U.S. patent application number 13/590767 was filed with the patent office on 2012-12-13 for inhibitors of udp-galactopyranose mutase thwart mycobacterial growth.
Invention is credited to Emily Carla DYKHUIZEN, Laura Lee KIESSLING, John F. MAY.
Application Number | 20120316208 13/590767 |
Document ID | / |
Family ID | 41217165 |
Filed Date | 2012-12-13 |
United States Patent
Application |
20120316208 |
Kind Code |
A1 |
KIESSLING; Laura Lee ; et
al. |
December 13, 2012 |
INHIBITORS OF UDP-GALACTOPYRANOSE MUTASE THWART MYCOBACTERIAL
GROWTH
Abstract
Compounds which inhibit microbial growth or attenuate the
virulence of pathogen microorganisms. Compounds of the invention
inhibit UDP-galactopyranose mutase (UGM) and have activity as
inhibitors of microbial growth of microorganisms which contain this
enzyme and particularly those microorganisms in which this enzyme
is responsible for the incorporation of galactofuranose residues,
particularly for uridine 5'-diphosphate (UDP) galactopyranose
mutase. Compounds of the invention inhibit UDP-galactopyranose
mutase (UGM) and have activity to attenuate virulence of pathogenic
microorganisms, including mycobacteria.
Inventors: |
KIESSLING; Laura Lee;
(Madison, WI) ; DYKHUIZEN; Emily Carla; (Madison,
WI) ; MAY; John F.; (Madison, WI) |
Family ID: |
41217165 |
Appl. No.: |
13/590767 |
Filed: |
August 21, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12430610 |
Apr 27, 2009 |
8273778 |
|
|
13590767 |
|
|
|
|
PCT/US2009/041719 |
Apr 24, 2009 |
|
|
|
12430610 |
|
|
|
|
61048080 |
Apr 25, 2008 |
|
|
|
61048080 |
Apr 25, 2008 |
|
|
|
Current U.S.
Class: |
514/370 ;
435/184; 548/194 |
Current CPC
Class: |
A61P 31/04 20180101;
A61P 31/00 20180101; C07D 417/12 20130101 |
Class at
Publication: |
514/370 ;
548/194; 435/184 |
International
Class: |
A01N 43/78 20060101
A01N043/78; A01P 1/00 20060101 A01P001/00; A61K 31/426 20060101
A61K031/426; A61P 31/04 20060101 A61P031/04; C07D 277/42 20060101
C07D277/42; C12N 9/99 20060101 C12N009/99 |
Goverment Interests
STATEMENT REGARDING GOVERNMENT SUPPORT
[0002] This invention was funded by the United States government
through National Institutes of Health grant A1063596. The U.S.
government has certain rights in this invention.
Claims
1. A compound of formula: ##STR00078## and salts thereof, where: n
is 0 or 1 to indicate the presence or absence of L.sub.1; L.sub.1,
if present, is an alkylene, alkenylene or alkyleneoxy linker having
1-6 carbon atoms; Y is CH.sub.2 or NR.sub.Y, where R.sub.Y is
hydrogen or a C1-C6 alkyl; W is --COOR.sub.W, --CON(R.sub.W).sub.2,
--SO.sub.2--OR.sub.W, --SO.sub.2--N(R.sub.W).sub.2,
--OPO.sub.3R.sub.W, --OP(OR.sub.W).sub.2R.sub.W, or a tetrazolyl
group, where each R.sub.W, independent of other R.sub.W, is
hydrogen, C1-C6 alkyl or an aryl or heteroaryl group having one or
two 5-member or 6-member rings; m is 1 or 2; the D ring is a 1,3-
or 1,2-substituted 5-member or 6-member ring, which is a
carbocyclic or heterocyclic ring; R.sup.1 represents no
substitution on the D ring or substitution on the D ring with one
or more C1-C3 alky, one or more C1-C3 haloalkyl, one or more
halogens or combinations thereof; the B and C rings are
independently selected from an aryl or heteroaryl group having one
or two rings; R.sub.B and R.sub.C represent no substitution on the
indicated ring or substitution on the indicated ring by one to five
non-hydrogen substituents selected from the group consisting of
halogen, nitro, cyano, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy,
sulfonamide (--SO.sub.2--N(R.sub.F).sub.2), azide, sulfonyl
(--SO.sub.2--R.sub.F), --COOR.sub.F, --COR.sub.F,
--CON(R.sub.F).sub.2, --N(R.sub.F).sub.2, - and C1-C6 haloalkyl
groups, including trifluoromethyl, trichloromethyl and
tribromomethyl groups, where R.sub.F is hydrogen or a C1-C6 alkyl
group; or wherein R.sub.B and R.sub.C substituents on two adjacent
carbons of a B or C ring together form a carbocyclic or
heterocyclic ring having 5 to 8 ring atoms; or R.sub.B is
-M-L.sub.2-Q-AR, where: AR is an aryl or heteroaryl group having at
least two rings; L.sub.2 is a linker group which is an alkylene,
cycloalkylene group, alkenylene group, or an alkyleneoxy group,
which contains 5-12 carbon atoms, or L.sub.2 is a linker group
which is a 5- or 6-member heterocyclene group containing one or two
O, N or S atoms or combinations thereof, a 6-member ring arylene,
or a 6-member ring heteroarylene group containing one or two O, N
or S atoms or combinations thereof; M and Q are chemical moieties
independently be selected from, --O--, --S--, --CO--, --NR.sub.M--,
carboxyl, amide, sulfonamide, thiourea, urea, carbonate,
guanidinium, carbamate, thiocarbamate, alkylene moieties or
combinations thereof, where R.sub.M is hydrogen or C1-C6 alkyl.
2. A compound of claim 1 having formula: ##STR00079## or salts
thereof, where n is 0 or 1; m is 1 or 2; Y is CH.sub.2 or NR.sub.Y
where R.sub.Y is hydrogen or a C1-C6 alkyl; W is --COOR.sub.W,
--CON(R.sub.W).sub.2, --SO.sub.2--OR.sub.W,
--SO.sub.2--N(R.sub.W).sub.2, --OPO.sub.3R.sub.W,
--OP(OR.sub.W).sub.2R.sub.W, or tetrazolyl, where each R.sub.W,
independent of other R.sub.W, is hydrogen, a C1-C6 alkyl, an aryl
or heteroaryl group.
3. A compound of claim 1 having formula: ##STR00080## or salts
thereof.
4. A compound of claim 3 wherein D is S.
5. A compound of claim 3 wherein R.sub.W is H, R.sub.Y is H and
R.sup.1 is H.
6. A compound of claim 3, where: n is 0 or 1 to indicate the
presence or absence of L.sub.1; L.sub.1, if present, is an
alkylene, alkenylene or alkyleneoxy linker having 1-6 carbon atoms;
Ry is hydrogen or a C1-C6 alkyl; R.sub.W is hydrogen, a C1-C6 alkyl
or an aryl or heteroaryl group having one or two 5-member or
6-member rings; D is S or O; R.sup.1 represents hydrogen, a 01-03
alkyl, a 01-03 haloalkyl, or a halogen; R.sub.B represents
substitution on the indicated ring by one to five non-hydrogen
substituents selected from the group consisting of halogen, nitro,
cyano, azide, --COOR.sub.F, --COR.sub.F, --CON(R.sub.F).sub.2,
--N(R.sub.F).sub.2, and C1-C6 haloalkyl groups, where R.sub.F is
hydrogen or a C1-C6 alkyl group; and R.sub.C represents
substitution on the indicated ring by a halogen at ring position 3,
and zero to four non-hydrogen substituents at positions 1, 2, 4,
and 5 selected from the group consisting of halogen, hydroxyl,
nitro, cyano, sulfonamide (--SO.sub.2--N(R.sub.F).sub.2), azide,
sulfonyl (--SO.sub.2--R.sub.F), --COOR.sub.F, --COR.sub.F,
--CON(R.sub.F).sub.2, --N(R.sub.F).sub.2, and C1-C6 haloalkyl
groups, where R.sub.F is hydrogen or a C1-C6 alkyl group.
7. A compound of claim 6 wherein L.sub.1 is an alkylene having 1 to
6 carbon atoms.
8. A compound of claim 6 wherein R.sub.W is H, and Ry is
hydrogen.
9. A compound of claim 6 wherein R.sub.B represents at least one
non-hydrogen substituent selected from the group consisting of
halogen, nitro, cyano, azide, and C1-C6 haloalkyl groups.
10. A compound of claim 1 wherein R.sub.B represents at least one
non-hydrogen substituent selected from the group consisting of
halogen, nitro, cyano, azide, and C1-C6 haloalkyl groups.
11. A compound of claim 1 wherein R.sub.C represents substitution
by a halogen at ring position 3 and R.sub.B represents substitution
by a halogen at ring position 3, substitution by two halogens at
ring positions 2 and 4, substitution by two halogens at ring
positions 1 and 3, substitution by a nitro group at ring position 2
or substitution by a hydroxyl group at ring position 3.
12. A compound of claim 1 wherein R.sub.C represents substitution
by a chlorine, iodine or bromine at ring position 3; and R.sub.B
represents substitution by chlorine, iodine or bromine at ring
position 3, substitution by two chlorines or fluorines at ring
positions 2 and 4, substitution by two chlorines or fluorines at
ring positions 1 and 3, substitution by a nitro group at ring
position 2, or substitution by a hydroxyl group at ring position
3.
13. A compound of claim 12 wherein R.sub.C represents substitution
by an iodine at ring position 3.
14. A compound of claim 1 wherein L.sub.1 is --CH.sub.2--.
15. A compound of claim 1 wherein R.sub.B and R.sub.C represent
substitution on the indicated ring by one to five non-hydrogen
substituents selected from the group consisting of halogen, nitro,
cyano, azide, and C1-C6 haloalkyl groups.
16. A compound of claim 1 wherein R.sub.B represents substitution
on the indicated ring by one to five halogen atoms.
17. A compound of claim 1 where W is --COOH; n is 1; L.sub.1 is an
alkylene linker having 1-6 carbon atoms; R.sub.B represents
substitution on the indicated ring by one to five non-hydrogen
substituents selected from the group consisting of halogen, nitro,
cyano, azide, and C1-C6 haloalkyl groups, where R.sub.F is hydrogen
or a C1-C6 alkyl group; and R.sub.C represents substitution on the
indicated ring by a halogen at ring position 3, and zero to four
non-hydrogen substituents, at ring positions 1, 2, 4, and 5,
selected from the group consisting of halogen, nitro, cyano, azide,
--N(R.sub.F).sub.2, and C1-C6 haloalkyl groups, where R.sub.F is
hydrogen or a C1-C6 alkyl group.
18. A method for inhibiting UGM which comprises the step of
contacting UGM with an amount of one or more compounds of claim 1
effective for inhibiting the enzyme.
19. A method for inhibiting the growth of a bacterium or a
mycobacterium which comprises contacting the organism or an
environment containing the organism with an effective amount of one
or more compounds of claim 1.
20. A method for treatment of an infection which comprises the step
of administering to a human or a non-human animal an effective
amount of one or more compounds of claim 1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. application Ser.
No. 12/430,610 filed Apr. 27, 2009, now allowed which in turn
claims the benefit of International Application PCT/US09/41719,
filed Apr. 24, 2009 which designates the U.S. and of U.S.
provisional application 61/048,080, filed Apr. 25, 2008. Each of
these applications is incorporated by reference in its entirety
herein.
BACKGROUND OF THE INVENTION
[0003] Galactofuranose (Galf) residues are present in many
pathogens. For example, they are essential components of the
arabinogalactan layer of mycobacteria. Mycobacteria cause a number
of diseases, the most deadly of these is tuberculosis (TB). Each
year, Mycobacterium tuberculosis is responsible for 8 million human
infections and 2 million deaths. (Tripathi, R. P.; Tewari, N.;
Dwivedi, N.; Tiwari, V. K. Med. Res. Rev. 2005, 25, 93-131.)
Strains have emerged that are resistant to most or all known
antibiotics. (Marris, E. Nature 2006, 443, 131.) Resistance can be
combated by developing an inhibitor with a new mechanism of action
against a known target. (Sullivan, T. J.; Truglio, J. J.; Boyne, M.
E.; Novichenok, P.; Zhang, X.; Stratton, C. F.; L.sub.1, H. J.;
Kaur, T.; Amin, A.; Johnson, F.; Slayden, R. A.; Kisker, C.; Tonge,
P. J. ACS Chem. Biol. 2006, 1, 43-53.) An alternative approach is
to identify novel targets. One such potential target is the
essential enzyme responsible for the incorporation of
galacto-furanose residues, uridine 5'-diphosphate (UDP)
galactopyranose mutase (UGM). (Lowary, T. L. Curr. Opin. Chem.
Biol. 2003, 7, 749-756.)
[0004] UGM uses a unique mechanism to catalyze the isomerization of
UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf)
(Scheme 1, where U is uracil). (Soltero-Higgin, M.; Carlson, E. E.;
Gruber, T. D.; Kiessling, L. L. Nat. Struct. Mol. Biol. 2004, 11,
539-543; Chad, J. M.; Sarathy, K. P.; Gruber, T. D.; Addala, E.;
Kiessling, L. L.; Sanders, D. A. R. Biochemistry 2007, 46,
6723-6732.) Since UGM is not a target of other tuberculosis drugs,
compounds that block UGM are expected to be effective against drug
resistant strains.
##STR00001##
[0005] The gene encoding UGM is essential for mycobacterial
viability; the identification of UGM inhibitors can validate it as
a therapeutic target. (Pan, F.; Jackson, M.; Ma, Y. F.; McNeil, M.
J. Bacteriol. 2001, 183, 3991-3998.) Moreover, Galf residues are
also found in some eukaryotes, including a number of pathogenic (to
humans and animals) eukaryotes, therefore, UGM inhibitors can also
provide insight into the role of Galf-containing oligosaccharides
in these organisms. (Beverley, S. M.; Owens, K. L.; Showalter, M.;
Griffith, C. L.; Doering, T. L.; Jones, V. C.; McNeil, M. R.
Eukaryotic Cell 2005, 4, 1147-1154.) Additionally, there is no
enzyme comparable to UGM in humans or other mammals increasing the
appeal of UGM inhibitors as useful therapeutics.
[0006] Most efforts to develop UGM inhibitors have focused on
UDP-sugar substrate analogs. (Caravano, A.; Dohi, H.; Sinay, P.;
Vincent, S. P. Chem.-Eur. J. 2006, 12, 3114-3123; Liautard, V.;
Christina, A. E.; Desvergnes, V.; Martin, O. R. J. Org. Chem. 2006,
71, 7337-7345; Ghavami, A.; Chen, J. J. W.; Pinto, B. M. Carbohydr.
Res. 2004, 339, 401-407; Lee, R. E.; Smith, M. D.; Pickering, L.;
Fleet, G. W. J. Tetrahedron Lett. 1999, 40, 8689-8692; Liautard,
V.; Desvergnes, V.; Martin, O. R. Org. Lett. 2006, 8, 1299-1302.)
Simple sugar derivatives, including galactopyranose or
galactofuranose analogs, bind weakly with affinities in the
millimolar range (Lee, R. E.; Smith, M. D.; Nash, R. J.; Griffiths,
R. C.; McNeil, M.; Grewal, R. K.; Yan, W. X.; Besra, G. S.;
Brennan, P. J.; Fleet, G. W. J. Tetrahedron Lett. 1997, 38,
6733-6736; Veerapen, N.; Yuan, Y.; Sanders, D. A. R.; Pinto, B. M.
Carbohydr. Res. 2004, 339, 2205-2217.) Inhibitors that incorporate
the uridine portion of the substrate bind substantially better,
with affinities that approximate that of UDP-Galp (K.sub.d=52
.mu.M) (Itoh, K.; Huang, Z. S.; Liu, H. W. Org. Lett. 2007, 9,
879-882; Caravano, A.; Vincent, S. P.; Sinay, P. Chem. Commun.
2004, 1216-1217; Caravano, A.; Mengin-Lecreulx, D.; Brondello, J.
M.; Vincent, S. P.; Sinay, P. Chem.-Eur. J. 2003, 9, 5888-5898;
Pan, W. D.; Ansiaux, C.; Vincent, S. P. Tetrahedron Lett. 2007, 48,
4353-4356; Scherman, M. S.; Winans, K. A.; Stern, R. J.; Jones, V.;
Bertozzi, C. R.; McNeil, M. R. Antimicrob. Agents Chemother. 2003,
47, 378-382.) These approaches have not yet afforded compounds that
block mycobacterial growth.
[0007] Certain non-substrate based molecules have been identified
as UMG ligands. For example, certain nitrofuranylamides have been
identified as inhibitors of UGM catalysis and mycobacterial growth.
(Tangallapally, R. P.; Yendapally, R.; Lee, R. E.; Hevener, K.;
Jones, V. C.; Lenaerts, A. J. M.; McNeil, M. R.; Wang, Y. H.;
Franzblau, S.; Lee, R. E. J. Med. Chem. 2004, 47, 5276-5283.)
Nevertheless, the UGM inhibition and antimycobacterial activity of
these compounds were not correlated, so they do not address the
utility of inhibiting UGM.
[0008] Published International application WO 2005/007625 (Lee et
al.), as well as published U.S. application 20050222408, relate to
certain heterocyclic amides with anti-tuberculosis activity. More
specifically, these patent documents relate to compounds of
formula:
##STR00002##
wherein A is selected from the group consisting of oxygen, sulfur,
and NR.sub.15, and R.sub.15 is selected from the group consisting
of H, alkyl, aryl, substituted alkyl, and substituted aryl; B, D,
and E are each independently selected from the group consisting of
CH, nitrogen, sulfur and oxygen; R.sub.1 is selected from the group
consisting of nitro, halo, alkyl ester, arylsulfanyl, arylsulfinyl,
arylsulfonyl and sulfonic acid; t is an integer from 1 to 3; and X
is a substituted amide. These patent documents are incorporated by
reference herein at least in part for the definitions of structural
elements of the above formula.
[0009] A high-throughput, fluorescence polarization (FP) screen was
reported to identify several compounds (including 1A-4A below) with
good IC50 values (.about.10.sup.-6 M) for the UGM from Klebsiella
pneumoniae (UGM.sub.kleb) or M. tuberculosis (UGM.sub.myco).
(Soltero-Higgin, M.; Carlson, E. E.; Phillips, J. H.; Kiessling, L.
L. J. Am. Chem. Soc. 2004, 126, 10532-10533.)
##STR00003##
[0010] A directed library containing a
5-arylidine-2-thioxo-4-thiazolidinone core has been reported to
identify factors influencing UGM ligand binding. (Carlson, E. E.;
May, J. F.; Kiessling, L. L. Chem. Biol. 2006, 13, 825-837.)
Several thiazolidinone derivatives were reported to be ligands for
both the UGM.sub.kleb and UGM.sub.myco homologs. The thiazolidinone
scaffold, however, reacts reversibly with biologically relevant
thiols in solution. Not surprisingly, inhibitors of this structural
class were reported to fail to block mycobacterial growth.
[0011] There remains a need in the art for small molecules that
exhibit antimicrobial activity, particularly against
Mycobacteria.
SUMMARY OF THE INVENTION
[0012] The present invention provides compounds which inhibit
microbial growth or attenuate the virulence of pathogen
microorganisms. In certain embodiments, compounds of the invention
inhibit UDP-galactopyranose mutase (UGM) and have activity as
inhibitors of microbial growth of microorganisms which contain this
enzyme and particularly those microorganisms in which this enzyme
is responsible for the incorporation of galactofuranose residues,
particularly for uridine 5'-diphosphate (UDP) galactopyranose
mutase. In certain embodiments, compounds of the invention inhibit
UDP-galactopyranose mutase (UGM) and have activity to attenuate
virulence of pathogenic microorganisms which contain this enzyme
and particularly those microorganisms in which this enzyme is
responsible for the incorporation of galactofuranose residues.
[0013] More specifically, the inhibitors of UGM of this invention
inhibit growth or attenuate virulence of microbial pathogens
including mycobacterium, for example, M. tuberculosis and M.
smegmatis and Klebsiella, for example, Klebsiella pneumoniae.
Additionally, UGM inhibitors of this invention can also inhibit UGM
of certain eukaryotic human and animal pathogens. Compounds of this
invention are useful for treatment of infections by prokaryotic and
eukaryotic pathogens. Compounds of this invention are useful in
human and veterinary treatment applications. Compounds of this
invention are useful for the treatment of tuberculosis. Compounds
of this invention are useful in combination therapy with other
antibiotics for the treatment of microbial infections, including
tuberculosis. Compounds of this invention are useful for the
treatment of multiple drug resistant microbial infections,
including multiple drug resistant tuberculosis.
[0014] The invention provides compounds of formula I:
##STR00004##
and salts thereof,
[0015] where: n is 0 or 1 to indicate the presence or absence of
L.sub.1; L.sub.1, if present, is an alkylene, alkenylene or
alkyleneoxy linker having 1-6 carbon atoms;
[0016] Y is CH.sub.2 or NR.sub.Y, where Ry is hydrogen or a C1-C6
alkyl;
[0017] W is --COOR.sub.W, --O--COR.sub.W, --CON(R.sub.W).sub.2,
--OCON(R.sub.w).sub.2, --SO.sub.2--OR.sub.W,
--SO.sub.2--N(R.sub.W).sub.2, --OPO.sub.3R.sub.W,
--OP(OR.sub.W).sub.2R.sub.W, or a tetrazolyl group, where each
R.sub.W, independent of other R.sub.W, is hydrogen, C1-C6 alkyl or
an aryl or heteroaryl group having one or two 5-member or 6-member
rings;
[0018] the D ring is a 1,3- or 1,2-substituted 5-member (when m is
1) or 6-member ring (when m is 2), which is a carbocyclic or
heterocyclic ring, including a heteroaryl, or an aryl group, where
the 1, 2 and 3 ring positions are as indicated above;
[0019] R.sup.1 represents no substitution on the D ring (where
R.sup.1 are all hydrogens) or substitution with one or more C1-C3
alky, one or more C1-C3 haloalkyl (including trihalomethyl groups),
one or more halogens or combinations thereof;
[0020] the B and C rings are independently selected from an aryl or
heteroaryl group having one or two rings;
[0021] R.sub.B and R.sub.C represent no substitution on the
indicated ring or substitution on that ring by one to five
non-hydrogen substituents selected from the group consisting of
halogen, nitro, cyano, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy,
sulfonamide (--SO.sub.2--N(R.sub.F).sub.2), azide, sulfonyl
(--SO.sub.2--R.sub.F), --COOR.sub.F, --O--COR.sub.E, --COR.sub.F,
--CON(R.sub.F).sub.2, --O--CON(R.sub.F).sub.2, --N(R.sub.F).sub.2,
- and C1-C6 haloalkyl groups, including trifluoromethyl,
trichloromethyl and tribromomethyl groups, where R.sub.F is
hydrogen or a C1-C6 alkyl group;
[0022] or wherein substituents on two adjacent carbons of a B or C
ring together form a carbocyclic or heterocyclic ring having 5 to 8
ring atoms and optionally wherein one or two of the ring members
are heteroatoms, particularly N, O or S, which 5- to 8-member ring
may be an aromatic ring, or
[0023] R.sub.B is -M-L.sub.2-Q-AR, where:
[0024] AR is an aryl or heteroaryl group having at least two
rings;
[0025] L.sub.2 is a linker group which can be an alkylene,
cycloalkylene, alkenylene or alkyleneoxy group, which contains 5-12
carbon atoms, or a 5- or 6-member heterocyclene group, containing
one or two O, N or S atoms or combinations thereof, a 6-member ring
arylene or a 6-member ring heteroarylene group, containing one or
two O, N or S atoms or combinations thereof;
[0026] M and Q are chemical moieties that function to covalently
link L between the B ring and AR, M and Q can independently be
selected from, --O--, --S--, --CO--, --NR.sub.M--, (where R.sub.M
is hydrogen or C1-C6 alkyl), carboxyl, amide, sulfonamide,
thiourea, urea, carbonate, guanidinium, carbamate, thiocarbamate,
alkylene moieties or combinations thereof. In the above formula the
numbering of the carbons on the B and C rings is indicated.
[0027] In specific embodiments, L.sub.2 is --(CH.sub.2).sub.a--,
--CH.sub.2--(CH.sub.2--O--CH.sub.2).sub.b--CH.sub.2--, or
[--(CH.sub.2).sub.c--O--].sub.e--(CH.sub.2).sub.d--, where .alpha.,
independently, is an integer ranging from 3-12 and more preferably
ranges from 5-12 and more preferably ranges from 6-10, b,
independently, is an integer ranging from 1-4 and more preferably
1, 2 or 3, c, independently, is an integer ranging from 1-6, and
preferably 1-4, d, independently, is an integer ranging from 1-6
and preferably 2-4 and e, independently, is an integer ranging from
1 to 4 and preferably 2 or 3. In other embodiments, L.sub.2 is a
cyclohexylene group, or a
--(CH.sub.2).sub.f-cyclo-C.sub.6H.sub.10--(CH.sub.2).sub.g-- group,
where f and g, independently are integers, from 1-6. In other
embodiments, L.sub.2 is a 5- or 6-member heterocyclene group,
containing one or two O, N or S atoms or combinations thereof, a
6-member ring arylene or a 5- or 6-member ring heteroarylene group,
containing one or two O, N or S atoms or combinations thereof and M
and Q are alkylene groups.
