U.S. patent application number 17/278625 was filed with the patent office on 2022-02-03 for 2'-halogenated-4'-thio-2'-deoxy-5-azacytidine analogs and use thereof.
This patent application is currently assigned to The USA, as represented by the Secretary, Department of Health and Human Services. The applicant listed for this patent is THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVIC, THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVIC. Invention is credited to Omar Diego Lopez, Joel Morris, Donn G. Wishka.
Application Number | 20220033387 17/278625 |
Document ID | / |
Family ID | |
Filed Date | 2022-02-03 |
United States Patent
Application |
20220033387 |
Kind Code |
A1 |
Morris; Joel ; et
al. |
February 3, 2022 |
2'-HALOGENATED-4'-THIO-2'-DEOXY-5-AZACYTIDINE ANALOGS AND USE
THEREOF
Abstract
Halogenated analogs of 5-aza-2'-deoxycytidine, such as
halogenated analogs of 5-aza-4'-thio-2'-deoxycytidine
(5-aza-T-dCyd) are described. Pharmaceutical compositions including
a halogenated analog and methods of using the halogenated analogs
to inhibit neoplasia are described. In some examples, the
halogenated analogs have a structure according to formula Ia, or a
stereoisomer, tautomer, or pharmaceutically acceptable salt
thereof: ##STR00001##
Inventors: |
Morris; Joel; (Rockville,
MD) ; Wishka; Donn G.; (Middletown, MD) ;
Lopez; Omar Diego; (Walkersville, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY,
DEPARTMENT OF HEALTH AND HUMAN SERVIC |
Bethesda |
MA |
US |
|
|
Assignee: |
The USA, as represented by the
Secretary, Department of Health and Human Services
Bethesda
MD
|
Appl. No.: |
17/278625 |
Filed: |
September 23, 2019 |
PCT Filed: |
September 23, 2019 |
PCT NO: |
PCT/US2019/052410 |
371 Date: |
March 22, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62736246 |
Sep 25, 2018 |
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International
Class: |
C07D 409/04 20060101
C07D409/04; A61K 45/06 20060101 A61K045/06 |
Claims
1. A compound according to formula Ia, or a stereoisomer, tautomer,
or pharmaceutically acceptable salt thereof: ##STR00025## where X
is halo; Y is hydrogen, halo, or deuterium; each R independently is
hydrogen or deuterium; and R.sup.a is hydrogen, deuterium, alkyl,
alkoxy, amino, or halo.
2. The compound of claim 1, wherein: (i) X is F; or (ii) Y is
hydrogen or F; or (iii) each R is hydrogen; or (iv) R.sup.a is
hydrogen; or (v) any combination of (i), (ii), (iii), and (iv).
3. The compound of claim 1 having a stereochemistry according to
any one of formulas IIa-Va or any combination thereof
##STR00026##
4. The compound of claim 1, wherein the compound is ##STR00027## or
any combination thereof.
5. The compound of claim 1, wherein the compound is ##STR00028## or
any combination thereof.
6. The compound of claim 1, wherein the compound is
4-amino-1-((2R,3S,4S,5R)-3-fluoro-4-hydro-5-(hydroxymethyl)tetrahydrothio-
phen-2-yl)-1,3,5-triazin-2(1H)-one: ##STR00029##
7. A pharmaceutical composition comprising a compound according to
claim 1 and a pharmaceutically acceptable carrier.
8. The pharmaceutical composition of claim 7, wherein the
pharmaceutical composition is formulated for intravenous, oral,
intraperitoneal, subcutaneous, rectal, or buccal
administration.
9. A method of inhibiting a neoplasia, comprising: contacting
neoplastic cells with an effective amount of a compound according
to claim 1.
10. The method of claim 9, wherein contacting the neoplastic cells
with the effective amount of the compound reduces proliferation of
the neoplastic cells.
11. The method of claim 9, wherein the compound is
4-amino-1-((2R,3S,4S,5S)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydroth-
iophen-2-yl-1,3,5-triazin-2(1H)-one: ##STR00030##
12. The method of claim 9, wherein contacting the neoplastic cells
with the effective amount of the compound comprises administering a
therapeutically effective amount of the compound to a subject
having or suspected of having a disease characterized at least in
part by presence of neoplastic cells.
13. The method of claim 12, wherein the disease is a cancer.
14. The method of claim 13, wherein the cancer is a cancer of the
kidney, bladder, breast, colon, endometrium, skin, blood, pancreas,
prostate, bone, liver, lung, esophagus, or central nervous
system.
15. The method of claim 12, wherein administering the
therapeutically effective amount of the compound to the subject
reduces a sign or symptom of the disease.
16. The method of claim 15, wherein: the sign or symptom of the
disease is a solid tumor and administering the therapeutically
effective amount of the compound to the subject reduces growth of
the solid tumor, reduces a volume of the solid tumor, reduces
metastasis of the solid tumor, or any combination thereof; or the
sign or symptom of the disease is an abnormal complete blood count
and administering the therapeutically effective amount of the
compound to the subject at least partially normalizes the complete
blood count.
17. The method of claim 12, wherein administering the
therapeutically effective amount of the compound comprises
administering a pharmaceutical composition comprising the
therapeutically effective amount of the compound to the
subject.
18. The method of claim 12, wherein administering comprises
intravenous, oral, intraperitoneal, subcutaneous, rectal, or buccal
administration.
19. The method of claim 12, further comprising administering a
second active agent to the subject.
20. The method of claim 19, wherein the second active agent is an
anti-cancer agent, an anti-inflammatory agent, an antimicrobial
agent, an antiviral agent, an anesthetic agent, or any combination
thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of the earlier filing
date of U.S. Provisional Application No. 62/736,246, filed Sep. 25,
2018, which is incorporated by reference in its entirety
herein.
FIELD
[0002] This disclosure concerns halogenated analogs of
5-aza-2'-deoxycytidine, such as halogenated analogs of
5-aza-4'-thio-2'-deoxycytidine (5-aza-T-dCyd), and methods of using
the halogenated analogs.
SUMMARY
[0003] Halogenated analogs of 5-aza-2'-deoxycytidine and methods of
using the halogenated analogs are disclosed. In some embodiments,
the compounds have a structure according to formula Ia, or a
stereoisomer, tautomer, or pharmaceutically acceptable salt
thereof:
##STR00002##
where X is halo; Y is hydrogen, halo, or deuterium; each R
independently is hydrogen or deuterium; and R.sup.a is hydrogen,
deuterium, alkyl, alkoxy, amino, or halo. In certain embodiments,
(i) X is F; or (ii) Y is hydrogen; or (iii) each R is hydrogen; or
(iv) R.sup.a is hydrogen; or (v) any combination of (i), (ii),
(iii), and (iv).
[0004] In some embodiments, the compound has a structure according
to any one of formulas IIa-Va, or any combination thereof:
##STR00003##
[0005] In any or all of compounds IIa-Va, X may be F, Y may be H or
F, each R may be H, and/or R.sup.a may be H. In certain
embodiments, the compound is:
##STR00004##
or any combination thereof.
[0006] A pharmaceutical composition may include at least one of the
disclosed compounds and a pharmaceutically acceptable carrier. In
some embodiments, the pharmaceutical composition is formulated for
intravenous, oral, intraperitoneal, subcutaneous, rectal, or buccal
administration.
[0007] A method of inhibiting a neoplasia includes contacting
neoplastic cells with an effective amount of a compound as
disclosed herein. In some embodiments, contacting the neoplastic
cells with the effective amount of the compound reduces
proliferation of the neoplastic cells.
[0008] In any or all embodiments, contacting the neoplastic cells
with the effective amount of the compound may include administering
a therapeutically effective amount of the compound to a subject
having or suspected of having a disease characterized at least in
part by presence of neoplastic cells, for example, a cancer. In
some examples, the cancer is a cancer of the kidney, bladder,
breast, colon, endometrium, skin, blood, pancreas, prostate, bone,
liver, lung, esophagus, or central nervous system. In any or all
embodiments, administering the therapeutically effective amount of
the compound to the subject may reduce a sign or symptom of the
disease. In one embodiment, the sign or symptom of the disease is a
solid tumor and administering the therapeutically effective amount
of the compound to the subject reduces growth of the solid tumor,
reduces a volume of the solid tumor, reduces metastasis of the
solid tumor, or any combination thereof. In another embodiment, the
sign or symptom of the disease is an abnormal complete blood count
and administering the therapeutically effective amount of the
compound to the subject at least partially normalizes the complete
blood count. In any or all embodiments, administering the
therapeutically effective amount of the compound may include
administering a pharmaceutical composition comprising the
therapeutically effective amount of the compound to the
subject.
[0009] The foregoing and other objects, features, and advantages of
the invention will become more apparent from the following detailed
description, which proceeds with reference to the accompanying
figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 is an exemplary synthesis scheme for
4-amino-1-((2R,3S,4S,5R)-fluoro-4-hydroxy-5-(hyoxymethyl)tettahydrothioph-
en-2-yl)-1,3,5-triazin-2(1H)-one (Compound 1).
[0011] FIG. 2 is an exemplary synthesis scheme for
4-oxyloxyphenylmethanethiol.
[0012] FIGS. 3A and 3B show GI50 (drug concentration resulting in a
50% reduction in net protein increase) data for Compound 1 and
5-aza-T-dCyd, respectively, against leukemia cell lines.
[0013] FIGS. 4A and 4B show GI50 data for Compound 1 and
5-aza-T-dCyd. respectively, against central nervous system (CNS)
cancer cell lines.
[0014] FIGS. 5A and 5B show GI50 data for Compound 1 and
5-aza-T-dCyd, respectively, against renal cancer cell lines.
[0015] FIGS. 6A and 6B show GI50 data for Compound 1 and
5-aza-T-dCyd, respectively, against non-small cell lung cancer cell
lines.
[0016] FIGS. 7A and 7B show GI50 data for Compound 1 and
5-aza-T-dCyd, respectively, against melanoma cell lines.
[0017] FIGS. 8A and 8B show GI50 data for Compound 1 and
5-aza-T-dCyd, respectively, against prostate cancer cell lines.
[0018] FIGS. 9A and 9B show GI50 data for Compound 1 and 5-T-dCyd,
respectively, against colon cancer cell lines.
[0019] FIGS. 10A and 10B show GI50 data for Compound 1 and
5-aza-T-dCyd, respectively against ovarian cancer cell lines.
[0020] FIGS. 11A and 11B show GI50 data for Compound 1 and
5-aza-T-dCyd, respectively, against breast cancer cell lines.
[0021] FIGS. 12A and 12B show HCT-116 human colon carcinoma
xenograft tumor volume and mouse body weight, respectively, over
time in mice administered 10 mg/kg Compound 1 (F-aza-TdCyd)
intraperitoneally QDx5, rest and repeat for 4 cycles; 400 mg/kg
Compound 1 intraperitoneally Q7Dx3; 240 mg/kg F-TdCyd intravenously
Q7Dx4; 1.5 mg/kg 5-aza-T-dCyd intraperitoneally QDx5, rest and
repeat for 4 cycles; or 150 mg/kg gemcitabine intraperitoneally
Q7Dx3.
[0022] FIGS. 13A and 13B show BL0382 human bladder carcinoma
xenograft tumor volume and mouse body weight, respectively, over
time in mice administered 8 mg/kg Compound 1 (F-aza-TdCyd)
intraperitoneally QDx5, rest for 3 cycles; 250 mg/kg Compound 1
intraperitoneally Q7Dx3; 200 mg/kg F-TdCyd intravenously Q7Dx3; 1.5
mg/kg 5-aza-T-dCyd intraperitoneally QDx5, rest and repeat for 3
cycles; 150 mg/kg gemcitabine intraperitoneally Q7Dx3; or 8 mg/kg
Compound 1 orally QDx5, rest for 3 cycles.
[0023] FIGS. 14A and 14B show OVCAR3 human ovarian carcinoma
xenograft tumor volume and mouse body weight, respectively, over
time in mice administered 8 mg/kg Compound 1 (F-aza-TdCyd)
intraperitoneally QDx5, rest for 3 cycles; 250 mg/kg Compound 1
intraperitoneally Q7Dx3; 200 mg/kg F-TdCyd intravenously Q7Dx3; 1.5
mg/kg 5-aza-T-dCyd intraperitoneally QDx5, rest and repeat for 3
cycles; 150 mg/kg gemcitabine intraperitoneally Q7Dx3; or 8 mg/kg
Compound 1 orally QDx5, rest for 3 cycles.
[0024] FIGS. 15A and 15B show NCI-H23 NSCLC human lung carcinoma
xenograft tumor volume and mouse body weight, respectively, over
time in mice administered 10 mg/kg Compound 1 (F-aza-TdCyd)
intraperitoneally QDx5, rest for 3 cycles; 80 mg/kg Compound 1
orally QDx5, rest for 3 cycles; 400 mg/kg Compound 1
intraperitoneally Q7Dx3; 240 mg/kg F-TdCyd intravenously Q7Dx3; 1.5
mg/kg 5-aza-T-dCyd intraperitoneally QDx5, rest for 3 cycles; or
150 mg/kg gemcitabine intraperitoneally Q7Dx3.
