Synthesis of podophyllotoxin linked β-carboline congeners as potential anticancer agents and DNA topoisomerase II inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.12.055

A series of new podophyllotoxin linked β-carboline congeners have been synthesized by coupling various substituted β-carboline acids with 4β-aminopodophyllotoxin. Evaluation of their anticancer activity against a panel of human cancer cell lines such as lung cancer (A549), prostate cancer (DU-145), MDA MB-231 (breast cancer), HT-29 (colon cancer) and HeLa (cervical cancer) suggested that 7i and 7j are the most cytotoxic compounds with IC₅₀ values of 1.07 ± 0.07 μM and 1.14 ± 0.16 respectively against DU-145 cell line. Further, detailed biological studies such as cell cycle analysis, topoisomerase II inhibition, Comet assay, DNA binding studies and docking studies have revealed that these congeners are DNA interacting topoisomerase II inhibitors.

Full text of DOI:10.1016/j.ejmech.2017.12.055

Accepted Manuscript
Synthesis of podophyllotoxin linked β-carboline congeners as potential anticancer
agents and DNA topoisomerase II inhibitors
Manda Sathish, Botla Kavitha, V. Laxma Nayak, Yellaiah Tangella, Ayyappan Ajitha,
Shalini Nekkanti, Abdullah Alarifi, Nagula Shankaraiah, Narayana Nagesh, Ahmed
Kamal
PII:
S0223-5234(17)31086-3
10.1016/j.ejmech.2017.12.055
EJMECH 10033
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 September 2017
Revised Date: 14 December 2017
Accepted Date: 15 December 2017
Please cite this article as: M. Sathish, B. Kavitha, V.L. Nayak, Y. Tangella, A. Ajitha, S. Nekkanti, A.
Alarifi, N. Shankaraiah, N. Nagesh, A. Kamal, Synthesis of podophyllotoxin linked β-carboline congeners
as potential anticancer agents and DNA topoisomerase II inhibitors, European Journal of Medicinal
Chemistry (2018), doi: 10.1016/j.ejmech.2017.12.055.
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ACCEPTED MANUSCRIPT
Synthesis of podophyllotoxin linked β-carboline congeners as potential
anticancer agents and DNA topoisomerase II inhibitors
Manda Sathish, Botla Kavitha, V. Laxma Nayak, Yellaiah Tangella, Ayyappan Ajitha,
Shalini Nekkanti, Abdullah Alarifi, Nagula Shankaraiah*, Narayana Nagesh*, Ahmed
Kamal*

