Synthesis and evaluation of 2-carboxy indole derivatives as potent and selective anti-leukemic agents

Article abstract of DOI:10.1016/j.ejmech.2019.111570

Despite the success achieved in the treatment of acute lymphoblastic leukemia (ALL), the search for new drugs featuring selectivity against leukemia cells and effectiveness to prevent relapsed ALL is still highly desirable. Here, we described the synthesi

Full text of DOI:10.1016/j.ejmech.2019.111570

Journal Pre-proof
Synthesis and evaluation of 2-carboxy indole derivatives as potent and selective anti-
leukemic agents
Nathália Moreno Cury, Rebeca Monique Capitão, Renan do Canto Borges de
Almeida, Leonardo Luís Artico, Juliana Ronchi Corrêa, Eric Francisco Simão dos
Santos, José Andrés Yunes, Carlos Roque Duarte Correia
PII:
S0223-5234(19)30700-7
DOI:
https://doi.org/10.1016/j.ejmech.2019.111570
EJMECH 111570
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 June 2019
Revised Date: 26 July 2019
Accepted Date: 28 July 2019
Please cite this article as: Nathá.Moreno. Cury, R.M. Capitão, R.d.C.B.d. Almeida, Leonardo.Luí. Artico,
J.R. Corrêa, E.F. Simão dos Santos, José.André. Yunes, C.R.D. Correia, Synthesis and evaluation of 2-
carboxy indole derivatives as potent and selective anti-leukemic agents, European Journal of Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.111570.
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Synthesis and Evaluation of 2-Carboxy Indole Derivatives as Potent and Selective
Anti-leukemic Agents
Nathália Moreno Cury,a,b,1 Rebeca Monique Capitão,c,1 Renan do Canto Borges de
Almeida,c Leonardo Luís Artico,a Juliana Ronchi Corrêa,a Eric Francisco Simão dos
Santos,c José Andrés Yunes,a,d*and Carlos Roque Duarte Correia3c*
aLaboratório de Biologia Molecular, Centro Infantil Boldrini, Campinas, SP 13083-210,
Brazil
bGraduate Program in Genetics and Molecular Biology, State University of Campinas,
Campinas, SP 13083-210, Brazil
cInstitute of Chemistry, State University of Campinas, Campinas, SP 13083-970, Brazil
dGenetics Department, Faculty of Medical Sciences, State University of Campinas,
Campinas, SP 13083-887, Brazil
* Correspondence to:
Carlos Roque Duarte Correia, e-mail: roque@iqm.unicamp.br
José Andrés Yunes, e-mail: andres@boldrini.org.br
1 These authors contributed equally to this work.

ABSTRACT
Despite the success achieved in the treatment of acute lymphoblastic leukemia
(ALL), the search for new drugs featuring selectivity against leukemia cells and
effectiveness to prevent relapsed ALL is still highly desirable. Here, we described the
synthesis of several novel 3-substituted and 3,6-disubstituted-2-carboalkoxy indoles
followed by the elucidation of their mechanism of action and in vivo anti-leukemia
efficacy. The synthesis of 3-substituted-2-carboalkoxy indoles relied on two Heck
arylations of methyl acrylate and methyl cinnamates respectively, to generate β,β-
disubstituted acrylates followed by an efficient Cadogan-Sundberg reaction of these
latter intermediates. The method developed led to the synthesis of twenty-one novel
functionalized indoles. Of these, indole 20 showed selective cytotoxicity against
leukemia cells at the nanomolar scale, and, therefore, it was selected for the
investigation of its mechanism of action. Indole 20 was found to target tubulin leading
to G2/M cell cycle arrest, DNA damage and apoptosis. Indole 20 decreased β-tubulin
protein in leukemia cells in a time-dependent manner and induced depolymerization of
the microtubule network in Hela cells, thus fully characterizing its microtubule
destabilizer activity. The connectivity map analysis of HL60 promyelocytic leukemia
cells treated with indole 20 revealed a transcriptional profile similar to that of cells
treated with prostaglandins, apparently due to the induction of cellular differentiation as
addressed by the expression of CD11 and CD14 markers. Finally, indole 20 given
intraperitoneally, at 10 mg/kg, 5x/week significantly prolonged the overall survival of
NOD/SCID mice transplanted with RS4;11 B-ALL cells.
Keywords: Indoles; Heck arylations; disubstituted acrylates; acute lymphoblastic
leukemia; tubulin inhibitor; apoptosis.

1. Introduction
The indole nucleus is a privileged heteroaromatic framework, which forms a vast
and important class of natural and synthetic biologically active compounds. Indole
containing compounds show a diversity of pharmacological activities including,
antimicrobials,
antifungal,
antivirals,
anti-inflammatory,
antidepressants,
anticholinergics, anti-migraine, anti-hypertensive, and in particular anticancer activity,
mostly linked to targeting tubulin [1–3]. The tubulin polymerization inhibition activity
[4] results in the microtubule network disruption, which interferes with the correct
mitotic spindle formation and consequently in the cell division process.
The vinca alkaloids vincristine and vinblastine, are examples of indolic
compounds used as anticancer agents since 1965 [5]. Vincristine has been used in the
treatment of acute lymphoblastic leukemia (ALL) and many other cancers for decades.
However, its neurotoxic effects, presumably caused by the disturbance of neurons’
microtubule dynamics, limit the dosage and administration regimen of this drug.
Recently, the use of liposomal vincristine to overcome this drawback has been
demonstrating promising results [6,7]. Despite the successful and consolidated
chemotherapy regimen of vincristine for the treatment of childhood ALL, 20% of
patients still relapse [8,9]. Moreover, the non-selectivity of currently available
chemotherapeutics to malignant cells leads to the undesirable adverse effects observed
in ALL patients under chemotherapy. Accordingly, new anti-leukemia agents are still
needed to seek better therapeutic indexes as well as selectivity to leukemia cells.
Due to the wide applicability of the indole nucleus in medicinal chemistry, many
methods have been developed over the last few decades for its synthesis [10,11]. Some
of the classical methods for the indole synthesis employ the reductive cyclization of
nitro aromatics intermediates, which include: the Bartoli indole synthesis [12], the
Reissert indole synthesis [13], and the Leimgruber and Batcho indole synthesis [14].
Regarding more recent synthetic methods, previous work from our group [15]
investigating a Cu-catalyzed 1,4-reduction of o-nitro substituted 3,3-diaryl acrylates by
chiral copper hydride cleanly provided 2-carboxymethyl-3-aryl indoles as the major
product. Due to the importance of aryl indoles as potential pharmacological leads, we
decided to investigate this transformation even further. From the onset of this research,
we realized that other reducing agents could be applied for the reduction of the o o-nitro
substituted 3,3-diaryl acrylates as well. Therefore, in an attempt to improve the indoles
synthesis (e.g. lower costs, higher yields) from 3,3-diaryl acrylates, and having in mind

the potential evaluation of the biological activity of novel 2-carboalkoxy-3-aryl indoles
as anticancer compounds, we explored new methods for their synthesis. An effective
method for indole synthesis, the Cadogan-Sundberg reaction [16], employs P(OEt)3 to
promote the deoxygenative cyclization of o-nitro cinnamates [17] to furnish 2-
substituted indoles. In view of its effectiveness and low cost, we decided to investigate
the Cadogan-Sundberg reaction for the synthesis of 3-aryl substituted indoles starting
from 3,3-diaryl acrylates. To the best of our knowledge, this is the first report of the use
of the Cadogan-Sundberg reaction to synthesize 3-substituted indoles (aryl, alkyl or
carboxymethyl) starting from β,β-substituted acrylates.
The method developed herein has allowed the synthesis of thirty 2-
carbomethoxy-3-substituted indoles, with twenty-one of them being novel compounds.
With this library available, their cytotoxic activity against leukemia cells was evaluated
in vitro. The indole displaying the highest efficiency and selectivity against leukemia
cells was selected as representative for a detailed investigation of its mechanisms of
action and in vivo activity.
2. Results and discussion
2.1. Chemistry
The synthesis of 2-carbomethoxy-3-arylindoles began with the Heck-Matsuda
arylation of methyl acrylate with arenediazonium salts containing an o-nitro substituent
employing 10 mol% of palladium acetate as catalyst to provide the corresponding
cinnamates 1-3 (Scheme 1) in 80–87% yield. The nitro cinnamates were then used as
starting material to a second Heck-Matsuda reaction employing 2 equivalents of a
distinct arenediazonium salt and 7.5 mol% palladium acetate as catalyst in methanol to
furnish the β,β-diaryl acrylate 4-15 in yields ranging from 29% to 89%. Then, the
Cadogan-Sundberg reductive cyclization of these β,β-diaryl acrylates, promoted by
P(OEt)3, allowed the effective synthesis of the 2-carbomethoxy-3-arylindoles 16-27 in
yields varying from 57% to 91%.