[0025] FIGS. 16A and 16B show HL-60 human leukemia xenograft tumor
volume and mouse body weight, respectively, over time mice
administered 10 mg/kg Compound 1 (F-aza-TdCyd) intraperitoneally
QDx5, rest for 3 cycles; 400 mg/kg Compound 1 intraperitoneally
Q7Dx3; 240 mg/kg F-TdCyd intravenously Q7Dx3; 1.5 mg/kg
5-aza-T-dCyd intraperitoneally QDx5, rest for 3 cycles; or 150
mg/kg gemcitabine intraperitoneally Q7Dx3.
DETAILED DESCRIPTION
[0026] Halogenated analogs of 5-aza-2'-deoxycytidine and methods of
using the halogenated analogs are disclosed. In some embodiments,
the compounds are halogenated analogs of
5-aza-4'-thio-2'-deoxycytidine (5-aza-T-dCyd). The disclosed
compounds may be useful for treating diseases characterized at
least in part by the presence of neoplastic cells, such as cancers.
Some embodiments of the disclosed halogenated analogs may be more
efficacious with respect to treating certain cancers, than the
corresponding non-halogenated aza compounds and/or corresponding
halogenated, but non-aza analogs.
I. DEFINITIONS AND ABBREVIATIONS
[0027] The following explanations of terms and abbreviations are
provided to better describe the present disclosure and to guide
those of ordinary skill in the art in the practice of the present
disclosure. As used herein, "comprising" means "including" and the
singular forms "a" or "an" or "the" include plural references
unless the context clearly dictates otherwise. The term "or" refers
to a single element of stated alternative elements or a combination
of two or more elements, unless the context clearly indicates
otherwise.
[0028] Unless explained otherwise, all technical and scientific
terms used herein have the same meaning as commonly understood to
one of ordinary skill in the art to which this disclosure belongs.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present disclosure, suitable methods and materials are described
below. The materials, methods, and examples are illustrative only
and not intended to be limiting. Other features of the disclosure
are apparent from the following detailed description and the
claims.
[0029] Unless otherwise indicated, all numbers expressing
quantities of components, molecular weights, percentages,
temperatures, times, and so forth, as used in the specification or
claims are to be understood as being modified by the term "about."
Accordingly, unless otherwise implicitly or explicitly indicated,
or unless the context is properly understood by a person of
ordinary skill in the art to have a more definitive construction,
the numerical parameters set forth are approximations that may
depend on the desired properties sought and/or limits of detection
under standard test conditions/methods as known to those of
ordinary skill in the art. When directly and explicitly
distinguishing embodiments from discussed prior art, the embodiment
numbers are not approximates unless the word "about" is
recited.
[0030] Although there are alternatives for various components,
parameters, operating conditions, etc. set forth herein, that does
not mean that those alternatives are necessarily equivalent and/or
perform equally well. Nor does it mean that the alternatives are
listed in a preferred order unless stated otherwise.
[0031] Definitions of common terms in chemistry may be found in
Richard J. Lewis, Sr. (ed.), Hawley's Condensed Chemical
Dictionary, published by John Wiley & Sons, Inc., 1997 (ISBN
0-471-29205-2). In order to facilitate review of the various
embodiments of the disclosure, the following explanations of
specific terms are provided:
[0032] Active agent: A drug, medicament, pharmaceutical,
therapeutic agent, nutraceutical, or other compound administered to
a subject to effect a change, such as treatment, amelioration, or
prevention of a disease or disorder or at least one symptom
associated therewith. The term active agent also includes
biological active agents such as proteins, antibodies, antibody
fragments, peptides, oligonucleotides, vaccines, and various
derivatives of such materials.
[0033] Alkyl: A hydrocarbon group having a saturated carbon chain.
The chain may be cyclic, branched or unbranched. Examples, without
limitation, of alkyl groups include methyl, ethyl, propyl, butyl,
pentyl, hexyl, heptyl, octyl, nonyl and decyl.
[0034] Alkoxy: A radical (or substituent) having the structure
--OR, where R is a substituted or unsubstituted alkyl. Methoxy
(--OCH.sub.3) is an exemplary alkoxy group. In a substituted
alkoxy, R is alkyl substituted with a non-interfering
substituent.
[0035] Anomer: An epimer (an isomer having a different
configuration at just one chiral carbon) occurring in cyclic
saccharides.
[0036] Co-administration: The terms "co-administration" and
"co-administering" refer to administration of a compound disclosed
herein with at least one other active agent within the same general
time period, and does not require administration at the same exact
moment in time (although co-administration is inclusive of
administering at the same exact moment in time). Thus,
co-administration may be on the same day or on different days, or
in the same week or in different weeks.
[0037] Effective amount or therapeutically effective amount: An
amount sufficient to provide a beneficial, or therapeutic, effect
to a subject or a given percentage of subjects.
[0038] Inhibit: As used herein, the term "inhibit" means to reduce
or prevent.
[0039] Neoplasia/neoplasm/neoplastic: A neoplasia is an abnormal
growth of tissue or blood cells. The abnormal growth may form a
solid mass or tumor. A neoplasm can be benign, in situ (e.g., a
non-invasive cancer or precancerous neoplasm) or malignant (e.g., a
cancer).
[0040] Pharmaceutically acceptable carrier: The pharmaceutically
acceptable carriers (vehicles) useful in this disclosure are
conventional. Remington: The Science and Practice of Pharmacy, The
University of the Sciences in Philadelphia, Editor, Lippincott,
Williams, & Wilkins, Philadelphia, Pa., 21.sup.st Edition
(2005), describes compositions and formulations suitable for
pharmaceutical delivery of one or more cyanine fluorophores as
disclosed herein.
[0041] In general, the nature of the carrier will depend on the
particular mode of administration being employed. For instance,
parenteral formulations usually comprise injectable fluids that
include pharmaceutically and physiologically acceptable fluids such
as water, physiological saline, balanced salt solutions, aqueous
dextrose, glycerol or the like as a vehicle. In some examples, the
pharmaceutically acceptable carrier may be sterile to be suitable
for administration to a subject (for example, by parenteral,
intramuscular, or subcutaneous injection). In addition to
biologically-neutral carriers, pharmaceutical compositions to be
administered can contain minor amounts of non-toxic auxiliary
substances, such as wetting or emulsifying agents, preservatives,
and pH buffering agents and the like, for example sodium acetate or
sorbitan monolaurate.
[0042] Pharmaceutically acceptable salt: A biologically compatible
salt of a disclosed cyanine fluorophores, which salts are derived
from a variety of organic and inorganic counter ions well known in
the art and include, by way of example only, sodium, potassium,
calcium, magnesium, ammonium, tetraalkylammonium, and the like; and
when the molecule contains a basic functionality, salts of organic
or inorganic acids, such as hydrochloride, hydrobromide, tartrate,
mesylate, acetate, maleate, oxalate, and the like. Pharmaceutically
acceptable acid addition salts are those salts that retain the
biological effectiveness of the free bases while formed by acid
partners that are not biologically or otherwise undesirable, e.g.,
inorganic acids such as hydrochloric acid, hydrobromic acid,
sulfuric acid, nitric acid, phosphoric acid, and the like, as well
as organic acids such as acetic acid, trifluoroacetic acid,
propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic
acid, malonic acid, succinic acid, fumaric acid, tartaric acid,
citric acid, benzoic acid, cinnamic acid, mandelic acid,
methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,
salicylic acid and the like. Pharmaceutically acceptable base
addition salts include those derived from inorganic bases such as
sodium, potassium, lithium, ammonium, calcium, magnesium, iron,
zinc, copper, manganese, aluminum salts and the like. Exemplary
salts are the ammonium, potassium, sodium, calcium, and magnesium
salts. Salts derived from pharmaceutically acceptable organic
non-toxic bases include, but are not limited to, salts of primary,
secondary, and tertiary amines, substituted amines including
naturally occurring substituted amines, cyclic amines and basic ion
exchange resins, such as isopropylamine, trimethylamine,
diethylamine, triethylamine, tripropylamine, ethanolamine,
2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine,
lysine, arginine, histidine, caffeine, procaine, hydrabamine,
choline, betaine, ethylenediamine, glucosamine, methylglucamine,
theobromine, purines, piperazine, piperidine, N-ethylpiperidine,
polyamine resins, and the like. Exemplary organic bases are
isopropylamine, diethylamine, ethanolamine, trimethylamine,
dicyclohexylamine, choline, and caffeine. (See, for example, S. M.
Berge, et al., "Pharmaceutical Salts," J. Pharm. Sci., 1977;
66:1-19, which is incorporated herein by reference.)
[0043] Stereochemistry: The three-dimensional spatial configuration
of a molecule.
[0044] Stereoisomer: Isomers that have the same molecular formula
and sequence of bonded atoms, but which differ only in the
three-dimensional orientation of the atoms in space.
[0045] Subject: An animal or human subjected to a treatment,
observation or experiment.
[0046] Tautomers: Constitutional isomers of organic compounds that
differ only in the position of the protons and electrons, and are
interconvertible by migration of a hydrogen atom. Tautomers
ordinarily exist together in equilibrium.
[0047] Treat/treatment: As used herein, the terms "treat" and
"treatment" mean to inhibit (reduce or prevent) at least one sign
or symptom associated with a condition, i.e., a disorder or
disease. With respect to a tumor, treating may mean reducing or
preventing tumor growth, reducing a tumor volume, and/or reducing
or preventing tumor metastasis. Treatment may, for example, produce
a reduction in severity of some or all clinical symptoms of the
tumor, a slower progression of the tumor (for example by prolonging
the life of a subject having the tumor), a reduction in the number
of tumor reoccurrence, an improvement in the overall health or
well-being of the subject, or by other parameters well known in the
art that are specific to the particular disorder or disease.
II. HALOGENATED ANALOGS OF 5-AZA-2'-DEOXYCYTIDINE
[0048] Halogenated analogs of 5-aza-2'-deoxycytidine and
stereoisomers thereof have a structure according to general formula
I:
##STR00005##
where X is halo (F, Cl, Br, or I), Y is hydrogen, halo (F, Cl, Br,
or I), or deuterium, Z is S or O, each R independently is hydrogen
or deuterium, and R.sup.a is hydrogen, deuterium, alkyl (e.g.,
C.sub.1-5 or C.sub.1-3 alkyl, such as methyl, ethyl, n-propyl, or
isopropyl) alkoxy, amino, or halo. In some embodiments, Z is S. In
any or all embodiments, X may be F. In any or all embodiments, Y
may be hydrogen or F. In any or all embodiments, each R may be
hydrogen. In any or all embodiments, R.sup.a may be hydrogen.
[0049] In some embodiments, the compound is a halogenated analog of
5-aza-4'-thio-2'-deoxycitidine (5-aza-T-dCyd) and has a structure
according to general formula Ia where X, Y, R, and R.sup.a are as
defined above:
##STR00006##
[0050] The halogenated analogs may have a stereochemistry according
to any one of formulas II-V or any combination thereof, where X, Y,
Z, R, and R.sup.a are as defined above:
##STR00007##
[0051] In some embodiments, Z is S, and the compound has a
structure according to any one of formulas IIa-Va, or any
combination thereof:
##STR00008##
[0052] In certain embodiments, the compound is a 0-anomer, i.e., a
compound according to formula II, IIa, III, or Ma.
[0053] Exemplary compounds include, but are not limited to:
##STR00009##
or any combination thereof.
[0054] In some embodiments, the compound is:
##STR00010##
or any combination thereof.
[0055] In certain embodiments, the compound is:
##STR00011##
4-amino-1-(('
3S,4S,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)terthiophen-2-yl)-1,3,5-tri-
azin-2(1H)-one
III. Pharmaceutical Compositions
[0056] This disclosure also includes pharmaceutical compositions
comprising at least one compound as disclosed herein. Some
embodiments of the pharmaceutical compositions include a
pharmaceutically acceptable carrier and at least one compound. The
pharmaceutical compositions can also include one or more additional
active ingredients such as anti-cancer agents, anti-inflammatory
agents, antiviral agents, antimicrobial agents, anesthetics, and
the like. The pharmaceutically acceptable carriers useful for these
formulations are conventional. Remington's Pharmaceutical Sciences,
by E. W. Martin, Mack Publishing Co., Easton, Pa., 19.sup.th
Edition (1995), for example, describes compositions and
formulations suitable for pharmaceutical delivery of the compounds
herein disclosed.
