One-Pot, Three-Component Assembly of Indoloquinolines: Total Synthesis of Isocryptolepine

Article abstract of DOI:10.1021/acs.joc.6b03084

Indolo[3,2-c]quinolones have been efficiently synthesized via an acid-mediated, one-pot, three-component condensation of arylhydrazines, o-aminoacetophenones, and triazines or nitriles. The synthetic application of this method is showcased by the concise synthesis of isocryptolepine alkaloid and a series of its synthetic analogues with demonstrated cancer cell antiproliferative activities.

Full text of DOI:10.1021/acs.joc.6b03084

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Article
One-Pot, Three-Component Assembly of
Indoloquinolines: Total Synthesis of Isocryptolepine
Alexander V. Aksenov, Dmitrii A Aksenov, Naila A Orazova, Nicolai A Aksenov, Georgii D Griaznov,
Annelise De Carvalho, Robert Kiss, Véronique Mathieu, Alexander Kornienko, and Michael Rubin
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b03084 • Publication Date (Web): 03 Mar 2017
Downloaded from http://pubs.acs.org on March 3, 2017
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One-Pot, Three-Component Assembly of Indoloquinolines: Total
Synthesis of Isocryptolepine
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Alexander V. Aksenov,*,a Dmitrii A. Aksenov,a Naila A. Orazova,a Nicolai A. Aksenov,a
Georgii D. Griaznov,a Annelise De Carvalho,b Robert Kiss,b Véronique Mathieu,b
Alexander Kornienko,c and Michael Rubin*,a,d
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aDepartment of Chemistry, North Caucasus Federal University, 1a Pushkin St., Stavropol 355009, Russia.
E-mail: alexaks05@rambler.ru
bLaboratoire de Cancérologie et de Toxicologie Expérimentale, Faculté de Pharmacie, Université Libre de
Bruxelles, Brussels, Belgium
cDepartment of Chemistry and Biochemistry, Texas State University, San Marcos, Texas 78666, USA
dDepartment of Chemistry, University of Kansas, 1251 Wescoe Hall Drive, Lawrence, KS 66045, USA.
E-mail: mrubin@ku.edu
ABSTRACT: Indolo[3,2-c]quinolones are efficiently synthesized via an acid-mediated, one-pot, three-
component condensation of arylhydrazines, ortho-aminoacetophenones, and triazines or nitriles.
Synthetic application of this method is showcased by the concise synthesis of isocryptolepine alkaloid
and a series of its synthetic analogs with demonstrated cancer cell antiproliferative activities.
Introduction
The indoloquinoline heterocyclic system is frequently found in natural products isolated
from plants used in traditional medicine.1 Due to the broad spectrum of biological and
pharmacological activities associated with compounds incorporating this structural skeleton, it
has been recognized as an important privileged scaffold in drug discovery.2 For example,
isocryptolepine (cryptosanguinolentine, 1aa), one of the four types of indoloquinoline
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alkaloids isolated from West African plant Cryptolepis sanguinolenta,3 has been investigated as
a promising antimalarialand anticancer agent (Figure 1).4 Due to its potential pharmacological
applications, it has been the target of numerous synthetic efforts. The majority of the total
syntheses of isocryptolepine and analogs reported to date exploited stepwise assembly of the
two heterocyclic rings. Typically, a quinoline precursor would be used as a platform for
installation of the pyrrole moiety,4d,5 although alternate disconnection approaches are also
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known, including iodine-mediated intramolecular coupling, electrocyclic cyclization,
transition metal-catalyzed tandem reactions,8 and various acid-mediated annulations.9 All
previously reported synthetic routes required multistep linear sequences with isolation and
purification of intermediate products, with the shortest known approach consisting of 3 steps
from commercially available materials.10 Herein we wish to report a metal-free three-
component synthesis of isocryptolepine and its bioactive analogs, that involves sequential
assembly of the indoloquinoline core in one-pot with single isolation operation.
Me
N
Me
N
Me
N
N
N
Me
N
N
N
isocryptolepine (1)
cryptolepine
isoneocryptolepine
neocryptolepine
Figure 1. Naturally indoloquinoline alkaloids isolated from Cryptolepis sanguinolenta.
Me
N
N
N
N
H
N
H
N
+ MeI
Me
isocryptolepine (1aa)
3
2
H2N
H
N
H2N
NHNH2
+
N
MeOC
N
H
7
6
5
4
+ HCHO
Scheme 1. Kundu’s retrosynthetic analysis of isocryptolepine involving formation of the
quinoline and indole rings via modified Pictet-Spengler protocol and Fisher indolization,
respectively.
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R2
HN
R2
HN
R1
R1
R4
R3
R4
R3
+
H+
N
O
NHNH2
N
H
A
Me
6
Me
HN
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R2
R5
R2
X
N
R1
R1
R5CO2H or R5CN
PPA
Fisher
PPA
B
R4
R4
N
H
N
R3
H
R3
10
11
R5
R2
R5
R2
H2O3P
6
N
7
R1
X
N
5
4a
4
8
Vilsmeier
6b
R1
6a
3
11a
R4
11b
PPA
X = O, NH
R4
10a
9
N
2
N
H
10
1
11
R3
R3
1
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Scheme 2. Synthetic approach to isocryptolepine analogs involving one-pot acylation of
indoles with carboxylic acids or nitriles
Results and Discussion
In 2009 Kundu and co-workers reported a novel and interesting synthetic approach to the
indoloquinoline core of isocryptolepine alkaloid, which involved installation of the quinoline
ring via oxidative Pictet-Spengler type reaction of N-arylmethanimine 3.11 The indole moiety in
the latter compound was assembled via Fisher indolization of hydrazone 5, prepared from
ketone 7 and hydrazine 6 (Scheme 1). Although this strategy featured 4 linear steps involving
isolation and purification of intermediate products (starting from commercially available
precursors) and provided ~50% overall yield,11,12 we felt that it can be greatly simplified and
significantly shortened through designing of a cascade or sequential one-pot transformation
protocol.
