Note: Descriptions are shown in the official language in which they were submitted.
This invention relates, in one aspect; to
platinum diamine compounds; in a further aspect, this
invention relates to a method for preparing such
compounds.
In a still further embodiment, this invention
also relates to compositions containing, as the active
inyredient, a platinum diamine compound.
Cis-Dichlorodiamineplatinum (also known as cls-
platinum) is a dia~ine compound which is a first-
1~ generation drug. The present-day knowledge concerning the
structural, functional and geometrical requirements with
platinum coordination compounds can be summarized by
reference to the dichlorodiamamine platinum originally
described by Rosenberg (B. Rosenberg et al., Nature 222,
385 (1969)~, as an example, and which is shown as
both cis- and trans- isomers are known and~exist in
aqueous solution in the 2+ oxidation state. Octahedral
4+ analogs with loosely bound ligands are also known, but
the main emphasis has been on the Pt(II) compounds. A
most relevant finding is that only the cls-analog
exhibits certain activity, the trans being very weakly
active if at all. This reflects on the modes of binding
to DNA. Cis-Pt(II) is a bifunctional reagent with square
planar geometry in which the chloride ligands are prone
~6~
-- 2 --
to substitution by incoming groups that are either in
large excess (water) or form thermodynamically more
stable links to platinum tDNA~. The amine groups are
both kinetically and thermodynamically inert to
substitution, but Eor their use as pharmaceutical
components, they must play a key role in secondary
binding to biological receptors such as DNA by H-bonding
forces. The nature of the amine has an influence on the
activity and possibly toxicity. For example, maximum
activity and markedly reduced toxicity is observed with
the nbutylamine derivative~ Tertiary amine derivatives
are inactive. Low solubility still poses a major problem
in intravenous administration of these drugs when cls
P5(II) is dissolved in water, the two chloride ligands
are sequentially exchanged for water or hydroxy depending
on the pH. At 37 the half-life for the completion of
this reaction is 1.7h with an activation enthalpy of
about 20 Kcal/mol. The aquated forms of cls-Pt(II)
appear to be important bioactive species as they would be
0 expected to exist inside the cell, because of a
favourable pH balance.
With the present invention, applicant has
developed novel compounds which are described in greater
detail hereinafter, but which are of the general formula
X \ ~ N - CH
Pt \
X N - CH
H2 12
wherein Rl and R2 are independently selected from alkyl
groups comprising n carbon atoms, wherein n equals 1 - 8
containing at least one lower alkoxy group of 1 - 3
carbon atoms, and X is a protective group, e.g., a
halogen.
In accordance with another aspect of the
present invention, there is provided a process of
preparation of a compound of the general formula
R
H2
X ~ N --- CH
Pt
~ \
X N - T ( ~H
H2
R2
wherein Rl and R2 are independently selected from a
carbon chain comprising n carbon atoms wherein n equals 1
- 8 containing at least one lower alkoxy group of 1 - 3
carbon atoms, and X is a protective group, e.g~, a
halogen, comprising reacting a compound of the formula
NH2 -- CE~ -- Rl
NH2 ~ CH - R2
~26~5~
-- 4
wherein R1 and R2 are defined as above, with K2PtX4,
wherein x is defined above. ln the above ~ormulae, X may
also be a mono- or dicarboxylic acid; suitable acidic
ligands or aqua groups may also be employed.
In greater detail of the process aspect, for
the preparation of the platinum coordination complexes
several methods can be used. The starting material may
be a potassium tetrachloroplatina-te (II) (K2PtC14). To a
clear solution of this salt is added directly the
l() organic-amine ligand in water in alcohol, and allowed to
stand at room temperature or heated up to 65C until the
color is changed from red-blood to orange-yellow or blue-
green (in some cases). This then is followed by standard
pocedures of separation, purification, identi~ication and
chemical analysis. Separation of the various complexes
is done also by chromatography and high pressure liquid
chromatography (both analytic and preparative). The
latter technique would appear to be a particularly potent
tool to investigate the reactions of platinum
coordination compounds with nucleotides and nucleic
acids.
