CHIRALITY 4:356-366 (1992)

Stereochemical Studies of Chiral H- 1 Antagonists of Histamine: The Resolution, Chiral Analysis, and Biological Evaluation of Four Antipodal Pairs A.F. CASY, A.F. DRAKE, C.R. GANELLIN, A.D. MERCER, AND C. W O N School of Pharmacy and PhamzacoloB, University of Bath, Bath (A.F.C., AD.M., C.U.), Deoartment of Chemishy, Barbeck College, London (AFD.),and Department of Chemishy, University College London, London (C.R.G.), England

ABSTRACT The resolution of the H-1 antihistamines chloropheniramine, dimethindene, carbinoxamine, and mebrophenhydramineis described. The optical purity of antipodal products is investigated by chiral HPLC (use of a1-acid glycoprotein and P-cyclodextrincolumns) and NMR (spectra of P-cyclodextrininclusion complexes). Configurationalrelationships among the group are reviewed and assignments are confirmed and extended by circular dichroism evidence. Affinity constants of antipodal pairs for guinea pig ileum and cerebellum sites, determined by gut bath and binding experiments respectively, are reported together with some in vivo tests in man for central effects. Results are discussed in terms of configurational requirements for activity and variations in antipodal potency ratios within the group. 0 1992 Wiley-Liss, Inc.

KEY WORDS: H-1 antihistamines, chiral HPLC, 0-cyclodextrin-inclusioncomplexes, NMR, circular dichroism, affinity constants

INTRODUCTION Potency differences between the members of antipodal pairs of chiral H-1 antagonists of histamine are well documented. Our decision to review and extend the published data stemmed from:

~ C H , C H , N M e , CHMe

R 1. absence of a coordinated study of a group of examples, i.e., use of a variety of chiral antagonists resolved and tested in the same investigation, 2. restriction of evidence of optical purity of antipodal pairs examined prior to 1980 to optical rotational values measured at a single wave length, 3. the need to seek and confirm configurational relationships among the various eutomers, and 4. the limitation of pharmacological evaluations to the blockade of peripheral receptors.

The resolution of a series of ruc-antihistamines was therefore undertaken, and methods additional to optical sought for the purpose of establishing the optical purity of the homochiral products. Pharmacological evaluations of antipodal pairs at both peripheral and central sites have been made and the results, together with those reported for related compounds, analysed with special reference to absolute geometry. Evidence of stereochemical relationships has been augmented by the results of circular dichroism studies.

Optical Resolutions Resolution of the RS-bases 1-4 was carried out by the classical procedure of recrystallizing diastereoisomeric salts o 1992 Wiley-Liss, Inc.

la-c

I H-CI

2

3@

o(cH,), N~

Q

e ,

Ph Me--S--O(CH,),NMe, I

CI

3

Br 4

formed with antipodes of tartaric acid or their derivatives. Details are given in the experimental section. Discrepancies in the magnitude of antipodal maleates of carbinoxamine and mebrophenhydramine throw doubt on the optical purities of Received for publication October 1, 1991; accepted February 9, 1992. Address reprint requests to C. Upton, School of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY, England. Resent address for A.D. Mercer: SmithKline Beecham Pharmaceuticals, Mundells, Welwyn Garden City, Herts AL7 IEY, England.

357

CHIRAL H-1 ANTAGONISTS OF HISTAMINE

C

B

0

I

80 105

Fig. 1. Enantiopac chromatograms for pheniramine maleate and chlorpheniramine maleate. Mobile phase: 8 mM phosphate buffer containing 0.1 M NaCl and 0.4% 2-propanol, pH 6.9 (Method 1).(RS)- and (-)-pheniramines were derived from (RS)-and ( - )-chlorpheniramine,respectively, by catalytic dehalogenation. Ordinate: response; abscissa: time (min), start point denoted by arrow, peak times shown on each band (applies to all chromatograms).

these materials. It can be seen (experimental)that corresponding antipodes of salts formed with resolving acids had [a] Values of similar magnitudes. The problem was not encountered for maleates of chlorpheniramine and dimethindene to which the same procedures of base liberation and maleate formation were applied. Since there is evidence from chiral HPLC and NMR that the isomeric carbinoxamine and mebrophenhydramine maleates have high degrees of optical purity, it must be assumed that discrepancies in the optical rotational data are a result of the differing nature of ion pairs formed between antipodes of their protonated bases and the maleate anion which in turn influences the optical properties of the bases. [a]Values of chiral bases are notoriously sensitive to a variety of factors including solvent and state of ionization.* Chromatographic Evidence of Optical Purity

