Design, synthesis and cytotoxic evaluation of nitric oxide-releasing derivatives of isosteviol

Article abstract of DOI:10.1111/cbdd.12956

Twenty-six novel isosteviol derivatives coupled with two types of nitric oxide (NO) donors (furoxans and NONOates) were synthesized and screened for cytotoxic activities against four human cancer cell lines with sunitinib as the positive control. The results showed that seven furoxan-based derivatives (8a, 8b, 8c, 8d, 8e, 9e, and 9f) exhibited desirable cytotoxic activities, while NONOate-based derivatives displayed poor potency because of unstability. Compared with sunitinib, compounds 8a and 8e were more active on all tested cell lines, especially in HCT116 (8a, IC₅₀?=?0.48?±?0.02?μm; 8e, IC₅₀?=?0.94?±?0.01?μm); compounds 8b and 8d were more potent on HCT116 (IC₅₀?=?3.39?±?0.06 and 3.29?±?0.03?μm), HepG2 (IC₅₀?=?1.05?±?0.03 and 5.37?±?0.08?μm), and SW620 (IC₅₀?=?1.33?±?0.02 and 4.11?±?0.05?μm) cell lines, and 8c exhibited higher activities on HepG2 cells with an IC₅₀?=?4.76?±?0.14?μm. NO-releasing experiment of compounds 8a–e, 17a, 18a, 19a, and 21a reminded us that NO-releasing amount of this series of isosteviol derivatives positively correlates with their cytotoxic activities.

Full text of DOI:10.1111/cbdd.12956

PROF. LI CHEN (Orcid ID : 0000-0002-6211-7672)
Received Date : 04-Jul-2016
Revised Date : 27-Nov-2016
Accepted Date : 17-Jan-2017
Article type
: Research Letter
Design, synthesis and cytotoxic evaluation of nitric oxide releasing
derivatives of isosteviol
Yan Liu1,2,†, Tingting Wang1,†, Yong Ling3, Na Bao1, Wei Shi1, Li Chen1,* and Jianbo Sun1,*
1Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, China
2Zhenjiang Institute for drug control, Zhenjiang 212000, China
3School of Pharmacy, Nantong University, Nantong 226019, China
*Corresponding authors: Li Chen, chenliduo@sohu.com; Jianbo Sun, sjbcpu@gmail.com
†Contributed to this work equally
Abstract: Twenty-six novel isosteviol derivatives coupled with two types of nitric oxide (NO) donors (furoxans and
NONOates) were synthesized and screened for cytotoxic activities against four human cancer cell lines with sunitinib as the
positive control. The results showed that seven furoxan-based derivatives (8a, 8b, 8c, 8d, 8e, 9e, and 9f) exhibited
desirable cytotoxic activities, while NONOate-based derivatives displayed poor potency because of unstability. Compared
with sunitinib, compounds 8a and 8e were more active on all tested cell lines, especially in HCT116 (8a, IC50 = 0.48 ± 0.02
μM; 8e, IC50 = 0.94 ± 0.01 μM); compounds 8b and 8d were more potent on HCT116 (IC50 = 3.39 ± 0.06 and 3.29 ± 0.03 μM),
HepG2 (IC50 = 1.05 ± 0.03 and 5.37 ± 0.08 μM) and SW620 (IC50 = 1.33 ± 0.02 and 4.11 ± 0.05 μM) cell lines, and 8c exhibited
higher activities on HepG2 cells with an IC50 = 4.76 ± 0.14 μM. NO-releasing experiment of compounds 8a-e, 17a, 18a, 19a
and 21a reminded us that NO-releasing amount of this series of isosteviol derivatives positively correlates with their
cytotoxic activities.
Keywords: isosteviol derivatives, NO donors, cytotoxic activities, NO-releasing amount
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Nitric oxide is an important signaling and effector molecule, which plays an important role in various physiological and
pathophysiological processes (1). It is considered to promote tumor angiogenesis at low concentrations and inhibit tumor
cells via different methods at high concentrations (2). NO donors refer to compounds that can release a certain amount of
NO by the action of enzymes and non-enzymes. Furoxans and diazeniumdiolates (NONOates) are two important types of
NO donors which can produce high levels of NO by known releasing processes (3, 4). In our previous studies, over two
hundred hybrids were designed and synthesized by attaching NO donors such as furoxan, NONOates and nitrate to
different natural products via various linkers, and among them, some showed desirable antitumor activity without injuring
normal cells (5-8).