The diamino compounds described herein can also
be prepared by other methods. Thus within each series,
the diamine derivative could be prepared by displacing
suitably protec-ted derivatives ~sulfonates, halides) with
azide ion or ammonia. This could be done on 4,5 and 6-
-- 5 --
carbon acyclic or cyclic derivatives (such as~lycosides). In the latter case, the glycosides could be
hydroly~ed and reduced to -the corresponding alditols.
The methyl ethers can be formed prior to the introduction
of the amines or after (in case of azides). Platinum
complexes can also be preparea from the diamino compounds
in a conven~ional manner if desired. Epoxides can also
be used as starting materials for the preparation of the
diazides or the diamines.
In greater detail of the present invention, the
compounds of the above formula have been found to possess
antimicrobial activity, making them suitable for use as
antimicrobial agents which can be combined with
pharamceutically acceptable carriers to form
antimicrobial compositions.
In addition to the above, the platinum diamine
compounds of the present inven-tion also have been Eound
to possess activity against cer-tain strains of certain
types of tumors. In this respect, the known cisplatinum
compound is, of itself, in a unique class of coordination
compounds, that has therapeutic activity against a wide
spectrum of human tumors, either alone or in combination
with other agents such as adriamycin. In fact, recently
the FDA approved cis-platinum Eor use in humans.
.~ '.~!
~Z~5~
-- 6
Although the mode of action is still unknown, it has been
found that cis-platinum and its analogs bind to the DNA
and disturb the normal functions of the cell. In another
hypothesis, the drug is believed to enhance antigenicity
so that tumors become more susceptible to destruc-tion by
the host's immune ~ystem. Regardless of the precise mode
of action, the efficacy and potential of cis-platinum as
an antitumor agent are of some significance. Although
cis-platinum exerts its preferential toxicity to tumor
1~ cells when compared to normal cells at a therapeutic
index which allows its clinical use, the situation is
still far from perfect and there are also serious side
effects to overcome. An improvernent is stil] sought in
therapeutic index (presently 2-2,5 mg/Kg). Among the
major drawbacks are nephrotoxicity, gastro-intestinal
problems and depression of the function of the bone
marrow thu~ producing fewer than normal white blood cells
and platelets. These have obviously limited the size of
the dose and continue to be a major impediment to the
routine use of cis-platinum in humans.
Presently, patients are induced to increase their
flow of urine by intravenous administration of 1-2 liters
of fluid. The drug is then administered intravenously
together with a diuretic. A most recent development, is
-- 7
the administra~ion of D-mannitol which is believed to
"flush out'l residual cisplatinum from the body by
coordination. These are nonetheless relati~ely harsh
treatments even in the face of life-threatening
situations.
There is at present a renewed interest in the
treatment of a variety of tumors with platinum
coordination compounds. (B. Rosenberg, Cancer treatment
reports 68, 1343 (1979); and Science, 192, 774 (1976;
Chem. Eng. News, Jan. 21, 1980, p.35)~ Reports on the
clinical status of clsPt(II) in cancer chemotherapy,
generally on extremely advanced cases, indicate promising
anticancer activity. (J. A. Gottieb and B. Drewinko,
Cancer Chemother. Rep., 59, 621 (1975)). For example,
researchers at Georgetown University in Washington have
reported remarkable success in treating brain tumors with
this drug (Chem. Eng. News, Oc-t. 6, 1980, p. 27). The
patients in this case were 10 children whose tumors had
resisted many forms of chemotherapy. Cis-Pt(II)~ alone
0 or ln combination has had success in the treatment of
testicular carcinoma (~. J. Wallace and D. J. ~igby,
Recent Results Cancer Res., 48, 167 (1974)), hand and
neck cancer (I. H. Krakoff and A. J. Lippman, Recent
Results Cancer Res., 48~ 183 (1974)), squamous cell
~2~
carcinoma, malignan-t lymphoma and endometrial carcinoma
(J. M. Hill et al, Cancer Chemother. Rep., 48, 145
(1974) and J. M. ~lill et al., Cancer Chemother. Rep.,
59 647 (1975), and ovarian adenocarcinoma (~. Wittshaw
and B. Care, Cancer Res., 48, 178 (1974).