ACCEPTED MANUSCRIPT
Synthesis of podophyllotoxin linked
β
-carboline congeners as potential
anticancer agents and DNA topoisomerase II inhibitors
Manda Sathish a, Botla Kavitha b, V. Laxma Nayak a, Yellaiah Tangella a, Ayyappan Ajitha c,
Shalini Nekkanti b, Abdullah Alarifi d, Nagula Shankaraiah*b, Narayana Nagesh*c, Ahmed
Kamal*a,b,d,e
aMedicinal Chemistry and Pharmacology, CSIR-Indian Institute of Chemical Technology,
Hyderabad 500 007, India
bDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and
Research (NIPER), Hyderabad 500 037, India
cCSIR-Centre for Cellular and Molecular Biology, Hyderabad 500 007, India
dCatalytic Chemistry Research Chair, Chemistry Department, College of Science, King Saud
University, Riyadh 11451, Saudi Arabia
eSchool of Pharmaceutical Education and Research (SPER), Jamia Hamdad University, New
Delhi, 110 062, India
Abstract: A series of new podophyllotoxin linked β-carboline congeners have been synthesized
by coupling various substituted β-carboline acids with 4β-aminopodophyllotoxin. Evaluation of
their anticancer activity against a panel of human cancer cell lines such as lung cancer (A549),
prostate cancer (DU-145), MDA MB-231 (breast cancer), HT-29 (colon cancer) and HeLa
(cervical cancer) suggested that 7i and 7j are the most cytotoxic compounds with IC50 values of
1.07±0.07 µM and 1.14±0.16 respectively against DU-145 cell line. Further, detailed biological
studies such as cell cycle analysis, topoisomerase II inhibition, Comet assay, DNA binding
studies and docking studies have revealed that these congeners are DNA interacting
topoisomerase II inhibitors.
Keywords: β-Carbolines; podophyllotoxin; topoisomerase II inhibition; cytotoxicity; docking.
*Corresponding authors. Phone: (+)91-40-27193157; Fax: (+)91-40-27193189; E-mail:
ahmedkamal@iict.res.in; nagesh@ccmb.res.in; shankar@niperhyd.ac.in
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1. Introduction
Topoisomerase-II (topo II) is a vital nuclear enzyme involved in various DNA processes and also
a major target for many drugs currently used in cancer chemotherapy [1]. Based on their
mechanism of action, these drugs can be classified into catalytic inhibitory compounds (CICs)
and interfacial poisons (IFPs) [2]. The CICs target the N-terminal ATPase domain of topo II and
inhibit it at an initial stage by stabilizing the enzyme-ligand-DNA ternery complex eg: ICRF 193
(I, Fig. 1) [3]. On the other hand, IFPs initiate the cleavage reaction and stop the re-ligation of
DNA, which leads to apoptosis eg: doxorubicin [4] (II) and amsacrine [2] (III) Fig. 1.
Podophyllotoxin (IV) is a well-known natural lignan which is extracted from the roots of
Podophyllotoxin peltatum. Podophyllotoxin (IV) and its derivatives are potent anticancer agents
acting through diverse mechanisms; the former is an inhibitor of microtubule assembly while the
latter are DNA interacting agents and topoisomerase inhibitors [5,6]. Many research groups have
explored the structure activity relationships (SARs) of podophyllotoxin based on the results from
several modifications of the ring systems (ring A, B, C, D and E) of podophyllotoxin (IV, Fig. 1)
[7]. Among all the structural changes, the modification at the C-4 position of C-ring has received
great attention and the resulting derivatives displayed dramatic improvement in DNA topo II
inhibition with significant anticancer activity [7]. These studies have led to the development of
semi-synthetic podophyllotoxin derivatives such as etoposide (F) and teniposide (G) [8,9]. From
the SAR studies, it is clear that the podophyllotoxin derivatives in subcellular level bind
selectively to the colchicine binding site of tubulin, and inhibit the formation of microtubule.
Interestingly, etoposide (V) as well as teniposide (VI) do not affect the tubulin polymerization
and are reported as irreversible DNA topo II inhibitors (Fig. 1) which indicates that bulky groups
like sugar moiety at C-4 position on the C-ring of podophyllotoxin may be responsible for the
topo II inhibition [10]. Due to the poor solubility of etoposide, a phosphate ester prodrug has
been developed (etopophos (VII), Fig. 1) [11] which is used in chemotherapy for Ewing's
sarcoma, Kaposi’s sarcoma, testicular cancer, lung cancer, lymphoma, glioblastoma multiforme
and nonlymphocytic leukemia. Although numerous anticancer drugs are currently in use, many
side effects like low blood pressure, hair loss, diarrhea, etc were accompanied with these drugs
[12]. Hence, development of new cancer chemotherapeutic drugs with fewer side effects is
highly desirable in the drug discovery process.
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<Insert Fig. 1>
β-Carboline alkaloids are another interesting class of molecules with a wide variety of
medicinal properties including anticancer activity [13]. Small molecules with β-carboline
scaffold such as Harman, harmine, norharman and harmaline are isolated from Peganum
harmala (Zygophillaceae, Syrian Rue) [14]. These molecules basically possess tricyclic planar
structures with suitable features for DNA interaction and enzyme inhibition activities like
topoisomerase inhibition [15]. Moreover, they produce their anticancer effect through different
modes of action, such as interacting with DNA (intercalation with DNA or external binding to
DNA) [16], MK-2 [17], DNA topo inhibition [18], kinesin Eg5 [19] and CDK [20]. However,
DNA interactive topoisomerase inhibition has been studied widely and gained much significance
in cancer chemotherapy as many of the anticancer drugs currently in use display these
mechanisms of action [21]. Azatoxin, a tetrahydro-β-carboline was rationally designed to inhibit
topo II and binds to DNA in a non-intercalative fashion [22]. Macdonald et al. [22] have
synthesized structurally azatoxin related amino acid linked β-carboline derivatives, which inhibit
topo II mediated DNA relaxation. Recently, we have also reported dithiocarbamate linked β-
carboline derivatives as DNA interactive topo II inhibitors with apoptosis ability [23a].
In search for newer anticancer agents with topoisomerase inhibition and in continuation
of our efforts towards search for new chemical entities (NCEs) in drug discovery, we replaced
the sugar moiety of etoposide with other bulky moieties like β-carboline and envisioned to
generate new conjugates with enhanced cytotoxic activity as well as DNA topo II inhibition.
Thus, we describe the synthesis of new podophyllotoxin linked β-carboline congeners (7a‒q) by
coupling of β-carboline acids with 4β-aminopodophyllotoxin through simple amidation (Fig. 2).
In this regard, we have synthesized 7a‒q and evaluated them for their cytotoxic activity, cell
cycle analysis, DNA topo II inhibition, DNA binding affinity and molecular docking studies.
<Insert Fig. 2>
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2. Results and discussion
2.1. Chemistry
The synthesis of podophyllotoxin linked β-carboline congeners (7a‒q) have been discussed
in Schemes 1 and 2. The intermediate β-carboline-3-carboxylic acids 5a‒q have been
synthesized as shown in Scheme 1. The L-tryptophan methyl ester (2) was prepared from L-
tryptophan (1) by employing SOCl2. Next, Pictet-Spengler condensation of 2 with a variety of
substituted benzaldehydes gave the corresponding methyl tetrahydro-β-carboline-3-carboxylates
3a‒q which were used directly for the aromatization by using trichloroisocyanuric acid to afford
the methyl-β-carboline-3-carboxylates 4a–q. These are further hydrolyzed in the presence of
aqueous NaOH solution to get β-carboline acids 5a–q in good yields and were employed in the
final peptide coupling reaction.
<Insert Scheme 1>
Finally, the synthesis of the target podophyllotoxin linked β-carboline congeners 7a‒q
have been achieved by the reaction of the intermediates 5a–q with 4β-aminopodophyllotoxin (6)
[23b] in presence of peptide coupling reagents such as EDCI and HOBt (Scheme 2).
<Insert Scheme 2>
2.2. Evaluation of Biological Activity
2.2.1. Cytotoxicity
The podophyllotoxin-linked β-carboline congeners 7a‒q have been initially assayed for
their in vitro cytotoxic activity against selected human cancer cell lines namely A549 (lung
cancer), DU-145 (prostate cancer), MDA MB-231 (breast cancer), HT-29 (colon cancer) and
HeLa (cervical cancer) using MTT assay [24]. All the congeners displayed moderate to good
cytotoxicity and most of these demonstrate high cytotoxicity compared to doxorubicin,
etoposide, podophyllotoxin and showed cytotoxicity selectively against DU-145 cells. However,
majority of these congeners such as 7d, 7e and 7i‒7q have exhibited sensible cytotoxicity with
IC50 values of < 10 µM against DU-145 cells. Moreover, the congeners 7i‒7p were found to be
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considerably cytotoxic with IC50 value of < 5 µM. Interestingly, the congeners 7i and 7j showed
promising cytotoxicity with IC50 value of 1.07 and 1.14 µM respectively against DU-145 cells.
Further to evaluate the effect of active congeners 7i and 7j, cell cycle analysis, topo II inhibitory
assay, DNA interaction studies and molecular docking studies were carried out.
<Insert Table 1>
2.2.2. Cell cycle analysis
In order to substantiate the results obtained from MTT assay as well as to understand the
effect of 7i and 7j on cell cycle progression, cell cycle assay was performed. On treatment of
DU-145 cells with 1.0 µM concentration of each congener 7i and 7j for 48 h, significant
increment in population of cells at S phase and G2/M phase (≈ 41% and ≈ 29% respectively) was
observed and compared to control cells (≈ 25% and ≈ 22%). However, moderate accumulation of
cells in subG1 phase (from ≈ 2% to 11%) and extreme decrement of cells in G0/G1 phase (≈ 51%
to ≈ 19%) was also observed. Moreover, the extent of population of cells at S and G2/M is
slightly high with 7i compared to 7j. The high accumulation of cells in S and G2/M phase on
treatment with 7i and 7j indicates their ability to stop cycle at cell division stage. Based on the
results observed, the active congeners 7i and 7j efficiently arrested the cell cycle at S and G2/M
phase. As reported earlier [25], the molecules that inhibit cell cycle at S and G2/M phase could
effectively inhibit topoisomerase II and lead to apoptosis of cancer cell. The cell cycle
histograms obtained for DU-145 on treatment of 7i and 7j at 1.0 µM concentrations each are
shown in Fig. 3 and the results are tabulated in Table 2.
<Insert Fig. 3>
<Insert Table 2>
2.2.3. DNA Topoisomerase II inhibition assay
The enzymes which control the topology of DNA are called topoisomerases and play a
crucial role in several important processes such as DNA replications, transcription, segregation
and recombination [26]. Basically, two classes of topoisomerases are known: type I and type II
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topoisomerases. The type I topoisomerase known to make nicks on the single strand of DNA
whereas type II topoisomerases are familiar for making nicks on both strands of DNA. The
compounds that interfere with DNA topo II, inhibit the enzyme activity and stop the DNA
replication which ultimately leads to cell death [27a]. Topo II inhibition assay was performed
using Topo II Drug Screening Kit (TG 1009, Topogen, USA). In this present study, catenated
DNA and topo II enzyme (5 units) were incubated with 10 µM etoposide for 30 min (lane 5, Fig.
4) and this was considered as positive control. In the control lane a linear DNA formation was
observed, indicating that it catalyses the cleavage and inhibit the DNA resealing process. This
indicates that etoposide functions as an interfacial poison (IFP). The kDNA which is catenated
remained near the well itself (lane 1, Fig. 4) and upon addition of 5 units of topo II the catenated
kDNA has converted mostly to linear form (line 2, Fig. 4). The catenated kDNA under similar
conditions was incubated with topo II and 10 µM of 7i and 7j (lane 3 and 4 respectively, Fig. 8),
did not observe the formation of linear DNA. However, most of the catenated DNA remained in
the well without entering into the gel, signifies the formation of stable topo II-kDNA-7i/7j
ternary complex. Hence, it indicates that 7i and 7j congeners will interfere with topo II enzyme
of catalytic activity and these are considered as CIC. This assay indicates that the 7i and 7j
would interfere with catalytic activity of topo II enzyme and act as catalytic inhibitors.
<Insert Fig. 4>
2.2.4. Comet assay
The topoisomerase II inhibition assay indicate that 7i and 7j are efficient in inhibiting the activity
of topoisomerase II. To study the role of these congeners in fragmenting the DNA, a single cell
based assay namely comet assay was performed. It is used to detect single-strand DNA breaks,
double-strand DNA breaks on treating the DU-145 cells with 7i and 7j comparing with
etoposide. In the present assay propidium iodide, a fluorescent DNA-binding molecule is used to
detect the extent of DNA damage. DU-145 cells were treated with 10 µM of 7i, 7j and etoposide,
and after lysis the samples were allowed to undergo electrophoresis. Cells grown in the absence
of any congeners is considered as control. It was observed that the DNA damage in presence of
7i, 7j and etoposide. However, there is no DNA damage observed in the control. Etoposide is
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well known topoisomerase II inhibitor which exhibits this inhibition activity through stabilizing
the DNA cleavable complex. It was reported earlier that etoposide will induce the formation of
open circular and linear DNA through the topoisomerase-DNA cleavable complex formation
[27b]. We speculate that 7i and 7j can also effectively damage the DNA like etoposide and
inhibit the activity of topoisomerase II. From the Figure 5, it is clear that the congeners 7i and 7j
are efficient in damaging the DNA equally.
<Insert Fig. 5>
2.2.5. DNA binding studies
To find out the nature of interaction of active congeners 7i and 7j with DNA, biophysical
studies such as UV-visible spectroscopy, fluorescence spectroscopy and circular dichroism
spectroscopy were carried out.
2.2.5.1. UV-visible spectroscopy. In order to examine the binding nature of active congeners 7i
and 7j to DNA, the UV absorption spectra of these congeners in the presence and absence of
increasing concentrations of CT-DNA were examined (Fig. 6). On addition of equal alliquots of
CT-DNA to the solution of these congeners (7i and 7j), absorption band at around 207 nm which
gradually decreased without any shift with incresing concentration of CT-DNA was observed.
From UV-visible titration data, the 7i and 7j congeners with CT-DNA binding constatnts were
found to be in the range of ≈ 1 x 104 M-1. Since the absorption band at 207 nm shows
hyperchromicity upon addition of DNA, it is evident that these congeners could bind to the
surface of DNA [28]. The hyperchromic effect may also be due to the electrostatic interaction
between 7i and 7j and the phosphates in the backbone at the periphery of the double helix CT-
DNA [29]. However, it is not possible to understand the binding nature of congeners with UV-
visible titration data alone. The representative UV-visible spectra obtained on interaction of
complex with CT-DNA is shown in Fig. 6.
<Insert Fig. 6>
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2.2.5.2. Fluorescence spectroscopy. Fluorescence titration is another helpful technique for
understanding the binding mode of small molecules with DNA and to study the electronic
environment around the DNA-complex at relatively lower concentrations [30]. As these
congeners show fluorescence properties, their interaction with CT-DNA could provide certain
information about the nature of their interactions with DNA. In this study, 7i and 7j emission
spectra show a prominent peak at 409 nm and small humps at 325 nm and 335 nm and on
addition of equal increments of CT-DNA solution to the solution of 7i and 7j, the
hypochromicity of fluorescence emission intensity at 409 was observed, which probably
indicates non-intercalative mode of binding such as electrostatic binding mode (surface binding
or grove binding) that leads to effective protection of 7i and 7j molecules by DNA. However, the
less intense fluorescence peaks intensity at 325 and 335 nm exhibited gradual enhancement with
the addition of CT-DNA. The enhancement in the fluorescence peaks at lower wavelength may
be due to partial intercalating portion of 7i and 7j with DNA. But the prominent fluorescence
emission at 409 nm clearly showed a gradual reduction in its intensity. Sometimes, the
fluorescence emission band hypochromicity may also be due to the quenching of fluorescence
emission by surrounding water molecules by absorbing the fluorescence emission energy. The
fluorescence spectra of 7i and 7j were shown in Fig. 7.
<Insert Fig. 7>
2.2.5.3. Circular dichroism spectroscopy. In order to study the effect of active congeners 7i
and 7j on DNA conformation, circular dichoism (CD) studies were performed. The CD spectra
of CT-DNA exhibited positive and negative bands at 275 nm and 245 nm respectively. In this
study, at an average both the congeners 7i and 7j demonstrated around 5-7 % hypochromicity of
positive CD band on treatment of CT-DNA. On addition of 10 µM of 7i and 7j to CT-DNA (1:1
ratio of congener:CT DNA), the positive CD band showed hypochromicity which indicates that
unwinding of CT-DNA due to the interaction of 7i and 7j with CT-DNA [31]. Next, on doubling
the concentration of congeners (20 µM 7i/7j:10 µM CT DNA), the positive band intensity was
further reduced. Based on these observations, CD studies indicates that 7i and 7j would interact
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with DNA and bring small changes in DNA conformation. The CD spectra of 7i and 7j with CT-
DNA is shown in Fig. 8.
<Insert Fig. 8>
2.3. Molecular docking studies
To visualize the binding mode of the title compounds, molecular docking studies were
performed with ATP-binding domain of human topoisomerase-IIα. The results of the docking
study are in correlation with the DNA topoisomerase-IIα inhibition assay in which, the
synthesized compounds occupied the ATP-binding active site with similar orientations and
formed strong interactions with several surrounding residues. Fig. 9 represents the docking pose
of 7i and 7j in the ATP binding pocket, where it is enclosed by Asn91, Asn95, Arg98, Gly124,
Ile125, Pro126, Ile141, Gly164, and Gly166 residues. The lactone oxygen of podophyllotoxin
ring system forms hydrogen-bonding with the side chain of Arg 98. Additionally, the methoxy
group in 7i and fluorine in 7j on the C-1 phenyl of β-carboline ring form hydrogen-bonding with
the side chain of Asn150. Further, the entry of ATP into the binding site is also hindered by the
interactions of the compounds with the Mg+2 ion and residues below the ATP binding site
(Asn46, Asp73, Ile78, Arg136 and Tyr165).
<Insert Fig. 9>
Molecular docking simulations were also carried out into the DNA duplex
d(CGCGAATTCGCG)2. The docked poses for top scored 7i and 7j, shown in Fig. 10, depict the
ability of the amide functionality to orient the molecules perfectly in a curved shape along length
of the minor groove. Further, the docked poses were stabilized by hydrogen bonding between the
indole NH-, amide group and the side chains of the DNA base pairs. The aromatic ring in the β-
carboline as well as the podophyllotoxin parts were also observed to be involved in side wise π–
π stacking with the base pairs. All these interactions strengthen the minor groove binding affinity
of the β-carboline-linked podophyllotoxin derivatives. These results are in agreement with the
DNA binding studies as well as the cytotoxicity data.
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<Insert Fig. 10>
3. Conclusion
In conclusion, we have synthesized a series of new β-carboline-podophyllotoxin
congeners by amidation of different β-carboline acids with 4β-aminopodophyllotoxin. The target
congeners contain two well established pharmacophoric groups such as β-carboline ring system,
podophyllotoxin and the congeners 7i and 7j found to be most active in the series. Further, these
assayed for cell cycle analysis and DNA topo II inhibitory activity using catenated kDNA.
However, these studies revealed that the 7i and 7j inhibit efficiently topo II enzymatic activity by
catalytic inhibition of topo II. The DNA binding studies disclosed that 7i and 7j are DNA
external binders and the docking studies correlated with DNA binding studies as well as topo II
inhibition studies. Overall, these studies are very important to understand the role of different
pharmacophores that are connected to β-carboline nucleus or podophyllotoxin nucleus for the
cytotoxic activity, DNA binding ability and DNA topo II inhibitory activity.
4. Experimental Section
4.1. Materials and methods
Chemical reagents were purchased from Sigma-Aldrich and used without further purification.
All the solvents were of commercial grade and were purified prior to use when necessary. 1H and
13C NMR experiments were performed on Avance (300 MHz, 400 MHz and 500 MHz)
spectrometers. Chemical shifts are measured relative to an internal standard tetramethylilane
(TM) set to δ 0 ppm and are reported in parts per million (ppm). Spin multiplicities are described
as s (singlet), br s (broad singlet), d (doublet), dd (double doublet), t (triplet), td (triple doublet),
q (quartet), or m (multiplet). Coupling constants are reported in hertz (Hz). TLC analyses were
performed with silica gel plates (0.25 mm, E. Merck, 60 F254) using iodine, KMnO4, and a UV
lamp for visualization. Specific rotations were measured on a Perkin-Elmer 341MC polarimeter.
Mass spectra were recorded by electrospray ionization mass spectrometry (ESI-MS). HRMS was
performed on a Varian QFT-ESI instrument. Melting points were determined on an
electrothermal melting point apparatus and are uncorrected. IR spectra were measured on Bruker
FT-IR Equinox 55 and Bruker TENSOR 27 instruments.
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4.2. Preparation of (S)-methyl 2-amino-3-(1H-indol-3-yl)propanoate (2) [32]
To the stirred solution of L-tryptophan (1, 1 mmol) in methanol (50 ml), thionyl chloride (8.02
o
mL, 1.1 mol) was added drop-wise at 0 C and continued stirring for 12 h at room temperature.
The excess amount of solvent was removed under vacuum and dried well. Then, the resulting
solid was dissolved in CH2Cl2, basified with saturated NaHCO3 solution and extracted with
excess amount of CH2Cl2. Then, the organic layer was dried over anhydrous Na2SO4 and
concentrated in vacuo to obtain L-tryptophan methyl ester (2). White solid; 19.62 g; 90% yield;
Mp: 102‒103 oC; 1H NMR (300 MHz, CDCl3) δ (ppm) 8.81 (bs, 1H), 7.58 (d, J = 7.90 Hz, 1H),
7.25 (d, J = 7.90 Hz, 1H), 7.14 (t, J = 6.90, 7.14 Hz, 1H), 7.08 (t, J = 6.90, 7.14 Hz, 1H), 6.90
(s, 1H), 3.84 (t, J = 4.90, 5.42 Hz, 1H), 3.73 (s, 3H), 3.25 (dd, J = 4.45, 13.8 Hz, 1H), 3.00 (dd,
J = 4.12, 13.8 Hz, 1H), 2.00 (s, 2H); ESI-MS: m/z 219 [M+H] +.
4.3. General reaction procedure for the preparation of compounds 4a‒q
To a mixture of L‒tryptophan ester (2, 1 mmol) and substituted benzaldeyde (1.1 mmol) in
EtOH, added catalytic p-TSA and refluxed for 12 h. After completion of the reaction, solvent
was removed in vacuo and the crude product 3a‒c (1.0 mmol) was taken in DMF and treated
with TEA (3.0 mmol), a solution of TCCA (1.1 mmol) in DMF was added dropwise at ‒20 oC.
The reaction mixture was allowed slowly to 0 oC and stirred at same temperature for 2 h. After
the completion of reaction, reaction mixture was quenched with ice water. The precipitate was
filtered, washed with water, and dried in vacuo to give the compound (4a‒q).
4.3.1. Methyl 1-(benzo[d][1,3]dioxol-5-yl)-9H-pyrido[3,4-b]indole-3-carboxylate (4a) [33a]
White solid; 92% yield; mp: 292–294 °C; 1H NMR (300 MHz, DMSO‒d6) δ (ppm): 8.89 (s, 1H),
8.42 (d, J = 7.9 Hz, 1H), 7.70 (d, J = 8.3 Hz, 1H), 7.61 (d, J = 7.1 Hz, 0.9H), 7.58–7.51 (m, 2H),
7.32 (t, J = 7.1 Hz, 1H), 7.18 (d, J = 8.3 Hz, 0.9H), 6.17 (s, 1.8H), 3.93 (s, 3H) the extra peaks at
7.45, 7.10, 6.14 are due to 10% minor rotamer; ESI-MS: m/z 347 [M+H]+.
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4.3.2. Methyl 1-(3,4,5-trimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxylate (4b)
[23a,33]
1
Yellow solid: 80% yield; mp: 229–230 °C; H NMR (300 MHz, CDCl3) δ (ppm): 9.38 (bs, 1H),
8.75 (s, 1H), 8.21(d, J = 7.5 Hz, 1H), 7.60 (d, J = 7.5 Hz, 1H), 7.56–7.61 (m, 1H), 7.32–7.38 (m,
1H), 6.94 (s, 2H), 4.03 (s, 3H), 3.84 (s, 3H), 3.77 (s, 6H); ESI-MS: m/z 393 [M+H]+.
4.3.3. Methyl 1-(4-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxylate (4c) [23,33a]
1
White solid; 95% yield; mp: 226–230 °C; H NMR (300 MHz, CDCl3) δ (ppm): 9.18 (bs, 1H),
8.80 (s, 1H), 8.17 (d, J = 7.7 Hz, 1H), 7.78 (d, J = 8.8 Hz, 2H), 7.57–7.55 (m, 2H), 7.36–7.33 (m,
1H), 6.88 (d, J = 8.6 Hz, 2H), 4.03 (s, 3H), 3.77 (s, 3H); ESI-MS: m/z 333 [M+H]+.
4.3.4. Methyl 1-(4-fluorophenyl)-9H-pyrido[3,4-b]indole-3-carboxylate (4d) [23a,33a]
1
White solid: 73% yield; mp: 195–198 °C; H NMR (500 MHz, CDCl3) δ (ppm): 4.06 (s, 3H),
7.22 (t, J = 8.7 Hz, 2H), 7.39 (t, J = 7.9 Hz, 1H), 7.63–7.55 (m, 2H), 7.92 (q, J = 5.3, 8.7 Hz,
2H), 8.22 (d, J = 7.8 Hz, 1H), 8.83 (bs, 1H), 8.87 (s, 1H); ESI-MS: m/z 321 [M+H]+.
4.3.5. Methyl 1-( p-tolyl)-9H-pyrido[3,4-b]indole-3-carboxylate (4e) [33a]
1
Yellow solid: 90% yield; mp: 192–194 °C; H NMR (500 MHz, DMSO‒d6) δ (ppm): 8.90 (s,
1H), 8.42 (d, J = 7.8 Hz, 1H), 7.93 (d, J = 9.1 Hz, 2H), 7.70 (d, J = 8.2 Hz, 1H), 7.60 (t, J = 8.2
Hz, 1H), 7.45 (d, J = 7.9 Hz, 2H), 7.32 (t, J = 7.9 Hz, 1H), 3.93 (s, 3H), 2.45 (s, 3H); ESI-MS:
m/z 317 [M+H]+.
4.3.6. Methyl 1-(3-fluorophenyl)-9H-pyrido[3,4-b]indole-3-carboxylate (4f) [33a]
Light yellow solid; 75% yield; mp: 224–228 °C; 1H NMR (300 MHz, CDCl3) δ (ppm): 11.16 (s,
1H), 8.95 (s, 1H), 8.44 (d, J = 7.9 Hz, 1H), 7.91–7.84 (m, 1H), 7.83–7.77 (m, 1H), 7.73–7.67 (m,
2H), 7.66–7.59 (m, 1H), 7.46–7.31 (m, 2H), 3.94 (s, 3H); ESI-MS: m/z 321 [M+H]+.
12

