Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
1
AMINOBENZOIC ACID DERIVATIVES FOR USE AS ANTI-INFLAMMATORY
AGENTS, ANTI-METASTATIC AGENTS AND/OR ANTICANCER AGENTS
FIELD OF THE DISCLOSURE
[0001] This disclosure relates to the field of active agents. More
particularly,
this disclosure relates to anti-inflammatory agents, anti-metastatic agents,
anti-
proliferative agents and anticancer agents. The present disclosure also
relates to
methods for treating cancers using these agents.
BACKGROUND OF THE DISCLOSURE
[0002] There are several methods used to treat cancer) The most
common are:
surgery, chemotherapy, radiation therapy, targeted therapy and immunotherapy.1-
6
Other procedures are based on stem cell transplant, photodynamic therapy, and
cryogenic therapy) Lasers are nowadays a useful tool during surgery of
localized
cancers. Many of these methods are quite effective. However, most present
important
side effects.7,8 Hence, the need to discover alternative therapeutics and
treatment
modalities. Particularly, compounds and treatment protocols that could
simultaneously attack cancer on diverse fronts (initiation, propagation,
metastasis etc.)
are of great interest.
[0003] Urothelial bladder cancer (UBC) is the fifth most common
malignancy
of all cancers in North America. Although most of detectable tumors are
initially non-
muscle-invasive and are generally curable by means of chirurgical resection,
27-30%
of them exhibit a lethal phenotype characterized by high histological grade
and
muscle invasion. Recent studies have also provided ample evidence that UBC
intravesical therapy response is influenced by infiltration of major
inflammatory cells
(mainly macrophages) and activation of key inflammatory mediators (including
the
cytokines TNFa and IL6 and the transcription factors NFKB and STAT3).
Considering the critical functions of inflammatory mediators in UBC growth,
dissemination and resistance to cell death, they may represent potential drug
targets to
improve the efficacy of immunotherapy and chemotherapy agents.
SUMMARY OF THE DISCLOSURE
[0004] According to one aspect, there are included compounds of
formula (I) :
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0
0
R3 ................õ(
\ Q
1
R4V.-:----'.......<N _____________ C 1 N
1
1 R2
RI
0
(I)
wherein
RI is H, alkyl or halogen;
R2 is H, or a substituted or unsubstituted member chosen from acetyl,
propiolyl, butyryl, isobutyryl and benzoyl;
Q is QA or QB;
R5
1
QA=
H
N
(
/
QB = R7;
= a single bond or a double bond;
R5 is H, or a substituted or unsubstituted member chosen from acetyl,
propiolyl, butyryl, isobutyryl and benzoyl;
R6 is H, Boc, or a substituted or unsubstituted member chosen from
acetyl, propiolyl, butyryl, isobutyryl and benzoyl;
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R7 is a substituted or unsubstituted member chosen from C1-C8 alkyl,
C3-C8 cycloalkyl, phenyl, furanyl, thiophenyl, pyridinyl, naphthyl,
quinolyl and isoquinolyl;
R3 and R4 are independently chosen from H, -SR8 and ¨NR9R10,
or R3 and R4 are joined together to form a 5-7 membered ring that
optionally comprises an heteroatom chosen from N, S and 0;
R8 is H, Ci -C8 alkyl, -(CH2)nNHBoc, or -(CH2)nNH2 wherein n = I to
6;
R9 is H or Ci-C8 alkyl;
Rio is H, Ci -C8 alkyl, acetyl, propiolyl, butyryl, isobutyryl, or benzoyl;
wherein R2, R5, R6 and R7, when substituted, are substituted with at
least one substituent chosen from -0R9, -F, -Cl, -Br, -I, acetyl,
propiolyl, butyryl, isobutyryl, benzoyl, -NO2, C1-C8 alkyl,
methoxycarbonyl-, or alkyloxycarbonyl-;
or an enantiomer, diastereoisomer, racemic mixture, pharmaceutically
acceptable
salt, solvate or prodrug thereof.
[0005] According to another aspect, there is provided compounds of
formulae
(IIA), (JIB) or (IIC):
N N 0
II1)n N
NR2Q NR20 NR
0 0 H 0
(IA) (IIB) (IIC)
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wherein
Q is QA or QB;
R5
QA
(
Qs = R7;
R2 is H, or a substituted or unsubstituted member chosen from acetyl,
propiolyl, butyryl, isobutyryl and benzoyl;
R5 is H, or a substituted or unsubstituted member chosen from acetyl,
propiolyl, butyryl, isobutyryl and benzoyl;
R6 is H, Boc, or a substituted or unsubstituted member chosen from
acetyl, propiolyl, butyryl, isobutyryl and benzoyl;
R7 is a substituted or unsubstituted member chosen from Ci -C8 alkyl,
C3-C8 cycloalkyl, phenyl, furanyl, thiophenyl, pyridinyl, naphthyl,
quinolyl and isoquinolyl;
R9 is H or Ci-C8 alkyl;
Xis 0, S or NR2,
n is 1,2 or 3;
wherein R2, R5, R6 and R7, when substituted, are substituted with at
least one substituent chosen from -0R9, -F, -Cl, -Br, -I, acetyl,
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propiolyl, butyryl, isobutyryl, benzoyl, -NO2, Ci-C8 alkyl,
methoxycarbonyl-, or alkyloxycarbonyl-;
or an enantiomer, diastereoisomer, racemic mixture, pharmaceutically
acceptable
salt, solvate or prodrug thereof.
10006] According to another aspect, there is provided a compound of formula
(IF)
0
H
0
H N ¨ N
0 R2 R11
(IF)
R2 is H, or a substituted or unsubstituted member chosen from acetyl,
propiolyl, butyryl, isobutyryl and benzoyl;
RH is a substituted or unsubstituted member chosen from phenyl,
phenylethenyl, furanyl, thiophenyl, pyridinyl, naphthyl, quinolyl and
isoquinolyl;
wherein R11, when substituted, are substituted with at least one
substituent chosen from ¨01212, -F, -Br, -I, acetyl, propiolyl,
butyryl, isobutyryl, benzoyl, -NO2, -CH20H, nitrophenyl,
phenylethenyl, -CF3, CI-Cs alkyl, C2-Cs alkenyl, C2-C8 alkynyl, C3-C8
cycloalkenyl, methoxycarbonyl- and alkyloxycarbonyl-;
R12 is H, acetyl, propiolyl, Cu-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl or
C3-C8 cycloalkenyl;
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or an enantiomer, diastereoisomer, racemic mixture, pharmaceutically
acceptable
salt, solvate or prodrug thereof.
[0007] According to another aspect, there is provided a compound of
formula
(IG)
0
0
¨ N
0 R2
______________________________________________________ kRi3
(IG)
---1¨=>R14
R15
R2 is H, or a substituted or unsubstituted member chosen from acetyl,
propiolyl, butyryl, isobutyryl and benzoyl;
R12 is H, acetyl, propiolyl, CI-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl or
C3-C8 cycloalkenyl;
RI3, RI4 and RI5 are each independently chosen from H, ¨0R12, -F, -Cl,
-Br, -I, acetyl, propiolyl, butyryl, isobutyryl, benzoyl, -NO2, C1-C8
alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C3-C8 cycloalkenyl,
methoxycarbonyl- and alkyloxycarbonyl-;
or an enantiomer, diastereoisomer, racemic mixture, pharmaceutically
acceptable
salt, solvate or prodrug thereof.
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[0008] According to another aspect, there is provided a compound of
formula
(IH)
0
I N . 0
----\ NHN=--\
0 R16
(IH)
wherein R16 is chosen from
2,4-dihydroxyphenyl 3,5-ditertbuty1-4- 4-methoxyphenyl
hydroxybenzaldehyde
2-hydroxy-3-methoxyphenyl 3,4-dimethoxyphenyl 4-allyloxyphenyl
2-furanyl 2-pyridyl 3-pyridyl
4-pyridyl phenylethenyl 4-chlorophenyl
4-fluorophenyl 4-trifluoromethylphenyl and 4-nitropheny I.
[0009] According to another aspect, there is included a composition
comprising a pharmaceutically acceptable carrier and at least one compound of
the
present disclosure.
[0010] According to another aspect, there is included a method for
treating
cancer or at least one cancer chosen from breast cancer, uterine cancer,
ovarian
cancer, prostate cancer, bladder cancer, and melanoma, the method comprising
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administering to a subject in need thereof an effective amount of at least one
compound of the present disclosure.
[0011] According to another aspect, there is included a method for
reducing
the risks of developing cancer or for reducing the risk of developing at least
one
cancer in a subject, the cancer being, for example, chosen from breast cancer,
uterine
cancer, ovarian cancer, prostate cancer, bladder cancer, and melanoma, the
method
comprising administering to the subject an effective amount of at least one
compound
of the present disclosure.
[0012] According to another aspect, there is included a method for
inhibiting
cancer cell growth, the method comprising administering to a subject in need
thereof
an effective amount of at least one compound of the present disclosure. For
example,
the cancer can be chosen from breast cancer, uterine cancer, ovarian cancer,
prostate
cancer, bladder cancer, and melanoma.
[0013] According to another aspect, there is included a method for
inhibiting
cancer cell growth, the method comprising contacting cancer cells with an
effective
amount of at least one compound of the present disclosure. For example, the
cancer
can be chosen from breast cancer, uterine cancer, ovarian cancer, prostate
cancer,
bladder cancer, and melanoma.
[0014] According to another aspect, there is included a method for
inhibiting
cancer tumor growth and/or cancer tumor size, the method comprising
administering
to a subject in need thereof an effective amount of at least one compound of
the
present disclosure. For example, the cancer can be chosen from breast cancer,
uterine
cancer, ovarian cancer, prostate cancer, bladder cancer, and melanoma.
[0015] According to another aspect, there is included a method for
decreasing
and/or preventing cancer tumor metastases, the method comprising administering
to a
subject in need thereof an effective amount of at least one compound of the
present
disclosure. For example, the cancer can be chosen from breast cancer, uterine
cancer,
ovarian cancer, prostate cancer, bladder cancer, and melanoma.
[0016] According to another aspect, there is included the use of at
least one
compound of the present disclosure for treating cancer or for treating at
least one
cancer chosen from breast cancer, uterine cancer, ovarian cancer, prostate
cancer,
bladder cancer, and melanoma.
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[0017] According to another aspect, there is included the use of at
least one
compound of the present disclosure for reducing the risks of developing cancer
or for
reducing the risks of developing at least one cancer chosen from breast
cancer, uterine
cancer, ovarian cancer, prostate cancer, bladder cancer, and melanoma.
[0018] According to another aspect, there is included the use of at
least one
compound of the present disclosure for inhibiting cancer cell growth. For
example,
the cancer can be chosen from breast cancer, uterine cancer, ovarian cancer,
prostate
cancer, bladder cancer, and melanoma.
[0019] According to another aspect, there is included the use of at
least one
compound of the present disclosure for inhibiting cancer tumor growth and/or
cancer
tumor size of at least one cancer chosen from breast cancer, uterine cancer,
ovarian
cancer, prostate cancer, bladder cancer, and melanoma.
[0020] According to another aspect, there is included the use of at
least one
compound of the present disclosure for decreasing and/or preventing cancer
tumor
metastases of at least one cancer chosen from breast cancer, uterine cancer,
ovarian
cancer, prostate cancer, bladder cancer, and melanoma.
[0021] According to another aspect, there is included the use of at
least one
compound of the present disclosure in the manufacture of a medicament for
treating
cancer or for treating at least one cancer chosen from breast cancer, uterine
cancer,
ovarian cancer, prostate cancer, bladder cancer, and melanoma.
[0022] According to another aspect, there is included the use of at
least one
compound of the present disclosure in the manufacture of a medicament for
reducing
the risks of developing cancer or for reducing the risks of developing at
least one
cancer chosen from breast cancer, uterine cancer, ovarian cancer, prostate
cancer,
bladder cancer, and melanoma.
[0023] According to another aspect, there is included the use of at
least one
compound of the present disclosure in the manufacture of a medicament for
inhibiting
cancer cell growth. For example, the cancer can be chosen from breast cancer,
uterine
cancer, ovarian cancer, prostate cancer, bladder cancer, and melanoma.
[0024] According to another aspect, there is included the use of at
least one
compound of the present disclosure in the manufacture of a medicament for
inhibiting cancer tumor growth and/or cancer tumor size. For example, the
cancer can
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be chosen from breast cancer, uterine cancer, ovarian cancer, prostate cancer,
bladder
cancer, and melanoma.
[0025] According to another aspect, there is included the use of at
least one
compound of the present disclosure in the manufacture of a medicament for
decreasing and/or preventing cancer tumor metastases. For example, the cancer
can be
chosen from breast cancer, uterine cancer, ovarian cancer, prostate cancer,
bladder
cancer, and melanoma.
BRIEF DESCRIPTION OF FIGURES
[0026] The following drawings represent in a non-limitative manner
examples
of specific embodiments in which:
Figs. 1(a) and 1(b) represents images (1(a)) and graphical analysis (1(b))
showing
Western blot analysis to determine the expression level of phosphorylated
STAT1 and
STAT3 in human macrophages (MO) pretreated for 30 min with vehicle (DMSO) or
compounds 4 and 5 (both at 0, 10 and 50 ?AM), and then washed and recovered
immediately (t= 0) or after 30 min of activation with either 50 U/mL IFNy or
25
ng/mL IL6. The ratio of phosphorylated/no phosphorylated proteins was
calculated
from densitometric analysis of each sample to evaluate the relative activation
of
pSTAT1 or pSTAT3. Compounds 4 and 5 efficiently inhibited IFNy-induced STAT1
activation and IL6-induced STAT3 activation. * p < 0.05 and ** p < 0.01 denote
significant differences between treatments;
Fig. 2(a) and 2(b) represents images (2(a)) and graphical analysis (2(b))
showing
flow cytometry analysis to determine the expression level of MHC-II and CD40
surface antigens in resting and IFNy-activated human macrophages untreated and
pretreated with compounds 4 (10 M) and 5 (25 'LIM). Compounds 4 and 5
efficiently
inhibited IFNy-induced CD40 and MHC-II expression. * p < 0.05 and ** p < 0.01
denote significant differences between treatments;
Fig. 3(a) and 3(b) represents images (3(a)) and graphical analysis (3(b))
showing
scratch wound healing assays to determine the motility of human macrophages
monolayers cultured for 3 h with vehicle (DMSO) or compounds 4 (10 M) and 5
(25
M), and then activated for 48 h with vehicle (PBS) or 25 ng/mL IL6. The images
of
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the scratch were acquired at t= 0 h and t= 48 h by fluorescence microscopy.
