Design, Synthesis and Biological Evaluation of Jahanyne Analogs as Cell Cycle Arrest Inducers

Jahanyne, a lipopeptide with a unique terminal alkynyl and OEP (2-(1-oxo-ethyl)-pyrrolidine) moiety, exhibits anticancer activity. We synthesized jahanyne and analogs modified at the OEP moiety, employing an α-fluoromethyl ketone (FMK) strategy. Preliminary bioassays indicated that compound 1b (FMK–jahanyne) exhibited decreased activities to varying degrees against most of the cancer cells tested, whereas the introduction of a fluorine atom to the α-position of a hydroxyl group (2b) enhanced activities against all lung cancer cells. Moreover, jahanyne and 2b could induce G0/G1 cell cycle arrest in a concentration-dependent manner.

Jahanyne was initially isolated by Suenaga et al. as a secondary metabolite from cyanobacteria in 2015 [18]. The data from the initial MTT assays revealed that jahanyne inhibited the growth of both HL60 cells and HeLa cells with an IC 50 (half-inhibitory concentration) value at the micromolar level and could induce apoptosis in HeLa cells. The bolaamphiphile-mimicking structure of jahanyne and its potent anticancer activity have attracted attention not only from the synthetic community but also from the biological community [19][20][21][22]. Chandrasekhar et al. first completed the total synthesis of desmethyl jahanyne [19]. Preliminary MTT biological assays of desmethyl jahanyne and synthetic intermediates indicated that the N-methyl group and terminal alkynyl fatty acid motifs were essential for maintaining jahanyne's inhibitory activities against cancer cells. The authors further found that, based on a cellular thermal shift assay, several jahanyne intermediates with fatty acid moieties possibly bound to the P2 binding groove on BCL-2 (B cell lymphoma 2), an antiapoptotic protein. Recently, Brimble et al. made a breakthrough in designing the first synthetic route to jahanyne using a modified Fmoc solid-phase synthetic strategy, which could effectively couple multiple sterically-hindered N-methylated amino acids without epimerization [20]. Our previous study also provided a convergent and efficient synthetic route supplying us with multi-milligram jahanyne for further biological assays [21]. In the meantime, Suenaga et al. isolated two new jahanyne analogs-jahanane and jahanene [22]-and they achieved their total syntheses. Surprisingly, the MTT assays indicated that the growth-inhibitory activity of jahanyne was only one-tenth of the previously reported activity. They also discovered that a small degree of unsaturation of the terminus of the fatty acid moiety increased growth-inhibitory activity, which might be attributed to the interaction between the P2 binding groove on BCL-2 and the aliphatic chain of the jahanyne family. Jahanyne was initially isolated by Suenaga et al. as a secondary metabolite from cyanobacteria in 2015 [18]. The data from the initial MTT assays revealed that jahanyne inhibited the growth of both HL60 cells and HeLa cells with an IC50 (half-inhibitory concentration) value at the micromolar level and could induce apoptosis in HeLa cells. The bolaamphiphile-mimicking structure of jahanyne and its potent anticancer activity have attracted attention not only from the synthetic community but also from the biological community [19][20][21][22]. Chandrasekhar et al. first completed the total synthesis of desmethyl jahanyne [19]. Preliminary MTT biological assays of desmethyl jahanyne and synthetic intermediates indicated that the N-methyl group and terminal alkynyl fatty acid motifs were essential for maintaining jahanyne's inhibitory activities against cancer cells. The authors further found that, based on a cellular thermal shift assay, several jahanyne intermediates with fatty acid moieties possibly bound to the P2 binding groove on BCL-2 (B cell lymphoma 2), an antiapoptotic protein.
Recently, Brimble et al. made a breakthrough in designing the first synthetic route to jahanyne using a modified Fmoc solid-phase synthetic strategy, which could effectively couple multiple stericallyhindered N-methylated amino acids without epimerization [20]. Our previous study also provided a convergent and efficient synthetic route supplying us with multi-milligram jahanyne for further biological assays [21]. In the meantime, Suenaga et al. isolated two new jahanyne analogs-jahanane and jahanene [22]-and they achieved their total syntheses. Surprisingly, the MTT assays indicated that the growth-inhibitory activity of jahanyne was only one-tenth of the previously reported activity. They also discovered that a small degree of unsaturation of the terminus of the fatty acid moiety increased growth-inhibitory activity, which might be attributed to the interaction between the P2 binding groove on BCL-2 and the aliphatic chain of the jahanyne family.
