Synthesis of Novel ent-Kaurane-Type Diterpenoid Derivatives Effective for Highly Aggressive Tumor Cells

We have designed and synthesized 6 ent-Kaurane-type diterpenoid derivatives containing α,β-unsaturated ketone moieties. In vitro, activity was evaluated against three human tumor cell lines and a rat myogenic cell line (HepG2, NSCLC-H292, SNU-1040, L6) by MTT assay. All the tested compounds exhibited comparable or higher activity than DDP and eriocalyxin B. Compounds 16, 17 and 18 are promising anti-tumor leads due to their cytotoxic potencies and higher selectivity, with SI values of 161.06, 47.80 and 128.20, respectively.


Introduction
Highly aggressive tumors are a major cause of death worldwide, accounting for 8.8 million deaths each year, and are estimated to cause 9.6 million deaths in 2018. Globally, about 1 in 6 deaths is due to cancer. Approximately 70% of deaths from cancer occur in low-and middle-income countries, according to the World Health Organization (WHO) [1].
Plants are normally an important source of new drugs [2]. In recent years, Isodon genus members have been promising leads due to their extensive pharmacological and physiological effects, such as inhibition of hepatitis viral replication [3] and bacterial infections of the lung or gut [4], as well as its antimalaria [5], antiinflammatory [6], and anti-tumor properties [7]. Hitherto, over 1200 ent-Kaurane-type diterpenoids with highly compact polycyclic ring systems have been isolated from Isodon genus, notably the compounds oridonin [8]. Natural Diterpenoid Isoferritin A (IsoA) [9], longikaurin [10], xerophilusins [11] (Figure 1, 1-4), which have been studied for anti-tumor activity. A common structural unit presented by these diterpenoid compounds is the functionalized bridged C/D α,β-unsaturated ketone moiety. Unfortunately, the use of these natural diterpenoids for anti-tumor was hampered by its moderate potency and complex oxygenated diterpenoid scaffold. Therefore, it is very necessary to find compounds with simple structure and stronger activity. In the preceding letter we reported the synthesis and biological evaluation of a series of eriocalyxin B derivatives ( Figure 2, 5-7) and found that eriocalyxin B derivatives bearing one or two α,β-unsaturated ketone units are potent antitumor agents [12]. It is noteworthy that the 7-hydroxyl group and the hydrogen bond between 6-hydroxyl group and 15-carbonyl group are not necessary for antitumor activity.

Chemistry
Herein we report the synthesis of ent-Kaurane-type diterpenoid derivatives containing two α,βunsaturated ketone moieties and their in vitro cytotoxic properties against three human tumor cell lines. Our synthesis began with reacting isobutyraldehyde with methyl vinyl ketone (MVK) in the presence of sulfuric acid which produced 4,4-dimethylcyclohex-2-enone (compound 8). Treatment of commercially available 1,3-cyclohexan-dione with ethanol in a reflux of benzene in the presence of p-toluene sulfonic acid resulted in 3-ethoxy-cyclohex-2-enone (compound 9) (86%). Compound 9 was treated sequentially with NaH and diethyl carbonate to directly produce β-ketoester. Treatment of βketoester with NaH and allyl bromide produced compound 10 (81%). The resulting compound 10 was reduced using NaBH4 followed by acidification to produce compound 11. Following the wellestablished Ma protocol [13], the key intermediate 12 (52%) was obtained (Scheme 1). In the preceding letter we reported the synthesis and biological evaluation of a series of eriocalyxin B derivatives ( Figure 2, 5-7) and found that eriocalyxin B derivatives bearing one or two α,β-unsaturated ketone units are potent antitumor agents [12]. It is noteworthy that the 7-hydroxyl group and the hydrogen bond between 6-hydroxyl group and 15-carbonyl group are not necessary for antitumor activity. In the preceding letter we reported the synthesis and biological evaluation of a series of eriocalyxin B derivatives ( Figure 2, 5-7) and found that eriocalyxin B derivatives bearing one or two α,β-unsaturated ketone units are potent antitumor agents [12]. It is noteworthy that the 7-hydroxyl group and the hydrogen bond between 6-hydroxyl group and 15-carbonyl group are not necessary for antitumor activity.

Chemistry
Herein we report the synthesis of ent-Kaurane-type diterpenoid derivatives containing two α,βunsaturated ketone moieties and their in vitro cytotoxic properties against three human tumor cell lines. Our synthesis began with reacting isobutyraldehyde with methyl vinyl ketone (MVK) in the presence of sulfuric acid which produced 4,4-dimethylcyclohex-2-enone (compound 8). Treatment of commercially available 1,3-cyclohexan-dione with ethanol in a reflux of benzene in the presence of p-toluene sulfonic acid resulted in 3-ethoxy-cyclohex-2-enone (compound 9) (86%). Compound 9 was treated sequentially with NaH and diethyl carbonate to directly produce β-ketoester. Treatment of βketoester with NaH and allyl bromide produced compound 10 (81%). The resulting compound 10 was reduced using NaBH4 followed by acidification to produce compound 11. Following the wellestablished Ma protocol [13], the key intermediate 12 (52%) was obtained (Scheme 1).
Deprotonation of 8 with LiHMDS followed by a reaction with compound 12 led to compound 13 as the major product, along with isomer 14 in 82% combined yield (dr = 60:40). The structures of 13 and 14 were confirmed by NMR spectral and X-ray crystallographic analysis ( Figure 3). Next, compound 13 was subjected to allylic oxidation with selenium dioxide (SeO 2 , CH 2 Cl 2 , t-BuOOH) and IBX oxidation to form compounds 15 and 16, respectively. Compound 17 (or 18) was provided from the diastereoisomer 14 under the same conditions as 15 (or 16) (Scheme 2).