[0028] In specific embodiments, W is --COOH. In specific
embodiments, W is --CON(H).sub.2. In specific embodiments, W is
--SO.sub.2--OH. In specific embodiments, W is
--SO.sub.2--N(H).sub.2. In specific embodiments, W is --OPO.sub.3H.
In specific embodiments, W is, --OP(OH).sub.2H or --OP(OR).sub.2H,
where R is an alkyl group. In specific embodiments, W is a
tetrazol-1-yl group.
[0029] In an embodiment, the invention provides compounds of
formula II:
##STR00005##
and salts thereof,
[0030] where n is 0 or 1;
[0031] Y is CH.sub.2 or NR.sub.Y where R.sub.Y is hydrogen or a
C1-C6 alkyl;
[0032] W is --COOR.sub.W, --O--CO--R.sub.W, --CON(R.sub.W).sub.2,
--O--CON(R.sub.W).sub.2, --SO.sub.2--OR.sub.W,
--SO.sub.2--N(R.sub.W).sub.2, --OPO.sub.3R.sub.W,
--OP(OR.sub.W).sub.2R.sub.W, or tetrazolyl, where each R.sub.W,
independent of other R.sub.W, is hydrogen, C1-C6 alkyl or an aryl
or heteroaryl group having one 5 or 6-member ring;
[0033] the D ring is a 1,3- or 1,2-substituted 5-member (m is 1) or
6-member ring (m is 2) which is carbocyclic or heterocyclic,
including heteroaryl, and aryl groups, where the 1, 2 and 3 ring
positions are as indicated above;
[0034] R.sup.1 represents no substitution on the D ring (R.sup.1
all hydrogen) or substitution with one or more C1-C3 alky, C1-C3
haloalkyl (including trihalomethyl groups) or halogens;
[0035] the B and C rings are independently selected from an aryl or
heteroaryl group having one or two rings;
[0036] R.sub.B and R.sub.C represents no substitution on the
indicated ring or substitution on that ring by one to five
non-hydrogen substituents selected from the group consisting of
halogen, nitro, cyano, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy,
sulfonamide (--SO.sub.2--N(R.sub.F).sub.2), azide, sulfonyl
(--SO.sub.2--R.sub.F), --COOR.sub.F, --COR.sub.F, --OCOR.sub.F,
--CON(R.sub.F).sub.2, --O--CON(R.sub.F).sub.2, --N(R.sub.F).sub.2,
- and C1-C6 haloalkyl groups, including trifluoromethyl,
trichloromethyl and tribromomethyl groups, where R.sub.F is
hydrogen or a C1-C6 alkyl group;
[0037] or wherein substituents on two adjacent carbons of a B or C
ring together form a carbocyclic or heterocyclic ring containing
having 5-8 members and optionally containing one or two
heteroatoms, particularly N, O or S, which 5-8 member ring may be
an aromatic ring or
[0038] R.sub.B includes the group:
-M-L.sub.2-Q-AR, where:
[0039] AR is an aryl or heteroaryl group having at least two
rings;
[0040] L.sub.2 is a hydrophobic linker group ranging in length from
5-12 atoms (preferably 6-10 atoms), which can be
--(CH.sub.2).sub.n--,
--CH.sub.2--(CH.sub.2--O--CH.sub.2).sub.b--CH.sub.2--, where a is
an integer ranging from 5-12 and preferably ranges from 6-10 and b
is 2-4, a cyclohexyl group, a 6-member heterocyclic group
containing one or two O, N or S groups, or a 6-member ring aryl or
a 6-member ring heteroaryl group;
[0041] M and Q are chemical moieties that function to covalently
link L.sub.2 between the B ring and AR, M and Q can independently
be selected from, --O--, --S--, --CO--, --NR.sub.M--, where R.sub.M
is hydrogen or C1-C6 alkyl, carboxyl, amide, sulfonamide, thiourea,
urea, carbonate, guanidinium, carbamate, and thiocarbamate
moieties. In the above formula the numbering of the carbons on the
B and C rings is indicated.
[0042] Exemplary D rings for formulas herein are illustrated in
FIGS. 1A, 1B and 1C which follow, in which each R.sup.1,
independently, is hydrogen, C1-C3 alkyl, C1-C3-haloalkyl or halogen
and R.sub.D is hydrogen or a C1-C6 alkyl.
[0043] In specific embodiments, both the B and C rings are
optionally substituted phenyl rings. In specific embodiments, the B
or the C ring can be selected from heteroaryl 6-member rings. In
specific embodiments, the B or C ring has one or two 5- or 6-member
rings. In specific embodiments, the B and C rings can be selected
from pyridyl, indolyl, purinyl, pyrazinyl, pyrimidinyl, thienyl,
benzofuranyl, naphthyl or benzothienyl rings.
[0044] In a specific embodiment, there is at least one non-hydrogen
substituent on the B ring or C ring. In a specific embodiment,
there is at least one non-hydrogen substituent on each of the B and
C rings.
[0045] D rings are preferably substituted at the 1 and 3 positions
as those positions are numbered in Formula II and may have one or
two additional substituents R.sup.1. D ring groups include, among
others, phenyl, thiazolyl, oxazolyl, imidazolyl, pyrazolyl,
pyrrolyl, pyridyl, pyridazinyl, pyrimidinyl, thienyl (thiophenyl),
furanyl, isoxazolyl, isothiazolyl, or tetrazolyl, including various
isomers thereof.
[0046] In specific embodiments, AR has from two to four 5 or
6-member rings. AR groups include, among others, optionally
substituted benzophenonyl, naphthalenyl, benzofuranyl,
benzothienyl, indolyl, xanthenyl, purinyl, quinolyl, biphenyl,
phenylxanthenyl, acridinyl, phenazinyl, phenoxazinyl,
phenothiazinyl, carbazolyl, and naphthyridinyl. In specific
embodiments, AR groups include, among others, non-substituted
benzophenonyl, naphthalenyl, benzofuranyl, benzothienyl, indolyl,
xanthenyl, purinyl, quinolyl, biphenyl, phenylxanthenyl, acridinyl,
phenazinyl, phenoxazinyl, phenothiazinyl, carbazolyl, or
naphthyridinyl groups, including various isomers thereof.
[0047] In specific embodiments, W is --COOH. In specific
embodiments, W is --CON(H).sub.2. In specific embodiments, W is
--SO.sub.2--OH. In specific embodiments, W is
--SO.sub.2--N(H).sub.2. In specific embodiments, W is --OPO.sub.3H.
In specific embodiments, W is, --OP(OH).sub.2H or --OP(OR).sub.2H,
where R is an alkyl group. In specific embodiments, W is a
tetrazol-1-yl group.
[0048] In specific embodiments, M and Q are selected from
--NR.sub.M--, --O-- or a thiourea moiety. In specific embodiments,
L.sub.2 is an alkenylene, --(CH.sub.2).sub.a-- as defined above and
in a more specific embodiment, a is 6, 7, 8 or 9.
[0049] In specific embodiments, AR groups are dyes including
various fluorescein dye groups, eosin dye groups, erthrosin dye
groups, rhodamine dye groups, bodipyl dye groups, or alexafluor 488
dye groups. In these embodiments, the compound containing the AR
dye group is formed by forming a covalent bond to a dye molecule.
Typically, such groups are formed by reaction of a dye molecule
containing a reactive functional group (e.g., an isothiocyanate
group). In specific embodiments, where AR is a dye, the Q moiety is
the group that results on reaction of the functional group on the
dye with a compatible reactive group on the linker. See Example 2
for specific examples. In specific examples, AR is a structure in
FIG. 2A or 2B.
[0050] In a specific embodiment, the substituent on the B ring is
the -M-L-Q-AR group at the 3-position.
[0051] In a specific embodiment there is at least one non-hydrogen
substituent on each of the B and C ring. In specific examples,
there is one substituent on the C ring and at least one substituent
on the B ring. In specific examples, there is 0 or 1 substituent on
the C ring and the substituent on the B ring is the -M-L-Q-AR group
at the 3-position.
[0052] In specific embodiments, the UGM inhibitors are compounds of
formulas IVA, IVB, IVC, IVD, IVE or IVF:
##STR00006##
and salts thereof,
[0053] where L.sub.1, n, R.sub.C, R.sup.1 and Rings B, C and D are
defined above;
[0054] the A ring is a 6-member aryl or heteroaryl ring having one,
two or three nitrogens; and
[0055] each R.sub.A is a hydrogen, halogen, nitro, cyano, hydroxyl,
C1-C6 alkyl, C1-C6 alkoxy, sulfonamide
(--SO.sub.2--N(R.sub.F).sub.2), azide, sulfonyl
(--SO.sub.2--R.sub.F), --COOR.sub.F, --COR.sub.F,
--CON(R.sub.F).sub.2, --O--CON(R.sub.F).sub.2, --N(R.sub.F).sub.2,
or C1-C6 haloalkyl groups, including trifluoromethyl,
trichloromethyl and tribromomethyl groups, where R.sub.F is
hydrogen or a C1-C6 alkyl group, and R.sub.B represents no
substitution on the indicated B ring or substitution on the B ring
by one to five non-hydrogen substituents selected from the group
consisting of halogen, nitro, cyano, hydroxyl, C1-C6 alkyl, C1-C6
alkoxy, sulfonamide (--SO.sub.2--N(R.sub.F).sub.2), azide, sulfonyl
(--SO.sub.2--R.sub.F), --COOR.sub.F, --COR.sub.F, --OCR.sub.F,
--CON(R.sub.F).sub.2, --O--CON(R.sub.F).sub.2, --N(R.sub.F).sub.2,
- and C1-C6 alkylhalide groups, including trifluoromethyl,
trichloromethyl and tribromomethyl groups, where R.sub.F is
hydrogen or a C1-C6 alkyl group or wherein substituents on two
adjacent carbons of the phenyl ring together form a carbocyclic or
heterocyclic ring containing having 5-8 members and optionally
containing one or two heteroatoms which 5-8 member ring may be an
aromatic ring or where R.sup.1 and one substituent on the B ring
are linked together to form a 5-8 member carbocyclic or
heterocyclic ring which may be an aromatic ring. The numbers of the
carbons on the B ring are indicated.
[0056] In a specific embodiment n is 1. In a specific embodiment,
L.sub.1 is an alkeneylene having one double bond.
[0057] In a specific embodiment, the D ring is one of D1-D42.
[0058] In a specific embodiment, there is at least one non-hydrogen
substituent on either the C or B ring. In another embodiment, there
is at least one non-hydrogen substituent on each of the C and B
rings.
[0059] In a specific embodiment, there is at least one non-hydrogen
substituent on either the A or B ring. In another embodiment, there
is at least one non-hydrogen substituent on each of the A and B
ring. In a specific embodiment, the A ring is a phenyl ring.
[0060] In another embodiment, the invention provides compounds of
formula VA, VB, VC or VD:
##STR00007##
and salts thereof, where variables are as defined for formulas I-IV
above and n is 0 or 1.
[0061] In specific embodiments of formulas VA and VB, the C ring is
a phenyl ring. In specific embodiments of formulas VA and VB, the B
ring is a phenyl ring. In specific embodiments of formulas VA and
VB, both the C and B rings are phenyl rings. In specific
embodiments of formulas VA and VB, when the C ring is a phenyl ring
and when n is 0, then R.sub.C is a non-hydrogen substituent and
there is at least one non-hydrogen substituent on the B ring and
when n is 1 there is at least one non-hydrogen substituent on
either the C or B ring.
[0062] In specific embodiments of formulas VC and VD, the A ring is
a phenyl ring. In specific embodiments of formulas VC and VD, the B
ring is a phenyl ring. In specific embodiments of formulas VC and
VD, both the A and B rings are phenyl rings. In specific
embodiments of formulas VC and VD, when A is a phenyl ring and when
n is 0 then R.sub.A is a non-hydrogen substituent and there is at
least one non-hydrogen substituent on the B ring and when n is 1
there is at least one non-hydrogen substituent on either the A or B
ring.
[0063] In another embodiment, the invention provides compounds of
formula VIA and VIB:
##STR00008##
and salts thereof,
[0064] where D, n, R.sup.1 and R.sub.B are as defined above for
formulas I-V above and A is an optionally substituted aryl or
optionally substituted heteroaryl group having one or two rings,
wherein optional substitution is substitution with one or more of
halogen, nitro, cyano, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy,
sulfonamide (--SO.sub.2--N(R.sub.F).sub.2), azide, sulfonyl
(--SO.sub.2--R.sub.F), --CO--OR.sub.F, --COR.sub.F--O--COR.sub.F,
--CON(R.sub.F).sub.2, --O--CON(R.sub.F).sub.2, --N(R.sub.F).sub.2,
- and C1-C6 haloalkyl groups, including trifluoromethyl,
trichloromethyl and tribromomethyl groups, where R.sub.F is
hydrogen or a C1-C6 alkyl group,
[0065] wherein: when n is 0, A is an optionally substituted
heteroaryl group or A is a phenyl group having the structure:
##STR00009##
where R.sub.A is as defined above or when n is 1, A is an
optionally substituted phenyl or heteroaryl group.
[0066] In specific embodiments of formulas VIA or VIB, ring B is a
phenyl ring. In specific embodiments of formulas VIA or VIB, A is
an optionally substituted phenyl ring and ring B is a phenyl ring.
In specific embodiments of formulas VIA or VIB, A is an optionally
substituted naphthyl group. In specific embodiments of formulas VIA
or VIB, A is an optionally substituted naphthyl group and the B
ring is a phenyl ring.
[0067] In another embodiment the invention provides compounds of
formula VIIA and VIIB:
##STR00010##
where variables are as defined above.
[0068] In specific embodiments of formulas VIIA/B, Q is thiourea,
and M is O or NR.sub.M. In specific embodiments of formulas VIIA/B,
AR is a dye group, particularly a fluorescein dye group. In
specific embodiments of formulas VIIA/B, AR is optionally
substituted naphthyl. In specific embodiments of formulas VIIA/B,
the B ring is a phenyl ring. In specific embodiments of formulas
VIIA/B, the C ring is a phenyl ring. In specific embodiments of
formulas VIIIA/B, R.sub.C represents substitution with a single
non-hydrogen substituent at the 3 position. In specific
embodiments, n is 1. In specific embodiments of formulas VIIA/B,
R.sup.1 is hydrogen.
[0069] In additional embodiments, the invention provides compounds
of formula I or II wherein R.sub.B is -M-L.sub.2-Q-AR and AR is
selected from AR groups 100-104 or 200-204. Also provided are
compounds of formula I and II where AR is
##STR00011##
In additional embodiments, the invention provides compounds of
formula I or II wherein R.sub.B represents two or more substituents
on the B ring, one of which is -M-L.sub.2-Q-AR.
[0070] In additional embodiments, the invention provides compounds
of formula X:
##STR00012##
where variable are as defined above.
[0071] In additional embodiments, the invention provides compounds
of formula XI:
##STR00013##
where variables are as defined above and n is 0 or 1.
[0072] In additional embodiments, the invention provides compounds
of formula XII:
##STR00014##
where variables are as defined above.
[0073] In additional embodiments, the invention provides compounds
of formula XIII:
##STR00015##
where variables are as defined above.
[0074] In additional embodiments, the invention provides compounds
of formula XIV:
##STR00016##
where variables are as defined above.
[0075] In specific embodiments of formulas X-XIV, the B ring and
the C ring are phenyl rings. In specific embodiments of formulas
X-XIV, AR is an optionally substituted naphthyl group. In specific
embodiments of formulas X-XIV, AR is an unsubstituted naphthyl
group. In specific embodiments of formulas X-XIV, AR is an AR group
selected from groups 100-104 or 200-204 (FIGS. 2A and 2B).
In specific embodiments of formulas X-XIV, AR is
##STR00017##
[0076] In specific embodiments of formulas X-XIV, Q is a thiourea
or urea. In other specific embodiments of formulas X-XIV, Q is
thiourea and AR is
##STR00018##
[0077] The invention further provides a method for inhibiting UGM
in vitro or in vivo by contacting a biological composition
comprising an active UGM with an amount of one or more of the
compounds of any of the formulas herein effective for inhibiting
UGM. In a specific embodiment, the inhibition of UGM is in vivo in
a prokaryote. In a specific embodiment, the inhibition of UGM is in
vivo in a eukaryote.
[0078] The invention also provides a method for inhibiting the
growth of a microorganism containing UGM by contacting the
microorganism with an amount of one or more of the compounds of any
of the formulas herein effective for inhibiting the growth of the
microorganism. In a specific embodiment the microorganism is a
human or veterinary pathogen. In a specific embodiment, the
microorganism is a bacterium. In another embodiment, the
microorganism is of the genus Mycobacterium. In more specific
embodiments, the microorganism is Mycobacterium tuberculosis or
Mycobacterium smegmatis. In another embodiment, the microorganism
is of the genus Klebsiella, including Klebsiella pneumoniae. The
microorganism can be a prokaryote or a eukaryote.
[0079] The invention also provides a method for attenuating the
virulence of a microorganism containing UGM by contacting the
microorganism with an amount of one or more of the compounds of any
of the formulas herein effective for attenuating virulence of the
microorganism. In a specific embodiment the microorganism is a
human or veterinary pathogen. In a specific embodiment, the
microorganism is a bacterium. In another embodiment, the
microorganism is of the genus Mycobacterium. In more specific
embodiments, the microorganism is Mycobacterium tuberculosis or
Mycobacterium smegmatis. In another embodiment, the microorganism
is of the genus Klebsiella, including Klebsiella pneumoniae. The
microorganism can be a prokaryote or a eukaryote.
[0080] The invention also provides a method for inhibiting the
growth of a microorganism containing UGM by contacting the
microorganism with an amount of one or more of the compounds of any
of the formulas herein effective for inhibiting UGM. In a specific
embodiment the microorganism is a human or veterinary pathogen. In
a specific embodiment, the microorganism is a bacterium. In another
embodiment, the microorganism is of the genus Mycobacterium. In
more specific embodiments, the microorganism is Mycobacterium
tuberculosis or Mycobacterium smegmatis. In another embodiment, the
microorganism is of the genus Klebsiella, including Klebsiella
pneumoniae. The microorganism can be a prokaryote or a
eukaryote.
[0081] The invention further provides a method of treating a human,
a non-human mammal or a non-human animal individual having or
believed to have an infection of a microorganism containing UGM by
administering to the individual an amount of one or more compounds
of any of the formulas herein effective for inhibiting the growth
of the microorganism. In specific embodiments, the microorganism is
a bacterium or a mycobacterium. In specific embodiments, the
mycobacterial infection is tuberculosis. In specific embodiments,
the microorganism is of the genus Mycobacterium or Klebsiella. In
additional embodiments, the microorganism is Mycobacterium
tuberculosis, Mycobacterium smegmatis or Klebsiella pneumoniae. The
pathogenic microorganism can be a prokaryote or a eukaryote.
[0082] The invention additionally provides a method for treating a
human, a non-human mammal or a non-human animal individual having
or believed to have an infection of a microorganism containing UGM
by administering an effective amount of a compound of this
invention of any of the formulas herein, in combination with an
antibiotic appropriate for treatment of the infection. Compounds of
this invention can enhance the effectiveness of art-known
antibiotics and are useful in combination therapy in addition to
such antibiotics.
[0083] The compounds of the present invention can, for example, be
employed in combination therapy with antibiotics, such as
ethambutol, isoniazid, rifampicin, and pyrazinamide. Such
combination therapy is particularly useful in the treatment of
mycobacterial infections.
[0084] The invention additional provides a medicament comprising
one or more compounds of the formulas herein effective for
inhibiting the growth of a microorganism or effective for
attenuating the virulence of a microorganism which contains UGM.
The invention additional provides a medicament comprising one or
more compounds of any of the formulas herein effective for
inhibiting UGM. In specific embodiments, the microorganism is a
bacterium or a mycobacterium. In specific embodiments, the
microorganism is of the genus Mycobacterium or Klebsiella. In
additional embodiments, the microorganism is Mycobacterium
tuberculosis, Mycobacterium smegmatis or Klebsiella pneumoniae.
[0085] The invention also provides a method of making a medicament
for treating an individual (human, mammal or animal) having a
bacterial or mycobacterial infection. In specific embodiments, the
mycobacterial infection is tuberculosis. In specific embodiments,
the microorganism is of the genus Mycobacterium or Klebsiella. In
additional embodiments, the microorganism is Mycobacterium
tuberculosis, Mycobacterium smegmatis or Klebsiella pneumoniae. In
a specific embodiment, the method of making a medicament includes
the step of combining an amount of a compound of any of the
formulas herein with a pharmaceutically effective carrier. In a
specific embodiment, the medicament is in a dosage form appropriate
for oral administration, topical administration or administration
by injection.
[0086] The invention also provides method for screening a small
molecule library for members which inhibit the growth of a
microorganism having UGM which comprises the step of identifying
those member of the library which exhibit a dissociation constant K
for the UGM enzyme of 100 .mu.M or less. In another embodiment, the
member is identified as exhibiting a dissociation constant K for
the UGM enzyme of 50 .mu.M or less. In another embodiment, the
member is identified as exhibiting a dissociation constant K for
the UGM enzyme of 25 .mu.M or less. In another embodiment, the
member is identified as exhibiting a dissociation constant K for
the UGM enzyme of 10 .mu.M or less. In further embodiments, the
microorganism is a mycobacterium, particularly Mycobacterium
tuberculosis. In additional embodiments the microorganism is of the
genus Klebsiella.
[0087] In specific embodiments, the invention also provides
compounds of any of the formulas herein which inhibit the growth of
a microorganism having UGM which exhibit a dissociation constant K
for the UGM enzyme of 100 .mu.M or less. In another embodiment, the
compound exhibits a dissociation constant K for the UGM enzyme of
50 .mu.M or less. In another embodiment, the compound exhibits a
dissociation constant K for the UGM enzyme of 25 .mu.M or less. In
another embodiment, the compound exhibits a dissociation constant K
for the UGM enzyme of 10 .mu.M or less. In further embodiments, the
microorganism is a Mycobacterium, particularly Mycobacterium
tuberculosis. In additional embodiments the microorganism is of the
genus Klebsiella.
[0088] Additional aspects and embodiments of the invention will be
apparent on review of the detailed description and examples.
BRIEF DESCRIPTION OF THE DRAWINGS
[0089] FIGS. 1A-1C illustrate exemplary D rings for formulas
herein.
[0090] FIG. 2A or 2B illustrate exemplary AR groups for formulas
herein.
[0091] FIG. 3 illustrates a comparison of the shape of a
thiazolidinone scaffold to that of a 2-aminothiazole scaffold using
an overlay of minimized thiazolidinone scaffold (top) and novel
2-aminothiazole scaffold (bottom). Exemplary chemical structures
are also illustrated.
[0092] FIGS. 4A and 4B illustrate the results of disc assays to
assess microbial growth inhibition by a 2-aminothiazole. Discs
applied with DMSO (negative control, FIG. 4A) or the illustrated
compound in DMSO (FIG. 4B) were placed on agar medium inoculated
with M. smegmatis. The zone of inhibition persisted throughout the
4 days of the assay.
[0093] FIGS. 5A and 5B are graphs illustrating exemplary binding
curves as determined by the fluorescence polarization assay.
[0094] FIG. 6 (pages 1 to 5) is a Table of exemplary binding data
as determined by the fluorescence polarization assay. Binding is
shown for the UGM isoform from K. pneumoniae (K) and M.
tuberculosis (M). Dissociation constants are given in units of
.mu.M. The data in FIG. 6 depicts the effects of varying the A ring
substitution.
[0095] FIG. 7 (pages 1 to 4) is a Table of exemplary binding data
as determined by the fluorescence polarization assay. Binding is
shown for the UGM isoform from K. pneumoniae (K) and M.
tuberculosis (M). Dissociation constants are given in units of
.mu.M. The data in FIG. 7 depicts the effects of varying the B ring
substitution in compounds of structure.
[0096] FIG. 8 is a graph illustrating the relationship between
UGM.sub.myco Activity and K.sub.d for exemplary aminothiazoles.
[0097] FIGS. 9A and 9B are representative dose-response inhibition
curves for two mycobacterial growth inhibitors whose structures are
illustrated. The inhibition curves look similar to the binding
curves depicted in FIGS. 5A and 5B, above. The IC.sub.50 values are
comparable, albeit slightly higher than the K.sub.d values. In the
graphs of these figures, conversion is the amount of UDP-Galp
formed divided by the amount of total UDP-Gal
(UDP-Galp+UDP-Galf).
[0098] FIGS. 10A and 10B illustrate an exemplary graph of enzyme
inhibition kinetics and a double reciprocal plot, respectively for
a compound of the invention.
[0099] FIG. 11, pages 1 to 3, provides a table of exemplary data
for the indicated compounds which compares K.sub.d (UGM.sub.myco),
relative UGM.sub.myco activity at 50 .mu.M and the qualitative
results of bacterial growth disc assays.
