Non-nucleoside inhibitors of human adenosine kinase: Synthesis, molecular modeling, and biological studies

Article abstract of DOI:10.1021/jm101438u

Adenosine kinase (AK) catalyzes the phosphorylation of adenosine (Ado) to AMP by means of a kinetic mechanism in which the two substrates Ado and ATP bind the enzyme in a binary and/or ternary complex, with distinct protein conformations. Most of the desc

Full text of DOI:10.1021/jm101438u

ARTICLE
pubs.acs.org/jmc
Non-Nucleoside Inhibitors of Human Adenosine Kinase: Synthesis,
Molecular Modeling, and Biological Studies
Stefania Butini,†,‡ Sandra Gemma,†,‡ Margherita Brindisi,†,‡ Giuseppe Borrelli,†,‡ Andrea Lossani,†,§
Anna Maria Ponte,†,§ Andrea Torti,†,§ Giovanni Maga,†,§ Luciana Marinelli,†,# Valeria La Pietra,†,# Isabella Fiorini,†,‡
Stefania Lamponi,†,‡ Giuseppe Campiani,*,†,‡ Daniela M. Zisterer,|| Seema-Maria Nathwani,|| Stefania Sartini,^
Concettina La Motta,^ Federico Da Settimo,^ Ettore Novellino,†,# and Federico Focher†,§
†European Research Centre for Drug Discovery and Development (NatSynDrugs), Universitꢀa di Siena, 53100 Siena, Italy
‡Dipartimento Farmaco Chimico Tecnologico, Via Aldo Moro, Universitꢀa di Siena, 53100 Siena, Italy
§Istituto di Genetica Molecolare, CNR, Via Abbiategrasso 207, 27100 Pavia, Italy
School of Biochemistry and Immunology, Trinity College, Dublin 2, Ireland
^Dipartimento di Scienze Farmaceutiche, Universitꢀa di Pisa, Via Bonanno, 6, 56126 Pisa, Italy
#Dipartimento di Chimica Farmaceutica e Tossicologica, Universitꢀa di Napoli Federico II, Via D. Montesano 49, 80131 Napoli, Italy
S Supporting Information
b
ABSTRACT: Adenosine kinase (AK) catalyzes the phosphorylation of
adenosine (Ado) to AMP by means of a kinetic mechanism in which the two
substrates Ado and ATP bind the enzyme in a binary and/or ternary
complex, with distinct protein conformations. Most of the described
inhibitors have Ado-like structural motifs and are nonselective, and some
of them (e.g., the tubercidine-like ligands) are characterized by a toxic
profile. We have cloned and expressed human AK (hAK) and searched for
novel non-substrate-like inhibitors. Our efforts to widen the structural
diversity of AK inhibitors led to the identification of novel non-nucleoside, noncompetitive allosteric modulators characterized by a
unique molecular scaffold. Among the pyrrolobenzoxa(thia)zepinones (4a-qq) developed, 4a was identified as a non-nucleoside
prototype hAK inhibitor. 4a has proapoptotic efficacy, slight inhibition of short-term RNA synthesis, and cytostatic activity on tumor
cell lines while showing low cytotoxicity and no significant adverse effects on short-term DNA synthesis in cells.
’ INTRODUCTION
been identified and cloned (A1, A2A, A2B, and A3) to date.8 Ado
behaves as an endogenously released neuromodulator of inter-
cellular signaling that reduces cell excitability during tissue stress
and trauma.8,9 In fact, it has been observed that the local Ado
concentration increases at sites of inflammation or tissue damage.10
Ado exerts its antinociceptive and anti-inflammatory effects by
functioning as a negative feedback signal whose responses tend to
decrease cellular activity, increase local nutrient supply, and restore
normal cellular functions.11 Moreover, Ado plays a neuroprotective
role during ischemical episodes and acts as an anticonvulsant in
spontaneous or induced seizures in animals or epileptic patients.11,12
Emerging evidence has focused upon the role of Ado on cell
death and differentiation. Extracellular Ado induces apoptosis in
human epithelial cancer cells originating from breast, colon, and
ovary via the intrinsic or mitochondrial pathway.13-15 Ado
uptake into cells through Ado transporters and its ensuing
conversion to AMP by AK increase the intracellular AMP
concentration, thus triggering the AMP-activated protein kinase.
Adenosine kinase (AK, EC 2.7.1.20) is a ubiquitous intracel-
lular purine metabolic enzyme that plays a key role in the control
of intra- and extracellular concentration of adenosine (Ado, 1,
Chart 1). Once inside the cell, Ado is rapidly phosphorylated to
Ado monophosphate (AMP) by AK, using the γ-phosphate of
the second substrate Mg/ATP2-. This maintains low intra- and
extracellular concentrations of Ado, since Ado uptake is driven by its
concentration gradient. While at low substrate concentrations the
phosphorylation reaction follows Michaelis-Menten kinetics, Ado
phosphorylation is subjected to uncompetitive substrate (Ado)
inhibition.1 This could probably be explained by hypothesizing
that Ado binds not only the catalytic site but also a regulatory
site with lower affinity, being responsible for phosphorylation rate
modulation.2-5
Ado has been characterized as a homeostatic modulator of
cellular activity and has several important roles in central and
peripheral nervous systems (CNS, PNS), where the fine-tuning
of its concentrations holds a particular physiological relevance.6,7
The multitude of Ado effects are mediated by the activation of
specific metabotropic receptors; four receptor subtypes have
Received: November 9, 2010
Published: February 14, 2011
r
2011 American Chemical Society
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Chart 1. Reference and Tile Compounds
Scheme 1. General Synthesis of Compounds 4a-kka
a Reagents and conditions: (a) (Het)ArSH or ArOH, NaH 60% in
mineral oil, dry THF, room temp, 9 h; (b) PdCl2(PPh3)2, CuI,
trimethylsilylacetylene, triethylamine (TEA), room temp, 9 h; (c)
K2CO3, MeOH, THF, room temp, 45 min.
Scheme 2. Synthesis of Iodo(thio)phenol Intermediatesa
Ado-induced cell death (apoptosis) may occur via both receptor-
mediated and non-receptor-mediated pathways.
Selective human AK (hAK) inhibitors may potentiate extracel-
lular Ado levels, enhancing the endogenous protective effect of
Ado.11 Accordingly, AK inhibitors have been shown to provide
antinociceptive, anti-inflammatory, and anticonvulsant activity in
animal models, thus suggesting their potential therapeutic utility for
pain, inflammation, epilepsy, and possibly other CNS and PNS
diseases associated with cellular trauma and inflammation.16,17
The reports of Ado-induced apoptosis have suggested a
potential role for AK inhibitors in cancer therapy.18 However,
the direct involvement of Ado in apoptosis is still controversial.
In fact, while some AK inhibitors described by Mlejnek and co-
workers19 prevented cells from going into apoptosis, other
authors demonstrated that Ado induces apoptosis in human
leukemia (HL-60) cells.20
In recent years we have attempted to develop novel proapop-
totic agents, interfering with tubulin or mitochondrial perme-
ability proteins and blocking the cell cycle in the G1 or G2/M
phase.21 Consequently, we selected the hAK as an interesting
target to develop novel potential proapoptotic agents. Potent AK
modulators have been recently described, most of them compe-
titively binding the Ado binding site and containing Ado-like
structural motifs.22 In order to reduce the nucleotide structural
similarities and to optimize the biological properties, recently a
systematic approach has been taken to dismantle the nucleoside
structure of the early inhibitors, from tubercidine-like com-
pounds to pyridopyrimidines and later to alkynylpyrimidines.16
To identify novel non-nucleoside hAK inhibitors (NNhAKIs), a
biological screening of our in house database of proprietary com-
pounds was performed. Compounds 2a-f and 3a,b (Chart 1) were
identified as interesting molecular hits that were taken as starting
points for structure-activity relationship (SAR) studies through the
synthesis of derivatives 4a-qq. Our efforts to widen the structural
diversity of hAK inhibitors so far reported16,22,23 led to the identi-
fication of novel noncompetitive modulators of the human enzyme,
a Reagents and conditions: (a) NaNO2, KI, urea, HCl, H2SO4, H2O,
0 ꢀC, 5 h; (b) NaBH4, MeOH, 3 h; (c) DABCO, Me2NC(S)Cl,
dimethylformamide (DMF), room temp, 1 h; (d) microwave, 220 ꢀC,
150 W, 10 min; (e) 3 N KOH, EtOH, 3 h.
characterized by a molecular scaffold not reminiscent of the
substrate Ado.
’ CHEMISTRY
The synthesis of compounds 4a-qq is reported in Schemes 1-5.
As outlined in Scheme 1, for the synthesis of compounds 4a-kk
(Chart 1 and Tables 2 and 3) the bromo derivative 524 wasusedasthe
alkylating agent of the appropriate (hetero)arylthiophenols and
phenols previously deprotonated with sodium hydride. Starting from
4l, application of the Sonogashira protocol furnished compound 4p
from which, after treatment with potassium carbonate in MeOH and
tetrahydrofuran (THF), compound 4q was obtained.
The noncommercially available thiols and phenols were
synthesized as reported in Schemes 2 and 3. The iodobenze-
nethiols needed for the synthesis of compounds 4l and 4bb were
obtained starting from 4-aminobenzenethiol 6a or from 4-amino-
2-chlorophenol 6b. Compounds 6a,b were subjected to a
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Scheme 3. Synthesis of Substituted Phenol Intermediatesa
Scheme 5. Synthesis of Compounds 4oo-qqa
a Reagents and conditions: (a) CBr4, PPh3, dry MeCN, reflux, 12 h; (b)
diethylamine, TEA, dry MeCN, reflux, 12 h; (c) pyrrolidine, TEA, dry
MeCN, reflux, 12 h.
Scheme 4. Synthesis of Compounds 4ll-nna
a Reagents and conditions: (a) (()-R-bromo-p-tolylacetic acid ethyl
ester, NaBH4, absolute EtOH, reflux, 8 h; (b) NaOH, THF/EtOH,
room temp, 12 h; (c) PCl5, dry DCM, reflux, 12 h; (d) ethyl iodide, KH
30% in mineral oil, dry THF, room temp, 15 h; (e) N-bromosuccini-
mide, 2,20-azodiisobutyronitrile (AIBN), CCl4, reflux, 2.5 h; (f) ArSH,
NaH 60% in mineral oil, dry THF, room temp, 12 h; (g) from 4pp,
PdCl2(PPh3)2, CuI, trimethylsilylacetylene, TEA, room temp, 9 h.
appropriate thiophenols, and the resulting intermediates were
immediately reduced with sodium borohydride. The racemic
secondary alcohols 17a-c were brominated with carbon tetra-
bromide/triphenylphosphine, and the bromides (18a-c) were
used to alkylate the 2-(pyrrol-1-yl)phenol.24 The esters 19a-c
were then hydrolyzed to the corresponding acids 20a-c which
underwent an intramolecular Friedel-Crafts cyclization giving
the final compounds (4ll-nn) as racemic mixtures.
The synthesis of compounds 4oo-qq is reported in Scheme 5.
The reduction-alkylation reaction of 2127 with the ethyl ester
of (()-R-bromo-p-tolylacetic acid in the presence of sodium
borohydride afforded the intermediate 22. After saponification of
the ester 22, the acid 23 was cyclized (24) as previously
described. The cyclic ketone 24 was C-6 alkylated (25) and
brominated (26) by means of a radicalic bromination. Com-
pounds 4oo and 4pp were obtained by reaction of the bromo
derivative 26 with 2-chlorobenzenethiol and 4-iodobenzenethiol
(9a), respectively, in the presence of sodium hydride. Finally,
application of Sonogashira protocol with trimethylsilylacetylene
on compound 4pp afforded compound 4qq.
a Reagents and conditions: (a) Ar-S-S-Ar/Ar-SH, NaBH4, NaH, dry THF,
25-60 ꢀC, 12 h; (b) PPh3, CBr4, MeCN, 50 ꢀC, 12 h; (c) 2-(pyrrol-1-yl)
phenol, NaH, dry THF, 40 ꢀC, 11 h; (d) NaOH, THF/EtOH 1:1, room
temp, 12 h; (e) PCl5, dry dichloromethane (DCM), reflux, 7 h.
Sandmayer reaction to give the corresponding iodo derivatives 7
and 8 (Scheme 2). Sodium borohydride reduction of the disul-
fide 7 furnished the 4-iodobenzenethiol 9a. The synthesis of the
S-aryl thiocarbamate 11 was performed by formation of the
O-aryl dimethylthiocarbamate 10 using dimethylthiocarbamyl chlo-
ride in the presence of 1,4-diazabicyclo[2.2.2]octane (DABCO).25
The Newman-Kwart rearrangement (11) was conveniently per-
formed under microwave irradiation. Finally, compound 11 was
treated with potassium hydroxide to afford 2-chloro-4-iodobenze-
nethiol 9b.
The m-ethylphenol derivatives (14 and 15) used for the
synthesis of compounds 4jj,kk were prepared starting from
3-(2-hydroxyethyl)phenol 12 (Scheme 3). Compound 12 was
brominated in the presence of carbon tetrabromide and triphe-
nylphosphine in acetonitrile. The obtained bromo derivative 13
was used as the alkylating agent for diethylamine and pyrrolidine
to give the phenols 14 and 15, respectively.
’ RESULTS AND DISCUSSION
Compounds 4ll-nn were synthesized as described in
Scheme 4. The bromo derivative ethyl 2-[4-(bromomethyl)-
phenyl]-2-oxoacetate 1626 was the alkylating agent for the
Cloning, Expression, and Biochemical Characterization of
hAK. Messenger RNA for hAK was identified in GenBank
(accession number BC003568). Amplified cDNA was obtained
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by using RT-PCR applied on total HeLaS-3 RNA with sense
and antisense primers as reported in the Experimental Section.
The amplified region, containing the complete hAK coding
sequence, was inserted into the multiple cloning site of pTrcHisA
(Invitrogen) to give the recombinant bacterial expression vector
pHis-hAK. His-tagged hAK sequence encodes for the complete
345 amino acids enzyme with a 6-His tag at its NH2 terminus.
Expression and purification of hAK was carried out as de-
scribed in Experimental Section. The enzyme was purified from
E. coli crude extract in a single chromatographic step by using a
Ni-NTA Superflow column. Recombinant hAK had a specific
activity of 38 500 units/mg (1 unit is defined as the amount of
enzyme catalyzing the formation of 1 nmol of AMP in 1 h at
37 ꢀC). Figure 1 of the Supporting Information shows the SDS-
PAGE of the purification of recombinant hAK (MW = 46 kDa).
In order to determine the kinetic parameters of recombinant
hAK, we incubated the enzyme in the presence of different
concentration of substrate (Ado) using ATP as phosphate donor
at 1 mM. Table 1 reports the experimental kinetic parameters of
the enzyme. In a substrate concentration range between 0.1 and 2
μM, recombinant hAK phosphorylates Ado following typical
Michaelis-Menten kinetics (Km = 0.4 μM).
Table 1. Kinetic Parameters of Recombinant hAK with ATP
as Phosphate Donor
a
a
Km
Vmax
kcat
kcat/Km
substrate
(μM)
(pmol s-1 μg-1
)
(s-1
)
(s-1 μM-1
)
adenosine
0.4 ( 0.1
10.7 ( 1.0
0.5
1.25
a kcat was calculated by using 46.0 kDa as the molecular mass of the
enzyme.
