U.S. patent application number 15/998620 was filed with the patent office on 2021-05-20 for antifungal compounds and uses thereof.
This patent application is currently assigned to Dana-Farber Cancer Institute, Inc.. The applicant listed for this patent is Centre Hospitalier Universitaire Vaudois, Dana-Farber Cancer Institute, Inc., The General Hospital Corporation, President and Fellows of Harvard College. Invention is credited to Haribabu Arthanari, Andras Pal Boeszoermenyi, Sara Jean Buhrlage, Anders Naar, Joy Luz Nishikawa, Dominique Sanglard, Gerhard Wagner.
Application Number | 20210147351 15/998620 |
Document ID | / |
Family ID | 1000005565006 |
Filed Date | 2021-05-20 |
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United States Patent
Application |
20210147351 |
Kind Code |
A9 |
Buhrlage; Sara Jean ; et
al. |
May 20, 2021 |
ANTIFUNGAL COMPOUNDS AND USES THEREOF
Abstract
Provided herein are compounds (e.g., compounds of Formulae (I),
(II), and (III)) which are anti-fungal agents and can be used in
the treatment of diseases, including infectious diseases. The
invention provides methods of treating diseases in a subject (e.g.,
infectious diseases such as fungal infections), and methods of
killing or inhibiting the growth of fungi in or on a subject or
biological sample. The compounds may be used in subjects, in
clinical settings, or in agricultural settings. ##STR00001##
Inventors: |
Buhrlage; Sara Jean;
(Somerville, MA) ; Naar; Anders; (Arlington,
MA) ; Wagner; Gerhard; (Brookline, MA) ;
Arthanari; Haribabu; (Cambridge, MA) ; Nishikawa; Joy
Luz; (Cambridge, MA) ; Boeszoermenyi; Andras Pal;
(Boston, MA) ; Sanglard; Dominique; (Epalinges,
CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Dana-Farber Cancer Institute, Inc.
Centre Hospitalier Universitaire Vaudois
President and Fellows of Harvard College
The General Hospital Corporation |
Boston
Lausanne
Cambridge
Boston |
MA
MA
MA |
US
CH
US
US |
|
|
Assignee: |
Dana-Farber Cancer Institute,
Inc.
Boston
MA
Centre Hospitalier Universitaire Vaudois
Lausanne
MA
President and Fellows of Harvard College
Cambridge
MA
The General Hospital Corporation
Boston
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20200140381 A1 |
May 7, 2020 |
|
|
Family ID: |
1000005565006 |
Appl. No.: |
15/998620 |
Filed: |
February 17, 2017 |
PCT Filed: |
February 17, 2017 |
PCT NO: |
PCT/US2017/018433 PCKC 00 |
371 Date: |
August 16, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62296529 |
Feb 17, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 31/10 20180101;
C07C 335/40 20130101 |
International
Class: |
C07C 335/40 20060101
C07C335/40; A61P 31/10 20060101 A61P031/10 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with government support under grant
number P01 GM047467 awarded by the National Institutes of Health.
The government has certain rights in the invention.
Claims
1. A compound of Formula (II): ##STR00074## or a pharmaceutically
acceptable salt thereof, wherein: R.sup.1 and R.sup.2 are
independently hydrogen, optionally substituted alkyl; or optionally
R.sup.1 and R.sup.2 are joined together with the intervening atoms
to form optionally substituted carbocyclyl or optionally
substituted heterocyclyl; provided that at least one of R.sup.1 and
R.sup.2 is not hydrogen; each instance of R.sup.N is hydrogen,
optionally substituted alkyl, or optionally substituted acyl, or a
nitrogen protecting group; each instance of R.sup.3 is
independently hydrogen, halogen, --CN, --SCN, --NO.sub.2,
--N.sub.3, optionally substituted alkyl, optionally substituted
alkenyl, optionally substituted alkynyl, optionally substituted
carbocyclyl, optionally substituted heterocyclyl, optionally
substituted aryl, optionally substituted heteroaryl, optionally
substituted acyl, optionally substituted sulfinyl, optionally
substituted sulfonyl, --OR.sup.3a, --N(R.sup.3b).sub.2, or
--SR.sup.3c; or optionally two R.sup.3 are joined together with the
intervening atoms to form optionally substituted carbocyclyl or
optionally substituted heterocyclyl; each instance of R.sup.3a is
independently hydrogen, optionally substituted alkyl, optionally
substituted alkenyl, optionally substituted alkynyl, optionally
substituted carbocyclyl, optionally substituted heterocyclyl,
optionally substituted aryl, optionally substituted heteroaryl,
optionally substituted acyl, or an oxygen protecting group; each
instance of R.sup.3b is independently hydrogen, optionally
substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl, optionally substituted carbocyclyl, optionally
substituted heterocyclyl, optionally substituted aryl, optionally
substituted heteroaryl, optionally substituted acyl, or a nitrogen
protecting group; or optionally two R.sup.3b are joined together
with the intervening atoms to form optionally substituted
heterocyclyl or optionally substituted heteroaryl; each instance of
R.sup.3c is independently hydrogen, optionally substituted alkyl,
optionally substituted alkenyl, optionally substituted alkynyl,
optionally substituted carbocyclyl, optionally substituted
heterocyclyl, optionally substituted aryl, optionally substituted
heteroaryl, optionally substituted acyl, or a sulfur protecting
group; and n is 1, 2, 3, 4, or 5.
2. A compound of Formula (III): ##STR00075## or a pharmaceutically
acceptable salt thereof, wherein: Y is a bond, optionally
substituted alkylene, --O--, --NR.sup.N--, or --S--; R.sup.1 and
R.sup.2 are independently hydrogen, optionally substituted alkyl,
optionally substituted alkenyl, optionally substituted alkynyl,
optionally substituted aryl, optionally substituted carbocyclyl,
optionally substituted heterocyclyl, or optionally substituted
heteroaryl; or optionally R.sup.1 and R.sup.2 are joined together
with the intervening atoms to form optionally substituted
carbocyclyl or optionally substituted heterocyclyl; each instance
of R.sup.N is hydrogen, optionally substituted alkyl, or optionally
substituted acyl, or a nitrogen protecting group; each instance of
R.sup.3 is independently hydrogen, halogen, --CN, --SCN,
--NO.sub.2, --N.sub.3, optionally substituted alkyl, optionally
substituted alkenyl, optionally substituted alkynyl, optionally
substituted carbocyclyl, optionally substituted heterocyclyl,
optionally substituted aryl, optionally substituted heteroaryl,
optionally substituted acyl, optionally substituted sulfinyl,
optionally substituted sulfonyl, --OR.sup.3a, --N(R.sup.3b).sub.2,
or --SR.sup.3c; or optionally two R.sup.3 are joined together with
the intervening atoms to form optionally substituted carbocyclyl or
optionally substituted heterocyclyl; each instance of R.sup.3a is
independently hydrogen, optionally substituted alkyl, optionally
substituted alkenyl, optionally substituted alkynyl, optionally
substituted carbocyclyl, optionally substituted heterocyclyl,
optionally substituted aryl, optionally substituted heteroaryl,
optionally substituted acyl, or an oxygen protecting group; each
instance of R.sup.3b is independently hydrogen, optionally
substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl, optionally substituted carbocyclyl, optionally
substituted heterocyclyl, optionally substituted aryl, optionally
substituted heteroaryl, optionally substituted acyl, or a nitrogen
protecting group; or optionally two R.sup.3b are joined together
with the intervening atoms to form optionally substituted
heterocyclyl or optionally substituted heteroaryl; each instance of
R.sup.3c is independently hydrogen, optionally substituted alkyl,
optionally substituted alkenyl, optionally substituted alkynyl,
optionally substituted carbocyclyl, optionally substituted
heterocyclyl, optionally substituted aryl, optionally substituted
heteroaryl, optionally substituted acyl, or a sulfur protecting
group; R.sup.4 is optionally substituted carbocyclyl, optionally
substituted heterocyclyl, optionally substituted aryl, or
optionally substituted heteroaryl; and n is 1, 2, 3, or 4.
3. The compound of claim 1, wherein R.sup.1 and R.sup.2 are joined
together with the intervening atoms to form optionally substituted
carbocyclyl or optionally substituted heterocyclyl.
4-9. (canceled)
10. The compound of claim 1, wherein at least one of R.sup.1 and
R.sup.2 is halogen.
11-15. (canceled)
16. The compound of claim 1, wherein one of R.sup.1 or R.sup.2 is
optionally substituted phenyl.
17. (canceled)
18. The compound of claim 1, wherein each instance of R.sup.N is
hydrogen.
19-21. (canceled)
22. The compound of claim 1, wherein at least one instance of
R.sup.3 is selected from the group consisting of halogen,
optionally substituted acyl, optionally substituted sulfinyl,
optionally substituted sulfonyl, and haloalkyl.
23-37. (canceled)
38. The compound of claim 1, wherein Ring A is of one of the
following formulae: ##STR00076##
39. The compound of claim 1, wherein the compound is of Formula
(II-a): ##STR00077## or a pharmaceutically acceptable salt thereof,
wherein: m is 1, 2, 3, or 4.
40-41. (canceled)
42. The compound of claim 1, wherein the compound is of one of the
following formulae: ##STR00078## or a pharmaceutically acceptable
salt thereof.
43-45. (canceled)
46. The compound of claim 1, wherein the compound is of one of the
following formulae: ##STR00079## or a pharmaceutically acceptable
salt thereof.
47. The compound of claim 1, wherein the compound is of the
following formula: ##STR00080## or a pharmaceutically acceptable
salt thereof.
48. (canceled)
49. The compound of claim 1, wherein the compound is of the
following formula: ##STR00081## or a pharmaceutically acceptable
salt thereof.
50. The compound of claim 2, wherein the compound is of one of the
following formula: ##STR00082## or a pharmaceutically acceptable
salt thereof.
51-52. (canceled)
53. The compound of claim 1, wherein the compound is of one of the
following formulae: ##STR00083## or pharmaceutically acceptable
salt thereof.
54. A compound selected from the group consisting of: ##STR00084##
and pharmaceutically acceptable salts thereof.
55. A pharmaceutical compositions comprising a compound of claim 1,
and a pharmaceutically acceptable excipient.
56-59. (canceled)
60. A method for treating a disease in a subject, the method
comprising administering to the subject a therapeutically effective
amount of a compound of claim 1, or a pharmaceutically acceptable
salt thereof.
61. The method of claim 60, wherein the disease is an infectious
disease.
62-72. (canceled)
73. A method of killing a fungus or inhibiting the growth of a
fungus, the method comprising contacting the fungus with an
effective amount of a compound of claim 1, or a pharmaceutically
acceptable salt thereof.
74-87. (canceled)
Description
RELATED APPLICATIONS
[0001] This application is a national stage filing under 35 U.S.C.
.sctn. 371 of International PCT Application, PCT/US2017/018433,
filed Feb. 17, 2017, which claims priority under 35 U.S.C. .sctn.
119(e) to U.S. Provisional Patent Application, U.S. Ser. No.
62/296,529, filed Feb. 17, 2016, the entire contents of each of
which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] Eukaryotic transcription activators stimulate the expression
of specific sets of target genes through recruitment of
co-activators such as the RNA polymerase II-interacting Mediator
complex (see. e.g., Conaway, R. C. & Conaway, J. W. Function
and regulation of the Mediator complex. Current opinion in genetics
& development 21, 225-230, doi:10.1016/j.gde.2011.01.013
(2011); Poss, Z. C., Ebmeier, C. C. & Taatjes, D. J. The
Mediator complex and transcription regulation. Critical reviews in
biochemistry and molecular biology 48, 575-608.
doi:10.3109/10409238.2013.840259 (2013)). Aberrant function of
transcription activators has been implicated in a number of
diseases. However, therapeutic targeting efforts have been hampered
by a lack of detailed molecular knowledge of the mechanisms of gene
activation by disease-associated transcription activators. An
activator-targeted three-helix bundle KIX domain was previously
identified in the human MED 15 Mediator subunit that is
structurally conserved in Gal11/Med15 Mediator subunits in fungi
(See, e.g., Thakur, J. K. et al. A nuclear receptor-like pathway
regulating multidrug resistance in fungi. Nature 452, 604-609,
doi:nature06836 [pii] 10.1038/nature06836 (2008); Yang, F. et al.
An ARC/Mediator subunit required for SREBP control of cholesterol
and lipid homeostasis. Nature 442, 700-704 (2006)). The Gal11/Med15
KIX domain engages pleiotropic drug resistance transcription factor
(Pdr1) orthologues, which are key regulators of the multidrug
resistance (MDR) pathway in S. cerevisiae and in the clinically
important human pathogen Candida glabrata (see, e.g., Paul, S.
& Moye-Rowley, W. S. Multidrug resistance in fungi: regulation
of transporter-encoding gene expression. Frontiers in physiology 5,
143. doi:10.3389/fphys.2014.00143 (2014); Prasad, R. & Goffeau,
A. Yeast ATP-binding cassette transporters conferring multidrug
resistance. Annual review of microbiology 66, 39-63,
doi:10.1146/annurev-micro-092611-150111 (2012)).
[0004] The prevalence of C. glabrata is rising, partly due to their
low intrinsic susceptibility to azoles, the most widely used
antifungal (see, e.g., Pfaller, M. A. et al. Variation in
susceptibility of bloodstream isolates of Candida glabrata to
fluconazole according to patient age and geographic location in the
United States in 2001 to 2007. J Clin Microbiol 47, 3185-3190,
doi:JCM.00946-09 [pii] 10.1128/JCM.00946-09 (2009); Pfaller, M. A.,
Messer, S. A., Moet, G. J., Jones, R. N. & Castanheira, M.
Candida bloodstream infections: comparison of species distribution
and resistance to echinocandin and azole antifungal agents in
Intensive Care Unit (ICU) and non-ICU settings in the SENTRY
Antimicrobial Surveillance Program (2008-2009). Int J Antimicrob
Agents 38, 65-69, doi:S0924-8579(11)00132-4
[pii]10.1016/j.ijantimicag.2011.02.016). Drug-resistant clinical
isolates of C. glabrata most commonly harbour point mutations in
Pdr1 that render it constitutively active suggesting that this
transcriptional activation pathway represents a linchpin in C.
glabrata MDR (see, e.g., Ferrari, S. et al. Gain of function
mutations in CgPDR1 of Candida glabrata not only mediate antifungal
resistance but also enhance virulence. PLoS Pathog 5, e1000268,
doi:10.1371/journal.ppat.1000268 (2009); Ferrari, S., Sanguinetti,
M., Torelli, R., Posteraro, B. & Sanglard, D. Contribution of
CgPDR1-regulated genes in enhanced virulence of azole-resistant
Candida glabrata. PloS one 6, e17589,
doi:10.1371/journal.pone.0017589 (2011); Silva, L. V. et al.
Milbemycins: more than efflux inhibitors for fungal pathogens.
Antimicrobial agents and chemotherapy 57, 873-886,
doi:10.1128/AAC.02040-12 (2013); Vermitsky, J. P. et al. Pdr1
regulates multidrug resistance in Candida glabrata: gene disruption
and genome-wide expression studies. Mol Microbiol 61, 704-722
(2006); Sanguinetti, M. et al. Mechanisms of azole resistance in
clinical isolates of Candida glabrata collected during a hospital
survey of antifungal resistance. Antimicrob Agents Chemother 49,
668-679, doi:49/2/668 [pii] 10.1128/AAC.49.2.668-679.2005 (2005);
Caudle, K. E. et al. Genomewide expression profile analysis of the
Candida glabrata Pdr1 regulon. Eukaryot Cell 10, 373-383,
doi:EC.00073-10 [pii] 10.1128/EC.00073-10). Therefore, new
anti-infectives are needed for the treatment of infectious
diseases, especially those exhibiting multi-drug resistance
(MDR).
SUMMARY OF THE INVENTION
[0005] As described herein, sequential biochemical and in vivo
high-throughput screens have been carried out to identify small
molecule inhibitors of the interaction of the C. glabrata Pdr1
activation domain with the C. glabrata Gal11A KIX domain. The
compounds can be used for treating diseases (e.g., infectious
diseases) and/or for killing or inhibiting the growth of fungi. In
certain embodiments, for example, the compounds inhibit
Pdr1-dependent gene activation and re-sensitize drug-resistant C.
glabrata to azole antifungals in vitro and in animal models for
disseminated and urinary tract C. glabrata infection. Determining
the NMR structure of the C. glabrata Gal11A KIX domain provided a
detailed understanding of the molecular mechanism of Pdr1 gene
activation and MDR inhibition by iKIX1. As described herein, the
feasibility of small molecule targeting of a transcription
factor-binding site in Mediator as a novel therapeutic strategy in
treating diseases (e.g., fungal infectious disease) has been
demonstrated.
[0006] In one aspect, the present invention provides compounds of
Formula (II):
##STR00002##
and pharmaceutically acceptable salts, hydrates, solvates,
polymorphs, co-crystals, tautomers, stereoisomers, isotopically
labeled derivatives, and prodrugs thereof, wherein R.sup.1,
R.sup.2, R.sup.3. R.sup.N, and n are as defined herein.
[0007] Also provided herein are compounds of Formula (III):
##STR00003##
and pharmaceutically acceptable salts, hydrates, solvates,
polymorphs, co-crystals, tautomers, stereoisomers, isotopically
labeled derivatives, and prodrugs thereof, wherein R.sup.1.
R.sup.2, R.sup.3, R.sup.4. R.sup.N, Y, and n are as defined
herein.
[0008] Exemplary compounds of the present invention include, but
are not limited to, the following:
##STR00004## ##STR00005##
and pharmaceutically acceptable salts, hydrates, solvates,
polymorphs, co-crystals, tautomers, stereoisomers, isotopically
labeled derivatives, and prodrugs thereof.
[0009] In another aspect, the present invention provides
pharmaceutical compositions comprising a compound provided herein,
or a pharmaceutically acceptable salt, hydrate, solvate, polymorph,
co-crystal, tautomer, stereoisomer, isotopically labeled
derivative, or prodrug thereof, and a pharmaceutically acceptable
excipient. The pharmaceutical composition may comprise an effective
amount (e.g., a therapeutically or prophylactically effective
amount)
[0010] In yet another aspect, the present invention provides uses
of the compounds and pharmaceutical compositions described herein
for the treatment of diseases (e.g., infectious diseases such an
fungal infections), for inhibiting the activity of a fungus in a
subject or biological sample, for the killing of a fungus in a
subject or biological sample, and/or for inhibiting the growth of a
fungus in a subject or a biological sample. The methods of use
provided herein may be carried out, for example, in or on a
subject, in a clinical setting, or in an agricultural setting
(e.g., on a plant). The methods of use provided herein may be
carried out in vitro or in vivo.
[0011] In yet another aspect, the present invention provides kits
(e.g., pharmaceutical packs) comprising a compound described
herein, or a pharmaceutically acceptable salt, hydrate, solvate,
polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled
derivative, or prodrug thereof, or a pharmaceutical composition
thereof. In certain embodiments, the kit comprises an addition
therapeutic agent (e.g., an anti-infective agent). In certain
embodiments, the kit further comprises instructions for use or
administration.
[0012] The details of certain embodiments of the invention are set
forth in the Detailed Description of Certain Embodiments, as
described below. Other features, objects, and advantages of the
invention will be apparent from the Definitions, Examples, Figures,
and Claims.
Definitions
Chemical Definitions
[0013] Definitions of specific functional groups and chemical terms
are described in more detail below. The chemical elements are
identified in accordance with the Periodic Table of the Elements,
CAS version. Handbook of Chemistry and Physics, 75.sup.th Ed.,
inside cover, and specific functional groups are generally defined
as described therein. Additionally, general principles of organic
chemistry, as well as specific functional moieties and reactivity,
are described in Organic Chemistry, Thomas Sorrell, University
Science Books, Sausalito, 1999; Smith and March, March's Advanced
Organic Chemistry. 5.sup.th Edition, John Wiley & Sons, Inc.,
New York, 2001; Larock, Comprehensive Organic Transformations, VCH
Publishers, Inc., New York, 1989; and Carruthers, Some Modern
Methods of Organic Synthesis, 3.sup.rd Edition, Cambridge
University Press, Cambridge, 1987.
[0014] Compounds described herein can comprise one or more
asymmetric centers, and thus can exist in various stereoisomeric
forms, e.g., enantiomers and/or diastereomers. For example, the
compounds described herein can be in the form of an individual
enantiomer, diastereomer or geometric isomer, or can be in the form
of a mixture of stereoisomers, including racemic mixtures and
mixtures enriched in one or more stereoisomer. Isomers can be
isolated from mixtures by methods known to those skilled in the
art, including chiral high pressure liquid chromatography (HPLC)
and the formation and crystallization of chiral salts; or preferred
isomers can be prepared by asymmetric syntheses. See, for example,
Jacques et al., Enantiomers, Racemates and Resolutions (Wiley
Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725
(1977); Eliel, E. L. Stereochemistry of Carbon Compounds
(McGraw-Hill, N Y, 1962); and Wilen, S. H., Tables of Resolving
Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ. of
Notre Dame Press, Notre Dame, Ind. 1972). The invention
additionally encompasses compounds as individual isomers
substantially free of other isomers, and alternatively, as mixtures
of various isomers.
[0015] Unless otherwise stated, structures depicted herein are also
meant to include compounds that differ only in the presence of one
or more isotopically enriched atoms. For example, compounds having
the present structures except for the replacement of hydrogen by
deuterium or tritium, replacement of .sup.19F with .sup.18F, or the
replacement of .sup.12C with .sup.13C or .sup.14C are within the
scope of the disclosure. Such compounds are useful, for example, as
analytical tools or probes in biological assays.
[0016] When a range of values is listed, it is intended to
encompass each value and sub-range within the range. For example
"C.sub.1-6 alkyl" is intended to encompass, C.sub.1, C.sub.2,
C.sub.3, C.sub.4, C.sub.5, C.sub.6, C.sub.1-6, C.sub.1-5,
C.sub.1-4, C.sub.1-3, C.sub.1-2, C.sub.2-6, C.sub.2-5, C.sub.2-4,
C.sub.2-3, C.sub.3-6, C.sub.3-5, C.sub.3-4, C.sub.4-4, C.sub.4-5,
and C.sub.5-6 alkyl.
[0017] The term "aliphatic" refers to alkyl, alkenyl, alkynyl, and
carbocyclic groups. Likewise, the term "heteroaliphatic" refers to
heteroalkyl, heteroalkenyl, heteroalkynyl, and heterocyclic
groups.
[0018] The term "alkyl" refers to a radical of a straight-chain or
branched saturated hydrocarbon group having from 1 to 10 carbon
atoms ("C.sub.1-10 alkyl"). In some embodiments, an alkyl group has
1 to 9 carbon atoms ("C.sub.1-9 alkyl"). In some embodiments, an
alkyl group has 1 to 8 carbon atoms ("C.sub.1-8 alkyl"). In some
embodiments, an alkyl group has 1 to 7 carbon atoms ("C.sub.1-7
alkyl"). In some embodiments, an alkyl group has 1 to 6 carbon
atoms ("C.sub.1-6 alkyl"). In some embodiments, an alkyl group has
1 to 5 carbon atoms ("C.sub.1-5 alkyl"). In some embodiments, an
alkyl group has 1 to 4 carbon atoms ("C.sub.1-4 alkyl"). In some
embodiments, an alkyl group has 1 to 3 carbon atoms ("C.sub.1-3
alkyl"). In some embodiments, an alkyl group has 1 to 2 carbon
atoms ("C.sub.1-2 alkyl"). In some embodiments, an alkyl group has
1 carbon atom ("C.sub.1 alkyl"). In some embodiments, an alkyl
group has 2 to 6 carbon atoms ("C.sub.2-6 alkyl"). Examples of
C.sub.1-6 alkyl groups include methyl (C.sub.1), ethyl (C.sub.2),
propyl (C.sub.3) (e.g., n-propyl, isopropyl), butyl (C.sub.4)
(e.g., n-butyl, tert-butyl, sec-butyl, iso-butyl), pentyl (C.sub.5)
(e.g., n-pentyl, 3-pentanyl, amyl, neopentyl, 3-methyl-2-butanyl,
tertiary amyl), and hexyl (C.sub.6) (e.g., n-hexyl). Additional
examples of alkyl groups include n-heptyl (C.sub.7), n-octyl
(C.sub.8), and the like. Unless otherwise specified, each instance
of an alkyl group is independently unsubstituted (an "unsubstituted
alkyl") or substituted (a "substituted alkyl") with one or more
substituents (e.g., halogen, such as F). In certain embodiments,
the alkyl group is an unsubstituted C.sub.1-10 alkyl (such as
unsubstituted C.sub.1-6 alkyl, e.g., --CH.sub.3 (Me), unsubstituted
ethyl (Et), unsubstituted propyl (Pr, e.g., unsubstituted n-propyl
(n-Pr), unsubstituted isopropyl (i-Pr)), unsubstituted butyl (Bu,
e.g., unsubstituted n-butyl (n-Bu), unsubstituted tert-butyl
(tert-Bu or t-Bu), unsubstituted sec-butyl (sec-Bu), unsubstituted
isobutyl (i-Bu)). In certain embodiments, the alkyl group is a
substituted C.sub.1-10 alkyl (such as substituted C.sub.1-6 alkyl,
e.g., --CF.sub.3, Bn).
[0019] The term "haloalkyl" is a substituted alkyl group, wherein
one or more of the hydrogen atoms are independently replaced by a
halogen, e.g., fluoro, bromo, chloro, or iodo. In some embodiments,
the haloalkyl moiety has 1 to 8 carbon atoms ("C.sub.1-8
haloalkyl"). In some embodiments, the haloalkyl moiety has 1 to 6
carbon atoms ("C.sub.1-6 haloalkyl"). In some embodiments, the
haloalkyl moiety has 1 to 4 carbon atoms ("C.sub.1-4 haloalkyl").
In some embodiments, the haloalkyl moiety has 1 to 3 carbon atoms
("C.sub.1-3 haloalkyl"). In some embodiments, the haloalkyl moiety
has 1 to 2 carbon atoms ("C.sub.1-2 haloalkyl"). Examples of
haloalkyl groups include --CHF.sub.2, --CH.sub.2F, --CF.sub.3,
--CH.sub.2CF.sub.3, --CF.sub.2CF.sub.3, --CF.sub.2CF.sub.2CF.sub.3,
--CCl.sub.3. --CFCl.sub.2, --CF.sub.2Cl, and the like.
[0020] The term "heteroalkyl" refers to an alkyl group, which
further includes at least one heteroatom (e.g., 1, 2, 3, or 4
heteroatoms) selected from oxygen, nitrogen, or sulfur within
(i.e., inserted between adjacent carbon atoms of) and/or placed at
one or more terminal position(s) of the parent chain. In certain
embodiments, a heteroalkyl group refers to a saturated group having
from 1 to 10 carbon atoms and 1 or more heteroatoms within the
parent chain ("heteroC.sub.1-10 alkyl"). In some embodiments, a
heteroalkyl group is a saturated group having 1 to 9 carbon atoms
and 1 or more heteroatoms within the parent chain ("heteroC.sub.1-9
alkyl"). In some embodiments, a heteroalkyl group is a saturated
group having 1 to 8 carbon atoms and 1 or more heteroatoms within
the parent chain ("heteroC.sub.1-8 alkyl"). In some embodiments, a
heteroalkyl group is a saturated group having 1 to 7 carbon atoms
and 1 or more heteroatoms within the parent chain ("heteroC.sub.1-7
alkyl"). In some embodiments, a heteroalkyl group is a saturated
group having 1 to 6 carbon atoms and 1 or more heteroatoms within
the parent chain ("heteroC.sub.1-6 alkyl"). In some embodiments, a
heteroalkyl group is a saturated group having 1 to 5 carbon atoms
and 1 or 2 heteroatoms within the parent chain ("heteroC.sub.1-5
alkyl"). In some embodiments, a heteroalkyl group is a saturated
group having 1 to 4 carbon atoms and 1 or 2 heteroatoms within the
parent chain ("heteroC.sub.1-4 alkyl"). In some embodiments, a
heteroalkyl group is a saturated group having 1 to 3 carbon atoms
and 1 heteroatom within the parent chain ("heteroC.sub.1-3 alkyl").
In some embodiments, a heteroalkyl group is a saturated group
having 1 to 2 carbon atoms and 1 heteroatom within the parent chain
("heteroC.sub.1-2 alkyl"). In some embodiments, a heteroalkyl group
is a saturated group having 1 carbon atom and 1 heteroatom
("heteroC.sub.1 alkyl"). In some embodiments, a heteroalkyl group
is a saturated group having 2 to 6 carbon atoms and 1 or 2
heteroatoms within the parent chain ("heteroC.sub.2-6 alkyl").
Unless otherwise specified, each instance of a heteroalkyl group is
independently unsubstituted (an "unsubstituted heteroalkyl") or
substituted (a "substituted heteroalkyl") with one or more
substituents. In certain embodiments, the heteroalkyl group is an
unsubstituted heteroC.sub.1-10 alkyl. In certain embodiments, the
heteroalkyl group is a substituted heteroC.sub.1-10 alkyl.
[0021] The term "alkenyl" refers to a radical of a straight-chain
or branched hydrocarbon group having from 2 to 10 carbon atoms and
one or more carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double
bonds). In some embodiments, an alkenyl group has 2 to 9 carbon
atoms ("C.sub.2-9 alkenyl"). In some embodiments, an alkenyl group
has 2 to 8 carbon atoms ("C.sub.2-8 alkenyl"). In some embodiments,
an alkenyl group has 2 to 7 carbon atoms ("C.sub.2-7 alkenyl"). In
some embodiments, an alkenyl group has 2 to 6 carbon atoms
("C.sub.2-6 alkenyl"). In some embodiments, an alkenyl group has 2
to 5 carbon atoms ("C.sub.2-8 alkenyl"). In some embodiments, an
alkenyl group has 2 to 4 carbon atoms ("C.sub.2-4 alkenyl"). In
some embodiments, an alkenyl group has 2 to 3 carbon atoms
("C.sub.2-3 alkenyl"). In some embodiments, an alkenyl group has 2
carbon atoms ("C.sub.2 alkenyl"). The one or more carbon-carbon
double bonds can be internal (such as in 2-butenyl) or terminal
(such as in 1-butenyl). Examples of C.sub.2-4 alkenyl groups
include ethenyl (C.sub.2), 1-propenyl (C.sub.3), 2-propenyl
(C.sub.3), 1-butenyl (C.sub.4), 2-butenyl (C.sub.4), butadienyl
(C.sub.4), and the like. Examples of C.sub.2-6 alkenyl groups
include the aforementioned C.sub.2-4 alkenyl groups as well as
pentenyl (C.sub.5), pentadienyl (C.sub.5), hexenyl (C.sub.6), and
the like. Additional examples of alkenyl include heptenyl
(C.sub.7), octenyl (C.sub.8), octatrienyl (C.sub.8), and the like.
Unless otherwise specified, each instance of an alkenyl group is
independently unsubstituted (an "unsubstituted alkenyl") or
substituted (a "substituted alkenyl") with one or more
substituents. In certain embodiments, the alkenyl group is an
unsubstituted C.sub.2-10 alkenyl. In certain embodiments, the
alkenyl group is a substituted C.sub.2-10 alkenyl. In an alkenyl
group, a C.dbd.C double bond for which the stereochemistry is not
specified (e.g., --CH.dbd.CHCH.sub.3 or
##STR00006##
may be an (E)- or (Z)-double bond.
[0022] The term "heteroalkenyl" refers to an alkenyl group, which
further includes at least one heteroatom (e.g., 1, 2, 3, or 4
heteroatoms) selected from oxygen, nitrogen, or sulfur within
(i.e., inserted between adjacent carbon atoms of) and/or placed at
one or more terminal position(s) of the parent chain. In certain
embodiments, a heteroalkenyl group refers to a group having from 2
to 10 carbon atoms, at least one double bond, and 1 or more
heteroatoms within the parent chain ("heteroC.sub.2-10 alkenyl").
In some embodiments, a heteroalkenyl group has 2 to 9 carbon atoms
at least one double bond, and 1 or more heteroatoms within the
parent chain ("heteroC.sub.2-9 alkenyl"). In some embodiments, a
heteroalkenyl group has 2 to 8 carbon atoms, at least one double
bond, and 1 or more heteroatoms within the parent chain
("heteroC.sub.2-8 alkenyl"). In some embodiments, a heteroalkenyl
group has 2 to 7 carbon atoms, at least one double bond, and 1 or
more heteroatoms within the parent chain ("heteroC.sub.2-7
alkenyl"). In some embodiments, a heteroalkenyl group has 2 to 6
carbon atoms, at least one double bond, and 1 or more heteroatoms
within the parent chain ("heteroC.sub.2-6 alkenyl"). In some
embodiments, a heteroalkenyl group has 2 to 5 carbon atoms, at
least one double bond, and 1 or 2 heteroatoms within the parent
chain ("heteroC.sub.2-5 alkenyl"). In some embodiments, a
heteroalkenyl group has 2 to 4 carbon atoms, at least one double
bond, and 1 or 2 heteroatoms within the parent chain
("heteroC.sub.2-4 alkenyl"). In some embodiments, a heteroalkenyl
group has 2 to 3 carbon atoms, at least one double bond, and 1
heteroatom within the parent chain ("heteroC.sub.2-3 alkenyl"). In
some embodiments, a heteroalkenyl group has 2 to 6 carbon atoms, at
least one double bond, and 1 or 2 heteroatoms within the parent
chain ("heteroC.sub.2-6 alkenyl"). Unless otherwise specified, each
instance of a heteroalkenyl group is independently unsubstituted
(an "unsubstituted heteroalkenyl") or substituted (a "substituted
heteroalkenyl") with one or more substituents. In certain
embodiments, the heteroalkenyl group is an unsubstituted
heteroC.sub.2-10 alkenyl. In certain embodiments, the heteroalkenyl
group is a substituted heteroC.sub.2-10 alkenyl.
