U.S. patent application number 15/255466 was filed with the patent office on 2016-12-22 for linked purine pterin hppk inhibitors useful as antibacterial agents.
The applicant listed for this patent is THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY, DEPT. OF HEALTH AND HUMAN SERVICES, THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY, DEPT. OF HEALTH AND HUMAN SERVICES. Invention is credited to Xinhua Ji, Gary X. Shaw, Genbin Shi.
Application Number | 20160368944 15/255466 |
Document ID | / |
Family ID | 45348793 |
Filed Date | 2016-12-22 |
United States Patent
Application |
20160368944 |
Kind Code |
A1 |
Shi; Genbin ; et
al. |
December 22, 2016 |
LINKED PURINE PTERIN HPPK INHIBITORS USEFUL AS ANTIBACTERIAL
AGENTS
Abstract
The disclosure provides linked purine pterin compounds and
analogues thereof that are novel HPPK inhibitors. The HPPK
inhibitors described herein are compounds and the pharmaceutically
acceptable salts thereof of general Formula I: (I). The variables,
e.g. A1 to A3, R1 to R4, L1, L2, B1, and B2 are described herein.
Compounds and salts of Formula I bind to HPPK with high affinity
and specificity. Pharmaceutical compositions containing an HPPK
inhibitor of Formula I and methods of treating a bacterial
infection in a patient by providing one or more HPPK inhibitors of
Formula I to the patient are also provided. Processes and
intermediates useful for preparing compounds of Formula I are also
provided. Methods of using the disclosed compounds to guide the
development of additional novel anti-bacterial agents are also
provided. ##STR00001##
Inventors: |
Shi; Genbin; (Frederick,
MD) ; Shaw; Gary X.; (North Potomac, MD) ; Ji;
Xinhua; (Frederick, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY,
DEPT. OF HEALTH AND HUMAN SERVICES |
Bethesda |
MD |
US |
|
|
Family ID: |
45348793 |
Appl. No.: |
15/255466 |
Filed: |
September 2, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14689449 |
Apr 17, 2015 |
9453019 |
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15255466 |
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13704857 |
Dec 17, 2012 |
9029344 |
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PCT/US2011/038744 |
Jun 1, 2011 |
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14689449 |
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61356213 |
Jun 18, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 475/04 20130101;
G16B 15/00 20190201; A61P 31/00 20180101; C07H 19/167 20130101;
C07H 19/16 20130101 |
International
Class: |
C07H 19/16 20060101
C07H019/16 |
Claims
1-2. (canceled)
3. A compound having the formula: ##STR00018## or a
pharmaceutically acceptable salt thereof, wherein: A.sub.1 is oxo;
A.sub.3 are each independently hydrogen, halogen, methyl, or
methoxy; R.sub.1 is methylene optionally substituted with oxo;
R.sub.2 is NH; R.sub.3 is absent or R.sub.3 is an amide,
heteroalkylene, cycloalkyl, heterocycloalkyl, phenyl, or 5- or
6membered heteroaryl; and R.sub.3 is unsubstituted or substituted
with 1 or more substituents independently chosen from hydroxyl,
halogen, amino, C.sub.1-C.sub.4alkyl, , C.sub.1-C.sub.4alkoxy,
mono- and di-C.sub.1-C.sub.4alkylamino, C.sub.1-C.sub.2haloalkyl,
and C.sub.1-C.sub.2haloalkoxy; R.sub.4 in a group ##STR00019## is a
5- or 6-membered monosaccharide ring; L.sub.1 is an alkylene linker
having from 1 to 4 carbon atoms, wherein L.sub.1 is unsubstituted
or substituted with 1 or more substituents independently chosen
from hydroxyl, halogen, amino, C.sub.1-C.sub.4alkyl,
C.sub.1-C.sub.4alkoxy, C.sub.1-C.sub.2haloalkyl, and
C.sub.1-C.sub.2haloalkoxy. L.sub.2 is an alkylene linker having
from 1 to 4 carbon atoms, optionally containing 1 heteroatom
selected from oxygen, nitrogen, sulfur, and phosphorus and
optionally containing 1 to 2 carbon-carbon double bonds, wherein
L.sub.2 is unsubstituted or substituted with 1 or more substituents
independently chosen from oxo, hydroxyl, halogen, amino,
C.sub.1-C.sub.4alkyl, C.sub.1-C.sub.4alkoxy,
C.sub.1-C.sub.2haloalkyl, and C.sub.1-C.sub.2haloalkoxy; and
B.sub.1 and B.sub.2 are independently chosen from hydrogen,
halogen, hydroxyl, amino, C.sub.1-C.sub.2alkyl,
C.sub.1-C.sub.2alkoxy, mono- or di-C.sub.1-C.sub.2alkylamino,
aminoC.sub.1-C.sub.2alkyl, C.sub.1-C.sub.2haloalkyl. and
C.sub.1-C.sub.2haloalkoxy.
4. (canceled)
5. A compound or salt thereof of claim 3, wherein each A.sub.3
methyl; and R.sub.1 is methylene substituted with oxo.
6-11. (canceled)
12. A compound or salt thereof of claim 5, wherein R.sub.4 is a 5
or 6-membered monosaccharide ring.
13. A compound or salt thereof of claim 12, wherein R.sub.4 is
d-ribose, d-arabinose, d-xylose or d-lyxose.
14. A compound or salt thereof of claim 13 wherein R.sub.4 is
d-ribose and B.sub.1 and B.sub.2 are both hydrogen.
15-17. (canceled)
18. A pharmaceutical composition comprising a compound or salt of
claim 3 and a pharmaceutically acceptable carrier.
19. A method of treating a condition responsive to HPPK modulation,
the method comprising providing a therapeutically effective amount
of compound of claim 3 to a patient having a condition response to
HPPK modulation.
20. The method of claim 19, wherein the condition is a bacterial
infection.
21-27. (canceled)
28. A compound or salt of claim 5, wherein B.sub.1 and B.sub.2 are
both hydrogen.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims priority from U.S. Provisional Appl.
No. 61/356,213, filed Jun. 18, 2010, which is hereby incorporated
by reference in its entirety.
FIELD OF THE DISCLOSURE
[0002] The disclosure provides linked purine pterin compounds that
are novel HPPK inhibitors and new intermediates for the synthesis
of such compounds. These linked purine pterin inhibitors bind to
HPPK with high affinity and specificity. Pharmaceutical
compositions containing HPPK inhibitors and methods of treating a
bacterial infection in a patient with one or more of the HPPK
inhibitors of the disclosure are also provided. Methods of using
the disclosed compounds and intermediates to guide the development
of additional novel anti-bacterial agents are also provided.
BACKGROUND
[0003] Folate cofactors are essential for life. Mammals derive
folates from their diet, whereas most microorganisms must
synthesize folates de novo. Therefore, the folate pathway is an
ideal target for developing anti-bacterial agents. For example,
inhibitors of two enzymes in the pathway, dihydropteroate synthase
and dihydrofolate reductase, are currently used as antibiotics.
6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (E.C. 2.7.6.3,
HPPK), a kinase responsible for an essential step in the
biosynthesis of folic acid, catalyzes the transfer of pyrophosphate
from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP) (FIG. 1)
(Shiota, T., 1984, in Chemistry and Biochemistry of Folates, R. T.
Blakley, and S. J. Benkovic, eds. pp. 121-134, New York: John Wiley
& Sons). No existing antibiotic is known to modulate HPPK
activity. Due to in-depth structural and mechanistic studies of
HPPK, this enzyme is well understood and therefore a good target
for novel anti-bacterial compounds.
[0004] Two types of HPPK inhibitors (FIG. 1) have been reported
(Derrick, J. P. (2008) The structure and mechanism of
6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase, in G. Litwack,
ed. Folic Acid and Folates, (Oxford, UK: Academic Press). Type 1
inhibitors are HP derivatives, including HP-1 (Hennig, M. et al.,
J. Mol. Biol. (1999) 287: 211-219; Wood, H. C. S. (1975) Specific
inhibition of dihydrofolate biosynthesis--A new approach to
chemotherapy, in Chemistry and Biology of Pteridines, W.
Pfleiderer, ed. (Berlin-New York: Walter de Gruyter)) and HP-3.
Type 2 inhibitors are bisubstrate analogues HP.sub.nA (n=2, 3, or
4) (Shi, G. et al., J. of Med. Chem. (2001) 44: 1364-1371). No
pharmaceutically useful HPPK inhibitors have been identified to
date. Thus, there remains a need for novel and useful HPPK
inhibitor anti-bacterial compounds. The present disclosure fulfills
this need and provides further advantages, which are set forth
below.
SUMMARY
[0005] Compound of Formula I
##STR00002##
and the pharmaceutically acceptable salts thereof are provided
herein. Within Formula I the variables, e.g. A.sub.1 to A.sub.3,
R.sub.1 to R.sub.4, L.sub.1, L.sub.2, B.sub.1, and B.sub.2 carry
the following definitions.
[0006] The cyclic group
##STR00003##
is a 5- or 6-membered heterocyclic ring in which X is nitrogen,
--N(A.sub.5)--, --C(A.sub.6)--, or --C(A.sub.6A.sub.7)--.
[0007] A.sub.1 is hydrogen, oxo, amino, or
aminoC.sub.1-C.sub.2alkyl.
