Activation of Phenyl 4-(2-Oxo-3-Alkylimidazolidin-1-yl)benzenesulfonates Prodrugs by CYP1A1 as New Antimitotics Targeting Breast Cancer Cells

Article abstract of DOI:10.1021/acs.jmedchem.7b00343

Prodrug-mediated utilization of the cytochrome P450 (CYP) 1A1 to obtain the selective release of potent anticancer products within cancer tissues is a promising approach in chemotherapy. We herein report the rationale, preparation, biological evaluation, and mechanism of action of phenyl 4-(2-oxo-3-Alkylimidazolidin-1-yl)benzenesulfonates (PAIB-SOs) that are antimicrotubule prodrugs activated by CYP1A1. Although PAIB-SOs are inert in most cells tested, they are highly cytocidal toward several human breast cancer cells, including hormone-independent and chemoresistant types. PAIB-SOs are N-dealkylated into cytotoxic phenyl 4-(2-oxo-3-imidazolidin-1-yl)benzenesulfonates (PIB-SOs) in CYP1A1-positive cancer cells, both in vitro and in vivo. In conclusion, PAIB-SOs are novel chemotherapeutic prodrugs with no equivalent among current antineoplastics and whose selective action toward breast cancer is tailored to the characteristic pattern of CYP1A1 expression observed in a large percentage of human breast tumors.

Full text of DOI:10.1021/acs.jmedchem.7b00343

Subscriber access provided by CORNELL UNIVERSITY LIBRARY
Article
Activation of Phenyl 4-(2-Oxo-3-alkylimidazolidin-1-yl)benzenesulfonates
Prodrugs by CYP1A1 as New Antimitotics Targeting Breast Cancer Cells
Sébastien Fortin, Xavier Charest-Morin, Vanessa Turcotte, Coraline Lauvaux,
Jacques Lacroix, Marie-France Côté, Stéphane Gobeil, and Rene C.-Gaudreault
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 23 May 2017
Downloaded from http://pubs.acs.org on May 23, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 76
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Activation of Phenyl 4-(2-Oxo-3-alkylimidazolidin-1-yl)benzenesulfonates Prodrugs
by CYP1A1 as New Antimitotics Targeting Breast Cancer Cells
†
,‡,*
†
†
§
Sébastien Fortin , Xavier CharestꢀMorin , Vanessa Turcotte , Coraline Lauvaux ,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
†
†
§,£
†,£,*
Jacques Lacroix , MarieꢀFrance Côté , Stéphane Gobeil and René C.ꢀGaudreault
†
CHU de Québec Research Centre, Oncology Division, Hôpital SaintꢀFrançois d’Assise,
1
0 rue de l’Espinay, Quebec City, QC, Canada G1L 3L5.
‡
Faculty of Pharmacy, Université Laval, Quebec City, QC, Canada G1V 0A6.
CHU de Québec Research Centre, Endocrinology and Nephrology Division, CHUL,
§
2
705 Boulevard Laurier, Quebec City, QC, Canada G1V 4G2.
£
Department of Molecular Medicine, Faculty of Medicine, Université Laval, Quebec
City, QC, Canada G1V 0A6.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 76
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Abstract
Prodrugꢀmediated utilization of the cytochrome P450 (CYP) 1A1 to obtain the selective
release of potent anticancer products within cancer tissues is a promising approach in
chemotherapy. We herein report the rationale, preparation, biological evaluation and
mechanism of action of phenyl 4ꢀ(2ꢀoxoꢀ3ꢀalkylimidazolidinꢀ1ꢀyl)benzenesulfonates
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(PAIBꢀSOs), that are antimicrotubule prodrugs activated by CYP1A1. Although PAIBꢀ
SOs are inert in most cells tested, they are highly cytocidal towards several human breast
cancer cells, including hormoneꢀindependent and chemoresistant types. PAIBꢀSOs are Nꢀ
dealkylated into cytotoxic phenyl 4ꢀ(2ꢀoxoꢀ3ꢀimidazolidinꢀ1ꢀyl)benzenesulfonates (PIBꢀ
SOs) in CYP1A1ꢀpositive cancer cells, both in vitro and in vivo. In conclusion, PAIBꢀ
SOs are novel chemotherapeutic prodrugs with no equivalent among current
antineoplastics, and whose selective action toward breast cancer is tailored to the
characteristic pattern of CYP1A1 expression observed in a large percentage of human
breast tumors.
ACS Paragon Plus Environment

Page 3 of 76
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Introduction
Breast cancer accounts for 25% of all female cancers, making it by far the most common
cancer among women. It is estimated that 1.7 million new cases of breast cancers were
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1, 2
diagnosed worldwide in 2012 and that they were responsible for over 520,000 deaths.
The 5ꢀyear relative survival rate for women diagnosed with localized breast cancer is
3
9
8.6%; survival declines to 83.8% for regional stage and 23.3% for distant stage.
Therefore, breast cancer is among today’s most pressing health problems worldwide.
Human cytochromes P450 (CYPs) are essential hemeꢀcontaining enzymes comprising 57
individual types distributed into 18 families and 43 subfamilies. They catalyze > 40
different redox reactions and are involved in the biosynthesis of various hormones and in
the phase 1 elimination of various xenobiotics, including drugs and environmental
pollutants. CYPs are ubiquitous but are mainly expressed in the liver and, to a lesser
4
extent, in other organs and tissues. CYPs are of utmost importance in drug development
strategies for optimizing bioavailability, tissue selectivity and pharmacological responses
5, 6
to new drugs. In fact, these proteins are such critical determinants of drug interactions
and adverse effects that the FDA now requires a full characterization of the induction and
inhibition characteristics of the CYPs involved in the metabolism of every drug submitted
7
for approval. Although CYPꢀdependent metabolism is often perceived as a hindrance to
drug development due to its effects on drug response, interactions and adverse effects,
CYPs are nonetheless essential shields against toxicity resulting from drug
7
bioaccumulation. CYPs are widely used in drug design and in the development of
8
ꢀ12
prodrugs that can be converted into the active drug forms in vivo.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 76
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Prodrugs are designed so as to overcome issues of undesirable physicochemical,
1
3
pharmacological, or pharmacodynamic properties. More recently, new promising
prodrugs have been designed to exploit their bioactivation within tumors expressing
specific CYPs, in keeping with the current principles of personalized medicine. In cancer
chemotherapy, this approach aims at disseminating potent cancer drugs locally and
selectively within sensitive tumors, thereby allowing maximum efficacy while reducing
systemic toxicity. The underlying principle of the latter approach resides in the vast
differences found in the expression level of the various CYP isoforms in tumors
compared to other tissues. However, the development of tumorꢀspecific, CYPꢀactivatable
prodrugs requires the selection of CYP isoforms expressed or inducible in the targeted
tumor, while being largely absent in adjacent normal tissues and distal organs. Many
examples of such potentially targetable isoforms have been reported so far, including
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
4, 15
16
17ꢀ22
CYP1B1
, CYP2J2 , CYP2S1, CYP2W1 and CYP1A1.
CYP1A1 is an extrahepatic isoform predominantly expressed in breast, lung, placenta,
23
intestine and skin. Under physiological conditions, CYP1A1 expression is usually
absent, but is readily induced via the activation of the aryl hydrocarbon receptor (AhR)
by several ligands such as halogenated dioxins (e.g. 2,3,7,8ꢀtetrachlorodibenzoꢀpꢀdioxin,
1
9, 24
Figure 1) and polycyclic aromatic hydrocarbons (e.g. benzo[a]pyrene).
Catabolism of
these environmental contaminants by CYP1A1 leads either to their detoxification or their
25
conversion into genotoxic derivatives that may trigger carcinogenesis. Several
compounds, including drugs such as omeprazole and sunitinib, induce CYP1A1
2
6, 27
expression via an AhRꢀindependent or ꢀdependent mechanism.
Recent microarray
and immunohistochemistry studies have shown that CYP1A1 is constitutively expressed
ACS Paragon Plus Environment

