Benzothiopyranoindole- and pyridothiopyranoindole-based antiproliferative agents targeting topoisomerases

Article abstract of DOI:10.1016/j.ejmech.2019.01.015

New benzothiopyranoindoles (5a-l) and pyridothiopyranoindoles (5m-t), featuring different combinations of substituents (H, Cl, OCH₃) at R²-R⁴ positions and protonatable R¹-dialkylaminoalkyl chains, were synthesized and biologically assayed on three human tumor cell lines, showing significant antiproliferative activity (GI₅₀ values spanning from 0.31 to 6.93 μM) and pro-apoptotic effect. Linear flow dichroism experiments indicate the ability of both chromophores to form a molecular complex with DNA, following an intercalative mode of binding. All compounds displayed a moderate ability to inhibit the relaxation activity of both topoisomerases I and II, reasonably correlated to their intercalative capacities. Cleavable assay performed with topoisomerase I revealed a significant poisoning effect for compounds 5g, 5h, 5s, and 5t. A theoretical model provided by hydrated docking calculations clarified the role of the R¹-R⁴ substituents on the topoisomerase I poison activity, revealing a crucial role of the R²-OCH₃ group.

Full text of DOI:10.1016/j.ejmech.2019.01.015

Accepted Manuscript
Benzothiopyranoindole- and pyridothiopyranoindole-based antiproliferative agents
targeting topoisomerases
Silvia Salerno, Valeria La Pietra, Mariafrancesca Hyeraci, Sabrina Taliani, Marco
Robello, Elisabetta Barresi, Ciro Milite, Francesca Simorini, Aída Nelly García-
Argáez, Luciana Marinelli, Ettore Novellino, Federico Da Settimo, Anna Maria Marini,
Lisa Dalla Via
PII:
S0223-5234(19)30021-2
DOI:
https://doi.org/10.1016/j.ejmech.2019.01.015
EJMECH 11022
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 October 2018
Revised Date: 21 December 2018
Accepted Date: 7 January 2019
Please cite this article as: S. Salerno, V. La Pietra, M. Hyeraci, S. Taliani, M. Robello, E. Barresi, C.
Milite, F. Simorini, Aí.Nelly. García-Argáez, L. Marinelli, E. Novellino, F. Da Settimo, A.M. Marini, L.
Dalla Via, Benzothiopyranoindole- and pyridothiopyranoindole-based antiproliferative agents targeting
topoisomerases, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.01.015.
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ACCEPTED MANUSCRIPT
Graphical abstract
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Benzothiopyranoindole- and pyridothiopyranoindole-based antiproliferative agents targeting
topoisomerases.
Silvia Salerno,a,‡ Valeria La Pietra,b,‡ Mariafrancesca Hyeraci,c Sabrina Taliani,a,* Marco Robello,d
Elisabetta Barresi,a Ciro Milite,e Francesca Simorini,a Aída Nelly García-Argáez,c,f Luciana
Marinelli,b,* Ettore Novellino,b Federico Da Settimo,a Anna Maria Marini,a Lisa Dalla Via.c
aDipartimento di Farmacia, Università di Pisa, Via Bonanno 6, 56126 Pisa, Italy.
bDipartimento di Farmacia, Università di Napoli “Federico II”, Via D. Montesano 49, 80131
Napoli, Italy.
cDipartimento di Scienze del Farmaco, Università di Padova, Via Marzolo 5, 35131 Padova, Italy.
dSynthetic Bioactive Molecules Section LBC, NIDDK, NIH, 8 Center Dr., 20982, Bethesda,
Maryland, USA.
eDipartimento di Farmacia, Università di Salerno, Via Giovanni Paolo II 132, 84084 Fisciano,
Salerno, Italy.
fFondazione per la Biologia e la Medicina della Rigenerazione T.E.S., Via Marzolo 13, 35131,
Padova, Italy.
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Abstract
New benzothiopyranoindoles (5a-l) and pyridothiopyranoindoles (5m-t), featuring different
combinations of substituents (H, Cl, OCH3) at R2-R4 positions and protonatable R1-
dialkylaminoalkyl chains, were synthesized and biologically assayed on three human tumor cell
lines, showing significant antiproliferative activity (GI50 values spanning from 0.31 to 6.93 µM) and
pro-apoptotic effect. Linear flow dichroism experiments indicate the ability of both chromophores
to form a molecular complex with DNA, following an intercalative mode of binding. All
compounds displayed a moderate ability to inhibit the relaxation activity of both topoisomerases I
and II, reasonably correlated to their intercalative capacities. Cleavable assay performed with
topoisomerase I revealed a significant poisoning effect for compounds 5g, 5h, 5s, and 5t. A
theoretical model provided by hydrated docking calculations clarified the role of the R1-R4
substituents on the topoisomerase I poison activity, revealing a crucial role of the R2-OCH3 group.
Keywords: benzothiopyranoindoles, pyridothiopyranoindoles, antiproliferative activity,
topoisomerases, topoisomerase I poisons.
Abbreviation: A-431, squamous carcinoma cell line; AD4, AutoDock (version 4.2); ADT,
AutoDockTools; m-AMSA, m-amsacrine; CPT, camptothecin; HeLa, cervix adenocarcinoma cell
line; GALS, genetic algorithm local search; LD, linear flow dichroism; MSTO-211H, biphasic
mesothelioma cell line; Topo I, type I topoisomerase; Topo II, type II topoisomerase.
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1. Introduction
A large percentage of the chemotherapeutic anticancer agents currently used belong to the category
of DNA-intercalating small molecules [1,2]. Some of them are valuable drugs for the treatment of
ovarian and breast cancers and acute leukemias, while many others are in different phases of
clinical trials [3-5]. Characteristic structural features of intercalating agents include the presence of
planar polyaromatic systems, which interact reversibly with DNA by insertion between base-pairs,
and elicit structural modifications leading to functional changes of transcription and replication
processes, culminating in cellular death [1,5].
Several DNA-intercalating agents are also able to affect topoisomerase catalytic activity, thus
producing permanent DNA strand breaks [1,6-10]. In humans, type I topoisomerase (Topo I) and
type II topoisomerase (Topo II), are nuclear enzymes essential to resolve topological problems that
occur during DNA transcription, replication, and chromosome segregation [11-15]. Thus, the
biological functions of topoisomerases are crucial for ensuring genomic integrity, and the ability to
interfere with these enzymes or generate enzyme-mediated damage is an effective strategy for
cancer therapy [16-18]. In particular, drugs that interfere with the catalytic cycle by trapping a
covalent intermediate, the cleavage complex, are commonly referred as poisons, while those that
suppress enzyme activity by other mechanisms are named catalytic inhibitors. In this regard, Topo I
and Topo II poisons represent a significant class of anticancer agents, forming the basis of many
multidrug chemotherapy protocols widely used in a broad spectrum of tumors [16,19-23].
Camptothecin (CPT, 1) [24], topotecan 2, and irinotecan 3 (Chart 1) represent the most important
Topo I poisons [19]. Drugs targeting Topo II include both poisons, (etoposide, doxorubicin, m-
amsacrine (m-AMSA) and mitoxantrone), and inhibitors (bisdioxopiperazines or novobiocin,
merbarone and the anthracycline aclarubicin) [23].
The two FDA-approved water-soluble derivatives 2 and 3 derived from efforts to improve the
toxicity profile and pharmacokinetics of 1, and are currently used for the treatment of solid tumors,
2 (Chart 1) for ovarian and lung cancers, and 3 (Chart 1) mainly for colon cancer [22]. Despite their
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efficiency in the clinic, current anticancer therapies with these CPT derivatives are limited by some
important drawbacks, including the emergence of drug-resistance [19,20,25]. Consequently,
significant investments have been expended and are currently being made, and several chemical
classes of non-CPT Topo I poisons were developed as promising antitumor drugs [16,23].
As part of our research program, devoted to the identification of new antiproliferative agents, we
studied several polyheterocyclic chromophores, structurally related to classes of DNA intercalating
agents [26-29]. Among them, the benzothiopyranoindoles 4a-v [30] (Chart 1) were extensively
studied, leading to the identification of a number of derivatives showing an antiproliferative activity
at low micromolar concentrations [30]. Furthermore, it was proven that the chromophore scaffold
itself does not possess any significant antiproliferative effect, unless a basic side chain on the indole
nitrogen atom is present. It was also demonstrated a significant intercalation ability, as well as the
capacity to inhibit the relaxation of supercoiled plasmid DNA mediated by Topo II in a dose-
dependent manner. Finally, for the most cytotoxic derivative 4e (Chart 1, X=OCH3, X'=H,
R1=(CH2)2N(CH3)2), a weak Topo II poisoning effect and the capacity to inhibit the Topo I-
mediated DNA relaxation were also demonstrated [30].
