Note: Descriptions are shown in the official language in which they were submitted.
WO94/19008 ~ g 2 ~ PCT~S94/00710
COMBINATIONS OF RETROVIRAL IN~IBITORS
This invention relates to novel combinations of
castanospermine esters and short peptide analogs that act
effectively in their combination in the treatment of
15 retroviral infections, particularly in the treatment of
acquired immune deficiency syndrome(AIDS) and related human
immunodeficiency viral (HIV) infections.
BACKGROUND OF THE INVENTION
Intense research over the last several years has been
devoted to develop treatments and cures for retroviral viral
25 infections in humans and in animals, and particularly for
acquired immune deficiency syndrome (AIDS) and AIDS related
complex (ARC). Today the public realizes the severe health
risk posed by the AIDS retrovirus and that the incidence of
ARC and AIDS in humans is increasing at an alarming rate.
30 Furthermore, survival beyond 5 years for those who have
contracted AIDS remains remote. Further, AIDS patients
whose immune systems have been seriously impaired by the
infection, suffer from numerous opportunistic infections and
proliferative disease including Pneumocystis carninii
35 pneumonia and Kaposi's sarcoma. No cure for AIDS is known
and current treatments are largely without adequate proof of
efficacy and have numerous untoward side effects. Due to
the severity of the disease, and with death the resultant
~ ~ 3 ~
WO94/19008 PCT~S94/00710
outcome from the disease, fear of AIDS has resulted in
social ostracism and discrimination against those having or
suspected of having the disease.
The AIDS virus belongs to a general class of viruses
known as retroviruses. As a class, many of the known
retroviruses are also oncogenic or tumor causing. Indeed
the first two human retroviruses discovered, denoted human
T-cell leukemia virus type I and type II or HTLV-I and II,
lO were found to cause rare leukemias in humans after infection
of T-lymphocytes. The third such human virus to be
discovered, HTLV-III, now referred to as HIV, was found to
cause cell death after infection of T-lymphocytes and has
been identified as the causative agent of ac~uired immune
5 deficiency syndrome (AIDS) and AIDS related complex (ARC).
Retroviruses are a class of ribonucleic acid (RNA)
containing viruses that replicate by using a reverse
transcriptase activity to form a strand of complementary DNA
20 (cDNA) from which a double stranded, proviral DNA is
produced. This proviral DNA is then incorporated into the
chromosomal DNA of the host cell making possible viral
replication by transcription of this integrated DNA and
translation of viral messenger RNA into proteins.
25 Replication of the virus occurs by synthesis of viral
genomic RNA and its assembly with glycosytated and non-
glycosylated viral proteins to form new viral particles.
Maturation of virions at the cell surface results in the
release of infectious virus progeny.
Retroviral proteins are generally synthesized as
polyproteins and virus encoded proteases are required to
cleave the precursor polyproteins to form the viral enzymes
and structural proteins. ~or example, the gag and gag-pol
35 precursor polyproteins of retroviruses are synthesized as
precursors of viral encoded enzymes and non-glycosylated
s~ructural proteins. Similarly, the enveiope protein o~ HIV
WO94/19008 ~ PCT~S94/00710
is a 160 kDa highly glycosylated precursor glycoprotein.
The envelope proteins are cleaved by a host-cell protease to
give a 120 kDa external glycoprotein (gp 120) and a
transmembrane glycoprotein (gp 41). The gp 120 protein
5 contains a high affinity binding site that recognizes the
CD4 ligand on CD4-positive human T-helper cells, the known
receptor for this virus.
The retroviral proteases also show certain commonality
10 by their inhibition by aspartyl protease-specific
inhibitors, Iyoko, et al. Nature 329, 654-67. Similarly,
amino acid sequencing of the retroviral proteases show they
possess sequence homology. Because of their mutual
structural and functional characteristics and their obligate
15 function, the aspartyl proteases serve as a potentially
interesting therapeutic target for intervention.
The correctly processed envelope glycoproteins of the
retroviruses play an important role in the virus life cycle,
20 which also offers a possible target for clinical
intervention. The envelope glycoproteins serve a role in
both the initial interaction of the virion and the target
host-cell and in the subsequent fusion of the viral envelope
and host-cell membranes during penetration. Certain esters
25 of castanospermine are useful in interfering with the
processing of the viral envelope glycoproteins and thereby
in preventing the initial virus-host cell interaction and
subsequent fusion.
WO94/19008 ~~ ~5 ~ ~ ~ 9 PCT~594/00710
The applicants have discovered that the combination of
aspartyl protease-specific inhibitors (formula II) with
glycoprotein processing inhibitors such as castanospermine
5 derivatives (formula I) result in significant improvements
in the inhibition of the HIV virus that would not be
expected.
SUMMARY OF THE INVENTION
The pharmaceutical compositions consist of the
glucosidase inhibitor of formula I and a viral aspartyl
protease inhibitor of formula II.
The pharmaceutical compositions of formula l and formula
lS II, and their pharmaceutically acceptable addition salts,
are novel and possess valuable pharmacological properties.
Often these compositions can act synergistically to
effectively inhibit HIV activities, and therefore, can be
used in the prophylaxis or treatment of viral infections,
20 particularly infections caused by HIV.
Objects of the present invention are the compositions of
formula I and II, and their aforementioned salts, for use as
therapeutically active substances, medicaments containing
25 said compounds and salts, their manufacture, and the use of
said compositions and salts in the control or prevention of
illnesses, especially in the treatment or prophylaxis of HIV
infections.
