The Separation of the Enantiomers of some Potassium Channel Activators on C l-Acid Glycoprotein: the Effect of Solute Conformation

R. Smith* / D. Smith / J. Evans / G. Stemp

SmithKline Beecham Pharmaceuticals, Coldharbour Road, The Pinnacles, Harlow, Essex, CM19 5AD, UK

Key Words t~l-Acid glycoprotein Potassium channel activators 3,4-Dihydro-2,2'-dimethyl-2H-l-benzopyran- enantiomers Arnide conformation

Summary The amide conformers of two compounds and their enantiomers have been separated by liquid chromatog- raphy on an O~l-acid glycoprotein column. The effects of organic modifier and pH on the separations obtained Were investigated. The conformers of one of the Compounds were separated micro-preparatively at low temperature on the same column using a D20-based eluent; D20 had no deleterious effects on the chroma- tography obtained. Some preliminary competition ex- Periments on one of the amides and a close analogue are presented.

Introduction

3,4-Dihydro-2,2-dimethyl-2H-l-benzopyrans such as 1- 4 and the related tetrahydronaphthalene 5 (Table I gives the structures) are potent antihypertensive agents Which relax smooth muscle by activating potassium Channels [1, 2]. These compounds contain two chiral Centres but the relative stereochemistry of these is fixed xn the trans configuration, so that each compound exists as only one pair of enantiomers. Previous work [3] has Shown that this series of closely-related compounds behave differently when chromatographed on an ~l-acid glycoprotein (CHIRAL-AGP) column with eluents of ~ ifferent pHs. This paper extends that work to consider

ow the conformations of these and two related COmpounds 6 and 7, which exist at room temperature as Separable conformers, affect their chromatographic behaviour.

Table I Structures of the potassium channel activators discussed in the text.

R2"" 0

Me

Compound R 1 R 2 R 3 X

1 CN -(CH2) 3- O 2 CN -(CH2) 4- O 3 CN H Me O 4 H H Me O 5 CN H Me CH 2 6 CN Me Me O 7 CN H H O

Experimental

Chemicals

The synthesis of compounds 1-7 has been described [4, 5, 6]. Methanol and propan-2-ol were from Romil Chemicals. Sodium phosphate buffers were prepared by dissolving the appropriate amount of sodium phos- phate dibasic heptahydrate (Aldrich) or sodium phos- phate monobasic monohydrate (Aldrich) in HPLC grade water or D20 (99.8 %, Fluorochem), adjusting to the required pH with concentrated orthophosphoric acid (Aldrich) and filtering through a Millipore Durap- ore membrane (0.45 gm). All mobile phases were degassed with helium before use.

HPLC

Chromatographic analyses were performed on a Wa- ters 991 photodiode array system equipped with a Waters M600 pump and a Gilson 231/401 autosampler. The primary detection wavelengths were 220 and 252 nm. Analyses were carried out on a CHIRAL-AGP

Chromatographia Vol. 36, 1993

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column (100 x 4.0 mm), supplied by Technicol (Stock- port, UK). Samples were prepared as 0.1 mg/ml solu- tions in eluent after prior dissolution in a small volume of methanol. Injection volumes were 5 gl.

Investigations of pH and modifier effects were per- formed at ambient temperature (approximately 21 ~ The collection of fractions from 6 with a D20-based eluent (1% propan-2-ol, pD 6.9) used a column temperature of approximately 6 ~ and a 100 gl injection volume from a 1 mg/ml solution. With this loading the separation of the enantiomers was lost but the separa- tion of the amide conformers was maintained. The micro-preparation of 7 was performed at a column temperature of approximately 21 ~ with a sample load of 8 gg. The mobile phase was 1/99 = IPA/0.01 M aqueous phosphate pH 7.0 at a flow rate of 0.9 ml/min. Three fractions were collected, corresponding to one enantiomer of both conformers, the other enantiomer of the minor conformer and, finally, the other enanti- omer of the major conformer. 100 gl re-injections were made from the fractions. Competition experiments were performed by adding an appropriate amount of 2, dissolved in propan-2-ol, to the aqueous portion of the mobile phase to give 1/99 propan-2-ol/0.01 M aqueous phosphate pH 3, + 0, 5, or 15 gM 2. The samples were prepared as before.

