CHIRALITY 27:538–542 (2015)

Short Communication Enantioseparation of the Six Important Intermediates of Homoharringtonine With Immobilized Cellulose Chiral Stationary Phase JIE ZHOU,1,2 QIUZHENG DU,1 FANG SUN,1 QIAN LIU,1 AND ZHENZHONG ZHANG1,2* 1 School of Pharmacy, Zhengzhou University, Zhengzhou, Henan, P.R. China 2 Key Laboratory of State Ministry of Education for Pharmaceutical Technology, Zhengzhou, Henan, P.R. China

ABSTRACT A new liquid chromatographic method has been developed for the chiral separation of the enantiomers of intermediates in the preparation of the ester side-chain of homoharringtonine. The enantiomers were separated by a Chiralpak IC (250 × 4.6 mm, 5 μm) in normal phase high-performance liquid chromatography (HPLC). Four compounds were baseline resolved. By comparing the chromatographs of racemates and single enantiomers of the six intermediates, the enantiomeric excess values of the single enantiomers were evaluated, and the elution orders of the enantiomers were established. Chirality 27:538–542, 2015. © 2015 Wiley Periodicals, Inc.

KEY WORDS: chiralpak IC; e.e. values; enantiomeric separation; homoharringtonine; HPLC Homoharringtonine (HHT) (as shown in Figure 1) is a cephalotaxus alkaloid obtained from the evergreen tree Cephalotaxus harringtonia present in China, and native to seven species of this genus.1 HHT has been clinically tested in advanced breast cancer and acute myeloid leukemia.2 As a result of nearly 40 years of intensive research, numerous strategies for the elaboration of the side-chain of HHT, or its cyclic equivalents, have been developed.3 However, there is a marked stereorequirement of the structure of the sidechains for both their antitumor activity and toxicity; thus, epi-HHT, which only differs from HHT in the configuration of the stereogenic center at C-2’, exhibited no significant antineoplastic activity.4 Manipulations in the side-chain of HHT may produce second-generation HHT analogs that may be devoid of the cardiovascular toxicities and may have broader or different antitumor efficacy profiles. Due to its structural feature and the unique bioactivity, HHT has become an attractive target for organic synthesis, so the determination of the structure and the enantiomeric excess (e.e.) values of the intermediates in the preparation of the ester side-chain of HHT is very important. There are many methods for determining the e.e. values, for example, polarimetry, high-performance liquid chromatography (HPLC), gas chromatography (GC), and nuclear magnetic resonance (NMR). However, there have been few reports for the separation and the determination the e.e. values of the intermediates of HHT. In this study, Chiralpak IC was used to determine the e.e. values of the single intermediates (as shown in Figure 2) and separate their racemates for the first time. Relative to similar columns, Chiralpak IC might possess advantages in terms of robustness and the range of mobile phase solvents that can be utilized.5–7 Its robustness and universal solvent resistance is conferred by immobilization. In HPLC, the most versatile mobile phases on Chiralpak IC are hexaneiso-propanol, hexane-ethanol, methyl t-butyl ether-based, and dichloromethane-based eluents. © 2015 Wiley Periodicals, Inc.

Herein we report the results of our studies concerning the effect of variation of experimental conditions and optimization of the chiral separation of the enantiomers of the six intermediates. The e.e. values of the enantiomers were also determined. EXPERIMENTAL Apparatus The HPLC instrument used in this study was an Agilent 1100 series apparatus (Palo Alto, CA). It was equipped with a quaternary pump, a vacuum degasser, a column oven, a multiple wavelength UV detector, an autosampler, and HP Chemstation software. The analysis was carried out with a Chiralpak IC (250 × 4.6 mm, 5 μm; Daicel, Japan) column.

Reagents HPLC-grade n-hexane, ethanol, and isopropanol were obtained from T&J Kermel Reagent (Tianin City, China). Racemates and single enantiomers of the six intermediates were prepared by Yang et al., who devoted themselves to the enantioselective synthesis of the side-chain of HHT and harringtonine. The synthetic scheme of HHT and harringtonine has been reported.8

Sample Preparation The 1.0 mg racemic compounds (1–6) were dissolved in 1.0 mL ethanol. The solutions were all filtered (0.45 μm) to prepare sample solution.

