), 3.74 (s, 3/5 3H), 4.26-4.58 (m, 3~, 6.76-7.12 (m, 6H), 7.13-7.35 (m, 2H~, 7.42-7.66 (m, 2~, 10.80 (br s, lH).
Analysis for C33H4sNsO3:
Theory: C, 70.81; H, 8.10; N, 12.51.
Found: C, 70.57; H, 8.05; N, 12.39.

An ~ltern~l;ve process for preparing the compounds of Formula I follows.

W O 97/33583 PCT~US97/035S5 Preparation of (R)-3-(lH-indol-3-yl)-2-(N-triphPnylmethylz~mino)propanoic acid, N-methylmorpholine salt (N-trityl-- ~-tryptophan N-methylmopholine salt).
Q ~ o ~ ~ o NH
H trityl To a one liter 4 neck flask equipped with merh~ni.~l stirrer, condensor, probe, and stopper, were added D-tryptophan (40.0 g, 0.196 mol), ~cet(~ ;le (240 1), and 1,1,1,3,3,3-hexamethyl~ 7~ne (39.5 g, 0 0.245 moV. The resulting .. i~ .a was he~tell to 50-60~C and stirred until homogeneous. In a separate beaker trityl l~hlori~ 60.06 g, 0.215 moV and ~ceton;frila (120 ml) were slurried. The slurry was added to the silylated tryptophan ... i xl ~. . e and the beaker was rinsed with 40 ml of ac~l~...il ~ ;1P To the re~r~;on ...;xl--.e N-methylmorpholine (23.7 ml, 21.8 15 g, 0.216 mol) was added and the resulting ~L~ a was stirred for one hour. The ~)lV~; ~SS ofthere~ onwasmo~ 0. ~dbychrom~Eraphy After s~l~4~r~ o~;less, water (240 ml) was added dropwise to the re~ct;on ...i~LI ~.a and the resulting ...ix~ ~.a was cooled to less than 10~C, stirred for thirty minutes, and filtered. The residue was washed with water, and then dried to obtain 108.15 grams (~99% yield) of the desired title product.
lH NMR (DMSO-d6) ~ 2.70 (m, lH), 2.83 (m, 2H), 3.35 (m, lH), 6.92-7.20 (m, 12H), 7.30-7.41 (m, 8H), 10.83 (s, lEl), 11.73 (br s, lH~.
Analysis for C30H26N2o2:
Theory: C, 80.69; H, 5.87; N, 6.Z7.
Found: C, 80.47; H, 5.92; N, 6.10.

Preparation of (R)-3-(lH-indol-3-yl)-N-(2-methoxybenzyl)-2-(N-triphenylmethyls~mino)prop~n~mi~lP

W O 97/33583 PCT~US97/0355S

O H3C ~
Q~ H ~
H trityl To a two liter 4 neck ~lask equipped with mer.h~nic~l stirrer, conll~n.cor, and ~7mocouple, under a nitrogen atmosphere, were added N-trityl-D-1;ryptophan N-methylmopho~ille salt (108.0 g, 0.196 moV, acetonit~ (800 ml), 2-chloro-4,6-dimethoxy-1,3,5-~i~7.ine (38.63 g, 0.22 mol), and N-methylmorpholine (29.1 ml). The resulting ..~ . e was stirred at ~nlhiant temp~la~ e un1il hnmo~eneous (about ten minutes)~
After about one hour, 2-methoxybenzyl~mine (29 ml) was 10 added. The resulting ~ e was heated to 35~C and m~int~ined at that tempela~ule ov~.rnight The progress of 1~e re~c1;o~ was m~ o~ ed by chrom~t~raphy. Water (750 ml) was then added IL~u~LJ~ise to the ref~ct;on mix~llre and ~he resulting rni~t.lre was cooled to less than 10~C, stirred for thirty minutes, and i~ltered. The residue was washed with water (about 1()0 ml), and then dried to obt~in the desired title product. (Yield:
87% and 91% in two runs) EDMS 565 ~
lH NMR (CDCl3) ~ 2.19 (dd, J=6.4 Hz, ~o=14.4 Hz, lH), 2.64 (d, J=6.5 Hz, lH), 3.19 (dd, J=4.3 Hz, ~=14.4 Hz, lH), 3.49 (m, lH), 3.63 (s, 3~, 3.99 2 o (dd, J=5.4 Hz, ~v=14.2 Hz, lH), 4.2~ (dd, ~=7.1 Hz, ~=14.2 Hz, lE~, 6.64 (d, J=2.1 Hz, lH), 6.80 (d, J=8.2 Hz, lH~, 6.91 (t, J=7.4 Hz, lH~, 7.06-7.38 (m, Zl H~, 7.49 (d, J=7.9 Hz, 1~, 7.75 (s, lH).
Analysis for C38H3sN3O2:
Theory: C, 80.68; H, 6.24; N, 7.43.
Found: C, 80.65; H, 6.46; N, 7.50.

Reduction of Carbonyl Q~ ~Rl ~ Rl H trityl R2 H trityl R2 Preparation of ~ 3-(lH-indol-3-yl)-1-[N-(2-methoxybenzyl)amino]-2-(N-triphenylmethyl~mino)propane RED-ALE9, [a 3.4 M, solution of sodium bis(2-methoxyethoxy)aluminum hydride in toluene] (535 ml, 1.819 moV, dissolved in anhydrous tetrahy~rul~.. (40~ ml) was slowly added using an ~ lition ~unnel to a refluxing solution of the acylation product, (R)-3-(lH-indol-3 -yl)-N-(2 -methoxybenzyl)-2-~N-triphenylmethyl~mino)prop~n~mi~l~ (228.6 g, 0.404 mols) produced supra.
10 in anhydrous tetrahy~,rula-l (1.0 L) under a nitrogen atmosphere. The re~rtion mi~t-lre became a purple solution. The res~rtion was qnenrhe~l after at least 20 hours by the slow s~ lihon of excess sa~,ulated Rochelle's salt solution (potassium sodium talLate tetrahydrate). Inne organic layer was isolated, washed with brine (2~, dried over anhydrous sodium 5 sulfate, filtered, and concentrated to an oil on a rotary evaporator. No further pllrific~ n was done and the product was used directly in the next step.

Acylation o~Secondary ~n~ine ~ ~R2 ~2 trityl Preparation of ~ 3~ indol-3-yl)-1-~N-(2-methoxybenzyl)-acetyl~mino}
2-(N-t.riphenylmethyl~minQ)propane To a ,s~irring solution of ~ )-3-(lH-indol-3-yl)-1-[N-(2-metl~o~ybenzyl)amino]-2-~N-triphenylmethyl~mino)propane (0.404 moV
in anhydrous tetrahy-L~o~u~ ~ (1.2 L) under a nitrogen atmosphere at 0~C
was added triethylamine (66.5 ml, 0.477 mol) and acetic anhydride (45.0 3 a ml, 0.477 moV. After 4 hours, the ~ ule was conc~ntrated on a rotary evaporator, redissolved in methylene ~hlori~le and ethyl acetate, washed with water (2X~ and brine (2~, dried over anhydrous sodium sulfate, filtered, and con-~Pnt-rated to a solid on a rotary evaporator. The resulting CA 02247822 l998-08-3l W O 97l33583 PCTrUS97/035S5 solid was dissolved in chloloLl~ and loaded onto silica gel 60 (230-400 mesh) and eluted wit,h a 1:1 Jnixtllre of ethyl acetate and h~ nes The product was then cryst~lli7.ed from an ethyl acetatelhe~nes ..l i xl . . e. The resulting product of ( R)-3-(lH-indol-3-yl)-1-lN-(2-5 methoxybenzyl)ace~yl~mino}-2-(N-triphenylmethy~ no)propane was crystallized and i.col~ted over three crops giving 208.97 grams (87% yield) of analytically pure mz~ter Analysis for C40H3sN3O2:
l~eory: C, 80.91; H, 6.62; N, 7.08.
lo Found: C, 81.00; H, 6.69; N, 6.94.
Deprotection ~ ~ ~R2 trityl .2 HCl Preparation of ~E2v)-2-anlino-3-(lH-indol-3-yl)-1-lN-(2-met~oxybenzyVacetyls~m;no~propane llihy(Lol~.hlr~

