Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
-~ ~327~37
IITLE OF THE INVENTION
MITOMYCIN DERIVATIVES HA~IN6 REDUCEP BONE MARRO~ TOXTCITY~
PROCESSES FOR THEIR PREPARAT~ON ~ND THE ~SES THEREOF
FIELD OF THE INYENTION:
The invention is in the field of pharmaceutical agents and the
uses thereof.
BACKGROUND OF THE INVENTION
The mitomycins are a family of compounds having the following
general formula (I):
.
H3C r~
~ -2- ~327~37
Mitomyc~ns A, B and C are related to one another as set forth in
Table 1 below, the designat;ons X, Y and Z being those of formula 1.
TABLE 1
MitomYcin: X Y Z
A -OCH3 -OCH3 -H
B -OCH3 -OH -CH3
C -~H2 -OCH3 -H
Mitomycins are derived from mitosane compounds having the follow-
ing skeleton (II~:
H~C ~
The mitosanes are formed during the cultivation of the micro-
organism $treptomvces caesPitosus in a liquid nutrient medium under
artificially controlled conditions. After separating the resulting
mycellium, the various mitomycins may be isolated from the latter by
active carbon or preferably non-ion exchange resin ~dsorption, organ;c
solvent extraction or chromatography on alumina, as disclosed in U.S.
Patent No. 3,650,578 to Hata et al~
Although the mitosanes are excellent antibiotics, they have
limited utility due to their toxicity to human blood ~see U.S. Patent
No. 3,450,705 to Matsui ~t al.). The relatively highly toxin nature
of the co~pounds has prompted seareh for derivatives of mitomycin to
~ncrease the antibiotic activity and to decrease toxicity.
A68. 2 .WP 082288
For exa~ple, Matsui et al., U.S. Patent No. 3,450,705, disclose
mitomycin compounds substituted at the 7-position with amino, lower
alkylamino, phenylamino, or pyridyl, and substituted at the la
position with haloalkanoyl, halobenzoyl, nitrobenzoyl, alkenoyl,
acetyl glycyl, sorboyl, or acetyl methionyl.
Matsui et al., U.S. Patent No. 3,558,651, disclose mitosane
derivatives comprising la-acyl-7-acyloxy-9a-methoxy compounds.
Certain mitomycins and mitomycin derivatives also possess
antitumor activity. Oboshi et al., Gann 58:315-321 (1967); Usubuchi
et al., Gann 58:307-313 (1967~; Matsui et al., J. Antibiotics XXI:189-
198 (1968); Japanese Patent No. 68 06 627 to Matsui et al. (Chemical
Abstracts 69:86986k (1968)); and Cheng et al., J. Med. Chem. 20:767-
770 (1977)-
While mitomycin C is active against a relatively broad spectrumof experimental tumors, its toxicity and myelosuppressive effects
limit its use in clinical practice ~MitomYcin 0: Current Status and
New Developments, Carter et al. (eds.), Academic Press, New York
(1979)). In preclinical and clinical studies, mitomycin C has shown
activity against a variety of murine and human neoplasms, but has also
shown severe, delayed bone marrow toxicity. Goldin, A., et al., NCI-
EORTC Svm wsium on Mitomyc _ C, Brussels, Belgium (1981).
In other studies, a combination of 5-fluorouracil, adriamycin and
mitomycin C was found to be effective for the treatment of patients
with advanced gastric and colorectal cancer. This regimen incor-
porated mitomycin C administration in a single dose schedule every two
months, to decrease the treatment-limiting delayed myelosuppressive
effects of the compound. Schein, P.S., et al., MitomYcin C Current
Status and New DeveloDment~, pp. 133-143, Carter e~ al. (eds.),
A~ademic Press, New York (1979~.
Numerous synthetic derivatives of mitomycin C have been prepared
in the hope of obtaining compounds with improved therapeutic proper-
ties. These derivatives include substitution on the aziridine ring,
carbamoyl, or acyl group substituticn on the hydroxymethyl side chain,
A68.2.WP 082288
4 1327~37
and replacement of the 7- substituent in the quinone ring with other
functional groups, especially substituted amines. However, as
disclosed by Remers, U.S. Patent No. 4,~68,676, nnne of these analogs
have emerged as a clinical agent, with the possible exception of the
7-hydroxy analog of the mitomycin C, which has been involved in a
recent study in Japan. This analog is asserted to be less leukopenic
than mitomycin C, but is also less potent. Also disclosed by Remers,
suDra, are totally synthetic m1tomycin analogs of the mitnsane type
(Mott et al., J. Med. Chem. 21:493 (1978)J, prepared mainly for their
antibacterial activity.
Kinoshita, S., et al., J. Med. ChemO 14:103-112 (1971), disclose
several derivatives of mitomycin substituted in the la, 7, and 9a
positions. In particular, compounds substituted at the la position
with sulfonyl, ortho-substituted benzoyl, and acyl derivatives were
reported.
Iyengar, B.S., et al., J. Med. Chem. 24:97~-981 (1981), disclose
a series of 31 mitomycin C and porfiromycin analogues with various
substituents at the 7- and 1a-positions. The most active substituents
at the 7-position included aziridine, 2-methylaziridine, propargyl-
amine, furfurylamine, methyl glycinate and 3-aminopyridine.
Iyengar, B., et al., J. Med. Chem. 26:16-20 (1983), disclose a
series of 7-substituted mitomycin C and porfiromycin derivatives and
the screening the~eof in standard antitumor systems. The authors
report that the 7- position controls the reduction of the quinone
ring, thus suggesting that it would be possible to alter the substitu-
tion of the 7- position to gain selectivity between normal cells and
certain cancer c~lls.
Iyengar, B.S., et al., J. Med. Chem. 26:1453-1457 (1g83),
disclose 20 mitomycin C analogues substltuted with secondary amines at
the 7-position. Eleven of these analogues were more aetive than
m1tomycin C against P388 murine leukemia and two of these cleven were
significantly less leukopenic. The authors report that no quantita-
tive correlation between antitumor activity and physiochemical
A68.2.WP 08?288
~ ~327~3~
s-
properties of the analogues was e~ident, although the relative ease of
quinone reduction may be related to activity.