Isosteviol is a tetracyclic diterpenoid with a beyerane skeleton, obtained by acid hydrolysis of crude glycoside fraction
extracted from Stevia rebaudiana Bertoni (9). Recently, isosteviol has attracted growing interest of scientists because of its
unique structure (10), easy accessibility and considerable pharmacological activities (11-13), especially in structural
modification. By far, chemical modifications have been focused on CH2 group at C-15 position, the carbonyl at C-16 position
and the COOH group at C-19 position. Compared with the leading compound, many derivatives displayed much better
potency, many of which could even be used as candidates for further antitumor agents study (14-19). The preliminary
structure activity relationships (SARs) illustrate that esterification of the COOH group, introduction of amine, oxime or
heterocycle moieties may remarkably enhance the cytotoxic activities. Moreover, given the fact that tumor cells display the
character of “addicted nitrogen” and increase the transferring rate of amino acid (20), we supposed that introducing amino
acid into the target compounds may enhance their selectivity to tumor cells.
In order to find more antitumor candidates and further investigate the SARs, novel series of hybrids coupled with furoxans
1
and NONOates were designed and synthesized here. Their structures were characterized by IR, H NMR and MS. The
cytotoxic activities were determined by MTT method in HepG2, HCT116, Huh7 and SW620 cell lines, respectively.
Results and discussion
Chemistry
This study describes two groups of isosteviol derivatives coupled with furoxans and NONOates. The general route for
isosteviol derivatives coupled with furoxans is outlined in Scheme 1. The important intermediates 1a-f were synthesized as
described previously (6). Esterification of isosteviol with ethyl bromide or benzyl chloride provided corresponding esters 2
and 3 in 85%~90%, which reacted with hydroxylamine hydrochloride to afford 4 and 5 in 82%~92%. Subsequently, 6 and 7
were prepared from the reaction of 4 or 5 and succinic anhydride in 85%~90%. Finally, compounds 6 or 7 was treated with
furoxan 1a-f in the presence of 4-dimethylamino pyridine (DMAP) and 1-ethyl-(3-dimethylamino-propyl)–carbodiimide
hydrochloride (EDCI) to generate target compounds 8a-f and 9a-f in 32~51%, respectively.
The synthetic routes of isosteviol derivatives conjugated with NONOates were summarized in Scheme 2. The
important NONOates intermediates 10a-b were prepared with pyrrolidine or diethylamine according to the
reported procedure in 45%~46% (21).
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Compound 4 was treated with p-toluenesulfonic acid in methyl cyanides at 90
for 1h under the protection of N2 to
afford 11 in 85%. Oxidation of 11 gave 12 in 72%, and then 13 was prepared by removing the benzyl of 12 with 10% Pd-C in
91%. Isosteviol, 6 or 13 were treated with dibromoalkanes bearing two to four carbons to generate 14a-c, 15a-c and 16a-b
in good yields (90~97%), respectively. Subsequently, intermediates 14a-c, 15a-c and 16a-b reacted with 10a or 10b
respectively in N2 at room temperature for 72 h to derive the target compounds 17a-c, 18a-c, 19a-c and 20a-c in 29%~45%.
Cytotoxic activities
The cytotoxic activities of these compounds were determined in vitro by MTT assay (22) against HCT116, Huh7, HepG2 and
SW620 cells using sunitinib as the positive control. The data were calculated and presented as IC50 values in Table 1. Five
furoxan-based derivatives of isosteviol showed higher cytotoxicity than the lead compound isosteviol, while
NONOate-based derivatives appeared to be inactive, which attributed to their lability. It was observed that the
NONOate-based derivatives changed their characters from yellow oil into white solid in a week, and the degeneration was
1
further proved by ESI-MS and H-NMR spectra. Since the bioactivity results of NONOate-based derivatives of isosteviol in
this test might be false negative, they were not listed in Table 1.
Table 1 IC50 (μM) values of partial target compounds.
IC50 a (μM)
Compd.
