U.S. patent application number 14/263218 was filed with the patent office on 2015-01-15 for anti-infective compounds.
This patent application is currently assigned to Institut Pasteur Korea. The applicant listed for this patent is INSERM (Institut National de la Sante et de la Recherche Medicale), Institut Pasteur Korea. Invention is credited to Priscille BRODIN, Thierry CHRISTOPHE, Monica CONTRERAS DOMINGUEZ, Fanny Anne EWANN, Denis Philippe Cedric FENISTEIN, Auguste GENOVESIO, Heekyoung JEON, Sunhee KANG, Eun Hye KIM, Jaeseung KIM, Saeyeon LEE, Ji Youn NAM, Zaesung NO, Eunjung PARK, Min Jung SEO.
Application Number | 20150018543 14/263218 |
Document ID | / |
Family ID | 41037902 |
Filed Date | 2015-01-15 |
United States Patent
Application |
20150018543 |
Kind Code |
A1 |
BRODIN; Priscille ; et
al. |
January 15, 2015 |
ANTI-INFECTIVE COMPOUNDS
Abstract
The present invention relates to 4H-pyrido[1,2-a]pyrimidin-4-one
compounds and their use in the treatment of bacterial infections,
in particular Tuberculosis.
Inventors: |
BRODIN; Priscille; (Paris,
FR) ; CHRISTOPHE; Thierry; (Pontarlier, FR) ;
NO; Zaesung; (Yongin-si, KR) ; KIM; Jaeseung;
(Seoul, KR) ; GENOVESIO; Auguste; (Paris, FR)
; FENISTEIN; Denis Philippe Cedric; (Amsterdam, NL)
; JEON; Heekyoung; (Seongnam-si, KR) ; EWANN;
Fanny Anne; (Haramont, FR) ; KANG; Sunhee;
(Yongin-si, KR) ; LEE; Saeyeon; (Yongin-si,
KR) ; SEO; Min Jung; (Seongnam-si, KR) ; PARK;
Eunjung; (Seoul, KR) ; CONTRERAS DOMINGUEZ;
Monica; (Saint-Louis, FR) ; NAM; Ji Youn;
(Cheongju, KR) ; KIM; Eun Hye; (Namyangju-si,
KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Institut Pasteur Korea
INSERM (Institut National de la Sante et de la Recherche
Medicale) |
Seongnam-si
Paris |
|
KR
FR |
|
|
Assignee: |
Institut Pasteur Korea
Seongnam-si
KR
INSERM (Institut National de la Sante et de la Recherche
Medicale)
Paris
FR
|
Family ID: |
41037902 |
Appl. No.: |
14/263218 |
Filed: |
April 28, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12999095 |
Mar 11, 2011 |
8785452 |
|
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PCT/EP2009/004379 |
Jun 17, 2009 |
|
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14263218 |
|
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61132285 |
Jun 17, 2008 |
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Current U.S.
Class: |
540/592 ; 435/32;
544/209; 544/350; 544/83; 546/275.4; 546/277.7; 546/280.4; 546/88;
548/144; 548/195; 548/323.5; 558/391; 564/20 |
Current CPC
Class: |
C07D 498/04 20130101;
C07D 223/24 20130101; A61P 31/04 20180101; C07D 417/12 20130101;
C07C 335/40 20130101; C12Q 1/18 20130101; C07C 255/66 20130101;
C07D 251/66 20130101; A61P 31/06 20180101; C07D 409/12 20130101;
C07D 233/84 20130101; C07D 401/12 20130101; C07D 271/113 20130101;
C07D 471/04 20130101; C07D 251/52 20130101 |
Class at
Publication: |
540/592 ;
548/144; 548/323.5; 546/275.4; 546/88; 564/20; 548/195; 546/280.4;
544/350; 546/277.7; 558/391; 544/209; 544/83; 435/32 |
International
Class: |
C12Q 1/18 20060101
C12Q001/18; C07D 233/84 20060101 C07D233/84; C07D 401/12 20060101
C07D401/12; C07D 471/04 20060101 C07D471/04; C07C 335/40 20060101
C07C335/40; C07D 223/24 20060101 C07D223/24; C07D 409/12 20060101
C07D409/12; C07D 498/04 20060101 C07D498/04; C07C 255/66 20060101
C07C255/66; C07D 251/52 20060101 C07D251/52; C07D 251/66 20060101
C07D251/66; C07D 271/113 20060101 C07D271/113; C07D 417/12 20060101
C07D417/12 |
Claims
1. A screening method comprising the steps of: (a) batch infection
of host cells with fluorescently labeled M. tuberculosis
mycobacteria; (b) removing free unbound mycobacteria; (c) adding
compounds that are to be tested to a multi-well plate; (d) seeding
said host cells infected with fluorescently labeled M. tuberculosis
mycobacteria into said multi-well plate containing said compounds;
(e) incubating said multi-well plate containing host cells infected
with fluorescently labeled M. tuberculosis mycobacteria and said
compounds; (f) fluorescently labeling said host cells; and (g)
analyzing said multi-well plate using automated confocal
microscopy.
2. The method of claim 1, wherein the screening method searches for
compounds that interfere with the multiplication of M. tuberculosis
within said host cells.
3. The method of claim 1, wherein the host cells are
macrophages.
4. The method of claim 3, wherein the macrophages are live
macrophages.
5. The method of claim 1, wherein the automated confocal
fluorescence microscopy measures intracellular mycobacterial
growth.
6. The method of claim 1, wherein step (g) comprises determining
for each compound a total host cell number, a percentage of
infected host cells; and/or a percent inhibition of infection.
7. The method of claim 6, wherein the host cells are
macrophages.
8. The method of claim 6, wherein step (g) further comprises
analyzing the total host cell number to determine if it is high or
low; and wherein (i) a low total cell number is indicative for the
compound toxicity and/or of the unrestricted growth of M.
tuberculosis inside macrophages; and/or (ii) a high (total) cell
number is indicative that the compound is not toxic and prevents
mycobacterial growth.
9. The method of claim 1, further comprising the step of using
control(s) selected from at least one of the following adding a
compound with known anti-tuberculosis activity instead of the
compounds in step (c) as a positive control; and adding DMSO or
antibiotic control(s) instead of the compounds in step (c) as
negative control.
10. The method of claim 9, wherein the compound with known
anti-tuberculosis activity is selected from isoniazid, rifampin,
ethionamide, and moxifloxacin.
11. The method of claim 1, wherein the M. tuberculosis mycobacteria
are labeled with GFP.
12. The method of claim 1, wherein the host cells are labeled with
SYTO 60.
13. The method of claim 1, wherein after step (b) an incubation
with an antibiotic is carried out.
14. The method of claim 13, wherein the antibiotic is amykacin.
15. The method of claim 1, wherein the screening method is used for
the high throughput screening (HTS) of large scale chemical
libraries.
16. A compound that interferes with the multiplication of M.
tuberculosis within host cells identified in the method of claim
1.
17. A compound that interferes with the multiplication of M.
tuberculosis within host cells identified in the method of claim
3.
18. The compound of claim 17, which is represented by formula
##STR00859##
19. A compound that interferes with the multiplication of M.
tuberculosis within host cells identified in the method of claim
9.
20. A compound that interferes with the multiplication of M.
tuberculosis within host cells identified in the method of claim
13.
Description
CROSS REFERENCE TO A RELATED APPLICATION
[0001] This application is a continuation application of Ser. No.
12/999,095, filed Mar. 11, 2011, now U.S. Pat. No. 8,785,452; which
is a National Stage Application of International Application Number
PCT/EP2009/004379, filed Jun. 17, 2009; which claims the benefit of
U.S. Provisional Application Ser. No. 61/132,285, filed Jun. 17,
2008; which are incorporated herein by reference in their
entirety.
FIELD OF INVENTION
[0002] The present invention relates to small molecule compounds
and their use in the treatment of bacterial infections, in
particular Tuberculosis.
BACKGROUND OF THE INVENTION
[0003] Tuberculosis (TB) as a disease continues to result in
millions of deaths each year. Inadequate use of chemotherapy has
led to an increasing number of drug resistant cases. This situation
is likely to worsen with the emergence of extremely resistant
strains to all currently known drugs (Van Rie and Enarson, 2006).
The internationally recommended TB control strategy, also referred
to as directly observed short-course chemotherapy (DOTS), relies on
a combination of five antibacterial agents to be taken for a
protracted period of more than six months
(http://www.who.int/tb/dots/en/). With the use of a mathematical
model, taking into consideration treatment duration and TB
dynamics, benefits of reduced treatment length were predicted to be
substantial and likely to greatly contribute to a reduced global TB
burden (Salomon et al., 2006).
[0004] Current chemotherapy consists of compounds that directly
target Mycobacterium tuberculosis bacillus, either by neutralizing
general information pathways and critical processes such as RNA
polymerization and protein synthesis inhibition or by interfering
with mycobacterial specific cell envelope synthesis. The most
widely used dedicated anti-tubercular drugs isoniazid, ethionamide
and pyrazinamide are pro-drugs that first require activation. As
active forms, they demonstrate inhibitory activity on a wide range
of mycobacterial targets, which have not yet been fully
characterized. As for other chronic infectious diseases like human
immunodeficiency virus, a multi-therapy approach, including drugs
that target a wide range of critical features of M. tuberculosis,
proved to be the most successful strategy to date. It is, thus,
likely that a combination of current drug inhibitors, having
different mechanisms of action against M. tuberculosis, will be the
solution for the control of the disease.
[0005] The most challenging approaches for discovering new anti-TB
drugs rely on screening for active compounds that target critical
features essential for the survival of the bacillus. Although there
is still a lack of understanding of the biological mechanisms
behind tubercle bacillus persistence, i.e. the location and state
of latent bacteria, in humans, M. tuberculosis is thought to reside
in primary granulomas under hypoxic conditions (Lenaerts et al.,
2007) as well as to hide within various types of cells (Houben et
al., 2006; Neyrolles et al., 2006). The bacillus mainly localizes
inside phagocytic cells, such as macrophages and dendritic cells,
and it has clearly been established that the tubercle bacillus
adopts a different phenotype in the host macrophage's phagosome
compared to growth in extracellular conditions (Rohde et al., 2007;
Schnappinger et al., 2003). Upon infection, an inflammatory
response is induced, thereby initiating recruitment of T
lymphocytes that release interleukins and cytokines, which in turn
activate the infected macrophages to enable the destruction of the
pathogen. Upon the appropriate trigger, the host macrophage is,
thus, able to eliminate the invading bacillus. This is further
supported by the fact that of the people that inhale M.
tuberculosis, more than 95% percent do not develop the disease,
suggesting that the human host response is sufficient in most cases
to thwart M. tuberculosis induced pathogenesis. This gives rise to
the hypothesis that small molecular compounds could mimic the
immune cell response signals and induce the host cells to clear the
mycobacteria.
[0006] Accordingly, it was an object of the present invention to
develop a phenotypic cell-based assay suitable for high throughput
screening that allows for the search of compounds that would
prevent M. tuberculosis multiplication inside the host macrophage.
Up to now, this type of investigation of the tubercle bacillus
growth within host cells relied on colony forming units (CFUs)
determination after host cell lysis followed by serial dilutions
and a 3-week incubation period required for bacterial growth on
agar plates. Luciferase-expressing mycobacteria have been shown to
be efficient in reducing the experiment duration, although cell
lysis and luciferin substrate addition steps are still required
(Arain et al., 1996). Also, these types of experiments are not
easily amenable to large scale screening.
[0007] It was another object of the present invention to identify
compounds effective against bacterial infections, in particular
compounds that would prevent M. tuberculosis multiplication inside
the host macrophage.
DESCRIPTION OF THE INVENTION
[0008] In one aspect, the present invention relates to compounds
having the general formula VIII:
##STR00001##
wherein
[0009] m is 0, 1, 2, or 3;
[0010] X.sub.3 is selected from the group comprising CH.sub.2, O, S
and NH;
[0011] X.sub.4 is selected from the group comprising halide, alkyl,
OR.sub.23, SR.sub.24 and NR.sub.25R.sub.26;
[0012] R.sub.20 is selected from the group comprising acyl, alkoxy,
alkyl, alkylamino, alkylcarboxylic acid, arylcarboxylic acid,
alkylcarboxylic alkylester, alkylene, alkylether, alkylhydroxy,
alkylthio, alkynyl, amido, amino, aryl, arylalkoxy, arylamino,
arylthio, carboxylic acid, cyano, cycloalkyl, carboxylic acid,
ester, halo, haloalkoxy, haloalkyl, haloalkylether, heteroaryl,
heteroarylamino, heterocycloalkyl and hydrogen, any of which is
optionally substituted;
[0013] R.sub.21 and R.sub.22 are each independently selected from
the group comprising alkoxy, alkyl, alkylamino, alkylene,
alkylether, alkylthio, alkynyl, amido, amino, aryl, arylether,
arylalkoxy, arylamino, arylthio, carboxy, cyano, cycloalkyl, ester,
halo, haloalkoxy, haloalkyl, heteroaryl, heteroarylamino,
heterocycloalkyl, hydroxyl, hydrogen, nitro, thio, sulfonate,
sulfonyl and sulfonylamino, any of which is optionally
substituted;
[0014] R.sub.23 is selected from the group comprising acyl, alkyl,
alkylamino, alkylene, alkynyl, aryl, arylalkoxy, arylamino,
arylthio, carboxy, cycloalkyl, ester, ether, haloalkyl, heteroaryl,
heteroarylamino, heterocycloalkyl, hydrogen, thio, sulfonate, and
sulfonylamino, any of which is optionally substituted;
[0015] R.sub.24 is selected from the group comprising alkyl,
alkylaryl, alkylene, alkynyl, aryl, cycloalkyl, ester, halo,
haloalkyl, heteroaryl, heterocycloalkyl, and hydrogen, any of which
is optionally substituted; and
[0016] R.sub.25 and R.sub.26 are each independently selected from
the group comprising acyl, alkyl, aminoalkyl, alkylene, alkylthio,
alkynyl, aryl, arylalkoxy, arylamino, arylthio, carboxy,
cycloalkyl, ester, ether, halo, haloalkoxy, haloalkyl,
haloalkylether, heteroaryl, heteroarylamino, heterocycloalkyl and
hydrogen, any of which is optionally substituted.
[0017] In general, the term "optionally substituted" as used herein
is meant to indicate that a group, such as alkyl, alkylen, alkynyl,
aryl, cycloalkyl, heterocycloalkyl, or heteroaryl, may be
unsubstituted or substituted with one or more substituents.
"Substituted" in reference to a group indicates that a hydrogen
atom attached to a member atom within a group is replaced.
[0018] In another aspect, the present invention relates to
compounds having the general formula VIIIa:
##STR00002##
wherein
[0019] X.sub.5 is selected from the group comprising CH.sub.2,
C.dbd.O and C.dbd.S;
[0020] Z.sub.1 and Z.sub.2 are each independently selected from the
group comprising alkoxy, alkyl, alkylamino, alkylene, alkylether,
alkylthio, alkynyl, amido, amino, aryl, arylether, arylalkoxy,
arylamino, arylthio, carboxy, cyano, cycloalkyl, ester, halo,
haloalkoxy, haloalkyl, heteroaryl, heteroarylamino,
heterocycloalkyl, hydroxyl, and hydrogen, or two groups are
connected each other to make five or six membered cyclic,
heterocyclic and heteroaryl rings, any of which is optionally
substituted;
[0021] R.sub.27 and R.sub.28 are each independently selected from
the group comprising alkoxy, alkylamino, alkylene, alkylether,
alkylthio, alkynyl, amido, amino, aryl, arylether, arylalkoxy,
arylamino, arylthio, carboxy, cyano, cycloalkyl, ester, halo,
haloalkoxy, haloalkyl, heteroaryl, heteroarylamino,
heterocycloalkyl, hydroxyl, hydrogen, nitro, thio, sulfonate,
sulfonyl and sulfonylamino, any of which is optionally
substituted;
[0022] R.sub.29 and R.sub.30 are each independently selected from
the group comprising alkoxy, alkyl, alkylamino, alkylene,
alkylether, alkylthio, alkynyl, amido, amino, aryl, arylether,
arylalkoxy, arylamino, arylthio, carboxy, cyano, cycloalkyl, ester,
halo, haloalkoxy, haloalkyl, heteroaryl, heteroarylamino,
heterocycloalkyl, hydroxyl, hydrogen, nitro, thio, sulfonate,
sulfonyl and sulfonylamino, or two groups are connected each other
to make five or six membered cyclic, heterocyclic, aryl, and
heteroaryl rings, any of which is optionally substituted.
[0023] The term "alkyl" as used herein is meant to indicate that a
group, such as substituted or non-substituted C.sub.1-C.sub.10
alkyl group which has the straight or branched chain.
[0024] The term "cycloalkyl" as used herein is meant to indicate
that a group, such as substituted or non-substituted cyclic
compound of C.sub.3-C.sub.8 ring structure.
[0025] The term "heteroaryl" as used herein is meant to indicate
that a group, such as substituted or non-substituted 5- to
9-membered aromatic compounds which have more than one heteroatom
of N, O, and S in the ring structure itself.
[0026] The term "optionally substituted" as used herein is meant to
indicates that a hydrogen atom attached to a member atom within a
group is possibly replaced by group, such as C.sub.1-C.sub.10
alkyl, halogen including fluorine, OH, NO.sub.2, OR.sub.31, CN,
NR.sub.31R.sub.32, COR.sub.31, SOR.sub.32, SO.sub.2R.sub.31,
SO.sub.2NR.sub.31, CR.sub.31.dbd.CR.sub.31R.sub.32,
CR.sub.31.dbd.NR.sub.32, aryl, aryloxy, C.sub.4-C.sub.10 heteroaryl
group, or --NR.sub.31--COR.sub.32, --O--COR.sub.31.
[0027] R.sub.31 and R.sub.32 are each independently selected from
the group comprising hydrogen, alkyl, alkyloxy, alkylamino,
alkylcarbonyl, alkylcarbonylamino, alkylcarbonyloxy,
alkylaminocarbonyl, alkyloxycarbonyl, cycloalkyl, cycloalkyloxy,
cycloalkylamino, cycloalkylcarbonyl, cycloalkylcarbonylamino,
cycloalkylcarbonyloxy, cycloalkylaminocarbonyl,
cycloalkyloxycarbonyl, heteroaryl, heteroaryloxy, heteroaryl amino,
heteroaryl carbonyl, heteroaryl carbonylamino, heteroaryl
carbonyloxy, heteroaryl aminocarbonyl, heteroaryl oxycarbonyl,
heteroaryl alkyl, heteroaryl alkyloxy, heteroaryl alkylamino,
heteroaryl alkylcarbonyl, heteroaryl alkylcarbonylamino, heteroaryl
alkylcarbonyloxy, heteroaryl alkylaminocarbonyl, heteroaryl
alkyloxycarbonyl, phenyl, phenyloxy, phenylamino, phenylcarbonyl,
phenylcarbonylamino, phenylcarbonyloxy, phenylaminocarbonyl, and
phenyloxycarbonyl, any of which is optionally substituted.
[0028] In another aspect, the present invention relates to
compounds having one of the formulas 125-301 as shown in Example 7,
preferably 132-135, 137, 139-140, 147, 151-152, 160, 163, 173, 180,
184-185, 193, 195, 199-201, 204, 206-222, 224, 226, 229, 231-243,
245-278, 280-286 and 290-301 as shown in Table 3. Particularly
preferred compounds are compounds having one of the formulas 133,
232 and 264 as shown in Table 3.
[0029] In one aspect, the present invention relates to compounds
having the general formula II:
##STR00003##
wherein
[0030] R.sub.5 and R.sub.6 are each independently selected from the
group comprising acyl, alkyl, alkylamino, alkylene, alkylthio,
alkynyl, aryl, arylalkoxy, arylamino, arylthio, carboxy,
cycloalkyl, ester, haloalkoxy, haloalkyl, heteroaryl,
heteroarylamino, heterocycloalkyl, hydroxyl, hydrogen, sulfonate
and sulfonyl, any of which is optionally substituted and
[0031] R.sub.7, R.sub.8 and R.sub.9 are each independently selected
from the group comprising alkoxy, alkyl, alkylamino, alkylene,
alkylthio, alkynyl, amido, amino, aryl, arylalkoxy, arylamino,
arylthio, carboxy, cyano, cycloalkyl, ester, halo, haloalkoxy,
haloalkyl, heteroaryl, heteroarylamino, heterocycloalkyl, hydroxyl,
hydrogen, nitro, thio, sulfonate, sulfonyl and sulfonylamino, any
of which is optionally substituted.
[0032] In another aspect, the present invention relates to
compounds with the general formula II, wherein R.sub.5 and R.sub.6
are connected, having the general formula IIa:
##STR00004##
wherein
[0033] n is 0, 1, 2, or 3;
[0034] Y and Z are each independently selected from the group
comprising CH.sub.2, CHOR.sub.10, CHNR.sub.10R.sub.11,
CR.sub.10R.sub.11 and NR.sub.10; and
[0035] R.sub.10 and R.sub.11 are each independently selected from
the group comprising acyl, alkyl, alkylamino, alkylene, alkylthio,
alkynyl, aryl, arylalkoxy, arylamino, arylthio, carboxy,
cycloalkyl, ester, haloalkoxy, haloalkyl, heteroaryl,
heteroarylamino, heterocycloalkyl, hydrogen, sulfonate and
sulfonyl, any of which is optionally substituted.
[0036] In another aspect, the present invention relates to
compounds having one of the formulas with the general
formula/scaffold II as shown in FIGS. 9-1 to 9-41, as well as one
of the formulas 1-123 as shown in Example 6, preferably 1-24,
26-34, 54, 56, 58-61, 63-64, 67, 90-101, 103-105, 107-109, 112,
114-116 and 118-121 as shown in Table 3. Particularly preferred
compounds are compounds having one of the formulas 4 and 24 as
shown in Table 3.
[0037] Preferably, the compounds as defined above have an
inhibitory activity, preferably an inhibitory activity above 65%,
on bacterial growth, preferably on the growth of M tuberculosis,
inside a host cell, preferably a macrophage, at a concentration
between 5-20 .mu.M, preferably less than 5 .mu.M.
[0038] In one aspect, the present invention relates to compounds as
defined above for use in the treatment of bacterial infections.
[0039] In one aspect, the present invention relates to compounds as
defined above for use in the treatment of Tuberculosis.
[0040] In one aspect, the present invention relates to a
pharmaceutical composition comprising a compound as defined
above.
[0041] In one aspect, the present invention relates to a method of
treatment of Tuberculosis, comprising the application of a suitable
amount of a compound as defined above to a person in need
thereof.
[0042] In another aspect, the present invention relates to
compounds having one of the general formulas/scaffolds I, III-VII
and IX-XX as shown in Table 2.
In one aspect, the present invention relates to compounds having
the general formula I:
##STR00005##
wherein
[0043] X.sub.1 and X.sub.2 are each independently selected from the
group comprising CH and NH;
[0044] R.sub.1 and R.sub.2 are each independently selected from the
group comprising alkoxy, alkyl, alkylamino, alkylene, alkylthio,
alkynyl, amido, amino, aryl, arylalkoxy, arylamino, arylthio,
carboxy, cyano, cycloalkyl, ester, halo, haloalkoxy, haloalkyl,
heteroaryl, heteroarylamino, heterocycloalkyl, hydroxyl, hydrogen,
nitro, thio, sulfonate, sulfonyl and sulfonylamino, any of which is
optionally substituted; and
[0045] R.sub.3 and R.sub.4 are each independently selected from the
group comprising alkoxy, alkyl, alkylamino, alkylene, alkylthio,
alkynyl, aryl, arylalkoxy, arylamino, arylthio, cyano, cycloalkyl,
haloalkoxy, haloalkyl, heteroaryl, heteroarylamino,
heterocycloalkyl and hydrogen, any of which is optionally
substituted.
[0046] In one aspect, the present invention relates to compounds
having the general formula III:
##STR00006##
wherein
[0047] R.sub.10 and R.sub.11 are each independently selected from
the group comprising alkoxy, alkyl, alkylamino, alkylene,
alkylthio, alkynyl, amido, amino, aryl, arylalkoxy, arylamino,
arylthio, carboxy, cyano, cycloalkyl, ester, halo, haloalkoxy,
haloalkyl, heteroaryl, heteroarylamino, heterocycloalkyl, hydroxyl,
hydrogen, nitro, thio, sulfonate, sulfonyl and sulfonylamino, any
of which is optionally substituted.
[0048] In another aspect, the present invention relates to
compounds having the general formula IV:
##STR00007##
wherein
[0049] A is an optionally substituted heteroaryl, naphthyl and
phenyl and
[0050] R.sub.12 is selected from the group comprising alkoxy,
alkyl, alkylamino, alkylene, alkylthio, alkynyl, amido, amino,
aryl, arylalkoxy, arylamino, arylthio, carboxy, cyano, cycloalkyl,
ester, halo, haloalkoxy, haloalkyl, heteroaryl, heteroarylamino,
heterocycloalkyl, hydroxyl, hydrogen, nitro, thio, sulfonate,
sulfonyl and sulfonylamino, any of which is optionally
substituted.
[0051] In one aspect, the present invention relates to compounds
having the general formula V:
##STR00008##
wherein
[0052] R.sub.13, R.sub.14 and R.sub.15 are each independently
selected from the group comprising alkoxy, alkyl, alkylamino,
alkylene, alkylthio, alkynyl, amido, amino, aryl, arylalkoxy,
arylamino, arylthio, carboxy, cyano, cycloalkyl, ester, halo,
haloalkoxy, haloalkyl, heteroaryl, heteroarylamino,
heterocycloalkyl, hydroxyl, hydrogen, nitro, thio, sulfonate,
sulfonyl and sulfonylamino, any of which is optionally
substituted.
[0053] In another aspect, the present invention relates to
compounds having the general formula VI:
##STR00009##
wherein
[0054] R.sub.16 is selected from the group comprising alkoxy,
alkyl, alkylamino, alkylene, alkynyl, amino, aryl, arylalkoxy,
arylamino, arylthio, cycloalkyl, haloalkoxy, haloalkyl, heteroaryl,
heteroarylamino, heterocycloalkyl, hydroxyl and hydrogen, any of
which is optionally substituted and
[0055] R.sub.17 is selected from the group comprising alkoxy,
alkyl, alkylamino, alkylene, alkylthio, alkynyl, amido, amino,
aryl, arylalkoxy, arylamino, arylthio, carboxy, cyano, cycloalkyl,
ester, halo, haloalkoxy, haloalkyl, heteroaryl, heteroarylamino,
heterocycloalkyl, hydroxyl, hydrogen, nitro, thio, sulfonate,
sulfonyl and sulfonylamino, any of which is optionally
substituted.
[0056] In one aspect, the present invention relates to compounds
having the general formula VII:
##STR00010##
wherein
[0057] R.sub.18 and R.sub.19 are each independently selected from
the group comprising alkoxy, alkyl, alkylamino, alkylene,
alkylthio, alkynyl, amido, amino, aryl, arylalkoxy, arylamino,
arylthio, carboxy, cyano, cycloalkyl, ester, halo, haloalkoxy,
haloalkyl, heteroaryl, heteroarylamino, heterocycloalkyl and
hydrogen, any of which is optionally substituted.
[0058] In another aspect, the present invention relates to
compounds having one of the formulas with the general formulas I,
III-VII and IX-XX as shown in FIGS. 9-1 to 9-41.
[0059] In one aspect, the present invention relates to a compound
listed in Table 1.
[0060] In one aspect, the present invention relates to compounds as
defined above for use in the treatment of bacterial infections.
[0061] In one aspect, the present invention relates to compounds as
defined above for use in the treatment of Tuberculosis.
[0062] In one aspect, the present invention relates to a
pharmaceutical composition comprising a compound as defined
above.
[0063] In one aspect, the present invention relates to a method of
treatment of Tuberculosis, comprising the application of a suitable
amount of a compound as defined above to a person in need
thereof.
[0064] In another aspect, the present invention relates to a
screening method comprising the steps of
(a) batch infection of host cells with fluorescently labeled M.
tuberculosis mycobacteria; (b) removing free unbound mycobacteria;
(c) adding compounds that are to be tested to a multi-well plate;
(d) seeding said host cells infected with fluorescently labeled M.
tuberculosis mycobacteria into said multi-well plate containing
said compounds; (e) incubating said multi-well plate containing
host cells infected with fluorescently labeled M. tuberculosis
mycobacteria and said compounds; (f) fluorescently labeling said
host cells; (g) analyzing said multi-well plate using automated
confocal microscopy.
[0065] The screening method according to the present invention
represents a phenotypic cell-based assay enabling the search for
drugs that interfere with the multiplication of M. tuberculosis
within host macrophages. The assay makes use of fluorescently
labeled living macrophages infected with fluorescently labeled
mycobacteria and uses automated confocal fluorescence microscopy to
measure intracellular mycobacterial growth. The assay has been
set-up for the high throughput screening (HTS) of large scale
chemical libraries.
BRIEF DESCRIPTION OF THE FIGURES AND TABLES
[0066] Reference is now made to the figures and tables, wherein
[0067] FIG. 1 shows the monitoring of tubercle bacillus
intracellular growth inside macrophages by automated confocal
microscopy: (a) Representative pictures of Raw264.7 cells infected
with M. tuberculosis H37Rv-GFP at different time points after
infection. (b) Image analysis: 1: Typical 2-color image; 2: Circled
object corresponds to detected cells; 3: Circled object corresponds
to bacterial aggregates; 4: Filled purple cells correspond to
infected cells. (c,d,e) Image-based quantification of the
percentage of infected cells and the mean number of cells from 2
hours to day 7 after infection with H37Rv-GFP at a multiplicity of
infection of 0.5 (gray square), 1 (black circle) and 2 (dark gray
triangle). Non-infected cells (black diamonds) were used as the
negative control;
[0068] FIG. 2 shows the pharmacological validation and MIC (minimal
inhibitory concentration) comparison of the reference drugs in the
in vitro growth fluorescence assay and the phenotypic cell-based
assay: (a) Representative pictures of infected cells in presence of
INH at 1 .mu.g/mL or DMSO control. (b,c,d) Dose-response of INH,
rifampin and ethionamide; black square and line corresponds to
growth inhibition in cell-based assay; gray circle and line
correspond to in vitro growth inhibition; shown is a representative
data set;
[0069] FIG. 3 shows assay automation validation of the phenotypic
cell-based assay: (a) Percent of M. tuberculosis infected cells
relative to 384-plate well-index. Black square, dark gray square,
gray square and open square correspond to INH 1 .mu.g/mL, rifampin
5 .mu.g/mL, PBS and DMSO control respectively. (b,c) Percent of M.
tuberculosis infected cells relative to INH and rifampin
concentration. Experiments were performed on four different plates
on two independent days;
[0070] FIG. 4 shows primary screening results for the phenotypic
cell-based assay and the in vitro growth assay for 26500 compounds:
(a) Percent inhibition based on infection ratio relative to each
compound and distribution. (b) Percent inhibition based on RFU
relative to each compound and distribution. (c) Comparison of
inhibition percentage for the phenotypic cell-based assay and the
in vitro growth assay for each compound;
[0071] FIG. 5 shows serial dilution results from the in vitro
growth fluorescence assay and the phenotypic cell-based assay:
Typical curves for compounds inhibiting (a,b,c) in vitro bacterial
growth (d,e,f) both in vitro and intracellular growth and (g,h,i)
intracellular growth only. (a,d,g) Infection ratio relative to
compound concentration. (b,e,h) Cell number relative to compound
concentration. (c,f,i) Relative fluorescence intensity relative to
compound concentration. Compound concentration is given in M;
[0072] FIG. 6 shows (a) a scheme of assay automation. (b) a
384-plate format description; (c) a 384-plate dose-response curve
description. A to P and a to b correspond to 2-fold serial dilution
of INH and Rifampin respectively with a starting concentration of
20 mg/mL in well A or a; RIF: Rifampin 5 .mu.g/mL, Cpd: compound,
INH100 1 .mu.g/mL, INH50 0.05 .mu.g/mL;
[0073] FIG. 7 shows the anti-tuberculosis effect of compounds 4 and
24 (5 .mu.M) on M. tuberculosis H37Rv-GFP in (a) Raw267.4 (10.sup.4
cells), (b) mouse bone marrow-derived macrophages and (c) human
primary macrophages differentiated with 50 ng/mL rhM-CSF
(1.5*10.sup.4) after 7 days of infection with MOI 2.5:1 (control
INH at 5 .mu.M);
[0074] FIG. 8 illustrates the colony forming units (CFUs) recovered
from macrophages at different time points after infection with M.
tuberculosis H37Rv. Either Raw264.7 cells (a) or murine BMDM (b)
were infected at an MOI of 1:1 and treated with the indicated
amount of pyridopyrimidione compound 232 (20 .mu.M) with DMSO, INH
(10 .mu.M) and RIF (10 .mu.M) as controls;
[0075] FIGS. 9-1 to 9-41 list 121 compounds which demonstrated an
inhibitory activity above 65% at 2 .mu.M without any apparent cell
toxicity at 20 .mu.M and consequently were selected for further
confirmation by ten 3-fold serial dilutions;
[0076] Table 1 lists 340 hits whose inhibitory activity was
confirmed in an intracellular (QIM) assay or an in vitro (QUM)
assay, wherein the abbreviation "QIM" stands for Quantification of
Intracellular Mycobacteria, the abbreviation "QUM" stands for
Quantification of in vitro grown Mycobacteria, and the abbreviation
"CellNb" stands for cell number;
[0077] Table 2 summarizes the independent/general molecular
scaffolds/formulas of the 121 hits listed in FIGS. 9-1 to 9-41;
[0078] Table 3 lists dinitrobenzamide and pyridopyrimidinone
derivatives (general scaffold II and VIII, respectively, see Table
2) with their respective inhibitory activities, wherein the numbers
in bold print refer to the compounds listed in Examples 6 and
7;
[0079] Table 4 shows the cytotoxicity and antibacterial spectrum of
dinitrobenzamide compounds 4 and 24 (see Table 3);
[0080] Table 5 shows the cytotoxicity and antibacterial spectrum of
pyridopyrimidinone compound 133 (see Table 3); and
[0081] Table 6 shows the frequency of spontaneous resistance for
representative dinitrobenzamide and pyridopyrimidinone
compounds.
EXAMPLES
[0082] The invention is now further described by reference to the
following examples which are intended to illustrate, not to limit
the scope of the invention.
Materials and Methods
Genetic Constructs and Mycobacterial Strains
[0083] A recombinant strain of M. tuberculosis H37Rv expressing the
green fluorescent protein (H37Rv-GFP) was obtained by
transformation of an integrative plasmid (Abadie et al., 2005;
Cremer et al., 2002). Within this plasmid, which is derived from
the Ms6 mycobacteriophage, the gfp gene is cloned and
constitutively expressed under the strong mycobacterial promoter
pBlaF. Electrocompetent cells for M. tuberculosis H37Rv-GFP were
prepared from 400 mL of a 15 days old Middlebrook 7H9 culture
(Difco, Sparks Md., USA) supplemented with
albumin-dextrose-catalase (ADC, Difco, Sparks Md., USA), glycerol
and 0.05% Tween 80. Bacilli were harvested by centrifugation at
3000 g for 20 min, washed twice with H.sub.2O at room temperature,
and resuspended in 1-2 mL of 10% glycerol at room temperature after
recentrifugation. 250 .mu.l of bacilli were mixed with green
fluorescent protein encoding plasmid and electroporated using a
Biorad Gene Pulser (Biorad). After electroporation, bacilli were
resuspended in medium and left one day at 37.degree. C.
Transformants were selected on Middlebrook 7H11 medium (Difco,
Sparks Md., USA) supplemented with oleic
acid-albumin-dextrose-catalase (OADC, Difco, Sparks Md., USA) and
50 .mu.g/mL hygromycin (Invitrogen, Carlsbad, Calif. USA). The
selected hygromycin-resistant and green fluorescent colonies
appeared after 3 weeks. A 100 mL culture of the H37Rv-GFP strain
was grown in Middlebrook 7H9-ADC medium supplemented with 0.05%
Tween 80 and 50 .mu.g/mL of hygromycin. Bacteria were harvested,
washed twice and suspended in 50 mM sodium phosphate buffer (pH
7.5). The bacteria were then sonicated and allowed to stand for 1
hour to allow residual aggregates to settle. The bacterial
suspensions were then aliquoted and frozen at -80.degree. C. A
single defrosted aliquot was used to quantify the CFUs (colony
forming units) prior to inoculation and typical stock
concentrations were about 2 to 5.times.10.sup.8 CFU/mL.
Host Cells
[0084] Mouse macrophage cell lines Raw 264.7 (ATCC #TIB-71),
J774A.1 (ATCC #TIB-67) or human monocytes (ATCC #TIB-202)
differentiated with 50 ng/mL PMA (Sigma) were grown in RPMI 1640
(Gibco) with 10% heat-inactivated fetal calf serum (Gibco).
Chemical Compounds
[0085] The small synthetic molecules from the screening libraries
were suspended in pure DMSO (Sigma, D5879-500 mL) at a
concentration of 10 mM (Master plates) in Corning 96 well clear
V-bottom polypropylene plates (Corning, #3956). The compounds were
then reformatted in Greiner 384 well V-shape polypropylene plates
(Greiner, #781280) and diluted to a final concentration of 2 mM in
pure DMSO. The compounds were kept frozen until use. For screening,
compound plates were incubated at room temperature until thawed.
The compounds were directly added into the assay plates from the
DMSO stock using an EVObird liquid handler (Evotec Technologies),
which transfers 250 n1 of compound twice to reach a final dilution
of 1:100. This one-step dilution reduces the risk of compound
precipitation in intermediate plates and allows for a low final
DMSO concentration (1%).
[0086] Positive control antibiotics (Isoniazid (Sigma, 13377-50G)
and Rifampin (Euromedex, 1059-8, 5 g)) as well as negative controls
(DMSO) were added manually in each plate in columns 1-2 and 23-24
(see FIG. 6 b for plate description).
[0087] A total of 26500 compounds were tested. These compounds came
from commercial libraries from Timtec (25000 from the ActiProbe
diverse library, 1000 from the Kinase inhibitors ActiTargK library
and 500 from the Protease inhibitors ActitargP library). The
screened compounds were selected based on high diversity and
drug-like properties (using Lipinski rule-of-five (Lipinski et al.,
2001)). They were first screened at one concentration (primary
screen, concentration=20 .mu.M). The "positive" compounds selected
from the primary screen were then confirmed by testing at 3
concentrations (20, 2 and 0.2 .mu.M) to identify the most active
and/or by ten 3-fold ten dilutions (from 20 .mu.M to 0.5 nM).
Macrophage Invasion Assay Set-Up
[0088] Cells were first seeded in 50 .mu.l at a density of 20,000
cells per well of a 384-well plate (Evotec technologies #781058)
for 16 hours and then infected with bacterial suspensions at a
multiplicity of infection (MOI) varying from 10:1 to 1:1
(bacteria:host cells). After 2 hours, cells were washed three times
with phosphate buffered saline (PBS) and the compounds diluted in
fresh culture medium were added. Cells were incubated at 37.degree.
C., 5% CO.sub.2 for up to seven days.
Macrophage Batch Infection Assay Scale-Up
[0089] Cells (1.5.times.10.sup.8 cells) were infected with
H37Rv-GFP suspension at a MOI of 1:1 in 300 mL for 2 hours at
37.degree. C. with shaking (100 rpm). After two washes by
centrifugation at 1100 rpm (Beckman SX4250, 165 g) for 5 min., the
remaining extracellular bacilli from the infected cells suspension
were killed by a 1 hour amykacin (20 .mu.M, Sigma, A2324-5G)
treatment. After a final centrifugation step, cells were dispensed
with the Wellmate (Matrix) into 384-well Evotec plates (#781058)
preplated with 10 .mu.l of the respective compound diluted in cell
medium. Infected cells were then incubated in the presence of the
compound for 5 days at 37.degree. C., 5% CO.sub.2. After five days,
macrophages were stained with SYTO 60 (Invitrogen, S11342) followed
by plate sealing and image acquisition. During screening, staining
of the live cells was carried out on a set of three plates every
two hours to limit cell death due to prolonged incubation with cell
chemical stain.
Image Acquisition and Data Analysis
[0090] Confocal images were recorded on an automated fluorescent
confocal microscope Opera.TM. (Evotec Technologies) using a
20.times.-water objective (NA 0.70), 488-nm and 635-nm lasers and a
488/635 primary dichroic mirror. Each image was then processed
using dedicated in-house image analysis software (1M). Parameters
determined were the total cell number and the number of infected
cells. Briefly, the algorithm first segments the cells on the red
channel using a sequence of processing steps as described elsewhere
(Fenistein et al., manuscript in press). It is generally based on a
succession of 1) thresholding the histogram of the original image
(3 classes K-means) 2) gaussian filtering the original image with a
standard deviation that is set equal to the cells' average radius,
3) searching for the local maxima of the filtered image that
provides cell centers as seeds for 4) region growing that defines
each cell's own surface and finally 5) removing extremely small
cells as potential artifacts or noise. This step provides the total
number of cells in the red channel. Infected cells are then defined
as those having at least a given number of pixels (usually 3) whose
intensity in the green channel is above a given intensity
threshold. The ratio of infected cells to the total number of cells
is the measure of interest (named infection ratio). For each well,
4 pictures were recorded and for each parameter, the mean of the
four images was used.
[0091] Data obtained from either the intracellular assay image
analysis or from the conventional antibacterial assay (see below)
were then processed using ActivityBase (IDBS) to calculate the
statistical data (% of inhibition, Z score for each compound, Z',
CV etc. for the control plates) and to store the data in an Oracle
database. Additional analyses with regards to both quality control
of the screens and hit identification were performed with various
software packages including Spotfire (Tibco) and Pipelinepilot
(Accelrys).
In Vitro Aerobic Bacterial Growth Assay
[0092] A frozen aliquot of M. tuberculosis H37Rv-GFP was diluted at
1.5.times.10.sup.6 CFU/mL in Middlebrook 7119-ADC medium
supplemented with 0.05% Tween 80. Greiner .mu.clear-black 384-well
plates (Greiner, #781091) were first preplated with 0.5 .mu.l of
compound dispensed by EVOBird (Evotec) in 10 .mu.l of Middlebrook
7H9-ADC medium supplemented with 0.05% Tween 80. 40 .mu.l of the
diluted H37Rv-GFP bacterial suspension was then added on top of the
diluted compound resulting in a final volume of 50 .mu.l containing
1% DMSO. Plates were incubated at 37.degree. C., 5% CO.sub.2 for 10
days after which GFP-fluorescence was recorded using a Victor 3
reader (Perkin-Elmer Life Sciences).
Macrophage Infection Assay and Image Analysis
[0093] Raw 264.7 (ATCC #TIB-71) (1.5*10.sup.8 cells) were infected
with H37Rv-GFP (Abadie et al., 2005, Cremer et al., 2002) in
suspension at a MOI of 1:1 for 2 hours at 37.degree. C. with
shaking. After two washes by centrifugation, the remaining
extracellular bacilli from the infected cell suspension were killed
by a 1 hour Amikacin (20 .mu.M. Sigma, A2324) treatment. After a
final centrifugation step, cells were dispensed into 384-well
Evotec plates (#781058) preplated with compounds and controls.
Infected cells were then incubated for 5 days at 37.degree. C., 5%
CO.sub.2. Murine Bone Marrow-Derived Macrophages (BMDM) were
produced as described previously (Brodin et al., 2006). Briefly,
cells were extracted from the femurs and tibia of 6 weeks old
female mice (C57BL/6, Orientbio) and cultivated in RPMI 1640 media
containing 10% heat-inactivated fetal calf serum (FCS) (both from
Gibco.RTM. at Invitrogen, Carlsbad, Calif.) and 10% L-929 cell
conditioned medium. Peripheral Blood Mononuclear Cells (PBMC) were
isolated from Buffy coat from healthy volunteers. Buffy coat
diluted in PBS supplemented with 1% FCS was treated with 15 ml of
Ficoll-Paque Plus (Amersham Biosciences, Sweden) and centrifuged at
2500.times.g for 20 min. PBMC were obtained by CD14.sup.+ bead
separation (Miltenyi Biotec, Germany), washed 3-times with PBS (1%
FCS) and transferred to 75 cm.sup.2 culture flasks containing RPMI
1640 media, 10% FCS and 50 ng/ml of recombinant-human macrophage
colony stimulating factor (R & D systems, Minneapolis). Six day
old adherent murine BMDM and PBMC derived human macrophages were
infected with H37Rv-GFP (Abadie et al., 2005) in suspension at a
MOI of 1:1 for 2 hours at 37.degree. C. and then extensively washed
and finally incubated with compounds or controls. After several
days, macrophages were stained with SYTO 60 (Invitrogen, S11342)
and image acquisition was performed on an EVOscreen-MarkIII fully
automated platform (PerkinElmer) integrated with an Opera.TM.
(20.times.-water objective, NA 0.70) and located in a BSL-3 safety
laboratory. Mycobacteria-GFP were detected using a 488-nm laser
coupled with a 535/50 nm detection filter and cells labeled with a
635-nm laser coupled with a 690/40 nm detection filter. Four fields
were recorded for each plate well and each image was then processed
using dedicated in-house image analysis software (IM) as described
elsewhere (Fenistein et al., in press).
Mycobacterial Strains and In Vitro Bacterial Growth Assay
[0094] Mycobacterium tuberculosis H37Rv, H37Ra and BCG Pasteur were
used as reference strains. All strains were diluted at
1.5.times.10.sup.6 CFU/mL in Middlebrook 7H9-ADC medium
supplemented with 0.05% Tween 80. 384-well plates (Greiner,
#781091) were first preplated with 0.5 .mu.l of compound dispensed
by EVOBird (Evotec) in 10 .mu.l of Middlebrook 7H9-ADC medium
supplemented with 0.05% Tween 80. Forty microliters of the diluted
H37Rv-GFP bacterial suspension was then added to the diluted
compound resulting in a final volume of 50 .mu.l containing 1%
DMSO. Plates were incubated at 37.degree. C., 5% CO.sub.2 for 10
days. Mycobacterial growth was determined by measuring
GFP-fluorescence using a Victor 3 reader (Perkin-Elmer Life
Sciences) for H37Rv-GFP or with resazurin method. Isoniazid at 0.05
.mu.g/mL and 1 .mu.g/mL (Sigma, 13377), Rifampin at 1 .mu.g/mL
(Euromedex) and DMSO were used as controls.
Cytotoxicity Assay
[0095] In order to address compound toxicity, seven cell lines
originating from different body tissues were cultivated in the
presence of 3-fold dilutions of compounds starting from 100 .mu.M.
After 5 days of culture, cell viability was assessed by the
resazurin test. Briefly, cells were incubated with 10 .mu.g/mL of
resazurin (Sigma-Aldrich St. Louis, Mo.) for 4 h at 37.degree. C.
under 5% CO.sub.2. Resofurin fluorescence (RFU) was measured as
indicated above. Percentage of toxicity on cells was calculated as
follows: Cytotoxicity
(%)=(RFU.sub.DMSO-RFU.sub.Blank)-(RFU.sub.compound-RFU.sub.blank)/(RFU.su-
b.DMSO-RFU.sub.Blank).times.100. Percentage of cytotoxicity was
plotted against compound concentration and the minimal toxic
concentration (MTC.sub.50) was determined by non-linear regression
analysis as the lowest compound concentration where fifty percent
toxicity was observed on the corresponding cell line.
Frequency of Spontaneous Resistance
[0096] The frequency of spontaneous mutations was determined on
7H10 plates containing increasing concentrations of
dintirobenzamide (0.2, 0.8, 1.6 and 3.2 .mu.g/ml) or
pyridopyrimidinone (0.4, 0.8, 1.6 and 3.2 .mu.g/ml) compounds.
