Identification of azepinone fused tetracyclic heterocycles as new chemotypes with protein kinase inhibitory activities

Article abstract of DOI:10.1016/j.tet.2016.03.048

The design and synthesis of small tetracyclic heterocycles which bear two new regioisomeric 2-carboxyethyl-1H-pyrrole-annulated indoloazepinone scaffolds is described. An azepinone motif, which is inherent in the structures of many well studied protein kinase inhibitors, serves as prominent structural feature of the new compounds. Concise access to the new regioisomeric tetracyclic derivatives was accomplished through amide coupling of appropriate pyrrole and indole precursors followed by an intramolecular Heck coupling reaction of the intermediate amide conjugates. Preliminary evaluation of newly synthesized tetracyclic molecules against a panel of protein kinases indicated their inhibitory activities and revealed promising selectivity profiles. The new compounds displayed no significant antiproliferative activity against MCF-7 cancer cells. Interestingly, derivative 19a exhibited selective TAK1 kinase inhibitory activity and figures as a promising chemotype for the discovery of new TAK1 inhibitors.

Full text of DOI:10.1016/j.tet.2016.03.048

Tetrahedron xxx (2016) 1e10
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Identification of azepinone fused tetracyclic heterocycles as new
chemotypes with protein kinase inhibitory activities
,
b
b
Vassiliki Psarra a, Manolis A. Fousteris a , Lothar Hennig , Marina Bantzi ,
*
Athanassios Giannis b, Sotiris S. Nikolaropoulos a
a Laboratory of Medicinal Chemistry, Department of Pharmacy, University of Patras, Patras GR-26500, Greece
b Institute of Organic Chemistry, Faculty of Chemistry and Mineralogy, University of Leipzig, Johannisallee 29, Leipzig 04103, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The design and synthesis of small tetracyclic heterocycles which bear two new regioisomeric 2-
carboxyethyl-1H-pyrrole-annulated indoloazepinone scaffolds is described. An azepinone motif, which
is inherent in the structures of many well studied protein kinase inhibitors, serves as prominent
structural feature of the new compounds. Concise access to the new regioisomeric tetracyclic derivatives
was accomplished through amide coupling of appropriate pyrrole and indole precursors followed by an
intramolecular Heck coupling reaction of the intermediate amide conjugates. Preliminary evaluation of
newly synthesized tetracyclic molecules against a panel of protein kinases indicated their inhibitory
activities and revealed promising selectivity profiles. The new compounds displayed no significant an-
tiproliferative activity against MCF-7 cancer cells. Interestingly, derivative 19a exhibited selective TAK1
kinase inhibitory activity and figures as a promising chemotype for the discovery of new TAK1 inhibitors.
Ó 2016 Elsevier Ltd. All rights reserved.
Received 18 November 2015
Received in revised form 29 February 2016
Accepted 14 March 2016
Available online xxx
Keywords:
Kinase inhibitors
Azepinone
Tetracyclic scaffolds
Heck coupling
TAK1 inhibitors
1. Introduction
discovery of novel lead compounds with potent and selective
protein kinase inhibitory activities remains an open challenge.
Protein kinases are key components of many cellular signaling
pathways and play a crucial role in vital cellular processes such as
cell cycle regulation, DNA replication, transcription, metabolism,
immune responses and nervous system functions.1 Deregulation of
protein kinase activity results in a wide range of pathological dis-
orders among which cancer plays a prominent role. Over the past
decades, a great deal of research, both in pharmaceutical industry
and in academia, has been directed towards the discovery of new
protein kinase inhibitors.2 As a result, a number of small molecules
have been licensed for the treatment of various types of cancer,
while many others are under clinical trials.3 Although, significant
progress has been achieved in this field, a large fraction of the
human kinome remains unexplored offering new opportunities for
drug discovery efforts.4
The majority of protein kinase inhibitors reported in the litera-
ture are small heterocycles targeting the ATP-binding site of the
enzymes. Despite their structural diversity, recent data of
fragment-based approaches indicate that their structures cover
a limited part of chemical space.5 Consequently, the identification
of new scaffolds which may be used as starting points for the
Many bioactive protein kinase inhibitors contain an azepinone
motif as part of their structure (Fig. 1). Amongst them, paullones
(1), a class of synthetic benzazepinones, have been established as
inhibitors of various kinase targets such as cyclin dependent ki-
nases (CDKs)6 and glycogen synthase kinase-3 ).7 Struc-
b (GSK-3b
tural modifications of the paullone basic scaffold switched the
selectivity profile leading to dual polo-like kinase 1 (PLK1)/vascular
endothelial growth factor-receptor 2 (VEGF-R2) inhibitors with
in vitro antiproliferative and antiangiogenetic activities.8 Further-
more, congener derivatives have been described with interesting
sirtuin
1
(SIRT1) modulatory,9 antitrypanosomal10 and anti-
leishmanial activity.11 Hymenialdisine (2), a pyrroloazepinone
natural product, has shown potent inhibitory activity against CDKs,
GSK-3b, casein kinase 1 (CK1) and checkpoint kinase (Chk) 1 and
2,12 as well as anti-inflammatory,13 neuroprotective and antioxi-
dant properties.14 Importantly, structural data confirm that azepi-
none motif influences strongly the binding mode of inhibitors to
the enzymes, acting as an anchor point and allowing the de-
velopment of crucial hydrogen bonds in the hinge region of the
ATP-binding site. Particularly, crystal structure analysis of alster-
paullone (1a)-GSK-3b complex demonstrated the formation of
three hydrogen bonds between the lactam group and amino acid
* Corresponding author. Tel.: þ30 2610 969321; fax: þ30 2610 969182;
e-mail address: manolisf@upatras.gr (M.A. Fousteris).
residues of the hinge region.15 Similarly, the azepinone moiety of
http://dx.doi.org/10.1016/j.tet.2016.03.048
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2
V. Psarra et al. / Tetrahedron xxx (2016) 1e10
scaffolds A and B (Scheme 1). This involved the amide bond for-
mation between appropriately substituted N-protected 2-iodo-1H-
indole-3-carboxylic acids and an N-protected 2-ethoxycarbonyl-
1H-pyrrole-4-methanamine followed by N-protection of the gen-
erated amide linkage. N-protection of both the heterocyclic pre-
cursors was considered important not only for the directed
substitution of the cores, but also for the following palladium-
catalyzed synthetic step. Subsequently, palladium-mediated intra-
molecular Heck coupling reaction between the 2-iodo-substituted
indole position and either the unsubstituted C(5) or the C(3) of the
pyrrole ring was expected to generate the fully protected tetracyclic
scaffold A or B, correspondingly. Finally, sequential removal of the
protective groups would afford the target compounds of scaffold
A or B.
Fig. 1. Structures of bioactive azepinone heterocycles and target scaffolds A and B.
hymenialdisine participates on the formation of two directed hy-
drogen bonds in the ATP-binding pocket of CDK2.12
Moreover,
a number of reported observations underline
remarkable bioactivities of structurally related azepinone fused
heterocycles. Latondunine A (3) corrected cystic fibrosis trans-
membrane conductance regulator (CFTR) trafficking to the cell
membrane via poly(ADP-ribose) polymerase-3 (PARP-3) inhibition
providing new therapeutic opportunities for cystic fibrosis.16
Consequently, the incorporation of a seven-membered lactam
moiety into a multicyclic scaffold might contribute to the identifi-
cation of new kinase inhibitor chemotypes with potential applica-
tions to other molecular targets.
Inspired by these observations and due to our interest in the
identification of new kinase interacting molecules,17 we have fo-
cused on the generation of new azepinone containing scaffolds18 as
a starting point for the discovery of potential kinase inhibitors.
Particularly, we envisaged the regioisomeric 2-carboxyethyl-1H-
pyrrole-annulated indoloazepinone scaffolds A and B (Fig. 1) as
interesting core structures for the induction of high affinity in-
teractions with the ATP-binding site of kinases. To explore the
scope and limitations of our synthetic methodology and gain initial
insights into structure activity relationships, the introduction of
two halogen substituents at the C(8) position of the new tetracyclic
molecules was planned. The target scaffolds can be considered
structurally related to paullone family. To date, a notable number of
paullone analogues have been synthesized and much attention has
been paid to introducing structural variations on the paullone
parent skeleton either modifying the benzazepinone segment or
replacing the indole core with other heteroaromatic rings.19 Nev-
ertheless, such regioisomeric pyrrole-annulated tetracyclic het-
erocycles have not been reported previously in the literature.
