5,6-dihydropyrrolo[2,1-a]isoquinolines as alternative of new drugs with cytotoxic activity

Article abstract of DOI:10.1248/cpb.c17-00409

In this study, the pyrrolo[2,1-a]isoquinolines 4a–n were synthesized in good yields in a three steps synthesis from the corresponding α,β-unsaturated esters starting materials. These compounds were tested on six human cancer cells lines to measure the cyt

Full text of DOI:10.1248/cpb.c17-00409

Chemical and Pharmaceutical Bulletin Advance Publication by J-STAGE
DOI:10.1248/cpb.c17-00409
Advance Publication
July 22, 2017
Chem. Pharm. Bull
Note
5,6-dihydropyrrolo[2,1-a]isoquinolines as alternative of new drugs with cytotoxic
activity
Rosa Ma. Chávez-Santos,a Paul E. Reyes-Gutiérrez,b Rubén O. Torres-Ochoa,b
Ma. Teresa Ramírez-Apana, Roberto Martínez,*a
a
Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior,
b
Ciudad Universitaria, 04510, Cd. México, México. Institute of Organic Chemistry and
Biochemistry, CAS; Flemingovo náměstí 542/2, 16610 Prague 6, Czech Republic.
* robmar@unam.mx
Ⓒ 2017 The Pharmaceutical Society of Japan

Abstract
In this study, the pyrrolo[2,1-a]isoquinolines 4a-n were synthesized in good yields in a three
steps synthesis from the corresponding α,β-unsaturated esters starting materials. These
compounds were tested on six human cancer cells lines to measure the cytotoxic activity as a
function of the electronic properties and aromaticity of the substituent at the C-2 position of
the pyrroloisoquinoline. Our results reveal that the cytotoxic activity could be explained in
terms of the distribution of electronic density across the ring joined to C-2. Also, this study
identified 3-hydroxy (4d) and 3-chloro (4j) derivatives with powerful cytotoxic activities The
IC50 values of these compounds were found to be comparable to those of the commercially
available Topotecan, Irinotecan, Etoposide, Tamoxifen, and Cisplatin.
Keywords: pyrrolo[2,1-a]isoquinolines; synthesis, cytotoxic activity.
Chemical and Pharmaceutical Bulletin Advance Publication

Introduction
Cancer is a leading cause of death worldwide, and according to the World Cancer Research
Fund International (GLOBOCAN 2012), an estimated 17.1 million new cancer cases and
10.0 million cancer-related deaths will occur in 2020, compared with 15.2 million and 8.9
million, respectively, in 2015.1) The most commonly diagnosed cancers worldwide are
cancers of the skin (melanoma), prostate, lung, breast, and colorectum.2) Cancer poses a
challenge to researchers searching for potent drugs that are capable of controlling cancer
growth while minimizing side effects or the development of drug resistance. Our group is
currently engaged in a program aimed at synthesizing novel heterocyclic compounds that
inhibit the growth of cancer cells. We recently synthesized the pyrroloisoquinolines I–IV
(Table 1)3) in a three-step protocol, and their cytotoxic activities were tested on six tumor cell
lines: PC-3 (human prostatic adenocarcinoma), U-251 (human glioblastoma), K-562 (human
chronic myelogenous leukemia), HCT-15 (human colorectal adenocarcinoma), MCF-7
(human mammary adenocarcinoma ), and SKLU-1 (human lung adenocarcinoma). The
results of this study (Table 1), allowed us to establish preliminary structure–activity
relationship (Figure 1) that revealed the importance of the aromatic substituent at the C-2
position, particularly when the substituent was a m-(cyclohexylmethylpiperazinamide) phenyl
(I), phenyl(II), or m-(amino)phenyl (III), substituent, in combination with an ethyl ester at
the C-1 position. Following up on these preliminary structure–activity studies, the present
work sought to synthesize the novel pyrroloisoquinolines 4a–c (Figure 2) to evaluate the
effects of modifying the cyclohexylmethylpiperazinyl moiety on the antiproliferative activity.
Compounds 4d–k (Figure 2) were evaluated to determine the role of the phenyl ring
substituent on the antiproliferative activity of the compound. Compounds 4l–n (Figure 2)
were evaluated to determine the effect of modifying the aromaticity of the C-2 substituent on
the antiproliferative activity.
Results and discussion
Chemistry
The synthetic route to compounds 4a-n is depicted in Chart 1. Compounds 4a-n were
synthetized via a three-step procedure starting from α,β-unsaturated esters 1a’,1b, 1c, 1d’,
1e-j, 1k’, 1l’, 1m and 1n based in our previous synthetic strategy for synthesis of 5,6-
pyrrolo[2,1-a]isoquinolines I-IV with slightly modifications.3) According with our synthetic
Chemical and Pharmaceutical Bulletin Advance Publication

