Sodium-Periodate-Mediated Harringtonine Derivatives and Their

Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. J. Nat. Prod. , Article ASAP. DOI:...
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Sodium-Periodate-Mediated Harringtonine Derivatives and Their Antiproliferative Activity against HL-60 Acute Leukemia Cells Seiichi Sakamoto,* Tomofumi Miyamoto, Kazuteru Usui, Hiroyuki Tanaka, and Satoshi Morimoto Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan S Supporting Information *

ABSTRACT: Harringtonine (HT) is a naturally occurring alkaloid isolated from the plant genus Cephalotaxus. It possesses antileukemic activity and has been clinically utilized for the treatment of acute leukemia and lymphoma. Sodium periodate (NaIO4) was reacted with HT to produce five HT derivatives including four novel compounds. Their antiproliferative activity against HL-60 acute promyelocytic leukemia cells revealed that the presence of the C-5′ methyl group enhances the antiproliferative activity because the IC50 values of the HT derivatives, including HT1 (5′-de-O-methylharringtonine), were at least 2000 times higher (>100 μM) than that of HT (∼47 nM). In addition, an indirect competitive enzyme-linked immunosorbent assay (icELISA) using a monoclonal antibody against HT (mAb 1D2) revealed that these antiproliferative activities were related to their cellular uptake. These results indicated that esterification of HT1 at the C-4′ carboxylic acid group may enhance the antiproliferative activity of HT.

H

leukemia cells,1−3 HT1−5 were investigated for their antiproliferative activities against HL-60 acute leukemia cells. The results revealed that the C-5' methyl group considerably enhanced the activity. In addition, the cellular uptake of HT and HT derivatives (HT1 and HT3) was investigated by icELISA using mAb against HT (mAb 1D2);12 this revealed that their uptake is related to antiproliferative activity. This study describes the synthesis, isolation, structure, reaction mechanism, and biological activity of HT derivatives produced by treatment with NaIO4.

arringtonine (HT; Figure 1), classified as a Cephalotaxus alkaloid, is mainly produced by the evergreen conifer tree of the Cephalotaxus genus. Owing to the strong cytotoxic activity of HT and homoharringtonine (HHT) against leukemia cells, including mouse L-1210 leukemia cells and P388 lymphocytic cells,1−3 these compounds have been well studied and used in China for the treatment of chronic myeloid leukemia (CML), acute myeloid leukemia (AML), and myelodysplastic syndrome (MDS).4−6 Furthermore, they have attracted substantial attention as antileukemia drugs for chemotherapy by the National Cancer Institute and the U.S. Department of Agriculture since the early 1970s. HHT was approved by the U.S. Food and Drug Administration in 2012 and used clinically for the treatment of adult patients with CML who are resistant or intolerant to tyrosine kinase inhibitors. This antileukemia drug is marketed as Synribo (omacetaxine mepesuccinate) by Teva Pharmaceutical Industries Ltd.7−11 In a recent study, we produced an HT-specific monoclonal antibody (mAb) to develop an indirect competitive enzymelinked immunosorbent assay (icELISA)12 and an immunochromatographic strip assay (ICA)13 that can be applied for sensitive and specific determination of HT in plant samples. In the process of preparing an immunogen to produce mAb, HT was found to be conjugated with carrier proteins, such as bovine serum albumin and human serum albumin (HSA) after treatment with sodium periodate (NaIO4).12 In the present study, five HT derivatives (HT1−5) were produced by NaIO4mediated reactions in the absence of a carrier protein. Because HT has been shown to possess strong cytotoxic activity against © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Structure Determination of CT1 and HT Derivatives. The chemical structures of cephalotaxine derivative (CT1) and HT2−5 were assigned using NMR, HR-ESITOFMS, IR, and UV data. The 1H and 13C NMR chemical shifts are described in ppm (δ) and summarized in Tables 1 and 2, respectively. CT1 displayed a sodium adduct ion at m/z 352.1219 [M + Na]+, indicative of a molecular formula of C18H19NO5. The IR spectrum showed an amide carbonyl absorption at 1650 cm−1. The 13C NMR spectrum of CT1 showed five methylene carbons (δC 31.0, 32.0, 38.7, 39.4, 102.2) and a carbonyl carbon (δC 176.8), which suggested that CT1 is an oxoderivative of cephalotaxine at C-8 or C-10. The COSY spectrum gave three spin−spin correlations from H-3 (δH 4.74) to H-4 (δH 3.73), H2-6 (δH 2.18, 2.20) to H2-7 (δH 2.06, Received: June 23, 2017