ACCEPTED MANUSCRIPT
4.3.7. Methyl 1-(4-(trifluoromethyl)phenyl)-9H-pyrido[3,4-b]indole-3-carboxylate (4g)
[23,33]
1
Pale yellow solid; 79% yield; mp: 250–252 °C; H NMR (300 MHz, DMSO–d6) δ (ppm): 3.94
(s, 3H), 7.32 (t, J = 7.5 Hz, 1H), 7.59 (t, J = 7.9 Hz, 1H), 7.68 (d, J = 8.1 Hz, 1H), 7.92 (d, J =
8.1 Hz, 2H), 8.23 (d, J = 7.7 Hz, 2H), 8.36 (d, J = 7.7 Hz, 1H), 8.93 (s, 1H), 11.99 (bs, 1H); ESI-
MS: m/z 371 [M+H]+.
4.3.8. Methyl 1-(naphthalen-1-yl)-9H-pyrido[3,4-b]indole-3-carboxylate (4h) [33b]
1
White solid: 95% yield; mp: 294–297 °C; H NMR (300 MHz, DMSO−d6) δ (ppm): 11.61 (s,
1H), 9.05 (s, 1H), 8.48 (d, J = 7.7 Hz, 1H), 8.16 (dd, J = 2.5, 6.8 Hz, 1H), 8.11 (d, J = 8.1 Hz,
1H), 7.78‒7.73 (m, 2H), 7.62‒7.53 (m, 4H), 7.44 (t, J = 6.9 Hz, 1H), 7.33 (t, J = 7.7 Hz, 1H),
3.90 (s, 3H); ESI-MS: m/z 353 [M+H]+.
4.3.9. Methyl 1-(2,5-dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxylate (4i)
Creamish solid; 80% yield; mp: 290–294 °C; 1H NMR (300 MHz, DMSO−d6) δ (ppm): 11.51 (s,
1H), 8.92 (s, 1H), 8.40 (d, J = 8.3 Hz, 1H), 7.64‒7.55 (m, 2H), 7.30 (t, J = 6.8 Hz, 1H),
7.22‒7.11 (m, 2H), 7.04 (d, J = 1H), 3.91 (s, 3H), 3.77 (s, 3H), 3.69 (s, 3H); ESI-MS: m/z 363
[M+H]+.
4.3.10. Methyl 1-(3,4-difluorophenyl)-9H-pyrido[3,4-b]indole-3-carboxylate (4j) [34]
o
Off-white solid; 78% yield; mp: 236–239 C; 1H NMR (300 MHz, DMSO‒d6) δ (ppm): 3.94 (s,
3 H), 7.32–7.37 (m, 1 H), 7.42–7.49 (m, 1 H), 7.60–7.78 (m, 4 H), 8.46 (d, J = 7.93 Hz, 1 H),
8.97 (s, 1H), 12.05 (bs, 1H); ESI-MS: m/z 339 [M+H]+.
4.3.11. Methyl 1-(3-chlorophenyl)-9H-pyrido[3,4-b]indole-3-carboxylate (4k) [34]
o
1
Pale yellow solid; 80% yield; mp: 209–211 C; H NMR (300 MHz, DMSO‒d6) δ (ppm): 3.94
(s, 3H), 7.27–7.32 (m, 1 H), 7.47- 7.71 (m, 4H), 7.98 (d, J = 10.38 Hz, 2H), 8.31 (s, 1 H), 8.87
(s, 1 H), 11.93 (bs, 1H); ESI-MS: m/z 337 [M+H]+.
13

