CA2103665C

CA2103665C - Oligopeptide antiretroviral agents

Info

Publication number: CA2103665C
Application number: CA002103665A
Authority: CA
Inventors: J. William Lown; Ronald George Micetich
Original assignee: University of Alberta
Current assignee: University of Alberta
Priority date: 1991-02-06
Filing date: 1992-02-05
Publication date: 2003-04-15
Anticipated expiration: 2012-02-05
Also published as: AU1224392A; ZA92864B; CA2103665A1; WO1992013838A1

Abstract

Oligopeptide antiretroviral agents are represented by the formula:

Description

ryv. 210365 OLIGOPEPTIDE ANTIRET$OVIRAL ACTS
FTELD OF THE ~NV N~ TION
This invention relates to oligopeptides which are particularly useful as antiretroviral agents.
HACK~ROt N_D_QF THE INVENTION
Various oligopeptide derivatives have demonstrated various medicinal uses, such as enzyme inhibitors as disclosed in United States Patent 4,483,850. It is also known that various oligopeptides have anti-tumor activity as disclosed in United States Patents 4,216,208 and 4,314,999. Antibiotic activity of oligopeptides is disclosed in United States Patent 4,454,065. Naturally occurring oligopeptides, netropsin and distamycin, have been discovered as having antiviral and anti-tumor activity. The chemical formulas for netropsin and distamycin are as follows:
112N ~
~ C-NEi-CH2C0-NH netropsin IIZN~~
C1- ~ ~~ CO-Ntt / NH2 i7 ~ ~~ CO-NH-CH2CH2-C ~ + C1-CH3 j \ 11H2 Compound 1 llco-Nlt distamycin CO-NH
t1 ~ ~ ~~ CO-NH NH2 C113 11 ~ ~ CO-NH-CH2-CHZ-C ~+ C1 CH3 i ~ \ NHZ

Compound 2 SUBSTIl'UTE SHEET

2103fiG5 WO 92/13838 PCT/CA92/0005~:;,~

2 ,.
These oligopeptides are disclosed in Julia, M., Preau--Joseph, N., C.R. Hebd-Seances, Acad. Sci. 1963, X57.
1115 and Arcamone, F.; Orezzi, P.G.: Barbier, W.;
Nicolella, V.. Penco,S.: G~~z. Chim. Ital., 1967, 97, 1097.
Netropsin and distamycin contain pyrrole moieties connected by peptide bonds and with side chains, at least one of which is positively charged; i.e., an amidine group, or a group of the guanidyl type.
Only distamycin has been used as a therapeutic agent as commercialized and sold under the trade mark STALLIMYCIN HYDROCHLORIDE in the form of s 1~ cream, ointment or paste. This composition has been used in the treatments of infections produced by herpes simplex, herpes zoster and vaccinia viruses. Topical application of distamycin has been limited due to its high cytotoxicity and a low therapeutic index which in the instance of treating the herpes virus is about 3.
U.S. Patent No. x,912,199 discloses oligopeptides containing pyrrole moieties which demonstrated significantly enhanced antiviral and anticancer activities as compared to the oligopeptides of the prior art.
According to this invention oligopeptides have been developed which have significantly enhanced antiretroviral activity compared to prior types of oligopeptides.
~USST1TUTE SHEET

SUMMARY OF THF INVENTION
According to an aspect of the invention, a compound represented by the formula I:
A-(NHCO)x-Het-(NHCO-Het)y-NH-R1-NH-(Het-CONH)Z-Het-(CONH)x-A (I) wherein A is a moiety bearing a positive charge and of a size which does not inhibit binding of said compound to nucleic acid sequences associated with the cellular action of retroviruses; R1 is a moiety derived from a dicarboxylic acid or a residue of carbonic acid; Het is a five-membered heterocyclic moiety; y and z are independently 0, 1, 2 or 3, x is 0 or 1, and pharmaceutically acceptable salts thereof, exhibit antiretroviral activity, especially against Human Immunodeficiency Virus and Hepititus B virus.
A process for preparing such compounds comprises reacting a cor~pound of the formula (II):
2 0 g-(NHCO)x-Het-(NHCO-Het)y-NH2 (II) i wherein x and y are as defined above; and 8 is the same as A or is a group with a nitrile, halogen or sulfide substituent; with a dicarboxylic acid of the formula (III) X-R1-X {III) wherein R1 is as defined above and X is halogen, imidazolide or other reactive moiety and converting 8 to A to form said moiety bearing a positive charge.

2103~G5 WO 92/13838 PCT/CA92/U005,.~

R~.EF DESCRIPTION OF THE DRAwrNGs Figure 1 is a graph showing a correlation between DNA
binding constants of linked oligopeptides (Ka,-) and observed inhibitory properties expressed in reciprocal IDSO values against Moloney Leukemia Virus reverse transcriptase (M1V-RT).
Figures 2-6 are graphs showing anti-HIV activity of several compounds of the present invention.
DETAILED DESCRIPTION OF THE PREFERR D 1"MBODIMENTS
Compounds according to this invention demonstrate significant antiretroviral activity. Although the actual biological mechanism of these compounds which cause antiretroviral activity is not fully understood, it is thought that the activity may be due to the compounds of this invention binding with nucleic acid sequences) associated with the cellular action of retroviruses to inactivate such nucleic acids which code for the retroviral activity. It has also been observed that the linked oligopeptides of the present invention are potent inhibitors of Moloney Leukemia Virus (MIV) reverse transcriptase, a potential indicator of anti-HIV activity.
See Figure 1.
The compounds of this invention have heterocyclic moieties, which may be the same or different, linked by a dicarboxylic acid derivative. Such linked heterocyclic moieties of this invention have significant unexpected activity compared to unlinked pyrrole moieties such as the naturally occurring netropsin and distamycin.
SUBSTITUTE SF-iEET

The compounds according to this invention are represented by the following formula:
A-(NHCO)x-Het-(NHCO-Het)y-NH-R1-NH-S (Het-CONH)z-Het-(CONH)x-A
l wherein A is a moiety bearing a positive charge and of a size which does not ir.zibit binding of said compounds to deoxyribonucleic acid sequences associated with the cellular action of retroviruses; R1 is a moiety derived from a dicarboxylic acid; Het is a five-me:abered heterocyclic moiety; R2, R3, R4 and RS may be attached to a ring carbon atom or hetero ring atom and are independently selected from C1-C6 alkyl and CHZ-O-R6, where R6 is a C1-CS alkyl; y and z are independently 0, 1, 2 or 3; x is 0 or 1; and pharmaceutically acceptable salts thereof.
The positively charged moiety at aach extremity'of the compound and identified as group A is preferably selected Iron the group of derivatives consisting of an amidine, a guanidine, secondary ammonium salts, sulfonium salts and phosphonium salts.
The selected amidine may have one or both nitrogen atoms of the amidine as a member of a five-membered cyclic structure. More particularly, the amidine derivative is represented by the formula:
/. NH2 -CpH2p-C ~ +
NHX
where p equals 0 to 5 and X is -H, -OH, -NH2, -Ca3, _C2H5~
-C~H~.

2~.0366~
w0 92/131338 PC~'/CA92/0005~~'~
The selected guanidine for substituent A may be represented by the formula:

-CpH2p-NH-C O

where p equals 0 to 9 and X equals -H, -OH, -NH2, -CH3, _C2H5~ _C3H7.
t~Then A is selected to be a quaternary, tertiary or secondary ammonium salt, it may be represented by the formula:
~CpH2p- NHqX~3-q) where p equals 1 to 5 and q equals 0 to 3 and X is an alkyl or alkenyl group of 1 to 3 carbon atoms.
When A is selected as a sultonium salt, it may be represented by the lormula:
-CpH2p-SXY
where p equals 0 to 5 and X and Y are alkyl or alkenyl groups of 1 to 3 carbon atoms.
In the heterocyclic moieties, Het may be the same in 2~ each moiety or may be different. Preferably, the Het group is selected trom the group consisting of a pyrrole, an imidazole, a triazole, a pyrazole, a thiazole, a thiophene, a turan, an oxazole and derivatives thereof.
Preferred ring carbon atom substituents are alkyl groups, and especially methyl groups, on the Het moiety, especially on thiazole rings.
StJ~STI'Tl~'TE SHEET

2103~s5 ~~0 92aasas rcrscA9~iooosi Preferred Het substituents are N-alkyl pyrrale having 1 to 6 carbon atoms in the alkyl group; N-alkyl imidazole having 1 to 6 carbon atoms in the alkyl group: alkyl pyra~ole having 1 to 6 carbon atoms in the alkyl group:
and alkyl triazole having 1 to 6 carbon atoms in the alkyl group. Preferably the N-alkyl pyrrole has 1 to 4 carbon atoms in the alkyl group, and especially in N-methyl pyrrole. Also preferred Het substituents are N-linked alkoxymethyl groups. The choiee of Het substituents will depend on their cellular uptake ability.
R2, R3, R4 and RS are linked to the N or C atom of the Het moiety and are independently C1-C6 alkyl or -CHZ-O-R6 where R~ is C1-C6 alkyl. It has been found that the longer the alkyl group in either structure is, the better the cellular uptake of the compound. The choice of substituent will depend on solubility properties:
solubility in pharmacologically acceptable solvents, such as water or DMSO, has been found to be higher with the methoxy substituents.
The linking group R1 is a derivative from carboxylic acid. R1 is represented generally by the formula:
-co-epH2p-co-where p equals any number from 1 to 22. Alternatively, Rl 2S may be a residue of carbonic acid, namely -C-: or R1 may be a residue of an aromatic dicarboxylic acid. The -CO-groups of the aromatic dicarboxylic acid residues may be in the ortho, mete or pare positions on the ring. The aromatic residues may be S to 6 C membered rings. The aromatic dicarboxylic acid may also be a six membered heterocylic ring containing a nitrogen atom.
$LdI~ST4TUTE SHEET

2103665 ~;-WO 92/13838 PCC/CA92J0005 .,'r other alternative structures for the linking group may be a residue of an unsaturated aliphatic dicarboxylic acid of the formula:
_CO_C~~2q_2)_CO_ where q equals any number from 2 to 22.
R1 may also be a residue of cycloalkane dicarboxylic acids of the formula:
-CO'CrH(2r-2) CO
where r equals any number from 3 to 7 and optionally may be fused to one or more three to seven C membered rings, preferably fused to one or two three to seven C membered rings.
R1 may also be a residue of cycloalkane dicarboxylic acids of the formula:
-CO-CSH~2s-4)-CO-where s equals any number from 3 to 7.
In a preferred compound of the present invention, A
is a moiety selected from the group consisting of an amidine, a guanidine, secondary ammonium salts, tertia~-y~
ammonium salts, quaternary ammonium salts, sulfonium salts and phosphonium salts.
In another preferred compound of the presen>;
invention, R2, R3, R4 and R~ are each a C1-C6 alkyl or R2, R3, R4 and R5 are the same and are a C1-C6 alkyl group or R2, R3, R4 and RS are each a methoxymethyl.
SUBSTITUTE Sf-~EE'6' 21 d 3 6 6 5 p~/CA92/OOQ51 ~'~:. WO 92l138~3 ~s ~,.~.

In another preferred compound of the present invention, R~ is or R' is a residue of a dicarboxylic acid of the formula -CO-CpH~-CO- where p equals 1 to 22. R, may also be preferably a residue of a dicarboxylic acid selected from the group consisting of : a residue of an unsaturated aliphatic dicarboxylic acid of the formula -CO-Ca°H~y.2-CO-where q equals 2; a residue of an aromatic dicarboxylic acid; and a residue of a cycloalkane dicarboxylic acid of the formula -CO-C,-H~.2-CO- where r equals 3 to 6.
In yet another preferred compound, R, is CO CO CO
~ CO 0.
C~CO ~ ~ ar CO
Preferably, R~ is a dicarboxylic acid residue of cyclopropane, a dicarboxylic acid residue of cyclopentane, or a dicarboxylic acid residue of cycl.ohexane.
The following are representative examples of the preferred compounds of the present invention.
N, N'-di[1-methyl-2-[1-methyl-2-carboximido(3 propionamidine)-4-pyrrole]-4-pyrrolyl]
terephthalamide dihydrochloride.
N, N'-di[1-methyl-2-[1-methyl-2-carboximido(3-propionamidine)-4-pyrrole]-4-pyrrolyl]
isophthalamide dihydrochloride.
N, N'-di[1-methyl-2-[1-methyl-2-ca~boximido(3 propionamidine)-4-pyrrole]-4-pyrrolyl]
fumaramide dihydrochloride.
N, N'-di[1-methyl-2-[1-methyl-2-carboximido(3-propionamidine)-4-pyrrole]-4-pyrrolyl]
maleamide dihydrochloride.
N, N'-di[1-methyl-2-[1-methyl-2-carboximido(3-propionamidine)-4-pyrrole]-4-pyrrolyl] traps 1,?.-cyclobutanamide dihydrochloride.
SUBSTITUTE SHEET' WO 92/13838 , PC.'I'1CA92/000=~;' N, N~-di(1-methyl-z-[1-methyl-z-car.boximido(3-propionamidine)-4-pyrrole]-4-pyrrolyl] tr__ ans 1,2-cyclobutanamide dihydrochloride.
The compound:
z N

t ,2 v _~
Z.
U

O
Z
a =2 O
'~~ N
-V
S <.
s ~
Z~U
O

_e~
O
~z -v ~.z N
x_ +' _n z U
SUBSTITUTE SHEET

~ 10 3 6 6 5 ~~.~~Ag2~o~m l~':; WO 92/13838 The compound:
a N
N

t O
r1 ~~ U

O
a ~ ~~
Z' v _-z -w _~
o~u i o~
xz z~"v r~
.= z / 2" V
O
xZ
'~ V
W
O
x.z.
N
a z N
x Z I
V
SUBSTITIDTE SHEET

210 ~56;~
wo 9aia~g3s Pc~icAy2iooos~~'' The compound:
_N
Z
,~.
.T. '~
d 'z' v mz \ _ ~
Z U
Z
O
M
Z
O'U
z x z / x Z'r V
w O
xz Z" V

xz x "u x.z N
x Z
$

..1 U
SU~S"P°ITdJTE SHEET

;~ ~=WO 92/13838 ~ 10 3 ~ G 5 pCTlCA92/i8f051 The compound:
a N
z' N
Z
O
Z U

_~
Z.
U

O
/~ ~ .
xi U
x~
o.u z xz i x Z'_ a xz 2 x U
xx i _x a x .z N
x Z
N
x z , a SI~SSTITUTE SHEET

2103~f 5 PC~'1CA92/0005 ';":;
WO 92/13838 ,:,:

The compound:
:J
N

N
Z
.2 O
n z U
:Z
O
U
~'Z
O
z' v o-c~
xz M
z'° ~
~o x /z M
/ z-' a xz M
~U
O
x ,z.
N
x z N
x z 1 .a V
aUI3STITdJTE SH~~T

21 a 3 6 6 5 pCT/CA92/00051 In cases where RI is a dicarboxylic acid derivative of an aliphatic hydrocarbon, the linker is referred to as flexible. Rigid linkers refer to cases in which R1 is carbonic acid or residues of aromatic, unsaturated aliphatic, cycloalkane and cycloalkene dicarboxylic acids.
Most preferred are those compounds in which R1 is a rigid linker. Examples of the flexible linked and rigid linked oligopeptides are set forth below.
FLEX~BL~LI~IKEd OL7 GOPEPTIDES
H~HN
~ R.

