Synthesis and evaluation of the anti-proliferative activity of diaryl-3-pyrrolin-2-ones and fused analogs

Article abstract of DOI:10.1016/j.bmcl.2016.11.076

Analogs containing a central 3-pyrrolin-2-one core with different methoxyphenyl and/or indole substituents were prepared and tested for anti-proliferative activity in U-937 cells. The most efficacious analogs were non-rigid, (non-fused) contained methoxyaryl groups located at the 4-position, and contained either methoxyaryl or indole groups located at the 3-position. Both the number of methoxy groups contained in the substituents and the particular location of the indole rings with respect to the lactam carbonyl had significant affects on anti-proliferative activity. This work provides a framework to better understand structure-activity relationships for inducing anti-proliferative activity in diaryl heterocyclic scaffolds.

Full text of DOI:10.1016/j.bmcl.2016.11.076

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of the anti-proliferative activity of diaryl-3-
pyrrolin-2-ones and fused analogs
b
b
b
Patricia Mowery a, , Fernando Banales Mejia , Courtney L. Franceschi , Maeve H. Kean ,
⇑
Deborah O. Kwansare b, Megan M. Lafferty b, Namita D. Neerukonda a, Carly E. Rolph a,b
,
Nathanyal J. Truax b, Erin T. Pelkey b,
⇑
a Department of Biology, Hobart and William Smith Colleges, Geneva, NY 14456, USA
b Department of Chemistry, Hobart and William Smith Colleges, Geneva, NY 14456, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Analogs containing a central 3-pyrrolin-2-one core with different methoxyphenyl and/or indole sub-
stituents were prepared and tested for anti-proliferative activity in U-937 cells. The most efficacious ana-
logs were non-rigid, (non-fused) contained methoxyaryl groups located at the 4-position, and contained
either methoxyaryl or indole groups located at the 3-position. Both the number of methoxy groups con-
tained in the substituents and the particular location of the indole rings with respect to the lactam car-
bonyl had significant affects on anti-proliferative activity. This work provides a framework to better
understand structure-activity relationships for inducing anti-proliferative activity in diaryl heterocyclic
scaffolds.
Received 29 October 2016
Revised 23 November 2016
Accepted 24 November 2016
Available online xxxx
Keywords:
Indoles
Lactams
Anti-cancer
MTT
Ó 2016 Elsevier Ltd. All rights reserved.
Certain structural motifs are repeatedly found in biologically
active small molecules. These reoccurring substructures1 or scaf-
folds2 were first coined as ‘‘privileged structures”3–6 by Evans. Priv-
ileged structures provide a backbone upon which chemical motifs
can be added to alter ligand specificity and activity. The presence of
two proximal aryl rings is part of three common privileged struc-
tural motifs (Fig. 1): biphenyl, diarylmethane, and vicinal diaryl
(two aryl groups located vicinal on a central ring). Diaryl-contain-
ing privileged structures have proven their importance in a num-
ber of drug discovery programs.6
Numerous biologically active molecules contain a privileged
structure of a central nitrogen heterocycle substituted with two
adjacent aryl groups (vicinal diaryl substructure) (Fig. 2).7 Two such
nitrogen heterocyclic ring systems, maleimides and 3-pyrrolin-2-
ones, have been studied in some detail. Polymethoxylated malei-
mides (e.g., 2),8 3-pyrrolin-2-ones (e.g., 3),9 and pyrroles (not
shown)10,11 have been investigated as cis-constrained analogs of
the promising anti-cancer agent, combretastatin A-4.12 SB-216763
(4) is an ATP-competitive inhibitor of glycogen synthase kinase.13
Bisindolemaleimide (5)14 is a potent inhibitor of protein kinase C,
which has inspired a large number of follow-up studies on bisin-
dolemaleimide analogs.15–17 Indole-substituted arylmaleimides
(e.g., 6)18 and aryl-3-pyrrolin-2-ones (e.g., 7)19 have shown anti-
angiogenic activity, while fused bisindolemaleimide 8 (arcyri-
aflavin A) and 3-pyrrolin-2-one 9 (K-252c) are natural products
with demonstrated antiviral20 and protein kinase inhibitory activ-
ity,21 respectively. Common aryl substituents found across this
sample of biologically active nitrogen maleimides and 3-pyrrolin-
2-ones include polymethoxyaryl groups and indole rings.
