Note: Descriptions are shown in the official language in which they were submitted.
~5644
1126-050-0
273/
Description
Galactose~-6 Nitroqen ~stard ~mpounds and their lses
Technical Field
-
Thls invention relates to nitrogen mustard
derivatives which possess good antitumor activity
against leukemia and other tumors and particularly to
nitrogen mustards derived from carbohydratesv
Backqround Art
Nitrogen mustards were among the first
chemotherapeutic agents rationally applied to the
treatment of tumors. In many ways, modern cancer
chemotherapy can be said to have begun with the
discovery of the clinical activity of certain nitroqen
mustards against lymphoid neoplasms during studies made
on the biological effects and therapeutic applications
of certain chemical warfare agents during World War
II. However, the high chemical reactivity of nitrogen
mustards and the high probability of nonselective
reaction with diverse nucleophilic centers available in
vivo result in numerous toxic side effects. In
particular, damage to bone marrow and other rapidly
dividing normal cells limits the use of basic nitrogen
mustards (such as nitrogen mustard itself: 2-chloro-N-
(2-chloroethyl)-N-methylethaneamine) limits the
usefulness of these compounds. In fact, the damaging
effects of nitrogen mustards on bone marrow provided
the initial clue which first suggested that the
~2456~14
--2
mustards might also affect the growth of lymphoid
tumors.
Nitrogen mustard (mechlorethamine) represents the
standard for comparison in the mustard class of
alkylating agents. It has substantial therapeutic
activity for a number of human tumors, including
Hodgkin's and non-Hodgkin`s lymphomas. It is actively
used in the treatment of Hodgkin's disease, as a
component of the MOPP regimen (DeVita et al, Annals
Internal Med. 73, 891-895, 1970). Approximately 70-80%
of patients are cured on this reqimen~ However, the
substantial acute and chronic bone marrow toxic
prcperties of nitrogen mustard serve as a severe
limiting factor in its clinical use, as discussed above
for the class in general. Many patients, at least 20~,
require a substantial reduction in dose (perhaps to
sub-therapeutic levels) because of extreme depression
of white blood cell count. With repeated courses of
treatment, there is a threat of cumulative injury with
a resultant state of chronic bone marrow hypoplasia.
L-PAM (L-phenylalanine mustard) has similar limitations
because of severe bone marrow toxicity. In contrast to
nitrogen mustard, the nadir of white blood cell count
is somewhat delayed with L-PAM, and the injury to the
bone marrow is more cumulative.
Numerous derivatives of nitro~en mustard have been
synthesized in an effort to reduce toxic effects while
retaining the desired chemotherapeutic activity. See,
for example, Burger`s Medicinal Chemistry 4th Fd~, Part
II, M.E. Wolff, Ed., John Wiley ~ Sons, New York,
(1979~, pages 619-633 for a review of chemotherapeutic
alkylating agents, most of which are derivatives of or
have structural features in common with nitrogen
~2~S64~
--3--
mustard.
A few amino qlucose mustards have been developed
in the more recent past and tested for antitumor
activity against P388 leukemia and L1210 leukemia
(Wampler et al, Can. Res. 35, 1903-1906 (1975)). These
compounds employed a glucose moiety carrying a nitrogen
mustard at position C-2. Reist et al, J. Amer. Chem.
Soc. 82, 2025-2029 (1960) discusses the synthesis of a
C-6 glucose mustard without mentioning its possible
biological activity. Other sugar mustards showing good
activity include l,6-d i-(2-chloroethyl) amino-1,6-
dideoxy-D-mannitol dihydrochloride, developed by Var ha
et al, J. Chem. Soc., 1957, 810-812.
However, none of these studies have indicated any
suppression of damage to bone marrow. Accordingly,
there remains a need for therapeutically active
compounds which retain their activity against rapidly
dividing tumor cells but which have reduced activity
against bone marrow.
Disclosure of the Invention
Accordingly, it is an object of the present
invention to provide a nitrogen mustard derivative
which retains high activity against tumor cells but
which spares bone marrow.
It is another object of this invention to provide
a method for synthesizing such compounds.