[0023] The term "alkynyl" refers to a radical of a straight-chain
or branched hydrocarbon group having from 2 to 10 carbon atoms and
one or more carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple
bonds) ("C.sub.2-10 alkynyl"). In some embodiments, an alkynyl
group has 2 to 9 carbon atoms ("C.sub.2-9 alkynyl"). In some
embodiments, an alkynyl group has 2 to 8 carbon atoms ("C.sub.2-8
alkynyl"). In some embodiments, an alkynyl group has 2 to 7 carbon
atoms ("C.sub.2-7 alkynyl"). In some embodiments, an alkynyl group
has 2 to 6 carbon atoms ("C.sub.2-6 alkynyl"). In some embodiments,
an alkynyl group has 2 to 5 carbon atoms ("C.sub.2-5 alkynyl"). In
some embodiments, an alkynyl group has 2 to 4 carbon atoms
("C.sub.2-4 alkynyl"). In some embodiments, an alkynyl group has 2
to 3 carbon atoms ("C.sub.2-3 alkynyl"). In some embodiments, an
alkynyl group has 2 carbon atoms ("C.sub.2 alkynyl"). The one or
more carbon-carbon triple bonds can be internal (such as in
2-butynyl) or terminal (such as in 1-butynyl). Examples of
C.sub.2-4 alkynyl groups include, without limitation, ethynyl
(C.sub.2), 1-propynyl (C.sub.3), 2-propynyl (C.sub.3), 1-butynyl
(C.sub.4), 2-butynyl (C.sub.4), and the like. Examples of C.sub.2-6
alkenyl groups include the aforementioned C.sub.2-4 alkynyl groups
as well as pentynyl (C.sub.5), hexynyl (C.sub.6), and the like.
Additional examples of alkynyl include heptynyl (C.sub.7), octynyl
(C.sub.8), and the like. Unless otherwise specified, each instance
of an alkynyl group is independently unsubstituted (an
"unsubstituted alkynyl") or substituted (a "substituted alkynyl")
with one or more substituents. In certain embodiments, the alkynyl
group is an unsubstituted C.sub.2-10 alkynyl. In certain
embodiments, the alkynyl group is a substituted C.sub.2-10
alkynyl.
[0024] The term "heteroalkynyl" refers to an alkynyl group, which
further includes at least one heteroatom (e.g., 1, 2, 3, or 4
heteroatoms) selected from oxygen, nitrogen, or sulfur within
(i.e., inserted between adjacent carbon atoms of) and/or placed at
one or more terminal position(s) of the parent chain. In certain
embodiments, a heteroalkynyl group refers to a group having from 2
to 10 carbon atoms, at least one triple bond, and 1 or more
heteroatoms within the parent chain ("heteroC.sub.2-10 alkynyl").
In some embodiments, a heteroalkynyl group has 2 to 9 carbon atoms,
at least one triple bond, and 1 or more heteroatoms within the
parent chain ("heteroC.sub.2-9 alkynyl"). In some embodiments, a
heteroalkynyl group has 2 to 8 carbon atoms, at least one triple
bond, and 1 or more heteroatoms within the parent chain
("heteroC.sub.2-8 alkynyl"). In some embodiments, a heteroalkynyl
group has 2 to 7 carbon atoms, at least one triple bond, and 1 or
more heteroatoms within the parent chain ("heteroC.sub.2-7
alkynyl"). In some embodiments, a heteroalkynyl group has 2 to 6
carbon atoms, at least one triple bond, and 1 or more heteroatoms
within the parent chain ("heteroC.sub.2-6 alkynyl"). In some
embodiments, a heteroalkynyl group has 2 to 5 carbon atoms, at
least one triple bond, and 1 or 2 heteroatoms within the parent
chain ("heteroC.sub.2-5 alkynyl"). In some embodiments, a
heteroalkynyl group has 2 to 4 carbon atoms, at least one triple
bond, and 1 or 2 heteroatoms within the parent chain
("heteroC.sub.2-4 alkynyl"). In some embodiments, a heteroalkynyl
group has 2 to 3 carbon atoms, at least one triple bond, and 1
heteroatom within the parent chain ("heteroC.sub.2-3 alkynyl"). In
some embodiments, a heteroalkynyl group has 2 to 6 carbon atoms, at
least one triple bond, and 1 or 2 heteroatoms within the parent
chain ("heteroC.sub.2-6 alkynyl"). Unless otherwise specified, each
instance of a heteroalkynyl group is independently unsubstituted
(an "unsubstituted heteroalkynyl") or substituted (a "substituted
heteroalkynyl") with one or more substituents. In certain
embodiments, the heteroalkynyl group is an unsubstituted
heteroC.sub.2-10 alkynyl. In certain embodiments, the heteroalkynyl
group is a substituted heteroC.sub.2-10 alkynyl.
[0025] The term "carbocyclyl" or "carbocyclic" refers to a radical
of a non-aromatic cyclic hydrocarbon group having from 3 to 14 ring
carbon atoms ("C.sub.3-14 carbocyclyl") and zero heteroatoms in the
non-aromatic ring system. In some embodiments, a carbocyclyl group
has 3 to 10 ring carbon atoms ("C.sub.3-10 carbocyclyl"). In some
embodiments, a carbocyclyl group has 3 to 8 ring carbon atoms
("C.sub.3-8 carbocyclyl"). In some embodiments, a carbocyclyl group
has 3 to 7 ring carbon atoms ("C.sub.3-? carbocyclyl"). In some
embodiments, a carbocyclyl group has 3 to 6 ring carbon atoms
("C.sub.3-6 carbocyclyl"). In some embodiments, a carbocyclyl group
has 4 to 6 ring carbon atoms ("C.sub.4-6 carbocyclyl"). In some
embodiments, a carbocyclyl group has 5 to 6 ring carbon atoms
("C.sub.5-6 carbocyclyl"). In some embodiments, a carbocyclyl group
has 5 to 10 ring carbon atoms ("C.sub.5-10 carbocyclyl"). Exemplary
C.sub.3-6 carbocyclyl groups include, without limitation,
cyclopropyl (C.sub.3), cyclopropenyl (C.sub.3), cyclobutyl
(C.sub.4), cyclobutenyl (C.sub.4), cyclopentyl (C.sub.5),
cyclopentenyl (C.sub.5), cyclohexyl (C.sub.6), cyclohexenyl
(C.sub.6), cyclohexadienyl (C.sub.6), and the like. Exemplary
C.sub.3-8 carbocyclyl groups include, without limitation, the
aforementioned C.sub.3-6 carbocyclyl groups as well as cycloheptyl
(C.sub.4), cycloheptenyl (C.sub.7), cycloheptadienyl (C.sub.7),
cycloheptatrienyl (C.sub.7), cyclooctyl (C.sub.8), cyclooctenyl
(C.sub.8), bicyclo[2.2.1]heptanyl (C.sub.7), bicyclo[2.2.2]octanyl
(C.sub.8), and the like. Exemplary C.sub.3-10 carbocyclyl groups
include, without limitation, the aforementioned C.sub.3-8
carbocyclyl groups as well as cyclononyl (C.sub.9), cyclononenyl
(C.sub.9), cyclodecyl (C.sub.10), cyclodecenyl (C.sub.10),
octahydro-1H-indenyl (C.sub.9), decahydronaphthalenyl (C.sub.10),
spiro[4.5]decanyl (C.sub.10), and the like. As the foregoing
examples illustrate, in certain embodiments, the carbocyclyl group
is either monocyclic ("monocyclic carbocyclyl") or polycyclic
(e.g., containing a fused, bridged or spiro ring system such as a
bicyclic system ("bicyclic carbocyclyl") or tricyclic system
("tricyclic carbocyclyl")) and can be saturated or can contain one
or more carbon-carbon double or triple bonds. "Carbocyclyl" also
includes ring systems wherein the carbocyclyl ring, as defined
above, is fused with one or more aryl or heteroaryl groups wherein
the point of attachment is on the carbocyclyl ring, and in such
instances, the number of carbons continue to designate the number
of carbons in the carbocyclic ring system. Unless otherwise
specified, each instance of a carbocyclyl group is independently
unsubstituted (an "unsubstituted carbocyclyl") or substituted (a
"substituted carbocyclyl") with one or more substituents. In
certain embodiments, the carbocyclyl group is an unsubstituted
C.sub.3-14 carbocyclyl. In certain embodiments, the carbocyclyl
group is a substituted C.sub.3-14 carbocyclyl.
[0026] In some embodiments, "carbocyclyl" is a monocyclic,
saturated carbocyclyl group having from 3 to 14 ring carbon atoms
("C.sub.3-14 cycloalkyl"). In some embodiments, a cycloalkyl group
has 3 to 10 ring carbon atoms ("C.sub.3-10 cycloalkyl"). In some
embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms
("C.sub.3-8 cycloalkyl"). In some embodiments, a cycloalkyl group
has 3 to 6 ring carbon atoms ("C.sub.3-6 cycloalkyl"). In some
embodiments, a cycloalkyl group has 4 to 6 ring carbon atoms
("C.sub.4-6 cycloalkyl"). In some embodiments, a cycloalkyl group
has 5 to 6 ring carbon atoms ("C.sub.5-6 cycloalkyl"). In some
embodiments, a cycloalkyl group has 5 to 10 ring carbon atoms
("C.sub.5-10 cycloalkyl"). Examples of C.sub.5-6 cycloalkyl groups
include cyclopentyl (C.sub.5) and cyclohexyl (C.sub.5). Examples of
C.sub.3-6 cycloalkyl groups include the aforementioned C.sub.5-6
cycloalkyl groups as well as cyclopropyl (C.sub.3) and cyclobutyl
(C.sub.4). Examples of C.sub.3-8 cycloalkyl groups include the
aforementioned C.sub.3-6 cycloalkyl groups as well as cycloheptyl
(C?) and cyclooctyl (C.sub.8). Unless otherwise specified, each
instance of a cycloalkyl group is independently unsubstituted (an
"unsubstituted cycloalkyl") or substituted (a "substituted
cycloalkyl") with one or more substituents. In certain embodiments,
the cycloalkyl group is an unsubstituted C.sub.3-14 cycloalkyl. In
certain embodiments, the cycloalkyl group is a substituted
C.sub.3-14 cycloalkyl.
[0027] The term "heterocyclyl" or "heterocyclic" refers to a
radical of a 3- to 14-membered non-aromatic ring system having ring
carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom
is independently selected from nitrogen, oxygen, and sulfur ("3-14
membered heterocyclyl"). In heterocyclyl groups that contain one or
more nitrogen atoms, the point of attachment can be a carbon or
nitrogen atom, as valency permits. A heterocyclyl group can either
be monocyclic ("monocyclic heterocyclyl") or polycyclic (e.g., a
fused, bridged or spiro ring system such as a bicyclic system
("bicyclic heterocyclyl") or tricyclic system ("tricyclic
heterocyclyl")), and can be saturated or can contain one or more
carbon-carbon double or triple bonds. Heterocyclyl polycyclic ring
systems can include one or more heteroatoms in one or both rings.
"Heterocyclyl" also includes ring systems wherein the heterocyclyl
ring, as defined above, is fused with one or more carbocyclyl
groups wherein the point of attachment is either on the carbocyclyl
or heterocyclyl ring, or ring systems wherein the heterocyclyl
ring, as defined above, is fused with one or more aryl or
heteroaryl groups, wherein the point of attachment is on the
heterocyclyl ring, and in such instances, the number of ring
members continue to designate the number of ring members in the
heterocyclyl ring system. Unless otherwise specified, each instance
of heterocyclyl is independently unsubstituted (an "unsubstituted
heterocyclyl") or substituted (a "substituted heterocyclyl") with
one or more substituents. In certain embodiments, the heterocyclyl
group is an unsubstituted 3-14 membered heterocyclyl. In certain
embodiments, the heterocyclyl group is a substituted 3-14 membered
heterocyclyl.
[0028] In some embodiments, a heterocyclyl group is a 5-10 membered
non-aromatic ring system having ring carbon atoms and 1-4 ring
heteroatoms, wherein each heteroatom is independently selected from
nitrogen, oxygen, and sulfur ("5-10 membered heterocyclyl"). In
some embodiments, a heterocyclyl group is a 5-8 membered
non-aromatic ring system having ring carbon atoms and 1-4 ring
heteroatoms, wherein each heteroatom is independently selected from
nitrogen, oxygen, and sulfur ("5-8 membered heterocyclyl"). In some
embodiments, a heterocyclyl group is a 5-6 membered non-aromatic
ring system having ring carbon atoms and 1-4 ring heteroatoms,
wherein each heteroatom is independently selected from nitrogen,
oxygen, and sulfur ("5-6 membered heterocyclyl"). In some
embodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatoms
selected from nitrogen, oxygen, and sulfur. In some embodiments,
the 5-6 membered heterocyclyl has 1-2 ring heteroatoms selected
from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6
membered heterocyclyl has 1 ring heteroatom selected from nitrogen,
oxygen, and sulfur.
[0029] Exemplary 3-membered heterocyclyl groups containing 1
heteroatom include, without limitation, azirdinyl, oxiranyl, and
thiiranyl. Exemplary 4-membered heterocyclyl groups containing 1
heteroatom include, without limitation, azetidinyl, oxetanyl, and
thietanyl. Exemplary 5-membered heterocyclyl groups containing 1
heteroatom include, without limitation, tetrahydrofuranyl,
dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl,
pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary
5-membered heterocyclyl groups containing 2 heteroatoms include,
without limitation, dioxolanyl, oxathiolanyl and dithiolanyl.
Exemplary 5-membered heterocyclyl groups containing 3 heteroatoms
include, without limitation, triazolinyl, oxadiazolinyl, and
thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing
1 heteroatom include, without limitation, piperidinyl,
tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary
6-membered heterocyclyl groups containing 2 heteroatoms include,
without limitation, piperazinyl, morpholinyl, dithianyl, and
dioxanyl. Exemplary 6-membered heterocyclyl groups containing 2
heteroatoms include, without limitation, triazinanyl. Exemplary
7-membered heterocyclyl groups containing 1 heteroatom include,
without limitation, azepanyl, oxepanyl and thiepanyl. Exemplary
8-membered heterocyclyl groups containing 1 heteroatom include,
without limitation, azocanyl, oxecanyl and thiocanyl. Exemplary
bicyclic heterocyclyl groups include, without limitation,
indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl,
tetrahydrobenzothienyl, tetrahydrobenzofuranyl, tetrahydroindolyl,
tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl,
decahydroisoquinolinyl, octahydrochromenyl, octahydroisochromenyl,
decahydronaphthyridinyl, decahydro-1,8-naphthyridinyl,
octahydropyrrolo[3,2-b]pyrrole, indolinyl, phthalimidyl,
naphthalimidyl, chromanyl, chromenyl, 1H-benzo[e][1,4]diazepinyl,
1,4,5,7-tetrahydropyrano[3,4-b]pyrrolyl,
5,6-dihydro-4H-furo[3,2-b]pyrrolyl,
6,7-dihydro-5H-furo[3,2-b]pyranyl,
5,7-dihydro-4H-thieno[2,3-c]pyranyl,
2,3-dihydro-1H-pyrrolo[2,3-b]pyridinyl,
2,3-dihydrofuro[2,3-b]pyridinyl,
4,5,6,7-tetrahydro-1H-pyrrolo[2,3-b]pyridinyl,
4,5,6,7-tetrahydrofuro[3,2-c]pyridinyl,
4,5,6,7-tetrahydrothieno[3,2-b]pyridinyl,
1,2,3,4-tetrahydro-1,6-naphthyridinyl, and the like.
[0030] The term "aryl" refers to a radical of a monocyclic or
polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system
(e.g., having 6, 10, or 14 .pi. electrons shared in a cyclic array)
having 6-14 ring carbon atoms and zero heteroatoms provided in the
aromatic ring system ("C.sub.6-14 aryl"). In some embodiments, an
aryl group has 6 ring carbon atoms ("C.sub.6 aryl"; e.g., phenyl).
In some embodiments, an aryl group has 10 ring carbon atoms
("C.sub.10 aryl"; e.g., naphthyl such as 1-naphthyl and
2-naphthyl). In some embodiments, an aryl group has 14 ring carbon
atoms ("C.sub.14 aryl"; e.g., anthracyl). "Aryl" also includes ring
systems wherein the aryl ring, as defined above, is fused with one
or more carbocyclyl or heterocyclyl groups wherein the radical or
point of attachment is on the aryl ring, and in such instances, the
number of carbon atoms continue to designate the number of carbon
atoms in the aryl ring system. Unless otherwise specified, each
instance of an aryl group is independently unsubstituted (an
"unsubstituted aryl") or substituted (a "substituted aryl") with
one or more substituents. In certain embodiments, the aryl group is
an unsubstituted C.sub.6-14 aryl. In certain embodiments, the aryl
group is a substituted C.sub.6-14 aryl.
[0031] The term "heteroaryl" refers to a radical of a 5-14 membered
monocyclic or polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic
ring system (e.g., having 6, 10, or 14 .pi. electrons shared in a
cyclic array) having ring carbon atoms and 1-4 ring heteroatoms
provided in the aromatic ring system, wherein each heteroatom is
independently selected from nitrogen, oxygen, and sulfur ("5-14
membered heteroaryl"). In heteroaryl groups that contain one or
more nitrogen atoms, the point of attachment can be a carbon or
nitrogen atom, as valency permits. Heteroaryl polycyclic ring
systems can include one or more heteroatoms in one or both rings.
"Heteroaryl" includes ring systems wherein the heteroaryl ring, as
defined above, is fused with one or more carbocyclyl or
heterocyclyl groups wherein the point of attachment is on the
heteroaryl ring, and in such instances, the number of ring members
continue to designate the number of ring members in the heteroaryl
ring system. "Heteroaryl" also includes ring systems wherein the
heteroaryl ring, as defined above, is fused with one or more aryl
groups wherein the point of attachment is either on the aryl or
heteroaryl ring, and in such instances, the number of ring members
designates the number of ring members in the fused polycyclic
(aryl/heteroaryl) ring system. Polycyclic heteroaryl groups wherein
one ring does not contain a heteroatom (e.g., indolyl, quinolinyl,
carbazolyl, and the like) the point of attachment can be on either
ring, i.e., either the ring bearing a heteroatom (e.g., 2-indolyl)
or the ring that does not contain a heteroatom (e.g.,
5-indolyl).
[0032] In some embodiments, a heteroaryl group is a 5-10 membered
aromatic ring system having ring carbon atoms and 1-4 ring
heteroatoms provided in the aromatic ring system, wherein each
heteroatom is independently selected from nitrogen, oxygen, and
sulfur ("5-10 membered heteroaryl"). In some embodiments, a
heteroaryl group is a 5-8 membered aromatic ring system having ring
carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring
system, wherein each heteroatom is independently selected from
nitrogen, oxygen, and sulfur ("5-8 membered heteroaryl"). In some
embodiments, a heteroaryl group is a 5-6 membered aromatic ring
system having ring carbon atoms and 1-4 ring heteroatoms provided
in the aromatic ring system, wherein each heteroatom is
independently selected from nitrogen, oxygen, and sulfur ("5-6
membered heteroaryl"). In some embodiments, the 5-6 membered
heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen,
and sulfur. In some embodiments, the 5-6 membered heteroaryl has
1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In
some embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom
selected from nitrogen, oxygen, and sulfur. Unless otherwise
specified, each instance of a heteroaryl group is independently
unsubstituted (an "unsubstituted heteroaryl") or substituted (a
"substituted heteroaryl") with one or more substituents. In certain
embodiments, the heteroaryl group is an unsubstituted 5-14 membered
heteroaryl. In certain embodiments, the heteroaryl group is a
substituted 5-14 membered heteroaryl.
[0033] Exemplary 5-membered heteroaryl groups containing 1
heteroatom include, without limitation, pyrrolyl, furanyl, and
thiophenyl. Exemplary 5-membered heteroaryl groups containing 2
heteroatoms include, without limitation, imidazolyl, pyrazolyl,
oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary
5-membered heteroaryl groups containing 3 heteroatoms include,
without limitation, triazolyl, oxadiazolyl, and thiadiazolyl.
Exemplary 5-membered heteroaryl groups containing 4 heteroatoms
include, without limitation, tetrazolyl. Exemplary 6-membered
heteroaryl groups containing 1 heteroatom include, without
limitation, pyridinyl. Exemplary 6-membered heteroaryl groups
containing 2 heteroatoms include, without limitation, pyridazinyl,
pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups
containing 3 or 4 heteroatoms include, without limitation,
triazinyl and tetrazinyl, respectively. Exemplary 7-membered
heteroaryl groups containing 1 heteroatom include, without
limitation, azepinyl, oxepinyl, and thiepinyl. Exemplary
5,6-bicyclic heteroaryl groups include, without limitation,
indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl,
isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl,
benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl,
benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl.
Exemplary 6,6-bicyclic heteroaryl groups include, without
limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl,
cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplary
tricyclic heteroaryl groups include, without limitation,
phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl,
phenothiazinyl, phenoxazinyl and phenazinyl.
[0034] Affixing the suffix "-ene" to a group indicates the group is
a divalent moiety, e.g., alkylene is the divalent moiety of alkyl,
alkenylene is the divalent moiety of alkenyl, alkynylene is the
divalent moiety of alkynyl, heteroalkylene is the divalent moiety
of heteroalkyl, heteroalkenylene is the divalent moiety of
heteroalkenyl, heteroalkynylene is the divalent moiety of
heteroalkynyl, carbocyclylene is the divalent moiety of
carbocyclyl, heterocyclylene is the divalent moiety of
heterocyclyl, arylene is the divalent moiety of aryl, and
heteroarylene is the divalent moiety of heteroaryl.
[0035] A group is optionally substituted unless expressly provided
otherwise. The term "optionally substituted" refers to being
substituted or unsubstituted. In certain embodiments, alkyl,
alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,
carbocyclyl, heterocyclyl, aryl, and heteroaryl groups are
optionally substituted. "Optionally substituted" refers to a group
which may be substituted or unsubstituted (e.g., "substituted" or
"unsubstituted" alkyl, "substituted" or "unsubstituted" alkenyl,
"substituted" or "unsubstituted" alkynyl, "substituted" or
"unsubstituted" heteroalkyl, "substituted" or "unsubstituted"
heteroalkenyl, "substituted" or "unsubstituted" heteroalkynyl,
"substituted" or "unsubstituted" carbocyclyl, "substituted" or
"unsubstituted" heterocyclyl, "substituted" or "unsubstituted" aryl
or "substituted" or "unsubstituted" heteroaryl group). In general,
the term "substituted" means that at least one hydrogen present on
a group is replaced with a permissible substituent, e.g., a
substituent which upon substitution results in a stable compound,
e.g., a compound which does not spontaneously undergo
transformation such as by rearrangement, cyclization, elimination,
or other reaction. Unless otherwise indicated, a "substituted"
group has a substituent at one or more substitutable positions of
the group, and when more than one position in any given structure
is substituted, the substituent is either the same or different at
each position. The term "substituted" is contemplated to include
substitution with all permissible substituents of organic
compounds, and includes any of the substituents described herein
that results in the formation of a stable compound. The present
invention contemplates any and all such combinations in order to
arrive at a stable compound. For purposes of this invention,
heteroatoms such as nitrogen may have hydrogen substituents and/or
any suitable substituent as described herein which satisfy the
valencies of the heteroatoms and results in the formation of a
stable moiety. The invention is not intended to be limited in any
manner by the exemplary substituents described herein.
[0036] Exemplary carbon atom substituents include, but are not
limited to, halogen, --CN, --NO.sub.2, --N.sub.3, --SO.sub.2H,
--SO.sub.3H, --OH, --OR.sup.aa, --ON(R.sup.bb).sub.2,
--N(R.sup.bb).sub.2, --N(R.sup.bb).sub.3.sup.+X.sup.-,
--N(OR.sup.cc)R.sup.bb, --SH, --SR.sup.aa, --SSR.sup.cc,
--C(.dbd.O)R.sup.aa, --CO.sub.2H, --CHO, --C(OR.sup.cc).sub.2,
--CO.sub.2R.sup.aa, --OC(.dbd.O)R.sup.aa, --OCO.sub.2R.sup.aa,
--C(.dbd.O)N(R.sup.bb).sub.2, --OC(.dbd.O)N(R.sup.bb).sub.2,
--NR.sup.bbC(.dbd.O)R.sup.aa, --NR.sup.bbCO.sub.2R.sup.aa,
--NR.sup.bbC(.dbd.O)N(R.sup.bb).sub.2, --C(.dbd.NR.sup.bb)R.sup.aa,
--C(.dbd.NR.sup.bb)OR.sup.aa, --OC(.dbd.NR.sup.bb)R.sup.aa,
--OC(.dbd.NR.sup.bb)OR.sup.aa,
--C(.dbd.NR.sup.bb)N(R.sup.bb).sub.2,
--OC(.dbd.NR.sup.bb)N(R.sup.bb).sub.2,
--NR.sup.bbC(.dbd.NR.sup.bb)N(R.sup.bb).sub.2,
--C(.dbd.O)NR.sup.bbSO.sub.2R.sup.aa, --NR.sup.bbSO.sub.2R.sup.aa,
--SO.sub.2N(R.sup.bb).sub.2, --SO.sub.2R.sup.aa,
--OSO.sub.2R.sup.aa, --S(.dbd.O)R.sup.aa, --OS(.dbd.O)R.sup.aa,
--Si(R.sup.aa).sub.3,
--OSi(R.sup.aa).sub.3--C(.dbd.S)N(R.sup.bb).sub.2,
--C(.dbd.O)SR.sup.aa, --C(.dbd.S)SR.sup.aa, --SC(.dbd.S)SR.sup.aa,
--SC(.dbd.O)SR.sup.aa, --OC(.dbd.O)SR.sup.aa,
--SC(.dbd.O)OR.sup.aa, --SC(.dbd.O)R.sup.aa,
--P(.dbd.O)(R.sup.aa).sub.2, --P(.dbd.O)(OR.sup.cc).sub.2,
--OP(.dbd.O)(R.sup.aa).sub.2, --OP(.dbd.O)(OR.sup.cc).sub.2,
--P(.dbd.O)(N(R.sup.bb).sub.2).sub.2,
--OP(.dbd.O)(N(R.sup.bb).sub.2).sub.2,
--NR.sup.bbP(.dbd.O)(R.sup.aa).sub.2,
--NR.sup.bbP(.dbd.O)(OR.sup.cc).sub.2,
--NR.sup.bbP(.dbd.O)(N(R.sup.bb).sub.2).sub.2, --P(R.sup.cc).sub.2,
--P(OR.sup.cc).sub.2, --P(R.sup.cc).sub.3.sup.+X.sup.-,
--P(OR.sup.cc).sub.3.sup.+X.sup.-, --P(R.sup.cc).sub.4,
--P(OR.sup.cc).sub.4, --OP(R.sup.cc).sub.2,
--OP(R.sup.cc).sub.3.sup.+X.sup.-, --OP(OR.sup.cc).sub.2,
--OP(OR.sup.cc).sub.3.sup.+X.sup.-, --OP(R.sup.cc),
--OP(OR.sup.cc).sub.4, --B(R.sup.aa).sub.2, --B(OR.sup.cc).sub.2,
--BR.sup.aa(OR.sup.cc), C.sub.1-10 alkyl, C.sub.1-10 perhaloalkyl,
C.sub.2-10 alkenyl, C.sub.2-10 alkynyl, heteroC.sub.1-10 alkyl,
heteroC.sub.2-10 alkenyl, heleroC.sub.2-10 alkynyl, C.sub.3-10
carbocyclyl, 3-14 membered heterocyclyl, C.sub.6-14 aryl, and 5-14
membered heteroaryl, wherein each alkyl, alkenyl, alkynyl,
heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl,
heterocyclyl, aryl, and heteroaryl is independently substituted
with 0, 1, 2, 3, 4, or 5 R.sup.dd groups; wherein X.sup.- is a
counterion;
[0037] or two geminal hydrogens on a carbon atom are replaced with
the group .dbd.O, .dbd.S, .dbd.NN(R.sup.bb).sub.2,
.dbd.NNR.sup.bbC(.dbd.O)R.sup.aa,
.dbd.NNR.sup.bbC(.dbd.O)OR.sup.aa,
.dbd.NNR.sup.bbS(.dbd.O).sub.2R.sup.aa, .dbd.NR.sup.bb, or
.dbd.NOR.sup.cc;
[0038] each instance of R.sup.aa is, independently, selected from
C.sub.1-10 alkyl, C.sub.1-10 perhaloalkyl, C.sub.2-10 alkenyl,
C.sub.2-10 alkynyl, heteroC.sub.1-10 alkyl,
heteroC.sub.2-10alkenyl, heteroC.sub.2-10alkynyl, C.sub.3-10
carbocyclyl, 3-14 membered heterocyclyl, C.sub.6-14 aryl, and 5-14
membered heteroaryl, or two R.sup.aa groups are joined to form a
3-14 membered heterocyclyl or 5-14 membered heteroaryl ring,
wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,
heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is
independently substituted with 0, 1, 2, 3, 4, or 5 R.sup.dd
groups;
[0039] each instance of R.sup.bb is, independently, selected from
hydrogen, --OH, --OR.sup.aa, --N(R.sup.cc).sub.2, --CN,
--C(.dbd.O)R.sup.aa, --C(.dbd.O)N(R.sup.cc).sub.2,
--CO.sub.2R.sup.aa, --SO.sub.2R.sup.aa,
--C(.dbd.NR.sup.cc)OR.sup.aa, --C(.dbd.NR.sup.cc)N(R.sup.cc).sub.2,
--SO.sub.2N(R.sup.cc).sub.2, --SO.sub.2R.sup.aa,
--SO.sub.2OR.sup.cc, --SOR.sup.aa, --C(.dbd.S)N(R.sup.cc).sub.2,
--C(.dbd.O)SR.sup.cc, --C(.dbd.S)SR.sup.cc,
--P(.dbd.O)(R.sup.aa).sub.2, --P(.dbd.O)(OR.sup.cc).sub.2,
--P(.dbd.O)(N(R.sup.cc).sub.2).sub.2, C.sub.1-10 alkyl, C.sub.1-10
perhaloalkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl,
heteroC.sub.1-10alkyl, heteroC.sub.2-10alkenyl,
heteroC.sub.2-10alkynyl, C.sub.3-10 carbocyclyl, 3-14 membered
heterocyclyl, C.sub.6-14 aryl, and 5-14 membered heteroaryl, or two
R.sup.bb groups are joined to form a 3-14 membered heterocyclyl or
5-14 membered heteroaryl ring, wherein each alkyl, alkenyl,
alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl,
heterocyclyl, aryl, and heteroaryl is independently substituted
with 0, 1, 2, 3, 4, or 5 R.sup.dd groups; wherein X.sup.- is a
counterion;
[0040] each instance of R.sup.cc is, independently, selected from
hydrogen, C.sub.1-10 alkyl, C.sub.1-10 perhaloalkyl, C.sub.2-10
alkenyl, C.sub.2-10 alkynyl, heteroC.sub.1-10 alkyl,
heteroC.sub.2-10 alkenyl, heteroC.sub.2-10 alkynyl, C.sub.3-10
carbocyclyl, 3-14 membered heterocyclyl, C.sub.6-14 aryl, and 5-14
membered heteroaryl, or two R.sup.cc groups are joined to form a
3-14 membered heterocyclyl or 5-14 membered heteroaryl ring,
wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,
heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is
independently substituted with 0, 1, 2, 3, 4, or 5 R.sup.dd
groups;
[0041] each instance of R.sup.dd is, independently, selected from
halogen, --CN, --NO.sub.2, --N.sub.3, --SO.sub.2H, --SO.sub.3H,
--OH, --OR.sup.cc, --ON(R.sup.ff).sub.2, --N(R.sup.ff).sub.2,
--N(R.sup.ff).sub.3.sup.+X.sup.-, --N(OR.sup.ee)R.sup.ff, --SH,
--SR.sup.ee, --SSR.sup.ee, --C(.dbd.O)R.sup.ee, --CO.sub.2H,
--CO.sub.2R.sup.ee, --OC(.dbd.O)R.sup.ee, --OCO.sub.2R.sup.ee,
--C(.dbd.O)N(R.sup.ff).sub.2, --OC(.dbd.O)N(R.sup.ff).sub.2,
--NR.sup.ffC(.dbd.O)R.sup.ee, --NR.sup.ffCO.sub.2R.sup.ee,
--NR.sup.ffC(.dbd.O)N(R.sup.ff).sub.2,
--C(.dbd.NR.sup.ff)OR.sup.ee, --OC(.dbd.NR.sup.ff)R.sup.ee,
--OC(.dbd.NR.sup.ff)OR.sup.ee,
--C(.dbd.NR.sup.ff)N(R.sup.ff).sub.2,
--OC(.dbd.NR.sup.ff)N(R.sup.ff).sub.2,
--NR.sup.ffC(.dbd.NR.sup.ff)N(R.sup.ff).sub.2,
--NR.sup.ffSO.sub.2R.sup.ee, --SO.sub.2N(R.sup.ff).sub.2,
--SO.sub.2R.sup.ee, --SO.sub.2OR.sup.ee, --SO.sub.2R.sup.ee,
--S(.dbd.O)R.sup.ee, --Si(R.sup.ee).sub.3, --OSi(R.sup.ee).sub.3,
--C(.dbd.S)N(R.sup.ff).sub.2, --C(.dbd.O)SR.sup.ee,
--C(.dbd.S)SR.sup.ee, --SC(.dbd.S)SR.sup.ee,
--P(.dbd.O)(OR.sup.ee).sub.2, --P(.dbd.O)(R.sup.ee).sub.2,
--OP(.dbd.O)(R.sup.ee).sub.2, --OP(.dbd.O)(OR.sup.ee).sub.2,
C.sub.1-6 alkyl, C.sub.1-6 perhaloalkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, heteroC.sub.1-6alkyl, heteroC.sub.2-6alkenyl,
heteroC.sub.2-6alkynyl, C.sub.3-10 carbocyclyl, 3-10 membered
heterocyclyl, C.sub.6-10 aryl, 5-10 membered heteroaryl, wherein
each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,
heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is
independently substituted with 0, 1, 2, 3, 4, or 5 R.sup.gg groups,
or two geminal R.sup.dd substituents can be joined to form .dbd.O
or .dbd.S; wherein X.sup.- is a counterion;
[0042] each instance of R.sup.ee is, independently, selected from
C.sub.1-6 alkyl, C.sub.1-6 perhaloalkyl, C.sub.2-6 alkenyl,
C.sub.2-6 alkynyl, heteroC.sub.1-6 alkyl, heteroC.sub.2-6alkenyl,
heteroC.sub.2-6 alkynyl, C.sub.3-10 carbocyclyl, C.sub.6-10 aryl,
3-10 membered heterocyclyl, and 3-10 membered heteroaryl, wherein
each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,
heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is
independently substituted with 0, 1, 2, 3, 4, or 5 R.sup.gg
groups;
[0043] each instance of R.sup.ff is, independently, selected from
hydrogen, C.sub.1-6 alkyl, C.sub.1-6 perhaloalkyl, C.sub.2-6
alkenyl, C.sub.2-6 alkynyl, heteroC.sub.1-6alkyl,
heteroC.sub.2-6alkenyl, heteroC.sub.2-6alkynyl, C.sub.3-10
carbocyclyl, 3-10 membered heterocyclyl, C.sub.6-10 aryl and 5-10
membered heteroaryl, or two R.sup.ff groups are joined to form a
3-10 membered heterocyclyl or 5-10 membered heteroaryl ring,
wherein each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,
heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is
independently substituted with 0, 1, 2, 3, 4, or 5 R.sup.gg groups;
and
[0044] each instance of R.sup.gg is, independently, halogen, --CN,
--NO.sub.2, --N.sub.3, --SO.sub.2H, --SO.sub.3H, --OH, --OC.sub.1-6
alkyl, --ON(C.sub.1-6 alkyl).sub.2, --N(C.sub.1-6 alkyl).sub.2,
--N(C.sub.1-6 alkyl).sub.3.sup.+X.sup.-, --NH(C.sub.1-6
alkyl).sub.2.sup.+X.sup.-, --NH.sub.2(C.sub.1-6
alkyl).sup.+X.sup.-, --NH.sub.3.sup.+X.sup.-, --N(OC.sub.1-6
alkyl)(C.sub.1-6 alkyl), --N(OH)(C.sub.1-6 alkyl), --NH(OH), --SH,
--SC.sub.1-6 alkyl, --SS(C.sub.1-6 alkyl), --C(.dbd.O)(C.sub.1-6
alkyl), --CO.sub.2H, --CO.sub.2(C.sub.1-6 alkyl),
--OC(.dbd.O)(C.sub.1-6 alkyl), --OCO.sub.2(C.sub.1-6 alkyl),
--C(.dbd.O)NH.sub.2, --C(.dbd.O)N(C.sub.1-6 alkyl).sub.2,
--OC(.dbd.O)NH(C.sub.1-6 alkyl), --NHC(.dbd.O)(C.sub.1-6 alkyl),
--N(C.sub.1-6 alkyl)C(.dbd.O)(C.sub.1-6 alkyl),
--NHCO.sub.2(C.sub.1-6 alkyl), --NHC(.dbd.O)N(C.sub.1-6
alkyl).sub.2, --NHC(.dbd.O)NH(C.sub.1-6 alkyl),
--NHC(.dbd.O)NH.sub.2, --C(.dbd.NH)O(C.sub.1-6 alkyl),
--OC(.dbd.NH)(C.sub.1-6 alkyl), --OC(.dbd.NH)OC.sub.1-6 alkyl,
--C(.dbd.NH)N(C.sub.1-6 alkyl).sub.2, --C(.dbd.NH)NH(C.sub.1-6
alkyl), --C(.dbd.NH)NH.sub.2, --OC(.dbd.NH)N(C.sub.1-6
alkyl).sub.2, --OC(NH)NH(C.sub.1-6 alkyl), --OC(NH)NH.sub.2,
--NHC(NH)N(C.sub.1-6 alkyl).sub.2, --NHC(.dbd.NH)NH.sub.2,
--NHSO.sub.2(C.sub.1-6 alkyl), --SO.sub.2N(CM, alkyl).sub.2,
--SO.sub.2NH(C.sub.1-6 alkyl), --SO.sub.2NH.sub.2,
--SO.sub.2C.sub.1-6 alkyl, --SO.sub.2OC.sub.1-6 alkyl,
--OSO.sub.2C.sub.1-6 alkyl, --SOC.sub.1-6 alkyl, --Si(C.sub.1-6
alkyl).sub.3, --OSi(C.sub.1-6 alkyl).sub.3, --C(.dbd.S)N(C.sub.1-6
alkyl).sub.2, C(.dbd.S)NH(C.sub.1-6 alkyl), C(.dbd.S)NH.sub.2,
--C(.dbd.O)S(C.sub.1-6 alkyl), --C(.dbd.S)SC.sub.1-6 alkyl,
--SC(.dbd.S)SC.sub.1-6 alkyl, --P(.dbd.O)(OC.sub.1-6 alkyl).sub.2,
--P(.dbd.O)(C.sub.1-6 alkyl).sub.2, --OP(.dbd.O)(C.sub.1-6
alkyl).sub.2, --OP(.dbd.O)(OC.sub.1-6 alkyl).sub.2, C.sub.1-6
alkyl, C.sub.1-6 perhaloalkyl, C.sub.2-6 alkenyl, C.sub.2-6
alkynyl, heteroC.sub.1-6alkyl, heteroC.sub.2-6alkenyl,
heteroC.sub.2-6alkynyl, C.sub.3-10 carbocyclyl, C.sub.6-10 aryl,
3-10 membered heterocyclyl, 5-10 membered heteroaryl; or two
geminal R.sup.gg substituents can be joined to form .dbd.O or
.dbd.S; wherein X.sup.- is a counterion.