The application of HPLC to the chiral analysis of the resolved antihistamines and other samples was investigated with *See ref. 8 for an example of the unique influence of the maleate counterion as compared to fumarate and chloride upon the 'H N M R features of antipodal dimethindene bases included in P-cyclodextrin.

varying degrees of success. Use of an al-acid glycoprotein column (Enantiopac)according to details given by Hermansson6 gave satisfactory separations in only a few cases and retention times were lengthy (about 2 h). Best results were obtained for (RS)-pheniramine(la)which gave baseline resolution (resolution factor Rs = 1.50).Material obtained by reductive dehalogenation of (RS)-chlorpheniramine (lb) gave a chromatogram similar to that of the (RS)-pheniraminesample (broad nonsymmetrical bands). The chromatogram of ( - )-pheniramine similarly derived from resolved ( - )-chlorpheniramine displayed a single band (Fig. 1).Hence prior conversion of halogenated pheniramines to the pheniramine parent extends the use of Enantiopac columns to assessing the optical purity of such derivatives. Improved resolution of (RS)-chlorpheniramine was achieved by use of a phosphate buffer containing tetrabutylammonium phosphate instead of propanol/ NaCL7 A sensitivity experiment was carried out under these conditions in which a solution of (- )-chlorpheniramine was spiked with increasing quantities of its (+)-antipode. The results showed that a 1%addition of the (+)-isomer was visible on the chromatogram but quantities below 1%would be indistinguishable from baseline noise. The resolution of antipodal

358

CASY ET AL.

peaks was also achieved for carbinoxamine,pheniramine, and its halogenated analogues under the same conditions, but not for (RS)-dimethindene or (RS)-mebrophenhydramine.Chromatograms of resolved materials showed single broad bands with little evidence of isomeric impurity. Experiments were also carried out using various types of cyclodextrin-bondedcolumns (p, y, and acetylated-p). Best results were obtained using a p-cyclodextrinbonded phase with (RS)-halogenated pheniramines and carbinoxamine but baseline resolution could not be achieved (Fig. 2) Retention times were of the order 20-40 min. Spiking experiments showed the 0.5% of the minor chlorpheniramine isomer was detectable (1YO for carbinoxamine). The chromatogramof ( - )-chlorpheniraminemaleate thus gave evidence of an optical purity of 99.5% or above; that of a ( + )-maleatesample displayed two bands of areas indicative of a 97( +):3( - )antipodal content.

resonances of the guest compound. Such signals, if sufficiently resolved give evidence of antipodal ratios.8 A summary of reported findings is given here. In the case of dimethindene(2), the pyridyl H-3' resonance is best applied to this end-it appears as a pair of well-resolved doublets of separation 15Hz in the 400MHZ spectrum of the (RS)-maleatecomplex (Fig. 3).The H-3' signals in the corresponding 270 M H z spectrum of antipodal maleates formed nearly symmetrical doublets with no evidence of antipodal impurity (no distortion of the slope of the lower field edge of the dextro signal, or the higher field edge of the lev0 signal). Spiking experiments were performed by recording inclusion spectra of the dextro antipode (10.5 mg) mixed with increasingamounts of the lev0 isomer and monitoring the appearance of the H-3' doublet. The limit of detection of the minor antipodewas found to be between 0.76 and 1.96%. On this basis, the resolved materials contained no more than 1% of isomeric impurity. In the case of (RS)-carbinoxamine(3) the benzylic CH signal was chosen for optical purity assessUse of IH NMR ment. In (RS)-complexesseparation of the pair of C-H singlets Inclusion of chiral antihistamines within p-cyclodextrin was greatest when y-cyclodextrinwas used as the complexing leads in many cases to duplication of certain of the 'H NMR agent (6.6 Hz separation at 270 MHZ, 9.8 Hz at 400 MHZ). In the

C

8

0

2 6 30

0

min Fig. 2. Chromatograms for the resolution of chlorpheniramine, brompheniramine and carbinoxamine maleates on a cyclobond I column. Mobile phase: 90% TEAA (2%): 10% ACN. The chromatogram of the (+)-chlorpheniramine sample reveals the presence of ~ 3 of%the levo antipode.

359

CHIRAL H-l ANTAGONISTS OF HISTAMINE

i

zzz=--,

ao

1.a

I

7- 6

I

I

7.2

1.4

**”

7 .O

Fig. 3. Part of the low field region of the 400 MHz ‘H NMR spectrum of (RStdimethindene maleate in DzOcontaining a 1 molar proportion (approx.) of P-cyclodextrin.The signal near 7.5 ppm shows clear duplication of the pyridyl3’-H resonances. Duplications of 4’-H and other aromatic signals are also apparent.

