A potent and highly efficacious Bcl-2/Bcl-xL inhibitor

Article abstract of DOI:10.1021/jm4001105

Our previously reported Bcl-2/Bcl-xL inhibitor, 4, effectively inhibited tumor growth but failed to achieve complete regression in vivo. We have now performed extensive modifications on its pyrrole core structure, which has culminated in the discovery of 32 (BM-1074). Compound 32 binds to Bcl-2 and Bcl-xL proteins with K_i values of <1 nM and inhibits cancer cell growth with IC₅₀ values of 1-2 nM in four small-cell lung cancer cell lines sensitive to potent and specific Bcl-2/Bcl-xL inhibitors. Compound 32 is capable of achieving rapid, complete, and durable tumor regression in vivo at a well-tolerated dose schedule. Compound 32 is the most potent and efficacious Bcl-2/Bcl-xL inhibitor reported to date.

Full text of DOI:10.1021/jm4001105

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Article
A Potent and Highly Efficacious Bcl-2/Bcl-xL Inhibitor
Angelo Aguilar, Haibin Zhou, Jianfang Chen, Liu Liu, Longchuan Bai, Donna McEachern,
Chao-Yie Yang, Jennifer L. Meagher, Jeanne A. Stuckey, and Shaomeng Wang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm4001105 • Publication Date (Web): 28 Feb 2013
Downloaded from http://pubs.acs.org on March 10, 2013
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A Potent and Highly Efficacious Bcl-2/Bcl-xL Inhibitor
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,§
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Angelo Aguilar , Haibin Zhou , Jianfang Chen , Liu Liu , Longchuan Bai , Donna
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McEachern , Chao-Yie Yang , Jennifer Meagher , Jeanne Stuckey and Shaomeng Wang *
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Comprehensive Cancer Center and Departments of Internal Medicine,
#
Pharmacology and Medicinal Chemistry, and Life Sciences Institute, University
of Michigan, 1500 E. Medical Center Drive, Ann Arbor, MI 48109-0934, USA
§
Equal contribution
*
To whom correspondence should be addressed
Tel: 734-615-0362
Fax: 734-647-9647
Email: shaomeng@umich.edu
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ABSTRACT: Our previously reported Bcl-2/Bcl-xL inhibitor, 4, effectively inhibited tumor
growth but failed to achieve complete regression in vivo. We have now performed extensive
modifications on its pyrrole core structure, which has culminated in the discovery of 32 (BM-
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074). Compound 32 binds to Bcl-2 and Bcl-xL proteins with K values of < 1 nM and inhibits
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cancer cell growth with IC50 values of 1-2 nM in four small-cell lung cancer cell lines sensitive
to potent and specific Bcl-2/Bcl-xL inhibitors. Compound 32 is capable of achieving rapid,
complete and durable tumor regression in vivo at a well-tolerated dose-schedule. Compound 32
is the most potent and efficacious Bcl-2/Bcl-xL inhibitor reported to date.
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INTRODUCTION
A common feature in many different types of human tumors is overexpression of the pro-
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survival Bcl-2 family members Bcl-2 and Bcl-xL, which make tumor cells resistant to
conventional cancer therapeutic agents. Therefore, it has been proposed that small-molecule
inhibitors of Bcl-2 and Bcl-xL may have a promising therapeutic potential for the treatment of
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human cancer.
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Figure 1: Chemical structures of 1 (ABT-737) , 2 (ABT-263) and our recently reported potent
and specific Bcl-2/Bcl-xL inhibitors.
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Compounds 1 and 2 represent two highly potent and specific Bcl-2/Bcl-xL inhibitors.
Preclinical studies have shown that 1 and 2 are effective as single agents against lymphomas,
chronic lymphoid leukemia (CLL) and a subset of small-cell lung cancer (SCLC) models, and
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can enhance the antitumor activity of conventional anticancer drugs and γ-irradiation in
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preclinical models of diverse tumor types. Compound 2 is currently in Phase I/II clinical trials,
where it has shown promising single-agent activity in patients with CLL and B-cell lymphomas.
Because design of Bcl-2 and Bcl-xL inhibitors involves targeting the interaction of Bcl-
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/Bcl-xL proteins with their pro-apoptotic binding partners such as BAD and BIM proteins, a
challenging task in drug discovery, very few new, potent, specific and bona fide small-molecule
inhibitors of this interaction have been reported, even after the discovery of 1 and 2. Recently,
we reported the structure-based design of a family of new, highly potent and specific Bcl-2/Bcl-
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xL inhibitors (Figure 1).
Our initial lead compound 3 binds to Bcl-2 and Bcl-xL with high
affinities and potently inhibits cell growth in cancer cell lines that are sensitive to 1 and 2, but it
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lacks chemical stability and fails to achieve significant in vivo antitumor activity. Subsequent
structure-based design and optimization of 3 led to compounds 4 and 5, which have excellent
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chemical stability, bind to Bcl-2 and Bcl-xL with K values of <1 nM and inhibit cancer cell
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growth with low nanomolar activity. While 5 effectively inhibits tumor growth and in fact
induces tumor regression in the H146 small-cell lung cancer model at its maximum tolerated
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dose (MTD), the tumor regression it caused was transient, suggesting further optimization was
needed toward our goal of developing a new class of Bcl-2/Bcl-xL inhibitors for cancer
treatment. Very recently, we have reported further structure-based optimization of compound 5,
with a focus on two regions in the molecule, which led to the successful discovery of a superior
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compound, 6 (BM-957). Compound 6 binds to Bcl-2 and Bcl-xL with K
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values < 1 nM and
inhibits tumor cell growth with IC values of 21-22 nM against H146 and H1417 small-cell
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cancer cell lines. Significantly, 6 achieved tumor regression in an animal model of human
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cancer.
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In the previous study, which yielded compound 6, we focused our modifications on the
nitro group and the soluble “tail” containing the N,N-dimethylamino group in compound 4. In the
present study, we report our further optimization of 4, with a focus on its 1H-pyrrole-3-
carboxylic acid core structure. Our efforts have culminated in the discovery of 32 (BM-1074),
which, based upon its cellular activity and in vivo efficacy, is arguably the most potent and
efficacious Bcl-2/Bcl-xL inhibitor discovered to date.
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Results and Discussion
Previously, we have shown that removal of the acid group from the pyrrole carboxylic
acid of 4, yielding compound 7, resulted in a >50-fold decrease in binding affinity to Bcl-2 and a
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modest decrease in binding affinity to Bcl-xL. Compound 7, at concentrations as high as 10 μM,
was found to be completely inactive in inhibition of cell growth in the H146 cancer cell line
(Table 1), suggesting that very high binding affinity to Bcl-2/Bcl-xL is clearly needed in order
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for small-molecule inhibitors to effectively inhibit cancer cell growth. Converting this acid
group into a methylamide (compound 8) has a modest negative effect on binding to Bcl-2 but has
no effect on binding to Bcl-xL (Table 1). Interestingly, compound 8 has an IC50 value of 36 nM
in the H146 cell line (Table 1), and is thus slightly more potent than compound 4, suggesting that
compound 8 has superior cell permeability compared to compound 4. These binding and cellular
data showed that modifications of the acid group of 4 can have a significant negative or positive
effect on binding to Bcl-2/Bcl-xL and on cellular activity. Accordingly, we have made additional
modifications at this position in order to further explore the structure-activity relationships and to
identify promising new compounds. All the designed and synthesized new compounds were
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tested with our standard fluorescence-polarization (FP) assays for their binding affinities to Bcl-
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and Bcl-xL proteins and for their cell growth inhibitory activity in the H146 small-cell lung
cancer cell line, which is sensitive to potent and bona fide Bcl-2/Bcl-xL inhibitors such as
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compounds 1-6, and the results are presented in Table 1.
