Synthesis, characterization and biological evaluation of novel dihydropyranoindoles improving the anticancer effects of HDAC inhibitors

Article abstract of DOI:10.3390/molecules25061377

The dihydropyranoindole scaffold was identified as a promising target for improving the anti-cancer activity of HDAC inhibitors from the preliminary screening of a library of compounds. A suitable methodology has been developed for the preparation of novel dihydropyranoindoles via the Hemetsberger indole synthesis using azido-phenylacrylates, derived from the reaction of corresponding alkynyl-benzaldehydes with methyl azidoacetate, followed by thermal cyclization in high boiling solvents. Anti-cancer activity of all the newly synthesized compounds was evaluated against the SH-SY5Y and Kelly neuroblastoma cells as well as the MDA-MB-231 and MCF-7 breast adenocarcinoma cell lines. Biological studies showed that the tetracyclic systems had significant cytotoxic activity at higher concentration against the neuroblastoma cancer cells. More importantly, these systems, at the lower concentration, considerably enhanced the SAHA toxicity. In addition to that, the toxicity of designated systems on the healthy human cells was found to be significantly less than the cancer cells.

Full text of DOI:10.3390/molecules25061377

molecules
Article
Synthesis, Characterization and Biological Evaluation
of Novel Dihydropyranoindoles Improving the
Anticancer Effects of HDAC Inhibitors
Murat Bingul 1,2,3 , Greg M. Arndt 2,4, Glenn M. Marshall 2,5, Belamy B. Cheung 2,6,
* ,
Naresh Kumar 1,* and David StC. Black 1,
*
1
School of Chemistry, UNSW Sydney, Sydney, NSW 2052, Australia; muratbingul1983@gmail.com
Children’s Cancer Institute, Lowy Cancer Research Centre, UNSW Sydney, Sydney, NSW 2052, Australia;
garndt@ccia.unsw.edu.au (G.M.A.); gmarshall@ccia.unsw.edu.au (G.M.M.)
School of Pharmacy, Dicle University, 21280 Diyarbakır, Turkey
ACRF Drug Discovery Centre for Childhood Cancer, Children’s Cancer Institute, Lowy Cancer Research
Centre, UNSW Sydney, Sydney, NSW 2052, Australia
Kids Cancer Centre, Sydney Children’s Hospital, Randwick, NSW 2031, Australia
School of Women’s and Children’s Health, UNSW Sydney, Sydney, NSW 2052, Australia
Correspondence: bcheung@ccia.unsw.edu.au (B.B.C.); n.kumar@unsw.edu.au (N.K.);
d.black@unsw.edu.au (D.S.B.); Tel.: +61-2-9385-2450 (B.B.C.); +61-2-9385-4698 (N.K.);
+61-2-9385-4657 (D.S.B.)
2
3
4
5
6
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Qiao-Hong Chen
Received: 28 February 2020; Accepted: 16 March 2020; Published: 18 March 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: The dihydropyranoindole scaffold was identified as a promising target for improving the
anti-cancer activity of HDAC inhibitors from the preliminary screening of a library of compounds.
A suitable methodology has been developed for the preparation of novel dihydropyranoindoles
via the Hemetsberger indole synthesis using azido-phenylacrylates, derived from the reaction of
corresponding alkynyl-benzaldehydes with methyl azidoacetate, followed by thermal cyclization in
high boiling solvents. Anti-cancer activity of all the newly synthesized compounds was evaluated
against the SH-SY5Y and Kelly neuroblastoma cells as well as the MDA-MB-231 and MCF-7 breast
adenocarcinoma cell lines. Biological studies showed that the tetracyclic systems had significant
cytotoxic activity at higher concentration against the neuroblastoma cancer cells. More importantly,
these systems, at the lower concentration, considerably enhanced the SAHA toxicity. In addition to
that, the toxicity of designated systems on the healthy human cells was found to be significantly less
than the cancer cells.
Keywords: dihydropyranoindole; anticancer; HDAC inhibitors; neuroblastoma; breast cancer
1. Introduction
Chemotherapy is one of the most widely used treatments for high-risk cancer patients [
range of well-known agents, namely Doxorubicin, Cyclophosphamide, Etoposide have been used
to combat various cancers in modern chemotherapeutic therapy [ ]. However, a major problem
faced in chemotherapy is resistance to commonly-used anti-cancer drugs [ ]. Therefore, development
1,2]. A
3,4
5
of novel anti-cancer chemotherapeutic agents is of utmost importance in the area of drug discovery
and development.
The histone deacetylase (HDAC) inhibitors are a class of chemotherapeutic agents that hold
promise in cancer therapy [
angiogenesis, induce cell differentiation and promote apoptosis in a number of cancer cell types [
Suberoylanilide hydroxamic acid (SAHA) is the first HDAC inhibitor to obtain meet FDA approval [10],
6
,7
]. HDAC inhibitors have been reported to suppress cell proliferation and
8,9].
Molecules 2020, 25, 1377; doi:10.3390/molecules25061377
www.mdpi.com/journal/molecules

Molecules 2020, 25, 1377
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and has been considered to be a highly promising anticancer therapeutic agent due to its potent
cytotoxic effect on a number of tumor cell types as well as low toxicity towards healthy normal
cells [11
several cancers [14
agents with different mechanisms of action has been considered to be more promising as the drug
resistance caused by single agent therapies may be overcome [14 16]. Due to the significant cytotoxic
–
13]. However, single agent treatment with SAHA has been found to be ineffective against
,
15]. On the other hand, the combination of SAHA with other chemotherapeutic
,
effects observed during clinical trials of SAHA in combination with a variety of chemotherapeutic
agents, many synthetic compounds have been produced and screened to identify molecules capable of
enhancing the cytotoxic activity of SAHA, while producing fewer side effects.
In order to provide a basis for this study, a subset (10,560 compounds) of the Walter & Eliza Hall
Institute (WEHI) compound library was screened to identify molecules that can act synergistically
with a clinical dose of SAHA to overcome drug resistance in SAHA-resistant MDA-MB-231 breast
cancer cell lines [17]. The compounds that reduced viability to <40% in the presence of SAHA but to
>70% in the absence of SAHA, with a difference of at least 55% between the two conditions have been
identified as hit molecules for their SAHA enhancing capability [17]. A structural analysis of the hit
molecules demonstrated that the main structural feature to be the presence of 5- or 6-membered fused
heterocyclic systems, most commonly containing one or more nitrogen atoms. Furthermore, these
fused systems typically involved 3 or 4 conjoined rings. Due to the ongoing experiments regarding the
hit molecules, the structures of these compounds are not discussed in this manuscript.
Identification of New Target Molecules
The starting point of the current work was the selection of a range of compounds exhibiting
structural similarity to the hits from compound library in order to expand the structural diversity of
the screening set. The six related monomeric dihydropyranoindoles
1–3 and dimeric furoquinolines
4–6
were selected in order to understand the effect of the indole and quinoline structures, as well as the
fused dihydropyran and furan rings, on biological activities (Figure 1). The molecules were tested
against SH-SY5Y neuroblastoma and MDA-MB-231 breast cancer cell lines, using the Alamar blue
(Resazurin) assay [18] to measure the cell viability. The same screening conditions were used with
WEHI screening. Briefly, the designated cells were exposed to 1
combination of SAHA and compound for 72 h.
µM SAHA, 10 µM compound and the
OMe
O
O
O
O
O
OMe
N
OMe
O
H
N
N
OMe
MeO
MeO
H
H
2
1
3
Br
O
O
O
N
O
O
N
O
O
N
O
O
4
Br
6
5
Figure 1. Six compounds from the NK library.
The in vitro assays revealed that the MDA-MB-231 were the more resistant cell lines than the
SH-SY5Y cell lines for the single and combination treatments of all compounds. The compounds
and , analogues of quinolines, displayed the lowest reduction on SH-SY5Y cell viability and also
no SAHA enhancement was found by the combination with SAHA (Figure S1A, see Supplementary
5
6

Molecules 2020, 25, 1377
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Materials). In the case of compound
6
, the single and combination treatments showed no inhibition of
MDA-MB-231 cell viability, while the 20% and 5% of inhibitions were obtained with the treatment
of compound alone and combination with 1 M SAHA (Figure S1B, see Supplementary Materials).
The compound was the most active ligand among the quinoline candidates, with 30% inhibition of
5
µ
4
SH-SY5Y cell viability in the absence of SAHA and 20% additional cytotoxic effect in the presence of
SAHA (Figure S1A, see Supplementary Materials). The designated compound displayed 25% reduction
on MDA-MB-231 cells, while no SAHA enhancement effect was obtained against the same cell line
(Figure S1B, see Supplementary Materials).
Overall,
was concluded that the indole heterocyclic systems
SAHA enhancement with the higher differential values between in the absence and presence of SAHA
In order to further validate these compounds and as viable lead structures, it was essential to
1,
3
and
4
were determined as the most promising compounds as SAHA enhancer and it
1
and were found to be a potential target for the
3
1,
3
4
determine their toxicity towards normal and non-malignant cells. The most effective ligands were
screened against the MRC-5 normal human lung fibroblast cells, as described for cytotoxic assay. This
screen showed that the toxicity of
of against the normal and cancer cells was not different (Figure S2, see Supplementary Materials).
The single treatment of compound showed the cytotoxic activity with the values of 33% and 28%
3 was found to be greater than 1 and 4 proposing that specificity
3
3
reduction against the SH-SY5Y and MDA-MB-231 cells respectively, while the toxicity of this compound
against the MRC-5 cells was 26% and 30% greater than the SH-SY5Y and MDA-MB-231 cancer cells.
However, compounds 1 and 4 displayed non-toxic behavior on the MRC-5 healthy human cells with
values of 103% and 98% viable cells.
Based on the results of screening against the cancer cells and toxicity study on normal cells,
the indole heterocyclic system
the enhancement of SAHA activity and compound
1
was identified as promising leads for further development on
was also determined to be non-toxic across
1
the healthy human cells. The main work described in this manuscript focused on the synthesis,
characterization and in vitro biological evaluation of a series of targeted compounds based on tricyclic
and tetracyclic dihydropyrano derivatives. The effectiveness of the novel compounds as single agents
and in combination with a clinical dose of SAHA was determined against neuroblastoma and breast
cancer cells.
2. Results and Discussion
The preparation of dihydropyranoindole systems was achieved by two synthetic methods,
and the generality of these pathways were discussed in this paper. 3,4-Dihydroxybenzaldehyde
7
has been given as an example for the representation of two possible methods which could
be used for the preparation of the desired pyranoindole methyl 5,10-dihydro-7H-dipyrano
[3,2-e:20,30-g]indole-6-carboxylate 10 (Figure 2). The first method was to prepare the compound
10 via methyl hydroxyindole-2-carboxylate
synthesis [19]. Methyl hydroxyindole-2-carboxylate
alkyne indole-ether which upon Claisen cyclization would afford the desired dihydropyranoindole
10. As an alternative pathway, aryl ether benzaldehyde 11 would be prepared via simple alkylation
of phenol with haloalkynes and the Hemetsberger indole synthesis would then be applied to build
8
which could be prepared by the Hemetsberger indole
8
on reacting with haloalkynes would give the
9
7
the indole moiety. It is anticipated that thermal cyclization would yield the desired compound 10 in
one step.

Molecules 2020, 25, 1377
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O
O
HO
HO
O
O
CO2Me
CO2Me
CO2Me
N
N
H
N
H
H
Method 1
HO
8
10
9
CHO
HO
7
Method 2
O
O
O
O
O
CHO
CO2Me
CO2Me
N
N
O
H
H
10
9
11
Figure 2. Two methods for the preparation of pyranoindole systems.
2.1. Preparation of Tetracyclic Dihydropyranoindole 10 Via (Method 1)
In the first synthetic method, methyl 5,6-dihydroxyindole-2-carboxylate
8 was generated via the
Hemetsberger indole synthesis by benzyl-protected benzaldehydes and the subsequent deprotection
afforded the corresponding hydroxyindoles. The 3,4-dihydroxybenzaldehyde was reacted with benzyl
bromide in the presence of potassium carbonate in acetone to afford the protected carbaldehydes 12 20
7
[
]
in 88% yield (Scheme 1). Treatment of 3,4-dibenzyloxy benzaldehyde 12 with methyl azidoacetate
in methanol, in the presence of strong basic environment (sodium methoxide) gave the vinyl azido
intermediate 13 in 59% yield. Thermal decomposition of the arylazido 13 was performed by heating
at reflux in xylene, generating the methyl 5,6-dibenzyloxyindole-2-carboxylate 14 in 71% yield.
Hydrogenolysis of the benzyl group was carried out by treating the compound 14 with 5% w/w
palladium on carbon under hydrogen atmosphere at room temperature for 2 h, to yield the desired
methyl 5,6-dihydroxyindole-2-carboxylate
with propargyl bromide in the presence of potassium carbonate in acetone (Scheme 1). The desired
dipropargyloxyindole intermediate was prepared in 61% yield. The Claisen cyclization of
8 [21] in 77% yield. The dihydroxyindole 8 was reacted
9
9
was explored in xylene, 1,2-dichlorobenzene and toluene. It was found that heating at reflux in
chlorobenzene gave the optimum result in terms of the completion of the reaction as well as the yield
and purity of the product. Using this approach, the desired tetracyclic dihydropyranoindole 10 was
isolated in 51% yield.
BnO
BnO
BnO
BnO
BnO
CHO
HO
HO
CHO
CO2Me
iii
ii
i
CO2Me
N3
N
H
BnO
14
13
7
12
iv
3
9
2
4
HO
HO
1
5
O
O
O
vi
v
CO2Me
CO2Me
CO2Me
6
N
H
N
11
N
O
H
H
7
8
8
10
10
9
Scheme 1. Reagents and conditions: (i) Benzyl bromide, K2CO3, DMF, reflux, overnight; (ii) Methyl
azidoacetate, NaOMe, anhyd. MeOH, <
10 ◦C, 4 h; (iii) Xylene, reflux, 2 h, (iv) 5% Pd/C, H2,
−
MeOH/THF, 2h, rt, (v) Propargyl bromide, K2CO3, acetone, reflux, 4 h, 61%, (vi) Chlorobenzene, reflux
2 h, 52%.