These and other objects of the invention as will
hereinafter become more readily apparent have been
accomplished by Drovid ing a compound of the formula
- ~L2~56~
--4--
rRo 1
X ~ OR'
wherein X is halogen, each R independently represents
H, a Cl-C4 alkyl group, a C2-C4 alkanoyl group, a
phosphate group, or a sulfate, sulfonate, or benzoate
group with the proviso that no more than one sulfate,
sulfonate, or benzoate group is present in said
compound; and R' represents R or a carbohydrate residue
derived from a carbohydrate having the formula R'OH
with the proviso that R' and the R on C-2 or both R
groups on C-3 and C-4 together can represent an
isopropylidene group; or a pharmaceutically acceptable
salt thereof.
Brief Description of the Drawinqs
A more complete appreciation of the invention and
many of the attendant advantages thereof will be
readily obtained by reference to the following detailed
description when considered in connection with the
accompanying drawings, wherein:
Figure 1 shows a reaction scheme of the synthesis
of galactose-C-6 nitrogen mustard.
Figure 2 shows a plot of in vivo effects on
peripheral leukocyte (WBC) count after administration
of nitrogen mustard (HN2; a control reference
compound~, L-phenylalanine mustard (L-PAM; a control
~Z~56~
--5--
compound), or the C-6-galactose mustard compound, an
indication of the effect of these compounds on bone
marrow cells which produce the peripheral leukocytes.
Best Mode for Carryinq Out the Invention
The present invention arose in part from the
discovery that galactose C-~ nitrogen mustard compounds
show full antitumor activity with reduced toxicity to
bone marrow. Galactose C-6 nitrogen mustards of the
invention are compounds having the formula:
X~
N
X ~ R ~ O
~O ~ OR'
OR
wherein X is halogen; each R independently represents
H, a Cl-C4 alkyl group, a C2-C4 alkanoyl group, a
phosphate group, or a sulfate, sulfonate, or benzoate
group with the proviso that no more than one sulfate,
sulfonate, or benzoate group is present in said
compound; and R' represents R or a carbohydrate residue
derived from a carbohydrate having the formula R'OH
with the proviso that R' and the R on C-2 or both R
groups on C-3 and C-4 together can represent an
isopropylidene group; or a pharmaceutically acceptable
salt thereof. Although all of these compounds retain
the desired chemotherapeutic activity, those which are
more hydrophilic and less lipophilic are preferred
since such compounds have less tendency to enter
tissues or organs hi~h in lipids, such as bone
marrow. Accordingly, compounds in which each R on the
galactose portion of the molecule is H are preferred.
~specially preferred are compounds in which both R and
R' all represent hydrogen atoms or in which R' is a
:~LZ~5644
carbohydrate residue. When R and R' do not represent
H, R and R' should be enzymatically cleavable in the
human or animal to which the compound is administered,
a result that can readily be detected by standard
metabolic studies.
When R or R' is a phosphate, benzoate, sulfate, or
sulfonate (i.e., when the compound is a phosphate,
benzoate, sulfate, or sulfonate ester), phosphate
groups are preferred. Sulfates, sulfonates and
benzoates are limited to one such functional group per
compound. No such limit is placed on the number of
phosphate groups. Preferred sulfonates are Cl-C4 alkyl
sulfonates, phenylsulfonates, and amino-substituted
phenylsulfonates, most preferably p-
aminophenylsulfonates.
In the mustard portion of the molecule, X is
preferably bromine or chlorine and most preferably
chlorine. However, X may also represent fluorine or
iodine, and each X may independently represent
different halogen atoms, although preferred compounds
are those in which both X represent the same halogen.
When R' represents a carbohydrate residue derived
from a carbohydrate having the formula R'OH (i.e., the
"OH" is one of the hydroxyl groups of the
carhohydrate), it is preferred that the carbohydrate be
a single 5- or 6- carbon sugar such as glucose,
galactose, mannose, ribose, fructose, xylose, or a
similar monosaccharide of the formula C5H1005 or
C6H1206. Most preferred are galactose, xylose,
mannose, and ribose. As shown in the formula set forth
above, the R' moiety is attached to the remainder of
the formula through a hemiacetal bond at C-l of the
~Z~564~
--7--
non-reducing galactose C-6 nitrogen mustard portion of
the molecule. As shown in the formula, compounds of
the invention can exist as either the
~ or ~anomer. Attachment of carbohydrate R'OH is
preferably through a C-4 or C-6 (most preferably C-6)
hydroxyl for a 6-carbon sugar and through a C-5
hydroxyl for a 5-carbon sugar.