N M R Spectroscopy

400.14 MHz 1H NMR spectra of samples of 6 and 7 in DMSO-d 6 were acquired on a Bruker AMX-400 fitted with a 5 mm tuneable inverse probe. The samples were maintained in the probe at 25 ~ using a Eurotherm variable temperature control unit. Spectra of 6 in D20 were acquired at 5 ~ All acquisitions were of 128- 512 scans in 32 kw complex points using 45 ~ pulses, and the data were processed using an exponential line broadening of 0.1-43.25 Hz prior to Fourier transforma- tion. The spectra in DMSO-d 6 were referenced to te- tramethylsilane at 0.0 ppm; those of 6 in D20 were ref- erenced to sodium trimethylsilylpropionate and the spectra of the fractions in D20 were externally refer- enced to the same absolute frequency.

Results

Experiments were performed on 6 and 7 to examine their chromatographic behaviour on CHIRAL-AGP, using a variety of mobile phase pHs and either methanol or propan-2-ol as organic modifier. Up to four peaks were observed for each of these two compounds, depending on the chromatographic conditions, as shown in an example chromatogram for 6 in Figure 1. These peaks were rationalised as the enantiomers of the two amide conformers known to exist in solution at room temper- ature from NMR spectroscopic evidence. In DMSO-d 6 solution, the ratio of the conformers of 6 is approxi- mately 40:60 at 25 ~ where the first value corresponds to the conformer with the pyran ring anti to the

.04_

A220

.02.

\ I t

2 4

Time (minutes) Figure 1 Example chromatogram of 6. Column, CHIRAL-AGP (100 • 4mm); mobile phase, 10/90 methanol/0.01 M aqueous sodium phosphate pH 3.0; flow rate 0.9 ml/min; 21 ~ UV detection at 220 r i m .

6

5 .

~ 4.

o e~ 2~

0 .

11 3

-.--E2--- 3 ,t 6 minor

a) - - ~ , - - 6 major -" 7 minor

--0-- 7 major

- I I

" ' " . . . .0 . . . . . . . . . . . . . . . . . . . . . . : = : : : : : " ~

~ D

2.5. b)

8 2.0

~o 1.5( , w

ca. 1.0: o (o i

. _ _ . . . . . . . . . . .~_ .__= ~ . _ _ . ~ _ . . . . . . . . . . . .

0.51 , , . ---. 3 4 5 6 7

pH Figure 2 Influence of buffer pH on (a) the retention and (b) the enantiose" lectivities for 1, 3, 6, and 7, using 1% propan-2-ol as organic modifier. Conditions as Figure 1 except 1/99 propan-2-ol/0.01 ld aqueous sodium phosphate at varying pH used as mobile phase.

116 Chromatographia Vol. 36, 1993

3.5. a)

r

3.0J i=, o

2.5,

m ~ ~ 2.01

~ 1.5!

I 1.0i ~

a

- 4 1 - = 1 ---s 3

6 minor - - ~ ' - - 6 major

7 minor - - 0 - - 7 major

Y = . . . . . . . . . . . . . . . . S S B : : : : : : : : : : : : = = . . . . . . . . . . . . , . = ~

4 5 6 7 2.2 b)

2"0 t ~ 1.6.

~ ~ O 1.6. ...- ~ 1.4

~ 1.2

1.0. " ' , . . . . . . . . ~1 . . . . . . . . . . . . . . . . . . . . . . . . . 1r

3 4 5 6 7 Figure 3 pH Influence of buffer pH on (a) the retention and (b) the enantiose- lectivities for I, 3, 6, and 7, using I0 % methanol as organic modifier. Conditions as Figure 1 except for varying mobile phase PFI.