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We have previously reported a sequential three-component heteroannulation reaction
leading to 3-aryl-2-quinolones that proceeds via a cascade transformation involving Fisher
indole synthesis from hydrazines 2 and involves the use of polyphosphoric acid (PPA) as the
reaction medium.13 We have found that the P2O5-water ratio can be accurately tuned to
optimal values (which usually range between 75 and 85%) to achieve the necessary acidity and
anhydride activity of the medium. PPA also efficiently phosphorylates amino and hydroxyl
groups in situ and converts them into good nucleofuges, facilitating cascade cationic
transformations. Furthermore, it was demonstrated that carboxylic acids in the presence of
PPA could serve as electrophiles in acylation reactions and related cyclization cascades.14 We
envisioned that these previously developed tools could be combined to craft a convenient route
to indoloquinolines 1. Thus, hydrazone 9 readily available from acetophenone 8 and hydrazine
6 (step A, Scheme 2) would undergo the Fisher reaction (step B) to produce indole 10. Next,
acylation of the aniline moiety with carboxylic acid 13 or nitrile 14 (see Scheme 3) would
afford amide (X = O) or amidine (X = NH) 11, which would spontaneously undergo a PPA-
assisted intramolecular Vilsmeier reaction to furnish indoloquinoline 1. Given our previous
successful experience with PPA-assisted indole synthesis from hydrazines and aceto-
phenones,10 we anticipated that all of the steps in the proposed transformation could be
performed in PPA in a sequential, one-pot mode. However, our attempts to engage ketones 8 in
a condensation reaction with hydrazines 6 in PPA medium were unproductive. Formation of 9
was accompanied by significant polymerization processes, possibly originating from intermol-
ecular homocondensation of ortho-aminosubstituted acetophenones 6. Accordingly, a classical
reaction medium, such as ethanol with catalytic amounts of acetic acid,12 was employed in the
first step to enable the desired cross-condensation to form hydrazones (9a-d). The latter,
isolated and purified by crystallization, were exposed to PPA to produce the corresponding
indoles 11, which were subsequently treated in situ with acetic (13b), benzoic (13c), or caproic
(13d) acids to provide isocryptolepine analogs 1ab-1bd in high yields (Scheme 3, Method I).
Likewise, the reaction of hydrazones 9a,b with nitriles 14b,c,e afforded the corresponding
indoloquinolines as sole products in good yields (Scheme 3, Method II). After the successful
preparation of substituted analogs, we attempted the synthesis of isocryptolepine via this
method. To obtain a product such as 1aa with the unsubstituted C6-position, it was necessary
to use formic acid (13a) as the electrophile. Unfortunately, formic acid was incompatible with
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the reaction conditions:15 all our efforts resulted in decomposition of the starting materials. To
overcome this limitation, we searched for an alternative electrophilic component that would
permit the installation of the unsubstituted C-6 in the indoloquinoline core. Our previous work
on PPA-assisted SEAr-type cyclizations with triazines16 inspired us to probe this heterocycle as
a formic acid surrogate. To this end, a purified sample of indole 11a, obtained as described
above, was treated with triazine 15a in PPA under the standard reaction conditions.
Gratifyingly, isocryptolepine (1aa) was produced as the sole product in 83% yield.
Furthermore, the PPA-mediated reaction between triazine 15a and various hydrazones (9a-g)
proceeded smoothly in one pot affording the corresponding indoloquinoline products in high
yields (Scheme 3, Method III). An analogous reaction with symmetric 2,4,6-trisubstituted 1,3,5-
triazines 15b-c gave indoloquinolines substituted at C-6 (Scheme 3).
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Figure 2. ORTEP drawing of compounds 1ea (CCDC 1510030, left) and 1fa (CCDC 1510031,
right): showing atom-labeling schemes and 50% probability amplitude displacement ellipsoids
(See SI for details)
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R3
R2
R5
R1
R4
N
R1
i (13, 14 or 15)
N
R4
N
H
PPA
N
Me HN
R2
R3
9
1
7
8
9
9a: R1 = R3 = R4 = H; R2 = Me; 9b: R1 = R2 = R3 = R4 = H; 9c: R2 = R3 = R4 = H; R1 = Me;
9d: R3 = R4 = H; R1 = R2 = Me; 9e: R1 = R2 = R4 = H; R3 = OMe;
9f: R1 = R2 = H; R3 = R4 = OMe; 9g: R1 = H; R2 = Me; R3 = R4 = OMe.
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R5
N
R5
R5 CO2H
13
13a: R5 = H; 13b: R5 = Me;
13c: R5 = Ph; 13d: R5 = n-C5H11
14b: R5 = Me; 14c: R5 = Ph; 14e: R5 = 3-Py;
15a: R5 = H; 15b: R5 = Me; 15c: R5 = Ph.
N
N
;
R5 CN
14
R5
15
Me
Me
N
Me
Me
Me
Ph
N
N
N
N
N
N
N
1ac: Method I, 82%
Method II, 73%
Method III, 74%
1aa: Method III, 83%
Method IIIa, 82%
1ab: Method I, 79%
Method II, 73%
1ad: Method I, 76%
H
N
Ph
N
Me
N
N
N
H
N
H
N
H
N
H
1ba: Method III, 81%
Method IIIa, 80%
1bc: Method I, 81%
Method II, 71%
Method III, 76%
1bb: Method I, 77%
Method II, 71%
Method III, 78%
1bd: Method I, 81%
N
Ph
N
Me
N
N
Me
Me
Me
N
N
H
N
H
N
H
N
H
1cc: Method I, 75%
Method Ia, 72%
1ca: Method III, 86%
1cb: Method I, 82%
1be: Method II, 63%
Me
N
Me
Me
Me
Ph
N
N
N
Me
Me
Me
N
H
N
N
N
OMe
1ea: Method III, 71%
Method IIIa, 67%
1da: Method III, 79%
1db: Method I, 77%
1dc: Method I, 71%
Method Ia, 68%
Me
N
N
OMe
OMe
N
H
N
OMe
OMe
1fa: Method III, 80%
Method IIIa, 77%
1ga: Method III, 81%
Method IIIa, 79%
Scheme 3. One-Step Synthesis of Isocryptolepine and Analogs from Hydrazones. Isolated yields
of purified products are shown. Carboxylic acids 13 are employed in Method I, nitriles 14 are
in Method II, triazines 15 in Method III. Methods Ia and IIIa employ a one-pot protocol
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starting directly from hydrazines 6 and acetophenones 8 without isolation of intermediate
hydrazones 9, but otherwise identical to Methods I and III, respectively.