HCT116
0.48 ± 0.02
3.39 ± 0.06
>25
Huh7
2.86 ± 0.01
5.13 ± 0.05
>25
HepG2
2.95 ± 0.05
1.05 ± 0.03
4.76 ± 0.14
5.37 ± 0.08
2.42 ± 0.15
>25
SW620
3.02 ± 0.04
1.33 ± 0.02
>25
8a
8b
8c
8d
3.29 ± 0.03
0.94 ± 0.01
>25
4.62 ± 0.03
1.73 ± 0.02
>25
4.11 ± 0.05
1.15 ± 0.02
>25
8e
8f
9a
>25
>25
>25
>25
9b
>25
>25
>25
>25
9c
>25
>25
>25
>25
9d
>25
>25
>25
>25
9e
22.3 ± 0.02
24.1 ± 0.15
>25
>25
>25
>25
9f
>25
>25
>25
Isosteviol
Sunitinib b
>25
>25
>25
5.62 ± 0.04
3.03 ± 0.02
7.78 ± 0.04
6.25 ± 0.06
a Inhibitory activity was assayed by exposure for 48 h.
b Positive control drug.
Compounds 8a-b and 8d-e displayed strong inhibition against the tested four cell lines. Especially, compounds 8a and 8e
were the promising antitumor agents superior to the positive control sunitinib. Moreover, compounds 8b and 8d exhibited
higher activities on HCT116, HepG2 and SW620 cell lines, and 8c showed stronger inhibition against HepG2 cells selectively.
Compounds 9e-f effected HCT116 cells selectively. Comparing the performance of the homologues, we derived the rule:
the ethyl esters 8a-e were potent while benzyl esters 9a-e were not, indicating that esters with smaller steric hindrance
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were preferred at C-19 position; compounds 8e and 9e were superior to 8c and 9c, respectively, indicating that branched
linker was superior to straight linker with the same number of carbons; compounds 8c and 9c with straight linker of four
carbons displayed the lowest activities among their homologues, indicating that over-prolong the length of aliphatic chain
weakened the potency in turn. Further work is required to summarize more definite SARs.
In vitro NO-releasing amount detection
To verify the contribution of NO to the antitumor activity, the levels of NO released in vitro by the tested compounds (8a-c,
17a, 18a, 19a and 21a) (Figure 1) were measured by Griess method (23). As expected, the amount of NO released by the
furoxan-based derivatives was generally higher than that of NONOate-based derivatives, and the latter were proved to
have already degraded. It was observed that the levels of NO were in positive correlation with the anti-tumor activities of
the corresponding derivatives. Thus, furoxan-based derivatives possessing high cytotoxicity can be partially explained by
the fact that higher level of NO contributes to the anti-tumor activity.
Conclusion
In summary, twenty-six novel NO-releasing derivatives of isosteviol coupled with furoxans and NONOates have been
designed and synthesized. The anti-tumor bioactivities of these derivatives were determined by the MTT assay in vitro
against HCT116, Huh7, HepG2 and SW620 cells. The majority of furoxan-based derivatives improved the anti-tumor activity
of isosteviol, which consists with our hypothesis. It was a pity that NONOate-based derivatives of isosteviol were proved to
have degraded before the antitumor test thus appeared to be inactive. Among all these isosteviol derivatives, compound
8a-8e showed promising antitumor activity, which were stronger than that of the positive control on one to four tested cell
lines. Subsequently, we detected the NO-releasing amount of nine derivatives that were tested bioactivities to investigate
whether it affects the antitumor activity of these derivatives. As a result, derivatives with higher potency released more
NO, which may partly certify the higher antitumor activity of furoxan-based derivatives depended on high concentration of
NO. Further research is ongoing to enhance the stability of NONOated-based derivatives and illustrate more SARs. These
findings will be valuable for further utilization of isosteviol derivatives in the therapeutic interventions of some cancers.
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Supporting Information
Experimental section, ESI-MS and 1H NMR of synthetic compounds
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Figure Legends
Scheme 1. Reagents and conditions: (i) EtBr or BnCl, KOH, DMSO, rt, 6h, 85%~90%; (ii) NH2OH·HCl, pyridine, rt, 24h,
82%~92%; (iii) succinic anhydride, DMAP, CH2Cl2, reflux, 12h, 85%~90%; (iv) EDCI, DMAP, CH2Cl2, rt, 6h, 32%~51%.
Scheme 2. Reagents and conditions: (i) CH3ONa, NO, 4atm, rt, 22h, 45%~46%; (ii) Ac2O, p-TsOH, N2, CH3CN, 90 , 1h, 85%; (iii) SeO2,
1,4-dixone, reflux, 18h, 72%; (iv) H2, 10% Pd/C, 24h, 91%; (v) Br(CH2)nBr (n=2, 3 or 4), K2CO3, DMF, rt, 6h, 90%~ 97%; (vi) N2, dry THF, dry
DMF, rt, 72h, 29%~45%.
Fig. 1. NO-releasing amount of partial target compounds.
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