U.S. patent application number 17/017475 was filed with the patent office on 2021-01-07 for imidazotetrazine compounds.
This patent application is currently assigned to The Board of Trustees of the University of Illinois. The applicant listed for this patent is The Board of Trustees of the University of Illinois. Invention is credited to Timothy M. Fan, Paul J. HERGENROTHER, Riley L. Svec.
Application Number | 20210002286 17/017475 |
Document ID | / |
Family ID | |
Filed Date | 2021-01-07 |
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United States Patent
Application |
20210002286 |
Kind Code |
A1 |
HERGENROTHER; Paul J. ; et
al. |
January 7, 2021 |
IMIDAZOTETRAZINE COMPOUNDS
Abstract
New synthetic methods to provide access to previously unexplored
functionality at the C8 position of imidazotetrazines. Through
synthesis and evaluation of a suite of compounds with a range of
aqueous stabilities (from 0.5 to 40 hours), a predictive model for
imidazotetrazine hydrolytic stability based on the Hammett constant
of the C8 substituent was derived. Promising compounds were
identified that possess activity against a panel of GBM cell lines,
appropriate hydrolytic and metabolic stability, and brain-to-serum
ratios dramatically elevated relative to TMZ, leading to lower
hematological toxicity profiles and superior activity to TMZ in a
mouse model of GBM.
Inventors: |
HERGENROTHER; Paul J.;
(Champaign, IL) ; Fan; Timothy M.; (Mahomet,
IL) ; Svec; Riley L.; (Urbana, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Board of Trustees of the University of Illinois |
Urbana |
IL |
US |
|
|
Assignee: |
The Board of Trustees of the
University of Illinois
Urbana
IL
|
Appl. No.: |
17/017475 |
Filed: |
September 10, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US2019/045986 |
Aug 9, 2019 |
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17017475 |
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62873669 |
Jul 12, 2019 |
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62778750 |
Dec 12, 2018 |
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62716390 |
Aug 9, 2018 |
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Current U.S.
Class: |
1/1 |
International
Class: |
C07D 487/04 20060101
C07D487/04; A61K 9/20 20060101 A61K009/20; A61K 9/48 20060101
A61K009/48; A61K 47/02 20060101 A61K047/02; A61K 47/24 20060101
A61K047/24; A61K 47/26 20060101 A61K047/26; A61K 47/36 20060101
A61K047/36; A61K 47/34 20060101 A61K047/34 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under Grant
No. R21-CA195149 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A compound of Formula I: ##STR00037## or a salt thereof; wherein
X is O or S; R.sup.1 is halo, --CN, --NO.sub.2,
--(C.sub.1-C.sub.6)alkyl, --C(.dbd.O)R.sup.a, phenyl, or a 5- or
6-membered heterocycle, wherein R.sup.a is H, halo,
--(C.sub.1-C.sub.6)alkyl, --(C.sub.3-C.sub.6)cycloalkyl, OR.sup.b,
SR.sup.b, or --NR.sup.bR.sup.c; wherein R.sup.b is H,
--(C.sub.1-C.sub.6)alkyl, or --(C.sub.3-C.sub.6)cycloalkyl; R.sup.c
is H, --(C.sub.1-C.sub.6)alkyl, or --(C.sub.3-C.sub.6)cycloalkyl;
or when R.sup.a is --NR.sup.bR.sup.c, Rb and R.sup.c taken together
optionally forms a heterocycle; R.sup.2 is
--(C.sub.1-C.sub.6)alkyl, --(C.sub.3-C.sub.6)cycloalkyl, propargyl,
phenyl, or a 5- or 6-membered heterocycle; and R.sup.3 is H,
--(C.sub.1-C.sub.6)alkyl, or --(C.sub.3-C.sub.6)cycloalkyl; wherein
each --(C.sub.1-C.sub.6)alkyl, --(C.sub.3-C.sub.6)cycloalkyl,
propargyl, phenyl, and 5- or 6-membered heterocycle are optionally
substituted with one or more substituents, and each
--(C.sub.1-C.sub.6)alkyl is unbranched or optionally branched.
2. The compound of claim 1 wherein R.sup.1 is halo,
--(C.sub.1-C.sub.6)alkyl, or --(C.sub.3-C.sub.6)cycloalkyl.
3. The compound of claim 1 wherein R.sup.1 is
--C(.dbd.O)--(C.sub.1-C.sub.6)alkyl,
--C(.dbd.O)--NH(C.sub.1-C.sub.6)alkyl, or
--C(.dbd.O)--N[(C.sub.1-C.sub.6)alkyl].sub.2.
4. The compound of claim 1 wherein R.sup.1 is a moiety of Formula
IB: ##STR00038## wherein W is O, S, or NR.sup.d; wherein R.sup.d is
H, --(C.sub.1-C.sub.6)alkyl, or --(C.sub.3-C.sub.6)cycloalkyl; V is
N or CR, wherein R.sup.X is H, --(C.sub.1-C.sub.6)alkyl, or
--(C.sub.3-C.sub.6)cycloalkyl; Y is N or CR.sup.Y, wherein R is H,
--(C.sub.1-C.sub.6)alkyl, or --(C.sub.3-C.sub.6)cycloalkyl; and Z
is N or CH.
5. The compound of claim 4 wherein R.sup.1 is i, ii, or iii:
##STR00039## wherein (i), (ii) and (iii) are optionally substituted
at position 4 or 5.
6. The compound of claim 1 wherein R.sup.1 is a para-substituted
phenyl.
7. The compound of claim 6 wherein the para-substituent is halo,
--CN, --CF.sub.3, --CF.sub.2CF.sub.3, or
--(C.sub.1-C.sub.6)alkyl.
8. The compound of claim 1 wherein X is O, R.sup.3 is H and R.sup.1
is --C(.dbd.O)--(C.sub.1-C.sub.6)alkyl,
--C(.dbd.O)--NH(C.sub.1-C.sub.6)alkyl, or
--C(.dbd.O)--N[(C.sub.1-C.sub.6)alkyl].sub.2.
9. The compound of claim 1 wherein X is O and R is
--C(.dbd.O)--(C.sub.1-C.sub.6)alkyl.
10. The compound of claim 1 wherein X is O, R.sup.2 is
--(C.sub.1-C.sub.6)alkyl, and R.sup.3 is H.
11. The compound of claim 1 wherein R.sup.2 is
--(C.sub.1-C.sub.6)alkyl and R.sup.3 is H.
12. The compound of claim 1 wherein R.sup.2 is propargyl or a
substituted phenyl.
13. The compound of claim 12 wherein the substituted phenyl is
substituted with halo, alkyl, alkoxy, phenoxy, dialkylamine, or
combination thereof.
14. The compound of claim 1 wherein the compound is:
##STR00040##
15. The compound of claim 1 wherein the compound of Formula I is a
compound of Formula IC: ##STR00041## wherein G.sup.1 is OCH.sub.3,
OCH.sub.2CH.sub.3, OPh, or N(CH.sub.3).sub.2.
16. The compound of claim 1 wherein the compound of Formula I is a
compound of Formula II: ##STR00042##
17. The compound of claim 16 wherein R.sup.2 is
--(C.sub.1-C.sub.6)alkyl and R.sup.3 is H.
18. The compound of claim 16 wherein R.sup.a is CH.sub.3,
CH.sub.2CH.sub.3, NHCH.sub.3, NHCH.sub.2CH.sub.3,
N(CH.sub.3).sub.2, N(CH.sub.2CH.sub.3).sub.2,
N(CH.sub.2CH.sub.2CH.sub.3).sub.2,
N(CH.sub.2CH.sub.2CH.sub.2CH.sub.3).sub.2,
N(CH.sub.2CH.sub.2).sub.2, N[(CH.sub.2CH.sub.2).sub.2], OCH.sub.3,
OCH.sub.2CH.sub.3, SCH.sub.3, or SCH.sub.2CH.sub.3.
19. The compound of claim 18 wherein the compound is:
##STR00043##
20. The compound of claim 16 wherein the compound is K-TMZ:
##STR00044##
21. The compound of claim 1 wherein the compound of Formula I is a
compound of Formula IIIA or IIIB: ##STR00045## wherein R.sup.Z is
H, halo, --(C.sub.1-C.sub.6)alkyl, or
--(C.sub.3-C.sub.6)cycloalkyl.
22. The compound of claim 21 wherein R is CH.sub.3 or
CH.sub.2CH.sub.3.
23. The compound of claim 21 wherein the compound is:
##STR00046##
24. A method of treating a cancer comprising administering to a
subject in need thereof a therapeutically effective amount of a
compound of claim 1, wherein the cancer is thereby treated.
25. The method of claim 20 wherein the cancer is glioblastoma
(GBM).
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of International
Application No. PCT/US2019/045986 filed Aug. 9, 2019, which claims
priority under 35 U.S.C. .sctn. 119(e) to U.S. Provisional Patent
Application Nos. 62/716,390 filed on Aug. 9, 2018, 62/778,750 filed
on Dec. 12, 2018, and 62/873,669 filed on Jul. 12, 2019, each of
which are incorporated herein by reference in their entirety.
BACKGROUND OF THE INVENTION
[0003] Glioblastoma multiforme (GBM) is the most prevalent,
infiltrative, and lethal primary malignant brain tumor, with only
10% of patients surviving five years..sup.1 The current
standard-of-care for GBM is gross surgical resection followed by
radiotherapy combined with temozolomide (TMZ), a small molecule DNA
alkylating agent. The antitumor effect of TMZ is ultimately
mediated through methylation of the O.sup.6-position of guanine
residues and subsequent mismatch repair-dependent cell
death..sup.2-6 Among the beneficial properties of TMZ are favorable
pharmacokinetics (including 100% oral bioavailability.sup.7),
non-enzymatic prodrug activation, and some accumulation in the
brain (cerebral spinal fluid:blood ratio of 17:83 in human cancer
patients.sup.8,9). TMZ provides a significant therapeutic benefit
to a subset of GBM patients. For example, in patients whose tumors
do not express O.sup.6-methylguanine DNA methyltransferase (MGMT),
an enzyme that removes O.sup.6-methylguanine lesions, TMZ extends
median survival to approximately two years..sup.10 Even in the era
of personalized anticancer therapy, TMZ remains frontline therapy
for oligodendrogliomas, diffuse astrocytic gliomas, and pleomorphic
xanthoastrocytomas in addition to GBM..sup.11 However, given the
ineffectiveness of TMZ against tumors expressing MGMT and the
inevitable recurrence of GBM after multimodal combination therapy,
there remains a significant clinical need for better treatment
strategies.
[0004] TMZ is a prodrug activated in aqueous solutions that
ultimately releases methyldiazonium, the active alkylating
component (Scheme 1(a)). The half-life of TMZ is .about.2 hours in
vivo and in aqueous solutions in vitro, and it has been suggested
that the drug has an increased rate of hydrolysis in the more
alkaline environment of gliomas, providing some selectivity for
cancerous vs. non-cancerous cells..sup.12-15 While this 2 hour
half-life enables TMZ to reach the central nervous system (CNS) and
release methyldiazonium, there is scarce information on the
relationship between half-life and anticancer activity;
specifically, it is unclear if 2 hours is optimal to maximize
therapeutic efficacy or if shorter (or longer) half-lives may
bolster its effect. Given the advantageous features of TMZ, an
understanding of the relationship between its structure, hydrolytic
stability, and anticancer activity.
[0005] While TMZ has been FDA approved for two decades, more
efficacious drugs for glioblastoma that have lower systemic
toxicity would be desirable. Therapeutic compounds that can reach
the entirety of the diffuse tumor in sufficient concentrations to
be effective are sought. Accordingly, there is a need for new
compounds that possess the desirable properties of TMZ, but have
better brain penetration, lower toxicity, and provide improved
patient survival rates.
SUMMARY
[0006] Herein is described the development of a model for that
accurately predicts the hydrolytic stability and half-life of
imidazotetrazines. This model was used to discover novel
imidazotetrazines with exceptional BBB penetration and superior
anticancer activity compared to TMZ, including in a murine model of
GBM.
[0007] Accordingly, this disclosure provides a compound of Formula
I:
##STR00001##
or a salt thereof; wherein
[0008] X is O or S;
[0009] R.sup.1 is halo, --CN, --NO.sub.2, --(C.sub.1-C.sub.6)alkyl,
--C(.dbd.O)R.sup.a, phenyl, or a 5- or 6-membered heterocycle,
wherein R.sup.a is H, halo, --(C.sub.1-C.sub.6)alkyl,
--(C.sub.3-C.sub.6)cycloalkyl, OR.sup.b, SR.sup.b, or
--NR.sup.bR.sup.c; wherein [0010] R.sup.b is H,
--(C.sub.1-C.sub.6)alkyl, or --(C.sub.3-C.sub.6)cycloalkyl; [0011]
R.sup.c is H, --(C.sub.1-C.sub.6)alkyl, or
--(C.sub.3-C.sub.6)cycloalkyl; or [0012] when R.sup.a is
--NR.sup.bR.sup.c, Rb and R.sup.c taken together optionally forms a
heterocycle;
[0013] R.sup.2 is --(C.sub.1-C.sub.6)alkyl,
--(C.sub.3-C.sub.6)cycloalkyl, alkynyl, phenyl, or a 5- or
6-membered heterocycle; and
[0014] R.sup.3 is H, --(C.sub.1-C.sub.6)alkyl, or
--(C.sub.3-C.sub.6)cycloalkyl;
wherein each --(C.sub.1-C.sub.6)alkyl,
--(C.sub.3-C.sub.6)cycloalkyl, alkynyl, phenyl, and 5- or
6-membered heterocycle are optionally substituted with one or more
substituents, and each --(C.sub.1-C.sub.6)alkyl is unbranched or
optionally branched.
[0015] This disclosure also provides a method of treating a cancer
comprising administering to a subject in need thereof a
therapeutically effective amount of the compound disclosed above,
wherein the cancer is thereby treated.
[0016] The invention provides novel compounds of Formulas
I-III(A/B/C), intermediates for the synthesis of compounds of
Formulas I-III, as well as methods of preparing compounds of
Formulas I-III. The invention also provides compounds of Formulas
I-III that are useful as intermediates for the synthesis of other
useful compounds. The invention provides for the use of compounds
of Formulas I-III for the manufacture of medicaments useful for the
treatment of bacterial infections in a mammal, such as a human.
[0017] The invention provides for the use of the compositions
described herein for use in medical therapy. The medical therapy
can be treating cancer, for example, bladder cancer, breast cancer,
colon cancer, endometrial cancer, kidney (renal) cancer, leukemia,
lung cancer, lymphoma, Non-Hodgkin's lymphoma, melanoma, pancreatic
cancer, prostate cancer, non-melanoma skin cancer, stomach cancer,
thyroid cancer, or brain cancers such as glioblastoma. The
invention also provides for the use of a composition as described
herein for the manufacture of a medicament to treat a disease in a
mammal, for example, cancer in a human. The medicament can include
a pharmaceutically acceptable diluent, excipient, or carrier.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] The following drawings form part of the specification and
are included to further demonstrate certain embodiments or various
aspects of the invention. In some instances, embodiments of the
invention can be best understood by referring to the accompanying
drawings in combination with the detailed description presented
herein. The description and accompanying drawings may highlight a
certain specific example, or a certain aspect of the invention.
However, one skilled in the art will understand that portions of
the example or aspect may be used in combination with other
examples or aspects of the invention.
[0019] FIG. 1. Hydrolytic stability of C8-substituted
imidazotetrazines. (a) The percentage of compound remaining after 2
hours plotted against the Hammett constant of its C8 substituent.
Compounds with hydrolytic stability similar to TMZ are enclosed in
the oval (see Table 1c).
[0020] FIG. 2. (a) Relevant brain:serum ratios of TMZ, Me-TMZ, and
DiMe-TMZ (25 mg/kg) were measured 5 minutes after IV injection into
mice. Values are the fold change of brain:serum ratio relative to
TMZ. In a second experiment, brain (b) and serum (c) concentrations
of TMZ and C8 analogs (25 mg/kg) were quantitated 5 minutes after
IV injection into mice. (d) Brain:serum ratios were calculated
based on (b) and (c) assuming a mouse blood volume of 58.5 mL/kg
(see Scheme 2b). Error is SEM, number of mice per cohort=3.
Statistical significance was determined by using a two-sample
Student's t-test (two-tailed test, assuming equal variance).
[0021] FIG. 3. Assessment of the hematological toxicity of
imidazotetrazines in vivo. Mice were administered a single IV dose
of 125 mg/kg imidazotetrazine. After 7 days, whole blood was
collected, and a complete blood count was obtained for each
individual mouse. (a) Total WBC count. Control vs. Ox-TMZ: P=0.7,
Control vs. K-TMZ: P=0.9. (b) Lymphocyte concentrations. Control
vs. Ox-TMZ: P=0.5, Control vs. K-TMZ: P=0.9. Error is SEM, number
of mice per cohort=4. Statistical significance was determined by
using a two-sample Student's t-test (two-tailed test, assuming
equal variance). The concentrations of other relevant blood
constituents are shown in FIG. 8.
