Semi-Synthesis of Harringtonolide Derivatives and Their Antiproliferative Activity

Harringtonolide (HO), a natural product isolated from Cephalotaxus harringtonia, exhibits potent antiproliferative activity. However, little information has been reported on the systematic structure−activity relationship (SAR) of HO derivatives. Modifications on tropone, lactone, and allyl positions of HO (1) were carried out to provide 17 derivatives (2–13, 11a–11f). The in vitro antiproliferative activity against four cancer cell lines (HCT-116, A375, A549, and Huh-7) and one normal cell line (L-02) was tested. Amongst these novel derivatives, compound 6 exhibited comparable cell growth inhibitory activity to HO and displayed better selectivity index (SI = 56.5) between Huh-7 and L-02 cells. The SAR results revealed that the tropone and lactone moieties are essential for the cytotoxic activities, which provided useful suggestions for further structural optimization of HO.


Introduction
Cephalotane-type diterpenoids are widely distributed in the plant of the Cephalotaxus genus [1][2][3][4][5], which is the sole member of the Cephalotaxaceae family. Harringtonolide (also named as hainanolide, HO) is the first member of cephalotane-type diterpenoids that was isolated and structurally identified in 1978 from the seeds of Cephalotaxus harringtonia by Buta and coworkers. Structurally, HO, as a typical cephalotane-type diterpenoid, has a tropone ring, a fused tetracarbocyclic skeleton (A−B−C−D ring), a bridged lactone (E-ring), and a tetrahydrofuran ring (F-ring) in the molecule. Right after its isolation, it was shown to possess potent plant growth inhibitory [6], antiviral [7], anti-inflammatory [8], and antiproliferative activities [1,8,9]. During the past decades, HO has attracted considerable attention of synthetic chemists because of its unique cage-like troponoid skeleton and remarkable biological effects [10][11][12][13][14][15][16][17][18]. However, great efforts have been mainly devoted to the total synthesis of HO. Therefore, almost all of these HO derivatives ( Figure 1) were extracted and separated from the kingdom of plants, such as hainanolidol with F-ringopening [19], cephanolide A with A-ring-contracted [20], fortalpinoid K without a complete tropone moiety [21], fortalpinoid J [21], cephinoids F-G [8], fortunolide B [22] and 10hydroxyharringtonolide [23] with hydroxy substitutions in HO, and 6-en-harringtonolide with a double bond adjacent to tropone unit [23]. Only 13-Bromoharringtonolide is the semi-synthetic HO derivative reported by Evanno and coworkers [9]. However, to our knowledge, detailed anti-proliferative structure-activity relationship (SAR) for HO (1) is still insufficient up to now. In order to enrich the chemical diversity of HO and discover pharmacologically interesting compounds, a series of novel HO derivatives were semisynthesized through substitution at tropone, selective reduction of lactone, and allylic oxidation. Moreover, the antiproliferative activity of these derivatives against the HCT-116 (human colon cancer), A375 (human melanoma cancer), A-549 (human lung adenocarci-HCT-116 (human colon cancer), A375 (human melanoma cancer), A-549 (human lung adenocarcinoma cancer), and Huh-7 (human hepatoma cancer) was evaluated, and their SARs were discussed in the present study.

Chemical Synthesis
Two C-15 substituted analogues (2 and 3) were synthesized to probe and define the importance of the C-15 substituent. Scheme 1 outlines the synthesis of target compounds 2 and 3. The reaction of 1 with NH2-NH2·H2O in EtOH at room temperature (rt) produced 2 in 96% yield. Further amidation of 2 with acetyl chloride and Et3N in CH2Cl2 at rt provided 3 in 92% yield. To explore the role of lactone, compounds with reduced lactone ring (4 and 5) were synthesized as shown in Scheme 2. Selective reduction of the lactone with DIBAL-H at −78 °C afforded lactol 4 in 24% yield, which subsequently underwent an esterification reaction with Ac2O to give compound 5 in 92% yield.

Chemical Synthesis
Two C-15 substituted analogues (2 and 3) were synthesized to probe and define the importance of the C-15 substituent. Scheme 1 outlines the synthesis of target compounds 2 and 3. The reaction of 1 with NH 2 -NH 2 ·H 2 O in EtOH at room temperature (rt) produced 2 in 96% yield. Further amidation of 2 with acetyl chloride and Et 3 N in CH 2 Cl 2 at rt provided 3 in 92% yield. HCT-116 (human colon cancer), A375 (human melanoma cancer), A-549 (human lung adenocarcinoma cancer), and Huh-7 (human hepatoma cancer) was evaluated, and their SARs were discussed in the present study.

