Synthesis and cytotoxic evaluation of novel indenoisoquinoline-propan-2-ol hybrids

Article abstract of DOI:10.1016/j.tetlet.2015.12.045

The synthesis of N-substituted indenoisoquinolines was performed by applying a two-step condensation between 2-carboxybenzaldehyde and phthalide, followed by treatment with various primary amines. N-allylindenoisoquinoline was subsequently selected as a s

Full text of DOI:10.1016/j.tetlet.2015.12.045

Accepted Manuscript
Synthesis and cytotoxic evaluation of novel indenoisoquinoline-propan-2-ol
hybrids
Tham Pham Thi, Lena Decuyper, Tan Luc Quang, Chinh Pham The, Tuyet Anh
Dang Thi, Ha Thanh Nguyen, Thuy Giang Le Nhat, Tra Nguyen Thanh, Phuong
Hoang Thi, Matthias D’hooghe, Tuyen Van Nguyen
PII:
S0040-4039(15)30460-3
DOI:
http://dx.doi.org/10.1016/j.tetlet.2015.12.045
TETL 47092
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
30 October 2015
2 December 2015
8 December 2015
Please cite this article as: Thi, T.P., Decuyper, L., Quang, T.L., The, C.P., Thi, T.A.D., Nguyen, H.T., Nhat, T.G.L.,
Thanh, T.N., Thi, P.H., D’hooghe, M., Nguyen, T.V., Synthesis and cytotoxic evaluation of novel
indenoisoquinoline-propan-2-ol hybrids, Tetrahedron Letters (2015), doi: http://dx.doi.org/10.1016/j.tetlet.
2015.12.045
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Synthesis and cytotoxic evaluation of novel indenoisoquinoline-propan-2-ol hybrids
Tham Pham Thi,a,b Lena Decuyper,c Tan Luc Quang,a Chinh Pham The,a,b Tuyet Anh Dang Thi,a Ha
Thanh Nguyen,a Thuy Giang Le Nhat,a Tra Nguyen Thanh,a Phuong Hoang Thi,a Matthias D’hooghe,c,*
Tuyen Van Nguyena,*
a Institute of Chemistry, Vietnam Academy of Science and Technology, 18-Hoang Quoc Viet, CauGiay,
Hanoi, Vietnam
b Thainguyen University of Science, Tanthinh, Thainguyen, Vietnam
c SynBioC Research Group, Department of Sustainable Organic Chemistry and Technology, Faculty of
Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium
Abstract
The synthesis of N-substituted indenoisoquinolines was performed by applying a two-step
condensation between 2-carboxybenzaldehyde and phthalide, followed by treatment with various
primary amines. N-Allylindenoisoquinoline was subsequently selected as a substrate for
hydroxybromination, providing 6-(3-bromo-2-hydroxy)indenoisoquinoline as a key intermediate for
derivatization in the lactam side chain. In this way,
a
series of 6-(2-
hydroxypropyl)indenoisoquinolines bearing various functional groups at the 3’-position were
prepared, which can be considered as novel indenoisoquinoline-propan-2-ol hybrid molecules.
Subsequent cytotoxic evaluation of 28 indenoisoquinolines against two human cancer cell lines (Hep-
G2 and KB) demonstrated a moderate to high antiproliferative activity displayed by 11
indenoisoquinolines thus synthesized. In particular, the introduction of the 2-hydroxypropyl side
chain was shown to be beneficial for the overall cytotoxic activity, pointing to the potential relevance
of these novel indenoisoquinoline-propan-2-ol hybrids.
Keywords
Indenoisoquinolines; β-Amino alcohols; 2-Carboxybenzaldehyde; Phthalide; Cytotoxicity
Indenoisoquinolines represent a versatile class of compounds, known for their anticancer activity
resulting from the inhibition of DNA topoisomerase I (Top1), a universal and essential enzyme for the
relaxation of supercoiled DNA during important cellular processes.1 Top1 had previously emerged as
a valuable molecular target for the development of anticancer agents,1e,1f,2 such as the pentacyclic
alkaloid camptothecin 1 (Figure 1) and its semisynthetic analogues.1f,1k,3 However, in spite of the
established anticancer activity, the application and efficiency of the camptothecins was hampered by
several pharmacological and clinical limitations, moving forward the class of the indenoisoquinolines
1

