Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
3~ 1
This is' a divisional application of copendin~ app1icatio r-
~Seri~l No. 392,323, filed Decer~er 15, 198].
BAC:~GROUND OF THE INVENTION
This invention is directed -to a -treatment oE
malignant -tumors in vivo using the compound 2-~-D-
ribo-fuxanosylthiazole-4-carboxamide and related deriva-~ives
such as its estexs.
Control of malignant tumors in rnan and animals still
remains as an unrealized goal. Within the last several decades,
understanding of malignancy has made significant progress;
however, conquering of the malignant disease state has not
been realized.
Con~entional therapy of both humans and other valuable
animal species inflicted with malignant tumors presently
includes surgical excising of the tumor, local radiation therapy
oiF the afflicted animal, and chemotherapy by administration
oF a chemctheraputic agent to the animal. The death of a
significant number of patien-ts inflicted with malignant tumors
is attributable no-t to the primary tumor bu-t instead to
metastasis o~ the primary tumor -to secondary sites in the host.
If a primary tumor is detected early, it normally can be
eliminated by surgery, radiation or chemotherapy or combinations
of these. The metastatic colonies of these primary tumors,
however, are exceedingly harder to detect and eliminate and the
unsuccessful managemen-t of them remains a serious medical problem.
Tumors are normally classified either as benign or
malignant. The mali~nant tumor is characterized -From the
benign by its ability to invade both surrounding -tissue and to
coloni2e distant sites via metas-tasis~ Certain organs are more
prone -to metastasis -than o-thers. Included in this qroup would
be the lung, the brain, the liver, the ovaries and -the adrenal
glands. It has further been suggested that both surgery
and radiation of a primary tumor in certain instances actually
promotes metastasis.
In view of the inability of current cancer therapy
to successfully control the malignant tumor and its metastasis,
it is evident that there exists a need for additional
chemotheraputic agents.
In a paper entitled Synthesis and Antiviral Activity
of Certain Thiazole C-Nucleosides, J. Med. Chem. 1977,
Volume 20, No. 2, 256, I and my co-workers disclosed the
synthesis of and certain preliminary in vitro antiviral
activity of the compounds 2-.beta.-D-ribofuranosylthiazole-4-
carboxamide and 2-(2,3,5-tri-O-acetyle-.beta.-D-ribofuranosyl)-
thiazole-4-carboxamide in an in vitro test system utilizing
three viruses, type 1 herpes simplex virus, type 3 para-
influenza virus and type 3 rhinovirus. The in vitro activity
of the compound 2-.beta.-D-ribofuranosylthiazole-4-carboxamide
against these three viruses was only moderate. With the
compound 2-(2,3,5-tri-O-acetyle-.beta.-D-ribofuranosyl)thiazole-4-
carboxamide, only moderate activity was seen with type 1
herpes simplex virus whereas with the type 3 parainfluenza
and the type 3 rhinoviruses activity was negative. While
certain marginal in vitro antiviral activity noted in the
preceding was seen, quite to the contrary, in vivo antiviral
testing for both 2-.beta.-D-ribofuranosylthiazole-4-carboxamide
and 2-(2,3,5-tri-O-acetyl-.beta.-D-ribofuranosyl)thiazole-4-
carboxamide, as judged by the number of test animal deaths,
was negative. In the in vivo tests, the number of deaths
for the test animals for both 2-.beta.-D-ribofuranosylthiazole-4-
carboxamide and 2-(2,3,5-tri-O-acetyl-.beta.-D-ribofuranosyl)-
- 2 -
-thiazole~4-carboxamide was equal to or exceeded the number
of deaths of -the p:Lacebo control arlimals indicatincJ tha-t
both of the compounds 2-~ D--ribofuranosylthiazole-4-carboxamide
and 2-(2~3,5-tri-O-ace-tyl- ~-D-ribofuranosyl~thiazole-4-
carboxamide demonstrated no useful in vivo antiviral activity.
With regard to the above noted in vitro antiviral
testing of both 2- ~-D-ribofuranosylthiazole-4-carboxamide
and 2-(2,3,5-tri-O-acetyl ~-D~ribvfuranosyl)thiazole-4-
carboxamide, these compounds were te~ted against viruses or
which the known antiviral compound RIBAVIRIN~ i5 known to
have positive antivir~. activity. In view of ~he preliminary
marginal in vitro activity of 2- ~-D-ribofuranosyl-thiazole-4
carboxamide against these test viruses, it was expected that
the spectrum of activity of 2- ~-D-ribofuranosylthiazole-4-
carboxamide would be similar to the spectrum of activity of
the compound RIBAVIRIN~. RIBAVIRIN is kno~n to be an active
in vi-tro an-tiviral agent and in vivo antiviral and is further
known to exhibit no siynificant antitumor ac-tivity~ Additionally,
cer-tain derivatives of RIBAVIRIN~ such as its 5' monophosphate
are also known to be inactive as antitumor compounds~ I-t was
reasonable to expect, in comparing the preliminary în vitro
antiviral activity of 2-D-ribofurallosylthiazole-4-carboxamide
with that of RIBAVIRIN~, that ~-~-D-ribofurano~ylthiazole-4-
carboxamide would exhibit positive in vivo antiviral activity
and negative ~ntitumor activity similar to RIBA~IRIN~. Totally,
contrary to this, the compound 2 ~-D-ribofuranosylthia~ole-
4 carboxamide possessed no useful in vivo antivi.ral. ~ctivity
and, quit.e unexpectedly, has demonstrated positive antitumor
ac-tivity.
_ ,~ , . ..
i~q~''3~ ~
I have found that t:he compound 2-~-D-ribofurano-
sylthiazole-4-carboxamide and its es-ters, including 2-(2,3,5-
-tri-O-acetyl~ D-riboEuranosyl)thiazole-4-carbo~a~ide and
2-(5~0 phosphoryl~-D-ribofuranosy:l)thiazole~-c~rboxamide
exhibi-t antitumor activities of such significance as to be
useful as antitumor agents in vivo.