[0045] The term "halo" or "halogen" refers to fluorine (fluoro,
--F), chlorine (chloro, --Cl), bromine (bromo, --Br), or iodine
(iodo, --I).
[0046] The term "hydroxyl" or "hydroxy" refers to the group --OH.
The term "substituted hydroxyl" or "substituted hydroxyl," by
extension, refers to a hydroxyl group wherein the oxygen atom
directly attached to the parent molecule is substituted with a
group other than hydrogen, and includes groups selected from
--OR.sup.aa, --ON(R.sup.bb).sub.2, --OC(.dbd.O)SR.sup.aa,
--OC(.dbd.O)R.sup.aa, --OCO.sub.2R.sup.aa,
--OC(.dbd.O)N(R.sup.bb).sub.2, --OC(.dbd.NR.sup.bb)R.sup.aa,
--OC(.dbd.NR.sup.bb)OR.sup.aa,
--OC(.dbd.NR.sup.bb)N(R.sup.bb).sub.2, --OS(.dbd.O)R.sup.aa,
--OSO.sub.2R.sup.aa, --OSi(R.sup.aa).sub.3, --OP(R.sup.cc).sub.2,
--OP(R.sup.cc).sub.3.sup.+X.sup.-, --OP(OR.sup.cc).sub.2,
--OP(OR.sup.cc).sub.3.sup.+X.sup.-, --OP(.dbd.O)(R.sup.aa).sub.2,
--OP(.dbd.O)(OR.sup.cc).sub.2, and --OP(.dbd.O)(N(R.sup.bb)).sub.2,
wherein X.sup.-, R.sup.aa, R.sup.bb, and R.sup.cc are as defined
herein.
[0047] The term "amino" refers to the group --NH.sub.2. The term
"substituted amino," by extension, refers to a monosubstituted
amino, a disubstituted amino, or a trisubstituted amino. In certain
embodiments, the "substituted amino" is a monosubstituted amino or
a disubstituted amino group.
[0048] The term "monosubstituted amino" refers to an amino group
wherein the nitrogen atom directly attached to the parent molecule
is substituted with one hydrogen and one group other than hydrogen,
and includes groups selected from --NH(R.sup.bb),
--NHC(.dbd.O)R.sup.aa, --NHCO.sub.2R.sup.aa,
--NHC(.dbd.O)N(R.sup.bb).sub.2,
--NHC(.dbd.NR.sup.bb)N(R.sup.bb).sub.2, --NHSO.sub.2R.sup.aa,
--NHP(.dbd.O)(OR.sup.cc).sub.2, and
--NHP(.dbd.O)(N(R.sup.bb).sub.2).sub.2, wherein R.sup.aa, R.sup.bb
and R.sup.cc are as defined herein, and wherein R.sup.bb of the
group --NH(R.sup.bb) is not hydrogen.
[0049] The term "disubstituted amino" refers to an amino group
wherein the nitrogen atom directly attached to the parent molecule
is substituted with two groups other than hydrogen, and includes
groups selected from --N(R.sup.bb).sub.2,
--NR.sup.bbC(.dbd.O)R.sup.aa, --NR.sup.bbCO.sub.2R.sup.aa,
--NR.sup.bbC(.dbd.O)N(R.sup.bb).sub.2,
--NR.sup.bbC(.dbd.NR.sup.bb)N(R.sup.bb).sub.2,
--NR.sup.bbSO.sub.2R.sup.gg, --NR.sup.bbP(.dbd.O)(OR.sup.cc).sub.2,
and --NR.sup.bbP(.dbd.O)(N(R.sup.bb).sub.2).sub.2, wherein
R.sup.aa, R.sup.bb, and R.sup.cc are as defined herein, with the
proviso that the nitrogen atom directly attached to the parent
molecule is not substituted with hydrogen.
[0050] The term "trisubstituted amino" refers to an amino group
wherein the nitrogen atom directly attached to the parent molecule
is substituted with three groups, and includes groups selected from
--N(R.sup.bb).sub.3 and --N(R.sup.bb).sub.3.sup.+X.sup.-, wherein
R.sup.bb and X.sup.- are as defined herein.
[0051] The term "sulfonyl" refers to a group selected from
--SO.sub.2N(R.sup.bb).sub.2, --SO.sub.2R.sup.aa, and
--SO.sub.2OR.sup.aa, wherein R.sup.aa and R.sup.bb are as defined
herein.
[0052] The term "sulfinyl" refers to the group --S(.dbd.O)R.sup.aa,
wherein R.sup.aa is as defined herein.
[0053] The term "acyl" refers to a group having the general formula
--C(.dbd.O)R.sup.X1, --C(.dbd.O)OR.sup.X1,
--C(.dbd.O)--O--C(.dbd.O)R.sup.X1, --C(.dbd.O)SR.sup.X1,
--C(.dbd.O)N(R.sup.X1).sub.2, --C(.dbd.S)R.sup.X1,
--C(.dbd.S)N(R.sup.X1).sub.2, and --C(.dbd.S)S(R.sup.X1),
--C(.dbd.NR.sup.X1)R.sup.X1, --C(.dbd.NR.sup.X1)OR.sup.X1,
--C(.dbd.NR.sup.X1)SR.sup.X1, and
--C(.dbd.NR.sup.X1)N(R.sup.X1).sub.2, wherein R.sup.X1 is hydrogen;
halogen; substituted or unsubstituted hydroxyl; substituted or
unsubstituted thiol; substituted or unsubstituted amino;
substituted or unsubstituted acyl, cyclic or acyclic, substituted
or unsubstituted, branched or unbranched aliphatic; cyclic or
acyclic, substituted or unsubstituted, branched or unbranched
heteroaliphatic; cyclic or acyclic, substituted or unsubstituted,
branched or unbranched alkyl; cyclic or acyclic, substituted or
unsubstituted, branched or unbranched alkenyl; substituted or
unsubstituted alkynyl; substituted or unsubstituted aryl,
substituted or unsubstituted heteroaryl, aliphaticoxy,
heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy,
heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy,
heteroalkylthioxy, arylthioxy, heteroarylthioxy, mono- or
di-aliphaticamino, mono- or di-heteroaliphaticamino, mono- or
di-alkylamino, mono- or di-heteroalkylamino, mono- or di-arylamino,
or mono- or di-heteroarylamino; or two R.sup.X1 groups taken
together form a 5- to 6-membered heterocyclic ring. Exemplary acyl
groups include aldehydes (--CHO), carboxylic acids (--CO.sub.2H),
ketones, acyl halides, esters, amides, imines, carbonates,
carbamates, and ureas. Acyl substituents include, but are not
limited to, any of the substituents described herein, that result
in the formation of a stable moiety (e.g., aliphatic, alkyl,
alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl, heteroaryl,
acyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido, nitro,
hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino,
alkylamino, heteroalkylamino, arylamino, heteroarylamino,
alkylaryl, arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy,
heteroalkyloxy, aryloxy, heteroaryloxy, aliphaticthioxy,
heteroaliphaticthioxy, alkylthioxy, heteroalkylthioxy, arylthioxy,
heteroarylthioxy, acyloxy, and the like, each of which may or may
not be further substituted).
[0054] The term "carbonyl" refers a group wherein the carbon
directly attached to the parent molecule is sp.sup.2 hybridized,
and is substituted with an oxygen, nitrogen or sulfur atom, e.g., a
group selected from ketones (--C(.dbd.O)R.sup.aa), carboxylic acids
(--CO.sub.2H), aldehydes (--CHO), esters (--CO.sub.2R.sup.aa,
--C(.dbd.O)SR.sup.aa, --C(.dbd.S)SR.sup.aa), amides
(--C(.dbd.O)N(R.sup.bb).sub.2,
--C(.dbd.O)NR.sup.bbSO.sub.2R.sup.aa,
--C(.dbd.S)N(R.sup.bb).sub.2), and imines
(--C(.dbd.NR.sup.bb)R.sup.aa, --C(.dbd.NR.sup.bb)OR.sup.aa),
--C(.dbd.NR.sup.bb)N(R.sup.bb).sub.2), wherein R.sup.aa and
R.sup.bb are as defined herein.
[0055] Nitrogen atoms can be substituted or unsubstituted as
valency permits, and include primary, secondary, tertiary, and
quaternary nitrogen atoms. Exemplary nitrogen atom substituents
include, but are not limited to, hydrogen, --OH, --OR.sup.aa,
--N(R.sup.cc).sub.2, --CN, --C(.dbd.O)R.sup.aa,
--C(.dbd.O)N(R.sup.cc).sub.2, --CO.sub.2R.sup.aa,
--SO.sub.2R.sup.aa, --C(.dbd.NR.sup.bb)R.sup.aa,
--C(.dbd.NR.sup.cc)OR.sup.aa, --C(.dbd.NR.sup.cc)N(R.sup.cc).sub.2,
--SO.sub.2N(R.sup.cc).sub.2, --SO.sub.2R.sup.cc,
--SO.sub.2OR.sup.cc, --SOR.sup.aa, --C(.dbd.S)N(R.sup.cc).sub.2,
--C(.dbd.O)SR.sup.cc, --C(.dbd.S)SR.sup.cc,
--P(.dbd.O)(OR.sup.cc).sub.2, --P(.dbd.O)(R.sup.aa).sub.2,
--P(.dbd.O)(N(R.sup.cc).sub.2).sub.2, C.sub.1-10 alkyl, C.sub.1-10
perhaloalkyl, C.sub.2-10 alkenyl, C.sub.2-10 alkynyl,
heteroC.sub.1-10alkyl, heteroC.sub.2-10alkenyl,
heteroC.sub.2-10alkynyl, C.sub.3-10 carbocyclyl, 3-14 membered
heterocyclyl, C.sub.6-14 aryl, and 5-14 membered heteroaryl, or two
R.sup.cc groups attached to an N atom are joined to form a 3-14
membered heterocyclyl or 5-14 membered heteroaryl ring, wherein
each alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl,
heteroalkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl is
independently substituted with 0, 1, 2, 3, 4, or 5 R.sup.dd groups,
and wherein R.sup.aa, R.sup.bb, R.sup.cc and R.sup.dd are as
defined above.
[0056] In certain embodiments, the substituent present on the
nitrogen atom is an nitrogen protecting group (also referred to
herein as an "amino protecting group"). Nitrogen protecting groups
include, but are not limited to, --OH, --OR.sup.aa,
--N(R.sup.cc).sub.2, --C(.dbd.O)R.sup.aa,
--C(.dbd.O)N(R.sup.cc).sub.2, --CO.sub.2R.sup.aa,
--SO.sub.2R.sup.aa, --C(.dbd.NR.sup.cc)R.sup.aa,
--C(.dbd.NR.sup.cc)OR.sup.aa, --C(.dbd.NR.sup.cc)N(R.sup.cc).sub.2,
--SO.sub.2N(R.sup.cc).sub.2, --SO.sub.2R.sup.cc,
--SO.sub.2OR.sup.aa, --SOR.sup.aa, --C(.dbd.S)N(R.sup.cc).sub.2,
--C(.dbd.O)SR.sup.cc, --C(.dbd.S)SR.sup.cc, C.sub.1-10 alkyl (e.g.,
aralkyl, heteroaralkyl), C.sub.2-10 alkenyl, C.sub.2-10 alkynyl,
heteroC.sub.1-10 alkyl, heteroC.sub.2-10 alkenyl, heteroC.sub.2-10
alkynyl, C.sub.3-10 carbocyclyl, 3-14 membered heterocyclyl,
C.sub.6-14 aryl, and 5-14 membered heteroaryl groups, wherein each
alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,
carbocyclyl, heterocyclyl, aralkyl, aryl, and heteroaryl is
independently substituted with 0, 1, 2, 3, 4, or 5 R.sup.dd groups,
and wherein R.sup.aa, R.sup.bb, R.sup.cc and R.sup.dd are as
defined herein. Nitrogen protecting groups are well known in the
art and include those described in detail in Protecting Groups in
Organic Synthesis, T. W. Greene and P. G. M. Wuts, 3.sup.rd
edition, John Wiley & Sons, 1999, incorporated herein by
reference.
[0057] For example, nitrogen protecting groups such as amide groups
(e.g., --C(.dbd.O)R.sup.aa) include, but are not limited to,
formamide, acetamide, chloroacetamide, trichloroacetamide,
trifluoroacetamide, phenylacetamide, 3-phenylpropanamide,
picolinamide, 3-pyridylcarboxamide, N-benzoylphenylalanyl
derivative, benzamide, p-phenylbenzamide, o-nitophenylacetamide,
o-nitrophenoxyacetamide, acetoacetamide,
(N'-dithiobenzyloxyacylamino)acetamide,
3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide,
2-methyl-2-(o-nitrophenoxy)propanamide,
2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,
3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethionine
derivative, o-nitrobenzamide and o-(benzoyloxymethyl)benzamide.
[0058] Nitrogen protecting groups such as carbamate groups (e.g.,
--C(.dbd.O)OR.sup.aa) include, but are not limited to, methyl
carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc),
9-(2-sulfo)fluorenylmethyl carbamate,
9-(2,7-dibromo)fluoroenylmethyl carbamate,
2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl
carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),
2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl
carbamate (Teoc), 2-phenylethyl carbamate (hZ),
1-(1-adamantyl)-1-methylethyl carbamate (Adpoc),
1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl
carbamate (DB-t-BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate
(TCBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),
1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2'-
and 4'-pyridyl)ethyl carbamate (Pyoc),
2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate
(BOC or Boc), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc),
allyl carbamate (Alloc), 1-isopropylallyl carbamate (Ipaoc),
cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc),
8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithio
carbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),
p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzyl
carbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzyl
carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl
carbamate, 2-methylthioethyl carbamate, 2-methylsulfonylethyl
carbamate, 2-(p-toluenesulfonyl)ethyl carbamate,
[2-(1,3-dithianyl)]methyl carbamate (Dmoc), 4-methylthiophenyl
carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc),
2-phosphonioethyl carbamate (Peoc), 2-triphenylphosphonioisopropyl
carbamate (Ppoc), 1,1-dimethyl-2-cyanoethyl carbamate,
m-chloro-p-acyloxybenzyl carbamate, p-(dihydroxyboryl)benzyl
carbamate, 5-benzisoxazolylmethyl carbamate,
2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc),
m-nitrophenyl carbamate, 3,5-dimethoxybenzyl carbamate,
o-nitrobenzyl carbamate, 3,4-dimethoxy-6-nitrobenzyl carbamate,
phenyl(o-nitrophenyl)methyl carbamate, t-amyl carbamate, S-benzyl
thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate,
cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl
carbamate, p-decyloxybenzyl carbamate, 2,2-dimethoxyacylvinyl
carbamate, o-(N,N-dimethylcarboxamido)benzyl carbamate,
1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,
1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,
2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl
carbamate, isobutyl carbamate, isonicotinyl carbamate,
p-(p'-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl
carbamate, 1-methylcyclohexyl carbamate,
1-methyl-1-cyclopropylmethyl carbamate,
1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate,
1-methyl-1-(p-phenylazophenyl)ethyl carbamate,
1-methyl-1-phenylethyl carbamate, 1-methyl-1-(4-pyridyl)ethyl
carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate,
2,4,6-tri-t-butylphenyl carbamate, 4-(trimethylammonium)benzyl
carbamate, and 2,4,6-trimethylbenzyl carbamate.
[0059] Nitrogen protecting groups such as sulfonamide groups (e.g.,
--S(.dbd.O).sub.2R.sup.aa) include, but are not limited to,
p-toluenesulfonamide (Ts), benzenesulfonamide,
2,3,6-trimethyl-4-methoxybenzenesulfonamide (Mtr),
2,4,6-trimethoxybenzenesulfonamide (Mtb),
2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),
2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),
4-methoxybenzenesulfonamide (Mbs),
2,4,6-trimethylbenzenesulfonamide (Mts),
2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),
2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc),
methanesulfonamide (Ms), P-trimethylsilylethanesulfonamide (SES),
9-anthracenesulfonamide,
4-(4',8'-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),
benzylsulfonamide, trifluoromethylsulfonamide, and
phenacylsulfonamide.
[0060] Other nitrogen protecting groups include, but are not
limited to, phenothiazinyl-(10)-acyl derivative,
N'-p-toluenesulfonylaminoacyl derivative, N'-phenylaminothioacyl
derivative, N-benzoylphenylalanyl derivative, N-acetylmethionine
derivative, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide,
N-dithiasuccinimide (Dts), N-2,3-diphenylmaleimide,
N-2,5-dimethylpyrrole, N-1,1,4,4-tetramethyldisilylazacyclopentane
adduct (STABASE), 5-substituted
1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted
1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted
3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,
N-[2-(trimethylsilyl)ethoxy]methylamine (SEM),
N-3-acetoxypropylamine,
N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary
ammonium salts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,
N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr),
N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),
N-9-phenylfluorenylamine (PhF),
N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino
(Fcm), N-2-picolylamino N*-oxide, N-1,1-dimethylthiomethyleneamine,
N-benzylideneamine, N-p-methoxybenzylideneamine,
N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine,
N--(N',N'-dimethylaminomethylene)amine, N,N'-isopropylidenediamine,
N-p-nitrobenzylideneamine, N-salicylideneamine,
N-5-chlorosalicylideneamine,
N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,
N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,
N-borane derivative, N-diphenylborinic acid derivative,
N-[phenyl(pentaacylchromium- or tungsten)acyl]amine, N-copper
chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine
N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide
(Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates,
dibenzyl phosphoramidate, diphenyl phosphoramidate,
benzenesulfenamide, o-nitrobenzenesulfenamide (Nps),
2,4-dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide,
2-nitro-4-methoxybenzenesulfenamide, triphenylmethylsulfenamide,
and 3-niiropyridinesulfenamide (Npys).
[0061] In certain embodiments, the substituent present on an oxygen
atom is an oxygen protecting group (also referred to herein as an
"hydroxyl protecting group"). Oxygen protecting groups include, but
are not limited to, --R.sup.aa, --N(R.sup.bb).sub.2,
--C(.dbd.O)SR.sup.aa, --C(.dbd.O)R.sup.aa, --CO.sub.2R.sup.aa,
--C(.dbd.O)N(R.sup.bb).sub.2, --C(.dbd.NR.sup.bb)R.sup.aa,
--C(.dbd.NR.sup.bb)OR.sup.aa, --C(.dbd.NR.sup.bb)N(R.sup.bb).sub.2,
--S(.dbd.O)R.sup.aa, --SO.sub.2R.sup.aa, --Si(R.sup.aa).sub.3,
--P(R.sup.cc).sub.2, --P(R.sup.bb).sub.3.sup.+X.sup.-,
--P(OR.sup.cc).sub.2, --P(OR.sup.cc).sub.3.sup.+X.sup.-,
--P(.dbd.O)(R.sup.aa).sub.2, --P(.dbd.O)(OR.sup.cc).sub.2, and
--P(.dbd.O)(N(R.sup.bb).sub.2).sub.2, wherein X.sup.-, R.sup.aa,
R.sup.bb, and R.sup.cc are as defined herein. Oxygen protecting
groups are well known in the art and include those described in
detail in Protecting Groups in Organic Synthesis, T. W. Greene and
P. G. M. Wuts, 3.sup.rd edition, John Wiley & Sons, 1999,
incorporated herein by reference.
[0062] Exemplary oxygen protecting groups include, but are not
limited to, methyl, methoxylmethyl (MOM), methylthiomethyl (MTM),
t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM),
benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM),
(4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM),
t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl,
2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl,
bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR),
tetrahydropyranyl (THP), 3-bromotetrahydropyranyl,
tetrahydrothiopyranyl, 1-methoxycyclohexyl,
4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl,
4-methoxytetrahydrothiopyranyl S,S-dioxide,
1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP),
1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,
2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,
1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,
1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fiuoroethyl,
2,2,2-trichloroethyl, 2-trimethylsilylethyl,
2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl,
p-methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), p-methoxybenzyl,
3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl,
2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl,
4-picolyl, 3-methyl-2-picolyl N-oxido, diphenylmethyl,
p,p'-dinitrobenzhydryl, 5-dibenzosuberyl, tri phenyl methyl,
.alpha.-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl,
di(p-methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl,
4-(4'-bromophenacyloxyphenyl)diphenylmethyl,
4,4',4''-tris(4,5-dichlorophthalimidophenyl)methyl,
4,4',4''-tris(levulinoyloxyphenyl)methyl,
4,4',4''-tris(benzoyloxyphenyl)methyl,
3-(imidazol-1-yl)bis(4',4''-dimethoxyphenyl)methyl,
1,1-bis(4-methoxyphenyl)-1'-pyrenylmethyl, 9-anthryl,
9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,
1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido,
trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl
(TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl
(DEIPS), dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS),
t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl,
triphenylsilyl, diphenylmethylsilyl (DPMS),
t-butylmethoxyphenylsilyl (TBMPS), formate, benzoylformate,
acetate, chloroacetate, dichloroacetate, trichloroacetate,
trifluoroacetate, methoxyacetate, triphenylmethoxyacetate,
phenoxyacetate, p-chlorophenoxyacetate, 3-phenylpropionate,
4-oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate
(levulinoyldithioacetal), pivaloate, adamantoate, crotonate,
4-methoxycrotonate, benzoate, p-phenylbenzoate,
2,4,6-trimethylbenzoate (mesitoate), methyl carbonate,
9-fluorenylmethyl carbonate (Fmoc), ethyl carbonate,
2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl
carbonate (TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec),
2-(triphenylphosphonio) ethyl carbonate (Peoc), isobutyl carbonate,
vinyl carbonate, allyl carbonate, t-butyl carbonate (BOC or Boc),
p-nitrophenyl carbonate, benzyl carbonate, p-methoxybenzyl
carbonate, 3,4-dimethoxybenzyl carbonate, o-nitrobenzyl carbonate,
p-nitrobenzyl carbonate, S-benzyl thiocarbonate,
4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate,
2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate,
o-(dibromomethyl)benzoate, 2-formylbenzenesulfonate,
2-(methylthiomethoxy)ethyl, 4-(methylthiomethoxy)butyrate,
2-(methylthiomethoxymethyl)benzoate,
2,6-dichloro-4-methylphenoxyacetate,
2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,
2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlonxliphenylacelate,
isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,
o-(methoxyacyl)benzoate, .alpha.-naphthoate, nitrate, alkyl
N,N,N',N'-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,
borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,
sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate
(Ts).
[0063] In certain embodiments, the substituent present on a sulfur
atom is a sulfur protecting group (also referred to as a "thiol
protecting group"). Sulfur protecting groups include, but are not
limited to, --R.sup.aa, --N(R.sup.bb).sub.2, --C(.dbd.O)SR.sup.aa,
--C(.dbd.O)R.sup.aa, --CO.sub.2R.sup.aa,
--C(.dbd.O)N(R.sup.bb).sub.2, --C(.dbd.NR.sup.bb)R.sup.aa,
--C(.dbd.NR.sup.bb)OR.sup.aa, --C(.dbd.NR.sup.bb)N(R.sup.bb).sub.2,
--S(.dbd.O)R.sup.aa, --SO.sub.2R.sup.aa, --Si(R.sup.aa).sub.3,
--P(R.sup.cc).sub.2, --P(R.sup.cc).sub.3.sup.+X.sup.-,
--P(OR.sup.cc).sub.2, --P(OR.sup.cc).sub.3.sup.+X.sup.-,
--P(.dbd.O)(R.sup.aa).sub.2, --P(.dbd.O)(OR.sup.cc).sub.2, and
--P(.dbd.O)(N(R.sup.bb).sub.2).sub.2, wherein R.sup.aa, R.sup.bb,
and R.sup.cc are as defined herein. Sulfur protecting groups are
well known in the art and include those described in detail in
Protecting Groups in Organic Synthesis. T. W. Greene and P. G. M.
Wuts, 3.sup.rd edition, John Wiley & Sons, 1999, incorporated
herein by reference.
[0064] A "counterion" or "anionic counterion" is a negatively
charged group associated with a positively charged group in order
to maintain electronic neutrality. An anionic counterion may be
monovalent (i.e., including one formal negative charge). An anionic
counterion may also be multivalent (i.e., including more than one
formal negative charge), such as divalent or trivalent. Exemplary
counterions include halide ions (e.g., F.sup.-, Cl.sup.-, Br.sup.-,
I.sup.-), NO.sub.3.sup.-, ClO.sub.4.sup.-, OH.sup.-,
H.sub.2PO.sub.4.sup.-, HCO.sub.3.sup.-, HSO.sub.4.sup.-, sulfonate
ions (e.g., methansulfonate, trifluoromethanesulfonate,
p-toluenesulfonate, benzenesulfonate, 10-camphor sulfonate,
naphthalene-2-sulfonate, naphthalene-1-sulfonic acid-5-sulfonate,
ethan-1-sulfonic acid-2-sulfonate, and the like), carboxylate ions
(e.g., acetate, propanoate, benzoate, glycerate, lactate, tartrate,
glycolate, gluconate, and the like), BF.sub.4.sup.-,
PF.sub.4.sup.-, PF.sub.6.sup.-, AsF.sub.6.sup.-, SbF.sub.6.sup.-,
B[3,5-(CF.sub.3).sub.2C.sub.6H.sub.3].sub.4].sup.-,
B(C.sub.6F.sub.5).sub.4.sup.-, BPh.sub.4.sup.-,
Al(OdC(CF.sub.3).sub.3).sub.4.sup.-, and carborane anions (e.g.,
CB.sub.11H.sub.12.sup.- or (HCB.sub.11Me.sub.5Br.sub.6).sup.-).
Exemplary counterions which may be multivalent include
CO.sub.3.sup.2-, HPO.sub.4.sup.2-, PO.sub.4.sup.3-,
B.sub.4O.sub.7.sup.2-, SO.sub.4.sup.2-, S.sub.2O.sub.3.sup.2-,
carboxylate anions (e.g., tartrate, citrate, fumarate, maleate,
malate, malonate, gluconate, succinate, glutarate, adipate,
pimelate, suberate, azelate, sebacate, salicylate, phthalates,
aspartate, glutamate, and the like), and carboranes.
[0065] As used herein, use of the phrase "at least one instance"
refers to 1, 2, 3, 4, or more instances, but also encompasses a
range, e.g., for example, from 1 to 4, from 1 to 3, from 1 to 2,
from 2 to 4, from 2 to 3, or from 3 to 4 instances, inclusive.
Other Definitions
[0066] As used herein, the term "salt" refers to any and all salts,
and encompasses pharmaceutically acceptable salts.
[0067] The term "pharmaceutically acceptable salt" refers to those
salts which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of humans and lower
animals without undue toxicity, irritation, allergic response, and
the like, and are commensurate with a reasonable benefit/risk
ratio. Pharmaceutically acceptable salts are well known in the art.
For example, Berge et al. describe pharmaceutically acceptable
salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19,
incorporated herein by reference. Pharmaceutically acceptable salts
of the compounds of this invention include those derived from
suitable inorganic and organic acids and bases. Examples of
pharmaceutically acceptable, nontoxic acid addition salts are salts
of an amino group formed with inorganic acids, such as hydrochloric
acid, hydrobromic acid, phosphoric acid, sulfuric acid, and
perchloric acid or with organic acids, such as acetic acid, oxalic
acid, maleic acid, tartaric acid, citric acid, succinic acid, or
malonic acid or by using other methods known in the art such as ion
exchange. Other pharmaceutically acceptable salts include adipate,
alginate, ascorbate, aspartate, benzenesulfonate, benzoate,
bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, formate, fumarate, glucoheptonate,
glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate,
hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate,
laurate, lauryl sulfate, malate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
oleate, oxalate, palmitate, pamoate, pectinate, persulfate,
3-phenylpropionate, phosphate, picrate, pivalate, propionate,
stearate, succinate, sulfate, tartrate, thiocyanate,
p-toluenesulfonate, undecanoate, valerate salts, and the like.
Salts derived from appropriate bases include alkali metal, alkaline
earth metal, ammonium, and N.sup.+(C.sub.1-4 alkyl).sub.4.sup.-
salts. Representative alkali or alkaline earth metal salts include
sodium, lithium, potassium, calcium, magnesium, and the like.
Further pharmaceutically acceptable salts include, when
appropriate, nontoxic ammonium, quaternary ammonium, and amine
cations formed using counterions such as halide, hydroxide,
carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate,
and aryl sulfonate.
[0068] It is also to be understood that compounds that have the
same molecular formula but differ in the nature or sequence of
bonding of their atoms or the arrangement of their atoms in space
are termed "isomers". Isomers that differ in the arrangement of
their atoms in space are termed "stereoisomers".
[0069] Stereoisomers that are not mirror images of one another are
termed "diastereomers" and those that are non-superimposable mirror
images of each other are termed "enantiomers". When a compound has
an asymmetric center, for example, it is bonded to four different
groups, a pair of enantiomers is possible. An enantiomer can be
characterized by the absolute configuration of its asymmetric
center and is described by the R- and S-sequencing rules of Cahn
and Prelog, or by the manner in which the molecule rotates the
plane of polarized light and designated as dextrorotatory or
levorotatory (i.e., as (+) or (-)-isomers respectively). A chiral
compound can exist as either individual enantiomer or as a mixture
thereof. A mixture containing equal proportions of the enantiomers
is called a "racemic mixture".
[0070] A "subject" to which administration is contemplated refers
to a human (i.e., male or female of any age group, e.g., pediatric
subject (e.g., infant, child, or adolescent) or adult subject
(e.g., young adult, middle-aged adult, or senior adult)) or
non-human animal. In certain embodiments, the non-human animal is a
mammal (e.g., primate (e.g., cynomolgus monkey or rhesus monkey),
commercially relevant mammal (e.g., cattle, pig, horse, sheep,
goat, cat, or dog), or bird (e.g., commercially relevant bird, such
as chicken, duck, goose, or turkey)). In certain embodiments, the
non-human animal is a fish, reptile, or amphibian. The non-human
animal may be a male or female at any stage of development. The
non-human animal may be a transgenic animal or genetically
engineered animal. The term "patient" refers to a human subject in
need of treatment of a disease.
[0071] The term "biological sample" refers to any sample including
tissue samples (such as tissue sections and needle biopsies of a
tissue); cell samples (e.g., cytological smears (such as Pap or
blood smears) or samples of cells obtained by microdissection);
samples of whole organisms (such as samples of yeasts or bacteria);
or cell fractions, fragments or organelles (such as obtained by
lysing cells and separating the components thereof by
centrifugation or otherwise). Other examples of biological samples
include blood, serum, urine, semen, fecal matter, cerebrospinal
fluid, interstitial fluid, mucous, tears, sweat, pus, biopsied
tissue (e.g., obtained by a surgical biopsy or needle biopsy),
nipple aspirates, milk, vaginal fluid, saliva, swabs (such as
buccal swabs), or any material containing biomolecules that is
derived from a first biological sample.