Scheme 1. Synthesis of the 2-carbomethoxy-3-aryl-indoles 16–27.
A similar synthetic strategy has also allowed the synthesis of C-3 alkyl and
carboxy substituted indoles by employing 3-alkyl or 3-carboxyl substituted methyl
acrylates (Scheme 2) as starting materials. The initial conditions for the Heck-Matsuda
arylation employing the 3-substituted methyl acrylates were carried out using 1.5
equivalents of the 4-methoxy-2-nitro arenediazonium tetrafluoroborate, 7.5 mol% of
palladium acetate under refluxing MeOH. Optimizations were performed for the
synthesis of some 3-alkyl cinnamates (32, 33, 34) in order to improve their yields. For
the 3-cyclohexyl cinnamate 32, it was necessary to use two equivalents of the 4-
methoxy-2-nitro benzenediazonium tetrafluoroborate. For cinnamate 33 (R = benzyl), it
was also necessary to use two equivalents of the aryl diazonium salt and 10 mol% of
Pd(OAc)2. Finally, for cinnamate 34 (R= CO2CH3) it was necessary to use two
equivalents of dimethyl fumarate to achieve reasonable yields of the Heck product.
The Cadogan-Sundberg reductive cyclization reaction of cinnamates 28-35 was
carried out as before to furnish the substituted indoles 36-40 and 43 in yields varying
from 50% to 95%. For indoles 41 (R = benzyl) and 42 (R = carboxymethyl) only
moderated to low yields were obtained (44% and 25%, respectively).
Scheme 2. Synthesis of 2-carbomethoxy-3-alky and 3-carboxyalkyl indoles.

As many of the synthesized indoles have functionalities, which allow significant
structural modifications, new analogues were constructed for biological evaluation
besides the indoles 16-27 and 36-43 (a total of 20 indoles). Derivatives of indole 20
were also synthesized by functionalization of its carboxyl group or by alkylation of the
indole nitrogen to provide the new indoles 44-50 (Scheme 3).
The synthesis of the 2-carboxyindole 44 was carried out by reacting the ester 20
with LiOH in a mixture of THF/MeOH/H2O at room temperature (pathway a). The
synthesis of amides 45 and 46 was performed by reacting 2-carboxyindole 44 with the
amines morpholine or phenylalanine in the presence of 1-ethyl-3-(3-
(dimethylamino)propyl)carbodiimide (EDC), hydroxybenzotriazole (HOBt), and
triethylamine as base (pathway b). It was also possible to generate new esters from 2-
carboxyindole 44 through its conversion to the corresponding acyl chloride and reacting
it with the appropriate alcohol to furnish the novel esters 47 and 48 (pathway c). N-
alkylations of indole 20 were also performed using K2CO3 as base and methyl and
benzyl iodides as alkylating agents to provide indoles 49 and 50 respectively (pathway
d).
Scheme 3. Synthesis of new derivatives from indole 20.a
aReagents and conditions: (a) LiOH
EDC·HCl, amine, Et3N, overnight; (c) anhydrous THF, oxalyl chloride/DMF, alkali metal alkoxide
·H2O, MeOH/H2O/THF, 48h, rt; (b) anhydrous DCM, HOBt,

(sodium etoxide or potassium tert-butoxide); (d) anhydrous DMF, K2CO3, alkyl halide (methyl iodide or
benzyl bromide).
The N-hydroxy indole 51 (Scheme 4) was prepared from 3,3-diaryl acrylate 8 by
reductive cyclization with SnCl2 in DMF. The product was obtained in a moderate yield
of 47%.
Scheme 4. Synthesis of N-hydroxy indole 51.
CF3
CF3
O
SnCl2·2H2O
O
OMe
DMF, r.t., 6 h
N
OMe
MeO
OH
47%
MeO
NO2
51
8
Finally, two other analogues of indole 20 were synthesized for evaluation
(Scheme 5). Cinnamate 52 possessing a methoxy group at position 5 of the indole ring
was prepared from 5-methoxy-2-nitro arenediazonium salt and methyl acrylate to
evaluate the influence of a methoxy group at that position. Cinnamate 53 (hydroxy
group at position 6) was prepared from 4-hydroxy-2-nitro arenediazonium salt and
methyl acrylate to evaluate the effect of a phenolic group in the antileukemic activity
when compared to a methoxy group. The synthesis of the indole 56 (methoxy group at
position 5) and 57 (hydroxy group at position 6) were performed as previously
described in Scheme 1 from 3,3-diaryl acrylate 54 and 55.
Scheme 5. Synthesis of 2-carbomethoxy-3-aryl-indoles 56 and 57.
2.2. Biological assays

2.2.1 Indoles cytotoxicity against leukemia cell lines
Initially, the 2-carbomethoxy-3-aryl-indoles 16-27 (Table 1) were tested for their
cytotoxic activity against two different leukemia cell lines. Aryl indoles 18, 19, and 20
showed activity, in a submicromolar range, against CEM cells with EC50 values of 0.33,
0.58, and 0.22 µM respectively. Aryl indoles 18 and 20 also showed a submicromolar
activity against the RS4;11 cell line with EC50 values of 0.20 and 0.30 µM. The
influence of different substituents at the C6 position of indole bearing distinct
electronics (OMe, H, CF3) was also tested. With the exception of indole 22 (Entry 7,
Table 1), all other indoles bearing a hydrogen atom or a CF3 group at the position 6
showed poor cytotoxicity (>100µM) against both CEM and RS4;11 cells (Entry 6, and
entries 8-12, Table 1). The presence of a methoxy group at the C-6 position (Entries 1-
5, Table 1) of aryl indoles 16-20 proved beneficial when compared to aryl indoles 21-
24, which exhibited low cytotoxicity, with the exception of aryl indole 22 with modest
in vitro anti-leukemia activity. The next step was to evaluate aryl indoles bearing a
methoxy group at the C6 position and to test the effect of other substituents at the C3
position of the indole moiety. This study was carried out with the C-3 alkyl and C-3
ester indoles 36-43. However, C-3 alkyl or C-3 esters groups at position 3 of the indole
nucleus did not improve the anti-leukemia activity, showing cytotoxicity at the
micromolar range (Entries 13-20, Table 1).
Table 1. The antileukemic activity of the 3-substituted-indoles.
CEM
RS4;11
Entry Compound R1
R2
EC50 (µM) EC50 (µM)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
OMe 2-OMe-Ph
1.90
10.0
0.33
0.58
0.20
10.5
85.6
0.20
3.80
0.30
16
17
18
19
20
21
22
23
24
25
26
27
36
37
38
OMe
OMe
4-F-Ph
4-Br-Ph
OMe 3,4-Cl-Ph
OMe 4-CF3-Ph
H
H
H
H
4-OMe-Ph
Ph
4-F-Ph
4-CF3-Ph
>100
35.8
>100
95.2
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
81.5
CF3 4-OMe-Ph
CF3
CF3
OMe
OMe
OMe
Ph
4-CF3-Ph
Me
Et
iPr

16
17
18
19
20
OMe
OMe
OMe
OMe
i Bu
Cy
Bn
16.6
80.2
8.70
>100
>100
37.8
>100
61.6
>100
>100
39
40
41
42
43
CO2Me
OMe CH2CO2Me
In view of the promising in vitro anti-leukemia activity displayed by the
carboxymethyl aryl indole 20, we synthesized the aryl indole derivatives 44-48, the N-
alkyl and N-hydroxy indoles 49-51, and the aryl indoles 59 and 60 (methoxy at C-5, and
a hydroxy group at C-6 respectively). The 2-carboxyl analog 44 (Entry 1, Table 2) and
the amides analogs 45 and 46 (Entries 2 and 3, Table 2) showed moderate activity for
both CEM and RS4;11 cells (19.8 to 52.0 µM; Table 2). However, these compounds
were much less active when compared to indole 20 (EC50 = 0.20 µM for CEM, and
0.30µM for RS4;11). Both esters 47 and 48 (Entries 4 and 5, Table 2), and the N-methyl
indole 49 and N-benzyl indole 50 (Entries 6 and 7) displayed decreased cytotoxicity
against both CEM and RS4;11 cells. However, the N-hydroxy indole derivative 51
exhibited potency similar to that of aryl indole 20 with EC50 values of 0.39 nM and 0.30
nM against CEM and RS4;11 cells (Entry 8, Table 2). Finally, the change in the
position of the methoxy group was deleterious to the cytotoxic activity with aryl indole
56 (OMe at C-5) showing an EC50 higher than 100 µM (Entry 9, Table 2). The aryl
indole 57 bearing a hydroxyl group at position 6 showed a moderate activity of ~10 µM
for both CEM and RS4;11 cells (entry 10, Table 2) indicating that, in comparison to aryl
indole 20, demethylation was deleterious to the anti-leukemia activity.
Table 2. Evaluation of the new analogs of aryl indole 20.
CF3
O
R3
N
R2
R1
Entry Compound
R1
R2
R3
CEM
RS4;11
EC50 (µM) EC50 (µM)
1
2
6-OMe
6-OMe
H
H
OH
19.8
29.7
24.4
47.6
44
45
3
6-OMe
H
52.0
43.1
46
4
5
6
6-OMe
6-OMe
6-OMe Me OMe
H
H
OEt
>100
>100
>100
>100
>100
>100
47
48
49
OtBu