10.sup.6, 10.sup.7 and 10.sup.8 CFU containing bacterial
suspensions were spread on compound containing agar plates. After
5-6 weeks at 37.degree. C., colonies were counted and frequency of
mutation was evaluated as the ratio of colonies relative to the
original inoculum. DMSO and INH were used as negative and positive
controls, respectively.
Example 1
Phenotypic Macrophage-Based Assay Set-Up and Automated Image
Quantification
[0097] To set-up the optimal conditions of M. tuberculosis
infection, Raw264.7 macrophages were first infected with
mycobacteria that constitutively express green fluorescent protein
(GFP) at different multiplicities of infection (MOI) followed by
kinetic analysis. Up to 7 days post bacillus infection, the host
live cells were daily labeled with the red chemical fluorescent dye
Syto60, and confocal images of live samples were acquired using an
automated confocal microscope. Typical images are displayed in FIG.
1a. During the first 24 hours, a few discrete weakly fluorescent
bacteria localized within the cells. By day 2, the average number
of cells had increased and mycobacteria had started to spread into
neighboring cells leading to zones of strongly fluorescent
bacteria. The localization of the green signal is always within a
distance of 5 .mu.m to that of the red cell signal and in most
cases actually overlaps with the cell signal. This confirms the
intracellular nature of the mycobacteria growth. By day 4, the cell
number has significantly diminished and the bacteria have formed
large, highly fluorescent aggregates, which cover almost the entire
image from day 5 onwards. As a control, non-infected cells grew up
to confluence at day 2 and remained alive until day 7.
[0098] In order to automatically quantify the intracellular
bacterial load, an in-house image analysis script was developed.
This script enables the automated quantification of the number of
cells and the percentage of infected cells, whereby an infected
cell is a cell containing at least three green pixels with an
intensity above a defined threshold (FIG. 1b). 2 hours after
infection, between 2 and 10% of Raw264.7 cells were found to harbor
a low number of bacilli (FIG. 1c). The percentage of infected
cells, hereafter named infection ratio, continued to increase from
72 hours post-infection reaching up to 70% at seven days post
infection. This increase in infection ratio correlated with an
increase in cell mortality (FIG. 1 d/e).
Example 2
Comparative Minimal Inhibitory Concentration of Known
Anti-Tubercular Drugs
[0099] To validate the assay set-up, the effect of current
anti-tuberculosis drugs on M. tuberculosis intracellular growth was
investigated. 2-fold serial dilutions of isoniazid (INH), rifampin
and ethionamide were performed, followed by testing on macrophages
that had previously been infected with M. tuberculosis H37Rv-GFP.
After 5 days of incubation, macrophages were stained, and images
acquired on an automated confocal microscope as described above. A
larger number of cells and a fewer number of bacteria are clearly
seen on pictures corresponding to samples that were incubated with
INH compared to the DMSO negative control. This shows that INH
prevents both intracellular M. tuberculosis growth and bacillus
mediated cytotoxicity (FIG. 2a). A clear inhibition dose-response
curve was obtained by image-extracted analysis (FIG. 2b). In
parallel, inhibition of M. tuberculosis H37Rv-GFP in vitro growth
by INH was monitored by recording green fluorescence intensity
under the same conditions. In both experiments, the minimal
inhibitory concentration (MIC) for INH was 0.1 .mu.g/mL, which is
in accordance with the MIC reported in the literature for
extracellular M. tuberculosis growth (Andries et al., 2005).
Similar results were obtained with the standard anti-tuberculosis
drugs ethionamide (FIG. 2c) and ethambutol (data not shown),
whereas for rifampin, there was a log-fold decrease in the MIC in
the cell-based assay compared to the in vitro assay (FIG. 2d). The
diminished efficacy of rifampin in the cell-based assay is likely
due to impaired cell penetration and further demonstrates that it
is the intracellular antibacterial activity that is being monitored
in this assay. Thus, adaptation of both the intracellular and the
in vitro M. tuberculosis growth assay for high throughput screening
(HTS) was performed.
Example 3
Assay Scale-Up and Validation
[0100] To simplify the protocol for FITS purposes, macrophages were
infected in batch with M. tuberculosis before being dispensed onto
the compounds. The batch infection was carried out with macrophages
in suspension at 37.degree. C. under mild shaking. Free unbound
mycobacteria were removed by washing three times with PBS and
differential centrifugation, as well as by an additional one-hour
incubation step with amykacin, an antibiotic known to selectively
kill extracellular microbes (FIG. 6a). M. tuberculosis infected
macrophages were then seeded in plates that had been previously
dispensed with the compounds, DMSO or antibiotic controls. The
day-to-day as well as plate-to plate reproducibility was first
tested. To this end, either serial dilutions of INH or rifampin
were dispensed into 8 plates along with the regular DMSO and
positive control (INH at 1 .mu.g/mL (MIC100) and at 0.05 .mu.g/mL
(MIC90) and rifampin at 1 .mu.g/mL) wells that were subsequently
seeded with infected cells. The same experiment was repeated over 2
consecutive days. After incubation for 5 days and macrophage
staining, pictures from each plate were acquired. The mean
infection ratio determined for the DMSO negative controls in each
plate for the 2 days of experiments was between 50% and 70%,
whereas for the INH and rifampin samples, the mean infection ratio
fell to below 20% (FIG. 3a). Despite some variation in the mean
infection ratio between the two experiments, the difference between
the INH-positive and DMSO-negative controls was above five-fold for
both days. P values calculated for each plate using a paired
t-student test also confirmed a significant difference between the
positive and negative controls (p<0.000001, data not shown). In
addition, the inventors performed an experiment to determine if
inhibitors of M. tuberculosis intracellular growth infection
dispensed in any well on the plate could be detected by performing
double-blind controls (spike of INH and rifampin at 3 different
concentrations). Indeed, one hundred percent of the spikes were
identified (data not shown). Taken together, these results prove
that the assay is sensitive enough to be able to identify
inhibitors under HTS conditions. Finally, the robustness of the
assay was checked by monitoring the dose-response of reference
compounds. Almost identical MICs for the antibiotic positive
controls were determined independent of the plate or the day of the
experiment (FIG. 3b/c). Calculated MICs from the image based
quantification of the infection ratio were 0.16+/-0.05 .mu.g/mL and
2.4+/-1.3 .mu.g/mL for INH and rifampin, respectively, and were
confirmed by CFU plating (data not shown). In parallel, the
extracellular growth assay was validated with a similar approach
(data not shown).
Example 4
Primary Screening of a Large Library of Small Synthetic Compounds
Using the Phenotypic Cell-Based Assay
[0101] A 26500 small molecule compound library, that was selected
for its high chemical diversity and drug-like properties according
to the Lipinski rules (Lipinski et al., 2001), was chosen as the
first library to be screened using the validated phenotypic
cell-based assay. The primary screen was carried out with compounds
at 20 .mu.M in singleton. The throughput was set to about 6000
compounds per working day encompassing 25 plates. The screening was
performed with Raw264.7 cells that had been expanded from frozen
stocks for ten days before infection with M. tuberculosis
H37Rv-GFP. To accept the screening results, the MICs obtained from
2 serial dilutions of INH and Rifampin processed at the beginning
and at the end of the screening day should show similar results
compared to the values obtained during the validation (see above).
Each screened plate is then accepted by the quality control
procedure if the window between DMSO and INH (1 .mu.g/ml) is higher
than 3 and the CV calculated for the 320 compounds present in each
plate is lower than 25. Such quality control criteria allow the
identification of hits with an activity higher than 75%.
Subsequently, the percent inhibition for each compound was
determined relative to the corresponding mean infection ratio
between 1 .mu.g/mL INH (100%) and DMSO (0%) in the same 384-well
plate. The percent inhibition distribution is centered around -20%
of inhibition (FIG. 4a). It was decided to select compounds that
have an inhibitory effect greater than 65% which corresponds to a
little less than 1.5% of the total compounds.
[0102] In parallel, the same compounds were only tested for their
inhibitory activity on the M. tuberculosis H37Rv-GFP bacterial
growth. The results from this assay, which are based on classical
fluorescence intensity, showed a higher degree of reproducibility
and the criteria for plate validation was set to a Z' value
(DMSO/INH) greater than 0.35. The throughput for this fluorescence
based assay was approximately 20,000 compounds per day. Compounds
that prevented M. tuberculosis growth in vitro with an inhibitory
effect above 65% were then selected as hits (1.4%) as they belong
to a clear independent population compared to the inactive
population centered to 0% (FIG. 4b).
[0103] The results gathered from the two different screenings were
then compiled and compared (FIG. 4c). Four different populations
could be identified: compounds that are i) active only on
extracellular bacteria, ii) active only on intracellular bacteria,
iii) active in both settings or iv) not active. 657 compounds
(2.5%) belonged to one of the first three categories and, thus,
were selected for further investigation.
[0104] An important parameter that can be measured during image
analysis is the total cell number, also referred to as cell
cytotoxicity. A low cell number can be the result of two
independent phenomena, the compound toxicity and M. tuberculosis
growth mediated cell toxicity. Indeed, at day 5 after infection
with M. tuberculosis, the cell number decreased to less than 100
cells per image compared to more than 500 cells per image for
uninfected cells (FIG. 1e). In contrast, a high cell number is
obtained only when the compound is not toxic and prevents
mycobacterial growth. This turns out to be a second relevant
measurement of a compound's anti-mycobacterial activity. However,
this criterion was not applied for the selection of hits from the
primary screen as a low cell number was found for only a few
compounds and the inventors wanted to avoid failing to select
highly active compounds that would later on prove to be active at
much lower concentrations despite a cell toxicity at 20 .mu.M. An
additional validation criterion of a Z' (DMSO/INH) value of the
total cell number greater than 0.2 was added for the following
screening steps.
Example 5
Confirmation of Screening Results, Dose-Response Analysis and Hit
Classification
[0105] The 657 selected hits were first confirmed at 3 different
concentrations, 20 .mu.M, 2 .mu.M and 0.2 .mu.M. For 340 hits the
activity was confirmed either at 20 .mu.M or 2 .mu.M, on the
intracellular or the in vitro assay (see Table 1). From this latter
list, 121 compounds demonstrated an inhibitory activity above 65%
at 2 .mu.M without any apparent cell toxicity at 20 .mu.M and
consequently were selected for further confirmation by ten 3-fold
serial dilutions (see FIGS. 9-1 to 9-41). All 121 compounds were
confirmed by serial dilution with a MIC ranging between 250 nM and
20 .mu.M. The results shown in FIG. 5 are representative of the
three types of behavior observed: most of the compounds exhibited a
clear dose response curve when activity was measured as infection
ratio (FIG. 5b/e/h). Compounds active on the bacilli level present
a similar activity in the extracellular assay (FIG. 5c/f) even if
the MIC is different from one assay to the other. A few compounds
don't present clear activity on the in vitro bacilli (FIG. 5i) and
may represent drugs acting through a cellular target or on a
bacilli target involved only during the infection process.
Furthermore, toxic compounds can be identified thanks to a dramatic
decrease in the cell number when the compound concentration
increases (FIG. 5d) and activity of non-toxic compounds are
validated by a dose response protective effect on the cell number
(FIG. 5a). Consequently cell number detection represents an
independent secondary assay in the same well as the primary assay.
The serial dilution results from all 121 compounds are presented in
FIGS. 9-1 to 9-41.
[0106] The 121 confirmed hits can be clustered as 20
independent/general scaffolds (Table 2). The number of compounds
for each scaffold varied, ranging from 1 to 69 molecules. The
molecules from the 69-compound scaffold share a common structure
which is similar to INH thereby validating the screening results.
One scaffold contains molecules that were only active in the
intracellular assay and its mechanism of action will be the focus
of further investigation.
Example 6
Derivatization of the Benzamide Compounds
[0107] The benzamide compounds (scaffold II; see Table 2) underwent
derivatization according to the methods outlined below (Schemes
1-7). Formation of the amide can be performed under general
conditions using EDC or DCC coupling reagents with acids instead of
acyl chloride. Resulting derivatives were examined for inhibitory
activity using the assay described above and the results are
summarized in Table 3.
##STR00011##
##STR00012##
General procedure for the synthesis of 2-phenoxyethyl
isoindoline-1,3-dione (A1)
[0108] To a solution of 2-(2-hydroxyethyl)isoindoline-1,3-dione
(1.68 mmol) in methylene chloride (10 mL) was added ADDP (1.68
mmol), triphenylphosphine (1.68 mmol) and phenol (3.18 mmol) and
stirred at room temperature. After stirring overnight, the reaction
mixture was diluted with methylene chloride (30 mL) and washed with
1 M NaOH aqueous solution (50 mL), and brine (50 mL). The organic
layer was dried over anhydrous MgSO.sub.4 and concentrated in
vacuo. The crude product was purified by silica gel flash column
chromatography (4:1 hexanes/ethyl acetate) and recrystallized from
a mixture of hexanes and ethyl acetate to give A1.
General Procedure for the Synthesis of N-(2-Phenoxyethyl)-Benzamide
(A2)
[0109] To a solution of A1 (1.14 mmol) in methanol (10 mL) was
added hydrazine monohydrate (1.42 mmol) and the resulting mixture
was refluxed under a nitrogen atmosphere. After 3 h, the reaction
mixture was allowed to cool to room temperature and concentrated in
vacuo. The residue was precipitated with methylene chloride (10
mL). The resulting precipitate was filtered through Celite and the
filtrate was concentrated in vacuo to afford an amine intermediate.
To a solution of the amine in methylene chloride (10 mL) was added
triethylamine (0.45 mmol) and a benzoylchloride (0.45 mmol) at
0.degree. C. and the resulting mixture was stirred at room
temperature. After 3 h, the reaction mixture was diluted with
methylene chloride (10 mL) and washed with 1 M HCl aqueous solution
(30 mL), saturated Na.sub.2CO.sub.3 aqueous solution (30 mL) and
brine (30 mL). The organic layer was dried over anhydrous
MgSO.sub.4 and concentrated in vacuo. The crude product was
purified by silica gel flash column chromatography (3:1
hexanes/ethyl acetate) and recrystallized from a mixture of hexanes
and ethyl acetate to give A2.
3,5-Dinitro-N-(2-phenoxyethyl)benzamide (1)
##STR00013##
[0111] .sup.1H NMR (400 MHz, Acetone-d.sub.6) .delta. 3.88 (t,
J=4.4 Hz, 2H), 4.21 (t, J=5.2 Hz, 2H), 6.89 (d, J=8.4 Hz, 3H), 7.24
(t, J=8.0 Hz, 2H), 8.78 (brs, 1H), 9.02 (d, J=2.0 Hz, 1H), 9.07 (d,
J=2.0 Hz, 2H); .sup.13C NMR (100 MHz, Acetone-d.sub.6) .delta.
40.1, 66.0, 114.5, 120.8, 127.6, 129.6, 137.8, 148.8, 158.9,
163.0.
N-(2-(2-Methoxyphenoxy)ethyl)-3,5-dinitrobenzamide (2)
##STR00014##
[0113] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.89 (s, 3H), 3.92
(dd, J=5.2, 10.4 Hz, 2H), 4.23 (t, J=4.8 Hz, 2H), 6.91-7.02 (m,
4H), 7.63 (brs, 1H), 9.02 (d, J=1.6 Hz, 2H), 9.14 (t, J=2.0 Hz,
1H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta.40.0, 56.1, 68.8,
112.2, 115.8, 121.0, 121.5, 122.9, 127.3, 137.8, 147.5, 148.6,
149.8, 162.6.
N-(2-(3-Methoxyphenoxy)ethyl)-3,5-dinitrobenzamide (3)
##STR00015##
[0115] .sup.1H NMR (400 MHz, Acetone-d.sub.6) .delta. 3.74 (s, 3H),
3.85 (dd, J=5.6 Hz, 4.8 Hz, 2H), 4.21 (t, J=5.2 Hz, 2H), 6.50 (m,
3H), 7.14 (t, J=8.4 Hz, 1H), 8.75 (brs, 1H), 9.04 (s, 1H), 9.08 (s,
2H); .sup.13C NMR (100 MHz, Acetone-d.sub.6) .delta. 40.1, 54.8,
66.1, 100.9, 106.5, 106.8, 120.9, 127.5, 130.0, 137.9, 148.8,
160.2, 161.2, 163.0.
N-(2-(4-Methoxyphenoxy)ethyl)-3,5-dinitrobenzamide (4)
##STR00016##
[0117] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.72 (s, 3H), 3.91
(dd, J=5.2, 10.8 Hz, 2H), 4.12 (t, J=4.8 Hz, 2H), 6.74-6.80 (m,
4H), 7.21 (brs, 1H), 8.95 (d, J=2.0 Hz, 2H), 9.07 (t, J=2.0 Hz,
1H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 40.4, 55.6, 66.8,
114.7, 115.4, 121.0, 127.2, 137.6, 148.5, 152.2, 154.3, 163.1;
LC-MS (ESI, m/z): 361 [M+H].sup.+.
N-(2-(2-Chlorophenoxy)ethyl)-3,5-dinitrobenzamide (5)
##STR00017##
[0119] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.97 (dd, J=5.2,
10.4 Hz, 2H), 4.25 (t, J=5.2 Hz, 2H), 6.93-6.95 (m, 2H), 7.19-7.24
(m, 2H), 7.35 (dd, J=1.2, 8.0 Hz, 1H), 8.98 (d, J=2.0 Hz, 2H), 9.12
(t, J=2.0 Hz, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 34.9,
63.0, 109.7, 116.2, 117.7, 118.2, 122.3, 123.1, 125.5, 132.6,
143.7, 148.7, 157.9.
N-(2-(3-Chlorophenoxy)ethyl)-3,5-dinitrobenzamide (6)
##STR00018##
[0121] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.97 (dd, J=5.6,
10.8 Hz, 2H), 4.19 (t, J=4.8 Hz, 2H), 6.80-6.98 (m, 4H), 7.24 (t,
J=8.0 Hz, 1H), 8.96 (d, J=2.0 Hz, 2H), 9.17 (t, J=2.0 Hz, 1H);
.sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 40.1, 66.4, 110.7,
115.0, 121.2, 121.7, 127.2, 130.4, 135.1, 137.6, 148.7, 158.8,
163.0.
N-(2-(4-Chlorophenoxy)ethyl)-3,5-dinitrobenzamide (7)
##STR00019##
[0123] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.96 (dd, J=5.6,
10.4 Hz, 2H), 4.17 (t, J=4.8 Hz, 2H), 6.78 (brs, 1H), 6.86 (dd,
J=2.4, 6.8 Hz, 2H), 7.23 (dd, J=2.0, 6.8 Hz, 2H), 8.96 (d, J=2.4
Hz, 2H), 9.17 (t, J=2.0 Hz, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3)
.delta.40.1, 66.5, 115.7, 121.2, 126.5, 127.2, 129.6, 137.6, 148.9,
156.8, 163.0.
N-(2-(2-Fluorophenoxy)ethyl)-3,5-dinitrobenzamide (8)
##STR00020##
[0125] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.97 (dd, J=5.2,
10.8 Hz, 2H), 4.25 (t, J=5.2 Hz, 2H), 6.91-7.06 (m, 4H), 7.39 (brs,
1H), 8.97 (d, J=2.0 Hz, 2H), 9.15 (t, J=2.0 Hz, 1H); .sup.13C NMR
(100 MHz, CDCl.sub.3) .delta. 40.1, 68.3, 115.7, 116.3 (d, J=20 Hz,
due to F), 121.1, 122.3 (d, J=7Hz, due to F), 124.6 (d, J=5 Hz, due
to F), 127.3, 137.6, 146.2, 148.6, 152.8 (d, J=250 Hz, due to F),
163.1; LC-MS (ESI, m/z): 350 [M+H].sup.+.
N-(2-(4-Fluorophenoxy)ethyl)-3,5-dinitrobenzamide (9)
##STR00021##
[0127] .sup.1H NMR (400 MHz, Acetone-d.sub.6) .delta. 3.88 (dd,
J=5.2, 10.8 Hz, 2H), 4.23 (t, J=5.2 Hz, 2H), 6.95-7.07 (m, 4H),
8.79 (brs, 1H), 9.07 (t, J=2.4 Hz, 1H), 9.11 (d, J=2.0 Hz, 2H).
N-(2-(4-Hydroxyphenoxy)ethyl)-3,5-dinitrobenzamide (10)
##STR00022##
[0129] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 3.66 (dd, J=5.6,
11.2 Hz, 2H), 4.06 (t, J=5.2 Hz, 2H), 6.65-6.68 (m, 2H), 6.76-6.80
(m, 2H), 8.91 (brs, 1H), 8.98 (t, J=2.0 Hz, 1H), 9.08 (d, J=2.4 Hz,
2H), 9.42 (brs, 1H); .sup.13C NMR (100 MHz DMSO-d.sub.6) .delta.
40.1, 66.9, 116.2, 116.4, 121.5, 128.2, 137.4, 148.8, 151.8, 152.0,
163.1.
N-(2-(3-(Trifluoromethoxy)phenoxy)ethyl)-3,5-dinitrobenzamide
(11)
##STR00023##
[0131] .sup.1H NMR (400 MHz, Acetone-d.sub.6) .delta. 3.89 (dd,
J=5.6, 11.2 Hz, 2H), 4.29 (t, J=5.6 Hz, 2H), 6.88 (d, J=6.0 Hz,
2H), 6.99 (d, J=8.0 Hz, 1H), 7.38 (t, J=8.4 Hz, 1H), 8.79 (brs,
1H), 9.05 (d, J=1.2 Hz, 1H), 9.08 (d, J=1.2 Hz, 2H); .sup.13C NMR
(100 MHz, Acetone-d.sub.6) .delta. 39.9, 66.7, 107.8, 113.1, 113.6,
120.9, 127.6, 130.9, 137.8, 148.9, 150.1, 160.2, 163.0.
N-(2-(4-(Trifluoromethoxy)phenoxy)ethyl)-3,5-dinitrobenzamide
(12)
##STR00024##
[0133] .sup.1H NMR (400 MHz, Acetone-d.sub.6) .delta. 3.88 (dd,
J=10.8 Hz, 5.2 Hz, 2H), 4.27 (t, J=5.6 Hz, 2H), 7.03 (dd, J=7.2,
2.0 Hz, 2H), 7.23 (d, J=8.8 Hz, 2H), 8.78 (brs, 1H), 9.04 (d, J=2.0
Hz, 1H), 9.08 (d, J=2.0 Hz, 2H); .sup.13C NMR (100 MHz,
Acetone-d.sub.6) .delta. 40.0, 66.8, 115.7, 120.9, 122.7, 127.6,
137.8, 142.7, 142.8, 148.9, 157.9, 163.1.
Methyl 4-(2-(3,5-dinitrobenzamido)ethoxy)benzoate (13)
##STR00025##
[0135] .sup.1H NMR (400 MHz, Acetone-d.sub.6) .delta. 3.81 (s, 3H),
3.91 (t, J=5.6 Hz, 2H), 4.33 (t, J=5.6 Hz, 2H), 7.00 (t, J=2.8 Hz,
1H), 7.03 (t, J=2.8 Hz, 1H), 7.90 (t, J=2.8 Hz, 1H), 7.92 (t, J=2.8
Hz, 1H), 8.78 (brs, 1H), 9.03 (t, J=2.4 Hz, 1H), 9.07 (d, J=2.4 Hz,
2H); .sup.13C NMR (100 MHz, Acetone-d.sub.6) .delta.39.9, 51.3,
66.5, 114.4, 120.9, 123.0, 127.6, 131.5, 137.8, 148.9, 162.8,
163.0, 166.1.
N-(2-(4-Aminophenoxy)ethyl)-3,5-dinitrobenzamidehydrochloride
(14)
##STR00026##
[0137] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 3.67 (d, J=5.2
Hz, 2H), 4.15 (t, J=5.2 Hz, 2H), 7.03 (d, J=1.6 Hz, 2H), 7.29 (d,
J=1.6 Hz, 2H), 8.91 (d, J=2.0 Hz, 1H), 9.04 (d, J=2.0 Hz, 2H), 9.52
(brs, 1H), 10.28 (brs, 3H); .sup.13C NMR (100 MHz, DMSO-d.sub.6)
.delta. 40.1, 66.1, 115.4, 120.8, 124.3, 124.5, 127.5, 136.7,
148.1, 157.8, 162.4.
N-(2-(4-(t-Butoxycarbonylamino)phenoxy)ethyl)-3,5-dinitrobenzamide
(15)
##STR00027##
[0139] .sup.1H NMR (400 MHz, Acetone-d.sub.6) .delta. 1.44 (s, 9H),
3.83 (m, 2H), 4.18 (m, 2H), 6.84 (dd, J=3.2, 9.2 Hz, 2H), 7.40 (d,
J=7.6 Hz, 2H), 8.15 (brs, 1H), 8.73 (brs, 1H), 9.03 (t, J=2.0 Hz,
1H), 9.08 (d, J=2.0 Hz, 2H); .sup.13C NMR (100 MHz,
Acetone-d.sub.6) .delta. 27.8, 40.1, 66.4, 78.9, 114.8, 119.9,
120.9, 127.6, 133.3, 137.9, 148.8, 153.2, 154.4, 163.0; LC-MS (ESI,
m/z): 469[M+Na].sup.+.
N-(2-(4-Methoxyphenoxy)ethyl)-3-nitrobenzamide (16)
##STR00028##
[0141] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.69 (s, 3H), 3.81
(dd, J=5.2, 10.4 Hz, 2H), 4.06 (t, J=5.6 Hz, 2H), 6.73-6.78 (m,
4H), 7.48 (brs, 1H), 7.53 (t, J=8.0 Hz, 1H), 8.13 (d, J=7.6 Hz,
1H), 8.24 (d, J=10.4 Hz, 1H), 8.56 (t, J=2.0 Hz, 1H); .sup.13C NMR
(100 MHz, CDCl.sub.3) .delta. 39.8, 55.4, 66.7, 114.4, 115.2,
121.9, 125.8, 129.5, 133.1, 135.7, 147.8, 152.3, 153.9, 165.2.
N-(2-(2-Fluorophenoxy)ethyl)-3-nitrobenzamide (17)
##STR00029##
[0143] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.92 (dd, J=5.6,
10.8 Hz, 2H), 4.23 (t, J=4.8 Hz, 2H), 6.90-7.09 (m, 4H and brs,
1H), 7.62 (t, J=8.0 Hz, 1H), 8.14 (d, J=8.0 Hz, 1H), 8.33 (d, J=8.0
Hz, 1H), 8.63 (t, J=2.0 Hz, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3)
.delta. 39.8, 68.3, 115.6, 116.6 (d, J=18.6 Hz, due to F), 122.3
(d, J=5.3 Hz, due to F), 124.7 (d, J=4.5 Hz, due to F), 126.0,
129.7, 133.0, 135.8, 146.3 (d, J=10.4 Hz, due to F), 148.1, 152.6
(d, J=245 Hz, due to F), 165.2.
N-(2-(4-Methoxyphenoxy)ethyl)benzamide (18)
##STR00030##
[0145] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.72 (s, 3H), 3.80
(dd, J=5.2, 10.8 Hz, 2H), 4.05 (t, J=5.6 Hz, 2H), 6.78-6.83 (m,
4H), 7.03 (brs, 1H), 7.35-7.45 (m, 4H), 7.74 (d, J=11.2 Hz, 1H);
.sup.13C NMR (100 MHz, CDCl.sub.3) .delta.9.4, 55.4, 67.1, 114.5,
115.2, 126.8, 128.3, 131.3, 134.1, 152.4, 153.9, 167.6.
N-(2-(4-Methoxyphenoxy)ethyl)-N-methyl-3,5-dinitrobenzamide
(19)
##STR00031##
[0147] (Two rotamers, 1:1) .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 3.18 (brs, 3H), 3.65 (brs, 1H), 3.75 (s, 3H), 3.94 (brs,
1H), 4.03 (brs, 1H), 4.27 (brs, 1H), 6.79-6.84 (brd, 4H), 8.55
(brs, 1H), 8.72 (brs, 1H), 9.04 (brs, 1H).
N-Ethyl-N-(2-(4-methoxyphenoxy)ethyl)-3,5-dinitrobenzamide (20)
##STR00032##
[0149] (Two rotamers, 1:1) .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 1.22-1.30 (m, 3H), 3.42 (brs, 1H), 3.63 (brs, 2H), 3.75 (s,
3H), 3.89 (brs, 1H), 4.01 (brs, 1H), 4.26 (brs, 1H), 6.80 (br, 4H),
8.53 (brs, 1H), 8.72 (brs, 1H), 9.04 (brs, 1H).
N-(3-(4-Methoxyphenoxy)propyl)-3,5-dinitrobenzamide (21)
##STR00033##
[0151] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.04-2.20 (m, 2H),
3.76 (t, J=6.0 Hz, 2H), 3.77 (s, 3H), 4.17 (t, J=5.2 Hz, 2H),
6.85-6.91 (m, 4H), 7.24 (brs, 1H), 8.96 (d, J=2.0 Hz, 2H), 9.16 (t,
J=102.0 Hz, 1H).
Methyl 4-(3-(3,5-dinitrobenzamido)propoxy)benzoate (22)
##STR00034##
[0153] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.21-2.24 (m, 2H),
3.77 (dd, J=6.0, 12.0 Hz, 2H), 3.89 (s, 3H), 4.24 (t, J=5.6 Hz,
2H), 6.95 (d, J=8.8 Hz, 2H), 7.04 (brs, 1H), 8.00 (d, J=8.8 Hz,
2H), 8.96 (d, J=2.0 Hz, 2H), 9.16 (s, 1H); .sup.13C NMR (100 MHz,
CDCl.sub.3) .delta. 28.4, 39.3, 52.0, 67.2, 113.9, 121.1, 123.3,
127.0, 131.8, 137.8, 148.6, 161.9, 162.5, 166.6.
N-(3-(2-Fluorophenoxy)propyl)-3,5-dinitrobenzamide (23)
##STR00035##
[0155] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.19-2.25 (m, 2H),
3.83 (dd, J=5.2, 11.2 Hz, 2H), 4.27 (t, J=5.2 Hz, 2H), 6.90-7.11
(m, 4H), 7.50 (brs, 1H), 8.99 (d, J=2.0 Hz, 2H), 9.16 (t, J=2.0 Hz,
1H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 28.2, 40.0, 69.5,
114.0, 116.3 (d, J=18 Hz, due to F), 120.9, 121.8 (d, J=7.4 Hz, due
to F), 124.7 (d, J=3.7 Hz, due to F), 127.2, 127.3, 138.1, 147.3
(d, J=245 Hz, due to F), 153.5, 162.7.
##STR00036##
General Procedure for the Synthesis of
N-(2-(benzyloxy)ethyl)-dinitrobenzamide (B2)
[0156] To a solution of 2-(2-hydroxyethyl)isoindoline-1,3-dione
(1.17 mmol) in dimethyl formamide (10 mL) was added sodium hydride
(2.34 mmol) and a benzyl bromide (1.40 mmol) at 0.degree. C. and
the resulting mixture was stirred at room temperature. After
stirring overnight, distilled water (50 mL) was added and the
resulting precipitate was collected by filtration to afford B1.
[0157] To a solution of B1 (0.85 mmol) in methanol (10 mL) was
added hydrazine monohydrate (0.85 mmol) and the resulting mixture
was refluxed under a nitrogen atmosphere. After 3 h, the reaction
mixture was allowed to cool to room temperature and concentrated in
vacuo. The residue was precipitated with methylene chloride (10
mL). The resulting precipitate was filtered off through Celite, and
the filtrate was concentrated in vacuo to afford an amine.
[0158] To a solution of the amine in methylene chloride (10 mL) was
added triethylamine (113 .mu.l, 0.81 mmol) and a benzoylchloride
(0.81 mmol) at 0.degree. C. and the resulting mixture was stirred
at room temperature. After 3 h, the reaction mixture was diluted
with methylene chloride (30 mL) and washed with 1 M HCl aqueous
solution (50 mL), saturated Na.sub.2CO.sub.3 aqueous solution (50
mL) and brine (50 mL). The organic layer was dried over anhydrous
MgSO.sub.4 and concentrated in vacuo. The crude product was
purified by silica gel flash column chromatography (3:1
hexanes/ethyl acetate) and recrystallized from a mixture of hexanes
and ethyl acetate to give B2.
N-(2-(Benzyloxy)ethyl)-3,5-dinitrobenzamide (24)
##STR00037##
[0160] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.68-3.72 (m, 4H),
4.55 (s, 2H), 6.75 (brs, 1H), 7.24-7.33 (m, 5H), 8.91 (d, J=2.0 Hz,
2H), 9.13 (t, J=2.0 Hz, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3)
.delta. 40.4, 68.1, 73.4, 121.0, 127.2, 128.0, 128.2, 128.7, 137.5,
138.0, 148.6, 162.7; LC-MS (ESI, m/z): 346[M+H].sup.+.
N-(2-(3-Methoxybenzyloxy)ethyl)-3,5-dinitrobenzamide (25)
##STR00038##
[0162] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.71-3.74 (m, 4H),
3.76 (s, 3H), 4.52 (s, 2H), 6.77-6.90 (m, 3H), 6.97 (brs, 1H), 7.23
(t, J=8.0 Hz, 1H), 8.91 (d, J=2.0 Hz, 2H), 9.12 (t, J=2.0 Hz, 1H);
.sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 40.5, 55.2, 68.2, 73.1,
113.1, 113.6, 120.0, 120.9, 127.2, 129.6, 137.8, 139.1, 148.5,
159.7, 162.8.
N-(2-(4-Methoxybenzyloxy)ethyl)-3,5-dinitrobenzamide (26)
##STR00039##
[0164] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.65-3.71 (m, 4H),
3.75 (s, 3H), 4.47 (s, 2H), 6.71 (brs, 1H), 6.84 (dd, J=6.8, 2.0
Hz, 2H), 7.23 (d, J=8.4 Hz, 2H), 8.87 (d, J=2.4 Hz, 2H), 9.13 (t,
J=2.0 Hz, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 40.5,
55.3, 67.8, 73.1, 114.0, 121.0, 127.1, 129.6, 130.0, 137.9, 148.6,
159.5, 162.7.
N-(2-(4-Chlorobenzyloxy)ethyl)-3,5-dinitrobenzamide (27)
##STR00040##
[0166] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.68-3.76 (m, 4H),
4.53 (s, 2H), 6.77 (brs, 1H), 7.25-7.32 (m, 4H), 8.91 (d, J=2.0 Hz,
2H), 9.15 (t, J=2.0 Hz, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3)
.delta. 40.4, 68.3, 72.6, 121.1, 127.2, 128.8, 129.2, 134.0, 136.0,
137.8, 148.6, 162.7.
N-(2-(3-chlorobenzyloxy)ethyl)-3,5-dinitrobenzamide (28)
##STR00041##
[0168] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.68-3.76 (m, 4H),
4.52 (s, 2H), 6.79 (brs, 1H), 7.17-7.29 (m, 4H), 8.91 (d, J=2.0 Hz,
2H), 9.13 (t, J=2.0 Hz, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3)
.delta. 40.4, 68.4, 72.5, 121.1, 125.8, 127.2, 127.8, 128.1, 129.2,
134.5, 137.8, 139.6, 148.6, 162.8.
N-(2-(4-Fluorobenzyloxy)ethyl)-3,5-dinitrobenzamide (29)
##STR00042##
[0170] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.68-3.76 (m, 4H),
4.53 (s, 2H), 6.74 (brs, 1H), 7.02-7.06 (m, 2H), 7.30-7.33 (m, 2H),
8.92 (d, J=2.0 Hz, 2H), 9.16 (t, J=2.0 Hz, 1H); .sup.13C NMR (100
MHz, CDCl.sub.3) .delta. 40.4, 68.1, 72.6, 115.5 (d, J=22 Hz, due
to F), 121.1, 127.1, 130.0 (d, J=8.2 Hz, due to F), 133.5 (d, J=3.0
Hz, due to F), 137.8, 148.6, 162.5 (d, J=245 Hz, due to F),
162.7.
N-(2-(2-Fluorobenzyloxy)ethyl)-3,5-dinitrobenzamide (30)
##STR00043##
[0172] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.75 (s, 4H), 4.64
(s, 2H), 7.07-7.17 (m, 3H), 7.29-7.39 (m, 1H and brs. 1H), 8.94 (d,
J=2.0 Hz, 2H), 9.17 (t, J=2.0 Hz, 1H).
3,5-Dinitro-N-(2-(4-(trifluoromethoxy)benzyloxy)ethyl)benzamide
(31)
##STR00044##
[0174] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.72-3.76 (m, 4H),
4.54 (s, 2H), 7.13 (d, J=8.0 Hz, 2H), 7.31-7.35 (m, 2H and brs,
1H), 8.94 (d, J=2.0 Hz, 2H), 9.08 (t, J=2.0 Hz, 1H); .sup.13C NMR
(100 MHz, CDCl.sub.3) .delta.0.4, 68.4, 72.2, 120.9, 121.0, 127.2,
129.1, 136.3, 137.7, 148.4, 148.7, 148.9, 162.9.
3,5-Dinitro-N-(2-(3-(trifluoromethyl)benzyloxy)ethyl)benzamide
(32)
##STR00045##
[0176] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.72-3.79 (m, 4H),
4.61 (s, 2H), 7.06 (brs, 1H), 7.45-7.55 (m, 4H), 8.93 (d, J=2.0 Hz,
2H), 9.10 (t, J=2.0 Hz, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3)
.delta. 40.4, 68.7, 72.4, 121.0, 124.1, 124.6, 124.7, 127.2, 129.0,
130.6 (q, J=32 Hz, due to F), 130.8, 137.7, 138.6, 148.6,
162.9.
Methyl 4-((2-(3,5-dinitrobenzamido)ethoxy)methyl)benzoate (33)
##STR00046##
[0178] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.71-3.74 (m, 4H),
3.84 (s, 3H), 4.55 (s, 2H), 7.29 (d, J=8.0 Hz, 2H and brs, 1H),
7.85 (d, J=8.0 Hz, 2H), 8.90 (d, J=2.0 Hz, 2H), 9.01 (t, J=2.0 Hz,
1H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 40.6, 52.2, 68.8,
72.6, 120.9, 127.3, 129.5, 129.7, 137.8, 142.9, 148.5, 163.0,
166.8.
4-((2-(3,5-Dinitrobenzamido)ethoxy)methyl)benzoic acid (34)
##STR00047##
[0180] .sup.1H NMR (400 MHz, Acetone-d.sub.6) .delta. 3.74 (t,
J=5.2 Hz, 2H), 3.81 (t, J=5.2 Hz, 2H), 4.72 (s, 2H), 7.56 (d, J=8.4
Hz, 2H) 7.72 (brs, 1H), 8.03 (d, J=8.4 Hz, 2H), 9.02 (d, J=2.0 Hz,
2H), 9.13 (t, J=2.0 Hz, 1H).
N-(2-(Benzyloxy)ethyl)benzamide (35)
##STR00048##
[0182] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.62-3.68 (m, 4H),
4.52 (s, 2H), 6.71 (brs, 1H), 7.24-7.49 (m, 8H), 7.73-7.76 (m, 2H);
.sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 39.7, 68.8, 73.1, 126.9,
127.8, 128.4, 131.3, 134.5, 137.8, 167.5.
N-(2-(3-(Trifluoromethyl)benzyloxy)ethyl)benzamide (36)
##STR00049##
[0184] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.63-3.70 (m, 4H),
4.56 (s, 2H), 6.72 (brs, 1H), 7.37-7.53 (m, 6H), 7.58 (s, 1H),
7.74-7.76 (m, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 39.7,
69.3, 72.3, 124.2, 124.3, 124.6, 126.9, 128.5, 128.9, 130.8, 131.5,
134.4, 139.0, 148.6, 167.6.
N-(2-(3-Chlorobenzyloxy)ethyl)benzamide (37)
##STR00050##
[0186] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.62-3.69 (m, 4H),
4.49 (s, 2H), 6.71 (brs, 1H), 7.17-7.50 (m, 7H), 7.75-7.77 (m, 2H);
.sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 39.7, 69.0, 72.2, 125.6,
126.8, 127.6, 127.8, 128.4, 129.7, 131.3, 134.3, 139.9, 167.4.
N-(2-(3-Chlorobenzyloxy)ethyl)-3,5-difluorobenzamide (38)
##STR00051##
[0188] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.64-3.69 (m, 4H),
4.52 (s, 2H), 6.54 (brs, 1H), 6.95 (tt, J=2.4, 11.2 Hz, 1H),
7.19-7.33 (m, 6H).
N-(2-(3-Chlorobenzyloxy)ethyl)-3,5-dichlorobenzamide (39)
##STR00052##
[0190] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 3.13 (t, J=5.2 Hz,
2H), 3.67 (t, J=5.2 Hz, 2H), 4.55 (s, 2H), 7.27-7.29 (m, 3H), 7.42
(s, 1H), 7.46, (s, 1H), 7.81 (s, 2H); .sup.13C NMR (100 MHz,
CDCl.sub.3) .delta.40.6, 67.2, 73.2, 127.0, 128.7, 128.8, 128.9,
130.7, 131.0, 135.4, 135.5, 141.4, 142.7, 171.5.
N-(2-(3-Chlorobenzyloxy)ethyl)-3,5-bis(trifluoromethyl)benzamide
(40)
##STR00053##
[0192] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.64-3.68 (m, 4H),
4.49 (s, 2H), 6.89 (brs, 1H), 7.15 (d, J=3.6 Hz, 1H), 7.21-7.24 (m,
2H), 7.27 (s, 1H), 7.95 (s, 1H), 8.18 (s, 2H); .sup.13C NMR (100
MHz, CDCl.sub.3) .delta.40.3, 68.37, 72.5, 121.6, 125.0, 125.1,
125.7, 127.8, 128.1, 129.9, 132.0, 134.5, 136.6, 139.8, 164.8.
N-(2-(3-Chlorobenzyloxy)ethyl)-3-methoxybenzamide (41)
##STR00054##
[0194] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.63 (d, J=3.6 Hz,
2H), 3.65 (d, J=3.6 Hz, 2H), 3.81 (s, 3H), 4.49 (s, 2H), 6.51 (brs,
1H), 7.01 (dd, J=8.0 Hz, 2.4 Hz, 1H), 7.16 (d, J=4.4 Hz, 1H), 7.28
(m, 3H), 7.25-7.34 (m, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3)
.delta. 39.9, 55.5, 69.2, 72.5, 112.4, 117.8, 118.7, 125.8, 127.8,
128.0, 129.6, 129.9, 134.5, 136.0, 140.0, 159.9, 167.5.
N-(2-(3-Chlorobenzyloxy)ethyl)-4-methoxybenzamide (42)
##STR00055##
[0196] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.62-3.66 (m, 4H),
3.82 (s, 3H), 4.49 (S, 2H), 6.48 (brs, 1H), 6.89 (d, J=8.8 hz, 2H),
7.17 (t, J=4.4 Hz, 2H), 7.24 (m, 1H), 7.32 (s, 1H), 7.71 (d, J=8.8
Hz, 2H) .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 39.8, 55.5,
69.4, 72.4, 113.8, 125.7, 126.8, 127.8, 128.0, 128.8, 129.8, 134.5,
140.1, 162.2, 167.1.
N-(2-(3-Chlorobenzyloxy)ethyl)-3-(trifluoromethoxy)benzamide
(43)
##STR00056##
[0198] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.62-3.68 (m, 4H),
4.49 (s, 2H), 6.62 (brs, 1H), 7.15 (dd, J=1.2, 8.8 Hz, 1H),
7.22-7.23 (m, 2H), 7.36 (t, J=1.2 Hz, 2H), 7.43 (t, J=8.4 Hz, 1H),
7.63 (dd, J=1.2, 4.4 Hz, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3)
.delta. 40.0, 69.0, 72.4, 119.3, 120.1, 123.8, 125.1, 125.7, 127.8,
128.0, 129.9, 130.1, 134.5, 136.6, 140.0, 149.4, 166.1.
N-(2-(3-Chlorobenzyloxy)ethyl)-4-(trifluoromethyl)benzamide
(44)
##STR00057##
[0200] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.62-3.68 (m, 4H),
4.49 (s, 2H), 6.71 (brs, 1H), 7.14-7.17 (m, 1H), 7.23-7.24 (m, 2H),
7.3 (s, 1H), 7.64 (d, J=8.0 Hz, 2H), 7.83 (d, 0.1=8.0 Hz, 2H);
.sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 40.0, 68.9, 72.4, 125.6
(q, J=3.7 Hz), 125.8, 127.5, 127.8, 128.1, 129.9, 138.1, 133.4,
134.5, 137.7, 140.0, 166.4.
N-(2-(3-Chlorobenzyloxy)ethyl)-3-(trifluoromethyl)benzamide
(45)
##STR00058##
[0202] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.62 (m, 4H), 4.46
(s, 2H), 6.96 (brs, 1H), 7.14-7.27 (m, 4H), 7.47 (t, J=7.2 Hz, 1H),
7.68 (d, J=3.2 Hz, 1H), 7.89 (d, J=3.2 Hz, 1H), 8.01 (s, 1H);
.sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 40.0, 68.9, 72.3, 122.4,
124.1, 125.7, 127.7, 127.9, 128.0, 129.1, 129.8, 130.3, 130.8,
134.4, 135.2, 140.0, 166.3.
Methyl 3-(2-(3-chlorobenzyloxy)ethylcarbamoyl)benzoate (46)
##STR00059##
[0204] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.62-3.69 (m, 4H),
3.89 (s, 3H), 4.48 (s, 2H), 6.71 (brs, 1H), 7.15-7.16 (m, 1H),
7.21-7.24 (m, 2H), 7.28 (s, 1H), 7.47 (t, J=4.0 Hz, 1H), 7.97 (d,
J=4.8 Hz, 1H), 8.11 (d, J=4.8 Hz, 1H), 8.35 (t, J=1.6 Hz, 1H);
.sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 40.0, 52.4, 69.0, 72.4,
125.7, 127.7, 127.8, 128.0, 128.9, 129.8, 130.5, 131.8, 132.4,
134.4, 134.8, 140.0, 166.3, 166.6.
Methyl 4-(2-(3-chlorobenzyloxy)ethylcarbamoyl)benzoate (47)
##STR00060##
[0206] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.62-3.66 (m, 4H),
3.90 (s, 3H), 4.48 (s, 2H), 6.65 (brs, 1H), 7.14-7.17 (m, 1H), 7.22
(d, J=5.2 Hz, 2H), 7.30 (s, 1H), 7.78 (d, J=8.0 Hz, 2H), 8.04 (d,
J=8.0 Hz, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 40.0,
52.4, 69.0, 72.4, 125.7, 127.1, 127.8, 128.1, 129.9, 132.7, 134.5,
138.4, 140.0, 160.3, 166.7.
N-(2-(3-Chlorobenzyloxy)ethyl)-3-nitrobenzamide (48)
##STR00061##
[0208] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.64 (m, 4H), 4.45
(s, 2H), 7.13-7.23 (m, 5H), 7.53 (m, 1H), 8.08 (d, J=6.8 Hz, 1H)
8.22 (d, J=6.8 Hz, 1H), 8.54 (s, 1H); .sup.13C NMR (100 MHz,
CDCl.sub.3) .delta. 40.1, 68.7, 72.2, 122.0, 125.6, 125.9, 127.5,
127.8, 129.7, 129.8, 133.1, 134.2, 136.0, 139.9, 148.0, 165.3.
N-(2-(3-Chlorobenzyloxy)ethyl)-4-nitrobenzamide (49)
##STR00062##
[0210] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.63 (m, 4H), 4.45
(s, 2H), 6.97 (brs, 1H), 7.12-7.25 (m, 4H), 7.87 (d, J=6.4 Hz, 2H),
8.15 (d, J=6.4 Hz, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta.