5
6
7
O
8
9
+
Me
H+
N
N
H
NHNH2
HN
Me
A
10
11
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Me HN
8
6
9
Me
N
NH
N
N
Me
N
Fisher
PPA
B
N
N
15a
C
N
H
HN
PPA
10
N
Me
H
N
16
N
N
H2N
Me
H2N
Me
D
N
N
N
E
N
H
N
H
18
17
N
Me
6
Me
NH
N
H2N
N
7
5
F
6a
6b
4
N
8
H
G
11a
11b
3
10a
10
11
9
-H
N
1
2
19
1aa: Isocryptolepine
N
N
Me
Me
N
N
H2N
H2N
N
N
I
H
H
-H
N
H
N
H
18
20
Me
N
N
N
H2N
1aa
N
H
21
Scheme 4. Synthetic approach to isocryptolepine analogs involving one-pot reaction of indoles
with 1,3,5-triazines.
Formation of the indoloquinoline core was unambiguously proved by single crystal X-ray
crystallography of compounds 1fa and 1ea (Figure 2). The proposed mechanism of this trans
formation is outlined in Scheme 3. First, addition of triazine to the mixture of indole and PPA
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produces species 16 (step C, Scheme 4). Ring opening of the dihydrotriazine (step D) and the
subsequent intramolecular electrophilic attack of the resulting linear species 17 at C-3 of the
indole (step E) furnishes the six-membered heterocyclic ring in 18. Finally, proton transfer
(step F), followed by neighboring group-assisted elimination (step G), affords isocryptolepine
1aa (Scheme 4). Alternatively, species 18 could experience deprotonation at C-6a (step H) to
provide intermediate product 20 further undergoing elimination and deprotonation sequence
(Scheme 4).
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With the two-linear step, two-pot triazine-based synthesis of isocryptolepine in hand, we
next challenged ourselves to develop a single pot approach to this natural product. We
reasoned that sub-stoichiometric amounts of acetic acid (13b) employed as a catalyst in step A
would compromise step C in the synthesis by competing with triazine and producing 6-methyl-
isocryptolepine analog 1ab (Scheme 2). On the other hand, formic acid (13a) would not
survive exposure to PPA, as noted above. An attempt to use weaker non-nucleophilic acids,
such as TFA also proved unfruitful, as the Fisher indolization (step B) did not proceed at all
under these conditions. This prompted us to probe the condensation of commercially available
6a and 8a in refluxing ethanol in the presence of catalytic amounts of HCOOH. The reaction
proceeded uneventfully and, upon completion, ethanol was evaporated to give crude
hydrazone 9a. The subsequent Fisher indolization was carried out in the same pot at 80 oC for
2 hrs, during which time all residual amounts of ethanol were chemically destroyed in the PPA
medium via facile dehydration and liberation of ethylene gas. Likewise, formic acid (13a) was
tracelessly decomposed by PPA. Using this modified one-pot approach, isocryptolepine
alkaloid (1aa) was obtained in 81% overall yield. Further optimization revealed that the
condensation step A could be performed in the absence of solvent and catalyst by simply
stirring neat reactants at 120 oC for 10 min. Subsequent addition of PPA, followed by triazine
(6a) shortly after, provided the natural product 1aa in 82% yield. Several analogs were also
prepared using this one-pot protocol, with yields comparable to those obtained using the two-
step method (Scheme 3, Methods Ia, IIIa).
Because isocryptolepine is investigated as a potential anticancer agent, we evaluated this
alkaloid and several of its synthetic analogs against a panel of six cancer cell lines that included
the murine B16F10 melanoma and the human MCF-7 breast carcinoma, HS683 oligodendro-
glioma as well as A549 non-small cell lung cancer, U373 glioblastoma and SKMEL-28
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melanoma cell lines (Table 1). The latter three cell lines have been shown to display various
degrees of resistance to pro-apoptotic insults17 and are thus good models of chemotherapy-
resistant cancers associated with dismal prognoses. Isocryptolepine (1aa) exhibited the mean
antiproliferative IC50 value of 0.8 µM, while its demethylated (R2 = H) synthetic analogues 1ea
and 1fa had the mean IC50’s of 1.2 mM each. Of note, analogue 1ga, differing from
isocryptolepine by the presence of two methoxy groups in the quinoline part of the molecule
(R4 = R5 = OMe), was more potent than the natural alkaloid showing the mean IC50 of 0.4 µM.
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Table 1. 50% in vitro growth inhibitory concentration (IC50 ; µMa) determined by the colorimetric MTT
assay.
Compound
carcinoma
glioma
melanoma
Mean + SEM
A549 MCF-7 U373
HS683 SKMEL-28 B16F10
1aa
1ba
1ea
1fa
0.6
8
1.0
27
1.3
16
0.4
4
0.9
24
0.7
7
0.8 ± 0.1
14 ± 4
0.7
0.7
0.4
2.0
2.3
0.5
1.2
0.8
0.6
0.3
0.5
0.3
2.2
2.3
0.4
0.7
0.5
0.3
1.2 ± 0.3
1.2 ± 0.4
0.4 ± 0.05
1ga
a Average concentration required to reduce the cell viability by 50% after 72 h treatment relative to a control
as determined by MTT assay. Each experiment was performed once in sextuplicates.
Conclusion
In conclusion, an expeditious metal-free approach to indoloquinolines involving a PPA-assisted
cascade twofold heteroannulation has been developed. Three alternate routes for quinolone
ring closure were demonstrated employing carboxylic acids, nitriles, and triazines as
electrophilic components. The developed method was used for an efficient and concise (a
single pot operation starting from inexpensive commercially available precursors) total
synthesis of alkaloid isocryptolepine. A rapid increase of molecular complexity with up to three
points of diversification allowed by this method presents a promising avenue for drug
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discovery. The initial structure-activity studies made possible through our synthetic approach
demonstrated the possibility of obtaining analogues more potent than the natural alkaloid.
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Experimental Part
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General Information
1H and 13C NMR spectra were recorded on a Bruker Avance-III spectrometer (400 or 100 MHz,
respectively) equipped with BBO probe in CDCl3 or DMSO-d6, using TMS as internal standard.
