A Combination Strategy to Inhibit Pim-1: Synergism between Noncompetitive and ATP-Competitive Inhibitors

Article abstract of DOI:10.1002/cmdc.201200480

Pim-1 is a serine/threonine kinase critically involved in the initiation and progression of various types of cancer, especially leukemia, lymphomas and solid tumors such as prostate, pancreas and colon, and is considered a potential drug target against these malignancies. In an effort to discover new potent Pim-1 inhibitors, a previously identified ATP-competitive indolyl-pyrrolone scaffold was expanded to derive structure-activity relationship data. A virtual screening campaign was also performed, which led to the discovery of additional ATP-competitive inhibitors as well as a series of 2-aminothiazole derivatives, which are noncompetitive with respect to both ATP and peptide substrate. This mechanism of action, which resembles allosteric inhibition, has not previously been characterized for Pim-1. Notably, further evaluation of the 2-aminothiazoles indicated a synergistic inhibitory effect in enzymatic assays when tested in combination with ATP-competitive inhibitors. A synergistic effect in the inhibition of cell proliferation by ATP-competitive and ATP-noncompetitive compounds was also observed in prostate cancer cell lines (PC3), where all Pim-1 inhibitors tested in showed synergism with the known anticancer agent, paclitaxel. These results further establish Pim-1 as a target in cancer therapy, and highlight the potential of these agents for use as adjuvant agents in the treatment of cancer diseases in which Pim-1 is associated with chemotherapeutic resistance.

Full text of DOI:10.1002/cmdc.201200480

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DOI: 10.1002/cmdc.201200480
A Combination Strategy to Inhibit Pim-1: Synergism
between Noncompetitive and ATP-Competitive Inhibitors
Mattia Mori,[a, b] Cristina Tintori,[a] Robert Selwyne Arul Christopher,[a] Marco Radi,[a]
Silvia Schenone,*[c] Francesca Musumeci,[c] Chiara Brullo,[c] Patrizia Sanitꢀ,[d]
Simona Delle Monache,[d] Adriano Angelucci,[d] Miroslava Kissova,[e] Emmanuele Crespan,[e]
Giovanni Maga,[e] and Maurizio Botta*[a, f]
Pim-1 is a serine/threonine kinase critically involved in the ini-
tiation and progression of various types of cancer, especially
leukemia, lymphomas and solid tumors such as prostate, pan-
creas and colon, and is considered a potential drug target
against these malignancies. In an effort to discover new potent
Pim-1 inhibitors, a previously identified ATP-competitive indol-
yl-pyrrolone scaffold was expanded to derive structure–activity
relationship data. A virtual screening campaign was also per-
formed, which led to the discovery of additional ATP-competi-
tive inhibitors as well as a series of 2-aminothiazole derivatives,
which are noncompetitive with respect to both ATP and pep-
tide substrate. This mechanism of action, which resembles allo-
steric inhibition, has not previously been characterized for
Pim-1. Notably, further evaluation of the 2-aminothiazoles indi-
cated a synergistic inhibitory effect in enzymatic assays when
tested in combination with ATP-competitive inhibitors. A syner-
gistic effect in the inhibition of cell proliferation by ATP-com-
petitive and ATP-noncompetitive compounds was also ob-
served in prostate cancer cell lines (PC3), where all Pim-1 inhibi-
tors tested in showed synergism with the known anticancer
agent, paclitaxel. These results further establish Pim-1 as
a target in cancer therapy, and highlight the potential of these
agents for use as adjuvant agents in the treatment of cancer
diseases in which Pim-1 is associated with chemotherapeutic
resistance.
Introduction
Proviral integration site for Moloney murine leukemia virus 1
(PIM1) is a proto-oncogene first identified as a preferential pro-
viral integration site in Moloney murine leukemia virus-induced
T-cell lymphomas.[1,2] PIM1 encodes the serine/threonine kinase
Pim-1, the first member described of a family that includes at
least two other kinases, Pim-2 and Pim-3, which are structurally
related to Pim-1.[2,3]
transformation and tumor progression, especially in hemato-
poietic malignancies and prostate carcinoma where it is
a marker of a poor prognosis.[6–11] Somatic mutations and chro-
mosomal translocations in PIM1 have been also identified in
primary central nervous system (CNS) lymphomas.[12] In addi-
tion, overexpression of Pim-1 in hemopoietic cells enhances
cell survival by protecting these cells from apoptosis induced
by cytokine withdrawal, glucocorticoids, and genotoxic stress.
In light of its oncogenic potential, Pim-1 is emerging as an im-
portant new target for drug discovery. Accordingly, research
into Pim-1 inhibitors has received growing attention in recent
years and has led to the discovery of small molecules charac-
Human Pim-1 is a 313-amino acid serine/threonine kinase
that possesses several biological functions in cell survival, pro-
liferation and differentiation, and its overexpression has been
observed in several human cancers.[4,5] Indeed, the gene that
encodes Pim-1 is a proto-oncogene implicated in early cell
[a] Dr. M. Mori,+ Dr. C. Tintori,+ Dr. R. S. A. Christopher, Dr. M. Radi,
Prof. M. Botta
[d] Dr. P. Sanitꢀ, Dr. S. Delle Monache, Dr. A. Angelucci
Dipartimento di Scienze Cliniche Applicate e Biotecnologiche
Universitꢀ dell’Aquila
Dipartimento di Biotecnologie, Chimica e Farmacia
Universitꢀ degli Studi di Siena
Via Vetoio (Coppito 2), 67100 Coppito, L’Aquila (Italy)
Via A. Moro 2, 53100 Siena (Italy)
E-mail: botta.maurizio@gmail.com
[b] Dr. M. Mori+
[e] M. Kissova, Dr. E. Crespan, Dr. G. Maga
Istituto di Genetica Molecolare (IGM-CNR)
Via Abbiategrasso 207, 27100 Pavia (Italy)
Dipartimento di Chimica e Tecnologie del Farmaco
Universitꢀ di Roma “La Sapienza”
Piazzale A. Moro 5, 00185 Roma (Italy)
[f] Prof. M. Botta
Sbarro Institute for Cancer Research and Molecular Medicine
Center for Biotechnology, College of Science and Technology
Temple University
[c] Prof. S. Schenone, Dr. F. Musumeci, Dr. C. Brullo
Dipartimento di Farmacia
BioLife Science Building, Suite 333, 1900 N 12th St., Philadelphia, PA 19122
Sezione di Chimica del Farmaco e del Prodotto Cosmetico
University of Genoa
(USA)
[+] Authors equally contributed to this work.
Viale Benedetto XV 3, 16132 Genova (Italy)
E-mail: schensil@unige.it
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201200480.
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ed to adopt a canonical orientation within the binding site,
being involved in the same putative pattern of interactions as
staurosporine within the Pim-1 binding site.
Here, derivatives of 1 were synthesized based on molecular
modeling studies and then assayed for their activity against
Pim-1. A structure–activity relationship study of indolyl-pyrro-
lone Pim-1 inhibitors is reported. Furthermore, still pursuing
the aim of identifying novel Pim-1 inhibitors, a structure-based
drug design approach was applied that led to the identifica-
tion of new hit compounds characterized by chemical scaffolds
different to that of compound 1 as well as other Pim-1 inhibi-
tors reported in literature. The approach described involved
the use of a large amount of recently published crystallograph-
ic data that was not available during previous studies.[16] Nota-
bly, two of the newly identified hit compounds showed an
atypical mechanism of action, being noncompetitive against
both ATP and the peptide substrate in enzymatic assays. This
mechanism of action might be approximated as allosteric in-
hibition of Pim-1. To the best of our knowledge, no allosteric
inhibitor of Pim-1 kinase has been reported so far, and as such,
the results of this research lay the foundation for the develop-
ment of noncompetitive Pim-1 inhibitors.
Compounds with significant inhibitory activity were also
evaluated for their in vitro antitumoral effects against prostate
cancer, for which Pim-1 is currently under investigation as a po-
tential therapeutic target.[21] Pim-1 inhibitors, at micromolar
concentrations, decrease proliferation of different prostate
cancer cells, and this effect was particularly evident in combi-
nation with the chemotherapeutic taxane, paclitaxel (PTX).
Moreover a synergistic antiproliferative effect was observed in
some cell lines when ATP-competitive and ATP noncompetitive
compounds were used in combination.