WO94/19008 ~1 5 ~ PCT~S94/00710
Compounds of formula I comprise structures of the
following formula:
HO ~
~ N (1)
R2O-
ORl OR
wherein R, R1 and R2 are independently hydrogen,
C1 l4 alkanoyl, Cl-Cl4 alkenoyl, cyclohexanecarbonyl,
Cl_6 alkoxyacetyl,
y~
Y"
naphthalenecarbonyl optionally substituted by methyl or
halogen; phenyl(C2 6 alkanoyl) wherein the phenyl is
optionally substituted by methyl or halogen; cinnamoyl;
pyridinecarbonyl optionally substituted by methyl or
halogen; dihydropyridine carbonyl optionally
substituted by Cl lo alkyl; thiophenecarbonyl
optionally substituted by methyl or halogen; or
furancarbonyl optionally substituted by methyl or
halogen; Y is hydrogen, Cl 4 alkyl, C1 4 alkoxy,
halogen, trifluoromethyl, C1-4 alkylsulfonyl, C1-4
alkylmercapto, cyano or dimethylamino; Y' is hydrogen,
C1-4 alkyl, C1 4 alkoxy, halogen or it is combined with
~ 35 Y to give 3,4-methylenedioxy; Y" is hydrogen, C1 4
alkyl, Cl_4 alkoxy or halogen; with R, Rl and R2 being
selected in such a way that at least one of them, but
W O 94/19008 ~ 5 ~ PCTrUS94/00710
not more than two of them, is hydrogen; or a pharmaceu-
tically acceptable salt of these compounds.
The Cl l4 alkanoyl groups referred to above can be
straight- or branched-chain or cyclic and can be
exemplified by formyl, acetyl, propionyl, butyryl,
isobutyryl, cyclopropanecarbonyl, hexanoyl, octanoyl
and decanoyl. The Cl-l4 alkenoyl groups referred to
above can be straight- or branched-chain or cyclic but
have at least one carbon-carbon double bond as
exemplified, propenoyl, butenoyl, isobutenoyl,
hexenoyl, octenoyl and decenoyl. The Cl 6 alkoxyacetyl
referred to above can be methoxy-acetyl, ethoxyacetyl
and butoxyacetyl. The halogens referred to above can
be exemplified by fluorine, chlorine, bromine or
iodine. The C2_6 alkanoyl groups referred to above can
be exemplified by acetyl, propionyl, butyryl,
isobutyryl, and hexanoyl. The Cl-4 alkyl groups
referred to above, whether alone or as part of an
alkoxy, an alkylsulfonyl or an alkylmercapto group, can
be straight- or branched-chain alkyl groups containing
up to 4 carbon atoms. Examples of various such groups
are methyl, ethyl, propyl, butyl, methoxy, ethoxy,
butoxy, methylsulfonyl, ethylsulfonyl, methylmercapto
and ethylmercapto. The phenyl(C2_6 alkanoyl) groups
referred to above can be exemplified by benzeneacetyl
and benzenepropionyl. The various naphthalenecarbonyl,
pyridinecarbonyl, thiophenecarbonyl and furancarbonyl
groups referred to above include the various position
isomers and these can be exemplified by naphthalene-l-
carbonyl, naphthalene-2-carbonyl, nicotinoyl,
isonicotinoyl, N-methyl-dihydro-pyridine-3-carbonyl,
thiophene-2-carbonyl, thiophene-3-carbonyl, furan-2-
carbonyl and furan-3-carbonyl. The naphthalene,
pyridine, thiophene and furan groups can be optionally
further substituted as indicated above.
~5~2~
W094/1gO08 PCT~S94/00710
Preferred compounds of the present invention are
those wherein R, Rl and R2 are 1 or 2 alkanoyl,
alkenoyl, or benzoyl groups with the benzoyl
substituted by Y, Y' and Y" as described above,
especially a Cl 4 alkanoyl or a benzoyl optionally
substituted with an alkyl or halogen.
More preferred are those compounds of formula 1
wherein one of R, Rl and R2 is alkanoyl or benzoyl,
especially a Cl 8 alkanoyl, Cl g alkenoyl, or a benzoyl
optionally substituted with an alkyl or halogen, and
the others are hydrogens. Even more preferred are
those compounds of formula 1 wherein one of R, Rl and
R2 is a Cl 8 alkanoyl, Cl g alkenoyl, or a benzoyl
optionally substituted with an alkyl or halogen,
especially a methyl, bromo, chloro, or fluoro group,
and the others are hydrogens.
Most preferred grouping are those compounds of
formula 1 wherein Rl is a Cl 8 alkanoyl, Cl g alkenoyl,
or benzoyl optionally substituted with an alkyl or
halogen, especially a methyl, bromo, chloro, or fluoro
group, most especially a methyl, bromo, chloro, or
fluoro group at the para position, and wherein R and R2
are each a hydrogen.
A most especially preferred grouping can be
designated by the substituents represented by the
following compounds:
1. [lS~ ,6B,7a,8B,8aB)]-octahydro-1,6,7,8-
indolizinetetrol 6-benzoate:
2. [lS-(1~,6B,7a,8B,8aB)]-octahydro-1,6,7,8-
indolizinetetrol 7-benzoate:
3. [lS-(1~,6B,7~,8B,8aB)]-octahydro-1,6,7,8-
indolizinetetrol 6-(4-methylbenzoate):
4. [lS-(lal6B~7a~8B~8aB)]-octahydro-l~6~7~8
indolizinetetrol 7-(4-bromobenzoate):
' WO94/19~8 PCT~S94/00710
-8- 2 ~
5. [lS-(1~,6B,7~,8B,8aB)]-octahydro-1,6,7,8-
indolizinetetrol 6,8-dibutanoate:
6. A castanospermine ester of Claim I which is
[lS-(1~,6B,7~,8B,8aB)]-octahydro-1,6,7,8-
indolizinetetrol 6-butanoate:
7. [lS~ 6B,7a,8B,8aB)]-octahydro-1,6,7,8-
indolizinetetrol 6-(2-furancarboxylate):
8. [lS-(1~,6B,7~,8B,8aB)]-octahydro-1,6,7,8-
indolizinetetrol 7-(2,4-dichlorobenzoate)
9. ~ls~ 6s~7a~8B~8aB)]-octahydro-l~6~7~8
indolizinetetrol 6-(3-hexenoate).
