New synthetic approach to paullones and characterization of their SIRT1 inhibitory activity

Article abstract of DOI:10.1039/c2ob06695e

A series of 7,12-dihydroindolo[3,2-d][1]benzazepine-6(5H)-ones (paullones) substituted at C9/C10 (Br) and C2 (Me, CF₃, CO₂Me) have been synthesized by a one-pot Suzuki-Miyaura cross-coupling of an o-aminoarylboronic acid and methyl 2-iodoindoleacetate followed by intramolecular amide formation. Other approaches to the paullone scaffold based on Pd-catalyzed C-H activation were unsuccessful. In vitro enzymatic assay with recombinant human SIRT-1 indicated a strong inhibitory profile for the series, in particular the analogue with a methoxycarbonyl group at C2 and a bromine at C9. These compounds are, in general, inducers of granulocyte differentiation of the U937 acute leukemia cell line and cause a marked increase in pre-G1 of the cell cycle.

Full text of DOI:10.1039/c2ob06695e

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PAPER
New synthetic approach to paullones and characterization of their SIRT1
inhibitory activity†
Sara Soto,a Esther Vaz,a Carmela Dell’Aversana,b,c Rosana Álvarez,*a Lucia Altucci*b,c
and Ángel R. de Lera*a
Received 6th October 2011, Accepted 5th December 2011
DOI: 10.1039/c2ob06695e
A series of 7,12-dihydroindolo[3,2-d][1]benzazepine-6(5H)-ones (paullones) substituted at C9/C10 (Br)
and C2 (Me, CF3, CO2Me) have been synthesized by a one-pot Suzuki–Miyaura cross-coupling of an
o-aminoarylboronic acid and methyl 2-iodoindoleacetate followed by intramolecular amide formation.
Other approaches to the paullone scaffold based on Pd-catalyzed C–H activation were unsuccessful. In
vitro enzymatic assay with recombinant human SIRT-1 indicated a strong inhibitory profile for the series,
in particular the analogue with a methoxycarbonyl group at C2 and a bromine at C9. These compounds
are, in general, inducers of granulocyte differentiation of the U937 acute leukemia cell line and cause a
marked increase in pre-G1 of the cell cycle.
Several crystal structures of SIRT enzymes – either uncom-
Introduction
plexed or bound to NAD+ analogs and the acetyl–lysine peptide
Sirtuins (Class III HDACs)1 are nicotinamide adenine dinucleo-
substrates – have been reported, including the structures of
tide (NAD+)-dependent protein deacetylases that share homology
with the yeast transcriptional repressor Sir2 (Silent Information
Regulator 2) and regulate a variety of cellular functions such as
conservation of the genome, longevity and metabolism of organ-
isms ranging from bacteria to eukaryotes.1–6 In contrast, other
histone deacetylases (Class I and II HDACs) are Zn+2-dependent
hydrolases and do not share sequence similarities with the
sirtuins.7–9
human orthologs SIRT2,12 SIRT3,13 SIRT514 and SIRT6.15
These systems contain a Rossmann fold domain for NAD+
binding and a smaller domain composed of α-helical and Zn-
binding modules. The NAD+ binding site is usually divided into
three binding pockets, the adenine ribose moiety in site A, the
nicotinamide ribose moiety in site B, and the nicotinamide het-
erocycle in site C, which lies deep inside the pocket.16 The nico-
tinamide part seems to be more flexible and the heterocycle has
been found to occupy alternative locations in several of the
crystal structures. The acetylated peptide binds in the major
groove of sirtuins, and is located in a tunnel that leads to the
NAD+ binding site, close to residues (His and Asn) that are
important for the catalytic activity. Binding of acetyl–lysine
induces a strained conformation for NAD+ (trapped in the crystal
structure of a Michaelis complex) that buries the nicotinamide
ring deep within the active site.16–18 The precise details of the
nicotinamide displacement step by the incoming nucleophile
(the amide oxygen of the Nε-acetyl–lysine group) to form an O-
alkylamidate intermediate17–19 by SN1–,20,21 or SN2–type reac-
tions are still subject to debate.22
In mammals, the sirtuin family consists of seven members,
SIRT1–7, which share a conserved catalytic domain of about
275 amino acids but differ in their cellular localization and their
function.1 SIRT1, SIRT2, SIRT3, SIRT5, SIRT6 and SIRT7 have
activity as NAD+-dependent deacetylases, while SIRT4 and
SIRT6 show ADP-ribosyl transferase activity.10 SIRT3 is also a
mitochondrial protein with tumour suppressor function and is
necessary to maintain mitochondrial integrity and metabolism
during stress.11
aDepartamento de Química Orgánica, Facultade de Química,
Universidade de Vigo, Lagoas-Marcosende, 36310 Vigo, Spain. E-mail:
qolera@uvigo.es, rar@uvigo.es; Fax: +34 986 811940; Tel: +34 986
812316
The identification of small molecule modulators of sirtuins
has provided additional valuable tools for understanding the
roles of these enzymes in various biological systems.6,17,23
Mechanism-based small molecule SIRT inhibitors have been
developed ranging from the simple Nε-(thio)acetyl lysines
(1,2),24,25 to lysine-containing peptides (3–6) (Fig. 1). The key
lysine residue of these peptides has been modified with the thio-
acetyl (4)26 carbamoyl (3),27 or Nε-thiocarbamoyl (6),28
bDipartimento di Patologia generale, Seconda Università degli Studi di
Napoli, Vico L. de Crecchio 7, 80138 Napoli, Italy.
E-mail: lucia.altucci@unina2.it
cCNR-IGB, via P. Castellino, Napoli, Italy
†Electronic supplementary information (ESI) available: Experimental
procedures, analytical and spectral characterization data for compounds
not included in the text. See DOI: 10.1039/c2ob06695e
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Fig. 1 Mechanism-based sirtuin inhibitors.
functional groups, and also L-2-amino-7-carboxamidoheptanoic
acid has replaced lysine in tetrapeptide 5.29
Fig. 2 Small-molecule sirtuin inhibitors.
A fairly large collection of compounds derived from or
inspired by natural product structures (7–9), such as 1030,31 and
11–17 have also been discovered by HTS or VLS protocols (Fig
2). For example, suramin (11), which has been co-crystallized
with SIRT5,14 and analogues are SIRT2-selective (IC50 = 93 nM
for 11),32 the tenovins 12 are SIRT1/2 inhibitors,33 whereas (S)-
6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide,
of cytokine-induced β-cell apoptosis, increasing their viability,
and it therefore has potential for therapeutic intervention in type-
1 diabetes.44 Moreover, kenpullone (replacing Klf4), in combi-
nation with the transcription factors Oct4, Sox2 and C-Myc, can
reprogramme MEFs into iPS cells and these colonies showed
features of pluripotent ES cells.45
Given their interesting structure and biological activities,
several groups have developed efficient syntheses to these com-
pounds. These include functionalization of the 2-arylindole core
structure built by traditional Fischer indole synthesis,39,46 the
cyclodehydration of an N-monosubstituted α-aminonitrile
obtained by Strecker reaction of a protected 2-amino-benzal-
dehyde with ethyl 2-aminocinnamate,47 the radical cyclization of
iodoacetamides48 and metal-catalyzed transformations. Among
the latter approaches the Heck reaction,49,50 oxidative coupling
after rhodium(III)-catalyzed C–H functionalization of acetamides
with alkynes,51 the borylation–Suzuki coupling52 and the Stille
reaction53 have all been employed inter- and/or intramolecularly
with various degrees of success.
The presence of a haloindole fragment in both kenpaullones
16 and EX527/EX243 13a,b (Fig. 2) is suggestive of a similar
pharmacophore in the interaction with SIRT enzymes. To our
knowledge there have been no follow-up studies on the epige-
netic activities of the paullones. In view of this we extended the
previous work by Jung and co-workers37 and set out to prepare
novel analogues of the basic 7,12-dihydroindolo[3,2-d][1]benza-
zepine-6(5H)-one skeleton substituted at the C2-aryl ring and
expanded the targets to the C9–Br (kenpaullones) and C10–Br
analogues.
EX243 13b34 is a nanomolar (IC50 = 120 nM) inhibitor (nicoti-
namide pocket binder) that shows about 23 times greater selec-
tivity for SIRT1 than for SIRT2.35 Nitrile AKG2 14 was
identified by docking a focused library into a model of human
Sirt236 and it was proposed to occupy pocket C, thus competing
for the nicotinamide part of NAD+.14–16 Kinase inhibitor Ro-
318220 15, on the other hand, was considered to inhibit sirtuins
by competing with the adenosine subpocket through the planar
ring system of the maleimide group whereas the polar thioami-
dine functional group occupies a second binding site.37 In the
same campaign directed towards finding novel structures within
those that target the adenosine binding pocket of enzymes or
receptors, N-benzylkenpaullone 16b and hydroxyamidineken-
paullone 17 were identified as SIRT inhibitors,37 and were
shown to compete for the NAD+ binding site.