Figure 1. Examples of known Pim-1 inhibitors recently reported in the litera-
ture.
terized by a high degree of chemical diversity (Figure 1). These
known inhibitors belong to different chemical classes, includ-
ing ruthenium-containing organometallic complexes, bis-indo-
lylmaleimides (e.g., staurosporine), imidazo[1,2-b]pyridazines
(e.g., K00135), pyridones, flavonoids (e.g., quercetagetin), ben-
zoisoxazoles (e.g., VX1), isoxazolo[3,4-b]quinoline-diones, indo-
lyl-pyrrolones, pyrrolo[2,3-a]carbazole, 3H-benzo[4,5]thieno[3,2- Results and Discussion
d]pyrimidin-4-ones, thiazolidinediones, triazolo[4,3-b]pyrid-
Indolyl-pyrrolone as a promising scaffold for Pim-1 inhibi-
tors
azines, triazolopyridazines (e.g., VX2), and dipheylindoles (e.g.,
VX3).[13,14]
A key role in the discovery of novel Pim-1 inhibitors has
been played by X-ray crystallography.[5] Interestingly, crystal
structures of Pim-1 have revealed an atypical sequence in the
hinge region and a unique conformation that distinguishes
this protein from other structurally related kinases. In detail,
Pim-1 is characterized by the presence of a proline residue at
position 123, thus the adenine nucleus of ATP cannot form the
second hydrogen bond with the hinge region that is con-
served in all other protein kinases. Because of the availability
of many crystal structures, molecular modeling methods have
been applied in recent years for the identification of Pim-1 in-
hibitors.[15–20]
In order to study the structure–activity relationships of indolyl-
pyrrolones as Pim-1 inhibitors, a series of analogues (2–19)
was synthesized and tested in vitro for their inhibitory activity
against Pim-1 (Table 1). Taking into account the results from
previous docking studies, modifications were introduced while
maintaining the central core. In detail, our recent theoretical
studies on indolyl-pyrrolone 1 highlighted the structural fea-
tures responsible for the binding of this compound within the
ATP binding site of Pim-1 kinase.[16]
Compound 1 is predicted to bind with the indolyl-pyrrolone
nucleus within the adenine region where it can engage in hy-
drogen bonds with Glu121, Asp128 and Val126 (Figure 2a).
Furthermore, hydrophobic contacts were predicted between
the phenyl group of the ligand and Leu44, Phe49, Val52,
termed hydrophobic region I, and the indole ring and Val52,
Ala65, Ile104, Ile185, termed hydrophobic region II. According
with these considerations, we decided to synthesize: 1) deriva-
tives bearing different hydrophobic substituents in the para or
meta position of the phenyl ring in order to better explore the
In this respect, a structure-based virtual screening approach
was recently employed in our group by applying different fil-
ters such as pharmacophore models, drug-like property calcu-
lations, and docking simulations.[16] As a result of this research,
indolyl-pyrrolone 1 (Figure 1) emerged as a new scaffold and
exhibited inhibitory activity in the micromolar range against
Pim-1.[16] Despite its peculiar structure, compound 1 is predict-
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Table 1. Structure and in vitro Pim-1 kinase inhibitory activities of com-
pounds 2–19.[a]
Compd
R1
R2
ID50 [mm]
Ki [mm]
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
H
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
C6H5
C6H4-4-Br
C6H4-4-CF3
C6H4-4-CH(CH3)2
C6H4-4-piperidin-1-yl
C6H4-4-C6H5
C6H4-3-OC6H5
CH2CH2C6H5
C6H5
74
41
68
20
10
17.78
9.86
16.35
4.81
2.40
4.33
6.73
n.d.
32.69
14.42
27.64
26.20
n.d.
18
28
@100
136
60
115
109
@100
21
@100
@100
33
C6H4-4-Br
C6H4-4-CF3
C6H4-4-CH(CH3)2
C6H4-4-piperidin-1-yl
C6H4-4-C6H5
C6H4-3-OC6H5
CH2CH2C6H5
C6H4-4-CH3
5.05
n.d.
n.d.
7.93
4.57
C6H4-4-OCH3
19
[a] Compounds exhibiting an ID50 value significantly greater than 100 mm
were not investigated further. Not determined (n.d.). Data are the mean
of three independent experiments. Standard deviation (SD) was lower
than 5%. ID50: concentration of the inhibitor that reduced the in vitro ac-
tivity of Pim-1 by 50% under the conditions described in the Experimen-
tal Section.
Figure 2. a) Predicted binding mode of the indolyl-pyrrolone 1. The new de-
rivatives were designed in order to explore whether further hydrophobic in-
teractions with hydrophobic regions I and II could influence the activity of
the derivatives against Pim-1. b) Predicted binding mode of indolyl-pyrro-
lone 6.
interactions with hydrophobic region I; 2) analogues with a me-
thoxy substituent in position 5 of the indole ring to investigate
the effect of an additional lipophilic group within hydrophobic
region II.
(22j) were reacted, in presence of a basic catalyst, at 808C for
a significantly longer period (14 h) to obtain compounds 6 and
14 (Entry 8, Table 2).
The synthesis of compounds 2–19 was accomplished via
a
one-pot, three-components Biginelli-like condensation
(Scheme 1). Presumably, the reaction mechanism includes the
formation of a Schiff base from the aldehyde and the amine,
followed by reaction with the enolic form of 2,4-dioxopentano-
ic acid ethyl ester to give the final product through an intra-
molecular ring closure.[22]
Table 2. Preliminary study of synthesis optimization using compounds 4
and 6 as model systems.
Entry Desired Catalyst[a] Solvent[b] Method[c] 20:21a:22:cat[d] Yield[e]
product
[%]
1
2
3
4
5
6
7
8
9
4
4
4
4
6
6
6
6
6
DIPEA
AcOH
AcOH
DIPEA
DIPEA
AcOH
AcOH
DIPEA
AcOH
DME
iPrOH
DME
DME
DME
iPrOH
DME
DME
iPrOH
MW
MW
MW
1:1:1:0.1[f]
39.6
A microwave (MW)-assisted organic synthesis approach was
followed for the synthesis of these compounds, since MW con-
ditions usually allow shorter reaction times than traditional
methods. For the synthesis of compounds 2–5, 7–13 and 15–
19, 2,4-dioxopentanoic acid ethyl ester (20), tryptamine (21a)
or 5-methoxytryptamine (21b), and the appropriate aldehyde
(22a–i) were reacted in the presence of a basic catalyst under
MW irradiation (Scheme 1A; Entry 1, Table 2).
1.25:1.23:1:17.5[g] 30.0
1:1:1:0.1
20.1
47.1
NP
NP
NP
Traditional 1:1:1:0.1
MW
MW
MW
1:1:1:0.1
1.25:1.23:1:17.5
1:1:1:0.1
Traditional 1:1:1:0.1
Traditional 1.25:1.23:1:17.5
20.5
15.0
[a] N,N-Diisopropylethylamine (DIPEA) and acetic acid (AcOH) are indicat-
ed as catalysts even where used in excess. [b] Dimethoxyethane (DME),
propan-2-ol (iPrOH). [c] Microwave (MW) conditions: 1308C, 5 min, 300 W;
traditional conditions: N2 atmosphere, 808C, 14 h. [d] Ratio of reagents
and catalyst (cat). Compound 22 refers to the appropriate derivative: i.e.,
22c for the synthesis of 4, and 22j for the synthesis of 6. [e] Isolated
yield of desired product. No product (NP) obtained. [f] Ratio selected ac-
cording to experimental procedure reported by Zou et al.[24] [g] Ratio se-
lected according to experimental procedure reported by Choi et al.[23]
In contrast, despite several attempts, the MW-assisted syn-
thesis of 4-piperidinyl derivatives 6 and 14 led to a mixture of
decomposition products, preventing the use of this technique
(Entries 5–7, Table 2). Therefore, these two compounds were
synthesized via a conventional method (Scheme 1B). 2,4-Di-
oxopentanoic acid ethyl ester (20), tryptamine (21a) or
5-methoxytryptamine (21b), and 4-piperidin-1-yl-benzaldehyde
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Scheme 1. Synthesis of A) compounds 2–5, 7–13, 15–19, and B) compounds 6 and 14. Reagents and conditions: a) N,N-Diisopropylethylamine (DIPEA),
dimethoxyethane (DME), microwave (300 W), 1308C, 5 min, 15–69%; b) DIPEA, DME, 808C, 14 h, 21% (6) and 15% (14).
The evident difference in yield between MW and traditional
synthesis for 4-piperidinyl derivatives prompted us to investi-
gate whether the trend was maintained also for other com-
pounds. 4-Trifluoromethyl derivative 4, which was obtained in
fair yields using MW synthesis (40%), was resynthesized using
a traditional protocol (Entry 4, Table 2). While the classical
method led to a slight increase in yield (47%), comparison of
the reaction times (5 min vs 14 h) indicated that MW irradia-
tion is generally preferable for the synthesis of these com-
pounds.
para position of the phenyl ring, as well as the introduction of
a methoxyphenyl moiety in the meta position, were favorable
for activity. The best compounds is this series were 5–8 with
ID50 values of 20, 10, 18, 28 mm and Ki values of 4.81, 2.40, 4.33,
6.73 mm, respectively.
Conversely, the introduction of a methoxy group in posi-
tion 5 of the indole ring (10–19) resulted to be detrimental to
activity (cf. 10–14, 16 with 2–6, 8, respectively). Only com-
pounds 15, 18, and 19, with ID50 values of 21, 33, 19 mm and Ki
values of 5.05, 7.93 and 4.57 mm, respectively, showed activities
comparable with the best compounds of the unsubstituted
series (5–8).
From docking studies, the proposed binding mode of the
active compounds was comparable with that of the hit com-
pound 1. Taken together, the results of the docking study and
biological evaluation indicate that hydrophobic region I can ac-
commodate both aliphatic and aromatic groups. For example,
the predicted binding mode of compound 6, the most active
indolyl-pyrrolone reported here, indicates that 6 establishes fa-
vorable CH–p interactions with the residue Phe49 (Figure 2b).