[0008] A.sub.2 is absent, hydrogen, or C.sub.1-C.sub.2alkyl.
[0009] A.sub.3 is absent, or one or two substituents independently
chosen from hydrogen, halogen, C.sub.1-C.sub.2alkyl, and
C.sub.1-C.sub.2alkoxy.
[0010] A.sub.5 is hydrogen or C.sub.1-C.sub.4alkyl.
[0011] A.sub.6 and A.sub.7 are independently hydrogen,
C.sub.1-C.sub.4alkyl, or C.sub.1-C.sub.4alkoxy.
[0012] R.sub.1 is methylene, optionally substituted with hydroxyl,
halogen, C.sub.1-C.sub.2alkyl, or oxo.
[0013] R.sub.2 is chosen from --NH--, --SH--, --S(.dbd.O)--,
--S(.dbd.O).sub.2--, --P(.dbd.O)--, and P(.dbd.O).sub.2--.
[0014] L.sub.1 is an alkylene linker having from 1 to 4 carbon
atoms, optionally containing 1 heteroatom selected from oxygen,
nitrogen, and sulfur, and optionally containing 1 to 2
carbon-carbon double bonds, wherein L.sub.1 is unsubstituted or
substituted with 1 or more substituents independently chosen from
hydroxyl, halogen, amino, C.sub.1-C.sub.4alkyl,
C.sub.1-C.sub.4alkoxy, C.sub.1-C.sub.2haloalkyl, and
C.sub.1-C.sub.2haloalkoxy.
[0015] R.sub.3 is absent or R.sub.3 is an amide, heteroalkylene,
cycloalkyl, heterocycloalkyl, phenyl, or 5- or 6-membered
heteroaryl; and R.sub.3 is unsubstituted or substituted with 1 or
more substituents independently chosen from hydroxyl, halogen,
amino, C.sub.1-C.sub.4alkyl, C.sub.1-C.sub.4alkoxy, mono- and
di-C.sub.1-C.sub.4alkylamino, C.sub.1-C.sub.2haloalkyl, and
C.sub.1-C.sub.2haloalkoxy.
[0016] L.sub.2 is absent or L.sub.2 is an alkylene linker having
from 1 to 4 carbon atoms, optionally containing 1 heteroatom
selected from oxygen, nitrogen, sulfur, and phosphorus and
optionally containing 1 to 2 carbon-carbon double bonds, wherein
L.sub.2 is unsubstituted or substituted with 1 or more substituents
independently chosen from oxo, hydroxyl, halogen, amino,
C.sub.1-C.sub.4alkyl, C.sub.1-C.sub.4alkoxy,
C.sub.1-C.sub.2haloalkyl, and C.sub.1-C.sub.2haloalkoxy.
[0017] R.sub.4 is a 5- or 6-membered monosaccharide ring or R.sub.1
is an alkylene or heteroalkylene linker having from 1 to 4 chain
atoms.
[0018] B.sub.1 and B.sub.2 are independently chosen from hydrogen,
halogen, hydroxyl, amino, C.sub.1-C.sub.2alkyl,
C.sub.1-C.sub.2alkoxy, mono- or di-C.sub.1-C.sub.2alkylamino,
aminoC.sub.1-C.sub.2alkyl, C.sub.1-C.sub.2haloalkyl, and
C.sub.1-C.sub.2haloalkoxy.
[0019] Pharmaceutical compositions comprising a compound or salt of
Formula I together with a pharmaceutically acceptable carrier are
further provided herein.
[0020] A method of treating a condition responsive to HPPK
modulation, comprising providing a therapeutically effective amount
of compound of any one of claims 1 to 17 to a patient having a
condition response to HPPK modulation is also provided herein.
Conditions responsive to HPPK modulation include bacterial
infections.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1. Scheme showing HPPK catalyzing the transfer of
pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin.
[0022] FIG. 2. The titration curve of (A) BSS10113 and (B) BSS10128
for binding studies.
[0023] FIG. 3. Stereo illustration of the (A) HPPK.cndot.BSS10113,
(B) HPPK.cndot.BSS10124, and (C) HPPK.cndot.BSS10128 structure.
Polypeptide chains are shown as a ribbon diagrams. Ligands are
shown as sticks and overlapped with the 2F.sub.o-F.sub.c annealed
omit maps contoured at 0.90.sigma. (nets in blue).
[0024] FIG. 4. Stereoview showing the superposition of (A) the
HPPK.cndot.BSS10113 (disclosed here) and HPPK.cndot.AMP.cndot.HPPP
(Protein Data Bank (PDB) entry 1RAO) structures and (B) the
HPPK.cndot.BSS10128 (disclosed here) and
HPPK.cndot.MgAMPCPP.cndot.HP (PDB entry 1Q0N) structures. Proteins
are shown as C.alpha. traces and ligands as stick models.
DETAILED DISCLOSURE
Terminology
[0025] It is helpful to provide definitions of certain terms to be
used herein, prior to setting out the disclosure in detail.
[0026] Presently disclosed compounds are described using standard
nomenclature. Unless defined otherwise, all technical and
scientific terms used herein have the same meaning as is commonly
understood by one of skill in the art to which this disclosure
belongs. Unless clearly contraindicated by the context each
compound name includes the free acid or free base form of the
compound as all pharmaceutically acceptable salts of the
compound.
[0027] The phrase "compounds of Formula I" encompasses all
compounds that satisfy Formula I, including any enantiomers,
racemates and stereoisomers, as well as all pharmaceutically
acceptable salts of such compounds and also includes all subgeneric
groups of Formula I, unless clearly contraindicated by the context
in which this phrase is used.
[0028] The terms "a" and "an" do not denote a limitation of
quantity, but rather denote the presence of at least one of the
referenced item. The term "or" means "and/or". The terms
"comprising", "having", "including", and "containing" are to be
construed as open-ended terms (i.e., meaning "including, but not
limited to"). Recitation of ranges of values are merely intended to
serve as a shorthand method of referring individually to each
separate value falling within the range, unless otherwise indicated
herein, and each separate value is incorporated into the
specification as if it were individually recited herein. The
endpoints of all ranges are included within the range and
independently combinable. All methods described herein can be
performed in a suitable order unless otherwise indicated herein or
otherwise clearly contradicted by context. The use of any and all
examples, or exemplary language (e.g., "such as"), is intended
merely to better illustrate and does not pose a limitation on the
scope of the disclosure unless otherwise claimed. No language in
the specification should be construed as indicating any non-claimed
element as essential to the practice as used herein. Unless defined
otherwise, technical and scientific terms used herein have the same
meaning as is commonly understood by one of skill in the art to
which this disclosure belongs.
[0029] An "active agent" means a compound (including a compound
disclosed herein), element, or mixture that when administered to a
patient, alone or in combination with another compound, element, or
mixture, confers, directly or indirectly, a physiological effect on
the patient. The indirect physiological effect may occur via a
metabolite or other indirect mechanism.
[0030] A dash ("--") that is not between two letters or symbols is
used to indicate a point of attachment for a substituent. For
example, --(C.dbd.O)NH.sub.2 is attached through carbon of the keto
(C.dbd.O) group.
[0031] A bond represented by a combination of a solid and dashed
line, i.e. , may be either a single or double bond.
[0032] "Alkyl" is a branched or straight chain saturated aliphatic
hydrocarbon group, having the specified number of carbon atoms,
generally from 1 to about 12 carbon atoms. The term
C.sub.1-C.sub.6alkyl indicates an alkyl group having from 1, 2, 3,
4, 5, or 6 carbon atoms. Other embodiments include alkyl groups
having from 1 to 8 carbon atoms, 1 to 4 carbon atoms or 1 or 2
carbon atoms, e.g. C.sub.1-C.sub.8alkyl, C.sub.1-C.sub.4alkyl, and
C.sub.1-C.sub.2alkyl. When C.sub.0-C.sub.n alkyl is used herein in
conjunction with another group, for example, (aryl)C.sub.0-C.sub.4
alkyl, the indicated group, in this case aryl, is either directly
bound by a single covalent bond (C.sub.0), or attached by an alkyl
chain having the specified number of carbon atoms, in this case 1,
2, 3, or 4 carbon atoms. C.sub.0-C.sub.n alkyl is used in
conjunction with heteroaryl, aryl, phenyl, cycloalkyl, and
heterocycloalkyl, e.g., (5- to 10-membered
heteroaryl)C.sub.0-C.sub.2alkyl, (aryl)C.sub.0-C.sub.2alkyl,
(phenyl)C.sub.0-C.sub.2alkyl,
(C.sub.3-C.sub.7cycloalkyl)C.sub.0-C.sub.4alkyl, and
(heterocycloalkyl)C.sub.0-C.sub.4alkyl. Examples of alkyl include,
but are not limited to, methyl, ethyl, n-propyl, isopropyl,
n-butyl, 3-methylbutyl, t-butyl, n-pentyl, and sec-pentyl.
[0033] "Alkylene" is a saturated organic radical of the formula
--(CH.sub.2).sub.n-- where n is the number of CH.sub.2 groups in
the alkylene radical. Alkylene radicals having from 1 to 6 carbons
or from 1 to 4 carbons are usually preferred. Likewise
"heteroalkylene" is a saturated organic radical of the formula
--(CH.sub.2).sub.n-- where n is the number of CH.sub.2 groups in
the chain and the alkylene chain is interrupted at one or more
points, usually one point, with a covalently bound heteroatom
selected from nitrogen, oxygen, sulfur, and phosphorous.