Herein, we described a straightforward methodology for the
effective synthesis of small tetracyclic heterocycles bearing the
regioisomeric scaffolds A and B via assembly of appropriate indole
and pyrrole precursors followed by an intramolecular palladium-
catalyzed Heck coupling reaction. Preliminary evaluation of
a number of newly synthesized derivatives indicated their kinase
inhibitory activities and revealed promising selectivity profiles
against different kinase targets.
Scheme 1. Retrosynthetic plan towards the construction of the scaffolds A and B.
The synthesis of the pyrrole precursor 7 is depicted in Scheme 2.
Initially, the N-protection of the known 4-formyl pyrrole derivative
420 was performed. An electron-donating protective group was
expected to promote the upcoming intramolecular palladium-
mediated coupling reaction enriching the electron density of the
pyrrole ring. Among the known electron-donating protective
groups, the benzyloxymethyl (BOM) group was chosen. Thus, 4 was
N-protected using benzyloxymethyl chloride (BOM-Cl) in the
presence of potassium tert-butoxide (t-BuOK) providing the N-BOM
protected intermediate 5 in very good yield (85%). Oximation of the
4-formyl group using hydroxylamine hydrochloride and sodium
carbonate furnished a mixture of E,Z-pyrrole-4-carbaldehyde ox-
imes 6 in excellent yield (91%). The two geometric isomers were
isolated and characterized, but full assignment of the structures
was not completed. The mixture of the E,Z-oximes was reduced
upon treatment with sodium borohydride-nickel chloride hexa-
hydrate giving the N-protected 1H-pyrrole-4-methanamine pre-
cursor 7 in moderate yield (42%), while the dimerized by-product 8
was also isolated in a small amount.
With the pyrrole precursor 7 in hand, our attention was turned
to the synthesis of the N-protected 2-iodo-1H-indole-3-carboxylic
acid precursor 11a (Scheme 2). The introduction of the N-BOM
group also on the indole fragment was considered indispensable for
facilitating the final cleavage of the protective groups and the
generation of the fully deprotected tetracyclic compounds. Thus,
treatment of the previously described 2-iodo-substituted indole
9a21 with BOM-Cl in the presence of sodium hydride furnished the
N-BOM protected indole 10a quantitatively which after alkaline
2. Results and discussion
Initially, a simple retrosynthetic plan was considered towards
the regioisomeric 2-carboxyethyl-1H-pyrrole-annulated tetracyclic
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V. Psarra et al. / Tetrahedron xxx (2016) 1e10
3
Scheme 2. Reagents and conditions: (i) t-BuOK, BnOCH2Cl, 18-Crown-6, THF, 0 ꢀC, 30 min, 85%; (ii) H2NOH$HCl, Na2CO3, EtOH/H2O, 90 ꢀC, 1 h, 91%; (iii) NiCl2$6H2O, NaBH4, MeOH,
0 ꢀC to rt, 1.5 h, 7¼42%, 8¼10%; (iv) NaH, BnOCH2Cl, THF, rt, overnight, 100%; (v) NaOH/H2O, MeOH, 65 ꢀC, overnight, 85%; (vi) 7, 4-DMAP, EDCI$HCl, DCM, 0 ꢀC to rt, overnight, 81%;
(vii) 4-DMAP, Boc2O, DCM, 0 ꢀC to rt, overnight, 97%; (viii) Pd(OAc)2, PPh3, Ag2CO3, DMF, 80 ꢀC, 30 min, 14a¼44%, 15a¼14%; (ix) TFA, DCM, 0 ꢀC to rt, 7 h for 16a and 5 h for 17a,
16a¼94%, 17a¼92%; (x) 10% Pd/C, HCOONH4, EtOH abs, 68 ꢀC, 1 h for 18a and 8 h for 19a, 18a¼91%, 19a¼85%.
hydrolysis provided the corresponding 2-iodo-1H-indole-3-
carboxylic acid precursor 11a in 85% yield.
Having already synthesized the precursors 7 and 11a, we fo-
cused on the construction of the corresponding tetracyclic scaffolds
1D (1H, 13C, APT) and 2D (H,H COSY, edited HSQC, HMBC) NMR
experiments. With these methods a complete assignment of all
chemical shifts to the corresponding hydrogen and carbon atoms
was possible. Especially the HMBC spectrum, showing cross cor-
relation signals between proton and carbon atoms connected via
two and three bonds, is very helpful to distinguish both isomers.
In both 16a and 17a the isolated proton of the pyrrole ring gives
a singlet in the 1H spectrum at 7.08 and 7.06 ppm, respectively,
each showing in HMBC couplings via three bonds to the carbon
atoms of the CH2 group in the corresponding seven-membered
lactam ring (38.02 and 37.43 ppm) and to the quaternary car-
bons in the pyrrole ring (130.40 and 125.69 ppm) (Fig. 2). In
contrary, compound 17a shows a three bond coupling of the
pyrrole ring proton (7.06 ppm) to the NeCH2eO methylene car-
bon (77.80 ppm), which is missed in compound 16a. In that case
this proton (7.08 ppm) correlates to the carbonyl group at
160.71 ppm.
A
and B. Amide coupling between 11a and 7 using N-(3-
dimethylaminopropyl)-N0-ethylcarbodiimide (EDCI) and 4-(dime-
thylamino)pyridine (4-DMAP) in DCM provided the amide conju-
gate 12a in a very good yield (81%) (Scheme 2). N-amide protection
was effected by treatment with Boc2O in the presence of 4-DMAP.
Then, the N-Boc protected amide 13a was subjected to palladium-
mediated intramolecular Heck coupling reaction. The cyclization
was carried out using palladium(II) acetate/PPh3 in the presence of
Ag2CO3 as base, a catalytic system which has previously used effi-
ciently in intramolecular Heck reactions for the synthesis of tri-
cyclic or tetracyclic nitrogen heterocycles.22 Under these
conditions, cyclization at the C(5) of the pyrrole ring resulted in the
formation of tetracyclic compound 14a in 44% yield, while cycli-
zation at the C(3) led to the regioisomeric product 15a in 14% yield.
Furthermore, cleavage of the N-Boc protective group by treatment
with trifluoroacetic acid (TFA) in DCM led to the corresponding
compounds 16a and 17a.
Subsequently, the complete cleavage of the N-BOM protective
groups from the tetracyclic derivatives 16a and 17a was attempted
(Scheme 2). To this purpose, compound 16a was subjected to cat-
alytic hydrogenolysis conditions over 10% Pd/C in the presence of
ammonium formate. The fully deprotected analogue 18a was ob-
tained in excellent yield (91%). Applying the same hydrogenolysis
The assignment for the structural elucidation of the regioiso-
meric products 16a and 17a was performed with a combination of
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V. Psarra et al. / Tetrahedron xxx (2016) 1e10
a broad range of temperatures, the 2-iodo-indole 26b obtained only
in very low yields (Table 1, entries 2e4) together with small amounts
of the N-deprotected 2-iodo derivative 9b. In an effort to improve the
formation of 26b and diminish the extended decomposition of 25b
which was occurredatelevatedtemperatures, excess (2equiv)ofLDA
was used and the temperature of the iodination reaction was main-
tainedsteadyatꢁ60ꢀC. Undertheseconditions, aslightlybetteryield
was recorded for 26b (14%), while a great amount of 25b (62%) was
recovered intact (Table 1, entry 5). Further increase of the LDA
amount(3 equiv)didnot improvetheformationof26b(Table 1, entry
6). Interestingly, increasing the reaction scale and prolonging the
iodination reaction time led to a remarkable improvement in the
yield of 26b (51%), whereas recovered 25b (23%) and deprotected
indole 9b (19%) were also isolated (Table 1, entry 7). Extending the
reaction time to almost 33 h caused no significant alteration in the
yield of 26b (46%) (Table 1, entry 8). Surprisingly, carrying out the
reaction on a much bigger scale, 26b was obtained in optimum yield
(81%) (Table 1, entry 9), possibly due to the more precise monitoring
of the reaction progress.
Removal of the N-Boc protective group upon treatment with TFA
furnished the deprotected indole 9b in 87% yield (Scheme 3). Fi-
nally, N-BOM protection followed by alkaline hydrolysis of the
methyl ester provided the desired 5-chloro-indole precursor 11b in
75% yield.