A s1;rring solution of (R)-3-(lH-indol-3-yl)-l-~-(2-2 û methoxybenzyl)acetyl ~minQ]-2-~-triphenylmethyl~mino)propane in two volumes of methylene rlllf ~fle was cooled to between -40~C and -50~C.
Anhydrous hydrogen l~hlnrille gas was added at such a rate that the temp~7la~ule of the re~c~;~)n ...ixl-.. e did not exceed 0~C. The r~S~c1;l n ~I~;x~ a was stirredfor 30 minutes to one hour at 0-10~C.
2 5 To this re~c~;on ~ . . e was addLed two volumes of methyl t-butyl ether and the resulting mi~ 7re was allowed to stir for 30 minutes to one hour at 0-10~C. Tl~e resulting crystal~ne solid was removed by filtration and ~en washed with methyl t-buty~ ether. The reaction product was dlied under vacuum at 50~C. (Yield >98%) 3 0 Analysis for C2lH2sN3O2 ~ 2 HCl:
Theory: C, 59.44; H, 6.41; N, 9.90.
Found: (~, 60.40; H, 6.60; N, 9.99.

W O 97/33583 PCTrUS97/03555 Preparation of 2-((4-cyclohexyl)pipera7in-l-yVacetic acid potassium salt - hydrate Cyclohe~yl~i~era_ine (10.0 g, 0.059 mol) was added to ten volumes of methylene ~hlori-l~ at room tempela~ule. To this m;~rt~lre was added sodium hydroxide (361 of a 2N snln~on, 0.072 mol) and tetrabutylammonium bromide (1.3 g, 0.004 mov. After the sll~lit;on of the sodium hydroxide and tetrabutylsmmonium bromide, methyl bromoacetate (7.0 ml, 0.073 moV was added and the res~tion ,~ - . e was stirred for four to six hours. The progress of the res.~ .n was mo.. i ~o. ed by gas chromstography.
The organic fr~ ;on was separated and the aqueous phase was back-P~rtr~rted with methylene fhlorirle The organic phases were c~mhine~l and washed twice with ~lPioni7erl water, once with saturated sodium bicarbonate sollltion, and then with brine. The organic phase was dried over msgnecium sulfate and the solvents were removed in vacuo to yield methyl 2-((4-cyclohexyl)piperazin-1-yl)s-re~ste as a ye~lowish oil.
The title compound was prepared by dissolving the methyl 2-2 o ((4-cyclohexyl)piperazin- 1-yl)acetate (10.0 g, 0.042 mol) in ten volumes of diethyl ether. This snll~tion was cooled to 15~C and then pots~ inm trimethyl.cils-noate (5.9 g, 0.044) was ~rl~.l This ...i~ .e was then stirred for four to six hours. The res-rtinn product was removed by filtration, washed twice with five volumes of diethyl ether, then washed 2 5 twice with five vnlnmf~c of heY~nes, and t,hen d~ied in a vacuum oven for 12-24 hours at ~;0~C.
Analysis for C12H21KN202 ~ 1.5 H20:
Theory: C, 49.63; H, 7.98; N, 9.65.
Found: C, 49.54; H, 7.72; N, 9.11.
Preparation of ~ 2-~N-(2-((4-cyclohexyVpiperazin-1-yl)acetyl)amino]-3-(lH-indol-3-yV-l-[N-(2-me~o~ybenzyl)acetylaminolpropane W O 97/33583 PCT~US97/03555 H O
N N

The title compound was prepared by first cooling 2-((4-cyclohexyVpiper~7in-l-yl)acetic acid pots-~ rn salt to a tempela~ule 5 between -8~C and -15~C in 5 volumes of ~nhydrous methylene r.hlori~
To this mi~rt~lre was added isobutylchlol.~ro~ te at a rate such that the tempe~a~uLa did not exceed -8~C. The resul1~g re~c1;on m~ re was stirred for about 1 hour, the temperature being m~intDined between -8~C
and - 15~C.
To this .. ;xI--.e was then added ~R)-2-amino-3-(lH-indol-3-yV-l-[N-(2-methoxybenzyl)acetyl~mino]propane dihy~Lo~ lori(l~ at such a rate that the tempela~,ul~ did not e~eee-l 0~C. Next added to this ~ e was N-methyl morpholine at a rate such that the tempela~ule did not exceed 0~C. This ~Lule was then s~red for about 1 hour at a tempela~ula between -15~C and -8~C.
The re~*nn was g~enrhed by the s~ li~nn of 5 vnlll~n~.e. of water. The organic layer was washed o~ce with a satu.al,ed sodium bicarbonate s~ t;on The organic phase was then dried over anhydrous potassium carbonate and f;l'~ ~ed to remove the drying age~t. To the 2 0 filtrate was then added 2 equivalents of conc~nt,rated hydror.hlorir, acid,followed by 1 volume of isopropyl s~lrohol The methylene rhl~ le was then ~rh~n~ed with isopropyl s~lrrhnl u~der vacuum by ~li.e~ ;on The final volume of isopropyl ~lu~hnl was then conc~n1rated to three v~lllmes by vacuum. The r~ n n~ was cooled to 20~~ to 25~C and the product was aUowed to cryst~lli7.e for at least one hour. I~e desired product was then recovered by f;ltration and washed with sllffir-i~nt iso~ rl alcohol to give a cololless filtrate. Inne crystal cake was then dried under vacuum at 50~C. MS 560 ~+1~).
lH NMR (CDCl3) ~ 1.09-1.28 (m, 5H), 1.64 ~d, J=10 Hz, lH~, 1.80-1.89 (m, 4H), 2.10 (s, 3H), 2.24-2.52 (m, 9H), 2.90 (s, 2H), 2.95 (d, J=7 Hz, lEl), 3.02 (d, J=7 Hz, lH), 3.12 (dd, J=5, 14 Hz, lH), 3.77 (s, 3H), 4.01 (dd, ~=10, 14 Hz, lH), 4.49 (ABq, J=17 Hz, 43 Hz, 2H), 4.56 (m, lH~, 6.79-6.87 (m, 3H), 7.05-7.24 (m, 4H), 7.34-7.41 (m, 2H), 7.67 (d, J=8 Hz, lH), 8.22 - (s, lH).
Analysis for C33H4sNsO3:
Theory: C, 70.81; H, 8.10; N, 12.51.
Found: C, 70.71; H, 8.21; N, 12.42.