[0100] FIG. 12A provides a table of exemplary MIC data.
[0101] FIG. 12B provides a graph illustrating the correlation
between MIC values and UGM inhibition.
[0102] FIGS. 13A and 13B are graphs illustrating determination of
K.sub.d for compound II for UGM.sub.myco and UGM.sub.kleb,
respectively.
DETAILED DESCRIPTION OF THE INVENTION
[0103] The invention is based at least in part on the
identification of certain small molecule inhibitors of
UDP-galactopyranose mutase (UGM) which have activity as inhibitors
of microbial growth of microorganisms which contain this enzyme and
particularly those microorganisms in which this enzyme is
responsible for the incorporation of galactofuranose residues:
uridine 5'-diphosphate (UDP) galactopyranose mutase.
[0104] In specific embodiments, the invention provides compounds of
any of formulas I, II, IVA, IVC, IVE, VA, VC, VIA, VIIA, and X-XIII
where the D ring is one of D1-D42. In specific embodiments, the D
ring is a five member ring. In specific embodiments, the D ring is
a five member ring having two different heteroatoms in the ring. In
specific embodiments, the D ring is a five member ring having one S
and one N in the ring. In specific embodiments, the D ring is any
of D1-D15, or D16-D29, or D30-42. In specific embodiments, the D
ring is a six member ring. In specific embodiments, the D ring is a
six member ring having one heteroatom in the ring. In specific
embodiments, the D ring is a six member ring having two heteroatoms
in the ring. In specific embodiments, the D ring is a six member
ring having three heteroatoms in the ring. In specific embodiments,
the D ring is a six member ring having one, two or three N in the
ring.
[0105] In specific embodiments, the invention provides compounds of
any of the formulas herein, wherein:
[0106] D ring is D1 or D4;
[0107] D ring is D1-D6;
[0108] D ring is D2 or D5;
[0109] D ring is D3 or D6;
[0110] D ring is D7-D15;
[0111] D ring is D7, D10 or D13;
[0112] D ring is D8, D11 or D14;
[0113] D ring is D9, D12 or D15;
[0114] D ring is D3, D6, D16, D18, D19, or D21;
[0115] D ring is D16 or D19;
[0116] D ring is D17 or D20;
[0117] D ring is D18 or D 21;
[0118] D ring is D28 or D29;
[0119] D ring is D30-D39;
[0120] D ring is D30, D31, D32, or D33;
[0121] D ring is D34-D39; or
[0122] D ring is D40-D42; and/or
[0123] R.sup.1 is hydrogen; or
[0124] R.sup.1 is a methyl group; and/or
[0125] R.sub.A is a hydrogen;
[0126] R.sub.A is a halogen;
[0127] R.sub.A is iodine;
[0128] R.sub.A is chlorine;
[0129] R.sub.A is bromine;
[0130] R.sub.A is hydroxide; or
[0131] R.sub.A is nitro, and/or
[0132] n is 1; or
[0133] n is 0; and/or
[0134] R.sub.B is substitution by five halogens on the B ring;
[0135] R.sub.B is substitution by five fluorines on the B ring;
[0136] R.sub.B is substitution by three halogens on the B ring;
[0137] R.sub.B is substitution by three chlorines on the B
ring;
[0138] R.sub.B is substitution by three bromines on the B ring;
[0139] R.sub.B is substitution by three fluorines on the B
ring;
[0140] R.sub.B is substitution by three iodines on the B ring;
[0141] R.sub.B is substitution by halogens at carbons 2, 3 and 4 on
the B ring;
[0142] R.sub.B is substitution by chlorines at carbons 2, 3 and 4
on the B ring;
[0143] R.sub.B is substitution by fluorines at carbons 2, 3 and 4
on the B ring;
[0144] R.sub.B is substitution by bromines at carbons 2, 3 and 4 on
the B ring;
[0145] R.sub.B is substitution by iodines at carbons 2, 3 and 4 on
the B ring;
[0146] R.sub.B is substitution by halogens at carbons 1, 2 and 3 on
the B ring;
[0147] R.sub.B is substitution by chlorines at carbons 2, 3 and 4
on the B ring;
[0148] R.sub.B is substitution by two halogens on the B ring;
[0149] R.sub.B is substitution by two chlorines on the B ring;
[0150] R.sub.B is substitution by two bromines on the B ring;
[0151] R.sub.B is substitution by two iodines on the B ring;
[0152] R.sub.B is substitution by two fluorines on the B ring;
[0153] R.sub.B is substitution by halogens at carbons 2 and 4 on
the B ring;
[0154] R.sub.B is substitution by fluorines at carbons 2 and 4 on
the B ring;
[0155] R.sub.B is substitution by chlorines at carbons 2 and 4 on
the B ring;
[0156] R.sub.B is substitution by bromines at carbons 2 and 4 on
the B ring;
[0157] R.sub.B is substitution by iodines at carbons 2 and 4 on the
B ring;
[0158] R.sub.B is substitution by halogens at carbons 1 and 3 on
the B ring;
[0159] R.sub.B is substitution by fluorines at carbons 1 and 3 on
the B ring;
[0160] R.sub.B is substitution by chlorines at carbons 1 and 3 on
the B ring;
[0161] R.sub.B is substitution by bromines at carbons 1 and 3 on
the B ring;
[0162] R.sub.B is substitution by iodines at carbons 1 and 3 on the
B ring;
[0163] R.sub.B is substitution by halogens at carbons 2 and 3 on
the B ring;
[0164] R.sub.B is substitution by fluorines at carbons 2 and 3 on
the B ring;
[0165] R.sub.B is substitution by chlorines at carbons 2 and 3 on
the B ring;
[0166] R.sub.B is substitution by bromines at carbons 2 and 3 on
the B ring;
[0167] R.sub.B is substitution by iodines at carbons 2 and 3 on the
B ring;
[0168] R.sub.B is substitution by one halogen on the B ring;
[0169] R.sub.B is substitution by one fluorine on the B ring;
[0170] R.sub.B is substitution by one chlorine on the B ring;
[0171] R.sub.B is substitution by one bromine on the B ring;
[0172] R.sub.B is substitution by one iodine on the B ring;
[0173] R.sub.B is substitution by one halogen on carbon 3 of the B
ring;
[0174] R.sub.B is substitution by one fluorine on carbon 3 of the B
ring;
[0175] R.sub.B is substitution by one chlorine on carbon 3 of the B
ring;
[0176] R.sub.B is substitution by one chlorine on carbon 2 of the B
ring;
[0177] R.sub.B is substitution by one chlorine on carbon 1 of the B
ring;
[0178] R.sub.B is substitution by one bromine on carbon 3 of the B
ring;
[0179] R.sub.B is substitution by one iodine on carbon 3 of the B
ring;
[0180] R.sub.B is substitution by one nitro group on the B
ring;
[0181] R.sub.B is substitution by one nitro group on carbon 2 of
the B ring;
[0182] R.sub.B is substitution by one nitro group on carbon 3 of
the B ring;
[0183] R.sub.B is substitution by one hydroxyl group on the B
ring;
[0184] R.sub.B is substitution by one hydroxyl group on carbon 3 of
the B ring;
[0185] R.sub.B is substitution by one methoxy group on the B
ring;
[0186] R.sub.B is substitution by one methoxy group on carbon 3 of
the B ring;
[0187] R.sub.B is substitution by one cyano group on the B
ring;
[0188] R.sub.B is substitution by one cyano group on carbon 3 of
the B ring;
[0189] R.sub.B is substitution by a C1-C6 alkyl group at carbon 3
of the B ring;
[0190] R.sub.B is substitution by a nitro group at carbon 2 and a
halogen at carbon 3 of the B ring;
[0191] R.sub.B is substitution by a nitro group at carbon 2 and a
chlorine at carbon 3 of the B ring;
[0192] R.sub.B is substitution by a C1-C3 alkyl group at carbon 1
and a halogen at carbon 3 of the B ring;
[0193] R.sub.B is substitution by a C1-C3 alkyl group at carbon 1
and a hydroxy at carbon 3 of the B ring;
[0194] R.sub.B is substitution including two different substituents
on the B ring; and/or
[0195] R.sup.1 attached to the thiazole ring and R.sub.B together
form:
##STR00019##
where X is selected from hydrogen, halogen, hydroxyl, methoxy,
nitro or cyano; X in the above formula is halogen; X in the above
formula is chlorine; X in the above formula is iodine; and/or
[0196] A is phenyl with an RA substituent at the 3 carbon of the A
ring;
[0197] A is optionally substituted 1-naphtyl;
[0198] A is optionally substituted 2-napthyl; or
[0199] A is
##STR00020##
and/or
[0200] the B ring is 1-napthyl;
[0201] the B ring is 2-napthyl; and/or
[0202] R.sub.C is substitution by five halogens on the C ring;
[0203] R.sub.C is substitution by five fluorines on the C ring;
[0204] R.sub.C is substitution by three halogens on the C ring;
[0205] R.sub.C is substitution by three chlorines on the C
ring;
[0206] R.sub.C is substitution by three bromines on the C ring;
[0207] R.sub.C is substitution by three fluorines on the C
ring;
[0208] R.sub.C is substitution by three iodines on the C ring;
[0209] R.sub.C is substitution by halogens at carbons 2, 3 and 4 on
the C ring;
[0210] R.sub.C is substitution by chlorines at carbons 2, 3 and 4
on the C ring;
[0211] R.sub.C is substitution by fluorines at carbons 2, 3 and 4
on the C ring;
[0212] R.sub.C is substitution by bromines at carbons 2, 3 and 4 on
the C ring;
[0213] R.sub.C is substitution by iodines at carbons 2, 3 and 4 on
the C ring;
[0214] R.sub.C is substitution by two halogens on the C ring;
[0215] R.sub.C is substitution by two chlorines on the C ring;
[0216] R.sub.C is substitution by two bromines on the C ring;
[0217] R.sub.C is substitution by two iodines on the C ring;
[0218] R.sub.C is substitution by two fluorines on the C ring;
[0219] R.sub.C is substitution by halogens at carbons 2 and 4 on
the C ring;
[0220] R.sub.C is substitution by fluorines at carbons 2 and 4 on
the C ring;
[0221] R.sub.C is substitution by chlorines at carbons 2 and 4 on
the C ring;
[0222] R.sub.C is substitution by bromines at carbons 2 and 4 on
the C ring;
[0223] R.sub.C is substitution by iodines at carbons 2 and 4 on the
C ring;
[0224] R.sub.C is substitution by halogens at carbons 1 and 3 on
the C ring;
[0225] R.sub.C is substitution by fluorines at carbons 1 and 3 on
the C ring;
[0226] R.sub.C is substitution by chlorines at carbons 1 and 3 on
the C ring;
[0227] R.sub.C is substitution by bromines at carbons 1 and 3 on
the C ring;
[0228] R.sub.C is substitution by iodines at carbons 1 and 3 on the
C ring;
[0229] R.sub.C is substitution by halogens at carbons 2 and 3 on
the C ring;
[0230] R.sub.C is substitution by fluorines at carbons 2 and 3 on
the C ring;
[0231] R.sub.C is substitution by chlorines at carbons 2 and 3 on
the C ring;
[0232] R.sub.C is substitution by bromines at carbons 2 and 3 on
the C ring;
[0233] R.sub.C is substitution by iodines at carbons 2 and 3 on the
C ring;
[0234] R.sub.C is substitution by one halogen on the C ring;
[0235] R.sub.C is substitution by one fluorine on the C ring;
[0236] R.sub.C is substitution by one chlorine on the C ring;
[0237] R.sub.C is substitution by one bromine on the C ring;
[0238] R.sub.C is substitution by one iodine on the C ring;
[0239] R.sub.C is substitution by one halogen on carbon 3 of the C
ring;
[0240] R.sub.C is substitution by one fluorine on carbon 3 of the C
ring;
[0241] R.sub.C is substitution by one chlorine on carbon 3 of the C
ring;
[0242] R.sub.C is substitution by one bromine on carbon 3 of the C
ring;
[0243] R.sub.C is substitution by one iodine on carbon 3 of the C
ring;
[0244] R.sub.C is substitution by one nitro group on the C
ring;
[0245] R.sub.C is substitution by one nitro group on carbon 2 of
the C ring;
[0246] R.sub.C is substitution by one nitro group on carbon 3 of
the C ring;
[0247] R.sub.C is substitution by one hydroxyl group on the C
ring;
[0248] R.sub.C is substitution by one hydroxyl group on carbon 3 of
the C ring;
[0249] R.sub.C is substitution by one cyano group on the C
ring;
[0250] R.sub.C is substitution by one cyano group on carbon 3 of
the C ring; and/or
[0251] R.sub.B includes substitution at the 3 position with
-M-L-Q-AR; where:
[0252] M is thiourea; and/or
[0253] Q is thiourea, and/or
[0254] M is --O-- or NR.sub.M--; and/or
[0255] M is --O-- or --NR.sub.M-- and Q is thiourea; and/or
[0256] L is --(CH.sub.2).sub.n--; and/or
[0257] M is --O-- or --NR.sub.M--, Q is thiourea and L is
--(CH.sub.2).sub.n--; and/or
[0258] AR is naphthyl
[0259] AR is benzofuranyl; or
compounds of formulas which combine any of the above
embodiments.
[0260] In specific embodiments, the invention provides compounds of
any formulas herein formula where n is 0, where:
[0261] R.sub.A is halogen and R.sub.B is hydroxyl at the 3 carbon
of the B ring;
[0262] R.sub.A is chlorine and R.sub.B is hydroxyl at the 3 carbon
of the B ring;
[0263] R.sub.A is halogen and R.sub.B is halogens at the 2 and 4
carbons of the B ring;
[0264] R.sub.A is chlorine and R.sub.B is halogens at the 2 and 4
carbons of the B ring;
[0265] R.sub.A is chlorine and R.sub.B is chlorines at the 2 and 4
carbons of the B ring;
[0266] R.sub.A is chlorine and R.sub.B is fluorines at the 2 and 4
carbons of the B ring;
[0267] R.sub.A is iodine and R.sub.B is halogens at the 2 and 4
carbons of the B ring;
[0268] R.sub.A is iodine and R.sub.B is chlorines at the 2 and 4
carbons of the B ring;
[0269] R.sub.A is iodine and R.sub.B is fluorines at the 2 and 4
carbons of the B ring;
[0270] R.sub.A is halogen and R.sub.B is halogen at the 3 carbon of
the B ring;
[0271] R.sub.A is halogen and R.sub.B is chlorine at the 3 carbon
of the B ring;
[0272] R.sub.A is halogen and R.sub.B is iodine at the 3 carbon of
the B ring;
[0273] R.sub.A is halogen and R.sub.B is bromine at the 3 carbon of
the B ring;
[0274] R.sub.A is halogen and R.sub.B is fluorine at the 3 carbon
of the B ring;
[0275] R.sub.A is chlorine and R.sub.B is chlorine at the 3 carbon
of the B ring;
[0276] R.sub.A is chlorine and R.sub.B is iodine at the 3 carbon of
the B ring;
[0277] R.sub.A is chlorine and R.sub.B is bromine at the 3 carbon
of the B ring;
[0278] R.sub.A is chlorine and R.sub.B is fluorine at the 3 carbon
of the B ring;
[0279] R.sub.A is bromine and R.sub.B is chlorine at the 3 carbon
of the B ring;
[0280] R.sub.A is bromine and R.sub.B is iodine at the 3 carbon of
the B ring;
[0281] R.sub.A is bromine and R.sub.B is bromine at the 3 carbon of
the B ring;
[0282] R.sub.A is bromine and R.sub.B is fluorine at the 3 carbon
of the B ring;
[0283] R.sub.A is iodine and R.sub.B is chlorine at the 3 carbon of
the B ring;
[0284] R.sub.A is iodine and R.sub.B is iodine at the 3 carbon of
the B ring;
[0285] R.sub.A is iodine and R.sub.B is bromine at the 3 carbon of
the B ring;
[0286] R.sub.A is iodine and R.sub.B is fluorine at the 3 carbon of
the B ring;
[0287] R.sub.A is halogen and R.sub.B is nitro at the 2 carbon of
the B ring;
[0288] R.sub.A is iodine and R.sub.B is nitro at the 2 carbon of
the B ring;
[0289] R.sub.A is halogen and R.sub.B is halogens at the 1 and 3
carbons of the B ring;
[0290] R.sub.A is chlorine and R.sub.B is halogens at the 1 and 3
carbons of the B ring;
[0291] R.sub.A is chlorine and R.sub.B is chlorines at the 1 and 3
carbons of the B ring;
[0292] R.sub.A is chlorine and R.sub.B is fluorines at the 1 and 3
carbons of the B ring;
[0293] R.sub.A is iodine and R.sub.B is halogens at the 1 and 3
carbons of the B ring;
[0294] R.sub.A is iodine and R.sub.B is chlorines at the 1 and 3
carbons of the B ring;
[0295] R.sub.A is iodine and R.sub.B is fluorines at the 1 and 3
carbons of the B ring;
[0296] R.sub.A is bromine and R.sub.B is halogens at the 1 and 3
carbons of the B ring;
[0297] R.sub.A is bromine and R.sub.B is chlorines at the 1 and 3
carbons of the B ring;
[0298] R.sub.A is bromine and R.sub.B is fluorines at the 1 and 3
carbons of the B ring;
[0299] R.sub.A is fluorine and R.sub.B is halogens at the 1 and 3
carbons of the B ring;
[0300] R.sub.A is fluorine and R.sub.B is chlorines at the 1 and 3
carbons of the B ring;
[0301] R.sub.A is fluorine and R.sub.B is fluorines at the 1 and 3
carbons of the B ring;
[0302] R.sub.A is halogen and R.sub.B is halogens at the 2 and 3
carbons of the B ring;
[0303] R.sub.A is chlorine and R.sub.B is halogens at the 2 and 3
carbons of the B ring;
[0304] R.sub.A is chlorine and R.sub.B is chlorines at the 2 and 3
carbons of the B ring;
[0305] R.sub.A is chlorine and R.sub.B is fluorines at the 2 and 3
carbons of the B ring;
[0306] R.sub.A is iodine and R.sub.B is halogens at the 2 and 3
carbons of the B ring;
[0307] R.sub.A is iodine and R.sub.B is chlorines at the 2 and 3
carbons of the B ring;
[0308] R.sub.A is iodine and R.sub.B is fluorines at the 2 and 3
carbons of the B ring;
[0309] R.sub.A is bromine and R.sub.B is halogens at the 2 and 3
carbons of the B ring;
[0310] R.sub.A is bromine and R.sub.B is chlorines at the 2 and 3
carbons of the B ring;
[0311] R.sub.A is bromine and R.sub.B is fluorines at the 2 and 3
carbons of the B ring;
[0312] R.sub.A is fluorine and R.sub.B is halogens at the 2 and 3
carbons of the B ring;
[0313] R.sub.A is fluorine and R.sub.B is chlorines at the 2 and 3
carbons of the B ring;
[0314] R.sub.A is fluorine and R.sub.B is fluorines at the 2 and 3
carbons of the B ring;
[0315] R.sub.A is halogen and R.sub.B is halogens at the 1, 2 and 3
carbons of the B ring;
[0316] R.sub.A is iodine and R.sub.B is halogens at the 1, 2 and 3
carbons of the B ring;
[0317] R.sub.A is chlorine and R.sub.B is halogens at the 1, 2 and
3 carbons of the B ring;
[0318] R.sub.A is bromine and R.sub.B is halogens at the 1, 2 and 3
carbons of the B ring;
[0319] R.sub.A is fluorine and R.sub.B is halogens at the 1, 2 and
3 carbons of the B ring;
[0320] R.sub.A is iodine and R.sub.B is chlorine at the 1, 2 and 3
carbons of the B ring;
[0321] R.sub.A is chlorine and R.sub.B is chlorine at the 1, 2 and
3 carbons of the B ring;
[0322] R.sub.A is bromine and R.sub.B is chlorine at the 1, 2 and 3
carbons of the B ring;
[0323] R.sub.A is fluorine and R.sub.B is chlorine at the 1, 2 and
3 carbons of the B ring;
[0324] R.sub.A is iodine;
[0325] R.sub.A is chlorine; or
[0326] R.sub.A is nitro and R.sub.B is halogen at the 3 carbon of
the B ring.
[0327] In specific embodiments, the C ring and the A ring are not
aryl sulfinyl, aryl sulfuryl, arylsulfonyl or aryl sulfonic acid
groups. In specific embodiments, the D ring is a ring other than a
thiazolidinone ring. In specific embodiments, the D ring is a ring
other than a furanyl ring.
[0328] In specific embodiments, the invention provides compound of
any formulas herein which exhibit K.sub.d (1M) on UGM.sub.myco of
100 or less. In other embodiments, the invention provides compound
of any formulas herein which exhibit Kd (microM) on UGM.sub.myco of
80 or less. In other embodiments, the invention provides compound
of any formulas herein which exhibit Kd (microM) on UGM.sub.myco of
60 or less.
[0329] In specific embodiments, the invention provides compounds of
any formulas herein which exhibit K.sub.d (.mu.M) as measured by
the fluorescence polarization assay on the UGM isoform from M.
tuberculosis of 80 or less. In other embodiments, the invention
provides compounds of formulas herein which exhibit K.sub.d (.mu.M)
as measured by the fluorescence polarization assay on the UGM
isoform from M. tuberculosis of 60 or less. In other embodiments,
the invention provides compounds of any formulas herein which
exhibit K.sub.d (.mu.M) as measured by the fluorescence
polarization assay on the UGM isoform from M. tuberculosis of 25 or
less.
[0330] In specific embodiments, the invention provides compounds of
any formulas herein which exhibit K.sub.d (.mu.M) as measured by
the fluorescence polarization assay on the UGM isoform from K.
pneumoniae of 80 or less. In other embodiments, the invention
provides compounds of any formulas herein which exhibit K.sub.d
(.mu.M) as measured by the fluorescence polarization assay on the
UGM isoform from K. pneumoniae of 60 or less. In other embodiments,
the invention provides compounds of any formulas herein which
exhibit K.sub.d (.mu.M) as measured by the fluorescence
polarization assay on the UGM isoform from K. pneumoniae of 25 or
less.
[0331] The invention further provides a method for inhibiting UGM
in vitro or in vivo by contacting a biological composition
comprising an active UGM with an amount of one or more of the
compounds of any of the formulas herein effective for inhibiting
UGM. In a specific embodiment, the inhibition of UGM is in vivo in
a prokaryote. In a specific embodiment, the inhibition of UGM is in
vivo in a eukaryote.
[0332] Compounds of the invention are useful for inhibiting the
growth of a microorganism containing UGM which is the enzyme
responsible for the conversion of UDP-galactopyranose to
UDP-galactofuranose. UGM is expected to be present in
microorganisms in which galactofuranose (Galf) residues are
present, for example in cell walls. Galactofuranose (Galf) residues
are present in many pathogenic microorganisms (Pedersen, L. L.;
Turco, S. J. Cell. Mol. Life. Sci. 2003, 60, 259-266.) The gene
encoding UGM is essential for mycobacterial viability (Pan, F.;
Jackson, M.; Ma, Y. F.; McNeil, M. J. Bacteriol. 2001, 183,
3991-3998) suggesting that Galf-containing glycoconjugates are
necessary components of the mycobacterial cell wall. Compounds of
the invention are useful for inhibiting the growth of
microorganisms containing galactofuranose residues, particularly
those having such residues in the cell wall and more particularly
pathogenic microorganisms containing galactofuranose residues.
[0333] Compounds of the invention are useful for inhibition of
growth of microorganisms of the genus Mycobacterium, particularly
including M. tuberculosis and M. smegmatis. Compounds of the
invention can also be employed to inhibit the growth of
Mycobacterium leprae, Mycobacterium bovis, Mycobacterium africanum,
Mycobacterium canetti, and Mycobacterium microti.
[0334] Compounds of the invention are useful for inhibition of the
growth of gram-negative bacteria and particularly those of the
genus Klebsiella and particularly K. pneumoniae. The compounds of
the invention can also be employed to inhibit the growth of
Klebsiella ozaenae, Klebsiella rhinoscleromatis, Klebsiella
oxytoca, Klebsiella planticola, Klebsiella terrigena, and
Klebsiella ornithinolytica. Klebsiellae are important pathogens in
nosocomial infections. The compounds of the invention are useful
for the treatment of nosocomial infections.
[0335] Compounds of the invention are useful for inhibition of the
growth of or for attenuation of the virulence of eukaryotic
pathogens, including yeast, fungi, protozoa and nematodes. The
compounds of the invention are useful for inhibiting the growth or
attenuating the virulence of, for example, pathogenic Aspergillus,
in particular Aspergillus fumagatus.
[0336] The invention further provides a method of screening for and
identifying small molecules which inhibit growth of bacteria which
contain the UGM enzyme and particularly those bacteria in which the
UGM enzyme is associated with forming the bacterial cell wall. The
method is particularly useful for screening for and identifying
small molecules which inhibit growth of members of the genus
Klebsiella and more particularly for K. pneumoniae.
