CA2114798A1 - Synthetic aryl polyamines as excitatory amino acid neurotransmitter antagonists - Google Patents

Abstract

Compounds having the formula R-(CH2)m-CO-R' wherein R is a 5 to 7 member carbocyclic system or an 8 to 11 member carbobicyclic system or any such system substituted with one or more of F, Cl, Br, OH, C1 to C4 alkyl, C1 to C4 alkoxy, CF3, phenyl, amino, C1 to C4 alkylamino and di(C1 to C4 alkyl)amino; m is 0 or 1; R' is -[NH(CH2)n]xNH2; each n is independently 2 to 5; and x is 1 to 6, and their pharmaceutically acceptable acid addition salts are potent excitatory amino acid neurotransmitter antagonists. These compounds are useful as mammalian psychotherapeutants, as the active ingredient in pharmaceutical compositions for treating conditions in mammals which are mediated by excitatory amino acid neurotransmitters and in the control of invertebrate pests.

Description

SYNTHETIC ARYL POLYAMINES AS EXCITATORY

Backaround of the Invention This invention relates to a class of aryl polyamines and the 5 pharmaceutically acceptable satts thereof which are antagonists of excitatory amino acid neurotransmitters. These neurotransmitters affect neuronal cells of a variety of organisms including invertebrates and vertebrates. The polyamines of the present invention are syn~etic analogs of certain polyamines found to be present in the venom of the Agelenopsis aPerta 10 spider. This invention also relates to the use of such polyamines and their salts in antagonizing excitatory amino acid neurotransmitters. These neurotransmitters affect cells such as cells in the neNous system of an organism. This invention further relates to the US2 of such polyamines and their salts in the ~eatment of excitatory amino acid neurotransmitter-15 mediated dis~ases and conditions in a mammal, in con~ol of invertebrat~
pests, and to compositions comprising said polyàmines and salts thereof. `~;This invention also relates to methods of producing such polyamines.

It has been reported that the venorn of the spider Aqelenopsis apertacontains at least two toxins which affect calciurn currents. Jackson, H., et 20 al., Soc. Neu. Sci. Abstr. 12:1078 (1987~. Those authors disclose a toxin, referred to therein as AG2, which has a molecular weight of less than 1,000 daltons and appears to suppress calcium currents in a broad range of tissues. Further, Jackson, H. et al., Soc. Neu. Sci. Abstr. 12:730 ~1986) report another toxin from AgelenoDsis aperta comprising a component of 2s about 6,000 M.W. That toxin is reported to block presynaptic transmission and it has been suggested that the toxin blocks calcium channels associated with the release of neurotransmitter.

W093/04036 2lr4798 PCI/US92/06482 Certain polyamines found to be present in the venom of the Agelenopsis aperta spider are disclosed in U.S. patent no. 5,037,846, filed April 28, 1989 and assigned to the assignees hereof. Those polyamines and the pharmaceutically acceptable salts thereof are disclosed therein æ
5 blockers of excitatory amino acid receptors in cells and one such polyamine, B1 therein, is also disclosed as a blocker of calcium channels.

Compounds which are excitatory amino acid neurotransmitter antagonists have a variety of utilities. Excitatory amino acid neurotransmitter antagonis~ are useful in the treatment of such conditions as stroks, cerebral lO ischemia, neuronal degeneratNe disorders such as Alzheimer's disease and epilepsy and as psychotherapeutants, among others. See Excitatorv Amino Acids In Health and Disease, D. Lodge, E., John Wiley and Sons Ltd., New York, NY 1988, the teachings of which are incorporated herein by reference.
Further, such compounds are useful in the study of the physiology of cells `
lS such as neuronal cells and in the control of invertebrate pes~.

Glutamate is the major excitatory neurotransmitter in mammalian brain. There has been a great deal of excitement in the past decade as the developing pharmacology of glutamate receptors has suggested their differentiation into several subtypes. The glutamate receptor subtype ~ -20 classified by the selective action of the exogenous agonist N-methyl-D-aspartate (NMDA) has been the subject of intense research since these receptors have been proposed to play a role in a variety of neurological pathologies including stroke, epilepsy, and neurodegenerative disorders such as Akheimers's disease. There are currently two broad classes of 25 NMDA receetor antagonists that are being aggressively pursued in search of clinically useful drugs. The first class consists of competitive antagonists which inter~ere with binding of glutamate to its receptor site. These compounds are characterized as highly polar compounds such as the phosphonate compounds AP7 and AP5. The highly charged structure of the 30 competitive agents render them unable to penetrate the blood/brain barrier W0 93/04036 . ~ `2 ~ g7 9'8 PCr/US92/06482 and limits their therapeutic utility. The second class consists of noncompetitive antagonists which block NMDA receptor function by acting at the ion channel associated with the NMDA receptor complex. These compounds include MK-801 and phencyclidine (PCP). The potential ~`
psychotomimetic effects of these classe of compounds are clear liabilities of the known drugs that work via these mechanisms.

Recently, a third class of antagonists have come under scrutiny, -based on the identi~lcation of new glutamate antagonists from spider venoms. Aryîamine structures isolated from the venom of AaelenoDsis `
o aperta that show potent and specific antagonism of mammalian NMDA ;
receptors are discîosed in U.S. patent appîication serial no. 554,311, filed July 17, 1990 and in U.S. patent no. 5,037,846, filed July 31, 1990. The arylamines isolated from Ageleno~sis aperta venom are composite structures built up from an aromatic acid and polyamine fragments bonded together by amide bonds. In these structures, some of the amines in the polyamine fragment are functionalked as N-hydroxy amines or quatemary ammonium salts. The chem cal structures of the arylamines are distinct from the aforementioned standard competitive agents, AP5 or AP7, and the non-competitive compound MK~01. For example, polyamine AGEL 416, -20 discîosed in aforementioned patent no. 5,037,846, is disclosed as having the following structure.
/~ H H H
~,N~/\~N N N/~--~NH

The mechanism of NMDA antagonism by these arylamines is also distinct from both the competitive and MK-801/PCP classes. Thus, spider venom arylamines provide a novel class of antagonist compounds at the 25 NMDA receptor.

WO 93/04036 ~ PCI'/US92/06482 Given the benefit of the disclosure herein with respect tO the naturally-occurring compounds it is now possible to obtain said compounds by methods other than through isolation/purification from whole venom of AaelenoPsis aDerta, and correspondingly, it is also possible to syn~esize ~:s analogous compounds of the same class which are not naturally-occurring. ~ ::

Summary of the Invention This application concerns a class of synthetic aryl polyamines of the formula R-(CH2)m-CO-P~', wherein R is a 5 to 7 member carbocyclic system or an 8 to 11 member carbobicyclic system, or any of the above -eystems substituted with one or more substituents inâependently selected from Ft Cl, Br, OH, C1 to C4 alkyl, C1 to C4 alkoxy, CF3, phenyl, amino, C1 to C4 -~
alkylamino and di(C1 to C4)alkylamino; m is 0 or 1; R' is -[NH(CH2)JXNH2; each n is independently 2 to 5; and x is 1 to 6.

This invention also relates those compounds of the formula R-(CH2)m-CO-R', or a pharmaceutically acceptable acid addition salt thereof :`
wherein R is a 5 to 7 member carbocyclic system or an 8 to 11 member ;carbobicyclic system, or any of the above systems substituted with one or more substituents independently selected from F, Cl, Br, OH, C1 to C4 alkyl, C1 to C4 alkoxy, CF3, phenyl, amino, C1 to C4 alkylamino and di(C1 to C4)alkylamino; m is 0 or 1;
f (cH2)nNH]yH
R' is-[NH(cH2)n]xN\
[(CH2)nNH]2H; each n is independently 2 to 5;x is 0 to 4;
y and z are each independently 1 to 5; and the sum of x and the greater of yandz is 1 toS.

This application further relates to compounds of the formula R-(CH2)m-CO-R', or a pharmaceutically acceptable acid addition salt thereof wherein R is a 5 to 7 member carbocyclic system or an 8 to 11 member carbobicyclic system, or any of the above systems substibJted with one or WO 93/04~36 PCI'~US92/06482 more substituents independently selected from F, Cl, Br, OH, C1 to ~4 alkyl, C1 to C4 alkoxy, CF3, phenyl, amino, C1 to C4 alkylamino and di (C1 to C4) alkylamino; m is 0 or 1; R is -(CH2).[NH(CH2)b]V\
-[NH(CH2)n~X~/ /N~(CH2)nNHl~H
(CH2)~[NH(CH2)Jy wherein each a is the same and is 2 to 5;
each b is the same and is 2 to 5; each n is independently 2 to 5; x is 0 to 3;
10 each y is the same and is 0 or 1; z is 0 to 3; and x + y + z is 0 to 4.

The carbocyclic systems of the present invention can be saturated, unsaturated or aromatic, with the aromatic systems being preferred. With regard to ~e monocyclic systems, phenyl is preferred. The bicyclic systems can be fused or bridged, with 9 or 10-membered fused systems being 15 preferred, for example, naphthyl and indene. Of the aforementioned bicyclics, naphthyl is particularly preferred.

