U.S. patent application number 16/848032 was filed with the patent office on 2020-09-10 for pharmacophore for trail induction.
This patent application is currently assigned to THE SCRIPPS RESEARCH INSTITUTE. The applicant listed for this patent is THE SCRIPPS RESEARCH INSTITUTE. Invention is credited to Nicholas T. JACOB, Kim D. JANDA, Jonathan W. LOCKNER.
Application Number | 20200283440 16/848032 |
Document ID | / |
Family ID | 1000004853505 |
Filed Date | 2020-09-10 |
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United States Patent
Application |
20200283440 |
Kind Code |
A1 |
JANDA; Kim D. ; et
al. |
September 10, 2020 |
PHARMACOPHORE FOR TRAIL INDUCTION
Abstract
There are disclosed imidazolinopyrimidinone compounds that have
activity to induce TRAIL gene expression in macrophages. There is
further disclosed a method for treating various cancers comprising
administering effective amounts of an imidazolinopyrimidinone
having the structure of Formula I herein.
Inventors: |
JANDA; Kim D.; (La Jolla,
CA) ; JACOB; Nicholas T.; (San Diego, CA) ;
LOCKNER; Jonathan W.; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE SCRIPPS RESEARCH INSTITUTE |
La Jolla |
CA |
US |
|
|
Assignee: |
THE SCRIPPS RESEARCH
INSTITUTE
La Jolla
CA
|
Family ID: |
1000004853505 |
Appl. No.: |
16/848032 |
Filed: |
April 14, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16359633 |
Mar 20, 2019 |
10633385 |
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16848032 |
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15126192 |
Sep 14, 2016 |
10239877 |
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PCT/US15/23362 |
Mar 30, 2015 |
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16359633 |
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61972689 |
Mar 31, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/519 20130101;
C07D 471/14 20130101 |
International
Class: |
C07D 471/14 20060101
C07D471/14; A61K 31/519 20060101 A61K031/519 |
Goverment Interests
STATEMENT OF GOVERNMENT SUPPORT
[0002] The present invention was made with government support under
HHSN27200700038C, AI077644, AI079436, and AI094348, awarded by the
National Institutes of Health. The U.S. government has certain
rights in the invention.
Claims
1. A compound of formula (I) ##STR00040## wherein Cyc is a 5- to
8-membered monocyclic heterocyclyl ring comprising one nitrogen
atom, with a group of formula Ar.sup.1--CR.sub.2-being bonded to
the ring nitrogen atom; Ar.sup.1 and Ar.sup.2 are each
independently aryl groups which are substituted with 0, 1, or 2 J
groups; R is independently H or (C1-C6)alkyl; J is independently
(C1-C6)alkyl, (C3-C9)cycloalkyl, (C3-C9)cycloalkyl(C1-C6)alkyl,
halo, or (C1-C6)haloalkyl; or a pharmaceutically acceptable salt
thereof.
2. The compound of claim 1, wherein the compound is within the
subgenus formula (IA) ##STR00041## or a pharmaceutically acceptable
salt thereof.
3. The compound of claim 2, wherein Ar.sup.1 and Ar.sup.2 are each
a phenyl group substituted with 0, 1, or 2 J groups; and, R at each
occurrence is independently H or (C1-C6)alkyl; or a
pharmaceutically acceptable salt thereof.
4. The compound of claim 1, wherein the compound is selected from
the group ##STR00042## or a pharmaceutically acceptable salt
thereof.
5. A method for treating cancer, comprising administering an
effective amount of a compound of formula (I) ##STR00043## wherein
Cyc is a 5- to 8-membered monocyclic heterocyclyl ring comprising
one nitrogen atom, with a group of formula Ar.sup.1--CR.sub.2-being
bonded to the nitrogen atom; Ar.sup.1 and Ar.sup.2 are aryl groups
which are substituted with 0, 1, or 2 J groups; R is independently
H or (C1-C6)alkyl; J is independently (C1-C6)alkyl,
(C3-C9)cycloalkyl, (C3-C9)cycloalkyl(C1-C6)alkyl, halo, or
(C1-C6)haloalkyl; or a pharmaceutically acceptable salt
thereof.
6. The method of claim 5, wherein the compound is a compound of
formula (IA) ##STR00044## or a pharmaceutically acceptable salt
thereof.
7. The method of claim 5, the compound of formula (IA), Ar.sup.1
and Ar.sup.2 is a phenyl group substituted with 0, 1, or 2 J
groups.
8. The method of claim 5, wherein the compound of formula (I) is
formula 2 ##STR00045##
9. The method of claim 5, wherein the cancer is selected from the
group consisting of ovarian, colon, breast, liver, pancreas,
gastro-intestinal, head- and neck, cervix, prostate, lung cancers,
melanomas, glioblastomas, myelomas, neuroblastic-derived CNS
tumors, monocytic leukemias, B-cell derived leukemias, T-cell
derived leukemias, B-cell derived lymphomas, T-cell derived
lymphomas, and mast cell derived tumors, and combinations thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 16/359,633, filed on Mar. 20, 2019, which is a
continuation of U.S. patent application Ser. No. 15/126,192, filed
on Sep. 14, 2016, which is a U.S. national stage application filed
under 35 U.S.C. .sctn. 371 from International Application Serial
No. PCT/US2015/023362, which filed on Mar. 30, 2015, and published
as WO 2015/153468 on Oct. 8, 2015, which claims the benefit of
priority to U.S. Provisional Application Ser. No. 61/972,689, filed
Mar. 31, 2014, which applications and publication are incorporated
by reference as if reproduced herein and made a part hereof in
their entirety, and the benefit of priority of each of which is
claimed herein.
BACKGROUND
[0003] Cancer immunosurveillance relies on various effector
functions of the immune system that can modify both induced and
spontaneous carcinogenesis. TRAIL is an immunosurveillence cytokine
critically involved in this process due to its ability to
selectively induce apoptosis in cancer cells over normal cells (S.
R. Wiley, K. Schooley, P. J. Smolak, W. S. Din, C. P. Huang, J. K.
Nicholl, G. R. Sutherland, T. D. Smith, C. Rauch, C. A. Smith,
Immunity 1995, 3, 673-682; A. Ashkenazi, V. M. Dixit, Science 1998;
H. Walczak, R. E. Miller, K. Ariail, B. Gliniak, T. S. Griffith, M.
Kubin, W. Chin, J. Jones, A. Woodward, T. Le, et al., Nat. Med.
1999, 5, 157-163; and A. Ashkenazi, R. C. Pai, S. Fong, S. Leung,
D. A. Lawrence, S. A. Marsters, C. Blackie, L. Chang, A. E.
McMurtrey, A. Hebert, et al., J. Clin. Invest. 1999, 104, 155-162).
The TRAIL gene is expressed in a variety of tissues and cells (S.
R. Wiley, K. Schooley, P. J. Smolak, W. S. Din, C. P. Huang, J. K.
Nicholl, G. R. Sutherland, T. D. Smith, C. Rauch, C. A. Smith,
Immunity 1995, 3, 673-682); including dendritic cells, natural
killer (NK) cells, and monocytes/macrophages (M. J. Smyth, K.