Table 2. IC50 (μM) on hAK for Reference Compounds
Inhibition Assays on the Recombinant hAK. Following our
previous experience with the synthesis of non-nucleoside inhibitors of
HIV-1 reverse transcriptase24,28-30 and hepatitis C virus nonstructural
protein 3 enzyme,31 in order to identify novel hAK non-nucleo-
side inhibitor hits, we screened various classes of polycyclic com-
pounds that include quinolines, pyrroloquinoxalines, benzazepinones,
benzodiazepinones, pyrrolobenzothiazepinones, pyrrolobenzoxaze-
pines, dibenzazepines, and dibenzothiazepines, all available in our
laboratories. Among the tested compounds we found that pyrrolo-
benzoxa(thia)zepinones showed an inhibitory activity in the high
micromolar range (2a-f, Chart 1), indicating such scaffolds as
suitable for the development of specific NNhAKIs (Chart 1). Thus,
the objective of this study was the development of a novel class of
noncompetitive NNhAKIs with the pyrrolobenzoxa(thia)zepinone
core and drawing of SARs for hAK inhibition related to the variation
of the substituents on the heterocyclic system. The hAK inhibition
potency of prototypic compounds is reported in Table 2.
Compounds 2a-e are the representative of a class of non-
nucleoside inhibitors of HIV-1 reverse trancriptase.24,30 The
benzothiazepine 2a proved to be less potent than the corre-
sponding C-6 monosubstituted benzoxazepine (2b) and benzox-
azepines bearing an alkyl chain (R) at C-6 (2c,d), probably
because of the different geometry of the polycyclic system. A
methyl group at the para position on the phenyl at C-6 was better
tolerated than at the meta position (2d vs 2e). The insertion of a
substituent (R00) on the pyrrole ring led to compound 2f32 that
displayed no activity at 10-3 M. The critical role of the geometry
of the system (and possibly also the addition of bulky groups at
C-5) was also confirmed by the lack of activity of esters 3a,b,33
presenting a planar and conjugated pyrrolobenzoxa(thia)zepine
system.
On the basis of the results obtained and with the aim to improve
enzyme inhibitory potency, we synthesized compounds 4a-qq
(Chart 1) presenting a pyrrolo[2,1-d]benzoxa(thia)zepinone scaf-
fold with a “T-shape” geometry and bearing a variety of substituents
at C-6 (Tables 3 and 4), so maintaining the core system and
geometry of compounds 2.
The SAR studies were widely explored with respect to the
introduction of substituted (hetero)arylthiomethyl and phenox-
ylmethyl functions at the para position of the C-6 pendant phenyl
a Each value is the mean of at least three determinations. Standard error
of the mean (SEM) is e10%.
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ring of both the pyrrolobenzoxazepinone and pyrrolobenzothia-
zepinone scaffolds. The effect of removal of the C-6 ethyl group
was also evaluated. All the compounds were tested as racemates,
but to evaluate a possible stereoselective mechanism of enzyme
inhibition, the racemic mixture of derivative 4s was resolved by
HPLC techniques into the pure (þ)- and (-)-enantiomers,
which were tested in the enzyme inhibition assays.
Table 3. IC50 (μM)a on hAK for Compounds 4a-cc
All the explored modifications of the structures of 6-ethyl-6-
phenylpyrrolobenzoxazepinones are grouped into the following
subsets of inhibitors: (i) arylmercaptomethyl dervatives (4a-cc,
Table 3), (ii) heteroarylmercaptomethyl derivatives (4dd-gg,
Table 4), (iii) phenoxy derivatives (4hh-kk, Table 4), (iv) C-6-
desethyl analogues (4ll-nn, Table 4), and (v) thiazepinone
analogues (4oo-qq, Table 4).
i. Arylmercaptomethyl Derivatives. A large series of 6-ethyl-
6-arylpyrrolobenzoxazepinones (4a-cc, Table 3) bearing sub-
stituted arylmercaptomethyl groups at the C-6 phenyl moiety in
para position was synthesized and tested. The effects of mono-,
di-, tri-, and tetrasubstitutions with electron-withdrawing
groups (EWGs) or electron-donating groups (EDGs) were
evaluated.
SARs analysis of the inhibition data obtained with the mono-
substituted compounds demonstrated that with respect to the
unsubstituted derivative 4a (IC50 = 1.2 μM), ortho substitution
with halogens is well tolerated (4a vs 4b,c). Similarly, the meta
position benefits of EWGs (compounds 4e-g,i displayed IC50
between 2.5 and 4.0 μM) rather than EDGs (4d,h). At the para
position, halogen substitution was associated with better po-
tency, following the trend F > Br > I (4j-l). Other EWGs (-NO2
and -OCF3, 4 m,n) slightly lowered inhibition potency. In line
with previous data, the presence of EDGs (such as the methoxy
and the alkyne, 4o-q) lowered potency by one order of
magnitude (4j vs 4o-q).
The arylmercaptomethyl moiety was also disubstituted lead-
ing to compounds 4r-bb. In this subseries of analogues when
the o-Cl functionality of 4c was accompanied by an o-methyl
group (4r), a compound 3 times less potent than 4c was
obtained. Disubstitution of the phenylmercapto moiety by halo-
gens (4t-z,bb,cc) was generally tolerated, with 4bb being the
best performing with an IC50 of 2.0 μM. Interestingly, the
inhibitory potency of 4bb was similar to that of its correspond-
ing aryloxy analogue 4hh (Table 4), thus suggesting that
bridging compounds with sulfur or oxygen does not inter-
fere with potency. As expected, substitution of halogens by
methoxy groups at C-2 and C-5 produced a weak inhibitor
(4aa) of hAK.
Analysis of pure enantiomers (þ)-4s and (-)-4s assays
indicated a steroselective interaction with the human enzyme,
the (þ)-enantiomer being about 5 times more potent than the
corresponding (-)-enantiomer (Table 3).
ii. Heteroarylmercaptomethyl Derivatives. A subset of com-
pounds was synthesized to evaluate the effect of substitution of
the arylmercaptomethyl group by five/six membered nitrogen
containing heterocycles (4dd-gg, Table 4). In general these
compounds showed single digit inhibitory potencies, with the
imidazole based analogue 4ee being the most potent of the
subseries (4ee, IC50 = 3.5 μM).
iii. Phenoxymethyl Analogues. A small series of C-6 phenox-
ymethylphenyl substituted derivatives (4hh-kk, Table 4) was
synthesized. Notably the 2-chloro-4-iodophenoxy substituted
analogue 4hh was a potent hAK inhibitor (IC50 = 1.2 μM),
almost 2-fold more potent than the sulfur bridged analogue 4bb.
a Each value is the mean of at least three determinations. SEM is e10%.
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Table 4. IC50 (μM)a on hAK for Compounds 4dd-qq and
Calculated Single pKa for Selected Compounds
Figure 1. In vitro effect of increasing concentrations of 4a on hAK
activity.
and tested (4ll-nn, Table 4). All compounds displayed a slightly
lower inhibitory potency with respect to their ethyl-substituted
counterparts (4ll vs 4a, 4 mm vs 4c, and 4nn vs 4g) that could be
either due to a different conformational preference for the
tricyclic scaffold or due to the lack of favorable hydrophobic
interaction of the ethyl chain with the enzyme.
v. Pyrrolobenzothiazepinone Derivatives. We also compared
the potency of compounds based on a pyrrolo[2,1-b][1,4]benzo-
xazepinone skeleton with those based on a pyrrolo[2,1-b][1,4]-
benzothiazepinone tricyclic system (4oo-qq, Table 4), which
again demonstrated that the replacement of the bridging oxygen
was not essential for potency. Interestingly, in this subseries of
compounds the introduction of an ethynylsilane moiety (4qq) in
para position led to a compound more potent than the corre-
sponding pyrrolobenzoxazepinone 4p.
Enzymatic Inhibition Kinetic Studies and Possible Site of
Interaction within the hAK. Interestingly, the majority of
compounds, tested as described in the Experimental Section,
showed inhibitory activity on hAK in the micromolar range. The
behavior of selected compounds 4a, 4e, and 4g (IC50 = 1.2, 4.0,
and 2.5 μM, respectively) was studied more deeply. In general
they showed hyperbolic curves of inhibition (Figure 1 for 4a)
which indicate a nonlinear mode of action.
The inhibitory activity of 4a, representative of the whole series,
was then evaluated in kinetic experiments with our cloned hAK in
order to understand its site of binding and the mechanism of
action.
It is known that two Ado binding sites exist on hAK, a catalytic
site with high affinity for Ado and a low affinity regulatory site,5
probably partially overlapping the ATP binding site responsible
for substrate inhibition by Ado.4
The hAK follows an obligatory ordered ternary complex
reactant kinetic pathway for catalysis where the Ado binds first
followed by ATP or Ado. Upon binding of the substrate Ado or
upon binding of the second substrate ATP, a conformational
rearrangement is observed. Indeed, native AK and AK complexed
to Ado exist in a protein conformation distinct from AK
complexed to Ado/ATP or to Ado/Ado (ternary complex).5
Thus, we can hypothesize that three different conformation/
kinetic forms of the hAK exists with different affinities for specific
ligands.
We tested the effect of 4a at different concentrations of both
Ado and ATP. The inhibitory potency of 4a was unaffected by
increasing concentrations of Ado (Figure 2) or ATP (data not
shown), indicating a noncompetitive mode of inhibition.
Molecular Modeling. To rationalize the noncompetitive
(Ado and ATP) mode of action combined with the micromolar
a Each value is the mean of at least three determinations. SEM is e10%.
b Apparent pKa values (ACD/Labs, version 12.0, Toronto Canada).
We also explored the tolerance of longer side chains, bringing or
not protonatable groups (4ii and 4jj,kk). While the hydrophilic
OH group of 4ii (IC50 = 8.0 μM) was still tolerated, the
protonatable and bulkier lateral chains of 4jj,kk determined a
dramatic drop of potency.
iv. C-6-Desethyl Derivatives. To establish the role of the ethyl
chain at C-6, a series of C-6-desethyl analogues were synthesized
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Figure 2. Effect of increasing concentrations of 4a on hAK activity at
different Ado concentrations: ([), 0.4, (9) 0.6, (0) 0.8, (]) 1.0, (b)
1.2, (O) 1.4 μM. Inner panel: IC50, determined for each Ado concen-
tration with Prism3 software through the formula y = v/[1 þ (x/IC50)],
where v is the enzyme velocity and x is the 4a concentration.
inhibitory activity found for the best modulators of the series, we
hypothesized the existence of an allosteric site, whose accessi-
bility may be dependent upon the conformational/kinetic state
of the enzyme. However, to date there is no direct evidence of the
existence of any allosteric site in hAK. So with the aim of
identifying a binding site other than the catalytic/regulatory
ones, the server Q-SiteFinder and the SiteMap software
(Schr€odinger) were both used either on a closed or on the open
enzyme conformation. The closed structure 1BX4 (PDB code),5
cocrystallized with two Ado molecules and possessing the higher
resolution, was selected together with the unique open structure
available in the PDB (PDB code 2I6B)34 which is complexed
with a pyrimidin-4-ylamine analogue. The server Q-SiteFinder
and the SiteMap software generated comparable results. Inter-
estingly, in the closed enzyme conformation, beside the Ado and
ATP binding sites, a shallow pocket, which is located between the
helices R3, R4 and the long carboxylic tail and which is upon the
Ado and ATP binding sites, was detected (see Figure 3).
Intriguingly, such a site is just partially present in the open
conformation of the enzyme because of the movement of the β4
sheet. As nowadays it is well-known that in enzymes synergy
between structure and plasticity results in the unique power of
these biocatalysts, a ligand able to bind such a pocket would
hamper the hAK conformational flexibility and in turn its
catalytic activity, thus behaving as hAK inhibitor. Although the
question of how enzymes achieve the catalytically competent
state has recently become approachable by experimental and
computational studies,35 this is beyond the scope of this work.
Herein, in order to verify the capability of 4a to preferentially
recognize the hypothesized allosteric site, a blind docking
comprising the entire closed conformation of the enzyme was
performed by means of the Autodock program. Amazingly,
although the ligand can potentially bind other sites, in the lowest
energy conformation family, 4a was found to be well anchored in
such a pocket (Figure 4). After the capability of 4a to bind the
putative allosteric site was probed, the Glide docking program
was subsequently used to gain insights into the binding mode of
4a at an atomic level (see Experimental Section for further
details).
Figure 3. Putative allosteric site (cyan) in the close conformation of the
hAK enzyme as identified by the Q-SiteFinder software. In the picture
are also visible the Ado binding site (green) which extends toward the
ATP binding site (violet).
Figure 4. Lowest energy conformation family of 4a resulting after the
blind docking. 4a can allocate in a site upon those of Ado, which are
discernible because they contain the substrate molecule, shown as sticks.
between the carboxylic group of the oxazepinone ring and the
backbone NH of F338 was also detected. On the other side of the
molecule, the phenylmercaptomethyl group inserts into a well-
defined pocket made up by Q74, Q79, H81, H107, and K341 and
establishes a T-shaped interaction with H81. The above-de-
scribed binding mode would be in line with the extensive SAR
studies previously discussed. In fact compounds (2a-e) lacking
the phenylmercaptomethyl moiety were all weaker hAK inhibi-
tors than 4a. Among them, 2b is about 80-fold less potent than
4a, as it lacks both the phenylmercaptomethyl moiety and the
C-6 ethyl group. In line with the hydrophobic interactions
In the best scored binding pose (Figure 5), 4a was found to
bind the allosteric site with a well-defined “T-shape” geometry
where the C-6 ethyl group lies between the T337 and the K71
residues and forms hydrophobic interactions with T and K side
chains, while the benzene ring establishes a cation-π interaction
with the K102 amine group (the distance between the aromatic
ring centroid and the K nitrogen atom is 5.8 Å). An H-bond
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Figure 6. Effect of 4a on short-term DNA (A) and RNA (B) syntheses
in cell culture (HeLaS-3). Nucleic acids and protein syntheses were
determined by measuring the incorporation of [3H]thymidine and
[3H]uridine, respectively, in acid precipitable materials following the
method described in Experimental Section: (b) 0 μM, (O) 10 μM, and
(0) 50 μM 4a. Each value is the mean of three determinations. SEM is
e10%.
Figure 7. Effect of 4a on HeLaS-3 cell Ado phosphorylation in vivo. For
experimental details see Experimental Section: (b) 0 μM, (O) 2.5 μM,
and (9) 25 μM 4a. Each value is the mean of three determinations. SEM
is e10%.
potency is also detectable for nitrogen containing electron-rich
heterocycles (4dd-gg, Table 4). Finally, docking studies on
compounds 4jj and 4kk suggest that the insertion in the phenyloxo-
methyl meta position of a N-protonated side chain would be not
fruitful because of the narrow end of the pocket and the proximity of
K341 protonated side chain. Thus, the binding mode of 4a sugges-
ted by the Glide docking program is in line with the experimentally
determined SARs and on one hand would sustain our hypothesis and
on the other hand would validate the proposed binding pose. However,
further experiments such as site-directed mutagenesis studies should be
performed in the future to ultimately validate our assumptions.