The 7,12-dihydroindolo[3,2-d][1]benzazepine-6(5H)-ones or
paullones were first discovered during efforts directed towards
finding inhibitors of cyclin–dependent kinases (CDKs, CDK-1,
-2 and -5) based on the structure of flavopiridol, a semisynthetic
flavonoid.38,39 Together with the in vitro antiproliferative
activity,40 some members of the paullone family showed antitu-
mour39,41 and antileishmanial42 activities. In addition, cazpaul-
lone (9-cyano-1-aza-paullone) and analogues are inhibitors of
glycogen synthase kinase-3 (GSK-3), an activity that might be
relevant for the treatment of diabetes.43 In fact, alsterpaullone
(the C9-nitroderivative) acted in small screenings as a suppressor
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Scheme 3 a. Pd(PPh3)4, NaHCO3, DME/H2O, reflux, 27, 14%; 28aa,
29%. b. Pd(OAc)2, dppf, CuCl, Cs2CO3, DMF; 100 °C for 24a; 28aa,
50%; 80 °C for 25a; 28aa, 84%.
Scheme 1 Retrosynthetic analysis of the 7,12-dihydroindolo[3,2-d][1]
benzazepine-6(5H)-one skeleton.
using 22a (and N-modified derivatives) and commercial 2-iodo-
aniline 23 as partners was successful. Among them, we tested
the phosphine-free palladium-catalyzed arylation of unsubsti-
tuted (NH)-indoles described by Sames64 and related methods
that use co-catalysis by other metals, such as Fagnou’s catalytic
oxidative procedure with AgOAc;65 Larrosa’s phosphine-free
Pd(OAc)2-Ag2O-mediated synthesis;66 and Albericio-Lavilla’s
post-synthetic modification of tryptophans in peptides assisted
by Pd(OAc)2-AgBF4.67 Alternatively, Gaunt’s reaction of N-acet-
Results and discussion
A rather straightforward retrosynthetic approach to the paullone
skeleton (28) was considered first and this involved a direct C–H
arylation54–57 of the corresponding indole acetates with aniline
derivatives followed by the intramolecular amide bond formation
(Scheme 1). This method was deemed more convenient than the
cross-coupling, since functionalization of the precursor(s) was
not required or could be restricted to one of the components.
Methyl indoleacetate 22a58 was prepared from the commercial
acid 21a after in situ preparation of the acid chloride. The same
step was used for the preparation of brominated analogues 22b59
and 22c60 from the acids 21b and 21c,61 which in turn were
obtained by hydrolysis of the corresponding nitriles 20b62 and
20c.61 These intermediates were produced from commercial 5-
and 6-bromoindoles 18b and 18c, respectively, in a three-step
sequence comprising the alkylation at C3 with Eschenmoser’s
salt, followed by N-methylation of 19b,c and sodium cyanide-
induced substitution of the non-isolated quaternary ammonium
salts (Scheme 2).60
68
ylindoles with aryliodonium salts catalyzed by Cu(OTf)2 also
failed to provide the desired coupled product either under intra-
molecular or intermolecular fashion. In all cases, the control
experiments afforded the reported C-2 substituted indoles. It is
likely that the presence of a substituent at C3 of the indole
moiety and ortho to the aniline unit makes the coupling sterically
more difficult than in the unsubstituted cases reported to date.
With 26a in hand (commercial or prepared from the bromide
23a), classical Suzuki coupling conditions (Pd(PPh3)4, NaHCO3,
DME/H2O) were assayed with 25a,69 which afforded, after 8 h
under reflux, a mixture of the primary coupling product 27 and
the paullone skeleton 28aa in 14 and 29% yield, respectively
(Scheme 3). Longer reaction times led to deterioration of the pro-
ducts. The reaction of the coupling partners in water/dioxane
with Ba(OH)2·8H2O, Pd(OAc)2, bis(cyclohexyl)biphenylphos-
phine at 100 °C52 led to ester hydrolysis and incomplete conver-
sions. Interestingly, when CuCl was added to the reaction
mixture containing Pd(OAc)2 and dppf as pre-catalyst in DMF,
presumably to favour B-to-Cu transmetallation,70 the paullone
skeleton 28aa was obtained in 84% yield. Both the Suzuki
cross-coupling and the intramolecular formation of the amide
took place in the same pot under the reaction conditions
(Scheme 3). We also confirmed that the coupling of the C2-bro-
moindole 24a required higher temperatures and afforded lower
yields than the corresponding C2-iodoindole 25a under other-
wise identical conditions.
Neither of the protocols reported for the direct selective C2-
arylation of indoles, a method pioneered by Ohta,63 assayed
We next turned our attention to C–C bond formation between
the indole and the aryl units using in particular a Suzuki–
Miyaura cross-coupling reaction.71 In order to select the best
strategy to generate the organoboronic acid (or derivatives)
partner, the reciprocal combination of functional groups on both
coupling components (at the C2-position of the indole and at the
ortho-position of the aniline) was investigated. Halogenation of
the indole derivatives 22a–c was straightforward using NBS and
radical initiation for X = Br (24a)72 and silver triflate-assisted
iodination was successful for X = I (25a–c) using anhydrous
AgOTf (Scheme 2).73
Scheme 2 a. Me2N+vCH2I−, 19 : 1 CH3CN/AcOH; b. MeI, EtOH,
25 °C; c. NaCN, DMF, H2O, 70 °C; 20b, 69%; 20c, 58% for the three
steps. d. KOH, H2O, 100 °C, then HCl, 0 °C, 21b, 97%; 21c, 75%;
e. SOCl2, HOBt, MeOH, 0 to 25 °C; 22a, 99%; 22b, 99%; 22c,
80%. f. NBS, (PhCO)2O, CCl4, 25 °C, 78%; g. AgOTf, I2, THF, 25a,
86%; 25b, 79%; 25c, 85%; h. PdCl2(dppf), CH2Cl2, HB(pin), Et3N,
dioxane, 100 °C; 26a, 84%; 26b, 63%; 26c, 58%; 26d, 68%.
The preparation of the C2-indolyl pinacolboronate by
Miyaura-type palladium-catalyzed borylation of N-benzyl-2-
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bromoindoles74 failed despite variations in the nature of the pre-
catalysts and ligands, heating in the range 80–110 °C or under
microwave irradiation or adding Et3N. In most cases the dehalo-
genated substrate was obtained (for the alternative preparation of
2-borylindoles, see ref 75). We then turned our attention to the
palladium-catalyzed borylation of ortho- iodoanilines.52 Despite
our efforts, we could not obtain the high yields reported for the
reaction starting from o-iodoanilines and their acetate deriva-
tives52 using the same conditions [(Pd(OAc)2, bis(cyclohexyl)
biphenylphosphine (1 : 4 ratio), Et3N and HB(pin) in dioxane at
80 °C; a yield of 18% at most was obtained with o-iodoaniline,
25% if purified by reverse phase chromatography using MeOH/
H2O as eluent] or modified reaction conditions [using Pd(OAc)2
and bis(cyclohexyl)biphenylphosphine or PdCl2(dppf)·CH2Cl2,
pinacolborane and Et3N in dioxane].76 On the other hand, the
borylation of o-iodoaniline 23a using Buchwald’s improved pro-
cedure77 with PdCl2(CH3CN)2, bis(cyclohexyl)biphenylpho-
sphine, Et3N and HB(pin) in dioxane at 110 °C was incomplete
and afforded the product 26a (Scheme 2) together with the deha-
logenated aniline in a 53% combined yield. Moreover, boronate
26a proved to be unstable to purification by column chromato-
graphy. Given this shortcoming, we decided to carry out the
combined borylation–Suzuki coupling sequentially without iso-
lation of the intermediate. To this end, treatment of 2-iodoaniline
23a with PdCl2(CH3CN)2, bis(cyclohexyl)biphenylphosphine
(1 : 4 ratio), Et3N and HB(pin) in dioxane at 110 °C,77 followed
by the appropriate work-up, afforded a residue that was immedi-
ately treated with methyl 2-iodoindoleacetate 25a followed by
the addition of Pd(OAc)2, dppf, CuCl and Cs2CO3 in DMF and
heating at 80 °C. The desired product was isolated in moderate
yield (56%), and small amounts of the unreacted starting indole
remained. Application of these conditions to methyl 5-bromo-2-
iodoindoleacetate 25b led to the desired product 28ba in a disap-
pointing 24% yield. Likewise, sequential borylation/Suzuki
cross-coupling starting from substituted anilines 26b–c led to the
corresponding paullones 28ab and 28ac in moderate yields
(31–33%).
Scheme 4 General synthesis of 7,12-dihydroindolo[3,2-d][1]benzaze-
pine-6(5H)-ones by one-pot Suzuki cross-coupling/amidation.