Takeda, Y. Hayakawa, J. J. Peschon, M. R. M. van den Brink, H.
Yagita, Immunity 2003, 18, 1-6.). Its gene expression is under
control of several transcriptional regulators, such as
transcription factors NF-.kappa.B and p53 (K. Kuribayashi, G.
Krigsfeld, W. Wang, J. Xu, P. A. Mayes, D. T. Dicker, G. S. Wu, W.
S. El-Deiry, Cancer Biol. Ther. 2008, 7, 2034-2038.). Reduction of
TRAIL expression by neutralizing antibodies and ablation of TRAIL
expression in mice lacking the TRAIL gene results in the
development of carcinogen-induced fibrosarcomas, sarcomas, and
lymphomas; especially in p53-deficient mice (E. Cretney, K. Takeda,
H. Yagita, M. Glaccum, J. J. Peschon, M. J. Smyth, J. Immunol.
2002; and K. Takeda, M. J. Smyth, E. Cretney, Y. Hayakawa, N.
Kayagaki, H. Yagita, K. Okumura, J. Exp. Med. 2002, 195, 161-169).
These data are also consistent with observations that change in
TRAIL expression in immune cells is associated with TRAIL
resistance in cancer cells (N. S. M. Azahri, M. M. Kavurma, Cell.
Mol. Life Sci. 2013, 70, 3617-3629). Thus, effectors of TRAIL
production in immune cells are of clinical relevance (M. J. Smyth,
K. Takeda, Y. Hayakawa, J. J. Peschon, M. R. M. van den Brink, H.
Yagita, Immunity 2003, 18, 1-6.) and could also be used as a means
to achieve a model system for studying the complex
immunosurveillance signaling system
SUMMARY
[0004] The invention is directed, in various embodiments, to a
compound and pharmaceutical composition comprising an effective
amount of a compound capable of inducing expression of TRAIL gene
in cells capable of expressing the TRAIL gene to produce the
cytokine TRAIL. TRAIL (a cytokine) can selectively induce apoptosis
in cancer cells over normal cells. Therefore, the present
disclosure provides a compound and pharmaceutical that is effective
for treating various cancers. Without being bound by theory, the
disclosed compound and pharmaceutical composition induces
expression of TRAIL.
[0005] In various embodiments, the invention is directed to a
compound of formula (I)
##STR00001##
wherein
[0006] Cyc is a single 5- to 8-membered heterocyclyl ring
comprising at least one nitrogen atom, with a group of formula
Ar.sup.1--CR.sub.2-being bonded to the nitrogen atom;
[0007] Ar.sup.1 and Ar.sup.2 are each independently selected aryl
groups which are independently substituted with 0, 1, or 2 J
groups;
[0008] each independently selected R is H or is (C1-C6)alkyl;
[0009] J is (C1-C6)alkyl, (C3-C9)cycloalkyl,
(C3-C9)cycloalkyl(C1-C6)alkyl, or halo;
[0010] or a pharmaceutically acceptable salt thereof.
[0011] The present disclosure provides a pharmaceutical composition
comprising a compound selected from the group consisting of
##STR00002##
In various embodiments, the compound used to induce TRAIL is
compound 2
##STR00003##
or a pharmaceutically acceptable salt thereof. The IUPAC name for
compound 2 is
7-benzyl-4-(2-methylbenzyl)-1,2,6,7,8,9-hexahydroimidazo[1,2-a]pyrido[3,4-
-e]pyrimidin-5(4H)-one.
[0012] The present disclosure provides a method for treating
various cancers, comprising administering to a patient an effective
amount of a compound of formula (I), such as compound 2. The method
for treating a broad spectrum of mammalian cancers, wherein the
broad spectrum of mammalian cancers to be treated is selected from
the group consisting of ovarian, colon, breast, liver, pancreas,
gastro-intestinal, head- and neck, cervix, prostate, lung cancers,
melanomas, glioblastomas, myelomas, neuroblastic-derived CNS
tumors, monocytic leukemias, B-cell derived leukemias, T-cell
derived leukemias, B-cell derived lymphomas, T-cell derived
lymphomas, and mast cell derived tumors, and combinations
thereof.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIG. 1 shows a) Induction of TRAIL mRNA in RAW cells treated
for 48 h with indicated dose of linear isomer (1) or angular isomer
2; b) Induction of TRAIL mRNA induction to 5 .mu.M compound 1 and
compound 2 for indicated times. c) Dose-dependent response to
compound 2a and compound 2b. d) Dose-dependent response to angular
(2) and compound 9. Compound 2a is a sample obtained from the NCI
repository, compound 2b is a compound synthesized herein; both were
shown to be a compound of structure 2.
[0014] FIG. 2 shows a comparison of the imidazolinopyrimidanone
structures of inactive compound 1 and active compound 2 with
respect to TRAIL expression. The structures of each were confirmed
by X-ray crystallographic analysis.
[0015] FIG. 3 shows the structure of constitutional isomer,
compound 9.
[0016] FIG. 4 shows comparative structures of compound 1 and
compound 2.
[0017] FIG. 5 shows the X-ray crystal structure obtained for
compound 2.
[0018] FIG. 6 shows the X-ray crystal structure obtained for
compound 9.
[0019] FIG. 7 shows a cell viability assay comparing the activity
of a 20 mM concentration of various compounds including Compound 2
(HIPPO) and compounds A through R herein.
DETAILED DESCRIPTION
[0020] The present disclosure provides a compound of formula
(I)
##STR00004##
wherein
[0021] Cyc is a 5- to 8-membered monocyclic heterocyclyl ring
comprising one nitrogen atom, with a group of formula
Ar.sup.1--CR.sub.2-being bonded to the ring nitrogen atom;
[0022] Ar.sup.1 and Ar.sup.2 are each independently aryl groups
which are substituted with 0, 1, or 2 J groups;
[0023] R is independently H or (C1-C6)alkyl;
[0024] J is independently (C1-C6)alkyl, (C3-C9)cycloalkyl,
(C3-C9)cycloalkyl(C1-C6)alkyl, halo, or (C1-C6)haloalkyl;
[0025] or a pharmaceutically acceptable salt thereof.
[0026] Preferably, the compound of formula (I) is a compound within
the subgenus formula (IA)
##STR00005##
or a pharmaceutically acceptable salt thereof.
[0027] More specifically, the compound of formula (IA) is a
compound wherein Ar.sup.1 and Ar.sup.2 are each a phenyl group
substituted with 0, 1, or 2 J groups; and,
[0028] R at each occurrence is independently H or (C1-C6)alkyl;
[0029] or a pharmaceutically acceptable salt thereof.
[0030] Preferably, the compound of formula (I) is compound 2
##STR00006##
[0031] or a pharmaceutically acceptable salt thereof.
[0032] In various embodiments, the invention provides a compound of
formula (I) that is not compound 2.