Five
fields were taken randomly for each different treatment. All observations were
performed at 5x magnification. Cell motility was expressed as percent (%) of
control
of motile cells at t= 48 h relative to motile cells at t= 0 h. Compounds 4 and
5
efficiently inhibited IFNy-induced STAT1 activation and IL6-induced STAT3
activation. * p < 0.05 and ** p < 0.01 denote significant differences between
treatments;
Fig. 4 is a graphical representation of NO production in the macrophage-like
J774A.1
cells following a pro-inflammatory stimulation by IFNy and TNFa after
pretreatment
with vehicle (DMSO) and the derivatives 4 and 5. Compounds 4 and 5 efficiently
inhibited combined IFNy/TNFa-induced NO synthesis. * p < 0.05 and ** p < 0.01
denote significant differences between treatments;
Fig. 5 is a graphical representation of NO production by the murine UBC cell
line
MB49-I following a pro-inflammatory stimulation by IFNy and TNFa after
pretreatment with vehicle (DMSO) and derivatives 4 and 5. Compounds 4 and 5 at
10
and 50 p,M efficiently inhibited combined IFNy/TNFa-induced NO synthesis. * p
<
0.05 and **p < 0.01 denote significant differences between treatments;
Fig. 6 is a graphical representation of relative cell viability on the murine
UBC cell
line MB49-I following a pro-inflammatory stimulation by IFNy and TNFa after
pretreatment with vehicle (DMSO) and anti-inflammatory derivatives 4 and 5 at
different concentrations. Compounds 4 and 5 at low doses (10, 25 and 37.5 1AM)
had
little or no effect on the anti-proliferative activity of combined IFNy and
TNFa in
MB49-I cells. * p < 0.05 denote significant differences between treatments
with
combined IFNy and TNFa plus compounds 4 or 5 versus combined IFNy and TNFa
alone;
Fig. 7 refers to relative cell viability on MB49 and MB49-I cell lines 24-hour
post
treatment (3h) with either vehicle (DMSO) or compound 4 at different
concentrations
(0, 10, 15, 20, 30, and 50 M). The negative effects of compound 4 on cell
survival
would not be caused by an increase in cell mortality of UBC MB49 or MB49-I
cells
but rather by stopping cell proliferation. * p < 0.05 denote significant
difference
compared to control (without compound 4);
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Figs. 8(a) and 8(b) refers to (8(a)) Western blot analysis: compound 4
efficiently
inhibited TNFa/NFKB and IL6/STAT3 signaling pathways in murine UBC MB49-I
cells at doses as lower as 10 1.1M; and (8(b)) Luciferase assay: compound 4 at
50 !AM
efficiently inhibited NFKB activation. MB49-I cells were transfected with an
NFKB-
responsive luciferase construct encoding the firefly luciferase reporter gene
under the
control of CMV promoter and tandem repeats of the NFKB transcriptional
response
element. *p <0.01 denote significant difference compared to control;
Figs. 9(a) and 9(b) refers to (9(a)) microinvasion assays: compound 4
efficiently
inhibited Ml-induced human UBC T24 cell invasion (matrigel in Boyden chamber);
and (9(b)) motility assays: compound 4 at 30 p.M efficiently inhibited 1L6-
induced
motility (scratch assay) in human UBC T24 cells. * p < 0.05 denote significant
difference compared to control (M1 DMS0);
Fig. 10 refers to cellular analysis of 3D spheroid-based tumor invasion
assays:
compound 4 at 30 }AM efficiently inhibited the invasiveness activity of MDA-MB-
241
cells, a highly invasive human breast cancer cell line, thus demonstrating
that the anti-
invasion effects of compound 4 is not cell-specific. * p < 0.01 denote
significant
difference compared to day 0;
Figs. 11(a), 11(b) and 11(c) refers to acute toxicity studies with episodic
intraperitoneal (i.p.) injections of compound 4 at different concentrations
(0, 90, 150,
and 300 1.1M) in C57B1/6J mice (n=4) during 3 weeks. Treatments have no
obvious
effect on normal development and viability (11(a)), as well as in organs
weight
(11(b)) and hematocrit (11(c));
Fig. 12 represents images showing liver histological analysis in C57B1/6J mice
at the
end of treatment by periodic i.p. injections of compound 4 at different
concentrations
(0, 90, 150, and 300 04) during 3 weeks. Treatment, even at higher dose, does
not
induce features of liver dysfunction such as hepatocellular injury,
inflammation,
fibrosis and steatosis (haematoxylin and eosin, 100 x and 400 x
magnification). These
results showed no evidence of toxicity from the anti-inflammatory treatment,
indicating that compound 4 concentrations up to 300 IAM were well tolerated in
these
mice;
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Fig. 13 refers to in vivo targeting study showing the effects of episodic i.p.
injections
of compound 4 at different concentrations (0, 90, and 150 p,M) in C57B1/6J
male mice
bearing MB49-I tumors ectopically implanted in the right flank of animals
(n=6).
Treatment at 150 1AM significantly affects tumor development of MB49-1 cells
subcutaneously implanted in mice. *p < 0.01 compared to control;
Figs. 14(a) and 14(b) refers to the anti-metastatic effects of episodic i.p.
injections of
compound 4 at different concentrations (0, 90, and 150 I.A.M) in C57B1/6J male
mice
bearing MB49-I tumors ectopically implanted in animals (n=6). Treatment at 150
M
significantly reduce the size of subcutaneously implanted MB49-1 tumors
(14(a)) and
the number of lung metastases (14(b)). Histological examination of lung
specimens
demonstrated that MB49-1 tumor cell infiltration induced multifocal fibrotic
lesions in
the lungs of control mice (as indicated by the arrows), whereas no metastatic
foci and
a well-alveolized normal histology was seen in the lungs of mice treated with
150 IAM
compound 4. *p < 0.01 compared to control;
Figs. 15(a) and 15(b) refers to in vivo targeting study showing the effects of
episodic
i.p. injections of compound 4 in C57B1/6J female mice bearing MB49-1 tumors
orthotopically implanted in the bladder of animals (n=6). (15(a)) Represents
images
of bladders from normal age-matched mice and bladders that were chirurgically
recovered at the end of the study (day 16 after tumor implantation) from
control (Ctl)
and treated tumor-bearing mice. (15(b)) Graphical representation showing i.p.
injections of compound 4 biweekly at 150 1.1M during two weeks completely
abolished the development of muscle-invasive MB49-I tumors (n=6). *p < 0.5
compared to Ctl.
Figs. 16(a) and 16(b) refers to the regulatory effects of pro-inflammatory IL6
and
TNFcc in the production of iNOS proteins (16(a)) and NO molecules (16(b)) by
MB49 and MB49-1 cells. These results shown that non-muscle invasive UBC MB49
cells produced low levels of iNOS and NO relative to highly invasive MB49-1
cells in
response to TNFoc but not IL6. These results strongly support the idea that NO
production via inflammatory pathway TNFa/NFKB may provide a permissive
environment for MB49-1 tumor progression in vivo. *p <0.01 compared to
control;
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Figs. 17(a), 17(b) and 17(c) refers to in vivo study showing loss of basal and
TNFa-
induced iNOS expression (17(a)) and NO production (17(b)) by shRNA affects
luciferase-expressing MB49-1 tumor development (17(c)) ectopically implanted
in the
bladder of C57B1/6J female mice. These results strongly suggest that MB49-I
tumors
is probably highly dependent on the presence of functional iNOS/NO system to
develop in C57B1/6J mice *p < 0.01 compared to control;
Fig. 18 refers to in vivo targeting study showing the effects of episodic i.p.
injections
of compound 4 at different concentrations (0, 90, and 150 pi,M) in C57B1/6J
male mice
bearing non-muscle invasive MB49 tumors ectopically implanted in the right
flank of
animals (n=6). Treatment at 90 and 150 [IM significantly reduce the size of
subcutaneously implanted MB49 tumors. These results shown that compound 4 is
highly efficient to stop the development of UBC tumors producing very low
levels of
iNOS protein and NO molecules. *p < 0.001 compared to Ctl;
Figs. 19(a) and 19(b) refers to the anti-inflammatory effects of compound 4 in
peritoneal macrophages after in vivo therapeutic targeting of ectopically
implanted
MB49 tumors in C57B1/6J male mice. Treatment at 90 and 150 1.1.M significantly
decrease NO production (19(a)) and urea synthesis and release (19(b)) in
peritoneal
macrophages. Peritoneal macrophages were recovered at the end of the study
(day 26
after tumor implantation). Polarization to pro-inflammatory M1 macrophages is
characterized by NO production through iNOS activity while urea synthesis
through
arginase-1 activity is related to polarization to anti-inflammatory M2
macrophages.
These results strongly suggest that in vivo therapeutic targeting of bladder
tumors with
compound 4 directly affects macrophage polarization in mice, probably via
induction
of an inflammatory anergy status, a process typically observed in normal
intestinal
macrophages. *p < 0.05 and ** p < 0.01 denote significant differences between
treatments.
Figs. 20(a) and 20(b) refers to structure-function relationship study on the
anti-
inflammatory activity of compound 4. (20(a)) Chemical structures of compounds
4
and 12. (20(b)) Graphical representation of LPS/IFN7-induced NO production in
MB49-I cells after pretreatment with vehicle (DMSO) and the compounds 4 (30
M)
and 12 at different concentrations (10, 20, 37.5 and 50 ?AM). Compound 12 even
at
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higher concentration does not affect LPS/IFNThinduced NO synthesis. These
results
shown that the double bond in the heterocycle of the left part of the molecule
is
important for the inhibitory effect of compound 4 on NO production by
macrophages
and tumor cells * p < 0.01 denote significant difference compared to positive
control
(DMSO + LPS /IFN7);
Figs. 21(a) and 21(b) refers to the comparative effects of compounds 4, 8, 10,
and 11
in NO production in MB49-1 cells. (21(a)) Chemical structures of compounds 4,
8, 10,
and 11; (21(b)) Graphical representation of IFN7/LPS-induced NO production in
MB49-1 cells after pretreatment with vehicle (DMSO) and the compounds 4, 8,
10,
and 11 at 20 and 37.5 j.tM. These results shown that compound 8 at the same
concentrations (20 and 37.5 i_tM) is more efficient than compound 4 to inhibit
NO
production. * p < 0.05 and ** p < 0.01 denote significant differences between
treatments;
Figs. 22(a) and 22(b) represents images (22(a)) and graphical analysis (22(b))
showing Western blot analysis to determine the expression level of
phosphorylated
STAT1 in murine macrophages (RAW 264.7 cells) pretreated for 90 min with
vehicle
(DMSO) or compounds 4, 8, 10, and 11 (at 20 and 37.5 M), and then washed and
recovered after 15 min of activation with 5 ng/mL IF1\17. The ratio of p-
STAT1/STAT1 was calculated from densitometric analysis of each sample to
evaluate
the relative activation of the transcription factor STAT1. These results shown
that
compound 8 is more efficient than other compounds to inhibit 1FNy-activated
STAT1
signaling pathway in RAW 264.7 cells. * p < 0.05 and ** p < 0.01 denote
significant
differences between treatments;
Figs. 23(a) and 23(b) represents images (23(a)) and graphical analysis (23(b))
showing Western blot analysis to determine the expression level of
phosphorylated
1KB in murine macrophages (RAW 264.7 cells) pretreated for 90 min with vehicle
(DMSO) or compounds 4, 8, 10, and 11 (at 20 and 37.5 M), and then washed and
recovered after 15 min of activation with 100 ng/mL LPS. The ratio of p-
IkB/IkB was
calculated from densitometric analysis of each sample to evaluate the relative
deactivation of IKB proteins, the negative regulator of the transcription
factor NFKB.
These results shown that all compounds are efficient to inhibit LPS-activated
NFKB
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signaling pathway in RAW 264.7 cells. * p < 0.05 and ** p <0.01 denote
significant
differences between treatments;
Figs. 24(a), 24(b) and 24(c) represents images and graphical analysis showing
the
comparative effects of compounds 4 and 8 in cell viability of murine UBC MB49-
I
cells (24(a)), murine macrophages RAW 264.7 cells (24(b)) and human
macrophages
THP-1 cells (24(c)). Cells were pretreated for 90 min, with vehicle (DMSO) and
compounds 4 and 8 at different concentrations, and then washed and counted
after a
24-hour period of incubation to estimate the number of viable cells. These
results
demonstrated that the cytotoxic effect of compound 8 is lower than that of
compound
4. * p < 0.05 denote significant differences between treatments.
Fig. 25 refers to the comparative anticancer activities of compounds 4 and 8
in the
development of MB49-I tumors ectopically implanted in C57B1/6J male mice. In
vivo
targeting study showing episodic i.p. injections of compound 8 during 18 days
is more
efficient than compound 4 to reduce the tumor volume of muscle-invasive MB49-I
tumors (n=6) at the same concentration (150 PM). Tumors were chirurgically
recovered at the end of the study (day 20 after tumor implantation). These
results
shown that compound 8 is more efficient than compound 4 to inhibit the
development
of highly invasive MB49-I tumors. * p < 0.05 and ** p < 0.01 denote
significant
differences between treatments.
Figs. 26(a) and 26(b) is a graphical representation of (26(a)) NO production
and
(26(b)) cell viability in the macrophage-like RAW 264.7 cells. (26(a)) Griess
reagent
and (26(b)) MTT assays were performed following a 24-h period of pro-
inflammatory
stimulation by IFNy (10 ng/mL) and LPS (100 ng/mL) after pretreatment with
control
(DMSO) and the molecules 1, 13a, 13b, and 13j. (26(a)) Compound 13j, but not
13a
nor 13b, efficiently inhibited combined IFN7/LPS-induced NO synthesis;
compared to
compound 1, compound 13j was 1.8 fold and 12 fold more efficient at 5 piM and
20
[iM, respectively. (26(b)) While compounds 13a, 13b, and 13j had little or not
positive effects on RAW 264.7 cell proliferation, compound 13j was 1.6 less
toxic at
20 [i.1VI compared to compound 1. * p < 0.05 and ** p < 0.01 denote
significant
difference between each treatment dose with similar dose of compound 1.
CA 3021417 2018-10-18
17
Figs. 27(a) and 27(b) is a graphical representation of (27(a)) NO production
and
27(b) cell viability in the macrophage-like RAW 264.7 cells. (27(a)) Griess
reagent
and (27(b)) MIT assays were performed following a 24-h period of pro-
inflammatory
stimulation by IFNy (10 ng/mL) and LPS (100 ng/mL) after pretreatment with
control
(DMSO) and the molecules 1, 13q, 13h, and 131. (27(a)) While compound 13q had
similar anti-inflammatory activity than compound 1, compounds 13h and 131 were
more efficient to inhibit combined IFNy/LPS-induced NO synthesis in RAW 264.7
cells; for instance, compared to compound 1, compound 131 was 2.7 fold and 7.5
fold
more efficient at 5 !AM and 10 ttM, respectively. (27(b)) While compounds 13q
and
131 had similar cytotoxic activity than compound 1, compound 13h was 1.5 more
toxic at 10 ItM compared to compound 1. * p < 0.05 and ** p < 0.01 denote
significant difference between each treatment dose with similar dose of
compound 1.
Figs. 28(a) and 28(b) represents images (28(a)) and graphical analysis (28(b))
showing Western blot analysis to determine the expression level of
phosphorylated
STAT3 in human triple negative breast cancer cell line MDA-MB-231. (a) Cells
were
pretreated for 30 min with vehicle (DMSO) or compounds 1, 13a and 13j (all at
0, 10,
20 and 30 ccM), and then washed and recovered after 15 min of activation with
20
ng/mL IL6. (b) The ratio of phosphorylated/no phosphorylated proteins was
calculated from densitometric analysis of each sample to evaluate the relative
activation of pSTAT1 or pSTAT3. These ratios were used to express the relative
inhibition rate of pSTAT3. Compounds 1 and 13j, but not compound 13a,
efficiently
inhibited IL6-induced STAT3 activation. However, compared to compound 1,
compound 13j was 1.6 fold and 3 fold more efficient at 20 ttM and 30 ttM,
respectively. * p < 0.01 denote significant differences between each treatment
dose
with control (DMSO).
Figs. 29(a) and 29(b) represents images (29(a)) and graphical analysis (29(b))
showing Western blot analysis to determine the expression level of
phosphorylated
1KB in human triple negative breast cancer cell line MDA-MB-231. (29(a)) Cells
were
pretreated for 30 min with vehicle (DMSO) or compounds 1, 13a and 13j (all at
0, 10,
20 and 30 ccM), and then washed and recovered after 5 min of activation with
25
ng/mL TNFa. (29(b)) The ratio of phosphorylated IKB/r3-actine proteins was
calculated from densitometric analysis of each sample to evaluate the relative
CA 3021417 2018-10-18
18
activation of 11(13. These ratios were used to express the relative inhibition
rate of 'KB.