In contrast, placement of a methoxy group in hydrophobic
region II is not favorable for activity. Despite the profitable hy-
drophobic interactions of the 5-methoxy group predicted to
form within the pocket, a moderate loss of activity was ob-
served for compounds 10–13 (cf. 2–5, respectively). This could
be due to the energy cost required for the 5-substituted un-
bound ligand to achieve the predicted bound conformation in
Since these reactions generally gave low yields, mainly due
to the formation of several byproducts with a Rf values very
similar to that of the desired compound, a preliminary study
was conducted to evaluate the best reaction conditions. Start-
ing from data reported in the literature, showing that ana-
logues of these compounds have been synthesized both
under acid and basic conditions,[23,24] several experiments were
performed in presence of acetic acid to verify the impact on
the yields both in MW-assisted (Entries 2–3, Table 2) and tradi-
tional synthesis (Entry 9, Table 2). Data showed that basic catal-
ysis gives the best results in both methods. Nevertheless,
yields have to be further improved, and an optimization study
is ongoing looking at other solvents and bases.
The synthesized compounds were assayed for their inhibito-
ry activity toward Pim-1 (Table 1). With regard to compounds
2–8, the substitution of the hydrogen with bromine, trifluoro-
methyl, isopropyl, piperidine-N-yl and phenyl groups in the
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solution. Indeed, we calculated that an energy cost of 13.05,
4.01, 2.67 and 4.63 kJmolꢀ1 is required for compounds 10, 11,
12 and 13, respectively, while the energy cost is approximately
0 kJmolꢀ1 for binding of the 5-unsubstituted analogues 2–5 in
the proposed conformation. On the other hand, a marked de-
crease in activity was observed for compounds 14 and 16 (cf.
5-unsubstituted derivatives 6 and 8), which is probably due to
steric hindrance caused by the R2 substituent preventing the
concurrent placement of the 5-methoxy group within the hy-
drophobic pocket.
lecular docking was performed with GOLD by using the Chem-
Score function that was able to successfully self-dock crystallo-
graphic ligands into their native receptor structures. The dock-
ing-based binding modes of 500 top scoring compounds were
visually analyzed, and molecules showing a similar interaction
pattern within hydrophobic regions I and II were deemed top
priority.
Twenty of the most interesting compounds (23–42) having
a reasonable chemical diversity against known Pim-1 inhibitors
were selected for further in vitro evaluations (for chemical
structures, see the Supporting Information). Six compounds
(23, 30, 33, 37, 40 and 41) were found to inhibit Pim-1 catalytic
activity at concentrations lower than 100 mm in an enzymatic
assay (Figure 3 and Table 3). Candidate hit compounds were
subsequently tested by means of enzymatic and kinetic assays
prior to being analyzed in prostate cancer (PC3) cells. In details,
compounds were investigated for competition against ATP or
peptide substrate, in order to elucidate the mechanism of in-
hibition. Curiously, while most hit compounds showed ATP and
peptide substrates competition, 2-aminothiazole 37 was non-
competitive either against the ATP or the peptide substrate.
Structure-based virtual screening
With the aim of identifying novel chemical scaffolds for
Pim-1 inhibitors, with substantial chemical diversity in contrast
to indolyl-pyrrolone 1, a structure-based virtual screening rely-
ing on pharmacophore screening and molecular docking was
performed. In contrast to the previous computational screen-
ing performed against Pim-1,[16] at the time of the study de-
scribed here, several additional crystallographic structures of
Pim-1 in complex with ATP-competitive inhibitors were avail-
able (see Supporting Information). Crystallographic structures
were classified into three groups based on the bound inhibitor
scaffold, interaction pattern within the Pim-1 active site, and
the conformation of the protein glycine-rich loop, which is also
known as the P-loop.[25]
Two main conformations of the P-loop were found in
Pim-1 X-ray crystal structures, characterized by two different
conformations of the Phe49 side chain: P-loop-in, where
Phe49 points toward the hinge loop by partially occupying
the adenylyl-imidodiphosphate (AMP-PNP) binding site; P-loop-
out, where Phe49 points toward the solvent, and the loop
adopts a more opened conformation.
Three main pharmacophores were generated for Pim-1/in-
hibitor complexes by using LigandScout 2.0,[26] namely pharm1
and pharm2 accounting for the P-loop-in protein conformation
and pharm3 accounting for the P-loop-out conformation (see
Supporting Information). These three pharmacophore models
were then used as three-dimensional queries to screen the
whole Asinex Ltd database (about 550000 compounds). The
FitValue was used as a measure of how well the ligand fits the
pharmacophore. Accordingly, 8810 molecules mapping pharm1
or pharm2 with a FitValue higher than 2.0 and 699 molecules
mapping pharm3 with a FitValue higher than 3.5 were selected
for subsequent docking studies.
Figure 3. Of the twenty virtual hits evaluated, the six Pim-1 inhibitors shown
here exhibited activity in an enzymatic assay. The full list of virtual hits 23–
42 are given in the Supporting Information.
The 2-aminothiazole series
Compounds filtered through the pharm1 or pharm2 pharma-
cophore models were docked toward the representative crys-
tallographic structure of the Pim-1 catalytic site having a
P-loop-in conformation (PDB: 2C3I).[27] Conversely, compounds
filtered through the pharm3 model were docked with the
Pim-1 catalytic site having a P-loop-out conformation (PDB:
1XR1).[28] These structures were selected because they have the
lowest root-mean-square deviation (RMSD) of active site atoms
with respect to all other ligand-bound Pim-1 crystallographic
structures available.[29] A conserved crystallographic water mol-
ecules was retained and considered as part of the receptor.
After protein relaxation by means of energy minimization, mo-
Allosteric sites on Pim-1 have not been hypothesized or identi-
fied yet. However, enzymatic assays and kinetic studies re-
vealed that compound 37 inhibits Pim-1 by an atypical non-
competitive mechanism that might be comparable with allo-
steric inhibition. With the aim of understanding possible mo-
lecular determinants responsible for the noncompetitive mech-
anism of inhibition, eight derivatives of 37 were purchased
from Asinex Ltd and tested in vitro (Figure 4).
Compounds 43–50 exhibited a range of inhibitory activities
against Pim-1, and most compounds were active at concentra-
tions of less than 130 mm (Table 4). Compound 43 in particular
showed the same mechanism of action already observed for
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Table 3. In vitro inhibitory activities of virtual hits selected for further
evaluation.[a]
Table 4. Pim-1 inhibitory activities of the 2-aminothiazole series.[a]
Compd
ID50 [mm]
Ki [mm]
Compd
ID50 [mm]
Ki [mm]
Compd
ID50 [mm]
Ki [mm]
43
2.65
5.00
26.80
111.00
127.00
207.00
122.00
206.00
2.65
1.20
6.44
n.d.
n.d.
n.d.
n.d.
n.d.
23
24
25
26
27
28
29
30
31
32
25.00
@100
@100
@100
@100
@100
@100
8.55
6.01
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
2.05
n.d.
n.d.
33
34
35
36
37
38
39
40
41
42
89.00
@100
@100
@100
10.00
@100
@100
12.54
21.39
n.d.
n.d.
n.d.
10.00
n.d.
44[b]
45[b]
46
47
48
49
50
n.d.
3.01
24.04
n.d.
[a] Compounds exhibiting an ID50 value of greater than 30 mm were not
investigated further. Not determined (n.d.). Data are the mean of three in-
dependent experiments. Standard deviation (SD) was lower than 5%.
[b] ATP-competitive.
@100
@100
100.00
@100
[a] Compounds exhibiting an ID50 value significantly greater than 100 mm
were not investigated further. Not determined (n.d.). Data are the mean
of three independent experiments. Standard deviation (SD) was lower
than 5%.
co-crystallization of 43 within the catalytic active site by soak-
ing was unsuccessful.[17] Based on this evidence, studies aimed
at the identification and characterization of the possible allo-
steric site of Pim-1 are currently going on.
37, being noncompetitive against ATP or the peptide substrate
(Figure 5 and Table 5). Notably, compound 43 showed a greater
potency than hit compound 37. Interestingly, compound 43
was previously investigated as a Pim-1 inhibitor in the work of
Pierce and coworkers, although they did not speculate on the
possible mechanism of inhibition but rather pointed out that
Synergistic effects of drugs targeting catalytic and allosteric
sites in the same enzyme have already been reported in the lit-
erature for nucleoside and non-nucleoside inhibitors of
HIV-1 reverse transcriptase.[30] To further confirm the noncom-
petitive mechanism of action of compound 43, we investigat-
ed the in vitro effect of a combination of 43 with competitive
Pim-1 inhibitors such as 40, 6, 1 and derivative 44, which is
structurally related to 43. For each combination, the interac-
tion index for 50% inhibition (I50) value resulted lower than 1,
thus indicating a synergistic effect between 43 with ATP-com-
petitive inhibitors (Table 6). Combinations of ATP-competitive
inhibitors 1 and 6, as well as noncompetitive inhibitors 43 and
37, were also performed, resulting in I50 values close to 1,
which indicate a nonsynergistic effect, as expected for inhibi-
tors that share the same mechanism of action.