The successful results obtained from the benzothiopyranoindoles 4a-v (Chart 1) [30] prompted the
synthesis of a library of new derivatives from this class (5a-t, Chart 1), in order to evaluate the
effect of various structural modifications on antiproliferative activity. In particular, while
maintaining the benzothiopyranoindole system, a methoxy group was inserted at the 3-position (R3)
of the chromophore and variously combined with an additional methoxy at the 2-position (R2)
and/or methoxy/chlorine substituent at the 7-position (R4), allowing the obtainment of the novel
compounds 5a-d and 5g-l (Scheme 1). In addition, to investigate the effect of a double substitution
with chlorine at either 3- and 7-positions of the planar moiety, derivatives 5e-f (Scheme 1) were
also synthesized. Finally, a small library of pyridothiopyranoindole isosters 5m-t (Scheme 1) were
studied; in this case, the introduction into the chromophore of a protonatable nitrogen atom could
provide an additional or alternative anchor point in the formation of the intercalation complex.
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All derivatives have been then functionalized with dialkylaminoalkyl chains (R1), considering the
crucial role of these basic moieties for the biological activity. To this regard we decided to focus on
the dimethylaminoethyl and diethylaminoethyl chains, as they characterized the most active
compounds of series 4 (Chart 1) [30].
The ability of the new benzothiopyranoindole (5a-l) and pyridothiopyranoindole (5m-t) derivatives
to exert an antiproliferative activity was evaluated by means of an inhibition growth assay on three
human tumor cell lines: cervix adenocarcinoma (HeLa), epidermoid carcinoma (A-431) and
biphasic mesothelioma (MSTO-211H). Linear flow dichroism (LD) experiments were performed to
assess the occurrence of a molecular complex with DNA. In addition, the ability of the compounds
to interfere with the catalytic activity of both Topo I and Topo II was evaluated.
Finally, a molecular modeling study was carried out to shed light on the interaction between Topo I
covalently bound to DNA and our poisons. Molecular determinants of our compounds regarding the
poison property towards Topo I have also been discussed.
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Chart 1. Structures of known (1-4) and newly synthesized (5) compounds targeting
Topoisomerases.
2. Results and discussion
2.1 Chemistry
The preparation of the 11-dialkylaminoalkyl-2,3,7-substituted-benzothiopyrano[3,2-b]indol-
10(11H)-ones 5a-l, and 6-dialkylaminoalkyl-8,9-substituted-pyrido[3',2':5,6]thiopyrano[3,2-
b]indol-5(6H)-ones 5m-t was performed following the synthetic procedure outlined in Scheme 1.
The key starting 3-hydroxymethylenebenzothiopyranone derivatives 6a-c [30,31] and 2,3-
dihydro-3-(hydroxymethylene)-4H-thiopyran[2,3-b]pyridin-4-one 6d [27], containing a methine
active group, gave the desired 3-phenylhydrazono intermediates 8a-j, in particularly good yields, by
coupling with the appropriate substituted diazonium salt, using the Japp-Klingemann reaction
[30,27,32]. Compounds 8a-j were directly converted to the desired indoles 9a-j with good to high
yields using the Fischer cyclization, by refluxing in ethanolic hydrogen chloride solution. All the
structures proposed for compounds 9a-j agreed with 1H-NMR spectral data, in which an interesting
feature was the low-field signal (δ≈12 ppm), which was exchangeable with D2O and was assigned
to the proton of the indole NH group. The target 11-N-dialkylaminoalkyl-benzo substituted
derivatives 5a-l and 6-dialkylaminoalkyl-pyrido derivatives 5m-t were obtained by reaction of
compounds 9a-j with the appropriate commercially available dialkylaminoalkylchloride
hydrochlorides in anhydrous DMF solution, in the presence of sodium hydride. In the alkylated
1
derivatives 5a-t H-NMR spectra, the exchangeable singlet, observed around 12 ppm as
1
characteristic of the indole NH proton of compounds 9a-j, disappears. In addition, the H-NMR
spectra of the alkylated derivatives 5a-t clearly show the aliphatic protons signals, whose
multiplicity is in agreement with the structures of the side chains introduced on the system.
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Scheme 1
H2N
R2
R3
7a: R2 = H, R3 = H
7b: R2= H, R3 = OCH3
7c: R2 = H, R3 = Cl
7d: R2 = OCH3, R3 = OCH3
7 a-d
i
O
S
O
S
H
Cl-
N N+
CHOH
R2
R3
N
N
R2
R3
ii
R4
R4
X
X
8 a-j
6 a-d
8a-9a: X = CH, R2 = H, R3 = OCH3, R4 = H
8b-9b: X = CH, R2 = H, R3 = OCH3, R4 = Cl
8c-9c: X = CH, R2 = H, R3 = Cl, R4 = Cl
6a: X = CH, R4 = H
6b: X = CH, R4 = Cl
6c: X = CH, R4 = OCH3
6d: X = N, R4 = H
8d-9d: X = CH, R2 = OCH3, R3 = OCH3, R4 = H
8e-9e: X = CH, R2 = OCH3, R3 = OCH3, R4 = Cl
iii
8f-9f: X = CH, R2 = OCH3, R3 = OCH3, R4 = OCH3
8g-9g: X = N, R2 = H, R3 = H, R4 = H
8h-9h: X = N, R2 = H, R3 = OCH3, R4 = H
8i-9i: X = N, R2 = H, R3 = Cl, R4 = H
8j-9j: X = N, R2 = OCH3, R3 = OCH3, R4 = H
O
S
H
O
R1
N
N
iv
R4
X
R2
R4
R2
X
S
R3
R3
9 a-j
5 a-t
5a: X = CH, R1 = (CH2)2(CH3)2, R2 = H, R3 = OCH3, R4 = H
5b: X = CH, R1 = (CH2)2(C2H5)2, R2 = H, R3 = OCH3, R4 = H
5c: X = CH, R1 = (CH2)2(CH3)2, R2 = H, R3 = OCH3, R4 = Cl
5d: X = CH, R1 = (CH2)2(C2H5)2, R2 = H, R3 = OCH3, R4 = Cl
5e: X = CH, R1 = (CH2)2(CH3)2, R2 = H, R3 = Cl, R4 = Cl
5f: X = CH, R1 = (CH2)2(C2H5)2, R2 = H, R3 = Cl, R4 = Cl
5g: X = CH, R1 = (CH2)2(CH3)2, R2 = OCH3, R3 = OCH3, R4 = H
5h: X = CH, R1 = (CH2)2(C2H5)2, R2 = OCH3, R3 = OCH3, R4 = H
5i: X = CH, R1 = (CH2)2(CH3)2, R2 = OCH3, R3 = OCH3, R4 = Cl
5j: X = CH, R1 = (CH2)2(C2H5)2, R2 = OCH3, R3 = OCH3, R4 = Cl
5k: X = CH, R1 = (CH2)2(CH3)2, R2 = OCH3, R3 = OCH3, R4 = OCH3
5l: X = CH, R1 = (CH2)2(C2H5)2, R2 = OCH3, R3 = OCH3, R4 = OCH3
5m: X = N, R1 = (CH2)2(CH3)2, R2 = H, R3 = H, R4 = H
5n: X = N, R1 = (CH2)2(C2H5)2, R2 = H, R3 = H, R4 = H
5o: X = N, R1 = (CH2)2(CH3)2, R2 = H, R3 = OCH3, R4 = H
5p: X = N, R1 = (CH2)2(C2H5)2, R2 = H, R3 = OCH3, R4 = H
5q: X = N, R1 = (CH2)2(CH3)2, R2 = H, R3 = Cl, R4 = H
5r: X = N, R1 = (CH2)2(C2H5)2, R2 = H, R3 = Cl, R4 = H
5s: X = N, R1 = (CH2)2(CH3)2, R2 = OCH3, R3 = OCH3, R4 = H
5t: X = N, R1 = (CH2)2(C2H5)2, R2 = OCH3, R3 = OCH3, R4 = H
Reagents and conditions: (i) HCl 18%/NaNO2/H2O/0 °C; (ii) AcONa, MeOH; (iii) EtOH HCl,
(iv) ClCH2CH2N(R)2 HCl, NaH, DMF,
;
.