Compounds 1 and 13j, but not compound 13a, efficiently inhibited IL6-induced
STAT3 activation at lower doses. However, compared to compound 1, compound 13j
was 3 fold and 2 fold more efficient at 20 [IM and 30 pIVI, respectively. * p
<0.05 and
** p < 0.01 denote significant differences between each treatment dose with
control
(DMSO).
Fig. 30 is a graphical representation of relative cell viability on human
triple negative
breast cancer cell line MDA-MB-231 after pretreatment with vehicle (DMSO) and
anti-inflammatory derivatives 1, 13a and 13j at different concentrations.
While all
compounds had cytotoxic activity against MDA-MB-231 cells, compounds 13a and
13j were less toxic at 20, 30, and 40 M compared to compound 1; for instance,
compared to compound 1, compound 13a was 1.4 fold, 2.5 fold, and 6.1 fold less
toxic at 20, 30, and 40 p.M, respectively. * p < 0.01 denote significant
difference
between each treatment dose with similar dose of compound 1.
DETAILED DESCRIPTION OF THE DISCLOSURE
[0027] The present disclosure concerns the discovery of small
aminobenzoic
acid derivatives showing anti-inflammatory, anti-metastatic, anti-
proliferative and
anticancer properties in vitro and in vivo. It describes the synthetic
methodology to
make these derivatives from readily available ortho-, meta- and para-benzoic
acid and
their biological applications for the treatment of a several types of cancers.
In
addition, this disclosure relates to different pharmaceutical compositions
comprising
these compounds. The compounds and the pharmaceutical composition of this
disclosure have been shown to possess anticancerous activity on various types
of
cancers. Furthermore, this disclosure provides novel treatment modalities
against
cancer. The unique biological properties of these compounds may be
advantageously
used to provide compounds with anticancer activity against cancers including
but not
limited to breast, prostate, ovarian and bladder cancers.
[0028] The term a "therapeutically effective amount", "effective
amount" or a
"sufficient amount" of a compound of the present disclosure is a quantity
sufficient to,
when administered to the subject, including a mammal, for example a human,
effect
beneficial or desired results, including clinical results, and, as such, an
"effective
amount" or synonym thereto depends upon the context in which it is being
applied.
CA 3021417 2018-10-18
19
For example, in the context of treating cancer, for example, it is an amount
of the
compound sufficient to achieve such treatment of the cancer as compared to the
response obtained without administration of the compound. The amount of a
given
compound of the present disclosure that will correspond to an effective amount
will
vary depending upon various factors, such as the given drug or compound, the
pharmaceutical formulation, the route of administration, the type of disease
or
disorder, the identity of the subject or host being treated, and the like, but
can
nevertheless be routinely determined by one skilled in the art. Also, as used
herein, a
"therapeutically effective amount", "effective amount" or a "sufficient
amount" of a
compound of the present disclosure is an amount which inhibits, suppresses or
reduces a cancer (e.g., as determined by clinical symptoms or the amount of
cancerous cells) in a subject as compared to a control.
[0029] The term "subject" as used herein includes all members of the
animal
kingdom including human. According to one embodiment, the subject is a human.
[0030] The term "alkyl" as used herein means straight and/or branched
chain,
saturated alkyl groups containing from one to n carbon atoms and includes
(depending
on the identity of n) methyl, ethyl, propyl, isopropyl, n-butyl, s-butyl,
isobutyl, t-
butyl, 2,2-dimethylbutyl, n-pentyl, 2-methylpentyl, 3-methylpentyl, 4-
methylpentyl,
n-hexyl and the like, wherein n is the maximum number of carbon atoms in the
group.
[0031] The expression "an alkyl component of a naturally occurring
amino
acid" as used herein refers to the portion of a naturally occurring amino acid
that is
comprised between the carbon of the carbonyl group of the amino acid and the
nitrogen atom of the amino acid.
[0032] The expression "compound(s) of the present disclosure" as used
in the
present document refers to compounds of formulae I, IA, IB, IC, ID, IE, IIA,
IIB and
IIC presented in the present disclosure, isomers thereof, such as
stereoisomers (for
example, enantiomers, diastereoisomers, including racemic mixtures) or
tautomers, or
to pharmaceutically acceptable salts, solvates, hydrates and/or prodrugs of
these
compounds, isomers of these latter compounds, or racemic mixtures of these
latter
compounds. The expression "compound(s) of the present disclosure" also refers
to
mixtures of the various compounds or variants mentioned in the present
paragraph.
CA 3021417 2018-10-18
20
[0033] The term "halogen" as used herein comprises fluoro, chloro,
bromo
and iodo.
[0034] It is to be clear that the present disclosure includes
isomers, racemic
mixtures, pharmaceutically acceptable salts, solvates, hydrates and prodrugs
of
compounds described therein and mixtures comprising two or more of such
compounds.
[0035] The compounds of the disclosure may have at least one
asymmetric
centre. Where the compounds according to the present document possess more
than
one asymmetric centre, they may exist as diastereomers. It is to be understood
that all
such isomers and mixtures thereof in any proportion are encompassed within the
scope of the present disclosure. It is to be understood that while the
stereochemistry of
the compounds of the present disclosure may be as provided for in any given
compound listed herein, such compounds of the disclosure may also contain
certain
amounts (for example less than 30%, less than 20%, less than 10%, or less than
5%)
of compounds of the present disclosure having alternate stereochemistry.
[0036] The term "suitable", as in for example, "suitable counter
anion" or
"suitable reaction conditions" means that the selection of the particular
group or
conditions would depend on the specific synthetic manipulation to be performed
and
the identity of the molecule but the selection would be well within the skill
of a
person trained in the art. All process steps described herein are to be
conducted under
conditions suitable to provide the product shown. A person skilled in the art
would
understand that all reaction conditions, including, for example, reaction
solvent,
reaction time, reaction temperature, reaction pressure, reactant ratio and
whether or
not the reaction should be performed under an anhydrous or inert atmosphere,
can be
varied to optimize the yield of the desired product and it is within their
skill to do so.
[0037] The expression "pharmaceutically acceptable" means compatible
with
the treatment of subjects such as animals or humans.
[0038] The expression "pharmaceutically acceptable salt" means an
acid
addition salt or basic addition salt which is suitable for or compatible with
the
treatment of subjects such as animals or humans.
[0039] The expression "pharmaceutically acceptable acid addition
salt" as
used herein means any non-toxic organic or inorganic salt of any compound of
the
CA 3021417 2018-10-18
21
present disclosure, or any of its intermediates. Illustrative inorganic acids
which form
suitable salts include hydrochloric, hydrobromic, sulfuric and phosphoric
acids, as
well as metal salts such as sodium monohydrogen orthophosphate and potassium
hydrogen sulfate. Illustrative organic acids that form suitable salts include
mono-, di-,
and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic,
glutaric,
fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic,
cinnamic and
salicylic acids, as well as sulfonic acids such as para-toluene sulfonic and
methanesulfonic acids. Either the mono or di-acid salts can be formed, and
such salts
may exist in either a hydrated, solvated or substantially anhydrous form. In
general,
the acid addition salts of the compounds of the present disclosure are more
soluble in
water and various hydrophilic organic solvents, and generally demonstrate
higher
melting points in comparison to their free base forms. The selection of the
appropriate salt will be known to one skilled in the art. Other non-
pharmaceutically
acceptable salts, e.g. oxalates, may be used, for example, in the isolation of
the
compounds of the present disclosure, for laboratory use, or for subsequent
conversion
to a pharmaceutically acceptable acid addition salt. In embodiments of the
present
disclosure, the pharmaceutically acceptable acid addition salt is the
hydrochloride salt.
[0040] The term
"pharmaceutically acceptable basic addition salt- as used
herein means any non-toxic organic or inorganic base addition salt of any acid
compound of the disclosure, or any of its intermediates. Acidic compounds of
the
disclosure that may form a basic addition salt include, for example, where R
is CO2H.
Illustrative inorganic bases which form suitable salts include lithium,
sodium,
potassium, calcium, magnesium or barium hydroxide. Illustrative organic bases
which form suitable salts include aliphatic, alicyclic or aromatic organic
amines such
as methylamine, trimethylamine and picoline or ammonia. The selection of the
appropriate salt will be known to a person skilled in the art. Other non-
pharmaceutically acceptable basic addition salts, may be used, for example, in
the
isolation of the compounds of the disclosure, for laboratory use, or for
subsequent
conversion to a pharmaceutically acceptable acid addition salt.
[0041] The
formation of a desired compound salt is achieved using standard
techniques. For example, the neutral compound is treated with an acid or base
in a
suitable solvent and the formed salt is isolated by filtration, extraction or
any other
suitable method.
CA 3021417 2018-10-18
22
[0042] The term
"solvate" as used herein means a compound of the present
disclosure, wherein molecules of a suitable solvent are incorporated in the
crystal
lattice. A suitable solvent is physiologically tolerable at the dosage
administered.
Examples of suitable solvents are ethanol, water and the like. When water is
the
solvent, the molecule is referred to as a "hydrate". The formation of solvates
of the
compounds of the present disclosure will vary depending on the compound and
the
solvate. In
general, solvates are formed by dissolving the compound in the
appropriate solvent and isolating the solvate by cooling or using an
antisolvent. The
solvate is typically dried or azeotroped under ambient conditions.
[0043] Compounds
of the present disclosure include prodrugs. In general,
such prodrugs will be functional derivatives of these compounds which are
readily
convertible in vivo into the compound from which it is notionally derived.
Prodrugs
of the compounds of the present disclosure may be conventional esters formed
with
available hydroxy, or amino group. For example, an available OH or nitrogen in
a
compound of the present disclosure may be acylated using an activated acid in
the
presence of a base, and optionally, in inert solvent (e.g. an acid chloride in
pyridine).
Some common esters which have been utilized as prodrugs are phenyl esters,
aliphatic
(C8-C24) esters, acyloxymethyl esters, carbamates and amino acid esters. In
certain
instances, the prodrugs of the compounds of the present disclosure are those
in which
one or more of the hydroxy groups in the compounds is masked as groups which
can
be converted to hydroxy groups in vivo. Conventional procedures for the
selection
and preparation of suitable prodrugs are described, for example, in "Design of
Prodrugs" ed. H. Bundgaard, Elsevier, 1985.
[0044] Compounds
of the present disclosure include radiolabeled forms, for
example, compounds labeled by incorporation within the structure 2H, 3H, 14C,
15N, or
a radioactive halogen such as 1251. A radiolabeled compound of the compounds
of the
present disclosure may be prepared using standard methods known in the art.
[0045] As used
herein, and as well understood in the art, "treatment" or
"treating" is an approach for obtaining beneficial or desired results,
including clinical
results. Beneficial or desired clinical results can include, but are not
limited to,
alleviation or amelioration of one or more symptoms or conditions,
diminishment of
extent of disease, stabilized (i.e. not worsening) state of disease,
preventing spread of
disease, delay or slowing of disease progression, amelioration or palliation
of the
CA 3021417 2018-10-18
23
disease state, and remission (whether partial or total), whether detectable or
undetectable. "Treatment" or "treating" can also mean prolonging survival as
compared to expected survival if not receiving treatment.
[0046] "Palliating" a disease or disorder, means that the extent
and/or
undesirable clinical manifestations of a disorder or a disease state are
lessened and/or
time course of the progression is slowed or lengthened, as compared to not
treating
the disorder.
[0047] In understanding the scope of the present disclosure, the term
"comprising" and its derivatives, as used herein, are intended to be open
ended terms
that specify the presence of the stated features, elements, components,
groups,
integers, and/or steps, but do not exclude the presence of other unstated
features,
elements, components, groups, integers and/or steps. The foregoing also
applies to
words having similar meanings such as the terms, "including", "having" and
their
derivatives. Finally, terms of degree such as "substantially", "about" and
"approximately" as used herein mean a reasonable amount of deviation of the
modified term such that the end result is not significantly changed. These
terms of
degree should be construed as including a deviation of at least 5% of the
modified
term if this deviation would not negate the meaning of the word it modifies.
[0048] In an embodiment of the present disclosure, there are included
compounds of formula (IA) :
0
0
R3
R2
R4
0
(IA)
wherein R2, R3, R4 and Q are as previously defined.
[0049] In another embodiment of the present disclosure, there are
included
compounds of formula (TB) :
CA 3021417 2018-10-18
24
0
R3 N.......................<
0
1
1
1 N
zi....,......< N ___ Q
R4
/
0 R2
(IB)
wherein R2, R3, R4 and Q are as previously defined.
[0050] In a further embodiment of the present disclosure, there are
included
compounds of formula (IC) :
0
H........
0
N R5
/
HV---------< \
0 R2 R6
(IC)
wherein R2, R5 and R6 are as previously defined.
[0051] In still a further embodiment of the present disclosure, there
are
included compounds of formula (ID) :
CA 3021417 2018-10-18
25
0
R3 0
N-N
R4
(
0 R2 R7
(ID)
wherein R2, R3, R4 and R7 are as previously defined.
[0052] In still a further embodiment of the present disclosure, there
are
included compounds of formula (IE) :
0
H
0
/N-N
H
(
0 R2 R7
(1E)
wherein R2 and R7 are as previously defined.
[0053] In still a further embodiment of the present disclosure,
R2 is H, unsubstituted member chosen from acetyl and propiolyl;
Q is QA;
R5 is H, unsubstituted member chosen from acetyl and propiolyl; and
R6 is Boc, H, or an unsubstituted member chosen from acetyl and
propiolyl.
[0054] In still a further embodiment of the present disclosure,
CA 3021417 2018-10-18
26
R2 is H or unsubstituted member chosen from acetyl and propiolyl; and
R7 is an unsubstituted member chosen from Cl-Cs alkyl, C3-C6
cycloalkyl, phenyl, furanyl, thiophenyl, pyridinyl, naphthyl, quinolyl
and isoquinolyl.
[0055] For example, in a further embodiment of the present
disclosure, the
compound of formula I is as previously defined with the proviso that the
compound is
different from
0 0 0
41\1 c _____ 0 NHNH2*HC1 and NHNH2*CF3CO211 0 0
0
[0056] However, according to an embodiment the above three excluded
compounds are not to be excluded from the scope of the various uses and
methods as
previously described.
[0057] According to another embodiment, the above three excluded
compounds are to be excluded from the scope of the various uses and methods as
previously described.
[0058] In still a further embodiment of the present disclosure, there
are
included compounds of the following formulas:
CA 3021417 2018-10-18
27
0
0 0 o ci
---1( NHNH-- (
. . 0
I N 0 _____________________________ 0
---1 --AC NHNH¨ /
0 CI 0 0 __
4a 4b
O 0
,--A . 0 0
I N )1"-R
N li
----- NHN
O Ir-R NEINBoc
0
0 R 1:)
8 (R = CH3) 10 (R = CH3)
O 0
* 0 ---A
N = 0
I N Boc
----\ N¨N ---.1 NHNHBoc
O R-i =o 0
OR 12
11 (R = CH3)
0
0
H
,N..._"--s4N * HN-NH
Boc 0 'Floc
13
[0059] For example, the compound can be chosen from
0 0 0
0 yt... .7-14
I N Iii
N 4. 0 I N . 0 ,Boc
---- CH3
NHN
0 IrCH3 NHNBoc ----\ N¨N
0 /1 0 H3C-- 0
0 H3C .(1) 0 H3C
O 0
--14
I N li 0 I N . 0
----\ HN-NH N¨N11
O ,31 and 1) H3C--i 0
H3C 0 H3C
[0060] For example, the compounds can be chosen from
CA 3021417 2018-10-18
28
0 0 0
0
()tcH3 * 1 N * ,Boc
NHNIrCH3 NHNBoc ----\ N¨N
0 0 0 H3C-- 0
0 H3C 0 0 FI3C
0
----A 0
I N 11 0 ---A
---- 0
HN-NH I N *
0 0 ---1 N¨NH
H3C 0 H3C- =O
01I3C
0 0
---I ----k
I N 1, 0 I N * 0
---- FIN-N
HN- \ N ii and
---1 *
\
0 OH 0 OCH3
OCH3 OH
[0061] For example, the compounds can be chosen from
O o o
o 0
. Boc
. 0 yi...c
NHN H . 3 NHNBoc N¨N
O )r-CH3
0 0 I I3C- 0
0 H3C '-0 0 H3C
O 0
.
