Asymmetric synthesis and enantioselective teratogenicity of 2-n-propyl-4-pentenoic acid (4-en-VPA), an active metabolite of the anticonvulsant drug, valproic acid

Toxicology Letters, 49 (1989) 4148

41

Elsevier

TOXLET

022 17

Asymmetric synthesis and enantioselective teratogenicity of 2-n-propyl-4-pentenoic acid (4-enVPA), an active metabolite of the anticonvulsant drug, valproic acid

Ralf-Siegbert

Hawk

and Heinz Nau

Institute for Toxicology and Embryopharmacology, Free University of Berlin, Garystrasse 5, D-1000 Berlin 33 (Germany ) (Received

4 April 1989)

(Revision

received 29 May 1989)

(Accepted

3 1 May 1989)

Key words: 2-n-Propyl-4-pentenoic teratogenicity;

Valproic

acid; Enantiomers:

Asymmetric

synthesis;

Enantioselective

acid

SUMMARY Previous

studies indicated

teratogenic

potencies.

so that the intrinsic therefore bolite

that the enantiomers

Unfortunately, teratogenic

potencies

studied the teratogenicity

of the human synthesis

termination

of the absolute

and its analogue

to be unstable

teratogen

valproic

were difficult

of 2-n-propyl-4-pentenoic acid (VPA).

configuration

injections

shown to be highly susceptible more teratogenic

and of the optical

during

to interference

(formation

purities

early organogenesis

EM12 exhibit differing

and racemised

considerably.

to elucidate.

We have

acid (Cen-VPA),

Both enantiomers

using (R)- and (S)-1-amino-2-(methoxymethyl)pyrrolidine

as single intraperitoneal nificantly

proved

of the pure enantiomers

of the enantiomers

and animal

asymmetric

of thalidomide

these substances

(RAMP;

is described.

a meta-

were prepared

They were administered

in the mouse (day 8 of gestation),

a period

with neural tube closure by VPA. 9( -)-Cen-VPA

of exencephaly)

and embryotoxic

(incidence

via

SAMP). The de-

was sig-

of embryolethality

and

fetal growth retardation) than R-(+)-4-en-VPA. The rate of neural tube defects (exencephaly) produced by the Senantiomer was about 4 times higher than that produced by the R-enantiomer. The S-enantiomer of 4-en-VPA

is the first analogue

show that racemic findings

mixtures

may lead to the development

and assist in studies of molecular

Address University

of VPA with higher teratogenic

may consist of enantiomers

for correspondence: of Berlin, Garystrasse

0378-4274/89/$3,50

of clinically

mechanisms

R.-S. Hauck,

potency

with greatly

useful drug enantiomers

in drug and chemical

Institute

than the parent drug. Our results

differing

teratogenic

These

potencies,

teratogenesis.

for Toxicology

and Embryopharmacology,

5, D-1000 Berlin 33, Germany.

@ 1989 Elsevier Science Publishers

potencies.

with low teratogenic

B.V. (Biomedical

Division)

Free

42

INTRODUCTION

The antiepileptic drug, valproic acid (2-n-propylpentanoic acid, VPA, usually administered as the sodium salt), is teratogenic in the mouse, rat, rabbit, hamster, monkey and, highly likely, also in the human [l-3]. Neural tube defects (spina bifida) are the most striking malformations observed in man (in about 1-2s of VPA-exposed cases), although a number of other major and minor defects (‘fetal valproate syndrome’) are apparently also present [&6]. Neural tube defects (exencephaly) can be induced by VPA administration during early organogenesis in the mouse [7, 81. Previous studies with VPA and a number of analogous substances demonstrated a high structural specificity of teratogenicity in this class of compounds [9]. On the other hand, the anticonvulsant activity of these substances showed a rather broad structural specificity [lo]. This enabled us to develop a substance (2-n-propyl-2-pentenoic acid, 2-en-VPA) with the desired anticonvulsant property, but low teratogenic potency [I 1, 121. Certain structural elements were shown to be necessary for expression of teratogenic activity [9]: the C-2 atom must possess sp3-hybridization and must be connected to a free carboxyl group as well as two alkyl groups. The most potent teratogens contain two unbranched alkyl groups with 3 carbon atoms (VPA and 2-n-propyl-4-pentenoic acid, 4-en-VPA). Extension of the two alkyl groups (2-n-propyland 2-n-butylhexanoic acid) reduced the teratogenic potency less than a shortening of these two groups (2-ethylpentanoic and 2-ethylbutyric acid). Introduction of one C = C double bond between C-4 and C-5 did not result in loss of teratogenic activity [9]. The structure of 4-en-VPA contains an asymmetric carbon atom in position 2 of the molecule. It is therefore possible that the two enantiomers of 4-en-VPA (Fig. 1) may exhibit differing teratogenic potencies. Previous studies with the thalidomide analogue EM 12 indeed showed that the S-EM 12 was more teratogenic in the marmoset monkey than the R-enantiomer [ 131. Unfortunately, considerable racemisation occurred with both enantiomers, so that it was not possible to determine the intrinsic teratogenic potency of each enantiomer [14, 151. The stereoselectivity of the teratogenicity of thalidomide itself remains controversial: no difference between the teratogenie potencies of the two enantiomers was found in the rabbit [16]; on the other hand, the S-enantiomer of thalidomide was reported to be highly teratogenic in rats and mice, while the R-enantiomer did not exhibit any teratogenic activity [17]. The stereoselectivity of thalidomide’s teratogenicity reported here is difficult to interpret