The introduction of fluorine into amino acids benefits medicinal chemistry in several ways, such as increasing biological profile, interfering with metabolism, and modulating conformation evidenced by pharmaceutical drugs [23][24][25]. Further, α-fluoromethyl ketone (FMK) has been successfully developed as an electrophilic warhead with minimal off-target effects in the The introduction of fluorine into amino acids benefits medicinal chemistry in several ways, such as increasing biological profile, interfering with metabolism, and modulating conformation evidenced by pharmaceutical drugs [23][24][25]. Further, α-fluoromethyl ketone (FMK) has been successfully developed as an electrophilic warhead with minimal off-target effects in the development of covalent inhibitors [26,27]. To perform a preliminary evaluation of the structure and activity relationship of OEP moiety and gain a better anticancer lead compound based on jahanyne, we synthesized jahanyne and its analogs (Scheme 1). Herein, we reported our work on synthesis and preliminary biological evaluation of new fluoro analogs of jahanyne. Jahanyne was initially isolated by Suenaga et al. as a secondary metabolite from cyanobacteria in 2015 [18]. The data from the initial MTT assays revealed that jahanyne inhibited the growth of both HL60 cells and HeLa cells with an IC50 (half-inhibitory concentration) value at the micromolar level and could induce apoptosis in HeLa cells. The bolaamphiphile-mimicking structure of jahanyne and its potent anticancer activity have attracted attention not only from the synthetic community but also from the biological community [19][20][21][22]. Chandrasekhar et al. first completed the total synthesis of desmethyl jahanyne [19]. Preliminary MTT biological assays of desmethyl jahanyne and synthetic intermediates indicated that the N-methyl group and terminal alkynyl fatty acid motifs were essential for maintaining jahanyne's inhibitory activities against cancer cells. The authors further found that, based on a cellular thermal shift assay, several jahanyne intermediates with fatty acid moieties possibly bound to the P2 binding groove on BCL-2 (B cell lymphoma 2), an antiapoptotic protein.
Recently, Brimble et al. made a breakthrough in designing the first synthetic route to jahanyne using a modified Fmoc solid-phase synthetic strategy, which could effectively couple multiple stericallyhindered N-methylated amino acids without epimerization [20]. Our previous study also provided a convergent and efficient synthetic route supplying us with multi-milligram jahanyne for further biological assays [21]. In the meantime, Suenaga et al. isolated two new jahanyne analogs-jahanane and jahanene [22]-and they achieved their total syntheses. Surprisingly, the MTT assays indicated that the growth-inhibitory activity of jahanyne was only one-tenth of the previously reported activity. They also discovered that a small degree of unsaturation of the terminus of the fatty acid moiety increased growth-inhibitory activity, which might be attributed to the interaction between the P2 binding groove on BCL-2 and the aliphatic chain of the jahanyne family.
The introduction of fluorine into amino acids benefits medicinal chemistry in several ways, such as increasing biological profile, interfering with metabolism, and modulating conformation evidenced by pharmaceutical drugs [23][24][25]. Further, α-fluoromethyl ketone (FMK) has been successfully developed as an electrophilic warhead with minimal off-target effects in the development of covalent inhibitors [26,27]. To perform a preliminary evaluation of the structure and activity relationship of OEP moiety and gain a better anticancer lead compound based on jahanyne, we synthesized jahanyne and its analogs (Scheme 1). Herein, we reported our work on synthesis and preliminary biological evaluation of new fluoro analogs of jahanyne. Scheme 1. Analogs of jahanyne modified at the OEP moiety. Scheme 1. Analogs of jahanyne modified at the OEP moiety.

Chemistry
Based on the synthetic route we previously developed [21], the modified OEP moiety could be introduced at a late stage. The FMK warhead was constructed, employing commercially available compound Boc-OEP (3) as the starting material. The α-deprotonation of 3 with LDA (lithium diisopropylamide) followed by the slow addition of Selectfluor provided us with the Boc-OEP-FMK (4) in moderate yield. Then, we reduced the keto group of 4 using NaBH 4 to provide 5 in 56% yield (Felkin model). Deprotection of Boc with TFA (trifluoroacetic acid) provided us with 6, which was subjected to a coupling reaction with the acid obtained from the hydrolysis of methyl ester of 7 (Scheme 2).