Preparation of 2-(4-(piperidin-1-yVpiperidin-l-yVacetic acid, potassium salt -- --+ BrcH2co2cH3 CO2CH3 CO2- K+
~CH2 CH2 N N

4-(l?iperi-lin-l-yl)pip~r~ ne (1.20 kg, 7.13 mol) was added to methylene f~hlorille (12.0 L) under a nitrogen ~t~no.sphere.
Tetrabutyl~mm-mium bromide (0.150 kg, 0.47 mov and sodium hydroxide (1.7 L of a 5 N solution, 8.5 moV were then added. The reaction ~ ,Ule W O 97l33583 PCTrUS97/035SS

was cooled to 10-15~C and methyl bromoacetate (1.17 kg, 7.65 mol) was added and the resulting miX~lre was stirred for a mininlu m of 16 hours.
DPioni~ed water (1.2 L) was then added to the n~ e and the layers separated. l~e aqueous layer was back-extracted with 5 methylene r.hlo~la (2.4 L). The organiC fr~ on.c were eomhined and washed with ~l~,ioni7.ed water (3 x 1.2 L), a sa~u~ated sodium bicarbonate .$ol~ on (1.1 L) and a saturated sodium r.hlori~l~ solu1ion (1.1 L). l~ne organic fraction was then dried over anhydrous magnesium sulfate and conr,~,nt.rated to an oil on a rotary evaporator to yield 1.613 kg (93.~%) of methyl 2-(4-(piperidin- 1-yl)piperidin- 1-yV~et~te A solution of methyl 2-~4-(piperidin-1-yl)piperidin-1-yl]~r,etDte (2.395 kg, 9.96 mol) in me~s~nol (2.4 L) was added to a solution of potassium hydroxide (~.662 kg, 10.0 ~nol @, 85% purity) in me~nnl (10.5 L) under a nitrogen atmosphere. The re~rtion ~ lule was heated 1~ to 45-50~C for a miniTnum of 16 hours.
A solvent eY~.h~n~e from metll~nnl to acetone (15.0 L) was p~.rfiorrnell on the solution on a rotary evaporator. This qolntinn was slowly cooled to room temperature over 16 hours. The resulting solids were iiltered, rinsed with acetone (5.0 L) and tllen dried to yield 2.471 kg (93.8%) of 2-(4-(piperidin-l-yVpiperidin-l-yVacetic acid, potD.c.qinm salt.
MS 265 ~M+l) Preparation of ~R)-3-(lH-indol-3-yv-l-[N-(2-m~~o~ybenzyl)acetylamino]~
2-[N-(2-(4-(piperidin- l-yl)piperidin- l-yl)acetyVamino]propane dihy~orl~lnri~(le trihydrate oc~3 /=\ I
~ Q~c~
+ ~ 0~ ~ ~
N ~ N
C N -c N-C~2 CO2 K+

~ 3 H2O

W O 97/33S83 PCT~US97/03~55 Under a nitrogen ~t~nosphere 2-(4-(piperidin-1-yVpiperidin-l-yVacetic acid, potassium salt (0.75 kg, 2.84 mov was added to methylene rhlnrilla (7.5 L). The resulting ~ule was cooled to -15 to -8~C and isobutyl chl~loru~ te (0.29 kg, 2.12 mol) was added at such a 5 rate so as to m~int~in the tempeldlu~e ofthe re~r~ion ~~ix~-~.e below -8~C.
After the ~ li*on the resulting re~t;?n ~ ,ure was stirred for 90 minutes between - 15 and -8~C.
The re~r,~;nn .. . i x~ ~ ~ ~ e was then cooled to -35~C and solid ~R)-2-amino-3-(lH-indol-3-yl)- l-[N-(2-me~oxybenzyvamino]propane dihy~Loal~lori~la (0.60 kg, 1.14 mol) was added at such a rate that the re~r~ n temp~la~u~ was m~intsine-l at less than -20~C. After the s~ lit;on, the re~r1~on mixt~lre was stirred for about one hour with the tempel&~u e being m~int~ined between -37~C and -20~C. The res-r~ir,n was qll~nrhed by the ~llition of ~laioni7.ed water (7.~ L). The reslr1;on ... i xl ~. . e was b~ified to pH 12.8-13.2 by the ~rlfli~ion of 5 N sodium hydroxide. The aqueous fraction was removed and retained. ~ ;ons~1 d~ioni7ed water (3.75 L) was added to the organic fraction as was s-lffiriant 5 N sodium hydroxide to re-adjust the pH to 12.8-13.2.
The two aqueous fr~r,~;nnc. were comhined, back-extracted with methylene rhlori~lP (1.5 L) and then discarded. The organic frs~r1;on were comhined and washed with ~l~ioni7.ed water (4 x 3.5 L). These extracts were combined, back-~xl ~ ~ ed with methylene rhlori~la (1.5 L), and then discarded. The two organic layers were comhined and washed with a saturated sodium rhlo~fla solution (3.7 L~.
The organic frs-r~inon was dried over anhydrous ms~gne~cillm sulfate, filtered, and solvent ~-Ynh~nEed from methylene rhln~lla to ~cetone (3.75 L) on a rotary evaporator. An aqueous solution of hy~Lorhlorir acid (0.48 L of 6 N solution, 2.88 moV and seed crystals (2 g) were added and mi~t~lre was stirred for 30-90 minutes. Acetone (13.2 L) 3 0 was then added and the slurry stirred for one hour. The resul1ing solid was then filtered, washed with acetone (2 x 1.4 1,), and d~ied to yield 633 g (90%) of ~R)-3-(lH-indol-3-yV-l-~N-(2-methoxybenzyVacetylamino]-2-lN
(2 -(4-(pipe~idin- l-yVpiperidin- 1 -yl)acetyl)amino]propane dihydrorhl o ri ~1 e trihydrate.

W O 97/33S83 PCT~US97/03555 Preparation of (R)-3-(lH-indol-3-yv-l-[N-(2-mef;hoxybenzyvacetylamin 2-[N-(2-(4-(piperi~lin-1-yl)piperidin-1-yl~acetyl)amino]propane flio~ e N ~ CH3 N~>--N~
~ 2 oxalate Into a 500 ml jacketed round bottom ~lask was placed 2-(4-(piperidin-1-yl)piperidin-1-yl)acetic acid, potassium salt (25.0 g, 94.5 mmol3 and 375 ml of N,N-Ilimetl-ylform~ e. The resul1ing slurry was cooled to -19~C and isobutylchlu~..r.~ te (12.9 g, 94.5 mmol) was added 10 over five mirlutes. The resulting n~i~ture was s1 irred for twenty minutes and then ~)-2-amino-3-(lH-indol-3-yl)-1-lN-(2-methoxybenzyVacetyl~minQ]propane dihydrorhl-ri-l~ (25.0 g, 58.1 mmoV, dissolved in 75 ml of anhydrous N,N-dimethylfortn~miflP~, was added over ten minutes.
The resulting m~ re is then cooled to 0~C, sti~red for about ten minutes, and then permi~tetl to warm to room temp~fd~,ule. The progress of the re~rtion was mo~ oled by c31r~ toE~raphy. High p~7 fo7mz~nce liquid chroms~to~raphy showed 99% CUI1~e~ ~,ion of the re~ct~n~e after ninety minutes.
The re~ct;on .. ;~ was part~1ionedbetween ethyl ~cet~te (375 ml) and a saturated sodium bicarbonate solution (375 ml). Inne aqueous layer was back extracted with 375 ml of ethyl acetate. The organic fr~c~ion~q were co nhine.l, washed with water (3 x 375 ml), and then dried over mag~esium sulfate. Potassium Ly~lro~de is then added to 25 the aqueous fraction from above and this resulting basi~ed solution is extracted with ethyl acetate. Th~s organic fraction is then d~ied over m~gnesium sulfate.
The comhined d~ied org~niC fr~rt~onc are then treated with a conc.ontrated oxalic acid solution. The resulting solids are filtered and W O 97/33583 PCTrUS97/03555 dried at 50~C om a vacuum oven to yield 23.5 grams of the desired intermediate.