Fig. 1. Stereochemical

structures

of R-( +)-Cen-VPA

and S-( -)-4-en-VPA

43

in light of the rapid racemisation

of the enantiomers

ent insensitivity of rats and mice to the teratogenic remains to be clarified.

discussed action

above and the appar-

of thalidomide

[18] and

We have therefore studied the teratogenic potency of the two enantiomers of 4-enVPA to determine whether stereochemical considerations play a role in drug-induced teratogenicity. A highly significant difference between the two enantiomers was observed. This is the first example of stereoselective teratogenicity of a synthetic compound which follows up the controversial reports on the effects of the unstable enantiomers of thalidomide and EM 12. MATERIALS

AND METHODS

Instruments NMR (‘H and 13C) spectra were recorded with a Bruker WH270 spectrometer at 270 MHz. ‘H and 13C chemical shifts are reported in parts per million relative to internal tetramethylsilane. The gas chromatographic determination of the chemical purities was performed on a Hewlett-Packard 5700A using a SE 30 column (1.80 m x 2 mm; Applied Science Lab.) and flame ionisation detection. The optical purities were determined on a Carlo Erba Fractovab 4160 gas chromatograph using a DB-210 capillary column (30 m x 0.25 mm; J&W Scientific) and nitrogen selective detection. The optical rotations were measured at 589 nm using a Perkin-Elmer 241 polarimeter. The elemental analyses were performed on a Perkin-Elmer elemental analyser 2400. Chemicals Allyliodide. allylbromide, dide, (s)-( -)-phenethylamine

diisopropylamine, diethyl-n-propylmalonate, methylioand valeraldehyde were supplied by Aldrich (Stein-

heim, F.R.G.); (s)-( -)-l-amino-2-(methoxymethyl)pyrrolidine (SAMP), (R)-( +)I-amino-2-(methoxymethyl)pyrrolidine (RAMP), butyllithium, silver nitrate, sodium borhydride (NaBH& dry diethylether and dry methanol were obtained from Merck (Darmstadt, F.R.G.); N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) was from Pierce (Rockford,

IL, U.S.A.).

Svnthesis of (+)-4-en-VPA as well as R-( +)- and S-(-)-4-en-VPA ( f )-2-n-Propyl-4-pentenoic acid was synthesised by alkylation of diethyl-n-propylmalonate with allylbromide. subsequent alkaline hydrolysis and decarboxylation according to known malonic ester synthetic procedures. Chemical purity of >99% was determined by gas chromatographic analysis of the TMS derivative produced by reaction with MSTFA. Following the method described by Enders and Eichenauer for the enantioselective a-alkylation of aldehydes [19], we first synthesised the hydrazones from RAMP and SAMP, and valeraldehyde. Their metallation was carried out with a freshly prepared solution of lithium diisopropylamide in dry diethylether (OC, 4 h). Subsequent alky-