Iyengar, B.S., et al., J._Med. Chem. 29.1864-1868 (1986),
disclose the preparation of 7-substituted a~ino 1,2-aziridinomito-
senes.. The authors reported that a ~e~hyl group on the aziridine
nitrogen gave increased potency. The 7-amino mitosene derivatives
which were difficult to reduce to hydroquinones ~ere essentially
inactive.
. Sami, S., et al., J~ Med. Chem. ?7:701-708 (1984)9 disclose a
series of 30 N7-phenyl-subst~tuted mitomycin C analogs. Two of the
compounds having pyrazolyl or aminopyridyl substituents at the 7-
position were disclosed as clearly superior to ~itomycin C in activity
against P388 murine leukemia~
Sami, T., et al., J. Med. Chem. ~:247-250 11979), also disclose
N-(2-chloroethyl)-N-nitrosocarbamoyl derivatives of glycosylamines,
including three disaccharide derivatives which exhibited strong
antitumor actiYity against leukemia 1210 in ~ice. In addition,
glucopyranos~ derivatives of N-nitrosoureas possess i~munogenic and
marrow-sparing properties. Anderson et al., Cancer Research 35:761-
765 (1975); Panasci et al., ~L_s~ln,_lcy~s~ 64:1103-1111 (1979).
In U.S. Patent No. 4,720,543, compounds ha~ing the following
general formula (III~ are disclosed:
1l ~ff2 ON~2
~l_f ~CH3 111
H~C~--N~ CS-~tlR 2
A68. 2 .IJP 082288
- 6 - ~L32~37
where
R1 is selected from the group consisting of NH2, C1-C4
alkoxy and a glycosyl residue; and
R2 is selected from hydrogen, C1-C4 alky], and a
glycosyl residue, with the proviso that either R
or Rz, but not both, contain a glycosyl group.
The compounds represented by formul.a III have
excellent antineoplastic activity and at the same time
possess reduced bone marrow toxicity and lower overall
toxicity.
Despite the above-listed mitomycin derivatives,
a need continues .: to exist for improved mitomycin
derivatives having good anti-neoplastic properties and low
bone marrow and overall toxicity.
SUMMARY OF THE INVENTION
The invention relates to a mitomycin derivative having
the following general formula (IV):
o o CH20-~-NH2
H
wherein
n is 0 or l;
Y is selected from the group consisting of
glucopyranosyl, galactopyranosyl,
mannopyranosyl, xylopvranosyl, cellobiosyl,
.; lactosyl, glucofuranosyl, maltosyl, and l,3-
cyclohexanediol-2-yl, or a hydroxyl-protected
peracetate derivative thereof;
.
D`
~ 1327~
R is hydrogen;
Rl is hydrogen, Cl-C4 alkyl or Cl-C4 alkyl substituted by phenyl,
hydroxyphenyl, indolyl, mercapto, C1-C4 alkylthio, hydroxy, carboxy,
amino, guanidino, imidazole or carbamyl; or
R and Rl together form a f~ve or six membered nitrogen containing
ring.
The invention also relates to a mitomycin derivative having the
following general foMmul a (V):
11
o CH20-~-NH2
R2 ~ ~ C-CH2-CHz-C-(N-CIH;T)n-NH-Y V
wherein n, R, R1 and Y are as defined above and R2 is NH~- or CH30-.
The invention also relates to a mitomycin derivative having the
following structural formul a (V I ):
t) CH20-C-~H2
R2 ~N -SCNH- R3 Vl
wherein
R2 is as defined above; and
R3 is a 2-(3-cyano-4-morpholinyl)-2-deoxypyranosyl saccharide or
a 2-~4-morpholinylJ-2-deoxypyranosyl saccharide.
The invention also relates t~ a process for preparing a mitomycin
derivatiYe havlng the formula (IV)
A68.2.~P 9~2288
-
~ 32 7~3~
o o ~H2~-C-NH2
Y-NH-(-~-~R~~~)n ~ IV
wherein
n is 1;
Y is selected from the group consisting of glucopyranosyl,
galactopyranosyl, mannopyranosyl, xylopyranosyl, cello-
biosyl, lactosyl, glucofuranosyl, maltosyl, and 1,3-cyclo-
hexanediol-2-yl, or a hydroxyl-protected peracetate deriva-
tive thereof,
R is hydrogen;
R1 is hydrogen, C1-C4 alkyl or C1 C4 alkyl substituted by phenyl,
hydroxyphenyl, indolyl, mercapto, C1-C4 alkylthio, hydroxy, carboxy,
amino, guanidino, imidazole or carbamyl; or
R and ~1 together form a five or six membered nitrogen containing
ring;
comprising:
(a) condensing an N-protected amino acid w;th an alcohol in the
presence of a dehydration reagent to give an activated ester,
(b) condensing the activated ester obtained in step (a) with an
amino compound to give a protectcd amino acid amino compound con-
jugate,
:: ~c) removing the protecting group of the protected amino acid-
amino compound conjugate obtained in s~ep (b) to give an amino acid-
amino compound con~usate, and
: ~ ld~ condensing the amino acid-amino compound conjugate obtained
in step (c) with mitomycin A to give the mitomycin derivative.
