Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~lL7~
ANTITUMOR 6-SULFENAMIDE, 6-S~LFINAMIDE AND
6-SULFONAMIDE PURINES, PURINE NUCLEOSIDES,
PURINE NUCLEOTIDES AND RELATED COMPOUNDS
TECHNICAh FIELD
This invPntion is directed to certain 6-sulfenamide, 6
sulfinamide, and 6-sulfonamide purines, purine nucleosides
and purir,e nucleotides including 3-deaza, 7-deaza and 8-aza
derivatives thereof, to their preparation and to using these
compounds to treat malignant tumors ln v1vo.
BACKGROUND ART
Certain antimetabolites are known useful cancer chemo-
therapeutic agents. One such antimetabolite chemothera-
peutic agent is 6-mercaptopurine. 6-Mercaptopurine was
initially found to be highly active against adenocarcinoma
and currently 6-mercaptopurine is utilized as a drug of
choice in the treatment of leukemia Its use in the treat-
ment of leukemia led to dramatic increases in controlling
this disease. Other useful antimetabolites are 6-thio~
guanine and 5-bromouracil. Nucleoside and nucleotide
analogs of these and other purines and pyrimidines had been
synthesized and tested as antitumor agents.
Purine and pyrimidine nucleosides and nucleotides are
ubiquitous throughout biological systems. It further
appears that most of the analogs of purines and pyrimidines
exert their biological activity only after conversion to a
corresponding nucleotide. In view of this, a number of
purine and pyrimidine nucleosides and nucleotides have been
synthesized and screened for their antitumor propertiesA
To be an effective chemotherapeutic agent a compound
must possess a number of desirable properties. First of
3~ all, it must, of course, be an active antitumor agent.
Coupled with this, it must not exhibit too great a host
toxicity or must exhibit reversible toxicity such that the
host is capable of surviving the chemotherapeutic treatment
., .
'
~3~7~ ~
regimen. optimally the chemotherapeutic agent shoul~ not
induce the development of drug resistant cell lines. The
in~lucement of drug resistant cell line occurs with certain
kno~-n chemotherapeutic agents, as for instance, 6-
mercaptopurine and cytosine arabinoside.
Further, effective chemotherapeutic agents need totransport to the site in the body inflicted with the
neoplastic condition~ Thus depending upon the type of
tumor, this requires thak chemotherapeutic agents be capable
of reaching tumor containing organs. This includ~s being
able to effectively penetrate the central nervous system by
crossing the blood brain barrier. As i9 evident by the
sparsity of clinically efective chemotherapeutic agents,
very few compounds possess a sufficient nu~ber of these
capabilities to be clinically useful.
Many effective chemotherapeutic agents require repeated
dosing in order to progressively diminish and kill the
neoplastic cell populations affecting the host. During
these repeated administrations of the chemotherapeutic agent
it is further advantageous for the agent to not develop
resistant cell lines. Because of the development of
resistant cells by certain drugs presently used in the
treatment of many neoplastic disease states, combinations o~
drugs are usually utilized. Thus, as resistant cells
develop to a first drug, treatment with a second or further
druy is often made in an attempt to effecti~ely treat the
drug resistant neoplastic cells.
We have found that certain 6-sulfenamide, 6-sulfinamide
and 6 sulfonamide purines, purine nucleosides and purine
nucleotides and related analogs exhibit one or more of the
properties discussed above, and further exhibit significant
antitumor activity so as to be useful as antitumor agents in
:: ~rivo.
DISCLOSURE OF THE INVENTION
The present inventior~ relates to a novel class of 6-
sulfenamide, 6-sulfinamide and 6-sulfonamide purines, purine
3 L31 7 2-9 ~
nucleosides, purine nucleotides and 3 and 7 deaza and 8 aza
derivative6 thereof and to their preparation and use as
: antitumor agents.
In accordance with the invention, disclosed are
compounds of the formula:
X
N ~ ~T
lo Z ~ G N
.
wherein Z is H or -NH2;
o O
ll ll
X i5 -S-NH2, -S-NH2 or -S-NH2;
; G, T and Q are C-H or N;
Y is H or an ~-pentofuranose or ~-pentofuranose of the
formula:
R2 ~p
Rl R3
wherein Rl and R2 independently are Hl OH, -O-acyl or
3s
4 ~ 3
OH f-
O=P-O- , or together R1 and R2 are O=P-O-
OH OH
and R3 and R4 are H or one of R3 or R~ is OH and the other
is H; provided that when Y is H, Z is -NH2; and
pharmaceutically acceptable salts thereof.
These compounds are useful as antitumor agents or they
are intermediates for compoullds which have these properties.
They can be used to treat an affscted host as~ for example a
mammalian host (i.e. a warm blooded host~ by serving as the
active ingredients of suitable pharmaceutical ccmpositions.
Additionally, in accordance to the invention, an
antitumor composition for the treatment o~ tumors in vivo
contains as its active ingredient a therapeutic effective
amount of a compound of the above formula.
Further, in accordance with the invention/ tumors in
warm blooded animals are treated by adm.inistering to the
animal in need thereof, a pharmaceutical compositi.on
containing as the active component therein a therapeutically
: effect amount of a compound of the above formula.
The method of the invention and khe antitumor
composition of the invention used therain, are effective in
bringing about regression, palliation, inhibition vf growth,
i 25 and remission of tumors.
Particularly useful are compound of the above ~ormula
wherein Y is a ~-pentofuranosa of the formula:
R2
Rl R3
Included in this group a.re 2-amirlo-9~-D~ribo~uranosyl-
9H-purine-6-sulfenamide (see compound 18), 2-amino-9-~-D-
ribofuranosyl-9H-purine-~-sulfinamide (see compound 19~, 2-
amino-9-9-D-ribofuranosyl-9H-purine-6-sulfonamide (see com-
~c~ 3
pound 20) and 2-amino-9 (2~-deoxy~ -er~thro-pentofUrano-
syl)-gH-purine-6-sulfinamide (see compound 23).
Exhibiting particularly useful antitumor properties is
the above 2-amino-9-~-D-ribofuranosyl-9H-purine-6-sulfin-
amide. This compound exhibits a particularly us~fulcombination of solubility, activity and the lack of
generating resistant cell lines as well as being able to
penetrate the central nervous system and b~ active in both
an oral and an injectable form.
For use in pharmaceutical compositions of the
invention, a pharmaceutical carrier would be utilized.
Preferably, the pharmaceutical carrier would be chosen to
allow for administration of a suitable concentration of the
active compounds of the invention either by oral
administration, ophthalmic administration, topical
administration, suppository administration or by suitable
injection as a solution or suspension into the effected
host. The dose and choice of administration of the active
compounds of the invention would depend upon the host
harboring the malignant tumor, the type of tumor, and the
tumor site. For injection, the active compounds of the
invention could he administered intravenously,
intramuscularly, intracerebrally, subcutaneously, or
intraperitoneally.
The compounds of the invention are especially useful in
treating carcinomas, sarcomas and leukemias. Included in
such a class are mammary, colon, bladder, lung, prostate,
stomach and pancreas carcinomas and lymphoblastic and
myeloid leukemias.
Other compounds of the invention are useful as
intermediates for the preparation of the active antitumor
compounds of the invention. Further certain of the
compounds of the invention are useful as prodrugs for other
active antitumor compounds of the invention.
BEST MODE FOR CARRYING OUT THE INVENTION
A group of 6-sulfenamide, 6-sulfinamide and 6-
sul~onamide purines, purine nucleosides and purlne
nucleotides and related analogs have been found to have
antitumor properties or be intermediates for compounds
having such antitumor properties. Included in this group of
compounds are purin~s which have been substituted on the
purine ring at the 2 position with an amino group and
various nucleosides and nucleotides as well as modification
of the purine ring at the 3, 7, and 8 positions forming
deaza and aza purines. Included in this group are the
ribo~uranosyl, the deoxyribofuranosyl and the arabino-
fuxanosyl nucleosides, the monophosphates of these nucleo-
sides and the 3',5'-cyclic phosphates of these nucleosides
and derivatives thereof. Included in the deaza and aza
purine compounds are the 3-deaza and the 7-deaza purine as
well as the 8-aza-7-deaza purine.
One particular compound, 2-amin~-9-~-D-ribofuranosyl
9H-purine-6-sulfinamide, has exhibited good ln vivo activity
coupled with an excellent dos~ response per~ormance while
penetrating the central nervous system and exhibiting a lack
of resistance cell generation. This compound is water
soluble and orally active. Further, it has demonstrated
activity against cells which have become resistant to other
chemotherapeutic agents.
The 6-sul~onamide analog of this compound exhibits many
of the properties o~ the 6-sul~inamide with the exception of
lack of oral actlvity and CNS penetration. The deoxy
derivative o~ this compound, i.e. the 2-deoxy-~-D-~y~h~-
pento~uranosyl derivative, also exhibits good activity with
increased water solubility.
While we do not wish to be bound by theory, it is
bel ieved that many purines, pyrimidines, and purine and
pyrimidine nucleosides exhibit their antitumor activity by
being enzymatically phosphorylated in situ to their 5'
phosphate derivative. Other enzyme systems are known which
convert the 5'-phosphate to a 3',5'-cyclic phosphate.
7 ~.3.7~
Additionally, ~sterases are known which cleave phosphates
and/or cyclic phosphates. In any event, activity has been
shown for compounds o~ the invention as bokh nucleosides and
nucleotides.
In addition to phosphate or cyclic phosphate
derivatives (phosphoryl ester prodrugs) the compounds o the
inventions can also be administer~d as acyl ester prodrugs
which are then also cleaved in vlvo to the parent compound.
Suitable acyl derivatives can be selected as, for example,
from formyl, acetyl, propionyl, butyryl, isobutyryl,
hexanoyl and benzoyl. Preferably acetyl is utilized. One
or more hydroxyl group on the nucleosides o~ the invention
can be suitable reacted to yield such a Cl-C8 acyl prodrug.
In performing the invention, a compound of the
invention or a selected derivative thereof, is appropriately
admixed with a suitable pharmaceutical carrier which may ~e
as simple as sterilized water or could be a complex carrier
having appropriate agents to suitably mimic certaill
biological environmental, i.e., pH or salt adjustment for
solution suitable for intravenous, intramuscular or other
injections, or other appropriate carrier manipulation for
different routes of administration of the compounds o~ the
invention.
In selecting a suitable pharmaceutical carrier,
consideration of the type o~ tumor, the site of the tumor
and the health and age of the host would be given.
Additionally, if a derivatized form of a compound o~ the
- invention i~ used, consideration of the chamical reactivity
of the derivative would also be given. Thus, if a phosphate
form o~ a compound of the invention is used in practicing
the invention, it might be used in the presence of a
suitable buffer or an acceptable pharmacelltical salt
thereof.
Acceptable salts of the phosphate moiety can be
selected from, but not necessarily limited to the group
consisting of alkali and alkaline earths, e.g. sodium,
potassium, calcium, magnesium, lithium, or ammonium and
substituted ammonium, trialklyammonium, dialkylammonium,
~ 3 ~ r7 ~
alklyammonium, e.g. triethylammonium, trimethylammonium,
diethylammonium, octylammonium, cetyltrimethylammonium and
cetylpyridium.
Since the compounds of the invention are water soluble
they could suitably be given to a host as a solution in a
suitable carrier. Alternately, however~ suspensions,
emulsions, or other formulations o~ the compounds of the
invention could be used where indicated. The pharmaceutical
carrier, in addition to having a solubilizing or suspending
agent therein, might include suitable diluents, ~uffers,
surface active agents or other similar agents as are
typically used in pharmaceutical carriers. However, the
total composition of the pharmaceutical carrier would be
chosen to be compatible with the site of the delivery, the
mode of delive~y, the concentration of the active ingredient
and other parameters as are standard in the pharmaceutical
arts.
The compounds of the invention would be suitably
admixed with the pharmaceutical carrier such that they would
be present in a composition of at least 0.1 percent Dy
weight of the total composition. Pre~erably, the compounds
of the invention would be present in a pharmaceutical
carrier at a concentration of about 10% to about 90% by
weight o~ the total composition.
A therapeutic effective amount of the compounds of the
invention, as will be evident from the biological responses
and solubilities given below, would be utilized in treatiny
an affec-ted host animal taking into consideration certain
parameters such as the type of tumor, the tumor site, the
form of administration of the compound, and the physical
size and condition of the host. In any event, the actual
amount should be sufficient to provide a
chemotherapeutically effective amount of the agent in the
host in a convenient carrier. This will be readily within
the ability of those skilled in the Art given the disclosure
herein.
The compounds of the invention can be given as single
doses or as multiple doses divided into sub-doses given
~ 3 .~ 7 i~
g
daily or over a period of days. As will be evident from th~
examples below, compounds of the invention exhibit certain
dose response curves and, as such, optimization o~ a dosage
schedule is well within the skill of the Art given the
disclosure herein.
; In novel processes of the invention, generally 6-
mercaptopurine derivatives as the purine base, nucleoside or
nucleotide are treated with chloramine to prepare the
corresponding 6-sulfenamides. The chloramine can be
prepared in situ by reacting ammonium hydroxide with sodium
hypochlorite. The 6-sulfenamides are then selectively
oxidized either to the 6-sulfinamide or fully oxidized to
the 6-sulfonamide compounds. Generally for selective
oxidation to the 6-sulfinamide, 1 eq. of an oxidizing agent
is utilized~ For full oxidation to the 6-sulfonamide,
further equivalents o~ the oxidizing agent are utilized.
Preferred as an oxidizing agent for process of the invention
is m-chloroperoxybenzoic acid.
j The above processes have been found useful for both
preparing free purines, purine nucleosides and purine
nucleotides of the invention. Typically, the 6-sulfinamide
is prepared utilizing 1 eq. of the above referred to _-
chloroperoxybenzoic acid, and the 6-sulfonamide is prepared
utilizing 4 eq. of m-chloroperoxybenzoic acid. It is
evident that 6-sulfonamide compounds can be prepared
directly ~rom the corre~ponding 6-sulfenamide compounds or
could be prepared via the 6-sulfinamide compounds as an
intermediate.
Schemes I and II illustrate the general reaction
schemes for pxeparation of compounds o~ the inventlon from
starting 6-mercaptopurine precursors. In scheme I, the
heterocycle utilized is a purine, whereas in scheme II
various deaza and aza heterocycles are depicted. In cross
reference between the schemes and the illustrative examples
which follow, the numbers in the parentheses following the
names that app~ar after each example refer to the compound
numbers and structures that appear in schemes I and II.
1 0 ~ J e~
SCHEME I
l H2 I Hz l H2
S S S=O O~S=O
HN ~N~ N ~ N~N
N I N I N 1 11 1
Y r Y Y
Z = -NH2
Y = -H
COMPOUND , COMPOUND --~ COMPOUND --~ COMPOUND
1 2 3 4
Z = -H
Y = -~-D~ribofuranosyl
COMPOUND ~ COMPOUND i COMPOUND i COMPOUND
5 6 7 8
Z = -H
Y = -~-D-arabinofuranosyl
COMPOUND ~ COMPOUND --~ COMPOUND --~ COMPOUND
9 10 11 1
. Z = -H
Y = -2-deoxy-~D-,erythro-pentofuranosyl
30COMPOUND --~ COMPOUND --~ COMPOUND --~ COMPOUND
. 13 ~4 ~5 16
3 5
.
, r~
11
Z = -NH2
Y = -~-D-ribofuranosyl
COMPOUND --~ COMPOUND --~ COMPOUND --~ COMPOUND
5~7 18 19 29
Z = -NH
Y = -2-~eoxy-~-D-erythro-pentofuranosyl
10COMPoUND , CoMPOUND , COMPOUND --~ COMPOUND
21 22 23 2
Z = -NH2
Y = -~-D~ribofuranosyl 5'-phosphate
COMPOUND --~ COMPOUND , COMPOUND
25 26 27
Z = NH2
Y = -~-D-ri~ofuranosyl 3',5'-cyclic phosphate
COMPOUND --~ COMPOUND --~ COMPOUND
28 ~9 30
Z = -NH
Y = -5-~eoxy-~-D-ribofurano~yl
COMPOUND --~ COMPOUND , COMPOUND --~ COMPOUND
~0 ~1 42 ~3
z = -NH
3 0 y a -2-~eoxy-~D-erythro-pentofuranosyl
COMPOUND --~ COMPOUND --~ COMPOUND ~-~ COMPOUND
~ 45 ~6 ~7
'
~ ~ 1 7 ~
12
Z = -NH
Y = -~-D-arabino~uranosyl
COMPOUND , COMPOUND --~ COMPOUND ~-~ COMPOUND
4~ 4g 50 51
Z = NH2
Y = 2,3,5-tri-O-acetyl-~-~-ribo~uranosyl
19
, 10
- COMPOUND
58
"
.,
: SCHEME II
7 I H2 yH,
. s s s=o o=s=o
--~T --> N ~ ~ N ~a,.~, ~Q
. 25 zJ~G il zJ~G N ZJ~ H ZJ~C' N
j r Y r r
.