Molecules 2020, 25, 1377
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The possible reaction mechanism could be explained as in Figure 3.
Methyl
5,6-bis(prop-2-yn-1-yloxy)-1H-indole-2-carboxylate
9
undergoes an initial Claisen rearrangement
to generate intermediate
derivative (intermediate
A
, which subsequently enolizes to produce 5,6-dihydroxyindole
B). A double hydride shift in intermediate gives the keto
B
intermediate
C, which undergoes an electrocyclic ring closing reaction to form methyl
5,10-dihydro-2H-dipyrano[3,2-e:20,30-g]indole-6-carboxylate 10.
H
HO
HO
O
O
HO
HO
enolize
CO2Me
CO2Me
CO2Me
N
N
N
H
H
H
H
9
intermediate A
intermediate B
electrolytic
reaction
O
O
O
O
CO2Me
CO2Me
N
H
N
H
intermediate C
10
Figure 3. Possible reaction mechanism.
The 1H-NMR spectrum of compound 10 in CDCl3 showed a singlet at 3.96 ppm corresponding
to the methyl ester protons, and two doublets of doublets at 4.91 and 4.96 ppm assigned to the two
CH2 groups. A multiplet at 5.87–5.97 ppm corresponded to H3 and H9, while another multiplet at
6.72–6.80 ppm corresponded to H4 and H8. Furthermore, H3 appeared as a doublet at 7.19 ppm and
the NH proton appeared as a broad singlet at δ 8.85 ppm. The DEPT 135 spectrum of compound 10
confirmed the structure of the molecule, displaying the loss of two CH carbon signals as a result of
cyclization of the alkyne group as well as the appearance of two negative signals at 64.68 and 64.72
ppm corresponding to the methylene carbon atoms in the product.
2.2. Alternative Pathway for the Preparation of Tetracyclic Dihydropyranoindole 10 (Method 2)
The alternative approach began with the reaction of 3,4-dihydroxybenzaldehyde
bromide under basic conditions (potassium carbonate), which afforded the propargyloxy benzaldehyde
11 22] in 87% yield. At this point, two synthetic strategies using benzaldehyde 11 as a key intermediate
7 with propargyl
[
were envisioned (Scheme 2). The first route involved Claisen cyclization to build the dihydropyrano
rings 15, followed by the application of the Hemetsberger indole synthesis to construct the indole scaffold
via the unsaturated azido intermediate 16 (Route 1). The cyclization of the aryl ether intermediate
11 was attempted by refluxing in high-boiling solvents such as xylene, toluene, 1,2-dichlorobenzene
and chlorobenzene (Scheme 2). In all cases, the novel compound 15 was afforded in low yields, with
unreacted starting material being recovered as the major product. The highest yield (35%) was obtained
by the use of chlorobenzene. In order to synthesize the desired tetracyclic dihydropyranoindole 10, the
standard Hemetsberger indole synthesis was applied to new intermediate 15. The condensation of
benzaldehyde 15 with methyl azidoacetate was carried out at low temperature (ice-salt bath) in the
presence of sodium methoxide. The addition of a small amount of crushed ice to the reaction mixture
resulted in the isolation of azido ester 16 as an oily compound which was found to be unstable at room
temperature, and was hence used in the next step without further purification. It was postulated that
the presence of the dihydropyran ring caused the generation of an unstable azido ester intermediate.

Molecules 2020, 25, 1377
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Thermal cyclization of the azido ester 16 was performed by heating at reflux in xylene, which afforded
the desired dihydropyranoindole 10 in 62% yield.
O
O
CO2Me
O
O
CHO
b
N3
16
15
c
Route 1
d
O
O
HO
HO
CHO
O
a
CHO
CO2Me
N
O
H
7
11
10
Route 2
b
O
CO2Me
d
O
O
CO2Me
N
O
H
N3
9
17
Scheme 2. Reagents and conditions: (a) Propargyl bromide, K2CO3, acetone, reflux, 4 h, (b) Methyl
azidoacetate, metallic Na, anhyd. MeOH, <
10 ◦C, 2 h (c) Chlorobenzene, reflux, 2 h, (d) Xylene, reflux,
−
4 h.
In the second route, it was anticipated that the indole heterocyclic system could be constructed
first, followed by the installation of dihydropyrano rings onto the indole moiety. Since the cyclization
of both aryl ether and unsaturated azide ester moieties require refluxing conditions in a high-boiling
solvent, it was predicted that the indole and dihydropyran rings could be prepared in one step from the
key azide intermediate 17 (Route 2). The benzaldehyde 11 was condensed with methyl azidoacetate
under strongly basic conditions at low temperature to generate the novel unsaturated stable azide
intermediate 17 in 63% yield (Scheme 2). Since xylene was the common solvent for the generation
of the indole moiety from the azido ester intermediate, the cyclization reaction was first carried out
in refluxing xylene. As anticipated, the thermal cyclization of intermediate 17 resulted in both the
decomposition of the azidocinnamate moiety and formation of the indole heterocyclic system, as well
as the cyclization of the propargyloxy moiety to furnish the fused pyran ring in a single step. Hence,
the desired tetracyclic dihydropyranoindole 10 was obtained in a yield of 72%.
Taken altogether, it was concluded that the first method is a less favorable synthetic pathway
consisting of six steps to prepare the desired dihydropyranoindole and also generating the lowest
of yield (51%) at the final step. In the case of the second method, route 1 was also found to be
an unfavorable synthetic method for further synthesis due to the low-yielding cyclization step to
generate the new tricyclic aldehyde 15, the formation of the unstable azido intermediate 16 from the
Hemestberger indole synthesis and the four step pathway for the desired dihydropyranoindole 10. On
the other hand, route 2 of the second strategy was deemed to be the most favorable synthetic route
towards dihydropyranoindoles, as it contained three steps to afford the desired dihydropyranoindole
and gave the highest yield of 72%. Thus, route 2 of the second method was chosen for further studies
in this study.

Molecules 2020, 25, 1377
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2.3. The Preparation of Monomeric Hydropyrano System
The synthetic strategy (Route 2) was extended to the synthesis of indole systems containing a
single fused dihydropyran ring. Hence, 4-hydroxy-3-methoxy benzaldehyde 18 was reacted with
propargyl bromide in the presence of potassium carbonate to give aryl ether 19a [23] in 84% yield,
which was then reacted with methyl azidoacetate under basic conditions to give azidocinnamate 20a in
76% yield (Scheme 3). Interestingly, thermal cyclization of the 20a in xylene afforded the corresponding
propargyloxy indole 21a in the yield of 68% instead of the expected dihydropyranoindole system 22a
Further heating of indole 21a in refluxing chlorobenzene afforded the desired dihydropyranoindole
22a in 77% yield.
.
MeO
O
CO2Me
MeO
O
CHO
MeO
HO
CHO
b
a
N3
20
R1
R1
18
19
R3
R2
R2
R3
Compound
22a
R1
H
H
R2
H
H
R3
H
c
4
3
22b
Me
H
d
5
MeO
O
MeO
2
CO2Me
CO2Me
22c
Me Me
6
N
N
1
O
H
H
9
R3
R1
R2
R1
R2
7
21
8
R3
22
Scheme 3. Reagents and conditions: (a) Propargyl bromide or 3-chloro-3-methylbut-1-yne or
1-bromobut-2-yne, K2CO3, acetone, reflux, 4 h, (b) Methyl azidoacetate, metallic Na, anhyd. MeOH,
<−10 ◦C, 2 h (c) Xylene, reflux, 4 h, (d) Chlorobenzene, reflux, 2 h.
The 1H-NMR spectrum of compound 22a in CDCl3 showed the presence of two singlets at 3.94
ppm and 3.96 ppm corresponding to the methoxy and methyl ester protons, and doublets of doublets
at 4.96 ppm corresponding to the CH2 protons. Protons H8 and H9 appeared as multiplets in a range
of 5.89–5.95 ppm and 6.73–6.79 ppm respectively. Furthermore, H4 appeared as a singlet at 7.02 ppm,
while H3 was appeared as a doublet at 7.13 ppm (J = 2.1 Hz) due to its coupling with the NH proton,
which resonated as a broad singlet at 8.94 ppm. The DEPT 135 spectrum showed the presence of a
negative peak at 65.79 ppm corresponding to a methylene carbon atom. Furthermore, the spectrum
revealed the absence of the alkyne CH signal due to cyclization of the alkyne moiety in indole 21a.
The synthesis of tricyclic dihydropyranoindole systems containing methyl substituents
on the fused dihydropyran ring at different positions was also investigated. To achieve
this, 4-hydroxy-3-methoxybenzaldehyde 18 was reacted with 1-bromobut-2-yne and
3-chloro-3-methylbut-1-yne in the presence of potassium carbonate to give the aryl ether
aldehydes 19b [24] and 19c [25] in 92% and 84% yields, respectively (Scheme 3). The aldehydes
19b and 19c were treated with methyl azidoacetate in the presence of sodium methoxide to give
azidocinnamates 20b and 20c in yields of 61% and 62%. Finally, the thermal cyclization of the
unsaturated azide 20b and 20c in refluxing xylene generated the desired product 22c in 74% yields,
while the indole ether 21b was isolated in 67% yield. The desired dihydropyranoindole 22b was
obtained in 49% yield, by the further cyclization of 21b in refluxing chlorobenzene.
In the 1H-NMR spectrum of methyl 5-methoxy-7,7-dimethyl-1,7-dihydropyrano[2,3-g]indole-2-
carboxylate 22c,the two methyl groups appeared as a singlet at 1.55 ppm, while H8 and H9 appeared
as doublet signals at 5.71 and 6.66 ppm (J = 9.7 Hz). Furthermore, H4 appeared as a singlet at 7.01 ppm
and H3 appeared as a doublet at 7.13 ppm (J = 2.1 Hz), confirming that cyclization occurred at C7. In
the 1H-NMR spectrum of the cyclization product 22b, the olefinic proton on the dihydropyran ring
appeared as multiplet signals in the ranges of 5.65–5.68 ppm, due to coupling with the neighboring

Molecules 2020, 25, 1377
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CH2 and CH3 groups. For compound 22b, the O-CH2 protons resonated as a multiplet at 4.77 ppm
and the methyl protons of the dihydropyran ring appeared as a quartet at 1.71 ppm.
The same synthetic route was applied to 3-hydroxy-4-methoxy benzaldehyde 23 in order
to generate an analogue with the dihydropyran ring fused at a different position. Thus,
hydroxybenzaldehyde 23 was reacted with propargyl bromide in the presence of potassium carbonate
to generate the intermediate ether 24a [26] in 87% yield, which upon reaction with methyl azidoacetate
in strongly basic conditions gave the azidocinnamate 25a in 72% yield (Scheme 4). Heating the
azidocinnamate 25a in refluxing xylene gave the propargyloxy indole 26a in 73% yield, which was
then cyclized in chlorobenzene to afford the desired dihydropyranoindole 27a in 74% yield.
The 1H-NMR spectrum of compound 27a in CDCl3 showed two singlet signals at 3.92 and 3.93
ppm corresponding to the methoxy and methyl ester protons, and doublets of doublets at 4.90 ppm
corresponding to the CH2 protons. H8 and H9 appeared as multiplets at 5.89–5.95 and 6.76–6.79 ppm
respectively. The H4 appeared as a singlet at 6.77 ppm, while H1 appeared as a doublet at 7.17 ppm
(J = 2.1 Hz). Moreover, the NH proton resonated as a broad singlet at δ 8.96 ppm. The formation of
the desired dihydropyranoindole was further confirmed by DEPT 135 spectroscopy which revealed
the presence of a negative peak at 65.71 ppm corresponding to the CH2 group accompanied by the
disappearance of the signal corresponding to the CH group of the starting alkyne 26a.
3-Hydroxy-4-methoxybenzaldehyde 23 was further treated with 1-bromobut-2-yne and
3-chloro-3-methylbut-1-yne under basic conditions to generate the corresponding intermediates
24b [24] and 24c [27] 89% and 82% yields, respectively (Scheme 4). The aryl ethers 24b and 24c were
condensed with methyl azidoacetate in the presence of sodium methoxide to generate the azidoacrylates
25b and 25c in 64% and 62% yields respectively. Thermal decomposition of the unsaturated azides
25b gave the indole ethers 26b in 71%, while the desired hydropyrano compound 27c was directly
isolated in 68% yield. The desired dihydropyranoindoles 27b was obtained in 54% yields respectively,
by the cyclization of 26b and in refluxing chlorobenzene. It was also of interest to construct new
dihydropyranoindole systems containing substituents other than a methyl group on a pyran ring. The
haloalkyne, 3-phenylprop-2-yn-1-ol, was treated with 3-hydroxy-4-methoxybenzaldehyde 23 in the
presence of potassium carbonate to produce the alkylated carbaldehyde 24d in 92% yield (Scheme 4).
The reaction between the aryl ether 24d with methyl azidoacetate afforded the unsaturated azido ester
25d in 64% yield. Finally, heating compound 25d in refluxing xylene afforded the corresponding indole
ether 26d in 74% yield, which further underwent thermal cyclization in chlorobenzene to furnish the
desired dihydropyranoindole 27d in 81% yield.
In the 1H-NMR spectrum of methyl 5-methoxy-7,7-dimethyl-3,7-dihydropyrano[3,2-e]indole-2-
carboxylate 27c, the two methyl groups appeared as a sharp singlet at 1.53 ppm. The H8 and H9 of the
dihydropyran ring appeared as two doublets at 5.73 and 6.68 ppm that coupled to each other with a
coupling constant of 9.7 Hz. H1 appeared as a doublet (J = 2.1 Hz) at 7.20 ppm while H4 was assigned
as a singlet at 6.79 ppm. In the 1H-NMR spectrum of the cyclization product 27b the olefinic protons
on the dihydropyran ring appeared as multiplet signals in range of 5.69–5.72, due to coupling with
the neighboring CH2 and CH3 groups. The characteristic O-CH2 protons resonated as a multiplet at
4.77 ppm, while the methyl group of the dihydropyran ring appeared at as a quartet 1.63 ppm. The
1H-NMR spectrum of compound 27d displayed the O-CH2 protons as a doublet at 4.85 ppm with a
coupling constant of 4.4 Hz, and the olefinic proton as a multiplet at 5.94–5.97 ppm. The aromatic
protons of phenyl ring appeared as multiplet in the range of 7.34–7.44 ppm. The H4 and H1 appeared
as two singlets at 5.98 and 6.88 ppm.
Similar attempts were made to the prepare methyl substituted tetracyclic dihydropyranoindole
compounds from the 3,4-dihydroxybenzaldehyde
7, 1-bromobut-2-yne and 3-chloro-3-methylbut-1-yne.
The benzaldehyde was alkylated with 2 equivalents of 1-bromobut-2-yne in the presence of potassium
7
carbonate to generate the novel aryl ether 29 in 87% yield (Scheme 5). The benzaldehyde 29 was then
treated with methyl azidoacetate under strongly basic conditions to generate the azido compound
30 in 63% yield, which subsequently underwent thermal decomposition in refluxing xylene to afford