As previously mentioned, cis Pt(II) treatment
has certain disadvantages. For example the ir is not
very soluble in water which renders its intravenous
administration problematic Some derivatives are known
1~ to maintain the desired levels of activity (T. A.
Connors, M.J. Cleare and K.R. Harrap, Cancer Treatment
REports 63, 1499 (1979)), but the solubility
characteristics were not noticeably improved; thus,
e.g., malonato (1,2-diaminocyclohexane) platinum (II)
has been found to be effective against a number of
animal tumors in addition to L1210 Leukemia (J. H.
Burchenal et al., Cancer Treatment Reports, 63, 1493
(1979)). Nephrotoxicity and other forms of toxicity
remain as the most serious drawback of c1s-Pt(II) and the
limited number of analogs tested so far. The synthesis
of different platinum coordination compounds with greater
water solubility, less renal toxicity and greater
antitumor activity is a goal that is still actively
sought by investigators tsee Roberts and Thomson below).
q. ~ 'R
,
.
- 9
The primary mode of action (J. J. Roberts and A.
J. Thomson, Prog. Nucl. Acid Res., Mol. Biol., 22, 71
(1979) and A. D. Kelman and H. J. Persesie, Cancer
Treatment Repor-ts, 63, 1445 (1979)) of cis-Pt(II) and its
derivatives is localized at the intracellular DNA level.
While the sites within the various bases to which cls-
Pt(II) binds and their relative importance to the
antitumor activity have not been conclusively discerned,
there is good evidence that DNA interstrand (A. D. Kelman
1~ and H. J. Persesie, Cancer Treatment Reports, 63, 1445
(1979)) cross-linking, most probably at adjacent guanine
pairs, or interstrand crosslinking (P. K. Ganguli and T.
Theophanides, Euchem Conference on Coordination Chemistry
and Cancer Chemotherapy, Toulouse, France, 1978; and Eur.
J. Biochem 101, 377 (1979)) at N(7) and C (6)-0 on
guanine may be responsible. The bou~d cls-Pt(II) drug
would result in template inactivation of DNA and the
accumulation of potentially lethal damage.
.,
~26~
- 10 -
With respect to their transport, again, little is
known except that the drug mu.st be able to penetrate the
cell membrane prior to exerting intra-cellular
tumoridical action. The active form must be electrically
neutral in the plasma prior to transport. Otherwise a
carrier-mediated mechanism has to be involved for which
there is no evidence (L. ~. Zwelling and K. W. Kohn,
Cancer Treatment Reports, 63, 1439 (1979)).
The new compounds of the present invention have
several advantages over known compounds, namely:
1. Superior water solubility
2. When used as pharmaceuticals, they have more
efficient exclusion Erom the body
3. Improved transport (maintaining charge
neutrality)
4. Possible oral activity
5. Possible different metabolism
6. Better balance oE hydrophobic and hydrophilic
character ~more eEfective transport and distribution in
plasma).
7. Interference with repair en~ymes which reverse
the drug-inflicted damage by an excision repair process
8. Improved biocompatibility in plasma and other
fluids
~5~ L
9. Inherent chirality (selective interaction with
certain receptors, membrane constituents and the DNA
helix itself).