In order to better characterize this class of non-nucleoside
noncompetitive inhibitors of hAK, we selected 4a as the refer-
ence compound for further biological studies.
Effect of 4a on in Vitro Short-Term DNA and RNA Synth-
eses and Cell Viability. In order to verify whether 4a shows any
effect in vitro on short time DNA and RNA syntheses, we
incubated HeLaS-3 cells in the presence of 10 and 50 μM 4a
as described in the Experimental Section. In our assay conditions, as
reported in Figure 6, 4a does not significantly affect DNA synthesis
but slightly interferes with RNA synthesis (IC50 = 50 μM).
Figure 5. (a) Putative binding pose of 4a in the allosteric site of hAK
shown as surface and colored based on molecular electrostatic potential
(MEP) as calculated by the PyMol program. (b) Putative binding pose
of 4a in the allosteric site of hAK shown as white cartoon. Interacting
residues are shown as sticks and colored by atom type, while the H-bond
is represented by a black dashed line.
formed by the ethyl group of 4a, compounds 4ll-nn are all less
potent inhibitors than 4a. In agreement with the importance of the
“T-shape” geometry for a proper interaction with the enzyme site
and in line with the limited space available for substitution on the
oxazepinone ring, ligands 3a,b are totally inactive. Taking into
account the newly synthesized compounds 4a-cc (Table 3), SARs
indicated that the presence of EDGs, such as methyl (4d) or
methoxy (4h), lowers the binding potency while the presence of
EWG such as fluorine or chlorine atoms in ortho or meta position
or iodine and bromine atoms in para position is well tolerated, and
this would be in line with the T-shaped interaction established with
H81 ring. Notably, insertion of other EWGs in para position, such
as -NO2 and -OCF3 (4m,n), slightly lowered inhibition potency
probably because of a major steric hindrance of these groups with
respect to the other above-mentioned EWG. Docking experiments
suggest that in 4d and/or 4h the lowered activity would not be due
to a steric factor but to the presence of an EDG such as a methyl
and a methoxy, which would disfavor the interaction of benzene
with H81 electron-rich imidazole ring. Accordingly, a slight loss in
In order to verify cellular permeability of compound 4a, HeLaS-3
cells were incubated with the tested compound and its ability to
inhibit in vitro phosphorylation of Ado was evaluated as described in
the Experimental Section. The amount of produced [3H]AMP was
measured after co-incubation with different concentrations of 4a at
different times (Figure 7). This experiment indicates that after
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Figure 10. HeLaS-3 (b, O) and Cen3tel (9, 0) cell growth in the
presence of 40 μM 4a. For experimental details, see Experimental
Section: (b, 9) control; (O, 0) 3 days in the presence of 4a.
Figure 8. Effect of different concentrations of 4a (b), and ddCyd (O)
as control, on K562 cell line after 7 days of exposure to the compounds.
Figure 11. HL-60 cell viability in the presence of vehicle (0.1% (v/v)
DMSO) or a range of concentrations of 4a as described in the
Experimental Section. Values represent the mean ( SEM for three
independent experiments.
Interestingly, we found that the cytostatic effect of the compound
did not last more than 24-48 h even when HeLaS-3 cells and
Cen3tel (fibroblast) cells36 were incubated longer than 48 h, in
presence of 40 μM 4a freshly added every 24 h, as well
(Figure 10).
Figure 9. HeLaS-3 cell growth in the presence of 50 μM 4a. For
experimental details, see Experimental Section: (b) control, (0) 1 day,
and (4) 2 days in the presence of 4a.
This fact would suggest that cells, after a short period in which
they are sensitive to the compound, are able to overcome the
inhibitory effect by specific mechanisms that may involve induc-
tion of metabolic inactivation, increase of hAK activity, or other
mechanisms of resistance. In order to evaluate these possibilities,
we cultivated HeLaS-3 cells for 3 days in the presence of the drug
(40 μM, added fresh every 24 h) and then we prepared cell crude
extract as described in the Experimental Section. Extracts from
treated and untreated cells were thus tested in vitro for both hAK
and 4a metabolizing activity. Our results demonstrated that 4a is
not degraded when incubated up to 20 h at 37 ꢀC in the presence
of crude extract, and no difference could be observed in hAK
activity between 4a treated and untreated cells (data not shown).
This suggests that another mechanism is involved in the 4a
resistance in cells incubated 24-48 h in the presence of the
compound.
We also performed cell viability assays in human promyelo-
cytic leukemia HL-60 cells. The cells were treated with different
concentrations of compound 4a for 72 h. As highlighted in
Figure 11, we determined that 4a was effective in reducing cell
viability with an IC50 of 34.17 μM.
We then explored if this reduction in HL60 cell viability
occurred as a result of apoptosis. We analyzed the DNA content
of the cells by flow cytometry. Cells found in the sub-G0/G1 peak
entering the cell, 4a is able to reduce the amount of phosphorylated
Ado, although less efficiently than with the isolated cloned enzyme.
Effect of 4a on Cell Cultures. To evaluate the effects of 4a on
cellular growth and viability, we exposed K562 (chronic myeloid
leukemia cells) to various concentrations of the compound. Cell
viability was measured by the dye exclusion assay described in the
Experimental Section.
For comparison, we also determined the effect of 20,30-
dideoxycytidine (ddCyd), a compound known to affect cell
growth and viability. As shown in Figure 8, 4a inhibits cell
growth and the drug concentration that inhibits the 50% of cell
growth is 1 μM, a value comparable to that of ddCyd (0.8 μM).
In both cases the compound blocks but does not kill the cells,
since no blue stained cell was counted.
In order to study the effect of 4a on cell growth, we incubated
HeLaS-3 cells for 2 days in the presence of the compound (50
μM). We then washed the cells and left them to grow in fresh
medium for an additional day. We found that once cells were
washed and incubated in fresh medium, the inhibitory effect of 4a
on cells was maintained for the first 24 h while at later times cells
rapidly recovered their normal growth rate (Figure 9).
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death or apoptosis.15,39 In order to assess the selectivity of our
non-nucleoside hAK inhibitors, we also evaluated their ability to
inhibit ADA. The enzymatic assay was performed on compounds
4a, 4c, 4g, 4j, and 4q (for details see Experimental Section). Data
reveal the almost complete lack of activity of these molecules
(Table 5), confirming the selectivity of the compounds for hAK.
These data allow us to exclude the inhibition of ADA by 4a as the
causative effect of apoptosis.
Cytotoxicity Studies. Cytotoxicity assays were performed to
establish the effect of 4a, 4c, 4g, 4k, and 4l on cell proliferation in
vitro. IC50 values on 3T3 murine cell line are given in Table 6 (for
details see the Experimental Section). The data confirm a low
cytotoxic potential for the tested compounds.
Figure 12. Effect of 4a on apoptosis. Cells were treated with vehicle
(0.1% DMSO) (Veh) or 25-100 μM 4a for 24, 48, and 72 h. Their
DNA content was then analyzed as described in the Experimental
Section. Cells with less than 2N DNA content were deemed apoptotic.
Values represent the mean ( SEM for three independent experiments.
(/) P < 0.05, (//) P < 0.005, and (///) P < 0.0005 were considered
statistically significant using paired Student's t-tests.
’ CONCLUSIONS
By a screening approach, we identified a new class of non-
nucleoside inhibitors of hAK with no affinity for ADA. The
described compounds are characterized by structural features not
reminiscent of the nucleoside structure. Through specific biolo-
gical investigation we also demonstrated that the compounds
were noncompetitive with both Ado and MgATP2-. Thus, these
benzoxa(thia)zepinones represent the first example of novel
non-nucleoside noncompetitive inhibitors of hAK behaving as
allosteric modulators of the human enzyme. This hypothesis was
investigated by in silico studies that allowed us to propose a
putative allosteric binding site for our molecules. The SAR trends
experimentally determined for our compounds on the human
enzyme could nicely be explained by the identified interaction in
the postulated allosteric pocket. To explore the role of hAK in cell
proliferation, we performed studies on living cells, highlighting that 4a
does not show significant adverse effect on short-term DNA synthesis.
Furthermore, our lead compound 4a, although it slightly inhibits
short-term RNA synthesis, shows a cytostatic effect on different
tumor cell lines, with almost no cytotoxic effect on normal cells.
In line with the described role played by Ado in the apoptotic
pathway, suggesting a potential role for hAK inhibitors in cancer
therapy, we also tested 4a to evaluate its biological activity on
leukemic (HL-60) cells. These studies demonstrated that 4a
potently reduced HL-60 cell viability (IC50 = 34.17 μM) by
acting as a potent proapoptotic agent.
Table 5. Percentage of Inhibition of ADA for Compounds 4a,
4c, 4g, 4j, and 4q
compd
% inhibition (100 μM)a
4a
4c
4g
4j
15 ( 2
6.5 ( 0.7
2.7 ( 0.2
13 ( 1.9
16 ( 3
4q
a Percentage inhibition values of ADA activity at 100 μM. Values are the
mean ( SEM of two determinations carried out in triplicate.
Table 6. Cytotoxic Effect on 3T3 Cells Expressed as IC50 for
Compounds 4a, 4c, 4g, 4k, and 4la
compd
IC50 (μM)
4a
4c
4g
4k
4l
117.5
97.8
44.6
47.6
43.5
’ EXPERIMENTAL SECTION
a Each value is the mean of three determinations. SEM is e10%.
Chemistry. Reagents were purchased from Aldrich and were used
as received. Reaction progress was monitored by TLC using Merck silica
gel 60 F254 (0.040-0.063 mm) with detection by UV. Merck silica gel
60 (0.040-0.063 mm) was used for column chromatography.
contained less than 2N DNA content and were considered
apoptotic. After 24 h of treatment, 50 and 100 μM 4a induced
statistically significant increases in percent apoptosis, 44% and
70%, respectively, compared to 0.8% in vehicle-treated controls
(Figure 12). After 48 h of treatment, the number of cells
undergoing apoptosis further increased to over 90% following
100 μM 4a compared to 0.7% in controls. Hence, we concluded
that 4a can function as a proapoptotic agent in HL-60 leukemia
cells.
Effect of Pyrrolobenzoxa(thia)zepinones on Adenosine
Deaminase (ADA) Activity. Besides AK, ADA is the other Ado
metabolic enzyme present in cells. ADA catalyzes the irreversible
Ado deamination to inosine. The role of AK is predominant in
the physiological inactivation of Ado because of its higher affinity
for the nucleoside (lower Km).37,38 However, several studies have
identified ADA inhibitors as potentiators of Ado induced cell
Melting points were determined in Pyrex capillary tubes using an
Electrothermal 8103 apparatus and are uncorrected. 1H NMR and 13
C
NMR spectra were recorded on Bruker 200 MHz or Varian 300 MHz
spectrometer with TMS as internal standard. Splitting patterns are
described as singlet (s), doublet (d), triplet (t), quartet (q), and broad
(br). The values of chemical shifts (δ) are given in ppm and coupling
constants (J) in hertz (Hz).
ES-MS and APCI-MS spectra were obtained from an Agilent 1100
series LC/MSD spectrometer and from a LCQDeca-THERMOFINNIGAN
spectrometer.
Elemental analyses were performed in a Perkin-Elmer 240C ele-
mental analyzer, and the results were within (0.4% of the theoretical
values, unless otherwise noted.
Yields refer to purified products and are not optimized. All moisture-
sensitive reactions were performed under argon atmosphere using oven-dried
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glassware and anhydrous solvents. All the organic layers were dried using
anhydrous sodium sulfate.
oil (71% yield): 1H NMR (200 MHz, CDCl3) δ1.03 (t, 3H, J= 7.5 Hz), 2.38
(m, 2H), 3.35 (s, 3H), 4.20 (s, 2H), 6.44 (m, 1H), 6.91-7.36 (m, 14H).
Anal. (C28H25NO2S) C, H, N.
(()-6-Ethyl-6-[4-[(3-fluorophenylthio)methyl]phenyl]-6,7-
dihydrobenzo[b]pyrrolo[1,2-d][1,4]oxazepin-7-one (4e). Com-
pound 4e was obtained following the same procedure described for com-
pound 4a. Purification was performed by means of flash chromatography
(EtOAc/n-hexane 1:9). Title compound was obtained as a pale yellow oil
(66% yield): 1H NMR (200 MHz, CDCl3) δ 1.03 (t, 3H, J = 7.5 Hz), 2.38
(m, 2H), 4.00 (s, 2H), 6.43 (m, 1H), 6.91-7.35 (m, 14H). Anal.
(C27H22FNO2S) C, H, N.
(()-6-Ethyl-6-[4-[(3-chlorophenylthio)methyl]phenyl]-6,7-
dihydrobenzo[b]pyrrolo[1,2-d][1,4]oxazepin-7-one (4f). Com-
pound 4f was obtained following the same procedure described for com-
pound 4a. Purification was performed by means of flash chromatography
(EtOAc/n-hexane 1:9). Title compound was obtained as a pale yellow oil
(71% yield): 1H NMR (200 MHz, CDCl3) δ 1.03 (t, 3H, J = 7.5 Hz), 2.38
(m, 2H), 4.00 (s, 2H), 6.40 (m, 1H), 6.90-7.31 (m, 14H). Anal.
(C27H22ClNO2S) C, H, N.
HPLC analytical chiral separation was performed by an AS Chir-
alpack analytical column (0.46 cm ꢀ 25 cm). The column used for the
semipreparative separation was a Chiralpack AS (1 cm ꢀ 25 cm)
containing as chiral stationary phase amylose tris[(S)-R-methylbenzyl-
carbamate] coated on 10 μm silica gel. Before and after use, column
functionality was tested with trans-stilbene oxide as standard compound.
All runs were performed in isocratic mode at 25 ꢀC with detection at
254 nm. Eluents were degassed by ultrasound treatment. Sample solu-
tions for semipreparative separation (approximately 1 mg/mL) were
prepared by dissolving the analytes in the eluent and filtering through
0.45 μm Acrodisc filters. The injection volume was 100 μL.
Optical rotation values were measured at room temperature operat-
ing at λ = 589 nm, corresponding to the sodium D line, and were
determined in a Perkin-Elmer polarimeter using a microcell (1 mg
sensitivity) containing the compound dissolved in 1 mL of chloroform.
For biological tests analytical grade reagents were exclusively used.
Bacterial media components were from Difco. Ni-NTA Superflow resin
was from Qiagen. Restriction and modification enzymes were from
Promega, Sigma, or Roche Molecular Biochemicals. Isopropylthio-
β-D-galactoside (IPTG) was from Sigma. [3H]Ado, 22 Ci/mmol, was
from Amersham.
(()-6-Ethyl-6-[4-[(3-trifluoromethylphenylthio)methyl]-
phenyl]-6,7-dihydrobenzo[b]pyrrolo[1,2-d][1,4]oxazepin-
7-one (4g). Compound 4g was obtained following the same
procedure described for compound 4a. Purification was performed
by means of flash chromatography (EtOAc/n-hexane 1:9). Title
(()-6-Ethyl-6-[4-[(phenylthio)methyl]phenyl]-6,7-dihydro-
benzo[b]pyrrolo[1,2-d][1,4]oxazepin-7-one (4a). Sodium hy-
dride (60% suspension in mineral oil, 22 mg, 0.550 mmol) was added
to a solution of thiophenol (50 mg, 0.530 mmol) in 3.0 mL of dry THF.