Molecules 2020, 25, 1377
9 of 24
the indole ether 31 in 65% yield. However, the cyclization of 31 could not be achieved in a number
of solvents, including chlorobenzene, toluene, xylene and 1,2-dichlorobenzene which resulted in the
formation of decomposed reaction mixture. The reaction between 3,4-dihydroxybenzaldehyde 7 and
3-chloro-3-methylbut-1-yne was investigated in an attempt to produce the dipropargyloxybenzaldehyde
intermediate 28 (Scheme 5). However, this reaction resulted in a black reaction mixture, presumably
due to decomposition.
R3
R3
R2
R2
R1
R1
O
CO2Me
O
CHO
HO
CHO
b
a
N3
MeO
MeO
MeO
25
24
23
c
R3
8
5
R2
R2
R1
R1
R3
9
7
1
d
O
O
6
CO2Me
CO2Me
2
N
N
MeO
MeO
H
H
3
4
26
27
Compound
R1
R2
R3
27a
27b
27c
27d
H
H
Me
H
H
H
Me
H
H
Me
H
Ph
Scheme 4. Reagents and conditions: (a) Propargyl bromide or 3-chloro-3-methylbut-1-yne
or1-bromobut-2-yne or 3-phenylprop-2-yn-1-ol, K2CO3, acetone, reflux, 4 h, (b) Methyl azidoacetate,
metallic Na, anhyd. MeOH, <−10 ◦C, 2 h (c) Xylene, reflux, 4 h, (d) Chlorobenzene, reflux, 2 h.
HO
CHO
HO
a
a
7
Me
Me
Me
O
Me
Me
O
O
CO2Me
O
O
CHO
b
CHO
N3
O
30
Me
Me
Me
29
Me
28
c
Me
Me
d
O
O
O
O
CO2Me
CO2Me
N
N
H
H
Me
32
31
Scheme 5. Reagents and conditions: (a) 1-Bromobut-2-yne or 3-Chloro-3-methyl-1-butyne, K2CO3,
acetone, reflux, 4 h, (b) methyl azidoacetate, metallic Na, anhdyrous MeOH,
10 ◦C, 2 h (c) Xylene,
reflux, 2 h, (d) Xylene, chlorobenzene, toluene, 1,2-dichlorobenzene reflux, overnight.
−

Molecules 2020, 25, 1377
10 of 24
2.4. Biological Study
2.4.1. Preliminary Biological Screening of Dihydropyranoindoles for SAHA Enhancement Activity
Novel dihydropyranoindole analogues were screened to determine the levels of SAHA
enhancement activity as well as their own cytotoxic profile against the SH-SY5Y and Kelly
neuroblastoma cells and the MDA-MB-231 and MCF-7 breast adenocarcinoma cell lines using the
Alamar blue (Resazurin) assay [18]. Briefly, the cells were allowed to attach for 24 h in a 96-well culture
plate before being exposed to the ligands at a concentration of 10 µM in DMSO for 72 h, either in the
presence or absence of SAHA. Comparative values for cell viability in each well were determined by a
Wallac 1420 Victor III spectrophotometer, which measured light absorbance in each well at 570 nm.
The mean and standard deviation (SD) values for each compound were calculated from at least three
replicate experiments. The anticancer activity of the compounds was evaluated by comparison to a
negative (DMSO) control. Figure 4 shows the tested novel dihydropyranoindoles synthesized in this
study and the compound 33 was selected from the NK library. In this assay, the cell lines were exposed
to SAHA, 10
µM of the compounds as well as the combination of SAHA with the compounds for 72 h.
O
MeO
CO2Me
O
CO2Me
CO2Me
CO2Me
N
H
O
CO2Me
N
O
N
H
MeO
H
27a
22a
33
Ph
CH3
MeO
CO2Me
O
O
N
O
CO2Me
H
N
N
MeO
MeO
CH3
H
H
27d
27b
22b
Me
Me
O
MeO
O
O
CO2Me
CO2Me
N
H
N
O
N
H
MeO
H
Me
Me
27c
22c
10
Figure 4. Synthesized and selected compounds.
According to the results from the screening, the neuroblastoma cells were found to be the
more sensitive cells towards the treatment of new pyranoindoles, while these compounds showed a
moderate effect on the breast cancer cell lines (Figures S3A,B and S4A,B, see Supplementary Materials).
MDA-MB-231 cells were determined as the most resistant cell line for the single as well as the
combination treatments (Figure S3A, see Supplementary Materials). The dihydropyranoindole 27c
exhibited the greatest cell viability inhibition with the values of 34% and 35% in the absence and
presence of SAHA against the MDA-MB-231 cells. The SAHA enhancement was achieved by the
additional reduction value of 20% across MDA-MB-231 viability. MCF-7 cells showed an identical
pattern of sensitivity. However, most of the dihydropyranoindoles were able to reduce the viability by
the value of 20% (Figure S3, see Supplementary Materials).
Compound 33 displayed the highest reduction with the 35% inhibition, while compounds 27c and
22c generated similar reductions with the values of 20% and 22% on the same cell line. Unfortunately
the combination of pyranoindoles with 1
values of 5% across the MCF-7 cell lines.
µM SAHA had very low SAHA enhancement by the average
It was observed that dihydropyranoindoles had a variety of impact on the viability of
neuroblastoma cells (Kelly and SH-SY5Y) with the single and combination treatments (Figure S4A,B,
see Supplementary Materials). Out of all the novel ligands synthesized, ligand 33 had the strongest
cytotoxic activity with the reduction value of 50% at 10 µM, while ligands 27c and 10 showed similar

Molecules 2020, 25, 1377
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inhibition behaviors by the average reduction value of 32% against Kelly cells for the single treatment.
The dihydropyranoindoles 27c and 33 displayed the enhancement of SAHA cytotoxicity with the
values of 24 and 22% respectively (Figure S4A, see Supplementary Materials). The value of 40%
SAHA enhancement was obtained by the combination of compound 33. However, the ligand 33 was
considered as self-toxic compound due to the reduction value for the single treatment. The levels of
individual cytotoxic efficiency against the SH-SY5Y were found to be higher than those observed on the
Kelly cells (Figure S4B, see Supplementary Materials). The compounds 27d and 10 showed the greatest
cytotoxic activity reducing 44% and 56% of the cells respectively. Most importantly, the enhancement
of SAHA activity was achieved by the treatment of almost all of the novel dihydropyranoindole
analogues showing at least 20% additional reduction to SAHA cytotoxicity. Although the highest
SAHA enhancement was obtained in the case of compound 10 with the value of 55%, it was not
considered as a promising enhancer since the single treatment also generated the greatest reduction
(56%) on the SH-SY5Y cell. The best enhancers were assigned as compounds 22a, 27a, 27c and 33 with
the reduction values of 25%, 29%, 30% and 31%respectively due to the lower toxicity but the higher
enhancement effects on SAHA cytotoxicity.
2.4.2. Determination of SAHA Enhancement Activity of Selected Dihydropyranoindoles at Lower
Concentrations
Since the levels of SAHA enhancement were found to be promising, further investigations were
carried out using five different concentrations (0.01, 0.1, 1, 10, 20 µM), using the Alamar blue (Resazurin)
assay [18] to determine whether the lower concentrations would provide higher enhancement of the
SAHA cytotoxicity or the cytotoxic manner would be dependent on the dose usage. The initial screening
revealed that the single treatment of dihydropyranoindoles 33
showed the highest cytotoxic efficiency against the Kelly cells. The designated compounds were treated
with the Kelly cells by the combinations of 0.5 M SAHA with the five different concentrations (0.01,
0.1, 1, 10, 20 M) and the results are shown at Figure S5A,C (see Supplementary Materials). In general,
the single treatment of these compounds provided lower cytotoxic activity at the concentrations of
0.01, 0.1 and 1 M compared with the values at 10 M and the cell viability differentials between
the absence and presence of SAHA were greater than those at 10 M concentrations, suggesting that
, 27c and 10 at a concentration of 10 µM
µ
µ
µ
µ
µ
the designated compounds behave as suitable SAHA enhancers (Figure S5A,C, see Supplementary
Materials). Similar SAHA enhancement pattern was observed at the concentrations of 0.01, 0.1 and
1 µM for all three compounds, while the combination of 1 µM compound 27c or 10 with the 0.5 µM
SAHA revealed the best SAHA enhancement with the values of 24% and 25% additional inhibition
(Figure S5B,C, see Supplementary Materials).
In addition to those two compounds, compound 33 showed the greatest viability differentials
with the values of 61%, 63% and 65% at the concentrations of 0.01, 0.1 and 1
enhancement was observed for all the concentrations. The lowest concentration of compound 33 (0.01
M) revealed a remarkable reduction with the value of 25%, while the best combination for the highest
µM and the desired SAHA
µ
SAHA enhancement was determined as a 10:0.5 ratio of compound and SAHA with the additional
reduction value of 55% (Figure S5 A, see Supplementary Materials). It was concluded that the highest
cell viability differentials was achieved by the use of nanomolar concentrations in the case of all three
compounds and the best SAHA enhancer was determined as compound 33.
Similarly, in the case of the SH-SY5Y cell line, further investigations were undertaken with 27a
33 and 27c which were chosen due to their highest additional effects on the SAHA cytotoxicity with
the lower toxicity behavior in the absence of SAHA. Compound 10 was also explored to determine
its cytotoxic behavior as well as the SAHA enhancement potential at lower concentrations. The
,
combinations of 1
µM SAHA with the five different concentrations (0.01, 0.1, 1, 10, 20 µM) of
designated compounds were treated with the SH-SY5Y cells and the results are shown at Figure S6A–D
(see Supplementary Materials).

Molecules 2020, 25, 1377
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The SAHA enhancement pattern was found to be identical on SH-SY5Y value for all cases of
combinations of 27a with SAHA. The highest viability differential was obtained in the ratio 1:10 of
SAHA to compound with the reduction value of 35% (Figure S6A, see Supplementary Materials). The
dose-dependent pattern of single treatments of 33 and 27c suggested that these compounds displayed
higher toxicity at 20
µM with the values of 45% and 55% reductions. Furthermore, the combinations
of these compounds at the concentrations of 0.01, 0.1 and 1
µM with SAHA enhanced the SAHA
cytotoxicity in a similar manner and the best combinations were found to be as the ratio 1:10 of SAHA
to compound with the additional reduction values of 28% and 33% (Figure S6B,C, see Supplementary
Materials). The greatest cytotoxicity was obtained at a concentration of 20 µM of 10 alone with the
reduction values of 88% on SH-SY5Y cell viability and the ratio of 1:20 SAHA to compound combination
provided 94% cell death (Figure S6D, see Supplementary Materials).
More importantly, at the lower concentrations (0.01, 0.1 and 1 µM) of 10, the cytotoxicity was
found to be lower with the average reduction value of 25%. Encouragingly, the biggest cell viability
differential was obtained at the ratio of 1:10 SAHA to compound with the value of 44%, and the greatest
SAHA enhancement was also found as 37% at the same combination. It was concluded that 10 was the
most cytotoxic compound at higher concentration (20 µM, 88% reduction) and also it was found to be
the best SAHA enhancer at the ratio of 1:10 SAHA to compound combination.
2.4.3. SAR Study of Selected Dihydropyranoindoles
These observations suggested an important structure-activity relationship among 27a, 33, 27c
and 10. That is, the incorporation of tetracyclic dihydropyranoindoles 33 and 10 resulted in lower cell
viability reductions at higher concentrations (20 µM) and also this system was found to offer the best
enhancement with the ratio of 1:10 SAHA to compound combinations against both neuroblastoma
cancer cells (Kelly and SH-SY5Y). The SAR analysis revealed that the location of dihydropyran ring on
the benzene ring was important for the cytotoxic efficiency of tricyclic dihydropyranoindoles, The
compounds 27c, an example of dihydropyrano[3,2-e]indole system, was found to be more potent than
the dihydropyranoindole 22c which is a member of dihydropyrano[2,3-g]indole scaffold against both
neuroblastoma cells (Kelly and SH-SY5Y). In comparison of two tricyclic dihydropyranoindoles 27c
and 22c at 20
µM, it was found that the compound 27c reduced 20% more SH-SY5Y cell with the
combination of 1
µM SAHA than compound 22c, while the 20% SAHA enhancement was achieved
using the combination of compound 27c and SAHA but no SAHA enhancement was found with the
compound 22c in the case of Kelly cells. Furthermore, the dihydropyrano[2,3-g]indole analogues 22a
and 22b displayed the lowest reduction on Kelly cells in the presence and absence of SAHA. Similar
results were observed in the case of SH-SY5Y cells with the exception of 21% additional reduction on
SAHA enhancement obtained by the use of 22a.
2.4.4. Toxicity Study Against Normal Cells
While selected dihydropyranoindoles showed potent SAHA enhancement activity against Kelly
and SH-SY5Y neuroblastoma and MCF-7 and MDA-MB-231 breast cancer cells, they must be markedly
less toxic against the healthy cells in order to be considered as potential SAHA enhancers. Thus, the
dihydropyranoindoles 27a, 33, 27c and 10 were also examined for their toxicity effects on the MRC-5
and WI-38 lung fibroblasts in order to determine whether these compounds exhibited selectivity for
tumor cells (Figure S7A–D, see Supplementary Materials). Comparison of the cytotoxic activity and
toxicity levels of the dihydropyranoindoles against cancer cells and the healthy cells revealed that
the cancer cells were more sensitive to the dihydropyranoindoles compared to the normal cells. The
toxicity of 1
respectively, while 27a alone displayed a similar pattern of toxicity against normal cells with values
of 10% for MRC-5 and 22% for WI-38 at a concentration of 10 M (Figure S7A, see Supplementary
µM of SAHA reduced the viability of MRC-5 and WI-38 cells with the value of 9% and 24%
µ
Materials). The viability of MRC-5 and WI-38 were reduced 21% and 16%, 3% and 26% by the use of
33 and 10 alone (Figure S7B,C, see Supplementary Materials), while the highest toxicity levels were

