Anal. Chem. 1995,67,2292-2295
Kinetic Analysis and Subambient Temperature Chromatography of an Active Ester John 0. Egekeze, Michael C. Danielski, Nelu Grinberg,* George B. Smiih, Daniel R. Sidler, Holly J. Perpall, Gary R. Bicker, and Pattkia C. Tway Merck Research Laboratories, R8OY-115, P.O.Box 2000,Rahway, New Jersey 07065-0914
The inherent instability of active esters provides a chromatographic challenge. A nonnal phase HPLC gradient was developed using nonprotic solvents and a diol stationary phase to determine the impurity profile of a mesylate which, under protic conditions, undergoes intramolecular ring closure to a cyclic ether. On-column cyclization of the analytewas found to be extensiveat room temperature but insiguificant at -30 "C.The on-column reaction rate was determined as a function of column temperature, and an Arrhenius activation energy was calculated. Advances in liquid chromatography, regarding the synthesis of stationary phases with controlled pores and particle sizes along with the development of chemically bonded phases, have led to explosive development in the field of analytical chemistry in the last decade. The speed, high efficiency, and easily manipulated selectivity of modem chromatographic systems continue to make this technique directly applicable in various fields. Production, identification, and impurity profile characterization of new compounds is performed routinely, consistently,and efficiently through the use of these chemically bonded phases. Chromatography has been applied in the pharmaceutical industry as a powerful analytical method for the determination of impurity profiles. However, when establishing the purity of a drug, care must be taken in interpreting chromatographic zones since not all of them may be due to impurities. Some chromatographic zones may be artifacts of the system due to degradation of the sample during the chromatographic process. We have reported several cases of such artifacts,'J and such phenomena have been extensively documented by othersS3 There are various sources of multiple zones in chromatography: chemical reactions," impurities present in the sample s ~ l u t i o ndiscontinuities ,~ in the stationary" and mobile phase^,^ formation of charged species and/or complexes! and equilibrium between speciesg It has been long recognized that a chemical reaction occurring during a chromatographicprocess can give rise to distorted peaks (1) Grinberg, N.; Bicker, G.; Tway, P.; Baiano, J. A J. Liq. Chromatogr. 1988, 11 (15), 3138. (2) Price, IC;Perpall, H.; Bicker, G.; Tway, P.; Grinberg, N.]. Liq. Chromatogr. 1990,13 (4), 2783. (3) Keller, R A; Giddings, J. C. J. Chromatogr. 1960,3, 205. (4) Zechmeister, L.; Cholnovky, L. Pnnciples and Practice of Chromatography: John Wiley & Sons: New York, 1951; p 5. (5) Strain, H. H. Ind. Eng. Chem. 1950,42, 1307. (6) Moore, S.; Stein, W. H. Annu. Rev. Biochem. 1952,23, 521. (7) Boman, H. G. Nature 1949,21, 215. (8) Landau, A; Fuerst, R; Awapar, R A Anal. Chem. 1951,23, 162. (9) Partridge, S. M.; Westall, R. G. Biochem. J. 1948,42, 238.
