Note: Descriptions are shown in the official language in which they were submitted.
WO 92/16494 ~ o ~ ~ PCT/GB92/00438
LONG CHAIN ANTIVIRAL COMPOUNDS
., .
This invention concems improvements in chemical compounds,
more especially it concerns compounds and pha~maceutical compositions. ln
5 particular it concerns compositions and compounds having activity in in vitro
tests on Human Immunodeficiency Virus-infected cells.
The disease known as Acquired lmmune Deficiency Syndrome
(AIDS) caused by infection by HlV has attracted immense research efforl
because of the effects of the disease on infected individuals and the dangers
of the disease spreading to a wider section of the population. In general.
~- although various chemo-therapeutic treatments have been advocated. and some
compounds have emerged as a potential basis ~or treatment. there is still a
need for alten~atives. In particular, most treatments such as the compound
. .
wo 92/16494 Pcr/GB92/00438
ii 8 - 2 -
known as AZT have a high toxicity to cells, and it would be desirable to
find compounds which are less toxic. In mam the development of resistance
to AZT has been identified as an additional clinical problem.
We have found a group of compounds which show protective
properties in m vitro screens of cells challenged with HIV- I and/or HIV-2 .
and are therefore indicated as having potential for the treatment of AIDS and
AIDS Related Complex and other viral and especially retroviral infections.
Accordingly, the present invention provides the use of compoundls defined
below, in pharrnaceutical compositions for treating HIV-infected patients. The
invention further provides pharmaceutical compositions comprising a said
compound in combination or association with a pharmaceutically acceptable
diluent or carrier, for the treatment of HIV-infected patients. The invention
may also be defined as the use of a said compound for the manufacture of
a medicarnent for the treatment of HlV-infected patients. The invention
further provides a process for the production of a phannaceutical composition
for the treatment of a HlV-infected patient, comprising the combination of
a compound as defined below with a phalmaceutically acceptable diluent or
carrier, and forrnulating said composition into a form suitable for
administration to said patient. The invention also provides a method of
treatment of an HIV-infected patient, comprising administering to said patient
an effective dose of a said compound. It is to be understood that treatment
includes prophylactic treatment of patients at risk. in view of the protective
properties observed. Whilst this description is especially directed to
- 25 combating HIV, this invention includes other aspects in which other diseases
may be treated, including for exarnple microbial infections.
,.
WO 92/l6494 ~ ; 8 PCr/GB92/00438
- 3 -
A 2.2'-dimer of cycla~n has been reported as being isolated as
a 2% by-product in the synthesis of cyclam (1.4.8.11-tetraazacyclotetradecane)
(Barefield et al . J C S Chem Comm (1981). 302). This compound was
stated to be insoluble in water. We believe that the insoluble 2.2'-bicyclam
is a rnixture of the 2R.2 R and 2S,2 S enantiomers: we have characterised
a soluble dimer which we believe to be the meso 2R.2' S isomer. The
6.6-bicyclam isomer has been reported by Pabbrizi et al, lnorg Chem 25.
2671 (1986). C~rtain N.N'-linked bicyclic compounds have been reported by
Ciampolini et a Inorg. Chem. 26, 3527 (1987). No biological activity has
been suggested for such compounds.
US Patent 4,156,683 discloses monocyclic and bicyclic
macrocyclic compounds, which are said to have biological activity in
regulating sodium~ potassium and calcium levels in mamtnals. Additionally.
a specific group of N-alkylated monocyclic compounds are said to possess
activity against A2 influenza viruses in a modified Hermann test on chick
fibroblast tissue. The single example mentioned of such N-alkylates/
monocyclic compounds is 4,13-dimethyl-1,7,10.16-tetra~,13-diazacyclo-
octadecane. It is also said that the preferred compounds, which form
complexes of greater stability, are those having three blidging chains between
bridgehead nitrogen atoms. that is fused bicyclic compounds.
It is also reported in Chemical Abstracts 88 1052925 (1978) that
;. certain fused triple ring heterocyclic compounds ha~te activity against A2
~ngland influenza virus in chick embryos.
wo 92/1~94 pcr/GB92/oo438
j 3 ~ ~ 4
Rowe~t et al have reported in Biochem J 1987, 245(3) 641-7
that some simple cyclic polyarnines reduce the infectivity of bactenophages.