Chart 1, the reaction between the corresponding aromatic aldehydes and ethyl
diethylphosphonoacetate, using a Horner–Wadsworth protocol4) gave the starting materials 1c,
1d’, 1e-j, 1l’, 1m, and 1n in excellent yields (Chart 2). The ethyl cinnamates 1a’ and 1b were
obtained from m-iodobenzoic acid or m-iodoaniline through a Heck reaction5) and a
subsequent amidation reaction6) with the corresponding cyclic amines (Chart 3). It should be
noted that all the α,β-unsaturated esters prepared were obtained exclusively as the (E)-
isomers in good yields (78–98%).
Treatment of the electrophilic alkenes 1a’–n with monomethylated TosMIC, prepared from
the commercially available TosMIC under phase transfer conditions according to van
Leusen’s protocol,7) afforded the 2,3,4-polysubstituted pyrroles 2a’–n in 70–96% yields. The
key intermediates 3a’–n were prepared by N-alkylation of the pyrroles 2a’–n using 2-bromo-
4,5-dimethoxyphenethyl 4-methylbenzenesulfonate as alkylating agent, which had been
prepared from 2-(3,4-dimethoxyphenyl)ethanol in the presence of NaH as a base in dry
DMSO. Finally, the N-alkyl-pyrroles 3a’–n were cyclized to the corresponding
tetrasubstituted-5,6-dihydropyrroloisoquinolines 4a–n using radical oxidative conditions in
the presence of tributyltin hydride (n-Bu3SnH) and dilauroyl peroxide (DLP) in toluene.8) The
nitro derivative was prepared via a palladium-catalyzed reaction9) used to obtain the tricyclic
framework 4g. This step was necessary due to the possibility of a denitration reaction in the
presence of tributyltin hydride (Chart 1, conditions iii’). The piperazinyl compound 4a was
prepared from compound 4a’ in ethanol using hydrazine hydrate as a deformylating agent.10)
The pyrroloisoquinoline 4d was prepared after hydrogenolysis of the benzyl ether 4d’.11) The
bromo derivative 4k was synthesized through a Sandmeyer reaction12) using the
corresponding aniline 4k’ as a precursor. Finally, the N-deprotection of pyrrole 4l’ was
achieved after applying the reductive conditions described by Sajiko,13) which afforded the
pyrroloisoquinoline 4l.
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Cytotoxic Activity and Structure-Activity Relationship(SAR) for compounds 4a-4m
We evaluated the effects of modifying the cyclohexylmethylpiperazinyl group in compound I
on the antiproliferative activities (Table 2). The removal of the methylcyclohexyl group from
our lead compound I decreased the antiproliferative activity of the synthesized compound 4a
in all cell lines compared to the activity of the lead compound I [Table 2, Entry 3]. These
results suggested that the methylcyclohexyl group is key to the cytotoxic activity. The
influence of the piperazine NH group on the activity of 4a was examined by synthesizing the
morpholinyl analog 4b. Surprisingly, 4b did not inhibit proliferation of any of the six cancer
lines tested (Table 2, Entry 4). On the other hand, the antiproliferative activity of N-
formylpiperazine 4a’ displayed inhibition levels minor to those displayed by 4a. These
results could be attributed to effect of the piperazinyl-NH group of 4a on the activity,
probably by forming a quaternary ammonium ion in situ.
Complete removal of the piperazine ring, in compound 4c (3-CONH2), provided a level of
growth inhibition in all cell lines tested that exceeded the inhibitory activity of 4a. The
antiproliferative activity of 4c was better than that of the lead compound I in the MCF-7 cell
line but lesser than in tested: PC-3, U-251, K-562, HCT-15 and SKLU-1 cell lines. The last
results suggested that the electronic properties of amide group of compound (4c) affected the
cytotoxicity to a greater degree than the electronic properties of cyclohexylmethyl-
piperazinyl group on I in the MCF-7 cancer cell line.
The electronic effects were further examined by introducing electron-donating groups (4d, 3-
OH; 4e, 3-OMe) or electron-withdrawing groups (4f, 3-CN; 4g, 3-NO2; 4h, 3-CF3) at the
meta position of the 2-benzene ring. The majority of these changes significantly increased the
inhibitory activity compared to the unsubstituted compound II in all cell lines (Table 3). By
the contrary, the inhibitory activity of compounds 4d-h was lesser than that the lead
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compound I in all cell lines, with exception in HCT-15 cell line were compound 4d was
twice times more active than I. Interestingly, compound 4d (3-OH) was the most active
derivative across three of the six cancer lines (Table 3). The nature of the –OH substituent
appeared to increase the activity due to the capacity of the substituent to form hydrogen
bonds.