400 MHz inclusion spectra (y-polymer)of samples resolved for this work, antipodal CH resonances formed sharp singlets which were symmetrical, merged smoothly with baseline noise, and showed little evidence of antipodal impurity. Spiking experiments, carried out as described for dimethindene, showed that 1%of the minor antipode could be detected by this means. The methine resonances of the carbinoxamine antipodal samples obtained from an industrial source clearly revealed these to be incompletely resolved. The A2B, aromatic signal of chlorpheniramine maleate proved most useful for optical purity assessments. It formed an apparent singlet (7.1 ppm) in the spectrum of the free maleate but split into two lines after complexation with P-cyclodextrin separated by 4.6 Hz at 400 MHZ. Only one of these lines was present in the spectrum of the (+)-antipode complex (Fig. 4) but the presence of antipodal impurity could not be excluded because of baseline complexity. A spectrum of the (+)-complex spiked with a small amount of the ( - )-complex clearly displayed the minor A2 B2 aryl signal (Fig. 4). The corresponding A2B2 aryl signal of brompheniramine (lc)(four lines, centres 7.2 and 7.0 ppm for free maleate, each half duplicated after complexation) served a similar purpose when judging the optical purity of resolved samples. Inclusion spectra of antipodal brompheniramines supplied by Schering displayed nonduplicated A2B2 resonances. The higher field half of the aromatic A2B2 resonance of (RS)-mebrophenhydramine (4) maleate was also clearly duplicated after inclusion within P-cyclodextrin.The spectrum of the included ( f )-antipode displayed a sharp doublet in this region indicative of its high degree of optical purity although quantification was again not possible (Fig. 5). It is of interest that diastereoisomeric ion pairs formed be-

C

1

PPH I

7 d

PPn

,

l ’ , , ’

r

:.4C

7.J5

PPM

7.d

Fig. 4. Aromatic 400 MHz ‘H NMR signals of chlorpheniramine maleate complexed with p-cyclodextrin in Dz0. (A) (RS)-guest; (B) (+)-antipode; (C) ( + )-antipode spiked with ( - )-antipode.

360

CASY ET AL.

chiral centres. If these requirements are met, antipodes of related chirality give rise to CD effects of identical sign and The CD spectra are presented as plots of wavelength against AE,the differential molar absorptivity of left and right circularly polarized light. When AE is positive (AL > AR) a Cotton Effect band of positive sign is obtained and vice versa (see Experimental section for details of procedure). All materials reported here contain aromatic chromophores close to the chiral centre. In addition to compounds resolved for B this work (carbinoxamine and mebrophenhydramine), antipodes of two further members of the diphenhydramine group were available for the CD study, namely neobenodine (6)and clemastine (7).The group clearly satisfies requirements (1)and (2) above. Hence the close correspondence of the CD spectra of ( + )-neobenodine HCl, ( + )-(RR)-clemastine fumarate and (-)-mebrophenhydramine maleate (Figs. 6-8) is evidence for the identical configuration of their benzylic chiral centres (5). The CD features of (-)-neobenodine, (-)-(SS)-clemastine and ( + )-mebrophenhydraminehad mirror image relationships to their respective antipodes. Spectra of clemastine and its three stereoisomers shown in Figure 7 demonstrate the lack of influence of the pyrrolidino chiral centre which is remote from the aromatic chromophore upon the CD bands. Since the absolute configurations of the clemastine group are known," the CD results provide evidence that all eutomers of the group have the (R)-configurationat their benzylic centre. This conclusion exPPll tends toBrompheniramine branes and [ 1251]iodobolpyramineas the radioligand (more sen-86 2.16 0.335 ( - )-Carbinommine sitive than [3H]mepyramine). Results expressed as inhibition - 12.9 1.12 0.050 ( + )-Carbinoxamhe -60 constant K, (nM) values are shown in Table 4. 1.71 0.234 ( - )-Mebrophenhydramine - 19 1.19 0.074 ( + )-Mebrophenhydramine ( - )-Dimethindeneshowed the greater affinity for both cerebellum and auricle sites (514 and 158 that of dextro isomer, respectively) while affinity differences between antipodal meAH values for other antihistamines are not available; magni- brophenhydramines were low ( 2 4 with lev0 the more strongly tudes found for eutomers of chlorpheniramine and brom- bound antipode). pheniramine are of the same order as those recorded for In Vivo Tests in Man (- )-hyoscyaminemethiodide ( - 126 kJ/mol) and ( - )-hyoscine The central effects of antipodal pairs of chlorpheniramine methidide (- 152 kJ/mol) at GP ileum sites. l3 In all cases the enthalpy change following binding of a distomer was several and dimethindene maleates (encapsulated in lactose) were asfold lower than that of a corresponding eutomer. pA2 Results sessed in human volunteers by a double blind study. A sum-