Preferred R' groups are those wherein x is 4 or 5 and ea~h n is independently 3 or 4. Particularly preferred R' groups are -[NH(CH2)3~5NH~ and-[NH(CH2)3]3NH(CH2)4NH(CH2)3NH2-The polyamines of this invention and the pharmaceutically acceptable salts thereof are antagonists of excitatory amino acid neurotransmitters.
Thus, said polyamines are useful in antagonizing such excitatory amino acid neurotransmitters, per se. The polyamines of this invention are also useful in Ule control of invertebrate pests and in the treatment of diseases and 2s conditions in a mammal mediated by excitatory amino acid neurotransmit-ters. Said po!yamines are useful also as mammalian psychotherapeutanCs.

This invention also concerns pharmaceutical compositions comprising said polyamines, methods of administering said polyamines and methods of making said polyamines.

WO 93/04036 PCI`/US92/06482 2-1;14.~798 --6-- . ' Detailed Description of the Invention A synthetic scheme for production of a polyamine of the formula ,/~ H H H
~i ~ ; N ,N ,N~/ H N 2 ~ :

H
isshown in Reaction SchemesAto C, below. ~ ~

.. . .

W093/04036 ~!11'479'8 PCI`/USD2/1)6482 ~E:ACTION SCHEME A

H2 2 ~ NC~

H : `
H2 N~ + Br~ ~N-BOC

~I) (II) BOC-I ~ ~N~CN
R H
( II I ) I IBOC]20 ~OC ' `' ' . BOC-N--N~
H
(IV) H2/Pd (ON) 2 ~ ~O~c BOC
BOC-N~ ' N~2 ~ NC~
H BOC
(V) 2 1 1 4 7 9 8 PCJ/US92tO6482 . . ' E~OC H
BOC-N--~N~N~ CN
BO~
(Vl ) [I~OC3 2 BOC BOC
BOC -N--N~~N ~ N~ CN
..
BOC
VII ) ¦ H2/Pd IOH) 2~ HOAC

BOC BOC
BOC-N--N~
H BOC
(YIII~

REACTION SCl~EME B

~ '~ HON/--H ~--N~l t IX) WO 93/04036 ~9!! ~147 9 8 PCll/U592/0648 I~EACTI ON_SCHEME C

IX + VIII

BOC BOC
H
~N ~ ~ N ~R N-BOC
W`NJ --- BOC H
1~ ~; ` ,.
(X) ¦ lBOC] 2 H
~3~N~,~ N~ N ~N N-BOC

BOC
tXI ) H H H
r R _N ~N ~R~ NN2 (XII1 :

WO 93/04036 ~ 2 1 1 4 7 9 8 PCI'/US92/06482 According to Reaction Scheme A, the polyamine intermediate compound of formula IV is prepared through a sequence of steps beginning with diaminobutane. Reaction conditions suitable to prepare the intermediate compound of formula Vlll according to Reaction Scheme A are 5 given in Example 5, parts 1 to 7. Reaction Scheme B illustrates a method for the preparation of the intermediate compound of formula IX. P(eaction -conditions suitable to prepare that intermediate are given in Example 5, part 8. Preparation of the polyamine compound of this invention of the formula Xll is shown in Reaction Scheme C. Reaction conditions suitable for the lo coupling of the intermediate compounds of formulae Vlll and IX and the subsequent preparation of the compound of formula Xll are given in Example 5, parts 1 to 11.

Thc polyamines of this invention reversibiy antagonize excitatory amino acid neurotransmitters, which neurotransmitters affect cells such as 15 cells in the nervous system of a variety of organisms including invertebrates and vertebrates. The term vertebrates as used throughout is meant to include mammals. The term invertebrates as used throughout is meant to include for example, insects, ectoparasites and endoparasites.

The ability of the polyamines of the present invention to antagonize 20 excitatory amino acid neurotransmitters is demonstrated by their ability to block N-methyl-D-æpartic acid (NMDA)-induced elevations of cGMP in neonatal rat cerebellums according to the following procedure. Cerebellums from ten 8-14 day old Wistar rats are quickly excised and placed in 4C
Krebs/bicarbonate buffer, pH 7.4 and then chopped in 0.5 mm x 0.5 mm 25 sections using a Mcllwain tissue chopper (The Nickle Laboratory Engineering Co., Gomshall, Surrey, England). The resulting pieces of cerebellum are transferred to 100 ml of Krebs/bicarbonate buffer at 37C ` -which is continuously equilibrated with 95:5 02/CO2. The pieces of cerebellum are incubated in such a manner for ninety minutes with three 30 changes of the buffer. The buffer then is decanted, the tissue centrifuged (1 WO 93/04036 2 1 1 47 9 ~ PCI/US92/0~i482 min., 3200 rpm) and the tissue resuspended in 20 ml of ~e Krebs/bicarbonate buffer. Then, 250 ~1 aliquots (approximately 2 mg) are removed and placed in 1.5 ml microfuge tubes. To those tubes are added 10 ~ul of the compound under study from a stock solution followed by 10 ~ul s of 2.5 mM solution of NMDA to start the reaction. The final NMDA
concentration is 100 ~M. Controls do not have NMDA added. The tubes are incubated for one minute at 37C in a shaking water bath and then 750 ul of a 50 mM Tris-CI, 5 mM EDTA solution is added to stop the reaction.
The tubes are placed immediately in a boiling water bath for five minutes.
10 The contents of each tube are then sonicated for 15 seconds using a probe sonicator set at power level three. Ten microliters are removed and the protein determined by the method of Lowry, Anal. Biochem. 100:201-220 (1979). The tubes are then centrifuged (5 min., 10,000 xg), 100 ~l of the supematant is removed and the level of cyclic GMP (cGMP) is assayed 5 using a New England Nuclear (Boston, Massachusetts) cGMP RIA assay according to ths method of the supplier. The data is reported as pmole cGMP generated per mg protein.

Further, the ability of the polyamines of the present invention to antagonize excitatory amino acid neurotransmitters is demonstrated by their 20 ability to block NMDA/glycine induced increæes in cytosolic free [Ca2~]j in dissociated cerebellar granule cells according to the following procedure.
Cerebellar granule cells are prepared from the cerebellum of 8 day old rats (Wilkin, G. P. et al., Brain Res:115: 181-199, 1976). Squares (1 cm2) of Aclar (Proplastics Inc., 5033 Industrial Ave., Wall, NJ, 07719) are coated wi~ poly- ;
25 L-lysine and placed in 12-well dishes that contain 1 ml of Eagles Basal Medium. The cells are dissociated and aliquots containing 6.25 x 10~ cells are added to each well containing the squares of Aclar. Cytosine-beta-D-arabino furanoside (final concentration 10 ~M) is added 24 hours post ~ -plating. The cells are used for fura2 analysis at 6, 7 and 8 days of culture.
30 The cells (attached to the Aclar squares) are transferred to 12-well dishes containing 1 ml of 2 ~uM fura2/AM (Molecular Probes Inc., Eugene, OR) in WO 93/04Q36 . ,~ . PCI`/US92/06482 ~14~ 9 8 --12-- -:
HEPES buffer (containing 0.1% bovine serum albumin, 0.1% de~ose, pH
7.4, magnesium-~re~). The cells are incubated for 40 minutes at 37C; the fura2JAM containing buffer was removed and replaced with 1 ml of the same buffer withou~ fura2/AM. To a quartz cuvette is added 2.0 ml of prewarmed (37C) buffer. The cells on the Aclar are placed in the cuvette and the cuvette is inserted in a thermostated (37C) holder equipped wi~ a magnetic stirrer and the fluorescence is measured with a fluorescence ~ ~ -spectrophotometer (Biomedical Instrument Group, University of Pennsyl-vania). The fluorescence signal is allowed to stabilize for about 2 minutes.

o An increase in cytosolic free calcium, represented by an increase in fluorescence, is produced by the addition of 50 ~uM NMDA and 1 ~M
glycine. Then 5-20 ~ul of a stock solution of the compound under study in phosphate-buffered saline (PBS, pH 7.4) at appropriate concentrations are added to the cuvette. Calibration of the fluorescent signals and fura2/AM
leakage correction are performed using the established procedures of Grynkiewicz, G. Et al., J. Biol. Chem. 260:3440 (1985). At the completion of each test, maximum fluorescence value (Fmax) is determined by addition of ionomyein (35 ~uM) and the minimum fluorescence value (Fmi~i) is dstermin- -ed by the subsequent addition of EGTA (12 mM) to chelate calcium.
Employing the foregoing procedure, the ability of a subject compound to antagonize excitatory amino acid neurotransmitters is shown to occur by a decrease in fluorescence upon addition of the subject compound.

The polyamines of this invention are useful in antagonizing excitatory amino acid neurotransmitters, per se. As such, the polyamines are also useful in the.control of invertebrate pests in the treatment of excitatory aminoacid nsurotransmitters-mediated diseases and conditions in a mammal such as stroke, cerebral ischemia, neuronal degenerative disorders such as Alzheimer's disease and epilepsy. Said polyamines aiso are useful as psychotherapeutants in a mammal and, further, in the study of the physiology of cells including, but not limited to, cells of the nervous system.