High-resolution mass spectra were registered with a Bruker Maxis spectrometer (electrospray
ionization, in MeCN solution, using HCO2Na–HCO2H for calibration). Melting points were
measured with a Stuart smp30 apparatus. All reactions were performed in oven-dried 5 mL
round-bottomed flasks open to the atmosphere, employing overhead stirring. Reaction
progress and purity of isolated compounds were controlled by TLC on Silufol UV-254 plates,
eluting with EtOAc. Hydrazones 4a-g were obtained from the corresponding arylhydrazines
and ortho-aminoacetophenones according to published literature procedures.12 All other
reagents and solvents were purchased from commercial vendors and used as received.
Experimental Procedures
General procedure for preparation of indoloquinolines 1 from arylhydrazones 9 (General
protocol A, combining Methods I, II, and III): Mixture of hydrazone 9 (1.0 mmol) and 80%
o
polyphosphoric acid (PPA, 2.0 g) was stirred at 100 C and acylating agent (13, 14 or 14, 1.2
o
o
mmol) was added. The mixture was stirred at 100 C for 30 min, then at 130 C for 90 min
(heating should be continued for 5 hr at 150 oC when nitriles 14 used as acylating agents or for
2 hr at 150 oC in reactions with 1,3,5-triphenyltriazine 15c). The reaction mixture was poured
out into water (50 mL) and neutralized with 25% aqueous ammonia. Formed precipitate was
filtered out. Usually this material is sufficiently pure for further application, but for analytical
purposes it can be additionally purified by Flash column chromatography on Silica gel, eluting
with mixture acetone-benzene (1:3) doped with ammonia (one drop of per 10 mL of eluent).
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General procedure for preparation of indoloquinoline 1 from arylhydrazines 6 and
acetophenones (7 or 8) (performing first step in ethanol/formic acid mixture, General
protocol B): Reaction vessel was charged with arylhydrazine 2 (1.0 mmol) and ortho-amino-
acetophenone (7 or 8) (1.0 mmol), ethanol (700 μL) and formic acid (20 mg). The mixture was
stirred at reflux for 30 min, and then the solvent was removed in vacuum. Polyphosphoric acid
(80% PPA, 2.0 g) was added, after which the reaction, post-reaction work up, and isolation of
the products were carried out in the same manner as described above in the procedure for
General protocol A.
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General procedure for preparation of indoloquinolines 1 from arylhydrazines 6 and
acetophenones (7 or 8) (performing first step in neat, General protocol C, combining Methods
Ia and IIIa): Reaction vessel was charged with arylhydrazine 6 (1.0 mmol) and ortho-
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aminoacetophenone (7 or 8) (1.0 mmol), and the mixture was heated at 120 C for 10 min.
Then, the mixture was cooled down to 100 oC, and polyphosphoric acid (80% PPA, 2.0 g) was
added. The reaction, post-reaction work up, and isolation of the products were carried out in
the same manner as described above in the procedure for General protocol A.
5-Methyl-5H-indolo[3,2-c]quinoline (isocryptolepine, 1aa):18 yield 193 mg (0.83 mmol,
83%) via Method III, 190 mg (0.82 mmol, 82%) via Method IIIa. Physical and spectral
properties listed below are measured for the sample obtained via Method III. Yellowish
crystals, m.p. 198-199 oC (EtOH), Rf 0.17 (EtOH); 1H NMR (400 MHz, CDCl3) δ 8.91 (dd, J = 8.3,
1.4 Hz, 1H), 8.23 (s, 1H), 8.00 (d, J = 8.1 Hz, 1H), 7.90 (d, J = 7.7 Hz, 1H), 7.70 – 7.62 (m, 1H),
7.61 – 7.55 (m, 2H), 7.55 – 7.49 (m, 1H), 7.31 – 7.26 (m, 1H), 4.02 (s, 3H); 13C NMR (100 MHz,
CDCl3) δ 155.1, 153.9, 135.6, 135.4, 129.4, 126.5, 125.5, 125.2, 125.2, 121.4, 120.7, 119.2, 119.2,
117.6, 116.0, 77.2, 42.5; FT IR (NaCl, cm-1): 2372, 2340, 1650, 1597, 1451, 1347, 1320, 1226,
1111, 745; HRMS (TOF-ES): Calcd. for C16H13N2 (M+H)+ 233.1073, found 233.1075 (0.7 ppm).
5,6-Dimethyl-5H-indolo[3,2-c]quinoline (1ab):19 yield 194 mg (0.79 mmol, 79%) via
Method I, 180 mg (0.73 mmol, 73%) via Method II. Physical and spectral properties listed
o
below are measured for the sample obtained via Method I. Yellowish crystals, m.p. 235 C
(EtOH), Rf 0.34 (EtOH); 1H NMR (400 MHz, DMSO) δ 8.75 (d, J = 7.9 Hz, 1H), 8.32 (dd, J = 13.1,
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8.5 Hz, 2H), 7.96 (t, J = 7.8 Hz, 1H), 7.83 (t, J = 7.1 Hz, 2H), 7.57 (t, J = 7.5 Hz, 1H), 7.40 (t, J = 7.5
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Hz, 1H), 4.27 (s, 3H), 3.30 (s, 3H); C NMR (101 MHz, DMSO) δ 154.6, 152.0, 149.4, 137.1,
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129.8, 126.7, 125.3, 125.2, 124.4, 121.5, 121.2, 120.2, 118.9, 118.1, 116.0, 36.2, 19.0; FT IR
(NaCl, cm-1): 2924, 2848, 1735, 1587, 1456, 1432, 1342, 1243, 1077; HRMS (TOF-ES): Calcd. for
C17H15N2 (M+H)+: 247.1230, found 247.1233 (1.4 ppm).
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5-Methyl-6-phenyl-5H-indolo[3,2-c]quinoline (1ac): yield 253 mg (0.82 mmol, 82%) via
Method I, 225 mg (0.73 mmol, 73%) via Method II, 228 mg (0.74 mmol, 74%) via Method III.