Page 5 of 76
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
28, 29
in breast tumors, contrasting with its low expression in adjacent breast tissue.
Moreover, CYP1A1 expression correlates with breast cancer grade and progression, and
is inducible in various breast tumor cell lines (e.g. MCF7, MDAꢀMBꢀ468, ZRꢀ75ꢀ1 and
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
19, 29
T47D).
CYP1A1 and AhR contribute in a coordinated manner to carcinogenesis,
3
0, 31
proliferation, survival, and chemoresistance of cancer cells.
In this regard, the
induction of CYP1A1 in breast cancer cells, resulting in the local conversion of prodrugs
into their active toxic form in situ, represents an innovative and promising strategy for
3
1ꢀ35
breast cancer chemotherapy
, irrespective of the hormonal status of the tumor
(
positivity for estrogen receptor (ER), progesterone receptor (PR) and the HER2
(
membraneꢀbound tyrosine kinase), i.e. the phenotype currently used clinically for
3
6
selecting the appropriate therapeutic regimen.
Insert Figure 1 and its Legend
We recently developed a subset of phenyl 4ꢀ(2ꢀoxoimidazolidinꢀ1ꢀyl)benzenesulfonates
PIBꢀSOs, Figure 1) as highly potent microtubuleꢀinterfering agents exemplified by
(
3
,4,5ꢀtrimethoxyphenyl 4ꢀ(2ꢀoxoimidazolidinꢀ1ꢀyl)benzenesulfonate (CEUꢀ602) and 3,5ꢀ
3
7
dimethoxyphenyl 4ꢀ(2ꢀoxoimidazolidinꢀ1ꢀyl)benzenesulfonate (CEUꢀ699).
Briefly,
38
PIBꢀSOs bind to the colchicineꢀbinding site at the interface between αꢀ and βꢀtubulins ,
exhibit antiproliferative activity at nanomolar concentrations in a wide range of tumor
cell lines, and arrest cell cycle progression in G2/M. Their cytotoxic activity remains
intact in cells resistant to paclitaxel and vinblastine and in cells overexpressing the Pꢀ
glycoprotein. Moreover, the antitumoral activity of PIBꢀSOs is equivalent to that of
cisplatin and combretastatin Aꢀ4 in human fibrosarcoma HTꢀ1080 cells grafted onto the
chorioallantoic membrane of developing chick embryos (or CAM assay), while
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 76
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
37
displaying minor toxicity in the chick embryo. We now report the design, preparation
and biological evaluation of phenyl 4ꢀ(2ꢀoxoꢀ3ꢀalkylimidazolidinꢀ1ꢀyl)benzenesulfonates
(
PAIBꢀSOs 6ꢀ68, Scheme 1) as novel prodrugs bioactivated by CYP1A1 in human breast
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
cancer cells.
Design of PAIB-SOs. Microtubules play pivotal roles in major functions such as mitosis,
3
9, 40
cell, motility, and cytoplasmic transport.
Antimicrotubule drugs are among the most
potent in the cancer armamentarium, and particularly for breast cancer chemotherapy,
4
1ꢀ43
and are thus prime candidates for the development of new cancer drugs.
However,
microtubuleꢀinterfering agents have deleterious effects such as bone marrow suppression,
hypersensitivity reactions, peripheral neuropathy, myalgia/arthralgia, as well as
gastrointestinal and renal toxicity. We thus developed a tumorꢀspecific drug delivery
approach to circumvent these limitations while improving the pharmacological properties
and tumor selectivity of antimicrotubule PIBꢀSOs. First, structureꢀactivity relationship
and molecular modeling studies showed that an unsubstituted amino group in the
phenylimidazolidinone moiety is essential for the cytocidal activity of PIBꢀSOs.
Moreover, various modifications, including the homologation of the imidazolidone
moiety of PIBꢀSOs, dramatically decrease affinity for the colchicineꢀbinding site, leading
3
7
to inactive compounds. The rationale of our PAIBꢀSO prodrug design relied on the
derivatization of the phenylimidazolidinone moiety with lower alkyl groups, thus
eliminating the cytotoxic activity of the resulting PIBꢀSO precursors. With such a design,
cytotoxicity can only be restored upon oxidative Nꢀdealkylation by CYP1A1, which is
frequently expressed and/or inducible in breast cancer tumors.
Results.
ACS Paragon Plus Environment

Page 7 of 76
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Chemistry. Scheme 1 summarizes the synthesis of PAIBꢀSOs. First, 1ꢀ
phenylimidazolidinꢀ2ꢀone was prepared quantitatively in two steps, as previously
3
7, 44
reported.
Then, NaH and the desired alkyl iodide were added to 1ꢀ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
phenylimidazolidinꢀ2ꢀone to yield 1ꢀalkylꢀ3ꢀphenylimidazolidinꢀ2ꢀones 69ꢀ72 in good to
very good yields. Moreover, we increased reaction yields to nearly quantitative levels by
adding NaH and the alkyl iodide portionwise every 8 h until reaction completion. The 4ꢀ
(3ꢀalkylꢀ2ꢀoxoimidazolidinꢀ1ꢀyl)benzenesulfonyl chlorides 73ꢀ76 were then obtained by
reacting compounds 69 to 72 with 7.5 molar equivalents of chlorosulfonic acid at 0 °C.
Finally, the PAIBꢀSOs 6ꢀ68 were prepared by addition of the appropriate phenol and
triethylamine to compounds 73ꢀ76 at room temperature.
Insert Scheme 1 and its Legend
Antiproliferative Activity. Using several human cancer cell lines, we first studied the
effects of the nature and position of aromatic ring B substituents and the homologation of
the alkyl groups Nꢀsubstituted on the phenylimidazolidinone moiety on the
antiproliferative activity of the corresponding PAIBꢀSOs. The antiproliferative activity
(IC ) of PAIBꢀSOs was first assessed in HTꢀ29 colon carcinoma, M21 skin melanoma,
5
0
and MCF7 estrogenꢀdependent breast carcinoma cells according to protocols
4
5
recommended by the NCI/NIH Developmental Therapeutics Program (Table 1). At
3
7
first, the aromatic ring B of known PIBꢀSOs substituted with either alkyl, methoxy,
nitro or halogen groups were converted into their corresponding PAIBꢀSOs 6ꢀ53 bearing
a methylꢀ, ethylꢀ and propylꢀsubstituted phenylimidazolidinone moiety. Table 1 shows
that with the exception in MCF7 cells of PAIBꢀSO 45, 48 and 49, whose aromatic ring B
is chloroꢀ or methoxyꢀsubstituted at position 3, 4 and/or 5, the antiproliferative activity of
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 76
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
PAIBꢀSOs decreased upon lengthening of the alkyl chain. These results indicate that Nꢀ
dealkylation does not occur with PAIBꢀSOs bearing either an Nꢀmethyl or Nꢀethyl group.
Remarkably, however, Nꢀpropylꢀ substituted PAIBꢀSOs 45, 48 and 49 displayed an IC50
value of 380, 270 and 49 nM, respectively, in MCF7 breast cancer cells, compared to a
range of 3 900 to 33 000 nM in colon carcinoma and skin melanoma cells. Thus, a ~2ꢀlog
selectivity ratio ([IC (reference cell line)]/[IC (sensitive cell line tested)] was observed
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
50
50
that favored a selectivity of action of all active PAIBꢀSOs towards MCF7 breast cancer
cells, relative to M21 and HTꢀ29 cells as well as all other nonꢀmammary tumor cell lines
tested so far. This marked selectivity strongly suggested the involvement of a selective
enzymatic Nꢀdealkylation of PAIBꢀSO 45, 48 and 49 generating the highly cytocidal
PIBꢀSO precursors in MCF7 cells to a much larger extent than in HTꢀ29 and M21 cells.
We next examined the effect of an Nꢀsubstituted butyl group on the cytotoxicity of the
resulting PAIBꢀSO prodrug towards breast cancer cells. As expected, substitution of the
phenylimidazolidinone moiety of 1, the most potent PIBꢀSO thus far identified, with a
butyl group (compound 67), induced strong and markedly selective cytotoxicity in MCF7
cells.
Insert Table 1 and its Legend
Finally, permutation of halogen and methoxy groups between positions 3, 4 and/or 5 of
ring B and substitution of the 2ꢀimidazolidone moiety with an Nꢀn-butyl group led to
PAIBꢀSOs 55 to 68. With the exception of PAIBꢀSO 55, 56 and 68, the latter series
exhibited IC50 values in the nanomolar range and selectivity ratio values in favor of
3
MCF7 cells ranging from 61 to > 5.3 x 10 . Furthermore, diꢀ or trisubstituted PAIBꢀSOs
(61ꢀ67) were more cytocidal and exhibited better breast cancer cell selectivity than their
ACS Paragon Plus Environment