[0337] The invention further provides a method of screening for and
identifying small molecules which inhibit growth of mycobacteria
which contain the UGM enzyme and particularly those mycobacteria in
which the UGM enzyme is responsible for the incorporation of
galactofuranose residues. The method is particularly useful for
screening for and identifying small molecules which inhibit growth
of members of the genus Mycobacterium and more particularly for M.
tuberculosis.
[0338] The term "microorganism" is used broadly herein to refer to
organisms too small to be seen with the naked human eye and
includes prokaryotes (e.g., bacteria and mycobacteria), single cell
and multiple cell eukaryotes, yeast, fungi and protozoa. More
specifically microorganisms upon which the compounds of this
invention act are human or non-human mammal pathogens. Pathogenic
protozoa include, among others, plasmodium, trypanosomes and
leishmania (e.g., Leishmania major, Trypanosoma cruizii.) Fungi
include cryptococcus and chlamydomonas (e.g., Cryptococcus
neoformans). Microorganism also includes parasitic nematodes.
[0339] In a specific embodiment, compounds of this invention of any
of the formulas herein can block incorporation of Galf into
polysaccharides essential for viability or virulence of pathogenic
microorganisms.
[0340] In specific embodiments, the invention provides compounds of
any formulas herein which are cell permeable.
[0341] The term "alkyl" refers to a monoradical of a branched or
unbranched (straight-chain or linear) saturated hydrocarbon and to
cycloalkyl groups having one or more rings. Unless otherwise
indicated preferred alkyl groups have 1 to 22 carbon atoms and more
preferred are those that contain 1-12 carbon atoms. Short alkyl
groups are those having 1 to 6 carbon atoms including methyl,
ethyl, propyl, butyl, pentyl and hexyl groups, including all
isomers thereof. Long alkyl groups are those having 8-22 carbon
atoms and preferably those having 12-22 carbon atoms. The term
"cycloalkyl" refers to cyclic alkyl groups having 3 to 22 and
preferably 5-10 carbon atoms having a single cyclic ring or
multiple condensed rings. Cycloalkyl groups include, by way of
example, single ring structures such as cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, cyclooctyl, and the like, or multiple ring
structures such as adamantanyl, and the like. Unless otherwise
indicated alkyl groups including cycloalkyl groups are optionally
substituted as defined herein.
[0342] The term "alkylene" refers to a diradical of a branched or
unbranched saturated hydrocarbon chain, which unless otherwise
indicated can have 1 to 15 carbon atoms, 1 to 12 carbon atoms, 1-6
carbon atoms, or 2-4 carbon atoms. This term is exemplified by
groups such as methylene (--CH.sub.2--), ethylene
(--CH.sub.2CH.sub.2--), more generally --(CH.sub.2).sub.n, where n
is 1-10 or more preferably 1-6 or n is 2, 3 or 4. Alkylene groups
may be branched, e.g., by substitution with alkyl group
substituents. Alkylene groups may be optionally substituted as
described herein. More specifically alkylene groups may be
substituted with one or more hydroxyl groups and/or one or more
halogen groups. Alkylene groups may have up to two non-hydrogen
substituents per carbon atoms. The term "alkenylene" refers to a
diradical of a branched or unbranched alkene. Alkenylene linkers
can contain one or more than one double bond. In specific
embodiments, alkenylene linkers contain 1 double bond or two double
bonds. Alkenylene linkers can unless otherwise indicated have 2 to
10 carbon atoms, or 2-6 carbon atoms, or 3-5 carbon atoms. The term
cycloalkylene relates to an alkylene group that contains a carbon
ring having 3-8 carbon atoms and preferably having 5 or 6 carbon
atoms.
[0343] The term alkoxy refers to the group --OR where R is an alkyl
group as defined above.
[0344] The term "alkyleneoxy" refers to a diradical of a branched
or unbranched saturated hydrocarbon chain in which one or more
--CH2-- groups are replaced with -0-, which unless otherwise
indicated can have 1 to 10 carbon atoms, or 1-6 carbon atoms, or
2-4 carbon atoms. This term is exemplified by groups such as
--CH.sub.2OCH.sub.2--, --CH.sub.2CH.sub.2OCH.sub.2CH.sub.2--,
--CH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.2CH.sub.2--, more
generally --((CH.sub.2).sub.x--O--).sub.a--(CH.sub.2).sub.y--,
where x and y are integers ranging from 1-6, and a is an integer
ranging from 1-3, as well as, --OCH.sub.2--CH.sub.2--O--,
--O--(CH.sub.2).sub.x--O--,
--O--(CH.sub.2).sub.xO--(CH.sub.2).sub.y--O--,
--O--((CH.sub.2)O--).sub.a--, or
--O--((CH.sub.2).sub.x--O--).sub.a--(CH.sub.2).sub.y--, where x and
y are integers ranging from 1-6, and a is an integer ranging from
1-3, Alkyleneoxy groups may be branched, e.g., by substitution with
alkyl group substituents.
[0345] The term "carbocyclic" is used generically herein to refer
to groups which contain a carbon ring which may be a saturated,
partially unsaturated or aromatic ring. Carbocyclic groups may
contain one or more than one carbon ring which ring may be a
cycloakyl, unsaturated cycloalkyl or aryl ring. Typically
carbocyclic rings include those having 3-12 carbon atoms in the
ring. Carbocyclic rings include those having two or more fused
rings, bicyclic rings, tricyclic ring etc. Preferred carbocyclic
rings have 6 to 12 carbon atoms. Unless otherwise indicated
carbocyclic groups are optionally substituted as defined
herein.
[0346] The term "heterocyclic" is also used generically herein to
refer to carbocyclic rings in which one or more ring carbons are
replaced with a heteroatom. The heterocyclic ring may contain a
carbon ring in combination with a heteroatom containing ring.
Heterocyclic groups can contain from one to six heteroatoms
including one, two, three or four hetero atoms. Preferred
heteroatoms are N, O or S (or NR' where R' is a hydrogen or an
optional substituent). Heterocyclic groups can be or contain
heteroaryl groups. Unless otherwise indicated heterocyclic groups
are optionally substituted as defined herein.
[0347] The term heterocyclene group relates to a diradical of a
heterocyclic group. Such groups can contain one or more
heteroatoms, particularly O, N or S atoms or combinations thereof.
A heterocylene group can, for example have a 5 or 6-member
heterocyclic ring.
[0348] The term "aryl" refers to a monoradical containing at least
one aromatic ring. The radical is formally derived by removing a H
from a ring carbon. Aryl groups contain one or more rings at least
one of which is aromatic. Rings of aryl groups may be linked by a
single bond or a linker group or may be fused. Exemplary aryl
groups include phenyl, biphenyl and naphthyl groups. Aryl groups
include those having from 6 to 30 carbon atoms and those containing
6-12 carbon atoms. Unless otherwise noted aryl groups are
optionally substituted as described herein.
[0349] The term "arylene" refers to a diradical containing at least
one aromatic ring. The diradical is formally derived by removing a
H from two different ring carbons, e.g., a phenylene, and more
specifically a 1,4-phenylene. Arylene groups include those having
from 6 to 30 carbon atoms and those containing 612 carbon atoms.
Unless otherwise noted arylene groups are optionally substituted as
described herein.
[0350] The term "heteroaryl" refers to a group that contains at
least one aromatic ring in which one or more of the ring carbons is
replaced with a heteroatom (non-carbon atom). To satisfy valence
the heteroatom may be bonded to H or a substituent groups. Ring
carbons may be replaced with --O--, --S--, --NR--, --N.dbd.,
--PR--, or --POR among others, where R is an alkyl, aryl,
heterocyclyl or heteroaryl group. Heteroaryl groups may include one
or more aryl groups (carbon aromatic rings) heteroaromatic and aryl
rings of the heteroaryl group may be linked by a single bond or a
linker group or may be fused. Heteroaryl groups include those
having aromatic rings with 5 or 6 ring atoms of which 1-3 ring
atoms are heteroatoms. Preferred heteroatoms are --O--, --S--,
--NR-- and --N.dbd.. Heteroaryl groups include those containing
5-12 ring atoms as well as those having 5 and 6 ring atoms. Unless
otherwise noted heteroaryl groups are optionally substituted as
described herein.
[0351] The term "heteroarylene" refers to a diradical containing at
least one heteroaromatic ring. The diradical is formally derived by
removing a H from two different ring carbons. Heteroarylene groups
include those having from 6 to 30 carbon atoms and those containing
6-12 carbon atoms. Unless otherwise noted heteroarylene groups are
optionally substituted as described herein.
[0352] The term tetrazolyl refers to groups derived formally from
tetrazole or substituted tetrazoles by removal of a hydrogen,
including:
##STR00021##
where R.sub.T is hydrogen or a non-hydrogen substituent, which can
be an optionally substituted alkyl or an optionally substituted
aryl group. In specific embodiments, R.sub.T are optionally
substituted alkyl groups, particularly those having 1-6 carbon
atoms, or optionally substituted aryl groups, particularly phenyl
groups.
[0353] Additional chemical group or moiety names employed herein
are intended to have their broadest meaning as understood in the
art.
[0354] Unless otherwise specified optional substitution means
substitution by one or more non-hydrogen substituents selected from
halogen, hydroxyl, amine, cyano, azide, nitro, isocyanate,
isothiocyanate, C1-C6 alkyl, C1-C3 alkyl, C1-C6 haloalkyl, C1-C3
haloalkyl, phenyl, benzyl, sulfate, phosphate, phosphonate,
carboxyl, sulfonyl, sulfonamide, and amide. All alkyl, aryl,
heteroaryl, heterocyclic, carbocyclic groups herein are optionally
substituted with one or more non-hydrogen substituents unless
otherwise specified. Substitution may be on one or more carbons or,
if feasible, on one or more heteroatoms, e.g., a nitrogen. The
number of substituents on such groups depends generally upon the
nature of the group, but includes substitution with one, two,
three, four, five or six substituents.
[0355] As to any of the groups herein which contain one or more
substituents, it is understood, that such groups do not contain any
substitution or substitution patterns which are sterically
impractical and/or synthetically non-feasible. In addition, the
compounds of this invention include all stereochemical isomers
arising from the substitution of these compounds.
[0356] The compounds of this invention may contain one or more
chiral centers. Accordingly, this invention is intended to include
racemic mixtures, diasteromers, enantiomers and mixture enriched in
one or more stereoisomer. The scope of the invention as described
and claimed encompasses the racemic forms of the compounds as well
as the individual enantiomers and non-racemic mixtures thereof.
[0357] Treatment methods of this invention comprise the step of
administering a effective amount of one or more compounds of this
invention, or a salt thereof to an individual (human and/or
non-human animal) to treat or prevent infection. The term
"effective amount," as used herein, refers to the amount of the
compound, that, when administered to the individual is effective to
at least partially treat or prevent infection, or to at least
partially ameliorate a symptom of infection. Infection herein
refers to infection by a microorganism which contains the enzyme
UGM. As is understood in the art, the effective amount of a given
compound will depend at least in part upon, the mode of
administration, any carrier or vehicle (e.g., solution, emulsion,
etc.) employed, the extent of damage and the specific individual to
whom the compound is to be administered (age, weight, condition,
sex, etc.). The dosage requirements needed to achieve the
"effective amount" vary with the particular compositions employed,
the route of administration, the severity of the symptoms presented
and the particular subject being treated. Based on the results
obtained in standard pharmacological test procedures, projected
daily dosages of active compound can be determined as is understood
in the art.
[0358] Compounds of this invention can be employed in unit dosage
form, e.g. as tablets or capsules. In such form, the active
compound or more typically a pharmaceutical composition containing
the active compound is sub-divided in unit dose containing
appropriate quantities of the active compound; the unit dosage
forms can be packaged compositions, for example, packaged powders,
vials, ampoules, pre-filled syringes or sachets containing liquids.
The unit dosage form can be, for example, a capsule or tablet
itself, or it can be the appropriate number of any such
compositions in package form.
[0359] Any suitable form of administration can be employed in the
method herein. The compounds of this invention can, for example, be
administered in oral dosage forms including tablets, capsules,
pills, powders, granules, elixirs, tinctures, suspensions, syrups
and emulsions. Oral dosage forms may include sustained release or
timed release formulations. The compounds of this invention may
also be administered topically, intravenously, intraperitoneally,
subcutaneously, or intramuscularly, all using dosage forms well
known to those of ordinary skill in the pharmaceutical arts.
[0360] The therapeutically active compounds of the invention can be
administered alone, but generally will be administered with a
pharmaceutical carrier selected upon the basis of the chosen route
of administration and standard pharmaceutical practice.
[0361] Pharmaceutical compositions and medicaments of this
invention comprise one or more compounds in combination with a
pharmaceutically acceptable carrier, excipient, or diluent. Such
compositions and medicaments are prepared in accordance with
acceptable pharmaceutical procedures, such as, for example, those
described in Remington's Pharmaceutical Sciences, 17th edition, ed.
Alfonoso R. Gennaro, Mack Publishing Company, Easton, Pa. (1985),
which is incorporated herein by reference in its entirety.
[0362] Pharmaceutically acceptable carriers are those carriers that
are compatible with the other ingredients in the formulation and
are biologically acceptable. Carriers can be solid or liquid. Solid
carriers can include one or more substances that can also act as
flavoring agents, lubricants, solubilizers, suspending agents,
fillers, glidants, compression aids, binders, tablet-disintegrating
agents, or encapsulating materials. Liquid carriers can be used in
preparing solutions, suspensions, emulsions, syrups and elixirs.
The active ingredient can be dissolved or suspended in a
pharmaceutically acceptable liquid carrier such as water (of
appropriate purity, e.g., pyrogen-free, sterile, etc.), an organic
solvent, a mixture of both, or a pharmaceutically acceptable oil or
fat. The liquid carrier can contain other suitable pharmaceutical
additives such as, for example, solubilizers, emulsifiers, buffers,
preservatives, sweeteners, flavoring agents, suspending agents,
thickening agents, colors, viscosity regulators, stabilizers or
osmo-regulators. Compositions for oral administration can be in
either liquid or solid form.
[0363] The compounds of the present inventions may form salts which
are also within the scope of this invention. Reference to a
compound of the formulas herein is understood to include reference
to salts thereof, unless otherwise indicated. The term "salt(s)",
as employed herein, denotes acidic and/or basic salts formed with
inorganic and/or organic acids and bases. In addition, when a
compound of a formula herein contains both a basic moiety, such as,
but not limited to an amine or a pyridine ring, and an acidic
moiety, such as, but not limited to, a carboxylic acid, zwitterions
("inner salts") may be formed and are included within the term
"salt(s)" as used herein. Pharmaceutically acceptable (i.e.,
non-toxic, physiologically acceptable) salts are preferred,
although other salts are also useful, e.g., in isolation or
purification steps which may be employed during preparation. Salts
of the compounds of the formula I may be formed, for example, by
reacting a compound of the formula I with an amount of acid or
base, such as an equivalent amount, in a medium such as one in
which the salt precipitates or in an aqueous medium followed by
lyophilization.
[0364] Exemplary acid addition salts include acetates (such as
those formed with acetic acid or trihaloacetic acid, for example,
trifluoroacetic acid), adipates, alginates, ascorbates, aspartates,
benzoates, benzenesulfonates, bisulfates, borates, butyrates,
citrates, camphorates, camphorsulfonates, cyclopentanepropionates,
digluconates, dodecylsulfates, ethanesulfonates, fumarates,
glucoheptanoates, glycerophosphates, hemisulfates, heptanoates,
hexanoates, hydrochlorides (formed with hydrochloric acid),
hydrobromides (formed with hydrogen bromide), hydroiodides,
2-hydroxyethanesulfonates, lactates, maleates (formed with maleic
acid), methanesulfonates (formed with methanesulfonic acid),
2-naphthalenesulfonates, nicotinates, nitrates, oxalates,
pectinates, persulfates, 3-phenylpropionates, phosphates, picrates,
pivalates, propionates, salicylates, succinates, sulfates (such as
those formed with sulfuric acid), sulfonates (such as those
mentioned herein), tartrates, thiocyanates, toluenesulfonates such
as tosylates, undecanoates, and the like.
[0365] Exemplary basic salts include ammonium salts, alkali metal
salts such as sodium, lithium, and potassium salts, alkaline earth
metal salts such as calcium and magnesium salts, salts with organic
bases (for example, organic amines) such as benzathines,
dicyclohexylamines, hydrabamines [formed with
N,N-bis(dehydro-abietyl)ethylenediamine], N-methyl-D-glucamines,
N-methyl-D-glucamides, t-butyl amines, and salts with amino acids
such as arginine, lysine and the like. Basic nitrogen-containing
groups may be quaternized with agents such as lower alkyl halides
(e.g., methyl, ethyl, propyl, and butyl chlorides, bromides and
iodides), dialkyl sulfates (e.g., dimethyl, diethyl, dibutyl, and
diamyl sulfates), long chain halides (e.g., decyl, lauryl, myristyl
and stearyl chlorides, bromides and iodides), aralkyl halides
(e.g., benzyl and phenethyl bromides), and others.
[0366] Compounds of the present invention, and salts thereof, may
exist in their tautomeric form, in which hydrogen atoms are
transposed to other parts of the molecules and the chemical bonds
between the atoms of the molecules are consequently rearranged. It
should be understood that all tautomeric forms, insofar as they may
exist, are included within the invention. Additionally, inventive
compounds may have trans and cis isomers and may contain one or
more chiral centers, therefore exist in enantiomeric and
diastereomeric forms. The invention includes all such isomers, as
well as mixtures of cis and trans isomers, mixtures of
diastereomers and racemic mixtures of enantiomers (optical
isomers). When no specific mention is made of the configuration
(cis, trans or R or S) of a compound (or of an asymmetric carbon),
then any one of the isomers or a mixture of more than one isomer is
intended. The processes for preparation can use racemates,
enantiomers, or diastereomers as starting materials. When
enantiomeric or diastereomeric products are prepared, they can be
separated by conventional methods, for example, by chromatographic
or fractional crystallization. The inventive compounds may be in
the free or hydrate form.
[0367] In addition, compounds of formulas herein may have prodrug
forms. As used herein, a prodrug is a compound that, upon in vivo
administration, is metabolized or otherwise converted to the
biologically, pharmaceutically or therapeutically active form of
the compound. To produce a prodrug, the pharmaceutically active
compound is modified such that the active compound will be
regenerated by metabolic processes. The prodrug may be designed to
alter the metabolic stability or the transport characteristics of a
drug, to mask side effects or toxicity, to improve the flavor of a
drug or to alter other characteristics or properties of a drug. By
virtue of knowledge of pharmacodynamic processes and drug
metabolism in vivo, those of skill in this art, once a
pharmaceutically active compound is known, can design prodrugs of
the compound. See for example a) Design of Prodrugs, edited by H.
Bundgaard, (Elsevier, 1985), and Methods in Enzymology, Vol. 42, at
pp. 309-396, edited by K. Widder, et. al. (Academic Press, 1985);
b) A Textbook of Drug Design and Development, edited by
Krosgaard-Larsen and H. Bundgaard, Chapter 5, "Design and
Application of Prodrugs," by H. Bundgaard, at pp. 113-191 (1991);
c) H. Bundgaard, Advanced Drug Delivery Reviews, Vol. 8, p. 1-38
(1992); and d) H. Bundgaard, et al., Journal of Pharmaceutical
Sciences, Vol. 77, p. 285 (1988).
[0368] Well-known methods for assessment of drugability can be used
to further assess active compounds of the invention for application
to given therapeutic application. The term "drugability" relates to
pharmaceutical properties of a prospective drug for administration,
distribution, metabolism and excretion. Drugability is assessed in
various ways in the art. For example, the "Lipinski Rule of 5" for
determining drug-like characteristics in a molecule related to in
vivo absorption and permeability can be applied (C. A. Lipinski, F.
Lombardo, B. W. Dominy, P. J. Feeney, Experimental and
computational approaches to estimate solubility and permeability in
drug discovery and development settings, Adv. Drug Del. Rev., 2001,
46, 3-26 and Arup K. Ghose, Vellarkad N. Viswanadhan, and John J.
Wendoloski, A Knowledge-Based Approach in Designing Combinatorial
or Medicinal Chemistry Libraries for Drug Discovery, J. Combin.
Chem., 1999, 1, 55-68.)
[0369] In general a preferred drug for oral administration exhibits
no more than one violation of the following rules:
[0370] (1) Not more than 5 hydrogen bond donors (e.g., nitrogen or
oxygen atoms with one or more hydrogens);
[0371] (2) Not more than 10 hydrogen bond acceptors (e.g., nitrogen
or oxygen atoms);
[0372] (3) Molecular weight under 500 g/mol and more preferably
between 160 and 480; or
[0373] (4) log P less than 5 and more preferably between -0.4 to
+5.6 and yet more preferably -1<log P<2. Methods for
calculating or experimentally determining Log P are well-known in
the art. Compounds of this invention preferred for therapeutic
application include those that do not violate one or more of 1-4
above.
[0374] Compounds of this invention preferred for therapeutic
application include those having log P less than 5 and more
preferably between -0.4 to +5.6 and yet more preferably -1<log
P<2.
[0375] When a group of substituents is disclosed herein, it is
understood that all individual members of that group and all
subgroups, including any isomers, enantiomers, and diastereomers of
the group members, are disclosed separately. When a Markush group
or other grouping is used herein, all individual members of the
group and all combinations and subcombinations possible of the
group are intended to be individually included in the disclosure. A
number of specific groups of variable definitions have been
described herein. It is intended that all combinations and
subcombinations of the specific groups of variable definitions are
individually included in this disclosure.
[0376] Compounds described herein may exist in one or more isomeric
forms, e.g., structural or optical isomers. When a compound is
described herein such that a particular isomer, enantiomer or
diastereomer of the compound is not specified, for example, in a
formula or in a chemical name, that description is intended to
include each isomers and enantiomer (e.g., cis/trans isomers, R/S
enantiomers) of the compound described individual or in any
combination. Additionally, unless otherwise specified, all isotopic
variants of compounds disclosed herein are intended to be
encompassed by the disclosure. For example, it will be understood
that any one or more hydrogens in a molecule disclosed can be
replaced with deuterium or tritium. Isotopic variants of a molecule
are generally useful as standards in assays for the molecule and in
chemical and biological research related to the molecule or its
use. Isotopic variants, including those carrying radioisotopes, may
also be useful in diagnostic assays and in therapeutics. Methods
for making such isotopic variants are known in the art.
[0377] Specific names of compounds are intended to be exemplary, as
it is known that one of ordinary skill in the art can name the same
compounds differently.
[0378] Molecules disclosed herein may contain one or more ionizable
groups [groups from which a proton can be removed (e.g., --COOH) or
added (e.g., amines) or which can be quaternized (e.g., amines)].
All possible ionic forms of such molecules and salts thereof are
intended to be included individually in the disclosure herein. With
regard to salts of the compounds herein, one of ordinary skill in
the art can select from among a wide variety of available
counterions those that are appropriate for preparation of salts of
this invention for a given application. In specific applications,
the selection of a given anion or cation for preparation of a salt
may result in increased or decreased solubility of that salt.
[0379] Every formulation or combination of components described or
exemplified herein can be used to practice the invention, unless
otherwise stated. The invention illustratively described herein
suitably may be practiced in the absence of any element or
elements, limitation or limitations which is not specifically
disclosed herein.
[0380] It is understood that this invention is not limited to the
particular methodology, protocols, cell lines, and reagents
described, as these may vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only, and is not intended to limit the scope of the
present invention which will be limited only by the appended
claims.
[0381] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
reference unless the context clearly dictates otherwise. Thus, for
example, reference to "a cell" includes a plurality of such cells
and equivalents thereof known to those skilled in the art, and so
forth. As well, the terms "a" (or "an"), "one or more" and "at
least one" can be used interchangeably herein.
[0382] Whenever a range is given in the specification, for example,
a temperature range, a time range, or a composition or
concentration range, all intermediate ranges and subranges, as well
as all individual values included in the ranges given are intended
to be included in the disclosure. It will be understood that any
subranges or individual values in a range or subrange that are
included in the description herein can be excluded from the claims
herein.
[0383] All patents and publications mentioned in the specification
are indicative of the levels of skill of those skilled in the art
to which the invention pertains. References cited herein are
incorporated by reference herein in their entirety to indicate the
state of the art as of their publication or filing date and it is
intended that this information can be employed herein, if needed,
to exclude specific embodiments that are in the prior art. For
example, when composition of matter are claimed, it should be
understood that compounds known and available in the art prior to
Applicant's invention, including compounds for which an enabling
disclosure is provided in the references cited herein, are not
intended to be included in the composition of matter claims
herein.
[0384] As used herein, "comprising" is synonymous with "including,"
"containing," or "characterized by," and is inclusive or open-ended
and does not exclude additional, unrecited elements or method
steps. As used herein, "consisting of" excludes any element, step,
or ingredient not specified in the claim element. As used herein,
"consisting essentially of" does not exclude materials or steps
that do not materially affect the basic and novel characteristics
of the claim. The broad term "comprising" is intended to encompass
the narrower consisting essentially of and the even narrower
consisting of: Thus, in any recitation herein of a phrase
"comprising one or more claim element" (e.g., "comprising A and B),
the phrase is intended to encompass the narrower, for example,
"consisting essentially of A and B" and "consisting of A and B."