as valuable alternatives.1f,4 NSC 314622 2 was developed as a lead compound5 and in the years
thereafter, many derivatives followed, two of which have been promoted into the stage of clinical
trials, indimitecan 3 and indotecan 4.6 In view of these promising results, medicinal chemistry
research on indenoisoquinolines is of present interest in order to obtain new inhibitors that (i)
overcome the limitation of instability and the resulting poor bioavailability of the camptothecins, (ii)
are more selective towards tumor cells versus normal cells and therefore have improved toxicity
profiles, and (iii) are less subject to the emergence of resistance and have improved
pharmacokinetics.7
Functionalized propanes are often part of biologically active agents. Particularly, the class of the β-
amino propanols consists of multiple representatives with inter alia antimalarial, anticancer, Src
kinase inhibiting, antimicrobial and antifungal properties.8 Famous examples include propranolol 5
and timolol 6, depicted in Figure 1. Both compounds are commercially available β-blockers,
prescribed for the treatment of hypertension and the eye disease glaucoma, respectively.9 Besides,
functionalized 3-amino-2-propanol derivatives have been evaluated as potent sodium channel
blockers for the treatment of stroke,10 while others represent green volatile corrosion inhibitors for
brass.11 In addition, amino propanols comprise important intermediates or precursors in the
chemical synthesis of therapeutically or biologically relevant compounds,12 concluding that this
constitutes a versatile class of functionalized propanes with promising applications in divergent
areas.
The nature of the indenoisoquinoline lactam side chains is known to have a significant impact on the
biological effects of these molecules.13 In that respect, this paper presents the synthesis of a variety
of novel indenoisoquinolines combining the indenoisoquinoline scaffold with the promising
functionalized 2-hydroxypropane unit, linked together via the indenoisoquinoline lactam nitrogen,
followed by a preliminary evaluation of the cytotoxic potential of these derivatives against two
human tumor cell lines. However, at first, a series of different indenoisoquinolines, N-functionalized
with diverse heteroatom-containing side chains, was synthesized in order to be able to compare the
influence of a functionalized 2-hydroxypropane unit with other side chains on the cytotoxicity in a
later stage. The main objective of this work thus comprised the synthesis of indenoisoquinoline-
propan-2-ol hybrids as new molecular entities and their preliminary cytotoxic evaluation to assess
the potential of these new scaffolds as templates for more elaborate studies (e.g. as Top1 inhibitors)
in the future.
2

Figure 1
The synthesis of the indenoisoquinoline skeleton has been performed by many methods in the
literature. Some recently reported procedures include (i) the condensation of primary amines with
the appropriately substituted indenoisochromenones,14 (ii) the Suzuki−Miyaura cross-coupling
followed by ring-closing metathesis,15 (iii) the oxidative cyclization of the cis acid resulting from the
condensation of homophthalic anhydrides and benzylidene Schiff bases,2e,14a,16 (iv) cobalt-catalyzed
dual annulation of o-halobenzaldimines with suitable alkynes,17 (v) the combinational photochemical
and carbocationic cyclization of adequately functionalized N-styryl benzamides,18 and (vi) lithiated
toluamide-benzonitrile cycloaddition followed by intramolecular radical cyclization.19 The synthesis
of the indenoisoquinolines was pursued in the present work by applying the first, widely used
method of Morrell et al.14d
In this way, the key starting material, benz[d]indeno[1,2-b]pyran-5,11-dione 10, or shortly
indenobenzopyran 10, was synthesized using a two-step methodology in a multi-gram scale.
Condensation of 2-carboxybenzaldehyde 7 with phthalide 8 in the presence of sodium methoxide in
methanol/ethyl acetate (2:1) under reflux furnished intermediate 9, which could, after dehydrative
acid-catalyzed lactonization in toluene, efficiently be converted to indenobenzopyran 10 in 58% yield
after recrystallization from ethyl acetate. This compound 10 is an excellent precursor for the efficient
generation of indenoisoquinolines 11 in high yields (81-96%) upon treatment with the appropriate
primary amines, as illustrated in Scheme 1 (Table 1).14d,20 In this way, the synthesis of four new
derivatives was efficiently performed, giving rise to a series of nine variously substituted
indenoisoquinolines at the lactam nitrogen atom. The molecular structures of the obtained
indenoisoquinolines 11 were confirmed by means of spectral data (1H NMR, 13C NMR, IR and MS),
some of which were identical to those reported in the literature.13c,14c,21b,25
3