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Table 1. Structure-activity relationship studies on the pyrrole ring of lead compound 4.
Binding Affinities
Cell growth inhibition
Compound ID
R1
R2
(IC ± SD, nM)
(IC ± SD, nM)
50
50
Bcl-2
Bcl-XL
H-146
61 ± 39
>1,000
4
7
8
9
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CF3
-Cl
-CO H
1.3 ± 0.2
99 ± 5
6 ± 1
2
-H
11 ± 6
6 ± 3
-CONHCH3
-CONH2
5 ± 1
36 ± 26
245 ± 17
12 ± 2
19 ± 5
14 ± 5
15 ± 1
9.4 ± 0.6
5 ± 1
1
0
7.5 ± 1.2
5.6 ± 0.6
0.9 ± 0.2
260 ± 93
271 ± 74
17 ± 6
11
66 ± 4
12
-CONHSO CH
38 ± 22
>1,000
2
3
1
1
3
4
-Cl
-CF3
-CN
12 ± 8
13 ± 2
4.5 ± 1.3
6 ± 2
>1,000
15
491 ± 160
496 ± 59
100 ± 17
1
1
6
7
-CO H
1.1 ± 0.6
1.5 ± 0.9
2
-CO H
4.7 ± 1.5
2
First, changing the N-methylamide in compound 8 to an amide group resulted in
compound 9, which is 4- and 2-times less potent than 8, respectively, in binding to Bcl-2 and
Bcl-xL. Compound 9 has an IC50 value of 245 nM in the H146 cell line and is thus 6-times less
potent than 8 in this cell line. Replacement of the methyl group in the N-methylamide of
compound 8 with a cis-3-hydroxy-3-methylcyclobutyl group or a 3-methylazetidine-3-ol group
resulted in compounds 10 and 11, respectively. Compounds 10 and 11 bind to Bcl-2 with
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affinities similar to that of compound 8 but both compounds are slightly less potent than 8 in
binding to Bcl-xL. While compounds 10 and 11 have very similar affinities to Bcl-2 and Bcl-xL
proteins, their activity in inhibition of cell growth in the H146 cell line differs by a factor of 6,
further indicating that modifications of this site can have a significantly different effect on
binding affinities to Bcl-2/Bcl-xL and cellular activity for this class of compounds.
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Compound 4
E129
R132
N136
R139
W137
L130
F191
Y101
L112
L108
V141
F146
Y195
F97
A104
F105
R100
E96
Bcl-xL
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Figure 2. Crystal structure of 4 (yellow) in a complex with Bcl-xL (gray) (PDB entry: 3SP7).
The red oval highlights space available for further modifications around the pyrrole ring. The
hydrogen bond/salt bridge of the acid group to Arg 132 is indicated by a dashed cyan line.
In the co-crystal structure of 4 in complex with Bcl-xL (Figure 2), the acid group in 4
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forms a hydrogen bond/salt bridge with Arg 132 in Bcl-xL, and we synthesized compound 12 to
test if the methylsulfonylamide, a bioisostere of the carboxylic acid, can replace the acid group in
4 and achieve high binding affinities to Bcl-2 and Bcl-xL and potent cellular activity in the H146
cell line. Compound 12 is as potent as 4 in binding to both Bcl-2 and Bcl-xL proteins, and has an
IC value of 38 nM in the H146 cell line, thus twice as potent as 4. These results show that the
5
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methylsulfonylamide group can indeed effectively replace the acid group, achieving not only
high binding affinities to Bcl-2/Bcl-xL, but also potent cellular activity.
In addition to their ability to form hydrogen bonds and salt bridges, acid and methyl-
sulfonylamide groups have strong electron withdrawing properties. To further define the
contributions of these groups for binding to Bcl-2 and Bcl-xL and cellular activity in compounds
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and 12, we synthesized compounds 13-15 to investigate if other electron withdrawing groups,
such as Cl, CF and CN, can effectively replace the acid group in compound 4 to achieve high
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binding affinities to Bcl-2 and Bcl-xL and potent cellular activity. Although compound 13 with
3
Cl and compound 14 with CF have similar potencies to Bcl-xL as compared to 4, both
compounds are 100-times less potent than 4 in binding to Bcl-2. Compounds 13 and 14 are also
>100-times less potent than 4 in inhibition of cell growth in the H146 cancer cell line.
Compound 15, in which the acid group in 4 has been replaced by a nitrile, is >10-times less
potent than 4 in binding to Bcl-2 but has a similar binding affinity to Bcl-xL, as compared to 4.
Compound 15 has an IC50 value of 496 nM in the H146 cell line, and is thus 8 times less potent
than 4.
In our co-crystal structure of 4 in complex with Bcl-xL (Figure 2), the methyl group in 4
(R
We thus synthesized compounds 16 and 17 to determine whether the methyl group can be
replaced by other small hydrophobic groups such as CF and Cl groups to achieve high binding
affinities to Bcl-2/Bcl-xL. As suggested by the co-crystal structure for compound 4, both 16 and
1 3
= CH ) is in close contact with the hydrophobic portion of the Glu129 side chain in Bcl-xL.
3
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7 bind to Bcl-2 and Bcl-xL with the same high affinities as compared to compound 4 (Table 1).
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In the H146 cell line, compound 17 is slightly less potent than 4, but 16 is 8-times less potent
than 4.
In all the synthesized compounds described here, the pyrrole was retained as the core
structure. We next designed and synthesized 5 classes of compounds to investigate if other 5-
membered heteroaromatic groups can effectively replace the pyrrole moiety in compound 4 and
achieve high binding affinities to Bcl-2/Bcl-xL and potent cellular activity. Since our data
showed that removal of the carboxylic acid in 4 greatly decreases its binding to Bcl-2 and
cellular activity while replacement of the carboxylic acid with a methylsulfonylamide group can
effectively maintain both high binding affinities to Bcl-2/Bcl-xL and potent cellular activity, we
have synthesized several new compounds possessing or lacking an acid or a
methylsulfonylamide group in each class of compounds. The binding and cellular data for these
compounds are summarized in Table 2.
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For each of these five different classes, compounds 23-27, containing either an acid or a
methylsulfonylamide group, show high binding affinities to both Bcl-2 and Bcl-xL proteins with
IC50 values of 3-7 nM. However, these compounds have a significantly weaker activity in
cellular assays than compounds 4 and 12. While compounds 26 and 27 have IC50 values of 541
nM and 199 nM, respectively, compounds 23, 24 and 25 have minimal activity at 1 μM.