10. [ls-(l~6B~7~8B~8aB)l-octahydro-l~6~7~8-
indolizinetetrol 6-octanoate.
~ s-(lQ~6s~7~8B~8aB)]-octahydro-l~6~7~8
indolizinetetrol 6-pentanoate.
Certain compounds are preferred. Amongst the
preferred compounds of formula I is [lS-
~ 6B~7~8s~8as)]-octahydro-1~6~7~8-indolizinetetrol 6-
butanoate.
Preparation, and preferred grouping of compounds of
compounds of formula I are also taught in the US Patent
No. 5,017,563, Issued May 21, 1991. Processes for the
preparation of Castanospermine are also taught in US
Patent No. 5,066,807, Issued Novem~er 19, 1991.
,~ .
WO94tl9008 21~51 2 ~ PCT~S94/00710
Compound of the formula II comprise structures of the
following formula:
_ Pl2 Pll
R1CNHCH CNHCHC-CF~C-NR5R6
O O O O
and the hydrates, isosteres and the pharmaceutically
acceptable salts thereof wherein
x is zero or one,
P1 is Q, or B, B being CH~(CH2)a-(O)b-(CH2)c-~ d
with the proviso that B is other than ~-hydroxy-
benzyl or ~-alkoxybenzyl,
a is zero, or l, 2 or 3,
b is zero or l,
c is zero or l, 2, 3, 4 or 5,
d is l or 2,
e is zero, l or 2,
Q is (CH2)d ~ (CH2)d~
P2 is C1_6 alkyl, cyclopentyl, cyclohexyl, hydroxy C1_6
alkylene,, ~ with T being H or C(O)R4,
N - T ,
CH2CONHR4, or CH2CONHR4;
R is hydrogen, -CH2CHO, hydroxy C1_6 alkylene, C1_6 alkoxy
C1 6alkylene, C1 6 alkyl, phenyl, ~ (R3)d or Q,
R1 is benzyloxy, C1 6 alkoxy, C1 6 alkyl, phenyl, benzyl,
phenethyl, fluorenylmethylenoxy, 2-isoquinolinyl, PDL,
WO 94/19008 ~.. rj~ ~ PCT/US94tO0710
--10--
f 2 (CH2)3lCH2~ ~I-(CH2)2-N-CH2fH2~ NHSO2R4, N(R4)(benZyl)
and N(R4)(PDL), with PDL being -(CH2)a-2-,3-, or
4-pyridyl, or ~-W-substituted benzyloxy with W being
nitro, OH amino, Cl_6 alkoxy, or hydroxy Cl 6 alkylene,
or halogeno,
R3 is Cl_6 allenyl Cl_6 alkoxy, Cl 6 alkoxy Cl 6 alkylene,
hydroxy Cl_6 alkylene, Cl_6 alkyl, H, or OH,
R4 is H, Cl_6 alkyl, phenyl or benzyl,
5 is H~ Cl 6 alkyl, OH, Cl_6 alkoxy,-(CH2)d ~ (V)el V
being OR4 or hydroxy Cl 6 alkylene, CH2Si(CH3)2(R3),
-(CH2)d-Q, PDL,_l_(cH2)2_0_cH2clH2,(cH ) ~ ,
CH2-< ~ ~ -tCl_6 alkylenet OR4 or -CH(Y)(Z), Y being
hydroxy Cl_6 alkylene, Cl_6 alkyl, or(CH2~-C6H4-~V)e,
and Z being CHO, CO2R4, C02NHR4 or(CH2~-OR4,
R6 is as defined for R5 with the proviso that R6 is other
than H when R5 is H, and when R5 and R6 are taken
together with nitrogen atom to which they are attached
form a heterocyclic moiety of the formulae:
WO94/19008 ~S ~ 2 9 PCT~S94/00710
-N(CH2)3lCH2' -N(cH2)4lcH2r -N(CH2)2OcH2clH2'
(a) (b) (c)
~3 H~
R7
(d) (e) (f)
lRl8 lRl8
-N(CH2)2N-CH2CH2, -NCH2C(CH2)2CH2, or -N-(CH2)2CCH2CH2,
CH(O)
(9) (h) (i)
~ R7 is CH2OR4 or C(O)NHR4,
R8 is (H,OH) or =O.
As is true for most classes of compounds found to be
useful in the pharmaceutical industry, certain subgeneric
25 groups and certain specific compounds are more preferred.