Thus, the broader word "comprising" is intended to provide specific
support in each use herein for either "consisting essentially of"
or "consisting of."
[0385] One of ordinary skill in the art will appreciate that
starting materials, reagents, synthetic methods, purification
methods, analytical methods, and assay methods, other than those
specifically exemplified can be employed in the practice of the
invention without resort to undue experimentation. All art-known
functional equivalents, of any such materials and methods are
intended to be included in this invention. The terms and
expressions which have been employed are used as terms of
description and not of limitation, and there is no intention that
in the use of such terms and expressions of excluding any
equivalents of the features shown and described or portions
thereof, but it is recognized that various modifications are
possible within the scope of the invention claimed. Thus, it should
be understood that although the present invention has been
specifically disclosed by examples, preferred embodiments and
optional features, modification and variation of the concepts
herein disclosed may be resorted to by those skilled in the art,
and that such modifications and variations are considered to be
within the scope of this invention as defined by the appended
claims.
[0386] All references cited herein are hereby incorporated by
reference to the extent that there is no inconsistency with the
disclosure of this specification. Some references provided herein
are incorporated by reference to provide details concerning sources
of starting materials; alternative starting materials, reagents,
methods of synthesis, purification methods, and methods of
analysis; as well as additional uses of the invention.
THE EXAMPLES
Example 1
Amino Thiozoles as UGM Inhibitors
[0387] Certain 5-arylidine-2-thioxo-4-thiazolidinone compounds were
found to serve as ligands for UGM homologs. The thiazolidinone
scaffold, however, reacts reversibly with biologically relevant
thiols in solution and UGM inhibitors of this structural class fail
to block mycobacterial growth (Carlson, E. E.; May, J. F.;
Kiessling, L. L. Chem. Biol. 2006, 13, 825-837.) Given the
reactivity of the thiazolidinones, an alternative scaffold that
would display functionality important for UGM binding, yet be inert
under physiological conditions was sought. Stable 2-aminothiazole
derivatives were investigated because it was believed that such
molecules would have a shape similar to that of thiazolidinones. In
addition, compounds of this class can be assembled efficiently, as
illustrated in the exemplary synthesis of Scheme 2 which includes
four synthetic steps with minimal purification.
[0388] Specifically, the methyl ester of the racemic phenylalanine
analog was converted to the thiourea using mild conditions.
(Kearney, P. C.; Fernandez, M.; Flygare, J. A. J. Org. Chem. 1998,
63, 196-200.) The product was obtained using the Hantzsch thiazole
synthesis, in which a thiourea generated from an aryl amino acid
and an .alpha.-bromo ketone were condensed. The desired products
were obtained after a single purification step (silica gel
chromatography) in good overall yields (50-70%). Using this
approach, 18 commercially available phenylalanine analogs and
23a-bromo ketones (King, L. C.; Ostrun, G. K. J. Org. Chem. 1964,
29, 3459-3461) were employed to generate 62 aminothiazoles.
##STR00022##
[0389] Members of the resulting focused library were screened
against UGM.sub.kleb and UGM.sub.myco using a previously described
fluorescence polarization assay..sup.20,21 (Soltero-Higgin, M.;
Carlson, E. E.; Phillips, J. H.; Kiessling, L. L. J. Am. Chem. Soc.
2004, 126, 10532-10533: Carlson, E. E.; May, J. F.; Kiessling, L.
L. Chem. Biol. 2006, 13, 825-837.) Twenty-five 2-aminothiazoles
were identified as good UGM ligands (K.sub.d<60 .mu.M). We
assessed the ability of selected compounds to inhibit
UGM.sub.myco(Carlson, E. E.; May, J. F.; Kiessling, L. L. Chem.
Biol. 2006, 13, 825-837) and found a correlation between binding
affinity and inhibitory activity Kinetic assays revealed that the
active 2-aminothiazoles function as competitive inhibitors with
respect to UDP-Galf.
[0390] With access to a new class of UGM.sub.myco inhibitors,
structural features which contribute to binding were explored. The
2-aminothiazoles and the thiazolidinone inhibitors possess aryl
substituents in similar relative orientations. The consequences of
perturbing this orientation were tested. Specifically, when a
phenylglycine rather than phenylalanine building block was used,
the resulting 2-aminothiazole was less potent (2-3 fold). Compounds
with halogen substituents on either aryl ring or both had increased
activity. In contrast, compounds bearing electron-rich rings were
less effective inhibitors. It was found that a greater variety of
substituents could be appended to the B than the A ring. Without
wishing to be bound by any particular theories, the results
indicate that the B ring occupies a region of the binding site with
fewer steric constraints. This observation is consistent with the
effects of aryl substituents on thiazolidinone derivative activity.
(Carlson, E. E.; May, J. F.; Kiessling, L. L. Chem. Biol. 2006, 13,
825-837.) These results suggest that the ring A occupies the uracil
binding pocket and aryl group B resides in the sugar binding pocket
of UMG.
A disc susceptibility test was used to evaluate several active as
well as inactive 2-aminothiazoles for growth inhibition of M.
smegmatis. Only compounds that were found to be UGM inhibitors,
blocked mycobacterial growth (FIG. 6).
[0391] To test for off-target effects, Escherichia coli
(BL21(DE3)), which lacks the gene encoding UGM,.sup.8,26 was
exposed to several mycobacterial growth inhibitors. None inhibited
E. coli growth. To further characterize the observed
antimycobacterial activity, minimum inhibitory concentrations
(MICs) were determined for five 2-aminothiazoles with different UGM
inhibitory activities. The MIC for the most potent UGM inhibitor
was 50 .mu.M, a value in the same range as the clinically used
antimycobacterial agents, ethambutol and rifampicin. (Mitchison, D.
A. Eur. Respir. J. 2005, 25, 376-379.) A direct relationship was
observed between UGM inhibitor potency and the MIC. This finding
suggests that the ability of the compounds to block mycobacterial
growth is related to their ability to inhibit UGM.
[0392] The results support the validity of UGM as a target for
antimycobacterial agents. The data indicate that compounds that
block UGM can serve as new therapeutic leads for antimycobacterial
agents. Our findings also highlight the utility of the
2-aminothiazole scaffold for targeting the UDP-sugar binding site
of UGM. The similarity between the 2-aminothiazole and compounds
found to inhibit other enzymes that act on nucleotide-sugar
substrates suggests this scaffold could yield inhibitors of other
UDP-sugar utilizing enzymes. (Helm, J. S.; Hu, Y.; Chen, L.; Gross,
B.; Walker, S. J. Am. Chem. Soc. 2003, 125, 11168-11169.)
Synthetic Procedures
[0393] Phenylalanine analogs were purchased from Chem-Impex
International, Inc. All other compounds and reagents were purchased
from Sigma-Aldrich Co. All compounds were used as received, with
the exception of solvents. Methanol was distilled from magnesium;
methylene chloride and diisopropylethylamine were distilled from
calcium hydride; and dimethylformamide (DMF) was used as biotech
grade (Aldrich). Flash chromatography was performed using silica
gel 60, 230-450 mesh (Sorbent Technologies). Analytical thin-layer
chromatography (TLC) was carried out on EM Science TLC plates
precoated with silica gel 60 F254 (250-.mu.m layer thickness).
Visualization of TLC plates was accomplished using a UV lamp.
[0394] .sup.1H NMR spectra were obtained using a Bruker AC-300 (300
MHz) or Varian MercuryPlus 300 (300 MHz), and .sup.13C NMR specta
were obtained using a Varian MercuryPlus 300 (300 MHz). Chemical
shifts are reported relative to residual solvent signals
(CDCl.sub.3): .sup.1H: .delta. 7.27, .sup.13C: .delta. 77.23;
(CD.sub.3OD): .sup.1H: .delta. 3.31, .sup.13C: .delta. 49.15.
.sup.1H NMR data are assumed to be first order with apparent
doublets and triplets reported as d and t, respectively. Multiplets
are reported as m and resonances that appear broad are designated
as br s. High-resolution electrospray ionization mass spectra
(HRESI-MS) were obtained on a Micromass LCT.
Preparation of representative compound:
3-(4-Chlorophenyl)-2-[4-(3,5-difluorophenyl)-thiazol-2-ylamino]-propionic
acid
##STR00023##
[0395] 2-Amino-3-(4-chlorophenyl)-propionic acid methyl ester
(1-1)
##STR00024##
[0396] 4-Chloro-DL-phenylalanine (1.00 g, 5.00 mmol) was dissolved
in methanol (30 mL) and cooled to 0.degree. C. Thionyl chloride
(2.92 mL, 40.0 mmol) was added dropwise. The mixture was allowed to
warm to room temperature and was heated at reflux for 4 h. The
solvent and reagents were removed in vacuo to yield the
hydrochloride salt 1-1 as a white powder in quantitative yield
(1.25 g, 5.00 mmol). .sup.1H NMR (300 MHz, CD.sub.3OD):
.delta.=7.37 (d, 2H, J=6.6 Hz), 7.27 (d, 2H, J=6.6 Hz), 4.34 (t,
1H, J=6.9 Hz), 3.81 (s, 3H), 3.26 (dd, 1H, J=14.4, 6.4 Hz), 3.18
(dd, 1H, J=14.4, 7.3 Hz); .sup.13C NMR (75 mHz, CD.sub.3OD):
.delta.=170.28, 134.87, 134.18, 132.11, 130.19, 54.99, 53.85,
38.81; ESI-MS calcd for C.sub.10H.sub.13ClNO.sub.2 [M+H].sup.+:
214.0635 found 214.0638.
2-[[[(ethoxycarbonyl)amino]thioxomethyl]amino]-3-(4-chlorophenyl)-propioni-
c acid methyl ester (1-2)
##STR00025##
[0397] Methyl ester 1-1 (394 mg, 1.58 mmol) was dissolved in
methylene chloride (4.0 mL) and diisopropylethylamine (0.82 mL,
4.73 mmol). The solution was cooled to 0.degree. C. and
ethoxycarbonyl isothiocyanate (0.21 mL, 1.73 mmol) was added
dropwise. The mixture was allowed to warm to room temperature and
was stirred 30 min. Methylene chloride (20 mL) was added, and the
organic layer was washed with water (20 mL), 10% aqueous HCl (20
mL), and saturated brine (20 mL). The organic layer was dried with
MgSO.sub.4, filtered and the solvent was removed in vacuo to yield
the product as a yellow oil in 92% yield (502 mg, 1.45 mmol).
.sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=10.10 (d, 1H, J=6.6 Hz),
8.11 (s, 1H), 7.27 (d, 2H, J=8.1 Hz), 7.10 (d, 2H, J=8.1 Hz), 5.25
(t, 1H, J=6.3 Hz), 4.23 (q, 2H, J=7.2 Hz), 3.73 (s, 3H), 3.32 (dd,
1H, J=14.1, 6.0 Hz), 3.20 (dd, 1H, J=13.8, 6.0 Hz), 1.30 (t, 3H,
J=7.2 Hz); .sup.13C NMR (75 mHz, CDCl.sub.3): .delta.=179.4, 170.8,
152.6, 134.2, 133.4, 130.8, 129.0, 63.2, 59.2, 53.6, 52.8, 36.8,
14.4; ESI-MS calcd for C.sub.14H.sub.17ClN.sub.2O.sub.4S
[M+Na].sup.+: 267.0495 found 267.0495.
3-(4-Chlorophenyl)-2-thioureido-propionic acid 1-3)
##STR00026##
[0398] Compound 1-2 (500 mg, 1.45 mmol) was dissolved in a 1:1
solution of 1 M NaOH and MeOH (6 mL). The solution was heated to
reflux for 30 min. The solution was acidified to a pH of 3 using 1
M HCl and then extracted using EtOAc (3.times.15 mL). The combined
organic extracts were dried with MgSO.sub.4 and filtered. The
solvent was removed in vacuo to yield 1-3 as a white solid in
quantitative yield (372 mg, 1.44 mmol). .sup.1H NMR (300 MHz,
CD.sub.3OD): .delta.=7.27 (d, 2H, J=8.4 Hz), 7.19 (d, 2H, J=8.4
Hz), 5.20 (m, 1H), 3.31 (dd, 1H, J=13.5, 6.5 Hz), 3.08 (dd, 1H,
J=13.5, 6.3 Hz); .sup.13C NMR (75 mHz, CD.sub.3OD): .delta.=185.0,
174.6, 136.9, 133.9, 132.2, 129.6, 59.4, 38.2; ESI-MS calcd for
C.sub.10H.sub.11ClN.sub.2O.sub.2S [M-H].sup.-: 257.0152 found
257.0155.
2-Bromo-3',5'-difluoro-acetophenone (1-4)
##STR00027##
[0399] A solution of CuBr.sub.2 (2.36 g, 10.6 mmol) in EtOAc (3 mL)
was heated to reflux. A solution of 3',5'-difluoroacetophenone
(1.00 g, 6.4 mmol) in CHCl.sub.3 (3 mL) was added dropwise. The
solution was stirred at reflux until all CuBr.sub.2 appeared to be
consumed (precipitate turns white), about 1 h. The solution was
filtered and concentrated, and the product was purified by silica
column chromatography (3.fwdarw.5% EtOAc in hexanes) to yield 1-4
in 61% yield (0.92 g, 3.91 mmol). .sup.1H NMR (300 MHz,
CDCl.sub.3): .delta.=7.49 (m, 2H), 7.07 (m, 1H), 4.38 (s, 2H);
.sup.13C NMR (75 mHz, CDCl.sub.3): .delta.=189.2, 165.0 (d, 1C,
J=12.4 Hz), 161.6 (d, 1C, J=11.5 Hz), 136.9 (t, 1C, J=7.9 Hz),
112.2 (d, 2C, J=26 Hz) 109.5 (t, 1C, J=25 Hz), 30.2; EI-MS calcd
for C.sub.8H.sub.5BrF.sub.2O [M+].sup.+: 233.9492 found
233.9484.
3-(4-Chlorophenyl)-2-[4-(3,5-difluoro-phenyl)-thiazol-2-ylamino]-propionic
acid (1-5)
##STR00028##
[0400] Thiourea 1-3 (20 mg, 77 .mu.mol) and .alpha.-bromoketone 1-4
(20 mg, 85 .mu.mol) were combined in DMF (150 .mu.L). The reaction
was stirred under N.sub.2 for 2 h and concentrated; the product was
purified by silica gel chromatography (2:1 hexanes/EtOAc.fwdarw.2:1
hexanes/EtOAc containing 2% AcOH) to yield 1-5 (19.8 mg, 50
.mu.mol) as a white powder in 65% yield. .sup.1H NMR (300 MHz,
CD.sub.3OD): .delta.=7.39 (dd, 2H, J=9, 2.4 Hz), 7.26 (s, 4H), 7.02
(s, 1H), 6.81 (tt, 1H, J=9.3, 2.1 Hz), 4.72 (dd, 1H, J=7.8, 5.1
Hz), 3.31 (dd, 1H, J=13.8, 5.4 Hz), 3.10 (dd, 1H, J=13.8, 8.1 Hz);
.sup.13C NMR (75 mHz, CD.sub.3OD): .delta.=173.2, 167.2, 164.4 (d,
1C, J=13.3 Hz), 160.9 (d, 1C, J=13.8 Hz), 147.1, 138.1 (t, 1C,
J=10.3 Hz), 136.6, 131.2, 131.0, 128.1, 108.3 (d, 2C, J=25.6 Hz),
104.8, 102.4 (t, 1C, J=25.0 Hz), 58.4, 36.2; ESI-MS calcd for
C.sub.18H.sub.13ClF.sub.2N.sub.2O.sub.2S [M-H].sup.-: 393.0276
found 393.0266.
[0401] Binding Data
[0402] The fluorescence polarization assay to determine binding
affinity was performed as described previously. (Carlson, E. E.;
May, J. F.; Kiessling, L. L. Chem. Biol. 2006, 13, 825-837.)
Representative binding curves are depicted in FIGS. 5A and 5B.
Exemplary binding data as determined by the fluorescence
polarization assay is provided in the Tables in FIG. 6, pages 1 to
4 and FIG. 7, pages 1 to 5. Binding is shown for the UGM isoform
from K. pneumoniae (K) and M. tuberculosis (M). Dissociation
constants are given in units of .mu.M. FIG. 6 depicts the effects
of varying the A ring substitution and FIG. 7 the effects of
varying the B ring substitution in compounds of structure:
##STR00029##
[0403] Activity Data
[0404] UGM.sub.myco inhibition was assessed using a previously
described HPLC assay to measure the extent of enzymatic conversion
of UDP-galactofuranose to UDP-galactopyranose. (Carlson, E. E.;
May, J. F.; Kiessling, L. L. Chem. Biol. 2006, 13, 825-837.)
[0405] The relative activity of UGM.sub.myco in the presence of
DMSO alone was compared to that in the presence of 50 .mu.M of
selected aminothiazoles. Enzymatic reaction conditions were as
follows: 50 .mu.M UDP-Galf, 4 .mu.M UGM.sub.myco and 20 mM DTT in
4% DMSO v/v. The mean activity of UGM.+-.the standard deviation are
shown in FIG. 8 for three replicates. The mean activities of
UGM.sub.myco in the presence of the aminothiazoles are presented in
order of decreasing binding affinities to highlight the
relationship between enzyme inhibition and binding affinity.
[0406] Representative dose-response inhibition curves for two
mycobacterial growth inhibitors at provided in FIGS. 9A and 9B. The
inhibition curves look similar to the binding curves depicted in
FIGS. 5A and 5B. The IC.sub.50 values are comparable, albeit
slightly higher than the K.sub.d values. In the graphs of these
figures, conversion is the amount of UDP-Galp formed divided by the
amount of total UDP-Gal (UDP-Galp+UDP-Galf). The data were fit to
the equation as previously described. (Soltero-Higgin, M.; Carlson,
E. E.; Phillips, J. H.; Kiessling, L. L., Identification of
inhibitors for UDP-galactopyranose mutase. J. Am. Chem. Soc. 2004,
126, 10532-10533.)
[0407] For inhibitor kinetic assays, the initial rate of
UGM.sub.myco was determined at various UDP-Galf concentrations in
the presence of either DMSO alone (4% v/v), 25 .mu.M inhibitor, or
50 .mu.M inhibitor. Enzymatic reaction conditions were as follows:
50 mM sodium phosphate buffer (pH 7), 20 mM sodium dithionite, 11
nM UGM.sub.myco, UDP-Galf, and inhibitor or DMSO in a total volume
of 60 .mu.L. Reactions were initiated upon UGM.sub.myco addition
and were quenched by the addition of an 60 .mu.L of 1:1
MeOH:CHCl.sub.3. Product formation was analyzed using a HPLC-based
assay. (Carlson, E. E.; May, J. F.; Kiessling, L. L. Chemistry
& Biology 2006, 13, 825-837.) FIGS. 10A and 10B illustrate an
exemplary graph of enzyme inhibition kinetics and a double
reciprocal plot, respectively, or compound:
##STR00030##
where K.sub.i=5.3 .mu.M.
[0408] Mycobacterial Growth Inhibition-Disc Assay
[0409] Sterile cloning discs (3 mm diameter) were obtained from
ThermoFisher Scientific, Inc. DMSO was obtained from Sigma-Aldrich
Chemical Co. To test for growth inhibition of Mycobacterium
smegmatis or Escherichia coli, LB (Luria-Bertani) medium was
inoculated with M. smegmatis (ATCC number 700084) or E. coli
BL21(DE3) and incubated at 37.degree. C. (about 2 days for M.
smegmatis, about 6 hours for E. coli). The culture was diluted to
OD.sub.600=0.2 in LB and was spread (100 .mu.L) on LB agar plates.
Cultures were allowed to soak into the plates for approximately one
hour. Then, sterile discs (3 mm diameter), to which either DMSO (2
.mu.L) or compound dissolved in DMSO (2 .mu.L, 16 nmol) had been
added, were placed onto the surface of the agar. Plates were
incubated overnight at 37.degree. C., then at room temperature for
several additional days. In both assays, a disc with kanamycin (2.5
.mu.g, 4.3 nmol) was included as a positive control.
[0410] The Table in FIG. 11, pages 1 to 3, provides exemplary data
for the indicated compounds which compares K.sub.d (UGM.sub.myco),
relative UGM.sub.myco activity at 50 .mu.M and the qualitative
results of bacterial growth disc assays. All aminothiazoles tested
that inhibited UGM.sub.myco, as determined by the activity assay,
also inhibited bacterial growth, whereas aminothiazoles that did
not show inhibition of UGM.sub.myco did not inhibit bacterial
growth.
[0411] Standard agar dilution technique to determine minimum
inhibitory concentration (MIC) values. MIC values for
representative aminothiazoles were determined using standard agar
dilution methods in 48-well plates. Each well contained 0.5 mL LB
agar medium with 3 .mu.L of compound dissolved in DMSO or with 3
.mu.L of DMSO alone as the control. Wells were inoculated with 10
.mu.L of M. Smegmatis culture that was diluted to give an
OD.sub.600 of 0.03. Plates were incubated at 37.degree. C. for 48
h. The MIC was defined as the lowest concentration of aminothiazole
at which no visible growth was observed. The MIC was determined
within 10 .mu.M. Exemplary MIC data are presented in the table of
FIG. 12A. A graph illustrating the correlation between MIC values
and UGM inhibition is provided in FIG. 12B.
Example 2
Potent Ligands for UGM that Exploit an Enzyme Subsite
[0412] This example relates to highly potent ligands for UGM which
are believed to access both the substrate binding pocket of UGM and
an adjacent site on the enzyme. In previous work, we identified
inhibitors of UGM using a high throughput screen based upon
fluorescence polarization (Soltero-Higgin, M.; Carlson, E. E.;
Phillips, J. H.; Kiessling, L. L. J. Am. Chem. Soc. 2004, 126,
10532-10533.). In designing the fluorescent probe employed in that
work (compound 1A, above) a certain fluorophore was coupled to UDP
through the diphosphate linkage. A hexanolamine linker was used to
minimize any destabilizing effect from the fluorescein moiety. The
probe was found to bind to UGM from Klebsiella pneumoniae
(UGM.sub.kleb) with an affinity of 0.10 .mu.M and to UGM from
Mycobacterium tuberculosis (UGM.sub.myco) with an affinity of 0.16
.mu.M; thus, its affinity is approximately 100-fold better than
that of UDP. The present work relates to the synthesis and
assessment of UGM ligands comprising multiple ring moieties, such
as fluorescein and the further assessment of the ability of such
compounds to inhibit microbial growth.
[0413] The effect of the length of the linker separating
fluorescein and the UDP moiety on binding was assessed. A panel of
UDP-fluorescein derivatives in which the linker length was varied
was synthesized. A series of amine-containing linkers were appended
through the pyrophosphate group to afford UDP derivatives with
alkyl linker lengths of two, four, six, eight, and ten methylene
units (Scheme 4). (Vincent, S. P.; Gastinel, L. N. Carbohydr. Res.
2002, 337, 1039-1042.) These linkers were assembled from
commercially available amino alcohols, which were first converted
to trifluoroacetamides 1a-1e. These compounds were treated with
dibenzyl phosphate to yield the phosphotriesters 2a-2e.
Hydrogenolysis of the benzyl groups afforded the phosphates 3a-3e
as the triethylamine salts, and these were coupled to uridine
5'-monophosphate (UMP)-N-methylimidazolide. The trifluoroacetamide
group was removed and UDP derivatives 4a-4-e were treated with
fluorescein isothiocyanate (FITC) to yield conjugates 5a-5e.
[0414] The affinities of the UDP-fluorescein conjugates for
UGM.sub.kleb and UGM.sub.myco were determined using fluorescence
polarization and are listed for compounds 5a-5e where n is 1, 3, 5,
7 or 9, respectively. UDP binds to UGM.sub.myco with a K.sub.d of
15 .mu.M and to UGM.sub.kleb with a K.sub.d of 26 .mu.M. The
dissociation constants of derivatives 5a-5e for either UGM.sub.kleb
or UGM.sub.myco depend on linker length. Specifically, compound 5a
(two methylene linker) binds poorly (K.sub.d>30 .mu.M);
conjugate 5b (four methylene linker) is slightly (5-fold) more
potent than UDP. Conjugate 5c (six methylene linker) binds 100-fold
better, while 5d (eight methylene linker) bound with the highest
affinity: 500-fold tighter than UDP. The eight methylene linker
seems to provide the necessary distance for optimal binding; the
affinity of 5e (ten methylene linker) for UGM is similar than that
of 5d.
##STR00031##
TABLE-US-00001 TABLE 1 n UGM.sub.myco K.sub.d(.mu.M) 1 >30 3 2.5
5 0.17 7 0.054 9 0.064 UDP 15
[0415] The dependence of binding affinity on linker length suggests
there is a subsite that the fluorophore occupies and the eight
methylene linker provides the means to access this site. Ligands
that exploit this subsite are highly potent.