In addition, the chiral, functional nature oE the
analogs may increase their effectiveness due to:
1. Added binding sites to the DN~ and intracellular
proteins
2. Presence oE auxiliary stabilizing structures
telectrotatic stabili~ation by polar groups)
ln 3. Presence oE masked aquasystems (physiologically
active form of cis-platinum.
The compositions oE the present invention having use
as anti-microbial compositions or for the treatment of
tumors may be formulated using conventional techniques so
as to include pharmaceutically acceptable carriers or
diluents.
The compositions may be formulated as, e.g.,
powders, solutions, or suspensions which have been
conventionally produced using acceptable diluents. The
~Q compositions may also be formulated for parenteral
administration by injection or continuous infusion when
used to treat tumours; such formulations for injection
may be present in unit dose form as ampoules in multiple-
dose contai~ers with added preservative.
.
~p~
- -
- 12 -
The compositions may also assume such forms as
suspensions, solutions or emulsions in oily or aqueous
carriers and may contain Eormulation aids, such as
suspending agents, stabilizers and/or dispersants.
~ lternatively, the active principle may even be
present in powder form Eor reconstitution before use with
a suitable carrier, for example, sterile, pyrogen-free
~ater.
Having thus generally described the invention,
10 reference will now be made to the accompanying examples,
illustrating preferred procedures.
~26~
-- 13 --
EXI~MPLE
l~2:5l6-Di-o-Isopropylidene-3~4-d_O-methanesulfonyl-D-
mannitol
A solution of 1,2:5,6-di-O-isopropylidene-D-
mannitol (8.4g, 32mmoles) in dry methylene chloride
(144mL) and triethylamine (llg,O.lmmole) is treated with
methanesulfonyl chloride (8.9g, 0.008mmole) dropwise at
OC under nitrogen atmosphere. After allowing the
mixture to warm up to room temperature, it is washed
ln abundantly with cold distilled water and the organic
phase is dried tsodium sulfate) and evaporated under
vacuo, to afford (yield 12g; 90%) of a colorless
crystalline product. M.P. 142-143C [~]D = -5, (C=l,
CHC13). For the preparation of the mannitol starting
compound, see E. Baer, Biochemical Preparations, 2, 31
1952 ).
EXAMPLE 2
3,4-Diazido-3,4-dideoxy-1,2:5,6-di-O-isopropylidene-D-
iditol
To a solution of 1,2:5,6-l~i-O-Isopropylidene-3,
4-di-O-methanesulfonyl-D-mannitol (12g, 28.7mmoles) in
dry toluene (300mL) is added tetrabutylammonium azide
t24g, 3eq.). The mixture is heated at reflux
temperature under stirring for 12 hours, poured into
~,
i8~
water t500mL) and extracted. TiIe organic extract is
washed with brine, dried (sodium sulfate), and
evapor~ted at 50C. Chromatography on silica gel (3:1
hexane-AcOEt) afEorded a pale yellow syrup (yield:
3.59g, 40~). Sublimation at 110C (0.3mm Hg) gave the
iditol crystalline product which melts at room temp.
[~D25 = ~ 130~ (C,l, CIIC13).
The deprotection of 3,4-Diazido-3,4-dideoxy-
1,2:5, 6-di-O-isopropylidene-D-iditol was executed under
1~ mild conditions (AcOH 120 ml, H20 60 ml, at 70C for 2
hours) to afford -the crystalline diazidopolyol
derivative.
EXAMPLE 3
3,4-Diazido-3,4-dideoxy-1,2:5,6-tetra-O-methyl-D-iditol
~ solution of the preceding diazido polyol
(500mg, 2.15mmoles) in dry dimethylformamide t7.5mL) is
transferred to a round bottom flask containing sodium
hydride t60~ oil dispersion, 0.688 g, 8eq.) in dry
dimethylformamide t5mL) at OC under nitrogen
~tmosphere. Upon ceasing of effervescence ( 45min),
methyl iodide (3.05g, 10 equiv.) is added dropwise. The
reaction mixture is then stirred at OC for 1 hr., and
left standing at r~om temperature overnight. The
mixture is neutralized with methanol followed by N
sulfuric acid. A large amount of
~,~
~Z658
- 15 -
saturated sodium chlo~ide solution is added to the
mixture, and then ~he mixture is extracted with ether.