In comparison, analogues (18-22) without either an acid or a methylsulfonylamide group
show at least a 10-fold weaker binding affinity to Bcl-2 than their corresponding analogues 23-
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7. Compounds 18-22 are also 5-10 times less potent in their binding to Bcl-xL than compounds
3-27. Consistent with their weaker affinities to both Bcl-2 and Bcl-xL, compounds 18-22 have
IC50 values of >10 µM in the H146 cell line in the cell growth inhibition assay, further
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emphasizing that very high binding affinities are needed in order for small molecule inhibitors of
Bcl-2 and Bcl-xL to achieve potent cellular activity.
Table 2. Structure-activity relationships for compounds in which other 5-membered hetero-
aromatic rings were used to replace the pyrrole in the initial lead compound 4.
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Cell growth
inhibition
Cell growth
inhibition
Binding Affinities
(IC50 ± SD, nM)
Binding Affinities
(IC50 ± SD, nM)
Compound
ID
Compound
ID
Core
Core
(
IC50 ± SD, nM)
(IC50 ± SD, nM)
Bcl-2
Bcl-XL
H-146
Bcl-2
Bcl-XL
H-146
4
1.3 ± 0.2
6 ± 1
61 ± 39
7
99 ± 5
11 ± 6
>10,000
12
23
24
25
26
27
0.9 ± 0.2
5 ± 1
38 ± 22
>1,000
18
46 ± 10
110 ± 4
18 ± 1
32 ± 9
15 ± 1
26 ± 3
22 ± 7
>10,000
>10,000
>10,000
>10,000
>10,000
3.8 ± 1.1 8.3 ± 0.7
5.4 ± 1.1 5 ± 2
1
2
2
2
9
0
1
2
>1,000
210 ± 28
140 ± 53
350 ± 146
7.1 ± 1.6 6.1 ± 2.2
3.7 ± 0.6 5.9 ± 1.7
3.8 ± 1.0 4.7 ± 0.2
>1,000
541 ± 76
199 ± 46
Judged by the binding affinities to Bcl-2 and Bcl-xL and the cellular activity in the H146
cell line, compounds 4 and 12 are two of the most potent and promising compounds and,
accordingly, we focused on these two compounds in our further optimization.
Based upon the co-crystal structure of compound 4 complexed with Bcl-xL (Figure 2),
the N-methyl group in 4 inserts into a hydrophobic pocket in Bcl-xL, which can, however,
accommodate a hydrophobic group larger than methyl. Indeed, our previous study showed that
2 3
replacement of the N-methyl group in 4 (R = CH , Table 3) with either an N-ethyl or N-
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isopropyl group yielded analogues 5 and 28 with high binding affinities to Bcl-2/Bcl-xL and
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significantly improved cellular activity (Table 3). In fact, compounds 5 and 28 are 8- and 20-
times more potent than compound 4 in inhibition of cell growth in the H146 cell line.
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To probe this site further and to identify the optimal group for binding to Bcl-2/Bcl-xL
and cellular activity, we designed and synthesized compounds 29 and 30 in which the N-methyl
group in compound 4 was replaced by either an N-n-propyl or N-n-butyl group, respectively.
Although both 29 and 30 have high binding affinities to Bcl-2 and Bcl-xL, they are several times
less potent than compounds 5 and 28 in their binding to Bcl-2. Consistent with their weaker
affinities to Bcl-2, compounds 29 and 30 are >5- and >10-times less potent than compounds 5
and 28, respectively, in inhibition of cell growth in the H146 cell line. Our data therefore showed
that the N-ethyl and N-isopropyl groups at this site in compounds 5 and 28 are most optimal for
achieving high binding affinities to Bcl-2 and Bcl-xL and potent cell growth inhibition of the
H146 cell line.
Since the methylsulfonylamide group can effectively replace the acid group in compound
4
and retain high binding affinities to Bcl-2/Bcl-xL and potent cellular activity, we next
synthesized compounds 31 and 32 in which the acid group in compounds 5 and 28 was replaced
by a methylsulfonylamide group (Table 3). As shown in Table 3, compounds 31 and 32 show
high binding affinities to both Bcl-2 and Bcl-xL proteins and are very potent in inhibition of cell
growth in the H146 cell line, and achieve IC50 values of 4.8 nM and 1.3 nM, respectively. To
assess their binding specificity, we tested compounds 31 and 32 for their binding affinity to Mcl-
7
1
in our optimized FP assay (Table 3). Our data showed that compounds 31 and 32 have no
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1
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1
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1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
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3
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5
5
5
5
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appreciable binding to Mcl-1 at concentrations as high as 2 µM. Hence, compounds 31 and 32
are potent and specific Bcl-2 and Bcl-xL inhibitors.
Table 3. Structure-activity relationship of analogues of compounds 4 and 12.
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
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
Cell Growth Inhibition
Binding Affinities (IC50 ± SD, nM)
COMPOUND
R1
R2
(IC50 ± SD, nM)
Bcl-2
Bcl-XL
Mcl-1
H-146
5
-OH
Et-
2 ± 1.6
6.6 ± 2.3
NT
8.1 ± 3.5
28
-OH
-OH
-OH
iPr-
Pr-
1.4 ± 0.5
10 ± 4
4.8 ± 0.1
8.2 ± 2.2
16 ± 2
NT
NT
3 ± 2.4
42 ± 5
2
3
9
0
Bu-
Et-
23 ± 2
NT
67 ± 5
31 (BM-1075) -NHSO CH
1.8 ± 0.3
1.8 ± 0.2
7.0 ± 1.8
6.9 ± 1.8
> 2000
> 2000
4.8 ± 0.9
1.3 ± 0.3
2
3
3
2 (BM-1074) -NHSO CH
iPr-
2
3
NT = not tested
Based upon their high binding affinities to Bcl-2 and Bcl-xL and potent cell growth
inhibitory activity in the H146 cell line, 31 and 32 represent promising new lead compounds for
further in vivo evaluation for their therapeutic potential.
First, we evaluated the maximum tolerated dose (MTD) of both compounds in severe
combined immunodeficient (SCID) mice. Both compounds administered intravenously (i.v.) in
mice at 15 mg/kg, daily, 5 days a week for 2 weeks, were found to be well tolerated and did not
cause significant weight loss (<5%) or other signs of toxicity during and after the treatment.
However, at 25 mg/kg, both compounds caused significant weight loss (~10%) and, therefore,
we concluded that 15 mg/kg dosed intravenously is the MTD for both drugs in SCID mice.
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We next tested compounds 31 and 32 for their ability to induce apoptosis at the MTD in
H146 xenograft tumors in SCID mice. A single dose of either compound at 15 mg/kg was
administered i.v. to SCID mice bearing the H146 xenograft tumors. Animals were sacrificed at 3
h, 6 h and 24 h time points, and tumor tissues were removed for western blot analysis for
cleavage of Poly ADP ribose polymerase (PARP) and caspase-3, two critical biochemical
apoptosis markers. The data in Figure 3 showed that both compounds induce robust cleavage of
PARP and caspase-3 at both 3 and 6-hr time-points in H146 tumor tissues, indicative of strong
apoptosis induction in vivo.