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2.2
Antiproliferative activity
The antiproliferative activity of the newly synthesized derivatives was evaluated in vitro on three
human tumor cell lines: HeLa (cervix adenocarcinoma), A-431 (squamous carcinoma), and MSTO-
211H (biphasic mesothelioma) cells. The results, expressed as GI50 values, i.e. the concentration
(µM) of compound able to produce 50% cell death with respect to the control culture, are reported
in Table 1. The potent antitumor agent ellipticine was used as a reference compound, in view of its
structural similarity to the newly synthesized derivatives 5a-t [30].
All the tested compounds exert a significant antiproliferative activity on the considered cell lines,
showing GI50 values ranging from 0.31 to 6.93 µM, which are in some cases comparable to those
obtained for ellipticine. In general, HeLa cells seem to be less sensitive than A431 and MSTO-
211H cell lines, which actually display the lower GI50 values. Moreover, in most cases, the
antiproliferative effect exerted by the compounds featuring a dimethylaminoethyl side chain at R1
appears higher with respect to that exerted by the corresponding analogues characterized by the
diethylaminoethyl group. In details, the most active benzothiopyranoindoles are 5g and 5k, while,
within the pyridothiopyranoindole subseries, the most cytotoxic derivatives are 5m and 5s.
Interestingly, from a structural point of view, all compounds are characterized by a
dimethylaminoethyl side chain, and three out of four (5g, 5k, 5s) bear methoxy groups at R2 and R3
positions.
Considering the benzothiopyranoindole derivatives (compounds 5a-l) an important role seems to be
played by the group at the R2-position. Indeed, the substitution of the hydrogen with a methoxy
group at this position induces a significant increase in cytotoxicity, as demonstrated by comparing
the antiproliferative activity of 5a and 5b with that exerted by 5g and 5h, respectively. Interestingly,
for compound 5g an increase in cytotoxicity of about 7-fold with respect to the congener 5a was
observed in all three human tumor cell lines taken into consideration. The addition of a further
methoxy substituent at R4 does not influence appreciably the antiproliferative profile of both 5g and
5h, as their analogues 5k and 5l show comparable GI50 values.
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Table 1. Antiproliferative activity of the new compounds 5a-t and ellipticine taken as reference.
GI50 (µM)
A-431
n
X
R1
R2
R3
R4
HeLa
MSTO-211H
5a
CH
CH2CH2N(CH3)2
H
OCH3
H
5.81± 0.59
2.24± 1.68
2.37± 0.30
5b
5c
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
CH2CH2N(CH2CH3)2
CH2CH2N(CH3)2
H
H
OCH3
OCH3
OCH3
Cl
H
Cl
Cl
Cl
Cl
H
2.79 ± 0.71
2.79 ± 0.95
3.21 ± 0.26
2.66 ± 0.74
4.31 ± 1.15
0.82 ± 0.04
1.77 ± 0.32
3.54 ± 0.41
4.55 ± 0.39
2.08 ± 0.09
2.67 ± 0.46
5.44 ± 0.48
1.76 ± 0.68
3.29 ± 1.71
0.33 ± 0.06
1.11 ± 0.38
3.19 ± 0.56
2.22 ± 0.80
0.48 ± 0.10
0.67 ± 0.14
0.54 ± 0.04
0.47 ± 0.02
1.42 ± 0.26
1.90 ± 0.21
2.00 ± 0.35
1.79 ± 0.05
0.71 ± 0.28
2.67 ± 1.66
0.65 ± 0.15
1.61 ± 0.28
2.96 ± 0.05
4.19 ± 0.14
1.64 ± 0.55
2.99 ± 0.11
0.31 ± 0.03
0.70 ± 0.05
1.82 ± 0.61
2.37 ± 0.48
0.44 ± 0.15
0.36 ± 0.05
1.08 ± 0.07
1.33 ± 0.06
0.75 ± 0.27
0.70 ± 0.06
2.36 ± 0.46
1.42 ± 0.09
0.35 ± 0.03
0.62 ± 0.01
0.77 ± 0.14
5d
CH2CH2N(CH2CH3)2
CH2CH2N(CH3)2
H
5e
H
5f
CH2CH2N(CH2CH3)2
CH2CH2N(CH3)2
H
Cl
5g
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
5h
CH2CH2N(CH2CH3)2
CH2CH2N(CH3)2
H
5i
Cl
Cl
5j
CH2CH2N(CH2CH3)2
CH2CH2N(CH3)2
5k
OCH3 0.74 ± 0.13
OCH3 1.75 ± 0.45
5l
CH2CH2N(CH2CH3)2
CH2CH2N(CH3)2
5m
5n
H
H
H
H
H
H
H
H
1.55 ± 0.05
2.57 ± 0.21
2.71 ± 0.53
2.61± 1.04
3.02 ± 0.82
4.20 ± 0.36
1.70 ± 0.01
6.93 ± 1.14
0.47 ± 0.12
N
CH2CH2N(CH2CH3)2
CH2CH2N(CH3)2
H
H
5o
N
H
OCH3
OCH3
Cl
5p
N
CH2CH2N(CH2CH3)2
CH2CH2N(CH3)2
H
5q
N
H
5r
N
CH2CH2N(CH2CH3)2
CH2CH2N(CH3)2
H
Cl
5s
N
OCH3
OCH3
OCH3
OCH3
5t
N
CH2CH2N(CH2CH3)2
Ellipticine
Nevertheless, the R2 position lacks to be determinant for cytotoxicity if a chlorine is present at R4.
Indeed, the insertion of a methoxy group at R2 in 5c and 5d to obtain 5i and 5j, does not induce any
appreciable change in the GI50 values. It could be speculated that the presence of the chlorine at R4
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provokes a detrimental effect, which prevails on the advantageous influence mediated by the
methoxy substituent at R2.
The comparison between the effect exerted by 5c and 5d with that of 5e and 5f, respectively,
highlighted no significant differences in cytotoxicity, suggesting for the R3 position a less relevant
role.
As regards the pyridothiopyranoindole derivatives 5m-t, the lower GI50 values are showed by the
unsubstituted 5m and by derivative 5s, characterized by the presence of two methoxy groups at R2
and R3 and by the dimethylaminoethyl side chain at R1. In particular, antiproliferative activity
induced by 5m, that is slightly decreased by the insertion of a methoxy group at R3 in compound
5o, is recovered when a further methoxy is present at R2-position, as in 5s. The detrimental effect of
the insertion of a chlorine inserted at position R3 appears confirmed also for pyridothiopyranoindole
subseries: in all three tested cell lines, the cytotoxic effect of 5q and 5r is lower with respect to that
obtained for 5m and 5n.
Finally, preliminary viability experiments performed on the non-tumorigenic mesothelial cells
Met5-A for some representative compounds, i.e. 5a, 5g, 5o and 5s, as expected, did not permit to
demonstrate any significant selectivity against tumor cells over the nonmalignant ones (data not
shown). These results are not surprising, as Topoisomerase poisons are all known to suffer from
limited tumor selectivity [23]. However, nowadays, a number of tumor delivering nanotechnologies
have been implemented and allow for a preferential tumor distribution (Trojan horse approach)
[23].
2.3 Evaluation of Cell Death
To investigate the cell death pathway, cytofluorimetric evaluations on HeLa cells labeled with
Annexin V and propidium iodide, and treated for 18 h with the most cytotoxic
benzothiopyranoindole 5g were performed. The results are shown as dot plots in Figure 1. The
incubation in the presence of 5g causes a dose-dependent decrease in viability and a concurrent
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increase in apoptotic cells. In detail, the percentage of viable cells diminishes from 91.6% (control)
to less than 50% at 80 µM concentration. Accordingly, the percentage of apoptotic cells, both early
and late apoptosis, increases from 7.8% to about 48% in the same experimental conditions.
Figure 1. Dual parameter cytogram of FITC-annexin V vs propidium iodide staining. HeLa cells
were incubated for 18 h in the absence (control) and in the presence of 5g at 20 and 80 µM
concentration as indicated.