Bacteriophages infect bacteria but do not cause any human disease. and this
paper does not discuss anti-viral activity in humans.
Certain tetrarnines have been synthesised and shown to be active
in tests indicating antimalalial activity. (Edwards et al, J Med Chem. 34.
No 2, 56911991]).
l 0 Our US Patent 5 ,021,409 discloses that linked cyclic
polyheterocyclic compounds have activity against HIV.
The present invention provides active compounds of the general
formula 1,
Z ~ ~)n ~ Y
in which each :Z and Y is independently a polyheteroalkyl chain having a
chain length of 9 to 32 members or a polyheterocyclic moiety having 'from
9 to 32 ring members, and each having from 3 to 8 heteroatoms, wherein
the heteroatoms are selected from nitrogen, oxygen and sulphur, provided that
at least one of Z and Y is a said chain,
A is a linking atom or group, and
n is O or an integer from I to 6.
The invention also encompasses acid addition salts and metal
2~ complexes of the compounds of formula 1.
3 ~
WO 92/16494 pcrtGB92/oo438
In the above forrnula, A may be alkylene, for example 1,3
propandiyl, unsaturated alkylene or a group selected from aryl, alkylaryl,
alkylarylalkyl, fused aryl, polyoxoethylene, carboxylate, esters ancl arnides, or
a nitrogen or sulphur atom. In a particularly preferred embodiment of the
5 invention, A is alkylene of I to 6 carbon atoms or is alkylphenylalkyl in
which each alkyl is of I to 6 carbon atoms and the phenyl ring is
unsubstituted or substituted by methoxy~ fluorine. chlorine, bromine or nitro~
and the alkyl groups are in the m- or p- positions relative to one another.
The chains or heterocycles Z and Y may be linked to the
remainder of the molecule through carbon or heteroatoms, for example linked
through C.C'~C,N' or N.N',
Each of Z and Y may contain nitrogen and/or oxygen and/or
15 sulphur heteroatoms; preferably the moieties contain nitrogen atoms with
optional further heteroatoms selected from oxygen and sulphur, A particular
embodiment of the invention relates to compounds in which all the chain or
nuclear heteroatoms are nitrogen atoms. A more preferred embodiment
relates to compounds in which each of Z and Y contain four nitrogen atoms.
The chains or cyclic moieties may be substituted or
unsubstituted, and may contain unsaturation. Suitable substituents may be
selected from halogens, especially fluorine, chlorine or bromine. -NH2. -OH~
;. -COOH, ester groups~ -CONH2 and alkyl or aryl groups~ eg of up to 10
25 carbon atoms, which themsel~es may be substituted by the aforementioned
substituents. Preferred chains are of 8 to 18 atoms. especially 8 to 16
atoms, preferably with 4 to 8 nitrogen heteroatoms; preferred cyclic moieties
WO92/16494 ~ `?~j 3;(~ pcr/GB92/oo438
- 6 -
are those of 10 to 24 ring members. especially 12 to 18 ring members, and
preferred numbers of nuclear nitrogen atoms are 4 ~o 6. It is convenient
that if two chains are linked. they are identicah
S The invention also includes what may be termed "pro-drugs".
that is protected forms of the linked compounds, which release the compound
after adminis~ration to a patient. For exarnple, the compound may carry a
protective group which is split off by hydrolysis in body fluids. e.g. in the
bloodstrearn, thus releasing active compound. A discussion of pro-drugs may
be found in "Smith and Williams' Introduction to the Principles of Drug
Design ", H J Smith, Wright, 2nd Edition, London 1988 .
The compounds of general formula I are believed to be novel.
They may be prepared by the man skilled in organic syntheses, using a
variety of methods analogous to those already known in the literature. Thus,
Ciampolini et al ~Inorg Chem 26, 3527[19871) have demonstrated the
syntheses of alkyl-linked bicyclams by condensation of N,N',Nn-tritosylcyclam
with bis (tosyloxy)alkanes or with bis(acyl chlorides). The syntheses of C.N'-
and N,N'-linked compounds of formula I may be perfonned in a similar
`~ 20 manner by reacting activated precursors (eg compounds substituted with alkyl
chains terminated with halides or activated carboxylates) with (N-l)-substitutedheterocycles (eg N,N'.N"-tritosylcyclam) or (N-l)-substituted polyazaalkanes
followed by deprotection.