Page 9 of 76
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
respective monosubstituted counterparts (55ꢀ60). Of note, compounds 63 and 65, which
bear a 3,5ꢀOMe and 3,5ꢀCl group, respectively, on ring B, and whose
phenylimidazolidinone moiety was Nꢀsubstituted with an n-butyl group exhibited IC50
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
values ≤ 60 nM and selectivity ratios > 1 x 10 with respect to MCF7 cells. In addition,
PAIBꢀSO 67 was the most potent, with an IC value of 10 nM towards MCF7 cells,
5
0
3
whereas compound 63 exhibited the highest selectivity against MCF7 cells (> 5.3 x 10 ).
The latter results suggested that at least some of the PAIBꢀSOs synthesized might exhibit
a remarkably more selective cytocidal activity towards breast tumor cells than in human
cancer cells of different histological origin. To further assess this hypothesis, compounds
4
9, 63 and 67 were evaluated using an array of breast cancer cell lines expressing various
phenotypes of clinical interest, including soꢀcalled tripleꢀnegative types (ERꢀ, PRꢀ and
ꢀ
ꢀ
ꢀ
HER2ꢀnegative, i.e. ER /PR /HER2 ; MDAꢀMBꢀ231 and MDAꢀMBꢀ468 cells),
+
+
low
ꢀ
ꢀ
high
46
ER /PR /HER2
(T47D cells) and ER /PR /HER2
(SKꢀBRꢀ3 cells). With the
exception of MDAꢀMBꢀ231 cells, which are insensitive to all PAIBꢀSOs tested, all breast
cancer cell lines evaluated so far were remarkably sensitive to the selected PAIBꢀSOs
ꢀ9
(with SKꢀBRꢀ3 > MDAꢀMBꢀ468 > T47D cells, Table 2), with IC values in the 10 to
5
0
ꢀ7
3
1
0 M range (2.0ꢀ240 nM) and selectivity ratios of 0.13ꢀ3.0 x 10 (relative to insensitive
MDAꢀMBꢀ231 cells), according to the cell line and/or PAIBꢀSO considered. Of note, we
assessed the effect of phenol red in culture medium on the activity of PAIBꢀSO 67 on
MDAꢀMBꢀ231, MDAꢀMBꢀ468, T47D, SKꢀBRꢀ3 and MCF7 cell lines and phenol red
does not impact significantly the antiproliferative activity of 67 in all cancer cell lines
tested.
Insert Table 2 and its Legend
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 76
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Determination of the Mechanism of Action. To confirm that the Nꢀdealkylation of
PAIBꢀSOs into PIBꢀSOs occurs in a enzymaticallyꢀmediated fashion, we assessed the
effect of PAIBꢀSO 67 on cell cycle progression and microtubule integrity in PAIBꢀSOꢀ
sensitive MDAꢀMBꢀ468 and MCF7 cell lines, in comparison to insensitive MDAꢀMBꢀ
231 cells. Figure 2 shows that PAIBꢀSO 67 blocks cell cycle progression in the G2/M
phase and disrupts the microtubule network of sensitive tumor cells (MDAꢀMBꢀ468 and
MCF7) in a manner similar to that observed with an equimolar concentration of 1, while
having no detectable effect in insensitive MDAꢀMBꢀ231 cells tested in parallel.
Insert Figure 2 and its Legend
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
To identify the tumorꢀspecific CYP isoform responsible for the Nꢀdealkylation of PAIBꢀ
SOs in breast cancer cells, sensitive MDAꢀMBꢀ468 cells were coincubated with PAIBꢀ
SO 67 and subtoxic concentrations of various CYP inhibitors (αꢀnaphthoflavone,
resveratrol, ketoconazole, quercetin, tacrine, capsaicin, aniline, curcumin, mefipristone,
pentachlorophenol and salicylamide). As shown in Figure 3, a 72 h incubation with either
αꢀnaphthoflavone, resveratrol, ketoconazole, quercetin, tacrine and capsaicin
dramatically reduced the cytocidal activity of PAIBꢀSO 67 towards MDAꢀMBꢀ468 cells,
while neither aniline, curcumin, mefipristone, pentachlorophenol nor salicylamide
affected the antiproliferative activity of the PAIBꢀSO. Interestingly, the inhibitors that
reduced the antiproliferative activity of PAIBꢀSO 67 are known nonꢀselective inhibitors
of several CYPs, including CYP1A1, whereas the subset of compounds that did not
4
7ꢀ53
interfere with the cytocidal effect of 67 do not inhibit the CYP1A1 isoform.
Insert Figure 3 and its Legend
ACS Paragon Plus Environment

Page 11 of 76
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
The evidence gathered so led us to determine the affinity of the most potent PAIBꢀSOs
for CYP1A1, along with their intracellular activation to the corresponding PIBꢀSO. As
shown in Table 2 and in Figure 4, CYP1A1 readily oxidizes all PAIBꢀSOs thus
evaluated. Moreover, CYP1A1 Nꢀdealkylates PAIBꢀSOs with various velocities, yielding
the corresponding hydroxylated intermediates. In addition, the ethoxyresorufinꢀOꢀ
deethylase assay for CYP1A1 activity showed that all PAIBꢀSOs studied inhibit the
CYP1A1ꢀmediated oxidation of 7ꢀethoxyresorufin to fluorescent resorufin, while PIBꢀ
SOs do not inhibit the latter reaction. These results showed that all PAIBꢀSOs studied are
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ꢀ7
ꢀ7
competitive CYP1A1 inhibitors, with K values ranging from 2.4 x 10 to 5.4 x 10 M.
i
Importantly, Figure 5 shows that a 4ꢀh incubation (maximum incubation time) of PAIBꢀ
SO 67 with CYP1A2, ꢀ1B1 and ꢀ3A4 did not yield detectable amounts of the parent PIBꢀ
SO 1; longer incubation times were not assessed because enzymes instability may
5
4, 55
occur.
Nonetheless, the incubation of PAIBꢀSO with CYP1A2 leads to a oxidized
intermediate. The results shown above thus confirm that the activation of PAIBꢀSOs
observed in breast cancer cells is related to CYP1A1 expression. However, several other
experiments should be conducted to study the activation and identify others CYPs
involved in the metabolization pathway of PAIBꢀSOs. Interestingly, incubation of 67
with human liver microsomes pooled from several donors showed that after 60 min.,
approximately 25% of the intact PAIBꢀSO and only 3.3% of the parent PIBꢀSO 1 were
found. These results confirmed that hepatic enzymes have the ability to metabolize
PAIBꢀSOs into several oxidized intermediates and only a small amount of PIBꢀSOs is
generated suggesting a relative resistance of PAIBꢀSOs to hepatic metabolism. Further
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 76
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
experiments are required to study the antineoplastic activity and the potential
hepatotoxicity of PAIBꢀSOs on animal models of human breast cancers.
Insert Figure 4 and 5 and their Legend
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
To further confirm that CYP1A1 is involved in the activation of PAIBꢀSOs in sensitive
breast cancer cells, we determined the effect of compound 67 on CYP1A1 mRNA
expression in MCF7 and MDAꢀMBꢀ231 cells. Indeed, PAIBꢀSO 67 did not significantly
increase the CYP1A1 mRNA levels after 18 and 24 h of treatment in insensitive MDAꢀ
MBꢀ231 cells while it led to a 2ꢀfold increase in CYP1A1 mRNA levels in sensitive
MCF7 cells after an 18ꢀh incubation, an effect which was transient, as seen with the
eventual decline in the mRNA level in the following 6ꢀh interval (Figure S1 in supporting
information). Next, we found that PAIBꢀSOs are inactive both on wildꢀtype human
fibrosarcoma HTꢀ1080 cells and HTꢀ1080 cells transfected with the empty vector
empty
pCMV6ꢀAC_Empty (HTꢀ1080
) that do not express CYP1A1 enzyme with a
cytocidal activity in the micromolar range (IC value of 5.5 to 82 ꢀM). However, when
5
0
HTꢀ1080 cells are stably transfected with pCMV6ꢀAC_CYP1A1 plasmid (HTꢀ
CYP1A1
1080
), they become sensitive to PAIBꢀSOs 49, 67 and 63 with a cytocidal activity
in the low nanomolar range (IC value of 13 to 31 nM), and a selectivity ratio ranging of
5
0
empty
empty
CYP1A1
2
,600ꢀ5,100 relative to HTꢀ1080
(i.e. IC50 (HTꢀ1080
)/IC50 (HTꢀ1080
),
Figure 6). On one hand, in the chick CAM assay, 1 used as positive control inhibited the
growth of HTꢀ1080 tumor cells tumors by 51% while reducing the growth of HTꢀ
CYP1A1
empty
1080
and HTꢀ1080
by 32% and 21%, respectively. On the other hand, PAIBꢀ
CYP1A1
SOs 67 inhibited the growth of HTꢀ1080
tumors by 49% while reducing the growth
of wildꢀtype HTꢀ1080 tumor only by 15% and does not reduce the growth of HTꢀ
ACS Paragon Plus Environment