Building on our expertise in preparing aryl-substituted 3-pyrro-
lin-2-ones,22–25 we systematically studied the effects of changing
the aryl groups around the central 3-pyrrolin-2-one ring on cancer
cell viability. In our study (Fig. 3), we prepared a small library of
analogs that differed in the following ways: (i) analogs with differ-
ent numbers (and locations) of aryl groups and methoxy sub-
stituents around the aryl periphery; (ii) analogs with indole
groups located at different positions; and (iii) cyclized analogs
(central ring fusion).
We started by preparing 4-aryl-substituted 3-pyrrolin-2-ones
from known tetramic acid tosylate 10 (Scheme 1).23 This starting
material has previously been proven to be viable in Suzuki-
Miyaura cross-coupling reactions. Indeed, treatment of 10 with
arylboronic and Pd(dppf)Cl2 in the presence of Cs2CO3 gave 11c
and 11d in modest yields.
The preparation of 3,4-diaryl-3-pyrrolin-2-ones started with
the synthesis of 3-aryl- and 3-indolyltetramic acids 15 adapting
our recently published method (Scheme 2).25 The three-step proce-
dure included DCC-mediated amide coupling of arylacetic acids 12
⇑
Corresponding authors.
E-mail addresses: mowery@hws.edu (P. Mowery), pelkey@hws.edu (E.T. Pelkey).
http://dx.doi.org/10.1016/j.bmcl.2016.11.076
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Mowery P., et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.11.076

2
P. Mowery et al. / Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Ar3–B(OH)2
Ar4
TsO
Pd(dppf)Cl2
Cs2CO3, THF
O
O
N
H
N
H
10
Ar4 = 3,4-dimethoxyphenyl (37%)
Ar4 = 3,4,5-trimethoxyphenyl (58%)
11c
11d
biphenyl
diarylmethane
vicinal diaryl
Fig. 1. ‘‘Privileged” diaryl-containing structural motifs.
Scheme 1. Synthesis of 4-aryl-3-pyrrolin-2-ones.
EtO
NH2•HCl
with ethyl glycinate giving amidoesters 13, Boc-protection to 14
with Boc2O promoted by DMAP, and a Dieckmann cyclization to
tetramic acids 15 promoted by t-BuOK. The tetramic acids 15 were
then converted into the corresponding tetramic acid triflates 16 by
treatment with Tf2O followed by removal of the Boc-protecting
group with TFA. Suzuki-Miyaura cross-coupling of 16 with aryl-
boronic acids or arylboronic acid pinacol esters gave the desired
3,4-diaryl-3-pyrrolin-2-ones 17 (Fig. 4). Given that there are two
different aryl/heteroaryl rings present in most of the analogs, the
compound numbers assigned to each analog include two letters
with each letter denoting one of the aryl rings. For example,
17ea is an analog with a ‘‘N-methylindol-3-yl” substituent at the
3-position and a ‘‘phenyl” substituent at the 4-position.
We have previously found that electron-rich diaryl-substituted
3-pyrrolin-2-ones can be transformed into their corresponding
ring fused analogs using the oxidant, phenyliodine(III)bis(trifluo-
roacetate) (PIFA).25 Using this strategy, benzo[a]carbazole 18ec
was prepared using an oxidative cyclization of 17ec (Scheme 3).
Treatment of 17ec with PIFA and BF3ÁEt2O at À40 °C gave 18ec in
70% yield. An additional ring fused analog, 18cc, was available from
a prior study.25
O
DCC, Et3N
DMAP, CH2Cl2
(82-93%)
O
EtO
O
Ar3
O
t-BuOK, THF
HO
(57-59%)
N
R1
Ar3
Boc2O
DMAP
CH2Cl2
(83-86%)
13 R1 = H
12
14 R1 = Boc
Ar3–B(OH)2 or Ar3–Bpin
Pd(PPh3)4,
1. Tf2O, Et3N
Ar3
HO
Ar3
TfO
CH2Cl2
Na2CO3, THF
2. TFA
(18-35%)
O
O
N
Boc
15
N
H
(yields in Figure 4)
16
Ar3 & Ar4 groups
a = phenyl
Ar4
Ar3
b = 4-methoxyphenyl
c = 3,4-dimethoxyphenyl
d = 3,4,5-trimethoxyphenyl
e = N-methylindol-3-yl
O
N
H
17
Scheme 2. Synthesis of 3,4-diaryl-3-pyrrolin-2-ones.