BRIEF SUMl~RY OF THE INVENTION
The compound 2-~-D-ribofuranosyltniazole~4-carboxamide
has been shown to exnibit significan-t antit7~mor activity in
vivoO The present invention relates to the use of this
compolmd and certain related derivatives in treating maligna~t
tumors in warm blooded animals. According to this invention,
the antitumor properties of 2-~-D-ribofuranosylthiazole~4
caxboxamide and its related esters are utilized ~y administerin~
to a warm blooded animal an effective amount of a pharmaceutical
composition containing as the active compound at least about
0~1 percent by weight, based on the total weight of the
composition, a compound of the structure:
2 0 2N >~\
N~,S
R30 _ / O \
\~ ~
R ?
wherein Rl and R2 are H or Cl-C18 aoyl and R3 is Hl Cl~C18 acyl
or HQ ?- and physioloaically acceptable sal~s -thereo.
- 4
7~
In .l more preferred ~roup of compounds Rl and R2 are ~1 or
,~
Cl C8 acyl and R3 is H, Cl~C8 acyl or ~ and physiologically
OH
aceeptable salts thereof.
Specifically noted for Rl, R2 and R3 as preferred
acyl groups are acetyl, propionyl, isobu-tyryl and b~nzoyl.
Specifically noted as acceptable salts are the alkalai metals
and ammonium or substituted ammonium salts sueh as sodium,
pota~sium and ammonium ~alts.
Preferrably, when Rl and R2 are H, R3 is OH, Cl-C8
aeyl or HO-?~ , and when Rl and R2 is Cl-C~ aeyl, R3 is Cl-C8 aeyl~
For use in the pharmaceutieal composition of the
invention, a pharmaceutieal carrier would be utili~ed sueh -that,
preferredly, the pharmaceutical carrier would be ehosen -to
allow administration of a suitable concentration of the ac-tive
compounds of the invention as solutions or suspensions by
injec~ion into an afflicted warm blooded animal. Depending on
the host harboring the malignant tumor, the type of tumor,
and the tumor site, administration by injeetion would be
intraveneously, intra~uscularly, intracerebrally~ subeutaneously,
or intraperitoneally.
Alternately r the eomposition of the invention mi~ht
suitably be formulated in appropriate pharmaeeutical earriers
allowing for adminis-tration by other routes such as oral
administration, ophthalmic administration, topical
administration or aclministration by suppository~
DETAILED DESCRlPTION
The parerlt compound of the invention, compound 2-~-U~
5 --
pC/ l''~' ''
~ ~g~ g ~
rlboEuranosylthiazo:le~4-carboxamicle, is preferredly
prepared as described in Example l. An al-ternate sy~-thesis
oE this compound appears in J. Org. Chem. r Vol~ 41~ ~0. 26
1976, 4074~
Cer-tain es-ters o:E 2-~-D-ribofuranosylthiazQ,le~4-
carboxamide, compound 1, such as 2-(2,3,5-tri-O-acet~l-~-D-
ri.bofuranosyl)thiazol.e-4-carboxamide, compound 2~ or 2-~5 O~
phosphoryl-~-D-ribofuraIlosyl~thiazole-4-carboxamide r compound
3, are prepared as described in Examples 2 an~ 3 res~ctively.
1~ Additional.ly, other esters, such as the monoester 2~ O-
acetyl-~-D-ribofuranosyl)thiazole-4-carboxamide, comp~und 4~
would be prepared as in the synthesis described in e~mple 4.
For other preferred carboxylic esters of the inventio~
substitution of acetic anhydrîde with a sui-ta~le an~ride
such as propionic anhydride, butyric anhydri~e or ~e~oic
anhydride is made. Alternately, the appropriate aci~ chloride
could be substituted for the acid anhydride.
The esters of compound 1 could assist in de~ivery
of the compound in ar. afflicted host. Such esters of the
compound could be formed by reacting olle or more of t~e
hydroxyl groups of the sugar moiety of compound 1 wit~ suitable
reversible blocking groups which could be cleaved in ~ivo from
the parent compound 1 by certain in situ chemical or ~nzymatic
reactions.
For reaction with the hydroxyl groups/ es-te~s such
as~ but not necessarily limited to, acyl and phosphor~l esters
would be cons.idered The acyl groups can be ~elected from a
group consisting of straight chain, branch chain, su~-tituted~
unsaturat.ed, saturated or aromatic acids such as, but not
~~ necessarily limited to, acetic, trifluoroacetic, propiLollic,
- 6
(~
n-butyric, isob-ltyric, valeric, caproict pelargonic, enan-thic
capyxilic, lactic, acrylic, propargylic, palmitic~ benzoic,
phthalic, sa:Licylic~ cinnamlc and naphthoic acids. If a
phosphoryl group is chosen, the phosphoryl ester could be as
a free acid or as a salt form. Acceptable salts of the
phcsphate moiety of the phosphoryl ester can be selected
from, but not necessarily limited to, the group consisting
of alkali and alkaline earths, e.g., sodium, potassium~
calcium, magnesium, lithium~ ammonium and subs-tituted
ammonium, -trialkylammonium~ dialky'lammonium, alkylammonium~
e.g., triethylammonium, trimethylammonium, diethylammonium~
octylammonium, cetyltrimethylammonium and cetylpyridium.
As preferred forms of the esters of the inventi~n,
compounds 2, 3 and 4 are mentioned. In addi-tion to these,
other tri-O-acyl esters such as the 2',3',5'~tri-O'ben~oyl
are mentioned. Additionally, other mono-es-ters such as the
5'-O-benzoyl is mentioned. Generally, for carboxylic esters
the preferred esters would include Cl C18 acyls. A more
preferred group includes Cl-C8 acylsO Preferredly, when
phosphoryl esters ~ere utilized, the phosphate groups would be
formed as a salt preferredly as a sodium salt or other alkali
metal s~lt or ammonium.
