Preclinical Activity of New [1,2]Oxazolo[5,4-e]isoindole Derivatives in Diffuse Malignant Peritoneal Mesothelioma

Article abstract of DOI:10.1021/acs.jmedchem.6b00777

A series of 22 derivatives of the [1,2]oxazolo[5,4-e]isoindole system were synthesized through an efficient and versatile procedure that involves the annelation of the [1,2]oxazole moiety to the isoindole ring, producing derivatives with a wide substitution pattern. The structure-activity relationship indicates that the N-4-methoxybenzyl group appears crucial for potent activity. In addition, the presence of a 6-phenyl moiety is important and the best activity is reached with a 3,4,5-trimethoxy substituent. The most active compound, bearing both the structural features, was able to inhibit tumor cell proliferation at nanomolar concentrations when tested against the full NCI human tumor cell line panel. Interestingly, this compound was effective in reducing in vitro and in vivo cell growth, impairing cell cycle progression and inducing apoptosis, as a consequence of the inhibition of tubulin polymerization, in experimental models of diffuse malignant peritoneal mesothelioma (DMPM), a rapidly lethal disease, poorly responsive to conventional therapeutic strategies.

Full text of DOI:10.1021/acs.jmedchem.6b00777

Article
pubs.acs.org/jmc
Preclinical Activity of New [1,2]Oxazolo[5,4‑e]isoindole Derivatives in
Diffuse Malignant Peritoneal Mesothelioma
Virginia Spano,† Marzia Pennati,‡ Barbara Parrino,† Anna Carbone,† Alessandra Montalbano,†
̀
Vincenzo Cilibrasi,† Valentina Zuco,‡ Alessia Lopergolo,‡ Denis Cominetti,‡ Patrizia Diana,†
Girolamo Cirrincione,† Paola Barraja,*,† and Nadia Zaffaroni*,‡
†Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche (STEBICEF), Universita
̀
degli Studi di Palermo, Via
Archirafi 32, 90123 Palermo, Italy
‡Molecular Pharmacology Unit, Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto
Nazionale Tumori, Via Amadeo 42, 20133 Milano, Italy
S
* Supporting Information
ABSTRACT: A series of 22 derivatives of the [1,2]oxazolo-
[5,4-e]isoindole system were synthesized through an efficient
and versatile procedure that involves the annelation of the
[1,2]oxazole moiety to the isoindole ring, producing
derivatives with a wide substitution pattern. The structure−
activity relationship indicates that the N-4-methoxybenzyl
group appears crucial for potent activity. In addition, the
presence of a 6-phenyl moiety is important and the best
activity is reached with a 3,4,5-trimethoxy substituent. The
most active compound, bearing both the structural features,
was able to inhibit tumor cell proliferation at nanomolar concentrations when tested against the full NCI human tumor cell line
panel. Interestingly, this compound was effective in reducing in vitro and in vivo cell growth, impairing cell cycle progression and
inducing apoptosis, as a consequence of the inhibition of tubulin polymerization, in experimental models of diffuse malignant
peritoneal mesothelioma (DMPM), a rapidly lethal disease, poorly responsive to conventional therapeutic strategies.
interest because they constitute an important class of natural
and synthetic products, many of which exhibit relevant
biological activities.7−17 Isoxazoles and [1,2]oxazoles are
nitrogen and oxygen containing heterocycles that belong to
the azoles family, which have aroused much interest as novel
anticancer agents. Among these, diaryl[1,2]oxazoles of type 1
(Chart 1) have been widely studied, showing very promising
results either as HSP-90 inhibitors18 or potent analogues of the
antitubulin compound combretastatin A4 (CA4).19,20
In the latter series of compounds, the 3,4,5-trimethoxy group
on one of the two aromatic rings was essential for the cytotoxic
activity especially in the presence of a nitro or an amino group
in α-position to the 4-methoxy on the other phenyl ring,
reaching the best antitubulin activity.
Derivatives of the 3,5-bis(3′-indolyl)[1,2]oxazole system,
recently reported by us as analogues of nortopsentins A-C,
exhibited in vitro cytotoxicity in the micromolar range.21
3-Amino benzo[d]isoxazoles potently inhibited both the
vascular endothelial growth factor receptor (VEGFR) and
platelet-derived growth factor receptor families of receptor
tyrosine kinases.22 Other 3-amino-benzo[d]isoxazoles displayed
potent inhibitory effects both at enzymatic and cellular levels
INTRODUCTION
■
Malignant mesothelioma is a rare tumor associated with
asbestos exposure that develops from mesothelial cells of the
serosal membranes, including pleura, peritoneum, tunica
vaginalis, and pericardium.1 Diffuse malignant peritoneal
mesothelioma (DMPM) accounts for approximately 20% of
all malignant mesotheliomas, with a yearly incidence in Europe
of 1−2 cases per million.2 DMPM prognosis is dismal. In
historical case series, standard therapy with palliative surgery,
systemic/intraperitoneal chemotherapy, and abdominal irradi-
ation showed to be ineffective, with a median survival of about
one year.2 More recently, a loco-regional strategy that combines
aggressive cytoreductive surgery (CRS) with hyperthermic
intraperitoneal chemotherapy (HIPEC) significantly improved
median survival up to 52−92 months in selected series of
patients, although half of them still experienced recurrence.3
For these patients, and for those who are not eligible to CRS
+HIPEC, the prognosis remains severe due to the lack of
effective alternative treatment options, highlighting the need to
develop new therapeutic strategies.
In a recent project aimed at developing new candidates for
the treatment of DMPM, we identified novel molecules based
on natural compound scaffolds.4−6 Small molecules have found
application in medicine, notably as cancer chemotherapeutic
agents. Among these, nitrogen heterocycles are of special
Received: May 23, 2016
© XXXX American Chemical Society
A
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The incorporation of heterocyclic rings into pharmaceutical
candidates is a major strategy to enhance activity and safety
profiles. As a consequence, for a long time, much of our
attention has been paid to the design and synthesis of pyrrolo-
fused systems.28−32 Several examples in the literature high-
lighted the indole nucleus as a valuable pharmacophore for
potent antimitotic agents,33,34 but less is known about isoindole
derivatives.35−37 We have already reported the synthesis of
heterocyclic systems in which the isoindole scaffold is
condensed with esatomic rings such as a pyrane,38 pyridine,39
and pyrimidine35,40 and with a pentatomic one such as
pyrazole41 producing compounds with interesting antitumor
activity.
Chart 1. [1,2]Oxazole Derivatives 1−6
A literature search revealed only two reports, from the same
research group, concerning the [1,2]oxazolo[5,4-e]isoindoles
ring system. Both reports described the same synthetic
approach, leading to the tricyclic system with limited yield
and poor possibility of obtaining derivatives with a wide
substitution pattern.42,43 Thus, as initial rationale for our work,
we decided to explore new synthetic strategies, planning the
annelation of an isoxazole ring to an isoindole moiety to
generate [1,2]oxazolo [5,4-e]isoindoles of type 6 (Chart 1). We
have recently reported that tricyclic compounds incorporating
the [1,2]oxazole unit fused to an indazole or an indole scaffold,
namely [1,2]oxazolo[5,4-e]indazoles 444 and [1,2]oxazolo[4,5-
g]indoles 5,45 which showed promising antitumor properties
(Chart 1). In particular, some derivatives of the latter class of
compounds demonstrated a potent growth inhibitory effect on
toward c-Met tyrosine kinase.23 Tricyclic[1,2]oxazoles of type 2
have been described as selective inhibitors of the multidrug
resistance protein MRP1.24 Finally, isoxazolo-quinazolines 3
emerged as potent modulators of kinases, such as MSK1 and
PERK.25
Drugs that interfere with tubulin dynamics (including
taxanes, vinca alkaloids, and combrestatins) are commonly
used in the clinic to treat a variety of cancers.26 In particular,
the vinca alkaloid vinorelbine has demonstrated clinical efficacy
in malignant pleural mesothelioma, representing a reasonable
alternative treatment option for the disease.27
a
Scheme 1. Synthesis of 1-Aryl Tetrahydro-4H-isoindol-4-ones 11a−g
a
Reagents and conditions: (a) DMFDMA, reflux, 30 min−1 h, 98−99%; (b) phenylglycine or 4-hydroxyphenylglycine or 3,4,5-
trimethoxyphenylglycine, AcONa·3H2O, ethanol, reflux, 1−2 h, (90−95%); (c) Et3N, Ac2O, reflux, 15−30 min, (75−90%); (d) 80% acetic acid,
37% HCl, rt−60 °C, 10−55 min, 5−80%; (e) 10% K2CO3, ethanol, rt, 2 min, (93%).
B
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a
Scheme 2. Synthesis of 5,7-Dihydro-4H-[1,2]oxazole[5,4-e]isoindoles 15a−v
a
Reagents and conditions: (a) for 12a−c,35,40 12d−k,n-p,r,s NaH, DMF, 0 °C to rt, 1 h then aralkyl halide at 0 °C to rt, 1−24 h, 52−93%, for 12m,
K2CO3, N-methylpyrrolidone, N2, rt, 1 h, then Cu(I)Br, rt 1 h, then 4-OMePhI, reflux, 48 h, 60%; (b) H2, 10% Pd/C, ethanol, rt, 24 h, 95−98%; (c)
10% K2CO3, ethanol, rt, 16 h, 96%; (d) NaH, THF, 0 °C to rt, 1 h then MeI at 0 °C to rt, 20 h, 88%; (e) t-BuOK, toluene, 0 °C to rt, 3 h then
HCOOEt, rt, 24 h, 60−88%; (f) TBDMAM, toluene, reflux, 24 h; (g) NH2OH·HCl, ethanol, reflux, 50 min, 57−84%; (h) BBr3, dichloromethane,
−78 °C to rt, 24 h, 84%.
the National Cancer Institute 60 human tumor cell line panel
(NCI-60) at concentrations in the micromolar to nanomolar
range. The most active compounds were particularly effective
against the leukemia subpanel, and the presence of a methoxy
substituted benzyl moiety at the indole nitrogen appeared
crucial for conferring remarkable cytotoxic activity to the
[1,2]oxazolo indole derivatives. On the basis of the above
observations, the present study was designed to explore further
chemical modifications of the five-membered heterocycle
nucleus. Herein, we describe the synthesis and biological
evaluation of new [1,2]oxazolo[5,4-e]isoindoles 6 as positional
isomers of compounds 5 (Chart 1), bearing aryl substituted
moieties including methoxy groups, in an the attempt to
synthesize new active drugs against mesothelioma.
moiety and 12a−c bearing a methyl, benzyl, and 4-
methoxybenzyl substituent, respectively, in position 2 were
conveniently prepared by a method previously reported by us
(Schemes 1 and 2).35,40
Several new tetrahydroisoindoles were prepared as outlined
in the Scheme 1 by a multistep sequence starting from
commercially available resorcine 7a and dimedone 7b, which
were converted into their enamino derivatives 8a,b in
quantitative yields upon heating the latter under reflux in
dimethylformamide dimethylacetal (DMFDMA) (99 and 98%
yields, respectively). 2-[(Dimethylamino)methylidene]-
cyclohexane-1,3-diones 8a,b were subsequently reacted in
refluxing ethanol with the commercially available phenylglycine,
3,4,5-trimethoxy phenylglycine, in turn prepared by a Strecker
reaction from 3,4,5-trimethoxybenzaldehyde46 or 4-hydroxy-
phenylglycine to give the enamino acids 9a−d (90−95%).
Cyclization in acetic anhydride and triethylamine of 9a−c gave
the expected dihydroisoindole 10a−c (75−90%). In the case of
9d, the cyclization process in acetic anhydride also determined
acetylation of the phenolic group, thus furnishing the 4-[2-
acetyl-4-(acetyloxy)-6,7-dihydro-2H-isoindol-1-yl]phenyl ace-
tate 10d (81%). Finally, tetrahydroisoindole-2-ones 11a−c
were obtained (70−80%) by deacetylation of the correspond-
RESULTS AND DISCUSSION
■
Chemistry. Our approach to the synthesis of the new ring
system consisted in the preparation of a number of
tetrahydroisoindole-4-ones of types 11 (Scheme 1) and 12
(Scheme 2); the latter could be further functionalized in α
position to the carbonyl group to have the access to key
synthons of type 13 and 14 ready for the annelation leading to
the [1,2]oxazole moiety. Ketones 11a bearing the 1-phenyl
C
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ing derivatives 10a−c by heating in aqueous acetic acid (80%)
and HCl (37%). Hydrolysis of 10d, performed under the same
conditions, led to several byproducts, making it difficult to
isolate the desired tetrahydroisoindole 11e. A strict temperature
control was crucial in order to obtain 11e with the best yield.
By using the same conditions as for its analogues (10−15 min,
60 °C), 10d gave a mixture of two deacetylated products: the 4-
acethoxyphenyl derivative 11e (12%) and the 4-hydroxyphenyl
one 11d (32%). Prolonged heating led to 11d (73%) as the
sole product, which was not useful for our purpose. To make
the deacetylation process smoother, it was conducted at room
temperature.
After 90 min, four products were isolated: 11d (17%), 11e
(19%), 11f (9%), and 11g (21%). Instead, after 50 min, 11d
(9%), 11e (23%), 11f (5%), and 11g (51%) were isolated with
the indicated yields. This finding was considered the best result
considering that 11e and 11g were preferred for our synthetic
purpose. Derivative 11g was readily converted into 11e (93%)
in 10% K2CO3.
Ketones 11a−c,e, bearing a free NH, were subjected to
nucleophilic reactions with aralkyl halides to give N-substituted
derivatives (Scheme 2). Thus, reaction in dimethylformamide
with benzyl bromide or substituted benzyl chlorides (2-
methoxybenzyl chloride, 3-methoxybenzyl chloride, 4-methox-
ybenzyl chloride, 2,3-dimethoxybenzyl chloride, 2,5-dimethox-
ybenzyl chloride, 3,4-dimethoxybenzyl chloride, 3,5-dimethox-
ybenzyl chloride, 3,4,5-trimethoxybenzyl chloride, 3-nitro-4-
methoxybenzyl chloride) in the presence of sodium hydride
gave derivatives 12a−k,m−q,s in 62−84% yields. Instead, the 4-
methoxyphenyl derivative 12m was obtained in 60% yield by a
modified Ulmann cross-coupling reaction using 4-methoxy
iodoanisole as an alkylating agent in the presence of
copper(I)bromide and potassium carbonate. Moreover, the 3-
nitro 4-methoxybenzyl substituted derivatives 12k and 12q
were subjected to catalytic reduction with 10% Pd on charcoal,
furnishing the corresponding amino derivatives 12l (98%) and
12r (95%). Hydrolysis in basic media (10% K2CO3) of 12s
furnished the 4-hydroxyphenyl derivative 12t (96%), which was
subsequently methylated with iodomethane to give 12u in good
yield (88%).
The synthesis of [1,2]oxazolo[5,4-e]isoindoles 15a−v has
been accomplished as outlined in the Scheme 2. The N-
substituted tetrahydroisoindoles 12a−s,u were properly
functionalized in α position to the carbonyl (Table 1). In
particular, tetrahydroisondoles 12a−j,m−p,s,u were subjected
to formylation, using ethyl formate as the formylating agent in
the presence of potassium t-buthoxide, to furnish the
corresponding hydroxymethyl derivatives 13a−j,m−p,s,u
(60−97%). Compound 13t was obtained as a byproduct
from the formylation of 12s, together with the expected formyl
derivative 13s.
Table 1. N-Substituted Tetrahydro-4H-isoindol-4-ones 12a−
u
a
R
R1
R2
yields (%)
12a
12b
12c
12d
12e
12f
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
H
H
H
H
H
H
90
90
85
71
64
62
93
62
76
84
83
98
60
76
88
88
74
95
64
96
88
Bn
H
4-OMeBn
H
3-OMeBn
H
2-OMeBn
H
2,3-(OMe)2Bn
2,5-(OMe)2Bn
3,4-(OMe)2Bn
3,5-(OMe)2Bn
3,4,5-(OMe)3Bn
3-NO2,4-OMeBn
3-NH2,4-OMeBn
4-OMePh
H
12g
12h
12i
H
H
H
12j
H
12k
12l
H
H
H
12m
12n
12o
12p
12q
12r
12s
12t
Bn
H
4-OMeBn
H
4-OMeBn
3,4,5-(OMe)3
3,4,5-(OMe)3
3,4,5-(OMe)3
4-OAc
3-NO2,4-OMeBn
3-NH2,4-OMeBn
4-OMeBn
4-OMeBn
4-OH
12u
4-OMeBn
4-OMe
a
Figures represent the yield obtained at the final reaction step.
By this versatile route, 21 derivatives of the [1,2]oxazolo[5,4-
e]isoindole system 15a−u were obtained (57−84%, Table 2).
Additionally, the 7-(4-methoxybenzyl)-6-phenyl-5,7-dihydro-
4H-[1,2]oxazolo[5,4-e]isoindole 15c was subjected to deme-
thylation of the methoxy functionality with BBr3, furnishing the
4-hydroxy analogue 15v (84%).
