Design, synthesis and structure-activity relationship studies of morpholino-1H-phenalene derivatives that antagonize Mcl-1/Bcl-2

Article abstract of DOI:10.1016/j.bmc.2014.09.047

We report herein characteristic studies of Mcl-1 and Bcl-2 dual inhibitors. It was found that a protruding carbonyl group forming hydrogen bond with R263 plays a predominant role compared with the hydrophobic group that occupies the p2 pocket. A series of dual inhibitors representing different parts of the morpholino-1H-phenalene were designed, synthesized and evaluated.

Full text of DOI:10.1016/j.bmc.2014.09.047

Bioorganic & Medicinal Chemistry 22 (2014) 5738–5746
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and structure–activity relationship studies of
morpholino-1H-phenalene derivatives that antagonize Mcl-1/Bcl-2
Xiangqian Li a,b, Xiaomeng Liang a, Ting Song a, Pengchen Su a, Zhichao Zhang a,
⇑
a State Key Laboratory of Fine Chemicals, School of Chemistry, Dalian University of Technology, Dalian, China
b Ocean University of China, 238 Songling Road, Qingdao, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 May 2014
Revised 19 September 2014
Accepted 23 September 2014
Available online 2 October 2014
We report herein characteristic studies of Mcl-1 and Bcl-2 dual inhibitors. It was found that a protruding
carbonyl group forming hydrogen bond with R263 plays a predominant role compared with the hydro-
phobic group that occupies the p2 pocket. A series of dual inhibitors representing different parts of the
morpholino-1H-phenalene were designed, synthesized and evaluated.
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Dual inhibitors
Hydrogen bond
Apoptosis
Mcl-1
Bcl-2
1. Introduction
cell death.14–17 However, it has been identified that the BH3 groove
of Mcl-1 is different with that of Bcl-2.18,19 Although some mole-
The programmed cell death (apoptosis) machinery follows sev-
eral well-organized cell signaling networks where one of the two
major pathways (i.e., the mitochondrial process or intrinsic path-
way) involves a highly regulated series of proteins.1–3 As the cen-
tral regulators of this pathway, Bcl-2 family members contain
from one to four Bcl-2 homology (BH) domains and can be broadly
divided into two classes: anti-apoptotic and pro-apoptotic pro-
teins.4,5 A balance between these two classes dictates a cell’s fate
and is mediated by the BH3 domain of the BH3-only proteins
inserting into a hydrophobic groove on the surface of anti-apopto-
tic proteins including Bcl-2, Bcl-xL, and Mcl-1.6
Overexpression of anti-apoptotic Bcl-2 family proteins occurs in
many human cancers, and therefore, these proteins are very attrac-
tive targets for the development of novel anticancer agents.7,8 One
of the most promising inhibitors to date is ABT-737 which inhibits
multiple members of the prosurvival proteins of the Bcl-2 family
except for Mcl-1.9,10 Accordingly, it is a poor killer of most cells,
except those that have reduced Mcl-1 levels, or in which Mcl-1
degradation has been induced.11 As such, a potent small-molecule
inhibitor of Bcl-2 family proteins should bind not only to Bcl-2 and
Bcl-xL, but also to Mcl-1 with high affinities.12,13
cules reported are capable to bind to both Mcl-1 and Bcl-2,7,20,21
it is rarely discussed the key features for a dual inhibitor. The
design of Mcl-1/Bcl-2 dual inhibitor is still a challenge.
Previously, we reported a small-molecule Mcl-1/Bcl-2 dual
inhibitor, 3-thiomorpholin-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-
carbonitrile (1).22,23 The binding mode of 1 with Bcl-2/Mcl-1 has
been illustrated by structure–activity relationship study. Based
on its structural information, we designed some novel dual inhib-
itors not only to provide more candidates for clinical development,
but also to reveal the crucial parts of compound 1. Guided by our
discovery, more potent Mcl-1/Bcl-2 dual inhibitors could be
designed and synthesized.
2. Chemistry
Compounds 2 and 2b were prepared according to the method
that we reported previously (Schemes 1 and 2).24 The synthetic
route of the 5-(phenylthio)isoindoline-1,3-dione derivatives (4a,
4b, 4d, and 4f) was outlined in Scheme 3.25,26 4-Bromophthalic
acid was prepared by bromination of phthalic anhydride, and it
was converted to 4-bromophthalimide as a white crystalline pow-
der by dehydration and imidization. The target compounds were
obtained by the reaction between 4-bromophthalimide and corre-
sponding thiophenol with K2CO3 at 130 °C in DMF. The syntheses
of 4c, 4e, 4g, and 4h were accomplished by the reaction of
Mcl-1 plays
a notable pro-survival role in vivo, and its
elimination in response to cytotoxic signals is critical in promoting
⇑
Corresponding author. Tel./fax: +86 411 84986032.
E-mail address: zczhang@dlut.edu.cn (Z. Zhang).
http://dx.doi.org/10.1016/j.bmc.2014.09.047
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

X. Li et al. / Bioorg. Med. Chem. 22 (2014) 5738–5746
5739
OH
O
O
Cl
N
N
a
a
5a, X=Br, R=benzyl
5b, X=Cl, R=butyl
N
N
X R
NK
O
N R
O
2
Scheme 5. Synthesis of 5a and 5b. Reagents and conditions: (a) DMF, 70 °C, 2 h.