..
G = N
:~ T = N
.~ Q = CH
,, Z = -NH2
Y = -~-D-ribo~uranosyl
COMPOUND --~ COMPOUND --~ COMPOUND
31 3z 33
: '
"'
11 3~ t~3,
G = CH
T = CH
Q = N
Z = -NH2
Y = -~-D-ribofuranosyl
COMPOUND --~ COMPOUND --~ COM.POUND
34 35 3
lo G - N
Q = CH
Z = -NX~
Y = -2-aeoxy-,B-D-exythro-pentofuranosyl
COMPOUND --~ COMPOUND ~ COMPOUND
37 3~ 3g
G = N
T = CH
Q = CH
Z = H
Y = -2-deoxy-~-D-erythro-pentofuranosyl
COMPOUND --~ COMPOUND - ~ COMPOUND --~ CO~POUND
52 53 s~ 55
G = N
T = N
Q = CH
Z = H
Y = -~-D-ribo~uranosyl
COMPOUND ~COMPOUND
~6 57
.
~3~7 ~J~
14
The following illustrative examples are given ~or the
preparation of compounds of the invention. Unless otherwise
indicated, the various startiny 6-mercaptopurine -ompounds
or other compounds utilized for the staxting materials were
obtained from suitable commercial sources. In these
illustrative examples, the preparation of the compounds of
the invention is accomplished utilizing the processes of the
invention.
1o EXAMPLE 1
2-Aminopurine-6-sulfenamide ~2).
To an ice-cold 5.25% sodium hypochlorite solution (33.8
mL) was added 7N NH40H (17.8 mL) and stirred for 10 minutes~
A solution of 2-aminopurine-6-thione 1, see A.G. Beaman and
15 R.K. Robins, J. Am. Chem. Soc., 83, 4038, (1961), (1.67 g,
22 mmol~ in 2N KOH (11 mL) was added and continued stirring
for 25 min at 0C. The mixture was allowed to stand at 0C
without stirring for 1.5 h. The precipitate was collected
by filtration and washed with small amount of water and EtO~
20 to obtain 1.45 g (36%) of the title compound, mp >250C: UV.
~max (pH 1) 325 nm (~ 6,400), 2~0 nm (sh): ~max (pH 7) 310
nm (~ 5,900), 237 nm (~ 6,800): ~max (pH 11) 312 nm (~
5,900): lH NMR (DMSO-d6): ~ 5.78 (br s, ~, NH2, exchangeable
in D2O), 7.73 ~s, 1, C8H): Anal. Calcd for C~H6N6S.1/2 H2O
25 (191.1~: C, 31.41: H, 3.27: N, 43.98: S, 16.79. Found: C,
31.89: H, 3.~7: N, 43.33: S, 17.19.
EXAMPhE 2
2-Aminopurin -6-sulfinamide (3)~
2-Aminopurine-6-suffenamide 2 (182 mg, 1 mmol) was
suspended in EtOH (lO0 mL) and cooled to 0C. m-
Chloroperoxybenzoic acid (~5%, 20~ mg, 1 mmol) was added
portionwise during 1 h with stirring, and stirring continued
~`
for additional 30 min. After filtration, the filtrate was
concentrated to half the volume in vacuo. Ethyl ether (50
mL) was added and allowed to stand in a refrigerator
overnight. The precipitate was collected by filtration and
washed with ether to obtain 115 mg (58%) of the d~sired
.
15 13.~7,^?~3.
compound, mp. >250C, : UV ~max (pH 1) 332 nm (~ 4,600~ 240
nm (sh): ~max (pH 7) 326 nm (~ 4~500) ~max (pH 11) 326 nm
(~ 4,200), 283 nm (~ 2,800): IR(KBr): 1140 (SO) cm 1 1H NMR
; (DMSO-d~ 6.59 (br s, 2,-SONH2, exchangeable in D2O),
6.57 (~r s, 2, NH2, exchangeable in D2O), 8.12 (s, 1, C8H),
12.50 (br 5, 1, NH, exchangeable in D2O). Anal. Calcd for
C5H6N6OS (198.21): Cl 30.30: H, 3.05: N, 42.40: S, 16.18.
Found: Cl 30.02: H, 2.82: Nl 42.64: S, 15.97.
EXAMPLE 3
2-Aminopurine-6-sulfonamide (4)
To a suspension of 2-aminopurine-6-sul~enamide 2 (500
mg, 2.7 mmol) in EtOH (250 mL) was added m-
chloroperoxybenzoic acid (85%, 2.25 g, 11 mmol) and stirred
for 1.5 h at room temperature. After filtrationl the
filtrate was evaporated to dryness. The residue was
triturated with ether and then purified on a silica gel
column using ethyl acetate : (EtOAc:H~O:l~PrOH, 4:2:1, upper
; phase) (90:10, v/v) as eluent. The precipitation from EtOH-
ether gave 162 mg (28%) of the title compound, mp >250C: UV
~max (pH 1) 338 nm ~ 4,200): ~max (P~ 7~ 329 nm (~ 4,000):
~max (pH 11) 325 nm (~ 4,100), 285 (2,800): IR (KBr) 1150
(S=O), 1320 tSO2) cm 1 lH NMR (DMSO-d6): ~ 6.67 (br s, 2,
NH2, exchangeable in D2O), 7.61 (br s, SO2NH2, exc:hangeable
in D2O), 8.29 (s, 1, C8H), 12.75 (br s, 1, NH, exchangeable
in D2O): Anal Calcd for C5H6N6O2S (214.21): C, 28.03: H,
2.82: N, 39.24: S, 14.97. Found: C, 2~.20: H, 2.72: N,
38.98: S, 15.03.
EXAMPLE 4
9-B-D~Ribofuranosy~-gH-purine-6-sulfenamlde ~6)
Commercial 0.77M sodium hypochlorite (5.25%, 15 mL) was
cooled to ~0C and added with stirring to similarly cooled
0.77M ammonium hydroxide (29%, 3.7 mL diluted to 40 mL with
H2O). The resulting solution of chloramine was mixed with a
solution of 9-~D-ribofuranosyl-9H-purine-6-thione 5 (2.84
g, 10 mmol) in 2M potas~ium hydroxide (5 mL) at <0C. The
mixture was stirred for 40 min until it had warmed to room
16 ~3~7~
temperature and the solvents were evaporated. The residue
was dissolved in MeOH (50 mL) and adsorbed onto silica gel
(2 g). The excess solvent was evaporated under reduced
pressure and the residue was loaded onto a silica gel column
( 3 x 40 cm) packed in CH2C12. The column was eluted ~ith
CH2C12:MeOH (8:2, 7:3, v/v). The appropriate homogeneous
fractions were combined and the solvents evaporated to give
6 as a foam (1.5 g, 50% yield): m.p. 100C: UV: ~max (pH 1)
301 nm (~ 11,100): ~max (pH 7) 288 nm (~ 8,700): ~ma~ (pH
lo 11) 288 nm (~ 9,500): 1H NMR (DMsO-d6): ~ 4.15 (s, 2, S-NH2,
exchanged with D2O), 6.00 (d, 1, J = 5.73 Hz, C1,H), 8.70
(s, 1, C2H), 8.77 (s, 1, C8H), and other sugar protons.
Anal- Calcd for Cl0H13N54S (299.3): C, 40-13- H, 4-38 N,
23.40: S, 10.71. Found: C, 40.29: H, 4.46: N, 23.10: S,
10.45.
EXAMPLE 5
9-~-D-Ribofuranosyl-9H-purine-6-sulfinamide (7).
To an ice-cooled stirred solution of 6 (0.299 g,
mmol) in ethanol (30 mL), a solution of _-
chloroperoxybenzoic acid (0.2 g, 1 mmol) in ethanol (10 mL)
was added dropwise during 10 min. After 40 min the solvent
was evaporated, the residue was disso~ved in MeOH (30 m~)
and adsorbed onto silica gel (10 g). The excess solvent was
evaporated under reduced pressure and the dry residue was
loaded onto a flash silica gel column (2 x 40 cm) packed in
CH2C12. The column was ~luted with CH2C12:MeOH (8.2 and
then 7:3, v/v). ~he appropriate homogeneous fractions were
combined and the solvents were evaporated to ~ive 7 as a
foam, m.p. 80C, (0.21 g, 67% yield). IR (KBr~: 1050 (vs,
S=O), 1330 (s, S=O), 3000~3600 (OH, NH2)cm 1 UV: ~max (pH
1) 272 nm (~ 3,600): ~max (pH 7) 273 nm ~f 4,100): ~m~x (pH
11) 273 nm (f 3,200): lH NMR (DMSO-d6): ~ 6.08 (d, 1, J =
5.4 Hz, C1,H), 6.6~ (s, 2, SONH2, exchanged with D2O), 9.00
(s, 1, C2H), 9.08 (~, 1, C8H), and other sugar protons.
Anal. Calcd ~or CloH13N5O5S.1/2 H2O (324.3): C~ 37.04: H,
4.32: N, 21.60: S, 9.88. Found: C, 37.43- H~ 4.53: N,
21.36: S, 9O97.
17 ~3~7~
EXAMPLE 6
9-~-D-Ribo~uranosyl-9H-purine-6-slllfonamide (8).
To a solution of 6 (0.299 g, 1 mmol) in ethanol (35 mL)
at room temperature, a solution of m-chloroperoxybenzoic
: acid (0.8 g, 4 eq.) in ethanol (20 mL) was added, with
stirring. After 30 min the ~eaction mixture was evaporated
and the residue was purified by flash column chromatography
and treated in the same way as described for 7, -to gi~e 8 as
a foam, (0.11 g, 33~ yield): IR (KBr): 1060, 1080 (s, S=O),
1340 (vs~ b, S02), 3000-3600 (OH, NH2)cm : ~V. ~max (pH 1)
275 nm (~ 14~000) ~max (pH 7) 275 nm (~ 12,900) ~max (pH
11) 272 nm (~ 17,800): lH NMR (DMSO-d6): ~ 6,10 (d, 1, ~ =
5.4 Hz, Cl,H), 7.80 (br s, 2, SO2NH2, exchanged with D2O),
9.04 (s, 1, C2H), 9.10 (s, 1, C8H) and other sugar protons.
Anal. CalCd for C10H13N56S-C2H5QH-1/2 2 ( C,
37.28: H, 5.18: N, 18.12: S, 8.28, Found: C, 37.24: H,
4.51: N, 18.26: S, 8.13.
EXAMPLE 7
9-~-D-Arabinofuranosyl-9H-purine-6-sulfenamide (10).
Commercial 0.77M sodium hypochlorite (5.25%, 46 mL) was
cooled to <0C and added with stirring to similarly cool~d
0.77M ammonium hydroxide (29%, 11.1 mL diluteA to 120 mL
with H2O) The resulting solution of chloramine was mixed
with a solution o~ 9~-D-arabinofuranosyl-9H-purine-6-thione
9 (8.52 g, 30 mmol) in 2M pota~ium hydroxide (15 mL) at
<OC. The mixture was stirred until it had warmed to room
.' temperature (40 min~. After 1 h the product that
crystallized out was filtered, washed with ethanol, dried at
room temperature and recrystallized from ethanol to give (5
g, 56% yield) of 10. m.p. 176-178C (dec.): W : ~max (pH 1)
295 nm ~ 6,000)~ ~ma~ (pH 7) 285 nm ~ 5,800~: ~max (P~ 11)
285 nm (~ 5,500) lH N~R lDMSO-d6): ~4.15 (s, 2, S-NH2,
excha~ged with D2O), 6.37 (d, 1, J = 5.19 Hz, C1,H), 8.50
(s, 1, C2H), 8071 (s, 1, C8H), and other sugar protons.
cd for Cl0H13Ns4S (299-3~: C, 40.13- H, 4,38 N
23.40: S, 10.71. Found: C, 39.94: H, 4.38: N, 22.90: 5,
,
".
.,
18 ~3172~1
11. 00.
EXAMPLE 8
9 ~ D-Arabinofuranosyl-9H-purine-6-sulfinamide (11).
To an ice cooled stirred solution of 10 (1.5 g, 5 mmol)
in ethanol:H2O (525 mL, 20:1, v/v), _-chloroperoxybenzoic
acid (1 g, 1 eq.) in ethanol (50 mL) was added dropwise
during 20 min. After 4 h the separated crystals were
filtered off, the filtrate was evaporated to dryness,
triturated with methanol, filtered, washed with methanol and
dried at room temperature to yield 11, (0.5 g, ~1% yield),
m.p. > 120C. The filtrate was evaporated and puri~ied by
chromatography as described for 6 to yiPld another crop of
11, (0.25 g, 15%: overall yield 46%). IR (KBr): 1060 (vs,
br, S = O), 1330 (s, S = O), 3000-3600 (NH2, OH) cm 1 W :
ax (pH 1) 272 nm (~ 3~000): ~max (pH
~max (pH 11) 272 nm (~ 1,700): lH NMR (DMSO-d6): ~ 6.46 (d,
1, J = 5.16 Hz, C1,H), 6.71 (s, 2, SONH2, exchanged with
D2O), 8.83 (s, 1, C2H), 9.06 (s, 1, C8~), and other sugar
protons. Anal. Calcd for CloH13N5O5S 0.3H2O (321-32): C,
37.38: H, 4.24: N, 21,80: S, 9.97. Found: C, 37.03: H,
4.19: N, 21.42: S, 10.37.
EXAMPLE 9
9-~-D-Arabinofuranos~9H~purine-6-sulfonamide (12).
To a solution of 10 (3.6 g, 12 mmol) in ~thanol (1200
mL) and water (80 mL) at room temperature was added m-
chloroperoxybenzoic acid (8.8 g, 4 eg.) with stirring. The
reaction mixture was left overnight at room temperaturec
The pr~cipitatPd product (12) was ~iltered, washed well with
ethanol to yield 3 g (75%) of 12. The filtrate was
concentrated to get another crop of (12), 0.3 g (6%) over-
all yield (81%): m~p. 160C (dec.): IR (KBr)- 1050 (s, S =
O), 1340 (V5, br, SO2), 3000-3600 (OH, NH2~cm 1 UV: ~max
(pH 1) 275 nm (~ 5,600): ~max (pH 7) 276 nm ~ 6,500): ~max
~pH 11) 274 nm (~ 6,900): 1H NMR (DMSU-d6): ~ 6~70 (d, 1, J
= 5.28 Hz, Cl,H), 7.85 (s, 2, SO2NH2; exchanged with D2O),
8.a8 (s, 1, C~N), 9.08 (s, 1, C8~), and other sugar protons.
19 ~3~72~
Anal. Calcd ~or CloH13N56S 1/2H20 (3
4.12: N, 20.5~: S, 9.41. Found: C, 35.63: H, 4.07: N,
20.27: S, 8097.
EXAMPLE 10
9-~2-Deoxy-~-D-erythro-pentofuranosyl)-9H-Purine-6-
sulfenamide (14~
Commercial 0.77M sodium hypochlorite (5.25~, 15 mL) was
cooled to <0C and added with stirring to similarly cooled
0.77M ammonium hydroxide (29%, 3.7 mL diluted to 40 mL with
~2) The resulting solution of chloramine was mixed with a
solution of 9-(2-deoxy-~-D-~ythro-pentofuranosyl)-9H-
purine-6-thione 13 (2.68 g, 10 mmol) in 2M potassium
- hydroxide ~5 mL) at <0~C. The mixture was stirred until it
had warmed to room temperature (50 min). The solvent~ were
evaporated and the residue was purified by ~lash
chromatography as described for 6 to give 14 as a foam (2.1
g~ 71% yield)- UV: ~max (pH 1) 300 nm (~,): ~max (pH
7) 288 nm (~8,200) ~ma~ (pH 11) 288 nm (~10,300~ 1H NMR
(DMSO-d~ 3.87 (s, 2, SNH2, exchanged with D2O), 6.43
(t, 1, J = 3.54 Hz, Cl,H), 8.75 (s, 1, C2H), 8-84 (s, 1,
C8H), and other sugar protons. Anal. Calcd for CloH13N5O3S
(283.3): C, 42.39: H, ~.62: N, 24.72: S, 11.32. Found: C,
42.12: H, 4.85: N, 24.48: S, 11.51.
EXAMPLE 11
~ =1_ Deoxy-~-D-erythro-pentofuranosy~l) 9_ pu_ ne 6-
sulfinamide ~15).
To an ice-cooied stirred solu~ion of 14 (0.368 g, 1.3
mmol) in ethanol (20 mL), _-chloroperoxybenzoic acid ~0.26
g, 1 eq.~ in ethanol (10 mL) was added dropwise during 10
min. The mixture was warmed to room temperature (90 min).