7
8
9
6-OMe Bn OMe
6-OMe OH OMe
>100
0. 39
>100
9.90
>100
0.30
>100
10.3
50
51
56
57
5-OMe
6-OH
H
H
OMe
OMe
10
Among the compounds with the highest cytotoxic activity against leukemia cell
lines, we chose indole 20 for further investigation including its mechanisms of action
since it showed no cytotoxicity against healthy T-lymphocytes up to a dose of 100 µM,
indicating selective cytotoxicity against leukemia cells (Figure 1).
Figure 1. Indole 20 presented selective cytotoxicity against leukemia cells. CEM,
RS4;11 cell lines and T-lymphocytes stimulated with phytohemagglutinin and
interleukin-2 overnight were incubated with decreasing doses of indole 20 for 48h and
cell viability was assessed using the MTT method.
2.2.2 Indole 20 targets tubulin leading to cell cycle arrest and apoptosis
To shed light on the mode of action of indole 20, we performed gene expression
profiling analysis in HL60 cells treated with 1 µM of indole 20 (EC90 dose - data not
shown) for 6 h. The HL60 acute promyelocytic leukemia cell line was chosen for the
gene expression assay to enable Connectivity Map (CMap) analysis [18]. Treatment of
HL60 cells with indole 20 resulted in the upregulation of 22 genes and downregulation
of 23 genes (-1.5 ≤ FC ≥ 1.5, p-value≤0.01, Supplemental Table 1). Gene Set
Enrichment Analysis (GSEA) [19] revealed a significant enrichment of G2/M
Checkpoint, Mitotic Spindle and NOTCH Signaling gene sets as well as a decrease in
the cholesterol homeostasis gene set as a result of indole 20 treatment (Figure 2).

Figure 2. GSEA enrichment plots. HL60 cells were treated with 1 µM of indole 20
for 6 h and gene expression data was analyzed using the GSEA platform. All the probe
sets/transcript clusters annotated with a Gene Symbol in the array were used in the
analysis. p, p-value; FDR, False Discovery Rate after 1,000 permutations by gene-set.
As expected, cell cycle analysis of HL60 cells treated with indole 20 manifested
G2/M arrest in a time-dependent manner, as well as the rising of a sub-G1 population of
cells indicative of apoptosis (Figure 3A). Apoptosis was further confirmed by an assay
detecting both phosphatidylserine exposure and cell membrane integrity. Indole 20
induced apoptosis in HL60, CEM, and RS4;11 leukemia cells after 18 hours of
treatment (Figure 3B).
To investigate whether cell cycle arrest induced DNA damage and then
apoptosis, RS4;11 cells were treated with indole 20 (Figure 4A) for 18 hours followed
by BrdU/PI (cell cycle), H2AX (DNA damage) and PARP (apoptosis) labeling. As

shown in Figure 4B, indole 20 treatment had no effect on S-phase proportion but caused
a significant increase in G2/M and the corresponding decrease in G1. Although it is not
well known how anti-mitotic drugs kill cancer cells, prolonged mitotic arrest triggers
cell death. Some of the cells divide unequally, i.e., producing aneuploid daughter cells
[20]. Indole 20 treatment resulted in a population of cells in between G1 and G2
(hereafter called sub-G2-like) which may be suggestive of unequal division.
Staining with anti-H2AX antibody allowed us to verify that DNA damage
occurred in 6.9%, 2.3% and 6.9% of each G1, sub-G2-like and G2 populations
respectively, while staining with PARP antibody revealed that apoptosis occurred in
7%, 3.5% and 1.5% of each G1, sub-G2-like and G2 populations respectively (data not
shown).
One possible interpretation of these results is that indole 20 induces DNA
damage in G2. Some of these cells die (PARP labeled) right away but some of them
proceed with division or unequal division, dyeing afterwards in G1 or sub-G2-Like.
That is why there is higher PARP labeling in G1 and sub-G2 than in G2.
Figure 3. Indole 20 promotes G2/M cell cycle arrest and apoptosis in leukemia
cells. In a time-dependent manner. For cell cycle analysis, HL60 cells were treated with
586 nM (EC50) and 1 µM (EC90) for 12 and 18 hours followed by PI staining. Cell cycle
phases were represented as sub-G0/G1 (apoptotic cells), G0/G1, S, and G2/M.
Apoptosis induced by indole 20. For apoptosis analysis, CEM, HL60, and RS4;11 cells
were treated with 300 nM of indole 20 for 18 hours followed by staining with Annexin
V and PI and flow cytometry analysis.

Figure 4. Indole 20 induces apoptosis of RS4;11 cells at G2/M and G0/G1 phases.
Cells were treated with (A) DMSO (vehicle) or (B) 300 nM of indole 20 for 18 hours
followed by labeling with 10 µM BrdU for 1 hr. The cells were then harvested and
analyzed by immunofluorescent staining and multicolor flow cytometric analysis.
BrdU-positive cells are color-gated light blue whereas BrdU-negative cells at G1 phase
and G2/M phase of the cell cycle are colored red and dark blue respectively. Cells
between G1 and G2 phases (sub-G2-like cells) and sub-G0/G1 are colored light green
and dark gray respectively.

It is well established that some microtubule-interacting agents, like vincristine
and vimblastine, promote cell cycle arrest by inhibiting tubulin polymerization. Free
tubulin, i.e. tubulin that is not incorporated into microtubules, negatively regulates the
levels of tubulin mRNA and tubulin protein [21]. Western blot analysis of RS4;11,
CEM and HL60 cells treated with indole 20 showed a markedly tubulin decrease along
time (Figure 6), indicating that this compound acts through inhibition of microtubule
polymerization. In addition, we could also observe an increase on p53 protein during
time (Figure 6). These results are in agreement with our previous data evidencing the
induction of cell cycle arrest in G2/M (Figure 3A), DNA damage (Figure 4B) and
apoptosis (Figures 3B and 4B), respectively.
Figure 6. Indole 20 decreases β-tubulin and increases p53 proteins in leukemia cell
lines. Western blotting analysis for β-tubulin and p53 proteins in RS4;11, CEM and
HL60 cells treated with 300 nM of indole 20 for, 0, 15, 30, 60 and 360 min. Lamin was
used as loading control.
To further confirm tubulin as the main target of indole 20, we performed the
immunofluorescence assay using HELA cells stained with anti β-tubulin antibody after
20 hours of treatment with indole 20 (Figure 7). Compared to vehicle-treated cells, 500
nM of indole 20 clearly induced the microtubule network depolymerization and the cell
cycle arrest, evidenced by the condensed chromosomes. In addition, we could observe
abnormal chromosomal segregation and fragmented centrosomal material which are

characteristic of microtubules degradation. At the dose of 1 µM of indole 20, we could
barely observe tubulin staining in some of the cells (Figure 7).
Figure 7. Effects of indole 20 on microtubule cytoskeleton of Hela cells. Hela cells
were incubated for 18 hours with DMSO (Control), 500 nM and 1 µM of indole 20.
Anti-β-tubulin antibodies and DAPI were used to stain microtubules (red), and DNA
(blue), respectively.
2.2.3 Indole 20 promotes leukemia cells differentiation
The gene expression profile of HL60 cells treated with indole 20 was compared
to the gene expression profiles obtained from hundreds of known drugs, available in the
Connectivity Map platform [22]. Indole 20 showed a gene expression signature similar
to that of prostaglandins (C01EA; Figure 8A). Prostaglandin J2 was shown to inhibit
microtubule polymerization [23]. Moreover, prostaglandins were shown to promote
rapid in vitro differentiation of HL60 cells along the granulocyte pathway [24].
Consistent with this fact, treatment of HL60 with indole 20 during 48 hours resulted in
an increased expression of the differentiation markers CD11b and CD14, in a dose-
dependent manner (Figure 8B). Retinoic acid and vitamin D3 treatments were used as
positive controls for CD11b and CD11b/CD14, respectively. CD11b levels, which is a
marker of the granulocyte pathway, begins to increase after 24h of indole 20 treatment
(Supplemental Figure 1), while cells positive for both CD11b and CD14 levels began to
appear later, after 48 hours of treatment (Figure 8B).
Of note, CMap analysis evidenced that indole 20 induced a gene expression
profile opposite to both apigenin, a well-known as an autophagy inducer [25], and
HDAC (histone deacetylases) inhibitors which also act inducing autophagy [26]
(Supplemental Figure 2). These results suggest that indole 20 may decrease or inhibit