[0033] The present disclosure further provides a method for
treating various cancers, comprising administering an effective
amount of a compound of formula (I)
##STR00007##
wherein
[0034] Cyc is a 5- to 8-membered monocyclic heterocyclyl ring
comprising one nitrogen atom, with a group of formula
Ar.sup.1--CR.sub.2-being bonded to the nitrogen atom;
[0035] Ar.sup.1 and Ar.sup.2 are aryl groups which are substituted
with 0, 1, or 2 J groups;
[0036] R is independently H or (C1-C6)alkyl;
[0037] J is independently (C1-C6)alkyl, (C3-C9)cycloalkyl,
(C3-C9)cycloalkyl(C1-C6)alkyl, halo, or (C1-C6)haloalkyl;
[0038] or a pharmaceutically acceptable salt thereof.
[0039] Preferably, the compound is a compound selected from the
subgenus of formula (IA)
##STR00008##
or a pharmaceutically acceptable salt thereof.
[0040] More preferably, in the compound of formula (IA), Ar.sup.1
and Ar.sup.2 is a phenyl group substituted with 0, 1, or 2 J
groups.
[0041] Most preferably the compound of formula (I) is compound
2
##STR00009##
[0042] In various embodiments, the invention provides a method for
treating various cancers with a compound of formula (I) wherein the
compound of formula (I) is not compound 2.
[0043] The method can be used for treating a broad spectrum of
mammalian cancers, wherein the broad spectrum of mammalian cancers
to be treated is selected from the group consisting of ovarian,
colon, breast, liver, pancreas, gastro-intestinal, head- and neck,
cervix, prostate, lung cancers, melanomas, glioblastomas, myelomas,
neuroblastic-derived CNS tumors, monocytic leukemias, B-cell
derived leukemias, T-cell derived leukemias, B-cell derived
lymphomas, T-cell derived lymphomas, and mast cell derived tumors,
and combinations thereof.
[0044] Another imidazolinopyrimidinone, (called compound 1 herein)
in disclosed in United States patent application 20120276088
published 1 Nov. 2012. This patent application discloses linear
compound 1 which is used for comparison purposes herein. We
synthesized compound 1 in four steps from 4-chloronicotinic acid
(Scheme 1).
##STR00010##
Synthesis of Compound 1
[0045] (a) SOCl.sub.2, 90.degree. C., 1 h, then
2-methylthioimidazoline hydroiodide, Et.sub.3N, CH.sub.2Cl.sub.2,
0.degree. C. to rt, 19 h, 96%; (b) 2-methylbenzylamine,
K.sub.3PO.sub.4, N,N-dimethylacetamide, reflux, 1 h, 79%; (c) 45
psi H.sub.2(g), PtO.sub.2, MeOH/TFA, rt, 5 h, 80%; (d)
benzaldehyde, Na(OAc).sub.3BH, AcOH, CH.sub.2Cl.sub.2, rt, 4 h,
87%.
[0046] Briefly, acylation of an activated carboxylic acid, followed
by a double displacement reaction, and subsequent hydrogenation and
reductive amination afforded compound 1 in 52% overall yield. This
structure of compound 1 was confirmed by mass spectrometry, nuclear
magnetic resonance (NMR) spectroscopic, and X-ray crystallographic
analyses (see Examples section).
[0047] The biological activity of compound 1 was measured by RT-PCR
analysis of TRAIL mRNA expression in the murine macrophage cell
line RAW 264.7. No change in TRAIL mRNA expression over controls
was observed, even at doses as high as 10 .mu.M (FIG. 1a) or with
prolonged exposure (FIG. 1b). As shown in FIG. 1, compound 2a,
(obtained from the NCI), exhibits the desired TRAIL bioactivity, as
did synthesized compound 2b, but synthesized compound 1 does not.
Therefore, there is a need in the art to create a biologically
active imidazolinopyrimidinone, which is the more angular compound
of formula (I), and in particular compound 2.
[0048] Compound 2 was prepared in three steps in 82% yield (Scheme
2). A synthetic product, termed herein compound 2b, was obtained,
and its structure confirmed as 2.
##STR00011##
Synthesis of Compound 2
[0049] (a) methyl chloroformate, Et.sub.3N, CH.sub.2Cl.sub.2,
0.degree. C. to rt, 44 h, 97%; (b) 2-methylbenzylamine, MeOH, AcOH,
reflux, 45 h, 87%; (c) NaOMe, MeOH, reflux, 18 h, 97%.
[0050] A mixture of guanidine 7 and
1-benzyl-4-oxopiperidine-3-carboxylate hydrochloride (8) in
refluxing methanol and sodium methoxide afforded 2b almost
exclusively; a trace amount of 1 was detected by .sup.1H NMR
following work-up of this reaction, but was removed by subsequent
purification. We rationalize this result by considering that the
imidazolinyl nitrogens of 7 possess both statistical and steric
advantages over the benzylic nitrogen of 7. Initial attack by
nitrogen at the ketone carbonyl of 8 affords an aminocarbinol
intermediate, which suffers intramolecular cyclocondensation to
provide synthetic sample 2b. Its structure 2 was confirmed by mass
spectrometry and NMR spectroscopy.
[0051] Compound 2, obtained as synthetic sample 2b was able to
induce TRAIL mRNA expression, as did repository compound 2a (FIG.
1c).
[0052] Therefore, angular compound 2 (shown by the inventors herein
to be the active TRAIL induction factor) has the structure
##STR00012##
Compound 1 (does not seem to be active) has the structure
##STR00013##
and the isomeric linear compound to have the structure 9
##STR00014##
Of these three compounds, only compound 2 exhibits the desired
TRAIL bioactivity.
[0053] X-ray crystal structures, taken as described in the Examples
section, are provided in the Figures.
[0054] These findings provide a structure-activity relationship
wherein the angular fusion of the tricyclic core is a necessity of
the pharmacophore for TRAIL induction in macrophages.
[0055] Our three-step synthesis of compound 2 began with the
preparation of carbamate 6 (T. Smejkal, D. Gribkov, J. Geier, M.
Keller, B. Breit, Chemistry 2010, 16, 2470-2478) and its conversion
to guanidine 7 (W. K. Fang, P. X. Nguyen, K. Chow, T. M.
Heidelbaugh, D. G. Gomez, M. E. Garst, S. C. Sinha, Allergan Inc.,
USA, 2011). If the 1,1-diamine is unsymmetrical, an isomeric
mixture of products is possible (see: J. V. Greenhill, M. J.
Ismail, P. N. Edwards, P. J. Taylor, J Chem Soc Perk T 2 1985,
1255-1264; C. Romano, E. Delacuesta, C. Avendano, F. Florencio, J.
Sainzaparicio, Tetrahedron 1988, 44, 7185-7192; F. Esser, K. H.
Pook, A. Carpy, Synthesis-Stuttgart 1990, 72-78). A mixture of
guanidine 7 and 1-benzyl-4-oxopiperidine-3-carboxylate
hydrochloride in refluxing methanol (with the aid of NaOMe)
afforded compound 2 almost exclusively; a trace amount of compound
1 was detected by .sup.1H NMR following work-up of this reaction.
We rationalize this result by considering that the imidazolinyl
nitrogens of 7 possess both statistical and steric advantages over
the benzylic nitrogen of 7. Initial attack by nitrogen at the
ketone carbonyl affords an aminocarbinol intermediate, which
suffers intramolecular cyclocondensation to provide 2.