The product which crystalli~ed out was filtered, washed with
ethanol, dried at room temperature to yield 15 (0.18 g, 46%
yield), m.p. 120C: IR (KBr~O 1060 (vs, S=O), 1360 ~S=o),
3000-3500 (NH2, OH)cm 1 UV: ~max (pH 1) 272 nm (~7,400):
~max (pH 7) 273 nm (~8,600~: ~max (pH 11) 274 nm (~9,100)-
H NMR (DMSO-d6): ~ 6.50 (t, 1, J = 6.60 Hz, Cl,H), 6.68
,
~. 3.~7,~
(s, 2, SONH2, exchanged with D2O), 8.94 (s, 1, C2H), g.06
(s, 1, C8H~, and other sugar protons. Anal. Calcd ~or
CloH13N5O4S (299.3): C, 40.13: H, 4.38: N, 23.40: S, 10.71.
Found: C, 40.39: H, 4.40: N, 23.32: S, 10.51.
EXAMPLE 12
9~ Deoxy-~-D~erythro-pentofuranosyl)-9H-purine-6-
sulfonamide (16).
To a ~tirred solution of 14 ~1.3 g, 4.6 mmol) in
ethanol (120 mL) was added a solution o~ m-
-chloroperoxybenzoic acid (3 g, 4 eq.) in ethanol t50 mL) at
room temperature. After 1 h the reaction mixture was
evaporated and the residue was purified by flash column
chromatography as described for 8 to yield 16, (0.6 g, 41%)
as a ~oam. IR (KBr): 1140 (s, S=O)~ 1320 (vs, SO2),
2800-3500 (NH2, OH)cm 1 UV: ~ma~ (pH 1) 275 nm (~5,800)o
~m~ ~pH 7) 275 nm (~7,600): ~max (pH 11) 273 nm (~7,900):
H NMR (DMSO-d6): ~ 6.53 (t, 1, J = 6.45 Hz, C1, H), 7.85
(s, 2, SO2NH2, exchanged with D2O), 9.00 (s, 1, C2H), 9.08
(s, 1, C8H), and other sugar protons. Anal. Calcd ~or
C1oH13N5O5S.lf2H2O (324.3): C, 37.03: H, 4.32: N, 21.60: S,
.88. Found: C, 36.67: H, 4.1~: ~, 22.01: S, 10.26.
, EXAMPLE '13
252-Amino-9-~-D-ribofuranosyl-9H-purine-6-sul~enamide
,' (1~)-
Sodium hypochlorite, 0.77M (76 mL, 0.532 mmol, ~reshly
opened bottle of commercial bleach) was placed in a
stoppered 1 L flask and the ~lask was submerged in an ice
30bath. Ammonium hydxoxide, 0.77M (20~ mL, 1.4 mmol) was
similarly cooled in an ice bath. Acetone was added to the
ice haths to obtain a temperature of <0C in both solutions.
The ammonia solution was then added rapidly to the bleach
solution and the flask was immediately stoppered. The
mixture was stirred in the cold (O to -5C) ~or approx. 15
min and then a suspension of thioguanosine 17 (15 g, 0.0501
mmol ~ in 2 N KOH was added quickly and rinsed into the
chloramine mixture with a small amount o~ water. The flask
."
.
21 ~3~
was immediately stoppered. The reaction mixture was
initially a clear yellow solution but after a few minutes a
white solid began separating. The reaction mixture was
stirred in the cold (0 to -5C) for 30 min and then the
solid was collected and washed with ethanol (50 mL). The
solid was further washed by suspension in ethanol (3 x 50
mL) and air dried to yield 11.7 g, (0.0372 mmol, 74%) of 18,
m.p. lg6-198C dec.: UV: ~max (pH 1) 332 nm (~3,000): ~max
~pH 7) 311 nm (~3,53: ~max ~pH 11) 311 nm (~3,500): 1H NMR
10 (DMS0-d6): ~ 3.91 (s, 2, SNH2, exchanged with D~0), 5.80
(d, 1, J = 5.97 Hz, C1'~), 6.50 (s, 2, NH2, exchanged with
D20), 8.18 (s, 1, C8H), and other sugar protons. Anal.
Calcd~ for CloH14N64S (314 ) C, 38.21: H, 4.49: N,
26.74: S, 10.20. Found: C, 38.16: H, 4.68: N, 26.49: S,
15 10.49.
EXAMPLE 14
2-Amino-9 ~-D-rlbofuranosyl-9H-purlne-6-sulfinamide
(19) -
A mixture of 2-amino-9-~-D-ribofurano~yl-9H-purine~6-
sulfenamide (18) tl-57 g, 0.005 mol), ethanol (700 mI,) and
water (50 mL) was vigorously stirred and cooled in an ice
bath. After the temperature o~ the suspension had decreased
to <10C, acetone was added to the ice bath to obtain a
temperature of <O~C. With continual stirring a solution of
commercially available (Aldrich Chem Co.) 3-
chloroperoxybenzoic ~cid (80-85%, 1.0 g, 0.0046-0.0049 mol)
in ethanol (40 mL) was added dropwise o~er a period o~
approx. 15 min. The reaotion flask was stoppered, the
mi~ture was allowed to stir and w~rm as th2 ice melted, and
then stirred at ambient temperature for a total reaction
time of 19 hr. The reaction mixture was filtered (Whatman
GF/A glass microfiber filter) to remove a trace o~
undissolved solid and then the filtrate was evaporated in
vacuo and ~t a temperature of <25~C to near dryness. The
product was washed ~rom the evaporation flask with acetone
(50-100 m~) and the solid was collected by filtration,
suspended in diethyl ether (50 mL), refiltered, dried under
22 ~ 3~72)~
vacuum at ambient temperature: (1.3 g, 0.0042 mol, 85%),
m.p. 183-1~5C dec. with prior sintering and darkening. IR
(KBr): 1040 (vs, S=O), 3000-3600 (NH2, OH)cm ~: UV: ~max
(pH 1) 333 nm (~2,900): ~max (pH 7) 326 nm (~10,700): ~max
~pH 11) 325 nm (~8,700): 1H NMR (DMSO-d6)~ ~ 5.85 ~d, 1, J
= 5.52 Hz, Cl,H), 6.49 (5, 2, SONH2, exchanged with D2O),
6.98 (s, 2, NH2, exchanged with D2O), 8.45 ts, 1, C8H), and
other sugar protons. Anal. Calcd. for Cl0Hl4N6o5s.l/2c2H5oH
(353.32): C, 37.39: H, ~.82: N, 23.80: S, 9 07. Found: C,
lV 37.29: H, 4.56: N, 23.78: S, 8.92.
EXAMPLE 15
2-Amino-9-~-D-ribofuranosyl-9H-purine-6-sulfonamide
(20).
To a stirred suspension of 18 (3.14 g, 10 mmol) in
EtOH:CH2C12 (800 mL, 3:1, v/v) at room temperature was added
a solution of m-chloroperoxybenzoic acid ~8 g, 4 eq.) in
ethanol (60 mL). After 4 hl the separated crystals were
filtered, washed with ethanol to get 2.55 g (74%) of a
20 mixture of 19 and 20. By fractional crystallization from
methanol, 20 (2 g, 64%) was obtained. Recrystalliæation of
20 from H2O-MeOH (100 mL, 8:2, v/v) gave colorless crystals
(1 g, 34%), m.p. 210C (dec.)- IR (KBr): 1320 (vs, SO2),
3000-3600 ~NH2, OH)cm 1 UV: ~max (pH 1) 332 nm
(~7 700): ~ x tPH 7) 328 nm (~8,600): ~max (pH 11)
(~12,700): 1H NMR (DMSO-d6): ~ 5.85 (d, 1, J = 5.85 Hz,
C1,H), 6.99 (s, 2, SO2NH2, exchanged with D2O), 7.52 (s, 2,
NH2, exchanged with D2O), 8.48 (s, 1, C3H), and other sugar
protons. Anal. Calcd. ~or C1oH14N6O6S (346.32): C, 34.68:
H, 4O07 N, 24.27: S, 9.26. Found: C, 34.49: H, 4.18: N,
24.09: S, 9.51.
EXAMPLE 16
2-Amino-9-(2-deoxy-~-D-erythro-pentofuranosyl ? -9H-
purine-6-sulfenamide (22).
Commercial 0.77M sodium hypochlorite (5025%, lS mL) was
cooled to <0C and added with stirring to similarly cooled
0.77M ammonium hydroxide (29~, 3.7 mL diluted to 40 mL with
'
.,
~3~2~
23
H20). The resulting solution of chloramine was mixed with a
solution of 2-amino-9-(2-deoxy-~-D-erythro-pentofuran~syl)-
9-H-purine-6-thione 21, prepared as per K. Ramasamy et al.,
J. Heterocycl. Chem, (in press), (2.83 g, lo mmol) in 2M
potassium hydroxide (5 mL3 at 0C. After ~0 min the
solvents were evaporated and the residue was dissolved in
MeOH (50 mL) and adsorbed onto silica gel (1 g). The excess
solvent was evaporated under reduced pressura and the
residue was loaded onto a silica gel column (4 x 15 cm)
packed in CH2Cl2. The column was eluted with CH2Cl2 MeOH
(8:2, 7:3, v/v). The appropriate homogeneous fractions were
combined and the solvents evaporated to yield 22 (2.6 g,
86%), m.p. 130C (dec~): UV: Xmax (pH 1) 328 nm (~11,700):
~max tPH 7) 309 nm (~10,600): ~max (pH 11) 309 nm (~10,900):
1H NMR (DMSO-d6): ~ 3.90 (s, 2, SNH2, exchanged with D20),
6.23 (t, 1, J = 6.60 Hz, C1,H), 6.50 (s, 2, NH2, exchanged
with D20), ~.16 (s, 1, C8~), and other sugar protons. Anal.
Calcd. for CloH14N603S (298-32): C, 40.27: H, 4.70: N,
28.19: S, 10.74. Found: C, 40.10: H, 4.40: N, 27089: S
10.53.
EXAMPLE 1 7
2~Amino-9-(2-deoxy-~-D-erythro~pento~ur3nosylL~H-
; purine-6-sulfinamide (23).
To an ice-cooled stirred suspension of 22 (0.298 g, 1
mmol) in ethanol (200 mL) and CH2C12 (50 mL), m-
chloroperoxybenzoic acid (0.5 g, 1 eq.) in ekhanol (30 mL)
was added dropwise during 15 min. A~ter 80 min the clear
solution of the reaction mixture was adsorbed onto silica
gel (1 g) and the excess solvent was evaporated under
reduced pressure and the residue loaded onto a silica gel
column (2.5 x 15 cm) packed in CH~Cl2. The column was
eluted with CH2C12:MeO~ (802, 75:25, v/v). The appropriate
homogeneous fractions were comhined and the solvent
evaporated to yield 23 (0.65 g, 69%), m.p. 80C (dec.): IR
(~Br): 1050 (vs, S=O), 3000-3600 (NH2, OH3cm l UV
(pH l) 339 n~ (~4,300) ~max (P~ 7)
~x ~pH 1l~ 326 n: (~6,000): 1H NNR (DMSO-d6~- ~ 6.27 (t,
24 ~s~
1, J = 6~75 Hz, C1IH), 6.51 (s, 2, SONH2, exchanged with
D2O), 6.g8 (s, 2, NH2, exchanged with D2O), B.43 (s, 1,
C8H), and other sugar protons. Anal. Calcd. ~or C1oHl~N6O~S
(314.32): C, 38.21: H, 4.49: N, 26.74: S, 10.20. Found.
C, 38.48: H, 4.83: N, 26.75: S, 10.21.
EXAMPLE 18
2-Amino~9-t2-deoxy-~-D-erythro-pentofuranosyl)-gH~
purine-6-sulfonamide (24).
To a stirred suspension of 22 (0.895 g, 3 mmol) in
ethanol (250 rnL) at room temperature was added m-
chloroperoxybenzoic acid (2.4 g, 4 eq.). After 6 h the
clear solution of the reaction mixture was evaporated to
dryness. The residue dissolved in EtOH (10 mL) and adsorbed
onto silica gel (1 g.). The excess solvent was evaporated
und2r reduced pressure and loaded onto a silica gel column
(2.5 x 15 cm) packed in CH2C12. The column was eluted with
CH2C12:MeOH (85:15, 8:2, v/v). The appropriate homogeneous
fractions were combined and the solvent evaporated to yield
24 (0.25 g, 25%) as semisolid: IR (KBr): 1350 (s, SO2),
3000-3600 (NH2, OH)cm 1 UV ~max (pH 1) 332 nm (~4,300)
~max (p~I 7} 329 nm (~5,200): ~max (pH 11) 320 nm (~6,500):
H NMR ~DMSO-d6): ~ 6.28 (t, 1, J = 6.75 Hz, C1lH~, 6~99
(s, 2, SO2NH2, exchanged with D2O), 7.52 (s, 2, NH~,
exchanged with D2O), 8.46 (s, 1, C8H), and other sugar
prvtons. Anal. Calcd. for C1oH14N6sS H2~1/2C2H5H
(330.32): C, 35.58: H, 5.12: N, 22.64: S, 8.63. Found: C',
35.49: H, 5.33: N, 22.57: S, 8.43.
EXAMPLE 19
2~Amino-9-~-D-ribofuranosylpurine-6-sul1nam de 5'~
Monophosphate Potassium Salt (27).
To an ice-cold 5.25% sodium hypochlorite solution (2.3
mL) was added 4N NH40H ~2 mL) and stirred for 10 min. 2-
Amino-9~~-D-ribo~uranosylpurine-6-thione 5'-Monophosphate
25, prepared as per M. Saneyoshi, Chem. Pharm. Bull!, 19,
493 (1971), ~569 mg, 1.5 mmol) in 2N KOH (0.75 mL) was added
and stirring continued for 2 h at 0C. The mixture was
~3~
evaporated to dryne~s and the residue was applied on XAD~4
column and eluted with water. The ~ractions containiny
desired compound were combined and evaporated to dryness to
obtain 420 mg of the sulfenamide dipotassium salt 26. The
white powder 26 (378 mg) was dissolved in 20 mL of water and
cooled to 0CO m-Chloroperoxybenzoic acid ~85~, 250 mg, 1.2
mmol) in MeOH tl0 mL) was added and stirred for 40 min.
After the filtration, the filtrate was concentrated to 3 mL
in vacuo and purified on XAD-~ column using water as eluent
to provide 145 mg of the titie hygroscopic compound 27:
m-p- >250~C UV ~max (pH 1) 332 nm: ~max (pH 7) 2
~max (pH 11) 320 nm: IR (KBr) 1045 (S=O) cm 1 lH NMR (DMSO-
d~ 5.88 (d, 1, C1,H, J = 6.0H~), 6.73 (br s, 2, NH2,
' exchangeable in D2O), 8.50 (s, 1, C8H) : FA~-MS (on
glycerol) ~/z 449 [M~H]+: 411 [M-K+H]~: (NaCl addition)
/z 494 EM+2Na]+, 472 [M+Na+H~+.
~ EXAMPLE 20
`, 2-Amino-9-~-D-ribofuranosylpurine_6-sulfenamlde 3'.5'-
cyclic ~hosphate (29).
Commercial 0.77M sodium hypochlorite (5.25%, 1.2 mL)
,was cooled to <0C and added with stirring to similarly
cooled 0.77M ammonium hydroxide (29%, 0.3 mL diluted to 3 mL
, with H2O). The resulting solution of chloramine was mixed
,;25 with a solution of 2-amino~9-~-D-ribofuranoSylpUrine-6-9-~
thione 3',5'-cyclic phosphate 28, prepared as per RoBo
Meyer et al, J. ~Y~l~ Nucleotide Res., 11 159 (1975), (0.3
g, 0083 mmol) in 2M potassium hydroxid~ (0.37 mL) at ~0C.
~The mixture was stirred for 1 h and the ~olvents were
,'30 evaporated. The residue was dissolved in MeOH and adsorbed
onto silica gel (1 g). The excess solvent was evaporated
under reduced pressure and the solids were loaded onto a
silica gel column (1.5 x 15 cm) packsd in CH2C12. The
column was eluted with CH2C12:MeOH (8:2, 4:6, v/v). The
~,35 appropriate homogeneous fractions were combined and the
'solvents evaporated to give the ~itle compound 29 (0O25 g,
,,~0%): m.p. Z65C (dec): UV. ~max (pH 1) 329 nm (c10,400):
~max (pH 7) 309 nm (~9,000)~ ~max (pH 11) 309 nm (~8~800)o
.
':
26 ~ 3~7~
lH NMR (DMSO-d6): ~ 3.93 (s, 2, S-NH2, exchanged with D2O),
5.82 (s, 1, C1~H), 6,58 (s, 2, NH2, exchanged with D2O),
~.08 (s, 1, C8H), and other sugar protons.