Chemistry
Based on the synthetic route we previously developed [21], the modified OEP moiety could be introduced at a late stage. The FMK warhead was constructed, employing commercially available compound Boc-OEP (3) as the starting material. The α-deprotonation of 3 with LDA (lithium diisopropylamide) followed by the slow addition of Selectfluor provided us with the Boc-OEP-FMK (4) in moderate yield. Then, we reduced the keto group of 4 using NaBH4 to provide 5 in 56% yield (Felkin model). Deprotection of Boc with TFA (trifluoroacetic acid) provided us with 6, which was subjected to a coupling reaction with the acid obtained from the hydrolysis of methyl ester of 7 (Scheme 2).

Chemistry
Based on the synthetic route we previously developed [21], the modified OEP moiety could be introduced at a late stage. The FMK warhead was constructed, employing commercially available compound Boc-OEP (3) as the starting material. The α-deprotonation of 3 with LDA (lithium diisopropylamide) followed by the slow addition of Selectfluor provided us with the Boc-OEP-FMK (4) in moderate yield. Then, we reduced the keto group of 4 using NaBH4 to provide 5 in 56% yield (Felkin model). Deprotection of Boc with TFA (trifluoroacetic acid) provided us with 6, which was subjected to a coupling reaction with the acid obtained from the hydrolysis of methyl ester of 7 (Scheme 2).

Biological Activity against Cancer Cells
The in vitro anticancer activities of the four compounds in diverse cancer cells were examined by a Cell Counting Kit-8 (CCK-8) assay.
As shown in Table 1, all the compounds displayed obvious inhibition against cancer cells with moderate to high potencies (IC 50 = 7.64 − 29.68 µM), whereas compound 2b presented the most promising activity against H820 cells. A previous study has shown that the natural and synthetic jahanyne exhibit suppression against HL60 cells with IC 50 values of 4.6 µM and 8.3 µM, respectively [18]. Similarly, the IC 50 value of the jahanyne (1a) synthesized by us against HL60 cells was 13.98 µM. When we applied the FMK strategy using a fluorine atom as a replacement for the hydrogen atom of OEP moiety (1b), the inhibitory activity was not improved compared with that of 1a, except in A549 cells. Compounds 2a and 2b, containing a hydroxyl group, showed decreased activities against HL60 cells, and compound 2b showed a two-fold decrease compared to 1a. Among H1688 cells, H1299 cells, and H820 cells, compounds 2a and 2b showed more potent activities than compounds 1a and 1b. Unfortunately, these compounds exhibited no significantly improved activities against A549 cells with IC 50 values ranging from 13.83 µM to 16.65 µM. It was worth noting that α-fluoromethyl of ketone groups caused decreased activities in varying degrees against H1688 cells, H1299 cells, and H820 cells, whereas the introduction of a fluorine atom to the α-position of the hydroxyl group enhanced activities against all lung cancer cells.

Compound 1a Induced G0/G1 Phase Arrest
To investigate whether the cell growth inhibitory effects of compounds 1a and 2b are caused by cell cycle progression, H820 cells were treated with compounds 1a and 2b at different concentrations (4,8,12, and 16 µM). The cell cycle was analyzed by flow cytometry after staining the DNA with propidium iodide (PI). As shown in Figure 2, after the treatment of compounds 1a and 2b for 48 h, the population of cells in the G0/G1 phase increased (78.37% and 78.46%, respectively) compared to the control (55.15%). Inversely, the S phase cell population dramatically decreased. Therefore, these results indicated that compounds 1a and 2b could induce G0/G1 phase arrest in a concentration-dependent manner.

Cell Apoptosis Effects of Compound 2b and 1a
To evaluate the cell apoptosis effects of compounds 1a and 2b, H820 cells were treated with compounds 1a and 2b at different concentrations (4,8,12, and 16 µM). The cell apoptosis was analyzed by flow cytometry after staining the DNA with 7-aminoactinomycin D (7AAD)/Annexin-V. As shown in Figure 3, compared with the control group, the average rate of apoptosis in H820 cells did not change, but there was a statistical difference. These results suggested that the cell growth inhibitory effects of compounds 1a and 2b were not via the induction of apoptosis.