Representative Cl-C4 alkyl and alkanoyl groups
include methyl, ethyl, t-butyl, acetyl, propionyl and
similar groups. Ethyl groups are most preferred. No
more than one methyl should be present in order to
minimize toxicity.
It is preferred that all R groups on C-2, C-3, and
C-4 be the same, although this is not essential.
When a compound of the invention is present as a
pharmaceutically acceptable salt, the salt will usually
be an acid addition salt formed by reacting an acid
with the amino group attached at C-6 of the galactose
portion of the molecule. The acid used to form the
addition salt can be either a mineral acid or an
organic acid. Examples of suitable mineral acids
include hydrochloric acid, hydroiodic acid, sulfuric
acid, and nitric acid. Suitable organic acids are
those having a dissociation constant sufficient to form
an acid addition salt. Examples of suitable organic
acids include citric acid and lactic acid.
A salt may also be formed at a phosphate or
sulfate functional group that is part of the
compound. Such salts would typically be alkali metal
salts; e.g., -po3HNa or -P03K2 instead of -P03H2 for R
or R'.
~`
~L2~569~4
Taking these preferences into consideration,
examples of preferred compounds of the invention
include the following:
~ample R' C C-3 C-4 X salt
1 H H H H Cl
2 galactose H H H Cl HCl
3 CH2CH3 -CH2CH3 -CH2CH3 -CH2CH3 Brcitrate
4 (CH~)7C= (CHl)~C= Cl
ribose -CH2CH3 -CH2CH3 -CH2-CH3 Brlactate
6 -P03H2 -PC3H2 -F03H2 -F03H2 Cl
O O O
7 H -CCH3 -CCH3 ~CoCH3 Cl HCl
8 H H -CCH3 -CCH3 Br
9 mannose -CH3 H H ClH ~ 3
xylose H H H Br
11 galactose H H H Br citrate
12 H H H H Br lactate
The procedures previously known for the
preparation of nitrogen mustard derivatives of glucose
are not suitable for producing the derivatives of
galactose that form the present invention. The
crystalline diisobenzylidine derivative of
glucofuranose is impractical with a galactose sugar.
The yields are extremely low and the isobenzylidene,
isopropylidene is extremely difficult to prepare. The
1,2,3,4-diisopropylidene galactose derivative used in
the method disclosed herein was selected based on its
high yield, the commercial availability of raw startinq
~245644
g
material, and the low cost of production (Reist et al,
J. Amer. Chem. Soc. 82, 2025-2023, 1960). However, all
compounds of the invention can be synthesized usinq the
following procedure and modifications thereof readily
apparent to those skilled in the art of carbohydrate
synthesis.
The first step in the general synthetic procedure
is the preparation of a 1,2,3,4-diisopropylidene
derivative of galactose. The reaction is generally
carried out in dry acetone under an inert atmosphere.
Zinc chloride or a similar catalyst is added along with
sulfuric acid, after which D-galactose is added. After
neutralization of the acid and addition of water, the
resulting 1,2,3,4-diisopropylidene derivative is
separated from the solvent. This product is then
dissolved in a polar aprotic solvent and treated with
sodium azide to give the 6-azido derivative. The 6-
azido derivative is reduced with a catalyst to give the
6-amino derivative, which was used for structure
verification since this is a known compound. The
tosylate derivative is treated with diethanolamine in
an amino exchange reaction to give the 6-bis-(2-
hydroxyethyl)amino derivative. This compound is
treated with thionyl chloride to give the 6-bis-(2-
chloroethyl)amino derivative. The final product in
which R and R' all represent H is obtained from this
compound by removal of the isopropylidene protective
groups. At this point, R and R' groups can be added
using standard techniques of carbohydrate chemistry.
General techniques of alkylation, acylation, and
formation of disaccharides are well known and are
described in many publications such as Vargha et al, J.