40.1, 68.7, 72.2, 123.6, 125.6, 127.5, 127.9, 128.2, 129.7, 134.3,
139.9, 140.0, 149.4, 165.6.
N-(2-(3-Chlorobenzyloxy)ethyl)-3-fluorobenzamide (50)
##STR00063##
[0212] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.56-3.61 (m, 4H),
4.43 (s, 2H), 6.66 (brs, 1H), 7.10-7.12 (m, 2H), 7.18-7.19 (m, 2H),
7.25 (s, 1H), 7.30-7.31 (m, 1H), 7.41-7.45 (m, 2H); .sup.13C NMR
(100 MHz, CDCl.sub.3) .delta. 39.9, 69.0, 72.4, 114.3 (d, J=23.0
Hz, due to F), 118.4 (d, J=20.8 Hz, due to F), 122.4 (d, J=3.0 Hz,
due to F), 125.7, 127.7, 128.0, 129.8, 130.2 (d, J=8.2 Hz, due to
F), 134.5, 136.7 (d, J=6.7 Hz, due to F), 140.0, 163.0 (d, J=245
Hz, due to F), 166.3 (d, J=3.0 Hz, due to F).
N-(2-(3-Chlorobenzyloxy)ethyl)-3-chlorobenzamide (51)
##STR00064##
[0214] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.64 (m, 4H), 4.49
(s, 2H), 6.52 (brs, 1H), 7.17 (d, J=3.2 Hz, 1H), 7.24 (s, 2H),
7.31-7.36 (m, 2H), 7.44 (d, J=3.6 Hz, 1H), 7.59 (d, J=7.6 Hz, 1H),
7.73 (s, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta.40.0, 69.1,
72.5, 125.1, 125.8, 127.5, 127.8, 128.1, 129.9, 130.0, 131.6,
134.6, 134.9, 136.3, 140.0, 166.3.
N-(2-(3-Chlorobenzyloxy)ethyl)-4-hydroxybenzamide (52)
##STR00065##
[0216] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.64 (s, 4H), 4.48
(s, 2H), 6.57 (brs, 1H), 6.84 (dd, J=2.0, 8.8 Hz, 2H), 7.17 (d,
J=3.2 Hz, 1H), 7.23 (d, J=3.2 Hz, 2H), 7.31 (s, 1H), 7.60 (dd,
J=2.0, 8.8 Hz, 2H), 8.22 (brs, 1H); .sup.13C NMR (100 MHz,
CDCl.sub.3) .delta. 40.0, 69.1, 72.5, 115.7, 125.4,
.quadrature.125.8, 127.8, 128.1, 129.0, 129.9, 134.5, 140.0, 160.2,
168.2.
N-(2-(3-Chlorobenzyloxy)ethyl)-3-hydroxybenzamide (53)
##STR00066##
[0218] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.65 (m, 4H), 4.49
(s, 2H), 6.64 (brs, 1H), 6.98 (d, J=8.0 Hz, 1H), 7.13 (d, J=8.0 Hz,
1H), 7.17-7.26 (m, 5H), 7.30 (s, 1H), 7.50 (s, 1H); .sup.13C NMR
(100 MHz, CDCl.sub.3) .delta. 40.0, 69.1, 72.5, 115.1, 117.8,
119.3, 125.9, 127.3, 128.1, 129.9, 130.0, 134.6, 135.4, 139.9,
157.2, 168.0.
##STR00067##
General Procedure for the Synthesis of
phenoxy-pyrrolidin-1-yl-methanone (C2)
[0219] To a solution of (S)-3-pyrrolidinol (10 mmol) and
triethylamine (11 mmol) in methylene chloride (50 mL) was added
benzoyl chloride (8.67 mmol) at 0.degree. C. The reaction
temperature was brought up to room temperature. After 2 h, the
reaction mixture was diluted with methylene chloride (50 mL) and
then washed with 0.5 M HCl aqueous solution (100 mL) and brine (100
mL). The organic layer was dried over anhydrous MgSO.sub.4 and
concentrated in vacuo. The crude product was purified by silica gel
flash column chromatography (2:1 hexanes/ethyl acetate) to give
C1.
[0220] To a solution of C1 (1.07 mmol) in methylene chloride (10
mL) was added ADDP (1.28 mmol), triphenylphosphine (1.28 mmol) and
a phenol (1.28 mmol) at room temperature. After stirring overnight,
the reaction mixture was diluted with methylene chloride (30 mL)
and washed with 1 M HCl aqueous solution (50 mL), saturated
Na.sub.2CO.sub.3 aqueous solution (50 mL) and brine (50 mL). The
organic layer was dried over anhydrous MgSO.sub.4 and concentrated
in vacuo. The crude was purified by silica gel flash column
chromatography (2:1 hexanes/ethyl acetate) and recrystallized from
a mixture of hexanes and ethyl acetate to give C2.
(R)-(3,5-Dinitrophenyl)(3-(4-methoxyphenoxy)pyrrolidin-1-yl)methanone
(54)
##STR00068##
[0222] (Two rotamers, 1:1 ratio), m.p. 124-125.degree. C.; .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. 2.11-2.19 (m, 1H), 2.30-2.34 (m,
1H), 3.54-3.64 (m, 1H), 3.72 & 3.76 (s, 3H), 3.81-3.99 (m, 3H),
4.86-4.94 (m, 1H), 6.74-6.84 (m, 4H), 8.68 & 8.75 (d, J=1.6 Hz,
2H), 9.05 & 9.08 (brs, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3)
.delta. 30.6, 32.4, 45.2, 47.7, 52.8, 54.8, 55.8, 55.9, 75.7,
115.0, 117.1, 117.3, 120.1, 120.2, 127.7, 127.9, 139.9, 140.0,
148.6, 150.4, 150.8, 154.8, 154.8, 164.7, 165.1; LC-MS (ESI, m/z):
388 [M+H].sup.+.
(R)-(3,5-Dinitrophenyl)(3-(4-fluorophenoxy)pyrrolidin-1-yl)methanone
(55)
##STR00069##
[0224] (Two rotamers, 1:1 ratio, 75%), a pale yellow solid; .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. 2.15-2.37 (m, 2H), 3.56-3.63 (m,
1H), 3.79-3.97 (m, 3H), 4.91-4.99 (m, 1H), 6.76-7.03 (m, 4H), 8.71
& 8.76 (d, J=1.6 Hz, 2H), 9.08 & 9.10 (brs, 1H); .sup.13C
NMR (100 MHz, CDCl.sub.3) .delta. 29.9, 32.3, 45.1, 47.7, 52.7,
54.8, 75.5, 77.0, 116.2, 116.5, 116.9, 117.0, 117.1, 120.1, 120.2,
127.7, 127.8, 139.8, 139.9, 148.6, 152.6, 152.9, 157.9 (d, J=245
Hz, due to F), 164.7, 165.0.
(R)--N-(4-(1-(3,5-Dinitrobenzoyl)pyrrolidin-3-yloxy)phenyl)acetamide
(56)
##STR00070##
[0226] (Two rotamers, 1:1 ratio, 63%), a yellow solid; .sup.1H NMR
(400 MHz, CDCl.sub.3+CD.sub.3OD) .delta. 1.96 & 1.99 (s, 3H),
2.03-2.27 (m, 2H), 3.45-3.50 (m, 1H), 3.69-3.83 (m, 3H), 4.83-4.91
(m, 1H), 6.64 & 6.74 (d, J=8.8 Hz, 2H), 7.26 & 7.33 (d,
J=8.8 Hz, 2H), 8.58 & 8.65 (d, J=2.0 Hz, 2H), 8.95-8.99 (m,
1H); .sup.13C NMR (100 MHz, CDCl.sub.3+CD.sub.3OD) .delta. 23.3,
23.4, 29.7, 32.0, 45.0, 47.6, 52.6, 54.6, 75.0, 76.4, 115.8, 115.9,
120.0, 121.9, 127.4, 127.5, 127.6, 127.7, 132.4, 132.5, 139.4,
148.4, 152.8, 153.1, 165.0, 165.3, 169.7.
(R)-(3,5-Dinitrophenyl)(3-(4-(trifluoromethoxy)phenoxy)pyrrolidin-1-yl)met-
hanone (57)
##STR00071##
[0228] (Two rotamers, 6:4 ratio, 67%), a white solid; .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 2.20-2.40 (m, 2H), 3.59-3.66 (m, 1H),
3.84-4.00 (m, 3H), 4.97-5.05 (m, 1H), 6.83 & 6.92 (d, J=8.8 Hz,
2H), 7.12 & 7.18 (d, J=8.8 Hz, 2H), 8.73 & 8.77 (d, J=2.0
Hz, 2H), 9.09 & 9.11 (d, J=2.0 Hz, 1H); .sup.13C NMR (100 MHz,
CDCl.sub.3) .delta. 29.8, 32.2, 45.1, 47.6, 52.6, 54.7, 75.2, 76.7,
116.4, 120.1, 122.8, 127.7, 127.8, 139.6, 139.7, 143.4, 148.5,
155.0, 155.2, 164.7, 164.9.
(R)-Methyl 4-(1-(3,5-dinitrobenzoyl)pyrrolidin-3-yloxy)benzoate
(58)
##STR00072##
[0230] (Two rotamers 1:1 ratio), .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 2.21-2.37 (m, 2H), 3.57-3.65 (m, 1H), 3.85 & 3.87 (s,
3H), 3.89-3.99 (m, 3H), 5.03-5.11 (m, 1H), 6.82 & 6.91 (d,
J=7.2 Hz, 2H), 7.93 & 7.99 (d, J=7.2 Hz, 2H), 8.70 & 8.75
(s, 2H), 9.07 & 9.09 (s, 1H); .sup.13C NMR (100 MHz,
CDCl.sub.3) .delta.22.1, 30.0, .quadrature. 32.4, 45.2, 47.7, 52.2,
52.8, 54.8, 74.9, 76.3, 115.0, 120.36, 123.7, 123.8, 127.8, 127.9,
132.0, 139.7, 148.6, 160.2, 160.5, 164.7, 166.7.
(R)-(3,5-Dinitrophenyl)(3-(2-fluorophenoxy)pyrrolidin-1-yl)methanone
(59)
##STR00073##
[0232] (Two rotamers 1:1 ratio), .sup.1H NMR (400 MHz, CD.sub.3OD)
.delta. 2.26-2.33 (m, 2H), 3.62-3.97 (m, 3H), 4.00 & 4.36 (s,
1H), 5.06 & 5.21 (s, 1H), 7.11 & 7.27 (m, 4H), 8.78 &
8.83 (d, J=2.0 Hz, 2H), 9.01 & 9.04 (d, J=2.0 Hz, 1H); .sup.13C
NMR (100 MHz, CD.sub.3OD) .delta. .quadrature.29.9, 31.9, 44.9,
52.3, 54.4, 77.2, 78.7, 116.62, 116.67, 116.80, 116.85, 117.8 (d,
J=20 Hz, due to F), 120.04 (d, J=3.7 Hz, due to F), 122.5, 122.6,
122.70, 122.77, 125.1, 125.15 (d, J=3.7 Hz, due to F), 127.80 (d,
0.1=7.4 Hz due to F), 127.9, 139.8, 153.6 (d, J=244 Hz, due to F),
165.4, 165.5.
(S)-Methyl-4-(1-(3,5-dinitrobenzoyl)pyrrolidin-3-yloxy)benzoate
(60)
##STR00074##
[0234] (Two rotamers 1:1 ratio), .sup.1H NMR (400 MHz,
Acetone-d.sub.6) .delta. 2.21-2.29 (m, 2H), 3.58 & 3.61 (s,
1H), 3.69 & 3.71 (s, 3H), 3.73-4.02 (m, 3H), 4.99 & 5.06
(s, 1H), 6.77-6.94 (m, 4H), 8.73 & 8.77 (s, 2H), 8.96 &
8.99 (s, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta.
.quadrature.29.9, 31.9, 44.1, 44.7, 52.2, 54.2, 55.1, 55.2, 76.0,
77.5, 114.82, 114.88, 117.2, 119.6, 127.7, 127.8, 140.5, 148.7,
151.1, 151.3, 154.7, 164.6, 164.7.
(S)-(3,5-dinitrophenyl)(3-(4-methoxyphenoxy)pyrrolidin-1-yl)methanone
(61)
##STR00075##
[0236] (Two rotamers 1:1 ratio), .sup.1H NMR (400 MHz,
Acetone-d.sub.6) .delta. 2.19-2.28 (m, 2H) 3.60-4.01 (m, 4H), 4.98
& 5.06 (s, 1H), 6.76-6.94 (m, 4H), 8.73 & 8.76 (s, 2H),
8.95 & 8.99 (s, 1H); .sup.13C NMR (100 MHz, Acetone-d.sub.6)
.delta. .quadrature.31.9, 44.7, 52.2, 54.2, 55.0, 55.1, 65.8, 75.9,
77.5, 114.81, 114.87, 117.2, 119.6, 127.7, 127.8, 128.6, 129.8,
140.4, 148.7, 151.3, 154.7, 164.6, 164.7.
(S)--N-(4-(1-(3,5-Dinitrobenzoyl)pyrrolidin-3-yloxy)phenyl)acetamide
(62)
##STR00076##
[0238] (Two rotamers 1:1 ratio), .sup.1H NMR (400 MHz,
Acetone-d.sub.6) .delta. 1.99 (s, 3H), 2.22-2.28 (m, 2H), 3.54-4.06
(m, 3H), 5.04 & 5.11 (s, 1H), 6.80 & 6.90 (d, J=8.8 Hz,
1H), 7.46-7.70 (m, 4H, brs, 1H), 8.73 & 8.76 (s, 2H), 8.95
& 8.99 (s, 1H); .sup.13C NMR (100 MHz, Acetone-d.sub.6)
.delta..quadrature.24.1, 24.2, 30.0, 32.2, 45.2, 47.7, 52.7, 54.7,
75.1, 76.6, 115.9, 120.0, 120.1, 127.7, 127.8, 128.7, 128.8, 131.6,
132.0, 132.4, 132.6, 132.7, 132.8, 139.7, 148.4, 153.1, 165.0,
169.1.
(S)-4-(1-(3,5-Dinitrobenzoyl)pyrrolidin-3-yloxy)benzoic acid
(63)
##STR00077##
[0240] (Two rotamers 1:1 ratio), .sup.1H NMR (400 MHz,
Acetone-d.sub.6) .delta. 2.31-2.42 (m, 2H), 3.61-3.65 (m, 1H),
3.75-4.06 (m, 3H), 5.19 & 5.28 (s, 1H), 7.02 & 7.13 (d,
J=8.8 Hz, 2H), 7.98 & 8.06 (d, J=8.8 Hz, 2H), 8.72 & 8.78
(d, J=2.0 Hz, 2H), 9.02 & 9.05 (s, 1H).
(S)-(3,5-Dinitrophenyl)(3-(2-fluorophenoxy)pyrrolidin-1-yl)methanone
(64)
##STR00078##
[0242] (Two rotamers, 1:1 ratio), .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 2.14-2.24 (m, 2H), 3.50-3.88 (m, 4H), 4.98
& 5.08 (s, 1H), 6.86-7.15 (m, 4H), 8.65 & 8.69 (s, 2H),
8.88 & 8.92 (s, 1H); .sup.13C NMR (100 MHz, DMSO-d.sub.6)
.delta. .quadrature.029.1, 31.1, 44.1, 51.5, 53.6, 76.4, 77.9,
115.7, 115.8, 115.9, 116.0, 117.1 (d, J=22.3 Hz, due to F), 119.2
(d, J=3.7 Hz, due to F), 121.7, 121.83, 121.88, 121.9, 124.2 (d,
J=3.7 Hz, due to F), 127.0, 139.0, 144.1, 144.4, 148.0, 152.8 (d,
J=242.6 Hz, due to F), 164.6, 164.7.
(R)-(3-(2-Fluorophenoxy)pyrrolidin-1-yl)(phenyl)methanone (65)
##STR00079##
[0244] (Two rotamers 1:1 ratio), .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 2.02-2.24 (m, 2H), 3.51-3.91 (m, 4H), 4.85 & 4.98 (s,
1H), 6.86-7.09 (m, 4H), 7.36-7.48 (m, 3H), 7.52 (d, J=5.2, 1H),
7.53 (d, J=5.2 Hz, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta.
.quadrature.30.3, 32.4, 44.3, 47.5, 52.1, 54.8, 78.0, 79.0, 116.8,
117.0, 117.9, 118.6, 122.6, 122.7, 122.9, 123.0, 124.6 (d, J=3.7 Hz
due to F), 127.2, 127.4, 128.5, (d, J=3.7 Hz, due to F), 130.1,
130.3, 136.7, 136.9, 144.7 (d, J=20.1 Hz due to F), 153.8 (d,
J=245.6 Hz, due to F), 155.2, 170.0, 170.2.
(R)-(3-(4-Methoxyphenoxy)pyrrolidin-1-yl)(phenyl)methanone (66)
##STR00080##
[0246] (Two rotamers, 1:1 ratio), .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 1.99-2.21 (m, 2H), 3.48-3.66 (m, 2H), 3.68 & 3.73 (s,
3H), 3.79-3.89 (m, 2H), 4.74 & 4.96 (s, 1H), 6.71 (s, 2H), 6.76
(s, 2H), 7.34 & 7.36 (d, J=5.6 Hz, 3H), 7.46 & 7.52 (d,
J=5.2 Hz, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 30.2,
32.3, 44.4, 47.6, 52.1, 54.8, 55.8, 55.9, 76.0, 114.9, 115.0,
.quadrature. 117.1, 117.3, 127.3, 127.4, 128.50, 128.54, 130.1,
130.2, 136.8, 137.0, 150.9, 151.1, 154.5, 154.6, 169.9, 170.2;
LC-MS (ESI, m/z): 298.1 [M+H].sup.+.
(R)-(3,5-Dinitrophenyl)(3-hydroxypyrrolidin-1-yl)methanone (67)
##STR00081##
[0248] (Two rotamers, 1:1 ratio), .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 1.98-2.11 (m, 2H), 3.23 (brs, 1H), 3.37-3.48 (m, 1H),
3.61-3.79 (m, 3H), 4.47 & 4.56 (s, 1H), 8.62 & 8.67 (s,
2H), 8.99-9.00 (m, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta.
.quadrature.33.0, 34.9, 45.1, 47.6, 55.5, 57.5, 69.4, 70.9, 120.1,
120.2, 127.8, 139.8, 139.9, 148.5, 165.1, 165.3.
(R)-(3-(3-Methoxyphenoxy)pyrrolidin-1-yl)(3-methoxyphenyl)methanone
(68)
##STR00082##
[0250] (Two rotamers, 1:1 ratio, 85%), a pale yellow liquid;
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.97-2.22 (m, 2H),
3.48-3.65 (m, 2H), 3.68 & 3.71 (s, 3H), 3.73 & 3.76 (s,
3H), 3.79-3.89 (m, 2H), 4.74-4.84 (m, 1H), 6.70-6.80 (m, 4H),
6.86-6.92 (m, 1H), 6.99 & 7.01 (s, 1H), 7.04 & 7.08 (s,
1H), 7.21-7.28 (m, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta.
30.2, 32.3, 44.5, 47.7, 52.2, 54.8, 55.6, 55.8, 76.0, 112.6, 112.8,
114.9, 115.0, 116.1, 116.6, 117.1, 117.2, 119.4, 119.6, 179.27,
129.32, 138.1, 150.9, 151.1, 154.5, 159.7, 169.8.
(R)-(3-(4-Methoxyphenoxy)pyrrolidin-1-yl)(3-methoxyphenyl)methanone
(69)
##STR00083##
[0252] (Two rotamers, 1:1 ratio, 83%), a pale yellow liquid;
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.97-2.22 (m, 2H),
3.48-3.65 (m, 2H), 3.68 & 3.71 (s, 3H), 3.73 & 3.76 (s,
3H), 3.79-3.89 (m, 2H), 4.72-4.84 (m, 1H), 6.70-6.80 (m, 4H),
6.86-6.92 (m, 1H), 6.99-7.08 (m, 2H), 7.21-7.28 (m, 1H); .sup.13C
NMR (100 MHz, CDCl.sub.3) .delta. 29.2, 32.1, 44.4, 47.6, 52.1,
54.4, 55.33, 55.62, 75.8, 113.4, 114.7, 116.9, 128.63, 128.75,
129.16, 129.32, 131.9, 150.9, 154.3, 160.9, 169.48, 169.79.
(R)-Methyl 3-(3-(4-methoxyphenoxy)pyrrolidine-1-carbonyl)benzoate
(70)
##STR00084##
[0254] (Two rotamers, 1:1 ratio, 87%), a pale yellow liquid;
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.99-2.24 (m, 2H),
3.45-3.65 (m, 2H), 3.67 & 3.71 (s, 3H), 3.75-3.82 (m, 2H), 3.86
& 3.87 (s, 3H), 4.74-4.86 (m, 1H), 6.72 & 6.80 (m, 4H),
7.40-7.67 (m, 1H), 7.66 & 7.71 (d, J=7.6 Hz, 1H), 8.04 (t,
J=9.0 Hz, 1H), 8.13 & 8.19 (s, 1H); .sup.13C NMR (100 MHz,
CDCl.sub.3) .delta. 29.9, 32.0, 44.3, 47.3, 52.2, 54.5, 55.5, 55.6,
75.7, 114.7, 114.8, 116.9, 117.0, 128.1, 128.2, 128.5, 128.6,
130.9, 134.0, 131.5, 131.6, 136.8, 136.9, 150.5, 150.7, 154.33,
154.38, 166.6, 168.6, 168.9.
(R)-Methyl 4-(3-(4-methoxyphenoxy)pyrrolidine-1-carbonyl)benzoate
(71)
##STR00085##
[0256] (Two rotamers, 1:1 ratio, 85%), a pale yellow liquid;
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.98-2.11 (m, 1H),
2.15-2.25 (m, 1H), 3.42-3.67 (m, 2H), 3.68 & 3.71 (s, 3H),
3.77-3.81 (m, 1H), 3.83-3.88 (m, 1H), 3.86 & 3.88 (s, 3H),
4.73-4.86 (m, 1H), 6.69-6.75 (m, 2H), 6.80 (s, 2H), 7.51 (d, J=8.0
Hz, 1H), 7.57 (d, J=8.4 Hz, 1H), 8.00 (d, J=8.4 Hz, 1H), 8.03 (d,
J=8.4 Hz, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 29.9,
32.0, 44.2, 47.2, 51.9, 52.2, 54.3, 55.5, 55.6, 75.6, 114.7, 114.8,
116.8, 117.0, 127.0, 127.1, 129.5, 129.6, 131.2, 131.3, 140.7,
140.8, 150.5, 150.7, 154.3, 154.4, 168.7, 168.9.
(R)-(3-(4-Methoxyphenoxy)pyrrolidin-1-yl)-3-(trifluoromethyl)phenyl)methan-
one (72)
##STR00086##
[0258] (Two rotamers, 1:1 ratio, 82%), a pale yellow liquid;
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.04-2.15 (m, 1H),
2.21-2.30 (m, 1H), 3.48-3.67 (m, 2H), 3.72 & 3.75 (s, 3H),
3.78-3.90 (m, 2H), 4.79-4.90 (m, 1H), 6.74-6.83 (m, 4H), 7.48-7.55
(m, 1H), 7.64-7.82 (m, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3)
.delta. 30.0, 32.1, 44.5, 47.5, 52.2, 54.6, 55.7, 55.8, 75.8,
114.8, 114.9, 117.0, 117.2, 124.2, 124.3, 129.0, 129.1, 130.4,
130.6, 137.3, 137.4, 150.6, 150.8, 154.5, 154.6, 168.3, 168.6.
(R)-(3-(4-Methoxyphenoxy)pyrrolidin-1-yl)(4-(trifluoromethyl)phenyl)methan-
one (73)
##STR00087##
[0260] (Two rotamers, 1:1 ratio, 55%), a pale yellow solid; .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. 2.03-2.06 (m, 1H), 2.20-2.25 (m,
1H), 3.49-3.70 (m, 2H), 3.72 & 3.75 (s, 3H), 3.81-3.88 (m, 2H),
4.72 & 8.89 (m, 1H), 6.74-6.83 (m, 4H), 7.23-7.50 (m, 4H);
.sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 30.0, 32.2, 44.5, 47.5,
52.2, 54.6, 55.7, 55.8, 75.8, 76.8, 114.9, 117.0, 117.2, 119.8,
120.1, 122.5, 122.6, 125.6, 125.8, 130.0, 130.1, 138.5, 149.1,
150.6, 150.9, 154.5, 168.2.
(R)-(3-(4-Methoxyphenoxy)pyrrolidin-1-yl)-3-(trifluoromethoxy)phenyl)metha-
none (74)
##STR00088##
[0262] (Two rotamers, 1:1 ratio, 67%), a yellow liquid; .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 2.01-2.23 (m, 2H), 3.43-3.68 (m, 2H),
3.69 & 3.72 (s, 3H), 3.72-3.83 (m, 2H), 4.75-4.88 (m, 1H),
6.72-6.82 (m, 4H), 7.58-7.66 (m, 4H); .sup.13C NMR (100 MHz,
CDCl.sub.3) .delta. 29.1, 30.1, 32.2, 38.9, 44.6, 47.6, 52.2, 54.7,
55.8, 75.9, 114.9, 115.0, 117.1, 117.3, 125.5, 125.6, 127.7, 128.8,
150.8, 151.0, 154.6, 154.7, 168.5, 168.6.
(R)-(3-(4-Methoxyphenoxy)pyrrolidin-1-yl)(3-nitrophenyl)methanone
(75)
##STR00089##
[0264] (Two rotamers, 1:1 ratio, 84%), a yellow liquid; .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 2.00-2.24 (m, 2H), 3.48-3.56 (m, 1H),
3.68 & 3.72 (s, 3H), 3.73-3.88 (m, 3H), 4.79-4.89 (m, 1H),
6.71-6.83 (m, 4H), 7.52-7.59 (m, 1H), 7.81 & 7.87 (d, J=7.6H,
1H), 8.22 (t, J=9.8 Hz, 1H), 8.32 & 8.38 (s, 1H); .sup.13C NMR
(100 MHz, CDCl.sub.3) .delta. 29.8, 32.0, 44.5, 47.4, 52.2, 54.5,
55.5, 55.6, 75.6, 77.0, 114.7, 114.8, 116.9, 117.0, 122.2, 122.3,
124.6, 124.7, 129.6, 133.1, 133.2, 138.0, 138.1, 147.8, 150.4,
150.6, 154.3, 154.4, 166.9, 167.2.
(R)-(3-(4-Methoxyphenoxy)pyrrolidin-1-yl)(4-nitrophenyl)methanone
(76)
##STR00090##
[0266] (Two rotamers, 1:1 ratio, 73%), a yellow solid; .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 2.01-2.31 (m, 2H), 3.44-3.69 (m, 2H),
3.72 & 3.75 (s, 3H), 3.80-3.90 (m, 2H), 4.79-4.90 (m, 1H),
6.72-6.82 (m, 4H), 7.63 & 7.70 (d, J=8.0 Hz, 2H), 8.22 &
8.24 (d, J=8.2 Hz, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta.
30.2, 31.9, 44.3, 47.2, 52.0, 55.5, 75.5, 76.7, 114.7, 114.8,
116.8, 116.9, 123.5, 128.0, 128.2, 128.6, 142.4, 142.5, 148.4,
150.3, 150.6, 154.3, 154.4, 167.3, 167.6.
(R)-(3-Fluorophenyl)(3-(4-methoxyphenoxy)pyrrolidin-1-yl)methanone
(77)
##STR00091##
[0268] (Two rotamers, 1:1 ratio, 78%), a pale yellow liquid;
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.01-2.11 (m, 1H),
2.12-2.42 (m, 1H), 3.48-3.69 (m, 2H), 3.71 & 3.74 (s, 3H),
3.78-3.87 (m, 2H), 4.76-4.88 (m, 1H), 6.72-6.82 (m, 4H), 7.05-7.36
(m, 4H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta.29.9, 32.0,
44.3, 47.4, 52.0, 54.5, 55.6, 75.7, 114.4, 114.8, 116.9, 117.1,
122.8, 122.9, 130.1, 130.2, 138.6, 138.7, 150.6, 150.8, 154.4,
154.5, 162.4 (d, J=245 Hz, due to F), 168.3, 168.5.
(R)-(3-Chlorophenyl)(3-(4-methoxyphenoxy)pyrrolidin-1-yl)methanone
(78)
##STR00092##
[0270] (Two rotamers, 1:1 ratio, 87%), a pale yellow liquid;
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.01-2.24 (m, 2H),
3.47-3.69 (m, 2H), 3.71 & 3.74 (s, 3H), 3.78-3.86 (m, 2H),
4.75-4.88 (m, 1H), 6.73-6.82 (m, 4H), 7.26-7.42 (m, 3H), 7.46 &
7.52 (s, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 29.9,
32.0, 44.3, 47.4, 52.0, 54.5, 55.6, 55.7, 75.7, 76.7, 114.7, 114.8,
116.6, 117.1, 125.1, 125.3, 127.3, 127.4, 129.7, 129.8, 130.0,
130.1, 134.3, 138.2, 138.3, 150.5, 150.7, 154.4, 168.1, 168.4
(R)-(3-Hydroxyphenyl)(3-(4-methoxyphenoxy)pyrrolidin-1-yl)methanone
(79)
##STR00093##
[0272] (Two rotamers, 1:1 ratio, 53%), a white liquid; .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 1.96-2.25 (m, 2H), 3.53-3.74 (m, 2H),
3.77 & 3.81 (s, 3H), 3.83-3.94 (m, 2H), 4.73 & 4.87 (m,
1H), 6.72-6.82 (m, 4H), 6.85-6.98 (m, 2H), 7.08-7.20 (m, 2H), 8.21
(brs, 1H);
(R)-(4-Hydroxyphenyl)(3-(4-methoxyphenoxy)pyrrolidin-1-yl)methanone
(80)
##STR00094##
[0274] (Two rotamers, 1:1 ratio, 37%), a white solid; .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 2.03-2.32 (m, 2H), 3.59-3.71 (m, 2H),
3.74 & 3.76 (s, 3H), 3.79-3.93 (m, 2H), 4.80-4.91 (m, 1H),
6.75-6.84 (m, 4H), 7.21-7.24 (m, 2H), 7.56 & 7.62 (d, J=8.0 Hz,
2H), 8.01 & 8.03 (brs, 1H).
(R)-(4-Hydroxy-3-nitrophenyl)(3-(4-methoxyphenoxy)pyrrolidin-1-yl)methanon-
e (81)
##STR00095##
[0276] (Two rotamers, 1:1 ratio, 63%), a yellow liquid; .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 2.01-2.14 (m, 1H), 2.25-2.27 (m, 1H),
3.56-3.65 (m, 2H), 3.72 & 3.74 (s, 3H), 3.81-3.91 (m, 2H),
4.81-4.89 (m, 1H), 6.76 (m, 4H), 7.16 (t, J=9.4 Hz, 1H), 7.78 &
7.84 (d, J=8.4 Hz, 1H), 8.29 & 8.37 (s, 1H); .sup.13C NMR (100
MHz, CDCl.sub.3) .delta. 29.9, 31.8, 45.0, 47.6, 52.6, 54.9, 55.9,
115.1, 117.2, 117.3, 120.4, 124.7, 125.0, 128.8, 133.1, 136.9,
137.0, 151.0, 154.7, 156.4, 166.9, 167.3.
(R)-(3,5-Dichlorophenyl)(3-(4-methoxyphenoxy)pyrrolidin-1-yl)methanone
(82)
##STR00096##
[0278] (Two rotamers, 1:1 ratio, 85%), a pale yellow liquid;
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.02-2.10 (m, 1H),
2.20-2.25 (m, 1H), 3.47-3.70 (m, 2H), 3.72 & 3.74 (s, 3H),
3.75-3.85 (m, 2H), 4.78-4.87 (m, 1H), 6.74-6.82 (m, 4H), 7.34-7.41
(m, 3H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 29.9, 32.0,
44.4, 47.4, 52.1, 54.4, 55.6, 55.7, 75.5, 114.8, 116.9, 125.6,
125.7, 130.0, 135.1, 139.2, 139.3, 150.4, 150.7, 154.4, 154.5,
166.7, 167.0.
(R)-(3,5-Difluorophenyl)(3-(4-methoxyphenoxy)pyrrolidin-1-yl)methanone
(83)
##STR00097##
[0280] (Two rotamers, 1:1 ratio, 75%), a yellow liquid; .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 2.01-2.27 (m, 2H), 3.48-3.67 (m, 2H),
3.71 & 3.74 (s, 3H), 3.77-3.85 (m, 2H), 4.78-4.88 (m, 1H),
6.73-6.87 (m, 5H), 6.99 & 7.06 (d, J=5.6 Hz, 2H); .sup.13C NMR
(100 MHz, CDCl.sub.3) .delta. 29.8, 32.0, 44.4, 47.3, 52.1, 54.9,
55.6, 75.6, 105.3, 105.4, 110.3, 110.4, 110.5, 110.7, 114.8, 116.9,
117.1, 150.2, 154.9, 162.4 (d, J=250 Hz, due to F), 162.5 (d, J=250
Hz, due to F), 167.0,
(R)-(3,5-Bis(trifluoromethyl)phenyl)(3-(4-methoxyphenoxy)pyrrolidin-1-yl)m-
ethanone (84)
##STR00098##
[0282] (Two rotamers, 1:1 ratio, 65%), a yellow liquid; .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 2.08-2.14 (m, 1H), 2.24-2.29 (m, 1H),
3.47-3.67 (m, 2H), 3.71 & 3.74 (s, 3H), 3.76-3.91 (m, 2H),
4.81-4.91 (m, 1H), 6.74-6.83 (m, 4H), 7.90-8.12 (m, 3H); .sup.13C
NMR (100 MHz, CDCl.sub.3) .delta. 29.8, 32.1, 44.6, 47.4, 52.3,
54.5, 55.6, 75.6, 114.8, 114.9, 116, 9, 117.2, 123.7, 124.3, 127.5,
127.7, 131.1, 132.1, 138.5, 138.6, 150.4, 150.7, 154.5, 154.7,
166.5, 166.8.
(R)-(3-(4-Methoxyphenoxy)pyrrolidin-1-yl)(pyridin-3-yl)methanone
(85)
##STR00099##
[0284] (Two rotamers, 1:1 ratio, 82%), a yellow solid; .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 2.00-2.10 (m, 1H), 2.16-2.24 (m, 1H),
3.48-3.58 (m, 1H), 3.64-3.73 (m, 1H), 3.67 & 3.69 (s, 3H),
3.73-3.85 (m, 2H), 4.75-4.85 (m, 1H), 6.69-6.78 (m, 4H), 7.25-7.31
(m, 1H), 7.78 & 7.83 (d, J=7.6 Hz, 1H), 8.57-8.61 (m, 1H), 8.71
& 8.77 (s, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta.
29.7, 31.9, 44.3, 47.2, 51.9, 54.4, 55.49, 55.53, 75.5, 114.66,
114.69, 116.8, 116.9, 123.1, 123.2, 132.3, 134.8, 134.9, 147.9,
148.1, 150.39, 150.63, 150.83, 150.89, 154.2, 154.3, 167.0,
167.3.
(R)-(3-(4-Methoxyphenoxy)pyrrolidin-1-yl)(pyridin-4-yl)methanone
(86)
##STR00100##
[0286] (Two rotamers, 1:1 ratio, 79%), a yellow solid; .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 2.04-2.23 (m, 2H), 3.46-3.67 (m, 2H),
3.70 & 3.72 (s, 3H), 3.73-3.90 (m, 2H), 4.78-4.88 (m, 1H),
6.76-6.82 (m, 4H), 7.34 (s, 1H), 7.40 (s, 1H), 8.66 (d, J=13.2 Hz,
2H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 29.6, 31.7, 44.0,
46.8, 51.7, 53.9, 55.3, 55.4, 75.3, 114.5, 114.6, 116.7, 116.8,
120.9, 121.0, 143.6, 143.7, 149.7, 150.2, 154.1, 154.2, 166.9,
167.1.
(R)-4-(3-(4-Methoxyphenoxy)pyrrolidine-1-carbonyl)pyridine 1-oxide
(87)
##STR00101##
[0288] (Two rotamers, 1:1 ratio, 97%), a yellow solid; .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 2.03-2.11 (m, 1H), 2.21-2.26 (m, 1H),
3.50-3.68 (m, 2H), 3.70 & 3.72 (s, 3H), 3.74-3.88 (m, 2H),
4.79-4.87 (m, 1H), 6.70-6.81 (m, 4H), 7.25-7.41 (m, 2H), 8.17-8.20
(m, 1H), 8.29 & 8.35 (brs, 1H); .sup.13C NMR (100 MHz,
CDCl.sub.3) .delta. 29.8, 32.0, 44, 7, 47.3, 52.3, 54.4, 55.7,
75.4, 114.8, 116.9, 117.0, 124.5, 126.0, 126.1, 135.7, 135.8,
137.9, 138.1, 140.1, 150.3, 154.5, 154.5, 164.1, 164.3.
(R)-4-(3-(4-Methoxyphenoxy)pyrrolidine-1-carbonyl)pyridine-1-oxide
(88)
##STR00102##
[0290] (Two rotamers, 1:1 ratio, 95%), a yellow solid; .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 2.03-2.10 (m, 1H), 2.22-2.27 (m, 1H),
3.52-3.68 (m, 2H), 3.70 & 3.72 (s, 3H), 3.76-3.83 (m, 2H),
4.80-4.87 (m, 1H), 6.70-6.79 (m, 4H), 7.40 (d, J=6.4 Hz, 1H), 7.47
(d, J=6.8 Hz, 1H), 8.13-8.18 (m, 2H); .sup.13C NMR (100 MHz,
CDCl.sub.3) .delta. 29.7, 32.1, 44.7, 47.3, 52.4, 54.4, 55.6, 75.4,
114.8, 116.9, 125.0, 125.1, 132.9, 133.0, 139.1, 150.3, 150.6,
154.4, 154.6, 165.3.
(R)-(3-(4-Methoxyphenoxy)pyrrolidin-1-yl)(pyrimidin-5-yl)methanone
(89)
##STR00103##
[0292] (Two rotamers, 1:1 ratio, 84%), a pale yellow solid; .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. 2.03-2.13 (m, 1H), 2.23-2.28 (m,
1H), 3.52-3.67 (m, 2H), 3.69 & 3.72 (s, 3H), 3.78-3.88 (m, 2H),
4.79-4.89 (m, 1H), 6.70-6.80 (m, 4H), 8.56 & 8.91 (s, 2H), 9.20
& 9.22 (s, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta.
29.8, 32.1, 44.7, 52.3, 54.4, 55.6, 55.7, 75.5, 114.8, 116.9, 117.0
130.2, 130.3, 150.3, 150.6, 154.5, 154.6, 155.5, 155.6, 159.4,
159.5, 164.5.
(3,5-Dinitrophenyl)(4-hydroxypiperidin-1-yl)methanone (90)
##STR00104##
[0294] .sup.1H NMR (400 MHz, Acetone-d.sub.6) .delta. 1.50-1.56 (m,
2H), 1.80-1.90 (m, 2H), 3.30-3.42 (m, 2H), 3.63 (brs, 1H),
3.94-4.05 (m, 3H), 8.61 (d, J=2.0 Hz, 2H), 8.95 (d, J=2.0 Hz, 1H);
.sup.13C NMR (100 MHz, Acetone-d.sub.6) .delta. 33.7, 34.5, 39.5,
44.9, 66.0, 119.1, 127.4, 140.2, 148.8, 165.1.
Methyl 4-(1-(3,5-dinitrobenzoyl)piperidin-4-yloxy)benzoate (91)
##STR00105##
[0296] .sup.1H NMR (400 MHz, Acetone-d.sub.6) .delta. 1.84 (brs,
2H), 1.96 (brs, 2H), 3.31 (brs, 1H), 3.59-3.74 (m, 2H), 3.77 (s,
3H), 3.84-3.96 (m, 1H), 4.63-4.66 (m, 1H), 6.81-6.85 (m, 2H),
7.87-7.90 (m, 2H), 8.50 (d, J=2.0 Hz, 2H), 8.97 (d, J=2.0 Hz,
1H).
(3,5-Dinitrophenyl)(4-(4-methoxyphenoxy)piperidin-1-yl)methanone
(92)
##STR00106##
[0298] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.85-1.98 (m, 4H),
3.35 (brs, 1H), 3.68-3.80 (m, 2H), 3.73 (s, 3H), 3.93 (brs, 1H),
4.49 (brs, 1H), 6.79 (d, J=8.4 Hz, 2H), 6.84 (d, J=8.4 Hz, 2H),
8.57 (s, 2H), 9.03 (s, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3)
.delta. 30.0, 31.2, 39.3, 44.6, 55.8, 71.9, 115.0, 117.9, 119.8,
127.5, 139.6, .quadrature. 148.7, 150.8, 154.6, 165.4.
N-(4-(1-(3,5-Dinitrobenzoyl)piperidin-4-yloxy)phenyl)acetamide
(93)
##STR00107##
[0300] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 1.62-1.96 (m,
4H), 1.97 (s, 3H), 3.48 (m, 3H), 3.93 (brs, 1H), 4.56 (s, 1H), 6.89
(d, J=8.4 Hz, 2H), 7.44 (d, J=8.4 Hz, 2H), 8.64 (s, 2H), 8.33 (s,
1H), 9.74 (s, 1H);
(3,5-Dinitrophenyl)(4-(2-fluorophenoxy)piperidin-1-yl)methanone
(94)
##STR00108##
[0302] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 1.70-2.10 (m,
4H), 3.39-4.11 (m, 4H), 4.59 (m, 1H), 6.86-6.92 (m, 1H), 7.01-7.15
(m, 3H), 8.60 (d, J=2.0 Hz, 2H), 8.89 (d, J=2.0 Hz, 1H); .sup.13C
NMR (100 MHz, DMSO-d.sub.6) .delta.31.1, 31.9, 45.5, 49.6, 75.0,
117.6 (d, .quadrature. J=18.6 Hz, due to F), 119.5, 120.5, 123.3
(d, J=6.7 Hz, due to F), 126.0 (d, J=3.7 Hz, due to F), 128.6,
140.6, 146.1, 149.8, 154.8 (d, J=242.6 Hz, due to F), 166.9.
(3,5-Dinitrophenyl)(4-(2-methoxyphenyl)piperazin-1-yl)methanone
(95)
##STR00109##
[0304] .sup.1H NMR (400 MHz, Acetone-d.sub.6) .delta. 3.02-3.12 (m,
4H), 3.62 (brs, 2H), 3.82 (s, 3H), 3.87 (brs, 2H), 6.85-6.95 (m,
4H), 8.68 (d, J=2.0 Hz, 2H), 8.96 (d, J=2.4 Hz, 1H); LC-MS (ESI,
m/z): 387 [M+H].sup.+.
(3,5-Dinitrophenyl)(4-(4-methoxyphenyl)piperazin-1-yl)methanone
(96)
##STR00110##
[0306] .sup.1H NMR (400 MHz, Acetone-d.sub.6) .delta. 3.08-3.17 (m,
4H), 3.68 (brs, 2H), 3.71 (s, 3H), 3.88 (brs, 2H), 6.82 (d, J=8.8
Hz, 2H), 6.93, (d, J=8.8 Hz, 2H), 8.69 (d, J=2.0 Hz, 2H), 8.98 (d,
J=2.0 Hz, 1H); .sup.13C NMR (100 MHz, Acetone-d.sub.6) .delta.
42.4, 47.7, 50.5, 50.9, 54.9, 114.4, 118.8, 119.3, 127.7, 139.9,
.quadrature. 145.6, 148.8, 154.5, 165.2; LC-MS (ESI, m/z): 387
[M+H].sup.+.
(4-(2-chlorophenyl)piperazin-1-yl)(3,5-dinitrophenyl)methanone
(97)
##STR00111##
[0308] .sup.1H NMR (400 MHz, Acetone-d.sub.6) .delta. 3.09-3.17 (m,
4H), 3.70 (brs, 2H), 3.94 (brs, 2H), 7.07 (t, J=7.6 Hz, 1H), 7.18
(d, J=8 Hz, 1H), 7.30 (t, J=8 Hz, 1H), 7.41 (d, J=8 Hz, 1H), 8.72
(s, 1H), 9.00 (s, 1H); .sup.13C NMR (100 MHz, Acetone-d.sub.6)
.delta. 43.3, 48.7, 51.6, 52.1, 120.0, 122.0, .quadrature. 125.3,
128.5, 128.9, 129.4, 131.4, 140.6, 149.6, 149.8, 166.1; LC-MS (ESI,
m/z): 391 [M+H].sup.+.
##STR00112##
General Procedure for the Synthesis of
t-butyl-benzyloxypyrrolidine-1-carboxylate (D1)
[0309] To a solution of (R)-tert-butyl
3-hydroxypyrrolidine-1-carboxylate (3.2 mmol) in dimethyl formamide
(10 mL) was added sodium hydride (3.2 mmol) and benzyl bromide (3.2
mmol) at 0.degree. C. and the resulting mixture was stirred at room
temperature. After stirring overnight, distilled water (50 mL) was
added and the resulting precipitate was collected by filtration to
afford D1.
General Procedure for the Synthesis of
benzyloxy-pyrrolidinyl-phenylmethanone (D2)
[0310] D1 (0.43 mmol) was dissolved in trifluoro acetic acid (5 mL)
and stirred at room temperature. After 1 h, the reaction mixture
was concentrated in vacuo to afford an amine. To a solution of the
amine in methylene chloride (5 mL) was added triethylamine (0.51
mmol) and a benzoylchloride (0.51 mmol) at 0.degree. C. and the
resulting mixture was stirred at room temperature. After 3 h, the
reaction mixture was diluted with methylene chloride (30 mL) and
washed with 1 M HCl aqueous solution (30 mL), saturated
Na.sub.2CO.sub.3 aqueous solution (30 mL) and brine (30 mL). The
organic layer was dried over anhydrous MgSO.sub.4 and concentrated
in vacuo. The crude product was purified by silica gel flash column
chromatography (3:1 hexanes/ethyl acetate) and recrystallized from
a mixture of hexanes and ethyl acetate to give D2.
(R)-(3-(Benzyloxy)pyrrolidin-1-yl)(3,5-dinitrophenyl)methanone
(98)
##STR00113##
[0312] (Two rotamers, 1:1 ratio, 23%), a white solid; .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 2.18-2.29 (m, 2H), 3.53-3.58 (m, 1H),
3.76-3.93 (m, 3H), 5.12-5.37 (m, 3H), 7.34-7.44 (m, 5H), 8.67 &
8.73 (d, J=1.6 Hz, 2H), 9.08 & 9.09 (d, J=1.6 Hz, 1H).
((R)-3-(3-Chlorobenzyloxy)pyrrolidin-1-yl)(3,5-dinitrophenyl)methanone
(99)
##STR00114##
[0314] (Two rotamers 3:1 ratio, 75%); .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 1.93-2.21 (m, 2H), 3.38-3.83 (m, 4H), 4.13-4.47
(m, 1H), 4.99 & 5.07 (s, 1H), 5.17 & 5.29 (s, 1H),
7.07-7.29 (m, 4H), 8.64 & 8.69 (s, 2H), 8.98 (s, 1H); .sup.13C
NMR (100 MHz, CDCl.sub.3) .delta. 29.8, 32.2, 45.1, 47.6, 52.3,
54.8, 70.3, 70.4, 76.4, 78.0, 120.0, 120.1, 125.5, 125.6, 127.5,
127.7, 127.8, 127.9, 128.1, 129.9, 134.5, 134.6, 139.7, 139.8,
139.9, 148.5, 164.7, 164.8.