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DOI: 10.1021/acs.jnatprod.7b00541 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 1. Chemical structures of CT, HT, and its derivatives.

was observed. These data suggested that the structure of HT4 was 11-nor-10-oxoharringtonine. The molecular formula of HT5 was determined to be C26H31NO10 from the sodium adduct molecular ion at m/z 540.1825. From the 1H and 13C NMR chemical shifts, assigned by COSY, TOCSY, HSQC, and HMBC experiments, and the UV bathochromic effect (302.4 nm), the pentacyclic core of HT5 was found to be identical to that of HT4, and the acyl moiety of HT5 was identical to that of HT2. Accordingly, the structure of HT5 was determined to be 5′-de-O-methyl-11-nor10-oxoharringtonine. Mechanism of NaIO4-Mediated Oxidative Reaction. Treatment of HT with NaIO4 in carbonate buffer led to the formation of lactams and carboxylic acid derivatives (HT1−5). The methyl ester of HT was selectively hydrolyzed by NaIO4 to give HT1 presumably due to steric hindrance of HT. With respect to the formation of the other HT derivatives (HT2−5), the mechanisms for the NaIO4-mediated oxidative transformations are illustrated in Scheme 1. The oxidation (C−H bond activation)15 of the α-methylene carbon followed by hydration of iminium ion A leads to hemiaminal B (path a), which undergoes further oxidation to give the corresponding γlactams HT2 and HT3. This mechanism may be similar to that postulated for cytochrome P-450 (CYP)-catalyzed oxidation of amines or RuO4 oxidation of activated methylene groups.16 In contrast, lactams HT4 and HT5 may be formed through intermediate C, which resulted from benzylic oxidation (path b). Thus, a subsequent oxidation of intermediate C may afford intermediate E via iminium ion D. Finally, intermediate E may undergo a ring-rearrangement process similar to that postulated for NaIO4-mediated oxidative transformation of 3benzazepin-1-one17 to furnish the corresponding δ-lactams HT4 and HT5. HL-60 Acute Leukemia Cell Proliferation Assay. The antiproliferative activity of Cephalotaxus alkaloid including derivatives HT1−5 against HL-60 acute leukemia cells was evaluated by a CellTiter 96 AQueous nonradioactive cell proliferation (MTS) assay (Promega, WI, USA), which is a colorimetric assay for assessing cellular metabolic activity.

2.15), and H2-10 (δH 3.15, 3.91) to H2-11 (δH 2.43, 3.49). HMBC correlations were observed between the carbonyl carbon and H2-6, H2-7, and H2-10 (Figure 2). These findings suggested that CT1 was an 8-oxo derivative of CT. The structure of HT1 was defined as 5′-de-O-methylharringtonine by comparison of the observed and reported 1H and 13C NMR spectroscopic data.14 The molecular formula of HT2 was determined to be C27H33NO10 from the sodium adduct molecular ion at m/z 554.2018. From the 1H and 13C NMR chemical shifts assigned by COSY, TOCSY, HSQC, and HMBC experiments, the pentacyclic core of HT2 was identical to that of HT3 and CT1, and the acyl moiety was assumed to be a de-O-methyl derivative of HT, because only one O-methyl group (δH 3.77, δC 58.5) and a carbonyl carbon at C-8 (δC 177.0) were observed in HT2. The structure of HT2 was defined as 5′-deO-methyl-8-oxoharringtonine. HR-ESITOFMS of HT3 showed the sodium adduct molecular ion at m/z 568.2294 [(M + Na) + for C28H35NO10], and this molecular formula is 14 mass units higher than that of HT. The 13C NMR spectroscopic data of the acyl moiety of HT3 were identical to those of HT, and those of the pentacyclic core were similar to those of CT1. The HMBC correlations were also observed between the carbonyl carbon (δC 176.8) and H2-6 (δH 2.22), H2-7 (δH 2.15, 2.20), and H2-10 (δH 3.14, 3.97). Accordingly, the structure of HT3 was determined to be 8-oxoharringtonine. HR-ESITOFMS of HT4 gave a sodium adduct molecular ion at m/z 554.2018, and the molecular formula was estimated to be C27H33NO10. The 1H and 13C NMR spectroscopic data suggested that the acyl moiety of HT4 was the same as that of HT and HT3; however, the pentacyclic core was different. The COSY and TOCSY spectra showed two spin−spin correlations from H-3 (δH 6.01) to H-4 (δH 4.14) and from H2-6 (δH 1.82, 1.89) and H2-7 (δH 1.94, 2.08) to H2-8 (δH 3.37, 4.17). The HMBC correlation between the carbonyl carbon (δC 164.2) and H-17 (δH 7.44) confirmed the C-10 location of the carbonyl group (Figure 2). In addition, a UV bathochromic effect (300.6 nm), which is derived from extended conjugation, B

