Synthesis and cytotoxic evaluation of novel indenoisoquinoline-substituted triazole hybrids

Article abstract of DOI:10.1016/j.bmcl.2016.05.092

The synthesis of various substituted triazole–indenoisoquinoline hybrids was performed based on a CuI-catalyzed 1,3-cycloaddition between propargyl-substituted derivatives and the azide-containing indenoisoquinoline. Besides, a variety of N-(alkyl)propargylindenoisoquinolines was used as substrates for the construction of triazole–indenoisoquinoline–AZT conjugated via a click chemistry-mediated coupling with 3′-azido-3′-deoxythymidine (AZT). Thus, twenty three new indenoisoquinoline-substituted triazole hybrids were successfully prepared and evaluated as cytotoxic agents, revealing an interesting anticancer activity of four triazole linker–indenoisoquinoline–AZT hybrids in KB and HepG2 cancer cell lines.

Full text of DOI:10.1016/j.bmcl.2016.05.092

Accepted Manuscript
Synthesis and cytotoxic evaluation of novel indenoisoquinoline-substituted tri-
azole hybrids
Tham Pham Thi, Thuy Giang Le Nhat, Thuong Ngo Hanh, Tan Luc Quang,
Chinh Pham The, Tuyet Anh Dang Thi, Ha Thanh Nguyen, Thu Ha Nguyen,
Phuong Hoang Thi, Tuyen Van Nguyen
PII:
S0960-894X(16)30602-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.05.092
BMCL 23951
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
8 March 2016
26 May 2016
31 May 2016
Please cite this article as: Thi, T.P., Nhat, T.G.L., Hanh, T.N., Quang, T.L., The, C.P., Thi, T.A.D., Nguyen, H.T.,
Nguyen, T.H., Thi, P.H., Nguyen, T.V., Synthesis and cytotoxic evaluation of novel indenoisoquinoline-substituted
triazole hybrids, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.
2016.05.092
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

SYNTHESIS AND CYTOTOXIC EVALUATION OF NOVEL INDENOISOQUINOLINE-
SUBSTITUTED TRIAZOLE HYBRIDS
Tham Pham Thia,b, Thuy Giang Le Nhata, Thuong Ngo Hanha, Tan Luc Quanga,c, Chinh Pham
Thea,d, Tuyet Anh Dang Thia, Ha Thanh Nguyena, Thu Ha Nguyena, Phuong Hoang Thia, Tuyen Van
Nguyena,*
aInstitute of Chemistry, Vietnam Academy of Science and Technology, 18-Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam
bThuyloi University, 175, Tay Son, Ha Noi, Vietnam
cHanoi Pedagogical University No.2
dThainguyen University of Science, Tanthinh, Thainguyen, Vietnam
* Corresponding author. Tel.: +84 917683979.
E-mail address: ngvtuyen@hotmail.com (T. Van Nguyen)
ABSTRACT
The synthesis of various substituted triazole-indenoisoquinoline hybrids was performed based on
a CuI-catalyzed 1,3-cycloaddition between propargyl-substituted derivatives and the azide-containing
indenoisoquinoline. Besides, a variety of N-(alkyl)propargylindenoisoquinolines was used as substrates
for the construction of triazole-indenoisoquinoline-AZT conjugated via a click chemistry-mediated
coupling with 3’-azido-3’-deoxythymidine (AZT). Thus, twenty three new indenoisoquinoline-
substituted triazole hybrids were successfully prepared and evaluated as cytotoxic agents, revealing an
interesting anticancer activity of four triazole linker-indenoisoquinoline-AZT hybrids in KB and HepG2
cancer cell lines.
Keywords: indenoisoquinolines, triazole, AZT, hybrids, cytotoxic agents
Pharmacophore hybridization, in which two or more bioactive entities are linked into a single
hybrid molecule have been known as an efficient and comfortable method to give new compounds with
anticancer properties. Molecular hybridization can deliver a synthetic advantage through selective
chemical modification of the more reactive entity within hybrid systems. Moreover, when bioactive
agents are combined, new hybrid structures might display both a biological and a synthetic benefit. In
the pursuit of potential new compounds, the selection of molecules with high biological activities for
hybridization is occuping an important role.
Thus, indenoisoquinolines are one of the key structural units, which have been attracting
considerable interest because of their various biological activities, particularly cytotoxicity against
human cancer cell lines.1 The lead compound of this class is NCS 314622 (1), which was at first
prepared in 1978 and was found to be a mammalian topoisomerase I (Top1) inhibitor.1b,2 Its cytotoxicity
profile revealed a strong resemblance with camptothecin derivatives by stabilizing its covalent complex
with DNA, the Top1-DNA cleavage complex (Top1cc), preventing further DNA religation and thus
leading to the accumulation of DNA breaks.3 During the past decade considerable effort has been made
to improve the potencies and pharmacokinetics properties of these indenoisoquinolinediones through the
contributions of the indenone ring, isoquinoline ring and lactam side chain.1f,1g,4-6 It was demonstrated
that the introduction of heterocycles, which possess a heteroatom capable of serving as a hydrogen-bond
acceptor at physiological pH (e.g., hydroxyl, imidazole), in the N-functionalized lactam chains

significantly enhances the biological activity.5a,5c,5d,7-14 Well-known examples are anticancer
topoisomerase I (Top1) inhibitors indotecan (LMP400, 2) and indimitecan (LMP776, 5), which were
ultimately promoted to clinical study at the National Cancer Institute.3d,15 Recently, hydroxylated
analogues of the indotecan and indimitecan 4-11, which contain various functionalized at A and D rings
and the imidazole or morpholine group in N-lactam side chain, have been prepared and found to be very
potent Top1 inhibitor and antiproliferative agents.9 All of them, unlike camptothecin, appear to be stable
and are powerful, cytotoxic Top1 poisons that induce long-lasting DNA breaks and overcome the drug
resistance issues associated with the camptothecins.3d,15,16 In addition, in our previous work, a novel of
indenoisoquinoline-propan-2-ols 12-15 with high cytotoxicity against KB and HepG2 was designed by
incorporating an N-functionalized three-carbon side chain, possessing a 2’-hydroxyl group, combined
with pyrolidinyl, piperazine and piperidine units.7
O
R3
O
O
O
O
R4
R1
R2
MeO
MeO
OH
N
R'
N
N
R
CH3
O
O
O
12-15
1
2-11
O
O
R3, R4 =
; R =
R =
; R1 = R2 = OMe (2);
N
O
R1 = OH, R2 = OMe (3);
R1 = OMe, R2 = OH (4).
