Synthesis and cytotoxic evaluation of novel amide-triazole-linked
triterpenoid-AZT conjugates
Tuyet Anh Dang Thi,a Nguyen Thi Kim Tuyet,a Chinh Pham The,a Ha Thanh Nguyen,b Cham Ba Thi,a
Hoang Thi Phuong,a Luu Van Boi,b Tuyen Van Nguyen,a, Matthias D’hooghec,*
a
Institute of Chemistry, Vietnam Academy of Science and Technology, 18-Hoang Quoc Viet, CauGiay, Hanoi,
Vietnam
b
Hanoi University of Science-Vietnam National University
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
A variety of triterpenoid propargyl amides, prepared from the corresponding acids, was used as substrates for
the construction of novel triazole-tethered triterpenoid-AZT conjugates via a click chemistry-mediated coupling
with azidothymidine (AZT). Thus, nineteen new hybrids were successfully prepared and evaluated as cytotoxic
agents, revealing an interesting anticancer activity of four triterpenoid-AZT hybrids on KB and Hep-G2 tumor
cell lines.
Keywords: Triterpenoids; AZT; Hybrids; Cytotoxic agents
Molecular hybridization of biologically active compounds has attracted considerable interest in
recent years because of the medicinal relevance of the resulting conjugates, which have been shown
to be useful building blocks in several drug discovery programs.1 Also in the field of cancer research,
pharmacophore hybridization has been explored successfully to construct new compounds with
anticancer properties. Within that concept, we recently examined the synthetic feasibility of
connecting triterpenoid acids (betulinic acid, oleanolic acid, ursolic acid and their derivatives) with
azidothymidine (AZT), affording hybrid structures A (Figure 1) via click chemistry-assisted coupling of
the corresponding triterpenoid propargyl esters and the azido group of AZT.2 Because of the
pronounced and diverse anticancer activities of triterpenoids3 and AZT4 as separate entities, and
given the fact that the conjunction of triterpenoids and azidothymidine (AZT) into hybrid molecules
has only been studied to a limited extent in the literature,5,6 the hypothesis of preparing triterpenoidAZT conjugates as novel cytotoxic agents was proposed and investigated. This study indeed revealed
a promising potential of triterpenoid-AZT hybrids A, showing a good cytotoxicity against KB and HepG2 cancer cell lines.2
In the present work, further structure-activity relationship studies were undertaken with regard to
these triazole-tethered triterpenoid-AZT hybrid compounds in order to assess the importance of the
linker moiety on the biological activity. In particular, the replacement of the ester unit in conjugates
A by an amido group in structures B (Fig. 1) was proposed to study the effect of oxygen with nitrogen
exchange, and thus the aim of this work was to develop a convenient synthetic route toward these

ngvtuyen@hotmail.com, tel: +84917683979; matthias.dhooghe@UGent.be, tel: +3292649394
1
new hybrids B and to evaluate their cytotoxicity profile. In addition, the synthetic scope was further
expanded by decoration (in casu acylation) at C3 of the pentacyclic backbone to provide a more
diverse library of compounds.
Figure 1. Ester-triazole- (A) and amide-triazole- (B) linked triterpenoid-AZT conjugates
The design of hybrid molecules requires a facile and straightforward synthetic method to connect the
parent entities. A powerful approach for the fusion of two chemical units into one hybrid molecule
comprises the Cu(I)-catalyzed azide alkyne Huisgen cycloaddition, which has gained major interest
due to its high efficiency and selectivity.7 In addition to the expedient “click” procedure for triazole
synthesis, the introduction of a triazole linker in hybrid compounds can also contribute to the overall
activity because of the known biological properties of functionalized triazoles as such.8
In analogy with the previously performed efficient synthesis of ester-triazole-tethered triterpenoidAZT hybrids employing triterpenoid propargyl esters,2 the application of a similar approach using
triterpenoid propargyl amides as coupling partners was evaluated in this work. To that end, betulinic
acid 1 was first converted into its propargyl amide 2a upon treatment with 1.5 equiv of propargyl
amine in DMF at room temperature for 12h in the presence of 1.5 equiv of DCC (N,N'dicyclohexylcarbodiimide), 1.5 equiv of HOBt (1-hydroxybenzotriazole) and 2.0 equiv of DIPEA (N,Ndiisopropylethylamine) (Scheme 1). Subsequent click reaction of the resulting alkyne 2a with 0.8
equiv of AZT in the presence of CuI (0.2 equiv) in t-BuOH at 70oC for 12h afforded triazole-tethered
AZT-betulinic acid conjugate 3a in 20% yield. Alternatively, transformation of betulinic acid 1 into its
propargyl amide was followed by esterification of the free C3 hydroxyl group utilizing 4.0 equiv of
succinic anhydride or glutaric anhydride in pyridine at 120oC for 12h to give amido esters 2b-c, which
were converted into new hybrids 3b-c in acceptable yields by means of CuI-promoted click chemistry
with AZT (Scheme 1).