[0072] The term "administer," "administering," or "administration"
refers to implanting, absorbing, ingesting, injecting, inhaling, or
otherwise introducing a compound described herein, or a composition
thereof, in or on a subject.
[0073] The terms "treatment," "treat," and "treating" refer to
reversing, alleviating, delaying the onset of, or inhibiting the
progress of a disease described herein. In some embodiments,
treatment may be administered after one or more signs or symptoms
of the disease have developed or have been observed. In other
embodiments, treatment may be administered in the absence of signs
or symptoms of the disease. For example, treatment may be
administered to a susceptible subject prior to the onset of
symptoms (e.g., in light of a history of symptoms and/or in light
of exposure to a pathogen). Treatment may also be continued after
symptoms have resolved, for example, to delay or prevent
recurrence.
[0074] The terms "condition," "disease," and "disorder" are used
interchangeably.
[0075] An "effective amount" of a compound described herein refers
to an amount sufficient to elicit the desired biological response.
An effective amount of a compound described herein may vary
depending on such factors as the desired biological endpoint, the
pharmacokinetics of the compound, the condition being treated, the
mode of administration, and the age and health of the subject. In
certain embodiments, an effective amount is a therapeutically
effective amount. In certain embodiments, an effective amount is a
prophylactic treatment. In certain embodiments, an effective amount
is the amount of a compound described herein in a single dose. In
certain embodiments, an effective amount is the combined amounts of
a compound described herein in multiple doses.
[0076] A "therapeutically effective amount" of a compound described
herein is an amount sufficient to provide a therapeutic benefit in
the treatment of a condition or to delay or minimize one or more
symptoms associated with the condition. A therapeutically effective
amount of a compound means an amount of therapeutic agent, alone or
in combination with other therapies, which provides a therapeutic
benefit in the treatment of the condition. The term
"therapeutically effective amount" can encompass an amount that
improves overall therapy, reduces or avoids symptoms, signs, or
causes of the condition, and/or enhances the therapeutic efficacy
of another therapeutic agent.
[0077] An "infectious disease" refers to any disease caused by a
pathogen (i.e., pathogenic microorganisms). An infectious disease
may be caused by bacteria, viruses, parasites, or fungi. An
infectious disease can be a microbial infection. A "microbial
infection" refers to an infection with a microorganism, such as a
fungus, bacteria or virus. In certain embodiments, the microbial
infection is an infection with a fungus, i.e., a fungal infection.
In certain embodiments, the microbial infection is an infection
with a virus, i.e., a viral infection. In certain embodiments, the
microbial infection is an infection with a bacteria, i.e., a
bacterial infection. Various microbial infections include, but are
not limited to, skin infections, GI infections, urinary tract
infections, genito-urinary infections, sepsis, blood infections,
and systemic infections. In certain embodiments, the infectious
disease is a bacterial infection. In certain embodiments, the
infectious disease is a viral infection. In certain embodiments,
the infectious disease is a microbial infection.
[0078] "Anti-infective agents" and the like include "antibiotics,"
"antibacterial agents" and "antifungal agents." Anti-infective
agents are agents that are capable of killing or inhibiting the
growth of pathogens (e.g., bacteria, viruses, parasites, or fungi).
In certain embodiments, the anti-infective agent is an
antibacterial agent. In certain embodiments, the anti-infective
agent is (-)-Florfenicol, Acetylsulfisoxazole, Actinonin, Amikacin
sulfate, Benzethonium chloride, Cephalosporins (e.g.
7-Aminocephalosporanic acid, 7-Aminodeacetoxycephalosporanic acid,
Cefaclor, Cefadroxil, Cefamnandole, Cefazolin, Cefepime, Cefixime,
Cefmenoxinme, Cefmetazole, Cefoperazone, Cefotaxime, Cefotetan,
Cefotiam, Cefoxitin, Cefpirome, Cefpodoxime proxetil, Cefsulodin,
Cefsulodin sodium, Ceftazidime, Ceftizoxime, Ceftriaxone,
Cefuroxime, Cephalexin, Cephaloridine, Cephalosporin C,
Cephalothin, Cephalothin sodium, Cephapirin, Cephradine),
Cetrimide, Chelerythrine, Chlorhexidine (e.g. Chlorhexidine
gluconate), Chlorhexidine acetate, Chlorhexidine gluconate,
Chlorothalonil, Ciprofloxacin (e.g. Enrofloxacin), Clarithromycin,
Ciavulanic acid (e.g. Amoxicillin-clavulanic acid), Clindamycin,
Co-Trimoxazole, Dichlorophene, Didecyldimethylanmnonium chloride,
Dihydrostreptomycin, Enoxacin, Ethambutol, Fleroxacin,
Furazolidone, Grepafloxacin hydrochloride, Levofloxacin, Linezolid,
Lomefloxacin, Methylisothiazolinone, Monolaurin, Oxolinic acid,
Pefioxacin, Penicillin (e.g. 6-Aminopenicillanic acid, Amoxicillin
(e.g. Amoxicillin-clavulanic acid), Ampicillin, Ampicillin sodium,
Azlocillin, Carbenicillin, Cefoxitin, Cephaloridine, Cloxacillin,
Dicloxacillin, Mecillinam, Methicillin, Mezlocillin, Nafcillin,
Oxacillin, Penicillin G, Penicillin G potassium, Penicillin G
procaine Penicillin G sodium, NT2 Penicillin V, Piperacillin,
Piperacillin-tazobactam, Sulbactam, Tazobactam, Ticarcillin),
Povidone-iodine, Rotproofing agents, Salinomycin, Sparfloxacin,
Spirocheticides (e.g. Arsphenamine, Neoarsphenamine), NT1
Sulbactam, NT1 Sulfaquinoxaline, NT1 Tetracyclines (e.g. Achromycin
V, Demeclocycline, Doxycycline, Doxycycline monohydrate,
Minocycline, Oxytetracycline, Oxytetracycline hydrochloride,
Tetracycline, Tetracycline hydrochloride), Thiamphenicol,
Tinidazole, Triclosan, Trovafloxacin, Tuberculostatics (e.g.
4-Aminosalicylic acid, AZD 5847, Anminosalicyiic acid,
Ethionanmide), Vidarabine, Zinc pyrithione, or Zirconium phosphate.
In certain embodiments, the additional pharmaceutical agent is an
antiviral agent. In certain embodiments, the additional
pharmaceutical agent is (-)-Oseltamivir, .beta.-D-Ribofuranose,
1-acetate 2,3,5-tribenzoate, 1-Docosanol, 2-Amino-6-chloropurine,
5-Iodo-2'-deoxyuridine, 6-Chloropurine, Abacavir sulfate,
Abacavir-epivir mixt., Acyclovir, Acyclovir sodium, Adefovir
dipivoxil, Amantadine (e.g. Amantadine hydrochloride), Amantadine
hydrochloride, anti-HIV agent (e.g. Abacavir, Amprenavir,
Atazanavir, Azidothymidine, Bryostatin (e.g. Bryostatin 1,
Bryostatin 10, Bryostatin 11, Bryostatin 12, Bryostatin 13,
Bryostatin 14, Bryostatin 15, Bryostatin 16, Bryostatin 17,
Bryostatin 18, Bryostatin 19, Bryostatin 2, Bryostatin 20,
Bryostatin 3, Bryostatin 4, Bryostatin 5, Bryostatin 6, Bryostatin
7, Bryostatin 8, Bryostatin 9), Dideoxycytidine, Dideoxyinosine,
Efavirenz, Indinavir, Lamivudine, Lopinavir, Nevirapine, Ritonavir,
Saquinavir, Stavudine, Tenofovir), Azauridine, ombivir,
Deoxynojirimycin, Docosanol, Fomivirsen sodium, Foscarnet,
Ganciciovir, Integrase inhibitors (e.g. 5CITEP, Chloropeptin I,
Complestatin, Dolutegravir, Elvitegravir, L 708906, L 731988, MK
2048, Raltegravir, Raltegravir potassium), MK 5172, MK 8742,
Palivizumab, Pegylated interferon alfa-2b, Phosphonoacetic acid,
Ribavirin, Simeprevir, Sofosbuvir, Tubercidin. Vidarabine, or virus
entry inhibitor (e.g. Enfuvirtide, Maraviroc). In certain
embodiments, the additional pharmaceutical agent is a fungicide. In
certain embodiments, the additional pharmaceutical agent is
(-)-Fumagillin, (-)-Metalaxyl, 1,2,5-Fluorocytosine, Acrisorcin,
Anilazine, Antifouling agent, Azoxystrobin, Benomyl, Bordeaux
mixture, Captan, Carbendazim, Caspofungin acetate, Chlorothalonil,
Clotrimazole, Dichlofluanid, Dinocap, Dodine, Fenhexamid,
Fenpropimorph, Ferbam, Fluconazole, Fosetyl A1, Griseofulvin,
Guanidine (e.g. Agmatine, Amiloride hydrochloride, Biguanide (e.g.
Imidodicarbonimidic diamide, N,N-dimethyl-,hydrochloride (1:1)
(e.g. Metformin hydrochloride), Metformin), Cimetidine,
Guanethidine, Guanfacine, Guanidine, Guanidinium, Methylguanidine,
Sulfaguanidine), Iprobenfos, Iprodione, Isoprothiolane,
Itraconazole, Ketoconazole, Mancozeb, Metalaxyl, Metiram,
Miconazole, Natamycin, Nystatin, Oxycarboxine.
Pentachloronitrobenzene, Prochloraz, Procymidone, Propiconazole,
Pyrazophos, Reduced viscotoxin A3, Salicylanilide, Tebuconazole,
Terbinafine. Thiabendazole, Thiophanate, Thiophanate methyl,
Triadimefon, Vinclozolin, or Voriconazole. In certain embodiments,
the additional pharmaceutical agent is a protozoacide. In certain
embodiments, the additional pharmaceutical agent is Amebicide,
Antimalarial (e.g. Artemisinin, Chloroquine (e.g. Chloroquine
phosphate), Mefloquine, Sulfadoxine), Coccidiostat, Leishmanicide,
Trichomonacide, or Trypanosomicide (e.g. Eflornithine). In certain
embodiments, the additional pharmaceutical agent is a parasiticide.
In certain embodiments, the additional pharmaceutical agent is
Anthelmintic (e.g. Abamectin, Dimethylformocarbothialdine,
Niclosamide, Schistosomicide), Protozoacide (e.g. Amebicide,
Antimalarial (e.g. Artemisinin, Chloroquine (e.g. Chloroquine
phosphate), Mefloquine, Sulfadoxine), Coccidiostat. Leishmanicide,
Trichomonacide, or Trypanosomicide (e.g. Eflornithine)). In certain
embodiments, the additional pharmaceutical agent is a .beta.-lactam
antibiotic. Exemplary .beta.-lactam antibiotics include, but are
not limited to: .beta.-lactamase inhibitors (e.g., avibactam,
clavulanic acid, tazobactam, sulbactam); carbacephems (e.g.,
loracarbef); carbapenems (e.g., doripenem, imipenem, ertapenem,
meropenem); cephalosporins (1st generation) (e.g., cefacetrile,
cefadroxil, cefalexin, cefaloglycin, cefalonium, cefaloridine,
cefalotin, cefapirin, cefatrizine, cefazaflur, cefazedone,
cefazolin, cefradine, cefroxadine, ceftezole, cephalosporin C);
cephalosporins (2nd generation) (e.g., cefaclor, cefamandole,
cefbuperzone, cefmetazole, cefonicid, ceforanide, cefotetan,
cefotiam, cefoxitin, cefminox, cefprozil, cefuroxime, cefuzonam);
cephalosporins (3rd generation) (e.g., cefcapene, cefdaloxime,
cefdinir, cefditorin, cefetamet, cefixime, cefmenoxime. cefodizime,
cefoperazone, cefotaxime, cefovecin, cefpimizole, cefpiramide,
cefpodoxime, cefiamere, ceftazidime, cefteram, ceftibuten,
ceftiofur, ceftiolene, ceftizoxime, ceftriaxone, latamoxef);
cephalosporins (4th generation) (e.g., cefepime, cefluprenam,
cefoselis, cefozopran, cefpirome, cefquinome, flomoxef);
cephalosporins (5th generation) (e.g., ceftaroline fosamil,
ceftobiprole, ceftolozane); cephems (e.g., cefaloram, cefaparole,
cefcanel, cefedrolor, cefempidone, cefetrizole, cefivitril,
cefmepidium cefoxazole, cefrotil, cefsulodin, cefsumide,
ceftioline, ceftioxime, cefuracetime, nitrocefin); monobactams
(e.g., aztreonam, carumonam, norcadicin A, tabtoxinine
.beta.-lactam, tigemonam); penicillins/penams (e.g., amoxicillin,
amoxicillin/clavulanate, ampicillin, ampicillin/flucloxacillin,
ampicillin/sulbactam, azidocillin, azlocillin, bacampacillin,
benzathine benzylpenicillin, benzathine phenoxymethylpenicillin,
carbenicillin, carindacillin, clometocillin, cloxacillin,
dicloxacillin, epicillin, flucloxacillin, hetacllin, mecillinam,
mezlocillin, meticillin, metampiciillin, nafcillin, oxacillin,
penamacillin, penicillin G, penicillin V, phenaticillin,
piperacillin, piperacillin/tazobactam, pivampicillin,
pivmeclillinam, procaine benzylpenicillin, propicillin,
sulbenicillin, talampicillin, temocllin, ticarcillin,
ticarcillin/clavulanate); and penems/carbapenems (e.g., biapenem,
doripenem, ertapenem, faropenem, imipenem, imipenem/cilastatin,
lenapenem, meropenem, panipenem, razupenem, tebipenem, thienamycin,
tomopenem). In certain embodiments, the additional pharmacetucial
agent is a non-.beta.-lactam antibiotic. Exemplary
non-.beta.-lactam antibiotics include, but are not limited to:
aminoglycosides (e.g., amikacin, dibekacin, gentamicin, kanamycin,
neomycin, netilmicin, tobramycin, paromomycin, sisomicin,
streptomycin, spectinomycin); ansamycins (e.g., geldanamycin,
herbimycin); glycopeptides (e.g., belomycin, dalbavancin,
oritavancin, ramoplanin, teicoplanin, telavancin, vancomycin);
glycylcyclines (e.g., tigecycline); lincosamides (e.g.,
clindamycin, lincomycin); lipopeptides (e.g., anidulafungin,
caspofungin, cilofungin, daptomycin, echinocandin B, micafungin,
mycosubtilin); macrolides (e.g., azithromycin, carbomycin A,
clarithromycin, dirithromycin, erythromycin, josmycin, kitasamycin,
midecamycin, oleandomycin, roxithromycin, solithromycin,
spiramycin, troleandomycin, telithromycin, tylosin); nitrofurans
(e.g., furazolidone, furylfuramide, nitrofurantoin, nitrofurazone,
nifuratel, nifurquinazol, nifurtoinol, nifuroxazide, nifurtimox,
nifurzide, ranbezolid); nitroimidazoles (e.g., metronidazole,
nimorazole, tinadazole); oxazolidinones (e.g., cycloserine,
linezolid, posizolid radezolid, tedizolid); polypeptides (e.g.,
actinomycin, bacitracin, colistin, polymyxin B); quinolones (e.g.,
balofloxacin, besifloxacin, cinoxacin, ciprofloxacin,
clinafloxacin, danofloxacin, delafloxacin, diflofloxacin, enoxacin,
enrofloxacin, fleroxacin, flumequine, gatifloxacin, gemifloxacin,
grepafloxacin, ibafloxacin, JNJ-Q2, levofloxacin, lomefloxacin,
marbofloxacin, moxifloxacin, nadifloxacin, nalidixic acid,
nemonoxacin, norfloxacin, ofloxacin, orbifloxacin, oxilinic acid,
pazufloxacin, pefloxacin, piromidic acid, pipemidic acid,
prulifloxacin, rosoxacin, rufloxacin, sarafloxacin, sparfloxacin,
sitafloxacin, temafloxacin, tosufloxacin, trovafloxacin);
rifamycins (e.g., rifamycin B, rifamycin S, rifamycin SV,
rifampicin, rifabutin, rifapentine, rifalazil, rifaximin);
sulfonamides (e.g., co-trimoxazole, mafenide, pediazole,
sulfacetamide, sulfadiazine, silver sulfadiazine, sulfadimidine,
sulfadimethoxine, sulfadoxine, sulfafurazole, sulfamethizole,
sulfamethoxazole, sulfamethoxypyridazine, sulfametopyrazine,
sulfametoxydiazine, sulfamoxole, sulfanilamide, sulfanitran,
sulfasalazine, sulfisomidine, sulfonamidochrysoidine,
trimethoprim); tetracyclines (e.g., 6-deoxytetracycline,
aureomycin, chlortetracycline, demeclocycline, doxycycline,
lymecycline, meclocycline, methacycline, minocycline,
oxytetracycline, PTK-0796, sancycline, rolitetracycline,
tetracycline, terramycin); tuberactinomycins (e.g.,
tuberactinomycin A, tuberactinomycin 0, viomycin, enviomycin,
capreomycin); arsphenamine; chloramphenicol; dalfoprisitin;
fosfomycin; fusidic acid; fidaxomycin, gramicidin; lysozyme;
mupirocin; platensimycin; pristinamycin; sparsomycin; quinupristin;
quinupristin/dalfopristin; teixobactin; and thiamphenicol.
[0079] Antifungal agents include azole antifungal agents. "Azole
antifungal agents" are antifungal agents that contain azole
moieties (e.g., imidazoles, triazoles, and thiazoles). Azole
antifungal agents include, but are not limited to, imidazoles
(e.g., Bifonazole, Butoconazole, Clotrimazole, Econazole.
Fenticonazole, Isoconazole, Ketoconazole. Luliconazole, Miconazole,
Omoconazole, Oxiconazole, Sertaconazole, Sulconazole, Tioconazole),
triazoles (e.g., Albaconazole, Efinaconazole, Epoxiconazole,
Fluconazole, Isavuconazole, Itraconazole, Posaconazole,
Propiconazole, Ravuconazole. Terconazole. Voriconazole), and
thiazoles (e.g., Abafungin).
BRIEF DESCRIPTION OF THE DRAWINGS
[0080] The accompanying drawings, which constitute a part of this
specification, illustrate several embodiments of the invention and
together with the description, serve to explain the principles of
the invention.
[0081] FIGS. 1A-ID show the discovery of inhibitors of the CgGal11A
KIX-CgPdr1AD interaction interface. FIG. 1A. Schematic of the
screening process. FIG. 1B, iKIX1 inhibits cell growth in a
concentration-dependent manner in the presence of 5 .mu.M
ketoconazole (KET); error bars represent means+/-s.d. from
duplicate plates. FIG. 1C. iKIX1 structure. FIG. 1D. FP titration
curve showing the interaction of CgGal11A KIX domain with CgPdr1
AD30 fitted to a K.sub.d of 319.7 nM.+-.9.5 nM (left), iKIX1
competes out CgPdr1 AD30 with an IC.sub.50 of 190.2 .mu.M.+-.4.1
.mu.M (right). The measured K.sub.1 and IC.sub.50 values were used
to calculate an apparent Ki of 18.1 .mu.M for iKIX1. Data represent
mean of two replicates and standard error from the fit is
shown.
[0082] FIGS. 2A-2D show elucidation of the CgGal11A KIX domain
structure; CgPdr1 AD and iKIX1 bind to a similar interface on the
CgGal11A KIX domain. FIG. 2A. Backbone representation of the 10
lowest energy NMR structures of CgGal11A KIX domain; backbone
RMSD.about.0.7 .ANG., left. Overlay of CgGal11A (purple) and S.
cerevisiae Gal11/Med15 (blue) KIX domains, with an overall RMSD of
2.0 .ANG., right (see, e.g., Krissinel. E. & Henrick, K.
Secondary-structure matching (SSM), a new tool for fast protein
structure alignment in three dimensions. Acta Crystallogr D Biol
Crystallogr 60, 2256-2268, doi:S0907444904026460 [pii]
10.1107/S0907444904026460 (2004)). FIG. 2B. Chemical shift
perturbations (CSPs) on the CgGal11A KIX domain in the presence of
CgPdr1 AD (red) or iKIX1 (blue). Residues coloured in red or blue
indicate a chemical shift perturbation greater than 2 s.d. Residues
highlighted in green (L19, L23 and L51) represent significant CSPs
in the side-chain methyl groups of an ILV labeled sample. FIG. 2C.
The iKIX1 and CgPdr1 AD target the hydrophobic groove on the
CgGal11A KIX domain, which is surrounded by a basic patch. Residues
H43, K54, K68, K78, R79, F47 and M72 present a positive
electrostatic surface enclosing the binding interface. FIG. 2D,
iKIX1 docked to the CgGal11A KIX domain, iKIX1 is depicted as red
sticks and spheres. Residues that experience significant methyl CSP
upon addition of iKIX1 are depicted as blue sticks.
[0083] FIGS. 3A-3G show iKIX1 blocks Gal11/Med15 recruitment and
upregulation of Pdr1 target genes. FIG. 3A, iKIX1 inhibits
ketoconazole (KET)-induced upregulation of luciferase activity in a
dose-responsive manner in a Sc pdr1.DELTA.pdr3.DELTA. strain
containing plasmid-borne CgPDR1 and 3.times.PDRE-luciferase. (UT):
untreated control; **P<0.001. FIGS. 3B and 3C, iKIX1 prevents
the ketoconazole (KET)-induced recruitment of Gal11/Med15/Mediator
to the upstream activating sequences (UAS) of the PDRE-regulated
promoter ScPDR5 (FIG. 3B), and ScPDR5 induction (FIG. 3C).
Representative experiment from two biological replicates (ChIP DNA
and RNA from same experiment) is shown. Error bars indicate s.d. of
technical replicates; ***P<0.00001, **P<0.0005, and
*P<0.01 by two-tailed Student's t-test. FIG. 3D, iKIX1 inhibits
ketoconazole (KET)-induced transcriptional upregulation of CgCDR1
and CgCDR2 in a CgPDR1 wild-type strain (SFY114). **P<0.005.
FIG. 3E. RNA-Seq analysis of a C. glabrata SFY114 (PDR1 wild-type)
strain pre-treated with iKIX1 or vehicle alone then induced with
ketoconazole (iKIX1+KET and KET, respectively). FIG. 3F, iKIX1
inhibits xenobiotic-induced CgPdr1 transcription in CgPdr1
gain-of-function mutants (amino acid changes indicated). Samples
shown were induced with ketoconazole. *P<0.05 and **P<0.01 as
compared to DMSO+ketoconazole control. FIG. 3G, iKIX1 inhibits
rhodamine 6G efflux in C. glabrata as compared to vehicle control.
* P<0.05, **P<0.005 as compared to DMSO+ketoconazole control.
Data represent the means of three biological replicates. Two-tailed
student's t-test used to determine P values; error bars represent
means+/-standard deviation.
[0084] FIGS. 4A-4D show iKIX1 as a co-therapeutic in models of C.
glabrata disseminated disease. FIG. 4A. Schematic showing CgPdr1
gain-of-function alterations in relation to putative functional
domains. DBD: DNA-binding domain, ID: inhibitory domain, MHR:
middle homology region, AD: activation domain. FIG. 4B, iKIX1
restores the efficacy of azoles towards CgPDR1 gain-of-function
mutants. Plates contained increasing concentrations of vehicle
control (DMSO) or iKIX1 to 150 .mu.M in the absence or presence of
fluconazole (FLU) or ketoconazole (KET). FIG. 4C, iKIX1 in
combination with fluconazole but not fluconazole alone
significantly extended survival of G. mellonella larvae injected
with CgPDR1.sup.L280F (SFY115, n=9). For SFY114, n=10. *P<0.05,
***P<0.001 as compared to PBS vehicle control. Statistical
differences measured using a log-rank (Mantel-Cox) test. FIG. 4D,
iKIX1 combination treatment with 25 mg/kg fluconazole (low FLU)
reduces fungal tissue burden in the kidney or spleen of mice
injected with CgPDR1 wild-type (SFY114); iKIX1 in combination with
100 mg/kg fluconazole (high FLU) reduces fungal tissue burden in
the kidney or spleen of mice injected with CgPDR1.sup.L280F
(SFY115). N=5 mice for each treatment condition; *P<0.05.
**P<0.005 and ***P<0.0001 as compared to no treatment.
Statistical differences measured using a Wilcoxon rank-sum test;
error bars represent means+/-standard deviation.
[0085] FIG. 5 shows a summary of quality statistics for the
ensemble of 10 structures calculated with AMBER explicit water
refinement and list of experimental restraints.
[0086] FIG. 6. Left: Table of compound libraries that were screened
using a fluorescence polarization assay at the Institute of
Chemistry & Cell Biology (ICCB) facility at Harvard Medical
School. Right: An S. cerevisiae viability screen identifies small
molecules that preferentially inhibit growth of S. cerevisiae in a
concentration-dependent manner in the presence of 5 .mu.M
ketoconazole (KET). Top hits from the screen are shown; OD.sub.600
values are the average of values from duplicate plates.
[0087] FIG. 7A. 2-dimensional representation of the H-bonding
network between the CgGal11A KIX domain and iKIX1 based on docking
studies. FIG. 7B. Chemical shift perturbations (CSPs) of ILV methyl
resonances. Left: .sup.1H-.sup.13C HSQC showing ILV methyl
resonances of CgGal11A KIX domain in presence (brown) and absence
(teal) of CgPdr1 AD (2-fold excess). Right: .sup.1H-.sup.13C HSQC
showing ILV methyl resonances of CgGal11A KIX domain in presence
(purple) and in absence (teal) of iKIX1 (4-fold excess). Three
leucines (L19, L23, L51) show significant CSPs in both spectra.
FIG. 7C. Sequence alignment of the C. glabrata Gal11A and S.
cerevisiae Gal11/Med15 KIX domains (see, e.g., Robert, X. &
Gouet, P. Deciphering key features in protein structures with the
new ENDscript server. Nucleic acids research 42, W320-324,
doi:10.1093/nar/gku316 (2014)).
[0088] FIG. 8A, iKIX1 prevents the ketoconazole (KET)-induced
recruitment of ScGal11/Med15/Mediator to the upstream activating
sequences (UAS) of the PDRE-regulated promoter ScSNQ2 and
transcriptional upregulation of ScSNQ2. FIG. 8B. HA-Pdr1 occupies
PDRE-regulated promoters of ScPDR5 and ScSNQ2 in the presence of 20
.mu.M iKIX1 or vehicle (DMSO) control prior to and following
ketoconazole (KET) addition. FIG. 8C. 20 .mu.M iKIX1 inhibits
ketoconazole-induced upregulation of ScPdr1 target genes ScPDR5 and
ScSNQ2 in the HA-Pdr1 strain. RNA was harvested concurrently with
representative chromatin immunoprecipitation experiment shown FIG.
8B at t=0 min. (DMSO, 20 .mu.M iKIX1) and t=15 min. after
ketoconazole induction (DMSO+KET, 20 .mu.M iKIX1+KET). Transcripts
are normalized to ScSCR1 and un-induced DMSO control. (a-c)
Representative experiment from two biological replicates is shown.
Error bars represent mean+/-s.d. of technical replicates;
*P<0.05, **P<0.01 and ***P<0.001 as calculated by
two-tailed Student's t-test. FIG. 8D. RNA-Seq analysis of a
wild-type S. cerevisiae strain (BY4741) pre-treated with iKIX1 or
vehicle alone then induced with ketoconazole (iKIX1+KET and KET,
respectively) demonstrate a blunted induction of Pdr1 target genes
following iKIX1 pre-treatment. Data represents means of three
biological replicates. FIG. 8E, iKIX1 pre-treatment does not
significantly alter the transcript levels of PDR1 or GAL11/GAL11A
in S. cerevisiae or C. glabrata after azole induction. Cells were
pre-incubated with vehicle (DMSO) or iKIX1 and then induced with 40
.mu.M ketoconazole (+KET) for 15 minutes before harvest. Average
value of three biological replicates is shown and error bars
represent mean+/-standard deviation; *P<0.05, **P<0.001 as
compared to DMSO or DMSO+KET control, calculated by two-tailed
Student's t-test.
[0089] FIGS. 9A-D. With iKIX1 pre-treatment, CgPdr1-dependent
transcription of (FIG. 9A) CgCDR1 and (FIG. 9B) CgYOR1 remains
repressed 120 minutes after ketoconazole induction. SFY114 (PDR1
wild-type) cells were pre-incubated with vehicle (DMSO) or iKIX1
and then induced with 40 .mu.M ketoconazole (+KET). Transcript
levels were assessed by quantitative RT-PCR prior to and for 120
minutes following ketoconazole induction. Transcript levels are
normalized to CgRDN25-1 and un-induced vehicle control (DMSO) at
t=0. FIG. 9C, iKIX1 treatment alone does not have significant
effects on CgPdr1 target gene induction either in the presence of
wild-type (SFY114) or gain-of-function mutant CgPDR1 (amino acid
alterations indicated). FIG. 9D. Table of average CgCDR1 delta Cp
values (Cp.sub.CgCD1-Cp.sub.CgRDN25-1) and corresponding standard
deviation for quantitative real-time PCR experiments. For all
panels of FIGS. 9A-9D, average value of three biological replicates
is shown and error bars represent+/-standard deviation; *P<0.05,
**p<0.01, and ***p<0.005 as compared to vehicle+ketoconazole
control. P values calculated using two-tailed Student's t-test.
[0090] FIG. 10 shows iKIX1 inhibits efflux of rhodamine 6G in PDR1
wild-type, PDR1.sup.L280F and PDR1.sup.Y584C strains. Data points
indicate mean of three biological replicates and error bars
represent mean+/-s.d.
[0091] FIGS. 11A and 11B, iKIX1 increases the sensitivity of Cg
strains bearing wild-type CgPDR1 to azole treatment. Two strains
bearing wild-type CgPDR1 alleles (SFY114. DSY759) were plated at
concentrations differing by ten-fold (10.times., 1.times.) on
plates containing increasing concentrations of (FIG. 11A) iKIX1 to
300 .mu.M in the presence or absence of 1 .mu.M ketoconazole (KETO)
or (FIG. 11B) iKIX1 to 250 .mu.M in the presence or absence of 50
.mu.M fluconazole (FLU). FIG. 11C, iKIX1 and ketoconazole (KET)
have additive effects on the growth of a CgPDR1 wild-type strain.
FIG. 11D, iKIX1 and ketoconazole (KET) synergistically inhibit the
growth of the CgPDR1.sup.L280F mutant. FIGS. 11C and 11D. The
EUCAST broth microdilution method.sup.27 was used to assess the
effects of iKIX1 and ketoconazole combination treatment. Growth, as
assessed by OD.sub.540, was normalized to no drug control. All
combination indices (CI) for the CgPDR1.sup.L280F mutant were less
than 1, indicating synergy. A representative of three biological
replicates is shown and the red line indicates a combination index
of 1.
[0092] FIGS. 12A-12H show electron-withdrawing groups in the
aromatic ring of iKIX1 complement the basic binding interface of
the CgGal11A KIX domain and thus play a key role in iKIX1 function.
A iKIX1 analog (A2) lacking electron-withdrawing groups increases
the IC.sub.50 in the FP assay (FIG. 12A) and abolishes activity in
the S. cerevisiae luciferase reporter assay (FIG. 12B), repression
of CgCDR1 expression (FIG. 12C), and synergistic C. glabrata cell
growth inhibition with azole antifungal agents (FIG. 12D). Error
bars in FIGS. 12B and 12C indicate mean+/-s.d. of technical
replicates (reads/real-time PCR reactions, respectively).
**P<0.005; statistical differences calculated using two-tailed
Student's t-test. FIG. 12E, iKIX1 inhibits viability of HepG2 cells
at concentrations>50 .mu.M. The mean of 3 biological replicates
is shown; error bars represent means+/-s.d. FIG. 12F, iKIX1
exhibits no effect on transcription of SREBP-target genes in HepG2
cells at concentrations up to 100 .mu.M. Biological duplicates were
assessed; representative experiment is shown and error bars
represent means+/-s.d. of technical (real-time PCR) replicates.
FIG. 12G. Mouse plasma stability of iKIX1 and mouse and human
microsomal stability of iKIX1, n=1. FIG. 12H. In vivo
pharmacokinetic parameters of iKIX1, n=3 mice per time point.
[0093] FIG. 13A. Clinical isolates DSY562/DSY565 (azole sensitive
and PDR1.sup.L280F azole-resistant strains, respectively) behave
similarly to SFY114/SFY115 (isogenic PDR1.sup.+ and PDR1.sup.L280F
strains, shown in FIG. 4d) in the mouse infection model, n=10 mice
for each treatment condition; *P<0.01, **P<0.005 and
***P<0.0001. FIG. 13B, iKIX1 combination treatment with
fluconazole reduces fungal tissue burdens in the spleen or kidney
of mice injected with C. glabrata PDR1.sup.P822L (SFY116). n=5 mice
for each treatment condition; **P<0.01 and *P<0.05. FIG. 13C.
100 mg/kg/day iKIX1 (high iKIX1) treatment of mice infected with
SFY93 (pdr1.DELTA.) significantly reduces fungal burden in a mouse
infection model (CFU/g kidney) alone as compared to SFY114
(PDR1.sup.+) or SFY115 (PDR1.sup.L280F). n=10 mice for each
treatment condition; *P<0.01, **P<0.005, ***P<0.0001. FIG.
13D. Mice infected with SFY114 (PDR1+), SFY115 (PDR1.sup.L280F) or
SFY93 (pdr1.DELTA.) were treated with low (10 mg/kg/day) iKIX1, low
fluconazole (low FLU; 25 mg/kg/day), fluconazole at 100 mg/kg/day
(FLU) or combination with the two, iKIX1 did not confer additional
reductions in CFU/g kidney with SFY93 infection. n=10 mice for each
treatment condition. ***P<0.0005. FIG. 13E, iKIX1 and
ketoconazole (KETO) reduce adherence of CgPDR1.sup.L280F (SFY116)
to CHO-Lec2 cells. Adherence is normalized to SFY114 DMSO control;
each column represents the average of 4 biological replicates.
*P<0.05 as compared to SFY114 DMSO control. FIG. 13F, iKIX1 (100
mg/kg/day) or fluconazole (FLU) significantly reduces fungal burden
in the bladder and kidney in a urinary tract infection model in
mice. n=15 mice were infected in each group and points at 0
log.sub.10 CFU/g organ fell below the detection limit of the method
(50 CFU/g organ). *P<0.05, **P<0.005. FIGS. 13A-13F.