Scheme 1. Synthesis of 2. Reagents and conditions: (a) CH3COOH, 120 °C, 8 h.
O
O
O
N
O
N
a
NH
NH
a
ClO4
N
N
OH
b
S
S
O
O
5c
Scheme 6. Synthesis of 5c. Reagents and conditions: (a) benzyl bromide, DMF,
K2CO3, 70 °C, 30 min.
2b
Scheme 2. Synthesis of 2b. Reagents and conditions: (a) DMF, POCl3, 80 °C, 3 h;
H2O, NaClO4; (b) NaOMe, MeOH, NCCH2CONH2, rt 2 h; reflux, 3 h; H2O.
O
O
NH
O
O
O
O
a
b
a
OH
OH
NH
O
O
N
H
Br
Br
O
O
O
O
5d
O
O
4a
, R=H
4b, R=CH3
4d
Scheme 7. Synthesis of 5d. Reagents and conditions: (a) benzylamine, DMF, K2CO3,
50 °C, 2 h.
R
d
c
NH
NH
, R=OCH3
4f
,
R=i-Pr
Br
S
O
O
O
O
Scheme 3. Synthesis of 4a, 4b, 4d and 4f. Reagents and conditions: (a) NaOH, H2O,
Br2, 90 °C, 6 h; HCl; (b) Ac2O, 140 °C, 2 h; (c) CO(NH2)2, 160 °C, 1 h; (d) 4-R-PhSH,
K2CO3,CuI, DMF, 130 °C, N2, 6 h.
a
NH
NH
O
O
O
6
O
Scheme 8. Synthesis of 6. Reagents and conditions: (a) N2H4ꢀH2O, C2H5OH, reflux,
O
O
4c, R=CH3
R
b
1 h.
a
4e
4g
4h
, R=i-Pr
, R=s-bu
, R=t-Am
NH
NH
NH
O2N
O
O
O
O
O
O
O
Scheme 4. Synthesis of 4c, 4e, 4g and 4h. Reagents and conditions: (a) HNO3,
H2SO4, 3 h; (b) 4-R-PhOH, K2CO3,CuI, DMF, 130 °C, N2, 18–30 h.
O
OH
OH
NH
NH2
N
N
O
c
N
b
O
a
O
7
corresponding phenol with 4-nitrophthalimide with K2CO3 at
130 °C in DMF (Scheme 4).
Scheme 9. Synthesis of 7. Reagents and conditions: (a) ClCH2COOH, KOH, H2O,
70 °C, 4 h; HCl; (b) Ac2O, 140 °C, 2 h; (c) CO(NH2)2, 110 °C, 1 h.
Compounds 5a–5c were synthesized according to Gabriel syn-
thesis (Schemes 5 and 6). Compound 5d was prepared by the reac-
tion of phthalimide and benzylamine with K2CO3 at 50 °C in DMF
(Scheme 7).27 Compound 6 was obtained by the reaction of phtha-
lic anhydride and hydrazine hydrate in ethanol under reflux for 1 h
(Scheme 8).28 Compound 7 was synthesized according to the route
described in Scheme 9, and the starting material phenylamine was
converted to 7 by three steps, substitution, dehydration and
imidization.29
evaluated using Fluorescence polarization assay (FPA) (Fig. 3a,
and b). The commerce thiomorpholine 3 could not bind to Mcl-1
at all, while 2 exhibited a weak affinity (Ki = 17.9 lM). Conse-
quently, two compounds (2a, 2b) were obtained by introducing a
benzene ring to enrich the complexity of 2. Although, the structure
of these two compounds, 2a and 2b, is very different from com-
pound 1, the effective carbonyl group that may form a hydrogen
bond with Arg263 was still reserved (Fig. 2, Scheme 2).
3. Results and discussion
FPA showed 2a binds to Mcl-1 and Bcl-2 with Ki values of
6.9
l
M and 11.6
l
l
M, respectively, while 2b binds to Mcl-1 with a
M, but shows no appreciable binding to Bcl-2
Our previous studies revealed that compound 1 is able to form a
hydrogen bond with Arg263 in Mcl-1 through its carbonyl group,
while the thiomorpholine inserts into the p2 pocket of Mcl-1
(Fig. 1a).23 The structural information suggested that not only the
hydrogen bonding but also hydrophobic interactions contribute
to the dual inhibition of Mcl-1 and Bcl-2. Here, we intended to
explore the significance of these two kinds of mimicking for the
Mcl-1/Bcl-2dual inhibitors.
Ki value of 9.51
(Table 1, Fig. 3a and b). Relatively high affinity of these two simply
fragments implies that all that is required for ligand-Arg263 inter-
action is an amide. The predicted binding model showed that one
of carbonyl groups in 2a forms a hydrogen bond with Arg263 in
Mcl-1 (Fig. 1b). Therefore, compound 2a was selected as a starting
point for the further optimization.