2
Scheme 1. Synthesis of betulinic acid-derived triterpenoid-AZT hybrids 3a-c
In order to further validate this synthetic strategy and to generate a small library of novel
triterpenoid-AZT conjugates, the same procedure as explained above was applied to triterpenoid
analogs of betulinic acid 1. First, ursolic acid 4 was deployed as a parent entity, and conversion of this
compound to the corresponding propargyl amide 5 followed by CuI-mediated click reaction with AZT
afforded hybrid product 6 in 64% yield (Scheme 2). Also triterpenoid acids 7, prepared from their
dicarboxylic acid precursors9 via initial Jones oxidation (CrO3) and subsequent decarboxylation of the
resulting -oxo acid intermediates, were transformed into N-propynyl amides 8 upon reaction with
propargyl amine in DMF in the presence of DCC, HOBt and DIPEA. The latter alkynes 8 were then
subjected to a Cu(I)-mediated click reaction protocol with AZT to result in the novel AZT conjugates
9a,b in good yields (Scheme 2).
3
Scheme 2. Synthesis of triterpenoid-AZT conjugates 6 and 9a,b
Reagents and conditions: (a) 1.5 equiv propargyl amine, 1.5 equiv DCC, 1.5 equiv HOBt, 2.0 equiv DIPEA, rt,
DMF, 12h; (b) 0.8 equiv AZT, 0.2 equiv. CuI, t-BuOH, 70°C, 12h
Analogously, amide 11 was prepared from hydroxy acid 1010 and esterified at the C3 hydroxyl group
with succinic anhydride, glutaric anhydride or methyl glutaric anhydride in pyridine to give
compounds 12a-c in good yields. Subsequent Huisgen cycloaddition between alkynes 12 and AZT
produced the desired new hybrids 13a-c in 50-60% yield. Amide 11 was also deployed directly as a
substrate for a click reaction with AZT, affording conjugate 13d in 48% yield (Scheme 3).
4
Scheme 3. Synthesis of triterpenoid-AZT hybrids 13a-d
Reagents and conditions: (a) 1.5 equiv propargyl amine, 1.5 equiv DCC, 1.5 equiv HOBt, 2.0 equiv DIPEA, rt,
DMF, 12h; (b) 0.8 equiv AZT, 0.2 equiv. CuI, t-BuOH, 70oC, 12h; (c) 4.0 equiv anhydride, pyridine, DMAP, 120oC,
12h
Further structural diversity was introduced by using triterpenoid acid 14, isolated from Acanthopanax
trifoliatus,11 as an eligible substrate for molecular hybridization. This acid 14 was converted to amide
15 upon treatment with propargyl amine in DMF, followed by conjugation with AZT by means of the
above-described click chemistry methodology. This approach resulted in novel triterpenoid-AZT
hybrid 16 in 67% yield (Scheme 4). Also compound 17, prepared from betulin,12 was converted to
amide 18 and treated with AZT to afford conjugate 19 in 67% yield (Scheme 4).