1w 5 w 10 w

TABLE 4. Binding parameters for antipodes of dimethindene tartrate and mebrophenhydramine maleate at guinea pig cerebellum and auricle sites Ki(nM) f SEa cerebellum K,(nM) f SEb Sample ( - )-Dimethindene ( )-Dimethindene ( )-Mebrophenhydramine ( - )-Mebrophenhydramine

+ +

a[3HJMepyramineradioligand, ref. (30). b[’25111cdobolpyramineradioligand, ref. (15).

Particulate fraction 0.20 45.2 10.2 0.28 0.29

f 0.06

f 1.35 f 0.1 f 0.02 f 0.02

Solubilized preparation 0.45 f 0.08 105.8 & 12.5 -

Cerebellum 0.028 14.4 0.58 0.27

f 0.01

f 1.8 f 0.03 f 0.02

Auricle 0.05 f 0.01 7.9 f 2.7 1.42 f 0.35 0.33 f 0.04

363

CHRAL H-1 ANTAGONIST3 OF HISTAMINE

mary of the results, reported in detail elsewhere,l6 is presented here. Performance, subjective assessments and sleep latencies were measured at 08 30,lO 00,11 00,and 12 30 h, with drug ingestion after the first session at 09 30 h. Each subject took, on separate occasions with intervals of at least four days, 10 mg ( + )- and ( - )-chlorpheniramine,5 mg ( + )- and ( - )-dimethindene maleate, 5 mg triprolidine as active control, and two placebos. Changes in the measures of central activity with ( - )-chlorpheniramineand ( + )-dimethindene(distomers at peripheral receptors) were not different from those with placebo. With ( + )-chlorpheniramine and ( - )-dimethindene (eutomers at peripheral receptors), however, marked central effects were observed in all the tests applied. Thus the more potent peripheral H-1 antagonist was also the more potent central sedative in the two sets of antipodes, results consistent with the notion that blockade of central H-1 receptors may cause sedation. EXPERIMENTAL Resolutions Optical rotations were measured at five wavelengths using an Optical activity polarimeter (1 dcm cell, c ~ 1 in%various solvents). Specific rotational data refer to materials which showed little relative change in this property on further crystallization. The recrystallization solvent was ethanol unless otherwise stated.

(RS)-Dimethindene(2)

(RS)-Dimethindene (1 mol) and L-(+)-tartaric acid (1 mol) in ethanol gave a crystalline (-)-tartrate which was recrystalized four times from the same solvent. The derived (-)-base was converted to the ( - )-acid maleate. Base enriched in ( + )-(2) from mother liquors was purified via the D-( -)-tartrate and (+)-acid maleate. The literature report on antipodes of (2) omits rotational data.4 Specific rotational data:

~~

~~

~~

589 nm

578 nm

546 nm

436

nm

365 nm

Form

Solvent

mp ('C)

L-Tartrate &Tartrate Maleate from L-tartrate".* Maleate from D-tartrate'

MeOH MeOH

-

-146 +144

-154 +155

-177 +178

-334 +338

-628 +631

MeOH

127-129

-207

-214

-243

-456

-847

MeOH

127-129

+198

+207

+240

+452

+840

"Unpurified bases in MeOH [ u g - 144 from L-tartrate, + 136 from 1)-tartrate. 'Found C, 70.57; H, 6.91; N, 6.86. C,H&O, requires C, 70.4; H. 6.50; N, 7.2%. 'Found: C, 70.55; H, 6.72: N, 6.39%.