As wou~d be appreciated by a skilled pr~rt;*oner the mixed 5 anhydride process will work in a number of org~nic solvents, in z~ ;on to the anhydrous N,N-dimethylfmmAmi-l~ depicted above. Representative eX~mI le,c of solvents which may be employed include ~cetonitrile, tetrahy~ru~ lirhloromet~ne The mixed anhydride process can be pf~!rform~d at tempel~ules below 0~C.
The oXSIl~tQ can be isolated from ethyl acetate as well as from other solvents, probably in~uding acetone, ~cetonitrile, and t-butyl methyl ether. The use of ox~lic acid is, how~v~r, very important for the pre. ;~ l ;nn as a large number of acids do not give a ~le. ;~ e. Among those acids ~ .ted, but found not s~ f~rtory for the processes of the 5 present invP-ntien, are citric, anhydrous hylllorl.lolir, tartaric, mandelic, trifluoroacetic, p-nitroben~)ir" phenoxyacetic, m~lPir, films~rir, Flllt~rir, ~ipic, me1~n~.cmlfnnir, p-toluenes..lfonir, pamoic, trans-1,2-cyclohexane dicarboxylic, ~llr~inir" pht~lir, trans-1,2-~ minocyr~loh~x~ne-N~N~N~N~
naph~l~n~dislllfonir" and 5-slllfn.s~lir,ylic acids. Only oxalic acid and 20 1,5-naph~l~ne dis~llfonir, acid reproducibly produced a solid.

Preparation of (R)-3-(lH-indol-3-yl)-1-[N-(2-me~nxybenzyVacetyl~nlino]
2-[N-(2-(4-~iperidin-1-yl)piperidin-1-yl)acetyl)~mino~propane dihydrorhl-)ri~l~ trihydrate H O
N ~ - N

-2 HCl ~3 H2O

Into a large beaker were added ~R)-3-(lH-indol-3-yl)-1-[N-(2-methoxybenzyl)acetylamino]-2-1N-(2-(4-(piperidin- 1-yl)piperidin- 1-W O 97133583 PCTrUS97/03555 yl)acetyl)amino]propane (lioY~l~te (13.4 g, 18.1 mmol), methylene ~hlo (~8.16 ml, 78.51 g), and water (118.59 ml). The resulting mixt~lre was stirred and the pH of the re~1;on ...~X(5.1~ was adjusted to 10-12 using 50% caustic.
The phases were separated and the organic phase was back extracted with water (101.44 ml). The organic fi action was tr~neferred to a j~r.ket~ round bottom flask and a solvent ~Y~.han~e was p~rform~d using about 23 volumes of ~ceton~. Portions of the ~cetc-ne were added to the product solution and the amount added was ~1;Q~ away. The lo progress of the solvent ~x~h~nge was mo~i~oled by Nl~. The amount of desired product was mo~itoled by high p~.rfi~rm~nce liquid chrom ~to~raphy~
E~ough water was added to bring the water cono~nt~ation to eleven percent and 1~he resulting ... i ,.l ~ . . c was heated to ~;5~C. Enough 15 conc~nt.rated hy~lro~ k~ric acid was added to lower the pH to 2.0 and the re~ n ~-~ was then p~ -i I led to cool to 37~C over 45 minutes.
The product solu1ion was seeded and p~ i l L~:d to stir for 10-30 minutes. The product solution was cooled to 19~C over two hours and acetone (ten equivalent volumes) was added over three hours, after which 2 0 time the re~ ;on rnixture was stirred for one to three hours, n~s~in~inin~
the tempelatu~e at 19~C. The product sol~ n was filtered and the residue was washed with 6.67 equivalents of acetone. The residue was then dried in a vacuum oven at 42~C to give the desired title product.

The other compounds of Formula I may be prepared essentially as (lesr.rihed above, employing correspon~ing :~la~ g m ~ ri r-l .c.

The biological efficacy of a compound believed to be ~e.;l ive 3 0 as a tachyl~in receptor antagonist may be c- r~firmed by employi~g an initial screening assay which rapidly and accurately measured the l~in~ing of the tested compound to known NK-l and NK-2 receptor sites.
Assays usefill for evaluating tachykinin receptor antagonists are well known in the art. ~::ee. e ~., J. Jukic, ~aL T-ife ~ on~eS~ 49:1463-1469 (1991); N. Kllr.h~rczyk, et al., Jollrn~l of Me~ir.in~l Ch~nlistry, W O 97/33583 PCTnUS97103555 36:1654-1661 (1993); N. Rouissi, ~L, Rio~ mical snd Ri~ y.
Rese~r~.h C- mmllni- ~tions~ 176:894-901 (1991).

NK-1 Receptor P~inllinF ~.cs~y ~ lioreceptor binding assays were parformed using a del;vaLive of a previously published protocol. D.G. Payan, et ~1.. Jollrnsl "f Tmmnnol(?~Fy, 133:3260-3265 (1984). In this assay an aliquot of IM9 cells (1 x 106 cells/tube in RPMI 1604 medium supplçmanted with 10%
10 fetal calf serum) was incubated with 20 pM l25I-labeled substance P in the presence of increasing competitor concentrations ~or 45 minutes at 4~C.
The IM9 cell line is a well-char~-t~ri7e-1 cell line which is readily avsilshla to the public. See~ e ~ nnf~ of f~e New y~rk Ar~ y of ~ ianra 190: 221-234 (1972); Nature a.-mllnr~), 251:443-444 (1974); Procee-lin~.~ of f~a N~ ns.1 ~rsrla~rly of ~riances (IJ~ 71:84-88 (1974). These cells were routinely cultured in RPMI 1640 supplemented with 60 ~g/ml gçntsmi~in sulfate and 10% fetal calf serum~
The res-f-*on was t~ te l by fil~rs*on through a glass 2 û fiber filter ha~vesting sy: ilem using filters previously soaked for 2û
minutes in 0.1% polyethyle..i...i..e Specific bin~ing of labeled substance P was deta~ i--ed in the presence of 20 nM unlabeled lig~n~l Many of the compounds employed in the methods of the 2~ present invention are also ~-;live antagoni~ts of the NK-2 l~e~tor~

NR-~ F9r~tor P~;nllinF ~.c.c~y The CHO-hNl~-2R cells, a CEO-derived cell line tr~ncfiorm~d 3 û with the human NK-2 receptor, expressing about 400,000 such receptors per cell, were grown in 75 cm2 ~lasks or roller bottles in minimAl essential medium (alpha mo(lif;rs~tion) with 10% fetal bovine serum. The gene sequence of the human NK-2 receptor is given in N.P. Gerard, et al., Jollrns~l of Rinln~ir.~l Ch~mi~try~ 265:20455-20462 (lggO).