[0057] The compounds according to Formula I disclosed herein can be
administered to subjects by a variety of routes, including by
parenteral, oral, or rectal routes. Parenteral routes include, but
are not limited to, intravenous, intraperitoneal, subcutaneous,
buccal, and sublingual routes. In other alternative embodiments,
the compounds can be administered ex vivo by direct exposure to
cells, tissues or organs originating from a subject. Ex vivo
administration may be useful, for example, to determine whether a
cell (e.g., a cancer cell) is responsive to administration of the
compound.
[0058] The pharmaceutical compositions may be in a dosage unit form
such as an injectable fluid, an oral delivery fluid (e.g., a
solution or suspension), a nasal delivery fluid (e.g., for delivery
as an aerosol or vapor), a semisolid form (e.g., a topical cream),
or a solid form such as powder, pill, tablet, or capsule forms.
[0059] In general, the nature of the carrier will depend on the
particular mode of administration being employed. For instance,
parenteral formulations usually contain injectable fluids that
include pharmaceutically and physiologically acceptable fluids such
as water, physiological saline, balanced salt solutions, aqueous
dextrose, glycerol or the like as a vehicle. For solid compositions
(for example, powder, pill, tablet, or capsule forms), conventional
non-toxic solid carriers can include, for example, pharmaceutical
grades of mannitol, lactose, starch, or magnesium stearate. In
addition to biologically-neutral carriers, pharmaceutical
compositions to be administered can contain minor amounts of
non-toxic auxiliary substances, such as wetting or emulsifying
agents, preservatives, and pH buffering agents and the like, for
example sodium acetate or sorbitan monolaurate.
[0060] To formulate the pharmaceutical compositions, the compounds
can be combined with various pharmaceutically acceptable additives,
as well as a base or vehicle for dispersion of the compound.
Desired additives include, but are not limited to, pH control
agents, such as arginine, sodium hydroxide, glycine, hydrochloric
acid, citric acid, and the like. In addition, isotonizing agents
(for example, sodium chloride, mannitol, sorbitol), adsorption
inhibitors (for example, Tween.RTM. 80 polyethylene sorbitol ester
or Miglyol.RTM. 812 triglycerides), solubility enhancing agents
(for example, cyclodextrins and derivatives thereof), stabilizers
(for example, serum albumin), and reducing agents (for example,
glutathione) can be included. Adjuvants, such as aluminum hydroxide
(for example, Amphogel, Wyeth Laboratories, Madison, N.J.),
Freund's adjuvant, MPL.TM. (3-O-deacylated monophosphoryl lipid A;
Corixa, Hamilton, Ind.) and IL-12 (Genetics Institute, Cambridge,
Mass.), among many other suitable adjuvants well known in the art,
can be included in the compositions. When the composition is a
liquid, the tonicity of the formulation, as measured with reference
to the tonicity of 0.9% (w/v) physiological saline solution taken
as unity, is typically adjusted to a value at which no substantial,
irreversible tissue damage will be induced at the site of
administration. Generally, the tonicity of the solution is adjusted
to a value of about 0.3 to about 3.0, such as about 0.5 to about
2.0, or about 0.8 to about 1.7.
[0061] The compounds can be dispersed in a base or vehicle, which
can include a hydrophilic compound having a capacity to disperse
the compound, and any desired additives. The base can be selected
from a wide range of suitable compounds, including but not limited
to, copolymers of polycarboxylic acids or salts thereof, carboxylic
anhydrides (for example, maleic anhydride) with other monomers (for
example, methyl (meth)acrylate, acrylic acid and the like),
hydrophilic vinyl polymers, such as polyvinyl acetate, polyvinyl
alcohol, polyvinylpyrrolidone, cellulose derivatives, such as
hydroxymethylcellulose, hydroxypropylcellulose and the like, and
natural polymers, such as chitosan, collagen, sodium alginate,
gelatin, hyaluronic acid, and nontoxic metal salts thereof. Often,
a biodegradable polymer is selected as a base or vehicle, for
example, polylactic acid, poly(lactic acid-glycolic acid)
copolymer, polyhydroxybutyric acid, poly(hydroxybutyric
acid-glycolic acid) copolymer and mixtures thereof. Alternatively
or additionally, synthetic fatty acid esters such as polyglycerin
fatty acid esters, sucrose fatty acid esters and the like can be
employed as vehicles. Hydrophilic polymers and other vehicles can
be used alone or in combination, and enhanced structural integrity
can be imparted to the vehicle by partial crystallization, ionic
bonding, cross-linking and the like. The vehicle can be provided in
a variety of forms, including fluid or viscous solutions, gels,
pastes, powders, microspheres and films for direct application to a
mucosal surface.
[0062] The compounds can be combined with the base or vehicle
according to a variety of methods, and release of the compounds can
be by diffusion, disintegration of the vehicle, or associated
formation of water channels. In some circumstances, the compound is
dispersed in microcapsules (microspheres) or nanocapsules
(nanospheres) prepared from a suitable polymer, for example,
isobutyl 2-cyanoacrylate (see, for example, Michael et al., J.
Pharmacy Pharmacol. 43:1-5, 1991), and dispersed in a biocompatible
dispersing medium, which yields sustained delivery and biological
activity over a protracted time.
[0063] The compositions of the disclosure can alternatively
contain, as pharmaceutically acceptable vehicles, substances as
required to approximate physiological conditions, such as pH
adjusting and buffering agents, tonicity adjusting agents, wetting
agents and the like, for example, sodium acetate, sodium lactate,
sodium chloride, potassium chloride, calcium chloride, sorbitan
monolaurate, and triethanolamine oleate. For solid compositions,
conventional nontoxic pharmaceutically acceptable vehicles can be
used which include, for example, pharmaceutical grades of mannitol,
lactose, starch, magnesium stearate, sodium saccharin, talcum,
cellulose, glucose, sucrose, magnesium carbonate, and the like.
[0064] Pharmaceutical compositions for administering the compounds
can also be formulated as a solution, microemulsion, or other
ordered structure suitable for high concentration of active
ingredients. The vehicle can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, liquid polyethylene glycol, and the
like), and suitable mixtures thereof. Proper fluidity for solutions
can be maintained, for example, by the use of a coating such as
lecithin, by the maintenance of a desired particle size in the case
of dispersible formulations, and by the use of surfactants. In many
cases, it will be desirable to include isotonic agents, for
example, sugars, polyalcohols, such as mannitol and sorbitol, or
sodium chloride in the composition. Prolonged absorption of the
compound can be brought about by including in the composition an
agent which delays absorption, for example, monostearate salts and
gelatin.
[0065] In certain embodiments, the compounds can be administered in
a time release formulation, for example in a composition which
includes a slow release polymer. These compositions can be prepared
with vehicles that will protect against rapid release, for example
a controlled release vehicle such as a polymer, microencapsulated
delivery system or bioadhesive gel. Prolonged delivery in various
compositions of the disclosure can be brought about by including in
the composition agents that delay absorption, for example, aluminum
monostearate hydrogels and gelatin. When controlled release
formulations are desired, controlled release binders suitable for
use in accordance with the disclosure include any biocompatible
controlled release material which is inert to the active compound
and which is capable of incorporating the compound and/or other
biologically active agent. Numerous such materials are known in the
art. Useful controlled-release binders are materials that are
metabolized slowly under physiological conditions following their
delivery (for example, at a mucosal surface, or in the presence of
bodily fluids). Appropriate binders include, but are not limited
to, biocompatible polymers and copolymers well known in the art for
use in sustained release formulations. Such biocompatible compounds
are non-toxic and inert to surrounding tissues, and do not trigger
significant adverse side effects, such as nasal irritation, immune
response, inflammation, or the like. They are metabolized into
metabolic products that are also biocompatible and easily
eliminated from the body.
[0066] Exemplary polymeric materials for use in the present
disclosure include, but are not limited to, polymeric matrices
derived from copolymeric and homopolymeric polyesters having
hydrolyzable ester linkages. A number of these are known in the art
to be biodegradable and to lead to degradation products having no
or low toxicity. Exemplary polymers include polyglycolic acids and
polylactic acids, poly(DL-lactic acid-co-glycolic acid),
poly(D-lactic acid-co-glycolic acid), and poly(L-lactic
acid-co-glycolic acid). Other useful biodegradable or bioerodable
polymers include, but are not limited to, such polymers as
poly(epsilon-caprolactone), poly(epsilon-caprolactone-CO-lactic
acid), poly(epsilon.-caprolactone-CO-glycolic acid),
poly(beta-hydroxy butyric acid), poly(alkyl-2-cyanoacrilate),
hydrogels, such as poly(hydroxyethyl methacrylate), polyamides,
poly(amino acids) (for example, L-leucine, glutamic acid,
L-aspartic acid and the like), poly(ester urea),
poly(2-hydroxyethyl DL-aspartamide), polyacetal polymers,
polyorthoesters, polycarbonate, polymaleamides, polysaccharides,
and copolymers thereof. Many methods for preparing such
formulations are well known to those skilled in the art (see, for
example, Sustained and Controlled Release Drug Delivery Systems, J.
R. Robinson, ed., Marcel Dekker, Inc., New York, 1978). Other
useful formulations include controlled-release microcapsules (U.S.
Pat. Nos. 4,652,441 and 4,917,893), lactic acid-glycolic acid
copolymers useful in making microcapsules and other formulations
(U.S. Pat. Nos. 4,677,191 and 4,728,721) and sustained-release
compositions for water-soluble peptides (U.S. Pat. No.
4,675,189).
[0067] The pharmaceutical compositions of the disclosure typically
are sterile and stable under conditions of manufacture, storage and
use. Sterile solutions can be prepared by incorporating the
compound in the required amount in an appropriate solvent with one
or a combination of ingredients enumerated herein, as required,
followed by filtered sterilization. Generally, dispersions are
prepared by incorporating the compound and/or other biologically
active agent into a sterile vehicle that contains a basic
dispersion medium and the required other ingredients from those
enumerated herein. In the case of sterile powders, methods of
preparation include vacuum drying and freeze-drying which yields a
powder of the compound plus any additional desired ingredient from
a previously sterile-filtered solution thereof. The prevention of
the action of microorganisms can be accomplished by various
antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
[0068] In accordance with the various treatment methods of the
disclosure, the compounds can be delivered to a subject in a manner
consistent with conventional methodologies associated with
management of the disorder for which treatment or prevention is
sought. In accordance with the disclosure herein, a
prophylactically or therapeutically effective amount of the
compound(s) is administered to a subject in need of such treatment
for a time and under conditions sufficient to inhibit and/or treat
a disease (e.g., a cancer) or one or more symptom(s) thereof.
[0069] The compounds can be administered to the subject by the oral
route or in a single bolus delivery, via continuous delivery (for
example, continuous transdermal, mucosal or intravenous delivery)
over an extended time period, or in a repeated administration
protocol (for example, by an hourly, daily or weekly, repeated
administration protocol). The therapeutically effective dosages of
the compounds can be provided as repeated doses within a prolonged
prophylaxis or treatment regimen that will yield clinically
significant results to alleviate one or more symptoms or detectable
conditions associated with a targeted condition as set forth
herein. Determination of effective dosages in this context is
typically based on animal model studies followed up by human
clinical trials and is guided by administration protocols that
significantly reduce the occurrence or severity of targeted disease
symptoms or conditions in the subject. Suitable models in this
regard include, for example, murine, rat, avian, porcine, feline,
non-human primate, and other accepted animal model subjects known
in the art.
IV. METHODS OF USE
[0070] In some embodiments, a compound according to formula I, or a
stereoisomer, tautomer, or pharmaceutically acceptable salt
thereof, inhibits a neoplasia when neoplastic cells are contacted
with an effective amount of the compound. Inhibition of a neoplasia
includes reducing a sign or symptom associated with the neoplasia,
such as a rate of growth or proliferation of neoplastic cells
relative to a rate of proliferation in the absence of the compound.
When the neoplasia is a solid tumor, inhibiting the neoplasia may
reduce or prevent tumor growth, reduce tumor volume, and/or reduce
metastasis of the solid tumor.
[0071] Neoplasias may be benign, in situ/precancerous, or
malignant. In some embodiments, the neoplasia is malignant--that
is, a cancer--and contacting the cancer cells with an effective
amount of the compound according to formula I reduces or prevents
proliferation of the cancer cells. Cancers that may be inhibited or
treated by administration of the disclosed compounds include, but
are not limited to, cancers of the kidney (renal cell), bladder,
breast (male and female), colon, endometrium, lung (including
bronchus), skin (including melanoma), blood (e.g., leukemia,
lymphoma (for example, non-Hodgkin's lymphoma), myeloma), pancreas,
prostate, bone, liver, lung, esophagus, and central nervous system
cancers.
[0072] Contacting neoplastic cells with the compound according to
formula I may be performed in vitro, in vivo, or ex vivo. When
ascertaining whether the compound might be effective against a
particular neoplasia, the cells may be contacted in vitro (for
example, in a cell culture) or ex vivo (for example, in a
biological sample obtained from a subject) to determine whether the
compound reduces or prevents cellular growth and/or
proliferation.