[0034] "Alkoxy" is an alkyl group as defined above with the
indicated number of carbon atoms attached through an oxygen bridge
(--O--). Examples of alkoxy include, but are not limited to,
methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, 2-butoxy,
t-butoxy, n-pentoxy, 2-pentoxy, 3-pentoxy, isopentoxy, neopentoxy,
n-hexoxy, 2-hexoxy, 3-hexoxy, and 3-methylpentoxy.
[0035] "Aryl" is an aromatic group containing only carbon in the
aromatic ring or rings. Such aromatic groups may be further
substituted with carbon or non-carbon atoms or groups. Typical aryl
groups contain 1 or 2 separate, fused, or pendant rings and from 6
to about 12 ring atoms, without heteroatoms as ring members. Where
indicated aryl groups may be substituted. Such substitution may
include fusion to a 5 to 7-membered saturated cyclic group that
optionally contains 1 or 2 heteroatoms independently chosen from N,
O, and S, to form, for example, a 3,4-methylenedioxy-phenyl group.
Aryl groups include, for example, phenyl, naphthyl, including
1-naphthyl and 2-naphthyl, and bi-phenyl. An "aryloxy" group is an
aryl group as described herein bound to the group it substitutes
via an oxygen bridge.
[0036] "Cycloalkyl" is a saturated hydrocarbon ring group, having
the specified number of carbon atoms. Monocyclic cycloalkyl groups
typically have from 3 to about 8 carbon ring atoms or from 3 to 7
(3, 4, 5, 6, or 7) carbon ring atoms. Cycloalkyl substituents may
be pendant from a substituted nitrogen or carbon atom, or a
substituted carbon atom that may have two substituents may have a
cycloalkyl group, which is attached as a spiro group. Examples of
cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, or
cyclohexyl as well as bridged or caged saturated ring groups such
as norbornane or adamantane.
[0037] "Haloalkyl" is both branched and straight-chain alkyl groups
having the specified number of carbon atoms, substituted with 1 or
more halogen atoms, up to the maximum allowable number of halogen
atoms. Examples of haloalkyl include, but are not limited to,
trifluoromethyl, difluoromethyl, 2-fluoroethyl, and
penta-fluoroethyl.
[0038] "Haloalkoxy" is a haloalkyl group as defined herein attached
through an oxygen bridge (oxygen of an alcohol radical).
[0039] "Halo" or "halogen" is any of fluoro, chloro, bromo, and
iodo.
[0040] "Heteroaryl" is a stable monocyclic aromatic ring having the
indicated number of ring atoms which contains from 1 to 3, or in
some embodiments from 1 to 2, heteroatoms chosen from N, O, and S,
with remaining ring atoms being carbon, or a stable bicyclic or
tricyclic system containing at least one 5- to 7-membered aromatic
ring which contains from 1 to 3, or in some embodiments from 1 to
2, heteroatoms chosen from N, O, and S, with remaining ring atoms
being carbon. Monocyclic heteroaryl groups typically have from 5 to
7 ring atoms. When the total number of S and O atoms in the
heteroaryl group exceeds 1, these heteroatoms are not adjacent to
one another. It is preferred that the total number of S and O atoms
in the heteroaryl group is not more than 2. It is particularly
preferred that the total number of S and O atoms in the aromatic
heterocycle is not more than 1. Examples of heteroaryl groups
include, but are not limited to, oxazolyl, pyranyl, pyrazinyl,
pyrazolopyrimidinyl, pyrazolyl, pyridizinyl, pyridyl, pyrimidinyl,
pyrrolyl, quinolinyl, tetrazolyl, thiazolyl, thienylpyrazolyl,
thiophenyl, triazolyl, benzo[d]oxazolyl, benzofuranyl,
benzothiazolyl, furanyl, imidazolyl, indolyl, and isoxazolyl.
[0041] "Heterocycloalkyl" is a saturated monocyclic group having
the indicated number of ring atoms and containing from 1 to about 3
heteroatoms chosen from N, O, and S, with remaining ring atoms
being carbon, or a saturated bicyclic ring system having at least
one N, O, or S ring atom with the remaining atoms being carbon.
Monocyclic heterocycloalkyl groups usually have from 4 to about 8
ring atoms. In some embodiments monocyclic heterocycloalkyl groups
have from 5 to 7 ring atoms.
[0042] The term "mono- and/or di-alkylamino" indicates secondary or
tertiary alkyl amino groups, wherein the alkyl groups are
independently chosen alkyl groups, as defined herein, having the
indicated number of carbon atoms. The point of attachment of the
alkylamino group is on the nitrogen. Examples of mono- and
di-alkylamino groups include ethylamino, dimethylamino, and
methyl-propyl-amino.
[0043] A "monosaccharide" is any of several carbohydrates, such as
tetroses, pentoses, and hexoses, which cannot be broken down to
simpler sugars by hydrolysis. When a monosaccharide is recited as a
component of Formula I a divalent radical of a monosaccharide is
intended. Such a monosaccharide is covalently bound through the
oxygen atoms of two of its hydroxyl groups to the purine nitrogen
and L.sub.2 (if present) or R.sub.3 (if L.sub.2 is absent) or
L.sub.1 (if both L.sub.2 and R.sub.3 are absent).
[0044] The term "substituted" means that any one or more hydrogens
on the designated atom or group is replaced with a selection from
the indicated group, provided that the designated atom's normal
valence is not exceeded. When the substituent is oxo (i.e., .dbd.O)
then 2 hydrogens on the atom are replaced. When an oxo group
substitutes aromatic moieties, the corresponding partially
unsaturated ring replaces the aromatic ring. For example a pyridyl
group substituted by oxo is a pyridone. Combinations of
substituents and/or variables are permissible only if such
combinations result in stable compounds or useful synthetic
intermediates. A stable compound or stable structure is meant to
imply a compound that is sufficiently robust to survive isolation
from a reaction mixture, and subsequent formulation into an
effective therapeutic agent. Unless otherwise specified
substituents are named into the core structure. For example, it is
to be understood that when aminoalkyl is listed as a possible
substituent the point of attachment of this substituent to the core
structure is in the alkyl portion.
[0045] Suitable groups that may be present on a "substituted"
position include, but are not limited to, e.g., halogen; cyano;
hydroxyl; nitro; azido; alkanoyl (such as a C.sub.2-C.sub.6
alkanoyl group such as acyl or the like); carboxamido; alkyl groups
(including cycloalkyl groups) having 1 to about 8 carbon atoms, or
1 to about 6 carbon atoms; alkenyl and alkynyl groups including
groups having one or more unsaturated linkages and from 2 to about
8, or 2 to about 6 carbon atoms; alkoxy groups having one or more
oxygen linkages and from 1 to about 8, or from 1 to about 6 carbon
atoms; aryloxy such as phenoxy; alkylthio groups including those
having one or more thioether linkages and from 1 to about 8 carbon
atoms, or from 1 to about 6 carbon atoms; alkylsulfinyl groups
including those having one or more sulfinyl linkages and from 1 to
about 8 carbon atoms, or from 1 to about 6 carbon atoms;
alkylsulfonyl groups including those having one or more sulfonyl
linkages and from 1 to about 8 carbon atoms, or from 1 to about 6
carbon atoms; aminoalkyl groups including groups having one or more
N atoms and from 1 to about 8, or from 1 to about 6 carbon atoms;
aryl having 6 or more carbons and one or more rings, (e.g., phenyl,
biphenyl, naphthyl, or the like, each ring either substituted or
unsubstituted aromatic); arylalkyl having 1 to 3 separate or fused
rings and from 6 to about 18 ring carbon atoms, with benzyl being
an exemplary arylalkyl group; arylalkoxy having 1 to 3 separate or
fused rings and from 6 to about 18 ring carbon atoms, with
benzyloxy being an exemplary arylalkoxy group; or a saturated,
unsaturated, or aromatic heterocyclic group having 1 to 3 separate
or fused rings with 3 to about 8 members per ring and one or more
N, O or S atoms, e.g., coumarinyl, quinolinyl, isoquinolinyl,
quinazolinyl, pyridyl, pyrazinyl, pyrimidinyl, furanyl, pyrrolyl,
thienyl, thiazolyl, triazinyl, oxazolyl, isoxazolyl, imidazolyl,
indolyl, benzofuranyl, benzothiazolyl, tetrahydrofuranyl,
tetrahydropyranyl, piperidinyl, morpholinyl, piperazinyl, and
pyrrolidinyl. Such heterocyclic groups may be further substituted,
e.g. with hydroxy, alkyl, alkoxy, halogen and amino.
[0046] A "dosage form" means a unit of administration of an active
agent. Examples of dosage forms include tablets, capsules,
injections, suspensions, liquids, emulsions, creams, ointments,
suppositories, inhalable forms, transdermal forms, and the
like.
[0047] "Pharmaceutical compositions" are compositions comprising at
least one active agent, such as a compound or salt of Formula I,
and at least one other substance, such as a carrier. Pharmaceutical
compositions meet the U.S. FDA's GMP (good manufacturing practice)
standards for human or non-human drugs.