2.4
Interaction with DNA
To investigate the mechanism of action responsible for the cytotoxic activity of the new
benzothiopyranoindole and pyridothiopyranoindole derivatives 5a-t, linear flow dichroism (LD)
experiments were performed. As an example, the LD spectra of an aqueous solution of salmon
testes DNA alone and in the presence of 5g and 5s at different [drug]/[DNA] ratios are shown in
Figure 2A and 2B, respectively. Compounds 5g and 5s were selected as representatives of the
benzothiopyranoindole and pyridothiopyranoindole subseries, respectively, in virtue of their high
antiproliferative activity (Table 1). The LD spectra of DNA in the presence of ellipticine were
reported as reference (Figure 2C).
The DNA LD spectrum shows the typical negative dichroic signal at 260 nm (lines a). In the
presence of test compounds at 0.02 and 0.04 [drug]/[DNA] ratios (lines b and c, respectively), the
occurrence of a negative dichroic signal at wavelengths higher (330-450 nm) than that due to DNA
bases indicates the ability of both the benzothiopyranoindole and pyridothiopyranoindole
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chromophores to form a molecular complex with DNA. Moreover, the negative sign of this LD
induced signal suggests an orientation of the planar chromophore parallel to the plane of DNA
bases, i.e. an intercalative mode of binding, in agreement with the spectra of DNA in the presence
of ellipticine, a well-known intercalative compound (Figure 2C). Similar behaviours have been
observed for all the new derivatives (Figures S2-S5, Supporting Information, SI).
B
A
0.0
-0.1
-0.2
-0.3
0.00
-0.05
-0.10
-0.15
-0.20
-0.25
-0.30
-0.35
0.00
-0.01
-0.02
-0.03
-0.04
0.00
-0.01
-0.02
-0.03
-0.04
300 350 400 450 500
wavelength (nm)
300 350 400 450 500
wavelength ((nm)
250 300 350 400 450 500
wavelength (nm)
250 300 350
400 450 500
wavelength (nm)
C
0.00
-0.05
-0.10
-0.15
-0.20
-0.25
-0.30
-0.35
0.000
-0.004
-0.008
-0.012
300 350 400 450 500
wavelength (nm)
250 300 350 400 450 500
wavelength (nm)
Figure 2. Linear flow dichroism (LD) spectra for compound 5g (A), 5s (B) and ellipticine (C) at
[drug]/[DNA] ratios = 0.00 (a), 0.02 (b) and 0.04 (c). [DNA]=1.9 x10-3M in ETN buffer.
2.5
Effect on Topoisomerase Activity
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Considering that DNA Topoisomerases constitute the molecular intracellular target of many
anticancer drugs, including known DNA intercalators, such as adriamycin, m-amsacrine (m-AMSA)
and mitoxantrone [5,18], the effect of the new derivatives on the relaxation of supercoiled plasmid
pBR322 DNA mediated by both Topo II and Topo I was also investigated.
In particular, Figure 3A and 3B report the effect of 5g and 5s on the relaxation of pBR322
supercoiled DNA catalyzed by Topo II, respectively. Both derivatives are able to inhibit the
formation of topoisomers catalyzed by the enzyme from supercoiled DNA (DNA). This effect is
concentration-dependent and both derivatives are able to induce a practically complete inhibitory
effect at 50 µM concentration.
Figure 3. Effect on relaxation of supercoiled pBR322 DNA mediated by Topo II. Supercoiled DNA
(DNA) was incubated with Topo II in the absence (topo II) and presence of 5g (A) or 5s (B) at
indicated concentrations (µM); 10 µM m-AMSA was used as reference.
A similar behavior was also observed by incubating Topo I with the same derivatives 5g and 5s
(Figure 4A and 4B, respectively). In particular, by increasing the concentration of the compounds, a
dose-dependent decrease in the number of topoisomers appears.
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Figure 4. Effect on relaxation of supercoiled pBR322 DNA by human recombinant Topo I.
Supercoiled DNA (DNA) was incubated with Topo I in the absence (topo I) and presence of 5g (A)
and 5s (B) at indicated concentrations (µM).
It is to underline that all the new derivatives, both benzothiopyranoindoles (5a-l) and
pyridothiopyranoindoles (5m-t) showed a concentration-dependent ability to inhibit the relaxation
activity of both Topo II and Topo I (data not shown), suggesting a possible relationship with the
shared intercalative capacity. Thus, it seems reasonable to assume that the intercalation of the planar
tetracyclic chromophore inside base pairs could interfere with the ability of both enzymes to relax
supercoiled DNA.
The most clinically relevant intercalative anticancer drugs are able to affect the catalytic cycle of
topoisomerases by stabilizing a covalent intermediate, named cleavable complex, thus converting
topoisomerase into a cellular poison. Notably, this ability, also referred as poisoning effect, is
retained of crucial importance for the anticancer properties of both anti-Topo II (e.g. doxorubicin
and m-AMSA) and anti-Topo I (e.g. 2 and 3) drugs.
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In this connection, further experiments were performed to verify the ability of the new derivatives
to promote the formation of the cleavable complex, in the presence of both Topo II and Topo I.
From an experimental point of view, the cleavable complex can be evidenced by the enzyme-
mediated formation of linear (Topo II) or nicked (Topo I) DNA from supercoiled one in the
presence of test compounds.
Figure 5 shows the results of the cleavable assay performed in the presence of Topo II incubated
with the benzothiopyranoindole 5g and the pyridothiopyranoindole 5s. The well-known Topo II
poison m-AMSA was used as reference compound. Both compounds are unable to induce any
poison effect up to 50 µM concentration, as demonstrated by the absence of linear DNA, which is
otherwise detectable in the presence of the reference drug already at 10 µM. Similar results were
obtained also for the other benzothiopyranoindole and pyridothiopyranoindole derivatives (data not
shown).
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Figure 5. Effect on the stabilization of covalent DNA-Topo II complex. Supercoiled pBR322 DNA
(DNA) was incubated with Topo II in the absence (topo II) and presence of 5g (A) or 5s (B) at
indicated concentration (µM); 10 µM m-AMSA was used as reference drug.
Figure 6 reports the results from the cleavable assay performed with Topo I in the presence of 5g
(A) and 5s (B), and the structurally related analogues 5h (A) and 5t (B), respectively. Compound 1
(CPT) was used as reference. The cleavable complex can be evidenced by the enzyme-mediated
formation of nicked DNA from supercoiled one in the presence of test compounds. As expected, 1
induces the formation of a significant amount of nicked DNA, i.e. stabilizes the cleavable complex.
Figure 6. Effect on the stabilization of covalent DNA-Topo I complex. Supercoiled pBR322 DNA
(DNA) was incubated with Topo I in the absence (topo I) and presence of 5g and 5h (A), 5s and 5t
(B) at indicated concentration (µM). 0.5 µM 1 (CPT) was used as reference.
Notably, also in the presence of 5g and 5s a dose-dependent poisoning effect is observed, as
demonstrated by the occurrence of nicked DNA. Interestingly, the evaluation of the effect on the
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stabilization of cleavable complex performed with all the new derivatives highlighted this ability
also for the corresponding analogues 5h and 5t, carrying the same methoxy substituents at R2 and
R3, but the diethylaminoethyl side chain at R1 (Figure 6A and 6B, respectively). It is noteworthy
that all the other benzothiopyranoindole and pyridothiopyranoindole derivatives appear unable to
exert any poisoning effect on Topo I (results not shown).
2.6
Molecular Modeling Studies
The analysis of human Topo I covalently bound to duplex DNA and in complex with 2 (PDB code:
1K4T) [33] or other inhibitors (SA315F, MJ238, AI-III-52, PDB codes: 1SEU, 1SC7, 1TL8),
revealed that there are certain strictly conserved contacts between the known ligands and the
protein, that can be either direct- or water-mediated- interactions. For this reason, we decided to run
docking calculations explicitly considering the water molecules, using the Autodock4.2 software
(AD4) [34]. In this regard, AD4 has been implemented with a new force field and with a hydration
docking method able to predict waters mediating ligand binding [35]. Such a protocol has been
already used by us to successfully reproduce the experimental binding conformation of 2 and to
rationalize the Topo I inhibitory activity of pyrazoloquinazoline derivatives [29]. The poisons 5g,
5h, 5s and 5t were docked in the high resolution (2.10 Å) crystal structure of the human Topo I in
complex with duplex DNA/2 (PDB code: 1K4T, see Experimental Section for the selection criteria)
[33] after removal of the poison. Analyzing, for each docked molecule, the lowest energy binding
pose in the most populated family, it emerges that 5g, 5h, 5s and 5t docks similarly. All the
obtained complexes were energy minimized as to allow the water molecules to adjust and the ligand
to optimize its geometries of interactions, and the protein to remove its internal bad contacts.