W093/04036 ; ~ 17~18 PCI`/US92/06482 - 1 3 - ;
Also within the scope of this invention are the pharmaceutically acceptable salts of the polyamines of this invention. Such salts are formed by methods well know to those skilled in the art. For example, acid addition salts of the polyamines can be prepared according to conventional methods. Acid addition salts of the polyamines such as hydrochloric and ~ ~
trifluoroacetic acid addition salts thereof are preferred. Hydrochloric acid ~ ~-addiffon salts of the polyamines are particularly preferred.
-:
When a polyamine or a pharmaceutically acceptable salt thereof of this invention is to be administered to a mammal, it can be administered o alone or in combination with pharmaceutically acceptable carriers or diluents in a pharmaceutical composition according to standard practice. The poly- -~
amines or pharmaceutically-acceptable salts thereof can be administered orally or parenterally. Parenteral administration includes intravenous, intramuscular, intraperitoneal, subcutaneous and topical administration.
:~
lS For oral use of a polyamine or a pharmaceutically acceptable salt thereof of this invention, the compound can be administered, for example, in the form of tablets or capsules, or as an aqueous solution or suspension. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch, and lubricating agents, such as magnesium stear- ` `
ate, are commonly added. For oral administration in capsule form, useful diluents are lactose and dried corn starch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agen~. If desired, sweetening or flavoring agents can be used.

For intramuscular, intraperitoneal, subcutaneous and intravenous use, 2s sterile solutions of the active ingredient are usually prepared, and the pH of the solutions is suitably adjusted and buffered. For intravenous use, total concen~ation of solutes can be controlled to render the preparation isotonic.

WO 93/04036 ~ 21 1 ~ 98 PCI'/US92/06482 When a polyamine or salt thereof of this invention is used in a human subject, the daily dosage will normally be determined by the prescribing physician. However, suitable dosages for the polyamines of this invention are from about 1 to 30 mg/kg/day. Moreover, the dosage will vary according to the age, weight and response of the individual patient, as well as the severity of the patients's symptoms and the potency of the particular compound being administered. Therefore, dosages outside the range given above are possible and are within the scope of this invention.

When a polyamine or salt thereof of this invention is used in control of invertebrate pests, said compound is administered to said invertebrate directly or provided to the environment of said invertebrate. For example, a compound of this invention can be sprayed as a solution onto said invertebrate. The amount of compound necessary for control of said invertebrate will vary according to the invertebrate and environmental conditions and will be determined by the person applying the compound.

When a polyamine or salt thereof ~f this invention is used in the physiological study of cells, said compound is administered to the cells according to methods well known to those skilled in the art. For example, said compound can be administered to cells in an appropriate physiological buffer. An appropriate concentration of the compounds of this invention for use in such studies is 100 /uM. However, the concentration ~f said compounds in such studies may be greater than or much less than 100 ~M.
The amount of the compound administered will be determined by the person skilled in the art according to the well known methods.

.
EXAMPLES
Procedure Types found in Experimentals (A) Protection of secondary amine with di-tert-butyl dicarbonate (B) Cyanoethylation of primary amine (C) Ca~tic hydrogenation of nitrile to primary amine WO 93/04036 ` 2 1 1 `I 7 9 8 PCI/US92/06482 (D) Amide bond-forming reactions (D1) Dimethylaminopropyl, ethyl carbodiimide/hydroxybenztriazole (D2) Dicyclohexylcarbodiimidelhydroxybenz~iazole (D3) Dimethylaminopropyl, ethyl carbodiimide/hydroxy benztriazoleltriethylamine (D4) Dicyclohexylcarbodiimide/hydroxysuccinimide (E) Deprotection of N-Boc substrates with HCI in dioxane -~
(F) Deprotection of N-Boc substrates with TFA (tr-~luoroacetic acid) (G) Alkylation of amines with N-Boc-3-bromopropylamine amDle 1 - PreDaration of H2Nl(CH2)~NHlX Polvamine Sids Chain Step 1 ^ Procedure Type B

H2N ~NH2 ~ H2N ~NH~CN (8) A 103 9 sample of 1,3-diaminopropane was combined with 45 ml of -~
MeOH wi~ stirring at 4C. Acrylonitrile (100 ml, 81 g, 1.1 eq) was dripped into ~e solution via pressure-equilibrating addition funnel over 90 minutes.
After 3 hrs, a 500 mg portion was removed and evaluated by 13C NMR; no 1,3-diaminoproprane was observed. The crude material, containing the product aminonitrile 8, was distilled under reduced pressure and three fractions were collected in the 100-125C temperature range of the distillation; all were clean enough to proceed to the reaction with di-tert-butyl carbonate and subsequent silica gel chromatographic pur~ication.

lH NMR (250 MHz, CDCI3) ~ 2.67 (t, 2H, J=6.6 Hz), 2.54-2.43 (m, 4H), 2.28 (~ 2H, J=6.6 Hz), 1.37 (m, 2H, J=6.7 Hz), t.05 (s, 3H); '3C NMR
(63.1 MHz, CDCI3) ~ 118.8, 46.9, 44.9, 40.2, 33.4, 18.5.

Ste~ 2 - Procedure Ty~e A
(8)--HN N~ CN (9) Boc Boc ~ ":

w093/04036 ~ ~ ` 21.1.47!~8 Pcr/uss2/064s2 To a solution of aminonitrile 8 (23 9, 0.18 mol) in 500 ml of me~ylene chloride at 0C was added di-tert-butyl dicarbonate (80 9, 0.36 mol, 2 eq).
The reaction mixture was stirred at room temperature for 1~ hours and treated wi~ an additional portion of di-tert-bu~yl dicarbonate (8 9, 0.036 5 mol). After stirring an additional 4 hours, the reaction was washed wi~ 1 N
KOH (2x60 ml), dried over K~CO3, filtered and concentrated in vacuo. The product was purified by flash chromatography (SiO2, 20--100% e~ylacetate in hexane) to provide the product N-Boc-nitrlle 9 as a clear oil (14 9, 24%
yield).

lH NMR (250 MHz, CDCI3) ~S 3.40 (t, 2H, J=6.7 Hz), 3.28 (t, 2H, J-6.6 Hz), 3.05 (bs, 2H), 2.63-2.46 (m, 2H), 1.70-i.56 (m, 2H), 1.42 (s, 9H), 1.38 (s, 9H); 13C NMR (63.1 MHz, CDCI3) ~ 155.8, 155.1, 118.5, 80.7, 78.9, 45.7, 44.4, 43.4, 32.4, 28.3, 28.2.

Step 3 - Procedure Type C
(9)--HN N~ NH2 (11) ~oc ~oc N-Boc nitrile 9, (49 9, 0.4 mol), 1000 ml acetic acid and 20 9 (20 u~%
Pd(OH)2) Pd(OH)2/C were placed in a 2.6 I Parr shaker bottle. The botUe was filled with hydrogen gas to 50 psi and shaken for 4 hours. The reaction was filtered through a 0.47 ~ filter paper and concentrated in vacuo. The residue was dissolved in 1.5 I CH2CI2 and washed with 1 N KOH (2x200 ml).
The base layers were ex~acted wi~ CH2CI2 (400 ml); the CH2CI2 layers were -~
combined, dried over K2CO3, filtered and concentrated in vacuo to yield ~e N-Boc amin- 11 as a clear colorless oil (43 9, 86% yield). , -lH NMR (250 MHz, CDCI3) ~ 3.28-3.12 (m, 2H), 3.11-3.00(m, 4H), 2.64 (t, 2H, J=7 Hz), 1.65-1.50 (m, 4H), 1.42 (s, 9H), 1.38 (s, 9H); 13C NMR
(63.1 MHz, CDCI3) ~ 155.8, 79.3, 78.5, 44.0, 43.4, 39.2, 32.3, 31.6, 28.5, ~ --28.2.

WO 93/04036 ~11 47g 8 PCI`/US92/06482 Step 4 - Procedure TyPe B
(11)--HN N~ NH CN (12) : .
~oc Boc A 38 9 sample of N-Boc amine 11 (0.114 mol) was combined with 6.7 g acrylonitrile (0.126 mol, 1.1 eq) in 60 ml methanol and stirred for 11 hours.
Solvents were removed to yield 43 9 (100% yield) of nitrile 12 as a clear 5 colorless oil which was used without further purification.

lH NMR (250 MHz, CDCI3) ~ 3.18 (bs, 4H), 3.03 (m, 2H), 2.85 (t, 2H, J=6.6 Hz), 2.57 (t, 2H, J=6.7 Hz), 2.45 (t, 2H, J=6.7 Hz), 1.72-1.53 (m, 4H), 1.41 (s, 9H), 1.38 (s, 9H); 13C NMR (63.1 MHz, CDCI3) ~155.9, 118.6, 79.6, 78.9, 46.3, 45.0, 43.9, 37.4, 28.8, 28.3, 18.6.

Steo 5 - Procedure Tvpe A
(t2)--HN N N~ CN ~13) ~oc ~oc ~oc A 43 9 sarnple of above-prepared nitrile 12 (0.114 mol) was combined with di-tert-butyl dicarbonat~ (25.6 9, 0.120 mmol, 1.05 eq) and 350 ml CH2CI2 at 0C and stirred for 9 hours. Thin layer chromatography (TLC) (EtOAc, KMnO4) showed no starting material remaining; reaction was 15 purified in the same manner as N-Boc-nitrile 9~ N-Boc-nitrile 13 was recovered a~ a clear, colorless oil (34 9, 63% yield)~

'H NMR (250 MHz, CDCI3) ~ 3.45 (t, 2H, J=6.6 Hz), 3.39-2.97 (m, 8H), 2.68-2.46 (2, 2H), 1.82-1.66 (m, 4H), 1~44 (s, 18H), 1.87 (s, 9H); 13C
NMR (75.7 MHz, CDCI3) ~S 155.9, ao.s, 79.7, 78.9, 46.5, 44.5, 43.9, 37.6, 20 28.4, 28.3, t6.9.