2292 Analytical Chemistry, Vol. 67,No. 73,July 7, 7995
and breakthrough curves.1o Many such reactions were reviewed recently by Jeng and Langer." Clearly, determining the kinetics of such reactions can help to establish proper chromatographic conditions so that extraneous peaks will be minimized. An undesired reaction may be mitigated by lowering the temperature at which the separation occurs. The influence of temperature on chromatographic separations has been extensively described in the literature. In general, temperature can have an effect on the mass transfer of the analyte between the mobile and stationary phases, and consequently can lead to improved separations.I2 Sheikh and Tou~hstone'~ reported improved separation of various steroids at subambient temperature using a nonaqueous mobile phase and a reversed phase column. Jinno et investigated the effect of lowering the temperature on the separation mechanism in reversed phase chromatography. The authors concluded that, at subambient temperature and with more than 9.8%water in the mobile phase, the retention mechanism was similar to that of a reversed phase separation at ambient temperature. Below 9.8%,the mechanism was found to be similar to that of a normal phase separation. Sentell et investigated the effect of temperature on the separation of polycyclic aromatic hydrocarbons under reversed phase conditions. The authors observed that the high bonding density of an octadecyl bonded phase afforded better selectivity than a low-density stationary phase. Also, shape selectivity was observed at 25 "C and below for both types of stationary phases. Greater selectivity at lower temperature was observed for the highdensity stationary phases. Van7 Hoff plots were linear for lowdensity stationary phases but nonlinear for high-density phases. This was due to a phase transition of the bonded phase from a liquid-like state to a more crystalline state at subambient temperatures. Henderson and HorvathI6and Henderson and M e l l ~showed '~ in early reports that lowering the temperature of separation of reversed phase systems retarded the cis-trans isomerization of proline-based peptides. Lowering the temperature of chromatographic separations has served to retard the decomposition of unstable mole~ules.~*J~ (10) Van Swaay, M. In Advances in Chromatography; Giddings, J. C., Keller. R. A, Eds.; Marcel Dekker, Inc.: New York, 1969 Vol. 8, p 364. (11) Jeng, C.-Y.; Langer, S. H. J. Chromatogr. 1992,589,1. (12) Snyder, L. R. J. Chromatogr. 1979,179,167. (13) Sheikh, S. U.; Touchstone, J. C. J. Liq. Chromatogr, 1987,10 (10, 2489. (14) Jinno, IC;Ohshima. T.; Hirata. Y. J. High Resolut. Chromatogr.,Chromatogr. Commun. 1982,5,621. (15) Sentell, K. B.; Henderson, A N. Anal. Chim. Acta 1991,246,139. (16) Henderson, D. E.; Horvath, Cs. J. Chromatogr. 1986,368, 203. (17) Henderson, D. E.; Mello, J. A./. Chromatogr. 1990,499,79.
0003-2700/95/0367-2292$9.00/0 0 1995 American Chemical Society
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Figure 1. Mesylation of the diol and cyclization of the active mesylate ester.
Active esters are common intermediatesin chemical synthesis, and their chromatography may be challenging because of their instability. On the other hand, their accurate quantitation may be essential in optimiig a synthesis. In this paper, we report the chromatographicseparation of an active mesylate, an intermediate in the synthesis of a leukotriene D4antagonist, whose tertiary alcohol moiety promotes cyclization to a cyclic ether. In order to quantitate the mesylate and examine its impurity profile, a normal phase gradient was designed using nonprotic solvents and a diol stationary phase column. It was discovered, however, that cyclization occurred on the stationary phase. The rate of oncolumn degradation was determined at several temperatures, and the chromatography was optimiied.
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EXPERIMENTAL SECTION
Solvents. Toluene, hexane, and ethyl acetate were purchased from EM Science (Gibbstown, NJ) . The solvents were dried over molecular sieves. Apparatus. Two HPLC systems were utilized for the analyses: a Spectra Physics P4000 ternary pump coupled with a Spectra system AS1000 autosampler (Spectra Physics, San Jose, CA) and a 759A absorbance detector (Applied Biosystems, Foster City, CA). A Beckman System Gold was used with a 126 programmable solvent module, a 167 scanning detector, and a 507 autosampler (Beckman, Fullerton, CA). The column was placed in a column jacket (Aura Industries, Staten Island, NY) containing a thermometer and connected to a constant temperature bath (Neslab Instruments, Newington, NH). Chromatogramswere processed using a PE Nelson version 1.8 data acquisition system (Cupertino, CA). IUMR Conditions. 'H NMR spectra were recorded using a Bruker AM 250 instrument. The compounds were collected from the chromatographic system. The solvent was removed and reconstituted in CDCh. The chemical shifts were reported in parts per million. Cyclization of the mesylate was readily apparent in the 'H NMR spectra due to a chemical shift of the mesylate methiie of 6 5.70 (dd,. I = 8.0,5.4 Hz,lH), while the cyclic ether methiie was shifted upfield to 6 4.57 (dd, 1= 10.7, 5.6 Hz,1H). The diol methine had a chemical shift of 6 4.69 (t, J = 6.4 Hz, 1H). (18) Henderson, D. E.; O'Connor, D. J. InAdvances in Chromutogmphy;Giddings, J. C., Gmshka, E., C a s , J., Brown, P. R, Eds.; Marcel Dekker, Inc.: New York, 1984; Vol. 23,p 65. (19) Hendenon, D. E.; O'Connor, D. J.; Kirby, J. F.; Sears, C. P., 111. J. Chromatogr. Sci. 1985,23, 477.