44

lation

with allyliodide

tenylidenamino)pyrrolidine

led to (2’-R and 2’-S)-2-methoxymethyl-1-(2-n-propyl-4-pen(Compounds

1 and 2) (- lOO”C, 6 h). Hydrazones

cleaved according to the methyliodide method and produced Without further purification they were oxidized to R-(+)

were

the a-chiral aldehydes. and S-(-)-4-en-VPA

(Compounds 3 and 4) using a freshly prepared suspension of silver-(I)-oxide. The carboxylic acids were finally purified by distillation. Analyses of the TMS derivatives by gas chromatography indicates a chemical purity of > 99% for both enantiomers. Compound I ‘H-NMR(CDCls): 6 = 0.90 (t,J=7 Hz, 3H, 3”-H), 1.27-1.49 (m, 4H, 1”-H, 2”-H), 1.762.00 (m, 4H, 3-H, 4-H), 2.16-2.35 (m, 3H,3’-H, 2-H) 2.71 (mc, lH, 5-H,), 3.29-3.48 (m, 3H, 5-Ht,, OCHz), 3.38 (s, 3H, OCHs), 3.56 (m. lH, 2-H), 4.96-5.11 (m,2H,5’-H), 5.80 (mc, lH, 4-H) 6.48 (d,J=7 Hz, lH, I’-H)..t3C-NMR (CDCl& 6= 13.97, 19.97, 21.88, 26.36, 35.26, 38.03, 41.73, 50.30, 58.95, 63.28, 74.59, 115.62, 136.64, 142.42. 1. Compound 2 ‘H and 13C-NMR spectra are identical with Compound Compound 3 ‘H-NMR (CDC13): 6=0.91 (t,J=8 Hz, 3H, 3’-H), 1.30-1.73 (m, 4H, I’-H, 2’-H), 2.20-2.54 (m, 3H, 2-H, 3-H), 5.02-5.15 (m, 2H, 5-H), 5.80 (mc, lH, 4-H), 12.05 (s, lH, COOH). 13C-NMR (CDC13): 6=13.84, 20.36, 33.63, 36.07, 45.02, 116.84, 135.18, 182.51. [I$,~~= +5.6 (c= 1.8, CHC13). CsHi402 (142.2): ca1cd.C 67.57 H 9.92; found C 67.49 H 9.87. 3. [~]p~~= Compound 4 ‘H and 13C-NMR spectra are identical with compound -5.5 (c = 1.7, CHC13). CsHi402

(142.2): calcd. C 67.57 H 9.92; found C 67.51 H 9.95.

Determination of absolute conjiguration (-)-Cen-VPA was transformed into 4-hydroxy-2-n-propylbutanoic acid via ozonolysis (methanol, -78°C) followed by reductive work-up (NaBH4). This compound was then cyclized in a mixture of acetic acid anhydride and pyridine (25”C, 12 h) to the known (s)-( +)-2-n-propyl-y-butyrolactone (Compound 5) [20]. Therefore the absolute configuration of (-)-Cen-VPA is S. Compound 5 ‘H-NMR (CDCls): d-0.97 (t,J=5.5 Hz, 3H, CHs), 1.361.52 (m, 3H, I’-H,, 2’-H), 1.79-2.04 (m, 2H, I’-Ht,, 3-H,), 2.342.45 and 2.48-2.60 (2m, 2H, 2-H and 3Hb), 4.20 (dt, Jt = J2=9 Hz, Js=7Hz, lH, 4-H,), 4.35 (dt, Jt = JZ=9 Hz, 5s = 3 Hz, lH, 4-H,,). [cr]nZ2= + 6.5 (c = 1.O, EtOH).

Determination of optical purity For determination of the enantiometric purity the chiral carboxylic acids were transformed into the diastereometric (s>-( -)-1-phenethylamide derivatives, which were analysed by gas chromatography. The racemate served as standard: R-( + )-Cen-VPA: 9 1 + 1% (R) S-( -)4-en-VPA: 91+ 1% (S)

45

Animul experiments Female mice (Han: NMRI 29-36 g) were mated between 6.00 and 9.00 a.m. The first 24 hours after conception were day 0 of gestation. The mice were fed an Altromin 1324 diet and given access to water ad libitum. Controlled conditions were maintained (room temperature 21+ 1“C, air moisture 50 2 5%); a 1Zhour lightdark cycle was employed inducing light from 10.00 a.m. to 10.00 p.m. and darkness from 10.00 p.m. to 10.00 a.m. The substances were injected intraperitoneally as their sodium salts during the active phase on day 8 between 7.00 a.m. and 9.00 a.m. [21, 221. The injected solutions in the applied dosages of 500 mg/kg (3.0 mmol/kg) and 450 mg/kg (2.7 mmol/kg) body weight were prepared as aqueous solutions of the sodium salts (10 ml/kg). On day 18 of gestation the implantations were counted and every living fetus was weighed individually and examined for exencephaly. RESULTS