Ester forms of compound 1, as is shown in the
examples herein, are useful in delivering the compoun~ to
the af~ected site in an affected host. As is shown in the
examplesj the tri-acetyl ester, compounc~ 2~ is indicated as
being an efEective antitumor agent when injected intra-
peri~oneally into an affected host. The described triacetyl
compound and any other acyl ester of compound 1 woulc~ ~e
c~p^.ctecl -to be hydrol.i7.ed to compollncl ] in certain bioloyicaL
fluids such as the acid environrne.lt of the s-tomach or an
environment which inclwcles an appropriate enZyMe capable o:~
in vivo enzymatic cleaY~cJ2 of ~he ester -to compound 1. While
I do not wish -to be bound by theory, if the phosphoryl ester
of compound I~ such as the 5' phosphate~ were used~ other
en2ymes present in vivo migh-t also be expected to appropriately
en~ymatically cleave the phosphate -to yield an in situ
delivery of compound lo Compound 3, the phosphoryl ester of
compound 1, as is shown in the examples~ is indicated as
being an effective antitumor a~ent when injected in an effected
hostO At this time it is not known whe-ther its ac-tivity is
expressed as the 5I phcsphate or whether it i~ enzymatically
cleaved to compound ]. Further, it is possible khat compound
1 might be promoted in situ by other enzymatic reactions to
compound 3. In any event, both compound 1 and compound 3 are
indicated as being effective in vivo antitumor ayents as is
indicated by the examples.
In performing the invention, compound 1, or a
selected ester form thereof, is appropriately mixed with a
suitable pharmaceutical carrier which may be as si~ple as
sterilized water or could be a complex carrier havin~ appropriate
agents to suita~ly mimic certain biological environments J
i.e~, pH and salt adjusted solution sui-table for i~traveneou~,
intramuscular or other injections.
In selecting a suitable pharmaceutiGal carrier,
consideration of the type of tumor/ the site of the tumor and
the health and age of the host would be gi~en. Ad~itionally,
if an ester :Eorm o compound 1 is used~ considerati~n of the
pc/~
chemical reactivity o:E the est-er would also he t3iven Thus,
carbo~ylic acyl ester wou:Ld preferredly be suspended or
solubilized in an appropriate non-clcidic medium. A phosphoryl
ester miyht be appropriately used in the presence of a suitable
buffer or as a salt as discussed above.
Preferredly, compound 1 or any of the other
~ompounds of the invention, would be mixed with an appropriate
pharmaceutical carrier such that compound l or a derivative
thereof would be suitably soluble in the carrier. Alternately,
however, suspensions, emulsions and other formulations of the
compounds of the invention could be used where indicated.
The pharmaceutical carrier, in addition to having a ~lubilizing
or suspending agent therein, might also include ~ui~a~le
dilutants, buffers, surface active agents and other similar
agents as are typically used in pharmaceu-tical carriers. The
total composition of the pharmaceutical carrier would, however,
be chosen to be compatible with the site of delivery, 1the
concentration of -the active ingredient and other parameters
as are s'andard in pharmaceutical industry.
Compound l, or the other compounds of the invention,
would be suitably admixed with the pharmaceutical carrier
such that i~ would be present in a concentrat.ion of at least
0.1 percent by weight of the total composition. Preferredly,
it would be present in the pharmaceutical carrier at a
concentration oE about 10% to about 90% by we-ght o~ the
total composition~
Effective amounts oE compound l, or the other
compounds of the invention, typically would range f.rom about
2~5 milligrams per kilogram (mg/Kg) of the total body weight
P~ P
~ 7 ~
oE the -treated warm blooded animal -to about 200 mg/K~ per day.
Preferredly, the ran~e would be from 12.5 mg/Kg to about 100
mg/Kg An even more preferred range would be from about 15
mg/K$ to about 50 mg~Kg. As with other fackors noted above,
the amount of compound utilized in treating an afflicted
animal would take into account parameters such as the type of
tumor~ the t~mor site 9 the form of administerin~ the compound
and the physical size and condition of the host~ In any event,
the actual amount should be sufficient to provide a chemothera-
peut.ically effective amount of the agent to the host in a
convenient vol~ne, which will be readily within the ability of
thos~ skilled in the art to determine given the disclosure
herein.
In at least one study, compound 1 of the in~ention
has been injected at dosages up to 2000 mg/Kg in-to tumor
bearing animals and no deaths of the animals were attributed
to the toxicity of compound 1 on the toxicity day of the test.
In a host which has been diagnosed as being terminally ill with
a malignant tumor excessive amounts beyond any toxicity range
might be indica-ted if there is ~ny probability of cure of
the terminally ill host as is commonly practiced in current
cancer chemotherapy.
As in the examples used for illustra-tive purposes
below, wherein a tumor bearing host was treated once daily
with the indicated test compound. Depending upon the clinical
situation, the daily dose of compound 1 or any o~ the other
ompounds of the invention~ might be similarly given; however,
the daily dose cou:ld also be broken up into sub-unit doses
-- 10 --
mab/ ,l~
which, in their totality, e~ual ~he daily dose~ Thus r for
example, at a 50 mg/Kg dose level the patient might be
appropriately treated follr times a day with doses of 12~5
rng/KgO
A composition used Eor inhibiting ma~ignant
tumors in warm blooded animals might be suitably pr pared by
adding compound 1 or any of the other compounds o the inven-
tion~ to a pharmacologically compatible solvent followed by
sterilization and packaging in appropriate sealable vials at
a known concentration. Appropriate doses of the compound are
then withdrawn from the vial and administered by injection to
-the host.
EXAMPLE 1
2-~-D-Ribofuranosylthia~ole~4--Carboxamide, COMPOUND 1
Ethyl 2-(2,3,5-tri V-benzoyl-~-D-ribofuranosyl)-
thiazole-4-carboxamide was utilized as prepared in Srivastova
et. al. J. Medu Chem. 1977, Volume 20, No. 2, 256.
concentrated solution of ethyl 2-(2/3~5-tri-o-benzyl-B-D-rib
furanosyl)thia~ole-4-carboxamide (5O0 g, 8.31 mmol) in
methanol (15 ml) was stirred with methanolic ammonia (satur-
ated at 0C, 100 ml) in a pressure bottle at room temperature
for 2 days. The solvent was evaporated and the residue wa~
chromatographed through a column (2.5 x 35 cm3 o silica gel
(100 g) packed in ethyl acetate. E-~ution of the column with a
solvent system (eth~l acetate-l propanol-water, 4:1:2; v~v;
top layer3 removed tne fast-moving methyl benzo~te and benz-
amide~ The slower moving, major, W and sugar-positive
fractions were collected and the solvent was evaporated in
vacuo. The residue (syrup), thus obtained, was readily
crys-tallized from e-th~nol-ethyl ace-tate to provide 106 g (74%~
of pure product, compound 1: mp 144-145 C.; la~ Sp-1'I.3
( 1 DMF); W ~ pHl 237 nm (8640); UV ~maXP
H ~R(Me2SOd~)~7.5 7.8 [S(br),2,CONH2]~ Me2SO-d6-D2O~-
4.g9(d,1,J=5 HZr Hle) t 8.25 ~s,ltH5)~ Anal. ~CgHl~N2O5S)
C,H,N,S.