Table 2. 5,7-Dihydro-4H-[1,2]oxazole[5,4-e]isoindoles 15a−
v
a
[1,2]
oxazoles
yields
(%)
Sbt
R
R1
R2
15a
15b
15c
15d
15e
15f
13a
13b
13c
13d
13e
13f
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
H
H
H
H
H
H
H
62
60
67
78
80
70
60
83
69
76
62
60
76
84
75
60
62
58
69
57
75
84
Bn
H
4-OMeBn
H
3-OMeBn
H
2-OMeBn
H
2,3-(OMe)2Bn
2,5-(OMe)2Bn
3,4-(OMe)2Bn
3,5-(OMe)2Bn
3,4,5-(OMe)3Bn
3-NO2,4-OMeBn
3-NH2,4-OMeBn
4-OMePh
H
15g
15h
15i
13g
13h
13i
H
H
H
15j
13j
H
15k
15l
14k
14l
H
H
15m
15n
15o
15p
15q
15r
15s
15t
13m
13n
13o
13p
14q
14r
13s
13t
H
H
Compounds 13m,s,t were unstable and were used as crude
products for the next step.
Bn
4-OMeBn
H
Only in the case of the 3-nitro 4-methoxybenzyl substituted
derivatives 12k,q and the 3-amino 4-methoxybenzyl substituted
derivatives 12l,r, the Bredereck reagent was used to convert
them into the α-enaminoketons 14k,l,q,r, which were not
isolated as pure compounds, and thus used as crude products
directly in the next step. Annelation of the [1,2]oxazole ring
was achieved reacting the key intermediates 13 and 14 with
hydroxylamine hydrochloride as 1,3 dinucleophile in refluxing
ethanol.
4-OMeBn
3,4,5-(OMe)3
3,4,5-(OMe)3
3,4,5-(OMe)3
4-OAc
3-NO2,4-OMeBn
3-NH2,4-OMeBn
4-OMeBn
4-OMeBn
4-OH
15u
15v
13u
15c
4-OMeBn
4-OMe
H
4-OHBn
a
Figures represent the yield obtained at the final reaction step.
D
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Biology. In Vitro Antiproliferative Activity of the Type 15
Compounds. All the synthesized compounds of type 15 were
submitted to the NCI and prescreened at a 10−5 M dose in the
full panel of 60 human cell lines of different tumor types47−49
(data not shown). Compounds 15b,c,e,g,h,l,o−r,t,u were
selected for further screenings at five concentration levels
(10−4−10−8 M), and their antiproliferative activity was defined
in terms of GI50 value (i.e., the molar concentration that
inhibits 50% net cell growth). Data analysis showed that almost
all compounds exhibited growth inhibitory effect against all
tested cell lines, with GI50 values in the micromolar to
nanomolar range. In particular, the 4-methoxybenzyl sub-
stitution at the benzyl bound to pyrrole nitrogen seems to be
crucial for the activity. In fact, derivatives 15c,l,p−r,t,u, showed
remarkable antiproliferative activity at submicromolar to
nanomolar concentrations. The most potent derivative was
15r, bearing a 3,4,5-trimethoxyphenyl substitution, for which
nanomolar GI50 values were observed in 46 out of 56 cell lines
(see Supporting Information, Tables S1 and S2 and Figures
S1−S10).
Western blot results showed that treatment with 15r derivative
markedly inhibited tubulin polymerization in STO and MP4
cells, as evidenced by the reduction in the polymerized
compared to soluble fraction of tubulin (Figure 2A). The
On the basis of the remarkable cytotoxic activity observed
with the 15r derivative in the NCI panel of human tumor cell
lines, including those from chemoresitant histotypes (i.e.,
melanoma, nonsmall cell lung cancer), the study was extended
to three additional cell lines, STO, MP4, and MP8 which were
established from clinical specimens of DMPM, a tumor type
highly resistant to conventional treatments. The antiprolifer-
ative activity of 15r was assessed by MTS assay following 72 h
exposure to increasing concentrations of the compound. A
dose- and time-dependent cell growth inhibition was
consistently observed in all cell models (Figure 1). STO cells
Figure 2. 15r derivative interferes with tubulin polymerization. (A)
Representative Western blot showing the soluble (S) or polymerized
(P) tubulin fraction in STO and MP4 cells after 24 h of exposure to
15r at the concentrations corresponding to the IC50 at 72 h.
Vinorelbine (Vin; 2 and 10 nM for STO and MP4, respectively) and
taxol (Tx; 1 and 20 nM for STO and MP4, respectively) were selected
as reference drugs due to their opposite mechanism of action on
tubulin polymerization. (B) In vitro assessment of tubulin polymer-
ization in the absence (unt) or presence of 15r, vinorelbine (Vin) and
taxol (Tx), used at 10 μM. A representative experiment of two is
shown.
direct interference of 15r with tubulin polymerization was
further demonstrated by monitoring in vitro changes in the
GTP-induced assembly of purified tubulin monomers. Specif-
ically, results indicated that tubulin polymerization was
markedly inhibited in samples treated with 15r with respect
to untreated controls (Figure 2B). Our findings also suggested
that the compound acts in a vinca alkaloid-like manner because
its effect on tubulin polymerization was superimposable to that
observed following exposure to vinorelbine but opposite to that
induced by taxol (Figure 2).
Figure 1. 15r derivative impairs DMPM cell growth. DMPM cells
were cultured for 72 h in the presence of increasing concentrations of
the compound and the cytotoxic activity was assessed by MTS assay.
Data are expressed as percentage values with respect to DMSO-treated
cells and represent the mean values SD (standard deviation) of three
independent experiments.
15r Derivative Promotes Cell-Cycle Arrest and Induces
Apoptosis. The biological effects consequent to 15r-induced
tubulin polymerization impairment and responsible for the
observed antiproliferative activity of the compound were
further investigated. Exposure to 15r induced a marked
perturbation in the distribution of the cells in the different
cell cycle phases (Figure 3A), as assessed by flow cytometry.
Specifically, drug treatment (IC50 and IC80) of asynchronously
growing STO and MP4 cells resulted in a time-dependent
accumulation of cells in the G2/M phase, with a concomitant
reduction in the percentage of cells in G1 and S compartments
(Figure 3A). The data are comparable to those obtained in the
same cell lines following exposure to drug interfering with
were highly responsive to the compound, as indicated by the
concentration required to inhibit growth by 50% (IC50) of 0.07
0.02 μM, whereas a slightly less pronounced and comparable
sensitivity to 15r was displayed by MP4 and MP8, with IC50
values of 0.16
0.05 and 0.13
0.04 μM, respectively.
Interestingly, 15r did not alter the growth of WI38 normal
human lung fibroblasts (IC50 > 100 μM).
Effects of 15r Derivative on Tubulin Polymerization. On
the basis of the evidence that several derivatives structurally
related to our [1,2]oxazolo[5,4-e]isoindole were previously
reported to interfere with tubulin polymerization,19,20 we
investigated whether 15r was able to affect tubulin dynamics.
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Figure 3. 15r derivative causes mitotic arrest in DMPM cells. (A) DMPM cells were exposed to 1% (v/v) DMSO (control cells; Ctr), derivative 15r
(IC50 and IC80), or taxol (Tx) and vinorelbine (Vin) (IC50) for 48 and 72 h, and the effect on the cell cycle distribution was assessed as described in
the Experimental Section. Data are reported as the percentage of cells in G1, S, and G2/M phases and represent the mean values of three
independent experiments; SDs were always within 5%. (B) Percentage of mitotic cells upon the exposure of DMPM cells to equitoxic concentrations
(IC50) of derivative 15r, vinorelbine (Vin; 2 and 13 nM for STO and MP8, respectively), and taxol (Tx; 1 and 18 nM for STO and MP8,
respectively). Data are expressed as mean values SD of three independent experiments.
tubulin dynamics, such as taxol and vinorelbine (Figure 3A). In
addition, fluorescence microscopy analysis of DMPM cells
stained with the Mitotic Protein Monoclonal no. 2 (MPM-2)
antibody, which recognizes the phosphorylated epitope in
specific phosphoproteins which are phosphorylated in the onset
of mitosis, showed the presence of 30−50% mitotic cells within
the overall cell population following 72 h exposure of STO and
MP8 cells to equitoxic (IC50) concentrations of 15r,
vinorelbine, and taxol (Figure 3B). Treatment of DMPM
cells with 15r also resulted in a marked and time-dependent
apoptotic response, as indicated by a significant increase in
caspase-3 catalytic activity, determined in vitro by the
hydrolysis of the specific fluorogenic substrate. Specifically, in
STO cells exposed for 72 h to derivative 15r (IC80), caspase-3
catalytic activity was 38.5-fold higher than that observed in
control samples (Figure 4). Consistently, a 35-fold increase in
the enzyme activity was observed in MP4 cells under the same
experimental conditions (Figure 4).
Antitumor Activity of 15r Derivative in STO Xenograft
Model. In the last step of the study, the in vivo antitumor
activity of 15r was evaluated in STO cells following
subcutaneous xenotransplantation into athymic nude mice. As
shown in Figure 5A, intraperitoneal administration of the
compound resulted in a statistical significant (P < 0.05) tumor
growth delay compared to control mice, with a maximum
tumor volume inhibition of 59%. Consistently, Ki67
immunohistochemical staining of tumor xenografts after the
last treatment showed a marked 15r-induced decline of cell
proliferation (Figure 5B). The compound was well tolerated
with minimal weight loss and no toxic deaths.
CONCLUSION
■
We have reported a versatile method for the synthesis of 5,7-
dihydro-4H-[1,2]oxazole[5,4-e]isoindoles involving the annela-
tion of the [1,2]oxazole moiety to the isoindole ring. Several
derivatives with a wide substitution pattern were efficiently
obtained and evaluated for their anticancer activity. The
structure−activity relationship indicated that the N-4-methox-
ybenzyl group appears crucial for the antiproliferative activity of
the compounds, with the most potent derivative being 15r,
bearing a 3,4,5-trimetoxy substituent. Indeed, in vitro studies
indicated that this compound induced significant antiprolifer-
ative effects in a large panel of human cancer cell lines and did
not appreciably interfere with the growth of human normal
fibroblasts, suggesting a preferential activity against malignant
cells. It is worth noting that the compound markedly impaired
the growth of three experimental models of DMPM, a rapidly
lethal malignancy, poorly responsive to chemotherapy.
The antiproliferative activity of 15r was found to rely on the
ability to impair microtubule assembly during mitosis, in a vinca
alkaloid-like manner, with a consequent cell cycle arrest at the
G2/M compartment and the induction of caspase-dependent
apoptosis. Most importantly, our results documented a
significant in vivo antitumor activity of 15r derivative at well-
F
DOI: 10.1021/acs.jmedchem.6b00777
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Journal of Medicinal Chemistry
Article
in DMSO-d6 or CDCl3 solution using a Bruker Avance II series 200
MHz spectrometer. Column chromatography was performed with
Merck silica gel (230−400 mesh ASTM) or a Buchi Sepacor
̈
chromatography module (prepacked cartridge system). Elemental
analyses (C, H, N) were within 0.4% of theoretical values and were
performed with a VARIO EL III elemental analyzer. The purity of all
the tested compounds was >95%, determined by HPLC (Agilent 1100
series). In particular, (NH4)2HPO4 0.4 M/acetonitrile 68:32 v/v was
used as eluent, with a flow rate of 1 mL/min, filtered on a cellulose
regenerated filter, cut off 0.2 μm, and samples injected in a C18
Gemini HPLC column. The purity was calculated by using a
calibration curve obtained for serially diluted concentrations of
compounds (Kmax = 254 nm) in the eluent and expressed as the
amount of intact molecules per unit mass.
Compounds 8a, 9a, 10a, 11a, 12a−c, and 13a−c were conveniently
prepared by a method previously reported by us.35,40
Synthesis of 2-[(Dimethylamino)methylidene]ciclohexane-1,3-
dione (8b). A solution of 7b (71 mmol) in N,N-dimethylformamide
dimethyl acetal (20 mL) was heated under reflux up to completeness
(30 min, TLC). After cooling, the solvent was evaporated at reduced
pressure. The solid obtained was used in the next step without further
purification. Brown solid; yield 98%; mp 86−87 °C. IR (cm−1) 1718
(CO), 1662 (CO). 1H NMR (200 MHz, DMSO-d6) δ 0.96 (s, 6H, 2 ×
CH3), 2.22 (s, 4H, 2 × CH2), 3.04 (s, 3H, CH3), 3.38 (s, 3H, CH3),
7.95 (s, 1H, CH). 13C NMR (50 MHz, DMSO-d6) δ 28.1 (2 × q),
44.2 (q), 47.7 (q), 51.7 (2t), 99.8 (s), 107.1 (s), 193.3 (s), 193.4 (s).
Anal. Calcd for C11H17NO2: C, 67.66; H, 8.78; N, 7.17. Found: C,
67.79; H, 8.45; N, 7.45.
General Procedure for the Synthesis of {[(2,6-
Dioxocyclohexylidene)methyl]amino}arylacetic Acid (9b−d). To a
solution of 8a,b (18 mmol) in ethanol (42 mL), a solution of the
suitable phenylglycine (21.6 mmol) and sodium acetate trihydrate
(2.94 g) in ethanol was added and the reaction mixture was heated
under reflux up to completeness (TLC). After cooling, the reaction
mixture was filtered and the filtrate was evaporated at reduced
pressure. To the residue, ice and water were added and the resulting
solution was acidified with HCl 6 M. The solid obtained was filtered
and dried.
Figure 4. 15r derivative induces apoptosis in DMPM cells. The
catalytic activity of caspase-3 was assessed by the in vitro hydrolysis of
the fluorogenic substrates (DEVD-pNA) after exposure of STO and
MP4 cells to 1% (v/v) DMSO (control cells; Ctr) or to 15r derivative.
Data are expressed as mean values
SD of three independent
experiments. ***P < 0.001, ** P < 0.01, * P < 0.05.
Data for {[(4,4-Dimethyl-2,6-dioxocyclohexylidene)methyl]-
amino}(phenyl)acetic Acid (9b). This compound was obtained from
reaction of 8b with phenylglycine after 1 h and 30 min. Pale-yellow
solid; yield 95%; mp 186−187 °C. IR (cm−1) 3408 (NH), 2954 (OH),
1720 (CO), 1651 (CO), 1585 (CO) cm−1. 1H NMR (200 MHz,
DMSO-d6) δ 0.95 (s, 6H, 2 × CH3), 2.23 (s, 2H, CH2), 2.33 (s, 2H,
CH2), 3.67 (s, 1H, OH), 5.70 (d, 1H, J = 7.3 Hz, CH), 7.36−7.50 (m,
5H, Ar), 8.07 (d, 1H, J = 14.1 Hz, CH), 11.60−11.70 (m, 1H, NH).
13C NMR (50 MHz, DMSO-d6) δ 27.8 (q), 28.2 (q), 50.5 (t), 50.8 (t),
63.4 (d), 107.5 (s), 127.3 (2 × d), 128.8 (d), 129.2 (2 × d), 136.8 (s),
156.6 (d), 170.6 (s), 194.5 (s), 194.6 (s), 198.3 (s). Anal. Calcd for
C17H19NO4: C, 67.76; H, 6.36; N, 4.65. Found: C, 67.57; H, 6.71; N,
4.36.
Figure 5. Efficacy of 15r derivative against DMPM xenografts. (A)
Tumor growth curves of STO cells subcutaneously injected into right
flank of nude mice. Mice (eight mice/group) were randomly grouped
to receive vehicle (Control) or 15r (5 mg/kg, q7dx4). The treatment
started 10 days after cell inoculum. Data are expressed as mean values
SE. (B) Representative Ki67 staining performed in FFPE sections of
tumors from the experimental groups reported in A. Images for one
representative mouse per group are shown. Original magnification:
×20.
Data for {[(2,6-Dioxocyclohexylidene)methyl]amino}(3,4,5-
trimethoxyphenyl)acetic Acid (9c). This compound was obtained
from reaction of 8a with 3,4,5-trimethoxyphenylglycine after 2 h. Pale-
yellow solid; yield 95%; mp 183−184 °C. IR (cm−1) 3182 (NH), 2939
1
(OH), 1714 (CO), 1697 (CO), 1651 (CO). H NMR (200 MHz,
DMSO-d6) δ 1.72−1.89 (m, 2H, CH2), 2.32−2.40 (m, 4H, 2 × CH2),
3.52 (s, 1H, OH), 3.67 (s, 3H, CH3), 3.78 (s, 6H, 2 × CH3), 5.55−
5.58 (m, 1H, CH), 6.68 (s, 2H, H-2′, H-6′), 8.09 (d, 1H, J = 13.8 Hz,
CH), 11.59−11.69 (m, 1H, NH). 13C NMR (50 MHz, DMSO-d6) δ
19.38 (t), 37.0 (t), 37.3 (t), 55.9 (2 × q), 60.0 (q), 63.6 (d), 104.7 (2
× d), 108.6 (s), 132.3 (s), 137.7 (s), 153.3 (2 × s), 156.9 (d), 170.6
(s), 195.2 (s), 198.9 (s). Anal. Calcd for C18H21NO7: C, 59.50; H,
5.83; N, 3.85. Found: C, 59.35; H, 5.99; N, 4.01.
tolerated doses in a DMPM xenograft model, suggesting a
possible therapeutic potential of the compound for the
management of the disease.