Cell Apoptosis Effects of Compound 2b and 1a
To evaluate the cell apoptosis effects of compounds 1a and 2b, H820 cells were treated with compounds 1a and 2b at different concentrations (4,8,12, and 16 μM). The cell apoptosis was analyzed by flow cytometry after staining the DNA with 7-aminoactinomycin D (7AAD)/Annexin-V. As shown in Figure 3, compared with the control group, the average rate of apoptosis in H820 cells did not change, but there was a statistical difference. These results suggested that the cell growth inhibitory effects of compounds 1a and 2b were not via the induction of apoptosis.

Cell Apoptosis Effects of Compound 2b and 1a
To evaluate the cell apoptosis effects of compounds 1a and 2b, H820 cells were treated with compounds 1a and 2b at different concentrations (4,8,12, and 16 μM). The cell apoptosis was analyzed by flow cytometry after staining the DNA with 7-aminoactinomycin D (7AAD)/Annexin-V. As shown in Figure 3, compared with the control group, the average rate of apoptosis in H820 cells did not change, but there was a statistical difference. These results suggested that the cell growth inhibitory effects of compounds 1a and 2b were not via the induction of apoptosis.
All reactions were carried out with dry solvents under anhydrous conditions under an argon atmosphere unless otherwise mentioned. Tetrahydrofuran (THF) was distilled from sodium-benzophenone ketyl before using. Reactions were checked by thin-layer chromatography (TLC) on silica gel plates and visualized by UV light and basic aqueous potassium permanganate or aqueous phosphomolybdic acid. Column chromatography was carried out using 200-300 mesh silica gel. Chromatography solvents were purchased from Tianjin Chemical Reagent (Tianjin, China).

Chemistry
Compound 7 was obtained following the procedure reported previously.

Cell Culture
Human leukemia cell line HL60 was purchased from the American Type Culture Collection (USA). Human lung cancer cell lines H820, H1299, and A549 were purchased from the National Infrastructure of Cell Line Resource (Beijing, China). The H1688 cell line was donated by Tianjin Medical University Cancer Institute and Hospital. These cancer cells were cultured in 1640 medium supplemented with 10% FBS and 100 units of penicillin-streptomycin under 5% CO 2 at 37 • C.

CCK-8 Assay
Cells were seeded with a density of 3000-10,000 cells/100 µL/well into a 96-well plate. After 18-24 h, the compounds were diluted to different concentrations and added to the 96-well plate. After incubation for 72 h, the CCK-8 reagent was added and incubated at 37 • C for an additional 1-4 h. The absorbance was measured at 450 nm using a microplate reader. The 50% inhibitory concentration (IC 50 ) values were calculated using GraphPad Prism 5 (GraphPad, San Diego, CA, USA).

Cell Cycle Assay
The H820 cells were seeded into a 6-well plate with a density of 5 × 10 5 cells/3 mL/well. Compounds 2b and 1a at different concentrations (0 µM, 4 µM, 8 µM, 12 µM, and 16 µM) were added and incubated for 48 h. After being collected, the cells were immobilized with 75% ethanol on ice for 2 h. The cells were washed twice with PBS and incubated with propidium iodide (PI; Sigma, St. Louis, MO, USA) for 15 min. The cells were analyzed by flow cytometry (NovoCyte, ACEA, San Diego, CA, USA).

Conclusions
By coupling and DMP oxidation, we synthesized jahanyne and its fluoro analogs. Preliminary bioassays showed that 2b showed increased activities towards H1688 cells, H1299 cells, and H820 cells compared to jahanyne. However, 1b (FMK-jahanyne) exhibited decreased activities to varying degrees against most of the cells tested. The results indicated that the unique terminal OEP moiety might not interact with its target protein in a covalent manner. Jahanyne and compound 2b behaved in a similar manner in regulating the G0/G1 phase cell cycle. With an alkynyl group, jahanyne itself could act as a probe to investigate its detailed mode of action. The target verification, together with its medicinal work, is ongoing and will be reported in due course.