[0073] In some embodiments, contacting neoplastic cells with the
effective amount of the compound comprises administering a
therapeutically effective amount of the compound to a subject
having or suspected of having a disease characterized at least in
part by presence of neoplastic cells. The subject may be a mammal,
such as a human or a non-human mammal (for example, a dog, cat,
horse, rabbit, or the like). In certain embodiments, the neoplastic
cells are malignant and the disease is a cancer. In any or all
embodiments, administering the therapeutically effective amount of
the compound to the subject may reduce a sign or symptom of the
disease. In some embodiments, the sign or symptom is a solid tumor
and administering the therapeutically effective amount of the
compound to the subject reduces growth of the solid tumor, reduces
a volume of the solid tumor, reduces metastasis of the solid tumor,
or any combination thereof. In other embodiments, the sign or
symptom may be an abnormal complete blood count and administering
the therapeutically effective amount of the compound to the subject
partially or fully normalizes the complete blood count. Other signs
of symptoms of a disease characterized at least in part by presence
of neoplastic cells include, but are not limited to, coughing,
shortness of breath, coughing up blood, swelling, pain, fever,
chills, frequent infections, itchy skin or rash, loss of appetite,
nausea, night sweats, persistent weakness, fatigue, shortness of
breath, bloating, changes in bowel or bladder habits, constipation,
diarrhea, bloody stools, rectal bleeding, jaundice, abnormal
vaginal bleeding, difficulty swallowing, voice changes, mouth
sores, dry mouth, flu-like symptoms, headaches, easy bruising or
bleeding, enlarged lymph nodes, and changes in appearance of a
mole.
[0074] Administering the therapeutically effective amount of the
compound may comprise administering a pharmaceutical composition
comprising the therapeutically effective amount of the compound to
the subject. Administration may be by any suitable route including,
but not limited to, intravenous, oral, intraperitoneal,
subcutaneous, rectal, or buccal administration.
[0075] The actual dosages of the compounds will vary according to
factors such as the disease indication and particular status of the
subject (for example, the subject's age, size, fitness, extent of
symptoms, susceptibility factors, and the like), time and route of
administration, other drugs or treatments being administered
concurrently, as well as the specific pharmacology of the agent for
eliciting the desired activity or biological response in the
subject. Dosage regimens can be adjusted to provide an optimum
prophylactic or therapeutic response. A therapeutically effective
amount is also one in which any toxic or detrimental side effects
of the compound is outweighed in clinical terms by therapeutically
beneficial effects.
[0076] Alternatively, effective dosages can be determined using in
vitro models. Using such models, only ordinary calculations and
adjustments are required to determine an appropriate concentration
and dose to administer a therapeutically effective amount of the
compound (for example, amounts that are effective to elicit a
desired immune response or alleviate one or more symptoms of a
targeted disease). In alternative embodiments, an effective amount
or effective dose of the compounds may simply inhibit or enhance
one or more selected biological activities correlated with a
disease or condition, as set forth herein, for either therapeutic
or diagnostic purposes.
[0077] A non-limiting range for a therapeutically effective amount
of a compound according to formula I within the methods and
formulations of the disclosure is from 0.0001 grams to 100 grams
for an adult human, such as from 0.001 grams to 50 grams, 0.01
grams to 25 grams, or 0.1 grams to 10 grams. In some embodiments,
the therapeutically effective amount is within a range of from
0.001 mg/kg body weight to 100 mg/kg body weight, such as 0.01
mg/kg body weight to 20 mg/kg body weight, 0.01 mg/kg body weight
to 10 mg/kg body weight 0.05 mg/kg to 5 mg/kg body weight, or 0.1
mg/kg to 2 mg/kg body weight. The therapeutically effective amount
may be administered in a single dose or split into two or more
doses administered over time. In some embodiments, the
therapeutically effective amount of the compound, or a
pharmaceutical composition comprising the compound, is administered
to a subject in a dosing regimen of once, twice, or three times
daily. The dosing regimen may include dosing holidays of 1-14
days.
[0078] Dosage can be varied by the attending clinician to maintain
a desired concentration at a target site (for example, systemic
circulation). Higher or lower concentrations can be selected based
on the mode of delivery, for example, oral delivery versus
intravenous or subcutaneous delivery. Dosage can also be adjusted
based on the release rate of the administered formulation, for
example, of sustained release oral versus injected particulate or
rectal suppository, and so forth.
[0079] In some embodiments, a second active agent may be
co-administered with the compound. The compound and the second
active agent may be administered either separately or together in a
single composition. The second active agent may be administered by
the same route or a different route. The compound and the second
agent may be administered concurrently or at separate times.
Separate administration may be performed in any sequence. If
administered concurrently, the compound and the second active agent
may be combined in a single pharmaceutical composition or may be
administered concurrently as two pharmaceutical compositions. The
second active agent may be, for example, an anti-cancer agent, an
anti-inflammatory agent, an antimicrobial agent, an antiviral
agent, an anesthetic agent, or the like.
[0080] Illustrative anti-cancer agents include, but are not limited
to, abiraterone, actinomycin D, altretamine, amifostine,
anastrozole, asparaginase, bexarotene, bicalutamide, bleomycin,
buserelin, busulfan, carboplatin, carmustine, chlorambucil
cisplatin, cladribine, clodronate, combretastatin A4,
cyclophosphamide, cyproterone, cytarabine, dacarbazine,
daunorubicin, degarelix, diethylstilbestrol, docetaxel,
doxorubicin, duocarmycin DM, epirubicin, ethinyl estradiol,
etoposide, exemestane, 5-fluorouracil, fludarabine, flutamide,
folinic acid, fulvestrant, gemcitabine, goserelin, ibandronic acid,
idarubicin, ifosfamide, irinotecan, lanreotide, lenalidomide,
letrozole, leuprorelin, medroxyprogesterone, megestrol, melphalan,
mesna, methotrexate, octreotide, pamidronate, pemetrexed,
mitocmycin, mitotane, mitoxantrone, oxaliplatin, paclitaxel,
pentastatin, pipbroman, plicamycin, procarbazine, raltitrexed,
stilbestrol, streptozocin, tamoxifen, temozolomide, teniposide,
topotecan, triptorelin, vinblastine, vincristine, vinorelbine, and
zolendronic acid.
V. EXAMPLES
Example 1
Synthesis of
4-amino-1-((2R,3S,4S,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydroth-
iophen-2-yl)-1,3,5-triazin-2(1H)-one
[0081] FIG. 1 is an exemplary synthesis scheme for
4-amino-1-((2R,3S,4S,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydroth-
iophen-2-yl)-1,3,5-triazin-2(1H)-one (Scheme 1). FIG. 2 is an
exemplary synthesis scheme for precursor
4-oxyloxyphenylmethanethiol (Scheme 2).
4-Octyloxyphenylmethanol (13)
##STR00012##
[0083] Preparation: A mixture of 4-hydroxybenzaldehyde (998.0 g,
8.17 mol), 1-bromooctane (1664.2 g, 8.617 mol) and potassium
carbonate (1164.2 g, 8.436 mol) in acetonitrile (8.0 L) was
refluxed overnight and cooled to ambient temperature. The solid was
filtered off, and the filtrate was concentrated under reduced
pressure to give 1959.0 g (102.3%) of crude 4-octyloxybenzaldehyde.
Product was dissolved in methanol (6.0 L) and sodium borohydride
(100.0 g, 2.64 mol) was added portion-wise to the formed solution
while keeping the temperature below 15.degree. C. The reaction
mixture was stirred at ambient temperature for 1 h. A solution of
sodium hydroxide (33.3 g, 832.5 mmol) in water (500 mL) was added,
followed by ethyl acetate (3.0 L) and brine (3.0 L). The organic
solution was separated, dried over sodium sulfate and evaporated
under reduced pressure. Heptane (2 L) was added to the residue and
the formed mixture was cooled to 4.degree. C. The resulting solid
was filtered off, washed with ice-cooled heptane and dried in
vacuum to give 1715.0 g (88.8%) of crude 4-octyloxyphenylmethanol
(13), which was used in the following step without further
purification.
[0084] .sup.1H NMR (300 MHz, CDCl.sub.3/TMS) d 7.26 (d, J=8.7 Hz,
2H); 6.87 (d, J=8.7 Hz, 2H); 4.59 (s, 2H); 3.95 (t, J=6.6 Hz, 2H);
1.73-1.81 (m, 3H); 1.30-1.48 (m, 10H); 0.89 (t, J=6.9 Hz, 3H)
ppm.
4-Octyloxyphenylmethanethiol (14)
##STR00013##
[0086] Preparation: A mixture of 4-octyloxyphenylmethanol (13)
(1716.2 g, 7.26 mol), concentrated HCl (1345 mL, 16.41 mol) and
acetonitrile (3.8 L) was stirred overnight at ambient temperature.
Then, thiourea (663.8 g, 8.72 mol) and acetonitrile (1.3 L) were
added. The mixture was heated to reflux for 2 h, cooled to room
temperature and kept overnight. A solution of sodium hydroxide
(12162.7 g, 29.07 mol) in water (2.3 L) was added. The mixture was
heated to reflux for 3 h and cooled to 10.degree. C. Concentrated
HCl (1.34 L) was added while keeping the temperature below
15.degree. C. The mixture was extracted with methyl t-butyl ether
(7 L). The extract was dried over magnesium sulfate and
concentrated under reduced pressure. Heptane (2.2 L) was added to
the residue, and the mixture was evaporated. Heptane (3.4 L) was
again added to the residue. The formed milky solution was kept
overnight and filtered through a silica gel pad (1.0 kg). The
filtrate was evaporated to give 1636.0 g (89.3%) of
4-octyloxyphenylmethanethiol (14).
[0087] .sup.1H NMR (300 MHz, CDCl.sub.3/TMS) d 7.23 (d, J=8 Hz,
2H); 6.83 (d, J=8 Hz, 2H); 3.95 (m, 2H); 3.72 (d, J=7.5 Hz, 2H);
1.75 (m, 3H); 1.25 (m, 9H); 0.87 (m, 3H) ppm.
((2R,3R,4S,5S)-3-(Benzoyloxy)-4-fluoro-5-hydroxytetrahydrofuran-2-yl)methy-
l benzoate (3)
##STR00014##
[0089] Preparation: To a solution of
(2R,3S,4R,5R)-5-((benzoyloxy)methyl)-3-fluorotetrahydrofuran-2,4-diyl
dibenzoate (2) (176 g, 379 mmol) in dichloromethane (600 mL) in a
3000 mL one neck round bottom flask under nitrogen was added
hydrobromic acid, 33% in acetic acid (131 mL, 796 mmol) and the
reaction was stirred 6 hours at rt. The solution was poured into a
vigorously stirred mixture of 1200 mL saturated sodium bicarbonate,
solid sodium bicarbonate (121 g, 1440 mmole) and 1200 mL
dichloromethane held at 0.degree. C. The layers were separated and
the organic layer was washed with 1.times.100 mL saturated sodium
bicarbonate, dried over anhydrous magnesium sulfate, and
concentrated in vacuo to provide 136 g (84%) of the intermediate
bromide as a pale yellow oil. .sup.1H NMR (400 MHz, Chloroform-d)
.delta. 8.14-7.99 (m, 3H), 7.66-7.45 (m, 2H), 7.50-7.43 (m, 1H),
7.48-7.37 (m, 1H), 6.62 (m, 1H), 5.59-5.47 (m, 1H), 4.87-4.64 (m,
2H) ppm.
[0090] The crude bromide was dissolved in DMF (600 mL) in a 3000 mL
one-neck round-bottom flask under ambient atmosphere. The solution
was treated with triethylamine (317 mL, 2274 mmol) and water (205
mL, 1.14E+04 mmol) and the reaction was stirred at room
temperature. The reaction showed an exotherm from 23 to 37.degree.
C. upon addition of water/triethylamine mixture. The reaction was
stirred for 1 h and then it was diluted with 2400 mL ethyl acetate.
After stirring vigorously with 1200 mL 50% saturated sodium
chloride, the layers were separated, and the organic layer was
washed with 3.times.500 mL 50% saturated sodium chloride. The
organic layer was filtered through anhydrous magnesium sulfate and
the filtrate was concentrated in vacuo to afford 116 grams (85%) of
the title compound (3) as a viscous amber oil. It was elected to
use the crude oil directly, without further purification.
[0091] NMR (400 MHz, Chloroform-d) .delta. 8.07-8.02 (m, 1H),
8.06-7.99 (m, 2H), 8.04-7.91 (m, 1H), 7.63-7.28 (m, 6H), 5.66 (dd,
J=10.4, 0.9 Hz, 1H), 5.46 (ddd, J=22.3, 4.5, 1.0 Hz, 1H), 5.21-5.06
(m, 1H), 4.78-4.54 (m, 4H) ppm.