A220

l 14

0 14

Time (minutes) Figure 4

Analytical separation of 6 using D20-based eluent. Conditions as Figure 1, except that the mobile phase was 1/99 propan-2-ol sodium phosphate pD 6.9 and the column temperature was 8 ~ Inset chromatogram shows equivalent separation in H20-based eluent. The three peaks observed in each case correspond, in order of elution, to the first and second enantiomers of the minor conformer of 6 and the major conformer of 6, the enantiomers of which were unresolved under these mobile phase conditions.

Carbonyl oxygen. The corresponding ratio for 7 is approximately 30:70, also at 25 ~ The barrier to rotation in 6 has been measured as approximately 18- 20 kcal/mol [5]. The capacity and enantioselectivity factors for the conformers of 6 and 7 are shown in Figures 2 and 3, for propan-2-ol and methanol as .tnodifier, respectively. Data for 1 and 3 are also included in the Figures to allow comparisons to be made.

q'o verify that the four peaks seen in chromatograms of 6 Corresponded to the enantiomers of the conformers, a micro-preparative separation of 6 was undertaken. Since the separated conformers were not expected to be Particularly stable with respect to interconversion, an eluent based on D20 was chosen for the micro-prepa- ration so that the fractions could be analysed directly by NMR spectroscopy (the ratio of the conformers of 6 in I320 is 30:70, as measured by NMR). A low column temperature was also used to preserve the identity of the fractions during the chromatography. An analytical Separation in the D20-based eluent is shown in Figure 4, together with an example chromatogram in the equivalent H20-based eluent. The Figure shows that the results in both eluents were similar, with capacity factors being rather longer in D20 but the enantiose-

lectivity for the minor enantiomers being the same in both eluents ((ZD20 = 1.67; (ZH20 = 1.69). The fractions containing the conformers were collected and analysed by 1H NMR spectroscopy [7]; the resulting spectra could be compared against spectra of the parent compound 6 in D20 to show that the second-eluting (major) conformer and first-eluting (minor) conformer had the conformations 6a and 6b, respectively, as shown in Figure 5.

Support for the interpretation that the conformers of 7 were separable by HPLC was obtained from collection and re-injection experiments. These showed that the separated conformers underwent fairly rapid intercon- version at ambient temperature, producing chromato- grams with conformer ratios similar to those of the starting mixture after only a few minutes. In an attempt to probe the nature of the binding of this series of compounds to oq-acid glycoprotein, competi- tion experiments were performed. In these experiments up to 15 IJ.M 2 was added to the mobile phase and either 3 or 6 was injected on to the column. The additions caused only slight reductions in capacity factors for the enantiomers of 3 and 6 and there was no effect (within experimental error) on the enantioselectivities for these compounds.

Chromatographia Vol. 36, 1993 117

H3 H 3

.m ,~ a,,on 0

M e ' ~ H4 ~ -'~ ~ Me- I "4 Me Me Me

6a 6b Figure 5

Conformations adopted by 6 in solution at room temperature; 6a = major conformer, 6b = minor.

Discussion

Earlier work [3] clearly showed the utility of CHIRAL- AGP in separating compounds from this class. Howev- er, the binding of these compounds to 0q-acid glyco- protein differed from case to case; compound 2 apparently underwent less enantioselective binding than did, for example, 3 under the same conditions, despite a longer retention for the former. For many series of compounds, changes in substituents may cause conformational changes as well as steric bulk/polarity changes, etc. However, for compounds 1-5 in protic solvents at room temperature the preferred conforma- tion is analogous to 6a [8,9]. Note that pyran ring inversion is disfavoured with the gem-dimethyl substi- tution, due to a 1,3 interaction with the amide substitu- ent and the x-ray crystallography shows only very small differences in the conformation of the pyran ring for the compounds studied [7]. Thus the differences in binding behaviour noted above appear to be due to changes in features other than conformation. The compounds 6 and 7 presented an opportunity to inves- tigate the effect of conformational changes on binding, as (a) their conformers persist in aqueous solution long enough to be separated and (b) there is a significant population in both forms. This behaviour can be contrasted with that of compounds such as 3 for which only one conformer is observed in NMR spectroscopy - that analogous to 6a. This difference between forma- mides and N-disubstituted acetamides on the one hand and N-monosubstituted acetamides on the other is consistent with published data [10].