For preparation of membranes, 30 con~lant roller bottle cultures were dissociated by w~-qhin~ each roller bot1~1e with 10 ml of Dulbecco's phosphate bu~e.ed saline (PBS) without calcium and magnesium, followed by s~ on of 10 ml of enzyme-free cell diqcoris~hon 5 so1ution ~PBS-based, from SpecialtyMedia, Inc.). After an ~ itional 15 minutes, the ~iqsori~qted cells were pooled and cantrifilged at 1,000 RPM
for 10 minutes in a rlinir~l centrifuge. MarnhraneS were prepared by homogani7 sl~nn of the cell pellets in 300 ml 50 mM l~is bu~fer, pH 7.4 with a Tekmarq~) homogenizer for 10-15 secon~q, followed by centrifugation at 12,000 RPM (20,000 x g) for 30 minutes using a Berkm~n JA-14~ rotor. The pellets were washed once using the above procedure. and the fin~l pellets were resuspended in 100-120 ml 50 mM:
I'lis bufEer, pH 7.4, and 4 ml aliquots stored frozen at -70~C. The protein conc~ntr~tion of this preparation was 2 mg/ml.
~s l~or the receptor bin-ling assay, one 4-ml aliquot of the CHO-hNK-2R mamhr~ne preparation was suspended in 40 ml of assay buffer cont~ining ~;0 mM Tlis, pH 7.4, 3 3mM m~ngz~nese rhloritl~o~o2%
bovine serwn albumim ~IBSA) and 4 ~lg/ml chyrnosts1;n A 200,ul volume of the homo~enate (40 ,ug ~ leill) was used per ~m~le The radioactive 20 ligand was tl2~I~iodohi~yl-neurokinin A ~New h:n~l~nd Nuclear, NEX-252),2200 Ci/mmol. The ligand was prepared in assay buffer at 20 nCi per 100 ~ he final concant~ ation iIl the assay was 20 pM.
Non-specific binding was ~l~le....i..ed using 1 ~IM eledoisin. Ten con~n~ations of e1edoisin from 0.1 to 1000 nM were used for a st~n-l~rd con~f~ntration-response curve.
All ~ m~ ; and st~n(l~rds were added to the incubation in 10 ~1 dimethylslllf~ e ~DMSO) for screening (single dose) or in 5 ~1 DMSO for ICso det~ ;on~ The order of ~ hon~ for incllh~*on was 190 or 195 ~ assay bufEer, 200 ,u1 homogenate, 10 or 5 ~1 sample in 3 0 DMSO, 100 ~1 r~(lin~ctive li~n~l The s~mrl~c were incubated 1 hr at room temperature and then filtered on a cell harvester through filters which had been preso~ked for two hours in 50 mM l~is bufEer, pH 7.7, cont~ining 0.!i% BSA. The filter was washed 3 ~mes with apprnxims~t~ly 3 ml of cold 50 mM Tris buffer, pH 7.7. The filter cir~es were then W O 97/33583 PCT~US97/03555 punched into 12 x 7~ mm poly~ly~ e tubes and counted in a g~mm~
counter.

It has been rl~t~rmined that the method of the present - 5 invention is eLCdclivd in treating m~mm~ , par1;rul~rly mi~flle aged women, Pxhihitin~ symptoms of inte. ~4~ cystitis andlor urethral syndrome. In this regard, the r.linir~l and local immune response to the compounds of the present invention is inves1;~ted in an open trail with 10 fam~le. intel~lilial cystitis p~tiPnt~, whose disease is (~ nosed 0 acco~ g to the consensus rritQri~ developed in 1987 at a Nz-tion~l Tns~itllt~.c of ~e~lt~ work-qhop. To make objective the symptoms and the r.linir.s-l response of the p~t;Pntc the present inventors scored (scale O to 2)the symptoms of frequency, urgency, nocturia, dysuria and ~u~1a~ubic pain, as flesrrihed in United States Patent 5,145,859, issued September 8, 1992, the entire cont~ntq of which are herein incorporated by larel~ce. A
compound of the present invention is ~rlmini.~tered as a single daily dose det~.rmined by a dose-titration test. Urinary int~rlaukin-2 inhihitory activity (TL-2-IN), a m~rk~.r of ce~l-mafli~te~ infl~mm~ti~m, is measured using a mu~ine in~~rlaukin-2 dep~n-lent cell line.
2 o The p~ti~ntS are reviewed for re~ rtit n in rl;nir,~l symptoms.
Drug side-effects are minim~l Urinary IL-2-IN activity before therapy co.. l;.. ~ t,he presence of cell-me~ tQd infk~mm~tion after 4 mon~ of therapy IL-2-IN a.;Livily is norm~l in most of the p~t;antc, regardless of the sevel;ly of symptoms, which in~lir~te,s that the compounds of Formula 2 5 I exerts an immunosuppressive effect. The data suggests that the compounds of Formula I can be an ~ffir~rinus, well-tolerated, collv~ient oral me~lir~tion for the tre~tm ~nt of intQ- sl; I ; ~l cystitis.
In ~ on, as more clearly demonstrated below in ~.~mp1e 2, the present inventors also observes .cimil~r responses in regard to the 3 0 tre~t~nent of urethral syndrome. As a result, the test data clearly in~lirsltqs that the compounds employed in the present invention can be effective therapeutic agents for the treatment of interstitial cystitis and/or urethral syndrome.
As a result, it has been found that compounds of Formula I
3 ~ are par~ir~ rly well-suited for the treatment of inte ~Lilial cystitis and/or W O 97/33583 PCT~US97/03S55 urethral syndrome becsuse they not only provide effective relief, are av~ hle for oral ~rlmini.qtration, and are relatively inexpensive. It has been disc~,veled that p~tiPntS receiving the compounds of Formula I
subst~nti~lly reduce the p~tllolngil~l conllition.c exhihited by these two 5 painful l~ Pr disorders, and are able to carry on t~heir daily activities in a relatively no~l existence in comp~rison with their pre-tre~nant state.
The present invention will be further d~.c~.rihed acco ~i~g to ~e following non-l i ...i l; ..~ ex~rnples.
F',~le 1 M~tqri~lc andMethods 15 ps~t;Pn~c. Inne ~ no.ci.q of intor~t~ l cystitis is s-.c.ci~ned to 10 fPm~le p~tiP.ntc, aged 23 to 51 years, in accordance with ~he consensus n~it~~
cstohliqhell at the N~ n~1 Tn~it~ .q of Fre~l~ workshop on inte~ ia cystitis, August, 1987 (Gille~water, J. Y. alld Wein, A. J: Sllmms~ry of the N~ion~l Institute of Art~r ti.c, Diabetes, Digestive and ~dIley 20 DiseasesWorkshoponInle~ l Cystitis,Ns~tinns~ stitutesofl~es~lt~, Bethesda, Md., Aug 28-29, 1987, J. Urol., 140:2()3, 1988), alld United States Patent 5,145,859:

Tn~ l Cys~ q: Crit~ for ni~Fnn.ci.c 2s Inclusion Crit~ri~ Exclusion Criteri~
Hunner's Ulcer (if present, less than 18 years old aut~m~ inclusion) benign or m~lign~nt t1lmor.~;
r~ ;ien, tuberculous, b5lrtrris~1 Positive Factors (at least 2 or cyclophosph~mille cystitis required for inclusion): v~giniti.c duration of symptoms c 1 year suprapubic, pelvic, urethral, gynecnlogic cancer vaginal or pP.rine~l pain urethral diver~ bl~l.ler W O 97/33583 PCTnUS97103SSS

or lower ulete. ~1 cs~
m~r llAt~onc at cystoscopy active herpes (HSV II) after bl~ r dist~n.cion waking frequency c 5 in 12 hrs.
(80 cm water pressure x 1 min.) nocturia < 2 neurogenic bl~ r dycfilnc*on decreased comI)li~nce on waking capacity ~ 400 ml, ~;y~on~etrogram absence of urgency with bl s~ ?r filling symptoms relieved by s~n*hiotics, urinary pain on bl~ r filling urinary analgesics or relieved by emptyin~ ~n~i.ceptics Cystometrics are p~rfnrm~d after cess~*on of other modes of therapy andprior to in~ tion of therapy: all ps~ti~ntc had a waking hls~ .r capacity of less than 350 ml (range 150 ml to 340 ml).