10
11
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31
32
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38
39
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Compound 32
Vehicle 3 h 6 h 24 h
Compound 31
6 h 24 h
3 h
1
2 3 4 5 6 7 8 9 10 11 12 13 14 Tumor #
PARP
Cl PARP
Caspase-3
Cl Cas-3
Figure 3. Western blot analysis of tumor tissues for cleavage of PARP and caspase-3. Mice were
treated with a single dose of compound 31 or 32 (15 mg/kg, i.v.), were sacrificed at 3, 6 and 24-h
time points and tumors were removed for western blot analysis. Cleavage of Cl PARP, cleaved
PARP; Cl Cas-3, cleaved caspase-3.
Based upon the strong apoptosis induction in vivo observed for both 31 and 32, we
evaluated their antitumor efficacy in the H-146 xenograft tumor model, and the results are shown
in Figure 4. Both compounds 31 and 32 show strong antitumor activity. While compound 31
achieves only partial tumor regression, 32 is capable of achieving rapid, complete and persistent
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tumor regression. At day 62, 18 days after the treatment was ended, none of the 8 mice treated
with 32 had measurable tumors, and at day 117, 74 days after the end of the treatment with 32,
four mice (50%) did not have measurable tumors. The average tumor size for the 8 mice was 47
0
1
2
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5
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8
9
0
1
2
3
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9
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0
3
3
mm at day 117, as compared to 130 mm at the start of the treatment (day 33). Furthermore, all
the mice treated with compound 32 suffered no significant weight loss (<5%) and did not show
other signs of toxicity during or after the treatment. In our previous study, we had tested
compound 5 for its antitumor activity in the H146 xenograft model and found that while 5
effectively inhibited tumor growth at 25 mg/kg, i.v., 5 days per week for 2 weeks, it failed to
8
achieve long-lasting tumor regression. Hence, compound 32 at 15 mg/kg is considerably more
effective than compound 5 at 25 mg/kg in inducing complete and persistent tumor regression in
the H146 xenograft model.
1
500
(a). Tumor growth
1200
Vehicle Control
3
2
9
00
00
Treatment
3
1
6
300
0
3
0
40
50
60
70
80
90 100 110 120
Days post Implantation
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(
b). Animal body weight
2
2
1
5
0
5
Vehicle Control
32
Treatment
3
1
10
11
12
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23
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29
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31
32
33
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52
53
54
55
56
57
58
59
60
3
0
40
50
60
70
80
90 100 110 120
Days post Implantation
Figure 4. Antitumor activity of compounds 31 and 32 in the H146 small-cell lung cancer
xenograft model in SCID mice. Tumors were grown to an average size of 126 mm and
compound 31 or 32 was administered at 15 mg/kg intravenously, daily, 5 days a week for 2
weeks. (a). Tumor growth. (b). Animal body weight.
3
To further define their anticancer activity, we next tested compounds 31 and 32 in three
additional small-cell lung cancer cell lines, known to be sensitive to 1 and 2, and the data are
summarized in Table 4. While all four compounds effectively inhibit cell growth in these three
cancer cell lines, compound 32 is the most potent of the compounds. Compound 32 has IC50
values of 1.0 nM, 1.4 nM and 2.3 nM in these three cancer cell lines and is >10-times more
potent than 2, and >50-times more potent than 1, against each of these three cell lines, based
upon their IC50 values.
Table 4. Cell growth inhibitory activity of compounds 31, 32, 1 and 2 in three small-cell lung
cancer cell lines.
IC50 ± SD (nM)
Cell Lines
1
2
31
32
H1963
H187
54.0±28.2
137.7±71.3
173.4±122.1
26.6±7.9
38.4±26.8
54.2±11.1
8.2±4.9
7.9±3.9
11.1±2.0
1.0±0.5
1.4±1.3
2.3±0.2
H1417
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6
Chemistry:
7
Initially, we employed a convergent synthesis (Method A) for the preparation of the
target molecules. The piperazine 38, embedded in our target molecules, was prepared in three
steps starting with the reaction of the aniline (33) and 4-fluoro-3-nitrobenzene-1-sulfonyl
0
1
2
3
4
5
6
7
8
9
0
1
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9
0
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0
7
chloride (34), forming the sulfonamide (35). Displacement of the activated fluorine in 35 with
1
1
(
R)-N ,N -dimethyl-4-(phenylthio)butane-1,3-diamine (36) and acid removal of the Boc
protecting group generated 38. The second fragment was designed as a variable scaffold, in
which diversity could be built upon to develop compounds for SAR. These scaffolds consisted
of various 5-membered hetero-aromatic rings with a 4-chlorophenyl and as a synthetic handle, an
adjacent 3-iodophenyl group, to which 38 could be attached. Accordingly, in this methodology,
7
, 10
38 was subjected to Buchwald-Hartwig coupling
with the synthesized scaffolds 9a, and 18a-
2
1a furnishing 9 and 18-21, respectively. However, this synthetic route was complicated by
poor yields of the target molecules, especially when the scaffolds contained a carboxylic acid or
bioisosteres and, ultimately, this precluded further use of this methodology for further studies.
7
, 8
Consequently, a stepwise synthesis (Method B), applicable to all scaffolds was carried
1
1
out. In this methodology, (4-nitrophenyl)piperazine (39) was subjected to Ullman coupling
with the synthetic scaffolds 4b, 5b, 12a, 13a, 14a, 15a, 16b, 17a, 22a, 23b, 24b, 27a, 28b, 29a,
3
0a, 31a, and 32a producing intermediates 4c, 5c, 12b, 13b, 14b, 15b, 16c, 17b, 22b, 23c, 24c,
7b, 28c, 29b, 30b, 31b, and 32b, respectively. Intermediates 25a and 26a were obtained using
2
the method described in Scheme 6. Base hydrolysis of ethyl ester intermediates 17b and 27b, or
low temperature acid hydrolysis of the tert-butyl ester intermediates 29b and 30b, gave the
corresponding carboxylic acids. Reduction of the nitro groups in these intermediates (17b-
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carboxylic acid, 27b-carboxylic acid, 29b-carboxylic acid, 30b-carboxylic acid, 4c, 5c, 12b,
1
3b, 14b, 15b, 16c, 22b, 23c, 24c, 25a, 26a, 28c, 31b, and 32b) by catalytic hydrogenation led
to aniline intermediates that were subjected to the same synthetic strategy used for the
preparation of 37, generating the target compounds 4, 5, 12-17 and 22-32. Decarboxylation of 4
by acid treatment generated target compound 7. Target compounds 8, 10, and 11 were obtained
by EDCI-mediated coupling to carboxylic acid 4 of methylamine, (1s,3s)-3-amino-1-
methylcyclobutanol, and 3-methylazetidine-3-ol, respectively.