Data for {[(2,6-Dioxocyclohexylidene)methyl]amino}(4-
hydroxyphenyl)acetic Acid (9d). This compound was obtained from
reaction of 8a with 4-hydroxyphenylglycine after 1 h. Light-yellow
solid; yield 90%; mp 210−211 °C. IR (cm−1) 3252 (NH), 2943 (OH),
EXPERIMENTAL SECTION
Chemistry. General Methods. All melting points were taken on a
Buchi melting point M-560 apparatus. IR spectra were determined in
■
̈
1
1
bromoform with Shimadzu FT/IR 8400S spectrophotometer. H and
2883 (OH), 1730 (CO), 1664 (CO), 1606 (CO). H NMR (200
13C NMR spectra were measured at 200 and 50.0 MHz, respectively,
MHz, DMSO-d6) δ 1.76−1.88 (m, 2H, CH2), 2.30 (t, 2H, J = 6.2 Hz,
G
DOI: 10.1021/acs.jmedchem.6b00777
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Journal of Medicinal Chemistry
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1
CH2), 2.39 (t, 2H, J = 6.2 Hz, CH2), 3.49 (s, 1H, OH), 5.54 (d, 1H, J
= 6.9 Hz, CH), 6.81 (d, 2H, J = 8.5 Hz, H-3′, H-5′), 7.16 (d, 2H, J =
8.5 Hz, H-2′, H-6′), 8.04 (d, 1H, J = 14.2 Hz, CH), 9.70 (s, 1H, OH),
11.58−11.68 (m, 1H, NH). 13C NMR (50 MHz, DMSO-d6) δ 19.3
(t), 37.0 (t), 37.3 (t), 62.9 (d), 108.5 (s), 115.9 (2 × d), 126.9 (s),
128.7 (2 × d), 156.8 (d), 157.8 (s), 171.0 (s), 195.1 (s), 198.8 (s).
Anal. Calcd for C15H15NO5: C, 62.28; H, 5.23; N, 4.84. Found: C,
62.01; H, 5.42; N, 4.28.
3251 (NH), 1658 (CO). H NMR (200 MHz, DMSO-d6) δ 1.92−
2.04 (m, 2H, CH2), 2.38 (t, 2H, J = 6.3 Hz, CH2), 2.87 (t, 2H, J = 6.3
Hz, CH2), 3.68 (s, 3H, CH3), 3.83 (s, 6H, 2 × CH3), 6.79 (s, 2H, H-
2′, H-6′), 7.46 (s, 1H, H-3), 11.88 (s, 1H, NH). 13C NMR (50 MHz,
DMSO-d6) δ 22.4 (t), 24.7 (t), 38.8 (t), 55.8 (2 × q), 60.0 (q), 103.0
(2 × d), 119.8 (d), 122.3 (s), 122.5 (s), 126.7 (s), 127.9 (s), 135.8 (s),
153.0 (2 × s), 194.2 (s). Anal. Calcd for C17H19NO4: C, 67.76; H,
6.36; N, 4.65. Found: C, 67.64; H, 6.22; N, 4.76.
General Procedure for the Synthesis of 2-Acetyl-1-phenyl-6,7-
tetrahydro-2H-isoindol-4-yl Acetate (10b−d). To a solution of 9b−d
(10 mmol) in acetic anhydride (30 mL), triethylamine was added
(7.17 mmol, 10 mL). The reaction mixture was heated under reflux up
to completeness (TLC). After cooling, the reaction mixture was
poured into water and ice. It formed a rubbery solid which was
decanted and then stirred with a saturated solution of Na2CO3 (50
mL). The solid obtained was filtered and dried.
Data for 2-Acetyl-6,6-dimethyl-1-phenyl-6,7-dihydro-2H-isoin-
dol-4-yl Acetate (10b). This compound was obtained from reaction
of 9b after 15 min. Yellow oil; yield 90%. IR (cm−1) 1747 (CO), 1722
(CO). 1H NMR (200 MHz, DMSO-d6) δ 1.03 (s, 6H, 2 × CH3), 2.24
(s, 3H, CH3), 2.31 (s, 2H, CH2), 2.39 (s, 3H, CH3), 5.33 (s, 1H, CH),
7.25−7.42 (m, 6H, H-3, Ar). 13C NMR (50 MHz, DMSO-d6) δ 20.7
(q), 24.2 (2 × q), 28.6 (q), 33.9 (t), 114.4 (d), 118.4 (s), 122.9 (s),
123.0 (s), 124.6 (d), 127.1 (d), 127.7 (2 × d), 129.4 (2 × d), 129.7
(s), 132.6 (s), 140.2 (s), 168.5 (s), 168.6 (s). Anal. Calcd for
C20H21NO3: C, 74.28; H, 6.55; N, 4.33. Found: C, 74.02; H, 6.79; N,
4.07.
General Procedure for the Synthesis of 1-Substituted-2,5,6,7-
tetrahydro-4H-isoindol-4-one (11d−g). To a solution of 10d (10
mmol) in AcOH (80%, 62 mL), HCl (37%, 5 mL) was added
dropwise. The reaction mixture was stirred at room temperature for 55
min. The reaction mixture was poured into water and ice. The solid
obtained was filtered and dried.
Data for 1-(4-Hydroxyphenyl)-2,5,6,7-tetrahydro-4H-isoindol-4-
one (11d). Pale-yellow solid; yield 9%; mp 243−244 °C. IR (cm−1)
1
3356 (NH), 2951 (OH), 1643 (CO). H NMR (200 MHz, DMSO-
d6) δ 1.89−2.02 (m, 2H, CH2), 2.36 (t, 2H, J = 6.1 Hz, CH2), 2.76 (t,
2H, J = 6.1 Hz, CH2), 6.84 (d, 2H, J = 8.6 Hz, H-3′, H-5′), 7.30−7.34
(m, 3H, H-2′, H-6′, H-3), 9.49 (s, 1H, OH), 11.70 (s, 1H, NH). 13C
NMR (50 MHz, DMSO-d6) δ 22.8 (t), 25.3 (t), 39.3 (t), 116.0 (2 ×
d), 119.5 (d), 121.3 (s), 122.8 (s), 124.0 (s), 127.4 (s), 127.5 (2 × d),
156.4 (s), 194.7 (s). Anal. Calcd for C14H13NO2: C, 73.99; H, 5.77; N,
6.16. Found: C, 74.07; H, 5.99; N, 5.92.
Data for 4-(4-Oxo-4,5,6,7-tetrahydro-2H-isoindol-1-yl)phenyl
Acetate (11e). Pale-brown solid; yield 21%, mp 217−218 °C. IR
1
(cm−1) 3289 (NH), 1736 (CO), 1655 (CO). H NMR (200 MHz,
Data for 2-Acetyl-1-(3,4,5-trimethoxyphenyl)-6,7-dihydro-2H-iso-
indol-4-yl Acetate (10c). This compound was obtained from reaction
of 9c after 30 min. Brown solid; yield 75%; mp 147−148 °C. IR
DMSO-d6) δ 1.91−2.03 (m, 2H, CH2), 2.28 (s, 3H, CH3), 2.38 (t, 2H,
J = 6.1 Hz, CH2), 2.82 (t, 2H, J = 6.1 Hz, CH2), 7.20 (d, 2H, J = 8.7
Hz, H-3′, H-5′), 7.44 (s, 1H, H-3), 7.54 (d, 2H, J = 8.7 Hz, H-2′, H-
6′), 11.92 (s, 1H, NH). 13C NMR (50 MHz, DMSO-d6) δ 20.8 (q),
22.3 (t), 24.7 (t), 39.3 (t), 120.1 (d), 122.1 (2 × d), 122.6 (s), 122.7
(s), 125.9 (s), 126.5 (2 × d), 130.0 (s), 148.6 (s), 169.3 (s), 194.1 (s).
Anal. Calcd for C16H15NO3: C, 71.36; H, 5.61; N, 5.20. Found: C,
71.22; H, 5.98; N, 5.01.
1
(cm−1) 1708 (CO), 1658 (CO). H NMR (200 MHz, DMSO-d6) δ
2.25 (s, 3H, CH3), 2.35 (s, 3H, CH3), 2.38−2.47 (m, 4H, 2 × CH2),
3.69 (s, 3H, CH3), 3.76 (s, 6H, 2 × CH3), 5.50 (t, 1H, J = 4.1 Hz,
CH), 6.61 (s, 2H, H-2′, H-6′), 7.31 (s, 1H, H-3). 13C NMR (50 MHz,
DMSO-d6) δ 18.5 (t), 20.7 (q), 22.9 (t), 24.3 (q), 55.9 (2 × q), 56.0
(d), 60.0 (q), 107.1 (2 × d), 114.0 (d), 119.0 (s), 123.5 (s), 128.0 (s),
128.9 (s), 136.8 (s), 141.7 (s), 152.4 (2 × s), 168.6 (s), 168.7 (s).
Anal. Calcd for C21H23NO6: C, 65.44; H, 6.02; N, 3.63. Found: C,
65.32; H, 6.13; N, 3.77.
Data for 4-[2-Acetyl-4-(acetyloxy)-6,7-dihydro-2H-isoindol-1-yl]-
phenyl Acetate (10d). This compound was obtained from reaction of
9d after 20 min. Brown solid; yield 81%; mp 86−87 °C. IR (cm−1)
1749 (CO), 1712 (CO), 1662 (CO). 1H NMR (200 MHz, DMSO-d6)
δ 2.25 (s, 3H, CH3), 2.28 (s, 3H, CH3), 2.34−2.47 (m, 7H, CH3, 2 ×
CH2), 5.51 (t, 1H, J = 4.3 Hz, CH), 7.12 (d, 2H, J = 8.6 Hz, H-3′, H-
5′), 7.10−7.35 (m, 3H, H-2′, H-6′, H-3). 13C NMR (50 MHz, DMSO-
d6) δ 18.4 (t), 20.7 (q), 20.9 (q), 22.9 (t), 24.1 (q), 114.0 (d), 114.7
(d), 119.2 (s), 121.0 (2 × d), 123.8 (s), 128.1 (s), 130.1 (s), 130.4 (2
× d), 141.6 (s), 149.4 (s), 168.6 (s), 168.7 (s), 169.1 (s). Anal. Calcd
for C20H19NO5: C, 67.98; H, 5.42; N, 3.96. Found: C, 68.15; H, 5.64;
N, 3.62.
Data for 2-Acetyl-1-(4-hydroxyphenyl)-2,5,6,7-tetrahydro-4H-iso-
indol-4-one (11f). Light-brown solid; yield 5%; mp 184−185 °C. IR
1
(cm−1) 2950 (OH), 1718 (CO), 1645 (CO). H NMR (200 MHz,
DMSO-d6) δ 1.84−1.96 (m, 2H, CH2), 2.41−2.52 (m, 7H, 2 × CH2,
CH3), 6.77 (d, 2H, J = 8.5 Hz, H-3′, H-5′), 7.10 (d, 2H, J = 8.5 Hz, H-
2′, H-6′), 7.95 (s, 1H, H-3), 9.59 (s, 1H, OH). 13C NMR (50 MHz,
DMSO-d6) δ 20.8 (t), 22.3 (t), 24.7 (q), 38.7 (t), 115.2 (2 × d), 122.3
(d), 122.2 (s), 122.7 (s), 125.9 (s), 130.0 (s), 131.2 (2 × d), 148.6 (s),
169.3 (s), 194.2 (s). Anal. Calcd for C16H15NO3: C, 71.36; H, 5.61; N,
5.20. Found: C, 71.44; H, 5.51; N, 5.31.
Data for 4-(2-Acetyl-4-oxo-4,5,6,7-tetrahydro-2H-isoindol-1-yl)-
phenyl Acetate (11g). Pale-yellow solid; yield 51%; mp 160−161 °C.
IR (cm−1) 1734 (CO), 1674 (CO), 1576 (CO). 1H NMR (200 MHz,
DMSO-d6) δ 1.85−1.97 (m, 2H, CH2), 2.29 (s, 3H, CH3), 2.43−2.55
(m, 4H, 2 × CH2), 2.60 (s, 3H, CH3), 7.14 (d, 2H, J = 8.6 Hz, H-3′,
H-5′), 7.34 (d, 2H, J = 8.6 Hz, H-2′, H-6′), 8.05 (s, 1H, H-3). 13C
NMR (50 MHz, DMSO-d6) δ 20.9 (q), 21.0 (t), 23.7 (t), 24.1 (q),
39.2 (t), 121.1 (2 × d), 122.9 (d), 123.0 (s), 127.8 (s), 127.9 (s), 129.6
(s), 130.3 (2 × d), 149.5 (s), 169.1 (s), 169.8 (s), 194.9 (s). Anal.
Calcd for C18H17NO4: C, 69.44; H, 5.50; N, 4.50. Found: C, 69.19; H,
5.87; N, 4.31.
General Procedure for the Synthesis of 2,5,6,7-Tetrahydro-4H-
isoindol-4-one (11e, 12t). To a solution of 11g/12s (1 mmol) in
ethanol (40 mL), an aqueous solution of K2CO3 (10%, 0.7 mL) was
added dropwise. The reaction mixture was stirred at room temperature
for 2 min for 11e and 16 h for 12t. Then to the reaction mixture water
was added and the resulting solution was extracted with ethyl acetate
(3 × 10 mL). The organic phase was dried over Na2SO4 and the
solvent evaporated at reduced pressure. The crude product was
purified by chromatography column using dichloromethane/ethyl
acetate (95/5) as eluent.
General Procedure for the Synthesis of 1-Substituted-2,5,6,7-
tetrahydro-4H-isoindol-4-one (11b,c). To a solution of 10b or 10c
(10 mmol) in AcOH (80%, 62 mL), HCl (37%, 5 mL) was added
dropwise. The reaction mixture was heated at 60 °C up to
completeness (TLC). After cooling, the reaction mixture was poured
into water and ice. The solid obtained was filtered and dried.
Data for 6,6-Dimethyl-1-phenyl-2,5,6,7-tetrahydro-4H-isoindol-
4-one (11b). This compound was obtained from reaction of 10b after
10 min. Brown solid; yield 80%; mp 235−236 °C. IR (cm−1) 3245
1
(NH), 1656 (CO). H NMR (200 MHz, DMSO-d6) δ 1.00 (s, 6H, 2
× CH3), 2.27 (s, 2H, CH2), 2.73 (s, 2H, CH2), 7.25 (t, 1H, J = 7.1 Hz,
Ar), 7.39−7.55 (m, 5H, H-3, Ar), 11.90 (s, 1H, NH). 13C NMR (50
MHz, DMSO-d6) δ 28.2 (2 × q), 36.3 (t), 52.3 (t), 119.9 (d), 121.2
(s), 121.3 (s), 121.6 (s), 125.6 (2 × d), 126.2 (d), 127.2 (s), 128.7 (2
× d), 132.3 (s), 193.7 (s). Anal. Calcd for C16H17NO: C, 80.30; H,
7.16; N, 5.85. Found: C, 80.52; H, 6.95; N, 6.01.
Derivative 4-(4-oxo-4,5,6,7-tetrahydro-2H-isoindol-1-yl)phenyl ac-
etate (11e) was prepared also according to this procedure in 93%
yield.
Data for 1-(3,4,5-Trimethoxyphenyl)-2,5,6,7-tetrahydro-4H-iso-
indol-4-one (11c). This compound was obtained from reaction of 10c
after 15 min. Brown solid; yield 70%; mp 185−186 °C. IR (cm−1)
H
DOI: 10.1021/acs.jmedchem.6b00777
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Data for 1-(4-Hydroxyphenyl)-2-(4-methoxybenzyl)-2,5,6,7-tetra-
hydro-4H-isoindol-4-one (12t). This compound was obtained from
reaction of 12s. Yellow oil; yield 96%. IR (cm−1) 2956 (OH), 1641
(CO). 1H NMR (200 MHz, CDCl3) δ 1.84−1.99 (m, 2H, CH2), 2.34
(t, 2H, J = 6.4 Hz, CH2), 2.53 (t, 2H, J = 6.4 Hz, CH2), 3.69 (s, 3H,
CH3), 5.03 (s, 2H, CH2), 6.79−6.90 (m, 6H, Ar), 7.09 (d, 2H, J = 8.5
Hz, Ar), 7.42 (s, 1H, H-3), 9.67 (s, 1H, OH). 13C NMR (50 MHz,
CDCl3) δ 21.4 (t), 24.7 (t), 39.0 (t), 49.8 (t), 55.0 (q), 113.8 (2 × d),
115.3 (2 × d), 120.5 (s), 121.4 (s), 122.2 (d), 123.7 (s), 128.4 (2 × d),
129.0 (s), 129.6 (s), 131.0 (2 × d), 156.9 (s), 158.5 (s), 193.8 (s).
Anal. Calcd for C22H21NO3: C, 76.06; H, 6.09; N, 4.03. Found: C,
75.94; H, 6.30; N, 4.16.
General Procedure for the Synthesis of 2-Substituted-2,5,6,7-
tetrahydro-4H-isoindol-4-ones (12). To a solution of 11a−c,e (1.35
g, 10 mmol) in anhydrous DMF (15 mL), NaH (0.24 g, 10 mmol) was
added at 0 °C and the reaction was stirred for 1 h at room
temperature. The suitable benzyl chloride (15 mmol) was added at 0
°C, and the reaction mixture was stirred at room temperature up to
completeness (TLC). Then the reaction mixture was poured into ice
and brine. The aqueous solution was extracted with dichloromethane
(3 × 50 mL). The organic phase was dried over Na2SO4 and the
solvent evaporated at reduced pressure. The crude product was
purified by chromatography column using dichloromethane/ethyl
acetate (98/2) as eluent.
