Synthesis and cytotoxic activity of nitric oxide-releasing isosteviol derivatives

Article abstract of DOI:10.1016/j.bmcl.2014.03.004

Fifteen novel hybrids containing diterpene skeleton and nitric oxide (NO) donor were prepared from isosteviol. All the compounds were tested on preliminary cytotoxicity, and the results showed that six target compounds (8c, 10b, 14a, 14c, 18c, and 18d) exhibited anti-proliferation activity on HepG2 cells, with 8c (IC₅₀ = 4.24 μM) and 18d (IC₅₀ = 2.75 μM) superior to the positive control CDDO-Me (2-cyano-3,12-dioxooleana-1, 9(11)-dien-28-acid methyl ester, IC₅₀ = 4.99 μM); eleven target compounds (8a-c, 9a-c, 10a-b, 14a, 14c, 18d) exhibited anti-proliferation activities on B16F10 cells at different levels, among them, seven compounds were more potent than comptothecin (IC₅₀ = 2.78 μM) and CDDO-Me (IC₅₀ = 5.85 μM), particularly, 10b (IC₅₀ = 0.02 μM) presented the strongest effect, which was selected as a candidate for further study.

Full text of DOI:10.1016/j.bmcl.2014.03.004

Accepted Manuscript
Synthesis and cytotoxic activity of nitric oxide-releasing isosteviol derivatives
Ting-ting Wang, Yan Liu, Li Chen
PII:
S0960-894X(14)00220-0
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.03.004
BMCL 21397
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
14 January 2014
20 February 2014
3 March 2014
Please cite this article as: Wang, T-t., Liu, Y., Chen, L., Synthesis and cytotoxic activity of nitric oxide-releasing
isosteviol derivatives, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.
2014.03.004
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Synthesis and cytotoxic activity of nitric oxide-releasing isosteviol derivatives
†
†
Ting-ting Wang a,b, , Yan Liu a, , Li Chen a,*
a
Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tongjiaxiang
Road, Nanjing 210009, China
b Zhenjiang Institute for drug control, 62 Nanxu Road, Zhenjiang 212000, China
ABSTRACT
Fifteen novel hybrids containing diterpene skeleton and nitric oxide (NO) donor were prepared from isosteviol. All
the compounds were tested on preliminary cytotoxicity, and the results showed that six target compounds (8c, 10b,
14a, 14c, 18c, and 18d) exhibited anti-proliferation activity on HepG2 cells, with 8c (IC50 = 4.24 μM) and 18d
(IC50 = 2.75 μM) superior to the positive control CDDO-Me (2-Cyano-3,12-dioxooleana-1,9(11)-dien-28-acid
methyl ester, IC50 = 4.99 μM); eleven target compounds (8a-c, 9a-c, 10a-b, 14a, 14c, 18d) exhibited
anti-proliferation activities on B16F10 cells at different levels, among them, seven compounds were more potent
than comptothecin (IC50 = 2.78 μM) and CDDO-Me (IC50 = 5.85 μM), particularly, 10b (IC50 = 0.02 μM) presented
the strongest effect, which was slected as a candidate for further study.
Keywords: isosteviol; furoxan; cytotoxicity; structural modification; NO-donor
Isosteviol (ent-16-oxobeyeran-19-oic acid, 1, Figure 1), bearing distinctive tetracyclic skeleton of
beyerane can be obtained by acid hydrolysis of stevioside in good yield.1, 2 Recently, isoteviol and its
derivatives have attracted considerable interest because of their wide biological activities including
antihyperglycemia,3 anti-hypertension,4 anti-inflammation,5 antitumor,6-9 antimicrobial activity10 and so
on. In the process of looking for potent antitumor derivatives of isosteviol, some structure activity
relationships (SARs) were summarized, for example, exo-methylene cyclopentanone and
a-methylenelactone are crucial structures to produce antitumor effects. The parent structures of the new
compounds in this paper were designed on the basis of the SARs above.
Meanwhile, nitric oxide (NO) played dual roles in the occurrence and development of tumor, i.e.
high concentration of NO was cytotoxic to tumor cells by leading to apoptosis and blocking spread
and migration, whereas relatively low level of NO promoted tumor growth and proliferation.11 NO
donors refer to compounds that can release a certain amount of NO by the action of enzymes and
non-enzymes, including organic nitrates, furoxans, metal-NO complexes, nitrosothiols, and
NONOates, etc. With the development of NO donors, NO-donating drugs gradually emerged and
developed rapidly. Among these, the most successful drug is NO-nonsteroidal anti-inflammatory
drugs (NO-NSAIDs), which reduced the side effect of NSAIDs on the one hand and increased the
anti-cancer effect on the other hand.12 Our group had discovered several novel NO-releasing
derivatives of natural compounds such as NO-oleanolic acid or NO-ursolic acid hybrids with
desirable cytotoxic activity. 13-17
Considering the SARs of isoteviol derivatives, the anti-proliferation activity of NO, and the
* Corresponding author. Tel.: +86 25 83271447
E-mail address: chenliduo12@gmail.com
† These authors contributed to this work equally.
1