N
RCN ' R_ ' 0 H NN
/
N

H HN
I . R_ I ~ / ~ H
C~i~ N
i~ I
O~a N~N~cN

R' -C0- ( CHI ) n-COR' R"--COCHZ CHZ COR "
R'°°~-COCH2CH2COR°'°
SIJ~S'Ti~'U'TE SHEE°f WO 92!13838 PC1'lCA92l044S ~:;°:'i;:

H~HN
-~~ H
N
N
H
CHI N NHa V ~ t 0 'NH= CI
H ~ HIV
~\ H
N~N~NH~ R
v ~IIf s CHI O 'NH= CI' NIHN
x' Rl--CO- ( CH2 ) n--COR1 3 Rg-~CO-Rl 4 RlCOCHZCO-R1 5 Rl-CO ( CH2 ) z CO-Rl 6 R1--CO(CH2)3C0-R1 7 R1--CO(CH2)4C0-R1 8 R1--CO(CH2)5C0-R1 10 9 Rl-CO ( CH2 ) 6C0--R1 10 Rl-CO (CH2 ) 7C0-R1 11 Rl-CO ( Ch2 ) $ CO-Rl 12 R1--CO (CH2 ) 9C0-Rl 13 R1--CO (CH2 ) LOCO-R1 14 R2--CO ( CH2 ) 2 CO-R2 15 R~--CO(CH2)2C0-R3 , 16 R3-CO ( CH2 ) 6C0-R3 17 R3-CO (CHZ ) 8C0-R3 18 R3-CO ( CH2 ) 2 2 CO-R3 SUBSTITUTE SHEET

i ~v~: WO 92/13838 PC~'/CA92/00051 Rigid J inked O~igopeptides H HN
it N
N
t1 CIl3 0 N' y N~~ -R1 CI13 '0 H HN
t1 N

N
Cll 3 ~ N
1 ~ e.1 =R
cII3 p .vNIl2 2 CO-R

traps 23aR=Rt 2 3 bR=Ra CO-R

CO-R

cis 24aR=R, IOR 24bR=R2 CO-R

CO-R
l9aR=R 25aR=R

I , traps l9bR=R2 25bR=Rz COR C O-R
COR

20aR=R~CO-R
traps 25aR=R~

2 0 2 6 bR=RZ
bR=RZ

NCO-R

COR CO-R
/

2laR=R~ trans27aR=R~
/ -COR

RCO 21 bR=R2 2 7 bR=R2 CO-R

~
CO-R

RCO 22aR=Rj cis 28aR=R~

~COR 22bR=RZ 28bR=Rz ~
CO-R

gUH~TITUTE SHEET

2~fl3665 WO 92/ 13838 PCT/CA92/Q0051~.

H HN
i1 N
N
i II

NII
CIt 3 ~
6113 c: l-0 ~I~N112 -R2 x x c~
n ~N N
n~\/CN =R3 N
Ctl3 N

~, COR2 / /
R2C0 - ~ ~ -' CORZ ~ \ ~ ,~ \ ~ i~ COR2 R2C0 CORz N
R CO -- ~ ~ "" COR2 / .
R CO / ~ "COR2 R2C0 ~ H ~'~COR2 R2CO~CORZ R2CO~COR2 RICO ~ COR2 R3CO~OR3 g8 39 SIJ~STITUTE SHEET

E'-':,r WO 92/1383~i 210 3 s s ~ Pcr,c~.~z,oo~si other preferred compounds include compounds o!
formula I wherein Het is pyrrole and x is 1; ~, is~

-CH2 CH2 -C ,~
+NH2 and R1 is a rigid linker (as defined above).
The heterocyclic moiety of the compounds of this invention may be linked in accordance with various processes by use of the dicarboxylic acid derivatives. In 1o accordance with one aspect of this invention, the process for providing such linkage comprises reacting a compound of the formula:
8- (NHCO) x-Het- (NliCO-Het) y-NEiZ

15 wherein x and y are as defined above; and ~ is the same as 1~ or is a group with a nitrils, halogen or sulfide substituent: with a dicarboxylic acid of the formula:

wherein R1 is as defined above and X is halogen, imidazolide or other reactive moiety and converting 8 to 7~ to form said moiety bearing a positive charge.
In the reactants, B may be generally represented by the formula:
Z_CPH2P_ where 2 is CN-, hal or XS: hal is a halogen ion, X is an alkyl or alkenyl group having 1 to 3 carbon atoms, and p equals 0 to 5.
SUBS1~1'TUTE St~EE'T

WO 92/13838 PCT/CA92/OOOSX:;,:,';;v ' 1 ~~ 1 is It is to be appreciated that B may also be identical to A in providing a charge group, for example, a guanidinium end group. In that instance, B has the general formula:

C-(NH)s-CPH2p-XHN+
wherein X is an alkyl having 1 to 3 carbon atoms or alkenyl group having 2 or 3 carbon atoms and p equmls 0 to 5 and s equals 0 or 1.
Compounds of the present invention which are asymmetrical around the linking group (i.e., wherein y and z are different in number) can be prepared by a two-step process, wherein the first step involves coupling a compound of the formula:
B-(NHCO)x-Het~(NHCO-Het)y-NHa wherein B, x and z are as defined above, with a dicarboxylic acid of the formula:

wherein Rl and X are as defined above (this coupling is generally with the use of equimolar amounts of the reactants). this is followed by coupling of a compound of the formula:
B-(NHCO)x-Het-(NHCO-Het)z-NH2 t i wherein B, x and z are as defined above, with the provisions that z is different than y.
StJSSTlThITE SHEET

21~366~
~;' ~' WO 92/ 13838 PCT/CA92/a0051 According to preferred embodiments of the invention, the following reaction schemes demonstrate preferred chemical pathways to the compounds of this invention having the various desired end groupso A - Preparation of Amidinium End Groud 2G~J-Cptl~ (r~'iICO)m Ilet-(NliCO-idet) ni'tEl2 + C1-R-Cl if r E IN
2 y (CDI-CPtd2p (NHCO)m tlet-(rifJCa-Ilet)~ NrJ-)aR
HZN
1. llcl/etaH
2. Nii~/Et0ll~ C1 ~-CpH2p-(N11C0)m°llet-(NF1C0-ilet)n-hltl- Z iZ
+
H ZN
B Pret~aration of Guanidinium End Groups t1 2N
C1 ~ Nit-CpH2p- iNllCO) m-filet- iNiICO-llet) n-N02 ~Nf1-C H - iNIlCO) -Fle t- (NilCO-lie t) n-NE12 reduce C1 ~ p 2P

1f2P1 C1-R-CJ~ ~ 'Ntl-Cpit2g-(NHCO)m-Elet-(NEiCO-tlet)n-Nll- R

XEIN+

$USSTITUTE SHtET

WO 92/13838 PC.TlCA92/(1(DOS~;, _C - Preparation of Ammonium Salt in End Group 2 C1-CPIIZP- (NHCO) m-Het- (NIICO-llet) n-NH2 + C1-R-C1 iPr2EtN
(C1-CpHZP- (NtICO) m-llet-(NiICO-ltet) n-Nt1°) 2R
lltlHy \ ( 3_~) ' (X(3-q)+NIIq-Cpli2P (NHCO)m-llet-(NIICO-ifet)~-N11-)2R

D - Preparation of Sulfonium Salts 2X5-CpH2P-(NHCO)m-Het-(NHCO-Het)n-NIIZ + C1-R-C1 iPrZEtN
-_ (XS-CPHZp- (NHCO) m-Het- (NH-CO-Het) ~-NFI-) 2R
YC1 (or Br) (XYS+-CPH2p-(NHCO)m Het-(NtiCO-Het)n-NH-JzR
SUBSTITUTE SHEET

PC 1'/CA92/00051 Reference may be made to J.W. Lown and K. Krowicki, J. Ora~Chem. 1985, ~"~c, 3774 regarding the synthesis op related types of pyrrole moieties such as the synthesis of distamycin. The general synthesis op the compounds according to this invention are based on the total synthesis op distamycin. Dipyrrole or tripyrrole peptides bearing an amino group and a side-chain containing a group (B) which is the nitrite, ammonium or sulfide as represented by the following formula:

CO-NH- B
N r . 2.3 CH3 r l0 are allowed to react at a temperatures of -35 to +l0'C, preferably about -20'C, With a dicarboxylic acid dichloride in the presence of a base or with a diimidazolfde of a dicarboxylic acid to give a bis-amide of the dicarboxylic acid. Tha resulting compound in the case of nitrilr is allowed to react at a temperature or 0 to +35'C, preterable +15' to +25'C, more preferably about +20'C, with ethanol in the presence of hydrochloric acid and then at a temperature op 0 to +35'C, preferably +15 to +25'C, moss preferably about +20'C, with ammonia (Dinner reaction) to generate an amidinium moiety in the final product, as exemplified by the above reaction scheme A.
As with reaction scheme D, the sulfide is methylated at a temperature of 0 to +35'C, preferable +15 to +25~C, more preferably about +20'C, to produce the corresponding ZS sulfonium salt.
The compounds op formula I, are useful as antiretroviral agents, especially against the Human Immunodeticiency Virus (HIV). Human patients suffering prom diseases caused by, for example, HIV, can be treated by administering to the patient a pharmaceutically SUBSTIT~1E SHEFT

~i03665 WO 92/ 13838 PCT/CA92lOn0 ~~,'~-'~~'.;
effective amount of one or more of the present compounds optionally, but preferably in' the presence of a pharmaceutically acceptable carrier or diluent. There may be also included pharmaceutically compatible binding 5 agents and/or adjuvant materials. The active materials can also be mixed with other active materials which do not impair the desired action and/or supplement the desired action. The active materials according to the present invention can be administered by any route, for example, 10 orally, parenterally, intravenously, intradermally, subcutaneously, rectally or topically, in a liquid or solid form. For injection purposes, the medium used may be a sterile liquid. As an injection medium, it is preferred to use water which contains the stabilizing 15 agents, solubilizing agents and/or buffers conventional in the case of injection solutions. Desirable additives include, for example, tartrate and borate buffers, ethanol, dimethylsulfoxide, complex forming agents (for example, ethylenediamine tetracetic acid), high molecular 20 weight polymers (for example, liquid polyethylene oxide) for viscosity regulation or polyethylene derivatives of sorbitan anhydrides. solid carrier materials include, for example, starch, lactose, mannitol, methylcellulose, talc, highly dispersed silicic acid, high molecular weight fatty acids (such as stearic acid), gelatin, agar, calcium phosphate, magnesium sterate, animal and vegetable fats or solid high molecular weight polymers (such as polyethylene glycol). Compositions .suitable for oral administration can, if desired, contain flavoring and/or sweetening agents.
A preferred mode of administration of the compounds of this invention is oral. Accordingly, the compounds may be formulated into capsule form or tablet form.
SUBSTITUTE SHEET

~'"°' WO 92/13838 The active materials according to the present invention can be employed in dosages and amounts which are conventional in the art. Thus., the materials can be used at a dosage range in humans of from about 1 to 200 mg/kg total body weight/day. A more preferred range lies between 1-30 mg/kg total body weight/day. The dosages may be administered at once, or may be divided into a number of smaller doses to be administered at varying intervals of time.
The in vj~t~-~ anti-HIV screening test results, performed at the United States National Cancer Institute, have shown that 23 of the present compounds are active.
Of the fifteen, ten are considered "active", and thirteen are determined "moderately active". Certain of the compounds screened for anti-AIDS activity at the NCI were determined to be "inactive". These compounds were ones wherein the R1 is -CO-(CH2)6-CO- or -CO-(CH2)8-CO-, A is mmidine, x is 1, Net is methylpyrrole, and y and a are 1, as well as compounds 9, 11, 15, 16, 18 and 37.
The therapeutic index of a compound is determined by dividing the inhibitory or lethal concentration for S0~ of the population (IC50) by the effective concentration for 50~ of the population (EC50). The therapeutic indexes for the particularly active compounds of the present invention range from 1.46 to 161.
As used in this invention, antiretroviral activity refers to the ability of a compound to inhibit the growth of a retrovirus. The retrovirus of primary importance with respect to the present invention is HIV. However, the present compounds may also exhibit antiretroviral activity towards other retroviruses as would be apparent by the suspected mechanism of action and other viruses which replicate or exhibit reverse transcription.
SUST~T~TE S~T

~~a~~s~
WO 92/t3R3R P'C'r/CA92/OOOSt°~~'~s O..CY
The compounds of the present invention should also be therapeutically eft~ctive~in the treatment o:f hepatitis B
viral infection in mammals, especially humans Similar to retroviruses (including HIV-1), the hepatitus B virus replicates by reverse transcription. In addition, hepatitus B virus putative viral polymerase share amino acid homology with reverse transcriptase of retroviruses and a comparison of the thirteen (13) hepadnavirus isolates determined that other conserved areas showing homolgy to corresponding regions of Type C retro virus.
Miller et al., Proc.Natl.7~cad.Sci. USA, Vol 83:2531-2535 (1986).
Since it is theorized that the activity of the compounds of the present invention may be due to the compounds binding with nucleic acid sequence(s) associated with the cellular action o~ rstroviruses to inactivate such nucleic acids which code for the retroviral activity, the compounds ors likely to inhibit binding with nucleic acid sequence(s) o! the hepatitus 8 virus associated with the cellular action of reverse transcription to inactivate such nucleic acids which code for the retroviral-like activity. Therapeuticmlly effective anti-hepatitus B
dosages would be the same as anti-HIV-1 dosage levels as z5 well as would the routes of administration.
The ability o! a compound to inhibit HIV may be measured by various experimental techniques. One such technique, currently employed by the United States National Cancer Institute to screen potential anti-HIV
compounds, involves the inhibition of the killing of HIV
infected T~ lymphocytes. Compounds of the present invention hav8 been tested for anti-HIV-1 activity in the NCI protocol; however, one skilled in the art would appreciate that the compounds should exhibit activity against. HIV-2 as well.
SIJ~STITIITE St~EET

'.j WO 92J13838 210 3 6 6 5 p~-~CA92J00(IS'1 Preferred embodiments of the invention are exemplified in the following Examples whieh are in no way to be construed as limiting the scope of the appended claims.
EX~"~LE 1 Compound of the formula I, where x=1, y and a each are 1;

A=CH2CH2C ~ Cl-;
~ +NH2 R1 equals -COCH2CH2C0-, was prepared. 1-Methyl-4-(1-methyl-4-aminopyrrole-2-carboxamido)-pyrrole-2-carboxamidopropionitrile (105 mg, 0.33 mmole) and 1-Pr2EtN
(diisopropylethylamine) (65u1, 0.16 mmole) in anhydraus THF (1 ml) was added and the mixture was allowed to reach room temperature. The solvents were evaporated to dryness and water was added. The resulting solid was collected and washed with hot MeOH to give 90 mg (77% yield) of the product m.p. 297°C. The latter vas suspended in anhydrous EtOH and saturated with HIC1 while cooling. After 1.5 hours at room temperature, the solvent was removed in vacuo and the residue was washed with dry ether then ethanol was added followed by some ammonia condensed into the solution. After 1 hour at room temperature, the solvent was removed and the residue was washed with MeOH, EtOH and hexane to afford 80 mg of a solid.
Recrystallization from a small volume of water gave a jelly-like precipitate which was washed with EtOH, hexane and dried to give 35 mg (35% yield) of pure product m.p.
283-285'C dec. 1H-NMR (DSMO-d6): d 2.60 (m, 4H), 3.60 (m, 2H), 3.83 (s, 6H), 6.92 (d, 2H),' 7.18 (d, 2H), 8.25 (t, SUBSTITUTE SHEET

~/~3~3~ P'Cf/~,~92/0005 ~'._'' .~: /Y

1H), $.70 (bs, 2H), 9.02 (bs, 2H), 9.93 and 9.97 (2s, 2H), MS-FAB (m/2):745 (M-C1-HC1)ø: Anal. Calcd. gor C,4H46C12N1406: C, 49.9, H. 5.7, N, 24.0, C1, 8.7, Found:
C, 50.7, H, 6.05, N, 22.9, C1, 8.7.