Within the concepts of this invention, it is to be found
that the preferred compounds are those wherein when Rs is H,
then:
Rl is benzyl, benzyloxy, 4-alkoxybenzyloxy, morpholyl,
~
SO2HN, (3-pyridyl)ethyl, isoquinolyl;
P2 is methylamid, isopropyl, cyclopentyl,
2-(4,4-difluoro)-pyrrolidyl, 2-hydroxy-2-propyl,
t-butyl;
Pl is piperonyl, 4-(benzyloxy)benzyl,
3-(benzyloxy)benzyl, (4-benzyloxy-3-methoxy)benzyl;
WO94/19008 ~loJ~ PCT~S94/00710
R6 is benzyl, piperonyl, CH2-pyridyl,
4-(benzyloxy)benzyl, morpholino, tetrahydroisoquinolyl,
4-(3-hydroxypropyl)benzyl,
OH
2-(3-hydroxypropyl)benzyl, and ~ ~, or -CH(Y)(Z)
with Y and Z both being as generally defined, but
particularly when Y is isopropyl, preferably in the D
configuration, or phenyl and when Z is benzyloxymethylene,
CHO, COOH, alkoxy or COOR4.
When R5 is other than H it is preferred that R5 be
methyl, 4-hydroxybutyl or 3-hydroxypropyl and that R6 be
benzoxy or benzyl, and when R5 and R6 form a heterocyclic
moiety with the nitrogen attached thereto, the heterocycle
is a perhydroiso~uinoline of (f),
-N~_~NCHO of(9),
and morpholino of (c) be the heterocyclic moieties. The
preferred specific compounds of formula II are those shown
in the chart below and those products exemplified herein.
WO94/19008 215 ~ 1 2 9 PCT~S94/00710
A most especially preferred grouping can be designated
by the substituents represented by the following compounds:
1. N-tertiary butyl-decahydro-2[R(R)-hydroxy-4-phenyl-
3(S)-[[N(2-quinolylcarbonyl)-L-asparaginyl]amino]-
butyl]-(4aS,8aS)-isoquinoline-3(S)-carboxamide;
2. [l(S)-[[3,3-difluoro-2,4,-dioxo-1-[[4-
(phenylmethyl)amino]butyl]aminocarbonyl]-2-
methylpropyl]carboamic acid, phenylmethyl ester; or
3. N-[4-(N-benzyloxycarbonyl-L-valyl)amino-2,2-
difluoro-1,3,-dioxo-5-(e-benzyloxy)phenyl-pentyl]-(0-
benzyl)-D-valinol.
The specific making and compounds of compounds of
formula II are further taught in the International
Application Published Under the Patent Cooperation Treaty,
~ International Publication Number W0 92/12123,
In the present invention, each compound in the
compositions compounds of formula I and II may occur with
asymmetric centers or may occur as racemates, racemic
mixtures and as individual diastereomers, with all isomeric
forms of the compounds being included in the present
invention.
~ The preferred compounds of formula I and formula II may
be selected in any combination from one group select from
formula I and one group selected from formula II. It is
recognized that such combinations would include, for
instance, a pharmaceutical composition comprising a
compound of formula I:
WO94119008 PCT~S94/00710
p~ 14-
HO ~
R2O ~ > Formula I
OR 1 OR
wherein Rl is a Cl g alkanoyl, Cl lo alkenoyl, Cl 8
alkoxyacetyl, or benzoyl optionally substituted with an
alkyl or halo~en group; or a pharmaceutically acceptable
15 salt thereof and a compound of formula II
_ Pl2 Pll
RlCNHCH CNHCHC-CF~C-NR5R6
o _ o ~ ~ Formuia 11
x
and the hydrates, isosteres and the pharmaceutically
acceptable salts thereof wherein R1 is benzyl
oxy, ~ SO2HN, (3-pyridyl)ethyl, isoquinolyl, 4-alkoxy-
benzyloxy, or morpholyl, P2 is isopropyl, cyclopentyl,
2-(4,4-difluoro)-pyrrolidyl, 2-hydroxy-2-propyl, t-butyl, P
is piperonyl, 4-(benzyloxy)benzyl, 3-(benzyloxy)benzyl,
(4-benzyloxy-3-methoxy)benzyl, when R5 is H, R6 is benzyl,
piperonyl, CH2-pyridyl, 4-(benzyloxy)benzyl, morpholino,
tetrahydroisoquinolyl, 4-(3-hydroxypropyl)benzyl,
OH
2-(3-hydroxypropyl)benzyl, and ~ ~, , or -CH(Y)(Z)
with Y and Z both being as generally defined, but
particularly when Y is isopropyl, preferably in the D
configuration, or phenyl and when Z is benzyloxymethylene,
WO94/19008 2~ ~ l 2~ PCT~S94/00710
-15-
CHO, COOH, alkoxy or COOR4, when R5 is other than H it is
preferred that R5 be methyl, 4-hydroxybutyl or 3-hydroxy-
propyl and that R6 be benzoxy or benzyl, and when R5 and R6
form a heterocyclic moiety with the
nitrogen attached thereto, it is preferred tha-N~-~NcHo
and morpholino be the heterocyclic moieties. It is further
understood further subgroupings from the above combination
may be made from the groups of formula I and II defined
herein.