The roles of the electronegativity and/or size of the halogen group on the antiproliferative
activity were investigated by synthesizing compounds 4i (3-F), 4j (3-Cl), and 4k (4-Br). As
shown in Table 3, the chloro derivative 4j was the most active of the halogenated compounds
on all the cancer cell lines tested but was lesser than that the lead compound I, with exception
in MCF-7 cell line were compound 4j was six times more active than I. These results suggest
that derivatization of C-2-phenyl group with an m-chloro substituent is suitable to obtain best
anticancer pyrroloisoquinoline compounds
Finally, the noteworthy results that have been obtained by studying bioisosteric compounds14)
led us to synthesize bioisosters of II by changing the benzene ring to a pyrrolo, a furan or a
pyridine ring, creating compounds 4l, 4m, and 4n, respectively. Our results demonstrate that
a bioisosteric modification of the C-2 benzene ring of compound II, gives compounds with
preserved cytotoxic activity. Moreover, this activity is enhanced by the presence of a furan
ring in the PC-3 cell line (compound 4m). However, the inhibitory activity of compounds 4l-
n was lesser than that the lead compound I in all cell lines tested. Likewise, the data for
compounds II, IV,4l-m in three of the six cancer lines U-251, HCT-15 and SKLU-1 indicate
that the cytotoxic effect was dependent of the kind of aromaticity of the substituent joined to
C-2. These findings confirmed that the cytotoxic activity could be explained in terms of the
distribution of electronic density across the ring joined to C-2.
The cytotoxic activities of our most active compounds 4d and 4j were compared with those
of the commercially available Topotecan,15) Irinotecan,16) Etoposide,17) Tamoxifen,18) and
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Cisplatin.19) As shown in Table 3, compound 4d (3-OH) was ten times more active than
cisplatin in the prostate PC-3 cell line, almost two thousand times more active than irinotecan
in the colon HCT-15 cell line. In the lung SKLU-1 cell line, compound 4d (3-OH) was thirty-
seven times more active than topotecan, six hundred forty-one times more active than
irinotecan and seventy-six times more active than etoposide. Compound 4j (3-Cl) showed
more activity in the U-251(CNS) cell line and leukemia K-562 cell line compared to the
reference etoposide. Finally, compound 4j (3-Cl) showed a higher activity than tamoxifen in
the breast cancer cell line MCF-7
Conclusion
Compounds 4a–4n were synthetized trough a practical synthetic route involving the van
Leusen’s pyrrole construction protocol and an intramolecular radical oxidative cyclization.
The inhibitory activities of compounds 4a–4n were evaluated using six cancer cell lines. Our
results reveal that the cytotoxic activity could be explained in terms of the distribution of
electronic density across the ring joined to C-2. Also, the present study enabled the discovery
of the novel 3-hydroxy 4d and 3-chloro 4j derivatives pyrroloisoquinoline compounds, which
displayed excellent cytotoxic activity. The IC50 values of these compounds were determined
and were found to compare satisfactorily with those of the commercially available drugs
topotecan, irinotecan, etoposide, tamoxifen, and cisplatin.
Experimental
Chemistry
All reported melting points were measured in open capillaries using a Mel-Temp apparatus.
1H-NMR spectra were recorded on a Avance III HD 700 MHz Brucker, Avance III HD 500
MHz Brucker, Avance 400 MHz Brucker, 300 MHz Jeol Eclipse, Fourier 300 MHz Brucker
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spectrometers in deuterated chloroform (CDCl3) solutions using TMS as the internal standard
(δ = 0 ppm), 13C-NMR spectra were recorded at 75, 100, 125, 150 and 175 MHz on the same
instruments. The chemical shifts are reported in the δ scale in parts per million (ppm). The
peak patterns are indicated as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet;
brs, broad signal. The coupling constants (J) are reported in Hertz (Hz). IR spectra were
obtained on a Magna-IR spectrometer. Mass spectra were recorded on Jeol JEM-AX505HA
spectrometer by electronic impact (EI) detection at 70 eV for low-resolution and on a Jeol
5X102A mass spectrometer (Jeol Ltd.) with fast atom bombardment (FAB+) and electronic
impact (EI) ionization detection for high-resolution measurements.
General procedure for synthesizing the pyrroles 2a’–2n
A solution containing the alkene (2.6 mmol) and 1-(1-isocyanoethylsulfonyl)-4-
methylbenzene (Me-TosMIC) (0.6 g, 2.9 mmol) in Et2O: DMSO (2:1, 15 mL) was added
dropwise to a suspension of NaH (0.23 g, 5.7 mmol, 60% dispersion in mineral oil) in dry