The organic extract is dried (sodium sulfate),
evaporated unaer ~acuum and the resultiny syrup
chromatographed on silica gel (35:65 ethyl
acetate-hexane) to afEord the pure product as a
colorless syrup; (yield: 554mg, 89%, [~D25 = 188 (C=l,
C~IC13).
EXAMPLE 4
3-4-Viamino-3,4-dideoxy-D-iditol
To a solution oE the diazido polyol in Example 2
(406 mg, 1.75 mmole) in methanol (30mL) and distilled
water (5mL) is added 10% palladium-on-charcoal (150mg)
suspended in water (5mL). The mixture is hydrogenated
under atmospheric pressure at room temperature for 24
hours (an infrared oE an aliquot is taken at this time
to insure disappearance oE the azide band). The
catalyst is then removed by filtration through celite,
washed with distilled water (5mL), and the filtrate
evaporated to dryness by co-distillation with absolute
ethanol. A white solid is obtained (yield 302mg, 85%);
recrystallization from hot ethanol/water aEEorded
colorless needles (yield of recrystallization: 50%).
M P 160-161C, [~]D25 = -104 (C=l, H20).
~ 2~5~ ~
-- 16 --
EXAMPLE 5
2,3-Diazido-2,3-dideox~-D-threitol
-
To a solution of the diazido polyol (Example 2)
(500mg, 2.15mmoles) in distilled water (lOmL) is added
sodium metaperiodate (92mg, 4.32mmoles, solid),
portionwise at OC, in the absence of light, the mixture
is stirred Eor 2 hours at oc. then tre~ted with ~odium
borohydride (eq.) in distilled water (lOml). After two
hours, the reaction is neutralized with N sulfuric acid,
10 decolorized with sodium thiosulfate and evaporated under
vacuum. The residue is dissolved in water (25mL) and
extracted with ethyl acetate (5 x 40 mL); the combined
organic extracts is decolorized with sodium thiosulfate,
dried and evaporated under vacuum to afford a colorless
oil (390mg, 96%). Thin layer chromatography of the oil
shows the presence of the diol 2,3-Diazido-2,3-dideoxy-
D-threitol and the triol 2,3-Diazido-2,3-dideoxy-D-
xylitol in e~ual proportion. The oil is recycled
through the oxidative cleavage and the reduction as
~ described above and the reaction mixture is processed
similarly to afford the diol 2r3-Diazido-2,3-dideoxy-D-
threitol in the form of white crystals;
recrystallization in chloroEorm/hexane yields 303mg,
(82%~ of product. M.P. 78-79C, [~]D = ~41.4
(C=0.5,Et20).
S~
- 17 -
EXAMPLE 6
2,3-Diazido-2,3-dideoxy-D-XylitOl
To a solution of the diazido polyol (Example 3)
~lg, 4.3mmol) in distilled water (25mL) is added sodium
metaperiodate (920mg, leq) in distilled water (lOmL),
portionwise at OC, in the absence of light. The
mixture is stirred 1 1/2 hours at OC, then treated with
s~dium boroh~dride (3 e~.) in distilled water (20mL).
~Eter 1/2 hour, the reaction is neutralized with I
sulfuric acid. (The mixture may be decolorized with a
few milliliters of saturated sodium thiosulfate), and
evaporated under vacuum. The residue is dissolved in
water (25mL) and extracted with ethyl acetate (5 x
lOOmL). The combined organic extract is decolorized
with sodium thiosulfate, dried, evaporated under vacuum
and chromatographed on Silica gel (85:15 ethyl acetate-
hexane) to afford the diol namely 2,3-Diazido-2,3-
dideoxy-D-threitol (69mg, 10%), and the
triol, namely 2,3-Diazido-2,3-dideoxy-D-xylitol (577mg;
66~ yield). [~]D 5 = -239 (C=1.35, CH3QH), IR ma~:
2100cm 1 (N3).