Physical and spectral properties listed below are measured for the sample obtained via Method
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III. Yellowish crystals, m.p. 258-262 C (benzene), Rf 0.27 (EtOAc/EtOH 2:1); H NMR (400
MHz, CDCl3) δ 8.76 (d, J = 7.9 Hz, 1H), 8.09 (d, J = 8.9 Hz, 1H), 7.96 (d, J = 8.2 Hz, 1H), 7.85 – 7.79
(m, 3H), 7.76 – 7.71 (m, 2H), 7.63 – 7.57 (m, 1H), 7.48 – 7.42 (m, 1H), 7.35 – 7.29 (m, 1H), 7.09 –
13
7.04 (m, 1H), 6.41 (d, J = 8.1 Hz, 1H), 4.32 (s, 3H); C NMR (101 MHz, CDCl3) δ 152.4, 145.4,
136.8, 132.4, 132.0, 131.4, 130.2 (2C), 130.1, 128.5 (2C), 128.3, 127.7, 127.3, 125.2, 122.8,
122.7, 120.7, 118.2, 115.5, 114.9, 54.8; FT IR (NaCl, cm-1): 3441, 3054, 2975, 1589, 1540, 1447,
1349, 1261, 1202, 1060, 1011; HRMS (TOF-ES): Calcd. for C22H17N2 (M+H)+: 309.1386, found
309.1385 (0.4 ppm).
5-Methyl-6-pentyl-5H-indolo[3,2-c]quinoline (1ad): yield 230 mg (0.76 mmol, 76%) via
o
Method I. Yellowish crystals, m.p. 340-341 C (EtOH), Rf 0.02 (petroleum ether/ EtOAc, 1:1);
1H NMR (400 MHz, CDCl3) δ 8.33 (d, J = 8.0 Hz, 1H), 7.92 (d, J = 8.1 Hz, 1H), 7.79 (d, J = 8.9 Hz,
1H), 7.56 – 7.40 (m, 3H), 7.26 – 7.16 (m, 2H), 4.26 (s, 3H), 3.33 (t, J = 8.2 Hz, 2H), 1.72 – 1.61 (m,
2H), 1.59 – 1.48 (m, 2H), 1.45 – 1.31 (m, 2H), 0.92 (t, J = 7.3 Hz, 3H); 13C NMR (101 MHz, CDCl3)
δ 156.2, 144.2, 142.6, 136.4, 131.7, 127.3, 126.9, 124.7, 123.0, 121.9, 120.7, 117.8, 116.0, 115.3,
113.2, 37.2, 32.0, 32.0, 27.4, 22.4, 14.0.; FT IR (NaCl, cm-1): 2945, 2869, 1666, 1583, 1546, 1439,
1339, 1239, 1188, 1098; HRMS (TOF-ES): Calcd. for C21H23N2 (M+H)+: 303.1856, found
303.1865 (2.9 ppm).
11H-Indolo[3,2-c]quinoline (1ba):20 yield 177 mg (0.81 mmol, 81%) via Method III, 174 mg
(0.80 mmol, 80%) via Method IIIa. Physical and spectral properties listed below are measured
o
for the sample obtained via Method III. Colorless crystals, m.p. 340-341 C (EtOH), Rf 0.19
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(EtOAc); H NMR (400 MHz, DMSO) δ 12.73 (s, 1H), 8.53 (dd, J = 8.0, 1.1 Hz, 1H), 8.32 (d, J = 7.8
Hz, 1H), 8.14 (d, J = 7.9 Hz, 1H), 7.78 – 7.65 (m, 3H), 7.55 – 7.45 (m, 1H), 7.34 (t, J = 7.3 Hz, 1H);
13C NMR (101 MHz, DMSO) δ 145.4, 144.8, 139.8, 138.8, 129.5, 128, 125.7, 125.5, 122.1, 121.9,
120.6, 120.1, 117.1, 114.3, 111.8, 39.5; FT IR (NaCl, cm-1): 3047, 2775, 1571, 1519, 1462, 1373,
1341, 1242, 1158, 933, 771, 740; HRMS (TOF-ES): Calcd. for C15H11N2 (M+H)+ 219.0917, found
219.0917 (0.1 ppm).
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6-Methyl-11H-indolo[3,2-c]quinoline (1bb):18 yield 179 mg (0.77 mmol, 77%) via Method I,
165 mg (0.71 mmol, 71%) via Method II, 181 mg (0.78 mmol, 78%) via Method III. Physical
and spectral properties listed below are measured for the sample obtained via Method III.
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Colorless crystals, m.p. 208-210 oC (EtOH); Rf 0.5 (EtOAc); H NMR (400 MHz, DMSO) δ 12.90
(s, 1H), 8.52 (dd, J = 8.0, 0.9 Hz, 1H), 8.22 (d, J = 8.0 Hz, 1H), 8.06 (d, J = 8.1 Hz, 1H), 7.76 – 7.70
(m, 2H), 7.67 – 7.63 (m, 1H), 7.52 (dd, J = 11.2, 4.0 Hz, 1H), 7.37 (dd, J = 11.1, 3.9 Hz, 1H), 3.10
(s, 3H); 13C NMR (101 MHz, DMSO) δ 154.0, 143.3, 140.3, 138.9, 128.6, 127.3, 125.4, 125.3,
122.3, 122.1, 121.6, 121.0, 116.0, 113.0, 112.0, 22.5; FT IR (NaCl, cm-1): 3054, 2932, 2853, 1599,
1555, 1452, 1359, 1114; HRMS (TOF-ES): Calcd. for C16H13N2 (M+H)+ 233.1073, found
233.1074 (0.2 ppm).
6-Phenyl-11H-indolo[3,2-c]quinoline (1bc):21 yield 238 mg (0.81 mmol, 81%) via Method I,
209 mg (0.71 mmol, 71%) via Method II, 223 mg (0.76 mmol, 76%) via Method III. Physical
and spectral properties listed below are measured for the sample obtained via Method III.
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Colorless crystals, m.p. 248-250 oC (EtOH), Rf 0.30 (petroleum ether/ EtOAc 1:1); H NMR (400
MHz, CDCl3) δ 12.24 (s, 1H), 8.33 (d, J = 8.3 Hz, 1H), 8.24 (dd, J = 8.2, 0.8 Hz, 1H), 7.94 (dd, J =
13
7.9, 1.5 Hz, 2H), 7.62 – 7.57 (m, 2H), 7.50 – 7.33 (m, 6H), 7.17 – 7.12 (m, 1H); C NMR (101
MHz, DMSO) δ 156.9, 145.3, 141.8, 140.3, 139.4, 129.1, 129.0 (2C), 128.9, 128.7, 128.6 (2C),
125.6, 125.6, 122.8, 121.9, 121.8, 120.8, 116.8, 113.4, 111.8; FT IR (NaCl, cm-1): 3172, 3054,
2917, 2843, 1560, 1530, 1501, 1501, 1452, 1359, 1320, 1241, 1222; HRMS (TOF-ES): Calcd. for
C21H15N2 (M+H)+: 295.1230, found 295.1237 (2.4 ppm).