EXAMPLE 2
2~(2,3~5-Tri-O-Acetyl~-D-Ribofuranosyl3-thiazole-4-
Carhoxamide, COMPOUND 2
Acetic anhydride (2.G ml) was added to an ice-
cold solution of compound 1 (1.04 g, 4 mmol) in anhydrous
pyridine tl6 m]) and the reaction solution was stirred at
room temperature for 17 h. The solvent was evaporated in
vacuo, the residue was dissolved in ethyl acetate, and the
solution was washed with water and dried (MgSO4) The ethyl
acetate portion was e~aporated in vacuo and the resi~ue thus
obtained was crys~alized from water to provide 1~4 g (90%~ o~
compound 2 as white needles; mp 103C;lH NMR ~CDC13) 2.1 (3
s, 9,tri-O-acetyl), 6.2 and 7.15 [pair of s~br), 2, CONH2J,
8-2 (s, 1, H5)- Anal- (ClsHl~N2O8s) C~H~N~S-
EXP~IPLE 3
2-(5-O-Phosphoryl-~-D~Ribofuranosyl)thiazole-4-Carboxamide
~2-~-D-Ribofuranosylthiazole-4-Carboxamide 5'~Phosphate),
COMPOUND 3
Water (151 mg, 8.4 mmol) was added carefully to a
solution (maintained at 0C with stirring) of freshly dis-
tilled phosphoryl chloride (2.0 g, 13.2 mmol), pyridine
~1~21 g, 14,4 mrnol) and acetonitrile (2 3 g, 56~7 mmol)
12 -
2-~-D--Ribofuranosy]-thiazole-4-caI^bo~lmide, compound 1, (clri~d
over P2O5 arld powdered, 800 mg~ :3.0 mmol) was added t~ the
solution and the xeaction mixture was stirred ~ontinuously
for ~l hrs. at 0C. The reaction mixture was poured into ice
water ~ca. 50 ml) and the pH was adjusted to 2.0 with 2N
sodium hydroxide. The solu-~ion was applied to a col~nn o~
activated charcoal (20 g3, and the column was washed thoroughly
with water until t~le elua~e was salt~free. The column was
eluted with a solution ethanol-water concentrated ammonlum
hydroxide (10^10:1) and the fractions (25 ml each) were
collected. The fractions containing pure (tlc, silica gel,
ace-tonitrile~0.1 N ammonium chloride (7:3)~ nucleotide, com-
pound 3, were collected and evaporated to dryn(ess under
vacuum. The anhydrous residue was dissolved in water and
passed through a column of Dowex* 50-W-X8 120-50 mesh, H form,
15 ml). The column was washed with water and the fraction
containing the nucleotide was collected. The solution was
concentrated to a small volume (5 ml) and passed through a
column of Dowex* 50W-X8 (20 ~ 50 mesh, Na form, 15 ml). The
column was washed with water. The nucleotide containing
fraction was lyophilized. The residue was triturated with
ethanol, collected by filtration and dried (P2O5)~ to provide
560 mg (47~) of compound 3 as monosodium dihy~rate in the
crystalline form.
AnaL. calcd~ for CgH]2N~O8PSNa-2H~O: C, 27~13;
H, 4.0g; N,7~04; P, 7.78; S, $.05. Found: C, 27~42; ~I, 3.87;
N, 7~07i P, 8.03; S, 8.41.
*trade mark
- 13 -^
mab/ ~'
4I'~
EXAMPLE 4
2-(5-O-Acetyl~-D-Ribofllranosyl)thiazole-4-Caxboxamide
COMPOUND 4
A solution of 2~(~,3-O-isopropylidene-~-D-ribo--
furanosyl)thiazole-4-carboxamide 11.5 g, 5 mmol) (prepared as
per Fuertes, et al; ~. Org. Chem., Volume 41, NO~ 26, 1976,
4074) in anhydrous pyridine (2Q ml) was chilled in an ice~
water bath and acetic anhydride (2.5 ml) was slowly added
with stirring. The reaction solukion was allowed to warm to
roo~ temperature and stirriny was continued for 15 h. The
solvent was evaporated in vacuo and the residue was dissolved
in ethyl acetate and washed with water. The ethyl acetate
portion was evaporated in vacuo and the residue was dissolved
in 80% acetic acid ~25 ml). The solution was heate~ on a steam
bath for 30 mins. and the solvent evaporated in ~acuo. The
residue was dissolved in ethyl acetate, washed once with
water and dried over MgSO4. The ethyl acetate portiorl was
evaporated and the crude product was passed through a column
of silica gel ~100 g, packed in chloroform) and eluted with
20~ (V/V) ethyl acetate in chloroEormr The nucleoside bearing
fractions were pooled and evaporated to yield 1~05 g (70%~
o compound 4. (CllH14N2O6S~o
As illus-trative examples of the use of compound 1
and other illustrative compounds of the invention 3 examples
5 through 12~ below, are given. In these examples~ the
efficacy of the compounds is demonstrated using the standaxd
tests against cextain malignant tumors. The tesks utilized
in these illu~trative examples were conducted by the Develop-
mental ~'herapeutics Program, Division of Cancer Treat~ent,
Nakîonal Cancer Institute. The tests were supervised by this
~4 --
agency utiliz:ing the:ir standarcl protocols and procedures. All
tests conformed to these protoco:Ls and all tests were evaluated
under -the criteria defined by these protocols. I'he fol]owin~
representative examples illustrate confirmed activity of the
illustrative compounds of the in~ention against screenin~
tumor systems of the National Cancer Institute.