Chem. Soc~, 1957, 805-809; Varqha et al, J. ChemO Soc.,
1957, 810-812; Horton, J. Org. Chem., 29, 1776-1782
~2~5644
--10--
(1964); and Suami et al, J. Med. Chem., 22, 247-250 (1979)
Compounds of the invention can be used to selectively
suppress cell division in hydrophilic tissue while minimizing
undesired side reactions in lyophilic tissue by administering
an amount of one or more compounds of the invention sufficient
to suppress cell division to a mammal. Freireich et al,
Cancer Chemo. Rept. 50, 219-244 (1966), compared the
quantitative toxicity of 18 anticancer drugs in six species
after correcting the data to a uniform schedule of treatment
for five consecutive days. This analysis demonstrated that
mouse, rat, dog, human, monkey, and man have essentially the
same maximum tolerated dose (MTD) when compared on a basis of
mg/m2 of body surface area. the study suggested that Phase I
clinical trials could be safely initiated at a d-.,e one-third
the animal MTD. The mouse was as useiul as ani other species
in this regard on which to base ,he calcul.liion. The
appropriate therapeutically effective dose for any compound of
the invention can therefore be determined readily by those
skilled in the art from simple experimentation with laboratory
animals, preferably mice, and will usually be within the range
from about 4 to about 8 mg/kg of body weight, preferably from
about 5 to about 6 mg/kg. Tumors, the growth of which may be
suppressed, include those listed in Holland and Frei, Cancer
Medicine, Lea and Febiger, Philadelphia, 1973. Tumors which
are preferred to be treated include those already known to be
sensitive to nitrogen mustard and L-PAM, such as Hodgkin's and
non-Hodgkin's lymphomas.
Although treatment of any mammal, (e.g., cattle,
horses, dogs, and cats) is encompassed by this
invention, treatment of humans is especially preferred.
The mode of administration of compounds of the
invention may be by any suitable route which delivers
the compound to the system being treated. For the
purposes of the present invention, the compounds may be
administered orally, topically, parenterally,
intraperitoneally, or by any other method which enables
the active ingredient to reach the site being
treated. The term parenteral as used herein includes
subcutaneous, intravenous, intramuscular, intrasternal,
and infusion techniques. Intravenous injection is the
preferred method of administration.
Compounds of the invention may be prepared into
pharmaceutical compositions containing the active
ingredient in a form suitable for any of the usages
previously described. For example, a pharmaceutical
composition suitable for oral use may be in the form
of, for example, a tablet, troche, lozenge, aqueous or
oral suspension, dispersible powder or granule,
emulsion, hard or soft capsule, syrup, or elixir.
Compositions intended for oral use may be prepared
according to any method known in the art for the
manufacture of pharmaceutical compositions and may
contain one or more agents selected from the group
consisting of sweetening, flavoring, coloring, and
preserving agents. Tablets containing the active
ingredient in admixture with non-toxic pharmaceutically
acceptable excipients may be prepared by any method
suitable for the manufacture of tablets. Excipients
may include, for example, inert diluents, such as
calcium carbonate or lactose; granulating and
:~LZ456~4
-12-
disintegrating agents, such as maize starch or algenic
acid; binding agents, such as starch and gelatin; and
lubricating agents, such as magnesium stearate and
talc. The tablets may be uncoated or they may be
coated by any known technique to delay disintegration
and absorption in the gastrointestinal tract and
thereby provide a sustained action over a long period.
Aqueous suspensions containing the active material
in admixture with excipients suitable for the
manufacture of aqueous suspensions may also be
prepared. Such excipients include suspending agents,
such as methyl cellulose, dispersing or wetting agents
such as lecithin and condensation products of an
alkylene oxide with a fatty acid, such as
polyoxyethylene stearate; or similar materials. The
agueous suspension may also contain a preservative,
such as p-hydroxybenzoate, a coloring agent, a
flavoring agent, or a sweetening agent.
Oily suspensions may be formulated by suspending
the active ingredient in a vegetable oil or mineral
oil. The oil suspensions may contain a thickening
agent, such as beeswax. Sweetening agents, flavoring
agents, and preserving agents, such as those described
above, may also be used.
Other pharmaceutical preparations may be prepared
by any of the techniques now known to the
pharmaceutical arts.
It is preferred that the compounds of the
invention, when in the form of pharmaceutical
preparations, are present in unit dosage forms. When
intended for human use, these amounts can easily be
S69L4
-13-
calculated from the dosage rates previously given by
assuming a body weight of 70 kg~ Accordingly, a
preferred unit-dose-containing pharmaceutical
preparation would contain from about 300 to about 375
mg of active ingredient. It will be understood,
however, that the specific dose level for any
particular patient will depend upon a variety of
factors including the activity of the specific compound
employed; the age, general health, sex, and diet of the
patient; the time of administration; the route of
administration; the rate of excretion; possible
synergistic effects with any other drugs being
administered; and the severity of the particular
disease being treated.