Following the above established sequence of reactions, we
succeeded to obtain the N-BOM protected 5-bromo-2-iodo-1H-in-
dole-3-carboxylic acid 11c starting from commercially available 5-
bromo-1H-indole 20c (Scheme 3). Notably, the 2-iodo-indole 26c
was obtained in optimum yield (79%) by adjusting the temperature
of the iodination reaction at ꢁ50 ꢀC.
Applying the conditions described in Scheme 2, the conjugation
between the indole precursors 11(bec) and the pyrrole fragment 7
furnished amides 12(bec) which were easily transformed to the
corresponding N-Boc protected derivatives 13(bec) in excellent
yields (Scheme 3). The protected amides 13b and 13c were sub-
mitted to intramolecular Heck coupling reaction providing the
regioisomeric cyclized C(8)-halogenated products, 14be15b and
14ce15c, respectively. Furthermore, treatment of compounds
14(bec) and 15(bec) with TFA in DCM resulted in the removal of
the N-Boc protective group affording compounds 16(bec) and
17(bec), respectively.
Finally, the removal of N-BOM groups from the tetracyclic com-
pounds 16(bec) and 17(bec), was investigated. The BOM-depro-
tection of bromo-derivative 16c was proven non-trivial. Catalytic
hydrogenolysis over 10% Pd/C in the presence of ammoniumformate
led not only to the cleavage of the BOM groups, but also to debro-
mination of the skeleton affording the unsubstituted derivative 18a
(Scheme 4). Classical hydrogenolysis using H2 over various catalysts
(10% Pd/C, Pd(OH)2, 5% Pt/C sulfided) neither prevent the debromi-
nation nor led to the removal of the BOM groups providing the
known intermediate 16a. Alternatively, the acidic cleavage of BOM-
protective groups of 16c was examined. Thus, various acidic condi-
tions such as p-TsOH/DCM, AlCl3/DCM or BBr3/DCM were used but
they resulted either in the recovery or decomposition of 16c. Pro-
longed treatment of 16c with TFA in a mixture of CHCl3/toluene
furnished the N,N0-di(hydroxymethyl) compound 27c in 60% yield.
Extensive efforts to eliminate the hydroxymethyl groups upon basic
treatment (aq K2CO3/acetone, 1 N aq NaOH/THF, CH3COONa/EtOH,
NH3 7 M in CH3OH/CH3OH, 40% Triton B in CH3OH/THF or CH3CN,
iPr2NEt/, ethylenediamine/CH3OH, Et3N/CH3OH) failed to afford the
fully deprotected compound 18c.
Fig. 2. HMBC spectra of compounds 16a and 17a.
conditions to 17a, the regioisomeric analogue 19a was also isolated
in very good yield (85%).
Prompted by these results, we decided to extend our study to
the synthesis of the regioisomeric C(8)-halogenated tetracyclic
analogues 16(bec) and 17(bec), respectively. To achieve our goal,
we first investigated the access to the 5-halogenated 2-iodo-1H-
indole-3-carboxylic acids 11(bec).
Thus, commercially available 5-chloro-indole 20b was trans-
formed easily to 1H-indole-3-carboxylic acid methyl ester 22b23 via
trichloroacetylation followed by basic methanolysis (Scheme 3).
Protection of the indole nitrogen atom with the electron-
withdrawing benzenesulfonyl group was expected to promote the
C(2) iodo-substitution. Benzenesulfonylation using standard con-
ditions provided the N-benzenesulfonylated indole 23b in 85%
yield which was introduced to C(2) iodination conditions. Initial
attempts to obtain the 2-iodo-substituted indole 24b using LDA/I2
or mesityllithium/1,2-diiodoethane (ICH2CH2I),24 led either to the
recovery of unreacted 23b or to its partial decomposition. These
results prompted us to replace the N-benzenesulfonyl group with
the N-tert-butyloxycarbonyl (N-Boc) group. Thus, N-Boc protected
indole 25b was obtained from 22b upon treatment with Boc an-
hydride and 4-DMAP in quantitative yield.
Various experimental conditions were investigated for the ef-
fective 2-iodo-substitution of substrate 25b (Table 1). Initially, lith-
iation of 25b was examined using equimolar amount of LDA at
ꢁ78 ꢀC. The attempt to trap the lithiated species using excess of I2
resulted in the recovery of unreacted 25b (Table 1, entry 1). Using
excess of ICH2CH2I and performing the iodination reaction over
Notably, treatment both of 16b and 16c with 6 N aq HCl in
ethanol provided a mixture of partially deprotected products. 1H
NMR analysis of the isolated compounds detected a halogenated
mono-N-BOM protected and a halogenated mono-N-hydroxy-
methyl compound. Nevertheless, all attempts to complete the
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V. Psarra et al. / Tetrahedron xxx (2016) 1e10
5
Scheme 3. Reagents and conditions: (i) Cl3CCOCl, C5H5N, THF, 0 ꢀC to rt, overnight, 21b¼95%, 21c25¼100%; (ii) KOH/H2O, MeOH, 85 ꢀC, 3 h, 22b¼83%, 22c26¼86%; (iii) R1¼SO2Ph:
NaH, PhSO2Cl, THF, rt, overnight, 23b¼85%; R1¼Boc: 4-DMAP, Boc2O, THF, 0 ꢀC to rt, 1 h, 25b¼99%, 25c¼96%; (iv) ICH2CH2I, LDA, THF, ꢁ78 ꢀC to ꢁ60 ꢀC for 26b and ꢁ78 ꢀC to ꢁ50 ꢀ
C
for 26c, overnight, 26b¼81%, 26c¼79%; (v) TFA, DCM, rt, overnight, 9b¼87%, 9c¼79%; (vi) NaH, BnOCH2Cl, THF, rt, overnight, 10b¼84%, 10c¼80%; (vii) NaOH/H2O, MeOH, 78 ꢀC,
overnight, 11b¼75%, 11c¼90%; (viii) 7, 4-DMAP, EDCI$HCl, DCM, 0 ꢀC to rt, overnight, 12b¼93%, 12c¼95%; (ix) 4-DMAP, Boc2O, DCM, 0 ꢀC to rt, overnight, 13b¼92%, 13c¼93%; (x)
Pd(OAc)2, PPh3, Ag2CO3, DMF, 80 ꢀC, 30 min, 14b¼36%, 15b¼26%, 14c¼39%, 15c¼13%; (xi) TFA, DCM, 0 ꢀC to rt, 4 h for 16b and 17b, 21.5 h for 16c and 16 h for 17c, 16b¼100%,
17b¼91%, 16c¼75%, 17c¼97%.
Table 1
2-Iodo-substitution of the N-Boc protected indole 25b
Entry
25b (mmol)
LDAa (equiv)
ICH2CH2Ib (equiv)
Iodination conditions
T (ꢀC)/time (h)
26b (%)
25b (%) recovered
9b (%)
1
2
3
4
5
6
7
8
9
65ꢂ10ꢁ3
65ꢂ10ꢁ3
65ꢂ10ꢁ3
65ꢂ10ꢁ3
65ꢂ10ꢁ3
65ꢂ10ꢁ3
323ꢂ10ꢁ3
323ꢂ10ꢁ3
2.5
1.1
1.1
1.1
1.1
2
3
2
2
2
I2c/3
3
3
3
3
3
3
3
3
ꢁ78/25/2.5
d
7
7
74
14
28
88
62
24
23
18
3
d
d
6
8
7
10
19
19
14
ꢁ78/25/25.5
ꢁ78/ꢁ30/10
ꢁ78/ꢁ50/13
ꢁ78/ꢁ60/13.5
ꢁ78/ꢁ60/10.5
ꢁ78/ꢁ60/22.5
ꢁ78/ꢁ60/33.5
ꢁ78/ꢁ60/28.5
9
14
10
51
46
81
a
LDA solution 2 M in THF/heptane/ethylbenzene; Lithiation conditions: e78 ꢀC/0.5 h.
1,2-diiodoethane solution 0.74 M in THF.
I2 solution 0.74 M in THF.
b
c
deprotection step by prolonged exposure of the mixture of the
partially deprotected products to 6 N aq HCl led to decomposition.