EXAMPLE 21
2-Amino-9-~-D-ribofuranosylpurine-6-sulfinamide 3',5'-
cyclic phosphate (30).
To an ice-cooled stirred suspension of 29 (0.13 g, 0.35
; mmol) in ethanol (20 mL), _-chloroperoxybenzoic acid (0.07
g, 1 eq.) in ethanol (5 mL) was added dropwise during 10
min. The reaction mixture was stirred for 5 h. The
precipitated product was filtered, washed with ethanol,
dried to yield the title compound 30, (0.1 g, 72%): IR
(KBr): 1030 (s, S=O), 1360 (~, SO2), 3000-3600 (OH,
NH2)cm UV: ~max (pH 1) 330 nm (9~000) ~max (pH 7) 325
nm (~4,500): ~max (pH 11~ 324 nm (~4,700): 1H NMR (DMSO-d6):
5.88 (s, 1, C1lH), 6.60 (s, 2, SONH2, exchanged with D2O~,
7.13 (s, 2, NH2, exchanged with D2O), 8.36 (s, 1, C8H), and
other sugar protons.
EXAMPLE 22
6-Amino-1-(2,3,5-tri-O-acetyl ~-D-ribofuranosyl)-
yrazolor3,4-dlpyrimidin-4-one.
A solution of acetic anhydride ~60 m1) and 4-
dimethylaminopyridine (300 mg) in dry dimethyl~ormamide (300
mL) was cooled below 10C. 6-Amino~ D-ribo~uranosyl-
pyrazolo[3,4-d]pyrimidill-4-one, prepared as per H.B. Cottam
et al, Nucleic Acid Research, ~1, 871 882 (1983), (6.0 g, 21
mmol) was added and stirred for 3 h below 10C. Methanol
(150 mL) was added and stood for 30 min at 0C. After the
removal of solvent ln vacuo, the residue was dissolved in
EtOAc (500 mL) and filtrated. The filtrate was wash~d with
water, dried over anhydrous Na2SO4, and evaporated to
dryness. The mixture was purified on silica gel column with
CH2Cl2:MeOH (97:3, v/v) as the solvent to yield 302 g (37%)
of title compound. The analytical sample was obtained by
crystallization from acetone-hexane : mp 191<193C: UV: ~max
~MeOH) 253 nm (:16,700): lH NM~ (DMSO-d6) ~2 00, 2.07 and
,.~
:
27 13~72~
2.09 ~3s, 9H, 3-CH3 of Ac), 6.10 (d, 1~, ~=3.6 Hz, C1,H),
6.81 (br s, 2, -NH2, exchangeable in D2O) 7.94(s, 1, C3H),
10.73(s, 1, NH) : Anal. Calcd for C16H1gN5O8 (409-35): C,
46.94: H, 4.68: N, 17.11. Found C, 47.03: H, 4.67: N, 16.90.
6-Amino-1-(2~3,5-tr~-o-acetyl-~-D-ribofuranos~~LL=
pyrazolo[3 4-d]pyrimidine-4-thlone.
6 Amino-1-(2,3,5-tri-O-~cetyl-~-D-ribo~uranosly)pyrazo-
1O-[3,4-d]pyrimidin-4-one (5.0 g, 12.2 mmol) and phosphorus
pentasulfide (3.5 g, 15.7 mmol) in anhydrous pyridine was
refluxed for 5 h. After removal of half volume of solvent
ln vacuo, the mixture was poured into 600 mL of water and
then extracted with CH2Cl2 (200 mL, 6 times). The combined
extract were washed with water, dried over anhydrous Na2SO~,
evaporated to dryness. The residue was purified on silica
gel column with CH2C12:MeOH (98:2, v/v) as solvent to yield
3.0 g (58%) of desired compound. mp 230<232C: UV ~max
(MeOH) 336 nm (~21,700), 272 nm (~10,700): lH NMR (DMSO-d6)
~2.00, 207, and 2.09 ~3s, 9H, 3-CH3 of Ac), 6.08 ~d, 1,
J=3.6 Hz, C1,H), 7.09 (br s, 2, NH2), 8.07 (s, 1, C3H),
12.16 (br s, 1, NH): Anal. Calcd for C16H19O7N5S (425.41):
C, 45.17: H, 4.50: N, 16.46: S, 7.54. Found: C, 45.23: H,
4.50: N, 16.30: S, 7.46.
256-Amino~ D-ribofuranosylpyr _o~l~LI~t~ ~y ~i ine-4-
thione. (31)
6-Amino-1-(2,3,5-tri-O-acetyl-~D-ribofuranosyl)pyrazo~
1O[3,4-~]pyrimidine-4-thione (2.4g, 5.6 mmol) was suspended
in MeOH (150 mL) and lN NaOMa was added to pH 10. The
mixture was refluxed for 8 h and maintained at pH 10 by
addition of lN NaOMe. A~ter cooled to room temperature, the
mixture was neutralized with Dowex 50[H+] resin and the
solvent was evaporated. The residue was purified on silica
gel column with CH2Cl2:MeOH (9:1, v/v) to yield 1.2 g (71%)
of the title compound. mp 222-224C: W ~max (pH 1) 328 nm
(5,900), 268 nm (~2,300), 237 nm ~6,300). ~max (pH 7)328
nm (~5,900), 268 nm (~2,6003, 237 nm (~6,700): ~max (P~ 11)
319 nm (~4,800), 276 nm (2,400), 236 nm (~6,100): 1H N~R
:
~ 3~7~ ~
(~MSO~d6) ~5.85 td, lH, J=4.5 Hz, C1,H), 7.01 (br s, 2, NH2~
exchangeable in D20), 7.~9 (g, 1, C3H), ~2.07 (s, 1, NH,
exchangeable in D20). Anal. Calcd for CloH13N504S (299.30):
C, 40.13: H, 4.38: N, 23.40: S, 10.71. Found: C, 39.88: H,
4.37: N, 23.12: S, 10.49.
6-Amino~ -D-ribofuranosylpyrazolo r 3,4-dlpyrimidine-4-
sulfenamide (32).
_
To aqueous 5.25% sodium hypochlorite solution (4.6 mL)
cooled to 0C was added 1.4N NH40H (12 mL~ and stirred ~or
10 min. 6 Amino-l-~-D-ribofuranosylpyrazolo[3,4-d]pyrimi-
dine-4-thione 31 (sOO mg, 3 mmol) in 2N KOH (1.5 mL) was
added and allowed to stand for 2 h at 0C. EtOH (15 mL) was
added to dissolve the gelatinous reaction mixture and
filtered. The filtrate was evaporated to dryness with a
small amount of silica gel. Purification of the residue on
silica gel with CH2Cl2:MeOH (6:~, v/v) gave 144 mg (15%) of
the title compound 32 : m.p. 145-150C (dec): W : ~m~x
(pH 1) 327 nm (~6,300): 253 nm (5,200), 236 nm (10,600):
~max ~pH 7) 303 nm (~6,500): 273 nm (5,500) 232 nm tl7,700):
~max tPH 11) 303 nm (~6,100): 274 nm (S,OOO), 232 nm
(16,000): lH NMR tDMSO-d6): ~4.74 ts, 2, SNH2,
exchangeable in D20), 5.99 (d, 1, J = 4.5 Hz, Cl,H)), 6.81
(s, 2, NH2, exchangeable in D20) 8.~ ts, 1, C3H): Anal.
Calcd ~or CloHl4N6o~s.l/4H2o- t318
N, 26.36: S, 10.06. Found: C, 37.88: H, 4.58: N, 25.95: S,
g.67.
EX~MPLE 23
6-Amino~ -D-ribofuranosylpyrazolor3,4 d]pyrimidine-4-
sulfinamlde t33)-
; A solution of 6 amino-1-~-D-ribofuranosylpyrazolo[3,4-
d]pyrimidine-4-sulfenamide t32) (100 mg, 0.32 mmol) in ~tOH
(40 mL) was cooled to 0C and m-chloroperoxybenzoic acid
(85%, 70 mg, 0.3~ mmol) in EtO~ t20 mL) was added dropwise
during 20 min. The mixture was concentrated to 5 mL in
vacuo below 10C, and then ether (30 mL) was added to yield
73 mg (69%) of desired compound 3~ : m.p. 158 162C tdec):
~ 3 ~
UV: ~max (pH 1) 327 nm (~3,500): 233 (13,000): ~max (pH 7
and 11) 323 nm (~4,700): 232 (~7,000): IR (K~r) 1065 (S=O)
cm 1 lH N~R ~DMSO-d~ 6.06 (d, 1, J = 5.0 Hz, Cl,H)
6.77 (s, 2, SONH2, exchangeable in D2O), 7.34 (br s, 2,
NH2, exchangeable in D2O), 8.27 (d, 1, C3H): Anal. Calcd
for CloHl4N6oss-l/3H2o (336-32) C, 35.71: H 4 39: N
24.99: S, 9.53. Found: C, 35.95: H, 4.21: N, 24.86: S,
8.93.
EXAMPLE 24
6-Amino~ -D-ribofuranosylimidazor4 5-c~pyridine-4-
sulfenamide (35).
~ queous sodium hypochlorite (5.25~, 4.6 mL, 3.2 mmol)
was cooled to 0C. Twelve mL of 1.4N ammonium hydroxide was
added and stirred for 10 min at 0C. A suspension of 6-
amino-l-~-D-ribofuranosylimidazo[4,5-c]pyridine-4(5H)-thione
34, prepared as per P.D. Cook and R. K. Robins, J. Orq.
Chem., 43, 189 ~197B), (900 mg, 3 mmol) in 2N potassium
hydroxide (1.5 mL) was added and stirred for 1.5 h at 0C.
The precipitate was collec~ed by filtration, washed with
water, EtOH, and acetone and dried at room temperature over
P2O5 to yield 660 mg of desired compound 35: m.p. 134 137C
(dec): UV: ~max (pH 1) 374 nm (~7,000): 26~ (5,400), 230
(21,400) : ~max (pH 7) 322 nm (~5,500), 261 (5,800) 223
25 (24,000) ~max (pH 11) 319 nm (~8,500), 223 (24,100) lH
NMR (DMSO-d6): ~3.70 (5, 2, exchangeable in D2O, SNH2),
5.61 (d, 1, J = 6.4 Hz, Cl,H), 5.63 (s, 2, exchangeable in
D2O, NH2), 6.25 (s, 1, C7H), 8.12 ~s, 1, C2H). Anal. Calcd
for CllH15N5O45.1/2H2O: C, 40.99. H, 5.00 N, 21.73: S,
30 9.95. Found: C, 41.12: H, 4.81: N, 21.43^ S, 10.23.
EXAMPLE 25
6-Amino-l-~-D-ribofuranosylimidazo[4~cJpyridin~ 4
sulfinamide (36).
To a solution of 6-amino~ D-ribofuranosylimida
zo[4,5-c]pyridine-4-sulfenamide 35 (~50 mg, 0.48 mmol) in
EtOH (60 mL) was added _-chloroperoxybenzoic acid (85~, 95
mg, 0.48 mmol) portionwise during 40 min at 0DC. After
~3~7~
stirring for an additional 10 min, the mixture was ~iltered.
The filtrate was concentrated to 10 mL and poured into ethyl
ether (40 mL). The title compound was obtained as
precipitate which was collected by filtration, washed wi~h
ethyl ether and dried at room temperature over P2O5 ln vacuo
to yield 105 mg (67%) : m.p. 171-176C: UV: ~max (pH 1)
344 nm (~3,400), 263 (3,150), 230 (19,900) ~max (pH 7) 317
nm (~3,100), 259 (2,g00) 225 (19,700): ~max (pH 11) 318 nm
(~3,200), 258 (3,000), 225 (19,900): IR (KBr): 1045 (S=O)
10 cm 1 1H NMR (DMSO-d6)o ~5.71 (d, 1, J = 6.1 Hz, Cl,H),
6.03 (s, 2, SONH2, exchangeable in D2O)~ ~6.33 (s, 2, NH2,
exchangeable in D2O), ~6.68 (s, lH, C7H), 8.36 (s, 1, C2H).
d for CllHl5N5Oss-l/2H2o (338-34) C~ 39O05 H
4.77: N, 20.69: S, 9.48. Found: C, 39.43: H, 4.56: N,
15 20.29: S, 9.43.
EXAMPLE 26
2-Amino-7-(2-deoxy-~-D-ervthro-pentofuranosyl)Pyrrolo-
[2,3 d~pyrimidine-4-sulfenamide (38).
Four mL of 5.25% aqueous sodium hypochlorite (2.8 mmol~
was cooled and added to 10 mL of 1.4N ammonium hydroxide.
After stirring for 30 min at 0C, 2-amino-7-(2-deoxy-~-D-
erythro-pentofuranosyl)pyrrolo[2,3-d]pyrimidine-4~thione 37
(0.78 g, 2.8 mmol) in 1.3 mL of 2N potassium hydroxide was
added and stirred for 1 h at 0C. The precipitate was
collected by ~iltration, washed wlth EtOH and dried at 25C
over P2O5 in vacuo to obtain 670 mg (81%) of the title
compound 38. m.p; 162-l64oc (dec): UV ~max (pH 1~
nm (~28,500~: 347 (5,600): ~max (pH 7) 234 nm (~35,400), 317
~10~400) ~max (P~ 11) 234 nm (~30,900), 318 (10,300). 1H
NMR (DMSO-d6): ~4.11 (s, 2, exchangeable in D2O, SNH2),
6.18 (s, 2, exchan~eable in D2OI NH2), 6.44 (dd, 1, J = 8.3
and 5.9 Hz, Cl,H), 6.61 (d, 1, J = 3.8 Hz, C5H), 6.18 ~d,
1, J = 3.8 Hz, C H). Anal. Calcd for C11H15N$O3S.1/4H2O:
6- -
35 (301.83): C, 43.77: H, 5.17: N, 23 20: S, 10.62. Found-
C~ 43.5g~ H, 4.95: N, 23.13: S, 10O32.
EXaNPLE 27
'
~3~7~ ~
?-Amino-7-(2-deoxy-~-D-erythro-pentofuranosyl)pyrrolo~
[2,3-d~pyrimidine-4-sulfinamide (39).
2-Amino-7-(2-deoxy-~-D-erythro-pentofuranosyl)pyrrolo-
[2,3-d]pyrimidine-4-sul~enamide 33 (300 mg, 1 mmol) was
suspended in EtOH (120 mL) and cooled to 0C. m-
Chloroperoxybenzoic acid (~5%, 100 mg, 1 mmol) in EkOH (30
mL) was added dropwise during 1.5 h. After stirring for an
additional 30 min at 0C, the mixture was concentrated to 10
mL in vacuo below 25C. Ethyl ether (lU0 mL) was added to
the concentrate solution and allowed to stand in khe
refrigerator overnight. The precipitate was collected by
filtration, washed with ethyl ether and dried at 25C under
reduced pressure to yield 110 mg (35~) o~ desired compound
39: m.p. 122C (dec~: W: ~max (pH 1) 352 nm (~3,100):
- 15 272 (3,100), 240 (21,100): ~max (pH 7) 336 nm (~4,800), 239
~ max (pH 11) 337 nm (~4,600), 239 (20 500) IR
(KBr) 1060 (S=O) cm 1 1H NMR (DMSO-d6): ~6.45 (s, 2,
exchangeable in D2O, SONH2), 6.49, (dd, 1, J = 8.3 and 5.9
Hz, Cl~H)~ 6.62 (s, 2, exchangeable in D2O, NH2), 6.73
; 20 (d, 1, J = 3.8 Hz, C5H), 7.38 (d, 1, J = 3-8 Hz, C6H).
Anal- Calcd for CllH15Ns4S: C, 42.16: H, 4.82 N, 22.35
S, 10.23. Found: C, 41.91: H, 4.86: N, 22.07: S, 9.91.
.
EX~MPLE 28
2-Amino-9-(5-deoxy-~ D-ribo~uranosyl!-9H-pur -6-
sulfenamide (41).
Commercial 0.77M sodium hypochlorite (5~25%, 3.2 mL~
was cooled to < 0C in an ice-salt bath and added with
stirring to similarly cooled 1.4~ ammonium hydroxide (29~,
0.8 mL diluted to 8 mL with water). The resulting solution
of the chloramine was mixed with a solution of 2~amino-9-(5--
deoxy-~-D-ribofuranosyl)~9H-6-thiopurine [E.J. Reist, P.~.