Data for 2-(3-Methoxybenzyl)-1-phenyl-2,5,6,7-tetrahydro-4H-
isoindol-4-one (12d). This compound was obtained from reaction
of 11a with 3-methoxybenzyl chloride after 2 h and 30 min. Yellow oil;
yield 71%. IR (cm−1) 1651 (CO). 1H NMR (200 MHz, DMSO-d6) δ
1.84−1.99 (m, 2H, CH2), 2.37 (t, 2H, J = 6.2 Hz, CH2), 2.56 (t, 2H, J
= 6.2 Hz, CH2), 3.63 (s, 3H, CH3), 5.16 (s, 2H, CH2), 6.46−6.51 (m,
2H, Ar), 6.75−6.80 (m, 1H, Ar), 7.12−7.48 (m, 6H, Ar), 7.55 (s, 1H,
H-3). 13C NMR (50 MHz, DMSO-d6) δ 21.4 (t), 24.7 (t), 38.9 (t),
50.5 (t), 54.9 (q), 112.6 (d), 112.8 (d), 119.0 (d), 120.8 (s), 123.3 (d),
124.6 (s), 127.5 (d), 128.6 (2 × d), 128.8 (s), 129.5 (2 × d), 129.6
(d), 131.0 (s), 139.2 (s), 159.2 (s), 193.8 (s). Anal. Calcd for
C22H21NO2: C, 79.73; H, 6.39; N, 4.23. Found: C, 79.57; H, 6.12; N,
4.52.
Data for 2-(2-Methoxybenzyl)-1-phenyl-2,5,6,7-tetrahydro-4H-
isoindol-4-one (12e). This compound was obtained from reaction
of 11a with 2-methoxybenzyl chloride after 1 h and 30 min. Yellow oil;
yield 64%. IR (cm−1) 1651 (CO). 1H NMR (200 MHz, DMSO-d6) δ
1.85−1.99 (m, 2H, CH2), 2.37 (t, 2H, J = 5.8 Hz, CH2), 2.57 (t, 2H, J
= 5.8 Hz, CH2), 3.69 (s, 3H, CH3), 5.11 (s, 2H, CH2), 6.60−6.64 (m,
1H, Ar), 6.81−6.97 (m, 2H, Ar), 7.20−7.46 (m, 7H, H-3, Ar). 13C
NMR (50 MHz, DMSO-d6) δ 21.5 (t), 24.7 (t), 38.9 (t), 45.8 (t), 55.2
(q), 110.7 (d), 120.4 (d), 120.7 (s), 123.1 (d), 124.3 (s), 125.4 (s),
127.5 (d), 127.6 (d), 128.5 (2 × d), 129.0 (d), 129.1 (s), 129.5 (2 ×
d), 130.9 (s), 156.0 (s), 193.8 (s). Anal. Calcd for C22H21NO2: C,
79.73; H, 6.39; N, 4.23. Found: C, 79.51; H, 6.61; N, 4.45.
Data for 2-(2,3-Dimethoxybenzyl)-1-phenyl-2,5,6,7-tetrahydro-
4H-isoindol-4-one (12f). This compound was obtained from reaction
of 11a with 2,3-dimethoxybenzyl chloride after 24 h. Yellow oil; yield
62%. IR (cm−1) 1653 (CO). 1H NMR (200 MHz, DMSO-d6) δ 1.87−
1.99 (m, 2H, CH2), 2.37 (t, 2H, J = 6.0 Hz, CH2), 2.57 (t, 2H, J = 6.0
Hz, CH2), 3.51 (s, 3H, CH3), 3.77 (s, 3H, CH3), 5.15 (s, 2H, CH2),
6.28 (m, 1H, Ar), 6.97 (d, 2H, J = 4.8 Hz, Ar), 7.29−7.47 (m, 6H, H-3,
Ar). 13C NMR (50 MHz, DMSO-d6) δ 21.5 (t), 24.7 (t), 38.9 (t), 45.9
(t), 55.6 (q), 59.6 (q), 112.5 (d), 119.3 (d), 120.7 (s), 123.2 (d), 124.1
(d), 124.4 (s), 127.5 (d), 128.2 (2 × d), 129.0 (s), 129.5 (2 × d),
130.9 (s), 131.0 (s), 145.5 (s), 152.1 (s), 193.8 (s). Anal. Calcd for
C23H23NO3: C, 76.43; H, 6.41; N, 3.88. Found: C, 76.75; H, 6.72; N,
3.52.
(t), 24.7 (t), 38.9 (t), 45.9 (t), 55.2 (q), 55.7 (q), 111.6 (d), 112.7 (d),
114.5 (d), 120.7 (s), 123.1 (d), 124.4 (s), 126.4 (s), 127.5 (d), 128.5
(2 × d), 129.0 (s), 129.5 (2 × d), 131.0 (s), 150.2 (s), 153.0 (s), 193.8
(s). Anal. Calcd for C23H23NO3: C, 76.43; H, 6.41; N, 3.88. Found: C,
76.72; H, 6.75; N, 3.57.
Data for 2-(3,4-Dimethoxybenzyl)-1-phenyl-2,5,6,7-tetrahydro-
4H-isoindol-4-one (12h). This compound was obtained from reaction
of 11a with 3,4-dimethoxybenzyl chloride after 24 h. Yellow oil; yield
62%. IR (cm−1) 1660 (CO). 1H NMR (200 MHz, DMSO-d6) δ 1.85−
1.97 (m, 2H, CH2), 2.35 (t, 2H, J = 6.0 Hz, CH2), 2.54 (t, 2H, J = 6.0
Hz, CH2), 3.57 (s, 3H, CH3), 3.68 (s, 3H, CH3), 5.09 (s, 2H, CH2),
6.46−6.50 (m, 2H, Ar), 6.82 (d, 1H, J = 8.7 Hz, Ar), 7.32−7.51 (m,
5H, Ar), 7.54 (s, 1H, H-3). 13C NMR (50 MHz, DMSO-d6) δ 21.8 (t),
25.2 (t), 39.4 (t), 50.9 (t), 55.7 (q), 55.9 (q), 111.7 (d), 112.1 (d),
120.1 (d), 121.1 (s), 123.6 (d), 124.0 (s), 125.1 (s), 128.0 (d), 129.1
(2 d), 130.2 (2 d), 130.3 (s), 131.7 (s), 148.6 (s), 148.9 (s), 194.3 (s).
Anal. Calcd for C23H23NO3: C, 76.43; H, 6.41; N, 3.88. Found: C,
76.69; H, 6.28; N, 3.39.
Data for 2-(3,5-Dimethoxybenzyl)-1-phenyl-2,5,6,7-tetrahydro-
4H-isoindol-4-one (12i). This compound was obtained from reaction
of 11a with 3,5-dimethoxybenzyl chloride after 2 h. Brown solid; yield
1
76%; mp 110−111 °C. IR (cm−1) 1653 (CO). H NMR (200 MHz,
DMSO-d6) δ 1.85−1.99 (m, 2H, CH2), 2.37 (t, 2H, J = 6.0 Hz, CH2),
2.56 (t, 2H, J = 6.0 Hz, CH2), 3.62 (s, 6H, 2 × CH3), 5.10 (s, 2H,
CH2), 6.06 (d, 2H, J = 2.2 Hz, Ar), 6.34 (t, 1H, J = 2.2 Hz, Ar), 7.29−
7.49 (m, 5H, Ar), 7.55 (s, 1H, H-3). 13C NMR (50 MHz, DMSO-d6) δ
21.4 (t), 24.7 (t), 38.9 (t), 50.5 (t), 55.0 (2 q), 98.9 (d), 105.1 (2 d),
120.8 (s), 123.4 (d), 124.6 (s), 127.5 (d), 128.6 (2 × d), 129.8 (s),
129.6 (2 × d), 131.0 (s), 140.0 (s), 160.5 (2 × s), 193.8 (s). Anal.
Calcd for C23H23NO3: C, 76.43; H, 6.41; N, 3.88. Found: C, 76.79; H,
6.71; N, 3.59.
Data for 1-Phenyl-2-(3,4,5-trimethoxybenzyl)-2,5,6,7-tetrahydro-
4H-isoindol-4-one (12j). This compound was obtained from reaction
of 11a with 3,4,5-trimethoxybenzyl chloride after 16 h. White solid;
1
yield 84%; mp 151−152 °C. IR (cm−1) 1651 (CO). H NMR (200
MHz, DMSO-d6) δ 1.85−1.97 (m, 2H, CH2), 2.35 (t, 2H, J = 6.6 Hz,
CH2), 2.54 (t, 2H, J = 6.6 Hz, CH2), 3.58 (s, 3H, CH3), 3.59 (s, 6H, 2
× CH3), 5.09 (s, 2H, CH2), 6.23 (s, 2H, H-2′, H-6′), 7.37−7.52 (m,
5H, Ar), 7.62 (s, 1H, H-3). 13C NMR (50 MHz, DMSO-d6) δ 21.4 (t),
24.7 (t), 39.9 (t), 50.8 (t), 55.6 (2 × q), 59.9 (q), 105.0 (2 × d), 120.7
(s), 123.2 (d), 124.7 (s), 127.5 (d), 128.6 (s), 128.7 (2 × d), 129.8 (2
× d), 131.3 (s), 132.9 (s), 136.7 (s), 152.7 (2 × s), 193.8 (s). Anal.
Calcd for C24H25NO4: C, 73.64; H, 6.44; N, 3.58. Found: C, 73.77; H,
6.29; N, 3.74.
Data for 2-(4-Methoxy-3-nitrobenzyl)-1-phenyl-2,5,6,7-tetrahy-
dro-4H-isoindol-4-one (12k). This compound was obtained from
reaction of 11a with 3-nitro-4-methoxybenzyl chloride after 1 h and 30
min. Brown solid; yield 83%; mp 196−197 °C. IR (cm−1) 1647 (CO),
1
1529 (NO2). H NMR (200 MHz, DMSO-d6) δ 1.86−1.98 (m, 2H,
CH2), 2.37 (t, 2H, J = 6.6 Hz, CH2), 2.54 (t, 2H, J = 6.6 Hz, CH2),
3.86 (s, 3H, CH3), 5.21 (s, 2H, CH2), 7.11−7.49 (m, 8H, Ar), 7.63 (s,
1H, H-3). 13C NMR (50 MHz, DMSO-d6) δ 21.3 (t), 24.6 (t), 38.9
(t), 49.3 (t), 56.6 (q), 114.5 (d), 120.9 (s), 123.3 (d), 123.8 (d), 124.9
(s), 127.7 (d), 128.5 (2 × d), 128.6 (s), 129.6 (2 × d), 129.8 (s), 130.8
(s), 133.2 (d), 138.6 (s), 151.3 (s), 193.9 (s). Anal. Calcd for
C22H20N2O4: C, 70.20; H, 5.36; N, 7.44. Found: C, 70.09; H, 5.48; N,
7.32.
Data for 2-Benzyl-6,6-dimethyl-1-phenyl-2,5,6,7-tetrahydro-4H-
isoindol-4-one (12n). This compound was obtained from reaction of
11b with benzyl bromide after 24 h. Light-brown solid; yield 76%; mp
122−123 °C. IR (cm−1) 1655 (CO). 1H NMR (200 MHz, DMSO-d6)
δ 0.96 (s, 6H, 2 × CH3), 2.25 (s, 2H, CH2), 2.46 (s, 2H, CH2), 5.21 (s,
2H, CH2), 6.87−6.92 (m, 2H, Ar), 7.15−7.46 (m, 8H, Ar), 7.56 (s,
1H, H-3). 13C NMR (50 MHz, DMSO-d6) δ 28.2 (2 × q), 35.3 (t),
50.7 (t), 52.6 (t), 119.8 (s), 123.2 (d), 123.3 (s), 123.4 (s), 126.7 (2 ×
d), 127.4 (d), 127.5 (d), 128.4 (2 × d), 128.5 (2 × d), 129.5 (2 × d),
129.6 (s), 130.9 (s), 137.7 (s), 193.4 (s). Anal. Calcd for C23H23NO:
C, 83.85; H, 7.04; N, 4.25. Found: C, 84.03; H, 6.84; N, 4.49.
Data for 2-(4-Methoxybenzyl)-6,6-dimethyl-1-phenyl-2,5,6,7-tet-
rahydro-4H-isoindol-4-one (12o). This compound was obtained from
Data for 2-(2,5-Dimethoxybenzyl)-1-phenyl-2,5,6,7-tetraydro-
4H-isoindol-4-one (12g). This compound was obtained from reaction
of 11a with 2,5-dimethoxybenzyl chloride after 2 h. Light-brown solid;
1
yield 93%; mp 125−126 °C. IR (cm−1) 1651 (CO). H NMR (200
MHz, DMSO-d6) δ 1.86−1.99 (m, 2H, CH2), 2.37 (t, 2H, J = 6.0 Hz,
CH2), 2.57 (t, 2H, J = 6.0 Hz, CH2), 3.59 (s, 3H, CH3), 3.63 (s, 3H,
CH3), 5.08 (s, 2H, CH2), 6.19 (s, 1H, Ar), 6.76−6.90 (m, 2H, Ar),
7.30−7.48 (m, 6H, H-3, Ar). 13C NMR (50 MHz, DMSO-d6) δ 21.4
I
DOI: 10.1021/acs.jmedchem.6b00777
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
reaction of 11b with 4-methoxybenzyl chloride after 1 h and 30 min.
J = 6.2 Hz, CH2), 2.69 (t, 2H, J = 6.2 Hz, CH2), 3.75 (s, 3H, CH3),
6.91 (d, 2H, J = 8.9 Hz, H-3′, H-5′), 7.04−7.34 (m, 7H, Ar), 7.53 (s,
1H, H-3). 13C NMR (50 MHz, DMSO-d6) δ 21.8 (t), 24.5 (t), 39.0
(t), 55.3 (q), 114.3 (2 × d), 121.6 (s), 123.8 (d), 125.2 (s), 125.7 (d),
125.8 (2 × d), 128.2 (2 × d), 128.6 (s), 129.2 (2 × d), 130.9 (s), 131.9
(s), 158.3 (s), 194.1 (s). Anal. Calcd for C21H19NO2: C, 79.47; H,
6.03; N, 4.41. Found: C, 79.55; H, 5.88; N, 4.54.
1
White solid; yield 88%; mp 107−108 °C. IR (cm−1) 1655 (CO). H
NMR (200 MHz, DMSO-d6) δ 0.95 (s, 6H, 2 × CH3), 2.24 (s, 2H,
CH2), 2.45 (s, 2H, CH2), 3.68 (s, 3H, CH3), 5.11 (s, 2H, CH2), 6.79
(d, 2H, J = 8.9 Hz, H-3′, H-5′), 6.86 (d, 2H, J = 8.9 Hz, H-2′, H-6′),
7.29−7.49 (m, 5H, Ar), 7.50 (s, 1H, H-3). 13C NMR (50 MHz,
DMSO-d6) δ 28.0 (2 × q), 35.3 (t), 50.1 (t), 52.6 (t), 55.0 (q), 113.9
(2 × d), 119.7 (s), 122.9 (d), 123.3 (s), 123.4 (s), 127.5 (d), 128.3 (2
× d), 128.6 (2 × d), 129.4 (s), 129.5 (s), 129.6 (2 × d), 131.0 (s),
158.5 (s), 193.3 (s). Anal. Calcd for C24H25NO2: C, 80.19; H, 7.01; N,
3.90. Found: C, 80.38; H, 6.82; N, 4.19.
Procedure for the Synthesis of 2-(4-Methoxybenzyl)-1-(4-
methoxyphenyl)-2,5,6,7-tetrahydro-4H-isoindol-4-one (12u). To a
solution of 12t (10 mmol) in THF (15 mL) NaH (0.24 g, 10 mmol)
was added at 0 °C, and the reaction was stirred for 1 h at room
temperature. Iodomethane (0.3 mL, 10 mmol) was added at 0 °C, and
the reaction mixture was stirred at room temperature for 20 h. Then
the reaction mixture was poured into ice and brine. The aqueous
solution was extracted with dichloromethane (3 × 50 mL). The
organic phase was dried over Na2SO4 and the solvent evaporated at
reduced pressure. The crude product was purified by chromatography
column using dichloromethane/ethyl acetate (98/2) as eluent. Yellow
Data for 2-(4-Methoxybenzyl)-1-(3,4,5-trimethoxyphenyl)-
2,5,6,7-tetrahydro-4H-isoindol-4-one (12p). This compound was
obtained from reaction of 11c with 4-methoxybenzyl chloride after 1 h.
1
White solid; yield 88%; mp 160−161 °C. IR (cm−1) 1691 (CO). H
NMR (200 MHz, DMSO-d6) δ 1.87−1.99 (m, 2H, CH2), 2.36 (t, 2H,
J = 6.1 Hz, CH2), 2.59 (t, 2H, J = 6.1 Hz, CH2), 3.68 (s, 6H, 2 ×
CH3), 3.69 (s, 3H, CH3), 3.70 (s, 3H, CH3), 5.12 (s, 2H, CH2), 6.50
(s, 2H, H-2′, H-6′), 6.84 (d, 2H, J = 8.9 Hz, H-3″, H-5″), 6.92 (d, 2H,
J = 8.9 Hz, H-2″, H-6″), 7.49 (s, 1H, H-3). 13C NMR (50 MHz,
DMSO-d6) δ 21.5 (t), 24.7 (t), 38.9 (t), 50.3 (t), 55.1 (q), 55.8 (2 ×
q), 60.0 (q), 107.0 (2 × d), 113.9 (2 × d), 120.5 (s), 122.6 (d), 124.4
(s), 126.4 (s), 128.3 (2 × d), 128.9 (s), 129.8 (s), 136.8 (s), 152.7 (2
× s), 158.5 (s), 193.8 (s). Anal. Calcd for C25H27NO5: C, 71.24; H,
6.46; N, 3.32. Found: C, 71.32; H, 6.34; N, 3.46.