Particularly, 5g (likewise 5h, 5s and 5t) docks at the DNA cleavage site (Figure 7), where its
benzothiopyrano fragment stacks with the non-cleaved strand bases (+1C and -1A) and the indole
ring stacks with the scissile strand site of the duplex DNA (+1G and -1T bases) similarly to other
CPT-like Topo I poisons. Noteworthy, a water bridge is found between the R3-methoxy oxygen and
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the K751 side chain and two H-bonds are detected: one between the 5g carbonyl oxygen and the
Topo I R364 side chain, and another between the 5g R2-methoxy oxygen and the amide of the N722
side chain. These two H-bonds are strictly conserved among the CPTs, indolocarbazoles, and
indenoisoquinolines poisons and, together with the extensive stacking interactions, are crucial for an
efficient bonding to the DNA-Topo I cleavage complex. In fact, mutations of the above mentioned
residues are known to confer resistance to CPT-like [36] and non-CPT-like poisons [37-39]. The
importance of these H-bonds would be in line with the inability to exert a poisoning effect of our
analogues 5a-5f and 5m-5r, which, lacking of a H-bond acceptor at the R2 position, are not able to
establish a contact with the N722 side chain. Furthermore, in the docking pose predicted for 5g (and
5s), the R1-dimethylaminoethyl branch is found in a small and rather shallow protein pocket in
proximity of the catalytic phosphotyrosine (pY) and the DNA phosphodiester backbone, where the
5g protonated amino group is able to establish a water-mediated contact with the D533 side chain (2
establishes a direct interaction). The size of the pocket is sufficient to fit the R1-diethylaminoethyl
moiety of the 5h and 5t derivatives, which are still able to establish the same water-mediated
interaction. However, given the lower cytotoxicity exerted by these two analogues on the three
tumor cell lines used in our assays, it seems that the larger diethylamino group (5h and 5t) would be
less tolerated with respect to the dimethylamino one, although other in-cell effects could also
explain the observed results.
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Figure 7. A zoomed-out (a) and zoomed-in (b) view of the minimized putative binding mode of 5g
(golden sticks) into the DNA (green ribbon) cleavage site of Topo I (blue ribbon). The protein
interacting residues and the DNA bases are depicted as light-blue and light-green sticks,
respectively. H-bonds are depicted as dashed black lines.
The results presented herein can also explain the catalytic inactivity of compounds 5i-5l. In fact,
according to the described binding pose, the R4 substituent of these derivatives is oriented toward
the non-scissile DNA strand, where several evidences indicate that a steric hindrance in the ligand is
extremely poorly tolerated (see also Figure S1, Supporting Information) [40]. Molecular docking of
a R4-substituted-compound, like 5k, confirmed in fact, that the ligand is not able to accommodate
into the binding pocket anymore and none of the aforementioned conserved H-bonds can be
established.
In conclusion, the theoretical model provided by hydrated docking calculations well clarified the
molecular determinants that translate DNA intercalation into Topo I poison activity. Importantly, it
has been revealed that only compounds possessing a H-bond acceptor at the R2-position can
effectively interact with the complex and that bulky substituents at the R4-position do not allow a
proper accommodation. The identification of these structural requirements can be useful for the
future development of novel series of antiproliferative agents acting as Topo I poisons.
3. Conclusions
As a continuation of our studies aimed to identify new antiproliferative agents, novel
benzothiopyranoindole (5a-l) and pyridothiopyranoindole (5m-t) derivatives featuring different
substitution patterns at R2-R4 positions and protonatable dialkylaminoalkyl chains at R1-position,
were synthesized and biologically evaluated on a panel of three human tumor cell lines, showing
evident cytotoxic (GI50 values in the low micromolar/submicromolar range) and pro-apoptotic
effects. Further investigations revealed that the tetracyclic chromophores allow a complexation with
DNA through an intercalative mode of binding and this ability could be considered responsible for
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the capacity of all derivatives to inhibit the DNA relaxation activity mediated by both Topo II and
Topo I. More interestingly, the presence of a methoxy group at R2-position of both chromophores
seems to play a crucial role in the ability to stabilize the cleavable complex formed by the catalytic
activity of Topo I, conferring Topo I poison properties. A theoretical model provided by hydrated
docking calculations clarified the molecular determinants responsible for the Topo I poison activity,
rationalizing that only compounds possessing a H-bond acceptor at the R2-position can effectively
interact with the complex and that bulky substituents at the R4-position do not allow a proper
accommodation.
The results obtained so far highlight the benzothiopyranoindole and the isoster
pyridothiopyranoindole systems as excellent scaffolds for the development of antiproliferative
agents, and furnish crucial indications about the structural requirements suitable for the future
design of novel efficacious Topo I poisons.
4. Experimental protocols
4.1. Chemistry
1
The uncorrected melting points were determined using a Reichert Köfler hot-stage apparatus. H
NMR and 13C NMR spectra were recorded on a Varian Gemini 200 spectrometer (1H, 200 MHz) in
13
dimethyl-d6 sulfoxide or on a Bruker AVANCE 400 (1H, 400 MHz; C, 100 MHz) in CDCl3. The
coupling constants (J) are given in Hertz. Accurate molecular weights of compounds were
confirmed by ESI mass spectrometry using a ThermoFisher Scientific Orbitrap XL mass
spectrometer in electrospray positive ionization modes (ESI-MS). Magnesium sulphate was used as
the drying agent. Evaporations were made in vacuo (rotating evaporator). Analytical TLC have
been carried out on Merck 0.2 mm precoated silica gel aluminium sheets (60 F-254). Silica gel 60
(230-400mesh) was used for column chromatography. Purity of the target inhibitors was
determined, using a Shimadzu LC-20AD SP liquid chromatograph equipped with a DDA Detector
at 288 nm and 340 nm (column C18 (250 mm-4.6 mm, 5 µm, Shim-pack)). The mobile phase,
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delivered at isocratic flow, consisted of acetonitrile (70–90%) and water (30–10%) and a flow rate
of 1.0 mL/min. All the compounds showed percent purity values of ≥ 95%. Reagents, starting
materials, and solvents were purchased from commercial suppliers and used as received. The
following compounds were obtained according to methods previously described: 3-
(hydroxymethylene)-2H-thiopyran-4(3H)-one 6a [31], 3-(hydroxymethylene)-7-methoxy-2H-
thiopyran-4(3H)-one 6b, and 3-(hydroxymethylene)-7-chloro-2H-thiopyran-4(3H)-one 6c [30], 2,3-
dihydro-3-(hydroxymethylene)-thiopyrano[2,3-b]pyridin-4(4H)-one, 6d, 3–(2-phenylhydrazono)-
2H-thiopyrano[2,3-b]pyridin-4(3H)-one
8g,
3–[2-(4-methoxyphenyl)hydrazono]-2H-
thiopyrano[2,3-b]pyridin-4(3H)-one 8h, 3–[2-(4-chlorophenyl)hydrazono]-2H-thiopyrano[2,3-
b]pyridin-4(3H)-one 8i, pyrido[3',2':5,6]thiopyrano[3,2-b]indol-5(6H)-one 9g, 9-methoxy-
pyrido[3',2':5,6]thiopyrano[3,2-b]indol-5(6H)-one, 9h and 9-chloro-pyrido[3',2':5,6]thiopyrano[3,2-
b]indol-5(6H)-one, 9i [27].
4.1.1. General synthetic procedure for 7-substituted-3-(2-phenylhydrazono)-2H-benzothiopyran-
4(3H)-ones 8a-f and 3–[2-(3,4-dimethoxyphenyl)hydrazono]thiopyrano[2,3-b]pyridin-4(3H)-one
8j.