Scheme 1 Synthesis of indenoisoquinolines 11a-i
The interesting properties of 3-aminopropan-2-ols and indenoisoquinolines as such, together with
the fact that an improvement of the biological activity resulting from specific lactam side chain
features has been observed previously, motivated the synthesis of indenoisoquinoline-propan-2-ols
as a new class of hybrid molecules. More specifically, the advantages associated with the
incorporation of an alkyl side chain containing two to four methylene units into the
indenoisoquinoline core structure has been described earlier.13c,14a,21a,22 Besides, multiple
indenoisoquinoline structure-activity relationships have demonstrated that the presence of hydrogen
bonding groups (e.g., hydroxyl) on the lactam side chains correlates well with an increase in
biological activity.14c,22b,22c In particular, the (S)-2,3-dihydroxypropyl lactam side chain has recently
been proven to exert significant influence on the Top1 poisoning and cytotoxic activity, as the 2’-
hydroxyl group may form a crucial hydrogen bond in the active site.2d,13a Moreover, a compound in
which the terminal hydroxyl group is replaced with an amino group, exerts even higher cytotoxic
activity.14c,22c Furthermore, it was demonstrated that the incorporation of heterocycles into the
indenoisoquinoline lactam side chain, possessing a heteroatom capable of serving as a hydrogen-
bond acceptor at physiological pH, generally provides good biological results.14d,22b,22c,23 All together,
these findings led to the hypothesis that an efficacious indenoisoquinoline could be designed by
incorporating an N-functionalized three-carbon side chain, possessing a 2’-hydroxyl group, combined
with a terminal hydroxyl, ester, sulfonyl or amino group, substituted or integrated in various
heterocycles. In that respect, the current work is the first in combining those extensively reported
and promising features, concretized by the formation of indenoisoquinoline-propan-2-ol hybrids as
novel structures.
For this purpose, we opted to derivatize N-allyl-substituted indenoisoquinoline 11g via
hydroxybromination using two equiv of bromine in water/dichloromethane (1:50). A mixture of 6-
4

(2,3-dibromopropyl)indenoisoquinoline
12
(10%)
and
6-(3-bromo-2-
hydroxypropyl)indenoisoquinoline 13 (77%) was formed, after which the latter was considered as the
central building block for conversion into a variety of O-, N- or S-based functional groups linked to
the 3’-carbon of the propyl side chain. Firstly, 6-(3-bromo-2-hydroxypropyl)indenoisoquinoline 13
was converted into 6-(3-amino/thio-2-hydroxypropyl)indenoisoquinolines 14 in moderate to good
yields (45-78%) after base-catalyzed nucleophilic substitution of the primary bromide by a series of
primary or secondary amines or primary thiols in acetone or DMF at 65°C (Table 1, Scheme 2).24
Furthermore, intermediate 13 could be converted to the corresponding 2,3-propanediol 15 via
K2CO3-catalyzed nucleophilic substitution by water. It should be mentioned, however, that 6-(2,3-
dihydroxypropyl)indenoisoquinolines 15 can also be synthesized via condensation of
indenobenzopyran 10 with the corresponding commercially available amino alcohols.14c Compound
15 was then further acylated using acetic and isobutyric anhydride in the presence of three equiv of
triethylamine to provide esters 16 in 62-65% yield. On the other hand, treatment of 2,3-propanediol
15 with tosyl or mesyl chloride resulted in the formation of the monosulfonylated diols 17 (54-58%).
A final option involved reaction of 6-(3-bromo-2-hydroxypropyl)indenoisoquinoline 13 with three
equiv of sodium azide in order to furnish the corresponding azide 18 in good yield (81%). The
1
synthesized indenoisoquinolines 12-18 were analyzed using H NMR, 13C NMR, MS and IR techniques
in order to confirm the proposed molecular frameworks. Additionally, single crystal X-ray analysis of
compound 13 secured the formation of this new scaffold (Figure 2).
5