Compound og the formula I, where x equals 1; y and z are each equal to l;

A=CH2CH2C ~ C1-;
+NH

R1 equals -CO- was prepared. 1-Methyl-4-(1-methyl-4-aminopyrrole-2-carboxamido)-pyrrole-2-carboxamidopropionitrile (315 mg, 1 mmole) and 81 mg of l,l'-carbonyldiimidazole were dissolved in 10 ml of anhydrous CH3CN and retluxed~ under argon for S minutes.
A solid forms which was collected to give 302 mg (88.6%
yield) of the pure product was treated with HC1 in EtOH
and then NH3 (as in Example 1). After the reaction was completed, the mixture was decanted from an insoluble residue. The solvent was removed in vacuo and the residue was dissolved in d ml of MeOH and an excess of CH3CN was added to precipitate the product which was collected and washed with l ml of Bold water whereupon it became jelly-like. The product was redissolved 1n MeOH and reprecipitated with CH3CN to give 216 mg (57% overall yield) of the pure compound m.p. 211-215'C; 1H-NMR
(DMS) -d6) : d 2. 64 (t, 2H) , 3 . S2 (q, 2H) , 3.84 (s, 6H) , 6.82, 6.94, 7.03, 7.20 (4d, 4H), $.25 (t, 1H), 8.73 (2s, 3H), 9.05 (s, 2H), 9.88 (s, 1H), MS-FAB: 690 (M-C1-HC1)+.
Anal. Calcd. for C31H42C12N1405 v C~ 48.9, H, 5.6, C1, 9.3, N, 25.7; Found C, 48.5, H, 5.7, C1, 9.7, N, 25.3.
SII~STOTUTE S~f EFT

~103~fi~

EXAMPLES 3 (A1 l~Np _j~,2 The following Examples illustrate the effect of altering the steric sine of the terminal group (in these cases trialkylammonium) on the basic Het block of the 5 general formula on the nucleic acid binding and antiviral efficacy. The effects were demonstrated on deoxyribonucleic acid to show that steric hindrance in the terminal group in ONA binding generally reduces antiviral activity of the compounds.
10 (A) 1-Methyl-4-(1-methyl-4-trimethylammonium-acetamidopyrrole-2-carboxamido)pyrrole-2-~arboxvamidoprioeionamidine ehlorid h~~~-~r~~~ ~d A solution of the precursor 1-methyl-4-(1-methyl-4-trimethylammonium-acetamido-pyrrole-2-carboxamido)pyrrole-15 . 2-carboxyamidopriopionitrils chloride (347 mg, 0.07 mmoles) in 5 ml o! absolute ethanol was treated with dry hydrogen chloride with cooling. Alter 2 hours, the solvent was removed in vacuo, 5 ml of absolute ethanol was added and dry NH3 gas passed into the solution. The solid 20 dissolved during 2 hours at room temperature, then the solution was,evaporated to dryness and extracted with hot isopropyl alcohol (100 ml). The extract was concentrated to ca. 10 ml, acetone added and the resulting precipitate callected, washed with acetone, and dried to vacuo to give 25 the product, 300 mg (85~ yield) as an amorphous hygroscopic solid, no definite m.p.; iH-Nr.d (OMSO-d6): d 2.67 (t, 2H), 3.31 (s, 9H), 3.52 (q, 2H), 3.82 and 3.87 (2s, 6H), 4.44 (s, 2H), 6.97 (d, 1H), 7.02 (d, 1H), 7.24 (d, 1H), 7.29 (d, 1H), 8.31 (t, 1H), 8.82 (be, 2H), 9.72 (be, 2H), 10.06 (s, 1H), 11.23 (5, 1H), TR (Nujol) vmaX' 1260, 1377, 1405, 1453, 1531, 1582, 1643, 1685, 3247 cm-1:
MS-FAB (m/t) 430 (M-HC1-C1)~'.
SIJ~STITIJTE SI-IEET

~I03fi65 WO 92/73838 p(.fi/~A92/0005 .~' ,:y<.

5ulfa~g. The sulfate corresponding to the product was prepared in order to obtain an analytically pure sample by precipitation from a methanolic solution of the above compound by means. of a large excess of tetraethylammonium sulfate,~m.p. 295°C: IR (Nujol) Amax' 1255, 1377, 1405, 1462, 1525, 1560, 1580, 1640, 1670, 3280 cm-1; MS-FA8 (to/z) 431 (M-HS04)+, 529 MH+: Anal. Calcd.
for C20H32N807S (528.59), C, 45.4, H, 6.1, N, 21.1, S, 6.1. Found: C, 45.0, H, 6.A, N, 20.7, S, 5.8.
(B) 1-Methyl-4-(1-methyl-4-trimethylammonium-acetamidopyrrole-2-carboxamido)pyrrole-2-carboxyamidoprio,Qionamidine chloride hydrochloride A solution of the precursor analogous to that of ~xample 3(A) (173 mg, 035 mmoles) in 10 ml of absolute ethanol was treated with dry hydrogen chloride with cooling. 'After 2 hours, the solvent was removed in vacuo and the residue dissolved in 10 n1 of absolute ethanol and treated with an excess of dry ammonia. After 2 hours at room temperature, the solvent was removed in vacuo and the residue dissolved in 5 ml of isopropyl alcohol; then the product was precipitated with ether. The solid was collected, washed with ether and dried at 100' in vacuo to afford the product 103 mg (59~ yield) m.p. 180' (dec);
iH-NMR (DMSO-d6): a 1.32 (t, 9H), 2.67 (t, 2H), 3.54 (m, 8H), 3.83 and 3.88 (2s, 6H), 4.32 (s, 2H), 6.96 (d, 1H), 7.01 (d, 1H), 7.21 (d, 1H), 7.30 (d, 1H), 8.28 (t, 1H) 8.80 and 9.10 (bs, 4H), 10.03 (s, 1H), 11.47 (s, 1H), IR
(Nujol): 1376, 1404, 1462, 1531, 1581, 1646, 1684, 3250 cm-l: MS-FAB (m/z): 981 (2M-HC1-C1)+, 473 (M-HC1-C1)'~.
The activities of ~xamples 3 (A) and 3 (B) expressed as minimum inhibitory concentration (pg/ml) against vaccinia virus were 20 and 300 respectively illustrating the effects of steric hindrance in DNA binding on reducing agent activity. The larger the terminal group, as ShBSTITIJTE S~iEET

.:.~ V~ 92/13Fi3Ed ~ 10 3 ~ G ~ Pcri~c~~aiooos~

demonstrated by compound 3(6), the lesser the activity;
hence the terminal group is of a selected size which will maintain nucleic acid sequence bonding desired antiretroviral activity.
The compound numbers referred to in the following examples correspond to the numbered structures in the "Detailed Description of the Invention" section.
1~XAMPLE 4 (A) 1-Methyl-4-(1-methyl-4-(1-methyl-4-aminopyrrole-2-l0 carboxamido)pyrrole-2-carboxamido)pyrrole-2-carboxamidopriopionitril~(Intermediate Compound?
1-Methyl°4-(1-methyl-4-(1-methyl-4-aminopyrrole-2-carboxamido)pyrrolie-2-carboxamidoJpyrrole-2-carboxamidopriopionitrile (town, W.J. and Krowicki, lc. , J.
Org. Chem. Vol. 50, p. 3774 (1985) and Krowicki, R. and Lows, W.J., J. Org. Chem., Vol. 52, p. 3493 (1987) (420 mg, 0.9 mmol) was reduced over 5t palladium on charcoal (260 mg) in a mixture of DMF (15 ml) and methanol (5 ml) at 45'. After the reduction the solvents were evaporated under seduced pressure. The residue was dissolved in a small amount of acetonitrile (2 ml) and an excess of ethyl acetate (20-30 ml) as added to precipitate some impurities. The filtrate was treated with an excess of hexane to precipitate a white pure product 9 (250 mg, 63.5 yield), m.p. 155-160'. iH-NMR (DMSO-d6): a 2.74 (t, 2H), 3.42 (Q, 2H), 3.76 (s, 3H), 3.85 and 3.87 overlapped with a bs (3s, 8H), 6.27 (d, 1H), 6.40 (d, 1H), 6.95 (d, 1H), 7.04 (d, 1H), 7.24 (2d, 2H), 8.37 (t, 1H) 9.66 (s, 1H), 9.96 (s, 1H)T IR (nujol): 1260, 1377, 1403, 1464, 1529, 1582, 1646, 2245, 3120, 3310 Cm-1: MS m/z 436.1981 (calcd. 436.1983). Analysis Calcd. for C19H26C1N603: C 52.3, H 6.0, C1 8.1, N 22.5. Found: C
52.3, H 6.0, C1 7.9, N 22Ø
SUBSTITUTE SHEET

2~.a3~65 WO 92/ 13838 PCT/CA92/000y' a,',', 2s (A) N,N'-Di(1-methyl-2-(1-methyl-2-carboxamido 0 o ucc' 'de The intermediate compound (105 mg, 0,33 mmol) and i-Pr2EtN (65 ~sL, 0.37 mmol) were dissolved in anhydrous acetonitrile (5 ml) and cooled to -20'C. Succinyl chloride (18 uL, 0.16 mmol) in anhydrous THF (1 ml) was added. The mixture was allowed to reach ambient temperature. The solvents were evaporated to dryness, l0 water was added, and the resulting solid was collected and washed with riot MeOH. The product was dissolved in DMF
and when placed on a TLC plate (Si02) with CHC13 + 15~
MeOH system it gave one spot. For analytical purposes, the product was purified by dissolution in a small amount of DMF and precipitation with a large amount of EtOH to give 90 mg (77~) of 15 m.p. 292'. 1H-NMR (DMSO-d6): 6 2.58 (s, 4H) 2.74 (t, 4H), 3.42 (q, 4#i), 3.83 (2s, 12H), 6.86, 6.93, 7.17 and 7.22 (4d, 2H each) , 8.35 (t, 2H) , 9.89 (s, 4H): IR (nujol): 1376, 1401, 1447, 1465, 1511, 1535, 1585, 1645, 2245, 3120, 3304 cm -1; MS (m. z. rel.
int.): 396.1543 (9.98) for CiQH20N604 which is (0=C=CH-M1~2)+. Analysis Calcd. for C34H38N1206: C 57.5, H 5.4, N 23.6. Found: C 57.8, N 5.4, N 23.3.
(B) N,N'-Di(1-methyl-2-(1-methyl-2-carboxamido (3-proprionamidine)-4-pyrrolej-4-pyrrolyl)succinamide dihvdrochloride (Compound 51 A suspension of the previous product (130 mg, 0.18 mmol) in 15 ml anhydrous EtOH was saturated with HC1 with cooling. After 1.5 hr. at r.t., the solvent was evaporated under redueed pressure. The residue was washed with dry ether, then ethanol was added followed by some NH3 condensed into the vessel. J4fter 1 hr at r.t. the solvents were removed and the residue was washed with MeOH, ~tOH and hexane to give 116 mg of a solid. The SU~STITUT~ SHEET

21(~3u~~
wo 9zi a 3s~~ ~cricA9ziooo~~

latter vas examined by TLC (SiA2) with MeO~I and a drop o!
formic acid and indicated formation of the product (Rf 0.3) containing some more polar impurity.
Recrystalliaation from a small amount of water gave a gel-s like precipitate which was washed with EtOH and hexane and dried dive to 50 mg (34% of pure 5a, m.p. 283-5' dec.
1H-NMR (DMSO-d6): d 2.60 (m, 8H) 3.50 (m, 4H), 3.83 (s, 12H), 6.92 (d, 4H), 7.1B (d, 4H), 8.25 (t, 2H), 8.70 (bs, 4H), 9.02 (bs, 4H) 9.93 and 9.97 (2s, 4H); IR (nujol):
1352, 1377, 1464, 1521, 1576, 1638, 1700, 3260 cm -1; MS-FAB (m/~): 745 (M-C1-HCl)+. Analysis Calcd. !or C34H46C12N1406' C 49.94, H 5.67, H 23.98, C1 8.67.
Found: C 50.3, H 6.05, N 22.90, C1 8.75.
~:XAMPLE 6 (A) N,N'-Di(1-methyl-2-(1-methyl-2-carboXamido ~~~~grj,onitrile)-4 ~yrrole,~ 4-nvrrolyllmalonamide The intermediate compound (315 mg, 1 mmol), malonic acid (52 mg, 0.5 mmol) and DCC 206 mg, 1 mmol) 'were stirred in acetonitrile (6 ml) for 2 hr at roam temperature and finally the mixture was heated briefly to boiling to complete the reaction. A solid which contained dicyclohexylurea was collected and the filtrate was extracted with DMF. The DMF solution was treated with water and the solid formed was recrystallized from a mixture o! acetonitrile (2 ml) and methanol (2 ml) to give pure compound (140 mg, 40~ yield), m.p. 925-30'. 1H-NMR
(DMSO-d6): 3 2.73 (t, 2x2H), °2.40 (q+s overlapped, 2x2H+2H), 3.83 and 3.86 (2s, 2x6H), 6.91 (2d, 2x2H), 7.18 and 7.22 (2d, 2x2H), 8.35 (t, 2xlH), 9.91 (s, 2xiH), 10.09 (s, 2xlH); IR (nujol): 1200, 1264, 1290, 1376, 1401, 1464, 1511, 1532, 1585, 1638, 1662, 2250, 3120, 3305 cm°1~
MS-FAB (m/z): 697 (MH+). Analysis Calcd. for c33H36N1206' C 56.9, H 5.2, N 24.1. Found: C 56.6, H 5.4, N 23.9.
SUBSTITUTE SHEET

~'O 92113838 PCC/CA92/0005~'~=' (B) N,N'-Di(1-methyl-2-(1-methyl-2-carboxamido (7-proprionamide)-4-pyrrole]-4-pyrrolyl)malonamide c m The compound of the previous synthesis (160 mg, 0.23 5 mmol) was suspended in dry ethanol and the mixture was saturated with dry halogen chloride. After 1.5 hr at room temperature, the solvent was rer~oved under reduced pressure. The residue was treated with dry ethanol and dry ammonia. After 1 hr the solution was decanted from 10 undissolved material and evaporated to dryness. The residue was dissolved in 2 ml of boiling water and an excess of acetonitrile was added to the hot solution. The precipitate was collected and washed with a small amount of water. The operation was repeated and pure compound 4 15 was collected, 100 mg (59% yield), m.p. 218-224'. The compound, it crystallized from water, precipitates in the form of a jelly. iH-NMR (DMSO-d6): d 2.63 (t, 2x2H), °3.35 (s overlapped with the peak of water), 3.50 (q, 2x2H), 3.80 and 3.83 (2s, 2x6H), 6.93 (s, 2x2H), 7.20 (s, 20 2x2H), 8.26 (t, 2xlH), 8.90 (bs, 2x4H), 9.96 (s, 2xlH), 10.28 (s, 2xiH). D20 exchange experiment showed the presence of malonyl protons at 43.30. IR (nujol): 1260, 1377, 1505, 1463, 1535, 1580, 1645, 3100, 3270 cm-1; MS-FAB (m/t) 731 (M-Cl-HC1)+. Analysis Calcd. for 25 C33H44N1406C12' C 49.3, H S.S, N 24.4, C1 8.8. Found:
C 49.0, H 5.7, N 27.0, C1 9Ø