To a solution of 4.5 mmol of the appropriate compound 6a-d in 30 mL of methanol was added an
aqueous saturated solution of 14.0 mmol of sodium acetate. After cooling at 0 °C, a solution of the
suitable diazonium salt, obtained from the appropriately substituted aniline 7a-d in 18%
hydrochloridric acid and sodium nitrite, was added dropwise in slight excess (6.0 mmol). An orange
precipitate was immediately formed, and the mixture was stirred for 30 min at room temperature.
The solid was collected and washed with water to give compounds 8a-f and 8j.
4.1.1.1. 3-[2-(4-Methoxyphenyl)hydrazono]-2H-benzothiopyrano-4(3H)-one (8a).Yield, 75 %; m. p.
123-125 °C; 1H-NMR (200 MHz, DMSO-d6): δ 3.77 (s, 3H), 4.09 (s, 2H), 6.76-6.83 (m, 3H), 6.87-
6.91 (m, 3H), 7.01 (d, J = 8.4 Hz, 2H), 13.81 (s, 1H, NH exch.) ppm.
4.1.1.2. 7-Chloro-3-[2-(4-methoxyphenyl)hydrazono]-2H-benzothiopyrano-4(3H)-one (8b). Yield,
1
70 %; m. p. 136-138 °C; H-NMR (200 MHz, DMSO-d6): δ 3.74 (s, 3H), 4.14 (s, 2H), 6.93-6.97
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(m, 2H), 7.29-7.43 (m, 3H), 7.61 (d, J = 2.4 Hz, 1H), 7.97 (d, J = 8.4 Hz, 1H), 13.82 (s, 1H, NH
exch.) ppm.
4.1.1.3. 7-Chloro-3-[2-(4-chlorophenyl)hydrazono]-2H-benzothiopyrano-4(3H)-one (8c). Yield, 80
%; m. p. 168-170 °C; 1H-NMR (200 MHz, DMSO-d6): δ 4.11 (s, 2H), 6.91-6.99 (m, 2H), 7.33-7.36
(m, 3H), 7.53 (d, J = 2.2 Hz, 1H), 7.97 (d, J = 8.4 Hz, 1H), 13.45 (s, 1H, NH exch.) ppm.
4.1.1.4. 3-[2-(3,4-Dimethoxyphenyl)hydrazono]-2H-benzothiopyrano-4(3H)-one (8d). Yield, 80%;
m. p. 129-131 °C; 1H-NMR (200 MHz, DMSO-d6): δ 3.78 (s, 3H), 3.84 (s, 3H), 4.10 (s, 2H), 6.76-
6.83 (m, 3H), 6.87-6.91 (m, 3H), 7.18 (s, 1H), 13.84 (s, 1H, NH exch.) ppm.
4.1.1.5. 7-Chloro-3-[2-(3,4-dimethoxyphenyl)hydrazono]-2H-benzothiopyrano-4(3H)-one (8e).
Yield, 65 %; m. p. 142-144 °C; 1H-NMR (200 MHz, DMSO-d6): δ 3.75 (s, 3H), 3.80 (s, 3H), 4.16
(s, 2H), 6.93-6.97 (m, 2H), 7.16 (s, 1H), 7.42 (dd, Jmin = 2.0 Hz, Jmax = 8.2 Hz, 1H), 7.63 (s, 1H),
7.98 (d, J = 8.6 Hz, 1H), 13.83 (s, 1H, NH exch.) ppm.
4.1.1.6. 7-Methoxy-3-[2-(3,4-dimethoxyphenyl)hydrazono]-2H-benzothiopyrano-4(3H)-one (8f).
1
Yield, 60 %; m. p. 138-140 °C; H-NMR (200 MHz, DMSO-d6): δ 3.73 (s, 3H), 3.81 (s, 3H), 3.86
(s, 3H), 4.09 (s, 2H), 6.81-6.94 (m, 4H), 7.67 (s, 1H), 7.90 (d, J = 8.4 Hz, 1H), 11.52 (s, 1H, NH
exch.) ppm.
4.1.1.7. 3–[2-(3,4-Dimethoxyphenyl)hydrazono]-2H-thiopyrano[2,3-b]pyridin-4(3H)-one (8j).
1
Yield, 60 %; m.p. 174-176 °C; H-NMR (200 MHz, DMSO-d6): δ 3.73 (s, 3H), 3.79 (s, 3H), 4.22
(s, 2H), 6.95-7.00 (m, 2H), 7.16 (s, 1H), 7.35-7.39 (m, 1H), 8.27 (dd, Jmin = 1.8 Hz, Jmax = 7.8 Hz,
1H), 8.57 (dd, Jmin = 1.6 Hz, Jmax = 4.6 Hz, 1H), 13.80 (s, 1H, NH exch.) ppm.
4.1.2. General synthetic procedure for substituted-benzothiopyrano[3,2-b]indol-10(11H)-ones 9a-f
and 8,9-dimethoxypyrido[3',2':5, 6]thiopyrano[3,2-b]indol-5(6H)-one 9j.
A solution of 4.0 mmol of the suitable substituted phenylhydrazone derivatives 8a-f, 8j in 20 mL of
ethanolic hydrogen chloride solution was refluxed for 20 min. After cooling the yellow/light-brown
precipitate was collected and washed with ethanol to give crude indoles 9a-f, 9j, which were
purified by recrystallization from DMF.
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4.1.2.1. 3-Methoxybenzothiopyrano[3,2-b]indol-10(11H)-one (9a). Yield, 70 %; m. p. > 300 °C;
1H-NMR (200 MHz, DMSO-d6): δ 3.84 (s, 3H), 7.16 (d, J = 9.0 Hz, 1H), 7.33 (s, 1H); 7.52 (d, J =
9.0 Hz, 1H), 7.62-7.76 (m, 2H), 8.01 (d, J = 8.4 Hz, 1H), 8.59 (d, J = 8.4 Hz, 1H), 12.39 (s, 1H, NH
exch.) ppm.
4.1.2.2. 7-Chloro-3-methoxybenzothiopyrano[3,2-b]indol-10(11H)-one (9b). Yield, 65 %. m. p. >
1
300 °C; H-NMR (200 MHz, DMSO-d6): δ 3.86 (s, 3H), 7.17 (dd, Jmin = 2.2 Hz, Jmax = 9.0
Hz, 1H), 7.33 (d, J = 2.0 Hz, 1H), 7.53 (d, J = 9.0 Hz, 1H), 7.67 (dd, Jmin = 2.2 Hz, Jmax
=
8.8 Hz, 1H), 8.25 (d, J = 1.8 Hz, 1H), 8.57 (d, J = 8.8 Hz, 1H), 12.47 (s, 1H, NH exch.)
ppm.
4.1.2.3. 3,7-Dichlorobenzothiopyrano[3,2-b]indol-10(11H)-one (9c). Yield, 40 %; m. p. > 300 °C.
1H-NMR (200 MHz, DMSO-d6): δ 7.51-7.71 (m, 3H), 8.09 (s, 1H), 8.30 (d, J = 1.8 Hz, 1H),
8.57 (d, J = 8.4 Hz, 1H), 12.79 (s, 1H, NH exch.) ppm.
4.1.2.4. 2,3-Dimethoxybenzothiopyrano[3,2-b]indol-10(11H)-one (9d). Yield, 75 %; m. p. > 300
1
°C; H-NMR (200 MHz, DMSO-d6): δ 3.87 (s 6H), 7.02 (s, 1H), 7.32 (s, 1H), 7.58-7.66 (m,
1H), 7.70-7.78 (m, 1H), 7.99 (d, J = 7.2 Hz, 1H), 8.59 (dd, Jmin = 1.2 Hz. Jmax = 7.8 Hz,
1H), 12,32 (s, 1H, NH exch.) ppm.
4.1.2.5. 7-Chloro-2,3-dimethoxybenzothiopyrano[3,2-b]indol-10(11H)-one (9e). Yield, 60 %.; m. p.
> 300 °C; 1H-NMR (200 MHz, DMSO-d6): δ 3.89 (s, 6H), 7.03 (s, 1H), 7.33 (s, 1H), 7.67 (dd,
Jmin = 2.0 Hz, Jmax = 8.8 Hz, 1H), 8.25 (d, J = 8.8 Hz, 1H), 8.57 (d, J = 9.0 Hz, 1H), 12.39
(s, 1H, NH exch.) ppm.