Hart, L~ Goodman and B.R. Baker, J. ~Eg~ Chem.~ 2~, 1557
(1961), 40, 0.56 g, 2 mmol] in 2M potassium hydroxide
solution (1 mL) at 0C. The mixture was stirred until it
had warmed to room temperature (~ 2 h~. After 3 h of
stirring, the clear reastion mixture was evaporated to
dryness. The residue was dissolved in methanol (20 mL),
-
32 ~33 7~3~
adsorbed onto silica gel (~ 2 g) and the excess solvent
evaporated under reduced pressure. The dry residue was
loaded onto a silica gel column (1.5 x 20 cm) packed in
dichloromethane. The column was eluted with
5 dichloromethane:methanol (85:15, 8:2, v/v). The appropriate
homogeneous ~ractions were pooled and the ~olvent evaporated
to yield 0.52 g (87%) of 41, mp 160-162C (dec.): UV ~ ma~
(p~ 1) 328 nm (~ 10,800): ~max (pH 7) 306 nm (~ 9~500): ~max
(pH 11) 308 nm (~ 10,300): 1H NMR (DMSO-d6): ~ 1.28 (d, 3,
lO CH3), 3.89 (s, 2, SN~12, exchanged with D2O), 5.74 td, 1, J =
5.22 Hz, C1,H), 6.51 (s, 2, NH2, exchanged with D2O), 8.12
(s, 1, C8H), and other sugar protons. Anal. Calcd. for
CloH14N6O3S (298.32): C, 40.26; H, 4.73; N, 28.17; S, 10.75.
Found: C, 40.49; H, 5.01; N, 27.85; S, 10.56.
EXAMPLE 29
2-Amino-9-(5-deoxy-~-D-ribofuranosvl)-9H-purine-6-
sulfinamide (42).
A solution of m-chloroperoxybenzoic acid (0.10 g, 0.5
20 mmo]) in ethanol (10 mL) was added dropwise to an ice-cooled
stirred solution of 41 (0.15 g, 0.5 mmol) in ethanol (25
mL), during 15 min. The reaction mixture was allowed to
stand at 0C overnight and then evap~rated to dryness under
reduced pressure. The residue was triturated with a mixture
25 of ethanol (2 mL) and ethyl ether (30 mL). The precipikated
crystalline product was collected by filtration and dried at
80C for 3 h to yield 70 ~Ig (45%) of the title compound, mp
> 100C (dec.)~ IR (KBr): 1050 (vs, s, S=O), 3100-3600
(NH2~ OH)cm : ~V: ~max (pH 1) 330 nm (~ 3~00): ~max (P~ 7)
( ~400) ~m~x ~pH 11) 321 nm (~ 4,300): lH N~R
(~SO-d6): ~ 1.30 (d, 3, C~3), 5.80 (d, 1, J = 5 25 Hæ,
C1,H), 6.51 (s, 2, SONH2, exchang~d with D2O), 6.98 (s, 2,
NH2, exchanged with D2O), 8.40 (s, l, C8H), and other sugar
protons. Anal. Calcd. for CloH14N~O4S (314.32): C, 38.21;
35 H, 4.49; N, 26.74; S, 10.20. Found: C, 37.98, H, 4.41; N,
26.51; S, 9.91.
EXA~P~E 30
33 ~3~7~
2-Amino-9-(5-deox~ D-ribofuranosyl~9H-purine-6-
sulfonamide ~43).
To a stirred solution of ~1 (0.30 g, 1 mmol) in ethanol
(35 mL) at room temperature was added _-chloroperoxybenzoic
acid (0.80 g, 4 mmol) and the mixture was allowed to stand
overnight. The reaction mixture was evaporated to dryness
and the residue was triturated with a mixture o~ ethanol (2
mL) and ethyl ~ther (20 mL)O After storing in the
refrigerator (~ 4C) overnight, the precipitated crystalline
product was collected by filtration and dried at 80C for
several hours to yield 0.17 g (52%) o~ the title compound,
mp > 90C. IR (XBr): 1160 ~s, S=Q), 1350 (vs, b, SO2),
3000-3600 (NH2, OH)cm 1; UV: ~max (pH 1) 331 nm (~ 5,400):
~max (pH 7) 326 nm (~ 5~500) ~max (pH 11) 318 (~ 6,500): H
; 15 NMR (DMSO-d6): ~ 1.30 (d, 3, CH3), 5.80 (d, 1, J = 5.13 Hz,
C1,H), 6.99 (s, 2, SO2NH2, exchanged with D2O), 7.54 (s, 2,
NH2, exchanged with D2O), 8.44 (s, 1, C8H), and other sugar
protons. Anal. Calcd. for CloH14N6O5S (330.32): C, 36.36;
H, 4.27; N, 25.45; S, 9.71. Found: C, 36.41; H, 4.55; N,
20 25.38; S, 10.08.
EXAMPLh 31
2-Amino-9-(2-deoxy-a-D-erythro-pentofuranosyl)-9H-
; purine-6-sulfenamide (45).
Commercial 0.77M sodium hypochlorite (5.25%, 15 mL) was
cooled to < 0C in an ice-salt bath and added with ~tirring
to similarly cooled 1.4M ammonium hydroxide (29%, 3.7 mL
diluted to 40 mL wlth water). The resulting solution of the
chloramine was mixed with a solution o~ 2-amino-9-(2-deoxy-
~-D-erythro--pentofuranosyl)-9H 6 thiopurine ~R.H. Iwamoto,
E.M. Acton an~ L. Goodman, J. ~ed. Chem., 6, ~8~ (lg63), 44,
2.83 g~ 10 mmol] in 2M potassium hydroxide solution (5 mL)
at 0C. The reaction mixture was stirred until it had
warmed to room temperature (about an hour)O The crystalline
material that deposited was collected by ~iltration, washed
with cold water (2 x 5 mL), followed by ethanol (10 mh~ and
air-dried to yiald 2.5 g (84%) o~ the title compound, mp
163 C (dec-): UV: ~max (pH 1) 328 nm (~ 9~700): ~max (pH 7)
34 :~3172~
t ,900): )~max (pH 11) 308 nm (~ 12,400): lH NMR
(DMSO-d6): ~ 3.98 (5, 2, SNH2, exchanged with D2O), 6.2ï
(dd, 1, J = 5.10 Hz, Cl,H), 6.49 (s, 2, NH2, exchanged with
D2O), 8.19 (5, 1, C8H), and other sugar protons. Anal.
Eor ClOH14N603S (298.32) C, 40.27; H, 4 70; M
28.19; S, 10.74. Found: C, 39.98; H, 4.70; N, 28.01; S,
10.79.
EXAMPLE 32
?-Amino-9-L2-deoxy-~-D-erythrO-pent
purine-6-sulfinamide (_).
A solution of m-chloroperoxybenzoic acid (0.50 g, 2.5
mmol) in ethanol (50 mL) was added dropwise to an ice-cooled
(0-5C), stirred solution of 45 (0.75 g, 2.5 mmol) in
15 ethanol (150 mL), during 15 min. The reaction mixture was
allowed to stand at room temperature overnight and the
crystalline product tl~at deposited was collected by
filtration. The product was washed with ethanol (2 x 15 mL)
and air-dried to yield 0.24 g (31%) of 4fi, mp 178C (dec.).
20 IR (~Br): 1040, 1300 (s, S=O), 3100-3600 (NH2, OH)cm l; UV:
~max (pH 1) 329 nm (~ 3,800): 3~max (1?H 7) 323 nm (~ 5,B00):
)~max (pH 11) 323 nm (~ 3,700)~ lH NMR ~DMSO-d6): ~ 6.27 (dd,
1~ J = 5-5 Hz, Cl~a), 6-50 (s, 2, SONH2, exchanged with
D2O), 6.94 (s, 2, NH2, exchanged with D20), 8O43 (s, 1,
25 C8H), and other sugar protons. Anal. Calcd. for CLoHl,~,N604S
(314.32): C, 38.21; H, 4.49; N, 26.74; S, lQ.20. Found: C,
38.34; H, 4.59; N, 26.47; S, 10.17.
,.
EXAMPLE 33
- 30 2 Amino-9-(2~deoxy-~-D-erythro-pentofuranosyl)-9H
purine 6-sulfonamide ~47).
~ o a stirred solution of 45 (0.75 g, 2.5 mmol~ in
ethanol (lS0 mL) at room temperature was added m-
chloroperoxybenzoic acid ~2.0 g, 10 mmol) and the mixture
35 was stirred for 3 h. Silica gel (~ 2 g) was added to the
clear reaction mixture and the excess solvent was evapora~ed.
under reduced pressure. The dry residue was loaded onto a
silica gel colu=n (1.5 x 20 cm) packed in dichloromethane.
.
The column was eluted with dichloromethane:methanol t85:15,
8:2, v/v). The appropriat2 homogeneous fractions were
pooled and the solvent evaporated to dryness. The residue
was crystallized from aqueous ethanol to yield 0.30 g (36~)
; 5 of the title compound, mp > lOODC. IR (KBr)~ 0, 1340
(vs SO2), 3000-3600 (NH2, OH)cm : UV: ~max (pH 1) 3
(~ 5~00) ~max (pH 7) 327 nm (~ 9,800): ~max (pH 11) 319 nm
(~ 10,500): lH NMR (DMSO-d6): ~ 6.27 (dd, 1, J = 5.37 Hz,
C1~H), 6.96 ~s, 2, SO2NH2, exchanged with D2O), 7.51 (5, 2,
NH2, exchanged with D2O), 8.4~ ts, 1, C8H), and other sugar
protons. Anal. Calcd. for CloH14N6O5S (330.32): C, 36.36;
H, 4.27; N, 25.45; S, 9.71. Found: C, 36.11, H, 4.25; N,
25.31; S, 10.08.
EXAMPLE 34
2-Amino-9-~-D~arabinofuranosyl-9H-purina-6-sul~enamide
(49)-
To an ice-cold solution of ammonium hydroxide (1.4N, 20
mL) was added 0.77M sodium hypochlorite solution (5.25~, 7.5
mL, 5.25 mmol) in one lot. The mixture was stirred at 0C
for 10 min. A solution o~ 2-amino-9-~-D-arabinofuranosyl-
9H purine-6-thione [W.W. Lee, A.P. Martinez, R.W. Blackford,
V.J. Bartuska, E.J. Reist and L. Goodman, J. Med~ Chem., 1~,
819 (1971), 48, 1.49 g, 5 mmol] in lN pota~sium hydroxide
solution (5 mL, 5 mmol) was added in one lot, and the
reaction mixture was stirred at 0C for 1 h. After allowing
the reaction mixture to warm up to 15C during 1 h, the
~lear solution was evaporated to dryne~s under reduced
pressure. The residue was purified by flash chromatography
over silica gel using dichloromethane -~ methanol gradient.
The homogeneou~ fractions were pooled and evaporated to
dryness. The residue was crystallized from a mixture of
dichloromethane and methanol to give 0.85 g (54%) o~ the
title compound, mp 190-192C. IR (KBr): 3200-3400 (NH2,
OH)cm 1 UV: ~max (pH 1) 227 nm (~ 26,400), 25~ (10,600),
328 (19,400): ~max (pH 7) ~22 nm ( 22,~00), 243 ~13,70~),
( 3~200) ~max (pH 11) 221 nm (~ 22,200), 243 (13 500)
308 (13,200): ld NMR (~MSO-d~ 4.09 (-, Z, SNd
., ,
.
,j
36 ~ 2~1
exchanged with D2O), 6~13 (d, 1, J = 4.0 Hz, C1,H), 6.50 (g,
2, NH2, Pxchanged with D2O), 7.99 (s, l/ C8H), an~ other
sugar protons. Anal. Calcd. for C1oH14N6O4S ~314.32) C,
38.21; H, 4.49; ~, 26.74; S, 10.20. Found: C, 38.40; H,
4.47; N, 26.53; S, 10.29.
EXAMPLE 35
2-Amino-9-~-D-axabln fu anosyl-9H-purine-6-sulfinamide
(50)-
A solution of ~9 (1.5 g, 4 7 mmol) in ethanol (350 m~
and water (50 mL) was cooled to 0C. To this cold solution
was added m chloroperoxybenzoic acid (80%, 0.90 g, 4.45
mmol) in ethanol (5Q mL) during 1.5 h. After th addition,
the reaction mixture was stirred at ice-bath temperature for
1O5 h. The solven~ was evaporated under reduced pressure
and the residue was dissolved in methanol (50 mL). Silica
gel (~ 5 g) was added and evaporated to dryness. The dried
silica gel was placed on top of a flash silica gel column
and the column was aluted with ethyl acetate -~ methanol
gradient. The pure compound crystallized out after
concentration of the homogeneous fractions. The product was
collected by filtration and dried to give 0.95 g (60%) of
the title compound, mp > 200~C (dec.): IR (KBr): 1120
(S=O), 3100-3600 (NH2, OH)cm 1 UV: ~max (pH 1) 220 nm t~
17,900~, 249 (7,600), 330 (5~10~) ~max (pH 7) ~25 nm (~
24,100), 248 (sh) (6,100), 323 (8,000): ~ma~ (p~l ~1) 224 nm
(~ 21,000), 244 (sh) (6,600), 322 (6,600): lH NMR (DMSO-d6):
~ 6.17 (d, l, J = 4.03 Hz, Cl,H), 6.50 (s, 2, SONH2,
exchanged with D2O), 6.97 (s, 2, NH2, exchanyed with D2O),
8.25 (s, l, C8H), and other sugar protons. Anal. Calcd. ~or
C1oH14N6O5S (330.32): C, 36.36; H, 4.27; N, 25.44; S, 9.71.
Found: C, 3~.65; H, 4.09; N, 25.19; S, 9.~6.
EXAMPLE 36
2-Amino-s-~-D arabinofuranosyl-9H~purine~6~sulfonamid
(51)-
To a stirred solution of 49 (0.46 g, 1.46 mmol) in
ethanol (250 mL) and water ~50 mL) was added m-
37 ~3~2~
chloroperoxybenzoic acid (1.0 g, 5.84 mmol) in ethanol (50
mL) dropwise during 1 h at room temperature. A~ter the
addition, the reaction mixture was stirred at room
temperature for 6 h and evaporated to dryness under reduced
5 pressure. The residue was purified by flash silica gel
chromatography using ethyl acetate -~ methanol as the
gradient. The homogeneous fractions were pooled and
evaporated to dryness to give 0.30 g (59~) of the title
compound, mp > 193C. IR (~Br): 1170 (S=O~, 1340 (SO2),
3100-3600 (NH2~ OH)cm 1 UV ~max (pH 1) 222 nm (~ 16,900),
( ~200)D ~max (pH 7) 225 nm (~ 17,300) 326 (4 goo)
~max (pH 11) 223 nm (~ 17,200), 320 (5,600): lH NMR (DMSO
d6): ~ 6.18 (d, 1, J = 4.3 Hz, Cl,H), 6.95 (s, 2, SO2NH?,
exchanged with D2O), 7.48 (br s, 2, NH2, exchanged with
D20), 8.27 (s, 1, C8H), and other sugar protons. Anal.
Calcd. for CloH~4N606S 1/2Et0A~ (390.37): C, 36.92; H, 4.65;
N, 21.52: S, 8.20. Found: C, 37.04; H, 4.32; N, 21.50; S,
8.41.
EXAMPLE 37
7-(2-De.oxy-B-D-erythro-~entofuranosyl)pyrrolo r 2,3-dl-
pyrimidine-4-sulfenamide (53).
To an ice-cold solution of ammonium hydroxide (1.4N, 8
mL) was added 0.77M sodium hypochlorite solution (5.25~, 3
mL, 2.1 mmol) in one lot. The mixture was stirred at 0~C
for 10 min. A solution o~ 7-(2 deoxy-~-D~erythro~
pentofuranosyl)pyrrolo[2,3-d]pyrimidine-4-thione [H.B.
Cottam, Z. Kazimierczuk, S. Geary, P.A~ McKernan, G.R.
Revankar and R.K. Robins, J. Med. Chem., 2B, 1461 (1985),
52, 0.53 g, 2 mmol] in lN potassium hydroxide solution t2
mL, 2 mmol) was added in one lot, and the reaction mixture
was stirred at 0C for 1 h. After allowing the reaction
mixture to warm up to 15C during 1 h, the clear solution
was evaporated to dryness under reduced pressure. The
residue was purified by flash chromatography over silica gel
using dichloromethane:methanol (95:5, v/v) as the elu~nt.
The homogeneous fractions were pooled and evaporated to
dryness. The residue was crystallized from s mixture of
"
38 ~3~7t~
methanol and dichloromethane to give 0.31 g (55~) o~ the
title compound, mp 153-155C. IR (KBr): 3200-3450 (NH~,
OH)cm 1 UV: ~max (pH 1~ Z66 nm (~ 9,900), 321 (22,900):
~ (pH 7) 295 nm (~ 11,200): ~max (pH 1 )
17,100): 1H NMR (DMSO-d6): ~ 4.29 (s, 2, SNH2, exchanged
with D2O), 6.62 (t, 1, J = 6.7 Hz, C1lH), ~.85 (d, 1, C5H),
7.71 (d, 1, C6H), 8.54 (s, 1, C2H), and other sugar protons.