0
9
( II
NHNH-i< ( NHNH2*HC1
O _____________________________________ 0 0
O 0
0
HN-NH N¨NH
O o 0 H3C- 0
H3C 0 H3c
o 0
N. 0 0
1---k
I N .
HN-N and HN-N
O \ li OH 0 0C113
\ . '
OCH3 OH
CA 3021417 2018-10-18
29
[0062] For example, the compounds can be chosen from
O 0
0
ip 0
NHNH2*I ICI
O ______________________________________ 0 0
O 0 0
1-14N = 0 0
=
NHN
CFHI33 NHNBoc and
N _N Boc
O 0 0 H3C-
0 H3C 0113c
[0063] For example, RH can be substituted or unsubstituted member
chosen
from phenyl, furanyl and thiophenyl.
[0064] For example, at least one of R13, R14 and R15 can be different
from H.
[0065] For example, at least one of R13, R14 and R15 is -OH.
[0066] For example, at least one of R13, R14 and R15 is -OCH3.
[0067] For example, at least one of R13, R14 and Ris is C2-C8 alkyl.
[0068] For example, at least one of R13, Ri4 and R15 is -0R12 in
which R12 is
C2-Cs alkenyl.
[0069] For example, the compound is chosen from
0
0
0 OCH3
OH
0
0 and
HN __ N
0 OH
OCH,
CA 3021417 2018-10-18
30
[0070] For example, said compound is chosen from
0
0
N
HN __ N
0
0
0 HO
HN¨N
0
HO OCH,
0
0
HN __ N
0 OH
0
0
N
FIN N
o/
0
0
j(1 0
FIN¨N
0
CA 3021417 2018-10-18
31
100711 For example, RI I can be chosen from
2,4-dihydroxyphenyl 3,5-ditertbuty1-4- 4-methoxyphenyl
hydroxybenzaldehyde
2-hydroxy-3-methoxyphenyl 3,4-dimethoxyphenyl 4-allyloxyphenyl
2-furanyl 2-pyridyl 3-pyridyl
4-pyridyl phenylethenyl 4-chlorophenyl
4-fluorophenyl 4-trifluoromethylphenyl and .. 4-nitrophenyl.
[0072] Schemes 1 to 4 represent examples of synthetic routes used for
the
preparation of the compounds of the present disclosure. The reaction
conditions of
each step are presented directly in the schemes.
[0073] Using para-amino benzoic acid (1) as the starting material
derivative 4
was made in a three-step reaction sequence (Scheme 1).9-11 Para-amino benzoic
acid
(1 or PABA) was first reacted with maleic anhydride (MA) in dry acetone to
give the
diacid (2) with 90% yield. Cyclisation to form the maleimide was accomplished
with
acetic anhydride and sodium acetate to give compound 3 with 89% yield after
hydrolysis of the mixed anhydride intermediate with water. Finally, activation
of acid
3 with iso-butyl chloroformate in the presence of pyridine was done followed
by
treatment with tert-butyl carbazate gave the desired anti-inflammatory
derivative 4
with 54% yield. Deprotection of 4 with hydrochloric acid in ether gave the
hydrochloride salt 5 with 46% yield after recrystallization. Example 1 shows
the
preparation of compounds 4 and 5. This reaction sequence can be used to
produce
other derivatives starting from unsubstituted or substituted ortho-, meta- and
para-
benzoic acid starting materials. Hence, this approach lead to the synthesis of
N'13-
(2,5-dioxo-2,5-dihydro-pyrrol-1-y1)-4-chloro-benzoy11-hydrazine carboxylic
acid tent-
butyl ester (4a) and N'44-(2,5-dioxo-2,5-dihydro-pyrrol-1-y1)-2-chloro-
benzoyl]-
CA 3021417 2018-10-18
32
hydrazine carboxylic acid tert-butyl ester (4b) shown in examples 2 and 3,
respectively.
0 0
1-1,1\1 =
0
a
HN
OH --0O2H1IVI N 111 OH OH
0
1 2 (90%) 3 (88%)
0 0
<14 () N 0 IN
NHNH¨ ( NHNII)*IICI
0 0 ___________________ 0
4 (54%) 5 (46%)
Reagents and conditions: a) Maleic anhydride (MA), dry acetone, methanol, 22
C, 1 h; b) I) Ac,O.
AcONa, 50 C, 2 h; 2) H20, 70 C, 2 h; c) 1) iso-butyl chloroformate, Et3N,
CH2Cl2, 0 C, I h and 22
C, 1 h; 2) tert-butyl carbazate, CH2Cl2, 22 C, 12 h; d) HCI, dioxane, 22 C,
5 h.
Scheme 1
100741 Further transformations of derivative 4 can lead to novel
compounds
with anti-inflammatory, anti-metastatic, anti-proliferative and anticancer
activities
(Scheme 2). However, catalytic hydrogenation of 4 led to compound 12 which
lost its
anti-inflammatory activity (see example 4). Otherwise, compound 4 is
transformed
into its trifluoroacetate salt 6 upon treatment with trifluoroacetic acid in
dichloromethane. The crude material 6 can be reacted either with acetyl
chloride or
acetic anhydride (or any relevant anhydride or acid chloride) to give
derivative 8. Of
note, derivatives 7 and 9 were likely produced but not isolated in example 5.
Alternatively, compound 4 can be acylated with a relevant anhydride or acid
chloride
to yield compound 10 and 11. Interestingly, using acetyl chloride, compound 4
was
transformed efficienly into the diacetylated derivative 11 (R = CH3) and, with
acetic
anhydride the main product of the reaction was the monoacetylated derivative
10 (R =
CH3). Derivative 10 can be deprotected to give 7 and derivative 11 can lead to
derivative 9. It is possible to produce efficienly compound 7 and 9 via a two-
step
sequence from 4.
CA 3021417 2018-10-18
33
- o
o 11 NHNHBoc
0 H
NHNHBoc a [ N '11 b HN¨N
NHNH2*CF3CO2H 0
4 6 7
+
0 0
0 0
b N *
N XR
NHNHBoc li
= e*
NHNIT¨R
0
0
12 0
0 0 =c' 8 (R = CH3)
0 e
+ N * Boc 0
NHNBoc N¨N. a 0 0
N li .1c,
(from 11 to yield 9) NNH 1\ 0
10 (R = CH3) 11 (R = CH3) 9 OR
Reagents and conditions: a) TFA, CH2C12, 22 C, 0.5 h; b) Relevant anhydride
or acid chloride, Et0Ac, 22 C, 30 mm to
2 h; c) H2, Pd/C, CH3OH, 22 C, 3 h.
Scheme 2
[0075] Scheme 3 presents
the methodology leading to 13 the alkylhydrazones
or arylhydrazones derivatives following the procedure described by Taha et
al.12
Accordingly, compound 5 can be treated with a relevant aldehyde (alkyl
aldehydes
(linear or branched), benzaldehyde or substituted benzaldehydes or other
arylaldehydes) under acidic conditions at reflux in butanol (or other solvent)
to give
the desired derivatives of general structure 13.
0 0
0 0
N li a .---1(
1 NV
NHNH2 --,..-
----- HN¨N=\
0 0 R
13
R = alkyl or aromatic (unsubstituted or substituted)
Reagents and conditions: a) RCHO, H+, Butanol, reflux, 1 to 5 h.
Scheme 3
CA 3021417 2018-10-18
34
[0076] On Scheme 4, derivative 4 (or any other maleimides described
herein)
can be reacted with an appropriate diene (butadiene (unsubstituted or
substituted),
cyclopentadiene, cyclohexadiene cycloheptadiene, furane, thiophene, pyrrole, N-
alkylpyrrole) to give the desired cycloadducts (Diets-Alder products) such as
14, 15
and 16. This reaction can be performed by heating the pure reagents (diene and
dienophile) either neat or in solution, with or without pressure as it is
described in
example 10.
H
N
HN¨Q
H 0
0 14 H NH-Q
a
or or 41) N
NI-I-Q
0 H 0
4 H X=O,S,NHorNR
N
NH-Q 15
H 0
n = I, 2 or 3
16
Relevant diene = butadiene (unsubstituted or substituted), cyclopentadiene,
cyclohexadiene, furane, thiophene, pyrrole, N-alkyl
pyrrole.
Reagents and conditions: a) Relevant diene, toluene, A, 3 h.
Scheme 4
[0077] As it can be appreciated by the skilled artisan, the above
synthetic
schemes are not intended to be a comprehensive list of all means by which the
compounds described and claimed in this application may be synthesized.
Further
methods can also potentially be used to prepare the compounds of the present
disclosure.
[0078] The compounds of the present disclosure may be modified by
appending appropriate functionalities to enhance selective biological
properties. Such
modifications are known in the art and include those which increase biological
penetration into a given biological system (e.g., blood, lymphatic system,
central
nervous system), increase oral availability, increase solubility to allow
administration
by injection, alter metabolism and alter rate of excretion.
CA 3021417 2018-10-18
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[0079] The compounds of the present disclosure may contain one or
more
asymmetric carbon atoms and thus may occur as racemates and racemic mixtures,
single enantiomer, diastereomeric mixtures and individual diastereoisomers.
All such
isomeric forms of these compounds are expressly included in the present
disclosure.
Each stereogenic carbon may be of the R or S configuration.
[0080] It has been found that the small aminobenzoic acid derivatives herein
disclosed display anti-inflammatory, anti-metastatic, anti-proliferative
and/or anti-
cancer properties as well as favorable toxicity profiles, and may be used for
example
in treating malignancies, for example muscle-invasive and superficial UBC
tumors,
for example in humans.
[0081] An aspect herein provided is a method for treating cancer or
at least
one cancer chosen from melanoma, breast cancer, uterine cancer, ovarian
cancer,
prostate cancer and bladder cancer, the method comprising administering to a
subject
in need thereof an effective amount of at least one compound as defined
herein,
[0082] Another aspect is a method for reducing the risks of
developing cancer
or for reducing the risk of developing at least one cancer in a subject, the
cancer
being, for example, chosen from melanoma, breast cancer, uterine cancer,
ovarian
cancer, prostate cancer and bladder cancer, the method comprising
administering to
the subject an effective amount of at least one compound as defined herein.
[0083] Yet a further aspect is a method for inhibiting cancer cell
growth, the
method comprising administering to a subject in need thereof an effective
amount of
at least one compound as defined herein.
[0084] For example, the cancer cell growth is inhibited by at least
about 10%,
about 20%, about 30%, about 40%, about 50%, about 60%, about 70% or about 80%
relative to an untreated subject.
[0085] Another aspect is a method for inhibiting tumor growth and/or
reducing tumor size, the method comprising administering to a subject in need
thereof
an effective amount of at least one compound as defined herein.
[0086] For example the tumor size is measured in weight and/or
volume.
CA 3021417 2018-10-18
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[0087] For example, the tumor growth and/or tumor size is decreased
by at
least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about
70%, about 80% or about 90% relative to an untreated subject.
[0088] For example, administration of an effective amount of at least
one
compound disclosed herein to a subject in need thereof increases time to
progression
of at least one cancer chosen from melanoma, breast cancer, uterine cancer,
ovarian
cancer, prostate cancer and bladder cancer.
[0089] For example, the compound herein disclosed is for use in
reducing
risks of developing at least one cancer chosen from melanoma, breast cancer,
uterine
cancer, ovarian cancer, prostate cancer and bladder cancer.
[0090] For example, the compound is for use in the treatment of at
least one
cancer chosen from melanoma, breast cancer, uterine cancer, ovarian cancer
prostate
cancer and bladder cancer.
[0091] For example, the compound is for use as an anticancer agent.
[0092] For example the compound is for use as an anti-inflammatory
agent.
[0093] For example the compound is for use as an anti-metastatic
agent.
[0094] As described herein, several compounds, namely derivatives 4,
5, 8,
10, 11, and 12, were tested on macrophages and UBC cells for their anti-
inflammatory, anti-metastatic and/or anti-cancer properties.
[0095] As shown herein, the compounds presently disclosed display
anti-
inflammatory properties.
[0096] Referring to Fig. 1 to 10, in vitro studies of human
macrophages and
UBC cells tested with compounds 4 and 5 were conducted to investigate the anti-
inflammatory properties of the compounds on pro-inflammatory cytokines. As
shown
in Fig. 1, IFNy-induced STAT1 activation and IL6-induced STAT3 activation were
decreased in pretreated human macrophages. Similarly, compounds 4 and 5 were
found effective in reducing IFNy-induced CD40 and MHC-II expressions, as shown
in
Fig. 2. Also, referring to Fig. 3, scratch wound healing assays were conducted
and
compounds 4 and 5 were found effective in inhibiting IFNy-induced STAT1
activation and IL6-induced STAT3 activation in human macrophages.
CA 3021417 2018-10-18
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[0097] An aspect provided is a method for decreasing anti-
inflammatory
properties of cancer cells, the method comprising contacting the cancer cells
with at
least one compound as defined herein.
[0098] For example the relative STAT1 activation of cells treated
with a
compound herein disclosed is decreased by at least about 10%, about 20%, about
30%, about 40%, about 50%, about 60%, about 70% or about 80% relative to
untreated cells.
[0099] For example the relative STAT3 activation of cells treated
with a
compound herein disclosed is decreased by at least about 10%, about 20%, about
30%, about 40%, about 50%, about 60%, about 70% or about 80% relative to
untreated cells.
[00100] For example the CD40 expression of cells treated with a
compound
herein disclosed is decreased by at least about 10%, about 20%, about 30%,
about
40%, about 50%, about 60%, about 70% or about 80% relative to untreated cells.
[00101] For example the MHC-II expression of cells treated with a
compound
herein disclosed is decreased by at least about 10%, about 20%, about 30%,
about
40%, about 50%, about 60%, about 70% or about 80% relative to untreated cells.
[00102] Referring now to Fig. 4 and 5, anti-inflammatory activity of
compounds 4 and 5 was assessed by measuring nitric oxide (NO) production in
murine macrophage-like cells stimulated with inflammatory signals, IFNy/TNFa.
Compounds 4 and 5 were found effective in reducing IFNy/TNFoc induced NO
production.
[00103] For example, NO production in cells treated with a compound
herein
disclosed is decreased by at least about 10%, about 20%, about 30%, about 40%,
about 50%, about 60%, about 70% or about 80% relative to untreated cells.
[00104] Referring to Fig. 6 and 7 it was demonstrated that compounds 4
and 5
have anti-proliferative properties in UBC cells stimulated with IFNy/TNFoc.
CA 3021417 2018-10-18
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[00105] An aspect provided is a method for inducing anti-proliferative
properties of cancer cells, the method comprising contacting the cancer cells
with at
least one compound as defined herein.
[00106] For example, the viability of cells treated with a compound
herein
disclosed is decreased by at least about 10%, about 20%, about 30%, about 40%,
about 50%, about 60%, about 70% or about 80% relative to untreated cells.
[00107] For example, the cancer cell growth is inhibited by at least
about 10%,
about 20%, about 30%, about 40%, about 50%, about 60%, about 70% or about 80%
relative to untreated cancer cells.