Chemical Synthesis
Two C-15 substituted analogues (2 and 3) were synthesized to probe and define the importance of the C-15 substituent. Scheme 1 outlines the synthesis of target compounds 2 and 3. The reaction of 1 with NH2-NH2·H2O in EtOH at room temperature (rt) produced 2 in 96% yield. Further amidation of 2 with acetyl chloride and Et3N in CH2Cl2 at rt provided 3 in 92% yield. To explore the role of lactone, compounds with reduced lactone ring (4 and 5) were synthesized as shown in Scheme 2. Selective reduction of the lactone with DIBAL-H at −78 °C afforded lactol 4 in 24% yield, which subsequently underwent an esterification reaction with Ac2O to give compound 5 in 92% yield. To explore the role of lactone, compounds with reduced lactone ring (4 and 5) were synthesized as shown in Scheme 2. Selective reduction of the lactone with DIBAL-H at −78 • C afforded lactol 4 in 24% yield, which subsequently underwent an esterification reaction with Ac 2 O to give compound 5 in 92% yield. In order to discover HO derivatives with diverse allyl alcohol replacements, allylic oxidation of HO with SeO2 was conducted. Interestingly, under the condition of Riley oxidation (SeO2/TBHP) at rt, compounds 6 with a double bond between C-6 and C-7 in 16% yield and 7 with A-ring contracted in 5% yield were produced as shown in Scheme 3. The structure of compound 6 is consistent with the natural product 6-en-harringtonolide [23] isolated from Cephalotaxus mannii as shown in Figure 1. The formation of compound 6 was likely based on the elimination of hydroxyl. A possible mechanism for the formation of 7 is presented in Scheme S1, which is meaningful to explore the conversion of tropone to benzene ring in cephalotane-type diterpenoids. Next, another route was designed to obtain our target compounds. At the beginning, HO (1) was treated with NBS/AIBN at 65 °C for 12 h to obtain compound 7α-Br harringtonolide (8) in 54% yield. Compound 8 was then subjected to hydrolysis with AgBF4 in acetone/H2O at 65 °C to produce 7β-OH harringtonolide (10) in 47% yield and 7α-OH harringtonolide (12) in 21% yield (Scheme 4). The β-orientation of 7-OH in compound 10 was deduced from the ROESY correlations ( Figure S1) of 7-H with 1-H and 10-H. The structure of compound 12 is identical to the natural product cephinoid F [8] isolated from Cephalotaxus lanceolata as shown in Figure 1. In order to discover HO derivatives with diverse allyl alcohol replacements, allylic oxidation of HO with SeO 2 was conducted. Interestingly, under the condition of Riley oxidation (SeO 2 /TBHP) at rt, compounds 6 with a double bond between C-6 and C-7 in 16% yield and 7 with A-ring contracted in 5% yield were produced as shown in Scheme 3. The structure of compound 6 is consistent with the natural product 6-en-harringtonolide [23] isolated from Cephalotaxus mannii as shown in Figure 1. The formation of compound 6 was likely based on the elimination of hydroxyl. A possible mechanism for the formation of 7 is presented in Scheme S1, which is meaningful to explore the conversion of tropone to benzene ring in cephalotane-type diterpenoids. In order to discover HO derivatives with diverse allyl alcohol replacements, allylic oxidation of HO with SeO2 was conducted. Interestingly, under the condition of Riley oxidation (SeO2/TBHP) at rt, compounds 6 with a double bond between C-6 and C-7 in 16% yield and 7 with A-ring contracted in 5% yield were produced as shown in Scheme 3. The structure of compound 6 is consistent with the natural product 6-en-harringtonolide [23] isolated from Cephalotaxus mannii as shown in Figure 1. The formation of compound 6 was likely based on the elimination of hydroxyl. A possible mechanism for the formation of 7 is presented in Scheme S1, which is meaningful to explore the conversion of tropone to benzene ring in cephalotane-type diterpenoids. Next, another route was designed to obtain our target compounds. At the beginning, HO (1) was treated with NBS/AIBN at 65 °C for 12 h to obtain compound 7α-Br harringtonolide (8) in 54% yield. Compound 8 was then subjected to hydrolysis with AgBF4 in acetone/H2O at 65 °C to produce 7β-OH harringtonolide (10) in 47% yield and 7α-OH harringtonolide (12) in 21% yield (Scheme 4). The β-orientation of 7-OH in compound 10 was deduced from the ROESY correlations ( Figure S1) of 7-H with 1-H and 10-H. The structure of compound 12 is identical to the natural product cephinoid F [8] isolated from Cephalotaxus lanceolata as shown in Figure 1. Next, another route was designed to obtain our target compounds. At the beginning, HO (1) was treated with NBS/AIBN at 65 • C for 12 h to obtain compound 7α-Br harringtonolide (8) in 54% yield. Compound 8 was then subjected to hydrolysis with AgBF 4 in acetone/H 2 O at 65 • C to produce 7β-OH harringtonolide (10) in 47% yield and 7α-OH harringtonolide (12) in 21% yield (Scheme 4). The β-orientation of 7-OH in compound 10 was deduced from the ROESY correlations ( Figure S1) of 7-H with 1-H and 10-H. The structure of compound 12 is identical to the natural product cephinoid F [8] isolated from Cephalotaxus lanceolata as shown in Figure 1.
Furthermore, to explore the effects of hydroxy group and its stereochemistry of HO on antiproliferative activity, a series of HO derivatives were designed in which the hydroxy at C-7 was modified as shown in Scheme 4. The cytotoxicity assay (vide infra) of these two compounds indicated that the stereochemistry at C-7 influences the cytotoxic activities with β-orientation favored. Thus, compound 10 was then coupled with acyl moieties to its 7β-OH position to afford a series of new ester derivatives 11a-11f. The 3,5,6trimethylpyrazine-2-carboxylic acid (TMPA), used in the synthesis of 11f, was obtained according to the previous report [24]. Interestingly, treatment of 8 with MeONa/MeOH at rt provided 9 with a methoxy substituent at tropone in 72% yield rather than allyl position, the generation of 9 may be through the rearrangement of allyl carbocation and subsequent replacement by methoxy anion (Scheme S2). The oxidation of hydroxy in compound 12 with Dess-Martin periodinane (DMP) in CH 2 Cl 2 at rt produced 13 in 78% yield.
Furthermore, to explore the effects of hydroxy group and its stereochemistry of HO on antiproliferative activity, a series of HO derivatives were designed in which the hydroxy at C-7 was modified as shown in Scheme 4. The cytotoxicity assay (vide infra) of these two compounds indicated that the stereochemistry at C-7 influences the cytotoxic activities with β-orientation favored. Thus, compound 10 was then coupled with acyl moieties to its 7β-OH position to afford a series of new ester derivatives 11a-11f. The 3,5,6trimethylpyrazine-2-carboxylic acid (TMPA), used in the synthesis of 11f, was obtained according to the previous report [24]. Interestingly, treatment of 8 with MeONa/MeOH at rt provided 9 with a methoxy substituent at tropone in 72% yield rather than allyl position, the generation of 9 may be through the rearrangement of allyl carbocation and subsequent replacement by methoxy anion (Scheme S2). The oxidation of hydroxy in compound 12 with Dess-Martin periodinane (DMP) in CH2Cl2 at rt produced 13 in 78% yield.