6-Pentyl-11H-indolo[3,2-c]quinoline (1bd): yield 233 mg (0.81 mmol, 81%) via Method I.
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Colorless crystals, m.p. 147-152 C (benzene), Rf 0.32 (petroleum ether/EtOAc 1:1); H NMR
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(400 MHz, CDCl3) δ 11.92 (s, 1H), 8.33 (dd, J = 21.9, 8.2 Hz, 2H), 8.19 (d, J = 7.6 Hz, 1H), 7.61 (t, J
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= 7.0 Hz, 2H), 7.52 – 7.36 (m, 3H), 3.58 (t, J = 7.9 Hz, 2H), 2.10 – 1.96 (m, 2H), 1.57 – 1.43 (m,
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2H), 1.35 – 1.24 (m, 2H), 0.80 (t, J = 7.2 Hz, 3H); C NMR (101 MHz, CDCl3) δ 159.9, 145.3,
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141.4, 139.2, 128.6, 128.5, 125.4, 125.3, 123.0, 122.0, 121.5, 121.4, 116.7, 113.7, 111.9, 38.1,
32.4, 28.9, 22.7, 14.1; FT IR (NaCl, cm-1): 3069, 2959, 2862, 1563, 1515, 1449, 1360, 1249;
HRMS (TOF-ES): Calcd. for C20H21N2 (M+H)+: 289.1699, found 289.1703 (1.4 ppm).
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6-(Pyridin-3-yl)-11H-indolo[3,2-c]quinoline (1be): yield 186 mg (0.63 mmol, 63%) via
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Method II. Сolorless crystals, m.p. 300-302 C (EtOH), Rf 0.24 (EtOAc), H NMR (400 MHz,
DMSO) δ 12.98 (s, 1H), 9.04 (d, J = 2.0 Hz, 1H), 8.83 (dd, J = 4.8, 1.6 Hz, 1H), 8.60 (dd, J = 8.2, 1.1
Hz, 1H), 8.26 (dt, J = 7.8, 1.9 Hz, 1H), 8.16 (d, J = 8.0 Hz, 1H), 7.83 – 7.66 (m, 4H), 7.47 (dd, J =
13
12.2, 5.5 Hz, 2H), 7.18 (t, J = 7.4 Hz, 1H); C NMR (101 MHz, DMSO) δ 152.5, 149.9, 149.4,
145.0, 141.1, 139.1, 136.4, 136.3, 129.5, 128.7, 126.0, 125.6, 123.6, 122.0, 121.4, 120.6, 120.5,
116.3, 112.1; FT IR (NaCl, cm-1): 3054, 2936, 2373, 2255, 1560, 1520, 1457, 1364, 1251, 1212,
1026; HRMS (TOF-ES): Calcd. for C20H14N3 (M+H)+: 296.1182, found 296.1188 (2.03 ppm).
8-Methyl-11H-indolo[3,2-c]quinoline (1ca):22 yield 200 mg (0.86 mmol, 86%) via Method
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III. Colorless crystals, m.p. 305-311 oC (EtOH), Rf 0.24 (petroleum ether/ EtOAc 4:1); H NMR
(400 MHz, DMSO) δ 12.59 (s, 1H), 9.54 (s, 1H), 8.50 (dd, J = 8.0, 1.1 Hz, 1H), 8.12 (d, J = 8.4 Hz,
2H), 7.77 – 7.64 (m, 2H), 7.61 (dd, J = 8.1, 4.6 Hz, 1H), 7.32 (d, J = 8.3 Hz, 1H), 2.52 (s, 3H); 13C
NMR (101 MHz, DMSO) δ 145.4, 144.7, 139.8, 137.0, 129.5, 129.4, 127.9, 126.9, 125.6, 122.1,
122.0, 119.8, 117.2, 114.1, 111.5, 21.2; FT IR (NaCl, cm-1): 3042, 2773, 2366, 1570, 1363, 1239;
HRMS (TOF-ES): Calcd. for C16H13N2 (M+H)+: 233.1073, found 233.1076 (1.3 ppm);
6,8-Dimethyl-11H-indolo[3,2-c]quinoline (1cb):23 yield 202 mg (0.82 mmol, 82%) via
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Method I. Colorless crystals, m.p. 270-272 oC (benzene), Rf 0.15 (EtOAc); H NMR (400 MHz,
DMSO) δ 12.58 (s, 1H), 8.45 (dd, J = 8.0, 1.0 Hz, 1H), 8.02 (d, J = 7.4 Hz, 2H), 7.76 – 7.56 (m, 4H),
7.32 (dd, J = 8.2, 1.0 Hz, 1H), 3.07 (s, 3H), 2.54 (s, 3H); 13C NMR (101 MHz, DMSO) δ 154.7,
144.9, 140.5, 137.5, 130.0, 128.7, 128.5, 126.9, 125.3, 123.0, 122.3, 121.8, 116.7, 113.2, 112.0,
24.8, 21.8; FT IR (NaCl, cm-1): 3157, 3059, 2922, 2775, 1594, 1569, 1516, 1442, 1359, 1300,
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1251, 1207, 1021, 1002; HRMS (TOF-ES): Calcd. for C17H15N2 (M+H)+: 247.1230, found
247.1236 (2.42 ppm).
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8-Methyl-6-phenyl-11H-indolo[3,2-c]quinoline (1сс): yield 231 mg (0.75 mmol, 75%) via
Method I, 222 mg (0.72 mmol, 72%) via Method Ia. Physical and spectral properties listed
below are measured for the sample obtained via Method I. Colorless crystals, m.p. 231-233 oC
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(EtOH); Rf 0.56 (petroleum ether/EtOAc 1:1); H NMR (400 MHz, CDCl3) δ 9.83 (br. s, 1H), 8.30
(d, J = 8.1 Hz, 1H), 8.20 (dd, J = 8.1, 0.9 Hz, 1H), 7.88 (dd, J = 7.8, 1.6 Hz, 2H), 7.67 (ddd, J = 8.4,
7.0, 1.4 Hz, 1H), 7.60 – 7.49 (m, 5H), 7.36 (d, J = 0.7 Hz, 1H), 7.24 (dd, J = 8.3, 1.2 Hz, 1H), 2.37 (s,
3H); 13C NMR (100 MHz, CDCl3) δ 156.6, 145.4, 141.4, 140.8, 137.2, 130.4, 129.8, 129.2,
129.1(2C), 128.6(2C), 128.6, 127.1, 125.7, 123.1, 122.0, 120.9, 116.4, 113.2, 111.1, 21.8; FT IR
(NaCl, cm-1): 2931, 2366, 1642, 1563, 1442, 1336, 1225, 1119; HRMS (TOF-ES): Calcd. for
C22H17N2 (M+H)+: 309.1386, found 309.1391 (1.5 ppm).