After being stirred for 2 h at room temperature under argon atmosphere,
a solution of the bromide 5 (200 mg, 0.500 mmol) in dry THF (4.0 mL)
was added. The mixture was stirredatroom temperature for12 h. Afterthe
reaction was quenched with a saturated aqueous solution of ammonium
chloride, THF was removed under reduced pressure. DCM was added,
and thesolution waswashed with brine. Thecombinedorganiclayers were
dried over sodium sulfate, filtered, and concentrated. The crude product
was purified by means of flash chromatography (EtOAc/n-hexane 1:10),
1
compound was obtained as a pale yellow oil (65% yield): H NMR
(200 MHz, CDCl3) δ 1.02 (t, 3H, J = 7.5 Hz), 2.38 (m, 2H), 4.03 (s,
2H), 6.45 (m, 1H), 6.91-7.47 (m, 14H). Anal. (C28H22F3NO2S)
C, H, N.
(()-6-Ethyl-6-[4-[(3-methoxyphenylthio)methyl]phenyl]-
6,7-dihydrobenzo[b]pyrrolo[1,2-d][1,4]oxazepin-7-one (4h).
Compound 4h was obtained following the same procedure described
for compound 4a. Purification was performed by means of flash chroma-
tography (EtOAc/n-hexane 1:9). Title compound was obtained as a pale
yellow oil (73% yield): 1H NMR (300 MHz, CDCl3) δ 1.03 (t, 3H, J = 7.2
Hz), 2.40 (m, 2H), 3.71 (s, 3H), 3.86 (s, 2H), 6.46 (m, 1H), 6.75 (m, 2H),
6.94-7.23 (m, 7H), 7.25-7.33 (m, 5H). Anal. (C28H25NO3S) C, H, N.
(()-6-Ethyl-6-[4-[(3-trifluoromethoxyphenylthio)methyl]-
phenyl]-6,7-dihydrobenzo[b]pyrrolo [1,2-d][1,4]oxazepin-7-
one (4i). Compound 4i was obtained following the same procedure
described forcompound 4a. Purification was performedby meansof flash
chromatography (EtOAc/n-hexane 1:9). Title compound was obtained
as a pale yellow oil (77% yield): 1H NMR (300 MHz, CDCl3) δ 1.01
(t, 3H, J = 7.3 Hz), 2.36 (m, 2H), 4.01 (s, 2H), 6.42 (m, 1H), 6.87-7.33
(m, 14H). Anal. (C28H22F3NO3S) C, H, N.
(()-6-Ethyl-6-[4-[(4-fluorophenylthio)methyl]phenyl]-6,7-
dihydrobenzo[b]pyrrolo[1,2-d][1,4]oxazepin-7-one (4j). Com-
pound 4j was obtained following the same procedure described for com-
pound 4a. Purification was performed by means of flash chromatography
(petroleum ether 40-60 ꢀC/DCM 2:1). Title compound was obtained as a
pale yellow oil (71% yield): 1H NMR (300 MHz, CDCl3) δ 1.04 (t, 3H,
J= 7.3 Hz), 2.40 (m, 2H), 3.91 (s, 2H), 6.46 (m,1H), 6.84-7.29 (m, 14 H).
Anal. (C27H22FNO2S) C, H, N.
(()-6-Ethyl-6-[4-[(4-bromophenylthio)methyl]phenyl]-6,7-
dihydrobenzo[b]pyrrolo[1,2-d][1,4]oxazepin-7-one (4k). Com-
pound 4k was obtained following the same procedure described for com-
pound 4a. Purification was performed by means of flash chromatography
(EtOAc/n-hexane 1:9). Title compound was obtained as a pale yellow oil
(70% yield): 1H NMR (300 MHz, CDCl3) δ 1.03 (t, 3H, J = 7.3 Hz), 2.39
(m, 2H), 4.00 (s, 2H), 6.43 (m, 1H), 6.88-7.30 (m, 14H). Anal.
(C27H22BrNO2S) C, H, N.
1,2-bis(4-Iodophenyl)disulfane (7). To a solution of 4-amino-
benzenethiol (6a) (192 mg, 1.53 mmol) in 1.4 mL of water, cooled to
0 ꢀC, concentrated HCl (1.5 mL), concentrated H2SO4 (600 μL), and
sodium nitrite (126 mg, 1.840 mmol) were added. After the mixture was
1
affording 4a as an amorphous white solid (72% yield): H NMR (200
MHz, CDCl3) δ 1.03 (t, 3H, J = 7.5 Hz), 2.38 (m, 2H), 3.99 (s, 2H), 6.45
(m, 1H), 6.91-7.28 (m, 15 H); 13C NMR (75 MHz, CDCl3) δ 9.1, 33.0,
37.0, 95.4, 112.5, 121.2, 122.7, 126.2, 126.4, 126.5, 126.7, 126.8, 127.8,
127.9, 128.1, 128.2, 129.3, 129.4, 129.5, 129.6, 133.6, 134.0, 136.5, 137.1,
138.4, 146.4, 191.8. Anal. (C27H23NO2S) C, H, N.
(()-6-Ethyl-6-[4-[(2-fluorophenylthio)methyl]phenyl]-6,7-
dihydrobenzo[b]pyrrolo[1,2-d][1,4]oxazepin-7-one (4b). Com-
pound 4b was obtained following the same procedure described for com-
pound 4a. Purification was performed by means of flash chromatography
(EtOAc/n-hexane 1:9). Title compound was obtained as a pale yellow oil
(61% yield): 1H NMR (200 MHz, CDCl3) δ 1.02 (t, 3H, J = 7.2 Hz) 2.37
(m, 2H), 3.98 (s, 2H), 6.45 (m, 1H), 6.92-7.28 (m, 14 H). Anal.
(C27H22FNO2S) C, H, N.
(()-6-Ethyl-6-[4-[(2-chlorophenylthio)methyl]phenyl]-6,7-
dihydrobenzo[b]pyrrolo[1,2-d][1,4]oxazepin-7-one (4c). Com-
pound 4c was obtained following the same procedure described for
compound 4a. Purification was performed by means of flash chromatogra-
phy (EtOAc/n-hexane 1:9). Title compound was obtained as a pale yellow
oil (57% yield): 1H NMR (300 MHz, CDCl3) δ 1.02 (t, 3H, J = 7.3 Hz),
2.38 (m, 2H), 4.04 (s, 2H), 6.45 (m, 1H), 6.92-7.37 (m, 14H); 13C NMR
(75 MHz, CDCl3) δ 8.6, 32.5, 37.3, 96.0, 112.0, 120.8, 121.9, 125.5, 125.8,
126.2, 127.2, 127.3, 128.0, 128.8 (2C), 129.8, 129.9, 134.0, 134.1, 134.2,
135.6, 136.6, 137.7, 146.8, 192.8. Anal. (C27H22ClNO2S) C, H, N.
(()-6-Ethyl-6-[4-[(3-methylphenylthio)methyl]phenyl]-6,7-
dihydrobenzo[b]pyrrolo[1,2-d][1,4]oxazepin-7-one (4d). Com-
pound 4d was obtained following the same procedure described for com-
pound 4a. Purification was performed by means of flash chromatography
(diethyl ether/n-hexane 1:4). Title compound was obtained as a pale yellow
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stirred at 0 ꢀC for 30 min urea (9 mg, 0.150 mmol) was added. The
mixture was stirred for another 15 min. A solution of potassium iodide
(508 mg, 3.060 mmol) in 30 mL of water was added, and the resulting
mixture was stirred for 5 h at 0 ꢀC. The disappearance of the starting
material was checked by thin layer chromatography (petroleum ether
40-60 ꢀC). The mixture was then taken up with EtOAc. The aqueous
phase was separated and extracted with EtOAc (3 ꢀ 80 mL). The
combined organic layers were washed with water, dried over sodium
sulfate, filtered, and evaporated. The residue was purified by means of
flash column chromatography (petroleum ether 40-60 ꢀC). Title
compound was obtained with a 62% yield. Spectroscopic data of title
compound are consistent with those reported in the literature.40
4-Iodobenzenethiol (9a). To an ice-cooled solution of com-
pound 7 in 2 mL of MeOH, sodium borohydride (18 mg, 0.480 mmol)
was added. After the mixture was stirred at 0 ꢀC for 3 h water was added
and MeOH was removed under reduced pressure. HCl (12 N) was
added to the mixture until a white precipitate formed, then EtOAc
was added. The aqueous phase was separated and extracted with EtOAc
(3 ꢀ 20 mL). The combined organic phases were dried over sodium
sulfate, filtered, and concentrated. The compound was carried on
without further purification (84% yield). Spectroscopic data of the title
compound are consistent with those reported in the literature.41
(()-6-Ethyl-6-[4-[(4-iodophenylthio)methyl]phenyl]-6,7-
dihydrobenzo[b]pyrrolo[1,2-d][1,4]oxazepin-7-one (4l). Com-
pound 4l was obtained following the same procedure described for com-
pound 4a. Purification was performed by means of flash chromatography
(EtOAc/n-hexane 1:1). Title compound was obtained as a pale yellow oil
extracted with diethyl ether (3 ꢀ 10 mL). The combined organic layers
were dried over sodium sulfate, filtered, and concentrated. Purification
was performed by means of flash chromatography (n-hexane/DCM 1:1).
Title compound was obtained as a pale yellow oil (85% yield): 1H NMR
(300 MHz, CDCl3) δ 0.26 (s, 9H), 1.05 (t, 3H, J = 7.0 Hz), 2.29-2.51
(m, 2H), 4.01 (s, 2H), 6.47 (s, 1H), 6.93-7.30 (m, 14H); 13C NMR (75
MHz, CDCl3) δ 0.2, 8.6, 32.7, 38.3, 95.0, 95.9, 104.9, 112.0, 120.8, 121.2,
121.8, 125.5, 125.8, 126.2, 127.1, 128.0, 128.7, 129.3, 132.4, 133.9, 134.3,
137.2 (2C), 137.6, 146.8, 192.7. Anal. (C32H31NO2SSi) C, H, N.
(()-6-Ethyl-6-[4-[(4-ethynylphenylthio)methyl]phenyl]-6,7-
dihydrobenzo[b]pyrrolo[1,2-d][1,4]oxazepin-7-one (4q). To a
solution of 4p (37 mg, 0.07 mmol) in 2 mL of MeOH and 360 μL of THF,
potassium carbonate (10 mg 0.08 mmol) was added. The resultant
suspension was stirred for 45 min. Then the solution was diluted with
water and extracted with diethyl ether (3 ꢀ 10 mL). The combined organic
layers were dried over sodium sulfate, filtered, and concentrated. Purifica-
tion was performed by means of flash chromatography (EtOAc/n-hexane
1:8). Title compound was obtained as a pale yellow oil (75% yield): 1H
NMR (300 MHz, CDCl3) δ 1.04 (t, 3H, J = 7.3 Hz), 2.29-2.51 (m, 2H),
3.09 (s, 1H), 4.03 (s, 2H), 6.46 (s, 1H), 6.92-7.33 (m, 14H); 13C NMR
(75 MHz, CDCl3) δ 8.6, 14.4, 32.6, 38.1, 77.9, 83.5, 95.9, 112.0, 120.8,
121.6, 125.5, 125.8, 126.2, 127.1, 128.0 (2C), 128.7 (2C), 129.2 (2C),
132.6, 134.0, 134.3, 137.1, 137.6, 137.8, 146.8, 192.7. Anal. (C29H23NO2S)
C, H, N.
(()-6-Ethyl-6-[4-[(2-methyl-6-chlorophenylthio)methyl]-
phenyl]-6,7-dihydrobenzo[b]pyrrolo[1,2-d][1,4]oxazepin-7-
one (4r). Compound 4r was obtained following the same procedure
described for compound 4a. Purification was performed by means of
flash chromatography (EtOAc/n-hexane 1:9). Title compound was
obtained as a pale yellow oil (64% yield): 1H NMR (300 MHz, CDCl3)
δ 1.00 (t, 3H, J = 7.3 Hz), 2.11 (s, 3H), 2.34 (m, 2H), 3.88 (s, 2H), 6.45
(m, 1H), 6.91-7.36 (m, 13H). Anal. (C28H24ClNO2S) C, H, N.
(()-6-Ethyl-6-[4-[(2,6-dichlorophenylthio)methyl]phenyl]-
6,7-dihydrobenzo[b]pyrrolo[1,2-d][1,4]oxazepin-7-one (4s).
Compound 4s was obtained following the same procedure described
for compound 4a. Purification was performed by means of flash chroma-
tography (diethyl ether/petroleum ether 40-60ꢀC 1:8). Titlecompound
was obtained as an amorphous white solid (77% yield): 1H NMR (300
MHz, CDCl3) δ 1.00 (t, 3H, J = 7.3 Hz), 2.34 (m, 2H), 4.00 (s, 2H), 6.45
(m, 1H), 6.96-7.14 (m, 6H), 7.21-7.47 (m, 7H). Anal. (C27H21Cl2-
NO2S) C, H, N.
1
(47% yield): H NMR (300 MHz, CDCl3) δ 1.05 (t, 3H, J = 7.3 Hz),
2.26-2.51 (m, 2H), 3.98 (s, 2H), 6.47 (m, 1H) 6.89-7.51 (m, 14H); 13C
NMR (75 MHz, CDCl3) δ 8.6, 32.7, 38.6, 91.7, 95.9, 112.1, 120.9, 121.9,
125.5, 125.9, 126.2, 127.1, 128.0, 128.8, 131.9, 134.0, 134.3, 136.2, 137.3,
137.6, 137.9, 146.8, 192.8. Anal. (C27H22INO2S) C, H, N.
(()-6-Ethyl-6-[4-[(4-nitrophenylthio)methyl]phenyl]-6,7-
dihydrobenzo[b]pyrrolo[1,2-d][1,4]oxazepin-7-one (4m). Com-
pound 4m was obtained following the same procedure described for com-
pound 4a. Purification was performed by means of flash chromatography
(EtOAc/n-hexane 1:4). Title compound was obtained as a pale yellow oil
1
(44% yield): H NMR (300 MHz, CDCl3) δ 1.01 (t, 3H, J = 7.4 Hz),
2.35 (m, 2H), 4.03 (s, 2H), 6.44 (m, 1H), 6.88-7.32 (m, 14H). Anal.
(C27H22N2O4S) C, H, N.
(()-6-Ethyl-6-[4-[(4-trifluoromethoxyphenylthio)methyl]-
phenyl]-6,7-dihydrobenzo[b]pyrrolo[1,2-d][1,4]oxazepin-7-
one (4n). Compound 4n was obtained following the same procedure
described for compound 4a. Purification was performed by means of
flash chromatography (EtOAc/n-hexane 1:9). Title compound was
obtained as a pale yellow oil (85% yield): 1H NMR (300 MHz, CDCl3)
δ 1.03 (t, 3H, J = 7.3 Hz), 2.40 (m, 2H), 3.98 (s, 2H), 6.44 (m, 1H),
6.90-7.35 (m, 14H). Anal. (C28H22F3NO3S) C, H, N.
(()-6-Ethyl-6-[4-[(4-methoxyphenylthio)methyl]phenyl]-
6,7-dihydrobenzo[b]pyrrolo[1,2-d][1,4]oxazepin-7-one (4o).