Anal. Calcd. for CllH14N4O3S (282.28) C, 46-80; H, 4.99; N,
19.84; S, 11.34. Found: C, 47.01; H, 4.63; N, 19.63; S,
11.52.
EXAMPLE 38
7-(2 Deoxy-~-D-erythro-pento~uranosyl)pyrrolo[2,3-dl-
pyrlmidine-4-sulfinamide (54).
To a solution of 53 (1.41 g, 5 mmol) in ethanol:water
(190:10, v/v), cooled to 0C in an ice bath was added m-
chloroperoxybenzoic acid (80%, 1.01 g, 5 mmol) in ethanol
(50 mL), dropwise during 1.5 h. The reaction mixture was
stirred at 0C for 1~5 h before the solvent was evaporated
under reduced pressure. The residue was dissolvsd in
ethanol (25 mL) and diluted with ethyl ether (150 mL) and
stored in the refrigerator ovarnight. The precipitated
solid was collected by filtration and drled to yield 1.0 g
(67~) of the title compound, mp 170-172C. IR(KBr): 1100
~S=O), 3200-3400 (NH2, OH)cm 1; UV: ~max (P~ 1) 231 nm (~
, (6~200)~ ~max (pH 7): 227 nm (~ 28,300), 285
(6,200), 302 (sh) (5,400): ~max (P~ 224 nm (~ 22,800~,
273 ~5,900), 301 (sh) (3,200): 1H NMR (DMSO-d6~: ~ 6.66 (s,
2, SONH~, exchanged with D2O), ~.71 (t, ~, J = 6.8 Hz,
Cl,H), 7.06 (d, 1, C5H), 7.97 (d~ 1, C6H)~ 8-86 (s, 1, ~2H)~
and other sugar protons. Anal. Calcd. for CllH14N~O4S
~298.28): C, 44.2g; H, 4.73; N, 18.77; S, 10.73. Found: C,
44.30; H, 4.49; N, 48O48; S, 10O91.
EXAMPLE 39
7-(2-Deoxy~-D~erythro-pentofuranosyl)pyrrolo r 2,3-dL-
pyrimidine~4-sulfonamide (55).
To a stirred solution of 53 (1.41 g, 5 mmol) in a
- .
3 9 :~ 3 ~ 7 ~ eJ ~
mixture of ethanol:water (300:50, v/v) was added m-
chloroperoxybenzoic acid (3.44 g, 20 mmol) in ethanol (50
mL) dropwise during 1.5 h at room temperature. A~ter the
addition, the reaction mixture was stirred ak room
5 temperature for 12 h and evaporated to dryness under reduced
pressure. The residue was dissolved in ethanol (50 mL),
mixed with silica gel (~ 5 g) and again evaporated to
dryness 1n va~uo. The dry residue was placed on top of a
flash silica gel column (5 x 30 cm). The column was eluted
successively with dichloromethane (1 L),
dichloromethane:acetone (1:1, 500 mL) and then
dichloromethane -~ methanol gradient. The appropriate
- homogeneous fractions were pooled and concentrated to about
50 mL, and stored in the refrigerator overnight. The
product that crystallized out was collect~d by filtration
and dried to yield 1.10 g (71%), mp 175-177C. IR (KBr):
1150 (S=O), 1350 (SO2), 3100-3600 (NH2, OH~cm 1 UV: ~max
(pH 1) 228 nm (~ 27,700), 284 (5,100), 310 (sh) (3~800):
~max (pH 7) 228 nm (~ 27,400), 285 (4,900), 308 (sh)
(3,800): ~max (P~ ll) 22~ nm (~ 25,800), ~84 (5,700): lH NMR
i~ (DMSO~d6): ~ 6.72 (t, 1, J = 7.2 Hz, C1,H), 6.92 (d, l,
C5H), 7.82 (br s, 2, SO2NH2, exchanged with D2O), 8.08 (d,
1, C6H), 8.9S (s, l, C2H), and other sugar protons. Anal.
~- for CllHl4N4sS (314-22): C, 42.04; H, 4.49; N
17.82; S, 10.18. Found: C, 42.07; H, 4.~6; N, 17.62; S,
10.15.
,
EXAMPLE 40
l=~ D Ribofuranosylpyrazolo~3 ~-d]pyrimidine-4 sulfen-
amide (57).
Commercial 0.77M sodium hypochlorite (5.25%, 8 mL) was
cooled to c 0C in an ice-salt bath and added with stirring
- to a similarly cooled 0.7M ammonium hydroxide (29%, 2 mL
diluted to 20 mL with water). The resulting solution of
chloramine was mixed with a solution of l-~-D~
ribofuranosylpyrazolo[3,4-d]pyrimidine-4(5H)-thione ~J~LoG~
Montero, G.A. Bhat, R.P. Panzica and L.B. Townsend, J.
Heterocycl. ChemO, 14, 483 (1977), 56, 1.42 g, 5 mmol] in 2M
,,,
. .
13:1 r~2~
potassium hydroxide solution (2.5 m~,) at 0C. The reaction
mixture was stirred until it had warmed to room temperature
(about an hour), and allowed to stand for 2 more hours. The
product that crystallized out was collected by ~iltration,
washed with cold ethanol (2 x 10 mL) and dried at room
temperature to yield 0.61 g (41%) of the title compound.
Recrystallization from ethanol:water (3:1) gave analytically
pure material of mp 166-169C- UV: ~max (P~ 1) 295 nm (~
28 000): ~ (pH 7) 293 nm (~ 24,000): ~max (pH 11)
( 21,000): 1H NMR (DMSO~d6): ~ 4.70 (s, 2, SNH2, exchanged
with D20), 6.24 (d, 1, J = 4.53 Hz, C1,H), 8.67 (s, 1, C3H),
8.75 (s, 1, C6H), and other sugar protons. Anal. Calcd. for
C1oH13N5O4S (299.3): C, 40.13; H, 4.3%; N, 23.40; S, 10.71.
Found: C, 40.35; H, 4~34; N, 23.2%; S, 10.79.
EXAMPLE 41
2-Amino-9-(2,3,5-tri~O-acetyl-~-D-ribofuranosyl)-9H-
urine-6-sulfinamide (58).
A mixture of dimethylaminopyridine (10 mg) and acetic
anhydride (1 mL) in anhydrous N,N-dimethylformamide (2 mL)
was cooled to -15C. 2-Amino-9-~-D-ribofuranosyl-9H-purine-
6 sulfinamide (19, 0.10 g, 0.3 mmol) was added and the
mixture was stirred for 40 min at -15C. The reaction was
quenched by the addition o~ methanol (4 mL) and the
~s resulting solution was stirred at 10C for 20 min and then
evaporated to dryness. The resi~ue was tritura~ed with
ethyl ether (10 mL) and the product was precipitated by the
addition of hexane to yield 0.102 g (75%) of the title
compound as amorphous solid. IR (KBr): 1050, 1095 (s,
S=O), 1745 (vs, C=O), 3200-3500 (NH2)cm 1 W : ~max (pH 1)
( 6~100) ~max (pH 7) 328 nm ( 6,700) ~m (pH 11)
321 nm (~ 7,000): 1H NMR (DMSO-d6): ~ 2.03-2.13 (3s, 9,
3COCH3), 6.15 ~d, 1, J = 3.5 Hz, Cl,H~, 6-51 (s, 2, SONH2,
exchanged with D2O), 7 . 07 ~S, 2, NH2, exchanged with D20~,
%.44 (s, 1, C8H), and other sugar protons. Anal. Calcd. for
C16H20N6O8S (456.43): C, 42,10; H, 4.42; N, 18.41; S, 7.03.
Found: C, 41.99, H, 4.47; N, 18.19; S, 6.79.
41 ~3~
As illustrative examples of use of the compounds of the
invention the followiny examples are given. In these
examples the efficacy of the compounds of the invention are
demonstrated using standard tests against certain malignant
tumors. These standard tests utilize protocols developed
under the auspices of the Developmental Therapeutics
Program, Division of Cancer Treatment, Mational Cancer
~nstitute, Bethesda, Maryland, U.S.A~ Except as otherwise
indicated the tests conform to thase protocols and are
evaluated utilizing criteria defined by the protocols.
For the purposes of these examples certain of standard
abbreviations are utilized as follows: ip
intraperitoneal: qd - Once a Day: bid - Twice a Day: tid -
Three Times a Day- qid - Four Times a Day: %T/C Percent
Treated Divided by Control: %I~S - Percent Increased Life
Span: inj - Injection.
For those tests whose results are indicated as % T/C
generally using NCI protocols for the L1210 tumor cell line,
a value greater than 125~ is considered as having activity.
For those tests expressed as a percent of the ori~inal
inoculum, values above 100 are considered inactive while
those below 100 are viewed as active. Values below 25% are
considered capable of producing effective therapy, those
below 10% are considered ~ood and those below 5% are
considered very good. This expresses the percent of the
cells which sur~ived treatment based upon the original cells
in the inoculum. Those tests expressed as increases in life
span (% ILS) indicate the increased life span of the drug
~reated group compared to a control group.
THERAPEUTIC EX~MPLE A
As an indicator of reproducible activity, compounds of
the invention and other known cancer chemotherapeutic agents
were screened against L1210 lymphoid leukemia in vivo
utilizing the mouse as a test species. Normal NCI protocols
for this test require 105 seed cells of the L1210 cell linP.
However for the purposes of tests with the L1210 cell line
in testing the compounds of the invention for antitumor
42 ~3~7~
activity, a log greater, i~e. 106 cells were utilized.
Table 1 demonstrates the results of inoculating mice
with 106 Ll210 seed cells and the spread of this tumor cell
line throughout the body to multiple organ systems in the
test species. As is indicative of Ta~le 1, at day 7 the
L1210 cellular population in multiple organ systems greatly
axceeded 105 cells in each of the organs assayed. It is
thus evident from Table 1 that if a chemotherapeutic agent
is to be effective against the ~1210 tumor line seeded at
106 i~ must reach all of the organ systems of the animal in
: view of spread of this neoplastic disease to all these organ
systems.
. 20
.
.
,~
~ ,
,:
3 ~ 3 ~
TABLE 1
VIABLE L1210 CELLS IN TISSUES OF BDF MICE
AFTER
INTRAPERITONEAL INOCULATION ON DAY ol
TISSUE POST INOCULATION DAY
1 7
BRAIN <100 >400,000
LUNG <100 >600,000
SPLE~N >6,000 <4,000,000
LIVER ~64,000 >120,000,000
BLOOD NONE >300~000
MARR0W NONE >500,000
1. Inoculated IP on day 0 with 1o6 L1210 cells.
THERAPEUTIC EXAMPLE B
Ta~les 2-a and 2-b illustrate a dose response for
Compound 19 against L1210 inoculated mice expressed as both
increased life span compared to control and as the number of
cells o~ the original inoculum surviving drug treatment. As
is evident from the various regimens of drug treatment shown
in Tables 2-a and 2-b, effective therapeutlc e~ects are
noted and a dose response to Compound 19 is evident. The
ef~icacy for Compound 19 seen in Tables 2-a and 2-b is
similar to that æeen Por Cytosine Arabinoside with the
exception that to see e~fects with CytosinQ Arabinoside like
those in Tables 2 a and 2-b for compound 19, Cytosine
Arabinoside must be given every three hours. Test results
are given utilizing standard protocols based on mean
survival time and are expressed as T/C percentages ~treated
animals/control animals).
'
'14 ~3~72~
TABLE 2-a
LIFE SPAN (g6T/C OF L1210 INOCULATEDl ~qICE T:REATED2 WITH
COMPOUND 19
5 DOSAGE SCHEDULE OF DELIVERY
(mg/kg/inj) (qd days indicated) (bid days indicated)
1,4,71,3,5,7 1-7 1,4,7 1,3,5,7 1-7
22 161~93 ~0
37 154 174 213 216236 233
62 193 223 239 249285 3613
104 230 236 3074 3115
173 269 2~g _ _
1. BDF1 female mice were inoculated ip on day O with 106
L1210 cells~
2 ~ Drug was delivered l~y the ip route.
3. Treatment group included 2 long term survivors which
~: were not included in the calculation of life span.
4. Treatment group included 1 long term survivor which was
not included in the calculation of life span.
5. Treatment group included 1 mouse which died from drug
toxicity and was not included in the calculation o~ life
span.
.,
~.
TABLE 2-b
L1210 CELLS SURVIVING DAY 7 TREATMENT WITH COMPOUND 19
(expressed as % of original inoculum)
DOSAGE DRUG DELIVERED DRUG DELIVERED
5 (mg/kg) (qd days .indicated~ (bid days indicated)
1,4,7 1,3,5,7 1-71,4,7 1,3,5,7 1-7
22 9211 594 343
10 37 15936 3077 115 87 17 22
62 594 50 13 5.6 0.3 2/5
104 29 17 1~5Cl l/5T2
173 1.1 0.2 _ _
____ -
1. Data expressed as number of long term survivors (Cures)
per the number of animals in test group.
2. Data expressed as number of toxic deaths (Toxic Doses)
per the number of animals in test group.
2~
~. 3 ~ 9 ~
46
'~HERAPEUTIC EXAMPLE C
In Example C the oral efficacy of Compound 19 was
compared to that for the dru~ when given intraperitoneally.
TABLE 3
RESPONSES OF L1210-INOCULATED BDFl MICE TO COMPOUND 19
GIVEN ORALLY AND INTRAPERITONEALLY
. ~ ~
DRUG INCREASED LIFE SPAN PRODUCED
DOSAGE BY COMPOUND 19 DELIVERED
~MG/KG/INJ) ORALLYINTRAPERITONEALLY
37 45 54
15 62 45 93
104 38 ~30
173 38 169
Mice were inoculated intraperitoneally on day 0 with a
million cells of murine leukemia Ll210. Drug was given qd
days 1, 4 and 7. Increased life span is the mean increase
in the treated group presented as a percentage of the mean
life span of control mice.
~ S~ 7~ ~
~7
THERAPEUTIC EX~MPLE D
In Example D Compound 19 is compared with 6-
Thioguanosine. As per the oral treatment of Compound 19
seen in Table 3, in Table 4 it is evident that Thioguanosine
has a flat dose response whereas Compound 19 shows a dose
response curve when injected intraperitoneally.
TABLE 4
RESPONSES OF L1210-INOCULAT~D BDFl MI OE TO
TREATMENT WITH 6-THIOGUANOSINE OR COMPOUND 19
DRUG INCREASED LIFE SPAN PRODUCED BY
DOSAGE
(MG/KG/INJ) 6-THIOGUANOSINE COMPOUND 19
8.1 48 NR
13 38 NR
22 42 NR
37 45 74
62 42 123
104 42 136
173 52 (toxic) 189
2 5 _ __ _
NR = Not Run
Mice were inoculated intraperitoneally on day 0 with a
million cells of murine leukemia L1210. ~rugs were given
qd days 1, 3, 5 ~and 7. Increased life span is presented
30 as a percentage o~ the mean life span of control mice.
, ~
.,
48 1 ~ 7,~
THERAPEUTIC EXAMPLE E
Further elucidation of the dose response of Compound 19
is shown in Table 5. A dose of 288 mg per kg represents the
maximum solubility of Compound 19 in water which was
utilized as the drug vehicle for this test. As is evident
from Table 5, Compound 19 shows an excellent dose response
curve when delivered only once on day 1 with significant
activity indicated at or above 37 mg per kg.
TABLE 5
RESPONSE OF MICEl INOCULATED WITH L1210 CELLS2
TO TREATMENT WITH COMPOUND 19
SCHEDULE OFDOSAGE T/C
DELIVERY (mg/kg) (%)
- -
qd: day 1 2884 TOX
173 167
104 15~
62 135
37 128
22 106
13 106
bid: day 1 62 184
tid: day 1 62 133
qid: day 1 62 194
---~
1. Each treatment yroup consisted of 3 female E3DF1 mice.
2. Mice were inoculated i.p. with L1210 cells (106 per
mouse) on day 0.
3. Dru~ delivery ~as by the i.p. route.
4. Maximum solubility.
; 35
49 :~3~
THERAPEUTIC EXAMPLE F
In Example F Compound 19 and other known cancer
chemotherapeutic agents were bioassayed for activity against
neoplastic cells in the brain utilizing the L1210 cell line
injected intracranially into test animals. In Tahle 6-a
Compound 19 is compared to a control and in Table 6-b
Compound 19 is compared to other known cancer
chemotherapeutic agents. ~5 iS evident from Table 6-a there
is a significant reduction in the number of neoplastic cells
in the brain of the test animal after i.p. infection with
Compound 19. This is indicative of both activity of
Compound 19 and the ability of Compound 19 to cross the
blood brain barrier. As is indicative of Table 6-b,
Compound l9 shows an excellent therapeutic effeet when
compared to known chemotherapeutic agents evaluated by this
test procedure. Only three known chemotherapeutic agents
out of the seven tested showed results approximately equal
to or better than those for Compound 19.