Step 6 - Procedure TyPe C
(13)--HN I N~--NH2 (1 Boc aOC Boc WO 93/04036 ` ' '' 2 1 1 4 7 9 8 PCI-/US92/06482 N-Boc-amine 14 was prepared from N-Boc-nitrile 13 as N-Boc-amine 11 was prepared from N-Boc-nitrile 9 in 99% yield (30 9).

1H NMR (300 MHz, CDCI3) ~ 3.32-2.94 (m, lOH), 2.62 (t, 2H, J=6.7 Hz), 1.76-1.52 (m, 6H), 1.39 (s, 18H), 1.37 (s, 9H), 1.2~ (s, 2H); 13C NMR
(63.1 MHz, CDCI3) ~155.5, 7g.5, 79.3, 45.5 43.7, 39.1, 37.3, 32.3, 28.3.

Step 7 - Procedure Tvpe B
HN N~N~ NH CN (15) 80c Boc ~oc Nitrile 15 was prepared from N-Boc-amine 14 as nitrile 12 was prepared from N-Boc-amine 11 in 90% yield.

1H NMR (300 MHz, CDCI3~ ~ 3.29-3.02 (m, 14H), ~.86 (t, 2H, J=6.7 10 Hz~, 2.57 (t, 2H, J=6.6 Hz), 2.46 (t, 2H, J=6.6 Hz), t.72-1.57 (m, 6H), 1.41 (s, 9H)~ 1.40 (s, 9H), 1.39 (s, 9H); 13C NMR p5.7 MHz, CDCI3) ~155.5, 155.0, 118.7, 79.6, 79.~, 46.7 46.0, 45.243.3, 38.0-36.9, 28.4, 18.7. '3C NMR
(300 MHz, CDCI3) of nitrile 15 is indistinguishable from ~at of ni~ile 12.
.
Step 8 - Procedure Type A
(15)--HN N ~N ~N CN (16~ :
~oc Boc ~oc 30c N-Boc-nitrile 16 was prepared from nitrile 15 as N-Boc-nitrile 13 was prepared from nitrile 12 as a clear colorless oil (30 9, 87% yield).

lH NMR (300 MHz, CDCI3) ~ 3.36 (t, 2H, J=6 Hz), 3.18-2.90 (m, 14H), 2.57-2.42 ~m, 2H), 1.72-1.48 (m, 6H), 1.40-1.28 (m, 27H).

Ste~ 9 - Procedure Ty~e C
(16)--HN N~N ~N NH2 (17) Boc Boc ~oc ~oc wog3/04036 ~ 1 1798 PCI/US92/06482 --19-- :
N-Boc-amine 17 was prepared from N-Boc-ni~ile 16 as N-Boc-amine 11 was prepared from N-Boc-nitrile 9 in 74% yield (2.61 9).

1H NMR (250 MHz, CDCI3) ~ 3.39-2.97 (m, 14H), 2.63 (t, 2H, J=6.6 Hz), 1.80-1.53 (m, 8H), 1.39 (s, 27H), 1.38 (s, 9H), 1.23 9 (s, 2H).

Step 10 - Procedure Type B
(17)~HN N~N~N N~ CN (18) Boc Boc ~oc ~Oc Ni~ile 18 was prepared from N-Boc-amine 17 as ni~ile 12 was prepared from N-Boc amine 1 t in 91% yield (19 g), and was used without fu~er purKication.

lH NMR (300 MHz, CDCI3) ~ 3.242.94 (m, t4H), 2.87 (t, 2H, J~6 10 Hz), 2.57 (t, 2H, J-6 Hz), 2.47 (t, 2H, J-6 Hz), 1.741.54 (m, 8H), 1.45-1.36 (m, 36H).
'~
Step 11 - Procedure Tvpe A
(18)--HN ~N N~N~N~ CN (19) ` -I
Boc Boc ~oc aoc Boc N-Boc-nitrile 19 was prepared from nitrile 18 as N-Boc-nitrile 13 was prepared from ni~ile 12 (16 9, 74% yield).

$5 'H NMR (250 MHz, CDCI3) ~ 3.43 (t, 2H, J=6.6 Hz), 3.28-3.02 (m, 16H), 2.62-2.50 (m, 2H), 1.801.56 (m, 8H), 1.43 (s, 9H), 1.42 (s, 9H), 1.41 (s, 18H), 1.39 (s, 9H).

SteD 12 - Procedure Type C ;
(19)--llN I N - ~N - ~N~ - NH2 (20) Boc Boc Boc Boc Boc N-Boc-amine 20 was prepared from N-Boc-nitrile 19 as N-Boc-amine 20 11 was prepared from N-Boc-nitrile 9 (17 9, 99% yield~.

wo 93/04036 ' ~ 2~1 ~7 9 8 Pcr/uss2/06~82 lH NMR (300 MHz, CDCI3) ~ 3.24-2.95 (m, 18H), 2.6 (t, 2H, J-6H), 1.72-1.52 (m, 10H), 1.42-1.32 (m, 45H).

Step 1~ Procedure Tvpe B
~20)--HN N ~N N ~N~/ NH CN (2 ~) sOc sOc sOc eOc sOc Nitrile 21 was prepared from N-Boc-amine 20 as nitrile 12 was 5 prepared from N-Boc-amine 11 in 99% yield.

1H NMR (250 MHz, CDCI3) ~ 3.32-3.03 (m, 16H), 2.91 (t, 2H, J=6.7 Hz~, 2.61 (t, 2H, J_6 Hz), 2.5t (t, 2H, J=6.6 Hz), 1.82-1.57 (m, 10H), 1.44 (s, 36H), 1.43 (s, 9H).
.. . .

Ste~ 14 - Procedure Tv~e A ~ -(21)--HN ~N ~N~N N ~N CN (22) ~oc ~oc aOc ~oc aOc B~
N-Boc-nitrile 22 was prepared from nitrile 21 as N-Boc-nitrile 13 was prepared from ni~ils 12 in 99% yield.

3.45 ppm (t, 2H, J.-6.6 Hz), 3.26-3.02 (m, 18H), 2.65-2.53 (m, 2H), ;
1.79-1.55 (m, 10H), t.43 (s, 9H), 1.42 (s, 9H~, 1.41 (s, 18H), 1.40 (s, 9H); 13C
NMR {'H} (250 MHz, CDCI3) ~155.2, 79.3, 44.7, 28.3, 28.3, 28.2.

Step 15 - Procedure Tvpe C
~22)--HN ~N~~N ~N ~N ~N ~NH2 ~oc Boc Boc ~oc ~oc Boc (23) N-Boc-amine 23 was prepared from N-Boc-nitril~ 22 as N-Boc-amine 11 was prepared from N-Boc-nitrile 9 (8 9, 99% yield).

'H NMR (300 MHz, CDCIJ ~ 3.32-2.98 (m, 22H), 2.65 (t, 2H, J=6.6 Hz), 1.78-1.53 (m, 12H), 1.41 (s, 54H), 1.40 (s, 9H).

WO 93J04036 . PCI /US92/064fl2 9~

Step 16 Procedure. Tv~e B
(23)--HN~N ~N ~N ~N ~N ~NH CN
~oc ~oc ~oc ~oc ~oe 30c (2~) . -Nitrile 24 was prepared from N-Boc-amine 23 as nitrile 12 was prepared from N-Boc-amine 11 in 99% yield.

lH NMR (250 MHz, CDCI3) ~ 3.32-.304 (m, 22H), 2.gO (t, 2H, ~=6.7 5 Hz), 2.61 (t, 2H, J_6 Hz), 2.53 (t, 2H, J=6.7 Hz~, 1.82-1.57 (m, 12H), 1.44 (s, 45H), 1.43 (s, 9H).

SteD 17 - Procedure TvDe A
(2,,)--HN ~N ~N ~N ~N N ~N--CN `
Boc Boc Boc 90c Boc Boc ~oc (25) ~`
N-Boc-nitrile 25 was, prepared from nitrile 24 as N-Boc-nitrile 13 was ~ ;:
prepared from nitrile 12 (5.5 9, 74% yietd).

o lH NMR (250 MHz, CDCI.~) ~S 3.45 (t, 2H, J _6.7 Hz), 3.40-2.97 (m, 24H), 2.66-2.51 (m, 2H), 1.80-1.55 (m, 12H), 1.541.30 (s, 63H).

Ster~ 18 - Procedure Tvr e C

(25)--HN ~N~~N ~N ~N ~N ~N ~NH2 ~ t I~OC Eloc 30C ~OC I~OC 10C (26) ~ .
N-Boc-amine 26 was prepared from N-Boc-nitrile 25 as N-Boc-amine ~.
11 was prepared from N-BoGnitrile 9 (5.01 9, 91% yield). - `

H NMR (250 MHz, CDCI3) o 3.30-2.97 (m, 26H), 2.60 (t, 2H, ~)=6 Hz), 1.81 1.57 (m, 14H), 1.53-1.28 (m, 63H). 13C NMR (250 MHz, CDCI3) ~155.2, 79.3, 44.7, 28.7, 28.4, 27.5.