N
N
N
(13);
(12);
R' =
N
; R1 = R2 = OMe (5);
R1 = OH, R2 = OMe (6);
R1 = OMe, R2 = OH (7).
N
N
(14);
(15).
NH
R1 = R2 = OMe; R =
; R3 = R4 = OH (8);
N
N
O
R1 = OH, R2 = OMe (9).
N
R =
; R3 = R4 = OH (10);
R1 = OH, R2 = OMe (11).
Another nitrogen-containing hetreocycle 1,2,3-triazole has been known as a part of biologically
active agent. Triazole has a high aromatic stabilization and high dipole moment, which might participate
actively in hydrogen bond formation and in dipole-dipole and π stacking interactions.17 Triazole is
relatively resistant to metabolic degradation,18 stable to acid and basic hydrolysis as well as reductive
and oxidative conditions. Plannar heteroaromatic triazole might lead to a more facile interaction with
DNA, proteins, or cells.19 Besides, triazole is easy synthesized by “click” chemistry and exhibits a
broad-spectrum of anticancer activities20 and antiproliferative properties.21 Therefore the combining
1,2,3-triazole with other pharmacophores becomes one of the most importance medicinal chemistry
strategies. By this way a number of compounds with potent antitumor activity have been synthesized
and are now available in the market such as tazobactam,22 cefatrizine23 and carboxyamidotriazole.24
From the multitude of contributions in the scientific literature, it is clear that indenoisoquinolines
and triazoles can be considered as valuable compounds, both from a medicinal and a synthetic point of
view. Morever, consideration for the chemical structure of indimitecan, the addition of one more
nitrogen atom to the imidazole ring could give more interesting bioactivities. All together, these findings
have led to the hypothesis that the introduction of triazole group into N-functionalized three-carbon side
chain of indenoisoquinoline, especially indenoisoquinoline-propan-2-ols, could give the promising
potent biological compounds. However, the combination of these two bioactive moieties into hybrid

molecules has not been reported in the literature so far. Having been inspired by the biological
importance of 1,2,3-triazoles and indenoisoquinoline-propan-2-ols as anticancer agents and in
continuation of our interest in pharmacophore hybirdization we herein reported the synthesis of novel
triazole-indenoisoquinolines hybrids. The anticancer activity evaluation results revealed that the 1,2,3-
triazole-indenoisoquinoline hybrids exhibited potent anticancer activity.
The strategies for the formation of five-membered heterocyclic ring systems contain 1,3-dipolar
cycloaddition reactions, which have gained major interest for several decades in view of the large
numbers of potential dipoles and dipolarophiles.25 As one such possibility, Cu(I)-catalyzed azide-alkyne
cycloaddition is a widely utilized, reliable and powerful way to form 1,4-disubstituted 1,2,3-triazole.26
In that respect, a synthetic approach toward novel triazole-indenoisoquinoline hybrids was devised
based on a CuI-catalyzed 1,3-cycloaddition between propargyl-substituted derivatives and the azide-
containing indenoisoquinoline.
In this way, the key starting material, 6-(3-azido-2-hydroxypropyl)indenoisoquinoline 16, or
shortly azidoindenoisoquinoline 16, was prepared by four-step methodology in our previous report.7
Azidoindenoisoquinoline 16 exhibits an excellent precusor for the efficient generation of triazole-
indenoisoquinoline hybrids 17a-n in high yields (60-80%) upon treatment with the appropriate 1-
propargyl derivative, as illustrated in scheme 1.27 This reaction was carried out in refluxing
tetrahydrofuran in the presence of N,N-diisopropylethylamine (DIPEA) and CuI. The chemical
structures of the obtained indenoisoquinolines 17a-n were confirmed by means of spectral data (1H
13
NMR, C NMR, IR and MS). In the previous work, it was shown that the hydroxy group in the N-
lactam side chain of indenoisoquinoline-propan-2-ols increases antiproliferative activity.7 In order to
persuade this hypothesis, the hydroxy group in side chain was acylated by treatment with acetic
anhydride to obtain esters 18a,b, respectively (Scheme 1).