DOI: 10.1021/acs.jnatprod.7b00541 J. Nat. Prod. XXXX, XXX, XXX−XXX

2.62 (m), 2.84 (dt, 7.0, 11.5)

2.32 (dd, 7.0, 14.5), 3.41 (ddd, 8.0, 12.0, 14.5) 6.66 (s) 6.64 (s) 5.86 (d, 1.0), 5.87 (d, 1.0)

10

11

C

(d, 0.5) (dd, 0.5, 9.0) (d, 9.0) (m), 2.20 (m) (m), 2.15 (m)

3.77 (3H, s)

2.43 (ddd, 1.5, 7.0, 14.5), 3.49 (ddd, 7.5 12.0, 14.5) 6.71 (s) 6.63 (s) 5.86 (d, 1.0), 5.87 (d, 1.0)

3.15 (m), 3.91 (dt, 7.0, 12.5)

4.79 4.74 3.73 2.18 2.06

CT1

*Overlapped chemical shifts were shown as asterisk.

5′ 1″ 2″ 4″ 5″

19 3′

3.71 (3H, s)

(brs) (d, 9.0) (d, 9.0) (m), 1.96 (m) (m), 1.79 (m) (m), 2.92 (dt, 3.5, 9.5)

5.01 4.71 3.65 1.86 1.71 2.61

1 3 4 6 7 8

14 17 18

CT

H (d, 1.0) (dd, 1.0, 10.0) (d, 10.0) (m), 2.03 (m) (m), 1.83 (m) (m) 2.95 (dt, 4.0, 9.5)

3.55 1.56 1.32 1.12 1.15

(3H, s) (2H, m) (m), 1.54 (m) (3H, s) (3H, s)

3.69 (3H,s) 1.87 (d, 16.0), 2.19 (d, 16.0)

2.44 (dd,7.0, 14.5), 3.21 (ddd, 7.0, 12.5, 14.0) 6.59 (s) 6.67 (s) 5.84 (d, 1.0), 5.89 (d,1.0)

2.63 (m), 2.88 (dt, 7.0, 12.0)

5.22 5.98 3.89 1.95 1.74 2.64

HT (brs) (brd, 9.5) (d, 9.5) (2H, m) (m), 2.19 (m)

1.53 1.27 1.10 1.13

(2H, m) (m), 1.51 (m) (3H, s) (3H, s)

3.77 (3H,s) 1.78 (d, 16.0), 2.03 (d, 16.0)

3.15 (dd, 7.0, 12.5), 3.96 (dt, 6.0, 13.0) 2.55 (dd, 6.0, 14.0), 3.31 (*) 6.65 (s) 6.67 (s) 5.86 (d, 1.5), 5.87 (d, 1.5)

4.99 5.88 3.99 2.20 2.13

HT2

Table 1. 1H NMR Spectroscopic Data of CT, CT1, HT, HT2, HT3, HT4, and HT5 (600 MHz, Methanol-d4) (d, 1.0) (dd, 1.0, 9.5) (d,9.5) (2H, m) (m), 2.20 (m)

3.77 (3H, s) 1.92 (d, 16.0), 2.22 (d, 16.0) 3.35 (3H, s) 1.56 (2H, m) 1.32 (m), 1.56 (m) 1.13 (3H, s) 1.16 (3H, s)

3.14 (dd, 6.5, 13.0), 3.97 (dt, 6.5, 13.0) 2.53 (dd, 6.0, 14.5), 3.29 (m) 6.65 (s) 6.66 (s) 5.86 (d, 1.0), 5.88 (d, 1.0)

5.02 6.03 4.03 2.22 2.15

HT3 (s) (d,7.0) (d,7.0) (m), 1.89 (m) (m), 2.08 (m) (m), 4.17 (*)

6.76 (s) 7.44 (s) 5.98 (d, 1.0), 6.01 (d, 1.0) 3.64 (3H, s) 2.03 (d, 15.5), 2.25 (d, 15.5) 3.62 (3H, s) 1.49 (m), 1.57 (m) 1.19 (m), 1.49 (m) 1.07 (3H, s) 1.10 (3H, s)