Finally, based on the in vitro results, preliminary structure–
activity relationship analysis for this series showed that molec-
ular dimension might play a key role in discriminating between
a noncompetitive and ATP-competitive mechanism of action.
Noncompetitive inhibition was observed for the smallest
2-aminothiazoles (37 and 43), while those with bulkier sub-
stituents were competitive against both ATP and the peptide
substrate. Phenolic hydroxy groups seem to improve the affini-
ty of a ligand for Pim-1, probably due to the formation of addi-
tional hydrogen-bond contacts.
Cell culture assays
Over the last decade, increasing evidence has been accumulat-
ing regarding the potential role of Pim-1 as a prognostic
marker in prostate cancer.[31–33] For this reason, the develop-
ment of effective Pim-1 inhibitors could have a great impact in
the future therapy of prostate cancer. To further investigate
the inhibition activity, as well as to probe their potential as an-
ticancer agents, the most potent Pim-1 inhibitors described
above were tested against a panel of prostate cancer cell lines:
PC3, DU145, LNCaP and 22RV1.
Figure 4. To gain insight into the structure–activity relationships and mecha-
nism of inhibition of hit compound 37, which exhibited good activity in
vitro against Pim-1, the eight 2-aminothiazoles shown here were purchased
from Asinex Ltd for further evaluation.
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Pim-1 is mainly involved in
promoting survival of cancer
cells under apoptosis-inducing
conditions, such as the presence
of chemotherapeutic agents.[31]
For this reason, our compounds
were also tested in combination
with the well-known antitumor
drug paclitaxel (PTX). This com-
bination provided a more potent
effect, especially for compound
40 that showed a 48% residual
viability when treated at 10 mm
in combination with PTX com-
pared with a 80% residual viabil-
ity when cells were treated with
PTX alone (Figure 6). When we
evaluated cell cycle distribution
and apoptosis by flow cytome-
try, the synergistic effect was
confirmed. In fact, the combina-
tion treatment 40+PTX induced
apoptosis in 31% of PC3 cells
compared with 10% when treat-
ed with PTX alone. It is worth
noting that these data are com-
parable with the percentage of
apoptosis (~30%) observed
under similar experimental con-
ditions by silencing Pim-1
mRNA.[31] Based on recent evi-
dences about the role of Pim-
1
in promoting cell survival
under conditions of cellular
stress and in response to tax-
anes, compounds 40, 43, and 6
could be promising adjuvant
drugs for the treatment of pros-
tate cancer and other malignan-
cies in which Pim-1 is associated
with chemotherapeutic resist-
ance.
Figure 5. Kinetic analysis of kinase reactions of Pim-1 in the presence of different concentration of test com-
~
!
~
!
*
*
*
*
*
:
pounds: a) 37 ( : 0 mm; : 5 mm; : 10 mm; : 20 mm), b) 43 ( : 0 mm; : 2 mm; : 3 mm; : 6 mm) and c) 6 (
~
!
*
0 mm; : 5 mm; : 10 mm; : 20 mm). Left-hand side: Variation of the reaction velocity of Pim-1 as a function of
ATP concentration at different fixed concentrations of test compound. Right-hand side: Variation of the reaction
velocity of Pim-1 as a function of peptide substrate concentration at different fixed concentrations of test com-
pound. Each reaction was performed as described in the Experimental Section; values are the mean of three inde-
pendent experiments, and error bars represent ꢁSD.
First, we evaluated the cytotoxicity of compounds 40, 43
and 6 by determining the residual viability of cells treated for
Conclusions
48 hours and comparing the results against untreated control
cells. Notably, all compounds exhibited an IC50 value below
50 mm in all cell lines (Table 7). Equimolar combination treat-
ment with compounds 40+43 and 6+43 gave rise to an evi-
dent decrease in IC50 value, particularly for the 40+43 combi-
nation in PC3 and DU145 cells, when compared with treatment
by the individual agents alone (Table 7). No synergistic effects
were observed in the other cancer cell lines tested (DU145,
LNCaP and 22RV1) when treated with the 43+6 combination
(Table 7). Calculation of the combination index(CI) for the IC50,
IC75 and IC90 values further confirmed the synergistic effects of
the 40+43 combination treatment in PC3 and DU145 cells
(Table 8).
Pim-1 is a member of the serine/threonine kinase family, which
is attracting much interest as drug targets for the pharmaco-
logical treatment of cancer. Despite evidence for the critical
role of Pim-1 in the replication and survival of cancer cells,
only a few molecules endowed with drug-like properties have
been developed so far. In this work, we made a step forward
in the characterization of Pim-1 inhibitors by expanding the in-
dolyl-pyrrolone scaffold, already identified as an ATP-competi-
tive Pim-1 inhibitor in a previous work.[16]
In our search for chemically diverse Pim-1 inhibitors, we per-
formed a virtual screening campaign that led to the identifica-
tion of ATP-competitive inhibitors, as well as some 2-amino-
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thiazole derivatives endowed with a noncompetitive
mechanism of Pim-1 inhibition. These compounds
were classified as possible allosteric inhibitors, being
noncompetitive with respect to both ATP and the
peptide substrate. In vitro evaluation against re-
combinant Pim-1 confirmed the inhibitory activity of
all hits identified in this work, and highlighted the
synergistic effect of ATP-competitive inhibitors and
2-aminothiazole derivatives, when administered in
combination.
Table 5. Effect of compounds 37, 43 and 6 at various concentrations (0–20 mm) on
the Km and Vmax values.[a]
Concentration
Compd 37
0 mm
5 mm
10 mm
20 mm
Vmax (ATP) [pmolminꢀ1
Km (ATP) [mM]
]
34.26ꢁ2.10 23.77ꢁ1.60 15.53ꢁ1.50
9.63ꢁ0.90
48.23ꢁ4.10 48.75ꢁ3.80 48.11ꢁ3.80 50.01ꢁ4.20
Vmax (peptide) [pmolminꢀ1
Km (peptide) [mm]
]
]
]
27.75ꢁ1.28 19.60ꢁ0.99 14.00ꢁ0.66
8.15ꢁ0.62
41.10ꢁ3.50 44.20ꢁ4.10 42.20ꢁ3.70 40.10ꢁ3.60
Compd 43
0 mm 2 mm 3 mm 6 mm
36.95ꢁ1.30 31.40ꢁ1.50 24.11ꢁ1.10 16.95ꢁ0.80
Vmax (ATP) [pmolminꢀ1
Km (ATP) [mM]
Vmax (peptide) [pmolminꢀ1
Km (peptide) [mm]
]
In vitro assays performed in a number of human
prostate cancer cell lines confirmed a broad activity
against the different cell lines tested for representa-
tive compounds. In addition, the synergistic effect of
a non-ATP competitive inhibitor was confirmed in
more highly metastatic cell lines, PC3 and Du145,
when used in combination with the most effective
ATP-competitive compound.
45.77ꢁ3.20 44.50ꢁ4.5
39.60ꢁ6.20 39.80ꢁ5.50
28.12ꢁ1.16 23.94ꢁ1.07 18.87ꢁ0.63 12.11ꢁ0.73
43.20ꢁ4.10 45.60ꢁ3.50 45.40ꢁ3.70 44.80ꢁ3.90
Compd 6
0 mm
5 mm
10 mm
20 mm
Vmax (ATP) [pmolminꢀ1
Km (ATP) [mM]
Vmax (peptide) [pmolminꢀ1
Km (peptide) [mm]
]
40.60ꢁ2.00 39.20ꢁ4.10 38.00ꢁ3.20
42.10ꢁ1.30
55.47ꢁ5.70 115.10ꢁ11.8 199.10ꢁ15.40 299.40ꢁ25.70
34.20ꢁ1.70 28.10ꢁ1.60 18.20ꢁ0.87
54.30ꢁ3.70 59.10ꢁ7.10 51.00ꢁ6.20
12.90ꢁ1.07
53.20ꢁ4.40
In summary, we have presented a new series of
valuable lead compounds with an ATP-competitive
mechanism of action, and the 2-aminothiazole scaf-
fold for noncompetitive, possibly allosteric, inhibitors
of Pim-1. To the best of our knowledge, this is the
[a] Data represent the mean ꢁ standard deviation (SD) of three independent experi-
ments.
first time that such a noncompetitive mechanism of inhibition
has been declared for this serine/threonine kinase family
member. In addition, since most potent Pim-1 inhibitors tested
in PC3 cell lines showed a synergistic effect with paclitaxel,
these derivatives could be promising adjuvant agents for the
treatment of cancer diseases in which Pim-1 is associated with
chemotherapeutic resistance.
Table 6. Synergistic interaction of 43 with compounds 40, 6, 1, 44.[a]
Compd
I50
43+40
43+6
43+1
43+44
43+37
6+1
0.50
0.59
0.74
0.76
0.92
0.99
[a] Interaction index for 50% inhibition (I50) was calculated according to
Equation (6). For full details, see the Experimental Section.