[0056] The K.sub.2CO.sub.3-mediated reaction of a 3-keto ester with
a 2-amino-2-oxazoline (a type of unsymmetrical 1,1-diamine) affords
a mixture of linear and angular products (I. Forfar, C. Jarry, M.
Laguerre, J. M. Leger, I. Pianet, Tetrahedron 1999, 55,
12819-12828). The authors accumulated empirical and theoretical
evidence to support the notion that "the endocyclic nitrogen atom
is the most nucleophilic and attacks the most electrophilic carbon
of the biselectrophile. A ring closure between the exocyclic
nitrogen atom and the second electrophilic center concludes the
bicyclic heterocycle synthesis." This is consistent with our own
observations in the synthesis of 7 via a similar strategy.
[0057] To reiterate the salient feature of the present synthesis,
by using sodium methoxide in refluxing methanol (M. F. Koehler, P.
Bergeron, E. Blackwood, K. K. Bowman, Y. H. Chen, G. Deshmukh, X.
Ding, J. Epler, K. Lau, L. Lee, L. Liu, C. Ly, S. Malek, J.
Nonomiya, J. Oeh, D. F. Ortwine, D. Sampath, S. Sideris, L. Trinh,
T. Truong, J. Wu, Z. Pei, J. P. Lyssikatos, J. Med. Chem. 2012, 55,
10958-10971), compound 2 is produced nearly exclusively. If the
condensation is performed in the presence of base and/or at higher
temperature, then sufficient means are available for statistically
and sterically more likely aminocarbinol intermediate to suffer
rapid intramolecular cyclocondensation leading to compound 2.
[0058] In addition, related compounds A through R were synthesized.
The characteristics of compounds A through R are provided in Table
1 below:
TABLE-US-00001 TABLE 1 Compound Label Structure Chemical Info A
##STR00015## Chemical Formula: C23H24N4O Molecular Weight: 372.47
Log P: 2.6 B ##STR00016## Chemical Formula: C24H26N4O Molecular
Weight: 386.50 Log P: 3.09 C ##STR00017## Chemical Formula:
C23H23BrN4O Molecular Weight: 451.37 Log P: 3.43 D ##STR00018##
Chemical Formula: C23H23ClN4O Molecular Weight: 406.91 Log P: 3.16
E ##STR00019## Chemical Formula: C17H20N4O Molecular Weight: 296.37
Log P: 0.87 F ##STR00020## Chemical Formula: C24H26N4O2 Molecular
Weight: 402.50 Log P: 2.47 G ##STR00021## Chemical Formula:
C17H20N4O Molecular Weight: 296.37 Log P: 0.87 H ##STR00022##
Chemical Formula: C18H22N4O Molecular Weight: 310.40 Log P: 1.35 I
##STR00023## Chemical Formula: C17H19BrN4O Molecular Weight: 375.27
Log F: 1.7 J ##STR00024## Chemical Formula: C17H19ClN4O Molecular
Weight: 330.82 Log P: 1.42 K ##STR00025## Chemical Formula:
C11H16N4O Molecular Weight: 220.28 Log P: -0.87 L ##STR00026##
Chemical Formula: C18H22N4O2 Molecular Weight: 326.40 Log P: 0.74 M
##STR00027## Chemical Formula: C17H19N3O Molecular Weight: 281.36
Log P: 2.29 N ##STR00028## Chemical Formula: C18H21N3O Molecular
Weight: 295.39 Log P: 2.78 O ##STR00029## Chemical Formula:
C17H18BrN3O Molecular Weight: 360.26 Log P: 3.12 P ##STR00030##
Chemical Formula: C17H18ClN3O Molecular Weight: 315.80 Log P: 2.85
Q ##STR00031## Chemical Formula: C11H15N3O Molecular Weight: 205.26
Log P: 0.56 R ##STR00032## Chemical Formula: C18H21N3O2 Molecular
Weight: 311.38 Log P: 2.17
[0059] As used herein, the singular forms "a," "an" and "the"
include plural referents unless the context clearly dictates
otherwise.
[0060] The term "about" as used herein, when referring to a
numerical value or range, allows for a degree of variability in the
value or range, for example, within 10%, or within 5% of a stated
value or of a stated limit of a range.
[0061] All percent compositions are given as weight-percentages,
unless otherwise stated.
[0062] The term "disease" or "disorder" or "malcondition" are used
interchangeably, and are used to refer to diseases or conditions
wherein TRAIL, such as inducing expression of the TRAIL gene in a
cell, plays a role in the biochemical mechanisms involved in the
disease or malcondition or symptom(s) thereof such that a
therapeutically beneficial effect can be achieved with an effective
amount or concentration of a synthetic ligand of the invention
adequate to induce expression of TRAIL and induce apoptosis, e.g.,
selectively in cancer cells. For example, the cancers to be treated
by the compounds of the present disclosure include a broad spectrum
of mammalian cancers, wherein the broad spectrum of mammalian
cancers to be treated is selected from the group consisting of
ovarian, colon, breast, lung cancers, myelomas,
neuroblastic-derived CNS tumors, monocytic leukemias, B-cell
derived leukemias, T-cell derived leukemias, B-cell derived
lymphomas, T-cell derived lymphomas, and mast cell derived tumors,
and combinations thereof.
[0063] The expression "effective amount", when used to describe
therapy to an individual suffering from a disorder, refers to the
quantity or concentration of a compound of the invention that is
effective to induce expression of TRAIL in the individual's
tissues.
[0064] The terms "halo" or "halogen" or "halide" by themselves or
as part of another substituent mean, unless otherwise stated, a
fluorine, chlorine, bromine, or iodine atom, preferably, fluorine,
chlorine, or bromine.
[0065] A "salt" as is well known in the art includes an organic
compound such as a carboxylic acid, a sulfonic acid, or an amine,
in ionic form, in combination with a counterion. For example, acids
in their anionic form can form salts with cations such as metal
cations, for example sodium, potassium, and the like; with ammonium
salts such as NH.sub.4.sup.+or the cations of various amines,
including tetraalkyl ammonium salts such as tetramethylammonium, or
other cations such as trimethylsulfonium, and the like. A
"pharmaceutically acceptable" or "pharmacologically acceptable"
salt is a salt formed from an ion that has been approved for human
consumption and is generally non-toxic, such as a chloride salt or
a sodium salt. A "zwitterion" is an internal salt such as can be
formed in a molecule that has at least two ionizable groups, one
forming an anion and the other a cation, which serve to balance
each other. For example, amino acids such as glycine can exist in a
zwitterionic form. A "zwitterion" is a salt within the meaning
herein. The compounds of the present invention may take the form of
salts. The term "salts" embraces addition salts of free acids or
free bases which are compounds of the invention. Salts can be
"pharmaceutically-acceptable salts." The term
"pharmaceutically-acceptable salt" refers to salts which possess
toxicity profiles within a range that affords utility in
pharmaceutical applications. Pharmaceutically unacceptable salts
may nonetheless possess properties such as high crystallinity,
which have utility in the practice of the present invention, such
as for example utility in process of synthesis, purification or
formulation of compounds of the invention. "Pharmaceutically or
pharmacologically acceptable" include molecular entities and
compositions that do not produce an adverse, allergic or other
untoward reaction when administered to an animal, or a human, as
appropriate. For human administration, preparations should meet
sterility, pyrogenicity, and general safety and purity standards as
required by FDA Office of Biologics standards.