(RS)-Carbinoxamine(3) (RS)-Carbinoxamine(1 mol) and L-( +)-tartaric acid (1 mol) in (RS)-Chlorpheniramine(lb) ethanol were seeded with rotoxamine-L-tartrate to give the (RS)-Chlorpheniraminewas resolved by treating the (RS)- ( + )-diastereoisomericsalt which was recrystallized four times. base (1 mol) with ( -)-di-p-toluoyl-L-tartaric acid (0.5 mol) and The derived (-)-base gave a (+)-hydrogen maleate. Base enhydrochloric acid (0.5 mol) in ethanol-water (3:4).After seed- riched in the ( + )-isomerfrom mother liquors was purified via ing the ( - )-diastereoisomer separated which was recrystal- the D-( - )-tartrate and ( - )-hydrogen maleate. lized three times from 50% aqueous ethanol. The recovered Specific rotational data: (+)-base was converted to the (+)-acid maleate and the salt recrystallized. Likewise, the (RS)-base(fresh or enriched in one isomer from mother liquors) and ( + )-di-p-toluoyl-D-tartaric acid gave the ( + )-diastereoisomer from which the ( - )-acid 589 578 546 436 365 maleate was derived. Form

rag Form

Solvent mp ('C)

Di-p-toluoyl1.-tartrate EtOH" 135 Di-p-toluoylD-tartrate EtOH 135 Maleate from DPTL-tartratek HzO 11S115 DMF Maleate from DF'T~-hrtrate H,O 112-114 DMF

589 nm

578 nm

-60.7b -65.0

436

546 nm

nm

-74.2 -157

+62.3' +65.6 +79.1 +153

365 nm

-320 +313

+23.26 +25.2 +27.0 +51.6 +lo4 +41.1e +44.1 +57.1 +lo4 +225 -24.1' -42.8g

-24.9 -28.7 -57.6 -113 -44.7 -54.8 -113 -239 ~~

"95%. bHunt2gives [ u g - 57.8 (c 1.7, EtOH). CHunt2gives +57.4 (c 1.1, EtOH). dHun? gives [ u g + 23.1 (c 1.2. H20). 'Scherim3 gives [ u g +44.3 (c 1.0, DMF); resolved with antipodal phenylsuccinic acids. /Hun? gives [ a E - 23.1 (c 1.2, H20). gScherico3 gives [af: -44.1 (c 1.0, DMF). 'Unpurified bases in EtOH [ u E + 25.6 from DPT-L-tartrate. - 19.8 from DPT-o-tamate.

[uE

Solvent mp('C)

L-Tartrateo MeOH o-Tartrateb MeOH Maleate from L-tartratec,d MeOH Maleate from D P T ~ t a r t r a t e ~MeOH

nm

nm

nm

nm

nm

142-143 +45.8 +45.8 +51.4 140-141 -46.0 -45.0 -52.0

+87.9 154 -89.0 -152

135-136

+40.8 +44.7 +49.7

+97.4

135-137

-47.8

-48.8

-56.9

- 103

+ 182 -189

'McNiell v a l ~ e s ' [uE5+38.4, ~: 37.2 (c 2, MeOH). bMcNeill value'7: [ug - 38.6 (c 2.0, MeOH). runpurifiedbases: - 8.0(c 1, EtOH) from L-tartrate; + 6.0 (c 2.2, EtOH) from wtartrate. dMcNeill value17: [ u g +41.2 (c 2, MeOH). Found (this work): C, 58.9 H, 5.6; N 6.4. C,H,CIN,O, requires C, 59.04; H, 5.7; N. 6.9%. eFound C, 58.95; H, 5.78; N, 6.8%.

[ag

(RS)-Mebrophenhydramine (4) (RS)-Mebrophenhydramine(1 mol) and ( - )-di-p-toluoyl-Ltartaric acid (1mol) in ethanol gave the (- )-diastereoisomeric salt which was recrystallized three times. The derived base yielded a ( - )-hydrogen maleate. Base recovered from mother liquors was purified by the di-p-toluoyl-D-tartrateand acid mal a t e salts (discrepancies in [u] magnitudes of the latter salts are discussed in the text). Specific rotational data:

364

CASY ET AL.

Dr. R.B. Barlow. Affinity constants were measured on the Form

589

578

546

436

Solvent mp("C) nm

nm

nm

nm

365 nm

-89.4 -87.3 -102

-204

-405

+91.2 +97.5 +112

+221

+432

Di-p-toluoylL-tartrate MeOH Di-p-toluoylD-tartrate MeOH Maleate from DPT-L-tartraten.b MeOH 144-146 Maleate from DPT-wtartratec MeOH 1 6 1 4 2

-20.9 +5.1

- 17.9 - 19.9 +7.1

+8.1

-30.9

-50.8

+ 18.2

+30.4

Wnpurified bases:[ag - 32.1 (c 5, MeOHI from DPT.L-tar&ate; + 28.0 from DPT-I)-tartrate. *Found C, 56.97; H, 5.56; N, 3.13. C.jlxBrNO5 requires C, 56.91;H, 5.64; N. 3.02%.