5,8-Dimethyl-5H-indolo[3,2-c]quinoline (1da): yield 194 mg (0.79 mmol, 79%) via Method
III. Yellowish crystals, m.p. 184-188 oC (benzene), Rf 0.07 (EtOH); 1H NMR (400 MHz, CDCl3) δ
8.84 (d, J = 7.8 Hz, 1H), 8.32 (s, 1H), 7.79 (d, J = 8.2 Hz, 1H), 7.67 – 7.43 (m, 4H), 7.24 (d, J = 8.3
Hz, 1H), 3.99 (s, 3H), 2.44 (s, 3H); 13C NMR (101 MHz, CDCl3) δ 151.3, 149.9, 136.6, 135.4,
130.6, 129.6, 128.1, 125.6, 125.4, 124.6, 120.3, 119.4, 117.5, 116.4, 116.2, 42.7, 21.8; FT IR
(NaCl, cm-1): 3049, 2917, 1653, 1609, 1452, 1334, 1227, 1124; HRMS (TOF-ES): Calcd. for
C17H15N2 (M+H)+: 247.1230, found 247.1236 (2.4 ppm).
5,6,8-Trimethyl-5H-indolo[3,2-c]quinoline (1db):19 yield 231 mg (0.75 mmol, 75%) via
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Method I. Yellowish crystals, m.p. 182-184 C (benzene), Rf 0.10 (EtOH); H NMR (400 MHz,
DMSO) δ 8.72 (dd, J = 7.9, 0.9 Hz, 1H), 8.07 (d, J = 8.6 Hz, 1H), 7.95 (s, 1H), 7.77 (t, J = 7.7 Hz,
1H), 7.64 (dd, J = 17.2, 7.8 Hz, 2H), 7.24 (d, J = 8.0 Hz, 1H), 4.09 (s, 3H), 3.16 (s, 3H), 2.51 (s, 3H);
13C NMR (101 MHz, DMSO) δ 152.3, 151.2, 148.6, 136.5, 129.1, 128.5, 126.3, 126.3, 124.6,
123.8, 121.1, 120.7, 118.0, 117.5, 115.3, 35.6, 21.6, 18.4; FT IR (NaCl, cm-1): 3062, 2897, 1590,
1435, 1429, 1336, 1243, 1215; HRMS (TOF-ES): Calcd. for C18H16N2 (M+H)+: 261,1386 found
261.1393 (2.7 ppm).
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5,8-Dimethyl-6-phenyl-5H-indolo[3,2-c]quinoline (1dc): yield 229 mg (0.71 mmol, 71%)
via Method I, 219 mg (0.68 mmol, 68%) via Method Ia. Physical and spectral properties listed
below are measured for the sample obtained via Method I. Yellowish crystals, m.p. 234-236 oC
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(benzene), Rf 0.15 (petroleum ether/EtOAc, 1:1); H NMR (400 MHz, CDCl3) 9.04 (dd, J = 8.0,
1.0 Hz, 1H), 7.85 (d, J = 8.2 Hz, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.75 – 7.59 (m, 5H), 7.48 (dd, J = 7.7,
1.6 Hz, 2H), 7.24 (dd, J = 8.1, 1.2 Hz, 1H), 6.26 (s, 1H), 3.84 (s, 3H), 2.26 (s, 3H); 13C NMR (101
MHz, CDCl3) δ 153.1, 147.7, 136.5, 134.0, 130.1, 129.6, 129.6 (2C), 129.3, 128.6 (2C), 128.3,
127.5, 126.4, 125.2, 125.0, 121.6, 120.4, 118.5, 117.2, 116.5, 37.1, 21.7; FT IR (NaCl, cm-1):
2931, 2366, 1659, 1439, 1339, 1260, 1032; HRMS (TOF-ES): Calcd. for C23H19N2 (M+H)+:
323.1543, found 323.1550 (2.2 ppm).
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2-Methoxy-11H-indolo[3,2-c]quinoline (1ea):9a yield 176 mg (0.71 mmol, 71%) via Method
III, 166 mg (0.67 mmol, 67%) via Method IIIa. Physical and spectral properties listed below
o
are measured for the sample obtained via Method III. Colorless crystals, m.p. 300-301 C
1
(EtOH), Rf 0.21 (EtOAc); H NMR (400 MHz, DMSO) δ 12.56 (s, 1H), 9.45 (s, 1H), 8.29 (d, J = 7.7
Hz, 1H), 8.04 (d, J = 9.1 Hz, 1H), 7.96 (s, 1H), 7.72 (d, J = 8.1 Hz, 1H), 7.49 (t, J = 8.1 Hz, 1H), 7.41
– 7.26 (m, 2H), 3.99 (s, 3H); 13C NMR (101 MHz, DMSO) δ 157.0, 142.2, 140.0, 139.4, 138.7, 130,
125.5, 121.9, 120.4, 120.1, 119.4, 117.7, 114.3, 111.8, 101.3, 55.6; FT IR (NaCl, cm-1): 3042,
2827, 1529, 1462, 1378, 1247, 1211, 949, 818, 740, 557; HRMS (TOF-ES): Calcd. for C16H13N2O
(M+H)+: 249.1022, found 249.1026 (1.6 ppm).