WO 94tl9008 ~ 5 ~ 2 9 PCTtUS94/OO'tlO
--16--
o ~ ~ ~ ~ o ~ ~ ~ ~ ~ \~
N N N N N L~ N QJ Q, Q Q~ \
ta c ~U c c c c c
m m m m )~"-
N N
C
~ a)
N N N :~1 >1 N N N N N N
C C C X X C C C C C C:
a) o o ~ aJ aJ ~ ~ a,
n ~ ~c s ~ n
C C :~ :~ ~ ~ a
o o x x x e e x x x x x x
o o o I I o o o o o o
N N N ~ >1 N N N N N N
C C - X X C C ~ C C C
O O ~ ~ ~ ~ ~ aJ
~ ~ Q ~ ~ ~ ~ ~ ~ ~ ~
~,~, _ _ _ _
N N
~r ~ ~ C C
~ qJ
D O
~rer
O O O O V O O ~ O O O O V
O O O O I O O o O O O O
S
:~
r, " ~ s
X X :D X X X X ~ V ~ V v X
o o Z o o o o o ,_, o ~ ~ o
o ~ ~ ~ ~ ~ ~ ~
N N ~ N N C N N ,, ~ ~ ' ~ cN
aJ a) \ a) a~ a) o o o a~
D (~ D D ~ D D ~ D
~ ~ ~~
Rl P2 Pl R5=~, R6
benzyloxy isopropyl 4-(benzyloxy)benzyl
CHO
(3-pyridyl)ethyl isopropyl 4-(benzyloxy)benzyl
~1 CHO
isoquinolyl isopropyl 4-(benzyloxy)benzyl l ~ oc~3 ~ r
benzyloxy isopropyl 4-(benzyloxy)benzyl ~OCH3
WO 94/19008 X~ PCT/US94/00710
--18--
~ :>. :>
N N N
C C C
~ O r o _ N
er ~r ~r
N N N" N !~ N N
C C C~- C ~ C C
Q Q QQ Q .D Q n
_~ X XX X X X X X
p~ O OO O O O O O
'~ N N N N N N
CC C C C C
J aJ O a) ~
Q QQ Q D Q Q
O O ~O O O O O
O O lO O O O O
~ O
S ~
~ ~,~, lJ >~ ~ N ~,
X X X~ X X C X
O O O~ O O Q O
N N N~ N N ~, N
C C C'~ C C C
, ~ a) s
Q
O
WO 94/19008 21~ 9 PCT/US94/00710
--19--
~O~ ~ ~ ~ ~
> ~ ~ >1 \I N N N N
~'~ O C O C O ) O C C C C
~ 'D Q ~') n ~
_ o -- o
U
u~In U'7 ~n In
N N N NN N N N
C C C C~ C C C
- -
x x x - ~ x x x x x
o o o p, o o o o o
N N N N N N N N
C C C C C C C C
D D D D D 1~ ~ D
,~ ~ ~ ~ O
>. V
OJ ~ ~ C ~ ~ Q~
L~ ~ ~ ~o
O O O O O ~ l ~ O
~ ~1 .
~ r
X X X Xa) a) X :~
O O O O ~ ~ O
N N N C~ N ~ ~ N Q,
C C C C
Q D D .D G Q~
r~ ~
K~ P2 P- R5~ ~6 L ,~
(3-pyridyl)ethyl isopropyl 4-(benzyloxy)benzyl R5=4-hydroxybutyl
R6=benzyl p~
benzyloxy isopropyl 4-(benzyloxy)benzyl R5=3-hydroxypropyl
R6=benzyl
(3-pyridyl)ethyl isopropyl 4-(benzyloxy)benzyl R5,R6= morpholyl
(3-pyridyl)ethylcyclopentyl 4-(benzyloxy)benzyl R5,R6= morpholyl
benzyloxy isopropyl 4-(benzyloxy)benzyl R5,R6= -N~_~NcHo
(3-pyridyl)ethyl isopropyl 4-(benzyloxy)benzyl tetrahydro- ~
isoquinolyl
benzyl methylamide benzyl perhydroiso-
WO94/19008 ~ 5 ~ PCT~S94/00710
.
-21-
DETAILED DESCRIPTION OF THE INVENTION
The pharmaceutical compositions of the present
invention are useful as inhibitors of retroviral proteases
and cellular y-glucosidase I which are required for virus
replication, particularly HIV-l and HIV-2, the prevention
or treatment of infection by the human immunodeficiency
virus (HIV), and the treatment of consequent pathological
conditions such as the acquired immunodeficiency syndrome
(AIDS) in mammals capable of being infected with HIV virus.
Treating AIDS (preventing infection by HIV or treating
infection by HIV) is defined as including, but not limited
to, treating a wide range of states of HIV infection: AIDS,
ARC (AIDS related complex), both symptomatic and
asymptomatic, and actual or potential exposure to HIV. For
example, the pharmaceutical compositions of this invention
are useful in preventing infection by HIV after suspected
past exposure to HIV by, e.g., blood transfusion,
accidental needle stick, or exposure to patient blood
during surgery.
For these purposes, the compounds of the present
invention may be administered orally, parenterally
( including subcutaneous injections, intravenous, intra-
muscular, transdermal, intrasternal injection or infusion
techniques), by inhalation spray, or rectally, in dosage
unit formulations containing convention non-toxic pharma-
ceutically acceptable carriers, adjuvants and vehicles.
Thus, in accordance with the present invention there isfurther provided a method of treating and a pharmaceutical
composition for treating HIV infection and AIDS. The treat-
ment involves administering to a patient in need of such
treatment a pharmaceutical composition comprising a pharma-
ceutical carrier and a therapeutically effective amount of
WOg4/1gO08 ~ PCT~S94/00710
-22-
a compounds of formula I and II of the present invention,
or a pharmaceutically acceptable salt thereof.
These pharmaceutical compositions may be in the form of
orally-administerable suspensions or tablets; nasal sprays;
sterile injectable preparations, for example, as sterile
injectable aqueous or oleagenous suspensions or
suppositories) or they may be administered transdermally.
When administered orally as a suspension, these
compositions are prepared according to techniques well
known in the art of pharmaceutical formulation and may
contain microcrystalline cellulose for imparting bulk,
alginic acid or sodium alginate as a suspending agent,
methylcellulose as a viscosity enhancer, and
sweetener/flavoring agents known in the art. As immediate
release tablets, these compositions may contain
microcrystalline cellulose, dicalcium phosphate, starch,
magnesium stearate and lactose and/or other excipients,
binders, extenders, disintegrants, diluents and lubricants
known in the art.