,,
TABLE 6-a
BIOASSAY OF L1210-INOCULATED BRAIN5
BDF1 mice were inoculated intracranially on day 0 with
lx105 L1210 cells. 24 hr later, on day 1, the mice were
25 injected ip with Compound 19 or 0.9~ NaCl. 2~ hr a~ter drug
` delivery the brains were collected, homogenized, and
injected ip into untreated mice. Each mouse received the
equivalent of half a brain. Thereafter, li~e span was
monitored and, using inoculum-response data as a base o~
comparison, estima~es were made oP the numbers of viable
i cells in trea~ed and control brains.
~ 30
_____________________.________.__________~__________________
cell/half brain day 2 change due to drug
logl0 cell number logl0cells
Compound 173 mg/kg 5.42 ~61016 -~.10 -92.08%
19
Control6.52 3295013
______________________________________________________________
.
TABLE 6-b
VIABLE L1210 CELLS IN MOUSE BRAIN 24-HR AFTER A SINGLE I.P.
TREATMENT WITH VARIOUS ANTICANCER DRUGS
DRU~ DOSAGE RESIDUAL CELLS
: 5 (mg/kg) (% o~ control brain)
Methotrexate 12 97
Adriamycin 3 89.5
6-Mercaptopurine160 37
Cytosine Arabinoside 1200 15
Cyclophosphamide140 11.5
Compound 19 173 7.9
Tiazofurin 1~00 2.9
BCNU 20 1.6
_ _ __ _
In Examples G through K Compound 19 was tested against
L1210 cell lines which had developed resistance to other
:known cancer chemotherapeutic agents. Depending upon th0
r~sistant cell line which was being tested and the mode o~
25 administration and/or treatment regimen, Compollnd 19 shQwed
activity against various cell lines which are resistant to
.other known chemotherapeutic agents.
" ~
,, .
,.............................. .
51 1~3~72~
THERAPEUTIC EXAMPLE G
In Example G Compound 19 was tested against both L1210
calls and L1210 cells which were resistant to 6-
Mercaptopurine, 6-Thioguanine and 6-Thioguanosine. As i5
evident from the different drug regimens shown, Compound 19
exhibited activity against the drug resistant cells. As was
note~ above, drugs in the 6-Mercaptopurine ~amily are
presently among the drugs of choice for treatment of
leukemia. It is thus avident from Table 7-a that Compound
19 is active against cells which have become resistant to
these drugs.
TABLE 7-a
15 L1210 AND L1210/6MP, 6TG CELLSl SURVIVING
TREATMENT WITH COMPOUND 19
DRUG DELIVERED2 DOSAGE CELL LINE
(schedule indicated) (mg/kg/inj) L1210 L1210/6MP,6TG3
6TGR
~0
qd: dayl 173 1.4 4.6
10~ 5.4 54.5
62 0.8 15.9
bid: day 1 62 0.8 15.9
tid: day 1 62 0.4 15.9
1. Expressed as % of day zero, intraperitoneal inoculum of
1x106 cells.
2. Drug delivery was by the intraperitoneal route.
3. 6MP = 6 Mercaptopurineo 6TG = 6-Thioguanine:
6TGR = 6-Thioguanosine.
.~
'
52 ~3~7 ~
Table 7-b shows the activity of Compound 19 against
cells resistant to the 6-Mercaptopurine ~amily of drugs
expressed as increased life span. ~s is eviden~ ~rom Table
7-b Compound 19 shows efficacy against these resistant cells
5 when the affected animal was treated intraperitoneally.
TABLE 7-b
ACTIVITY OF COMPOUND 19 AGAINST L1210 AND L1210/6MP,6TG
WHEN
DELIVERED ORALLY OR INTRAPERITONEALLY
Drug Tumor
Dosage Schedule Route L1210 L1210/6MP,6TG
mg/kg/in;
%ILS1 Rcp2 ILS RCP
,~
37bid 1,3,5,7 ip 79 (.32) 66 (~28)
oral 49 (3.73) 0
. 62bid 1,4,7 ip 88 (.14) 75 (.09)
oral 40 (8.48)
1. % Increase Life Span.
2. Residual C~ll Population.
..
.
,,
53 :~ 3 ~ 3
THERAPEUTIC EXAMPLE H
In Example H, the results o~ which are shown in Table
8, Cytosine Arabinoside resistant L1210 cells were treated
with Compound 19. As is evident ~rom Table 8, Compound 19
5 showed greater efficacy against this resistant cell line
than it did to the parent non-drug resistant L1210 ce'ls.
This is indicative cf 'collateral activity' of compounds of
the invention against resistant cells.
TABLE 8
L1210 AND L1210/AR~-C CELLS1 SURVIVING
TREATMENT WITH COMPOUND 19
15 DRUG DELIVERED2 DOSAGE CELL LINE
(qd days indicated) (mg/kg/inj) L1210 Ll210/ARA-C
1 173 1.4 o.o23
l, 4, 7 173 l.1 0.6
1, 4, 7 104 29.0 1.7
: 25
.. . .
1. ~xpressed as % o~ day zero, intraperitoneal inoculum of
lx106 cells.
2. Drug delivery was by the intraperitoneal route.
3. Indicative of 'collateral activity'.
1 ~ 11. Pl ~ p~l ,~,,
54
THERAPEUTIC' EXAMPLE I
In Exam]ple I Compound 19 was tested against
M~thotrexat2 resistant L1210 cells. Depending upon the dose
level and the dose regimen, activity can be seen against
these Methotrexate resistant cells.
TABLE 9
L1210 AND L1210/MTX CELLSl SURVIVING TREATMENT
WITH COMPO~ND 19
DRUG DELIVERED2 DOSAGE CELL LINE
15 (qd days indicated) (mg/kg/day) L1210 Ll210/MTX
1 1731.4 1.6
1, 4, 7 1731.1 33~8
l, 4, 7 10429.0 238.4
l. Expressed as ~ of day zerQ, intraperitoneal inoculum o~
lx106 cells.
2. Drug delivery was by intraperitoneal route.
3. MTX - Methokrexate.
~ 3 ~ 7 ~
THERAPEUTIC EX~MPLE J
In Example J, the results of which are shown in TahlP
10 below, Compound 19 was tested against 5-~luorouracil
resistant cells. Large dosages of Cvmpound 19 were highly
active against these resistant cells.
TABLE 10
L1210 AND L1210/5FU4 CELLSl SURVIVING TREATMENT
WITH COMPOUND 19
DRUG DELIVERED2 DOSAGE CELL LINE
15(qd days indicated)(mg/ky/day) L1210 L1210/FU
1-7 104 0.043 3
201-7 62 13 933
1, 3, 5, 7 173 0.213
1, 3, 5, 7 104 17 525
1. Expressed as % of day zero, intraperitoneal inoculum o~
lx106 cells.
2. Drug delivery was by intraperitoneal route.
3. Treatment group included 1 long term survivor which was
30 not included in the calculation of lifa span.
4. 5FU = 5-Fluorouracil.
Compound 19 has not been found to generate resistant
cell lines as per the other known cancer chemotherapeutic
agents listed in Tables 7 through 10 above. However,
Compound l8 does generate drug resistant cell lines.
..
56 .~ 3 ~ P~
THERAPEUTIC EXAMPLE K
In Example K, the results of which are shown in Table
11 below, Compound lg was tested against drug resistant cell
lines developed against Comp~und 18. As is evident from the
results shown in Table 11, Compound 19 differs from Compound
18 only by the state of oxidation between the sulfenamide o~
Compound 18 and the sulfinamide of Compound 19. As is
evident from Table 11 Compound 19 .is e~fective against those
L1210 cell lines which have developed resistance against
Compound 18. Thus, while Compound l8 may mimic 6-
Thioguanosine in that it generates drug resistant cells, themode of action of Compound 19 is bel.ieved to be completely
different as is expressed by its activity seen in Example G
against 6-Thioguanosine resistant cell lines and its
activity in Example K against Compound 18 resistant cell
lines.
TABLE ll
20L1210 AND L1210/DRUG RESISTANT CELLSl SURVIVING TREATMENT
WITH COMPOUND 19
DRUG DELIVERED2DOSAGE CELL LIIIE
(qd days indicated) (mg/kg/day) L1210 L1210/COMPOUND 18
25qd: day 1 173 1.4 7~5
104 5.4 17.9
62 8.5 100.0
bid: day 1 62 0.8 27.4
1. Expressed as % of day zero, intraperitoneal inoculum of
1x106 cells.
2. Drug delivery was by intraperitoneal route A
5'7 ~ 3 1 ~2~:3.
THERAPEUTIC EXAMPLE L
In this Example Compounds 1~ and 19 were tes~ed
singularly and then in combination first given bokh toyether
and then given in different orders. As is evident from the
5 result tabulated in Table 12 both Compounds 18 and 19
induced increases in life span in 'he test animals with the
activity of the compounds given simultaneously or
sequentially being similar or even inferior to that seen for
- Compound 19 by itself.
TABLE 12
15 COMBINED DRUG TREATMENT OF L1210: COMPOUND 19 AND COMPOUND 18
Schedule of Delivery1 %ILS2
Day 1 Day 2
; 20
Compound 19 88
Compound 18 51
Compound 19
and 58
Compound 18
Compound 19 Compound 18 91
Compound 18 Compound 19 84
,~
35 1. Dosages: Compound 19 173 mg/kg
Compound 18 22 mg/kg.
2. ~ Increase Life Span.
,
58 13~7~
THERAPEUTIC EXAMPLE M
In Example M a further example was run similar to that
of Exhibit L except that Compound 19 was utilized in
conjunction with the known chemotherapeutic agent
Tiazofurin. As is evident ~`rQm the results tabulated in
Table 13 increased activity i5 seen when Compound 19 and
Tiazofurin are given sequentially. Sequence dependency was
also observed with the best result being produced when
Compound 19 preceded Tiazofurin.
TABLE 13
COMBINED DRUG TREATMENT QF Ll210: COMPOUND 19 AND TIAZOFURIN
Schedule of Delivery1 %ILS2
Day 1 Day 2
_
Compound 19 81
Tiazofurin 44
Compound 19
and 69
Tiazofurin
Tiazofurin Compound 19 100
Compound 19 Tiazofurin 150
1. Dosages: Compound 19 173 mg/kg
Tiazo~urin 22 mg/ky
2. Increase Life Span
s
THERAPEUTIC EXAMPLE N
In Example N other compounds of the invention were
tested against L1210 cells. All of the compounds listed in
Table 14 exhibit activity. Further, Table 14 shows thP
maximum solubility (in water unless otherwise indicated) and
the maximum tolerated dose. Activity in Table 14 a is
tabulated as both increases in life span and as cells
surviving treatment and that in table 14-b as T/C.
59 ~3~7~
TABLE 14-a
R~SPONSE OF L1210 INOCULATED BDF1 MICE
TO COMPOUNDS OF THE INVENTION
COMPOUND MAX. SO~. MAX. TOLERATED ~ILS CELLS
# DOSAGE DOSAGE SURVIVING
( MG/ XG ) t MG/ ~G ) TREATMENT
(% OF DAY 0 INNOC.)
62 62 28 40
38 173 173 29 27
800 104 33 16
24 480 288 - 34 17
12 173 173 39 13
8 480 173 43 6.4
22 173 173 47 6.0
29 480 480 59 1.7
23 4~0 173 59 2.0
288 288 59 1.7
2 104(NaOH) 104 63 2.0
2 62(DMSO) 37 66 0.8
14 800 288 66 1.0
6 480 480 67 2.8
16 800 173 69 1.0
18 2~ 22 85 0.3
1. Ahove data resulted ~rom single QD day 1 treatment of
BDF1 mice on day 0 with 1o6 cell L~210. Both cell
inoculation and treatment were IP.
2. In water unless otherwise indicated.
` :
~3~
TABLE 14-b
RESPONSE OF L1210 INOCULATED BDFl MICE
TO COMPOUNDS OF THE INVENTION
COMPOUND MAX. S~I,. T/C
# DOSAGE
( MG/ KG )
42 104(NaOH~ 172
43 480 ~.30
10 ~5 480 172
800 140
288 125
1. Above data resulted from single QD day 1 treatment of
BDFl mice on day 0 with 106 cell L1210. Both cell
inoculation and treatment were IP.
2. In water unless otherwise indicated.
Compound 19 was also tested against a variety of solid
tumors. No activity was noted against B-16 melanoma, Lewis
lung carcinoma or human lung carcinoma LX-1. Activity,
however, was noted in a variety of other solid tumors as per
examples 0 through S below.
THERAPEUTIC EXAMPLE O
In this exa~ple Compound 19 was tested against
reticulum cell sarcoma ~5076. For this and certain other
tests below, test results are shown as ~T/~C. Utilizing
this protocol the difference in tumor weight be~or
,~ treatment and after treatment of the treated animals
compared to the control animals is determined. As is seen
in Table 15, Compound 19 exhibited activity against this
cell line and shows a dose response for this aotivity.
61 ~3~
TABLE 15
RESPONSE OF RETICULUM CELL SARCOMA M5076
TO TREATMENT WITH COMPOUND 19
SCHEDULE OF DOSAGETUMOR WT3 (Mean + lSD5 ~T/~C
5 DELIVERY2 ~mg/kg/inj) staging4evaluation
(qd days indicated) day day
1,3,5,7,9,11 173.0 35~ + 81 409 + 385 5.0
138.4 355 + 79855 + 325 45.4
110.8 396 + 1161121 + 343 65.8
0 396 + 104149~3 + 438
_
1. C57B1/6 female mice (7/group) were inoculated s.c~ with
1x106 M5076 cells on day 0.
2. Drug was delivered by the i.p. route.
3. Tumor weight was estimated from caliper measurements
using the formula: tumor wt. ~mg) = w21/4. 45.
4. Treatment was initiated on staging day (day 15
post inoculation).
5. Day 12 post initiation of treatment.
62 ~ 3 .~ 7 ~
THERAPEUTIC EXAMPLE P
In Example P Compound 19 was tested against human
mammary carcinoma MX-l. As per the results tabulated in
Table 16, Compound 19 exhibited a dose response against this
solid tumor.
TABLE 16
RESPONSE OF HUMAN MAMMARY CARCINOMA MX
TO TREATMENT WITH COMPOUND 1~
SCHEDULE OF DOSAGE TUMOR WT3 (Mean + lSD) ~T/~C4
DELIVERY2 (mg/kg/inj) staging5 evaluation6
lS (qd days day day
indicated)
:
1,3,5,7,9,11 ~73.0 323 + 127 489 + 104 17.6
138.4 330 + 11~ 884 + 415 58.9
110.8 321 + 132 ~43 + 2g7 66.1
o 325 + 112 1266 ~ 695
1. CD-l nu/nu female mice (7 per group) were implanted s.c.
with fragments (<25 mg ea.) of MX-1 carcinoma on day o.
2. Drug delivery was by the i.p. route.
3. Tumor weight was estimated from caliper measurements
using the formula: tumor wt (mg) = w21/4.45.
4. NCI guidelines suggest a ~T/~C < 20% for demonstration
of moderate activity.
5. Treatment was initiated on staging day (day 17 post
implant).
6. Day 15 post initiation of treatment.
.
;
.
63
~3~7,~ ~
THERAPEUTIC EXAMPLE Q
In Example Q Compound 19 was tested against Colon 26
Adenocarcinoma. Except when given once a day in the regimen
on days 1, 4 and 7, for the other dosages and test regimens
Compound 19 exhibited acti~ity against this tumor.
TABLE 17
RESPONSE OF MICEl BEARING COLON 26 ADENOCARCINOMA2
TO TREATMENT WITH COMPOUND 19
SCHEDULE OF DOSAGE MEDIAN SURVIVAL TIME T/C4
DELIVERY3 (mg/kg/inj) ~days post inoculation) (%)
15 qd: day5 1, 4, 7 173 31 135
104 31 13
qd: days 1,3,5,7 173 42 183
104 34 148
20 qd: days 1-7 104 38 165
62 40 174
bid: days 1,4,7 104 175
bid: days 1,4,7 62 45 196
1. Each treatment yroup consisted of 11 female C'DF1 mice.
2. 3X106 cells of Colon 26 Adenocarcinoma were implanted
i.p. on day 0.
3. Drug delivery was by the i.p. route.
4. NCI guidelines suggest a T/C > 150% for demonstration of
significant activity.
5. Treatment group included 6 toxic deaths.
64 3 3~.72~:~
THERAPEUTIC EXAMPLE R
Compound 19 was ~urther tested against Human Colon
Adenocarcinoma CX-1. For comparison purposes the activity
of other clinically active antitumor agents is shown in
Table 18-a. Activity ayainst this tumor system is indicated
at a ST/~C value of less than 20.