1
oil; yield 88%. IR (cm−1) 1647 (CO). H NMR (200 MHz, DMSO-
d6) δ 1.85−1.99 (m, 2H, CH2), 2.35 (t, 2H, J = 6.4 Hz, CH2), 2.51 (t,
2H, J = 6.4 Hz, CH2), 3.69 (s, 3H, CH3), 3.79 (s, 3H, CH3), 5.05 (s,
2H, CH2), 6.81 (d, 2H, J = 9.0 Hz, Ar), 6.87 (d, 2H, J = 9.0 Hz, Ar),
6.99 (d, 2H, J = 8.7 Hz, Ar), 7.22 (d, 2H, J = 8.7 Hz, Ar), 7.45 (s, 1H,
H-3). 13C NMR (50 MHz, DMSO-d6) δ 21.4 (t), 24.7 (t), 38.9 (t),
49.8 (t), 55.0 (q), 55.1 (q), 113.9 (2 × d), 114.0 (2 × d), 120.6 (s),
122.4 (d), 123.1 (s), 124.1 (s), 128.4 (2 × d), 128.6 (s), 129.5 (s),
131.0 (2 × d), 158.5 (s), 158.6 (s), 193.8 (s). Anal. Calcd for
C23H23NO3: C, 76.43; H, 6.41; N, 3.88. Found: C, 76.34; H, 6.59; N,
3.73.
Procedure for the Synthesis of 2-(3-Amino-4-methoxybenzyl)-1-
substituted-2,5,6,7-tetrahydro-4H-isoindol-4-one (12l,r). To a sol-
ution of 12k,q (1.5 mmol) in ethanol, palladium 10% on carbon was
added and the reaction mixture was stirred under hydrogen
atmosphere for 24 h. The solution was filtered, and the filtrate was
evaporated at reduced pressure. The crude product was purified by
chromatography column using dichloromethane/ethyl acetate (90/10)
as eluent.
Data for 2-(4-Methoxy-3-nitrobenzyl)-1-(3,4,5-trimethoxyphen-
yl)-2,5,6,7-tetrahydro-4H-isoindol-4-one (12q). This compound was
obtained from reaction of 11c with 4-methoxy-3-nitro-benzyl chloride
after 1 h. Brown solid; yield 74%; mp 192−193 °C. IR (cm−1) 1697
(CO), 1531 (NO2). 1H NMR (200 MHz, DMSO-d6) δ 1.86−1.99 (m,
2H, CH2), 2.36 (t, 2H, J = 6.0 Hz, CH2), 2.57 (t, 2H, J = 6.0 Hz,
CH2), 3.69 (s, 3H, CH3), 3.71 (s, 6H, 2 × CH3), 3.86 (s, 3H, CH3),
5.21 (s, 2H, CH2), 6.50 (s, 2H, H-2′, H-6′), 7.27 (s, 2H, Ar), 7.37 (s,
1H, Ar), 7.62 (s, 1H, H-3). 13C NMR (50 MHz, DMSO-d6) δ 21.3 (t),
24.6 (t), 39.0 (t), 49.5 (t), 55.8 (2 × q), 56.7 (q), 60.0 (q), 107.1 (2 ×
d), 114.5 (d), 120.7 (s), 123.0 (d), 123.9 (d), 124.7 (s), 126.2 (s),
128.7 (s), 130.1 (s), 133.4 (d), 137.0 (s), 138.6 (s), 151.3 (s), 152.9 (2
× s), 193.8 (s). Anal. Calcd for C25H26N2O7: C, 64.37; H, 5.62; N,
6.01. Found: C, 64.44; H, 5.79; N, 5.93.
Data for 2-(3-Amino-4-methoxybenzyl)-1-phenyl-2,5,6,7-tetrahy-
dro-4H-isoindol-4-one (12l). This compound was obtained from
reaction of 12k. White solid; yield 98%; mp 168−169 °C. IR (cm−1)
Data for 4-[2-(4-Methoxybenzyl)-4-oxo-4,5,6,7-tetrahydro-2H-
isoindol-1-yl]phenyl Acetate (12s). This compound was obtained
from reaction of 11e with 4-methoxybenzyl chloride after 24 h. Light-
brown solid; yield 64%; mp 84−85 °C. IR (cm−1) 1655 (CO), 1620
1
3458−3375 (NH2), 1649 (CO). H NMR (200 MHz, DMSO-d6) δ
1.87−1.99 (m, 2H, CH2), 2.36 (t, 2H, J = 6.6 Hz, CH2), 2.56 (t, 2H, J
= 6.6 Hz, CH2), 3.70 (s, 3H, CH3), 4.72 (s, 2H, NH2), 4.96 (s, 2H,
CH2), 6.10 (dd, 1H, J = 8.1, 2.1 Hz, Ar), 6.32 (d, 1H, J = 2.1 Hz, Ar),
6.65 (d, 1H, J = 8.1 Hz, Ar), 7.29−7.44 (m, 6H, H-3, Ar). 13C NMR
(50 MHz, DMSO-d6) δ 21.5 (t), 24.7 (t), 38.9 (t), 50.4 (t), 55.2 (q),
110.2 (d), 112.1 (d), 114.8 (d), 120.6 (s), 123.0 (d), 124.3 (s), 127.4
(d), 128.5 (2 × d), 128.9 (s), 129.6 (2 × d), 129.7 (s), 131.0 (s), 137.7
(s), 145.7 (s), 193.7 (s). Anal. Calcd for C22H22N2O2: C, 76.28; H,
6.40; N, 8.09. Found: C, 76.39; H, 6.61; N, 7.92.
1
(CO). H NMR (200 MHz, DMSO-d6) δ 1.85−1.98 (m, 2H, CH2),
2.29 (s, 3H, CH3), 2.36 (t, 2H, J = 6.1 Hz, CH2), 2.55 (t, 2H, J = 6.1
Hz, CH2), 3.68 (s, 3H, CH3), 5.10 (s, 2H, CH2), 6.79 (d, 2H, J = 8.9
Hz, Ar), 6.87 (d, 2H, J = 8.9 Hz, Ar), 7.20 (d, 2H, J = 8.6 Hz, Ar), 7.35
(d, 2H, J = 8.6 Hz, Ar), 7.50 (s, 1H, H-3). 13C NMR (50 MHz,
DMSO-d6) δ 20.9 (q), 21.4 (t), 24.7 (t), 39.3 (t), 50.1 (t), 55.0 (q),
113.9 (2 × d), 120.6 (s), 122.0 (2 × d), 123.1 (d), 124.8 (s), 127.9 (s),
128.5 (2 × d), 129.3 (s), 129.6 (s), 130.7 (2 × d), 149.7 (s), 158.6 (s),
169.1 (s), 193.8 (s). Anal. Calcd for C24H23NO4: C, 74.02; H, 5.95; N,
3.60. Found: C, 74.33; H, 5.61; N, 3.73.
Data for 2-(3-Amino-4-methoxybenzyl)-1-(3,4,5-trimethoxy-
phenyl)-2,5,6,7-tetrahydro-4H-isoindol-4-one (12r). This compound
was obtained from reaction of 12q. White solid; yield 95%; mp 164−
1
165 °C. IR (cm−1) 3452−3373 (NH2), 1650 (CO). H NMR (200
Synthesis of 2-(4-Methoxyphenyl)-1-phenyl-2,5,6,7-tetrahydro-
4H-isoindol-4-one (12m). To a solution of 11a (20 mmol) in N-
methyl-2-pyrrolidone (40 mL), anhydrous K2CO3 (20 mmol) was
added under nitrogen atmosphere and the reaction mixture was stirred
at room temperature for 1 h. Then Cu(I)Br (40 mmol) was added and
the reaction mixture was stirred at room temperature for 1 h, and
finally 4-iodoanisole (70 mmol) was added. The reaction mixture was
heated under reflux for 48 h. After cooling, HCl (5%, 20 mL) was
added and the mixture was stirred for 1 h, then ethyl acetate (20 mL)
was added and the mixture was stirred for further 30 min. Then the
resulting solution was filtered through Celite and washed with ethyl
acetate (20 mL). The organic layer was stirred for 1 h with ice and
brine, separated, dried over Na2SO4, and the solvent evaporated at
reduced pressure. The crude product was purified by chromatography
column using dichloromethane/ethyl acetate (95/5) as eluent. Light-
MHz, DMSO-d6) δ 1.90−1.99 (m, 2H, CH2), 2.35 (t, 2H, J = 5.8 Hz,
CH2), 2.61 (t, 2H, J = 5.8 Hz, CH2), 3.67 (s, 6H, 2 × CH3), 3.68 (s,
3H, CH3), 3.71 (s, 3H, CH3), 4.76 (s, 2H, NH2), 4.97 (s, 2H, CH2),
6.15−6.20 (m, 1H, Ar), 6.36 (s, 1H, Ar), 6.51 (s, 2H, H-2′, H-6′), 6.70
(d, 1H, J = 8.1 Hz, Ar), 7.41 (s, 1H, H-3). 13C NMR (50 MHz,
DMSO-d6) δ 21.5 (t), 24.8 (t), 39.2 (t), 50.7 (t), 55.3 (q), 55.7 (2 ×
q), 60.0 (q), 106.9 (2 × d), 110.9 (d), 111.4 (d), 111.9 (d), 112.1 (s),
114.5 (d), 114.7 (s), 120.6 (s), 124.2 (s), 130.4 (s), 136.9 (s), 137.8
(s), 145.7 (s), 152.7 (2 × s), 194.0 (s). Anal. Calcd for C25H28N2O5:
C, 68.79; H, 6.47; N, 6.42. Found: C, 68.65; H, 6.39; N, 6.60.
Procedure for the Synthesis of 5-(Hydroxymethylidene)-2-
substituted-2,5,6,7-tetrahydro-4H-isoindol-4-ones (13). To a sus-
pension of t-BuOK (3.7 g, 36 mmol) in anhydrous toluene (30 mL), a
solution of the suitable derivative 12 (12 mmol) in anhydrous toluene
(40 mL) was added dropwise under N2 at 0 °C. After 3 h stirring at
room temperature, the reaction was cooled at 0 °C and a solution of
1
brown solid; yield 60%; mp 159−160 °C. IR (cm−1) 1657 (CO). H
NMR (200 MHz, DMSO-d6) δ 1.92−2.05 (m, 2H, CH2), 2.44 (t, 2H,
J
DOI: 10.1021/acs.jmedchem.6b00777
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
ethyl formate (2.91 mL, 36 mmol) in anhydrous toluene (20 mL) was
added and the mixture was kept stirring at room temperature for 24 h,
then the solvent was removed at reduced pressure. The residue was
dissolved in water, and the solution was washed with diethyl ether.
The acqueous solution was then acidified with HCl 6 M and the
precipitate was filtered and dried, in absence the solution was extracted
with dichloromethane (3 × 30 mL). The organic phase was dried over
Na2SO4 and the solvent evaporated at reduced pressure. The crude
product was purified by chromatography column using dichloro-
methane as eluent.
109.5 (s), 110.4 (d), 111.2 (d), 119.8 (d), 120.3 (s), 122.9 (d), 123.4
(s), 127.8 (d), 128.6 (2 × d), 129.2 (s), 129.9 (s), 130.1 (2 × d), 131.1
(s), 148.6 (s), 149.0 (s), 165.8 (d), 186.7 (s). Anal. Calcd for
C24H23NO4: C, 74.02; H, 5.95; N, 3.60. Found: C, 74.38; H, 6.20; N,
3.41.
Data for 2-(3,5-Dimethoxybenzyl)-5-(hydroxymethylidene)-1-
phenyl-2,5,6,7-tetrahydro-4H-isoindol-4-one (13i). This compound
was obtained from reaction of 12i. Yellow oil; yield 60%. IR (cm−1)
2933 (OH), 1633 (CO). 1H NMR (200 MHz, CDCl3) δ 2.49 (t, 2H, J
= 6.1 Hz, CH2), 2.63 (t, 2H, J = 6.1 Hz, CH2), 3.71 (s, 6H, 2 × CH3),
4.97 (s, 2H, CH2), 6.14 (s, 2H, Ar), 6.33−6.35 (m, 1H, Ar), 7.21−7.40
(m, 6H, H-3, Ar), 7.53 (s, 1H, CH), 14.37 (bs, 1H, OH). 13C NMR
(50 MHz, CDCl3) δ 21.1 (t), 25.8 (t), 51.5 (t), 55.3 (2 × q), 99.5 (d),
105.2 (2 × d), 109.5 (s), 113.6 (s), 120.5 (s), 123.2 (d), 123.3 (s),
127.9 (d), 128.6 (2 × d), 130.1 (2 × d), 131.0 (s), 139.4 (s), 161.1 (2
× s), 166.1 (d), 186.6 (s). Anal. Calcd for C24H23NO4: C, 74.02; H,
5.95; N, 3.60. Found: C, 74.33; H, 6.22; N, 3.38.
Data for 5-(Hydroxymethylidene)-2-(3-methoxybenzyl)-1-phe-
nyl-2,5,6,7-tetrahydro-4H-isoindol-4-one (13d). This compound
was obtained from reaction of 12d. Brown solid; yield 72%; mp
1
110−111 °C. IR (cm−1) 2935 (OH), 1633 (CO). H NMR (200
MHz, CDCl3) δ 2.49 (t, 2H, J = 6.3 Hz, CH2), 2.62 (t, 2H, J = 6.3 Hz,
CH2), 3.73 (s, 3H, CH3), 5.02 (s, 2H, CH2), 6.51−6.61 (m, 2H, Ar),
6.76−6.81 (m, 1H, Ar), 7.16−7.42 (m, 7H, H-3, Ar), 7.53 (s, 1H,
CH), 14.39 (bs, 1H, OH). 13C NMR (50 MHz, CDCl3) δ 21.2 (t),
25.8 (t), 51.4 (t), 55.2 (q), 109.5 (s), 112.9 (d), 113.1 (d), 119.3 (d),
120.5 (s), 123.1 (d), 123.3 (s), 127.9 (d), 128.6 (2 × d), 128.7 (s),
129.9 (d), 130.0 (2 × d), 131.0 (s), 138.6 (s), 159.9 (s), 166.1 (d),
186.6 (s). Anal. Calcd for C23H21NO3: C, 76.86; H, 5.89; N, 3.90.
Found: C, 76.72; H, 6.12; N, 4.19.
Data for 5-(Hydroxymethylidene)-1-phenyl-2-(3,4,5-trimethoxy-
benzyl)-2,5,6,7-tetrahydro-4H-isoindol-4-one (13j). This compound
was obtained from reaction of 12j. Light-brown solid; yield 88%; mp
1
146−147 °C. IR (cm−1) 2935 (OH), 1633 (CO). H NMR (200
MHz, CDCl3) δ 2.50 (t, 2H, J = 6.1 Hz, CH2), 2.63 (t, 2H, J = 6.1 Hz,
CH2), 3.73 (s, 6H, 2 × CH3), 3.81 (s, 3H, CH3), 4.98 (s, 2H, CH2),
6.17 (s, 2H, H-2′, H-6′), 7.23−7.47 (m, 6H, CH, Ar), 7.52 (s, 1H, H-
3), 14.38 (d, 1H, J = 5.2 Hz, OH). 13C NMR (50 MHz, CDCl3) δ 21.1
(t), 25.8 (t), 51.9 (t), 56.0 (2 × q), 60.9 (q), 104.4 (2 × d), 109.5 (s),
120.4 (s), 122.9 (d), 123.6 (s), 127.9 (d), 128.6 (2 × d), 129.9 (s),
130.1 (2 × d), 131.1 (s), 132.3 (s), 137.5 (s), 153.4 (2 × s), 165.8 (d),
186.8 (s). Anal. Calcd for C25H25NO5: C, 71.58; H, 6.01; N, 3.34.
Found: C, 71.67; H, 5.89; N, 3.18.
Data for 5-(Hydroxymethylidene)-2-(2-methoxybenzyl)-1-phe-
nyl-2,5,6,7-tetrahydro-4H-isoindol-4-one (13e). This compound
was obtained from reaction of 12e. Yellow oil; yield 71%. IR (cm−1)
2933 (OH), 1637 (CO). 1H NMR (200 MHz, CDCl3) δ 2.49 (t, 2H, J
= 6.7 Hz, CH2), 2.63 (t, 2H, J = 6.7 Hz, CH2), 3.77 (s, 3H, CH3), 5.05
(s, 2H, CH2), 6.73−6.89 (m, 3H, Ar), 7.21−7.45 (m, 7H, H-3, Ar),
7.50 (s, 1H, CH), 14.42 (bs, 1H, OH). 13C NMR (50 MHz, CDCl3) δ
21.2 (t), 25.9 (t), 46.6 (t), 55.3 (q), 109.6 (s), 110.3 (d), 120.2 (s),
120.7 (d), 123.1 (s), 123.3 (d), 125.2 (s), 127.7 (d), 128.5 (2 × d),
128.7 (d), 129.3 (d), 130.0 (2 × d), 130.1 (s), 131.2 (s), 156.7 (s),
165.6 (d), 186.9 (s). Anal. Calcd for C23H21NO3: C, 76.86; H, 5.89; N,
3.90. Found: C, 76.52; H, 6.24; N, 4.15.
Data for 2-Benzyl-5-(hydroxymethylidene)-6,6-dimethyl-1-phe-
nyl-2,5,6,7-tetrahydro-4H-isoindol-4-one (13n). This compound was
obtained from reaction of 12n. Brown solid; yield 72%; mp 117−118
°C. IR (cm−1) 2958 (OH), 1624 (CO). 1H NMR (200 MHz, CDCl3)
δ 1.19 (s, 6H, 2 × CH3), 2.51 (s, 2H, CH2), 5.05 (s, 2H, CH2), 6.96−
7.00 (m, 2H, Ar), 7.18−7.42 (m, 9H, H-3, Ar), 7.62 (s, 1H, CH),
15.19 (bs, 1H, OH). 13C NMR (50 MHz, CDCl3) δ 29.1 (2 × q), 36.1
(t), 51.4 (t), 118.4 (s), 119.5 (s), 122.3 (s), 122.4 (s), 123.2 (d), 126.9
(2 × d), 127.8 (d), 127.9 (d), 128.5 (2 × d), 128.8 (2 × d), 130.0 (2 ×
d), 130.8 (s), 130.9 (s), 137.0 (s), 165.5 (d), 187.1 (s). Anal. Calcd for
C24H23NO2: C, 80.64; H, 6.49; N, 3.92. Found: C, 80.29; H, 6.20; N,
4.14.