the cellular self-degradative process of autophagy. Additional experiments are
warranted to investigate the role of indole 20 in autophagy.
Figure 8. Indole 20 induces differentiation of HL60 leukemia cells. A. Cmap
analysis based on ATC code showed that indole 20 presented a drug-induced gene
expression profile similar to prostaglandins. B. HL60 cells were treated with vehicle
(DMSO), EC20, EC50 and EC90 doses of indole 20, 2 µM of ATRA or 100 nM of VD3
for 48 hours. ATRA, Trans retinoic acid; VD3, vitamin D3.
2.2.4 Indole 20 anti-leukemic activity in ALL engrafted NOD/SCID mice
The in vivo anti-leukemia effect of indole 20 was evaluated in NOD/SCID mice
transplanted with RS4;11 leukemia cells. Ten animals were randomized in control
(vehicle-treated) and indole 20-treated groups. Treatment initiated when the human
ALL percentage in the peripheral blood of half of the animals reached >1,5%, featuring
an advanced stage of the disease. Animals were treated with vehicle (1% PVP, 4%
DMSO and 95% PBS) or indole 20 at 10 mg/kg, given intraperitoneally once a day, 5x
per week. Indole 20 was well tolerated by mice during the whole experiment. As shown

in Figure 9, NOD/SCID mice treated with indole 20 showed a significant increase in
survival compared to controls.
Figure 9. Indole-20-prolonged survival of RS4;11 engrafted NOD/SCID mice.
Kaplan Meyer survival curves of mice following the treatment with 10 mg/kg of indole
20 (n=5) or vehicle (n=5) administrated 5x per week, intraperitoneally. Curves were
compared by the log-rank test. Dashed line marks the time when leukemia engraftment
at ≥ 1.5% in peripheral blood was documented and treatment was initiated.
2.2.5 Discussion
It is a well-known fact that indole molecules acting as inhibitors of tubulin
polymerization are considered potential new anticancer agents. Here, we show the
synthesis of the new indole 20, which showed anti-proliferative, and pro-apoptotic
activity, at the nanomolar range, against acute leukemia cells. Indole 20 promoted
microtubule depolymerization and cell cycle arrest at G2/M suggesting that our
structural modifications in the indole ring did not affect its well-known molecular target
in cancer cells. However, an attractive feature of indole 20 was its selectivity against
leukemia cells, showing no apparent cytotoxicity against normal T-lymphocytes, even
at 100 µM. On the contrary, vincristine, one of the main drugs used in the treatment of
ALL, presents a narrow therapeutic window regimen due to its high toxicity [6,7]. The
selective cytotoxicity of indole 20 against leukemia cells in comparison to T-
lymphocytes may be due to a particular tubulin isoform composition of leukemia cell
[27,28].
Microarray expression analysis of HL60 cells treated with indole 20 confirmed
the upregulation of mitotic spindle and G2/M checkpoints gene sets, as expected for a
tubulin-binding agent. However, indole 20 transcriptional profile showed also similarity
to prostaglandins, probably due to upregulation of NOTCH signaling [24] and induction
of cell differentiation.

CMap analysis also showed a transcriptional profile opposite to apigenin and
HDAC inhibitors. Both classes of drugs are known to induce autophagy in TF1 and
AML1-ETO leukemia cells, respectively [25,26]. Thus, we speculated that indole 20
may have an autophagy repression activity. It is well known that autophagy has
cytoprotective roles to guarantee cell survival [29]. Autophagy activation has been
described as a mechanism of resistance to dexamethasone [30] and its inhibition can
overcome this resistance in lymphoid malignant cells [31]. Moreover, inactivation of
autophagy potentiated the efficacy of AraC treatment as well as reduced functional
leukemia initiation cells in murine myeloid leukemia [32]. Further studies are warranted
to verify the effects of indole 20 on autophagy.
Indole 20 prolonged the overall survival of NOD/SCID mice transplanted with a
pre B-ALL cell line when given at a dose of 10 mg/kg/5x per week. Of note, indole 20
treatment started when leukemia cells were ≥ 1.5% on peripheral blood, which in this
model corresponds to a high stage leukemia burden in the bone marrow and spleen [33].
These preliminary results revealed that indole 20 had a positive effect on survival, even
when used as a single agent against ALL. However, further in vivo studies are necessary
to better establish the correct schedule of indole 20 administration and its combination
with other chemotherapeutic agents.
3. Conclusions
We have developed a versatile and high yielding method for the synthesis of several
new 2-carboxyindoles starting from β,β-acrylates. This synthetic method allowed the
construction of a library of over thirty different 2-carbomethoxy-3-substituted indoles
with a diversity of groups at the C3 position such as aryl, alkyl, and carboxymethyl
groups. The new active compound methyl 6-methoxy-3-(4-(trifluoromethyl) phenyl)-
1H-indole-2-carboxylate (indole 20) was found to target tubulin leading to G2/M cell
cycle arrest, DNA damage and apoptosis featuring selectivity against leukemia cells.
The Connectivity map analysis of HL60 promyelocytic leukemia cells treated with
indole 20 revealed a transcriptional profile similar to that of cells treated with
prostaglandins. Moreover, indole 20 given intraperitoneally, at 10 mg/kg, 5x/week
significantly prolonged the overall survival of NOD/SCID mice transplanted with
RS4;11 B-ALL cells. These results indicate that indole 20 is a promising lead for the
development of new agents for the treatment of acute lymphoblastic leukemia.

4. Experimental section
4.1 Chemistry
General Information. Solvents used for chromatography were distilled prior to use.
The other reagents were used without further purification unless otherwise stated. The
purification of reaction products was performed by flash chromatography using silica
gel (220−440 mesh) and EtOAc/hexane mixtures as the eluent. Analytical thin-layer
chromatography (TLC) was performed employing silica gel 60 F254 plates. Visualization
was accomplished with UV light. NMR analyses were performed on 250, 400, 500 or
1
600 MHz NMR spectrometers. Chemical shifts (δ) for H and 13C-NMR spectra are
given in ppm. The residual solvent signals were used as references for 1H and 13C-NMR
spectra and the chemical shifts converted to the TMS scale. Data were reported as
follows: chemical shift (δ), multiplicity, coupling constant (J) in Hertz and integrated
relative intensity. High-resolution mass spectrometry (HRMS) was performed by direct
infusion in the mass spectrometer using electrospray ionization with quadrupole time-
of-flight or orbitrap as an analyzer (ESI-Q-TOF or ESI-orbitrap, respectively). Melting
points (Mp) were determined on a capillary melting point apparatus and are
uncorrected. The arenediazonium tetrafluoroborates were prepared according to the
literature [34]. Methyl (E)-4-phenylbut-2-enoate and methyl (E)-3-cyclohexyl acrylate
starting materials were synthesized according to the literature and the spectroscopic data
matches the values reported in the literature [35,36]. Other acrylate derivatives were
commercially available in its pure E configuration and have been used without previous
purification.
General Procedure A: Synthesis of of Cinnamates. To a round-bottom flask equipped
with a magnetic stir bar, a solution of methyl acrylate (15 mmol) in methanol (60 mL)
was kept under stirring. Next, half of the o-nitro substituted arenediazonium
tetrafluoroborate (5 mmol), and palladium acetate (0.5 mmol) were added. After stirring
at room temperature for 15 minutes, the other half (5 mmol) of the o-nitro substituted
arenediazonium tetrafluoroborate (5 mmol) and additional palladium acetate (0.5 mmol)
were added. The mixture was stirred at r.t. until complete consumption of the
arenediazonium salt. The reaction mixture was then filtered through a short pad of silica