4.1.2.6. 2,3,7-Trimethoxybenzothiopyrano[3,2-b]indol-10(11H)-one (9f). Yield, 80 %; m. p. > 300
°C; 1H-NMR (200 MHz, DMSO-d6): δ 3.87(s, 6H), 3.94 (s, 3H), 7.02 (s, 1H), 7.23 (dd, Jmin = 2.4
Hz, Jmax = 8.8 Hz, 1H), 7.26 (s, 1H), 7.48 (d, J = 2.4 Hz, 1H), 8.50 (d, J = 8.8 Hz, 1H), 12.20
(s, 1H, NH exch.) ppm.
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4.1.2.7. 8,9-Dimethoxypyrido[3',2':5,6]thiopyrano[3,2-b]indol-5(6H)-one (9j). Yield, 55 %; m. p. >
1
300 °C. H-NMR (200 MHz, DMSO-d6): δ 3.88 (s 6H), 7.03 (s, 1H), 7.38 (s, 1H), 7.68 (dd,
Jmin = 4.8 Hz, Jmax = 7.8 Hz, 1H), 8.86-8.90 (m, 2H), 12.42 (s, 1H, NH exch.) ppm.
4.1.3. General synthetic procedure for substituted-11-dialkylaminoalkyl-benzothiopyrano
indol-10 11H -ones 5a-l and substituted 6-dialkylaminoalkyl-pyrido[3',2':5,6]thiopyrano[3,2-
b]indol-5(6H)-ones 5m-t.
[3,2-
b
]
(
)
The appropriate compound 9a-j (0.70 mmol) was added in small portions to a stirred suspension of
sodium hydride (60% dispersion in mineral oil, 0.104 g, 2.60 mmol) in 10 mL of anhydrous DMF,
under nitrogen atmosphere. The reaction mixture was stirred at room temperature for 2 h and then
was supplemented with 1.03 mmol of the appropriate dialkylaminoalkyl chloride hydrochloride and
heated at 100 °C for 12-24 h (TLC analysis). After cooling, the obtained suspension was added with
water and left to stand at room temperature overnight. The crude products precipitated were
collected and purified by recrystallization from DMF.
4.1.3.1. 11-[2-(Dimethylamino)ethyl]-3-methoxybenzothiopyrano[3,2-b]indol-10(11H)-one (5a).
Yield, 30 %; m. p. 107-109 °C; 1H NMR (400 MHz, CDCl3): δ 2.17 (s, 6H), 2.53 (t, J = 6.6 Hz,
2H), 3.91 (s, 3H), 5.03 (t, J = 6.6 Hz, 2H), 7.17 (d, J = 2.4 Hz, 1H), 7.24-7.27 (m, 1H),
7.53-7.59 (m, 2H), 7.64 (td, Jmin = 1.4 Hz, Jmax = 8.0 Hz, 1H), 7.77 (d, J = 8.0 Hz, 1H),
13
8.67 (dd, Jmin = 1.6 Hz, Jmax = 8.0 Hz, 1H) ppm; C-NMR (100 MHz, CDCl3): δ 42.86, 43.23
(2C), 55.68, 56.05, 101.04, 112.03, 119.41, 120.50, 123.09, 126.20, 127.02, 127.61, 128.88, 130.98,
132.40, 134.70, 136.05, 155.06, 173.35 ppm; HRMS (ESI) m/z calculated for
C20H21N2O2S([M+H]+) 353.1324, found: 353.1307. Anal. C20H20N2O2S (C, H, N).
4.1.3.2. 11-[2-(Diethylamino)ethyl]-3-methoxybenzothiopyrano[3,2-b]indol-10(11H)-one (5b).
Yield, 45 %; m. p. 103-105 °C; 1H NMR (400 MHz, CDCl3): δ 1.12 (t, J = 7.2 Hz, 6H), 2.75 (q,
J = 7.2 Hz, 4H), 2.69 (t, J = 6.4 Hz, 2H), 3.92 (s, 3H), 5.03 (t, J = 6.4 Hz, 2H), 7.15 (d, J
= 2.0 Hz, 1H), 7.22 (dd, Jmin = 2.0 Hz, Jmax = 9.2 Hz, 1H), 7.51-7.61 (m, 3H), 7.74 (dd,
25

ACCEPTED MANUSCRIPT
Jmin = 0.8 Hz, Jmax = 8.0 Hz, 1H), 8.72 (dd, Jmin = 1.2 Hz, Jmax = 8.0 Hz, 1H) ppm; 13C-
NMR (100 MHz, CDCl3): δ 14.67 (2C), 42.83 (2C), 51.08, 55.61, 56.09, 100.71, 112.50, 119.36,
120.03, 123.02, 126.07, 127.01, 128.16, 129.08, 130.83, 132.65, 135.13, 136.09, 154.82, 173.33
ppm; HRMS (ESI) m/z calculated for C22H25N2O2S([M+H]+) 381.1637, found: 381.1621. Anal.
C22H24N2O2S (C, H, N).
4.1.3.3. 11-[2-(Dimethylamino)ethyl]-7-chloro-3-methoxybenzothiopyrano[3,2-b]indol-10(11H)-
one (5c). Yield, 30 %; m. p. 99-101 °C; 1H NMR (400 MHz, CDCl3): δ 2.30 (s, 6H), 2.45 (t, J =
7.2 Hz, 2H), 3.92 (s, 3H), 4.89 (t, J = 7.4 Hz, 2H), 7.11 (d, J = 2.4 Hz 1H), 7.20 (dd, Jmin
= 2.4 Hz, Jmax = 9.2 Hz, 1H), 7.45 (dd, Jmin = 2.0 Hz, Jmax = 8.8 Hz 1H), 7.53 (d, J = 9.2
Hz, 1H), 7.71 (d, J = 2Hz, 1H), 8.61 (d, J = 8.8 Hz, 1H) ppm; 13C-NMR (100 MHz, CDCl3):
δ 43.73, 45.14 (2C), 56.00, 56.41, 100.40, 112.37, 117.59, 119.42, 120.08, 122.71, 126.00, 126.65,
127.62, 130.58, 131.09, 135.09, 137.32, 154.78, 173.31 ppm; HRMS (ESI) m/z calculated for
C20H20ClN2O2S ([M+H]+) 387.0934, found: 387.0911. Anal. C20H19ClN2O2S (C, H, N).
4.1.3.4. 11-[2-(Diethylamino)ethyl]-7-chloro-3-methoxybenzothiopyrano[3,2-b]indol-10(11H)-one
1
(5d). Yield, 35 %; m. p. 95-97 °C; H NMR (400 MHz, CDCl3): δ 0.78 (t, J = 7.2 Hz, 6H),
3.06-3.09 (q, 4H), 3.10 (t, J = 6.6 Hz, 2H), 3.89 (s, 3H), 5.23 (t, J = 6.6 Hz, 2H); 7.10 (d,
J = 2.0 Hz, 2H), 7.47 (dd, Jmin = 2.0 Hz, Jmax = 8.8 Hz, 2H), 7.73 (d, J = 1.6 Hz, 1H), 8.60
(d, J = 8.8 Hz, 1H) ppm; 13C-NMR (100 MHz, CDCl3): δ 14.61 (2C), 47.03 (2C), 51.47, 56.00,
56.47, 100.56, 112.71, 117.83, 119.24, 120.15, 122.36, 126.17, 126.86, 127.43, 130.38, 131.01,
134.87, 137.21, 154.63, 173.29 ppm; HRMS (ESI) m/z calculated for C22H24ClN2O2S ([M+H]+)
415.1247, found: 415.1234. Anal. C22H23ClN2O2S (C, H, N).
4.1.3.5. 11-[2-(Dimethylamino)ethyl]-3,7-dichlorobenzothiopyrano[3,2-b]indol-10(11H)-one (5e).
Yield, 40 %; m. p. 183-185 °C; 1H-NMR (400 MHz, CDCl3): δ 2.99 (s, 6H), 3.51 (t, J = 6.4 Hz,
2H), 5.44 (t, J = 6.6 Hz, 2H), 7.46-7.54 (m, 2H), 7.74-7.83 (m, 2H), 8.13 (d, J = 9.0 Hz,
1H), 8.58 (d, J = 9.0 Hz, 1H) ppm; 13C-NMR (100 MHz, CDCl3): δ 43.64, 45.71 (2C), 57.03,
26

ACCEPTED MANUSCRIPT
100.89, 111.94, 117.98, 119.86, 121.02, 123.45, 126.33, 127.59, 128.45, 130.86, 131.15, 134,03,
137.17, 155.04, 172.81 ppm; HRMS (ESI) m/z calculated for C19H17Cl2N2OS ([M+H]+) 391.0439,
found: 391.0420. Anal. C19H16Cl2N2OS (C, H, N).