Page 13 of 76
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
empty
CYP1A1
1
080
cells. Therefore, PAIBꢀSO 67 showed in vivo selectivity towards HTꢀ1080
cells in the chick CAM assay, while exhibiting no or only marginal toxicity towards
chick embryos. The weak antitumor activity of PAIBꢀSO 67 on wildꢀtype HTꢀ1080 cells
tumors could also involve another CYP or enzyme capable of bioactivating PAIBꢀSO not
identified yet. It could be due also to the inhibition of various processes in the formation
and growth of tumors in this model other than the bioactivation of PAIBꢀSO into
antimicrotubule agents. Finally, PAIBꢀSO 67 seemed unaffected by potential resistance
profile of HTꢀ1080 cells since it reached approximatively the maximum antitumor value
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
37, 44, 56, 57
observed using that model.
Insert Figure 6 and its Legend
Discussion
Pharmacological strategies based on the CYP enzyme family represent a relevant and
promising approach for the development of new and highly selective tumorꢀtargeting
3
4
drugs exploiting CYPꢀdependent bioactivation of prodrugs. Such finely designed
strategies might in principle improve treatment regimens and efficacy while reducing the
deleterious effects associated with nonꢀtarget drug action. However, this concept has
been notoriously difficult to implement, since its therapeutic success depends on several
1
3, 58
prerequisites.
First, the CYP isoform selected for activating the prodrug must be
overexpressed or inducible in the tumor while remaining expressed at negligible levels in
offꢀtarget tissues. Second, the design of the prodrug must aim at optimal physicochemical
and pharmacokinetic properties to allow its efficient diffusion into the tumors, while its
oxidation must release a cytocidal metabolite locally. Third, such a prodrug should
exhibit a high affinity and selectivity towards a specific CYP isoform, and the parent
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 76
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
drug released upon enzymatic activation must bind its target(s) selectively and readily,
with minimal entry into the general circulation. Finally, the prodrug should not or only
partially be a substrate for other CYP isoenzymes to maximally reduce potential offꢀ
target toxicity.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CYP1A1 was identified as one of the most promising isoforms for the development of
3
4
new cancer prodrugs for the specific treatment of human breast carcinomas. Indeed,
CYP1A1 is an extrahepatic enzyme that is constitutively expressed in several breast
1
9
cancer tumors, but largely absent in healthy adjacent tissues. It is also involved in
carcinogenesis, in tumor aggressiveness and in the induction of cancer cell defense
mechanisms. Over the last decade, three families of antitumor prodrugs, namely
precursors of duocarmycin, benzothiazoles and aminoflavones have been developed
based on the concept of CYP1A1 bioactivation. Aminoflavones and benzothiazoles
prodrugs induce CYP1A1 expression while duocarmycin bioprecursors do not. They are
converted into potent alkylating metabolites that covalently bind to DNA and/or other
21, 31, 59
macromolecules.
While duocarmycin bioprecursors (1ꢀ(chloromethyl)ꢀ1,6ꢀ
dihydropyrrolo[3,2ꢀe]indolꢀ3(2H)ꢀyl)(5ꢀmethoxyꢀ1Hꢀindolꢀ2ꢀyl)methanone (3, ICT2700)
21
are still undergoing preclinical studies , extensive structureꢀactivity relationship studies
of aminoflavones and benzothiazoles have singled out two lead compounds exhibiting
high activity and selectivity towards human breast cancer cells both in vitro and in vivo,
59, 60
for subsequent clinical studies.
The lysyl benzothiazole 2ꢀ[4ꢀaminoꢀ3ꢀmethylphenyl]ꢀ
5
ꢀfluorobenzothiazole lysylamide dihydrochloride (4, NSC710305, Phortress) and the
lysyl
aminoflavone
(2S)ꢀ2,6ꢀdiaminoꢀNꢀ[4ꢀ(5ꢀaminoꢀ6,8ꢀdifluoroꢀ7ꢀmethylꢀ4ꢀ
oxochromenꢀ2ꢀyl)ꢀ2ꢀfluorophenyl]hexanamide (5, NSC710464) have been assessed as
ACS Paragon Plus Environment

Page 15 of 76
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
61
prodrugs in clinical trials (Figure 1) . Compound 5 is now in Phase II studies to assess
+
its efficacy in tripleꢀnegative and ER breast carcinomas, and is currently in preclinical
6
2
evaluation for kidney and ovarian cancers. Of note, dosage limitation was observed in
Phase I with 5 due to pulmonary toxicity. On the other hand, the clinical evaluation of 4
has been discontinued, as Phase I studies failed to determine a maximal tolerated dose
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
6
3
and displayed lung and liver toxicity.
The preclinical results obtained for 3, 4 and 5 as well as the Phase II evaluation of 5 have
shown that a CYPꢀactivated prodrug approach holds much promise for the development
of innovative, highly selective cancer therapies. This prompted us to develop PAIBꢀSOs
as a new class of cancer prodrugs relying on a CYPꢀbased activation strategy. This study
supports our hypothesis that PAIBꢀSOs are potent anticancer prodrugs with a high
selectivity towards a subset of established breast tumor cells. A subset of these prodrugs
induce CYP1A1 mRNA expression to a moderate and significant extent in sensitive cells,
and exhibit a high affinity and selectivity for activation via this CYP isoform. The other
CYP isoenzymes examined so far did not release the expected PIBꢀSO parent drug. The
design of PAIBꢀSOs relies on a genuine molecular scaffold that can be easily prepared,
purified and modified, and whose synthesis can be scaled up at low cost. Moreover, since
their core molecular structure is unrelated to aminoflavone, benzothiazole and
duocarmycin prodrugs, PAIBꢀSOs exhibit distinctive physicochemical and
biopharmaceutical properties that might circumvent the clinical limitations of 4 and 5.
The CYP1A1ꢀdependent antimicrotubule action of PAIBꢀSOs, which is unique to this
class of CYPꢀactivated prodrugs to the best of our knowledge, might present advantages
over the other prodrugs mentioned above. Indeed, microtubuleꢀdisrupting agents such as
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 76
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
paclitaxel and vinblastine have been used successfully for breast cancer therapy without
the pulmonary toxicity that impaired the use of the other CYP1A1ꢀdependent prodrugs in
clinical trials. Furthermore, the offꢀtarget toxicity of the PAIBꢀSOs might be expected to
be lower than with other CYP1A1ꢀactivatable drugs that potentially affect a wider
spectrum of biological targets due to their reactive character. Indeed, cytoskeletonꢀ
disrupting drugs such as nocodazole are known to progressively downregulate CYP1A1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
64
expression. Unlike nocodazole, which acts indirectly by disrupting the microtubule
transport system that is responsible for CYP1A1 induction by polycyclic aromatic
65
hydrocarbons via the AhR , PAIBꢀSOs, the prodrug form of PIBꢀSOs, might affect
CYP1A1 expression via a twoꢀpronged mechanism. Thus, PAIBꢀSOs likely upregulate
CYP1A1 expression via the microtubule transport system and the AhR, a process that
sustains itself via a selfꢀamplified induction loop in the case of polycyclic aromatic
hydrocarbons that interact with the AhR but are inactive towards the microtubule
3
7
transport system. However, CYP1A1 upregulation by PAIBꢀSOs is expected to be shortꢀ
lived, unlike with other inducers, as it is allosterically inhibited by the direct inactivation
3
7
of microtubules by PIBꢀSOs. In turn, the retroinhibitory action of PIBꢀSOs on the
CYP1A1 induction loop initially afforded by PAIBꢀSOs progressively suppresses further
release of PIBꢀSOs, thereby leading to an autoregulatory mechanism of action for the
PAIBꢀSO prodrug.
In vitro studies have shown that CYP1A1 is a striking example of strictly ligandꢀ
dependent transcriptional control. Whereas CYP1A1 expression is virtually negligible in
cells unexposed to inducing agents, it is strikingly induced upon ligandꢀdependent
4
activation of the AhR by a number of pollutants or pharmaceuticals. Ligandꢀbound AhR
ACS Paragon Plus Environment