(2R,3R,4S)-4-Fluoro-2-hydroxy-5,5-bis((4-(octyloxy)benzyl)thio)pentane-1,3-
-diyl dibenzoate (4)
##STR00015##
[0093] Preparation: A 2,000 mL one-neck round-bottom flask under
nitrogen was charged with
((2R,3R,4S,5S)-3-(benzoyloxy)-4-fluoro-5-hydroxytetrahydrofuran-2-yl)meth-
yl benzoate (3) (58 g, 161 mmol) and
(4-(octyloxy)phenyl)methanethiol (102 g, 402 mmol) (14) in 500 mL
dichloromethane. The solution was cooled to 0.degree. C. and was
treated dropwise with boron trifluoride diethyletherate (39.7 mL,
322 mmol) in 50 mL dichloromethane. The reaction was stirred for
1.5 h at 0.degree. C. and was added rapidly drop-wise to an
ice-cooled solution of potassium carbonate (150 g, 1.08 moles) in
600 mL water layered with 200 mL dichloromethane. The mixture was
filtered through celite 503, and the layers were separated. The
organic layer was dried over anhydrous magnesium sulfate, and the
filtrate was concentrated in vacuo to an amber oil. The oil was
diluted with hexane, loaded onto a 125 G RediSep.RTM. Rf column
(Teledyne ISCO) and was eluted over a 340 G UltraSil SNAP cartridge
(Biotage, Charlotte, N.C.) with a 0-20% ethyl acetate/hexane
gradient into 27 mL fractions on a Biotage.RTM. system. Fractions
28-100 were combined and concentrated to afford 85 g (62%) of the
title compound (4) as a golden oil.
[0094] .sup.1H NMR (400 MHz, Chloroform-d): .delta. 8.03-7.93 (m,
2H), 7.94-7.86 (m, 2H), 7.61-7.49 (m, 2H), 7.40 (m, 4H), 7.19-7.10
(m, 2H), 6.97-6.88 (m, 2H), 6.71-6.62 (m, 2H), 6.57-6.48 (m, 2H),
5.65 (ddd, J=26.9, 7.7, 1.7 Hz, 1H), 5.01 (ddd, J=45.2, 9.1, 1.8
Hz, 1H), 4.45 (dd, J=11.7, 2.9 Hz, 1H), 4.34-4.17 (m, 2H),
3.97-3.62 (m, 8H), 2.73 (d, J=6.2 Hz, 1H), 1.80-1.65 (m, 4H),
1.49-1.32 (m, 4H), 1.37-1.20 (m, 17H), 0.92-0.81 (m, 6H).
(2S,3R,4S)-2-(2-chloroacetoxy)-4-fluoro-5,5-bis((4-(octyloxy)benzyl)thio)p-
entane-1,3-diyl dibenzoate (5)
##STR00016##
[0096] Preparation: An oven-dried 1,000 mL three-neck round-bottom
flask under nitrogen was charged with triphenylphosphine (38.7 g,
148 mmol) and 340 mL anhydrous THF. The solution was cooled to
-15.degree. C. and was treated dropwise with 40 wt % diethyl
azodicarboxylate in toluene (67.2 mL, 148 mmol). The mixture was
stirred 1 h as it became heavy with precipitate. The slurry was
treated rapidly drop-wise with
(2R,3R,4S)-4-fluoro-2-hydroxy-5,5-bis((4-(octyloxy)benzyl)thio)pentane-1,-
3-diyl dibenzoate (4) (25 g, 29.5 mmol) in 60 mL anhydrous THF. The
resulting suspension was treated dropwise with chloroacetic acid
(13.94 g, 148 mmol) in 100 mL anhydrous THF. The reaction became
briefly homogeneous and then slurried as a white solid precipitated
out. The reaction was stirred overnight as the cooling bath
expired. The volatiles were removed in vacuo to give a pale yellow
paste. The paste was slurried with 900 mL 2:1 hexane/tBuOMe (methyl
tert-butyl ether) and the insoluble material was removed by
filtration. The filtrate was concentrated in vacuo to give 48 g a
crude yellow oil. The crude material was dissolved in hexane with a
small amount of dichloromethane, was loaded onto a 100 G UltraSil
SNAP cartridge and was eluted over a 120 g Zip.RTM. Sphere
cartridge (Biotage, Charlotte, N.C.) with a 0-20% ethyl
acetate/hexane gradient on a Biotage.RTM. system. Fractions 4-21
were combined and concentrated to afford 21.70 g (80%) of the title
compound (5) as a yellow oil.
[0097] .sup.1H NMR (400 MHz, Chloroform-d) .delta. 8.02-7.86 (m,
4H), 7.63-7.49 (m, 2H), 7.48-7.33 (m, 4H), 7.19-7.11 (m, 2H),
6.97-6.86 (m, 2H), 6.70-6.60 (m, 2H), 6.63-6.52 (m, 2H), 5.92 (ddd,
J=25.5, 6.2, 2.4 Hz, 1H), 5.54 (td, J=6.3, 3.2 Hz, 1H), 4.78 (ddd,
J=46.0, 8.5, 2.5 Hz, 1H), 4.61-4.44 (m, 2H), 4.37 (ddd, J=12.5,
6.3, 1.2 Hz, 1H), 4.02-3.64 (m, 10H), 1.81-1.66 (m, 4H), 1.49-1.36
(m, 4H), 1.41-1.20 (m, 16H), 0.99-0.80 (m, 6H).
(2S,3R,4S)-4-Fluoro-2-hydroxy-5,5-bis((4-(octyloxy)benzyl)thio)pentane-1,3-
-diyl dibenzoate (6)
##STR00017##
[0099] Preparation: A 1000 mL one-neck round-bottom flask under
nitrogen at ambient temperature was charged with
(2S,3R,4S)-2-(2-chloroacetoxy)-4-fluoro-5,5-bis((4-(octyloxy)benzyl)thio)-
pentane-1,3-diyl dibenzoate (5) (21.70 g, 23.49 mmol),
dichloromethane (205 mL) and methanol (240 mL). The yellow solution
was treated with thiourea (17.88 g, 235 mmol) which caused rapid
decolorization of the yellow mixture as the suspension thinned and
then became heavier. The suspension was treated dropwise with
2,6-lutidine (2.74 mL, 23.49 mmol) in 35 mL dichloromethane. The
heavy suspension gradually achieved near homogeneity within 15
minutes of the lutidine addition. Stirring was continued overnight
at room temperature for a total of 23 hours. The reaction was
neutralized with citric acid (4.51 g, 23.49 mmol) in water (52 mL),
the mixture was diluted with 1 L dichloromethane, and the layers
were separated. The organic layer was washed with 1.times.250 mL
1:115% aqueous citric acid and saturated sodium chloride. The
layers were separated and the organic layer was dried over
anhydrous magnesium sulfate, and concentrated in vacuo to give 21 g
of a pale paste. The organic component of the residue was dissolved
in 1:3 dichloromethane/hexane, was loaded onto a 100 G UltraSil
SNAP cartridge, and was eluted over a 120 G Zip.RTM. Sphere
cartridge with a 0-20% ethyl acetate/hexane gradient into 27 mL
fractions on a Biotage.RTM. system. Fractions 24-54 were combined
and concentrated to afford 16.98 g (85%) of the title compound (6)
as a pale oil.
[0100] 1H NMR (400 MHz, Chloroform-d) .delta. 8.03-7.92 (m, 4H),
7.60-7.49 (m, 2H), 7.45-7.34 (m, 4H), 7.12-6.98 (m, 4H), 6.74-6.59
(m, 4H), 5.72 (m, 1H), 5.02 (ddd, J=46.6, 6.3, 4.4 Hz, 1H),
4.40-4.28 (m, 2H), 3.94 (m, 1H), 3.89-3.67 (m, 8H), 2.64 (d, J=6.4
Hz, 1H), 1.71 (m, 4H), 1.49-1.38 (m, 4H), 1.41-1.20 (m, 18H),
0.99-0.80 (m, 6H).
(2S,3R,4S)-4-Fluoro-2-((methylsulfonyl)oxy)-5,5-bis((4-(octyloxy)benzyl)th-
io)pentane-1,3-diyl dibenzoate (7)
##STR00018##
[0102] Preparation: A 1000 mL one-neck round bottom flask under
nitrogen was charged with
(2S,3R,4S)-4-fluoro-2-hydroxy-5,5-bis((4-(octyloxy)benzyl)thio)pentane-1,-
3-diyl dibenzoate (6) (16.95 g, 20.01 mmol) and dichloromethane
(200 mL). The solution was cooled to 0.degree. C., treated dropwise
with methanesulfonyl chloride (2.03 mL, 26.0 mmol) in 20 mL
dichloromethane, followed by drop-wise addition of triethylamine
(3.63 mL, 26.0 mmol) in 20 mL dichloromethane. The reaction was
stirred for 72 h as the cooling bath expired. The mixture was
washed successively with 1.times.100 mL water, 1.times.100 mL
saturated sodium bicarbonate, and 1.times.100 mL 1:1 saturated
sodium chloride/15% aqueous citric acid. The organic layer was
dried over anhydrous magnesium sulfate and concentrated in vacuo to
give 17.99 g (97%) of the title compound as a viscous amber oil.
Material was used without further purification.
[0103] .sup.1H NMR (400 MHz, Chloroform-d) .delta. 7.99 (m, 4H),
7.70-7.49 (m, 2H), 7.49-7.35 (m, 4H), 7.21-7.10 (m, 2H), 7.01-6.85
(m, 2H), 6.77-6.64 (m, 2H), 6.62-6.50 (m, 2H), 5.92 (m, 1H),
5.22-5.14 (m, 1H), 4.85 (m, 1H), 4.68-4.56 (m, 1H), 4.44 (m, 1H),
3.93-3.62 (m, 10H), 2.93 (s, 3H), 1.72 (h, J=6.8 Hz, 4H), 1.50-1.18
(m, 19H), 0.98-0.84 (m, 6H). .sup.19F NMR (376 MHz, Chloroform-d)
.delta. -188.51 (ddd, J=45.7, 25.7, 11.3 Hz).
((2R,3S,4S)-3-(benzoyloxy)-4-fluoro-5-((4-(octyloxy)benzyl)thio)tetrahydro-
thiophen-2-yl)methyl benzoate (8)
##STR00019##
[0105] Preparation: A 3000 mL three-neck round-bottom flask under
nitrogen was charged with
(2S,3R,4S)-4-fluoro-2-((methylsulfonyl)oxy)-5,5-bis((4-(octyloxy)benzyl)t-
hio)pentane-1,3-diyl dibenzoate (7) (59 g, 63.8 mmol), anhydrous
acetonitrile (1000 mL), triethylamine (17.78 mL, 128 mmol), and
tetrabutylammonium iodide (47.1 g, 128 mmol). The reaction mixture
was heated to reflux for 96 h. The reaction was allowed to cool to
rt and the mixture was then poured into 400 mL silica gel (230-400
mesh) and concentrated to dryness. The plug was chromatographed
over a 340 G UltraSil SNAP cartridge, eluting with a 0-15% ethyl
acetate/hexane gradient on a Biotage.RTM. system. Fractions 28-69
were combined and concentrated to give 27.6 g (71%) of the title
compound (8) as a viscous oil that crystallized under vacuum to
give a cream colored solid. .sup.19F-NMR indicated a 15:1 C-1
.alpha./.beta. mixture.
[0106] .sup.1H NMR (400 MHz, Chloroform-d) .delta. 8.02-7.90 (m,
3H), 7.65-7.47 (m, 2H), 7.47-7.33 (m, 4H), 7.29-7.19 (m, 3H),
6.86-6.76 (m, 2H), 5.97 (ddd, J=9.9, 5.0, 3.9 Hz, 1H), 5.15 (dt,
J=50.5, 4.7 Hz, 1H), 4.69-4.42 (m, 3H), 3.97-3.82 (m, 4H),
3.80-3.69 (m, 1H), 1.75 (m, 2H), 1.50-1.19 (m, 10H), 0.95-0.82 (m,
3H). .sup.19F NMR (376 MHz, Chloroform-d) -187.14 (ddd, J=50.5,
16.6, 9.9 Hz).
(2R,3S,4S)-4-Fluoro-2-(hydroxymethyl)-5-((4-(octyloxy)benzyl)thio)tetrahyd-
rothiophen-3-ol (9)
##STR00020##
[0108] Preparation: 7M Ammonia in methanol (MeOH) (187 mL, 1310
mmol) was added to a solution of
((2R,3S,4S)-3-(benzoyloxy)-4-fluoro-5-((4-(octyloxy)benzyl)thio)tetrahydr-
othiophen-2-yl)methyl benzoate (8) (10 g, 16.37 mmol) in
dichloromethane (50 mL, 16.37 mmol) and the resulting mixture was
stirred at rt. After stirring for 65 h at room temperature the
volatiles were removed in vacuo to afford a pasty solid. The solid
was stirred with hexane (60 mL) for 1 h at room temperature. The
fine white solid was collected, washed with hexane, and dried on
the filter to afford 6.60 g (100%) of the title compound (9) as a
white solid.