Chromatographic Condition The basic solvent of the mobile phase was n-hexane, with isopropanol or ethanol as the polarity modifier. The mobile phase was filtered with a 0.45 μm solvent filter and ultrasonically degassed. The detection wavelengths of the six compounds were all set at 220 nm. The volume of sample injected was 5 μL or 10 μL. The dead time was determined by injecting 1, 3, 5-tert-butyl benzene as a nonretained marker. *Correspondence to: Zhenzhong Zhang, School of Pharmacy, Zhengzhou University, Zhengzhou, Henan 450001, P.R. China. E-mail: [email protected] Received for publication 19 November 2014; Accepted 17 March 2015 DOI: 10.1002/chir.22451 Published online 21 May 2015 in Wiley Online Library (wileyonlinelibrary.com).

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SIX IMPORTANT INTERMEDIATES OF HOMOHARRINGTONINE

Hydrogen bonding, π–π, dipole–dipole stacking, and steric interactions, hydrophobic interactions between analytes and chiral stationary phase (CSP) played an important role in the mechanism of enantiorecognition.9 By increasing the alcohol content of the mobile phase, its eluting strength increased, the hydrophobic interactions between analytes and CSP were weakened, and the resolution was decreased. Therefore, the logical approach to improving chiral resolution was to allow longer retention. However, in cases when retention times were longer, further decreasing the organic modifier content in the mobile phase might produce baseline separation only with very long retention times or might not work at all. This latter phenomenon could be explained by an excessive increase in peak widths noticed at long retention times occasionally counterbalancing any benefit with regard to selectivity.10 By mobile phase modified with ethanol, the column could afford shorter retention times than with isopropanol, but the resolution was decreased.

Fig. 1. The structure of homoharringtonine.

Effect of Column Temperature

Fig. 2. The structure of the single intermediates.

RESULTS AND DISCUSSION Effect of Alcohols Modifier

The effect that the content of mobile phase modifiers (ethanol or isopropanol) had on the enantioselectivity of the six racemic compounds is shown in Table 1.

The effect that the column temperature had on the six racemic compounds was studied (as shown in Table 2). Several authors have reported improved resolution on polysaccharide-based CSP when the column was operated at subambient temperature11,12 (e.g., compounds (1), (2), (3), (5), and (6)). But in the current study, it was found that the resolution of a compound might be increased by the increasing temperature (e.g., compound (4)).

TABLE 1. Effect of content of alcohols on the enantioselectivity of the six racemic compounds Isopropanol (modifier)

Ethanol (modifier)

tR1 (min)

tR2 (min)

α

RS

%

tR1 (min)

tR2 (min)

α

RS

%

(1)

— 14.629 8.529

— 18.946 9.831

— 5 10 20 2* 5 10 5 10 20 30 40 5 10 20 30 40 30 40 10 20 30 40

9.593 9.735 6.377

40.057 17.999

— 1.09 0.59 — 1.65 0.59 — — 7.36 5.78 4.65 4.21 — 13.05 8.56 6.34 5.38 — 14.00 — 4.68 3.76 3.29

8.721 8.858 6.028

(2)

— 1.37 1.24 — 1.21 1.13 — — 1.35 1.29 1.26 1.25 — 1.81 1.66 1.59 1.50 — 2.24 — 1.24 1.21 1.20

1.15 1.15 1.11 — — — — 1.20 1.17 1.15 1.13 1.13 1.42 1.34 1.27 1.13 1.21 1.65 1.63 1.73 1.14 1.13 1.11

1.85 1.09 0.55 — — — — 4.92 3.71 2.86 2.51 2.41 10.23 6.15 4.13 2.51 2.47 10.13 9.26 13.71 2.89 2.24 1.81

5* 5 10 20 — 5 10 5 10 20 30 40 5 10 20 30 40 30 40 10 20 30 40

Compound

5.764 46.865 20.045 10.572 >100

(3) 35.401 20.068 14.169 11.979

46.856 25.14 17.129 14.261 >100

(4) 47.123 19.233 12.168 9.237

83.01 29.977 17.511 12.324 >100

(5) 26.403

55.383 >100

(6) 31.945 20.312 14.136

38.953 24.167 16.421

4.939 — 8.949 6.458 26.447 16.135 10.484 8.382 7.373 58.791 22.947 11.111 8.382 6.645 14.259 11.574 37.915 15.563 10.450 8.169