and purified by flash column chromatography using hexane/ethyl acetate (80:20) as
eluent.
General Procedure B: Heck Matsuda of Cinnamates. A round-bottom flask equipped
with a magnetic stir bar was charged with the cinnamate ester (1 mmol), Pd(OAc)2 (7.5
mol%, 0.075 mmol), and methanol (6 mL). After vigorous stirring at room temperature,
the corresponding arenediazonium tetrafluoroborate (2 mmol) was added in one portion.
The reaction mixture was immersed in oil bath and heated to reflux until TLC indicated
complete consumption of the cinnamate. After cooling, the mixture was filtered
through a short pad of silica and purified by flash column chromatography using
hexane/ethyl acetate (80:20) as an eluent.
General Procedure C: Synthesis of indoles. In a sealed tube were added the β,β-
substituted acrylate (1 mmol) and triethyl phosphite (0.79 mL, 4 mmol). The reaction
was heated at 160°C in an oil bath for 24 hours. After cooling to rt, the crude reaction
mixture was mixed with silica gel, and the resulting slurry was place on the top of a
flash column chromatography (silica gel), and purified using hexane/ethyl acetate
(80:20) as eluent to provide the corresponding indoles.
Methyl (E)-3-(4-methoxy-2-nitrophenyl)acrylate (1). Prepared using general
procedure
A
from methyl acrylate and 4-methoxy-2-nitrobenzenediazonium
1
tetrafluoroborate to provide the compound 1 as a yellow solid (80% yield). H NMR
(500 MHz, CDCl3) δ 8.04 (d, J = 15.8 Hz, 1H), 7.58 (d, J = 8.7 Hz, 1H), 7.50 (d, J = 2.7
Hz, 1H), 7.17 (dd, J = 8.8, 2.6 Hz, 1H), 6.31 (d, J = 15.8 Hz, 1H), 3.91 (s, 4H), 3.82 (s,
4H). 13C NMR (126 MHz, CDCl3) δ 166.5, 160.9, 149.4, 139.5, 130.0, 122.5, 121.1,
120.0, 109.5, 56.1, 51.9. CAS No.: 103986-96-5.
Methyl (E)-3-(2-nitrophenyl)acrylate (2). Prepared using general procedure A from
methyl acrylate and 2-nitrobenzenediazonium tetrafluoroborate to provide the
compound 2 as a light yellow solid (87% yield). 1H NMR (500 MHz, CDCl3) δ 8.12 (d,
J = 15.8 Hz, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.69–7.61 (m, 2H), 7.55 (ddd, J = 8.6, 6.6,
13
2.2 Hz, 1H), 6.37 (d, J = 15.8 Hz, 1H), 3.83 (s, 3H). C NMR (126 MHz, CDCl3) δ
166.2, 148.4, 140.1, 133.5, 130.3, 129.1, 124.9, 122.9, 52.0. CAS No.: 612-43-1.

Methyl (E)-3-(2-nitro-4-(trifluoromethyl)phenyl)acrylate (3). Prepared using general
procedure A from methyl acrylate and 4-trifluoromethyl-2-nitrobenzenediazonium
tetrafluoroborate to provide the compound 3 as a light yellow solid (86% yield). Mp 58-
59 °C. 1H NMR (500 MHz, CDCl3) δ 8.33 (s, 1H), 8.12 (d, J =15.8 Hz, 1H), 7.91 (d, J
13
= 8.1 Hz, 1H), 7.78 (d, J = 8.1 Hz, 1H), 6.43 (d, J = 15.9 Hz, 1H), 3.85 (s, 4H). C
NMR (126 MHz, CDCl3) δ 165.7, 148.1, 138.7, 134.1, 132.7, 132.5, 130.2, 130.0,
124.9, 123.6, 122.4, 121.5, 52.2. HRMS (ESI/Q-TOF) m/z [M + H]+ calcd for
C11H9F3NO4: 276.1907; found 276.1909.
Methyl (E)-3-(4-methoxy-2-nitrophenyl)-3-(2-methoxyphenyl)acrylate (4). Prepared
using general procedure B from compound 1 and 2-methoxybenzenediazonium
tetrafluoroborate to provide the compound 4 as a light yellow solid (78% yield). Mp
118-119 °C. 1H NMR (600 MHz, CDCl3) δ 7.62 (d, J = 2.6 Hz, 1H), 7.29 (ddd, J = 8.4,
7.4, 1.8 Hz, 1H), 7.20 (d, J = 8.5 Hz, 1H), 7.14 (dd, J = 8.5, 2.6 Hz, 1H), 7.09 (dd, J =
7.7, 1.8 Hz, 1H), 6.90 (d, J = 8.8 Hz, 1H), 6.87 (dd, J = 7.5, 1.1 Hz, 1H), 6.59 (s, 1H),
13
3.89 (s, 3H), 3.75 (s, 3H), 3.60 (s, 3H). C NMR (151 MHz, CDCl3): δ 166.3, 159.2,
157.6, 150.7, 148.3, 132.2, 131.0, 130.6, 128.0, 120.7, 120.5, 119.4, 111.7, 108.6, 55.8,
55.6, 51.4. HRMS (ESI/Q-TOF) m/z [M + H]+calcd for C18H18NO6: 344.1129; found
344.1122.
Methyl (Z)-3-(4-fluorophenyl)-3-(4-methoxy-2-nitrophenyl)acrylate (5). Prepared
using general procedure B from compound 1 and 4-fluorobenzenediazonium
tetrafluoroborate to provide the compound 5 as a light yellow solid (55% yield). Mp 98-
100 °C.1H NMR (400 MHz, CDCl3) δ 7.71 (d, J = 2.5 Hz, 1H), 7.32–7.29 (m, 2H), 7.22
(dd, J = 8.5, 2.6 Hz, 1H), 7.15 (d, J = 8.5 Hz, 1H), 7.01 (t, J = 8.6 Hz, 2H), 6.41 (s, 1H),
3.92 (s, 3H), 3.60 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 166.0, 163.4 (d, J = 251 Hz),
159.9, 153.0, 148.6, 135.0, 134.9, 132.0, 129.6, 129.5, 126.6, 120.2, 116.4 (d, J = 21.9
Hz), 115.9, 115.7, 109.4, 56.0, 51.6. HRMS (ESI-Orbitrap) m/z [M + H]+ calcd for
C17H15FNO5: 332.09288; found 332.09285. Also, [M + Na]+ calcd for C17H14FNO5Na:
354.07482; found 354.07489.
Methyl (Z)-3-(4-bromophenyl)-3-(4-methoxy-2-nitrophenyl)acrylate (6). Prepared
using general procedure B from compound 1 and 4-bromobenzenediazonium
tetrafluoroborate to provide the compound 6 as a light yellow solid (57% yield). Mp