Statistical differences were measured using a Mann-Whitney/Wilcoxon
rank-sum test as compared to no treatment control; error bars
represent means+/-standard deviation.
[0094] FIG. 14 shows model of iKIX1 function as a co-therapeutic in
combination with an azole, blocking the azole-induced recruitment
of Gal11/Med15-Mediator to Pdr1 target genes upon azole-treatment
and preventing the upregulation of Pdr1 target genes, including
those which encode drug efflux pumps.
[0095] FIGS. 15-18. Top Row. Compound names and structures. Second
Row. Spot plating. Candida glabrata PDR1 gain-of-function mutants
are sensitive to 200 .mu.M fluconazole in the presence of compound
gradients. Plates with compound gradients from approximately 0 to
150 .mu.M are shown on top, plates with compound gradients from
approximately 0 to 150 .mu.M in the presence of 200 .mu.M
fluconazole (+FLU) are below. Isogenic Candida glabrata PDR1
gain-of-function mutants used in the assays are as follows:
L280F-CgPDR1.sup.L280F (SFY115), R376W-CgPDR1.sup.R376W (SFY101),
T588A-CgPDR1.sup.T588A (105), Y584C-CgPDR1.sup.Y584C (SFY111),
D1082G-CgPDR1.sup.D1082G (SFY103). Spot plating methods: Strains
were inoculated and grown in YPD at 30.degree. C. with shaking
overnight and 5 mL of YPD was inoculated with 20 .mu.L of overnight
culture. Strains were grown to log phase at 30.degree. C. with
shaking and then diluted to an OD.sub.600 of 0.0004, 3 .mu.L of
cell suspension was spotted on plates containing gradients with
increasing concentration of compound and a fixed concentration of
fluconazole, as indicated. Plates were incubated at 30.degree. C.
and growth was assessed after 48 hours. Third Row. Luciferase
assays. Compounds reduce azole-induced PDRE-dependent luciferase
gene transcription to a similar degree as iKIX1. Y-axis represents
luciferase activity, normalized to vehicle (DMSO)+ketoconazole
control. Strains were incubated with 5 .mu.M or 50 .mu.M compound
in the presence of 40 .mu.M ketoconazole (KET). Luciferase assay
methods: An S. cerevisiae pdr1.DELTA.::KANpdr3.DELTA.::KAN strain
bearing plasmids carrying CgPDR1-1 and PDRES-luciferase was used
for the luciferase assays. Strains were grown overnight in YPD and
then washed twice with sterilized Milli-Q water before being
resuspended to an OD600 of 0.8 in YP. After 20 hours, cultures were
split and iKIX1 or DMSO alone was added to final concentrations as
indicated. At the same time, ketoconazole to a final concentration
of 40 .mu.M (resuspended in ethanol) or ethanol alone (vehicle)
were added to cultures. After 24 hours of treatment, an equal
volume of 1 mM d-luciferin (sodium salt) in 0.1 M sodium citrate
buffer at pH 5.0 was added to 100 .mu.L aliquots. Luminescent
signal was acquired over 10 seconds and RLU (relative light units)
presented are normalized to growth (assessed by OD.sub.600)) and
untreated controls. Fourth Row. Compounds reduce azole-induced
transcription of CgPdr1 target CgCDR1 in a strain bearing wild-type
CgPDR1 (SFY114) to a similar degree as iKIX1. Y-axis represents
CDR1 transcription, normalized to vehicle (DMSO)+ketoconazole
control. Strains were pre-incubated with 30 .mu.M compound before
being induced with 40 .mu.M keotconazole (KET) for 15' prior to
harvest. Transcription assays and quantitative real-time PCR
methods: Cells were grown in YPD at 30.degree. C. with shaking 24
hours then washed twice in 2.times. volume of Milli-Q water before
being resuspended in YP to an OD.sub.600 of 0.8. Cells were grown
with shaking overnight (.about.16 hours) before splitting and
treatment with vehicle (DMSO) or iKIX1 to concentrations indicated.
Cells were grown with shaking another 8 hours before harvest of
non-azole induced samples. Remaining cultures were induced to a
final concentration of 40 .mu.M ketoconazole and harvested after 15
minutes and subsequent time points, if shown. For harvest and
preparation of RNA, cells equivalent to an OD.sub.600 of 0.4 were
centrifuged at 4,000 rpm for 4 minutes and then resuspended in 700
.mu.L Trizol. Cells in Trizol were bead beat with 0.4 mL glass
beads for 2.times.30 seconds and then protocol was followed
according to manufacturer's instructions. One .mu.g of RNA was used
for DNase I treatment (Roche) and subsequent cDNA synthesis using
Roche Transcriptor First Strand cDNA synthesis kit. cDNA was
diluted 5.times. in water and 2.5 .mu.L was used per reaction with
Roche SYBR green. Quantitative real-time PCR reactions were run and
analyzed on a Roche LightCycler 480 system. Sc transcripts were
normalized to ScSCR1 and untreated, uninduced DMSO control; Cg
transcripts were normalized to CgRDN25-1 and untreated, uninduced
DMSO control.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
[0096] Provided herein are compounds (e.g., Compounds of Formula
(I). (II), and (III)), and pharmaceutically acceptable salts,
hydrates, solvates, polymorphs, co-crystals, tautomers,
stereoisomers, isotopically labeled derivatives, and prodrugs
thereof, and pharmaceutical compositions thereof. In certain
embodiments, the compounds are inhibitors of the interaction of the
C. glabrata Pdr1 activation domain with the C. glabrata Gal11A KIX
domain. The compounds can therefore be used for treating diseases
(e.g., infectious diseases) and/or for killing or inhibiting the
growth of pathogens (e.g., fungi). In certain embodiments, for
example, the compounds inhibit Pdr1-dependent gene activation and
re-sensitize drug-resistant fungi (e.g., C. glabrata) to
antifungals (e.g., azole antifungals). In another aspect, the
present invention provides kits comprising the compounds or
pharmaceutical compositions provided herein.
Compounds
[0097] Provided herein are compounds of Formula (I):
##STR00007##
and pharmaceutically acceptable salts, hydrates, solvates,
polymorphs, co-crystals, tautomers, stereoisomers, isotopically
labeled derivatives, and prodrugs thereof, wherein:
[0098] R.sup.1 and R.sup.2 are independently hydrogen, optionally
substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl, optionally substituted aryl, optionally
substituted carbocyclyl, optionally substituted heterocyclyl, or
optionally substituted heteroaryl; or optionally R.sup.1 and
R.sup.2 are joined together with the intervening atoms to form
optionally substituted carbocyclyl or optionally substituted
heterocyclyl;
[0099] each instance of R.sup.N is hydrogen, optionally substituted
alkyl, or optionally substituted acyl, or a nitrogen protecting
group;
[0100] each instance of R.sup.3 is independently hydrogen, halogen,
--CN, --SCN, --NO.sub.2, --N.sub.3, optionally substituted alkyl,
optionally substituted alkenyl, optionally substituted alkynyl,
optionally substituted carbocyclyl, optionally substituted
heterocyclyl, optionally substituted aryl, optionally substituted
heteroaryl, optionally substituted acyl, optionally substituted
sulfinyl, optionally substituted sulfonyl, --OR.sup.3a,
--N(R.sup.3b).sub.2, or --SR.sup.3; or optionally two R.sup.3 are
joined together with the intervening atoms to form optionally
substituted carbocyclyl or optionally substituted heterocyclyl;
[0101] each instance of R.sup.3a is independently hydrogen,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted carbocyclyl,
optionally substituted heterocyclyl, optionally substituted aryl,
optionally substituted heteroaryl, optionally substituted acyl, or
an oxygen protecting group;
[0102] each instance of R.sup.3b is independently hydrogen,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted carbocyclyl,
optionally substituted heterocyclyl, optionally substituted aryl,
optionally substituted heteroaryl, optionally substituted acyl, or
a nitrogen protecting group; or optionally two R.sup.3b are joined
together with the intervening atoms to form optionally substituted
heterocyclyl or optionally substituted heteroaryl;
[0103] each instance of R.sup.3c is independently hydrogen,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted carbocyclyl,
optionally substituted heterocyclyl, optionally substituted aryl,
optionally substituted heteroaryl, optionally substituted acyl, or
a sulfur protecting group; and
[0104] n is 1, 2, 3, 4, or 5.
[0105] In certain embodiments, a compound of the invention is not
one of the following:
##STR00008## ##STR00009## ##STR00010## ##STR00011## ##STR00012##
##STR00013## ##STR00014## ##STR00015## ##STR00016##
##STR00017##
[0106] In certain embodiments, the compound of Formula (I) is of
Formula (I-a):
##STR00018##
or a pharmaceutically acceptable salt, hydrate, solvate, polymorph,
co-crystal, tautomer, stereoisomer, isotopically labeled
derivative, or prodrug thereof, wherein:
[0107] each instance of n is independently 0, 1, 2, 3, 4, or 5.
[0108] In certain embodiments, the compound of Formula (I) is of
the following formula:
##STR00019##
or a pharmaceutically acceptable salt, hydrate, solvate, polymorph,
co-crystal, tautomer, stereoisomer, isotopically labeled
derivative, or prodrug thereof.
[0109] In certain embodiments, the compound of Formula (I) is of
the following formula:
##STR00020##
or a pharmaceutically acceptable salt, hydrate, solvate, polymorph,
co-crystal, tautomer, stereoisomer, isotopically labeled
derivative, or prodrug thereof.
[0110] In certain embodiments, the compound of Formula (I) is of
one of the following
##STR00021##
or a pharmaceutically acceptable salt, hydrate, solvate, polymorph,
co-crystal, taulomer, stereoisomer, isotopically labeled
derivative, or prodrug thereof.
[0111] In certain embodiments, the compound of Formula (I) is of
one of the following formulae:
##STR00022##
or a pharmaceutically acceptable salt, hydrate, solvate, polymorph,
co-crystal, tautomer, stereoisomer, isotopically labeled
derivative, or prodrug thereof.
[0112] Also provided herein are compounds of Formula (II):
##STR00023##
and pharmaceutically acceptable salts, hydrates, solvates,
polymorphs, co-crystals, tautomers, stereoisomers, isotopically
labeled derivatives, and prodrugs thereof, wherein:
[0113] R.sup.1 and R.sup.2 are independently hydrogen, halogen, or
optionally substituted alkyl, or optionally R.sup.1 and R.sup.2 are
joined together with the intervening atoms to form optionally
substituted carbocyclyl or optionally substituted heterocyclyl;
[0114] provided that at least one of R.sup.1 and R.sup.2 is not
hydrogen;
[0115] each instance of R.sup.N is hydrogen, optionally substituted
alkyl, or optionally substituted acyl, or a nitrogen protecting
group;
[0116] each instance of R.sup.3 is independently hydrogen, halogen,
--CN, --SCN, --NO.sub.2, --N.sub.3, optionally substituted alkyl,
optionally substituted alkenyl, optionally substituted alkynyl,
optionally substituted carbocyclyl, optionally substituted
heterocyclyl, optionally substituted aryl, optionally substituted
heteroaryl, optionally substituted acyl, optionally substituted
sulfinyl, optionally substituted sulfonyl, --OR.sup.3a,
--N(R.sup.3b).sub.2, or --SR.sup.3c; or optionally two R.sup.3 are
joined together with the intervening atoms to form optionally
substituted carbocyclyl or optionally substituted heterocyclyl;
[0117] each instance of R.sup.3a is independently hydrogen,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted carbocyclyl,
optionally substituted heterocyclyl, optionally substituted aryl,
optionally substituted heteroaryl, optionally substituted acyl, or
an oxygen protecting group;
[0118] each instance of R.sup.3b is independently hydrogen,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted carbocyclyl,
optionally substituted heterocyclyl, optionally substituted aryl,
optionally substituted heteroaryl, optionally substituted acyl, or
a nitrogen protecting group; or optionally two R.sup.3b are joined
together with the intervening atoms to form optionally substituted
heterocyclyl or optionally substituted heteroaryl;
[0119] each instance of R.sup.3c is independently hydrogen,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted carbocyclyl,
optionally substituted heterocyclyl, optionally substituted aryl,
optionally substituted heteroaryl, optionally substituted acyl, or
a sulfur protecting group; and
[0120] n is 1, 2, 3, 4, or 5.
[0121] In certain embodiments. R.sup.1 and R.sup.2 are
independently hydrogen or optionally substituted alkyl; or
optionally R.sup.1 and R.sup.2 are joined together with the
intervening atoms to form optionally substituted carbocyclyl or
optionally substituted heterocyclyl; provided that at least one of
R.sup.1 and R.sup.2 is not hydrogen.
[0122] In certain embodiments, the compound of Formula (II) is of
Formula (II-a):
##STR00024##
or a pharmaceutically acceptable salt, hydrate, solvate, polymorph,
co-crystal, tautomer, stereoisomer, isotopically labeled
derivative, or prodrug thereof, wherein:
[0123] m is 1, 2, 3, or 4.
[0124] As generally defined herein, m is 1, 2, 3, or 4. In certain
embodiments, m is 1. In certain embodiments, m is 2. In certain
embodiments, m is 3. In certain embodiments, m is 4.
[0125] In certain embodiments, the compound of Formula (II) is of
one of the following formulae:
##STR00025##
or a pharmaceutically acceptable salt, hydrate, solvate, polymorph,
co-crystal, tautomer, stereoisomer, isotopically labeled
derivative, or prodrug thereof.
[0126] In certain embodiments, the compound of Formula (II) is of
one of the following
##STR00026##
or a pharmaceutically acceptable salt, hydrate, solvate, polymorph,
co-crystal, tautomer, stereoisomer, isotopically labeled
derivative, or prodrug thereof.
[0127] In certain embodiments, the compound of Formula (II) is of
one of the following formulae:
##STR00027##
or a pharmaceutically acceptable salt, hydrate, solvate, polymorph,
co-crystal, tautomer, stereoisomer, isotopically labeled
derivative, or prodrug thereof.
[0128] In certain embodiments, the compound of Formula (II) is of
one of the following formulae:
##STR00028##
or a pharmaceutically acceptable salt, hydrate, solvate, polymorph,
co-crystal, tautomer, stereoisomer, isotopically labeled
derivative, or prodrug thereof.
[0129] In certain embodiments, the compound of Formula (II) is of
one of the following formulae:
##STR00029##
or a pharmaceutically acceptable salt, hydrate, solvate, polymorph,
co-crystal, tautomer, stereoisomer, isotopically labeled
derivative, or prodrug thereof.
[0130] Also provided herein are compounds of Formula (III):
##STR00030##
and pharmaceutically acceptable salts, hydrates, solvates,
polymorphs, co-crystals, tautomers, stereoisomers, isotopically
labeled derivatives, and prodrugs thereof, wherein:
[0131] Y is a bond, optionally substituted alkylene, --O--,
--NR.sup.N--, or --S--;
[0132] R.sup.1 and R.sup.2 are independently hydrogen, optionally
substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl, optionally substituted aryl, optionally
substituted carbocyclyl, optionally substituted heterocyclyl, or
optionally substituted heteroaryl; or optionally R.sup.1 and
R.sup.2 are joined together with the intervening atoms to form
optionally substituted carbocyclyl or optionally substituted
heterocyclyl;
[0133] each instance of R.sup.N is hydrogen, optionally substituted
alkyl, or optionally substituted acyl, or a nitrogen protecting
group;
[0134] each instance of R.sup.3 is independently hydrogen, halogen,
--CN, --SCN, --NO.sub.2, --N.sub.3, optionally substituted alkyl,
optionally substituted alkenyl, optionally substituted alkynyl,
optionally substituted carbocyclyl, optionally substituted
heterocyclyl, optionally substituted aryl, optionally substituted
heteroaryl, optionally substituted acyl, optionally substituted
sulfinyl, optionally substituted sulfonyl, --OR.sup.3a,
--N(R.sup.3b).sub.2, or --SR.sup.3; or optionally two R.sup.3 are
joined together with the intervening atoms to form optionally
substituted carbocyclyl or optionally substituted heterocyclyl;
[0135] each instance of R.sup.3a is independently hydrogen,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted carbocyclyl,
optionally substituted heterocyclyl, optionally substituted aryl,
optionally substituted heteroaryl, optionally substituted acyl, or
an oxygen protecting group;
[0136] each instance of R.sup.3b is independently hydrogen,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted carbocyclyl,
optionally substituted heterocyclyl, optionally substituted aryl,
optionally substituted heteroaryl, optionally substituted acyl, or
a nitrogen protecting group; or optionally two R.sup.3b are joined
together with the intervening atoms to form optionally substituted
heterocyclyl or optionally substituted heteroaryl;
[0137] each instance of R.sup.3c is independently hydrogen,
optionally substituted alkyl, optionally substituted alkenyl,
optionally substituted alkynyl, optionally substituted carbocyclyl,
optionally substituted heterocyclyl, optionally substituted aryl,
optionally substituted heteroaryl, optionally substituted acyl, or
a sulfur protecting group;
[0138] R.sup.4 is optionally substituted carbocyclyl, optionally
substituted heterocyclyl, optionally substituted aryl, or
optionally substituted heteroaryl; and
[0139] n is 1, 2, 3, or 4.
[0140] In certain embodiments, the compound of Formula (III) is of
the following formula:
##STR00031##
or a pharmaceutically acceptable salt, hydrate, solvate, polymorph,
co-crystal, tautomer, stereoisomer, isotopically labeled
derivative, or prodrug thereof.
[0141] In certain embodiments, the compound of Formula (III) is of
one of the following formulae:
##STR00032##
or a pharmaceutically acceptable salt, hydrate, solvate, polymorph,
co-crystal, tautomer, stereoisomer, isotopically labeled
derivative, or prodrug thereof.
[0142] In certain embodiments, the compound of Formula (III) is of
one of the following formulae:
##STR00033##
or a pharmaceutically acceptable salt, hydrate, solvate, polymorph,
co-crystal, tautomer, stereoisomer, isotopically labeled
derivative, or prodrug thereof.
[0143] In certain embodiments, the compound of Formula (III) is of
one of the following formulae:
##STR00034##
or a pharmaceutically acceptable salt, hydrate, solvate, polymorph,
co-crystal, tautomer, stereoisomer, isotopically labeled
derivative, or prodrug thereof.
[0144] In certain embodiments, the compound of Formula (III) is of
one of the following formulae:
##STR00035##
or a pharmaceutically acceptable salt, hydrate, solvate, polymorph,
co-crystal, tautomer, stereoisomer, isotopically labeled
derivative, or prodrug thereof.
[0145] Also provided herein are compounds of one of the following
formulae:
##STR00036##
and pharmaceutically acceptable salts, hydrates, solvates,
polymorphs, co-crystals, tautomers, stereoisomers, isotopically
labeled derivatives, or prodrugs thereof.
[0146] In certain embodiments, the compound is selected from the
group consisting of:
##STR00037##
and pharmaceutically acceptable salts, hydrates, solvates,
polymorphs, co-crystals, tautomers, stereoisomers, isotopically
labeled derivatives, and prodrugs thereof.
[0147] As generally defined herein, R.sup.1 is hydrogen, optionally
substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl, optionally substituted aryl, optionally
substituted carbocyclyl, optionally substituted heterocyclyl, or
optionally substituted heteroaryl. In certain embodiments, R.sup.1
is hydrogen. In certain embodiments, R.sup.1 is optionally
substituted alkyl. In certain embodiments, R.sup.1 is optionally
substituted alkenyl. In certain embodiments, R.sup.1 is optionally
substituted alkynyl. In certain embodiments, R.sup.1 is optionally
substituted aryl. In certain embodiments, R.sup.1 is optionally
substituted carbocyclyl. In certain embodiments, R.sup.1 is
optionally substituted heterocyclyl. In certain embodiments,
R.sup.1 is optionally substituted heteroaryl.
[0148] As generally defined herein, R.sup.2 is hydrogen, optionally
substituted alkyl, optionally substituted alkenyl, optionally
substituted alkynyl, optionally substituted aryl, optionally
substituted carbocyclyl, optionally substituted heterocyclyl, or
optionally substituted heteroaryl. In certain embodiments, R.sup.2
is hydrogen. In certain embodiments, R.sup.1 is optionally
substituted alkyl. In certain embodiments, R.sup.2 is optionally
substituted alkenyl. In certain embodiments, R.sup.2 is optionally
substituted alkynyl. In certain embodiments, R.sup.2 is optionally
substituted aryl. In certain embodiments, R.sup.2 is optionally
substituted carbocyclyl. In certain embodiments, R.sup.2 is
optionally substituted heterocyclyl. In certain embodiments,
R.sup.2 is optionally substituted heteroaryl.
[0149] In certain embodiments, at last one of R.sup.1 and R.sup.2
is not hydrogen. In certain embodiments, both R.sup.1 and R.sup.2
are not hydrogen.
[0150] In certain embodiments, R.sup.1 and R.sup.2 are joined
together with the intervening atoms to form optionally substituted
carbocyclyl or optionally substituted heterocyclyl. In certain
embodiments, R.sup.1 and R.sup.2 are joined together with the
intervening atoms to form optionally substituted carbocyclyl. In
certain embodiments, R.sup.1 and R.sup.2 are joined together with
the intervening atoms to form optionally substituted heterocyclyl.
In certain embodiments, R.sup.1 and R.sup.2 are joined together
with the intervening atoms to form optionally substituted
cyclopropyl. In certain embodiments, R.sup.1 and R.sup.2 are joined
together with the intervening atoms to form optionally substituted
cyclobutyl. In certain embodiments, R.sup.1 and R.sup.2 are joined
together with the intervening atoms to form optionally substituted
cyclopentyl. In certain embodiments, R.sup.1 and R.sup.2 are joined
together with the intervening atoms to form optionally substituted
cyclohexyl. In certain embodiments, R.sup.1 and R.sup.2 are joined
together with the intervening atoms to form:
##STR00038##
In certain embodiments, R.sup.1 and R.sup.2 are joined together
with the intervening atoms to form:
##STR00039##
In certain embodiments, R.sup.1 and R.sup.2 are joined together
with the intervening atoms to form:
##STR00040##
In certain embodiments, R.sup.1 and R.sup.2 are joined together
with the intervening atoms to form:
##STR00041##
In certain embodiments, R.sup.1 and R.sup.2 are joined together
with the intervening atoms to form:
##STR00042##
[0151] In certain embodiments, at least one of R.sup.1 and R.sup.2
is optionally substituted phenyl. In certain embodiments, at least
one of R.sup.1 and R.sup.2 is unsubstituted phenyl. In certain
embodiments, R.sup.1 is phenyl and R.sup.2 is hydrogen.
[0152] In certain embodiments, at least one of R.sup.1 and R.sup.2
is halogen (e.g., --Cl, --Br, --I, --F). In certain embodiments,
both R.sup.1 and R.sup.2 are independently halogen. In certain
embodiments, R.sup.1 and R.sup.2 are independently hydrogen or
C.sub.1-4 alkyl; provided that at least one of R.sup.1 and R.sup.2
is not hydrogen. In certain embodiments. R.sup.1 and R.sup.2 are
independently selected from the group consisting of hydrogen,
methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, and
sec-butyl, each of which is unsubstituted; provided that at least
one of R.sup.1 and R.sup.2 is not hydrogen. In certain embodiments,
R.sup.1 and R.sup.2 are both independently selected from the group
consisting of methyl, ethyl, n-propyl, iso-propyl, n-butyl,
iso-butyl, and sec-butyl, each of which is unsubstituted. In
certain embodiments, R.sup.1 and R.sup.2 are both unsubstituted
methyl.
[0153] As generally defined herein, each instance of R.sup.3 is
independently hydrogen, halogen, --CN, --SCN, --NO.sub.2,
--N.sub.3, optionally substituted alkyl, optionally substituted
alkenyl, optionally substituted alkynyl, optionally substituted
carbocyclyl, optionally substituted heterocyclyl, optionally
substituted aryl, optionally substituted heteroaryl, optionally
substituted acyl, optionally substituted sulfinyl, optionally
substituted sulfonyl, --OR.sup.3, --N(R.sup.3b).sub.2, or SR.sup.3;
or optionally two R.sup.3 are joined together with the intervening
atoms to form optionally substituted carbocyclyl or optionally
substituted heterocyclyl. In certain embodiments, at least one
instance of R.sup.3 is hydrogen. In certain embodiments, at least
one instance of R.sup.3 is halogen. In certain embodiments, at
least one instance of R.sup.3 is --CN. In certain embodiments, at
least one instance of R.sup.3 is --SCN. In certain embodiments, at
least one instance of R.sup.3 is --NO.sub.2. In certain
embodiments, at least one instance of R.sup.3 is --N.sub.3. In
certain embodiments, at least one instance of R.sup.3 is optionally
substituted alkyl. In certain embodiments, at least one instance of
R.sup.3 is optionally substituted alkenyl. In certain embodiments,
at least one instance of R.sup.3 is optionally substituted alkynyl.
In certain embodiments, at least one instance of R.sup.3 is
optionally substituted carbocyclyl. In certain embodiments, at
least one instance of R.sup.3 is optionally substituted
heterocyclyl. In certain embodiments, at least one instance of
R.sup.3 is optionally substituted aryl. In certain embodiments, at
least one instance of R.sup.3 is optionally substituted heteroaryl.
In certain embodiments, at least one instance of R.sup.3 is
optionally substituted acyl. In certain embodiments, at least one
instance of R.sup.3 is optionally substituted sulfinyl. In certain
embodiments, at least one instance of R.sup.3 is optionally
substituted sulfonyl. In certain embodiments, at least one instance
of R.sup.3 is --OR.sup.3a. In certain embodiments, at least one
instance of R.sup.3 is --N(R.sup.3b).sub.2. In certain embodiments,
at least one instance of R.sup.3 is --SR.sup.3c. In certain
embodiments, two R.sup.3 are joined together with the intervening
atoms to form optionally substituted carbocyclyl or optionally
substituted heterocyclyl.
[0154] In certain embodiments, at least one instance of R.sup.3 is
halogen (e.g., --Cl, --Br, --I, --F). In certain embodiments, at
least one instance of R.sup.3 is --Cl. In certain embodiments, at
least one instance of R.sup.3 is --I. In certain embodiments, at
least one instance of R.sup.3 is --Br. In certain embodiments, at
least one instance of R.sup.3 is --F.
[0155] In certain embodiments, at least one instance of R.sup.3 is
optionally substituted alkyl. In certain embodiments, at least one
instance of R.sup.3 is haloalkyl. In certain embodiments, at least
one instance of R.sup.3 is trihalomethyl. In certain embodiments,
at least one instance of R.sup.3 is trifluoromethyl (--CF.sub.3).
In certain embodiments, at least one instance of R.sup.3 is
optionally substituted C.sub.1-6 alkyl. In certain embodiments, at
least one instance of R.sup.3 is optionally substituted C.sub.1-3
alkyl. In certain embodiments, at least one instance of R.sup.3 is
unsubstituted C.sub.1-3 alkyl. In certain embodiments, at least one
instance of R.sup.1 is selected from the group consisting of
methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, and
sec-butyl. In certain embodiments, at least one instance of R.sup.3
is methyl.
[0156] In certain embodiments, at least one instance of R.sup.3 is
--OR.sup.3a. In certain embodiments, at least one instance of
R.sup.3 is --O--C.sub.1-6 alkyl. In certain embodiments, at least
one instance of R.sup.3 is --O--C.sub.1-3 alkyl. In certain
embodiments, at least one instance of R.sup.3 is --OCH.sub.3. In
certain embodiments, at least one instance of R.sup.3 is
--O-Ph.
[0157] In certain embodiments, at least one instance of R.sup.3 is
optionally substituted phenyl. In certain embodiments, at least one
instance of R.sup.3 is unsubstituted phenyl.
[0158] In certain embodiments, at least one instance of R.sup.3 is
an electron-withdrawing group. In certain embodiments, at least two
instances of R.sup.3 are electron-withdrawing groups. In certain
embodiments, at least one instance of R.sup.3 is selected from the
group consisting of halogen, optionally substituted acyl,
optionally substituted sulfinyl, optionally substituted sulfonyl,
and haloalkyl. In certain embodiments, at least one instance of
R.sup.3 is selected from the group consisting of halogen,
optionally substituted acyl, and haloalkyl. In certain embodiments,
at least two instances of R.sup.3 are selected from the group
consisting of halogen, optionally substituted acyl, optionally
substituted sulfinyl, optionally substituted sulfonyl, and
haloalkyl. In certain embodiments, at least two instances of
R.sup.3 are --F, --Cl, --Br, or --I. In certain embodiments, at
least two instances of R.sup.1 are --Cl. In certain embodiments,
one instance of R.sup.3 is --Cl, and another instance of R.sup.3 is
--F. In certain embodiments, one instance of R.sup.3 is --Cl, and
another instance of R.sup.3 is --CF.sub.3. In certain embodiments,
one instance of R.sup.3 is --Cl, and another instance of R.sup.3 is
methyl. In certain embodiments, one instance of R.sup.3 is --Cl,
and another instance of R.sup.3 is --OCH.sub.3.
[0159] As generally defined herein, each instance of R.sup.3a is
independently hydrogen, optionally substituted alkyl, optionally
substituted alkenyl, optionally substituted alkynyl, optionally
substituted carbocyclyl, optionally substituted heterocyclyl,
optionally substituted aryl, optionally substituted heteroaryl,
optionally substituted acyl, or an oxygen protecting group.
[0160] As generally defined herein, each instance of R.sup.3b is
independently hydrogen, optionally substituted alkyl, optionally
substituted alkenyl, optionally substituted alkynyl, optionally
substituted carbocyclyl, optionally substituted heterocyclyl,
optionally substituted aryl, optionally substituted heteroaryl,
optionally substituted acyl, or a nitrogen protecting group; or
optionally two R.sup.3b are joined together with the intervening
atoms to form optionally substituted heterocyclyl or optionally
substituted heteroaryl.
[0161] As generally defined herein, each instance of R.sup.3c is
independently hydrogen, optionally substituted alkyl, optionally
substituted alkenyl, optionally substituted alkynyl, optionally
substituted carbocyclyl, optionally substituted heterocyclyl,
optionally substituted aryl, optionally substituted heteroaryl,
optionally substituted acyl, or a sulfur protecting group.
[0162] As generally defined herein, n is 0, 1, 2, 3, 4, or 5. In
certain embodiments, n is 0. In certain embodiments, n is 1. In
certain embodiments, n is 2. In certain embodiments, n is 3. In
certain embodiments, n is 4. In certain embodiments, n is 5.
[0163] In certain embodiments, Ring A is of one of the following
formulae:
##STR00043##
[0164] In certain embodiments, Ring A is of one of the following
formulae:
##STR00044##
[0165] As generally defined herein, Y is a bond, optionally
substituted alkylene, --O--, --NR.sup.N--, or --S--. In certain
embodiments, Y is a bond. In certain embodiments, Y is optionally
substituted alkylene. In certain embodiments, Y is --O--. In
certain embodiments, Y is --NR.sup.N--. In certain embodiments, Y
is --S--.
[0166] As generally defined herein, R.sup.4 is optionally
substituted carbocyclyl, optionally substituted heterocyclyl,
optionally substituted aryl, or optionally substituted heteroaryl.
In certain embodiments, R.sup.4 is optionally substituted
carbocyclyl. In certain embodiments, R.sup.4 is optionally
substituted heterocyclyl. In certain embodiments, R.sup.4 is
optionally substituted aryl. In certain embodiments, R.sup.4 is or
optionally substituted heteroaryl. In certain embodiments. R.sup.4
is optionally substituted phenyl. In certain embodiments, R.sup.4
is unsubstituted phenyl.
[0167] As generally defined herein, each instance of R.sup.N is
hydrogen, optionally substituted alkyl, or optionally substituted
acyl, or a nitrogen protecting group. In certain embodiments, at
least one instance of R.sup.N is hydrogen. In certain embodiments,
at least one instance of R.sup.N is optionally substituted alkyl.
In certain embodiments, at least one instance of R.sup.N is
optionally substituted acyl. In certain embodiments, at least one
instance of R.sup.N is a nitrogen protecting group. In certain
embodiments, each R.sup.N is hydrogen.
Pharmaceutical Compositions, Kits, Administration, Methods of Use,
and Uses
[0168] Provided herein are pharmaceutical compositions comprising a
compound provided herein (e.g., a compound of Formula (I), (H), or
(III)), or a pharmaceutically acceptable salt, hydrate, solvate,
polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled
derivative, or prodrug thereof, and a pharmaceutically acceptable
carrier or excipient. In certain embodiments, the compound
described herein is provided in an effective amount in the
pharmaceutical composition. In certain embodiments, the effective
amount is a therapeutically effective amount. In certain
embodiments, the effective amount is a prophylactically effective
amount.
[0169] Pharmaceutical compositions described herein can be prepared
by any method known in the art of pharmacology. In general, such
preparatory methods include bringing the compound described herein
(i.e., the "active ingredient") into association with a carrier or
excipient, and/or one or more other accessory ingredients, and
then, if necessary and/or desirable, shaping, and/or packaging the
product into a desired single- or multi-dose unit.
[0170] Pharmaceutical compositions can be prepared, packaged,
and/or sold in bulk, as a single unit dose, and/or as a plurality
of single unit doses. A "unit dose" is a discrete amount of the
pharmaceutical composition comprising a predetermined amount of the
active ingredient. The amount of the active ingredient is generally
equal to the dosage of the active ingredient which would be
administered to a subject and/or a convenient fraction of such a
dosage, such as one-half or one-third of such a dosage.
[0171] Relative amounts of the active ingredient, the
pharmaceutically acceptable excipient, and/or any additional
ingredients in a pharmaceutical composition described herein will
vary, depending upon the identity, size, and/or condition of the
subject treated and further depending upon the route by which the
composition is to be administered. The composition may comprise
between 0.1% and 100% (w/w) active ingredient.
[0172] Pharmaceutically acceptable excipients used in the
manufacture of provided pharmaceutical compositions include inert
diluents, dispersing and/or granulating agents, surface active
agents and/or emulsifiers, disintegrating agents, binding agents,
preservatives, buffering agents, lubricating agents, and/or oils.
Excipients such as cocoa butter and suppository waxes, coloring
agents, coating agents, sweetening, flavoring, and perfuming agents
may also be present in the composition.
[0173] Exemplary diluents include calcium carbonate, sodium
carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate,
calcium hydrogen phosphate, sodium phosphate lactose, sucrose,
cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol,
inositol, sodium chloride, dry starch, cornstarch, powdered sugar,
and mixtures thereof.