2
4
6
8
10
12
14
16
18
b
Time (min)
Figure 2. Chromatogram of a mesylate sample. Temperature, 25 "C. For other chromatographic conditions, see the Experimental Section. The diol peak corresponds to unreacted material prior to the chromatography.
Chromatography. A Chromegabond Diol 2 5 x 0.46-cm i.d. column (ES Industries, Berlin, NJ) was used with a 15min linear gradient from 15:85 to 5:95 (AB v/v), followed by a l(lmin hold at the final composition [where A consisted of toluene-#-hexane (1:3 v/v) and B ethyl acetate]. Analytes were dissolved in ethyl acetate at a concentration of 0.5 mg/mL, and 5 p L was injected into the HPLC system. Compounds were detected by W at 320 nm, at which all of the compounds of interest exhibited an absorbance maximum, characteristic of the quinoline functionality, with the same molar response. RESULTS AND DISCUSSION In the synthesis of leukotriene D4 antagonist MK-0476, diol 1 was reacted with mesyl chloride to obtain mesylate 2. Under
protic conditions, undesired cyclization to 3 occurred during the chromatography (Figure 1). A secondary elimination product, 4, with a double bond in the a position to the R group, formed also, but more slowly. Accurate quantitation of 2 was important. Careful determination of 2 in reaction mixtures established the exact amount of reactants to be used in the next step of the synthesis. Initially, using reversed phase chromatography, 2 disappeared completely with formation of 3 and 4. Therefore, a normal phase gradient was developed using a diol stationary phase and a mobile Analytical Chemistty, Vol. 67,No. 13, July 1, 1995
2293
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phase consisting of the aprotic solvents hexane, toluene, and ethyl acetate. With a column temperature of 25 "C, chromatograms of the mesylate included sharp peaks of 1 and 2 and a broad peak of 3 (Figure 2). When the eluted 3 was collected and reinjected, a sharp peak was observed with the early retention time of 4.5 min, identified as 3 by NMR It was assumed that injected 2 remained at or near the top of the column, cyclizing all the while to the rapidly eluting 3,until the gradient was polar enough to allow the unreacted 2 to elute. Thus the 3 formed from 2 on the column appeared as broad peak. Complete cyclization occurred when the gradient program was delayed for 15 min. The chromatogram of Figure 2 was examined from 3.8 min to the appearance of the diol peak at -12 min. To establish the kinetics of cyclization, the mesylate peak was collected and reinjected. A chromatogram like that shown in Figure 2 was obtained again. An estimated baseline was drawn from the beginning of the broad 3 peak to the end of the 2 peak, and perpendiculars (hi) were drawn at regular intervals. The his were assumed to be proportional to the amount of uncyclized 2 on the column at the corresponding retention time, and the retention time and reaction time scales were assumed to be equal. Plots of In hi versus t were linear (12 = 0.997) and thus consistent with pseudo-first-order cyclization:
ln{[21/[2,11= -kt
(1)
Upon lowering the column temperature from 25 to 10 "C, the cyclic ether peak decreased signilicantly, indicating slower cyclization ( F i r e 3). Applying a similar mathematical treatment as above, a series of plots were generated in the range of 10-30 "C. The slopes represent the rate constant of cyclization at the respective temperatures (Table 1). As expected, the slope of the lines decreases as the temperature decreases. A flow rate study showed that the rate constant did not change over a flow rate range of 0.5-2 mL/min. 2294 Analytical Chemistry, Vol. 67,No. 13, July 1, 1995
A
Table 1. Influence of Temperature on the Rate Constant of Cyclization
T 10 15 20 25 30
k (min-l)
SD (min-l)O
0.10 0.13 0.16 0.22 0.32
0.011 0.011 0.015 0.014 0.011
Standard deviation.