The invention now being generally described, the
same will be better understood by reference to certain
specific examples which are included herein for
purposes of illustration only and are not intended to
be limiting of the invention or any embodiment thereof,
unless specified.
Example 1: SYNTHESIS OF 6-BIS-(2-CHLOROETHYL)AMINO-6-
DEOXY-D-GALACTOPYRANOSE HYDROCHLORIDE (F)
A synthesis scheme for the production of
galactose-C-6-nitrogen mustard is shown in Figure 1.
I. Preparation of Compound A; 1,2,3,4-
Diisopropylidene-6-0-p-Tolylsufonyl-
~-D-Galactopyranose
Anhydrous zinc chloride (43.2g) was rapidly added
to a flask containing 450 ml of dry acetone under a dry
nitrogen atmosphere with viqorous stirring.
- ~Z4L56~L~
-14-
Concentrated sulfuric acid (1.7'1 ml) was added
dropwise; then 36g of D-galactose ~anhydrous) was added
and stirring continued for 4 1/2 hours. A suspension of
anhydrous sodium carbonate (72g) in 126 ml of water was
added in small portions. The suspension was filtered
and washed with several portions of acetone. The
filtrate and washings were combined and evaporated in
vacuo to remove the acetone. The upper oily layer was
then extracted with ether, and the ether was dried over
anhydrous sodium sulfate. Then ether was removed in
vacuo to give a syrup-like liquid (46g) of the 1,2,3,4-
diisopropylidene-~-D-galactopyranose which was
purified by distillation (vacuum).
This distilled product (30g) and 33g of p-
toluenesulfonylchloride were added to 60 ml pyridine
(dried over molecular sieves) in a flask protected from
moisture and kept at rocm temperature overnight. Water
was added to dissolve the pyridine ~Cl, and additional
water was added with stirring until crystallization was
complete. The crystals were washed with water and
recrystallized from hot ethanol after treatment with
charcoal to give compound A, m.p. 101-102C, NMR
(Acetone-d6), tosyl group 7.4-7.8~; -D anomeric
protons 5 3~ Jl 2= 4.8cps.
II. Preparation of 1,2,3,4-Diisopropylidene-6-azido-6-
deoxy- ~D-galactopyranose; Compound B
Compound A (18.6g) was dissolved in 150ml of
dimethylformamide (dried over molecular sieves) in a
flask protected from moisture. Sodium azide (7.8g) was
added with stirring, and the mixture was heated at 120
C for 36 hours. The mixture was then evaporated to
dryness in vacuo, and the residue was suspended in
56~9~
-15-
water and methylene chloride; The aqueous layer was
then separated. The methylene chloride layer was
washed several times with water and dried over
anhydrous sodium sulfate. Then the methylene chloride
was removed in vacuo to give a syrupy liquid. NMR
(CDC13): no tosyl group in region 7.4-7.8~; IR
(ETOAC): N3, 2090 cm strong band. This is compound s.
Compound B ~49) was dissolved in ethyl acetate
(200 ml) along with 0.4g of 5% Pd/C and hydrogenated at
1 atmosphere and room temperature for 5 hours to give
the amine derivative, compound C. IRl (ETOAC): broad
band at 2800-3400cm~l and disappearance of band at 2090
cm 1. This is compound C.
III. Preparation of 1,2,3,4-Diisopropylidene-6-Bis-(2-
Hydroxyethyl)amino-6-Deoxy-~-D-Galactopyranose;
Compound D
Twenty-one grams of compound A were suspended into
225 ml of freshly distilled diethanolamine and heated
at 150-160 C for 4-41/2 hours under a dry nitrogen
atmosphere with vigorous stirring. The amber colored
viscous mixture was cooled to room temperature and
added to 800 ml of methylene chloride. The methylene
chloride solution was washed with 250 ml portions of
water 2X and back extracted with fresh methylene
chloride (100 ml). The methylene chloride extracts
were combined and dried over anhydrous sodium
sulfate. Concentration in vacuo gave 16g of an amber
syrup. ~MR (CDC13): disappearance of tosyl group at
7.4-7.8~.