Scheme 4. Synthesis of triterpenoid-AZT conjugates 16 and 19
Reagents and conditions: (a) 1.5 equiv propargyl amine, 1.5 equiv DCC, 1.5 equiv HOBt, 2.0 equiv DIPEA, rt,
DMF, 12h; (b) 0.8 equiv AZT, 0.2 equiv. CuI, t-BuOH, 70oC, 12h.
In another approach toward functionalization at the periphery of these scaffolds, inspired by the
chemical structure of the triterpenoid-based drug bevirimat, different chloroformyl esters (2 equiv)
were used for the acylation of betulinic acid 1 at the C3 hydroxyl group in CH2Cl2 in presence of a
catalytic amount of DMAP to give diesters 20a-c after 24h. The latter triterpenoid acids 20 were
further converted to the corresponding amides 21 by using propargyl amine in DMF at room
temperature for 12h in the presence of 1.5 equiv of DCC, 1.5 equiv of HOBt, and 2.0 of equiv DIPEA.
Cycloaddition between alkynes 20 and AZT then afforded the desired new hybrids 22a-c in
acceptable yields (Scheme 5).
5
Scheme 5. Synthesis of triterpenoid-AZT conjugates 22a-c
Finally, the same strategy was applied to triterpenoid 10, implying the synthesis of amides 24a-b via
diesters 23a-b, followed by click chemistry-assisted conjugation with AZT. This approach resulted in
novel triterpenoid-AZT hybrids 25a-b (esters) and 26a-b (acids) in moderate yields (Scheme 6).
Scheme 6. Synthesis of triterpenoid-AZT conjugates 25a,b and 26a,b
In summary, the construction of 19 novel amide-triazole-tethered triterpenoid-AZT conjugates was
performed successfully. The structural identity of all new products was confirmed by detailed 1H
NMR, 13C NMR, IR and MS analysis.
6
In the next part, these triterpenoid compounds were subjected to in vitro biological assessment
against two human tumor cell lines (KB, Hep-G2) in order to evaluate their potential as cytotoxic
agents, and the results are summarized in Table 1. These results indicate that most of the derivatives
possess at least moderate cytotoxic activity, and some AZT conjugates even display a promising
activity profile. In particular, four hybrid AZT-triazole triterpenoids (19, 22b, 22c, 25b) show good
cytotoxicity against KB and three triterpenoid-AZT hybrids (19, 22b, 22c) exhibit considerable
cytotoxicity against Hep-G2 cancer cell lines with IC50-values < 10 µM, pointing to the potential
interest in this new class of hybrid molecules. Hybrid 22c can be identified as the most promising
compound with IC50-values below 5 µM against both cell lines (4.6 and 3.5 µM, respectively), only
slightly higher than those of the reference compound ellipticine, thus representing a suitable
template for further elaboration and optimization toward potent cytotoxic agents.
Table 1. Cytotoxicity evaluation of triterpenoid compounds
Entry
Compound
IC50 (µM) KB
IC50
(µM)
Hep-G2
Entry
Compound
IC50 (µM) KB
IC50
(µM)
Hep-G2
1
1
27.5
23.9
15
13d
>171
>171
2
3a
126.1
92.5
16
14
46.8
58.0
3
3b
92.0
80.8
17
16
>168
>168
4
3c
68.4
65.0
18
17
>249
39.3
5
4
31.5
42.9
19
19
8.1
5.9
6
6
25.1
23.8
20
22a
25.6
26.9
7
7a
7.7
11.2
21
22b
8.1
6.6
8
7b
51.4
70.5
22
22c
4.6
3.5
9
9a
32.5
31.4
23
25a
>151
79.0
10
9b
>168
>168
24
25b
8.3
>143
11
10
180.5
274.8
25
26a
>153
30.6
12
13a
34.5
51.4
26
26b
>146
18.7
13
13b
23.6
27.9
27
AZT
>479
>479
14
13c
20.5
29.3
28
Ellipticine
1.2
1.2
When the newly prepared amide-containing hybrids B are compared with their ester-linked
counterparts A2, it appears that replacement of oxygen by nitrogen in the most potent esters A gives
rise to a drop in cytotoxic activity (Table 2). As can be seen, the nitrogen analogs (hybrids 3a, 13a,
13d and 16) display less potent cytotoxic effects as compared to the corresponding oxygen
counterparts (with IC50-values < 10 µM). This observation might be attributable to the fact that esters
are more readily hydrolyzed biochemically as compared to amides, meaning that the cytotoxic
effects are (partly) caused by the hydrolysis products of the initial conjugates.