On the other hand, exposure of the regioisomeric halogenated
derivatives 17b and 17c to 6 N aq HCl in ethanol led to the isolation of
the desired deprotected compounds 19b and 19c in 62% and 91%,
respectively (Scheme 5). Unexpectedly, in both reactions a haloge-
nated mono-N-EOM protected derivative (28b or 28c) was formed,
possiblyviaacidcatalyzedtransetherificationof thecorrespondingN-
hydroxymethyl intermediates. The latter were easily converted to the
desired deprotected products upon furthertreatment with6 NaqHCl.
A preliminary study was conducted concerning the in vitro in-
hibitory activities of the new tetracyclic compounds 18a and
19(aec) against an extended panel of protein kinases (MRC-PPU
Express Screen, International Centre for Kinase profiling, University
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V. Psarra et al. / Tetrahedron xxx (2016) 1e10
Scheme 4. Reagents and conditions: (i) 10% Pd/C, HCOONH4, abs. EtOH, 68 ꢀC, 1 h; (ii) Classical hydrogenolysis conditions (see text for details); (iii) TFA, CHCl3/toluene (1/2), 98 ꢀC,
25 h, 27c¼60%.
skeleton may contribute to the inhibitory profile of the new com-
pounds. Although a limited number of substituents were in-
troduced at C(8), it is pointed out that this particular position is
correlated probably with the selective activity against different
enzymes. Thus, a detailed structure-activity relationship study
around the basic scaffold B will define the structural requirements
for optimized TAK1 inhibition. To this direction, compound 19a
may serve as a starting point of a medicinal chemistry campaign
aiming at the discovery of new TAK1 inhibitors.
Finally, the antiproliferative activity of derivatives 18a and
19(aec) against an MCF-7 breast cancer cell line, was investigated.
The compounds were tested in a concentration range from 1
mM
to 500 M (each concentration was checked three times) utilizing
m
MCF-7 cells and 20% DMSO as positive and negative controls,
respectively. Unfortunately, none of the compounds showed
any significant antiproliferative activity (See Fig. S1, Electronic
Supplementary data).
3. Conclusions
In summary, we have developed the synthesis of small tetra-
cyclic heterocycles, derivatives of the two new regioisomeric 2-
carboxyethyl-1H-pyrrole-annulated indoloazepinone scaffolds A
and B, via sequential amide conjugation of appropriate indole and
pyrrole precursors and intramolecular Heck coupling reaction.
Preliminary kinase selectivity profiling data suggest that the aze-
pinone containing scaffolds A and B can serve as new chemotypes
for the identification of new protein kinase inhibitors. Importantly,
derivative 19a figures as a promising selective compound for the
discovery of new TAK1 inhibitors.
Scheme 5. Reagents and conditions: (i) 6 N aq HCl, EtOH abs, sealed tube, 130 ꢀC, 2 h,
19b¼62%, 19c¼91%, 28b¼33%, 28c¼7%; (ii) 6 N aq HCl, EtOH abs, sealed tube, 130 ꢀC,
3.5 h, 19b¼56%, 19c¼54%.
of Dundee).27 The compounds were screened at a single concen-
tration of 10 mM (in duplicate) and the kinase remaining activity (%)
was determined. As it is presented in Table 2, compound 18a
showed more than 80% inhibition of AMPK activity, while it also
displayed good PIM1 (62%) kinase. Interestingly, the regioisomeric
derivative 19a proved the most selective among the tested com-
pounds displaying a 78% decrease of TAK1 activity, while it did not
exhibit any significant inhibitory effect against the rest of the kinase
targets. Considering the detected inhibition of TAK1 activity by 18a
was up to 58%, it is concluded that the framework of the tetracyclic
scaffold may affect the compound’s selectivity. Furthermore, the
C(8)-halogenated derivatives 19b and 19c presented a quite similar
activity profile against PLK1, MST2, TRKA, VEG-FR and SmMLCK
kinase (64e76%). Notably, TAK1 activity was reduced in only 28%
and 39% by 19b and 19c correspondingly, indicating that except for
the framework also the substitution pattern of the tetracyclic
4. Experimental section
4.1. General
All reagents were obtained commercially from Alfa Aesar,
Sigma-Aldrich or Merck and used without further purification.
Reactions involving moisture-sensitive reactants were conducted
in flame-dried glassware under an atmosphere of argon. Reagents
and anhydrous solvents were transferred via syringe. Analytical TLC
was performed on Merck Silica gel 60 F254 on precoated silica gel
plates. Visualization was accomplished under UV light (254 and
365 nm), by exposure to iodine vapours and by use of Seebach
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V. Psarra et al. / Tetrahedron xxx (2016) 1e10
7
Table 2
Kinase selectivity profiling of compounds 18a and 19(aec) screened at a concentration of 10 mM (in duplicate) against a panel of 50 kinases. The results are displayed as the
mean percentage of kinase remaining activity (%) with a standard deviation (SD). Remaining kinase activities >75% have been omitted for clarity (See Table S1, Electronic
Supplementary data)
Kinase
%
SD
Kinase
%
SD
Kinase
%
SD
Kinase
%
SD
AMPK(hum)
Aurora B
PLK1
PIM1
TAK1
19
31
36
38
42
45
47
49
51
55
62
65
65
66
66
67
71
72
73
74
4
9
4
10
21
3
0
11
7
2
1
2
5
3
3
0
1
4
15
2
PLK1
MST2
TrkA
VEG-FR
SmMLCK
IRAK4
TBK1
TAK1
MLK3
Aurora B
RSK1
RIPK2
CHK2
SGK1
AMPK(hum)
HIPK2
24
25
28
28
30
39
47
61
62
63
66
67
68
71
72
73
0
2
3
1
4
9
2
33
0
0
1
8
5
9
2
1
MST2
TrkA
PLK1
SmMLCK
VEG-FR
IRAK4
TBK1
MLK3
RIPK2
TAK1
CHK2
CAMKKb
AMPK (hum)
RSK1
24
25
25
31
36
38
51
67
68
72
73
73
75
75
2
4
5
0
6
11
7
3
TAK1
TrkA
VEG-FR
PLK1
22
66
71
74
4
8
5
6
MLK3
TBK1
MARK3
CAMK1
CHK2
CAMKKb
VEG-FR
PDK1
DYRK1A
RSK1
MST2
5
28
11
0
5
1
SmMLCK
MSK1
SGK1
IGF-1R
staining solution. Flash column chromatography was performed on
silica gel (SDS 60A, 40e63 m). Melting points were determined on
(4 equiv) were added under argon atmosphere. The reaction mix-
ture was stirred at 80 ꢀC for the indicated time. After cooling, it was
diluted with H2O and extracted with EtOAc (3ꢂ). The combined
organic layers were washed with brine (1ꢂ), dried over anhydrous
Na2SO4, filtered and the solvents were removed under reduced
pressure. The crude residue was purified by silica gel flash column
chromatography using either mixtures of n-hexane/EtOAc or tolu-
ene/EtOAc to afford the corresponding regioisomeric tetracyclic
derivatives 14(aec) and 15(aec).
m
an Electrothermal IA9200 digital melting point apparatus in cap-
illary tubes and are uncorrected. The IR spectra were recorded on
a FTIR Jasco spectrophotometer. NMR spectra (1H and 13C) were
recorded on a Brucker DPX 400 MHz (400 MHz for 1H, 100 MHz for
13C) or a Bruker Avance III High-Definition four-channel 700 MHz
(700 MHz for 1H, 176 MHz for 13C) spectrometer. Also, NMR spectra
(1H, 13C, APT, H,H COSY, edited HSQC, and HMBC) were recorded on
a VARIAN MERCURY 400 plus (400 MHz for 1H, 100 MHz for 13C)
and a VARIAN MERCURY 300 plus (300 MHz for 1H, 75 MHz for 13C)
4.2.1. 5-(tert-Butyl) 2-ethyl 1,11-bis(benzyloxymethyl)-6-oxo-1,4,6,11-
tetrahydro-5H-pyrrolo[20,30:5,6]azepino[4,3-b]indole-2,5-dicarboxylate
(14a) and 5-(tert-Butyl) 1-ethyl 2,11-bis(benzyloxymethyl)-6-oxo-
2,4,6,11-tetrahydro-5H-pyrrolo[30,40:5,6]azepino[4,3-b]indole-1,5-
dicarboxylate (15a). Compounds 14a and 15a were synthesized
according to the general procedure from 13a (120 mg, 0.154 mmol) in
dry DMF (3.3 mL) using Pd(OAc)2 (3.11 mg, 0.014 mmol), PPh3 (8.1 mg,
0.031 mmol) and Ag2CO3 (170 mg, 0.616 mmol). The reaction time was
30 min. The crude residue was purified by silica gel flash column
chromatography (n-hexane/EtOAc 90:10 to 80:20). 14a (44 mg, 44%),
yellow solid; mp 122.7e123.6 ꢀC; Rf (n-hexane/EtOAc 7:3) 0.48; IR
spectrometer. The chemical shifts (d) are reported in parts per
million (ppm) and the residual solvent signal is used as an internal
standard. The following abbreviations are used for the proton
spectra multiplicities: singlet (s), doublet (d), triplet (t), quartet (q),
multiplet (m), broad signal (br) and combinations thereof. Coupling
constants (J) are given in Hertz (Hz). Electron-spray ionization (ESI)
mass spectra were recorded at 30V, on a Micromass-Platform LC
spectrometer using methanol or acetonitrile as solvent. High res-
olution mass spectra were obtained on a Bruker Daltonics ESI-FT-
ICR-MS ‘APEX II’ [7T] mass spectrometer.