A68.2.WP 082288
~27~37
The invention also relates to a process for the
preparation of a mitomycin derivative having the formula
(VI)o
o
1 CH2o_~_NH2 VI
R2 ~ CNH-R3
O
wherein
R2 is NH2 or CH30-; and
R3 is a 2-(3-cyano-4-morpholinyl)-2-deoxy pyranosyl
saccharidç;
comprising
(a) condensing bis(acetaldehyde-2-yl) ether with a 2-
amino-2-deoxy saccharide in the presence of a salt of
cyanoborohydride to give a 2-deoxy-2-(3-cyano-4-
morpholinyl) pyranosyl saccharide and a 2-deoxy-4-
morpholinyl pyranosyl saccharide;
(b) separation of the 2-deoxy-2-(3-cyano-4-
morpholinyl) pyranosyl saccharide from the 2-deoxy-4-
morpholinyl pyranosyl saccharide obtained in step (a);
(c) reaction of the 2-deoxy-2-(3-cyano-4-morpholinyl)
. pyrano yl saccharide obtained in step (b) with an acetyl
halide to give a 2-deoxy 1-halo-2-(3-cyano-4-morpholinyl)
peracetyl pyranosyl saccharide;
(d) treatment of the 2-deoxy-1-halo-2-(3-cyano-4-
morpholinyl) peracetyl pyranosyl saccharide obtained in
step (c) with silver thiocyanate to give a pyranosyl
saccharide-1-thiocyanate;
11~'
- lo - 1 ~ 2 7 ~ ~7
(e) reaction of the pyranosyl saccharide-l-
thiocyanate obtained in step (d) with mitomycin C or
mitomycin A to give a mitomycin C- or mitomycin A-pyranosyl
saccharide peracetate carbothioamide; and
(f) hydrolysis of the acetate groups of the
mitomycin-C-pyranosyl saccharide peracetate obtained in
step (e) to give the mitomycin derivative.
The invention also relates to a process for the
preparatian of a mitomycin derivative having the following
formula (VI)
CH20-C-NH2
CH3~N_CNH-R3 Vl
wherein
R2 is NH2- or CH30-; and
R3 is a (4-morpholinyl)-2-deoxy pyranosyl saccharide;
comprising
(a~ condensation of bis(acetaldehyde-2-yl) ether with
a 2-amino-2-deoxy pyransoyl saccharide in the presence of
a salt of cyanoborohydride to give a 2-deoxy-2-(3-cyano-4-
morpholinyl) pyranosyl saccharide and a 2-deoxy-2-(4-
morpholinyl~ paranosyl saccharide;
(b) separation of said 2-deoxy-2-(4-morpholinyl)
pyranosyl saccharide from said 2-deoxy-2-~3-cyano-4-
morpholinyl) pyranosyl saccharide obtained in step (a)i
327937
(c) reaction of the 2-deoxy-2-(4-morpholinyl pyranosyl
saccharide obtained in step (b) with an acetyl halide to
give a 2-deoxy-1-halo-2-(4-morpholinyl) peracetyl pyranosyl
saccharide;
(d) treatment of the 2-deoxy-1-halo-~-(4-morpholinyl)
peracetyl pyranosyl saccharide obtained in step (c) with
silver thiocyanate to give a pyranosyl saccharide-l-
thiocyanate;
(e) reaction of the pyranosyl saccharide-l-thiocyanate
obtained in step (d) with mitomycin A o~ C to give a
mitomycin A- or C-pyranosyl saccharide percetate
carbothioamide; and
(f) hydrolysis of the acetate groups of the mitomycin-
C-pyranosyl saccharide peracetate obtained in step (e) to
give the mitomycin derivative.
The invention also relates to a process for the
preparation of a mitomycin derivative having the following
formula (V)
O CH O-C-NH2
CH3 ~ 3 C-tH2-cll2-c-(-~-clH-c)n-NH-y
wherein
n is 9 or 1;
Y is selected from the group consisting of
glucopyranosyl, galactopyranosyl,
: ~ mannopyranosyl, xylopyranosyl, cellobiosyl,
lactosyl, glucofuranosyl, maltosyl, and 1,3-
cyclohexanediol-2-yl;
R is hydrogen;
R1 is hydrogen, C1-C4 alkyl or C1-C4 alkyl substituted
by phenyl, hydroxyphenyl, indolyl, mercapto, C1-C4
alkylthio, hydroxy, carboxy, amino, guanidino, imidazole or
carbamyl; or
R and R1 together form a five or six mem}}ered nitrogen
containing ring;
-12- ~327~37
R2 is NH2-;
comprising:
(a) condensation of mitomycin C with succinic anhydride under
basic conditions to give mitomycin C-la-succinic acid ester;
(b) condensation of the ~;tomycin C-la-succinic acid ester
obtained in step (a~ with a compound of the Formula (VII).
~(N-cH-~)n-N~l-YP VII
wherein R, R1 and n are defined above and YP is a hydroxyl-protected
saccharide selected from the group consisting of the hydroxyl-pro-
tected derivatives of glucopyranosyl, galactopyranosyl, mannopyrano-
syl, xylopyranosyl 9 cellobiosyl, lactosyl, glucofuranosyl, maltosyl,
and 1,3-cyclohexamediol-2-yl;
~ c~ removal of the hydroxyl protecting groups to give the
mitomycin derivative.
The invention also relates to a process for the preparation of a
mitomycin derivat;ve having the following formula (V):
O cH20-C-NH~ p o n
CH3 ~ ~ / C-CHz-CH2-C-(-N-CH-C~-NH-Y V
wherein
n is 0 or 1;
Y is selected from the group consisting of glucopyranosyl,
g al actopyranosyl, mannopyranosyl, xylopyranosyl, cello-
biosyl, lactosyl, glucofuranosyl, maltosyl, and 1,3-cyclo-
hexanediol-2-yl;
A68.2.WP 082288
_13- 13~7037
R is hydrogen;
R1 is hydrogen, C1-C4 alkyl or C1-C4 alkyl substituted by phenyl,
hydroxyphenyl, indolyl, mercapto, C1-C4 alkylthio, hydroxy, carboxy,
amino, guanidino, imidazole or carbamyl; or
R and Rl together form a five or six membered nitrogen containing
ring;
R2 is CH30-;
comprising:
(a) condensation of mitomycin A with succinic anhydride under
basic conditions to give mitomycin A-la-succinic acid ester;
(b~ condensat;on of the mitomycin A-la-succinic acid ester
obtained in step ~a) with a compound of the Formula (VII)
Il P
~(N-cH-c)n-NH-y VII
R R1
wherein R, R1 and n are as defined above and YP is a hydroxyl-pro-
tected saccharide selected from the group consist;ng of the hydroxyl-
protected derivatives of glucopyranosyl, galactopyranosyl, manno-
pyranosyl, xylopyranosyl, cellob;osyl, lactosyl, glucofuranosyl,
maltosyl, and 1,3-cyclohexanediol-2-yl;
(c) removal of the hydroxyl protecting groups to give the
mitomycin derivative.