Experimental Section
Computational methods
Molecular modeling studies on indolyl-pyrrolone derivatives:
Docking studies of all indolyl-pyrrolone compounds were per-
formed within the ATP binding site of Pim-1 (PDB: 1YHS)[25] using
the software package Gold version 4.1.[34] Compounds were first
processed with the Schrçdinger LigPrep tool (version 25225) to
generate separate files for all possible enantiomers and protona-
tion states at physiological pH. Chemscore was chosen as the fit-
ness function; the genetic algorithm parameter settings were em-
ployed using the search efficiency set at 100%, and 30 runs were
carried out for each ligand. The compounds were docked exclu-
Table 7. Inhibitory activities of individual and combined compounds
against prostate cancer cell lines.
Compd
IC50 [mm]
PC3
DU145
LNCaP
22RV1
6
40
43
40+43[b]
6+43[b]
34.0ꢁ2.3
21.0ꢁ3.4
30.4ꢁ2.7
12.1ꢁ4.1
14.1ꢁ3.7
26.3ꢁ1.6
22.4ꢁ3.2
47.3ꢁ6.5
10.6ꢁ6.2
37.7ꢁ5.4
41.0ꢁ5.5
20.8ꢁ3.3
27.5ꢁ2.5
13.8ꢁ3.3
54.6ꢁ4.6
32.6ꢁ4.3
14.4ꢁ3.2
32.6ꢁ3.4
15.3ꢁ4.5
26.0ꢁ3.5
1
sively as the enolic tautomer shown (as suggested by H NMR anal-
ysis). Conformational search of compounds 2–5 and 10–13 was
performed with the OPLS 2005 force field and using a GB/SA im-
plicit solvent model.[35–36]
[a] IC50 values were determined after 48 h incubation. Data represent the
mean ꢁ standard deviation (SD) of three independent experiments.
[b] Compound combined in a 1:1 ratio.
Virtual screening: 37 crystallographic structures of Pim-1 in com-
plex with inhibitors were downloaded from the Protein Data Bank
(for a full list, see the Supporting Information). Common inhibitor
scaffolds were analyzed and divided into three groups. A represen-
tative pharmacophore model was then generated for each group
by LigandScout (version 3.0).[26] Pharmacophore models were then
converted in a format compatible with Discovery Studio 2.5 (Accel-
rys), which was used to screen the whole Asinex Ltd databases
(Gold, Platinum, Synergy, Emerald and LeadLike collections). The
“Best Flexible” search routine was applied to screen the database.
Table 8. Combination index (CI) values calculated for the combination
treatment of 40+43 corresponding to the IC50, IC75 and IC90 values.
CI (IC50)
CI (IC75)
CI (IC90)
PC3
DU145
0.79
0.56
0.76
0.47
0.82
0.41
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Figure 6. Prostate cancer cells (PC3) were treated with selected Pim-1 inhibitors in combination with paclitaxel (PTX). a) Residual viability of PC3 cells after
treatment for 48 h with compounds 43, 40 and 6 at 1 mm and 10 mm, and in combination with PTX (0.1 mm). b) Evaluation by flow cytofluorimetry of the pro-
portion (%) of apoptotic cells (sub-G1) and cell cycle distribution in PC3 cells treated for 48 h with compounds 43, 40 and 6 at 10 mm, and in combination
with PTX (0.1 mm). Data represent the mean percentage of three different experiments with SD shown. * P<0.05 according to a Student’s t-test.
Conformational ensembles were generated with an energy thresh-
old of 20 kcalmolꢀ1 from the local minimized structure, and a maxi-
mum limit of 255 conformers per molecule. The “Search 3D Data-
base” protocol was used for mapping ligands to pharmacophores,
whereas the “Ligand Pharmacophore Mapping” protocol was used
to align selected ligands to the query pharmacophore and for cal-
culating the relative FitValue.
Chemistry
All commercially available reagents were purchased from Sigma–
Aldrich and were used as received. Anhydrous reactions were run
under a positive pressure of dry N2. Flash chromatography was per-
formed on columns packed with Merck silica gel 60, 23–400 mesh.
Thin-layer chromatography (TLC) was carried out using Merck TLC
silica gel 60 F254 plates. Melting points (mp) were determined
1
PDB crystallographic structures 2C3I[27] and 1XR1[28] were selected
for docking and prepared by means of the “Protein Preparation
Wizard” implemented in MAESTRO 9.0.[37] The protonation and tau-
tomerization states of the residues were assigned for pH 7.0ꢁ1.0.
Protein–ligand complexes were subjected to energy minimization
using the OPLS-2005 force field until geometric convergence was
achieved (RMSD=0.3 ꢂ).[36]
with a Bꢃchi 530 apparatus and are uncorrected. H NMR spectra
were recorded at 400 MHz in [D6]DMSO on a Bruker Avance
DPX400 spectrometer. Chemical shifts (d) are reported in parts per
million (ppm) relative to tetramethylsilane (TMS) as the internal
standard. Coupling constants (J) are reported in Hz, and splitting
patterns are described using the following abbreviations: singlet
(s), doublet (d), triplet (t), quartet (q), quintet (quint), sextet (sx),
multiplet (m), and broad (br). IR spectra were recorded on Perkin-
Elmer 398 spectrophotometer using KBr pellets. Mass spectrometry
(MS) data were obtained with an Agilent 1100 LC/MSD VL system
(G1946C) at a flow rate of 0.4 mLminꢀ1 using a binary solvent
system of MeOH/H2O (95:5). Data were acquired using electrospray
ionization (ESI) in the positive mode, scanning over the mass range
50–1500. UV detection was monitored at 254 nm, and the follow-
ing ion source parameters were used: drying gas flow=
9 mLminꢀ1; nebulizer pressure=40 psig; drying gas temperature=
3508C. All target compounds possessed a purity of ꢂ95%, which
was verified by elemental analyses through comparison with theo-
retical values. Elemental analyses (C, H, N) were performed in
house using a Carlo Erba 1106 Elemental Analyzer, and data are
within 0.4% of the theoretical values.
The GOLD 4.1 docking program was chosen to perform automatic
docking simulation of selected compounds and to evaluate the in-
teraction energy between such compounds and the enzyme.[38]
The ChemScore function was used with default settings. The bind-
ing site was centered on the crystallographic ligand and had
a radius of 10 ꢂ.
Molecules selected by virtual screening were purchased from
Asinex Ltd. The purity of the purchased molecules was reported by
Asinex Ltd to range from 93 to 99%. To check this, we performed
a HPLC analysis of compounds after shipping and storage, and we
found the purities (92–98%) to be comparable to those reported
1
by Asinex Ltd. H NMR spectra were provided by Asinex Ltd, fur-
ther confirming the purity of the samples and the structures of the
compounds. The structures of all compounds purchased from
Asinex Ltd are shown in the Supporting Information.
Microwave (MW) irradiation experiments: Microwave irradiation
experiments were conducted with a CEM Discover Synthesis Unit
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(CEM Corp., Matthews, NC, USA). The instrument consists of a con-
tinuous focused microwave power delivery system with operator-
selectable power output (0–300 W). The temperature of the vessel
contents was monitored with a calibrated infrared temperature
control unit mounted under the reaction vessel. All experiments
were performed with a stirring option, whereby the contents of
the vessel are stirred by means of a rotating magnetic plate locat-
ed below the floor of the microwave cavity and a Teflon-coated
magnetic stir bar in the vessel.
H]+; Anal. calcd for C25H26N2O3: C 74.60, H 6.51, N 6.96, found: C
74.42, H 6.67, N 7.09.
4-Acetyl-5-(1,1’-biphenyl-4-yl)-3-hydroxy-1-[2-(1H-indol-3-yl)eth-
yl]-1,5-dihydro-2H-pyrrol-2-one (7): brown–orange solid (59 mg,
1
21.6%); mp: 203–2068C; H NMR (400 MHz, [D6]DMSO): d=2.00 (s,
3H, COCH3), 2.79–2.90 (m, 4H, CH2CH2), 3.70 (m, 1H, CH), 5.10 (br
s, 1H, NH, disappears with D2O), 6.79–6.83 (m, 1H, indole H-2),
6.93–7.00 (m, 4H, Ar-H), 7.18–7.34 (m, 5H, Ar-H), 7.51–7.59 (m, 4H,
Ar-H), 10.71 ppm (br s, 1H, OH, disappears with D2O); IR (KBr): n˜ =
3405–3020 (OH), 3387 (NH), 1662 (CO), 1638 cmꢀ1 (CON); MS
(ESI+): m/z 438 [M+H]+; Anal. calcd for C28H24N2O3: C 77.04, H
5.54, N 6.42, found: C 77.10, H 5.67, N 6.59.
General procedure for the synthesis of 5-substituted derivatives
2–5, 7–13 and 15–19: A solution of 20 (100.0 mg, 0.630 mmol) in
DME (4 mL) was treated with appropriate derivative 21
(0.630 mmol), appropriate aldehyde 22a–i (0.630 mmol), and
DIPEA (8.2 mg, 0.063 mmol), and the mixture was stirred at 1308C
under microwave irradiation (300 W) for 5 min. After cooling,
EtOAc (5 mL) was added, and the solution was washed with satu-
rated aq NH4Cl (5 mL), water (5 mL), and brine (5 mL), then dried
(MgSO4), filtered and concentrated in vacuo. For products 2, 4 and
9, the compound was purified by crystallization from CH2Cl2/n-
hexane (1:4). For products 3, 5, 7, 8, 10–13 and 15–19, the com-
pounds were first purified by column chromatography with gradi-
ent elution (CH2Cl2/MeOH/Et3N, 100:0:0!95:5:1), and then recrys-
tallized from CH2Cl2/n-hexane (1:4).