AND DISCUSSION

Single injections of S-4-en-VPA during the sensitive stage of early mouse organogenesis (day 8 of gestation) were more teratogenic than comparable doses of R4-en-

TABLE

I

TERATOGENICITY

OF THE ENANTIOMERS

AND THE RACEMIC

MIXTURE

OF 4-EN-VPA

IN MICE Substance

Dose*

Number

Number

Embryo-

mg/kg (mmol/kg)

of litters

of live

lethality

fetuses

9( - )-

450

4-en-VPA

(2.7)

R-( + )4-en-VPA

450 (2.7)

9( - )-

500

4-en-VPA

(3.0)

R-( + )-

500

4-en-VPA

(3.0)

Exencephaly (sg of live

weight(g)

total implants)

fetuses)

(mean + SD)

56**

0.93 kO.06

13

1.04+_0.11

(% of

11

SO

32

8

84

8

9

64

33

70**

1.01+0.13

7

75

19

17

I .04 f 0.04

1.07*0.10

( * )-4-en-

500

VPA

(3.0)

13

115

I7

35

Control***

-

10

126

6

0

*Single i.p. dose of the sodium salts per kg body weight on the morning **Significantly

different

***3.0 mmol aqueous

Fetal

from R-Cen-WA NaCl/kg.

induced

exencephaly

1.14&0.05

of day 8 of gestation.

rate (PC 0.005; ,$-test).

46 Exencephaiy (%) S-C-)4-en-‘/PA

VPA

Fig. 2. Comparison of the rates of neural tube defects produced by VPA [data from Ref. 231 with those produced by the enantiomers of 4-en-VPA (single i.p. dose of 500 mg of the sodium salts per kg body weight on day 8 of gestation).

VPA: at both dosing levels selected the S-enantiomer was significantly more teratogenie (formation of exencephaly) as well as embryotoxic (incidence of embryolethality and growth retardation) than the R-enantiomer (Table I). Although both enantiomers were teratogenic, the rates of exencephaly produced by the S-enantiomer were about 4 times higher than those produced by comparable doses of the R-enantiomer. At the dose level of 500 mg/kg the exencephaly rate induced by the racemate of 4-en-VPA (( 3-)4-en-VPA) was between the rates induced by the enantiomers (Table I). Maternal toxicity (sedation, induction of sleep) produced by R- and S-4-en-VPA was comparable, and lower than the toxicity observed after administration of VPA. The exencephaly and resorption rates varied from litter to litter, but no extreme values (‘outliers’) were observed. The teratogenic potency of S-4-en-WA was higher and that of the R-4-en-VPA lower than that of the parent drug VPA (Fig. 2). S-Cen-VPA is therefore the first compound with a teratogenic potency exceeding that of VPA. The introduction of a C = C double bond (increase of electron density) between carbons 4 and 5 of valproic acid therefore results in an increase (S-4-en-VPA) or decrease (R-4-en-VPA) of teratogenic potency. For expression of high teratogenic potency, one but not both of the alkyl groups must have a functional group of increased electron density in the 4-position, and these two alkyl groups must have a certain stereochemical configuration (Fig. 1). If C=C double bonds are introduced into the 4-position of both alkyl groups, then the teratogenicity is abolished (4,4’-dien-VPA) [9]. Our results clearly indicate the enantioselective teratogenicity of optically active compounds. As demonstrated here with 4-en-VPA, racemic mixtures may consist of enantiomers with greatly differing teratogenic potencies. Our findings should prove particularly important to studies on molecular mechanisms in drug and chemical teratogenesis, and in the development of clinically useful enantiomers with low toxicity.

47

Further

experiments

4-en-VPA tiomers,

show

whether

the enantioselective

teratogenicity

is the result of differences

between

the intrinsic

of the two enan-

or whether

must

pharmacokinetic

activities

of

factors also play a role.

ACKNOWLEDGEMENTS

This study was supported by the Deutsche Forschungsgemeinschaft (Grant No. C-6, SFB 174). The authors thank K.-D. Graske for the determination of the absolute configuration and Ms. S. Drinkwater for preparation of the manuscript. REFERENCES 1 Robert. 2,937.

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