[0048] "Pharmaceutically acceptable salts" includes derivatives of
the disclosed compounds in which the parent compound is modified by
making inorganic and organic, non-toxic, acid or base addition
salts thereof. The salts of the present compounds can be
synthesized from a parent compound that contains a basic or acidic
moiety by conventional chemical methods. Generally, such salts can
be prepared by reacting free acid forms of these compounds with a
stoichiometric amount of the appropriate base (such as Na, Ca, Mg,
or K hydroxide, carbonate, bicarbonate, or the like), or by
reacting free base forms of these compounds with a stoichiometric
amount of the appropriate acid. Such reactions are typically
carried out in water or in an organic solvent, or in a mixture of
the two. Generally, non-aqueous media like ether, ethyl acetate,
ethanol, isopropanol, or acetonitrile are preferred, where
practicable. Salts of the present compounds further include
solvates of the compounds and of the compound salts.
[0049] Examples of pharmaceutically acceptable salts include, but
are not limited to, mineral or organic acid salts of basic residues
such as amines; alkali or organic salts of acidic residues such as
carboxylic acids; and the like. The pharmaceutically acceptable
salts include the conventional non-toxic salts and the quaternary
ammonium salts of the parent compound formed, for example, from
non-toxic inorganic or organic acids. For example, conventional
non-toxic acid salts include those derived from inorganic acids
such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric,
nitric and the like; and the salts prepared from organic acids such
as acetic, propionic, succinic, glycolic, stearic, lactic, malic,
tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic,
phenylacetic, glutamic, benzoic, salicylic, mesylic, esylic,
besylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic,
methanesulfonic, ethane disulfonic, oxalic, isethionic,
HOOC--(CH.sub.2).sub.n--COOH where n is 0-4, and the like.
[0050] The term "carrier" applied to pharmaceutical compositions of
the disclosure refers to a diluent, excipient, or vehicle with
which an active compound is provided. An excipient is an inactive
ingredient useful in preparing a pharmaceutical composition that is
generally safe, non-toxic, and neither biologically nor otherwise
undesirable, and includes an excipient that is acceptable for
veterinary use as well as human pharmaceutical use.
[0051] A "patient" is a human or non-human animal in need of
medical treatment. Medical treatment can include treatment of an
existing condition, such as a disease or disorder, prophylactic or
preventative treatment, or diagnostic treatment. In some
embodiments the patient is a human patient.
[0052] "Providing" means giving, administering, selling,
distributing, transferring (for profit or not), manufacturing,
compounding, or dispensing.
[0053] "Providing a compound of Formula I with at least one
additional active agent" means the compound of Formula I and the
additional active agent(s) are provided simultaneously in a single
dosage form, provided concomitantly in separate dosage forms, or
provided in separate dosage forms for administration separated by
some amount of time that is within the time in which both the
compound of Formula I and at least one additional active agent are
within the blood stream of a patient. The compound of Formula I and
the additional active agent need not be prescribed for a patient by
the same medical care worker. The additional active agent or agents
need not require a prescription. Administration of the compound of
Formula I or the at least one additional active agent can occur via
any appropriate route, for example, oral tablets, oral capsules,
oral liquids, inhalation, injection, suppositories or topical
contact.
[0054] "Treatment" as used herein includes providing a compound of
Formula I, either as the only active agent or together with at
least one additional active agent sufficient to: (a) inhibiting the
disease, i.e., arresting its development; and (b) relieving the
disease, i.e., causing regression of the disease. Bacterial
infections, such as bacterial infections and fungal infections, are
included in the diseases treated with a compound of Formula I.
"Treating" and "treatment" also means providing a therapeutically
effective amount of a compound of Formula I, as the only active
agent or together with at least one additional active agent to a
patient having a bacterial infection.
[0055] A "therapeutically effective amount" of a compound of
Formula I or composition of this disclosure means an amount
effective, when administered to a patient, to provide a therapeutic
benefit such as an amelioration of symptoms, e.g., an amount
effective to decrease the symptoms of a bacterial infection. For
example a patient having a bacterial infection may present elevated
levels of certain liver enzymes or an elevated white blood cell
count. A therapeutically effect amount is thus an amount sufficient
to provide a significant reduction in elevated liver enzyme levels
or white blood cell count, or an amount sufficient to provide a
return of the liver enzyme levels or white blood cell count to the
normal range. A therapeutically effective amount is also an amount
sufficient to prevent a significant increase or significantly
reduce the detectable level of bacterial particles or
anti-bacterial antibodies in the patient's blood, serum, or
tissues.
[0056] A "significant reduction" in the detectable level of
bacterial particles or anti-bacterial antibodies is any detectable
reduction that is statistically significant in a standard
parametric test of statistical significance such as Student's
T-test, where p<0.05.
Chemical Description
[0057] Formula I includes all subformulae thereof. In certain
situations, the compounds of Formula I may contain one or more
asymmetric elements such as stereogenic centers, stereogenic axes
and the like, e.g. asymmetric carbon atoms, so that the compounds
can exist in different stereoisomeric forms. These compounds can
be, for example, racemates or optically active forms. For compounds
with two or more asymmetric elements, these compounds can
additionally be mixtures of diastereomers. For compounds having
asymmetric centers, it should be understood that all of the optical
isomers and mixtures thereof are encompassed. In addition,
compounds with carbon-carbon double bonds may occur in Z- and
E-forms, with all isomeric forms of the compounds being included in
the present disclosure. In these situations, single enantiomers,
i.e., optically active forms, can be obtained by asymmetric
synthesis, synthesis from optically pure precursors, or by
resolution of the racemates. Resolution of the racemates can also
be accomplished, for example, by conventional methods such as
crystallization in the presence of a resolving agent, or
chromatography, using, for example using a chiral high pressure
liquid chromatography (HPLC) column.
[0058] Where a compound exists in various tautomeric forms, the
disclosure is not limited to any one of the specific tautomers, but
rather includes all tautomeric forms.
[0059] The present disclosure is intended to include all isotopes
of atoms occurring in the present compounds. Isotopes include those
atoms having the same atomic number but different mass numbers. By
way of general example, and without limitation, isotopes of
hydrogen include tritium and deuterium and isotopes of carbon
include .sup.11C, .sup.13C, and .sup.14C.
[0060] Certain compounds are described herein using a general
formula that includes variables, e.g., A.sub.1 to A.sub.3, R.sub.1
to R.sub.4, L.sub.1, L.sub.2, B.sub.l, and B.sub.2. Unless
otherwise specified, each variable within such a formula is defined
independently of other variables. Thus, if a group is said to be
substituted, e.g., with 0-2 R*, then the group may be substituted
with up to two R* groups and R* at each occurrence is selected
independently from the definition of R*. Also, combinations of
substituents and/or variables are permissible only if such
combinations result in stable compounds.
[0061] In addition to compounds of Formula I as described above,
the disclosure also includes compounds of Formula I in which one or
more of the following conditions is met for the variables in
Formula I. Compounds having any combination of the variable
definitions set forth below that result in a stable compound are
included in the disclosure.
[0062] Thus in addition to compounds of Formula I, the disclosure
provides compounds and salts thereof of Formula II.
##STR00004##
[0063] Within Formula II the following conditions are met.
[0064] A.sub.2 is absent or hydrogen.
[0065] A.sub.3 is one or two substituents independently chosen from
hydrogen and C.sub.1-C.sub.2alkyl; and
[0066] X is N or --C(A.sub.6)--.
[0067] The remaining variables carry the definitions set forth for
Formula I.
[0068] Also provided herein are compounds and salts thereof of
Formula III and IV.
##STR00005##
[0069] Within Formula III and IV the following conditions are
met.
[0070] A.sub.3 are each independently hydrogen, halogen, methyl, or
methoxy.
[0071] R.sub.3 is a 5- or 6- membered heterocycloalkyl group having
1 or 2 nitrogen ring atoms with remaining ring atoms being carbon,
and R.sub.3 is unsubstituted or substituted with 1 or more
substituents independently chosen from hydroxyl, halogen, amino,
C.sub.1-C.sub.2alkyl, and C.sub.1-C.sub.2alkoxy.
[0072] R.sub.4 is a 5- or 6-membered monosaccharide ring.
[0073] The remaining variables carry the definitions set forth for
Formula I.
[0074] The disclosure also provides compounds and salts thereof of
Formula I in which one or more of the following conditions are
met.
[0075] A.sub.3 is hydrogen, halogen, methyl, or methoxy,
[0076] R.sub.3 is a 5- or 6- membered heterocycloalkyl group having
1 or 2 nitrogen ring atoms with remaining ring atoms being carbon,
wherein R.sub.3 is unsubstituted or substituted with 1 or more
substituents independently chosen from hydroxyl, halogen, amino,
and C.sub.1-C.sub.2alkyl, C.sub.1-C.sub.2alkoxy; and
[0077] R.sub.4 is a 5 or 6-membered monosaccharide ring.
[0078] A.sub.1 is oxo and each A.sub.3 is independently chosen from
hydrogen and methyl.
[0079] R.sub.1 is methylene optionally substituted with
--(C--O)--.
[0080] R.sub.2 is --NH--.