5.30 6.01 4.14 1.82 1.94 3.37

HT4

1.52 1.24 1.06 1.08

(2H, m) (m), 1.53 (m) (3H, s) (3H, s)

6.72 (s) 7.45 (s) 6.01 (d, 1.5), 6.04 (d, 1.5) 3.63 (3H, s) 1.98 (d,17.0), 2.08 (d, 17.0)

5.26 (s) 6.08 (d, 7.5) 4.12 (d, 7.5) 1.85 (m), 1.89 (m) 1.94 (m), 2.06 (m) 3.39 (ddd, 4.5, 10.5, 12.5), 4.12 (*)

HT5

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DOI: 10.1021/acs.jnatprod.7b00541 J. Nat. Prod. XXXX, XXX, XXX−XXX

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C NMR Spectroscopic Data of CT, CT1, HT, HT2, HT3, HT4, and HT5 (125 MHz, Methanol-d4)

C

CT

CT1

HT

HT2

HT3

HT4

HT5

1 2 3 4 5 6 7 8 10 11 12 13 14 15 16 17 18 19 1′ 2′ 3′ 4′ 5′ 1″ 2″ 3″ 4″ 5″

97.7 (d) 163.2 (s) 74.3 (d) 57.5 (d) 72.1 (s) 43.9 (t) 20.7 (t) 54.4 (t) 49.6 (t) 32.3 (t) 135.3 (s) 130.7 (s) 113.7 (d) 147.8 (s) 147.2 (s) 110.9 (d) 101.9 (t) 58.7 (q)

100.1 (d) 165.2 (s) 74.4 (d) 60.0 (d) 73.3 (s) 31.0 (t) 38.7 (t) 176.8 (s) 39.4 (t) 32.0 (t) 134.4 (s) 130.5 (s) 113.5 (d) 148.2 (s) 147.5 (s) 111.2 (d) 102.2 (t) 58.0 (q)

100.8 (d) 159.9 (s) 75.4 (d) 56.5 (d) 72.2 (s) 44.4 (t) 20.8 (t) 54.6 (t) 49.6 (t) 32.2 (t) 134.5 (s) 129.9 (s) 114.0 (d) 148.2 (s) 147.3 (s) 110.9 (d) 102.1 (t) 57.8 (q) 174.6 (s) 75.8 (s) 43.9 (t) 171.8 (s) 52.0 (q) 35.1 (t) 37.5 (t) 70.7 (s) 28.5 (q) 29.9 (q)

102.1 (d) 163.1 (s) 74.5 (d) 58.2 (d) 73.0 (s) 32.0 (t) 38.5 (t) 177.0 (s) 40.3 (t) 32.0 (t) 132.3 (s) 130.6 (s) 114.0 (d) 147.4 (s) 147.1 (s) 110.5 (d) 101.9 (t) 58.5 (q) 174.2 (s) 75.9 (s) 44.4 (t) 171.9 (s)

103.0 (d) 161.9 (s) 75.1 (d) 57.4 (d) 72.9 (s) 30.8 (t) 38.8 (t) 176.8 (s) 39.7 (t) 31.7 (t) 133.5 (s) 129.8 (s) 113.8 (d) 148.7 (s) 147.7 (s) 111.1 (d) 102.4 (t) 58.4 (q) 174.4 (s) 75.8 (s) 44.3 (t) 171.7 (s) 50.0 (q) 35.1 (t) 37.5 (t) 70.7 (s) 28.7 (q) 29.8 (q)

105.2 (d) 161.1 (s) 79.0 (d) 45.7 (d) 72.0 (s) 37.3 (t) 20.9 (t) 44.1 (t) 164.2 (s)

104.7 (d) 163.7 (s) 78.3 (d) 46.0 (d) 71.8 (s) 37.8 (t) 20.9 (t) 44.1 (t) 160.7 (s)

123.9 (s) 130.7 (s) 110.3 (d) 152.6 (s) 149.0 (s) 107.8 (d) 103.3 (t) 58.0 (q) 174.4 (s) 75.9 (s) 44.4 (t) 171.7 (s) 52.1 (q) 35.1 (t) 37.6 (t) 70.7 (s) 28.6 (q) 29.7 (q)

123.5 (s) 130.6 (s) 110.2 (d) 152.8 (s) 149.2 (s) 107.9 (d) 103.4 (t) 57.9 (q) 175.5 (s) 76.3 (s) 45.0 (t) 175.5 (s)

35.0 37.6 70.7 28.7 29.7

(t) (t) (s) (q) (q)