Figures 2 and 3 demonstrate that the amide conformers of 6 and 7 do indeed behave differently on cxl-acid glycoprotein. For example, the enantiomers of 6b were separable at all pHs tried and with both organic modifiers, whereas the enantiomers of 6a were separa- ble only at pH 3. Similar constrasts were observed for the conformers of 7. The major conformer of 7 does appear to behave in a similar fashion to its close analogue 3, but there is no such simple relationship between the behaviour of 6a and 3. The data confirm the observation [11] that minor changes in structure can have profound effects on chromatographic behaviour, even if the molecules retain the same conformation and, additionally, that conformation is an important factor to be considered in making structure/behaviour

comparisons. The behaviour of 6 in D20-based eluent was interesting in that the separation achieved was very similar to that obtained in the analogous H20-based eluent. Although the conditions used precluded comment on the separa- tion of the enantiomers of 6a, the exchange of the acidic protons on ul-acid glycoprotein for deuterons had no effect on the enantioselectivity observed for the enan- tiomers of 6b. It would be interesting to investigate further the use of D20, to establish whether it has similarly little effect on the separations of other analytes, including cations and anions.

The results of the competition experiments were unexpected; it had been thought that 2 would compete for the same binding sites as 3 and 6, resulting in a alteration of capacity and possibly also enantioselectiv- ity factors for the latter compounds. Mobile phase modifiers have been observed to have this effect, even at the low concentrations used here [12], although further experiments (at, for example, higher concentra- tions of 2) need to be performed to allow interpretation of these results.

Conclusions

Solute conformation as well as solute structure plays a large part in determining chromatographic behaviour on (xl-acid glycoprotein and it should not be neglected when rationalising chiral discrimination on CHIRAL- AGP. Replacing an aqueous mobile phase with one based on D20 maintained enantioselectivity during a micro-preparative separation and this approach may be useful in investigating the role of hydrogen bonding in chiral discrimination on this column and/or in allowing validation of chiral separations by NMR spectroscopY. Initial experiments have shown little effect on the enantioselectivities of two of the compounds studied when chromatographed in the presence of a third in the mobile phase.

References

[1] T.C Hamilton, S.W. Weir, A.H. Weston, Brit. J. Pharmacol' 88, 103 (1986).

[2] J.M. Evans, G. Stemp, Chem. in Britain 2"/, 439 (1991).

118 Chromatographia Vol. 36, 1993

[3] J.M. Evans, R.J. Smith, G. Stemp, J. Chromatogr. 623, 163 (1992).

[4] V.A. Ashwood, R.E. Buckingham, E Cassidy, J.M. Evans, E.A. Faruk, T.C. Hamilton, D.J. Nash, G. Stemp, K. Willcocks, J. Med. Chem. 29, 2194 (1986).

[5] V.A. Ashwood, F. Cassidy, M.C. Coldwell, J.M. Evans, T.C. Hamilton, D.R. Howlett, D.M. Smith, G. Stemp, J. Med. Chem. 33, 2667 (1990).

[6] V.A. Ashwood, E Cassidy, J.M. Evans, S. Gagliardi, G. Stemp, J. Med. Chem. 34, 3261 (1991).

[7] ZM. Evans, to be published. [8] F. Cassidy, J.M. Evans, D.M. Smith, G. Stemp, C. Edge, D.J.

Williams, J. Chem. Soc., Chem. Commun., 377 (1989).

[9] W.A. Thomas, LW.A. Whitcombe, J. Chem. Soc., Chem. Commun., 528 (1990).

[10] W.E. Stewart, T.H. Siddall, Ili, Chem. Rev. 70, 517 (1970). [11] J. Hermansson, G. Schill, in "High Performance Liquid

Chromatography", P.R. Brown, R.A. Hartwick, eds., Wiley- Interscience, New York, 1989, p. 337.

[12] M. Eriksson, J. Hermansson, J. Chromatogr. 519, 285 (1990).

Received: Sep 15, 1992 Revised manuscript received: Dec 22, 1992 Accepted: Jan 6, 1993

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