The invention also relates to pharmaceutical compositions
comprising a therapeutically effective amount of the mitomycin
derivatives of the invention together with a pharmaceutically accep-
table carrier.
The invention also relates to methods for the treatment of
bacterial inf2ctions compr;sing admin;stering the pharmaceutical
compositions of the invention to an animal.
The invention also relates to methods for the treatment of cancer
by suppress~ng growth of ca~cer cells susceptible to growth suppres-
A68.2.~P 082288
-14- ~27~37
sion comprising administering the pharmaceutical compositions of the
invention to an animal.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The synthetic preparation of the mitomycin deri~atives of the
invention ha~e, as their starting poin~, mi~omycin C. Mitomycin C may
be prepared according to the methods generally disclosed in Cheng et
al., J. Med. Chem. 2~:767-770 ~1977~. Alternatively, mitomycin C can
be obtained from mitomycin A by treatment of mitomycin A with a
methanolic-ammonia solution as described by Matsui, M., et al., J.
Antibiotics XXI:189 (1968~.
The mitomycin derivatives of Formula IV, wherein n=O (X), may he
obtained by displacement of the methoxy group of mitomycin A (VIII)
with the amino group of an amino compound, for example, glucosamine
(Y-NH2; (IX)) under basis conditions in a polar organic solvent to
give the N7 substituted mitomycin derivative (X) (see Scheme I below).
Amino compounds (Y-NH2) which may be substituted at the 7-
position include, but are not limited to glucosamine, galactosam;ne,
mannosamine, xylosamine, eellob;osamine, maltosamine and 2-amino-1,3-
cyclohexanediol and the hydroxyl-protected peracetate derivatives
thereof. Preferably, the saccharide comprising the group "Y" is
substituted at the 2-position with the amino group. Polar organic
solvents which may be used ln the practice of the invention include
methanol, ethanol, propanol, dimethylsulfoxide, and dimethylformamide.
Suitable bases fsr providing the basic conditions of the reaction
include alkylamines such as C1-C3 trialkyl amines, diisopropylethyl-
amine, 1,8-d~azabicyclo~5.4.0]undec-7-ene ~DBU) and dimethylamino-
pyr;dine (DMAP). In general, mitomycin A and the amino derivative are
present in a 1:1 molar ratio, although excess amino derivative may be
present. Sufficient base is present in the reaction mixture to insure
that the reaction remains basic throughout.
A68.2.WP 082288
1327~7
-15-
Preferred mitomycin derivatives having Formula X include N7-(2-
deoxyglucopyranosyl)mitomycin C, N7-(2-deoxygalactopyranosyl)mitomycin
C, N7-(tetraacetyl-2-deoxyglucopyranosyl~mitomycin C, and N7-(tetra-
acetyl-2-deoxygalactopyranosyl~mitomycin C.
Scheme I
o CH~-C-NH2
NHz ~H Mitomycin C
1. NaOH/MeOH
2. CH2N2
o ~H2D-C-NH2
CH30 ~ " OCH3
CH ~ N ~ 'NH Mitvmycin A
¦ Y-NH2 IX
o SH~O- C-NH2
Y-NH ~H X
A6B . 2 . ~P 082 288
~ ~7~37
-16-
The amino acid linked mitomycin derivatives of formula IV,
wherein n=l, may be prepared (Scheme II) from mitomycin A by condensa-
tion of the N-protected amino acid, for example, the N-benzyloxy-
carbonyl derivative (XI)~ with an alcohol such as N-hydroxysuc-
cinamide, which is capable of generating an activated ester, and a
dehydrating reagent to give the activated ester (XII). Dehydrating
reagents which may be used ln this process include, but are not
limited to dicyclohexylcarbodiimide (DCC) and diethylazodicarboxylate
(DEAD) and triphenylphosphine. Treatment of the activated ester (XII)
with any of the above-listed amino compounds (IX) gives the protected
amino acid-amino compound conjugate ~XIII). Removal of the protecting
group, for example, by hydrogenolysis of the N-benzyloxycarbonyl
group, gives the free amino derivative (XIV). Compound (XIV) may then
be condensed with mitomycin A (VIII) by displacement of -OCH3 as
described abnve to give the amino acid linked mitomycin derivative
(IV).
Preferred mitomycin derivatives having Formula (IV~, wherein n=l,
Rl = H and R ~ H include N7-[1[(2-deoxy-2-glucopyranosyl)amino]
carbonyl]methyl] mitomycin C and N7-[~[(tetraacetyl-2-deoxy-2-glucopy-
ranosyl)amino]carbonyl]methyl]mitomycin C.
A68.2.WP 082288
17 132~
Scheme I I
l~aO~~N~ 01~ CC els2~N--I:a-C-O-N~
N~ ~ ~ R R
¦ Y-NH2, IX
Pd/Cg 8a
H ,~1 R
XIV l;~J!I Xlll
P
cH R cH2o c NH~
3~ H
~1111
Q o ~H~o-~-hH2
~-NH-(-~ -N-~n~,
~H3~ N~"~H
IY
A68. 2 .WP 08228
-18- ~3~7~
Mitomycin derivatives having Formula (V), wherein ~ is NH2 and n
is O ~Formula (XV), below), may be prepared (Scheme III) by condensa-
tion of mitomycin C. (XVI) with succinic anhydride to give the amide
~XVII) which may then be condensed with the hydroxyl-protected amino
derivative YP-NH2 (XVIII) using any of the above-listed dehydrating
reagents followed by deprotection to give (XV). Protec~ing groups for
the amino derivative include, but are not limited to, C2-C4 acyl
esters.
Preferred mitomyein derivatives hav~ng Formula (XV) include N1-
[[2-[[(2-deoxy-2-glucopyranosyl)amino~carbonyl]ethyl]carbonyl]
mitomycin C.