(A) N,N'-Di(1-methyl-2-(1-methyl-2-carboxamido 13-proprionitrilel-4-nyrrolel-4-p rolyl?urea 30 The intermediate compound (365 mg, 1.16 mmol) and 1,1'carbonyldiimidazole (94 mg, 0.58 mmol) were allowed to reaet in boiling acetonitrile (3 ml). A solid which formed was collected, washed with acetonitrile to give 350 mg (88.6% yield) of pure product, m.p. 296-7~, ig-~
SIJ~STITUTE SHEET

rv" wo ~zit3s~s ~ ~ ~ 3 ~ s ~ ~cricA9ziooos~

3.88 (s, 6H}, 6.80 (d, 2H), 6.92 (d, 2H), 7.02 (d, 2H), 7.21 (d, 2H), 8.12 (a, 2H), 8.25 (t, 2H), 9.81 (a, 2N)a IR
(nujol): 1199, 1217, 1252, 1378, 1409, 1436, 1455, 1504, 1544, 1589, 1621, 1$53, 1672, 2240, 3270, 3424 cm°1; MS-FAB (m/z): 655 (Pgi+). Analysis Calcd.: C 56.9, H 5.2, N
25.7. Found: C 56.6, H 5.4, N 25.5.
(B) N,N'-Di(1-methyl-2-(1-methyl-2-carboxaroido (3-propionamidine)-4-pyrrole~-4-pyrrolyl}urea dihvdrochloride (Compound 31 The compound synthesized in the previous step (116 mg, 0.25 mmol) was suspended in dry ethanol and the solution saturated with HC1. dlfter 2 hr the solvent was evaporated in vacuo and the residue treated with dry ammonia in ethanol for 1 hour. The mixture was decanted from an insoluble residue and the solution evaporated to dryness. The residue was dissolved in 2 ml of methanol and an excess of acetonitrile was added to precipitate the product. The latter was collected and washed with 1 ml of water when it became jelly-like. It was redfssolved in methanol and precipitated with aeetonitrile to give the compound (3) (117 mg, 61.6 yield), m.p. 211-215°. 1H-NM3t (DMSO-d6): d 2.64 (t, 4H), 3.52 (q, 4H), 3.84 (2s, 12H), 6.82 (d, 2H), 6.94 (d, 2H), 7.03 (d, 2H), 7.20 (d, 2H), 8.73 (2s averlapped, 6H), 9.05 (s, 4H), 9.88 (s, 2H); IR
(Nujol): 1264, 1377, 1402, 1439, 1489, 1531, 1583, 1640, 1689, 3088, 3279 cm-1; MS-FAB (m/z): 690 (M-C1-HC1)+.
Analysis Calcd. for C31H42C12N1405~ C 48.9. H 5.6, Cl 9.3, N25.7. Found: C 48.5, H 5.7, C1 9.7, N 25.3.
SUBSTITUTE SHEET

WO 92/13838 PCT/CA92/0005~ ~~e:':' ~'.A%' A~~_.P ~~
(A) N,N'-Oi(1-methyl-2-carboicamido(3-proprionitrile)-4-o -4- O
J~dipic acid (29.2 mg, 0.2 mmol) in acetonitrile (0.5 ml) was treated with pivaloyl chloride (50 t~L, 0.4 mmol) and Hunig's base (160 ~cL, 0.9 mmol) and then compound 7 (126 mg, 0.42 mmol) in DMF (0.5 ml) was added. Jlfter a half hour at room temperature the mixture was evaporated to dryness under reduced pressure. 'The residue was washed with water and hot acetonitrile. The solid was dissolved in hot DMF and precipitated with an excess of acetonitrile to give the compound (95 mg, 61t yield) , m.p. 244'46' dec.
1H-NMR (DMSO-d6): 6 1.60 (s, 4H), 2.27 (s, 4H), 2.74 (t, 4H), 3.40 (q, 4H), 3.83 (2s, 12H), 6.86 (s, 2H), 6.93 (s, 2H), 7.17 (s, 2H), 7.22 (s. 2H), 8.78 (t. 2H). 9.82 (s, 2H) , 9.91 (s, 2H: IR (Hugol) : 1376, 1400, 1~64, 1s13, 1333, 1s85, 1641, 22s8, 3294 cm-1; I!S-F11B (m/z) : '718 (H'') , 739 (Nx~) ; l~naiysis caicd. c se.s, H s.7, N 22.7.
Found: C s8.9, H s.9, N 22.s.
(B) t~,N'-Di(1-methyl-Z-(1-methyl-2-carboxamido(3-proprionamidina)-4-pyrrolsj-4-pyrrolyl)adipamide dihvdrochl~ride (Compound 7) The compound synthesized in the previous step (320 mg, 0.43 mmol) was treated under Pinner reaction conditions as in Example 7 above. lifter evaporation of solvents, water (3.5 ml) was added and a crystalline substance was collected to give (7) (215 mg, 58.7 yield), m.p. 195-6'. 1H-NMR (DMSO-d6): d 1.60 (s, 4H), 2,27 (s, 4H), 2.62 (t, 4H), 3.52 (q, 4H), 3.80 (2s, 12H), 6.88 (d, 2H), 6.95 (d, 2H), 7.18 and 7.20 (2d, 4H), 8.25 (t, 2H) 8.70 (s, 4H), 9.00 (s, 4H), 9.00 (s, 4H), 9.92 (s, 2H); IR
SU~ST1TUT~ S~E~T

~z~~s~~
W0 92/13338 PC'I~/CA92/04051 (Nujal): 1208, 1261, 1377, 1404. 1463, 1531, 1579, 1b.:1, 1691, 3256 cm-1; MS-FAB m/z 773 (M°NC1-C1)ø1 hnalysis Calcd.: C 51.1, M 6.0, N 23.2, C1 8.4. Found: C 50.9, H 6.2, N 23.6, C1 8.8.
S ~XAMPI,E 9 N,N'-Di(1-methyl-2-(1-methyl-2-carboxamida(3-gropionitrile)-4-,p,yrrol~e~-4-pvr-olyl malemide The intermediate compound (15a mg, 0.5 mmol) and malefic anhydride (49 mg, 0.5 mmol) were heated in acetonitrile (5 ml) at 50' for 3 minutes. Another portion of the intermediate compound (158 mg) was added and the solution was evaporated to dryness. The residual solid was dissolved in DMF (2 ml) and DCC (103 mg, 0.5 mmol) was added, and the mixture was set aside overnight at room temperature. Two drops of water were added and the solution was filtered. Then an excess of water precipitated the crude product. The product was collected and chromatographed on silica gel with chloroform and 15~
of methanol providing yellow fractions. These were combined and evaporated, and the residue recrystallized from acetone to give the product (100 mg, 56.5 yield), m.p. 250-2'. hnalytical data for this and related compounds is given in Table I.
(8) N,N'-Di(1-methyl-2-carboxamido(3-proprionamidine)-4-pyrrole)-4-pyxrolyi)maleamide dihydrochlo~ide y ompound Zg~
The product obtained in the previous step (170 mg, 0.24 mm) was treated under Pinner reaction conditions as in Example 3. The completed reaction mixture was evaporated to dryness and the residue dissolved in ethanol. Controlled addition of isopropanol provided selective precipitation of impurities. The mother liquor was evaporated and the residue was dissolved in methanol SU~51't°T~J'fE SHEET

WO 92/13838 PCT/CA92/Om05 .f'~

and precipitation with acetonitrile gave pure compound (14) (166 mg, 85% yield), m,p. 217'.

(A) N,N'-Di(1-methyl-2-(1-methyl-2-carboxamido(3 proprionitrile)-4-pyrrolej-4-pyrrolyl)trans ~vc ~prODYld~~arboxamide (Compound 8a) The synthesis and characterization of compounds 3, 4 and 5 have been reported (KroWicki, K. et al, J. Med.
Chem., Vol. 31, p. 341 (1988)). Trans-cyclopropyldicarboxylic acid (59 mg, 0.45 mmole) and 1,1'-carbonyldiimidazole (146 mg, 0.7 mmole) in acetonitrile (2.5 ml) were heated under rellux until the evolution of carbon dioxide ceased. To the cooled solution the appropriate amine (284 mg, 0.9 mmole) and 0.8 ml of DMF
were added and the mixture was stirred for 2 hr at room temperature (the product partially precipitated) and was evaporated to dryness under reduced pressure. The residue was washed with acetonitrile, aqueous R2C03 then water to give 8a, Z89 mg (88.6% yield) m.p. 312' dec.
(8) N,N'-Di(1-methyl-2-(1-methyl-2-carboxamide(3-proprionamidine)-4-pyrrolej,-4-pyrrolyl)trans-cylcopropyldicarboxamide dihydrochloride jComrsound Bbl Compound 8a (216 mg, 0.7 mmole) was treated under Dinner reaction conditions as described previously. The linal reaction mixture was evaporated to dryness and the residue was extracted with hot propanol (150 ml). The extract was evaporated to dryness and the residue dissolved in methanol l ml, and an excess of acetonitrile was added to precipitate the produet 8b, 170 mg (68.5%
yield) m.p. 210' (softens).
SIIBST'ITIJ~'E S~EET' 2103~~5 . : wo ~zi~zs~~ ~cricA9zio~osy ~XAMP;~F 11 Commercially available acid chlorides for the linker groups were used directly without further purification.
otherwise, the appropriate acid chlorides were prepared 5 from the acids according to the following procedure: An acid and a drop of dimethylformamide was heated in thionyl chloride (5 to 10 mole in excess) to 55-65'C for 30 to 45 min until a homogeneous liquid was obtained. The excess of the chlorinating agent was removed by evaporation. A
10 small amount of methylene chlcride was added to the crude acid chloride then evaporated. The diacid dichloride was then dissolved in methylene chloride or THF and aliquots were taken and used fox coupling reactions.
F~X~ 12 15 Distamycin A (50 mg, 0.09 mmol) was dissolved in 4 mL
of methanol. To this yellow solution was added 100 ~L of concentrated hydrochloric acid. The solution was stirred for 6-8 h and the reaction progress was followed by TLC
(methanol: acetic acid, 100:5). Ths solvent was evaporated 20 and' the crude product was redissolved in methanol and precipitated with ether. The product was secrystallized in this way twice more. The supernatant was decanted and the residual solid was dried~in vacuo. The final product was obtained as an otf-white solid 50 mg (89~ yield).
2 s ExpriPLE 13 Bis-distamycin (Compound 15) A solution of succinyl dicarbonyl dichloride (9.28 mg, 0.046 moral) in 5 mL of tetrahydrofuran was added to a solution of deformyl distamycin (48 mg, 0.09 mmol) and 30. dissiopropylethylamine (Hunig's base, 16 uL, 0.09 mmol) in 3 mL of dimethylformamide cooled to 0'C. After 10 min, a solution of Hunig's base (16 pL, 0.09 mmol) in 3 mL of THF
was added to the reaction solution. The resulting mixture was stirred overnight. The solvent was evaporated and the 35 crude product was recrystallized from methanol and ethe:.
SIJRST~TIJTE SHEET

w0 32/t3838 PCT/CA92/OOOSL.
' ~ 36 The final product was obtained as a light yellow solid in 68~ yield. m.p. 210'C; 1H-Mgt, 2.48 (COCH2CH2C0, 4H, s), 2.56 (2xCg2C(NH2)2C1, 4H, tr, J = 6 Hz), 7.50 (2xC0NHCji2, 4H, q, J = 6 Hz), 3.80 (2xNCH3, 6H, S), 3.82 (2xNCH3, 6H, s). 3.83 (2xNCH3, 6H, s), 6.90 (2xpy-CH, 2H, d, J = 2 Hz), 6.94 (2xpy-CH, 2H, d, J = 2 Hz), 7.04 (2xpy-CH, 2H, d, J
= 2 Hz), 7.14 (2xpy-CH, ZH, D, J = 2 Hz), 7.18 (2xpy-CH, 2H, d, J = 2 Hz), 7.22 (2xpy-CH, 2H, d, J = 2 Hz), 8.24 (2xCONf~CH2, 2H, tr, J = 6 Hz), 8.74 [2xC(NH2)2C1, 4H, s), 9.04 (2xC(NH2)2C1, 4H, S), 9.93 (5xpy-NHCO, 5H, s), 9.96 (py-NHCO, 1H, s); MS (FAB), 989 (M-2xCl-H, 0.34).

Bis-distamycin (Co~apound 16) A solution of hexan-1,6-dicarbonyl dichloride (9,28 mg, 0.046 mmol) in 5 mL of tetrahydroturan was added to a solution of detormyl distamycin (48 mg, 0.09 mmol) and dissiopropylethylamine (Hunig~s bass, 16 ~L, 0.09 mmol) in 3 mL of dimethyllormamide cooled to 0'C. litter 10 min, a solution of Hunig~s base (16 ~cL, 0.09 mmol) in 3 mL of THF was added to the reaction solution. The resulting mixture was stirred overnight. The solvent was evaporated and the crude product was recrystallized from methanol and ether. The final product was obtained as a light yellow solid in 78~ yield, m.p., 210'C; iH-NMR, 1.28 (4,5-suber-CH2, 4H, m), 1.57 (3,6-suber-CH2, 4H, m), 2.23 (2m7-suber-CH2, 4H, tr, J = 7 Hz), 2.63 (2xCg2C(NH2)2C1, 4H, tr, J =
6 Hz), 3.49 (2xCONHCF~2, 4H, m), 3.80 (2xNCH3, 6H, s), 3.81 (2xNCH3, 6H, s), 3.83 (2xNCH3, 6H, s), 6.88 (2xpy-CH, 2H, d, J = 2Hz), 6.94 (2xpy-CH, 2H, d, J = 2Hz), 7.05 (2xpy-cH, 2H, d, J = 2 Hz) , 7.15 (2xpy-CH, 2H, d, J = 2 Hz) , 7.18 (2xpy-CH, 2H, d, J = 2 Hz), 7.23 (2xpy-CH, 2H, d, J
= 2 Hz), 8.25 (2xC0NgCH2, 2H, m), 8.72 (2xC(NH2)2C1, 4H, s), 9.03 (2xC(NH2)2C1, 4H, s), 9.86 (2xpy-NHCO, 2H, s), 9.92 (4xpy-NHCO, 4H, s); MS (FAB), 1045 (M-2xC1-H, 0.38).
SUBSTITUTE S~EFT