Molecules 2020, 25, 1377
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obtained in the case of 27c with the reduction values of 20% and 43% (Figure S7D, see Supplementary
Materials). The combination treatments were found to be quite identical to the reduction values of
single treatments of each compound. These observations demonstrated that 27a, 33 and 10 were either
less toxic or slightly less toxic than SAHA, while 27c displayed more toxicity than SAHA for both
normal cells.
3. Materials and Methods
3.1. General Information
Commercially available reagents were purchased from Fluka (Sydney, NSW, Australia), Aldrich
(Sydney, NSW, Australia), Acros Organics (Morris Plains, NJ, USA), Alfa Aesar (Lancashire, UK) and
Lancaster (Lancashire, UK) and purified if necessary. The synthetic procedures have been reported for
all compounds as general methods and appropriate references have been given for known compounds.
1H (300 MHz) and 13C-NMR (75 MHz) spectra were obtained in the designated solvents on a DPX
300 spectrometer (Bruker, Sydney, NSW, Australia). Melting points were measured using a Mel-Temp
melting point apparatus and are uncorrected. Infrared spectra were recorded on Avatar Series FT-IR
spectrophotometer as KBr disks (Thermo Nicolet, Waltham, MA, USA). Ultraviolet spectra were
measured using a Cary 100 spectrophotometer (Varian, Santa Clara, CA, USA) in the designated
) in cm−1M−1
solvents and data reported as wavelength (λ) in nm and adsorption coefficient (ε .
High-resolution [ESI] mass spectra were recorded by the UNSW Bioanalytical Mass Spectrometry
Facility, on an Orbitrap LTQ XL (Thermo Scientific, Waltham, MA, USA) ion trap mass spectrometer
using a nanospray (nano-electrospray) ionization source.
3.1.1. GP-1: General Procedure for the Benzylation of Dihydroxybenzaldehydes
A solution of hydroxybenzaldehyde (1 equiv.), anhydrous potassium carbonate (per hydroxyl
group, 1 equiv.) and benzyl bromide (per hydroxyl group, 1 equiv.) in anhydrous DMF (100 mL)
was heated at reflux until TLC analysis showed consumption of the starting aldehyde (9 h). Upon
cooling to room temperature, the reaction mixture was diluted with water (300 mL) and the resulting
precipitate was collected via filtration and washed with water (2
was recrystallized from dichloromethane and n-hexane to give the title compound.
×
250 mL). Upon drying, the residue
3.1.2. GP-2: General Procedure for the Preparation of Vinyl-Azido Intermediates
A solution of sodium methoxide was prepared via the portion-wise addition of metallic sodium
(17 equiv.) to anhydrous methanol (30 mL) with stirring under nitrogen. A dropping funnel was
attached to the reaction flask and charged with the corresponding aldehyde (1 equiv.) and methyl
azidoacetate (10 equiv.) in methanol (15 mL). The contents of the funnel were added dropwise to the
sodium methoxide solution over 1.5 h under a nitrogen atmosphere. Once the addition was completed,
the reaction mixture was warmed to 5 ◦C, where it remained for 4 h. The heterogeneous mixture was
poured into crushed ice. The resulting precipitate was filtered, washed with water and dried to give
the title compound. The crude product was used in the next step without further purification.
3.1.3. GP-3: General Procedure for the Preparation of Methyl Benzyloxyindole-2-carboxylates
The vinyl azido intermediate (6.66 mmol) was dissolved in xylene (50 mL) and the reaction
mixture was heated under reflux for 6 h. After refluxing, the solvent was evaporated under reduced
pressure and the remaining residue was extracted with boiling hexane. Upon cooling, the resulting
solid was filtered to give crude product which was recrystallized from dichloromethane and n-hexane
to give the title compound.

Molecules 2020, 25, 1377
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3.1.4. GP-4: General Procedure for the Hydrogenolysis of Methyl Dibenzyloxyindole-2-carboxylate
After vacuum/H2 cycles to remove air from the reaction flask, a stirred mixture of benzyloxyindole
(1 mmol) and 5% Pd/C (10% w/w) in THF/MeOH (1:1, 15 mL) was exposed to a hydrogen atmosphere
(1 atm) and stirred at room temperature for 2 h. The reaction mixture was filtered through a pad of
Celite® and the filtrate was then concentrated under reduced pressure. The crude product was purified
by flash column chromatography (SiO2), eluted with dichloromethane to give the title compounds.
3.1.5. GP-5: General Procedure for the Synthesis of Propargyloxybenzaldehydes:
Propargyl bromide (per hydroxyl group, 1.2 equiv.) was added to a mixture of potassium carbonate
(per hydroxyl group, 1 equiv.) and hydroxybenzaldehyde (1 equiv.) in acetone. The reaction mixture
was heated under reflux with stirring until no more starting material remained (~30 h). The reaction
mixture was cooled to room temperature and Et2O (100 mL) was added. The ethereal solution was
washed with NaOH (1 N, 3
reduced pressure and recrystallized from dichloromethane/n-hexane to give the compound.
×
50 mL). The organic layer was dried over MgSO4, concentrated under
3.1.6. GP-6 General Procedure for the Synthesis of Dihydropyranoindoles:
A solution of alkyne indole ethers (1.04 mmol) in chlorobenzene (20 mL) was heated under reflux
until TLC analysis showed consumption of the starting indole (12 h). The heating was discontinued and
the solvent was evaporated under reduced pressure. The crude product was purified using flash column
chromatography (SiO2), eluted with 30% dichloromethane/n-hexane, to give the dihydropyranoindole.
Methyl 2-azido-3-(3,4-dibenzyloxyphenyl)-propenoate (13) The title compound was prepared as described
in GP-2 from 3,4-dibenzyloxybenzaldehyde (12) (2.95 g, 9.3 mmol) and methyl azidoacetate (10.69 g, 93
mmol) in anhydrous methanol (30 mL) to give the product (2.27 g, 59%) as a pale yellow granular solid;
m.p. 114–116 ◦C; IR (KBr): vmax 2917, 2119, 1701, 1683, 1590, 1508, 1432, 1379, 1233, 1201, 1130, 999,
802, 728 cm−1; UV-vis (CH3CN):
5.25 (s, 4H, 2
O-CH2), 6.83 (s, 1H, CH=C), 7.35–7.52 (m, 12H, ArH), 7.64 (d, J = 2.2 Hz, 1H, H20);
13C-NMR: (75.6 MHz, CDCl3): 52.7 (CO2Me), 70.9 (CH2), 71.3 (CH2), 113.8 (C20), 116.6 (C50), 123.3
H=C), 125.4 (C60), 125.7 (ArC), 126.6 (ArC), 127.1 (2
ArC), 127.2 (2 ArC), 127.8 (ArC), 127.9 (ArC),
128.5 (2 ArC), 128.6 (2 O2Me);
ArC), 136.8 (C10), 137.1 (CH= ), 148.3 (C40), 150.1 (C30), 164.1 (
HRMS (+ESI): Found m/z 438.1439 [M + Na]+, C24H21N3O4Na requires 438.1439.
λ δ 3.91 (s, 3H, CO2Me),
max325 (24,500); 1H-NMR: (300 MHz, CDCl3):
×
δ
(C
×
×
×
×
C
C
Methyl 5,6-dibenzyloxyindole-2-carboxylate (14) The title compound was prepared as described in GP-3
from methyl 2-azido-3-(30,40-dibenzyloxyphenyl)propenoate (13) (2.76 g, 6.66 mmol) in xylene (50 mL)
to give the product (1.84 g, 71%) as a yellow granular solid; m.p. 148–150 ◦C; IR (KBr): vmax 3309, 2942,
2871, 2113, 1680, 1627, 1519, 1488, 1452, 1353, 1288, 1246, 1208, 1144, 1000, 918, 905, 839, 794 cm−1
;
UV-vis (CH3CN): λmax 208 nm (ε δ 3.93 (s,
72,400 cm−1 M−1), 320 (31,700); 1H-NMR (300 MHz, CDCl3):
3H, CO2Me), 5.21 (s, 2H, O-CH2), 5.24 (s, 2H, O-CH2), 7.00 (s, 1H, H4), 7.01 (s, 1H, H7), 7.23 (d, J = 2.1
Hz, 1H, H3), 7.33–7.51 (m, 10H, ArH), 8.73 (bs, 1H, NH); 13C-NMR (75.6 MHz, CDCl3): 51.8 (CO2Me),
71.3 (CH2), 72.0 (CH2), 97.0 (C7), 107.0 (C4), 108.8 (C3), 121.0 (ArC), 126.0 (C2), 127.0 (ArC), 127.2 (2
×
ArC), 127.3 (ArC), 127.7 (2
×
ArC), 127.8 (ArC), 128.4 (ArC), 128.5 (ArC), 132.4 (ArC), 137.0 (ArC),
137.4 (2 ArC), 145.8 (C5), 149.9 (C6), 162.1 (
×
C
O2Me); HRMS (+ESI): Found m/z 410.1364 [M + Na]+,
C24H21NO4Na requires 410.1363.
Methyl 5,6-dihydroxyindole-2-carboxylate (8) The title compound was prepared as described in GP-4 from
methyl 5,6-dibenzyloxyindole-2-carboxylate (14) (0.387 g, 1.0 mmol) and 5% Pd/C catalyst (40 mg) in
methanol/THF mixture (15 mL) to give the product (159 mg, 77%) as yellow solid; m.p. 256–258 ◦C;
IR (KBr): vmax 3437, 3315, 2953, 2107, 1653, 1632, 1531, 1506, 1437, 1311, 1283, 1230, 1198, 1139, 992,
937, 849, 825, 767 cm−1; UV-vis (CH3CN):
λ
max
203 nm (
ε
42,100 cm−1
3.82 (s, 3H, CO2Me), 6.79 (d, J = 0.8 Hz, 1H, H4), 6.88 (s, 1H, H3), 6.90 (d, J = 0.8
Hz, 1H, H7), 8.84 and 9.17 (bs, each 1H, OH), 11.28 (bs, 1H, NH); 13C-NMR (75.6 MHz, CDCl3): 52.0
M
−1), 318 (27,000);1H-NMR
(300MHz, d6-DMSO):
δ