Page 17 of 76
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
65
is translocated to the nucleus, via microtubules and other cytoskeletal elements , where it
4
heterodimerizes with the aryl hydrocarbon nuclear translocator. The latter heterodimer
then interacts with xenobiotic DNA response elements present within the CYP1A1
promoter, thereby activating CYP1A1 transcription and expression. An intriguing
hypothesis for the suppression of CYP1A1 induction observed in MCF7 cells incubated
with compound 67 for intervals > 18 h might be an allosteric interference with AhR
nuclear translocation by the steady release and accumulation of the active PIBꢀSO parent
drug upon sustained CYP1A1 induction, due to disruption of the microtubule bundles.
The cytocidal activity of PAIBꢀSOs was found to be independent from the hormone
receptor status of target breast cancer cells. Our previous studies have also shown that
PIBꢀSOs released upon CYP1A1 activation exhibit intact cytocidal activity in cancer
cells resistant to colchicine, paclitaxel, and vinblastine or overexpressing the Pꢀ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
7
glycoprotein. These observations suggest that PAIBꢀSOs might be useful as a secondꢀ
line breast cancer therapy after failure of endocrine therapy, or for the chemotherapy of
the more aggressive tripleꢀnegative tumors, which have a poorer prognosis. In addition to
their potential as a firstꢀline therapy, PAIBꢀSOs might also be considered as an
alternative strategy against tumors that have become resistant to current microtubuleꢀ
disrupting drugs.
2
1, 59, 60
Thus, in contrast with prodrugs of alkylating agents
, despite PAIBꢀSOs being
potential ligands of the AhR, their action is selfꢀlimited by the AhR/microtubuleꢀ
dependent system of CYP1A1 induction by retroinhibition via an autoregulatory
inactivation of the latter system. Therefore, as a new class of CYP1A1ꢀdependent
prodrugs, PAIBꢀSOs present the advantages of acting selectively in tissues displaying a
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 76
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
basal level of CYP1A1 expression, and via the disruption of microtubules in a specific
and selfꢀcontained manner. These features suggest that such drugs may offer especially
attractive pharmacological properties due to the highly localized and selective character
of their mechanism of action.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Conclusion
In conclusion, we developed new and promising antimicrotubule prodrugs that are highly
selective and cytocidal in the low nanomolar range toward several breast cancer cell lines
representative of the cancer tumors diagnosed in clinic. Their mechanism of action is not
dependent on the hormone receptor status and is based on their Nꢀdealkylation by
CYP1A1 into highly potent microtubuleꢀdisrupting agents. Moreover, PAIBꢀSO 67
showed also selectivity toward HTꢀ1080 cells stably transfected with CYP1A1 in the
CAM assay, with only minimal toxicity towards chick embryos. PAIBꢀSOs thus
represent a promising and innovative approach for a highꢀquality oncological new drug
target for the treatments of breast cancers that become unresponsive to currently used
chemotherapy. Finally, this work opens the door for the design of new antimitotic and
antineoplastic prodrugs targeting CYP1A1, and is paving the way to the identification of
other cancer tissues in which CYP1A1 is overexpressed or induced, and the development
of new prodrugs using this strategic approach.
Experimental Section
Biological Methods & Cell Culture. HTꢀ29 human colon carcinoma, MCF7 human
breast carcinoma, MDAꢀMBꢀ231 human breast carcinoma, MDAꢀMBꢀ468 human breast
adenocarcinoma, T47D human breast ductal carcinoma, SKꢀBRꢀ3 human breast
adenocarcinoma and HTꢀ1080 human fibrosarcoma cells were purchased from the
ACS Paragon Plus Environment

Page 19 of 76
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
American Type Culture Collection (Manassas, VA). M21 human skin melanoma cells
were kindly provided by Dr. David Cheresh (University of California, San Diego School
of Medicine). All cell lines were maintained in DMEM containing NaHCO , high glucose
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
concentration, glutamine, phenol red (or not) and sodium pyruvate (Hyclone, Logan, UT)
supplemented with 5% calf serum (Life Technologies). Cultures of HTꢀ1080 cells stably
CYP1A1
transfected with CYP1A1 (HTꢀ1080
) were maintained in the presence of the
selective agent (1% G418). All cells were grown at 37 °C in a 5% CO , moistureꢀ
2
saturated atmosphere.
Antiproliferative Activity Assay. The antiproliferative activity of PAIBꢀSOs was
assessed using the procedure described by the National Cancer Institute for its drug
45
screening program, with slight modifications . The 96ꢀwell microtiter plates were
3
3
seeded with 75 µL of tumor cell suspension (for HTꢀ29, 5.0 x 10 cells; M21, 3.5 x 10
3
3
CYP1A1
3
cells; MCF7, 7.5 x 10 cells; HTꢀ1080, 3.0 x 10 cells; HTꢀ1080
, 3.0 x 10 cells;
3
3
3
MDAꢀMBꢀ231, 3.0 x 10 cells; MDAꢀMBꢀ468, 5.0 x 10 cells; T47D, 3.0 x 10 cells;
3
SKꢀBRꢀ3, 3.0 x 10 cells) in the appropriate medium. Plates were incubated for 24 h.
Drugs freshly solubilized in DMSO were added in fresh medium, and 75 µL aliquots
5
containing a range of concentrations (from 0.75 to 1.0 x 10 nM) of the drug were then
added to the plates, and cells incubated for the indicated time period (48 or 72 h). Cells
were then stained with sulforhodamine B to determine the number of viable cells. Briefly,
cells were fixed by addition of cold trichloroacetic acid to the wells (10% (w/v) final
concentration), for 30 min at 4 °C. Plates were washed five times with tap water and
dried. Fifty µl of a sulforhodamine B solution (0.1% (w/v) in 1% acetic acid) were added
to each well, and the plates were incubated for 15 min at room temperature. Unbound dye
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 76
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
was removed by washing the cell plates five times with 1% acetic acid. Bonded dye was
solubilized in 10 mM Tris base, and the absorbance was then determined using a ꢁQuant
Universal microplate spectrophotometer (Biotek, Winooski, VT) using an optimal
wavelength (530ꢀ565 nm), according to color intensity. Experiments were performed at
least twice with triplicate cultures. The IC assay was considered valid when the relative
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
standard deviation was < 10%.
Cell Cycle Progression. After incubating MCF7, MDAꢀMBꢀ468 and MDAꢀMBꢀ231
5
cells (2.5 x 10 ) with the drugs at a 1ꢀ, 2ꢀ and 5ꢀfold equivalent of the respective IC
5
0
value for 24 h, cells were trypsinized, washed with PBS, resuspended in 250 µL of PBS,
fixed by the addition of 750 µL of iceꢀcold EtOH under agitation, and stored at ꢀ20 °C
until analysis. Prior to FACS analysis, cells were washed with PBS and resuspended in
5
00 µL of PBS containing 2 µg/mL 4’,6’ꢀdiamidinoꢀ2ꢀphenylindole (DAPI). Cell cycle
distribution of fixed cell suspensions was analyzed using an LSR II flow cytometer (BD
Biosciences, Franklin Lakes, NJ).
Fluorescence Microscopy. MDAꢀMBꢀ468, MCF7 and MDAꢀMBꢀ231 cells were seeded
5
at 1 x 10 cells per well in sixꢀwell plates containing 22ꢀµm glass coverslips coated with
fibronectin (10 µg/mL), and incubated for 24 h. Tumor cells were incubated either with
PAIBꢀSO 67 and its PIBꢀSO counterpart 1 at 2ꢀ and 5ꢀfold equivalents of their respective
IC value, or with (0.5 % DMSO) for 24 h. Cells were then washed twice with PBS and
5
0
fixed with 3.7% formaldehyde in PBS for 10 min. After two washes with PBS, the cells
were permeabilized with saponin (0.1% in PBS) and blocked with 3% (w/v) BSA in PBS
for 45 min at 37 °C. The cells were then incubated for 2 h at room temperature with an
antiꢀβꢀtubulin monoclonal antibody (clone TUB 2.1, 1:200) in a solution containing 0.1%
ACS Paragon Plus Environment