WO 93/04036 , PCI'/US92/06482 ExamPIe 2 0 ~NH--~--NH NH NH ~--NH--~NH2 F ~

Ste~ 1. Amide Bond Formation - Proeedure Tvpe D1 In a 50 ml one neck RBF were combined 0.19 9 ferrocene carbo)~ylic acid (0.82 mmol, 1.1 eq), 0.12 9 hydroxybenzotriazole (0.89 mmol, 1.2 eq), 5 0.17 g 1-(3-Dimethylaminopropyl~-3-ethylearbodiimide (HCI salt, 0.90 mrnol, 1.2 eq) and 10 ml CH2CI2 with stirring under dry N2 atmosphere. - After 30 minutes, 0.61 g of N-Boc-amine 27 (0.75 mmol, 1.0 eq., see Example 5a herein for preparation) was added to the solution. TLC (2x MeOH, 12) indicated N-Boc-amine had been consumed after 2 hours. The reaction was 10 diluted to 4Q0 ml with EtO~c and washed with pH 4 buffer (2 x 25 ml), 25 ml H20, 1 N KOH (2 x 25 ml), 25 H20 and 5û ml brine. The EtOAc layer was ~ -dried over Na2SO~, filtered and the solvents were removed to yield 712 mg (93%) of product as an orange 3il.

1H NMR (250 MHz, CDCI3) ~ 4.78 ~s, 2H), 4.32 (t, 2H, J--1.8 Hz), 4.19 15 (s, 5H), 3.45-3.04 (m, 20H), 1.83-1.59 (m, 12H), 1.50 (s, 9H), 1.45 (s, 18H).1.44 (s, 9H), 1.43 (s, 9H).

Step 2 Polyamine De~rotection - Procedure Type F
Trifluoroacetic acid (30 ml) was degassed with a dry N2 bubble s~eam (via Teflon tubing) in a 100 ml one neek RBF at 0C. The ferrocene 20 carboxamide polyamine of step 1 above (712 mg, 0.69 mmol~ was dissolved in 2 ml CH2CI2 and transferred to the stirring TFA with 3 x 2 ml CH2CI2 rinses. After 30 minutes, the ice bath was removed; aRer an additional 30 minutes, the solvents were removed under reduced pressure, then by Hi-Vac. The reddish brown oil remaining was mashed with Et20 (3 x 30 ml); a 25 yellow solid formed and was collected under positive N2 pressure on a porosity ~B~ ~rit. The solid was rinsed with ether and the residual ether was wo 93/04036 2 l 1 ~ 7 9 8 Pcr/uss2/06482 driven off by positive N2 pressure to yield 690 mg (g3% yieldj of product as a solid.

lH NMR (DMSO) ~ 4.71 (t, 2H, J=1.73 Hz), 4.38 (t, 2H, J--2 Hz), 4.15 (s, 5H), 3.28-3.21 (m, 2H), 3.03-2.81 (m, 18H), 2.04-1.72 (m, 8H), 1.61-1.50 5 (m, 4H); 13C NMR (250 MHz, D20) 177.3, 78.5, 76.1, 74.3, 49.7, 48.1, 47.3., 47.2, 47.1, 39.3, 38.8, 28.5, 26.5, 25.5, 25.4. HPLC ~not less than) 96.08%
pure; Novapak C18 column, 540% CH3CNI2%/r H20 over 60 minutes, detected at 230 nm, elution time: 23.2 minutes. HP~MS (FAB): (M + H) calculated for C2,H48N~,O, 529.3328845; found 529.33172 10 Example 3 o ~NII --Nh --N~----NH~NlJ ~NH, SteD 1. Amide Bond Formation - Procedure TvPe D3 The hydrochloride salt of 2-pyridylacetic acid (0.105 g, 0.60 mmol, 1.0) was combined with 0.16 ml TEA (1.15 mmol, 2 eq) and 4 ml CH2CI2.
After 10 minutes, DEC (0.12 9, 0.62 mmol, 1.0 eq) and 0.09 9 HOBt (0.66 mmol, 1.1 eq) were added and the mixture was stirred for 2 hours. N-Boc-amina 27 (0.44 9, 0.54 mmol, 0.9 eq) was added and reaction stirred an additional 10 hours. TLC (2 x MeOH, 12) isldicated the N-Boc-amine had been consumed. The reaction was diluted to 400 ml with EtOAc, washed with lN KOH (40 ml), brine (50 ml) and dried over MgSO4. Th~ EtOAc solution was filtered and the solvents removed to yield 0.4 9 of a clear, green oil. The crude material was chromatographed from 10 g silica gel slurried in EtOAc with EtOAc as eluent. Appropriate fractions were combined ~d the solvents wsre removed to yield 0.20 9 (40% yield) of a clear, light green oil.
, 2 5 lH N M R (250 M H z, C D Cl3) ~ 8.59~3.52 ~m, lH), 7.76-7.68~ (m, l H), 7.62-7.49 (m, lH), 7.37 (d, lH, J = 8 Hz), 3.78 (s, 2 H), 3.31-3.02 (m, 20 H).
1.80-1.56 (m, 12 H), 1.56-1.34 (m, 45 H).

W093/04036 i 211`~79~3 PCI/US92/06482 Step 2~ Polyamine Deprotection - Procedure Type F
Trifluoroacetic acid (30 ml) was continuously degæsed with a dry N2 bubble stream (via Teflon tubing) in a 100 ml one neck RBF at room temperature. The 2-pyridylacetamide of step 1 above (180 mg) was dissolved in 2 ml CH2CI2 and transferred to the stirring TFA. After 1 hour, the solvents were removed under reduced pressure and the residue was placed in Hi-Vac. The residue was mashed with ether (3 x 30 ml); a white solid formed and was collected under positive N2 pressure on a porosity ~Bu frit. Remaining ether was removed by positive N2 pressure; 169 mg (91% ` ~ -yield) of the product was isolated.
.... .
lH NMR (250 MHz, D2O) ~ 8.48 (d, 'H, J-15 Hz), 7.98 (t, lH, J=6 ~--Hz), 7.51-7.47 (m, 2H), 3.86 (s, 2H), 3.32 (t, 2H, J_9 Hz), 3.17-2.96 (m, 18H), 2.17-1.98 (m, 6H), 1.98-1.81 (m, 2H), 1.81-1.69 (m, 4H). -Example 4 .~

~ IR~IN~ N----NN~ IIN~IIN~ ~ ~

S ~p ~ Amide Bond For~ation - Procedure TvDe D4 -4Biphenylacetic acid (53 mg, 0.25 mmol, 1.2 eq) was combined with 5 ml CH2CI2, 84 ~ul triethylamine (0.6 mmol, 3 eq) 70 mg dicyclohexylcarbo-diimide (0.34 mmol, 1.6 eq), 11 mg hydroxysuccinimide (0.09 mmol, mol/O) and 175 mg N-Boc-amine 27 (0.21 mmol, 1.0 eq). TLC (2 x MeOH, -~
~o KMnO~) indicated the N-Boc-amine had been consumed after 16 hours. The reaction was diluted to 100 ml with CH2CI2 and washed with aqueous 20%
NH~OH (2 x 100 ml). The base layers were extracted with CH2CI2 (3 x 50 ml); all the CH2CI2 fractions were combined, then washed with brine (50 ml) dried over K2CO3, filtered and the solvents removed to yield 281 mg 2s (>100% yield) crude material. Pure product was isolated via flash silica gel chromatography (12 9 slurried in CH2CI2 and eluted with a 0-10%
MeOH/CH2CI2 gradient) as a white, waxy, solid (190 mg, 88% yield).

wo 93/04036 ~ 7 9 8 Pcr/US92/06482 'H NMR t250 MHz, CDCI3) ~ 7.56-7.50 ~m, 4H), 7.43-7.28 tm, 5H), 3.56 (s, 2H), 3.26-2.98 ~m, 20H), 1.78-1.52 (m, 12Ht, 1.48-1.36 (m, 45H).

Step 2. Poiyamine DeprotectiQn - Procedure Type F
Triflouroacetic acid (30 ml) was degasssed with a continuous 1~
bubble stream (via Teflon tubing~ at 0C. The biphenylacetamine of step 1 above (150 mg, 0.15 mmol) was added as a dry powder to the stirring TFA. After 40 minutes, the ice bath was removed; after an additional 20 minutes th~ solvents were removed under reduced pressure, then Hi-Vac.
o After 2 hours, the resulting tan oil was mashed with Et20 ~3 x 30 ml); a white solid formed and was collected under positive N2 pressure on a porosity "C" frit. Th~ solid was dissolved in water, rinsed ~hrough the frit and freeze dried to yield 136 mg (99% yield) of product as a white solid.

'H NMR ~300 MHz, D~0) ~ 7.62-7.56 (m, 5H), 7.4 (t, 2H, J=7.5 Hz), 7.38-7.31 (m, 2H), 3.45 (s, 2H), 3.13 ~t, 2H, J=6.7 Hz), 3.02-2.80 (m, 18H), 2.00-1.54 (mt 12H).