Examples
General Procedures
[0066] All reactions were carried out under an argon atmosphere
with dry solvents using anhydrous conditions unless otherwise
stated. Chemicals were purchased from Acros Organics, Oakwood
Products, and Sigma-Aldrich. They were used as received unless
otherwise noted. Dry dichloromethane (CH.sub.2Cl.sub.2) was
obtained via distillation over calcium hydride (CaH.sub.2). Dry
methanol (MeOH) was obtained via distillation over magnesium
turnings. Reagents were purchased at the highest commercial quality
and used without further purification, unless otherwise stated.
Yields refer to chromatographically and spectroscopically (.sup.1H
NMR) homogeneous materials, unless otherwise stated. Reactions were
monitored by thin layer chromatography (TLC) carried out on 0.25 mm
E. Merck silica gel plates (60F-254) using UV light as the
visualizing agent, or basic aqueous potassium permanganate
(KMnO.sub.4), and heat as developing agent. E. Merck silica gel
(60, particle size 0.040-0.063 mm) was used for flash column
chromatography. Preparative thin layer chromatography (PTLC)
separations were carried out on 0.50 mm E. Merck silica gel plates
(60F-254). Concentration of organic solvents was performed on a
rotary evaporator under reduced pressure followed by further
evacuation using a dual stage mechanical pump. NMR spectra were
recorded on Bruker DRX-600, DRX-500, and AMX-400 instruments and
calibrated using residual undeuterated solvent as an internal
reference (CHCl.sub.3 @ .delta. 7.26 ppm .sup.1H NMR, .delta. 77.16
ppm .sup.13C NMR; CD.sub.3OD @ .delta. 4.87 ppm .sup.1H NMR,
.delta. 49.00 ppm 13C NMR). The following abbreviations (or
combinations thereof) were used to explain .sup.1H NMR
multiplicities: s=singlet, d=doublet, t=triplet, m=multiplet,
br=broad. High-resolution mass spectra (HRMS) were recorded on
Agilent LC/MSD TOF mass spectrometer by electrospray ionization
time-of-flight reflectron experiments. IR spectra were recorded on
either a PerkinElmer Spectrum 100 FTIR spectrometer with ATR
accessory or a Jasco 480 Plus FTIR spectrometer. Melting points
were recorded on a Fisher-Johns 12-144 melting point apparatus and
are uncorrected.
Synthetic Procedures
##STR00033##
[0067]
(4-Chloropyridin-3-yl)(2-(methylthio)-4,5-dihydro-1H-imidazol-1-yl)-
methanone (3)
[0068] A mixture of 4-chloronicotinic acid (1.00 g, 6.35 mmol) and
SOCl.sub.2 (15 mL) was stirred at 90.degree. C. for 1 h. Removal of
SOCl.sub.2 by rotary evaporation gave 4-chloronicotinic acid
chloride hydrochloride as a pale yellow solid, which was placed
under argon balloon, cooled to 0.degree. C., and dissolved in
CH.sub.2Cl.sub.2 (45 mL). A solution of 2-methylthio-2-imidazoline
hydriodide (1.32 g, 5.40 mmol) and Et.sub.3N (2.92 mL, 20.95 mmol)
in CH.sub.2Cl.sub.2 (75 mL) was added via cannula. The pale amber
solution was stirred at room temperature overnight. After 19 h,
CH.sub.2Cl.sub.2 (150 mL) was added and the resulting solution
washed with saturated aqueous NaHCO.sub.3 (2.times.100 mL) and
brine (2.times.100 mL). The organic layer was dried
(Na.sub.2SO.sub.4) and concentrated in vacuo. Purification by
silica gel chromatography (19:1 CH.sub.2Cl.sub.2/MeOH) afforded 3
(1.32 g, 96%) as a pale yellow syrup.
[0069] R.sub.f=0.19 (silica gel, 19:1 CH.sub.2Cl.sub.2/MeOH)
[0070] IR (neat) .nu..sub.max 1661, 1574, 1377, 1200, 903, 724
cm.sup.-1
[0071] .sup.1H NMR (600 MHz, CDCl.sub.3) .delta. 8.56 (d, J=5.5 Hz,
1H), 8.54 (s, 1H), 7.37 (d, J=5.2 Hz, 1H), 4.15-3.65 (m, 2H), 3.93
(t, J=8.3 Hz, 2H), 2.37 (s, 3H)
[0072] .sup.13C NMR (150 MHz, CDCl.sub.3) .delta. 162.1, 151.9,
148.6, 131.9, 124.7, 54.1, 48.5, 15.6
[0073] HRMS (ESI-TOF) calcd. for C.sub.10H.sub.10ClN.sub.3OSH.sup.+
[M+H.sup.+] 256.0306, found 256.0309
##STR00034##
10-(2-Methylbenzyl)-2,3-dihydroimidazo[1,2-a]pyrido[4,3-d]pyrimidin-5(10H-
)-one (4)
[0074] A mixture of 3 (1.30 g, 5.08 mmol), 2-methylbenzylamine
(1.89 mL, 15.25 mmol), powdered K.sub.3PO.sub.4 (1.08 g, 5.08
mmol), and N,N-dimethylacetamide (10 mL) was heated at reflux for 1
h. The resulting mixture was cooled and partitioned between
CH.sub.2Cl.sub.2 (30 mL) and H.sub.2O (30 mL). The organic layer
was dried (Na.sub.2SO.sub.4) and concentrated in vacuo.
Purification by silica gel chromatography (19:1
CH.sub.2Cl.sub.2/MeOH) and trituration with cold hexanes afforded 4
(1.17 g, 79%) as a white solid.
[0075] m.p. 182-188.degree. C. (hexanes)
[0076] R.sub.f=0.32 (silica gel, 19:1 CH.sub.2Cl.sub.2/MeOH)
[0077] IR (neat) .nu..sub.max 1674, 1634, 1591, 1455, 1400, 1284,
747 cm.sup.-1
[0078] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 9.15 (s, 1H), 8.45
(d, J=5.9 Hz, 1H), 7.23 (d, J=7.4 Hz, 1H), 7.19 (t, J=7.4 Hz, 1H),
7.11 (t, J=7.4 Hz, 1H), 6.84 (d, J=7.7 Hz, 1H), 6.55 (d, J=5.9 Hz,
1H), 5.21 (s, 2H), 4.20 (t, J=8.9 Hz, 2H), 3.96 (t, J=8.9 Hz, 2H),
2.41 (s, 3H)
[0079] .sup.13C NMR (150 MHz, CDCl.sub.3) .delta. 158.0, 154.6,
151.0, 150.2, 147.7, 135.0, 131.6, 131.0, 127.8, 126.7, 124.2,
111.9, 107.9, 50.2, 46.7, 45.3, 19.2
[0080] HRMS (ESI-TOF) calcd. for C.sub.17H.sub.16N.sub.4OH.sup.+
[M+H.sup.+] 293.1397, found 293.1397
##STR00035##
10-(2-Methylbenzyl)-2,3,6,7,8,9-hexahydroimidazo[1,2-a]pyrido[4,3-d]pyrim-
idin-5(10H)-one (5)
[0081] A mixture of 4 (300 mg, 1.03 mmol), PtO.sub.2 (60 mg), MeOH
(3 mL), and TFA (3 mL) was hydrogenated (45 psi) in a Parr shaker
for 5 h. The mixture was filtered through a Celite pad to remove
catalyst, then concentrated in vacuo. The colorless syrup was
dissolved in 1:1 EtOAc/H.sub.2O (40 mL), made basic by addition of
2 M NaOH (10 mL), and layers were separated. The aqueous layer was
extracted with EtOAc (40 mL). The combined organic layers were
washed with brine (20 mL), dried (Na.sub.2SO.sub.4), and
concentrated in vacuo. Purification by silica gel chromatography
(19:1:0.1 CH.sub.2Cl.sub.2/MeOH/NH.sub.4OH) afforded 5 (244 mg,
80%) as a white solid.