O
O
O
(b)
(a)
N
N
OH
OH
N
N
OAc N
N
R
R'
N
N3
N
N
O
O
O
16
17a-n
18a: R' = CH2OAc
18b: R' = C6H4NHAc
Scheme 1: Synthesis of triazole-indenoisoquinoline hybrids 17a-n and 18a,b. Reagents and conditions: (a) 1.1
equiv ethynyl derivatives, 0.2 equiv DIPEA, 0.1 equiv CuI, THF, reflux, 24 h; (b) 3 equiv Ac2O, 2 equiv Et3N,
DMF, rt, 24 h.
As a part of our ongoing work, the introduction of bioactive moieties into N-lactam side chain of
indenoisoquinolines is also studied in this letter. 3’-Azido-3’-deoxythymidine (AZT, zidovudine) has
known as a nucleoside reverse transcriptase inhibitor used for the treatment of HIV infections. AZT has
also been exhibited pronounced anticancer activity, especially in combination with other antitumor
agents, for example, such as 5-fluorouracil, cisplatin, paclitaxel28 and triterpenoids.29,30 Thus,
considering the documented anticancer activity of indenoisoquinolines, AZT and functionalized triazole,
it is reasonable to suggest that the construction of triazole-indenoisoquinoline-AZT hybrids might show
good cytotoxicity activities. For this purpose, N-propargyl indenoisoquinoline 20, prepared from
indenobenzopyran 19,7 was treated with AZT in tert-BuOH in the presence of CuI at 70oC to afford
1,2,3-triazole-indenoisoquinoline-AZT conjugate 21 in good yield (Scheme 2). On the other hand, in

order to investigate the subtituted group on N-lactam side chain and containing triazole bridge the
synthesized indenoisoquinoline acids 22a-e were esterified by propargyl bromide in DMF/THF (1:1) in
the presence of Cs2CO3 to furnish the corresponding esters 23a-e, which were conjugated with AZT by
the above-described click chemistry methodology. This approach resulted in novel indenoisoquinoline-
AZT hybrids 24a-e in a good yield (Scheme 2).31 The chemical structures of indenoisoquinolines 20-24
were confirmed using 1H NMR, 13C NMR, MS and IR techniques.
O
H3C
NH
HO
N
O
O
O
O
(a)
(b)
O
N
O
N
N
N
O
19
O
N
20
O
(c)
21
O
N
H3C
HO
NH
O
O
O
O
(d)
(e)
O
N
N
R
R
N
O
O
O
N
O
O
O
OH
N
N
22a-e
23a-e
R
O
O
CH
24a-e
R = CH2 (a), C6H5CH2CH (b), iso-C3H7CH2CH(c), HOCH2CH (d),
H2C
(e).
N
H
Scheme 2: Synthesis of triazole-indenoisoquinoline-AZT hybrids 21 and 24a-e. Reagents and conditions: (a) 1.1
equiv propargyl amine, CH2Cl2, 40oC, 24 h; (b) 0.9 equiv AZT, 0.2 equiv CuI, tert-BuOH, 70oC, 24 h; (c) 1.1
equiv amino acid, CH2Cl2, 40oC, 2-8 h; (d) 1.1 equiv propargyl bromide, 1.1 equiv Cs2CO3, DMF:THF (1:1), rt,
2-6 h; (e) 0.8 equiv AZT, 0.2 equiv DIPEA, 0.1 equiv CuI, THF, reflux, 24 h.
In continuation of our interest on hybridization of indenoisoquinolines via triazole linker, another
bioactive coupling partner concerns imatinib and analogs, tyrosine-kinase inhibitors used in the
treatment of multiple cancers, most notably chronic myelogenous leukemia,32 have been taken into
account. So the triazole hybrid between N-propargyl indenoisoquinoline 20 and imatinib derivative 4-
(azidomethyl)-N-(4-methyl-3-(4-pyridin-3-yl)pyrimidin-2-ylamino)phenyl)benzamide
25
was
synthesized as outlined in scheme 3.
O
N
N
NH
O
N
N
O
O
NH
N
N
+
N
N
H
N
N
O
N
CH3
N3
N
N
O
CH3 H
20
26
25
Scheme 3: Synthesis of triazole-indenoisoquinoline hybrid 26. Reagents and condition: 0.9 equiv 25, 0.2 equiv
CuI, tert-BuOH, 70oC, 24 h.

Table 1
Synthesis and cytotoxicity evaluation (IC50) of triazole-indenoisoquinoline hybrids 17-26
Cytotoxicity (µM)
Cytotoxicity (µM)
Compound R or R’:
Yield, %
Compound R or R’:
Entry
1
Entry
14
Yield, %
O
KB
HepG2
0.74
KB
HepG2
2.29
HO-CH2-
2.86
2.89
17a: 70
N
CH2
O
17n: 79
AcOCH2-
18a: 75
O
2
3
4
6.28
11.19
10.46
4.27
11.86
9.75
15
16
17
18.61
> 234
0.79
19.04
>234
0.64
CH2
O
Br
17b: 60
O
AcHN
CH2
O
MeO
18b: 77
21: 75
17c: 55
O
CH2
O
Cl
17d: 46
H2N
5
6
15.33
89.89
31.96
69.76
18
19
CH2
24a: 75
26.2
0.49
26.2
0.48
17e: 75
TsHN
C6H5CH2CH
24b: 85
17f: 77
NH2CH2-
17g: 50
TsNHCH2-
17h: 78
7
8
9
50.03
115.31
14.07
195.43
115.31
9.04
20
21
22
iso-C3H7CH2CH
24c: 82
0.75
0.35
2.25
1.18
HOCH2CH
24d: 78
CH3CONHCH2-
17i: 80
21.63
84.50
CH
H2C
N
H
24e: 80
26: 70
O
10
11
123.23
56.74
22.1
23
24
1.56
42.0
2.33
45.0
N
H
17j: 70
O
43.83
16
CH2
N
H
Br
17k: 50
BrCH2-
17l: 60
12
13
0.79
1.56
0.76
10.2
25
26
AZT
> 479*
3.12**
9.17***
1.26
> 479
1.46
Elipticine
N CH2
O
17m: 71
*KB-CCL-17 (mouth epidermoid carcinoma)29,30,33
**KB (nasopharynx carcinoma)34
***KB-3-1 (cervix epidermoid carcinoma)35

In the next part of this work, the newly prepared compounds were subjected to in vitro biological
assessment against two human cancer cell lines (KB-CCL-17, HepG2-HB-8065)33 in order to evaluate
their potential as cytotoxic agents, and the results are summarized in Table 1. These results indicated
that most of the derivatives possess at least moderate cytotoxic activity, and some triazole-
indenoisoquinoline conjugates even display a promising activity profile. It is important to note that these
separate pharmacophores display considerably less potent cytotoxic activities (IC50-values ranging from
42 to > 400 µM) as compared to the most promising conjugates 17l, 21 and 24b (IC50-values between
0.4 and 1 µM) showing a reasonable activity against both cancer cells.