Based on our predicted binding model for 2a, we designed and
synthesized a series of 5-substituted compounds, 4a–4h, using
Firstly, 1 was deconstructed into small pieces 2 and 3 (Fig. 2 and
Scheme 1). The binding affinities (Ki) of these compounds were

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X. Li et al. / Bioorg. Med. Chem. 22 (2014) 5738–5746
(b)
(a)
(c)
(e)
R263
R263
P2 pocket
P2 pocket
P2 pocket
(d)
R263
R263
P2 pocket
(f)
R263
R263
P2 pocket
P2 pocket
Figure 1. Predicted binding models of Mcl-1 in complex with (a) 1, (b) 4f, (c) 4h, (d) 5c, (e) 6 and (f) 7. Carbon, hydrogen, oxygen, nitrogen, and sulfur atoms in all designed
compounds and in surface of Mcl-1 are in gray, white, red, blue, and yellow, respectively. Hydrogen bonds are depicted as dashed lines in green.
O
substituent group resulted in no binding (Fig. 3a and b, and
HN
Table 1). The most potent compound 4f binds to Mcl-1 and Bcl-2
with Ki values of 298 nM and 1.1 lM, respectively. 4h showed no
binding.
O
O
N
O
2a
O
C
HN
N
C
N
N
Analysis of the predicted binding models showed the isopropyl
of 4f can mimic the iso-butyl group of Leu62 in the Bim BH3 pep-
tide to fill the p2 pocket of Mcl-1, while one of the carbonyl groups
forms a hydrogen bond with Arg263. As shown in Figure 1b, the
position and orientation of 4f in the BH3 groove is very similar
to that of 1. 4-tert-amylphenoxy of 4h, however, cannot be accom-
modated well inside the p2 pocket because of steric hindrance
(Fig. 1c). Comparing to 1, the 4 series compounds hold the very dif-
ferent structure but the similar binding mode and affinity. Com-
pounds 4f and 1 shared the same carbonyl group and similar
hydrophobic groups, both of which are proposed to contribute to
the binding.
C
HN
2
N
S
H
N
2b
1
S
3
Figure 2. Deconstruction of 1 into fragments by maintaining key groups (high-
lighted in red).
benzenethiol, 4-methylbenzenethiol, 4-methylphenol, 4-methoxy-
benzenethiol, 4-isopropylphenol, 4-isopropylbenzenethiol, 4-sec-
butylphenol, and 4-tert-pentylphenol as the substituent groups
(Schemes 3 and 4). In our previous SAR studies, we successfully
occupied the p2 pocket of Mcl-1/Bcl-2 by using these groups.23
Very similar to the survey of these substituent groups in the previ-
ous study, progressive increase in steric bulk of the group resulted
in the corresponding increase in binding affinity, and overlarge
To further identify the direct binding ability of 4f and 4h to Mcl-
1, isothermal titration calorimetry (ITC) was performed (Fig. 4a and
b). In consistent with their Ki values showed in Table 1, 4f exhib-
ited a Kd value of 0.632 lM, illustrating the binding to Mcl-1, while
4h shows no appreciable binding to Mcl-1, presumably due to the
added steric bulk of the neopentyl group. It further confirmed that
forming hydrogen bond with R263 plays the most important role
in binding to Mcl-1.

X. Li et al. / Bioorg. Med. Chem. 22 (2014) 5738–5746
5741
Figure 3. Competitive binding curves of designed compounds to (a) Mcl-1 and (b) Bcl-2 proteins as determined using a fluorescence-polarization assay.
We tried to explore the striking feature of the carbonyl group
for the binding of these compounds to the targets. Firstly, we found
the carbonyl group protrudes from the molecular platform. We
then referred to the three dimension structure of Bim in a complex
with Mcl-1 (Fig. 5a and b). We found that the Arg263 is surrounded
by four residues, Val253, Asp256, Val258 and Asn260. Arg263 is
flush with these four residues. Accordingly, Asp67 in Bim that
forms salt bridge with Arg263 protrudes from the backbone of
the peptide (Fig. 5b). As such, we hypothesized that an exposed
hydrogen-bond donor should be a specific feature for 1 and the 4
series as a dual inhibitor. Consequently, we referred to the pan-
Bcl-2 inhibitors TW-37 and TM-179.20,30 These compounds also
hold a protruding pyrogallol group, which forms hydrogen bond
with Arg263 in Mcl-1 and Arg146 in Bcl-2.
Encouraged by the above findings, we designed compounds
5a–5d to further verify that a protruding hydrogen-bond donor
plays a critical role in this binding model. Three groups of different
sizes were introduced to N-position of 2a and 4f (Schemes 5–7).
Our FP-based binding assay determined that these four compounds
showed no appreciable binding to Mcl-1 or Bcl-2 at the concentra-
flexibility substituent such as the butyl of 5b, could hampered
the hydrogen bond.
From the predicted binding model, we found 5c could still
occupy the P2 pocket, though it loses the hydrogen bond
(Fig. 1d). Additionally, the single hydrophobic group itself, such
as compound 3, did not show any affinity, while compound 2
which held a six-ring as simple as thiomorpholine showed a weak
but appreciable binding. These data indicated that the carbonyl
group may play a more important role in this binding model than
the hydrophobic groups. This result is inconsistent with the Bim
BH3 alanine mutations reported by W. Douglas Fairlie.18 The
D67A mutant which destroyed the interaction with Arg263 had
significant effects on Mcl-1 binding, but no significant effects were
observed with L62A mutant which occupied p2 pocket in Mcl-1.