:
Thus~ the invention further provides a process for the production
of compourlds of formula I. comprising reacting a compound of formula 11
or 111,
wo 92/16494 ~ ~ d pcr/GB92/oo438
Z - (A),~ - X 11
Z _ Xl lll
in which Z. A and n are as defined above. and
Xl is a reac~ive atom or group
S with a compound of forrnula IV or V. respectivelly,
X2 - Y IV
X2 ~A)n - Y V
in which Y. A and n are as defined above, and
X2 is a reactive atom or group~
under conditions such that a compound of formula I is forrned and the
reactive atoms or groups Xl and x2 are split off. It will be realised that
it may be desirable to protect other react~ve sites on the moieties Z and Y,
for example amino nitrogen atoms, from participation in unwanted reactions,
and this may be done by methods well known to the skilled synthetic
chemist, fol1Owed by deprotection. Such process variants are to be
understood as being within the scope of the invention.
The compounds are indicated for the treatment of viral
infections, especially retrovirus infections and particularly HIV infections, and
the compounds of formula 1, are to be be considered as active compounds
for the pharmaceutical compositions, processes for making the same and
methods of treatment mentioned above. In these aspects of the invention.
it is to be understood that rneso forrns. enantiorners and resolved optically
active forms of the compounds of formula 1. are included. Also. it is to be
considered within the invention. compounds of forrnula I diluted with
non-toxic or with other active substances. Acid addition salts, for example
hydrochlorides. and non-toxic labile metal complexes of the compounds of
WO 92/16494 ~ ~ PCr/GB92/00438
forrnula I are also active compounds according to the present invention.
Non-toxic in the present context has to be considered with reference to the
prognosis for the infected patient without treatment. Zinc and nickel
complexes are especial]y indicated. whereas less labile metal atoms such as
cobalt and rhodium are less preferred because of likely lower selectivity.
The invention will now be described by way of example only.
- EXAMPLE I
a) N-TosYI-3-asnino~roPanoic acid
To a mixture of 3-aminopropanoic acid ( 1 equivalent) in
water/dioxane ( 1/1 ) was added a solution of NaOH (3 equivalents) in water
(50% solution). To the vigorously stisred reaction mixture tosyl chloride (1.1
equivalents) was added portion-wise over 3 hours. The reaction mixture was
stirred at room temperature for 6 hours. The reaction mixture was washed
with ether and the aqueous phase was acidified to pH 2.0 with concentrated
HCI. The acidffled solution was extracted with ethyl acetate three times.
The combined organic extracts were washed with brine, dried over magnesium
sulphate, filtered and concentrated to give the desired product as a white
solid: m p 118.5-120C; yield 85%.
b) SuccinimidYI N-tosyl-3-arninopropanoate
To a solutiosl of N-tosyl-3-arninopropanoic acid ( I equivalent)
and N-hydroxysuccinimide (I equivalent) in ethyl acetate was added dropwise
a solution of dicyclohexylcarbodiimide (I equivalent) in ethyl acetate. The
reaction mixture was stirred at room temperature for 4 hours during which
time copious amounts of a white precipitate formed. The solids
wO 92/16494 2 :~ ;v ~; t3 ~) ~ pcr/GB92/oo438
g
(dicyclohexylurea by-product) were removed by filtration and the filtrate was
concentrated to drynes~ to give a thick colourless viscous oil The oil was
treated with ether/ethyl acetate ~2/1) tO cause precipitation of a white solid:
yield 72 %; m p 126-129 C .
c) rneso- 1,2~3 ~4-tetraaminobutane
The tetraamine was synthesised from meso-elythritol according
to the method of R L Weller, J Org Chem 49, 5150 ~1984).
d) meso-1,2,3,4-tetrakis~N-~N-(3-tosvlarnidolpro~anamid~)-
butane
To a solution of 1~2,3 ,4-meso-tetraaminobutane ( I equivalent)
in dimethylforrnarnide was added dropwise a solution of succinirnidyl
N-tosyl-3-aminopropanoate (8 equivalents) in dimethylformamide. The reaction
mixture was heated at 50C for 5days. The DMF was removed under
reduced pressure and disso1ved in THF/lN NaOH and stirred overnight during
which time a white/yellow solid precipitated which was removed by filtration.