ACCEPTED MANUSCRIPT
4.3.12. Methyl 1-(4-chlorophenyl)-9H-pyrido[3,4-b]indole-3-carboxylate (4l) [33b]
o
1
Yellow solid: 93% yield; mp: 268−272 C; H NMR (300 MHz, CDCl3 + DMSO−d6) δ (ppm):
8.81 (s, 1H), 8.23 (d, J = 7.7 Hz, 1H), 8.01 (d, J = 8.3 Hz, 1H), 7.64 (d, J = 8.3 Hz, 1H),
7.59‒7.51 (m, 3H), 7.27 (t, J = 7.2 Hz, 1H), 6.92 (d, J = 7.4 Hz, 1H), 3.93 (s, 3H); ESI-MS: m/z
337 [M+H]+.
4.3.13. Methyl 1-(2-methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxylate (4m) [33a]
White solid; 88% yield; mp: 208–210 °C; 1H NMR (500 MHz, CDCl3) δ (ppm): 8.91 (d, J = 0.4
Hz, 1H), 8.75 (bs, 1H), 8.22 (d, J = 7.9 Hz, 1H), 7.79 (dd, J = 1.8, 7.6 Hz, 1H), 7.60–7.56 (m,
1H), 7.53 (d, J = 8.1 Hz, 1H), 7.50–7.46 (m, 1H), 7.37–7.34 (m, 1H), 7.20–7.17 (m, 1H), 7.12 (d,
J = 7.6 Hz, 1H), 4.05 (s, 3H), 3.84 (s, 3H); ESI-MS: m/z 333 [M+H]+.
4.3.14. Methyl 1-phenyl-9H-pyrido[3,4-b]indole-3-carboxylate (4n) [33a]
White solid: 88% yield; mp: 257–259 °C; 1H NMR (300 MHz, DMSO‒d6) δ (ppm): 8.92 (s, 1H),
8.42 (d, J = 7.9 Hz, 1H), 8.02 (d, J = 7.0 Hz, 2H), 7.73–7.54 (m, 5H), 7.33 (t, J = 7.7 Hz, 1H),
3.93 (s, 3H); ESI-MS: m/z 303 [M+H]+.
4.3.15. Methyl 1-(pyridin-3-yl)-9H-pyrido[3,4-b]indole-3-carboxylate (4o) [33a]
White solid: 90% yield; mp: 233–236 °C; 1H NMR (300 MHz, DMSO‒d6) δ (ppm): 9.21 (s, 1H),
8.98 (s, 1H), 8.77 (d, J = 3.8 Hz, 1H), 8.46 (d, J = 7.9 Hz, 1H), 8.42–8.36 (m, 1H), 7.73–7.60 (m,
3H), 7.35 (t, J = 7.7 Hz, 1H), 3.94 (s, 3H); ESI-MS: m/z 304 [M+H]+.
4.3.16. Methyl 1-(3-phenoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxylate (4p)
o
Creamish solid; 65% yield; mp: 191‒195 C; 1H NMR (300 MHz, DMSO‒d6) δ (ppm): 11.4 (s,
1H), 8.76 (s, 1H), 8.12 (d, J = 7.7 Hz, 1H), 7.73 (d, J = 7.7 Hz, 1H), 7.66‒7.58 (m, 2H),
7.52‒7.44 (m, 2H), 7.30‒7.22 (m, 3H), 7.06‒6.97 (m, 4H), 3.93 (s, 3H); ESI-MS: m/z 395
[M+H]+.
14

ACCEPTED MANUSCRIPT
4.3.17. Methyl 1-(3,4-dimethoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxylate (4q)
o
Creamish solid; 82% yield; mp: 198‒202 C; 1H NMR (300 MHz, DMSO‒d6) δ (ppm): 8.83 (s,
1H), 8.35 (d, J = 7.7 Hz, 1H), 7.69 (d, J = 8.3 Hz, 1H), 7.60 (d, J = 7.9 Hz, 1H), 7.57‒7.51 (m,
2H), 7.32 (t, J = 7.7 Hz, 1H), 7.18 (d, J = 8.8 Hz, 1H), 3.91 (s, 3H), 3.87 (s, 3H), 3.85 (s, 3H);
ESI-MS: m/z 363 [M+H]+.
4.4. General procedure for the synthesis of compounds 5a‒q
To the stirred suspension of compound 4a‒q (1 mmol) in MeOH was added aqueous solution of
o
NaOH (2 mmol) and stirred for 6 h at 70 C. The solvent was evaporated in vacuo and the
obtained residue was acidified with 10% citric acid solution and the resulted precipitate was
filtered, washed with ethanol and dried. These well dried solid products 5a‒q were employed for
directly for next step without any further purification.
4.5. General procedure for the synthesis of compounds 7a‒q
To a stirred solution of compound 5a‒q (1 mmol) and compound 6 [23c] (1 mmol) in DCM were
o
added EDCI (1.2 mmol), HOBt (1.2 mmol) and TEA (3 mmol) at 0 C and stirred for 16 h at
room temperature. The reaction mixture was quenched with ice cold water and extracted with
DCM, the combined organic phases were dried over Na2SO4 and concentrated under reduced
pressure to get crude products 7a‒q which were purified by silica gel column chromatography by
using EtOAc/n-hexane.
4.5.1.
1-(Benzo[d][1,3]dioxol-5-yl)-N-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-
5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-9H-pyrido[3,4-
b]indole-3-carboxamide (7a)
o
1
White solid; 80% yield; mp: 190−195 C; [α]25 : –67.448 (c = 0.14, CHCl3); H NMR
D
(500 MHz, CDCl3) δ (ppm): 8.91 (s, 1H), 8.78 (s, 1H), 8.32 (d, J = 7.7 Hz, 1H), 8.22 (d, J = 7.9
Hz, 1H), 7.61 (t, J = 8.0 Hz, 1H), 7.56 (d, J = 8.0 Hz, 1H), 7.41‒7.36 (m, 2H), 7.34 (d, J = 1.5
Hz, 1H), 7.01 (d, J = 7.9 Hz, 1H), 6.87 (s, 1H), 6.57 (s, 1H), 6.35 (s, 2H), 6.07 (s, 2H), 5.98 (d, J
15

ACCEPTED MANUSCRIPT
= 3.9 Hz, 2H), 5.54 (dd, J = 4.4 Hz, 7.78 Hz, 1H), 4.68 (d, J = 4.5 Hz, 1H), 4.54 (dd, J = 7.0 Hz,
9.0 Hz, 1H), 3.99 (t, J = 9.4 Hz, 1H), 3.82 (s, 3H), 3.77 (s, 6H), 3.15‒3.08 (m, 1H), 3.05 (dd, J =
4.5, 14.1 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ (ppm): 174.5, 165.8, 152.6, 148.5, 148.3, 147.6,
141.2, 140.7, 139.1, 137.2, 134.9, 132.4, 131.6, 130.4, 129.2, 129.0, 122.1, 122.0, 121.9, 121.1,
113.8, 111.9, 110.1, 109.3, 108.8, 108.7, 108.3, 101.5, 101.3, 69.2, 60.7, 56.2, 48.1, 43.9, 41.9,
37.7; IR (KBr): 3373.72, 2923.96, 2358.96, 1776.87, 1660.26, 1588.36, 1488.58, 1334.81,
1237.33 cm−1; ESI-MS: m/z 728 [M+H]+; HRMS: m/z calcd. for C41H34N3O10 728.22442
[M+H]+, found 728.22440.
4.5.2.
N-((5S,5aS,8aR,9R)-8-Oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,8a,9-
hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-1-(3,4,5-trimethoxyphenyl)-9H-
pyrido[3,4-b]indole-3-carboxamide (7b)
o
1
White solid; 85% yield; mp: 185−190 C; [α]25 : –74.560 (c = 0.17, CHCl3); H NMR
D
(500 MHz, CDCl3) δ (ppm): 8.91 (s, 1H), 8.85 (bs, 1H), 8.38 (d, J = 7.3 Hz, 1H), 8.24 (d, J = 7.9
Hz, 1H), 7.66–7.60 (m, 1H), 7.58 (d, J = 8.0 Hz, 1H), 7.40 (t, J = 6.7 Hz, 1H), 7.04 (s, 2H), 6.90
(s, 1H), 6.57 (s, 1H), 6.35 (s, 2H), 5.95 (dd, J = 1.0 Hz, 17.5 Hz, 2H), 5.48 (dd, J = 4.5 Hz, 7.17
Hz, 1H), 4.65 (d, J = 4.7 Hz, 1H), 4.57(dd, J = 7.3 Hz, 9.15 Hz, 1H), 4.04 (t, J = 9.4 Hz, 1H),
3.94 (s, 3H), 3.89 (s, 6H), 3.82 (s, 3H), 3.77 (s, 6H), 3.16–3.08 (m, 1H), 3.04 (dd, J = 4.8, 14.3
13
Hz, 1H); C NMR (75 MHz, CDCl3) δ (ppm): 174.4, 165.7, 153.6, 152.5, 148.9, 148.1, 147.4,
141.3, 140.8, 138.9, 138.7, 135.1, 134.8, 133.0, 132.4, 130.9, 130.4, 129.3, 129.0, 122.0, 121.1,
113.8, 111.9, 110.0, 109.2, 108.1, 105.5, 101.4, 69.3, 60.8, 60.6, 56.2, 56.1, 48.3, 43.7, 41.8,
37.7; IR (KBr): 3362.11, 2936.86, 2835.20, 2359.43, 1776.56, 1660.92, 1586.48, 1504.75,
1329.00, 1234.02 cm−1; ESI-MS: m/z 774 [M+H]+; HRMS: m/z calcd. for C43H40N3O11
774.26628 [M+H]+, found 774.26640.
4.5.3.
1-(4-Methoxyphenyl)-N-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-
5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-9H-pyrido[3,4-
b]indole-3-carboxamide (7c)
o
1
White solid; 90% yield; mp: 190−195 C; [α]25 : –68.411 (c = 0.10, CHCl3); H NMR
D
(500 MHz, CDCl3) δ (ppm): 8.90 (s, 1H), 8.76 (s, 1H), 8.37 (d, J = 7.9 Hz, 1H), 8.23 (d, J = 7.9
16