[0416] In view of the dramatic effect of linker length on ligand
binding the affect of the chemical nature of the linker was
assessed. Compound 4d, which is lacking a fluorophore, has a
similar affinity for UGM as UDP. These data suggest that the linker
has little influence on binding in the absence of the
fluorophore.
[0417] To test whether the chemical composition of the linker
contributes to the affinity of the fluorophore-containing UDP
derivatives, we analogs possessing oligoethyleneglycol linker were
synthesized. Triethylene glycol and tetraethylene glycol were
monomesylated and converted to the corresponding azide (Vincent, S.
P.; Gastinel, L. N. Carbohydr. Res. 2002, 337, 1039-1042.). The
azides were reduced to the corresponding amine via catalytic
hydrogen and converted to UDP-fluorescein conjugates 5f and 5 g
(Scheme 5).
[0418] For UGM.sub.myco, 5f bound with 20-fold greater affinity
than UDP, but its affinity was more than 10-fold lower than the
conjugate with the alkyl linker of similar length (compound 5d,
eight methylene linker). A similar trend was observed with
UGM.sub.kleb. This discrepancy in the affinity of the oligoethylene
glycol conjugate and the alkyl conjugate of similar length could be
due to differences in conformation affecting their length in
solution (Karlsson, L.; Asbrink, L.; Fridh, C.; Lindholm, E.;
Svensson, A. Phys. Scr. 1980, 21, 170-172.) Specifically, the
gauche effect will influence the conformation of oligoethylene
glycol linkers. Still, conjugate 5 g with a longer oligoethylene
glycol linker did not exhibit improved binding to either
UGM.sub.kleb or UGM.sub.myco. These results indicate that the
nature of the linker plays a role in the binding of UDP-fluorescein
compounds to UGM.
##STR00032##
[0419] To investigate whether binding of the fluorophore alone
could be detected, fluorescein derivative 6, which possesses a
linker from FITC and octanolamine, was synthesized (Scheme 6).
Interestingly, no binding was observed at concentrations up to 34
.mu.M of either UGMmyco or UGMkleb. Similarly, no binding was
observed when UGM was first incubated with 200 .mu.M of UDP. This
result indicates that the UDP moiety and the fluorophore must be
linked. Given that fluorescein contributes significantly to
binding, other aryl substituents were investigated. Specifically,
UDP-octanolamine 4d was appended to naphthyl isothiocyanate to form
conjugate 7 (Scheme 7). While the affinity of naphthyl conjugate 7
for UGM was 10-fold less than the corresponding fluorescein
conjugate 5d, it was 25-fold greater than UDP alone. These results
suggest that the naphthyl group can occupy the subsite exploited by
the fluorescein group.
[0420] As described above, certain 2-aminothiazoles were found to
be competitive inhibitors of UGM. Experiments were performed to
assess whether the affinity of 2-aminothiozoles for UGM could be
increased by exploiting the identified subsite. As a starting
point, analogs of 2-aminothiazole 8, which has a pentyl
substitutent, and has an UGM affinity of 15 .mu.M, was synthesized.
A fluorescein group was attached to this 2-aminothiozole via
elaboration of the pentyl group. As illustrated in Scheme 8 an
analog with an eight methylene linker terminating in an amine was
prepared, for the conjugation of a fluorophore.
[0421] More specifically, the synthesis of aminothiazole 11 began
with a Friedel-Crafts acylation of 8-phenyl-1-octanol to produce
the acetophenone. (Hernandezgallegos, Z.; Lehmann, P. A. J. Med.
Chem. 1990, 33, 2813-2817.) The alcohol was converted to the azide
through the mesylate followed by a-bromination with CuBr.sub.2 to
provide 9. (King, L. C.; Ostrun, G. K. J. Org. Chem. 1964, 29,
3459-3461.) The .alpha.-bromoketone 9 was cyclized with the
thiourea derived from 4-chloro phenylalanine to generate
2-aminothiazole 10. The azide was reduced with catalytic
hydrogenation to afford the amine, which was conjugated to
fluorescein isothiocyanate (FITC). The affinity of fluorescein
conjugate 11 is 0.38 .mu.M for UGM.sub.kleb and 0.30 .mu.M for
UGM.sub.myco. Thus, this substituted aminothiazole binds between 40
and 50-fold more tightly than thiazole 8 alone.
[0422] In summary, we have identified highly potent UGM ligands,
having binding affinities of .about.50 nM., which possess aryl
substituents linked to an aminothiazole, which are believed to
access a subsite present on the UGM from K. pneumoniae and M.
tuberculosis.
General Synthetic Procedures
[0423] All reagents were purchased from Sigma-Aldrich Co., except
for octanolamine and decanolamine, which were purchased from TCI
America. All compounds were used as received. Methanol (MeOH) was
distilled from magnesium, methylene chloride (CH.sub.2Cl.sub.2) and
diisopropylethylamine (DIEA) were distilled from calcium hydride,
and dimethyl formamide (DMF) was used as biotech grade
(Sigma-Aldrich Co.).
[0424] Flash chromatography was performed using silica gel 60,
230-450 mesh (Sorbent Technologies). Analytical thin-layer
chromatography (TLC) was carried out on EM Science TLC plates
precoated with silica gel 60 F254 (250-.mu.m layer thickness).
Visualization of TLC was accomplished using a UV lamp.
[0425] .sup.1H NMR spectra were obtained using a Bruker AC-300 (300
mHz) or Varian MercuryPlus 300 (300 MHz), and .sup.13C NMR specta
were obtained using a Varian MercuryPlus 300 (300 MHz). Chemical
shifts are reported relative to residual solvent signals
(CDCl.sub.3): .sup.1H: 37.27, .sup.13C: .delta. 77.23;
(CD.sub.3OD): .sup.1H: .delta. 3.31, .sup.13C: .delta. 49.15;
(DMF-d.sub.7): .sup.1H: .delta. 2.92, .sup.13C: .delta. 34.89;
(D.sub.2O): .sup.1H: .delta. 4.79. .sup.1H NMR data are assumed to
be first order with apparent doublets and triplets reported as d
and t, respectively. Multiplets are reported as m and resonances
that appear broad are designated as br.
##STR00033##
##STR00034##
##STR00035##
##STR00036##
[0426] High-resolution electrospray ionization mass spectra
(HRESI-MS) were obtained on a Micromass LCT. LC-MS (ESI) were
obtained using a Shimadzu LCMS-2010 (Columbia, Md.) equipped with
two pumps (LC-10Advp), controller (SCL-10Avp), autoinjector
(SIL-10Advp), UV diode array detector (SPD-M10Avp), and single
quadrupole analyzer (by electrospray ionization, ESI). The LC-MS is
interfaced with a PC running the Shimadzu LCMS solution software
package (Version 2.04 Su2-H2). A Supelco (Bellefonte, Pa.) 15
cm.times.2.1 mm C-18 wide pore reverse phase column was used for
all LC-MS analyses. Standard reverse phase HPLC conditions were
used as follows: flow rate=200 mL/min; mobile phase A=0.1% formic
acid; mobile phase B=0.1% formic acid in acetonitrile, 50-95% B
over 7 min. UV spectra were recorded using an HP-8452 UV-Vis
spectrometer running UV Visible Chemstation software. High
performance liquid chromatography (HPLC) was performed on a C18
reverse phase column using water (A) and acetonitrile (B) (both
buffered with 0.02% trifluoroacetic acid) as the elution solvents
at 10 mL/min. Compound elution was detected by UV absorbance at
range 200-600 nm.
[0427] Cation-exchange resin Dowex 50WX8-200 (H+ form, strongly
acidic) was purchased from Aldrich and converted to the appropriate
salt form prior to use. Uridine 5'-monophosphate (5'-UMP) disodium
salt was purchased from Sigma and converted to the triethylammonium
salt (1.4 eq by .sup.1H NMR) prior to coupling reactions by
stirring with Dowex 50WX8-200 (NEt.sub.3H.sup.+ form) overnight.
The resin was removed by filtration and washed with H.sub.2O.
Combined filtrates were lyophilized to produce the
UMP-Et.sub.3NH.sup.+ salt as a fluffy white solid.
[0428] General Procedure I:
[0429] The installation of the trifluoracetamide protecting
group.
[0430] A solution of amino alcohol (1.0 eq) and triethylamine (2.5
eq) in MeOH was cooled to 0.degree. C. Trifluoroacetic anhydride
(1.4 eq) was added dropwise under an argon atmosphere and the
reaction was allowed to warm to room-temperature. The solution was
stirred 4 h, concentrated, and the product was purified by silica
gel chromatography to afford 1.
##STR00037##
[0431] 2-Trifluoroacetamido-1-ethanol (1a): Following general
procedure I, ethanolamine (2.0 g, 32.74 mmol) was combined with
triethylamine (11.4 mL, 81.86 mmol) and trifluoroacetic anhydride
(6.47 mL, 45.84 mmol) in MeOH (30 mL). Purification by silica gel
chromatography (2:3 hexanes/EtOAc) yielded 4.86 g (94%) of la as a
white solid (Lokhov, S. G.; Podyminogin, M. A.; Sergeev, D. S.;
Silnikov, V. N.; Kutyavin, I. V.; Shishkin, G. V.; Zarytova, V. P.
Bioconjugate Chem. 1992, 3, 414-419.)
##STR00038##
[0432] 4-Trifluoroacetamido-1-butanol (1b): Following general
procedure I, butanolamine (2.0 g, 22.44 mmol) was combined with
triethylamine (7.82 mL, 56.09 mmol) and trifluoroacetic anhydride
(4.44 mL, 24.10 mmol) in MeOH (20 mL). Purification by silica gel
chromatography (2:3 hexanes/EtOAc) yielded 4.03 g (97%) of 1b as a
pale yellow oil (Lokhov, S. G.; Podyminogin, M. A.; Sergeev, D. S.;
Silnikov, V. N.; Kutyavin, I. V.; Shishkin, G. V.; Zarytova, V. P.
Bioconjugate Chem. 1992, 3, 414-419.)
##STR00039##
[0433] 6-Trifluoroacetamido-1-hexanol (1c): Following general
procedure I, hexanolamine (2.0 g, 17.06 mmol) was combined with
triethylamine (5.95 mL, 42.67 mmol) and trifluoroacetic anhydride
(3.37 mL, 23.89 mmol) in MeOH (15 mL). Purification by silica gel
chromatography (2:3 hexanes/EtOAc) yielded 3.35 g (92%) of 1c as a
white solid (Vincent, S. P.; Gastinel, L. N. Carbohydr. Res.
##STR00040##
[0434] 8-Trifluoroacetamido-1-octanol (1d): Following general
procedure I, octanolamine (2.5 g, 17.21 mmol) was combined with
triethylamine (6.0 mL, 43.04 mmol) and trifluoroacetic anhydride
(3.4 mL, 24.10 mmol) in MeOH (15 mL). Purification by silica gel
chromatography (2:3 hexanes/EtOAc) yielded 4.09 g (98%) of 1d as a
white solid. .sup.1H (300 MHz, CD.sub.3OD): .delta. 3.61 (t, 2H,
J=6.6 Hz), 3.34 (t, 2H, J=7.2), 1.63 (m, 4H), 1.42 (m, 8H);
.sup.13C (75 mHz, CD.sub.3OD): .delta. 158.61 (q, J=81.5 Hz),
117.58 (q, J=284.6 Hz), 63.02, 40.79, 33.88, 30.51, 30.48, 30.30,
29.85, 27.79, 28.88; ESI-MS calcd for
C.sub.10H.sub.18F.sub.3NO.sub.2 [M+Na].sup.+: 264.1197 found
264.1186.
##STR00041##
[0435] 10-Trifluoroacetamido-1-decanol (1e): Following general
procedure I, decanolamine (0.50 g, 2.89 mmol) was combined with
triethylamine (1.0 mL, 7.21 mmol) and trifluoroacetic anhydride
(0.57 mL, 4.04 mmol) in MeOH (3 mL). Purification by silica gel
chromatography (2:3 hexanes/EtOAc) yielded 0.722 g (99%) of 1e as a
white solid. .sup.1H (300 MHz, CD.sub.3OD): .delta. 3.55 (t, 2H,
J=5.4 Hz), 3.28 (t, 2H, J=7.2 Hz), 1.55 (m, 4H), 1.35 (m, 10H).
.sup.13C (75 mHz, CD.sub.3OD): .delta. 82.99, 40.74, 33.83, 30.62,
30.54, 30.25, 29.78, 27.77, 26.92; ESI-MS calcd for
C.sub.12H.sub.22F.sub.3NO.sub.2 [M+Na].sup.+: 292.1500 found
292.1507.
##STR00042##
[0436] 8-Trifluoroacetamido-3,6-dioxa-1-octanol (1f):
8-azido-3,6-dioxa-1-octanol (Amvamzollo, P. H.; Sinay, P.
Carbohydr. Res. 1986, 150, 199-212) (0.7 g, 4.0 mmol) was
synthesized from triethylene glycol following the reported
procedure (Bertozzi, C. R.; Bednarski, M. D. J. Org. Chem. 1991,
56, 4326-4329). The azide was combined with Pd/C (150 mg) in MeOH
(8.0 mL) and stirred 12 h under H.sub.2 (1 atm). The suspension was
filtered over celite, and the filtrate was concentrated to afford
the amine (0.590 g, 4.0 mmol) in quantitative yields (Sato, H.;
Hayashi, E.; Yamada, N.; Yatagai, M.; Takahara, Y. Bioconjugate
Chem. 2001, 12, 701-710.). Following general procedure I,
2-[2-(2-aminoethoxy)ethoxy]ethanol (0.590 g, 3.95 mmol) was
combined with triethylamine (1.378 mL, 9.89 mmol) and
trifluoroacetic anhydride (0.782 mL, 5.54 mmol) in MeOH (5 mL).
Purification by silica gel chromatography (EtOAc) yielded 0.576 g
(60%) of if as a white solid. .sup.1H (300 MHz, CD.sub.3OD):
.delta. 7.87 (br s, 1H), 3.73 (m, 2H), 3.68-3.60 (m, 10H), 3.55 (t,
2H, J=4.5 Hz), 3.32 (br s, 1H); .sup.13C (75 mHz, CD.sub.3OD):
.delta. 157.6 (q, J=36.6 Hz), 116.1 (q, J=286.1 Hz), 72.68, 70.35,
70.24, 68.90, 61.52, 39.84; ESI-MS calcd for
C.sub.8H.sub.14F.sub.3NO.sub.4 [M-H].sup.-: 244.0797 found
244.0795.
##STR00043##
[0437] 11-Trifluoroacetamido-3,6,9-trioxa-1-undecanol (1g):
11-azido-3,6,9-trioxa-1-undecane (0.6 g, 2.7 mmol) was synthesized
from tetraethylene glycol following the reported procedure
(Bertozzi, C. R.; Bednarski, M. D. J. Org. Chem. 1991, 56,
4326-4329.). The azide was combined with Pd/C (150 mg) in MeOH (7.0
mL) and stirred 12 h under H.sub.2 (1 atm). The suspension was
filtered over celite and the filtrate was concentrated to afford
the amine (0.520 g, 2.7 mmol) in quantitative yields (Xie, H. Z.;
Braha, O.; Gu, L. Q.; Cheley, S.; Bayley, H. Chem. Biol. 2005, 12,
109-120.) Following general procedure I,
2-[2-(2-[2-aminoethoxy]ethoxy)ethoxy]ethanol (0.50 g, 2.59 mmol)
was combined with triethylamine (0.902 mL, 6.48 mmol) and
trifluoroacetic anhydride (0.51 mL, 3.62 mmol) in MeOH (3 mL).
Purification by silica gel chromatography (EtOAc) yielded 0.343 g
(46%) of 1 g as a white solid. .sup.1H (300 MHz, CD.sub.3OD):
.delta. 3.69 (t, 2H, J=3.9 Hz), 3.64 (m, 12H), 3.57 (t, 2H, J=3.5
Hz). .sup.13C (75 mHz, CD.sub.3OD): .delta. 157.7 (q, J=37.1 Hz),
118.1 (q, J=285.8 Hz), 72.56. 70.78, 70.46, 70.15, 69.82, 69.56,
61.34, 39.95; ESI-MS calcd for C.sub.10H.sub.18F.sub.3NO.sub.5
[M-H].sup.-: 288.1059 found 288.1060.
[0438] General Procedure II:
[0439] Coupling of dibenzyl phosphate and alcohol 1.
[0440] Dibenzyl phosphate (1.5 eq) was dissolved in DMF and
CH.sub.2Cl.sub.2, and the resulting solution was cooled to
0.degree. C. Oxalyl chloride (3.0 eq) was added dropwise under an
argon atmosphere. The solution was allowed to warm to
room-temperature, and was stirred for 1 h. The solvent was removed
in vacuo and the remaining material was azeotroped with toluene.
The resulting viscous liquid was dissolved in CH.sub.2Cl.sub.2 and
added dropwise to a flask containing 1 and 4 .ANG. molecular sieves
(ca. 5-10) in pyridine at 0.degree. C. The solution was stirred
under an argon atmosphere for 1 h at 0.degree. C. and then for 3 h
at room-temperature. The solvent was removed in vacuo and the
product was purified by silica gel chromatography (1:1.fwdarw.1:2
Hexane/EtOAc) to afford 2.
##STR00044##
1-O-(Dibenzyl phosphoryloxy)-2-trifluoroacetamido-1-ethanol
(2a)
[0441] Following general procedure II, dibenzyl phosphate (1.79 g,
6.45 mmol) was dissolved in DMF (10 .mu.L) and CH.sub.2Cl.sub.2 (25
mL) and combined with oxalyl chloride (1.13 mL, 12.9 mmol). After
concentration, the compound was redissolved in CH.sub.2Cl.sub.2 (5
mL) and added to la (675 mg, 4.3 mmol) in pyridine (10 mL) and 4
.ANG. molecular sieves to yield 621 mg (35%) of 2a as an off-white
solid. .sup.1H (300 MHz, CDCl.sub.3): .delta. 7.35 (m, 10H), 5.03
(ABX system, 4H, J.sub.AB=15 Hz, J.sub.AP=J.sub.BP=11.7 Hz), 4.04
(pentet, 2H, J=4.8 Hz), 3.50 (q, 2H, J=5.1). .sup.13C (75 mHz,
CDCl.sub.3): .delta. 135.45, 129.00, 128.12, 70.02, 65.7, 40.49.
ESI-MS calcd for C.sub.18H.sub.19F.sub.3NO.sub.5P [M+H].sup.+:
418.1031 found 418.1019.
##STR00045##
1-O-(Dibenzyl phosphoryloxy)-4-trifluoroacetamido-1-butanol
(2b)
[0442] Following general procedure II, dibenzyl phosphate (1.79 g,
6.45 mmol) was dissolved in DMF (10 .mu.L) and CH.sub.2Cl.sub.2 (30
mL) and combined with oxalyl chloride (1.13 mL, 12.9 mmol). After
concentration, the compound was redissolved in CH.sub.2Cl.sub.2 (5
mL) and added to 1b (796 mg, 4.3 mmol) in pyridine (10 mL) and 4
.ANG. molecular sieves to yield 777 mg (41%) of 2b as a clear oil.
.sup.1H (300 MHz, CDCl.sub.3): .delta. 7.35 (m, 10H), 5.01 (ABX
system, 4H, J.sub.AB=15 Hz, J.sub.AP=J.sub.BP=11.7 Hz), 3.97 (q,
2H, J=6 Hz), 3.28 (q, 2H, J=6), 1.70 (m, 4H). .sup.13C (75 mHz,
CDCl.sub.3): .delta. 135.90, 128.84, 128.16, 116.54 (q, J=284.6
Hz), 69.60, 67.42, 39.97, 27.49, 25.08; ESI-MS calcd for
C.sub.20H.sub.23F.sub.3NO.sub.5P [M+Na].sup.+: 468.1164 found
##STR00046##
1-O-(Dibenzyl phosphoryloxy)-6-trifluoroacetamido-1-hexanol
(2c)
[0443] Following general procedure II, dibenzyl phosphate (1.79 g,
6.45 mmol) was dissolved in DMF (10 .mu.L) and CH.sub.2Cl.sub.2 (25
mL) and combined with oxalyl chloride (1.13 mL, 12.9 mmol). After
concentration, the compound was redissolved in CH.sub.2Cl.sub.2 (5
mL) and added to 1c (917 mg, 4.3 mmol) in pyridine (10 mL) and 4
.ANG. molecular sieves to yield 884 mg (43%) of 2c as a clear oil
(16).
##STR00047##
1-O-(Dibenzyl phosphoryloxy)-8-trifluoroacetamido-1-octanol
(2d)
[0444] Following general procedure II, dibenzyl phosphate (1.73 g,
6.22 mmol) was dissolved in DMF (10 .mu.L) and CH.sub.2Cl.sub.2 (25
mL) and combined with oxalyl chloride (1.08 mL, 12.4 mmol). After
concentration, the compound was redissolved in CH.sub.2Cl.sub.2 (5
mL) and added to 1d (1.00 g, 4.15 mmol) in pyridine (10 mL) and 4
.ANG. molecular sieves to yield 966 mg (46%) of 2d as a clear oil.
.sup.1H (300 MHz, CDCl.sub.3): .delta. 7.31 (m, 10H), 5.00 (ABX
system, 4H, J.sub.AB=15 Hz, J.sub.AP=J.sub.BP=11.7 Hz), 3.96 (q,
2H, J=6.6 Hz), 3.31 (q, 2H, J=6.6 Hz), 1.55 (m, 4H), 1.27 (m, 8H).
.sup.13C (75 mHz, CDCl.sub.3): .delta. 135.92, 128.63, 127.96,
69.33, 67.98, 39.99, 30.09, 28.91, 26.58, 25.29; ESI-MS calcd for
C.sub.24H.sub.31F.sub.3NO.sub.5P [M+Na].sup.+: 524.1790 found
##STR00048##
1-O-(Dibenzyl phosphoryloxy)-10-trifluoroacetamido-1-decanol
(2e)
[0445] Following general procedure II, dibenzyl phosphate (775 mg,
2.78 mmol) was dissolved in DMF (10 .mu.L) and CH.sub.2Cl.sub.2 (15
mL) and combined with oxalyl chloride (0.50 mL, 5.57 mmol). After
concentration, the compound was redissolved in CH.sub.2Cl.sub.2 (5
mL) and added to 1e (500 mg, 1.86 mmol) in pyridine (5 mL) and 4
.ANG. molecular sieves to yield 578 mg (59%) of 2e as a clear oil.
.sup.1H (300 MHz, CDCl.sub.3): .delta. 7.34 (m, 10H), 5.01 (ABX
system, 4H, J.sub.AB=15 Hz, J.sub.AP=J.sub.BP=11.7 Hz), 3.97 (q,
2H, J=6 Hz), 3.33 (q, 2H, J=6.6), 1.70 (m, 4H), 1.25 (m, 12H).
.sup.13C (75 mHz, CDCl.sub.3): .delta. 135.99, 128.62, 127.98,
69.33, 66.18, 40.09, 30.25, 29.44, 29.30, 29.19, 29.02, 28.97,
26.10, 25.37; ESI-MS calcd for C.sub.26H.sub.35F.sub.3NO.sub.5P
[M+Na].sup.+: 552.2103 found 552.2094.
##STR00049##
1-O-(Dibenzyl
phosphoryloxy)-8-trifluoroacetamido-3,6-dioxa-1-octanol (2f)
[0446] Following general procedure II, dibenzyl phosphate (981 mg,
3.53 mmol) was dissolved in DMF (20 .mu.L) and CH.sub.2Cl.sub.2 (15
mL) and combined with oxalyl chloride (0.62 mL, 7.05 mmol). After
concentration, the compound was redissolved in CH.sub.2Cl.sub.2 (5
mL) and added to if (500 mg, 2.35 mmol) in pyridine (5 mL) and 4
.ANG. molecular sieves to yield 623 mg (53%) of 2f as a clear oil.
.sup.1H (300 MHz, CDCl.sub.3): .delta. 7.34 (m, 10H), 5.04 (ABX
system, 4H, J.sub.AB=15 Hz, J.sub.AP=J.sub.BP=11.7 Hz), 4.12-4.09
(m, 2H), 3.64 (t, 2H, J=5.1 Hz), 3.61-3.54 (m, 6H), 3.47 (q, 2H,
J=4.8 Hz); .sup.13C (75 mHz, CDCl.sub.3): .delta. 157.5 (q, J=36.5
Hz), 135.9 (d, J=7.1 Hz), 135.8, 128.7, 128.0, 116.0 (q, J=286.4
Hz), 70.7, 70.4, 70.0 (d, J=6.5 Hz), 69.4 (d, J=5.1 Hz), 68.8, 66.9
(d, J=5.9 Hz), 39.94; ESI-MS calcd for
C.sub.22H.sub.27F.sub.3NO.sub.7P [M-H].sup.-: 504.1399 found
504.1420.