The filtrate was washed with water and dried over magneshlm sulphate to
give 12.0g of a light yellow solid; m p 195-197C; yield 70%; mass spec.
e) meso- 1,2 , 3 ,4-tetrakis(N-mesyl-N ' -tosYl- 13 diarnino-
proPYl)butane)
To a solution of tetraarnide-tetratosylate ( I equivalent) as
synthsised above in tetrahydrofuran was added borane.tetrahydrofuran complex
(I M: 10 equivalents). The reaction mixture was heated at reflux for 16
hours. The reaction mixture was cooled to room temperature and excess
borane was quenched by the addition of methanol. The solution was
WO 92/16494 pcr/GB92/oo438
~ ^; r~ 10
concentrated to dryness and more methanol was added and the solution was
re-concentrated The residue was treated with 10 % HCl and then basified
to pH 12 with 10 N ~laOH. The cloudy solution was extracted with ethyl
acetate. The combined organic extracts were dried over magnesium sulphate.
S filtered and concentrated to give an off-white solid. This product is
approximately 50% desired tetraa~nine and was used directly.
The solid was dissolved in dichloromethane and triethylamine
(6 equivalents) was added. To the homogeous so}ution a solution of mesyl
chloride (4.5 equivalent) in dichlorornethane was added dropwise and the
reaction mixture was stirred overnight at room temperature. Dichloromethane
and water were added and the phases were separated. The organic phase
was washed with 10% HCI and brine, dried over magnesium sulphate and
concentrated to give a white amorphous solid. The solid was dissolved in
ethyl acetate and passed through a column of silica gel using ethyl acetate
as eluant. The eluate was concentrated to dryness to give the desired
product as an arnorphous solid; overall yield 30~; mass spectrum '1275
(14%), llg7 (100%), 559 (13%).
f) meso-1,2,3,4-tetrahs(N-1,3-diaminopropYl)butane
The tetrarnesyltetratosylate was dissolved in 48% HBr/HOAc and
heated at 100C for 48 hours. The solvents were removed under reduced
pressure to give a brown oil. The oil was dissolved in a minimum amount
of concentrated HCI and concentrated to give a brown amorphous oil. The
product was characterised by its mass spectrum. and designated compound A.
WO 92/16494 ~, ~ iJ ~ 8 pcr/GB92/oo438
Other compounds considered to be significant in the present
invention are:
4 ,4-( 1 . 3-propanediyl)-bis- 1 .4 . 8 ~ I I -tetraazadodecane
2~4' -( I ~3-propanediyl)-2-rac-tetraazacyclotetradecyl~ -(1~4~8.1 1 )-tetraazadodecane
1 .4 ' -( I . 3-propanediyl )- I -tetraazacyclotetradecyl-4 -~ I .4 . 8 . I I )-tetraa7aundecane
2.4' -(1 .3-propanediyl)-rac-2-eetraazacyclotetradecyl-4 -(1,4.8.11)-tetraazaundecane
I .4 -(1 .3-propanediyl~ tetraazacyclotetradecyl-4 -( I .4, 8, I I )-tetraazadodecane
Additionally, the following compounds may be synthesised using
methods analogous to those illustrated above and those described in the
literature, and are indicated for further testing in the biological fields
described below.