2 ~. 0 3 6 6 5 p~/c~~~~'~~s' ,7 Bis-distamycin (compound 17) A solution of octan-1,8-dicarbonyl dichloride (9.28 mg, 0.046 mmol) in 5 mL of tetrahydrofuran was added to a solution of deformyl distamycin (48 mg, 0.09 mmol) and dissiopropylethylamine (Hunig's base, 16 ~L, 0.09 moral) in 3 mL of dimethylformamide cooled to 0'C. After 10 min. a solution of Hunig's base (16 ~L, 0.09 mmol) in 3 mL of THF
was added to the reaction solution. The resulting mixture was stirred overnight. The solvent was evaporated and the crude product was recrystallized from methanol and ether.
The final product was obtained as a light yellow solid in 65~ yield. m.p., 198-202'C; iH-Nt~, 1.26 ((4,3,6,'7-seba-CH2, SH, m), 4H, tr, J ~ 6 Hzj, 1.55 ((3,8-seba-CH2), 4H, mj, 2.22 (2,9-seba-CH2), 4H, tr, J = 8 H2j, 2.61 (2xCji2C(NH2)2C1, tr, J = ~ Hzj, 3.48 (2xCOMHCji2, dH, m), 3.80 (2xNCH3, 6H, s), 3.81 (2xNCH3, 6H, s), 3.83 (2xPJCH3, 6H, s), 6.89 (2xpy-CH, 2H, d, J = 2Hz), 6.95 (2xph-CH, ZH, d, J = 2Hz), 7.05 (2xpy-CH, 2H, d, J = 2 Hz), 7.15 (2xpy-CH, 2H, d, J ' 2 Hz), 7.18 (2xpy-CH, 2H, d, J ~ 2 Hz), 7.22 (2xpy-CH, 2H, d, J ~ 2 HZ), 8.23 (2xCONHpy-2H, m), 8.65 (2xC(NH2)2C1, 4H, sj, 8.99 (2xC(NH2)2C1, 4H, sj, 9.82 (2xpy-NHCO, 2H, s), 9.91 (4xpy-NtiCO, 4H, s): MS (FAB), 104 (m~-2xC1-H, 0.08) .
EXAMPLE
His-distamycin (Compound 18) A solution of docosane-1,22-dicarbonyl dichloride (9.28 mg, 0.046 mmol) in 5 mL of tetrahydrofuran was added to a solution of detormyl distamycin (48 mg, O.OQ mmol) and dissiopropylethylamine (Hunig's base, 16 uL, 0.09 mmol) in 3 mL of dimethylformamide eooled to 0'C. After 10 min, a solution of Hunig's base (16 uL, 0.09 mmol) in 3 mL of THF was added to the reaction solution. The resulting mixture was stirred overnight. The solvent was evaporated and the crude product was recrystallited from methanol and ether. The final product was obtained as a SU~ST1T~.JTE Si~EE'T

w0 92/13838 PCT/CA92/0005:

light yellow solid in 73~ yield. m.p., 215°C; 1H-NMR, 1.23 (4,5,...20,21-tetraco-CH2, 36H, s), 1.55 (3,22-tetraco-CH2, 4H, m), 2.21 (2,23-tetracv-CH2, 4H, tr, J
7 Hz), 2.62 (2xCg2C(NH2)2C1, 4H, tr, J = 6 HzJ, 3.50 (2xCONHCt32, 4H, tr, J = 6 Hz), 3.80 (2xNCH3, 6H, s), 3.82 (2xNCH3, 6H, s), 3.84 (2xNCH3, 6H, s), 6.89 (2xpy-CH, 2H, d, J = 2Hz), 6.94 (2xpy-CH, 2H, d, J = 2 Hz), 7.05 (2xpy-CH, 2H, d, J = 2 Hz) , 7. 15 (2xpy-CH, 2H, d, J' a 2 Hz) , 7.19 (2xpy-CH, 2H, d, J = 2 Hz), 7.23 (2xpy-CH, 2H, d, J
= 2 Hz), 8.25 (2xCON~CH2, 4H, tr, J = 6 Hz), 8.72 (2xC(NHZ)ZCl, 4H, sJ, 9.02 (2xC(MH2)2C1, 4H, sJ, 9.83 (2xpy-NHCO, 2H, s), 9.92 (4xpy-NHCO, 4H, s): MS (FAB), 1270 (~-2xcl-H, o.lo).

Bis-distamycin (Compound Z9) A solution of benzene-1,4-dicarbonyl dichloride (9.28 mg, 0.046 mmol) in 5 a~L of tatrahydrofuran vas added to a solution of deformyl distamycin (48 mg, 0.09 mmol) and dissiopropylethylamine (Hunig's base, 16 ~L, 0.09 mmol) in 3 mL of dimethylformamide cooled to 0'C. J~fter 10 min, a solution of Hunig's base (16 uL, 0.09 mmol) in 7 mL of THF
was added to the reaction solution. The resulting mixture was stirred overnight. The solvent was evaporated and the crude product was~recrystallized from methanol and ether.
The final product was obtained as a light yellow solid in 77t yield, m.p., >300'C: 1H-NI~t, 2.63 (2xCg2C(NH2)2C1, 4H, tr, J = 6 HzJ, 3.50 (2xCONHCF~2, 4H,. tr, J = 6 Hz), 3.82 (2xNCH3, 6H, s), 3.86 (2xNCH3, 6H, s), 3.90 (2xNCH3, 6H, s),,6.97 (2xpy-CH, 2H, d, J = 1.6 Hz), 7.09 (2xpy-CH, 2H, d, J = 1.6 Hz), 7.15 (2xpy-CH, 2H, d, J = 1.6 Hz), 7.20 (2xpy-CH, 2H, d, J m 1.6 Hz), 7.26 (2xpy-CH, 2H, d, J = 1.6 Hz), 7.38 (2xpy-CH, 2H, d, J = 1.6 Ht), 8.10 (aromatic-CH, 4H, s), 8.25 (2xCON~iCH2, 2H, tr, J = 6 Hz), 8.65 (2xC(NH2)2C1, 4H, sJ, 9.01 (2xC(NH2)2C1, 4H, sJ, 9.95 (2xpy-NHCO, 2H, s), 10.03 (2xpy-NHCO, 2H, s), 10.57 (2xpy-NHCO, 2H, s)o' (CD30D), 2.71 (2XCH_2C(NH2)2C1, 4H, tr, J
SUBSTITUTE ~~IEET

~':v WO 92/13838 210 3 6 6 5 PCf/CA92/001051 .. <

7 HzJ, 3.65 (2xCONHCg2, 4H, tr, J ~~ 7 Hz), 3.87 (2xNCH3, 6H, s), 3.91 (2xNCH3, 6H, s), 3.95 (2xNCH3, 6H, s), 6.90 (2xph-CH, 2H, d, J = 1.8 HZ) , 6.98 (2xpy-CH, 2H, d, J =
1.8 Hz), 7.07 (2xpy-CH, 2H, d, J ~ 1.8 Hz), 7.16 (2xpy-CH, 2H, d, J = 1.8 Hz), 7.20 (2xpy-CH, 2H, d, J = 1.8 Hz), 7.34 (2xpy-CH, 2H, d, J = 1.8 Hz), 8.04 (aromatic-CH, 4H, s); MS (FA8), 1037 (M-2xC1-H, 0.05).
~XAMPL~ 18 His-distamycin (Compound 30) l0 A solution og benzene-1,3-dicarbonyl dichloride (9.28 mg, 0.046 mmolj in 5 mL o! tetrahydroturan was added to a solution of defonayl distamycin (48 mg, 0.09 mmol) and dissiopropylethylamine (Hunig's base, 16 ~aL, 0.09 mmol) in 3 mL of dimethylformamide cooled to 0'C. d~tter 10 m.in, a solution of Hunig's base (16 ~L, 0.09 mmol) in 3 mL of THF
was added to the reaction solution. Tha resulting mixture was stirred overnight. Ths solvent was evaporated and the crude product was recrystallized troa methanol and ether.
The final product was obtained as a light yellow solid in 68~ yield, m.p. , 240'Co 1H-Nt~t, 2.61 [2xCg2C(NH2) 2C1, 4H, tr, J = 6 Hz), 3.48 (2xGONHCg2, 4H, tr, J = 6 Hz), 3.80 (2xNCH3, 6H, s), 7.86 (2xHCH3, 6H, s), 3.91 (2xNCH3, 6H, s), 6.97 (2xpy-CH, 2H, d, J = 1.6 Hzj, 7.09 (2xpy-CH, 2H, d, J = 1.6 Hz), 7.16 (2xpy-CH, 2H, d, J = 1.6 Hz), 7.20 (2xpy-CH, 2H, d, J = 1.b Ht), 7.25 (2xpy-CH, 2H, d, J =
1.6 Hz), 7.38 (2xpy-CH, 2H, d, J = 1.6 Hzj, 7.66 (5-aromatic-CH, 1H, tr, J = 7.5 Hz), 8.10 (4,6-aromatic -CH, 2H, d, J1 = 8 Hz)t 8.21 (2-aronatic-CH, 1H, br, s): 8.21 (2xCONhCH2, 2H, br, s), 8.58 [2xCy2C(NH2)2C1, 4H, tr, J =
7 Hzj, 3.64 (2xCONHCg2, 4H, tr, J = 7 Hz), 3.88 (2xNCH3, 6H, s), 3.90 (2xNCH3, 6H, s), 3.94 (2xNCH3, 6H, s), 6.89 (2xpy-CH, 2H, d, J = 1.8 Hz), 6.97 (2xph-CH, 2H, d, J s 1.8 H2), x.07 (2xpy-cH, 2H, d, J = 1.a x2), x.20 (2xpy-cH, 2H, d, J = 1.8 Hz), 7.33 (2xpy-CH, 2H, d, J = 1.8 Hz), SU~~~'iT'UT~ SHEET.

WO 91/13838 PC1 /CA92/0005~T~, J', 7.65 (5-aromatic-CH, 1H, tr, J = 7.5 Hz), 8.08 (4,6-aromatic-CH, 2H, d,d, J1 = 7.5 Hz, Ja = 2 Hz), 8.47 (2-aromatic-CH, 1H, br, tr, J = 2 Hz); riS (FAH), 1077 (-2xC1-H, 0.43).
5 ~~pT~,E 1~
His-distamycin (Compound 31) A solution of benzene-1,2°dicarbonyl diehloride (9.28 mg, 0.046 mmol) in 5 mL of tetrahydrofuran was added to a solution of deformyl distamycin (48 mg, 0.09 mmol) and l0 dissiopropylethylamine (Hunig's base, 16 ~L, 0.09 mmol) in 3 mL of dimethyltormamide cooled to 0'C. After 10 min, m solution of Hunfg's base (16 ~L, 0.09 mmol) in 3 mL of THF
was added to the reaction solution. The resulting mixture was stirred overnight. The solvent was evaporated and the 15 crude product was recrystallized from methanol and ether.
The final product was obtained as a light yellow solid in 83~ yield. m.p., 245'Ct 1H-Nl~t (CD3pD), 2.71 (2xCgaC(NH2)2C1, 4H, tr,.J - 6 Hzj, 3.53 (3xCONHCg2, 4H, tr, J = 6 Hz), 3.87 (2xNCH3, 6H, s), 3.88 (2xNCH3, 6H, s), 20 3.90 (2xNCH3, 6H, s) , 6.89 (2xpy-CH, ~H, d, J = 2 Hz) , 6.91 (2xpy-CH, 2H, d, J = 2 Hz), 6.97 (2xpy-CH, 2H, d, J
= 2 Hz), 7.15 (2xpy-CH, 2H, d, J = 2 Hz), 7.18 (2xpy-CH, 2H, d, J = 2 Hz), 7.24 (2xpy-CH, 2H, d, J = 2 Hz), ?.60 (2xm-aromatic-CH, 2H, q, J = 3 Hz) , 7. 68 (2xo-aromatic-CH, 25 aH, q, J = 3 Hz): MS (FAH), 103? (m-2xC1-H, 0.69).
EXAIdPLE 20 ais-distamycin (Compound 32) A solution of 3,5-pyridine dicarbonyl dichloride (9.28 mg, 0.046 mmolj in 5 mL of tetrahydrofuran was added 30 to a solution of deformyl distamycin (48 mg, 0.09 mr~ol) and dissiopropylethylamine (Hunig's base, 16 ~cL, 0.09 mmol) in 3 mL of dimethylformamide cooled to 0'C. After 10 min, a solution of Hunig°s base (16 ~L, 0.09 mmol) in 7 mL of THF ryas added to the reaction solution. The 35 resulting mixture was stirred overnight. The solvent vac Sl»S~'tT~°~ ~ S~E~T

i~'O 92i 13838 ... ~ ~ ~ 3 ~ ~ ~ ~cr~cA9ze~oosa al evaporated and the crude product was recrystallized Prom methanol and ether. The final product was obtained as a light yellow solid m.p. 2S0'C in 88t yield. m.p., 250'C;
1H-NMR, 2.52 (2xC1(2C(NH2)2C1, 4H, m], 3.48 (2xCONHC$2, 4H, m), 3.81 (2xNCH3, 6H, s), 3.85 (2xNCH), 6H, s), 3.88 (2xNCH3, 6H, s), 7.90 (2xNCH~, 6H, s), 6.96 (2xpy-CH, 2H, m), 7.09 (2xpy-CH, 2H, d, J = 2 Hz), 7.17 (py-CH, 1FI, d, J = 2 Hz), 7.19 (2xpy-CH, 2H, d, J = 2 Hz), 7.25 (2xpy-CH, 2H, d, J = 2 HZ), 7.29 (py-CH, 1H, m), 7.40 (py-CH, 1H, m), 7.42 (py-CH, 1H, m), 8.23 (2xCON~CH2, 2H, m), 8.25 (3-py-CH, 1H, d, J = 8 Hx), 8, 54 (4-py-CH, 1H, m), 8.64 [2xC(NH2)2C1, 4H, s], 8.99 [2xC(NH2)2C1, 4H, s], 9.20 (96-py-CH, 1H, m), 9.95 (2xpy-N'HCO, 2H, s), 10.04 (2xpy-NHCO, 2H, s), 10.94 (py-NHCO, 1H, s), 11.00 (py-NHCO, 1H, s);
(CD30D), 2.72 (2xCg2C(NH2)2C1, 4H, tr, J = 6 H2], 3.65 (2xCONHCg2, 4H, tr, J = 6 Hz), 3.87 (2xNCH3, 6H, s), 3.91 (2XNCH3, 6H, s), 3.94 (NCH3, 3H, s), 3.954 (NCH3, 3H, s), 6.90 (2xpy-CH, 2H, d, J = 2 Hz), 6.98 (2xpy-CH, 2H, d, J
= 2 Hz), 7.07 (py-CH, 1H, d, J = 2 Hz), 7.10 (py-CH, 1H, d, J = 2 Hz), 7.15 (2xpy-CH, 2H, d, J = 2 Hz), 7.20 (2xpy-CH, 2H, d, J = 2 Hz), 7.34 (py-CH, 1H, d, J = 2 Hz), 7.41 (py-CH, 1H, d, J = 2 Hz), 8.27 (3-py-CH, 1H, d, J = 8 Hz), 8.44 (4-py-CH, 1H, m), 9.17 (6-py-CH, 1H, m): MS (FAB), 1038 (M-2xC1-H, 0.03).
as ~xAM~LE 2~
Bis-distampcin (Compound 33) A solution of pyridine-3,6-dicarbonyl dichloride (9.28 mg, 0.046 mmol) in 5 mL of tetrahydrofuran was added to a solution of deformyl distamycin (48 mg, 0.09 mmol) and dissiopropylethylamine (Hunig's base, 16 uL, 0.09 mmol) in 3 mL of dimethylformamide cooled to 0'C. After 10 min, a solution of Hunig's base (16 ~cL, 0.09 mmol) in 3 mL of THF was added to the reaction solution. The resulting mixture was stirred overnight. The solvent was evaporated and the crude product was recrystallized from methanol and ether. The final product was obtained as a aUIBSTITUTE SHEET