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
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39
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43
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60
Preparation of the 2-methylfuran scaffold 27a, and the variously substituted 1H-pyrrole
scaffolds 4b, 5b, 9a, 12a, 13a, 14a, 15a, 16b, 28b, 29a, 30a, 31a, 32a is outlined in Scheme 2.
8
Intermediates 44a-d were prepared using an established synthetic strategy in which
condensation of the β-ketoesters 40a-c with 3-iodobenzaldehyde (41a) produced intermediates
4
2a-c, respectively, and the bromo-analogue (42d) was prepared by the condensation of β-
ketoester (40a) with 3-bromobenzaldehyde (41b). Compounds 42a-d were subjected to a Stetter
8
, 12
reaction with 4-chlorobenzaldehyde furnishing 44a-d.
The furan intermediate (27a) was
prepared by heating 44a with HCl, and AcOH in EtOH. The 2-trifluoromethyl-1H-pyrrole
intermediate (16a) was prepared by condensation of intermediate (44b) with methylamine. Paal-
Knorr cyclization of 44a with methylamine, of 44d with methylamine, and 44c with ethylamine,
n-propylamine, n-butylamine, or iso-propylamine furnished pyrroles 4a, 46, 5a, 28a, 29a, and
13
30a, respectively. Base hydrolysis of the ethyl ester intermediates 4a, 16a or low temperature
acid hydrolysis of the tert-butyl ester intermediates 5a, 28a furnished the corresponding 1H-
8
pyrrole-3-carboxylic acid scaffolds 4b, 16b and 5b, 28b, respectively.
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1
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1
2
2
2
2
2
2
2
2
2
2
3
3
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3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 1. The convergent Method A and stepwise Method B for the preparation of 4, 5, and 7-
3
2
ID
b, 4c, 4 a
Core
R
ID
Core
R
ID
Core
R
ID
Core
R
R
N
4
14a, 14b, 14
15a, 15b, 15
16b, 16c, 16
17a, 17b
17
21a, 21
N
5b, 5c, 5 a
R
N
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
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7 a
O
28b, 28c, 28a
22a, 22b, 22
23b, 23c, 23
24b, 24c, 24
25a, 25
8 a
29a, 29b
29
Cl
R
R
9a, 9
N
Cl
N
O
OH
10
30a, 30b
30
N
H
11
18a, 18
19a, 19
20a, 20
26a, 26
27a, 27b
27
R
O
1
2a, 12b, 12
3a, 13b, 13
31a, 31b, 31
32a, 32b, 32
R
O
1
o
Reagents and conditions: (a) Pyridine, 34, 0 C, 15 min.; (b) DMF, 36, DIEA, overnight; (c) DCM, TFA, rt; (d) 2:1
o
Toluene:DMF, Pd(dba) , NaOtBu, P(tBu) ; (e) CuI, L-proline, K CO , DMSO, 90 C overnight; (f) NaOH, 1:1:1 1,4-
2
3
2
3
o
dioxane:EtOH:H
2
O, reflux (g) (1) DCM, H
2
SO
4
(conc.), 0 C 10 min, (2) saturated NaHCO
MeOH, 1 atm, 15 min; (i) TFA, 10 min; (j) amine, EDCI, HOBt, DIEA, DCM, rt overnight. ( previously reported ,
3
; (h) 10% Pd-C/H8
2
,
a
b
c
synthesis described in Scheme 6, the I = Br)
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Scheme 2. Synthesis of variously substituted 1H-pyrroles and 2-methyl furan scaffolds
O
O
O
O
R1
O
OR2
O
O
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
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52
53
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A
a
b
R1
OR2
R1
OR2
A
O
O
A
Cl
41a, A = I
1b, A = Br
Cl
4
0a, R = Me, R = Et
42a, R = Me, R = Et, A = I
44a, R = Me, R = Et, A = I
1
2
1
2
1 2
4
43
40b, R = CF , R = Et
42b, R = CF , R = Et, A = I
44b, R = CF , R = Et, A = I
1 3 2
1
3
2
1
3
2
40c, R = Me, R = tBu
42c, R = Me, R = tBu, A = I
44c, R = Me, R = tBu, A = I
1
2
1
2
1 2
4
2d, R = Me, R = Et, A = Br
44d, R = Me, R = Et, A = Br
1
2
1
2
O
O
R1
R1
X
R3
OR2
OH
X
X
I
I
I
Cl
4b, R = Me, X = NMe
Cl
Cl
4a, R = Me, R = Et, X = NMe
c
e
g
4
4a
45, X = NMe, R = H
1
2
1
3
h
i
j
d
c
2
7a, R = Me, R = Et, X = O
13a, X = NMe, R = Cl
1
2
3
O
e
f
9a, X = NMe, R =
4
4b
4c
16a, R = CF , R = Et, X = NMe
16b, R = CF , X = NMe
3
NH2
CN
1
3
2
1
3
i
i
k
5
2
a, R = Me, R = tBu, X = NEt
9a, R = Me, R = tBu, X = NPr
30a, R = Me, R = tBu, X = NBu
8a, R = Me, R = tBu, X = NiPr
5b, R = Me, X = NEt
1
1
2
15a, X = NMe, R =
3
c
4
1
2
O
O
O
1
2
f
S
2
28b, R = Me, X = NiPr
N
H
CH3
1
2
1
12a, X = NMe, R =
3
O
R3
O O
S
3
1a, X = NEt, R =
N
CH3
3
N
H
Br
O
O
O
3
2a, X = NiPr, R =
S
3
N
CH3
H
Cl
47, R = -H
c
g
l
m
4
4d
46, R = -CO Et
48, R = -I
14a, R = -CF3
3
3
2
3
3
o
Reagents and conditions: (a) piperidine, 41a or 41b, AcOH, toluene reflux; (b) 43, thiazolium catalyst, Et
h; (c) (i) primary amine, MeOH, overnight (ii) 2N HCl, 5 min; (d) AcOH, HCl (conc.), EtOH, 70 C 4h; (e) NaOH,
:1:1 1,4-dioxane:EtOH:H O, reflux; (f) (1) DCM, H SO (conc.), 0 C 10 min, (2) saturated NaHCO ; (g) TFA,
2 2 4 3
DCM, rt overnight; (h) ammonia (0.5 M in 1,4-dioxane), EDCI, HOBt, DIEA, DCM, rt overnight; (i) (1) (COCl)2,
DCM, cat. DMF, reflux 1h (2) methanesulfonamide, DMAP, ClCH CH Cl, reflux overnight; (j) NCS, DMF, rt
3
N, 70 C,
o
5
1
o
2
2
o
overnight; (k) pyridine, TFAA, 1,4-dioxane, 0 C, 3h rt; (l) NIS, DMF, rt overnight; (m) Methyl 2,2-difluoro-2-
fluorosulfonyl)acetate, CuI, DMF, 100 C overnight.
o
(
EDCI-catalyzed coupling of ammonia to the carboxylic acid 4b furnished the 1H-pyrrole-
3
-carboxamide scaffold 9a, but the same conditions failed to produce the N-(methylsulfonyl)-
carboxamide (12a) which was instead prepared by formation of the acid chloride of 4b and
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heating it with excess methanesulfonamide. In a similar manner used for the preparation of 12a,
3
1a and 32a were prepared from 5b and 28b, respectively. Scaffold 13a was prepared in two
steps starting with the acid-mediated decarboxylation of 4b followed by chlorination at the 3-
position with N-chlorosuccinamide (NCS). The 3-trifluoromethyl-1H-pyrrole scaffold 14a was
prepared from 46 in three steps. Acid-mediated decarboxylation of 46 generated intermediate 47
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which was iodinated at the 3-position with N-iodosuccinamide (NIS), producing 48.