[0174] Exemplary granulating and/or dispersing agents include
potato starch, corn starch, tapioca starch, sodium starch
glycolate, clays, alginic acid, guar gum, citrus pulp, agar,
bentonite, cellulose, and wood products, natural sponge,
cation-exchange resins, calcium carbonate, silicates, sodium
carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone),
sodium carboxymethyl starch (sodium starch glycolate),
carboxymethyl cellulose, cross-linked sodium carboxymethyl
cellulose (croscarmellose), methylcellulose, pregelatinized starch
(starch 1500), microcrystalline starch, water insoluble starch,
calcium carboxymethyl cellulose, magnesium aluminum silicate
(Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and
mixtures thereof.
[0175] Exemplary surface active agents and/or emulsifiers include
natural emulsifiers (e.g., acacia, agar, alginic acid, sodium
alginate, tragacanth, chondrux, cholesterol, xanthan, pectin,
gelatin, egg yolk, casein, wool fat, cholesterol, wax, and
lecithin), colloidal clays (e.g., bentonite (aluminum silicate) and
Veegum (magnesium aluminum silicate)), long chain amino acid
derivatives, high molecular weight alcohols (e.g., stearyl alcohol,
cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene
glycol distearate, glyceryl monostearate, and propylene glycol
monostearate, polyvinyl alcohol), carbomers (e.g., carboxy
polymethylene, polyacrylic acid, acrylic acid polymer, and
carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g.,
carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl
cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,
methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene
sorbitan monolaurate (Tween.RTM. 20), polyoxyethylene sorbitan
(Tween.RTM. 60), polyoxyethylene sorbitan monooleate (Tween.RTM.
80), sorbitan monopalmitate (Span.RTM. 40), sorbitan monostearate
(Span.RTM. 60), sorbitan tristearate (Span.RTM. 65), glyceryl
monooleate, sorbitan monooleate (Span.RTM. 80), polyoxyethylene
esters (e.g., polyoxyethylene monostearate (Myrj.RTM. 45),
polyoxyethylene hydrogenated castor oil, polyethoxylated castor
oil, polyoxymethylene stearate, and Solutol.RTM.), sucrose fatty
acid esters, polyethylene glycol fatty acid esters (e.g.,
Cremophor.RTM.), polyoxyethylene ethers, (e.g., polyoxyethylene
lauryl ether (Brij.RTM. 30)), poly(vinyl-pyrrolidone), diethylene
glycol monolaurate, triethanolamine oleate, sodium oleate,
potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium
lauryl sulfate, Pluronic.RTM. F-68, poloxamer P-188, cetrimonium
bromide, cetylpyridinium chloride, benzalkonium chloride, docusate
sodium, and/or mixtures thereof.
[0176] Exemplary binding agents include starch (e.g., cornstarch
and starch paste), gelatin, sugars (e.g., sucrose, glucose,
dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.),
natural and synthetic gums (e.g., acacia, sodium alginate, extract
of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks,
carboxymethylcellulose, methylcellulose, ethylcellulose,
hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl
methylcellulose, microcrystalline cellulose, cellulose acetate,
poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum.RTM.),
and larch arabogalactan), alginates, polyethylene oxide,
polyethylene glycol, inorganic calcium salts, silicic acid,
polymethacrylates, waxes, water, alcohol, and/or mixtures
thereof.
[0177] Exemplary preservatives include antioxidants, chelating
agents, antimicrobial preservatives, antifungal preservatives,
antiprotozoan preservatives, alcohol preservatives, acidic
preservatives, and other preservatives. In certain embodiments, the
preservative is an antioxidant. In other embodiments, the
preservative is a chelating agent.
[0178] Exemplary antioxidants include alpha tocopherol, ascorbic
acid, acorbyl palmitate, butylated hydroxyanisole, butylated
hydroxytoluene, monothioglycerol, potassium metabisulfite,
propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite,
sodium metabisulfite, and sodium sulfite.
[0179] Exemplary chelating agents include
ethylenediaminetetraacetic acid (EDTA) and salts and hydrates
thereof (e.g., sodium edetate, disodium edetate, trisodium edetate,
calcium disodium edetate, dipotassium edetate, and the like),
citric acid and salts and hydrates thereof (e.g., citric acid
monohydrate), fumaric acid and salts and hydrates thereof, malic
acid and salts and hydrates thereof, phosphoric acid and salts and
hydrates thereof, and tartaric acid and salts and hydrates thereof.
Exemplary antimicrobial preservatives include benzalkonium
chloride, benzethonium chloride, benzyl alcohol, bronopol,
cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol,
chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin,
hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol,
phenylmercuric nitrate, propylene glycol, and thimerosal.
[0180] Exemplary antifungal preservatives include butyl paraben,
methyl paraben, ethyl paraben, propyl paraben, benzoic acid,
hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium
benzoate, sodium propionate, and sorbic acid.
[0181] Exemplary alcohol preservatives include ethanol,
polyethylene glycol, phenol, phenolic compounds, bisphenol,
chlorobutanol, hydroxybenzoate, and phenylethyl alcohol.
[0182] Exemplary acidic preservatives include vitamin A, vitamin C,
vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic
acid, ascorbic acid, sorbic acid, and phytic acid.
[0183] Other preservatives include tocopherol, tocopherol acetate,
deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA),
butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl
sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium
bisulfite, sodium metabisulfite, potassium sulfite, potassium
metabisulfite, Glydant.RTM. Plus. Phenonip.RTM., methylparaben,
Germall.RTM. 115, Germaben.RTM. II, Neolone.RTM., Kathon.RTM., and
Euxyl.RTM..
[0184] Exemplary buffering agents include citrate buffer solutions,
acetate buffer solutions, phosphate buffer solutions, ammonium
chloride, calcium carbonate, calcium chloride, calcium citrate,
calcium glubionate, calcium gluceptate, calcium gluconate,
D-gluconic acid, calcium glycerophosphate, calcium lactate,
propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium
phosphate, phosphoric acid, tribasic calcium phosphate, calcium
hydroxide phosphate, potassium acetate, potassium chloride,
potassium gluconate, potassium mixtures, dibasic potassium
phosphate, monobasic potassium phosphate, potassium phosphate
mixtures, sodium acetate, sodium bicarbonate, sodium chloride,
sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic
sodium phosphate, sodium phosphate mixtures, tromethamine,
magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free
water, isotonic saline, Ringer's solution, ethyl alcohol, and
mixtures thereof.
[0185] Exemplary lubricating agents include magnesium stearate,
calcium stearate, stearic acid, silica, talc, malt, glyceryl
behanate, hydrogenated vegetable oils, polyethylene glycol, sodium
benzoate, sodium acetate, sodium chloride, leucine, magnesium
lauryl sulfate, sodium lauryl sulfate, and mixtures thereof.
[0186] Exemplary natural oils include almond, apricot kernel,
avocado, babassu, bergamot, black current seed, borage, cade,
camomile, canola, caraway, carnauba, castor, cinnamon, cocoa
butter, coconut, cod liver, coffee, corn, cotton seed, emu,
eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd,
grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui
nut, lavandin, lavender, lemon, litsea cubeba, macademia nut,
mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange,
orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed,
pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood,
sasquana, savoury, sea buckthorn, sesame, shea butter, silicone,
soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut,
and wheat germ oils. Exemplary synthetic oils include, but are not
limited to, butyl stearate, caprylic triglyceride, capric
triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360,
isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol,
silicone oil, and mixtures thereof.
[0187] Liquid dosage forms for oral and parenteral administration
include pharmaceutically acceptable emulsions, microemulsions,
solutions, suspensions, syrups and elixirs. In addition to the
active ingredients, the liquid dosage forms may comprise inert
diluents commonly used in the art such as, for example, water or
other solvents, solubilizing agents and emulsifiers such as ethyl
alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl
alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,
dimethylformamide, oils (e.g., cottonseed, groundnut, corn, germ,
olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl
alcohol, polyethylene glycols and fatty acid esters of sorbitan,
and mixtures thereof. Besides inert diluents, the oral compositions
can include adjuvants such as wetting agents, emulsifying and
suspending agents, sweetening, flavoring, and perfuming agents. In
certain embodiments for parenteral administration, the conjugates
described herein are mixed with solubilizing agents such as
Cremophor.RTM., alcohols, oils, modified oils, glycols,
polysorbates, cyclodextrins, polymers, and mixtures thereof.
[0188] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions can be formulated according to
the known art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation can be a
sterile injectable solution, suspension, or emulsion in a nontoxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that can be employed are water, Ringer's solution, U.S.P.,
and isotonic sodium chloride solution. In addition, sterile, fixed
oils are conventionally employed as a solvent or suspending medium.
For this purpose any bland fixed oil can be employed including
synthetic mono- or di-glycerides. In addition, fatty acids such as
oleic acid are used in the preparation of injectables.
[0189] The injectable formulations can be sterilized, for example,
by filtration through a bacterial-retaining filter, or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved or dispersed in sterile water
or other sterile injectable medium prior to use.
[0190] In order to prolong the effect of a drug, it is often
desirable to slow the absorption of the drug from subcutaneous or
intramuscular injection. This can be accomplished by the use of a
liquid suspension of crystalline or amorphous material with poor
water solubility. The rate of absorption of the drug then depends
upon its rate of dissolution, which, in turn, may depend upon
crystal size and crystalline form. Alternatively, delayed
absorption of a parenterally administered drug form may be
accomplished by dissolving or suspending the drug in an oil
vehicle.
[0191] Compositions for rectal or vaginal administration are
typically suppositories which can be prepared by mixing the
conjugates described herein with suitable non-irritating excipients
or carriers such as cocoa butter, polyethylene glycol, or a
suppository wax which are solid at ambient temperature but liquid
at body temperature and therefore melt in the rectum or vaginal
cavity and release the active ingredient.
[0192] Solid dosage forms for oral administration include capsules,
tablets, pills, powders, and granules. In such solid dosage forms,
the active ingredient is mixed with at least one inert,
pharmaceutically acceptable excipient or carrier such as sodium
citrate or dicalcium phosphate and/or (a) fillers or extenders such
as starches, lactose, sucrose, glucose, mannitol, and silicic acid,
(b) binders such as, for example, carboxymethylcellulose,
alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia,
(c) humectants such as glycerol, (d) disintegrating agents such as
agar, calcium carbonate, potato or tapioca starch, alginic acid,
certain silicates, and sodium carbonate, (e) solution retarding
agents such as paraffin. (f) absorption accelerators such as
quaternary ammonium compounds, (g) wetting agents such as, for
example, cetyl alcohol and glycerol monostearate, (h) absorbents
such as kaolin and bentonite clay, and (i) lubricants such as talc,
calcium stearate, magnesium stearate, solid polyethylene glycols,
sodium lauryl sulfate, and mixtures thereof. In the case of
capsules, tablets, and pills, the dosage form may include a
buffering agent.
[0193] Solid compositions of a similar type can be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycols and the like. The solid dosage forms of
tablets, dragees, capsules, pills, and granules can be prepared
with coatings and shells such as enteric coatings and other
coatings well known in the art of pharmacology. They may optionally
comprise opacifying agents and can be of a composition that they
release the active ingredient(s) only, or preferentially, in a
certain part of the intestinal tract, optionally, in a delayed
manner. Examples of encapsulating compositions which can be used
include polymeric substances and waxes. Solid compositions of a
similar type can be employed as fillers in soft and hard-filled
gelatin capsules using such excipients as lactose or milk sugar as
well as high molecular weight polethylene glycols and the like.
[0194] The active ingredient can be in a micro-encapsulated form
with one or more excipients as noted above. The solid dosage forms
of tablets, dragees, capsules, pills, and granules can be prepared
with coatings and shells such as enteric coatings, release
controlling coatings, and other coatings well known in the
pharmaceutical formulating art. In such solid dosage forms the
active ingredient can be admixed with at least one inert diluent
such as sucrose, lactose, or starch. Such dosage forms may
comprise, as is normal practice, additional substances other than
inert diluents, e.g., tableting lubricants and other tableting aids
such a magnesium stearate and microcrystalline cellulose. In the
case of capsules, tablets and pills, the dosage forms may comprise
buffering agents. They may optionally comprise opacifying agents
and can be of a composition that they release the active
ingredient(s) only, or preferentially, in a certain part of the
intestinal tract, optionally, in a delayed manner. Examples of
encapsulating agents which can be used include polymeric substances
and waxes.
[0195] Dosage forms for topical and/or transdermal administration
of a compound described herein may include ointments, pastes,
creams, lotions, gels, powders, solutions, sprays, inhalants,
and/or patches. Generally, the active ingredient is admixed under
sterile conditions with a pharmaceutically acceptable carrier or
excipient and/or any needed preservatives and/or buffers as can be
required. Additionally, the present disclosure contemplates the use
of transdermal patches, which often have the added advantage of
providing controlled delivery of an active ingredient to the body.
Such dosage forms can be prepared, for example, by dissolving
and/or dispensing the active ingredient in the proper medium.
Alternatively or additionally, the rate can be controlled by either
providing a rate controlling membrane and/or by dispersing the
active ingredient in a polymer matrix and/or gel.
[0196] Suitable devices for use in delivering intradermal
pharmaceutical compositions described herein include short needle
devices. Intradermal compositions can be administered by devices
which limit the effective penetration length of a needle into the
skin. Alternatively or additionally, conventional syringes can be
used in the classical mantoux method of intradermal administration.
Jet injection devices which deliver liquid formulations to the
dermis via a liquid jet injector and/or via a needle which pierces
the stratum corneum and produces a jet which reaches the dermis are
suitable. Ballistic powder/particle delivery devices which use
compressed gas to accelerate the compound in powder form through
the outer layers of the skin to the dermis are suitable.
[0197] Formulations suitable for topical administration include,
but are not limited to, liquid and/or semi-liquid preparations such
as liniments, lotions, oil-in-water and/or water-in-oil emulsions
such as creams, ointments, and/or pastes, and/or solutions and/or
suspensions. Topically administrable formulations may, for example,
comprise from about 1% to about 10% (w/w) active ingredient,
although the concentration of the active ingredient can be as high
as the solubility limit of the active ingredient in the solvent.
Formulations for topical administration may further comprise one or
more of the additional ingredients described herein.
[0198] A pharmaceutical composition described herein can be
prepared, packaged, and/or sold in a formulation suitable for
pulmonary administration via the buccal cavity. Such a formulation
may comprise dry particles which comprise the active ingredient and
which have a diameter in the range from about 0.5 to about 7
nanometers, or from about 1 to about 6 nanometers. Such
compositions are conveniently in the form of dry powders for
administration using a device comprising a dry powder reservoir to
which a stream of propellant can be directed to disperse the powder
and/or using a self-propelling solvent/powder dispensing container
such as a device comprising the active ingredient dissolved and/or
suspended in a low-boiling propellant in a sealed container. Such
powders comprise particles wherein at least 98% of the particles by
weight have a diameter greater than 0.5 nanometers and at least 95%
of the particles by number have a diameter less than 7 nanometers.
Alternatively, at least 95% of the particles by weight have a
diameter greater than 1 nanometer and at least 90% of the particles
by number have a diameter less than 6 nanometers. Dry powder
compositions may include a solid fine powder diluent such as sugar
and are conveniently provided in a unit dose form.
[0199] Low boiling propellants generally include liquid propellants
having a boiling point of below 65.degree. F. at atmospheric
pressure. Generally the propellant may constitute 50 to 99.9% (w/w)
of the composition, and the active ingredient may constitute 0.1 to
20% (w/w) of the composition. The propellant may further comprise
additional ingredients such as a liquid non-ionic and/or solid
anionic surfactant and/or a solid diluent (which may have a
particle size of the same order as particles comprising the active
ingredient).
[0200] Pharmaceutical compositions described herein formulated for
pulmonary delivery may provide the active ingredient in the form of
droplets of a solution and/or suspension. Such formulations can be
prepared, packaged, and/or sold as aqueous and/or dilute alcoholic
solutions and/or suspensions, optionally sterile, comprising the
active ingredient, and may conveniently be administered using any
nebulization and/or atomization device. Such formulations may
further comprise one or more additional ingredients including, but
not limited to, a flavoring agent such as saccharin sodium, a
volatile oil, a buffering agent, a surface active agent, and/or a
preservative such as methylhydroxybenzoate. The droplets provided
by this route of administration may have an average diameter in the
range from about 0.1 to about 200 nanometers.
[0201] Formulations described herein as being useful for pulmonary
delivery are useful for intranasal delivery of a pharmaceutical
composition described herein. Another formulation suitable for
intranasal administration is a coarse powder comprising the active
ingredient and having an average particle from about 0.2 to 500
micrometers. Such a formulation is administered by rapid inhalation
through the nasal passage from a container of the powder held close
to the nares.
[0202] Formulations for nasal administration may, for example,
comprise from about as little as 0.1% (w/w) to as much as 100%
(w/w) of the active ingredient, and may comprise one or more of the
additional ingredients described herein. A pharmaceutical
composition described herein can be prepared, packaged, and/or sold
in a formulation for buccal administration. Such formulations may,
for example, be in the form of tablets and/or lozenges made using
conventional methods, and may contain, for example, 0.1 to 20%
(w/w) active ingredient, the balance comprising an orally
dissolvable and/or degradable composition and, optionally, one or
more of the additional ingredients described herein. Alternately,
formulations for buccal administration may comprise a powder and/or
an aerosolized and/or atomized solution and/or suspension
comprising the active ingredient. Such powdered, aerosolized,
and/or aerosolized formulations, when dispersed, may have an
average particle and/or droplet size in the range from about 0.1 to
about 200 nanometers, and may further comprise one or more of the
additional ingredients described herein.
[0203] A pharmaceutical composition described herein can be
prepared, packaged, and/or sold in a formulation for ophthalmic
administration. Such formulations may, for example, be in the form
of eye drops including, for example, a 0.1-1.0% (w/w) solution
and/or suspension of the active ingredient in an aqueous or oily
liquid carrier or excipient. Such drops may further comprise
buffering agents, salts, and/or one or more other of the additional
ingredients described herein. Other opthalmically-administrable
formulations which are useful include those which comprise the
active ingredient in microcrystalline form and/or in a liposomal
preparation. Ear drops and/or eye drops are also contemplated as
being within the scope of this disclosure.
[0204] Although the descriptions of pharmaceutical compositions
provided herein are principally directed to pharmaceutical
compositions which are suitable for administration to humans, it
will be understood by the skilled artisan that such compositions
are generally suitable for administration to animals of all sorts.
Modification of pharmaceutical compositions suitable for
administration to humans in order to render the compositions
suitable for administration to various animals is well understood,
and the ordinarily skilled veterinary pharmacologist can design
and/or perform such modification with ordinary experimentation.
[0205] Compounds provided herein are typically formulated in dosage
unit form for ease of administration and uniformity of dosage. It
will be understood, however, that the total daily usage of the
compositions described herein will be decided by a physician within
the scope of sound medical judgment. The specific therapeutically
effective dose level for any particular subject or organism will
depend upon a variety of factors including the disease being
treated and the severity of the disorder; the activity of the
specific active ingredient employed; the specific composition
employed; the age, body weight, general health, sex, and diet of
the subject; the time of administration, route of administration,
and rate of excretion of the specific active ingredient employed;
the duration of the treatment; drugs used in combination or
coincidental with the specific active ingredient employed; and like
factors well known in the medical arts.
[0206] The compounds and compositions provided herein can be
administered by any route, including enteral (e.g., oral),
parenteral, intravenous, intramuscular, intra-arterial,
intramedullary, intrathecal, subcutaneous, intraventricular,
transdermal, interdermal, rectal, intravaginal, intraperitoneal,
topical (as by powders, ointments, creams, and/or drops), mucosal,
nasal, bucal, sublingual; by intratracheal instillation, bronchial
instillation, and/or inhalation; and/or as an oral spray, nasal
spray, and/or aerosol. Specifically contemplated routes are oral
administration, intravenous administration (e.g., systemic
intravenous injection), regional administration via blood and/or
lymph supply, and/or direct administration to an affected site. In
general, the most appropriate route of administration will depend
upon a variety of factors including the nature of the agent (e.g.,
its stability in the environment of the gastrointestinal tract),
and/or the condition of the subject (e.g., whether the subject is
able to tolerate oral administration). In certain embodiments, the
compound or pharmaceutical composition described herein is suitable
for topical administration to the eye of a subject.
[0207] The exact amount of a compound required to achieve an
effective amount will vary from subject to subject, depending, for
example, on species, age, and general condition of a subject,
severity of the side effects or disorder, identity of the
particular compound, mode of administration, and the like. An
effective amount may be included in a single dose (e.g., single
oral dose) or multiple doses (e.g., multiple oral doses). In
certain embodiments, when multiple doses are administered to a
subject or applied to a tissue or cell, any two doses of the
multiple doses include different or substantially the same amounts
of a compound described herein. In certain embodiments, when
multiple doses are administered to a subject or applied to a tissue
or cell, the frequency of administering the multiple doses to the
subject or applying the multiple doses to the tissue or cell is
three doses a day, two doses a day, one dose a day, one dose every
other day, one dose every third day, one dose every week, one dose
every two weeks, one dose every three weeks, or one dose every four
weeks. In certain embodiments, the frequency of administering the
multiple doses to the subject or applying the multiple doses to the
tissue or cell is one dose per day. In certain embodiments, the
frequency of administering the multiple doses to the subject or
applying the multiple doses to the tissue or cell is two doses per
day. In certain embodiments, the frequency of administering the
multiple doses to the subject or applying the multiple doses to the
tissue or cell is three doses per day. In certain embodiments, when
multiple doses are administered to a subject or applied to a tissue
or cell, the duration between the first dose and last dose of the
multiple doses is one day, two days, four days, one week, two
weeks, three weeks, one month, two months, three months, four
months, six months, nine months, one year, two years, three years,
four years, five years, seven years, ten years, fifteen years,
twenty years, or the lifetime of the subject, tissue, or cell. In
certain embodiments, the duration between the first dose and last
dose of the multiple doses is three months, six months, or one
year. In certain embodiments, the duration between the first dose
and last dose of the multiple doses is the lifetime of the subject,
tissue, or cell. In certain embodiments, a dose (e.g., a single
dose, or any dose of multiple doses) described herein includes
independently between 0.1 .mu.g and 1 .mu.g, between 0.001 mg and
0.01 mg, between 0.01 mg and 0.1 mg, between 0.1 mg and 1 mg,
between 1 mg and 3 mg, between 3 mg and 10 mg, between 10 mg and 30
mg, between 30 mg and 100 mg, between 100 mg and 300 mg, between
300 mg and 1,000 mg, or between 1 g and 10 g, inclusive, of a
compound described herein. In certain embodiments, a dose described
herein includes independently between 1 mg and 3 mg, inclusive, of
a compound described herein. In certain embodiments, a dose
described herein includes independently between 3 mg and 10 mg,
inclusive, of a compound described herein. In certain embodiments,
a dose described herein includes independently between 10 mg and 30
mg, inclusive, of a compound described herein. In certain
embodiments, a dose described herein includes independently between
30 mg and 100 mg, inclusive, of a compound described herein.
[0208] Dose ranges as described herein provide guidance for the
administration of provided pharmaceutical compositions to an adult.
The amount to be administered to, for example, a child or an
adolescent can be determined by a medical practitioner or person
skilled in the art and can be lower or the same as that
administered to an adult.
[0209] A compound or composition, as described herein, can be
administered in combination with one or more additional agents
(e.g., therapeutically and/or prophylactically active agents). The
compounds or compositions can be administered in combination with
additional agents that improve their activity (e.g., activity
(e.g., potency and/or efficacy) in treating a disease in a subject
in need thereof, in preventing a disease in a subject in need
thereof, in reducing the risk to develop a disease in a subject in
need thereof), improve bioavailability, improve safety, reduce drug
resistance, reduce and/or modify metabolism, inhibit excretion,
and/or modify distribution in a subject or cell. It will also be
appreciated that the therapy employed may achieve a desired effect
for the same disorder, and/or it may achieve different effects. In
certain embodiments, a pharmaceutical composition described herein
including a compound described herein and an additional
pharmaceutical agent shows a synergistic effect that is absent in a
pharmaceutical composition including one of the compound and the
additional pharmaceutical agent, but not both.
[0210] The compound or composition can be administered concurrently
with, prior to, or subsequent to one or more additional
pharmaceutical agents, which may be useful as, e.g., combination
therapies. Pharmaceutical agents include therapeutically active
agents. Pharmaceutical agents also include prophylactically active
agents. Pharmaceutical agents include small organic molecules such
as drug compounds (e.g., compounds approved for human or veterinary
use by the U.S. Food and Drug Administration as provided in the
Code of Federal Regulations (CFR)), peptides, proteins,
carbohydrates, monosaccharides, oligosaccharides, polysaccharides,
nucleoproteins, mucoproteins, lipoproteins, synthetic polypeptides
or proteins, small molecules linked to proteins, glycoproteins,
steroids, nucleic acids, DNAs, RNAs, nucleotides, nucleosides,
oligonucleotides, antisense oligonucleotides, lipids, hormones,
vitamins, and cells. In certain embodiments, the additional
pharmaceutical agent is a pharmaceutical agent useful for treating
and/or preventing a disease. Each additional pharmaceutical agent
may be administered at a dose and/or on a time schedule determined
for that pharmaceutical agent. The additional pharmaceutical agents
may also be administered together with each other and/or with the
compound or composition described herein in a single dose or
administered separately in different doses. The particular
combination to employ in a regimen will take into account
compatibility of the compound described herein with the additional
pharmaceutical agent(s) and/or the desired therapeutic and/or
prophylactic effect to be achieved. In general, it is expected that
the additional pharmaceutical agent(s) in combination be utilized
at levels that do not exceed the levels at which they are utilized
individually. In some embodiments, the levels utilized in
combination will be lower than those utilized individually.
[0211] The additional pharmaceutical agents include, but are not
limited to, anti-proliferative agents, anti-cancer agents,
anti-angiogenesis agents, anti-inflammatory agents,
immunosuppressants, anti-bacterial agents, anti-viral agents,
cardiovascular agents, cholesterol-lowering agents, anti-diabetic
agents, anti-allergic agents, contraceptive agents, and
pain-relieving agents. In certain embodiments, the additional
pharmaceutical agent is an anti-infective agent. In certain
embodiments, the additional agent is an antifungal agent. In
certain embodiments, the additional agent is an azole antifungal
agent. Examples of anti-infective agents, including azole
antifungal agents, are provided herein.
[0212] Also encompassed by the disclosure are kits (e.g.,
pharmaceutical packs). The kits provided may comprise a
pharmaceutical composition or compound described herein and a
container (e.g., a vial, ampule, bottle, syringe, and/or dispenser
package, or other suitable container). In some embodiments,
provided kits may optionally further include a second container
comprising a pharmaceutical excipient for dilution or suspension of
a pharmaceutical composition or compound described herein. In some
embodiments, the pharmaceutical composition or compound described
herein provided in the first container and the second container are
combined to form one unit dosage form.
[0213] Thus, in one aspect, provided are kits including a first
container comprising a compound or pharmaceutical composition
described herein. In certain embodiments, the kits are useful for
treating a disease in a subject in need thereof. In certain
embodiments, the kits are useful for preventing a disease in a
subject in need thereof. In certain embodiments, the kits are
useful for reducing the risk of developing a disease in a subject
in need thereof. In certain embodiments, a kit described herein
further includes instructions for using the kit. A kit described
herein may also include information as required by a regulatory
agency such as the U.S. Food and Drug Administration (FDA). In
certain embodiments, the information included in the kits is
prescribing information. In certain embodiments, the kits and
instructions provide for treating a disease in a subject in need
thereof. In certain embodiments, the kits and instructions provide
for preventing a disease in a subject in need thereof. In certain
embodiments, the kits and instructions provide for reducing the
risk of developing a disease in a subject in need thereof. A kit
described herein may include one or more additional pharmaceutical
agents described herein as a separate composition.
Methods of Treatment and Use
[0214] Provided herein is a method for treating a disease in a
subject in need thereof, the method comprising administering to the
subject a compound provided herein (e.g., a compound of Formula
(I), (II), or (III)), or a pharmaceutically acceptable salt,
hydrate, solvate, polymorph, co-crystal, tautomer, stereoisomer,
isotopically labeled derivative, or prodrug thereof, or a
pharmaceutical composition thereof. Also provided are uses of a
compound provided herein (e.g., a compound of Formula (I), (II), or
(II)), or a pharmaceutically acceptable salt, hydrate, solvate,
polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled
derivative, or prodrug thereof, or a pharmaceutical composition
thereof, for the manufacture of a medicament for treating a disease
in a subject.
[0215] In certain embodiments, the disease is an infectious
disease. In certain embodiments, the disease is a microbial
infection. In certain embodiments, the disease is a fungal
infection.
[0216] In certain embodiments, the infectious disease is caused by
a fungus belonging to a genus selected from the group consisting of
Aspergillus, Blastomyces, Candida, Coccidioides, Cryptococcus,
Fusarium. Histoplasma, Malassezia, Microsporum, Mucor,
Paracoccidioide, Pneumocvstis, Pseudallescheria, Rhizopus,
Scedosporium, Sporothrix, Siachybotrys, Saccharomyces,
Trichophyton, and Trichosporonphylum; or caused by a fungus
belonging to a phylum selected from the group consisting of
Ascomycota, Basidiomyvcota, Chytridiomycota, Glomeromycota, and
Zygomycota.
[0217] In certain embodiments, the infectious disease is caused by
a fungus selected from the group consisting of C. albicans, C.
glabrata, C. krusii, C. rugosa, C. parapsilosis, C. tropicalis, C.
dubliniensis, C. lusitaniae, C. guilliermondii, C. famata, C.
kefyr, C. pelliculosa, C. lipolytica, C. inconspicua, C. sake, C.
lambica, C. norvegensis, C. zeylanoides. Aspergillus terreus, A.
clavatus, A. fumigatus, A. niger, A. flavus, Saccharomyces
cerevisiae, Blastomyces dermatitidis. Coccidioides immitis,
Coccidioides posadasii, Cryptococcus neoformans, C. gattii, C.
albidus, C. laurentii, C. uniguttulas, E. floccosum, Fusarium
graminearum, Fusarium oxysporum, fsp. cubense, a member of the
Fusarium solani complex, Fusarium oxysporum, Fusarium
verticillioides, Fusarium proliferatum, Histoplasma capsulatum,
Malassezia furfur, M. circinelloides, Paracoccidioides
brasiliensis, Penicillium marneffei, Pichia anomala, Pichia
guilliermondi, Pneumocystis carinii, Pneumocystis jirovecii,
Pseudallescheria boydii, Rhizopus orvzae, Rhodotorula rubra,
Scedosporium apiospermum, Schizophyllum commmue, Sporothrix
schenckii, Trichophyton mentagrophytes, Trichophyton rubrunm,
Trichophyton vernrucosum, Trichophyton tonsurans, Trichophyton
violaceum, Trichosporon asahii, Trichosporon culaneum, Trichosporon
inkin, and Trichosporon mucoides.
[0218] In certain embodiments, the infection disease is caused by a
Candida species. In certain embodiments, the infectious disease is
caused by C. glabrata. In certain embodiments, the infectious
disease is caused by S. cerevisiae.
[0219] Also provided herein is a method for inhibiting the activity
of a fungus in a subject or a biological sample, the method
comprising administering to the subject or contacting the
biological sample with a compound provided herein (e.g., a compound
of Formula (I), (II), or (III)), or a pharmaceutically acceptable
salt, hydrate, solvate, polymorph, co-crystal, tautomer,
stereoisomer, isotopically labeled derivative, or prodrug thereof,
or a pharmaceutical composition thereof. Also provided are uses of
a compound provided herein (e.g., a compound of Formula (I), (H),
or (III)), or a pharmaceutically acceptable salt, hydrate, solvate,
polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled
derivative, or prodrug thereof, or a pharmaceutical composition
thereof, for the manufacture of a medicament for inhibiting the
activity of a fungus in a subject.
[0220] Also provided herein is a method for killing a fungus or
inhibiting the growth of a fungus in a subject or a biological
sample, the method comprising administering to the subject or
contacting the biological sample with a compound provided herein
(e.g., a of Formula (I), (II), or (III)), or a pharmaceutically
acceptable salt, hydrate, solvate, polymorph, co-crystal, tautomer,
stereoisomer, isotopically labeled derivative, or prodrug thereof,
or a pharmaceutical composition thereof. Also provided are uses of
a compound provided herein (e.g., a compound of Formula (I), (II),
or (III)), or a pharmaceutically acceptable salt, hydrate, solvate,
polymorph, co-crystal, tautomer, stereoisomer, isotopically labeled
derivative, or prodrug thereof, or a pharmaceutical composition
thereof, for the manufacture of a medicament for killing a fungus
or inhibiting the growth of a fungus.
[0221] In certain embodiments, the method is carried out in an
agricultural setting (e.g., on a plant). In certain embodiments,
the method is carried out in a clinical setting. In certain
embodiments, the method is carried out in or on a subject. In
certain embodiments, the method is carried out in or on a human
subject.
[0222] In certain embodiments, the fungus belongs to a genus
selected from the group consisting of Aspergillus, Blasomyces,
Candida, Coccidioides, Cryptococcus, Fusarium, Histoplasma,
Malassezia. Microsporum. Mucor, Paracoccidioide, Pneumocystis,
Pseudallescheria, Rhizopus, Scedosporium, Sporothrix, Stachybotrys,
Saccharomyces, Trichophyton, and Trichosporonphylum; or caused by a
fungus belonging to a phylum selected from the group consisting of
Ascomycota, Basidiomycota, Chytridiomycota, Glomeromycota, and
Zygomycota.
[0223] In certain embodiments, the fungus selected from the group
consisting of C. albicans, C. glabrata, C. krusii, C. rugosa, C.
parapsilosis, C. tropicalis, C. dubliniensis, C. lusitaniae, C.
guilliermondii, C. famata, C. kefyr, C. pelliculosa, C. lipolytica,
C. inconspicua, C. sake, C. lambica, C. norvegensis, C.
zeylanoides. Aspergillus terreus, A. clavatus, A. fumigatus, A.
niger, A. flavus, Saccharomyces cerevisiae, Blastomyces
dermatitidis, Coccidioides immitis, Coccidioides posadasii,
Cryptococcus neoformans, C. gattii, C. albidus, C. laurentii, C.
uniguttulas, E. floccosum. Fusarium graminearum, Fusarium
oxysporum, fsp. cubense, a member of the Fusarium solani complex,
Fusarium oxvsporum, Fusarium verticillioides, Fusarium
proliferatum, Histoplasma capsulatum, Malassezia furfur, M.
circinelloides, Paracoccidioides brasiliensis, Penicillium
marneffei, Pichia anomala, Pichia guilliermondi, Pneumocystis
carinii, Pneumocystis jirovecii, Pseudallescheria boydii, Rhizopus
oryzae, Rhodotorula rubra, Scedosporium apiospermum, Schizophyllum
commune, Sporothrix schenckii, Trichophyton mentagrophytes,
Trichophyton rubrum, Trichophyton verrucosum, Trichophyton
tonsurans, Trichophyton violaceum, Trichosporon asahii,
Trichosporon cutaneum, Trichosporon inkin, and Trichosporon
mucoides.