When the borylation reaction was performed under the con-
ditions reported by Hashimoto et al.78 using PdCl2(dppf)·CH2-
Cl2, the yield of the reaction product 26a–d was considerably
improved (Scheme 2). Further optimization of the cross-coupling
confirmed the requirement for 1 equivalent of CuCl, since the
use of substoichiometric quantities led to longer reaction times
and lower yields. Improved yields also resulted when the reac-
tion time was shortened (2 h instead of 12 h), the work-up was
carried out with ammonium chloride and the residue was
purified by crystallization (Scheme 3). Applying these optimized
conditions to the coupling of the fragments 25a–c and 26a–d led
to the 7,12-dihydroindolo[3,2-d][1]benzazepine-6(5H)-ones
28xx in good-to-excellent yields (Scheme 4). Of these com-
pounds, 28aa and 28ba are known.79 Starting from the bromi-
nated indoles chemoselective coupling at the C2-I position was
exclusively obtained.
Fig. 3 Effects of paullones on cell cycle and granulocytic differen-
tiation in the U937 acute myeloid leukemia cell line. A: Cytofluorimetric
cell cycle analysis of U937 cells after treatment with the synthetic com-
pounds at 5 μM and 50 μM for 30h. B: Cytofluorimetric differentiation
analysis. CD11c expression after treatment with paullones at 5 μM and
50 μM for 30 h; MS-275 (5 μM) was used as positive control. The data
represent the average value of independent triplicates.
carried out in the acute myeloid leukemia cell line U937 to
assess the anti-proliferative potential and the alteration of cell
cycle induced by the synthetic compounds. Compared to the
vehicle-treated cells, the paullones produced some significant
alterations on the cell cycle (Fig. 3A). In particular, the com-
pounds at 50 μM caused an increase of the pre-G1 phase. More-
over, some of these compounds induced granulocytic
Biological evaluation
In vitro and in vivo tests were performed to evaluate the inhibi-
tory activity of these novel paullones. The latter tests were
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Fig. 5 In vitro cdc2 activity assay. The cdc2 inhibitory activity of com-
pounds 28 was tested at 50 μM. The inhibition value was reported as
percentage of residual activity in comparison to the control without com-
pound incubation and to the known cdc2 inhibitor olomoucine at 10 μM
concentration. The data represent the average value of independent
triplicates.
amide formation to generate the 7,12-dihydroindolo[3,2-d][1]
benzazepine-6(5H)-one skeleton.
SAR studies are only available for the CDK inhibition39,80
and for antileishmanial activity42 but not for SIRT inhibition or
other epigenetic modulatory activities of the paullones. We tested
the ability of the series of compounds to alter the cell cycle and
induce differentiation of the acute myelogenous cell line U937
and carried out an in vitro enzymatic assay with recombinant
human sirtuin-1. The analogue of kenpaullone with a methoxy-
carbonyl group at C2 is more than one order of magnitude more
potent than N-benzylkenpaullone, thus showing that substituents
at the anilide ring can indeed improve the SIRT1 inhibitory
profile of this scaffold relative to other activities such as the inhi-
bition of CDC2/Cdk1.
Fig. 4 In vitro SIRT1 activity analysis. The SIRT1 inhibitory activity
of paullones was tested at 50 μM. The inhibition value was reported as
percentage of residual activity in comparison to the control without com-
pound incubation and to the known SIRT1 inhibitor EX-527 13a. The
data represent the average value of independent triplicates.
differentiation of U937 cells after treatment at 5 μM and 50 μM
for 30 h (Fig. 3B), as indicated by the increased expression of
the granulocytic differentiation cell-surface marker CD11c using
FACS analysis.
In vitro assays with recombinant human sirtuin-1 revealed that
paullones are potent inhibitors of SIRT1 (Fig. 4A). On average
they reduce SIRT1 activity by up to 45%. For the most potent
inhibitor of the series, 28bc, an IC50 of 0.215 μM was deter-
mined (Fig. 4B), and therefore this compound surpasses the
activity of N-benzylkenpaullone 16b (Fig. 2, IC50 = 8 μM).
As the paullones have been reported as CDK inhibitors38,39,43
we tested the synthesized compounds in an enzymatic assay with
Cdc/Cdk1. As seen in Fig. 5, all the compounds were roughly
equally active as inhibitors, with a potency similar to kenpaul-
lone (28ba). Therefore compound 28bc is more selective for
SIRT1 than for Cdk1, and this finding suggests that additional
studies, in combination with Molecular Modeling, are warranted
to increase the selectivity profile of the series.
Experimental section
General procedures
Reagents and solvents were purchased as reagent-grade and used
without further purification unless otherwise stated. Solvents
were dried according to standard methods and distilled before
use or dispensed from a Puresolv™ solvent purification system
of Innovative Technology, Inc. All reactions were performed in
oven-dried or flame-dried glassware under an inert atmosphere
of Ar unless otherwise stated. Chromatography refers to flash
chromatography (FC) on SiO2 60 (230–400 mesh) from Merck,
head pressure of ca. 0.2 bar. TLC: UV254 SiO2-coated plates
from Merck, visualization by UV light (254 nm) or by spraying
with a 15% ethanolic phosphomolybdic acid solution. NMR
spectra were recorded in a Bruker AMX400 (400.13 MHz and
100.61 MHz for proton and carbon respectively) spectrometer at
298 K with residual solvent peaks as internal reference and the
chemical shifts are reported in δ [ppm], coupling constants J are
given in [Hz] and the multiplicities assigned with DEPT exper-
iments and expressed as follows: s = singlet, d = doublet, t =
Conclusion
A novel synthesis of paullones substituted at C9/C10 (Br) and
C2 (for the synthesis of other members with substituents at C9
and C2, see ref. 42) has been developed and is based on a one-
pot Suzuki–Miyaura cross-coupling of an o-aminoarylboronic
acid and a C2-iodoindoleacetic acid followed by intramolecular
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triplet, q = quartet, m = multiplet. For fluorinated compounds, a
second multiplicity corresponding to the 13C–19F coupling is
given in parenthesis together with the JC–F coupling constant.
COSY, HMBC and HSQC methods were used to establish atom
connectivities. Electrospray ionization (ESI) mass spectra were
recorded on a Bruker APEX3 instrument. Infrared spectra (IR)
were obtained on a JASCO FT/IR-4200 infrared spectrometer.
Peaks are quoted in wave numbers (cm−1) and their relative
intensities are reported as follows: s = strong, m = medium, w =
weak. Melting points were measured in a Stuart Scientific appar-
atus. Elemental analyses were carried out in a Fisons EA-1108
elemental analyzer. Irradiation with microwaves was performed
using a CEM DISCOVER apparatus.
indoles at C2, the reaction of methyl 2-(6-bromo-1H-indol-3-yl)
acetate 22c (250 mg, 0.93 mmol), AgOTf (287.5 mg,
1.12 mmol; 23.9 mg, 0.09 mmol) and I2 (236.7 mg, 0.93 mmol)
in THF (7.8 mL) afforded, after purification by column chrom-
atography (silicagel, from 90 : 10 to 70 : 30 hexane/EtOAc),
compound 25c as a light brown solid (312.7 mg, 85%). m.p.:
135–137 °C (hexane/ethyl acetate). 1H-NMR (400.13 MHz,
CDCl3): δ 8.21 (br, 1H, NH), 7.38 (d, J = 1.8 Hz, 1H, H7), 7.37
(d, J = 8.4 Hz, 1H, H4), 7.19 (dd, J = 8.4, 1.6 Hz, 1H, ArH),
3.70 (s, 3H, CH3), 3.69 (s, 2H, CH2) ppm. 13C-NMR
(100.61 MHz, CDCl3): δ 171.4 (s), 139.3 (s), 126.2 (s), 123.5
(d), 119.3 (d), 116.2 (s), 115.3 (s), 113.3 (d), 80.4 (s), 52.2 (q),
32.8 (t) ppm. HRMS (ESI+): calcd. for C11H981BrINNaO2 ([M
+ Na]+), 417.8739; found 417.8732. Calcd. for C11H9
-
79
BrINNaO2 ([M + Na]+), 415.8754; found 415.8752. IR (NaCl):
ν 3323 (br, NH), 3000 (w, C–H), 2949 (w, C–H), 1723 (s,
2-(5-Bromo-1H-indol-3-yl)acetic acid 21b. To a solution of 2-
(5-bromo-1H-indol-3-yl)acetonitrile 20b (0.86 g, 3.68 mmol) in
MeOH (4.6 mL) was added a solution of KOH (1.67 g,
29.78 mmol) in water (14.9 mL) and the mixture was heated at
100 °C for 2 h. The reaction was diluted with water, cooled to
0 °C and treated with a 6 M aqueous solution of HCl until pH
1. The solid was filtered, washed twice with water and dried
under high vacuum to give compound 21b as a white solid
(1.037 g, 97%). m.p.: 144–146 °C (methanol). 1H-NMR
(400.13 MHz, CD3OD): δ 7.67 (d, J = 1.8 Hz, 1H, ArH), 7.25
(d, J = 8.6 Hz, 1H, ArH), 7.19 (s, 1H, ArH), 7.17 (dd, J = 8.6,
1.8 Hz, 1H, ArH), 3.67 (s, 2H, CH2) ppm. 13C-NMR
(100.61 MHz, CD3OD): δ 176.1 (s), 136.7 (s), 130.5 (s), 126.3
(d), 125.2 (d), 122.2 (d), 113.9 (d), 113.1 (s), 108.9 (s), 31.8 (t)
ppm. HRMS (ESI+): calcd. for C10H881BrNNaO2 ([M + Na]+),
277.9610; found, 277.9608. Calcd. for C10H879BrNNaO2 ([M +
Na]+), 275.9631; found, 275.9629. IR (NaCl): ν 3500–3100 (br,
OH), 3418 (s, NH), 3085 (w, C–H), 1703 (s, CvO), 1456 (m),
CvO), 1437 (m), 1329 (m), 801 (m) cm−1
.