In order to further confirm that Arg263 in Mcl-1 (Arg146 in
Bcl-2) plays a predominant role for dual inhibitors to capture, other
two compounds 6 and 7 were designed and synthesized (Schemes
8 and 9). On the premise of maintaining the protruding carbonyl
group, the crucial part of 6 binding to Arg263 becomes a stable
six-membered ring, while that of 7 becomes a flexible ring.
tion as high as 100
l
M (Fig. 3a and b). As expected, the docking
FP-based binding assays showed that compound 6 has Ki of 8.13
and 12.5 M to Mcl-1 and Bcl-2, respectively, while 7 was deter-
mined to bind to Mcl-1 and Bcl-2 with Ki values of 10.7 M and
22.1 M, respectively. Although the structures of these two com-
pounds have changed a lot compared with 1, both of them still
maintain the dual inhibition of Mcl-1 and Bcl-2. The protruding
hydrogen bond donor in the proper position is the determinant.
lM
study showed that 5c lost the hydrogen bond with Arg263 of
Mcl-1 (Fig. 1d). It illustrated that a hydrogen-bond donor protrud-
ing from the molecular platform should be necessary for the for-
mation of the hydrogen bond in this binding model. When a
steric hindrance is available, either the big rigid substituent groups
such as the phenyl and benzyl of 5a, 5c and 5d, or the small
l
l
l

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X. Li et al. / Bioorg. Med. Chem. 22 (2014) 5738–5746
Table 1
Structure and binding affinities of small-molecule inhibitors to Mcl-l and Bcl-2 as determined by FPAs
Compounds
Structure
Kia (Mcl-1)
Kia (Bcl-2)
O
CN
2
17.9
NA
l
M
NA
HN
H
N
3
NA
S
O
NH
2a
6.9 0.8
9.5 1.3
4.6 1.2
l
l
l
M
M
M
11.6 1.6
l
M
O
H
N
O
2b
4a
12%, [100
l
M]
CN
O
NH
3.2 0.6
3.1 0.7
l
M
M
S
O
O
NH
4b
4c
4d
4e
4f
3.69 0.7
l
M
l
S
O
O
NH
2.7 0.5
l
M
3.39 1.7
l
M
O
O
O
O
NH
2.63 0.8
l
M
3.6 1.2
l
M
S
O
S
O
O
NH
783 120 nM
298 130 nM
331 160 nM
1.87 0.5
lM
O
O
NH
1.1 0.2
l
M
O
O
NH
4g
4h
1.42 0.3
lM
O
O
O
NH
5%, [100
l
M]
9%, [100
l
M]
O
O
O
N
5a
29%, [100
l
M]
34%, [100 lM]
O

X. Li et al. / Bioorg. Med. Chem. 22 (2014) 5738–5746
5743
Table 1 (continued)
Compounds
Structure
Kia (Mcl-1)
Kia (Bcl-2)
O
N
5b
5c
15%, [100
43%, [100
l
M]
M]
27%, [100
l
M]
O
O
N
O
l
l
44%, [100
lM]
S
O
NH
O
5d
36%, [100
M]
16%, [100
lM]
N
H
O
O
NH
NH
6
8.13 1.2
l
M
M
12.49 1.4 lM
O
NH
7
10.7 1.9
l
22.1
4 lM
N
O
a
Values were measured by FPA for inhibition constant (Ki). The values are the mean SD of three independent experiments.
Time (min)
Time (min)
0
10
20
30
0
10
20
30
(a) 0.10
(b) 0.10
0.00
0.00
-0.10
-0.20
-0.30
-0.40
-0.50
-0.60
-0.10
-0.20
-0.30
-0.40
-0.50
-0.60
-0.70
0.00
-0.70
0.00
-2.00
-4.00
-6.00
-8.00
-2.00
-4.00
-6.00
N
K
ΔH
ΔS
0.928
5.32E4
-3926
0.031Sites
-1
N
K
0.962
0.0245Sites
-1
8.14E5 M
20.58 cal/mol
1.58E6 1.13E7M
ΔH -7738 68.32 cal/mol
ΔS 2.65 cal/mol/deg
8.55 cal/mol/deg
0.0
0.5
1.0
1.5
2.0
0.0
0.5
1.0
1.5 2.0
Molar Ratio
Molar Ratio
Figure 4. The binding affinities determined by ITC of (a) 4f and (b) 4h to Mcl-1, respectively. The upper panels display the raw titration data. Each point on the curves in the
lower panels represent.

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X. Li et al. / Bioorg. Med. Chem. 22 (2014) 5738–5746
(a)
(b)
D256
D256
V258
R263
D67
R263
V258
N260
D67
V253
V253
L62
Figure 5. Positions of residues around R263 in a Ribbon diagram of the Bim/Mcl-1 complex (a) front view, (b) side view.
Analysis of the binding model of 6 and 7 to Mcl-1 (Fig. 1e and f)
confirmed that one of the carbonyls of them can form hydrogen
bond with Arg263 in Mcl-1.