[0081] L.sub.1 is an alkylene linker having from 2 to 4 carbon
atoms, wherein L.sub.1 is unsubstituted or substituted with 1 or
more substituents independently chosen from hydroxyl, halogen,
amino, C.sub.1-C.sub.4alkyl, C.sub.1-C.sub.4alkoxy,
C.sub.1-C.sub.2haloalkyl, and C.sub.1-C.sub.2haloalkoxy; and
[0082] L.sub.2 is an alkylene linker having from 1 to 2 carbon
atoms, containing 1 heteroatom selected from oxygen, nitrogen, and
sulfur, wherein L.sub.2 is unsubstituted or substituted with 1 or
more substituents independently chosen from hydroxyl, halogen,
amino, C.sub.1-C.sub.4alkyl, C.sub.1-C.sub.4alkoxy,
C.sub.1-C.sub.2haloalkyl, and C.sub.1-C.sub.2haloalkoxy.
[0083] The total number of carbon atoms in the L.sub.1 and L.sub.2
alkylene linkers is from 3 to 5.
[0084] L.sub.1 is an alkylene linker having from 2 to 3 carbon
atoms, wherein L.sub.1 is unsubstituted or substituted with 1 or
more substituents independently chosen from halogen and methyl;
and
[0085] L.sub.2 is an alkylene linker of the formula --SCH.sub.2--,
wherein L.sub.2 is unsubstituted or substituted with one or more
substituents independently chosen from halogen and methyl.
[0086] R.sub.3 is a piperidinyl, piperazinyl, or pyrrolidinyl ring;
each of which R.sub.3 is unsubstituted or substituted with 1 or
more substituents independently chosen from hydroxyl, halogen,
C.sub.1-C.sub.2alkyl, and C.sub.1-C.sub.2alkoxy.
[0087] R.sub.4 is a 5- or 6-membered monosaccharide ring.
[0088] R.sub.4 is d-ribose, d-arabinose, d-xylose, or d-lyxose.
[0089] R.sub.4 is d-ribose and B.sub.1 and B.sub.2 are both
hydrogen.
[0090] Also provided are compounds and salts thereof of Formula
V
##STR00006##
[0091] Within Formula V the following conditions are met.
[0092] A.sub.1 is oxo.
[0093] A.sub.2 is absent or hydrogen.
[0094] A.sub.3 is one or two substituents independently chosen from
hydrogen and C.sub.1-C.sub.2 alkyl.
[0095] X is N or --C(A.sub.6)--.
[0096] X.sub.1 and X.sub.2 are independently CH or N.
[0097] R.sub.1 is methylene optionally substituted with
(C.dbd.O)--.
[0098] L.sub.1 is an alkylene linker having from 2 to 4 carbon
atoms, wherein L.sub.1 is unsubstituted or substituted with 1 or
more substituents independently chosen from hydroxyl, halogen,
amino, C.sub.1-C.sub.4alkyl, C.sub.1-C.sub.4alkoxy,
C.sub.1-C.sub.2haloalkyl, and C.sub.1-C.sub.2haloalkoxy.
[0099] L.sub.2 is an alkylene linker having from 1 to 2 carbon
atoms, containing 1 heteroatom selected from oxygen, nitrogen, and
sulfur, wherein L.sub.2 is unsubstituted or substituted with 1 or
more substituents independently chosen from hydroxyl, halogen,
amino, C.sub.1-C.sub.4alkyl, C.sub.1-C.sub.4alkoxy,
C.sub.1-C.sub.2haloalkyl, and C.sub.1-C.sub.2haloalkoxy.
[0100] B.sub.1 and B.sub.2 are independently hydrogen or
methyl.
Synthetic Methods and Intermediates
[0101] This disclosure includes synthetic methods for producing
compounds of Formula I. The disclosure also includes synthetic
intermediates useful for producing compounds of Formula I.
Particularly this disclosure includes compounds of Formula A
##STR00007##
[0102] Within Formula A, A.sub.1 is hydrogen, oxo, amino, or
amino(C.sub.1-C.sub.2alkyl); A.sub.2 is hydrogen or
C.sub.1-C.sub.2alkyl; A.sub.3 is one or two substituents
independently chosen from hydrogen and C.sub.1-C.sub.2alkyl; and
A.sub.4 is a formyl group or C.sub.1-C.sub.4alkylester. The
disclosure includes intermediate compounds 25 and 26.
##STR00008##
[0103] Synthetic methods for preparing intermediate compounds of
Formula A, including compounds 25 and 26 are provided in Scheme 3.
Compound 25 is prepared by methyl group direct oxidation of the
6-methyl pterin with SeO.sub.2, for which experimental details are
provided in Example 2. Compound 26 is prepared by bromination of
the 6-methyl pterin followed by hydrolysis to the 6-carboxylic acid
ethyl ester, for which experimental details are provided in Example
3.
Pharmaceutical Preparations
[0104] Compounds disclosed herein can be administered as the neat
chemical, but are preferably administered as a pharmaceutical
composition. Accordingly, pharmaceutical compositions comprising a
compound or pharmaceutically acceptable salt of Formula I, together
with at least one pharmaceutically acceptable carrier are provided
herein. The pharmaceutical composition may contain a compound or
salt of Formula I as the only active agent, or may contain one or
more additional active agents.
[0105] Compounds disclosed herein may be administered orally,
topically, parenterally, by inhalation or spray, sublingually,
transdermally, via buccal administration, rectally, as an
ophthalmic solution, or by other means, in dosage unit formulations
containing conventional pharmaceutically acceptable carriers. The
pharmaceutical composition may be formulated as any
pharmaceutically useful form, e.g., as an aerosol, a cream, a gel,
a pill, a capsule, a tablet, a syrup, a transdermal patch, or an
ophthalmic solution. Some dosage forms, such as tablets and
capsules, are subdivided into suitably sized unit doses containing
appropriate quantities of the active components, e.g., an effective
amount to achieve the desired purpose.
[0106] Carriers include excipients and diluents and must be of
sufficiently high purity and sufficiently low toxicity to render
them suitable for administration to the patient being treated. The
carrier can be inert or it can possess pharmaceutical benefits of
its own. The amount of carrier employed in conjunction with the
compound is sufficient to provide a practical quantity of material
for administration per unit dose of the compound.
[0107] Classes of carriers include, but are not limited to binders,
buffering agents, coloring agents, diluents, disintegrants,
emulsifiers, flavorants, glidents, lubricants, preservatives,
stabilizers, surfactants, tableting agents, and wetting agents.
Some carriers may be listed in more than one class, for example
vegetable oil may be used as a lubricant in some formulations and a
diluent in others. Exemplary pharmaceutically acceptable carriers
include sugars, starches, celluloses, powdered tragacanth, malt,
gelatin; talc, and vegetable oils. Optional active agents may be
included in a pharmaceutical composition, which do not
substantially interfere with the activity of the compounds
described herein.
[0108] The pharmaceutical compositions can be formulated for oral
administration. These compositions contain between 0.1 and 99
weight % (wt. %) of a hydrazone or a diacyl hydrazine compound and
usually at least about 5 wt. % of a hydrazone or a diacyl hydrazine
compound. Some embodiments contain from about 25 wt. % to about 50
wt. % or from about 5 wt. % to about 75 wt. % of the hydrazone or
diacyl hydrazine compound.
Methods of Treatment
[0109] Methods of treating bacterial infections by providing an
effective amount of a compound of the invention to a patient having
a bacterial infection are provided. A compound as described herein
may be provided as the only active agent or may be provided
together with one or more additional active agents. Bacterial
infections that may be treated with compounds of Formula I include,
but are not limited to, Escherichia coli, Yersinia pestis, Bacillus
anthracis, Francisella tularensis, Staphylococcus aureus,
Enterococcus faecalis, Mycobacterium tuberculosis, and Helicobacter
pylori infections.
[0110] The pharmaceutical combinations disclosed herein are useful
for treating bacterial infections in human and non-human patients.
Non-human patients include, for example, livestock animals and
companion animals.
[0111] An effective amount of a pharmaceutical combination as
provided by this disclosure may be an amount sufficient to (a)
cause a regression of the bacterial infection; or (b) cause a cure
of a bacterial infection such that bacterial particles, or
anti-bacterial antibodies, can no longer be detected in a
previously infected patient's blood or plasma. An amount of a
pharmaceutical composition needed to inhibit the progress or cause
a regression of a bacterial infection, includes an amount effective
to stop the worsening of symptoms of the infection or reduce the
symptoms experienced by an infected patient. Alternatively a halt
in progression or regression of infections may be indicated by any
of several markers for the disease. For example, a lack of increase
or reduction in the number of bacterial particles in the patient's
blood or serum, or a lack of increase or reduction in the number of
circulating anti-bacterial antibodies in a patient's blood, or
return to normal for the patient's white blood cell count are
markers of a halt in progression or regression of bacterial
infection.
[0112] An effective amount of a combination described herein will
also provide a sufficient concentration of the active agents in the
concentration when administered to a patient. A sufficient
concentration of an active agent is a concentration of the agent in
the patient's body necessary to prevent or combat the infection.
Such an amount may be ascertained experimentally, for example by
assaying blood concentration of the agent, or theoretically, by
calculating bioavailability. The amount of an active agent
sufficient to inhibit viral infection in vitro may be determined
with a conventional assay for viral infectivity such as a replicon
based assay, which has been described in the literature.