Analytical samples of the final products were provided after
semi-preparative Reverse Phase HPLC using a Millipore Waters 501
solvent delivery system with a Waters Spherisorb S5 ODS2 column
(KBr): nmax 1711, 1678, 1219, 1138, 1091 cmꢁ1
;
1H NMR (CDCl3,
8.45e8.42 (m, 1H), 7.48 (dq, J¼4.7 Hz, J¼2.5 Hz, 1H),
400 MHz):
d
7.44e7.40 (m, 2H), 7.30e7.26 (m, 4H), 7.12e7.06 (m, 3H), 7.02 (dd,
J¼6.4 Hz, J¼3.1 Hz, 2H), 6.98 (dd, J¼7.5 Hz, J¼1.9 Hz, 2H), 6.04 (d,
J¼9.8 Hz, 1H), 5.61 (d, J¼10.5 Hz, 1H), 5.48 (dd, J¼10.2 Hz, J¼6.9 Hz,
2H), 5.24 (d, J¼15.2 Hz, 1H), 4.46e4.38 (m, 2H), 4.29 (d, J¼11.9 Hz, 1H),
4.21 (q, J¼7.1 Hz, 2H), 4.02e3.96 (m, 2H), 1.56 (s, 9H), 1.45 (t, J¼7.1 Hz,
(5
m
m, 250ꢂ10 mm) and a Waters Lambda-Max Model 481 LC
Spectrometer (
l
254 nm). The mobile phase consisted of acetoni-
trile containing 0.08% TFA (solvent A) and water containing 0.08%
TFA (solvent B). Elution conditions are provided for each compound
(flow rate 3.0 mL/min). Purity of the final products was determined
by a combination of 1H, 13C NMR and HLPC techniques and was
found to be >95%.
3H); 13C NMR (CDCl3,100 MHz):
d 164.6,160.7,152.0,137.9,136.2,136.1,
132.1, 130.5, 128.6, 128.6 (2ꢂC), 128.3 (2ꢂC), 128.3, 127.9, 127.8 (2ꢂC),
127.8 (2ꢂC), 126.9, 126.4, 124.9, 123.4, 122.8, 118.3, 114.2, 111.5, 83.1,
75.4, 74.8, 70.6, 70.6, 60.8, 41.3, 28.3 (3ꢂC), 14.5. MS (ESIþ) (m/z):
650.33 [MþH]þ; HRMS-ESI (m/z): [MþNa]þ calcd for C38H39N3O7:
672.26802, found: 672.26749, [2MþNa]þ calcd for C38H39N3O7:
1321.54682, found: 1321.54536. 15a (14 mg, 14%), white solid; mp
55e56.6 ꢀC; Rf (n-hexane/EtOAc 7:3) 0.36; IR (KBr): nmax 1705, 1670,
4.2. General procedure for the synthesis of the tetracyclic
derivatives 14(aec) and 15(aec)
To a solution of an appropriate precursor 13(aec) (1 equiv) in
dry DMF, Pd(OAc)2, (0.09 equiv), PPh3 (0.2 equiv) and Ag2CO3
1211, 1138, 1105, 617 cmꢁ1 1H NMR (CDCl3, 400 MHz):
; d 8.37 (dd,
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8
V. Psarra et al. / Tetrahedron xxx (2016) 1e10
J¼6.7 Hz, J¼1.9 Hz, 1H), 7.6 (dd, J¼6.7 Hz, J¼1.5 Hz, 1H), 7.39e7.28 (m,
7H), 7.19 (s, 1H), 7.16e7.14 (m, 3H), 6.9e6.87 (m, 2H), 5.81 (d,
J¼10.2 Hz, 1H), 5.77 (d, J¼11.1 Hz, 1H), 5.71 (d, J¼10.2 Hz, 1H), 5.45 (d,
J¼11.1 Hz, 1H), 5.21 (d, J¼14.9 Hz, 1H), 4.50 (s, 2H), 4.19 (q, J¼7.1 Hz,
2H), 4.02 (d, J¼11.9 Hz, 1H), 3.96 (d, J¼14.8 Hz, 1H), 3.86 (d, J¼11.9 Hz,
1H), 1.53 (s, 9H), 1.13 (t, J¼7.1 Hz, 3H); 13C NMR (CDCl3, 100 MHz):
J¼10.9 Hz, 1H), 5.63 (d, J¼10.9 Hz, 1H), 5.54 (d, J¼10.1 Hz, 1H), 5.20 (d,
J¼15.4 Hz, 1H), 4.34 (dd, J¼9.1 Hz, J¼7.1 Hz, 2H), 4.24 (q, J¼7.1 Hz, 2H),
4.14e4.06 (m, 2H), 3.95 (d, J¼11.6 Hz,1H),1.45 (s, 9H),1.37 (t, J¼7.1 Hz,
3H); 13C NMR (CD3COCD3,100 MHz):
d 164.6, 160.9, 152.8, 137.6,137.6,
137.5, 134.7, 131.2, 130.9, 129.1 (2ꢂC), 128.9 (2ꢂC), 128.7, 128.5 (2ꢂC),
128.4, 128.4 (2ꢂC), 128.2, 128.2, 127.9, 125.4, 118.8, 116.7, 114.9, 113.3,
82.7, 76.7, 75.8, 70.9, 70.7, 61.2, 41.5, 28.2 (3ꢂC), 14.7; MS (ESIþ) (m/z):
750.15 [MþNa]þ; HRMS-ESI (m/z): [MþH]þ calcd for C38H38BrN3O7:
728.19659, found: 728.19673. 15c (32 mg, 13%), white solid; mp
136.4e138 ꢀC; Rf (n-hexane/EtOAc 7:3) 0.43; IR (KBr): nmax 1703, 1672,
d
165.4, 160.2, 152.3, 136.8, 136.7, 136.7, 136.5, 128.8 (2ꢂC), 128.40
(2ꢂC), 128.4, 128.3, 128.0, 128.0 (2ꢂC), 127.6 (2ꢂC), 125.0, 124.2, 124.2,
122.8, 122.4, 121.2, 119.8, 111.9, 111.5, 83.0, 77.7, 74.6, 70.7, 69.9, 61.2,
40.5, 28.3 (3ꢂC), 14.1; MS (ESIþ) (m/z): 672.58 [MþNa]þ; HRMS-ESI
(m/z): [MþH]þ calcd for C38H39N3O7: 650.28608, found: 650.28543.