[0109] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 7.24-7.16 (m,
2H), 6.90-6.81 (m, 2H), 5.68 (d, J=5.1 Hz, 1H), 5.00 (t, J=5.5 Hz,
1H), 4.85 (dt, J=51.4, 4.9 Hz, 1H), 4.49 (dd, J=16.1, 4.5 Hz, 1H),
4.20 (m, 1H), 3.92 (t, J=6.5 Hz, 2H), 3.91-3.75 (m, 2H), 3.68 (m,
1H), 3.40-3.28 (m, 1H), 3.09 (dddd, J=7.6, 6.1, 4.7, 0.9 Hz, 1H),
1.68 (m, 2H), 1.46-1.33 (m, 2H), 1.28 (m, 8H), 0.90-0.82 (m,
3H).
[0110] .sup.13C NMR (101 MHz, DMSO-d.sub.6) .delta. 158.15, 131.59,
130.42, 129.75, 128.59, 127.86, 114.79, 99.37, 97.49, 75.29, 75.06,
67.81, 64.27, 64.25, 53.22, 53.19, 50.32, 50.13, 36.00, 35.97,
31.67, 29.18, 29.13, 29.10, 25.97, 22.51, 14.38.
[0111] .sup.19F-NMR (400 MHz, DMSO-d.sub.6) .delta. -186.62 (dddd,
1F)
(3S,4S,5R)-3-Fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrothiophen-2-yl
acetate (10)
##STR00021##
[0113] Preparation: A 500 mL one-neck round bottom flask under
nitrogen was charged with
(2R,3S,4S)-4-fluoro-2-(hydroxymethyl)-5-((4-(octyloxy)benzyl)thio)tetrahy-
drothiophen-3-ol (37.5 g, 93 mmol) and acetic acid (225 mL, 3972
mmol). The colorless liquid suspension was treated with
diacetoxymercury (44.5 g, 140 mmol) in a single portion and the
reaction mixture was stirred for 24 h at room temperature. The
mixture was diluted with 800 mL heptane and the insoluble material
was removed by filtration. The filter cake was washed with 300 mL
dichloromethane (DCM) followed by 600 mL heptane and the filtrate
was concentrated in vacuo to a pale amber oil. The crude material
was taken up in a minimum amount of DCM, was loaded onto a 50 G
UltraSIL.RTM. SNAP.RTM. cartridge (Biotage, Uppsala, Sweden) and
was eluted over a 100 G UltraSIL.RTM. SNAP.RTM. cartridge and with
a 0-50% ethyl acetate/DCM gradient. Fractions 27-92 were combined
and concentrated to afford 17.59 g (90%) of the title compound (10)
as a 1:1 mixture of anomers.
[0114] .sup.1H NMR (400 MHz, Chloroform-d) .delta. 6.02-5.88 (m,
1H), 5.09 (dd, J=7.1, 4.5 Hz, 0.2H), 5.00-4.92 (m, 0.5H), 4.83 (dd,
J=9.3, 4.7 Hz, 0.3H), 4.50 (ddd, J=12.1, 9.3, 8.0 Hz, 0.5H), 4.33
(dt, J=14.7, 7.5 Hz, 0.5H), 3.87-3.68 (m, 2H), 3.54 (dtd, J=7.9,
5.1, 1.3 Hz, 0.5H), 3.24 (ddd, J=7.9, 5.3, 4.5 Hz, 0.5H), 2.88 (s,
1H), 2.11 (m, 3H).
[0115] .sup.19F NMR (376 MHz, Chloroform-d) .delta. -188.81 (dt,
J=51.3, 15.2 Hz), -193.39 (dd, J=50.9, 12.2 Hz).
(3S,4S,5R)-4-((tert-butyldiphenylsilyl)oxy)-5-(((tert-butyldiphenylsilyl)o-
xy)methyl)-3-fluorotetrahydrothiophen-2-yl acetate (11)
##STR00022##
[0117] Preparation:
(3S,4S,5R)-3-Fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrothiophen-2-yl
acetate (10) (11.85 g, 56.4 mmol) was dissolved in DMF (180 mL) in
a 1,000 mL one neck round bottom flask under nitrogen. The solution
was treated with imidazole (9.59 g, 141 mmol) followed by
tert-butyldiphenylchlorosilane (43.4 mL, 169 mmol) and the reaction
mixture was stirred overnight at room temperature. The reaction was
diluted with 1,000 mL ethyl acetate and extracted with 1.times.500
mL 50% saturated sodium chloride followed by 3.times.150 mL 50%
saturated sodium chloride. The organic layer was dried over
anhydrous magnesium sulfate and concentrated in vacuo to a
colorless oil. The crude material was dissolved in a minimum amount
of hexane, was loaded onto a 340 G UltraSil SNAP cartridge, and was
eluted with a 0-15% ethyl acetate/hexane gradient into 27 mL
fractions on a Biotage.RTM. system. Fractions 27-66 were combined
and concentrated to afford 36.7 g (95%) of the title compound (11)
as a colorless oil.
[0118] .sup.1H NMR (400 MHz, Chloroform-d) .delta. 7.78-7.68 (m,
4H), 7.65-7.46 (m, 4H), 7.46-7.20 (m, 12H), 6.07-5.87 (m, 1H),
5.15-4.86 (m, 1H), 4.37-4.19 (m, 1H), 3.81 (qd, J=6.1, 3.1 Hz,
0.4H), 3.64 (m, 1H), 3.41 (m, 1H), 3.25 (ddd, J=24.0, 10.3, 8.5 Hz,
1H), 2.17 (s, 2H), 1.88 (s, 1H), 1.07 (s, 9H), 0.94 (s, 9H).
.sup.19F NMR (376 MHz, Chloroform-d) .delta. -184.98 (dt, J=49.3,
14.2 Hz), -190.33--190.64 (m).
4-Amino-1-((2R,3S,4S,5R)-4-((tert-butyldiphenylsilyl)oxy)-5-(((tert-butyld-
iphenylsilyl)oxy)methyl)-3-fluorotetrahydrothiophen-2-yl)-1,3,5-triazin-2(-
1H)-one (12)
##STR00023##
[0120] Preparation: HMDS (21.97 mL, 105 mmol) was added to
azacytosine (2.91 g, 26.2 mmol) and ammonium sulfate (0.115 g,
0.873 mmol), and the resulting mixture was stirred at 130.degree.
C. for 20 h. Excess HMDS was removed under reduced pressure and the
remaining residue was suspended in 1,2-dichloroethane (50 mL).
Meanwhile, hydrobromic acid (33% in acetic acid) (3.45 mL, 20.96
mmol) was added to a solution of
(3S,4S,5R)-4-((tert-butyldiphenylsilyl)oxy)-5-(((tert-butyldiphenylsilyl)-
oxy)methyl)-3-fluorotetrahydrothiophen-2-yl acetate (11) (6 g, 8.73
mmol) in 1,2-dichloroethane (50 mL) and the resulting mixture was
stirred at 0.degree. C. for 2 h. The reaction was quenched with the
addition of 75 mL saturated sodium bicarbonate and the mixture was
stirred vigorously for 15 min. The layers were separated and the
organic layer was dried over anhydrous magnesium sulfate. The
solution of intermediate bromide was added to the suspension of
silylated azacytosine in a single portion and the white suspension
was warmed to 84.degree. C. The suspension became a light slurry as
the reaction came to temperature. The reaction was stirred for 5 h
at 84.degree. C. The reaction was cooled to rt overnight, diluted
with 75 mL saturated sodium bicarbonate, and stirred for 10
minutes. The slurry was filtered through a bed of Celite.RTM.
diatomaceous earth and the filter pad was washed with fresh
dichloromethane. The layers were separated and the organic layer
was dried over anhydrous magnesium sulfate, and concentrated in
vacuo to give 5.3 g of an off-white foam. The crude material was
dissolved in a minimum amount of dichloromethane, was loaded onto a
50 G UltraSil SNAP cartridge, and was eluted into 27 mL fractions
with a 10-65% ethyl acetate/dichloromethane gradient on a
Biotage.RTM. system. Fractions 19-33 were combined and concentrated
to afford 2.7 g (42%) of a white foam. H-NMR indicated the material
is a 2:1 .beta./.alpha. mixture.
[0121] The white foam was dissolved in 10 mL absolute ethanol
(EtOH) and was stirred for 2 h at rt as a fine white solid
crystallized from the mixture. The solid was collected, washed with
a small amount of EtOH, and dried on the filter to give 630 mg (9%)
of pure C-1 alpha anomer as a white solid. The mother liquor was
concentrated in vacuo to give 2.07 grams (32%) of the title
compound 12 as a white foam (>20:1 C-1 Beta anomer). This
material was used without additional fractionation.
[0122] Proton, C-1 Alpha Anomer: .sup.1H NMR (400 MHz,
Chloroform-d) .delta. 8.85 (s, 1H), 7.53 (ddd, J=14.6, 8.0, 4.2 Hz,
6H), 7.48-7.25 (m, 14H), 6.75 (s, 1H), 6.10 (dd, J=14.0, 1.8 Hz,
1H), 5.72 (s, 1H), 5.09 (dt, J=46.7, 2.0 Hz, 1H), 4.30 (m, 1H),
3.97-3.88 (m, 1H), 3.47 (dd, J=10.2, 6.6 Hz, 1H), 3.43-3.33 (m,
1H), 0.96 (m, 18H).
[0123] Fluorine, C-1 Alpha Anomer: .sup.19F NMR (376 MHz,
Chloroform-d) .delta. -177.38 (dt, J=46.8, 12.5 Hz).
[0124] Proton, C-1 Beta Anomer: .sup.1H NMR (400 MHz, Chloroform-d)
.delta. 8.41 (d, J=2.6 Hz, 1H), 7.68-7.52 (m, 6H), 7.57-7.42 (m,
4H), 7.47-7.28 (m, 10H), 7.04-6.99 (m, 1H), 6.80 (dd, J=24.5, 3.6
Hz, 1H), 5.69 (s, 1H), 4.81 (dt, J=50.8, 2.8 Hz, 1H), 4.47 (m, 1H),
3.70-3.65 (m, 1H), 3.60-3.44 (m, 2H), 1.10 (s, 9H), 0.89 (s,
9H).
[0125] Fluorine, C-1 Beta Anomer: .sup.19F NMR (376 MHz,
Chloroform-d) .delta. -194.16 (ddd, J=50.5, 24.6, 7.4 Hz).
4-Amino-1-((2R,3S,4S,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrothi-
ophen-2-yl)-1,3,5-triazin-2(1H)-one (1)
##STR00024##
[0127] Preparation 1:
4-Amino-1-((2R,3S,4S,5R)-4-((tert-butyldiphenylsilyl)oxy)-5-(((tert-butyl-
diphenylsilyl)oxy)methyl)-3-fluorotetrahydrothiophen-2-yl)-1,3,5-triazin-2-
(1H)-one (12) (5.9 g, 7.98 mmol) was dissolved in tetrahydrofuran
(45 mL, 555 mmol) in a 200 mL one-neck round-bottom flask under
nitrogen. The solution was treated with tetra-n-butylammonium
fluoride on silica gel (21.16 g, 29.1 mmol) and was stirred at rt
for 1 h. The reaction mixture was treated with 18 g silica gel
(230-400 mesh) and concentrated to dryness. The solid plug was
chromatographed over a 50 gram UltraSil.RTM. SNAP.RTM. cartridge
while eluting with a 0-12.5% (10% MeOH in IPA)/dichloromethane
gradient (0-50% of a 25% (10% MeOH in IPA)/dichloromethane polar
phase) into 27 mL fractions on a Biotage.RTM. system. Fractions
33-84 were combined and concentrated to afford 1.83 g of a pasty
solid. The solid was triturated with 20 mL acetonitrile, was
collected by filtration and was dried on the filter to afford 1.48
g (71%) of the title compound (1) an off-white solid.
[0128] .sup.1H NMR (400 MHz, DMSO-d6) .delta. 8.78 (d, J=1.3 Hz,
1H), 7.68-7.60 (m, 2H), 6.29 (dd, J=11.6, 5.4 Hz, 1H), 5.92 (d,
J=5.2 Hz, 1H), 5.34 (t, J=5.2 Hz, 1H), 5.00 (dt, J=50.7, 5.8 Hz,
1H), 4.27 (m, 1H), 3.68 (m, 2H), 3.23 (q, J=5.5 Hz, 1H) ppm.