4-Methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)
aniline 26b. General procedure for the borylation of anilines. To
a solution of 2-iodo-4-methylaniline 23b (1.0 g, 4.29 mmol) in
1,4-dioxane (11.0 mL) were added Et3N (2.4 mL, 17.16 mmol),
PdCl2(dppf)·CH2Cl2 (175.2 mg, 0.21 mmol) and pinacolborane
(1.9 ml, 12.87 mmol). The reaction mixture was heated to
100 °C for 5 h. After cooling down to ambient temperature, the
reaction mixture was filtered through Celite eluting with EtOAc.
After removal of the solvent under vacuum, the residue was
purified by column chromatography (silicagel, from 90 : 10 to
60 : 40 hexane/AcOEt), to afford compound 26b as a white solid
(628.4 mg, 63%). m.p.: 71–73 °C (hexane/ethyl acetate).
1H-NMR (400.13 MHz, CD3OD): δ 7.34 (d, J = 1.6 Hz, 1H,
ArH), 6.97 (dd, J = 8.2, 1.9 Hz, 1H, ArH), 6.55 (d, J = 8.2 Hz,
1H, ArH), 4.82 (s, 2H, NH2), 2.15 (s, 3H, CH3), 1.29 (s, 12H,
4×CH3) ppm. 13C-NMR (100.61 MHz, CD3OD): δ 152.8 (s),
137.6 (d), 134.5 (d), 126.8 (s), 116.6 (d), 112.5 (s), 84.6 (s, 2x),
25.3 (q, 4x), 20.6 (q) ppm. HRMS (ESI+): calcd. for
C13H21BNO2 ([M + H]+), 234.1662; found 234.1666. IR
(NaCl): ν 3483 (m, NH), 3385 (m, NH), 2980 (m, C–H), 2928
792 (m) cm−1
.
Methyl 2-(5-bromo-2-iodo-1H-indol-3-yl)acetate 25b. A sol-
ution of I2 (236.7 mg, 0.93 mmol) in THF (5.5 mL) was added
dropwise to a solution of methyl 2-(5-bromo-1H-indol-3-yl)
acetate 22b (250 mg, 0.93 mmol) and AgOTf (287.5 mg,
1.12 mmol) in THF (2.3 mL). Then, a second portion of AgOTf
(23.9 mg, 0.09 mmol) was added and the mixture was stirred at
ambient temperature for 30 min before the addition of an
aqueous saturated solution of Na2S2O3. The mixture was
extracted with EtOAc (2x) and the combined organic layers were
washed with brine, dried and the solvent was evaporated under
vacuum to afford, after purification by column chromatography
(silicagel, from 90 : 10 to 50 : 50 hexane/AcOEt), compound 25b
as a white solid (291 mg, 79%). m.p.: 115–118 °C (hexane/ethyl
(w, C–H), 1621 (s), 1496 (s), 1357 (s, B–O), 1146 (s) cm−1
.
Methyl 4-amino-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)benzoate 26c. Following the general procedure described
above for the borylation of anilines, the reaction of methyl 4-
amino-3-iodobenzoate 23c (277.1 mg, 1 mmol), Et3N (557 μL,
4 mmol), PdCl2(dppf)·CH2Cl2 (40.8 mg, 0.05 mmol) and pina-
colborane (435 μL, 3 mmol) in 1,4-dioxane (2.6 mL) afforded,
after purification by column chromatography (silicagel, from
90 : 10 to 50 : 50 hexane/EtOAc), compound 26c as a white solid
(160.6 mg, 58%). m.p.: 167–169 °C (hexane/ethyl acetate).
1H-NMR (400.13 MHz, DMSO-d6): δ 8.05 (d, J = 2.1 Hz, 1H,
ArH), 7.71 (dd, J = 8.7, 2.2 Hz, 1H, ArH), 6.63 (d, J = 8.7 Hz,
1H, ArH), 6.24 (br, 1H, NH), 3.74 (s, 3H, CH3), 1.30 (s, 12H,
4×CH3) ppm. 13C-NMR (100.61 MHz, DMSO-d6): δ 166.0 (s),
158.3 (s), 138.9 (d), 133.6 (d), 115.5 (d), 113.6 (s), 108.0 (s),
83.5 (s, 2x), 51.1 (q), 24.5 (q, 4x) ppm. HRMS (ESI+): calcd.
for C14H21BNO4 ([M + H]+), 278.1558; found 278.1554. IR
(NaCl): ν 3481 (m, NH), 3374 (m, NH), 2981 (m, C–H), 1702
(s, CvO), 1608 (s), 1430 (m), 1371 (m, B–O), 1282 (s), 1246
1
acetate). H-NMR (400.13 MHz, CDCl3): δ 8.28 (br, 1H, NH),
7.64 (s, 1H, ArH), 7.2–7.1 (m, 1H, ArH), 7.1–7.0 (m, 1H, ArH),
3.71 (s, 3H, CH3), 3.67 (s, 2H, CH2) ppm. 13C-NMR
(100.61 MHz, CDCl3): δ 171.4 (s), 137.3 (s), 128.9 (s), 125.3
(d), 120.6 (d), 114.6 (s), 113.6 (s), 111.8 (d), 81.4 (s), 52.2 (q),
32.7 (t) ppm. HRMS (ESI+): calcd. for C11H981BrINNaO2 ([M
79
+ Na]+), 417.8733; found 417.8730. Calcd. for C11H9
-
BrINNaO2 ([M + Na]+), 415.8754; found 415.8750. IR (NaCl):
ν 3326 (br, NH), 3000 (w, C–H), 2949 (w, C–H), 1723 (s,
CvO), 1438 (m), 1334 (m), 795 (m) cm−1
.
Methyl 2-(6-bromo-2-iodo-1H-indol-3-yl)acetate 25c. Follow-
ing the general procedure described above for the iodination of
(s), 1146 (s) cm−1
.
2106 | Org. Biomol. Chem., 2012, 10, 2101–2112
This journal is © The Royal Society of Chemistry 2012

View Article Online
2-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-4-(trifluoro-
methyl)aniline 26d. Following the general procedure described
above for the borylation of anilines, the reaction of 2-iodo-4-
(trifluoromethyl)aniline 23d (1.0 g, 3.48 mmol), Et3N (1.9 mL,
13.94 mmol), PdCl2(dppf)·CH2Cl2 (142.3 mg, 0.17 mmol) and
pinacolborane (1.3 mL, 10.45 mmol) in 1,4-dioxane (8.9 mL)
afforded, after purification by column chromatography (silicagel,
from 90 : 10 to 20 : 80 hexane/EtOAc), compound 26d as a
white solid (676 mg, 68%). m.p.: 115–117 °C (hexane/ethyl
(18.8 mg, 0.19 mmol), Pd(OAc)2 (2.1 mg, 0.01 mmol) and dppf
(10.6 mg, 0.02 mmol) in DMF (1.9 mL) afforded, after purifi-
cation by recrystallization, compound 28ba as a brown solid
(55.4 mg, 89%). m.p.: >300 °C (ethanol/hexane). 1H-NMR
(400.13 MHz, DMSO-d6): δ 11.81 (s, 1H, NH), 10.11 (s, 1H,
NH), 7.91 (d, J = 1.5 Hz, 1H, ArH), 7.74 (d, J = 7.3 Hz, 1H,
ArH), 7.5–7.4 (m, 2H, ArH), 7.3–7.2 (m, 3H, ArH), 3.52 (s, 2H,
CH2) ppm. 13C-NMR (100.61 MHz, DMSO-d6): δ 171.4 (s),
136.0 (s), 135.6 (s), 133.9 (s), 128.4 (d), 128.2 (s), 126.9 (d),
124.4 (d), 123.6 (d), 122.3 (s), 122.2 (d), 120.3 (d), 113.3 (d),
111.6 (s), 107.1 (s), 31.3 (t) ppm. MS (EI): m/z (%) 328 (M+,
81Br, 100), 326 (M+, 79Br, 91), 299 (98), 297 (100), 218 (94).