[00108] Referring now to Fig. 8, compound 4 was shown to inhibit
TNFa/NFKB and IL6/STAT3 signaling pathways in murine UBC cells. Compound 4
was also tested in human UBC cells and was shown to inhibit 1L6-induced
motility, as
shown in Fig. 9. Similarly, referring to Fig. 10, a human breast cancer cell
line was
treated with compound 4 which demonstrated anti-invasion properties, thus
demonstrating that compound 4 is not cell-specific.
[00109] For example, the TNFa/NFKB and/or IL6/STAT3 signaling pathways
of cells treated with a compound herein disclosed is decreased by at least
about 10%,
about 20%, about 30%, about 40%, about 50%, about 60%, about 70% or about 80%
relative to untreated cells.
[00110] For example, the invasiveness of cells with a compound herein
disclosed is decreased by at least about 10%, about 20%, about 30%, about 40%,
about 50%, about 60%, about 70% or about 80% relative to untreated cells.
[00111] In vivo studies were also conducted to evaluate the
anticancer, anti-
proliferative and anti-metastatic activities of compound 4 in ectopic and
orthotopic
murine models of UBC.
[00112] Acute toxicity studies in mice, as shown in Fig. 11,
demonstrated that
treatment with compound 4 had no obvious effect on normal development and
viability, as well as in organ weight and hematocrit, suggesting tolerability
in the
mice. Similarly, referring to Fig. 12, no evidence of toxicity related to
compound 4
CA 3021417 2018-10-18
39
was found in liver histological analyses in mice treated with various
concentrations of
compound 4.
[00113] For example, subjects treated with a compound disclosed herein
have a
similar body weight relative to a comparable untreated subject. "Similar body
weight"
as used herein means no greater than a 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or
1% difference in body weight loss.
[00114] For example, subjects treated with a compound disclosed herein
have a
similar hematocrit relative to a comparable untreated subject. "Similar
hematocrit" as
used herein means no greater than a 5%, 4%, 3%, 2% or 1% difference in
hematocrit
level.
[00115] As demonstrated herein, the compounds herein disclosed display
anti-
proliferative and anti-metastatic activity.
[00116] Referring to Fig. 13 and 14, male mice implanted ectopically
with
UBC tumors and treated with compound 4 saw a significant decrease in tumor
proliferation and in the number of metastases relative to untreated mice. In
female
mice bearing orthotopically implanted urothelial bladder cancer tumors, as
shown in
Fig. 15, anti-proliferative properties of compound 4 were also seen. More
particularly,
the tumor development was stopped after two weeks of treatment. Similarly,
mice
ectopically implanted with urothelial bladder cancer tumors and treated with
compound 4 saw a significant reduction of the tumor size, as illustrated in
Fig. 18.
[00117] For example, tumors treated with a compound disclosed herein
have a
decrease in volume and/or a decrease in tumor growth of at least about 10%,
about
20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80% or about
90% relative to untreated tumors.
[00118] Another aspect is a method for decreasing and/or preventing
tumor
metastases, the method comprising administering to a subject in need thereof
an
effective amount of at least one compound disclosed herein.
CA 3021417 2018-10-18
40
[00119] For example, the number of tumor metastases is decreased by at
least
about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,
about 80% or about 90% relative to an untreated subject.
[00120] Referring to Fig. 19, further in vivo murine studies of anti-
inflammatory properties of compound 4 were conducted and a significant
decrease in
NO production and urea synthesis was found in peritoneal macrophages.
[00121] Derivatives of compound 4 were also found to display anti-
inflammatory, anticancer, anti-proliferative and anti-metastatic properties.
Additional
in vitro and in vivo studies of compound 4 derivatives, compounds 8, 10 and 11
were
conducted in macrophages and UBC cells.
[00122] Fig. 21 shows that these compounds were effective in inhibiting
NO
production. The compounds were also effective in inhibiting the IFN7-activated
STAT1 signaling pathway and the LPS-activated NFK13 signaling pathway in
macrophage cells (Fig. 22 and 23).
[00123] For example, the LPS-activated NFic13 signaling pathway of
cells
treated with a compound herein disclosed is decreased by at least about 10%,
about
20%, about 30%, about 40%, about 50%, about 60%, about 70% or about 80%
relative to untreated cells.
[00124] Referring now to Fig. 25, compounds 4 and 8 were found to have
anti-
proliferative activity in ectopically implanted UBC tumors in male mice.
[00125] For example, the compound is compound 4.
[00126] For example, the compound is compound 8.
[00127] For example, the compound is compound 10.
[00128] For example, the compound is compound 11.
[00129] For example, the subject is a mammal. For example, the subject
is a
human.
[00130] For example, the cell is in vitro.
CA 3021417 2018-10-18
41
[00131] For example, the cell is in vivo.
[00132] For example, the cell is a cancer cell.
[00133] For example, the cancer is melanoma, uterine cancer, ovarian
cancer,
prostate cancer or bladder cancer.
[00134] For example, the bladder cancer is superficial UBC or muscle
invasive
UBC.
[00135] For example the tumor is a melanoma tumor, uterine tumor,
ovarian
tumor, prostate tumor or bladder tumor.
[00136] For example, the bladder tumor is a superficial UBC tumor or a
muscle
invasive UBC tumor.
[00137] For example, the methods disclosed herein are effective for
presenting
the development of superficial UBC into muscle invasive UBC tumor.
[00138] For example, the at least one compound as defined in the
present
disclosure can be used in the manufacture of a medicament for decreasing
and/or
preventing pathological humoral and cell-mediated inflammation of at least one
disease chosen from inflammatory diseases (Crohn's disease, rheumatoid
arthritis,
asthma, coeliac disease, glomerulonephritis, hepatitis, inflammatory bowel
disease,
reperfusion injury or transplant rejection); pregnancy complications
(preeclampsia or
preterm birth); or gynecological pathologies (endometriosis, uterine
leiomyoma,
polycystic ovary syndrome or recurrent miscarriage).
[00139] In the present disclosure, the following abbreviations are
used:
Abbreviation Meaning
Ac20 Acetic anhydride
AcONa Sodium acetate
CH2Cl2 Dichloromethane
Boc t-Butyloxycarbonyl
Et20 Diethyl ether
Et0Ac Ethyl acetate
Et3N Triethyl amine
Hour
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42
MCI Chlorhydric acid
i.p. Intraperitoneal
m Meta
MA Maleic anhydride
Me0H Methanol
min Minute
mmol Millimole
MTT 3-(4,5-Dimethylthiazol-2-y1)-2,5-
diphenyltetrazolium
bromide
NMR Nuclear magnetic resonance
o Ortho
p Para
Phe Phenyl
s.c. Subcutaneously
TLC Thin layer chromatography
TFA Trifluoroacetic acid
UBC Urothelial bladder cancer
EXAMPLES
[00140] This section also describes the synthesis of several compounds
that are
presented in this document. These examples are not to be construed as limiting
the
scope of the present disclosure in any way.
Materials and Methods - Chemistry
[00141] Anhydrous reactions were performed under an inert atmosphere,
the
set-up assembled and cooled under dry nitrogen. Unless otherwise noted,
starting
material, reactant and solvents were obtained commercially and were used as
such or
purified and dried by standard means.13 Organic solutions were dried over
magnesium
sulfate, evaporated on a rotatory evaporator and under reduced pressure. All
reactions
were monitored by UV fluorescence, or staining with iodine. Commercial TLC
plates
were Sigma T 6145 (polyester silica gel 60 A, 0.25mm). Flash column
CA 3021417 2018-10-18
43
chromatography was performed according to the method of Still and co-workers
on
Merck grade 60 silica gel, 230-400 mesh.' All solvents used in chromatography
had
been distilled prior to use.
[00142] The infrared spectra were taken on a Nicolet Impact 420 FT-IR.
Mass
spectral assays were obtained using a MS model 6210, Agilent technology
instrument.
The high resolution mass spectra (HRMS) were obtained by TOF (time of flight)
using ESI (electrospray ionization) using the positive mode (ES1+).
(Plateforme
analytique pour molecules organiques de FUniversite du Quebec a Montreal).
[00143] Nuclear magnetic resonance (NMR) spectra were recorded on a
Varian
200 MHz NMR apparatus. Samples were dissolved in deuterochloroform (CDC13),
deuteroacetone (acetone-d6) or deuterodimethylsulfoxide (DMSO-d6) for data
acquisition using tetramethylsilane or chloroform as internal standard (TMS, 8
0.0
ppm for 1H-NMR and CDC13 877.0 ppm for 13C-NMR). Chemical shifts (8) are
expressed in parts per million (ppm), the coupling constants (J) are expressed
in hertz
(Hz). Multiplicities are described by the following abbreviations: s for
singlet, d for
doublet, dd for doublet of doublets, t for triplet, dt for doublet of
triplets, q for quartet,
dq for doublet of quartets, m for multiplet, #m for several multiplets, br for
a broad
signal.
[00144] The following compounds were prepared from a relevant
aminobenzoic acid derivative using the procedures summarized in schemes 1, 2,
3 or
4.
Example 1. Preparation of N'44-(2,5-dioxo-2,5-dihydro-pyrrol-1-y1)-benzoyll-
hydrazine carboxylic acid tert-butyl ester (4), its hydrochloric acid
salt (5) and its trilluoroacetic acid salt (6)
Step A. Synthesis of 4-(3-carboxy-acryloylamino)-benzoic acid (2)
[00145] 4-Aminobenzoic acid (1, 5.34 g, 38.93 mmol) was dissolved in
dry
acetone (12 mL) to which was added methanol (1 mL). Maleic anhydride (1.05
eq.)
dissolved in dry acetone was added to the first solution. The reaction mixture
was
stirred for a period of 2 h allowing sufficient time for the complete
precipitation of the
CA 3021417 2018-10-18
44
diacid 2. The precipitate was filtered and washed twice with acetone (2 x 2
mL) and
dried in a desiccator overnight. The crude diacid 2 (9.16 g, 90%) was
sufficiently pure
to be use without further purification at the next step. IR (v, cm-1): 3500-
2500
(CO2H), 1686 cm-1 (C=0); 11-1 NMR (DMSO-d6, (5 ppm): 12.79 (br s, 2 H, 2 x
CO2H), 10.58 (s, 1H, NH), 7.89 and 7.71 (2 x d, J=8.6 Hz, 4H, aromatic), 6.48
and
6.30 (2 x d, J=12.2 Hz, 2H, maleimide); 13C NMR (DMSO-d6, (5 ppm): 167.4,
167.3,
164.1, 143.2, 132.1, 130.9 (2), 130.6, 126.0, 119.2 (2); ESI+ HRMS: (M+H)+
calculated for CI iHioN05 = 236.0553; found = 236.0558.
Step B. Synthesis of 4-(2,5-dioxo-2,5-dihydro-pyrrol-1-y1)-benzoic
acid (3)
[00146] The diacid 2 (2.01 g, 8.54 mmol) was treated with acetic
anhydride
(4.0 mL, 36.28 mmol) and anhydrous sodium acetate (350 mg, 4.27 mmol) and the
mixture heated at 50 C for 2 h. Afterwards, the solution was evaporated to
dryness
and stirred with water at 70 C for a period of 2 h. The resulting precipitate
was
filtered and dried in a desiccator overnight to yield 1.65 g (89%) of
maleimide 4. The
spectral data of this derivative correspond to those reported in the
literature.9,11 IR (v,
cm-1): 3475-2600 (CO2H), 1715 (C=0), 1704 (C=0); NMR
(acetone-d6, 6 ppm):
8.14 and 7.57 (2 x d, J=8.6 Hz, 4H, aromatic), 7.08 (s, 2H, maleimide); BC NMR
(acetone-d6, 6 ppm): 169.3 (2), 166.2, 136.2, 134.7 (2), 130.1 (2), 129.3,
125.8 (2);
ESI+ HRMS: (M+H)+ calculated for CliF18N04 = 218.0448; found = 218.0447.
Step C. Synthesis of N44-(2,5-dioxo-2,5-dihydro-pyrrol-1-y1)-benzoy1]-
hydrazine carboxylic acid tert-butyl ester (4)
[00147] Derivative 4 was synthesized using a modified procedure
reported by
Willner et al.15 as it is also described by Lau et al. and Hamelin-Morrissette
et al.9-11
A cooled suspension (0 C) of molecule 3 (211 mg, 0.97 mmol) in methylene
chloride
(4.5 mL) was treated with triethylamine (190 ?AL, 1.36 mmol) and isobutyl
chloroformate (175 !IL, 1.34 mmol). The mixture was stirrred for I h at 0 C
and at
room temperature (22 C) for about 1 h. Afterwards, tert-butyl carbazate (128
mg,
CA 3021417 2018-10-18
45
0.97 mmol) dissolved in methylene chloride (0.8 mL) was added dropwise to the
mixture and stirred for an additional 12 h at 22 C. The reaction mixture was
diluted
with ethyl acetate (55 mL) and methylene chloride (20 mL) and washed twice
with
saturated NaHCO3 (2 x 50 mL), twice with 0.1 N HCI (2 x 50 mL), twice with
saturated NaC1 (2 x 50 mL), and finally with H20 (50 mL). The organic phase
was
dried (MgSO4) and evaporated to give crude derivative 4. The product was
purified by
flash chromatography, using a mixture of hexanes / acetone (3/2), to yield 173
mg
(54%) of 4. The spectral data of this derivative correspond to those reported
in the
literature.10 IR (v, cm-1): 3360-3240 (NH), 3087 (C=C), 2988 (CH, aliphatic),
1733
(C=0), 1706 (C=0); 1H NMR (acetone-d6, 6 ppm): 9.05 (s, 1H, NH), 8.02 and 7.53
(2
x d, J=8.6 Hz, 4H, aromatic), 7.07 (s, 2H, maleimide), 2.84 (br s, 1H, NH),
1.45 (s,
9H, 3 x CH3); 13C NMR (acetone-d6, 6 ppm): 169.3 (2), 166.0, 155.7, 135.1,
134.6
(2), 131.6, 127.9 (2), 125.9 (2), 79.6, 27.5 (3); ES1+ HRMS: (M+Na)+
calculated for
Ci6H17N3Na05 = 354.1060; found = 354.1072; (M -2-methylpropene +H)+ calculated
for Ci2Hi1N305 = 276.0620; found = 276.0627.
Step D. Synthesis of 4-(2,5-dioxo-2,5-dihydro-pyrrol-1-y1)-benzoic
acid
hydrazide hydrochloride (or 4-maleimidbenzoic acid hydrazide hydrochloride)
(5)
1001481 The hydrolysis of 4 was performed using a similar procedure
reported
by Heindel et al. for the cleavage of maleimidoacetic acid (tert-
butyloxycarbonyl)
hydrazide with hydrochloric acid to form maleimidoacetic acid hydrazide
hydrochloride.16 To a solution of 4 (2.41 g, 7.27 mmol) dissolved in dry
dioxane (30
mL) was added a solution of hydrochloric acid (60 mL, 1.0 M in diethyl ether,
60
mmol). The mixture was stirred at room temperature for a period of 5 hours.
Afterwards, 150 mL of hexanes were added to complete the precipitation of the
hydrochloride salt 5. The crude precipitated was filtered, washed with hexanes
and
recrystallized twice with a mixture of methanol / isopropyl alcohol / hexanes
(8 / 3 /
10) to yield 1.7 g (46%) of the desired material. IR (v, cm-1): 3200-2500
(CO2H),
3269 (NH), 1702 (C=0), 1693 (C=0); 11-1 NMR (DMSO-d6, 6 ppm): 8.06 and 7.52
(2 x d, J=8.8 Hz, 4H, aromatic), 7.21 (s, 2H, maleimide); 13C NMR (DMSO-d6,
CA 3021417 2018-10-18
46
6 ppm): 170.0 (2), 165.6, 135.9, 135.4 (2), 129.6, 129.0 (2), 126.8 (2); ESI+
HRMS:
(M+H)+ calculated for CI iHioN303 = 232.0717; found = 232.0717 and ESI+ HRMS:
(M+H)+ calculated for C11FIIICIN303 = 268.0483; found = 268.0483.