[0082] m.p. 170-174.degree. C. (MeOH)
[0083] R.sub.f=0.12 (silica gel, 19:1:0.1
CH.sub.2Cl.sub.2/MeOH/NH.sub.4OH)
[0084] IR (neat) .nu..sub.max 3287, 1660, 1627, 1605, 1472, 1293,
919 cm.sup.-1
[0085] .sup.1H NMR (600 MHz, CDCl.sub.3) .delta. 7.20-7.14 (m, 3H),
6.92-6.90 (m, 1H), 4.98 (s, 2H), 4.05 (t, J=9.4 Hz, 2H), 3.82 (t,
J=9.4 Hz, 2H), 3.68 (t, J=1.9 Hz, 2H), 2.95 (t, J=5.8 Hz, 2H), 2.30
(s, 3H), 2.28-2.25 (m, 2H), 1.66 (br s, 1H)
[0086] .sup.13C NMR (150 MHz, CDCl.sub.3) .delta. 160.0, 152.8,
147.2, 134.6, 133.8, 130.7, 127.4, 126.8, 123.7, 106.6, 49.9, 46.0,
45.2, 42.7, 42.2, 25.5, 19.1
[0087] HRMS (ESI-TOF) calcd. for C.sub.17H.sub.20N.sub.4OH.sup.+
[M+H.sup.+] 297.1710, found 297.1709
##STR00036##
7-Benzyl-10-(2-methylbenzyl)-2,6,7,8,9,10-hexahydroimidazo[1,2-a]pyrido[4-
,3-d]pyrimidin-5(3H)-one (1)
[0088] A solution of 5 (230 mg, 0.78 mmol) and benzaldehyde (103
.mu.L, 1.02 mmol) in CH.sub.2Cl.sub.2 (2.5 mL) was treated with
AcOH (76 .mu.L, 1.35 mmol) and Na(OAc).sub.3BH (267 mg, 1.26 mmol)
at room temperature. The mixture was stirred for 4 h, then diluted
with CH.sub.2Cl.sub.2 (10 mL) and washed with saturated aqueous
NaHCO.sub.3 (10 mL). The aqueous layer was extracted with
CH.sub.2Cl.sub.2 (10 mL). The combined organic layers were dried
(Na.sub.2SO.sub.4) and concentrated in vacuo. Purification by
silica gel chromatography (19:1 CH.sub.2Cl.sub.2/MeOH) afforded 1
(261 mg, 87%) as a white solid.
[0089] m.p. 166-168.degree. C. (MeOH)
[0090] R.sub.f=0.25 (silica gel, 19:1 CH.sub.2Cl.sub.2/MeOH)
[0091] IR (neat) .nu..sub.max 2866, 2358, 2339, 1616, 1456, 983
cm.sup.-1
[0092] .sup.1H NMR (600 MHz, CDCl.sub.3) .delta. 7.37-7.28 (m, 4H),
7.26-7.14 (m, 4H), 6.93-6.91 (m, 1H), 4.98 (s, 2H), 4.06 (t, J=9.4
Hz, 2H), 3.84 (t, J=9.4 Hz, 2H), 3.64 (s, 2H), 3.38 (s, 2H), 2.54
(t, J=5.7 Hz, 2H), 2.37 (t, J=5.5 Hz, 2H), 2.29 (s, 3H)
[0093] .sup.13C NMR (150 MHz, CDCl.sub.3) .delta. 159.8, 152.9,
147.1, 137.6, 134.6, 133.7, 130.7, 129.2, 128.5, 127.5, 127.4,
126.8, 123.7, 105.7, 62.1, 49.9, 49.6, 48.6, 46.4, 45.3, 26.1,
19.1
[0094] HRMS (ESI-TOF) calcd. for C.sub.24H.sub.26N.sub.4OH.sup.+
[M+H.sup.+] 387.2179, found 387.2189
##STR00037##
Methyl 2-(methylthio)-4,5-dihydro-1H-imidazole-1-carboxylate
(6)
[0095] A solution of 2-methylthio-2-imidazoline hydriodide (12.21
g, 50 mmol) and Et.sub.3N (16 mL, 115 mmol) in CH.sub.2Cl.sub.2 (50
mL) at 0.degree. C. was treated with methyl chloroformate (5.0 mL,
65 mmol) dropwise. The mixture was allowed to warm to room
temperature and stirred overnight. After 44 h, the mixture was
diluted with EtOAc (200 mL), stirred, then filtered to remove
insoluble salts. The salts were rinsed with EtOAc (50 mL). The
filtrate was concentrated in vacuo, affording 6 (8.47 g, 97%) as a
white solid.
[0096] R.sub.f=0.33 (silica gel, 19:1 CH.sub.2Cl.sub.2/MeOH)
[0097] IR (neat) .nu..sub.max 1717, 1576, 1429, 1378, 1218, 1023,
758 cm.sup.-1
[0098] .sup.1H NMR (600 MHz, CDCl.sub.3) .delta. 3.92-3.85 (m, 4H),
3.78 (s, 3H), 2.41 (s, 3H)
[0099] .sup.13C NMR (150 MHz, CDCl.sub.3) .delta. 159.7, 152.5,
53.9, 53.2, 47.5, 15.2
[0100] HRMS (ESI-TOF) calcd. for
C.sub.6H.sub.10N.sub.2O.sub.2SH.sup.+ [M+H.sup.+] 175.0536, found
175.0539
##STR00038##
N-(2-Methylbenzyl)-4,5-dihydro-1H-imidazol-2-amine (7)
[0101] A solution of 6 (1.5 g, 8.61 mmol) and 2-methylbenzylamine
(1.08 mL, 8.74 mmol) in MeOH (48 mL) was treated with AcOH (4.8
mL). The solution was stirred at a gentle reflux. After 45 h, the
solution was cooled to room temperature and concentrated in vacuo.
The residue was dissolved in CH.sub.2Cl.sub.2 (100 mL), washed with
1 M NaOH (55 mL), brine (55 mL), dried over Na.sub.2SO.sub.4,
filtered, and concentrated in vacuo. Trituration with cold
CH.sub.3CN afforded 7 (1.42 g, 87%) as a white solid.