Data for 2-(2,3-Dimethoxybenzyl)-5-(hydroxymethylidene)-1-
phenyl-2,5,6,7-tetrahydro-4H-isoindol-4-one (13f). This compound
was obtained from reaction of 12f. Brown solid; yield 70%; mp 115−
1
116 °C. IR (cm−1) 2935 (OH), 1633 (CO). H NMR (200 MHz,
CDCl3) δ 2.49 (t, 2H, J = 6.2 Hz, CH2), 2.64 (t, 2H, J = 6.2 Hz, CH2),
3.62 (s, 3H, CH3), 3.85 (s, 3H, CH3), 5.06 (s, 2H, CH2), 6.47 (d, 1H,
J = 7.5 Hz, Ar), 6.84−7.02 (m, 2H, Ar), 7.27−7.45 (m, 6H, H-3, Ar),
7.51 (s, 1H, CH), 14.39 (bs, 1H, OH). 13C NMR (50 MHz, CDCl3) δ
21.1 (t), 25.8 (t), 46.7 (t), 55.7 (q), 60.2 (q), 109.5 (s), 112.4 (d),
120.2 (s), 120.5 (d), 123.1 (d), 124.3 (d), 127.8 (d), 128.5 (2 × d),
128.6 (s), 130.1 (2 × d), 130.2 (s), 130.7 (s), 131.1 (s), 146.4 (s),
152.6 (s), 165.7 (d), 186.8 (s). Anal. Calcd for C24H23NO4: C, 74.02;
H, 5.95; N, 3.60. Found: C, 74.32; H, 6.12; N, 3.39.
Data for 2-(2,5-Dimethoxybenzyl)-5-(hydroxymethylidene)-1-
phenyl-2,5,6,7-tetrahydro-4H-isoindol-4-one (13g). This compound
was obtained from reaction of 12g. Yellow oil; yield 63%. IR (cm−1)
2929 (OH), 1641 (CO). 1H NMR (200 MHz, CDCl3) δ 2.49 (t, 2H, J
= 6.3 Hz, CH2), 2.63 (t, 2H, J = 6.3 Hz, CH2), 3.66 (s, 3H, CH3), 3.72
(s, 3H, CH3), 5.02 (s, 2H, CH2), 6.32 (s, 1H, Ar), 6.76 (s, 2H, Ar),
7.25−7.44 (m, 6H, H-3, Ar), 7.52 (s, 1H, CH), 14.40 (bs, 1H, OH).
13C NMR (50 MHz, CDCl3) δ 21.1 (t), 25.8 (t), 46.6 (t), 55.7 (q),
Data for 5-(Hydroxymethylidene)-2-(4-methoxybenzyl)-6,6-di-
methyl-1-phenyl-2,5,6,7-tetrahydro-4H-isoindol-4-one (13o). This
compound was obtained from reaction of 12o. Brown solid; yield 60%;
1
mp 150−151 °C. IR (cm−1) 2958 (OH), 1624 (CO). H NMR (200
MHz, CDCl3) δ 1.18 (s, 6H, 2 × CH3), 2.50 (s, 2H, CH2), 3.77 (s,
3H, CH3), 4.97 (s, 2H, CH2), 6.80 (d, 2H, J = 8.8 Hz, H-3′, H-5′),
6.93 (d, 2H, J = 8.8 Hz, H-2′, H-6′), 7.18−7.45 (m, 6H, H-3, Ar), 7.61
(s, 1H, CH), 15.18 (bs, 1H, OH). 13C NMR (50 MHz, CDCl3) δ 29.1
(2 × q), 36.1 (t), 51.0 (t), 55.3 (q), 114.2 (2 × d), 118.5 (s), 119.4 (s),
122.3 (s), 122.4 (s), 123.0 (d), 127.8 (d), 128.5 (2 × d), 128.6 (2 ×
d), 128.9 (s), 130.1 (2 × d), 130.8 (s), 131.0 (s), 159.2 (s), 165.4 (d),
187.1 (s). Anal. Calcd for C25H25NO3: C, 77.49; H, 6.50; N, 3.61.
Found: C, 77.11; H, 6.27; N, 3.86.
Data for 5-(Hydroxymethylidene)-2-(4-methoxybenzyl)-1-(3,4,5-
trimethoxyphenyl)-2,5,6,7-tetrahydro-4H-isoindol-4-one (13p). This
compound was obtained from reaction of 12p. Yellow oil; yield 65%.
55.8 (q), 109.5 (s), 111.3 (d), 113.2 (d), 115.2 (d), 120.2 (s), 123.1
(s), 123.4 (d), 126.3 (s), 127.7 (d), 128.5 (2 × d), 128.6 (s), 130.1 (2
× d), 131.2 (s), 150.9 (s), 153.6 (s), 165.8 (d), 186.8 (s). Anal. Calcd
for C24H23NO4: C, 74.02; H, 5.95; N, 3.60. Found: C, 74.22; H, 6.22;
N, 3.35.
1
IR (cm−1) 2956 (OH), 1622 (CO). H NMR (200 MHz, CDCl3) δ
2.51 (t, 2H, J = 6.7 Hz, CH2), 2.64 (t, 2H, J = 6.7 Hz, CH2), 3.72 (s,
6H, 2 × CH3), 3.78 (s, 3H, CH3), 3.88 (s, 3H, CH3), 5.00 (s, 2H,
CH2), 6.38 (s, 2H, H-2′, H-6′), 6.83 (d, 2H, J = 8.8 Hz, H-3″, H-5″),
6.96 (d, 2H, J = 8.8 Hz, H-2″, H-6″), 7.35 (s, 1H, H-3), 7.53 (s, 1H,
CH), 14.37 (s, 1H, OH). 13C NMR (50 MHz, CDCl3) δ 21.2 (t), 25.8
(t), 51.1 (t), 55.3 (q), 56.0 (2 × q), 60.9 (q), 100.1 (s), 107.2 (2 × d),
109.4 (s), 114.2 (2 × d), 120.3 (s), 122.9 (d), 123.1 (s), 126.4 (s),
128.2 (2 × d), 129.2 (s), 129.9 (s), 153.1 (2 × s), 159.2 (s), 165.9 (d),
186.7 (s). Anal. Calcd for C26H27NO6: C, 69.47; H, 6.05; N, 3.12.
Found: C, 69.59; H, 5.92; N, 3.26.
Data for 2-(3,4-Dimethoxybenzyl)-5-(hydroxymethylidene)-1-
phenyl-2,5,6,7-tetrahydro-4H-isoindol-4-one (13h). This compound
was obtained from reaction of 12h. Brown solid; yield 65%; mp 136−
1
137 °C. IR (cm−1) 2933 (OH), 1635 (CO). H NMR (200 MHz,
CDCl3) δ 2.48 (t, 2H, J = 6.1 Hz, CH2), 2.62 (t, 2H, J = 6.1 Hz, CH2),
3.74 (s, 3H, CH3), 3.84 (s, 3H, CH3), 4.98 (s, 2H, CH2), 6.45 (s, 1H,
Ar), 6.55−6.60 (m, 1H, Ar), 6.76 (d, 1H, J = 8.2 Hz, CH2), 7.22−7.46
(m, 6H, H-3, Ar), 7.51 (s, 1H, CH), 14.41 (bs, 1H, OH). 13C NMR
(50 MHz, CDCl3) δ 21.1 (t), 25.8 (t), 51.4 (t), 55.8 (q), 55.9 (q),
K
DOI: 10.1021/acs.jmedchem.6b00777
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Journal of Medicinal Chemistry
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Data for 5-(Hydroxymethylidene)-2-(4-methoxybenzyl)-1-(4-me-
thoxyphenyl)-2,5,6,7-tetrahydro-4H-isoindol-4-one (13u). This
compound was obtained from reaction of 12u. Yellow oil; yield
60%. IR (cm−1) 2965 (OH), 1626 (CO). 1H NMR (200 MHz,
CDCl3) δ 2.45−2.63 (m, 4H, 2 × CH2), 3.78 (s, 3H, CH3), 3.84 (s,
3H, CH3), 4.94 (s, 2H, CH2), 6.80 (d, 2H, J = 6.7 Hz, Ar), 6.93 (dd,
4H, J = 11.4, 2.4 Hz, Ar), 7.15 (d, 2H, J = 8.9 Hz, Ar), 7.30 (s, 1H, H-
3), 7.52 (d, 1H, J = 5.8 Hz, CH), 7.52 (d, 1H, J = 5.8 Hz, OH). 13C
NMR (50 MHz, CDCl3) δ 21.1 (t), 25.8 (t), 50.9 (t), 55.3 (q), 55.4
(q), 109.5 (s), 114.0 (2 × d), 114.2 (2 × d), 120.3 (s), 122.5 (d),
123.0 (s), 123.3 (s), 128.6 (2 × d), 128.9 (s), 129.8 (s), 131.4 (2 × d),
159.2 (s), 159.3 (s), 165.7 (d), 186.8 (s). Anal. Calcd for C24H23NO4:
C, 74.02; H, 5.95; N, 3.60. Found: C, 74.33; H, 5.61; N, 3.73.
Procedure for the Synthesis of 5-[(Dimethylamino)methylidene]-
2-substituted-2,5,6,7-tetrahydro-4H-isoindol-4-ones (14). To a
solution of the suitable ketone 12 (5.3 mmol) in anhydrous toluene
(10 mL), the Bredereck reagent, t-butoxy-bis-(dimethylamino)-
methane (TBDMAM) (3.31 mL, 16 mmol) was added under nitrogen
atmosphere and the reaction mixture was heated under reflux for 24 h.
After cooling, the solvent was evaporated at reduced pressure. The
crude product was used in the next step without any further
purification.
Anal. Calcd for C23H20N2O2: C, 77.51; H, 5.66; N, 7.86. Found: C,
77.69; H, 5.35; N, 8.10.
Data for 7-(2-methoxybenzyl)-6-phenyl-5,7-dihydro-4H-[1,2]-
oxazolo[5,4-e]isoindole (15e). This compound was obtained from
1
reaction of 13e. White solid; yield 80%; mp 133−134 °C. H NMR
(200 MHz, DMSO-d6) δ 2.64−2.68 (m, 4H, 2 × CH2), 3.71 (s, 3H,
CH3), 5.10 (s, 2H, CH2), 6.56−6.61 (m, 1H, Ar), 6.80−6.96 (m, 2H,
Ar), 7.18−7.46 (m, 7H, H-8, Ar), 8.42 (s, 1H, H-3). 13C NMR (50
MHz, DMSO-d6) δ 19.3 (t), 20.2 (t), 45.5 (t), 55.2 (q), 108.9 (s),
109.6 (s), 110.5 (d), 116.9 (d), 118.4 (s), 120.4 (d), 126.2 (s), 127.0
(d), 127.5 (d), 128.5 (2 × d), 128.7 (d), 129.4 (2 × d), 130.3 (s),
131.1 (s), 149.1 (d), 155.8 (s), 162.4 (s). Anal. Calcd for C23H20N2O2:
C, 77.51; H, 5.66; N, 7.86. Found: C, 77.75; H, 5.39; N, 8.03.
Data for 7-(2,3-Dimethoxybenzyl)-6-phenyl-5,7-dihydro-4H-
[1,2]oxazolo[5,4-e]isoindole (15f). This compound was obtained
1
from reaction of 13f. White solid; yield 70%; mp 142−143 °C. H
NMR (200 MHz, DMSO-d6) δ 2.60−2.68 (m, 4H, 2 × CH2), 3.54 (s,
3H, CH3), 3.77 (s, 3H, CH3), 5.13 (s, 2H, CH2), 6.22−6.31 (m, 1H,
Ar), 6.92−7.01 (m, 2H, Ar), 7.27−7.47 (m, 6H, H-8, Ar), 8.42 (s, 1H,
H-3). 13C NMR (50 MHz, DMSO-d6) δ 19.3 (t), 20.2 (t), 45.6 (t),
55.6 (q), 59.6 (q), 109.0 (s), 109.6 (s), 112.2 (d), 116.9 (d), 118.4 (s),
119.0 (d), 124.1 (d), 127.5 (d), 128.5 (2 × d), 129.5 (2 × d), 130.3
(s), 131.0 (s), 131.8 (s), 145.3 (s), 149.1 (d), 152.1 (s), 162.3 (s).
Anal. Calcd for C24H22N2O3: C, 74.59; H, 5.74; N, 7.25. Found: C,
74.69; H, 5.42; N, 7.37.
General Procedure for the Synthesis of [1,2]Oxazolo[5,4-
e]isoindoles (15). To a solution of the suitable hydroxymethyleneke-
tones 13 or enaminoketones 14 (5 mmol) in ethanol (15 mL),
hydroxylamine hydrochloride (0.38 g, 5.5 mmol) was added and the
reaction mixture was heated under reflux for 50 min. After cooling, the
solvent was evaporated at reduced pressure. The crude product was
purified by chromatography column using dichloromethane as eluent.
Data for 7-Methyl-6-phenyl-5,7-dihydro-4H-[1,2]oxazolo[5,4-e]-
isoindole (15a). This compound was obtained from reaction of 13a.
White solid; yield 62%; mp 145−146 °C. 1H NMR (200 MHz,
DMSO-d6) δ 2.50−2.74 (m, 4H, 2 × CH2), 3.59 (s, 3H, CH3), 7.29 (s,
1H, H-8), 7.33−7.52 (m, 5H, Ar), 8.40 (s, 1H, H-3). 13C NMR (50
MHz, DMSO-d6) δ 19.9 (t), 20.9 (t), 35.4 (q), 109.1 (s), 109.6 (s),
118.0 (d), 118.7 (s), 127.8 (d), 129.0 (2 × d), 129.9 (2 × d), 130.5
(s), 131.5 (s), 149.5 (d), 163.0 (s). Anal. Calcd for C16H14N2O: C,
76.78; H, 5.64; N, 11.19. Found: C, 77.09; H, 5.78; N, 10.83.
Data for 7-Benzyl-6-phenyl-5,7-dihydro-4H-[1,2]oxazolo[5,4-e]-
isoindole (15b). This compound was obtained from reaction of 13b.
White solid; yield 60%; mp 139−140 °C. 1H NMR (200 MHz,
DMSO-d6) δ 2.60−2.78 (m, 4H, 2 × CH2), 5.17 (s, 2H, CH2), 6.92
(d, 2H, J = 7.4 Hz, H-2′, H-6′), 7.19−7.47 (m, 9H, H-8, Ar), 8.42 (s,
1H, H-3). 13C NMR (50 MHz, DMSO-d6) δ 19.8 (t), 20.7 (t), 50.7
(t), 109.5 (s), 110.2 (s), 117.5 (d), 119.2 (s), 127.0 (2 × d), 127.7 (d),
128.0 (d), 128.9 (2 × d), 129.0 (2 × d), 130.0 (2 × d), 130.6 (s),
131.6 (s), 138.9 (s), 149.6 (d), 162.9 (s). Anal. Calcd for C22H18N2O:
C, 80.96; H, 5.56; N, 8.58. Found: C, 81.21; H, 5.87; N, 8.30.
Data for 7-(4-Methoxybenzyl)-6-phenyl-5,7-dihydro-4H-[1,2]-
oxazolo[5,4-e]isoindole (15c). This compound was obtained from
Data for 7-(2,5-Dimethoxybenzyl)-6-phenyl-5,7-dihydro-4H-
[1,2]oxazolo[5,4-e]isoindole (15g). This compound was obtained
1
from reaction of 13g. White solid; yield 60%; mp 120−121 °C. H
NMR (200 MHz, DMSO-d6) δ 2.62−2.67 (m, 4H, 2 × CH2), 3.57 (s,
3H, CH3), 3.66 (s, 3H, CH3), 5.07 (s, 2H, CH2), 6.13 (d, 1H, J = 2.9
Hz, Ar), 6.74−6.89 (m, 2H, Ar), 7.29−7.48 (m, 6H, H-8, Ar), 8.42 (s,
1H, H-3). 13C NMR (50 MHz, DMSO-d6) δ 19.3 (t), 20.2 (t), 45.6
(t), 55.2 (q), 55.6 (q), 109.0 (s), 109.6 (s), 111.4 (d), 112.2 (d), 114.0
(d), 116.9 (d), 118.5 (s), 127.3 (s), 127.5 (d), 128.5 (2 × d), 129.4 (2
× d), 130.3 (s), 131.1 (s), 149.1 (d), 150.0 (s), 153.1 (s), 162.3 (s).
Anal. Calcd for C24H22N2O3: C, 74.59; H, 5.74; N, 7.25. Found: C,
74.44; H, 5.99; N, 7.54.