Compound 4o was obtained following the same procedure described
for compound 4a. Purification was performed by means of flash chroma-
tography (EtOAc/n-hexane 1:7). Title compound was obtained as a pale
yellow oil (55% yield): 1H NMR (300 MHz, CDCl3) δ 1.05 (t, 3H, J = 7.3
Hz), 2.40 (m, 2H), 3.78 (s, 3H), 3.86 (s, 2H), 6.46 (m, 1H), 6.72 (m, 2H),
6.94-7.12 (m, 7H), 7.22-7.29 (m, 5H). Anal. (C28H25NO3S) C, H, N.
(()-6-Ethyl-6-[4-[[4-[2-(trimethylsilyl)ethynyl]phenylthio]-
methyl]phenyl]-6,7-dihydrobenzo[b]pyrrolo[1,2-d][1,4]oxa-
zepin-7-one (4p). In a sealed tube containing a suspension of
compound 4l (54 mg, 0,098 mmol) in TEA (1 mL) with dichlorobis-
(triphenylphosphine)palladium(II) (3 mg, 0.005 mmol) and copper
iodide (1 mg, 0,005 mmol), trimethylsilylacetylene (17 μL, 0.12 mmol)
was added dropwise. The resulting mixture was stirred for 12 h at room
temperature. After that time the solution was diluted with water and
Resolution of (()-6-Ethyl-6-[4-[(2,6-dichlorophenylthio)-
methyl]phenyl]-6,7-dihydrobenzo[b]pyrrolo[1,2-d][1,4]oxa-
zepin-7-one (4s). A sample solution of (()-4s (1 mg/mL) was
prepared by dissolving the compound in the eluent (n-hexane 98%,
EtOH 2%, TEA 0.2%) and filtering through 0.45 μm Acrodisc filters. The
injection volume was 100 μL. The racemic mixture was resolved at room
temperature using n-hexane 98%, EtOH 2%, and TEA 0.2% as eluent
(flow rate of the mobile phase 3.8 mL/min). The effluent was monitored
at (λ) 589 nm, and the enantiomers (-)-4s and (þ)-4s were eluted with
retention times of 13.5 and 15.08 min, respectively. Fractions were
collected by using a Vario2000 fraction collector, and solutions were
dried with a vacuum pump. Purity of the enantiomers was evaluated by
analytical HPLC using the same mobile phase (flow rate of 0.8 mL/min),
and the enantiomers (-)-4s and (þ)-4s were eluted with retention times
of 13.8 and 15.30 min, respectively. (-)-4s and (þ)-4s 1H NMR data are
identical to those of (()-4s. (-)-4s [R]20D -18.7ꢀ (c 0.4, chloroform).
Anal. (C27H21Cl2NO2S) C, H, N. (þ)-4s [R]20 þ18.5ꢀ (c 0.4,
D
chloroform). Anal. (C27H21Cl2NO2S) C, H, N.
(()-6-Ethyl-6-[4-[(2,3-dichlorophenylthio)methyl]phenyl]-
6,7-dihydrobenzo[b]pyrrolo[1,2-d][1,4]oxazepin-7-one (4t).
Compound 4t was obtained following the same procedure described for
compound 4a. Purification was performed by means of flash chromatog-
raphy (EtOAc/n-hexane 1:5). Title compound was obtained as an
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Journal of Medicinal Chemistry
ARTICLE
amorphous white solid (54% yield): 1H NMR (300 MHz, CDCl3)
δ 1.02 (t, 3H, J = 7.3 Hz), 2.38 (m, 2H), 4.06 (s, 2H), 6.45 (m, 1H),
6.97-7.14 (m, 5H), 7.21-7.38 (m, 8H). Anal. (C27H21Cl2NO2S) C, H, N.
(()-6-Ethyl-6-[4-[(2,5-dichlorophenylthio)methyl]phenyl]-
6,7-dihydrobenzo[b]pyrrolo[1,2-d][1,4]oxazepin-7-one (4u).
Compound 4u was obtained following the same procedure described for
compound 4a. Purification was performed by means of flash chromatogra-
phy (DCM/petroleum ether 40-60 ꢀC 1:2). Title compound was obtained
as an amorphous white solid (66% yield): 1H NMR (300 MHz, CDCl3)
δ 1.02 (t, 3H, J = 7.2 Hz), 2.38 (m, 2H), 4.05 (s, 2H), 6.45 (m, 1H),
6.94-7.13 (m, 5H), 7.21-7.35 (m, 8H). Anal. (C27H21Cl2NO2S) C, H, N
(()-6-Ethyl-6-[4-[(2,4-dichlorophenylthio)methyl]phenyl]-
6,7-dihydrobenzo[b]pyrrolo[1,2-d][1,4]oxazepin-7-one (4v).
Compound 4v was obtained following the same procedure described
for compound 4a. Purification was performed by means of flash chroma-
tography (diethyl ether/petroleum ether 40-60 ꢀC 1:8). Title compound
was obtained as an amorphous white solid (74% yield): 1H NMR (300
MHz, CDCl3) δ 1.03 (t, 3H, J = 7.3 Hz), 2.38 (m, 2H), 4.03 (s, 2H), 6.46
(m, 1H), 6.94-7.37 (m, 13H). Anal. (C27H21Cl2NO2S) C, H, N.
(()-6-Ethyl-6-[4-[(3,4-dichlorophenylthio)methyl]phenyl]-
6,7-dihydrobenzo[b]pyrrolo[1,2-d][1,4]oxazepin-7-one (4w).
Compound 4w was obtained following the same procedure described for
compound 4a. Purification was performed by means of flash chromatog-
raphy (diethyl ether/petroleum ether 40-60 ꢀC 1:8). Title compound
was obtained as an amorphous white solid (72% yield): 1H NMR (300
MHz, CDCl3) δ 1.01 (t, 3H, J = 7.3 Hz), 2.37(m, 2H), 4.01 (s, 2H), 6.45
(m, 1H), 6.93-7.13 (m, 6H), 7.21-7.46 (m, 7H). Anal. (C27H21Cl2-
NO2S) C, H, N
(()-6-Ethyl-6-[4-[(2,4-difluorophenylthio)methyl]phenyl]-
6,7-dihydrobenzo[b]pyrrolo[1,2-d][1,4]oxazepin-7-one (4x).
Compound 4x was obtained following the same procedure described for
compound 4a. Purification was performed by means of flash chroma-
tography (diethyl ether/n-hexane 1:9). Title compound was obtained as
a pale yellow oil (53% yield): 1H NMR (300 MHz, CDCl3) δ 1.00 (t, 3H,
J = 7.3 Hz), 2.36 (m, 2H), 3.90 (s, 2H), 6.44 (m, 1H), 6.66 (m, 1H), 6.80
(m, 1H), 6.92-7.32 (m, 11H). Anal. (C27H21F2NO2S) C, H, N.
(()-6-Ethyl-6-[4-[(2-chloro-4-fluorophenylthio)methyl]-
phenyl]-6,7-dihydrobenzo[b]pyrrolo[1,2-d][1,4]oxazepin-
7-one (4y). Compound 4y was obtained following the same pro-
cedure described for compound 4a. Purification was performed by
means of flash chromatography (diethyl ether/petroleum ether 40-
60 ꢀC 1:9). Title compound was obtained as an amorphous white solid
(67% yield): 1H NMR (300 MHz, CDCl3) δ 1.02 (t, 3H, J = 7.3 Hz),
2.37 (m, 2H), 4.02 (s, 2H), 6.41 (m, 1H), 6.91-7.36 (m, 13H). Anal.
(C27H21ClFNO2S) C, H, N.
(()-6-Ethyl-6-[4-[(3-chloro-4-fluorophenylthio)methyl]-
phenyl]-6,7-dihydrobenzo[b]pyrrolo[1,2-d][1,4]oxazepin-
7-one (4z). Compound 4z was obtained following the same proce-
dure described for compound 4a. Purification was performed by
means of flash chromatography (EtOAc/petroleum ether 40-60 ꢀC
1:9). Title compound was obtained as an amorphous white solid (65%
yield): 1H NMR (300 MHz, CDCl3) δ 1.03 (t, 3H, J = 7.3 Hz), 2.34
(m, 2H), 4.00 (s, 2H), 6.38 (m, 1H), 6.88-7.32 (m, 13H). Anal.
(C27H21ClFNO2S) C, H, N.
(()-6-Ethyl-6-[4-[(2,5-dimethoxyphenylthio)methyl]phenyl]-
6,7-dihydrobenzo[b]pyrrolo[1,2-d][1,4]oxazepin-7-one (4aa).
Compound 4aa was obtained following the same procedure described for
compound 4a. Purification was performed by means of flash chromatography
(EtOAc/n-hexane 1:9). Title compound was obtained as a pale yellow oil
(70% yield): 1H NMR (300 MHz, CDCl3) δ 1.01 (t, 3H, J = 7.2 Hz),
2.33 (m, 2H), 3.81 (s, 3H), 3.83 (s, 3H), 4.20 (s, 2H), 6.64 (m, 2H),
6.78 (m, 1H), 6.91 (m 1H), 7.15-7.20 (m, 4H), 7.25-7.30 (m, 3H),
7.37, (m, 1H), 7.46, (m, 1H), 7.55 (m, 1H). Anal. (C29H27NO4S)
C, H, N
2-Chloro-4-iodophenol (8). Starting from 4-amino-2-chloro-
phenol 6b the title compound was prepared according to the procedure
described for 7. The crude was purified by means of flash chromatog-
raphy (EtOAc/n-hexane 1:5) (55% yield). Spectroscopic data of the title
compound are consistent with those reported in the literature.42
O-2-Chloro-4-iodophenyl-N,N-dimethylcarbamothioate
(10). A solution of compound 8 (124 mg, 0.490 mmol) in DMF (600
μL) was treated with DABCO (110 mg, 0.980 mmol) and dimethylthio-
carbamyl chloride (96 mg, 0.780 mmol). The mixture was stirred at room
temperature for 1 h. Then it was diluted with EtOAc and poured into ice-
water. The layers were separated, and the aqueous phase was extracted
with EtOAc (3 ꢀ 50 mL). The combined organic extracts were washed
with water and with brine. The combined organic layers were dried over
sodium sulfate, filtered, and concentrated. The crude was purified by
means of flash chromatography (EtOAc/n-hexane 1:5). Title compound
1
was obtained with a 63% yield: H NMR (300 MHz, CDCl3) δ 3.37
(s, 3H), 3.45 (s, 3H), 6.89 (d, 1H, J = 8.5 Hz), 7.63 (d, 1H, J = 8.5 Hz),
7.76 (s, 1H); 13C NMR (75 MHz, CDCl3) δ 39.2, 43.8, 90.2, 127.1, 129.1,
136.8, 138.7, 150.2, 186.1.
S-2-Chloro-4-iodophenyl-N,N-dimethylcarbamothioate
(11). A sealed tube containing compound 10 (105 mg, 0.31 mmol)
was inserted into the cavity of the microwave apparatus and heated
neat at 220 ꢀC, employing a microwave power of 150 W for 10 min.
After the mixture was cooled, the crude was taken up with DCM and
purified by means of flash chromatography (DCM). Title compound
was obtained with a 82% yield: 1H NMR (300 MHz, CDCl3) δ 3.03
(s, 3H), 3.10 (s, 3H), 7.28 (d, 1H, J = 8.2 Hz) 7.60 (d, 1H, J = 8.2 Hz),
7.84 (s, 1H); 13C NMR (75 MHz, CDCl3) δ 37.3, 96.1, 128.7, 136.6,
138.6, 139.3, 140.5, 164.7.
2-Chloro-4-iodobenzenethiol (9b). Compound 11 (87 mg,
0.250 mmol) was dissolved in EtOH (400 μL) and treated with 3 N
aqueous KOH (292 μL). The mixture was heated at reflux for 3 h, and
then it was allowed to cool to room temperature. After removal of EtOH
under reduced pressure, the aqueous solution was acidified with 3 N HCl
until pH ∼ 3 and extracted with EtOAc (3 ꢀ 100 mL). The combined
organic layers were dried over sodium sulfate, filtered, and concentrated.
The compound was used in the following step without further purifica-
tion (80% yield): 1H NMR (300 MHz, CDCl3) δ 3.89 (s, 1H), 7.05 (d,
1H, J = 8.2 Hz), 7.43 (d, 1H, J = 8.2 Hz), 7.69 (s, 1H).
(()-6-Ethyl-6-[4-[(2-chloro-4-iodophenylthio)methyl]-
phenyl]-6,7-dihydrobenzo[b]pyrrolo[1,2-d][1,4]oxazepin-7-
one (4bb). Compound 4bb was obtained following the same proce-
dure described for compound 4a. Purification was performed by means
of flash chromatography (chloroform/n-hexane 1:1). Title compound
was obtained as an amorphous white solid (55% yield): 1H NMR (300
MHz, CDCl3) δ 1.03 (t, 3H, J = 7.3 Hz), 2.27-2.50 (m, 2H), 4.03
(s, 2H), 6.45 (m, 1H), 6.77-7.40 (m, 12H) 7.67 (s, 1H); 13C NMR
(75 MHz, CDCl3) δ 8.6, 29.9, 32.5, 37.1, 77.5, 90.2, 95.9, 112.0, 120.9,
121.9, 125.5, 125.9, 126.2, 127.1, 128.1, 128.8, 130.8, 133.9, 134.2, 134.8,
135.8, 136.2, 137.9, 146.8, 192.7. Anal. (C27H21ClINO2S) C, H, N
(()-6-Ethyl-6-[4-[(2,3,5,6-tetrafluorophenylthio)methyl]-
phenyl]-6,7-dihydrobenzo[b]pyrrolo[1,2-d][1,4]oxazepin-7-
one (4cc). Compound 4cc was obtained following the same proce-
dure described for compound 4a. Purification was performed by means
of flash chromatography (diethyl ether/n-hexane 1:9). Title compound
was obtained as an amorphous white solid (45% yield): 1H NMR (300
MHz, CDCl3) δ 1.02 (t, 3H, J = 7.3 Hz), 2.36 (m, 2H), 4.02 (s, 2H), 6.42
(m, 1H), 6.86-7.35 (m, 11H). Anal. (C27H19F4NO2S) C, H, N.
(()-6-Ethyl-6-[4-[(2-pyridylthio)methyl]phenyl]-6,7-dihy-
drobenzo[b]pyrrolo[1,2-d][1,4]oxazepin-7-one (4dd). Com-
pound 4dd was obtained following the same procedure described for
compound 4a. Purification was performed by means of flash chromato-
graphy (EtOAc/n-hexane 1:9). Title compound was obtained as a
pale yellow oil (75% yield): 1H NMR (200 MHz, CDCl3) δ 1.03
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Journal of Medicinal Chemistry
ARTICLE
(t, 3H, J = 7.5 Hz), 2.38 (m, 2H), 4.35 (s, 2H), 6.43 (m, 1H), 6.93-7.48
(m, 13H), 8.39 (m, 1H). Anal. (C26H22N2O2S) C, H, N.
ture was cooled to room temperature, the solvent was removed under
reduced pressure. The crude was purified by means of flash chromatog-
raphy (MeOH/chloroform 1:9). Title compound was obtained as a
yellow solid (71% yield). Spectroscopic data of title compound are
consistent with those reported in the literature.44
(()-6-Ethyl-6-[4-[(2-imidazolylthio)methyl]phenyl]-6,7-di-
hydrobenzo[b]pyrrolo[1,2-d][1,4]oxazepin-7-one (4ee). Com-
pound 4ee was obtained following the same procedure described for com-
pound 4a. Purification was performed by means of flash chromatography
(EtOAc/n-hexane 4:6). Title compound was obtained as a pale yellow oil
(78% yield): 1H NMR (200 MHz, CDCl3) δ 1.02 (t, 3H, J = 7.5 Hz), 2.36
(m, 2H), 4.12 (s, 2H), 6.43 (t, 1H), 6.97-7.39 (m, 12H), 12.9 (br s, 1H).