4-Acetyl-3-hydroxy-1-[2–1H-indol-3-yl)ethyl]-5-(3-phenoxyphen-
yl)-1,5-dihydro-2H-pyrrol-2-one (8): brown–orange solid (116 mg,
1
40.7%); mp: 205–2088C; H NMR (400 MHz, [D6]DMSO): d=2.00 (s,
3H, COCH3), 2.50–2.80 (m, 4H, CH2CH2), 3.65 (m, 1H, CH), 4.97 (br
s, 1H, NH, disappears with D2O), 6.76–6.81 (m, 1H, indole H-2),
6.83–6.87 (m, 4H, Ar-H), 6.94–7.03 (m, 5H, Ar-H), 7.22–7.26 (m, 4H,
Ar-H), 10.71 ppm (br s, 1H, OH, disappears with D2O); IR (KBr): n˜ =
3380–3000 (OH), 3385 (NH), 1667 (CO), 1641 cmꢀ1 (CON); MS
(ESI+): m/z 454 [M+H]+; Anal. calcd for C28H24N2O4: C 74.32, H
5.35, N 6.19, found: C 74.50, H 5.71, N 6.47.
4-Acetyl-3-hydroxy-1-[2–1H-indol-3-yl)ethyl]-5-(2-phenylethyl)-
1,5-dihydro-2H-pyrrol-2-one (9): yellow–brown solid (127 mg,
59.7%); mp: 193–1968C; H NMR (400 MHz, [D6]DMSO): d=1.80 (t,
4-Acetyl-3-hydroxy-1-[2-(1H-indol-3-yl)ethyl]-5-phenyl-1,5-dihy-
dro-2H-pyrrol-2-one (2): yellow–brown solid (157 mg, 69.3%); mp:
189–1908C; H NMR (400 MHz, [D6]DMSO): d=2.01 (s, 3H, COCH3),
1
1
J=6.2 Hz, 2H, CH2CH2C6H5), 2.10 (t, J=6.2 Hz, 2H, CH2CH2C6H5),
2.18 (s, 3H, COCH3), 2.78–2.84 (m, 2H, NCH2CH2), 2.93–2.95 (m, 2H,
NCH2CH2), 3.84–3.91 (m, 1H, CH), 4.27 (br s, 1H, NH, disappears
with D2O), 6.86–6.90 (m, 1H, indole H-2), 6.96–7.05 (m, 3H, Ar-H),
7.11–7.26 (m, 3H, Ar-H), 7.46–7.48 (m, 3H, Ar-H), 10.80 ppm (br s,
1H, OH disappears with D2O); IR (KBr): n˜ =3390–3010 (OH), 3397
(NH), 1665 (CO), 1643 cmꢀ1 (CON); MS (ESI+): m/z 339 [M+H]+;
Anal. calcd for C24H24N2O3: C 74.21, H 6.23, N 7.21, found: C 74.07,
H 5.98, N 7.10.
2.70–2.92 (m, 4H, CH2CH2), 3.63–3.70 (m, 1H, CH), 5.00 (br s, 1H,
NH, disappears with D2O), 6.82–6.84 (m, 1H, indole H-2), 6.86–6.95
(m, 3H, Ar-H), 7.06–7.07 (m, 3H, Ar-H), 7.18–7.24 (m, 3H, Ar-H),
10.69 ppm (br s, 1H, OH, disappears with D2O); IR (KBr): n˜ =3455–
3000 (OH), 3398 (NH), 1660 (CO), 1648 cmꢀ1 (CON); MS (ESI+): m/z
361 [M+H]+; Anal. calcd for C22H20N2O3: C 73.32, H 5.59, N 7.77,
found: C 73.11, H 5.43, N 7.52.
4-Acetyl-5-(4-bromophenyl)-3-hydroxy-1-[2-1H-indol-3-yl)ethyl]-
1,5-dihydro-2H-pyrrol-2-one (3): brown–orange solid (53 mg,
1
4-Acetyl-3-hydroxy-1-[2-(5-methoxy-1H-indol-3-yl)ethyl]-5-
phenyl-1,5-dihydro-2H-pyrrol-2-one (10): brown–orange solid
(47 mg, 15.3%); mp: 192–1968C; 1H NMR (400 MHz, [D6]DMSO):
d=1.95 (s, 3H, COCH3), 2.56–2.81 (m, 4H, CH2CH2), 3.64 (s, 3H,
OCH3), 3.70–3.72 (m, 1H, CH), 6.59–6.60 (m, 1H, indole H-2), 6.61–
6.62 (m, 2H, Ar-H), 6.72–6.94 (m, 3H, Ar-H), 7.11–7.20 (m, 3H, Ar-H),
10.56 ppm (s, 1H, OH, disappears with D2O); IR (KBr): n˜ =3425–
3000 (OH+NH), 1665 (CO), 1651 cmꢀ1 (CON); MS (ESI+): m/z 491
[M+H]+; Anal. calcd for C23H22N2O4: C 70.75, H 5.68, N 7.17, found:
C 70.86, H 5.91, N 7.11.
19.0%); mp: 192–1948C; H NMR (400 MHz, [D6]DMSO): d=1.97 (s,
3H, COCH3), 2.57–2.83 (m, 4H, CH2CH2), 3.63–3.71 (m, 1H, CH), 5.89
(br s, 1H, NH, disappears with D2O), 6.82–6.87 (m, 1H, indole H-2),
6.94–7.01 (m, 3H, Ar-H), 7.22–7.24 (m, 3H, Ar-H), 7.36–7.38 (m, 2H,
Ar-H), 10.70 ppm (br s, 1H, OH, disappears with D2O); IR (KBr): n˜ =
3450–3100 (OH), 3401 (NH), 1665 (CO), 1643 cmꢀ1 (CON); MS
(ESI+): m/z 440 [M+H]+; Anal. calcd for C22H19BrN2O3: C 60.15, H
4.36, N 6.38, found: C 60.41, H 4.38, N 6.42.
4-Acetyl-3-hydroxy-1-[2-(1H-indol-3-yl)ethyl]-5-[4-(trifluorometh-
yl)phenyl)-1,5-dihydro-2H-pyrrol-2-one (4): yellow–brown solid
(107 mg, 39.6%); mp: 220–2258C; 1H NMR (400 MHz, [D6]DMSO):
d=1.20 (s, 3H, COCH3), 2.79–3.01 (m, 4H, CH2CH2), 3.80–3.85 (m,
1H, CH), 6.86–6.92 (m, 1H, indole H-2), 7.00–7.05 (m, 3H, Ar-H),
7.21–7.29 (m, 3H, Ar-H), 7.43–7.45 ppm (m, 2H, Ar-H); IR (KBr): n˜ =
3427 (NH), 3105 (OH), 1682 (CO), 1638 cmꢀ1 (CON); MS (ESI+): m/z
429 [M+H]+; Anal. calcd for C23H19F3N2O3: C 64.48, H 4.47, N 6.54,
found: C 64.80, H 4.32, N 6.84.
4-Acetyl-5-(4-bromophenyl)-3-hydroxy-1-[2-(5-methoxy-1H-
indol-3-yl)ethyl]-1,5-dihydro-2H-pyrrol-2-one (11): brown–orange
solid (64 mg, 21.9%); mp: 196–1988C; 1H NMR (400 MHz,
[D6]DMSO): d=1.91 (s, 3H, COCH3), 2.56–2.83 (m, 4H, 2CH2), 3.63
(s, 3H, OCH3), 3.70–3.74 (m, 1H, CH), 5.05 (br s, 1H, NH, disappears
with D2O), 6.60–6.62 (m, 1H, indole H-2), 6.70–6.95 (m, 3H, Ar-H),
7.03–7.13 (m, 2H, Ar-H), 7.35–7.38 (m, 2H, Ar-H), 10.58 ppm (s, 1H,
OH, disappears with D2O); IR (KBr): n˜ =3340–3005 (OH+NH), 1684
(CO), 1642 cmꢀ1 (CON); MS (ESI+): m/z 470 [M+H]+; Anal. calcd
for C23H21N2O4Br: C 58.86, H 4.52, N 5.97, found: C 58.73, H 4.76, N
5.67.