2,3-Dimethoxy-11H-indolo[3,2-c]quinoline (1fa):9a yield 222 mg (0.80 mmol, 80%) via
Method III, 214 mg (0.77 mmol, 77%) via Method IIIa. Physical and spectral properties listed
below are measured for the sample obtained via Method III. Colorless crystals, m.p. 320-321
1
oC (EtOH), Rf 0.05 (EtOAc); H NMR (400 MHz, DMSO) δ 12.37 (s, 1H), 9.38 (s, 1H), 8.24 (d, J =
7.8 Hz, 1H), 7.93 (s, 1H), 7.67 (d, J = 8.1 Hz, 1H), 7.53 (s, 1H), 7.45 (t, J = 7.6 Hz, 1H), 7.29 (t, J =
7.5 Hz, 1H), 4.00 (s, 3H), 3.95 (s, 3H); 13C NMR (101 MHz, DMSO) δ 150.6, 148.7, 142.2, 142.0,
139.7, 138.7, 125.1, 122.1, 120.2, 119.8, 113.5, 111.5, 111.1, 109.2, 101.2, 55.7, 55.5; FT IR
(NaCl, cm-1): 3005, 838,2367,2346, 1629, 1493, 1425, 1263, 1226, 1200, 1179, 834, 751; HRMS
(TOF-ES): Calcd. for C17H15N2O2 (M+H)+: 279.1128, found 279.1122, (2.1 ppm).
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2,3-Dimethoxy-5-methyl-5H-indolo[3,2-c]quinoline (1ga): yield 237 mg (0.81 mmol, 81%)
via Method III, 231 mg (0.79 mmol, 79%) via Method IIIa. Physical and spectral properties
listed below are measured for the sample obtained via Method III. Yellowish crystals, m.p. 276-
1
278 oC (EtOH), Rf 0.13 (EtOH); H NMR (400 MHz, DMSO) δ 9.28 (s, 1H), 8.11 (s, 1H), 8.09 (d, J
= 7.7 Hz, 1H), 7.73 (d, J = 8.0 Hz, 1H), 7.43 (s, 1H), 7.40 (d, J = 7.8 Hz, 1H), 7.20 (t, J = 7.4 Hz, 1H),
4.29 (s, 3H), 4.04 (s, 3H), 4.01 (s, 3H); 13C NMR (100 MHz, DMSO) δ 155.1, 152.9, 151.3, 148.1,
136.7, 131.1, 125.5, 125.1, 120.4, 119.6, 119.3, 117.9, 115.4, 103.5, 99.6, 56.1, 55.8, 42.6; FT IR
(NaCl, cm-1): 3005, 2361, 2340, 1639, 1514, 1430, 1352, 1273, 1226, 1054, 813; HRMS (TOF-
ES): Calcd. for C18H17N2O2 (M+H)+: 293.1285, found 293.1282 (0.8 ppm).
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Cancer Cell Growth Inhibition
Material and Methods (Cell culture and growth inhibitory evaluation).
Cell lines used to evaluate the growth inhibitory effects of the compounds were obtained from
the American Type Culture Collection (ATCC, Manassas, VA, USA), the European Collection of
Cell Culture (ECACC, Salisbury, UK) and the Deutsche Sammlung von Mikroorganismen und
Zellkulturen (DSMZ, Braunschweig, Germany). The human cell lines breast carcinoma MCF-7
(DSMZ ACC107), oligodendroglioma Hs683 (ATCC HTB138), non-small cell lung cancer A549
(DSMZ ACC107), glioblastoma U373 cells (ECACC 08061901), melanoma SKMEL-28 (ATCC
HTB72) and the murine melanoma B16F10 (ATCC CRL-6475) cells were cultured in RPMI
supplemented with 10% FBS (GIBCO code 10270106), 4mM glutamine (Lonza code BE17-
605E), 100 µg/mL gentamicin (Lonza code 17-5182), and penicillin-streptomycin (200
units/ml and 200 µg/ml) (Lonza code 17-602E). Cell lines were cultured in flasks, maintained
and grown at 37o C, 95% humidity, 5% CO2. Antiproliferative effects of the compounds on
these cell lines were evaluated through the colorimetric assay MTT.24 Briefly, cells were
trypsinized and seeded in 96 well plates. Prior to treatment compounds were dissolved in
DMSO at a concentration of 10mM. After 24h, cells were treated with the compounds at
different concentrations ranging from 10nM to 100 µM or left untreated for 72 h. Cell viability
was estimated by means of the MTT - (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (Sigma, Bornem, Belgium) mitochondrial reduction into formazan in living cells as
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previously described (Mosmann, 1983). The optical density of the untreated control was
normalized as 100% of viable cells allowing determination of the concentration that reduced
their global growth by 50%. The results are shown in Table 1.
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Acknowledgements
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This project received financial support from the Ministry of Education and Science of the
Russia in the framework of the State Assignment to the Higher Education Institutions (grant
#4.1196.2017/ПЧ). Financial support from the Russian Science Foundation (grant #14-13-
01108, Chemistry) is gratefully acknowledged.
Supporting Information
1H, 13C NMR, and HRMS spectral charts for new compounds, X-ray crystallography
information, and CIF files. This material is available free of charge via the Internet at
http://pubs.acs.org.
Notes and references
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(13) (a) Aksenov, A. V.; Smirnov, A. N.; Aksenov, N. A.; Aksenova, I. V.; Frolova, L. V.; Kornienko,
A.; Magedov, I. V.; Rubin, M. Chem. Commun. 2013, 49, 9305; (b) Aksenov, A. V.; Smirnov, A.
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Nguyen, G.; Johnston, R. K.; Rubin, M.; Carvalho, A. De; Kiss, R.; Mathieu, V.; Lefranc, F.;
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(16) (a) Aksenov, A. V.; Borovlev, I. V.; Aksenova, I. V.; Pisarenko, S. V.; Kovalev, D. A.
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Tetrahedron Lett. 2008, 49, 707; (b) Aksenov, A. V.; Lyahovnenko, A. S.; Aksenova, I. V.;
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N. A.; Lyakhovnenko, A. S.; Aksenova, I. V. Synthesis 2009, 3439.
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One-Pot, Three-Component Assembly of Indoloquinolines: Total
Synthesis of Isocryptolepine
Alexander V. Aksenov,*,† Dmitrii A. Aksenov,† Naila A. Orazova,† Nicolai A. Aksenov,† Georgii D.
Griaznov,† Annelise De Carvalho,‡ Robert Kiss,‡ Véronique Mathieu,‡ Alexander Kornienko,§ and Michael
Rubin*,†,
∥
TOC graphics for jo-2016-03084
O
R4
R4
R3
H
N
N
Me
+
NH2
R3
R1
HN
R2
R1
one-pot
R5
N
R2
R5
N
N
Isocryptolepine
and analogs
R5
R5
N
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