A
trifluoromethyl group was selectively installed at the 3-position of the iodopyrrole by Cu(I)
1
4
catalyzed coupling with methyl 2,2-difluoro-2-(fluorosulfonyl)acetate, producing 14a. The 1H-
15
pyrrole-3-carbonitrile scaffold 15a was prepared by dehydration of pyrrole-3-carboxamide 9a.
Scheme 3. Synthesis of imidazole scaffolds 18a and 23b
Reagents and conditions: (a) toluene, reflux; (b) TosMIC, K
overnight; (d) (i) KOt-Bu, THF, ClPO(OEt) , (ii) ethyl isocyanoacetate, KOt-Bu; (e) (i) NaOH, 1:1:1 1,4-
, reflux 3h (2) methanesulfonamide, DMAP, ClCH CH Cl, reflux
2 3
CO , MeOH, DME; (c) EDCI, HOBt, DIEA, DCM, rt
2
dioxane:EtOH:H
overnight.
2
O, reflux, (ii) (1) SOCl
2
2
2
Imidazole scaffolds 18a and 23b were prepared as outlined in Scheme 3. The imidazole
8a was synthesized by a two-step 1,3-dipolar addition of toluenesulfonylmethyl isocyanide
1
1
6
(
TosMIC) with the imine 50. Compound 50 was prepared by reaction of 4-chloroaniline (49)
2
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with 41a under Dean-Stark conditions. The imidazole (23b) was prepared using a procedure
1
7
described by Yang, et al., for the construction of imidazol[1,5-a][1,4]benzodiazapines. EDCI
coupling of 49 with 3-iodobenzoic acid (51) generated the amide 52, which was treated with
diethyl chlorophosphate to generate the unstable iminophosphate which was condensed under
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49
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53
54
55
56
57
58
59
60
1
7
basic conditions with ethyl isocyanoacetate to produce the imidazole 23a. Base hydrolysis of
the ethyl ester (23a) produced its corresponding acid that was converted to its acid chloride and
reacted with methanesulfonamide to generate the imidazole scaffold (23b).
The pyrazole scaffolds (19a and 24b) were prepared using a previously established
1
8
method. Treatment of 53a or 53b with N,N-dimethylformamide dimethylacetal (54) generated
the enaminones (55a and 55b) which, condensed with 4-chlorophenylhydrazine (56), produced
1
8
the pyrazoles (19a and 24a), respectively. Base hydrolysis of the ethyl ester (24a) produced
the corresponding carboxylic acid that was converted to its acid chloride and reacted with
methanesulfonamide to produce the pyrazole scaffold 24b.
Scheme 4. Synthesis of pyrazole scaffolds 19a and 24b
Reagents and conditions: (a) toluene, 54, reflux; (b) 56, EtOH, reflux; (c) (i) NaOH, 1,4-dioxane:EtOH:H
v/v/v), reflux, (ii) (1) SOCl , reflux 3h (2) methanesulfonamide, DMAP, ClCH CH Cl, reflux overnight
2
O (1:1:1
2
2
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The pyrazoles 20a and 21a and the isoxazole 22a were prepared from a common
intermediate, the enaminone (59). 3-Iodophenylacetic acid (57) was converted to its acid
chloride by refluxing in thionyl chloride. Friedel-Craft acylation of chlorobenzene with 3-
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iodophenylacetyl chloride, using a standard method , resulted in removal of the iodine atom
o
from 58 and, to avoid this problem, the reaction was diluted and carried out at 0 C, producing
compound 58 as the major product. Treatment of ketone 58 with 54 produced the enaminone
(
59). Condensation of methyl hydrazine with 59 produced a mixture of 2 regio-isomers that
2
0
were separated to give the pyrazoles (20a and 21a). Condensation of 59 with hydroxylamine
21
produced only one isoxazole (22a).
Scheme 5. Synthesis of pyrazoles 20a and 21a, isoxazole 22a, and 2-chloropyrrole 17a
o
Reagents and conditions: (a) (i) SOCl
NH NHCH , EtOH, reflux; (d) NH OH.HCl, Na
cyanoacetate, K CO , acetone; (g) 4M HCl in 1,4-dioxane; (h) MeI, K
2
, reflux 2h, (ii) AlCl
3
, chlorobenzene, 0 C, 2h; (b) 54, toluene, reflux 2h; (c)
2
3
2
2
CO , 2:1 MeOH:H
3
2
O, AcOH, reflux; (e) AcOH, Br
CO , DMF.
2
, DCM; (f) 2-
2
3
2
3
The 2-chloropyrrole (17a) was prepared in four steps starting from intermediate 58.
Compound 58 was treated with Br in AcOH to produce the α-bromoketone (60), reacted with
ethyl 2-cyanoacetate under basic conditions to produce compound 61, treatment of which with
2
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M HCl in 1,4-dioxane formed the 2-chloropyrrole (62), which was methylated with CH I under
2
2
basic conditions to give the ethyl ester (17a).
The pyrazole acids (25a and 26a) were synthesized following the procedure outlined in
Scheme 6. Condensation of 63 with methyl hydrazine produced two regio-isomers, (64a and
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59
60
2
3
24
6
4b), which were separated and were treated separately with NIS, producing 65a and 65b. In
parallel, the pinacol boronic ester (68) was prepared by displacement of the activated fluorine in
4
-fluoronitrobenzene (67) with the piperazine (66). This boronic ester was subjected to Suzuki
2
4
coupling with 65a or 65b.
The Suzuki coupling condition also resulted in hydrolysis of the
methyl ester and furnished the pyrazole acids 25a and 26a.
Scheme 6. Synthesis of pyrazole acid intermediates 25a and 26a
o
Reagents and conditions: (a) NH
overnight; (d) Pd(PPh , K CO , 2:1 DME/H
2
NHCH
3
, AcOH, EtOH; (b) NIS, CAN, CH
O reflux, 2h.
3 2 3
CN, 70 C; (c) K CO , DMSO, rt
3
)
4
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3
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Summary:
In the present study, we have optimized compound 4, which is a potent Bcl-2/Bcl-xL
inhibitor but was unable to achieve tumor regression in animal models of human cancer. In
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contrast to our previous study , in which our modifications were focused on the nitro group and
the soluble thiophenyl-containing “tail” group in compound 4, the present study was centered on
the pyrrole core structure, which has a significant effect on binding affinities to Bcl-2/Bcl-xL and
cellular activity in cancer cells. Our optimization efforts have yielded compound 32 which binds
to Bcl-2 and Bcl-xL with K values <1 nM. Similar to other initial lead compounds, compound
i
3
2 does not show any appreciable binding to Mcl-1 protein at concentrations as high as 2 µM.