Cytotoxicity
All the synthesized derivatives of HO were tested for their antiproliferative activity against four human cancer cell lines, including the HCT-116, A375, A-549, and Huh-7 cell lines, using the MTT assay. Cisplatin was used as a positive control, while lead compound HO (1) was also included in the study for comparison. IC50 values (50% inhibition concentration of cell viability) of the tested compounds were summarized in Table 1.

Cytotoxicity
All the synthesized derivatives of HO were tested for their antiproliferative activity against four human cancer cell lines, including the HCT-116, A375, A-549, and Huh-7 cell lines, using the MTT assay. Cisplatin was used as a positive control, while lead compound HO (1) was also included in the study for comparison. IC 50 values (50% inhibition concentration of cell viability) of the tested compounds were summarized in Table 1.
The selectivity index is crucial for drug development, because it could mediate side effects, including tissues toxicity [25,26]. Therefore, according to cytotoxic activity results, compounds 6 and 10 with potent cytotoxicity in Huh-7 cells were selected to investigate the selectivity between normal and cancer cells. These two compounds were tested on human normal hepatic L-02 cells with HO as control. The results are listed in Table 2. Interestingly, compound 6 displayed obvious selectivity between Huh-7 and L-02 cells with SI = 56.5 compared with the parent compound 1 with SI = 2.8.