Symptom Eval l~tion The symptom scores (total score range:
O to 10) form the basis for the ev~ ;on of lA.~AI~ nt efficacy. The sev~.;ly of each symptom is ~c.ci~ned a nllm~ l value, as follows:

Symptom Sev~l;ly Survey Symptom Description Score Frequency voids once every 3 to 5 hours 0 (daytime) voids once every 1 to 2 hours voids more than once every hour 2 Urgency urge to void equal to actual o frequency urge to void exceeds actual frequency conct~nt. urge to void 2 Nocturia no noctllri~, or 1 void nightly 0 = .=

W O 97/33583 PCTnUS97/03555 nocturia 2 to 4 times night~y more than 4 times nightly 2 Dysuria no dysu~ia o intermitt~nt dysuria dysu:~a with each voi(l 2 Suprapubic pain no pain o (ab~ minO- int~rmitt~nt pain p~rine~l) constant pain 2 At the 1ime of ~ nosi.s, and lbefore any tres~t~n~.nt any patient who f~lls wi1~hin the par~n-eters of the inclusion of exr.lll.ci~n ~l~sr.rirtors of the NIH workshop con~nsus r.riferi~ (above~ will score at 5- least a "4" on this ~U~ y (frequency c l; urgency < l; noct~ c l; a~d either dysuria or ~u~la~ubic pain~ 1).

Ur-n~? Cnll~r1;fn~ Urine spefim~.n-s are ctllectedfrom all p~ .nts before and ~ rin~ therapy. Voided urine is ceIltrifuged at 1000 x g for 10 minutes at 4~C and the sup~ t~nt separated from the se~limPnt The urine supern~nt is subiected to 0. 2 ~1 filtration (c~ lose~et~te) at 4~C to remove any b~rt~~i~ and debris, and a 1 ml aliquot is removed for cre~nine measurement (CR~ATIN~E II ANALY~ ~, Berkm~n Instr~lmen~s, Inc., Brea, C~lifn? nis-). The supern~t~ntiS ultraiiltered ~inQt 3 x volume i~ pho.sph~e-Lufl~led sali~le OEBS~ with 0.1 ~g~ml albu in (~ , St. Louis, Missouri) using a ~iltration device (5,000 MVV
cut off; ~micnn~ Deavers, M~qs~.husetts). The conc~n~rated sup~rnflt~nt is dialyzed using 3,600 MW cutoff tubi~lg, shell frozen with dry ice, and v~uu~ lyophili7.ed The powder is stored at -20~C.
Measurement of IL-2-IN As;l,ivi~y. The hio~q.q~y for IL-2-IN
is modified from the method for measuring IL-2 activity ~.s-~.rihed by GiDis and associates. S. Gil~s, et al., "T-Cell alvw~h Factor: Parameters Of Pro~lllr1;nn And A Qll~ntit~tive Microassay For Activity, Jollrn~l of TmmllnnlnFy. 120:2027, (1978). The murLne rL-2-depPn~Pnt Cy~OtOxiC T-cell line (CTLL-N) is derived fromthe CT-6 cell line. J. Kuqllg~mi, ~aL, 'Inte~;n~l Immune Reactivity To Tnterleukin-2 Differs Among ~rohn's W O 97/33583 PCTrUS97/03555 Disease, Ulc~laLive Colitis And Controls", Gastro~nterolo~y, 97:1 (1989).
The CTLL-Ns are m ~intsined in liquid culture using a 1:1 ..- i ~l u .~e of Roswell Park Memorial Institute (;RPMI) 1640 and Dulbecco's Modified F'.s~gle.s Medium ~DMEM; 4.5 g/L glucose) media supplamantad with 2.9 mg/ml glucose, 9.4 mM HEPES buffer, 1.9 mg/ml ~ .. ;.. e, 289 ~Lg/ml a~ e, 0.12 M non-essential amino acids, 5 x 10-5 M 2-mercaptoethz~nnl, 4.5% fetal bovine serum, 90 units/l p~nil~illin, 90 llg/l streptomycin, 22 ~lg/ml fungi~.one, 0.45 mg/ml g~ntqmirin and 20 units/ml of human recomhin~nt IL-2.
The CTLL-Ns are washed and suspended at a crnc~ntration of 10~5/ml in the culture media. Assays are p~rformed in ~ lic~te, as follows: a serial dilution of the sample aliquot (50 ,ul), a 1:10 dilution of the human recomhin~nt IL-2 standard and 10-4 CTLI,-Ns (100 1ll) are placed in microliter wells. The microliter plates are incubated in a h~lmi-lified 6% C02 ~tmosphere at 37~C. for 24 hrs, and the cells are pulsed at the 19th hour with 1 ,uCi/well of methyl-tritiated thymi-line (specific a ;Livi~y 6.7 Ci/~, New F',ngl~nd Nuclear,I. E. Dupont, Boston, Ms~.css-rhusetts).
The c~llc are cl llect~d onto glass filter paper discs. The discs 2 0 are placed in .srint~ tion fluid and thymi-line uptake is measured by liquid srintill~ti~n spectrophotometry. IL-2 inhihitory activity is c~l~nl~ted by motlified probit analysis.
The prql; ra- ~ ~ ;on "m~ximum" iS the tri~ ~ted thymidine uptake c~ncell by 1~he amount of ~-J~ ous IL-2 a :Livil,y in the control 2 5 microliter wells, assessed in quadrllr lir~te for each assay. The proliL'elaLion ~minimum" is derived from lowest amount of ~i~ ted thymi(line uptake caused by the IL-2 inhihitor st~nll~rd. The probit calclll~tion corle-,~ed for minor interassay v~ri~t;nnq of thymilline uptake in control wells, and pçrmitt~d interassay comp~riconc of inhi~itor 3 0 activity ~mong the u~ine s~m~les By this tre~tmPnt of the data, the c~lrlll~tsd value of IL-2 inhihitory activity in lyophil~zed urine samples varies less than 10% from assay to assay. IL-2-IN activity is expressed in units/mg urine cre~tinine (U/mg u.c.). II.-2-IN activity is less than 0.05 U/mg u.c. inthe uriIle of healthy adults. J. FlPisrhm~nn, et al., .Jollrn~l of Riolo~irz3l Re~ulators ~ndHomeostaticA~ents, 4:73, (1990).

W O 97/33S83 PCTnUS97/03SS5 M~lir.~*t~n ~q.siFnm~ntS~ p~ti~.nt~ are treated initis3lly with a total daily dose of 30 mg, which is ~-lmini.~tered as a sing1e7 e~ n~l release tablet.
Ps~ti~nt Ml nit~)rin~ P~tientq are interviewed and blood pressure measured twice mon~ly during the ~rst 2 montl .~ of therapy, during the first 2 montbs after a dose ~sc~ r, and then once nlontllly thereafter. The symptom seve ;~y score at each interview is based on the 10 patient's exp~rif~.nrç~C fillrinF the previous 24 hours.

In ~ lition to the tre~ .nt of ps~ ntc with interstitial 15 cystitis, p~ti~ntc with the urethral sy~droIne have been treated with a compound of Formula I, using 1~he titration test and Lle~ .nt protocol d~srrihed in United States Patent 5,145,859. .~ r to the data of ~ mpJe 1, the positive response to the co~pounds ofthe present invention in ~his limited study supports the hypothesis that the 2 o urethralsyndrome and interstitial cystitis are both part of the same disease spe~ ,perhaps as v~ n~c of re~1ex sympathetic dy~lLvphy.