ACCEPTED MANUSCRIPT
Hz, 1H), 7.83 (d, J = 8.8 Hz, 2H), 7.63–7.58 (m, 1H), 7.55 (d, J = 8.0 Hz, 1H), 7.42–7.36 (m,
1H), 7.12 (d, J = 8.6 Hz, 2H), 6.87 (s, 1H), 6.56 (s, 1H), 6.35 (s, 2H), 5.97 (dd, J = 1.2 Hz, 4.73
Hz, 2H), 5.55 (dd, J = 4.4 Hz, 7.78 Hz, 1H), 4.66 (d, J = 4.5 Hz, 1H), 4.54 (dd, J = 7.0 Hz, 9.15
Hz, 1H), 4.00 (t, J = 9.3 Hz, 1H), 3.90 (s, 3H), 3.82 (s, 3H), 3.77 (s, 6H), 3.16–3.08 (m, 1H),
3.05 (dd, J = 4.7, 17.1 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ (ppm): 174.5, 165.8, 160.4, 152.5,
148.3, 147.6, 141.4, 140.6, 139.1, 135.0, 134.9, 132.3, 130.9, 130.2, 129.9, 129.5, 129.2, 128.9,
122.1, 122.0, 121.1, 114.6, 113.6, 111.8, 110.0, 109.3, 108.2, 101.5, 69.2, 60.7, 56.1, 55.4, 48.0,
43.8, 42.0, 37.6; IR (KBr): 3375.23, 2935.01, 2836.43, 2359.73, 1776.14, 1660.14, 1588.75,
1509.74, 1322.33, 1247.07 cm−1; ESI-MS: m/z 714 [M+H]+; HRMS: m/z calcd. for C41H36N3O9
714.24515 [M+H]+, found 714.24452.
4.5.4.
1-(4-Fluorophenyl)-N-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-
5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-9H-pyrido[3,4-
b]indole-3-carboxamide (7d)
o
1
White solid; 89% yield; mp: 325−330 C; [α]25 : –75.887 (c = 0.13, CHCl3); H NMR
D
(500 MHz, CDCl3) δ (ppm): 8.94 (s, 1H), 8.74 (s, 1H) 8.30 (d, J = 7.9 Hz, 1H), 8.24 (d, J = 7.7
Hz, 1H), 7.87 (dd, J = 5.3, 8.6 Hz, 2H), 7.65−7.60 (m, 1H), 7.56 (d, J = 8.0 Hz, 1H), 7.40 (t, J =
7.0 Hz, 1H), 7.29 (t, J = 8.6 Hz, 2H), 6.87 (s, 1H), 6.57 (s, 1H), 6.35 (s, 2H), 5.97 (d, J = 1.8 Hz,
2H), 5.55 (dd, J = 4.5, 7.7 Hz, 1H), 4.67 (d, J = 4.7 Hz, 1H), 4.54 (dd, J = 7.3, 9.0 Hz, 1H), 3.99
(t, J = 9.9 Hz, 1H), 3.8 (s, 3H), 3.77 (s, 6H), 3.16−3.07 (m, 1H), 3.04 (dd, J = 4.7, 14.1 Hz, 1H);
13C NMR (75 MHz, CDCl3) δ (ppm): 174.4, 165.6, 163.2 (d, J = 249.7 Hz), 152.6, 148.3, 147.6,
140.7, 140.5, 139.3, 137.2, 135.0, 134.9, 133.6, 132.4, 130.6, 130.1 (d, J = 8.2 Hz), 129.2, 122.1,
121.2, 116.3 (d, J = 22.0 Hz), 114.1, 111.8, 110.0, 109.3, 108.2, 101.5, 69.2, 60.7, 56.2, 48.0,
43.8, 42.0, 37.7; IR (KBr): 3374.81, 2936.00, 2359.32, 1777.12, 1664.91, 1588.01, 1507.94,
1322.51, 1231.92 cm−1; ESI-MS: m/z 702 [M+H]+; HRMS: m/z calcd. for C40H33FN3O8
702.22517 [M+H]+, found 702.22460.
17

ACCEPTED MANUSCRIPT
4.5.5.
N-((5S,5aS,8aR,9R)-8-Oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,8a,9-
hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-1-(p-tolyl)-9H-pyrido[3,4-b]indole-
3-carboxamide (7e)
o
1
White solid; 87% yield; mp: 210−215 C; [α]25D : –83.798 (c = 0.13, CHCl3); H NMR
(300 MHz, CDCl3) δ (ppm): 8.91(s, 1H), 8.87 (s, 1H), 8.38 (d, J = 7.7 Hz, 1H), 8.22 (d, J = 8.1
Hz, 1H), 7.77 (d, J = 7.7 Hz, 2H), 7.64−7.51 (m, 2H), 7.42−7.34 (m, 3H), 6.85 (s, 1H), 6.55 (s,
1H), 6.34 (s, 2H), 5.96 (d, J = 3.0 Hz, 2H), 5.53 (dd, J = 4.1, 7.3 Hz, 1H), 4.65 (d, J = 3.9 Hz,
1H), 4.53 (dd, J = 6.0, 8.1 Hz, 1H), 4.00 (t, J = 9.0 Hz, 1H), 3.82 (s, 3H), 3.76 (s, 6H), 3.18−2.98
13
(m, 2H), 2.44 (s, 3H); C NMR (75 MHz, CDCl3) δ (ppm): 174.5, 165.7, 152.6, 148.3, 147.6,
141.6, 140.6, 139.5, 139.2, 137.2, 135.0, 134.9, 134.7, 132.3, 130.2, 129.9, 129.2, 129.0, 128.0,
122.2, 122.1, 121.1, 113.8, 111.8, 110.0, 109.3, 108.2, 101.5, 69.2, 60.7, 56.1, 48.0, 43.8, 41.9,
37.7, 21.3; IR (KBr): 3373.06, 2931.76, 2359.42, 1777.00, 1660.86, 1588.78, 1484.95, 1321.52,
1233.35 cm−1; ESI-MS: m/z 698 [M+H]+; HRMS: m/z calcd. for C41H36N3O8 698.25024 [M+H]+,
found 698.24987.
4.5.6.
1-(3-Fluorophenyl)-N-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-
5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-9H-pyrido[3,4-
b]indole-3-carboxamide (7f)
o
1
White solid; 86% yield; mp: 315−320 C; [α]25 : –62.679 (c = 0.11, CHCl3); H NMR
D
(300 MHz, CDCl3) δ (ppm): 8.96 (s, 1H), 8.74 (s, 1H), 8.34–8.22 (m, 2H), 7.74–7.53 (m, 5H),
7.41 (t, J = 6.7 Hz, 1H), 7.26–7.20 (m, 1H), 6.87 (s, 1H), 6.58 (s, 1H), 6.36 (s, 2H), 5.98 (d, J =
2.2 Hz, 2H), 5.55 (dd, J = 4.5, 7.5 Hz, 1H), 4.69 (d, J = 4.5 Hz, 1H), 4.55 (dd, J = 6.7, 8.3 Hz,
1H), 4.00 (t, J = 9.8 Hz, 1H), 3.83 (s, 3H), 3.78 (s, 6H), 3.21–2.97 (m, 2H); 13C NMR (75 MHz,
CDCl3) δ (ppm): 174.4, 165.5, 163.5, 163.2, 163.1 (d, J = 248.0 Hz), 162.7, 152.5, 148.3, 147.6,
139.2, 135.0, 134.9, 132.3, 130.8 (d, J = 8.7 Hz), 130.7, 129.2 (d, J = 17.5 Hz), 123.8, 122.1,
121.9, 121.3, 116.2 (d, J = 19.7 Hz), 115.3 (d, J = 21.9), 114.4, 111.9, 110.1, 109.2, 108.1,
101.5, 69.2, 60.7, 56.1, 48.1, 43.8, 41.9, 37.6; IR (KBr): 3385.36, 3279.05, 3066.81, 2893.94,
1776.79, 1664.75, 1587.36, 1489.69, 1446.85, 1320.33, 1223.65 cm−1; ESI-MS: m/z 702
[M+H]+; HRMS: m/z calcd. for C40H33FN3O8 702.22517 [M+H]+, found 702.22491.
18

ACCEPTED MANUSCRIPT
4.5.7.
N-((5S,5aS,8aR,9R)-8-Oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,8a,9-
hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-1-(4-(trifluoromethyl)phenyl)-9H-
pyrido[3,4-b]indole-3-carboxamide (7g)
o
1
White solid; 88% yield; mp: 333−338 C; [α]25 : –63.425 (c = 0.15, CHCl3); H NMR
D
(500 MHz, CDCl3) δ (ppm): 8.98 (s, 1H), 8.79 (bs, 1H), 8.26 (t, J = 7.7 Hz, 2H), 8.01 (d, J = 8.0
Hz, 2H), 7.86 (d, J = 8.2 Hz, 2H), 7.67–7.61 (m, 1H), 7.57 (d, J = 8.0 Hz, 1H), 7.42 (t, J = 7.7
Hz, 1H), 6.87 (s, 1H), 6.57 (s, 1H), 6.35 (s, 2H), 5.98 (s, 2H), 5.55 (dd, J = 4.7, 7.7 Hz, 1H), 4.67
(d, J = 4.8 Hz, 1H), 4.55 (dd, J = 7.3, 9.1 Hz, 1H), 3.99 (t, J = 9.4 Hz, 1H), 3.82 (s, 3H), 3.77 (s,
13
6H), 3.16–3.08 (m, 1H), 3.03 (dd, J = 4.8, 14.3 Hz, 1H); C NMR (75 MHz, CDCl3) δ (ppm):
174.4, 165.5, 152.5, 148.3, 147.6, 140.8, 140.8, 139.8, 139.3, 137.1, 135.1, 134.9, 132.3, 130.9,
129.3, 129.0, 128.6, 126.2 (q, J = 4.3 Hz), 122.1, 121.9, 121.4, 114.6, 111.9, 111.0, 109.2, 108.1,
101.5, 69.1, 60.7, 56.1, 48.1, 43.8, 41.9, 37.6; IR (KBr): 3375.21, 2937.41, 1776.72, 1664.96,
1589.35, 1492.66, 1323.59, 1234.85 cm−1; ESI-MS: m/z 752 [M+H]+; HRMS: m/z calcd. for
C41H33F3N3O8 752.22197 [M+H]+, found 752.22215.
4.5.8.
1-(Naphthalen-1-yl)-N-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-
5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-9H-pyrido[3,4-
b]indole-3-carboxamide (7h)
o
1
White solid; 89% yield; mp: 210−214 C [α]25 : –61.425 (c = 0.18, CHCl3); H NMR (300
D
MHz, CDCl3) δ (ppm): 9.02 (s, 1H), 8.48 (s, 1H), 8.36 (d, J = 7.5 Hz, 1H), 8.27 (d, J = 8.3 Hz,
1H), 7.93 (dd, J = 8.3, 15.1 Hz, 2H), 7.78‒7.72 (m, 2H), 7.59 (q, J = 8.3, 15.8 Hz, 2H), 7.50 (t, J
= 7.5 Hz, 1H), 7.45‒7.36 (m, 3H), 6.85 (s, 1H), 6.42 (s, 1H), 6.27 (s, 2H), 5.93 (d, J = 11.3 Hz,
2H), 5.50 (dd, J = 4.5, 7.5 Hz, 1H), 4.53 (t, J = 8.3 Hz, 1H), 4.4 (d, J = 4.5 Hz, 1H), 4.00 (t, J =
13
9.8 Hz, 1H), 3.79 (s, 3H), 3.71 (s, 6H), 3.13‒3.00 (m, 1H), 2.86 (dd, J = 5.2, 15.1 Hz, 1H); C
NMR (75 MHz, CDCl3) δ (ppm): 174.6, 165.6, 152.4, 148.0, 147.4, 141.1, 140.6, 138.8, 136.2,
134.8, 134.2, 133.9, 132.1, 131.0, 130.0, 129.5, 129.0, 128.9, 128.5, 127.4, 126.8, 126.3, 125.3,
125.2, 122.0, 121.9, 121.0, 114.1, 111.9, 110.0, 109.1, 107.9, 101.4, 69.2, 60.6, 56.0, 47.9, 43.5,
41.6, 37.6; IR (KBr): 3366.37, 2929.85, 2359.88, 1776.45, 1666.68, 1504.04, 1233.30, 1126.17
cm−1; ESI-MS: m/z 734 [M+H]+; HRMS: m/z calcd. for C44H36N3O8 734.25024 [M+H]+, found
734.25005.
19