(734 mg, 5.4 mmol) was stirred for 3 h at 80 °C. The mixture was
put on ice (30 g) and conc. NaClO4 solution was added. The result-
ing precipitate was filtered off and washed with diluted NaClO4
solution. The product was used in the next step without further
disposal.
4. Conclusions
The solid was added to a solution of sodium methoxide (0.162 g,
3.0 mmol) in 13 mL of CH3OH. Then cyanoacetamide (0.10 g,
3.28 mmol) was added. The mixture was stirred for 2 h and then
refluxed 3 h during which time a yellow solid separated. Water
was added, and the mixture was acidified and filtered to remove
a yellow solid which was washed with water, ethanol, ether, and
then hexane to give crude product. The crude product was recrys-
tallized from ethanol.
In summary, our study demonstrates although both Arg263
(Arg146 in Bcl-2) and p2 pocket are the hotspots of Mcl-1 and
Bcl-2, the Arg263 in Mcl-1 (Arg146 in Bcl-2) shows the predomi-
nant requirement for the dual inhibitors to capture. The appropri-
ate hydrogen bonding donors need to protrude from the molecular
platform, while isopropyl and sec-butyl shows proper size and
hydrophobic property to occupy the p2 pocket. The molecules that
well occupy both of these two sites could gain good dual inhibition.
Yield: 392 mg, 61%. 1H NMR (400 MHz, CDCl3): d: 12.97 (s, 1H),
8.30 (s, 1H), 8.02 (s, 1H, Ar-H), 7.49–7.52 (d, J = 8.0 Hz, 2H), 7.41–
7.43 (m, 3H). TOF MS (EI+): C12H8N2O, found 196.06. Mp: 232–
233 °C.
5. Experimental section
5.1. Materials and methods
5.2.3. General procedure for the preparation of 4a, 4b, 4d and 4f
To sodium hydroxide (2.4 g, 0.06 mol) dissolved in water
(20 mL) was added phthalic anhydride (4.44 g, 0.03 mol). When
complete solution was obtained, bromine (9.4 g, 3 mL, 0.06 mol)
was incrementally added, while stirring, over 1 h. The reaction
mixture was heated to 90 °C and allowed to react under reflux
for 6 h. After standing for 10 h, the white solids that crystallized
out of solution were filtered, washed with cold water, and analyzed
as monosodium 4-bromophthalic acid salt. The total product was
dissolved in hot water, and the pH was adjusted to about 1.5 by
addition of concentrated hydrochloric acid. The resulting solution
was evaporated to dryness on a rotary evaporator and extracted
with acetone to give 4-bromophthalic acid. Yield: 5.0 g, 68%, mp:
164–166 °C.
All commercial reagents were purchased and used without fur-
ther purification or distillation unless otherwise stated. 1H NMR
was obtained with Bruker AV-400 spectrometer with chemical
shifts reported as ppm (in CDCl3, TMS as internal standard). The
following abbreviations are used for multiplicity of NMR signals:
s = singlet, d = doublet, t = triplet. q = quartet, m = multiplet,
br = broad. High-resolution mass spectra (HRMS) were obtained
on HPLC-Q-Tof MS (Micro) spectrometer. Column chromatography
was performed on silica gel 200–300 mesh. Purityof all final prod-
ucts was determined by analytical HPLC to be P95%. HPLC purity
of compounds was measured with a normal phase HPLC (XBridge
C18, 4.6 ꢁ 150 mm, 5
lM) with two diverse wavelength detection
systems.
A solution of 4-bromophthalic acid (5 g, 0.02 mol) and acetic
anhydride (30 mL) was heated for 2 h at 140 °C. The reaction mix-
ture was cooled to room temperature and the excess of acetic
anhydride was removed under reduced pressure. The residue was
washed with petroleum ether and then 4-bromophthalic anhy-
dride was obtained as white solid. Yield: 3.8 g, 84%, mp: 104–
106 °C.
4-Bromophthalic anhydride (2.26 g, 0.01 mol) was added to
urea (6.0 g, 0.1 mol). Then the solid mixture was heated till molten,
and maintained at 160 °C for 2 h. After cooled, the solid was
washed with 100 mL water and recrystallized with ethanol
(100 mL). 4-Bromophthalimide was obtained as white solid. Yield:
1.32 g, 58%, mp: 230–233 °C.
5.2. Synthesis
5.2.1. 2-Hydroxynicotinonitrile (2)
Acetic acid (20 mL) was added in three portions to 2-chloronic-
otinonitrile (1.38 g, 10 mmol) under stirring at room temperature.
Then, the temperature raised to 120 °C, and kept for 8 h. After
cooled to room temperature, the resulting precipitation was fil-
tered. 2-hydroxynicotinonitrile was obtained as white needle
crystal.
Yield: 68%, 1H NMR (400 MHz, CDCl3): d: 10.12 (s, 1H), 7.93 (d,
J = 8.0 Hz, 1H), 7.72 (d, J = 8.0 Hz, 1H), 6.34 (m, 1H).TOF MS (EI+):
C6H4N2O, found 121.03. Mp: 224–225 °C.