The invention has been (l~sr.rihed with r~,~..ce to the pl~elled embo~lim~,nt Obviously, mo~ifir,~*f nc and alterations will 25 occur to others upon a reading and llndp-re-tsn~ing of this sperifir.~tion Itis ;nt~nlle~l to inrlllde all su~ morlifir~tionq and alterations in.ql~f~r as they come within the scope of the appended claims or ~he equivalents thereo~

3 0 VVhile it is pos.cihle to ~(lmini.eter a compound employed in the methods of this invention directly wil~out any form~ *cm, the compounds are usually ~rlmini.ct.~red in the form of ph~ ceutical compo.ci~';onc comprising a ph~rn~el-*r~lly acceptable excipient and at least one active ingredient. These compo.qitionR can be a-l rnini.qtered by a 3 5 : variety of routes including oral, rectal, trans-lerm ~l, subcutaneous, W O 97133583 PCT~US97/0355S

intravenous, intram11-qc~11sr, and intrsns..es-1 Many of the compounds employed in the methods of this invention are effective as both inject-sh1~
and oral compocitionq. Such composit;on.C are prepared in a msnnPr well known in the phsrlnsreutical art and co.u~l;se at least one active 5 compound. ~ee. e ~, REMINGToN~s PHARMACEUTICAL SCIENCES, (16th ed. 1980).
In msking the compoeitinnc employed in the present invention the active ingredient is usually mixed with an ~X~ipi~nt, diluted by an e~riI i~nt or enclosed within such a carrier which can be in the form 10 of a capsule, sachet, paper or other contsinçr. When the ~xripiPnt serves as a diluent, it can be a solid, semi-solid, or liquid mst~~s.1, which acts as a vehicle, csrri~r or medium for the active ingredient. l~us, the compo.qitionc can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, susp~ncionc, em1~1cion.c, solutions, syrups, 15 aelosols (as a solid or in a liquid medium), oin~nçn~C contsining for eY-smp1e up to 10% by weight of the active compound, soft and hard gelatin capsules, sUppocitories~ sterile inject-sh1e s~ ti- ~.c, and sterile packaged powders.
In preparing a form~11s.*on, it may be necesc~ry to mill the 2 o active compound to provide the appropriate particle size prior to comhining with the other ingredients. If the active compound is substsnti~s-lly insoluhle, it or~insri1y is milled to a particle size of less than 200 mesh. ~f the active compound is s11hstsntially water soluble, the particle size is norms.11y adjusted by mi11ing to provide a substSm~i~s11y 25 ..~ira~ ) distribution in the form~11st;on, e.g. about 40 mesh.
Some PY~mplçs of st1itsh1e eY~ipi~ntc include lactose, dextrose, sucrose, sorbitol, msnnitr~1, starches, gum srsris., calcium phosphate, ~91~n9tQs, trsgscsntll, gelatin, c~s.1~ lm .ci1ir"stQ, mi~ sl-slline ce11111Ose, polyvinylpyrro1irl-ne, c~11111Ose, water, syrup, 3 0 and methyl cellulose. The form.11st;onc can ~ i*on~11y include:
l11hrirsting agents such as tlc, ms.~ne.cinm stearate, and mi~eral oil;
wetting agents; emulsi~ing and suspending agents; preserving agents such as methyl- and propylhy~o~yl~en7.0stQ.c; sweetening agents; and flavo ing agents. The compositionc of t,he invention can be formulated so 3 5 as to provide quick, s11~tsined or delayed release of the active ingredient W O 97/33S83 PCTrUS97/03555 after s~minictration to the patient by eInploying procedures known in the art.
The compositions are preferably formulated in a unit dosage form, each dosage contsining from about 0.05 to about 100 mg, more 5 usually about 1.0 to about 30 mg, of the active ingredient. The term "unit dosage form" refers to physically discrete units sllit-shle as u~i~aly dosages for huma~ subjects and other msmmsl~, each unit contDinin~ a pre-l~Pr-nined quantity of active ms~erisl c~lrul~qted to produce the desired therapeutic effect, in s~sori~t;on with a sllifshle phsrm~sceutica 10 e~ ~ipi~.nt The active compounds are ger ~-r~lly ~e~;l.ive over a wide dosage range. For e7r~s-rnples, dosages per day nn~m~slly fall within the range of about 0.01 to about 30 mg/kg of body weight. In the treatment of adult hllm~nc, the range of about 0.1 to about 15 mg/kg/day, in single or 15 divided dose, is especially ~ ed. How~ver, it will be understood that the ~mount of the compound ~ lly ~ ini~tered wi~l be det~rmin~d by a physician, in the light of the relevant ~L.;~ tsnces~ including the c-n-li';on to be treated, the chosen route of ~liminiQt~hc~n~ the actual compound or compou~ds ~flmini~t~red, the age, weight, and response of 2 o the individual patient, and the s~v~;ly of the patient's symptoms, and thel~o~ e 1~he above dosage ranges are not intsn~ 1 to limit the scope of the invention i~ ally way. In some inQt~nseS dosage levels below 1~he lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing any 2 5 harmfill side effect, provided that such larger doses are first divided into several cm~llPr doses for ~mini.ctration throughout the day.

F- rm~ nn Prel;>ar~ti-m 1 3 o Hard gelatiIl capsules cont~ining the follow~ng ingredients are prepared:

Quantity Tn~re~ nt ~ slll 3 5 Active Ingredient(s) 30 0 W O 97/33583 PCT~US97/03S55 Starch 305 0 M~gne.~ium stearate 6.0 The above ingredients are mixed and filled into hard gelatin capsules in 340 mg q~ntities-Forrnnls~ti- n Pre~slr~tif)n A tablet formula is prepared using the ingredients below:

Quantity Tn ~Fre~i~nt. ~n ~/table~
Active Ingredient(s) 25.0 Cellulose, mi~ L~lline 200.0 Cc~lloi~l~l silicon flioxi~l~ 10.0 Stearic acid 5.0 The components are blended and co~lessed to form tablets, each w~ighinE~ 240 mg.

W O 97/33583 PCT~US97103S55 Form~ h-m Pre~ t.inn 3 A dry powder inhs~l~r form~ t;on is prepared cont~ining the following components:

Tn~re~liPn~. WP.i~ %
Active Ingredient(s) 5 Lactose 95 The active ~ re is mixed with the lactose and the ~ - e is added to a dry powder inhs~ling appliance.

Form~ ;on Pre~ r~t.i--n 4 Tablets, each contsinin~ 30 mg of active ingredient, are prepared as follows:

Quantity 2 o Tn~re~i~nt ~m~ h Active Ingre-lient(s) 30.0 mg Starch 45.0 mg 2 5 Mi~,c~ t411ine cP.ll~ e35.0 mg Polyvinyl~yl~lo~ )np (as 10% solution in water) 4.0 mg 3 0 Sodium carboxymethyl starch 4.5 mg Magnesium stearate 0.5 mg Talc 1.0 m~

CA 02247822 l998-08-3l W 097133583 PCTrUS97/03555 Total 120 mg The active ingredient, starch and cellulose are passed through a No. 20 mesh U.S. sieve and mixed thoroughly. The solution of 5 polyvinylpyrr~ ne is mixed with the resultant powders, which are then passed through a 16 mesh U.S. sieve. The granules so produced are dried at 50-60~C and passed through a 16 mesh U.S. sieve. The sodium carboxymethyl starch, m~Psium stearate, and talc, previously passed through a No. 30 mesh U.S. sieve, are then added to the granules which, 10 after mixing, are compressed on a tablet m~rhinP to yield t~blets each wPiFhing 120 mg.