##STR00050##
1-O-(Dibenzyl
phosphoryloxy)-11-trifluoroacetamido-3,6,9-trioxa-1-undecanol
(2g)
[0447] Following general procedure II, dibenzyl phosphate (433 mg,
1.56 mmol) was dissolved in DMF (10 .mu.L) and CH.sub.2Cl.sub.2 (10
mL) and combined with oxalyl chloride (0.27 mL, 3.11 mmol). After
concentration, the compound was redissolved in CH.sub.2Cl.sub.2 (5
mL) and added to 1 g (300 mg, 1.04 mmol) in pyridine (5 mL) and 4
.ANG. molecular sieves to yield 499 mg (87%) of 2 g as a clear oil.
.sup.1H (300 MHz, CDCl.sub.3): .delta. 7.34 (m, 10H), 5.04 (ABX
system, 4H, J.sub.AB=15 Hz, J.sub.AP=J.sub.BP=11.7 Hz), 4.14 (m,
2H), 3.64 (t, 2H, J=5.0 Hz), 3.62-3.55 (m, 10H), 3.50 (q, 2H, J=5.1
Hz). .sup.13C (75 mHz, CDCl.sub.3): .delta. 157.5 (q, J=37.2 Hz),
136.0 (d, J=6.1 Hz), 128.7, 128.1, 116.1 (q, J=286.3 Hz), 70.8,
70.7, 70.6, 70.4, 70.1 (d, J=6.8 Hz), 68.8, 66.9 (d, J=5.9 Hz),
39.9; ESI-MS calcd for C.sub.24H.sub.31F.sub.3NO.sub.8P
[M+Na].sup.+: 572.1637 found 572.1654.
[0448] General Procedure III: Hydrogenolysis of phosphotriester
2.
[0449] Phosphotriester 2 (1 eq) was dissolved in 3:2 MeOH/EtOAc
with triethylamine (1 eq). Pd/C was added and the suspension was
stirred under H.sub.2 (1 atm) for 12 h. The suspension was filtered
through celite and the filtrate was concentrated to yield phosphate
3 as the triethylammonium salt.
##STR00051##
2-Trifluoroacetamido-ethanol-1-phosphate triethylammonium salt
(3a)
[0450] Following general procedure III, phosphotriester 2a (367 mg,
0.88 mmol) was dissolved in 3:2 MeOH/EtOAc (15 mL) and
triethylamine (0.12 mL), and combined with Pd/C (184 mg) and
H.sub.2 to yield the triethylammonium salt of 3a (290 mg, 97%) as a
white solid. .sup.1H (300 MHz, CD.sub.3OD): .delta. 3.98 (q, 2H,
J=6.9), 3.52 (t, 2H, J=5.4 Hz), 3.18 (q, 6H, J=7.2 Hz), 1.31 (t,
9H, J=7.5 Hz); .sup.13C (75 mHz, CD.sub.3OD): .delta. 63.78, 47.52,
41.93, 9.10; ESI-MS calcd for C.sub.4H.sub.7F.sub.3NO.sub.5P [M-H]:
235.9936 found 235.9947.
##STR00052##
4-Trifluoroacetamido-butanol-1-phosphate triethylammonium salt
(3b)
[0451] Following general procedure III, phosphotriester 2b (392 mg,
0.88 mmol) was dissolved in 3:2 MeOH/EtOAc (15 mL) and
triethylamine (0.12 mL), and combined with Pd/C (196 mg) and
H.sub.2 to yield the triethylammonium salt of 3b (339 mg, 97%) as a
white solid. .sup.1H (300 MHz, CD.sub.3OD): .delta. 3.85 (q, 2H,
J=6.3), 3.25-3.20 (m, 2H), 3.14 (q, 6H, J=7.5 Hz), 1.74-1.54 (m,
4H), 1.27 (t, 9H, J=7.2 Hz); .sup.13C (75 mHz, CD.sub.3OD): .delta.
64.35, 46.31, 39.26, 27.76, 25.24, 7.89; ESI-MS calcd for
C.sub.6H.sub.11F.sub.3NO.sub.5P [M-H].sup.-: 264.0249 found
264.0240.
##STR00053##
6-Trifluoroacetamido-hexanol-1-phosphate triethylammonium salt
(3c)
[0452] Following general procedure III, phosphotriester 2c (441 mg,
0.93 mmol) was dissolved in 3:2 MeOH/EtOAc (15 mL) and
triethylamine (0.13 mL), and combined with Pd/C (220 mg) and
H.sub.2 to yield the triethylammonium salt of 3c (351 mg, 96%) as a
clear oil (Vincent, S. P.; Gastinel, L. N. Carbohydr. Res. 2002,
337, 1039-1042.)
##STR00054##
8-Trifluoroacetamido-octanol-1-phosphate triethylammonium salt
(3d)
[0453] Following general procedure III phosphotriester 2d (400 mg,
0.80 mmol) was dissolved in 3:2 MeOH/EtOAc (12 mL) and
triethylamine (0.11 mL), and combined with Pd/C (200 mg) and
H.sub.2 to yield the triethylammonium salt of 3d (320 mg, 95%) as a
white solid. .sup.1H (300 MHz, CD.sub.3OD): .delta. 3.92 (q, 2H,
J=6.6), 3.26 (t, 2H, J=7.5), 3.20 (q, 6H, J=7.2 Hz), 1.70-1.50 (m,
4H), 1.40-1.29 (m, 17H); .sup.13C (75 mHz, CD.sub.3OD): .delta.
67.23, 47.74, 40.70, 30.17, 29.79, 27.71, 26.64, 18.78, 17.30,
9.18; ESI-MS calcd for C.sub.10H.sub.19F.sub.3NO.sub.5P
[M-H].sup.-: 320.0875 found
##STR00055##
10-Trifluoroacetamido-decanol-1-phosphate triethylammonium salt
(3e)
[0454] Following general procedure III, phosphotriester 2e (554 mg,
1.02 mmol) was dissolved in 3:2 MeOH/EtOAc (15 mL) and
triethylamine (0.15 mL), and combined with Pd/C (270 mg) and
H.sub.2 to yield the triethylammonium salt of 3e (460 mg, 98%) as a
white solid. .sup.1H (300 MHz, CD.sub.3OD): .delta. 3.77 (q, 2H,
J=6.6), 3.19 (t, 2H, J=7.2 Hz), 3.09 (q, 6H, J=7.2 Hz), 1.59-1.43
(m, 4H), 1.33-1.20 (m, 21H); .sup.13C (75 mHz, CD.sub.3OD): .delta.
66.22, 47.48, 40.74, 30.63, 30.53, 30.50, 30.45, 30.29, 29.80,
27.79, 26.94, 9.09; ESI-MS calcd for
C.sub.12H.sub.22F.sub.3NO.sub.5P [M-H].sup.-: 348.1188 found
348.1198.
##STR00056##
8-trifluoroacetamido-3,6-dioxa-octanol-1-phosphate triethylammonium
salt (3f)
[0455] Following general procedure III, phosphotriester 2f (550 mg,
1.09 mmol) was dissolved in MeOH (10 mL) and triethylamine (0.15
mL), and combined with Pd/C (275 mg) and H.sub.2 to yield the
triethylammonium salt of 3f (462 mg, 99%) as a white solid. .sup.1H
(300 MHz, CD.sub.3OD): .delta. 3.81 (td, 2H, J=6.1, 4.4 Hz),
3.50-3.40 (m, 8H), 3.29 (t, 2H, J=5.3 Hz), 3.00 (q, 6H, J=7.0 Hz),
1.35 (t, 9H, J=7.1 Hz); .sup.13C (75 mHz, CD.sub.3OD): .delta.
159.2 (q, J=36.2 Hz), 117.7 (q, J=285.1 Hz), 72.2 (d, J=7.2 Hz),
71.7, 71.5, 69.9, 65.6 (d, J=5.6 Hz), 47.5, 40.9, 9.3; ESI-MS calcd
for C.sub.8H.sub.15F.sub.3NO.sub.7P [M-H].sup.-: 324.0460 found
324.0471.
##STR00057##
11-trifluoroacetamido-3,6,9-trioxa-undecanol-1-phosphate
triethylammonium salt (3g)
[0456] Following general procedure III, phosphotriester 2g (450 mg,
0.82 mmol) was dissolved in MeOH (9 mL) and triethylamine (0.12
mL), and combined with Pd/C (225 mg) and H.sub.2 to yield the
triethylammonium salt of 3 g (397 mg, 99%) as a pasty, white solid.
.sup.1H (300 MHz, CD.sub.3OD): .delta. 3.92-2.90 (m, 2H), 3.60-3.47
(m, 12H), 3.37 (t, 2H, J=5.1 Hz), 3.08 (q, 6H, J=7.1 Hz), 1.21 (t,
9H, J=6.8 Hz); .sup.13C (75 mHz, CD.sub.3OD): .delta. 117.7 (q,
J=284.9 Hz), 72.0, 71.7, 71.6, 71.5, 69.9, 66.0, 47.8, 40.9, 9.3;
ESI-MS calcd for C.sub.10H.sub.19F.sub.3NO.sub.8P [M-H].sup.-:
368.0722 found 368.0721.
[0457] General Procedure IV: Coupling of phosphate 3 to uridine
5'-monophosphate (UMP) and trifluoracetamide removal.
[0458] 5'-UMP (triethylammonium salt, 1.0 eq) was suspended in
acetonitrile (3 mL) and cooled to 0.degree. C. Dimethylaniline (4
eq) and triethylamine (2 eq) were added under an argon atmosphere.
A pre-cooled solution of trifluoroacetic anhydride (6 eq) in
acetonitrile (1 mL) was added dropwise over several minutes to
yield a transparent, pink solution. The reaction was stirred at
0.degree. C. for 15 min and the solvent was removed in vacuo. The
residue was resuspended in acetonitrile (3 mL) and stirred at
0.degree. C. with 4 .ANG. molecular sieves (ca. 5-10) under an
argon atmosphere. Triethylamine (5 eq) and 1-methyl imidazole (5.3
eq) were added dropwise and the solution turned bright yellow.
Phosphate 3 (0.63 eq) was dissolved in acetonitrile (1 mL) and
added dropwise to the solution. The reaction was stirred at
0.degree. C. for 1 h, and room-temperature for 3 h. The reaction
was quenched with 15 mL H.sub.2O and extracted with
CH.sub.2Cl.sub.2 (15 mL). The organic layer was extracted with
H.sub.2O (15 mL) and the combined aqueous layers were combined and
concentrated, and the product was purified by silica gel
chromatography. The resulting product was treated with 3 M ammonium
hydroxide to deprotect the trifluoroacetamide moiety. The solvent
was evaporated in vacuo and the product was lyophilized to yield
diphosphate 4 as the ammonium salt.
##STR00058##
Uridine 5'-diphosphoethanolamine, ammonium salt (4a)
[0459] Following general procedure IV, UMP.Et.sub.3NH.sup.+ (102
mg, 0.238 mmol) was activated with dimethyl aniline (120 .mu.L,
0.952 mmol), triethylamine (50 .mu.L, 0.476 mmol) and
trifluoroacetic anhydride (200 .mu.L, 1.428 mmol). The activated
UMP was then reacted with 1-methylimidazole (100 mL, 1.26 mmol),
triethylamine (130 .mu.L, 1.19 mmol) and phosphate 3a (50 mg, 0.15
mmol). The product was purified by silica gel chromatography (5:4:1
CHCl.sub.3/MeOH/1 M ammonium acetate) to yield the ammonium salt of
the diphosphate as an off white solid. The diphosphate was stirred
with 3 M ammonium hydroxide (10 mL) for 2 h under N.sub.2, the
solvent was removed in vacuo and the product was lyophilized to
yield the ammonium salt of uridine 5'-diphosphoethanolamine 4a (33
mg, 45%) as an off-white solid. .sup.1H (300 MHz, D.sub.2O):
.delta. 7.98 (d, 1H, J=8.1 Hz), 6.03 (d, 1H, J=4.6 Hz), 6.01 (d,
1H, J=8.5 Hz), 4.42 (m, 2H), 4.33 (br s, 1H), 4.30-4.22 (m, 5H),
3.35 (t, 2H, J=6.9 Hz), 1.92 (s, 4H); .sup.13C (75 mHz, D.sub.2O):
.delta. 152.1, 141.7, 102.8, 88.8, 83.3 (d, J=8.0 Hz), 73.9, 69.8,
65.1 (d, J=7.7 Hz), 62.5, 36.8, 23.5; ESI-MS calcd for
C.sub.11H.sub.19N.sub.3O.sub.12P.sub.2 [M-H].sup.-: 446.0366 found
446.0348.
##STR00059##
[0460] Uridine 5'-diphosphobutanolamine, ammonium salt (4b)
[0461] Following general procedure IV, UMP.Et.sub.3NH.sup.+ (102
mg, 0.238 mmol) was activated with dimethyl aniline (120 .mu.L,
0.952 mmol), triethylamine (50 .mu.L, 0.476 mmol) and
trifluoroacetic anhydride (200 .mu.L, 1.428 mmol). The activated
UMP was then reacted with 1-methylimidazole (100 mL, 1.26 mmol),
triethylamine (130 .mu.L, 1.19 mmol) and phosphate 3b (55 mg, 0.15
mmol). The product was purified by silica gel chromatography (5:4:1
CHCl.sub.3/MeOH/1 M ammonium acetate) to yield the ammonium salt of
the diphosphate as an off white solid. The diphosphate was stirred
with 3 M ammonium hydroxide (10 mL) for 2 h under N.sub.2, the
solvent was removed in vacuo and the product was lyophilized to
yield the ammonium salt of uridine 5'-diphosphobutanolamine 4b (46
mg, 58%) as an off-white solid. .sup.1H (300 MHz, D.sub.2O):
.delta. 7.96 (d, 1H, J=8.1 Hz), 6.01 (d, 1H, J=4.7 Hz), 6.00 (d,
1H, J=8.4 Hz), 4.39 (br s, 2H), 4.31 (br s, 1H), 4.31-4.22 (m, 2H),
4.04-4.00 (m, 2H), 3.68 (br s, 2H), 1.96 (s, 4H), 1.78 (m, 4H);
.sup.13C (75 mHz, D.sub.2O): .delta. 165.1, 150.6, 140.1, 101.2,
87.1, 81.7, 72.2, 68.2, 63.5, 62.1, 37.8, 25.2 (d, J=6.6 Hz), 22.2;
ESI-MS calcd for C.sub.13H.sub.23N.sub.3O.sub.12P.sub.2
[M+Na].sup.+: 498.0655 found 498.0648.
##STR00060##
[0462] Uridine 5'-diphosphohexanolamine, ammonium salt (4c):
Following general procedure IV, UMP.Et.sub.3NH.sup.+ (102 mg, 0.238
mmol) was activated with dimethyl aniline (120 .mu.L, 0.952 mmol),
triethylamine (50 .mu.L, 0.476 mmol) and trifluoroacetic anhydride
(200 .mu.L, 1.428 mmol). The activated UMP was then reacted with
1-methylimidazole (100 mL, 1.26 mmol), triethylamine (130 .mu.L,
1.19 mmol) and phosphate 3c (6 mg, 0.15 mmol). The product was
purified by silica gel chromatography (5:4:1
CHCl.sub.3/MeOH/H.sub.2O) to yield the triethylammonium salt of the
diphosphate as an off-white solid. The diphosphate was stirred with
3 M ammonium hydroxide (10 mL) for 2 h under N.sub.2, the solvent
was removed in vacuo and the product was lyophilized to yield the
ammonium salt of uridine 5'-diphosphohexanolamine 4c (56 mg, 71%)
as an off-white solid (Barker, R.; Shaper, J. H.; Hill, R. L.;
Olsen, K. W. J. Biol. Chem. 1972, 247, 7135.)
##STR00061##
[0463] Uridine 5'-diphosphooctanolamine, ammonium salt (4d):
Following general procedure IV, UMP.Et.sub.3NH.sup.+ (102 mg, 0.238
mmol) was activated with dimethyl aniline (120 .mu.L, 0.952 mmol),
triethylamine (50 .mu.L, 0.476 mmol) and trifluoroacetic anhydride
(200 .mu.L, 1.428 mmol). The activated UMP was then reacted with
1-methylimidazole (100 mL, 1.26 mmol), triethylamine (130 .mu.L,
1.19 mmol) and phosphate 3d (65 mg, 0.15 mmol). The product was
purified by silica gel chromatography (5:4:1
CHCl.sub.3/MeOH/H.sub.2O) to yield the triethylammonium salt of the
diphosphate as an off-white solid. The diphosphate was stirred with
3 M ammonium hydroxide (10 mL) for 2 h under N.sub.2, the solvent
was removed in vacuo and the product was lyophilized to yield the
ammonium salt of uridine 5'-diphosphooctanolamine 4d (78 mg, 89%)
as an off-white solid. .sup.1H (300 MHz, D.sub.2O): .delta. 8.67
(s, 1H), 7.98 (d, 1H, J=7.9 Hz), 7.44 (s, 1H), 6.0-5.97 (m, 2H),
4.37-4.29 (m, 2H), 4.37 (d, 1H, J=3.9 Hz), 4.29-4.18 (m, 3H),
3.98-3.92 (m, 2H), 2.99 (t, 2H, J=8.4 Hz), 2.75 (s, 4H), 1.68-1.61
(m, 4H), 1.38-1.30 (m, 8H); .sup.13C (75 mHz, D.sub.2O): .delta.
168.8, 154.4, 144.3, 137.5, 125.5, 122.0, 105.3, 91.0, 85.8 (d,
J=5.4 Hz), 76.4, 72.1, 69.6 (d, J=3 Hz), 67.5, 42.1, 41.3, 38.0,
32.2 (d, J=4.0 Hz), 30.5, 29.2, 27.9, 27.2; ESI-MS calcd for
C.sub.17H.sub.31N.sub.3O.sub.12P.sub.2 [M+Na]: 554.1281 found
554.1257.
##STR00062##
[0464] Uridine 5'-diphosphodecanolamine, ammonium salt (4e):
Following general procedure IV, UMP.Et.sub.3NH.sup.+ (102 mg, 0.238
mmol) was activated with dimethyl aniline (120 .mu.L, 0.952 mmol),
triethylamine (50 .mu.L, 0.476 mmol) and trifluoroacetic anhydride
(200 .mu.L, 1.428 mmol). The activated UMP was then reacted with
1-methylimidazole (100 mL, 1.26 mmol), triethylamine (130 .mu.L,
1.19 mmol) and phosphate 3e (70 mg, 0.15 mmol). The product was
purified by silica gel chromatography (CHCl.sub.3/MeOH/H.sub.2O
12/6/1) to yield the triethylammonium salt of the diphosphate as an
off-white solid. The diphosphate was stirred with 3 M ammonium
hydroxide (10 mL) for 2 h under N.sub.2, the solvent was removed in
vacuo and the product was lyophilized to yield the ammonium salt of
uridine 5'-diphosphodecanolamine 4e (66 mg, 76%) as an off-white
solid. .sup.1H (300 MHz, D.sub.2O): .delta. 8.77 (br s, 1H, 8.11,
(d, 1H, J=8.0 Hz), 7.57 (br s, 2H), 6.09-6.04 (m, 2H), 4.47-4.42
(m, 2H), 4.34 (br s, 3H), 4.04 (s, 4H), 4.02 (m, 2H), 3.11 (t, 2H,
J=7.6 Hz), 1.78 (t, 2H, J=6.2 Hz), 1.69 (t, 2H, J=6.7 Hz); .sup.13C
(75 mHz, 10:1 D.sub.2O/d.sub.6-acetone): .delta. 165.9, 151.8,
142.1, 102.9, 88.8, 83.6 (d, J=8.5 Hz), 74.3, 70.1, 66.9 (d, J=5.3
Hz), 65.3, 39.9, 35.7, 29.2, 29.1, 28.8, 27.2, 26.2, 25.6; ESI-MS
calcd for C.sub.19H.sub.35N.sub.3O.sub.12P.sub.2 [M+H].sup.+:
560.1774 found 560.1777.
##STR00063##
Uridine 5'-diphospho-3,6-dioxa-octanolamine, ammonium salt (4f)
[0465] Following general procedure IV, UMP.Et.sub.3NH.sup.+ (150
mg, 0.35 mmol) was activated with dimethyl aniline (177 .mu.L, 1.40
mmol), triethylamine (98 .mu.L, 0.70 mmol) and trifluoroacetic
anhydride (297 .mu.L, 2.10 mmol). The activated UMP was then
reacted with 1-methylimidazole (171 mL, 1.86 mmol), triethylamine
(244 .mu.L, 1.75 mmol) and phosphate 3f (94 mg, 0.22 mmol). The
product was purified by silica gel chromatography
(CHCl.sub.3/MeOH/H.sub.2O 12/6/1) to yield the triethylammonium
salt of the diphosphate as an off-white solid. The diphosphate was
stirred with 3 M ammonium hydroxide (10 mL) for 2 h under N.sub.2,
the solvent was removed in vacuo and the product was lyophilized to
yield the ammonium salt of uridine
5'-diphospho-3,6-dioxa-octanolamine 4f (98 mg, 78%) as an off-white
solid. .sup.1H (300 MHz, D.sub.2O): .delta. 7.96 (d, 1H, J=8.2 Hz),
6.01 (d, 1H, J=4.2 Hz), 5.99 (d, 1H, J=7.8 Hz), 4.39 (m, 2H), 4.30
(m, 1H), 4.15-4.09 (m, 2H), 3.80 (t, 2H, J=6.0 Hz), 3.56 (br s,
8H), 3.25 (t, 2H, J=5.1 Hz); .sup.13C (75 mHz, D.sub.2O): .delta.
166.3, 151.9, 141.8, 102.8, 88.7, 83.3 (d, J=8.2 Hz), 73.9, 70.2,
70.2, 69.8, 69.6, 66.6, 65.3, 65.1 (d, J=7.7 Hz), 39.3; ESI-MS
calcd for C.sub.15H.sub.28N.sub.3O.sub.14P.sub.2 [M+H].sup.+:
536.1047 found 536.1060.
##STR00064##
Uridine 5'-diphospho-3,6,9-trioxa-undecanolamine, ammonium salt
(4g)
[0466] Following general procedure IV, UMP.Et.sub.3NH.sup.+ (147
mg, 0.344 mmol) was activated with dimethyl aniline (174 .mu.L,
1.376 mmol), triethylamine (96 .mu.L, 0.688 mmol) and
trifluoroacetic anhydride (292 .mu.L, 2.064 mmol). The activated
UMP was then reacted with 1-methylimidazole (168 mL, 1.823 mmol),
triethylamine (240 .mu.L, 1.770 mmol) and phosphate 3g (102 mg,
0.220 mmol). The product was purified by silica gel chromatography
(CHCl.sub.3/MeOH/H.sub.2O 12/6/1) to yield the triethylammonium
salt of the diphosphate as an off-white solid. The diphosphate was
stirred with 3 M ammonium hydroxide (10 mL) for 2 h under N.sub.2,
the solvent was removed in vacuo and the product was lyophilized to
yield the ammonium salt of uridine
5'-diphospho-3,6,9-trioxa-undecanolamine 4 g (95 mg, 70%) as an
off-white solid. .sup.1H (300 MHz, D.sub.2O): .delta. 7.93 (d, 1H,
J=8.1 Hz), 5.97 (d, 1H, J=4.2 Hz), 5.95 (d, 1H, J=8.1 Hz),
4.39-4.33 (m, 2H), 4.28 (br s, 1H), 4.10 (br s, 2H), 3.78 (t, 2H,
J=5.4 Hz), 3.72 (br s, 12H), 3.23 (t, 2H, J=5.3 Hz); .sup.13C (75
mHz, D.sub.2O): .delta. 166.2, 151.8, 141.8, 102.7, 88.8, 73.8,
70.2, 69.7, 69.6, 69.5, 66.5, 65.3 (d, J=7.5 Hz), 39.3; ESI-MS
calcd for C.sub.17H.sub.31N.sub.3O.sub.15P.sub.2 [M-H].sup.-:
578.1152 found 578.1130.
[0467] General Procedure V: Conjugation of uridine 5'-diphospho
(UDP)-alcoholamine 4 to fluorescein-5-isothiocyanate (FITC).
[0468] UDP-alcoholamine 4 (1.0 eq) was combined with FITC (1.5 eq)
in 2:1 DMF/0.1 M NaHCO.sub.3. The reaction mixture was stirred for
2 h and concentrated. The product was purified by silica gel
chromatography (12:10:1 CHCl.sub.3/MeOH/H.sub.2O) to yield the
sodium salt of UDP-fluorescein conjugate 5 as a yellow solid.
##STR00065##
UDP-ethanolamine-fluorescein conjugate (5a)
[0469] Following general procedure V, UDP-ethanolamine 4a (5 mg,
9.6 .mu.mol) was combined with FITC (5.6 mg, 14.4 .mu.g) in 2:1
DMF/0.1M NaHCO.sub.3 (150 .mu.L) to yield the sodium salt of
UDP-ethanolamine-fluorescein conjugate 5a (5.3 mg, 64%) as a yellow
solid. .sup.1H (300 MHz, 5:1 D.sub.2O:d.sub.7-DMF): .delta. 7.94
(m, 1H), 7.73 (d, 1H, J=6.4 Hz), 7.24-7.20 (m, 3H), 6.75-6.71 (m,
5H), 5.92-5.90 (m, 2H), 4.36-4.31 (m, 2H), 4.27-4.22 (m, 5H), 3.20
(br s, 2H); .sup.13C (75 MHz, 5:1 D.sub.2O:d.sub.7-DMF): .delta.