31.344 18.366 11.597 9.116 7.935 82.376 29.784 13.326 9.116 7.387 21.381 16.938 63.339 17.356 11.395 8.756

tR1, tR2: retention times; α: separation factor; RS: resolution factor; “—”: retention time is too long (>100 min). *The volume of sample injected was 5 μL. The other injection volume was 10 μL. Chromatographic conditions: the basic solvent of mobile phase was n-hexane, the column temperature was at 25 °C with a flow rate of -1 0.8 mL · min . Chirality DOI 10.1002/chir

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TABLE 2. Effect of temperature on the enantioselectivity of the racemic compounds Compound (1)

(2)

(3)

(4)

(5)

(6)

tR1(min)

tR2(min)

α

RS

Temperature (°C)

9.181 8.904 8.721 8.598 8.408 47.212 42.277 40.057 34.698 31.691 7.886 7.716 7.373 7.294 6.931 7.049 6.867 6.645 6.593 6.308 13.661 11.748 11.574 11.059 10.206 12.119 11.454 10.450 10.070 9.639

10.122 9.816 9.593 9.461 9.253 55.985 49.728 46.865 41.049 37.296 8.588 8.359 7.935 7.838 7.388 7.922 7.660 7.387 7.313 6.923 21.308 17.359 16.938 15.873 14.143 13.356 12.568 11.395 10.934 10.418

1.16 1.15 1.15 1.15 1.14 1.19 1.19 1.21 1.18 1.17 1.15 1.13 1.13 1.13 1.12 1.20 1.21 1.21 1.22 1.22 1.72 1.64 1.63 1.60 1.55 1.13 1.13 1.13 1.12 1.11

1.89 1.88 1.85 1.82 1.78 2.02 1.77 1.65 1.30 1.10 2.92 2.43 2.41 2.40 2.05 2.32 2.36 2.47 2.55 2.56 9.34 9.35 9.26 9.06 8.60 2.34 2.34 2.24 2.20 2.15

15 20 25 30 35 15 20 25 30 35 15 20 25 30 35 15 20 25 30 35 15 20 25 30 35 15 20 25 30 35

Chromatographic conditions: compounds (3), (4), and (5): n-hexane-ethanol (60/40, V/V); compound (1): n-hexane-ethanol (95/5, V/V); compound (2): n-hexane-isopropanol (98/2, V/V); compound (6): n-hexane-ethanol (70/30, V/V). The injection volume of (1) and (2) was 5 μL; for (3), (4), (5), and (6), -1 it was 10 μL. The flow rate was 0.8 mL · min .

Parameters were calculated by Equation

13

ΔR, SΔH (KJ/mol)

ΔR,SΔS (J/(mol · K))

(1)

-0.055

-0.060

(2)

-0.070

-0.079

(3)

-0.082

-0.168

(4)

0.069

0.416

(5)

-0.400

-0.874

(6)

-0.068

-0.128

Compound

Here, α = k’2/k’1, α was separation factor, R is the gas constant, and T is the temperature in K, ΔR, SΔH and ΔR, SΔS were enthalpy change and entropy change, respectively. With lnα for the vertical axis Y, 1 / T abscissa X, Y on X mapping. The regression equations are shown in Table 3. ΔR, SΔH and ΔR,SΔS were obtained from the slope and intercept of the straight lines, respectively (as reported in Table 3). Over the temperature ranges of 288–308 K, for compounds (1), (2), (3), (5) and (6), |ΔR,SΔH | > |TΔR,SΔS |, so the chiral separation process was controlled by enthalpy. For compound (4), |ΔR,SΔH | < |TΔR,SΔS |, so the chiral separation process was controlled by entropy. The results showed that the column temperature should be carefully controlled for optimum chiral separation of enantiomers retention. And 25 °C was closer to room temperature, so the other parameters were optimized at 25 °C. Effect of Flow Rate

The effect the flow rate had on the six racemic compounds was studied from 0.4 to 1.2 mL · min-1 (as shown in Table 4).