[0022] FIG. 4. Imidazotetrazines were added to T98G cells with or
without 3 h pre-treatment of O6BG (100 .mu.M). IC50 values after
7-day incubation and fold changes between (+/-) O6BG treatments are
reported. P-values between IC50 values (+/-) O6BG<0.02 for all
compounds. Error is SEM, n.gtoreq.3. Statistical significance was
determined by using a two-sample Student's t-test (two-tailed test,
assuming equal variance).
[0023] FIG. 5. Evaluation of imidazotetrazines in intracranial
mouse models of GBM. GBM Br23c oncospheres were intracranially
implanted into female athymic nude mice. Treatment was started 5
days post implantation. (a) Mice were administered 15 mg/kg TMZ or
an equimolar dose of Me-TMZ (16.1 mg/kg) or DiMe-TMZ (17.2 mg/kg)
orally once-per-day, 5.times./week for 7 weeks. Control vs. TMZ:
P=0.0014, TMZ vs. DiMe-TMZ: P=0.061, TMZ vs. Me-TMZ: P=0.016. (b)
Mice were administered 15 mg/kg TMZ or an equimolar dose of
DiMe-TMZ (17.2 mg/kg) or K-TMZ (14.9 mg/kg) orally once-per-day for
5 total doses. Control vs. TMZ: P=0.0007, DiMe-TMZ vs. TMZ: P=0.7,
K-TMZ vs. TMZ: P=0.055. Compounds were formulated in 10% PEG in PBS
immediately prior to each treatment. Number of mice per treatment
cohort.gtoreq.5. Survival curves were compared using log-rank
test.
[0024] FIG. 6. Western blot for MGMT status of all cell lines
used.
[0025] FIG. 7. The hydrolytic stabilities of TMZ and K-TMZ assessed
in saline at pH 7.0, 7.4, and 8.0 by calculating the percentage of
parent compound remaining after 2 h at 37.degree. C.
[0026] FIG. 8. Assessment of the hematological toxicity of
imidazotetrazines in vivo. Mice were treated with a single IV dose
of 125 mg/kg imidazotetrazine and a complete blood count was
obtained for each mouse after 7 days. (a) Neutrophil concentrations
(b) RBC concentrations (c) Platelet concentrations (see Table
2b).
[0027] FIG. 9. GBM oncosphere Br23c cells were intracranially
implanted into female athymic nude mice. Treatment was started 5
days post implantation. Mice were administered compound 14 (12.8
mg/kg, equimolar to 15 mg/kg TMZ) orally once-per-day for 5 doses.
Compound was formulated in 10% PEG in PBS. n.gtoreq.5. This
experiment was run alongside that presented in FIG. 5b; the control
group is the same for FIG. 5b and FIG. 9.
[0028] FIG. 10. Development pathway of MGMT-Independent
Imidazotetrazines. Graph of brain:serum ratio: mice were
administered 25 mg/kg compound IV. After 15 min, mice were
sacrificed, and blood and brain were collected. The concentration
of drug in each was quantified by LC-MS/MS. N.gtoreq.3 mice per
cohort, error is SEM. *P<0.05, **P<0.01.
DETAILED DESCRIPTION
[0029] Even in the era of personalized medicine and immunotherapy,
temozolomide (TMZ), a small molecule DNA alkylating agent, remains
the standard-of-care for glioblastoma (GBM). TMZ has an unusual
mode-of-action, spontaneously converting to its active component
via hydrolysis in vivo. While TMZ has been FDA approved for two
decades, it provides little benefit to patients whose tumors
express the resistance enzyme MGMT and gives rise to systemic
toxicity through myelosuppression. TMZ was first synthesized in
1984, but certain key derivatives have been inaccessible due to the
chemical sensitivity of TMZ, precluding broad exploration of the
link between imidazotetrazine structure and biological activity.
Therefore, discerning the relationship between the hydrolytic
stability and anticancer activity of imidazotetrazines, with the
objectives of identifying optimal timing for prodrug activation and
developing suitable compounds with enhanced efficacy via increased
blood-brain barrier penetrance was sought.
[0030] This work necessitated the development of new synthetic
methods to provide access to previously unexplored functionality
(such as aliphatic, ketone, halogen, and aryl groups) at the C8
position of imidazotetrazines. Through synthesis and evaluation of
a suite of compounds with a range of aqueous stabilities (from 0.5
to 40 hours), a predictive model for imidazotetrazine hydrolytic
stability based on the Hammett constant of the C8 substituent was
derived. Promising compounds were identified that possess activity
against a panel of GBM cell lines, appropriate hydrolytic and
metabolic stability, and brain-to-serum ratios dramatically
elevated relative to TMZ leading to lower hematological toxicity
profiles and superior activity to TMZ in a mouse model of GBM. This
work points a clear path forward for the development of novel and
effective anticancer imidazotetrazines.
[0031] While the amide at C8 of TMZ had been suggested in the past
to be essential for activity,.sup.2,16 conflicting reports have
since indicated that alternate functionality may be tolerated at
this position..sup.17-19 Indeed, an analysis led to a belief that
strategic substitutions at C8 could be used to tune the hydrolytic
stability of imidazotetrazines, and that in doing so a suite of
compounds with a range of half-lives could be constructed. In
addition to varying the stability of the prodrug, alterations at
the C8 position could lead to compounds that retain the favorable
pharmacokinetic properties of TMZ but have increased CNS
penetrance. An imidazotetrazine with enhanced blood-brain barrier
(BBB) penetrance will exhibit lower systemic toxicity and allow for
higher and more efficacious dosing regimens since the dose-limiting
toxicity of TMZ (myelosuppression) is not
CNS-related..sup.7,20,21
Definitions
[0032] The following definitions are included to provide a clear
and consistent understanding of the specification and claims. As
used herein, the recited terms have the following meanings. All
other terms and phrases used in this specification have their
ordinary meanings as one of skill in the art would understand. Such
ordinary meanings may be obtained by reference to technical
dictionaries, such as Hawley's Condensed Chemical Dictionary
14.sup.th Edition, by R. J. Lewis, John Wiley & Sons, New York,
N.Y., 2001.
[0033] References in the specification to "one embodiment", "an
embodiment", etc., indicate that the embodiment described may
include a particular aspect, feature, structure, moiety, or
characteristic, but not every embodiment necessarily includes that
aspect, feature, structure, moiety, or characteristic. Moreover,
such phrases may, but do not necessarily, refer to the same
embodiment referred to in other portions of the specification.
Further, when a particular aspect, feature, structure, moiety, or
characteristic is described in connection with an embodiment, it is
within the knowledge of one skilled in the art to affect or connect
such aspect, feature, structure, moiety, or characteristic with
other embodiments, whether or not explicitly described.
[0034] The singular forms "a," "an," and "the" include plural
reference unless the context clearly dictates otherwise. Thus, for
example, a reference to "a compound" includes a plurality of such
compounds, so that a compound X includes a plurality of compounds
X. It is further noted that the claims may be drafted to exclude
any optional element. As such, this statement is intended to serve
as antecedent basis for the use of exclusive terminology, such as
"solely," "only," and the like, in connection with any element
described herein, and/or the recitation of claim elements or use of
"negative" limitations.
[0035] The term "and/or" means any one of the items, any
combination of the items, or all of the items with which this term
is associated. The phrases "one or more" and "at least one" are
readily understood by one of skill in the art, particularly when
read in context of its usage. For example, the phrase can mean one,
two, three, four, five, six, ten, 100, or any upper limit
approximately 10, 100, or 1000 times higher than a recited lower
limit. For example, one or more substituents on a phenyl ring
refers to one to five, or one to four, for example if the phenyl
ring is disubstituted.
[0036] As will be understood by the skilled artisan, all numbers,
including those expressing quantities of ingredients, properties
such as molecular weight, reaction conditions, and so forth, are
approximations and are understood as being optionally modified in
all instances by the term "about." These values can vary depending
upon the desired properties sought to be obtained by those skilled
in the art utilizing the teachings of the descriptions herein. It
is also understood that such values inherently contain variability
necessarily resulting from the standard deviations found in their
respective testing measurements. When values are expressed as
approximations, by use of the antecedent "about," it will be
understood that the particular value without the modifier "about"
also forms a further aspect.
[0037] The terms "about" and "approximately" are used
interchangeably. Both terms can refer to a variation of .+-.5%,
.+-.10%, .+-.20%, or .+-.25% of the value specified. For example,
"about 50" percent can in some embodiments carry a variation from
45 to 55 percent, or as otherwise defined by a particular claim.
For integer ranges, the term "about" can include one or two
integers greater than and/or less than a recited integer at each
end of the range. Unless indicated otherwise herein, the terms
"about" and "approximately" are intended to include values, e.g.,
weight percentages, proximate to the recited range that are
equivalent in terms of the functionality of the individual
ingredient, composition, or embodiment. The terms "about" and
"approximately" can also modify the endpoints of a recited range as
discussed above in this paragraph.
[0038] As will be understood by one skilled in the art, for any and
all purposes, particularly in terms of providing a written
description, all ranges recited herein also encompass any and all
possible sub-ranges and combinations of sub-ranges thereof, as well
as the individual values making up the range, particularly integer
values. It is therefore understood that each unit between two
particular units are also disclosed. For example, if 10 to 15 is
disclosed, then 11, 12, 13, and 14 are also disclosed,
individually, and as part of a range. A recited range (e.g., weight
percentages or carbon groups) includes each specific value,
integer, decimal, or identity within the range. Any listed range
can be easily recognized as sufficiently describing and enabling
the same range being broken down into at least equal halves,
thirds, quarters, fifths, or tenths. As a non-limiting example,
each range discussed herein can be readily broken down into a lower
third, middle third and upper third, etc. As will also be
understood by one skilled in the art, all language such as "up to",
"at least", "greater than", "less than", "more than", "or more",
and the like, include the number recited and such terms refer to
ranges that can be subsequently broken down into sub-ranges as
discussed above. In the same manner, all ratios recited herein also
include all sub-ratios falling within the broader ratio.
Accordingly, specific values recited for radicals, substituents,
and ranges, are for illustration only; they do not exclude other
defined values or other values within defined ranges for radicals
and substituents. It will be further understood that the endpoints
of each of the ranges are significant both in relation to the other
endpoint, and independently of the other endpoint.
[0039] One skilled in the art will also readily recognize that
where members are grouped together in a common manner, such as in a
Markush group, the invention encompasses not only the entire group
listed as a whole, but each member of the group individually and
all possible subgroups of the main group. Additionally, for all
purposes, the invention encompasses not only the main group, but
also the main group absent one or more of the group members. The
invention therefore envisages the explicit exclusion of any one or
more of members of a recited group. Accordingly, provisos may apply
to any of the disclosed categories or embodiments whereby any one
or more of the recited elements, species, or embodiments, may be
excluded from such categories or embodiments, for example, for use
in an explicit negative limitation.
[0040] The term "contacting" refers to the act of touching, making
contact, or of bringing to immediate or close proximity, including
at the cellular or molecular level, for example, to bring about a
physiological reaction, a chemical reaction, or a physical change,
e.g., in a solution, in a reaction mixture, in vitro, or in
vivo.
[0041] An "effective amount" refers to an amount effective to treat
a disease, disorder, and/or condition, or to bring about a recited
effect. For example, an effective amount can be an amount effective
to reduce the progression or severity of the condition or symptoms
being treated. Determination of a therapeutically effective amount
is well within the capacity of persons skilled in the art. The term
"effective amount" is intended to include an amount of a compound
described herein, or an amount of a combination of compounds
described herein, e.g., that is effective to treat or prevent a
disease or disorder, or to treat the symptoms of the disease or
disorder, in a host. Thus, an "effective amount" generally means an
amount that provides the desired effect.
[0042] Alternatively, the terms "effective amount" or
"therapeutically effective amount," as used herein, refer to a
sufficient amount of an agent or a composition or combination of
compositions being administered which will relieve to some extent
one or more of the symptoms of the disease or condition being
treated. The result can be reduction and/or alleviation of the
signs, symptoms, or causes of a disease, or any other desired
alteration of a biological system. For example, an "effective
amount" for therapeutic uses is the amount of the composition
comprising a compound as disclosed herein required to provide a
clinically significant decrease in disease symptoms. An appropriate
"effective" amount in any individual case may be determined using
techniques, such as a dose escalation study. The dose could be
administered in one or more administrations. However, the precise
determination of what would be considered an effective dose may be
based on factors individual to each patient, including, but not
limited to, the patient's age, size, type or extent of disease,
stage of the disease, route of administration of the compositions,
the type or extent of supplemental therapy used, ongoing disease
process and type of treatment desired (e.g., aggressive vs.
conventional treatment).
[0043] The terms "treating", "treat" and "treatment" include (i)
preventing a disease, pathologic or medical condition from
occurring (e.g., prophylaxis); (ii) inhibiting the disease,
pathologic or medical condition or arresting its development; (iii)
relieving the disease, pathologic or medical condition; and/or (iv)
diminishing symptoms associated with the disease, pathologic or
medical condition. Thus, the terms "treat", "treatment", and
"treating" can extend to prophylaxis and can include prevent,
prevention, preventing, lowering, stopping or reversing the
progression or severity of the condition or symptoms being treated.
As such, the term "treatment" can include medical, therapeutic,
and/or prophylactic administration, as appropriate.
[0044] As used herein, "subject" or "patient" means an individual
having symptoms of, or at risk for, a disease or other malignancy.
A patient may be human or non-human and may include, for example,
animal strains or species used as "model systems" for research
purposes, such a mouse model as described herein. Likewise, patient
may include either adults or juveniles (e.g., children). Moreover,
patient may mean any living organism, preferably a mammal (e.g.,
human or non-human) that may benefit from the administration of
compositions contemplated herein. Examples of mammals include, but
are not limited to, any member of the Mammalian class: humans,
non-human primates such as chimpanzees, and other apes and monkey
species; farm animals such as cattle, horses, sheep, goats, swine;
domestic animals such as rabbits, dogs, and cats; laboratory
animals including rodents, such as rats, mice and guinea pigs, and
the like. Examples of non-mammals include, but are not limited to,
birds, fish and the like. In one embodiment of the methods provided
herein, the mammal is a human.
[0045] As used herein, the terms "providing", "administering,"
"introducing," are used interchangeably herein and refer to the
placement of the compositions of the disclosure into a subject by a
method or route which results in at least partial localization of
the composition to a desired site. The compositions can be
administered by any appropriate route which results in delivery to
a desired location in the subject. The compositions described
herein may be administered with additional compositions to prolong
stability and activity of the compositions, or in combination with
other therapeutic drugs.
[0046] The terms "inhibit", "inhibiting", and "inhibition" refer to
the slowing, halting, or reversing the growth or progression of a
disease, infection, condition, or group of cells. The inhibition
can be greater than about 20%, 40%, 60%, 80%, 90%, 95%, or 99%, for
example, compared to the growth or progression that occurs in the
absence of the treatment or contacting.
[0047] The term "substantially" as used herein, is a broad term and
is used in its ordinary sense, including, without limitation, being
largely but not necessarily wholly that which is specified. For
example, the term could refer to a numerical value that may not be
100% the full numerical value. The full numerical value may be less
by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%,
about 7%, about 8%, about 9%, about 10%, about 15%, or about
20%.
[0048] As used herein, the term "substituted" or "substituent" is
intended to indicate that one or more (for example, 1-20 in various
embodiments, 1-10 in other embodiments, 1, 2, 3, 4, or 5; in some
embodiments 1, 2, or 3; and in other embodiments 1 or 2) hydrogens
on the group indicated in the expression using "substituted" (or
"substituent") is replaced with a selection from the indicated
group(s), or with a suitable group known to those of skill in the
art, provided that the indicated atom's normal valency is not
exceeded, and that the substitution results in a stable compound.