114-115 °C. 1H NMR (400 MHz, CDCl3) δ 7.71 (d, J = 2.6 Hz, 1H), 7.46 (d, J = 8.7 Hz,
1H), 7.24 (dd, J = 8.5, 2.6 Hz, 1H), 7.21 (d, J = 8.6 Hz, 2H), 7.17 (d, J = 8.5 Hz, 1H),
13
6.44 (s, 1H), 3.92 (s, 3H), 3.60 (s, 3H). C NMR (101 MHz, CDCl3) δ 165.7, 159.9,
152.8, 148.4, 137.6, 131.9, 131.8, 129.0, 126.1, 124.3, 120.0, 116.9, 109.26, 55.9, 51.6.
HRMS (ESI/Q-TOF) m/z [M + H]+ calcd for C17H15BrNO5: 392.0128; found 392.0142.
Methyl
(Z)-3-(3,4-dichlorophenyl)-3-(4-methoxy-2-nitrophenyl)acrylate
(7).
Prepared using general procedure
B
from compound and 3,4-
1
dichlorobenzenediazonium tetrafluoroborate to provide the compound 7 as a white solid
1
(56% yield). Mp 133-134 °C. H NMR (500 MHz, CDCl3) δ 7.73 (d, J = 2.7 Hz, 1H),
7.41 (d, J = 8.4 Hz, 1H), 7.38 (d, J = 2.2 Hz, 1H), 7.23 (dd, J = 8.5, 2.6 Hz, 1H), 7.17
(dd, J = 8.5, 2.2 Hz, 1H), 7.14 (d, J = 8,5 Hz, 1H), 6.43 (s, 1H), 3.94 (s, 3H), 3.61 (s,
3H).13C NMR (126 MHz, CDCl3):δ 165.5, 160.0, 151.6, 148.4, 138.7, 134.0, 133.1,
131.8, 130.6, 129.2, 126.6, 125.5, 120.2, 117.8, 109.4, 55.9, 51.7. HRMS (ESI/Q-TOF)
m/z [M + H]+ calcd for C17H14Cl2NO5: 382.0244; found 382.0241.
Methyl (Z)-3-(4-methoxy-2-nitrophenyl)-3-(4-(trifluoromethyl)phenyl)acrylate (8).
Prepared using general procedure B from compound 1 and 4-trifluoromethyl
benzenediazonium tetrafluoroborate to provide the compound 8 as a light yellow solid
(65% yield). Mp 114-115 °C.1H NMR (400 MHz, CDCl3) δ 7.77 (s, 1H), 7.62 (d, J =
8.1 Hz, 2H), 7.48 (d, J = 8.2 Hz, 2H), 7.28 (m, 1H), 7.21 (d, J = 8.6 Hz, 1H), 6.53 (s,
1H), 3.97 (s, 3H), 3.65 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 165.7, 160.2, 152.6,
148.6, 142.3, 132.0, 131.6 (q, J = 32.8 Hz), 128.0, 126.0, 125.7 (q, J = 3.5 Hz), 124.0
(q, J = 272 Hz), 120.2, 118.6, 109.5, 56.0, 51.7. HRMS (ESI/Q-TOF) m/z [M + H]+
calcd for C18H15F3NO5: 382.0897; found 382.0888.
Methyl (Z)-3-(4-methoxyphenyl)-3-(2-nitrophenyl)acrylate (9). Prepared using
general procedure
B
from compound
2
and 4-methoxybenzenediazonium
tetrafluoroborate to provide the compound 8 as a light yellow solid (57% yield). Mp
148-149 °C. 1H NMR (600 MHz, CDCl3) δ 8.20 (d, J = 8.2 Hz, 1H), 7.67 (m, 1H), 7.57
(m, 1H), 7.26 (m, 3H), 6.84 (d, J = 9.0 Hz, 2H), 6.42 (s, 1H), 3.80 (s, 3H), 3.57 (s, 3H).
13C NMR (151 MHz, CDCl3) δ 166.1, 161.1, 153.6, 147.8, 135.2, 133.3, 131.0, 130.5,
129.0, 128.8, 124.6, 114.2, 114.1, 55.4, 51.4. HRMS (ESI/Q-TOF) m/z [M + H]+ calcd
for C17H16NO5: 314.1023; found 314.1020.

Methyl (Z)-3-(2-nitrophenyl)-3-phenylacrylate (10). Prepared using general
procedure B from compound 2 and benzenediazonium tetrafluoroborate to provide the
compound 10 as a light yellow solid (39% yield). Mp 115-116 °C. 1H NMR (500 MHz,
CDCl3) δ 8.14 (dd, J = 8.3, 1.3 Hz, 1H), 7.60 (td, J = 7.5, 1.3 Hz, 1H), 7.50 (td, J = 8.7,
7.4, 1.5 Hz, 1H), 7.28–7.25 (m, 2H), 7.25-7.23 (m, 3H), 7.21 (dd, J = 7.6, 1.4 Hz, 1H),
13
6.42 (s, 1H), 3.51 (s, 3H). C NMR (126 MHz, CDCl3) δ 165.9, 154.0, 147.8, 138.1,
134.9, 133.3, 131.1, 129.9, 128.9, 128.7, 127.5, 124.7, 116.4, 51.5. HRMS (ESI/Q-
TOF) m/z [M + H]+ calcd for C16H14NO4: 284.0917; found 284.0906.
Methyl (Z)-3-(4-fluorophenyl)-3-(2-nitrophenyl)acrylate (11). Prepared using
general procedure B from compound 2 and 4-fluorobenzenediazonium tetrafluoroborate
to provide the compound 11 as a light yellow solid (89% yield). Mp 98-99 °C. 1H NMR
(500 MHz, CDCl3) δ 8.22 (dd, J = 8.3, 1.0 Hz, 1H), 7.69 (td, J = 7.5, 1.2 Hz, 1H), 7.62–
7.55 (m, 1H), 7.34–7.29 (m, 2H), 7.27 (dd, J = 7.6, 1.3 Hz, 1H), 7.06–6.99 (m, 2H),
6.43 (s, 1H), 3.58 (s, 3H).13C NMR (126 MHz, CDCl3) δ 165.8, 163.8 (d, J = 251 Hz),
153.0, 147.8, 134.8, 134.5, 134.4, 133.5, 131.0, 129.6 (d, J = 8.5 Hz), 129.2, 124.9,
116.4, 115.9 (d, J = 21.9 Hz), 51.6. HRMS (ESI/Q-TOF) m/z [M + H]+ calcd for
C16H13FNO4: 302.0823; found 302.0825.
Methyl (Z)-3-(2-nitrophenyl)-3-(4-(trifluoromethyl)phenyl)acrylate (12). Prepared
using general procedure B from compound 2 and 4-trifluoromethylbenzenediazonium
tetrafluoroborate to provide the compound 12 as a light yellow solid (40% yield). Mp
114-115 °C. 1H NMR (400 MHz, CDCl3) δ 8.25 (dd, J = 8.2, 1.3 Hz, 1H), 7.72 (td, J =
7.5, 1.3 Hz, 1H), 7.63–7.59 (m, 3H), 7.45 (d, J = 8.0 Hz, 2H), 7.29 (dd, J = 7.6, 1.5 Hz,
1H), 6.52 (s, 1H), 3.60 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 165.5, 152.6, 147.8,
141.8, 134.2, 133.7, 131.7 (q, J = 32.8 Hz), 131.1, 129.5, 127.9, 125.8 (q, J = 3.7 Hz),
125.0, 123.9 (q, J = 272 Hz), 118.5, 51.8. HRMS (ESI/Q-TOF) m/z [M + H]+ calcd for
C17H13F3NO4: 352.0791; found 352.0788.
Methyl
(Z)-3-(4-methoxyphenyl)-3-(2-nitro-4-(trifluoromethyl)phenyl)acrylate
from compound and 4-
(13). Prepared using general procedure
B
3
methoxybenzenediazonium tetrafluoroborate to provide the compound 13 as a light
1
yellow solid (49% yield). Mp 142-144°C. H NMR (500 MHz, CDCl3) δ 8.46 (d, J =

1.7 Hz, 1H), 7.91 (dd, J = 8.1, 1.7 Hz, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.22 (d, J = 8.9 Hz,
13
2H), 6.86 (d, J = 8.9 Hz, 2H), 6.46 (s, 1H), 3.81 (s, 3H), 3.59 (s, 3H). C NMR (126
MHz, CDCl3) δ 166.0, 161.4, 152.3, 147.9, 139.0, 132.0, 131.4 (q, J = 34.2 Hz), 129.7
(q, J = 3.9 Hz), 129.0, 122.9 (q, J = 273 Hz), 122.0 (q, J = 3.8 Hz), 114.7, 114.3, 55.4,
51.6. HRMS (ESI/Q-TOF) m/z [M + H]+ calcd for C18H15F3NO5: 382.0877; found
382.0880.
Methyl (Z)-3-(2-nitro-4-(trifluoromethyl)phenyl)-3-phenylacrylate (14). Prepared
using general procedure B from compound 3 and benzenediazonium tetrafluoroborate to
provide the compound 14 as a light yellow solid (29% yield). Mp 132-134 °C. 1H NMR
(500 MHz, CDCl3) δ 8.52(d, J = 1.7 Hz, 1H), 7.96 (dd, J = 8,0, 1.0 Hz, 1H), 7.48 (d, J =
8.0 Hz, 1H), 7.42–7.37 (m, 3H), 7.34-7.32 (m, 2H), 6.57 (s, 1H), 3.65 (s, 3H). 13C NMR
(126 MHz, CDCl3) δ 165.85, 152.8, 148.0, 138.8, 137.5, 132.2, 131.7 (q, J = 34.3 Hz),
130.5, 129.8 (q, J = 3.3 Hz), 129.0, 127.6, 125.1 (q, J = 273 Hz), 122.2 (q, J = 3.8 Hz),
117.1, 51.8. HRMS (ESI/Q-TOF) m/z [M + H]+ calcd for C17H13F3NO4: 352.0791;
found 352.0802.
Methyl
(Z)-3-(2-nitro-4-(trifluoromethyl)phenyl)-3-(4(trifluoromethyl)phenyl)
acrylate (15). Prepared using general procedure B from compound 3 and 4-
trifluoromethylbenzenediazonium tetrafluoroborate to provide the compound 15 as a
light yellow solid (44% yield). Mp 1221-124 °C. 1H NMR (600 MHz, CDCl3) δ 8.50 (d,
J = 1.9 Hz, 1H), 7.96 (dd, J = 8.0, 1.7 Hz, 1H), 7.62 (d, J = 8.2 Hz, 2H), 7.46 (d, J = 8.0
Hz, 1H), 7.43 (d, J = 8.2 Hz, 2H), 6.57 (s, 1H), 3.63 (s, 3H). 13C NMR (126 MHz,
CDCl3) δ 165.4, 151.3, 147.8, 137.9, 132.1 (q, J = 32.8 Hz), 132.1, 130.1 (q, J = 3.2
Hz), 127.9, 126.0 (q, J = 3.6 Hz), 123.8 (q, J = 272 Hz), 122.4 (q, J = 3.8 Hz), 122.8 (q,
J = 272 Hz), 119.1, 52.0. HRMS (ESI/Q-TOF) m/z [M + H]+ calcd for C18H12F6NO4:
420.0665; found 420.0669.
Methyl 6-methoxy-3-(2-methoxyphenyl)-1H-indole-2-carboxylate (16). Prepared
using general procedure C from compound 4 to provide the compound 16 as a white
1
solid (83% yield). Mp 162-165 °C. H NMR (500 MHz, CDCl3) δ 8.89 (s, 1H), 7.40-
7.38 (m, 2H), 7.37 (d, J = 8.9 Hz, 1H), 7.08 (dd, J = 7.4, 1.1 Hz, 1H), 7.05 (dd, J = 8.5,
1.1 Hz, 1H), 6.87 (d, J = 2.2 Hz, 1H), 6.82 (dd, J = 8.9, 2.3 Hz, 1H), 3.90 (s, 3H), 3.79
(s, 3H), 3.78 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 162.5, 159.1, 157.4, 136.8, 132.1,