The mixture of 4-bromophthalimide (225 mg, 1 mmol), 4-R-
PhSH (3 mmol), K2CO3 (136 mg, 1 mmol) and CuI (10 mg) was
added to DMF (10 mL). The solution was heated at 130 °C for 6 h
under the protection of nitrogen. After cooled, water (30 mL) was
added and the precipitation was filtered, washed with water. The
5.2.2. 5-Phenyl-2-hydroxynicotinonitrile (2b)
A
mixture of dimethylformamide (1.96 g, 27 mmol) and
phosphoryl chloride (2.47 g, 16 mmol) and 2-phenylacetic acid

X. Li et al. / Bioorg. Med. Chem. 22 (2014) 5738–5746
5745
product was purified by column chromatography on silica gel
using dichloromethane/acetone (60:1).
(d, J = 4.0 Hz, 2H), 1.67 (m, 2H), 1.31 (s, 6H), 0.73 (t, J = 8.0 Hz,
3H). TOF MS (EI+): C19H19NO3, found 309.13. Mp: 140–142 °C.
5.2.3.1. 5-(Phenylthio)isoindoline-1,3-dione (4a). Yield: 85 mg,
38%. 1H NMR (400 MHz, CDCl3): d 7.92 (s, 1H, NH), 7.69 (d,
J = 8.0 Hz, 1H), 7.52 (q, J = 8.0 Hz, 2H), 7.50 (d, J = 8.0 Hz, 1H),
7.46 (m, 4H). TOF MS (EI+): C14H9NO2S, found 255.03. Mp: 169–
171 °C.
5.2.5. General procedure for the preparation of 5a and 5b
A mixture of phthalimide potassium (500 mg, 2.7 mmol), and
benzyl bromide or 1-butyl chloride (6.0 mmol) was stirred in
DMF (10 mL) for 2 h at 70 °C. After cooled to room temperature,
the solution was poured into 20 mL cooled water, stilled for 10 h.
The precipitation was filtered, washed with water and recrystal-
lized with ethanol/H2O (20 mL/20 mL).
5.2.3.2. 5-(p-Tolylthio)isoindoline-1,3-dione (4b). Yield: 140 mg,
52%. 1H NMR (400 MHz, CDCl3): d 7.79 (s, 1H, NH), 7.66 (d,
J = 8.0 Hz, 1H), 7.43 (m, 4H), 7.27 (d, J = 8.0 Hz, 2H), 2.42 (s, 3H).
TOF MS (EI+): C15H11NO2S, found 269.05. Mp: 154–156 °C.
5.2.5.1. 2-Benzylisoindoline-1,3-dione (5a). Yield: 300 mg, 46%,
white needle crystal. 1H NMR (400 MHz, CDCl3): d 7.83 (m, 2H),
7.68 (m, 2H), 7.31 (d, J = 4.0 Hz, 2H), 7.27 (d, J = 8.0 Hz, 1H), 7.24
(s, 1H), 4.84 (s, 2H, CH2). TOF MS (EI+): C15H11NO2, found 237.08.
Mp: 117–118 °C.
5.2.3.3. 5-(4-Methoxyphenylthio)isoindoline-1,3-dione (4d).
Yield: 121 mg, 42%. 1H NMR (400 MHz, CDCl3): d 7.91 (s, 1H,
NH), 7.66 (d, J = 8.0 Hz, 1H), 7.48 (d, J = 8.0 Hz, 2H), 7.38 (m, 2H),
6.99 (d, J = 8.0 Hz, 2H), 3.87 (s, 3H). TOF MS (EI+): C15H11NO3S,
found 285.05. Mp: 157–159 °C.
5.2.5.2. 2-Butylisoindoline-1,3-dione (5b). Yield: 155 mg, 28%,
white solid. 1H NMR (400 MHz, CDCl3) d: 7.84 (m, J = 8.0 Hz, 2H),
7.71 (m, J = 8.0 Hz, 2H), 3.69 (t, 2H, CH2), 1.66 (m, 2H, CH2), 1.38
(m, 2H, CH2), 0.95 (t, 3H, CH3). TOF MS (EI+): C12H13NO2, found
203.09. Mp: 33–35 °C.
5.2.3.4. 5-(4-Isopropylphenylthio)isoindoline-1,3-dione (4f).
Yield: 110 mg, 37%. 1H NMR (400 MHz, CDCl3): d 8.09 (s, 1H, NH),
7.67 (d, J = 8.0 Hz, 1H), 7.48 (m, 4H), 7.32 (d, J = 8.0 Hz, 2H), 2.96
(m, 1H), 1.29 (d, J = 4.0 Hz, 6H). TOF MS (EI+): C17H15NO2S, found
297.08. Mp: 151–152 °C.
5.2.6. 2-Benzyl-5-(4-isopropylphenylthio)isoindoline-1,3-dione
(5c)
A mixture of 5-(4-isopropylphenylthio)isoindoline-1,3-dione
(4f) (148 mg, 0.5 mmol), benzyl bromide (171 mg, 1.0 mmol), and
K2CO3 (136 mg, 1.0 mmol) was stirred in DMF (10 mL) for 30 min
at 70 °C. After cooled to room temperature, the solution was poured
into 20 mL cooled water and extracted with ethyl acetate. The
resulting product was purified by column chromatography on silica
gel using ethyl acetate/petroleum ether (1:20) as the mobile phase.