Molecules 2020, 25, 1377
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(CO2Me), 97.3 (C7), 105.4 (C4), 108.4 (C3), 120.3 (ArC), 125.0 (C2), 133.2 (ArC), 142.3 (C5), 146.7 (C6),
162.5 (CO2Me); HRMS (+ESI): Found m/z 230.0423 [M + Na]+, C10H9NO4Na requires 230.0424.
Methyl 5,6-bis(prop-2-yn-1-yloxy)-1H-indole-2-carboxylate (9) The title compound was prepared as
described in GP-5 from methyl 5,6-dihydroxyindole-2-carboxylate (8) (993 mg, 4.8 mmol), potassium
carbonate (1.32 g, 9.6 mmol) and propargyl bromide (1.36 g, 10.6 mmol) in acetone to give the product
(828 mg, 61%) as a yellow solid;m.p. 170–172 ◦C; IR (KBr): νmax 3332, 3285, 2939, 2925, 2865, 2292,
2108, 1720, 1684, 1625, 1520, 1480, 1435, 1380, 1365, 1280, 1247, 1203, 1150 1024, 986, 892, 819, 805, 759,
720, 672 cm−1; UV-vis (CH3CN):
CDCl3): 2.55 (t, J = 1.6 Hz, 1H, CH
J = 2.4 Hz, 2H, O-CH2), 4.85 (d, J = 2.4 Hz, 2H, O-CH2), 7.10 (d, J = 0.7 Hz, 1H, H7), 7.16 (q, J = 0.9
Hz, 1H, H3), 7.31 (s, 1H, H4), 8.86 (bs, 1H, NH), 13C-NMR (75.6 MHz, CDCl3):
51.8 (CO2Me), 57.0
λmax 209 nm (
ε
28,300 cm−1 M−1), 313 (18,000); 1H-NMR (300 MHz,
δ
≡
C), 2.57 (t, J = 1.6 Hz, 1H, CH
≡
C), 3.95 (s, 3H, CO2Me), 4.82 (d,
δ
(O-CH2), 57.5 (O-CH2), 75.7 (C≡CH), 75.7 (C≡CH), 78.6 (C≡CH), 79.1 (C≡CH), 97.2 (C7), 107.2 (C4),
108.9 (C3), 121.3 (aryl C), 126.4 (C2), 132.4 (aryl C), 144.2 (C4), 148.2 (C5), 162.1 (CO2Me); HRMS (+ESI):
Found m/z 306.0736 [M + Na]+, C16H13NO4Na requires 306.0737.
2,9-Dihydropyrano[3,2-h]chromene-5-carbaldehyde (15) The title compound was prepared as described
in GP-6 from 3,4-bis(prop-2-yn-1-yloxy)benzaldehyde (11) (214 mg, 1.04 mmol) in chlorobenzene (20
mL) to give the product (77 mg, 35%) as a white solid; m.p. 102–104 ◦C; IR (KBr): νmax 1680, 1570,
1490, 1440, 1340, 1301, 1205, 1102, 995, 945, 900, 898, 861, 745 cm−1; UV-vis (CH3CN):
27,400 cm−1 M−1), 268 (18,800), 305 (17,500); 1H-NMR (300 MHz, CDCl3):
2H, O-CH2), 4.79 (dd, J = 3.0, 1.4 Hz, 2H, O-CH2), 6.97 (d, J = 8.7 Hz, 2H, H4 and H7), 7.37 (s, 1H,
H6), 7.40–7.42 (m, 2H, H3 and H8), 9.80 (s, 1H, CHO); 13C-NMR (75.6 MHz, CDCl3):
71.3 (O-CH2),
λ
max 225 nm (
ε
δ
4.40 (dd, J = 3.0, 1.4 Hz,
δ
71.5 (O-CH2), 119.6 (C6), 120.6 (aryl C), 121.6 (C4), 121.8 (C7), 125.3 (aryl C), 126.5 (C3), 126.9 (C8),
127.1 (C5), 147.9 (aryl C), 154.3 (aryl C), 190.7 (CHO); HRMS (+ESI): Found m/z 237.0533 [M + Na]+,
C13H10O3Na requires 237.0528.
3,4-Bis(but-2-yn-1-yloxy)benzaldehyde (29) The title compound was prepared as described in GP-5 from
3,4-dihydroxybenzaldehyde (7) (660 mg, 4.8 mmol), potassium carbonate (1.32 g, 9.6 mmol) and
1-bromobut-2-yne (1.40 g, 10.6 mmol) in acetone to give the product (1.01 g, 87%) as a white solid;
m.p. 86–88 ◦C; IR (KBr): νmax 1688, 1582, 1499, 1431, 1375, 1248, 1201, 1125, 987, 920, 857, 797, 729
cm−1; UV-vis (CH3CN):
λ
max
228 nm (
ε
31,500 cm−1
C-CH3), 4.79 (q, J = 2.3 Hz, 2H, O-CH2), 4.82 (q, J = 2.3 Hz, 2H,
O-CH2), 7.17 (d, J = 8.2 Hz, 1H, H5), 7.52 (dd, J = 8.2, 1.9 Hz, 1H, H6), 7.57 (d, J = 1.9 Hz, 1H, H2),
9.89 (s, 1H, CHO); 13C-NMR (75.6 MHz, CDCl3):
3.6 (C- H3), 3.7 (C- H3), 57.2 (2 O-CH2), 73.1
M
−1), 272 (22,200), 303 (14,800); 1H-NMR (300
MHz, CDCl3):
δ
1.85–1.87 (m, 6H, 2
×
δ
C
C
×
(
C
-CH3), 73.3 (
C
-CH3), 84.6 (C≡C(CH3)), 84.9 (C≡C(CH3)), 112.0 (C5), 112.7 (C2), 126.4 (C6), 130.3 (aryl
C), 147.9 (C40), 152.9 (C30), 190.8 (
requires 265.0835.
C
HO); HRMS (+ESI): Found m/z 265.0840 [M + Na]+, C15H14O3Na
4-Methoxy-3-((3-phenylprop-2-yn-1-yl)oxy)benzaldehyde (24d) The title compound was prepared as
described in GP-5 from 3-hydroxy-4-methoxybenzaldehyde 23 (730 mg, 4.8 mmol), potassium carbonate
(662 mg, 4.8 mmol) and 3-phenylprop-2-yn-1-yl-4-methylbenzenesulfonate (1.66 g, 5.8 mmol) in acetone
to give the product (1.17 g, 92%) as a white solid; m.p. 112–114 ◦C; IR (KBr): νmax 1673, 1579, 1506,
1430, 1378, 1256, 1221, 1165, 1130, 1012, 931, 874, 810, 754, 686 cm−1; UV-vis (CH3CN):
λ
max
232 nm (ε
45,500 cm−1 M−1), 272 (21,500), 302 (14,800); 1H-NMR (300 MHz, CDCl3):
δ 4.00 (s, 3H, OMe), 5.08 (s,
2H, O-CH2), 7.03 (d, J = 8.2 Hz, 1H, H5), 7.30–7.33 (m, 3H, ArH), 7.34–7.45 (m, 2H, ArH), 7.54 (dd, J =
8.2, 1.9 Hz, 1H, H6), 7.68 (d, J = 1.9 Hz, 1H, H2), 9.90 (s, 1H, CHO); 13C-NMR (75.6 MHz, CDCl3):
56.2 (OMe), 57.5 (O-CH2), 83.0 (C≡C(Ph)), 88.0 ( -Ph), 110.9 (C5), 112.1 (C2), 122.1 (aryl C), 127.1 (2
aryl CH), 127.8 (aryl CH), 128.8 (C6), 130.0 (aryl C), 131.9 (2
(CHO); HRMS (+ESI): Found m/z 289.0837 [M + Na]+, C17H14O3Na requires 289.0835.
δ
C
×
×
aryl CH), 147.5 (C4), 155.0 (C3), 190.7

Molecules 2020, 25, 1377
16 of 24
Methyl (Z)-2-azido-3-(3,4-bis(prop-2-yn-1-yloxy)phenyl) acrylate (17) The title compound was prepared as
described in GP-2 from 3,4-bis(prop-2-yn-1-yloxy)benzaldehyde (11) (558 mg, 2.6 mmol) and methyl
azidoacetate (2.99 g, 26.0 mmol) in anhydrous methanol (30 mL) to give the product (582 mg, 63%)
as a pale yellow granular solid; m.p. 114–116 ◦C; IR (KBr): νmax 2954, 2123, 1709, 1594, 1506, 1433,
1378, 1239, 1133, 1081, 1009, 793, 745, 683 cm−1; UV-vis (CH3CN):
1H-NMR (300 MHz,CDCl3):
2.57 (t, J = 2.4 Hz, 1H, CH C), 2.59 (t, J = 2.4 Hz, 1H, CH
CO2Me), 4.83 (d, J = 2.4 Hz, 4H, 2
O-CH2), 6.91 (s, 1H, CH=C), 7.09 (d, J = 8.5 Hz, 1H, H50), 7.44 (dd,
λ
max 327 nm (
ε
19,100 cm−1 M−1);
δ
≡
≡
C), 3.92 (s, 3H,
×
J = 8.5, 2.0 Hz, 1H, H60), 7.77 (d, J = 2.0 Hz, 1H, H20), 13C-NMR (75.6 MHz, CDCl3):
δ 52.8 (CO2Me),
56.6 (OMe), 57.0 (2
×
O-CH2), 76.1 (C≡CH), 76.2 (C≡CH), 77.2 (C≡CH), 77.4 (C≡CH), 113.8 (C50), 116.6
(C20), 123.9 (
C
H=C), 125.3 (C60), 125.6 (aryl C), 146.9 (aryl C), 148.5 (aryl C), 164.1 (
CO2Me); HRMS
could not be determined due to the unstable properties of the compound
Methyl (Z)-2-azido-3-(4-methoxy-3-(prop-2-yn-1-yloxy)phenyl)acrylate (25a) The title compound was
prepared as described in GP-2 from 4-methoxy-3-(prop-2-yn-1-yloxy)benzaldehyde (24a) (494 mg,
2.6 mmol) and methyl azidoacetate (2.99 g, 26.0 mmol) in anhydrous methanol (30 mL) to give the
product (537 mg, 72%) as a pale yellow granular solid; m.p. 116–118 ◦C; IR (KBr): νmax 2951, 2101,
1698, 1592, 1507, 1432, 1379, 1254, 1214, 1139, 1084, 1016, 809, 745 cm−1; UV-vis (CH3CN):
λ
235 nm
max
(ε
16,600 cm−1
M
−1), 330 (27,100); 1H-NMR (300 MHz, CDCl3):
δ
2.58 (t, J = 2.4 Hz, 1H, CH C), 3.94 (s,
≡
3H, OMe), 3.94 (s, 3H, CO2Me), 4.83 (d, J = 2.4 Hz, 2H, O-CH2), 6.90 (s, 1H, CH=C), 6.93 (d, J = 8.5
Hz, 1H, H50), 7.43 (dd, J = 8.5, 2.0 Hz, 1H, H60), 7.76 (d, J = 2.0 Hz, 1H, H20); 13C-NMR (75.6 MHz,
CDCl3):
123.4 (
δ
52.8 (CO2Me), 55.9 (OMe), 56.8 (O-CH2), 76.0 (C≡CH), 77.4 (C≡CH), 111.2 (C50), 116.1 (C20),
C
H=C), 125.6 (C60), 125.0 (aryl C), 126.1 (CH= ), 146.3 (C40), 150.8 (C30), 164.1 (
C CO2Me); HRMS
(+ESI): Found m/z 310.0800 [M + Na]+, C14H13N3O4Na requires 310.0798.
Methyl (Z)-2-azido-3-(3-methoxy-4-(prop-2-yn-1-yloxy) phenyl)acrylate (20a) The title compound was
prepared as described in GP-2 from 3-methoxy-4-(prop-2-yn-1-yloxy)benzaldehyde (19a) (494 mg, 2.6
mmol) and methyl azidoacetate (2.99 g, 26.0 mmol) in anhydrous methanol (30 mL) to give the product
(567 mg, 76%) as a pale yellow granular solid; m.p. 120–122 ◦C; IR (KBr): νmax 2920, 2122, 1704, 1592,
1509, 1450, 1345, 1243, 1206, 1141, 1008, 922, 804, 764 cm−1; UV-vis (CH3CN):
λ
323 nm (
ε
17,500
C), 3.94 (s, 3H, OMe), 3.95 (s, 3H,
CO2Me), 4.83 (d, J = 2.4 Hz, 2H, O-CH2), 6.91 ( s, 1H, CH=C), 7.06 (d, J = 8.5 Hz, 1H, H50), 7.43 (dd, J =
max
cm−1 M−1); 1H-NMR (300 MHz, CDCl3):
δ
2.56 (t, J = 2.4 Hz, 1H, CH
≡
8.5, 1.9 Hz, 1H, H60), 7.56 (d, J = 1.9 Hz, 1H, H20); 13C-NMR (75.6 MHz, CDCl3):
δ
52.8 (CO2Me), 55.9
H=C), 124.4 (C60),
(OMe), 56.5 (O-CH2), 76.2 (C≡CH), 77.4 (C≡CH), 113.3 (C50), 113.5 (C20), 123.7 (
C
125.5 (aryl C), 127.3 (CH=C CO2Me); HRMS could not be determined
), 147.9 (C30), 149.1 (C40), 164.1 (
due to the unstable properties of the compound.
Methyl (Z)-2-azido-3-(3,4-bis(but-2-yn-1-yl)oxy)phenyl) acrylate (30) The title compound was prepared as
described in GP-2 from 3,4-bis(but-2-yn-1-yloxy)benzaldehyde (29) (629 mg, 2.6 mmol) and methyl
azidoacetate (2.99 g, 26.0 mmol) in anhydrous methanol (30 mL) to give the product (555 mg, 63%) as a
pale yellow granular solid; m.p. 124–126 ◦C; IR (KBr): νmax 2918, 2119, 1712, 1687, 1590, 1506, 1432,
1373, 1314, 1243, 1128, 992, 804, 745 cm−1; UV-vis (CH3CN):
(300 MHz, CDCl3): 1.87 (t, J = 2.3 Hz, 6H, 2 C-CH3), 3.93 (s, 3H, CO2Me), 4.79 (q, J = 2.3 Hz, 2H,
O-CH2), 4.82 (q, J = 2.3 Hz, 2H, O-CH2), 6.91 (s, 1H CH=C), 7.05 (d, J = 8.5 Hz, 1H, H50), 7.44 (dd, J =
8.5, 1.8 Hz, 1H, H60), 7.73 (d, J = 2.0 Hz, 1H, H20); 13C-NMR (75.6 MHz, CDCl3):
3.7 (2 C- H3), 52.8
(CO2Me), 57.1 (OMe), 57.2 (O-CH2), 57.5 (O-CH2), 73.9 (2×C-CH3), 84.2 (C≡C(CH3)), 84.9 (C≡C(CH3)),
113.2 (C50), 116.0 (C20), 125.3 ( H=C), 125.8 (C60), 126.5 (aryl C), 126.6 (CH= ), 147.9 (C40), 148.8 (C3),
λmax 307 nm (ε
14,500 cm−1 M−1); 1H-NMR
δ
×
δ
×
C
C
C
164.2 (CO2Me); HRMS could not be determined due to the unstable properties of the compound.
Methyl (Z)-2-azido-3-(3-(but-2-yn-1-yloxy)-4-methoxyphenyl) acrylate (25b) The title compound was
prepared as described in GP-2 from 3-(but-2-yn-1-yloxy)-4-methoxybenzaldehyde (24b) (530 mg, 2.6
mmol) and methyl azidoacetate (2.9 g, 26.0 mmol) in anhydrous methanol (30 mL) to give the product
(500 mg, 64%) as a pale yellow granular solid; m.p. 96–98 ◦C; IR (KBr): νmax 2951, 2101, 1698, 1592,