7
Table 2. Comparison of the most potent ester conjugates A2 with their nitrogen analogs B in terms of
their cytotoxic activity
Compound A
IC50 (µM)
KB
IC50 (µM)
Hep-G2
5.9
Compound B
IC50 (µM)
KB
IC50 (µM)
Hep-G2
7.0
126.1
92.5
7.3
7.8
34.5
51.4
34
7.3
>171
>171
136.5
8.5
>168
>168
On the other hand, it seems that transformation of the C3 hydroxyl group into an ester moiety is
beneficial for the biological activity of the amide-containing conjugates B, as exemplified by Oacylated hybrids 19, 22b, 22c and 25b. Thus, from a structure-activity relationship point of view, it
might be of interest for prospective research to use the parent triterpenoid acids, acylated at the C3OH with methyl 3-chloro-3-oxopropanoate or methyl 4-chloro-4-oxobutanoate, for hybridization
with AZT by means of an ester-triazole linker and to evaluate their cytotoxicity profile.
Finally, it is also important to note that the parent pharmacophores display considerably less potent
cytotoxic activities as compared to the most promising conjugates 19, 22b and 22c (IC50-values
between 3.5 and 8.1 µM), with triterpenoid acid 7a as the only exception showing a reasonable
activity against both cancer cells. These biological results clearly indicate the added value of merging
AZT and triterpenoids into single hybrid compounds in terms of their cytotoxic properties.
In conclusion, different triterpenoid acids were transformed into their propargyl amides and
subsequently used as substrates for a CuI-catalyzed 1,3-cycloaddition with AZT to produce a set of
novel amide-triazole-tethered triterpenoid-AZT conjugates as counterparts for previously evaluated
ester-linked analogs. Cytotoxic analysis of these hybrids and their triterpenoid precursors revealed
moderate to good cytotoxic activities against two human tumor cell lines (KB, Hep-G2), and four
8
representatives of the newly prepared class of triterpenoid-AZT conjugates displayed a promising
potential for further elaboration toward novel anticancer agents. Amide-triazole-linked hybrids
seemed to be less potent then the corresponding ester-triazole-linked analogs, but O-acylation at the
C3-OH appeared to result in good cytotoxic activities of the amide-linked conjugates, affording an
eligible hit molecule for further investigation. The parent pharmacophores also showed considerably
less potent cytotoxic activities as compared to the most promising conjugates, pointing to the added
value of merging AZT and triterpenoids into single hybrid compounds in terms of their antiproliferative activity.
Acknowledgments
The authors are indebted to the Vietnamese National Foundation for Science and Technology
Development (NAFOSTED, code: 104-01-2012-02) and to Ghent University – Belgium (BOF) for
financial support.
References
1
(a) Decker, M. Curr. Med. Chem. 2011, 18, 1464-1475. (b) Tsogoeva, S. B. Mini-Rev. Med. Chem. 2010, 10, 773793. (c) Muregi, F. W.; Ishih, A. Drug Dev. Res. 2010, 71, 20-32. (d) Meunier, B. Acc. Chem. Res. 2008, 41, 69-77.
(e) Vandekerckhove, S.; D’hooghe, M. Bioorg. Med. Chem. 2013, 21, 3643-3647.
2
Dang Thi, T. A.; Kim Tuyet, N. T.; Pham The, C.; Thanh Nguyen, H.; Ba Thi, C.; Doan Duy, T.; D’hooghe, M.; Van
Nguyen, T. Bioorg. Med. Chem. Lett. 2014, 24, 5190-5194.