Scheme
2
Transformation
of
allylindenoisoquinoline
11g
towards
6-(2-
hydroxypropyl)indenoisoquinolines 12-18
Figure 2 Single crystal X-ray analysis of compound 13
Table 1 Synthesis and cytotoxicity evaluation (IC50) of indenoisoquinoline derivatives 11-18
Entry R or X
Compound: Yielda
Cytotoxicity (µM)
Entry
R or X
Cytotoxicity (µM)
Compound: Yielda
KB
1.42
> 379
Hep-G2
1.62
> 379
KB
Hep-G2
32.9
1
2
Ellipticine
16
31.4
14db: 66%
11a: 86%13c
6

3
> 362
> 391
> 384
> 420
> 449
> 446
> 294
1.82
> 362
309
17
18
19
20
21
22
23
24
25
26
27
28
29
0.82
73.2
2.35
> 128
15.3
2.50
1.51
26.6
10.6
54.9
4.33
57.1
42.0
0.69
68.3
3.44
> 128
14.8
2.99
1.19
27.1
8.33
52.1
7.19
52.1
45.0
14eb: 60%24
11b: 83%20
11c: 83%21b
4
14fb: 64%
14gb: 68%
5
> 384
> 420
> 449
> 446
> 294
1.41
11d: 81%25
11e: 96%
6
14hb: 65%
14ib: 45%
7
11f: 81%21b
11g: 92%21b
8
14jb: 65%
14kb: 49%
9
11h: 82%
11i: 86%
10
11
12
13
14
15
-
15b: 69%14c
-
3.40
2.05
12b: 10%
16ab: 62%
16bb: 65%
-
5.20
3.30
13b: 77%
0.82
0.47
17ab: 54%
17bb: 58%
14ab: 78%
14bb: 73%
1.79
2.23
1.67
1.15
-
18b: 81%
14cb: 76%
7