[0224] In certain embodiments, the fungus is a Candida species. In
certain embodiments, the fungus is C. glabrata. In certain
embodiments, the fungus S. cerevisiae.
[0225] In certain embodiments, the methods provided herein further
comprise administering to a subject, or contacting a plant or
biological sample, with an additional agent. In certain
embodiments, the additional agent is an anti-infective agent. In
certain embodiments, the additional agent is an antifungal agent.
In certain embodiments, the additional agent is an azole antifungal
agent.
Examples
Antifungal Hit Identification
[0226] Based on the previous findings that deletion of the KIX
domain of Saccharomyces cerevisiae GAL11 or Candida glabrata GAL11A
abrogates Pdr1-dependent transcriptional responses and xenobiotic
tolerance, it was hypothesized that the CgPdr1-CgGal11A interaction
interface might serve as a promising target for novel anti-MDR
compounds (see, e.g., Thakur, J. K. et al. A nuclear receptor-like
pathway regulating multidrug resistance in fungi. Nature 452,
604-609, doi:nature06836 [pii] 10.1038/nature06836 (2008)). A
fluorescently tagged CgPdr1 activation domain (AD) was used in an
in vitro fluorescence polarization (FP) screen of .about.140,000
chemically diverse compounds to identify small molecules that block
the interaction between the CgGal11A KIX domain and the CgPdr1 AD
(FIG. 6) (see. e.g., Roehrl, M. H., Wang, J. Y. & Wagner, G. A
general framework for development and data analysis of competitive
high-throughput screens for small-molecule inhibitors of
protein-protein interactions by fluorescence polarization.
Biochemistry 43, 16056-16066 (2004)). Based on the high degree of
conservation between S. cerevisiae and C. glabrata, the top hits
from the FP screen with an azole growth inhibition screen in S.
cerevisiae were considered to identify hits with in vivo efficacy
(FIG. 1A). Compounds that reproducibly inhibited growth in a
concentration-dependent manner only in the presence of ketoconazole
(FIG. 6) were identified. The most potent compound is referred to
as iKIX1 (FIG. 1B, 1C). In vitro binding studies revealed that the
Kd of the CgPdr1 AD for the CgGal11A KIX domain is 0.32 .mu.M and
the apparent Ki for iKIX1 is 18 .mu.M (FIG. 1D).
[0227] To facilitate the elucidation of the mechanism of action of
iKIX1, the high-resolution solution structure of the CgGal11A KIX
domain with a backbone RMSD of 0.7 .ANG. was determined (FIGS.
2A-2D and FIG. 5; PDB #4D7X). The CgGal11A KIX domain has 51%
sequence identity and 61% similarity with the S. cerevisiae
Gal11/Med15 KIX domain with an overall RMS deviation of 2.0 .ANG.
(FIG. 2A and FIG. 7C). The CgGal11A KIX domain forms a three-helix
bundle harbouring an extensively hydrophobic core and a short helix
at the N-terminus (FIG. 2A). Interaction interfaces of the CgGal11A
KIX domain with the CgPdr1 AD and iKIX1 (FIG. 2B) were determined
by chemical shift perturbation (CSP) analysis. The CgPdr1 AD and
iKIX1 target the same large hydrophobic groove harboured by the
three helices. Residues from all three helices constitute the
interaction interface, and titration of an ILV-methyl labeled
CgGal11A KIX domain reveals large CSPs on the three leucines (L19,
L23 and L51) upon addition of CgPdr1 AD and iKIX1 (FIG. 7B). The
basic interaction interface on the KIX domain complements the
acidic residues of CgPdr1 AD (FIG. 2C). Residues of the CgGal11A
KIX domain that interact with CgPdr1 AD and iKIX1 overlap strongly,
suggesting direct competitive binding as the mechanism of
inhibition. Docking of iKIX1 to the CgGal14A KIX domain suggests
extensive hydrogen bonding and hydrophobic interactions between
iKIX1 and KIX domain residues (FIG. 2D and FIG. 7A), matching the
interaction interface mapped by CSP analysis.
[0228] To assess the in vivo effects of iKIX1 on Pdr1-dependent
transcription, a strain was initially utilized in which the two S.
cerevisiae PDR1 orthologues (ScPDR1 and ScPDR3) are deleted and
which carries a plasmid expressing CgPDR1, and a heterologous
luciferase gene driven by 3 pleiotropic drug response elements
(PDREs). Luciferase activity was strongly induced by ketoconazole
treatment; iKIX1 co-treatment was able to block this induction in a
concentration-dependent manner (FIG. 3A).
[0229] A chromatin immunoprecipitation (ChIP) assay was used to
examine Gal11/Med15 recruitment to Pdr1-regulated target genes in
S. cerevisiae after iKIX) treatment. Gal11/Med15 was rapidly
recruited to the promoters of the Pdr1 target genes PDR5 and SNQ2
after ketoconazole addition; in contrast, ketoconazole-induced
recruitment of Gal11/Med15 was abrogated when the cells were
pre-treated with iKIX1 (FIG. 3B and FIG. 8A), iKIX1 did not impede
the constitutive occupancy of Pdr1 at the same Pdr1-regulated
target genes (FIG. 88). Consistent with the ChIP data, iKIX1
strongly inhibited azole-induced transcription of ScPdr1 target
genes (FIG. 3C, FIG. 8A, FIG. 80.
[0230] Next, the effect of iKIX1 on the transcription of C.
glabrata Pdr1-regulated genes involved in drug efflux and MDR
(CgCDR1. CgCDR2 and CgYOR1) was determined. CgPdr1 targets were
strongly up-regulated after ketoconazole treatment (see, e.g.,
Vermitsky, J. P. et al. Pdr1 regulates multidrug resistance in
Candida glabrata: gene disruption and genome-wide expression
studies. Mol Microbiol 61, 704-722 (2006); Ferrari, S.,
Sanguinetti, M., Torelli, R., Posteraro, B. & Sanglard, D.
Contribution of CgPDR1-regulated genes in enhanced virulence of
azole-resistant Candida glabrata. PLoS One 6, e17589,
doi:10.1371/journal.pone.0017589). However, pre-treatment with
iKIX1 reduced target gene induction in a durable and
concentration-dependent manner (FIG. 3D and FIG. 9A, FIG. 9B).
Treatment with iKIX1 alone did not significantly affect Pdr1-target
gene induction (FIG. 9C, FIG. 9D).
[0231] Next generation RNA sequencing (RNA-Seq) was employed to
query the genome-wide effects of iKIX1 and azole treatments alone
and in combination on the transcriptome in both S. cerevisiae and
in C. glabrata. In accord with previous reports, azole treatment
up-regulates Pdr1-dependent genes in both yeasts, such as the drug
efflux pumps ScPDR5 and CgCDR1 (see, e.g., Ferrari, S.,
Sanguinetti, M., Torelli, R.. Posteraro, B. & Sanglard, D.
Contribution of CgPDR1-regulated genes in enhanced virulence of
azole-resistant Candida glabrata. PLoS One 6, e17589.
doi:10.1371/journal.pone.0017589; DeRisi, J. et al. Genome
microarray analysis of transcriptional activation in multidrug
resistance yeast mutants. FEBS Lett 470, 156-160,
doi:S0014-5793(00)01294-1 [pii] (2000)). Combined azole and iKIX1
treatment strongly blunted expression of many azole-activated and
Pdr1-dependent genes in both S. cerevisiae and C. glabrata (FIG.
3E, FIG. 8D). consistent with prior data and the proposed mechanism
of action of iKIX1, iKIX1 alone affected very different sets of
genes in S. cerevisiae and C. glabrata. Treatment of S. cerevisiae
and C. glabrata cells with iKIX1 did not significantly alter the
expression of PDR1 or GAL11/MED15 after azole treatment (FIG. 8E).
Together, these findings suggest that the primary mechanism of
synergistic antifungal effects of iKIX1 with azoles is through
blocking the azole-stimulated and Pdr1-dependent drug efflux
pathway.
[0232] To ascertain iKIX1 efficacy in azole-resistant C. glabrata
strains, the effects of iKIX1 was examined on CgPdr1 target gene
expression in a set of isogenic strains with gain-of-function
CgPDR1 mutations originally identified in azole-resistant C.
glabrata clinical isolates (see, e.g., Ferrari, S. et al. Gain of
function mutations in CgPDR1 of Candida glabrata not only mediate
antifungal resistance but also enhance virulence. PLoS Pathog 5,
e1000268, doi:10.1371/journal.ppat.1000268 (2009)), iKIX1 reduced
azole-induced transcription of CgPdr1 target genes (e.g., CgCDR1)
in a concentration-dependent manner in all strains tested (FIG. 3F
and FIG. 9D).
[0233] To investigate whether these transcriptional effects
translated to functional effects on drug efflux rates, the
fluorescent compound rhodamine 60, a substrate of the i efflux
pump, was utilized (see, e.g., Sanglard, D., Ischer. F., Calabrese,
D., Majcherczyk, P. A. & Bille, J. The ATP binding cassette
transporter gene CgCDR1 from Candida glabrata is involved in the
resistance of clinical isolates to azole antifungal agents.
Antimicrobial agents and chemotherapy 43, 2753-2765 (1999); Silva,
L. V. et al. Milbemycins: more than efflux inhibitors for fungal
pathogens. Antimicrob Agents Chemother 57, 873-886,
doi:AAC.02040-12 [pii]10.1128/AAC.02040-12). Maximum efflux rates
were significantly decreased in PDR1 wild-type or gain-of-function
strains pre-treated with iKIX1, as compared to vehicle control
(FIG. 3D and FIG. 10).
[0234] Due to its ability to reduce efflux pump gene expression and
pump activity, it was predicted that iKIX1 could restore
azole-sensitivity to CgPDR1 gain-of-function mutant strains.
Isogenic C. glabrata strains with wild-type or single
gain-of-function alterations across CgPdr1 (FIG. 9A) were tested
for their sensitivity to fluconazole or ketoconazole on gradient
plates with increasing concentrations of iKIX1 or vehicle. As
expected, a CgPDR1 wild-type strain was sensitive to both
fluconazole and ketoconazole, whereas CgPDR1 gain-of-function
mutant strains grew robustly in the presence of azoles, iKIX1
restored azole-sensitivity to PDR1 gain-of-function mutant strains
in a concentration-dependent manner (FIG. 4B). CgPDR1 wild-type
strains also exhibited increased growth inhibition in the presence
of both iKIX1 and azole versus single agents alone (FIG. 11A. FIG.
11B).
[0235] Based on the strong combination effect of azoles and iKIX1
in the CgPDR1.sup.L280F mutant follow-up studies were focused on
this mutant strain. To investigate whether azoles and iKIX1 act in
a synergistic or additive manner in CgPDR1 wild-type and
CgPDR1.sup.L280F mutant strains, growth in checkerboard assays was
assessed with ketoconazole and iKIX1. In the wild-type CgPDR1
strain, the combination of ketoconazole and iKIX1 was additive
(FIG. 11C). However, the CgPDR1.sup.L280F mutant exhibited
synergistic growth inhibition with iKIX1 and ketoconazole
combination treatment, with combination indices <1 (FIG. 11D).
in concordance with the spot-plating assay.
[0236] A limited analysis was carried out exploring the chemical
space around the iKIX1 scaffold using commercial and custom
synthesized iKIX1 analogs, identifying several compounds that lost
activity in all assays; one analog (A2) is shown in Extended Data
FIG. 7a-d. This example, together with data from iKIX1 analogs and
the docked structure of iKIX1 to the CgGal11A KIX domain, supports
a model where iKIX1 engages the core of the KIX domain using an
array of hydrophobic and hydrogen bond contacts.
[0237] Two metazoan model systems were utilized to evaluate the
potential utility of iKIX1 as a co-therapeutic with fluconazole to
treat disseminated C. glabrata infection. The larvae of the moth
Galleria mellonella has been used as a model to test the
pathogenicity of a wide variety of human pathogens (see, e.g.,
Arvanitis, M., Glavis-Bloom, J. & Mylonakis, E. Invertebrate
models of fungal infection. Biochimica et biophysica acta 1832,
1378-1383, doi:10.1016/j.bbadis.2013.03.008 (2013)). A G.
mellonella survival assay was utilized to determine the virulence
of C. glabrata PDR1 wild-type or PDR1.sup.L280F strains in the
presence of fluconazole, iKIX1, or a combination of the two (FIG.
4C). Larvae were injected with C. glabrata and a single injection
of fluconazole (50 mg/kg), iKIX1 (25 mg/kg). a combination of the
two, or vehicle; survival was monitored every 24 hours. G.
mellonella injected with wild-type CgPDR1 was sensitive to
fluconazole alone, and exhibited no significant alterations in
survival with a fluconazole-iKIX1 combination. However, in G.
mellonella larvae injected with a CgPDR1.sup.L280F strain, whereas
the single agents fluconazole or iKIX1 did not significantly
increase survival compared to vehicle, co-treatment with iKIX1 and
fluconazole significantly increased survival (P<0.001).
[0238] Prior to mammalian studies, the potential toxicity of iKIX1
was evaluated in mammalian cells (FIG. 12E, FIG. 12F). Human HepG2
cells treated with iKIX1 revealed toxicity only at high
concentrations of iKIX1 (IC50.about.100 .mu.M), iKIX1 had no effect
on the transcription of SREBP-target genes at concentrations up to
100 .mu.M, indicating its specificity for the fungal Gal11/Med15
KIX domain (see. e.g., Yang. F. et al. An ARC/Mediator subunit
required for SREBP control of cholesterol and lipid homeostasis.
Nature 442, 700-704 (2006)). Also assessed was the in vitro
stability and in vivo mouse pharmacokinetics of iKIX1 and found
that iKIX1 exhibited favorable drug-like properties and in vivo
exposure in these studies (FIG. 13G, FIG. 13H).
[0239] To evaluate the therapeutic potential of iKIX1 and azole
antifungal co-therapy in a mammalian model, an established mouse
model of disseminated fungal disease was used (see, e.g., Silva, L.
V. et al. Milbemycins: more than efflux inhibitors for fungal
pathogens. Antimicrobial agents and chemotherapy 57, 873-886,
doi:0.1128/AAC.02040-12 (2013)). Mice were inoculated with C.
glabrata by tail-vein injection and were dosed peritoneally
once-daily with 100 mg/kg fluconazole (high FLU), 100 mg/kg iKIX1,
a combination of the two, or vehicle alone. After 7 days, mice
injected with a CgPDR1 wild-type strain exhibited significantly
reduced tissue fungal burden in the kidney and spleen following
fluconazole treatment alone; iKIX1 co-treatment did not result in
further reductions (FIG. 4D). In contrast, in mice injected with
the azole-resistant CgPDR1.sup.L280F strain, only co-treatment with
iKIX1 and fluconazole resulted in significant (.about.10-fold)
reductions in fungal burdens in the kidney and spleen (P<0.0001)
(FIG. 4D). Similar results were observed with the clinically
isolated CgPDR1.sup.+ and CgPDR1.sup.L280F strains DSY562 and
DSY565 (FIG. 13A). Consistent with previous studies, the fungal
burden in mice infected with the CgPDR1.sup.L280F strain was higher
than those infected with wild-type CgPDR1 strains, suggesting that
PDR1 mutant strains may be more virulent in vivo (see, e.g., Silva,
L. V. et al. Milbemycins: more than efflux inhibitors for fungal
pathogens. Antimicrobial agents and chemotherapy 57, 873-886,
doi:10.1128/AAC.02040-12 (2013)). Similar but less pronounced
results were found in mice injected with a CgPDR1.sup.P822L strain
(FIG. 13B). When mice were injected with a CgPDR1 wild-type strain
and dosed with 25 mg/kg fluconazole (low FLU) alone or in
combination with iKIX1, fluconazole alone poorly reduced tissue
burden, whereas combination treatment resulted in significant
(.about.10-fold) reductions in fungal burdens in both organs
(P<0.0001) (FIG. 4D). These results suggest that iKIX1
combination treatment with azole may be therapeutically desirable
even in the absence of CgPDR1 gain-of-function mutations. Mice
infected with a Cgpdr1 null strain were more sensitive to iKIX1
alone; unlike mice infected with CgPDR1.sup.- or CgPDR1.sup.L280F
strains, low doses of iKIX1 did not further reduce fungal burden in
Cgpdr1 null infections (FIG. 13C, FIG. 13D).
[0240] CgPDR1 gain-of-function mutations are also known to control
adherence to host cells. As previously observed, a PDR1.sup.L280F
mutant increased relative adherence to epithelial cells as compared
to a PDR1 wild-type strain (see, e.g., Vale-Silva, L., Ischer, F.,
Leibundgut-Landmann, S. & Sanglard, D. Gain-of-function
mutations in PDR1, a regulator of antifungal drug resistance in
Candida glabrata, control adherence to host cells. Infect Immun 81,
1709-1720, doi:IAI.00074-13 [pii] 10.1128/IAI.00074-13).
Strikingly, iKIX1 treatment alone reduced adherence to levels
similar to a PDR1 wild-type strain (FIG. 13E). Ketoconazole alone
or co-treatment with iKIX1 also reduced relative adherence to
levels comparable to a PDR1 wild-type strain. To assess the role of
iKIX1 in modulating adhesion in an infection model, a mouse model
of urinary tract infection was used (see, e.g., Chen, Y. L. et al.
Convergent Evolution of Calcineurin Pathway Roles in
Thermotolerance and Virulence in Candida glabrata. G3 (Bethesda) 2,
675-691, doi:10.1534/g3.112.002279 GGG_002279 [pii]). In both the
bladder and kidney, iKIX1 alone was sufficient to decrease fungal
load after infection with either a PDR1 wild-type strain or a
PDR1.sup.L280F strain (FIG. 13F), suggesting that iKIX1 may indeed
modulate adhesion.
[0241] The proportion of azole-resistant C. glabrata (up to 20% in
the US) and the emergence of multidrug resistance (approximately
40% of echninocandin-resistant isolates are azole-resistant) argues
for the need for novel treatments that can target these resistant
populations (see, e.g., Farmakiotis, D., Tarrand, J. J. &
Kontoyiannis, D. P. Drug-Resistant Candida glabrata Infection in
Cancer Patients. Emerg Infect Dis 20, 1833-1840,
doi:10.3201/eid2011.140685; Pfaller, M. A. et al. Frequency of
decreased susceptibility and resistance to echinocandins among
fluconazole-resistant bloodstream isolates of Candida glabrata. J
Clin Microbiol 50, 1199-1203, doi:JCM.06112-11 [pii]
10.1128/JCM.06112-11). The results herein demonstrate that small
molecule disruption of the interaction between the CgGal11A KIX
domain and the CgPdr1 activation domain is a therapeutically
tractable method for treating infectious diseases (e.g., fungal
infections), including resensitizing azole-resistant C. glabrata to
standard azole antifungal treatment (FIG. 14).
Additional Biological Data
TABLE-US-00001 [0242] TABLE 1 Luciferase Luciferase CgCDR1 activity
activity qRT-PCR (5 .mu.M + (50 .mu.M + (30 .mu.M in CgCDR1 keto)
keto) PDR1 WT) qRT-PCR Compound (% DMSO + (% DMSO + (% DMSO + (%
DMSO + Structure MW keto) keto) keto) keto) ##STR00045## 329.2 62
25 5 10 .mu.m - 12% 30 .mu.m - 5% ##STR00046## 286.71 54 30 -- 10
.mu.m - 74% 30 .mu.m - 42% ##STR00047## 336.72 40 21 -- 10 .mu.m -
10% 30 .mu.m - 3% ##STR00048## 303.17 45 25 -- 10 .mu.m - 17% 30
.mu.m - 7% ##STR00049## 302.18 77 28 -- 30 .mu.m - 3% ##STR00050##
-- 86 38 -- 30 .mu.m - 1% ##STR00051## -- 53 14 -- 10 .mu.m - 10%
30 .mu.m - 2% ##STR00052## -- 52 26 -- 10 .mu.m - 15% 30 .mu.m -
6%
[0243] iKIX1 and analogs that show antifungal activity in Candida
glabrata also show antifungal activity in other clinically relevant
pathogenic Candida species including Candida albicans, Candida
krusei, Candida tropicalis and Candida parapsilosis. Antifungal
susceptibility testing was carried out using broth dilution assays
which examined effects of two-fold dilutions of compounds on
cellular growth. The minimum inhibitory concentration (MIC) was
defined as the drug concentration at which the optical density was
equal to or decreased more than 50% from that of the drug-free
culture. MIC shown in the table are in .mu.M. (Table 2 and Table
3).
TABLE-US-00002 TABLE 2 yeast Candida Candida Candida Candida
glabrata glabraia albicans albicans strain DSY562 DSY565 SC5314
DSY2621 deleted for clinical efflux isolate, azole transporters
clinical isolate, resistant, ATCC and compound/ azole sensitive,
PDR1.sup.1.280F strain calcineurin analog compound structure PDR1
wild type mutant SC5314 subunit A iK1X1 ##STR00053## 12.5 12.5 12.5
12.5 A02 (inactive analog, negative control) ##STR00054## >100
>100 >100 >100 A18 ##STR00055## 6.25 12.5 6.25 6.25 A52-8
##STR00056## 3.125 6.25 3.125 3.125 DFCI-1 ##STR00057## 6.25 6.25
6.25 12.5 A03 ##STR00058## 12.5 25 25 25 A07 ##STR00059## 3.125
3.125 3.25 3.25 A16 ##STR00060## 3.125 6.25 6.25 6.25
TABLE-US-00003 TABLE 3 yeast Candida Candida Candida Saccharomyces
krusei tropicalis parapsilosis cerevisiae strain DSY471 DSY472
DSY473 DSY2094 compound/ ATCC strain ATCC strain ATCC strain analog
compound structure 6258 75/44508 22019 -- iK1X1 ##STR00061## 6.25
6.25 25 12.5 A02 (inactive analog, negative control) ##STR00062##
>100 >100 >100 >100 A18 ##STR00063## 6.25 12.5 25 12.5
A52-8 ##STR00064## 3.125 3.125 12.5 3.125 DFCI-1 ##STR00065## 3.125
6.25 12.5 6.25 A03 ##STR00066## 12.5 12.5 50 12.5 A07 ##STR00067##
6.25 3.25 6.25 3.125 A16 ##STR00068## 3.25 3.25 12.5 3.125
Biological Materials and Methods
Fluorescence Polarization-Based High-Throughput Screen
[0244] A fluorescein-tagged 30 amino acid C-terminal activation
domain of CgPdr1 (FITC-LGTLDEFVNKGDLNELYNSLWGDLFSDVYL (SEQ ID NO:
1)) CgPdr1 AD30 was purchased as a synthetic peptide from
Pepide2.0. The CgGal11A KIX domain was expressed as a His.sub.6-GST
fusion protein and purified by affinity chromatography with Ni-NTA
resin (Qiagen) and size exclusion chromatography (Sephadex 75,
Pharmacia). The K.sub.d for CgPdr1 AD30 binding to CgGal11A KIX was
determined to be 320 nM by fluorescence polarization (FP) assay.
For small molecule screening, fluorescein-CgPdr1 AD30 was held at a
concentration of 30 nM and the GST-tagged CgGal11A KIX was at a
concentration of 1 .mu.M (above the Kd). The screen was carried out
in duplicate and the volume in each well was 25 .mu.L.
Hit Identification
[0245] The Z-score for Fluorescence Polarization and Total
Fluorescence was determined for each individual plate. To filter
out false positives arising from the auto-fluorescence of the
compounds and allow strict filtering for the initial cherry picks,
the mean was calculated only from wells without compound. Standard
deviation of Fluorescence Polarization was calculated using the
readings from the entire plate, whereas standard deviation of Total
Fluorescence was calculated using wells without compound. For
cherry picking for the in vivo screen only those compounds that
exhibited a Z-score greater than 4 in Fluorescence Polarization,
and a Z-score of less than 3 in Total Fluorescence, with values
consistent between both samples, were considered.
CgPdr1 AD Kd and Titration with iKIX1
[0246] To measure the dissociation constant (Kd) of CgPdr1 AD30,
FITC labeled CgPdr1 AD30 was held at a concentration of 30 nM and
the GST-tagged CgGal11A KIX was prepared with concentrations
ranging from 0 to 300 .mu.M. The IC50 for iKIX-1 binding was
measured with GST-tagged CgGal11A KIX held at a constant 3 .mu.M
concentration, iKIX-1 was titrated from 0 to 1000 .mu.M and 30 nM
FITC-labeled CgPdr1 AD30 was added subsequently with an HP D300
digital dispenser (Tecan Group. Maennerdorf, Switzerland). All
experiments were carried out in duplicate and the s.d. is reported
at each point on the plot.
Analysis of CgPdr1 AD and iKIX1 Binding to CgGal11A KIX
[0247] The FP titration curve of the CgPdr1 AD30 was fitted to
GraphPad Prisms (La Jolla, Calif., USA) One site-total binding
equation (equation 1).
Y = B max * X ( K d + X ) + NS * X + Background ( 1 )
##EQU00001##
[0248] Where Bmax is the maximum specific binding, Kd is the
equilibrium binding constant. NS is the slope of nonspecific
binding and Background is the amount of nonspecific binding. The
curve of the competition assay with iKIX1 was fitted to a decaying
exponential (equation 2) in MATLAB (Natick, Mass., USA) and a 190.2
.mu.M IC.sub.50 was obtained.
Y=A*e.sup.(-b*x.sup.n.sup.)+c (2)
[0249] Subsequently, this IC.sub.50 was used to calculate an
apparent inhibition constant (Ki) of 18.1 .mu.M for iKIX1 binding;
according to a procedure described by Cer and colleagues (see,
e.g., Cer R Z, Mudunuri U, Stephens R, Lebeda F J. IC50-to-Ki: a
web-based tool for converting IC.sub.50 to Ki values for inhibitors
of enzyme activity and ligand binding. Nucleic acids research.
2009; 37(Web Server issue):W441-5. PMCID: 2703898) (equation
3).
K i = I 50 L 50 K d + P 0 K d + 1 ( 3 ) ##EQU00002##
[0250] Where Ki is the inhibitor concentration, IC.sub.50 is the
concentration of the free inhibitor (iKIX1) at 50% inhibition, L50
is the concentration of the free ligand (CgPdr1 AD30) at 50%
inhibition, K.sub.d is the dissociation constant of
fluorescein-CgPdr1 AD30 to GST-tagged CgGal11A KIX and P.sub.0 is
the concentration of the free protein at 0% inhibition.
Cell Growth Inhibition Screen
[0251] S. cerevisiae SEY6210 wild-type strains were grown in YPD (U
% yeast extract, 2% bacto peptone, 2% dextrose) at 30.degree. C.
with shaking overnight until saturation. The next day, cultures
were inoculated to an OD.sub.600 of 0.0007 and grown for 17 hours,
to an OD.sub.600.about.0.5, 384-well plates were set up in
duplicate with either YPD alone or with YPD containing a final
concentration of 5 .mu.M ketoconazole. 384-well plates contained 5
two-fold dilutions of each compound, corresponding to a final
concentration range of 20 .mu.g/mL to 1.25 .mu.g/mL. Wells were
inoculated with cells to a final OD.sub.600 of 0.00025 in YPD. To
identify compounds that potentiate azole inhibition of cell growth,
cells were allowed to grow for another 45 hours followed by
OD.sub.600 measurement on an Envision plate reader (Perkin
Elmer).
NMR Methods
[0252] The sequence corresponding to the CgGal11A KIX domain was
cloned into a pET24b plasmid with an N-terminal His6-tag and was
transformed into E. coli BL21 (DE3) cells. Cells were grown in
.sup.15N, .sup.15N/.sup.13C enriched minimal media at 37 C. The
cells were induced at an OD.sub.600 of 0.7 with 1 mM isopropyl
.beta.-D-1-thiogalactopyranoside at 25 C. The cells were then lysed
by sonication after addition of 1 mg/mL lysozyme. The protein was
affinity purified using Ni-NTA resin (Qiagen) and further purified
by fast protein liquid chromatography using a size exclusion column
(Sephadex 75. Pharmacia). All NMR samples were in PBS buffer (10 mM
Na2HPO.sub.4, 2 mM K2HPO.sub.4, 137 mM NaCl, 2.7 mM KCl, 1 mM EDTA
and 0.01% NaN.sub.3), pH 6.5, unless otherwise stated. The samples
were measured at a concentration of approximately 800 .mu.M.
[0253] Backbone assignments were obtained by the standard set of
triple resonance experiments (HNCA/HNCOCA, HNCACB/CBCACONH,
HNCO/HNCACO) and side-chain resonances were assigned using HCCONH
and CCONH experiments in H.sub.2O and HCCH-TOCSY in .sup.2H.sub.2O.
Distance constrains were obtained from .sup.15N- and
.sup.13C-dispersed Nuclear Overhauser Enhancement (NOE) experiments
with mixing times of 90 milliseconds and 80 milliseconds,
respectively. Stereo-specific methyl assignments were obtained with
a stereo-specific methyl (ILV) labeling strategy developed by the
Boisbouvier group (see, e.g., Gans P, Hamelin O, Sounier R, Ayala
I, Dura M A, Amero C D, et al. Stereospecific isotopic labeling of
methyl groups for NMR spectroscopic studies of
high-molecular-weight proteins. Angewandte Chemie. 2010; 49(11):
1958-62).
Structure Calculation and Refinement
[0254] Peak volumes were integrated and converted to distance
restraints in the CcpNmr software suite (see, e.g., Vranken W F,
Boucher W, Stevens T J, Fogh R H, Pajon A, Llinas M. et al. The
CCPN data model for NMR spectroscopy: development of a software
pipeline. Proteins. 2005; 59(4):687-96). One hundred structures
were calculated with CYANA and the 10 lowest energy structures were
selected for AMBER refinement in explicit water (see, e.g., Guntert
P. Automated NMR structure calculation with CYANA. Methods in
molecular biology. 2004: 278:353-78). The refinement was performed
through the WeNMR web-interface with the AMBER99SB force field,
TIP3PBOX and a box distance of 10 .ANG.ngstrom (.ANG.) (see, e.g.,
Bertini I, Case D A, Ferella L, Giachetti A, Rosato A. A
Grid-enabled web portal for NMR structure refinement with AMBER.
Bioinformatics. 2011; 27(17):2384-90). The quality of the CgGal11A
KIX structure was analyzed and validated with the protein structure
validation software suite PSVS, the common interface for NMR
structure generation (CING) and Procheck-NMR (see, e.g.,
Bhattacharya A, Tejero R, Montelione G T. Evaluating protein
structures determined by structural genomics consortia. Proteins.
2007; 66(4):778-95: Doreleijers J F, Sousa da Silva A W, Krieger E,
Nabuurs S B, Spronk C A, Stevens T J. et al. CING: an integrated
residue-based structure validation program suite. Journal of
biomolecular NMR, 2012; 54(3):267-83. PMCID: 3483101; Laskowski R
A, MacArthur M W, Moss D S, Thornton J M. PROCHECK: a program to
check the stereochemical quality of protein structures. Journal of
Applied Crystallography, 1993; 26(2):283-91). 96.1% of dihedral
(.PHI. and .psi.) angles occupy the core region of the Ramachandran
plot, and 3.6% are in the allowed, 0.3% in the generously allowed
and none in the disallowed region.
Chemical Shift Perturbation (CSP) Analysis
[0255] CSPs in .sup.1H-.sup.15N HSQCs and .sup.1H-.sup.13C ILV
HSQCs of CgGal11A KIX domain were calculated according to equations
4a and 4b, respectively.
CSP=[(.DELTA.proton shifts){circumflex over ( )}+(.DELTA.nitrogen
shifts*0.2){circumflex over ( )}]{circumflex over ( )}0.5 (4a)
CSP=[(.DELTA.proton shifts){circumflex over ( )}+(.DELTA.carbon
shifts*0.3){circumflex over ( )}]{circumflex over ( )}0.5 (4b)
[0256] Residues that experienced a CSPs larger than two the
standard deviation were considered to be significant and plotted on
the structure of the CgGal11A KIX domain. Residues with significant
.sup.1H-.sup.15N CSPs are (L23, M24, N27, I29, N30, G31, T35, T36,
A37, M40, H43, A44, A49, L51, K54, M65, K68, I69, M72, R73, T75,
R76, R79, E82, S83) and (L23, M24, D25, 126, N27, 129, N30, T35,
T36, A37, H43, A44, L51, K66, R73, T75, E82) for the CgPdr1AD and
iKIX1, respectively. L19, L23 and L51 showed significant ILV CSPs
in both titrations.
Ligand Docking
[0257] The position of the ligand binding cavity site on the
CgGal11A KIX domain was obtained by center of mass calculation of
the amino acid residues 10-30, 35-54 and 59-83. In the next step,
partial atomic charges and other force field parameters were
assigned to the CgGal11A KIX domain with the AMBER 14 force field
(see, e.g., D. A. Case J T B, R. M. Betz, D. S. Cerutti, T. E.
Cheatham, III. T. A. Darden, R. E. Duke. T. J. Giese, H. Gohlke. A.
W. Goetz, N. Homeyer, S. Izadi, P. Janowski, J. Kaus, A. Kovalenko,
T. S. Lee, S. LeGrand, P. Li, T. Luchko, R. Luo, B. Madej, K. M.
Merz, G. Monard, P. Needham, H. Nguyen, H. T. Nguyen. I. Omelyan,
A. Onufriev, D. R. Roe, A. Roitberg, R. Salomon-Ferrer, C. L.
Simmerling, W. Smith, J. Swails, R. C. Walker. J. Wang, R. M. Wolf,
X. Wu, D. M. York and P. A. Kollman. AMBER 2015. University of
California, San Francisco 2015). Partial atomic charges and other
force field parameters were assigned to iKIX-1 with the AM1-BCC
method and GAFF, respectively (see, e.g., Jakalian A, Jack D B,
Bayly C I. Fast, efficient generation of high-quality atomic
charges. AM1-BCC model: II. Parameterization and validation.
Journal of computational chemistry. 2002; 23(16): 1623-41; Wang J,
Wolf R M, Caldwell J W, Kollman P A. Case D A. Development and
testing of a general amber force field. Journal of computational
chemistry. 2004; 25(9):1157-74). Docking and scoring of iKIX1 with
CgGal11A KIX was carried out using the Pardock and BAPPL modules of
Sanjeevini, an automated, linux based freely accessible drug design
software suite (see, e.g., Gupta A, Gandhimathi A, Sharma P.