Compound 32 potently inhibits cancer cell growth in the H146 small-cell lung cancer cell line
and achieves an IC50 value of 1.3 nM. Compound 32 also potently inhibits cell growth inhibition,
with IC50 values of 1.0-2.3 nM, in three other small-cell lung cancer cell lines which were known
to be sensitive to 1 and 2. In direct comparison, compound 32 is >10- and >50-times more
potent than 2 and 1, respectively, against these cancer cell lines. Significantly, compound 32
achieves rapid, complete and persistent tumor regression in the H146 xenograft tumor model in
mice at a well-tolerated dose-schedule. Taken together, these data show that compound 32 is
arguably the most potent and efficacious Bcl-2/Bcl-xL inhibitor reported to date and warrants
extensive evaluation as a potential clinical development candidate for the treatment of human
cancer.
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Experimental:
General Information. Unless otherwise stated, all reactions were performed under a nitrogen
atmosphere in dry solvents under anhydrous conditions and all commercial reagents were used as
supplied without further purification. NMR spectra were obtained on a Bruker 300 UltraShield
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59
60
1
13
spectrometer at a H frequency of 300 MHz and C frequency of 75 MHz. Chemical shifts (δ)
are reported in parts per million (ppm) relative to an internal standard. The final products were
purified by a C18 reverse phase semi-preparative HPLC column with solvent A (0.1% of TFA in
water) and solvent B (0.1% of TFA in CH CN) as eluents. All final compounds have purity ≥
3
9
5% as determined by Waters ACQUITY UPLC. Synthesis of compounds 4, 5, 7, 8, 28 and
8
their intermediates were reported previously.
8
Ethyl 2-acetyl-4-(4-chlorophenyl)-3-(3-iodophenyl)-4-oxobutanoate (44a)
Ethyl acetoacetate 44a (2.27g, 17.4 mmol), 3-iodobenzaldehyde 41a (4.04g, 17.4 mmol),
piperidine (70 µL), and acetic acid (200 µL) were dissolved in toluene (10 ml) and refluxed with
azeotropic removal of water using a Dean-Stark apparatus. After reaction overnight, the solution
was cooled, diluted with EtOAc, washed sequentially with 1.0M HCl, saturated sodium
bicarbonate, and brine, then dried over sodium sulfate. Purification by column chromatography
(9:1 hexane:EtOAc) provided 5.3 g of the product 42a as a mixture of isomers. Triethylamine
(1.55 mL) was added to a slurry of 42a (5.3 g, 15.4 mmol), 4-chlorobenzaldehyde 43 (2.16 g,
1
5.4 mmol), and 3-ethyl-5-(2-hydroxyethyl)-4-methylthiazolium bromide (0.583 g, 2.3 mmol)
and the mixture was stirred with heating at 70 ºC until 42a was consumed. After cooling to room
temperature, the mixture was diluted with EtOAc, washed sequentially with 1.0M HCl, saturated
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sodium bicarbonate, and brine, and dried over sodium sulfate. The EtOAc was removed in
vacuo and provided 7.5 g of crude 44a which was used without further purification.
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Ethyl 5-(4-chlorophenyl)-4-(3-iodophenyl)-1,2-dimethyl-1H-pyrrole-3-carboxylate (4a) .
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A 2.0M solution of methylamine in MeOH (35 mL, 70 mmol) was added to compound 44a (7.5
g, 15.4 mmol) dissolved in 5 mL of MeOH. After 24 hours the solution was acidified with 1.0 M
HCl and the compound was extracted with EtOAc. The EtOAc solution was washed with brine,
dried over sodium sulfate and concentrated in vacuo to provide crude 4a. Purification by column
chromatography (5:1 hexane:EtOAc) provided 4.9 g (59 % after 3 steps) of 4a.
Ethyl
5-(4-chlorophenyl)-4-(3-iodophenyl)-2-methylfuran-3-carboxylate
(27a).
Concentrated HCl (25 mL), and glacial acetic acid (5 mL) were added to a solution of 44a (1.0 g,
2.06 mmol) in EtOH ( 25 mL) and the mixture was heated to 70 ºC. After 4 hours, the reaction
was quenched with saturated sodium bicarbonate, extracted with EtOAc, washed with brine,
dried over sodium sulfate, filtered, and the EtOAc was removed in vacuo to produce crude 27a.
1
Purification by column chromatography provided 834 mg (96% yield) of 27a. H-NMR (300
3
MHz, CDCl ) δ ppm 7.74-7.65 (m, 2H), 7.29-7.17 (m, 5H), 7.11 (t, J = 7.74 Hz, 1H), 4.09 (q, J =
1
3
7
1
1
3
.13 Hz, 2H), 2.68 (s, 3H), 1.06 (t, J = 7.15 Hz, 3H); C-NMR (75 MHz, CDCl ) δ ppm 163.83,
59.06, 146.84, 139.22, 136.78, 135.95, 133.65, 130.11, 129.63, 128.88(2C), 128.59,
26.93(2C), 121.23, 115.78, 94.03, 60.22, 14.39, 14.08.
Ethyl
44b) Starting with the ethyl trifluoroacetoacetate (40b), compound 44b was prepared according
to the procedure described for the preparation of compound 44a.
2-(2-(4-chlorophenyl)-1-(3-iodophenyl)-2-oxoethyl)-4,4,4-trifluoro-3-oxobutanoate
(
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Ethyl
5-(4-chlorophenyl)-4-(3-iodophenyl)-1-methyl-2-(trifluoromethyl)-1H-pyrrole-3-
carboxylate (16a). Starting with 44b, compound 16a was prepared in a similar manner
1
described for the preparation of compound 4a. H-NMR (300 MHz, CDCl ) δ ppm 7.54-7.45
3
10
11
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17
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19
20
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25
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49
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(
m, 2H), 7.35 (d, J = 8.51 Hz, 2H), 7.10 (d, J = 8.50 Hz, 2H), 7.01-6.85 (m, 2H), 4.22 (q, J = 7.14
+
Hz, 2H), 3.58 (s, 3H), 1.18 (t, J = 7.13 Hz, 3H); ESI-MS m/z 533.83 (M+H) .
tert-Butyl 2-acetyl-4-(4-chlorophenyl)-3-(3-iodophenyl)-4-oxobutanoate (44c). Starting with
tert-butyl acetoacetate (40c), compound 44c was prepared according to the procedure described
for the preparation of compound 44a.