[0113] Methods of treatment include providing certain dosage
amounts of a compound of Formula I to a patient. Dosage levels of
each active agent of from about 0.1 mg to about 140 mg per kilogram
of body weight per day are useful in the treatment of the
above-indicated conditions (about 0.5 mg to about 7 g per patient
per day). The amount of active ingredient that may be combined with
the carrier materials to produce a single dosage form will vary
depending upon the patient treated and the particular mode of
administration. Dosage unit a compound of Formula I. In certain
embodiments 25 mg to 500 mg, or 25 mg to 200 mg of a compound of
Formula I are provided daily to a patient. Frequency of dosage may
also vary depending on the compound used and the particular disease
treated. However, for treatment of most infectious disorders, a
dosage regimen of 4 times daily or less is preferred and a dosage
regimen of 1 or 2 times daily is particularly preferred.
[0114] It will be understood, however, that the specific dose level
for any particular patient will depend upon a variety of factors
including the activity of the specific compound employed, the age,
body weight, general health, sex, diet, time of administration,
route of administration, and rate of excretion, drug combination
and the severity of the particular disease in the patient
undergoing therapy.
Packaged Formulations
[0115] Methods comprising providing a compound or salt of Formula I
in a container together with instructions for using the composition
to treat a patient suffering from a bacterial infection are
included herein.
[0116] Packaged pharmaceutical combinations are also included
herein. Such packaged combinations include a compound of Formula I
in a container together with instructions for using the combination
to treat or prevent a viral infection, such as a bacterial
infection, in a patient.
[0117] The packaged pharmaceutical combination may include one or
more additional active agents.
Combination Methods
[0118] Pharmaceutical compositions and methods of treatment in
which a compound or salt of Formula I is provided together with one
or more additional active agents are included herein. In certain
embodiments the active agent (or agents) is an anti-bacterial
compound such as an antibiotic. The compound of Formula I and an
additional active agent may be: (1) co-formulated and administered
or delivered simultaneously in a combined formulation; (2)
delivered by alternation or in parallel as separate formulations;
or (3) by any other combination therapy regimen known in the art.
When delivered in alternation therapy, the methods disclosed herein
comprise administering or delivering the compound of Formula I and
an additional active agent sequentially, e.g., in separate
solution, emulsion, suspension, tablets, pills or capsules, or by
different injections in separate syringes. In general, during
alternation therapy, an effective dosage of each active ingredient
is administered sequentially, i.e., serially, whereas in
simultaneous therapy, effective dosages of two or more active
ingredients are administered together. Various sequences of
intermittent combination therapy may also be used.
Crystal Structures of Compound 13, 24, or 28 in Complex with
HPPK.
[0119] HPPK in complex with 13, 24, or 28 (HPPK.cndot.BSS10113,
HPPK.cndot.BSS10124, or HPPK.cndot.BSS10128) was crystallized
(Table 1).
TABLE-US-00001 TABLE 1 Crystallization Conditions
HPPK.cndot.BSS10113 HPPK.cndot.BSS10124 HPPK.cndot.BSS10128 Protein
Solution HPPK (mg/mL) 10 10 10 BSS10113 saturated BSS10124
Saturated BSS10128 Saturated Tris-HCl [mM (pH)] 20 (8.0) 20 (8.0)
20 (8.0) Reservoir Solution PEG 3350 [% (w/v)] 25 25 20
CH.sub.3COONH.sub.4 (mM) 200 200 Bis-Tris [mM (pH)] 100 (6.5) 100
(8.5) HEPES [mM (pH)] 100 (7.5) Crystals Appear (days) 7 7 14 Final
size (days) 14 14 21 Shape Thin plate Thin plate Thin plate
Dimension (mm) 0.15 .times. 0.10 .times. 0.005 0.10 .times. 0.05
.times. 0.005 0.15 .times. 0.10 .times. 0.005
[0120] The structures of the three complexes were determined (Table
2).
TABLE-US-00002 TABLE 2 Crystals, X-ray Diffraction Data, and
Structures HPPK.cndot.BSS10113 HPPK.cndot.BSS10124
HPPK.cndot.BSS10128 Crystal Space group C2 P2.sub.12.sub.12
P2.sub.12.sub.12 79.98 52.91 53.00 Unit cell parameters: a (.ANG.)
b (.ANG.) 52.77 70.98 70.64 c (.ANG.) 36.69 36.38 36.25 .beta.
(.degree.) 102.70 90 90 Matthews coefficient (.ANG..sup.3/Da) 2.1
1.9 1.9 Data Statistics Overall (last shell) Overall (last shell)
Overall (last shell) Resolution (.ANG.) 30-2.00 (2.07-2.00) 30-1.89
(1.96-1.89) 30-1.88 (1.95-1.88) Unique reflections 9255 (695) 10823
(808) 10756 (794) Redundancy 6.6 (5.9) 6.4 (3.5) 6.3 (4.0)
Completeness (%) 90.8 (70.1) 93.9 (72.1) 92.9 (69.8)
R.sub.merge.sup.a 0.081 (0.238) 0.082 (0.472) 0.074 (0.325)
I/.sigma. 20.8 (5.5) 19.3 (2.1) 20.2 (3.1) Structure Solution
Method Fourier synthesis Fourier synthesis Fourier synthesis Model
PDB 1EQM PDB entry 3ILJ HPPK.cndot.BSS10124 Refinement Statistics
Overall (last shell) Overall (last shell) Overall (last shell)
Resolution (.ANG.) 30-2.00 (2.13-2.00) 30-1.89 (1.99-1.89) 30-1.88
(1.98-1.88) Unique reflections 9247 (1202) 10792 (1178) 10753
(1052) Completeness (%) 90.7 (72.0) 93.9 (74.0) 92.8 (72.0) Data in
the test set 824 (107) 1000 (109) 919 (98) R-work 0.158 (0.168)
0.216 (0.271) 0.205 (0.242) R-free 0.208 (0.260) 0.276 (0.328)
0.270 (0.278) Structure Statistics Protein non-H atoms/B
(.ANG..sup.2) 1448/34.7 1422/31.0 1388/25.6 Heterogen atoms/B
(.ANG..sup.2) 45/49.1 47/34.1 48/40.8 Water oxygen atoms/B
(.ANG..sup.2) 107/43.1 107/39.3 94/30.9 Rmsd Bond lengths (.ANG.)
0.008 0.005 0.008 Bond angles (.degree.) 0.957 0.778 0.972
Coordinate error (.ANG.) 0.29 0.14 0.29 Ramachandran plot.sup.b
Favored regions (%) 98.7 97.3 97.3 Disallowed regions (%) 0.0 0.0
0.0 .sup.aR.sub.merge = .SIGMA.|(I - <I>)|/.SIGMA.(I), where
I is the observed intensity. .sup.bObtained using Ramachandran data
(Lovell et al., Proteins (2003) 50: 437-450).
[0121] The 2.0-.ANG. structure of HPPK.cndot.BSS10113 contains 1
HPPK, 1 BSS10113, 1 ethylene glycol molecule, and 107 water
molecules (FIG. 3A), in which the overall conformation of HPPK is
similar to that observed in the HPPK.cndot.MgADP complex (PDB entry
1EQM), in which Loop 3 moves dramatically away from the active
center.
[0122] The 1.89-.ANG. structure of HPPK.cndot.BSS10124 contains 1
HPPK, 1 BSS10124, 1 acetate ion, and 89 water molecules (FIG. 3B),
in which the overall conformation of HPPK is similar to that
observed in the HPPK-HP-MgAMPCPP complex (PDB entry 1Q0N). However,
part of Loop 3 in the HPPK.cndot.BSS10124 structure is disordered;
no electron density was observed for residues 83-86.
[0123] The 1.88-.ANG. structure of HPPK.cndot.BSS10128 contains 1
HPPK, 1 BSS10128, 1 acetate ion, and 94 water molecules (FIG. 3C),
in which the overall conformation of HPPK is similar to the
HPPK.cndot.BSS10124 complex with disordered residues 83-86.
[0124] The disclosure includes crystals of HPPK and inhibitors. For
example the disclosure includes the following HPPK/inhibitor
crystals: A crystal of HPPK and BSS10113 consisting of a crystal
space group C2 with unit cell dimensions of a=79.98, b=52.77, and
c=36.69; A crystal of HPPK and BSS10124 consisting of a crystal
space group P2.sub.12.sub.12 with unit cell dimensions of a=52.91,
b=70.98, and c=36.38; and a crystal of HPPK and BSS10128 consisting
of a crystal space group P2.sub.12.sub.12 with unit cell dimensions
of a=53.00, b=70.64, and c=36.25. The disclosure also includes a
composition of matter comprising a crystal structure of HPPK with
an HPPK ligand disposed therein. In certain instances the HPPK
ligand contains a pterin moiety and/ or is a compound of Formula
I.
[0125] The disclosure includes a method for a identifying a
molecule that binds to HPPK, the method comprising the steps of (a)
providing a molecular target selected from the BSS10113, BSS10124,
or BSS10128 binding site of HPPK, (b) using the molecular target to
identify a candidate molecule that can bind to one or more said
molecular targets; and (c) producing the candidate molecule
identified in step (b). The molecular target selected from the
BSS10113, BSS10124, or BSS10128 binding site of HPPK is obtained
from the atomic coordinates of the HPPK-BSS10113, HPPK-BSS10124, or
HPPK-BSS10128 supplied in Appendices 1, 2, and 3 or is a molecular
model derived from the atomic coordinates supplied in these
Appendices. The method may include the additional step of repeating
steps (a) through (c) to identify and produce a modified candidate
molecule having high binding affinity for HPPK or high potency
relative to the candidate molecule.