Discussion
Combined with previous reports [8,21], systematic SAR of the HO analogues as antiproliferative agents (Figure 2) was discussed as follows based on above-described results. (1) From the screening results in Table 1, it was observed that substitution with a bromine atom, an amino or a methoxy group at tropone (2-bromoharringtonolide [9] and compounds 2-3, 9), lack of tropone ring (fortalpinoid K [21]), and A-ring contraction of tropone (cephanolide A [20] and compound 7) in HO led to losing of cytotoxic activities.
(2) Reduction of lactone in HO also led to losing of cytotoxic activities as observed in the compounds 4 and 5. These results indicated that the tropone and lactone moieties are essential for the cytotoxic activities. (IC 50 = 0.61 and 1.25 µM, respectively) but obviously increased the selectivity between Huh-7 and L-02 cells, as observed in Table 2. The selectivity index of compound 6 (SI = 56.5) was 20 times higher than HO (SI = 2.8). (5) Carbonyl adjacent to the tropone motif led to losing of cytotoxic activities, as observed in 13 (IC 50 > 50 µM against tested cells).
HO (IC50 = 1.67 μM) or led to losing of cytotoxic activities as observed in compounds 11b and 11d (IC50 > 50 μM against tested cells). The compounds 11a, 11c, 11e, and 11f also showed weak cytotoxic activities against three other cancer cell lines. This could be attributed to the steric hindrance that affecting the binding of compounds to their targets [27,28]. (4) The presence of a double bond adjacent to the tropone unit had no obvious effects on the cytotoxic activities against HCT-116 and Huh-7 cells as observed in the cases of compound 6 (IC50 = 0.86 and 1.19 μM, respectively) compared with HO (IC50 = 0.61 and 1.25 μM, respectively) but obviously increased the selectivity between Huh-7 and L-02 cells, as observed in Table 2. The selectivity index of compound 6 (SI = 56.5) was 20 times higher than HO (SI = 2.8). (5) Carbonyl adjacent to the tropone motif led to losing of cytotoxic activities, as observed in 13 (IC50 >50 μM against tested cells). According to the SAR analysis of HO derivatives, compound 6 exhibited the most potent antiproliferative activity and low toxicity. Our studies indicated that analogue 6 could be further investigated as an antitumor drug candidate. Additionally, there is an urgent need for thorough research to understand the mechanism of action and targets of this kind of analogues.

Chemistry
HO, used as starting material, was isolated from Cephalotaxus fortunei Hook. f. by our group, and its structure was identified by ESI-MS and NMR. All reagents were purchased from Energy Chemical (Shanghai, China) and Aladdin (Shanghai, China) and were used without any further purification. Thin-layer chromatography (TLC) was performed using silica gel plates (GF254, Qingdao Marine Chemical Ltd., Qingdao, China) and visualized under ultraviolet (UV) light (254 nm). Silica gel column chromatography was performed using 200-300 mesh (Qingdao Marine Chemical Ltd., Qingdao, China).
NMR spectra were recorded on Bruker Avance III-500 and Bruker Avance III-600 spectrometers (Bruker, Karlsruhe, Germany) at ambient temperature using TMS as the internal standard. High-resolution electrospray ionization (HRESI) mass spectra were carried out using an Agilent 6520B Q-TOF mass spectrometer (Agilent Technologies, Santa According to the SAR analysis of HO derivatives, compound 6 exhibited the most potent antiproliferative activity and low toxicity. Our studies indicated that analogue 6 could be further investigated as an antitumor drug candidate. Additionally, there is an urgent need for thorough research to understand the mechanism of action and targets of this kind of analogues.

Chemistry
HO, used as starting material, was isolated from Cephalotaxus fortunei Hook. f. by our group, and its structure was identified by ESI-MS and NMR. All reagents were purchased from Energy Chemical (Shanghai, China) and Aladdin (Shanghai, China) and were used without any further purification. Thin-layer chromatography (TLC) was performed using silica gel plates (GF254, Qingdao Marine Chemical Ltd., Qingdao, China) and visualized under ultraviolet (UV) light (254 nm). Silica gel column chromatography was performed using 200-300 mesh (Qingdao Marine Chemical Ltd., Qingdao, China).

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.