[0102] R.sub.f=0.14 (silica gel, 9:1:0.1
CH.sub.2Cl.sub.2/MeOH/NH.sub.4OH)
[0103] IR (neat) .nu..sub.max 2862, 2358, 1684, 1635, 1521, 1349,
1238 cm.sup.-1
[0104] .sup.1H NMR (600 MHz, CD.sub.3OD) .delta. 7.25-7.15 (m, 4H),
4.34 (s, 2H), 3.61 (s, 4H), 2.32 (s, 3H)
[0105] .sup.13C NMR (150 MHz, CD.sub.3OD) .delta. 163.0, 161.5,
137.4, 136.7, 131.4, 128.8, 128.5, 127.2, 46.2, 45.8, 18.9
[0106] HRMS (ESI-TOF) calcd. for C.sub.11H.sub.15N.sub.3H.sup.+
[M+H.sup.+] 190.1339, found 190.1344.
##STR00039##
7-Benzyl-4-(2-methylbenzyl)-1,2,6,7,8,9-hexahydroimidazo[1,2-a]pyrido[3,4-
-e]pyrimidin-5(4H)-one (2)
[0107] A mixture of methyl 1-benzyl-4-oxopiperidine-3-carboxylate
hydrochloride, 8, (568 mg, 2.0 mmol) and 7 (795 mg, 4.2 mmol) was
treated with a solution of sodium methoxide in MeOH (0.5 M, 3.0 mL,
1.5 mmol). The mixture was stirred at a gentle reflux overnight.
After 18 h, the reaction was cooled to room temperature, diluted
with CH.sub.2Cl.sub.2 (50 mL), washed with brine (20 mL), dried
over Na.sub.2SO.sub.4, filtered, and concentrated in vacuo.
Purification by silica gel chromatography (19:1
CH.sub.2Cl.sub.2/MeOH) afforded 2 (753 mg, 97%) as a pale yellow
solid.
[0108] m.p. 132-135.degree. C. (MeOH)
[0109] R.sub.f=0.25 (silica gel, 19:1 CH.sub.2Cl.sub.2/MeOH)
[0110] IR (neat) .nu..sub.max 2750, 2358, 1646, 1616, 1487, 1296,
738 cm.sup.-1
[0111] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta. 7.33 (m, 5H), 7.11
(m, 4H), 5.05 (s, 2H), 3.89 (m, 4H), 3.67 (s, 2H), 3.32 (s, 2H),
2.68 (m, 2H), 2.51 (m, 2H), 2.40 (s, 3H)
[0112] .sup.13C NMR (150 MHz, CDCl.sub.3) .delta. 161.6, 153.4,
145.8, 137.7, 135.7, 134.4, 130.4, 129.3, 128.6, 127.5, 127.0,
126.0, 125.4, 102.1, 62.5, 50.7, 49.7, 48.3, 47.1, 43.3, 27.0,
19.4
[0113] HRMS (ESI-TOF) calcd. for C.sub.24H.sub.26N.sub.4OH.sup.+
[M+H.sup.+] 387.2179, found 387.2166
TABLE-US-00002 TABLE 1 Comparison of 13C NMR chemical shifts for
compounds 1, 2, and 9 JWL JWL NCI MK (1) (2) (2) (9) 159.8 161.6
161.5 160.7 152.9 153.4 153.4 160.4 147.1 145.8 145.8 154.5 137.6
137.7 137.6 138.4 134.6 135.7 135.7 137.0 133.7 134.4 134.3 133.6
130.7 130.4 130.3 130.9 129.2 129.3 129.3 129.3 128.5 128.6 128.6
129.0 127.5 127.5 127.5 128.5 127.4 127.0 126.9 128.2 126.8 126.0
126.0 127.3 123.7 125.4 125.3 126.3 105.7 102.1 102.2 109.2 62.1
62.5 62.4 62.7 49.9 50.7 50.5 50.1 49.6 49.7 49.6 49.6 48.6 48.3
48.3 46.9 46.4 47.1 47.1 44.5 45.3 43.3 43.3 40.6 26.1 27.0 26.9
32.4 19.1 19.4 19.4 19.3 Spectra were recorded at 150 MHz in
CDCl3.
X-Ray Crystal Structures
[0114] The X-ray crystal structures of compounds 2 (as synthetic
sample 2b) and 9 were obtained. The parameters are given below, and
the structures obtained provided in FIGS. 5 and 6,
respectively.
Compound 2 (Also Called HIPPO)
[0115] The single crystal X-ray diffraction studies were carried
out on a Bruker X8 APEX II Ultra CCD diffractometer equipped with
Mo K.alpha. radiation (.lamda.=0.71073). A
0.18.times.0.16.times.0.08 mm clear colorless plate of 2 was
mounted on a Cryoloop with Paratone oil. Data were collected in a
nitrogen gas stream at 100 K using .omega. scans.
Crystal-to-detector distance was 50 mm using 5 s exposure time with
a 1.0.degree. scan width. Data collection was 99.9% complete to
25.00.degree. in 0. A total of 14019 reflections were collected
covering the indices, -11<=h<=10, -11<=k<=11,
-19<=l<=18. 4833 reflections were found to be symmetry
independent, with a Rint of 0.0391. Indexing and unit cell
refinement indicated a primitive, triclinic lattice. The space
group was found to be P-1. The data were integrated using the
Bruker SAINT software program and scaled using the SADABS software
program. Solution by direct methods (SHELXT) produced a complete
phasing model consistent with the proposed structure.
[0116] All non-hydrogen atoms were refined anisotropically by
full-matrix least-squares (SHELXL). All hydrogen atoms were placed
using a riding model. Their positions were constrained relative to
their parent atom using the appropriate HFIX command in SHELXL.
[0117] Crystallographic data are summarized below. Full metrical
parameters are available from the CCDC under number 981022. See
FIG. 5.
Crystal Data and Structure Refinement for Compound 2
TABLE-US-00003 [0118] Identification code Janda01 (2) Empirical
formula C.sub.24 H.sub.26 N.sub.4O Molecular formula C.sub.24
H.sub.26 N.sub.4O Formula weight 386.49 Temperature 100 K
Wavelength 0.71073 .ANG. Crystal system Triclinic Space group P-1
Unit cell dimensions a = 8.1173(11) .ANG. .alpha. =
85.638(3).degree. b = 8.4320(11) .ANG. .beta. = 85.045(3).degree. c
= 14.6360(19) .ANG. .gamma. = 83.059(3).degree. Volume 988.5(2)
.ANG..sup.3 Z 2 Density (calculated) 1.298 Mg/m.sup.3 Absorption
coefficient 0.082 mm.sup.-1 F(000) 412 Crystal size 0.18 .times.
0.16 .times. 0.08 mm.sup.3 Crystal color, habit colorless plate
Theta range for data collection 2.439 to 29.252.degree. Index
ranges -11 <= h <= 10, -11 <= k <= 11, -19 <= l
<= 18 Reflections collected 14019 Independent reflections 4833
[R(int) = 0.0391] Completeness to theta = 99.9% 25.000.degree.