4 ~4 ' -( I ,2-ethanediyl)-bis- 1 ,4, 8, I I -tetraazadodecane
4 ,4 ' -(1 ,4-butanediyl)bis- 1,4, 8 ,1 1 -tetraazadodecane
4.4' -( I ,5-pentanediyl)-bis- 1,4,8,1 1 -tetraazadodecane
4.4' -( I .6-hexanediyl)-bis- 1,4,8,1 I-tetraazadodecane
2 ,4 ' -( I ,2-ethanediyl)-rac-2-tetraazacyclotetradecyl-4 ' -( I ,4, 8, I I )-tetraazaundecane
2,4' -(1 ,4-butanediyl)-rac-2-tetraazacyclotetradecyl-4' -( I ,4,8, I I )-tetraazaundecane
2.4' -(1,5-pentanediyl)-rac-2-tetraazacyclotetradecyl-4 -(1.4.8.1 1)-tetraazaundecane
2.4' -( I .6-hexanediyl)-rac-2-tetraazacyclotetradecyl-4 ' -( I .4, 8 . I I )-tetraazaundecane
1.4'-( I ,2-ethanediyl)- 1 -tetraazacyclotetradecyl-4' -(1.4,8, I I )-tetraa~aundecane
1.4' -( I .4-butanediyl)- 1 -tetraazacyclotetradecyl-4' -(1,4,8.1 1 )-tetraazaundecane
I ,4 -( I ,5-pentanediyl)- 1 -tetraazacyclotetradecyl-4 -( I ,4 ,8.1 1 )-tetraazaundecane
1.4' -( I .6-hexanediyl~- 1 -tetraazacyclotetradecyl-4 -( I .4.8.1 1 )-tetraazaundecane
wO 92/16494 PCr/GB92/00438
~`~33~ - 12 -
2 ,4' -( I,2-ethanediyl)-2-rac-tetraazacyclotetradecyl-4' -~1 ,4~8. I 1 )-tetraazadodecane
2 .4 ' -( I .4-butanediyl )-2 -rac-tetraazacyclotetradecyl-4 ' -(1 .4 . 8, I I )-tetraazadodecane
2.4' -(1.S-pentanediyl)-2-rac-tetr~7acyclotetradecyl~'-(1.4.8.1 1)-tetraazadodecane
2.4' -( I .6-hexanediyl)-2-rac-tetraazacyclotetradocyl-4' -(1.4.8.1 1 )-tetraazadodecane
s
I .4 ' -( I .2-ethanediyl)- 1 -tetraazacyclotetradecyl-4 ' -( I ,4, 8 ,1 1 )-tetraazadodecane
! .4 ' -(1 ,4-butanediyl)- 1 -tetraazacyclotetradecyl-4 ' -( I ,4, B ,1 1 )-tetraazadodecane
I .4 -( I .5-pentanediyl)- 1 -tetraazacyclotetradecyl-4 ' -( I ,4, 8 . I I )-tetraazadodecane
1 .4 -( I, 6-hexanediyl)- 1 -tetraa~acyclotetradecyl-4 ' -( I ,4 . 8 . I I )-tetraazadodecane
EXAMPI E 2
a) To a solution of p-dibromoxylene (2.0g, 7.54mmol) and
K2 CO3 (524mg, 3 . 77mmol) in 20ml dry CH3 CN was added tritosylcyclam
(l.Og, 1.506mmol) in 10ml of dry CH3CN dropwise over 45 minutes at
room temperature. After ~he reaction mixture had been stirred for 2 hours
at room temperature, the solution was concentrated, taken up in CH2 Cl2
(ISOml), washed with H20 (10mi) and dried over MgSO4. The organic layer
was concentrated and purified by chromatography on silica gel (elu~ion with
5% EtOAc/hexane to 60% EtOAc/hexane) to afford 980mg (77%) of
1-bromomethyl-4~ (4,8,1I-tritosyl-l,4,8,1I tetraazacyclotetradecyl)methyl)-
benzene as a white solid.
b) To a solution of N.N'-Bis(3-aminopropyl)ethylenediamine
(771mg. 4.42mmol) K2CO3 (17Smg. 1.26mmol) in 20ml of CH3CN was
added 950mg. 0.532mmol of the product of step a) in 10ml of dry CH3CN
dropwise over I hour at room temperature. After the reaction mixture had
been stirred for 3 hours at room temperature. the solution was filtered and
wo 92/16494 ~ .~ U ~ pcr/GB92/oo43B
the filtrate was concentrated. The excess tetraamine was removed by
distillation of the concentrated filtrate on a Kugel Rohr apparatus ( 100C:
O.lmm Hg) and the residual oil was then tal¢en up in lOOml of chloroforrn
and washed with 10% sat Na2CO3 three times. The organic layers were
combined~ dried over K2 C03 and concentra~ed to afford a white solid
950mg (90%), as an approximate 2:1 mixture of 1~ (4,8.1 I-tritosyl-
I . 4 . 8 . I I -tetraazacyclotetradecyl )methyl)-4- ( I -( I ,5, 8, 1 2-tetraazadodecyl )-
methyl)benzene and 1-(1-(4,8,11-tritosyl-1,4,8.1 I-tetraazacyclotetradecyl)-
me~hyl)-4-(s-( 1.5 . 8 .1 2-tetraazadodecyl)methyl)benzene .