.sup.13C NMR (101 MHz, dmso) .delta. 165.87, 158.07, 158.04,
153.63, 96.79, 94.87, 73.44, 73.21, 61.28, 61.26, 57.44, 57.27,
51.97, 51.93. .sup.19F NMR (376 MHz, DMSO-d6) .delta. -192.78 (dt,
J=50.6, 11.7 Hz). Melting Point: 208-209.degree. C., d. Combustion
Analysis: Calculated: C, 36.64; H, 4.23; F, 7.24; N, 21.36; S,
12.22. Found: C, 36.66; H, 4.14; F, 7.04; N, 21.08; S, 12.27.
[0129] Preparation 2:
4-Amino-1-((2R,3S,4S,5R)-4-((tert-butyldiphenylsilyl)oxy)-5-(((tert-butyl-
diphenylsilyl)oxy)methyl)-3-fluorotetrahydrothiophen-2-yl)-1,3,5-triazin-2-
(1H)-one (12) (12.2 g, 16.51 mmol) was dissolved in methanol
(anhydrous) (110 mL, 16.51 mmol) in a 200 mL one-neck round bottom
flask under nitrogen. The solution was treated with ammonium
fluoride (5.20 g, 140 mmol) and was stirred at 60.degree. C.
Thin-layer chromatography (30% MeOH/DCM) after 2 h @ 60.degree. C.
showed complete consumption of starting material. The mixture was
warmed to 60.degree. C. for a total of 3 h. Reaction was cooled to
room temperature, was combined with 12 g silica gel (230-400 mesh),
and the mixture was concentrated to dryness. The crude plug was
eluted over a 25 G UltraSil.RTM. SNAP.RTM. cartridge with a 5-25%
MeOH/DCM gradient into 27 mL fractions. Fractions 12-66 were
combined and concentrated to afford 4.8 g of a white solid. The
solid was triturated with 20 mL absolute EtOH for 1 h and the white
solid was collected, washed with MeOH followed by diethyl ether and
was dried on the filter overnight to afford 2.40 g (55%) of the
title compound (1) as a fine white solid. The mother liquor was
concentrated in vacuo to give 2.05 g of a white foam that appeared
to contain primarily the decomposition product of the title
compound by TLC and LCMS. This residue was dissolved in MeOH, was
treated with 10 mL Florisil.RTM. adsorbent (60-100 mesh, Sigma
Aldrich), and was concentrated to dryness. The plug was
chromatographed over a 25 G UltraSil.RTM. SNAP.RTM. cartridge,
eluting with a 5-25% MeOH/DCM gradient into 27 mL fractions.
Fractions 15-22 were combined and concentrated to give 360 mg of a
white foam. Crystallization of the foam from MeCN afforded an
additional 264 mg (6%) of the title compound (1) as a fine white
solid.
[0130] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 8.74 (d, J=1.3
Hz, 1H), 7.64-7.57 (m, 2H), 6.23 (dd, J=11.5, 5.4 Hz, 1H), 5.89 (d,
J=5.2 Hz, 1H), 5.31 (t, J=5.2 Hz, 1H), 4.96 (dt, J=50.7, 5.9 Hz,
1H), 4.22 (dq, J=11.5, 5.7 Hz, 1H), 3.71-3.55 (m, 2H), 3.17 (q,
J=5.5 Hz, 1H). .sup.13C NMR (101 MHz, DMSO-d.sub.6) .delta. 165.88,
158.08, 158.05, 153.65, 96.78, 94.86, 73.42, 73.19, 61.26, 57.43,
57.26, 51.94, 51.90 ppm.
[0131] .sup.19F NMR (376 MHz, DMSO-d.sub.6) .delta. -192.77 (dt,
J=50.8, 11.7 Hz).
[0132] LC/MS: [M+H]=263.1, 98.2% purity.
Example 2
NCI60 Human Tumor Cell Line Anticancer Drug Screen
[0133] Embodiments of the disclosed compounds may be screened for
anticancer activity using the NCI60 human tumor cell line
anticancer drug screen (see, e.g., Shoemaker, Nature Reviews Cancer
2006, 6:813-823;
https://dtp.cancer.gov/discovery_development/nci-60/handling.htm).
Standard Operating Procedures for Sample Preparation:
[0134] General: NCI60 testing is performed in two parts: first a
single concentration is tested in all 60 cell lines at a single
dose of 10.sup.-5 molar or 15 .mu.g/ml. If the results obtained
meet selection criteria, then the compound is tested again in all
60 cell lines in 5.times.10 fold dilutions with the top dose being
10.sup.-4 molar or 150 .mu.g/ml. Compounds accepted for NCI60
testing are prepared for both 1-dose and 5-dose testing at the same
time.
[0135] Agents Received: Synthetics and pure compounds with known
molecular weight and macromolecules and compounds without molecular
weights--Aliquots of agents identified for testing are weighed and
transferred into pre-weighed glass vials. Compounds are solubilized
in these vials. Except when specifically noted, all agents are
stored in a -70.degree. C. freezer. Crude Natural Products--crude
natural product extracts are plated on detachable polypropylene
(PP) 96-well microtiter plates. Plates are dried and stored at
-20.degree. C. until called up for 1-dose testing. Based on the
results of the 1-dose testing, those samples selected for 5-dose
testing are rearranged from 88 wells to 72 wells per 96 well plate
with a column of standard agent, Adriamycin, NSC 123127, and a new
platemap is created and uploaded for assignment. Extracts are then
solubilized at 40 mg/ml in DMSO or water.
[0136] Concentration Requirements--1-dose/cancer in vitro program:
DMSO:glycerol 9:1 (unless otherwise noted) at a concentration of 4
mM for the one dose assay and 40 mM for the 5-dose assay. In both
cases the solution is diluted 1:400, giving a High Test
concentration of 10 or 100 .mu.M respectively. Synthetic agents
(macromolecules) without a molecular weight are prepared in
DMSO:glycerol 9:1 (unless otherwise noted) at a concentration of 6
and 60 mg/ml which is diluted 1:400, giving a High Test
concentration of 15 and 150 .mu.g/ml. Natural products crude
extracts which are organic solvent soluble are prepared in DMSO,
while those which were produced by aqueous extraction are
solubilized in water, both at 40 mg/ml.
[0137] Volume Requirements--prescreen/cancer in vitro program: The
cancer screen requires 100 .mu.l for 1 log, 5-dose dilutions of
regular compounds and 75 .mu.l for 1-dose testing. 1-dose testing
is done at 1/10th the high concentration of 5-dose testing, so the
volume requirement is 210 .mu.l+20% at 40 mM for compounds with
molecular weights or 210 .mu.l+20% at 60,000 .mu.g/ml=250 .mu.l for
compounds without molecular weights (macromolecules) (i.e. less
than 10 mg for MW=1000 or 15 mg for compounds tested as
weight/volume).
[0138] Compound Special Instructions: Special instructions, (e.g.
oxygen sensitive, light sensitive) can change the handling of the
agent according to the instructions.
[0139] Fresh Compounds: Compounds that are identified as needing to
be prepared fresh before use are solubilized no more than one hour
before serial dilution. It is serial diluted on a TECAN Freedom 200
(two drugs/plate), transferred to a column plate and stored under
nitrogen in a desiccator box until delivered to the testing
lab.
Solubilization Standard Operating Procedures
[0140] Entering Information into the NPSG TECAN System: Prior to
beginning the solubilization procedure, information is entered into
the NPSG TECAN (Visual Basic instrument control and front end to
ORACLE) system for each compound to be solubilized by the TECAN
Freedom 200. A set of 72 compounds are assigned to a plateset by
entering the shiplist numbers. A shiplist is loaded into TECAN
software which looks up quantity and MW (from DIS Oracle tables)
and calculates volume of solvent to be added to each vial to get
constant concentration (40 mM or 60,000 .mu.g/ml) and adjusts
concentrations if insufficient material for 1-dose and a test &
one retest (75 .mu.l @ 4 mM & 200 .mu.l @ 40 mM+20% or 75 .mu.l
@ 6,000 .mu.g/ml & 200 .mu.l @ 60,000 .mu.g/ml+20%). A Platemap
(defines which compound is in which well) for prescreen is uploaded
via ORADIS to ORACLE PLATEWELL table.
[0141] Supplies and Equipment: Vials are put on the TECAN table in
shiplist order as designated on the PLATEMAP printout. TECAN
Freedom robot adds appropriate volume of solvent (methanol/ethyl
acetate/methyl-t-butyl ether, 6:3:1) to each vial to give constant
concentration. Technician inspects each vial individually and
sonicates, warms, etc. to achieve solution keeping the time of
exposure less than two hours. Plate Preparation prior to drug
solution transfer: Technician prepares three 96 well PP detachable
well plates (one for 1-dose and two for 60 cell testing): 100 .mu.l
of 10% glycerol in isopropanol is added to each well. [After drug
solution addition and vacuum drying, this leaves 10 .mu.L glycerol
per well.]TECAN mixes drug solution in vial once then transfers 40
.mu.L (400 .mu.M) of drug solution into each well of the 96 well
detachable plate for 1-dose and 400 .mu.L (4,000 .mu.M) into 96
well PP detachable well plates for full 60 cell screen. All plates
are transferred to the SpeedVac system for drying. All solvent is
removed by high vacuum without heating leaving a residue of
glycerol plus drug in bottom of the well. Dry plates are stored @
-70.degree. C. until called for test, typically 1-3 weeks.
Untransferred residue in glass vials is dried in a SpeedVac and
stored dry at -70.degree. C. and can be used if additional
retesting is required.
[0142] For 1-dose 60 cell testing: On the day of or the day before
drug addition to growing cells in tissue culture, a strip of
standards (adriamycin, NSC 123127 prepared and stored the same as
the compounds) is added to the detachable well plate, and 90 .mu.l
DMSO is added to each well (4 mM solution), and mixed/sonicated and
75 .mu.l is transferred, using a 12 channel hand pipettor, to a 12
channel reservoir plates (column plates), which is sealed and
stored under nitrogen in a desiccator box until delivered to
testing lab. The labels are placed at the right and the left of the
front of the reservoir plate. It will be the first and the last NSC
number in the row. Rows are transferred from detachable plate to
columns 3-12 of column plates. Plates are sealed and stored under
nitrogen no more than 24 hours prior to drug addition.
[0143] For 5-dose 60 cell testing: On the day of drug addition to
growing cells in tissue culture, 90 DMSO is added to each well (40
mM solution), and mixed/sonicated on the shaker of the TECAN
Freedom 200. Tubes are then placed on a TECAN Freedom 200 (two
drugs/plate), and serial diluted/transferred to column plates which
is sealed and stored under nitrogen in a desiccator box until
delivered to testing lab. The plate labels are printed by the SATO
thermal transfer printer utilizing the ORACLE front end program
option AA-Expid NSC labels. The labels are placed at the right and
the left of the front of the reservoir plate, drug one by column
one and drug two by column twelve.
[0144] Vehicle Selection--Synthetics: The vehicles of choice are
DMSO and water. Most agents are solubilized using one of these two
vehicles. Other vehicles are used at the request of the supplier or
based upon past testing methods. Agents utilizing volatile solvents
as a vehicle are labeled `Fresh` and are prepared within an hour of
screening addition. Currently, all synthetic agents for
Prescreen/Cancer screening are prepared in DMSO:glycerol 9:1,
unless another vehicle is indicated. When water is indicated, the
compound is solubilized in either distilled water or in cell
culture media (RPMI 1640) without serum. All solubilizations
requiring THF, ethanol, methanol, or other volatile solvents are
prepared fresh to reduce evaporation.
[0145] Minimum Volume Requirements: The goal of solubilization is
to deliver the highest requested concentration of an agent for the
screening process. However, the number of vials required by the
program screening the agent determines the minimum amount of
vehicle that can be added. If the amount of material is
insufficient to create the required number of aliquots, the
concentration is dropped to ensure an appropriate volume is
met.
[0146] Volume Requirements--1-dose/cancer in vitro program: The
cancer screen requires 100 .mu.l for 1 log, 5-dose dilutions for
the regular compounds. For compounds solubilized on the TECAN for
both 1-dose and five-dose cancer assays, a minimum volume of 250
.mu.l is needed, enough for the initial 1-dose assay, a test and a
retest in the 5-dose assay.
[0147] Solubility Codes: The agents will not always solubilize to a
clear solution absent of particles. Therefore the solution is
described via a code best describing the solubility of the agent in
the vehicle. It is the clarity of the solution that is being
evaluated. Presence or absence of color is not accounted for in the
solubility codes.
NCI60 Screening Methodology
[0148] NCI60 Cell One-Dose Screen:
[0149] General Description: All compounds submitted to the NCI 60
Cell screen are tested initially at a single high dose (10-5 M) in
the full NCI 60 cell panel. Only compounds which satisfy
pre-determined threshold inhibition criteria in a minimum number of
cell lines will progress to the full 5-dose assay. The threshold
inhibition criteria for progression to the 5-dose screen was
selected to efficiently capture compounds with anti-proliferative
activity based on careful analysis of historical DTP screening
data. The threshold criteria may be updated as additional data
becomes available.