Step E. Synthesis of 4-(2,5-dioxo-2,5-dihydro-pyrrol-1-y1)-benzoic
acid
hydrazide trifluoroacetic acid salt (6)
[00149] A solution of 4 (106 mg, 0.32 mmol) dissolved in
trifluoroacetic acid
(0.5 mL) was stirred at 0 C for a period of 30 minutes. Afterwards, the
excess
trifluoroacetic acid was removed under vacum at 22 C to give compound 6
quantitatively. IR (v, cm-1): 3500-2500 (CO2H), 3277 (NH), 1710 (C=0); 1H NMR
(DMSO-d6, 6 ppm): 11.62 (br s, 1H, NHNH3+CF3CO2-), 8.63 (br s, NH3), 8.01 and
7.56 (2 x d, J=8.5 Hz, 4H, aromatic), 7.24 (s, 2H, maleimide); 13C NMR (DMSO-
d6,
6 ppm): 169.6 (2), 165.4, 135.5, 135.0 (2), 129.4, 128.4 (2), 126.5 (2).
Example 2. Preparation of N'43-(2,5-dioxo-2,5-dihydro-pyrrol-1-y1)-4-chloro-
benzoyll-hydrazine carboxylic acid tert-butyl ester (4a)
Following the procedure of Example 1, steps A ¨ C described above using 3-
amino-4-
chloro benzoic acid as the starting material instead of 4-amino benzoic acid
derivative
4a was prepared efficiently.
Step A. Synthesis of 3-(cis-3-carboxy-acryloylamino)-4-chloro benzoic
acid
(2a)
[00150] Spectral data for 2a: IR (v, cm-1): 3500-2500 (CO2H), 3310
(NH),
1696 (C=0); 1H NMR (DMSO-d6, 6 ppm): 13.08 (s, 2H, 2 x CO2H), 10.13 (s, 1H,
NH), 8.37 (s, 1H, aromatic) 7.72 and 7.61 (2 x d, 2H, J = 10.0 Hz, aromatic),
6.59 and
6.34 (2 x d, 2H, 1 = 12.0 Hz, maleimide); 13C MNR (DMSO-d6, 6 ppm): 167.5,
166.7, 164.1, 135.0, 131.6, 131.2 (2), 130.4 (2), 127.2 (2).
CA 3021417 2018-10-18
47
Step B. Synthesis of 3-(2,5-dioxo-2,5-dihydro-pyrrol-1-y1)-4-chloro
benzoic
acid (3a)
[00151] Spectral data for 3a: IR (v, cm-1): 3300-2500 (CO2H), 3490
(amine),
1674 (C=0). 1H NMR (acetone-d6, 6 ppm): 8.1 (m, 2H, aromatic), 7.76 (d, 1H, J
=
8.0 Hz, aromatic), 7.14 (s, 2H, maleimide) 13C NMR (acetone-d6, 6 ppm): 168.7
(2),
165.0, 143.0, 137.84, 134.9 (2), 132.3, 131.6, 130.6, 130.2.
Step C. Synthesis of N'-[3-(2,5-dioxo-2,5-dihydro-pyrrol-1-y1)-4-
chloro-
benzoy1]-hydrazine carboxylic acid tert-butyl ester (4a)
[00152] Spectral data for 4a: IR (v, cm-1): 3494 (amine), 3090 (C=C),
2974
(aliphatic), 1717 (C=0). 111 NMR (acetone-d6, 6 ppm): 9.71 (s, 1H, NH), 8.05
(dd,
I H, J = 8.2 Hz and J = 1.8 Hz, aromatic) 7.95 (d, 1H, J = 1.8 Hz, aromatic),
7.75 (d,
1H, J = 8.6 Hz, aromatic), 7.11 (s, 2H, maleimide), 2.91 (s, 1H, NH), 1.43 (s,
9H, 3 x
CH3); 13C NMR (acetone-d6, 6 ppm): 169.5 (2), 166.0, 156.5, 135.0 (2), 134.0
(2),
130.0, 130.7, 129.0 (2), 80.0, 28.1(3).
Example 3. Preparation of N'44-(2,5-dioxo-2,5-dihydro-pyrrol-1-y1)-2-chloro-
benzoy1]-hydrazine carboxylic acid tert-butyl ester (4b)
Following the procedure of Example 1, steps A ¨ C described above using 4-
amino-2-
chloro benzoic acid as the starting material instead of 4-amino benzoic acid
derivative
4b was prepared efficiently.
Step A. Synthesis of 4-(cis-3-carboxy-acryloylamino)-2-chloro benzoic
acid
(2b)
CA 3021417 2018-10-18
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[00153] Spectral
data for 2b : IR (v, cm-'): 3500-2500 (CO2H), 3262 (NH),
1689 (C=0); 'H NMR (DMSO-d6, 6 ppm): 13.8 (s, 2H, 2 x CO2H), 10.7 (s, 1H, NH),
7.85 (m, 2H, aromatic) 7.55 (dd, 1H, J = 10.0 Hz and J = 2.0 Hz, aromatic),
6.45 and
6.31 (2 x d, 2H, J = 12.0 Hz, maleimide); 13C MNR (DMSO-d6, 6 ppm): 168.0,
166.0, 164.0, 143.0, 136.5, 135.0, 132.0, 131.5, 126.0, 121.5, 119.2.
Step B.
Synthesis of 3-(2,5-dioxo-2,5-dihydro-pyrrol-1-y1)-4-chloro benzoic
acid (3b)
[00154] Spectral
data for 3b : IR (v, cm-'): 3300-2500 (CO2H), 3470 (amine),
1723 (C=0). 11-1 NMR (acetone-d6, 6 ppm): 7.90 (d, 1H, J = 8.6 Hz, aromatic),
7,67
(d, 1H, J = 2.2 Hz, aromatic), 7.50 (dd, 1H, J = 8.3 Hz and J = 2.0 Hz,
aromatic) 7.11
(s, 2H, maleimide); '3C NMR (acetone-d6, 6 ppm): 168.0 (2), 165.0, 138.0,
136.0 (2),
135.0, 132.0, 128.0, 124.0, 118Ø
Step C.
Synthesis of N43-(2,5-dioxo-2,5-dihydro-pyrrol-1-y1)-4-chloro-
benzoyll-hydrazine carboxylic acid tert-butyl ester (4b)
[00155] Spectral
data for 4b : IR (v, cm-'): 3473 (amine), 3090 (C=C), 2984
(aliphatic), 1708 (C=0). 'H NMR (acetone-d6, 6 ppm): 9.03 (s, 1H, NH), 7.65(d,
1H,
J = 8.0 Hz, aromatic) 7.59 (d, 1H, J = 1.6 Hz, aromatic), 7.45 (dd, 1H, J =
8.2 and J =
1.8 Hz aromatic), 7.08 (s, 2H, maleimide), 2.96 (s, 1H, NH), 1.47 (s. 9H, 3 x
CH3);
13C NMR (acetone-d6, 6 ppm): 169.0 (2), 166.0, 156.0, 135.0 (2), 134.6, 133.0,
131.0,
127.9(2), 125.9, 80.0, 27.5 (3).
Example 4. Preparation of N44-(2,5-
dioxo-pyrrolidin-1-y1)-benzoyll-
hydrazine carboxylic acid tert-butyl ester (12)
[00156]
Maleimide 4 (103 mg, 0.31 mmol) was dissolved in methanol (1 mL)
to which was added 5% Pd/C (14 mg). Some hydrogen gas was bubbled during 30
CA 3021417 2018-10-18
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seconds into the mixture. The suspension was stirred vigorously under a
hydrogen
atmosphere for a period of 3 hours. Of note, a longer period of time is
required on a
larger scale. Afterwards, the suspension was filtered on silica with a mixture
of
hexanes / acetone (3/2) as the eluent, to yield 87 mg (84%) of 12. IR (v, cm-
1): 3400-
3100 (NH), 2981 (CH), 1703 (C=0); 111 NMR (acetone-d6, 6 ppm) : 9.54 (br s,
1H,
NH), 8.00 and 7.45 (2 x d, J=8.6 Hz, 4H, aromatic), 1.45 (s, 9H, 3 x CH3); 13C
MNR
(acetone-d6, 6 ppm) 176.2 (2), 165.9, 155.7, 136.1, 132.4, 127.8 (2), 126.7
(2), 79.6,
28.2, 27.5; ESI+ HRMS: (M+H)+ calculated for Ci6H20N305 = 334.1397; found =
334.1391 and ESI+ HRMS: (M+H -C4H9)+ calculated for Ci2H:2N305 = 278.0771;
found = 278.0769.
Example 5. Preparation of 4-(2,5-dioxo-2,5-dihydro-pyrrol-1-y1)-benzoic acid
N'-diacetyl-hydrazide (8, R = CH3)
1001571 To a
solution of crude 6 (444 mg, 1.28 mmol) dissolved in
dichloromethane (10 mL) was added triethylamine (1.07 mL, 780 mg, 7.7 mmol)
and
acetic anhydride (0.61 mL, 659 mg, 6.4 mmol). The mixtured was stirred at 22
C for
about 30 minutes. Afterwards, the organic phase was diluted with ethyl acetate
(75
mL) directly into an extraction funnel. The organic phase was washed
successively
with a 5% sodium bicarbonate aqueous solution (50 mL), with a 10% sodium
chloride
aoous solution (50 mL) and finnally with water (50 mL). The organic phase was
dried
with anhydrous magnesium sulfate, filtered and evaporated to the crude
material (111
mg). The product was purified by flash column chromatography using a mixture
of
hexanes / acetone (7/3) to give 75 mg (18%) of the desired material 8, R =
CH3). Of
note, different anhydrides or alkaloyl chlorides can be used to produce
analogs of this
specific derivative. IR (v, cm-1): 3200 (NH), 1702 (C=0), 1662 (C=0); 'H NMR
(acetone-d6, 6 ppm): 10.15 (br s, 1H, NH), 8.09 and 7.61 (2 x d, J = 8.6 Hz,
4H,
aromatic), 7.10 (s, 2H, maleimide), 2.41 (s, 6H, 2 x CH3); 13C NMR (acetone-
d6,
6 ppm): 171.1 (2), 169.3 (2), 166.0, 135.8, 134.7 (2), 130.8, 128.2 (2), 126.1
(2), 24.2
(2). ESI+ HRMS: (M+H)+ calculated for C151-114N305 = 316.0928; found =
316.0945
and ESI+ HRMS: (M+H -Ac)+ calculated for Ci3Hi2N304 =275.08252; found =
275.0856.
CA 3021417 2018-10-18
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Example 6. Preparation of N-acetyl-AP-14-(2,5-dioxo-2,5-dihydro-pyrrol-1-y1)-
benzoy11-hydrazinecarboxylic acid tert-butyl ester (10, R = CH3)
[00158] To a solution of 4 (100 mg, 0.30 mmol) dissolved in
dichloromethane
(3 mL) was added triethylamine (252 p.L, 183 mg, 1.81 mmol) and acetic
anhydride
(71 mt, 76.7 mg, 0.75mmo1). The mixtured was stirred at 22 C for about 30
minutes.
Afterwards, the organic phase was diluted with ethyl acetate (25 mL) directly
into an
extraction funnel. The organic phase was washed successively with a 5% sodium
bicarbonate aqueous solution (10 mL), with a 10% sodium chloride aoous
solution (10
mL) and finnally with water (20 mL). The organic phase was dried with
anhydrous
magnesium sulfate, filtered and evaporated to the crude material (123 mg). The
product was purified by flash column chromatography using a mixture of hexanes
/
acetone (4/1) to give 22 mg (20%) of the desired material 10, R = CH3). It is
noteworthy, that a longer reaction time (2 hours) lead to higher yield of the
desired
material. However, some diacetylated product (11) is also present as a side
product.
IR (v, cm-1): 3286 (NH), 1753 (C=0), 1711 (C=0), 1652 (C=0); 1H NMR (CDCI3,
6 ppm): 8.28 (br s, 1H, NH), 7.90 and 7.50(2 x d, J = 8.6 Hz, 4H, aromatic),
6.89 (s,
2H, maleimide), 2.61 (s, 3H, CH3), 1.51 (s, 9H, 3 x CH3); 13C NMR (CDCI3, 6
ppm):
170.5, 168.9 (2), 165.2, 151.0, 135.0, 134.4 (2), 130.9, 128.3 (2), 125.7 (2),
84.8, 27.8
(3), 25.5; ESI+ HRMS: (M+Na)+ calculated for C181-119N3Na06 = 396.1166; found
=
396.1165 and ESI+ HRMS: (M+H -tert-Boc)+ calculated for C13F112N304 =
274.0822;
found = 274.0824.
Example 7. Preparation of N,N'-diacetyl-N'44-(2,5-dioxo-2,5-dihydro-pyrrol-
1-y1)-benzoy1]-hydrazinecarboxylic acid tert-butyl ester (11, R =
CH3)
[00159] To a solution of 4 (100 mg, 0.30 mmol) dissolved in
dichloromethane
(3 mL) was added triethylamine (252 JAL, 183 mg, 1.81 mmol) and acetyl
chloride (54
60 mg, 0.75 mmol). The mixtured was stirred at 22 C for about 2 hours.
CA 3021417 2018-10-18
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Afterwards, the organic phase was diluted with ethyl acetate (25 mL) directly
into an
extraction funnel. The organic phase was washed successively with a 5% sodium
bicarbonate aqueous solution (10 mL), with a 10% sodium chloride aoous
solution (10
mL) and finally with water (20 mL). The organic phase was dried with anhydrous
magnesium sulfate, filtered and evaporated to the crude material (118 mg). The
product was purified by flash column chromatography using a mixture of hexanes
/
acetone (4/1) to give 100 mg (80%) of the desired material 11, R = CH3). IR
(v, cm-1):
3193 (NH), 1701 (C=0), 1660 (C=0); 1H NMR (CDCI3, 6 ppm): 7.68 and 7.50(2 x d,
J = 8.6 Hz, 4H, aromatic), 6.89 (s, 2H, maleimide), 2.49 and 2.48 (2 x s, 6H,
2 x CH3),
1.50 (s, 9H, 3 x CH3); 13C NMR (CDCI3, 6 ppm): 170.2, 169.9 (2), 168.8 (2),
150.2,
134.6, 134.4 (2), 132.9, 128.2 (2), 125.0 (2), 85.9, 27.8 (3), 25.4, 24.6;
ESI+ HRMS:
(M+Na)+ calculated for C201-121N3Na07 = 438.1272; found = 438.1281 and ESI+
HRMS: (M+H -Ac and -tert-Boc)+ calculated for C13H12N304 = 274.0822; found =
274.0833.
Example 8. Preparation of N'-{4-13-
(2-tert-butoxycarbonylamino-
ethylsulfany1)-2,5-dioxo-pyrrolidin-1-y11-benzoyl}-
hydrazinecarboxylic acid tert-butyl ester (13)
[00160] To a
solution of 4 (115 mg, 0.34 mmol) dissolved in methanol (3 mL)
was added 2-(Boc-amino) ethanethiol (70 L, 73 mg, 0.41 mmol). The mixture was
stirred at 22 C for 2 hours and at 50 C for I hour. The organic phase was
diluted with
ethyl acetate (30 mL) directly into an extraction funnel and washed
successively with
a 5% sodium bicarbonate aqueous solution (2 x 10 mL) and with water (2 x 20
mL).