Molecules 2020, 25, 1377
17 of 24
1507, 1431, 1379, 1254, 1139, 1016, 810, 745 cm−1; UV-vis (CH3CN):
λ
max 327 nm (
ε
25,700 cm−1 M−1);
1H-NMR (300 MHz, CDCl3):
δ
1.89 (t, J = 2.3 Hz, 3H, CH3), 3.93 (s, 3H, OMe), 3.94 (s, 3H, CO2Me),
4.78 (q, J = 2.3 Hz, 2H, O-CH2), 6.91 (d, J = 8.5 Hz, 1H, H50), 6.92 (s, 1H, CH=C), 7.41 (dd, J = 8.5, 2.0
Hz, 1H, H60), 7.73 (d, J = 2.0 Hz, 1H, H20); 13C-NMR (75.6 MHz, CDCl3):
δ 3.7 (C-CH3), 52.8 (CO2Me),
55.9 (OMe), 57.4 (O-CH2), 73.7 (
(C60), 125.9 (aryl C), 126.0 (CH=
C
-CH3), 84.3 (C≡C(CH3)), 111.0 (C50), 115.6 (C20), 123.3 (
CH=C), 125.6
C
), 146.7 (C40), 150.7 (C30), 164.2 (
CO2Me); HRMS (+ESI): Found m/z
324.0944 [M + Na]+, C15H15N3O4Na requires 324.0955.
Methyl (Z)-2-azido-3-(4-(but-2-yn-1-yloxy)-3-methoxyphenyl)acrylate (20b) The title compound was
prepared as described in GP-2 from 4-(but-2-yn-1-yloxy)-3-methoxybenzaldehyde (19b)(530 mg,
2.6 mmol) and methyl azidoacetate (2.99 g, 26.0 mmol) in anhydrous methanol (30 mL) to give the
product (477 mg, 61%) as a pale yellow granular solid; m.p. 94–96 ◦C; IR (KBr): νmax 2920, 2120, 1702,
1592, 1509, 1434, 1377, 1244, 1140, 1011, 801, 763 cm−1; UV-vis (CH3CN):
λ
max 324 nm (
ε
14,900 cm−1
1
M
−1); H-NMR (300 MHz, CDCl3):
δ
1.87 (t, J = 2.3 Hz, 3H, CH3), 3.94 (s, 3H, OMe), 3.94 (s, 3H,
CO2Me), 4.77 (q, J = 2.3 Hz, 2H, O-CH2), 6.90 (s, 1H, CH=C), 7.05 (d, J = 8.5 Hz, 1H, H50), 7.38 (dd, J =
8.5, 1.8, Hz, 1H, H60), 7.53 (d, J = 1.8 Hz, 1H, H20); 13CNMR (75.6 MHz, CDCl3):
(CO2Me), 55.9 (OMe), 57.1 (O-CH2), 73.5 (
C), 124.5 ( C CO2Me); HRMS (+ESI):
δ 3.7 (C-CH3), 52.8
C
-CH3), 84.4 (C≡C(CH3)), 112.9 (C50), 113.3 (C20), 123.4 (aryl
C
H=C), 125.8 (C60), 126.8 (CH= ), 14.34 (C30), 149.0 (C40), 164.2 (
Found m/z 324.0955 [M + Na]+, C15H15N3O4Na requires 324.0955.
Methyl (Z)-2-azido-3-(4-methoxy-3-((3-phenylprop-2-yn-1-yl) oxy)phenyl)acrylate (25d) The title compound
was prepared as described in GP-2 from 4-methoxy-3-((3-phenylprop-2-yn-1-yl)oxy)benzaldehyde
(
24d) (692 mg, 2.6 mmol) and methyl azidoacetate (2.99 g, 26.0 mmol) in anhydrous methanol (30 mL)
to give the product (632 mg, 67%) as a pale yellow granular solid; m.p. 118–120 ◦C; IR (KBr): νmax
2921, 2827, 2119, 1674, 1612, 1580, 1506, 1431, 1378, 1275, 1232, 1129, 1008, 966. 873, 809, 753, 686 cm−1
UV-vis (CH3CN): 234 nm (
57,700 cm−1 −1), 272 (22,600), 306 (21,800); 1H-NMR (300 MHz,
3.93 (s, 3H, OMe), 3.96 (s, 3H, CO2Me), 5.04 (d, J = 6.0 Hz, 2H, O-CH2), 6.91 (s, 1H, CH C),
6.92 (d, J = 8.0 Hz, 1H, H50), 7.09 (dd, J = 8.0, 1.8 Hz, 1H, H60), 7.27–7.33 (m, 3H, ArH), 7.43–7.46 (m,
;
λ
max
ε
M
CDCl3):
δ
≡
2H, ArH), 7.85 (d, J = 1.8 Hz, 1H, H20); 13C-NMR (75.6 MHz, CDCl3):
(O-CH2), 83.9 (
126.1 (aryl C), 128.3 (2
(C30), 164.2 (
δ 52.6 (CO2Me), 55.9 (OMe), 57.5
C
-Ph), 87.5 (C≡C(Ph)), 111.1 (C50), 116.0 (C20), 120.4 (aryl C), 125.7 (
C
H=C), 126.0 (C60),
×
aryl CH), 128.6 (CH=
C
), 130.6 (aryl CH), 131.9 (2
aryl CH), 146.7 (C40), 150.9
×
C
O2Me); HRMS (+ESI): Found m/z 386.1099 [M + Na]+, C20H17N3O4Na requires 386.1117.
Methyl (Z)-2-azido-3-(4-methoxy-3-((2-methylbut-3-yn-2-yl) oxy)phenyl)acrylate (25c) The title compound
was prepared as described in GP-2 from 4-methoxy-3-((2-methylbut-3-yn-2-yl)oxy)benzaldehyde (24c
(567 mg, 2.6 mmol) and methyl azidoacetate (2.99 g, 26.0 mmol) in anhydrous methanol (30 mL) to give
the product (507 mg, 62%) as a pale yellow granular solid; m.p. 122–124 ◦C; IR (KBr): νmax 2988, 2835,
2094, 1691, 1619, 1565, 1506, 1425, 1374, 1253, 1138, 1084, 1027, 962. 891, 799, 756, 687 cm−1; UV-vis
)
(CH3CN):
×
λ
max
238 nm (
ε
16,800 cm−1
C-CH3), 2.59 (d, J = 0.9 Hz, 1H, CH
6.90 (d, J = 8.5 Hz, 1H, H50), 7.45 (dd, J = 1.9, 8.5 Hz, 1H, H60), 8.16 (d, J = 2.0 Hz, 1H, H20);13C-NMR
M
−1), 328 (35,400); 1H-NMR (300 MHz, CDCl3):
δ 1.70 (s, 6H, 2
≡
C), 3.87 (s, 3H, OMe), 3.92 (s, 3H, CO2Me), 6.91 (s, 1H, CH=C),
(75.6 MHz, CDCl3):
δ
29.3 (2
×
C-
C
H3), 52.7 (CO2Me), 55.7 (OMe), 73.6 (C≡CH), 74.1 (
C
-(CH3)2), 86.1
(
C≡CH), 111.5 (C50), 123.2 (C20), 124.8 (
C C
H=C), 125.7 (C60), 125.9 (aryl C), 127.4 (CH= ), 144.2 (C40),
153.9 (C30), 164.2 (
338.1117.
C
O2Me); HRMS (+ESI): Found m/z 338.1099 [M + Na]+, C16H17N3O4Na requires
Methyl (Z)-2-azido-3-(3-methoxy-4-((2-methylbut-3-yn-2-yl)oxy)phenyl)acrylate (20c) The title compound
was prepared as described in GP-2 from 3-methoxy-4-((2-methylbut-3-yn-2-yl)oxy)benzaldehyde (19c
(567 mg, 2.6 mmol) and methyl azidoacetate (2.1 g, 26. mmol) in anhydrous methanol (30 mL) to give
the product (524 mg, 62%) as a pale yellow granular solid; m.p. 116–118 ◦C; IR (KBr): νmax 2975, 2802,
2112, 1703, 1635, 1565, 1512, 1400, 1387, 1212, 1100, 1052, 998, 957, 888, 765, 745, 677 cm−1; UV-vis
)
(CH3CN):
λmax 328 nm (
ε
17,100 cm−1 M−1); 1H-NMR (300 MHz, CDCl3):
δ
1.72 (s, 6H, 2
×
C-CH3),

Molecules 2020, 25, 1377
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2.60 (d, J = 0.9 Hz, 1H, CH
≡
C), 3.89 (s, 3H, OMe), 3.93 (s, 3H, CO2Me), 6.91 (s, 1H, CH=C), 7.33 (dd, J =
8.5, 1.9 Hz, 1H, H60), 7.45 (d, J = 8.5 Hz, 1H, H50), 7.52 (d, J = 1.9 Hz, 1H, H20); 13C-NMR (75.6 MHz,
CDCl3):
δ
29.4 (2
×
C-
C
H3), 52.8 (CO2Me), 55.9 (OMe), 73.9 (C≡CH), 73.9 (
C
-(CH3)2) 85.8 (C≡CH),
113.9 (C50), 121.5 (C20), 123.8 (
C
H=C), 123.9 (C60), 125.7 (aryl C), 128.5 (CH= ), 146.1 (C30), 152.0 (C40),
C
164.1 (CO2Me); HRMS (+ESI): Found m/z 338.1104 [M + Na]+, C16H17N3O4Na requires 338.1117.
Methyl 6-methoxy-5-(prop-2-yn-1-yloxy)-1H-indole-2-carboxylate (26a) The title compound was prepared
as described in GP-3 from methyl (Z)-2-azido-3-(3-(but-2-yn-1-yloxy)-4-methoxyphenyl)acrylate (25a
(596 mg, 2.08 mmol) in xylene (20 mL) to give the product (196 mg, 73%) as a yellow solid; m.p. 164–166
◦C; IR (KBr): νmax 3325, 3259, 2953, 2925, 2123, 1671, 1624, 1520, 1480, 1440, 1367, 1320, 1249, 1208, 1185,
)
1146, 1005, 890, 833, 766, 694 cm−1; UV-vis (CH3CN):
1H-NMR (300 MHz, CDCl3):
2.55 (t, J = 2.4 Hz, 1H, CH
4.82 (d, J = 2.4 Hz, 2H, O-CH2), 6.90 (d, J = 0.8 Hz, 1H, H4), 7.16 (q, J = 0.9 Hz, 1H, H3), 7.28 (s, 1H,
H7), 8.91 (bs, 1H, NH), 13C-NMR (75.6 MHz, CDCl3):
51.8 (CO2Me), 56.0 (OMe), 57.3 (O-CH2), 75.7
λmax 208 nm (
ε
40,600 cm−1 M−1), 318 (28,700);
δ
≡
C), 3.95 (s, 3H, OMe), 3.96 (s, 3H, CO2Me),
δ
(C≡CH), 78.7 (C≡CH), 94.0 (C7), 106.6 (C4), 109.0 (C3), 120.2 (aryl C), 125.9 (C2), 132.9 (aryl C), 143.7
(C6), 150.6 (C5), 162.3 (
282.0742.
C
O2Me); HRMS (+ESI): Found m/z 282.0725 [M + Na]+, C14H13NO3Na requires
Methyl 5-methoxy-6-(prop-2-yn-1-yloxy)-1H-indole-2-carboxylate (21a) The title compound was prepared
as described in GP-3 from methyl (Z)-2-azido-3-(4-(but-2-yn-1-yloxy)-3-methoxyphenyl)acrylate (20a
(596 mg, 2.08 mmol) in xylene (20 mL) to give the product (183 mg, 68%) as a yellow solid; m.p. 152–154
◦C; IR (KBr): νmax 3325, 3241, 2999, 2937, 2113, 1681, 1638, 1522, 1475, 1452, 1360, 1280, 1237, 1211,
)
1195, 1143, 1003, 924, 843, 819, 761, 675 cm−1; UV-vis (CH3CN):
λ
209 nm (
ε
35,800 cm−1
C), 3.95 (s, 3H, OMe), 3.96 (s, 3H,
CO2Me), 4.85 (d, J = 2.4 Hz, 2H, O-CH2), 7.08 (d, J = 0.8 Hz, 1H, H4), 7.11 (s, 1H, H7), 7.15 (q, J = 0.9 Hz,
1H, H3), 8.87 (bs, 1H, NH); 13C-NMR (75.6 MHz, CDCl3):
51.8 (CO2Me), 56.2 (OMe), 56.9 (O-CH2),
M
−1), 308
max
(21,400); 1H-NMR (300 MHz, CDCl3):
δ
2.57 (t, J = 2.4 Hz, 1H, CH
≡
δ
76.1 (C≡CH), 77.4 (C≡CH), 96.8 (C7), 103.0 (C4), 108.7 (C3), 121.4 (aryl C), 126.1 (C2), 131.6 (aryl C),
146.6 (C5), 147.6 (C6), 162.2 (
requires 282.0742.
C
O2Me); HRMS (+ESI): Found m/z 282.0726 [M + Na]+, C14H13NO3Na
Methyl 5,6-bis(but-2-yn-1-yloxy)-1H-indole-2-carboxylate (31) The title compound was prepared as
described in GP-3 from methyl (Z)-2-azido-3-(3,4-bis(but-2-yn-1-yloxy)phenyl)acrylate (30) (704 mg,
2.08 mmol) in xylene (20 mL) to give the product 210 mg, 65%) as a yellow solid; m.p. 146–148 ◦C; IR
(KBr): νmax 3332, 2912, 2294, 2120, 1682, 1644, 1519, 1434, 1379, 1244, 1204, 1145, 984, 891, 819, 765 cm−1
;
UV-vis (CH3CN):
(q, J = 2.4 Hz, 6H, 2
Hz, 2H, O-CH2), 7.08 (d, J = 0.7 Hz, 1H, H4), 7.16 (q, J = 0.9 Hz, 1H, H3), 7.25 (s, 1H, H7), 9.03 (bs,
1H, NH); 13C-NMR (75.6 MHz, CDCl3):
3.7 (2 C- H3), 51.8 (CO2Me), 57.4 and 57.8 (O-CH2), 73.9
-CH3), 74.2 (
λmax 210 nm (
ε δ 1.87
39,600 cm−1 M−1), 317 (24,900); 1H-NMR (300 MHz, CDCl3):
×
C-CH3), 3.96 (s, 3H, CO2Me), 4.76 (q, J = 2.4 Hz, 2H, O-CH2), 4.79 (q, J = 2.4
δ
×
C
(
C
C
-CH3), 83.8 (C≡C(CH3)), 84.2 (C≡C(CH3)), 96.5 (C7), 106.1 (C4), 108.9 (C3), 120.9 (aryl
C), 126.0 (C2), 132.5 (aryl C), 144.4 (C5), 148.5 (C6), 162.3 (CO2Me); HRMS (+ESI): Found m/z 334.1046
[M + Na]+, C18H17NO4Na requires 334.1050.
Methyl 5-(but-2-yn-1-yloxy)-6-methoxy-1H-indole-2-carboxylate (26b) The title compound was prepared as
described in GP-3 from methyl (Z)-2-azido-3-(3-(but-2-yn-1-yloxy)-4-methoxyphenyl)acrylate (25b
(626 mg, 2.08 mmol) in xylene (20 mL) to give the product (201 mg, 71%) as a yellow solid; m.p. 160–162
◦C; IR (KBr): νmax 3324, 3258, 3009, 2918, 2795, 2123, 2108, 1672, 1600, 1520, 1490, 1425, 1400, 1385, 1346,
)
1249, 1208, 1185, 1146, 1005, 980, 890, 833, 766 cm−1; UV-vis (CH3CN):
M
λ
max
208 nm (
ε
27,700 cm−1
1.88 (q, J = 2.4 Hz, 3H, C-CH3), 3.95 (s, 3H, OMe),
3.96 (s, 3H, CO2Me), 4.77 (q, J = 2.4 Hz, 2H, O-CH2), 6.88 (d, J = 0.7 Hz, 1H, H4), 7.16 (q, J = 0.9 Hz, 1H,
H3), 7.23 (s, 1H, H7), 8.82 (bs, 1H, NH); 13C-NMR (75.6 MHz, CDCl3):
5.2 (C- H3), 53.2 (CO2Me),
57.4 (OMe), 59.1 (O-CH2), 75.5 (
-CH3), 85.3 (C≡C), 95.2 (C7), 107.1 (C4), 110.4 (C3), 121.7 (aryl C),
−1), 319 (18,900);1H-NMR (300 MHz, CDCl3):
δ
δ
C
C