Hybrid triazole-indenoisoquinolines 17a,b,l,m,n, 21, 24b,c,d and 26 exhibit potent cytotoxicity
against both cell lines with IC50 < 10 µM, pointing that the introduction of functionalized 1,2,3-triazole
into N-lactam side chain of indenoisoquinoline actually enhances biological activities. On the other
hand, as compared to compounds 17a,e containing 2-hydroxypropane side chain, the synthesized
compounds 18a,b by acylation display low cytotoxicity against both cell lines with IC50-values ranging
from 18 to > 234 µM. It is demontrated that the acylation of hydroxyl and amine groups decreases
cytotoxicity activity of these hybrids.
The triazole-indenoisoquinoline hybrids with AZT and imatinib derivative also possess a high
cytotoxic against both cell lines with IC50 < 1 µM. Moreover, hybrid 24b can be identified as the most
promising compound with IC50-values below 0.5 µM against both cell lines (0.49 and 0.48 µM,
respectively), thus representing a suitable template for further elaboration and optimization toward
potent cytotoxic agents. These biological results clearly indicate the added value of merging AZT and
indenoisoquinoline into single hybrid compounds in terms of their anticancer properties.
In conclusion, 6-(3- azido-2-hydroxypropyl)indenoisoquinoline was converted to various triazole-
indenoisoquinoline hybrids by CuI-catalyzed 1,3-cycloaddition. In addition, a serie of new triazole-
indenoisoquinoline-AZT hybrids was prepared starting from diverse propargyl indenoisoquinolines.
Subsequently, the 23 triazole-indenoisoquinoline hybrids thus prepared were subjected to in vitro
cytotoxic evaluation against two human cancer cell lines, resulting in moderate to high bioactivity
values of 10 representatives, especially those comprising the AZT unit. The potential relevance of this
novel class of indenoisoquinolines, incorporating the promising triazole in lactam 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) for financial support.
References and notes
1. For recent articles, see: (a) Song, Y.; Shao, Z.; Dexheimer, T. S.; Scher, E. S.; Pommier, Y.; Cushman, M. J. Med. Chem.
2010, 53, 1979; (b) Kohlhagen, G.; Paull, K. D.; Cushman, M.; Nagafuji, P.; Pommier, Y. Mol. Pharmacol. 1998, 54, 50; (c)
Ryckebusch, A.; Garcin, D.; Lansiaux, A.; Goossens, J.-F.; Baldeyrou, B.; Houssin, R.; Bailly, C.; Hénichart, J.-P. J. Med.
Chem. 2008, 51, 3617; (d) Kim, S. H.; Oh, S. M.; Song, J. H.; Cho, D.; Le, Q. M.; Lee, S. H.; Cho, W. J.; Kim, T. S. Bioorg.
Med. Chem. 2008, 16, 1125; (e) Van, H. T. M.; Le, Q. M.; Lee, K. Y.; Lee, E.-S.; Kwon, Y.; Kim, T. S.; Le, T. N.; Lee, S.
H.; Cho, W. J. Bioorg. Med. Chem. Lett. 2007, 17, 5763; (f) Morrell, A.; Placzek, M. S.; Parmley, S.; Grella, B.; Antony, S.;
Pommier, Y.; Cushman, M. J. Med. Chem. 2007, 50, 4388; (g) Morrell, A.; Placzek, M. S.; Steffen, J. D.; Antony, S.;
Agama, K. K.; Pommier, Y.; Cushman, M. J. Med. Chem. 2007, 50, 2040; (h) Cho, W.-J.; Le, Q. M.; Hue, T. M. V.; Lee, K.

Y.; Kang, B. Y.; Lee, E. S.; Lee, S. K.; Kwon, Y. Bioorg. Med. Chem. Lett. 2007, 17, 3531; (i) Pommier, Y.; Cushman, M.
Mol. Cancer Ther. 2009, 8, 1008; (j) Cinelli, M. A.; Morrell, A.; Dexheimer, T. S.; Scher, E. S.; Pommier, Y.; Cushman, M.