ACCEPTED MANUSCRIPT
4.5.9.
1-(2,5-Dimethoxyphenyl)-N-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-
5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-9H-pyrido[3,4-
b]indole-3-carboxamide (7i)
o
1
White solid; 85% yield; mp: 197−202 C; [α]25 : –82.881 (c = 0.11, CHCl3); H NMR
D
(500 MHz, CDCl3) δ (ppm): 8.96 (s, 1H), 8.86 (s, 1H), 8.41 (d, J = 7.4 Hz, 1H), 8.25 (d, J = 7.9
Hz, 1H), 7.60 (t, J = 8.2 Hz, 1H), 7.55 (d, J = 8.0 Hz, 1H), 7.38 (t, J = 7.9 Hz, 1H), 7.20 (d, J =
3.0 Hz, 1H), 7.12 (d, J = 9.0 Hz, 1H), 7.05 (dd, J = 3.2, 9.0 Hz, 1H), 6.89 (s, 1H), 6.56 (s, 1H),
6.35 (s, 2H), 5.96 (dd, J = 1.2, 13.1 Hz, 2H), 5.51 (dd, J = 4.5, 7.4 Hz, 1H), 4.65 (d, J = 4.7 Hz,
1H), 4.56 (dd, J = 7.0, 9.0 Hz, 1H), 4.03 (t, J = 9.3 Hz, 1H), 3.83 (s, 3H), 3.80 (s, 3H), 3.77 (2×s,
13
9H), 3.16–3.07 (m, 1H), 3.04 (dd, J = 4.7, 14.1 Hz, 1H); C NMR (75 MHz, CDCl3) δ (ppm):
174.4, 165.8, 153.3, 152.5, 150.7, 147.5, 140.6, 139.1, 138.9, 136.2, 134.9, 132.4, 130.1, 129.3,
128.9, 121.9, 120.8, 116.8, 116.5, 114.5, 113.9, 111.8, 110.1, 109.2, 108.1, 101.4, 69.3, 60.7,
57.6, 56.1, 55.5, 48.2, 43.8, 41.9, 37.7; IR (KBr): 3373.66, 2935.90, 2834.68, 1776.54, 1665.91,
1588.06, 1492.02, 1328.28, 1226.50 cm−1; ESI-MS: m/z 744 [M+H]+; HRMS: m/z calcd. for
C42H38N3O10 744.25572 [M+H]+, found 744.25549.
4.5.10.
1-(3,4-Difluorophenyl)-N-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-
5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-9H-pyrido[3,4-
b]indole-3-carboxamide (7j)
o
1
White solid; 89% yield; mp: 290−294 C; [α]25 : –79.205 (c = 0.15, CHCl3); H NMR
D
(500 MHz, CDCl3) δ (ppm): 8.97 (s, 1H), 8.80 (s, 1H), 8.23 (t, J = 8.3 Hz, 2H), 7.64 (t, J = 7.1
Hz, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.45–7.39 (m, 3H), 7.00–6.94 (m, 1H), 6.87 (s, 1H), 6.59 (s,
1H), 6.36 (s, 2H), 5.99 (d, J = 5.0 Hz, 2H), 5.54 (dd, J = 4.5, 7.6 Hz, 1H), 4.70 (d, J = 4.7 Hz,
1H), 4.55 (dd, J = 7.1, 9.1 Hz, 1H), 3.99 (t, J = 9.4 Hz, 1H), 3.82 (s, 3H), 3.77 (s, 6H), 3.16–3.08
13
(m, 1H), 3.05 (dd, J = 4.7, 14.3 Hz, 1H); C NMR (75 MHz, CDCl3 + DMSO–d6) δ (ppm):
173.8, 165.1, 162.4 (d, J = 248.1 Hz), 162.2 (d, J = 248.6 Hz), 151.7, 147.2 (d, J = 45.5 Hz),
141.4, 140.3, 137.9, 136.3, 134.5 (d, J = 18.7 Hz), 131.6, 130.2, 128.4 (d, J = 36.8 Hz), 120.8 (d,
J = 7.7 Hz), 120.0, 113.9, 112.1, 111.2 (d, J = 25.8 Hz), 108.9 (d, J = 55.5 Hz), 107.5, 103.7,
103.3, 103.0, 100.8, 68.4, 59.8, 55.4, 47.4, 43.2, 41.0, 37.0; IR (KBr): 3386.54, 3273.54,
20

ACCEPTED MANUSCRIPT
2906.49, 2841.55, 2359.81, 2341.69, 1778.36, 1665.07, 1597.09, 1448.54 cm−1; ESI-MS: m/z
720 [M+H]+; HRMS: m/z calcd. for C40H32F2N3O8 720.21575 [M+H]+, found 720.21600.
4.5.11.
1-(3-Chlorophenyl)-N-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-
5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-9H-pyrido[3,4-
b]indole-3-carboxamide (7k)
o
1
White solid; 81% yield; mp: 312−316 C; [α]25 : –41.860 (c = 0.04, CHCl3); H NMR (300
D
MHz, DMSO–d6) δ (ppm): 11.44 (s, 1H), 8.82 (s, 1H), 8.36 (d, J = 7.7 Hz, 1H), 8.12 (d, J = 7.7
Hz, 1H), 7.88–7.77 (m, 2H), 7.57 (d, J = 8.1 Hz, 1H), 7.54–7.37 (m, 3H), 7.30–7.20 (m, 1H), 6.8
(s, 1H), 6.48 (s, 1H), 6.27 (s, 2H), 5.90 (d, J = 3.3 Hz, 2H), 5.45 (dd, J = 4.5, 7.5 Hz, 1H), 4.58
(d, J = 4.5 Hz, 1H), 4.43 (t, J = 7.5 Hz, 1H), 3.91 (t, J = 9.8 Hz, 1H), 3.70 (s, 3H), 3.68 (s, 6H),
3.19–3.04 (m, 2H); 13C NMR (75 MHz, CDCl3 + DMSO–d6) δ (ppm): 174.1, 165.5, 152.1,
147.9, 147.2, 141.4, 139.5, 139.3, 138.2, 136.7, 136.5, 134.9, 134.7, 134.2, 131.9, 130.2, 129.9,
128.9, 128.4, 126.7, 121.3, 120.3, 114.0, 112.3, 109.6, 108.9, 107.8, 101.2, 68.9, 60.3, 55.8, 47.7,
43.5, 41.5, 37.4; IR (KBr): 3389.00, 2924.46, 2851.72, 1777.13, 1663.54, 1589.73, 1492.98,
1461.89, 1329.27, 1233.18 cm−1; ESI-MS: m/z 718 [M+H]+; HRMS: m/z calcd. for
C40H33ClN3O8 718.19562 [M+H]+, found 718.19587.
4.5.12.
1-(4-Chlorophenyl)-N-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-
5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-9H-pyrido[3,4-
b]indole-3-carboxamide (7l)
o
1
White solid; 80% yield; mp: 215−220 C; [α]25 : –76.682 (c = 0.14, CHCl3); H NMR
D
(500 MHz, CDCl3) δ (ppm): 8.94 (s, 1H), 8.74 (s, 1H), 8.29 (d, J = 7.7 Hz, 1H), 8.24 (d, J = 7.7
Hz, 1H), 7.82 (d, J = 8.3 Hz, 2H), 7.66−7.60 (m, 1H), 7.59−7.55 (m, 3H), 7.42−7.38 (m, 1H),
6.86 (s, 1H), 6.57 (s, 1H), 6.35 (s, 2H), 5.98 (s, 2H), 5.54 (dd, J = 4.5, 7.7 Hz, 1H), 4.67 (d, J =
4.8 Hz, 1H), 4.54 (dd, J = 7.3, 9.1 Hz, 1H), 3.99 (t, J = 9.4 Hz, 1H), 3.82 (s, 3H), 3.77 (s, 6H),
3.15−3.07 (m, 1H), 3.03 (dd, J = 4.8, 14.4 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ (ppm): 174.5,
165.6, 152.5, 148.3, 147.6, 140.8, 140.2, 139.2, 137.1, 135.9, 135.3, 135.0, 134.9, 132.3, 130.6,
129.5, 129.4, 129.2, 129.1, 122.1, 122.0, 121.2, 114.2, 111.9, 110.0, 109.2, 108.2, 101.5, 69.2,
60.7, 56.1, 48.1, 43.8, 42.0, 37.7; IR (KBr): 3363.54, 2933.74, 2359.78, 1776.21, 1661.25,
21