TABLE 18-a
ACTIVITY OF CLINICALLY ACTIVE ANTITUMOR AGENTS AGAINST CX-1
NSC DRUG ACTIVITY ~ATING
# ~T/ISC2
740 Methotrexate 66
752 6-Thioguanine 81
15 755 6-Mercaptopurine 9g
3053 Actinomycin D 73
3088 Chlorambucil 60
8806 Melphalan 101
13875 Hexamethylmelamine 85
19893 5-Fluorouracil 88
26271 Cyclophosphamide 113
26980 Mitomycin C 62
20 45388 DTIC 92
49842 Vinblastine 119
63878 Cytosine arabinoside 73
67574 Vincristine 89
77213 Procarbazine 60
79037 CCNU 77
95441 Methyl CCNU 83
119875 Cis-Platinum 66
123127 Adriamycin 72
125066 Bleomycin 51
178248 Chlorozo~ocin 75
409962 BCNIJ 63
.
1. Data taken ~rom~: Ao Goldin, et al, Current Results O~
30 The Screening Program Of The Division O~ Cancer
Tr~atment, National Cancer Institute, Euro~ J. Cancer,
Vol. 17, 129-142, (1981).
2. Activity indicated at ~T/~C < 20.
`:
In a like manner Compound 19 was tested against this
tumor system with the results shown in Tabl~ 18-b. As is
shown, at the i73 mg level when drug was glven on ~sys
.
.,
~3~
1,4,7,10 and ~3, activity against this tumor sy~tem is
demonstrated.
` TABLE 18-b
RESPONSE OF HUMAN COLON ADENOCARCINOMA CS-l
TO TREATMENT WITH COMPOUND 19
SCHEDULE ~F DOSAGE TUMOR W~3 (mean + lSD~
DELIVERY mg/kg~in;) staging evaluation ~T/~C4
(qd days indicated) day day
lo 1,3,5,7,9 173.0 22g + 141 364 ~ 254 (16) 39.9
1,4,7,10,13173.0 226 + 125 279 + 121 (17) 15.8
1,3,5,7 173.0 222 + 79 302 + 1~4 (12) 38.5
1,4,7,10 173.0 226 + 103 402 + 187 (16) 52.1
1,3,5,7,9,11138.4 220 ~ 80 358 + ~25 (15) 46~6
1,4,7,10,13,16 138.4 215 + 81296 + 186 (12) 38.9
1,3,5,7,9 138.4 226 + 120 27~ + 114 (12~ 25.0
1,4,7,10,13138.4 225 + 104 375 + 155 (15) 50.7
~0
0 218 + 81 426 + 195 (12) _ _
0 218 + 81 514 + 252 (15) _ _
0 218 + 81 556 + ~38 (16) _ _
0 218 + 81 553 + 231 (17)
1. CD-1 nu/nu female mice (6 per group) were lmplanted s.c.
with fragments (<25 mg ea.) of CX-1 adenocarcinoma on
day 0.
2. Drug delivery was by the i.p. route.
3. Tumor weight was estimated from caliper measurements
using the formula: tumor wt (mg)=w21/4.45.
4. NCI guidelines suggest a T/C < 20% for demonstration of
moderate activity.
5. Treatment was initiated on staging day (day 33 post
implant).
6. The day of occurxence (shown in parenthesis~ of optimum
~/~ % between day 12 and day 21 post initiation of
treatment.
., ,
66 ~3~7~
It is indicative that activity against this tumor
system is possible utiliæing optimum dose scheduling o~
Compound 19 against this tumor system.
THERAPEUTIC EXAMPLE S
Compound 19 was also tested against Murine Glioma 261.
In this test activity is indicated at levels of T/C below
42%. As is shown in Table 19, Compound 19 is active at
various doses against this tumor system.
TABLE 19
RESPONSE OF MURINE GLIOMA 261l TO TREATMENT
WITH COMPOUND 19
SCHEDULE OF DOSAGE ATTAINED TUMOR WT3 T/C5
DELIVERY2(mg/kg/inj)(mg) (~)
qd: days 1-9 104 5 + 54 1.4
62 137 + 12~ 39.3
37 119 + 105 34.1
22 80 + 130 22.9
O 349 + 139
1. C57B1/6 male mice (6/group) were implanted s.c. with
~ra~nents (<25 Tng/ea) of Glioma 261 on day 0.
2. Drug was delivered by the i.p. routeO
3. On day lO post implant, tumor sizes were estimated from
caliper measUrementS USing the ~ormula: tumor wt(mg) =
w21/4.45.
4. Mean + lSD.
j 5. Activity indicated at or below T/C of 42%~
,
67 ~ 3~7,~
THERAPEUTIC EXAMPLE T
As an example of activity among the dif~erent members
of the oxidation series repre~ented by sulfenamides,
: sulfinamides and sulfonamides comparison of activity of
Compounds 18, 19, and 20 is shown in Table l9-a and is
summarized in Table 19-b. As is evident from the summaries
in Table l9-bo Compounds 18 and 19 effectively cross the
blood brain barrier and thus are active intracranially
whereas at the high oxidation state of Compound 20 no
intracranial activity i5 seen. Oral activity is seen for
both Compounds 18 and 19 but not present in Compound 20O
Contrasted to this is activity against resistant cells
wherein Compounds 19 and 20 are active but Compound 18 in
; fact shows no activity. As was shown in Example K, Table 11
above, Compound 19 in fact was active against cells which
developed resistance to Compound 18.
.
.,
~.
. 30
'',
'
'.'
1 ~l 7~
6~
TABLE 19-a
Activity against ~ctivity against Activity against
I. c. I.P. L1210 cellsI.P. L1210 cells
L1210 cells with oral drugresistant to
administration 6MP, 6TG, 6TGR
I.~ Cell
Dosage Kill Schedule Dosage T/C Schedule Dosage T/C
~mg/kg) (x) (mg/kg) (%) (mg/kg) (%)
_______________________________________________________________
Compound 18
22 57.3qd:dl 22 153.0 qd:dl 22 93~8
13 1~7.0
8 14~.0
Compound 19
173 92.1gd:dl,4,7 173 138.5qd:dl 173 144.5
104 13~.5 104 122.2
62 144.6 62 127.3
37 144.6 bid:dl 104 150.0
bid:dl,3,5,7 37 148.6 62 133.3
bid:dl,4,7 62 140.0tid:dl 62 133.3
- bid:dl,3,5,7 37 165.6
0 bid:l,4,7 62 175.0
Compound 20
62 0.0 bid:dl,4,762 103.3bid:dl,4,7 62 163.3
37 103.3 37 130.0
22 103.3 22 ~06.7
The different compounds of the sulfinamide, sulfenamide and
sulfcnamide series ~s is demonstrated by Compound 18, 19 and
20 show various advantages and disadvantages with the
sulfinamide Compound 19 having optimization o~ certain
properties. Compound lg shares the best properties of both
Compounds 18 and 20
~3~72~.
TABLE l9-b
ACTIVITY VS. ORAL ACTIVITYACTIVITY VS.
Io~ CE~LS ~1210/6MP:6TG:6TGR
Compound 18 + +
Compound 19 ~ + +
10 Compound 20 - - +
Compound 19 exists as two enantiomers. The above test
results for Compound 19 were done on the racemic mixture of
these enantiomers. Further, separation of these enantiomers
to a high degree of (but not absolute) puriky has been
effected. The separate enantiomers have been independently
tested and further tested as contrived mixtures of known
amounts of the enantiomers. Of the two purified
enantiomers, enantiomer B shows a higher solubility than
enantiomer A and the racemic mixture exhibits solubility
characteristics of enantiomer B exhibiting solubility ak
about 17.3 mg per ml~ In contrast to this enantiomer A
exhibits solubility of about 3.7 mg per ml. The two
enantiomers present comparable activity, however, because of
the solubility of enantiomer A is less than enantiomer B,
tests with enanti~mer A have been done at a much lower
dosage level.
.,
.,
"
.
,
7o 131 J ~ 9 ~
THERAPEUTIC EXAMPLE U
In this example the enantiomers of Compound 19 labeled
enantiomer A and enantiomer B were tested independent of one
another. The results o~ these tests are indicated utilizing
two different protocols as are shown in Tables 20-a and 20-
b. Enantiomer A shows greater ackivity with respect to
enantiomer B because of solubility differences.
TABLE 20-a
LIFE SPAN (%T/C) OF L1210 INOCULATEDl MICE TREATED2
WITH COMPOUND 19 ENANTIO~ER A OR ENANTIOMER B
DRUG DOSAGE T/C
(mg/kg) ~%)
Compound 19
Enantiomer A 37 108.7
Enantiomer B 173 144.9
1~ BDF1 female mice were inoculated i.p. on day 0 with 106
L1210 cells.
2.Drug was administered i.p. dq:day 1.
TABLE 20-b
Ll210 CELLS1 SURVIVING TREATMENT2 WITH
COMPOUND 19 ENANTIOMER A OR ENANTIOMER B
~RUG DOSAGE % OF ORIGINAL
(mg/kg) YNOCULUM
- - _ _
Compound 19
Enantiomer A 37 182.8
Enantiomer B 173 7.5
1. BDF1 female mice were inoculated i.p. on day O with 106
L1210 cells.
2. Drug was administered i.p. dq:day l.
71 13~7~ ~
THERAPEUTIC EXAMPLE V
In this example different regimens of dosages ~or
enantiomer A of Compound 19 were utilized and the results
were tabulated in Table 21. The percent of contamination o~
enantiomer A with enantiomer B and with a ~urther
contaminate comprising Guanosine and Compound 20 was tested
using HPLC. As is evident from ~able 21, when the dosage of
enantiomer A was divided into multiple dose regimens
effective therapy was indicated.
TABI,E 21
EFFECT OF COMPOUND 19 ENANTIOMER-A ON T~E MEANS LIFE SPAN OF
BDFl MICE INOCULATED I.P. WITH lX10 CELLS OF L1210
Drug ScheduleTotal Drug % ILS Cells
Delivered of (mg/kg) Surviving
(mg/kg/inj) Delivery Delivered as Treatment
A B Contam- (% o~
inant original
inoc.)
37qd, day 1 35.47 0.63 0.9022 64
37bid, day 1 70O94 1.26 1.8044 10
25 37tid, day 1 106.41 1.89 2.7072
37qid, day 1 14~.88 2.52 3.6072
37qid, day 1 177.35 3.15 4.50 0 tox
As shown, all injections were made on day lo For mice
treated more than once, treatment was completed within 20
minutes. Enzntiomer A was 35.~7% A and 1.71% B: thus, these
two enantiomers comprised 97.58% of the material delivered.
The A-B ratio was 95.87:1.71. There were 3 mice in each
treatment group.
72
THERAPEUTIC EXAMPLE W
In this example, concocted mixtures of enantiomers A
and B were made. In addition, these mixtures were
contaminated with small amount of contaminant~ comprising
5 Guanosine and Compound 20. As is indicative from the
results shown in Table 22, activity does not reside with
only one or the other of the two enantiomers but is
apparently optimized in mixtures of the enantiome.rs. It is
presently believed a 50/50 mixture of the enantiomers of
10 compound 19 is suggested for use in antitumor compositions
of this compound.
TABLE 22
EFFECTS OF VARIOUS RATIOS OF COMPOUND l9-ENANTIOMERS A AND B
15 ON THE MEAN LIFE SPAN ~F BDFl MICE INOCULATED I.P. WITH
lX10 CELLS OF L1210
Drug A/B Ratio Total Drug (mg/kg) g6 lLS Cells
Delivered Delivered as Surviving
(mg/kg/inj) A B Contam- Treatment
inant (% of
original
inoc.)
62 90/1055.586. 42 1.74 14 ~0
104 70/3073.5331.13 3.50 20 58
25 173 50/5087.1785.83 5.09 57 (2 tox) 2
173 30/7059. 03113. 97 6.3:L 31 19
173 10/9019.0~153.92 4.14 37 11
173 90/10156.9116.09 ~.88 62 (2 tox)
173 70/30121.2651.74 5.83 43 (4 tox) 7
173 50/5087.6985.48 5.10 62 ~2 ~ox~ 1
173 30/705~ .74118. 26 3. 29 40 8
173 10/9019. 36153.6d, 3.44 29 25
A11 treatments were made qd, day 1. Each trPatment group
consisted of 5 mice: the postinoculation li~e span of these
treated mice was compared w.ith that OI 9 control mice that
were injected with a 0.9% solution of NaCl.
73 ~ 7~
For delivery to a host inflicted with a neoplastic
disease compounds of the invention can be ~ormulated in
various formulations to prepare pharmaceutical compositions
containing the compounds o~ the invention as active
ingredients. The following illustrative examples are given
fcr the formulations of such pharmaceutical compositions
utilizing Compound 19 as the illustrative compound. In
these examples, Pharmaceutical Preparative Example
illustrates the use of the compounds of the invention in
injectables suitable for intravenous or other types of
injection into the host animal. Pharmaceutical Preparative
Example 2 is directed to an oral syrup preparation,
Pharmaceutical Preparative Examples 3 to an oral capsule
preparation and Pharmaceutical Preparative Example 4 to oral
tablets. Pharmaceutical Preparative Example 5 is directed
to use of the compounds of the invention in suitable
suppositories. For Pharmaceutical Preparative Examples 1
through 5, the ingredients are listed followed by the
method~ of preparing the compositionO
PHARMACEUTICAL PREPARATIVE EXAMPLE 1
INJECTABLES
.
Compound 19 250 mg - 1000 mg
Water for Injectio~ USP q.s.
- - -
Compound 19 is dissolved in the water and passed
through a 0.22; filter. The filtered so~ution is added to
ampoules or vials, sealed and sterilized.
;
-
~ 3 ~l 7 ~
PHARMACEUTICAL PREPARATIVE EXAMPLE 2
SYRUP
250 mg Active ingredient/5 ml syrup
~
Compound 19 50.0 g
Purified Water USP q.s. or 200 ml
Cherry Syrup q.s. ad 1000 ml
_ ,
Compound 19 is dissolved in the water and to thissolution the syrup is added with mild stirring.
PHARMACEUTICAL PREPARATIVE EXAMPLE 3
CAPSULES
100 mg 250 mg or 500 mg
Compound 19 500 g
A~ ~ Lactose WSP, Anhydrous q.s. or 200 g
-' Sterotex Powder HM 5 g
Combine compound 19 and the Lactose in a twin-shell
blender equipped with an int~nsifier bar. Tumble blend Eor
two minutes, followed by blending for one minute wikh the
intensifier bar and then tumble blend again for one minute.
A portion of the blend is then mixed with the Sterotex
Powder, passed throuyh a #30 screen and added back to the
remainder o~ the blend. The mixed ingredients are then
blended for one minute, blended for the intensifier bar for
thirty seconds and tumble blended for an additional minute.
Appropriate sized capsules are filled with 141 mg, 352.5 mg
or 705 mg of the blend, respectively, for the 100 mg, 260 mg
and 500 mg containing capsules.
.
~ ~ fr~ de r~r ~
~3~ 3
PHARMACEUTICAL PREPARATIVE EXAMPLE 4
TABLETS
100 mg, 200mg or 500 mg
Compound 19 500 g
Corn Starch NF 200.0 g
Cellulose Micxocrystalline 46.0 g
10 Sterotex Powder HM 4-0 g
Purified Water q.s. or 300.0 ml
Combine the corn starch, the cellulose and Compound 19
together in a planetary mixer and mix for two minutes. Add
the water to this cor~bination and mix for one minute. The
resulting mix is spread on trays and dried in a hot air oven
at 50 C. until a moisture level of 1 to 2 percent is
obtained. The dried mix is then milled with a Fitzmill
through a #RH2B screen at medium speed. Th~ Sterotex Powder
is added to a portion of the mix and passed through a #30
screen and added back to the milled mixture and the total
blended for five minutes by drum rolling. Compressed tables
of 150 mg, 375 mg and 750 mg respectively, of the total mix
are formed with appropriate sized punches ~or the 100 my,
25 250 mg or 500 mg containing tables.
PHA~U~ACEUTICAL PREPARATIVE EXAMPLE 5
SUPPOSITORIES
250 mg, 500 mg or 1000 mg per 3 g
__ _ _
Compound 19 250 mg 500 mg 1000 mg
Polyethylene Glycol 1540 1925 mg 1750 mg 1400 mg
35 Polyethylene Glycol 8000 825 mg 750 mg ~00 mg
:
Melt the Polyethylene Glycol 1540 and the Polyethylene
Glycol 8000 together at 60C. and dissolve Compollnd 19 into
~3~72~
76
the melt. Mold thi~ total at 25 C. into appropriate
suppositories .
~5