For purposes of the Eollowing examples, the abbre-
viation IP stands for intraperitoneal and IV stands for intra-
venousO The mean and median survival times are calculated
in instruction 14 (revised 6/78) of the Screening Data Su~mary9
Devel3pmental Therapeutics Prog~am, Division of ~ancer '~reat-
ment, National Cançer Ins-titute. The contents of this
Screening Data Summ,ary including appropriate revisions are
herein incorporated by ref~rence.
In the illustrated examples below, the vehicle
used as carrier for the drug was, injected (minus any drug
therein) into the control animals at the same level of use o~
the yehicle in the drug treated animals in order to eliminate
any vehicle effect of the tests.
EXAMPLE 5
As an indicator of reproducible activity, compou~d
1 of the invention was screened against L-1210 lymphoid leuke
mia in yivo using CD2Fl male mice as the testing species.
The parameter o~ efficacy chosen was based upon the median
suxvival time of the animals treated wi-th the drug vs. appr~-
priate control group animals. Both drug treated animals and
control ~roup animals were inoculated IP ~itA 10 seed cells
o~ 1210 lymphoid leukemia in Ascitic fluid~
One day after tumor inoculation, the drug ~roup
-- 15 ~ ~ ~ a a,t
of animals was pl~ced on a reqimell of trecltrnent of compound 1
at the dose levels as no-ted below, table 1. The druy tr~ated
group of animals was inocula-ted once daily for five days at
the doses noted by IP injection of the test compound appro-
priately d-luted with water.
Day six was chosen as an indication of druy
toxicity. In this example, all drug treated animals survi~;ed
- through day six. Death of drug treated ani~als after day six
was, there~ore, attributed to tumor death$ and n~t d~u~
1~t~xi~i`ty~
The median death day of the control group was day
8~5. As noted in table 1 below, the median death day of the
dru~ treated group was longer at all levels of drug tested and
was si~nificantly longer at greater than 50 mg/Kg (amount of
drug/wei~ht of test animal). The results shown in table 1
below indicate that in this multiple dose assay, t~e drug
showed positive activity. A percent of drug treated animals/-
control anin)als greater than 1~5% is taken as positive drug
activity.
TABI,E 1
Percent
Dru~ DoseTreated Group Control Group Treated ~ni~als/
mg~KgSurvi~val Time Survival Time Control Animals
200 14O3 8,5 168%
100 12.~ 1~9%
11~0 129%
10.2 1~%
12 7 5 9.5 111%
- 16
EXAMPLE 6
Compound2r 2-(2~3,5 t:ri-O-ace-tyl-~D-ribofurano-
syl)thiazo]e-4-carboxamide was screened in a manner similar
to that shown in Example 5, howeve.r, ~he tumor system used
as the test tumor was P388 lymphocytic leukemia. 106 seed
cells were used to initiate the tumor in bo-th the control
group and the drug treated group of animals. The same strain
of ~ice was used exoept female mice were substituted for
male~. Test results were based on mean survival time and are
expressed as T/C percentages (treated ani.mals~contxol animals~
as per Example 5.
In the drug treated animals, treatmen~ was
initiated one day after tumor inocu]ation and the drug was
given at the dose levels noted below in table 2. Drug treat-
ment was fo:r nine days and drug toxici.ty, as in Example 5,
was measured on day six. At the 100 mg~Kg level, one animal
did not survive the toxicity cut-off date.
Average day of death for the control group was
10.2 days whereas at the lowes-t level of drug treatment -the
~0 treated animals su.rYived or more than 15 days. As with
Ex~mple 5, 125% increase in longevity of treated animals over
oontrol animals was taken as indicative of positive drug
response.
TABI,E 2
Peroent
Drug Dose Treated Group Control Group Trea-ted Animals/
mg/Kg Survival Time Survival Time Control Animals
2~0 1~.3 10.2 17~%
1~0 18.0 176~
15.3 150%
mab/~ll
Compound 1 is al.so indica-ted as being active
agains-t P38g lymphocy-tic leu~emia as per Examples 6a, b, an~
c, ancl 2-(2,3,5-tri-0-acetyl. ~-D-ribofuranosyl)thiazole 4-
carboxamide, compound 2, is indicated also as being active
agains-t P388 lymphocyti.c leukemi.a as per Example 7. In both
of these examples, the compound successfully passed the DN
2 ~Decision Network) criteria of the National Cancer Institute
Testing. For examples 7 and 8, CD2Fl femaie mice were used
and challenged with P388 lymphocytic leukemia tumors. The
median survival time of the drug -treated ~nimals w~s compared
to appropriate control animals and based on this criteria both
of the compounds -tested were considered as active antitumor
agents~ The test period was for thirty days in both Examples
7 and 8.
For Examples 7 and 8, as well as Examples 9 and
10 below, any animal of the d.rug treated group which survived
beyond the end of the testing period was then evaluated and
placed in one of three groups. The first group wa.s designated
as cured, meaning the animal was successfully cured of the
tumor. I'he second group designation was no--takes, meanins
survival of the animal was considered to be due to failure
of tumor implant. The remaining group was designated as tumor
survivors meaning the animals lived beyond the test cut-off
date but could not be classified as either cured or no-takes.
For both Examples ~ and 8, thirty anim~ls were
used as -the control group and six animals each were used at
each dose level indicated in tables 3 and 4 below in the dru~
treated groups. In bo-th Examples 7 and 8, for both the
control group and the dru~ treated groups, tumors were induced
- 18 ~
by IP i~oculation of -tumor seed cells on day ~ero followed
by initiation of drug t~eatment orl day one. For bvth E~amples
7a and 8, sallne Wit~l -tween/80 was used as the dru~ vehicle.
~or Examples 7b and 7c, water was used as the druy vehicle.
In both ~he control grQUp and the drug treated
group in Examples 7 and 8, the test animals were inoculated
on day zero IP with 106 seed cells of P33B lymphocytio
leukemia. In both Examples 7 and 8, treatment of the drug
~roup was started on day one and the drug was given IP once
daily for nine days. Day six was utilized as the cut-off date
for de~th attributable to toxicity of the drug. In only one
i~nstance, in Example 7b, was animal mortality attribute~ to
drug toxicity. Efflcacy of treatment was determined by
comparing median survival time of the drug treated animals
compared to median survival time of the control animals, and
is expressed as percentage increase of treated animals/control
animals (T/C1 as per Example 5.