HRMS (EI): calcd. for C16H1181BrN2O ([M]+), 328.0034;
found, 328.0047. Calcd. for C16H1179BrN2O ([M]+), 326.0055;
found, 326.0056. IR (NaCl): ν 3217 (br, NH), 1642 (m, CvO),
1401 (w), 676 (w) cm−1. Purity: 90% (RPHPLC-ESI, Sunfire™
C18 5 μm, 46 × 250 mm, gradient A/B, 0 : 100 to 100 : 0,
30 min, 1 mL min−1, tR = 18 min, A: CH3CN/HCOOH 99 : 1,
B: H2O/HCOOH 99 : 1).74
1
acetate). H-NMR (400.13 MHz, CD3OD): δ 7.69 (d, J = 1.7
Hz, 1H, ArH), 7.34 (dd, J = 8.7, 2.0 Hz, 1H, ArH), 6.66 (d, J =
8.6 Hz, 1H, ArH), 4.82 (br s, 2H, NH2), 1.35 (s, 12H, 4×CH3)
ppm. 13C-NMR (100.61 MHz, CD3OD): δ 158.8 (s), 134.9 (d)
3
3
(q, JC–F = 3.8 Hz), 130.3 (d) (q, JC–F = 3.5 Hz), 126.7 (s) (q,
1JC–F = 269.2 Hz, CF3), 118.4 (s) (q, JC–F = 32.3 Hz, C-CF3),
2
115.3 (d), 110.1 (s), 85.3 (s, 2x), 25.2 (q, 4×CH3) ppm. HRMS
(ESI+): calcd. for C13H18BF3NO2 ([M + H]+), 288.1380; found
288.1383. IR (NaCl): ν 3473 (m, NH), 3378 (w, NH), 2981 (w,
C–H), 1627 (m), 1374 (s, B–O), 1320 (s, B–O), 1149 (s), 1101
(s) cm−1
.
10-Bromo-7,12-dihydroindolo[3,2-d]benzazepin-6-(5H)-one
28ca. Following the general procedure described above for the
synthesis of kenpaullone analogues, the reaction of methyl 2-(6-
bromo-2-iodo-1H-indol-3-yl)acetate 25c (75.0 mg, 0.19 mmol),
7,12-Dihydroindolo[3,2-d]benzazepin-6(5H)-one 28aa. General
procedure for the synthesis of kenpaullone analogues. In a
Schlenk tube, methyl 2-(2-iodo-1H-indol-3-yl)acetate 25a
(60 mg, 0.19 mmol), 2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)aniline 26a (62.6 mg, 0.29 mmol), Cs2CO3 (248.2 mg,
0.76 mmol), CuCl (18.8 mg, 0.19 mmol), Pd(OAc)2 (2.1 mg,
0.01 mmol), dppf (10.6 mg, 0.02 mmol) and anhydrous DMF
(1.9 mL) were added. After the mixture was purged, the tube
was closed and heated to 80 °C for 2 h. The reaction was cooled
down, EtOAc and a saturated aqueous solution of NH4Cl were
added and the mixture was stirred for 30 min. After extracting
with EtOAc, the combined organic layers were washed with
H2O, dried (Na2SO4) and the solvent was evaporated. The
residue was recrystallized in EtOH and washed with hexane to
afford compound 28aa as a brown solid (43.6 mg, 92%). m.p.:
2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline
26a
(62.6 mg, 0.29 mmol), Cs2CO3 (248.2 mg, 0.76 mmol), CuCl
(18.8 mg, 0.19 mmol), Pd(OAc)2 (2.1 mg, 0.01 mmol) and dppf
(10.6 mg, 0.02 mmol) in DMF (1.9 mL) afforded, after purifi-
cation by recrystallization, compound 28ca as a brown solid
(51.7 mg, 83%). m.p.: >300 °C (ethanol/hexane). 1H-NMR
(400.13 MHz, DMSO-d6): δ 10.12 (s, 1H, NH), 7.73 (d, J = 7.2
Hz, 1H, ArH), 7.65 (d, J = 8.4 Hz, 1H, ArH), 7.59 (s, 1H, ArH),
7.4–7.3 (m, 1H, ArH), 7.3–7.2 (m, 2H, ArH), 7.20 (d, J = 8.2
Hz, 1H, ArH), 3.51 (s, 2H, CH2) ppm. 13C-NMR (100.61 MHz,
DMSO-d6): δ 171.3 (s), 138.1 (s), 135.5 (s), 133.3 (s), 128.3 (d),
126.8 (d), 125.5 (s), 123.6 (d), 122.3 (s), 122.2 (d), 121.9 (d),
119.7 (d), 114.6 (s), 113.8 (d), 107.6 (s), 31.4 (t) ppm. MS (EI):
m/z (%) 328 (M+, 81Br, 100), 326 (M+, 79Br, 68), 299 (91), 297
(93), 218 (81). HRMS (EI): calcd. for C16H1181BrN2O ([M]+),
328.0034; found, 328.0025. Calcd. for C16H1179BrN2O ([M]+),
326.0055; found, 326.0043. IR (NaCl): ν 3263 (br, NH), 2923
(w, C–H), 1656 (s, CvO), 1430 (w), 1162 (w) cm−1. Purity:
96% (RPHPLC-ESI, Sunfire™ C18 5 μm, 46 × 250 mm, gradi-
ent A/B, 0 : 100 to 100 : 0, 30 min, 1 mL min−1, tR = 18 min, A:
CH3CN/HCOOH 99 : 1, B: H2O/HCOOH 99 : 1).74
1
>300 °C (ethanol/hexane). H-NMR (400.13 MHz, DMSO-d6):
δ 11.58 (br, 1H, NH), 10.08 (br, 1H, NH), 7.75 (d, J = 6.6 Hz,
1H, ArH), 7.66 (d, J = 7.8 Hz, 1H, ArH), 7.44 (d, J = 8.1 Hz,
1H, ArH), 7.37 (td, J = 7.7, 1.4 Hz, 1H, ArH), 7.3–7.2 (m, 2H,
ArH), 7.18 (t, J = 7.5 Hz, 1H, ArH), 7.08 (t, J = 7.5 Hz, 1H,
ArH), 3.51 (s, 2H, CH2) ppm. 13C-NMR (100.61 MHz, DMSO-
d6): δ 171.5 (s), 137.3 (s), 135.3 (s), 132.4 (s), 127.9 (d), 126.8
(d), 126.5 (s), 123.6 (d), 122.8 (s), 122.2 (d), 122.0 (d), 119.0
(d), 117.8 (d), 111.4 (d), 107.5 (s), 31.5 (t) ppm. HRMS (ESI+):
calcd. for C16H13N2O ([M + H]+), 249.1022; found, 249.1022.
IR (NaCl): ν 3220 (br, NH), 2922 (w, C–H), 2851 (w, C–H),
1641 (s, CvO), 1400 (w) cm−1. UV (MeOH): λmax 228,
314 nm. Purity: 90% (RPHPLC-ESI, Sunfire™ C18 5 μm, 46 ×
2-Methyl-7,12-dihydroindolo[3,2-d]benzazepin-6-(5H)-one
28ab. Following the general procedure described above for the
synthesis of kenpaullone analogues, the reaction of methyl 2-(2-
iodo-1H-indol-3-yl)acetate 25a (60 mg, 0.19 mmol), 4-methyl-
250 mm, gradient A/B, 0 : 100 to 100 : 0, 30 min, 1 mL min−1
,
tR = 16 min, A: CH3CN/HCOOH 99 : 1, B: H2O/HCOOH
2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline
26b
99 : 1).74
(66.6 mg, 0.29 mmol), Cs2CO3 (248.2 mg, 0.76 mmol), CuCl
(18.8 mg, 0.19 mmol), Pd(OAc)2 (2.1 mg, 0.01 mmol) and dppf
(10.6 mg, 0.02 mmol) in DMF (1.9 mL) afforded, after purifi-
cation by recrystallization, compound 28ab as a brown solid
9-Bromo-7,12-dihydroindolo[3,2-d]benzazepin-6-(5H)-one
28ba. Following the general procedure described above for the
synthesis of kenpaullone analogues, the reaction of methyl 2-(5-
bromo-2-iodo-1H-indol-3-yl)acetate 25b (75.0 mg, 0.19 mmol),
1
(40.9 mg, 82%). m.p.: 229–232 °C (ethanol/hexane). H-NMR
(400.13 MHz, DMSO-d6): δ 11.54 (s, 1H, NH), 9.98 (s, 1H,
NH), 7.64 (d, J = 7.7 Hz, 1H, ArH), 7.56 (s, 1H, ArH), 7.42 (d,
J = 8.0 Hz, 1H, ArH), 7.2–7.1 (m, 3H, ArH), 7.06 (t, J = 7.4 Hz,
2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)aniline
26a
(62.6 mg, 0.29 mmol), Cs2CO3 (248.2 mg, 0.76 mmol), CuCl
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 2101–2112 | 2107

View Article Online
1H, ArH), 3.47 (s, 2H, CH2), 2.38 (s, 3H, CH3) ppm. 13C-NMR
(100.61 MHz, DMSO-d6): δ 171.3 (s), 137.3 (s), 133.1 (s),
132.6 (s), 132.5 (s), 128.6 (d), 126.9 (d), 126.4 (s), 122.6 (s),
122.1 (d), 121.9 (d), 119.0 (d), 117.8 (d), 111.3 (d), 107.3 (s),
31.5 (t), 20.4 (q) ppm. HRMS (ESI+): calcd. for C17H15N2O
([M + H]+), 263.1179; found, 263.1167. IR (NaCl): ν 3235 (br,
([M]+), 342.0191; found, 342.0194. Calculated for
C17H1379BrN2O ([M]+), 340.0211; found, 340.0224. IR (NaCl):
ν 3207 (br, NH), 2923 (w, C–H), 1646 (s, CvO), 1409 (w),
1221 (w) cm−1. Purity: 96% (RPHPLC-ESI, Sunfire™ C18
5 μm, 46 × 250 mm, gradient A/B, 0 : 100 to 100 : 0, 30 min,
1 mL min−1, tR = 19 min, A: CH3CN/HCOOH 99 : 1, B: H2O/
HCOOH 99 : 1).