For oral administration the compositions of formula l
can be formulated into solid or liquid preparations such as
25 capsules, pills, tablets, troches, lozenges, melts, powders,
solutions, suspensions, or emulsions. The solid unit dosage
forms can be a capsule which can be of the ordinary hard- or
soft-shelled gelatin type containing, for example,
surfactants, lubricants, and inert fillers such as lactose,
sucrose, calcium phosphate, and cornstarch. In another
embodiment the compounds of this invention can be tableted
with conventional tablet bases such as lactose, sucrose, and
cornstarch in combination with binders such as acacia,
cornstarch, or gelatin, disintegrating agents intended to
35 assist the break-up and dissolution of the tablet following
administration such as potato starch, alginic acid, corn
starch, and guar gum, lubricants intended to improve the
wo 94tl9008 ~ I 2 g PCT~S94/00710
flow of tablet granulations and to prevent the adhesion of
tablet material to the surfaces of the tablet dies and
punches, for example, talc, stearic acid, or magnesium,
calcium, or zinc stearate, dyes, coloring agents, and
5 flavoring agents intended to enhance the aesthetic qualities
of the tablets and make them more acceptable to the patient.
Suitable excipients for use in oral liquid dosage forms
include diluents such as water and alcohols, for example,
ethanol, benzyl alcohol, and the polyethylene alcohols,
10 either with or without the addition of a pharmaceutically
acceptably surfactant, suspending agent, or emulsifying
agent.
When administered by nasal aerosol or inhalation, these
compositions are prepared according to techniques well
known in the art of pharmaceutical formulation and may be
prepared as solutions in saline, employing benzyl alcohol
or other suitable preservatives, absorption promoters to
enhance bioavailability, fluorocarbons, and/or other
solublizing or dispersing agents known in the art.
The compositions including formula l and 2 may also be
administered parenterally, that is, subcutaneously,
intravenously, intramuscularly, or interperitoneally, as
25 injectable dosages of the compound in a physiologically
acceptable diluent with a pharmaceutical carrier which can
be a sterile liquid or mixture of liquids such as water,
saline, aqueous dextrose and related sugar solutions, an
alcohol such as ethanol, isopropanol, or hexadecyl alcohol,
30 glycols such as propylene glycol or polyethylene glycol,
glycerol ketals such as 2,2-dimethyl-1,3-dioxolane-4-
methanol, ethers such as poly(ethyleneglycol) 400, an oil, a
fatty acid, a fatty acid ester or glyceride, or an
acetylated fatty acid glyceride with or without the addition
5 of a pharmaceutically acceptable surfactant such as a soap
or a detergent, suspending agent such as pectin, carbomers,
methylcellulose, hydroxypropylmethylcellulose, or
WO94/19008 ~ 5~q ~ PCT~S94100710
-24-
carboxymethylcellulose, or emulsifying agent and other
pharmaceutical adjuvants. Illustrative of oils which can be
used in the parenteral formulations of this invention are
those of petroleum, animal, vegetable, or synthetic origin,
5 for example, peanut oil, soybean oil, sesame oil, cottonseed
oil, corn oil, olive oil, petrolatum, and mineral oil.
Suitable fatty acids include oleic acid, stearic acid, and
isostearic acid. Suitable fatty acid esters are, for
example, ethyl oleate and isopropyl myristate. Suitable
lO soaps include fatty alkali metal, ammonium, and
triethanolamine salts and suitable detergents include
cationic detergents, for example, dimethyl dialkyl ammonium
halides, alkyl pyridinium halides, and alkylamines acetates;
anionic detergents, for example, alkyl, aryl, and olefin
15 sulfonates, alkyl, olefin, ether, and monoglyceride
sulfates, and sulfosuccinates; nonionic detergents, for
example, fatty amine oxides, fatty acid alkanolamides, and
polyoxyethylenepolypropylene copolymers; and amphoteric
detergents, for example, alkyl-beta-aminopropionates, and 2-
alkylimidazoline quarternary ammonium salts, as well as
mixtures.
When rectally administered in the form of
suppositories, these compositions may be prepared by mixing
the drug with a suitable non-irritating excipient, such as
cocoa butter, synthetic glyceride esters or polyethylene
glycols, which are solid at ordinary temperatures, but
liquidize and/or dissolve in the rectal cavity to release
the drug.
The pharmaceutically acceptable addition salts, either
cation or anion salts, are those salts that are not
substantially toxic at the dosage administered to achieve
the desired effect and do not independently possess
significant pharmacological activity. The conversion of the
composition of formula I and II may independently or jointly
be formulated as addition salts. Illustratively, cation
WO94/19008 2 1 ~ 5 ~ ~ 9 PCT~S94/00710
salts include those of alkali metals, as for example, sodium
and potassium; alkaline earth metals, such as calcium and
magnesium; light metals of Group IIIA including aluminum;
and organic primary, secondary and tertiary amines, as for
5 example, trialkylamines, including triethylamine, procaine,
dibenzylamine, l-ethenamine, N,N'-dibenzylethylenediamine,
dihydroabiethylamine, N-(lower)alkylpiperidine, and any
other suitable amine. Sodium salts are preferred. An
acceptable acid addition salt may be carried out by treating
lO such compounds in a conventional manner with an inorganic
acid or example a hydrobromic acid, sulfphuric acid, nitric
acid, phosphoric acid etc., or with an organic acid such as
acetic acid, citric acid, maleic acid, fumaric acid,
tartaric acid, methanesulfonic acid, p-toluenesulphonic
15 acid.