previous research results of our group, we designed and synthesized isoteviol derivatives with active
fragments such as oxime, latone, exo-methylene cyclopentanone and tricycles via structural
modification or skeleton reconstruction and furoxan connection to the intermediates above through
different linkers. All the compounds were tested on preliminary cytotoxicity against human
hepatocellular liver carcinoma cell line (HepG2) and highly metastatic melanoma cells (B16F10) in
vitro by MTT method.18
This paper describes two kinds of isosteviol derivatives with furoxan linked to C19 carboxylic
acid and C16 with D-ring opened or not, repectively. Compounds 8a-c 19, 9a-c and 10a-b belong to one
group. The general route of these derivatives is outlined in Scheme 1. Acid hydrolysis of stevioside
produced 1 with a yield of 72%. Baeyer-Villiger reaction of 1 gave 2 in 59%, subsequently, 3 was
synthesized in four steps by the reported process.6 The three compounds above were treated
respectively with thionyl chloride under ice bath to prepare acyl chloride 4a-c, and then glycine methyl
ester were added with triethylamine (TEA) as the catalyst to give 5a-c. Removal of methyl ester were
carried out in the methanol solution of sodium hydroxide for 3 h, and then acidification of the solution
afforded 6a-c in 80~93% yields. Finally, 6a-c reacted with compounds 7a-d in the presence of
4-dimethylamino pyridine (DMAP) and 1-ethyl-(3-dimethylamino-propyl)-carbodiimide hydrochloride
(EDCI) to generate target compounds 8a-c, 9a-c, and 10a-b in 37~49% yields. In this route, important
intermediates 7a-d were prepared as described previously.13
The synthetic approaches employed to prepare 14a-c and 18a-d are outlined in Scheme 2. Firstly,
compound 11 was prepared according to the reference in three steps.7 Reduction of 11 with sodium
borohydride in ethanol at 0°C gave 12 (90%), and then 12 was treated with succinic anhydride to
prepare compound 13. 13 reacted with compounds 7a-c in the presence of DMAP and EDCI to
generate target compounds 14a-c. Methyl esterification of 1 gave 15, which reacted with
hydroxylamine hydrochloride under room temperature (r.t.) for 5 h to afford 16. 17 and target
compounds 18a-d were synthesized as described above.
All the derivatives of isosteviol synthesized in this study were screened for the cytotoxicity
against HepG2 and B16F10 cells in vitro by MTT assay. CDDO-Me (one of the most potent
antitumor triterpenoid derivatives) 20 and comptothecin were selected to be the positive control drugs.
The IC50 values were used to determine the growth inhibition. As shown in Table 1, most of the
derivatives showed higher cytotoxicity than the lead compound isosteviol. Except 18a-c, all the
active compounds exhibited stronger potency on B16F10 cells than on HepG2. By comparing effects
of the derivatives against the tested cell lines, some patterns of selectivity may be summarized: 8a-b
with isosteviol as the parent part and 9a-c with lactone as the parent skeleton were not active on
HepG2 cells, however, they exhibited potency on B16F10 cells, which suggested selectivity of the
anti-proliferation activity; 18a-c with oxime as the linker, showed no effects on B16F10 cells but
were potent on HepG2 cells, suggesting that the oximes with medium length of aliphatic chain may
be selective to HepG2 cells.
Comparing the performance of the homologues, we derived the rule: 8a-b were not potent on
HepG2 cells while 8c 21 is potent on both cell lines, indicating that the butyne part may be important for
the potency; 10a plays better role on HepG2 cells than 10b, whereas 10b showed better cytotoxicity on
B16F10 cell lines than 10a, indicating that chain extension may reduce the cytotoxicity on HepG2 cells,
on the contrary, it may increase the cytotoxicity on B16F10 cell lines; 18d showed the best potency
2