34.8 38.1 70.9 28.6 29.6

(t) (t) (s) (q) (q)

study, HT1 (5′-de-O-methylharringtonine) and HT3 (8oxoharringtonine), in which CR of mAb 1D2 against HT1 and HT3 was found to be 3.4% and 1133.2%, respectively,12 were selected as representative HT derivatives because their structural differences with HT are simple. For this analysis, HL-60 cells (5.0 × 106) were treated with HT, HT1, and HT3, each at final concentrations of 1, 10, and 100 μM, and incubated in a CO2 incubator for 3 h at 37 °C. After washing the cells, they were treated with radio-immunoprecipitation assay (RIPA) buffer and subjected to icELISA for detection. Interestingly, HT1 was not detected at all and only low levels of HT3 were detected by icELISA, although high levels of HT were detected (Table 4), corresponding well to their respective antiproliferative activities. Thus, the weak antiproliferative activities of HT1 and HT3 were related to their lower levels of uptake into HL-60 cells. These results suggested that the esterification of the C-4′ carboxylic acid moiety of HT1 with other alcohols, such as EtOH, propyl alcohol, or n-BuOH, may increase its antiproliferative activity because the activity of HT was completely suppressed by hydrolysis of the methyl ester moiety. In conclusion, five HT derivatives (HT1−5) were found to be produced by NaIO4-mediated reactions with HT, four of which were novel compounds. To date, NaIO4 has been reported to catalyze unique and unpredictable oxidative reactions, as has been well summarized by Sudalai et al.15 Our study revealed another utility of NaIO4 as a hydrolyzing or oxidative reagent involving HT in the field of chemical synthesis. Moreover, subsequent antiproliferative activity tests of these HT derivatives against HL-60 acute leukemia cells revealed that much of the strong antiproliferative activity of

Figure 2. COSY and HMBC key correlations of CT1 and HT4.

Because the number of viable cells reflects NADH-dependent cellular oxido-reductase, which transforms TMS to formazan, possessing a maximum absorbance at 490 nm, antiproliferative activity can be evaluated by calculating IC50 values based on that absorbance. Upon using this approach, HHT (∼22 nM) and HT (∼47 nM) exhibited strong activity, although CT (∼61.2 μM) showed weak activity (Table 3). These results corresponded to previous reports identifying IC50 in HL-60 cells.18−20 Interestingly, the antiproliferative activity of the other HT derivatives was at least 2000 times lower than that of HT. The structures of HT1 and HT3 are simply different from that of HT in terms of a methyl group at C-5′ and a carbonyl group at C-8, suggesting that the presence of Me-5′ is essential for the activity. Uptake of HT and HT Derivatives (HT1 and HT3) by HL-60 Cells. To gain more insight into their antiproliferative activity, the uptake levels of HT and its derivatives (HT1 and HT3) in HL-60 cells by icELISA using mAb 1D2 were investigated. By taking advantage of wide cross-reactivity (CR) of mAb 1D2 against HT derivatives, these can be quantitatively analyzed by icELISA using mAb 1D2 (Figure 3). In the present D

DOI: 10.1021/acs.jnatprod.7b00541 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Scheme 1. Proposed Reaction Mechanism for the NaIO4-Mediated Oxidative Transformations of HT

silica gel 60F254 and TLC silica gel 60 RP-18 F254s (Merck, Darmstadt, Germany). HT (98%) was purchased from LKT Laboratories, Inc. (St. Paul, MN, USA). HHT (98%) and CT (98%) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA) and Toronto Research Chemicals (Toronto, ON, Canada), respectively. Fetal bovine serum (FBS) was purchased from Nichirei Bioscience Inc. (Tokyo, Japan) and heat-inactivated at 56 °C for 30 min for cell culture. All other chemicals were standard commercial products of analytical-reagent grade. Synthesis and Isolation of HT Derivatives by NaIO4Mediated Reaction. The products obtained by NaIO4-mediated reactions were investigated by large-scale synthesis, isolation, and structure determination, in which a reaction similar to that utilized for preparing HT−carrier protein conjugates was used without carrier proteins. Briefly, HT (90.1 mg) and NaIO4 (90.2 mg) were dissolved in 40% MeOH (22 mL) and distilled H2O (22 mL), respectively. The HT solution was slowly added dropwise to the NaIO4 solution, and the mixture was added to 50 mM carbonate buffer solution (pH 9.6, 44 mL), followed by stirring at room temperature for 20 h. After the MeOH was evaporated under vacuum, the reaction products were isolated by reverse-phase chromatography using Cosmosil 75C18OPN (Nacalai Tesque, Kyoto, Japan) with various concentrations of MeCN (30−50%) and silica gel 60 (0.063−0.200 mm) (Merck, Darmstadt, Germany) with a combination of CHCl3 and MeOH containing 1% ammonia to give five compounds, HT1 (25.5 mg), HT2 (24.8 mg), HT3 (11.3 mg), HT4 (2.5 mg), and HT5 (6.1 mg), and recovered HT (6.6 mg). Considering the initial amount of HT (90.1 mg) and the obtained amount of reaction products (76.8 mg), the recovery rate was ∼85%. Utilizing the same conditions, CT1 (29.9 mg) was obtained from CT (50.0 mg) via a NaIO4-mediated reaction. 5′-De-O-methyl-8-oxoharringtonine (HT2): white, amorphous solid; [α]25D −101 (MeOH; c 0.7); UV (MeOH) λmax 289.2 nm