((R)-3-(2-Fluorobenzyloxy)pyrrolidin-1-yl)(3,5-dinitrophenyl)methanone
(100)
##STR00115##
[0316] (Two rotamers 1:1 ratio), .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 2.02-2.30 (m, 2H), 3.50 & 3.52 (s, 1H), 3.63-3.94 (m,
3H), 4.24 & 4.33 (s, 1H), 4.48 & 4.56 (d, J=12.0 Hz, 1H),
4.65 (s, 1H), 6.99-7.44 (m, 4H), 8.69 & 8.75 (s, 2H), 9.10 (s,
1H).
((R)-3-(3-(Trifluoromethyl)benzyloxy)pyrrolidin-1-yl)(3,5-dinitrophenyl)me-
thanone (101)
##STR00116##
[0318] (Two rotamers 2:1 ratio), .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 2.06-2.29 (m, 2H), 3.53 & 3.55 (s, 1H), 3.78-3.96 (m,
3H), 4.27 & 4.35 (s, 1H), 4.51 & 4.62 (d, J=12.4 Hz, 1H),
4.65 (s, 1H), 7.47-7.62 (m, 4H), 8.69 & 8.74 (s, 2H), 9.07 (s,
1H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 29.7, 32.1, 45.1,
47.7, 52.4, 54.9, 70.4, 70.5, 76.7, 78.2, 120.1, 124.1, 124.3,
124.83, 124.87, 127.7, 127.8, 129.2, 130.8, 130.9, 138.7, 138.8,
139.7, 139.8, 148.5, 165.0.
(R)-(3,5-Dinitrophenyl)(3-(pyridin-4-ylmethoxy)pyrrolidin-1-yl)methanone
(102)
##STR00117##
[0320] (Two rotamers, 1:1 ratio, 75%), a brown oil; .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 1.99-2.24 (m, 2H), 3.49-3.92 (m, 4H),
4.20-4.28 (m, 1H), 4.41-4.61 (m, 2H), 7.14-7.24 (m, 2H), 8.49-8.56
(m, 2H), 8.67 & 8.70 (d, J=1.6 Hz, 2H), 9.04 (d, J=1.6 Hz, 1H);
.sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 29.7, 32.3, 45.1, 47.6,
52.3, 54.8, 69.4, 69.5, 76.9, 78.5, 120.1, 121.6, 121.7, 121.8,
127.7, 127.8, 139.8, 139.9, 146.6, 146.8, 148.5, 150.1, 150.2,
164.7.
##STR00118##
General Procedure for the Synthesis of
aminopyrrolidinyl-phenyl-methanone (E3)
[0321] To a solution of (S)-(+)-N-Boc-3-pyrrolidinol (2.67 mmol)
and triethylamine (4.01 mmol) in methylene chloride (50 mL) was
added methansulfonyl chloride (4.01 mmol) under ice-bath and the
resulting mixture was further stirred at 4.degree. C. After 2 h,
the residue was diluted with methylene chloride (50 mL) and washed
with water (100 mL) and brine (100 mL). The organic layer was dried
over anhydrous MgSO.sub.4 and concentrated in vacuo. The crude
product was purified by silica gel flash column chromatography (2:1
hexanes/ethyl acetate) to give E1.
[0322] A solution of E1 (0.75 mmol) and an amine (3.75 mmol) was
stirred at 100.degree. C. After stirring overnight, the residue was
dissolved in methylene chloride (30 mL) and washed with water (30
mL) and brine (30 mL). The organic layer was dried over anhydrous
MgSO.sub.4 and concentrated in vacuo. The crude product was
purified by silica gel flash column chromatography (1:1
hexanes/ethyl acetate) to give E2.
[0323] To a solution of E2 (0.96 mmol) in methylene chloride (20
mL) was added trifluoroacetic acid (0.5 mL). After 3 h, the solvent
was removed in vacuo. The reaction mixture was dissolved in
methylene chloride (20 mL) and cooled to 0.degree. C. Triethylamine
(4.83 mmol) and a benzoyl chloride (1.05 mmol) was added. After 2
h, the residue was diluted with methylene chloride (20 mL) and
washed with water (40 mL) and brine (40 mL). The organic layer was
dried over anhydrous MgSO.sub.4 and concentrated in vacuo. The
crude was purified by silica gel flash column chromatography (1:1
hexanes/ethyl acetate) to give E3.
(R)-(3,5-Dinitrophenyl)(3-(4-methoxyphenylamino)pyrrolidin-1-yl)methanone
(103)
##STR00119##
[0325] (Two rotamers, 1:1 ratio, 63%), a brown solid; .sup.1H NMR
(400 MHz, CDCl.sub.3+CD.sub.3OD) .delta. 1.93-2.01 (m, 1H),
2.14-2.30 (m, 1H), 3.26-3.30 & 3.44-3.50 (m, 1H), 3.54-3.72 (m,
2H), 3.61 & 3.68 (s, 3H), 3.80-3.91 (m, 1H), 3.95-4.05 (m, 1H),
6.43 & 6.55 (d, J=8.8 Hz, 2H), 6.62 & 6.70 (d, J=8.8 Hz,
2H), 8.58 & 8.67 (d, J=2.4 Hz, 2H), 8.95-8.99 (m, 1H); .sup.13C
NMR (100 MHz, CDCl.sub.3+CD.sub.3OD) .delta. 30.4, 32.4, 45.2,
47.9, 52.6, 53.0, 54.4, 55.0, 55.8, 55.9, 115.0, 115.1, 115.2,
115.3, 120.1, 127.6, 127.7, 139.6, 140.5, 140.7, 148.5, 148.6,
152.8, 152.9, 165.2, 165.4.
(R)-(3-(4-Butoxyphenylamino)pyrrolidin-1-yl)(3,5-dinitrophenyl)methanone
(104)
##STR00120##
[0327] (Two rotamers, 1:1 ratio, 54%), a brown solid; m.p.
118-120.degree. C.; .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.
0.83-0.98 (m, 3H), 1.39-1.52 (m, 2H), 1.61-1.76 (m, 2H), 2.02-2.05
(m, 1H), 2.24-2.41 (m, 1H), 3.33-3.37 & 3.50-3.63 (m, 2H),
3.66-4.13 (m, 6H), 6.47 & 6.60 (d, J=8.4 Hz, 2H), 6.70 &
6.78 (d, J=8.4 Hz, 2H), 8.66 & 8.74 (s, 2H), 9.05 & 9.08
(s, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 14.0, 14.1,
19.4, 19.5, 30.9, 31.6, 31.7, 32.9, 45.3, 47.9, 52.8, 53.4, 54.6,
55.2, 68.5, 68.6, 115.0, 115.2, 116.0, 116.2, 120.2, 127.8, 127.9,
139.9, 140.1, 140.4, 148.6, 152.7, 164.9, 165.1.
(R)-(3,5-Dinitrophenyl)(3-(4-phenoxyphenylamino)pyrrolidin-1-yl)methanone
(105)
##STR00121##
[0329] (Two rotamers, 1:1 ratio, 60%), a brown solid; .sup.1H NMR
(400 MHz, CDCl.sub.3+CD.sub.3OD) .delta. 2.00-2.06 (m, 1H),
2.18-2.35 (m, 1H), 3.32-3.35 & 3.48-3.54 (m, 1H), 3.61-3.78 (m,
2H), 3.82-4.12 (m, 2H), 6.47 & 6.60 (d, J=8.8 Hz, 2H),
6.77-6.97 (m, 5H), 7.17, 7.24 (m, 2H), 8.63 & 8.69 (d, J=1.6
Hz, 2H), 9.01 & 9.04 (s, 1H); .sup.13C NMR (100 MHz,
CDCl.sub.3+CD.sub.3OD) .delta. 30.6, 32.5, 45.3, 47.9, 52.3, 53.0,
54.0, 55.1, 114.5, 114.8, 117.4, 117.5, 120.2, 121.3, 121.4, 122.4,
122.5, 127.7, 127.8, 129.7, 139.6, 142.8, 143.0, 148.6, 148.8,
165.2, 165.3.
(R)-(3,5-Dinitrophenyl(3-(4-hydroxyphenylamino)pyrrolidin-1-yl)methanone
(106)
##STR00122##
[0331] (Two rotamers, 1:1 ratio, 83%), a yellow solid; .sup.1H NMR
(400 MHz, DMSO-d.sub.6) .delta. 1.78-1.89 (m, 1H), 2.03-2.15 (m,
1H), 3.12-3.17 (m, 1H), 3.37-3.45 (m, 1H), 3.52-3.95 (m, 3H),
5.15-5.23 (m, 1H), 6.36-6.56 (m, 4H), 8.38 & 8.44 (brs, 1H),
8.64 & 8.67 (s, 2H), 8.81 & 8.84 (s, 1H); .sup.13C NMR (100
MHz, DMSO-d.sub.6) .delta. 29.6, 31.3, 44.6, 46.9, 51.6, 51.9,
53.3, 54.1, 113.8, 114.2, 115.6, 115.7, 119.4, 127.4, 127.5, 139.6,
139.7, 140.3, 140.4, 148.0, 148.1, 148.5, 148.7, 164.2.
(R)-(3,5-Dinitrophenyl)-3-(phenylamino)pyrrolidin-1-yl)methanone
(107)
##STR00123##
[0333] (Two rotamers, 1:1 ratio, 80%), a red solid; .sup.1H NMR
(400 MHz, CDCl.sub.13+CD.sub.3OD) .delta. 1.99-2.04 (m, 1H),
2.17-2.33 (m, 1H), 3.28-3.31 & 3.57-3.95 (m, 4H), 4.04-4.11 (m,
1H), 6.46-6.48 (m, 1H), 6.59-6.70 (m, 2H), 7.02-7.14 (m, 2H), 8.60
& 8.67 (s, 2H), 8.98 & 9.01 (s, 1H); .sup.13C NMR (100 MHz,
CDCl.sub.3+CD.sub.3OD) .delta. 30.3, 32.2, 45.1, 47.8, 51.7, 52.8,
53.3, 54.9, 113.2, 113.5, 118.3, 118.4, 120.0, 127.6, 127.7, 129.4,
139.5, 146.3, 146.4, 148.4, 148.5, 165.1, 165.3.
(R)-(3,5-Dinitrophenyl)(3-(pyridin-2-ylamino)pyrrolidin-1-yl)methanone
(108)
##STR00124##
[0335] (Two rotamers, 1:1 ratio, 70%), a yellow solid; .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 2.00-2.44 (m, 2H), 3.38-4.11 (m, 4H),
4.38 & 4.50 (m, 1H), 6.36 & 6.44 (d, J=8.4 Hz, 1H), 6.57
& 6.64 (t, J=6.0 Hz, 1H), 7.37 & 7.44 (t, J=7.8 Hz, 1H),
7.98 & 8.11 (d, J=5.2 Hz, 1H), 8.67 & 8.73 (s, 2H), 9.05
& 9.09 (s, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta.
30.4, 32.6, 45.1, 47.7, 51.7, 52.9, 55.3, 76.7, 101.8, 108.3,
113.7, 119.9, 127.7, 137.5, 137.7, 147.8, 147.9, 148.3, 148.4,
157.2, 157.4, 164.8, 164.9.
(R)-(3-(Cyclohexylamino)pyrrolidin-1-yl)(3,5-dinitrophenyl)methanone
(109)
##STR00125##
[0337] (Two rotamers, 1:1 ratio, 69%), a pale yellow solid; .sup.1H
NMR (400 MHz, CDCl.sub.3+CD.sub.3OD) .delta. 0.99-1.35 (m, 6H),
1.60-1.98 (m, 5H), 2.15-2.32 (m, 1H), 2.39-2.57 (m, 1H), 3.24-3.60
(m, 2H), 3.63-3.73 (m, 2H), 3.81-3.91 (m, 1H), 8.73 & 8.78 (s,
2H), 9.10 (s, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3+CD.sub.3OD)
.delta. 24.7, 24.8, 24.9, 25.6, 25.7, 30.6, 32.4, 33.3, 33.4, 45.2,
47.7, 52.6, 54.5, 54.8, 54.9, 55.1, 119.7, 127.4, 127.5, 139.5,
148.3, 164.9, 165.0.
(R)--N-Cyclohexyl-N-(1-(3,5-dinitrobenzoyl)pyrrolidin-3-yl)-3,5-dinitroben-
zamide (110)
##STR00126##
[0339] (Two rotamers, 1:1 ratio, 15%), a white solid; .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 1.01-1.22 (m, 3H), 1.62-1.86 (m, 6H),
2.18-2.26 (m, 1H), 2.74-2.89 (m, 1H), 3.30-3.35 (m, 1H), 3.50-3.78
(m, 2H), 3.97-4.19 (m, 4H), 8.51 & 8.56 (s, 2H), 8.74 (s, 2H),
9.09-9.10 (m, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 24.8,
24.9, 25.3, 25.5, 27.3, 30.0, 31.8, 45.6, 48.7, 48.9, 50.0, 53.6,
54.8, 60.5, 119.8, 120.0, 126.7, 127.8, 139.8, 140.1, 140.2, 140.4,
148.6, 148.2, 164.4, 164.7, 166.6, 166.7.
(R)-(3-(4-Methoxyphenylamino)pyrrolidin-1-yl)(phenyl)methanone
(111)
##STR00127##
[0341] (Two rotamers, 1:1 ratio, 75%), a pale yellow solid; .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. 1.84-1.88 (m, 1H), 2.08-2.32 (m,
1H), 3.26-3.34 & 3.49-4.03 (m, 5H), 3.69 & 3.72 (s, 3H),
6.48 & 6.50 (d, J=6.4 Hz, 2H), 6.71 & 6.76 (d, J=6.4 Hz,
2H), 7.36-7.51 (m, 5H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta.
30.7, 32.5, 44.5, 47.7, 52.6, 52.7, 54.2, 55.2, 55.8, 55.9, 114.7,
114.9, 115.0, 127.2, 128.3, 130.1, 136.7, 140.8, 141.0, 152.6,
170.0.
(R)-(3-(3-Chlorobenzylamino)pyrrolidin-1-yl)(3,5-dinitrophenyl)methanone
(112)
##STR00128##
[0343] (Two rotamers, 1:1 ratio, 32%) as a pale yellow solid;
.sup.1H NMR (400 MHz, CDCl.sub.3+CD.sub.3OD) .delta. 1.83-1.89 (m,
1H), 2.01-2.08 & 2.14-2.19 (m, 1H), 2.75 (brs, 1H), 3.15-3.19
& 3.35-3.83 (m, 7H), 7.05-7.23 (m, 4H), 8.58 & 8.67 (d,
J=2.0 Hz, 2H), 8.97-8.99 (m, 1H); .sup.13C NMR (100 MHz,
CDCl.sub.3+CD.sub.3OD) .delta. 30.4, 32.1, 45.2, 47.7, 51.2, 51.4,
52.4, 54.9, 55.2, 57.5, 119.8, 126.0, 126.2, 127.2, 127.3, 127.5,
127.6, 127.8, 128.0, 129.7, 129.8, 134.1, 134.2, 139.5, 139.6,
141.3, 141.7, 148.2, 148.3, 164.7, 164.8.
(R)--N-(3-Chlorobenzyl)-N-(1-(3,5-dinitrobenzoyl)pyrrolidin-3-yl)-3,5-dini-
trobenzamide (113)
##STR00129##
[0345] (Two rotamers, 1:1 ratio, 44%), a white solid; .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 2.26-2.35 (m, 2H), 3.56-4.05 (m, 4H),
4.57-4.65 (m, 3H), 7.06-7.15 (m, 2H), 7.24-7.35 (m, 2H), 8.50-8.62
(m, 4H), 8.97-9.02 (m, 2H); LC-MS (ESI, m/z): 599 [M+H].sup.+.
(R)-(3-(Benzylamino)pyrrolidin-1-yl)(3,5-dinitrophenyl)methanone
(114)
##STR00130##
[0347] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.59 (brs, 1H),
1.87-1.94 (m, 1H), 2.06-2.24 (m, 1H), 3.20 (dd, J=4.8, 10.4 Hz,
0.5H), 3.46-3.89 (m, 6.5H), 7.15-7.36 (m, 5H), 8.63 (d, J=2.0 Hz,
1H), 8.71 (d, J=2.0 Hz, 111), 9.03 (t, J=2.0 Hz, 0.5H), 9.06 (t,
J=2.0 Hz, 0.5H).
(R)-(3,5-Dinitrophenyl)(3-(3-(trifluoromethyl)benzylamino)pyrrolidin-1-yl)-
methanone (115)
##STR00131##
[0349] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.51 (brs, 1H),
1.89-1.94 (m, 1H), 2.10-2.28 (m, 1H), 3.24 (dd, J=5.2, 10.0 Hz,
0.5H), 3.45-3.92 (m, 6.5H), 7.40-7.61 (m, 4H), 8.65 (d, J=2.0 Hz,
1H), 8.72 (d, J=2.0 Hz, 1H), 9.06 (t, J=2.0 Hz, 0.5H), 9.08 (t,
J=2.0 Hz, 0.5H).
(R)-(3,5-Dinitrophenyl)-3-(2-fluorobenzylamino)pyrrolidin-1-yl)methanone
(116)
##STR00132##
[0351] (Two rotamers, 1:1 ratio, 75%), a yellow solid; .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 1.89-1.94 (m, 1H), 2.11-2.25 (m, 1H),
3.22-3.89 (m, 7H), 6.93 & 7.02 (t, J=8.6 Hz, 2H), 7.20 &
7.33 (m, 2H), 8.66 & 8.72 (d, J=2.0 Hz, 2H), 9.06 (s, 1H);
.sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 30.7, 32.5, 45.3, 47.8,
51.3, 51.5, 52.7, 55.1, 55.4, 57.7, 115.1, 115.3, 119.8, 119.9,
127.6, 127.7, 129.4, 129.6, 135.4, 135.5, 139.8, 148.3, 148.4,
162.0 (d, J=245 Hz, due to F), 162.1 (d, J=245 Hz, due to F),
164.5, 164.6.
(R)-(3,5-Dinitrophenyl)(3-(2-fluorobenzylamino)pyrrolidin-1-yl)methanone
hydrochloride (117)
##STR00133##
[0353] (Two rotamers, 1:1 ratio, 92%), a white solid; .sup.1H NMR
(400 MHz, CD.sub.3OD+D.sub.2O) .delta. 2.24-2.35 (m, 1H), 2.48-2.63
(m, 1H), 3.48-4.34 (m, 7H), 7.13 & 7.24 (t, J=8.6 Hz, 2H), 7.47
& 7.58 (q, J=7.0 Hz, 2H), 8.73 & 8.8 (d, J=2.0 Hz, 2H),
9.16 (brs, 1H); .sup.13C NMR (100 MHz, CD.sub.3OD+D.sub.2O) .delta.
28.1, 29.7, 45.5, 50.6, 50.7, 51.9, 56.6, 57.7, 81.1, 117.0, 117.1,
127.7, 128.6, 128.7, 133.2, 133.3, 139.0, 147.1, 149.7, 167.5,
167.6.
(R)-(3,5-Dinitrophenyl)(3-(pyridin-4-ylmethylamino)pyrrolidin-1-yl)methano-
ne (118)
##STR00134##
[0355] (Two rotamers, 1:1 ratio, 69%), a yellow solid; .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 1.80 (br, 1H), 1.88-2.23 (m, 2H),
3.23-3.89 (m, 7H), 7.17 & 7.26 (d, J=5.2 Hz, 2H), 8.45 &
8.52 (d, J=5.6 Hz, 2H), 8.65 & 8.69 (d, J=2.0 Hz, 2H), 9.04 (t,
J=2.0 Hz, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 30.2,
32.4, 44.7, 47.7, 52.4, 54.8, 55.9, 76.0, 115.0, 115.1, 117.2,
117.4, 124.4, 124.5, 127.0, 129.1, 130.6, 130.8, 137.5, 137.7,
150.8, 151.0, 154.7, 154.8, 168.5, 168.8.
##STR00135##
General Procedure for the Synthesis of
(R)--N-benzoylpyrrolidinyl-benzamide (F5)
[0356] To a solution of F1 (3.77 mmol) in DMF (15 mL) was added
sodium azide (11.00 mmol) and the resulting mixture was warmed to
70.degree. C. After 3 h, the solvent was removed in vacuo,
dissolved in ethylacetate (50 mL) and washed with water (50 mL) and
brine (50 mL). The organic layer was dried over anhydrous
MgSO.sub.4 and concentrated in vacuo. The crude product was
purified by silical gel flash column chromatography (1:1
hexanes/ethyl acetate) to give F2.
[0357] To a solution of F2 (2.68 mmol) was added 10% palladium on
activated carbon and stirred overnight under hydrogen atmosphere.
The reaction mixture was filtered using cellite 545 and the
resulting filtrate was concentrated in vacuo to give F3.
[0358] To a solution of F3 (0.77 mmol) and triethylamine (1.16
mmol) in methylene chloride (10 mL) was added benzoyl chloride
(1.00 mmol) under ice bath. The reaction mixture was brought up to
room temperature. After 2 h, the reaction mixture was diluted with
methylene chloride (20 mL) and washed with water (30 mL) and brine
(30 mL). The organic layer was dried over anhydrous MgSO.sub.4 and
concentrated in vacuo. The crude product was purified by silica gel
flash column chromatography (2:1 hexanes/ethyl acetate) to give
F4.
[0359] To a solution of F4 (0.59 mmol) in methylene chloride (10
mL) was added trifluoroacetic acid (0.5 mL) and stirred at room
temperature. After 3 h, the solvent was removed in vacuo. The crude
product was dissolved in methylene chloride (10 mL) and
triethylamine (0.41 mL, 2.96 mmol) was added. The reaction mixture
was cooled to 0.degree. C. and then 3,5-dichlorobenzoyl chloride
(0.65 mmol) was added. The resulting mixture was brought up to room
temperature. After 2 h, the solvent was removed in vacuo and the
crude residue was purified by silica gel flash column
chromatography (1:1 hexanes/ethyl acetate) to give F5.
(R)--N-(1-(3,5-Dinitrobenzol)pyrrolidin-3-yl)-3-(trifluoromethoxy)benzamid-
e (119)
##STR00136##
[0361] (Two rotamers, 1:1 ratio, 67%), a pale yellow solid; .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. 2.07-2.18 (m, 1H), 2.29-2.40 (m,
1H), 3.49-3.60 (m, 1H), 3.68-3.76 (m, 1H), 3.87-3.98 (m, 2H),
4.60-4.74 (m, 1H), 7.19-7.60 (m, 5H), 8.51 & 8.59 (s, 2H), 8.91
& 8.96 (s, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta.
29.9, 32.5, 45.3, 48.0, 49.2, 50.8, 51.9, 54.8, 119.9, 120.0,
120.2, 124.3, 125.7, 127.6, 130.2, 135.7, 136.0, 139.4, 148.4,
148.5, 149.2, 164.9, 165.0, 166.5, 166.6.
(R)--N-(1-(3,5-Dinitrobenzoyl)pyrrolidin-3-yl)-4-methoxybenzamide
(120)
##STR00137##
[0363] (Two rotamers, 1:1 ratio, 0.19 g, 76%), a white solid;
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.08-2.15 (m, 1H),
2.35-2.47 (m, 1H), 3.47-4.08 (m, 4H), 3.81 & 3.84 (s, 3H),
4.62-4.64 & 4.77-4.78 (m, 1H), 6.45 & 6.50 (brs, 1H), 6.82
& 6.88 (d, J=8.4 Hz, 2H), 7.62 & 7.72 (d, J=8.4 Hz, 2H),
8.62 & 8.71 (s, 2H), 9.04 & 9.08 (s, 1H); .sup.13C NMR (100
MHz, CDCl.sub.3) .delta..quadrature. 30.0, 32.8, 45.2, 48.0, 48.9,
50.5, 52.2, 55.2, 55.6, 60.6, 113.9, 120.2, 125.6, 126.1, 127.7,
127.8, 129.0, 139.5, 148.5, 162.7, 164.9, 165.0, 167.4.
(R)-3-Chloro-N-(1-(3,5-dinitrobenzoyl)pyrrolidin-3-yl)benzamide
(121)
##STR00138##
[0365] (Two rotamers, 1:1 ratio, 66%), a pale yellow solid; .sup.1H
NMR (400 MHz, DMSO-d.sub.6) .delta. 1.94-2.20 (m, 2H), 3.33-3.83
(m, 4H), 4.42-4.55 (m, 1H), 7.43-7.60 (m, 2H), 7.71-7.90 (m, 2H),
8.66 & 8.69 (d, J=2.0 Hz, 2H, brs, 1H), 8.83-8.86 (m, 1H);
.sup.13C NMR (100 MHz, DMSO-d.sub.6) .delta. 29.3, 31.5, 44.6,
47.0, 48.4, 49.9, 51.1, 53.3, 119.4, 119.5, 126.2, 126.3, 127.0,
127.1, 127.5, 130.2, 130.3, 131.1, 133.0, 133.1, 136.1, 136.3,
139.5, 139.6, 148.0, 164.0, 164.1, 165.0, 165.1.
(S)-1-(3,5-Dinitrobenzoyl)pyrrolidin-3-yl methanesulfonate
(122)
##STR00139##
[0367] (Two rotamers, 1:1 ratio, 92%), a white solid; m.p.
138-140.degree. C.; .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.
2.25-2.46 (m, 2H), 3.03 & 3.10 (s, 3H), 3.59-3.67 &
3.75-4.03 (m, 4H), 5.28-5.40 (m, 1H), 8.68 & 8.73 (s, 2H), 9.08
(s, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 31.1, 33.6,
38.9, 39.0, 44.7, 47.2, 53.3, 55.3, 78.2, 78.6, 120.5, 127.8,
127.9, 139.3, 148.7, 164.8, 165.0; LC-MS (ESI, m/z): 360
[M+H].sup.+.
(R)-1-(3,5-Dinitrobenzoyl)pyrrolidin-3-yl methanesulfonate
(123)
##STR00140##
[0369] (Two rotamers, 1:1 ratio, 89%), a white solid; .sup.1H NMR
(400 MHz, CDCl.sub.3+CD.sub.3OD) .delta. 2.16-2.32 (m, 2H), 2.94
& 3.02 (s, 3H), 3.50-3.91 (m, 4H), 5.19-5.30 (m, 1H), 8.58
& 8.63 (s, 2H), 8.97 (s, 1H); .sup.13C NMR (100 MHz,
CDCl.sub.3+CD.sub.3OD) .delta. 30.7, 33.1, 38.3, 38.4, 44.5, 46.9,
53.0, 55.0, 78.5, 79.0, 120.1, 127.6, 139.0, 148.4, 164.9,
165.0.
Example 7
Derivatization of the Pyridopyrimidinone Compounds
[0370] The pyridopyrimidinone compounds (scaffold VIII; see Table
2) underwent derivatization according to the methods outlined below
(Schemes 8-10). Resulting derivatives were examined for inhibitory
activity using the assay described above and the results are
summarized in Table 3.
##STR00141##
General Procedure for the Synthesis of G1
[0371] 2-Amino-3-picoline (1.0 mmol) was dissolved in diethyl
malonate (1.0 mmol). The solution was heated to 1701' for 12 h.
After cooling, the dark residue was triturated with
CH.sub.2Cl.sub.2 (10 mL). The residual pale solid was collected by
filtration and washed with CH.sub.2Cl.sub.2 to give G1.
General Procedure for the Synthesis of G2
[0372] To a DMF (2.0 mL) was added POCl.sub.3 (3.0 mmol) at
0.degree. C. After the mixture was stirred at 0 for 40 min, a
solution of G1 (1.0 mmol) in DMF (2.0 mL) was added and stirred at
80.degree. C. for 1 h. The mixture was cooled and concentrated in
vacuo. The residue was diluted with water and extracted with
CH.sub.2Cl.sub.2 (10 mL.times.3). The combined organic layers were
washed with brine, dried over MgSO.sub.4 and concentrated. The
residue was purified by flash column chromatography to give G2.
General Procedure for the Synthesis of G3
[0373] To a stirred solution of G2 (1.0 mmol) in THF (2.0 mL) was
added Et.sub.3N (2.0 mmol). The mixture was cooled to 0.degree. C.
After 5 min, an amine (1.0 mmol) was added dropwise and the mixture
was stirred at room temperature overnight. The reaction mixture was
diluted with CH.sub.2Cl.sub.2 (10 mL) and washed with brine (10
mL). The organic layer was dried over anhydrous MgSO.sub.4 and
concentrated in vacuo. The crude product was purified by flash
column chromatography to give G3.
General Procedure for the Synthesis of G4
[0374] G2 (0.5 mmol) was dissolved in 10.4 mL of tert-butyl alcohol
and 2.5 mL of 2-methyl-2-butene. A solution of sodium chlorite
(4.59 mmol) and sodium dihydrogenphosphate (3.46 mmol) in 4.2 mL of
water was added dropwise. The reaction mixture was stirred at room
temperature overnight. Volatile components were then removed under
vacuum, and the residue was dissolved in 10 ml of water and
extracted with two 10 ml portions of hexane. The aqueous layer was
acidified to pH=3 with HCl(aq) and extracted with 10 mL portions of
methylene chloride. The combined organic layers were washed with 20
mL of cold water, dried and concentrated to give G4.
General Procedure for the Synthesis of G5 from G3
[0375] G3 (36.6 .mu.mol) was dissolved in 760 .mu.l of tert-butyl
alcohol and 180 .mu.l of 2-methyl-2-butene. A solution of sodium
chlorite (335 .mu.mol) and sodium dihydrogenphosphate (253 .mu.mol)
in 300 .mu.l of water was added dropwise. The reaction mixture was
stirred at room temperature overnight. Volatile components were
then removed under vacuum and the residue was dissolved in 10 ml of
water and extracted with two 10 ml portions of hexane. The aqueous
layer was acidified to pH=3 with HCl(aq) and extracted with 10 ml
portions of methylene chloride. The combined organic layers were
washed with 20 ml of cold water, dried and concentrated to give
G5.
General Procedure for the Synthesis of G5 from G4
[0376] To a stirred solution of G4 (1.0 mmol) in DMF (2.0 mL) was
added Et.sub.3N (2.0 mmol) and amine (1.5 mmol) and the mixture was
stirred at 60.degree. C. overnight. The reaction mixture was
diluted with CH.sub.2Cl.sub.2 (10 mL) and washed with brine (10
ml). The organic layer was dried over anhydrous MgSO.sub.4 and
concentrated in vacuo. The crude product was purified by
recrystallization from a mixture of hexanes and methylene chloride
to give G5.
General Procedure for the Synthesis of G6
[0377] The solution of 2-amino-3-picoline (4.0 mmol) in a solution
of CH.sub.2Cl.sub.2 (3 mL) and dried pyridine (1 mL) was added
dropwise at room temperature to a stirred solution of ethyl
3-chloro-3-oxo-propionate (5.3 mmol) in CH.sub.2Cl.sub.2 (3 mL) (an
exothermic reaction with emission of white fume occurred during the
addition). The resulting warm mixture was stirred at room
temperature for 30 min and then poured into 30 mL of cold water; an
excess of sodium carbonate was carefully added with stirring and
the mixture was further stirred at room temperature for 1 h. The
organic layer was then collected and the aqueous phase was
extracted several times with CH.sub.2Cl.sub.2. The combined organic
layers were washed with water, dried over anhydrous
Na.sub.2SO.sub.4, and concentrated in vacuo. The crude product was
purified by flash column chromatography to give G6.
General Procedure for the Synthesis of G7
[0378] A mixture of G6 (1.83 mmol), POCl.sub.3 (0.5 mL) and
polyphosphoric acid (137 mg) was heated with stirring at
130.degree. C. for 3 h. After cooling, anhydrous ethanol was added
and the mixture was refluxed for 30 min, then allowed to cool. The
mixture was treated with aqueous sodium carbonate and exhaustively
extracted with CH.sub.2Cl.sub.2 (10 mL.times.3). The combined
layers were washed with water (10 mL), brine (10 mL), dried over
MgSO.sub.4, filtered and concentrated in vacuo. The crude product
was purified by flash column chromatography to give G7.
General Procedure for the Synthesis of G8
[0379] To a solution of G6 (1 mmol) in DMF (0.96 mL) was added
potassium carbonate (5.0 mmol) followed by phenol (1.94 mmol).
After 12 h at 100.degree. C., the solution was allowed to cool to
23.degree. C. The reaction mixture was washed with H.sub.2O (50
mL), and the aqueous layer was extracted with CH.sub.2Cl.sub.2 (20
mL.times.3). The combined organic layers were washed with 1N HCl
(20 mL.times.2), filtered, and concentrated in vacuo. The crude
product was purified by flash column chromatography to give G8.
General Procedure for the Synthesis of G9
[0380] To DMF (2.0 mL) was added POCl.sub.3 (3.0 mmol) at 0.degree.
C. After the mixture was stirred at 0.degree. C. for 40 min, a
solution of G8 (1.0 mmol) in DMF (2.0 mL) was added and stirred at
80.degree. C. for 1 h. The mixture was cooled and concentrated in
vacuo. The residue was diluted with water and extracted with
CH.sub.2Cl.sub.2 (10 mL.times.3). The combined organic layers were
washed with brine, dried over MgSO.sub.4 and concentrated. The
residue was purified by flash column chromatography to give G9.
Ethyl 3-(3-methylpyridin-2-ylamino)-3-oxopropanoate (124)
##STR00142##
[0382] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.25 (t, J=7.0 Hz,
3H), 2.25 (s, 3H), 3.45 (s, 2H), 4.20 (q, J=7.2 Hz, 2H), 7.47 (d,
J=8.4 Hz, 1H), 8.03 (d, J=8.4 Hz, 1H), 8.07 (s, 1H), 9.67 (brs,
1H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 13.9, 17.7, 42.6,
61.7, 113.8, 129.3, 138.8, 147.6, 148.8, 163.5, 168.4.
2-Hydroxy-9-methyl-4H-pyrido[1,2-a]pyrimidin-4-one (125)
##STR00143##
[0384] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 2.48 (s, 3H),
5.44 (s, 1H), 7.20 (t, J=7.0 Hz, 1H), 7.87 (d, J=6.8 Hz, 1H), 8.84
(d, J=6.8 Hz, 1H), 11.52 (brs, 1H).
2-Hydroxy-8-methyl-4H-pyrido[1,2-a]pyrimidin-4-one (126)
##STR00144##
[0386] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 2.50 (s, 3H),
4.88 (s, 1H), 7.20-7.24 (m, 2H), 8.85 (d, J=6.8 Hz, 1H), 11.98 (br
s, 1H); .sup.13C NMR (100 MHz, DMSO-d.sub.6) .delta. 20.6, 80.3,
114.4, 117.1, 127.7, 146.7, 153.5, 155.3, 162.3.
2-Chloro-9-methyl-4H-pyrido[1,2-a]pyrimidin-4-one (127)
##STR00145##
[0388] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.57 (s, 3H), 6.45
(s, 1H), 7.12 (t, J=7.0 Hz, 1H), 7.68 (d, J=6.8 Hz, 1H), 8.93 (d,
J=6.8 Hz, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 18.0,
102.3, 115.8, 125.7, 134.7, 136.9, 150.0, 157.6, 157.9.
2-Chloro-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbaldehyde
(128)
##STR00146##
[0390] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.64 (s, 3H), 7.30
(t, J=7.0 Hz, 1H), 7.92 (d, J=7.2 Hz, 1H), 9.10 (d, J=6.4 Hz, 1H),
10.42 (s, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 17.7,
107.3, 117.7, 127.0, 135.6, 140.6, 150.0, 156.4, 160.2, 187.1.
2-Chloro-8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbaldehyde
(129)
##STR00147##
[0392] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.59 (s, 3H), 7.24
(d, J=7.2 Hz, 1H), 7.52 (s, 1H), 9.09 (d, J=7.2 Hz, 1H), 10.40 (s,
1H).
2-Chloro-7-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbaldehyde
(130)
##STR00148##
[0394] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 2.32 (s, 3H),
7.49 (d, J=8.8 Hz, 1H), 7.78 (d, J=8.8 Hz, 1H), 8.79 (s, 1H), 10.16
(s, 1H).
2-Chloro-6-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbaldehyde
(131)
##STR00149##
[0396] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.11 (s, 3H), 6.98
(d, J=7.2 Hz, 1H), 7.51 (d, J=8.8 Hz, 1H), 7.79 (t, J=8.0 Hz, 1H),
10.29 (s, 1H).
9-Methyl-4-oxo-2-(phenylamino)-4H-pyrido[1,2-a]pyrimidine-3-carbaldehyde
(132)
##STR00150##
[0398] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.44 (s, 3H), 6.89
(t, J=6.8 Hz, 1H), 7.11 (t, J=7.2 Hz, 1H), 7.34 (t, J=7.6 Hz, 2H),
7.62 (d, J=6.4 Hz, 1H), 7.76 (d, J=8.0 Hz, 2H), 8.80 (d, J=6.8 Hz,
1H), 10.27 (s, 1H), 11.67 (brs, 1H); .sup.13C NMR (100 MHz,
CDCl.sub.3) .delta. 18.1, 94.6, 113.6, 121.8, 124.2, 125.9, 128.7,
133.6, 138.1, 138.9, 152.5, 153.8, 160.2, 190.2.
2-(3-Chlorophenylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbal-
dehyde (133)
##STR00151##
[0400] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.50 (s, 3H), 6.97
(t, J=6.8 Hz, 1H), 7.08 (d, J=8.0 Hz, 1H), 7.25 (t, J=8.0 Hz, 1H),
7.42 (d, J=8.0H, 1H), 7.69 (d, J=6.8 Hz, 1H), 8.18 (s, 1H), 8.84
(d, J=6.8 Hz, 1H), 10.27 (s, 1H), 11.72 (brs, 1H).
9-Methyl-4-oxo-2-(3-(trifluoromethoxy)phenylamino)-4H-pyrido[1,2-a]pyrimid-
ine-3-carbaldehyde (134)
##STR00152##
[0402] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.50 (s, 3H), 6.99
(t, J=7.0 Hz, 1H), 7.36 (t, J=8.0 Hz, 1H), 7.42 (d, J=8.0 Hz, 1H),
7.70 (d, J=6.8 Hz, 1H), 8.16 (s, 1H), 8.88 (d, J=8.0 Hz, 1H), 10.32
(s, 1H), 11.86 (brs, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3)
.delta. 18.0, 94.7, 114.2, 114.7, 116.5, 119.7, 126.1, 129.7,
133.8, 139.4, 139.7, 149.4, 152.6, 157.0, 160.1, 190.4.
9-Methyl-4-oxo-2-(3-(trifluoromethyl)phenylamino)-4H-pyrido[1,2-a]pyrimidi-
ne-3-carbaldehyde (135)
##STR00153##
[0404] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.49 (s, 1H), 6.98
(t, J=6.8 Hz, 1H), 7.37 (d, J=7.6 Hz, 1H), 7.45 (d, J=7.6 Hz, 1H),
7.61 (d, J=8.0 Hz, 1H), 7.70 (d, J=6.0 Hz, 1H), 8.61 (s, 1H), 8.87
(d, J=6.8 Hz, 1H), 10.30 (s, 1H), 11.85 (brs, 1H).
2-(4-tert-Butylphenylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-ca-
rbaldehyde (136)
##STR00154##
[0406] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.32 (s, 9H), 2.48
(s, 3H), 6.89 (t, J=7.0 Hz, 1H), 7.37 (d, J=8.4 Hz, 1H), 7.62 (d,
J=6.8 Hz, 1H), 7.73 (d, J=8.8 Hz, 1H), 8.81 (d, J=7.2 Hz, 1H),
10.30 (s, 1H), 11.68 (br s, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3)
.delta. 18.2, 31.3, 34.3, 94.6, 113.5, 121.4, 125.6, .quadrature.
125.9, 133.6, 135.6, 138.8, 147.2, 152.6, 156.7, 160.4, 190.2.
2-(3-Chlorobenzylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbal-
dehyde (137)
##STR00155##
[0408] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.40 (s, 3H), 4.80
(d, J=6.0 Hz, 2H), 6.87 (t, J=7.0 Hz, 1H), 7.24-7.26 (m, 3H), 7.37
(s, 1H), 7.59 (d, J=6.8 Hz, 1H), 8.79 (d, J=7.2 Hz, 1H), 10.34
(brs, 1H), 10.30 (s, 1H).
9-Methyl-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbaldehyde
(138)
##STR00156##
[0410] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.30 (s, 3H), 3.65
(d, J=2.4 Hz, 4H), 3.72 (d, J=3.2 Hz, 4H), 6.74-6.77 (m, 1H), 7.49
(d, J=6.8 Hz, 1H), 8.62 (d, J=7.2 Hz, 1H), 10.01 (s, 1H); .sup.13C
NMR (100 MHz, CDCl.sub.3) .delta. 17.6, 49.5, 67.0, 95.9, 112.9,
125.7, 133.0, 138.1, 150.5, 158.4, 162.3, 186.2
2-(4-(2-Chlorophenyl)piperazin-1-yl)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimi-
dine-3-carbaldehyde (139)
##STR00157##
[0412] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.41 (s, 3H), 3.19
(t, J=4.8 Hz, 4H), 3.92 (t, J=4.6 Hz, 4H), 6.82 (t, J=7.0 Hz, 1H),
6.98 (t, J=7.6 Hz, 1H), 7.04 (d, J=7.2 Hz, 1H), 7.21 (t, J=7.6 Hz,
1H), 7.36 (d, J=7.6 Hz, 1H), 7.55 (d, J=6.4 Hz, 1H), 8.73 (d, J=6.8
Hz, 1H), 10.15 (s, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta.
17.6, 49.3, 51.4, 96.1, 112.7, 120.5, 124.0, 125.8, 127.6, 128.8,
130.6, 133.0, 137.8, 148.7, 150.5, 158.6, 162.5, 186.4.
2-(3,4-Dihydroisoquinolin-2(1H)-yl)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimid-
ine-3-carbaldehyde (140)
##STR00158##
[0414] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.43 (s, 3H), 3.05
(t, J=5.8 Hz, 2H), 4.03 (t, J=5.8 Hz, 2H), 4.73 (s, 2H), 6.78 (t,
J=7.0 Hz, 1H), 7.06-7.17 (m, 4H), 7.52 (d, J=6.8 Hz, 1H), 8.70 (d,
J=7.6 Hz, 1H), 10.21 (s, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3)
.delta. 17.6, 28.7, 46.3, 52.0, 96.1, 112.5, 125.8, 126.2, 126.6,
128.4, 133.0, 133.9, 134.6, 137.5, 150.3, 158.6, 162.3, 186.7.
2-(Isobutylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbaldehyde
(141)
##STR00159##
[0416] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 0.95 (d, J=4 Hz,
6H), 1.90 (m, 1H), 2.37 (s, 3H), 3.41 (t, J=6.8 Hz, 2H), 6.76 (t,
J=6.8 Hz, 1H), 7.24-7.52 (m, 1H), 8.69 (dd, J=0.8, 7.2 Hz, 1H),
9.67 (brs, 1H), 10.22 (s, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3)
.delta. 17.9, 20.4, 28.7, 48.1, 94.4, 112.5, 125.9, 133.2, 138.1,
152.8, 159.5, 160.7, 190.2.
2-(Diethylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbaldehyde
(142)
##STR00160##
[0418] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.25 (t, J=6.8 Hz,
6H), 2.36 (s, 3H), 3.65 (q, J=6.8 Hz, 4H), 6.72 (t, J=6.8 Hz, 1H),
7.47 (d, J=6.8 Hz, 1H), 8.65 (d, J=6.4 Hz, 1H), 10.12 (s, 1H);
.sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 13.2, 17.7, 45.3, 96.2,
112.2, 125.8, 133.0, 137.3, 150.2, 158.5, 162.6, 186.9.
2-(Cyclohexylmethylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carb-
aldehyde (143)
##STR00161##
[0420] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 0.93-1.02 (m, 2H),
1.11-1.25 (m, 3H), 1.57-1.77 (m, 6H), 2.36 (s, 3H), 3.43 (t, J=6.0
Hz, 2H), 6.75 (t, J=7.2 Hz, 1H), 7.50 (d, J=7.2 Hz, 1H), 8.67 (d,
J=6.8 Hz, 1H), 9.65 (brs, 1H), 10.21 (s, 1H); .sup.13C NMR (100
MHz, CDCl.sub.3) .delta. 17.9, 26.0, 26.5, 31.1, 38.2, 47.0, 94.4,
112.5, 125.8, 133.2, 138.0, 152.8, 159.4, 160.6, 190.2
2-Chloro-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic
acid (144)
##STR00162##
[0422] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 2.58 (s, 3H),
7.53 (t, J=7.0 Hz, 1H), 8.14 (d, J=7.2 Hz, 1H), 8.97 (d. J=6.8 Hz,
1H), 13.53 (brs, 1H); .sup.13C NMR (100 MHz, DMSO-d) .delta. 16.7,
108.1, 117.1, 125.6, 133.3, 138.7, 148.2, 152.0, 154.6, 163.9.
2-Chloro-7-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic
acid (145)
##STR00163##
[0424] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 2.49 (s, 3H),
7.76 (d, J=8.8 Hz, 1H), 8.11 (d, J=8.8 Hz, 1H), 8.89 (s, 1H), 13.46
(br s, 1H).
2-Chloro-6-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic
acid (146)
##STR00164##
[0426] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 3.00 (s, 3H),
7.19 (d, J=7.6 Hz, 1H), 7.52 (d, J=8.0 Hz, 1H), 7.92 (t, J=8.0 Hz,
1H), 13.35 (br s, 1H).
9-Methyl-4-oxo-2-(phenylamino)-4H-pyrido[1,2-a]pyrimidine-3-carboxylic
acid (147)
##STR00165##
[0428] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.50 (s, 3H), 6.70
(dd, J=6.8, 7.2 Hz, 1H), 7.15 (dd, J=7.2, 7.2 Hz, 1H), 7.37 (dd,
J=7.2, 7.6 Hz, 2H), 7.65 (d, J=6.8 Hz, 1H), 7.76 (d, J=8.4 Hz, 2H),
8.76 (d, J=7.2 Hz, 1H), 11.70 (brs, 1H), 14.31 (s, 1H).
2-(3-Chlorophenylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbox-
ylic acid (148)
##STR00166##
[0430] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 2.55 (s, 3H),
7.04 (t, J=7.0 Hz, 1H), 7.12 (d, J=8.0 Hz, 1H), 7.28 (J=8.0 Hz,
1H), 7.71 (d, J=8.0 Hz, 1H), 8.17 (s, 1H), 8.79 (d, J=7.6 Hz, 1H),
11.78 (brs, 1H).
2-(3-Chlorophenylamino)-8-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbox-
ylic acid (149)
##STR00167##
[0432] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.49 (s, 3H), 6.93
(d, J=7.6 Hz, 1H), 7.12 (d, J=7.6 Hz, 1H), 7.25-7.29 (m, 2H), 7.46
(d, J=7.2 Hz, 1H), 7.96 (s, 1H), 8.76 (d, J=7.2 Hz, 1H), 11.72 (br
s, 1H), 14.19 (s, 1H).
2-(3-Chlorophenylamino)-7-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbox-
ylic acid (150)
##STR00168##
[0434] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.41 (s, 3H), 7.12
(d, J=8.0 Hz, 1H), 7.27 (t, J=8.6 Hz, 1H), 7.41 (d, J=8.8 Hz, 1H),
7.47 (d, J=7.6 Hz, 1H), 7.96 (s, 1H), 8.68 (s, 1H), 11.70 (br s,
1H), 14.28 (s, 1H).
2-(3-Chlorophenylamino)-6-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbox-
ylic acid (151)
##STR00169##
[0436] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.03 (s, 3H), 6.70
(d, J=6.8 Hz, 1H), 7.10 (d, J=8.0 Hz, 1H), 7.23-7.27 (m, 2H), 7.44
(d, J=8.0 Hz, 1H), 7.56 (t, J=8.0 Hz, 1H), 7.91 (s, 1H), 11.76 (br
s, 1H), 14.37 (s, 1H).