Anal. (C24H21N3O2S) C, H, N.
(()-6-Ethyl-6-[4-[(2-pyrazylthio)methyl]phenyl]-6,7-dihy-
drobenzo[b]pyrrolo[1,2-d][1,4]oxazepin-7-one (4ff). Compound
4ff was obtained following the same procedure described for compound 4a.
Purification was performed by means of flash chromatography (EtOAc/n-
hexane 1:9). Title compound was obtained as a yellow oil (64% yield): 1H
NMR (200 MHz, CDCl3) δ 1.03 (t, 3H, J = 7.5 Hz), 2.39 (m, 2H), 4.08 (s,
2H), 6.44 (m, 1H), 6.91-7.36 (m, 10H), 8.59-8.63 (m, 3H). Anal.
(C25H21N3O2S) C, H, N.
(()-6-Ethyl-6-[4-[(2-pyrimidylthio)methyl]phenyl]-6,7-di-
hydrobenzo[b]pyrrolo[1,2-d][1,4]oxazepin-7-one (4gg). Com-
pound 4gg was obtained following the same procedure described for com-
pound 4a. Purification was performed by means of flash chromatography
(EtOAc/n-hexane 1:9). Title compound was obtained as a yellow oil (69%
yield): 1H NMR (200 MHz, CDCl3) δ 1.01 (t, 3H, J = 7.5 Hz), 2.38 (m,
2H), 4.06 (s, 2H), 6.45 (m, 1H), 6.91-7.39 (m, 11H), 8.61-8.65 (m, 2H).
Anal. (C25H21N3O2S) C, H, N.
(()-6-Ethyl-6-[4-[(2-chloro-4-iodophenyloxy)methyl]phenyl]-
6,7-dihydrobenzo[b]pyrrolo[1,2-d][1,4]oxazepin-7-one (4hh).
Compound 4hh was obtained following the same procedure described for
compound 4a and using the appropriate phenol derivative. Purification was
performed by means of flash chromatography (diethyl ether/petroleum ether
40-60 ꢀC 1:5). Title compound was obtained as an amorphous white solid
(48% yield): 1H NMR (300 MHz, CDCl3) δ 1.04 (t, 3H, J = 7.3 Hz), 2.28-
2.52 (m, 2H), 5.06 (s, 2H), 6.45-6.47 (m, 1H), 6.60 (d, 1H, J = 8.5 Hz),
6.96-7.44 (m, 11H), 7.67 (d, 1H, J = 2.3 Hz); 13C NMR (75 MHz, CDCl3)
δ 8.6, 29.9, 32.4, 70.6, 77.4, 82.7, 95.9, 112.0, 116.1, 120.9, 121.9, 124.8, 125.5,
125.9, 126.2, 126.9, 127.2, 128.1, 134.0, 136.1, 136.7, 138.6, 146.8, 154.3, 192.8.
Anal. (C27H21ClINO3) C, H, N.
(()-6-Ethyl-6-[4-[3-(2-hydroxy)ethylphenoxymethyl]phenyl]-
6,7-dihydrobenzo[b]pyrrolo[1,2-d][1,4]oxazepin-7-one (4ii).
Compound 4ii was obtained following the same procedure described for
compound 4a and using the appropriate phenol derivative (12). Purifica-
tion was performed by means of flash chromatography (EtOAc). Title
compound was obtained as an amorphous white solid (56% yield): 1H
NMR (200 MHz, CD3OD), δ 1.03 (t, 3H, J = 7.0 Hz), 2.40 (m, 2H), 2.54
(t, 2H, J = 7.1 Hz), 3.70 (t, 2H, J = 7.1 Hz), 4.95 (s, 2H), 6.47-6.51
(m, 1H),6.63-6.79(m,3H),6.93-7.46 (m, 11H); ESI-MS m/z[Mþ H]þ
454 (100), [M þ Na]þ 476 [M þ K]þ 492. Anal. (C29H27NO4) C, H, N.
3-(2-Bromoethyl)phenol (13). 3-(2-Hydroxyethyl)phenol (12)
(1.0 g, 7.24 mmol) was dissolved in 40.0 mL of dry acetonitrile. Then
triphenylphosphine (2.1 g, 7.96 mmol) and carbon tetrabromide (2.7 g,
7.96 mmol) were added. The reaction mixture was refluxed under argon
atmosphere for 12 h. After cooling to room temperature, the reaction
was quenched with 15% aqueous NaOH. The solvent was removed
under reduced pressure, and the residue was extracted with EtOAc (3 ꢀ
100 mL). The organic layers were dried over sodium sulfate, filtered, and
concentrated. The crude was purified by means of flash chromatography
(EtOAc/n-hexane 2:1). Title compound was obtained as a colorless oil
(55% yield). Spectroscopic data of title compound are consistent with
those reported in the literature.43
3-[2-(Pyrrolidin-1-yl)ethyl]phenol (15). Compound 15 was
obtained following the same procedure described for compound 14
and using pyrrolidine. Purification was performed by means of flash
chromatography (MeOH/chloroform 1:9). Title compound was ob-
tained as a yellowish solid (60% yield). Spectroscopic data of title
compound are consistent with those reported in the literature.44
(()-6-Ethyl-6-[4-[3-(2-diethylamino)ethylphenoxymethyl]-
phenyl]-6,7-dihydrobenzo[b]pyrrolo[1,2-d][1,4]oxazepin-7-
one (4jj). Compound 4jj was obtained following the same procedure
described for compound 4a and using the appropriate phenol derivative
(14). Purification was performed by means of flash chromatography
(MeOH/chloroform 1:19). Title compound was obtained as an amor-
phous white solid (65% yield): 1H NMR (200 MHz, CDCl3), δ 1.05 (t,
3H, J = 6.8 MHz), 1.22 (t, 6H, J = 7.2 Hz), 2.24-2.55 (m, 2H), 2.83-2.94
(m, 8H), 4.96 (s, 2H), 6.45 (m, 1H), 6.75-6.81 (m, 3H), 6.97-7.43
(m,11H);ESI-MSm/z[Mþ H]þ 509 (100). Anal. (C33H36N2O3) C,H,N.
(()-6-Ethyl-6-[4-[3-(2-pyrrolidin-1-yl)ethylphenoxymethyl]-
phenyl]-6,7-dihydrobenzo[b]pyrrolo[1,2-d][1,4]oxazepin-7-
one (4kk). Compound 4kk was obtained following the same proce-
dure described for compound 4a and using the appropriate phenol
derivative (15). Purification was performed by means of flash chroma-
tography (MeOH/chloroform 1:19). Title compound was obtained as
an amorphous white solid (70% yield): 1H NMR (200 MHz, CDCl3), δ
1.00 (t, 3H, J = 6.9 Hz), 1.59 (m, 4H), 2.24-2.29 (m, 2H), 2.41 (m, 4H),
2.65-2.69 (m, 4H), 4.91 (s, 2H), 6.39 (m, 1H), 6.74-7.36 (m, 14H);
ESI-MS m/z [M þ H]þ 507 (100). Anal. (C33H34N2O3) C, H, N.
(()-Ethyl 2-[4-[(Phenylthio)methyl]phenyl]-2-hydroxya-
cetate (17a). To a solution of thiophenol (103 μL, 1.00 mmoL) in
dry THF (2.0 mL), sodium borohydride (18.0 mg, 0.460 mmol) was
added. After the mixture was stirred for 30 min at room temperature, a
solution of compound 16 (292 mg, 0.92 mmol) in 5.5 mL of dry THF
was added dropwise. Then additional sodium borohydride (18.0 mg,
0.460 mmol) and sodium hydride (24 mg, 1.0 mmol) were added to the
mixture. The mixture was allowed to stir for 12 h at 60 ꢀC under argon
atmosphere. After the mixture was cooled to room temperature, a
saturated aqueous ammonium chloride solution was added. THF was
removed under reduced pressure, and the residue was extracted with
DCM (2 ꢀ 100 mL). The combined organic layers were dried over
sodium sulfate, filtered, and concentrated. Purification was performed by
means of flash chromatography (EtOAc/n-hexane 1:3). Pure title
compound was obtained as an amorphous white solid (65% yield): 1H
NMR (300 MHz, CDCl3) δ 1.22 (t, 3H, J = 7.0 Hz), 3.45 (d, 1H, J = 5.7
Hz), 4.11-4.29 (m, 4H), 5.13 (d, 1H, J = 5.7 Hz), 7.18-7.40 (m, 9H);
ESI-MS m/z [M þ Na]þ 325 [M þ K]þ 341 (100).
(()-Ethyl 2-[4-[(2-Chlorophenylthio)methyl]phenyl]-2-hy-
droxyacetate (17b). Compound 17b was obtained following the
same procedure described for compound 17a and using the appropriate
tiophenol derivative. Purification was performed by means of flash
chromatography (EtOAc/n-hexane 1:4). Title compound was obtained
as an amorphous white solid (68% yield): 1H NMR (300 MHz, CDCl3) δ
1.22 (t, 3H, J = 7.0 Hz), 3.44 (d, 1H, J = 5.6 Hz), 4.14-4.30 (m, 4H), 5.11
(d, 1H, J = 5.6 Hz), 7.07-7.40 (m, 8H); ESI-MS m/z [M þ H]þ 337
(100).
(()-Ethyl 2-[4-[(3-Trifluoromethylphenylthio)methyl]-
phenyl]-2-hydroxyacetate (17c). Compound 17c was obtained fol-
lowing the same procedure described for compound 17a and using the
appropriate thiophenol derivative. Purification was performed by means of
flash chromatography (EtOAc/n-hexane 1:4). Title compound was ob-
3-[2-(Diethylamino)ethyl]phenol (14). Bromide 13 (359 mg,
1.79 mmol) was dissolved in 15.0 mL of dry acetonitrile. To the refluxing
solution, diethylamine (187 μL, 1.79 mmol) and TEA (301 μL, 2.15
mmol) were added, and the mixture was refluxed for 12 h. After the mix-
1
tained as an amorphous white solid (72% yield): H NMR (300 MHz,
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Journal of Medicinal Chemistry
ARTICLE
CDCl3) δ 1.22 (t, 3H, J = 6.9 Hz), 3.43 (d, 1H, J = 5.6 Hz), 4.08-4.32 (m,
4H), 5.10 (d, 1H, J = 5.6 Hz), 7.20-7.48 (m, 8H); ESI-MS m/z [Mþ H]þ
371 (100).
solution of NaOH (3.34 mL, 4.180 mmol) was added dropwise. The mixture
was allowed to stir at room temperature for 12 h. Then the solvents were
removed under reduced pressure and EtOAc was added. After the first
extraction, the aqueous phase was treated with 6 N HCl until the acid
precipitated. The aqueous phase was then extracted with EtOAc (3 ꢀ 200
mL). The combined organic layers were dried over sodium sulfate, filtered,
and concentrated. Title compound was obtained as an amorphous yellow
solid and was used in the following step without further purification (99%
yield): 1H NMR (300 MHz, CDCl3) δ 4.10 (s, 2H), 5.55 (s, 1H), 6.32 (m,
2H), 6.98-7.38 (m, 15H), 11.23 (br s, 1H).
(()-2-[2-(1H-Pyrrol-1-yl)phenoxy]-2-[4-[((2-chlorophenyl-
thio)methyl)]phenyl]acetic Acid (20b). Compound 20b was
obtained following the same procedure described for 20a. Title com-
pound was obtained as an amorphous yellow solid and was used in the
following step without further purification (98% yield): 1H NMR (300
MHz, CDCl3) δ 4.11(s, 2H), 5.56 (s, 1H), 6.33 (m, 2H), 6.95-7.40 (m,
14H), 9.56 (br s, 1H).
(()-Ethyl 2-Bromo-2-[4-[(phenylthio)methyl]phenyl]-
acetate (18a). To a solution of compound 17a (243 mg, 0.800
mmol) in acetonitrile (10.0 mL), triphenylphosphine (232 mg, 0.880
mmol) and carbon tetrabromide (294 mg, 0.880 mmol) were added.
After 2 h at room temperature, the mixture was stirred for 12 h at 50 ꢀC.
After the mixture was cooled to room temperature, a 5% NaOH aqueous
solution was added. Acetonitrile was removed under reduced pressure,
and the residue was extracted with EtOAc (3 ꢀ 100 mL). The combined
organic layers were dried over sodium sulfate, filtered, and concentrated.
The crude product was purified by means of flash chromatography
(EtOAc/n-hexane 1:6). Title compound was obtained as a pale yellow
oil (51% yield): 1H NMR (300 MHz, CDCl3) δ 1.27 (t, 3H, J = 6.9 Hz),
4.10 (s, 2H), 4.23 (m, 2H), 5.31 (s, 1H), 7.19-7.29 (m, 6H), 7.47
(d, 2H, J = 7.5 Hz), 7.56 (m, 1H).
(()-Ethyl 2-[4-[(2-Chlorophenylthio)methyl]phenyl]-2-bro-
moacetate (18b). Compound 18b was obtained following the same
procedure described for compound 18a. Purification was performed by
means of flash chromatography (EtOAc/n-hexane 1:6). Title compound
was obtained as a pale yellow oil (59% yield): 1H NMR (300 MHz, CDCl3)
δ 1.28 (m, 3H, J = 7.1 Hz), 4.13 (s, 2H), 4.21-4.29 (m, 2H), 5.31 (s, 1H),
7.13-7.49 (m, 8H).
(()-2-[2-(1H-Pyrrol-1-yl)phenoxy]-2-[4-[((3-trifluoromethyl-
phenylthio)methyl)]phenyl]acetic Acid (20c). Compound 20c
was obtained following the same procedure described for 20a. Title
compound was obtained as an amorphous yellow solid and was used in
the following step without further purification (99% yield): 1H NMR (300
MHz, CDCl3) δ 4.10(s, 2H), 5.55 (s, 1H), 6.32 (m, 2H), 6.98-7.41
(m, 14H), 9.73 (br s, 1H).
(()-Ethyl 2-[4-[(3-Trifluoromethylphenylthio)methyl]-
phenyl]-2-bromoacetate (18c). Compound 18c was obtained
following the same procedure described for compound 18a. Purification
was performed by means of flash chromatography (EtOAc/n-hexane
1:6). Title compound was obtained as a pale yellow oil (57% yield):
1H NMR (300 MHz, CDCl3) δ 1.27 (t, 3H, J = 7.0 Hz), 4.13 (s, 2H),
4.15-4.25 (m, 2H), 5.31 (s, 1H), 7.26-7.49 (m, 8H).