c) The mixture produced in step b) (420mg, 0.448mmol) was
added to 1 2ml of acetic acid and 6ml of 48 %aq HBr (Aldrich) and the
solution was stirred for 30 hours at 110-120C. The red solution was cooled
to room temperature and concentrated to a paste. Acetic acid (lOml) was
added and the resulting solid was collected by filtration through a sintered
glass funnel. The tan-white solid was washed with acetic acid (20ml) and
ether (20ml) and dried under vacuum overnight (60C/0.4mm Hg~. A tan-
white solid was thus obtained (400mg, 80%) as an approxinate 2:1 mixture
of 1-(1-(1,4,8,1 1-tetraazacyclotetradecyl)methyl)-4-(1-(1,5,8,12-tetraaza-
dodecyl)methyl,~benzene octahydrobromide and 1-(1-(1,4,8,1 I-te~raazacyclotetra-decyl)methyl)~-(5-( 1 ,5, 8, 1 2-tetraazadodecyl)methyl)benzene octahydrobromide .
The products were separated by chromatography. and their structures were
confirrned.
. .
WO 92/16494 ~ pcr/GB92/oo438
~- ~g,`S~ ~'J 3 ~' 1 4 -
~,,
EXAMPLE 3
a) To a solution of triethylenetetraamine (346mg. 2.37mmol)
and K2 C03 (83mg, 0.592mmol) in 20ml of dry CH3 CN was added Lhe
S product of step a) of Example 2 (250mg. 0.296mmol) in I Oml of dr~
CH3 CN dropwise over 1 hour at room temperature. After the reaction
mixture had been stirred for 16 hours at room temperature, the solution was
filtered and the filtrate was concentrated. The excess tetraarnine was removed
by distillation of the concentrated filtrate on a Kugel Rohr apparatus (100C:
O. Imm Hg) and the residual oil was taken up in lOOml of chlorofol'rn and
washed with 10 % sat Na2 CO3 three times. The organic layers were
combined, dried over K2C03 and concentrated to af~ord a white solid 220mg
(82%), as an approximate 2:1 mixture of 1-(1-(1,4,7,10-tetraazadecy])methyl)-
4-(1-(1-(4,8,1 1-tritosyl-1,4,8,1 1-tetraazacyclotetradecyl)methyl)benzerle and
1-(1-(4, 8, I I -tritosyl- 1,4, 8,1 1 -tetraazacyclotetradecyl)methyl)-4-(4-( 1 ,4,7,10-
tetraazadecyl)methyl)benzene .
b) The product from step a) above (220mg, 0.242mmol) was
added to 8ml of acetic acid and 6rnl of 48 % aq HBr (Aldrich) and the
solution was stirred for 30 hours at 110-120C. The red solution was cooled
to room temperature and concentrated to a paste. Acetic acid (lOml) was
added and the resulting solid was collected by filtration through a sintered
glass funnel. The solid was washed with acetic acid (20ml) and ether (30ml)
and dried under vacuum overnight ~60C/0.4mm Hg). A tan-white solid was
wo 92/16494 ~ 8 Pcr/Gs92/Oû438
- 15 -
thus obtained (120mg, 46%) as an approximate 2:1 mixture of 1-~1-(1,4,7,10-
tetraazadecyl)methyl)-4-(1~ 4~8,11-tetraazacyclotetradecyl)methyl)benzene
octahydrobromide and 1-(1-(1~4~8~11-tetra~7acyclotetradecyl)methyl)-4-(4-
( I ~4 ~7 ~ 10-tetraazadecyl)methyl)benzene, The products were separated using
S chromatography, and their structures were confinned.