EXAMPLE 7
General Methylation Procedure
A solution of polyol (2.15rrlmoles) in dry dimethyl
formamide (75m~) is transferred to a round bottorn flask
- 1,8 -
containillg sodium hydride (20 equiv) in dry dimethyl
formamide (5mL) at OC under nitrogen atmosphere. Upon
ceasing of effervescence (45 min) methyl iodide (10
equiv) is added dropwise. The reaction mixture is
stirred at o~C for 1 hour, and le~t standing at room
temperature overnight. After the reaction is finished,
the mixture is neutralizeA with methanol Eollowed by N
sul~uric acid. The organic phase is processed as usual
to give a syrup. Chromatography on silica gel using
11) 35:65 v/v ethyl acetate-hexane as the eluant gives the
pure desired product. Ethylation was performed with
ethyl iodide.
EXAMPLE 8
2,3-diamino-2,3-dideoxy-1,4-di-O-ethyl-D-
threitol, 2,3-diamino-2,3-dideoxy-1,4-di-O-methyl-D-
threitol, 2,3-diamino-2,3-dideoxy-1:4,5-tri-O-methyl-D-
xylitol and 3,4-diamino-3,4-dideoxy-1,2:5,6-tetra-O-
methyl-D-iditol are prepared from the analogous diazido
polyol by methylation following the process of Example
~0 7, and subsequent reduction of diazido polymethyl ether
using hydrogenation in the presence of palladium-on-
charcoal.
` ~
~5~
- 19 -
EXAMPLE 9
_s-Pt co~plex formation
The complex formation of diamines 3,4-Diamino-
3l4dideoxy-D-iditol~2~3-Diamino-2~3-dideoxy-D-threitol;
2,3-Diamino-2,3-dideoxy-D-xylitol, Methyl 2,3-dia~ino-
2,3-dideoxy-~-D-allopyranoside and O-methyl analogs with
tetrachloroplatinate are carried out according to the
~ollowing protocol; the substrate is dissolved in an
a~lueous solution oE the platinum salt. The resulting
homogeneous solution is left standing at room
temperature in the absence of light, resulting in the
slow crystallization of the desired complex. For
example, the ligand 3,4-Diamino-3,4-dideoxy-D-iditol
gives pure crystaline needles cis-~3,4-Diamino-3,4-
dideoxy-D-iditol) dichloroplatinum ~II) whose structure
was confirmed by single crystal X-ray crystallography.
EXAMPLE 10
2,3-Diazido-2,3-dideoxy-1:4-di-O-ethyl-D-threitol
~ solution of diol ~0.2g, 1.2mmoles) in dry
20 DMF(2.5ml) is transferred to a round bottom flask
containing sodium hydride l60~ dislpersion, 4eq.) in dry
~MF(2.5ml) at OC under nitrogen atmosphere. Upon
:,~
-- 20 --
ceasing of eEfervescence (30min), ethyl iodide (0.4ml,
~eq.) is added dropwise. The reaction mixture is then
stirred at 0C for 1 hrs, and left standing at roolr
temperature overnight. A large amount of saturated
soaium chloride solution is added and the mixture is
extracted with ether. The organic phase is evaporated
under ~vacuum. The resulting syrup is chromatographed on
s,ilica ~el (~:1 Hexane: AcOEt) to aEford the pure
product as a colorless syrup; yield: 0.2318g, 876.