To determine the potency of the various analogs, compounds
were tested on the human promonocytic cell line, U-937,26 via
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide] assay over 48 h.27 IC50 values were defined as the analog
concentration at which cell viability reduced to 50% compared to
mock-treated cells. The biological data for the compounds are pre-
sented in a graphical format in two figures: (i) indole-containing
analogs (Fig. 5) and (ii) polymethoxyaryl-containing analogs
(Fig. 6). The analogs with the most potent IC50 values within each
and 17dd (11
were statistically equivalent (p > 0.35).
In examining the results with the indole-containing analogs
(Fig. 5), it is clear that the location of the indole substituent on
the 3-pyrrolin-2-one central core ring impacts cell viability. Ana-
logs with indole substitution at C4, 11e28 and 17ce, were inactive,
l
M
3). The IC50 values for these three compounds
as defined as an IC50 > 100 lM. Two of the analogs with indole sub-
figure included one indole-containing analog, 17ec (10
and two polymethoxyaryl-containing analogs, 17bb (13
l
l
M
M
2),
2)
stitution at C3, 17ec (10
lM
2) and 17ed (38
lM
9), showed
Fig. 2. Biologically active diaryl-substituted maleimides and 3-pyrrolin-2-ones.
this work
number of aryl rings
indole rings
ring fusion
Me
number of methoxy groups
Me
N
MeO
MeO
OMe
MeO
N
analogsto be studied
4
3
O
O
O
O
N
H
N
H
N
H
N
H
aryl-substituted
3-pyrrolin-2-ones
(i)
(ii)
(iii)
Fig. 3. Proposed aryl-substituted 3-pyrrolin-2-one targets for anti-proliferative studies.
Please cite this article in press as: Mowery P., et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.11.076

P. Mowery et al. / Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
3
activity. The inactivity of 17ea indicated that a polymethoxyaryl
substituent at C4 was required. The number of methoxy groups
also appears consequential as dimethoxy analog 17ec was margin-
ally more active than 17ed (p = 0.091). Finally, the fused analog
18ec showed lower activity compared to unfused 17ec (p < 0.02).
This indicates that a rigid cyclic structure is less active than a more
flexible (non-ring fused) structure.
OMe
OMe
MeO
MeO
OMe
Me
N
O
O
N
H
N
H
17ad
17ce
(64%)
(47%)
To explore the importance of the indole ring and to further ana-
lyze how varying the number of methoxyaryl substitutions
impacts function, a series of polymethoxyaryl-containing analogs
were also explored. The results for these compounds were less
straight-forward (Fig. 6). Mono-substituted analogs, 11c and 11d,
showed no activity. On the other hand, all of the 3,4-disubstituted
3-pyrrolin-2-ones showed activity. In comparing trimethoxyaryl-
OMe OMe
MeO
OMe
OMe
MeO
MeO
OMe
OMe
O
O
N
N
H
17da
(76%)
H
17dd
(54%)
containing
(11 3) was more active than mono(trimethoxyphenyl) ana-
logs 17da (33 3) and 17ad (84 8) (p 6 0.004). Decreas-
ing the number of methoxy groups around the periphery had
differential effects. Analog 17cc (57 13), which contains four
methoxy groups, had much lower activity compared to 17dd
(p < 0.05). On the other hand, 17bb (13 2) with two methoxy
groups had statistically the same activity as the analog with six
methoxy groups 17dd (11 3) (p > 0.55). As seen previously,
analogs,
bis(trimethoxyphenyl)
analog
17dd
lM
l
M
lM
MeO
MeO
R
Me
N
Me
N
lM
O
lM
O
N
N
H
H
17ea
17ecR = H (21%)
lM
(39%)
17edR =OMe (40%)
ring fusion was detrimental to activity. Fused analog 18cc showed
no activity.
