Novel three-component synthesis and antiproliferative properties of diversely functionalized pyrrolines

Article abstract of DOI:10.1016/j.bmcl.2008.01.019

Diversely substituted 2-pyrrolines have been prepared by a novel multicomponent process involving a reaction of various N-(aryl- and alkylsulfonamido)-acetophenones with aldehydes and malononitrile. While the reaction is highly regioselective, it is not s

Full text of DOI:10.1016/j.bmcl.2008.01.019

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 1392–1396
Novel three-component synthesis and antiproliferative properties
of diversely functionalized pyrrolines
a
b
a
Igor V. Magedov,a,b, Giovanni Luchetti, Nikolai M. Evdokimov, Madhuri Manpadi,
Wim F. A. Steelant,a Severine Van slambrouck,a Paul Tongwa,c
Mikhail Yu. Antipinc,d and Alexander Kornienkoa,*
*
aDepartment of Chemistry, New Mexico Institute of Mining and Technology, Socorro, NM 87801, USA
bDepartment of Organic Chemistry, Timiryazev Agriculture Academy, Moscow 127550, Russia
cDepartment of Natural Sciences, New Mexico Highlands University, Las Vegas, NM 87701, USA
dInstitute of Organoelement Compounds, Russian Academy of Sciences, Moscow, Russia
Received 30 November 2007; revised 27 December 2007; accepted 2 January 2008
Available online 11 January 2008
Abstract—Diversely substituted 2-pyrrolines have been prepared by a novel multicomponent process involving a reaction of various
N-(aryl- and alkylsulfonamido)-acetophenones with aldehydes and malononitrile. While the reaction is highly regioselective, it is not
stereoselective, generating a mixture of cis and trans 2-pyrrolines. A number of analogs from both cis and trans 2-pyrroline libraries
were found to have antiproliferative activity in human cancer cell lines.
ꢀ 2008 Elsevier Ltd. All rights reserved.
Nitrogen-containing five-membered heterocycles, such
as pyrrolines, are common structural scaffolds in natural
products and pharmaceutical agents.1,2 They have also
received a lot of attention as intermediates in the synthe-
ses of biologically active pyrroles and pyrrolidines.3
Examples of medicinally important pyrroline-based
compounds include protein kinase inhibitor stauro-
sporine (1)4 and geranylgeranyltransferase inhibitor 25
(Fig. 1).
H
N
HO2C
O
N
F
O S O
N
N
O
MeO
NHMe
Me
1
2
Although many methods to synthesize pyrrolines ex-
ist,2,6 the importance of these heterocyclic structures in
medicinal and synthetic chemistry provides strong impe-
tus to the development of novel synthetic routes that
may lead to pyrroline derivatives inaccessible with the
previously developed chemistry.7 As part of an effort
aimed at developing multicomponent reactions (MCRs)
leading to the preparation of privileged medicinal scaf-
folds,8 we investigated reactions of N-(arylsulfonami-
do)-acetophenones with aldehydes and malononitrile.
Figure 1. Structures of selected biologically active pyrrolines.
Specifically, we found that refluxing these three compo-
nents in ethanol in the presence of Et3N leads to the for-
mation of mixtures of densely functionalized cis and
trans 2-pyrrolines (Scheme 1).
R3
R1
R3
R3
NC
H2N
NC
H2N
O
CHO
EtOH
R1
R1
Keywords: Multicomponent synthesis; Antiproliferative; Apoptosis;
Heterocycles.
N
Et3N
reflux
N
CN
CN
NH
O
O
O
O
O
O
S
R2
O
O
S
R2
S
*
R2
Corresponding authors. Tel.: +1 575 835 6886; fax: +1 575 835 5364
(I.V.M.); tel.: +1 575 835 5884; fax: +1 575 835 5364 (A.K.); e-mail
addresses: imagedov@gmail.com; akornien@nmt.edu
Scheme 1. Three-component synthesis of 2-pyrrolines.