Suitable indicated groups include, e.g., alkyl, alkenyl, alkynyl,
alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl,
heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino,
alkylamino, dialkylamino, trifluoromethylthio, difluoromethyl,
acylamino, nitro, trifluoromethyl, trifluoromethoxy, carboxy,
carboxyalkyl, keto, thioxo, alkylthio, alkylsulfinyl,
alkylsulfonyl, and cyano. Additionally, non-limiting examples of
substituents that can be bonded to a substituted carbon (or other)
atom include F, Cl, Br, I, OR', OC(O)N(R').sub.2, CN, CF.sub.3,
OCF.sub.3, R', O, S, C(O), S(O), methylenedioxy, ethylenedioxy,
N(R').sub.2, SR', SOR', SO.sub.2R', SO.sub.2N(R').sub.2,
SO.sub.3R', C(O)R', C(O)C(O)R', C(O)CH.sub.2C(O)R', C(S)R',
C(O)OR', OC(O)R', C(O)N(R').sub.2, OC(O)N(R').sub.2,
C(S)N(R').sub.2, (CH.sub.2).sub.0-2NHC(O)R', N(R')N(R')C(O)R',
N(R')N(R')C(O)OR', N(R')N(R')CON(R').sub.2, N(R')SO.sub.2R',
N(R')SO.sub.2N(R').sub.2, N(R')C(O)OR', N(R')C(O)R', N(R')C(S)R',
N(R')C(O)N(R').sub.2, N(R')C(S)N(R').sub.2, N(COR')COR', N(OR')R',
C(.dbd.NH)N(R').sub.2, C(O)N(OR')R', or C(.dbd.NOR')R' wherein R'
can be hydrogen or a carbon-based moiety, and wherein the
carbon-based moiety can itself be further substituted. When a
substituent is monovalent, such as, for example, F or Cl, it is
bonded to the atom it is substituting by a single bond. When a
substituent is more than monovalent, such as O, which is divalent,
it can be bonded to the atom it is substituting by more than one
bond, i.e., a divalent substituent is bonded by a double bond; for
example, a C substituted with O forms a carbonyl group, C.dbd.O,
wherein the C and the O are double bonded. Alternatively, a
divalent substituent such as O, S, C(O), S(O), or S(O).sub.2 can be
connected by two single bonds to two different carbon atoms. For
example, O, a divalent substituent, can be bonded to each of two
adjacent carbon atoms to provide an epoxide group, or the O can
form a bridging ether group between adjacent or non-adjacent carbon
atoms, for example bridging the 1,4-carbons of a cyclohexyl group
to form a [2.2.1]-oxabicyclo system. Further, any substituent can
be bonded to a carbon or other atom by a linker, such as
(CH.sub.2).sub.n or (CR'.sub.2).sub.n wherein n is 1, 2, 3, or
more, and each R' is independently selected.
[0049] The term "halo" or "halide" refers to fluoro, chloro, bromo,
or iodo. Similarly, the term "halogen" refers to fluorine,
chlorine, bromine, and iodine.
[0050] The term "alkyl" refers to a branched or unbranched
hydrocarbon having, for example, from 1-20 carbon atoms or a range
in between (such as 2-8 or 3-8 carbons), and often 1-12, 1-10, 1-8,
1-6, or 1-4 carbon atoms. As used herein, the term "alkyl" also
encompasses a "cycloalkyl", defined below. Examples include, but
are not limited to, methyl, ethyl, 1-propyl, 2-propyl (iso-propyl),
1-butyl, 2-methyl-1-propyl (isobutyl), 2-butyl (sec-butyl),
2-methyl-2-propyl (t-butyl), 1-pentyl, 2-pentyl, 3-pentyl,
2-methyl-2-butyl, 3-methyl-2-butyl, 3-methyl-1-butyl,
2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl, 2-methyl-2-pentyl,
3-methyl-2-pentyl, 4-methyl-2-pentyl, 3-methyl-3-pentyl,
2-methyl-3-pentyl, 2,3-dimethyl-2-butyl, 3,3-dimethyl-2-butyl,
hexyl, octyl, decyl, dodecyl, and the like. The alkyl can be
unsubstituted or substituted, for example, with a substituent
described below. The alkyl can also be optionally partially or
fully unsaturated. As such, the recitation of an alkyl group can
include both alkenyl and alkynyl groups in various embodiments. An
alkynyl group can be, for example, acetylene (--C.ident.CH),
propargyl (--CH.sub.2C.ident.CH), butynyl (e.g.,
--CH.sub.2CH.sub.2C.ident.CH or --CH.sub.2C.ident.CCH.sub.3), or
other alkynyl groups having 5-10 carbon atoms. The alkyl can be a
monovalent hydrocarbon radical, as described and exemplified above,
or it can be a divalent hydrocarbon radical (i.e., an
alkylene).
[0051] The term "cycloalkyl" refers to cyclic alkyl groups of, for
example, from 3 to 10 carbon atoms having a single cyclic ring or
multiple condensed rings. Cycloalkyl groups include, by way of
example, single ring structures such as cyclopropyl, cyclobutyl,
cyclopentyl, cyclooctyl, and the like, or multiple ring structures
such as adamantyl, and the like. The cycloalkyl can be
unsubstituted or substituted. The cycloalkyl group can be
monovalent or divalent, and can be optionally substituted as
described for alkyl groups. The cycloalkyl group can optionally
include one or more cites of unsaturation, for example, the
cycloalkyl group can include one or more carbon-carbon double
bonds, such as, for example, 1-cyclopent-1-enyl,
1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,
1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, and the
like.
[0052] The term "heterocycloalkyl" refers to a saturated or
partially saturated monocyclic, bicyclic, or polycyclic ring
containing at least one heteroatom selected from nitrogen, sulfur,
oxygen, preferably from 1 to 3 heteroatoms in at least one ring.
Each ring is preferably from 3 to 10 membered, more preferably 4 to
7 membered. Examples of suitable heterocycloalkyl substituents
include pyrrolidyl, tetrahydrofuryl, tetrahydrothiofuranyl,
piperidyl, piperazyl, tetrahydropyranyl, morpholino, 1,3-diazapane,
1,4-diazapane, 1,4-oxazepane, and 1,4-oxathiapane. The group may be
a terminal group or a bridging group.
[0053] The term "aryl" refers to an aromatic hydrocarbon group
derived from the removal of at least one hydrogen atom from a
single carbon atom of a parent aromatic ring system. The radical
attachment site can be at a saturated or unsaturated carbon atom of
the parent ring system. The aryl group can have from 6 to 30 carbon
atoms, for example, about 6-10 carbon atoms. In other embodiments,
the aryl group can have 6 to 60 carbons atoms, 6 to 120 carbon
atoms, or 6 to 240 carbon atoms. The aryl group can have a single
ring (e.g., phenyl) or multiple condensed (fused) rings, wherein at
least one ring is aromatic (e.g., naphthyl, dihydrophenanthrenyl,
fluorenyl, or anthryl). Typical aryl groups include, but are not
limited to, radicals derived from benzene, naphthalene, anthracene,
biphenyl, and the like. The aryl can be unsubstituted or optionally
substituted.
[0054] The term "heteroaryl" refers to a monocyclic, bicyclic, or
tricyclic ring system containing one, two, or three aromatic rings
and containing at least one nitrogen, oxygen, or sulfur atom in an
aromatic ring. The heteroaryl can be unsubstituted or substituted,
for example, with one or more, and in particular one to three,
substituents, as described in the definition of "substituted".
Typical heteroaryl groups contain 2-20 carbon atoms in the ring
skeleton in addition to the one or more heteroatoms. Examples of
heteroaryl groups include, but are not limited to, 2H-pyrrolyl,
3H-indolyl, 4H-quinolizinyl, acridinyl, benzo[b]thienyl,
benzothiazolyl, .beta.-carbolinyl, carbazolyl, chromenyl,
cinnolinyl, dibenzo[b,d]furanyl, furazanyl, furyl, imidazolyl,
imidizolyl, indazolyl, indolisinyl, indolyl, isobenzofuranyl,
isoindolyl, isoquinolyl, isothiazolyl, isoxazolyl, naphthyridinyl,
oxazolyl, perimidinyl, phenanthridinyl, phenanthrolinyl,
phenarsazinyl, phenazinyl, phenothiazinyl, phenoxathiinyl,
phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyranyl,
pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl,
quinazolinyl, quinolyl, quinoxalinyl, thiadiazolyl, thianthrenyl,
thiazolyl, thienyl, triazolyl, tetrazolyl, and xanthenyl. In one
embodiment the term "heteroaryl" denotes a monocyclic aromatic ring
containing five or six ring atoms containing carbon and 1, 2, 3, or
4 heteroatoms independently selected from non-peroxide oxygen,
sulfur, and N(Z) wherein Z is absent or is H, O, alkyl, aryl, or
(C.sub.1-C.sub.6)alkylaryl. In some embodiments, heteroaryl denotes
an ortho-fused bicyclic heterocycle of about eight to ten ring
atoms derived therefrom, particularly a benz-derivative or one
derived by fusing a propylene, trimethylene, or tetramethylene
diradical thereto.
EMBODIMENTS OF THE INVENTION
[0055] This disclosure provides a compound of Formula I:
##STR00002##
or a salt thereof; wherein
[0056] X is O or S;
[0057] R.sup.1 is halo, --CN, --NO.sub.2, --(C.sub.1-C.sub.6)alkyl,
--C(.dbd.O)R.sup.a, phenyl, or a 5- or 6-membered heterocycle,
wherein R.sup.a is H, halo, --(C.sub.1-C.sub.6)alkyl,
--(C.sub.3-C.sub.6)cycloalkyl, OR.sup.b, SR.sup.b, or
--NR.sup.bR.sup.c; wherein [0058] R.sup.b is H,
--(C.sub.1-C.sub.6)alkyl, or --(C.sub.3-C.sub.6)cycloalkyl; [0059]
R.sup.c is H, --(C.sub.1-C.sub.6)alkyl, or
--(C.sub.3-C.sub.6)cycloalkyl; or [0060] when R.sup.a is
--NR.sup.bR.sup.c, Rb and R.sup.c taken together optionally forms a
heterocycle;
[0061] R.sup.2 is --(C.sub.1-C.sub.6)alkyl,
--(C.sub.3-C.sub.6)cycloalkyl, alkynyl, phenyl, or a 5- or
6-membered heterocycle; and
[0062] R.sup.3 is H, --(C.sub.1-C.sub.6)alkyl, or
--(C.sub.3-C.sub.6)cycloalkyl;
wherein each --(C.sub.1-C.sub.6)alkyl,
--(C.sub.3-C.sub.6)cycloalkyl, alkynyl, phenyl, and 5- or
6-membered heterocycle are optionally substituted with one or more
substituents, and each --(C.sub.1-C.sub.6)alkyl is unbranched or
optionally branched.
[0063] In some embodiments, the phenyl and --(C.sub.1-C.sub.6)alkyl
are each independently substituted with, for example but not
limited to, halo (e.g., one or more chloro or fluoro), alkoxy, or
aminoalkyl. In some other embodiments the substituents do not
include a phenyl group, or the molecular weight of the substituent
is less than about 100, about 90, about 80, about 70, about 60, or
about 50. In yet other embodiments, both R.sup.b and R.sup.c cannot
be H.
[0064] In other embodiments, R.sup.1 is
--C(.dbd.O)--(C.sub.1-C.sub.6)alkyl,
--C(.dbd.O)--NH(C.sub.1-C.sub.6)alkyl, or
--C(.dbd.O)--N[(C.sub.1-C.sub.6)alkyl].sub.2. In further In other
embodiments, X is O, R.sup.3 is H and R.sup.1 is
--C(.dbd.O)--(C.sub.1-C.sub.6)alkyl,
--C(.dbd.O)--NH(C.sub.1-C.sub.6)alkyl, or
--C(.dbd.O)--N[(C.sub.1-C.sub.6)alkyl].sub.2. In yet other
embodiments, X is O and R.sup.1 is
--C(.dbd.O)--(C.sub.1-C.sub.6)alkyl. In additional embodiments, X
is O, R.sup.2 is --(C.sub.1-C.sub.6)alkyl, and R.sup.3 is H. In
some other embodiments, R.sup.2 is --(C.sub.1-C.sub.6)alkyl and
R.sup.3 is H. In various other embodiments, R.sup.2 is propargyl or
a substituted phenyl. In some embodiments, X is O and R.sup.3 is
H.
[0065] In various embodiments, R.sup.1 is a moiety of Formula
IB:
##STR00003##
wherein [0066] W is O, S, or NR.sup.d; wherein R.sup.d is H,
--(C.sub.1-C.sub.6)alkyl, or --(C.sub.3-C.sub.6)cycloalkyl; [0067]
V is N or CR.sup.x, wherein R.sup.x is H, --(C.sub.1-C.sub.6)alkyl,
or --(C.sub.3-C.sub.6)cycloalkyl; [0068] Y is N or CR.sup.y,
wherein R.sup.y is H, --(C.sub.1-C.sub.6)alkyl, or
--(C.sub.3-C.sub.6)cycloalkyl; and [0069] Z is N or CH.
[0070] In various other embodiments, R.sup.1 is one of:
##STR00004##
wherein the 5-membered heterocyclic moiety R.sup.1 is optionally
substituted (at one or the other of the carbon atoms CH, thereby
modifying that carbon to C-substituent, wherein the substituent is
a substituent as defined herein).
[0071] In further embodiments, R.sup.1 is i, ii, or iii:
##STR00005##
wherein (i), (ii) and (iii) are optionally substituted at position
4 or 5.
[0072] In additional embodiments, R.sup.1 is a para-substituted
phenyl, wherein the molecular weight of each substituent is less
than about 300, about 200 or about 100 daltons. In yet other
embodiments, the para-substituent is halo, --CN, --CF.sub.3,
--CF.sub.2CF.sub.3, or --(C.sub.1-C.sub.6)alkyl. In some other
embodiments, R.sup.1 is halo, --(C.sub.1-C.sub.6)alkyl, or
--(C.sub.3-C.sub.6)cycloalkyl.
[0073] In various additional embodiments, the substituted phenyl is
substituted with halo, alkyl, alkoxy, phenoxy, amine, alkylamine,
dialkylamine, or combination thereof.
[0074] In other embodiments, the compound is:
##STR00006##
[0075] In additional embodiments, the compound of Formula I is a
compound of Formula IC:
##STR00007##
wherein G.sup.1 is halo, alkyl, alkoxy, phenoxy, or dialkylamine.
In some embodiments G.sup.1 is OCH.sub.3, OCH.sub.2CH.sub.3, OPh,
or N(CH.sub.3).sub.2.
[0076] In various other embodiments, the compound of Formula I is a
compound of Formula II:
##STR00008##
[0077] In additional embodiments, R.sup.2 is
--(C.sub.1-C.sub.6)alkyl and R.sup.3 is H. In yet other
embodiments, the compound is K-TMZ:
##STR00009##
[0078] In further embodiments, R.sup.a is CH.sub.3,
CH.sub.2CH.sub.3, NHCH.sub.3, NHCH.sub.2CH.sub.3,
N(CH.sub.3).sub.2, N(CH.sub.2CH.sub.3).sub.2,
N(CH.sub.2CH.sub.2CH.sub.3).sub.2,
N(CH.sub.2CH.sub.2CH.sub.2CH.sub.3).sub.2,
N(CH.sub.2CH.sub.2).sub.2, N[(CH.sub.2CH.sub.2).sub.2O], OCH.sub.3,
OCH.sub.2CH.sub.3, SCH.sub.3, or SCH.sub.2CH.sub.3. In other
embodiments, the compound is:
##STR00010##
[0079] In additional embodiments, the compound of Formula I is a
compound of Formula IIIA or IIIB:
##STR00011##
wherein R.sup.z is H, halo, --(C.sub.1-C.sub.6)alkyl, or
--(C.sub.3-C.sub.6)cycloalkyl. In other embodiments, R.sup.z is
CH.sub.3 or CH.sub.2CH.sub.3. In yet other embodiments, the
compound is:
##STR00012##
[0080] This disclosure additionally provides a method of treating a
cancer comprising administering to a subject in need thereof a
therapeutically effective amount of the compound of any formula
described herein, wherein the cancer is thereby treated. In other
additional embodiments, the cancer is glioblastoma (GBM).
[0081] In some embodiments, a composition comprises the compounds
disclosed above and a second active agent. In other embodiments,
the second active agent is a procaspase-3 activator, for example
PAC-1:
##STR00013##
[0082] In yet further embodiments, the disclosed compounds herein
and the second active agent are administered to a subject
concurrently or sequentially for the treatment of a cancer. In some
additional embodiments, the disclosed compound and the second
active agent are concurrently administered to the subject. In other
embodiments, the disclosed compound and the second active agent are
sequentially administered to the subject. In some other
embodiments, the disclosed compound is administered to the subject
before the second active agent. In yet more embodiments, the
disclosed compound is administered to the subject after the second
active agent.
[0083] In some embodiments, the concentration of the disclosed
compounds herein is about 1 nM to about 10 .mu.M, or corresponding
mg active agent/kg body weight of the subject, as would be
recognized by one of skill in the art. In yet other embodiments,
the concentration of the second active agent is about 1 nM to about
1 .mu.M.
[0084] This disclosure provides ranges, limits, and deviations to
variables such as volume, mass, percentages, ratios, etc. It is
understood by an ordinary person skilled in the art that a range,
such as "number1" to "number2", implies a continuous range of
numbers that includes the whole numbers and fractional numbers. For
example, 1 to 10 means 1, 2, 3, 4, 5, . . . 9, 10. It also means
1.0, 1.1, 1.2. 1.3, . . . , 9.8, 9.9, 10.0, and also means 1.01,
1.02, 1.03, and so on. If the variable disclosed is a number less
than "number10", it implies a continuous range that includes whole
numbers and fractional numbers less than number10, as discussed
above. Similarly, if the variable disclosed is a number greater
than "number10", it implies a continuous range that includes whole
numbers and fractional numbers greater than number10. These ranges
can be modified by the term "about", whose meaning has been
described above.