[0150] Interpretation of One-Dose Data: The One-dose data will be
reported as a mean graph of the percent growth of treated cells and
will be similar in appearance to mean graphs from the 5-dose assay.
The number reported for the One-dose assay is growth relative to
the no-drug control, and relative to the time zero number of cells.
This allows detection of both growth inhibition (values between 0
and 100) and lethality (values less than 0). This is the same as
for the 5-dose assay, described below. For example, a value of 100
means no growth inhibition. A value of 40 would mean 60% growth
inhibition. A value of 0 means no net growth over the course of the
experiment. A value of -40 would mean 40% lethality. A value of
-100 means all cells are dead. Information from the One-dose mean
graph is available for COMPARE analysis.
[0151] NCI60 Cell Five-Dose Screen:
[0152] Compounds which exhibit significant growth inhibition in the
One-Dose Screen are evaluated against the 60 cell panel at five
concentration levels.
[0153] The human tumor cell lines of the cancer screening panel are
grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM
L-glutamine. For a typical screening experiment, cells are
inoculated into 96 well microtiter plates in 100 .mu.L at plating
densities ranging from 5,000 to 40,000 cells/well depending on the
doubling time of individual cell lines. After cell inoculation, the
microtiter plates are incubated at 37.degree. C., 5% CO2, 95% air
and 100% relative humidity for 24 h prior to addition of
experimental drugs.
[0154] After 24 h, two plates of each cell line are fixed in situ
with TCA, to represent a measurement of the cell population for
each cell line at the time of drug addition (Tz). Experimental
drugs are solubilized in dimethyl sulfoxide at 400-fold the desired
final maximum test concentration and stored frozen prior to use. At
the time of drug addition, an aliquot of frozen concentrate is
thawed and diluted to twice the desired final maximum test
concentration with complete medium containing 50 .mu.g/ml
gentamicin. Additional four, 10-fold or 1/2 log serial dilutions
are made to provide a total of five drug concentrations plus
control. Aliquots of 100 .mu.L of these different drug dilutions
are added to the appropriate microtiter wells already containing
100 .mu.L of medium, resulting in the required final drug
concentrations.
[0155] Following drug addition, the plates are incubated for an
additional 48 h at 37.degree. C., 5% CO2, 95% air, and 100%
relative humidity. For adherent cells, the assay is terminated by
the addition of cold TCA. Cells are fixed in situ by the gentle
addition of 50 .mu.L of cold 50% (w/v) TCA (final concentration,
10% TCA) and incubated for 60 minutes at 4.degree. C. The
supernatant is discarded, and the plates are washed five times with
tap water and air dried. Sulforhodamine B (SRB) solution (100
.mu.L) at 0.4% (w/v) in 1% acetic acid is added to each well, and
plates are incubated for 10 minutes at room temperature. After
staining, unbound dye is removed by washing five times with 1%
acetic acid and the plates are air dried. Bound stain is
subsequently solubilized with 10 mM Trizma.RTM. base (Tris base),
and the absorbance is read on an automated plate reader at a
wavelength of 515 nm. For suspension cells, the methodology is the
same except that the assay is terminated by fixing settled cells at
the bottom of the wells by gently adding 50 .mu.L of 80% TCA (final
concentration, 16% TCA). Using the seven absorbance measurements
[time zero, (Tz), control growth, (C), and test growth in the
presence of drug at the five concentration levels (Ti)], the
percentage growth is calculated at each of the drug concentrations
levels. Percentage growth inhibition is calculated as:
[(Ti-Tz)/(C-Tz)].times.100 for concentrations for which
Ti>/=Tz
[(Ti-Tz)/Tz].times.100 for concentrations for which Ti<Tz
[0156] 50% (GI50) is calculated from [(Ti-Tz)/(C-Tz)].times.100=50,
which is the drug concentration resulting in a 50% reduction in the
net protein increase (as measured by SRB staining) in control cells
during the drug incubation. The drug concentration resulting in
total growth inhibition (TGI) is calculated from Ti=Tz. The LC50
(concentration of drug resulting in a 50% reduction in the measured
protein at the end of the drug treatment as compared to that at the
beginning) indicating a net loss of cells following treatment is
calculated from [(Ti-Tz)/Tz].times.100=-50. Values are calculated
for each of these three parameters if the level of activity is
reached; however, if the effect is not reached or is exceeded, the
value for that parameter is expressed as greater or less than the
maximum or minimum concentration tested.
[0157] FIGS. 3A-3B show GI50 data for Compound 1
(4-amino-1-42R,3S,4S,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydroth-
iophen-2-yl)-1,3,5-triazin-2(1H)-one) and 5-aza-T-dCyd,
respectively, against leukemia cell lines. FIGS. 4A and 4B show
GI50 data for Compound 1 and 5-aza-T-dCyd, respectively, against
central nervous system (CNS) cancer cell lines. FIGS. 5A and 5B
show GI50 data for Compound 1 and 5-aza-T-dCyd, respectively,
against renal cancer cell lines. FIGS. 6A and 6B show GI50 data for
Compound 1 and 5-aza-T-dCyd, respectively, against non-small cell
lung cancer cell lines. FIGS. 7A and 7B show GI50 data for Compound
1 and 5-aza-T-dCyd, respectively, against melanoma cell lines.
FIGS. 8A and 8B show GI50 data for Compound 1 and 5-aza-T-dCyd,
respectively, against prostate cancer cell lines. FIGS. 9A and 9B
show GI50 data for Compound 1 and 5-aza-T-dCyd, respectively,
against colon cancer cell lines. FIGS. 10A and 10B show GI50 data
for Compound 1 and 5-aza-T-dCyd, respectively, against ovarian
cancer cell lines. FIGS. 11A and 11B show GI50 data for Compound 1
and 5-aza-T-dCyd, respectively, against breast cancer cell
lines.
Example 3
Xenograft Studies
[0158] Xenograft studies were performed in mice with tumors of
HCT-116 human colon carcinoma cells, BL0382 human bladder carcinoma
cells, OVCAR3 human ovarian carcinoma cells, NCI-H23 NSCLC human
lung carcinoma cells, and HL-60 human leukemia cells. Human tumor
xenografts were generated in 4- to 6-week-old female athymic nude
mice (nu/nu NCr) or NSG mice by subcutaneous injection of tumor
cells (HL-60, NCI-H23, OVCAR-3, HCT-116) grown in vitro using RPMI
1640 with 10% fetal bovine serum and 2 mM 1-glutamine. For the
patient-derived xenograft (PDX) model, BL0382F1232, tumor fragments
were serially passaged from donor mice as described for other
xenograft models (Plowman et al., "Human tumor xenograft models in
NCI drug development." In: Teicher, B. A. (Ed.), Anticancer Drug
Development Guide: Preclinical Screening, Clinical Trials and
Approval. Humana Press, Totowa, N.J., pp. 101-125; 1997). The PDX
donor tumors were produced by implantation of tumor material
received from human patients into NSG mice. The resulting tumors
were serially passaged in NSG mice and fragments were cryopreserved
for subsequent establishment of newly tumored animals from the
archived material. The mice were housed in an AAALACi (Association
for Assessment and Accreditation of Laboratory Animal Care
International) accredited facility with food and water provided ad
libitum. When tumors reached the predetermined starting weight
(staging weight), the animals were randomized into experimental
groups and treatment was initiated. Groups included a vehicle
control group as well as the drug-treated groups. Drug doses were
selected based upon prior experience or newly conducted mouse
tolerability studies as described elsewhere (ibid.). Tumors were
monitored by bidirectional caliper measurements and the tumor
weights were calculated as tumor weight (mg)=(tumor length in
mm.times.tumor width in mm.sup.2)/2. Data collection and analysis
was performed using the StudyLog software program Study Director
(Studylog Systems, Inc., South San Francisco, Calif.).
[0159] The drug dosing was as follows:
TABLE-US-00001 Cell Line Drug Amount Route Schedule HCT-116
F-aza-TdCyd 10 mg/kg IP QDx5, rest and repeat for 4 cycles* 400
mg/kg IP Q7Dx3 F-TdCyd 240 mg/kg IV Q7Dx4 5-aza-T-dCyd 1.5 mg/kg IP
QDx5, rest and repeat for 4 cycles gemcitabine 150 mg/kg IP Q7Dx3
BL0382 F-aza-TdCyd 8 mg/kg IP QDx5, rest for 3 cycles 250 mg/kg IP
Q7Dx3 8 mg/kg PO QDx5, rest for 3 cycles F-TdCyd 200 mg/kg IV Q7Dx3
5-aza-T-dCyd 1.5 mg/kg IP QDx5 rest for 3 cycles gemcitabine 150
mg/kg IP Q7Dx3 OVCAR3 F-aza-TdCyd 8 mg/kg IP QDx5, rest for 3
cycles 250 mg/kg IP Q7Dx3 8 mg/kg PO QDx5, rest for 3 cycles
F-TdCyd 200 mg/kg IV Q7Dx3 5-aza-T-dCyd 1.5 mg/kg IP QDx5 rest for
3 cycles gemcitabine 150 mg/kg IP Q7Dx3 NCI-H23 F-aza-TdCyd 10
mg/kg IP QDx5, rest for 3 cycles NSCLC 80 mg/kg PO QDx5, rest for 3
cycles 400 mg/kg IP Q7Dx3 F-TdCyd 240 mg/kg IV Q7Dx3 5-aza-T-dCyd
1.5 mg/kg IP QDx5, rest for 3 cycles gemcitabine 150 mg/kg IP Q7Dx3
HL-60 F-aza-TdCyd 10 mg/kg IP QDx5, rest for 3 cycles 400 mg/kg IP
Q7Dx3 F-TdCyd 240 mg/kg IV Q7Dx3 5-aza-T-dCyd 1.5 mg/kg IP QDx5,
rest for 3 cycles gemcitabine 150 mg/kg IP Q7Dx3 IP =
intraperitonally, IV = intravenously, PO = orally *Each cycle is
one week. Thus, a "QDx5" cycle includes two days of rest.
[0160] Serial measurements of tumor volume and body weight were
obtained post tumor implant. The results are shown in FIGS. 12A and
12B (HCT-116 human colon carcinoma cells), 13A and 13B (BL0382
human bladder carcinoma cells), 14A and 14B (OVCAR3 human ovarian
carcinoma cells), 15A and 15B (NCI-H23 NSCLC human lung carcinoma
cells), and 16A and 16B (HL-60 human leukemia cells).
Example 4
Treatment with a Halogenated 5-Aza-T-dCyd Analog
[0161] A subject having, or suspected of having a disease, that may
be treated with a halogenated 5-aza-T-dCyd analog is identified.
The disease may be a disease characterized at least in part by the
presence of neoplastic cells. In some instances, the disease is a
cancer. The subject may be selected based on a clinical
presentation and/or by performing tests to demonstrate presence of
a disease, such as a cancer, characterized at least in part by
presence of neoplastic cells. In some examples, the subject may
have a solid tumor or a blood cancer.
[0162] The subject is treated by administering a halogenated
5-aza-T-dCyd analog, or a stereoisomer, tautomer, or
pharmaceutically acceptable salt thereof at a dose determined by a
clinician to be therapeutically effective. In some examples, the
compound is
4-amino-1-((2R,3S,4S,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydroth-
iophen-2-yl)-1,3,5-triazin-2(1H)-one. The compound is administered
by any suitable means, such as parenteral (e.g., intravenous,
intra-arterial, subcutaneous, intramuscular) or intrathecal
injection or by oral administration. Treatment efficacy may be
assessed by conventional means, e.g., prevention of tumor growth,
reduction in tumor growth, reduction in or lack of metastasis,
normalization of blood cell counts, and the like. In some examples,
assessment is performed by computed tomography (CT), magnetic
resonance imaging (MRI), or positron emission tomography (PET)
scans.
[0163] A therapeutically effective amount of a second active agent
may be co-administered with the compound. The compound and the
second active agent may be administered either separately or
together in a single composition. The second active agent may be
administered by the same route or a different route. If
administered concurrently, the compound and the second active agent
may be combined in a single pharmaceutical composition or may be
administered concurrently as two pharmaceutical compositions. The
second active agent may be, for example, an anti-cancer agent, an
anti-inflammatory agent, an antimicrobial agent, an antiviral
agent, an anesthetic agent, or the like.
[0164] In view of the many possible embodiments to which the
principles of the disclosed invention may be applied, it should be
recognized that the illustrated embodiments are only preferred
examples of the invention and should not be taken as limiting the
scope of the invention. Rather, the scope of the invention is
defined by the following claims. We therefore claim as our
invention all that comes within the scope and spirit of these
claims.
* * * * *
References