ether (5 mL). The mixture was stirred at room temperature for 1 h, then H2O (15 mL) was
added dropwise and the product was extracted with EtOAc (3x30 mL). The organic layer was
washed with H2O and brine (3x10 mL), dried with Na2SO4, and evaporated in vacuo. The
residue was purified by flash column chromatography on silica gel to furnish the respective
pyrrole. Physical and spectroscopic data of all compounds 2á-2n are reported in
supplementary material.
General procedure for the synthesis of the N-alkylpyrroles 3a’–3n
NaH (0.2 g, 5.7 mmol, 60% dispersion in mineral oil) was added portionwise to a solution of
the corresponding pyrrole (2.3 mmol) and 2-bromo-4,5-dimethoxyphenethyl 4-methyl
benzenesulfonate (1.9 g, 4.5 mmol) in dry dimethyl sulfoxide (10 mL). The mixture was
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stirred at room temperature for 6 h, EtOAc (20 mL) was added, and the solution was washed
with water and brine (3 x 10 mL). The organic layer was dried with Na2SO4 and evaporated
in vacuo. The residue was purified by flash column chromatography on silica gel to furnish
the respective N -alkylpyrrole. Physical and spectroscopic data of all compounds 3á-3n are
reported in supplementary material.
General procedure for synthesizing the 5,6-dihydropyrrolo[2,1-a]isoquinolines 4a’, 4b,
4c, 4d’, 4e, 4f, 4j, 4k’, 4l’, 4m, 4n.
To a refluxing solution of the N-alkylpyrrole in degassed dry toluene (10 mL), a solution of
n-Bu3SnH (1.0 mL, 3.7 mmol) in toluene (5 mL) was added dropwise (syringe pump) over 7
h. During that time, solid dilauroyl peroxide (DLP) was added portionwise (1.49 g, 3.7 mmol,
0.11 g/30 min). The solvent was removed under reduced pressure and the crude residue was
purified by flash column chromatography on silica gel. Hexane was first added to remove the
n-Bu3SnBr, then hexane–EtOAc–Et3N (70:25:5 to 50:45:5). Physical and spectroscopic data
of all compounds 4á-4n are reported in supplementary material.
Cell Culture and Assay for Cytotoxic Activity
Cell culture and assay for activity PC-3, U-251, K-562, HCT-15, MFC-7, and SKUL-1, were
supplied by The National Cancer Institute(NCI), U.S.A. The cytotoxicity of tumors cells with
the test compounds was determined using the protein-binding dye sulforhodamine B(SBR) in
microculture assay to measure cell growth.20 The cell lines were cultured in RPMI-1640
(Sigma Chemical Co., Ltd., St. Louis, MO, U.S.A.) supplemented with 10% fetal bovine
serum which was purchased from Invitrogen Corporation, 2 mM L-glutamine, 10000
units/mL of penicillin G, 10000 μg/mL streptomycin and 0.25 μg/mL Fungizone(Gibco).
They were maintained at 37oC in a 5% CO2 atmosphere with 95% humidity. For the assay,
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5104 cell/mL (K-562, MCF-7), 7510 cell/ mL (U-251, PC-3) and 10104 cell/mL (SKLU-1,
HCT-15), and 100 mL/well of these cells suspension was seeded in a 96-well microtiter
plates and incubated to allow for cell attachment. After 24 h, 100 μL of each test compounds
and positive substances were added to each well. Later 48 h, adherent cell cultures were fixed
in situ by adding 50 mL od cold 50% (w/v) trichloroacetic acid (TCA) and incubated for 60
min at 4oC. The supernatant was discarded and the plates were washed three times with water
and air dried. Cultured fixed with TCA were stained for 30 min with 100 μL of 0.4% SRB
solution. Protein-bounded dye was extraxted with 10mM unbuffered tris base and the optical
densities were read on a Microplate Reader Synergy HT (Elx 808, BIO-TEK Instruments,
Inc.), with a test wavelength of 515 nm. Results were expressed as IC50 values, they were
calculated according to the protocol of Monks, were a dose-response curve was plotted for
each compound, and the concentration giving 50% inhibition (IC50) was estimated from non-
linear regression equations. The IC50 value (mean standard error (S.E). 21
Acknowledgments
This work was supported by Conacyt (Consejo Nacional de Ciencia y Tecnología), Project
No. 131295 and Fellowship No. 223141. Financial support from the DGAPA, UNAM
(project PAPIIT IN213407) is gratefully acknowledged. We also thank R. Patiño, N. Esturau,
A. Peña, E. Huerta, B. Quiroz, and C. García, L. Velasco, J. Pérez, and V. Labastida (CIQ,
UAEM) for technical support. During the experimentation process Rosa María Chávez
Santos was a Conacyt Graduate Scholarship holder (No. 223441).
Conflict of Interest The authors declare no conflict of interest
Supplementary Materials The online version of this article contains supplementary
materials
Chemical and Pharmaceutical Bulletin Advance Publication