Results and Discussion
[0085] Construction of C8-Substituted Imidazotetrazines.
[0086] The inclusion of an amide at the C8 position of TMZ is
largely an artifact of the original synthesis of imidazotriazenes
and imidazotetrazines. Both dacarbazine and TMZ are derived from
precursor 4-diazoimidazole-5-carboxamide (1, Scheme 1b). The
remarkable stability of this diazo species, reportedly >2.5
years at room temperature,.sup.22 permitted its use for exploratory
chemistry where other diazoimidazole species (such as
4-diazoimidazole (2)) simply decomposed..sup.23 Thus, the initial
synthesis of dacarbazine in 1962 and TMZ in 1984 involved the
quenching of 1 with dimethylamine.sup.24 or the cyclization of 1
with methyl isocyanate,.sup.25 respectively, and the primary amide
moiety remained. Over time, there have been suggestions that this
amide is critical for anticancer activity. Such claims were
supported by theoretical studies suggesting that a hydrogen bond
donor at C8 is required for activity,.sup.2,16 but clouding the
picture is a conflicting structure-activity relationship (SAR)
adopted from derivatives of a related compound (mitozolomide) in
non-CNS cancer models..sup.26 There are considerable challenges to
establishing a general synthetic route that can be used to
construct novel derivatives at the C8 position; these synthetic
challenges have hindered the development of new imidazotetrazines,
and in the absence of new compounds and biological data, the
outdated SAR has persisted.
##STR00014## ##STR00015##
Challenges of replacing C8 amide include: aqueous sensitivity; base
sensitivity; diazoimidazole degradation; and poor substitutes for
CH.sub.3NCO.
[0087] Key challenges to making novel imidazotetrazines include
sensitivity to protic solvents or basic reagents, instability of
intermediate diazo species, and the lack of efficient reagents to
install the N3 methyl. The sensitivity of the prodrug to conditions
involving base or water (at pH>6) renders the tetrazinone
unstable to many practical cross-coupling or reducing conditions.
Another challenge, as alluded to above, is the instability of
intermediate diazo species. The privileged
4-diazoimidazole-5-carboxamide (1, Scheme 1b) readily precipitates
out of solution as a pure, stable compound; however, other
4-diazoimidazoles (such as 2) remain in aqueous solution, are
exceptionally prone to decomposition, and are sensitive to heat,
shock, and often light..sup.23 Finally, installation of the
N3-methyl group in the initial route to TMZ was achieved via
cyclization with methyl isocyanate..sup.25 Methyl isocyanate,
however, is a poisonous gas and no longer commercially available.
As such, alternate routes.sup.27 or less effective alternatives to
methyl isocyanate such as N-succinimidyl N-methylcarbamate or
N-methylcarbamic chloride must be used that reduce the yield of the
cyclizations.
[0088] To provide access to certain derivatives of the C8 amide, an
exploration of these types of compounds was begun by modifying an
established route, largely developed for mitozolomide..sup.28 This
sequence begins with a hydrolysis of the amide of TMZ to carboxylic
acid 3 (Scheme 2a), which can then be converted to the acid
chloride. From this intermediate, substitution with various
nucleophiles provides products in high yields. This route was used
to synthesize amide, ester, and thioester derivatives 4-10 (Scheme
2a). Additionally, an established reaction was employed to install
a cyano group (11) directly from TMZ (Scheme 2a)..sup.29 The
creation of a structurally diverse panel of C8 analogs, however,
would require novel synthetic routes, especially for those with
aliphatic, ketone, halogen, and aryl groups; such substituents have
not been described at this position in the .about.35 year history
of TMZ. Thus, an aliphatic group at C8 was introduced via
diazotization of 5-amino-4-methylimidazole 12 to diazo species 13
and subsequent cyclization with methyl isocyanate surrogate
N-methylcarbamoyl chloride to afford C8-methyl derivative 14
(Scheme 2b).
[0089] Although various amides, esters, and thioamides had been
installed at C8, ketones were entirely absent, perhaps
unsurprisingly since initial attempts to use Grignard or
alkyllithium reagents led to complete degradation of the
tetrazinone ring. Thus, a stepwise cyclization was utilized to
synthesize methyl ketone derivative 17 from its disubstituted
precursor 16, obtained upon hydrolytic degradation of
6-methylpurine N-oxide (15).sup.30 (Scheme 2c). Bromine and
chlorine substituents were directly incorporated at C8 in moderate
yields upon a decarboxylative halogenation of intermediate 3
employing Dess-Martin periodinane and the respective
tetraethylammonium salt (compounds 18 and 19, Scheme 2a). This
strategy had not previously been applied to imidazoles and endows
potential points of diversity in addition to representing novel
derivatives themselves. Using 18 as a cross coupling partner,
however, was unsuccessful due to the basic, aqueous conditions
required. Instead, a Suzuki coupling fashioned
5-nitro-4-phenylimidazole (21) from the 5-nitro-4-bromoimidazole
(20) precursor, which could be subsequently reduced to
corresponding amine 22 and cyclized to the phenyl-substituted
imidazotetrazine as above (Scheme 2d). This method supplied 23 as
well as a small series of p-substituted aryl derivatives 24-26.
Finally, heterocyclic compounds 27 and 28 (Scheme 2e) were
synthesized upon cyclization of the C8 amide or thioamide,
respectively; an analogous route had been utilized to introduce
bulkier 4-substituted oxazoles and thiazoles at the C8
position,.sup.19 but not smaller methyl groups.
[0090] Anticancer Activity of C8 Substituted Imidazotetrazines.
[0091] With a suite of imidazotetrazines in hand, each compound was
evaluated against a panel of human GBM cell lines (Table 1a, Table
1b). Cell lines were selected to include those expressing and
lacking MGMT (FIG. 6) and, consistent with literature reports,
those with negligible MGMT expression were sensitive to TMZ
(IC.sub.50.about.50 .mu.M or less) whereas those with significant
MGMT expression were resistant (IC.sub.50>300 .mu.M).
Amide-substituted derivatives 4-8 as well as ester (9) and
thioester (10) derivatives had activity comparable to TMZ in the
MGMT-deficient U87 and D54 cell lines. Notably, the retention of
activity for disubstituted amide (5-8) and ester (9)
imidazotetrazines confirms that a hydrogen bond donor is not
required at C8. In U118MG and T98G MGMT-expressing GBM cells, more
potent activity was observed for these derivatives compared to
TMZ.
[0092] Ketone analog 17 was also effective against MGMT-deficient
cell lines, demonstrating that an amide is not required at the C8
position. Compounds completely lacking a carbonyl, such as 14, 19,
23, and 27 proved to be as (or more) potent than TMZ in the absence
of MGMT and significantly more potent in cell lines expressing
MGMT. Methyl (14) and phenyl (23) substitutions were the most
active across all cell lines. Cyano derivative 11 and carboxylic
acid derivative 3 were inactive in all tested cell lines
(>7-fold less potent than TMZ), even in the absence of MGMT. In
addition to these canonical adherent GBM cell lines, most analogs
were more active than TMZ in the patient-derived U3054MG GBM cell
line cultured under serum-free stem cell conditions..sup.31
##STR00016## ##STR00017##
[0093] This disclosure therefore also provides methods for
preparing compounds of the formulas described herein using the
methods outlined in the schemes above, wherein any alkyl (e.g.,
methyl) or phenyl of the structures in the schemes can be replaced
with other substituents, such as alkyl or phenyl groups, including
substituted alkyl and/or phenyl groups. Substituents on the alkyl
and/or phenyl groups can be one or more substituents as recited in
the definition of a substituent above.
[0094] In various embodiments, the invention provides a method of
preparing a substituted imidazotetrazine of a formula described
herein comprising the steps outlined in Scheme 2 above, for
example, the steps as substantially shown in part a), part b), part
c), or part d) of Scheme 2, wherein any alkyl or phenyl group shown
in the relevant scheme can be illustrated as a general R group that
is defined as alkyl or phenyl, each optionally substituted and/or
optionally unsaturated (for the alkyl). For example, C-8
substituted imidazotetrazines can be prepared by the method of
Scheme 2, part b) wherein the methyl substituent of imidazole 12 is
a different alkyl group, such as a (C.sub.2-C.sub.10)alkyl,
optionally branched and/or optionally substituted, and the N-methyl
group of product 14 can have various alkyl or phenyl groups on the
corresponding nitrogen in place of the methyl of that N-methyl
group. Various alkynyl groups can be present in the final product,
for example, at locations C-8 and/or N-2. Relevant imidazole
starting materials can be purchased commercially or prepared by
known methods. Numerous methods for preparing substituted
imidazoles are well-known in the art.
TABLE-US-00001 TABLE 1a Panel of C8-substituted imidazotetrazines
and associated IC.sub.50 values (.mu.M) in multiple GBM cell lines.
Cell lines were incubated with compound for 7 days then viability
was assessed using the Alamar Blue assay. Error is SEM, n .gtoreq.
3. Prl = pyrrolidine. A table with additional compounds (Table 1b)
and a Western blot for MGMT status of all cell lines used (FIG. 6)
are disclosed herein. ##STR00018## MGMT - - + + + Compound R U87
D54 U118MG T98G U3054MG TMZ CONH.sub.2 51 .+-. 8 12 .+-. 1 322 .+-.
7 660 .+-. 10 370 .+-. 40 3 COO.sup.- 320 .+-. 7 130 .+-. 8 370
.+-. 20 321 .+-. 8 ND 4 (Me-TMZ) CONHMe 49 .+-. 7 11 .+-. 1 280
.+-. 20 580 .+-. 20 290 .+-. 20 5 (DiMe-TMZ) CONMe.sub.2 40 .+-. 20
12 .+-. 5 130 .+-. 30 250 .+-. 60 132 .+-. 6 6 CONEt.sub.2 80 .+-.
30 13 .+-. 2 80 .+-. 10 160 .+-. 40 ND 7 CON(n-Bu).sub.2 27 .+-. 6
11 .+-. 2 62 .+-. 2 140 .+-. 20 ND 8 CO(Prl) 17 .+-. 3 12 .+-. 4
136 .+-. 8 186 .+-. 5 ND 9 COOEt 66 .+-. 3 7 .+-. 1 180 .+-. 20 236
.+-. 10 ND 10 COSEt 64 .+-. 21 8 .+-. 1 165 .+-. 4 327 .+-. 4 ND 11
CN 500 .+-. 60 91 .+-. 6 670 .+-. 20 >1000 870 .+-. 60 14 Me 3
.+-. 1 6 .+-. 3 7 .+-. 1 6 .+-. 1 ND 17 (K-TMZ) COMe 44 .+-. 6 18
.+-. 1 115 .+-. 9 240 .+-. 20 125 .+-. 4 19 Cl 15 .+-. 4 21 .+-. 4
60 .+-. 20 60 .+-. 10 87 .+-. 4 23 Ph 9 .+-. 1 7 .+-. 1 3 .+-. 1 14
.+-. 1 18 .+-. 1 27 (Ox-TMZ) 5-Me-Oxaz 27 .+-. 4 9 .+-. 1 70 .+-.
20 100 .+-. 20 123 .+-. 6 28 4-Me-Thiaz 9 .+-. 1 8 .+-. 1 12 .+-. 2
31 .+-. 4 ND
[0095] Hydrolytic Stability of C8 Substituted
Imidazotetrazines.
[0096] The principal aspect governing the anticancer activity of
imidazotetrazines is the hydrolytic activation of the prodrug. As
depicted in Scheme 1(a), TMZ has a half-life of .about.2 hours in
humans..sup.7 This timeline allows the intact prodrug to reach the
brain and release the active methylating component prior to
elimination. Beyond TMZ, the relationship between imidazotetrazine
stability and anticancer activity is unknown; that is, while
hydrolytic activation is required for cancer cell death, the
optimal timing of this event is unclear both in vitro and in vivo.
Towards this end, the hydrolytic stability of each new compound was
assessed in buffered saline, which mimics in vivo conditions (in pH
7.4 PBS TMZ has a half-life of 119 minutes, FIG. 1). A HPLC assay
was developed to quantify the fraction of intact prodrug remaining
in solution after 2 hours at pH 7.4, 37.degree. C. The results of
this experiment suggest that electronic substituent effects at C8
directly translate through the bicycle to C4, the site of
hydrolysis. The magnitude of this effect was dramatic, with
stabilities ranging from 0% to 97% remaining after 2 hours
depending on the substituent at the C8 position (FIG. 1). Since the
group at C8 appeared to have such a clear influence on the aqueous
stability of the prodrug, its Hammett constant (.sigma..sub.p) was
plotted against the percent remaining after 2 hours. As shown in
FIG. 1, an obvious relationship exists between these two
parameters, suggesting that .sigma.p can be used to accurately
predict the stability of C8-substituted imidazotetrazines. Among
those compounds possessing substituents with similar electronics
(0.23<.sigma..sub.p<0.50) to a primary amide
(.sigma..sub.p=0.36) were amide derivatives 4 and 5, ketone
derivative 17, and chloro derivative 19. Each had measured
half-lives within an hour of TMZ in PBS at pH 7.4 (Table 1c). On
either extreme were cyano analog 11 (.sigma..sub.p=0.66), with a
half-life of 0.5 h, and methyl derivative 14 (.sigma..sub.p=-0.17),
which remained in its prodrug form the longest with a half-life of
40 hours. The same assay was used to confirm that hydrolysis
remained pH-dependent for C8-substituted derivatives (e.g. K-TMZ
17, FIG. 7).
TABLE-US-00002 TABLE 1b Panel of C8-substituted imidazotetrazines
and associated 7-day IC.sub.50 values (.mu.M) in multiple GBM cell
lines; the four compounds below were tested, and this supporting
table is a complement to Table 1a. Cell viability was assessed
using the Alamar Blue assay. Error is SEM, n .gtoreq. 3.
##STR00019## MGMT - - + + Compound R U87 D54 U118MG T98G TMZ
CONH.sub.2 51 .+-. 8 12 .+-. 1 322 .+-. 7 660 .+-. 10 18 Br 26 .+-.
7 20 .+-. 1 80 .+-. 20 60 .+-. 10 24 C.sub.6H.sub.4-4-F 13 .+-. 2
ND ND 18 .+-. 2 25 C.sub.6H.sub.4-4-CF.sub.3 16 .+-. 1 ND ND
>100 26 C.sub.6H.sub.4-4-Cl 9 .+-. 1 7 .+-. 1 5 .+-. 1 19 .+-.
3
TABLE-US-00003 TABLE 1c Half-lives of select C8 derivatives in PBS
(pH 7.4, 37.degree. C.). Compound t.sub.1/2 (h) 11 0.57 .+-. 0.03
17 (K-TMZ) 1.20 .+-. 0.10 TMZ 1.98 .+-. 0.01 4 (Me-TMZ) 2.70 .+-.
0.10 5 (DiMe-TMZ) 2.80 .+-. 0.20 25 2.90 .+-. 0.30 27 (Ox-TMZ) 3.00
.+-. 0.10 19 3.10 .+-. 0.10 23 27 .+-. 3 14 40 .+-. 1
[0097] Relationship Between Hydrolytic Stability and Anticancer
Activity.
[0098] Methyl and phenyl derivatives 14 and 23 were consistently
the most potent compounds in each of the tested cell lines (Table
1a). Interestingly, they also possessed electron-donating
substituents and, accordingly, the greatest aqueous stability (FIG.
1), suggesting that a longer-lived prodrug is favorable for
efficacy in cell culture. The opposite effect was observed for
compound 11, which was the least stable in solution. Even in U87
cells lacking MGMT, it exhibited a ten-fold loss of activity
compared to TMZ, suggesting that there is a critical threshold of
aqueous stability below which hydrolysis occurs too quickly to
methylate target DNA. Compounds with hydrolytic stabilities similar
to TMZ such as 4, 5, 17, 19, and 27 retained activity in culture.
Notably, ketone derivative 17 was equipotent to TMZ even with a
shorter aqueous half-life, indicating that compounds with
.sigma..sub.p.about.0.50 can still retain marked anticancer
activity.
[0099] Liver Microsome Stability.
[0100] TMZ fortuitously possesses several ideal pharmacokinetic
properties including avoidance of primary metabolism..sup.7 To
assess whether modification or replacement of the amide at C8 would
lead to significant metabolic liabilities, the stabilities of
select compounds were assessed after 2 hours in the presence of
mouse liver microsomes. Prodrug hydrolysis was accounted for by
including control runs that did not contain liver microsomes. The
slightly acidic pH of the working solution resulted in enhanced
stability of TMZ compared to incubation in PBS alone. Predictably,
TMZ was insensitive to metabolic perturbation as its instability
was entirely accounted for by hydrolysis (Table 2a). The addition
of methyl(s) to the amide (compounds 4 and 5) resulted in some
susceptibility to the effects of the microsomes, and this effect
was amplified for larger amide substitutions (compound 7), which
demonstrated improved aqueous stability but markedly less stability
in liver microsomes. Ketone 17 and chloro 19 were generally stable
to oxidative metabolism, suggesting that for these compounds the
hydrolysis could drive the pharmacokinetics in vivo, similar to
TMZ.