1205, 1141, 1101, 607 cmꢁ1 1H NMR (CD3COCD3, 400 MHz):
; d 8.46
(dd, J¼2.0 Hz, J¼0.5 Hz, 1H), 7.71 (dd, J¼8.7 Hz, J¼0.5 Hz, 1H), 7.57 (s,
1H), 7.48 (dd, J¼8.7 Hz, J¼2.0 Hz,1H), 7.34e7.27 (m, 5H), 7.18e7.16 (m,
3H), 6.93e6.91 (m, 2H), 5.96e5.93 (m, 2H), 5.83 (d, J¼10.1 Hz, 1H),
5.50 (d, J¼11.3 Hz, 1H), 5.23 (d, J¼15.2 Hz, 1H), 4.56 (s, 2H), 4.22 (q,
J¼7.1 Hz, 2H), 4.15e4.08 (m, 2H), 3.94 (d, J¼12.1 Hz, 1H), 1.48 (s, 9H),
4.2.2. 5-(tert-Butyl) 2-ethyl 1,11-bis(benzyloxymethyl)-8-chloro-6-oxo-
1,4,6,11-tetrahydro-5H-pyrrolo[20,30:5,6]azepino[4,3-b]indole-2,5-
dicarboxylate (14b) and 5-(tert-Butyl) 1-ethyl 2,11-bis(benzyloxymethyl)-
8-chloro-6-oxo-2,4,6,11-tetrahydro-5H-pyrrolo[30,40:5,6]azepino[4,3-b]
indole-1,5-dicarboxylate (15b). Compounds 14b and 15b were syn-
thesized according to the general procedure from 13b (168 mg,
0.21 mmol) in dry DMF (4.5 mL) using Pd(OAc)2 (4.2 mg, 0.019 mmol),
PPh3 (11 mg, 0.042 mmol) and Ag2CO3 (231.6 mg, 0.84 mmol). The
reaction time was 30 min. The crude residue was purified by silica gel
flash column chromatography (toluene/EtOAc 98:2). 14b (52 mg, 36%),
yellow crystalline solid; mp 63e64.7 ꢀC; Rf (toluene/EtOAc 95:5) 0.58;
1.12 (t, J¼7.1 Hz, 3H); 13C NMR (CD3COCD3, 100 MHz):
d 165.3, 160.6,
152.9, 139.2, 138.5, 137.7, 136.5, 131.0, 129.2 (2ꢂC), 129.0 (2ꢂC), 128.6,
128.5, 128.4 (2ꢂC), 128.3 (2ꢂC), 127.1, 126.5, 125.2, 125.1, 121.1, 120.9,
116.1, 114.7, 111.4, 82.6, 78.8, 75.3, 71.2, 70.3, 61.7, 40.8, 28.2 (3ꢂC),
14.3; MS (ESIþ) (m/z): 750.03 [MþNa]þ; HRMS-ESI (m/z): [MþH]þ
calcd for C38H38BrN3O7: 728.19659, found: 728.19647.
IR (KBr): nmax 1714, 1672, 1221, 1138, 1093 cmꢁ1
;
1H NMR (CD3OD,
8.20 (d, J¼1.9 Hz, 1H), 7.51 (d, J¼8.8 Hz, 1H), 7.33 (dd,
4.3. General procedure for the synthesis of the tetracyclic
derivatives 18a and 19a
400 MHz):
d
J¼8.8 Hz, J¼2.1 Hz, 1H), 7.17e7.14 (m, 4H), 6.96 (dd, J¼5.0 Hz, J¼1.8 Hz,
3H), 6.91e6.89 (m, 2H), 6.81e6.79 (m, 2H), 6.03 (d, J¼10.2 Hz,1H), 5.68
(d, J¼10.8 Hz, 1H), 5.37 (d, J¼10.8 Hz, 1H), 5.28 (d, J¼10.2 Hz, 1H), 5.10
(d, J¼15.4 Hz, 1H), 4.33 (qd, J¼7.1 Hz, J¼3.3 Hz, 2H), 4.22 (d, J¼12.2 Hz,
1H), 4.08e3.94 (m, 3H), 3.71 (d, J¼11.9 Hz, 1H), 1.46 (s, 9H), 1.36 (t,
A mixture of 16a or 17a (1 equiv), 10% Pd/C (0.3 equiv) and
HCOONH4 (10.6 equiv) in EtOH abs was heated to reflux for the
indicated time. The resulting reaction mixture was filtered through
a Celite pad and the filtrate was concentrated under reduced
pressure. The crude residue was partitioned between a saturated
solution of NaHCO3 and EtOAc. The two phases were separated and
the aqueous phase was extracted with EtOAc (2ꢂ). The combined
organic layers were washed with brine (1ꢂ), dried over anhydrous
Na2SO4, filtered and evaporated under reduced pressure. The
resulting solid residue was purified by recrystallization with EtOAc
and drops of n-hexane to afford the corresponding fully depro-
tected tetracyclic products 18a or 19a.
J¼7.1 Hz, 3H); 13C NMR (CD3OD, 100 MHz):
d 166.6, 161.8, 152.9, 137.6,
137.3, 135.3, 131.3, 130.8, 130.1, 129.4 (2ꢂC), 129.2 (2ꢂC), 129.0, 129.0,
128.9, 128.7 (2ꢂC), 128.6 (2ꢂC), 128.3, 128.3, 126.0, 122.4, 119.2, 114.6,
113.6, 84.4, 76.6, 75.8, 71.5, 71.0, 61.9, 42.5, 28.3 (3ꢂC), 14.7; MS (ESIþ)
(m/z): 706.19 [MþNa]þ; HRMS-ESI (m/z): [MþH]þ calcd for
C
38H38ClN3O7: 684.24710, found: 684.24671. 15b (37 mg, 26%), white
solid; mp 130.6e131.8 ꢀC; Rf (toluene/EtOAc 95:5) 0.37; IR (KBr): nmax
1
1703, 1672, 1209, 1140, 1101, 590 cmꢁ1; H NMR (CD3OD, 400 MHz):
d
8.20 (d, J¼1.8 Hz, 1H), 7.68 (d, J¼8.8 Hz, 1H), 7.45 (s, 1H), 7.34 (dd,
J¼8.7 Hz, J¼2.1 Hz, 1H), 7.3e7.19 (m, 5H), 7.12 (dd, J¼5.0 Hz, J¼1.0 Hz,
3H), 6.85e6.83 (m, 2H), 5.90 (d, J¼10.2 Hz, 1H), 5.82 (d, J¼11.2 Hz, 1H),
5.70 (d, J¼10.2 Hz, 1H), 5.38 (d, J¼11.2 Hz, 1H), 5.17 (d, J¼15.2 Hz, 1H),
4.50 (d, J¼3.2 Hz, 2H), 4.20e4.15 (m, 2H), 4.10 (d, J¼12.4 Hz, 1H), 3.99
(d, J¼15.1 Hz, 1H), 3.90 (d, J¼12.4 Hz, 1H), 1.51 (s, 9H), 1.10 (t, J¼7.1 Hz,
4.3.1. Ethyl
6-oxo-4,5,6,11-tetrahydro-1H-pyrrolo[20,30:5,6]azepino
[4,3-b]indole-2-carboxylate (18a). Compound 18a was synthesized
according to the general procedure from 16a (6 mg, 0.011 mmol)
upon treatment with 10% Pd/C (3.5 mg) and HCOONH4 (7.35 mg,
0.117 mmol) in EtOH abs (0.34 mL). The reaction time was 1 h 18a
(3.1 mg, 91%), yellow solid; mp 227.1 ꢀC (dec); Rf (n-hexane/EtOAc
3H); 13C NMR (CDCl3,100 MHz):
d 165.0, 160.1, 152.3,137.7,136.7, 136.3,
135.1, 129.4, 128.8, 128.7 (2ꢂC), 128.4 (2ꢂC), 128.3, 128.1, 127.9 (2ꢂC),
127.6 (2ꢂC), 125.1, 124.5, 124.2, 121.9, 120.8, 119.9, 112.6, 111.4, 83.1,
77.8, 74.8, 70.8, 70.0, 61.3, 40.4, 28.3 (3ꢂC), 14.1; MS (ESIþ) (m/z):
706.07 [MþNa]þ; HRMS-ESI (m/z): [MþH]þ calcd for C38H38ClN3O7:
684.24710, found: 684.24690.