(()-Ethyl 2-[2-(1H-Pyrrol-1-yl)phenoxy]-2-[4-[(phenylthio)-
methyl]phenyl]acetate (19a). To a solution of 2-(pyrrol-1-yl)-
phenol29 (80.0 mg, 0.500 mmol) in dry THF (4.0 mL), sodium
hydride (14.0 mg, 0.560 mmol) was added. The mixture was stirred at
room temperature for 30 min. Then compound 18a (204.0 mg, 0.560
mmol) dissolved in 6.5 mL of dry THF was added dropwise, and the
mixture was allowed to stir at 40 ꢀC for 11 h. After the mixture was
cooled, a saturated aqueous solution of ammonium chloride was
added to the mixture. THF was removed under reduced pressure, and
the residue was extracted with DCM (3 ꢀ 60 mL). The combined
organic layers were dried over sodium sulfate, filtered, and concen-
trated. The crude product was purified by means of flash chroma-
tography (EtOAc/n-hexane 1:13). Title compound was obtained as a
yellow oil (56% yield): 1H NMR (300 MHz, CDCl3) δ 1.16 (t, 3H, J =
7.0 Hz), 4.08-4.19 (m, 4H), 5.50 (s, 1H), 6.33 (m, 2H), 6.98-7.38
(m, 15H).
(()-Ethyl 2-[2-(1H-Pyrrol-1-yl)phenoxy]-2-[4-[(2-chloro-
phenylthio)methyl]phenyl]acetate (19b). Compound 19b
was obtained following the same procedure described for compound
19a. Purification was performed by means of flash chromatography
(EtOAc/n-hexane 1:13). Title compound was obtained as a yellow oil
(65% yield): 1H NMR (300 MHz, CDCl3) δ 1.17 (t, 3H, J = 7.0 Hz),
4.12-4.16 (m, 4H), 5.51 (s, 1H), 6.33 (m, 2H), 6.95-7.40 (m, 14H).
(()-Ethyl 2-[2-(1H-Pyrrol-1-yl)phenoxy]-2-[4-[(3-trifluoro-
methylphenylthio)methyl]phenyl]acetate (19c). Compound
19c was obtained following the same procedure described for compound
19a. Purification was performed by means of flash chromatography
(EtOAc/n-hexane 1:13). Title compound was obtained as a yellow oil
(61% yield): 1H NMR (300 MHz, CDCl3) δ 1.16 (m, 3H, J = 7.0 Hz),
4.13-4.17 (m, 4H), 5.50 (s, 1H), 6.34 (m, 2H), 6.99-7.43 (m, 14H).
(()-2-[2-(1H-Pyrrol-1-yl)phenoxy]-2-[4-[(phenylthiomethyl)-
phenyl]acetic Acid (20a). To a solution of compound 19a (1.854 g,
4.180 mmol) in 50.0 mL of a THF/EtOH 1:1 mixture, a 5% aqueous
(()-6-[4-[(Phenylthio)methyl]phenyl]-6,7-dihydrobenzo-
[b]pyrrolo[1,2-d][1,4]oxazepin-7-one (4ll). To a refluxing solu-
tion of 20a (1.620 g, 3.900 mmol) in dry DCM (40.0 mL), PCl5 (894
mg, 4.290 mmol) was added. The resulting mixture was stirred under
reflux for 7 h. After the mixture was cooled to room temperature,
a saturated aqueous solution of sodium bicarbonate was added. The
residue was extracted with DCM (3 ꢀ 100 mL). The combined organic
layers were dried over sodium sulfate, filtered, and concentrated. The
crude was purified by means of flash chromatography (EtOAc/n-
hexane 1:9). Title compound was obtained as an amorphous white
solid (80% yield): 1H NMR (300 MHz, CDCl3) δ 4.08 (s, 2H), 5.54 (s,
1H), 6.51 (m 1H), 7.11-7.42 (m, 15H). Anal. (C25H19NO2S) C, H, N.
(()-6-[4-[(2-Chlorophenylthio)methyl]phenyl]-6,7-dihy-
drobenzo[b]pyrrolo[1,2-d][1,4]oxazepin-7-one (4mm). Com-
pound 4mm was obtained following the same procedure described for 4ll.
The crude product was purified by means of flash chromatography (EtOAc/
n-hexane 1:9). Title compound was obtained as an amorphous white solid
(69%yield):1H NMR (300 MHz, CDCl3) δ4.13 (s, 2H), 5.52 (s, 1H), 6.51
(m 1H), 7.06-7.42 (m, 14H). Anal. (C25H18ClNO2S) C, H, N.
(()-6-[4-[(3-Trifluoromethylphenylthio)methyl]phenyl]-
6,7-dihydrobenzo[b]pyrrolo[1,2-d][1,4]oxazepin-7-one (4nn).
Compound 4nn was obtained following the same procedure described for
4ll. The crude product was purified by means of flash chromatography
(EtOAc/n-hexane 1:9). Title compound was obtained as an amorphous
1
white solid (55% yield): H NMR (300 MHz, CDCl3) δ 4.10 (s, 2H),
5.53 (s, 1H), 6.52 (m 1H), 7.08-7.45 (m, 14H). Anal. (C26H18F3NO2S)
C, H, N.
(()-r-[[2-(1H-Pyrrol-1-yl)phenyl]thio]-p-tolylacetic Acid
Ethyl Ester (22). A suspension of 21 (2.50 g, 11.43 mmol) in absolute
EtOH (45.0 mL) was heated to reflux. Sodium borohydride (550 mg,
14.50 mmol) was then added in portions over 30 min. Then the mixture
was allowed to cool to room temperature and (()-R-bromo-p-tolyla-
cetic acid ethyl ester (4.0 g, 15.80 mmol) in 20.0 mL of absolute EtOH
was added dropwise. After 8 h water was added to the mixture, and
EtOH was removed under reduced pressure. The residue was extracted
with chloroform (3 ꢀ 150 mL). The combined organic layers were dried
over sodium sulfate, filtered, and concentrated. The crude was purified
by means of flash chromatography (chloroform/n-hexane 1:1). Title
compound was obtained as an amorphous white solid (79% yield): 1H
NMR (300 MHz, CDCl3), δ 1.13 (t, 3H, J = 7.1 Hz), 2.35 (s, 3H), 4.09
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Journal of Medicinal Chemistry
ARTICLE
(m, 2H), 4.43 (s, 1H), 6.43(t, 2H, J = 1.8 Hz), 7.01 (t, 2H, J = 1.9 Hz),
7.14 (d, 2H, J = 7.2 Hz), 7.28-7.39 (m, 5H), 7.61 (d, 1H, J = 7.3 Hz).
(()-r-[[2-(1H-Pyrrol-1-yl)phenyl]thio]-p-tolylacetic Acid
(23). Compound 23 was obtained following the same procedure
described 20a. Title compound was obtained as a yellowish amorphous
solid and was used in the following step without further purification
(99% yield): 1H NMR (300 MHz, CDCl3) δ 2.26 (s, 3H), 4.36 (s, 1H),
6.39 (t, 2H, J = 1.8 Hz) 7.00-7.58 (m, 10 H), 9.69 (br s, 1H).
(()-6-(p-Tolyl)pyrrolo[2,1-d][1,5]benzothiazepin-7-(6H)-
one (24). Compound 24 was obtained following the same procedure
described for 4ll. The crude product was purified by means of flash
chromatography (EtOAc/n-hexane 1:7). Title compound was obtainedas
an amorphous white solid (65% yield): 1H NMR (300 MHz, CDCl3) δ
2.25 (s, 3H), 4.79 (s, 1H), 6.52 (m, 1H), 6.95-7.46 (m, 10H).
(()-6-Ethyl-6-(p-tolyl)pyrrolo[2,1-d][1,5]benzothiazepin-
7-(6H)-one (25). A solution of 24 (150 mg, 0.490 mmol) in dry THF
(5.0 mL) was added dropwise to a suspension of potassium hydride
(35% in mineral oil, 63 mg, 0.540 mmol) in dry THF (1.5 mL). The
mixture was stirred for 2 h at room temperature. Then iodoethane (223
μL, 2.760 mmol) was slowly added and the mixture was allowed to stir at
room temperature for 15 h. After that time a saturated aqueous solution
of ammonium chloride was added and THF was removed under reduced
pressure and the residue was extracted with DCM (3 ꢀ 100 mL). The
combined organic layers were dried over sodium sulfate, filtered, and
concentrated. The product was purified by means of flash chromatog-
raphy (DCM/petroleum ether 40-60 ꢀC 1:1). Pure title compound was
obtained as an amorphous white solid (70% yield): 1H NMR (300 MHz,
CDCl3) δ 1.06 (t, 3H, J = 7.3 Hz), 2.15-2.29 (m, 4H), 2.42-2.59
(m, 1H), 6.41 (m, 1H), 6.81-7.42 (m, 10H).
(()-6-Ethyl-6-(4-bromomethylphenyl)pyrrolo[2,1-d][1,5]-
benzothiazepin-7(6H)-one (26). To a solution of 25 (100 mg,
0.300 mmol) in carbon tetrachloride (8.5 mL), N-bromosuccinimide
(59 mg, 0.330 mmol) and a catalytic amount of AIBN were added. The
mixture was stirred for 2.5 h under reflux. After the mixture was cooled to
room temperature, succinimide was removed by filtration and the
solvent was evaporated under reduced pressure. The residue was taken
up with DCM, and the solution was washed with brine. The organic layer
was dried over sodium sulfate, filtered, and concentrated. The product
was purified by means of flash chromatography (EtOAc/n-hexane 1:7).
Title compound was obtained as an amorphous white solid (85% yield):
1H NMR (300 MHz, CDCl3) δ 1.08 (t, 3H, J = 7.4 Hz), 2.11-2.30
(m, 1H), 2.44-2.63 (m, 1H), 4.28 (s, 2H), 6.44 (m, 1H), 6.97-7.48
(m, 10H).
(()-6-Ethyl-6-[4-[(2-chlorophenylthio)methyl]phenyl]-6,7-
dihydrobenzo[b]pyrrolo[1,2-d][1,4]thiazepin-7-one (4oo).
Compound 4oo was obtained following the same procedure described
for compound 4a. Purification was performed by means of flash
chromatography (EtOAc/n-hexane 1:10). Title compound was ob-
tained as an amorphous white solid (82% yield): 1H NMR (300 MHz,
CDCl3) δ 1.07 (t, 3H, J = 7.2 Hz), 2.18-2.28 (m, 1H), 2.47-2.56
(m, 1H), 3.95 (s, 2H), 6.44 (m, 1H), 6.94-7.17 (m, 12H), 7.33-7.35
(m, 1H), 7.41-7.46 (m, 1H). Anal. (C27H22ClNOS2) C, H, N.
(()-6-Ethyl-6-[4-[(4-iodophenylthio)methyl]phenyl]-6,7-
dihydrobenzo[b]pyrrolo[1,2-d][1,4]thiazepin-7-one (4pp).
Compound 4pp was obtained following the same procedure described
for compound 4a. Purification was performed by means of flash
chromatography (petroleum ether 40-60 ꢀC/diethyl ether 5:1). Title
compound was obtained as an amorphous white solid (70% yield): 1H
NMR (300 MHz, CDCl3) δ 1.07 (t, 3H, J = 7.0 Hz), 2.04-2.27
(m, 1H), 2.47-2.57 (m, 1H), 3.88 (s, 2H), 6.43-6.45 (m, 1H), 6.85-7.19
(m, 10H), 7.33-7.35 (m, 1H), 7.45-7.52 (m, 3H); 13C NMR (75 MHz,
CDCl3) δ 10.4, 34.1, 38.2, 68.7, 91.3, 111.2, 122.2, 124.0, 126.2, 126.6,
128.1, 128.1, 128.5, 128.9, 131.4, 135.5, 135.9, 136.4, 136.5, 137.9, 139.1,
142.04, 189.4. Anal. (C27H22INOS2) C, H, N.
(()-6-Ethyl-6-[4-[[4-[2-(trimethylsilyl)ethynyl]phenylthio]-
methyl]phenyl]-6,7-dihydrobenzo[b]pyrrolo[1,2-d][1,4]thia-
zepin-7-one (4qq). Compound 4qq was obtained following the same
procedure described for compound 4p. Purification was performed by
means of flash chromatography (EtOAc/n-hexane 1:8). Title compound
was obtained as an amorphous white solid (80% yield): 1H NMR (300
MHz, CDCl3) δ 0.24 (s, 9H), 1.07 (t, 3H, J = 7.3 Hz), 2.17-2.24
(m, 1H), 2.50-2.55 (m, 1H), 3.91 (s, 2H), 6.42-6.45 (m, 1H), 6.93-7.34
(m, 13H), 7.46-7.48 (m, 1H); 13C NMR (75 MHz, CDCl3) δ 0.2, 10.3,
34.1, 37.9, 68.7, 95.0, 104.9, 111.2, 120.9, 122.1, 124.0, 126.2, 126.6, 128.0,
128.5, 128.8, 128.9, 129.9, 132.3 (2C), 135.5, 135.9, 136.5, 137.4, 139.1,
142.0. Anal. (C32H31NOS2Si) C, H, N.
Construction of Recombinant Bacterial Expression Vector
for hAK. An amount of 1 μg of total RNA extracted from HeLaS-3 cells
using the Rneasy Mini kit (Quiagen) was retrotranscribed by using RT-
PCR method. cDNA of hAK was then amplified by using the following
specific primers: 50-GGAGGGCGGATCCATGACGT CAGTCA-
GAG-30 (primer 1, sense) and 50-GTGTTTTAAGCTTTTCCATC
AGTGGAAGTC-30 (primer 2, antisense), presenting the NheI and
HindIII restriction sites, respectively. The amplified region (1070 bp
long) containing the complete hAK coding sequence was inserted into
the multiple cloning site of pTrcHisA (Invitrogen). His-tagged hAK
sequence encodes for the complete 345 amino acids enzyme containing
a 6-His tag at its NH2 terminus.
Expression and Purification of Recombinant hAK from
Bacterial Cells. Expression and purification of the hAK were carried
out as described by the manufacturer of the Ni-NTA Superflow resin
(Quiagen). Briefly, a fresh overnight saturated culture of E. coli (DH5R
strain) transformed with pHis-hAK was diluted 1:100 in 1 L of 2XYT
broth containing ampicillin (60 μg/mL) and incubated at 25 ꢀC with
shaking. At 0.6 OD600 IPTG was added to a final concentration of
0.4 mM, and the culture was incubated for a further 2 h at 37 ꢀC. The
bacterial cells pellet was resuspended in four volumes of lysis buffer
(50 mM sodium phosphate, pH 8.0, 300 mM NaCl, 10 mM imidazole,
1 mM phenylmethylsulfonyl fluoride, and 1 mg/mL lysozyme) and
incubated on ice for 30 min. Cells were then homogenized and sonicated
on ice, and the lysate was centrifuged at 10000g for 30 min at 4 ꢀC. The
supernatant was loaded onto a Ni-NTA Superflow column (1 mL). The
column was then washed at a flow rate of 0.25 mL/min with lysis buffer
and then with 50 mM sodium phosphate buffer, pH 8.0, containing
300 mM NaCl and 20 mM imidazole. The protein was then step-eluted
with 250 mM imidazole in 50 mM sodium phosphate buffer, pH 8.0, and
300 mM NaCl. Fractions were collected for enzymatic activity analysis.