0960-894X/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.019

I. V. Magedov et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1392–1396
1393
In our proposed mechanistic pathway 2-pyrrolines result
from an initial attack of the a-carbon of the acetophe-
none moiety onto the double bond of the intermediate
Knoevenagel adduct, followed by cyclization (Fig. 2).
However, this requires extensive experimentation
involving crystallization from various combinations of
solvents and no general separation method was found.
We reasoned that by modulating the steric and elec-
tronic nature of the three substituents R1, R2, and R3,
it would be possible to significantly enrich the product
mixtures in either cis or trans pyrrolines to facilitate
separation. To this end, we prepared 14 N-(sulfon-
amido)-acetophenones incorporating electron-rich,
electron-poor, and neutral aromatic as well as aliphatic
groups at both sulfonamide and acetophenone parts of
the molecule,10 and utilized them in reactions with three
aromatic aldehydes also differing in their electronic
character.11 The ratios of cis and trans pyrrolines were
In contrast, the initial attack of the sulfonamidic nitro-
gen with the subsequent cyclization, which would lead
to the formation of 3-pyrrolines, does not occur. It
could be speculated that due to the higher acidity of
the sulfonamidic nitrogen, the concentration of the N-
deprotonated species is higher. However, in accordance
with the Curtin–Hammett principle, the faster Michael
addition of the C-deprotonated intermediate channels
the reaction toward the formation of 2-pyrrolines.
1
directly obtained from H NMR spectra of the crude
The X-ray crystallographic analysis of the separated cis
and trans pyrroline products provided unambiguous
proof of our structure assignments (Fig. 3).9
reaction mixtures and are shown in Table 1. Although
the reaction was successful with all combinations of
R1, R2, and R3, including the use of heteroaromatic
(ct-16–ct-18) and aliphatic sulfonamido groups (ct-19–
ct-24), a slight preponderance of the trans product
(1:1.5–1:2) remained highly conserved irrespective of
the differences in the electronic and steric nature of the
three substituents.
The yields of the stereoisomeric mixtures generally
exceed 90% and, in most cases, the product mixtures
can be separated into individual cis and trans pyrroline
components on the basis of their dissimilar solubilities.
CN
N
CN
R3
R3
NC
NC
R3
NC
NC
R3
R1
slow
R1
fast
R1
R1
N
N
N
HN
N
O
O
O
O S O
O S O
O
O S O
O S O
R2
R2
R2
R2
C-attack leads to 2-pyrrolines
N-attack leads to 3-pyrrolines
R3
H2N
O
CN
R3
NC
H2N
R1
R1
N
N
O
O S O
O S O
R2
R2
Figure 2. Proposed mechanistic interpretation of the formation of 2-pyrrolines.
R1 = C6H5, R2 = p-O2N-C6H5, R3 = C6H5
Figure 3. X-ray structures of selected cis and trans pyrroline products.