Molecules 2020, 25, 1377
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127.1 (C2), 134.0 (aryl C), 145.5 (C6), 151.9 (C5), 163.7 (
CO2Me); HRMS (+ESI): Found m/z 296.0884 [M +
Na]+, C15H15NO4Na requires 296.0899.
Methyl 6-(but-2-yn-1-yloxy)-5-methoxy-1H-indole-2-carboxylate (21b) The title compound was prepared as
described in GP-3 from methyl (Z)-2-azido-3-(4-(but-2-yn-1-yloxy)-3-methoxyphenyl)acrylate (20b
(626 mg, 2.08 mmol) in xylene (20 mL) to give the product (190 mg, 67%) as a yellow solid; m.p. 164–166
◦C; IR (KBr): νmax 3324, 3224, 3005, 2936, 2835, 2215, 2108, 1680, 1584, 1495, 1455, 1433, 1362, 1285, 1243,
)
1228, 1146, 1045, 988, 946, 846, 819, 760, 672 cm−1; UV-vis (CH3CN):
λ
210 nm (
ε
37,100 cm−1
1.87 (q, J = 2.4 Hz, 3H, C-CH3), 3.94 (s, 3H, OMe), 3.96 (s,
3H, CO2Me), 4.80 (q, J = 2.4 Hz, 2H, O-CH2), 7.06 (d, J = 0.7 Hz, 1H, H4), 7.08 (s, 1H, H7), 7.14 (q, J =
0.9 Hz, 1H, H3), 9.01 (bs, 1H, NH); 13C-NMR (75.6 MHz, CDCl3):
3.7 (C- H3), 51.8 (CO2Me), 56.1
(OMe), 57.4 (O-CH2), 73.8 (
M
−1),
max
319 (22,300); 1H-NMR (300 MHz, CDCl3):
δ
δ
C
C
-CH3), 84.3 (C≡C(CH3)), 96.2 (C7), 102.7 (C4), 108.7 (C3), 121.0 (aryl C),
125.8 (C2), 131.8 (aryl C), 146.1 (C5), 148.0 (C6), 162.3 (
Na]+, C15H15NO4Na requires 296.0889.
CO2Me); HRMS (+ESI): Found m/z 296.0883 [M +
Methyl
The
6-methoxy-5-((3-phenylprop-2-yn-1-yl)oxy)-1H-indole-2-carboxylate
compound was prepared as described in GP-3 from
(
26d
)
title
methyl
(Z)-2-azido-3-(4-methoxy-3-((3-phenylprop-2-yn-1-yl)oxy)phenyl)acrylate (25d) (754 mg, 2.08
mmol) in xylene (20 mL) to give the product (257 mg, 74%) as a yellow solid; m.p. 138–140 ◦C; IR
(KBr): νmax 3326, 2984, 2924, 2740, 2113, 1675, 1645, 1520, 1480, 1439, 1381, 1247, 1195, 1145, 996, 845,
823, 761, 672 cm−1; UV-vis (CH3CN):
λ
max
203 nm (
ε
58,900 cm−1
3.95 (s, 3H, OMe), 3.98 (s, 3H, CO2Me), 5.04 (s, 2H, O-CH2), 6.90 (s, 1H, H4), 7.17 (q, J
= 0.9 Hz, 1H, H3), 7.31 (s, 1H, H7), 7.32–7.36 (m, 3H, ArCH), 7.44–7.47 (m, 2H, ArCH), 8.84 (bs, 1H,
NH); 13C-NMR (75.6 MHz, CDCl3):
51.8 (CO2Me), 56.0 (OMe), 58.2 (O-CH2), 84.1 (C≡C(Ph)), 87.4
-Ph), 93.9 (C7), 106.6 (C4), 109.0 (C3), 120.3 (aryl C), 122.4 (aryl C), 125.8 (C2), 128.2 (2 aryl CH),
O2Me); HRMS (+ESI):
M
−1), 318 (22,200); 1H-NMR (300
MHz, CDCl3):
δ
δ
(C
×
128.5 (aryl CH), 131.8 (2
×
aryl CH), 132.8 (aryl C), 144.0 (C6), 150.7 (C5), 162.2 (C
Found m/z 336.1221 [M + H]+, C20H18NO4 requires 336.1236.
Methyl 5,10-dihydro-7H-dipyrano[3,2-e:20,30-g]indole-6-carboxylate
(
10
)
Method 2:
Route
2
The title compound was prepared as described in GP-3 from methyl
(Z)-2-azido-3-(3,4-bis(prop-2-yn-1-yloxy)phenyl) acrylate (
9
) (646 mg, 2.08 mmol) in xylene
3415,
(20 mL) to give the product (61.4 mg, 72%) as a yellow solid; m.p. 168–170 ◦C; IR (KBr):
ν
max
3328, 2947, 2831, 2341, 2116, 1674, 1640, 1575, 1524, 1486, 1441, 1400, 1274, 1213, 1158, 1135, 1025, 1000,
920, 813, 755 cm−1; UV-vis (CH3CN): 24,100 cm−1 −1), 269 (13,800), 360 (13,000);
225 nm (
3.96 (s, 3H, CO2Me), 4.91 (dd, J = 3.8, 1.8 Hz, 2H, O-CH2), 4.94 (dd, J =
3.8, 1.8 Hz, 2H, O-CH2), 5.87–5.97 (m, 2H, H3 and H9), 6.72–6.80 (m, 2H, H4 and H8), 7.19 (d, J =
2.1 Hz, 1H, H5), 8.85 (s, 1H, NH); 13C-NMR (75.6 MHz, CDCl3):
52.0 (CO2Me), 65.7 (O-CH2), 65.8
(O-CH2), 106.5 (C5), 107.0 (aryl C), 115.3 (aryl C), 118.8 (C4), 119.3 (C8), 120.8 (aryl C), 121.8 (C3), 121.9
λ
max
ε
M
1H-NMR (300 MHz, CDCl3):
δ
δ
(C9), 126.5 (C6), 128.8 (aryl C), 138.1 (aryl C), 141.7 (aryl C), 162.3 (CO2Me); HRMS (+ESI): Found m/z
306.0736 [M + Na]+, C16H13NO4Na requires 306.0737.
This compound was also prepared by the methods described below. Method 1: The title compound
was prepared as described in GP-6 from methyl 5,6-bis(prop-2-yn-1-yloxy)-1H-indole-2-carboxylate
(16) (118 mg, 0.42 mmol) in chlorobenzene (30 mL) to give the product (43.5 mg, 51%) as a yellow solid.
Method 2: Route 1 The title compound was prepared as described in GP-6 from methyl
(Z)-2-azido-3-(2H,10H-pyrano[4,3-h]chromen-5-yl)acrylate (17) (646 mg, 2.08 mmol) in xylene (20 mL)
to give the product (52.9 mg, 62%) as a yellow solid.
Methyl 5-methoxy-3,7-dihydropyrano[3,2-e]indole-2-carboxylate (27a) The title compound was prepared as
described in GP-6 from methyl 6-methoxy-5-(prop-2-yn-1-yloxy)-1H-indole-2-carboxylate (26a) (108
mg, 0.42 mmol) in chlorobenzene (30 mL) to give the product (80 mg, 74%) as a yellow solid; m.p.
166–168 ◦C; IR (KBr):
νmax 3325, 2948, 2839, 2340, 2110, 1677, 1637, 1515, 1439, 1271, 1193, 1143, 1098,

Molecules 2020, 25, 1377
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998, 931, 883, 808, 752 cm−1; UV-vis (CH3CN):
λ
212 nm (
ε
37,400 cm−1
M
−1), 271 (13,300), 322
max
(30,600); 1H-NMR (300 MHz, CDCl3):
δ
3.92 (s, 3H, OMe), 3.93 (s, 3H, CO2Me), 4.90 (dd, J = 3.7, 1.7 Hz,
2H, O-CH2), 5.89–5.95 (m, 1H, H8), 6.76–6.79 (m, 1H, H9), 6.79 (s, 1H, H4), 7.17 (d, J = 2.1 Hz, 1H, H1),
8.96 (s, 1H, NH); 13C-NMR (75.6 MHz, CDCl3):
51.8 (CO2Me), 56.0 (OMe), 65.7 (O-CH2), 93.8 (C4),
106.1 (C1), 114.9 (aryl C), 118.3 (aryl C), 121.6 (C9), 121.8 (C8), 126.0 (C2), 132.2 (aryl C), 139.3 (aryl
δ
C), 148.8 (C5), 162.2 (
282.0737.
C
O2Me); HRMS (+ESI): Found m/z 282.0731 [M + Na]+, C14H13NO4Na requires
Methyl 5-methoxy-1,7-dihydropyrano[2,3-g]indole-2-carboxylate (22a) The title compound was prepared as
described in GP-6 from methyl 5-methoxy-6-(prop-2-yn-1-yloxy)-1H-indole-2-carboxylate (21a) (108
mg, 0.42 mmol) in chlorobenzene (30 mL) to give the product (83 mg, 77%) as a yellow solid; m.p.
188–190 ◦C; IR (KBr): νmax 3327, 2944, 2827, 2366, 2106, 1677, 1528, 1444, 1306, 1221, 1148, 1101, 989, 955,
831, 755 cm−1; UV-vis (CH3CN):
λ
max
226 nm (
ε
28,100 cm−1
3.94 (s, 3H, OMe), 3.96 (s, 3H, CO2Me), 4.96 (dd, J = 3.8, 1.8 Hz, 2H, O CH2),
5.89–5.95 (m, 1H, H8), 6.75–6.79 (m, 1H, H9), 7.02 (s, 1H, H4), 7.13 (d, J = 2.1 Hz, 1H, H3), 8.94 (s, 1H
NH); 13C-NMR (75.6 MHz, CDCl3):
51.9 (CO2Me), 56.2 (OMe), 65.7 (O-CH2), 103.1 (C4), 107.1 (aryl
M
−1), 290 (16,700), 339 (18,400); 1H-NMR
(300 MHz, CDCl3):
δ
δ
C), 109.2 (C3), 119.3 (aryl C), 120.8 (C8), 121.0 (C9), 126.0 (C2), 128.7 (aryl C), 137.6 (aryl C), 145.2 (C5),
162.3 (CO2Me); HRMS (+ESI): Found m/z 282.0738 [M + Na]+, C14H13NO4Na requires 282.0737.
Methyl 5-methoxy-9-methyl-3,7-dihydropyrano[3,2-e]indole-2-carboxylate (27b) The title compound was
prepared as described in GP-6 from methyl 5-(but-2-yn-1-yloxy)-6-methoxy-1H-indole-2-carboxylate
(
26b) (114 mg, 0.42 mmol) in chlorobenzene (30 mL) to give the product (62 mg, 54%) as a yellow solid;
m.p. 196–198 ◦C; IR (KBr): νmax 3310, 2948, 2925, 2833, 2106, 1675, 1640, 1517, 1495, 1439, 1366, 1274,
1265, 1194, 1146, 1080, 1004, 960, 880, 815, 765, 672 cm−1; UV-vis (CH3CN): 28,900 cm−1
213 nm (
1.63 (s, 3H, C-CH3), 3.94 (s, 3H, OMe),
3.95 (s, 3H, CO2Me), 4.77 (q, J = 2.4 Hz, 2H, O-CH2), 5.69–5.72 (m, 1H, H8), 6.87 (s, 1H, H1), 7.23 (s,
1H, H4), 8.82 (bs, 1H, NH); 13C-NMR (75.6 MHz, CDCl3):
22.6 (C- H3), 51.8 (CO2Me), 56.1 (OMe),
65.2 (O-CH2), 93.8 (C4), 109.3 (C1), 117.4 (aryl C), 117.9 (aryl C), 118.3 (C8), 126.5 (C2), 132.0 ( -CH3),
O2Me); HRMS (+ESI): Found m/z 296.0887 [M + Na]+,
λ
max
ε
M
−1), 267 (9,500), 322 (24,200); 1H-NMR (300 MHz, CDCl3):
δ
δ
C
C
132.9 (aryl C), 140.4 (aryl C), 149.0 (C5), 162.2 (
C15H15NO4Na requires 296.0893.
C
Methyl 5-methoxy-9-methyl-1,7-dihydropyrano[2,3-g]indole-2-carboxylate (22b) The title compound was
prepared as described in GP-6 from methyl 6-(but-2-yn-1-yloxy)-5-methoxy-1H-indole-2-carboxylate
(
21b) (114 mg, 0.42 mmol) in chlorobenzene (30 mL) to give the product (56 mg, 49%) as a yellow solid;
m.p. 176–178 ◦C; IR (KBr): νmax 3410, 2980, 2930, 2845, 1695, 1645, 1606, 1534, 1445, 1430, 1385, 1375,
1232, 1189, 1139, 1096, 1040, 998, 981, 927, 857, 744, 712cm−1; UV-vis (CH3CN):
max 224 nm ( 32,500
cm−1 M−1), 286 (22,200), 337 (23,500); 1H-NMR (300 MHz, CDCl3):
1.71 (s, 3H, C-CH3), 3.94 (s, 3H,
OMe), 3.96 (s, 3H, CO2Me), 4.77 (q, J = 1.6 Hz, 2H, O-CH2), 5.65–5.68 (m, 1H, H8), 7.05 (s, 1H, H4),
7.14 (d, J = 2.1 Hz, 1H, H3), 8.81 (bs, 1H, NH), 13C-NMR (75.6 MHz, CDCl3):
20.8 (C- H3), 51.8
(CO2Me), 56.2 (OMe), 65.4 (O-CH2), 103.2 (C4), 108.9 (C3), 109.9 (aryl C), 117.7 (C8), 121.9 (aryl C),
126.0 (C2), 128.7 ( -CH3), 129.3 (aryl C), 144.2 (aryl C), 145.4 (C5), 162.1 ( O2Me); HRMS (+ESI): Found
m/z 296.0886 [M + Na]+, C15H15NO4Na requires 296.0893.
λ
ε
δ
δ
C
C
C
Methyl
title
5-methoxy-9-phenyl-3,7-dihydropyrano[3,2-e]indole-2-carboxylate
compound was prepared as described in GP-6
(
27d
)
The
methyl
from
6-methoxy-5-((3-phenylprop-2-yn-1-yl)oxy)-1H-indole-2-carboxylate (26d) (140 mg, 0.42 mmol) in
chlorobenzene (30 mL) to give the product (113 mg, 81%) as a yellow solid; m.p. 188–190 ◦C; IR (KBr):
νmax 3311, 2949, 2816, 2114, 1684, 1639, 1598, 1515, 1500, 1438, 1400, 1360, 1260, 1241, 1193, 1141, 1042,
1011, 995, 920, 823, 759, 703 cm−1; UV-vis (CH3CN):
λ
max
274 nm (
ε
11,600 cm−1
3.82 (s, 3H, OMe), 4.00 (s, 3H, CO2Me), 4.85 (d, J = 4.4 Hz, 2H, O-CH2),
5.94–5.97 (m, 1H, H8), 5.98 (s, 1H, H4), 6.88 (s, 1H, H1), 7.30–7.34 (m, 2H, ArH), 7.41–7.44 (m, 3H, ArH),
8.76 (bs, 1H, NH), 13C-NMR (75.6 MHz, CDCl3):
51.7 (CO2Me), 58.1 (OMe), 65.1 (O-CH2), 94.2 (C4),
M
−1), 326 (26,400);
1H-NMR (300 MHz, CDCl3):
δ
δ