S~heme IIl
Il O O
O CH O-C-NH
CH3 ~ CH3 ~ C-tHZ-CH2 C-~H
XVI XVlI
/1. )P-N~ XV I I I
2. Depro ec~
CH O~ H
H2 CHZ C-NH Y
~v
A68. 2 .WP 082~88
~7~
-19-
Mitomycin derivatives hav;ng Formula (V), wherein R2 is -OCH3 and
n is O ~Fnrmula ~XIX~, below), may be prepared (Scheme IV) by treat-
ment of mitomyc~n C (XVI) with sodium methoxide in absolute methanol
~o give mitomycin A (VIII) followed by condensation with succinic
anhydride to give the mitomycin A-la-succinic acld ester (XX).
Condensation of the carboxylic ac~d group of (XX) with the hydroxyl-
protected amino derivative YP-NH2 (XVIII), as described above,
followed by deprotection g~ves (XIX).
Preferred mitomycin derivatives having Formula XVIII include Nl-
[[2-[~(2-deoxy-2-~lucopyranosyl)aminoJcarbonyl]ethyl]carbonyl]
mitomycin A.
Scheme IY
o CH20-c-NH2 R
NH2 ~ 1. NaOHIMe~
~Xvl)
O ~ cS
o CH~o_C_~H
C~O ¦ ~ ~CH3
-cH2-c-NH-Y O
~3 ~ ~ O CH20-C~NH,2
XIX p ~ ~3~ ,0 ~C~3 ~ ~
1. Y -NH XVI I I \ ~ CH~-cH2 ~-~H
2. Depro~ect ~H3 ~N~; N
XX
A68.2.iP 082288
,;
~ ~.327~7
-2~-
The mitomycin derivatives of Formula YI may be prepared according
to the sequence depicted in Scheme Y. Treatment of 3,4-dihydroxy-
tetrahydrofuran (XXI) with aqueous sodium periodate in a polar organic
solvent gives bis(acetaldehyde-2-yl) ether (XXII) which may be con-
densed with a 2-amino-2-deoxy-saccharide (XXIII) in the presence of a
salt of cyanoborohydride to give a mixture of 2-deoxy-2-(3-cyano-4-
morpholino) saccharide (~XXIVa), ~ - -CN), and 2-deoxy-4-morpholinyl
saccharide ((XXIVb), Q ~ -H) which may be separated, for example, by
column chromatography. Sal~s of cyanoborohydride may include any of
the alkali metal salts of cyanoborohydride, preferably sodium cyanobo-
rohydride. Treatment of the saccharide deriva~ive (XXIVa) with an
acetyl halide gives the 2-deoxy-1-halo-(4-morpholinyl) peracetyl
saccharide which may be reacted with silver thiocyanate to give a 1-
thiocyanate saccharide (XXV). Condensation of the thiocyanate (XXV)
with mitomycin C (XVI) gives the mitomycin C-saccharide peracetate
carbothioamide (XXVI). Deacylation of (XXVI), for example, with
methanolic ammonia, gives (VI) (Q = -CN or H).
Preferred mitomycin derivatives having Formula VI include 2-(3-
cyano-4-morpholinyl) 2-deoxyglucopyranosyl mitomycin-la-carbothioamide
~nd 2-(3-cyano 4-morpholinyl)-2-deoxygalactopyanosylmitomycin-la-
carbothioamide.
A68.2.WP 082288
-21- ~3~71~7
Scheme V
f ~20H C132C~13
1~0 ~ N~194 ~,eB~ O ~ ~ ~ N~ 3e~ ~ y
~C:H2~ 30 BO~OH I~O~OH
XXI XXII N~12~C~
XXIII ~ J
XXIV
1. AcBr
2. AgSC~I
NH2~--N 2 ~O NCS
l ~Q\ ~NHC- Mitomycin C ~?Ac
/ ~A~ c~ ~
A~ ~, . ~N~ Q
rQ ~ XXV ~oJ
~o~ \ NH3lcH3oH
XXVI
N~32- co-~H2
CH2~H ~33~ 2
NHC-~p~LCH3
~0~
A68 . 2 . WP 082288
~3~7~
-22-
The compounds of the invention may be present as pharmaceutically
acceptable salts. Among the preferred an;onic counter ions are those
of the halides (derived from hydrohalic acids~, such as chloride,
brom;de, or fluoride. Other anions include sulfonate, or D-toluene-
sulfonate.
As an antibiotio, the compounds of the present invention are
useful aga;nst all microorgan;sms susceptible to the anti-bacterial
action of the parent compounds, these microorganisms including, but
not limited to, Pseudomonas, $taDhYlococcus, Sarcinia, D;plococcus,
Str~ptococcus, CorYnebacterium t ~emo~hi1us, Escherichia, Klebsiella,
Proteus, Salmonella, Shiaella, Brucella, MYcobacterium, Nocardia,
SaccharomYces, Cand;da, Penicill;um, and As~erqillusO Specific
microorganism treatable with the compounds of the present invention
include Pseudomonas aer~q~_osa, Staphvlococcus aureus, StaphYlococcus
albus, StaDhvlococcus c treus, Sarcina lutea, Diplococcus Dneumoniae,
Streptococcus hemol Yti CU$, strePtococcus lactis, CorYnebacterium
diphtheriae, Hemophilus pertussis, Escherichia coli, Klebsiella
Pneumoniae, Proteus vulqaris, Salmonella _~yPhosa, Salmonella Para-
~Yeh~, Shiqella dYsenteriae, Brucella abortus, Brucella meqatherium,
Brucella mYcoides, Brucella anthracius, MYcobacterium ATCC_ 607,
MvcQbacterium avium, Mvcobacterium Dhlei, Nocardia asteroides,
SaccharomYces cervisiae, Candida albicans, Penicillium qlacum, and
AsPergillus niqer.
The mitomycin derivatives of the present invention are useful in
vitro as antiseptics, i.e. ~or disinfecting. The compounds are also
useful top;cally and internally as therapeutic agents in combating
pathogenic bacteria, e.g. in cases of staphylodermatitis, bacterial
pneumoniae, leptopserosis, rickettsiosis, salmonellosis, and the like.
Typ;callyl for top kal application, the mitomycins of this
invention are applied in compositions having concentrations in the
range of 0.01 to l000 ug~ml.
As antineoplastic agents, the compounds of the present invention
are useful in treating a variety of cancers, including, but not
A68.2.WP 082288
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limited to, those cancers susceptible to cell growth suppression by
the parent compounds. Treatment o~ cancers with the parent compounds
are described in the following references:
Driscoll, J.S. ~t al., Cancer ChemotheraDY Rep. 4:1 (1974).