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I. V. Magedov et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1392–1396
Table 1. Ratios of cis to trans pyrrolines for different combinations of
substituents R1, R2, and R3 in the starting materials
Table 2. Antiproliferative effect of pyrroline mixtures
Mixture
% Cell viabilitya
MCF-7/AZ
72
R3
R3
NC
NC
HeLa
ct-1
ct-2
81
97
4
1
2
2
3
3
1
4
4
1
2
1
1
2
2
1
2
2
1
1
2
3
3
4
3
1
3
4
1
4
2
4
4
4
3
2
4
3
4
1
2
3
2
4
4
3
4
2
2
1
4
2
2
2
3
2
2
2
4
2
R1
R1
H2N
H2N
80
45
50
61
93
58
72
97
45
54
72
50
69
90
45
57
78
38
27
90
81
58
72
35
54
31
73
80
73
N
N
O
O
ct-3
ct-4
70
72
O S O
O S O
R2
R2
ct-5
ct-6
86
Pyrroline R1
mixture
R2
R3
Ph
cis: trans
ratio
103
65
ct-7
ct-8
91
ct-1
ct-2
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
p-O2N–C6H5
p-O2N–C6H5
p-O2N–C6H5
p-F–C6H5
1:2
ct-9
108
69
p–MeO–C6H5 1:2
p-O2N–C6H5 1:1.4
ct-10
ct-11
ct-12
ct-13
ct-14
ct-15
ct-16
ct-17
ct-18
ct-19
ct-20
ct-21
ct-22
ct-23
ct-24
ct-25
ct-26
ct-27
ct-28
ct-29
ct-30
ct-3
ct-4
65
Ph
1:1.6
105
36
ct-5
ct-6
p-F–C6H5
p-F–C6H5
p–MeO–C6H5 1:1.8
p-O2N–C6H5 1:1.7
98
ct-7
ct-8
p-Me–C6H5
p-Me–C6H5
p-Me–C6H5
Ph
1:1.9
115
79
p–MeO–C6H5 1:1.7
p-O2N–C6H5 1:1.7
ct-9
84
ct-10
ct-11
ct-12
ct-13
ct-14
ct-15
2,4,6-i-Pr–C6H5 Ph
1:1.7
104
54
2,4,6-i-Pr–C6H5 p-MeO–C6H5 1:2
2,4,6-i-Pr–C6H5 p-O2N–C6H5 1:1.9
46
p-MeO–C6H5
p-MeO–C6H5
p-MeO–C6H5
Ph
1:1.9
107
102
49
p-MeO–C6H5 1:1.6
p-O2N–C6H5 1:1.7
102
45
ct-16
ct-17
ct-18
Ph
Ph
Ph
Ph
1:1.2
S
S
S
36
45
82
98
p-MeO–C6H5 1:1.7
p-O2N–C6H5 1:1.4
82
a % Remaining cell viability after 48 h of treatment with indicated
pyrroline mixtures at the final concentrations of 100 lM relative to
DMSO control. Data are means SD of two independent experi-
ments, each performed in four replicates, determined by MTT assay.
ct-19
ct-20
ct-21
ct-22
ct-23
ct-24
ct-25
ct-26
ct-27
ct-28
ct-29
ct-30
Ph
Ph
Ph
Ph
Ph
Ph
Bu
Bu
Ph
1:1.7
p-MeO–C6H5 1:2
p-O2N–C6H5 1:1.4
Bu
Me
Ph
1:1.4
Me
Me
p-MeO–C6H5 1:1.5
p-O2N–C6H5 1:1.3
mixtures were then separated into the individual stereo-
isomerically pure cis and trans pyrroline components (to
give c and t series), which were further evaluated in these
cell lines at three concentrations (Fig. 4). Although the
activity profiles are similar for the two cell lines, HeLa
cells are more responsive. The GI50 values of the most
active pyrrolines fall in the range between 50 and
5 lM in this cell line. Another interesting observation
is that in most of the active mixtures both cis and trans
pyrroline products showed antiproliferative effect,
although it is more pronounced in the t series (e.g.,
t-25 vs c-25, t-26 vs c-26, t-26 vs c-26 in both cell lines).
p-Br–C6H5
p-Br–C6H5
p-Br–C6H5
p-Me–C6H5
p-MeO–C6H5
p-O2N-C6H5
Ph
Ph
Ph
Ph
Ph
Ph
1:1.6
1:1.5
1:1.5
1:1.6
1:1.4
1:1.6
p-MeO–C6H5 p-Me-C6H5
p-MeO–C6H5 p-MeO-C6H5
p-MeO–C6H5 p-O2N–C6H5
The synthesized pyrroline library was biologically tested
for its effect on the proliferation of two cancer cell lines,
HeLa and MCF7/AZ, as models for human cervical and
breast adenocarcinomas, respectively. Because of the labor-
intensive separation of the stereoisomeric pyrroline
mixtures, the initial tests were performed with these mix-
tures. The corresponding cells were treated with the test
pyrrolines at final concentrations of 100 lM for 48 h
and cell viability was assessed through measurements
of mitochondrial dehydrogenase activities using the
MTT method.12 The percent of remaining cell viability
for each pyrroline mixture is shown in Table 2.