4-Acetyl-3-hydroxy-1-[2-(1H-indol-3-yl)ethyl]-5-(4-isopropylphen-
yl)-1,5-dihydro-2H-pyrrol-2-one (5): brown–orange solid (69 mg,
27.3%); mp: 188–1918C; 1H NMR (400 MHz, [D6]DMSO): d=1.10–
1.13 (m, 6H, 2CH3 isoprop), 2.05 (s, 3H, COCH3), 2.54–2.62 (m, 1H,
CH isoprop), 2.79–2.88 (m, 4H, CH2CH2), 3.63–3.70 (m, 1H, CH),
5.00 (br s, 1H, NH, disappears with D2O), 6.84–6.86 (m, 1H, indole
H-2), 6.89–6.93 (m, 4H, Ar-H), 6.95–6.98 (m, 4H, Ar-H), 10.73 ppm
(br s, 1H, OH, disappears with D2O; IR (KBr): n˜ =3400–3090 (OH),
3392 (NH), 1668 (CO), 1653 cmꢀ1 (CON); MS (ESI+): m/z 403 [M+
4-Acetyl-3-hydroxy-1-[2-(5-methoxy-1H-indol-3-yl)ethyl]-5-[4-(tri-
fluoromethyl)phenyl]-1,5-dihydro-2H-pyrrol-2-one (12): brown–
orange solid (76 mg, 26.3%); mp: 228–2328C; 1H NMR (400 MHz,
[D6]DMSO): d=1.99 (s, 3H, COCH3), 2.56–2.82 (m, 4H, 2CH2), 3.60
(s, 3H, OCH3), 3.71 (m, 1H, CH), 5.09 (br s, 1H, NH, disappears with
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D2O), 6.68–6.72 (m, 1H, indole H-2), 6.95–7.13 (m, 3H, Ar-H), 7.24–
7.26 (m, 2H, Ar-H), 7.51–7.52 (m, 2H, Ar-H), 10.55 ppm (s, 1H, OH,
disappears with D2O); IR (KBr): n˜ =3400–3080 (OH+NH), 1678
(CO), 1635 cmꢀ1 (CON); MS (ESI+): m/z 459 [M+H]+; Anal. calcd
for C24H21N2O4F3: C 62.88, H 4.62, N 6.11, found: C 62.77, H 4.77, N
5.98.
4-Acetyl-3-hydroxy-1-[2-(5-methoxy-1H-indol-3-yl)ethyl]-5-(4-
methoxyphenyl)-1,5-dihydro-2H-pyrrol-2-one (19): brown–orange
solid (135 mg, 51.0%); mp: 198–1998C; 1H NMR (400 MHz,
[D6]DMSO): d=2.38 (s, 3H, COCH3), 2.90–3.02 (m, 4H, 2CH2), 3.75
(s, 3H, C6H4-4-OCH3), 3.87 (s, 3H, OCH3), 4.21–4.33 (m, 1H, CH),
6.78–6.93 (m, 1H, indole H-2), 7.00–7.17 (m, 3H, Ar-H), 7.18–7.26
(m, 2H, Ar-H), 7.29–7.36 (m, 2H, Ar-H), 8.58 ppm (br s, 1H, OH, dis-
appears with D2O); IR (KBr): n˜ =3395–3000 (OH), 3382 (NH), 1675
(CO), 1652 cmꢀ1 (CON); MS (ESI+): m/z 422 [M+H]+; Anal. calcd
for C24H24N2O5: C 68.56, H 5.75, N 6.66, found: C 68.49, H 5.77, N
6.81.
4-Acetyl-3-hydroxy-5-(4-isopropylphenyl)1-[2-(5-methoxy-1H-
indol-3-yl)ethyl]-1,5-dihydro-2H-pyrrol-2-one (13): brown–orange
solid (41 mg, 15.0%); mp: 194–1968C; 1H NMR (400 MHz,
[D6]DMSO): d=1.07–1.09 (m, 6H, 2CH3 isoprop), 1.48–1.52 (m, 1H,
CH isoprop), 1.90 (s, 3H, COCH3), 2.55–2.75 (m, 4H, 2CH2), 3.64 (s,
3H, OCH3), 4.04–4.06 (m, 1H, CH), 5.02 (br s, 1H, NH, disappears
with D2O), 6.60–6.74 (m, 1H, indole H-2), 6.94–7.01 (m, 3H, Ar-H),
7.05–7.13 (m, 2H, Ar-H), 7.59–7.62 (m, 2H, Ar-H), 10.55 ppm (s, 1H,
OH, disappears with D2O); IR (KBr): n˜ =3400–3000 (OH+NH), 1684
(CO), 1651 cmꢀ1 (CON); MS (ESI+): m/z 434 [M+H]+; Anal. calcd
for C26H28N2O4: C 72.20, H 6.53, N 6.48, found: C 72.48, H 6.81, N
6.39.
General procedure for the synthesis of derivatives 6 and 14: A
solution of 20 (100.0 mg, 0.630 mmol) in DME (4 mL) was treated
with appropriate derivative 21 (0.630 mmol), 22j (118 mg,
0.630 mmol), and DIPEA (8.2 mg, 0.063 mmol), and the mixture
was stirred for 14 h at 808C under a nitrogen atmosphere. After
cooling, EtOAc (5 mL) was added, and the solution was washed
with saturated aq NH4Cl (5 mL), water (5 mL), brine (5 mL), then
dried (MgSO4), filtered and concentrated in vacuo. The crude mate-
rial was first purified by column chromatography (silica gel 70–
230 mesh) with gradient elution (CH2Cl2/MeOH/Et3N, 100:0:0!
95:5:1), and the resultant brown–orange solid was further purified
by recrystallization from CH2Cl2/n-hexane (1:4).
4-Acetyl-5-(1,1’-biphenyl-4-yl)-3-hydroxy-1-[2-(5-methoxy-1H-
indol-3-yl)ethyl]-1,5-dihydro-2H-pyrrol-2-one (15): brown–orange
solid (74 mg, 25.0%); mp: 207–2108C; 1H NMR (400 MHz,
[D6]DMSO): d=1.90 (s, 3H, COCH3), 2.42–2.88 (m, 4H, 2CH2), 3.58
(s, 3H, OCH3), 3.69–3.71 (m, 1H, CH), 5.10 (br s, 1H, NH, disappears
with D2O), 6.59–6.63 (m, 1H, indole H-2), 6.97–7.19 (m, 4H, Ar-H),
7.24–7.34 (m, 4H, Ar-H), 7.36–7.51 (m, 4H, Ar-H), 10.59 ppm (s, 1H,
OH, disappears with D2O); IR (KBr): n˜ =3425–3070 (OH+NH), 1678
(CO), 1631 cmꢀ1 (CON); MS (ESI+): m/z 468 [M+H]+; Anal. calcd
for C29H26N2O4: C 74.66, H 5.62, N 6.00, found: C 74.51, H 5.91, N
5.84.
4-Acetyl-3-hydroxy-1-[2-1H-indol-3-yl)ethyl]-5-(4-piperidin-1-yl-
phenyl)-1,5-dihydro-2H-pyrrol-2-one (6): brown–orange solid
(57 mg, 20.5%); mp: 195–1998C; 1H NMR (400 MHz, [D6]DMSO):
d=1.40–1.45 (m, 2H, CH2 pip), 1.48–1.52 (m, 4H, 2CH2 pip), 2.05 (s,
3H, COCH3), 2.75–2.55 (m, 4H, CH2CH2), 2.95–3.00 (m, 4H, 2CH2N
pip), 3.60 (m, 1H, CH), 4.99 (br s, 1H, NH, disappears with D2O),
6.75–6.83 (m, 1H, indole H-2), 6.85–6.89 (m, 3H, Ar-H), 6.94–6.98
(m, 3H, Ar-H), 7.21–7.23 (m, 2H, Ar-H), 10.69 ppm (br s, 1H, OH,
disappears with D2O); IR (KBr): n˜ =3430–3080 (OH+NH), 1650
(CO), 1610 cmꢀ1 (CON); MS (ESI+): m/z 445 [M+H]+; Anal. calcd
for C27H29N3O3: C 73.11, H 6.59, N 9.47, found: C 72.99, H 6.43, N
9.35.
4-Acetyl-3-hydroxy-1-[2-(5-methoxy-1H-indol-3-yl)ethyl]-5-(3-
phenoxyohenyl)-1,5-dihydro-2H-pyrrol-2-one (16): brown–orange
solid (46 mg, 15.1%); mp: 214–2178C; 1H NMR (400 MHz,
[D6]DMSO): d=1.97 (s, 3H, COCH3), 2.51–2.82 (m, 4H, 2CH2), 3.63
(s, 3H, OCH3), 3.67–3.69 (m, 1H, CH), 6.60–6.63 (m, 1H, indole H-2),
6.76–6.94 (m, 4H, Ar-H), 7.00–7.13 (m, 4H, Ar-H), 7.21–7.25 (m, 4H,
Ar-H), 10.56 ppm (s, 1H, OH, disappears with D2O); IR (KBr): n˜ =
3380–3050 (OH+NH), 1684 (CO), 1641 cmꢀ1 (CON); MS (ESI+): m/z
484 [M+H]+; Anal. calcd for C29H26N2O5: C 72.18, H 5.43, N 5.81,
found: C 71.95, H 5.48, N 5.70.