1~ EXAMPLE 11
2,3-Diamino-2,3-dideox~-1,4-di-0-ethyl-D-threitol
To a solution oE 2,3-diazido-2,3-dideoxy-1,4-di-
0-ethyl-D-threitol(0.2318g, 1.0166mmoles) in methanol
(5.5ml) is added palladium-on-activated-charcol(10%, Pd,
lOOmg) suspended in distilled water(3.5ml). The mixture
is hydrogenated under atmospheric pressure at room
temperature for 24 hours. (An infrared of an aliquot is
taken at this time to ensure disappearance of the
azide). The catalyst is then removed by filtration
~0 through celite and evaporated under vacuum then
coevaporated with ethanol to dryness to afford the
product as a colorless syrup 0.1415g, 80% yield).
`~
- 2l -
EX~MPL~ 12
cis-(2~3-Diamino-2~3-dideoxy-lr4-di-o-ekhyl-D-threitol)-
dichloroplatinum( II )
2,3-Diamino-2,3-dideoxy-1,4-di-0-ethyl-D-
threitol(O.1415g) is transferred, using distilled
water(O.3ml), into a solution containing K2PtC14(leq.)
in distilled water(O.3ml). The mixture is stirred
m~nually until homogeneous, and is left standing in the
dar~. ~fter a few hours the slow crystallization process
1~ begins to give yellow needles. The crystals are
Eiltered, washed with a little blt oE cold water and
dried by suction of air, for several hours, to afford
y~llow crystals (0.1612g, yield 45%).
EXAMPLE 13
cis-(2,3-Diamino-2,3-dideoxy-1,4,5-tri-0-methyl-D-
xylitol)-dichloroplatinum(II)
2~3-Diamino-2~3-dideoxy-l~4~5-tri-o-methyl-D-
~ylitol (2.6 g) is transferred, using distilled water
(1~5 ml), into a solution containing K2PtC14 (leq.,
5.6g) in distilled water (7 ml). The mixture is stirred
manually until homogeneous, and is left standing in
darkness. ~fter a few hours the slow crystallization
i~
- 2~ -
process begins in the Eorm of pale yellow fine crystals.
The crystals are filtered, washed with a little cold
water, and dried under a suction of air Eor several
hours to afford pale yellow fine crystals (2.5g, yield
~10~ ) .
EXAMPLE 14
cis-(2~3-Diamino-2,3-dideoxy~1~4-di-O-methyl-D-
threitol)dichloroplatinum(II)
To a solution of 2,3-diamino-2,3-dideoxy-1,4-di-
O-methyl-D-threitol (0.7541 g) in distilled water (5 ml)
was added K2PtC14 (1 eq., 2.1 g). The mixture is
stirred manuaLly until homogeneous, and is left standing
in darkness. After a few hours, the slow
cr~stallization process begins in the form of yellow
needles. The crystals are filtered, washed with a
little cold water, and dried under a suction o~f air for
several hours, the afford yellow crystals (1.2 g, yield
60%)
'~
-
~2~5 !3~ ~
- 23 -
EXAMPL~ 15
The product of Example 3 was hydrogenated in the
manner oE the pre~eaing examples to yield the
corresponding diamino compound; yield is 91~. [~]D25 =
-262 (in methanol). This was then converted to the
corresponding dichloroplatinum complex compound by
procedures described above to yield a product having a
~nelting point oE 286-287C. (with decomposition); [~]D 5
- -25.2 in water.
In addition to the compounds prepared above, the
corresponding L-series can be prepared in an identical
manner.
~.
~265~
- 2~ -
Varlous of the compounds of the present invention
were tested for antimicrobial activity. Testing was
carrie~ out to determine the activity based on the
growth of micro organisms tmeasured) and the mini~um
inhibitory concentration determined Eor the respective
compounds.
The method employed 96 well microtiter plates were
prepared by adding 100 ~1 appropriate medium to each
well. 50 ~1 aliquots of the diluted test compound are
a~ded to each well in a manner resulting in the required
dilutions and the proper controls. The test organisms
are innoculated into each well in a volume of 10 ~1.