Yield: 118 mg, 60%, white solid. 1H NMR (400 MHz, CDCl3) d:
7.66 (d, J = 8.0 Hz, 1H), 7.48–7.40 (m, 5H), 7.30–7.27 (m, 4H),
7.24 (d, J = 8.0 Hz, 1H), 4.80 (d, J = 4.0 Hz, 2H, CH2), 2.95 (m, 1H),
1.28 (d, J = 4.0 Hz, 6H). TOF MS (EI+): C24H21NO2S, found 387.13.
Mp: 94–96 °C.
5.2.4. General procedure for the preparation of 4c, 4e, 4g and 4h
To a stirred, cold (ice-bath) solution of 22 mL of fuming aqueous
nitric acid and 50 mL of concentrated aqueous sulfuric acid was
added, portionwise, 14.7 g (0.1 mol) of phthalimide. The mixture
was allowed to warm to room temperature. After about 3 h, com-
plete solution was obtained. The yellow solution was slowly
poured, with stirring, onto 200 g ice. The crude product which pre-
cipitated was collected by filtration and 4-nitrophthalimide was
recrystallized from ethanol to afford 8.74 g (45.5%) as yellow solid.
Mp: 198–202 °C.
The mixture of 4-nitrophthalimide (192 mg, 1 mmol), 4-R-
PhOH (3 mmol), K2CO3 (136 mg, 1 mmol) and CuI (10 mg) was
added to DMF (10 mL). The solution was heated at 130 °C for
18–30 h under the protection of nitrogen. After cooled, water
(30 mL) was added and the precipitation was filtered, washed with
water. The product was purified by column chromatography on
silica gel using dichloromethane/acetone (60:1).
5.2.7. N1,N2-Dibenzylphthalamide (5d)
A mixture of phthalimide (147 mg, 1.0 mmol), benzylamine
(321 mg, 3.0 mmol), and K2CO3 (136 mg, 1.0 mmol) was stirred in
DMF (10 mL) for 2 h at 50 °C. After cooled to room temperature,
the solution was poured into 20 mL cooled water. The resulting
precipitate was filtered, washed with water.
5.2.4.1. 5-(p-Tolyloxy)isoindoline-1,3-dione (4c). Yield: 104 mg,
41%. 1H NMR (400 MHz, CDCl3): d 7.70 (s, 1H, NH), 7.27 (m, 2H),
7.21 (m, 2H), 6.97 (m, 2H), 6.74 (d, J = 8.0 Hz, 1H), 2.38 (s, 3H).
TOF MS (EI+): C15H11NO3, found 253.07. Mp: 161–162 °C.
Yield: 267 mg, 77%, white solid. 1H NMR (400 MHz, CDCl3) d:
7.60 (d, J = 8.0 Hz, 2H), 7.45 (d, J = 8.0 Hz, 2H), 7.35–7.28 (m,
10H), 6.96 (s, 2H), 4.51 (d, J = 4.0 Hz,
22H20N2O2, found 344.14. Mp: 174 – 177 °C.
4
H). TOF MS (EI+):
C
5.2.4.2.
5-(4-Isopropylphenoxy)isoindoline-1,3-dione
(4e).
5.2.8. 2,3-Dihydrophthalazine-1,4-dione (6)
Yield: 50 mg, 18%. 1H NMR (400 MHz, CDCl3): d 7.80 (s, 1H), 7.59
(s, 1H, NH), 7.52 (d, J = 8.0 Hz, 1H), 7.43(d, J = 8.0 Hz, 1H), 7.36 (d,
J = 8.0 Hz, 2H), 7.06 (d, J = 8.0 Hz, 2H), 2.91 (m, 1H), 1.27 (d,
J = 8.0 Hz, 6H). TOF MS (EI+): C17H15NO3, found 282.06. Mp: 157–
159 °C.
Phthalic anhydride (1.48 g, 10 mmol) was dissolved in ethanol
(20 mL), then to this mixture was added hydrazine hydrate
(2 mL, 50%, 20 mmol) dropwise. The reaction mixture was refluxed
1 h. After cooled to room temperature, the white solid was col-
lected through filtration.
Yield: 1.32 g, 82%, white solid. 1H NMR (400 MHz, CDCl3) d:
11.52 (s, 2H), 8.10 (m, 2H), 7.90 (m, 2H). TOF MS (EI+): C8H6N2O2,
found 161.04. Mp: >300 °C.
5.2.4.3.
5-(4-sec-Butylphenoxy)isoindoline-1,3-dione
(4g).