EXAMPLE 7a
In this example drug treated animals were injected
IP with the dose level noted in Table 3 below. Six animals
were treated at each dose level. No control animals survived
beyond day 18 with a median death date of day 12~5~ The
median death day of the drug treated animals is as shown in
Table 3a below. At the 50 mg~Kg level, one drug treated
animal survi~ed and was ~udged as a no-take.
EXAMPLE 7b
This example was performed as per Example 7a at
dos~ leYels as noted in Table 3b below A sur~ivor at bot~
the 700 and 800 my~Kg level was judged as cured~ No controls
~ 19 -
Iilab/~ ,` t~Q~
survived beyond day :L2 and the mean death day of -the control
group was day 11.
E~AMPLE 7c
This example was run as per Example 7a above at
dose levels noted in Table 3c be:Low. All controls were dead
~y day 14 with a mean death date of day 11.9~ At the 500
mg/Kg level, one animal was judged as a cure.
EXAMPLE 8
Compound 2, 2-(2 ! 3,5-tri-0-acetyl ~-D-ribofuranosyl~-
thiazole-4-carboxamide was tested as per Example 7a above at
dose levels noted in Table 4 below. No controls survived be-
yond day lB with an average death date of day 12.6. At the
50 mg/Kg level, one surv~`ving animal was judged as a no take.
Both compounds 1 and 2 are indicated as being active
antitumor agents in the multiple dose studies noted in Examples
7 and 8.
TABLE 3a
Percen-t
Drug Dose Treated Group Control Group Treated Animals/
mg/Kg Sur~ival Time Survival Time Control Animals
400 20.3 12.6 161%
200 1~.0 150%
100 18~3 1~5%
15.3 121%
~5 14.3 ~13%
12.5 13.9 110%
TABLE 3b
Percen~
Drug Dose Treated Group Control Group Treated Anima~s/
mg/Kg Survival Time Survival Time Control Animals
800 2700 11.~ 2~5%
700 ~7.0 2~5%
~00 2S,8 ~3~%
500 21.~ 190%
400 24.7 2~4
300 21.8 198
-- :20 --
mctb/~
~ ~3~ ~t7~ ~
TABLE 3c
Perc~nt
Dr-lg Dose Treated Group Control Group Trea~ed Animals/
mg/Kg Survival Time Survival Time Con-trol ~nimals
800 11.8 ~1.9 99~
700 lOo ~36~i
600 28 ~ 3 ;23~%
5G0 25~0 210~
40G 24.0 201%
3~0 2300 lg3
TABLE 4
Percent
Drug Dose Treated Group Control Group Treated Animals/
mg/Kg Survival Time Survival Time Con~rol Animals
400 2~.3 1~.6 161
1~ 200 19~0 150
lOQ 1~.3 1~5%
15.3 1~1%
14.3 113%
12.5 13.7 ~10~
Compound 1 is indicated as being active against
L-1210 lymphoid leukemia as per Example 9, and successfully
passed the DN 2 criteria of the National Cancer Institute
testing. For Example 9a and 9b, CD2Fl male mi.ce were used and
challenged with L-1210 lymphoid leukemia. The mean survival
~ime of the test animals was co~pared to appropriate control
animals and based on this cri.teria, compound 1 was considered
as an active an~itumor agent. The test period was for thirty
days. Test results are expressed as T/C as per Example 5.
In Examp].e 9a, 24 control animals were used and
six test animals at each drug dose level as is indicated below
in table 9a. I.n ~xample 9b, forty control animals were used
and ten test animals each at drug dose levels as shown in
table 9b helow. For both the control ~roup and the drug test
group, tumo~s were induced by IP inoculation of tumor seed
~ells on day zexo followed by initiation of drug tre~tment on
day one. For Example 9a, water was used as -the druc~ vehicle
, .1
~ 3~
and for Exarnple 9b saline was used as the druy vehicle
In bo-th -the cont:rol ~roups and the dru~J treated
groups in Examp1es 9a and 9b, the test animals were inoculated
on day zero IP with 10 seed cel:Ls of L~1210 lymphoi.d leukemia.
.~or Example 9a~ drug -treatment was started on day one and
cvmpound 1 given once daily Eor nine days. Day five was
utilized as the cut-o~f date for death at-tributable to toxicity
of the drug. In only one instance in Ex2mple 9~ was mortality
attributed to drug toxicity r Efficacy of treatment was deter-
mined by comparing mean survival time of drug treated animals
with mean survival time of tne con-trol animals and is expressed
as percentage increase of treated animals/control a~imals (T~C)
as per Example 5.
EXAMPLE 9a
In this example, the drug treated animals wer~
injected IP with dose levels noted in table 5a below. Six
animals were treated at each dose levelO No control animals
survived beyond day ten with a mean death date of day 9.7.
The mean death day of the drug treated animals is as shown in
table 5a below.
EXAMPLE 9b
In this example, druy treated animals were injected
IP with the dose level noted in table 5b below. Ten animals
were treated at each dose leYel. No control animals sur~ived
beyond day ten with a mean death date of day 9Ø The mean
death day of the treated animals is as shown in table Sb below.
TABLE 5a
Percent
Drug Do~e Trea-ted Group Control Group Treated Animals/
mg/Kg 5urvival Time Survival Time Colltrol Animals
400 lg.7 3.7 19~%
200 15.3 1~7%
100 1~.0 144%
13.2 136~
12.8 131%
T~BLE 5b
Percent
Drug Dose Treated Group Control ~roup Trea-ted Animals~
mg/KgSurvival Time Survival TimeControl Animals
800 15~4 ~.0 171%
600 20.7 ~30%
400 20.2 224%
2~0 16.4 182%
100 16~5 183%
Compound 1 is indicated as being active against
I,ewis lung carcinoma as per ~xample 10 and successfully passed
the DN 2 criteria of the National Cancer Institut~ Testing~
E'or Example 10, B6D~Fl male mice were used and challenged with
Lewis lung carcinoma. The median survival time of the test
animals was compared to appropriate control animals and based
on this criteria compound 1 was considered as an effective
antitumor agent.