The trays are incubated at 37C for 16-24 hours for
bacteria; yeast and fungi containing trays are incubated
at 28 for 36-48 hours. The plates are read either with
Dynatech MR 600 plate reader or manually.
In the following tables the indicated codès are
used:
109050 is cis-dichlorodiamine platinum (II);
~0 114551 is cis-(3,4-diamino-3,4-dideoxy-1,2:5,6-tetra-
O-methyl-D-iditol)dichloroplatinum (II);
115448 is cis-(2,3-diamino-2,3-dideoxy-1,4-di-O-
methyl-D-threitol~dichloroplatinum (II);
~ 9~
- 25 -
115449 is cis-(2r3-diamino-2~3-dideoxy-l~4~5-tri
methyl-D-xylitol)dichloropl~tinum (II);
123597 is cis-(2,3-diamino-2~3-dideoxy-1,4-di-O-
ethyl-D-threitol)dichloroplatinum (II).
TABLE I
~ntimicrobial ~Cti~Ji t~
Organism
Salmonella Alcaligenes
C~mpound Typhimurium Viscolachs
lo 1145511000 333
115448333 333
1154491000 333
The next series of tables (II, III, IV) exhibit ln
vitro lack of cross resistance to strains sensitive (S)
and resistant (R) to cisplatinum were tested against the
compounds (L1210DPP is a resis-tant strain); t~he results
are ~iven at ICso in Mg/ml tII), ID50 in uM (III) and
ID50 in Mg/ml (IV).
8~ :
- 26 -
TABLE II
ICso (~g/ml)
train
_ ~ound L1210S L1210R
109050 0.21 0.53
109050~ 0.24 2.98
~14551 15.14 15.60
1154~18 5.35 1.01
115~49 6.35 2.30
115449~ 7.36 6.98
# degree of resistance to cis platinum increased by
second test.
The lack of cross resistance is noticeable.
TABLE III
ID50 (~M)
Strain
Compound L1210S L1210R
109050 0.8 9.9
114551 . 30.1 31.0
~ 115449 16.5 15.7
123597 13.4 14.6
Again, the lack of cross resistance is apparent.
r~gLE IV
I D 5 0 ( ~I g/ml )
Strain
Compound L1210S L1210R L1210DPPP388S P388R
109050Q.25 3.25 4.91 0.20 4.85
11455115.~ 15.6 - 27.9 27.0
115~8 5.35
11~4497.36 6.98
1235975.90 6.42 7.21 - 48.0
Lack of cross resistance shown to 114551 by L1210
and P388 strains, and 115449 and 123597 to L1210
strains.
The cells were incubated at 37C in a suitable
medium te.g., RPM11640 supplemented with 10% fetal calf
serum) with various concentrations of test age~t, and
counted a~ter 72 hours for assessment.
Numerous in vivo tests were run against L1210
leukemia, M5076 sarcoma, and PV induced sarcoma (PV-
239).
114551 had activity against L1210 intraperitoneal
implants but less than 10~ of cis platinum, using
intraperi~oneal treatment 115448 and 115449 had activity
.~
- 28 -
~gail~st intraperitoneal implants of M5076 sarcoma using
intraperitoneal treatment, but this was less active than
cis platinum using intraperitoneal treatment.
114551 was not very active againt subcutaneous
implants of M5076 sarcoma, which closely mimics the
clinical situation.
11~551 and 115448 when tested against PV induced
s~reoma were ineffective. 115449 was toxic to the
subjects at all doses tested.
1~ 114551, 115449 were tested against L1210 and
L1210/DPP in vivo, using intraperitoneal implants both
showed better activity against L1210 than L1210/DPP.
Similar testing oE 115448 and 115449 against the
same two strains under similar conditions gave similar
results.
115448 when tested against P388 and P388/p~P showed
better activity against P388 than P388/DPP.