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FIGURES
Figure 1. Preliminary SAR of pyrroloisoquinolines I-IV and designed compounds
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Figure 2. Proposed compounds to be synthesized and evaluated to determine the activity
effects of the structural modifications relative to the activities of structures I–IV.
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CHARTS
Chart 1. Synthetic route of compounds 4a-n. Reagents and conditions: (i) NaH, CH3-
TosMIC, diethyl ether: DMSO (2:1), 0 °C to RT.; (ii) NaH, DMSO, 2-bromo-4,5-
dimethoxyphenethyl 4-methylbenzenesulfonate, RT; (iii) n-Bu3SnH, DLP, Toluene, reflux;
(iii’) Pd(OAc)2, PPh3, Et3N, CH3CN, reflux; (iv) Hydrazine/EtOH, 60 °C; (v, vii) H2, Pd/C
EtOH, RT; (vi) t-BuONO, CuBr2, CH3CN, 0 °C.
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Chart 2. Synthetic route to the starting compounds 1c, 1d’, 1e–j, 1l’, 1m, and 1n. Reagents
and conditions: (i) ethyl 2-(diethoxyphosphoryl)acetate, NaH, DMF –60°C
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Chart 3. Synthetic route to compounds 1a’, 1b and 1k’. Reagents and conditions: (i) ethyl
acrylate, Pd(OAc)2, PPh3, Et3N, CH3CN, reflux; (ii) DCC, DCM, amine, RT.
Chemical and Pharmaceutical Bulletin Advance Publication

TABLES
Table 1. The IC50 values (mM) of compounds I to IV in the six cancer cell lines.a
a Results are expressed as IC50 values in units of μM ± standard error. The values indicate the
mean calculated from experiments conducted in triplicate
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Table 2 Effects of modifying the cyclohexylmethylpiperazinyl group on the antiproliferative
activitya
a Results are expressed as IC50 values in units of μM ± standard error. The values indicate the
mean calculated from experiments conducted in triplicate. N.A.: this compound was not
active. The bold numbers represent the highest activities of the compounds tested.
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Table 3 Effects of meta-substituent on 3-phenyl moiety on the antiproliferative activity and
comparison with the activities of commercially available drugs a
a Results are expressed as IC50 values in units of μM ± standard error. The values indicate the
mean calculated from experiments conducted in triplicate. The bold numbers represent the
highest activities of the compounds tested.
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Table 4. Effects of aromaticity of C-2 substituent on the antiproliferative activitya
a Results are expressed as IC50 values in units of μM ± standard error. The values indicate the
mean calculated from experiments conducted in triplicate. The bold numbers represent the
highest activities of the compounds tested.
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