NH), 2922 (w, C–H), 1646 (s, CvO), 1509 (w), 1408 (w) cm−1
Purity: 98% (RPHPLC-ESI, Sunfire™ C18 5 μm, 46 ×
250 mm, gradient A/B, 0 : 100 to 100 : 0, 30 min, 1 mL min−1
.
,
Methyl 6-oxo-5,6,7,12-tetrahydroindolo[3,2-d]benzazepin-2-
carboxylate 28ac. Following the general procedure described
above for the synthesis of kenpaullone analogues, the reaction of
tR = 16 min, A: CH3CN/HCOOH 99 : 1, B: H2O/HCOOH
99 : 1).
methyl
2-(2-iodo-1H-indol-3-yl)acetate
25a
(60
mg,
9-Bromo-2-methyl-7,12-dihydroindolo[3,2-d]benzazepin-6-
(5H)-one 28bb. Following the general procedure described
above for the synthesis of kenpaullone analogues, the reaction of
methyl 2-(5-bromo-2-iodo-1H-indol-3-yl)acetate 25b (75.0 mg,
0.19 mmol), 4-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-
lan-2-yl)aniline 26b (66.6 mg, 0.29 mmol), Cs2CO3 (248.2 mg,
0.76 mmol), CuCl (18.8 mg, 0.19 mmol), Pd(OAc)2 (2.1 mg,
0.01 mmol) and dppf (10.6 mg, 0.02 mmol) in DMF (1.9 mL)
afforded, after purification by recrystallization, compound 28bb
as a brown solid (44.7 mg, 69%). m.p.: >300 °C (ethanol/
0.19 mmol), methyl 4-amino-3-(4,4,5,5-tetramethyl-1,3,2-dioxa-
borolan-2-yl)benzoate 26c (79.1 mg, 0.29 mmol), Cs2CO3
(248.2 mg, 0.76 mmol), CuCl (18.8 mg, 0.19 mmol), Pd(OAc)2
(2.1 mg, 0.01 mmol) and dppf (10.6 mg, 0.02 mmol) in DMF
(1.9 mL) afforded, after purification by recrystallization, com-
pound 28ac as a brown solid (50.0 mg, 86%). m.p.: >300 °C
1
(ethanol/hexane). H-NMR (400.13 MHz, DMSO-d6): δ 10.45
(s, 1H, NH), 8.39 (s, 1H, ArH), 7.92 (d, J = 7.8 Hz, 1H, ArH),
7.68 (d, J = 7.5 Hz, 1H, ArH), 7.46 (d, J = 7.5 Hz, 1H, ArH),
7.36 (d, J = 8.1 Hz, 1H, ArH), 7.2–7.1 (m, 1H, ArH), 7.1–7.0
(m, 1H, ArH), 3.90 (s, 3H, CH3), 3.58 (s, 2H, CH2) ppm.
13C-NMR (100.61 MHz, DMSO-d6): δ 171.3 (s), 165.6 (s),
139.1 (s), 137.6 (s), 131.4 (s), 128.3 (d), 128.2 (d), 126.3 (s),
124.4 (s), 122.5 (s), 122.4 (d), 122.1 (d), 119.1 (d), 118.0 (d),
111.5 (d), 107.9 (s), 52.0 (q), 31.6 (t) ppm. MS (EI): m/z (%)
306 (M+, 75), 305 (45), 277 (100), 218 (30). HRMS (EI): calcd.
for C18H14N2O3 ([M]+), 306.1004; found, 306.1006. IR (NaCl):
ν 3313 (br, NH), 2923 (w, C–H), 1707 (s, CvO), 1671 (s,
CvO), 1435 (m), 1257 (m) cm−1. Purity: 83% (RPHPLC-ESI,
Sunfire™ C18 5 μm, 46 × 250 mm, gradient A/B, 0 : 100 to
100 : 0, 30 min, 1 mL min−1, tR = 17 min, A: CH3CN/HCOOH
99 : 1, B: H2O/HCOOH 99 : 1).
1
hexane). H-NMR (400.13 MHz, DMSO-d6): δ 10.03 (s, 1H,
NH), 7.90 (s, 1H, ArH), 7.56 (s, 1H, ArH), 7.39 (d, J = 8.5 Hz,
1H, ArH), 7.27 (dd, J = 8.6, 1.5 Hz, 1H, ArH), 7.22 (d, J = 7.7
Hz, 1H, ArH), 7.15 (d, J = 8.3 Hz, 1H, ArH), 3.49 (s, 2H, CH2),
2.38 (s, 3H, CH3) ppm. 13C-NMR (100.61 MHz, DMSO-d6): δ
171.8 (s), 136.4 (s), 134.6 (s), 133.8 (s), 133.2 (s), 129.6 (d),
128.7 (s), 127.5 (d), 124.9 (d), 122.7 (d), 122.6 (s), 120.8 (d),
113.8 (d), 112.1 (s), 107.5 (s), 31.8 (t), 20.9 (q) ppm. MS (EI):
m/z (%) 342 (M+, 81Br, 94), 340 (M+, 79Br, 97), 313 (98), 311
(100), 233 (22), 231 (36). HRMS (EI): calcd. for
C17H1381BrN2O ([M]+), 342.0191; found, 342.0205. Calcd. for
C17H1379BrN2O ([M]+), 340.0211; found, 340.0222. IR (NaCl):
ν 3223 (br, NH), 2921 (w, C–H), 1646 (s, CvO), 1502 (w),
1302 (w), 973 (w) cm−1. Purity: 95% (RPHPLC-ESI, Sunfire™
C18 5 μm, 46 × 250 mm, gradient A/B, 0 : 100 to 100 : 0,
30 min, 1 mL min−1, tR = 19 min, A: CH3CN/HCOOH 99 : 1,
B: H2O/HCOOH 99 : 1).
Methyl 9-bromo-6-oxo-5,6,7,12-tetrahydroindolo[3,2-d]benza-
zepin-2-carboxylate 28bc. Following the general procedure
described above for the synthesis of kenpaullone analogues, the
reaction of methyl 2-(5-bromo-2-iodo-1H-indol-3-yl)acetate 25b
(75.0 mg, 0.19 mmol), methyl 4-amino-3-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)benzoate 26c (79.1 mg, 0.29 mmol),
Cs2CO3 (248.2 mg, 0.76 mmol), CuCl (18.8 mg, 0.19 mmol),
Pd(OAc)2 (2.1 mg, 0.01 mmol) and dppf (10.6 mg, 0.02 mmol)
in DMF (1.9 mL) afforded, after purification by recrystallization,
compound 28bc as a brown solid (32.6 mg, 44%). m.p.:
10-Bromo-2-methyl-7,12-dihydroindolo[3,2-d]benzazepin-6-
(5H)-one 28cb. Following the general procedure described
above for the synthesis of kenpaullone analogues, the reaction of
methyl 2-(6-bromo-2-iodo-1H-indol-3-yl)acetate 25c (75.0 mg,
0.19 mmol), 4-methyl-2-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-
lan-2-yl)aniline 26b (66.6 mg, 0.29 mmol), Cs2CO3 (248.2 mg,
0.76 mmol), CuCl (18.8 mg, 0.19 mmol), Pd(OAc)2 (2.1 mg,
0.01 mmol) and dppf (10.6 mg, 0.02 mmol) in DMF (1.9 mL)
afforded, after purification by recrystallization, compound 28cb
as a brown solid (52.5 mg, 81%). m.p.: >300 °C (ethanol/
1
>300 °C (ethanol/hexane). H-NMR (400.13 MHz, DMSO-d6):
δ 12.00 (s, 1H, NH), 10.46 (s, 1H, NH), 8.38 (s, 1H, ArH), 7.94
(ap. s, 2H, ArH), 7.41 (d, J = 8.6 Hz, 1H, ArH), 7.36 (d, J = 8.5
Hz, 1H, ArH), 7.30 (d, J = 8.8 Hz, 1H, ArH), 3.90 (s, 3H, CH3),
3.59 (s, 2H, CH2) ppm. 13C-NMR (100.61 MHz, DMSO-d6): δ
171.2 (s), 165.5 (s), 139.4 (s), 136.2 (s), 132.9 (s), 128.8 (d),
128.3 (d), 128.1 (s), 124.8 (d), 124.5 (s), 122.3 (d), 122.0 (s),
120.5 (d), 113.5 (d), 111.8 (s), 107.5 (s), 52.1 (q), 31.4 (t) ppm.