Table 3. Antiproliferative Activity of HT Derivatives against HL-60 Acute Leukemia Cells compound

IC50 (μM)a

CT HHT HT HT1 HT2 HT3 HT4 HT5

61.2 ± 8.7 0.022 ± 0.001 0.047 ± 0.004 >100 >100 >100 >100 >100

The values represent mean ± SD from triplicate tests.

a

HT is contributed by the presence of the methyl group at C-5′, raising the possibility that the esterification of the C-4′ carboxylic acid moiety with other alcohols may enhance the antiproliferative activity compared with that of HT. NaIO4 may open the door to the development of new drug candidates for the treatment of acute leukemia and lymphoma in the future.

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EXPERIMENTAL SECTION

General Experimental Procedures. NMR spectra were recorded on a Varian INOVA 600 operating at 600 MHz for 1H NMR and 125 MHz for 13C NMR in methanol-d4. HR-ESITOFMS data were acquired with a micrOTOF II mass spectrometer (Bruker Daltonics, Bremen, Germany). Optical rotations were measured with a DIP-370 digital polarimeter (Jasco, Tokyo, Japan) at 25 °C. IR and UV spectra were measured with an FT/IR-410 spectrophotometer (Jasco, Tokyo, Japan) and V-630 UV−vis spectrophotometer (Jasco, Tokyo, Japan), respectively. TLC analysis was conducted using TLC E

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Figure 3. Calibration curves for the determination of (A) HT, (B) HT1, and (C) HT3 by icELISA using mAb 1D2. Concentrations of mAb 1D2 and HT−HSA conjugates were 500 ng/mL and 1 μg/mL, respectively. A0 and A correspond to the absorbance in the absence and presence of HT, respectively. Table 1; 13C NMR, Table 2; HR-ESITOFMS m/z [M + Na]+ 554.2018 (calcd for C27H33NNaO10, 554.2002). 5′-De-O-methyl-11-nor-10-oxoharringtonine (HT5): white, amorphous solid; [α]25D −129 (MeOH; c 0.2); UV (MeOH) λmax 302.4 nm (log ε 2.31); IR (KBr) νmax 3415 (br), 1739, 1597, 1463 cm−1; 1H NMR, Table 1; 13C NMR, Table 2; HR-ESITOFMS m/z [M + Na]+ 540.1825 (calcd for C26H31NNaO10, 540.1846). 8-Oxocephalotaxine (CT1): white, amorphous solid; [α]25D −116 (MeOH; c 0.7); UV (MeOH) λmax 290.0 nm (log ε 3.91); IR (KBr) νmax 3287 (br), 2929, 1650, 1489 cm−1; 1H NMR, Table 1; 13C NMR, Table 2; HR-ESITOFMS m/z [M + Na]+ 352.1219 (calcd for C18H19NNaO5, 352.1161). HL-60 Acute Leukemia Cell Proliferation Assay. HL-60 cells (human promyelocytic leukemia cells) were cultured in RPMI-1640 medium (Gibco, Manchester, UK) supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL), at 37 °C in a humidified incubator and a 5% CO2 atmosphere. Cell viability was determined by the MTS assay in accordance with a slightly modified version of the manufacturer’s protocol. Briefly, HL-60 cells were seeded in 96-well plates at a density of 2.0 × 104 cells/mL (180 μL/ well) in the presence of increasing concentrations of CT, HHT, HT, and HT1−5 (20 μL/well) for 62 h, after which 10 μL of MTS reagent was added to each well. The absorbance at 492/620 nm of the reaction product, soluble formazan, was measured after 2 h using a microplate reader (Multiskan FC microplate photometer; Thermo Fisher Scientific, Inc., Waltham, MA, USA). IC50 values were calculated with dose−response data using linear regression (Microsoft Excel), in which the curve was drawn by plotting viability against the logarithm of concentration of test compounds.