Data for 7-(3,4-Dimethoxybenzyl)-6-phenyl-5,7-dihydro-4H-
[1,2]oxazolo[5,4-e]isoindole (15h). This compound was obtained
1
from reaction of 13h. White solid; yield 83%; mp 122−123 °C. H
NMR (200 MHz, DMSO-d6) δ 2.59−2.65 (m, 4H, 2 × CH2), 3.58 (s,
3H, CH3), 3.68 (s, 3H, CH3), 5.07 (s, 2H, CH2), 6.46−6.51 (m, 2H,
Ar), 6.82 (d, 1H, J = 8.1 Hz, Ar), 7.34−7.51 (m, 6H, H-8, Ar), 8.41 (s,
1H, H-3). 13C NMR (50 MHz, DMSO-d6) δ 19.8 (t), 20.6 (t), 50.6
(t), 55.7 (q), 55.9 (q), 109.4 (s), 110.1 (s), 111.4 (d), 112.1 (d), 117.3
(d), 119.2 (s), 119.7 (d), 128.0 (d), 129.1 (2 × d), 130.1 (2 × d),
130.5 (s), 130.9 (s), 131.8 (s), 148.5 (s), 148.9 (s), 149.7 (d), 162.9
(s). Anal. Calcd for C24H22N2O3: C, 74.59; H, 5.74; N, 7.25. Found:
C, 74.34; H, 5.69; N, 7.59.
1
Data for 7-(3,5-Dimethoxybenzyl)-6-phenyl-5,7-dihydro-4H-
reaction of 13c. White solid; yield 67%; mp 115−116 °C. H NMR
[1,2]oxazolo[5,4-e]isoindole (15i). This compound was obtained
(200 MHz, DMSO-d6) δ 2.51−2.65 (m, 4H, 2 × CH2), 3.68 (s, 3H,
CH3), 5.07 (s, 2H, CH2), 6.78 (d, 2H, J = 8.2 Hz, H-3′, H-5′), 6.88 (d,
2H, J = 8.2 Hz, H-2′, H-6′), 7.30−7.49 (m, 6H, H-8, Ar), 8.41 (s, 1H,
H-3). 13C NMR (50 MHz, DMSO-d6) δ 19.3 (t), 20.2 (t), 49.7 (t),
55.0 (q), 108.9 (s), 109.7 (s), 113.8 (2 × d), 116.7 (d), 118.6 (s),
127.5 (d), 128.1 (2 × d), 128.5 (2 × d), 129.6 (2 × d), 130.0 (s),
130.1 (s), 131.2 (s), 149.1 (d), 158.4 (s), 162.4 (s). Anal. Calcd for
C23H20N2O2: C, 77.51; H, 5.66; N, 7.86. Found: C, 77.75; H, 6.00; N,
7.56.
1
from reaction of 13i. White solid; yield 69%; mp 158−159 °C. H
NMR (200 MHz, DMSO-d6) δ 2.63−2.69 (m, 4H, 2 × CH2), 3.62 (s,
6H, 2 × CH3), 5.08 (s, 2H, CH2), 6.08 (d, 2H, J = 2.2 Hz, Ar), 6.34 (t,
1H, J = 2.2 Hz, Ar), 7.31−7.50 (m, 6H, H-8, Ar), 8.41 (s, 1H, H-3).
13C NMR (50 MHz, DMSO-d6) δ 19.3 (t), 20.2 (t), 50.2 (t), 55.0 (2
× q), 98.7 (d), 104.7 (2 × d), 109.0 (s), 109.7 (s), 117.0 (d), 118.7
(s), 127.5 (d), 128.6 (2 × d), 129.6 (2 × d), 130.1 (s), 131.1 (s), 140.7
(s), 149.1 (d), 160.5 (2 × s), 162.3 (s). Anal. Calcd for C24H22N2O3:
C, 74.59; H, 5.74; N, 7.25. Found: C, 74.40; H, 5.82; N, 7.52.
Data for 7-(3,4,5-Trimethoxybenzyl)-6-phenyl-5,7-dihydro-4H-
[1,2]oxazolo[5,4-e]isoindole (15j). This compound was obtained
Data for 7-(3-Methoxybenzyl)-6-phenyl-5,7-dihydro-4H-[1,2]-
oxazolo[5,4-e]isoindole (15d). This compound was obtained from
1
reaction of 13d. White solid; yield 78%; mp 153−154 °C. H NMR
1
from reaction of 13j. White solid; yield 76%; mp 186−187 °C. H
(200 MHz, DMSO-d6) δ 2.64−2.67 (m, 4H, 2 × CH2), 3.64 (s, 3H,
CH3), 5.13 (s, 2H, CH2), 6.46−6.53 (m, 2H, Ar), 6.75−6.80 (m, 1H,
Ar), 7.16 (t, 1H, J = 7.9 Hz, Ar), 7.29−7.49 (m, 6H, H-8, Ar), 8.41 (s,
1H, H-3). 13C NMR (50 MHz, DMSO-d6) δ 19.3 (t), 20.2 (t), 50.12
(t), 54.9 (q), 109.0 (s), 109.7 (s), 112.3 (d), 112.6 (d), 117.0 (d),
118.7 (d), 127.5 (d), 128.6 (2 × d), 129.5 (2 × d), 129.6 (d), 130.1
(s), 131.1 (s), 139.9 (s), 149.1 (d), 159.2 (s), 159.3 (s), 162.3 (s).
NMR (200 MHz, DMSO-d6) δ 2.62−2.67 (m, 4H, 2 × CH2), 3.57 (s,
3H, CH3), 3.60 (s, 6H, 2 × CH3), 5.06 (s, 2H, CH2), 6.22 (s, 2H, H-
2′, H-6′), 7.36−7.53 (m, 6H, H-8, Ar), 8.42 (s, 1H, H-3). 13C NMR
(50 MHz, DMSO-d6) δ 19.3 (t), 20.1 (t), 50.4 (t), 55.6 (2 × q), 59.9
(q), 104.5 (2 × d), 108.9 (s), 109.6 (s), 116.9 (d), 118.8 (s), 127.6
(d), 128.7 (2 × d), 129.7 (2 × d), 129.9 (s), 131.4 (s), 133.6 (s), 136.5
L
DOI: 10.1021/acs.jmedchem.6b00777
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(s), 149.1 (d), 152.7 (2 × s), 162.4 (s). Anal. Calcd for C25H24N2O4:
C, 72.10; H, 5.81; N, 6.73. Found: C, 72.19; H, 5.68; N, 6.65.
Data for 7-(4-Methoxy-3-nitrobenzyl)-6-phenyl-5,7-dihydro-4H-
[1,2]oxazolo[5,4-e]isoindole (15k). This compound was obtained
from reaction of 14k. Pale-yellow solid; yield 62%; mp 174−175 °C.
IR (cm−1) 1532 (NO2). 1H NMR (200 MHz, DMSO-d6) δ 2.63−2.67
(m, 4H, 2 × CH2), 3.85 (s, 3H, CH3), 5.17 (s, 2H, CH2), 7.12−7.49
(m, 9H, H-8, Ar), 8.43 (s, 1H, H-3). 13C NMR (50 MHz, DMSO-d6)
δ 19.3 (t), 20.1 (t), 49.0 (t), 56.6 (q), 109.1 (s), 109.9 (s), 114.5 (d),
116.9 (d), 119.0 (s), 123.5 (d), 127.7 (d), 128.6 (2 × d), 129.6 (2 ×
d), 130.0 (s), 130.5 (s), 130.9 (s), 132.9 (d), 138.7 (s), 149.2 (d),
151.2 (s), 162.2 (s). Anal. Calcd for C23H19N3O4: C, 68.82; H, 4.77;
N, 10.47. Found: C, 69.01; H, 4.65; N, 10.56.
(s), 130.5 (s), 136.9 (s), 149.1 (d), 152.7 (2 × s), 158.4 (s), 162.4 (s).
Anal. Calcd for C26H26N2O5: C, 69.94; H, 5.87; N, 6.27. Found: C,
70.08; H, 5.75; N, 6.40.
Data for 7-(4-Methoxy-3-nitrobenzyl)-6-(3,4,5-trimethoxyphen-
yl)-5,7-dihydro-4H-[1,2]oxazolo[5,4-e]isoindole (15q). This com-
pound was obtained from reaction of 14q. White solid; yield 62%;
mp 184−185 °C. IR (cm−1) 1535 (NO2). 1H NMR (200 MHz,
DMSO-d6) δ 2.65−2.69 (m, 4H, 2 × CH2), 3.69 (s, 3H, CH3), 3.71 (s,
6H, 2 × CH3), 3.86 (s, 3H, CH3), 5.18 (s, 2H, CH2), 6.52 (s, 2H, Ar),
7.28 (s, 2H, Ar), 7.39 (s, 1H, H-8), 7.46 (s, 1H, Ar), 8.42 (s, 1H, H-3).
13C NMR (50 MHz, DMSO-d6) δ 19.2 (t), 20.0 (t), 49.2 (t), 55.8 (2
× q), 56.7 (q), 60.0 (q), 107.1 (2 × d), 109.0 (s), 109.7 (s), 114.5 (d),
116.6 (d), 118.9 (s), 123.6 (d), 126.4 (s), 130.0 (s), 130.8 (s), 133.1
(d), 137.0 (s), 138.7 (s), 149.2 (d), 151.2 (s), 152.9 (2 × s), 162.3 (s).
Anal. Calcd for C26H25N3O7: C, 63.54; H, 5.13; N, 8.55. Found: C,
63.42; H, 5.22; N, 8.61.
Data for 2-Methoxy-5-[(6-phenyl-4,5-dihydro-7H-[1,2]oxazolo-
[5,4-e]isoindol-7-yl)methyl]aniline (15l). This compound was ob-
tained from reaction of 14l. White solid; yield 60%; mp 157−158 °C.
1
IR (cm−1) 3455−3372 (NH2). H NMR (200 MHz, DMSO-d6) δ
Data for 2-Methoxy-5-{[6-(3,4,5-trimethoxyphenyl)-4,5-dihydro-
7H-[1,2]oxazolo[5,4-e]isoindol-7-yl]methyl}aniline (15r). This com-
pound was obtained from reaction of 14r. White solid; yield 58%; mp
2.64−2.69 (m, 4H, 2 × CH2), 3.70 (s, 3H, CH3), 4.71 (s, 2H, NH2),
4.94 (s, 2H, CH2), 6.11 (d, 1H, J = 7.9 Hz, Ar), 6.34 (s, 1H, Ar), 6.65
(d, 1H, J = 7.9 Hz, Ar), 7.26−7.54 (m, 6H, H-8, Ar), 8.41 (s, 1H, H-
3). 13C NMR (50 MHz, DMSO-d6) δ 19.3 (t), 20.2 (t), 50.0 (t), 55.2
(q), 108.8 (s), 109.6 (s), 110.2 (d), 112.0 (d), 114.6 (d), 118.3 (s),
127.5 (d), 127.8 (d), 128.5 (2 × d), 128.7 (s), 129.5 (2 × d), 130.1
(s), 130.5 (s), 131.1 (s), 137.4 (s), 145.7 (d), 162.4 (s). Anal. Calcd
for C23H21N3O2: C, 74.37; H, 5.70; N, 11.31. Found: C, 74.30; H,
5.85; N, 11.48.
1
145−146 °C. IR (cm−1) 3453−3371 (NH2). H NMR (200 MHz,
DMSO-d6) δ 2.67−2.72 (m, 4H, 2 × CH2), 3.66 (s, 6H, 2 × CH3),
3.68 (s, 3H, CH3), 3.71 (s, 3H, CH3), 4.87−5.02 (m, 4H, CH2, NH2),
6.22 (d, 1H, J = 8.1 Hz, Ar), 6.40 (s, 1H, Ar), 6.52 (s, 2H, Ar), 6.71 (d,
1H, J = 8.1 Hz, Ar), 7.29 (s, 1H, H-8), 8.42 (s, 1H, H-3). 13C NMR
(50 MHz, DMSO-d6) δ 19.3 (t), 20.3 (t), 50.3 (t), 55.4 (q), 55.7 (2 ×
q), 60.0 (q), 107.0 (2 × d), 108.7 (s), 109.3 (s), 110.4 (d), 111.9 (d),
114.4 (d), 116.7 (d), 118.2 (s), 126.5 (s), 130.1 (s), 131.0 (s), 136.8
(s), 137.4 (s), 145.6 (s), 149.1 (d), 152.7 (2 × s), 162.5 (s). Anal.
Calcd for C26H27N3O5: C, 67.66; H, 5.90; N, 9.10. Found: C, 67.81;
H, 5.77; N, 9.01.
Data for 7-(4-Methoxyphenyl)-6-phenyl-5,7-dihydro-4H-[1,2]-
oxazolo[5,4-e]isoindole (15m). This compound was obtained from
1
reaction of 13m. White solid; yield 76%; mp 146−147 °C. H NMR
(200 MHz, CDCl3) δ 2.77 (t, 2H, J = 6.5 Hz, CH2), 2.89 (t, 2H, J =
6.5 Hz, CH2), 3.80 (s, 3H, CH3), 6.81 (d, 2H, J = 8.9 Hz, H-3′, H-5′),
7.03−7.28 (m, 8H, H-8, Ar), 8.13 (s, 1H, H-3). 13C NMR (50 MHz,
CDCl3) δ 20.0 (t), 20.9 (t), 55.4 (q), 109.6 (s), 111.4 (s), 114.2 (2 ×
d), 117.3 (d), 120.0 (s), 126.8 (2 × d), 126.9 (d), 128.1 (2 × d), 129.7
(2 × d), 130.0 (s), 131.6 (s), 132.9 (s), 148.8 (d), 158.3 (s), 163.1 (s).
Anal. Calcd for C22H18N2O2: C, 77.17; H, 5.30; N, 8.18. Found: C,
77.26; H, 5.09; N, 8.32.
Data for 4-[7-(4-Methoxybenzyl)-5,7-dihydro-4H-[1,2]oxazolo-
[5,4-e]isoindol-6-yl]phenyl Acetate (15s). This compound was
obtained from reaction of 13s. Yellow oil; yield 69%. IR (cm−1)
1
1625 (CO). H NMR (200 MHz, CDCl3) δ 2.67−2.72 (m, 4H, 2 ×
CH2), 3.78 (s, 3H, CH3), 3.82 (s, 3H, CH3), 5.01 (s, 2H, CH2), 6.78−
6.84 (m, 2H, Ar), 6.90−7.02 (m, 3H, H-8, Ar), 7.14−7.22 (m, 2H,
Ar), 7.34−7.42 (m, 2H, Ar), 8.08 (s, 1H, H-3). 13C NMR (50 MHz,
CDCl3) δ 20.0 (t), 20.6 (t), 55.2 (q), 55.3 (q), 69.8 (t), 109.1 (s),
110.6 (s), 114.0 (2 × d), 115.6 (d), 118.7 (s), 128.2 (2 × d), 128.7 (s),
129.3 (2 × d), 129.9 (s), 130.4 (s), 131.3 (2 × d), 148.8 (d), 158.4 (s),
159.0 (s), 159.5 (s), 163.4 (s). Anal. Calcd for C25H22N2O4: C, 72.45;
H, 5.35; N, 6.76. Found: C, 72.65; H, 5.12; N, 6.54.
Data for 7-Benzyl-4,4-dimethyl-6-phenyl-5,7-dihydro-4H-[1,2]-
oxazolo[5,4-e]isoindole (15n). This compound was obtained from
1
reaction of 13n. White solid; yield 84%; mp 149−150 °C. H NMR
(200 MHz, DMSO-d6) δ 1.19 (s, 6H, 2 × CH3), 2.54 (s, 2H, CH2),
5.18 (s, 2H, CH2), 6.87−6.97 (m, 2H, Ar), 7.15−7.47 (m, 9H, H-8,
Ar), 8.52 (s, 1H, H-3). 13C NMR (50 MHz, DMSO-d6) δ 28.2 (2 ×
q), 36.2 (t), 50.2 (t), 109.9 (s), 117.0 (d), 118.1 (s), 118.9 (s), 119.0
(s), 126.3 (2 × d), 127.2 (d), 127.5 (d), 128.5 (2 × d), 128.6 (2 × d),
129.5 (2 × d), 130.8 (s), 131.0 (s), 138.4 (s), 147.6 (d), 160.7 (s).
Anal. Calcd for C24H22N2O: C, 81.33; H, 6.26; N, 7.90. Found: C,
81.53; H, 6.15; N, 8.08.
Data for 4-[7-(4-Methoxybenzyl)-5,7-dihydro-4H-[1,2]oxazolo-
[5,4-e]isoindol-6-yl]phenol (15t). This compound was obtained
from reaction of 13t. Yellow oil; yield 57%. IR (cm−1) 2962 (OH).
1H NMR (200 MHz, CDCl3) δ 2.65−2.72 (m, 4H, 2 × CH2), 3.77 (s,
3H, CH3), 4.94 (s, 2H, CH2), 6.77−6.97 (m, 7H, H-8, Ar), 7.08−7.13
(m, 2H, Ar), 7.28 (s, 1H, OH), 8.10 (s, 1H, H-3). 13C NMR (50 MHz,
CDCl3) δ 20.0 (t), 20.6 (t), 50.3 (t), 55.3 (q), 109.2 (s), 110.4 (s),
114.0 (2 × d), 114.1 (2 × d), 115.5 (2 × d), 118.7 (s), 123.2 (s), 128.3
(2 × d), 130.0 (s), 130.7 (s), 131.4 (d), 148.7 (d), 156.2 (s), 159.0 (s),
163.7 (s). Anal. Calcd for C23H20N2O3: C, 74.18; H, 5.41; N, 7.52.
Found: C, 74.39; H, 5.13; N, 7.69.