2-(3-Fluorophenylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbox-
ylic acid (152)
##STR00170##
[0438] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.54 (s, 3H),
6.81-6.87 (m, 1H), 7.03 (t, J=7.2 Hz, 1H), 7.28-7.31 (m, 2H), 7.71
(d, J=6.8 Hz, 1H), 7.89 (d, J=10.4 Hz, 1H), 8.79 (d, J=7.2 Hz 1H),
11.83 (b s, 1H), 14.26 (br s, 1H).
9-Methyl-4-oxo-2-(3-(trifluoromethyl)phenylamino)-4H-pyrido[1,2-a]pyrimidi-
ne-3-carboxylic acid (153)
##STR00171##
[0440] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.54 (s, 3H), 7.05
(t, J=7.0 Hz, 1H), 7.40 (d, J=7.6 Hz, 1H), 7.47 (t, J=8.0 Hz, 1H),
7.61 (d, J=8.0 Hz, 1H), 7.73 (d, J=6.8 Hz, 1H), 8.58 (s 1H), 8.81
(d, J=6.8 Hz, 1H), 11.91 (br s, 1H).
9-Methyl-4-oxo-2-(3-(trifluoromethoxy)phenylamino)-4,1-pyrido[1,2-a]pyrimi-
dine-3-carboxylic acid (154)
##STR00172##
[0442] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.58 (s, 3H), 7.00
(d, J=8.0 Hz, 1H), 7.05 (t, J=7.0 Hz, 1H), 7.36 (t, J=8.0 Hz, 1H),
7.42 (d, J=8.0 Hz, 1H), 7.72 (d, J=6.8 Hz, 1H), 8.09 (s, 1H), 8.81
(d, J=7.2 Hz, 1H), 11.89 (br s, 1H), 14.26 (br s, 1H).
9-Methyl-2-(3-nitrophenylamino)-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxy-
lic acid (155)
##STR00173##
[0444] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 2.60 (s, 3H),
7.40 (t, J=7.0 Hz, 1H), 7.73 (t, J=8.2 Hz, 1H), 7.96 (d, J=7.6 Hz,
1H), 8.02 (d, J=7.6 Hz, 1H), 8.13 (d, J=6.8 Hz, 1H), 8.90 (d, J=7.2
Hz, 1H), 9.33 (s, 1H), 11.84 (br s, 1H), 14.43 (br s, 1H).
2-(3-(Methoxycarbonyl)phenylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidi-
ne-3-carboxylic acid (156)
##STR00174##
[0446] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.57 (s, 3H), 3.92
(s, 3H), 7.052 (t, J=6.8 Hz, 1H), 7.43 (t, J=8.0 Hz, 1H), 7.71 (t,
J=7.0 Hz, 2H), 7.82 (d, J=8.0 Hz, 1H), 8.79 (d, J=6.8 Hz, 1H), 8.83
(s, 1H), 11.83 (br s, 1H), 14.28 (br s, 1H).
2-(3-Hydroxyphenylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbo-
xylic acid
##STR00175##
[0448] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 2.55 (s, 3H), 6.61
(d, J=8.0 Hz, 1H), 7.15-7.24 (m, 3H), 7.34 (s, 1H), 7.88 (d, J=6.8
Hz, 1H), 8.82 (d, J=7.2 Hz, 1H).
2-(4-Hydroxyphenylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbo-
xylic acid (158)
##STR00176##
[0450] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 2.45 (s, 3H), 6.81
(d, J=8.8 Hz, 2H), 7.10 (t, J=7.0 Hz, 1H), 7.57 (d, J=8.8 Hz, 1H),
7.81 (d, J=6.8 Hz, 1H), 8.78 (d, J=7.2 Hz, 1H), 11.26 (br s,
1H).
2-(4-tert-Butylphenylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-ca-
rboxylic acid (159)
##STR00177##
[0452] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.33 (s, 9H), 2.49
(s, 3H), 6.95 (t, J=7.0 Hz, 1H), 7.37 (d, J=7.2 Hz, 2H), 7.63 (d,
J=5.6 Hz, 1H), 7.69 (d, J=6.8 Hz, 2H), 8.71 (d, J=6.8 Hz, 1H),
11.64 (br s, 1H) 14.31 (br s, 1H); .sup.13C NMR (100 MHz,
CDCl.sub.3) .delta. 18.2, 31.3, 34.4, 85.3, 114.1, 121.3, 125.5,
125.7, 133.6, 135.4, 138.2, 147.4, 150.2, 157.0, 161.8, 169.7.
2-(3-Chlorobenzylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbox-
ylic acid (160)
##STR00178##
[0454] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.38 (s, 3H), 4.83
(d, J=6.0 Hz, 2H), 7.17 (t, 0.1=7.0 Hz, 1H), 7.32-7.40 (m, 3H),
7.50 (s, 1H), 7.89 (d, J=6.8 Hz, 1H), 8.68 (d, J=7.2 Hz, 1H), 9.82
(d, J=6.2 Hz, 1H), 14.25 (br s, 1H).
2-(Diethylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic
acid (161)
##STR00179##
[0456] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.32 (t, J=6.8 Hz,
6H), 2.41 (s, 3H), 3.68 (q, J=6.8 Hz, 4H), 6.67 (t, J=7.2 Hz, 1H),
7.38 (d, J=6.8 Hz, 1H), 8.71 (d, J=7.2 Hz, 1H), 14.08 (s, 1H);
.sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 13.8, 17.8, 45.4, 96.2,
112.2, 125.8, 133.0, 137.3, 150.2, 158.5, 162.6, 171.6.
2-(Isobutylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic
acid (162)
##STR00180##
[0458] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 0.97 (d, J=6.8 Hz,
6H), 1.93-1.99 (m, 1H), 2.40 (s, 3H), 3.43 (t, J=6.4 Hz, 2H), 6.84
(t, J=7.2 Hz, 1H), 7.53 (d, J=6.4 Hz, 1H), 8.62 (d, J=7.6 Hz, 1H),
9.52 (brs, 1H), 14.12 (s, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3)
.delta. 17.9, 20.4, 28.7, 48.6, 84.8, 113.2, 125.7, 133.2, 137.5,
150.5, 159.7, 162.0, 169.9.
2-(Cyclohexylmethylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carb-
oxylic acid (163)
##STR00181##
[0460] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 0.98-1.05 (m, 2H),
1.13-1.24 (m, 3H), 1.60-1.79 (m, 6H), 2.42 (s, 3H), 3.45 (t, J=6.4
Hz, 2H), 6.83 (t, J=7.2 Hz, 1H), 7.54 (d, J=6.8 Hz, 1H), 8.62 (d,
J=7.2 Hz, 1H), 9.57 (brs, 1H), 14.13 (s, 1H); .sup.13C NMR (100
MHz, CDCl.sub.3) .delta. 18.0, 26.0, 26.2, 31.2, 38.2, 47.4, 84.8,
113.2, 125.7, 133.2, 137.5, 150.5, 159.6, 162.0, 170.0.
2-(Cyclohexylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic
acid (164)
##STR00182##
[0462] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.19-1.42 (m, 5H),
1.56-1.60 (m, 2H), 1.70-1.76 (m, 2H), 1.94-1.98 (m, 2H), 2.38 (s,
3H), 6.79 (t, J=6.8 Hz, 1H), 7.51 (d, J=6.8 Hz, 1H), 8.56 (d, J=6.8
Hz, 1H), 9.42 (d, J=6.8 Hz, 1H), 14.14 (s, 1H); .sup.13C NMR (100
MHz, CDCl.sub.3) .delta. 17.8, 24.7, 25.7, 32.6, 50.0, 84.7, 113.1,
125.6, 133.1, 137.4, 150.5, 158.5, 162.0, 169.9.
2-(Cyclopentylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxyli-
c acid (165)
##STR00183##
[0464] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.54-1.67 (m, 4H),
1.73-1.78 (m, 2H), 2.04-2.10 (m, 2H), 2.42 (s, 3H), 4.51 (q, J=6.8
Hz, 1H), 6.83 (t, J=6.8 Hz, 1H), 7.53 (d, J=6.8 Hz, 1H), 8.59 (d,
J=6.8 Hz, 1H), 9.47 (d, J=6.8 Hz, 1H), 14.15 (s, 1H); .sup.13C NMR
(100 MHz, CDCl.sub.3) .delta. 18.0, 24.1, 33.3, 53.0, 84.8, 113.3,
125.7, 133.3, 137.5, 150.5, 158.9, 162.0, 169.9.
2-(Cycloheptylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxyli-
c acid (166)
##STR00184##
[0466] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.23-1.57 (m, 4H),
1.59-1.68 (m, 4H), 1.69-1.74 (m, 2H), 1.98-2.04 (m, 2H), 2.43 (s,
3H), 4.30-4.36 (m, 1H), 6.83 (t, J=6.8 Hz, 1H), 7.53 (d, J=6.8 Hz,
1H), 8.64 (d, J=6.8 Hz, 1H), 9.53 (d, J=6.8 Hz, 1H), 14.19 (s, 1H);
.sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 18.0, 24.6, 28.1, 34.7,
52.3, 84.8, 113.1, 125.8, 133.2, 137.4, 150.4, 158.3, 162.1,
170.0.
2-(1-(tert-Butoxycarbonyl)piperidin-4-ylamino)-9-methyl-4-oxo-4H-pyrido[1,-
2-a]pyrimidine-3-carboxylic acid (167)
##STR00185##
[0468] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.51 (s, 9H),
1.61-1.65 (m, 2H), 2.01-2.03 (m, 2H), 2.42 (s, 3H), 2.99-3.05 (m,
2H), 3.98-4.00 (m, 2H), 4.26-4.33 (m, 1H), 6.88 (t, J=7.2 Hz, 1H),
7.58 (d, J=6.8 Hz, 1H), 8.67 (d, J=7.2 Hz, 1H), 9.56 (d, J=6.8 Hz),
14.12 (s, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 17.9,
28.6, 31.6, 48.5, 66.4, 79.9, 85.0, 113.5, 125.9, 133.2, 137.8,
150.6, 154.9, 158.9, 162.0, 169.9.
2-(2-(4-Fluorophenoxy)ethylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-
e-3-carboxylic acid (168)
##STR00186##
[0470] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.44 (s, 3H), 4.01
(t, J=5.6 Hz, 2H), 4.15 (t, J=5.6 Hz, 2H), 6.83-6.96 (m, 5H), 7.59
(d, J=6.8 Hz, 1H), 8.68 (d, J=7.2 Hz, 1H), 9.81 (brs, 1H), 14.01
(s, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 18.0, 40.5,
67.1, 85.3, 113.6, 115.8, 115.9, 116.0, 116.1, 125.9, 133.2, 137.9,
150.6, 154.8, 159.8, 161.9, 169.7.
9-Methyl-4-oxo-2-(2-(4-(trifluoromethoxy)phenoxy)ethylamino)-4H-pyrido[1,2-
-a]pyrimidine-3-carboxylic acid (169)
##STR00187##
[0472] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.44 (s, 3H), 4.03
(t, J=5.6 Hz, 2H), 4.18 (t, J=5.6 Hz, 2H), 6.90 (d, J=9.2 Hz, 2H),
6.91 (t, J=6.8 Hz, 1H), 7.11 (d, J=9.2 Hz, 2H), 7.60 (d, J=6.8 Hz,
1H), 9.70 (d, J=7.2 Hz, 1H), 9.82 (brs, 1H), 14.08 (s, 1H);
.sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 18.0, 40.5, 66.9, 77.4,
85.4, 113.7, 115.7, 122.6, 126.0, 133.2, 138.0, 155.8, 157.6,
159.9, 162.0, 169.0, 170.4.
9-Methyl-2-morpholino-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic
acid (170)
##STR00188##
[0474] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.42 (s, 3H), 3.65
(t, J=4.8 Hz, 4H), 3.74 (t, J=4.8 Hz, 4H), 6.86 (t, J=6.8 Hz, 1H),
7.51 (d, J=6.8 Hz, 1H), 8.67 (d, J=6.8 Hz, 1H), 13.98 (s, 1H);
.sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 18.1, 58.4, 64.8, 97.5,
113.6, 124.6, 132.6, 136.0, 148.1, 160.5, 161.7, 171.3.
2-(3,4-Dihydroisoquinolin-2(1H)-yl)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimid-
ine-3-carboxylic acid (171)
##STR00189##
[0476] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.45 (s, 3H), 3.03
(t, J=5.8 Hz, 2H), 4.08 (m, 2H), 4.73 (m, 2H), 6.83 (t, J=7.0 Hz,
1H), 7.06-7.18 (m, 4H), 7.52 (d, J=6.8 Hz, 1H), 8.60 (d, J=7.2 Hz,
1H), 13.73 (br s, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta.
17.6, 28.5, 46.1, 52.4, 86.4, 113.0, 125.5, 126.1, 126.2, 126.6,
128.4, 132.9, 133.7, 134.4, 136.8, 148.1, 159.9, 163.2, 165.3.
2-(4-(2-Chlorophenyl)piperazin-1-yl)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimi-
dine-3-carboxylic acid (172)
##STR00190##
[0478] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.44 (s, 3H), 3.19
(t, J=4.8 Hz, 4H), 3.96 (m, 4H), 6.87 (t, J=7.0 Hz, 1H), 6.98 (t,
J=7.6 Hz, 1H), 7.02 (d, J=8.4 Hz, 1H), 7.20 (t, J=7.8 Hz, 1H), 7.36
(d, J=8.0 Hz, 1H), 7.55 (d, J=6.8 Hz, 1H), 8.66 (d, J=7.2 Hz, 1H),
13.74 (br s, 1H).
2-(3-Chlorophenylamino)-8-(4-methylpiperazin-1-yl)-4-oxo-4H-pyrido[1,2-a]p-
yrimidine-3-carboxylic acid (173)
##STR00191##
[0480] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.2.34 (s, 3H), 2.53
(t, J=4.8 Hz, 4H), 3.54 (t, J=4.8 Hz, 4H), 6.34 (d, J=2.8 Hz, 1H),
6.55 (dd, J=2.8, 8.4 Hz, 1H), 7.04 (d, J=7.2 Hz, 1H), 7.22 (t,
J=8.0 Hz, 1H), 7.49 (dd, J=1.6, 8.0 Hz, 1H), 7.86 (t, J=2.0 Hz,
1H), 8.53 (d, J=8.4 Hz, 1H), 11.5 (s, 1H), 14.18 (s, 1H); .sup.13C
NMR (100 MHz, CDCl.sub.3) .delta. 46.1, 46.4, 54.4, 83.6, 98.8,
105.1, 120.0, 121.9, 124.0, 128.8, 129.9, 134.4, 139.9, 151.4,
155.6, 158.2, 161.8, 170.2.
##STR00192## ##STR00193##
General Procedure for the Synthesis of H2
[0481] 2-Amino-3-picoline (1.0 mmol) was dissolved in diethyl
ethoxymethylenemalonate (1.0 mmol). The solution was heated to
170.degree. C. for 12 h. After cooling, the dark residue was
triturated with EtOAc (10 mL). The residual pale solid was
collected by filtration and washed with EtOAc to give II.
General Procedure for the Synthesis of H2
[0482] To a stirred solution of H1 (0.43 mmol) in H.sub.2O (3.0 mL)
and EtOH (1.0 mL) was added LiOH (0.86 mmol). The mixture was
stirred at room temperature for 3 h. The reaction mixture was
diluted with CH.sub.2Cl.sub.2 (10 mL) and washed with 1 N HCl (10
ml). The organic layer was dried over anhydrous MgSO.sub.4 and
concentrated in vacuo. The crude product was purified by flash
column chromatography to give H2.
General Procedure for the Synthesis of H3
[0483] To a stirred solution of H1 (0.38 mmol) in THF (2.0 mL) was
added LiAlH.sub.4 (0.57 mmol) at 0.degree. C. The reaction mixture
was stirred at 0.degree. C. for 3 h. After reaction was completed,
1N NaOH (2 mL) was added dropwise. The mixture was diluted with
CH.sub.2Cl.sub.2 (10 mL) and washed with H.sub.2O (10 ml). The
organic layer was dried over anhydrous MgSO.sub.4 and concentrated
in vacuo. The crude product was purified by flash column
chromatography to give H3.
General Procedure for the Synthesis of H4
[0484] To a stirred solution of H3 (95 .mu.mol) in CH.sub.2Cl.sub.2
(1.0 mL) was added NaHCO.sub.3 (285 .mu.mol) and Dess-Martin
Periodinane (114 .mu.mol) at 0.degree. C. The mixture was stirred
at 0.degree. C. for 1 h. The reaction mixture was filtered off and
concentrated in vacuo. The crude product was purified by flash
column chromatography to give H4.
General Procedure for the Synthesis of H5
[0485] To a stirred solution of 2-Amino-pyridine (10.6 mmol) in
xylene (10.0 mL) was added diethyl ethoxymethylenemalonate (21.2
mmol). The mixture was stirred at 140 for 3 hr. After reaction was
completed, the residual pale solid was collected by filtration and
washed with diethyl ether to give H5.
General Procedure for the Synthesis of H6
[0486] To a stirred solution of H5 (0.42 mmol) in THF (5.0 mL) was
added triethylamine (0.63 mmol) and p-toluenesulfonylchloride (0.46
mmol) at 0.degree. C. The reaction mixture was stirred at room
temperature for overnight. After reaction was completed, the
mixture was diluted with CH.sub.2Cl.sub.2 (40 mL) and washed with
1N HCl (50 ml), saturated NaHCO.sub.3 (50 ml) and brine (50 ml).
The organic layer was dried over anhydrous MgSO.sub.4 and
concentrated in vacuo. The crude product was purified by flash
column chromatography to give H6.
General Procedure for the Synthesis of 117
[0487] To a stirred solution of H6 (0.25 mmol) in THF (1.2 mL) was
added triethylamine (0.5 mmol) and an amine (0.26 mmol) at
0.degree. C. The reaction mixture was stirred at room temperature
for overnight. After reaction was completed, the mixture was
diluted with CH.sub.2Cl.sub.2 (10 mL) and washed with 1N HCl (10
ml), saturated NaHCO.sub.3 (10 ml) and brine (10 ml). The organic
layer was dried over anhydrous MgSO.sub.4 and concentrated in
vacuo. The crude product was purified by flash column
chromatography to give 117.
General Procedure for the Synthesis of H8
[0488] To a stirred solution of H7 (0.27 mmol) in ethylene glycol
(3.0 mL) was added methylamine (2 N solution in THF 1.3 mL). The
mixture was stirred at 150.degree. C. for 3 hr. The reaction
mixture was added with ethylacetate (10 mL) and the residual pale
solid was collected by filtration and washed with EtOAc. The crude
product was purified by flash column chromatography to give H8.
General Procedure for the Synthesis of H9
[0489] To a stirred solution of H5 (2.13 mmol) in MeOH (8.0 mL) was
added Pd/C (113 mg). The mixture was stirred at room temperature
under H.sub.2 for 3 h. After reaction was completed, the reaction
mixture was filtered off and concentrated in vacuo. The crude
product was recrystallized with EtOAc and hexane (1:4) to give
H9.
General Procedure for the Synthesis of H10
[0490] To a stirred solution of H9 (0.42 mmol) in CH.sub.2Cl.sub.2
(5.0 mL) was added triethylamine (0.63 mmol) and
p-toluenesulfonylchloride (0.46 mmol) at 0.degree. C. The reaction
mixture was stirred at room temperature for overnight. After
reaction was completed, the mixture was diluted with
CH.sub.2Cl.sub.2 (40 mL) and washed with 1N HCl (50 ml), saturated
NaHCO.sub.3 (50 ml) and brine (50 ml). The organic layer was dried
over anhydrous MgSO.sub.4 and concentrated in vacuo. The crude
product was purified by flash column chromatography
(Hexane:EtOAc=1:2) to give H10.
General Procedure for the Synthesis of H11
[0491] To a stirred solution of H10 (0.25 mmol) in THF (2.0 mL) was
added triethylamine (0.5 mmol) and an amine (0.37 mmol) at
0.degree. C. The reaction mixture was stirred at room temperature
for overnight. After reaction was completed, the mixture was
diluted with CH.sub.2Cl.sub.2 (10 mL) and washed with 1N HCl (10
ml), saturated NaHCO.sub.3 (10 ml) and brine (10 ml). The organic
layer was dried over anhydrous MgSO.sub.4 and concentrated in
vacuo. The crude product was purified by flash column
chromatography (Hexane:EtOAc=1:1) to give H11.
General Procedure for the Synthesis of H12
[0492] A solution of G3 (1.0 mmol), an amine (1.1 mmol) and
triethylamine (2.0 mmol) in THF (2 mL) was refluxed for 1 h and
cooled to room temperature. The solvent was evaporated to dryness,
which was extracted with CH.sub.2Cl.sub.2 (20 mL.times.3). The
reaction mixture was washed with 5% sodium bicarbonate. The organic
layer was dried (MgSO.sub.4), filtered, and concentrated in vacuo.
The crude product was purified by flash column chromatography to
give H12.
General Procedure for the Synthesis of H13
[0493] To a solution of G3 (1.1 mmol), an amine (1.0 mmol) in
CH.sub.2Cl.sub.2 (5 mL) were added sodium triacetoxyborohydride
(2.0 mmol) and glacial acetic acid (2.0 mmol) at room temperature
for 20 h. The reaction mixture was added saturated ammonium
chloride solution and stirred for 10 min. The reaction mixture was
extracted with CH.sub.2Cl.sub.2 (20 mL). The organic layer was
dried (MgSO.sub.4), filtered, and concentrated in vacuo. The crude
product was purified by flash column chromatography to give
H13.
Ethyl 9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylate
(174)
##STR00194##
[0495] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.39 (t, J=7.2 Hz,
3H), 2.62 (s, 3H), 4.39 (q, J=7.2 Hz, 2H), 7.20 (t, J=7.2 Hz, 1H),
7.77 (d, J=7.2 Hz, 1H), 9.05 (s, 1H), 9.16 (d, J=7.2 Hz, 1H);
.sup.13C NMR (100 MHz, CDCl.sub.3) 14.6, 18.2, 61.2, 105.3, 116.8,
127.0, 135.9, 138.2, 155.3, 158.4, 165.0, 189.1.
9-Methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid
(175)
##STR00195##
[0497] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.56 (s, 3H), 7.12
(t, J=6.8 Hz, 1H), 7.79 (d, J=6.8 Hz, 1H), 8.87 (s, 1H), 9.21 (d,
J=7.2 Hz), 14.13 (s, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3)
.delta.18.3, 110.9, 117.1, 128.1, 137.6, 141.1, 155.0, 157.1,
158.3, 171.3.
3-(Hydroxymethyl)-9-methyl-4H-pyrido[1,2-a]pyrimidin-4-one
(176)
##STR00196##
[0499] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.51 (s, 3H), 3.27
(brs, 1H), 4.66 (s, 2H), 7.01 (t, J=6.8 Hz, 1H), 7.51 (d, J=6.8 Hz,
1H), 8.32 (s, 1H), 8.87 (s, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3)
18.2, 44.1, 111.2, 117.9, 127.1, 135.7, 139.8, 153.9, 155.6,
158.2.
9-Methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbaldehyde (177)
##STR00197##
[0501] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.63 (s, 3H), 7.29
(t, J=7.2 Hz, 1H), 7.86 (d, J=7.2 Hz, 1H), 8.85 (s, 1H), 9.14 (d,
J=7.2 Hz, 1H), 10.33 (s, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3)
.delta. 18.2, 110.9, 117.5, 126.7, 136.5, 139.5, 153.1, 155.6,
158.1, 188.5.
Ethyl 2-hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylate
(178)
##STR00198##
[0503] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.42 (t, J=7.2 Hz
3H), 4.45 (q, J=7.2 Hz, 2H), 7.13 (ddd, J=1.2, 6.8, 7.2 Hz, 1H),
7.49 (d, J=8.8 Hz, 1H), 7.82-7.86 (m, 1H), 9.00 (d, J=7.2 Hz, 1H),
13.64 (brs, 1H, NH); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta.
14.2, 62.3, 87.1, 115.3, 125.1, 128.7, 140.3, 148.4, 152.6, 155.5,
171.7.
2-Hydroxy-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylic acid
(179)
##STR00199##
[0505] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.50 (s, 3H), 6.70
(dd, J=6.8, 7.2 Hz, 1H), 7.15 (dd, J=7.2, 7.2 Hz, 1H), 7.37, (dd,
J=7.2, 7.6 Hz, 1H), 7.65 (d, J=6.8 Hz, 1H), 7.76 (d, J=8.4 Hz, 1H),
8.76 (d, J=7.2 Hz, 1H), 11.70 (brs, 1H), 14.31 (s, 1H).
Ethyl
4-oxo-2-(phenylamino)-4H-pyrido[1,2-a]pyrimidine-3-carboxylate
(180)
##STR00200##
[0507] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.45 (t, J=7.2 Hz,
3H), 4.44 (q, J=7.2 Hz, 2H), 6.93 (dd, J=6.8, 6.8 Hz, 1H),
7.29-7.36 (m, 3H), 7.65-7.68 (m, 3H), 8.97 (d, J=7.2 Hz, 1H), 11.39
(brs, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 14.4, 61.0,
85.5, 113.6, 122.5, 124.2, 124.5, 128.4, 128.6, 138.4, 139.0,
151.6, 155.9, 159.5, 169.6.
Ethyl
2-(3-hydroxyphenylamino)-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxyl-
ate (181)
##STR00201##
[0509] .sup.1H NMR (400 MHz, CDCl.sub.3+CD.sub.3OD) .delta. 1.38
(t, J=7.0 Hz, 3H), 4.37 (q, J=7.2 Hz, 2H), 6.56-6.58 (m, 1H), 6.92
(dd, J=6.8, 7.2 Hz, 1H0, 7.05 (d, J=8.4 Hz, 1h0, 7.12 (dd, J=8.0,
8.0 Hz, 1H), 7.26 (m, 1H), 7.31 (d, J=8.8 Hz, 1H), 7.66 (dd, J=7.2,
7.6 Hz, 1H), 8.90 (d, J=7.2 Hz, 1H), 11.22 (brs, 1H).
Ethyl
2-(2-hydroxyphenylamino)-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxyl-
ate (182)
##STR00202##
[0511] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.45 (t, J=7.2 Hz,
3H), 4.45 (q, J=6.8 Hz, 2H), 6.90 (dd, J=7.2, 8.0 Hz, 1H),
7.05-7.08 (m, 2H), 7.13 (dd, J=7.6, 8.4 Hz, 2H), 7.37 (d, J=8.4 Hz,
1H), 7.81 (dd, J=7.6, 8.0 Hz, 1H), 9.03 (d, J=6.8 Hz, 1H), 11.52
(brs, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) 14.4, 61.3, 114.7,
120.1, 120.5, 122.9, 124.4, 127.0, 127.1, 129.0, 140.8, 149.3,
151.1, 158.6, 169.5.
Ethyl
2-(3-nitrophenylamino)-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylat-
e (183)
##STR00203##
[0513] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.46 (t, J=6.4 Hz,
3H), 4.45 (q, J=7.2 Hz, 2H), 7.05 (ddd, J=1.2, 6.8, 6.8 Hz, 1H),
7.43 (d, J=8.8 Hz, 1H), 7.47 (dd, J=8.0, 8.4 Hz, 2H), 7.77-7.82 (m,
2H), 7.93-7.96 (m, 1H), 8.97-8.98 (m, 1H), 9.04 (dd, J=0.8, 7.2 Hz,
1H), 11.74 (brs, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) 14.4,
61.3, 86.1, 114.5, 116.9, 118.4, 124.7, 127.4, 128.6, 129.2, 139.8,
148.5, 151.5, 155.7, 159.5, 169.6.
Ethyl 4-oxo-2-phenoxy-4H-pyrido[1,2-a]pyrimidine-3-carboxylate
(184)
##STR00204##
[0515] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.38 (t, J=7.2 Hz,
3H), 4.42 (q, J=7.2 Hz, 2H), 7.15-7.17 (m, 3H), 7.24 (d, J=6.4 Hz,
1H), 7.36-7.41 (m, 3H), 7.77 (ddd, J=1.6, 6.8, 6.8 Hz, 1H), 9.10
(dd, J=0.8, 6.8 Hz, 1H); ); .sup.13C NMR (100 MHz, CDCl.sub.3)
.delta. 14.2, 61.3, 115.7, 121.8, 125.3, 128.5, 129.2, 128.7,
150.3, 152.5, 156.7, 164.1, 165.0.
Ethyl
2-(3-fluorophenoxy)-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylate
(185)
##STR00205##
[0517] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.37 (t, J=7.0 Hz,
3H), 4.40 (q, J=6.8 Hz, 2H), 6.91-6.98 m, 3H), 7.19 (ddd, J=1.2,
7.2, 7.2 Hz, 1H), 7.32-7.36 (m, 1H), 7.39 (d, J=8.8 Hz, 1H),
7.78-7.82 (m, 1H), 9.10 (d, J=6.8 Hz, 1H); .sup.13C NMR (100 MHz,
CDCl.sub.3) .delta. 14.2, 61.4, 94.6, 109.8, 110.0, 112.2, 112.4,
115.9, 117.5, 117.6, 125.3, 128.5, 129.8, 129.9, 139.9, 150.3,
153.3, 156.6, 161.6, 163.8, 164.0, 164.5.
Ethyl
4-oxo-2-(3-(trifluoromethyl)phenoxy)-4H-pyrido[1,2-a]pyrimidine-3-ca-
rboxylate (186)
##STR00206##
[0519] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.39 (t, J=7.2 Hz,
3H), 4.43 (q, J=7.0 Hz 2H), 7.21 (dd, J=6.8, 6.8 Hz, 1H), 7.38 (d,
J=8.0 Hz, 2H), 7.47-7.52 (m, 2H), 7.81 (dd, J=7.2, 8.4 Hz, 1H),
9.12 (d, J=6.8 Hz, 1H).
Methyl
2-chloro-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carboxylate
(187)
##STR00207##
[0521] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.56 (s, 3H), 3.93
(s, 3H), 7.19 (t, J=7.2 Hz, 1H), 7.75 (d, J=6.8 Hz, 1H), 8.91 (d,
J=7.2 Hz, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 17.1,
52.8, 108.0, 116.7, 126.1, 134.9, 138:3, 149.1, 155.1, 155.2,
164.2.
Methyl
2-(3-chlorophenylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-
-carboxylate
##STR00208##
[0523] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.51 (s, 3H), 3.99
(s, 3H), 6.94 (t, J=7.0 Hz, 1H), 7.09 (d, J=7.6 Hz, 1H), 7.27 (d,
J=8.4 Hz, 1H), 7.41 (d, J=8.0 Hz, 1H), 7.64 (d, J=6.8 Hz, 1H), 8.18
(s, 1H), 8.91 (d, J=7.2 Hz, 1H), 11.52 (br s, 1H); .sup.13C NMR
(100 MHz, CDCl.sub.3) .delta. 18.0, 52.1, 85.3, 113.7, 119.6,
121.9, 123.5, 126.4, 129.4, 133.2, 134.1, 138.4, 139.9, 151.0,
156.2, 158.6, 170.1.
Methyl
2-(3-chlorobenzylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-
-carboxylate (189)
##STR00209##
[0525] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.35 (s, 3H), 3.92
(s, 3H), 4.77 (d, J=6.0 Hz, 2H), 6.80 (t, J=6.8 Hz, 1H), 7.20-7.24
(m, 3H), 7.34 (s, 3H), 7.50 (d, J=6.8 Hz, 1H), 8.82 (d, J=7.2 Hz,
1H), 9.69 (br s, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta.
17.8, 44.4, 51.8, 84.6, 112.6, 125.5, 126.4, 127.2, 127.7, 129.7,
132.7, 134.3, 137.6, 141.1, 151.3, 156.4, 160.8, 170.1.
Ethyl
2-hydroxy-4-oxo-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidine-3-carb-
oxylate (190)
##STR00210##
[0527] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.36 (t, J=7.2 Hz,
3H), 1.82-1.93 (m, 4H), 2.86 (t, J=6.8 Hz, 2H), 3.84 (t, J=6.0 Hz,
2H), 4.39 (q, J=7.2 Hz, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3)
.delta. 14.4, 18.9, 21.9, 32.2, 43.0, 62.4, 90.9, 159.8, 165.1,
171.7, 173.5.
Ethyl
4-oxo-2-(tosyloxy)-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidine-3-c-
arboxylate (191)
##STR00211##
[0529] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.25 (t, J=7.2 Hz,
3H), 1.79-1.91 (m, 4H), 2.41 (s, 3H), 2.79 (t, J=6.4 Hz, 2H), 3.84
(t, J=6.4 Hz, 2H), 4.25 (q, J=7.2 Hz, 2H), 7.31 (d, J=8.0 Hz, 2H),
7.89 (d, J=8.0 Hz, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta.
14.2, 18.8, 21.6, 21.9, 31.8, 43.6, 61.9, 104.2, 129.1, 129.7,
134.2, 145.8, 159.4, 160.8, 162.0, 162.2.
Ethyl
4-oxo-2-(phenylamino)-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrimidine--
3-carboxylate (192)
##STR00212##
[0531] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.40 (t, J=7.2 Hz,
3H), 1.80-1.92 (m, 4H), 2.80 (t, J=6.8 Hz, 2H), 3.87 (t, J=6.0 Hz,
2H), 4.36 (q, J=7.2 Hz, 2H), 7.08 (t, J=7.2 Hz, 1H), 7.29 (t, J=7.2
Hz, 2H), 7.53 (d, J=7.6 Hz, 2H), 11.2 (s, 1H); .sup.13C NMR (100
MHz, CDCl.sub.3) .delta. 14.6, 19.2, 22.2, 32.2, 42.4, 61.0, 88.4,
122.9, 124.4, 128.8, 138.4, 160.5, 160.8, 162.2, 169.8.
Ethyl
2-(3-chlorophenylamino)-4-oxo-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyr-
imidine-3-carboxylate (193)
##STR00213##
[0533] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.32 (t, J=7.2 Hz,
3H), 1.76-1.88 (m, 4H), 2.76 (t, J=6.8 Hz, 2H), 3.78 (t, J=6.0 Hz,
2H), 4.29 (q, J=7.06 (dd, J=7.2 Hz, 2H), J=1.2, 8.0 Hz, 1H), 7.27
(t, J=8.0 Hz, 1H), 7.51 (dd, J=1.2, 8.0 Hz, 1H), 7.58 (d, J=2.0 Hz,
1H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 14.3, 18.6, 22.1,
32.1, 42.6, 61.1, 81.4, 111.2, 111.7, 113.0, 128.4, 140.4, 149.6,
158.7, 161.12, 163.2, 170.4.
Ethyl
4-oxo-2-(3-(trifluoromethyl)phenylamino)-6,7,8,9-tetrahydro-4H-pyrid-
o[1,2-a]pyrimidine-3-carboxylate (194)
##STR00214##
[0535] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.45 (t, J=7.2 Hz,
3H), 1.88-1.97 (m, 4H), 2.87 (t, J=6.4 Hz, 2H), 3.93 (t, J=5.6 Hz,
2H), 4.41 (q, J=7.2 Hz, 2H), 7.35 (t, J=7.2 Hz, 1H), 7.35 (d, J=7.6
Hz, 1H), 7.67 (d, J=7.6 Hz, 1H), 8.05 (s, 1H), 11.2 (s, 1H);
Ethyl
2-(2-hydroxyphenylamino)-4-oxo-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]py-
rimidine-3-carboxylate 195)
##STR00215##
[0537] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.40 (t, J=7.2 Hz,
3H), 1.81-1.94 (m, 4H), 2.65 (t, J=6.8 Hz, 2H), 3.65 (t, J=6.0 Hz,
2H), 4.18 (q, J=6.8 Hz, 2H), 6.85 (t, J=7.2 Hz, 1H), 7.00 (d, J=7.2
Hz, 1H), 7.06-7.12 (m, 2H), 9.98 (s, 1H), 11.3 (s, 1H); .sup.13C
NMR (100 MHz, CDCl.sub.3) .delta. 14.6, 18.8, 21.9, 31.6, 42.6,
61.3, 88.4, 120.2, 120.7, 124.5, 127.1, 127.2, 149.1, 159.4, 159.5,
163.0, 169.6.
Ethyl
2-(3-hydroxyphenylamino)-4-oxo-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]py-
rimidine-3-carboxylate (196)
##STR00216##
[0539] .sup.1H NMR (400 MHz, CDCl.sub.3+MeOD-d.sub.4) .delta. 1.26
(t, J=7.2 Hz, 3H), 1.71-1.81 (m, 4H), 2.72 (t, J=6.4 Hz, 2H), 3.74
(t, J=6.4 Hz, 2H), 4.23 (q, J=7.2 Hz, 2H), 6.47 (d, J=7.6 Hz, 1H),
6.88 (d, J=8.0 Hz, 1H), 6.99 (d, J=8.0 Hz, 1H), 7.02 (t, J=2.0 Hz,
1H); .sup.13C NMR (100 MHz, CDCl.sub.3+MeOD-d.sub.4) .delta. 14.2,
18.8, 21.9, 31.8, 42.4, 60.9, 79.8, 109.8, 111.6, 114.0, 129.4,
139.4, 149.7, 159.3, 160.2, 163.1, 169.6.
Ethyl
2-(4-hydroxyphenylamino)-4-oxo-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]py-
rimidine-3-carboxylate (197)
##STR00217##
[0541] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 1.21 (t, J=7.2
Hz, 3H), 1.67-1.80 (m, 4H), 2.65 (t, J=6.8 Hz, 2H), 3.65 (t, J=6.0
Hz, 2H), 4.18 (q, J=7.2 Hz, 2H), 6.68 (d, J=8.8 Hz, 2H), 7.25 (d,
J=8.8 Hz, 2H), 9.29 (s, 1H), 10.7 (s, 1H); .sup.13C NMR (100 MHz,
CDCl.sub.3) .delta. 14.9, 18.9, 21.9, 32.1, 42.3, 60.4, 87.2,
115.7, 125.0, 130.1, 154.9, 159.4, 160.6, 163.3, 169.6.
2-(3-Chloro-4-fluorophenylamino)-9-methyl-4-oxo-4H-pyrido[1,2-a]pyrimidine-
-3-carboxamide (198)
##STR00218##
[0543] mp=218.degree. C. (decomp.); .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 2.97 (d, J=4.8 Hz, 3H), 4.41 (s, 3H), 6.89 (dd,
J=7.2 Hz, 7.2 Hz, 1H), 6.97 (dd, J=1.2 Hz, 8.0 Hz, 1H), 7.05 (dd,
J=8.8 Hz, 8.8 Hz, 1H), 7.40-7.44 (m, 1H), 8.46-8.51 (m, 2H), 8.82
(d, J=2.0 Hz, 1H), 12.98 (s, 1H);
(E)-2-(3-Chlorophenylamino)-3-((cyclohexylimino)methyl)-4H-pyrido[1,2-a]py-
rimidin-4-one (199)
##STR00219##
[0545] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.23-1.37 (m, 3H),
1.41-1.50 (m, 2H), 1.56-1.59 (m, 1H), 1.73-1.76 (m, 4H), 3.16-3.22
(m, 1H), 6.85 (ddd, J=1.2, 6.8, 6.8 Hz, 1H), 6.94 (ddd, J=0.8, 1.2,
8.0 Hz, 1H), 7.14 (dd, J=8.0, 8.0 Hz, 1H), 7.38 (ddd, J=0.8, 1.2,
8.0 Hz, 1H), 7.54-7.58 (m, 1H), 7.90-7.91 (m, 1H), 8.83 (s, 1H),
8.85 (dd, J=0.8, 1.2 Hz, 1H), 13.40 (brs, 1H); .sup.13C NMR (100
MHz, CDCl.sub.3) .delta. 24.4, 25.6, 34.9, 68.4, 91.6, 113.4,
119.2, 121.2, 123.0, 124.7, 127.6, 129.5, 134.2, 137.6, 140.8,
150.6, 156.3, 157.0, 158.3.
(E)-2-(3-Chlorophenylamino)-3-((3-chlorophenylimino)methyl)-4H-pyrido[1,2--
a]pyrimidin-4-one (200)
##STR00220##
[0547] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.01 (dd, J=0.8,
1.2, 8.0 Hz, 1H), 7.28 (d, J=8.4 Hz, 1H), 7.29 (dd, J=2.0, 4.0 Hz,
1H), 7.33 (d, J=8.0 Hz, 1H), 7.44 (d, J=8.8 Hz, 1H), 7.52 (ddd,
J=0.8, 1.2, 8.0 Hz, 1H), 7.17-7.76 (m, 1H), 8.02-8.04 (m, II), 8.98
(dd, J=0.8, 6.8 Hz, 1H), 9.17 (s, 1H), 12.94 (brs, 1H); .sup.13C
NMR (100 MHz, CDCl.sub.3) .delta. 92.6, 114.0, 119.5, 119.8, 121.8,
123.9, 125.0, 125.7, 128.0, 129.7, 130.2, 134.4, 134.8, 138.7,
140.1, 151.3, 151.8, 157.0, 158.0, 158.9.
2-(3-Chlorophenylamino)-3-((cyclopentylamino)methyl)-4H-pyrido[1,2-a]pyrim-
idin-4-one (201)
##STR00221##
[0549] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.54-1.57 (m, 2H),
1.74-1.83 (m, 4H), 2.05-2.08 (m, 2H), 3.23-3.24 (m, 1H), 4.19 (s,
2H), 6.93-6.98 (m, 2H), 7.11-7.15 (m, 1H), 7.32 (d, J=8.4 Hz, 1H),
7.51 (dd, J=2.0, 8.4 Hz, 1H), 7.61-7.65 (m, 1H), 7.74-7.75 (m, 1H),
8.73 (d, J=7.2 Hz, 1H).
2-(3-Chloroophenylamino)-3-((cyclohexylamino)methyl)-4H-pyrido[1,2-a]pyrim-
idin-4-one (202)
##STR00222##
[0551] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.20-1.35 (m, 4H),
1.66-1.72 (m, 2H), 1.86-1.89 (m, 2H), 2.23-2.39 (m, 2H), 3.12-3.18
(m, 1H), 6.93 (ddd, J=1.2, 6.8, 7.2 Hz, 1H), 6.99 (ddd, J=0.8, 1.2,
7.6 Hz, 1H), 7.20 (dd, J=8.0, 8.0 Hz, 1H), 7.25 (d, J=8.8 Hz, 1H),
7.52-7.57 (m, 1H), 7.61 (dd, J=1.2, 8.0 Hz, 1H), 7.84-7.85 (m, 1H),
8.76 (d, J=6.4 Hz, 1H), 9.77 (brs, 1H); .sup.13C NMR (100 MHz,
CDCl.sub.3) .delta. 24.6, 25.0, 41.2, 57.9, 88.9, 114.6, 119.2,
121.1, 122.8, 124.6, 127.3, 129.4, 133.7, 137.3, 140.8, 149.6,
157.2, 158.8.
2-(3-Chlorophenylamino)-3-((cycloheptylamino)methyl)-4H-pyrido[1,2-a]pyrim-
idin-4-one (203)
##STR00223##
[0553] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.40-1.59 (m, 6H),
1.72-1.81 (m, 4H), 2.18-2.23 (m, 2H), 3.07-3.12 (m, 1H), 4.05 (m,
2H), 6.82 (ddd, J=1.2, 6.8, 6.8 Hz, 1H), 6.91 (dd, J=1.2, 8.0 Hz,
1H), 7.14 (dd, J=8.0, 8.0 Hz, 1H), 7.44-7.49 (m, 2H), 7.78-7.80 (m,
1H), 8.70 (d, J=6.8 Hz, 1H), 10.00 (brs, 1H); .sup.13C NMR (100
MHz, CDCl.sub.3) .delta. 23.8, 32.3, 41.5, 59.7, 89.7, 114.2,
118.7, 120.6, 122.4, 124.4, 127.2, 129.3, 133.7, 136.8, 140.9,
149.4, 157.2, 158.2.
2-(3-Chlorophenylamino)-3-((isopropylamino)methyl)-4H-pyrido[1,2-a]pyrimid-
in-4-one (204)
##STR00224##
[0555] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.25 (s, 3H), 1.26
(s, 3H), 2.30-3.06 (m, 1H), 4.05 (s, 2H), 6.87 (dd, J=6.4, 7.2 Hz,
1H), 6.95 (d, J=7.2 Hz, 1H), 7.17 (dd, J=8.0, 8.0 Hz, 1H), 7.32 (d,
J=8.8 Hz, 1H), 7.41 (d, J=8.0 Hz, 1H), 7.54 (dd, J=7.2, 7.2 Hz,
1H), 7.81 (s, 1H), 8.83 (d, J=6.8 Hz, 1H); .sup.13C NMR (100 MHz,
CDCl.sub.3) .delta. 22.1, 41.7, 48.9, 91.5, 113.7, 118.2, 120.1,
122.2, 124.6, 127.5, 129.5, 134.1, 136.2, 141.2, 149.5, 157.4,
157.8.
2-(3-Chlorophenylamino)-3-((cyclohexylamino)methyl)-8-(4-methylpiperazin-1-
-yl)-4H-pyrido[1,2-a]pyrimidin-4-one (205)
##STR00225##
[0557] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.20-1.34 (m, 3H),
1.71-1.91 (m, 3H), 1.92-2.04 (m, 2H), 2.20 (s, 3H), 2.23-2.36 (m,
6H), 3.04-3.10 (m, 5H), 4.01 (s, 2H), 5.87 (s, 1H), 6.55 (s, J=8.0
hz, 1H), 6.90 (d, J=8.0 Hz, 1H), 7.14 (t, J=8.0 Hz, 1H), 7.62 (d,
J=7.6 Hz, 1H), 7.84 (s, 1H), 8.46 (d, J=7.6 Hz, 1H), 9.59 (s, 1H);
.sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 24.9, 25.3, 30.2, 41.2,
46.1, 46.3, 54.2, 58.4, 86.2, 98.9, 106.5, 119.3, 121.0, 122.3,
128.3, 129.5, 133.9, 141.9, 150.8, 154.8, 157.7, 158.9.
##STR00226##
General Procedure for the Synthesis of J1
[0558] To a solution of an aldehyde (0.9 mmol) in methanol (0.5 mL)
was added NaBH.sub.4 (1.35 mmol) at room temperature. After
stirring 1 h, the reaction mixture was diluted with methylene
chloride (10 mL) and washed with brine (10 ml). The organic layer
was dried over MgSO.sub.4 and concentrated in vacuo. The crude
product was purified by recrystallization from a mixture of hexanes
and ethyl acetate to give J1.
General Procedure for the Synthesis of J2
[0559] To a stirred solution of an ester (0.06 mmol) in THF (1.0
mL) was added LiAlH.sub.4 (0.09 mmol). The reaction mixture was
stirred at room temperature for 1 hr. After reaction was completed,
H.sub.2O (0.1 mL) was added dropwise. The reaction mixture was
filtered off and concentrated in vacuo. The crude product was
purified by flash column chromatography to give J2.
General Procedure for the Synthesis of J3
[0560] To a stirred solution of J1 or J2 (0.19 mmol) in
CH.sub.2Cl.sub.2 (0.6 mL) was added triethylamine (0.38 mmol) and a
benzoyl chloride (0.28 mmol) at 0.degree. C. The reaction mixture
was stirred at room temperature for 1 h. After reaction was
completed, the mixture was diluted with CH.sub.2Cl.sub.2 (10 mL)
and washed with brine (10 ml). The organic layer was dried over
anhydrous MgSO.sub.4 and concentrated in vacuo. The crude product
was purified by flash column chromatography (Hexane:EtOAc=2:1) to
give J3.
