Note: Descriptions are shown in the official language in which they were submitted.
WO 93/04036 211 47 9 8 PCI/US92/06482
--1--
SYNTHETIC ARYL POLYAMINES AS EXCITATORY
AMINO ACI13 NEUROTRANSMITTER ANTAGONISTS
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
--2--
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
--3--
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)
WO 93/04036
2 1 1 4 7 9 8 PCJ/US92tO6482
8--
. . '
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
BOC 80C
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
--10--
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
-14-
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
-15-
(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
--16--
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
--17--
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
--18--
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
--20--
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~
--21--
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
3 8
--22--
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
-23-
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
--24--
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
--25--
'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
--26--
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
--27--
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
--30--
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
--31--
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
--33--
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
--34--
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
--35--
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
--36--
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
--37--
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
--38--
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
--39--
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.