Scheme 1. Synthesis of compound 8 and compound 12.
Deprotonation of 8 with LiHMDS followed by a reaction with compound 12 led to compound 13 as the major product, along with isomer 14 in 82% combined yield (dr = 60:40). The structures of 13 and 14 were confirmed by NMR spectral and X-ray crystallographic analysis ( Figure 3). Next, compound 13 was subjected to allylic oxidation with selenium dioxide (SeO2, CH2Cl2, t-BuOOH) and IBX oxidation to form compounds 15 and 16, respectively. Compound 17 (or 18) was provided from the diastereoisomer 14 under the same conditions as 15 (or 16) (Scheme 2). Deprotonation of 8 with LiHMDS followed by a reaction with compound 12 led to compound 13 as the major product, along with isomer 14 in 82% combined yield (dr = 60:40). The structures of 13 and 14 were confirmed by NMR spectral and X-ray crystallographic analysis ( Figure 3). Next, compound 13 was subjected to allylic oxidation with selenium dioxide (SeO2, CH2Cl2, t-BuOOH) and IBX oxidation to form compounds 15 and 16, respectively. Compound 17 (or 18) was provided from the diastereoisomer 14 under the same conditions as 15 (or 16) (Scheme 2).

Biological Evaluation
The cytotoxic properties of all newly synthesized compounds were evaluated in vitro against three human tumor cell lines ((HepG2, NSCLC-H292, SNU-1040) and one rat myotubes cell line (L6) by MTT assay. Cisplatin (DDP) and eriocalycin B (5) were used as reference drugs. The results are summarized in Table 1. (IC50 value is defined as the concentrations corresponding to 50% growth inhibition). Remarkably, all the tested compounds exhibited comparable or even higher activity than their natural references eriocalyxin B and DDP. Although results are preliminary, we found a clear structure-activity relationship: the α,β-unsaturated ketone moiety is necessary for potency. Particularly for compound 16 (IC50 = 0.33; 1.69; 2.44 μM) and 18 (IC50 = 0.56; 1.35; 3.01 μM), which display two α,β-unsaturated ketone moieties, these had stronger in vitro potencies and cytotoxic activities to DDP. To obtain insight into the cytotoxic potential of these new compounds on normal cells, the effect of compounds 13, 14, 15, 16, 17, 18 and DDP (cisplatin) were evaluated in human liver carcinoma cell (HepG2) and rat myotubes (L6) cells. All the tested analogues exhibited higher selectivity than DDP. Especially, compound 16, which had more potency and selectivity than DDP and eriocalyxin B, with an SI (selective index, IC50 of normal cells/IC50 of tumor cells) value of 161.06. Interestingly, the two epimers 16, 18 showed no significant difference in their anticancer activities.

Biological Evaluation
The cytotoxic properties of all newly synthesized compounds were evaluated in vitro against three human tumor cell lines ((HepG2, NSCLC-H292, SNU-1040) and one rat myotubes cell line (L6) by MTT assay. Cisplatin (DDP) and eriocalycin B (5) were used as reference drugs. The results are summarized in Table 1. (IC 50 value is defined as the concentrations corresponding to 50% growth inhibition). Remarkably, all the tested compounds exhibited comparable or even higher activity than their natural references eriocalyxin B and DDP. Although results are preliminary, we found a clear structure-activity relationship: the α,β-unsaturated ketone moiety is necessary for potency. Particularly for compound 16 (IC 50 = 0.33; 1.69; 2.44 µM) and 18 (IC 50 = 0.56; 1.35; 3.01 µM), which display two α,β-unsaturated ketone moieties, these had stronger in vitro potencies and cytotoxic activities to DDP. To obtain insight into the cytotoxic potential of these new compounds on normal cells, the effect of compounds 13, 14, 15, 16, 17, 18 and DDP (cisplatin) were evaluated in human liver carcinoma cell (HepG2) and rat myotubes (L6) cells. All the tested analogues exhibited higher selectivity than DDP. Especially, compound 16, which had more potency and selectivity than DDP and eriocalyxin B, with an SI (selective index, IC 50 of normal cells/IC 50 of tumor cells) value of 161.06. Interestingly, the two epimers 16, 18 showed no significant difference in their anticancer activities.

General Method for Synthesis of 15 and 17
To a stirred solution of 13 (1.0 eq) in DCM, SeO 2 (0.9 eq) was added in one portion, and a solution of t-BuOOH (5.5 M in decane, 3 eq) was added dropwise. The solution was vigorously stirred at room temperature overnight, and then the mixture was filtered through a short pad of silica gel washed with EtOAc. The filtrate was concentrated under vacuum and purified by flash column chromatography to provide allylic alcohol 15.
penicillin and 100 mg/mL streptomycin) was supplemented. Cells are inoculated in 96-well plate and cultured for 24 h. After attached to the culture bottle wall cells were divided cells into three groups, (1) control group; (2) positive control group; (3) experiment group. The administration concentrations of Cisplatin (DTT) and compounds were 0.1 µM-10 µM. After 24 h, 10% MTT was added and cells were culture for 3.5 h. Then the culture medium was discard and 150 µL DMSO was added. Eventually, after incubation for 10 min by shaking in the dark at room temperature, the absorbance was detected at 490 nm using a microplate reader, and the difference was analyzed by prism 7 software.

Conclusions
In conclusion, we have designed and synthesized several compounds bearing two α,β-unsaturated ketone moiety. Their in vitro activities were evaluated against HepG2, NSCLC-H292, SUM-1040 tumor cell lines and an L6 myoblast cell line, with IC 50 values ranging from 0.33 to 115.17 µM. This study has revealed that compounds 16, 17 and 18 are promising anti-tumor leads due to the stronger cytotoxic potencies and higher selectivity they displayed than their natural counterparts.