J. Med. Chem. 2008, 51, 4609; (k) Pommier, Y. Nat. Rev. Cancer 2006, 6, 789.
2. Mark, C.; Cheng, L. J. Org. Chem. 1978, 43, 3781.
3. (a) Khadka, D. B.; Cho, W.-J. Expert Opin. Ther. Pat. 2013, 23, 1033; (b) Staker, B. L.; Feese, M. D.; Cushman, M.;
Pommier, Y.; Zembower, D.; Stewart, L.; Burgin, A. B. J. Med. Chem. 2005, 48, 2336; (c) Pommier, Y.; Marchand, C. Nat.
Rev. Drug Disc. 2012, 11, 25; (d) Pommier, Y.; Cushman, M. Mol. Cancer Ther. 2009, 8, 1008.
4. (a) Morrell, A.; Antony, S.; Kohlhagen, G.; Pommier, Y.; Cushman, M. J. Med. Chem. 2006, 49, 7740; (b) Nagarajan, M.;
Morrell, A.; Ioanoviciu, A.; Antony, S.; Kohlhagen, G.; Agama, K. K.; Hollingshead, M.; Pommier, Y.; Cushman, M. J.
Med. Chem. 2006, 49, 6283; (c) Nagarajan, M.; Morrell, A.; Antony, S.; Kohlhagen, G.; Agama, K. K.; Pommier, Y.;
Ragazzon, P. A.; Garbett, N. C.; Chaires, J. B.; Hollingshead, M.; Cushman, M. J. Med. Chem. 2006, 49, 5129; (d) Morrell,
A.; Placzek, M. S.; Parmley, S.; Antony, S.; Dexheimer, T. S.; Pommier, Y.; Cushman, M. J. Med. Chem. 2007, 50, 4419.
5. (a) Morrell, A.; Antony, S.; Kohlhagen, G.; Pommier, Y.; Cushman, M. Bioorg. Med. Chem. Lett. 2006, 16, 1846; (b)
Xiao, X.; Antony, S.; Kohlhagen, G.; Pommier, Y.; Cushman, M. Bioorg. Med. Chem. 2004, 12, 5147; (c) Nagarajan, M.;
Xiao, X.; Antony, S.; Kohlhagen, G.; Pommier, Y.; Cushman, M. J. Med. Chem. 2003, 46, 5712; (d) Nagarajan, M.; Morrell,
A.; Fort, B. C.; Meckley, M. R.; Antony, S.; Kohlhagen, G.; Pommier, Y.; Cushman, M. J. Med. Chem. 2004, 47, 5651.
6. (a) Xiao, X.; Antony, S.; Pommier, Y.; Cushman, M. J. Med. Chem. 2005, 48, 3231; (b) Morrell, A.; Jayaraman, M.;
Nagarajan, M.; Fox, B. M.; Meckley, M. R.; Ioanoviciu, A.; Pommier, Y.; Antony, S.; Hollingshead, M.; Cushman, M.
Bioorg. Med. Chem. Lett. 2006, 16, 4395; (c) Nagarajan, M.; Morrell, A.; Ioanoviciu, A.; Antony, S.; Kohlhagen, G.;
Agama, K.; Hollingshead, M.; Pommier, Y.; Cushman, M. J. Med. Chem. 2006, 49, 6283; (d) Morrell, A.; Antony, S.;
Kohlhagen, G.; Pommier, Y.; Cushman, M. J. Med. Chem. 2006, 49, 7740; (e) Morrell, A.; Placzek, M.; Parmley, S.;
Antony, S.; Dexheimer, T. S.; Pommier, Y.; Cushman, M. J. Med. Chem. 2007, 50, 4419; (f) Morrell, A.; Placzek, M. S.;
Steffen, J. D.; Antony, S.; Agama, K.; Pommier, Y.; Cushman, M. J. Med. Chem. 2007, 50, 2040.
7. Pham Thi, T.; Decuyper, L.; Luc Quang, T.; Pham The, C.; Dang Thi, T. A.; Nguyen, H. T.; Le Nhat, T. G.; Nguyen
Thanh, T.; Hoang Thi, P.; D’hooghe, M.; Van Nguyen, T. Tetrahedron Lett. 2016, 57, 466.
8. Zhang, X.; Wang, R.; Zhao, L.; Lu, N.; Wang, J.; You, Q.; Li, Z.; Guo, Q. Bioorg. Med. Chem. Lett. 2012, 22, 1276.
9. Cinelli, A. A.; Reddy, P. V. N.; Lv, P-C.; Liang, J-H.; Chen, L.; Agama, K.; Pommier, Y.; Breemen, R. B.; Cushman, M.
J. Med. Chem. 2012, 55, 10844.
10. Beck, D. E.; Agama, K.; Marchand, C.; Chergui, A.; Pommier, Y.; Cushman, M. J. Med. Chem. 2014, 57, 1495.
11. Nguyen, X. T.; Abdelmalak, M.; Marchand, C.; Agama, K.; Pommier, Y.; Cushman, M. J. Med. Chem. 2015, 58, 3188.
12. Conda-Sheridan, M.; Narasimha Reddy, P. V. N.; Morrell, A.; Cobb, B. T.;Marchand, C.; Agama, K.; Chergui, A.;
Renaud, A.; Stephen, A. G.; Bindu, L. K.; Pommier, Y.; Cushman, M. J. Med. Chem. 2013, 56, 182.
13. Lv, P.-C.; Agama, K.; Marchand, C.; Pommier, Y.; Cushman, M. J. Med. Chem. 2014, 57, 4324-4336.
14. 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.