Fig. 4. 3,4-Diaryl-3-pyrrolin-2-one analogs synthesized.
MTT assays are an indirect measure of cell viability via mito-
chondrial activity and have been shown to vary in correlation with
actual cell counts, depending on the analog tested.29 To confirm the
validity of the MTT findings, direct live cell counts over 48 h were
measured using analog IC50 concentrations (Fig. 7); if the activity is
analogous then approximately 50% of the cells should be viable
after 48 h. We chose 17ec and 17dd to study live cell counts given
their low IC50 values and their representation of the two divergent
classes of analogs studied. By 48 h (the same incubation length for
the MTT assay), 61% 6 of 17ec and 51% 15 of 17dd treated cells
were alive, confirming the reliability and consistency of the MTT
assay for analogs with varying structural attributes.
Me
N
MeO
MeO
MeO
MeO
Me
N
PIFA, BF3•Et2O
CH2Cl2, –40°C
O
O
N
H
N
H
17ec
(70%)
18ec
Scheme 3. PIFA-mediated oxidative cyclization.
4-substituted
3,4-disubstituted
ring fused
Me
N
O
N
H
17ea
> 100 µM (n = 2)
Me
N
MeO
MeO
OMe
MeO
Me
N
Me
N
Me
N
OMe
MeO
O
O
O
O
N
H
N
H
N
N
H
17ec
H
11e
18ec
17ce
> 100 µM (n = 5)
48 µM 9 (n = 4)
> 100 µM
(n = 3)
10 µM
2
(n = 4)
MeO
MeO
OMe
Me
N
O
N
H
17ed
38 µM 9 (n = 3)
Fig. 5. Cytotoxicity results of indole-containing analogs on U-937 cells. U-937 cells were treated with compounds for 48 h followed by MTT treatment. Averages and standard
errors based on a minimum of three independent runs with eight replicates each; n = number independent runs.
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4
P. Mowery et al. / Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
4-substituted
3,4-disubstituted
ring fused
MeO
OMe
OMe
MeO
OMe
O
O
N
H
N
H
17da
17bb
33 µM
3
(n = 3)
13 µM 2 (n = 4)
OMe OMe
MeO
MeO
OMe
OMe
MeO
MeO
OMe
OMe
MeO
MeO
MeO
OMe
OMe
MeO
O
O
O
N
O
N
N
H
N
H
11c
H
H
17cc
18cc
17dd
57 µM 13
(n = 6)
> 100 µM (n = 4)
> 100 µM (n = 4)
11 µM 3 (n = 3)
MeO
MeO
OMe
MeO
MeO
OMe
O
O
N
H
N
H
17ad
11d
84 µM
8
(n = 4)
> 100 µM (n = 3)
Fig. 6. Cytotoxicity results of polymethoxyaryl-containing analogs on U-937 cells. U-937 cells were treated with compounds for 48 h followed by MTT treatment. Averages
and standard errors based on a minimum of three independent runs with eight replicates each; n = number independent runs.
trimethoxy-substituted analog (17dd) or methoxy-substituted
analog (17bb). These findings may reflect that the two classes of
compounds target different cellular proteins, and future studies
identifying analog targets may clarify the initial structure-function
findings.
Acknowledgments
Financial support of this research from the NSF (RUI: 1362813
to ETP), Hobart and William Smith Colleges, Drs. Carey and Cohen
(MHK, MML), Dr. Edward Franks, and the Patchett Family (CAR,
CLF) is gratefully acknowledged. We thank Dr. Bradley Cosentino
(Hobart and William Smith Colleges) for statistical conversations.
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.11.
076.
Fig. 7. Direct cell counts of analogs on U-937 cells. U-937 cells were treated with
compounds for 48 h at their IC50 value as determined by MTT assay. Samples were
removed periodically and direct cell viability was measured by trypan exclusion.
Percentage alive was calculated relative to mock-treated cells. Averages and
standard errors based on two independent runs, each in triplicate.
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