ACCEPTED MANUSCRIPT
1588.90, 1491.26, 1321.61, 1233.44 cm−1; ESI-MS: m/z 718 [M+H]+; HRMS: m/z calcd. for
C40H33ClN3O8 718.19562 [M+H]+, found 718.19565.
4.5.13.
1-(2-Methoxyphenyl)-N-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-
5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-9H-pyrido[3,4-
b]indole-3-carboxamide (7m)
o
1
White solid; 80% yield; mp: 175−180 C; [α]25 : –92.488 (c = 0.06, CHCl3); H NMR
D
(500 MHz, CDCl3) δ (ppm): 8.96 (s, 1H), 8.71 (s, 1H), 8.39 (d, J = 7.9 Hz, 1H), 8.23 (d, J = 7.7
Hz, 1H), 7.63 (dd, J = 1.2, 7.4 Hz, 1H), 7.59 (t, J = 7.7 Hz, 1H), 7.55−7.50 (m, 2H), 7.36 (t, J =
7.6 Hz, 1H), 7.20−7.14 (m, 2H), 6.87 (s, 1H), 6.54 (s, 1H), 6.35 (s, 2H), 5.96 (d, J = 7.1 Hz, 2H),
5.54 (dd, J = 4.5, 7.7 Hz, 1H), 4.64 (d, J = 4.7 Hz, 1H), 4.56−4.52 (m, 1H), 4.01 (t, J = 9.6 Hz,
1H), 3.87 (s, 3H), 3.82 (s, 3H), 3.77 (s, 6H), 3.12−3.06 (m, 1H), 3.03 (dd, J = 4.7, 14.3 Hz, 1H);
13C NMR (75 MHz, CDCl3) δ (ppm): 174.5, 165.8, 156.5, 152.5, 148.2, 147.5, 140.5, 139.5,
138.9, 137.0, 136.2, 134.9, 132.4, 132.3, 130.7, 129.7, 129.2, 128.8, 126.7, 122.0, 121.7, 120.7,
113.9, 112.0, 111.7, 109.9, 109.3, 108.0, 101.4, 69.2, 60.7, 56.2, 56.1, 47.9, 43.8, 41.8, 37.6; IR
(KBr): 3363.08, 2926.21, 2836.52, 2363.35, 1775.32, 1665.79. 1587.42, 1489.80, 1320.34 cm−1;
ESI-MS: m/z 714 [M+H]+; HRMS: m/z calcd. for C41H36N3O9 714.24515 [M+H]+, found
714.24414.
4.5.14.
N-((5S,5aS,8aR,9R)-8-Oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,8a,9-
hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-1-phenyl-9H-pyrido[3,4-b]indole-3-
carboxamide (7n)
o
1
White solid; 89% yield; mp: 314−318 C; [α]25 : –61.655 (c = 0.14, CHCl3); H NMR
D
(500 MHz, CDCl3) δ (ppm): 8.94 (s, 1H), 8.72 (s, 1H), 8.36 (d, J = 7.9 Hz, 1H), 8.25 (d, J = 7.9
Hz, 1H), 7.90–7.86 (m, 2H), 7.64–7.59 (m, 3H), 7.57–7.53 (m, 2H), 7.42–7.38 (m, 1H), 6.87 (s,
1H), 6.56 (s, 1H), 6.35 (s, 2H), 5.97 (dd, J = 1.2, 6.4 Hz, 2H), 5.55 (dd, J = 4.4, 7.7 Hz, 1H), 4.67
(d, J = 4.7 Hz, 1H), 4.54 (dd, J = 7.0, 9.0 Hz, 1H), 4.00 (t, J = 9.3 Hz, 1H), 3.82 (s, 3H), 3.77 (s,
13
6H), 3.16–3.08 (m, 1H), 3.05 (dd, J = 4.7, 14.3 Hz, 1H); C NMR (75 MHz, CDCl3) δ (ppm):
174.5, 165.7, 152.5, 1448.2, 1447.6, 141.4, 140.7, 139.2, 137.5, 137.1, 135.1, 134.9, 132.3,
130.4, 129.2, 129.1, 129.0, 128.2, 122.0, 121.1, 114.0, 111.8, 110.0, 109.3, 108.2, 101.4, 69.2,
22

ACCEPTED MANUSCRIPT
60.7, 56.2, 48.0, 43.8, 41.9, 37.7; IR (KBr): 3375.91, 2906.72, 2838.34, 2359.25, 1776.23,
1664.68, 1587.65, 1493.72, 1326.28, 1231.98 cm−1; ESI-MS: m/z 684 [M+H]+; HRMS: m/z
calcd. for C40H34N3O8 684.23459 [M+H]+, found 684.23421.
4.5.15.
N-((5S,5aS,8aR,9R)-8-Oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,8a,9-
hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-1-(pyridin-3-yl)-9H-pyrido[3,4-
b]indole-3-carboxamide (7o)
o
1
White solid; 83% yield; mp: 210−215 C; [α]25 : –66.417 (c = 0.12, CHCl3); H NMR
D
(500 MHz, CDCl3) δ (ppm): 10.41 (s, 1H), 9.34 (d, J = 1.5 Hz, 1H), 9.01 (s, 1H), 8.67 (dd, J =
1.5, 4.8 Hz, 1H), 8.31−8.22 (m, 3H), 7.64−7.52 (m, 3H), 7.43−7.39 (m, 1H), 6.88 (s, 1H), 6.58
(s, 1H), 6.35 (s, 2H), 5.98 (d, J = 1.6 Hz, 2H), 5.56 (dd, J = 4.5, 7.7 Hz, 1H), 4.68 (d, J = 4.7 Hz,
1H), 4.56 (dd, J = 7.3, 9.1 Hz, 1H), 4.01 (t, J = 9.4 Hz, 1H), 3.83 (s, 3H), 3.77 (s, 6H), 3.17−3.09
13
(m, 1H), 3.05 (dd, J = 4.8, 14.3 Hz, 1H); C NMR (75 MHz, CDCl3) δ (ppm): 174.4, 165.5,
152.6, 149.2, 148.8, 148.3, 147.6, 141.7, 139.2, 137.9, 137.2, 136.7, 135.6, 134.8, 134.2, 132.4,
131.0, 129.3, 124.4, 129.2, 122.0, 121.8, 121.1, 114.8, 112.1, 110.0, 109.3, 108.2, 101.5, 69.2,
60.7, 56.1, 48.1, 43.8, 42.0, 37.6; IR (KBr): 3372.38, 2935.22, 2359.45, 1776.36, 1660.54,
1588.61, 1482.84, 1330.73, 1233.79 cm−1; ESI-MS: m/z 685 [M+H]+; HRMS: m/z calcd. for
C39H33N4O8 685.22984 [M+H]+, found 685.22920.
4.5.16.
N-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,8a,9-
hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-1-(3-phenoxyphenyl)-9H-
pyrido[3,4-b]indole-3-carboxamide (7p)
o
1
White solid; 89% yield; mp: 197−200 C; [α]25 : –68.652 (c = 0.14, CHCl3); H NMR
D
(500 MHz, CDCl3) δ (ppm): 8.93 (s, 1H), 8.73 (s, 1H), 8.28 (d, J = 7.9 Hz, 1H), 8.23 (d, J = 7.9
Hz, 1H), 7.64−7.59 (m, 2H), 7.55 (t, J = 7.6 Hz, 2H), 7.51 (t, J = 2.2 Hz, 1H), 7.41−7.34 (m,
3H), 7.20−7.13 (m, 2H), 7.04 (dd, J = 1.0, 8.6 Hz, 2H), 6.86 (s, 1H), 6.58 (s, 1H), 6.36 (s, 2H),
5.97 (dd, J = 1.2, 9.6 Hz, 2H), 5.54 (dd, J = 4.7, 7.7 Hz, 1H), 4.68 (d, J = 4.8 Hz, 1H), 4.53 (dd, J
= 7.4, 9.1 Hz, 1H), 3.97 (dd, J = 9.4, 10.6 Hz, 1H), 3.83 (s, 3H), 3.78 (s, 6H), 3.15−3.06 (m, 1H),
3.00 (dd, J = 4.8, 14.3 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ (ppm): 174.4, 165.6, 158.2, 156.4,
152.5, 148.2, 147.5, 140.6, 139.1, 137.1, 134.9, 132.3, 130.6, 130.5, 129.8, 129.2, 129.1, 123.9,
23