In Exarnple 10, forty control animals were used and
ten test animals each at dose levels indicated below in Ta~le
6. For both the control group and the drug treated group~
tu~oxs were induced by IV in~ection on day zero followed by
initiating of drug treatment on day one. For Example 10 water
was used as the drug ~ehicle.
In both the control group and the drug treated
~roup in Example 10, the animals were inoculated on day ~ero
with a homo~enate of 10 seed cells of Lewis lung carcinoma.
'For Example 10, drug treatment was started on day one and
compound 1 gi-ven once daily for nine days. Day ~ive was
utilized as the cut-off date for deaths attributable to toxicity
of thP dru~. There was no mortality attributable to drug
toxicity in this example~ Efficacy of treatment was de-termined
by comparing median survlval time of dru~ treated animals with
mab~','
~ 3,~Ji~
median survival time of the control animals and is expressed
as pe~rcentac3e increase of treatecl animals/con-trol animals
(T/C) as per Example 5.
The test period was for sixty days and ~t the end
of the sixty day perivd all animals surviving in the test
yroups were evaluated as either cured, no-takes~ or tumor sur-
vivors as per Example 5 a~ove.
EXAMPLE 10
In th.is example, the drug treat.ed animals were
injected IP with the dose level noted in table 6 below. Ten
animals were treated at each dose level. No control animals
survived beyond day 23 with a medi.an death date of day 18.4.
At the test levels of 400, 200 and 25 mg/Kg all test animals
survive~ tIIe s xty day test period~ Because of this f.~ct, the
T/C ratio noted in table 6 below is a constant figure based
on assigning survival day rate of sixty to the treated animal.s
and a median death date of 18.4 days to the control animals~
In Example 10 at both the 200 and 400 my~Kg level,
all ten surviving test animals were judged as cured. At the
100 mg/Kg level, there were eight cures and one tumor survivor
with one death noted on day 46. At the 50 mg/Kg level, there
were nilIe cures and one death on day 47.
Compound 1 i.s indicated as being an active antitumor
agent in the multiple dose studies noted in Example 1
TABLE 6
Percent
~rug Dose Treated Group Control Group Treated Animals~
mg/Kg Survival Time Survival Time Control Animals
~00 60 18.~ 326%
20~ 60 326%
100 60 32~%
326~
3~%
As is shown in Example 10 above, compound 1 shows
outstanding activity against Lewis l.ung carcinoma. Lewis lun~
carcinoma i~ an excellent example of a metastatic tumor
system. The tests and control animals of Example 10 were
inoculated 1~ with a homogenate of the -tumor. Dramatic expres-
sion oE this tumor is then seen in the lungs. As was noted
- ~4 -
mab/l~
p.revi.ous:Ly, ~he ahil:ity to m~tastasize is a property -that uni.q~lely charac-
terizes a n~alignan-t tu~or f.rom a beniyn tumor. In Example 10, n~t only
was -~he median survival time of d~lg treated animals dralratically extended
but, a-t the cessation of the -test period, except at one dose level, at
least 80% cures w~re no-ted and at two of those levels lOQ~ cures were
present.
:~X~MELE 11
Compound 3~ 2-(5-O-phosphoryl-~-D-riboEuranosyl~thia~ole-4-
carboxamide is i~licated as being active against L-1210 Lymphoid leukemia
æ per e~mnple lla and llb. These exc~mples were per~ormed essentiall~
as per exa~ple 9 c~bove except as noted. T~e compourld test ~osage levels
axe as no-ted in tables 7a and 7b belcw Eor Example lla a~d llb respec-tive7y.
In these exlmples, ~hirty-six control an~als wexe used and six tes~ ~n~m~
each at the drug dose levels as is shawn in tables 7a and 7b~ Saline
was used as the drug vehicle. No drug -toxicity was nobed in the test
~ni~ for either exampl~s lla or llb. No control ~n;~l~ survived
beyond day 10 in example lL~ with a mean death date of day 8~3 ~nd ~eyond
day ele~en in ~x~rle llb with a mean death date of day 10~1.
For bo~h the control groups and the drug test gro~sg tu~rs
were ;n~ur.~ by IP inocula-tion of tumor seed cells on day 2ero follcwed by
i~ tiation of drug -trea~ ~ t on day one wherein compolmd 3 was given once
daily for 5 days. Test results are expressed as T/C as per example 5.
TABLE 7a
Percent
Dl'Ug Dose Treated Group Control Group Treated Animals/
mg/Kg Survival Time Survival Time Con-trol Animals
80~ 16.3 ~.3 196%
400 15~2 183%
200 21.5 259%
100 15.5 1~6%
11.3 136%
TABLE 7b
Percent
Dru~ Dose Treated Group Control Group Treated Animals/
mg/Kg Sur~i~al Time Survival Time Control Animals
6~Q 16.2 10.1 160%
400 15.7 15~%
~00 14.7 145%
100 12.3 121~
13.0 128%
EXAMPIE 12
~or Example 12, co~pound 1 was given IP to a ~roup of
AKD2F~ r~ce which were i~flicted by intercra~ial inoculation with L~;s
- 25 -
..~,
rnah/, ~
~ 7,~
l~mg seed cells to est~ lish brain tu~.ors. The resul-ts in Table 8 bel~w
indicate that the IP inoculation of the afflic-ted ani~ls with cQmpound 1
resulted in reduction of the brain tumors indicating succ,essful crossing
of the blood brain barrier by compound 1 follawing the IP in~ection lnto
the afflic-ted allLmals~
For this -test, 32 control An;mAl~ were used and no control
~n;~ . survived beyond day eleven with a mean death date of da~ 9.6 for
the controls~ Eight test ~ni~r~l~ were used for each drug dose le~el with
the exception of the 300 mg/Kg level as is shown in table 8 ~elow~ Water
wa5 used as the drug vehicle. For both the control ~roup and ~he -test
gro~p~ .~3rs were induced on day zeno with i~itiation cf dru~ treatment
on day one where compoun~ 1 was given once daily ~or nin2 days. Test
results are expressed as T/C as per example 5.