4.1.3.6. 11-[2-(Diethylamino)ethyl]-3,7-dichlorobenzothiopyrano[3,2-b]indol-10(11H)-one (5f).
Yield, 40 %; m. p. 137-139 °C; 1H NMR (400 MHz, CDCl3): δ 1.53 (t, J = 7.2 Hz, 6H), 3.27 (q,
J = 7.2 Hz, 4H), 3.44 (t, J = 6.4 Hz, 2H), 5.46 (t, J = 6.6 Hz, 2H), 7.50-7.520 (m, 2H),
7.60 (d, J = 8.4 Hz, 1H), 7.80 (d, J = 2.0 Hz, 1H), 8.26 (d, J = 8.4 Hz,1H), 8.60 (d, J = 9.0
Hz, 1H) ppm; 13C-NMR (100 MHz, CDCl3): δ 14.63 (2C), 47.70 (2C), 52.02, 57.14, 101.58,
113.70, 117.94, 119.79, 120.06, 123.64, 126.46, 127.40, 128.48, 130.45, 131.09, 134.05, 137.12,
154.83, 172.84 ppm; HRMS (ESI) m/z calculated for C21H21Cl2N2OS ([M+H]+) 419.0752, found:
419.0730. Anal. C21H20Cl2N2OS (C, H, N).
4.1.3.7.
11-[2-(Dimethylamino)ethyl]-2,3-dimethoxybenzothiopyrano[3,2-b]indol-10(11H)-one
1
(5g). Yield, 25 %; m. p. 120-122 °C; H NMR (400 MHz, CDCl3): δ 2.93 (s, 6H), 2.56 (t, J =
6.8 Hz, 2H), 4.01 (s, 3H), 4.19 (s, 3H), 5.42 (t, J = 6.8 Hz, 2H), 7.10 (s, 1H), 7.56 (t, J =
7.0 Hz, 1H), 7.64 (td, Jmin = 1.4 Hz, Jmax = 8.0 Hz, 1H), 7.76-7.79 (m, 2H); 8.66 (dd, Jmin
= 1.2 Hz, Jmax = 8.0 Hz, 1H) ppm; 13C-NMR (100 MHz, CDCl3): δ 41.30 (2C), 43.83, 56.52,
57.32 57.90, 93.91, 100.25, 115.16, 119.86, 126.34, 126.61, 127.05, 128.54, 130.84, 135,82,
135.81, 135.92, 146.97, 153.01, 172.23 ppm; HRMS (ESI) m/z calculated for C21H23N2O3S
([M+H]+) 383.1429, found: 383.1408. Anal. C21H22N2O3S (C, H, N).
4.1.3.8. 11-[2-(Diethylamino)ethyl]-2,3-dimethoxybenzothiopyrano[3,2-b]indol-10(11H)-one (5h).
Yield, 40 %; m. p. 105-107 °C; 1H NMR (400 MHz, CDCl3): δ 1.24 (t, J = 7.2 Hz, 6H), 2.91 (q,
J = 7.2 Hz, 4H), 3.12 (t, J = 7.2 Hz, 2H), 3.98 (s, 3H), 4.08 (s, 3H), 5.12 (t, J = 7.2 Hz,
2H), 7.07 (s, 1H), 7.34 (s, 1H), 7.51 (td, Jmin = 1.2 Hz, Jmax = 8.0 Hz, 1H), 7.58 (td, Jmin
=
1.4 Hz, Jmax = 8.0 Hz, 1H), 7.71 (d, J = 7.6 Hz, 1H), 8.68 (dd, Jmin = 1.4 Hz, Jmax = 8.2 Hz,
1H) ppm; 13C-NMR (100 MHz, CDCl3): δ 10.75 (2C), 47.40 (2C), 52.74, 56.50, 56.64, 57.26,
27

ACCEPTED MANUSCRIPT
93.50, 100.48, 115.42, 119.92, 126.18, 126.56, 127.02, 128.93, 130.70, 132.56, 135.72, 135.86,
146.62, 152.88, 172.29 ppm; HRMS (ESI) m/z calculated for C23H27N2O3S ([M+H]+) 411.1742,
found: 411.1725. Anal. C23H26N2O3S (C, H, N).
4.1.3.9.
11-[2-(Dimethylamino)ethyl]-7-chloro-2,3-dimethoxybenzothiopyrano[3,2-b]indol-
1
10(11H)-one (5i). Yield, 25 %; m. p. 170-172 °C; H NMR (400 MHz, CDCl3): δ 2.43 (s, 6H),
2.80 (t, J = 7.4 Hz, 2H), 3.98 (s, 3H), 4.02 (s, 3H), 4.95 (t, J = 7.4 Hz, 2H), 6.97 (s, 1H),
7.03 (s, 1H), 7.42 (dd, Jmin = 2.0 Hz, Jmax = 8.8 Hz, 1H), 7.65 (d, J = 2.0 Hz, 1H), 8.59 (d,
13
J = 8.8 Hz, 1H) ppm; C-NMR (100 MHz, CDCl3): δ 43.90, 45.77 (2C), 56.48, 56.55, 59.28,
92.58, 100.33, 115.29, 118.62, 125.85, 126.33, 126.55, 130.39, 130.96, 135.41, 136.91, 136.97,
146.43, 152.52, 171.29 ppm; HRMS (ESI) m/z calculated for C21H22ClN2O3S ([M+H]+) 417.1040,
found: 417.1020. Anal. C21H21ClN2O3S (C, H, N).
4.1.3.10.
11-[2-(Diethylamino)ethyl]-7-chloro-2,3-dimethoxybenzothiopyrano[3,2-b]indol-
1
10(11H)-one (5j). Yield, 30 %; m. p. 134-136 °C; H NMR (400 MHz, CDCl3): 1.52 (t, J = 7.2
Hz, 6H), 3.25 (q, J = 7.2 Hz, 4H), 3.46 (t, J = 7.4 Hz, 2H), 3.99 (s, 3H), 4.02 (s, 3H), 5.41
(t, J = 7.6 Hz, 2H), 7.05 (s, 1H), 7.48 (dd, Jmin = 2.0 Hz, Jmax = 8.8 Hz, 1H), 7.72 (d, J =
13
2.0 Hz, 1H), 7.89 (s, 1H), 8.59 (d, J = 8.8 Hz, 1H) ppm; C-NMR (100 MHz, CDCl3): δ 8.42
(2C), 40.85 (2C), 51.44, 56.59 (2C), 58.20, 94.28, 100.04, 115.14, 119,04, 125.94, 126.23, 126.99,
130.21, 131.04, 135.75, 137.29, 137.45, 147.17, 153.84, 171.39 ppm; HRMS (ESI) m/z calculated
for C23H26ClN2O3S ([M+H]+) 445.1353, found: 445.1334. Anal. C23H25ClN2O3S (C, H, N).
4.1.3.11. 11-[2-(Dimethylamino)ethyl]-2,3,7-trimethoxybenzothiopyrano[3,2-b]indol-10(11H)-one
1
(5k). Yield, 50 %; m. p. 143-145 °C; H NMR (400 MHz, CDCl3): δ 2.47 (s, 6H), 2.87 (t, J = 7.4
Hz, 2H), 3.92 (s, 3H), 3.98 (s, 3H), 4.03 (s, 3H), 4.99 (t, J = 7.6 Hz, 2H), 7.02-7.07 (m, 4H), 8.60
13
(d, J = 8.4 Hz, 1H) ppm; C-NMR (100 MHz, CDCl3): δ 43.74, 45.69 (2C), 55.86, 56.56, 56.68,
59.25, 92.77, 100.49, 108.95, 114.87, 115.44, 118.44, 126.45 (2C), 130.72, 135.12, 137.90, 146.29,
152.19, 161.31, 172.02 ppm; HRMS (ESI) m/z calculated for C22H25N2O4S ([M+H]+) 413.1535,
found: 413.1519. Anal. C22H24N2O4S (C, H, N).
28