Our preliminary investigation of the biological mecha-
nism underlying the antiproliferative properties of the
pyrroline library points to an exciting possibility of dis-
similar modes of action for the cis and trans pyrroline
series. The library members c-25 and t-25 were evaluated
for their ability to induce apoptosis in Jurkat cells (a
model for human T-cell leukemia) using a flow cytomet-
ric annexin-V/propidium iodide assay. In contrast to
compound t-25, which shows little effect in this assay,
treatment of Jurkat cells with pyrroline c-25 produces
large populations of dead cells as well as cells manifest-
ing apoptotic phenotype (Table 3).
We were pleased to find that many pyrroline mixtures
exerted an antiproliferative effect in both cell lines with
some of them decreasing cell viability below 50%. These

I. V. Magedov et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1392–1396
1395
Figure 4. Antiproliferative effect of the individual cis and trans pyrrolines. % Remaining cell viability of HeLa (top) and MCF7/AZ (bottom) cells
after 48 h of treatment with indicated pyrrolines at the final concentrations of 100, 50, and 5 lM relative to DMSO control. Data are means SD of
two independent experiments, each performed in four replicates, determined by MTT assay.
Table 3. Apoptosis inducing and cell killing properties of pyrroline
c-25
Compound
% Apoptosisa
% Live cellsb
c-25 (50 lM)
t-25 (50 lM)
Podophyllotoxin (5 lM)
30
3
2
2
56
96
22
4
3
1
1
63
a % Apoptotic Jurkat cells after 48 h of treatment with indicated
compounds relative to DMSO control.
b % Live Jurkat cells after 48 h of treatment with indicated compounds
relative to DMSO control. Data are means SD of two independent
experiments, each performed in three replicates, determined by flow
cytometric Annexin-V/propidium iodide assay.
Figure 5. Cell counting assay performed with c-25 and t-25. Effect of c-
25 (50 lM) and t-25 (50 lM) on the growth of Jurkat cells estimated by
Trypan Blue dye exclusion method. The error bars represent data from
multiple counts.
It appears that c-25 exerts its antiproliferative effect
through induction of apoptosis in cancer cells, while
t-25 is strictly growth inhibitory and lacks cell killing
properties. This conclusion is consistent with the results
of a cell counting assay, which revealed that the number
of Jurkat cells decreased after 48 h as compared with the
original quantity, when the cells were treated with c-25.
In contrast, treatment with t-25 only blocked the cell
growth (Fig. 5).13
underway to make the reaction stereoselective and study
the biology of these compounds in more detail, as it ap-
pears that multiple biological activities reside in these
novel pyrroline libraries. The results of these studies will
be reported in a full article elsewhere.
In conclusion, a novel three-component reaction, lead-
ing to the synthesis of diversely substituted cis and trans
2-pyrrolines, was discovered and a library of these com-
pounds was prepared. Both cis and trans pyrrolines were
found to exhibit growth inhibitory properties in a num-
ber of human cancer cell lines. The preliminary investi-
gation of the biological mechanism of action revealed
that the modes of action of the two series of stereoiso-
meric pyrrolines may be different. Further work is
Acknowledgments
U.S. National Institutes of Health (Grants CA-99957
and RR-16480) are gratefully acknowledged for finan-
cial support of this work. G.L. thanks Artem S. Kireev
for his invaluable assistance with experimental tech-
niques and general guidance in the laboratory. P.T.

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I. V. Magedov et al. / Bioorg. Med. Chem. Lett. 18 (2008) 1392–1396
Data Centre and the allocated deposition numbers are
CCDC 669040 and 669041, respectively. The detailed
structure descriptions will be published elsewhere.
and M.Yu.A. are grateful to NSF/DMR (Grant
0420863) for the acquisition of X-ray single crystal dif-
fractometer and to the Distributed Nanomaterials Char-
acterization Network in the framework of New Mexico
NSF EPSCoR Nanoscience initiative. We thank Profes-
sors Tatiana V. Timofeeva and Snezna Rogelj for their
kind assistance with X-ray crystallography and flow
cytometry, respectively.