The organic phase was dried with anhydrous magnesium sulfate, filtered and
evaporated to the crude material (191 mg). The product was purified by flash
column
chromatography using a mixture of hexanes / acetone (3/2) to give 107 mg (61%)
of
the desired material 13 [see 0055]. IR (v, cm-1): 3300 (NH), 1707 (C=0), 1680
(C=0); 1H NMR (CDCI3, 6 ppm): 8.80 (br s, IH, NH), 7.86 and 7.34 (2 x d, J =
8.2
Hz, 4H, aromatic), 6.90 (br s, 1H, NH), 5.07 (br s, 1H, NH), 3.98 (1H, m, -CHS-
) 2.6-
3.6 (several m, 6H, 3 x -CH2-), 1.50 and 1.45 (2 x s, 18H, 2 x 3 x CH3); 13C
NMR
(CDC13, 6 ppm): 175.4, 173.2, 165.8, 155.9 (2), 134.8, 131.7, 128.2 (2), 126.4
(2),
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82.1, 79.7, 39.4, 38.9, 36.1, 32.7, 28.4 (3), 28.2 (3); ESI+ FIRMS: (M+Na)+
calculated for C23H321\14Na07S = 531.1884; found = 531.1881.
Example 9. General procedure for the preparation of derivatives of general
structure 13 (see scheme 3)
[00161] Following the procedure described by Taha et a/.12 compound 5
can be
treated with a relevant aldehyde (alkyl aldehydes (linear or branched),
benzaldehyde
or substituted benzaldehydes) under acidic conditions at reflux in butanol (or
other
solvent) to give the desired alkylhydrazones or benzoylhydrazones derivatives.
Example 10. General procedure for the preparation of derivatives 14, 15 or 16
(see scheme 4)
[00162] Derivative 4 (or any other maleimides described herein) can be
reacted
with an appropriate diene (butadiene (unsubstituted or substituted),
cyclopentadiene,
cyclohexadiene cycloheptadiene, furane, thiophene, pyrrole, N-alkylpyrrole) to
give
the desired cycloadducts (Diels-Alder products) such as for example 14, 15 and
16.
This reaction can be performed by heating the pure reagents (diene and
dienophile)
either neat or in solution, with or without pressure.
Example 11. Specific procedure for the preparation of hydrazone 13a with R =
4-hydroxy-3-methoxyphenyl (see scheme 3)
[00163] The intermediate 4-(2,5-dioxo-2,5-dihydro-pyrrol-1-y1)-benzoic
acid
hydrazide trifluoroacetic acid salt (6) was prepared as described in example
1, step E
using the following quantities: derivative 4 (100 mg, 0.303 mmol),
trifluoroacetic acid
250 j.iL, dichloromethane (1 mL). The intermediate 6 was dissolved in
dichloromethane (2 mL) and treated with 4-hydroxy-3-methoxybenzaldehyde
(vanillin, 46.1 mg, 0.303 mmol) and sodium bicarbonate (25.4 mg, 0.303 mmol).
CA 3021417 2018-10-18
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Dichloromethane (2-3 mL) was used to dissolve the aldehyde stuck to the
reaction
vessel wall. The reaction was stirred at room temperature for 12 hours.
Afterwards,
the reaction mixture was transferred into a separatory funnel containing ethyl
acetate
(50 mL). The organic phase was washed twice with 3% aqueous solution of sodium
bicarbonate (2 x 20 mL) and with water (3 x 15 mL). The organic phase was
dried
with anhydrous magnesium sulfate, filtered and evaporated to the crude
hydrazone
13a, R = 4-hydroxy-3-methoxyphenyl (79 mg, 71%). The product was homogeneous
by thin layer chromatography. 1H NMR (DMSO-d6, 6 ppm): 11.71 (br s, 1H, NH),
9.54 (br s, 1H, OH), 8.32 (br s, 1H, -CH=N-), 7.97 and 7.49 (2 x d, J = 8.2
Hz, 4H,
aromatic), 7.32 (br s, 1H, aromatic), 7.21 (s, 2H, maleimide), 7.09 (br d, J =
7.8 Hz,
1H, aromatic) 6.83 (d, J = 8.2 Hz, 1H, aromatic), 3.82 (s, 3H, OCH3); ESI+
HRMS:
(M+H)+ calculated for CI9H16N305= 366.1084; found = 366.1076.
[00164] Using iso-vanillin instead of vanillin the hydrazone 13b with R
= 3-
hydroxy-4-methoxyphenyl was obtained: 1H NMR (DMSO-d6, 6 ppm): 11.71 (br s,
1H, NH), 9.30 (br s, 1H, OH), 8.29 (br s, 1H, -CH=N-), 7.98 (d, J = 8.2 Hz,
2H,
aromatic), 7.49 (d, J = 8.6 Hz, 2H, aromatic), 7.27 (d, J = 1.5 Hz, 1H,
aromatic), 7.22
(s, 2H, maleimide), 7.04 (dd, J = 1.6 and 8.2 Hz, 1H, aromatic) 6.96 (d, J =
8.2 Hz,
1H, aromatic, 3.80 (s, 3H, OCH3); ESI+ HRMS: (M+H)+ calculated for C191-
116N305
= 366.1084; found = 366.1058.
[00165] With benzaldehyde the hydrazone 13c, R = phenyl was obtained:
1H NMR (DMSO-d6, 6 ppm): 11.90 (s, 1H, NH), 8.45 (s, 1H, CH=N), 8.00 and 7.47
(2 d, J=8.6 Hz, 4H, aromatic,), 7.73 and 7.45 (2 m, 5H, H aromatique), 7.21
(s, 2H,
maleimide); ESI+ FIRMS: (M+H)+ calculated for Ci8HI4N303 = 320.1030; found =
320.1028.
[00166] With 2-hydroxybenzaldehyde the hydrazone 13d, R = 2-
hydroxyphenyl was obtained: 1H NMR (DMSO-d6, 6 ppm): 12.14 (s, 1E1, NH),
11.23 (s, 1H, OH), 8.64 (s, 1H, CH=N), 8.03 and 7.52 (2d, 4H, H aromatic ,
J=8.6
Hz), 7.57 (d hidden, 1H, H-6), 7.30 (t, 1H, H-4, J=6.3 Hz), 7.22 (s, 2H,
maleimide),
6.93 (d, 1H, H-3, J=7.8 Hz), 6.88 (t hidden, 1H, H-5); ESI+ HRMS: (M+H)+
calculated for Ci8Hi4N304 = 336.0979; found = 336.0973.
[00167] With 3-hydroxybenzaldehyde the hydrazone 13e, R = 3-
hydroxyphenyl was obtained: 1H NMR (DMSO-d6, 6 ppm): 11.84 (s, 1H, NH), 9.62
CA 3021417 2018-10-18
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(s, 1H, OH), 8.35 (s, 1H, CH=N), 7.99 and 7.50 (2d, 4H, aromatic, J=8.0 Hz),
7.22 (s,
2H, maleimide), 7.23-7.11 (m, 3H, H-4-5-6), 6.82 (d, 1H, H-2, J=6.6 Hz); ESI+
HRMS: (M+H)+ calculated for CI8H141\1304 = 336.0979; found = 336.0978.
[00168] With 4-hydroxybenzaldehyde the hydrazone 13f, R = 4-
hydroxyphenyl was obtained: 'H NMR (DMSO-d6, 6 ppm): 11.70 (s, 1H, NH), 9.93
(s, 1H, OH), 8.34 (s, 1H, CH=N), 7.98 and 7.49 (2d, 4H, aromatic , J=8.4 Hz),
7.56
and 6.83 (2d, 4H, H-2-3-5-6, J=8.2 Hz), 7.21 (s, 2H, maleimide); ESI+ HRMS:
(M+H)+ calculated for Ci8Hi4N304 = 336.0979; found = 336.0978.
[00169] With 2,4-dihydroxybenzaldehyde the hydrazone 13g, R = 2,4-
dihydroxyphenyl was obtained: 1H NMR (DMSO-d6, 6 ppm): 11.95 (s, 1H, NH),
11.41 (s, 1H, OH ortho), 9,97 (s, 1H, OH para), 8.49 (s, I H, CH=N), 8.02 and
7.48(2
d, 4H, aromatic , J=8.4 Hz), 7.33 and 6.37 (2 d, 2H, H-5-6, J=8.2 Hz), 7.21
(s, 2H,
maleimide), 6.31 (s, 1H, H-3); ESI+ HRMS: (M+H)+ calculated for C18H14N305 =
352.0928; found = 352.0923.
[00170] With 4-hydroxy-3,5-ditertbutylbenzaldehyde the hydrazone 13h, R
= 3,5-ditertbuty1-4-hydroxybenzaldehyde was obtained: H NMR (DMSO-d6,
6 ppm): 11.68 (s, 1H, NH), 8.37 (s, 1H, CH=N), 7.98 and 7.47 (2 d, 4H,
aromatic ,
J=8.6 Hz), 7.47 (s, 2H, H-2-6), 7.21 (s, 2H, maleimide), 1.40 (s, 18H,
C(CH3)3); ESI+
HRMS: (M+H)+ calculated for C26H30N304 = 448.2231; found = 448.2224.
[00171] With 4-methoxybenzaldehyde the hydrazone 13i, R = 4-
methoxyphenyl was obtained: 11-1 NMR (DMSO-d6, 6 ppm): 11.78 (s, 1H, NH),
8.39 (s, 1H, CH=N), 7.99 and 7.02 (2 d, 4H, H-2-3-5-6, J=8.6 Hz), 7.67 and
7.49 (2 d,
4H, aromatic , J=8.6 Hz), 7.21 (s, 2H, maleimide), 3.80 (s, 3H, OCH3); ESI+
HRMS:
(M+H)+ calculated for Ci9H16N304 = 350.1135; found = 350.1127.
[00172] With 2-hydroxy-3-methoxybenzaldehyde the hydrazone 13j, R = 2-
hydroxy-3-methoxyphenyl was obtained: 11-1 NMR (DMSO-d6, 6 ppm): 12.11 (s,
I H, NH), 10.90 (s, 1H, OH), 8.65 (s, 1H, CH=N), 8.01 and 7.53 (2 m, 4H,
aromatic),
7.21 (s, 2H, maleimide), 7.01 a 6.86 (m, 3H, H-4-5-6), 3,80 (s, 3H, OCH3);
ESI+
HRMS: (M+H)+ calculated for Ci9H16N305 = 366.1084; found = 366.1075.
[00173] With 3,4-dimethoxybenzaldehyde the hydrazone 13k, R = 3,4-
dimethoxyphenyl was obtained: 11-1 NMR (DMSO-d6 and D20, 6 ppm): 11.78 (s,
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55
1H, NH), 8.34 (s, 1H, CH=N), 7.97 and 7.48 (2 d, 4H, aromatic, J=8.6 Hz), 7.40
(s,
1H, H-2), 7.18 and 7.00 (2 d, 2H, H-5-6, J=8.2 Hz), 7.15 (s, 2H, maleimide),
3.79 (2
s, 6H, 2 x OCH3). ESI+ HRMS: (M+H)+ calculated for C20Hi8N305 = 380.1241;
found = 380.1234.
[00174] With 4-
allyloxybenzaldehyde the hydrazone 131, R = 4-
allyloxyphenyl was obtained: 'H NMR (DMSO-d6, 6 ppm): 11.78 (s, 1H, NH), 8.38
(s, 1H, CH=N), 7.99 and 7.49 (2 d, 4H, aromatic, J=8.6 Hz), 7.66 and 7.03 (2
d, 4H,
H-2-3-5-6, J=8.6 Hz), 7.21 (s, 2H, maleimide), 6.04 (m, 1H, H-8), 5.33 (m, 2H,
H-9),
4.61 (d, 2H, H-7, J=5.08 Hz); ESI+ HRMS: (M+H)+ calculated for C21Hi8N304 =
376.1292; found = 376.1282.
[00175] With 2-
furaldehyde the hydrazone 13m, R = 2-furanyl was
obtained: 11-INMR (DMSO-d6, 6 ppm): 11.83 (s, 1H, NH), 8.33 (s, 1H, CH=N),
7.96
and 7.52 (2 d, 4H, aromatic , J=8.2 Hz), 7.84 (s, 1H, H-3), 7.21 (s, 2H,
maleimide),
6.93 (d, 1H, H-4, J=3.1 Hz), 6.63 (s, 1H, 1-1-5); ESI+ HRMS: (M+H)+ calculated
for
Ci6H i2N304 = 310.0822; found = 310.0820.
[00176] With
pyridine-2-carbaldehyde the hydrazone 13n, R = 2-pyridyl
was obtained: 11-1 NMR (DMSO-d6, 6 ppm): 12.09 (s, 1H, NH), 8.61 (m, 1H, H-3),
8.47 (s, 1H, CH=N), 8.01 and 7.52 (2 d, 4H, aromatic, J=7.10 Hz), 7.89 (m, 2H,
H-5-
6), 7.43 (m, 1H, H-4), 7.22 (s, 2H, maleimide); ESI+ HRMS: (M+H)+ calculated
for
CI7F113N403 = 321.0982; found = 321.0977.
[00177] With
pyridine-3-carbaldehyde the hydrazone 13o, R = 3-pyridyl
was obtained: 12.07 (s, 1H, NH), 8.86 (s, 1H, H-2), 8.60 (d, 1H, H-4, J=3.0
Hz), 8.50
(s, 1H, CH=N), 8.16 (d, 1H, H-6, J=7.1 Hz), 8.01 and 7.51 (2d, 4H, aromatic ,
J=8.0
Hz), 7.51 (t hidden, 1H, H-5), 7.22 (s, 2H, maleimide); ESI+ HRMS: (M+H)+
calculated for Ci7Hi3N403= 321.0982; found = 321.0979.
[00178] With
pyridine-4-carbaldehyde the hydrazone 13p, R = 4-pyridyl
was obtained: NMR (DMSO-
d6, 6 ppm): 12.29 (s, 1H, NH), 8.72 (d, 2H, H-3-5,
J=1.9 Hz), 8.47 (s, 1H, CH=N), 8.00 and 7.53 (2 d, 4H, aromatic, J=6.6 Hz),
7.82 (s,
2H, H-6-2); ESI+ HRMS: (M+H)+ calculated for C17Hi3N403 = 321.0982; found --
321.0974.
[00179] With
cinnamaldehyde the hydrazone 13q, R = phenylethenyl was
obtained: 114 NMR (DMSO-d6, 6 ppm): 11.80 (s, 1H, NH), 8.21 (s, 1H, CH=N),
7.97
CA 3021417 2018-10-18
56
and 7.49 (2 d, 4H, aromatic , J=7.8 Hz), 7.60 (d, 2H, H-2-6, J=6.3 Hz), 7.36
(m, 3H,
H-3-4-5), 7.18 (s, 2H, maleimide), 7.04 (m, 2H, H-7-8); ESI+ HRMS: (M+H)+
calculated for C20H16N303 = 346.1186; found = 346.1178.
[00180] With 4-chlorobenzaldehyde the hydrazone 13r, R = 4-chlorophenyl
was obtained: H NMR (DMSO-d6, 6 ppm): 11.96 (s, 1H, NH), 8.43 (s, 1H, CH=N),
8.00 and 7.51 (2 d, 4H, aromatic, J=8.0 Hz), 7.76 and 7.51 (2 d, 4H, H-2-3-5-
6, J=7.8
Hz), 7.22 (s, 1H, maleimide); ESI+ HRMS: (M+H)+ calculated for C181113C1N303 --
354.0640; found = 354.0630.
[00181] With 4-fluorobenzaldehyde the hydrazone 13s, R = 4-fluorophenyl
was obtained: H NMR (DMSO-d6, 6 ppm): 11.91 (s, 1H, NH), 8.44 (s, 1H, CH=N),
8.00 and 7,50 (2 d, 4H, aromatic, J=6.4 Hz), 7.79 (s, 2H, H-2-6), 7,29 (s, 2H,
H-3-5),
7.21 (s, 2H, maleimide); ESI+ HRMS: (M+H)+ calculated for Ci8Hi3EN303 =
338.0935; found = 338.0926.