Table 4. Uptake Levels of HT, HT1, and HT3 by HL-60 Acute Leukemia Cells Determined by icELISA Using MAb 1D2 HT (μM)

amount of HT (ng)/5 × 106 cells

uptake rate (%)

1 10 100 HT1 (μM)

177 325 1864 amount of HT1 (ng)/5 × 106 cells

3.34 0.61 0.35 uptake rate (%)

1 10 100 HT3 (μM)

n.d. n.d. n.d. amount of HT3 (ng)/5 × 106 cells

n.d. n.d. n.d. uptake rate (%)

1 10 100

5 10 95

0.09 0.02 0.02

(log ε 4.22); IR (KBr) νmax 3430 (br), 2971, 1743, 1653, 1505, 1489 cm−1; 1H NMR, Table 1; 13C NMR, Table 2; HR-ESITOFMS m/z [M + Na]+ 554.2018 (calcd for C27H33NNaO10, 554.2002). 8-Oxoharringtonine (HT3): white, amorphous solid; [α]25D −82 (MeOH; c 0.5); UV (MeOH) λmax 289.6 nm (log ε 4.45); IR (KBr) νmax 3421 (br), 2971, 1748, 1654, 1505, 1489 cm−1; 1H NMR, Table 1; 13C NMR, Table 2; HR-ESITOFMS m/z [M + Na]+ 568.2294 (calcd for C28H35NNaO10, 568.2159). 11-nor-10-Oxoharringtonine (HT4): white, amorphous solid; [α]25D −110 (MeOH; c 0.1); UV (MeOH) λmax 300.6 nm (log ε 1.33); IR (KBr) νmax 3421 (br), 1746, 1600, 1463 cm−1; 1H NMR, F

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Journal of Natural Products

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Uptake of HT and HT Derivatives (HT1 and HT3) by HL-60 Cells. HL-60 cells were suspended in 25 cm2 plastic tissue culture flasks (Nunc Thermo Fisher Scientific, Waltham, MA, USA) at a density of 5.0 × 105 cells/mL (10 mL) and incubated in a CO2 incubator for 1 h. The samples (HT, HT1, and HT3) were added to HL-60 cells at final concentrations of 1, 10, and 100 μM and further incubated for 3 h. The HL-60 cells were collected by centrifugation at 3000 rpm for 5 min at room temperature, after which they were resuspended in phosphate-buffered saline (PBS; 10 mL) for washing. This washing step was repeated three times for the complete removal of compounds around the cells. Next, the cells were resuspended with RIPA buffer consisting of 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and 1% NP 40 at a density of 1.0 × 107 cells/mL and incubated on ice for 20 min. After centrifugation at 14 000 rpm for 20 min at 4 °C, the supernatants were stored at −20 °C until use. icELISA Using mAb 1D2. The levels of uptake of HT, HT1, and HT3 by HL-60 cells were analyzed by icELISA, an assay previously developed using an mAb 1D2 (Figure 3).12 Briefly, HT−HSA conjugates were immobilized on the surface of each well of an immunoplate (Nunc, Maxisorb, Roskilde, Denmark) by incubating (2 μg/mL, 100 μL/well) in 50 mM carbonate buffer (pH 9.6) for 1 h at 37 °C. Surfaces were blocked with PBS containing 5% skimmed milk (300 μL/well), to prevent nonspecific binding of proteins, for 1 h at 37 °C. Subsequently, various concentrations of HT (0.38−195 ng/ mL), HT1 (12.2 ng/mL−6.25 μg/mL), and HT3 (0.024−12.2 ng/ mL) (50 μL/well), or serially diluted supernatant as mentioned above (50 μL/well), were incubated with mAb 1D2 (500 ng/mL, 50 μL/ well) for 1 h at 37 °C. The plate was treated with a 5000-fold diluted solution of goat F(ab) anti-mouse IgG H&L (HRP) (100 μL/well) for 1 h at 37 °C to detect mAb 1D2 binding to immobilized HT− HSA conjugates. In the last step, color was developed by addition of the substrate solution, which comprised 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (0.3 mg/mL) in 0.1 M citrate buffer (pH 4.0) supplemented with 0.003% (v/v) H2O2 (100 μL/well) for 15 min at 37 °C. The absorbance at 405 nm of the developed color was measured using a Multiskan microplate reader, as described above. PBS containing Tween-20 (PBST, 0.05%) was used to wash the plate three times between each step.