21Q366~
WO 92/ 13838 PCT/CA92/0005 ...'~:"'~

light yellow solid in 74% yield. m.p., 260'C: 1H-NMR, 2.62 (2xCH2C(HH2)2C1, 4H, tr, J = 6Hz), 3.50 (2xCONHCH2, 4H, q, J n 6Nz), 3.81 (2xNCH3, 6H, s), 3.85 (2xNCH3, 6H, s), 3.90 (2xNCH3, 6H, s), 6.96 (2xpy-CH, 2H, d, J ~~ 2 Hz), 7.08 (2xpY-CH 2H, d, J ~ 2 Hz, 7:16 (2xpy-CH, 2H, d, J =
2 Hz), 7.18 (2xpy-CH, 2H, d, J.~~ 2 Hz), 7.26 (2xpy-CH, 2H, d~ J ~ 2 Hz), 7.39 (2xpy-CH, 2H, d, J ~ 2 Hz), 8.23 (2xCONHCH2, 2H, tr, J = 6 Hz), 8.59 (2xC(NH2)2C1, 4H, sj, 8.87 (4-py-CH, 1H, br,s), 8.98 (2xC(NH2)2C1, 4H, sJ, 9.24 (2,6-py-CH, 2H, d, J = 2 Hz), 9.94 (2xpY-NHCO, 2H, s), 10.05 (2xpy-NHCO, 2H, s), 10.83 (2xpy-NHCO, 2H, s):
(CD30D), 2.71 (2xCH2C(NH2)2C1, 4H, tr, J ~ 6 Hz], 3.64 (2xCONHCH2, 4H, tr, J ~ 6 Hz), 3.87 (2xNCH3, 6H, s), 3.99 (2xNCH3, 6H, s), 4.02 (2xNCH3, 6H, s), b.88 (2xpy-CH, 2H, s), 6.96 (2xpy-CH, s),.7.07 (2xpy-CH, 2H, s), 7.15 (2xpy-CH, 2H, s), 7.19 (2xpy-CH, 2H, s), 7.75 (2xpy-CH, 2H, s), 8.82 (4-py-CH, 1H, s), 9.17 (2,5-py-CH, 2H, s): MS (FA8), (M-2xCL-H, 0.15).
EXAM
Bis-distamycin tCompound 34) R, solution o! pyridine-2,6-dicarbonyl dichloride (9.28 mg, 0.046 mmol) in 5 mL oI tetrahydroturan was added to a solution of delormyl distamycin (48 mg, 0.09 mmol) and dissiopropylethylamine (Hunig's base, 16 ~L, 0.09 mmol) in 3 mL of dimethyltormamide cooled to 0'C. Rlter 10 min, a solution of Hunig's base (16 ~cL, 0.09 mmol) in 3 mL of THF was added to the reaction solution. The resulting mixture was stirred overnight. The solvent was evaporated and the crude product was recrystallized from methanol and ether. The final product was obtained as a light yellow solid in 54% yield. m.p., >260°C; 1H-NMR, 2.62 (2xCH2(NH2)2C1, 4H, tr, J = 6 HzJ, 3.50 (2xCONHCH2, 4H, m), 3.82 (2xNCH3, 6H; s), 3.86 (2xNCH3, 6H, s), 3.90 (2xNCH3, 6H, s), 6.97 (2xpy-CH, 2H, d, J = 2 Hz), 7.08 (2xpy-CH, 2H, d, J = 2 Hz), 7.15 (2xpy-CH, 2H, d, J = 2 Hz), 7.18 (2xpy-CH, 2H, d, J = 2 Hz), 7.25 (2xpy-CH, 2H, WO 92113838 2 ~. D 3 6 6 5 ~~/CA92/00051 d, J = 2 Hz), 7.39 (2xpy-CH, 2H, d, J = 2 Hz), 8.23 (2xCONHCH2, 2H, tr, J ' 6 Hz, 8.56 (2xC(NH2)2C1, 4H, sJ, 8.85 (4°py-CH, 1H, tr, J = 2 Hz), 8.96 (2xC(NH2)2C1, 4H, s), 9.24 (3,5-py-CH,~2H, d, J = 2 Hz), 9.94 (2xpy-NHCO, 2H, s), 10.04 (2xpy-NHCO, 2H, s), 10.81 (2xpy-NHCO, 2H, s); MS (F'A9), 1038 (M-2xCl-H, 0.25).

His-distamycin (Compound 35) A solution of rans-1,2-cyclobutane-dicarbonyl dichloride (9.28 mg, 0.046 mmol) in 5 mL of tetrahydrofuran was added to a solution of deformyl distamycin (48 mg, 0.09 mmol) and dissiopropylethylamine (Hunig's base, 16 ~L, 0.09 mmol) in 3 mL of dimethyllormamide cooled to 0'C. Alter 10 min, a solution 0! Hunig's base (16 ~cL, 0.09 mmol) in 3 mL of THF was added to the reaction solution. The resulting mixture was stirred overnight. The solvent was evaporated and the crude product was recrystallized from methanol and ether.
The linal product was obtained as a light yellow solid in 78~ yield. m.p., >230'C; 1H-NMR, 2.05 (3,4-cyclobutane-CH2, 4H, m), 2.60 (2xCH2C(NH2)2C1, 4H, tr, J = 6 Hz], 3.38 (1,2-cyclobutane-CH, 2H, m), 3.49 (2xCONHCH2, 4H, tr, J =
6 Hz), 3.79 (2xNCH3, 6H, s), 3.84 (2xNCH3, 6H, s), 3.85 (2xNCH3, 6H, s), 6.88 (2xpy-CH, 2H, d, J = 1.8 Hz), 6.97 (2xpy-CH, 2H, d, J = 1.8 Hz), 7.05 (2xpy-CH, 2H, d, J =
1.8 Hz), 7.17 (2xpy-CH, 2H, d, J = 1.8 HZ), 7.21 (2xpy-CH, 2H, d, J = 1.8 Hz) , 7.23 (2xpy-CH, 2H, d, J = 1.8 Hz) , 8.22 (2xCONHCH2, 2H, tr, J = 6 Hz),~8.55 (2xc(NH2)2C1, 4H, s), 8.96 (2xC(NH2)2C1, 4H, sJ, 9.88 (2xpy-NHCO, 2H, s), 9.94 (4xpy-NHCO, 2H, s); (CD30D), 2.20 (3,4-cyclobutane-CH2, 4H, m), 2.71 (2XCH2, 4H, (r, J = 7 HZ), 3.49. (1,2-cyclobutane-CH, 2H, m), 3.64 (2xCH2C(NH2)2C1, 4H, tr, J =
7 Hz), 3.87 (2xNCH3, 6H, s), 3.89 (2xNCH3, 6H, s), 3.90 (2xNCH3, 6H, s), 6.84 (2xpy-CH, 2H, d, J = 2 Hz), 6.89 SUBSTITUTE SHEET

~1~3~~5 WO 92/13838 FCT/CA92/U(DOS~~;~

(2xpy-CH, 2H, d, J = 2 Hz) , 6.95 (2xpy-CH, 2H, d, J = 2 Hz), 7.15 (2xpy-CH, 2H, d, J~= 2 Hz), 7.18 (2xpy-CH, 2H, d, J = 2 Hz), 7.20 (2xpy-CH, 2H, d, J = 2 Ht); MS (FAS), , 1015 (M-2xC1-H, 1.06) . , FXAMPL~ 24 His-distamycia (Compound 36) h solution of maleic-dichloride (9.28 mg, 0.046 m~~nol) in 5 mL of tetrahydrofuran was added to a solution of deformyl distamycin (48 mg, 0.09 mmol) and dissiopropylethylamine (Hunig's base, 16 ~L, 0.09 mmol) in 3 mL of dimethylformamide cooled to 0'C. After 10 min, a solution of Hunig's base (16 ~ L, 0.09 mmol) in 3 mL of THF
was added to the reaction solution. The resulting mixture was stirred overnight. The solvent was evaporated and the crude product was recrystallized from methanol and ether.
The final product vas obtained as a light yellow solid in 33~ yield. m.p., >255'C: 1H-NMR, 2.61 (2xCH2C(NH2)2C1, 4H, tr, J =6 Ht,, 3.50 (2xCONHCH2, 4H, q, J = 6 Hz), 3.82 (2xNCH3, 6H, s), 3.85 (2xNCH3 6H, s), 3.87 (2xNCH3 6H, x),6.97 (2xpy-CH, 2H, tr, J = 2 Hz), 7.07 (2xpy-CH, 2H, d, J = 2 HZ), 7.10 (-CH=CH-, 2H, S)~, 7.18 (2xpy-CH, 2H, s), 7.24 (2xpy-CH, 2H, d, J = 2 Hz), 7.35 (2xpy-CH, 2H, d, J
2 Hz), 8.23 (2xC0NHCH2, 2H, tr, J ~ 6 Hz), 8.66 (zxc(Nx2)2c1, cx, s~, s.94 (2xc(rtH2)2e1, 4H, s,, 9.93 (2xpyNHCO, 2H, s), 9.99 (2xpy-NHCO, 2H, s), 10.54 (2xpy NHCO, 2H, s) " (CD30D), 2.72 (2xCH2C(NH2)2C1, 4H, tr, J =
6 Hz), 3.65 (2xCONHCH2, dH, tr, J = 6 Hz), 3.88 (2xNCH3, 6H, s), 3.90 (2xNCH3, 6H, s), 3.92 (2xNCH3, 6H, s), 6.91 (2xpy-CH, 2H, tr, J a 2 Ht), 6.98 (2xpy-CH, 2H, d, J ~~ 2 Hz), 7.09 (-CH=CH-, 2H, s), 7.16 (2xpy-CH, 2H, d, J = Z
Hz), 7.19 (2xpy-CH, 2H, d, J = 2 Hz), 7.37 (2xpy-CH, 2H, d, J = 2 Hz); MS (FAS), 987 (M-2xC1-H, 0.27).
SUBSTITUTE SHEET

2 ~ 0 3 s 6 5 PCT/CA92/00051 llis-distamycin (Compound 37) A solution of fumaroyl-dichloride (9.28 mg, 0.046 mmol) in 5 mL of tetrahydrofuran was added to a solution 5 of deformyl distamycin (48 mg, 0.09 mmolj and dissiopropylethylamine (Hunig's base, 16 ~sL, 0.09 mmol) in 3 mL of dimethylformamide cooled to 0'C. After 10 min, a solution of Hunig's base (16 ~L, 0.09 mmol) in 3 mL of THF
was added to the reaction solution. The resulting mixture 10 was stirred overnight. The solvent evaporated and the crude product was obtained as a light yellow solid in 67~
yield. m.p., >280'C; 1H-Nt~t, 2.61 (2xCH2C(NH2)2Ci, 4H, tr, J = 6 HzJ, 3.48 (2xCONHCH2, 4H, tr, J = 6 Hzj; 3.80 (2xNCH3, 6H, s), 3.84 (2xNCH3, 6H, s), 3.86 (2xNCH3, 6H, 15 s). 6.35 (-CH=CH-, 2H, s), 6.84-7.84 (l2xpy-CH, 12H, m), 8.2d (2xCONHCH2, 2H, tr, J s 6 Hz), 8.58-9.50 (2xC(NH2)2C1, 8H, br, sj, 9.93 (2xpy-NHCO, 2H, s), 9.97 (2xpy-MiCO, 2H, S), 9.98 (2Xpy-NHCO, 2H, S); (CD30D), 2.66 (2xCH2C(NJHZ)2C1, 4H, tr, J = 6 Hz), 3.58 (2xCONHCH2, 4H, 20 tr, J ~ 6 Hz) , 3.79 (2xNCH3, 6H, s) , 3.82 (2xNCH3, 6H, s) , 3.84 (2xNCH3, 6H, s), 6.26 (-CH=CH-, 2H, s), 6.83 (2xpy CH, 2H, d, J = 2 Ht) , 6.87 (2xpy-CH, 2H, d, J = 2 Hz) , 6.91 (2xpy-CH, d, J = 2 Hzj, 7.13 (2xpy-CH, 2H, d, J ~ 2 Hz), 7.17 (2xpy-CH, 2H, d, J = 2 Hz), 7.27 (2xpy-CH, 2H, 25 d, J = 2 Hz); MS (FA8), no M+1 peak.
~?CAMPLE 26 His-distamycin 35 (Compound 38) A solution of trans-5,6-bicyclo(2,2,1]-hept-2-ene dicarbonyl dichloride (9.28 mg, 0:046 mmol) in 5 mL op 30 tetrahydrofuran was added to a solution of deformyl di'stamycin (48 mg, 0.09 mmol) and dissiopropylethylamine (Hunig's base, 16 ~L, 0.09 mnol) in 3 mL of dimethylformamide cooled to 0'C. After 10 min, a solution' of Hunig's base (16 ~L, 0.09 mmol) in 3 mL of THF was 35 added to the reaction solution. The resulting mixture was stirred overnight. The solvent was evaporated and the ~~~~~s~
13838 P~CT/CA9210005(''~;
4s crude product was recrystallized lrom methanol and ether.
The linal product was obtained as a-light yellow solid in 53~ yield. m.p., 260°C: iH-NMR,,1.31 (7-bicyclohept, 1H, s), 1.86 (7-bicyclohept, 1H, d,vJ = 7 Hz), 2.76 (5-endo-bicyclohept, 1H, d, J = 8 Hz), 2.93 (4-bicyclohept, 1H, s), 3.35 (1-bicyclohept, 1H, s), 3.50 (6-exo-cyclohept, 1H, s), 3.50 (2xCH2C(NH2)2C1, 4H, mJ, 3.81 (3xNCH3, 9H, s), 3.85 (3xNCH3, 9H, s), 5.98 (3-bicyclohept, 1H, d,d, J
= 2.5 Hz), 5,30 (2-bicyclohept, 1H, d,d, J = 2.5 Hz), 6.86 (py-CH, 1H, d, J = 2 Hz), 6.91 (py-CH, 1H, d, J = 2 Hz), 6.97 (2xpy-CH, 2H, d, J = 2 Hz), 7.06 (2xpy-CH, 2H, d, J
= 2 Hz), 7.13 (py-CH, 1H, d, J = 2 Hz), 7.18 (2xpy-CH, 2H, d, J = 2 Hz), 7.19 (py-CH, 1H, d, J = 2 Hz), 7.23 (2xpy-CH, 2H, tr, J = 2 Hz), 8.24 (2xCONHCH2, 2H, m), 8.57 is t2xc(NH2)2c1, 4H, m~ a.97 t23xc(NH2)2c1, 4x, m~, 9.88 (py-NHCO, 1H, m), 9.92 (4xpy-CH, 4H, m), 10.11 (Ipy-NHCO, 1H, m): (CD30D), 1.47 (7-bfcyclohept, 1H, d, J = 8 Hz), 1.94 (7-bicyclchept, 1H, d, J - 8 Hz) , 2.71 (2xcH2c(r~H2) 2c1, 4H, d, J = 6 Hz), 2.77 (5-endo-bicyclohept, 1H, d, J = 4 Hz), 7.04 (4-bicyclohept, 1H, s), 5.47 (6-sxo-bicyelohept, 1H,), 3.64 (2xCONHCH2, 4H, tr, J ~ 6 Hz), 3.87 (2xNCH3, 9H, s), 3.89 (NCH3, 3H, s), 3.90 (2xHCH3, 6H, s), 6.08 (3-bicyclohept, 1H, d, J = 2.5 Hz), 6.37 (2-bicyclohept, 1H, d, J = 2.5 Hz), 6.82 (py-CH, 1H, d, J = 2 Hz), 6.83 (py-CH, 1H, d, J = 2 Hz), 6.89 (2xpy-CH, 2H, d, J = 2 Hz), 6.95 (2xpy-CH, 2H, d, J = 2 Hz), 7.11 (py-CH, 1H, d, J =
2 Hz), 7.14 (2xpy-CH, 2H, d, J = 2 Hz), 7.17 (3xpy-CH, 3H, tr, J = 2 Hz); MS (FAB), 1053 (M-2xC1-H, 0.21).
SUESSTITUTE SHEET