Examnle 5 l H-lndole-3-acetamide-N-( 1 6-amino-4 8 .1 3-triazahexadec- 1 -yl steD 1 H2N H2 + NC~

H 2N--~ ~ CN
''' (l) The compound of formula I was prepared from diaminobutane and acrylonitrile according to the published procedure of Yamamoto, Hisashi, J. Am. Chem. Soc. 103:6133-6136 (1981).

WO 93/04036 ' 2 1 1 ~ 7 ~ ~ PCr/US92/06482 Step 2 H2 N~CN + Br~' N~
~I) (1~) soc-N--N~~N~ CN
H H
(III) To a solution of N-cyanoethyl-1,4-diaminobutane (6.44 g, 0.0457 -mol) in aeetonitrile (200 ml) under a nitrogen atmosphere was added :~
KF/Celite ~11 9) followed by the dropwise addition over a 7 hour period of 5 N-ltert-butoxycarbonyl)-3-bromopropylamine(10.87 9, 0.0457 mol). The reaction was allowed to stir for 16 hours at ambi-ent temperature and was then heated to 70C for 24 hours. The reaction was allowed to cool and was filtered and concentrated in vacuo. The residue was dissolved in CH2Cl2 (200 ml), washed with 1 N NaOH (100 ml), dried and 10 concentrated jn v~cvo to afford crude product which was chromato~raphed on silica gel (using 9:1 CH2Cl2JMeOH) to afford 3.32 ~ :
of amine lll.

'H NMR ll::DCl3) ~1.19 1.59 ~m, 17H), 2.42 (t, J=6.6 Hz, 2H), 2.44-2.58 (m, 6H), 2.82 (t, J = 6.6 Hz, 2H), 3.08 (m, 2H), 5.22 (br s, lH); 13C NMR (CDCl3) ~18.68, 27.70, 27.74, 28.42, 29.94, 39.16, 45.03, 47.68, 48.99, 49.65, 78.78, 118.75, 156.11; HR FABMS
observed lM + H) mlz=299.2434, C1sH31N402 (req 299.2447).

steP 3 H
BOC-I~~N~N~CN + lBOC] 2O
H
(III) BOC

BOC-I N ~ N CN
H BOC
tlY) w0 93/04036 ` ~ 1'7 9$ Pcr/US92/06482 Under nitrogen atmosphere, 4.7 9 ~15.8 mmoles) of compound of formula lll, prepared as described in step 2, above, was dissolved in 150 ml of dichloromethane. Then, 7.56 9 (34.7 mmoles) of di-tert-butyldicarbonate were added and the reaction mixture was stirred s overnight at room temperature. The mixture was then concentrated in vacuo and chromatographed on 400 9 of silica gel using 50:50 ethylacetate/hexane solvent. The fractions were monitored by TLC -~50:50 ethylacetate/hexane~. The reactions containing the product of formula IV were combined and concentrated in vacuo to yield 7.9 9 of 10 product as an oil.
.. . . . . . . . .
H NMR (CDCI3) ~ 1.20-1.59 Im, 33H), 2.55 (m, 2H), 3.01-3.37 ~m, 8H), 3.39 (t, J=6.6 Hz, 2H), 5.25 (br s, lH); 13C NMR ~ 17.21, 25.73, 25.94, 28.22, 28.24, 28.27, 37.91, 43.78, 44.24, 46.60, 47.95, 78.96, 79.57, 80.44, 155.01, 155.75, 155.98; HR FABMS
15 observed (M + H) m/z=499.3501, C25H~,N40" (req 499.3496).
BOC
BOC-N~--N~~N~ CN
BOC BOC
( IV) BOC

~OC-N--, N "NH 2 H BOC
(V) To 125 ml of acetic acid under a nitrogen atmosphere were added 7.85 9 ~15.8 mmoles) of compound of formula IV, prepared as described in step 3, above, and 6.5 9 of Pd(OH2)/carbon. The mixture was 20 hydrogenated at 50 p.s.i. for 2 hours. The catalyst was removed by filtration and the filter cake was washed well with acetic acid. The filtrate was concentrated, taken up in 250 ml dichloromethane, washed twice with 100 ml of lN NaOH and dried over K2CO3. The solution was WO 93/04036 ~i~ 2 1 1 1 7 9 ~ PCI`/US92/06482 --2~--filtered and the filtrate was concentrated in vacuo to yield 7.8 9 of compound of formula V.

lH NMR (CDCI3) ~ 1.2~1.59 (m, 35H), 2.14 (s, 2H), 2.61 ~t, J=6.7 Hz, 2H), 2.98-3.14 (m, 10H~, 5.22 (br s, 1H); 13C NMR (t::DCI3) 5 25.89, 28.42, 31.38, 32.36, 37.55, 38.95, 43.95, 46.65, 79.34, 79.48, 155.65, 156.03; HR FABMS observed (M + H) m/z=503.3804, C25Hs,N"06 (req mlz=503.3809).

Step 5 V + NC~ - - --BOC-2, N~~N ~N
H BOC
(VI) Under nitro~en atmosphere, 7.i5 ~ (14.2 mmoles) of compound of 10 formula V, prepared as described in step 4, above, was dissolved in 150 ml of methanol. Then, 1.03 ml l15.6 mmoles) of acrylonitrile was added and the reaction was stirred 72 hours at room temperature. The reaction mixture was then concentrated, reconcentrated three times from dichloromethane and stripped of solvent in vacuo to yield 7.65 9 of 5 product of formula Vl as an oil.

'H NMR ~CDCI3) ~ 1.26.-1.73 (m, 36H), 2.44 lt, J= 6.7 Hz, 2H), 2.54 (t, J=6.7 Hz, 2H), 2.83 (t, .)=6.7 Hz, 2H), 3.00-3.16 (m, 10tl), 5.24 ~br s, 11~ 3C NMR (CDCI3) ~ 18.64, 25.84, 28.09, 28.43, 28.74 37.84, 44.18, 44.68, 45.14, 46.29, 46.73, 46.85, 49.70, 78.90, 20 79.29, 79.46, 118.52, 155.84, 155.98; IIR FABMS observed (M + H) m/z = 556.4064, C28H54Ns0~, (req mlz = 556.4074).

W093~04036 ' ``~ 47g8 PCI/USg2/06482 --2g--SteD 6 VI + 1 BOC ] 2 --BOC BOC

BOC-N~ , N~ CN
H BOC
~VII) Under nitrogen atmosphere, 6.45 9 ~11.6 mmoles) of compound of formula \~I, prepared as described in step 5, above, was dissolved in 125 ml of dichloromethane. To that solution were added 2.6 9 (12 mmoies) 5 of di-t-butyldicarbonate and the reaction mixture was s~irred ovemight at room temperature. The mixture was then concentrated in vacuo and chromatographed on 400 9 of silica gel using 50:50 ethylacetate/hexane eluent. The product fractions were combined and concentrated to yield 6.6 ~ of product for formula Vlll as an oil.

lo 'H NMR (CDCI3) ~1.26-1.73 (m, 44H), 3.03-3.24 (m, 14H), 3.42 (t, d=6.6 Hz, 2H), 5.25 ~br s, lH); 13C NMP~ ~CDCI3) ~17.20, 25.88, 27.83, 28.12, 28.35, 28.45, 28.77, 37.87, 43.~1, 44.20, 44.~7, 46.27, 46.88, 78.94, 79.42, 79.50, 80.54, 117.91, 154.96, 155.44, 155.74, 155.99; HR FABMS observed (M + H) m/z = 656.4579, 15 C33H~,2N508 ~re~ m/z=656.4598).

St~D 7 VI ~ ~ BOC-N , N ~ ' ~N~2 BOC
. . .
~VIII ) - To 150 ml of acetic acid under a nitrogen atmosphere were added 6.6 9 ~10.1 mmoles) of compound of formula Vll, prepared as described in step 6, above, and 6 9 of Pd~OH)2/carbon. The mixture was 20 hydrogenated at 50 p.s.i. for 2 hours. The catalyst was removed by wog3/04036 ~ 211~79~ PCI'/US92/06482 filtration and the filter cake was washed well with acetic acid. The filtrate was concentrated, taken up in 200 ml of dichloromethane, washed twice with 100 ml lN NaOH and dried over K2CO3. The solution was filtered and the filtrate was concentrated in vacuo to yield 6.5 9 of 5 product of formula Vlll.

lH NMR (CDCI3) 1.28-1.71 (m, 46H~, 2.16 (br s, 2H), 2.65 (t, J =
6.7 Hz, 2H), 3.01-3.18 (m, 14H), 5.24 (br s, lH); 13C NMR ~CDCI3) 25.85, 27.66, 28.45, 28.76, 39.10, 44.21, 44.91, 46.80, 79.27, 79.46, 155.41, 155.67, 155.99; HR FABMS observed (M ~ H) 10 m/z=660.4914, C33No~N508 (req 660.4911).

Step 8 . .