Jayaram B. ParDOCK: an all atom energy based Monte Carlo docking
protocol for protein-ligand complexes. Protein and peptide letters.
2007; 14(7):632-46; Jain T, Jayaram B. An all atom energy based
computational protocol for predicting binding affinities of
protein-ligand complexes. FEBS letters. 2005:579(29):6659-66;
Jayaram B, Singh T. Mukherjee G, Mathur A, Shekhar S. Shekhar V.
Sanjeevini: a freely accessible web-server for target directed lead
molecule discovery. BMC bioinformatics. 2012; 13 Suppl 17:S7.
PMCID: 3521208). The Pardock docking software samples a number of
configurations of the ligand in the binding pocket of the protein
and finally provides the top energetically favorable configurations
of the ligand bound to the KIX domain. These protein-ligand
complexes are then subjected to energy minimization in vacuum with
1000 steps of steepest descent and 1500 steps of conjugate gradient
methods using the SANDER module of AMBER 14. The binding free
energy of iKIX1, estimated through docking and scoring, is -9.30
kcal/mol.
Chromatin Immunoprecipitation (ChIP) Assays
[0258] ChIP was performed according to standard procedures (see.
e.g., McConnell A D, Gelbart M E, Tsukiyama T. Histone fold protein
Dls1p is required for Isw2-dependent chromatin remodeling in vivo.
Mol Cell Biol. 2004; 24(7):2605-13. PMCID: 371119). Briefly, cells
were grown to saturation then washed 2 times with sterilized
Milli-Q (Millipore) water, resuspended to an OD.sub.600 of 0.8 in
YP (1% yeast extract, 2% bacto peptone) and then grown for 6 hours
at 30.degree. C. with shaking in the presence of DMSO (vehicle) or
iKIX1. Cultures were induced with ketoconazole to a final
concentration of 40 .mu.M and harvested at the times indicated. 2
mL of culture was harvested at 0' and 15' for matched transcription
samples; RNA was prepared as described below. Cells were fixed with
1% formaldehyde (final) for 15 minutes with swirling and then
quenched with 125 mM glycine (final) for 5 min. with swirling.
Cells were pelleted, washed with TBS+125 mM glycine and then TBS.
Cells were lysed in buffer containing 50 mM HEPES, pH 7.5, 140 mM
NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 1 mM EDTA and
protease inhibitors and 0.5 mm glass beads. Glass beads were
removed by centrifugation before cell lysate was sonicated to
obtain 150 to 400 base pair fragments. Chromatin from clarified
extracts was immunoprecipitated with HA.11 clone 16B12 monoclonal
antibody (Covance MMS--101R) and protein G Dynabeads (Thermo Fisher
Scientific) or protein G Dynabeads alone then washed with lysis
buffer, high salt lysis buffer, wash buffer and TE, twice each
before being eluted from beads in elution buffer at 65.degree. C.
for 30 min. Crosslinks were reversed in eluates at 65.degree. C.
for 5 hours in the presence of RNase A to 0.2 mg/mL followed by
proteinase K treatment 0.2 .mu.g/mL for 2 hours at 42.degree. C.
DNA was cleaned and eluted into TE using the Qiagen QIAquick PCR
purification kit. No significant Gal11/Med15-HA or HA-Pdr1
association was observed with control genomic region (chr1) or in
the absence of HA antibody.
Luciferase Assay
[0259] An S. cerevisiae pdr1.DELTA.::KANpdr3.DELTA.::KAN strain
bearing plasmids carrying CgPDR1-1 and PDRES-luciferase was used
for the luciferase assays. Strains were grown overnight in YPD and
then washed twice with sterilized Milli-Q water before being
resuspended to an OD.sub.600 of 0.8 in YP. After 20 hours, cultures
were split and iKIX1 or DMSO alone was added to final
concentrations as indicated. At the same time, ketoconazole to a
final concentration of 40 .mu.M (resuspended in ethanol) or ethanol
alone (vehicle) were added to cultures. After 24 hours of
treatment, an equal volume of 1 mM d-luciferin (sodium salt) in 0.1
M sodium citrate buffer at pH 5.0 was added to 100 .mu.L aliquots
(see, e.g., Leskinen P, Virta M, Karp M. One-step measurement of
firefly luciferase activity in yeast. Yeast. 2003; 20(13):1109-13).
Luminescent signal was acquired over 10 seconds and RLU (relative
light units) presented are normalized to growth (assessed by
OD.sub.600) and untreated controls.
Spot Plating
[0260] Strains were inoculated & grown in YPD at 30.degree. C.
with shaking overnight and 5 mL of YPD was inoculated with 20 .mu.L
of overnight culture. Strains were grown to log phase at 30.degree.
C. with shaking and then diluted to an OD.sub.600 of 0.0004, 3
.mu.L of cell suspension was spotted on plates containing gradients
with increasing concentration of iKIX1 or vehicle (DMSO), and a
fixed concentration of ketoconazole or fluconazole, as indicated.
Gradients were made as previously described (see. e.g., Katzmann D
J, Hallstrom T C, Voet M, Wysock W, Golin J, Volckaert G, et al.
Expression of an ATP-binding cassette transporter-encoding gene
(YOR1) is required for oligomycin resistance in Saccharomyces
cerevisiae. Mol Cell Biol. 1995; 15(12):6875-83. PMCID: 230942).
Plates were incubated at 30.degree. C. and growth was assessed
after 48 hours.
Minimal Inhibitory Concentration (MIC) Assays
[0261] iKIX1--ketoconazole drug interactions were assessed by a
broth microdilution checkerboard assay as described in EUCAST
document 7.1 (see, e.g., EUCAST definitive document EDef 7.1:
method for the determination of broth dilution MICs of antifungal
agents for fermentative yeasts. Clin Microbiol Infect. 2008;
14(4):398-405). Absorbance was assessed using a Molecular Devices V
max Kinetic Microplate Reader at a wavelength of 540 nm.
Combination indices were calculated as follows:
CI=(C.sub.IKIX1/IC.sub.IKIX1)+(C.sub.AZ/IC.sub.AZ); where
IC.sub.IKIX1 and IC.sub.AZ are concentrations of iKIX1 alone or
ketoconazole alone, respectively, that result in greater than 50%
inhibition of growth as compared to untreated control and iKIX1 and
C.sub.AZ are the concentrations of iKIX1 and ketoconazole in
combination that provide the same effect (greater than 50%
inhibition of growth as compared to untreated control). A CI of
less than 1 is taken to indicate synergy.
RNA-Seq Experiments
[0262] Cells were grown in YPD at 30.degree. C. with shaking
overnight (24 hours) then washed twice in 2.times. volume of
Milli-Q water before being resuspended in YP to an OD.sub.600 of
0.8. Cells were grown with shaking overnight (.about.16 hours)
before splitting and treatment with vehicle (DMSO) or 10 .mu.M
iKIX1. Cells were grown with shaking another 8 hours before harvest
of non-azole induced samples. Remaining cultures were induced to a
final concentration of 40 .mu.M ketoconazole and harvested after 15
minutes by centrifugation at 4,000 rpm for 4 minutes at 4.degree.
C. Media was aspirated and cells were snap frozen on dry ice. RNA
isolation was performed as previously described (see, e.g., Martin
R, Moran G P, Jacobsen I D, Heyken A, Domey J, Sullivan D J, et al.
The Candida albicans-specific gene EED1 encodes a key regulator of
hyphal extension. PLoS One.6(4):e18394. PMCID: 3075580); RNA was
then DNase treated using recombinant DNasel. RNase-free (Roche)
according to manufacturer's instructions. Samples were prepared in
triplicate and multiplexed RNA-Seq libraries were constructed using
PolyA selection and the NEBNext Ultra Directional RNA Library Prep
Kit for Illumina (New England Biolabs). Sequencing was carried out
on Illumina HiSeq 2500, resulting in approximately 17.68 million of
50 bp paired-end reads per sample. STAR aligner was used to map
sequencing reads to transcripts in the reference genomes (S288C for
Saccharomyces cerevisiae and draft genome from isolate DSY562 for
Candida glabrata) (see, e.g., Dobin A, Davis C A, Schlesinger F,
Drenkow J, Zaleski C, Jha S, et al. STAR: ultrafast universal
RNA-seq aligner. Bioinformatics.29(1):15-21. PMCID: 3530905). Read
counts for individual transcripts were produced with HTSeq-count,
followed by the estimation of expression values and detection of
differentially expressed transcripts using EdgeR (see, e.g., Anders
S, Pyl P T, Huber W. HTSeq--a Python framework to work with
high-throughput sequencing data. Bioinformatics.31(2):166-9. PMCID:
4287950; Robinson M D, McCarthy D J, Smyth G K. edgeR: a
Bioconductor package for differential expression analysis of
digital gene expression data. Bioinformatics.26(1): 139-40. PMCID:
2796818). The data discussed in this publication have been
deposited in NCBI's Gene Expression Omnibus and are accessible
through GEO series accession number GSE74361 (see, e.g., Edgar R,
Domrachev M, Lash A E. Gene Expression Omnibus: NCBI gene
expression and hybridization array data repository. Nucleic Acids
Res. 2002; 30(1):207-10. PMCID: 99122).
Efflux Assays
[0263] Cells were grown in YPD at 30.degree. C. with shaking
overnight (24 hours) then washed twice in 2.times. volume of
Milli-Q water before being resuspended in YP to an OD.sub.600 of
0.8. Cells were treated with vehicle or iKIX1 and grown with
shaking for 16 hours. Equivalent numbers of cells (as assessed by
OD.sub.600=3) were concurrently induced with 5 .mu.M of
ketoconazole and loaded with 10 .mu.M of rhodamine 6G for 30
minutes at 30.degree. C. with shaking in the dark. Untreated cells
were placed on ice and not treated with ketoconazole or rhodamine
6G. All cells were placed on ice and washed twice with cold YP
containing either 5 .mu.M ketoconazole+vehicle (DMSO) or 5 .mu.M
ketoconazole+30 .mu.M iKIX1 to remove excess rhodamine 6G. Cells
were resuspended in room temperature YP with either 5 .mu.M
ketoconazole+vehicle (DMSO) or 5 .mu.M ketoconazole+30 .mu.M iKIX1
and 100 .mu.L was transferred to a 96-well plate. An Envision 2103
Multilabel plate reader was used to assess RFU (relative
fluorescence units; excitation 485, emission 535) over 60 minutes
at 7.5 minute intervals. Efflux rate was calculated as RFU per
second with 7.5 minute windows. Experiments were performed in
triplicate, with duplicate readings for each sample; error bars
indicate s.d.
Transcription Assays and Quantitative Real-Time PCR
[0264] Cells were grown in YPD at 30.degree. C. with shaking 24
hours then washed twice in 2.times. volume of Milli-Q water before
being resuspended in YP to an OD.sub.600 of 0.8. Cells were grown
with shaking overnight (.about.16 hours) before splitting and
treatment with vehicle (DMSO) or iKIX1 to concentrations indicated.
Cells were grown with shaking another 8 hours before harvest of
non-azole induced samples. Remaining cultures were induced to a
final concentration of 40 .mu.M ketoconazole and harvested after 15
minutes and subsequent time points, if shown. For harvest and
preparation of RNA. cells equivalent to an OD.sub.600 of 0.4 were
centrifuged at 4,000 rpm for 4 minutes and then resuspended in 700
.mu.L Trizol. Cells in Trizol were bead beat with 0.4 mL glass
beads for 2.times.30 seconds and then protocol was followed
according to manufacturer's instructions. One .mu.g of RNA was used
for DNase I treatment (Roche) and subsequent cDNA synthesis using
Roche Transcriptor First Strand cDNA synthesis kit. cDNA was
diluted 5.times. in water and 2.5 .mu.L was used per reaction with
Roche SYBR green. Quantitative real-time PCR reactions were run and
analyzed on a Roche LightCycler 480 system. Sc transcripts were
normalized to ScSCR1 and untreated, uninduced DMSO control; Cg
transcripts were normalized to CgRDN25-1 and untreated, uninduced
DMSO control.
Viability and Transcription Assays in Mammalian HepG2 Cells
[0265] HepG2 (ATCC HB-8065) cell viability was assessed using the
CellTiter-Glo assay kit (Promega). HepG2 cells (N=4,500) were
seeded in each well in 100 .mu.L MEM+10% FBS in 96-well CellBIND
(Corning) plates. After 24 hours, iKIX1 was added to final
concentrations indicated for a final volume of 150 .mu.L. HepG2
cells were incubated with iKIX1 for another 72 hours before
CellTiter-Glo signal was assessed with an Envision 2103 Multilabel
plate reader according to the manufacturer's instructions. HepG2
cells were not authenticated or tested for mycoplasma contamination
following receipt from ATCC.
[0266] For transcription assays, HepG2 cells were seeded in MEM+10%
FBS on 12-well poly-D-lysine-coated plates at a concentration of
175,000 cells per mL. After cells attached (4 hours), media was
aspirated and iKIX1 was added in fresh MEM+10% FBS to final
concentrations indicated. Cells were treated with iKIX1 for 24
hours before RNA was isolated using Trizol according to
manufacturer's instructions. Quantitative real-time RT-PCR was
carried out as described above.
Plasma Stability and Microsomal Stability Analysis
[0267] Microsome stability assays were performed as previously
described (see, e.g., Choi J Y, Calvet C M, Gunatilleke S S, Ruiz
C, Cameron M D, McKerrow J H, et al. Rational development of
4-aminopyridyl-based inhibitors targeting Trypanosoma cruzi CYP51
as anti-chagas agents. J Med Chem. 56(19):7651-68. PMCID: 3864028);
for plasma stability, 100% freshly drawn plasma in LiHeparin was
used with similar sampling and sample preparation with no addition
of microsomes, buffer or NADPH.
In Vivo Pharmacokinetic Analysis of iKIX1
[0268] Male Swiss albino mice were dosed via tail vein (IV;
intravenous, solution in 5% NMP and 10% solutol in saline, dose: 2
mg/kg) or via oral gavage (PO; suspensions in 0.5% w/v Na CMC with
0.1% v/v Tween-80 in water). Blood and brain samples were collected
at 0, 0.083 (for IV only), 0.25, 0.5, 1, 2, 4, 6 (for PO only), 8,
12, and 24 hours for the IV and PO groups. The blood samples were
collected from sets of three mice at each time point. Plasma
samples were separated by centrifugation and stored below
-70.degree. C. until analysis. Brain samples were homogenized using
ice-cold phosphate buffer saline (pH 7.4) and homogenates were
stored below -70.degree. C. until analysis. All samples were
processed and analyzed by C/MS/MS (LLOQ, 2.03 ng/mL for plasma and
10.16 ng/mL for brain). Pharmacokinetic parameters were calculated
using the noncompartmental analysis tool of Phoenix WinNonlin
(version 5.3).
Galleria mellonella Survival Assays
[0269] Galleria mellonella larvae were injected with
5.times.10.sup.6 CFUs of PDR1 wild-type (SFY114, 10 larvae per
group) or PDR1.sup.L280F (SFY115, 9 larvae per group) Candida
glabrata and a single injection of PBS alone, iKIX1 alone (25
mg/kg), fluconazole alone (50 mg/kg) or a combination of
fluconazole and iKIX1. Each larva was injected through the last
right proleg with 40 .mu.L of a cell suspension using a Myjector
U-100 Insulin syringe (Terumo Europe). Compound solutions (40
.mu.L) were injected through remaining prolegs one hour after
infection. As controls, groups of non-infected larvae were injected
with the combination of fluconazole and iKIX1, or not injected with
compounds. Larvae were incubated in the dark at 30.degree. C. and %
survival was assessed every 24 hours.
Yeast Adherence to Epithelial Cells
[0270] Adherence to epithelial cells was tested as previously
described (see, e.g., Vale-Silva L, Ischer F, Leibundgut-Landmann
S, Sanglard D. Gain-of-function mutations in PDR1, a regulator of
antifungal drug resistance in Candida glabrata, control adherence
to host cells. Infect Immun. 81(5): 1709-20. PMCID: 3648025).
Briefly, epithelial Chinese hamster ovary modified cell line Lec2
(CHO-Lec2; ATCC CRL1736) was cultured in high-glucose minimum
essential medium (MEM, Life Technologies) with L-glutamine,
supplemented with 100 U/mL penicillin, 100 .mu.g/mL streptomycin
(Life Technologies), and 10% FBS (Life Technologies). CHO-Lec2
cells were not authenticated or tested for mycoplasma contamination
following receipt from ATCC. Log-phase epithelial cells were seeded
in 24-well plates at a density of 1.0.times.10.sup.5 cells/well in
1 mL of culture medium and allowed to grow to full confluence at
37.degree. C. in humid atmosphere with 5% CO.sub.2, typically for
72 hours. To prepare C. glabrata suspensions for infection,
overnight cultures of test strains were diluted in fresh medium
containing 30 .mu.M iKIX1 or the same volume of DMSO (vehicle
controls) and grown for a minimum of two generations to mid-log
phase. Ketoconazole was added to indicated samples for the last 15
minutes of incubation at a concentration of 40 .mu.M. Log-phase
yeast cultures were washed and resuspended in PBS. Epithelial cell
monolayers were infected with 1:1 mixed yeast suspensions
containing 3.0.times.10.sup.5 yeast cells and the plates were
centrifuged at 200.times.g for 1 minute. Co-cultures were incubated
at 37.degree. C. in humid atmosphere with 5% CO.sub.2 for 30
minutes and nonadherent yeasts were removed by washing. Adherent
yeasts were recovered by lysis of the epithelial cells in 0.1%
Triton X-100 and plated onto YPD agar plates for quantification of
CFUs. YPD agar alone and YPD agar plates containing 30 .mu.g/mL of
fluconazole were used to distinguish between azole-susceptible and
azole-resistant yeast strains.
Mouse Urinary Tract Infection Model
[0271] Urinary tract infection (UTI) experiments were performed
following a previously described model (see, e.g., Chen Y L,
Konieczka J H, Springer D J, Bowen S E, Zhang J, Silao F G, et al.
Convergent Evolution of Calcineurin Pathway Roles in
Thermotolerance and Virulence in Candida glabrata. G3
(Bethesda).2(6):675-91. PMCID: 3362297). Briefly, for tissue burden
experiments each C. glabrata strain was grown in 10 mL of YPD broth
under agitation for 18 hours at 37.degree. C. After growth, cells
were centrifuged, washed and resuspended in 10 mL of sterile PBS,
and then adjusted to reach a concentration of 5.times.10.sup.9 C.
glabrata mL.sup.-1. For each strain, a group of 10
isoflurane-anaesthetized mice were infected via intra-urethral
catheterization (polyethylene catheter, .about.4 cm long; outer
diameter, 0.61 mm; Becton Dickinson. Sparks. Md., USA) using 100
.mu.L of the corresponding C. glabrata cell suspension for each
animal. Mice were sacrificed 7 days after the transurethral
challenge, and, for each animal, bladders and kidney pairs were
harvested, weighed and homogenized in 1 and 5 mL of sterile saline,
respectively. C. glabrata inocula and burdens were enumerated by
performing serial dilutions and counting CFUs on YPD agar. The C.
glabrata detection limits were 50 and 10 CFU mL.sup.-1 for kidneys
and bladder homogenates, respectively. For all experimental groups,
counts of CFU were analysed by unpaired I-test, and a P-value of
less than 0.05 was considered to be significant. For treatment
experiments the same procedure was used but animals were inoculated
intraperitoneally with fluconazole (100 mg/kg/day) and/or iKIX1
(100 mg/kg/day).
Mouse Disseminated Fungal Burden Model
[0272] Experiments were carried out as previously described (27).
Unless otherwise indicated, mice were injected with 100 mg/kg/day
iKIX1. Low fluconazole concentration was 25 mg/kg/day and high
fluconazole concentration was 100 mg/kg/day. Low iKIX1 treatment
was 10 mg/kg/day.
Mouse Studies
[0273] Female BALB/c mice (20 to 25 g) purchased from Harlan Italy
S.r.l (San Pietro al Natisone, Udine, Italy) and inbred in-house
were used for mouse studies. The animal experiments were performed
under a protocol approved by the Institutional Animal Use and Care
Committee at Universita Cattolica del S. Cuore, Rome, Italy (Permit
number: 721, Feb. 10, 2013) and authorized by the Italian Ministry
of Health, according to Legislative Decree 116/92, which
implemented the European Directive 86/609/EEC on laboratory animal
protection in Italy. Veterinarians of the Service for Animal
Welfare routinely checked animal welfare. Statistical differences
were measured using a Wilcoxon Mann-Whitney test and P<0.05 was
considered statistically significant.
Statistics
[0274] Unless otherwise indicated, in vitro experiments were
carried out with at least three biological replicates and graphs
represent mean+/-s.d. Unless otherwise indicated, statistical
differences were measured using an unpaired, two-tailed Student's
t-test. P<0.05 was considered as statistically significant. The
number of animals used in this study was calculated hypothesizing a
prevalence of infection of 99% and a precision of 0.005, using the
formula n=t2P1-PD2, where n is the numerosity of the sample, t the
distribution, P the prevalence and D the precision. Animals were
excluded from analysis if one of the following humane endpoint was
reached: body weight (decrease of more than 20% at baseline), body
temperature (hypothermia extremely important), matted hair (still
expected in the systemic model), posture and behavior (eg.
lethargy). After infection with C. glabrata strains the animals
were randomized in the control and the different treatment groups.
Tissue burden evaluation was carried out in a blinded manner, the
researcher who performed the tissue burden evaluation did not know
if the analyzed organs were from treated or control mouse
groups.
SYNTHESIS OF COMPOUNDS
Synthesis of Compound iKIX1
##STR00069##
[0276] 5.00 g (24.4 mmol) 3,4-dichlorophenyl isothiocyanate
(Oakwood Chemical) and 2.42 g (24.4 mmol) cyanoacetohydrazide
(Acros) were suspended in 50 ml benzene and the reaction mixture
was heated with stirring to reflux for 12 hours. The solvent was
filtered off and the solid product was recrystallized twice from
methanol to yield 2.55 g (8.42 mmol, 35% yield) of white fluffy
crystals. ESI-MS m/z 302.93 (M+H); 1H-NMR (400 MHz, dmso) .delta.
10.37 (s, 1H), 9.97 (s, 1H), 9.79 (br, 1H), 7.88 (br, 0.3H), 7.77
(br, 0.7H), 7.60 (d, 1H, J=8.7 Hz), 7.46 (dd, 1H, J=8.7 Hz. J=2.5
Hz), 3.72 (s, 2H); 13C-NMR (100 MHz, dmso) .delta. 178.6, 164.1,
152.1, 151.6, 150.5, 150.0, 144.4, 139.5, 130.4, 116.1.
Synthesis of SB1-A-01
##STR00070##
[0277]
2-(2-Cyanoacetyl)-N-(3,4-dichlorophenyl)hydrazinecarbothioamide
(SB1-A-01)
[0278] The mixture of SM-1-1 (200 mg, 0.98 mmol) and SM-1-2 (200
mg, 2.02 mmol) in EtOH (15 mL) was stirred at 65.degree. C. for 4
h, then concentrated to remove the solvent. The residue was
purified by prep-HPLC (C18 column, CH.sub.3CN/H.sub.2O. containing
0.05% TFA) to obtain the SB1-A-01 (white solid, 100 mg, yield:
34%). HPLC: 100% (254 nm); LCMS (m/z): 303 [M+H].sup.+; .sup.1H NMR
(DMSO-d.sub.6, 400 MHz): .delta. 10.40 (s, 1H), 10.00 (s, 1H),
9.90-9.70 (br s, 1H), 7.79 (s, 1H), 7.62 (d, J=8.4 Hz, 1H), 7.47
(dd, J.sub.1=8.4 Hz, J.sub.2=2.4 Hz, 1H), 3.74 (s, 2H) ppm.
Synthesis of SB1-A-03
##STR00071##
[0279] 1-Chloro-2-fluoro-4-isothiocyanatobenzene (SB1-A-03-1)
[0280] To a solution of SM-3-1 (300 mg, 2.06 mmol), TEA (2 mL) in
THF (20 mL) was added CSCl.sub.2 (0.3 mL, 3.91 mmol) dropwise, then
stirred at r.t overnight. After completion, the mixture was
concentrated, the residue was dissolved in ethyl acetate (100 mL),
and washed with brine (50 mL.times.2). The organic layer was dried
with Na.sub.2SO.sub.4, filtered, concentrated to obtain brown
sticky oil SB-A-03-1 (330 mg, yield: 85%), which was used directly
without further purification.
N-(4-Chloro-3-fluorophenyl)-2-(2-cyanoacetyl)hydrazinecarbothoamide
(SB1-A-03)
[0281] The mixture of SB1-A-03-1 (330 mg, 1.76 mmol). SM-3-2 (180
mg, 1.82 mmol) in EtOH (20 mL) was stirred at 85.degree. C. for 4
h. then concentrated to removed the solvent. The residue was
purified by prep-HPLC (C18 column, CH.sub.3CN/H.sub.2O, containing
0.05% TFA) to obtain SB1-A-03 (white solid 180 mg, yield: 36%).
HPLC: 100% (254 nm); LCMS (m/z): 287 [M+H]+; .sup.1H NMR
(DMSO-d.sub.6, 400 MHz): .delta. 10.39 (s, 1H), 9.98 (s, 1H), 9.86
(br s, 1H), 7.68 (d, J=9.6 Hz, 1H), 7.57 (t, J=8.8 Hz, 1H), 7.29
(dd, J.sub.1=8.8 Hz, J.sub.2=1.6 Hz, 1H), 3.76 (s, 2H) ppm.
Synthesis of SB1-A-07
##STR00072##
[0282] N-(3-Chloro-4-(trifluoromethyl)phenyl)-2-(2-cyanoacetyl)
hydrazinecarbothioamide (SB1-A-07)
[0283] The mixture of SB1-A-07-1 (396 mg, 1.67 mmol), SM-7-2 (166
mg, 1.67 mmol) in EtOH (20 mL) was stirred at 85.degree. C. for 3
h, then concentrated to remove the solvent. The residue was
purified by prep-HPLC (C18 column, CH.sub.3CN/H.sub.2O, containing
0.05% TFA) to obtain SB1-A-07 (white solid, 170 mg, yield: 30%).
HPLC: 96.17% (254 nm); LCMS (m/z): 333 [M+H].sup.+; .sup.1H NMR
(DMSO-d.sub.6, 400 MHz): .delta. 10.45 (bs, 1H), 10.17 (s, 1H),
9.91 (br s, 1H), 8.00 (s, 1H), 7.85 (d, J=8.4 Hz, 1H), 7.73 (d,
J=8.8 Hz, 1H), 3.76 (s, 2H) ppm.
2-Chloro-4-isothiocyanato-1-(trifluoromethyl)benzene
(SB1-A-07-1)
[0284] To a solution of SM-7-1 (340 mg, 1.74 mmol). NaHCO.sub.3
(438 mg, 5.21 mmol). H.sub.2O (5 mL) and DCM (20 mL) was added
CSCl.sub.2 (0.20 mL, 2.63 mmol) dropwise at 0.degree. C., then the
mixture was stirred for 4 h. after completion, the reaction mixture
was diluted with DCM (200 mL), and washed with brine (50 mL), the
organic phase was dried with Na.sub.2SO.sub.4, filtered, and
concentrated to remove the solvent to obtain SB1-A-07-1 (light
brown solid, 400 mg, yield: 97%).
Synthesis of SB1-A-18
##STR00073##
[0285] 1,2-Dichloro-4-isothlocyanatobenzene (SB1-A-18-1)
[0286] To a solution of 3,4-dichloroaniline (300 mg, 1.86 mmol) and
NaHCO.sub.3 (781 mg, 9.3 mmol) in H.sub.2O (10 mL) and DCM (20 mL)
was added thiophosgene (424 mg, 3.73 mmol) at 0.degree. C., the
mixture was stirred overnight. After completion, the reaction
mixture was extracted with DCM (50 mL), and washed with water, the
organic phase was concentrated under reduced pressure to obtain
SB1-A-18-1 (300 mg crude product) used for the next step
directly.
N-(3,4-Dichlorophenyl)hydrazinecarbothioamide (SB1-A-18-2)
[0287] The mixture of 1,2-dichloro-4-isothiocyanatobenzene (200 mg,
0.985 mmol) and NH.sub.2NH.sub.2--H.sub.2O (60%) (63 mg, 1.18 mmol)
in DCM (10 mL) was stirred at r.t for 2 h, after completion, the
reaction mixture was concentrated under reduced pressure to obtain
SB1-A-18-2 (231 mg, 100%) which was used for the next step
directly. LCMS (m/z): 236.0 (M+H).sup.-.
1-Cyanocyclopropanecarbonyl Chloride (SB1-A-18-3)
[0288] To a solution of 1-cyanocyclopropanecarboxylic acid (80 mg,
0.72 mmol) and oxalyl dichloride (136 mg, 1.08 mmol) in DCM (5 mL)
was added a drop of DMF at 0.degree. C., then the mixture was
stirred for 2 h. after completion the reaction mixture solution was
used for next step directly.
2-(1-Cyanocyclopropanecarbonyl)-N-(3,4-dichlorophenyl)hydrazinecarbothioam-
ide (SB1-A-18)
[0289] To a solution of
N-(3,4-dichlorophenyl)hydrazinecarbothioamide (150 mg, 0.63 mmol)
and pyridine (249 mg, 3.15 mmol) in DCM (10 mL) was added the above
solution of 1-cyanocyclopropanecarbonyl chloride (4 mL) at
0.degree. C., then the mixture was stirred for 1 h. then
concentrated under reduced pressure to remove the solvent, the
residue was purified by prep-HPLC (C18 column, CH.sub.3CN/H.sub.2O,
containing 0.05% TFA) to obtain SB1-A-18 (white solid, 7 mg, yield:
4%). HPLC: 100% (254 nm); LCMS (m/z): 329.0 [M+H]*; .sup.1H NMR
(DMSO-d.sub.6 400 MHz): .delta. 10.34 (s, 1H), 9.87 (s, 1H), 7.78
(s, 1H), 7.61 (d, J=9.2 Hz, 1H), 7.47-7.50 (m, 1H), 1.59-1.70 (m,
4H) ppm.
EQUIVALENTS AND SCOPE
[0290] In the claims articles such as "a," "an," and "the" may mean
one or more than one unless indicated to the contrary or otherwise
evident from the context. Claims or descriptions that include "or"
between one or more members of a group are considered satisfied if
one, more than one, or all of the group members are present in,
employed in, or otherwise relevant to a given product or process
unless indicated to the contrary or otherwise evident from the
context. The invention includes embodiments in which exactly one
member of the group is present in, employed in, or otherwise
relevant to a given product or process. The invention includes
embodiments in which more than one, or all of the group members are
present in, employed in, or otherwise relevant to a given product
or process.
[0291] Furthermore, the invention encompasses all variations,
combinations, and permutations in which one or more limitations,
elements, clauses, and descriptive terms from one or more of the
listed claims is introduced into another claim. For example, any
claim that is dependent on another claim can be modified to include
one or more limitations found in any other claim that is dependent
on the same base claim. Where elements are presented as lists,
e.g., in Markush group format, each subgroup of the elements is
also disclosed, and any element(s) can be removed from the group.
It should it be understood that, in general, where the invention,
or aspects of the invention, is/are referred to as comprising
particular elements and/or features, certain embodiments of the
invention or aspects of the invention consist, or consist
essentially of, such elements and/or features. For purposes of
simplicity, those embodiments have not been specifically set forth
in haec verba herein.
[0292] It is also noted that the terms "comprising" and
"containing" are intended to be open and permits the inclusion of
additional elements or steps. Where ranges are given. endpoints are
included. Furthermore, unless otherwise indicated or otherwise
evident from the context and understanding of one of ordinary skill
in the art, values that are expressed as ranges can assume any
specific value or sub-range within the stated ranges in different
embodiments of the invention, to the tenth of the unit of the lower
limit of the range, unless the context clearly dictates
otherwise.
[0293] This application refers to various issued patents, published
patent applications, journal articles, and other publications, all
of which are incorporated herein by reference. If there is a
conflict between any of the incorporated references and the instant
specification, the specification shall control. In addition, any
particular embodiment of the present invention that falls within
the prior art may be explicitly excluded from any one or more of
the claims. Because such embodiments are deemed to be known to one
of ordinary skill in the art, they may be excluded even if the
exclusion is not set forth explicitly herein. Any particular
embodiment of the invention can be excluded from any claim, for any
reason, whether or not related to the existence of prior art.
[0294] Those skilled in the art will recognize or be able to
ascertain using no more than routine experimentation many
equivalents to the specific embodiments described herein. The scope
of the present embodiments described herein is not intended to be
limited to the above Description, but rather is as set forth in the
appended claims. Those of ordinary skill in the art will appreciate
that various changes and modifications to this description may be
made without departing from the spirit or scope of the present
invention, as defined in the following claims.
Sequence CWU 1
1
5130PRTArtificial SequenceSynthetic polypeptideMISC_FEATUREModified
by FITC 1Leu Gly Thr Leu Asp Glu Phe Val Asn Lys Gly Asp Leu Asn
Glu Leu1 5 10 15Tyr Asn Ser Leu Trp Gly Asp Leu Phe Ser Asp Val Tyr
Leu 20 25 30243PRTSaccharomyces cerevisiae 2Val Gln Asp Lys Asp Thr
Leu Ser Asn Ala Glu Arg Ala Lys Asn Val1 5 10 15Asn Gly Leu Leu Gln
Val Leu Met Asp Ile Asn Thr Leu Asn Gly Gly 20 25 30Ser Ser Asp Thr
Ala Asp Lys Ile Arg Ile His 35 40343PRTCandida glabrata 3Met Ser
Ser Lys Glu Thr Ile Pro Met His Gln Arg Ser Gln Asn Val1 5 10 15Ala
Glu Leu Leu Thr Val Leu Met Asp Ile Asn Lys Ile Asn Gly Gly 20 25
30Asp Ser Thr Thr Ala Glu Lys Met Lys Val His 35
40442PRTSaccharomyces cerevisiae 4Ala Lys Asn Phe Glu Ala Ala Leu
Phe Ala Lys Ser Ser Ser Lys Lys1 5 10 15Glu Tyr Met Asp Ser Met Asn
Glu Lys Val Ala Val Met Arg Asn Thr 20 25 30Tyr Asn Thr Arg Lys Asn
Ala Val Thr Ala 35 40543PRTCandida glabrata 5Ala Lys Ser Phe Glu
Ala Ala Leu Phe Glu Lys Ser Ser Ser Lys Glu1 5 10 15Glu Tyr Gln Lys
Thr Met Lys Ser Lys Ile Asp Ala Met Arg Ser Thr 20 25 30Arg Asp Lys
Arg Lys Arg Glu Ser Val Gly Ser 35 40
* * * * *