~Z~56~
--16-
IV~ Preparation of 1,2,3,4-Diisopropylidene-6-Bis-(2-
Chloroethy~)amino-6-Deoxy- ~-D-Galactopyranose;
Compound E
Compound D (2g) was dissolved in 25 ml of dry
methylene chloride and 6 ml of thionyl chloride,
refluxed for 15 minutes, and immediately evaporated to
dryness in vacuo followed by repeated co-evaporation
with fresh dry methylene chloride. A sample of this
product was set aside for NMR analysis which was
consistent with the proton splitting pattern for
haloethyl compounds (a triplet of triplets,
2.5-3.9 ~). Isopropylidene methyl groups were also
indicated by peaks at 1.3-1. 5~.
V. Preparation of 6-Bis-(2-Chloroethyl)amino-6-Deoxy-
D-Galactopyranose Hydrochloride; Compound F
Without further purification compound E (l.Og) was
added to lOml of 6N HCl in a small flask fitted with a
reflux condenser and refluxed for 10 minutes. The
solution was then cooled to room temperature, extracted
2X with methylene chloride, and treated with
charcoal. The aqueous solution was lyophilized to give
450 mg of pale white crystals, which were
hydroscopic. NMR (D20): 3.4-4.6~, aalactose and N-
(CH2CH2Cl)2 protons; HDO, 4.8~; 5.3 and 5.6~, alpha
and beta anomeric protons respectively; disappearance
of isopropylidene protons (methyl groups) at
1.3-1.5~. Anal.: Calcd. for CloH2005NC13. 4H20: C,
29.05; H, 6.78; N, 3.38; Found: C, 28.43; H, 6.03; N,
3.28.
Example 2: ANIMAL STUDIES
Normal CD2Fl male mice, 6-9 weeks old, were used
for initial studies to determine toxic single doses of
564a~L
-17-
compound F in mice. Doses of the compound in the range
of the LDlo dose (single intraperitoneal dose that
produces toxic deaths in 10% of the normal mice) were
then tested for antitumor activity against several
murine tumor systems. The LDlo dose was then evaluated
in normal CD2Fl mice for effects on the hemapoietic
system.
retermination of toxic doses of the com~ound in nonmal mice
Normal CD2Fl male mice were used to determine the
LDlo dose (single intraperitoneal dose toxic to 10% of
normal mice) for the compound. Various concentrations
of the compound were prepared immediately prior to use
by dissolving the drug in physiological saline (on ice) `
and administering intraperitoneally (i.p.) in a volume
of 0.1 ml/10 grams body weight. The normal mice were
then observed for up to 45 days post drug ~~
administration to determine deaths due to acute and
chronic (up to 45 days) drug toxicity.
Results are summarized in the following table:
Dose (mq/kg) Dose (~mol/k~) Deaths due to druq toxicity
14 33.3 0/10 (LDo)
15.5 37 1/10 (LDlo)
18 43 2/10
22.5 53.4 7/10
27 64 10/10 ~LD100)
The approximate LDlo dose of the compound (single i.p.
dose toxic to 10% of normal mice) was 15.5 mg/kg or 37
~ol/kg; single doses in this range were used to
evaluate antitumor activity in two murine tumor
systems.
~2~644
-18-
Determination of murine antitumor activity
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 nitroyen mustard
(HN2) and L-phenylalanine mustard ~L-PAM). The water-
soluble dru~s (the galactose mustard compound and
nitrogen mustard) were dissolved in saline (on ice)
immediately prior to use and administered i.p. in a
volume of 0.1 ml/10 grams body weight. L-PAM was
dissolved in one milliliter of ethanol containinq
40 ~1 of concentrated HCl, and this was added to a
0.3% solution of hydroxypropyl cellulose (HPC) in
saline to give a final concentration of 4~ ethanol and
96% HPC - containing saline.
Each drug was administered to groups of 10 CD2Fl
male mice on day one after implantation of 1 x 106 P388
leukemia cells i.p. (in a volume of 0.10 ml). The P388
antileukemic activity of the test galactose mustard
compound was assessed by mean survival days, percentage
increased life span (~ILS), and number of survivors
after 45 days. The percentage ILS was calculated as
follows:
%ILS = (T-C)/C x 100 where T is the mean survival days of
the treated mice and C is the mean survival days
of the untreated mice.