2:8) 0.22; IR (KBr): nmax 3281, 1687, 1610, 1205 cmꢁ1
(DMSO-d6, 700 MHz):
;
1H NMR
d
12.05 (s, 1H), 11.5 (s, 1H), 8.11 (d, J¼7.9 Hz,
1H), 7.51 (t, J¼4.8 Hz, 1H), 7.49 (d, J¼8.1 Hz, 1H), 7.17 (dt, J¼54.4 Hz,
J¼7.9 Hz, 2H), 6.87 (d, J¼2.3 Hz, 1H), 4.31 (q, J¼7.1 Hz, 2H), 4.02 (d,
J¼4.8 Hz, 2H), 1.32 (t, J¼7.1 Hz, 3H); 13C NMR (DMSO-d6, 176 MHz):
d
167.3, 160.3, 135.8, 130.8, 128.3, 127.3, 124.4, 122.9, 122.9, 122.0,
4.2.3. 5-(tert-Butyl) 2-ethyl 1,11-bis(benzyloxymethyl)-8-bromo-6-oxo-
1,4,6,11-tetrahydro-5H-pyrrolo[20,30:5,6]azepino[4,3-b]indole-2,5-
dicarboxylate (14c)and 5-(tert-Butyl) 1-ethyl 2,11-bis(benzyloxymethyl)-
8-bromo-6-oxo-2,4,6,11-tetrahydro-5H-pyrrolo[30,40:5,6]azepino[4,3-b]
indole-1,5-dicarboxylate (15c). Compounds 14c and 15c were syn-
thesized according to the general procedure from 13c (300 mg,
0.35 mmol) in dry DMF (7.45 mL) using Pd(OAc)2 (7.1 mg,
0.032 mmol), PPh3 (18.4 mg, 0.07 mmol) and Ag2CO3 (386 mg,
1.4 mmol). The reaction time was 23 h. The crude residue was purified
by silica gel flash column chromatography (toluene/EtOAc 98:2). 14c
(100 mg, 39%), white solid; mp 54.8e56 ꢀC; Rf (n-hexane/EtOAc 7:3)
120.8, 113.4, 111.4, 108.4, 60.1, 37.0, 14.4; MS (ESIþ) (m/z): 353.70
[Mþ2Na-2H]þ; HRMS-ESI (m/z): [MþH]þ calcd for C17H15N3O3:
310.11862, found: 310.11855; HPLC: Isocratic elution AcN (þ0.08%
TFA)/H2O (þ0.08% TFA)¼70/30; tR¼5.488 min; purity¼96.1%.
4.3.2. Ethyl 6-oxo-4,5,6,11-tetrahydro-2H-pyrrolo[30,40:5,6]azepino[4,3-
b]indole-1-carboxylate (19a). Compound 19a was synthesized
according to the general procedure from 17a (12 mg, 0.022 mmol)
upon treatment with 10% Pd/C (6.96 mg) and HCOONH4 (14.5 mg,
0.23 mmol) in EtOH abs (0.67 mL). The reaction time was 8 h 19a
(5.75 mg, 85%), white solid; mp 126.4 ꢀC; Rf (n-hexane/EtOAc 2:8) 0.14;
0.52; IR (KBr): nmax 1712, 1672, 1221, 1136, 1091, 603 cmꢁ1; H NMR
IR (KBr): nmax 3406, 1682, 1614, 1205 cmꢁ1
;
1H NMR (DMSO-d6,
1
(CD3COCD3, 400 MHz):
7.51 (dd, J¼8.7 Hz, J¼2.0 Hz,1H), 7.34e7.22 (m, 5H), 7.15e7.08 (m, 3H),
7.04e7.02 (m, 1H), 6.97e6.95 (m, 1H), 6.20 (d, J¼10.1 Hz, 1H), 5.93 (d,
d
8.47 (d, J¼2.0 Hz, 1H), 7.63 (d, J¼8.8 Hz, 1H),
700 MHz):
d
12.11 (br s, 1H), 11.43 (br s, 1H), 8.05 (d, J¼8.0 Hz, 1H), 7.65
(t, J¼5.0 Hz, 1H), 7.53 (d, J¼8.1 Hz, 1H), 7.18 (t, J¼7.5 Hz, 1H), 7.14 (d,
J¼3.0 Hz, 1H), 7.1 (t, J¼7.5 Hz, 1H), 4.35 (q, J¼7.1 Hz, 2H), 3.96 (d,
Please cite this article in press as: Psarra, V.; et al., Tetrahedron (2016), http://dx.doi.org/10.1016/j.tet.2016.03.048

V. Psarra et al. / Tetrahedron xxx (2016) 1e10
9
J¼4.9 Hz, 2H), 1.33 (t, J¼7.1 Hz, 3H); 13C NMR (DMSO-d6, 176 MHz):
a sealed tube. After the completion of the reaction, the mixture was
neutralized with 25% NH3 and the solvents were removed with
evaporation under reduced pressure. The residue was extracted with
EtOAc (3ꢂ). The combined organic phases were washed with brine
(1ꢂ), dried over anhydrous Na2SO4, filtered and concentrated under
reduced pressure. The crude residue was purified by silica gel flash
column chromatography using DCM/MeOH 99:1 to afford com-
pounds 19c and 28c. 19c (8.5 mg, 91%), white solid; mp 257.4 ꢀC
(dec); Rf (n-hexane/EtOAc 3:7) 0.23; IR (KBr): nmax 3288, 1678, 1622,
d
167.8, 160.8, 135.3, 133.1, 128.0, 124.9, 122.5, 121.5, 120.4, 120.3, 120.0,
117.6, 111.4, 108.8, 60.6, 36.3, 14.2; MS (ESIþ) (m/z): 310.48 [MþH]þ;
HRMS-ESI (m/z): [MþH]þ calcd for C17H15N3O3: 310.11862, found:
310.11881, [MþNa]þ calcd for C17H15N3O3: 332.10056, found:
332.10054, [2MþNa]þ calcd for C17H15N3O3: 641.21190, found:
641.21144; HPLC: Isocratic elution AcN (þ0.08% TFA)/H2O (þ0.08%
TFA)¼70/30; tR¼7.156 min; purity¼97.2%.
4.4. Procedures for the synthesis of the tetracyclic derivatives
1203 cmꢁ1; 1H NMR (DMSO-d6, 700 MHz):
d 12.18 (br s, 1H), 11.59 (br
19(bec) and 28(bec)
s, 1H), 8.19 (d, J¼2.0 Hz, 1H), 7.75 (t, J¼5.0 Hz, 1H), 7.53 (d, J¼8.6 Hz,
1H), 7.3 (dd, J¼8.6 Hz, J¼2.0 Hz, 1H), 7.16 (d, J¼3.0 Hz, 1H), 4.34 (q,
J¼7.1 Hz, 2H), 3.97 (d, J¼4.9 Hz, 2H), 1.32 (t, J¼7.1 Hz, 3H); 13C NMR
4.4.1. Ethyl 8-chloro-6-oxo-4,5,6,11-tetrahydro-2H-pyrrolo[30,40:5,6]
azepino[4,3-b]indole-1-carboxylate (19b) and compound 28b. To a so-
lution of 17b (7.66 mg, 0.013 mmol) in EtOH abs (2.6 mL), 6 N aq
HCl (1.91 mL) was added and the mixture was heated at 130 ꢀC for
2 h in a sealed tube. After the completion of the reaction, the
mixture was neutralized with 25% aq NH3 and the solvents were
evaporated under reduced pressure. The residue was extracted
with EtOAc (3ꢂ). The combined organic phases were washed with
brine (1ꢂ), dried over anhydrous Na2SO4, filtered and concen-
trated under reduced pressure. The crude residue was purified by
silica gel flash column chromatography using DCM/MeOH 99:1 to
afford compounds 19b and 28b. 19b (2.76 mg, 62%), white solid;
mp 257.4 ꢀC (dec); Rf (n-hexane/EtOAc 3:7) 0.23; IR (KBr): nmax
(DMSO-d6, 176 MHz):
d
167.4, 160.6, 134.4, 134.1, 129.7, 124.9 (2ꢂC),
123.4, 120.1, 119.7, 117.9, 113.6, 113.0, 108.3, 60.7, 36.3, 14.1; MS (ESIþ)
(m/z): 410.35 [MþNa]þ; HRMS-ESI (m/z): [MþNa]þ calcd for
C17H14BrN3O3: 388.02913, found: 388.02876; HPLC: Isocratic elution
AcN (þ0.08% TFA)/H2O (þ0.08% TFA)¼70/30; tR¼8.688 min;
purity¼98.2%. 28c (0.72 mg, 7%), yellow solid; mp 220.1 ꢀC (dec); Rf
(n-hexane/EtOAc 3:7) 0.34; IR (KBr): nmax 2924, 1699, 1616, 1224,
1103 cmꢁ1 1H NMR (CD3COCD3, 400 MHz):
; d 11.46 (br s, 1H),
8.42e8.41 (m, 1H), 7.56 (d, J¼8.6 Hz, 1H), 7.35e7.34 (m, 1H), 7.33 (d,
J¼2.0 Hz, 1H), 6.95 (br s, 1H), 5.74 (s, 2H), 4.43 (q, J¼7.1 Hz, 2H), 4.15
(d, J¼4.3 Hz, 2H), 3.49 (q, J¼7.0 Hz, 2H), 1.38 (t, J¼7.1 Hz, 3H), 1.12 (t,
J¼7.0 Hz, 3H); 13C NMR (CD3COCD3, 176 MHz):
d 168.5, 162.5, 135.2,
3331, 1685, 1639, 1217 cmꢁ1
;
1H NMR (DMSO-d6, 700 MHz):
131.2, 126.4, 126.0, 125.3, 124.7, 124.2, 119.8, 114.5, 114.0, 113.9, 110.3,
79.8, 64.8, 62.3, 37.4, 15.2, 14.3; MS (ESIþ) (m/z): 468.41 [MþNa]þ;
HRMS-ESI (m/z): [MþH]þ calcd for C20H20BrN3O4: 446.07100, found:
446.07060, [2Mþ3H]þ calcd for C20H20BrN3O4: 893.14927, found:
893.13245;
d
12.18 (br s, 1H), 11.59 (br s, 1H), 8.03 (d, J¼2.1 Hz, 1H), 7.74 (t,
J¼5.0 Hz, 1H), 7.57 (d, J¼8.6 Hz, 1H), 7.19 (dd, J¼8.6 Hz, J¼2.2 Hz,
1H), 7.15 (d, J¼3.0 Hz, 1H), 4.35 (q, J¼7.1 Hz, 2H), 3.97 (d, J¼4.9 Hz,
2H), 1.32 (t, J¼7.1 Hz, 3H); 13C NMR (DMSO-d6, 176 MHz):
d 167.5,
160.7, 134.6, 133.8, 129.1, 125.0, 124.9, 122.4, 120.4, 120.2, 119.8,
117.9, 113.1, 108.4, 60.7, 36.3, 14.1; MS (ESIþ) (m/z): 366.37
[MþNa]þ; HRMS-ESI (m/z): [MþH]þ calcd for C17H14ClN3O3:
344.07965, found: 344.07946, [MþNa]þ calcd for C17H14ClN3O3:
366.06159, found: 366.06140, [2MþNa]þ calcd for C17H14ClN3O3:
709.13396, found: 709.13352; HPLC: Isocratic elution AcN (þ0.08%
TFA)/H2O (þ0.08% TFA)¼70/30; tR¼8.296 min; purity¼97.6%. 28b
(1.74 mg, 33%), yellow solid; mp 220.8 ꢀC (dec); Rf (n-hexane/
4.4.2.1. Deprotection of compound 28c. To a solution of 28c
(2.07 mg, 4.6 mmol) in EtOH abs (0.92 mL), 6 N aq HCl (0.66 mL) was
added and the mixture was heated at 130 ꢀC for 3.5 h in a sealed
tube. After the completion of the reaction, the mixture was neu-
tralized with 25% aq NH3 and the solvents were evaporated under
reduced pressure. The residue was extracted with EtOAc (3ꢂ). The
combined organic phases were washed with brine (1ꢂ), dried over
anhydrous Na2SO4, filtered and concentrated under reduced pres-
sure. The crude residue was purified by silica gel flash column
chromatography using DCM/MeOH 99:1 to afford compound 19c
(0.96 mg, 54%).
EtOAc 3:7) 0.34; IR (KBr): nmax 2924, 1682, 1614, 1222, 1095 cmꢁ1
;
1H NMR (CD3COCD3, 400 MHz):
d
11.45 (br s, 1H), 8.25 (d,
J¼2.5 Hz, 1H), 7.6 (d, J¼8.6 Hz, 1H), 7.34 (s, 1H), 7.21 (dd, J¼8.6 Hz,
J¼2.2 Hz, 1H), 6.95 (br s, 1H), 5.74 (s, 2H), 4.43 (q, J¼7.1 Hz, 2H),
4.15 (d, J¼4.3 Hz, 2H), 3.49 (q, J¼7.0 Hz, 2H), 1.38 (t, J¼7.1 Hz, 3H),
1.12 (t, J¼7.0 Hz, 3H); 13C NMR (CD3COCD3, 176 MHz):
d 168.5,
4.5. Protein kinase profiling
162.6, 135.3, 134.9, 130.6, 126.8, 126.0, 124.7, 124.2, 123.8, 122.2,
119.8, 113.6, 110.4, 79.8, 64.8, 62.3, 37.4, 15.2, 14.3; MS (ESIþ) (m/z):
424.50 [MþNa]þ; HRMS-ESI (m/z): [MþNa]þ calcd for
Compounds 18a and 19(aec) were profiled against a panel of 50
kinases (Exrpess Screen) at the International Centre for Kinase
Profiling, Division of Signal Transduction Therapy, University of
Dundee (http://www.kinase-screen.mrc.ac.uk/).27
C
C
20H20ClN3O4: 424.10345, found: 424.10307, [2MþNa]þ calcd for
20H20ClN3O4: 825.21769, found: 825.21684.
4.4.1.1. Deprotection of compound 28b. To a solution of 28b
(1.79 mg, 4.45
mmol) in EtOH abs (0.89 mL), 6 N aq HCl (0.65 mL)
4.6. Antiproliferative activity
was added and the mixture was heated at 130 ꢀC for 3.5 h in
a sealed tube. After the completion of the reaction, the mixture was
neutralized with 25% aq NH3 and the solvents were evaporated
under reduced pressure. The residue was extracted with EtOAc
(3ꢂ). The combined organic phases were washed with brine (1ꢂ),
dried over anhydrous Na2SO4, filtered and concentrated under re-
duced pressure. The crude residue was purified by silica gel flash
column chromatography using DCM/MeOH 99:1 to afford com-
pound 19b (0.86 mg, 56%).
Cell proliferation was measured using a cell proliferation ELISA,
BrdU assay kit (Roche, Cat. No. 11669915001). The assay was per-
formed according to the instructions. Therefore, 12000 MCF-7 cells
were seeded in each well of a 96 well microplate (Greiner bio-one,
flat bottomed, white,
medium was removed after 24 h and the cells were incubated for
additional 24 h with 100 l of different concentrations of the
compounds (500, 250, 100, 50, 10, 5 and 1 M), while after 4 h the
mclear) with a final volume of 100 ml. The
m
m
cells were incubated with 5-bromo-20-deoxyuridine (BrdU) label-
ling solution. Though, the solvent concentration was always lower
than 0.5% (v/v). After removal of the medium the cells were fixed
and incubated with anti-BrdU-POD solution. In the end, the cells
were washed, incubated with the substrate solution and the
4.4.2. Ethyl
8-bromo-6-oxo-4,5,6,11-tetrahydro-2H-pyrrolo[30,40:5,6]
azepino[4,3-b]indole-1-carboxylate (19c) and compound 28c. To a solu-
tion of 17c (15 mg, 0.024 mmol) in EtOH abs (4.8 mL), 6 N aq HCl
(3.59 mL) was added and the mixture was heated at 130 ꢀC for 2 h in
Please cite this article in press as: Psarra, V.; et al., Tetrahedron (2016), http://dx.doi.org/10.1016/j.tet.2016.03.048

10
V. Psarra et al. / Tetrahedron xxx (2016) 1e10
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Sausville, E. A. Cancer Res. 1999, 59, 2566e2569.
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8. Egert-Schmidt, A. M.; Dreher, J.; Dunkel, U.; Kohfeld, S.; Preu, L.; Weber, H.;
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www.seedrug.upatras.gr), is acknowledged for access to 700 MHz
NMR Instrumentation at University of Patras. We thank Dr. D.
Vachliotis and Dr. G. Magoulas (Center of Instrumental Analysis,
University of Patras, Greece) for recording NMR and MS spectra.
This research has been co-financed by the European Union
(European Social Fund e ESF) and Greek national funds through the
Operational Program “Education and Lifelong Learning” of the
National Strategic Reference Framework (NSRF) - Research Funding
Program: Heracleitus II. Investing in knowledge society through the
European Social Fund.
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Supplementary data
Supplementary data (Synthetic procedures and spectroscopic
data for compounds 4, 5, 6, 7, 8, 21(bec), 22(bec), 23b, 25(bec),
26(bec), 9(aec), 10(aec), 11(aec), 12(aec), 13(aec), 16(aec),
17(aec). Full kinase selectivity profiling data for compounds 18a
and 19(aec). Antiproliferative activity data for compounds 18a and
19(aec). Copies of 1D- and 2D-NMR spectra of compounds 16a and
17a associated with this article can be found in the online version,
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Please cite this article in press as: Psarra, V.; et al., Tetrahedron (2016), http://dx.doi.org/10.1016/j.tet.2016.03.048