EXAMPLF 4
a) To a solution of N,N'-Bis(3-~ninopropyl)ethylenediamine-
tetratosylate (467mg, 0.592mmol) and KzC03 (33mg, 0~236mmol) in 15ml
of dry DMF was added lOOrng (OH 18mmol) of the product of step a) of
Example 2 in lOml of dry DMF dropwise over I hour at 60C. After the
reaction mixture had been stirred for 2 hours at 60 C the solution was
cooled to room temperature and concentrated~ The residual oil was taken up
IS in CH2CI2 (50ml) and washed with (Sml)~ The organic layer was dried over
MgS04 and concentrated~ The residue was purified by chromatography on
silica gel (elution 2% MeOH; 98% CH2C12) to afford l lOmg (60%~ of
I -( I -( I ,4.8.11 -tetratosyl- I ,4,8,11 -tetraazacyclote~radecyl)methyl)4-(1 -( I ,5,8,12-
tetratosyl-1,5,8,12-tetraazadodecyl)methyl)benzene, a white solid~
b) 80mg (O.OSlrnmol) of the product of step a) above was
added to 6m} acetic acid and 4ml of 48% aq HBr (Aldrich) and the solution
was stirred for 48 hours at 110-120C. The brown solution was cooled to
.? .. room temperature and concentrated to a paste. Acetic acid (8ml) was added
and the resulting solid was collected by filtration through a sintered glass
funneh The solid was washed with acetic acid ~lOml) and ether (lOml) and
dried under vacuum overnight (60~/0~4mm Hg~ A tan-white solid 50mg
Wo 92/16494 , ,~ ,3 - 16 - pcrlGBg2/oo438
t88%), of 1-(1-(1,4,8,11-tetraazacyclotetradecyl)meth~l)-4-(1-(1.5.8.12-
tetraazadodecyl)- methyl)benzene octahydrobromide was thus obtained and its
structure was confirrned.
Characterised samples of compound A were tested in the
standard in vitro tests, described below.
The compound of the invention was tested in a screen by the
Ml~ method (J.Virol. Methods 120: 309-321 [1988]). MT-4 cells (2.5 x
104 / well) were challenged with HIV-1 (HTLV-IIIB) or HIV-2 (LAV-2 ROD)
at a concerltration of 100 CCID50 and incubated in the presence of various
concentrations of the test compounds, which were added immediately after
challenge with the virus. After 5 days culture at 37C in a CO2 incubator,
the number of viable cells was assessed by the MTT (tetrazolium) method.
Antiviral activity and cytotoxicity of the compounds are expressed in the table
below as ED50 (ug/ml) and CD50 (ug/ml), respectively. The potential
therapeutic usefulness was assessed by cslculating a Selectivity Index (Sl~
corresponding to the ratio of CD50 to ED50. A control test was performed
using the known anti-HIV treatment AZT.
In the table below, the compounds screened were sompound A
and the known compound AZT.
WO 92/16494 ~ J ~ pcr/GB92/oo438
TA}3LE
Compound Virus CDsoED50 Sl
A HIV-I >50039 > 13
HIV-2 >50039 > 13
AZT (Comparison) HIV-I> I <0.008 > 125
In this field of study. i~ is considered that any compound
exhibiting a Selectivity Index of greater than S has the potential for further
study. HIV is one of the most challenging viruses to combat~ and the results
given above provide. an indication of activity against other retroviruses and
against other vimses in general. The compounds of the invention are also
to be considered for activity against microorganisms, such as bacteria and
especially against the organisms causing malaria.
The active compounds may be adminis~ered in the form of
pharmaceutical compositions formulated according to well known principles
and incorporated the compound, preferably in unit dose fonn, in combination
with a pharmaceuticaily acceptable diluent or excipient~ Such compositions
may be in the form of solutions or suspensions for injection, or irrigation or
be in capsule, tablet. dragee, or other solid composition or as a solution or
suspension for oral administration or forrnulated into pessaries or suppositories
or sustained release fonns of any of the above for implantation. Suitable
diluents, calTiers. excipients and other components are known. lt may be
desirable also to fo~mulate a composition ~or topical administration such as
an ointment or crearn. The compounds of the invention may be used, in the
forrn of a composition or alone, and possibly carried on a finely divided
WO 92/16494 ~ 3 - 18 - PCr/GB92/00438
form of a composition or alone. and possibly carried on a finely divided
support. as a coating on devices which in use contact body fluids. to
discourage transmission of viral infections. Exarnples of devices to be
considered in this aspect of the invention are surgical devices and gloves and
S contraceptions such as condoms. and other items. appliances. wound dressings
and coverings. implements etc generally to be considered as devices according
to this aspect of the invention.
The pharmaceutical compositions according to the invention may
contain unit dosages deterrnined in accordance with conventional
pharmacological methods. suitably to provide active compounds in the dosage
range in humans of from 0.1 to 10û mg/kg body weight per day. in a single
dose or in a number of smaller doses. Preferred dosage ranges are 1 to 30
mg/kg body weight per day. Other active compounds may be used in the
i S compositions or such active compounds or supplernenta! therapy may be
included in a course of treatrnent.