MS (EI): m/z (%) 386 (M+, 81Br, 97), 384 (M+, 79Br, 97), 358
(20), 357 (96), 356 (22), 355 (100). HRMS (EI): calcd. for
C18H1381BrN2O3 ([M]+), 386.0089; found, 386.0075. Calcd. for
C18H1379BrN2O3 ([M]+), 384.0110; found, 384.0122. IR
(NaCl): ν 3312 (br, NH), 2922 (s, C–H), 2851 (m, C–H), 1706
(s, CvO), 1670 (s, CvO), 1434 (m), 1256 (m) cm−1. Purity:
1
hexane). H-NMR (400.13 MHz, DMSO-d6): δ 10.05 (s, 1H,
NH), 7.64 (d, J = 8.3 Hz, 1H, ArH), 7.58 (s, 1H, ArH), 7.55 (s,
1H, ArH), 7.2–7.1 (m, 3H, ArH), 3.48 (s, 2H, CH2), 2.38 (s, 3H,
CH3) ppm. 13C-NMR (100.61 MHz, DMSO-d6): δ 171.1 (s),
138.0 (s), 133.4 (s), 133.2 (s), 132.6 (s), 128.9 (d), 126.8 (d),
125.4 (s), 122.1 (d), 122.0 (s), 121.8 (d), 119.6 (d), 114.4 (s),
113.7 (d), 107.4 (s), 31.3 (t), 20.2 (q) ppm. MS (EI): m/z (%)
342 (M+, 81Br, 98), 340 (M+, 79Br, 99), 313 (96), 311 (100), 233
(18), 231 (32). HRMS (EI): calculated for C17H1381BrN2O
2108 | Org. Biomol. Chem., 2012, 10, 2101–2112
This journal is © The Royal Society of Chemistry 2012

View Article Online
96% (RPHPLC-ESI, Sunfire™ C18 5 μm, 46 × 250 mm, gradi-
ent A/B, 0 : 100 to 100 : 0, 30 min, 1 mL min−1, tR = 18 min, A:
CH3CN/HCOOH 99 : 1, B: H2O/HCOOH 99 : 1).
(75.0 mg, 0.19 mmol), 2-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-
lan-2-yl)-4-(trifluoromethyl)aniline 26d (82.0 mg, 0.29 mmol),
Cs2CO3 (248.2 mg, 0.76 mmol), CuCl (18.8 mg, 0.19 mmol),
Pd(OAc)2 (2.1 mg, 0.01 mmol) and dppf (10.6 mg, 0.02 mmol)
in DMF (1.9 mL) afforded, after purification by recrystallization,
compound 28bd as a brown solid (59.8 mg, 80%). m.p.:
Methyl 10-bromo-6-oxo-5,6,7,12-tetrahydroindolo[3,2-d]ben-
zazepin-2-carboxylate 28cc. Following the general procedure
described above for the synthesis of kenpaullone analogues, the
reaction of methyl 2-(6-bromo-2-iodo-1H-indol-3-yl)acetate 25c
(75.0 mg, 0.19 mmol), methyl 4-amino-3-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)benzoate 26c (79.1 mg, 0.29 mmol),
Cs2CO3 (248.2 mg, 0.76 mmol), CuCl (18.8 mg, 0.19 mmol),
Pd(OAc)2 (2.1 mg, 0.01 mmol) and dppf (10.6 mg, 0.02 mmol)
in DMF (1.9 mL) afforded, after purification by recrystallization,
compound 28cc as a brown solid (36.4 mg, 50%). m.p.:
1
>300 °C (ethanol/hexane). H-NMR (400.13 MHz, DMSO-d6):
δ 10.50 (s, 1H, NH), 8.10 (s, 1H, ArH), 7.98 (d, J = 1.4 Hz, 1H,
ArH), 7.75 (dd, J = 8.4, 1.4 Hz, 1H, ArH), 7.44 (dd, J = 8.5, 2.4
Hz, 2H, ArH), 7.32 (dd, J = 8.6, 1.6 Hz, 1H, ArH), 3.62 (s, 2H,
CH2) ppm. 13C-NMR (100.61 MHz, DMSO-d6): δ 171.3 (s),
138.6 (s), 136.2 (s), 132.5 (s), 128.0 (s), 125.0 (d), 124.7 (d)
3
3
(d, JC–F = 3.3 Hz), 124.1 (d) (d, JC–F = 3.8 Hz), 124.1 (s) (q,
1JC–F = 271.8 Hz, CF3), 123.7 (s), 122.8 (d), 122.3 (s), 120.6
(d), 113.5 (d), 111.8 (s), 108.0 (s), 31.3 (t) ppm. MS (EI): m/z
(%) 396 (M+, 81Br, 89), 394 (M+, 79Br, 100), 393 (43), 368 (17),
367 (98), 366 (21), 365 (96). HRMS (EI): calcd. for
C17H1081BrF3N2O ([M]+), 395.9908; found, 395.9894. Calcd.
for C17H1079BrF3N2O ([M]+), 393.9929; found, 393.9919. IR
(NaCl): ν 3293 (br, NH), 2925 (m, C–H), 1659 (s, CvO), 1317
(m), 1124 (m) cm−1. Purity: 84% (RPHPLC-ESI, Sunfire™
C18 5 μm, 46 × 250 mm, gradient A/B, 0 : 100 to 100 : 0,
30 min, 1 mL min−1, tR = 20 min, A: CH3CN/HCOOH 99 : 1,
B: H2O/HCOOH 99 : 1).
1
>300 °C (ethanol/hexane). H-NMR (400.13 MHz, DMSO-d6):
δ 10.48 (s, 1H, NH), 8.37 (s, 1H, ArH), 7.95 (d, J = 8.1 Hz, 1H,
ArH), 7.68 (d, J = 8.2 Hz, 1H, ArH), 7.61 (s, 1H, ArH), 7.37 (d,
J = 8.5 Hz, 1H, ArH), 7.22 (d, J = 8.6 Hz, 1H, ArH), 3.91 (s,
3H, CH3), 3.59 (s, 2H, CH2) ppm. 13C-NMR (100.61 MHz,
DMSO-d6): δ 171.2 (s), 165.5 (s), 139.2 (s), 138.3 (s), 132.3 (s),
128.7 (d), 128.2 (d), 125.4 (s), 124.5 (s), 122.2 (d), 122.1 (d),
122.0 (s), 119.9 (d), 115.0 (s), 113.9 (d), 108.0 (s), 52.1 (q),
31.5 (t) ppm. MS (EI): m/z (%) 386 (M+, 81Br, 100), 384 (M+,
79Br, 100), 358 (20), 357 (88), 356 (22), 355 (90). HRMS (EI):
calcd. for C18H1381BrN2O3 ([M]+), 386.0089; found, 386.0084.
Calcd. for C18H1379BrN2O3 ([M]+), 384.0110; found, 384.0112.
IR (NaCl): ν 3301 (br, NH), 2924 (w, C–H), 2852 (w, C–H),
1703 (m, CvO), 1670 (s, CvO), 1434 (w), 1287 (w), 1257 (w)
cm−1. Purity: 80% (RPHPLC-ESI, Sunfire™ C18 5 μm, 46 ×
10-Bromo-2-(trifluoromethyl)-7,12-dihydroindolo[3,2-d]benza-
zepin-6-(5H)-one 28cd. Following the general procedure
described above for the synthesis of kenpaullone analogues, the
reaction of methyl 2-(6-bromo-2-iodo-1H-indol-3-yl)acetate 25c
(75.0 mg, 0.19 mmol), 2-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-
lan-2-yl)-4-(trifluoromethyl)aniline 26d (82.0 mg, 0.29 mmol),
Cs2CO3 (248.2 mg, 0.76 mmol), CuCl (18.8 mg, 0.19 mmol),
Pd(OAc)2 (2.1 mg, 0.01 mmol) and dppf (10.6 mg, 0.02 mmol)
in DMF (1.9 mL) afforded, after purification by recrystallization,
compound 28cd as a brown solid (67.0 mg, 89%). m.p.:
250 mm, gradient A/B, 0 : 100 to 100 : 0, 30 min, 1 mL min−1
,
tR = 18 min, A: CH3CN/HCOOH 99 : 1, B: H2O/HCOOH
99 : 1).