Absorption correction Semi-empirical from equivalents Max. and min.
transmission 0.0976 and 0.0673 Refinement method Full-matrix
least-squares on F.sup.2 Data/restraints/parameters 4833/0/263
Goodness-of-fit on F2 1.027 Final R indices R1 = 0.0433, wR2 =
0.1082 [I > 2sigma(I)] R indices (all data) R1 = 0.0697, wR2 =
0.1181 Extinction coefficient n/a Largest diff. peak and hole 0.320
and -0.204 e..ANG..sup.-3
[0119] A colorless crystal of compound 9 was mounted on a Cryoloop
with Paratone oil and data was collected at 100 K on a Bruker APEX
II CCD with Mo K.sub.a radiation (generated from a Mo rotating
anode). Data was corrected for absorption with SADABS and structure
was solved by direct methods.
[0120] All non-hydrogen atoms were refined anisotropically by
full-matrix least-squares on F.sup.2 and all hydrogen atoms were
placed in calculated positions with appropriate riding
parameters.
Highest peak 0.20 at 0.4224 0.6962 0.1821 [0.63 A from C9] Deepest
hole -0.23 at 0.0912 0.4660 0.3644 [0.93 A from C17]
Crystallographic parameters are summarized below. Full metrical
parameters are available from the CCDC under number 981024. See
FIG. 6.
Crystal Data and Structure Refinement for Compound 9
TABLE-US-00004 [0121] Identification code Janda03 (9) Empirical
formula C.sub.24 H.sub.26 N.sub.4 O Molecular formula C.sub.24
H.sub.26 N.sub.4 O Formula weight 386.49 Temperature 100 K
Wavelength 0.71073 .ANG. Crystal system Triclinic Space group P-1
Unit cell dimensions a = 5.6439(18) .ANG. .alpha. =
93.194(9).degree. b = 10.537(4) .ANG. .beta. = 91.021(6).degree. c
= 16.502(5) .ANG. .gamma. = 96.745(5).degree. Volume 972.8(6)
.ANG..sup.3 Z 2 Density (calculated) 1.319 Mg/m.sup.3 Absorption
coefficient 0.083 mm.sup.-1 F(000) 412 Crystal size 0.22 .times.
0.02 .times. 0.02 mm.sup.3 Crystal color, habit colorless rod Theta
range for data collection 1.95 to 26.34.degree. Index ranges -6
<= h <= 6, -13 <= k <= 12, -20 <= l <= 18
Reflections collected 10564 Independent reflections 3904 [R(int) =
0.0507] Completeness to theta = 25.00.degree. 99.9% Absorption
correction multi-scan/SADABS Max. and min. transmission 0.9983 and
0.9820 Refinement method Full-matrix least-squares on F.sup.2
Data/restraints/parameters 3904/0/263 Goodness-of-fit on F.sup.2
1.003 Final R indices [I > 2sigma(I)] R1 = 0.0430, wR2 = 0.0942
R indices (all data) R1 = 0.0719, wR2 = 0.1068 Largest diff. peak
and hole 0.201 and -0.229 e..ANG..sup.-3
Biological Methods
Cell Culture Methods:
[0122] RAW 264.7 cells (ATCC TIB-71) were maintained in growth
medium of Dulbecco's Modified Eagle's Medium (DMEM with 4.5 g/L
glucose and pyruvate, Gibco BRL, Invitrogen Corp., USA)
supplemented with L-glutamine, penicillin/streptomycin,
non-essential amino acids (100.times. stocks, Invitrogen Corp.), 10
mM HEPES, pH 7.4 (1 M stock, Invitrogen), and 10% Fetal Bovine
Serum (FBS, Hyclone); (V. V. Kravchenko, R. J. Ulevitch, G. F.
Kaufmann, Methods Mol. Biol. 2011, 692, 133-145).
RNA Rt-PCR Experiments:
[0123] Cells were plated in 6-well plates (Corning Costar 3506)
diluted 1:5 in 3 mL growth medium, media was changed after cells
had adhered. After 12 h incubation, cells were treated with
described concentration of compound in DMSO, and incubated in the
presence of that compound or vehicle for the described amount of
time. At this time, media was removed and cells were treated with
TRIzol reagent (Life Technologies), and RNA extracted via included
protocol. RNA concentration determined using a Hitachi U-2000
UV-Vis Spectrophotometer and samples diluted to 12 .mu.g/5 .mu.L in
H2O. This solution was diluted 1:5 in H.sub.2O and 1 .mu.L of this
solution was mixed with 50 .mu.L of RT-PCR reaction mixture (Qiagen
Onestep RT-PCR kit) and TRAIL primers.
Mouse:
[0124] mTRAIL-F: 5'-GACACCATTTCTACAGTTCCAG-3' (SEQ ID NO. 1),
mTRAIL-R: 5'-CGGATAGCTGGTGTACTTGTAG-3' 3' (SEQ ID NO. 2).
Human:
[0125] hTrail-F2: 5'-ACAGACCTGCGTGCTGATCGTG-3' 3' (SEQ ID NO. 3)
(exon 1) hTrail-R2: 5'-ACGAGCTGACGGAGTTGCCAC-3' 3' (SEQ ID NO. 4)
(exon 2).
[0126] RT-PCR was run on an Applied Biosystems Gene Amp 9700 PCR
system. RT-PCR products were analyzed on 5.5% acrylamide gel in TAE
buffer (T. Maniatis, E. F. Fritsch, J. Sambrook, Molecular Cloning:
a Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring
Harbor, 1989).
[0127] RAW 264.7 cells were plated at 500 cell/well in Costar
96-well plates (Corning Inc, NY) in phenol-free Dulbecco's Modified
Eagle's Medium (DMEM with 4.5 g/L glucose, Gibco BRL, Invitrogen
Corporation, USA) supplemented with 10% fetal bovine serum (Gibco
BRL, Invitrogen Corp., USA), L-glutamine, pyruvate,
penicillin/streptomycin, and nonessential amino acids (100.times.
stocks from Invitrogen). After 4 hours, cells were then treated in
triplicate with vehicle, lysis buffer, 20 .mu.M of compound 2
(HIPPO), or 20 .mu.M of 18 derivative compounds (A through R) as
listed in Table 1, above. After 48 hours, cell viability was
assessed by colorimetric XTT formazan assay (Cell Signaling Tech.)
according to manufacturer protocol. Relative absorbance was
normalized to the vehicle treated cells (negative control) and
lysis buffer treated cells (positive control) using Prism 5 for Mac
(GraphPad). FIG. 7 summarizes the results of this assay. FIG. 7
shows a comparison of compounds A through R against HIPPO in their
ability to attenuate proliferation of RAW 264.7 cancer cells.
Compounds A through F exhibit similar activity to HIPPO,
demonstrating that modification of the substituent of the amide
nitrogen outside the tricyclic core is well tolerated and
represents an auxophore.
Sequence CWU 1
1
4122DNAArtificial SequenceA synthetic primer 1gacaccattt ctacagttcc
ag 22222DNAArtificial SequenceA synthetic primer 2cggatagctg
gtgtacttgt ag 22322DNAArtificial SequenceA synthetic primer
3acagacctgc gtgctgatcg tg 22421DNAArtificial SequenceA synthetic
primer 4acgagctgac ggagttgcca c 21
* * * * *