a After purification by column chromatography (SiO2).
b Obtained as a racemic mixture.
Subsequently, as compared to indenoisoquinolines 11a-i lacking a 2-hydroxypropane side chain, the
synthesized indenoisoquinolines 12-18 were evaluated in terms of their cytotoxicity profile against
two human cancer cell lines, KB and Hep-G2.26,27 Ellipticine, a compound with known cytotoxic
activity, was added as a reference compound. The results of this biological assessment, summarized
in Table 1, clearly indicate that the majority of the compounds exhibit at least moderate cytotoxicity
against both cancer cell lines. Eleven of the prepared indenoisoquinolines display IC50 values of
around or less than 5 µM (11i, 12, 13, 14a,b,c,e,g,j,k, 17a). Furthermore, it should be noticed that
derivatives 11i and 14a,c,e,k exhibit equal cytotoxic activity as compared to the reference compound
ellipticine, with 4-methylpiperidin-1-yl- and piperazin-1-ylindenoisoquinolines 14a and 14e being the
most promising (IC50 values of 0.82 and 0.47 µM and 0.82 and 0.69 µM respectively against KB and
Hep-G2).
In conclusion, the present paper described an efficient synthetic approach toward nine variously
substituted indenoisoquinolines with regard to the lactam nitrogen, starting from the two-step
condensation of 2-carboxybenzaldehyde and phthalide, followed by the reaction with the
appropriate primary amines. In addition,
a
series of new functionalized 6-(2-
hydroxypropyl)indenoisoquinolines was prepared via diverse methods of functionalization starting
from 6-(3-bromo-2-hydroxypropyl)indenoisoquinoline. Subsequently, the 28 indenoisoquinolines
thus prepared were subjected to in vitro cytotoxic evaluation against two human cancer cell lines,
resulting in moderate to high bioactivity values of 11 representatives, especially those comprising the
3-amino-2-hydroxypropyl unit. The potential relevance of this novel class of indenoisoquinolines,
incorporating the promising β-amino alcohol side chain, was thus proven, pointing to the importance
of further, more elaborate biological assays and optimization of the most promising compounds.
Acknowledgements
The authors are indebted to the Vietnamese National Foundation for Science and Technology
Development (NAFOSTED, code: 104.01-2014.38), Vietnam Academy of Science and Technology
(Vast -04.07/14-15) and to Ghent University – Belgium (BOF) for financial support.
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W.-J. Bioorg. Med. Chem. Lett. 2007, 17, 5763-5767. (b) Cho, W.-J.; Le, Q. M.; Van, H. T. M.;
Lee, K. Y.; Kang, B. Y.; Lee, E.-S.; Lee, S. K.; Kwon, Y. Bioorg. Med. Chem. Lett. 2007, 17, 3531-
3534. (c) Cho, W.-J.; Park, M.-J.; Imanishi, T.; Chung, B.-H. Chem. Pharm. Bull. 1999, 47, 900-
902.
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6-(6-Methoxypyridin-3-yl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione
(11b):
6-
Methoxypyridin-3-amine (275 mg, 1.1 equiv) was added to a solution of indeno[1,2-
c]isochromene-5,11-dione (10; 500 mg, 2.02 mmol) in anhydrous CH2Cl2 (20 mL). The solution
was allowed to stir at room temperature for 24h. Afterwards, the reaction mixture was
washed with brine (20 mL). Drying of the organic phase (MgSO4), filtration of the drying agent
and evaporation of the solvent in vacuo afforded indenoisoquinoline 11b as a racemic
mixture, which was then purified by column chromatography on silica gel (Hexane-EtOAc,
1
8:2) to obtain an analytically pure sample; Red crystals; M.p. 253-254°C; yield: 83%. H NMR
(500 MHz, CDCl3): δ = 8.72 (d, J = 8.0 Hz, 1 H), 8.34 (d, J = 8.0 Hz, 1 H), 8.21 (d, J = 2.5 Hz, 1 H),
7.77 (t x d, J = 1.0, 8.0 Hz, 1 H), 7.65 (d x d, J = 2.5, 8.5 Hz, 1 H), 7.57 (d, J = 8.0 Hz, 1 H), 7.49 (t
x d, J = 2.5, 8.0 Hz, 1 H), 7.26 (t, J = 2.5, 7.5 Hz, 1 H), 7.10 (t, J = 1.0, 8.0 Hz, 1 H), 7.01 (d, J =
8.5 Hz, 1 H), 5.80 (d, J = 2.5 Hz, 1 H), 4.08 (s, 3 H). 13C NMR (125 MHz, CDCl3): δ = 190.5, 164.6,
163.8, 155.3, 146.4, 138.8, 137.0, 134.6, 134.3, 132.8, 132.7, 130.8, 128.7, 127.7, 127.4,
123.8, 123.7, 123.0, 122.2, 112.0, 108.5, 54.1. IR (KBr): 3076, 2943, 1789, 1693, 1662, 1606,
1573, 1548, 1485, 1411, 1375, 1182, 1012, 817, 759 cm-1. MS: m/z (%) = 355 (100) [M+ + 1].
HRMS (ESI): m/z [M + H]+ calcd for C22H15N2O3: 355.1077; found: 355.1081.
(21)
(22)
(a) Cushman, M.; Jayaraman, M.; Vroman, J. A.; Fukunaga, A. K.; Fox, B. M.; Kohlhagen, G.;
Strumberg, D.; Pommier, Y. J. Med. Chem. 2000, 43, 3688-3698. (b) Wawzonek, S. Org. Prep.
Proced. Int. 1982, 14, 163-168.
(a) Antony, S.; Jayaraman, M.; Laco, G.; Kohlhagen, G.; Kohn, K. W.; Cushman, M.; Pommier,
Y. Cancer Res. 2003, 63, 7428-7435. (b) Nagarajan, M.; Morrell, A.; Ioanoviciu, A.; Antony, S.;
Kohlhagen, G.; Agama, K.; Hollingshead, M.; Pommier, Y.; Cushman, M. J. Med. Chem. 2006,
49, 6283-6289. (c) Nagarajan, M.; Xiao, X.; Antony, S.; Kohlhagen, G.; Pommier, Y.; Cushman,
M. J. Med. Chem. 2003, 46, 5712-5724.
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(24)
Nagarajan, M.; Morrell, A.; Fort, B. C.; Meckley, M. R.; Antony, S.; Kohlhagen, G.; Pommier, Y.;
Cushman, M. J. Med. Chem. 2004, 47, 5651-5661.
6-(2-Hydroxy-3-(piperazin-1-yl)propyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (14e): A
mixture of piperazine (100 mg, 1.5 equiv) and K2CO3 (215 mg, 2 equiv) was stirred for 30 min
11