Page 21 of 76
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
saponin and 3% BSA in PBS. The cells were washed twice with PBS containing 0.05%
Tween 20 and nuclei were then stained using antiꢀmouse IgG Alexa Fluor 488
(Molecular Probes, Eugene, OR; 1:1000) and DAPI (1.25 ng/mL final concentration) in
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
blocking buffer for 1 h at 37 °C. Cells were then mounted on a microscope slide
overnight with slow fade reagent (DakoCytomation, Carpinteria, CA) before analysis
under an Olympus BX51 microscope. Images were acquired as 8 bitꢀtagged image format
files with a Q imaging RETIGA EXI digital camera using the Image Pro Express
software.
Stable Transfection of CYP1A1 in HT-1080 Cells. The pCMV6ꢀAC_CYP1A1 and
pCMV6ꢀAC_Empty plasmids (Origene, Rockville, MD) were stably transfected in HTꢀ
1080 cells using the antibiotic G418 (geneticin, Thermo Scientific, MA) as selection
agent and Effectene® (Qiagen) as transfection reagent. Briefly, HTꢀ1080 cells were
6
seeded (1 x 10 ) in 100ꢀmm dishes containing DMEM (plus 5% calf serum) and
incubated for 24 h at 37 °C and 5% CO . Cells were then transfected with 2000 ng of
2
plasmid using Effectene® according to the manufacturer’s protocol and incubated for 24
h. Culture media was then renewed and G418 added at 1000 µg/mL (optimal
concentration determined experimentally) until cells were selected. Positive clones were
harvested and stored at ꢀ180 °C until needed.
Empty
CYP1A1
Western Blot Analysis of CYP1A1. HTꢀ1080, HTꢀ1080
and HTꢀ1080
cells
were washed in iceꢀcold PBS and harvested in Laemmli 1 x buffer (4.6% SDS; 20%
glycerol; 125 mM Tris pH 6.8 and 5% βꢀmercaptoethanol). The protein concentration
6
6
was determined using the Lowry method. Twenty micrograms of total protein were
subjected to electrophoresis in 10% bisacrylamide gels. Proteins were then transferred
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 76
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
onto nitrocellulose membranes and incubated with 5% nonꢀfat dry milk and TBST (TBS,
pH 7.4 containing 0.1% Tween 20) for 1 h at room temperature, followed by the rabbit
antiꢀCYP1A1 polyclonal antibody (scꢀ20772, Santa Cruz Biotechnology, CA) for 16 h at
room temperature (1:500). Membranes were washed with TBST and incubated with
peroxidaseꢀconjugated immunoglobulins (1:5000) in TBST for 1 h at room temperature.
After washing the membranes with TBST, immunoblotted proteins were detected using
an enhanced chemiluminescence detection reagent kit. Anti βꢀactinꢀHRP (1:10 000)
(Santa Cruz Biotechnology, CA) was used to normalize the amount of protein per sample
analyzed in each well.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Determination of the Relative Affinity of PAIB-SOs for CYP1A1. The K value was
i
determined using the ethoxyresorufinꢀOꢀdeethylase (EROD) assay, which measures the
inhibition of the conversion of 7ꢀethoxyresorufin (7ꢀER, SigmaꢀAldrich, Oakville, ON,
Canada) into fluorescent resorufin. Potassium phosphate buffer, human CYP1A1
supersomes containing reductase and NADPHꢀregenerating system solutions A and B
were supplied by Corning Discovery Labware (Mississauga, ON, Canada). Briefly, drugs
and 7ꢀER freshly solubilized in DMSO were diluted in potassium phosphate buffer to
final concentrations from 32 to 31 600 nM and from 94 to 3 000 nM for drugs and 7ꢀER,
respectively. The final concentration of DMSO was kept constant at 0.35% (49 mM).
Then, 50 µL of drug solution and 50 µL of 7ꢀER were added successively in 96ꢀwell
microtiter plates and incubated at 37 °C for 5 min. The enzymatic reaction was next
initiated by adding 50 µL of NADPHꢀregenerating system solution A (3%), NADPH
regenerating system solution B (0.6%) and 0.15 pmol of CYP1A1 supersomes in a final
volume of 150 µL. The enzymatic transformation of 7ꢀER into resorufin was monitored
ACS Paragon Plus Environment

Page 23 of 76
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
every minute for a 15ꢀmin interval at 37 °C using a Fluoroskan Ascent Plate Reader
(
(
LabSystems, ThermoFisher Scientific; λ = 544 nm, λ = 590 nm). The initial velocity
exc em
V was calculated for each condition using the linear portion of the curve (1ꢀ5 min).
0
)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Then, the maximal velocity (Vmax) of the reaction and the MichaelisꢀMenten constant
(K ) were calculated using a LineweaverꢀBurk plot. The K value was derived from the
m
i
MichaelisꢀMenten equation in the form:
ꢇ
ꢃꢄꢅ(ꢀꢆꢈꢉ)
ꢁꢄꢊꢋ
ꢀ
ꢀ
ꢀ
=
ꢅꢌꢅ ꢅ+ꢅꢁ
.
ꢁꢂ
[ꢍ]
ꢄꢊꢋ
Oxidation of PAIB-SOs by Various CYPs. Human supersomes CYP1A1, CYP1A2,
CYP1B1, CYP3A4 (including B5) and Human Liver S9 Fraction microsome pools from
human liver donors (20 mg/mL) including NADPH CYP reductase, potassium phosphate
(0.5 M) and NADPHꢀregenerating system solutions A and B, were supplied by BD
Biosciences (Mississauga, ON, Canada). Briefly, drugs freshly solubilized at 10 mM in
DMSO were further diluted to 1 mM in acetonitrile. Then, 710 µL of purified water, 200
µL of potassium phosphate buffer, 50 µL of NADPHꢀregenerating System Solution A, 10
µL of NADPHꢀregenerating System Solution B and 10 µL of the substrate (in
acetonitrile) were mixed and warmed to 37 °C for 5 min. The enzymatic reaction was
next initiated by adding 20 µL of freshly thawed human supersomes (1 nmol/mL) to the
mixture. At 0, 15, 30, 60, 120 and 240 min, 100 µL of the mixture were withdrawn, to
which 100 µL of acetonitrile were added, and the sample was mixed and kept on ice until
HPLC analysis. Samples removed at the end of the experimental period (240 min) were
further analyzed by HPLCꢀMS using internal standards of PAIBꢀSOs and PIBꢀSOs.
HPLC analysis was performed using a Prominence LCMSꢀ2020 system with binary
solvent equipped with a UV/visible photodiode array and an APCI probe (Shimadzu,
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 76
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Columbia, MD). All the MS spectra were recorded in positive ionization mode. All
compounds were eluted within 25 min using an MeOH/H O linear gradient (1.0 mL/min)
2
on a Zorbax SBꢀC18 reversedꢀphase column (5 µm, 4.6 x 250 mm) equipped with a
Zorbax XDB C18 precolumn (5ꢀµm, 4 x 4 mm).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Quantitative PCR Analysis of CYP1A1 mRNA. MCFꢀ7 and MDAꢀMBꢀ231 cells were
treated with 10 nM of PAIBꢀSO 67, and CYP1A1 expression was determined at 18 and 24
h. Total RNA was extracted with TriReagent (Sigma) following the manufacturer’s
instructions. Reverse transcription was performed with MꢀMuLV reverse transcriptase
(NEB), random hexamers and oligo(dT). Four ng of cDNA were analyzed in
quadruplicate for each data point using iTAQ Universal Sybr Green Supermix (BioRad).
The following cycling program was performed using a CFX384 RealTimeSystem
(BioRad): 95°C for 30 sec and 40 cycles at 95°C for 5 sec, followed by 60°C for 30 sec.
ꢀꢂꢂCt
67
Relative expression was determined with the 2
method , with ACTB and B2M as
control genes for normalization. The primers used were: (ACTB), Fꢀ
TCAAGATCATTGCTCCTCCTGAG and RꢀACATCTGCTGGAAGGTGGAGA;
(B2M), FꢀACTGAATTCACCCCCACTGA and RꢀCCTCCATGATGCTGCTTACA; and
(CYP1A1), FꢀCCCAACCCTTCCCTGAATG and RꢀTTCTCCTGACAGTGCTCAATC.
CYP1A1
CAM Tumor Assay. CYP1A1ꢀtransfected (HTꢀ1080
) and wildꢀtype human HTꢀ
1080 fibrosarcoma cells were used to assess the antitumor activity of PAIBꢀSO 67 and 1
in the CAM assay. Briefly, fertilized chicken eggs purchased from Couvoirs Victoriaville
(Victoriaville, QC, Canada) were incubated for 10 d in a ProꢀFI egg incubator (Lyon
Electric, Chula Vista, San Diego, CA) fitted with an automatic egg turner before being
transferred to a RollꢀX static incubator for the rest of the incubation time. The eggs were
ACS Paragon Plus Environment