128.9, 122.9, 122.7, 122.6, 122.5, 120.5, 120.2, 112.1, 110.9, 93.5, 55.6, 51.6. HRMS
(ESI/Q-TOF) m/z [M + H]+ calcd for C18H18NO4: 312.1236; found 312.1235.
Methyl 3-(4-fluorophenyl)-6-methoxy-1H-indole-2-carboxylate (17). Prepared using
general procedure C from compound 5 to provide the compound 17 as a white solid
(64% yield). Mp 184-186 °C. 1H NMR (500 MHz, CDCl3) δ 8.85 (s, 1H), 7.52–7.50 (m,
2H), 7.45 (d, J = 8.8 Hz, 1H), 7.19–7.11 (m, 2H), 6.85 (d, J = 1.8 Hz, 1H), 6.83 (dd, J =
8.8, 2.2 Hz, 1H), 3.88 (s, 3H), 3.81 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 162.4 (d, J =
246 Hz), 162.3, 159.6, 136.9, 132.2 (d, J = 8.0 Hz), 129.5, 124.0, 122.6, 122.4, 121.4,
115.0 (d, J = 22.0 Hz), 112.7, 93.6, 55.7, 51.8. HRMS (ESI-Orbitrap) m/z [M + H]+
calcd for C17H15FNO3: 300.10305; found 300.10278. Also [M + Na]+ calcd for
C17H14FNO3Na: 322.08499; found 322.08441.
Methyl 3-(4-bromophenyl)-6-methoxy-1H-indole-2-carboxylate (18).
Prepared
using general procedure C from compound 6 to provide the compound 18 as a white
solid (57% yield). Mp 193-195 °C. 1H NMR (600 MHz, CDCl3) δ 8.93 (s, 1H), 7.60 (d,
J = 8.4 Hz, 1H), 7.47 (d, J = 8.8 Hz, 1H), 7.44 (d, J = 8.4 Hz, 1H), 6.87 (d, J = 2.1 Hz,
13
1H), 6.85 (dd, J = 8.8, 2.3 Hz, 1H), 3.90 (s, 3H), 3.84 (s, 3H). C NMR (151 MHz,
CDCl3) δ 162.1, 159.4, 136.8, 132.4, 132.1, 131.1, 123.5, 122.3, 122.0, 121.4, 121.3,
112.7, 93.5, 55.6, 51.7. HRMS (ESI/Q-TOF) m/z [M + H]+ calcd for C17H15BrNO3:
360.0230; found 360.0210.
Methyl 3-(3,4-dichlorophenyl)-6-methoxy-1H-indole-2-carboxylate (19). Prepared
using general procedure C from compound 7 to provide the compound 19 as a white
solid (91% yield). Mp 227-230 °C. 1H NMR (500 MHz, CDCl3) δ 8.87 (s, 1H), 7.64 (d,
J = 2.0 Hz, 1H), 7.51 (d, J = 8.2 Hz, 1H), 7.44 (d, J = 9.5 Hz, 1H), 7.38 (dd, J = 8.3, 2.0
Hz, 1H), 6.85 (d, J = 3.9 Hz, 1H), 6.84 (d, J = 3.4 Hz, 1H), 3.88 (s, 3H), 3.82 (s, 3H).
13C NMR (126 MHz, CDCl3) δ 161.9, 159.5, 136.7, 133.6, 132.2, 131.9, 131.3, 129.9,
129.8, 122.1, 121.9, 121.5, 113.0, 93.5, 55.6, 51.8. HRMS (ESI/Q-TOF) m/z [M + H]+
calcd for C17H14Cl2NO3: 350.0345; found 350.0349.
Methyl 6-methoxy-3-(4-(trifluoromethyl)phenyl)-1H-indole-2-carboxylate (20).
Prepared using general procedure C from compound 8 to provide the compound 20 as a
1
white solid (75% yield). Mp 196-197 °C. H NMR (600 MHz, CDCl3) δ 8.96 (s, 1H),

7.73 (d, J = 8.2 Hz, 2H), 7.70 (d, J = 8.1 Hz, 2H), 7.47 (d, J = 8.8 Hz, 1H), 6.89 (d, J =
13
2.2 Hz, 1H), 6.87 (dd, J = 8.8, 2.3 Hz, 1H), 3.91 (s, 3H), 3.84 (s, 3H). C NMR (151
MHz, CDCl3) δ 162.1, 159.6, 137.5, 136.9, 130.9, 129.4 (q, J = 32.2 Hz), 125.0 (q, J =
3.6 Hz), 124.5 (q, J = 272 Hz), 123.37, 122.36, 122.15, 121.67, 113.07, 93.65, 55.7,
51.9. HRMS (ESI/Q-TOF) m/z [M + H]+ calcd for C18H15F3NO3: 350.0999; found
350.0976.
Methyl 3-(4-methoxyphenyl)-1H-indole-2-carboxylate (21).[37] Prepared using
general procedure C from compound 9 to provide the compound 21 as a white solid
1
(82% yield). Mp 161-162 °C (lit. 162–164 °C). H NMR (400 MHz, CDCl3) δ 9.01 (s,
1H), 7.67 (dd, J = 8.2, 1,0 Hz, 1H), 7.55–7.51 (m, 2H), 7.46 (m, 1H), 7.39 (ddd, J = 8.2,
6.8, 1.1 Hz, 1H), 7.18 (ddd, J = 8.1, 6.8, 1.1 Hz, 1H), 7.04 (m, 2H), 3.91 (s, 3H), 3.86
(s, 3H). 13C NMR (101 MHz, CDCl3) δ 162.4, 158.9, 135.8, 131.7, 128.0, 125.9, 125.6,
124.3, 122.2, 121.8, 120.8, 113.4, 111.7, 55.3, 51.8. HRMS (ESI/Q-TOF) m/z [M + H]+
calcd for C17H16NO3: 282.1125; found 282.114.
Methyl 3-phenyl-1H-indole-2-carboxylate (22).[37] Prepared using general procedure
C from compound 10 to provide the compound 22 as a white solid (95% yield). Mp
132-135 °C (lit. 132-135 °C). 1H NMR (400 MHz, CDCl3) δ 9.06 (s, 1H), 7.67 (dd, J =
8.2, 1.0 Hz, 1H), 7.59 (m, 2H), 7.52–7.46 (m, 3H), 7.44–7.38 (m, 2H), 7.18 (ddd, J =
8.1, 6.9, 1.1 Hz, 1H), 3.85 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 162.4, 135.8, 133.4,
130.6, 127.9, 127.3, 125.9, 124.4, 122.4, 121.8, 120.9, 111.7, 51.8. HRMS (ESI/Q-
TOF) m/z [M + H]+ calcd for C16H14NO2: 252.1019; found 252.1020.
Methyl 3-(4-fluorophenyl)-1H-indole-2-carboxylate (23). Prepared using general
procedure C from compound 11 to provide the compound 23 as a white solid (78%
yield). Mp 154-155 °C. 1H NMR (250 MHz, CDCl3) δ 9.11 (s, 1H), 7.60 (d, J = 8.1 Hz,
1H), 7.56 – 7.50 (m, 2H), 7.45 (d, J = 8.3 Hz, 1H), 7.41 – 7.34 (m, 1H), 7.20 – 7.13 (m,
13
3H), 3.84 (s, 3H). C NMR (63 MHz, CDCl3) δ 162.5, 162.3 (d, J = 246 Hz), 135.9,
132.3 (d, J = 8.0 Hz), 129.4, 129.4, 128.0, 126.2, 123.5, 122.6, 121.6, 121.2, 115.0 (d, J
= 22.0 Hz), 111.9, 52.0. HRMS (ESI/Q-TOF) m/z [M + H]+ calcd for C16H13FNO2:
270.0925; found 270.0925.