15. Antony, S.; Agama, K. K.; Miao, Z. H.; Takagi, K.; Wright, M. H.; Robles, A. I.; Varticovski, L.; Nagarajan, M.;
Morrell, A.; Cushman, M.; Pommier, Y. Cancer Res. 2007, 67, 10397−10405.
16. Chrencik, J. E.; Staker, B. L.; Burgin, A. B.; Pourquier, P.;Pommier, Y.; Stewart, L.; Redinbo, M. R. J. Mol. Biol. 2004,
339, 773-784.
17. Whiting M.; Muldoon J., Lin, Y. C.; Silverman, S. M.; Lindstron, W.; Olson, A. J.; Kolb, H. C.; Finn, M. G.; Sharpless,
K. B.; Elder, J. H.; et al. Angrew. Chem. Int. Ed. 2006, 45, 1435-1439.
18. Tornoe, C. W.; Sanderson, S. J.; Mottram, J. C.; Coombs, G. H.; Meldal, M. J. Comb. Chem. 2004, 6, 312-324.
19. Wang, S.; Wang, Q.; Wang, Y.; Liu, L.; Weng, X.; Li, G.; Zhang, X.; Zhou, X. Bioorg. Med. Chem. Lett. 2008, 18, 6505.
20. For recent articles, see: (a) Chen, Y.; Lopez-Sanchez, M.; Savoy, D. N.; Billadeau, D. D.; Dow, G. S.; Kozikowski, A. P.
J. Med. Chem. 2008, 51, 3437; (b) He, R.; Chen, Y.; Chen, Y.; Ougolkov, A. V.; Zhang, J-S.; Savoy, D. N.; Billadeau, D. D.;
Kozikowski, A. P. J. Med. Chem. 2010, 53, 1347; (c) Stefely, J. A.; Palchaudhuri, R.; Miller, P. A.; Peterson, R. J.; Moraski,
G.C.; Hergenrother, P. J.; Miller, M. J. J. Med. Chem. 2010, 53, 3389; (d) Sun, H.; Liu, L.; Lu, J.; Bai, L.; Li, X.;
Nikolovska-Coleska, Z.; McEachern, D.; Yang, C-Y.; Qiu, S.; Yi, H.; Sun, D.; Shaomeng Wang, S. J. Med. Chem. 2011, 54,
3306; (e) Suzuki, T.; Ta, Y.; Ri, M.; Bando, M.; Gotoh, A.; Itoh, Y.; Tsumoto, H.; Tatum, P. R.; Mizukami, T.; Nakagawa,
H.; Iida, S.; Ueda, R.; Shirahige, K.; Miyata, N. J. Med. Chem. 2012, 55, 9562; (f) Kang, X.; Hu, J.; Gao, Z.; Ju, Y.; Xu, C.
Med. Chem. Commun. 2012, 3, 1245; (g) Majeed, R.; Sangwan, P. L.; Chinthakindi, P. K.; Khan, I.; Dangroo, N. A.; Thota,
N.; Hamid, A.; Sharma, P. R.; Saxena, A. K.; Koul, S. Eur. J. Med. Chem. 2013, 63, 782; (h) Pertino, M. W.; Lopez, C.;

Theoduloz, C.; Schmeda-Hirschmann, G. Molecules 2013, 18, 7661; (i) Antimonova, A. N.; Petrenko, N. I.; Shakirov, M.
M.; Rybalova, T. V.; Frolova, T. S.; Shul’ts, E. E.; Kukina, T. P.; Sinitsyna, O. I.; Tolstikov, G. A. Chem. Nat. Compounds
2013, 49, 657; (j) Rashid, S.; Ahmad Dar, B.; Majeed, R.; Hamid, A.; Ahmad Bhat, B. Eur. J. Med. Chem. 2013, 66, 238; (k)
Thomopoulou, P.; Sachs, J.; Teusch, N.; Mariappan, A.; Gopalakrishnan, J.; Schmalz, H-G. ACS Med. Chem. Lett. 2016, 7,
188.
21. For recent articles, see: (a) Buckle, D. R.; Outred, D. J.; Rockell, C. J. M.; Smith, H.; Spicer, B. A. J. Med. Chem. 1983,
26, 251; (b) Buckle, D. R.; Rockell, C. J. M.; Smith, H.; Spicer, B. A. J. Med. Chem. 1986, 29, 2262; (c) Sanghvi, Y. S.;
Bhattacharya, B. K.; Kini, G. D.; Matsumoto, S. S.; Larson, S. B.; Jolley, W. B.; Robins, R. J.; Revankar, G. R. J. Med.
Chem. 1990, 33, 336; (d) Alvarez, R.; Velazquez, S.; San-Felix, A.; Aquaro, S.; De Clercq, E.; Perno, C.-F.; Karlsson, A.;
Balzarini, J.; Carmarasa, M. J. J. Med. Chem. 1994, 37, 4185; (e) Genin, M. J.; Allwine, D. A.; Anderson, D. J.; Barbachyn,
M. R.; Emmert, D. E.; Garmon, S. A.; Graber, D. R.; Grega, K. C.; Hester, J. B.; Hutchinson, D. K.; Morris, J.; Reischer, R.
J.; Ford, C. W.; Zurenko, G. E.; Hamel, J. C.; Schaadt, R. D.; Stapert, D.; Yagi, B. H. J. Med. Chem. 2000, 43, 953; (f) Aher,
G.; Pore, V. S.; Mishra, N. N.; Kumar, A.; Shukla, P. K.; Sharma, A.; Bhat, M. K. Bioorg. Med. Chem. Lett. 2009, 19, 759;
(g) Nordstrøm, L. U.; Sironi, J.; Aranda, E.; Maisonet, J.; Perez-Soler, R.; Wu, P.; Schwartz, E. L. ACS Med. Chem. Lett.