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00541. H and

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REFERENCES

(1) Powell, R. G.; Weisleder, D.; Smith, C. R., Jr.; Wolff, I. A. Tetrahedron Lett. 1969, 10, 4081−4084. (2) Powell, R. G.; Weisleder, D.; Smith, C. R., Jr.; Rohwedder, W. K. Tetrahedron Lett. 1970, 11, 815−818. (3) Powell, R. G.; Weislede, D.; Smith, C. R. J. Pharm. Sci. 1972, 61, 1227−1230. (4) Cao, P. S.; Liu, X.; Chin, J. Hematol. 1990, 11, 425. (5) Feldman, E. J.; Seiter, K. P.; Ahmed, T.; Baskind, P.; Arlin, Z. A. Leukemia 1996, 10, 40−42. (6) O’Brien, S.; Kantarjian, H.; Keating, M.; Beran, M.; Koller, C.; Robertson, L. E.; Hester, J.; Rios, M. B.; Andreeff, M.; Talpaz, M. Blood 1995, 86, 3322−3326. (7) Chen, Y.; Li, S. OncoTargets Ther. 2014, 7, 177−186. (8) Gandhi, V.; Plunkett, W.; Cortes, J. E. Clin. Cancer Res. 2014, 20, 1735−1740. (9) Wetzler, M.; Segal, D. Curr. Pharm. Des. 2011, 17, 59−64. (10) Al Ustwani, O.; Griffiths, E. A.; Wang, E. S.; Wetzler, M. Expert Opin. Pharmacother. 2014, 15, 2397−2405. (11) Quintas-Cardama, A.; Kantarjian, H.; Cortes, J. Cancer 2009, 115, 5382−5393. (12) Sakamoto, S.; Yusakul, G.; Tsuneura, Y.; Putalun, W.; Usui, K.; Miyamoto, T.; Tanaka, H.; Morimoto, S. Analyst 2017, 142, 1140− 1148. (13) Sakamoto, S.; Yusakul, G.; Nuntawong, P.; Kitisripanya, T.; Putalun, W.; Miyamoto, T.; Tanaka, H.; Morimoto, S. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2017, 1048, 150−154. (14) Takano, I.; Yasuda, I.; Nishijima, M.; Hitotsuyanagi, Y.; Takeya, K.; Itokawa, H. Phytochemistry 1996, 43, 299−303. (15) Sudalai, A.; Khenkin, A.; Neumann, R. Org. Biomol. Chem. 2015, 13, 4374−4394. (16) (a) Legacy, C. J.; Wang, A.; O’Day, B. J.; Emmert, M. H. Angew. Chem., Int. Ed. 2015, 54, 14907−14910. (b) Chiavarino, B.; Cipollini, R.; Crestoni, M. E.; Fornarini, S.; Lanucara, F.; Lapi, A. J. Am. Chem. Soc. 2008, 130, 3208−3217. (c) Piperno, A.; Chiacchio, U.; Iannazzo, D.; Giofrè, S. V.; Romeo, G.; Romeo, R. J. Org. Chem. 2007, 72, 3958−3960. (17) Möhrle, H.; Rohn, C.; Westle, G. Pharmazie 2006, 61, 391− 399. (18) Takemura, Y.; Ohnuma, T.; Chou, T. C.; Okano, T.; Holland, J. F. Cancer Chemother. Pharmacol. 1985, 14, 206−210. (19) Kantarjian, H. M.; Talpaz, M.; Santini, V.; Murgo, A.; Cheson, B.; O’Brien, S. M. Cancer 2001, 92, 1591−1605. (20) Efferth, T.; Sauerbrey, A.; Halatsch, M. E.; Ross, D. D.; Gebhart, E. Naunyn-Schmiedeberg's Arch. Pharmacol. 2003, 367, 56− 67.

ASSOCIATED CONTENT

1

Article

NMR spectra of CT, CT1, HT, HT2, HT3,

HT4, and HT5 (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel and Fax: +8192-642-6581. ORCID

Seiichi Sakamoto: 0000-0001-6871-522X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The research was funded by the Qdai-jump Research Program, Wakaba Challenge of Kyushu University. This work was also funded by a Grant-in-Aid for Young Scientists (B) of the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number JP17K15466. G

DOI: 10.1021/acs.jnatprod.7b00541 J. Nat. Prod. XXXX, XXX, XXX−XXX