ACCEPTED MANUSCRIPT
122.6, 122.0, 121.9, 121.1, 119.2, 119.0, 118.1, 114.2, 111.8, 110.0, 109.2, 108.1, 101.4, 69.1,
60.7, 56.1, 48.0, 43.8, 41.9, 37.6; IR (KBr): 3347.51, 2903.88, 2361.01, 1775.65, 1662.69,
1581.60, 1486.75, 1439.03, 1320.10, 123.42 cm−1; ESI-MS: m/z 776 [M+H]+; HRMS: m/z calcd.
for C46H38N3O9 776.26080 [M+H]+, found 776.26049.
4.5.17.
1-(3,4-Dimethoxyphenyl)-N-((5S,5aS,8aR,9R)-8-oxo-9-(3,4,5-trimethoxyphenyl)-
5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl)-9H-pyrido[3,4-
b]indole-3-carboxamide (7q)
1
White solid; 81% yield; mp: 195−200 oC; [α]25 : –62.679 (c = 0.06, CHCl3); H NMR
D
(500 MHz, CDCl3) δ (ppm): 8.89 (s, 1H), 8.75 (s, 1H), 8.40 (d, J = 7.4 Hz, 1H), 8.24 (d, J = 7.8
Hz, 1H), 7.62 (t, J = 7.4 Hz, 1H), 7.56 (d, J = 8.0 Hz, 1H), 7.47 ( dd, J = 1.7, 8.1 Hz, 1H), 7.39
(t, J = 7.5 Hz, 1H), 7.37 (d, J = 1.7 Hz, 1H), 7.07 (d, J = 8.3 Hz, 1H), 6.90 (s, 1H), 6.57 (s, 1H),
6.35 (s, 2H), 5.96 (d, J = 12.2 Hz, 2H), 5.49 (dd, J = 4.4, 7.2 Hz, 1H), 4.65 (d, J = 4.7 Hz, 1H),
4.56 (dd, J = 7.4, 8.9 Hz, 1H), 4.03 (t, J = 9.7 Hz, 1H), 3.97 (s, 3H), 3.89 (s, 3H), 3.82 (s, 3H),
13
3.77 (s, 6H), 3.16−3.08 (m, 1H), 3.04 (dd, J = 4.7, 14.1 Hz, 1H); C NMR (75 MHz, CDCl3) δ
(ppm): 174.4, 165.8, 152.5, 150.1, 149.6, 148.2, 147.5, 141.2, 140.6, 139.1, 137.1, 135.0, 134.8,
132.4, 130.3, 130.2, 129.3, 129.0, 122.2, 121.1, 120.3, 113.6, 111.8, 111.6, 111.4, 110.1, 109.2,
108.1, 101.5, 69.3, 60.7, 56.1, 56.0, 55.9, 48.2, 43.8, 41.9, 37.8; IR (KBr): 3359.82, 2931.73,
2836.63, 1776.63, 1665.57, 1588.31, 1512.45, 1485.38, 1329.72, 1234.69 cm−1; MS (ESI): 744
[M+H]+; HRMS(ESI): m/z calcd. for C42H38N3O10 744.25572, found 744.25549 [M+H]+.
4.6. Biology
4.6.1. Cytotoxic assay
The cytotoxic activity of the 7a‒q was determined using MTT assay [24]. 1×104 cells/well were
seeded in 100µl DMEM or MEM, supplemented with 10% FBS in each well of 96-well
microculture plates and incubated for 24 h at 37 °C in a CO2 incubator. After 24 h of incubation,
all the synthesized congeners were added to the cells and incubated for 48 h. After 48 h of drug
treatment, 10 µl MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) (5
mg/mL) was added to each well and the plates were further incubated for 4 h. Then the
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supernatant from each well was carefully removed, the formazon crystals were dissolved in 100
µl of DMSO and absorbance at 570 nm wavelength was recorded.
4.6.2. Cell cycle analysis
Flow cytometric analysis (FACS) was performed to evaluate the distribution of the cells in
different cell cycle phases. DU-145 cells were incubated with 7i and 7j congeners at 1 µM
concentration for 48 h. Untreated cells were considered as control sample. Untreated and treated
cells were harvested, washed with PBS, fixed in ice cold 70% ethanol and stained with
propidium iodide (Sigma Aldrich). Cell cycle was performed by using Becton Dickinson FACS
Caliber (USA).
4.6.3. DNA Topo II inhibition assay
In order to determine the effect of 7i and 7j in topoisomerase II inhibition and the formation
efficiency of decatenated kDNA was studied using the protocol mentioned in topoisomerase II
Drug Screening Kit (TG 1009, Topogen, USA). Topoisomerase II inhibition was assayed using
the ATP dependent decatenation of kDNA and all the reactions were carried out in 20 µl and
contained 120 mM KCl, 50 mM Tris–HCl, pH 8, 10 mM MgCl2, 0.5 mM dithiothreitol, 0.5 mM
ATP, 30 mg/ml bovine serum albumin, 200–300 ng of kDNA, and topoisomerase II. The amount
of topoisomerase II (5 units) was adjusted in preliminary experiments to decatenate
approximately 100% of the kDNA under these assay conditions. The reactions samples were
incubated at 37 oC for 30 min and terminated by the addition of 2 µl of a stop buffer containing
o
10% (w/v) SDS and 2µl of 0.5 mg/ml proteinase-K and incubated for 10 min at 37 C. After
completion of the reaction, the products in the reaction mixture were separated by 1% agarose
gel and visualized after staining with ethidium bromide (0.2l g/ml). The gels were run at 100 V
for about 40 min and visualized under UV transillumination (BIO RAD gel doc XR+, USA).
4.6.4. Comet assay
The DU-145 cells were seeded in 60 mm dishes and were treated with etoposide and 7i and 7j
hybrids at a concentration for 10 µM concentration for 24 h. Briefly, about 25,000 cells were
embedded in agarose and deposited in microscope slides. The slides were incubated for 2 hours
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in lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH 10, 10% DMSO, 1% Triton X-
100) followed by 3 washes in neutralizing buffer (0.4 M Tris–HCl, pH 7.5). Electrophoresis was
carried out for 20 min at 40 V in 0.5% TBE buffer (pH 8). During electrophoresis the damaged
DNA migrates away from the nucleus towards anode. Slides were then stained with a 10 mg/mL
propidium iodide solution for 30 min. Images from were taken in fluorescent microscope at 20x
magnification.
4.6.5. DNA binding studies
4.6.5.1. UV-Visible spectroscopy
UV-visible absorption spectra were recorded using Perkin Elmer ABI 35 Lambda
o
Spectrophotometer (Waltham, MA, USA) at 25 C. All the titrations were carried out in
polystyrene cuvettes to minimize binding of conjugates to the surface of the cuvettes. Then, 50
µM of 7i and 7j stock solution was prepared in DMSO and 25.0 µM of CT-DNA in 100 mM
Tris-HCl (pH 7.0). About 1 ml of 25.0 µM conjugate solution was taken in a 1 cm path length
cuvette and 5 µl of DNA was added each titration. All the stock solutions used were freshly
prepared before commencing the experiment, titration was carried out until saturation of
absorbance occurs and the absorption spectra recorded in the range of 200 nm to 400 nm.
4.6.5.2. Fluorescnece titration.
Fluorescence emission spectra were measured at 25 oC using a Hitachi F7000 spectrofluorimeter
(Maryland, USA) using a 1 cm path length quartz cuvette. Initially, quartz cuvettes was
thoroughly washed with distilled water and dilute nitric acid (approximately 0.1 N) to minimize
non-specific binding of the conjugates to the surface of the cuvette. Throughout the fluorescence
experiment, concentration of the 7i and 7j were kept constant (10 µM) and titrated with equally
increasing concentrations of CT-DNA (multiples of 0.5 µM). Fluorescence spectra were
recorded after each addition of CT-DNA to the fluorescent cuvette. The complexes were excited
at 230 nm and emission spectra for each titration was recorded up to 600 nm. The average of
three scans was taken after recording three individual spectrum.
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4.6.5.3. Circular dichorism (CD) studies.
DNA conformational studies were carried out on a JASCO 815 CD spectropolarimeter (Jasco,
Tokyo, Japan). CD Spectroscopic studies were performed to study the change in DNA
conformation brought by 7i and 7j molecules on interaction with CT-DNA at micro molar
concentration range. The CT-DNA solution was prepared in 100 mM Tris-HCl (pH 7.0) and 10
µM of CT-DNA added about 10.0 µM and 20 µM ( 1:1 and 1:2 ratio of DNA:complex) of each
solution containing 7i and 7j molecules was added and CD spectra was recorded from 200 nm to
350 nm using 1 mm path length cuvette. The spectra were averaged over 3 scans.
4.6.6. Docking studies
The active congeners 7i and 7j were built in Maestro 10.4 [35], prepared using Ligprep 3.6 [36]
and geometrically minimized with Macromodel 11.0 [37] followed by conformational analysis.
Truncated Newton Conjugate Gradient minimization was used with 500 iterations and
convergence threshold of 0.05 kJ/mol. The DNA topoisomerase-IIα (PDB ID: 1ZXM) and
d(CGCGAATTCGCG)2 (PDB ID: 1DNH) duplex were prepared using protein preparation
wizard [38]. The Glide XP 6.9 [39] algorithm was employed using a grid box volume of 10x 10x
10 Å. Briefly, Glide approximates a systematic search of positions, orientations and
conformations of the ligand in the receptor binding site using a series of hierarchical filters.
Upon completion of each docking calculation, 100 poses per ligand were generated and the best
docked structure was chosen using a GlideScore (Gscore) function.
Acknowledgments
The authers acknowledge the Council of Scientific and Industrial Research (CSIR), New Delhi
(India), for financial support under the 12th Five Year Plan project “Affordable Cancer
Therapeutics (ACT)” (CSC0301). M.S. and Y.T. are also thankful to the University Grant
Commission (UGC), Delhi for providing the research fellowships and we extend our
appreciation to the International Scientific Partnership Program ISPP at King Saud University for
funding this research work through ISPP#0054.
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References
[1] (a) Chikamori, K.; Grozav, A. G.; Kozuki, T.; Grabowski, D.; Ganapathi, R.; Ganapathi,
M. K.; Curr. Cancer. Drug Targets 2010, 10, 758; (b) Christodoulou, M.S.; Zarate, M.;
Ricci, F.; Damia, G.; Pieraccini, S.; Dapiaggi, F.; Sironi, M.; Lo Presti, L.; García-
Argáez, A. N.; Dalla Via, L.; Passarella, D. Eur. J. Med. Chem. 2016, 118, 79; (c) Kwon,
H. B.; Park, C.; Jeon, K. H.; Lee, E.; Park, S. E.; Jun, K. Y.; Kadayat, T. M.; Thapa, P.;
Karki, R.; Na, Y.; Park, M. S.; Rho, S. B.; Lee, E. S.; Kwon, Y. J. Med. Chem. 2015, 58,
1100; (d) Yao, B. L.; Mai, Y. W.; Chen, S. B.; Xie, H. T.; Yao, P. F.; Ou, T. M.; Tan, J.
H.; Wang, H. G.; Li, D.; Huang, S. L.; Gu, L. Q.; Huang, Z. S. Eur Eur. J. Med. Chem.
2015, 92, 540; (e) Bailly, C. Chem. Rev. 2012, 112, 3611; (f) Luniewski, W.; Wietrzyk,
J.; Godlewska, J.; Switalska, M.; Piskozub, M. Peczynska-Czoch, W.; Kaczmarek, L.
Bioorg. Med. Chem. Lett. 2012, 22, 6103; (g) Ferri, N.; Radice, T.; Antonino, M.;
Beccalli, E. M.; Tinelli, S.; Zunino, F.; Corsini, A.; Pratesi, G.; Ragg, E. M.; Gelmi, M.
L.; Contini, A. Bioorg. Med. Chem. 2011, 19, 5291; (h) Wu, A.; Xu. Y.; Qian, X.
Monatsh. Chem. 2010, 141, 893; (i) Pommier, Y.; Leo, E.; Zhang, H.; Marchand, C.
Chem. Biol. 2010, 17, 421. (j) Ferri, N.; Beccalli, E. M.; Contini, A.; Corsini, A.;
Antonino, M.; Radice, T.; Pratesi, G.; Tinelli, S.; Zunino, F.; Gelmi, M. L. Biorg. Med.
Chem. 2008, 16, 1691; (k) Kamal, A.; Narayan Reddy , B. S.; Suresh Kumar Reddy, G.;
Ramesh, G. Bioorg. Med. Chem. Lett. 2002, 12, 1933.
[2] Nitiss, J. L. Nat. Rev. Cancer. 2009, 9, 338.
[3] Iwai, M.; Hara, A.; Andoh, T.; Ishida, R. FEBS Lett. 1997, 406, 267.
[4] Pommier, Y.; Leo, E.; Zhang, H.; Marchand, C. Chem. Biol. 2010, 17, 421.
[5] You, Y. Curr. Pharm. Des. 2005, 11, 1695.
[6] (a) Cortese, F.; Bhattacharyya, B.; Wolf, B. J. Biol. Chem. 1977, 252, 1134; (b) Desbène,
S.; Giorgi-Renault, S. Curr. Med. Chem. Anti-Cancer Agents 2001, 1, 71.
[7] (a) Beers, S. A.; Imakura, Y.; Dai, H. J.; Li, D. H.; Cheng, Y. C.; Lee, K. H. J. Nat. Prod.
1988, 51, 901; (b) Laatsch, H.; Ernst, B. P.; Noltemeyer, M. Liebigs Ann. 1996, 731.
[8] Hande, K. R. Eur. J. Cancer. 1998, 34, 1514.
[9] Emmenggger, H.; Stahelin, H.; Rutschmann, J.; von Wartburg, A. Arzneimittel Forsch
1961, 11, 327.
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