Molecules 2020, 25, 1377
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109.4 (C1), 117.0 (aryl C), 117.5 (aryl C), 125.0 (C2), 120.4 (C8), 127.9 (aryl CH), 128.3 (2
×
arylCH), 128.5
(2
×
aryl CH), 132.8 (aryl C), 139.4 (C-Ph), 139.4 (aryl C), 141.1 (aryl C), 149.0 (C5), 162.1 (CO2Me);
HRMS (+ESI): Found m/z 358.1057 [M + Na]+, C20H17NO4Na requires 358.1055.
Methyl
The
5-methoxy-7,7-dimethyl-3,7-dihydropyrano[3,2-e]
compound was prepared as described
indole-2-carboxylate
in GP-3 from
25c (654 mg, 2.08
(
27c
)
title
methyl
(Z)-2-azido-3-(4-methoxy-3-((2-methylbut-3-yn-2-yl)oxy)phenyl)acrylate
mmol) in xylene (20 mL) to give the product (220 mg, 68%) as a yellow solid; m.p. 144–146 ◦C; IR
(
)
(KBr): νmax 3316, 2962, 2975, 2111, 1681, 1625, 1600, 1510, 1439, 1400, 1366, 1270, 1230, 1192, 1131, 1075,
999, 934, 887, 818, 754, 728 cm−1; UV-vis (CH3CN):
λ
max
219 nm (ε M
40,000 cm−1 −1), 271 (13,500), 323
(31,100); 1H-NMR (300 MHz, CDCl3):
5.73 (d, J = 9.7 Hz, 1H, H8), 6.68 (d, J = 9.7 Hz, 1H, H9), 6.79 (s, 1H, H4), 7.19 (d, J = 2.1 Hz, 1H, H1),
9.00 (bs, 1H, NH); 13C-NMR (75.6 MHz, CDCl3):
27.3 (2 C- H3), 51.7 (CO2Me), 58.1 (OMe), 76.2
-(CH3)2), 93.1 (C4), 106.1 (C1), 113.4 (aryl C), 118.2 (aryl C), 119.3 (C9), 125.7 (C2), 130.6 (C8), 132.0
δ
1.53 (s, 6H, 2
×
CH3), 3.92 (s, 3H, OMe), 3.95 (s, 3H, CO2Me),
δ
×
C
(
C
(aryl C), 138.1 (aryl C), 149.5 (C5), 162.3 (
C16H17NO4Na requires 310.1050.
C
O2Me); HRMS (+ESI): Found m/z 310.1044 [M + Na]+,
Methyl
The
5-methoxy-7,7-dimethyl-1,7-dihydropyrano[2,3-g]
compound was prepared as described
indole-2-carboxylate
in GP-3 from
20c (654 mg, 2.08
mmol) in xylene (20 mL) to give the product (203 mg, 74%) as a yellow solid; m.p. 158–160 ◦C; IR
(KBr): 3338, 2955, 2919, 2845, 2111, 1676, 1640, 1595, 1534, 1444, 1370, 1350, 1310, 1265, 1217, 1195,
1134, 1099, 1050, 985, 960, 880, 826, 760, 714 cm−1; UV-vis (CH3CN):
(
22c
)
title
methyl
(Z)-2-azido-3-(3-methoxy-4-((2-methylbut-3-yn-2-yl)oxy)phenyl)acrylate
(
)
ν
max
λ
max
226 nm (
ε
29,600 cm−1
CH3), 3.93 (s, 3H, OMe), 3.96
(s, 3H, CO2Me), 5.71 (d, J = 9.7 Hz, 1H, H8), 6.66 (d, J = 9.7 Hz, 1H, H9), 7.02 (s, 1H, H4), 7.13 (d, J = 2.1
Hz, 1H, H3), 8.95 (bs, 1H, NH); 13C-NMR (75.6 MHz, CDCl3):
27.4 (2 C- H3), 51.8 (CO2Me), 56.5
(OMe), 76.6 ( -(CH3)2), 103.3 (C4), 105.8 (aryl C), 109.3 (C3), 116.8 (C9), 120.6 (aryl C), 125.7 (C2), 128.9
M
−1),
262 (15,200), 341 (19,000); 1H-NMR (300 MHz, CDCl3):
δ
1.55 (s, 6H, 2
×
δ
×
C
C
(aryl C), 129.8 (C8), 142.3 (aryl C), 149.9 (C5), 162.4 (
CO2Me); HRMS (+ESI): Found m/z 288.1228 [M +
H]+, C16H18NO4 requires 288.1236.
3.2. Cell Biology Techniques
The SH-SY5Y and Kelly human neuroblastoma cell lines were generously donated by Dr. J.
Biedler (Memorial Sloan-Kettering Cancer Center, New York, NY, USA). The MDA-MB-231 and MCF-7
breast cancer cell lines were purchased from the American Type Culture Collection. All cell lines
were cultured under standard conditions at 37 ◦C in 5% CO2 as an adherent monolayer in Dulbecco’s
modified Eagle’s medium supplemented with l-glutamine (DMEM) (Invitrogen, Waltham, MA, USA)
and 10% fetal calf serum (FCS) (Thermo Fisher Scientific, Waltham, MA, USA).
Method for Cell Viability Assays
Cell viability was measured by the standard Alamar blue assay, as previously described [18].
Briefly, cells were allowed to attach for 24 h in 96-well culture plates. The cells were then continuously
exposed to serial dilutions of the hydrazide-hydrazone derivatives for 72 h, either in the presence
or absence of SAHA (0.5 or 1
µM), with five replicate wells for each determination. Cell viability
was determined by the addition of 22
µL of Alamar blue reagent, recorded at comparative 0 h
and 5 h values, using a Wallac 1420 Victor III spectrophotometer (GMI, Ramsey, MN, USA), which
measured light absorbance in each well at 570 nm. The cell viability of each plate was calculated as
a percentage compared to matched DMSO controls (0.5%). The mean (+/’SEM) is shown for three
independent experiments.

Molecules 2020, 25, 1377
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3.3. Statistical Analysis
Results of the cell viability studies were statistically analyzed using the two-tailed, unpaired
Student’s t-test. Results are expressed as mean values with 95% confidence intervals.
4. Conclusions
The studies presented in this study have contributed to the investigation of the potential of
dihydropyranoindoles to act as SAHA enhancers for the treatment of neuroblastoma and breast cancer
cells. To the best of our knowledge, this is the first study which was carried out on the combination
of SAHA with dihydropyranoindoles. The desired tricyclic and tetracyclic dihydropyranoindoles was
achieved by the reaction of corresponding hydroxybenzaldehydes with haloalkynes followed by the
application of the Hemetsberger indole synthesis to yield the related indoles. Claisen cyclization
in the presence of chlorobenzene afforded the desired compounds in good yields. Some of the
dihydropyranoindoles were successfully synthesized from the corresponding azido intermediates in one
step by the thermal decomposition step of the Hemetsberger indole synthesis. The biological studies
revealed that the breast cancer cells displayed significant resistance towards the combination treatments
of the dihydropyranoindoles compared to the neurobalstoma cells. It was also found that tetracyclic
analogues of dihydropyranoindoles 33 and 10 were found to be more favorable for the enhancement
of SAHA activity, while only dihydropyrano[3,2-e]indole tricyclic systems 27a and 27c resulted in the
additional reduction of the SAHA cytotoxicity against the neuroblastoma cell lines. Taken altogether, the
tetracyclic dihydropyranoindole 10 was determined as the most cytotoxic compound at the concentration
20 µM, while the lower concentration of the designated compound displayed the best enhancement on
SAHA activity with the value of 44% additional reduction at the ratio of 1:10 SAHA to compound.
Supplementary Materials: The following figures are available online: Figure S1 Cell viability of A) SH-SY5Y, B)
MDA-MB-231 cancer cells treated with the selected six compounds 1–6 (10 µM) over 72 h. Error bars represent
mean values (
and (10 M) against SH-SY5Y, MDA-MB-231 and MRC-5 cell lines after 72 h exposure. Error bars represent
mean values ( S.D.) for three independent determinations. Figure S3. Cell viability of A) MCF-7 p < 0.01 B)
MDA-MB-231 p < 0.05 breast cancer cells treated with 10 M compounds over 72 h, in the presence or absence
of 1 M of SAHA. Error bars represent mean values ( S.D.) for three independent determinations Figure S4
Cell viability of A) Kelly p 0.05 (ns) B) SH-SY5Y p < 0.0005 neuroblastoma cancer cellstreated with 10
compounds over 72 h, in the presence or absence of 1 M of SAHA. Error bars represent mean values ( S.D.) for
three independent determinations. Figure S5. Cell viability of KELLY neuroblastoma cancer cells treated with
compounds A) 33 p < 0.01 B) 27c p < 0.05 and C) 10 p < 0.05 at different concentrations (0.01, 0.1, 1, 10, 20 M)
over 72 h in the absence and presence of SAHA. Error bars represent mean values ( S.D.) for three independent
determinations. Figure S6. Cell viability of SH-SY5Y neuroblastoma cancer cells treated with compounds A) 27a
p < 0.0005 B) 33, p < 0.0005 C) 10, p < 0.01 and D) 27c, p < 0.01 at different concentrations (0.01, 0.1, 1, 10, 20 M)
over 72 h in the absence and presence of SAHA. Error bars represent mean values ( S.D.) for three independent
determinations. Figure S7. Comparative toxicity of compounds A) 27a, p < 0.05 B) 33, p < 0.05 C) 10, p < 0.05
and D) 27c, p < 0.0005 (10 M) against cancer cells (Kelly, SH-SY5Y, MCF-7 and MDA-MB-231) and human lung
±
S.D.) for three independent determinations. Figure S2. Comparative toxicity of compounds 1, 3
4
µ
±
µ
µ
±
.
≥
µM
µ
±
µ
±
,
µ
±
µ
fibroblasts (WI-38 and MRC-5) cell lines after 72 h exposure. Error bars represent mean values (
±
S.D.) for three
independent determinations.
Author Contributions: N.K., D.S.B., G.M.M. and B.B.C. conceived and designed the experiments, G.M.A. provided
the data from library screening, M.B. performed the experiments, analyzed, interpreted the data and wrote the
paper, N.K. and G.M.M. supported financially. All authors have read and agree to the published version of
the manuscript.
Funding: This research was funded by Australian Research Council Discovery Project, grant number DP180100845.
Acknowledgments: We thank the University of New South Wales, and the Australian Research Council (ARC)
for funding to N.K. and D.S.B. Mass spectrometric analysis for this work was carried out at the Bioanalytical
Mass Spectrometry Facility, UNSW and was supported in part by infrastructure funding from the New South
Wales Government as part of its co-investment in the National Collaborative Research Infrastructure Strategy.
This research was supported by Australian Postgraduate Research Scholarships, University of NSW, Program
Grants from the NHMRC Australia, Cancer Institute NSW Australia and Cancer Council NSW Australia. The
Children’s Cancer Institute Australia for Medical Research is affiliated with the University of NSW and Sydney
Children’s Hospital.
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2020, 25, 1377
23 of 24
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Sample Availability: Samples of the compound are not available from the authors.
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