TABLE 8
Percent
Drug Dose Treated Group ContrQl Group Treated Animalsf
mg~Kg Survival Time Survival Time Control Animals
800 21O3 9.6 221%
700 20.3 211%
600 20.3 211%
~0 5 213%
150 20.5 197%
~5 19.0 185%
37.5 16.0 1~6%
25.0 16.6 172%
In a significant number of disease states of the brain of
both pa~h~nic and host dysfunction origins, treatment is inhibited b~
the lack of drug transfer across the blood brain barrier. In. certain in-
stances wherein appropriate -treatment of a disease state is k2~0wn, com~
plications can arise in treating these diseases when they are located
intercra~ially because of the lack of ~ransfer across ~he blocd brain
b~rrier of effective concentrations of appropriate che~otheraputic agen~s.
The indication, as seen in table 8, that coI~?~und 1 success~ ~ly crosses
t~e blood brain barrier i5 thus very prorrlising for the trea-bment of brain
- 26 -
h ~
DJ~
t~nors.
~ he :Eollcwinc3 representat:i,ve e,~ mples, 13 throuyh 17, are
gi.ven for the fo~ lations of the active csmpo~md of the inven-ti.on in
illustrative pharmaceutical compositions utiliziny illustrative carriers~
In these examples~ ex~nple 13 illustrat:es the use of the compou~lds of the
inven-tion in injectables suitable for intra~enous or o~he~ type5 of injec-
tion into the host anLmaL. Example 14 is directed to an oral syrup prepar-
ation. Example 15 to an oral capsule p.reparation and E~3mple 16 to oral
tablets. Exa~ple 17 is di.r.ec-t~d to use of the compoun~s of the invention
in suitable suppositories. For Examples 13 through 17 the in~redients
a~e listed follcwed by ~he me-thods of preparing the co~çc~ition.
E~MPLE 13
INJECTABLES
Example 13a Csmpound 1
Qo~pound 1 250mg - 1000 mg
Water ~or Injectio~ USP q.s.
Cc~npound 1 is dissolved in the water and passecl through a
0.22 ~ filter. Ihe -filtered solution is added to ampoules or vials, sealed
and sterilized.
Example 13b Compound 3
Canpound 3 as a Sodium Salt 250 mg - 1000 mg
Water for Injection USP q.s.
~repared as per Example 3a, above.
E~AMPLE 14
SYRUP
Example 14a Compound 1
250 mg Active ingredient/5 ml syrup
Comp~l~nd 1 sOg
Purified Water USP 200 ml
Cherry Syr~p ~s. or lOOO ml
Cc~npow1d 1 is clissolved in the wa-ter and to ~his solu~ion
the syrup is added with mild s-tirring.
Example 14b Compound 3
250 mg Ac-tive ingred:Lerlt/5 ml syrup
Compound 3 as a Sodium Salt 50.0 g
Purified Water USP q.s. or 200 ml
Cherry Syrup q.s. ad 1000 ml
Prepared as per Example 14a above.
EXAMPLE 15
CAPSULES
Example 15a Compound 1
100 mg, 250 mg or 500 mg
Compound 1 50U g
Lactose USP, Anhydrous qOs. or 200 g
Stero-tex* Powder HM 5 g
Combine compound 1 and the Lactose in a t~in-shell
blender equipped with an intensifier bar~ Tumble ble~d for
two minutes, Eollowed by blending for one minute with ~he
intensifier bar and then tumble blend again for one mi~ute. A
port~on of the blend i~s then mixed with the Sterotex* ~owder,
passed through a #30 screen and added back to the rema~Lnder o~
the blend. The mixed ingredients are then blended for one
minute, blended with -the intensifier bar for thirty seconds
and tumble bl.ended for an additional minute Appropri~te si~ed
capsules are filled with 141 mg, 352 5 mg or 705 mg of the
blend, respectively~ for the 100 mg, 250 mg an~ 500 mg con~
taining capsules.
EXAMPLE 15b
Example 15b Compound 2
100 mg, 250 mg or 500 mg
Compound 2
*trade marJc 28 -
3~
I,actose USP, Anhydrous q.s. or 200 g
Sterotex* Powder ~IM 5 ~J
Mix and fill as per Example 15 a.
Example 15c Compound 4
100 mg, 250 mg or 500 mg
Compound 4 5~0 g
Lactose USP~ ~nhydrous q.sO or 20Q g
5terotex* Powder HM 5 g
Mix and fill as per Example 15a
EX~MPLE 16
TABLETS
Example 16a Compound 1
100 mgt 200 mg or 500 mg
Compound 1 500 g
Corn Starch NF 200 g
Cellullose, Microcrystalline 46.0 g
Sterotex* Powder HM 4.0 g
Purified Water q.s. or 300 ml
Combine the corn starch, the cellulose and Compoun~
1 -together .in a planetary mixer and mix for two minutes~ Add
the water to this combination and mix for one minute. The
resulting mix is spread on trays and dried in a hot air oven
at 50C until a moisture level of 1 to ? percent is o~tained.
The dried mix is then milled with a Fitzmill through a ~RH2B
screen at medium speed. The Sterotex~ Powder is adde~ to a
po~tion o~ the mix and passed through a #30 screen, and added
~ack to the milled mixture and the total blended for five
minutes by drum rol.lincJ. Compressed tablets oE 150 m~, 375 mcJ
~ 29 -
*trade mark
rn;~ 3
7 ~
and 750 mg respec-t:ivel~, of the tota:l mix a:re fo~led with
appropriate sized punches for ~.he ~00 mg, 250 mg or 500 mg con-
taining tablet~.
EXAMPL:E 17
SUPPOSITORIES
Example 17a Compound 1
250 mg, 500 mg or 1000 mg pex 3 g
Compound 1 250 mg500 mg1000 mg
Polyethylene Glycol 1925 my1750 mg 1400 mg
1540
Polyethylene Glycol 825 mg750 mg 600 mg
~00.0
Melt the Polyethylene Glycol 1540 and the Poly-
ethylene Glycol 8000 together at 60C and dissolve Compound 1
into the melt. ~old this total at 25~C into appropriate
suppositories.
Example 17b Compound 2
250, 500, 1000 mg pex 3 g
Compound 2 250 mg 500 mg 1000 mg
Polyethylene Glycol 1925 mg1750 mg 1400 mg
154~
Polyethylerle Glycol ~25 mg750 mg 600 mg
~~
Prepare as per Example 17a a~ove.
- 30 -
mab/~ n