3
(a) Santos, R. C.; Salvador, J. A.; Marín, S.; Cascante, M. Bioorg. Med. Chem. 2009, 17, 6241-6250. (b) Santos,
R. C.; Salvador, J. A.; Marín, S.; Cascante, M.; Moreira; J. N.; Dinis, T. C. Bioorg. Med. Chem. 2010, 15, 43854396. (c) Kommera, H.; Kaluderović, G. N.; Kalbitz, J.; Paschke, R. Arch. Pharm. 2010, 343, 449-457. (d) Dang, Z.;
Ho, P.; Zhu, L.; Qian, K.; Lee, K.-H.; Huang, L.; Chen, C.-H. J. Med. Chem. 2013, 56, 2029-2037. (e) Kommera, H.;
Kaluderović, G. N.; Dittrich, S.; Kalbitz, J.; Dräger, B.; Mueller, T.; Paschke, R. Bioorg. Med. Chem. Lett. 2010, 20,
3409-3412. (f) Kommera, H.; Kaluđerović, G. N.; Kalbitz, J.; Paschke, R. Invest. New Drugs 2011, 29, 266-272. (g)
Mukherjee, R.; Kumar, V.; Srivastava, S. K.; Agarwal, S. K.; Burman, A. C. Med. Chem. 2006, 6, 271-279. (h)
Santos, R. C.; Salvador, J. A.; Marín, S.; Cascante, M.; Moreira, J. N.; Dinis, T. C. Bioorg. Med. Chem. 2010, 18,
4385-4396. (i) Liu, W. K.; Ho, J. C.; Cheung, F. W.; Liu, B. P.; Ye, W. C.; Che, C. T. Eur. J. Pharmacol. 2004, 13, 7178. (j) Sultana, N.; Ata, A.; J. Enzyme Inhib. Med. Chem. 2008, 23, 739-756. (k) Pollier, J.; Goossens, A.
Phytochemistry 2012, 77, 10-15. (l) Li, H.; He, N.; Li, X.; Zhou, L.; Zhao, M.; Jiang, H.; Zhang, X. Oncol. Lett. 2013,
6, 885-890. (m) Rashid, S.; Dar, B. A.; Majeed, R.; Hamid, A.; Bhat, B. A. Eur. J. Med. Chem. 2013, 66, 238-245.
(n) Sultana, N. J. Enzyme Inhib. Med. Chem. 2011, 26, 616-642. (o) Liu, M. C.; Yang, S. J.; Jin, L. H.; Hu, D. Y.; Xue,
W.; Song, B. A.; Yang, S. Eur. J. Med. Chem. 2012, 58, 128-135. (p) Meng, Y.; Song, Y.; Yan, Z.; Xia, Y. Molecules
2010, 15, 4033-4040.
4
(a) Andreuccetti, M.; Allegrini, G.; Antonuzzo, A.; Malvaldi, G.; Conte, P. F.; Danesi, R.; Del Tacca, M.; Falcone,
A. Eur. J. Cancer 1996, 32A, 1219-1226. (b) Johnston, J. S.; Johnson, A.; Gan, Y.; Wientjes, M. G.; Au, J. L. Pharm.
Res. 2003, 20, 957-961. (c) Chen, C.; Zhang, Y.; Wang, Y.; Huang, D.; Xi, Y.; Qi, Y. Anticancer Drugs 2011, 22, 435443. (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, 232-237. (e) Multani, A. S.; Furlong, C.; Pathak, S.
Int. J. Oncol. 1998, 13, 923-925. (f) Chen, P.; Cai, X.; Cao, F.; Li, F.; Lei, J.; Deng, S. Shandong Yiyao 2012, 52, 6971. (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, 232-237. (h) Scanlon, K. J.; Funato, T.; Pezeshki,
B.; Tone, T.; Sowers, L. C. Cancer Commun. 1990, 2, 339-343. (i) Klann, R. C.; Holbrook, C. T.; Nyce, J. W.
Anticancer Res. 1992, 12, 781-787. (j) Morgan, R. J. Jr.; Newman, E. M.; Sowers, L.; Scanlon, K.; Harrison, J.;
Akman, S.; Leong, L.; Margolin, K.; Niland, J.; Raschko, J.; Somlo, G.; Carroll, M.; Chow, W.; Tetef, M.; Hamasaki,
V.; Yen, Y.; Doroshow, J. H. Cancer Chemother. Pharmacol. 2003, 51, 459-464.