at room temperature in 5 mL of DMF. Then, 6-(3-bromo-2-hydroxypropyl)-5H-indeno[1,2-
c]isoquinoline-5,11(6H)-dione (13; 300 mg, 0.78 mmol) was added, after which the reaction
was carried out at 65°C for 12h. Afterwards, the reaction mixture was poured in water and
extracted with EtOAc (3 x 5 mL). The combined organic phases were washed with water (10
mL) and brine (10 mL), dried (MgSO4), filtrated and evaporated in vacuo to afford the crude
product as a racemic mixture, which was then purified by means of column chromatography
on silica gel (Hexane-EtOAc, 1:1) to obtain the analytically pure compound 14e: red oil; Rf
0.61 (Hexane-EtOAc, 3:7); yield: 60%. 1H NMR (CD3OD, 500 MHz): δ = 8.55 (d, J = 8.5 Hz, 1 H),
8.17 (d, J = 8.0 Hz, 1 H), 7.87 (d, J = 7.5 Hz, 1 H), 7.68 (d x d x d, J = 1.0, 8.0, 15 Hz, 1 H), 7.48-
7.35 (m, 4 H), 4.72-4.60 (m, 1 H), 4.49-4.41 (m, 1 H), 4.25-4.21 (m, 1 H), 3.13-3.11 (m, 2 H),
2.78-2.59 (m, 8 H). 13C NMR (CD3OD, 125 MHz): δ = 192.0, 165.3, 138.7, 136.0, 135.0, 134.4,
133.5, 132.0, 129.1, 128.2, 125.6, 124.4, 124.2, 123.6, 109.4, 67.6, 63.1, 54.7, 52.1 (2 x C),
50.1, 45.0 (2 x C). IR (KBr): 3469, 3276, 3045, 2958, 2858, 1681, 1544, 1496, 1425, 1317,
1204, 1139, 799 cm-1. MS: m/z (%) = 390 (100) [M+ + 1]. HRMS (ESI): m/z [M + H]+ calcd for
C23H24N3O3: 390.1812; found: 390.1818.
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(26)
Strumberg, D.; Pommier, Y.; Paull, K.; Jayaraman, M.; Nagafuji, P.; Cushman, M. J. Med.
Chem. 1999, 42, 446-457.
Cell culture and cell viability assay. Two human cancer cell lines (epidermoid carcinoma cell
line (KB) and hepatoma carcinoma cell line (HepG2)), obtained from the American Type
Culture Collection (USA) ATCC, were used for cytotoxic evaluation. The cells were grown in
RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and
100 µg/mL streptomycin at 37°C in a humidified atmosphere (95% air and 5% CO2). The
exponentially growing cells were used throughout the experiments. The inhibitory effects of
the compounds on the growth of the human cancer cell lines were determined by measuring
the metabolic activity using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
(MTT) assay.27 Briefly, human cancer cell lines (1×105 cells/mL) were treated for 3 days with a
series of concentrations of the compounds (in DMSO): 0.125, 0.5, 2.0, 8.0, 32.0, and 128.0
µg/mL. After incubation, 0.1 mg MTT solution (50 µL of a 2 mg/mL solution) was added to
each well, and the cells were then incubated at 37°C for 4 h. The plates were centrifuged at
1000 rpm for 10 min at room temperature, and the media were then carefully aspirated.
Dimethylsulfoxide (150 µL) was added to each well to dissolve the formazan crystals. The
plates were read immediately at 540 nm on a microplate reader (TECAN GENIOUS). All the
experiments were performed three times, and the mean absorbance values were calculated.
The results are expressed as the percentage of inhibition that produced a reduction in the
absorbance by the treatment of the compounds compared to the untreated controls. A dose-
12

response curve was generated, and the inhibitory concentration of 50% (IC50) was
determined for each compound as well as each cell line.
(27) (a) Carmichael, J.; DeGraff, W. G.; Gazdar, A. F.; Minna, J. D.; Mitchell, J. B. Cancer Res. 1987,
47, 936-942. (b) Mosmann, T. J. Immunol. Methods 1983, 65, 55-63.
13

Graphical abstract
Synthesis and cytotoxic evaluation of novel N-substituted indenoisoquinolines
14