171.1, 164.4, 155.7, 150.1, 140.5, 140.2, 130.1, 118.7, 112.9,
101.8, 101.2, 87.3, 81.8, 72.5, 68.3, 63.7, 63.2, 53.1, 41.2;
ESI-MS calcd for C.sub.32H.sub.30N.sub.4O.sub.17P.sub.2S
[M-2H].sup.-2: 417.0323 found 417.0313.
##STR00066##
UDP-butanolamine-fluorescein conjugate (5b)
[0470] Following general procedure V, UDP-butanolamine 4b (10 mg,
20 .mu.mol) was combined with FITC (12 mg, 30 .mu.mol) in 2:1
DMF/0.1 M NaHCO.sub.3 (200 .mu.L) to yield the sodium salt of
UDP-ethanolamine-fluorescein conjugate 5b (11 mg, 62%) as a yellow
solid. .sup.1H (300 MHz, 5:1 D.sub.2O:d.sub.7-DMF): .delta. 7.96
(br s, 1H), 7.88 (d, 1H, J=8.6 Hz), 7.65 (m, 1H), 7.10 (m, 1H),
6.97 (d, 2H, J=9.2 Hz), 6.63 (d, 2H, J=1.8 Hz), 6.60 (d, 2H, J=9.8
Hz), 5.87-5.82 (m, 2H), 4.25 (br s, 2H), 4.13 (br s, 3H), 3.93 (br
s, 2H), 3.52 (br s, 2H), 1.63 (br s, 4H); .sup.13C (75 MHz, 5:1
D.sub.2O:d.sub.7-DMF): .delta. 182.8, 174.0, 168.0, 158.5, 154.0,
144.3, 133.5, 121.0, 115.7, 105.6, 105.1, 91.0, 86.0, 76.4, 72.8,
68.7, 67.5, 56.9, 45.1, 30.2, 27.5; ESI-MS calcd for
C.sub.34H.sub.34N.sub.4O.sub.17P.sub.2S [M-2H].sup.-2: 431.0479
found 459.0502.
##STR00067##
UDP-hexanolamine-fluorescein conjugate (5c)
[0471] Following general procedure V, UDP-hexanolamine 4c (20 mg,
38 .mu.mol) was combined with FITC (22 mg, 57 .mu.mol) in 2:1
DMF/0.1 M NaHCO.sub.3 (200 .mu.L) to yield the sodium salt of
UDP-hexanolamine-fluorescein conjugate 5c (13.7 mg, 40%) as a
yellow solid (Soltero-Higgin, M.; Carlson, E. E.; Phillips, J. H.;
Kiessling, L. L. J. Am. Chem. Soc. 2004, 126, 10532-10533.)
##STR00068##
UDP-octanolamine-fluorescein conjugate (5d)
[0472] Following general procedure V, UDP-octanolamine 4d (3.0 mg,
5.4 .mu.mol) was combined with FITC (3.2 mg, 8.1 .mu.mol) in 2:1
DMF/0.1 M NaHCO.sub.3 (200 .mu.L) to yield the sodium salt of
UDP-octanolamine-fluorescein conjugate 5d (4.7 mg, 90%) as a yellow
solid. .sup.1H (300 MHz, 5:1 D.sub.2O:d.sub.7-DMF): .delta.
8.14-8.10 (m, 1H), 7.88 (d, 1H, J=7.9 Hz), 7.34 (d, 1H, J=7.9 Hz),
7.23 (d, 2H, J=9.8 Hz), 6.79 (m, 4H), 6.10-6.06 (m, 2H), 4.46-4.43
(m, 2H), 4.33 (br s, 3H), 4.04 (m, 2H), 3.70 (br s, 2H), 1.73-1.68
(m, 4H), 1.47 (br s, 8H); .sup.13C (75 MHz, 5:1
D.sub.2O:d.sub.7-DMF): .delta. 156.1, 150.6, 140.9, 130.3, 119.5,
112.1, 102.2, 101.7, 87.4, 82.7, 73.0, 69.1, 65.5, 64.1, 53.4,
41.6, 28.0, 27.6, 25.6, 24.4; ESI-MS calcd for
C.sub.38H.sub.42N.sub.4O.sub.17P.sub.2S [M-2H].sup.-2: 459.0792
found 459.0776.
##STR00069##
UDP-decanolamine-fluorescein conjugate (5e)
[0473] Following general procedure V, UDP-decanolamine 4e (5 mg,
8.4 .mu.mol) was combined with FITC (5 mg, 12.6 .mu.mol) in 2/1
DMF/0.1 M NaHCO.sub.3 (200 .mu.L) to yield the sodium salt of
UDP-decanolamine-fluorescein conjugate 5e (6.6 mg, 81%) as a yellow
solid. .sup.1H (300 MHz, 5:1 D.sub.2O:d.sub.7-DMF): .delta. 8.13
(br s, 1H), 7.90 (d, 1H, J=7.7 Hz), 7.63 (br s, 1H), 7.00 (br s,
1H), 6.73 (m, 2H), 6.66 (s, 2H), 6.54 (m, 2H), 5.88-5.84 (m, 2H),
4.25 (br s, 2H), 4.15 (br s, 2H), 3.83 (br s, 2H), 3.39 (br s, 2H),
1.44 (br s, 2H), 1.13 (br s, 4H), 1.03 (s, 8H); .sup.13C (75 MHz,
5:1 D.sub.2O:d.sub.7-DMF): .delta. 179.2, 169.6, 164.3, 153.0,
150.4, 140.6, 128.9, 121.8, 116.9, 114.5, 110.6, 101.8, 101.5,
87.4, 82.4, 72.9, 68.8, 65.5, 64.0, 53.3, 43.3, 41.5, 28.2, 28.1,
27.9, 27.4, 25.5, 24.4; ESI-MS calcd for
C.sub.40H.sub.46N.sub.4O.sub.17P.sub.2S [M-2H].sup.-2: 473.0949
found 473.0965.
##STR00070##
UDP-3,6-dioxa-octanolamine-fluorescein conjugate (5f)
[0474] Following general procedure V, UDP-3,6-dioxa-octanolamine 4f
(5 mg, 8.8 .mu.mol) was combined with FITC (5.1 mg, 13.2 .mu.mol)
in 2:1 DMF/0.1 M NaHCO.sub.3 (200 .mu.L) to yield the sodium salt
of UDP-3,6-dioxa-octanolamine-fluorescein conjugate 5f (5.6 mg,
66%) as a yellow solid. .sup.1H (300 MHz, d.sub.7-DMF): .delta.
8.25 (br s, 1H), 7.81 (br s, 1H), 7.26 (d, 1H, J=8.7 Hz), 6.91-6.66
(m, 9H), 5.90 (m, 2H), 4.34-4.26 (m, 2H), 4.18 (br s, 3H), 4.10 (br
s, 2H), 3.80-3.66 (m, 10H); .sup.13C (75 MHz, d.sub.7-DMF): 181.8,
168.7, 153.2, 151.4, 143.0, 141.0, 130.4, 127.8, 119.2, 116.9,
114.6, 102.6, 102.5, 88.7, 84.8, 83.6, 74.0, 70.6, 70.3, 69.1,
66.7, 53.9, 44.0, 42.1, 17.9, 16.6, 12.1 .delta. ESI-MS calcd for
C.sub.36H.sub.38N.sub.4O.sub.19P.sub.2S [M-2H].sup.-2: 461.0574
found.
##STR00071##
UDP-3,6,9-trioxa-undecanolamine-fluorescein conjugate (5g)
[0475] Following general procedure V,
UDP-3,6,9-trioxa-undecanolamine 4 g (2 mg, 3.3 .mu.mol) was
combined with FITC (2.0 mg, 5.0 .mu.mol) in 2:1 DMF/0.1 M
NaHCO.sub.3 (200 .mu.L) to yield the sodium salt of
UDP-3,6,9-trioxa-undecanolamine-fluorescein conjugate 5 g (2.5 mg,
76%) as a yellow solid. .sup.1H (300 MHz, 5:1
D.sub.2O:d.sub.7-DMF): .delta. 7.91 (d, 1H, J=8.3 Hz), 7.86 (d, 1H,
J=2.0 Hz), 7.65 (dd, 1H, 8.3, 2.1 Hz), 7.32 (dd, 2H, J=9.3, 4.0
Hz), 7.27 (d, 1H, J=8.2 Hz), 6.78 (dt, 2H, J=9.1, 2.2 Hz), 6.72 (s,
2H), 5.95-5.91 (m, 2H), 4.38-4.31 (m, 2H), 4.24 (br s, 3H), 4.15
(br s, 2H), 3.86-3.79 (m, 14H); .sup.13C (75 MHz, 5:1
D.sub.2O:d.sub.7-DMF): .delta. 167.4, 160.4, 160.0, 154.1, 144.1,
143.0, 141.6, 134.3, 133.4, 123.6, 120.4, 117.4, 113.2, 105.8,
105.1, 91.1, 85.6 (d, J=4.8 Hz), 76.4, 72.6, 72.5, 72.3, 72.1,
71.4, 68.8, 67.8, 67.5; ESI-MS calcd for
C.sub.38H.sub.42N.sub.4O.sub.20P.sub.2S [M-2H].sup.-2: 483.0705
found 483.0720.
##STR00072##
Octanolamine-fluorescein conjugate (6)
[0476] 8-amino-1-octanol (11.2 mg, 0.077 mmol) was combined with
FITC (10 mg, 0.026 mmol) in MeOH (0.3 mL) and stirred at
room-temperature for 1 h. The product was purified by silica gel
chromatography (4:1 CH.sub.2Cl.sub.2/MeOH containing 1% H.sub.2O)
to yield control compound 6 (13.6 mg, 98%) as an orange solid.
.sup.1H (300 MHz, CD.sub.3OD): .delta. 8.12 (d, 1H, J=1.7 Hz), 7.74
(dd, 1H, J=8.3, 1.6 Hz), 7.15 (d, 1H, J=8.2 Hz), 6.77 (d, 2H, J=8.8
Hz), 6.67 (d, 2H, J=2.3 Hz), 6.56 (dd, 2H, J=8.8, 2.2 Hz),
3.60-3.52 (m, 4H), 1.68-1.63 (m, 4H), 1.56-1.51 (m, 2H), 1.38 (m,
8H); .sup.13C (75 mHz, CD.sub.3OD): .delta. 171.8, 155.2, 142.6,
131.0, 115.3, 112.6, 103.8, 63.2, 63.1, 33.8, 30.7, 30.6, 30.1,
28.2, 27.1); ESI-MS calcd for C.sub.29H.sub.30N.sub.2O.sub.6S
[M-H].sup.-: 533.1825 found 533.1846.
##STR00073##
UDP-octanolamine-naphthyl conjugate (7)
[0477] UDP-octanolamine 4d (25 mg, 50 .mu.mol) was combined with
2-naphthyl isothiocyanate (20 mg, 100 .mu.mol) in 3:1 DMF/0.1 M
NaHCO.sub.3 (200 .mu.L). The reaction was stirred 1 h and
concentrated. The product was purified by silica gel chromatography
(12:10:1 CHCl.sub.3/MeOH/H.sub.2O) to yield the sodium salt of
UDP-octanolamine-naphthyl conjugate 7 (30.9 mg, 83%) as an
off-white solid: .sup.1H (300 MHz, d.sub.7-DMF): .delta. 8.05 (d,
1H, J=8.4 Hz), 8.00-7.91 (m, 2H), 7.59-7.46 (m, 5H), 5.98-5.93 (m,
2H), 4.36-4.33 (m, 2H), 4.19 (br s, 3H), 3.91 (br s, 2H), 3.51 (t,
2H, J=6.6 Hz), 1.54 (br s, 2H), 1.48 (br s, 2H), 1.24-1.15 (m, 8H);
.sup.13C (75 mHz, d.sub.7-DMF): .delta. 164.4, 160.4, 150.8, 141.3,
133.9, 128.0, 127.5, 126.5, 126.3, 125.5, 125.1, 112.8, 117.6,
115.1, 101.9, 87.4, 83.1, 73.3, 69.4, 65.4, 64.3, 44.1, 28.4, 28.3,
28.1, 25.8, 24.7; ESI-MS calcd for
C.sub.28H.sub.38N.sub.4O.sub.12P.sub.2S [M-2H].sup.-2: 357.0763
found 357.0776.
##STR00074##
[0478] 2-Bromo, 4'-(8-azido-octyl)acetophenone (9), see Scheme
9
[0479] Aluminum chloride (3.25 g, 24 mmol) in CH.sub.2Cl.sub.2 (25
mL) was cooled to 0.degree. C. To the pre-cooled solution,
8-phenyl-1-octanol (0.50 g, 2.4 mmol) in CH.sub.2Cl.sub.2 (2 mL)
was added dropwise. Upon dropwise addition of acetyl chloride
(0.343 mL, 4.8 mmol), the solution turned yellow. The solution was
stirred for 12 h at room-temperature, after which it was poured
into a mixture of ice and concentrated HCl. The mixture was stirred
for 2 h until all salts were dissolved. The organic layer was
separated, washed with saturated NaHCO.sub.3 and brine, dried and
concentrated. The product was purified by silica gel chromatography
(10%.fwdarw.30% ethyl acetate in hexanes) to yield the acetophenone
(0.352 g, 1.42 mmol) as an off-white solid in 59% yield. .sup.1H
(300 MHz, CDCl.sub.3): .delta. 7.74 (d, 2H, J=8.1 Hz), 7.12 (d, 2H,
J=8.1 Hz), 3.49 (t, 2H, J=6.7 Hz), 2.51 (t, 2H, J=7.5 Hz), 2.43 (s,
3H), 1.49-1.41 (m, 4H), 1.20 (m, 8H); .sup.13C (75 mHz,
CDCl.sub.3): .delta. 197.9, 148.6, 134.7, 128.4, 128.3, 62.4, 35.8,
32.5, 30.9, 29.3, 29.2, 29.0, 26.3, 25.7; EI-MS calcd for
C.sub.16H.sub.24O.sub.2 [M+]: 248.1776 found 248.1777.
[0480] Triethylamine (0.296 mL, 2.12 mmol) was added to a solution
of acetophenone (0.262 g, 1.06 mmol) in CH.sub.2Cl.sub.2 (10 mL).
Mesyl chloride (0.165 mL, 2.12 mmol) was added dropwise, and the
solution was stirred 1 h under N.sub.2. The pink solution was
washed with brine, dried, filtered and concentrated. The crude
product was combined with sodium azide in DMF (8 mL) and stirred
for 12 h at 80.degree. C. A solution of 10% EtOAc in hexanes (100
mL) was added to the reaction, which was washed twice with brine
(50 mL), filtered and evaporated. The product was purified by
silica gel chromatography (5%.fwdarw.10% ethyl acetate in hexanes)
to yield the azide (0.243 g, 0.889 mmol) as an oil in 84% yield.
.sup.1H (300 MHz, CDCl.sub.3): .delta. 7.77 (d, 2H, J=8.1 Hz), 7.15
(d, 2H, J=8.2 Hz), 3.13 (t, 2H, J=6.8 Hz), 2.55 (t, 2H, J=7.6 Hz),
2.46 (s, 3H), 1.55-1.43 (m, 4 h), 1.22 (m, 8H); .sup.13C (75 mHz,
CDCl.sub.3): .delta. 197.6, 148.6, 135.0, 128.6, 128.5, 51.4, 39.9,
31.0, 29.3, 29.1, 29.0, 28.8, 26.7, 26.5; ESI-MS calcd for
C.sub.16H.sub.23N.sub.3O [M+Na]: 296.1739 found 296.1753.
[0481] A solution of CuBr.sub.2 (76.3 mg, 0.342 mmol) in EtOAc (1
mL) was heated to reflux. A solution of acetophenone in CHCl.sub.3
(1 mL) was added dropwise. The solution was stirred at reflux until
all CuBr.sub.2 appeared to be consumed (precipitate turns white).
The solution was filtered, concentrated, and the product purified
by silica gel chromatography (2%.fwdarw.4% EtOAc in hexanes) to
yield 2-bromo, 4'-(8-azido-octyl)acetophenone 9 in 43% yield (32.7
mg, 0.093 mmol). .sup.1H (300 MHz, CDCl.sub.3): .delta. 7.89 (d,
2H, J=8.4 Hz), 7.28 (d, 2H, J=7.3 Hz), 4.43 (s, 2H), 3.25 (t, 2H,
4.8 Hz), 2.65 (m, 2H), 1.59 (m, 4H), 1.33 (m, 8H); .sup.13C (75
mHz, CDCl.sub.3): .delta. 191.2, 150.0, 148.9, 129.1, 128.7, 77.1,
36.2, 31.2, 31.1, 29.5, 29.3, 23.2, 29.0, 26.9; ESI-MS calcd for
C.sub.16H.sub.23N.sub.3O [M+Na].sup.+: 374.0844 found 374.0854.
##STR00075##
3-(4-Chlorophenyl)-2-[4-(8-azido-octyl-phenyl)-thiazol-2-ylamino]-propion-
ic acid (10)
[0482] Thiourea (see Example 1) (26.4 mg, 0.102 mmol) and
.alpha.-bromoketone 9 (32.7 mg, 0.093 mmol) were combined in DMF
(300 .mu.L). The reaction was stirred under nitrogen for 2 h and
concentrated. The product was purified by silica gel chromatography
(3:1 hexanes/EtOAc.fwdarw.2:1 hexanes/EtOAc containing 2% AcOH) to
yield 10 (17 mg, 0.033 mmol) as a white solid in 36% yield. .sup.1H
(300 MHz, CDCl.sub.3): .delta. 7.54 (d, 2H, J=7.7 Hz), 7.17 (m,
6H), 6.45 (s, 1H), 4.24 (m, 1H) 3.35 (dd, 1H, J=14.2, 5.3 Hz), 3.25
(t, 2H, J=6.7 Hz), 3.15 (dd, 1H, J=13.3, 6.2 Hz), 2.62 (t, 2H,
J=7.5 Hz), 1.59 (m, 4H), 1.32 (m, 8H); .sup.13C (75 mHz,
CDCl.sub.3): .delta. 174.3, 169.9, 143.8, 135.7, 132.9, 131.2,
129.0, 128.8, 126.3, 99.4, 61.9, 51.7, 37.7, 35.9, 31.4, 29.5,
29.4, 29.3, 29.0, 26.9: ESI-MS calcd for
C.sub.26H.sub.30ClN.sub.5O.sub.2S [M-H].sup.-: 510.1730 found
510.1741.
##STR00076##
Aminothiazole-fluorescein conjugate (11)
[0483] Azide 10 (12 mg, 23 .mu.mol) was combined with
Pd(OH).sub.2/C (6 mg) in 4:1 MeOH/CHCl.sub.3 (0.5 mL) and stirred
12 h under H.sub.2 (1 atm). The suspension was filtered over celite
and the filtrate was concentrated to yield the amine as a white
solid (11.3 mg, 23 .mu.mol) in quantitative yields. .sup.1H (300
MHz, CD.sub.3OD): .delta. 7.47 (d, 2H, J=7.9 Hz), 7.47 (d, 2H,
J=7.9 Hz), 7.27 (m, 7H), 3.42 (dd, 1H, J=14.3, 3.9 Hz), 3.11 (dd,
1H, J=14.3, 8.7 Hz), 2.86 (t, 2H, J=7.1 Hz), 2.62 (t, 2H, J=7.7
Hz), 1.60 (m, 4H), 1.32 (m, 8H); .sup.13C (75 mHz, CD.sub.3OD):
.delta. 170.5, 170.3, 145.5, 140.2, 134.9, 133.2, 131.0, 129.1,
128.7, 126.4, 125.8, 60.4, 39.7, 39.6, 36.8, 35.4, 31.2, 29.1,
29.0, 27.4, 26.3; ESI-MS calcd for
C.sub.26H.sub.32ClN.sub.3O.sub.2S [M+H].sup.+: 486.1982 found
486.1969.
[0484] The amine (11.3 mg, 23 .mu.mol) was combined with
fluorescein isothiocyanate (10.4 mg, 35 .mu.mol) and DIEA (12
.mu.L, 69 .mu.mol) in DMF (400 .mu.L). The solution was stirred 1.5
h and concentrated. The product was purified using HPLC (gradient
50-70% ACN/H.sub.2O) to yield 11 as an orange solid (16.2 mg, 18.5
.mu.mol) in 80% yield. .sup.1H (300 MHz, CD.sub.3OD): .delta. 8.11
(s, 1H), 7.70 (d, 1H, J=8.1 Hz), 7.51 (d, 2H, J=7.8 Hz), 7.20 (s,
4H), 7.15 (d, 2H, J=8.5 Hz), 7.08 (d, 1H, J=8.2 Hz), 6.71 (d, 2H,
J=8.5 Hz), 6.68 (d, 2H, J=2.1 Hz), 6.54 (dd, 2H, J=8.8, 2.4 Hz),
4.66 (dd, 1H, J=8.4, 5.0 Hz), 3.53 (m, 2H), 3.30 (dd, 1H, J=14.4,
5.0 Hz), 3.05 (dd, 1H, J=14.0, 8.2 Hz), 2.56 (t, 2H, J=7.8 Hz),
1.60-1.57 (m, 4H), 1.31 (br s, 8H); .sup.13C (75 mHz, CD.sub.3OD):
.delta. 182.5, 173.4, 170.4, 170.1, 154.6, 136.5, 133.6, 131.8,
130.5, 129.6, 129.3, 126.9, 126.0, 114.1, 144.0, 103.2, 60.4, 60.3,
37.7, 36.3, 32.1, 30.1, 30.0, 29.8, 29.6, 27.6; LC/MS (ESI) (m/z)
[M+H].sup.+ calcd 875.3, found 875.3.
UGM Binding
[0485] Fluorescence Polarization Binding Assay:
[0486] Serial dilutions of dialyzed UGM (maximum concentration was
typically 30 .mu.M) was incubated with 15 nM of fluorescent
compounds 5a-5f, 6 or 11 in 50 mM sodium phosphate buffer, pH 7.0
at 25.degree. C. Final volumes were 30 .mu.L in 384 well black
microtiter plates (Costar). Fluorescence polarization was analyzed
using a Wallac EnVision plate reader. Data were fit to
y=m1+((m2-m1)*x m3)/(m4 m3+x m3); m2=maximum FP signal, m1=minimum
FP signal, m3=slope, m4=binding constant (KaleidaGraph, Synergy
Software).
[0487] Fluorescence polarization inhibition assay:
[0488] The fluorescence polarization inhibition assay was performed
as previously described (22). Reactions contained 580 nM
UGM.sub.myco or 500 nM UGM.sub.kleb and 15 nM of the fluorescent
probe 5c in 50 mM sodium phosphate buffer, pH 7.0 at 25.degree. C.
Final volumes were 30 .mu.L in 384 well black microtiter plates
(Costar). Serial dilutions of UDP, 4d or 7 were added to the wells.
Fluorescence polarization was analyzed using a Wallac EnVision
plate reader. Data were fit to y=m1+((m2-m1)*x m3)/(m4 m3+x m3);
m2=maximum FP signal, m1=minimum FP signal, m3=slope, m4=apparent
binding constant (KaleidaGraph, Synergy Software). To determine
K.sub.d values, the apparent binding constant was then subjected to
K.sub.app=K.sub.d(1+(I)/K.sub.I), where I=concentration of the
fluorescent probe and K.sub.I=binding affinity of the fluorescent
probe to UGM. For UGM.sub.myco I=15 nM and K.sub.I=160 nM. For
UGM.sub.kleb I=15 nM and K.sub.I=100 nM. Data for exemplary
compounds is provided in Table 2. FIGS. 13A and 13B are graphs
illustrating determination of K.sub.d for compound II for
UGM.sub.myco (FIG. 13A) and UGM.sub.kleb (FIG. 13B).
##STR00077##
TABLE-US-00002 TABLE 2 Exemplary Results of Fluorescence
Polarization UMG Inhibition Assay Compound K.sub.d (UMG.sub.myco)
[.mu.M] K.sub.d (UMG.sub.kleb) [.mu.M] 11 0.295 .+-. 0.066 0.385
.+-. 0.035 7 0.610 .+-. 0.109 0.583 .+-. 0.036 6 >30 >30 5a
>30 >30 5b 2.51 .+-. 0.28 1.94 .+-. 0.15 5c 0.166 .+-. 0.014
0.193 .+-. 0.011 5d 0.054 .+-. 0.006 0.045 .+-. 0.002 5e 0.064 .+-.
0.004 0.070 .+-. 0.002 5f 0.575 .+-. 0.016 0.767 .+-. 0.017 5g 1.07
.+-. 0.10 1.20 .+-. 0.14 4d 32.0 .+-. 3.4 37.7 .+-. 1.5 UDP 15.1
.+-. 1.7 26.0 .+-. 1.7
* * * * *