Dosage levels of the order of 0.02 to 5.0 or lO.0 grams
per day of the composition are useful in the treatment or
prevention of the above-indicated conditions, with oral
doses two to five times higher. ~or example, infection by
HIV is effectively treated by the administration of from lO
to 50 milligrams of the compound per kilogram of body
weight from one to three times per day. It will be
understood, however, that the specific dose level and
frequency of dosage for any particular patient may be
varied and will depend upon a variety of factors including
the activity of the specific compounds employed, the
metabolic stability and length of action of compounds in
combination with each other, the age, body weight, general
health, sex, diet, mode and time of administration, rate of
excretion, drug combination the severity of the particular
condition, and the host undergoing therapy.
The present invention is also directed to combinations
of the HIV protease-inhibitory compounds with one or more
agents useful in the treatment of AIDS, such as, for
example, with known antiviral agents suitable for treating
HIV l and HIV 2 viral infections, e.g., a ester of
WO94/19008 ~ PCT~S94/00710
-26-
castanospermine of formula I with a viral protease
inhibitor of formula II. For instance, the present
invention includes the use of a glycoprotein processing
inhibitor of formula I and a aspartyl protease specific
inhibitor of formula II and for the preparation of a
pharmaceutical formulation for simultaneous, separate or
sequential use for treating an HIV infection wherein the
said compounds of formula l are
HO ~
N
R20 -- -< > Formula I
~
OR1 OR
20 wherein Rl is a C1 8 alkanoyl, Cl lo alkenoyl, Cl_8
alkoxyacetyl, or benzoyl optionally substituted with an
alkyl or halogen group; or a pharmaceutically acceptable
salt thereof and said compound of formula II is
P2 P
R1CNHCH CNHCHC-CF2C-NR5R6
O O O O
Formula 11
and the hydrates, isosteres and the pharmaceutically
acceptable salts thereof wherein Rl is benzyl
oxy, ~ SO2HN, (3-pyridyl)ethyl, isoquinolyl, 4-alkoxy-
benzyloxy, or morpholyl, P2 is isopropyl, cyclopentyl,
2-(4,4-difluoro)-pyrrolidyl, 2-hydroxy-2-propyl, t-butyl, P
is piperonyl, 4-(benzyloxy)benzyl, 3-(benzyloxy)benzyl,
(4-benzyloxy-3-methoxy)benzyl, when R5 is H, R6 is benzyl,
WO94/19008 2 I S 5 1 Z 9 PCT~594/00710
piperonyl, CH2-pyridyl, 4-(benzyloxy)benzyl, morpholino,
tetrahydroisoquinolyl, 4-(3-hydroxypropyl)benzyl,
5 2-(3-hydroxypropyl)benzyl, and ~ t~ r -CH(Y)(Z)
with Y and Z both being as generally defined, but
particularly when Y is isopropyl, preferably in the D
configuration, or phenyl and when Z is benzyloxymethylene,
CHO, COOH, alkoxy or COOR4, when R5 is other than H it is
preferred that R5 be methyl, 4-hydroxybutyl or 3-hydroxy-
propyl and that R6 be benzoxy or benzyl, and when R5 and R6
form a heterocyclic moiety with the
nitrogen attached thereto, it is preferred tha -N~_~NCHO
and morpholino be the heterocyclic moieties. It is further
understood ~urther subgroupings from the above combination
may be made from the groups of formula I and II defined
herein.
A preferred composition containing the compounds of
formula I and II are those compositions which contain at
least 10% of the compounds of formula I or those
compositions that contain at least 10% of the compounds of
formula II.
The compounds of this invention may be assayed for
their inhibition of HIV replication using the following
published techniques.
EXPERIMENTAL PROCEDURES
The following examples illustrate various aspects of
this invention. The following information on reagents, cell
lines, virus strains and assays describe the usefulness of
the previously described compositions. Further methods and
procedures dealing with the assays are known in the art.
WO94/19008~ 9 PCT~S94/00710
-28-
To study the effects of the described compositions the
growth of HIV-l was used to study the treatment of AIDS as a
useful model for HIV.
5 Druq Combination Assay
For the investigation of drug combinations, the MTT cell
viability assay (Pauwels et al., J. Virol. Methods, 1988,
20, 309-321) was used. Various drug combinations were
achieved by creating chequerboards with one compound being
lO titrated horizontally with the second compound being
titrated vertically across a 96 well microtitre plate using
multichannel pipettes. The use of six microtiter plates was
required for each assay (only inner 60 wells) with
quadruplicate wells for each drug combination. Doubling
15 dilutions or half log dilutions, with the end rows left drug
free, were usually made.
MT-4 cells infected with lO0 TCIDso of HIV-lRF per 5 x
104 cells were added to each well at a concentration of 5 x
20 104 cells per well and after incubation at 37OC of six days,
lOul of acidified isopropanol was added and the plates read
at 540nm using a Multiscan MCC/340 spectrophotometer (Flow
Laboratories). The raw plate data were captured onto floppy
disc using Ultroterm (LKB). Subsequent data reduction was
5 performed using Excel (Microsoft). This enabled the mean
O.D. values for each drug combination to be calculated and a
series of l9 dose response curves, i.e. the dose response of
each drug at a fixed concentration of the other to be
generated with minimal user intervention. The top four left
30 hand corner wells which received the highest concentration
of each drug were used as a positive control (i.e. cells
viable totally protected) and the four bottom right hand
corner wells without any drug were used as the negative
control (i.e. total cell death).