Lead Compounds For Novel Anti-Bacterial Agents
[0126] Among the three compounds presented here, BSS10113 and
BSS10128 are stable. BSS10124 is not stable, but is obviously
stabilized in the HPPK.cndot.BSS10124 complex. Hence, BSS10113 and
BSS10128 are the lead compounds for novel anti-bacterial agents
targeting HPPK.
[0127] The catalytic trajectory of HPPK can be described by five
consecutive states: apo-HPPK, HPPK.cndot.MgATP,
HPPK.cndot.MgATP.cndot.HP, HPPK.cndot.AMP.cndot.HPPP, and
HPPK.cndot.HPPP (Blaszczyk et al., Structure (2004) 12: 467-475).
Among the five states, the protein in HPPK.cndot.MgATP and
HPPK.cndot.AMP.cndot.HPPP exhibits the same conformation, which is
also observed in the HPPK.cndot.BSS10113 complex (FIG. 4A). The
protein in the HPPK.cndot.BSS10128 complex displays the
conformation observed in the HPPK.cndot.MgATP.cndot.HP complex
(FIG. 4B). Both complexes show stable conformations of HPPK,
suggesting that the structure-based design is accurate.
[0128] Also provided herein is a method for identifying a molecule
that inhibits HPPK, the method comprising: (a) providing a
molecular model comprising one or more HPPK target regions
(protein-inhibitor interaction sites) selected from (i) the
BSS10113 binding site, (ii) the BSS10124 binding site, and (iii)
the BSS10128 binding site (1) from the atomic co-ordinates for HPPK
in complex with BSS10113, BSS10124, or BSS10128; and (b) using the
molecular model to identify a candidate molecule that can bind to
the molecular model. The atomic coordinated of HPPK in complex with
BSS10113, BSS10124, or BSS10128 are provided with this application
as Appendices 1, 2, and 3, respectively. In certain embodiments
this method further comprises producing the candidate molecule
identified in step (b) and can also include determining whether the
produced candidate molecule inhibits HPPK.
[0129] The protein-inhibitor interaction sites can be derived with
Accelrys' Discovery Studio suite, e.g., Discovery Studio, version
2.1; Accelrys: San Diego, Calif., 2008. Fragment searching, by
which a candidate molecule may be identified can be conducted, for
example, within the interaction site boxes using the De Novo
Evolution and De Novo Link protocols (Accelrys) and the ZINC
databases, Irwin and Shoichet, J. Chem. Inf. Model. (2005)
45(1):177-82.
EXAMPLES
ABBREVIATIONS
[0130] The following abbreviations are used in the reaction schemes
and synthetic examples, which follow. This list is not meant to be
an all-inclusive list of abbreviations used in the application as
additional standard abbreviations, which are readily understood by
those skilled in the art of organic synthesis, may also be used in
the synthetic schemes and examples. [0131] AMPCPP
.alpha.,.beta.-methyleneadenosine 5'-triphosphate [0132] DCM
Dichloromethane [0133] DMF Dimethyl formamide [0134] EtOH Ethanol
[0135] HP 6-hydroxymethyl-7,8-dihydropterin [0136] HPPK
6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase [0137] MR
molecular replacement [0138] NBS N-bromosuccinamide [0139] PDB
Protein Data Bank [0140] TFA trifluoroacetic acid [0141] T-HYDRO
t-butyl hydroperoxide
General Experimental Information
[0142] The compound 2',3'-isopropylideneadenosine (1, Scheme 1) was
purchased from TCI America. All other chemicals were purchased from
Sigma-Aldrich. Starting materials and solvents were used without
further purification. Anhydrous reactions were conducted under a
positive pressure of dry N.sub.2. Reactions were monitored by thin
layer chromatography (TLC) on Baker-flex Silica Gel IB-F (J. T.
Baker). Final compounds and intermediates were purified by flash
chromatography performed on Teledyne ISCO Combiflash Rf system
using RediSep Rf columns. Ion exchange chromatography was performed
using strata Scx (50 .mu.m particle size, 70 .ANG. pore) resin
cartridges. Preparative high pressure liquid chromatography (HPLC)
was conducted using a Waters 600E system using a Waters 2487 dual
.lamda. absorbance detector and Phenomenex C.sub.18 columns (250
mm.times.21.2 mm, 5 .mu.m particle size, 110 .ANG. pore) at a flow
rate of 10 mL/min. A binary solvent systems consisting of A=0.1%
aqueous TFA and B=0.1% TFA in acetonitrile was employed with the
gradients as indicated. .sup.1H and .sup.13C NMR data were obtained
on a Varian 400 MHz spectrometer and are reported in ppm relative
to TMS and referenced to the solvent in which the spectra were
collected. Mass spectra were measured with Agilent 1100 series
LC/Mass Selective Detector, Agilent 1200 LC/MSD-SL system and
Thermoquest Surveyor Finnigan LCQ deca. All compounds tested were
at least 95% pure by LCMS and NMR.
General Scheme for Preparation of Compound 13.
##STR00009## ##STR00010## ##STR00011##
[0143] General Scheme for Preparation of Compounds 22 and 24
##STR00012##
[0144] General Scheme for Preparation of Compounds 25, 26,
and28
##STR00013##
EXAMPLE 1
Synthesis of
2-Amino-6-[2-{4-[5-(6-amino-purin-9-yl)-3,4-dihydroxy-tetrahydro-furan-2--
ylmethylsulfanyl]-piperidin-1-yl}-ethylamino)-methyl]-3H-pteridin-4-one
(13).
##STR00014##
[0146] In Scheme 1, above, isopropylideneadenosine 1 is used to
synthesize toluenesulfonyl isopropylideneadenosine, 2
(2',3'-O-isopropylidene-5'-O-toluene-p-sulfonyl adenosine). An
anhydrous pyridine solution of commercially available
2',3'-isopropylideneadenosine 1 is shaken with p-toluenesulfonyl
chloride. In a separate reaction, the synthon
4-acetylsulfanyl-piperidine-1-carboxylic acid tert-butyl ester 4
was synthesized using the method of Plettenburg in which potassium
thioacetate and 4-bromo-piperidine 3 are heated in DMF. According
to modified procedures based on an existing protocol (Isakovic et
al., Bioorg. & Med. Chem. Lett. (2009) 19: 2742-2746), synthon
4 is reacted with sodium methoxide to form the thiol, followed by
the reaction with 2 to give
4-[6-(6-Amino-purin-9-y1)-2,2-dimethyl-tetrahydro-furo[3,4-d][1,3-
]dioxol-4-ylmethylsulfanyl]-piperidine-1-carboxylic acid tert-butyl
ester 5. Under the TFA/DCM condition, cleavage of the BOC
protection group and subsequent reaction of 6 with
(2-bromo-ethyl)-carbamic acid tert-butyl ester provides
intermediate 7. The deprotection of 7 yields 8, which contains an
amino group used to link 8 to the pterin moiety.
[0147] 2,4-Diamino-6-(hydroxymethyl)pteridine hydrochloride 9 is
treated with dibromotriphenylphosphorane in N,N-dimethylacetamide
to give 10, which in 48% hydrobromic acid is converted through
hydrolytic deamination to provide 11.
[0148] To a solution of
2-[1-(2-Amino-ethyl)-piperidin-4-ylsulfanylmethyl]-5-(6-amino-purin-9-yl)-
-tetrahydro-furan-3,4-diol (100.0 mg, 0.244 mmol, 1 eq) (8) and
potassium carbonate (337.9 mg, 2.44 mmol, 10 eq) in 20 mL
dimethylacetamide, 2-amino-6-bromomethyl-3H-pteridin-4-one (87.0
mg, 0.244 mmol, 1 eq) (11) was added and then stirred at room
temperature for 24 hours. It was evaporated in high vacuum and the
residue dissolved in water methanol mixture and purified by HPLC to
give compound 13 (71.0 mg, 0.122 mmol, 50%) of a yellowish powder.
MS (ESI) calculated for C.sub.24H.sub.32N.sub.12O.sub.4S
[M+H]+585.24, found 585.1
EXAMPLE 2
2-Amino-7,7-dimethyl-4-oxo-3,4,7,8-tetrahydro-pteridine-6-carbaldehyde
(25)
##STR00015##
[0149] General Description of Scheme 2
[0150] Compound 22,
2-amino-7,8-dihydro-6,7,7-trimethylpteridin-4(3H)-one, used in the
synthesis of 25, 26, and 28, was synthesized by a procedure of
Al-Hassan (Al-Hassan et al., (1985) 1: 1645-1659). This procedure
is based on a classical method for the preparation of
7,8-dihydropteridines, the condensation of .alpha.-aminoketone with
halopyrimidinone followed by reductive stylization. Accordingly, 22
was prepared by the condensation of
2-amino-5-nitro-6-chloropyrimidin-4-one (19) with
3-amino-3-methylbutan-2-one semicarbazone (17) under basic
conditions. Compound 22 can be converted to 23 by Stuart's method
(U.S. Pat. No. 4,036,961, which is hereby incorporated by
references for its teaching regarding pteridine synthesis) using
bromine in acetic acid solution (Scheme 2), or oxidized by
SeO.sub.2 in DMF to give 25 (Scheme 3).