among its homologues, but lost the selectivity. Further work is required to summarize more definite
SARs.
In summary, we synthesized two groups of novel NO-releasing derivatives of isosteviol with five
different scaffolds that inhibited the growth of HepG2 and B16F10 cell lines. The derivatives showed
better selectivity to B16F10. Compounds 8c (IC50 = 4.24 μM) and 18d (IC50 = 2.75 μM) had significant
cytotoxicity against HepG2 compared to CDDO-Me against HepG2 but showed no selectivity. Seven
derivatives were more potent than comptothecin (IC50 = 2.78 μM) and CDDO-Me (IC50 = 5.85 μM)
against B16F10. Among them, 10b (IC50 = 0.02 μM) presented the strongest effect. Interestingly, the
oxime series 18a-c (IC50 = 5.16~33.42 μM) exhibited moderate potency and selectivity on HepG2. The
lactone series 9a-c (IC50 = 2.0~5.21 μM) exhibited desirable activity and selectivity on B16F10. Further
research is ongoing to illustrate more SARs, and optimize the potency and selectivity. Our findings will
be of value for further utilization of isosteviol derivatives in the therapeutic interventions of some
human cancers.
Acknowledgements
This study was supported by grant from the Fundamental Research Funds for the Central
Universities (Program No. JKY2011042) and the “QingLan” Project of Jiangsu Department of
Education.
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3

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18. In vitro cytotoxicity study: Cells were cultured in DMEM (GIBCO) medium in a 5% CO2 humidified
atmosphere at 37°C. Cell cytotoxicity was assayed by MTT method. Briefly, cells were seeded in
96-well tissue culture plates and incubated with compounds (20 l) for 48 h at 37°C, 5% CO2.
Following treatment, MTT (0.5 mg/mL) was added. After an additional 4 h of incubation, the reaction
was terminated by removal of the supernatant. Then 150μL of DMSO (SIGMA) was added to each
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immunoassay instrument (Model 680, BIO-RAD, American). Then the inhibitory percentage of each
compound at various concentrations to the cell proliferation was calculated.
19. General procedure for the synthesis of the target compounds 8a–c: SOCl2 (1.0 mL, 14.00 mmol) was
added dropwise with stirring to the flask with 1, 2, or 3 at 0°C, and then one drop of anhydrous DMF
was added to the mixture. Two hours later, yellow solid was obtained after SOCl2 was removed in
vacuo. To the solution of NH2CH2COOMe (65 mg, 0.52 mmol) in 2 .0 mL of dry CH2Cl2, the solid was
added slowly with stirring at 0°C. When finished, TEA (1.5 mL, 1.10 mmol) was added and the
mixture was stirred at r.t.. Four hours later, 8.0 mL of CH2Cl2 was added, and the mixture was washed
with saturated brine for three times, and then dried by anhydrous sodium sulfate overnight. After
filtration and evaporation in vacuo, 160mg of yellow oil was got, which was then dissolved in
anhydrous methanol (1.0 mL). To the mixture 0.5 mL of 2 N NaOH was added. After stirring at r.t. for
two hours, water was added and the mixture was adjusted to pH = 1.5 with concentrated HCl.
Subsquently, the mixture was filtrated, washed and dried to obtain 6a-c. m.p. 189~191°C. A solution of
6a-c, 7a-c, EDCl, DMAP in dry CH2Cl2 (10.0 mL) was stirred at r.t.. Six hours later, the mixture was
concentrated and purified by rapid chromatography [Acetone/petroleum ether = 1/2 (V/V) ] to yield
8a-c.
20. Honda, T. Yoshizawa, H. Sundararajan, C. David, E.; Lajoie, M. J.; Favaloro, F. G. ; Jr.; Janosik, T.; Su,
X. Honda, Y.; Roebuck, B. D.; Gribble, G. W. J. Med .Chem. 2011, 54, 1762.
21. Analytical data for 8c: Yellow oil, ESI-MS: 668.3 [M+H]+; IR (KBr, cm-1): 3421, 2924, 2853, 1615,
1552, 1454, 1360, 1166;1H NMR (300 MHz, CDCl3), δ: 0.78 (s, 3H, CH3), 1.02 (s, 3H, CH3), 1.23 (s,
3H, CH3), 4.10 (s, 2H, NHCH2), 4.37 (s, 2H, OCH2), 5.13 (s, 2H, OCH2), 6.19 (brs, 1H, CONH), 7.65
(t, 2H, J = 8.0 Hz, ArH), 7.77 (t, 1H, J = 6.3 Hz, ArH), 8.10 (d, 2H , J = 8.0 Hz, ArH). , J = 8.3 Hz),
7.93 (s, 1H, ArH),8.26 (dd, 1H, J = 1.4, 8.3, ArH), 8.70 (s, 1H, NH). 13C NMR (75 MHz, CDCl3):
221.10, 177.10, 169.59, 154.46, 145.65, 135.75, 129.7, 128.65, 83.31, 79.18, 76.60, 58.81, 58.54,
57.47, 54.72, 54.22, 52.64, 50.99, 48.71, 48.38, 43.79, 41.65, 4117, 40.12, 39.49, 38.09, 37.27, 29.98,
22.09, 20.33, 19.84, 19.07, 13.54
22. Analytical data for 10a: ESI-MS: 656.2 [M+H]+; IR (KBr, cm-1): 3453, 2929, 2852, 1728, 1620, 1554,
1453, 1171;1H NMR (300 MHz, CDCl3), δ: 0.69 (s, 3H, CH3), 1.01 (s, 3H, CH3), 1.24 (s, 3H, CH3),
4.13 (d, 2H, J = 6.1 Hz, NHCH2), 4.62-5.01 (m, 4H, OCH2CH2O), 5.55 (s, 1H, =CH2), 6.05 (s, 1H,
=CH2), 6.30 (t, 1H, J = 5.2 Hz, NH), 7.64 (t, 2H, J = 9.0 Hz, ArH), 7.77 (t, 1H, J = 7.5 Hz, ArH), 8.06
(d, 2H, J = 8.7 Hz, ArH). 13C NMR (75 MHz, CDCl3): 210.72, 177.03, 170.31, 158.36, 154.17, 142.25,
137.10, 135.76, 129.76, 128.54, 116.29, 68.61, 61.41, 57.19, 56.89, 53.44, 46.76, 43.84, 43.74, 41.14,
40.74, 38.73, 38.16, 37.82, 30.03, 29.69, 22.19, 21.02, 20.11, 19.11, 12.54.
4