WO94/19008 21 ~ ~ ~ 2 9 PCT~S94/00710
-29-
Analysis of Druq Combinations by Isobolic Method
From the dose response curves, the IC50 for each drug,
either alone or in combination with a fixed concentration of
5 the other, was calculated and isobolograms plotted (Suhnel,
Antiviral Research 13, 23-40(1990)). It was possible to
determine from the shape of the isobole whether the drugs
had a synergistic relationship (concave), additive effect
(linear) or showed anatagonism (convex). Additionally a
lO combination index (CI) was calculated (Suhnel, l990).
The table provides ED50 values computed from dose
response lines for each compound in the presence of a fixed
5 concentration of the other compound.
From these data was determined the combination index
(CI) from the formula:-
CI = (D)l + (D~2 + a (D)1 (D)2
(DX)l (DX)2 (Dx)1 (Dx)2
where a = l for mutually non-exclusive agents
and (DX)l = IC 50 of drug l above;
(DX)2 = IC 50 of drug 2 above;
(D)l and (D)2 = concentrations of drugs l and 2
in combination giving 50% inhibition;
If it is:
<l (synergism);
CI = l (zero interaction);
>l (antagonism).
(ref: J. Suhnel, Antiviral Research 13 (l990), 23-40.)
WO94/19008 ~ PCT~S94/00710
-30-
EXAMPLE 1
ANTIVIRAL ACTIVITY OF RO-31-8959 AND MDL7369 WITH BUCAST
Three protease inhibitors MDL73669 (Tables lA and lB),
Ro-31-8959 (Tables 2A and 2B) and ~DL 74538 (Tables 3A and
3B) were investigate in combination with the glycoprotein
processing inhibitor MDL 28574 (BUCAST). The method used to
test the effectiveness of the combination was assessed by
10 looking at the various concentrations of the compounds in
combination on antiviral activity determined by using the
cell viability assay previously described. The method of
study of these compounds allowed for identification of
synergistic interactions by analysis of the data values for
5 a Combination Index (C.I.).
The following abbreviations apply to the following
compounds:
MDL73669 = [1(S)-[[3,3-difluoro-2,4,-dioxo-1-[[4-
(phenylmethoxy)phenyl]methyl]-4-
[(phenylmethyl)amino]butyl]aminocarbonyl]-2-
methylpropyl]carbamic acid, phenylmethyl ester.
MDL74538 = N-[4-(N-benzyloxycarbonyl-L-valyl)amino-2,2-
difluoro-1,3,-dioxo-5-(4-benzyloxy)phenyl-pentyl]-(O-
benzyl)-D-valinol.
MDL28574 = (Bucast)[lS-(1~,6B,7~,8B,8a~)]-octahydro-
1,6,7,8-indolizinetetrol 6-butanoate
Ro-31-8959 = N-tertiary butyl-decahydro-2[2(R)-hydroxy-
4-phenyl-3(S)-[[N(2-quinolylcarbonyl)-L-
asparaginyl]amino]-butyl]-(4aS,8aS)-isoquinoline-3(S)-
carboxamide.
WO94/19008 Z~ 5] 2q PCT~S94/00710
Table lA and lB: Concentrations of MDL73669 and BMDL28574,in combination and alone required to give 50~ protection of
MT-4 cells infected with ~IV-l~ and the combination indices
calculated from these values.
Fixed Conc IC 50
MDL 73669 MDL 28574 C.l.*
(~M) (~M)
0 100
0.0001 100
0.0003 100
0.001 45 0.45
0.003 25 0.25
0.010 21 0.22
0.03 32 0.37
lS 0.1 20 0.4
0.3 30 0.95
*The combination (C.I.) indices
indicate synergy
Fixed Conc IC 50
MDL 28574 MDL 73669 C.l.*
(~M) (~M)
0 0.6
0.1 0.55
0.3 0.6
l 0.5 0.85
3 0.5 0.89
0.42 0.87
0.002 0.3
1 00
300
~The combination (C.I.) indices
indicate synergy
WO94/19008 - r ~ PCT~S94/00710
-32-
Table 2A and 2B: Concentrations of Ro-31-8959 and MDL
28574, in combination and alone required to give 50%
protection of MT-4 cells infected with ~IV-l~ and the
combination indices calculated from these values.
Fixed Conc IC 50
Ro-31-8959 MDL 28574 C.l.*
(nM) (~M)
0 100
0.1 50 0.52
l o 0.3 60 0.66
12 0.28
3 6.8 0.52
1 0
1 00
I he combinationindices (C.l.) indicate
synergy
Fixed Conc IC 50
MDL 28574 Ro-31-8959 C.l.*
(~M) (nM)
0 6.9
0.1 6.7
0.3 9.0
6.1
3 12
1.4 0.32
0.38 0.37
1 00
300
Y I he combmatlonlndlces ~C.l.) Indlcate
synergy
WO94/19008 2~ PCT~S94/00710
Table 3A and 3B: Concentrations of 74538 and MDL 28574, in
combination and alone required to give 50% protection of MT-
4 cells infected with HIV-l~ and the combination indices
calculated from these values.
Fixed Conc IC50
MDL 74538 MDL 28574 C.l.*
(nM) (~M)
0 23
o.1 20 0.87
0.3 9.5 0.42
1.0 25 1.1
3.0 8.2 0.43
10.0 6.2 0.39
30.0 3.0 0.48
~ I he comblnatior (C.l.) indicate synergy.
Fixed Conc IC 50
MDL 28574 MDL 74538 C.l.*
(I~M) (nM)
0 100
3.0 30 0.47
10 0 2 0.46
~ I he combinatlor (C.l.) indlcate synergy.