N~'O 92/ 13R3H 2 ~ 0 ~ G 6 5 ~~~~A~2'°~'~' 4?
~,XAMPLE 27 His-Lexitropsin (Compound 39) A solution of malefic-dichloride (9.28 mg, 0.046 mmol) in 5 mL of tetrahydrofuran was added to a solution of 3 (1-methyl-4-(4-amino-1-methylimidazole-2 earboxamido)imidazole-2-earboxamidojpropionamidine hydrochloride (48 mg, 0.09 mmol) and dissiopropylethylamine (Hunig's base, 16 ~L, 0.09 mmol) in 3 mL of dimethylformamide cooled to 0°C. After 10 min, a solution of Hunig"s base (16 ~aL, 0.09 mmol) fn 3 mL of THF
was added to the reaction solution. The resulting mixture was stirred overnight. The solvent was evaporated and the crude product was recrystallized from methanol and ether.
The final product was obtained as a light yellow solid in ?3% yield. m.p., >250'C: iH-N'Mlt, 1.86 (2xCH2CH2CH2, 4H, q, J = 8 Hz), 3.00 (2xCH2N(CH3)2, 4H, tr, J a 8 Haj, 3.30 (2xCONHCH2, 4H, m), 3.96 (2xNCH3, 6H, s), 4.02 (2xHCH3, 6H, s), 7.28 (-CH~CH-, 2H, s), ?.54 (2xim-CH, 2H, s), ?.67 (2xim-CH, 2H, s), 8.52 (2xCONHCH2, 2H, tr, J ' 6 Hz), 9.43 (2xpy-NHCO, 2H, s), 11.01 (2xpy-NHCO, 2H, s); MS (FA,B), ??? (M-2xC1-H, 3.11).
Other compounds shown in Table I were similarily prepared and their analytical and physical data are summarized therein.
2 5 ,EXAM LE 2 8 Drug-DNA binding constants of the compounds of the present invention were estiaated. To 2 mL of Tris-EDTA
buffer, pH 8, containing 1.3 ~cM ethidium bromide, calf thymus DNA was added to give a final concentration of 1.35 pM. The fluorescence was measured after equilibration for a few minutes, using a Turner model 430 spectrofluorometer (Turner Amsco Instruments, Carpinteria, CA) equipped with a 150 w xenon lamp, at an excitation wavelength of 525 nm and an emission wavelength of 600 nm. Aliquots of concentrated drug solutions were added and the 2103~6~
WO 92/ 13838 ~'~.'T/CA92/OOOSI,' 4~
fluorescence measured. Controls were performed to show that the drugs themselves did not interfere with the fluorescence measurements at the levels employed. From a plot of the decreased fluorescence of the ethidium-DNA
complex with increase dose of drug, the concentration of drug needed to reduce the 'fluorescence by 50~ was determined and used to calculate a reslative binding constant for the drug, given the binding constant of ethidium to be 107 M-1 under similar conditions.
The results of binding tests are shown in Table II
and in Table III.

Compounds of the present invention were tested for anti-Moloney murine leukemfa virus (MLV). The method utilized was adapted from Rowe et al (1970) and Lin et al (1987).
The following materials were utilized in the method:
- Retroviruses: rauscher - lITCC 998 moloney LT (V) - d~TCC 190 heukosis-sarcoma complex - ATCC 245 - cells.: SC-1 - ATCC CRL 1404 XC - ATCC CCF. 165 minimum essential medium (eagle) with Hanks Bss supplemented with 10$ fet 1 bovine serum, 100 1U ml-~
penicillin G, i00 ugml ~ streptomycin, 2.5 ugml-1 amphotericin B and non-essential amino acids (Sigma M2025).
- Dulbecco's modified eagles medium, supplemented with 10~ fetal bovine with 5k fetal bovine serum, 100 ~Oml-1 penicillin G, 100 ugxnl-1 streptomycin and 2.5 ugml-amphotericin B.
- minimum essential medium (eagle) with earles salt' supplemented with 5t fetal bovine serum, 100 lOml-1 penicillin G, 100 ugml streptomycin and nori-essential aminoacids (Sigma M2025).
e'a t.9 B ~'s'T' 1'T' ~! °r E ~ t~l ~ E'E°

r ~v w0 ~zi ~ 3838 ~ ~ ~ t~ ~ ~ ~ PCT/CA92/00051 - phosphate buffered saline.
- crystal violet dye.
- 24 well plates, compounds dissolved in DMSO (or water) to 2-20 ugml-1 then further diluted in 5$ FEtS-MEM.
Stock cell cultures were prepared in the 10% FBS-Dulbecco. To prepare 24 well plates for experiments, 0.8m1 of 3.S x 10a SC1 cells ml-1 were added to each well one day in advance. This was using the 5% FBS-MEM. 0.1 ml of each compound dilution, in triplicate, was added to a well in the plate. 0.1 ml of 20-40 p.f.u. of a~oloney virus was added to each well of the plate. Those plates were shaken on a mechanical shaker at 0, 30 and 60 minutes. They were incubated !or 5 days at 37~C in a 5~
Co2 incubator. The medium was removed and plates were subjected to ultraviolet light (175 W cm2 at surface) !or three minutes.
0.8 ml o! 2 x 105 XC Bells ml-1 were added to each well using the 10~ FBS-Hanks mem. The plates were incubated at 37~C, 5~ C02 !or 4 days, but the medium was replaced after ~ days. The medium was removed, the wells were washed With pbs and 0.25 ml o! O.OSt crystal violet was added to each well for 2 hours. The plates were washed, dried and the plaques counted.
MIC50 values were calculated using thp formula -k inh'bition reater than 50%- 0%
% inhibition greater than 50%-~t inhibition less t:~an 50%
to give the interpolative values between two dilutions.
The results o! the test are shown in Tables IV and V
and demonstrate comparative anti--MLV activity between compounds of the present invention and AZT and DDC.
~UBSTiTUTE SHED-f-WC~ 92/13838 PC.'f/~A92/0(?OSa Compounds of the present~invention were tested for anti-HIV activity by the National Cancer Institute (NIH, Bethesda). The procedure used by the National Cancer 5 Institute is described in Weislow, O.W. et al, ,T. N~,~, Cancer Inst., Vol. 81, pages 577-586 (1989). NCI uses this procedure to test for agents active against Human Immunodeficiency Virus (HIV) and is designed to detect agents acting at any stage of the virus reproductive 10 cycle. The assay basically involves the killing of T4 lymphocytes by HIV. Small amounts of HIV are added to cells, and a complete cycle of virus reproduction is necessary to obtain the required cell killing. Agents that interact with virions, cells, or virus gene-products 15 to interfere with viral activities will protect cells from cytolysis. The system is automated in several features to accommodate large numbers of candidate agents and is generally designed to detect anti-HIV activity. However, compounds that degenerate or are rapidly metabolized in 20 the culture conditions may not show~activity in this screen. All tests are compared with at least one positive (e. g., A2T-treated) control done at the same time under identical conditions. The procedure is set forth below:
1. Candidate agent is dissolved in dimethyl sulfoxide 25 (unless otherwise instnacted) then diluted 1:100 in cell culture medium before preparing serial half-1og10 dilutions. T4 lymphocytes (CEM eell line) are added and after a brief interval HIV-1 is added, resulting in a 1:200 final dilution of the compound.
30 Uninfected cells with the compound serve as a toxicity control, and infected and uninfected cells without the compound serve as basic controls.
2. Cultures are incubated at 37' in a 5% carbon dioxide atmosphere for 6 days.
~USSTITUTE SHEET

F~', WO 92/13838 ~ 10 3 6 6 5 ~'CT/CA9Z/A0051 3. The tetratolium salt, XTT, is added to all wells, and cultures are incubated to allow tormzan color development by viable cells.
t. Individual wells are analyzed spectrophotometrically to quantitate tormazan production, and fn addition are viewed microscopically for detection of viable cells and contia°mation of protective activity.
5. Drug-treated virus-infected cells are compared with drug-tested nonintected cells and with, other to appropriate controls (untreated infected and untreated nonintected cells, drug-containing wells without cells, etc.) on the same plate.
6. Data are reviewed in comparison with other tests dons at th4 sane tine and a determination about activity is sade.
The test results for five of the active compounds are set forth in the Figures 2-6 and the corresponding Tables VI-X below and test results of the compounds of the present invention are compilated in Table XI.
SUBST'ITU'TE SHEET

2103~~~
WO 92/13838 , PCT/CJ~.92/O(i0 Table I. Analytical and physical data on linked netropsins and their, precursors Camp. Yield (51 m.p.a Formula Ana~,yrsis 15 85 210' C46H58H1808C12C,H,N,C1 16 76 210" C50H60N1808C12C,H,N,C1 17 84 198-202 C52H64N1808C12C,H,N,CL

18 69 215 C66H92N1808C12C,H,N,CL

19a 99 305-6 C38H38N1206 C,H,N

19b 64 262-8' C38H46N1406C12C,H,N,C1 20a 95 278-82' C38H38N1206 C,H,N, 20b 78 248-50 C38H46N14o6C12C,H,N,C1 21a 84.7 289-90 C34H36N1206 C,H,N

21b 58 295 C34H44N1406C12C,H,N,C1 22a 56.5 250-2' C34H36N1206 C,H,N, 22b 85 217' C34H44N1406C12C,H,N,C1 233 88.6 312' (deC) C35H38N1206 C,H,N

23b 68.5 210' (softens) C35H46N1406C12C,H,N,C1 24a 59 175 C H N 0 C H,N

24b 70.6 204 (softens) Cg5H46N1406C12C,H,N,Cl C

ON

25b 77 238' (softens) 40 CL C,H,N,C1 C
H
N

26a 70 165-8' C37~42~12~6 C,H,N, 26b 46 231' C37H50N1406C12C,H,N,C1 27a 82.6 189' C38H44N1206 C,H,N

27b 61 201' (softens) C38H52N1406C12C,H,N,C1 28a 54 175' C H N 0 C,H,N

28b 23 198' C38H52N1406C12C,H,N,C1 29 77 >300 C50H58N1808C12C,H,N,CL

30 68 240 C5pH58N1808C12C,H,N,CL

31 83 245 C50H58N1808C12C,H,N,CL

32 88 250 C49H57N1908C12C,H,N,CL

33 74 260 C49H57N1908C12C,H,N,CL

34 54 260 C49H57N1908C12C,H,N,CL

35 78 230 C4gH6pN1808C12C,H,N,CL

36 33 255 C46H56N1808C12C,H,N,CL

37 67 280 C46H56N1808C12C,H,N,CL

38 53 260 C51H62N1808C13C,H,N,CL

39 73 250 C34H~ON1606C12C,H,N,CL

a. Uncorrected.
b. All compounds gave satisfactory elemental analyses within 0.4% of the calculated values and exhibited 1H-NMR, IR and MS data consistent with the structures.
~UF3&TITUTE SHEET

'CVO 92/ i 383t~
.,. ~ PC1'/CA92/00051 TABLE II. Relative binding constants for natural and linked oligopeptides R1C0(CH2 )nC0-R1 to cnlf thymus DNA determined by ethidium displacement assay.a Comeound ~ QNA Binding Constant (M-1) 1 -° 1.9 x 10~
2 -- o.s x l07 3 0 5.6 x 10~
4 1 3.6 x 107 5 2 7.2 x 107 1.2 x 10~
6 2.5 x 10~
10 7 0.9 x 10~

11 8 1.7 x 10' 12 9 1.9 x 10~

13 10 2.2 x 107 aBased on a binding constant of ethidium of 107 P1-1 under similar conditions of temperature, pH and ionic strength.
Binding constant values represent the average of repeat measurements.
bNumber of CH2 units in the linker in R1-CO(CH2)nC0-R1.
TJ~eLE III. Relative binding constants for cis and trans bis-netropsins to poly(dA-dT) determined by the ethidium displacement assay.a Compound ~C -rr~ 1, ap~

1 9.4 x 10, 2 6.3 x 10 19b 4 20b .
x 10 4.9 x 107 21b 4.9 x 107 22b 3.8 x 107 23b 5.3 x 107 24b 4.4 x 107 25b 5.6 x 107 26b 3.1 x 107 27b 4.0 x 107 aBased on a binding constant of ethidium of 9.5 x 106 M-1 under similar conditions of temperature, pH and ionic strength. Binding constants represent the average of repeat measurements.
SU~STITI~TE SHEET

2103~G~
WO 92/ 138:38 PCT/CA92/0005~~;;'~'.