Table 1
In vitro anti-proliferation activities of isosteviol derivatives against two human cancer cell lines
Compo IC50 a (μM)
HepG2
Compoun IC50 a (μM)
unds
ol
ds
B16F10
>100
HepG2
B16F10
isostevi >100b
14a
6.44±0.04
1.98±0.16
8a
8b
8c
>50
2.76±0.02
8.06±0.03
0.25±0.01
3.52±0.07
2.05±0.01
5.21±0.03
1.01±0.01
14b
ND
>50
ND c
14c
7.54±0.04
30.24±0.15
33.42±0.96
5.16±0.05
2.75±0.02
4.99±0.02
3.84±0.02
ND
4.24±0.03
ND
18a
9a
9b
9c
18b
ND
ND
18c
ND
ND
18d
2.12±0.01
5.85±0.04
10a
7.13±0.02
CDDO-
Med
10b
38.56±0.14
0.016±0.004
Comptoth
-
2.78±0.04
ecind
a Inhibitory activity was assayed by exposure for 48 h.
b Data expressed in terms of IC50 value were obtained by dose dependent response.
c ND: not detectable. d Positive control drug.

Figure 1. Structure of isosteviol.

Scheme 1. Reagents and conditions: (i) mCPBA, H2SO4, 0°C to r.t., 24 h, 59%; (ii) four steps by
the reported process;6 (iii) SOCl2, ice bath, 2 h; (iv) NH2CH2COOMe, TEA, r.t., 4 h; (v) NaOH, MeOH,
r.t., 3 h, HCl, 80~95%; (vi) EDCI, DMAP, dry CH2Cl2, r.t., 6 h, 37~49%.

Scheme 2. Reagents and conditions: (i) three steps as described; 7 (ii) NaBH4, EtOH, 1 h, 90%; (iii)
succinic anhydride, DMAP, dry CH2Cl2, reflux, 12 h, 85%; (iv) EDCI, DMAP, dry CH2Cl2, rt, 6h,
39~45%; (v) SOCl2, MeOH, 6 h, 43%; (vi) NH2OH·HCl, pyridine, rt, 5 h, 80%.

Graphical abstract
Synthesis and cytotoxic activity of nitric oxide-releasing
isosteviol derivatives
Ting-ting Wang, Yan Liu, Li Chen*
8c IC50 = 4.24 μM; 10b IC50 = 0.02 μM
14a IC50 =1.98μM; 14c IC50 = 3.84μM
18a-c IC50 = 5.16~33.42 μM; 18d IC50 = 2.75 μM