2015, 6, 134.
22. Bennet, I. S.; Brooks, G.; Broom, N. J. P.; Calvert, S. H.; Coleman, K.; Francois, I. J. Antibiot. 1991, 44, 969.
23. Vatmurge, N. S.; Hazra, B. G.; Pore, V. S.; Shirazi, F.; Chavan, P. S.; Deshpande, M. V. Bioorg. Med. Chem. Lett. 2008,
18, 2043.
24. Soltis, M. J.; Yeh, H. J.; Cole, K. A.; Whittaker, N.; Wersto, R. P.; Kohn, E. C. Drug Metab. Dispos. 1996, 24, 799.
25. (a) Huisgen, R. Padwa, A., Ed.; John Wiley & Sons: New York, 1984, 1, 1; (b) Gothelf, K. V.; Jørgensen, K. A. Chem.
Rev. 1998, 98, 863.
26. Hein, J. E.; Fokin, V. V. Chem. Soc. Rev. 2010, 39, 1302.
27. 6-(2-hyroxy-3-(4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)propyl)-5H-indeno[1,2-c]isoquinoline-5,11(6H)-dione (17a):
Propargyl alcohol (1.1 equiv) was added to a solution of 6-(3-azido-2-hydroxypropyl)-5H-indeno[1,2-c]isoquinoline-
5,11(6H)-dione (16) (1.0 equiv), DIPEA (0.2 equiv) and CuI (0.1 equiv) in THF (20 mL). The solution was stired and heated
to reflux for 24 h. Afterwards, the tetrahydrofuran was removed under reduced pressure and the residue was extracted with
ethyl acetate. The combined organic phased was washed with brine, dried over MgSO4 and concentrated in vacuum to give
crude products 17a. The target hybrids 17a were obtained by column chromatography purification on silica gel using n-
hexane-ethyl acetate (8 : 2, v/v) as an eluent. Yield: 70%. M.p. 240oC. IR (KBr) cm-1: 3420, 3221, 2872, 1660, 1601, 1580,
1502, 1430, 1415.9, 1306, 1264, 1056, 971, 838, 756. 1H-NMR (DMSO-d6, 500 MHz): 8.56 (1H, d, J = 9.0 Hz), 8.2 (1H, d,
J = 8.0 Hz), 7.99 (1H, s, H-triazol), 7.79-7.82 (1H, m), 7.50-7.73 (2H, m), 7.41-7.44 (3H, m), 5.75(1H, bs, OH), 5.25 (1H,
bs, OH), 4.59 -4.69 (3H, m), 4.55 (2H, s, CH2OH), 4.40-4.44 (2H, m). 13C-NMR (DMSO-d6, 125 MHz): 190.1, 162.8, 157.4,
147.7, 137.1, 134.3, 134.0, 133.4, 131.9, 130.9, 128.0, 127.0, 124.7, 124.1, 122.8, 122.5, 122.2, 107.1, 67.0, 55.0, 53.0, 47.9.
28. (a) Andreuccetti, M.; Allegrini, G.; Antonuzzo, A.; Malvaldi, G.; Conte, P. F.; Danesi, R.; Del Tacca, M.; Falcone, A.
Eur. J. Cancer 1996, 32A, 1219; (b) Johnston, J. S.; Johnson, A.; Gan, Y.; Wientjes, M. G.; Au, J. L. Pharm. Res. 2003, 20,
957; (c) Chen, C.; Zhang, Y.; Wang, Y.; Huang, D.; Xi, Y.; Qi, Y. Anticancer Drugs. 2011, 22, 435; (d) Mattson, D. M.;
Ahmad, I. M.; Dayal, D.; Parsons, A. D.; Aykin-Burns, N.; Li, L.; Orcutt, K. P.; Spitz, D. R.; Dornfeld, K. J.; Simons, A. L.
Free Radical Biol. Med. 2009, 46, 232; (e) Multani, A. S.; Furlong, C.; Pathak, S. Int. J. Oncol. 1998, 13, 923; (f) Chen, P.;
Cai, X.; Cao, F.; Li, F.; Lei, J.; Deng, S. Shandong Yiyao. 2012, 52, 69; (g) Mattson, D. M.; Ahmad, I. M.; Dayal, D.;
Parsons, A. D.; Aykin-Burns, N.; Li, L.; Orcutt, K. P.; Spitz, D. R.; Dornfeld, K. J.; Simons, A. L. Free Radical Biol. Med.
2009, 46, 232; (h) Scanlon, K. J.; Funato, T.; Pezeshki, B.; Tone, T.; Sowers, L. C. Cancer Commun. 1990, 2, 339; (i) Klann,
R. C.; Holbrook, C. T.; Nyce, J. W. Anticancer Res. 1992, 12, 781; (j) Morgan, R. J., Jr.; Newman, E. M.; Sowers, L.;
Scanlon, K.; Harrison, J.; Akman, S.; Leong, L.; Margolin, K.; Niland, J.; Raschko, J., et al. Cancer Chemother. Pharmacol.
2003, 51, 459.
29. Dang Thi, T. A.; Kim Tuyet, N. T.; Pham The, C.; Nguyen, H. T.; Ba Thi, C.; Doan Duy, T.; D’hooghe, M.; Van
Nguyen, T. Bioorg. Med. Chem. Lett. 2014, 24, 5190.