5
Bori, I. D.; Hung, H. I.; Qian, K.; Chen, C. H.; Morris-Natschke, S. L.; Lee, K. H. Tetrahedron Lett. 2012, 53, 19871989.
9
6
(a) 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, 782-792. (b) Kang, X.; Hu, J.; Gao, Z.; Ju, Y.; Xu, C. Med.
Chem. Commun. 2012, 3, 1245-1249. (c) Cheng, K.; Liu, J.; Sun, H.; Xie J. Chem. Biodiversity 2010, 7, 690-697. (d)
Cheng, K.; Liu, J.; Liu, X.; Li, H.; Sun, H.; Xie, J. Carbohydr. Res. 2009, 344, 841-850. (e) Cheng, K.; Liu, J.; Sun, H.;
Bokor, E.; Czifrak, K.; Konya, B.; Toth, M.; Docsa, T.; Gergely, P.; Somsak, L. New J. Chem. 2010, 34, 1450-1464.
(f) Pertino, M. W.; Lopez, C.; Theoduloz, C.; Schmeda-Hirschmann, G. Molecules 2013, 18, 7661-7674. (g)
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, 657-664. (h) Rashid, S.; Ahmad Dar, B.;
Majeed, R.; Hamid, A.; Ahmad Bhat, B. Eur. J. Med. Chem. 2013, 66, 238-245.
7
Hein, J. E.; Fokin, V. V. Chem. Soc. Rev. 2010, 39, 1302-1315.
8
For a few examples, see: (a) De Las Heras, F. G.; Alonso, R.; Alonso, G. J. J. Med. Chem. 1979, 22, 496-501. (b)
Alonso, R.; Camarasa, M. J.; Alonso, G.; De Las Heras, F. G. Eur. J. Med. Chem. 1980, 15, 105-109. (c) Cho, S.; Oh,
S.; Uma, Y.; Jung, J.-H.; Hamc, J.; Shin, W.-S.; Lee, S. Bioorg. Med. Chem. Lett. 2009, 19, 382-385. (d) Bilat, A. B.;
Bhaska, P. R.; Agrawal, S. K.; Saxena, A. K.; Sampath Kumar, H. M.; Qazi, G. N. Eur. J. Med. Chem. 2008, 43,
2067-2072. (e) Kádár, Z.; Molnár, J.; Schneider, G.; Zupkó, I.; Frank, E. Bioorg. Med. Chem. 2012, 20, 1396-1402.
(f) Duan, Y.-C.; Ma, Y.-C.; Zhang, E.; Shi, X.-J.; Wang, M.-M.; Ye, X.-W.; Liu, H.-M. Eur. J. Med. Chem. 2013, 62,
11-19.
9
Van Loc, T.; Van Sung, T.; Kamperdick, C; Adam, G. J. Pract. Chem. 2000, 342, 63-71.
10
Kim, S. H.; Chen. Z.; Tuyen Nguyen, V.; Pezzuto, Z. M.; Qui, S.; Lu, Z. Synth. Commun. 2003, 27, 1607-1612.
11
(a) Lischewski, M.; Ty, P. D.; Kutschabsky, L.; Pfeiffer, D.; Phiet, H. V.; Preiss, A.; Sung, T. V.; Adam, G.
Phytochemistry 1985, 24, 2355-2357. (b) Kiem, P. V.; Minh, C. V.; Dat, N. T.; Lee, J. J.; Kim, Y. H. Chem. Pharm.
Bull. 2003, 51, 1432-1435.
12
Šarek, J.; Klinot, J.; Džubák, P.; Klinotová, E.; Nosková, V.; Křeček, V.; Kořínková, G.; Thomson, J. O.;
Janošt'áková, A.; Wang, S.; Parsons, S.; Fischer, P. M.; Zhelev, N. Z.; Hajdúch, M. J. Med. Chem. 2003, 46, 54025415.
10