Page 25 of 76
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
kept at 37 °C in a 60% relative humidity atmosphere for the entire incubation period. On
day 10, using a hobby drill (Dremel, Racine, WI), a hole was drilled on the side of the
egg, and a negative pressure was applied to create a new air sac. A window was opened
on this new air sac and was covered with transparent adhesive tape to prevent
contamination. A freshly prepared suspension (40 µL) of HTꢀ1080 human fibrosarcoma
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
CYP1A1
5
or HTꢀ1080
(3.5 x 10 cells/egg) cells was applied directly onto the freshly exposed
CAM tissue through the window. On day 11, the drugs dissolved in DMSO (40 mM)
were extemporaneously diluted in the excipient (cremophor/99% EtOH/PBS,
6
.25/6.25/87.5 v/v). The concentration of DMSO in the excipient was kept below 0.5% to
minimize toxicity and osmotic stress. The drug dissolved in 100 µL of excipient was
injected intravenously into 10-12 eggs. The eggs were incubated until day 17, at which
time the embryos were euthanized at 4 ºC, followed by decapitation. Tumors were
collected, and the tumor wet weight was recorded. Drugs were assessed twice in two
independent experiments (total of 20ꢀ24 eggs). The number of dead embryos and signs of
toxicity from the different groups were recorded.
General Chemical Procedures. Proton NMR spectra were recorded on a Bruker AMꢀ
3
00 spectrometer (Bruker, Germany). Chemical shifts (δ) are reported in parts per
million. IR spectra were recorded on a Magna FTꢀIR spectrometer (Nicolet Instrument
Corporation, Madison, WI). Uncorrected melting points were determined using an
®
Electrothermal melting point apparatus. HPLC analysis was performed using a
Prominence LCMSꢀ2020 system with binary solvent equipped with a UV/visible
photodiode array and an APCI probe (Shimadzu, Columbia, MD). All the MS spectra
were recorded in positive ionization mode. Compounds were eluted within 10 min on an
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 76
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Alltech Alltima C18 reversedꢀphase column (5 ꢁm, 250 mm × 4.6 mm) equipped with an
Alltech Alltima C18 precolumn (5 ꢁm, 7.5 mm × 4.6 mm) with an MeOH/H O linear
2
gradient (1.0 mL/min). The purity of the final compounds synthesized was > 95%. All
reactions were performed under a dried Ar atmosphere. All chemicals were supplied by
Aldrich Chemicals (Milwaukee, WI) or VWR International (MontꢀRoyal, QC, Canada)
and used as received unless specified otherwise. Liquid flash chromatography was
performed on silica gel F60 (60 Å, 40-63 ꢁm) supplied by Silicycle (Québec, Canada)
using an FPX flash purification system (Biotage, Charlottesville, VA) and the indicated
solvent mixture (expressed as v/v ratios). Solvents and reagents were used without prior
purification unless specified otherwise. The progress of all reactions was monitored by
TLC using precoated 60 F254 silica gel plates (VWR International). The chromatograms
were viewed under UV light at 254 and/or 275 nm.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Preparation of Compounds 6-68
The appropriate sulfonyl chloride derivative (8.00 mmol, compounds 73ꢀ76) was
suspended in dry CH Cl (10 mL), and the selected phenol (8.00 mmol) and then
2
2
triethylamine (8.00 mmol) were successively added dropwise. The mixture was stirred at
room temperature for 48 h, and then acidified with 1 N HCl (10 mL) and extracted thrice
with CH Cl (10 mL). The combined CH Cl extracts were washed successively with 1 N
2
2
2
2
NaOH (20 mL) and brine (20 mL), dried over sodium sulfate, filtered, and the filtrate
dried under vacuum. The residue was purified by flash chromatography on silica gel.
2
-Tolyl
4-(3-Methyl-2-oxoimidazolidin-1-yl)benzenesulfonate
(6).
Flash
chromatography (CH Cl to CH Cl /EtOAc (95:5)). Yield, 93%; white solid; mp: 157ꢀ
2
2
2
2
ꢀ1
1
1
58 °C. IR ν: 2928 (Ar), 1701 (C=O) cm . H NMR (CDCl ): δ 7.74ꢀ7.66 (m, 4H, Ar),
3
ACS Paragon Plus Environment