Synthesis of Compound 25
[0151] Compound 25 (Scheme 3) is an intermediate useful in the
preparation of compounds of Formula I. To synthesis 25, a solution
of 22 (207 mg, 1.0 mmol) in DMF (10 mL) and pyridine (105 uL, 1.30
mmol) was treated with SeO.sub.2 (145 mg, 1.30 mmol) and stirred at
room temperature for 5 h. The reaction was then heated to
80.degree. C. for 15 min. The solvent was evaporated under high
vacuum and the residue purified by flash chromatography (silica
gel, methanol:dichloromerhane=2:8) to give 25 (199 mg, 0.9 mmol,
90%) as a yellowish powder. MS (ESI) calculated for C9H11N5O2
[M+H]+222.09, found 222.1.
EXAMPLE 3
Synthesis OF 6-Carboxylic Ethyl
Ester-7,7-dimethyl-7,8-dihydropterin (26)
##STR00016##
[0153] Compound 26 is an intermediate useful in the preparation of
compounds of Formula I. To synthesize 26, compound 22 (0.5 g, 2.4
mmol) is dissolved in 80 mL ethanol in a heavy wall pressure
vessel. Bromine (0.43 mol, 8.4 mmol) is dropped into the solution.
The pressure vessel is sealed with Teflon bushing. The solution is
heated overnight at 120.degree. C. The ethanol is evaporated and
the residue purified by column chromatography. The desired
compound, 26, is obtained as a yellow solid (35% yield).
[0154] N-Bromosuccinimide (NBS) may be used in the above procedure
in place of bromine, as the bromination agent.
[0155] In an alternate procedure compound 22 (0.5 g, 2.4 mmol) and
NBS (1.28 g, 7.2 mmol) are dissolved in 20 mL ethanol. The reaction
mixture is heated in a Biotage microwave initiator for 5-25
minutes. The ethanol is evaporated and the residue purified by
column chromatography. Compound 26 is obtained as a yellow solid
(52% yield).
EXAMPLE 4
2-Amino-7,7-dimethyl-4-oxo-3,4,7,8-tetrahydro-pteridine-6-carboxylic
acid
(2-{4-[5-(6-amino-purin-9-yl)-3,4-dihydroxy-tetrahydro-furan-2-ylmethylsu-
lfanyl]-piperidin-1-yl}-ethyl)-amide (28).
##STR00017##
[0157] Compound 28 was synthesized by 25 and 8 using the procedure
developed by Yoo and Li (J. Am. Chem. Soc. (2006) 128: 13064-13065)
using copper-silver catalysis and aqueous tert-butyl hydroperoxide
(Method A) or by 26 and 8 using the disclosed procedure herein with
a significantly improved yield (Method B).
Synthesis of Compound 28 (Method A)
[0158] Compound 8 (12.3 mg, 0.03 mmol, 1.5 eq) was mixed with CuI
(0.0388 mg, 0.0002 mmol, 1.0 mol %), AgIO.sub.3 (0.057 mg, 0.0002
mmol, 1.0 mol %) and CaCO.sub.3 (2.2 mg, 0.022 mmol, 1.1 eq) in DMF
(0.2 mL). Compound 25 (4.5 mg, 0.020 mmol, 1.0 eq) and T-HYDRO.RTM.
(70 wt % in H.sub.2O, 0.00315 mL, 0.022 mmol, 1.1 eq) was added
under an inert atmosphere (N.sub.2) at room temperature. The
reaction was allowed to stir for overnight at 40.degree. C. The
crude reaction was purified by HPLC (H.sub.2O:methanol=2:3) to
provide 28 (3.77 mg, 0.006 mmol, 30%) as a pale yellow solid. MS
(ESI) calculated for C.sub.26H.sub.36N.sub.12O.sub.5S [M+H]+629.27,
found 629.1.
Synthesis of Compound 28 (Method B)
[0159] To a solution of compound 26 (265 mg, 1 mmol, 1 eq) methanol
(5 mL) was added a solution of sodium hydroxide (2M, 2 mmol, 2 eq).
After stiffing for two hours, the reaction mixture was acidified to
pH=2 with 1M HCl, the product was precipitated, the precipitate
washed once with water and then dried to obtain
2-Amino-7,7-dimethyl-4-oxo-3,4,7,8-tetrahydro-pteridine-6-carboxylic
acid (27) (213 mg, 0.9 mmol, 90%). MS (ESI) calculated for
C9H11N5O3 [M+H]+238.09, found 238.1
[0160] To a solution of compound 27 (190 mg, 0.8 mmol, 1 eq),
O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HATU) (334.6, 0.88 mmol, 1.1 eq), and compound
8 (327.2 mg, 0.8 mmol, 1 eq) in anhydrous DMF (100 mL) was added
DIPEA (4.18 uL, 2.4 mmol, 3 q). After 18 h, the solvent was
evaporated under high vacuum, the reaction residue was purified by
HPLC (H2O: Methanol=2:3) to provide 28 (377 mg, 0.6 mmol, 75%) as a
pale yellow solid. MS (ESI) calculated for C26H36N12O5S
[M+H]+629.27, found 629.1.
EXAMPLE 5
Data Acquisition, Structure Solution, and Refinement
[0161] X-ray diffraction data were collected at 100K with an MARCCD
detector mounted at the synchrotron Beamline 22 of the Southeast
Regional Collaborative Access Team (SER-CAT) at the Advanced Photon
Source, Argonne National Laboratory. Data processing was carried
out with the HKL2000 program suite (Otwinowski, Z. and Minor, W.,
Methods in Enzymology, (1997) 276: 307-326). The details of data
collection and processing, and structure solution and refinement
are summarized in Table 2. The structures were solved and refined
with PHENIX (Adams, P. D., Acta Crystallogr. D. (2002) 58:
1948-1954). For Fourier synthesis, multiple conformations of amino
acid residues, ligands, and solvent molecules were removed from the
starting models. All graphics effort, including model building and
rebuilding, was carried out with COOT (Emsley, P. and Cowtan, K.
(2004) Acta Crystallogr. D. 60: 2126-2132). The structures were
verified with annealed omit maps and the geometry of finalized
structures was assessed using PROCHECK (Laskowski, R. A. et al.,
(1993) J. Appl. Crystallogr. 26: 283-291) and WHAT IF (Vriend, G.
(1990) J. Mol. Graph 8: 42-56, 29). Illustrations were prepared
with PyMOL (DeLano Scientific LLC.).
EXAMPLE 6
Biochemical Studies
[0162] Binding studies were carried out at room temperature.
Protein (HPPK) and ligand stock solutions were made in 100 mM
Tris-HCl, pH 8.3, and their concentrations were determined
spectrophotometrically using the following extinction coefficients:
21600 M.sup.-1 cm.sup.-1 at 280 nm for HPPK, 7000 M.sup.-1
cm.sup.-1 at 366 nm for the pteridone bisubstrate inhibitors such
as compound 13, and 7124 M.sup.-1 cm.sup.-1 at 380 nm for the
tetrahydropteridine bisubstrate inhibitors such as 28. A 3-mL
dilute inhibitor solution in a fluorometric cuvette was titrated
with the protein stock solution. Fluorescence was measured on a
Horiba Jobin Yvon FluoroMax-4 fluorometer. The excitation
wavelength and slit were 364-380 and 2-3 nm, respectively, and the
emission wavelength and slit were 450-482 and 2-5 nm, respectively.
A few HPPK preparations showed some fluorescence at the excitation
and emission wavelengths. For these HPPK preparations, a control
experiment, in which a 3-mL buffer solution was titrated with the
protein solution, was performed. The control data was subtracted
from the titration data. The corrected titration data was then
analyzed by nonlinear least-squares regression using the software
Origin and the equation:
L t = L 0 V 0 V 0 + .DELTA. V a nd E t = E 0 .DELTA. V V 0 +
.DELTA. V ##EQU00001##
where F.sub.obs is the observed fluorescence, .epsilon..sub.f and
.epsilon..sub.b are the fluorescence coefficients of the ligand in
the free and protein-bound states, respectively, L.sub.t is the
total concentration of the ligand, and E.sub.t is the total
concentration of HPPK. L.sub.t and E.sub.t were varied during the
titration process according to the following expressions:
F obs = f L t + ( b - f ) ( L t + E t + K d - ( L t + E t + K d ) 2
- 4 E t L t ) 2 ##EQU00002##
where E.sub.0 is the concentration of the HPPK stock solution,
L.sub.0 is the initial concentration of the ligand, V.sub.0 is the
initial volume of the titration, and .DELTA.V is the total volume
of the added HPPK solution.
[0163] The titration curves for 13 and 28 of binding analysis is
shown in FIG. 2. The K.sub.d value was obtained by nonlinear
least-squares analysis. It was 0.59 (0.19) .mu.M for 13 and 0.33
(0.05) .mu.M for 28. Compound 24 was not as stable as 13 and 28,
and therefore, its K.sub.d was not determined.
[0164] While specific embodiments have been shown and described,
various modifications and substitutions can be made thereto without
departing from the spirit and scope of the invention. Accordingly,
it is to be understood that the present invention has been
described by way of illustration and not limitations.
* * * * *