4-Acetyl-3-hydroxy-1-[2-(5-methoxy-1H-indol-3-yl)ethyl]-5-(4-pi-
peridin-1-ylphenyl)-1,5-dihydro-2H-pyrrol-2-one (14): brown–
orange solid (46 mg, 15.3%); mp: 203–2078C; 1H NMR (400 MHz,
[D6]DMSO): d=1.40–1.47 (6H, 3CH2 pip), 1.85 (s, 3H, COCH3), 2.75–
2.95 (m, 8H, 2CH2 +2CH2N pip), 3.63 (s, 3H, OCH3), 3.70–3.72 (m,
1H, CH), 6.60–6.62 (m, 1H, indole H-2), 6.72–6.78 (m, 3H, Ar-H),
6.92–6.98 (m, 2H, Ar-H), 7.11–7.13 (m, 2H, Ar-H), 10.57 ppm (br s,
1H, OH, disappears with D2O); IR (KBr): n˜ =3420–3040 (OH+NH),
1682 (CO), 1637 cmꢀ1 (CON); MS (ESI+): m/z 475 [M+H]+; Anal.
calcd for C28H31N3O4: C 71.01 H 6.60, N 8.87, found: C 70.88, H 6.72,
N 8.74.
4-Acetyl-3-hydroxy-1-[2-(5-methoxy-1H-indol-3-yl)ethyl]-5-(2-
phenylethyl)-1,5-dihydro-2H-pyrrol-2-one (17): brown–orange
solid (58 mg, 22.0%); mp: 200–2038C; 1H NMR (400 MHz,
[D6]DMSO): d=1.90–2.12 (m, 4H, CH2CH2C6H5), 2.20 (s, 3H, COCH3),
2.66–2.96 (m, 4H, 2CH2), 3.66 (s, 3H, OCH3), 3.84–3.91 (m, 1H, CH),
6.58–6.64 (m, 1H, indole H-2), 6.80–6.98 (m, 3H, Ar-H), 7.04–7.14
(m, 3H, Ar-H), 7.18–7.23 (m, 2H, Ar-H), 10.57 ppm (br s, 1H, OH,
disappears with D2O); IR (KBr): n˜ =3380–3000 (OH+NH), 1675
(CO), 1636 cmꢀ1 (CON); MS (ESI+): m/z 419 [M+H]+; Anal. calcd
for C25H26N2O4: C 71.75, H 6.26, N 6.69, found: C 71.82, H 6.47, N
6.54.
Biology
Cell culture and viability analysis: The biological activity of Pim-1 in-
hibitors was tested in human prostate cancer cell lines PC3,
DU145, LNCaP and 22RV1. Cell lines were originally obtained from
the American Type Culture Collection (ATCC) (Rockville, MD, USA).
Cells were cultured according to the conditions indicated by the
cell depository. Cell growth was analyzed by a cell counting assay.
Prostate cancer cells were seeded onto six-well plates (1ꢄ105 cells/
well), and 24 h later, the medium was replaced with fresh medium
(untreated control cultures) or medium containing test compound
at the desired concentration. After 48 h incubation, cells were col-
lected by trypsinization, centrifuged and resuspended in a known
amount of culture medium. Aliquots of cell suspension were
counted by a Neubauer hemocytometric chamber. Viability was as-
4-Acetyl-3-hydroxy-1-[2-(5-methoxy-1H-indol-3-yl)ethyl]-5-(4-
methylphenyl)-1,5-dihydro-2H-pyrrol-2-one (18): brown–orange
solid (137 mg, 54.0%); mp: 200–2018C; 1H NMR (400 MHz,
[D6]DMSO): d=2.27 (s, 3H, C6H4-4-CH3), 2.37 (s, 3H, COCH3), 2.93–
3.05 (m, 4H, 2CH2), 3.85 (s, 3H, OCH3), 4.23–4.37 (m, 1H, CH), 6.84–
6.95 (m, 1H, indole H-2), 7.00–7.28 (m, 3H, Ar-H), 7.30–7.35 (m, 2H,
Ar-H), 7.38–7.41 (m, 2H, Ar-H), 8.59 ppm (br s, 1H, OH, disappears
with D2O); IR (KBr): n˜ =3390–3000 (OH), 3385 (NH), 1671 (CO),
1647 cmꢀ1 (CON); MS (ESI+): m/z 406 [M+H]+; Anal. calcd for
C24H24N2O4: C 71.27, H 5.98, N 6.93, found: C 71.48, H 6.25, N 7.02.
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sessed by the trypan blue dye exclusion test. IC50 values and com-
bination index (CI) data were calculated using the CalcuSyn soft-
ware 2.1 (BioSoft, Cambridge, UK).
two inhibitors at a fixed molar ratio (R=[inhibitor 1]/[inhibitor 2]),
D1 and D2 values were calculated from the D50 value derived from
Equation (4), with (D1 +D2)=D50 and D1 =RD2.
Flow cytometric analysis: The cell cycle progression and apoptosis
was evaluated by flow cytometry according to standard proce-
dures. Briefly, cells (1ꢄ106) were washed with phosphate-buffered
saline (PBS) and fixed for 30 min with 70% v/v EtOH at 48C. After
washing with PBS, the cells were incubated in 1 mL of DNA stain-
ing solution (PBS containing 200 mgmLꢀ1 of RNAse A, 20 mgmLꢀ1
of propidium iodide, and 0.1% v/v Triton X-100) at RT for 1 h. Sus-
pended cells were analyzed on a FACScan instrument (Becton–
Dickinson, Franklin Lakes, NJ, USA) using the Cell Quest software
3.1 (Becton–Dickinson). Analysis was performed by evaluating at
least 10000 cells for each sample. All measurements were per-
formed using the same instrument settings. Apoptotic cells were
detected by a quantifiable peak in sub-G1 phase corresponding to
the red fluorescent light emitted by subdiploid cell nuclei.
m
m
E ¼ ðEconð½Iꢃ=D50Þ Þ=ð1 þ ðD50=½Iꢃ Þ
ð4Þ
Expected D1, D2, and Di values for the combination of i drugs
under the null reference hypothesis of no interaction were derived
by inserting estimated D50 and m values for each drug in the com-
bination in the specific form of the Lowe additivity equation
[Eq. (5)], which assumes that Equation (4) is appropriate for each
drug individually.[39]
1=m1
1=m2
1 ¼ðD1=ðD50ð1ÞðE=ðEconꢀEÞÞ Þ þ ðD2=ðD50ð2ÞðE=ðEconꢀEÞÞ Þþ
1=mi
. . . þ ðDi=ðD50ðiÞðE=ðEconꢀEÞÞ
Þ
ð5Þ
The interaction index (I) was then calculated according to Beren-
baum using Equation (6), where D1 and D2 are the concentrations
of the drugs giving 50% inhibition when tested in the combination
D1 +D2, and D50(1) and D50(2) are the concentrations of each drug
giving 50% inhibition when tested individually. A value for I<1 in-
dicates synergy, I>1 indicates antagonism, and I=1 indicates addi-
tivity, according to the Lowe additivity model. The null reference
hypothesis of no interaction [Eq. (6)] corresponded to I=1.
Inhibition assay: Recombinant Pim-1 was purchased from Upstate
(Millipore). Reactions were performed according to the manufactur-
er’s protocol. Each compound was tested in the presence of
500 mm ATP and 100 mm peptide substrates (RBER-GSK3 14-27, Pro-
Qinase) and in the presence of 0.01 mg active Pim-1, 0.33 pmol
[
g32P]ATP, 10 mm magnesium acetate, 8 mm 3-(N-morpholino)pro-
panesulfonic acid (MOPS)/sodium hydroxide (pH 7), 1 mm ethyl-
enediaminetetraacetic acid (EDTA), and 10% DMSO in a final
volume of 10 mL. After 5 min at 308C, the reaction was stopped by
adding 2 mL of 3% phosphoric acid. Aliquots (10 mL) were then
transferred into a P30 Filtermat (PerkinElmer), washed five times
with 75 mm phosphoric acid and once with acetone for 2 min. The
filter was dried and transferred to a sealable plastic bag, and scin-
tillation cocktail (4 mL) was added. Spotted reactions were read in
a scintillation counter. The ID50 values were obtained according to
Equation (1), where v is the measured reaction velocity, V is the ap-
parent maximal velocity in the absence of inhibitor, I is the inhibi-
tor concentration, and the ID50 is the 50% inhibitory dose.
I ¼ D =D50ð1Þ þ D =D50ð2Þ
ð6Þ
ð1Þ
ð2Þ
All of the analyses are based on the results of three independent
experiments for each drug combination, and the standard devia-
tion (SD) for each parameter estimate is indicated.
Acknowledgements
The authors thank the University of Siena (Italy) for support of
the research presented here. This work has been partially sup-
ported by the Italian Cancer Research Association (AIRC) (grant
no. IG12084) to G.M.
n ¼ V=ð1 þ ðI=ID50ÞÞ
ð1Þ
Inhibition mechanism: Pim-1 kinase activity assays were performed
in the presence of different fixed amounts of inhibitor with varying
substrate (ATP or peptide) concentrations. Data were analyzed ac-
cording to the Michaelis–Menten method. Ki values, expressed as
a concentration (mm), toward recombinant human Pim-1 were cal-
culated according to Equation (2) for competitive inhibition toward
ATP and peptide substrates, and Equation (3) for noncompetitive
inhibition, where [SATP] and [Spep] are the concentration of compet-
ing substrate (ATP and peptide, respectively). Each experiment was
performed in triplicate, and mean values were used for interpola-
tion. Curve fitting was performed with the program GraphPad
Prism 5.00.
Keywords: cancer · medicinal chemistry · noncompetitive
inhibition · Pim-1 · organic synthesis
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Received: October 19, 2012
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