2-(Trifluoromethyl)-7,12-dihydroindolo[3,2-d]benzazepin-6-
(5H)-one 28ad. Following the general procedure described
above for the synthesis of kenpaullone analogues, the reaction of
1
>300 °C (ethanol/hexane). H-NMR (400.13 MHz, DMSO-d6):
methyl
2-(2-iodo-1H-indol-3-yl)acetate
25a
(60
mg,
δ 10.50 (s, 1H, NH), 8.09 (s, 1H, ArH), 7.72 (d, J = 8.5 Hz, 1H,
ArH), 7.69 (d, J = 8.3 Hz, 1H, ArH), 7.63 (s, 1H, ArH), 7.45 (d,
J = 8.3 Hz, 1H, ArH), 7.23 (d, J = 8.5 Hz, 1H, ArH), 3.61 (s,
2H, CH2) ppm. 13C-NMR (100.61 MHz, DMSO-d6): δ 171.3
0.19 mmol), 2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-4-
(trifluoromethyl)aniline 26d (82.0 mg, 0.29 mmol), Cs2CO3
(248.2 mg, 0.76 mmol), CuCl (18.8 mg, 0.19 mmol), Pd(OAc)2
(2.1 mg, 0.01 mmol) and dppf (10.6 mg, 0.02 mmol) in DMF
(1.9 mL) afforded, after purification by recrystallization, com-
pound 28ad as a brown solid (59.5 mg, 99%). m.p.: >300 °C
3
(s), 138.5 (s), 138.3 (s), 131.9 (s), 125.4 (s), 124.7 (d) (d, JC–F
3
1
= 3.1 Hz), 124.0 (d) (d, JC–F = 4.0 Hz), 124.2 (s) (q, JC–F
=
266.5 Hz, CF3), 123.7 (s), 122.8 (d), 122.3 (s), 122.2 (d), 120.0
(d), 115.3 (s), 114.0 (d), 108.6 (s), 31.4 (t) ppm. MS (EI): m/z
(%) 396 (M+, 81Br, 91), 394 (M+, 79Br, 100), 393 (42), 368 (17),
367 (95), 366 (18), 365 (95). HRMS (EI): calcd. for
C17H1081BrF3N2O ([M]+), 395.9908; found, 395.9893. Calcd.
for C17H1079BrF3N2O ([M]+), 393.9929; found, 393.9915. IR
(NaCl): ν 3276 (br, NH), 2925 (w, C–H), 1658 (s, CvO), 1322
(s), 1123 (s) cm−1. Purity: 92% (RPHPLC-ESI, Sunfire™ C18
5 μm, 46 × 250 mm, gradient A/B, 0 : 100 to 100 : 0, 30 min,
1 mL min−1, tR = 20 min, A: CH3CN/HCOOH 99 : 1, B: H2O/
HCOOH 99 : 1).
1
(ethanol/hexane). H-NMR (400.13 MHz, DMSO-d6): δ 10.48
(s, 1H, NH), 8.10 (s, 1H, ArH), 7.8–7.7 (m, 2H, ArH), 7.5–7.4
(m, 2H, ArH), 7.3–7.2 (m, 1H, ArH), 7.11 (t, J = 7.5 Hz, 1H,
ArH), 3.60 (s, 2H CH2) ppm. 13C-NMR (100.61 MHz, DMSO-
d6): δ 171.5 (s), 138.3 (s), 137.6 (s), 131.0 (s), 126.3 (s), 124.3
3
1
(d) (d, JC–F = 3.3 Hz), 124.2 (s) (q, JC–F = 274.3 Hz, CF3),
3
123.9 (d) (d, JC–F = 3.6 Hz), 123.7 (s), 122.9 (s), 122.8 (d),
122.7 (d), 119.3 (d), 118.2 (d), 111.5 (d), 108.5 (s), 31.6 (t) ppm.
MS (EI): m/z (%) 316 (M+, 58), 315 (35), 288 (18), 287 (100),
286 (7). HRMS (EI): calcd. for C17H11F3N2O ([M]+), 316.0823;
found, 316.0826. IR (NaCl): ν 3253 (br, NH), 2923 (s, C–H),
2854 (m, C–H), 1654 (s, CvO), 1322 (m), 1127 (m) cm−1
.
Compounds
9-Bromo-2-(trifluoromethyl)-7,12-dihydroindolo[3,2-d]benza-
zepin-6-(5H)-one 28bd. Following the general procedure
described above for the synthesis of kenpaullone analogues, the
reaction of methyl 2-(5-bromo-2-iodo-1H-indol-3-yl)acetate 25b
MS-275 (Bayer-Schering AG), EX-527 (Alexis-Biochemicals)
and paullones were dissolved in DMSO (Sigma-Aldrich) and
used at 5 μM or 50 μM.
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 2101–2112 | 2109

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Cell lines
Cdc2 kinase activity assay
U937 (human leukemic monocyte lymphoma cell line-ATCC)
were grown in RPMI 1640 medium (Euroclone) supplemented
with 10% heat-inactivated FBS (Euroclone), 1% glutamin
(Lonza), 1% penicillin/streptomycin (Euroclone) and 0.1% gen-
tamycin (Lonza), at 37 °C in air and 5% CO2.
Cdc2 kinase activity assay was performed using the MESACUP
cdc2 kinase Assay Kit (Medical and Biological Laboratories Co,
Ltd.). 20 × 106 U937 cells were collected by centrifugation after
16 h stimulation with colcemid at a concentration of 0.02 μg
mL−1. U937 were lysed by adding lysis Buffer (50 mM Tris HCl
pH 7.5, 0.5 M NaCl, 5 mM EDTA, 2 mM EGTA, 0.01%
TritonX, 50 mM β-mercaptoethanol, 25 mM β-glycerophosphate,
1 mM sodium orthovanadate, 1 mM PMSF, 0.05 mg mL−1 leu-
peptin) and by sonication for 25 s at 400W cm−2. Cell extract
was separated by centrifugation at 105 × g for 1 h at 4 °C. U937
samples were preincubated with the paullones or reference com-
pound olomoucine (Promega), at 50 μM and 10 μM concen-
Cell cycle analysis
2.5 × 105 U937 cells were collected by centrifugation after 30 h
stimulation with reference compounds or paullones at 5 μM or
50 μM. The cells were resuspended in 500 μL of hypotonic
buffer (0.1% NP-40, 0.1% sodium citrate, 50 μg ml−1 PI,
RNAse A) and incubated in the dark for 30 min. The analysis
was performed by FACS-Calibur (Becton Dickinson) using the
Cell Quest Pro software (Becton Dickinson) and ModFit LT
version 3 software (Verity). The experiment was performed in
triplicate.
tration respectively, for
2 h at 37 °C in a specific
phosphorylation reaction mixture (1X cdc2 Reaction Buffer,
1mM ATP). Then the samples were incubated with Cdc2 kinase
substrate, biotinylated MV peptide, at 30 °C for 30 min. Follow-
ing supplier’s instruction, the final reaction mixtures were trans-
ferred to microwell strip coated with Monoclonal Antibody
(4A4) for ELISA cdc2 kinase activity detection. The activity of
cdc2 was measured by horseradish peroxidase substrate emission
at 492 nm with a microplate reader (TECAN Infinite M200).
Granulocytic differentiation analysis
2.5 × 105 U937 cells were collected by centrifugation after 30 h
stimulation with reference compound MS-275 at 5 μM concen-
tration or paullones at 5 μM and 50 μM. The cells were washed
with PBS and incubated in the dark at 4 °C for 30 min with
10 μL of PE-conjugated anti-CD11c surface antigen antibody or
with 10 μL of PE-conjugated IgG, in order to define the back-
ground signal. At the end of the incubation the samples were
washed again and resuspended in 500 μL of PBS containing
0.25 μg mL−1 PI. The analysis was performed by FACS-Calibur
(Becton Dickinson) using the Cell Quest Pro software (Becton
Dickinson). The experiment was performed in triplicate and PI
positive cells were excluded from the analysis.
Acknowledgements
The authors are grateful to the European Union (EPITRON,
LSHC-CT-2005-518417), the MICINN-Spain (SAF2010-17935-
FEDER to A.R. de L.; FPU Fellowship to S.S.), the Xunta de
Galicia (Grant 08CSA052383PR from DXI+D+i; Consolidación
2006/15 from DXPCTSUG; INBIOMED, to A.R. de L.), Minis-
tero Italiano dell’Istruzione Università e Ricerca (Programmi di
Ricerca Scientifica di Rilevante Interesse Nazionale n°
PRIN2009PX2T2E to L.A.) and Associazione Italiana per la
ricerca contro il cancro (AIRC to L.A.) for financial support.
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