[00182] With 4-trifluoromethylbenzaldehyde the hydrazone 13t, R = 4-
trifluoromethylphenyl was obtained: 'El NMR (DMSO-d6, 6 ppm): 12.09 (s, 1H,
NH), 8.51 (s, 1H, CH=N), 8.01 and 7.51 (2 d, 4H, aromatic, J=8.2 Hz), 7.95 and
7.81
(2 d, 4H, H-2-3-5-6, J=7.9 Hz), 7.22 (s, 2H, maleimide); ESI+ HRMS: (M+H)+
calculated for Ci9Hi3F3N303 = 388.0904; found = 388.0897.
[00183] With 4-nitrobenzaldehyde the hydrazone 13u, R = 4-nitrophenyl
was obtained: 'H NMR (DMSO-d6, 6 ppm): 11.41 (s, 1H, NH), 7.90 and 7.48(2 d,
4H, aromatic , J=8.2 Hz), 7.67 (d, 1H, CH=N, J=5.1 Hz), 7.20 (s, 2H,
maleimide),
2.48 (septet, 1H, H-1), 1.07 (d, 6H, H-2-3, J=6.7 Hz); ESI+ HRMS: (M+H)+
calculated for Ci8Hi3N405= 365.0880; found = 365.0875.
[00184] General structure of hydrazones 13a-13u (see scheme 3)
13a, R = 4-hydroxy-3- 13b, R = 3-hydroxy-4- 13c, R = phenyl
methoxyphenyl methoxyphenyl
13d, R = 2-hydroxyphenyl 13e, R = 3-hydroxyphenyl 13f, R = 4-
hydroxyphenyl
CA 3021417 2018-10-18
57
13g, R = 2,4- 13h, R
= 3,5-ditertbuty1-4- 131, R = 4-methoxyphenyl
dihydroxyphenyl hydroxybenzaldehyde
13j, R = 2-hydroxy-3- 13k, R = 3,4- 131, R
= 4-allyloxyphenyl
methoxyphenyl dimethoxyphenyl
13m, R = 2-furanyl 13n, R = 2-pyridyl 13o, R = 3-pyridyl
13p, R = 4-pyridyl 13q, R = phenylethenyl 13r, R = 4-chlorophenyl
13s, R = 4-fluorophenyl 13t, R = 4- 13u, R = 4-nitrophenyl
trifluoromethylphenyl
Materials and Methods - Biology
In vitro studies (for derivatives 4, 5, 8, 10, 11, and 12, etc.)
[00185] Cell culture ¨ general: Biological assays were performed using
the
human monocytic cell line THP1, the murine macrophage-like cell lines J774A.1
and
RAW 264.7, and the murine UBC cell lines MB49 and MB49-1. The cells were
maintained in RPMI medium supplemented with 10% heat-inactivated fetal bovine
serum (FBS) and containing 1 mM sodium pyruvate, 10 mM 4-(2-
hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) and 50 p,g/mL gentamycin
(referred as 10% FBS RPMI-1640). The cells were maintained at 37 C in a
moisture-
saturated atmosphere containing 5% CO2.
THP1 cells, RAW 264.7 cells and J774A.1 cells are the most widely used cell
lines to investigate the function and differentiation of monocytes and
macrophages in
response to various inflammatory mediators.17,18 Undifferentiated THPI cells
resemble primary monocytes/macrophages isolated from healthy donors or donors
CA 3021417 2018-10-18
58
with inflammatory diseases, such as diabetes mellitus and atherosclerosis.I9
After
treatment with phorbol esters, THPI cells differentiate into macrophage-like
cells
which mimic native monocyte-derived macrophages in several respects.2 As we
previously described, green fluorescent protein-expressing (GFP)-THP1 cells
were
cultured for 18 h in 50 nM phorbol 12-myristate 13-acetate to induce monocyte-
to-
macrophage differentiation.2I-23 The cell lines RAW 264.7 and J774A.1 were
kindly
provided by Dr Tatiana Scorza (Universite du Quebec a Montreal, Canada). These
cell lines are macrophage-like cell models which produce large amount of NO in
response to IFNy, TNFet, bacterial infection and bacterial products, such as
lipopolysaccharide (LPS),I7 The cell line MB49-I is a highly invasive and
tumorigenic
UBC cell model that was developed by successive in vivo passages of MB49
primary
tumors.24
[00186] Cell signaling studies: THP1-derived human macrophages
(750x103
cells/mL) and MB49-I cells (500x103 cells/mL) were pretreated for 30 min with
vehicle (DMSO), compounds 4 and 5, at 10, 25 or 50 IAM; and then washed and
recovered immediately (t= 0 min) or after 30 min of activation with 50 U/mL
IFNy,
25 ng/mL IL6, or 10 ng/mL TNFa. Cell lysates were prepared and analyzed by
immunoblotting as described.2I-23'25 Briefly, protein samples were resolved by
SDS-
PAGE under reducing conditions and transferred onto a PVDF membrane. Blots
were
first probed with rabbit polyclonal antibodies against phospho-STAT1 (pSTAT1),
pSTAT3, or p-Ix13 (all at 1:2000) overnight at 4 C. Blots were then incubated
with
HRP-conjugated goat anti-rabbit IgG Ab (1:3000) for 1 h at room temperature.
The
same blots were stripped and then probed with anti-STAT1 or anti-STAT3 Abs
(both
at 1:1000). In both cases, probed molecules were visualized using an
enhancement
chemiluminescence detection kit (Thermo Fisher Scientific).
[00187] Luciferase assay. To assess the transcriptional activity of
NFKI3 in
response to TNFa, MB49-I cells cultured in monolayers (7.5x104 cells/well in
24-
well plates) were transiently transfected with an expression vector allowing
the
transcription of the firefly luciferase reporter gene under the control of CMV
promoter and tandem repeats of the NFKB transcriptional response element. The
luciferase gene encodes a 61-kDa enzyme that oxidizes D-luciferin in the
presence of
ATP, oxygen, and MC, yielding a fluorescent product that can be quantified by
measuring the released light. Transfection was performed using Opti-MEM
reduced
CA 3021417 2018-10-18
59
serum media and Lipofectamine 2000 (Thermo Fisher Scientific). Then, MB49-I
cells
were pretreated for 30 min with vehicle (DMSO) and compound 4 (at 10 uM or 30
M); and then washed and recovered after 24 h of activation with 10 ng/mL or 50
ng/mL TNFoc. Cell lysates were mixed with Luciferase assay reagent (a mixture
of
luciferine, coenzyme A, ATP and Mg2+) and the light produced was measured
using a
luminometer.
[00188] Surface antigen expression analysis. To study membrane receptor
expression, THP1-derived human macrophages (750x104 cells/mL) were pretreated
for 3 h with DMSO or compounds 4 and 5, and then left untreated (control) or
treated
for 48 h with 50 U/mL IFNy. The expression level of MHC-II and CD40 was
evaluated by flow cytometry as described.21-23
[00189] Motility assays. The in vitro scratch wound healing assay was
performed to study the effects of compounds 4 and 5 in 1L6-induced macrophage
cell
migration, as described.26 Briefly, THP1-derived human macrophages (750x103
cells/mL) were seeded into 24-well tissue culture plate to reach ¨70-80%
confluence
as a monolayer. The cell monolayers were scraped in a straight line in one
direction to
create a "scratch" with a p200 pipet tip. To obtain the same field during the
image
acquisition, another straight line was scratched perpendicular to the first
would line to
create a cross in each well. Cell debris were removed and the edges of the
scratch
were smoothed by washing the cells once with 1 mL of Hank's buffer. Cell
monolayers were pretreated for 3 h with vehicle (DMSO) or compounds 4 (10 M)
and 5 (25 p.M), and then left untreated (control) or treated for 48 h with 25
ng/mL
IL6. Using the cross as reference points the plate was placed under an
inverted
fluorescence microscope, and the images of the scratch were acquired at t= 0 h
and t=
48 h. The number of motile cells was determined using Java-based image
processing
program ImageJ (National Institutes of Health) and relative cell motility was
expressed as percent (%) of control of motile cells at t= 48 h relative to
motile cells
within the initial wound (at t= 0 h).
[00190] Microinvasion assays. Invasion was measured by assessment of
human
UBC T24 cell migration rate through an artificial basement membrane in a
modified
Boyden chamber (HTS Transwell System). Briefly, the membrane consisted of a
polycarbonate filter with 0.4 um pores diameter and was coated on ice with
Matrigel
CA 3021417 2018-10-18
60
(BD Biosciences) diluted 1:2 (v/v) in serum-free culture media. T24 cells (50
x 103
cells) were seeded into the Matrigel coat in the upper well of the chamber,
while
polarized MI macrophages (50 x 103 cells) were seeded in the lower well. The
polycarbonate filter with 0.4 pim pores diameter is too small to left the
cells move to
the bottom of the lower wells. Thus, non-invading cells into the matrigel coat
were
removed and cells that migrated down to the polycarbonate filter were stained
with
Hoescht 33258 dye after a 48-h incubation period, visualized by fluorescence
microscopy and counted.
[00191] Cellular analysis of 3D spheroid-based tumor invasion assays.
Cell
preparation and spheroid formation were performed following the manufacturer
instructions (Corning). Briefly, MDA-MB-231 cells were harvested and diluted
to a
concentration of 5 x 104 cells/mL in (complete) culture media supplemented
with 10%
serum. 100 pilL, of cell suspension was then pipetted to appropriate 96 well
spheroid
formation plate and placed at 37 C/5% CO2. Spheroid formation was monitored
every 24 hours. The typical cell aggregation period was 48 hours. Upon
spheroid
formation completion, 70 iaL of complete medium was removed from each well,
and
replaced by 70 tL of serum-free medium. This was repeated on a daily basis to
create
a serum starvation period of 48 hours. Matrigel matrix (100 [it) was then
added to
each well. The plate was centrifuged at 300 x g for 5 minutes in a swinging
bucket
centrifuge. The plate was then transferred to a 37 C/5% CO2 incubator for one
hour
to initiate gel formation. Following the 1-hour gel formation incubation
period,
DMSO or compound 4 was added to appropriate wells during 60 minutes. After
pretreatment, the media was removed and replaced by complete culture media,
and
then the media was changed every day for a period of 4 days. Brightfield
images at
day 0 and day 4 was used to measure invasive protrusion (invadopodia)
extending
away from the tumoroid and into the extracellular matrix. The role of these
protrusions is the breakdown of Matrigel matrix, thus aiding the invasion
process.
These protrusions were identified, and their area and perimeter properly
quantified
using the ImageJ software (National Institutes of Health).
1001921 Evaluation of NO and Urea production by chemical methods. The
MB49-I cells and the J774A.1 cells (25x103 cells/well) were grown and
pretreated, as
indicated, with various anti-inflammatory derivatives, precursors and mono-
functional
derivatives for a period of 3 h. Afterwards the cells were washed twice with
10% FBS
CA 3021417 2018-10-18
61
RPMI-1640 and then activated to produce NO for a period of 24 h with cytokines
INFy and TNFcc. NO production was measured using the Griess reagent method as
previously described.27 This method involves the detection of nitrite ions
(NO2-)
formed by the spontaneous oxidation of NO under physiological conditions.
According the manufacture procedure (Life Technologies; # G-7921), equal
volumes
of sulfanilic acid and N-(1-naphthyl)ethylenediamine are mixed together to
form the
Griess reagent. In the presence of NO2-, sulfanilic acid is converted to a
diazonium
salt, which in turn is coupled to N-(1-naphthyl)ethylenediamine to produce a
pink
coloration that is measured with a spectrophotometer (Biotek, synergy HT) at
548 nm.
In the same manner, urea synthesis through arginase-1 activity was measured
using
commercially available kit.
[00193] Evaluation of cell proliferation by the MTT assay. To evaluate
the anti-
proliferative activity, cell viability/proliferation MTT assays were performed
as
previously described.21-23,25,28 Briefly, MB49-I cells (5x103 cells/well) were
plated in
96-well plates in 100 [it 10% FBS RPMI-1640 and cultured for 24 h at 37 C and
5%
CO2. Cells were pretreated for a period of 3 h with vehicle (DMSO) or
derivatives 1
and 1A at 0, 10, 25, 37.5, and 50 p,M, and then incubated for 24 h in the
absence or
the presence of INFy and TNFa. At the end of the culture period, 10 1.1L of 5
mg/mL
methylthiazolyldiphenyl-tetrazolium bromide (MTT) solution was added to each
well.
After a 3-h incubation period with MTT reagent, 100 1.11L of MTT
solubilization buffer
(10% SDS in 10 mM HC1) was added and plates were placed overnight in the cell
incubator before absorbance measure. The optical density was read at 580 nm
using
the Microplate Reader Manager (from Bio-Rad Laboratories).
[00194] Statistical analyses. For all biological assays, data were
presented as
mean 1 SD from three independent experiments. Data were analyzed by one-way
ANOVA followed by Bonferonni post-test using Prism software, version 3.03
(GraphPad, San Diego, CA). p values of < 0.05 were considered to indicate
statistical
significance.
In vivo studies (for derivatives 4 and 8)
CA 3021417 2018-10-18
62
[00195] Ectopic UBC model. Male C57BL/6J mice (6-8 weeks old), each
weighing 15-18 g, were used for the experiments (supplied by Charles River).
The
mice were housed with free access to food and water on a 12:12 h light:dark
cycle
with the room temperature maintained at 21 C. MB49 cells, MB49-1 cells, and
iNOS-
deficient MB49-I cells (5 x 104 in 100 j.L PBS) were injected subcutaneously
(s.c.)
into the right flank of the mice. Growth rates of the s.c. tumors were
monitored. The
size of tumors was determined every 3 days for 24-28 days using a digital
caliper and
by measuring luciferin luminescence at days 15 and 25 using the IVIS imaging
system. A blinded observer measured tumor length and width. The volume of the
tumor was calculated from the formula: Length x width2 x 0.52, where length
and
width were tumor diameters measured with calipers in mutually perpendicular
directions. At a tumor size of approximately 10 mm3 the mice were divided into
different groups. A control group received PBS as treatment. As indicated,
other
groups were treated at different doses (90, 150 or 300 1.1M) with an
intraperitoneal
(i.p.) injection of compounds 4 or 8 every 3-4 days for 18-20 days.24,27
[00196] Orthotopic UBC model. Female C57BL/6J mice (6-8 weeks old),
each
weighing 15-18 g, were anesthetized and a 24-gauge Teflon catheter was
introduced
into the bladder lumen through the urethra. To prepare the bladder for tumor
implantation, a point lesion was induced in the bladder wall by
electrocauterization.
Then, 1 x 105 of MB49-I cells in 100 uI of PBS were instilled into the
bladder. When
indicated, control mice were electrocauterized and PBS was instilled into the
bladder.
Syringes containing the cell suspension were maintained into the catheter for
20 min
to allow cell attachment to the bladder wal1.24,27
[00197] Statistical analyses. Mann¨Whitney test was used to compare
tumor
growth, using Prism software, version 3.03 (GraphPad, San Diego, CA). p values
of <
0.05 were considered to indicate statistical significance.
The results from the in vitro and in vivo experiments are presented in figures
1 to 25.
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1001981 The
present disclosure has been described with regard to specific
examples. The description was intended to help the understanding of the
present
disclosure, rather than to limit its scope. It will be apparent to one skilled
in the art
that various modifications may be made to the present disclosure without
departing
from the scope of the present disclosure as described herein, and such
modifications
are intended to be covered by the present document.
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