Kojima, R., et al., Cancer Chemotherauv Rep. 3:111 (1972).
Sugiura, K., Cancer Res. 19:438 (1959).
Oboshi, S., et al., Gann ~:315 (1967).
Sugiura, K., Cancer Chemotherapv Re~. 13:51 (1961).
Venditti, J.M., et al., Advances in Cancer ChemotheraDv, pp. 201-
209 (1978) Editors: H. Umezawa et al., Japan Soc. Press, ~okyo/Univ.
Park Press, Balt;more.
Usubuchi, I., et al., ~Q 58:307 (1967).
Typical cancers treated by the mitomycin derivatives of this
invention include, but are not limited to gastric and pancreatic
neoplasms (Schein, P.S. et al., in M;tomYcin C: Curr*nt Status and New
DeveloPments, pp. 133-143, Carter et al. Eds., Academic Press, New
York (1979)~. Other cancers that may be treated using the compounds
of the invention include lung, breast, anal, colorectal, head and
neck, and melanoma.
The compounds of the invention are also active against the
following tumor systems: Leukemia L-1210, Leukemia P388~ P1534
leukemia, Friend Virus Leukemia, Leukemia L4946, Mecca lymphosarcoma,
Gardner lymphosarcoma, Ridgway Osteogenic sarcoma, Sarcoma 180
(ascites~, Wagner osteogenic sarcoma, Sarcoma T241, Lewis lung
carcinoma, Carcinoma 7555 CD8F, Mammary Carcinoma, Colon 38, Carcinoma
1025, Ehrlich carcinoma (ascites & solid1, Krubs 2 carcinoma ~as-
cites), Bashford carclnoma 63, Adenocarcinoma E 0771, B16 Melanoma,
Hardin-Passey melanoma, 6~10m2 26, Miyona adenocarcinoma, Walker
carcinosarcoma 256, Flexner-Jobling carcinoma, Jensen sarcoma,
Iglesias sarcoma, Ig12sias ovarian tumor, Murphy-Sturn lymphosarcoma,
Yoshida sarcoma, Dunning leukemia, Rous chicken sarcoma, and Crabb
hamster sarcoma.
A68.2.WP ` 082288
~ 24- ~327037
The pharmaceutical compositions of the present invention may be
administered by any means that achieve their intended purpose. For
example, admin;stration may be by parenteral, subcutaneous, intra-
venous, intramuscular, intraperitoneal, transdermal, or buccal routes.
Alternatively, or concurrently, administration may be by the oral
route. The dosage administered will be dependent upon the age,
health, and weight of the recipient, kind of concurrent treatment, if
any, frequency of treatment, and the nature of the effect desired.
Compositions within the ssope of this invention include all
compositions wherein the mitomycin derivatiYe is contained in an
amount effective to achieve its intended purpose. While individual
needs vary, determ;nation of optimal ranges of effective amounts of
each component is ~ith the skill of the art. Typical dosage forms
contain 10 to 300 ~mole/kg animal of the mitomycin derivative, or an
equivalent amount of the pharmaceutically acceptable salt thereof.
In addltion to the pharmacologically active compounds, the new
pharmaceutical preparations may contain suitable pharmaceutically
acceptable carriers comprising excipients and auxiliaries which
facilitate processing of the active compounds into preparations which
can be used pharmaceutically. Preferably, the preparations, particu-
larly those preparations which can be administered orally and which
can be used for the preferred type of administration, such as tablets,
dragees? and capsules, and also preparations which can be administered
rectally, such as suppositories, as well as suitable solutions for
administration by iniection or orally, contain from about n.ol to 99
percent, preferably from about 25 to 75 percent of active compound(s),
together with the excipient.
The pharmaceutical preparations of the present invention are
manufactured in a manner which is itself known, fur example, by means
of conventional mixing, granulating, dragee-makiny, ~issolving, or
lyophilizing processes. Thus, pharmaceutical preparatiuns for oral
use can be obtained by combining the active cumpounds with solid
excipients, optionally grinding the resulting mixture and processing
A68.2.WP 082288
- ~ 1327~3~
-25-
the mixture of granulcs9 after adding suitable auxiliaries, if desired
or necessary, to obtain tablets or dragee cores.
Suitable excipients are, in particular, fillers such as sac-
charides, for example lactose or sucrose, mannitol or sorbitol,
cellulose preparations and/or calcium phosphates, for example tri-
calc;um phosphate or calcium hydrogen phosphate, as well as binders
such as starch paste, using, for example9 ma ke starch, wheat starch,
rice starch, potato starch, gelatin, tragacanth, methyl cellulose,
hydroxypropylmethylccllulose, sodium carboxymethylcellulose, and/or
polyvinyl pyrrolidone. If desired, disintegrating agents may be added
such as the above-mentioned starches and also carboxymethyl-starch,
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof, such as sodium alg;nate. Auxiliaries are, above all, flow-
regulating agents and lubricants, for example, silica, talc, steric
acid or salts thereof, such as magnesium stearate or calcium stearate,
and/or polyethylene glycol. Dragee cores are provided with suitable
coatings which, if desired, are res1stant to gastric juices. For this
purpose, concentrated saccharide solutions may be used, which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone, poly-
ethylene glycol and/or titanium dioxide, lacquer solutions and
suitable organic solvents or solvent mixtures. In order to produce
coatings resistant to gastric juices, solutions of suitable cellulose
preparations such as acetylcellulose phthalate or hydroxypropymethyl-
cellulose phthalate, are used. Dye stuffs or pigments may be added to
the tablets Dr dragee coatings, for example, for identification or in
order to characterize combinations of active compound doses.
Other pharmaceutical preparations which can be used orally
include push-fit capsules made of gelatin, as well as soft, sealed
capsules made of gelatin and a plasticizer such as glycerol or
sorbitol. ~he push-fit capsules can contain the active compounds in
the form of granules which may be mixed with fillers such as lactose,
binders such as starches, and~or lubricants such as talc or magnesium
stearate and, optionally, stabilizers. In soft capsules, the active
A68.2.WP 082288
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-26- ~32~37
compounds are preferably dissolved or suspended in suitable liquids,
such as fatty oils, or liquid paraffin. In addition, stabilizers may
be added.