[0101] Blood-Brain Barrier Penetrance.
[0102] It has been reported that >98% of small molecule drugs do
not penetrate the BBB,.sup.32 making TMZ unusual, especially
amongst anticancer agents. In humans, TMZ is rapidly absorbed and
reaches the brain in minutes with cerebral spinal fluid
concentrations averaging 20% of those in the plasma;.sup.8,9 the
accumulation of even more drug in the brain by increasing the BBB
penetrance may be a viable strategy to increase efficacy against
CNS-based tumors. To predict the BBB penetrance of the novel
imidazotetrazines, log BB values were calculated (cLogBB) based on
a formula utilizing cLogP and total polar surface area..sup.33 When
applied across a consistent drug scaffold, these types of in silico
metrics have been used reliably to predict relative changes in BBB
penetrance as well as other biological phenomenon,.sup.34-38 though
not always reflective of absolute concentrations. The cLogBB value
for TMZ is -1.58 (Table 2a). Replacing the primary amide led to
marked increases in the cLogBB and larger predicted brain:blood
ratios relative to TMZ. Importantly, cLogBB does not account for
molecular weight, making one wary of analogs with large,
hydrophobic functionality (e.g. 7) even if they possess attractive
predicted values. A more comprehensive metric, the CNS
multiparameter optimization (MPO) tool.sup.39,40 was also employed
to gauge prospective BBB permeabilities. CNS MPO scores span from 0
to 6.0 based on the optimal ranges of 6 physicochemical properties.
Though TMZ has an agreeable MPO of 4.9, higher scores were achieved
for the C8 analogs, which in several cases reached the top
desirability value (Table 2a). The more favorable cLogBB and CNS
MPO values predicted for the panel suggests that certain
derivatives may achieve higher drug concentrations in the brain
than TMZ.
[0103] The BBB penetrance of top compounds (those with favorable
anticancer activity, appropriate hydrolytic and liver microsome
stability, and predicted BBB penetrance, Scheme 2b) was thus
assessed in vivo. In an initial experiment, Me-TMZ (4) and DiMe-TMZ
(5) were tested head-to-head with TMZ to explore whether alkylation
of the C8 amide could confer increased brain:blood ratios. Mice
were administered 25 mg/kg drug intravenously and sacrificed 5
minutes after injection. The serum and perfused brain samples were
immediately acidified to prevent prodrug degradation before the
drug concentration within each compartment was quantitated by
LC-MS/MS. After 5 minutes, drug concentrations in the brain were
significantly elevated for analogs Me-TMZ and DiMe-TMZ versus TMZ,
a >3-fold increase in brain:serum ratio for each compound (FIG.
2a). The equivalent brain:serum ratios for Me-TMZ and DiMe-TMZ is
likely due to the fast metabolism of the dimethylated amide to its
monomethylated counterpart. This preliminary experiment suggested
that other derivatives with higher predicted BBB penetrance may
lead to greater brain permeability in vivo. Accordingly, compounds
Ox-TMZ (27) and K-TMZ (17) were evaluated head-to-head with
DiMe-TMZ and TMZ. After 5 minutes, each derivative had accumulated
numerically higher concentrations in the brain than TMZ (FIG. 2b).
When paired with the corresponding serum concentrations (FIG. 2c),
TMZ had a relative brain:serum ratio of 0.23.+-.0.03 ng/g:ng/mL,
comparable to the few other TMZ biodistribution experiments in
murine systems..sup.41,42 Assigning average mouse blood volumes to
equate units, TMZ had an absolute brain:serum ratio of 8:92, while
Ox-TMZ and K-TMZ boasted brain:serum ratios of 55:45 and 69:31,
respectively. (FIG. 2d). The dramatic differences in drug
partitioning suggest that replacing the amide at C8 is a viable
strategy to significantly increase local drug concentration in the
brain relative to the blood, which may increase effectiveness
against brain tumors and also reduce hematological toxicity.
##STR00020##
TABLE-US-00004 TABLE 2a Metabolic stability, cLogBB, and CNS MPO
values for relevant C8 analogs. Stability Stability Compound (2h,
Microsomes) (2h, No Microsomes) cLogBB CNS MPO Propranolol 68 .+-.
2% 102 .+-. 3% ND ND TMZ 87 .+-. 6% 86 .+-. 4% -1.58 4.9 4 (Me-TMZ)
86 .+-. 1% 93 .+-. 1% -1.34 5.7 5 (DiMe-TMZ) 81 .+-. 2% 92 .+-. 3%
-1.18 6.0 6 81 .+-. 1% 95 .+-. 2% -1.07 6.0 7 1 .+-. 1% 98 .+-. 3%
-0.78 5.6 17 (K-TMZ) 70 .+-. 1% 77 .+-. 3% -1.08 6.0 19 91 .+-. 3%
91 .+-. 1% -0.72 6.0 23 44 .+-. 2% 103 .+-. 5% -0.56 5.7 27
(Ox-TMZ) 71 .+-. 1% 95 .+-. 4% -1.19 5.9 The metabolic stability
was assessed in mouse liver microsomes. Compounds were incubated in
microsomes for 2 h, then the percentage remaining was quantified
relative to t0. Experiments assessing stability in the absence of
microsomes were identical but replaced liver microsomes with PBS.
Error is SEM, n .gtoreq. 2. Internal standard = N3-propyl TMZ. CNS
MPO = Central Nervous System Multiparameter Optimization Score.
[0104] Assessment of Hematological Toxicity.
[0105] The elevated brain concentrations and dramatically decreased
serum concentrations (FIG. 2b, FIG. 2c) observed upon treatment
with Ox-TMZ and K-TMZ compared to TMZ indicated that these
C8-modified imidazotetrazines attenuate the dose-limiting
hematological toxicity observed for TMZ in the clinic. To test this
hypothesis, mice were treated with a single dose of 125 mg/kg TMZ,
Ox-TMZ, or K-TMZ intravenously; this dose of TMZ induces non-lethal
toxicity in mice..sup.43,44 Seven days post-treatment, whole blood
was collected and complete blood counts were obtained for each
individual mouse. Expectedly, a dose of 125 mg/kg TMZ led to white
blood cell (WBC) depletion relative to control mice (FIG. 3a),
suggestive of drug-induced myelosuppression. Both lymphocyte (FIG.
3b) and neutrophil (FIG. 8a) concentrations were decreased in
TMZ-treated mice. Conversely, treatment with 125 mg/kg of Ox-TMZ or
K-TMZ did not produce myelosuppression. Total WBC, lymphocyte, and
neutrophil counts for mice treated with these compounds were
equivalent to those of control mice. Notably, the novel
imidazotetrazines did not give rise to other hematological symptoms
such as red blood cell (RBC) toxicity (FIG. 8b) or thrombocytopenia
(FIG. 8c) and did not lead to weight loss 7 days post-treatment
(Table 2b).
TABLE-US-00005 TABLE 2b Cohort weights of mice prior to treatment
and at the time of blood collection 7 days post-treatment. Compound
Pre-treatment (g) 7 days Post (g) Control 31 .+-. 1 32 .+-. 1 TMZ
31 .+-. 1 32 .+-. 1 K-TMZ 30 .+-. 1 31 .+-. 1 Ox-TMZ 30.7 .+-. 0.1
31.6 .+-. 0.3 Error is SEM, number of mice per cohort = 4 (see FIG.
8).
[0106] Novel Imidazotetrazines Induce Alkylation-Mediated Cancer
Cell Death.
[0107] The cytotoxicity of TMZ is mediated by methylation of
O.sup.6 guanine; subsequent single- and double-strand breaks and
apoptosis are facilitated by the mismatch repair system..sup.2-6 To
assess if the novel imidazotetrazines kill through the same
mechanism, O.sup.6-methylguanine adducts were quantitated in U87
cells treated with 100 or 1000 .mu.M of each imidazotetrazine.
After 8 hours of incubation with compound, the genomic DNA was
isolated, quantified, and hydrolyzed to its constituent
deoxyribonucleosides, which were quantitated via LC-MS/MS analysis.
Dose dependent increases in the concentration of O.sup.6-methylated
deoxyguanosine were observed for TMZ as well as each of the lead
compounds (Table 2c), indicating that DNA methylation is occurring.
Further confirmation that the novel compounds remain DNA alkylators
was obtained upon pre-treatment with MGMT inhibitor
O.sup.6-benzylguanine (O6BG). O6BG is a pseudosubstrate for MGMT
that quenches cellular stores of the enzyme, leading to the
persistence of O.sup.6-methylguanine DNA adducts. Pre-incubation of
MGMT-expressing T98G cells with O6BG (100 .mu.M) led to an
eight-fold enhancement in cytotoxicity for TMZ (FIG. 4), consistent
with literature reports..sup.45,46 Similarly, DiMe-TMZ, Ox-TMZ, and
K-TMZ demonstrated a significant increase in activity when
administered after O6BG, suggesting that O.sup.6-methylguanine
lesions are the cause of cell death.
[0108] Novel Imidazotetrazines have Superior Activity in Mouse
Models of GBM.
[0109] The increased BBB penetrance observed for amide derivatives
(Me-TMZ and DiMe-TMZ) relative to TMZ suggested that greater drug
concentrations in the brain might lead to greater efficacy in an
intracranial tumor model. GBM oncosphere lines were chosen for
these studies as they more accurately recapitulate the genetic and
histopathological features of human GBM than traditional adherent
cell lines, which are passaged in serum and typically grow as
compact masses in vivo..sup.47 The Br23c GBM oncosphere cell line
does not express MGMT, was sensitive to TMZ and the novel
C8-substituted imidazotetrazines (Table 2d), and was thus chosen as
the model system. Mice implanted intracranially with these cells
were administered 15 mg/kg TMZ or the equimolar equivalent of
Me-TMZ or DiMe-TMZ once-per-day, 5.times./week via oral gavage. As
expected, TMZ significantly increased median survival compared with
vehicle (FIG. 5a). Mice treated with both Me-TMZ and DiMe-TMZ,
however, outperformed TMZ and increased median survival by 24% and
46%, respectively, suggesting that increasing the BBB-permeability
of imidazotetrazine prodrugs is a viable strategy to improve
efficacy. In a second experiment, K-TMZ was selected for evaluation
due to its most favorable brain:blood ratio (FIG. 2d). Mice
intracranially implanted with Br23c cells were treated with K-TMZ
(via oral gavage), which led to an extended median survival of more
than 50 days past TMZ-treated mice, and showed greater efficacy
even compared to DiMe-TMZ (FIG. 5b). Importantly, methyl derivative
14, which has excellent efficacy in cell culture but an extended
(40 hour) half-life in aqueous solution, had no effect in this in
vivo model (FIG. 9) suggesting that dramatically elongated
half-lives are detrimental in vivo, likely due to compound
clearance prior to hydrolytic activation.
TABLE-US-00006 TABLE 2c The concentration of O.sup.6-methylguanine
was measured in U87 cells (10 .mu.g DNA) after treatment with 100
or 1000 .mu.M of imidazotetrazine for 8 hours. IC.sub.50 Values
(.mu.M) Compound (-) O6BG (+) O6BG Fold Change TMZ 660 .+-. 10 81
.+-. 8 8 DiMe-TMZ 250 .+-. 60 25 .+-. 9 10 Ox-TMZ 100 .+-. 20 5
.+-. 1 20 K-TMZ 240 .+-. 20 32 .+-. 4 8
CONCLUSION
[0110] Despite being known since 1984, FDA approved since 1999, and
reaching $1 billion in sales in 2009, TMZ remains the only approved
imidazotetrazine anticancer drug; this likely stems from the lack
of generalized syntheses for this class of compounds prohibiting
conventional medicinal chemistry campaigns. Herein is reported new
synthetic methods that enable the construction of novel
C8-substituted imidazotetrazines that were previously inaccessible.
Evaluation of these compounds in systematic, head-to-head assays
led to the definitive conclusion that the C8 amide is not required
for anticancer activity, and indeed compounds lacking an H-bond
donor or acceptor (or both) at C8 can still retain activity
comparable to TMZ against cancer cells in culture. Unmoored from
the necessity of an amide at C8, a panel of imidazotetrazines was
synthesized, varying this position. Strikingly, the electronic
properties of the substituent at C8 has a dramatic effect on the
activation of the corresponding prodrug, a previously undefined
phenomenon. The relationship derived herein between the hydrolytic
stability of imidazotetrazines and the electronics at C8 allows the
stability of the prodrug to be tuned by employing easily accessible
.sigma..sub.p values, enabling the rational design of TMZ
derivatives that have similar stabilities in vivo and facilitating
investigation into the optimal timing of imidazotetrazine prodrug
activation.
[0111] From this work it appears that compounds with very short
half-lives (such as 11, t.sub.1/2=0.57 h) simply hydrolyze too
rapidly, releasing methyl diazonium prior to accumulation in the
DNA microenvironment and diminishing anticancer activity. Thus, for
activity against cancer cells in culture, a half-life of 1 h or
greater is optimal. Conversely, compounds that have very long
half-lives (such as 14 or 23, t.sub.1/2>20 h) can be distinctly
more potent than TMZ in cell culture as the prodrug has ample time
to distribute to the nucleus before conversion to the active
methylating agent. However, these compounds with markedly increased
hydrolytic stabilities are less likely to be useful in vivo as
elimination through alternate pathways (excretion of the intact
prodrug, oxidative metabolism, etc.) will occur before activation
to the alkylating species. This hypothesis accounts for the lack of
in vivo efficacy of compound 14.
[0112] A hallmark of GBM is its invasion into surrounding brain
tissue at an early stage, making cure via surgical resection
unachievable. As such, there is an obvious clinical need for
improved compounds that can reach the entirety of the diffuse tumor
in sufficient concentrations to be effective. Importantly, the data
herein shows that the BBB-penetrance of imidazotetrazines can be
improved through modifications at the C8 position. The dramatically
enhanced brain:serum distribution of Ox-TMZ and K-TMZ, in
particular, could provide substantial improvement over TMZ for
treatment of CNS cancers. Both of these compounds retain the
favorable features of TMZ (timely prodrug activation, stability to
liver microsomes) while also accumulating higher drug concentration
in the brain and reduced concentration in the blood. It was
hypothesized that partitioning the imidazotetrazine more to the
site of the tumor and less to the compartment responsible for
adverse effects would expand the therapeutic window by enhancing
anticancer activity while simultaneously reducing systemic
toxicity. Myelotoxicity occurs in .about.20% of TMZ-treated
patients, is the major dose-limiting toxicity,.sup.48 and is
exacerbated in elderly and female GBM patients..sup.49,50 Ox-TMZ
and K-TMZ demonstrated significantly less in vivo toxicity to WBCs
compared to TMZ, likely a direct result of the increased
partitioning to the CNS. Imidazotetrazines such as these with lower
toxicity profiles could permit elevated dosing schedules and
additional anticancer efficacy, and/or make this drug class
accessible to more patients.
[0113] Other imidazotetrazines of various composition in the
literature have failed to improve median survival head-to-head
compared to TMZ in preclinical models, despite promising results in
cell culture..sup.42,51,52 Only one derivative has outperformed TMZ
in an intracranial murine model of GBM, bestowing a modest 10%
increase in median survival..sup.53 Clearly, the interplay between
retaining the favorable properties that have kept TMZ as frontline
treatment for GBM and modulating its structure is not trivial. The
data reported herein now suggest that imidazotetrazines may be
substantially modified without losing these advantages, and indeed
such new compounds can have dramatically enhanced in vivo efficacy.
TMZ remains the gold-standard for treating the most aggressive
brain tumors, shows promise against brain metastases from other
cancers,.sup.54 and its predictable activity (based on clinical
biomarkers) has recently led to advocation for an expanded use of
TMZ in the management of diverse cancer types..sup.55 As such, the
novel imidazotetrazines reported here could hold considerable
promise for treatment of GBM and other cancers.
TABLE-US-00007 TABLE 2d 7-day IC50 values (.mu.M) for TMZ and lead
C8-substituted imidazotetrazines in the Br23c GBM oncosphere cell
line. Cell viability was assessed using the Alamar Blue assay.
Error is SEM, n .gtoreq.0 3. ##STR00021## Compound R MGMT - Br23C
TMZ CONH.sub.2 5.2 .+-. 0.2 4 (Me-TMZ) CONHMe 6 .+-. 1 5 (DiMe-TMZ)
CONMe.sub.2 6 .+-. 1 17 (K-TMZ) COMe 5.2 .+-. 0.3
General Synthetic Methods
[0114] The invention also relates to methods of making the
compounds and compositions of the invention. The compounds and
compositions can be prepared by any of the applicable techniques of
organic synthesis, for example, the techniques described herein.
Many such techniques are well known in the art. However, many of
the known techniques are elaborated in Compendium of Organic
Synthetic Methods (John Wiley & Sons, New York), Vol. 1, Ian T.