Form~ n Pre~ration 5 Capsules, each ContsininF 40 mg of medicament are made as follows:

Quantity Tn~re~iPnt (~n~ sule) 2 o Active Ingredient(s~ 40.0 mg Starch 109.0 mg Ms~neqillm stearate 1.0 m~
Total 150.0 mg The active ingredient, cPll~llo~se, starch, and m~np~ium ste~r~te are hlPnllp~l~ passed through a No. 20 mesh U.S. sieve, and filled 3 o into hard gelatin capsules in 150 mg qll~n*t;P.~

W O 97/33583 PCTnUS97/03555 Fo~nnl~tinn Prepsration 6 Suppositories~ each contsining 25 mg of active ingredient are made as follows:
Tn~re(li~nt .Amtnnt Active Ingredient(s) 25 mg Saturated fatty acid glycRrid~s to 2,000 mg The active ingredient(s) is passed through a No. 60 mesh U.S. sieve and suspended in the ssturs~ed fatty a~d glycPri-l~?.s previously melted using the minimum heat neC~s~s-ry~ The ...;~ .e is then poured i~to a suppository mold of nomins-l 2.0 g capacity and allowed to cool.
Fnrtnnls.1;nn Prep-s-rs.*tn 7 Susp~n~inn~, each contsining 50 mg of me-licsmPnt per 5.0 ml dose are made as follows:

TnprelliP.nt ~mmlnt Active Ingredient(s) ~;0.0 mg x~n~sn gum 4.0 mg Sodium carboxymethyl cellulose (11%) Microcrystalline cellulose ~89%) 50.0 mg Sucrose 1.75 g Sodium benzoate 10.0 mg Elavor and Color q.v.

3 5 P--rifie-i water to 5.0 ml W O 97/33583 PCTrUS97/03555 The me-lir-~mPnt sucrose and xanthan gum are hlf~n(l~d, passed through a No. 10 mesh U.S. sieve, and then mixed with a previously made solution of the miclo~ l,alline c~ lose and sodium 5 carboxymethyl ceJlulose in water. The sodium b~n~os~A, flavor, and color s~e A~lu~Ad with Qome of t,hv water a~d adde~l~31;~. Snffl~t water is then added to produce the required volume.

F-rmnl~ti~n Pre~r~t;-n 8 Capsules, each cont~ining 15 mg of me-~ic~ment, are made as follows:

1 5 Quantity TnFrerlif~.nt (mFlrs~sllle) Active Ingredient(s) 15.0 mg Starch 407.0 mg Magnesium stearate 3.0 mg Total 425.0 mg The active ingredient(s), cellulose, starch, and m~gne.qium stearate are blended, passed through a No. 20 mesh U.S. sieve, and ~led into hard gelatin capsules in 426 mg quantities.

W O 97/33583 PCT~US97tO3~5 F~rln~ n Pre~aration 9 An intravenous forInlll~*nn may be prepared as follow~:

Tn~re~i~nt Ql]~nt;t~y Active Ingredient(s) 250.0 mg Isotonic saline 1000 ml F~ tinn Pre~ )n 10 A topical form~ tion may be prepared as follows:
Tn~re~ nt. Q~n1;t~T
Active Ingredient(s) 1-10 g Emul~ing Wax 30 g Iiiquid P~. ~rr..................................... 20 g VVhite Soft Para~in to 101) g 25 The white soft p~rr;.- is heated until molten. The liquid pal.qrr;.- and emulsi~ging wax are incorporated and stirred un1il dissolved. Inne active ingredient is added and s1;rring is conlinued until dispersed. The mi~tllre is then cooled until solid.

W O 97/33583 PCT~S97/0355S

Fnrmnl~fi-)n PreDaration 11 t Sublingual or buccal tablets, each cont~ining 10 mg of active ingredient, may be prepared as follows:
Quantity Tn~redient Per T~hl~t Active Ingredient(s) 10.0 mg Glycerol 210.5 mg Water 143.0 mg Sodium Citrate 4.5 mg Polyvinyl ~lcohol 26.5 mg Polyvinyl~yllo~ ne 15.5 m~
Total 410.0 mg The ~ly~ vl, water, sodium citrate, polyvinyl ~lcohol, and polyvinyll.y-loli-lone are ~mixed together by continuous stirring and ms-int~inin~ the temp~la~ule at about 90~C. VVhen the polymers have gone into solution, the solution is cooled to about 50-55~C and the 25 me(lic~mentisslowly~lmiYed. The homogenous ...ixl~ is pouredinto for_s made of an inert m~t~ri~l to produce a drug-cont~inin~ rliffil.cinn ms~frix having a ~irkness of about ~-4 mm. lThis diffusion m~frix is then cut to form individual t~hlet~ having the a~ riate size.

3 o Another pl~el~d form~ fion employed in the methods of the present invention employs trans-lPrm~l delivery devices ("patches").
Such tr~n~ l patches may be used to provide cont,inuous or cont;nuous infusion of the compounds of the present invention in controlled amounts. The construction and use of transrlerm~l patches for the delivery of ph~rm~ceutica~ agents is well known in the art. See, e.F., CA 02247822 l998-08-3l U.S. Patent 5,023,252, issued June 11, 1991, herein incorporated by ~e. ellce. Such patches may be constructed for continuous, pulsatile, or on ~1am~n d delivery of phz-r~n ~ceuticA~ agents.
Frequently, it will be lla.cirAhl~ or necessary to introduce the 5 phAr~nAreutical composition to the brAin, either directly or in~ e~;~y.
Direct techniques usually involve pl~ramant of a drug delivery c~etçr into the host's v~ntrir~ r ~y~e~ to bypass the blood-brain barrier. One such implA~table delivery ~y~ , used for the transport of hiolo~icAl factors to specific ~nAtomirAl regions of the body, is ~la~rrihed in U.S.
1 o Patent 5,011,472, issued April 30, 1991, which is herein incorporated by ~e~ ce.
~ direct terhniques, which are generally pl~erLed, usually involve form~llAting 1~e composi~ion.~ to provide for d~ug l~tqntiAtion by the conversion of hydrophilic drugs into ~ipid-soluble d~ugs or prodrugs.
15 T.AtantiAti~n is gçnerAlly achieved through blocking of the hy~o~y, carbonyl, sulfate, and prin~ary Amine groups present on the drug to render the drug more lipid soluble and ~manAhle to transportation across the blood-brain barrier. Al+qrn~tively, the delivery of }Jly~ hilic drugs may be enh~nced by intra-arte~ial ;nfil.~ion of hypertonic solutions which 2 0 can ~An ~ ntly open the blood-brain hArriar -

Claims (6)

We Claim:

1. A method for the treatment or prevention of interstitial cystitis or urethral syndrome in a mammal which comprise administering to a mammal in need thereof an effective amount of a compound of the formula where R1 and R2 are independently selected from the group consisting of hydrogen, methyl, methoxy, chloro, and trifluoromethyl, with the proviso that no more than one of R1 and R2 can be hydrogen; and Y is , , , , , , N-Ra, or CH-NR b R c, where R a, R b, and R c are independently selected from the group consisting of hydrogen and C1-C6 alkyl;
or a pharmaceutically acceptable salt or solvate thereof.

2. A method as claimed in Claim 1 employing (R)-3-(1H-indol-3-yl)-1-[N-(2-methoxybenzyl)acetylamino]-2-[N-(2-(4-piperidin-1-yl)piperidin-1-yl)acety)amino]propane or a pharmaceutically acceptable salt or solvate thereof.

3. A method as claimed in Claim 2 employing (R)-3-(1H-indol-3-yl)-1-[N-(2-methoxybenzy)acetylamino]-2-[N-(2-(4-(piperidin-1-yl)piperidin-1-yl)acetyvamino]propane dihydrochloride trihydrate.

4. A compound as described in any one of Claims 1 to 3, for use in treating or preventing interstitial cystitis or urethral syndrome.

5. A pharmaceutical formulation containing, as an active ingredient, a compound as described in any one of Claims 1 to 3, adapted for use in the treatment or prevention of interstitial cystitis or urethral syndrome.

6. The use of a compound as described in any one of Claims 1 to 3, for the manufacture of a medicament for the treatment or prevention of interstitial cystitis or urethral syndrome.