30. Dang Thi, T. A. ; Kim Tuyet, N. T.; Pham The, C.; Nguyen, H. T.; Ba Thi, C.; Phuong, H. T.; Boi, L. T.; Van Nguyen,
T.; D’hooghe, M. Tetrahedron Lett. 2015, 56, 218.
31. (1-((2S,3S,5R)-5-(hydroxymethyl)-2-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-3-yl)-1H-1,2,3-
triazol-4-yl)methyl 2-(5,11-dioxo-5,11-dihydro-6H-indeno[1,2-c]isoquinolin-6-yl)-3-phenylpropanoate (24b):
Prop-2-yn-1-yl 2-(5,11-dioxo-5,11-dihydro-6H-indeno[1,2-c]isoquinolin-6-yl)-3-phenylpropanoate (23b) (1.0 equiv) was

added to a solution of AZT (0.8 equiv), DIPEA (0.2 equiv) and CuI (0.1 equiv) in THF (20 mL). The solution was stired and
heated to reflux for 24 h. Afterwards, the tetrahydrofuran was removed under reduced pressure and the residue was extracted
with ethyl acetate. The combined organic phased was washed with brine, dried over MgSO4 and concentrated in vacuum to
give crude products 24b. The target hybrids 24b were obtained by column chromatography purification on silica gel using n-
hexane-ethyl acetate (8 : 2, v/v) as an eluent. Yield: 85%. M.p. 182oC. IR (KBr) cm-1: 3670, 3211, 2988, 2850, 1752, 1691,
1
1648, 1608, 1572, 1499, 1413, 1270, 1065, 759. H-NMR (DMSO-d6, 500 MHz): 11.35 (1H, s, NH), 8.52 (1H, d, J = 6.5
Hz), 8.29 (1H, s, H-triazol), 8.2(1H, d, J = 6.5Hz), 7.81 (3H, s), 7.57 (1H, s), 7.40 (1H, s), 7.36 (2H, s), 6.92 (4H, s), 6.40
(1H, s), 5.93 (1H, d, J = 5.5 Hz, OH), 5.29-5.36 (4H, m), 4.17 (1H, s), 3.51-3.67 (4H, m), 2.66 (2H, s), 1.81 (3H, s). 13C-
NMR (DMSO-d6, 125 MHz): 189.7, 168.0, 163.7, 162.4, 156.1,150.4, 141.7, 136.2, 136.0, 135.8, 134.6, 133.5, 133.3, 131.5,
131.1, 129.4 (2C), 128.0 (2C), 127.9, 127.6, 126.7, 124.6, 123.7, 122.7, 122.4, 109.6, 107.4, 84.4, 83.9, 60.6, 60.4, 59.2,
58.2, 37.1, 33.6, 12.2.
32. (a) Castaner, J. et al. Drugs Fur. 2001, 6, 1765; (b) Lyseng-Wilhamson, K.; Jarvis, B. Drugs. 2001, 61, 303; (c)
Zimmermann, J.; Buchdunger, E.; Mett, H.; Meyer, T.; Lydon, N. B. Bioorg. Med. Chem. Lett. 1997, 7, 187; (d)
Zimmermann, J. et al. Med. Res. Rev. 2001, 21, 499; (e) Druker, B. J.; Moshe, T.; Debra, J. R.; Bin, P.; Buchdunger, E.;
Ford, J. M.; Lydon, N. B.; Kantarjian, H.; Capdeville, R.; Ohno-Jones, S.; Sawyers, C. L. N. Engl. J. Med. 2001, 344, 1031;
(f) Gao, H.; Lee, B-N.; Talpaz, M.; Donato, N. J.; Cortes, J. E.; Katarjian, H. M.; Reuben, J. M. Leukemia. 2005, 19, 1905.
33. Cell culture and cell viability assay: Two human cancer cell lines epidermoid carcinoma cell line ((KB)-ATCC®CCL-
17™) and hepatoma carcinoma cell line ((HepG2)-ATCC®HB-8065TM), 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. 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-
response curve was generated, and the inhibitory concentration of 50% (IC50) was determined for each compound as well as
each cell line.
34. Celewicz, L.; Jóźwiak, A.; Ruszkowski, P.; Laskowska, H.; Olejnik, A.; Czarnecka, A.; Hoffmann, M.; Hładoń, B.
Bioorg. Med. Chem. 2011, 19, 6375.
35. Wang, J.; Wang, Y.-J.; Chen, Z.-S.; Kwon, C.-H. Bioorg. Med. Chem. 2014, 22, 5747.

GRAPHICAL ABSTRACT
SYNTHESIS AND CYTOTOXIC EVALUATION OF NOVEL INDENOISOQUINOLINE-SUBSTITUTED TRIAZOLE HYBRIDS
O
H3C
NH
O
O
HO
R'
O
N
O
O
N
OH
OH
N
N
R
N
N3
N
O
O
N
N
O
O
O
N
N
R = CH2OH
IC50 (KB) = 2.86 M
IC50 (HepG2) = 0.74 M
O
R' = CH2
IC50 (KB) = 0.79 M
IC50 (HepG2) = 0.64 M
R = CH2Br
IC50 (KB) = 0.79 M
IC50 (HepG2) = 0.76 M
R' = CH(CH2Ph)C(O)OCH2
IC50 (KB) = 0.49 M
IC50 (HepG2) = 0.48 M