N0~ _ ~0--N~

IIX) Under a nitrogen atmosphere, 1.75 9 (10 mmoles) of indole acetic acid, 1.15 9 (10 mmoles) of N-hydroxysuccinimide and ~.06 9 (10 mmoles) of dicyclohexylcarbodiimide were added to 75 ml of 15 tetrahydrofuran. The reactlon mixture was stirred at room temperature and a precipitate formed after about 5 minutes. After about 1.5 hours, the precipitate was removed by filtration, the filter cake was washed with 75 ml of tetrahydrofuran and the cake was air dried to yield 1.84 9. The combined filt~rate was concentrated, taken up in ethylacetate and filtered, 20 washing the filter with ethylacetate. The filtrate was concentrated to yield a foam. The foam was triturated with 75 ml of diethylether to yield a hard gum. Then, about 30 ml of ethylacetate was added followed by ethyl ether. The solids were isolated by filtration, washed with diethyl ether and dried under nitrogen to yield 1.74 9 of product of formula IX. It WO 93/04036 ' ' ` ~ ` 2 ~ 1 47 9 ~ PCI'/US92/06482 was found that an additional 0.47 9 of product could be obtained by treating the mother liquor with petroleum ether.

Ste~ 9 IX + VIII
BOC aoc H
N ~N~ NBoc N~

(X) Under nitrogen atmosphere, 0.33 9 (5 mmoles) of compound of s formula Vlll, prepared as described in step 7, above, was dissolved in 10 ml of dichloromethane with stirring and then 0.136 9 (5 mmoles) of compound of formula IX, prepared as described in step 8, above, were added. The rea~tion was stirred overnight at room temperatur~. The reaction mixture was then diluted out to 35 ml with dichloromethane, 10 washed with 1t) ml of 0.5N NaOH, dried over K2CO3, and concentrated.
The concentrate was chromatographed on silica ~el usin~ 4:1 ethylacetate/hexane. The product fractions were concentrated to yield 0.37 9 of a white foam containing product of formula X with some ethylacetate present.

SteD 10 X + lBOCl 2 BOC BOC
H
~N ~ N N~~

BOC
(XI ) Under nitrogen atmosphere, 0.37 9 (0.45 mmoles) of compound of formula X, prepared as described in step 9, above, was dissolved in 10 ml o~ dichloromethane. Then 0.218 9 ~1 mmole) of di-t-butyldicarbonate were added followed by 12 ml (0.1 mmole) of 4-(N,N-dimethylamino)-WO 93/04036 -32- PCl`~US92/06482 pyridine. The reaction was stirred al room temperature for 1 hour then allowed to stand overnight. The reaction mixture was chromato~raphed on silica ~el usin~ 4:1 ethylacetate/hexan~ and the product fractions were concentrated to yield 0.32 ~ of product of formula Xl as a white foarn.

Step 1 1 XI
R
~N~N ~ NN2 (XII) Under nitrogen atmosphere, 0.32 ~ tO.35 ~nmoles) of compound of formula Xl, prepared as described in part 10, above, were added to 15 ml of trifluoroacetic acid and stirred for 15 minutes. The reaction mixture was then concentrated in vacuo and triturated with disthyle~her ~o yield lO Q.30 9 of product as a white powder.

By an analogous process, N-Boc amine 27, having the following structure, was made.
HN~ - N - ~N~~N~ - N - - NH2 30c ~oc 30c 9OC ~oc (2J) Steps 1 to 7, above, were followed, resulting in the N-Boc amine of formula Vlll.

15 Step 8a (Vl11)_ HN~ N ~N .~N~ NH CN
Boc ~oc Boc Boc (Xlll) , Nitrile Xlll was prepared from N-Boc amine Vlll as nitrile Vl was prepared from N-B~c amine V (Example 5, Step 5), giving 1.00 g of product t93% yield).

WO 93/04036 P~/USg2/06482 '7 !~ 8 1H NMR (CDCI3) ~1.26-1.66 (m, 47H), 2.45 (t, J=6.6 Hz, 2H), 2.56 (t, J=6.7 Hz, 2H), 2.85 (t, J=6.6 Hz, 2H), 3.01-3.30 (m, 14H), 5.25 (br s, 1 H);
13C NMR (CDCI3) ~18.68, 25.9~, 28.46, 28.48, 37.49, 44.19, 44.88, 45.16, 46.73, 78.93, 79.32, 79.44, 118.70, 155.46, 155.61, 156.04; HR FABMS
5 observed (M + H) m/z = 713.5191, C33H~gN~08 (req m/z = 713.5177).

SteD 9a ~XI11)_HN~ N~N ~N N~--CN
Boc ~oc lloc Boc ~oc (X IY) N-Boc ni~ile XIV wæ prepared from nitrllQ Xlll via amine protection using procedure A.

Step 10a N-Boc arninc 27 was prepared by hydrogenation of N-Boc ni~ile XIV
as N-Boc arnine ~/lll was prepared from N-Boc nitrile Vll (st~p 7 of ~is exarnple).

ExamDles 6 and 7 Starting with polyamine 20 and ~e appropriate R-acetic ffor m=1) or l5 carboxylic (for m=0) acid, compounds having the structure R(CH2)mCO[NH(CH2)J5NH2-5TFA were prepared via procedure D1 followed by procedure F.

Example m R
6 0 ferrocene 2 o 7 1 3-indole W093/04036 1 i,.. 21147:9~ PCI'/US92/06482 ExamDles 8 to 29 Starting wiUl polyamine 27 and the appropriate R-acetic or carboxylic acid, compounds having ths structure R(CH2)mCO[NH(CH2)J3NH(CH2)4NH(CH2)3NH2-5HCI ffrom procedure E) or 5 R(CH2)mCO[NH(CH2)J3NH(CH2)~NH(CH2)3NH2-5TFA ffrom procedure F) were prepared via the following procedures.

amDle m R Procedures 8 0 ferrocene D1, then F
9 0 2-pyridine D3, then F
o 10 0 3-pyridine D3, then F
11 0 4pyridine D3, then F
12 1 2-pyridine D3, then F
13 1 3-pyridine D3, then F
14 1 4pryidine D3, then F
S 15 0 2-quinoline Dl, then F
16 0 3-quinoline D2, then F
17 1 3-indole Dl, then E
18 t 3-(~hydroxyindole) D2, then F
19 1 3-(4-hydroxyinGole) D2, then E
1 3-(5-bromoindole) D2, then F
21 1 3-(4fluoroindole) D2, then F
22 0 2-(5-fluoroindole) D2, then F
23 1 2-(5-fluoroindole) D2, then F
24 1 3-(5-methoxyindole) D2, then F
2s 25 0 2-quinoxaline D2, then F
26 0 hydroquinone D2, then F
27 0 ~resorcinol D2, then F
28 1 p-biphenyl D4, then F
29 1 2-naphthalene D2, then F

30 ExamDles 30 and 31 Starting with polyamine 17 and the appropriate R-acetic or carboxylic acid, compounds having the structure R(CH2)mCO[NH(CH2~J4NH2-4TFA
were prepared via procedure Dl followed by procedure F~

wo 93/04036 2 1 1 4 7 9 8 Pcr/uss2/064s2 ExamPle m R
0 ferrocene 31 1 3-indole ExamDles 32 and 33 s Starting with polyamine 14 and the appropriate R-acetic or carboxylic acid, compounds having the structure R(CH2)mCO[NH~CH2)3]3NH2 3TFA
were prepared via procedure D1 followed by procedure F.

ExamDle m R
32 0 ferrocene o 33 1 3-indole Examples 34 and 35 Starting wi~ polyamine 11 and the appropriate R-acetic or carboxylic acid, compounds having ~e structure R(CH2)mCO[NH(CH2)3]2NH2 2TFA
were prepared via procedure D1 followed by procedure F.

ExamDle m R ::
34 0 ferrocene 1 3-indole Examples 36 and 37 Starting with polyamine 7 and the appropriate R-acetic or carboxylic 20 acid, compounds having ~e structure R(CH2)mCONH(CH2)3NH2 TFA were prepared via procedure D1 followed by procedure F.

Example m R
36 0 ferrocene 37 1 3-indole WO93/04036 ~ 2i1~79~ PCl/US92/06482 Examples 38 and 39 Starting with polyamine 23 and the appropriate R-acetic or carboxylic acid, compounds having the structure R(CH2)mCOENH(CH2)3]~NH2-6TFA
were prepared via procedure Dl followed by procedure F.

5 am~le m R
38 0 ferrocene 39 1 3-indole ExamPles 40 and 41 Starting with polyamine 26 and ~e appropria~e R-acetic or earboxylic lO acid, compounds having ~e structure R(t::H2)mCO[NH(CH2)3]~NH2-7TFA
were prepared via procedure D1 followed by procedure F.

Example m R
~o 0 ferrocene 41 1 3-indole :.

Example~42 /~ H /~\NH
~,N~ N~N NH2 Step 1 H
/--NH ~oc NC~N~NH2 -- NC~N~N NHBoc H H
To a solution of N-cyanoethyl-1,~diaminobutane (6.44 9, 0.0457 mol) in acetonitrile (200 ml) under a nitrogen atmosphere was added KF/Celite (11 9) followed by ~e dropwise addition, over 7 hours, of N-(tert-20 butoxycarbonyl)-3-bromopropylamine (10.87 9, 0.0457 mol). The reaction mixture was stirred for 16 hours at ambient temperature, was heated to WO 93/Q4036 ;~ 7 ~ ~ PCr/US92/06482 70C for 24 hours and was then cooled, filtered and concentrated in vacuo.
The residue was dissolved in CH2Ci2 (700 ml), washed with 1 N NaOH (100 ml), dried and concentrated in vacuo, affording a crude product which was chromatographed on silica gel (9:1 CH~CI2:MeOH), yielding 3.32 g of the 5 branched nitrile product.

lH NMR (CDCI3) ~1.31-1.65 (m, 27H~, 2.36-2.44 (m, 6H), 2.49 (t, J=6.6 Hz, 2H~, 2.61 (m, 2H), 2.90 (t, ~1=6.6 Hz, 2H), 3.09-3.16 (m, 4H), 5.28 (br s, 2H); '3C NMR (CDCI3) ~18.74, 24.60, 27.05, 27.96, 28.50, 39.52, 45.14, 49.07, 52.21, 53.76, 78.90, 118.79, 156.09; HR FABMS obseNed (M +
10 H) m/z = 456.3549, C23H,~,N5O4 (req mlz = 456.3550).