TABLE I~.
Toxicity_','~D50 Activity, -1) T.I.,TD50/
~o~mpound dug mL 1 MIC50 (u4 mL MIC50---29 >100.00 3.98 >25.13 30 >100.00 >50.0 2.0 31 >100.00 79.63 >1.26 32 >100.00 15.93 >6.28 33 >100.00 >100.00 --34 >100.0A 22.74 >4.40 35 87.50 >50.0 1.7 36 100.00 0.16 625.00 37 84.29 11.21 7.52 38 >100.00 22.04 >4.54 39 >100.00 >100.00 --AZT >100.00 0.0014 >7.14x105 DDC >100.00 0.74 >135.14 TaHLE 9. Inhibition of Moloney marine leukemia (MLV) associated reverse transciptase activity by linked.
ou ga ~Q5 b~g(mL1 (average;SD1 4 1 39.~ ~ 13.9 4 2 25.2 11.4 8 5 72.5 t 7.69 9 6 21.3 6.1 10 7 34.2 0.9 11 8 20.3 9.2 12 9 10.3 7.5 13 10 9.1 ~ 6.7 23b -- 7,0 3.6 24b -- 30.4 ~ 19.3 25b -- 21.8 9.2 26b -- 45.9 11.3 27b - 29.1 6.0 28b -- 63.8 41.0 Aurintricarb oxylicacid 1.42 0.26 aNumber of CH2 groups in linker in R1-CO(CH2)nC0-R1.
b50% inhibitory dose, measured after 120 min incubation of the reaction mixtures. (MLV: lot 804-845-8A; (3H-methyl)dTTP at 10 ~sC; (specific activity: 30 Curies/mmol) per 250 ~L of reaction mixture.) SIJ~SI~T~'TE Si~EET

~ 10 3 G G 5 PCTlCA92/OU051 ~;.',':W'O 92/13838 ThIILE VI
Results of the compound N,N'-di(1-methyl-2-(1-methyl-2-carboxamido(3-propionamidine)-4-pyrrole]-4-pyrrolyl]
terephthalamide dihydrochloride.
INFECTEDUNINFECTED
SUMMI\RY DOSE RESPONSERESPONSE

~ of ~t of Index Concentration(Molar) Control Control IC50 (Molar)>1.79 x 5.68 0'9 39.64 88.62 10'5 x EC50 (Molar)2.08 x 10'61.79 x10'8 33.00 92.56 TI50 (IC/EC)>8.59 X 5.68 x10-~ 25.70 94.69 1.79 x10'7 29.97 92.72 5.67 x10'7 28.86 88.21 1.79 x10-6 51.73 136.50 5.66 x10'6 134.54 164.46 1.79 xlo-S 167.25 19z.17 5 TnBL$ vaI
Results of the compound N,N'-di[1-methyl-2-[1-methyl-2-carboxamido(3-propionamidine)-4-pyrrole]-4-pyrrolyl]
isophthalamide dihydrochloride.
INFECTEDUNINFECTED
SUMMl,RY DOSE RESPONSERESPONSE

~ of ~ of Index concentration(Molar) control Control IC50 (Molar)2.84 x 10-44.28x 10-748.47 116.38 EC50 (Molar)3.55 x 10'61.35x 10'629.25 131.95 TI50 (IC/EC)8.00 x 10+14.27x 10'664.76 123.64 1.35x 10'5117.45 117.76 4.26x 10-5120.68 123.27 1.34x 10-463.57 142.80 4.26x 10-40.02 -0.29 1.34x 10-34.26 10.39 StJBfiTiTUTE SHEET

2~o~ss ~'O 92/13838 PGT/CA92/0005 ~;

TAHLE VIII
Results of the compound N,N'-di[1-methyl-2-[1-methyl-2-carboxamido(3-propionamidine)-4-pyrrole)-4-4pyrrolyl]
fumaride di)wdrochloride.
INFECTEDUNINFECTED
SUMMARY DOSE RESPONSERESPONSE

of ~ of Index concentration(Molar) control control IC50 (Holar)>3.30 x 1.05x10-8 18.00 83.05 EC50 (Molar)1.67 x 10'63.32x10-8 22.96 87.56 TI50 (IC/EC)x1.97 x 1.05x107 26.15 90.26 10+1 3.31x10-7 28.00 86.21 1.04x10-6 38.90 83.87 3.31x10'6 96.78 151.83 1.04x10-5 150.24 154.79 3.30x10-5 132.20 138.63 Results of the compound N,N'-di(1-methyl-2-[1-methyl-2-carboxamido(3-propionamidine)-4-pyrrole]-4-pyrrolyl]maleamide dihydroclaloride.
INFECTEDUNINFECTED
SUMMARY DOSE RESPONSERESPONSE

of Index Concentration(Molar) Control Control IC50 (Molar)1.63 x 10'43.04x107 28.75 100.16 EC50 (Molar)4.04 x 10'69.61x10'7 32.14 100.02 TI50 (IC/EC)4.04 x 10+13.03x106 4A.03 103.19 9,59x106 115.19 112.20 3.03xl05 114.32 112.05 9.58X10-5 84.02 94.18 3.02X10-4 0.29 -0.86 9.57x10-4 0.14 5.40 ~UBSTrTUTE SHEET

WO 92/1383t~ ~ ~ ~ ~ ~ ~ '~ PCf/CA92/UOOSl TAHLE X
Results of the compound N,N'-di[1-methyl-2-[1-methyl-2-carboxamido(3-propicnamidine)-4-pyrrole)-4-pyrrolyl]
trams 1,2-cyclobutaneamide dihydrochloride.
INFECTEDUNINFECTED
SUMMARY DOSE RESPONSERESPONSE

~~ of ; of Index Concentration(Molar) Control Gontr~1 ICSO (Mular)1.67 x 10-42.94x 10-?42.92 90.95 EC50 (Molar)1.79 x 10-69.29x 10-?59.0? 102.41 TI50 (IC/EC)1.20 x 10+22.93x 10-677.00 116.36 9.28x 10'680.26 145.18 2.93X 10'S110.05 174.62 9.27X 10-'90.52 101.95 2.92X 104 0.04 0.54 9.25x 10'4I 972 I 8'29 TABLE XI
The following Table XI shows the results of anti-HIV-1 data on the oligopeptides of the present invention and their anti-HIV-1 activity is designated as inactive, moderate or active. Compounds 19b, 20b, 21b, 25b, 29, 30, l0 32, 34 and 36 are designated as active.
~UI~STITUTE SHEET

WC) 92/13838 PGT/CA92/0005'=~;' TABLE ~I Anti-HIV-1 Activity Compound ~C50(uM) .~1'C50 Activitv*

3 83.5 11.9 Moderate 7.p1.~

5 75.3 12 6.3~. Moderate 8 64.8 5.3 12.1 Moderate 9 _- _- -- Inactive 51.1 2.1 24.1 Moderate 11 -- -- -- Inactive 10 12 S7 3.9 1.46 Moderate 13 78 6.6 11.7 Moderate 41 41 1.0 Inactive 16 >100 -- -- Inactive 17 29 14 2 Moderate 15 18 >12p -- -- Inactive 19b 17.9 1.2114.8 Active 20b 284 3.5580 Active 21b 33 1.3724.1 Active 22b 199 0.35566 Active 23b 9.3 3.442.7 Moderate 24b 257 42.56.1 Moderate 25b 68.2 0.42161 Active 26b 168 46.33.6 Moderate 27b 181 5.6 32.4 Moderate 29 4.7 0.3912 Active 140 21 6.6 Active 32 69 1.6 43 Active 33 69 9.8 7.p Moderate 34 140 13 11 Active 30 35 71 16 4.5 Moderate 36 207 10.419.8 Active 37 35 _- __ Inactive * National Cancer Institute Designation _ ~UBS'T'1TUTE SHEET

Claims

WE CLAIM:

1. Use of a compound of the formula:

wherein:
A is a moiety bearing a positive charge and of a size which does not inhibit binding of said compound to nucleic acid sequences associated with the cellular action of Human Immunodeficiency Virus;

R1 is a moiety derived from a residue of carbonic acid or a residue of a dicarboxylic acid selected from the group consisting of:
(i) a residue of a dicarboxylic acid of the formula -CO-Cp-H2p-CO
where p equals an integer from 1 to 22:
(ii) a residue of an unsaturated aliphatic dicarboxylic acid of the formula -CO-Cq-H2q-2-CO
where q equals an integer from 2 to 22;
(iii) a residue of an aromatic dicarboxylic acid;
(iv) a residue of a cycloalkane dicarboxylic acid of the formula -CO-Cr-H2r-2-CO-where r equals an integer from 3 to 7; and (v) a residue of a cycloalkene dicarboxylic acid of the formula -CO-C3-H2s-4-CO
where s equals an integer from 3 to 7;

Het is pyrrole;
x is 0 or 1;
y is 0, 1, 2 or 3;
z is 0, 1, 2 or 3;

R2, R3; R4 and R5 are attached to a ring atom other than carbon and are independently selected from the group consisting of C1-C6 alkyl and -CH2-O-R6, where R6 is a C1-C6 alkyl.;
and salts thereof;
for treating HIV.

2. Use according to claim 1, wherein A is a moiety selected from the group consisting of an amidine, a guanidine, secondary ammonium salts, tertiary ammonium salts, quaternary ammonium salts, sulfonium salts and phosphonium salts.

3. Use according to claim 1, wherein R2, R3, R4 and R5 are each a C1-C6 alkyl.

4. Use according to claim 1, wherein R2, R3, R4 and R5 are the same and are a C1-C6 alkyl group.

5. Use according to claim l, wherein R2, R3, R4 and R;
are each a methoxymethyl.

6. Use according to claim l, wherein R1 is

7. Use according to claim 1, wherein R1 is a residue of a dicarboxylic acid of the formula -CO-CpH2p-CO- where p equals an integer from 1 to 22.

8. Use according to claim l, wherein R1 is a residue of a dicarboxylic acid selected from the group consisting of:
a residue of an unsaturated aliphatic dicarboxylic acid of the formula -Co-Cq-H2q-2-CO- where q equals 2; a residue of an aromatic dicarboxylic acid; and a residue of a cycloalkane dicarboxylic acid of the formula -CO-Cr-H2r-2-CO- where r equals an integer from 3 to 6.

9. Use according to claim 1, wherein the compound is N,N'-di[1-methyl-2-[1-methyl-2-carboximido(3-propionamidine)-4-pyrrole]-4-pyrrolyl] terephthalamide dihydrochloride.

10. Use according to claim 1, wherein the compound is N,N'-di[1-methyl-2-[1-methyl-2-carboximido(3-propionamidine)-4-pyrrole]-4-pyrrolyl] isophthalamide dihydrochloride.

11. Use according to claim 1, wherein the compound is N,N'-di[1-methyl-2-[1-methyl-2-carboximido(3-propionamidine)-4-pyrrole]-4-pyrrolyl] fumaramide dihydrochloride.

12. Use according to claim 1, wherein the compound is N,N'-di[1-methyl-2-[1-methyl-2-carboximido(3-prapionamidine)-4-pyrrole]-4-pyrrolyl] maleamide dihydrochloride.

13. Use according to claim 1, wherein the compound is N,N'-di[1-methyl-2-[1-methyl-2-carboximido(3-propionamidine)-4-pyrrole]-4-pyrrolyl] trans 1, 2-cyclobutanamide dihydrochloride.

14. Use according to claim l, wherein the compound is:

15. Use according to claim l, wherein the compound is:

16. Use according to claim l, wherein the compound is:

and R is

17. Use according to claim 1, wherein the compound is:

<IMG'S>

18. Use according to claim 1, wherein the compound is:

<IMG'S>

19. Use according to claim 1, wherein said residue of cycloalkane dicarboxylic acid of the formula -CO-Cr-H2R-2-CO- is fused to one or more three to seven membered C rings.

20. Use according to claim 1, wherein the anti-HIV
effective dose is in a range of 1 to 200 mg/kg body weight per day.

21. A compound exhibiting activity against Human Immunodeficiency Virus represented by the formula:

<IMG'S>

wherein R1 is a moiety derived from a residue of a dicarboxylic acid selected from the group consisting of:
a residue of a C6 aromatic dicarboxylic acid: a residue of an unsaturated aliphatic dicarboxylic acid of the formula CO-Cq-H2q-2-CO- where q equals 2; a residue of a cyeloalkane dicarboxylic acid of the formula Co-Cr-H2r_2-CO where r equals an integer from 3 to 6 optionally fused to one or more three to seven C membered rings, and A, x, y and z are as defined in claim 1 and R2, R3, R4 and RS are attached to a ring atom other than carbon and are independently selected from the group consisting of C2-C6 alkyl and -CH2-0-R6, where R6 is a C1-C6 alkyl.

22. she compound of claim 21, wherein R1 is

23. The compound of claim 21, wherein Rl is

24. The compound of claim 21, wherein Rl is a dicarboxylic acid residue of cyclopropane.

25. The compound of claim 21, wherein R1 is a dicarboxylic acid residue of cyclopentane.

26. The compound of claim 21, wherein Rl is a dicarboxylic acid residue of cyclohexane.

27. The compound of claim 21, wherein R1 is CO n CO

28. The compound of claim 21, wherein R1 is CO n CO

29. A pharmaceutical composition suitable for the treatment of Human Immunodeficiency virus infections, comprising a compound of the formula:
A- (NHCO) X-Het- (NHCO-Het) y-NH-R1-NH-(Het-CONH)Z-Het-(CONH)X-A

wherein:
A is a moiety bearing a positive charge and of a size which does not inhibit binding of said compound to nucleic acid sequences associated with the cellular action of Human Immunodeficiency Virus;
R1 is a moiety derived from a residue of carbonic acid or a residue of dicarboxylic acid selected from the group consisting of:
(i) a residue of a dicarboxylic acid of the formula -CO-CpH2p-CO
where p equals an integer from 1 to 16 (ii) a residue of an unsaturated aliphatic dicarboxylic acid of the formula -CO-Cq-H2q-2-CO-where q equals an integer from 2 to 16;
(iii)a residue of an aromatic dicarboxylic acid;
(iv) a residue of a cycloalkane dicarboxylic acid of the formula -CO-Cr-H2r-2-CO-where r equals an integer from 3 to 7; and (v) a residue of a cycloalkene dicarboxylic acid of the formula -CO-CS-H2s-4-CO
where s equals an integer from 3 to 7;
Het is pyrrole;
x is 0 or l;
y is 0, 1, 2 or 3;
z is 0, l, 2 or 3;
R2, R3,R4, and R5 are attached to a ring atom other than carbon and are independently selected from the group consisting of C2-C6 alkyl and -CH2-O-R6 where R6 is a C1-C6 alkyl; and salts thereof, in a pharmaceutically acceptable carrier.

30. A process for the preparation of a compound of the formula:

wherein:
A is a moiety bearing a positive charge and of a size which does not inhibit binding of said compound to nucleic acid sequences associated with the cellular action of Human Immunodeficiency Virus;
R1 is a moiety derived from a residue of carbonic acid or a residue of a dicarboxylic acid selected from the group consisting of:
(i) a residue of a dicarboxylic acid of the formula -CO-Cp-H2p,-CO
where p equals an integer from 1 to 16;
(ii) a residue of an unsaturated aliphatic dicarboxylic acid of the formula -CO-Cq-H2q-2-CO-where q equals an integer from 2 to 16;
(iii) a residue of an aromatic dicarboxylic acid;
(iv) a residue of a cycloalkane dicarboxylic acid of the formula -CO-Cr-H2r-2-CO-where r equals an integer from 3 to 7; and (v) a residue of a cycloalkene dicarboxylic acid of the formula -CO-CS-H2s-4-CO
where s equals an integer from 3 to 7;
Het is a five membered heterocyclic moiety selected from the group consisting of a pyrrole, an imidazole, a triazole, a pyrazole, a thiazole, a thiophene, a furan and an oxazole;
x is 0 or 1;
y is 0, 1, 2 or 3;
z is 0, 1, 2 or 3;
R2, R3, R4 and R5 are attached to a ring atom other than carbon and are independently selected from the group consisting of C2-C6 alkyl and -CH2-O-R6 where R6 is a C1-C6 alkyl;
and salts thereof, comprising the steps of:
reacting a compound of the formula (II) with a dicarboxylic acid of the formula (III) (III) and converting B to A to form said moiety bearing a positive charge, wherein;
x, y and R1 are as defined above;
B is the same as A or is a group with a nitrile, halogen or sulfide substituent; and X is a halogen, imidazolide or other reactive moiety.

31. A pharmaceutical composition according to claim 29, wherein said residue of cycloalkane dicarboxylic acid of the formula -CO-Cr-H2r-2-CO- is fused to one or more three to six C membered rings.

32. A proves according to claim 30; wherein said residue -CO-Cr-2-CO- is fused to a three to seven C membered ring.

33. Use according to claim 1, wherein A is an amidine represented by the formula:

where p is an integer from 0 to 5 and X is selected from the group consisting of -H; -OH, -NH2, -CH3, -C2H5 and -C3H7.

34. Use according to claim 1, wherein A is a guanidine represented by the formula:

where p is an integer from 0 to 5 and X is selected from the group consisting of -H, -OH; -NH2, -CH3, -C2H5 and -C3H7.

35. Use according to claim 1, wherein A is an ammonium salt represented by the formula:
-CpH2p-~NHqX(3-q) where p is an integer from l to 5 and q is an integer from 0 to 3 and X is an alkyl or alkenyl group of 1 to 3 carbon atoms.

36. Use according to claim 1, wherein A is a sulfonium salt represented by the formula:
-CpH2p-SXY
where p is an integer from 0 to 5 and X and Y are alkyl or alkenyl groups of 1 to 3 carbon atoms.