Possible pharmaceutical preparations which can be used rectally
include, for example, suppos;tories, which consist of a combination of
the active compounds with a suppository base. Suitable suppository
bases are, for example, natural or synthetic triglycerides, or
paraffin hydrocarbons. In addition, it is also possible to use
gelatin rectal capsules which consist of a combination of the active
compounds with a base~ Possible base materials include, for example,
liquid triglycerides, polyethylene glycols, or paraffin hydrocarbons.
Suitable formulat;ons for parenteral administration include
aqueous solutions of the active compounds in water-soluble form, for
example, water-soluble salts. In addition, suspensions of the active
compounds as appropriate oily injection suspensions may be adminis-
tered. Suitable lipophilic solYents or vehicles include fatty oils,
for example, sesame oil, or synthetic fatty acid esters, for example,
ethyl oleate or triglycerides. Aqueous injection suspensions may
contain substances which increase the viscosity of the suspension
include, for example, sodium carboxymethyl cellulose, sorbitol, and/or
dextran. Optionally, the suspension may also contain stabilizers.
The following examples are illustrative, but not limiting, of the
method and compositions of the present invention. Other suitable
modifications and adaptations of the variety of conditions and
parameters normally encountered in clinical therapy and which are
obvious to those skilled in the art are within the spirit and scope of
the invention.
A68.2.WP 082288
~'~2~3~
-27-
EXAXPLES
Exam~le 1: Preparation of M;$omYcin A
Mitomycin C (50 mg, 0.15 mmol) was dissolved in 3 ml of a
solution of 50% methanol and 50% 0.1 N NaOH and stirred at room
temperature for 18 hrs. After completion of the reaction (TLC,
CHCl3:MeOH, 10:1), the reaction mixture was quenched with dry ice to
neutralize sodium hydroxide. The mixture was then freeze-dried in
vacuo, and the mitosane compound was removed with methanol. The
methanol solution was concentrated in vacuo to dryness, and thP
residue was redissolved in a minimum amount of methanol and then
precipitated with ether to give 20 mg of a red-purplish powder. ~his
was dissolved in 15 ml of ethyl acetate and cooled to 5~C, treated
with diazomethane (etherial solution of diazomethane was prepared
according to the procedure of Arndt, Ora. SYnthesis, Collective Volume
Il, pp. 165-167~, and stirred for 20 minutes (TLC, CHCl3:MeOH, 10:1).
After completion of the reaction, the solvent was first removed under
water aspirator reduced pressure and then dried in vacuo. The residue
was recrystallized from ether to give 18 mg of reddish needles, m.p.
159-160~C. TLC (EtOAc:acetone, 1:1) gave one spot Rf = 0.91, and
(acetone:benzene, 4:1) one spot R~ s 0.48. UV (methanol3 216 and 358
mu. NMR (acetone-d6, middle peak nf acetone at 2.10~, ~, 5.94 (br,
2H); 4.76 (dd, 1H~; 4.38 (t, lH); 4.07 (s, 3H~; 3.96 (d, lH)i 3.54
(dd, lH); 3.41 (d, lH); 3.35 (d, lH); 3.25 Is, 3H); 2.99-2.264 (mmm);
2.87 (s); 1.640 ~s, 3H).
Example 2: Prepar~tion of N7-(2-deoxvqlucopYranosyl)mitomvcin O
To a solution of mitomycin A ~10 mg, 0.028 mmol) in absolute
methanol was added a methanolic solution of glucosamine HOl (70 mg,
0.325 mmol) and diisopropylethylamine ~100 ul~. This mixture was
stirred under N2 atmosphere at room temperature until the reaction was
A68.2.~P 082288
-28- ~ 3~3~
complete by TLC (EtOAc: acetone, 1 1) at which time (10 hrs.) the
solution had changed color from reddish to dark purple. The solution
was concentrated by evaporation with a N2 s~ream and chromatoyraphed
on a preparative silica plate eluted with acetone-ethyl acetate (1:1).
The purple band remaining close to the origin was scraped off and
eluted with methanol. Further purification by HPLC (C18 reversed
phase, semi-preparative column, methanol: O.lN phosphate buffer, 1:1)
gave a purple powder, NMR (D20) ~ 5.32 ~d, lH~ saccharide anomeric H~;
3.85 (s, 3H, 9a-OCH3); and the disappearance of singlet at 4.09
(Matsui7 M., et al., J. Antibiot. 21:189 (1968~; Cheng, L., and
Remers, W.A., J. Med. Chem. 20:767 (1977); Vyas, D.M., e~ al., J. Ora.
Chem. 51:4307 (1986)).
Example 3: Preparation of N-(2~ DihvdroxYcYclohexyl~qlYcinamide
To a solution of N-benzyloxycarbonylglycine (3 g, 14.3 mmol) in
dioxane was added N-hydroxysuccinimide (1.65 9, 14.3 mmol) and N,N-
dicyclohexylcarbodiimide ~2.96 y, 14.3 mmol) with cooling. The
reaction mixture was stirred at 0-5C for one hour and allowed to
stand under refrigeration overnight. The urea precipitate was removed
by suction filtration and the filtrate was concentrated in vacuo to
dryness. The yellowish residue was recrystallized from ethyl acetate-
ether to give an 84% yield of the glycine activated ester mp. 112-
114C. NMR ~CHC13).
The above-prepared activated ester of glycine (25 9, O.C08 mol)
was dissolved in 15 ml of dry DMF (dimethyl formamide), chilled to
below S-C, and 2-amino-l~3-cyclohexanediol (2.1~ 9, 0.016 mol) in DMF
was added drop-wise with stirring under N2 atmosphere. After comple-
tion of the reaction (TLC, CHC13:MeOH~ , DMF was removed under
reduced pressure and the result~ng solid residue was crystallized from
ethyl acetate to give white crystals in 84% yield, m.p. 170-172~C.