Harrison and Shuyen Harrison, 1971; Vol. 2, Ian T. Harrison and
Shuyen Harrison, 1974; Vol. 3, Louis S. Hegedus and Leroy Wade,
1977; Vol. 4, Leroy G. Wade, Jr., 1980; Vol. 5, Leroy G. Wade, Jr.,
1984; and Vol. 6, Michael B. Smith; as well as standard organic
reference texts such as March's Advanced Organic Chemistry:
Reactions, Mechanisms, and Structure, 5.sup.th Ed. by M. B. Smith
and J. March (John Wiley & Sons, New York, 2001), Comprehensive
Organic Synthesis; Selectivity, Strategy & Efficiency in Modern
Organic Chemistry, in 9 Volumes, Barry M. Trost, Ed.-in-Chief
(Pergamon Press, New York, 1993 printing)); Advanced Organic
Chemistry, Part B: Reactions and Synthesis, Second Edition, Cary
and Sundberg (1983); Protecting Groups in Organic Synthesis, Second
Edition, Greene, T. W., and Wutz, P. G. M., John Wiley & Sons,
New York; and Comprehensive Organic Transformations, Larock, R. C.,
Second Edition, John Wiley & Sons, New York (1999).
[0115] A number of exemplary methods for the preparation of the
compounds of the invention are provided below. These methods are
intended to illustrate the nature of such preparations are not
intended to limit the scope of applicable methods.
[0116] Generally, the reaction conditions such as temperature,
reaction time, solvents, work-up procedures, and the like, will be
those common in the art for the particular reaction to be
performed. The cited reference material, together with material
cited therein, contains detailed descriptions of such conditions.
Typically, the temperatures will be -100.degree. C. to 200.degree.
C., solvents will be aprotic or protic depending on the conditions
required, and reaction times will be 1 minute to 10 days. Work-up
typically consists of quenching any unreacted reagents followed by
partition between a water/organic layer system (extraction) and
separation of the layer containing the product.
[0117] Oxidation and reduction reactions are typically carried out
at temperatures near room temperature (about 20.degree. C.),
although for metal hydride reductions frequently the temperature is
reduced to 0.degree. C. to -100.degree. C. Heating can also be used
when appropriate. Solvents are typically aprotic for reductions and
may be either protic or aprotic for oxidations. Reaction times are
adjusted to achieve desired conversions.
[0118] Condensation reactions are typically carried out at
temperatures near room temperature, although for non-equilibrating,
kinetically controlled condensations reduced temperatures
(0.degree. C. to -100.degree. C.) are also common. Solvents can be
either protic (common in equilibrating reactions) or aprotic
(common in kinetically controlled reactions). Standard synthetic
techniques such as azeotropic removal of reaction by-products and
use of anhydrous reaction conditions (e.g. inert gas environments)
are common in the art and will be applied when applicable.
[0119] Protecting Groups. The term "protecting group" refers to any
group which, when bound to a hydroxy or other heteroatom prevents
undesired reactions from occurring at this group and which can be
removed by conventional chemical or enzymatic steps to reestablish
the hydroxyl group. The particular removable protecting group
employed is not always critical and preferred removable hydroxyl
blocking groups include conventional substituents such as, for
example, allyl, benzyl, acetyl, chloroacetyl, thiobenzyl,
benzylidene, phenacyl, methyl methoxy, silyl ethers (e.g.,
trimethylsilyl (TMS), t-butyl-diphenylsilyl (TBDPS), or
t-butyldimethylsilyl (TBS)) and any other group that can be
introduced chemically onto a hydroxyl functionality and later
selectively removed either by chemical or enzymatic methods in mild
conditions compatible with the nature of the product.
[0120] Suitable hydroxyl protecting groups are known to those
skilled in the art and disclosed in more detail in T. W. Greene,
Protecting Groups In Organic Synthesis; Wiley: New York, 1981
("Greene") and the references cited therein, and Kocienski, Philip
J.; Protecting Groups (Georg Thieme Verlag Stuttgart, New York,
1994), both of which are incorporated herein by reference.
[0121] Protecting groups are available, commonly known and used,
and are optionally used to prevent side reactions with the
protected group during synthetic procedures, i.e. routes or methods
to prepare the compounds by the methods of the invention. For the
most part the decision as to which groups to protect, when to do
so, and the nature of the chemical protecting group "PG" will be
dependent upon the chemistry of the reaction to be protected
against (e.g., acidic, basic, oxidative, reductive or other
conditions) and the intended direction of the synthesis.
Pharmaceutical Formulations
[0122] The compounds described herein can be used to prepare
therapeutic pharmaceutical compositions, for example, by combining
the compounds with a pharmaceutically acceptable diluent,
excipient, or carrier. The compounds may be added to a carrier in
the form of a salt or solvate. For example, in cases where
compounds are sufficiently basic or acidic to form stable nontoxic
acid or base salts, administration of the compounds as salts may be
appropriate. Examples of pharmaceutically acceptable salts are
organic acid addition salts formed with acids that form a
physiologically acceptable anion, for example, tosylate,
methanesulfonate, acetate, citrate, malonate, tartrate, succinate,
benzoate, ascorbate, .alpha.-ketoglutarate, and
.beta.-glycerophosphate. Suitable inorganic salts may also be
formed, including hydrochloride, halide, sulfate, nitrate,
bicarbonate, and carbonate salts.
[0123] Pharmaceutically acceptable salts may be obtained using
standard procedures well known in the art, for example by reacting
a sufficiently basic compound such as an amine with a suitable acid
to provide a physiologically acceptable ionic compound. Alkali
metal (for example, sodium, potassium or lithium) or alkaline earth
metal (for example, calcium) salts of carboxylic acids can also be
prepared by analogous methods.
[0124] The compounds of the formulas described herein can be
formulated as pharmaceutical compositions and administered to a
vertebrate or mammalian host, such as a human patient, in a variety
of forms. The forms can be specifically adapted to a chosen route
of administration, e.g., oral or parenteral administration, by
intravenous, intramuscular, topical or subcutaneous routes.
[0125] The compounds described herein may be systemically
administered in combination with a pharmaceutically acceptable
vehicle, such as an inert diluent or an assimilable edible carrier.
For oral administration, compounds can be enclosed in hard- or
soft-shell gelatin capsules, compressed into tablets, or
incorporated directly into the food of a patient's diet. Compounds
may also be combined with one or more excipients and used in the
form of ingestible tablets, buccal tablets, troches, capsules,
elixirs, suspensions, syrups, wafers, and the like. Such
compositions and preparations typically contain at least 0.1% of
active compound. The percentage of the compositions and
preparations can vary and may conveniently be from about 0.5% to
about 60%, about 1% to about 25%, or about 2% to about 10%, of the
weight of a given unit dosage form. The amount of active compound
in such therapeutically useful compositions can be such that an
effective dosage level can be obtained.
[0126] The tablets, troches, pills, capsules, and the like may also
contain one or more of the following: binders such as gum
tragacanth, acacia, corn starch or gelatin; excipients such as
dicalcium phosphate; a disintegrating agent such as corn starch,
potato starch, alginic acid and the like; and a lubricant such as
magnesium stearate. A sweetening agent such as sucrose, fructose,
lactose or aspartame; or a flavoring agent such as peppermint, oil
of wintergreen, or cherry flavoring, may be added. When the unit
dosage form is a capsule, it may contain, in addition to materials
of the above type, a liquid carrier, such as a vegetable oil or a
polyethylene glycol. Various other materials may be present as
coatings or to otherwise modify the physical form of the solid unit
dosage form. For instance, tablets, pills, or capsules may be
coated with gelatin, wax, shellac or sugar and the like. A syrup or
elixir may contain the active compound, sucrose or fructose as a
sweetening agent, methyl and propyl parabens as preservatives, a
dye and flavoring such as cherry or orange flavor. Any material
used in preparing any unit dosage form should be pharmaceutically
acceptable and substantially non-toxic in the amounts employed. In
addition, the active compound may be incorporated into
sustained-release preparations and devices.
[0127] The active compound may be administered intravenously or
intraperitoneally by infusion or injection. Solutions of the active
compound or its salts can be prepared in water, optionally mixed
with a nontoxic surfactant. Dispersions can be prepared in
glycerol, liquid polyethylene glycols, triacetin, or mixtures
thereof, or in a pharmaceutically acceptable oil. Under ordinary
conditions of storage and use, preparations may contain a
preservative to prevent the growth of microorganisms.
[0128] Pharmaceutical dosage forms suitable for injection or
infusion can include sterile aqueous solutions, dispersions, or
sterile powders comprising the active ingredient adapted for the
extemporaneous preparation of sterile injectable or infusible
solutions or dispersions, optionally encapsulated in liposomes. The
ultimate dosage form should be sterile, fluid and stable under the
conditions of manufacture and storage. The liquid carrier or
vehicle can be a solvent or liquid dispersion medium comprising,
for example, water, ethanol, a polyol (for example, glycerol,
propylene glycol, liquid polyethylene glycols, and the like),
vegetable oils, nontoxic glyceryl esters, and suitable mixtures
thereof. The proper fluidity can be maintained, for example, by the
formation of liposomes, by the maintenance of the required particle
size in the case of dispersions, or by the use of surfactants. The
prevention of the action of microorganisms can be brought about by
various antibacterial and/or antifungal agents, for example,
parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the
like. In many cases, it will be preferable to include isotonic
agents, for example, sugars, buffers, or sodium chloride. Prolonged
absorption of the injectable compositions can be brought about by
agents delaying absorption, for example, aluminum monostearate
and/or gelatin.
[0129] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in the
appropriate solvent with various other ingredients enumerated
above, as required, optionally followed by filter sterilization. In
the case of sterile powders for the preparation of sterile
injectable solutions, methods of preparation can include vacuum
drying and freeze-drying techniques, which yield a powder of the
active ingredient plus any additional desired ingredient present in
the solution.
[0130] For topical administration, compounds may be applied in pure
form, e.g., when they are liquids. However, it will generally be
desirable to administer the active agent to the skin as a
composition or formulation, for example, in combination with a
dermatologically acceptable carrier, which may be a solid, a
liquid, a gel, or the like.
[0131] Useful solid carriers include finely divided solids such as
talc, clay, microcrystalline cellulose, silica, alumina, and the
like. Useful liquid carriers include water, dimethyl sulfoxide
(DMSO), alcohols, glycols, or water-alcohol/glycol blends, in which
a compound can be dissolved or dispersed at effective levels,
optionally with the aid of non-toxic surfactants. Adjuvants such as
fragrances and additional antimicrobial agents can be added to
optimize the properties for a given use. The resultant liquid
compositions can be applied from absorbent pads, used to impregnate
bandages and other dressings, or sprayed onto the affected area
using a pump-type or aerosol sprayer.
[0132] Thickeners such as synthetic polymers, fatty acids, fatty
acid salts and esters, fatty alcohols, modified celluloses, or
modified mineral materials can also be employed with liquid
carriers to form spreadable pastes, gels, ointments, soaps, and the
like, for application directly to the skin of the user.
[0133] Examples of dermatological compositions for delivering
active agents to the skin are known to the art; for example, see
U.S. Pat. No. 4,992,478 (Geria), U.S. Pat. No. 4,820,508
(Wortzman), U.S. Pat. No. 4,608,392 (Jacquet et al.), and U.S. Pat.
No. 4,559,157 (Smith et al.). Such dermatological compositions can
be used in combinations with the compounds described herein where
an ingredient of such compositions can optionally be replaced by a
compound described herein, or a compound described herein can be
added to the composition.
[0134] Useful dosages of the compounds described herein can be
determined by comparing their in vitro activity, and in vivo
activity in animal models. Methods for the extrapolation of
effective dosages in mice, and other animals, to humans are known
to the art; for example, see U.S. Pat. No. 4,938,949 (Borch et
al.). The amount of a compound, or an active salt or derivative
thereof, required for use in treatment will vary not only with the
particular compound or salt selected but also with the route of
administration, the nature of the condition being treated, and the
age and condition of the patient, and will be ultimately at the
discretion of an attendant physician or clinician.
[0135] In general, however, a suitable dose will be in the range of
from about 0.5 to about 100 mg/kg, e.g., from about 10 to about 75
mg/kg of body weight per day, such as 3 to about 50 mg per kilogram
body weight of the recipient per day, preferably in the range of 6
to 90 mg/kg/day, most preferably in the range of 15 to 60
mg/kg/day.
[0136] The compound is conveniently formulated in unit dosage form;
for example, containing 5 to 1000 mg, conveniently 10 to 750 mg,
most conveniently, 50 to 500 mg of active ingredient per unit
dosage form. In one embodiment, the invention provides a
composition comprising a compound of the invention formulated in
such a unit dosage form.
[0137] The compound can be conveniently administered in a unit
dosage form, for example, containing 5 to 1000 mg/m.sup.2,
conveniently 10 to 750 mg/m.sup.2, most conveniently, 50 to 500
mg/m.sup.2 of active ingredient per unit dosage form. The desired
dose may conveniently be presented in a single dose or as divided
doses administered at appropriate intervals, for example, as two,
three, four or more sub-doses per day. The sub-dose itself may be
further divided, e.g., into a number of discrete loosely spaced
administrations.
[0138] The desired dose may conveniently be presented in a single
dose or as divided doses administered at appropriate intervals, for
example, as two, three, four or more sub-doses per day. The
sub-dose itself may be further divided, e.g., into a number of
discrete loosely spaced administrations; such as multiple
inhalations from an insufflator or by application of a plurality of
drops into the eye.
[0139] The compounds described herein can be effective anti-tumor
agents and have higher potency and/or reduced toxicity as compared
to TMZ. Preferably, compounds of the invention are more potent and
less toxic than TMZ, and/or avoid a potential site of catabolic
metabolism encountered with TMZ, i.e., have a different metabolic
profile than TMZ.
[0140] The invention provides therapeutic methods of treating
cancer in a mammal, which involve administering to a mammal having
cancer an effective amount of a compound or composition described
herein. A mammal includes a primate, human, rodent, canine, feline,
bovine, ovine, equine, swine, caprine, bovine and the like. Cancer
refers to any various type of malignant neoplasm, for example,
colon cancer, breast cancer, melanoma and leukemia, and in general
is characterized by an undesirable cellular proliferation, e.g.,
unregulated growth, lack of differentiation, local tissue invasion,
and metastasis.
[0141] The ability of a compound of the invention to treat cancer
may be determined by using assays well known to the art. For
example, the design of treatment protocols, toxicity evaluation,
data analysis, quantification of tumor cell-kill, and the
biological significance of the use of transplantable tumor screens
are known.
[0142] The following Examples are intended to illustrate the above
invention and should not be construed as to narrow its scope. One
skilled in the art will readily recognize that the Examples suggest
many other ways in which the invention could be practiced. It
should be understood that numerous variations and modifications may
be made while remaining within the scope of the invention.
EXAMPLES
Example 1. Experimental Information for Biological Data
[0143] Cell Culture and Reagents.
[0144] All cell lines were grown in a 37.degree. C., 5% CO.sub.2,
humidified environment, in media containing 1%
penicillin/streptomycin. Cell culture conditions are as follows:
traditional cell lines U87 and T98G were grown in EMEM with 10%
FBS. Traditional cell lines D54 and U118MG were grown in DMEM with
10% FBS. HGCC patient-derived cell line U3054MG.sup.1 was cultured
under serum-free stem cell conditions (1:1 neurobasal: DMEM/F12
media supplemented with B27, N2, hEGF, and hFGF). GBM oncosphere
cell line Br23c.sup.2 was cultured with the NeuroCult NS-A
proliferation kit (Stem Cell Technologies) supplemented with
0.0002% heparin, hEGF, and hFGF. Temozolomide (TMZ) was purchased
from AK Scientific. TMZ analogs were synthesized as described
below. Compounds were dissolved in DMSO (1% final concentration,
Fisher Chemical) for cell culture studies.
[0145] Cell Viability Assays.
[0146] Cells were harvested, seeded in a 96-well plate and allowed
to adhere. After three hours, compound was added to each well in
DMSO (1% final concentration). Cells were incubated for seven days
before viability was assessed by the Alamar Blue Assay. Raptinal
(20 .mu.M) was used as a dead control.
[0147] Mouse Liver Microsome Stability Assay.
[0148] A mixture of PBS (pH 7.4), NADPH regenerating system
solution A (Corning Life Sciences), and NADPH regenerating system
solution B (Corning Life Sciences) was incubated at 37.degree. C.
in a shaking incubator for 5 min. Next, compound was added in DMSO
(final concentration 50 .mu.M, 0.5% DMSO) before ice-cold mouse
liver microsomes (Thermo Fisher, male CD-1 mice, pooled) were added
(final protein concentration of 1 mg/mL). An aliquot was
immediately removed, quenched with an equal volume of 100 .mu.M
internal standard and 0.5% hydrochloric acid in ice-cold
acetonitrile, and centrifuged at 13,000 rcf for 3 min. The
supernatant was diluted 1:5 in ddH.sub.2O and analyzed by LC-MS.