tert-Butyl 5-(4-chlorophenyl)-1-ethyl-4-(3-iodophenyl)-2-methyl-1H-pyrrole-3-carboxylate
(
5a). Ethylamine (11.7 mL, 2.0 M in MeOH, 23.40 mmol) was added to a solution of compound
4c (3.0 g, 5.85 mmol) in MeOH (20 mL). After 24 hours, the solution was acidified with 1.0 M
4
HCl, stirred briefly and the compound was extracted with EtOAc. The EtOAc solution was
washed sequentially with saturated NaHCO , brine, then dried over Na SO and concentrated in
3
2
4
vacuo to provide crude 5a. Purification by column chromatography (using a gradient of hexane
1
and DCM, the product eluted at 100% DCM) provided 1.30 g (43% yield) of 5a. H-NMR (300
3
MHz, CDCl ) δ ppm 7.53 (t, J = 1.63 Hz, 1H), 7.44 (dt, J = 1.39, 7.76 Hz, 1H), 7.26 (d, J = 8.50
Hz, 2H), 7.07 (d, J = 8.49 Hz, 2H), 6.93 (dt, J = 1.34, 7.66 Hz, 1H), 6.84 (t, J = 7.72 Hz, 1H),
13
3
.80 (q, J = 7.18 Hz, 2H), 2.61 (s, 3H), 1.27 (s, 9H), 1.14 (t, J = 7.18 Hz, 3H); C-NMR (75
MHz, CDCl
3
) δ ppm 165.26, 140.07, 138.85, 135.37, 134.70, 134.12, 132.78(2C), 130.50,
29.73, 129.66, 129.16, 128.75(2C), 122.64, 113.05, 93.37, 79.83, 39.18, 28.26(3C), 16.25,
1.47.
1
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tert-Butyl
5-(4-chlorophenyl)-4-(3-iodophenyl)-1-isopropyl-2-methyl-1H-pyrrole-3-
carboxylate (28a). Starting with iso-propylamine and 44c, compound 28a was prepared
according to the procedure described for the preparation of compound 5a. In this case the
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reaction was heated in a sealed tube. H-NMR (300 MHz, CDCl ) δ ppm 7.50 (t, J =1.39 Hz,
3
1H), 7.42 (dt, J = 1.30, 7.67 Hz, 1H), 7.25 (d, J = 8.41 Hz, 2H), 7.05 (d, J = 8.39 Hz, 2H), 6.92-
6
7
.87 (m, 1H), 6.82 (t, J = 7.69 Hz, 1H), 4.33 (sept, J = 7.07 Hz, 1H), 2.69 (s, 3H), 1.41 (d, J =
1
3
.09 Hz, 6H), 1.25 (s, 9H); C-NMR (75 MHz, CDCl ) δ ppm 165.35, 139.97, 139.05, 135.17,
3
134.57, 134.14, 133.16(2C), 131.21, 130.26, 129.53, 129.11, 128.56(2C), 122.48, 113.91, 93.34,
9.83, 48.81, 28.18(3C), 22.48(2C), 13.06.
7
tert-Butyl
5-(4-chlorophenyl)-4-(3-iodophenyl)-2-methyl-1-propyl-1H-pyrrole-3-
carboxylate (29a). Starting with n-propylamine and 44c, compound 29a was prepared
1
according to the procedure described for the preparation of compound 5a. H-NMR (300 MHz,
3
CDCl ) δ ppm 7.53 (t, J = 1.56 Hz, 1H), 7.46-7.41 (m, 1H), 7.25 (d, J = 8.48 Hz, 2H), 7.05 (d, J
= 8.49 Hz, 2H), 6.94-6.89 (m, 1H), 6.84 (t, J = 7.69 Hz, 1H), 3.75-3.66 (m, 2H), 2.59 (s, 3H),
13
1.51 (sex, J = 7.69 Hz, 2H), 1.27 (s, 9H), 0.75 (t, J = 7.42 Hz, 3H); C-NMR (75 MHz, CDCl
3
)
δ ppm 165.28, 140.07, 138.86, 135.71, 134.66, 133.99, 132.76(2C), 130.55, 130.00, 129.65,
29.15, 128.71(2C), 122.53, 112.94, 93.37, 79.81, 45.96, 28.23(3C), 24.24, 11.67, 11.28.
1
tert-Butyl 1-butyl-5-(4-chlorophenyl)-4-(3-iodophenyl)-2-methyl-1H-pyrrole-3-carboxylate
(
30a). Starting with n-butylamine and 44c, compound 30a was prepared according to the
1
procedure described for the preparation of compound 5a. H-NMR (300 MHz, CDCl
3
) δ ppm
7.53 (s, 1H), 7.43 (d, J = 7.75 Hz, 1H), 7.25 (d, J = 8.38 Hz, 2H), 7.05 (d, J = 8.40 Hz, 2H), 6.92
(
d, J = 7.60 Hz, 1H), 6.84 (t, J = 7.70 Hz, 1H), 3.79-3.69 (m, 2H), 2.60 (s, 3H), 1.47 (quin, J =
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.68 Hz, 2H), 1.27 (s, 9H), 1.14 (sex, J = 7.28 Hz, 2H), 0.78 (t, J = 7.31 Hz, 3H); C-NMR (75
MHz, CDCl ) δ ppm 165.27, 140.07, 138.87, 135.66. 134.65, 133.98, 132.78(2C), 130.53,
3
1
29.97, 129.65, 129.13, 128.68(2C), 122.53, 112.95, 93.35, 79.79, 44.15, 32.98, 28.23(3C),
9.97, 13.71, 11.66.
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Ethyl 2-acetyl-3-(3-bromophenyl)-4-(4-chlorophenyl)-4-oxobutanoate (44d). Starting with
-bromobenzaldehyde (41b) and ethyl acetoacetate (40a), compound 44d was prepared
3
according to the procedure described for the preparation of compound 44a.
Ethyl 4-(3-bromophenyl)-5-(4-chlorophenyl)-1,2-dimethyl-1H-pyrrole-3-carboxylate (46).
Starting with 44d, compound 46 was prepared in the manner described for the preparation of
1
compound 4a. H-NMR (300 MHz, CDCl ) δ ppm 7.31 (s, 1H), 7.29-7.20 (m, 3H), 7.07-6.90
3
1
3
(
m, 4H), 4.07 (q, J = 7.1 Hz, 2H), 3.39 (s, 3H), 2.61 (s, 3H), 1.03 (t, J = 7.1 Hz, 3H); C-NMR
75 MHz, CDCl ) δ ppm 165.55, 138.25, 136.80, 134.06, 133.84, 132.45(2C), 130.55, 129.96,
29.39, 128.93, 128.80, 128.68(2C), 122.51, 121.17, 111.21, 59.39, 31.92, 14.02, 11.87.
(
3
1
8
5
-(4-Chlorophenyl)-4-(3-iodophenyl)-1,2-dimethyl-1H-pyrrole-3-carboxylic acid (4b) .
NaOH (8.2 g, 204.3 mmol) was added to a solution of 4a (4.9 g, 10.2 mmol) in 300 ml of 1:1:1
dioxane, EtOH, and H O, and the solution was heated at reflux until no compound 4a was
2
observed by TLC. After cooling, the reaction was slowly neutralized with 1M HCl and the
compound was extracted with EtOAc. The EtOAc solution was washed with brine, dried over
Na SO and concentrated in vacuo to produce compound 4b as a pale solid which was not further
2
4
purified.
2
9
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