Methyl 3-(4-(trifluoromethyl)phenyl)-1H-indole-2-carboxylate (24).[37] Prepared
using general procedure C from compound 12 to provide the compound 24 as a white
solid (87% yield). Mp 174-175 °C (lit. 175-177 °C). 1H NMR (400 MHz, CDCl3) δ 9.12
(s, 1H), 7.75 (d, J = 8.7 Hz, 2H), 7.70 (d, J = 8.1 Hz, 2H), 7.62 (dd, J = 8.2, 1.0 Hz,
1H), 7.50 (d, J = 8.0 Hz, 1H), 7.42 (ddd, J = 8.3, 6.9, 1.1 Hz, 1H), 7.21 (ddd, J = 8.1,
6.9, 1.1 Hz, 1H), 3.87 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 162.2, 137.4, 135.8,
131.0, 129.4 (q, J = 32.3 Hz), 127.7, 126.3, 125.0 0 (q, J = 3.6 Hz), 124.5 (q, J = 272
Hz), 123.2, 122.9, 121.5, 112.0, 52.1. HRMS (ESI/Q-TOF) m/z [M + H]+ calcd for
C17H13F3NO2: 320.0893; found 320.0872.
Methyl 3-(4-methoxyphenyl)-6-(trifluoromethyl)-1H-indole-2-carboxylate (25).
Prepared using general procedure C from compound 13 to provide the compound 25 as
a white solid (83% yield). Mp 225-226 °C. 1H NMR (400 MHz, CDCl3) δ 9.16 (s, 1H),
7.76 (m, 2H), 7.49 (m, 2H), 7.39 (dd, J = 8.6, 1.5 Hz, 1H), 7.05 (m, 2H), 3.92 (s, 3H),
13
3.88 (s, 3H). C NMR (101 MHz, CDCl3) δ 162.1, 159.4, 134.6, 131.7, 130.2, 127.79,
126.16, 124.8 (q, J = 272 Hz), 124.21, 122.75, 117.4 (q, J = 3.0 Hz), 113.7, 109.5 (q, J
= 4.5 Hz), 55.5, 52.2. HRMS (ESI/Q-TOF) m/z [M + H]+ calcd for C18H15F3NO3:
350.0999; found 350.0979.
Methyl 3-phenyl-6-(trifluoromethyl)-1H-indole-2-carboxylate (26).[38] Prepared
using general procedure C from compound 14 to provide the compound 26 as a white
solid (80% yield). Mp 189-190 °C (lit. 192-193 °C). 1H NMR (500 MHz, CDCl3) δ 9.23
(s, 1H), 7.75 (s, 1H), 7.72 (d, J = 8.6 Hz, 1H), 7.53 (d, J = 6.7 Hz, 2H), 7.48 (t, J = 7.6
Hz, 2H), 7.48–7.42 (m, 1H), 7.37 (dd, J = 8.6,1.5 Hz, 1H), 3.84 (s, 3H). 13C NMR (126
MHz, CDCl3) δ 162.0, 134.4, 130.5, 130.0, 128.1, 127.7, 124.8, 124.7 (q, J = 273 Hz),
124.20, 122.6, 117.4 (q, J = 3.3 Hz), 109.5 (q, J = 4.6 Hz), 52.1. HRMS (ESI/Q-TOF)
m/z [M + H]+ calcd for C17H13F3NO2: 320.0893; found 320.0880.
Methyl 6-(trifluoromethyl)-3-(4-(trifluoromethyl)phenyl)-1H-indole-2-carboxylate
(27). Prepared using general procedure C from compound 15 to provide the compound
27 as a white solid (71% yield). Mp 169-170 °C. 1H NMR (400 MHz, CDCl3) δ 9.35 (s,
1H), 7.80 (s, 1H), 7.76 (m, 2H), 7.72 (d, J = 8.0 Hz, 1H), 7.69–7.67 (m, 2H), 7.43 (dd, J
13
= 8.5, 1.5 Hz, 1H), 3.89 (s, 3H). C NMR (101 MHz, CDCl3) δ 161.8, 136.6, 134.5,
131.0, 129.9, 129.8 (q, J = 32.3 Hz), 128.3 (q, J = 32.3 Hz), 125.2 (q, J = 3.6 Hz), 124.7

(q, J = 272 Hz), 124.5 (q, J = 272 Hz), 122.7, 122.26, 118.0 (q, J = 3.4 Hz), 109.8 (q, J
= 4.5 Hz), 52.42. HRMS (ESI/Q-TOF) m/z [M + H]+ calcd for C18H12F6NO2: 388.0767;
found 388.0774.
Methyl (E)-3-(4-methoxy-2-nitrophenyl)but-2-enoate (28). Prepared using general
procedure B from methyl (E)-but-2-enoate and 4-methoxy-2-nitrobenzenediazonium
tetrafluoroborate to provide the compound 28 as a light yellow solid (90% yield). Mp
1
50-52 °C. H NMR (400 MHz, CDCl3) δ 7.51 (d, J = 4.0 Hz, 1H), 7.19 (d, J = 8.0 Hz,
1H), 7.12 (dd, J = 8.0, 4.0Hz, 1H), 5.77 (q, J = 4Hz, 1H), 3.88 (s, 3H), 3.74 (s, 3H),
2.42 (d, J = 4Hz, 3H).13C NMR (101 MHz, CDCl3) δ 166.4, 159.6, 155.0, 147.8, 131.6,
130.8, 119.7, 118.6, 109.4, 56.0, 51.2, 20.5. HRMS (ESI/Q-TOF) m/z [M + H]+ calcd
for C12H14NO5: 252.0866; found 252.0852.
Methyl (E)-3-(4-methoxy-2-nitrophenyl)pent-2-enoate (29). Prepared using general
procedure B from methyl (E)-pent-2-enoate and 4-methoxy-2-nitrobenzenediazonium
tetrafluoroborate to provide the compound 29 as a light yellow solid (77% yield). Mp
59-60 °C. 1H NMR (500 MHz, CDCl3) δ 7.54 (d, J = 2.4 Hz, 1H), 7.16 (d, J = 8.5 Hz,
1H), 7.14–7.12 (m, 1H), 5.70 (s, 1H), 3.88 (s, 3H), 3.73 (s, 3H), 2.90 (q, J = 7.5 Hz,
2H), 0.97 (t, J = 7.6 Hz, 3H).13C NMR (126 MHz, CDCl3) δ 166.3, 161.2, 159.8, 148.0,
131.7, 130.2, 119.6, 118.1, 109.6, 56.1, 51.4, 26.8, 12.8. HRMS (ESI-Orbitrap) m/z [M
+ H]+ calcd for C13H16NO5: 266.10230; found 266.10233. Also, [M + Na]+ calcd for
C13H15NO5Na: 288.08424; found 288.08423.
Methyl (E)-3-(4-methoxy-2-nitrophenyl)-4-methylpent-2-enoate (30). Prepared
using general procedure B from methyl (E)-4-methylpent-2-enoate and 4-methoxy-2-
nitrobenzenediazonium tetrafluoroborate to provide the compound 30 as an orange oil,
(50% yield). 1H NMR (400 MHz, CDCl3) δ 7.51 (s, 1H), 7.11 (d, J = 1.3 Hz, 2H), 5.63
(s, 1H), 4.14-4.03 (m, 1H), 3.88 (s, 3H), 3.72 (s, 3H), 1.00 (d, J = 6.9 Hz, 6H).13C NMR
(126 MHz, CDCl3) δ166.0, 164.0, 159.4, 148.7, 131.3, 127.9, 119.2, 118.8, 109.2, 56.0,
51.3, 31.0, 21.5. HRMS (ESI-Orbitrap) m/z [M + H]+ calcd for C14H18NO5: 280.11795;
found 280.11789. Also [M + Na]+ calcd for C14H17NO5Na: 302.09989; found
302.09979.