10. The substituted N-(sulfonamido)-acetophenones were
prepared in the following way. To the solution of the
hydrochloride of a required aminomethylaryl ketone
(30 mmol) in H2O (30 mL) was added a corresponding
sulfonyl chloride (30 mmol) in acetone (70 mL) dropwise
with stirring at 10 ꢁC. Et3N (65 mmol) was then added
with vigorous stirring over 1 h. The reaction mixture was
further stirred for additional 1.5 h at the same temperature
and the precipitate was collected by vacuum filtration. The
filtrate was reduced in volume to one-half of the original
and additional precipitate was collected by filtration. The
precipitates were combined and recrystallized from etha-
nol. The yields were in the range of 86–92%.
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pyrrolines c-1 and t-1: To a solution of benzaldehyde
(95 mg, 0.9 mmol), malononitrile (59 mg, 0.9 mmol)
in EtOH (5 mL) were added Et3N (0.05 mL) and
N-(p-nitrobenzenesufonamido)-acetophenone
(290 mg,
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¨
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yield a mixture of pyrrolines c-1 and t-1. The mixture was
suspended in MeCN (25 mL) and refluxed for 30 min.
After cooling the suspension to room temperature the
solid was isolated by filtration and washed with methanol
(3 · 2 mL) to yield pure cis pyrroline c-1 as a yellow solid
(124 mg, 29%). The filtrate was evaporated to dryness to
give pure trans pyrroline t-1 as a yellow solid (265 mg,
62%). c-1: mp 201 ꢁC (MeCN); 1H NMR (DMSO-d6) d
8.53 (d, J = 8.8 Hz, 2H), 8.38 (d, J = 8.8 Hz, 2H), 7.65 (d,
J = 7.7 Hz, 2H), 7.49 (t, J = 7.4 Hz, 1H), 7.32 (d,
J = 7.4 Hz, 1H), 7.17 (s, 2H), 6.97 (s, 2H), 6.91 (s, 1H),
6.38 (d, J = 11.0 Hz, 1H), 4.77 (d, J = 11.0 Hz, 1H); 13C
NMR (DMSO-d6) d 194.2, 155.0, 151.3, 143.0, 136.4,
135.5, 134.0, 129.9, 128.8, 128.3, 128.0, 125.5, 118.2, 67.5,
62.7, 47.1. HRMS m/z (ESI) calcd for C24H19N4O5S
(M+H+) 475.1076, found 475.1058. t-1: mp 194 ꢁC
(MeCN); 1H NMR (DMSO-d6) d 8.47 (d, J = 8.3 Hz,
2H), 8.34 (d, J = 8.3 Hz, 2H), 7.90 (d, J = 7.4 Hz, 2H),
7.74 (t, J = 6.6 Hz, 1H), 7.59 (d, J = 7.4 Hz, 2H), 7.29 (s,
2H), 7.22 (t, J = 7.2 Hz, 1H), 7.11 (d, J = 7.2 Hz, 2H), 6.51
(d, J = 7.2 Hz, 2H), 5.56 (s, 1H), 3.83 (s, 1H); 13C NMR
(DMSO-d6) d 193.3, 154.4, 151.4, 142.5, 141.6, 135.0,
133.5, 129.9, 129.6, 128.3, 127.2, 125.6, 118.1, 71.0, 62.0,
47.0. HRMS m/z (ESI) calcd for C24H19N4O5S (M+H+)
475.1076, found 475.1079.
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pyrrolines in the cell culture to the corresponding pyrroles,
because in this case the biological effects of trans and cis
pyrrolines would be expected to be similar.
9. The crystal structures of these cis and trans pyrrolines
have been deposited at the Cambridge Crystallographic