P388 antileukemic activity of the test galactose
mustard compound was compared to that achieved with two
clinically used nitrogen mustard compounds: nitrogen
mustard (HN2) and L-phenylalanine mustard (L-PAM). The
results are summarized in the following table:
~Z~6~
--19--
Antitumor Activity A~ainst P388 Leukemia
Dose DoseMean survival
Drug (mq/kg) ( ~ol/kg) (days)_ ~ILS
gal. mustard 15.5* 37 19.5 107
compound 18~ 43 15.9 69
nitrogen 2.9* 15.1 15.0 60
mustard (HN2) 3.5~ 18.2 12.3 31
L-phenyl 12* 39 >21.5 >129
alanine
mustard 15~ 48.8 >19.1 >103
(L-PAM)
* approximate LDlo dose
approximate LD20 or LD25 dose
When antitumor activities against the murine P388
leukemia (a single LDlo dose administered i.p. on day
one after implantation of 106 P388 cells
intraperitoneally) are compared, overall survivals for
the galactose mustard compound were significantly
greater (p<0.05) than survivals for nitrogen mustard.
Four out of 30 mice receiving the LDlo dose of L-PAM
(12 mg/kg) were long term survivors, living more than
45 days; there were no long term survivors for either
nitrogen mustard or the galactose mustard compound.
The Ehrlich ascites tumor system was used as an
additional murine tumor system for confirmation of
therapeutic activity. Ehrlich ascites was maintained
intraperitoneally in female BALB,/C mice. Treatment as
described in the previous section was administered on
day one after implantation of 2 x 106 cells/0.10 ml in
male BD2Fl mice (6-9 weeks old). The mean survival of
untreated tumored mice was 15 days. On days 29 and 40,
the number of mice surviving was:
~2~564~
-20-
~ug ~se (mg/kg) Nm~er of surviving mice
day 29 day 40
gal. mustard 15.5 8~0 6~0
nitro~en
mustard (HN2) 2.9 7~0 7~0
L-PA~ 12 8~0 6/10
These initial studies with the Ehrlich ascites suggest
that the galactose mustard compound has antitumor
activity comparable to nitrogen mustard and L-PAM in
this system.
Determination of the effects of the compound on the
hematopoietic svstem in mice ` -`~
Effects on peripheral leukocyte (WBC) count.
Measurement of peripheral leukocyte (WBC) count
was performed using a 20- ~ sample of retro-orbital
sinus blood obtained from normal CD2Fl male mice on
days 3, 4, 5 or lO following i.p. administration of
doses in the range of the LDlo; groups of 10 mice were
treated with drug as described previously. Blood
samples obtained were diluted in 9.98 ml of Isoton (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 differential counts were performed
on Wright-stained smears. WBC and absolute neutrophil
counts are expressed as a percentage of values from
control mice receiving drug vehicle only.
Figure 2 presents the results of the peripheral
leukocyte (WBC) depression study. The nadir for the
3LZ~56~
-21-
galactose mustard compound occurred on day 3 after drug
administration and was 74~ of control. For nitrogen
mustard, the nadir WBC also occurred on day 3 and was
57% of control. For the galactose compound, the WBC
count recovered to control values by day 4, while
animals receiving nitrogen mustard did not recover to
control values until day 5. L-PAM produced a more
prolonged WBC nadir, with WBC counts reduced to
approximately 40-45~ of control from days 3 through
5. By day 10 after drug administration, the WBC counts
for the L-PAM treated mice had only recovered to 77~ of
control. Absolute neutrophil counts for the galactose
compound were not significantly different from control
(94~ of control). In contrast, nitrogen mustard
produced a nadir absolute neutrophil count of 70% of
control on day 3, and L-PAM produced a nadir absolute
neutrophil count of 50~ of control on day 4. In
summary, the galactose compound produced minimal WBC
depression and no significant decrease in absolute
neutrophil count at the LDlo dose that produced
antitumor activity against the murine P388 leukemia
superior to that achieved with a comparable LDlo dose
of nitrogen mustard.
The invention now being fully described, it will
be apparent to one of ordinary skill in the art that
many changes and modifications can be made thereto
without departing from the spirit or scope of the
invention as set forth herein.