The reactions were incubated at 37.degree. C. in a shaking
incubator for 2 h. A second aliquot was removed, quenched and
diluted as before and analyzed by LC-MS. The ratio of the areas of
analyte: internal standard at 2 hours was compared to the ratio at
t.sub.0 to determine the percentage of compound remaining. Analysis
was performed on an Agilent 6230 LC/MS TOF system with a 1.8 .mu.m,
2.1.times.50 mm Agilent ZORBAX Eclipse Plus C18 column. Internal
standard=N3-propyl TMZ.
[0149] O.sup.6-Methyldeoxyguanosine Quantitation.
[0150] U87 cells were plated at 1.times.10.sup.6 c/w in a 6-well
plate before they were treated with compound at the indicated
concentration (1% final concentration DMSO). After 8 h incubation,
the cells were harvested and pelleted. Genomic DNA was extracted
using the DNeasy Blood & Tissue Kit (Qiagen, ID: 69504). DNA
was then precipitated using the following procedure: 1/10 v/v 3M
sodium acetate (pH 5.2) and 2.5.times. v/v ethanol was added to
each sample which was then kept at -80.degree. C. for 1 h. The
mixture was centrifuged at max at 4.degree. C. for 30 min and
decanted to afford a pellet of DNA, which was re-suspended in
ddH.sub.2O containing 10 mM tris base (pH 7.5) and 1 mM EDTA. The
concentration of DNA in each sample was quantified measuring
absorbance on a NanoDrop 2000 UV-Vis Spectrophotometer (Thermo
Fisher). DNA (10 .mu.g) from each sample was added to DNA
hydrolysis buffer.sup.3 and incubated at 37.degree. C. for 6 h.
Hydrolyzed samples were then submitted for LC-MS/MS quantitation.
Samples were analyzed with a 5500 QTRAP LC/MS/MS system (AB Sciex)
with a 1200 series HPLC system (Agilent).
[0151] In Vivo Blood-Brain Barrier Permeability.
[0152] All experimental procedures were reviewed and approved by
the University of Illinois Institutional Animal Care and Use
Committee. CD-1 IGS mice were administered compound in 1% DMSO
(FIG. 2a) or 10% DMSO (FIG. 2b-d) in PBS at 25 mg/kg via lateral
tail vein injection. Five minutes post injection, mice were
sacrificed, and blood was collected by lacerating the right auricle
with iris scissors. An 18-gauge angiocatheter was inserted through
the left ventricle, and all residual circulatory volume was removed
by perfusing 0.9% saline solution via an analog peristaltic pump.
Blood samples were immediately centrifuged at 13,000 rcf for five
minutes and the supernatant collected and acidified with 8.5%
aqueous H.sub.3PO.sub.4. Brains were harvested from the cranial
vault, acidified with 0.3% aqueous H.sub.3PO.sub.4 and flash
frozen. Homogenized brain samples were centrifuged twice at 13,000
rcf for ten minutes and supernatant and tissue debris were
separated. The resultant supernatant was analyzed, along with
plasma, by LC-MS/MS to determine compound concentrations. In order
to calculate absolute brain:serum ratios (ng drug.sub.brain:ng
drug.sub.serum), a mouse blood volume of 58.5 mL/kg was assumed for
each mouse.
[0153] In Vivo Efficacy Models.
[0154] Human GBM Br23c stem-like neurosphere cells were
intracranially implanted in female athymic nude mice (150,000
cells/mouse). Beginning day 5 after implantation of the tumor
cells, drugs were formulated in 10% PEG 400 in saline and 15 mg/kg
TMZ (or equimolar dose of C8 analog) was administered via oral
gavage once-per-day for 7 weeks (FIG. 5a) or once-per-day for 5
total treatments (FIG. 5b). TMZ and C8 analogs were dissolved fresh
for each use. Mice were observed daily for any signs of
deterioration, neurotoxicity, or movement disorders. They were
inspected for signs of pain and distress, as in accordance with the
Johns Hopkins Animal Care and Use Guidelines. If the symptoms
persisted and resulted in debilitation, the animals were euthanized
according to protocol.
[0155] Assessment of Hematological Toxicity.
[0156] Male CD-1 IGS mice (n=4 mice/group) were administered a
single dose of 125 mg/kg compound intravenously. Imidazotetrazines
were formulated with SBE.beta.CD in sterile water immediately prior
to injection. Seven days post-treatment, mice were humanely
sacrificed and whole blood was collected for assessment of total
white blood cells, lymphocytes, neutrophils, platelets, and red
blood cells.
Example 2. Synthetic Methods
[0157] Materials and Methods.
[0158] Chemical reagents were purchased from commercial sources and
used without further purification. Flash chromatography was
performed using silica gel (230-400 mesh). Anhydrous solvents were
dried after being passed through columns packed with activated
alumina under positive pressure of nitrogen. Unless otherwise
noted, all reactions were carried out in oven-dried glassware with
magnetic stirring under nitrogen atmosphere. .sup.1H and .sup.13C
NMR spectra were recorded on Bruker 500 (500 MHz, .sup.1H; 125 MHz,
.sup.13C) or Varian Unity Inova 500 (500 MHz, .sup.1H) MHz
spectrometers. Spectra are referenced to residual chloroform
(.delta.=7.26 ppm, .sup.1H; 77.16 ppm, .sup.13C) or dimethyl
sulfoxide (.delta.=2.50 ppm, .sup.1H; 39.52 ppm, .sup.13C).
Multiplicities are indicated by s (singlet), d (doublet), t
(triplet), q (quartet), m (multiplet), and br (broad). Coupling
constants J are reported in Hertz (Hz). High resolution mass
spectrometry (HRMS) was performed on a Waters Q-Tof Ultima or
Waters Synapt G2-Si instrument with electrospray ionization (ESI)
or electron impact ionization (EI).
[0159] Preparation and Characterization of C8 Analogs.
[0160] Experimental information for compounds 3,.sup.4 9,.sup.5
11,.sup.6 15,.sup.7 16,.sup.8 29,.sup.9 31,.sup.10 and 33.sup.6 has
been previously reported.
General Scheme for Preparation of Amide, Ester, and Thioester
Derivatives 4-10:
##STR00022##
[0162] General Procedure for Preparation of 4-10.
[0163] In an oven-dried 25 mL round bottom flask, acyl chloride 29
(148.6 mg, 0.70 mmol, 1 eq.) was dissolved in anhydrous THF (2.8
mL, 0.25 M). Methylamine (33% w/w in ethanol, 0.09 mL, 0.73 mL,
1.05 eq.) was then added and the reaction was stirred for 3 h at
room temperature. When complete, the reaction was stopped and the
solvent was evaporated. The crude solid was purified by flash
silica gel chromatography (100% ethyl acetate) to yield 98.3 mg
(68%) of pure 4 as a white solid.
[0164] Experimental data for compounds 3, 9, and 29 has been
published..sup.4,5,9
##STR00023##
N,3-dimethyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-carboxam-
ide (4, Me-TMZ)
[0165] .sup.1H NMR (500 MHz, d-DMSO) .delta. 8.84 (s, 1H), 8.45 (d,
J=4.9 Hz, 1H), 3.86 (s, 3H), 2.81 (d, J=4.8 Hz, 3H). .sup.13C (125
MHz, d-DMSO) .delta. 160.13, 139.23, 134.27, 130.54, 128.44, 36.14,
25.80. HRMS (ESI) calc. for C.sub.7H.sub.8N.sub.6O.sub.2Na,
[M+Na].sup.+: 231.0606, found: 231.0608.
N,N,3-trimethyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-carbox-
amide (5, DiMe-TMZ)
[0166] 76% yield as a white solid.
[0167] .sup.1H NMR (500 MHz, d-DMSO) .delta. 8.81 (s, 1H), 3.85 (s,
3H), 3.06 (s, 6H). .sup.13C NMR (125 MHz, d-DMSO) .delta. 161.76,
139.22, 133.57, 132.05, 128.59, 38.12, 36.05, 34.84. HRMS (ESI)
calc. for C.sub.8H.sub.11N.sub.6O.sub.2, [M+H]: 223.0938, found:
223.0943.
N,N-diethyl-3-methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-c-
arboxamide (6)
[0168] 91% as a white solid.
[0169] .sup.1H NMR (500 MHz, d-DMSO) .delta. 8.81 (s, 1H), 3.84 (s,
3H), 3.49 (q, J=7.1 Hz, 2H), 3.38 (q, J=7.0 Hz, 2H), 1.18 (t, J=7.1
Hz, 3H), 1.11 (t, J=7.0 Hz, 3H). .sup.13C NMR (125 MHz, d-DMSO)
.delta. 161.35, 139.24, 133.54, 132.72, 128.45, 42.53, 36.01,
14.43, 12.80.
[0170] HRMS (ESI) calc. for C.sub.10H.sub.15N.sub.6O.sub.2,
[M+H].sup.+: 251.1256, found: 251.1250.
N,N-dibutyl-3-methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-c-
arboxamide (7)
[0171] 85% yield as a white solid.
[0172] .sup.1H NMR (500 MHz, d-DMSO) .delta. 8.80 (s, 1H), 3.84 (s,
3H), 3.45 (m, 2H), 3.34 (m, 2H), 1.59 (m, 2H), 1.49 (m, 2H), 1.35
(h, J=7.4 Hz, 2H), 1.11 (h, J=7.4 Hz, 2H), 0.94 (t, J=7.4 Hz, 3H),
0.76 (t, J=7.4 Hz, 3H). .sup.13C NMR (125 MHz, d-DMSO) .delta.
161.74, 139.23, 133.35, 132.80, 128.42, 47.66, 44.41, 35.99, 30.54,
29.20, 19.65, 19.17, 13.79, 13.55. HRMS (ESI) calc. for
C.sub.14H.sub.23N.sub.6O.sub.2, [M+H].sup.+: 307.1882, found:
307.1881.
3-methyl-8-(pyrrolidine-1-carbonyl)imidazo[5,1-d][1,2,3,5]tetrazin-4(3H)-o-
ne (8)
[0173] 55% yield as pale-yellow solid.
[0174] .sup.1H NMR (500 MHz, d-DMSO) .delta. 8.81 (s, 1H), 3.85 (s,
3H), 3.63 (m, 2H), 3.53 (m, 2H), 1.88 (m, 4H). .sup.13C NMR (125
MHz, d-DMSO) .delta. 159.73, 139.21, 134.07, 132.54, 128.33, 48.05,
46.09, 36.05, 25.80, 23.63. HRMS (ESI) calc. for
C.sub.10H.sub.13N.sub.6O.sub.2, [M+H].sup.+: 249.1100, found:
249.1105.
S-ethyl
3-methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-carbo-
thioate (10)
[0175] 92% as a white solid.
[0176] .sup.1H NMR (500 MHz, d-DMSO) .delta. 8.86 (s, 1H), 3.89 (s,
3H), 3.02 (q, J=7.4 Hz, 2H), 1.28 (t, J=7.4 Hz, 3H). .sup.13C NMR
(125 MHz, d-DMSO) .delta. 184.57, 138.95, 133.80, 131.55, 129.19,
36.50, 22.15, 14.70. HRMS (ESI) calc. for
C.sub.8H.sub.10N.sub.5O.sub.2S, [M+H].sup.+: 240.0555, found:
240.0551.
3,8-dimethylimidazo[5,1-d][1,2,3,5]tetrazin-4(3H)-one (14)
##STR00024##
[0178] Procedure.
[0179] To a 15 mL round bottom flask, 4-methyl-1H-imidazol-5-amine
dihydrochloride 12 (44.2 mg, 0.3 mmol, 1 eq.) was added and
dissolved in 1M HCl (0.4 mL, 0.65 M) before sodium nitrite (26.2
mg, 0.4 mmol, 1.5 eq.) in water (0.4 mL, 0.65 M) was added at
0.degree. C. in the dark. The solution was stirred 30 minutes then
concentrated and azeotroped twice with toluene to afford crude
diazo 13. To the crude diazo suspended in ethyl acetate (1.3 mL,
0.2 M), anhydrous triethylamine (0.08 mL, 0.6 mmol, 2.2 eq.) and
methylcarbamic chloride (79 mg, 0.8 mmol, 3.2 eq.) were added in
the dark. The reaction was stirred overnight before being purified
via flash silica gel chromatography (4:1 hexanes:ethyl acetate) to
afford 2.4 mg (6%) 14 as a pale yellow solid. Note: To minimize
decomposition of the crude diazo species, concentration was done
(without heating) in the dark as quickly as possible.
[0180] .sup.1H NMR (500 MHz, d-CHCl.sub.3) .delta. 8.35 (s, 1H),
3.94 (s, 3H), 2.66 (s, 3H). .sup.13C NMR (125 MHz, d-CHCl.sub.3)
.delta. 139.71, 139.64, 132.51, 127.94, 35.76, 12.53. HRMS (E)
calc. for C.sub.6H.sub.7N.sub.5O, [M].sup.+: 165.0651, found:
165.0654.
8-acetyl-3-methylimidazo[5,1-d][1,2,3,5]tetrazin-4(3H)-one (17,
K-TMZ)
##STR00025##
[0182] Procedure.
[0183] To an oven-dried 25 mL round bottom flask, 16 (186 mg, 0.89
mmol, 1 eq.) and N-succinimidyl N-methylcarbamate (321 mg, 1.86
mmol, 2.1 eq.) were added and suspended in anhydrous acetonitrile
(1.5 mL, 0.6 M). Next, under nitrogen, dry triethylamine (0.34 mL,
2.4 mmol, 2.7 eq.) was added slowly and the solution was stirred
overnight at room temperature. Upon completion, the mixture was
concentrated and purified by silica gel flash chromatography (100%
dichloromethane to 4:1 dichloromethane:methanol) to afford 106 mg
(66%) of intermediate 16a as a gold solid.
[0184] .sup.1H NMR (500 MHz, d-DMSO) .delta. 8.17 (br s, 1H), 7.97
(s, 1H), 7.56 (s, 2H), 3.36 (d, J=4.5 Hz, 3H), 2.73 (s, 3H).
[0185] In a 15 mL round bottom flask, LiCl (802 mg, 19 mmol, 36
eq.) was dissolved in distilled water (1.3 mL, 0.4 M) and AcOH
(0.10 mL, 5.3 M) and stirred for thirty minutes until the exotherm
dissipated. Intermediate 16a (96.3 mg, 0.53 mmol, 1 eq.) was added
in one portion and stirred for thirty minutes. The suspension was
then cooled to 0.degree. C. in an ice bath before a solution of
NaNO.sub.2 (57 mg, 0.8 mmol, 1.5 eq.) in a minimal amount of
distilled water was added dropwise. The resultant mixture was
stirred at 0.degree. C. for 30 minutes, then warmed to room
temperature and stirred an additional 5 hours. Upon completion, the
reaction mixture was diluted with CH.sub.2Cl.sub.2 and the organic
layer was separated. The aqueous layer was extracted with
dichloromethane (.times.6) and the combined organic layers were
dried over sodium sulfate and concentrated to yield crude solid
which was purified by flash silica chromatography (1:1 ethyl
acetate:hexanes) to afford 36 mg (35%) of 17 as a white solid.
[0186] .sup.1H NMR (500 MHz, d-DMSO) .delta. 8.86 (s, 1H), 3.90 (s,
3H), 2.68 (s, 3H). .sup.13C NMR (125 MHz, d-DMSO) .delta. 191.47,
139.01, 135.56, 133.35, 129.11, 36.43, 28.31. HRMS (ESI) calc. for
C.sub.7H.sub.8N.sub.5O.sub.2, [M+H].sup.+: 194.0678, found:
194.0683.
[0187] Experimental data for intermediates 31, 15, and 16 has been
published..sup.7,8,10
8-bromo-3-methylimidazo[5,1-d][1,2,3,5]tetrazin-4(3H)-one (18)
##STR00026##
[0189] Procedure.
[0190] To a stirred suspension of Dess-Martin periodinane (477 mg,
1.12 mmol, 2.2 eq) in anhydrous CH.sub.3CN (2.6 mL, 0.2 M),
tetraethylammonium bromide (240 mg, 1.12 mmol, 2.2 eq) was added.
Reaction was stirred 5 min at room temperature before 3 (100 mg,
0.51 mmol, 1 eq) was added. The resultant reaction mixture was
heated at 50.degree. C. for 2 h. Upon completion, the solvent was
concentrated under reduced pressure to give the crude product that
was purified by flash silica gel chromatography (9:1 hexanes:ethyl
acetate) to afford 73 mg (58%) of 18 as a white solid.
[0191] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 8.37 (s, 1H), 3.98
(s, 3H). .sup.13C NMR (125 MHz, CDCl.sub.3) .delta. 138.74, 133.32,
128.56, 117.16, 36.43. HRMS (ESI) calc. for
C.sub.5H.sub.5N.sub.5OBr, [M+H].sup.+: 229.9677, found:
229.9684.
8-chloro-3-methylimidazo[5,1-d][1,2,3,5]tetrazin-4(3H)-one (19)
##STR00027##
[0193] Procedure.