SteP 2 ~--NH Boc :
NC` N~N NH ~oc ~oc To a solution of the branched amin~ produced in Stap 1, abov~ (150 mg, 0.33 mmol), in dichlor~methane (5 ml) under a nitrogen atmosphere was added di-tert-butyldicarbonate (78 mg, 0.36 mmol). The reaction mix~ure was stirred overnight and was concen~ated under reduced pressure, affording a crude product which was chromatographed on silica gel (9:1 CH2CI2:MeOH), yielding 126 mg (69% yield) of the N-Boc-nitrile product.

SteP 3 ~--N H Boc H,N~N~N N H ~oc Boc To a solution of the N-Boc-nib ile produced in Step 2, above (126 mg, 0.277 mmol)t in acetic acid (20 ml) was added Pd(OH)21carbon (200 mg) and ~e reaction mixture was hydrogenated for 2 hours at 80 PSI of hydrogen, filtered through celite and concentrated in vacuo. The crude WO 93/04036 2 1 1 ~7 9 ~ PCI'/US92/06482 product was taken up into dichloromethane (30 ml) and was washed with 1N NaOH (2 x 10 ml), dried over potassium carbonate, filtered and concentrated under reducsd pressure, affording 129 mg (100% yield) of the desired N-Boc-amine.

Step 4 /~ H /~NHBoc ~N~N~ /N NH Boc N O

Under a nitrogen atmosphere, the N-Boc-amine produce in ';tep 3, above (28 mg, 0.05 mmol), in dichloromethane (2` ml), was combined with indoleacetic acid hydroxysuccinimide ester (14.1 mg, 0.05 mmol). The reaction mixture was stirred overnight and wæ diluted with CH2CI2 (25 ml) 10 and washed with saburated sodîum bicarbon~t~ (2 x 5 ml). The organic ex~act was dried over potassium carbonate, conen~ated in vacuo and chromatographed on silica gel (9:1 CH2CI2:MeOH followed by 9:1 :Q1 CH2CI2:MeOH:PrNH2), affording 28 mg (78% yieid) of the desired N-Boc-protected product.

Step 5 ~~N NH2 4TFA

N O

The indole prepared in Step 4, above (28 mg, 0.039 mmol), was dissolv~d in trifluoroacetic acid (3 ml) and was stirred fw 1 hour. The reaction mixture was concentrated under reduced pressure and triturated in diethyl ether to afford 19 mg (~6% yield) of the desired product.

WO 93/04036 2 1 1 4 7 9 8 PCI`/US92/06482 preDaratio-n A
Under nitrogen atmosphere, 34.5 9 (157.6 mmoles) of 3- -bromopropylamine-HBr in 600 ml of N,N-dimethy~ormamide was stirred. To - that solution was added 34.4 9 (157.6 mmoles) of di-tert-butyldicarbonate 5 followed by 32.3 ml (236 mmoles) triethylamine. A precipitate formed immediately. The reaction was stirred overnight. The reaction mixtu!e was then diluted to 1.5 liters with ethylacetate, washed once with 500 ml of 1 N
HCI, three times with 500 ml water, once with brine and dried over Na2/SO4.
After concentration, ~e product wæ chromatographed on 800 9 silica gel 10 using 4:1 hexanelethylacetate and the fractions were monitored by hexane/ethylacebte and the fractions were monitored by TLC (KMNOJI2).
The fractions containing the product were combined, concentrated in vacuo, chased twice with 50 ml dichlorome~ane and purged with high vacuum to yield 25.8 9 of the product of this preparation.

Claims (15)

1. A process for preparing a compound of the formula R-(CH2)m-CO-R' wherein:
R is a 5 to 7 member carbocyclic system or an 8 to 11 member carbobicyclic system, or any of the above systems substituted with one or more substituents independently selected from F, Cl, Br, OH, C1 to C4 alkyl, C1 to C4 alkoxy, CF3, phenyl, amino, C1 to C4 alkylamino and di(C1 to C4 alkyl)amino;
m is 0 or 1 ;
R' is-[NH(CH2)n]xNH2;
each n is independently 2 to 5; and x is 1 to 6, comprising:
(a) reacting a compound of the formula R with a compound of the formula Boc-NH(CH2)n[N(Boc)(CH2)n]x-1N(Boc)H, wherein Boc represents t-butyloxycarbonyl and R, n and x are as defined above, in the presence of a carbodiimide-based reagent in conjunction with an amide bond-forming catalyst in a reaction-inert solvent; and (b) removal of the t-butyloxycarbonyl groups by treatment with an organic or inorganic acid.

2. A process according to Claim 1 wherein said carbodiimide-based reagent is selected from dimethylaminopropyl, ethyl carbodiimide and dicyclohexylcarbodiimide and said amide bond-forming catalyst is selected from hydroxybenztriazole and hydroxysuccinimide.

3. A process according to Claim 1 or 2 wherein said reaction-inert solvent is dichloromethane and said organic or inorganic acid is selected from trifluoroacetic acid and hydrochloric acid.

4. A process according to one of Claims 1 to 3 wherein:
x is 5; and each n is independently 3 or 4.

5. A process according to one of Claims 1 to 4 wherein:
R is phenyl or phenyl monosubstituted with F, Cl, Br, OH or MeOH;
and m is 1.

6. A process according to one of Claims 1 to 5 wherein R' is -[NH(CH2)3]3-NH(CH2)4-NH(CH2)3-NH2.

7. A compound of the formula R-(CH2)m-CO-R', or a pharmaceutically acceptable acid addition salt thereof wherein:
R is a 5 to 7 member carbocyclic system or an 8 to 11 member carbobicyclic system, or any of the above systems substituted with one or more substituents independently selected from F, Cl, Br, OH, C1 to C4 alkyl, C1 to C4 alkoxy, CF3, phenyl, amino, C1 to C4 alkylamino and di(C1 to C4 alkyl)amino;
m is 0 or 1;
R' is -[NH(CH2)n]xNH2;
each n is independently 2 to 5; and x is 1 to 6.

8. A compound according to Claim 7 wherein:
x is 5; and each n is independently 3 or 4.

9. A compound according to Claim B wherein:
R is phenyl or phenyl monosubstituted with F, Cl, Br, OH or MeOH;
and m is 1.

10. A compound according to Claim 8 wherein R' is -[NH(CH2)3]3-NH(CH2)4-NH(CH2)3-NH2.

11. A compound of the formula R-(CH2)m-CO-R', or a pharmaceutically acceptable acid addition salt thereof wherein:
R is a 5 to 7 member carbocyclic system or an 8 to 11 member carbobicyclic system, or any of the above systems substituted with one or more substituents independently selected from F, Cl, Br, OH, C1 to C4 alkyl, C1 to C4 alkoxy, CF3, phenyl, amino, C1 to C4 alkylamino and di(C1 to C4 alkyl)amino;
m is 0 or 1;
R' is each n is independently 2 to 5;
x is 0 to 4;
y and z are each independently 1 to 5; and the sum of x and the greater of y and z is 1 to 5.

12. A compound of the formula R-(CH2)m-CO-R', or a pharmaceutically acceptable acid addition salt thereof wherein:
R is a 5 to 7 member carbocyclic system or an 8 to 11 member carbobicyclic system, or any of the above systems substituted with one or more substituent independently selected from F, Cl, Br, OH, C1 to C4 alkyl, C1 to C4 alkoxy, CF3, phenyl, amino, C1 to C4 alkylamino and di(C1 to C4 alkyl)amino;
m is 0 or 1;
R' is wherein:

each a is the same and is 2 to 5;
each b is the same and is 2 to 5;
each n is independently 2 to 5;
x is 0 to 3;
each y is the same and is 0 or 1;
z is 0 to 3; and x + y + z is 0 to 4.

13. A pharmaceutical composition for antagonizing mammalian excitatory acid neurotransmitters comprising a mammalian excitatory amino acid neurotransmitter antagonizing amount of a compound according to Claim 7, 8 or 10 or a pharmaceutically acceptable acid addition salt thereof and a pharmaceutically acceptable diluent or carrier.

14. A pharmaceutical composition for antagonizing mammalian excitatory amino acid neurotransmitters comprising a mammalian excitatory amino acid neurotransmitter antagonizing amount of a compound according to Claim 9 or a pharmaceutically acceptable acid addition salt thereof and a pharmaceutically acceptable diluent or carrier.

15. A use of a compound according to Claim 7, 8, 9 or 10 or a pharmaceutically acceptable acid addition salt thereof in the preparation of a pharmaceutical composition for antagonizing excitatory amino acid neurotransmitters in a mammal.