Yield: 193 mg, 62%. 1H NMR (400 MHz, CDCl3):
d
7.79 (d,
J = 8.0 Hz, 1H), 7.69 (s, 1H, NH), 7.30 (m, 2H), 7.24 (m, 2H), 7.01
(d, J = 4.0 Hz, 2H), 2.64 (m, 1H), 1.62 (m, 2H), 1.27 (d, J = 4.0 Hz,
3H), 0.85 (t, J = 8.0 Hz, 3H). TOF MS (EI+): C18H17NO2S, found
295.12. Mp: 146–147 °C.
5.2.9. 4-Phenylpiperazine-2,6-dione (7)
A mixture of phenylamine (186 mg, 2 mmol), 2-chloroacetic
acid (470 mg, 5 mmol) and KOH (560 mg, 10 mmol) was stirred
in H2O (20 mL) for 4 h at 70 °C. After being cooled down, the
mixture was adjusted to acidic with 0.1 N HCl. The precipitate
was collected by suction filtration, washed with cold water and
dried. Yield: 263 mg, 63%.
5.2.4.4. 5-(4-tert-Pentylphenoxy)isoindoline-1,3-dione (4h).
Yield: 40 mg, 13%. 1H NMR (400 MHz, CDCl3): d 7.88 (s, 1H, NH),
7.79 (d, J = 4.0 Hz, 1H), 7.37 (d, J = 4.0 Hz, 2H), 7.28 (m, 2H), 7.01

5746
X. Li et al. / Bioorg. Med. Chem. 22 (2014) 5738–5746
A
solution of 2,20-(phenylazanediyl)diacetic acid (418 mg,
protein bank in the RCSB. The 3D structures of the inhibitors were
generated using Chembio3D Ultra 11.0 followed by energy minimi-
zation. AutoDock 4.0 program equipped with ADT was used to per-
form the automated molecular docking.
2 mmol) and acetic anhydride (10 mL) was heated for 2 h at
140 °C. The reaction mixture was cooled to room temperature
and the excess of acetic anhydride was removed under reduced
pressure. The residue was used in the next step without further
disposal.
Acknowledgments
The residue (4-phenylmorpholine-2,6-dione) was added to urea
(1.8 g, 30 mmol), then, the solid mixture was heated till molten.
The molten reaction was stirred for 1 h at 110 °C and cooled. Acetic
ether (20 mL) was added. The organic layer was washed with brine
and dried with MgSO4. Concentration of the organic layer afforded
the desired crude product. After evaporation under reduced pres-
sure, the product was purified by column chromatography on silica
gel using dichloromethane/acetone (60:1).
The work was supported by the National Natural Science Foun-
dation of China (21372036, 81272876, 81430083, 21402022).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.09.047.
Yield: 0.167 mg, 44%. 1H NMR (400 MHz, CDCl3): d 8.32 (s, 1H),
7.33 (t, J = 12.0 Hz, 2H), 7.02 (t, J = 8.0 Hz, 1H), 6.35 (d, J = 4.0 Hz,
2H), 4.10 (s, 4H). TOF MS (EI+): C10H10N2O2, found 190.07. Mp:
157–158 °C.
References and notes
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5.3.1. Regents, plasmid and antibodies
A 21-residue Bid BH3 peptide (residues 79–99) bearing a 6-car-
boxyfluorescein succinimidyl ester fluorescence tag (FAM-Bid) was
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human Bcl-2 protein was purchased from Santa Cruz Biotechnol-
ogy (sc-4096, Santa Cruz). Caspase colorimetric protease assay kit
was purchased from Keygen Biotech (KGA202, Keygen, China).
pEGFP-C1/Mcl-1 plasmid and pET28a(+)/Mcl-1 plasmid were con-
structed in our Lab. Lipofectamine™ 2000 Transfection Reagent
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Bcl-2 antibody (sc-783, Santa Cruz) and rabbit antibody against
Mcl-1 (BS1220, Bioworld).
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5.3.2. Fluorescence polarization-based binding assay (FPA)
For the competitive binding assay for Bcl-2 protein, FAM-Bid
peptide (10 nM) and Bcl-2 protein (200 nM) were preincubated
in the assay buffer (25 mM Tris, pH 8.0; 150 mM NaCl). Next, serial
dilutions of compounds were added. After a 30-min incubation, the
polarization values were measured using the Spectra Max M5
Detection System in a black 96-well plate. Saturation experiments
determined that FAM-Bid binds to the Bcl-2 protein with a Kd value
of 112 nM. For Mcl-1, assays were performed in the same manner
as that for Bcl-2 with the following exceptions: 50 nM Mcl-1 and
10 nM FAM-Bid peptide were used in the assay buffer. FAM-Bid
peptide binds to the Mcl-1 protein with a Kd value of 13 nM.
5.3.3. Isothermal titration calorimetry (ITC) assay
Isothermal titration calorimetry (ITC) was performed using
ITC200 (Microcal). Experiments were performed in 20
8.0, 150 mM NaCl, 1% DMSO at 25 °C. For evaluating Kd value of
compounds, titrations consisted of 12 ꢁ 3 L injections of com-
pound at 300 M into Mcl-1 (30 M). All sample data obtained
lM Tris pH
l
l
l
after control data corrections were analyzed to fit to a one-site
model. For control ITC experiments, the sample cells were filled
with assay buffer and the compound solution was added. This pro-
cess was identical to that for protein samples.
5.4. Molecular docking
The 3D structures of the human Mcl-1 (hMcl-1; PDB ID: 2NLA)
and human Bcl-2 (Bcl-2; PDB ID: 1GJH) were obtained from the