3-(Hydroxymethyl)-2-(phenylamino)-4H-pyrido[1,2-a]pyrimidin-4-one
(206)
##STR00227##
[0562] .sup.1H NMR (400 MHz, CDCl.sub.3+CD.sub.3OD) .delta. 4.80
(s, 2H), 6.87-6.90 (m, 1H), 8.03 (dd, J=7.2, 7.6 Hz, 1H), 7.27 (dd,
J=7.6, 8.0 Hz, 2H), 7.53-7.58 (m, 3H), 8.36 (brs, 1H), 8.82 (d,
J=6.8 Hz, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3+CD.sub.3OD)
.delta. 56.0, 94.80, 94.85, 113.8, 121.1, 121.2, 123.2, 123.3,
124.5, 127.5, 128.6, 136.4, 138.9, 139.0, 149.7, 157.1, 158.0,
158.1.
2-(3-Chlorophenylamino)-3-(hydroxy
methyl)-4H-pyrido-[1,2-a]pyrimidin-4-one (207)
##STR00228##
[0564] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 4.95 (d, J=6.4 Hz,
2H), 6.93 (t, J=6.8 Hz, 1H), 7.05 (d, J=8.0 Hz, 1H), 7.38 (t, J=4.4
Hz, 2H), 7.42 (s, 1H), 7.63 (t, J=6.8 Hz, 1H), 7.81 (t, J=1.6 Hz,
1H), 8.20 (s, 1H), 8.92 (d, J=7.2 Hz, 1H).
2-(3-Fluorophenylamino)-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimidine-3-car-
baldehyde (208)
##STR00229##
[0566] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 4.94 (s, 2H), 6.94
(t, J=6.0 Hz, 2H), 7.17 (d, J=8.0 Hz, 1H), 7.43 (d, J=8.8 Hz, 2H),
7.63 (t, J=7.2 Hz, 2H), 7.70 (d, J=9.2 Hz, 1H), 8.26 (s, 1H), 8.93
(d, J=7.2 Hz, 1H).
3-(Hydroxymethyl)-2-(3-(trifluoromethyl)phenylamino)-4H-pyrido[1,2-a]pyrim-
idin-4-one (209)
##STR00230##
[0568] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 4.99 (s, 2H), 6.99
(d, J=6.0 Hz, 2H), 7.32 (d, J=8.0 Hz, 1H), 7.43 (d, J=7.6 Hz, 2H),
7.69 (brs, 2H), 8.06 (s, 1H), 8.27 (s, 1H), 8.96 (d, J=7.6 Hz,
1H).
3-(Hydroxymethyl)-2-(3-(trifluoromethoxy)phenylamino)-4H-pyrido[1,2-a]pyri-
midin-4-one (210)
##STR00231##
[0570] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 4.95 (d, J=6.4 Hz,
2H), 6.84 (t, J=6.8 Hz, 1H), 6.92 (d, J=6.8 Hz, 1H), 7.30-7.34 (m,
3H), 7.59 (t, J=7.2 Hz, 1H), 7.86 (s, 1H), 8.36 (s, 1H), 8.87 (d,
J=6.4 Hz, 1H),
Methyl
3-(3-(hydroxymethyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-ylamino)benz-
oate (211)
##STR00232##
[0572] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.92 (s, 3H), 4.99
(d, J=6.4 Hz, 2H), 6.96 (t, J=7.2 Hz, 1H), 7.38-7.42 (m, 2H), 7.63
(t, J=7.8 Hz, 1H), 7.75 (d, J=7.6 Hz, 1H), 7.88 (d, J=8.0 Hz, 1H),
8.21 (s, 1H), 8.25 (brs, 1H), 8.96 (d, J=7.6 Hz, 1H).
3-(3-(hydroxymethyl)-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-ylamino)benzoic
acid (212)
##STR00233##
[0574] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 4.73 (s, 1H), 5.74
(s, 2H), 7.19 (t, J=7.2 Hz, 1H), 7.38-7.42 (m, 2H), 7.45 (d, J=7.6
Hz, 1H), 7.86 (t, J=8.4 Hz, 1H), 8.00 (d, J=8.0 Hz, 1H), 8.19 (s,
1H), 8.82 (s, 1H), 8.89 (d, J=6.8 Hz, 1H).
2-(4-Chlorophenylamino)-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimidin-4-one
(213)
##STR00234##
[0576] .sup.1H NMR (400 MHz, DMSO) .delta. 4.05 (d, J=7.2 Hz, 2H),
7.37 (d, J=8.8Hz, 2H), 7.44 (d, J=8.8 Hz, 1H), 7.75 (d, J=6.8 Hz,
2H), 7.88 (t, J=8.8 Hz, 1H), 8.81 (s, 1H), 8.88 (d, J=6.4 Hz,
1H).
2-(2-Chlorophenylamino)-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimidin-4-one
(214)
##STR00235##
[0578] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 5.01 (d, J=5.6 Hz,
2H), 6.97-7.01 (m, 3H), 7.26-7.29 (m, 1H), 7.42 (t, J=8.8 Hz, 2H),
7.66 (t, J=7.2 Hz, 1H), 8.41 (t, J=5.2 Hz, 1H), 8.53 (s, 1H), 8.99
(d, J=6.8 Hz, 1H).
3-(Hydroxymethyl)-2-(3-hydroxyphenylamino)-4H-pyrido[1,2-a]pyrimidin-4-one
(215)
##STR00236##
[0580] .sup.1H NMR (400 MHz, CDCl.sub.3+CD.sub.3OD) .delta. 4.81
(s, 2H), 6.53 (d, J=8.0 Hz, 1H), 6.99 (dd, J=6.8, 6.8 Hz, 1H), 7.04
(d, J=8.0 Hz, 1H), 7.12 (dd, J=6.8, 6.8 Hz, 1H), 7.18 (s, 1H), 7.42
(d, J=9.6 Hz, 1H), 7.64 (dd, J=6.8, 8.8 Hz, 1H), 8.88 (d, J=7.2 Hz,
1H).
3-(Hydroxymethyl)-2-(4-hydroxyphenylamino)-4H-pyrido[1,2-a]pyrimidin-4-one
(216)
##STR00237##
[0582] .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. 4.83 (s, 2H), 6.77
(dd, J=2.0, 8.8 Hz, 2H), 7.04 (dd, J=6.8, 6.8 Hz, 1H), 7.32 (d,
J=8.8 Hz, 1H), 7.34-7.67 (m, 2H), 7.67-7.73 (m, 1H), 8.84 (d, J=6.8
Hz, 1H).
3-(Hydroxymethyl)-2-(2-hydroxyphenylamino)-4H-pyrido[1,2-a]pyrimidin-4-one
(217)
##STR00238##
[0584] .sup.1H NMR (400 MHz, CDCl.sub.3+CD.sub.3OD) .delta. 3.71
(s, 1H), 4.86 (s, 2H), 6.88 (ddd, J=1.6, 7.6, 8.0 Hz, 1H), 6.93
(dd, J=1.6, 8.0 Hz, 1H), 6.98 (ddd, J=1.6, 7.2, 8.0 Hz, 1H(, 7.05
(ddd, J=1.2, 6.8, 6.8 Hz, 1H), 7.43 (d, J=8.8 Hz, 1H), 7.69-7.73
(m, 2H), 8.91 (dd, J=0.8, 6.8 Hz, 1H).
2-(2,6-Dichlorophenylamino)-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimidin-4--
one (218)
##STR00239##
[0586] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 5.03 (d, J=6.0 Hz,
2H), 6.96 (t, J=7.2 Hz, 1H), 7.16 (t, J=7.6 Hz, 2H), 7.2 (s, 1H),
7.39 (d, J=8.0 Hz, 2H), 7.56 (t, J=7.6 Hz, 1H), 7.77 (s, 1H), 8.96
(d, J=7.2 Hz, 1H).
2-(3,5-Dichlorophenylamino)-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimidin-4--
one (219)
##STR00240##
[0588] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 4.97 (d, J=6.0 Hz,
2H), 7.01-7.04 (m, 2H), 7.50 (t, J=6.8 Hz, 1H), 7.60 (s, 2H), 7.71
(t, J=8.4 Hz, 2H), 8.24 (s, 1H), 8.98 (d, J=7.2 Hz, 1H).
2-(3,5-Difluorophenylamino)-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimidin-4--
one (220)
##STR00241##
[0590] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 4.99 (d, J=6.0 Hz,
2H), 6.52 (t, J=8.8 Hz, 1H), 7.05 (t, J=5.6 Hz, 2H), 7.29 (d, J=2.0
Hz, 2H), 7.51 (s, 1H), 7.72 (t, J=7.6 Hz, 1H), 8.30 (s, 1H), 8.99
(d, J=6.4 Hz, 1H).
2-(2,6-Dimethylphenylamino)-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimidin-4--
one (221)
##STR00242##
[0592] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.23 (s, 6H), 5.02
(d, J=6.4 Hz, 2H), 6.92 (t, J=6.8 Hz 1H), 7.12 (s, 3H), 7.20 (d,
J=8.8 Hz, 1H), 7.33 (s, 1H), 7.53 (t, J=6.8 Hz, 1H), 8.94 (d, J=6.4
Hz, 1H).
3-(Hydroxymethyl)-2-phenoxy-4H-pyrido[1,2-a]pyrimidin-4-one
(222)
##STR00243##
[0594] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.31 (brs, 1H),
4.86 (s, 2H), 7.03-7.09 (m, 3H), 7.13-7.18 (m, 1H), 7.28-7.34 (m,
3H), 7.58-7.62 (m, 1H), 8.94-8.96 (m, 1H); .sup.13C NMR (100 MHz,
CDCl.sub.3) .delta.6.0, 99.7, 115.2, 121.7, 125.1, 125.3, 127.4,
129.3, 136.8, 149.2, 152.8, 159.6, 164.0.
2-(3-Fluorophenoxy)-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimidin-4-one
(223)
##STR00244##
[0596] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.62 (brs, 1H),
4.78 (s, 2H), 6.78-6.85 (m, 3H), 7.02 (ddd, J=1.2, 6.8, 7.2 Hz,
1H), 7.18-7.23 (m, 1H), 7.25 (d, J=9.2 Hz, 1H), 7.57-7.62 (m, 1H),
8.89 (d, J=6.8 Hz, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta.
55.3, 99.7, 109.4, 109.6, 111.7, 111.9, 115.2, 117.2, 117.3, 125.0,
127.3, 129.7, 129.8, 137.0, 149.0, 153.5, 153.6, 159.4, 161.4,
163.6, 163.8.
2-(3-Chlorophenoxy)-3-(hydroxymethyl)-4-pyrido[1,2-a]pyrimidin-4-one
(224)
##STR00245##
[0598] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.51 (t, J=6.4 Hz,
1H), 4.79 (d, J=6.4 Hz, 2H), 6.95-6.98 (m, 1H), 7.04 (dd, J=6.8,
7.2 Hz, 1H), 7.08-7.10 (m, 1H), 7.20 (dd, J=8.4, 8.8 Hz, 1H), 7.27
(d, J=8.8 Hz, 1H), 7.59-7.63 (m, 1H), 8.91 9dd, J=0.4, 7.2 Hz, 1H);
.sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 53.3, 55.4, 99.7, 115.3,
120.1, 122.2, 125.1, 127.4, 129.8, 134.3, 137.0, 153.2, 159.2,
163.6.
3-(Hydroxymethyl)-2-(phenylamino)-6,7,8,9-tetrahydro-4H-pyrido[1,2-a]pyrim-
idin-4-one (225)
##STR00246##
[0600] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.85-1.93 (m, 4H),
2.15 (s, 2H), 2.84 (t, J=6.8 Hz, 2H), 3.87 (t, J=6.2 Hz, 2H), 7.06
(t, J=7.0 Hz, 1H), 7.26 (t, J=7.0 Hz, 2H), 7.51 (d, J=7.4 Hz, 2H),
11.2 (s, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 14.6,
19.2, 22.2, 32.2, 42.4, 88.4, 122.9, 124.4, 128.8, 138.4, 160.5,
160.8, 162.2.
2-(3-Chlorophenylamino)-3-(hydroxymethyl)-6,7,8,9-tetrahydro-4H-pyrido[1,2-
-a]pyrimidin-4-one (226)
##STR00247##
[0602] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 1.23-1.34 (m,
2H), 1.38-1.51 (m, 4H), 2.35-2.41 (m, 2H), 3.98-4.05 (m, 2H), 4.12
(s, 2H), 7.17-7.22 (m, 2H), 7.31 (t, J=2.0 Hz, 1H), 7.36 (t, J=8.0
Hz, 1H), 7.77 (s, 1H); .sup.13C NMR (100 MHz, DMSO-d.sub.6) .delta.
15.1, 23.1, 31.4, 42.4, 59.2, 61.4, 65.7, 122.8, 123.9, 125.6,
131.6, 134.3, 139.4, 157.9, 164.3
3-(Hydroxymethyl)-2-(3-(trifluoromethyl)phenylamino)-6,7,8,9-tetrahydro-4H-
-pyrido[1,2-a]pyrimidin-4-one (227)
##STR00248##
[0604] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 1.19-1.38 (m,
2H), 1.48-1.54 (m, 2H), 1.70-1.73 (m, 2H), 2.38 (t, J=12.8 Hz, 1H),
3.98-4.06 (m, 2H), 4.13 (s, 2H), 7.47 (d, J=7.6 Hz, 1H), 7.52 (d,
J=8.8 Hz, 1H), 7.55-7.59 (m, 2H), 7.83 (s, 1H); .sup.13C NMR (100
MHz, DMSO-d.sub.6) .delta.14.3, 22.2, 30.5, 41.5, 58.4, 77.9,
119.8, 121.2, 127.0, .quadrature. 129.8, 130.1, (d, J=26.8 due to
CF.sub.3), 138.2, 146.1, 157.1, 163.6, 169.1.
3-(Hydroxymethyl)-2-(2-hydroxyphenylamino)-6,7,8,9-tetrahydro-4H-pyrido[1,-
2-a]pyrimidin-4-one (228)
##STR00249##
[0606] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.78-1.94 (m, 4H),
2.13-2.23 (m, 2H), 2.61 (t, J=6.0 Hz, 1H), 3.98-4.05 (m, 2H), 4.12
(s, 2H), 6.81 (t, J=7.2 Hz, 1H), 6.89 (d, J=7.2 Hz, 1H), 6.98-7.12
(m, 2H), 10.11 (s, 1H), 11.3 (s, 1H); .sup.13C NMR (100 MHz,
CDCl.sub.3) .delta. 14.3, 21.4, 31.3, 42.1, 61.1, 87.7, 121.2,
126.4, 128.3, 128.6, 151.1, 161.3, 162.5, 163.7, 169.4.
3-(Hydroxymethyl)-2-(3-hydroxyphenylamino)-6,7,8,9-tetrahydro-4H-pyrido[1,-
2-a]pyrimidin-4-one (229)
##STR00250##
[0608] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.41-1.61 (m, 4H),
1.62-1.77 (m, 2H), 2.72 (t, J=10.0 Hz, 1H), 3.78-3.95 (m, 2H), 4.17
(s, 2H), 6.43 (d, J=7.6 Hz, 1H), 6.81 (d, J=8.0 Hz, 1H), 6.87 (d,
J=8.0 Hz, 1H), 6.98 (t, J=2.0 Hz, 1H); .sup.13C NMR (100 MHz,
CDCl.sub.3) .delta. 14.2, 21.8, 31.9, 42.4, 60.1, 79.8, 109.8,
111.6, 114.0, 129.4, 139.4, 149.7, 159.3, 160.2, 163.1.
3-(Hydroxymethyl)-2-(4-hydroxyphenylamino)-6,7,8,9-tetrahydro-4H-pyrido[1,-
2-a]pyrimidin-4-one (230)
##STR00251##
[0610] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 1.21-1.45 (m,
4H), 1.63-1.71 (m, 2H), 2.34 (t, J=12.8 Hz, 1H), 3.98-4.05 (m, 2H),
4.19 (s, 2H), 6.75 (d, J=8.8 Hz, 2H), 7.00 (d, J=8.8 Hz, 2H);
.sup.13C NMR (100 MI-Hz, DMSO-d.sub.6) .delta. 14.9, 21.9, 32.1,
42.3, 60.4, 87.2, 115.7, 125.0, 130.1, 154.9, 159.4, 160.6,
163.3.
3-(Hydroxymethyl)-9-methyl-2-(phenylamino)-4H-pyrido[1,2-a]pyrimidin-4-one
(231)
##STR00252##
[0612] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.40 (s, 3H), 2.97
(brs, 1H), 4.93 (s, 2H), 6.89 (t, J=6.8 Hz, 1H), 7.11 (t, J=7.2 Hz,
1H), 7.34 (t, J=7.6 Hz, 2H), 7.62 (d, J=6.4 Hz, 1H), 8.02 (d, J=8.0
Hz, 2H), 8.73 (d, J=6.8 Hz, 1H).
2-(3-Chlorophenylamino)-3-(hydroxymethyl)-9-methyl-4H-pyrido[1,2-a]pyrimid-
in-4-one (232)
##STR00253##
[0614] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.43 (s, 3H), 3.06
(t, J=6.4 Hz, 1H), 4.92 (d, 6.4 Hz, 2H), 6.69 (d, J=7.0 Hz, 1H),
7.03 (d, J=7.6 Hz, 1H), 7.23 (t, J=8.0 Hz, 1H), 7.29 (d, J=8.0 Hz,
1H), 7.44 (d, J=6.8 Hz, 1H), 8.03 (s, 1H), 8.38 (s, 1H), 8.71 (d,
J=7.2 Hz, 1H).
2-((3-Chlorophenyl)(methyl)amino)-3-(hydroxymethyl)-9-methyl-4H-pyrido[1,2-
-a]pyrimidin-4-one (233)
##STR00254##
[0616] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.51 (s, 3H), 4.09
(t, J=6.8 Hz, 1H), 4.12 (d, J=7.2 Hz, 2H), 6.95 (t, J=7.0 Hz, 1H),
7.04-7.06 (m, 2H), 7.20 (t, J=8.4 Hz, 1H), 7.54 (d, J=6.8 Hz, 1H),
8.84 (d, J=7.2 Hz, 1H).
2-((3-Chlorophenyl)(methyl)amino)-3-(methoxymethyl)-9-methyl-4H-pyrido[1,2-
-a]pyrimidin-4-one (234)
##STR00255##
[0618] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.49 (s, 3H), 3.01
(s, 3H), 4.04 (s, 3H), 6.91 (t, J=7.0 Hz, 1H), 7.08 (d, J=8.4 Hz,
1H), 7.12 (d, J=7.2 Hz, 1H), 7.20 (s, 1H), 7.26 (t, J=8.0 Hz, 1H),
7.52 (d, J=6.8 Hz, 1H), 8.86 (d, J=7.2 Hz, 1H).
3-(Hydroxymethyl)-9-methyl-2-(3-(trifluoromethoxy)phenylamino)-4H-pyrido[1-
,2-a]pyrimidin-4-one (235)
##STR00256##
[0620] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.40 (s, 3H), 3.15
(t, J=6.2 Hz, 1H), 4.93 (d, J=6.4 Hz, 1H), 6.67 (t, J=7.0 Hz, 1H),
6.91 (d, J=8.0 Hz, 1H), 7.25-7.27 (m, 1H), 7.32 (t, J=8.2 Hz, 1H),
7.43 (d, J=6.8 Hz, 1H), 7.98 (s, 1H), 8.51 (s, 1H), 8.72 (d, J=6.8
Hz, 1H).
3-(Hydroxymethyl)-2-(3-hydroxyphenylamino)-9-methyl-4H-pyrido[1,2-a]pyrimi-
din-4-one (236)
##STR00257##
[0622] .sup.1H NMR (400 MHz, CDCl.sub.3+CD.sub.3OD) .delta. 2.44
(s, 3H), 4.75 (s, 2H), 6.45 (dd, J=2.4, 8.0 Hz, 1 h), 6.84 (dd,
J=6.8, 6.8 Hz, 1H), 7.06 (dd, J=8.0, 8.4 Hz, 1H), 7.11 (dd, J=2.0,
2.4 Hz, 1H), 7.17 (dd, H=2.0, 8.0 Hz, 1H), 7.45 (d, J=6.8 Hzm 1H),
8.72 (d, J=7.2 Hz, 1H).
3-(Hydroxymethyl)-2-(4-hydroxyphenylamino)-9-methyl-4H-pyrido[1,2-a]pyrimi-
din-4-one (237)
##STR00258##
[0624] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.40 (s, 3H), 4.94
(d, J=4.8 Hz, 1H), 6.81-6.84 (m, 3H), 7.46 (d, J=7.2 Hz, 1H), 7.50
(d, J=8.8 Hz, 2H), 7.84 (s, 1H), 8.82 (d, J=7.2 Hz, 1H).
2-(4-tert-Butylphenylamino)-3-(hydroxymethyl)-9-methyl-4H-pyrido[1,2-a]pyr-
imidin-4-one (238)
##STR00259##
[0626] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.34 (s, 9H), 2.40
(s, 3H), 3.07 (t, J=6.2 Hz, 1H), 4.91 (d, J=6.4 Hz, 2H), 6.61 (t,
J=6.8 Hz, 1H), 7.34 (d, J=7.2 Hz, 2H), 7.38 (d, J=6.8 Hz, 1H), 8.21
(br s, 1H), 8.69 (d, J=7.2 Hz, H).
2-(3-Chlorobenzylamino)-3-(hydroxymethyl)-9-methyl-4H-pyrido[1,2-a]pyrimid-
in-4-one (239)
##STR00260##
[0628] .sup.1H NMR (400 MHz, CDCl.sub.3+CD.sub.3OD) .delta. 2.31
(s, 3H), 3.02 (s, 1H), 4.68 (d, J=5.6 Hz, 2H), 4.70 (s, 2H), 6.70
(dd, J=5.6, 6.0 Hz, 1H), 6.74 (dd, J=6.8, 7.2 Hz, 1H), 7.11-7.20
(m, 3H), 7.31 (s, 1H), 7.38 (d, J=6.8 Hz, 1H), 8.66 (d, J=6.8 Hz,
1H); .sup.13C NMR (100 MHz, CDCl.sub.3+CD.sub.3OD) .delta. 17.7,
44.2, 44.3, 55.8, 93.1, 93.2, 112.6, 125.4, 125.5, 126.9, 127.5,
129.5, 132.6, 134.0, 134.9, 141.7, 149.45, 149.47, 157.4, 159.10,
159.16.
3-(Hydroxymethyl)-2-(isobutylamino)-9-methyl-4H-pyrido[1,2-a]pyrimidin-4-o-
ne (240)
##STR00261##
[0630] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 0.96 (d, J=6.8 Hz,
6H), 1.88-1.95 (m, 1H), 2.34 (s, 3H), 3.13 (brs, 1H), 3.32 (t,
J=6.0 Hz, 2H), 4.78 (d, J=6.0 Hz, 2H), 6.08 (brs, 1H), 6.72 (t,
J=6.8 Hz, 1H), 7.37 (d, J=6.8 Hz, 1H), 8.66 (d, J=6.8 Hz, 1H);
.sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 17.9, 20.5, 28.9, 48.6,
57.1, 92.5, 112.1, 126.0, 132.5, 134.6, 149.6, 157.1, 159.5.
2-(Diethylamino)-3-(hydroxymethyl)-9-methyl-4H-pyrido[1,2-a]pyrimidin-4-on-
e (241)
##STR00262##
[0632] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.22 (t, J=6.8 Hz,
6H), 2.35 (s, 3H), 3.41 (s, 1H), 3.63 (q, J=6.8 Hz, 4H), 4.44 (s,
2H), 6.65 (t, J=7.2 Hz, 1H), 7.31 (d, J=6.8 Hz, 1H), 8.68 (d, J=7.2
Hz, 1H) .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 13.9, 17.7,
44.0, 67.0, 92.2, 111.7, 125.8, 132.5, 134.4, 148.1, 160.7,
160.8.
2-(Cyclohexylmethylamino)-3-(hydroxymethyl)-9-methyl-4H-pyrido[1,2-a]pyrim-
idin-4-one (242)
##STR00263##
[0634] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 0.95-0.98 (m, 2H),
1.18-1.23 (m, 3H), 1.58-1.79 (m, 6H), 2.42 (s, 3H), 3.27 (t, J=6.4
Hz, 2H), 3.85 (brs, 1H), 4.74 (m, 2H), 6.21 (t, J=7.2 Hz, 1H), 6.68
(d, J=6.8 Hz, 1H), 7.33 (d, J=7.2 Hz, 1H), 8.57 (d, J=7.2 Hz, 1H);
.sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 17.9, 26.2, 26.7, 31.3,
38.4, 47.5, 56.9, 92.8, 112.0, 126.0, 132.3, 134.5, 149.4, 156.9,
159.5.
3-(Hydroxymethyl)-9-methyl-2-morpholino-4H-pyrido[1,2-a]pyrimidin-4-one
(243)
##STR00264##
[0636] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.01 (brs, 1H),
2.43 (s, 3H), 3.62 (t, J=4.8 Hz, 4H), 3.78 (t, J=4.8 Hz, 4H), 4.62
(s, 2H), 6.85 (t, J=6.8 Hz, 1H), 7.46 (d, J=6.8 Hz, 1H), 8.76 (d,
J=6.8 Hz, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 17.9,
49.7, 58.9, 67.1, 95.5, 113.3, .quadrature. 125.2, 133.4, 135.0,
148.2, 160.6, 161.7.
3-(Hydroxymethyl)-9-methyl-2-morpholino-4H-pyrido[1,2-a]pyrimidin-4-one
hydrochloride (244)
##STR00265##
[0638] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.43 (s, 3H), 3.42
(s, 1H), 3.62 (t, J=4.8 Hz, 4H), 3.78 (t, J=4.8 Hz, 4H), 4.62 (s,
2H), 6.85 (t, J=6.8 Hz, 1H), 7.46 (d, J=6.8 Hz, 1H), 8.76 (d, J=6.8
Hz, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) .delta. 17.9, 49.7,
58.9, 67.1, 98.5, 113.3, 125.2, 133.4, 135.0, 148.2, 160.6,
161.7.
7-Bromo-2-(3-chlorophenylamino)-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimidi-
n-4-one (245)
##STR00266##
[0640] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 4.78 (s, 2H),
5.37 (s, 1H), 7.12 (dd, J=1.6 Hz, 8.4 Hz, 1H), 7.32 (d, J=8.0 Hz
1H), 7.42 (dd, J=1.6 Hz, 8.4 Hz, 1H), 7.54 (dd, J=0.8 Hz, 8.0 Hz,
1H), 7.64 (d, J=8.0 Hz 1H), 7.91 (d, J=2.0 Hz, 1H), 8.47 (s, 1H),
8.71 (s, 1H);
2-(3-Chlorophenylamino)-3-(hydroxymethyl)-7-methoxy-4H-pyrido[1,2-a]pyrimi-
din-4-one (246)
##STR00267##
[0642] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 3.86 (s, 3H),
4.70 (s, 2H), 5.22 (s, 1H), 7.02 (dd, J=0.8 Hz, 8.0 Hz, 1H),
7.28-7.32 (m, 1H), 7.41 (dd, J=1.2 Hz, 9.6 Hz, 1H), 7.58 (dd, J=0.8
Hz, 8.0 Hz, 1H), 7.64-7.68 (m, 1H), 7.87 (d, J=2.0 Hz, 1H), 8.36
(s, 1H), 8.69 (s, 1H)
2-(3-Chlorophenylamino)-3-(hydroxymethyl)-8-methoxy-4H-pyrido[1,2-a]pyrimi-
din-4-one (247)
##STR00268##
[0644] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 3.92 (s, 3H),
4.62 (s, 2H), 5.07 (s, 1H), 6.71 (d, J=2.8 Hz, 1H), 6.83 (dd, J=2.8
Hz, 8.0 Hz, 1H), 7.01 (d, J=8.0 Hz, 1H), 7.28 (dd, J=8.0 Hz, J=8.0
Hz, 1H), 7.62 (d, J=8.0 Hz, 1H), 7.76 (d, J=2.0 Hz, 1H), 8.62 (s,
1H), 8.71 (d, J=8.0 Hz, 1H); .sup.13C NMR (100 MHz, DMSO-d.sub.6)
54.8, 57.3, 93.8, 101.5, 109.3, 120.0, 120.9, 122.5, 129.5, 130.7,
133.4, 142.2, 151.9, 156.9, 157.8, 166.2.
8-Chloro-2-(3-chlorophenylamino)-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimid-
in-4-one (248)
##STR00269##
[0646] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 4.68 (s, 2H), 5.14
(brs, 1H), 7.03 (dd, J=1.2, 8.0 Hz, 1H), 7.19 (dd, J=2.4, 7.6 Hz,
1H), 7.28 (t, J=8.0 Hz, 1H), 7.54, (d, J=2.0 Hz, 1H), 7.58 (dd,
J=1.2, 8.4 Hz, 1H), 7.57 (t, J=2.0 Hz, 1H), 8.78 (d, J=8.0 Hz,
1H).
2-(3-Chlorophenylamino)-3-(hydroxymethyl)-8-(methylamino)-4H-pyrido[1,2-a]-
pyrimidin-4-one (249)
##STR00270##
[0648] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.81 (s, 3H), 3.85
(s, 2H), 6.02 (s, 1H), 6.32 (d, J=7.6 Hz, 1H), 6.93 (d, J=2 Hz,
1H), 7.12 (t, J=8.0 Hz, 1H), 7.38 (d, J=8.0 Hz, 1H), 7.81 (s, 1H),
8.42 (s, 1H), 9.93 (s, 1H).
2-(3-Chlorophenylamino)-8-(diethylamino)-3-(hydroxymethyl)-4H-pyrido[1,2-a-
]pyrimidin-4-one (250)
##STR00271##
[0650] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 1.23 (t, J=6.8 Hz,
6H), 3.44 (q, J=6.8 Hz, 4H), 3.99 (s, 2H), 4.82 (t, J=2.1 Hz, 1H),
6.29 (d, J=2.1 Hz, 1H), 6.54 (dd, J=2.4, 8.4 Hz, 1H), 6.92 (d, J=2
Hz, 1H), 7.21 (t, J=8.0 Hz, 1H), 7.81 (d, J=2.4 Hz, 1H), 8.06 (t,
J=2.0 Hz, 1H), 8.85 (d, J=8.4 Hz, 1H), 9.71 (s, 1H); .sup.13C NMR
(100 MHz, CDCl.sub.3) .delta. 12.7, 20.0, 44.7, 92.8, 97.1, 104.0,
118.9, 120.7, 121.9, 128.5, 129.5, 134.1, 142.8, 150.6, 151.9,
158.3, 159.2.
3-(Hydroxymethyl)-8-morpholino-2-(phenylamino)-4H-pyrido[1,2-a]pyrimidin-4-
-one (251)
##STR00272##
[0652] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 3.43 (s, 4H),
3.67 (s, 4H), 4.59 (d, J=5.2 Hz, 2H), 5.05, (t, J=4.8 Hz, 1H), 6.41
(d, J=2.0 Hz, 1H), 6.95 (t, J=7.2 Hz, 1H), 7.00 (dd, J=2.8, 8.4 Hz,
1H), 7.25 (t, J=8.0 Hz, 2H), 7.64 (d, J=7.6 Hz, 2H), 8.38 (s, 1H),
8.69 (d, J=8.0 Hz, 1H); .sup.13C NMR (100 MHz, DMSO-d.sub.6)
.delta. 46.5, 55.1, 66.3, 91.5, 99.1, 105.4, 121.3, 122.6, 128.5,
129.1, 140.9, 151.4, 155.0, 156.7, 158.5.
2-(3-Fluorophenylamino)-3-(hydroxymethyl)-8-morpholino-4H-pyrido[1,2-a]pyr-
imidin-4-one (252)
##STR00273##
[0654] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 3.46 (s, 4H),
3.68 (s, 4H), 4.59 (d, J=5.2 Hz, 2H), 5.06, (t, J=5.2 Hz, 1H), 6.47
(d, J=2.4 Hz, 1H), 6.74 (t, J=7.2 Hz, 1H), 7.03 (dd, J=2.8, 8.0 Hz,
1H), 7.26 (t, J=7.2 Hz, 1H), 7.64 (d, J=8.0 Hz, 1H), 7.79 (d,
J=12.4 Hz, 1H), 8.52 (s, 1H), 8.60 (d, J=8.0 Hz, 1H); .sup.13C NMR
(100 MHz, DMSO-d.sub.6) .delta. 45.8, 54.2, 65.6, 91.3, 98.4,
105.0, 108.0 (d, J=20 Hz, due to F), 116.0, 128.0, 129.8 (d, J=10
Hz, due to F), 142.1 (d, J=11 Hz, due to F), 150.6, 154.4, 156.1,
157.4, 161.0, 163.3.
2-(3-Chlorophenylamino)-3-(hydroxymethyl)-8-morpholino-4H-pyrido[8,2-a]pyr-
imidin-4-one (253)
##STR00274##
[0656] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 3.45 (t, J=5.6
Hz, 4H), 3.69 (t, J=5.6 Hz, 4H), 4.58 (d, J=5.2 Hz, 2H), 5.01 (t,
J=5.2 Hz, 1H), 6.42 (d, J=2.8 Hz, 1H), 6.98 (d, J=8.0 Hz, 1H), 7.05
(dd, J=2.8, 8.0 Hz, 1H), 7.26 (t, J=8.0 Hz, 1H), 7.64 (d, J=8.0 Hz,
1H), 7.80 (t, J=2.0 Hz, 1H), 8.48 (s, 1H), 8.60 (d, J=8.0 Hz, 1H);
.sup.13C NMR (100 MHz, DMSO-d.sub.6) .delta.5.4, 53.6, 65.7, 84.7,
98.6, 105.3, 117.8, 118.7, 119.8, 127.1, 130.2, 129.2, 141.8,
149.7, 153.0, 155.3, 157.4; LC-MS (ESI, m/z): 386 [M+H].sup.+.
3-(Hydroxymethyl)-8-(4-methylpiperazin-1-yl)-2-(phenylamino)-4H-pyrido[1,2-
-a]pyrimidin-4-one (254)
##STR00275##
[0658] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.34 (s, 3H), 2.52
(t, J=5.2 Hz, 4H), 3.43 (t, J=5.2 Hz, 4H), 4.88 (s, 2H), 5.28 (s,
1H), 6.37 (s, 1H), 6.55 (d, J=8.0 Hz, 1H), 7.05 (t, J=7.2 Hz, 1H),
7.33 (t, J=7.6 Hz, 2H), 7.60 (d, J=7.6 Hz, 2H), 7.91 (s, 1H), 8.64
(d, J=8.0 Hz, 1H).
2-(3-Chlorophenylamino)-3-(hydroxymethyl)-8-(4-methylpiperazin-1-yl)-4H-py-
rido[1,2-a]pyrimidin-4-one (255)
##STR00276##
[0660] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.14 (s, 3H), 2.38
(t, J=4.4 Hz, 4H), 3.45 (t, J=4.4 Hz, 4H), 3.56 (s, 2H), 6.41 (d,
J=2.4 Hz, 1H), 6.95 (dd, J=1.6, 8.0 Hz, 1H), 7.01 (dd, J=2.4, 8.0
Hz, 1H), 7.27 (t, J=8.0 Hz, 1H), 7.50 (d, J=1.6 Hz, 1H), 8.0 (d,
J=8.0 Hz, 1), 10.4 (s, 1H), 14.18 (s, 1H); .sup.13C NMR (100 MHz,
CDCl.sub.3) .delta. 45.6, 51.6, 54.0, 55.0, 85.3, 98.3, 105.1,
117.7, 118.5, 121.0, 127.9, 130.3, 133.0, 142.1, 150.8, 154.1,
156.4, 157.8; LC-MS (ESI, m/z): 400[M+H].sup.+.
2-(3-Fluorophenylamino)-3-(hydroxymethyl)-8-(4-methylpiperazin-1-yl)-4H-py-
rido[1,2-a]pyrimidin-4-one (256)
##STR00277##
[0662] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.35 (s, 3H), 2.54
(t, J=4.4 Hz, 4H), 3.48 (t, J=4.8 Hz, 4H), 4.87 (s, 2H), 5.23 (s,
1H), 6.42 (s, 1H), 6.60 (d, J=8.4 Hz, 1H), 6.73 (t, J=8.4 Hz, 1H),
7.12 (d, J=8.4 Hz, 1H), 7.19 (d, J=8.4 Hz, 1H), 7.71-7.75 (m, 1H),
8.04 (s, 1H), 8.71 (d, J=8.0 Hz, 1H).
2-(3-Chlorophenylamino)-3-(hydroxymethyl)-8-methyl-4H-pyrido[1,2-a]pyrimid-
in-4-one (257)
##STR00278##
[0664] Colorless solid, mp 235.degree. C. (decomp.); .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 2.42 (s, 3H), 4.07 (q, J=7.2 Hz, 2H),
7.03 (d, J=8.8 Hz, 2H), 7.26 (t, J=8.0 Hz, 2H), 7.46 (d, J=8.4 Hz,
1H), 7.84 (t, J=2.0 Hz, 1H), 8.79 (d, J=7.2 Hz, 2H).
2-(4-Chlorophenylamino)-3-(hydroxymethyl)-8-methyl-4H-pyrido[1,2-a]pyrimid-
in-4-one (258)
##STR00279##
[0666] Colorless solid, mp 227.degree. C. (decomp.); .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 2.42 (s, 3H), 4.10 (s, 2H), 6.85 (d,
J=7.2 Hz, 1H), 7.23-7.28 (m, 4H), 7.87 (d, J=6.8 Hz, 2H), 8.94 (d,
J=7.6 Hz, 1H).
2-(4-Fluorophenylamino)-3-(hydroxymethyl)-8-methyl-4H-pyrido[1,2-a]pyrimid-
in-4-one (259)
##STR00280##
[0668] Colorless solid, mp 232.degree. C. (decomp.); .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 2.42 (s, 3H), 4.12 (s, 2H), 6.85 (d,
J=6.8 Hz, 1H), 7.05 (t, J=8.4 Hz, 2H), 7.21 (s, 1H), 7.31-7.38 (m,
2H), 7.85 (q, J=4.8 Hz, 2H), 8.94 (d, J=7.2 Hz, 1H).
2-(3,4-Dichlorophenylamino)-3-(hydroxymethyl)-8-methyl-4H-pyrido[1,2-a]pyr-
imidin-4-one (260)
##STR00281##
[0670] Colorless solid, mp 230.degree. C. (decomp.); .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 2.44 (s, 3H), 4.09 (s, 2H), 6.89 (d,
J=7.2 Hz, 1H), 7.26 (s, 1H), 7.36 (d, J=8.8 Hz, 1H), 7.76 (d, J=8.4
Hz, 1H), 8.24 (d, J=2.4 Hz, 1H), 8.95 (d, J=7.2 Hz, 1H), 9.71 (s,
1H).
2-(3-Chloro-4-fluorophenylamino-3-(hydroxymethyl)-8-methyl-4H-pyrido[1,2-a-
]pyrimidin-4-one (261)
##STR00282##
[0672] Colorless solid, mp 225.degree. C. (decomp.); .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 2.43 (s, 3H), 4.09 (s, 2H), 6.88 (d,
J=7.2 Hz, 1H), 7.11 (t, J=8.8 Hz, 1H), 7.27 (s, 1H), 7.69-7.73 (m,
1H), 8.12 (d, J=6.8 Hz, 1H), 8.95 (d, J=7.2 Hz, 1H), 9.71 (s,
1H).
9-Chloro-2-(3-chlorophenylamino)-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimid-
in-4-one (262)
##STR00283##
[0674] Colorless solid, mp 230.degree. C. (decomp.); .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 4.95 (d, J=6.0 Hz, 2H), 6.80 (t,
J=7.2 Hz, 1H), 7.06 (d, J=8.0 Hz, 1H), 7.27 (d, J=8.4 Hz, 1H), 7.46
(d, J=8.0 Hz, 1H), 7.78 (d, J=7.2 Hz, 1H), 8.18 (t, J=2.4 Hz, 1H),
8.43 (s, 1H), 8.81 (d, J=7.2 Hz, 1H).
2-(3-Chlorophenylamino)-3-(hydroxymethyl)-9-(trifluoromethyl)-4H-pyrido[1,-
2-a]pyrimidin-4-one (263)
##STR00284##
[0676] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 4.77 (s, 2H),
7.11-7.13 (m, 1H), 7.32 (dd, J=7.2, 7.2 Hz, 1H), 7.35 (dd, J=8.0,
8.0 Hz, 1H), 7.48-7.50 (m, 1H), 8.13-8.14 (m, 1H), 8.41 (d, J=7.2
Hz, 1H), 9.12 (dd, J=1.2, 7.2 Hz, 1H).
2-(3-Chlorophenylamino)-9-fluoro-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimid-
in-4-one
##STR00285##
[0678] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.4.76 (s, 1H),
5.31 (brs, 1H), 7.11-7.13 (m, 1H), 7.18-7.23 (m, 1H), 7.38 (dd,
J=8.0, 8.0 Hz, 1H), 7.63-7.65 (m, 1H), 7.86 (dd, J=8.4, 8.8 Hz,
1H), 8.12-8.13 (m, 1H), 8.73 (d, J=7.2 Hz, 1H), 8.96 (brs, 1H).
2-(4-Chlorophenylamino)-9-fluoro-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimid-
in-4-one (265)
##STR00286##
[0680] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 4.72 (s, 2H),
5.30 (brs, 1H), 7.15-7.20 (m, 1H), 7.41-7.44 (m, 2H), 7.79-7.82 (m,
2H), 7.84-7.86 (m, 1H), 8.72 (d, J=7.2 Hz, 1H), 8.92 (brs, 1H).
9-Fluoro-2-(4-fluorophenylamino)-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyrimid-
in-4-one (266)
##STR00287##
[0682] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 4.75 (s, 2H),
5.25 (brs, 1H), 7.13-7.25 (m, 3H), 7.73-7.77 (m, 2H), 7.80-7.85 (m,
1H), 8.72 (d, J=7.2 Hz, 1H), 8.84 (brs, 1H).
2-(3-Chloro-4-fluorophenylamino)-9-fluoro-3-(hydroxymethyl)-4H-pyrido[1,2-
-a]pyrimidin-4-one (267)
##STR00288##
[0683] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.4.74 (s, 2H),
5.24 (brs, 1H), 7.18-7.22 (m, 1H), 7.39-7.44 (m, 1H), 7.65-7.69 (m,
1H), 7.83-7.87 (m, 1H), 8.20-8.22 (m, 1H), 8.72 (d, J=7.2 Hz, 1H),
8.91 (brs, 1H).
2-(3,4-Difluorophenylamino)-9-fluoro-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyr-
imidin-4-one (268)
##STR00289##
[0685] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 4.75 (s, 2H),
5.26 (brs, 1H), 7.17-7.22 (m, 1H), 7.39-7.49 (m, 1H), 7.84-7.88 (m,
1H), 8.08-8.14 (m, 1H), 8.73 (m, J=7.2 Hz, 1H), 8.93 (brs, 1H).
2-(3,4-Dichlorophenylamino)-9-fluoro-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyr-
imidin-4-one (269)
##STR00290##
[0687] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 4.75 (s, 2H),
5.27 (brs, 1H), 7.19-7.23 (m, 1H), 7.60 (d, J=8.8 Hz, 1H), 7.7 (dd,
J=2.8, 8.8 Hz, 1H), 7.85-7.89 (m, 1H), 8.83 (d, J=2.8 Hz, 1H), 8.73
(d, J=8.8 Hz, 1H), 9.00 (brs, 1H).
2-(1H-Indol-5-ylamino)-9-fluoro-3-(hydroxymethyl)-4H-pyrido[12-a]pyrimidin-
-4-one (270)
##STR00291##
[0689] m.p=184-185.degree. C.; .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. 4.70 (d, J=5.2 Hz, 2H), 5.18 (t, J=5.2 Hz, 1H), 6.35 (s,
1H), 7.00-7.04 (m, 1H), 7.23 (dd, 0.1=2 Hz, 8.8 Hz, 1H), 7.28-7.32
(m, 2H), 7.68 (dd, J=8 Hz, J=8 Hz, 1H), 7.82 (s, 1H), 8.61 (s, 1H),
8.64 (d, J=6 Hz, 1H), 10.98 (s, 1H); .sup.13C NMR (100 MHz,
DMSO-d.sub.6) .delta.5.2, 94.6, 101.7 (d, J=5.2 Hz, due to F),
111.6, 112.1 (d, J=7.4 Hz, due to F), 113.7, 118.0, 119.8 (d,
J=17.1 Hz, due to F), 124.2 (d, J=4.4 Hz, due to F), 126.5, 128.2,
131.9, 133.5, 151.6, 154.1, 156.3, 157.6.
3-(Hydroxymethyl)-9-methoxy-2-(phenylamino)-4H-pyrido[1,2-a]pyrimidin-4-on-
e (271)
##STR00292##
[0691] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 3.93 (s, 3H),
4.71 (d, J=5.2 Hz, 2H), 5.29 (t, J=5.2 Hz, 1H), 6.97-7.01 (m, 1H),
7.06-7.10 (m, 1H), 7.27-7.32 (m, 3H), 7.83 (d, J=8.4 Hz, 2H), 8.47
(d, J=7.2 Hz, 1H), 8.68 (s, 1H).
3-(Hydroxymethyl)-9-methoxy-2-(phenylamino)-4H-pyrido[1,2-a]pyrimidine-4-t-
hione (272)
##STR00293##
[0693] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 3.98 (s, 3H), 4.11
(d, J=7.2 Hz, 2H), 6.88 (t, J=8.0 Hz, 2H), 7.04 (t, J=7.2 Hz, 1H),
7.31 (t, J=7.2 Hz, 2H), 7.82 (d, J=7.6 Hz, 2H), 7.98 (s, 1H), 8.59
(d, J=5.6 Hz, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3) 26.9, 57.1,
94.2, 111.8, 112.7, 119.9, 121.1, 123.3, 128.9, 139.8, 143.7,
151.3, 155.6, 158.6.
2-(3-Chlorophenylamino)-3-(hydroxymethyl)-9-methoxy-4H-pyrido[1,2-a]pyrimi-
din-4-one (273)
##STR00294##
[0695] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 3.94 (s, 3H),
4.68 (s, 2H), 6.99 (d, J=7.6 Hz, 1H), 7.09 (dd, J=7.2 Hz, J=7.2 Hz,
1H), 7.25-7.29 (m, 2H), 7.56 (d, J=8.0 Hz, 1H), 8.42 (s, 1H), 8.45
(d, J=6.8 Hz, 1H), 8.77 (s, 1H).
2-(4-Chlorophenylamino)-3-(hydroxymethyl)-9-methoxy-4H-pyrido[1,2-a]pyrimi-
din-4-one (274)
##STR00295##
[0697] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 3.90 (s, 3H),
4.65 (d, J=5.2 Hz, 2H), 5.19 (t, J=5.2 Hz, 1H), 7.03 (dd, J=7.2 Hz,
7.6 Hz, 1H), 7.23 (d, J=7.6 Hz, 1H), 7.29 (d, J=8.8 Hz, 2H), 7.85
(d, J=9.2 Hz, 2H), 8.42 (d, J=7.2 Hz, 1H), 8.72 (s, 1H).
2-(4-Fluorophenylamino)-3-(hydroxymethyl)-9-methoxy-4H-pyrido[1,2-a]pyrimi-
din-4-one (275)
##STR00296##
[0699] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 3.91 (s, 3H),
4.69 (d, J=5.2 Hz, 2H), 5.19 (t, J=5.2 Hz, 1H), 7.06 (t, J=6.8 Hz,
1H), 7.13 (t, J=8.8 Hz, 1H), 7.25 (d, J=7.6 Hz, 1H), 7.83-7.86 (m,
1H), 8.45 (dd, J=1.2 Hz, 7.2 Hz, 1H), 8.66 (s, 1H).