Page 27 of 76
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
7
.12ꢀ7.07 (m, 3H, Ar), 6.98ꢀ6.95 (m, 1H, Ar), 3.82ꢀ3.77 (m, 2H, CH ), 3.53ꢀ3.48 (m, 2H,
2
13
CH ), 2.89 (s, 3H, CH ), 2.05 (s, 3H, CH ). C NMR (CDCl ): δ 157.1, 148.4, 145.8,
2
3
3
3
1
31.6, 129.5, 127.7, 126.9, 126.9, 122.3, 116.1, 43.5, 42.0, 31.0, 16.3. MS (APSI+) m/z
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
found 347.1; C H N O S (M + H) expected, 347.1.
1
7
19
2
4
2-Ethylphenyl 4-(3-Methyl-2-oxoimidazolidin-1-yl)benzenesulfonate (7). Flash
chromatography (CH Cl to CH Cl /EtOAc (95:5)). Yield, 92%; white solid; mp: 126ꢀ
2
2
2
2
1
128 °C. IR ν: 2878 (Ar), 1703 (C=O) cmꢀ1. H NMR (CDCl ): δ 7.76ꢀ7.67 (m, 4H, Ar),
3
7
.19ꢀ7.05 (m, 3H, Ar), 6.98ꢀ6.96 (m, 1H, Ar), 3.81ꢀ3.76 (m, 2H, CH ), 3.52ꢀ3.47 (m, 2H,
2
13
CH ), 2.88 (s, 3H, CH ), 2.48 (q, 2H, J = 7.6 Hz, CH ), 1.08 (t, 3H, J = 7.6 Hz, CH ). C
2
3
2
3
NMR (CDCl ): δ 157.1, 147.9, 145.8, 137.3, 129.8, 129.5, 127.8, 127.1, 126.8, 122.1,
3
+
1
16.1, 43.5, 42.0, 31.0, 22.8, 14.1. MS (APSI+) m/z found 361.1; C H N O S (M + H)
1
8
21
2
4
expected, 361.1.
2
-Methoxyphenyl 4-(3-Methyl-2-oxoimidazolidin-1-yl)benzenesulfonate (8). Flash
chromatography (CH Cl to CH Cl /EtOAc (9:1)). Yield, 61%; white solid; mp: 134ꢀ136
2
2
2
2
ꢀ1 1
°C. IR ν: 2897 (Ar), 1707 (C=O) cm . H NMR (CDCl ): δ 7.76ꢀ7.63 (m, 4H, Ar), 7.19ꢀ
3
7
.09 (m, 2H, Ar), 6.87ꢀ6.79 (m, 2H, Ar), 3.82ꢀ3.77 (m, 2H, CH ), 3.54ꢀ3.48 (m, 5H, CH2
2
1
3
and CH ), 2,89 (s, 3H, CH ). C NMR (CDCl ): δ 157.1, 151.8, 145.6, 138.5, 129.7,
3
3
3
1
3
2
28.0, 127.9, 124.0, 120.6, 115.8, 112.7, 55.6, 43.6, 42.0, 31.0. MS (APSI+) m/z found
+
63.1; C H N O S (M + H) expected, 363.1.
17 19
2
5
,4-Dimethylphenyl 4-(3-Methyl-2-oxoimidazolidin-1-yl)benzenesulfonate (9). Flash
chromatography (CH Cl to CH Cl /EtOAc (95:5)). Yield, 51%; white solid; mp: 159ꢀ
2
2
2
2
ꢀ1 1
161 °C. IR ν: 2899 (Ar), 1706 (C=O) cm . H NMR (CDCl ): δ 7.72ꢀ7.64 (m, 4H, Ar),
3
6
.90ꢀ6.79 (m, 3H, Ar), 3.81ꢀ3.76 (m, 2H, CH ), 3.52ꢀ3.46 (m, 2H, CH ), 2.87 (s, 3H,
2
2
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 76
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
13
CH ), 2.23 (s, 3H, CH ), 1.99 (s, 3H, CH ). C NMR (CDCl ): δ 157.1, 146.2, 145.8,
3
3
3
3
136.7, 132.2, 131.1, 129.5, 127.7, 127.4, 122.0, 116.1, 43.5, 42.0, 30.9, 20.8, 16.2. MS
+
(
APSI+) m/z found 361.1; C H N O S (M + H) expected, 361.1.
18
21
2
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
,4,5-Trichlorophenyl 4-(3-Methyl-2-oxoimidazolidin-1-yl)benzenesulfonate (10).
Flash chromatography (CH Cl to CH Cl /EtOAc (95:5)). Yield, 84%; white solid; mp:
2
2
2
2
ꢀ1
1
1
81ꢀ183 °C. IR ν: 2937 (Ar), 1711 (C=O) cm . H NMR (CDCl and MeOD): δ 7.76ꢀ
3
7.65 (m, 4H, Ar), 7.41ꢀ7.38 (m, 2H, Ar), 3.84ꢀ3.78 (m, 2H, CH ), 3.53ꢀ3.48 (m, 2H,
2
13
CH ), 2.86 (s, 3H, CH ). C NMR (CDCl and MeOD): δ 157.1, 146.3, 144.2, 131.6,
2
3
3
1
4
2
31.5, 131.3, 129.9, 126.8, 126.4, 125.7, 116.3, 43.5, 42.0, 30.8. MS (APSI+) m/z found
+
35.0; C H Cl N O S (M + H) expected, 435.0.
16
14
3
2
4
,4,6-Trichlorophenyl 4-(3-Methyl-2-oxoimidazolidin-1-yl)benzenesulfonate (11).
Flash chromatography (CH Cl to CH Cl /EtOAc (95:5)). Yield, 73%; white solid; mp:
2
2
2
2
ꢀ1
1
1
7
3
1
85ꢀ187 °C. IR ν: 3707 (Ar), 1715 (C=O) cm . H NMR (CDCl and MeOD): δ 7.83ꢀ
3
.80 (m, 2H, Ar), 7.68ꢀ7.65 (m, 2H, Ar), 7.26 (s, 2H, Ar), 3.82ꢀ3.77 (m, 2H, CH ), 3.51ꢀ
2
13
.46 (m, 2H, CH ), 2.83 (s, 3H, CH ). C NMR (CDCl and MeOD): δ 157.2, 146.1,
2
3
3
42.3, 132.7, 130.8, 129.7, 129.1, 128.3, 116.2, 43.5, 42.0, 30.8. MS (APSI+) m/z found
+
435.0; C H Cl N O S (M + H) expected, 435.0.
16
14
3
2
4
3
-Tolyl
4-(3-Methyl-2-oxoimidazolidin-1-yl)benzenesulfonate
(12).
Flash
chromatography (CH Cl to CH Cl /EtOAc (95:5)). Yield, 99%; white solid; mp: 123ꢀ
2
2
2
2
ꢀ
1 1
1
7
3
1
25 °C. IR ν: 2926 (Ar), 1694 (C=O) cm . H NMR (CDCl ): δ 7.71ꢀ7.64 (m, 4H, Ar),
3
.12ꢀ6.99 (m, 2H, Ar), 6.82 (s, 1H, Ar), 6.69ꢀ6.67 (m, 1H, Ar), 3.80ꢀ3.75 (m, 2H, CH2),
1
3
.51ꢀ3.46 (m, 2H, CH ), 2.87 (s, 3H, CH ), 2.25 (s, 3H, CH ). C NMR (CDCl ): δ
2
3
3
3
57.1, 149.6, 145.8, 140.0, 129.6, 129.2, 127.8, 127.1, 123.0, 119.1, 116.0, 43.5, 41.9,
ACS Paragon Plus Environment

Page 29 of 76
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
+
3
1.0, 21.2. MS (APSI+) m/z found 347.1; C H N O S (M + H) expected, 347.1.
17
19
2
4
3
-Methoxyphenyl 4-(3-Methyl-2-oxoimidazolidin-1-yl)benzenesulfonate (13). Flash
chromatography (CH Cl to CH Cl /EtOAc (95:5)). Yield, 92%; white solid; mp: 111ꢀ
2
2
2
2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ꢀ
1 1
1
13 °C. IR ν: 2897 (Ar), 1713 (C=O) cm . H NMR (CDCl ): δ 7.71ꢀ7.63 (m, 4H, Ar),
3
7.13ꢀ7.08 (m, 1H, Ar), 6.75ꢀ6.71 (m, 1H, Ar), 6.54ꢀ6.48 (m, 2H, Ar), 3.78ꢀ3.74 (m, 2H,
13
CH ), 3.67 (s, 3H, CH ), 3.51ꢀ3.45 (m, 2H, CH ), 2.87 (s, 3H, CH ). C NMR (CDCl ): δ
2
3
2
3
3
160.4, 157.0, 150.5, 145.9, 129.9, 129.6, 126.9, 116.1, 114.3, 112.9, 108.4, 55.5, 43.5,
+
4
1.9, 31.0. MS (APSI+) m/z found 363.1; C H N O S (M + H) expected, 363.1.
17
19
2
5
3
-Fluorophenyl 4-(3-Methyl-2-oxoimidazolidin-1-yl)benzenesulfonate (14). Flash
chromatography (CH Cl to CH Cl /EtOAc (95:5)). Yield, 99%; white solid; mp: 175ꢀ
2
2
2
2
ꢀ1
1
1
77 °C. IR ν: 2902 (Ar), 1697 (C=O) cm . H NMR (CDCl and MeOD): δ 7.65ꢀ7.58
3
(m, 4H, Ar), 7.18ꢀ7.11 (m, 1H, Ar), 6.89ꢀ6.83 (m, 1H, Ar), 6.68ꢀ6.64 (m, 2H, Ar), 3.79ꢀ
1
3
3
.74 (m, 2H, CH ), 3.49ꢀ3.44 (m, 2H, CH ), 2.81 (s, 3H, CH ). C NMR (CDCl and
2 2 3 3
MeOD): δ 164.2, 160.9, 157.3, 150.2, 150.0, 145.9, 130.4, 130.3, 129.6, 126.5, 118.1,
1
18.1, 116.2, 114.3, 114.1, 110.5, 110.2, 43.5, 42.0, 30.7. MS (APSI+) m/z found 351.1;
+
C H FN O S (M + H) expected, 351.1.
16
16
2
4
3-Chlorophenyl 4-(3-Methyl-2-oxoimidazolidin-1-yl)benzenesulfonate (15). Flash
chromatography (CH Cl to CH Cl /EtOAc (9:1)). Yield, 81%; white solid; mp: 105ꢀ106
2
2
2
2
ꢀ1 1
°
C. IR ν: 2900 (Ar), 1705 (C=O) cm . H NMR (CDCl ): δ 7.67 (s, 4H, Ar), 7.19ꢀ7.15
3
(
m, 2H, Ar), 7.02 (s, 1H, Ar), 6.83ꢀ6.79 (m, 1H, Ar), 3.81ꢀ3.75 (m, 2H, CH ), 3.52ꢀ3.47
2
1
3
(
m, 2H, CH ), 2.87 (s, 3H, CH ). C NMR (CDCl ): δ 157.0, 150.0, 146.1, 134.8, 130.4,
2
3
3
129.6, 127.4, 126.3, 123.0, 120.7, 116.1, 43.5, 41.9, 31.0. MS (APSI+) m/z found 367.1;
+
C H ClN O S (M + H) expected, 367.1.
16
16
2
4
ACS Paragon Plus Environment