The relationship between the rate constant k and the temperature is given by the Arrhenius equation:
ln k = -Ea/RT + constant where Ea is the activation energy and R is the gas constant. According to eq 2, a plot of In k vs the reciprocal of the absolute temperature defines a straight line of slope -Ea/R. Indeed, a straight line was obtained (Z = 0.991), and the activation energy E, obtained from the slope of the graph was 9.3 kcal/mol, which corresponds approximately to a doubling of the rate with a 10 "C temperature increase.2O Figure 4 shows a -30 "C chromatogram of a synthetic mixture of 2 and its known impurities originating in the synthesis. No cyclization occurred at -30 "C in a l@min delay of the gradient program, and 2 was recovered quantitatively. The peak area of all the components of the chromatogram remained unchanged compared to a gradient without isocratic hold. The second elimination product, 4, observed in the chromatogram was not produced during chromatography but rather originated as a byproduct of the reaction. Since the chromatography under the optimized conditions produced a rugged separation, the next step was to establish the stability of the mesylate in the solvent solution. An ethyl acetate solution of (20) Adamson, A W. A Textbook ofPhpical Chemisty; Academic Press: New
York. 1973; p 639.
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Figure 5. Chromatogram of an actual sample of mesylate originated from the reaction mixture. For chromatographic conditions, see the Experimental Section.
0
0
5
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15
Time (min) Figure 4. Chromatogram of a synthetic mixture of mesylate and known impurities. For chromatographic conditions, see the Experimental Section. 2 was kept at room temperature and injected repeatedly over a
24h period. The mesylate peak area counts were recorded, and no changes occurred over the period of time investigated. S i c e 2 was stable in aprotic solution at room temperature, the next step was to investigate what factors were responsible for the cyclization of the mesylate in the column. The column used in our experimentsconsisted of a diol stationary phase. S i c e the recovery of the compoundsfrom the column was quantitative, the cyclization of the mesylate seemed to be due to residual silanols in the column. To prove this, the same gradient conditions were used, along with a silica gel stationary phase at 25 "C. Compound 3 formed, while 2 disappeared. It was concluded that the on-column reaction of the mesylate was silicacatalyzed cyclization. Figure 5 shows the separation at -30 "C of an actual reaction mixture from the synthesis of 2. Under these conditions, the reproducibility of the retention time for the components was 0.36% in day-today use of the same column, and 2%variation was observed when different columns were used.
CONCLUSIONS
The chromatographic analysis of an active ester was optimized. Chromatograms at several column temperatures revealed oncolumn cyclization of the ester. Pseudo-first-order rate constants were obtained, and an Arrhenius energy was calculated. The kinetic analysis justified the low-temperature chromatographic assay, making the on-column cyclization insignificant. The procedure demonstrated that a rate constant can be obtained from a single chromatographic experiment. An accurate chromatographic analysis resulted for samples from the synthesis of the activated ester. ACKNOWLEWMENT
The authors would like to thank Prof. Karen Sentell of the Department of Chemistry of The University of Vermont and Dr. B. Feibush of ES Industries for fruitful discussions during the preparation of this work. Received for review November 28, 1994. Accepted April 17, 1995.@ AC941134W e Abstract published
in Advance ACS Abstracts, June 1, 1995.
Analytical Chemistry, Vol. 67, No. 13, July 1, 7995
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