The enzyme, collected from peak fractions, was dialyzed against 50 mM
Tris-HCl, pH 7.5, 1 mM 1,4-dithiothreitol (DTT) and then frozen in
liquid nitrogen until used.
Adenosine Kinase Assays. hAK was assayed with a radiochemi-
cal method which measures the formation of [3H]AMP from [3H]Ado.
The enzyme was incubated at 37 ꢀC in 25 μL of a mixture containing 32
mM Tris-HCl (pH 7.5), 20 mM KCl, 0.1 mM MgCl2, 1 mM ATP, and
0.5 μM [3H]Ado (2200 cpm/pmol). The reaction was terminated by
spotting 20 μL of the incubation mixture on a 25 mm diethylaminoethyl
paper disk (DE-81 paper; Whatman). The disk was washed twice in an
excess of 1 mM ammonium formate, pH 3.6, in order to remove
unconverted nucleoside, once in distilled water, and finally in ethanol.
Radioactive AMP was estimated by scintillation counting in 1 mL of
BetaMax scintillating fluid (ICN Pharmaceutical). One unit (U) is
defined as the amount of enzyme catalyzing the formation of 1 nmol
of AMP in 1 h at 37 ꢀC.
In inhibition studies, increasing concentrations of each compound
were tested in duplicate in two independent assays, as described above.
Stock solutions of compounds, in 100% DMSO, were diluted in water to
a final DMSO concentration of 1%. Controls without inhibitor were also
incubated in the presence of 1% DMSO.
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Journal of Medicinal Chemistry
ARTICLE
In Vitro Ado Phosphorylation. When hAK activity was deter-
mined in vitro, HeLaS-3 cells were grown in Dulbecco’s modified Eagle
medium (DMEM) with fetal calf serum at 37 ꢀC. After trypsinization,
they were resuspended in DMEM without calf serum at 106 cells/mL
(100 μL) and incubated at 37 ꢀC in the presence of 0, 10, or 50 μM 4a
and 3 μCi of [3H]Ado (25 Ci/mmol). At times 0, 10, 20, 30, and 40 min,
80 μL samples of culture were spotted on 25 mm DE-81 (Whatman)
filters, washed, and counted as above-described.
Assay of hAK Activity in HeLaS-3 Crude Extract. HeLaS-3
cells (2.5 ꢀ 106), grown in DMEM, were washed in phosphate buffered
saline (PBS), centrifuged (1000g), and resuspended in 10 mM Hepes,
pH 7.3, 1 mM DTT, and 0.5 mM PMSF. After 30 min on ice, cells were
disrupted by dounce homogenization and centrifuged 20 min at 10000g.
Supernatant (protein concentration of 6.0 mg/mL) was used as HeLa
crude extract. In hAK assay, as described above, 2 μL of HeLaS-3 crude
extract replaced recombinant hAK.
Measurement of Viability in HL-60 Cells. Human promyelo-
cytic leukemia HL-60 cells were obtained from the ECACC. Cells were
maintained in RPMI-1640 medium enhanced with GlutaMAX-I and
supplemented with 10% fetal bovine serum, 50 units/mL penicillin, and
50 μg/mL streptomycin (all from Gibco, Invitrogen, Carlsbad, CA,
U.S.). Cells were maintained in a humidified incubator at 37 ꢀC in 5% CO2.
Cell proliferation was monitored using AlamarBlue dye (BioSource,
Invitrogen, Carlsbad, CA, U.S.) which changes to a fluorescent state in
the reduced environment of living cells. Cells (40 000 cells/well) were
seeded on 96-well plates for 24 h and then treated with vehicle (0.1%
(v/v) DMSO) or a range of concentrations of 4a compound for 72 h.
AlamarBlue (final concentration 10% (v/v)) was added and incubated
for a further 4.5 h at 37 ꢀC. Fluorescence was measured at an excitation
wavelength of 544 nm and an emission wavelength of 590 nm using a
SpectraMax Gemini spectrofluorometric plate reader (Molecular De-
vices, Sunnyvale, CA). The results were expressed as the percentage cell
viability relative to vehicle-treated control cells (100%). Dose-response
curves were plotted, and IC50 values (concentration of drug resulting in
50% reduction in cell viability) were obtained using Prism GraphPad 4.
Determination of Apoptosis in HL-60 Cells. Following treat-
ment with vehicle (0.1% DMSO) or a range of concentrations of 4a for
24, 48, or 78 h, cells were harvested by centrifugation at 800g for 10 min.
Cell pellets were resuspended in 200 μL of PBS and fixed by a dropwise
addition of 2 mL of ice-cold 70% (v/v) ethanol/PBS while gently
vortexing. Following overnight fixation at -20 ꢀC the cells were again
centrifuged to remove the ethanol and resuspended in PBS supplemen-
ted with 0.5 mg/mL RNase A and 0.15 mg/mL propidium iodide (PI).
Cells were incubated in the dark at 37 ꢀC for 30 min. The PI fluorescence
was measured on a linear scale using a FACS Calibur flow cytometer
(Becton Dickinson, San Jose, CA, U.S.). Data collections (10 000 events
per sample) were gated to exclude cell debris and cell aggregates. PI
fluorescence was proportional to the amount of DNA present in each
entity and therefore indicated the stage of the cell cycle. Cells in G0/G1
were diploid (2N DNA content). Cells in the S phase had DNA contents
between 2N and 4N. Cells in G2/M were tetraploid (4N DNA content),
while apoptotic cells were hypoploid and contained <2N DNA (pre-G1).
Polyploid cells had a DNA content of >4N. All data were recorded and
analyzed using the CellQuest software (Becton Dickinson, San Jose, CA,
U.S.). Statistical analysis was performed using Prism GraphPad 4. P
values were obtained using two-tailed paired student’s t tests. P < 0.05
was considered significant.
In Vitro Incorporation of [3H]Thymidine and [3H]Uridine.
HeLaS-3 cells were grown in DMEM with fetal calf serum at 37 ꢀC. After
trypsinization, they were resuspended in DMEM without calf serum at 106
cells/mL (100 μL) and incubated at 37 ꢀC in the presence of 0, 10, or 50
μM 4a (or ddCyd) and 5 μCi of either [3H]thymidine (25 Ci/mmol) or
[3H]uridine (22 Ci/mmol). At times 0, 10, 20, 30, and 40 min, 80 μL
samples of culture were spotted on 25 mm GF/C (Whatman) filters which
were then washed three times (10 min) in 5% trichloroacetic acid, twice in
ethanol and dried. Remaining radioactivity was measured as described above.
Measurement of Cell Viability: Dye Exclusion Assay.
Chronic myeloid leukemia cells, from K562 cell line grown in Roswell
Park Memorial Institute (RPMI) 1640 medium containing 10% fetal calf
serum, were seeded in a 96-well microplate at 5000 cells/mL (150 μL/
well). After 15 h at 37 ꢀC in 5% CO2, an amount of 50 μL/well of serial
dilutions of the compound to test was added. The microplate, sealed
with Saran wrap to limit evaporation of the medium and diffusion of
metabolic carbon dioxide, was then incubated for 7 days in the above
conditions. Then an amount of 50 μL of the cell suspension was
removed from the well and mixed with 50 μL of tryptan blue solution
(0.5% in PBS). Both viable cells (not stained) and dead cells (blue
stained) were then counted on a B€urker cell counter. Each point was
done in triplicate, and the number of cells counted in each well is the
average of four determinations.
Measurement of Cell Growth. HeLaS-3 and Cen3tel cells (8 ꢀ
104) were incubated in multiwell plates at 37 ꢀC in 2 mL of DMEM
supplemented with 10% fetal calf serum, 5 mM L-glutamine, and 50 μg/
mL Gentamycin. Cells were left in the presence or in absence (control)
of 40 μM 4a for 3 days (medium and compound were replaced every
day). Sample of the cultures were counted every day. In order to evaluate
the capability of the cells to recover from the cytostatic effect of the
compound, cells were washed and left growing for 2 days and then
counted. Cell counting was performed after trypsinization.
Analysis of Possible 4a Intracellular Degradation. 4a (1
mM) was incubated at 37 ꢀC for 0, 1, 5, and 20 h in 175 μL of HeLaS-3
crude extract, prepared as described above. After incubation, samples
were frozen and lyophilized. In each tube, 50 μL of cold (-20 ꢀC)
MeOH was added. The mixtures were then vortexed for 10 min and
centrifuged at 10000g for 15 min. Supernatants was transferred into new
tubes. MeOH extraction was repeated on each pellet. The two supernatants
of each incubation time were combined, lyophilized, and resuspended in
25 μL of 5% DMSO. An amount of 20 μLofthesesolutionswasanalyzedby
reverse phase HPLC, as previously described,45 in order to evaluate the total
amount of 4a present in the samples.
Adenosine Deaminase Enzymatic Assay46. ADA type IX
from bovine spleen (150-200 U/mg) and Ado were purchased from
Sigma Chemical Co. All other chemicals were of reagent grade. The
activity of ADA was determined spectrophotometrically by monitoring
for 2 min the change in absorbance at 262 nm, which is due to the
deamination of Ado catalyzed by the enzyme. The change in Ado
concentration/min was determined using a Beckman DU-64 kinetics
software program (Solf Pack TM module). ADA activity was assayed at
30 ꢀC in a reaction mixture containing 50 μM Ado, 50 mM potassium
phosphate buffer, pH 7.2, and 0.3 nM enzyme solution in a total volume
of 500 μL. The inhibitory activity of the newly synthesized compounds
was assayed by adding 100 μL of the inhibitor solution to the reaction
mixture described above. All the inhibitors were dissolved in water, and
the solubility was facilitated by using DMSO, whose concentration never
exceeded 4% in the final reaction mixture. To correct for the none-
nzymatic change in Ado concentration and the absorption by the test
compounds, a reference blank containing all of the above assay
components except the substrate was prepared. The inhibitory effect
of the new derivatives was routinely estimated at 100 μM. Results are
expressed as the mean ( SEM of percentage inhibition values, obtained
through two determinations carried out in triplicate.
Cytotoxicity Assay. The cytotoxicity of the different compounds
was evaluated using the murine fibroblast cell line (3T3 cells). Cell
proliferation was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-di-
phenyltetrazolium bromide assay. The data processing included the
Student’s t test with p < 0.05 taken as significance level.
Molecular Modeling. Ligand Binding Site Prediction. Two pro-
grams were used for the ligand binding site prediction: QSiteFinder
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dx.doi.org/10.1021/jm101438u |J. Med. Chem. 2011, 54, 1401–1420

Journal of Medicinal Chemistry
ARTICLE
(http://www.bioinformatics.leeds.ac.uk/qsitefinder) and SiteMap from
Schr€odinger. The first is a server for ligand binding site prediction and
works by binding hydrophobic (CH3) probes to the protein and finding
clusters of probes with the most favorable binding energy. These clusters
are placed in rank order of the likelihood of being a binding site
according to the sum total binding energies for each cluster. The
programs were both used on either the closed (PDB code 1BX4) or
the open (PDB code 2I6B) enzyme conformation as retrieved from the
Protein Data Bank and produced comparable results.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: 0039-0577-234172. Fax: 0039-0577-234333. E-mail:
campiani@unisi.it.
’ ACKNOWLEDGMENT
This work was partially supported by FIRB Grant
RBAU01LSR4_001 (to F.F.).
Docking Simulations. The Autodock program (version 4.0)47 was
used just for the blind docking, although it has previously demonstrated
to be successful also in the prediction of enzyme inhibitors binding
modes.48-50 The inhibitor structure of 4a (S and R enantiomers) were
first generated through the Dundee PRODRG2 Server (http://davapc1.
bioch.dundee.ac.uk/prodrg). For the protein setup, to create initial
coordinates for docking studies, all water molecules of the crystal
structures were removed and excluded from calculations while the two
cocrystallized Ado were retained accordingly with the noncompetitive
behavior of 4a with regard to adenosine and/or ATP. The docking area
has been defined by a box large enough to comprise the entire protein
and centered on Ser 65 CR. Grids points of 126 ꢀ 126 ꢀ 126 with 0.531
Å spacing were calculated around the docking area for all the ligand atom
types using AutoGrid4. One-hundred separate docking calculations
were performed. Each docking calculation consisted of 25 ꢀ 106 energy
evaluations using the Lamarckian genetic algorithm local search (GALS)
method. A low-frequency local search according to the method of Solis
and Wets was applied to docking trials to ensure that the final solution
represents a local minimum. Each docking run was performed with
a population size of 150, and 300 rounds of Solis and Wets local search
were applied with a probability of 0.06. A mutation rate of 0.02 and a
crossover rate of 0.8 were used to generate new docking trials for
subsequent generations. The docking results from each of the 100
calculations were clustered on the basis of root-mean square deviation
(rmsd = 2.0 Å) between the Cartesian coordinates of the ligand atoms
and were ranked on the basis of the free energy of binding. The pocket
detected as putative allosteric binding site by the Autodock program was
further investigated with a subsequent “focused” docking step in order to
characterize the 4a and its derivatives binding modes. The “focused”
docking was performed with the aid of Glide software.51 The latter was
preferred over the Autodock, as all our derivatives can exist in two
different stereoisomers (one chiral carbon) and two different conforma-
tions (flipping of the pyrrolobenzoxa(thia)zepinone scaffold) and,
different from Autodock, Glide is able to generate all these variations
for each derivative and analyze all of them in a single docking run. The
inhibitor structures were first generated through the Dundee
PRODRG2 server. Then geometry optimized ligands were prepared
using Lig-Prep 2.3 as implemented in Maestro. The target protein was
prepared through the Protein Preparation Wizard of the graphical user
interface Maestro and the OPLS-2001 force field. Water molecules were
removed. Hydrogen atoms were added, and minimization was performed
until the rmsd of all heavy atoms was within 0.3 Å of the crystal-
lographically determined positions. The binding pocket was identified
byplacing a 15 Å cube centeredonthe masscenter of thebestranked pose
of 4a, obtained from AutoDock4 blind docking. Molecular docking
calculations were performed with the aid of Glide 5.5 in standard precision
(SP) mode, using Glidescore for ligand ranking. For multiple ligand
docking experiments, an output maximum of 5000 ligand poses per
docking run with a limit of 100 poses for each ligand was adopted.
’ ABBREVIATIONS USED
AK, adenosine kinase; Ado, adenosine; AMP, adenosine mono-
phosphate;CNS, central nervous system;PNS, peripheral nervous
systems; hAK, human adenosine kinase; HL, human leukemia;
NNhAKIs, non-nucleoside human adenosine kinase inhibitors;
SAR, structure-activity relationship; THF, tetrahydrofuran;TEA,
triethylamine; DABCO, 1,4-diazabicyclo[2.2.2]octane;DMF, di-
methylformamide; DCM, dichloromethane; AIBN, 2,20-azodiiso-
butyronitrile; HeLaS-3, human epithelial carcinoma cell line
subclone S3; EWG, electron-withdrawing group;EDG, electron-
donating group; ddCyd, 20,30-dideoxycytidine; DMSO, dimethyl-
sulfoxide;ADA, adenosine deaminase; IPTG, isopropylthio-β-D-
galactoside; DTT, 1,4-dithiothreitol; DMEM, Dulbecco’s modified
Eagle medium; PBS, phosphate buffered saline; RPMI, Roswell Park
Memorial Institute; PI, propidium iodide
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