Data for 7-(4-Methoxybenzyl)-4,4-dimethyl-6-phenyl-5,7-dihy-
dro-4H-[1,2]oxazolo[5,4-e]isoindole (15o). This compound was
obtained from reaction of 13o. White solid; yield 75%; mp 166−
1
167 °C. H NMR (200 MHz, DMSO-d6) δ 1.18 (s, 6H, 2 × CH3),
2.52 (s, 2H, CH2), 3.68 (s, 3H, CH3), 5.09 (s, 2H, CH2), 6.88 (d, 2H,
J = 9.0 Hz, H-3′, H-5′), 6.80 (d, 2H, J = 9.0 Hz, H-2′, H-6′), 7.28−
7.49 (m, 6H, H-8, Ar), 8.51 (s, 1H, H-3). 13C NMR (50 MHz,
DMSO-d6) δ 28.2 (2 × q), 36.2 (t), 49.7 (t), 55.0 (q), 109.0 (s), 113.8
(2 × d), 116.7 (d), 118.2 (s), 118.8 (s), 118.9 (s), 127.5 (d), 127.9 (2
× d), 128.6 (2 × d), 129.6 (2 × d), 130.2 (s), 130.7 (s), 131.1 (s),
147.6 (d), 158.4 (s), 160.7 (s). Anal. Calcd for C25H24N2O2: C, 78.10;
H, 6.29; N, 7.29. Found: C, 78.45; H, 5.99; N, 7.43.
Data for 7-(4-Methoxybenzyl)-6-(3,4,5-trimethoxyphenyl)-5,7-
dihydro-4H-[1,2]oxazolo[5,4-e]isoindole (15p). This compound was
obtained from reaction of 13p. White solid; yield 60%; mp 136−137
°C. 1H NMR (200 MHz, DMSO-d6) δ 2.67−2.71 (m, 4H, 2 × CH2),
3.68 (s, 6H, 2 × CH3), 3.69 (s, 3H, CH3), 3.70 (s, 3H, CH3), 5.09 (s,
2H, CH2), 6.52 (s, 2H, H-2″, H-6″), 6.85 (d, 2H, J = 8.7 Hz, H-3′, H-
5′), 6.94 (d, 2H, J = 8.7 Hz, H-2′, H-6′), 7.35 (s, 1H, H-8), 8.41 (s,
1H, H-3). 13C NMR (50 MHz, DMSO-d6) δ 19.3 (t), 20.2 (t), 50.0
(t), 55.1 (q), 55.8 (2 × q), 60.0 (q), 107.0 (2 × d), 108.8 (s), 109.5
(s), 113.9 (2 × d), 116.7 (d), 118.5 (s), 126.5 (s), 128.0 (2 × d), 130.1
Data for 7-(4-Methoxybenzyl)-6-(4-methoxyphenyl)-5,7-dihydro-
4H-[1,2]oxazolo[5,4-e]isoindole (15u). This compound was obtained
1
from reaction of 13u. White solid; yield 75%; mp 136−137 °C. H
NMR (200 MHz, CDCl3) δ 2.66−2.73 (m, 4H, 2 × CH2), 3.78 (s, 3H,
CH3), 3.84 (s, 3H, CH3), 4.95 (s, 2H, CH2), 6.81 (d, 2H, J = 8.8 Hz,
Ar), 6.91−6.98 (m, 5H, H-8, Ar), 7.18 (d, 2H, J = 8.8 Hz, Ar), 8.08 (s,
1H, H-3). 13C NMR (50 MHz, CDCl3) δ 20.0 (t), 20.6 (t), 50.3 (t),
55.2 (q), 55.3 (q), 109.1 (s), 110.6 (s), 113.9 (2 × d), 114.1 (2 × d),
115.6 (d), 118.7 (s), 123.8 (s), 128.2 (2 × d), 130.0 (s), 130.5 (s),
131.3 (2 × d), 148.8 (d), 159.0 (s), 159.1 (s), 163.4 (s). Anal. Calcd
for C24H22N2O3: C, 74.59; H, 5.74; N, 7.25. Found: C, 74.72; H, 5.89;
N, 7.12.
Synthesis of 4-[(6-Phenyl-4,5-dihydro-7H-[1,2]oxazolo[5,4-e]-
isoindol-7-yl)methyl]phenol (15v). To a solution of 15c (0.42
mmol) in anhydrous dichloromethane (10 mL) BBr3 (1.26 mmol)
was added at −78 °C. Then the reaction mixture was stirred at room
temperature for 24 h. Then the reaction mixture was added with
M
DOI: 10.1021/acs.jmedchem.6b00777
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
methanol and the solvent was evaporated at reduced pressure. The
crude product was purified by chromatography column using
dichloromethane/ethyl acetate (98/2) as eluent. White solid; yield
vinorelbine. Tubulin polymerization was started by incubation at 37
°C and followed by absorbance readings at 340 nm (POLARstar
OPTIMA) every min up to 60 min.
1
84%; mp 205−206 °C. IR (cm−1) 3161 (OH). H NMR (200 MHz,
Cell Cycle Distribution Analysis. Both adherent and floating cells
were fixed in 70% EtOH and incubated at 4 °C for 30 min in staining
solution containing 50 μg/mL of propidium iodide, 50 mg/mL of
RNase, and 0.05% Nonidet-P40 in PBS. Samples were analyzed with
BD Accuri c6 flow cytometer (Becton Dickinson). At least 30000
events were read, and histograms were analyzed using the CellQuest
software according to the Modfit model (Becton Dickinson).
Fluorescence Microscopy Analysis. Cells seeded on glass coverslips
were treated for 72 h with equitoxic (IC50) concentrations of 15r,
taxol, and vinorelbine and then fixed in 2% paraformaldehyde for 30
min and permeabilized with ice-cold methanol for 20 min at −20 °C.
After blocking with PBS containing 1% bovine serum albumin, cells
were probed overnight with a mouse anti-MPM-2 antibody (Upstate
Biotechnology) at room temperature, followed by a 1 h incubation at
room temperature with goat antimouse AlexaFluor594 dye secondary
antibody (Life Technologies). Images were acquired using a Leika
fluorescence microscope (Leica). The percentage of mitotic cells was
determined by scoring at least 300 cells for each sample run in
duplicate.
Apoptosis Analysis. The catalytic activity of caspase-3 was
measured in the same cellular samples as the ability to cleave the
specific substrate N-acetyl-Asp-Glu-Val-Asp-pNA (DEVD-pNA) by
means of the APOPCYTO/caspase-3 kit (MBL), according to
manufacturer’s instructions. Briefly, cells were washed, pelleted, and
lyzed according to the manufacturer’s instructions. Total protein and
the specific fluorogenic substrate N-acetyl-Asp-Glu-Val-Asp-pNA
(DEVD-pNA) were mixed for 1 h at 37 °C and transferred to 96-
well microtiter plates. The hydrolysis of the specific substrates was
monitored by a spectrofluorometer (POLARstar OPTIMA) with 380
nm excitation and 460 nm emission filters. Results were expressed as
relative fluorescence units (rfu).
In Vivo Experiments. Female nude mice (6−8 weeks-old, purchased
from Charles River) were maintained in laminar flow rooms, keeping
temperature and humidity constant. The mice had free access to food
and water. Experimental protocols were approved by the Ethics
Committee for Animal Experimentation of the Fondazione IRCCS
Istituto Nazionale Tumori of Milan according to institutional
guidelines that are in compliance with national and international
laws and policies. Exponentially growing STO cells were subcuta-
neously injected into the right flank mice (107 cells/flank). Eight mice
for each experimental group were used. Compound 15r was dissolved
in a mixture of DMSO (10%) and saline solution (90%) and delivered
ip every 7 days for 4 weeks (q7dx4) starting from the tenth day after
cell inoculum at a dose of 5 mg/kg. Tumor growth was followed by
weekly measurements of tumor diameters with a Vernier caliper, and
tumor volume (TV) was calculated according to the formula: TV
(mm3) = d2 × D/2 where d and D are the shortest and the longest
diameter, respectively. The efficacy of the drug treatment was assessed
as tumor volume inhibition percentage (TVI %) in treated versus
control mice, calculated as TVI% = 100 − (mean TV treated/mean
TV control × 100). The toxicity of the drug treatment was determined
as body weight loss and lethal toxicity.
CDCl3) δ 2.72−2.75 (m, 4H, 2 × CH2), 4.96 (s, 2H, CH2), 5.69 (s,
1H, OH), 6.76 (d, 2H, J = 8.7 Hz, H-3′, H-5′), 6.90 (d, 2H, J = 8.7 Hz,
H-2′, H-6′), 7.00 (s, 1H, H-8), 7.24−7.41 (m, 5H, Ar), 8.10 (s, 1H, H-
3). 13C NMR (50 MHz, CDCl3) δ 20.0 (t), 20.7 (t), 50.5 (t), 108.8
(s), 109.3 (s), 110.6 (s), 115.7 (2 × d), 116.1 (d), 119.1 (s), 127.7 (d),
128.5 (2 × d), 128.6 (2 × d), 129.7 (2 × d), 130.1 (s), 130.8 (s), 131.6
(s), 148.8 (d), 155.6 (s). Anal. Calcd for C22H18N2O2: C, 77.17; H,
5.30; N, 8.18. Found: C, 77.30; H, 5.44; N, 7.95.
Biology. Drugs. For in vitro studies, vinorelbine (purchased from
Pierre Fabre Pharma), taxol (Paclitaxel; purchased from Santa Cruz
Biotechnology), and 15r were completely dissolved in 1% dimethyl
sulfoxide (DMSO), stored at −20 °C, and then diluted in complete
culture medium immediately before use at the appropriate
concentration. For in vivo studies, 15r was dissolved in DMSO 10%
and diluted in saline solution.
Cell Culture. Human DMPM cell lines (STO, MP4, and MP8) were
established from surgical specimens of patients admitted to
Fondazione IRCCS Istituto Nazionale dei Tumori of Milan. The
normal human lung fibroblast cell line (WI38) was obtained from the
American Type Culture Collection (ATCC). Cells were maintained in
the logarithmic growth phase as a monolayer in DMEM F12 (STO,
MP4, and MP8) and DMEM (WI38) media (Lonza) supplemented
with 10% heat-inactivated fetal bovine serum, in a humidified
incubator at 37 °C with a supply of 5% CO2/95% air atmosphere.
Cell lines are tested fortnightly for the absence of Mycoplasma and
periodically (every six months) monitored for DNA profile of short
tandem repeats analysis by the AmpFISTR Identifiler PCR
amplification kit (Applied Biosystems).
Cell Proliferation Assay. After harvesting in the logarithmic growth
phase, 4500 cells/50 μL were plated in 96-well flatbottomed microtiter
plates (EuroClone) for 24 h and then treated with increasing
concentrations of derivative 15r (0.1−100 μM) for 72 h. Control cells
received vehicle alone (0.01% DMSO). Studies were performed in
eight replicates and repeated at least three times independently. At the
end of drug exposure, the antiproliferative potential was determined
with the CellTiter 96 AQueous One Solution Cell Proliferation Assay
(MTS, purchased from Promega) according to the manufacturer’s
protocols. Optical density was read at 490 nm on a microplate reader
(POLARstar OPTIMA, BMG Labtech GmbH), and the results were
expressed as a percentage relative to DMSO-treated cells. Dose−
response curves were created, and IC50 and IC80 values (i.e.,
concentrations able to inhibit cell growth by 50% and 80%,
respectively) were determined graphically from the curve for each
compound.
Tubulin Polymerization Assays. Cells were seeded in 6-well plates
(Eppendorf) and were exposed the next day to the 15r derivative for
24 h at concentrations corresponding to the IC50 at 72 h. Samples
were then processed for the tubulin polymerization assay.50,51 To
separate cytosolic and cytoskeletal-associated proteins, cells were
rinsed twice in PIPES−EGTA−MgCl2 (PEM) buffer (85 mmol/L
PIPES, pH 6.94; 10 mmol/L EGTA; 1 mmol/L MgCl2; 2 mol//L
glycerol; 1 mmol/L phenylmethylsulfonyl fluoride; 0.1 mmol/L
leupeptin; 1 μmol/L pepstatin; 2 μg/mL aprotinin), lysed at room
temperature for 10 min with PEM buffer supplemented with 0.1% v/v
Triton X-100, and rinsed in PEM buffer. The Triton X-100-soluble
fractions were then diluted 3:1 with 4× SDS-PAGE sample buffer. The
insoluble material that remained attached to the dish was scraped into
SDS-PAGE sample buffer containing protease inhibitors. Proteins were
separated by SDS-PAGE, and tubulin distribution was analyzed by
immunoblotting using anti α-tubulin antibody (Sigma-Aldrich).
The effect of 15r on the microtubule assembly/disassembly process
was determined using the tubulin polymerization assay (Cytoskele-
ton), according to the manufacturer’s instruction. Briefly, purified
porcine brain tubulin was resuspended in ice-cold buffer (80 mmol/L
PIPES at pH 6.9, 2 mmol/L MgCl2, 0.5 mmol/L EGTA, 1 mmol GTP,
10% glycerol) in the presence of 10 μmol/L 15r derivative, taxol, and
To evaluate the proliferation rate, at the end of the experiment,
tumor specimens were fixed in 10% buffered formalin and paraffin-
embedded (FFPE). Five-micrometer sections of FFPE samples were
used and stained, according to standard protocols, with the primary
antibodies anti-Ki67.
Statistical Analysis. Statistical evaluation of data was done with the
two-tailed Student’s t test. Ps < 0.05 was considered statistically
significant.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.6b00777.
N
DOI: 10.1021/acs.jmedchem.6b00777
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Cirrincione, G. Synthesis and antiproliferative activity of thiazolyl-bis-
pyrrolo[2,3-b]pyridines and indolyl-thiazolyl-pyrrolo[2,3-c]pyridines,
nortopsentin analogues. Mar. Drugs 2015, 13, 460−492.
Overview of the results of the NCI in vitro human tumor
cell line screen for derivatives 15b,c,e,g,h,l,o−r,t,u; in
vitro activity of compounds 15b,c,e,g,h,1,o−r,t,u in
individual tumor cell lines; dose response curves and
mean graph of compounds 15b,c,e,g,l,o,p,r,t,u (PDF)
Molecular formula strings (CSV)
(9) Barraja, P.; Diana, P.; Lauria, A.; Montalbano, A.; Almerico, A.
M.; Dattolo, G.; Cirrincione, G.; Viola, G.; Dall’Acqua, F. Pyrrolo[2,3-
h]quinolinones: synthesis and photo-chemotherapic activity. Bioorg.
Med. Chem. Lett. 2003, 13, 2809−2811.
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AUTHOR INFORMATION
Corresponding Authors
*Phone: +39-091-23896806. E-mail: paola.barraja@unipa.it.
*Phone: +39-02-23903260. E-mail: nadia.zaffaroni@
istitutotumori.mi.it.
Author Contributions
V.S. and M. P. contributed equally to this work. P.B. and N.Z.
share senior authorship.
A.; Cirrincione, G.; Brun, P.; Palu, G.; Castagliuolo, I.; Dall’Acqua, F.;
̀
■
Vedaldi, D.; Salvador, A. Synthesis of pyrrolo[3,2-h]quinolinones with
good photochemotherapeutic activity and no DNA damage. Bioorg.
Med. Chem. 2010, 18, 4830−4843.
(11) Barraja, P.; Caracausi, L.; Diana, P.; Montalbano, A.; Carbone,
A.; Salvador, A.; Brun, P.; Castagliuolo, I.; Tisi, S.; Dall’Acqua, F.;
Vedaldi, D.; Cirrincione, G. Pyrrolo[3,2-h]quinazolines as photo-
chemotherapeutic agents. ChemMedChem 2011, 6, 1238−1248.
̀
(12) Barraja, P.; Diana, P.; Spano, V.; Montalbano, A.; Carbone, A.;
Parrino, B.; Cirrincione, G. An efficient synthesis of pyrrolo[3′,2′:4,5]-
thiopyrano[3,2-b]pyridin-2-one: a new ring system of pharmaceutical
interest. Tetrahedron 2012, 68, 5087−5094.
Notes
The authors declare no competing financial interest.
̀
(13) Carbone, A.; Parrino, B.; Barraja, P.; Spano, V.; Cirrincione, G.;
ACKNOWLEDGMENTS
This work was financially supported by Ministero dell’Istru-
■
Diana, P.; Maier, A.; Kelter, G.; Fiebig, H.-H. Synthesis and
antiproliferative activity of 2,5-bis(3′-indolyl)pyrroles, analogues of
the marine alkaloid nortopsentin. Mar. Drugs 2013, 11, 643−654.
zione dell’Universita
recipient of an Associazione Italiana per la Ricerca sul Cancro
̀
e della Ricerca (MIUR). DC is the
̀
(14) Parrino, B.; Carbone, A.; Ciancimino, C.; Spano, V.;
Montalbano, A.; Barraja, P.; Cirrincione, G.; Diana, P.; Sissi, C.;
Palumbo, M.; Pinato, O.; Pennati, M.; Beretta, G.; Folini, P.; Matyus,
P.; Balogh, B.; Zaffaroni, N. Water-soluble isoindolo[2,1-a]quinoxalin-
6-imines: In vitro antiproliferative activity and molecular mecha-
nism(s) of action. Eur. J. Med. Chem. 2015, 94, 149−162.
(AIRC) fellowship (#16360).
ABBREVIATIONS USED
■
CRS, cytoreductive surgery; DMPM, diffuse malignant
peritoneal mesothelioma; HIPEC, hyperthermic intraperitoneal
chemotherapy; MPM-2, mitotic protein monoclonal 2; NCI,
National Cancer Institute
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Pyrrolo[3,4-d][1,2,3]-triazolo[1,5-a]pyrimidine. New Tricyclic Ring
Systems of Biological Interest. J. Heterocycl. Chem. 2000, 37, 747−750.
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