Regression equation lnα = 61.701/T- 0.0674 (R = 0.8943) lnα = 74.839/T-0.0841 (R = 0.9413) lnα = 94.299/T-0.1927 (R = 0.8717) lnα = -73.483/T + 0.439 (R = 0.9477) lnα = 413.58/T- 0.9018 (R = 0.9691) lnα = 78.315/T-0.1461 (R = 0.8745)

TABLE 4. Effect of flow rate on the enantioselectivity of the racemic compounds Compound (1)

(2)

(3)

(4)

as below:

ln α ¼ -ΔR;S ΔH=RT þ ΔR;S ΔS=R

Chirality DOI 10.1002/chir

TABLE 3. Thermodynamic data of the six racemic compounds

(5)

(6)

tR1(min)

tR2(min)

α

RS

Flow rate (mL · min-1)

17.524 11.717 8.721 7.005 5.853 82.097 52.215 40.057 30.904 26.685 14.739 9.855 7.373 5.894 4.920 15.161 8.896 6.645 5.300 4.428 23.566 15.825 11.574 9.384 7.846 21.687 14.516 10.450 8.615 7.223

19.294 12.901 9.593 7.707 6.458 99.988 62.737 46.865 37.116 31.687 15.850 10.613 7.935 6.348 5.310 16.677 9.902 7.387 5.885 4.916 34.710 23.354 16.938 13.876 11.618 23.717 15.866 11.395 9.412 7.892

1.16 1.15 1.15 1.15 1.15 1.23 1.21 1.21 1.20 1.20 1.12 1.12 1.13 1.12 1.13 1.17 1.21 1.21 1.20 1.21 1.64 1.64 1.63 1.63 1.63 1.13 1.13 1.13 1.13 1.13

2.07 1.98 1.85 1.76 1.70 1.68 1.66 1.65 1.56 1.40 2.60 2.61 2.41 2.30 2.08 3.36 2.67 2.47 2.30 2.21 11.16 10.21 9.26 8.48 7.86 2.63 2.50 2.24 2.16 2.03

0.4 0.6 0.8 1.0 1.2 0.4 0.6 0.8 1.0 1.2 0.4 0.6 0.8 1.0 1.2 0.4 0.6 0.8 1.0 1.2 0.4 0.6 0.8 1.0 1.2 0.4 0.6 0.8 1.0 1.2

Chromatographic conditions: compounds (3), (4), and (5): n-hexane-ethanol (60/40, V/V); compound (1): n-hexane-ethanol (95/5, V/V); compound (2): n-hexane-isopropanol (98/2, V/V); compound (6): n-hexane-ethanol (70/30, V/V). The injection volume of (1) and (2) was 5 μL; for (3), (4), (5), and (6), it was 10 μL. The column temperature was 25 °C.

With an increased flow rate of the mobile phase, decreased resolution was observed. By the Van Deemter chromatography theory, the vertical proliferation played a major role at a lower flow rate, and the mass transfer resistance played a major role at a higher rate. In order to reduce the analytical time, and to get better separation, a flow rate of 0.8 mL · min-1 was used.

SIX IMPORTANT INTERMEDIATES OF HOMOHARRINGTONINE

541

Fig. 3. HPLC chromatograms of the racemic compounds.

Fig. 4. Chromatograms of the single enantionmers of the six single compunds.

The chromatograph of the six compounds is shown in Figure 3. Determination of the e.e. values

The e.e. values of the single enantiomers of the six important intermediates were determined (as shown in Figure 4). The results showed that the first effluent of compounds (1), (4), and (5) was determined as S-isomer, and the after effluent as R-isomer. For compounds (2), (3), and (6), the first effluent was determined as R-isomer. The e.e. values of the single enantiomers of the six intermediates were determined (e.e. 95.5%, e.e. 87.7%, e.e. 99.8%, e.e. 99.9%, e.e. 91.5%, and e.e. 91.6%, respectively). CONCLUSION

The enantiomers of the six intermediates in the preparation of the ester side-chain of HHT were first separated with Chiralpak IC in normal phase HPLC. The optimum chromatographic conditions were as follows: compound (1): n-hexaneethanol (95/5, V/V), RS = 1.85; compound (2): n-hexaneisopropanol (98/2, V/V), RS = 1.62; compounds (3), (4), and (5) was n-hexane-ethanol (60/40, V/V), RS = 2.41, RS = 2.47, and RS = 9.26, respectively; compound (6): n-hexane- ethanol (70/30, V/V), RS = 2.24. The elution order and the e.e. values of the six intermediates were also determined. For compounds (1), (4), and (5), the first effluent was determined as S-isomer; for compounds (2), (3), and (6), the first effluent was determined as R-isomer. Their e.e. values were 95.5%, 87.7%, 99.8%, 99.9%, 91.5%, and 91.6%, respectively.

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