NMR (D20): ~ 7.45 ~s, 5H, aromatic H); 5.20 (s, 2H, benzylic-CH2);
3.95 (s, 2H, -CO-CH2^NH2); 3.6 (t, lH, ClH of cyclohexane ring); 3.45
A68.2.WP 082288
~L327~37
-29-
(m, 2H, C2H and C6H of cyclohexane ring); 2.0, 1.8 and 1.35 (m,m,m, 2
to 1 to 3H; C3, C4 and C5 hydrogens of cyclohexane). The product
comprises the N-protected benzyloxycarbonyl derivative of N-(2,6-
dihyroxycyclohexyl)glycinamide.
N-protected benzyloxycarbonyl N-(2,6-dihydroxycyclohexyl)
glycinamide (39, 0.033 mol) was dissolved in 100 ml of absolute
ethanol with a molar equivalent of 10% HCl. Hydrogenolysis with 5%
Pd/C at 30 psi, removal of the catalyst over celite, and subsequent
evaporation of solvents in vacuo yielded a pale brownish solid which
was triturated with ether and recrystallized from ethyl acetate and
ether, m.p. 207-210C. NMR (320~ ~, 3.65 (t, 3H, ClH of cyclohexane
ring); 3.55 (m, 2H, C2H and C6H of cyclohexane~; 3.4 (s, 2H, -C0-CH2-
NH2); 2.05, 1.80, and 1.38 (m,m,m, 2 to 1 to 3H9 hydrogens of C3, C4
and C5 of cyclohexane~.
ExamDle 4
Animal Studies
The compound N7-(2-deoxyglucopyranosyl)mitomycin C, prepared
according to Example 2, was evaluated for both murine P388 leukemia
antitumor activity and toxicity to bone marrow in normal mice.
A. Determination of murine antitumor actiyitY
The murine P388 leukemia system, maintained intraperitoneally in
female DBA/2 mice, was used to evaluate antitumor activity. This
tumor was selected because of its known sensitivity to the parent
compound, mitomycin C (Driscoll ~ ancer ChemotheraDv ReDorts
4:1 (1974~). N7-(2-deoxyylucopyranosyl~mitomycin C was dissolved in
sterile water (at 4-C) immediately prior to administration. Mitomycin
C was dissolved in ethanol, and the resultant solution was adjusted to
5% ethanol, 95% sterile water.
A68.2.WP 082288
~2~
Each compound was administered ;ntraperitoneally to groups of
CD2F1 male mice on Day 1 after intraperitoneal implantation of 1 x 106
P388 leukemia cells. The P388 antileukemic activity of the test
compound was assessed by mean survival days and percentage increased
life span (ILS). The % ILS was calculated as follows:
%ILS ~ (T-C3/C x 100;
where ~ is the mean survival days of the treated mice and C is
the mean survival days of the untreated mice.
P388 antitumor activi$y for N7-(2-deoxyglucopyranosyl)mitomycin
C9 in comparison with the parent mitomycin C, is summarized in Table
2:
Table 2
Antitumor Activitv Against P388 Leukemia
DruqDose (mg/kq~ %ILS Mean Survival (davs)
N7-(2- Sa 42% 14.2
deoxygluco-
pyranosyl)
mitomycin C13.5a 61% 16.1
Mitomycin C 4.5b ~1% 18.1
Control C 10 . 0
aLDo dose
bApproximate LD1~ dose
CTreated with drug vehicle
B. Determination of the effects of N7-(?-deoxYqlucoP.Yranosyl)
mitomycin C on the hematopoietic svstem in mice
Measurement of peripheral 7eukocyte (WBC) count was performed
us;ng a 20-ul sample of retro-or~ital sinus blood obtalned from normal
CD2F1 male ~ice on Day 3 ~ollowing i.p. administration of 13.5 mg/kg
of N7-~2-deoxyglucopyranosyl~mitomycin C or 4.5 mg/kg of mitomycin C.
Afi8.2.WP 082288
_31_ ~327~37
Blood samples obtained were diluted in 9.98 ml of Iso~on (a neutral,
isotonic buffer solution) and counted in a Coulter counter after lysis
with Zapoglobin ~an enzyme solution which lyses red blood cells, but
not white blood cells). WBC counts are expressed as a percentage of
~alues fro~ control mice receiving drug vehicle only. The results are
summarized in the following table:
Table 3
In vivo Murine WBC DePression
WBC Count on Day 3
Dose (as Dercent of control)
N7-(deoxy- 13.5 mg/kg ~4%
glucopyranosyl)
mitomycin C
Mitomycin C 4.5 mg/kg 56-66%
In summary, these in vivo studies demonstrate that N7-(2-deoxy-
glucopyranosyl)mitomycin C has significant activity against the murine
P388 tumor system, at doses producing no sign;f;cant bone marrow
toxicity, as determined by depression of peripheral leukocyte ~WBC)
count.
Example 5: Antibacterial Activitv
N7-(2-deoxyglucopyranosyl)mitomycin C was evaluated for activity
against Gram-negatiYe bacteria, in a comparative study with the parent
mitnmycin C. Minimum inhibition concentration (M.I.C.~ against a
Gram-negative strain of bacteria (HB101) was estimated by the dilution
methnd, with graded concentrations of drug added to agar at 37-40C.
N7-(2-deoxyglucopyranosyl)m;tomycin C was dissolved in 50% sterile
water-50% ethanol at 4-C, and mitomycin C was dissolved in ethanol.
The agar, containing drug, quickly solidified at room temperature, and
A68.2.WP 082288
-32- ~3~37
the bacteria were plated immediately. After 24 hours at 37C, the
agar plates were observed for inhibition of bacterial grow~h. The
results are summarized in Table 4.
Table 4
M.l.C.
Compound Çram-neqative Bacteria
N7-(2-deoxyglucopyranosyl) 1.66-3.3 ~cg/ml
~itomycin C
Mitomycin C 0O3 -0.5 mcg/ml
.
Having now fully described this invention, it will be understood
by those of skill in the art that the same can be pe-rformed within a
wide and equivalent range of conditions, formulations and other
parameters without affecting the scope of the invention or any
embodiment thereof.
A68.2.~P 082288