3-(Hydroxymethyl)-9-methoxy-2-(4-(trifluoromethoxy)phenylamino)-4H-pyrido[-
1,2-a]pyrimidin-4-one (276)
##STR00297##
[0701] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 3.96 (s, 3H),
4.67 (d, J=4.0 Hz, 2H), 5.20 (s, 1H), 7.07 (dd, J=7.2 Hz, J=7.2 Hz,
1H), 7.23 (s, 1H), 7.27 (d, J=8.0 Hz, 2H), 7.95 (dd, J=8.8 Hz,
J=8.8 Hz, 2H), 8.45 (d, J=7.6 Hz, 1H), 8.78 (s, 1H).
3-(Hydroxymethyl)-9-methoxy-2-(4-(trifluoromethyl)phenylamino)-4H-pyrido[1-
,2-a]pyrimidin-4-one (277)
##STR00298##
[0703] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 3.97 (s, 3H),
4.72 (s, 2H), 5.32 (s, 1H), 7.14, (dd, J=7.2 Hz, 7.2 Hz, 1H), 7.33
(d, J=7.6 Hz, 1H), 7.64 (d, J=8.8 Hz, 2H), 8.11 (d, J=8.8 Hz, 2H),
8.49 (d, J=7.2 Hz, 1H), 9.09 (s, 1H).
2-(3-Chloro-4-fluorophenylamino)-3-(hydroxymethyl)-9-methoxy-4-pyrido[1,2--
a]pyrimidin-4-one (278)
##STR00299##
[0705] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 3.95 (s, 3H),
4.69 (d, J=4.8 Hz, 2H), 5.16 (t, J=4.8 Hz, 1H), 7.10 (dd, J=7.2 Hz,
7.2 Hz, 1H), 7.30 (dd, J=0.8 Hz, 8.0 Hz, 1H), 7.32 (dd, J=9.2 Hz,
9.2 Hz, 1H), 7.61-7.65 (m, 1H), 8.46 (dd, J=0.8 Hz, 7.2 Hz, 1H),
8.59 (dd, J=2.8 Hz, 7.2 Hz, 1H), 8.76 (s, 1H).
2-(3,4-Difluorophenylamino)-3-(hydroxymethyl)-9-methoxy-4H-pyrido[1,2-a]py-
rimidin-4-one (279)
##STR00300##
[0707] m.p=231.degree. C. (decomp.); .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. 3.92 (s, 3H), 4.66 (s, 2H), 5.17 (brs, 1H),
7.07 (dd, J=7.2 Hz, 7.2 Hz, 1H), 7.26-7.33 (m, 2H), 7.39-7.41 (m,
1H), 8.34-8.40 (m, 1H), 8.44 (d, J=7.2 Hz, 1H), 8.74 (s, 1H);
.sup.13C NMR (100 MHz, DMSO) .delta. 54.1, 56.8, 95.2, 109.1,
113.4, 116.0 (d, J=3.8 Hz, due to F), 116.8, 118.7, 137.5 (d, J=9.7
Hz, due to F), 143.2 (d, J=11.9 Hz, due to F), 145.6, 147.5 (d,
J=13.4 Hz, due to F), 149.9 (d, J=13.4 Hz, due to F), 150.6,
155.5.
2-(3-Chloro-4-hydroxyphenylamino)-3-(hydroxymethyl)-9-methoxy-4H-pyrido[1,-
2-a]pyrimidin-4-one (280)
##STR00301##
[0709] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 3.93 (s, 3H),
4.68 (s, 2H), 5.14 (s, 1H), 6.99 (d, J=8.4 Hz, 1H), 7.06 (dd, J=7.2
Hz, 7.2 Hz, 1H), 7.26 (dd, J=1.2 Hz, 8.0 Hz, 1H), 7.38 (dd, J=1.2
Hz, 8.0 Hz, 1H), 8.25 (d, J=2.8 Hz, 1H), 8.45 (dd, J=1.2 Hz, 7.2
Hz, 1H), 8.52 (s, 1H), 9.79 (s, 1H).
2-(3,4-Dichlorophenylamino)-3-(hydroxymethyl)-9-methoxy-4H-pyrido[1,2-a]py-
rimidin-4-one (281)
##STR00302##
[0711] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 3.93 (s, 3H),
4.66 (d, J=5.2 Hz, 2H), 5.16 (d, J=5.2 Hz, 1H), 7.09 (t, J=7.2 Hz,
1H), 7.29 (d, J=6.8 Hz, 1H), 7.48 (d, J=8.8 Hz, 1H), 7.64 (dd,
J=2.8 Hz, 8.8 Hz, 1H), 8.44 (d, J=7.2 Hz, 1H), 8.67 (d, J=2.8 Hz,
1H), 8.82 (s, 1H).
3-(Hydroxymethyl)-9-methoxy-2-(4-methyl-3-(trifluoromethyl)phenylamino)-4H-
-pyrido[1,2-a]pyrimidin-4-one (282)
##STR00303##
[0713] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 2.49 (t, J=2.0
Hz, 3H due to CF.sub.3), 3.93 (s, 3H), 4.70 (d, J=4.8 Hz, 2H), 5.19
(t, J=4.8 Hz, 1H), 7.10 (t, J=7.2 Hz, 1H), 7.29 (dd, J=1.2 Hz, 8.0
Hz, 1H), 7.32 (d, J=8.4 Hz, 1H), 7.74 (dd, J=1.6 Hz, 8.0 Hz, 1H),
8.46 (dd, J=1.2 Hz, 6.8 Hz, 1H), 8.81 (s, 1H), 8.85 (d, J=2.0 Hz,
1H).
2-(4-Fluoro-3-(trifluoromethyl)phenylamino)-3-(hydroxymethyl)-9-methoxy-4H-
-pyrido[1,2-a]pyrimidin-4-one (283)
##STR00304##
[0715] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta.3.92 (s, 3H),
4.68 (d, J=5.2 Hz, 2H), 5.12 (t, J=5.2 Hz, 1H), 7.07 (dd, J=7.2 Hz,
7.2 Hz, 1H), 7.27 (d, J=7.2 Hz, 1H), 7.37-7.42 (m, 1H), 7.86-7.88
(m, 1H), 8.43 (d, J=7.2 Hz, 1H), 8.87 (s, 1H), 8.99-9.00 (m,
1H).
2-(2,3-Dihydro-1H-inden-5-ylamino)-3-(hydroxymethyl)-9-methoxy-4H-pyrido[1-
,2-a]pyrimidin-4-one (284)
##STR00305##
[0717] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 1.97-2.05 (m,
2H), 2.79 (t, J=7.6 Hz, 2H), 2.85 (t, J=7.6 Hz, 2H), 3.92 (s, 3H),
4.69 (d, J=5.6 Hz, 2H), 5.26 (t, J=5.6 Hz, 1H), 7.04 (dd, J=7.2 Hz,
1H), 7.12 (d, J=8.4 Hz, 1H), 7.24 (dd, J=0.8 Hz, 7.6 Hz, 1H), 7.46
(dd, J=2.0 Hz, 8.0 Hz, 1H), 7.82 (s, 1H), 8.45 (dd, J=1.2 Hz, 7.2
Hz, 1H), 8.59 (s, 1H).
2-(Benzo[d][1,3]dioxol-5-ylamino)-3-(hydroxymethyl)-9-methoxy-4H-pyrido[1,-
2-a]pyrimidin-4-one (285)
##STR00306##
[0719] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 3.91 (s, 3H),
4.68 (d, J=5.2 Hz, 2H), 5.21 (t, J=5.2 Hz, 1H), 5.98 (s, 2H), 6.84
(d, J=8.4 Hz, 1H), 7.05-7.07 (m, 1H), 7.26 (dd, J=1.2 Hz, 8.0 Hz,
1H), 7.82 (d, J=2.0 Hz, 1H), 8.46 (d, J=2.0 Hz, 1H), 8.45 (dd,
J=1.2 Hz, 7.2 Hz, 1H), 8.56 (s, 1H).
2-(2,3-Dihydrobenzo[b][1,4]dioxin-6-ylamino)-3-(hydroxymethyl)-9-methoxy-4-
H-pyrido[1,2-a]pyrimidin-4-one (286)
##STR00307##
[0721] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 3.92 (s, 3H),
4.19-4.24 (m, 4H), 4.67 (d, J=5.2 Hz, 2H), 5.19 (t, J=5.2 Hz, 1H),
6.77 (d, J=8.8 Hz, 1H), 7.05 (dd, J=7.2 Hz, 7.2 Hz, 1H), 7.12 (dd,
J=2.4 Hz, 8.4 Hz, 1H), 7.26 (d, J=6.8 Hz, 1H), 7.64 (d, J=2.4 Hz,
1H), 8.46 (dd, J=2.0 Hz, 7.2 Hz, 1H), 8.47 (s, 1H).
3-(Hydroxymethyl)-9-methoxy-2-(1-methyl-1H-indol-5-ylamino)-4H-pyrido[1,2--
a]pyrimidin-4-one (287)
##STR00308##
[0723] m.p=195-197.degree. C.; .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. 3.82 (s, 3H), 3.97 (s, 3H), 4.77 (d, J=5.2 Hz, 2H), 5.28
(t, J=5.2 Hz, 1H), 6.42 (d, J=3.0 Hz, 1H), 7.09 (dd, J=7.2, 7.6 Hz,
1H), 7.28-7.30 (m, 1H), 7.33 (d, J=3.0 Hz, 1H), 7.41 (d, J=8.8 Hz,
1H), 7.46 (dd, J=2.0, 8.8 Hz, 1H), 8.18 (d, J=2.0 Hz, 1H), 8.52
(dd, J=1.2, 6.8 Hz, 1H), 8.62 (br s, 1H).
3-(Hydroxymethyl)-9-methoxy-2-(1-methyl-1H-benzo[d]imidazol-5-ylamino)-4H--
pyrido[1,2-a]pyrimidin-4-one (288)
##STR00309##
[0725] m.p=186.degree. C. (decomp.); .sup.1H NMR (400 MHz,
DMSO-d.sub.6) .delta. 3.87 (s, 3H), 3.98 (s, 3H), 4.79 (d, J=5.6
Hz, 2H), 5.31 (t, J=5.6 Hz, 1H), 7.08 (dd, J=7.2, 7.2 Hz, 1H), 7.28
(dd, J=0.8, 7.6 Hz, 1H), 7.50 (d, J=8.8 Hz, 1H), 7.56 (dd, J=2.0,
8.8 Hz, 1H), 8.13 (s, 1H), 8.34 (d, J=1.6 Hz, 1H), 8.53 (dd, J=0.8,
7.2 Hz, 1H), 8.73 (br s, 1H).
3-(Hydroxymethyl)-9-methoxy-2-(1-methyl-1H-indazol-5-ylamino)-4H-pyrido[1,-
2-a]pyrimidin-4-one (289)
##STR00310##
[0727] m.p=205.degree. C. (decomp.); .sup.1H NMR (400 MHz, DMSO-d)
.delta. 3.40 (s, 3H), 4.08 (s, 3H), 4.78 (d, J=4.8 Hz, 2H), 5.28
(t, J=5.0 Hz, 1H), 7.12 (dd, J=7.2, 7.6 Hz, 1H), 7.32 (1H, J=1.2,
7.6 Hz, 1H), 7.62 (d, J=9.0 Hz, 1H), 7.68 (dd, J=2.0, 9.0 Hz, 1H),
8.04 (m, 1H), 8.07 (d, J=1.2 Hz, 1H), 8.53 (dd, J=1.2, 6.8 Hz, 1H),
8.75 (br s, 1H).
9-(Difluoromethoxy)-2-(4-fluorophenylamino)-3-(hydroxymethyl)-4H-pyrido[1,-
2-a]pyrimidin-4-one (290)
##STR00311##
[0729] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 4.67 (d, J=5.2
Hz, 2H), 5.14 (t, J=5.2 Hz, 1H), 7.07-7.11 (m, 3H), 7.17 (t, J=74
Hz due to F.sub.2, 1H), 7.63-7.69 (m, 3H), 8.71 (d, J=7.2 Hz, 1H),
8.75 (s, 1H).
2-(4-Chlorophenylamino)-9-(difluoromethoxy)-3-(hydroxymethyl)-4H-pyrido[1,-
2-a]pyrimidin-4-one (291)
##STR00312##
[0731] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 4.69 (d, J=5.6
Hz, 2H), 5.23 (t, J=5.2 Hz, 1H), 7.13 (dd, J=7.2 Hz, 7.2 Hz, 1H),
7.23 (t, J=74 Hz, 1H, due to F.sub.2), 7.30-7.33 (m, 2H), 7.72-7.75
(m, 3H), 8.75 (dd, J=1.2 Hz, 7.2 Hz, 1H), 8.86 (s, 1H);
9-(Difluoromethoxy)-2-(3,4-difluorophenylamino)-3-(hydroxymethyl)-4H-pyrid-
o[1,2-a]pyrimidin-4-one (292)
##STR00313##
[0733] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 4.70 (d, J=5.2
Hz, 2H), 5.22 (s, 1H), 7.16 (dd, J=7.2 Hz, J=7.2 Hz, 1H), 7.26 (t,
J=74 Hz, due to F2, 1H), 7.33-7.38 (m, 2H), 7.75 (d, J=7.2 Hz, 1H),
8.12 (dd, J=7.6 Hz, 12.8 Hz, 1H), 8.76 (d, J=6.8 Hz, 1H), 8.90 (s,
1H); LC-MS (ESI, m/z): 370[M+H].sup.+.
2-(3,4-Dichlorophenylamino)-9-(difluoromethoxy)-3-(hydroxymethyl)-4H-pyrid-
o[1,2-a]pyrimidin-4-one (293)
##STR00314##
[0735] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 4.68 (s, 2H),
5.19 (s, 1H), 7.15 (t, J=7.2 Hz, 1H), 7.24 (t, J=74 Hz, due to
F.sub.2, 1H), 7.47-7.57 (m, 2H), 7.72 (d, J=7.2 Hz, 1H), 8.32 (d,
J=2.4 Hz, 1H), 8.73 (dd, J=1.6 Hz, 7.2 Hz, 1H), 8.92 (s, 1H).
2-(3-Chloro-4-fluorophenylamino)-9-(difluoromethoxy)-3-(hydroxymethyl)-4H--
pyrido[1,2-a]pyrimidin-4-one (294)
##STR00315##
[0737] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 4.68 (d, J=4.0
Hz, 2H), 5.18 (s, 1H), 7.15 (dd, J=7.2 Hz, 7.2 Hz, 1H), 7.24 (t,
J=74 Hz, 1H, due to F.sub.2), 7.32 (dd, J=9.2 Hz, 9.2 Hz, 1H),
7.50-7.54 (m, 1H), 7.73 (d, J=7.6 Hz, 1H), 8.22 (dd, J=2.8 Hz, 6.8
Hz, 1H), 8.74 (dd, J=1.2 Hz, 7.2 Hz, 1H), 8.86 (s, 1H).
2-(1H-Indol-5-ylamino)-9-(difluoromethoxy)-3-(hydroxymethyl)-4H-pyrido[1,2-
-a]pyrimidin-4-one (295)
##STR00316##
[0739] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 4.72 (d, J=4.8
Hz, 2H), 5.23 (t, J=4.8 Hz, 1H), 6.34 (s, 1H), 7.05-7.09 (m, 1H),
7.23 (dd, J=8.8 Hz, 8.8 Hz, 1H), 7.25 (t, J=74.4 Hz, 1H due to
F.sub.2), 7.31-7.33 (m, 2H), 7.68 (d, J=7.2 Hz, 1H), 7.93 (s, 1H),
8.70 (s, 1H), 8.73 (d, J=1.2 Hz, 1H), 10.99 (s, 1H).
2-(3-chlorophenylamino)-3-(hydroxymethyl)-6,8-dimethyl-4H-pyrido[1,2-a]pyr-
imidin-4-one (296)
##STR00317##
[0741] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 2.32 (s, 3H), 2.40
(s, 3H), 3.55 (s, 2H), 6.78 (s, 1H), 7.06 (d, J=2.0 Hz, 1H), 7.21
(dd, J=8.0 Hz, J=8.0 Hz, 1H), 7.39 (d, J=8.4 Hz, 1H), 7.69 (d,
J=2.0 Hz, 1H), 7.71 (s, 1H), 9.60 (s, 1H); LC-MS (ESI, m/z): 330
[M+H].sup.+.
7,9-Dichloro-2-(3-chlorophenylamino)-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyr-
imidin-4-one (297)
##STR00318##
[0743] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 4.65 (s, 2H),
5.70 (d, J=7.6 Hz, 1H), 7.29 (dd, J=8.0 Hz, J=8.0 Hz, 1H), 7.57
(dd, J=8.0 Hz, J=8.0 Hz, 1H), 8.25 (s, 1H), 8.32 (d, J=2.0 Hz, 1H),
8.76 (d, J=2.0 Hz, 1H).
2-(3-Chlorophenylamino)-7,9-difluoro-3-(hydroxymethyl)-4H-pyrido[1,2-a]pyr-
imidin-4-one (298)
##STR00319##
[0745] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 4.69 (d, J=4.8 Hz,
2H), 5.31 (t, J=4.8 Hz, 1H), 7.06 (dd, J=1.2 Hz, 8.0 Hz, 1H), 7.32
(t, J=8.0 Hz, 1H), 7.56 (dd, J=1.2 Hz, 8.0 Hz, 1H), 8.02 (s, 1H),
8.18-8.23 (m, 1H), 8.68 (t, J=2.0 Hz, 1H), 8.90 (s, 1H).
(4-Oxo-2-(phenylamino)-4H-pyrido[1,2-a]pyrimidin-3-yl)methyl
benzoate (299)
##STR00320##
[0747] m.p=178-179.degree. C.; .sup.1H NMR (400 MHz, DMSO-d.sub.6)
.delta. 5.66 (s, 2H), 6.96 (ddd, J=1.2, 1.2, 6.8 Hz, 1H), 7.06-7.10
(m, 1H), 7.33-7.44 (m, 5H), 7.53-7.56 (m, 1H), 7.61-7.65 (m, 1H),
7.72 (m, 2H), 8.12 (dd, J=1.2, 8.4 Hz, 1H), 9.14 (brs, 1H).
(4-Oxo-2-(phenylamino)-4H-pyrido[1,2-a]pyrimidin-3-yl)methyl
acetate (300)
##STR00321##
[0749] m.p=160-161.degree. C.; .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 2.13 (s, 3H), 6.92 (dd, J=6.8, 7.2 Hz, 1H), 7.04-7.08 (m,
1H), 7.30-7.37 (m, 3H), 7.59-7.66 (m, 3H), 8.91 (brs, 1H), 8.94 (d,
J=7.2 Hz, 1H).
(4-Oxo-2-(phenylamino)-4H-pyrido[1,2-a]pyrimidin-3-yl)methyl
isobutyrate (301)
##STR00322##
[0751] m.p=161-163.degree. C.; .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. 1.17 (d, J=7.2 Hz, 6H), 2.62-2.65 (m, 1H), 6.94 (dd, J=6.8,
7.2 Hz, 1H), 7.04-7.08 (m, 1H), 7.31-7.38 (m, 3H), 7.60-7.67 (m,
3H), 8.95 (brs, 1H), 8.95 (d, J=6.8 Hz, 1H).
Example 8
Additional Studies on Dinitrobenzamide Compounds
[0752] Two representative molecules, compounds 4 and 24, were
re-synthesized in-house and subjected to conventional CFU-based
activity testing in primary macrophages (FIG. 7). A ten-fold
decrease in the number of CFUs, similar to that seen with INH, was
observed for both compounds five days after infection on three
different cell lines. This confirms the potency of this series of
compounds.
[0753] To address the issue of toxicity, compounds 4 and 24 were
tested on a panel of five cell lines derived from different body
tissue. Cells were incubated with increasing amounts of compound
and cell viability was assessed with resazurin after 5 days of
co-incubation. Percentage cytotoxicity was determined by taking as
a reference the resofurin fluorescence measured by DMSO containing
wells. The concentration where fifty percent of the cells died was
defined as the Minimal Toxic Concentration (MTC.sub.50). Both
compounds 4 and 24 showed no cytotoxicity against the panel of cell
lines suggesting this series of compounds to be promising new
anti-tuberculosis drugs (Table 4).
[0754] To gain insight into the possible specificity of activity of
compounds 4 and 24, analysis of the broad antimicrobial spectrum
was undertaken and showed that the effect of these dinitrobenzamide
derivatives was mainly restricted to actinomycetes with the most
potent activity observed against Mycobacterium (Table 4). Of
particular importance, the tested DNB were also highly active
against multidrug-resistant (MDR) and extensively drug-resistant
(XDR) clinical isolates, suggesting that they might act on
different targets than current antituberculosis compounds.
[0755] Mutation frequency of M. tuberculosis H37Rv was determined
for compounds 4 and 24. Increasing numbers of bacteria grew on 7H10
agar medium supplemented with different concentrations of
compounds. After a 6-week growth, colonies were counted in order to
evaluate the proportion of spontaneous mutational frequency (Table
6). For compound 4, 1.times.10.sup.-6 and 1.times.10.sup.-8
frequencies of resistance were found at 0.2 .mu.g/ml and 3.2
.mu.g/ml, respectively. Spontaneous mutational rate was therefore
calculated to be 1.times.10.sup.-7. For compound 24, at 0.2
.mu.g/ml and 3.2 .mu.g/ml, frequency of mutation was
7.times.10.sup.-7 and 1.times.10.sup.-8, respectively which
corresponds to a mean frequency of 3.5.times.10.sup.-7. Overall,
these values were superior to frequency of mutation observed for
INH-resistant mutants (3.times.10.sup.-6). These results, thus,
demonstrate that this class of compounds result in a low frequency
of mutation.
Example 9
Additional Studies on Pyridopyrimidinone Compounds
[0756] Table 5 shows the minimal inhibitory concentration (MIC) of
one representative compound, 133, on different Mycobacterial
species. While it has no effect on the fast growing Mycobacterium
smegmatis mc.sup.2, it was able to inhibit typical laboratory
strains such as H37Rv, H37Ra and BCG Pasteur with an MIC of 2
.mu.M. More importantly, the antimicrobial activity of 133 was also
tested against clinical isolates strains of mycobacteria. The MIC
values for multi-drug-resistant (MDR-TB) and
extensive-drug-resistant (XDR-TB) isolates strains were within the
micromolar range.
[0757] To address the issue of toxicity, compound 133 was tested on
a panel of seven cell lines derived from different body tissue.
Cells were incubated with increasing amounts of compound and cell
viability was assessed with resazurin after 5 days of
co-incubation. Percentage of cytotoxicity was determined by taking
as a reference the resofurin fluorescence measured by DMSO
containing wells. The concentration where fifty percent of the
cells died was defined as the Minimal Toxic Concentration
(MTC.sub.50). Compound 133 showed no cytotoxicity for all tested
cell lines up to 100 .mu.M (Table 5). The selectivity index, which
consists of the ratio between antitubercular activity and
cytotoxicity was therefore above 50 for both extracellular and
intracellular mycobacteria suggesting this series of compounds to
be promising new anti-tuberculosis drugs.
[0758] The effect of this series of compounds on primary
macrophages was further determined. Host cells that had priory been
incubated with compound 232 harbored fewer bacteria compared to
DMSO control and were more abundant at day 5 after infection as
shown in FIG. 8. Similar data were obtained for compound 133 (data
not shown). Conventional CFU determination was then performed seven
days after infection to quantify the remaining bacterial load. A
ten-fold decrease in the number of CFUs, similar to that seen with
INH, was observed for both compounds on both human and mouse cells
(FIG. 8). This confirms the potency of this series of
compounds.
[0759] Mutation frequency of M. tuberculosis H37Rv was determined
for compound 264. Increasing numbers of bacteria grew on 7H10 agar
medium supplemented with different concentrations of compound.
After a 6-week growth, colonies were counted in order to evaluate
the proportion of spontaneous mutational frequency (Table 6).
Compound 264 gave frequencies of resistance of 3.4.times.10.sup.-6
and 8.times.10.sup.-6 at 0.4 and 0.8 .mu.g/ml, respectively, and
2.times.10.sup.-8 at both 1.6 .mu.g/ml and 3.2 .mu.g/ml.
Accordingly, spontaneous mutational rate was calculated to be
7.times.10.sup.-7. Overall, these values are better than the
frequency of mutation observed for INH (2.9.times.10.sup.-6). These
results, therefore, demonstrate that this class of compounds result
in a low frequency of mutation.
[0760] One of the current challenges for TB drug discovery is the
identification of compounds that are active against persistent
bacteria. Although the location and state of latent bacteria
remains a matter of debate, one commonly shared hypothesis for
mycobacterial persistence is that M. tuberculosis bacilli are able
to survive in macrophages for prolonged periods of time and, unlike
other bacteria, are able to actively replicate. The intraphagosomal
profile of M. tuberculosis is complex; a large variety of genes are
over-expressed and timely regulated and are also dependent on
environmental factors. Altogether, this makes the identification of
one specific tubercle factor that could be selected as the ideal
target difficult. Consequently, non-target cell-based assays are a
critical tool in the search of intracellular M. tuberculosis
inhibitors.
[0761] Investigation of bacillus growth inhibitors within
macrophages has long been limited due to cumbersome CFU plating,
slow bacillus growth, safety requirements and difficulties in
setting-up appropriate infection conditions. As a consequence, this
approach was always used as a secondary assay after the initial
selection of compounds that are active on in vitro extracellular
growth. With the advent of automated confocal microscopy, the above
mentioned limitations could be readdressed and the inventors show
the feasibility of large scale compound screening. It was decided
to perform suspension macrophage batch infection in order to
minimize the steps and to meet safety requirements. To this end,
careful attention was paid to the removal of the extracellular
non-phagocytosed mycobacteria. The centrifugation conditions used
during the wash steps were set up in order to recover only the
infected cells and discard most of the extracellular bacteria. By
microscopy the inventors confirmed that unbound mycobacteria
represented less than 10% of the total bacterial load (data not
shown). Mycobacteria are able to grow independently of host cells
and consequently any remaining extracellular bacilli would greatly
compromise the validity of the inventors' model. To this end, an
additional amikacin treatment step was added to the protocol to
further eliminate any remaining mycobacteria. Thus with the
optimized protocol, there is almost no non-phagocytosed
mycobacteria left by the time compound is added. The obtained
results also demonstrate that it is specifically the effect on the
intracellular mycobacteria that is being measured with compound
treatment. Indeed, the inventors observed a weak inhibition with
rifampin, an antibiotic that is known to poorly penetrate cells.
The 50-fold reproducible decrease in MIC for rifampin in the
intracellular assay compared to the in vitro growth assay proved
that the targeted bacteria are not extracellular. Otherwise no
difference would have been seen in MIC between the two assays.
Similarly, compounds able to inhibit mycobacterial growth in the
phenotypic cell-based assay, but not the in vitro growth assay were
also identified. In addition, the fact that the compounds are mixed
with previously infected cells should decrease the chance for the
identification of primary infection inhibitors. However, such
compounds may still be identified as blockers of neighboring cell
infection.
[0762] Compared to a conventional CFU-plating method, the
microscopy based detection of fluorescent bacteria is not
sufficiently sensitive to distinguish between dead and live bacilli
as the GFP signal is stable for several days. Indeed, at a high
concentration of INH, rifampin or active compound, there is always
10% of the cells that appear to be infected, which is similar to
the initial infection ratio. Surprisingly, no CFU could be
recovered after plating such samples. Owing to the fact that latent
bacilli are able to recover growth (Cho et al., 2007), the
microscopy-detected bacilli must be dead bacilli rather than latent
bacilli. Thus, the inventors' assay detects compounds that
interfere with bacilli growth within macrophages.
[0763] As it is well established and confirmed (FIG. 1a),
macrophages are able to support high bacterial loads which end up
encompassing a large part of the cell cytoplasm and eventually lead
to macrophage cell death. It is obvious when M. tuberculosis is the
infectious agent compared to BCG (Bacille Calmette-Guerin), which
even at high MOI fails to induce much cytotoxicity (data not
shown). Taking this into account, it was decided to set the data
acquisition at day 5 post-infection when the cell number in the
DMSO samples had significantly decreased relative to the
antibiotically protected controls. Thus, monitoring cell number was
an additional parameter enabling the inventors to confirm the
compound's antibacterial activity.
[0764] Unlike direct fluorescence based assays, analysis for
image-based assays proved to be much more variable. Several
parameters that are inherent to the biology of the assay partially
explain the lower Z'-values that are usually accepted for HTS
validation. The remaining fluorescent dead bacilli do not have much
of an impact on the Z'-value, rather the variability in the
infection ratio for the DMSO controls seems to account for the
discrepancy. Also of importance is the fact that, upon infection,
the macrophages had a tendency to migrate which in turn led to a
heterogeneous set of images (FIG. 2a). However, the aim of the
primary screen was to identify compounds fully active at a
concentration of 20 .mu.M. Thus, for this purpose, a positive Z'
for the infection ratio (INH/DMSO) was considered an acceptable
value. The best proof of the validity of the hit selection
according to the present invention comes from the subsequent serial
dilution analysis, whereby almost 100% of the hits were confirmed.
For each of the hits, a nicely fitted dose-response curve for the
infection ratio was obtained as well as for the non-toxic compound
in terms of cell number. Again, cell number brought an additional
confirmation of the results that is totally independent of green
fluorescence emission and GFP expression.
[0765] Obviously compounds found to be active against both
intracellular and in vitro M. tuberculosis growth are the most
promising. The best inhibitors isolated from this library have an
inhibitory activity within the same range as INH. Further structure
activity relationship studies will contribute to determine if their
activity could be improved. In the course of another study using
this phenotypic cell-based model, MIC down to the ng/mL scale was
obtained for compounds with known in vitro antibacterial efficacy
showing that compounds with a lower MIC than INH can be identified
by the assay according to the present invention (data not shown).
Of utmost interest are the compounds that are active only in the
intracellular bacteria assay as they are likely to have a new
mechanism of action independent of the infecting strain suggesting
that they may also be active on the non-curable
multi-drug-resistant (MDR)-strains.
[0766] Taken together, the above results show that monitoring M.
tuberculosis growth with automated fluorescence microscopy is
highly robust and reliable and that this method enables fast
selection of potent anti-TB compounds.
TABLE-US-00001 TABLE 1 QIM QIM QIM QUM QUM QUM QIM QIM QIM Confirm
Confirm Confirm Confirm Confirm Confirm Primary Confirm Confirm
Confirm Primary % % % Primary % % % QIM CellNb CellNb CellNb QIM %
Inhibition Inhibition Inhibition QUM % Inhibition Inhibition
Inhibition ID Structure CellNb 20 uM 2 uM 0.2 uM Inhibition 20 uM 2
uM 0.2 uM Inhibition 20 uM 2 uM 0.2 uM IPK00000132 ##STR00323##
88.3 113.8 208.5 241.4 25.4 50.7 0.2 10.3 99.9 89.1 41.2 43.9
IPK00000190 ##STR00324## 435.5 317.6 173.6 190.0 91.9 96.5 13.2
12.7 2.9 42.3 29.4 34.6 IPK00000203 ##STR00325## 77.0 148.0 92.0
241.4 -28.5 -12.3 7.7 -2.0 99.7 69.7 52.4 32.5 IPK00000217
##STR00326## 235.5 249.8 541.6 472.3 26.6 24.5 70.4 54.5 98.9 49.7
67.4 56.9 IPK00000287 ##STR00327## 350.3 412.9 246.1 315.9 65.9
66.0 -1.3 11.4 -13.8 36.4 36.1 45.9 IPK00000301 ##STR00328## 373.5
248.3 457.3 232.6 88.2 40.4 77.1 4.7 98.7 43.0 82.8 46.4
IPK00000389 ##STR00329## 72.5 103.0 200.6 265.3 27.7 84.6 86.6 20.1
100.3 67.0 76.5 44.0 IPK00000390 ##STR00330## 78.0 133.4 75.6 142.3
15.7 67.9 43.2 2.2 99.7 72.7 68.8 44.0 IPK00000391 ##STR00331##
63.0 128.8 148.9 220.9 31.6 76.4 36.8 2.9 99.6 76.7 41.4 46.6
IPK00000635 ##STR00332## 424.3 328.8 320.9 262.8 97.6 65.2 22.8
17.7 42.0 43.4 41.9 23.7 IPK00000731 ##STR00333## 61.3 166.5 308.8
393.1 -28.2 25.8 14.7 45.7 76.6 80.8 33.0 41.0 IPK00000802
##STR00334## 305.8 484.5 218.8 306.6 83.2 98.0 9.3 0.7 34.8 98.2
31.9 36.4 IPK00000812 ##STR00335## 396.3 248.0 225.6 292.9 64.1
78.4 18.9 2.7 97.2 48.0 39.7 36.5 IPK00000933 ##STR00336## 314.5
333.6 475.9 264.8 79.9 56.9 92.5 8.5 30.3 69.8 44.5 31.8
IPK00000941 ##STR00337## 345.8 446.5 488.3 257.8 92.9 99.6 92.8
19.6 97.7 99.4 59.6 28.3 IPK00000942 ##STR00338## 376.5 255.0 473.5
326.3 93.1 97.9 92.3 21.3 92.6 100.0 50.3 28.8 IPK00000978
##STR00339## 454.5 376.1 414.4 325.5 102.6 88.6 50.2 17.0 39.5
100.1 61.1 30.0 IPK00001006 ##STR00340## 322.8 380.9 344.9 412.0
81.0 81.8 18.0 17.1 98.4 39.2 43.1 39.5 IPK00001119 ##STR00341##
190.8 279.1 80.0 248.8 60.0 47.6 31.6 9.7 90.5 43.1 93.2 67.2
IPK00001165 ##STR00342## 145.5 201.1 336.5 259.6 6.5 41.9 23.4 4.7
100.0 96.3 40.6 39.8 IPK00001367 ##STR00343## 358.0 457.3 545.1
452.5 98.7 66.7 102.1 84.1 98.7 69.7 90.2 49.4 IPK00001368
##STR00344## 276.3 438.8 528.8 400.1 65.0 67.2 101.8 50.9 98.9 77.6
77.1 50.4 IPK00001369 ##STR00345## 327.5 443.3 532.1 405.8 91.3
58.6 104.9 68.9 99.2 62.4 79.1 51.2 IPK00001370 ##STR00346## 309.3
518.5 510.9 412.6 88.3 103.0 98.1 50.5 98.4 67.6 79.4 46.3
IPK00001371 ##STR00347## 358.8 377.4 544.3 476.5 102.9 97.5 105.2
86.3 99.4 68.4 92.2 51.6 IPK00001372 ##STR00348## 355.5 457.3 541.6
448.5 82.2 100.6 103.7 63.2 100.0 98.0 73.9 57.7 IPK00001536
##STR00349## 146.5 168.0 286.0 331.0 38.1 36.1 -4.8 15.8 100.3 79.5
59.4 41.9 IPK00001600 ##STR00350## 93.0 300.3 265.4 278.8 -19.0
37.9 19.4 3.2 90.5 68.0 48.6 47.1 IPK00001605 ##STR00351## 192.0
282.0 148.9 206.8 67.9 43.4 0.8 -2.2 98.7 70.0 42.5 36.6
IPK00001865 ##STR00352## 218.0 256.4 218.3 256.6 23.1 39.2 14.0
28.0 99.9 99.0 54.0 36.4 IPK00001866 ##STR00353## 63.3 130.4 296.6
258.1 -26.8 16.7 0.4 8.6 100.6 99.6 72.0 39.3 IPK00001882
##STR00354## 106.8 184.1 209.0 443.0 -57.3 4.1 17.6 42.7 99.3 67.6
41.9 41.1 IPK00001897 ##STR00355## 314.0 553.9 299.8 288.5 76.7
83.8 20.0 7.3 10.3 36.1 33.9 40.5 IPK00001984 ##STR00356## 402.0
610.5 329.0 287.9 88.8 94.6 2.2 7.8 -10.5 43.7 36.8 46.7
IPK00002187 ##STR00357## 405.0 609.0 403.4 305.3 90.2 96.5 29.8
15.4 2.7 41.9 42.9 49.1 IPK00002233 ##STR00358## 372.0 517.1 472.3
315.6 66.0 90.0 33.5 30.3 96.0 74.2 45.4 35.7 IPK00002443
##STR00359## 203.8 205.0 349.0 352.1 5.8 59.1 58.3 50.4 75.8 67.7
40.1 34.7 IPK00002772 ##STR00360## 333.3 238.1 440.4 267.0 82.6
100.0 77.7 29.3 100.0 100.9 97.6 35.1 IPK00002774 ##STR00361##
366.0 435.8 478.8 268.5 87.2 96.9 83.3 -2.7 98.8 100.4 97.5 43.5
IPK00002777 ##STR00362## 378.0 322.3 315.3 218.1 89.1 86.3 47.7
-12.6 99.6 100.4 96.8 40.2 IPK00002778 ##STR00363## 332.0 499.4
543.5 366.0 79.6 96.3 99.3 33.0 98.8 78.2 97.5 71.7 IPK00002785
##STR00364## 315.0 224.8 487.4 429.3 100.3 40.1 99.4 83.0 99.6 71.7
97.9 97.2 IPK00002791 ##STR00365## 410.8 325.6 434.1 295.3 89.0
46.1 98.5 14.2 99.3 75.0 97.6 65.8 IPK00002835 ##STR00366## 315.3
308.9 478.6 489.8 94.5 17.8 102.4 64.5 99.8 48.3 98.1 94.0
IPK00003316 ##STR00367## 283.0 303.9 573.9 296.1 81.9 92.3 69.6 5.0
43.4 61.3 42.0 34.1 IPK00003361 ##STR00368## 188.3 111.8 434.1
210.8 31.6 62.9 13.8 16.2 94.9 67.1 37.6 51.6 IPK00003556
##STR00369## 226.0 524.3 313.1 247.1 83.3 89.0 18.8 9.1 71.9 65.6
39.6 41.5 IPK00003558 ##STR00370## 104.0 279.9 330.0 292.3 -51.3
2.8 34.4 5.1 87.2 65.3 46.4 45.1
IPK00003607 ##STR00371## 142.3 164.4 293.9 267.4 27.7 59.5 32.0
17.2 96.4 70.6 47.9 42.3 IPK00004014 ##STR00372## 95.5 330.0 262.3
321.4 -38.6 18.1 15.6 20.4 97.7 68.4 46.8 38.7 IPK00004145
##STR00373## 324.5 243.8 527.9 437.4 88.6 87.2 103.2 56.7 44.7
100.5 47.4 38.4 IPK00004146 ##STR00374## 320.0 347.1 542.6 386.9
81.2 93.3 101.2 41.8 98.3 57.9 81.4 41.1 IPK00004147 ##STR00375##
362.8 345.8 516.1 479.8 102.4 95.5 101.8 74.5 91.8 99.5 82.3 43.8
IPK00004148 ##STR00376## 315.3 347.6 508.4 414.6 84.5 88.4 97.5
55.9 96.4 38.6 84.5 43.7 IPK00004149 ##STR00377## 336.5 338.1 535.4
394.6 83.8 94.5 102.3 56.4 98.9 39.4 85.5 44.4 IPK00004150
##STR00378## 296.3 315.9 515.5 396.3 81.6 90.8 102.2 66.3 99.2 77.7
94.3 52.0 IPK00004151 ##STR00379## 351.3 350.6 505.6 368.0 89.0
94.0 102.9 55.1 98.4 70.9 85.2 40.8 IPK00004152 ##STR00380## 262.5
362.8 523.5 451.8 75.6 93.7 103.2 89.7 99.0 56.0 93.0 53.1
IPK00004153 ##STR00381## 426.8 431.1 523.3 197.8 100.2 97.0 98.1
18.8 96.9 44.4 82.2 15.1 IPK00004207 ##STR00382## 198.0 596.4 616.9
259.4 34.7 107.1 104.0 12.4 93.4 98.0 96.5 70.8 IPK00004230
##STR00383## 193.8 376.8 658.0 384.8 73.7 95.9 75.2 11.0 85.2 95.8
62.9 39.1 IPK00004272 ##STR00384## 152.0 199.9 96.1 227.8 11.9 57.6
-1.9 2.5 82.5 67.7 43.7 39.6 IPK00004277 ##STR00385## 367.0 425.5
255.3 352.6 104.9 48.9 7.7 22.4 99.2 67.1 51.3 33.5 IPK00004278
##STR00386## 194.5 341.9 311.5 322.6 73.9 91.7 33.0 25.8 98.7 67.1
56.4 42.9 IPK00004293 ##STR00387## 321.8 451.8 532.9 387.9 108.6
105.5 105.1 49.3 96.6 98.5 76.0 44.3 IPK00004295 ##STR00388## 143.5
433.9 494.6 493.0 64.7 98.8 106.0 70.4 96.6 53.1 82.0 42.2
IPK00004296 ##STR00389## 216.3 477.0 472.5 491.4 84.3 105.4 101.1
77.4 94.3 97.2 83.3 43.3 IPK00004297 ##STR00390## 307.8 483.0 502.4
312.9 99.7 103.4 99.2 19.2 97.0 98.8 73.9 40.1 IPK00004298
##STR00391## 350.0 554.9 494.5 279.6 102.3 105.4 95.3 19.0 30.3
82.9 48.5 31.8 IPK00004299 ##STR00392## 364.0 488.3 567.3 378.4
102.7 106.3 104.1 43.4 97.9 97.1 79.3 51.8 IPK00004300 ##STR00393##
333.3 413.1 537.9 371.0 104.4 101.8 101.4 37.5 98.3 98.6 70.6 51.0
IPK00004301 ##STR00394## 287.8 448.5 584.3 385.1 86.6 102.6 103.6
56.3 98.6 98.8 74.2 47.4 IPK00004302 ##STR00395## 229.5 422.1 483.0
476.3 81.1 99.8 98.7 71.3 98.4 98.2 82.6 48.4 IPK00004305
##STR00396## 228.8 494.4 502.8 469.0 97.1 103.8 101.6 80.2 99.4
98.8 94.4 51.4 IPK00004306 ##STR00397## 200.3 436.0 520.1 213.5
81.4 98.0 98.1 -0.4 99.8 98.5 75.2 38.5 IPK00004307 ##STR00398##
303.5 416.6 541.6 392.0 104.3 98.6 102.5 68.4 99.7 98.9 79.2 44.8
IPK00004308 ##STR00399## 255.5 367.6 486.8 397.6 87.3 95.1 102.7
72.9 77.2 63.9 63.5 39.8 IPK00004309 ##STR00400## 258.5 459.3 516.8
420.0 95.8 102.4 100.5 59.3 99.5 98.4 80.1 49.1 IPK00004310
##STR00401## 176.8 441.6 509.8 367.0 14.3 98.3 92.7 28.5 98.7 66.9
73.1 46.5 IPK00004311 ##STR00402## 366.0 514.6 532.3 350.4 104.4
104.5 99.5 40.3 97.5 70.9 76.7 43.5 IPK00004312 ##STR00403## 195.0
406.3 500.3 432.0 69.9 100.0 104.6 56.5 98.5 69.8 84.5 48.9
IPK00004313 ##STR00404## 177.8 468.8 469.0 269.9 49.2 104.2 101.6
17.8 98.7 98.5 75.0 48.9 IPK00004326 ##STR00405## 347.3 430.3 523.3
413.6 109.4 102.1 102.2 72.4 99.5 98.6 89.6 52.7 IPK00004327
##STR00406## 125.8 438.6 549.8 459.1 47.6 102.0 105.2 87.8 99.2
98.8 86.3 67.0 IPK00004328 ##STR00407## 326.5 408.3 482.8 332.6
94.9 95.9 87.1 11.2 12.2 69.7 38.8 43.0 IPK00004329 ##STR00408##