[0194] To a stirred suspension of Dess-Martin periodinane (477 mg,
1.12 mmol, 2.2 eq.) in anhydrous CH.sub.3CN (2.6 mL, 0.2 M),
tetramethylammonium chloride (123 mg, 1.12 mmol, 2.2 eq.) was
added. The reaction was stirred 5 min at room temperature before 3
(100 mg, 0.51 mmol, 1 eq.) was added. The resultant reaction
mixture was heated at 50.degree. C. for 2 hours. Upon completion,
the solvent was concentrated under reduced pressure to give the
crude product that was purified by flash silica chromatography (9:1
hexanes:ethyl acetate) to afford 43 mg (45%) of 19 as a white
solid.
[0195] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 8.33 (s, 1H), 3.98
(s, 3H). .sup.13C NMR (125 MHz, CDCl.sub.3) .delta. 138.74, 130.84,
129.81, 127.25, 36.37. HRMS (ESI) calc. for
C.sub.5H.sub.5N.sub.5OCl, [M+H].sup.+: 186.0183, found:
186.0186.
General Scheme for Preparation of Aryl Derivatives 23-26:
##STR00028##
[0197] General Procedure for Preparation of 23-26. (a) Suzuki
Coupling.
[0198] A mixture of 4-bromo-5-nitro-1H-imidazole 20 (400 mg, 2.08
mmol, 1 eq.), phenyl boronic acid (507 mg, 4.17 mmol, 2 eq.),
XPhosPdG.sub.1 (164 mg, 0.2 mmol, 0.1 eq.) and K.sub.3PO.sub.4
(1.32 g, 6.24 mmol, 3 eq.) under nitrogen was suspended in degassed
1:1 H.sub.2O:dioxane (16 mL, 0.13 M). The resulting mixture was
stirred at 110.degree. C. for 16 h. The reaction was cooled to room
temperature and H.sub.2O was added. The aqueous layer was extracted
.times.3 with ethyl acetate and the combined organic layers were
dried over Na.sub.2SO.sub.4 and concentrated. The residue obtained
was purified by flash silica gel chromatography (100% ethyl
acetate) to afford crude product 21 that was used for next step
without further purification.
[0199] (b) Nitro Reduction.
[0200] Crude 21 was dissolved in dry MeOH (10 mL, 0.2 M) containing
10% Pd/C before H.sub.2 (1 atm) was introduced. The reaction was
stirred for 16 h at room temperature before the catalyst was
filtered over Celite. The filtrate was concentrated under reduced
pressure and purified by flash silica gel chromatography (95:5
DCM:MeOH) providing compound 22 that was used for next step without
further purification.
[0201] (c) Cyclization.
[0202] To a suspension of intermediate 22 in 1 M HCl (2.9 mL, 0.7
M) at 0.degree. C. was added a pre-formed solution of NaNO.sub.2
(186 mg, 2.7 mmol, 1.3 eq.) in H.sub.2O (2.9 mL, 0.9 M) dropwise.
The resultant mixture was stirred at 0.degree. C. in the dark for
30 min. Upon completion, the solvent was evaporated, and the crude
diazo compound was dissolved in ethyl acetate (9.6 mL, 0.2 M)
before triethylamine (544 .mu.L, 4.6 mmol, 2 eq.) and
methylcarbamic chloride (1010 mg, 10.8 mmol, 5.2 eq.) were added.
The reaction mixture was stirred at room temperature for 16 h
protected from light. Upon reaction completion, the solvent was
concentrated under reduced pressure and the residue was purified by
flash silica gel chromatography (9:1 hexanes:ethyl acetate) to
afford 28 mg (6%) of pure 23 as a white solid.
3-methyl-8-phenylimidazo[5,1-d][1,2,3,5]tetrazin-4(3H)-one (23)
##STR00029##
[0204] Product was obtained using general procedure. White solid,
6% yield (4 steps).
[0205] .sup.1H NMR (500 MHz, d-DMSO) .delta. 8.84 (s, 1H),
8.31-8.29 (m, 2H), 7.57-7.53 (m, 2H), 7.44 (tt, J=7.4, 1.3 Hz, 1H),
3.85 (s, 3H). .sup.13C NMR (125 MHz, CDCl.sub.3) .delta. 140.02,
137.02, 132.30, 131.88, 129.86, 129.48, 129.43, 127.07, 36.29. HRMS
(ESI) calc. for C.sub.11H.sub.10N.sub.5O, [M+H].sup.+: 228.0885,
found: 228.0878.
8-(4-fluorophenyl)-3-methylimidazo[5,1-d][1,2,3,5]tetrazin-4(3H)-one
(24)
##STR00030##
[0207] Product was obtain using general procedure. Yellow solid, 3%
yield (4 steps).
[0208] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 8.46 (s, 1H),
8.44-8.40 (m, 2H), 7.24-7.19 (m, 2H), 4.01 (s, 3H). .sup.13C NMR
(125 MHz, CDCl.sub.3) .delta. 164.57, 162.58, 139.20 (d, J=86.4 Hz,
1C), 131.00, 129.43 (d, J=8.3 Hz, 1C), 128.56, 127.26 (d, J=3.3 Hz,
1C), 116.00 (d, J=21.6 Hz, 1C), 35.97. HRMS (ESI) calc. for
C.sub.11H.sub.9FN.sub.5O, [M+H].sup.+: 246.0791, found:
246.0788.
3-methyl-8-(4-(trifluoromethyl)phenyl)imidazo[5,1-d][1,2,3,5]tetrazin-4(3H-
)-one (25)
##STR00031##
[0210] Product was obtained using the general procedure. Yellow
solid, 5% yield (4 steps).
[0211] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 8.52 (d, J=8.2 Hz,
2H), 8.48 (s, 1H), 7.75 (d, J=8.2 Hz, 2H), 4.02 (s, 3H). .sup.13C
NMR (125 MHz, CDCl.sub.3) .delta. 139.34, 137.89, 134.30, 131.91,
131.03 (q, J=32.3 Hz, 1C), 128.77, 127.61, 125.80 (q, J=3.8 Hz,
1C), 122.96, 36.15. HRMS (ESI) calc. for
C.sub.12H.sub.9N.sub.5OF.sub.3, [M+H].sup.+: 296.0759, found:
296.0754.
8-(4-chlorophenyl)-3-methylimidazo[5,1-d][1,2,3,5]tetrazin-4(3H)-one
(26)
##STR00032##
[0213] Product was obtained using the general procedure. Yellow
solid, 1.2% yield (4 steps).
[0214] .sup.1H NMR (500 MHz, d-DMSO) .delta. 8.86 (s, 1H), 8.30
(dt, J=9.25, 2.5 Hz, 2H), 7.62 (dt, J=9.25, 2.5 Hz, 2H), 3.86 (s,
3H). .sup.13C NMR (125 MHz, d-DMSO) .delta. 139.92, 135.67, 133.97,
132.45, 130.75, 130.00, 129.63, 128.62, 36.37. HRMS (ESI) calc. for
C.sub.11H.sub.9N.sub.5OCl, [M+H].sup.+: 262.0496, found:
262.0489.
3-methyl-4-oxo-N-(2-oxopropyl)-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-
-8-carboxamide (32)
##STR00033##
[0216] Procedure.
[0217] To 29 (447 mg, 2.09 mmol, 1 eq.) and 2-aminoacetophenone
hydrochloride (229 mg, 2.09 mmol, 1 eq.) was added DMF (4.4 mL,
0.47 M) and pyridine (0.9 mL). The reaction mixture was stirred for
16 h at room temperature. Water was added and the aqueous layer was
extracted .times.5 with ethyl acetate. The combined organic layers
were dried over Na.sub.2SO.sub.4 and concentrated. The residue
obtained was purified by flash silica gel chromatography (100%
ethyl acetate) to afford 266 mg (51%) of 32 as an orange solid.
[0218] .sup.1H NMR (500 MHz, d-DMSO) .delta. 8.87 (s, 1H), 8.59 (t,
J=5.7 Hz, 1H), 4.17 (d, J=5.7 Hz, 2H), 3.88 (s, 3H), 2.15 (s, 3H).
.sup.13C NMR (125 MHz, d-DMSO) .delta. 204.55, 160.15, 139.65,
135.10, 130.18, 129.09, 49.57, 36.67, 27.52. LC-MS (ESI) calc. for
C.sub.9H.sub.11N.sub.6O.sub.3 [M+H].sup.+: 251.0893, found:
251.09.
3-methyl-8-(5-methyloxazol-2-yl)imidazo[5,1-d][1,2,3,5]tetrazin-4(3H)-one
(27, Ox-TMZ)
[0219] Procedure:
[0220] Intermediate 32 (266 mg, 1.06 mmol, 1 eq.) was added to
phosphoryl chloride (6.5 mL, 0.16 M) and the stirred mixture was
heated at 110.degree. C. for 3 h. Upon completion, ice water was
added, and the aqueous layer was extracted .times.4 with ethyl
acetate. The combined organic layers were dried over
Na.sub.2SO.sub.4 and concentrated. The residue obtained was
purified by flash silica gel chromatography (100% ethyl acetate) to
afford 80 mg (32%) of the product 27 as a yellow solid.
[0221] .sup.1H NMR (500 MHz, d-DMSO) .delta. 8.89 (s, 1H), 7.13 (br
d, J=1.2 Hz, 1H), 3.87 (s, 3H), 2.44 (d, J=1.2 Hz, 3H). .sup.13C
NMR (125 MHz, d-DMSO) .delta. 154.15, 150.47, 139.64, 133.64,
130.43, 126.15, 125.36, 36.56, 11.17. HRMS (ESI) calc. for
C.sub.9H.sub.9N.sub.6O.sub.2, [M+H].sup.+: 233.0782, found:
233.0787.
[0222] The route to 4-substituted oxazol-2-yls at the C8 position
of imidazotetrazines is known,.sup.11 however, the synthesis of
compound 27 via intermediate 32 had never been reported.
3-methyl-4-oxo-3,4-dihydroimidazo[5,1-d][1,2,3,5]tetrazine-8-carbothioamid-
e (28)
##STR00034##
[0224] Procedure.
[0225] To a solution of 33 (550 mg, 2.6 mmol, 1 eq.) in
acetonitrile (40 mL, 0.07 M) was added .alpha.-Bromo acetone (220
.mu.L, 2.6 mmol, 1 eq.) and the solution was stirred at room
temperature for 18 h. Upon completion, the reaction was stopped,
and the precipitate was filtered and purified by flash silica gel
chromatography (4:6 hexanes:ethyl acetate) to afford 167 mg (26%)
of the desired product 28 as a yellow solid.
[0226] .sup.1H NMR (500 MHz, d-DMSO) .delta. 8.87 (s, 1H), 7.46 (d,
J=0.9 Hz, 1H), 3.86 (s, 3H), 2.48 (d, J=0.9 Hz, 3H). .sup.13C NMR
(125 MHz, d-DMSO) .delta. 158.63, 154.58, 139.75, 131.84, 131.80,
130.24, 116.56, 36.52, 17.45. HRMS (ESI) calc. for
C.sub.9H.sub.9N.sub.6OS, [M+H].sup.+: 249.0559, found:
249.0559.
[0227] The route to 4-substituted thiazol-2-yls at the C8 position
of imidazotetrazines is known,.sup.11 however, the synthesis of
compound 28 had never been reported. Experimental data for
intermediate 33 has been published..sup.6
Example 3. Synthesis of Arene, Propargyl, and Diazoalkane
Compounds
[0228] Arene and propargyl substituted imidazotetrazines can be
prepared as follows.
##STR00035##
wherein G.sup.1 is OCH.sub.3, OCH.sub.2CH.sub.3, OPh,
N(CH.sub.3).sub.2, propargyl, or a substituent as defined
herein.
[0229] TMZ is a non-explosive, weighable surrogate for
diazomethane. TMZ and other imidazotetrazines can be used as
synthetic diazoalkane precursors as illustrated below.
##STR00036##
Example 4. Pharmaceutical Dosage Forms
[0230] The following formulations illustrate representative
pharmaceutical dosage forms that may be used for the therapeutic or
prophylactic administration of a compound of a formula described
herein, a compound specifically disclosed herein, or a
pharmaceutically acceptable salt or solvate thereof (hereinafter
referred to as `Compound X`):
TABLE-US-00008 (i) Tablet 1 mg/tablet `Compound X` 100.0 Lactose
77.5 Povidone 15.0 Croscarmellose sodium 12.0 Microcrystalline
cellulose 92.5 Magnesium stearate 3.0 300.0 (ii) Tablet 2 mg/tablet
`Compound X` 20.0 Microcrystalline cellulose 410.0 Starch 50.0
Sodium starch glycolate 15.0 Magnesium stearate 5.0 500.0 (iii)
Capsule mg/capsule `Compound X` 10.0 Colloidal silicon dioxide 1.5
Lactose 465.5 Pregelatinized starch 120.0 Magnesium stearate 3.0
600.0 (iv) Injection 1 (1 mg/mL) mg/mL `Compound X` (free acid
form) 1.0 Dibasic sodium phosphate 12.0 Monobasic sodium phosphate
0.7 Sodium chloride 4.5 1.0 N Sodium hydroxide solution q.s. (pH
adjustment to 7.0-7.5) Water for injection q.s. ad 1 mL (v)
Injection 2 (10 mg/mL) mg/mL `Compound X`(free acid form) 10.0
Monobasic sodium phosphate 0.3 Dibasic sodium phosphate 1.1
Polyethylene glycol 400 200.0 0.1 N Sodium hydroxide solution q.s.
(pH adjustment to 7.0-7.5) Water for injection q.s. ad 1 mL (vi)
Aerosol mg/can `Compound X` 20 Oleic acid 10
Trichloromonofluoromethane 5,000 Dichlorodifluoromethane 10,000
Dichlorotetrafluoroethane 5,000 (vii) Topical Gel 1 wt. % `Compound
X` 5% Carbomer 934 1.25% Triethanolamine q.s. (pH adjustment to
5-7) Methyl paraben 0.2% Purified water q.s. to 100 g (viii)
Topical Gel 2 wt. % `Compound X` 5% Methylcellulose 2% Methyl
paraben 0.2% Propyl paraben 0.02% Purified water q.s. to 100 g (ix)
Topical Ointment wt. % `Compound X` 5% Propylene glycol 1%
Anhydrous ointment base 40% Polysorbate 80 2% Methyl paraben 0.2%
Purified water q.s. to 100 g (x) Topical Cream 1 wt. % `Compound X`
5% White bees wax 10% Liquid paraffin 30% Benzyl alcohol 5%
Purified water q.s. to 100 g (xi) Topical Cream 2 wt. % `Compound
X` 5% Stearic acid 10% Glyceryl monostearate 3% Polyoxyethylene
stearyl ether 3% Sorbitol 5% Isopropyl palmitate 2% Methyl Paraben
0.2% Purified water q.s. to 100 g
[0231] These formulations may be prepared by conventional
procedures well known in the pharmaceutical art. It will be
appreciated that the above pharmaceutical compositions may be
varied according to well-known pharmaceutical techniques to
accommodate differing amounts and types of active ingredient
`Compound X`. Aerosol formulation (vi) may be used in conjunction
with a standard, metered dose aerosol dispenser. Additionally, the
specific ingredients and proportions are for illustrative purposes.
Ingredients may be exchanged for suitable equivalents and
proportions may be varied, according to the desired properties of
the dosage form of interest.
CITATIONS
[0232] (1) Stupp, R.; Mason, W.; van den Bent, M. J.; Weller, M.;
Fisher, B. M.; Taphoorn, M. J. B.; Belanger, K.; Brandes, A. A.;
Marosi, C.; Bogdahn, U.; Curschmann, J.; Janzer, R. C.; Ludwin, S.
K.; Gorlia, T.; Allgeier, A.; Lacombe, D.; Cairncross, J. G.;
Eisenhauer, E.; Mirimanoff, R. O. (2005) Radiotherapy plus
Concomitant and Adjuvant Temozolomide for Glioblastoma. N. Engl. J.
Med. 352, 987-996. [0233] (2) Denny, B. J.; Wheelhouse, R. T.;
Stevens, M. F.; Tsang, L. L.; Slack, J. a. (1994) NMR and Molecular
Modeling Investigation of the Mechanism of Activation of the
Antitumor Drug Temozolomide and Its Interaction with DNA.
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[0298] While specific embodiments have been described above with
reference to the disclosed embodiments and examples, such
embodiments are only illustrative and do not limit the scope of the
invention. Changes and modifications can be made in accordance with
ordinary skill in the art without departing from the invention in
its broader aspects as defined in the following claims.
[0299] All publications, patents, and patent documents are
incorporated by reference herein, as though individually
incorporated by reference. No limitations inconsistent with this
disclosure are to be understood therefrom. The invention has been
described with reference to various specific and preferred
embodiments and techniques. However, it should be understood that
many variations and modifications may be made while remaining
within the spirit and scope of the invention.
* * * * *