415.5 454.4 567.1 489.3 111.0 100.2 105.2 71.9 98.3 98.6 76.8 51.3
IPK00004330 ##STR00409## 305.0 503.3 575.8 251.1 107.2 102.5 100.8
-5.6 98.0 97.9 69.7 32.8 IPK00004331 ##STR00410## 334.0 442.5 526.9
321.1 94.6 100.4 101.5 43.8 98.7 52.7 69.8 24.0 IPK00004332
##STR00411## 164.0 452.4 425.4 481.6 60.5 102.5 94.5 72.0 98.9 99.1
78.1 37.0 IPK00004333 ##STR00412## 270.8 522.8 515.1 362.8 83.0
104.4 94.5 31.3 98.4 98.8 59.3 34.8 IPK00004335 ##STR00413## 245.5
461.5 484.3 335.4 104.0 100.9 94.4 31.6 96.1 67.1 75.7 39.9
IPK00004362 ##STR00414## 393.8 523.9 561.9 502.1 98.2 104.5 98.8
63.7 99.4 98.3 71.0 42.9 IPK00004383 ##STR00415## 89.0 252.3 303.8
296.4 -80.9 31.3 17.1 22.6 99.7 99.2 50.6 34.2 IPK00004420
##STR00416## 135.0 219.0 278.5 303.0 -67.5 30.7 6.3 24.5 99.6 98.5
52.4 40.3 IPK00004441 ##STR00417## 126.8 307.0 377.5 260.0 -120.3
29.9 34.0 18.0 98.6 84.0 41.4 37.1 IPK00004501 ##STR00418## 145.0
273.8 328.5 305.8 -9.3 30.6 24.3 19.3 99.3 66.8 57.6 41.3
IPK00004678 ##STR00419## 193.3 284.4 388.9 335.3 73.3 88.9 36.5
13.9 100.8 99.1 65.2 36.5 IPK00004680 ##STR00420## 238.8 389.1
277.8 353.3 76.9 64.6 3.1 38.6 99.2 73.5 41.8 44.4
IPK00004683 ##STR00421## 204.0 379.6 437.5 355.0 75.8 61.7 91.7
37.2 100.1 69.6 97.1 44.3 IPK00004686 ##STR00422## 129.3 285.6
305.0 300.1 26.6 40.8 15.0 17.3 100.2 70.3 41.2 38.3 IPK00004687
##STR00423## 239.0 343.3 411.1 328.6 74.4 56.3 61.3 14.4 99.2 62.5
70.4 44.2 IPK00004692 ##STR00424## 303.5 309.6 325.4 304.1 90.2
93.4 8.1 31.2 17.0 53.1 38.4 42.2 IPK00004706 ##STR00425## 290.8
440.0 365.8 300.0 57.3 69.9 17.6 35.0 97.4 76.4 41.9 41.1
IPK00004715 ##STR00426## 281.8 432.3 482.3 298.0 69.7 85.8 61.1
13.3 99.3 97.7 36.4 36.8 IPK00004716 ##STR00427## 280.5 309.1 549.8
297.5 69.4 85.1 77.9 6.0 100.4 98.3 39.0 32.0 IPK00004717
##STR00428## 152.3 196.3 536.1 328.1 57.6 88.5 69.7 46.5 99.3 98.6
36.5 37.7 IPK00004849 ##STR00429## 253.8 281.3 457.3 354.8 84.5
92.9 41.7 25.8 32.6 57.7 34.3 40.3 IPK00004871 ##STR00430## 56.5
98.1 331.5 255.1 62.1 70.0 22.6 4.5 85.2 72.7 44.3 39.8 IPK00004899
##STR00431## 217.8 211.4 494.5 311.4 76.8 73.5 40.3 8.6 84.7 72.0
36.4 43.6 IPK00004900 ##STR00432## 108.0 212.4 503.6 349.5 32.1
74.2 72.2 24.4 86.2 73.9 38.7 48.1 IPK00004903 ##STR00433## 205.3
481.3 525.4 370.8 62.6 48.6 56.0 23.4 94.5 69.1 43.7 41.3
IPK00004920 ##STR00434## 155.5 157.0 405.8 296.1 71.5 88.6 54.8
36.9 28.3 27.0 31.5 36.0 IPK00005250 ##STR00435## 44.0 117.6 289.6
339.1 45.0 76.4 10.8 33.8 101.0 70.4 28.8 30.9 IPK00005275
##STR00436## 48.8 290.3 265.9 256.8 -77.4 10.1 13.3 14.7 85.6 66.9
43.8 43.3 IPK00005778 ##STR00437## 177.0 292.4 203.8 252.1 -10.9
35.5 12.6 4.5 93.9 86.2 39.0 43.7 IPK00005792 ##STR00438## 165.3
197.4 225.8 237.3 -30.7 27.2 23.0 26.2 89.8 81.6 29.1 33.5
IPK00005820 ##STR00439## 344.8 278.0 458.8 295.1 98.1 44.3 66.6
13.3 55.8 68.0 57.9 42.5 IPK00005821 ##STR00440## 452.5 453.1 525.3
341.6 90.9 56.0 75.7 24.8 52.3 41.2 54.6 47.6 IPK00005829
##STR00441## 75.5 224.8 432.0 499.8 63.0 31.5 103.7 77.6 99.2 47.5
93.4 52.2 IPK00005830 ##STR00442## 315.8 435.3 483.4 325.6 95.5
70.8 98.3 21.0 84.5 48.8 72.8 47.0 IPK00006324 ##STR00443## 188.3
183.6 232.5 327.4 49.6 68.7 16.7 17.4 96.2 74.8 38.1 37.1
IPK00006503 ##STR00444## 197.8 172.1 227.0 299.3 68.2 73.2 1.1 14.7
-16.3 38.1 35.2 29.1 IPK00006751 ##STR00445## 142.3 164.5 301.5
367.1 75.2 73.2 17.9 11.0 37.4 59.5 39.9 39.0 IPK00006760
##STR00446## 164.3 278.3 179.1 326.4 63.4 46.6 25.8 17.7 99.7 65.3
46.4 40.8 IPK00006761 ##STR00447## 182.0 478.4 340.5 305.0 84.3
68.5 29.2 14.3 99.3 70.6 44.5 39.6 IPK00006887 ##STR00448## 156.8
183.6 221.4 295.9 73.1 71.4 -12.0 -0.6 92.9 79.3 31.0 38.1
IPK00007311 ##STR00449## 147.8 224.4 168.9 256.1 49.1 56.1 -10.5
6.4 84.0 67.2 33.5 42.2 IPK00007329 ##STR00450## 230.5 193.1 551.9
315.5 78.6 86.6 55.1 1.3 94.1 74.9 73.9 50.0 IPK00007368
##STR00451## 202.3 242.6 557.3 378.6 88.2 92.6 74.7 16.9 94.9 73.4
69.2 53.5 IPK00007369 ##STR00452## 246.5 465.9 375.5 335.9 87.0
32.3 14.0 21.8 80.9 72.9 44.3 37.2 IPK00007370 ##STR00453## 234.0
539.8 523.0 348.3 88.6 103.0 56.7 17.8 96.3 74.9 68.1 49.8
IPK00007371 ##STR00454## 218.3 534.3 467.8 244.5 87.5 65.2 29.8
-4.5 8.3 45.3 59.6 44.5 IPK00007722 ##STR00455## 84.5 253.9 380.5
314.1 -33.0 46.6 33.3 35.6 95.4 68.6 45.7 45.0 IPK00007830
##STR00456## 388.3 540.5 495.6 250.6 108.5 105.0 97.0 8.2 96.6 69.7
62.0 39.2 IPK00007853 ##STR00457## 386.0 523.3 489.9 298.6 84.0
106.6 95.6 17.6 98.3 68.4 73.8 39.3 IPK00007886 ##STR00458## 462.0
583.0 531.4 373.4 96.9 107.5 88.3 32.7 98.5 68.0 74.5 37.9
IPK00007913 ##STR00459## 294.0 528.5 493.6 217.9 69.6 101.6 97.8
8.4 97.0 97.8 68.5 36.6 IPK00007915 ##STR00460## 383.3 470.9 477.4
274.6 82.4 97.9 90.5 13.4 97.1 98.6 72.6 41.5 IPK00007988
##STR00461## 181.8 368.8 289.8 254.3 -69.2 28.1 7.5 0.9 92.2 88.3
34.9 39.3 IPK00008001 ##STR00462## 409.8 469.1 387.1 360.5 14.3
56.6 27.4 20.1 96.1 70.4 47.4 39.5 IPK00008024 ##STR00463## 188.8
321.0 492.1 323.8 0.2 23.0 67.6 13.0 99.5 73.0 62.4 45.3
IPK00008036 ##STR00464## 79.8 251.4 559.6 514.4 -91.1 26.0 75.8
70.0 98.9 71.9 68.7 59.7 IPK00008037 ##STR00465## 110.8 393.6 491.4
377.0 -44.0 36.0 62.1 55.4 99.6 78.6 80.6 63.1 IPK00008038
##STR00466## 307.5 371.6 337.3 264.9 21.0 3.4 32.1 13.3 98.2 73.8
58.7 52.3 IPK00008039 ##STR00467## 99.0 377.1 591.3 418.3 38.3 9.2
73.0 51.8 96.3 68.2 66.6 54.7 IPK00008069 ##STR00468## 123.5 328.0
358.9 289.4 -42.5 16.5 37.6 27.5 99.1 71.8 47.6 46.9 IPK00008081
##STR00469## 363.3 482.0 559.0 405.8 94.7 99.5 104.9 66.3 55.0 69.5
48.4 50.8 IPK00008389 ##STR00470## 38.8 268.3 255.5 217.4 73.5 59.1
19.5 2.6 98.7 72.9 48.1 46.3 IPK00008599 ##STR00471## 67.3 261.3
322.0 267.5 37.1 49.5 4.5 2.2 84.3
72.0 47.7 49.5 IPK00009117 ##STR00472## 191.5 276.8 254.8 319.9
75.6 75.7 35.4 50.6 -11.9 22.4 38.8 42.6 IPK00009149 ##STR00473##
110.5 228.6 265.4 388.5 90.5 95.1 9.3 30.6 94.1 65.5 33.4 37.3
IPK00009438 ##STR00474## 126.3 367.8 286.9 271.6 46.0 6.0 13.6 -9.3
97.2 68.6 54.6 41.3 IPK00009507 ##STR00475## 388.3 551.8 348.6
377.4 70.9 75.8 8.0 19.2 -3.7 39.6 46.1 45.0 IPK00010207
##STR00476## 116.0 124.8 516.3 476.1 94.7 84.7 99.5 79.6 99.2 100.1
84.3 77.4 IPK00010236 ##STR00477## 193.3 194.8 224.0 291.8 65.6
66.5 4.4 13.2 -1.6 16.8 32.7 36.3 IPK00010252 ##STR00478## 179.8
175.9 145.9 219.5 70.4 68.1 -7.0 12.6 18.3 19.1 40.8 36.7
IPK00010328 ##STR00479## 134.3 164.6 419.5 263.8 71.1 71.3 49.5
11.6 5.0 48.2 47.6 39.8 IPK00010376 ##STR00480## 47.8 122.4 109.5
143.1 85.7 74.5 -1.7 -13.2 45.6 50.2 50.0 39.0 IPK00010378
##STR00481## 263.5 409.1 244.8 236.5 94.6 98.0 3.5 -3.9 90.8 94.0
34.8 31.6 IPK00010407 ##STR00482## 243.5 222.5 575.9 330.4 82.3
80.2 65.1 37.5 35.1 58.0 45.1 42.5 IPK00010411 ##STR00483## 203.8
370.3 278.4 240.4 89.7 83.3 19.9 10.2 36.1 52.8 42.4 26.9
IPK00010413 ##STR00484## 115.8 446.5 313.3 260.6 68.8 89.6 25.2
10.1 3.7 37.9 29.0 44.7 IPK00010420 ##STR00485## 29.5 81.9 153.1
230.0 65.2 34.6 -2.9 5.3 67.1 90.6 56.2 40.4 IPK00010467
##STR00486## 183.0 211.4 332.9 283.1 33.8 73.7 16.1 11.9 67.7 86.2
34.2 36.4 IPK00010519 ##STR00487## 374.3 425.6 566.4 521.1 98.7
105.8 104.1 88.5 98.7 66.4 93.6 70.3 IPK00105020 ##STR00488## 316.5
397.9 547.6 482.3 97.1 96.4 103.8 83.8 99.6 66.0 96.0 66.6
IPK00010547 ##STR00489## 125.8 172.4 468.0 270.4 60.1 50.7 68.1
13.0 75.5 95.5 54.7 47.3 IPK00010555 ##STR00490## 181.3 538.8 402.4
272.8 89.3 91.5 55.5 13.0 29.2 62.2 35.1 18.8 IPK00010556
##STR00491## 225.0 169.6 442.0 371.5 65.7 86.9 79.9 28.0 36.1 68.0
39.5 36.4 IPK00010570 ##STR00492## 315.5 573.6 226.4 237.8 61.2
84.6 4.7 16.8 70.7 80.7 53.8 41.8 IPK00010630 ##STR00493## 146.5
171.3 409.1 233.5 65.7 67.2 28.5 9.3 19.1 61.2 36.4 17.1
IPK00010790 ##STR00494## 494.3 577.5 498.0 339.3 79.3 89.6 52.8
23.7 7.0 42.6 39.6 37.4 IPK00010827 ##STR00495## 171.0 369.9 332.0
321.6 54.7 44.1 19.0 14.1 95.0 71.0 47.6 49.0 IPK00010878
##STR00496## 200.3 287.6 400.0 339.8 78.8 68.8 38.3 28.2 20.9 43.1
39.2 38.4 IPK00010900 ##STR00497## 87.8 171.8 348.5 312.4 -7.8 8.2
23.8 15.6 98.4 72.2 55.2 47.6 IPK00010999 ##STR00498## 183.0 170.0
174.3 332.1 8.5 28.1 20.3 22.6 101.3 99.5 90.5 29.6 IPK00011016
##STR00499## 179.5 204.9 224.6 288.0 59.0 86.0 6.0 8.1 99.4 96.3
36.7 44.2 IPK00011017 ##STR00500## 155.5 173.4 221.5 292.3 55.6
71.6 12.9 23.9 92.6 97.3 47.2 44.1 IPK00011079 ##STR00501## 462.3
459.5 260.1 337.0 72.9 83.4 22.8 44.6 19.7 49.5 32.4 34.0
IPK00011267 ##STR00502## 250.8 383.9 356.4 188.8 -36.5 32.6 27.6
8.2 91.3 68.9 59.6 43.8 IPK00011280 ##STR00503## 152.0 183.0 189.0
279.9 41.1 55.6 10.7 7.7 98.3 72.1 32.5 35.9 IPK00011305
##STR00504## 162.0 107.9 309.0 264.5 55.4 56.4 31.6 10.0 74.9 67.9
45.3 41.6 IPK00011377 ##STR00505## 319.8 523.4 437.4 319.8 60.9
74.3 48.4 26.6 66.7 82.9 59.5 45.0 IPK00011401 ##STR00506## 163.5
236.9 198.4 233.3 3.2 -1.5 7.3 18.3 73.9 71.1 51.4 53.6 IPK00011705
##STR00507## 656.5 578.0 540.9 503.5 79.8 80.5 45.4 49.9 12.9 46.3
44.5 39.6 IPK00011714 ##STR00508## 546.0 548.6 349.0 329.5 69.1
66.5 15.6 11.2 100.8 98.6 82.8 33.9 IPK00012262 ##STR00509## 131.5
92.6 328.4 254.9 62.8 65.1 26.2 13.0 90.7 67.9 37.7 26.4
IPK00012302 ##STR00510## 411.8 347.8 427.0 270.5 76.8 78.2 45.3
24.6 23.9 32.0 38.2 42.1 IPK00012303 ##STR00511## 540.8 467.6 319.6
234.1 78.3 72.1 12.0 -8.6 21.2 34.7 35.8 36.5 IPK00012330
##STR00512## 296.8 390.9 495.6 263.1 77.2 64.1 72.5 22.6 20.9 37.0
36.6 38.0 IPK00012390 ##STR00513## 205.0 226.5 374.3 230.8 73.1
87.7 26.7 -6.0 35.1 49.4 43.2 40.5 IPK00012392 ##STR00514## 135.8
127.8 409.8 277.3 45.3 15.4 32.3 12.0 96.4 77.0 46.7 31.5
IPK00012443 ##STR00515## 168.3 110.6 305.5 279.0 35.6 64.7 26.2
15.5 96.6 66.0 42.2 44.0 IPK00012454 ##STR00516## 503.3 591.0 268.3
320.1 75.5 75.0 16.4 21.3 22.7 38.9 37.0 34.8 IPK00012464
##STR00517## 501.5 421.5 343.1 256.5 79.9 78.2 54.7 22.6 -0.6 25.1
31.1 33.9 IPK00012465 ##STR00518## 490.8 577.0 416.9 267.0 77.1
94.3 45.0 7.2 5.2 38.6 38.6 36.9 IPK00012508 ##STR00519## 261.5
254.5 476.3 388.9 72.8 65.1 46.9 28.2 74.4 65.1 32.8 36.9
IPK00012515 ##STR00520## 233.8 153.1 186.4 237.5 87.1 88.7 4.0 4.4
95.2 55.2 40.3 40.9 IPK00012522 ##STR00521## 265.5 254.5 194.1
274.0 75.4 82.0 -11.9 3.1 81.0 73.2 40.0 38.5
IPK00012561 ##STR00522## 55.5 112.0 293.5 280.8 44.1 85.8 15.7 2.8
89.0 25.3 26.3 31.2 IPK00012633 ##STR00523## 123.3 86.8 126.8 270.9
80.7 73.4 12.5 24.9 98.7 99.6 66.8 41.4 IPK00012673 ##STR00524##
524.3 271.6 149.0 225.6 85.7 60.8 5.7 3.3 78.1 76.0 55.4 35.5
IPK00012837 ##STR00525## 519.8 402.6 312.4 274.4 43.8 26.5 8.4 6.5
90.9 71.8 95.9 90.6 IPK00012972 ##STR00526## 479.5 540.9 272.3
289.8 61.8 75.4 16.8 14.1 98.1 69.4 44.9 46.3 IPK00012991
##STR00527## 436.3 477.9 426.5 372.0 -36.0 38.2 25.4 25.1 69.3 74.8
51.4 52.1 IPK00013026 ##STR00528## 476.8 385.9 270.4 340.1 66.3
29.9 14.5 17.8 100.0 70.1 79.7 53.0 IPK00013054 ##STR00529## 539.5
507.4 515.3 384.9 66.7 65.8 45.8 25.3 34.8 45.1 56.7 46.5
IPK00013302 ##STR00530## 194.8 255.4 472.4 360.5 -44.8 37.2 27.6
18.1 90.2 68.1 49.0 35.7 IPK00013346 ##STR00531## 473.3 534.5 397.5
243.9 69.6 94.4 45.3 22.5 40.1 57.0 38.3 40.2 IPK00013450
##STR00532## 560.0 526.9 490.5 255.9 99.7 100.7 70.4 0.1 95.1 64.5
71.7 34.1 IPK00013451 ##STR00533## 415.5 559.0 545.1 423.9 90.2
101.5 66.7 34.9 84.3 48.7 56.3 51.4 IPK00013462 ##STR00534## 207.8
406.9 296.6 308.6 -56.1 25.5 11.5 13.0 89.8 68.6 44.8 46.8
IPK00013463 ##STR00535## 275.3 294.1 416.9 316.6 -18.3 12.6 23.9
33.9 94.1 67.3 54.0 49.9 IPK00013528 ##STR00536## 337.5 255.6 223.4
219.5 53.3 26.6 17.5 2.9 99.0 73.2 56.9 46.4 IPK00013812
##STR00537## 480.8 501.4 420.9 343.8 96.8 104.7 92.8 16.4 98.8 67.5
90.8 37.7 IPK00013840 ##STR00538## 569.8 575.8 285.0 230.3 75.0
86.4 35.4 2.8 -1.0 52.4 47.7 44.6 IPK00013843 ##STR00539## 514.0
521.3 361.5 276.6 76.3 78.9 25.3 6.6 16.0 47.9 22.9 48.3
IPK00013917 ##STR00540## 199.8 195.3 414.0 411.5 72.9 65.8 38.1
40.8 33.8 70.4 54.4 52.3 IPK00014081 ##STR00541## 208.5 384.4 460.8
276.8 94.6 91.8 51.8 11.0 12.0 37.9 35.4 35.4 IPK00014087
##STR00542## 269.5 421.0 551.0 245.0 92.2 91.1 71.8 5.9 17.0 41.7
41.3 30.4 IPK00014108 ##STR00543## 529.8 625.5 270.9 200.4 67.5
84.0 11.7 4.6 13.8 34.5 47.8 41.3 IPK00014158 ##STR00544## 191.5
167.0 239.1 184.0 58.2 59.2 -2.2 -14.1 92.7 95.9 45.3 42.9
IPK00014161 ##STR00545## 308.3 337.6 378.3 225.5 4.2 50.8 23.0 10.3
87.4 83.4 46.7 45.5 IPK00014217 ##STR00546## 635.8 581.1 361.8
340.4 84.3 82.3 18.5 28.7 14.6 54.3 35.1 42.9 IPK00014218
##STR00547## 167.8 132.6 372.4 234.3 73.2 79.4 20.6 0.5 -1.3 38.3
39.8 39.4 IPK00014345 ##STR00548## 581.8 625.4 467.4 282.6 81.2
83.5 60.3 14.4 11.5 72.5 50.0 38.3 IPK00014422 ##STR00549## 262.5
377.4 452.9 318.4 -9.8 30.0 38.5 16.6 72.1 82.4 38.7 35.9
IPK00014691 ##STR00550## 167.5 132.9 201.1 229.5 57.0 70.0 57.2 4.4
99.5 100.7 40.9 40.7 IPK00014698 ##STR00551## 101.3 167.8 283.5
264.0 55.8 26.5 26.2 -2.8 100.3 70.3 35.1 37.6 IPK00014717
##STR00552## 81.0 177.6 186.5 330.8 11.9 40.8 47.2 22.9 98.7 74.3
40.4 45.1 IPK00014754 ##STR00553## 45.3 388.4 316.0 364.1 -10.9
10.1 13.0 26.0 92.6 66.8 48.9 42.9 IPK00014798 ##STR00554## 67.5
82.4 375.9 250.9 6.3 72.3 40.7 15.4 99.3 98.1 37.2 39.9 IPK00014804
##STR00555## 48.0 63.8 313.4 317.6 21.8 59.3 26.8 17.7 98.0 67.6
46.8 48.9 IPK00014811 ##STR00556## 333.0 577.0 347.5 341.8 65.4
86.6 25.3 31.9 14.0 36.6 43.7 49.4 IPK00014844 ##STR00557## 58.8
364.3 268.6 264.9 -15.1 8.2 14.9 11.0 98.4 67.1 43.4 41.7
IPK00014864 ##STR00558## 379.3 516.6 427.3 261.9 72.5 71.4 40.3 3.7
29.2 42.0 35.3 46.5 IPK00014865 ##STR00559## 47.8 73.8 338.0 247.8
41.2 29.1 17.1 6.4 99.1 65.1 43.4 43.1 IPK00014902 ##STR00560##
97.0 406.3 130.0 250.3 23.5 19.1 49.7 13.5 99.1 66.8 69.8 45.8
IPK00014944 ##STR00561## 259.5 465.9 298.1 200.1 57.6 47.1 21.2
-10.4 100.0 70.4 43.0 27.1 IPK00014978 ##STR00562## 36.0 328.1
331.9 289.0 15.2 3.6 12.3 9.6 99.6 74.4 56.2 49.9 IPK00015041
##STR00563## 373.8 539.6 366.1 285.3 78.3 83.3 24.2 5.2 31.8 44.6
46.3 35.9 IPK00015048 ##STR00564## 61.0 333.6 209.5 349.3 -96.3
16.1 -2.3 26.4 95.6 69.8 46.4 52.3 IPK00015085 ##STR00565## 416.3
397.3 299.6 230.3 72.3 72.2 33.8 5.1 23.0 93.2 58.8 40.4
IPK00015536 ##STR00566## 183.8 182.1 201.4 232.0 67.8 74.0 43.7
-8.6 88.3 68.3 29.0 33.2 IPK00015751 ##STR00567## 321.3 227.9 377.4
393.8 96.0 46.2 100.9 39.7 99.3 47.4 82.2 47.2 IPK00015755
##STR00568## 108.3 277.0 278.0 396.4 22.0 19.9 77.5 39.0 97.6 77.0
55.2 45.1 IPK00015849 ##STR00569## 96.0 266.6 168.3 248.6 -3.0 4.8
-0.8 8.4 66.6 69.1 44.3 43.2 IPK00016045 ##STR00570## 436.3 371.4
285.5 296.5 66.0 70.7 43.2 31.7 17.6 25.1 30.9 45.0 IPK00016132
##STR00571## 100.3 65.1 194.0 245.4 -37.9 17.8 25.1 9.4 99.1 63.7
95.8 52.1
IPK00016327 ##STR00572## 534.3 263.4 474.9 393.4 69.5 17.9 71.8
33.5 86.4 45.6 55.6 47.7 IPK00016351 ##STR00573## 447.5 377.5 491.4
335.8 89.4 50.0 82.7 40.8 92.9 68.6 57.2 52.8 IPK00016352
##STR00574## 446.5 473.3 447.6 428.4 86.2 70.7 61.4 43.7 83.0 64.6
46.4 44.3 IPK00016362 ##STR00575## 246.5 599.8 485.8 451.9 81.3
107.2 59.4 49.8 95.9 68.1 54.8 47.8 IPK00016364 ##STR00576## 486.3
576.1 394.9 302.8 74.9 74.2 26.7 14.7 20.8 63.3 47.4 49.9
IPK00016367 ##STR00577## 517.5 481.6 520.1 340.0 68.1 99.5 70.7
27.4 76.2 65.3 44.7 47.9 IPK00016393 ##STR00578## 74.0 123.6 249.4
339.0 66.7 89.9 36.3 45.5 30.4 50.1 34.2 31.3 IPK00016452
##STR00579## 202.3 171.8 204.8 286.8 64.7 80.0 -2.2 0.1 91.8 86.8
36.5 38.7 IPK00016754 ##STR00580## 60.3 117.3 184.8 248.0 19.5 16.5
27.7 23.2 74.3 98.3 34.3 33.3 IPK00016810 ##STR00581## 106.5 185.3
137.1 294.4 38.0 65.6 17.9 8.2 100.2 54.4 38.1 43.5 IPK00016831
##STR00582## 178.8 254.9 159.9 256.1 83.9 78.1 20.6 15.7 15.9 29.3
38.8 32.3 IPK00016832 ##STR00583## 167.5 254.9 352.9 312.6 75.7
81.9 37.2 18.3 50.5 29.5 31.5 35.6 IPK00016930 ##STR00584## 220.0
291.1 313.0 269.3 56.2 73.7 12.4 8.8 99.3 99.4 62.8 42.8
IPK00016942 ##STR00585## 106.0 218.3 410.4 361.9 -64.0 76.9 32.9
8.5 95.2 45.0 44.0 44.8 IPK00016968 ##STR00586## 97.8 114.9 183.8
289.1 -5.0 -5.3 -5.9 16.8 99.1 65.3 43.9 45.5 IPK00016976
##STR00587## 62.0 137.6 188.0 301.6 -71.9 16.7 2.1 16.4 99.6 100.3
31.4 30.7 IPK00016986 ##STR00588## 119.3 174.4 191.5 359.9 0.5 13.0
14.4 31.1 100.9 100.3 43.7 34.8 IPK00016996 ##STR00589## 64.8 140.4
134.5 193.4 -50.2 14.7 27.5 17.0 103.8 100.6 68.8 34.4 IPK00017027
##STR00590## 59.0 118.9 319.5 499.9 -72.4 18.9 8.1 46.9 80.7 76.3
34.0 33.5 IPK00017033 ##STR00591## 143.0 353.9 525.0 501.1 49.5
96.3 102.5 92.7 103.5 100.2 97.4 75.7 IPK00017072 ##STR00592## 67.0
72.1 321.0 275.8 -29.5 -21.3 27.0 17.4 76.0 82.7 30.3 5.9
IPK00017127 ##STR00593## 283.3 176.9 201.4 276.8 95.2 95.2 -1.5 9.0
99.2 82.5 59.9 40.9 IPK00017146 ##STR00594## 55.0 108.5 82.0 236.5
24.3 72.1 0.9 11.0 100.0 59.5 73.5 39.6 IPK00017184 ##STR00595##
30.0 93.9 129.5 298.4 -79.6 13.3 29.4 16.9 99.0 98.8 46.1 31.0
IPK00017234 ##STR00596## 40.8 69.5 158.4 298.4 -82.4 12.5 18.2 7.7
98.2 69.9 45.1 37.8 IPK00017235 ##STR00597## 104.3 185.4 470.5
345.6 0.9 59.9 55.2 28.5 101.4 81.2 53.6 41.1 IPK00017254
##STR00598## 92.0 221.3 190.0 226.6 4.1 40.4 14.5 11.4 82.1 92.0
40.3 36.2 IPK00017306 ##STR00599## 54.3 314.4 224.9 308.4 44.0 15.8
9.2 26.9 74.8 73.6 58.5 44.2 IPK00017345 ##STR00600## 113.0 304.0
305.5 292.8 14.9 19.2 25.3 24.8 103.7 71.5 72.0 46.7 IPK00017527
##STR00601## 44.3 177.0 131.8 302.8 -69.7 25.8 -3.3 25.1 95.8 70.7
46.5 39.9 IPK00017824 ##STR00602## 111.8 140.4 158.6 199.5 -26.4
-4.1 6.9 1.3 74.0 97.0 37.9 34.0 IPK00017905 ##STR00603## 124.3
190.0 194.5 352.4 -31.7 16.6 30.0 27.5 101.0 100.6 81.6 24.9
IPK00017949 ##STR00604## 187.0 257.9 163.8 300.4 65.7 78.5 -8.9
17.6 16.2 54.5 44.4 48.0 IPK00018011 ##STR00605## 447.8 451.5 309.9
309.1 68.2 69.7 26.0 9.7 14.8 28.9 40.6 44.8 IPK00018016
##STR00606## 217.0 234.4 323.8 332.3 46.1 69.8 12.6 13.5 93.8 68.9
41.3 43.5 IPK00018017 ##STR00607## 199.3 229.5 377.5 265.0 63.2
34.5 20.2 8.6 93.8 72.0 29.6 50.6 IPK00018076 ##STR00608## 467.8
565.3 289.9 347.6 70.6 92.1 37.5 43.8 90.0 63.8 40.8 32.9
IPK00018456 ##STR00609## 256.3 215.1 391.5 342.5 85.1 72.7 45.0
34.4 63.3 67.3 46.9 46.5 IPK00019245 ##STR00610## 219.8 386.4 376.8
334.0 88.8 65.4 22.0 16.2 -9.0 42.7 43.3 42.7 IPK00019259
##STR00611## 470.5 551.3 344.4 246.3 65.3 81.1 30.1 10.8 -8.7 41.9
31.7 42.7 IPK00019376 ##STR00612## 355.5 420.4 497.1 520.4 81.4
97.4 100.4 91.9 98.6 68.4 85.6 63.9 IPK00019599 ##STR00613## 305.0
488.8 537.1 477.8 79.1 69.5 105.7 96.1 98.8 74.2 97.1 70.8
IPK00019853 ##STR00614## 129.5 217.9 192.3 210.1 58.5 41.9 10.2 1.8
97.3 66.2 29.5 34.9 IPK00019854 ##STR00615## 153.0 189.3 394.4
309.5 38.6 77.9 27.3 20.3 88.8 55.5 44.6 31.6 IPK00019856
##STR00616## 131.5 278.1 272.5 276.0 15.1 64.7 28.7 21.2 95.9 68.3
39.0 34.3 IPK00019970 ##STR00617## 227.5 256.5 301.6 345.8 70.5
69.6 11.2 23.0 -8.2 39.8 43.5 49.3 IPK00020016 ##STR00618## 265.0
430.5 238.4 235.3 73.3 76.8 2.7 1.6 -16.9 50.3 42.3 40.2
IPK00020047 ##STR00619## 146.0 309.6 294.9 370.5 6.3 19.1 3.5 15.8
90.3 95.0 58.9 37.1 IPK00020208 ##STR00620## 283.0 279.9 354.1
396.1 65.7 67.4 18.1 23.7 4.6 28.7 38.5 39.4 IPK00020522
##STR00621## 280.0 319.8 414.0 313.6 85.1 69.7 33.2 6.2 38.2 47.4
50.0 45.5
IPK00020542 ##STR00622## 205.3 314.8 430.8 330.6 71.5 69.3 58.7
19.9 44.7 47.0 65.1 51.8 IPK00020853 ##STR00623## 166.8 232.5 319.1
275.9 71.6 23.3 65.1 -4.0 46.0 56.4 57.3 44.0 IPK00021074
##STR00624## 191.8 304.9 425.6 344.3 76.4 20.2 88.9 32.6 49.9 43.9
66.8 50.0 IPK00021079 ##STR00625## 273.8 255.0 350.9 353.6 77.2
28.7 71.5 25.1 43.5 48.6 58.2 49.2 IPK00021083 ##STR00626## 200.8
190.5 320.4 213.9 70.4 48.2 71.4 -5.0 49.8 52.8 62.8 50.6
IPK00021926 ##STR00627## 548.0 218.4 476.1 435.6 91.1 51.4 98.6
63.8 98.6 46.0 84.5 45.3 IPK00021927 ##STR00628## 586.3 384.6 513.9
443.3 96.9 57.4 104.8 78.1 98.7 70.3 85.0 47.6 IPK00021928
##STR00629## 623.3 240.8 480.5 433.8 106.5 46.2 98.1 57.6 98.0 40.9
83.1 42.4 IPK00021929 ##STR00630## 623.5 423.8 502.5 221.3 107.4
67.6 95.1 18.5 99.3 32.9 84.5 46.5 IPK00021930 ##STR00631## 700.8
300.0 564.3 356.9 103.5 93.0 101.1 13.0 99.5 99.7 82.9 44.9
IPK00022200 ##STR00632## 148.3 279.5 218.5 282.6 74.4 86.2 29.9 3.6
82.3 19.7 26.8 30.8 IPK00022204 ##STR00633## 181.3 124.9 210.8
261.9 65.4 85.8 69.0 40.1 54.9 78.1 21.1 30.0 IPK00022232
##STR00634## 124.8 114.3 227.8 294.8 16.2 63.2 53.1 14.4 102.4 96.7
47.9 40.7 IPK00022459 ##STR00635## 60.3 95.1 248.2 333.3 72.9 86.2
41.8 14.9 30.0 31.5 37.7 45.7 IPK00022846 ##STR00636## 492.8 261.5
431.4 376.6 22.5 37.8 67.1 38.9 97.5 41.0 47.1 25.0 IPK00022950
##STR00637## 488.0 322.5 352.8 412.9 60.1 83.6 21.6 24.6 99.6 28.1
39.7 36.6 IPK00022972 ##STR00638## 300.3 386.8 483.6 322.8 69.4
90.7 83.2 19.6 16.9 30.0 45.7 27.1 IPK00023002 ##STR00639## 223.5
277.6 453.1 388.4 -7.7 40.5 78.8 19.0 95.7 41.9 44.6 40.5
IPK00023461 ##STR00640## 156.8 207.9 209.1 292.6 -61.0 -1.6 -5.8
14.6 67.9 76.3 42.7 37.4 IPK00023509 ##STR00641## 91.8 251.8 346.0
276.6 46.2 36.4 32.6 3.9 98.8 45.4 70.1 22.4 IPK00023512
##STR00642## 39.8 254.0 325.5 333.5 -67.9 26.9 31.9 34.9 99.4 71.1
48.7 40.9 IPK00023891 ##STR00643## 379.0 382.4 145.6 202.6 99.3
27.8 78.4 -6.0 96.7 70.1 43.2 28.6 IPK00024037 ##STR00644## 132.8
156.5 116.5 213.9 17.0 67.5 11.6 -10.9 104.1 76.2 37.9 30.6
IPK00024172 ##STR00645## 175.3 102.4 487.5 421.1 -43.3 28.0 67.1
53.6 100.4 97.2 92.6 68.9 IPK00024412 ##STR00646## 136.0 257.8
249.4 270.0 29.9 11.8 60.1 0.5 98.4 75.7 66.8 42.3 IPK00024744
##STR00647## 584.0 332.8 158.6 174.5 89.1 97.1 17.5 12.3 -12.2 23.8
30.8 34.7 IPK00024871 ##STR00648## 150.8 256.0 486.9 334.6 79.5
79.0 73.4 21.5 99.2 41.6 70.4 40.5 IPK00024912 ##STR00649## 130.8
124.3 138.9 172.9 23.6 53.2 41.8 7.8 94.3 86.2 56.8 41.7
IPK00024914 ##STR00650## 404.3 211.3 407.6 332.9 45.0 68.6 41.9
21.0 96.3 37.4 40.3 43.3 IPK00024984 ##STR00651## 163.3 173.8 295.4
325.3 78.2 80.9 -4.9 21.4 86.0 64.6 46.3 45.8 IPK00025149
##STR00652## 162.5 282.1 327.6 285.9 27.0 15.0 18.0 0.8 92.6 65.1
55.1 44.5 IPK00025180 ##STR00653## 321.0 322.1 217.8 294.5 25.5
38.2 9.2 3.9 77.6 69.2 64.3 48.0 IPK00025412 ##STR00654## 179.8
351.4 306.5 225.3 30.3 13.2 13.1 -11.6 90.9 66.4 43.8 43.0
IPK00025425 ##STR00655## 243.3 367.0 356.3 356.9 32.5 18.5 20.0
20.5 100.5 75.6 53.2 49.1 IPK00025546 ##STR00656## 192.3 279.6
499.5 408.3 25.0 53.1 54.7 32.6 96.3 68.7 64.7 31.8 IPK00025761
##STR00657## 285.0 226.9 381.8 314.1 71.5 79.9 42.1 15.8 31.8 60.5
28.1 33.2 IPK00025807 ##STR00658## 142.3 174.8 380.6 240.9 49.9
72.7 26.2 9.4 98.4 47.9 36.8 37.0 IPK00025935 ##STR00659## 64.5
78.8 262.1 335.9 66.0 68.8 12.0 5.3 10.9 28.4 42.5 40.2 IPK00025978
##STR00660## 115.8 194.3 271.1 292.8 4.9 58.8 11.0 14.9 91.4 83.6
25.0 30.2 IPK00026207 ##STR00661## 242.0 309.8 461.8 383.3 80.3 7.9
89.0 40.1 99.0 34.3 61.4 45.9 IPK00026239 ##STR00662## 93.3 273.3
164.5 349.1 2.1 29.5 48.7 20.4 98.5 75.4 60.0 43.8
TABLE-US-00002 TABLE 2 Scaffold Number of Scaffold Name Coding
Compounds Scaffold Structure Isonicotinohydrazides I 69
##STR00663## Benzamides II 19 ##STR00664## Thiazolhydrazides III 6
##STR00665## Hydrazinecarbothioamides IV 5 ##STR00666##
Furancarbohydrazides V 4 ##STR00667## Thiophenes VI 3 ##STR00668##
Pyrazole-pyridines VII 2 ##STR00669## Pyridopyrimidinone VIII 1
##STR00670## One hit compound IX 1 ##STR00671## One hit compound X
1 ##STR00672## One hit compound XI 1 ##STR00673## One hit compound
XII 1 ##STR00674## One hit compound XIII 1 ##STR00675## One hit
compound XIV 1 ##STR00676## One hit compound XV 1 ##STR00677## One
hit compound XVI 1 ##STR00678## One hit compound XVII 1
##STR00679## One hit compound XVIII 1 ##STR00680## One hit compound
XIX 1 ##STR00681## One hit compound XX 1 ##STR00682##
TABLE-US-00003 TABLE 3 Compound QIM (.mu.M) QUM (.mu.M)
##STR00683## 1 +++ +++ ##STR00684## 2 ++ +++ ##STR00685## 3 ++ +++
##STR00686## 4 ++ +++ ##STR00687## 5 ++ +++ ##STR00688## 6 ++ +++
##STR00689## 7 ++ +++ ##STR00690## 8 +++ +++ ##STR00691## 9 ++ +++
##STR00692## 10 ++ +++ ##STR00693## 11 +++ +++ ##STR00694## 12 +++
+++ ##STR00695## 13 ++ +++ ##STR00696## 14 ++ ++ ##STR00697## 15 ++
+++ ##STR00698## 16 + + ##STR00699## 17 + + ##STR00700## 18 + +
##STR00701## 19 + ++ ##STR00702## 20 + + ##STR00703## 21 ++ +++
##STR00704## 22 ++ +++ ##STR00705## 23 +++ +++ ##STR00706## 24 +++
+++ ##STR00707## 26 ++ +++ ##STR00708## 27 +++ +++ ##STR00709## 28
+++ +++ ##STR00710## 29 +++ +++ ##STR00711## 30 +++ +++
##STR00712## 31 +++ +++ ##STR00713## 32 +++ +++ ##STR00714## 33 ++
+++ ##STR00715## 34 + + ##STR00716## 54 ++ +++ ##STR00717## 56 + ++
##STR00718## 58 + +++ ##STR00719## 59 ++ +++ ##STR00720## 60 + +++
##STR00721## 61 + +++ ##STR00722## 63 + ++ ##STR00723## 64 ++ +++
##STR00724## 67 + + ##STR00725## 90 + + ##STR00726## 91 ++ +++
##STR00727## 92 + +++ ##STR00728## 93 + ++ ##STR00729## 94 ++ +++
##STR00730## 95 + ++ ##STR00731## 96 ++ ++ ##STR00732## 97 + +++
##STR00733## 98 + ++ ##STR00734## 99 + ++ ##STR00735## 100 + +++
##STR00736## 101 + ++ ##STR00737## 103 ++ +++ ##STR00738## 104 ++
+++ ##STR00739## 105 +++ ++ ##STR00740## 107 ++ +++ ##STR00741##
108 ++ +++ ##STR00742## 109 + + ##STR00743## 112 + ++ ##STR00744##
114 + ++ ##STR00745## 115 + ++ ##STR00746## 116 + ++ ##STR00747##
118 + ++ ##STR00748## 119 + +++ ##STR00749## 120 + +++ ##STR00750##
121 + ++ ##STR00751## 132 + ++ ##STR00752## 133 ++ +++ ##STR00753##
134 + ++ ##STR00754## 135 + ++ ##STR00755## 137 + + ##STR00756##
139 + + ##STR00757## 140 + + ##STR00758## 147 + + ##STR00759## 151
++ + ##STR00760## 152 + + ##STR00761## 160 + + ##STR00762## 163 + +
##STR00763## 173 + + ##STR00764## 180 + + ##STR00765## 184 + +
##STR00766## 185 + ++ ##STR00767## 193 + + ##STR00768## 195 + +
##STR00769## 199 + ++ ##STR00770## 200 + + ##STR00771## 201 + +
##STR00772## 204 + + ##STR00773## 206 +++ +++ ##STR00774## 207 +++
+++ ##STR00775## 208 +++ +++ ##STR00776## 209 +++ +++ ##STR00777##
210 +++ +++ ##STR00778## 211 + +++ ##STR00779## 212 + +
##STR00780## 213 + +++ ##STR00781## 214 + ++ ##STR00782## 215 + +
##STR00783## 216 + + ##STR00784## 217 + +++ ##STR00785## 218 + +
##STR00786## 219 + + ##STR00787## 220 ++ +++ ##STR00788## 221 + +
##STR00789## 222 + + ##STR00790## 224 + + ##STR00791## 226 + +
##STR00792## 229 + + ##STR00793## 231 +++ +++ ##STR00794## 232 +++
++ ##STR00795## 233 + + ##STR00796## 234 + + ##STR00797## 235 +++
+++ ##STR00798## 236 ++ +++ ##STR00799## 237 + +++ ##STR00800## 238
+ + ##STR00801## 239 + + ##STR00802## 240 ++ ++ ##STR00803## 241 +
+ ##STR00804## 242 + + ##STR00805## 243 + +
##STR00806## 245 + ++ ##STR00807## 246 + + ##STR00808## 247 + +++
##STR00809## 248 + +++ ##STR00810## 249 + + ##STR00811## 250 + +
##STR00812## 251 + + ##STR00813## 252 + + ##STR00814## 253 + +
##STR00815## 254 + + ##STR00816## 255 + + ##STR00817## 256 + +
##STR00818## 257 +++ +++ ##STR00819## 258 +++ +++ ##STR00820## 259
++ +++ ##STR00821## 260 + ++ ##STR00822## 261 +++ +++ ##STR00823##
262 + + ##STR00824## 263 + + ##STR00825## 264 ++ +++ ##STR00826##
265 +++ +++ ##STR00827## 266 + +++ ##STR00828## 267 +++ +++
##STR00829## 268 + ++ ##STR00830## 269 ++ ++ ##STR00831## 270 +++
+++ ##STR00832## 271 + +++ ##STR00833## 272 + + ##STR00834## 273
+++ +++ ##STR00835## 274 + +++ ##STR00836## 275 + +++ ##STR00837##
276 + ++ ##STR00838## 277 + ++ ##STR00839## 278 +++ +++
##STR00840## 280 + +++ ##STR00841## 281 + +++ ##STR00842## 282 + +
##STR00843## 283 + + ##STR00844## 284 + +++ ##STR00845## 285 + +++
##STR00846## 286 + +++ ##STR00847## 290 + + ##STR00848## 291 + +
##STR00849## 292 + + ##STR00850## 293 + + ##STR00851## 294 + +
##STR00852## 295 ++ +++ ##STR00853## 296 + + ##STR00854## 297 + +
##STR00855## 298 + + ##STR00856## 299 ++ +++ ##STR00857## 300 ++
+++ ##STR00858## 301 ++ +++ Activity range: +++ indicates <5 uM,
++ indicates between 5-20 uM, + indicates >20 uM
TABLE-US-00004 TABLE 4 Compounds 4 24 Cytotoxicity Host Cells Range
of MTC.sub.50 (.mu.M) SK-N-SH-Brain >100 >100
HepG2-Hepatocytes >100 >100 MRC5-Lung >100 >100 BJ-Skin
>100 >100 HEK293-Kidney >100 >100 Antibacterial
activity & Specificity Range of MICs for Strains/Isolates Type
Origin Number multiple strains (.mu.M) Mycobacterium M.
tuberculosis Drug Sensitive Tissue 1 0.38 0.31 clinical
isolates.sup.1 RIF.sup.R Sputum 2 0.05 0.08 Tissue 2 0.02-0.05 0.08
INH.sup.R RIF.sup.R Strep.sup.R Sputum 1 0.1 0.08 Tissue 3 0.05-0.1
0.04-0.08 XDR Sputum 5 0.02-0.05 0.04-0.08 Tissue 0 0.05-0.1 0.08
MDR Sputum 3 0.05-0.1 0.04-0.08 Tissue 5 0.05-0.1 0.04-0.08 M.
tuberculosis H37Rv 0.6 0.6 laboratory strains H37Ra 1.2 1.3 Beijing
1237 0.3 0.1 M. bovis BCG BCG Tokyo 1.2 0.6 BCG Pasteur 1.2 1.2 M.
smegmatis mc.sup.2 155 1.2 0.6 Gram-negative Acinetobacter
baumannii, Escherichia coli, Enterobacter cloacae, E. aerogenes,
>250 >250 Klebsiella oxytoca, Pseudomonas aeruginosa,
Salmonella enteridis, Vibrio mimicus Gram-positive Staphylococcus
aureus, S. epidermis, S. capitis, S. xylosus, Micrococcus >250
>250 luteus, Listeria innocua, Lactobacillus gallinarum, group G
Streptococcus, Streptococcus agalactiae, S. pyogenes, Enterococcus
faecalis, E. faecium, E. gallinarum, Bacillus pumilus
Corynebacterium C. striatum 27 27 C. jeikeium 2.7 2.7 Fungi Candida
albicans, C. glabrata, C. parapsilosis >250 >250 INH:
Isoniazid, RIF: Rifampin, Strep: Streptomycin, .sup.Rresistant.
.sup.1The clinical isolates were isolated either from resected lung
tissue or sputum specimen, which were collected from active
tuberculosis in-patients from the National Masan Tuberculosis
Hospital during October 2003 to March 2007.
TABLE-US-00005 TABLE 5 Compounds 133 Cytotoxicity Range of Host
Cells MTC.sub.50 (.mu.M) SK-N-SH-Brain >100 HepG2-Hepatocytes
>100 MRC5-Lung >100 BJ-Skin >100 HEK293-Kidney >100
Jurkat-T-cell >100 THP-1-Monocytes >100 Primary BMDM >100
Primary human macrophages >100 Antibacterial activity &
Specificity Range of MICs for multiple Strains/Isolates Type Origin
Number strains (.mu.M) Mycobacterium M. tuberculosis Drug Sensitive
Sputum 2 5->20 clinical isolates.sup.1 Tissue 2 2.5-5 RIF.sup.R
Sputum 1 2.5 Tissue 1 1.2 INH.sup.R RIF.sup.R Strep.sup.R Sputum 3
0.3-1.2 Tissue 1 1.2 XDR Sputum 4 0.6-2.5 Tissue 5 0.3-5 MDR Sputum
3 0.3-1.2 Tissue 1 1.2 M. tuberculosis H37Rv 2 laboratory strains
H37Ra 2 BCG Pasteur-Tokyo 2 M. smegmatis mc.sup.2 155 >100
Gram-negative Acinetobacter baumannii, Escherichia coli,
Enterobacter NE cloacae, E. aerogenes, Klebsiella oxytoca,
Pseudomonas aeruginosa, Salmonella enteridis, Vibrio mimicus
Gram-positive Staphylococcus aureus, S. epidermis, S. capitis, S.
xylosus, NE Micrococcus luteus, Listeria innocua, Lactobacillus
gallinarum, group G Streptococcus, Streptococcus agalactiae, S.
pyogenes, Enterococcus faecalis, E. faecium, E. gallinarum,
Bacillus pumilus Corynebacterium C. striatum NE C. jeikeium Fungi
Candida albicans, C. glabrata, C. parapsilosis NE INH: Isoniazid,
RIF: Rifampin, Strep: Streptomycin, .sup.Rresistant. .sup.1The
clinical isolates were isolated either from resected lung tissue or
sputum specimen, which were collected from active tuberculosis
in-patients from the National Masan Tuberculosis Hospital during
October 2003 to March 2007. NE: No effect up to 100 .mu.g/mL
equivalent to 320 .mu.M. The antimicrobial spectrum was performed
on clinical isolates from CHU d'Angers, France.
TABLE-US-00006 TABLE 6 Concentration Bacteria inoculum (CFU)
Frequency of Compound (.mu.g/ml) 10.sup.5 10.sup.6 10.sup.7
10.sup.8 resistance 4 0.2 -- -- 12 >100 1 .times. 10.sup.-6 0.8
-- -- -- <100 1.6 -- -- -- <100 3.2 -- -- -- 1 1 .times.
10.sup.-8 24 0.2 -- -- 7 >100 7 .times. 10.sup.-7 0.8 -- -- --
<100 1.6 -- -- -- >100 3.2 -- -- -- 1 1 .times. 10.sup.-8
INH-control 10 ND ND 33 ND 3 .times. 10.sup.-6 Concentration
Bacteria inoculum (CFU) Frequency of Compound (.mu.g/ml) 10.sup.6
10.sup.7 10.sup.8 resistance 264 0.4 -- 37 306 3.4 .times.
10.sup.-6 0.8 -- 5 117 8 .times. 10.sup.-6 1.6 -- -- 22 2 .times.
10.sup.-8 3.2 -- -- 2 2 .times. 10.sup.-8 INH-control 10 -- 4 18
2.9 .times. 10.sup.-6 ND: not done; --: no colonies
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