N-substituted amino acid derivatives with hyperalphalipoproteinaemic activity

Eur.

J. Med.

Chem.

0 Elsevier, Paris

23 (1988)

523

523-531

Original

paper

N-Substituted amino acid derivatives with hyperalphalipoproteinaemic activity Keith H. BAGGALEY+, Robin FEARS, Harry FERRES**, Graham R. GEEN, Ian K. HATTON+*, L. John A. JENNINGS and A. William R. TYRRELL Beecham Pharmaceuticals Research Division, Biosciences Research Centre, Great Burgh, Yew Tree Bottom Road, Epsom, Surrey, KT18 5XQ, England (Received

November

27,

1987,

accepted

May

9, 1988)

Summary A series of N-substituted amino acid derivatives was synthesized and the compounds were evaluated for their effects on serum total cholesterol, HDL cholesterol and triglycerides in experimental animals. Hyperalphalipoproteinaemic activity was found for some of the compounds tested, especially BRL 26314 (2) and related 3-aryl-Z[(arylmethyl)]aminopropionic acids. Structure-activity relationships are discussed. RCsumi! Aminoacides N-substituCs g activitk hyperalphalipoprotCinCmique. Une se’rie de derives aminoacides N-substitues a e’te’synthe’tise’e et les produits ont e’te’e’valuespour leurs efSetssur le cholesterol se’rique total, le cholesterol LHD (lipoprote’ine de haute densite’) et les triglycerides chez les animaux de laboratoire. Une activite’ hyperalphalipoprot&nt?mique a e’te’trouvtfe chez quelques produits examinks, en particulier chez le BRL 26314 (2) et les acides aryl[(arylmPthyl)]-3-amino-2-propioniques apparent&. Les relations structure-activite’ ont tte’ discute’es. N-sabstituted

amino acid derivatives / hyperalphalipoproteinaemic

activity

Introduction

The role of circulating lipoprotein-cholesterol in the development of atherosclerosis and coronary heart disease has been a subject for considerable debate, since the publication of the early Framingham population studies. It has recently been established, however, that a pharmacological decrease in the concentration of LDL cholesterol in high-risk men is associated with reduction of coronary morbidity and mortality [I]. There is also now epidemiological and experimental evidence to suggest that, by contrast, the concentration of HDL is a negative risk factor for mortality and that this protective function is attributable to participation by the lipoprotein in reverse cholesterol transport. What has not yet been proven is whether or not intervention to raise HDL will also decrease coronary heart disease. Some of the established cholesterol-lowering drugs raise HDL but the effect is usually small; various other agents have been described with an effect on HDL, usually as part of a broader pharmacological action [2]. More specifically, a series of phenylenebis-(oxy)-bis[2,2-dimethyl

+Present *Author

address: to whom

Beecham Pharmaceuticals correspondence

should

Research Division,

be addressed.

pentanoic acids] was developed for lipoprotein-modulating activity and the structure-activity relationships for increasing HDL were described [3]. However, there were undesirable concomitant increases in plasma triglycerides and liver weight. In general, the interpretation of lipoprotein changes in rats can be difficult because of inter-species differences in metabolism [4]. The relevance of lipoprotein changes in animals to the prediction of a clinical response has been discussed elsewhere [5], but studies in our laboratories, using standard compounds and various experimental diets, suggest that rats can be used for primary screening in order to select compounds for detailed measurements on cholesterol turnover and atherosclerosis. We had discovered that appropriately substituted Nbenzyl derivatives of 4-aminobenzoic acid showed interesting biological activity [6]. In the present work, we report the synthesis and structure-activity relationships of a series of N-substituted amino acid derivatives (Tables I-V), some of which were found to have a potent hyperalphalipoproteinaemic effect in experimental animals. This series of compounds arose from structural variations to the above series and more detailed studies on one of these compounds (2, BRL 26314) have already been reported [5].

Brockham

Park, Betchworth,

Surrey, RH3 7AJ, England.

Table I. 3-Aryl-2-[(aryl)methyl]aminopropionic

acids.0

Activity, % changee Serum HDL Serum total SerWl cholesterol cholesterol triglyceride

Y

Prepn. method=

H

Af

4-Cl

A

4-Br

A

4-F

Af

228229O

11

Cl6HlbPNC2

0

-3

+3

+15

+2

3-CF3

Af

208210°

37

C1+'16F3~Oz

o

+51

+47h

-4lh

+2oh

mP, OC 217220°g 229230' 226228O

Yield,b %

formula=

Dietd

49

ClhH1?'2

0

+17

+9

-6

+16

57

ClbH16ClN%

S

+s+

+3oh

-21

+9

63

C16H16BrN02

S

+6bh

+uh

-40

-8

Body wt. gain

-II

H

3-OH

A

214216'j

30

Cl6H17~~3

s

-7

+2

-6

.z

H

Z-Cl

A

191192O

13

C16HlbClN02

s

0

+8

-29"

0

17

H

2-cF3

A

2oo"

16

C17H16F3%

S

+13

+a

+16

-2

20

CMH~~C~F'Z

S

+&ah

+4sh

-4lh

-19

50

Cl7Hl7N'k

0

f10

+a

-3lh

+33h

-13

E!

H

3,4-Cl2

A

207.5 -209O

22

H

3,4-OCH20-

A

226'

-20

H

2,4-Cl2

A

186188O

20

Cl6Hl5ClP'2

S

+2

+6

-7

-3

22

H

2,6-Cl2

A

176176.5'l

l8

CI~HISC~~N'+

S

-3

+46"

-13

-4

22

4-OCH3

4-Cl

A

224225'

36

Cl7HlaClN03

0

-20

-4

-6

-1

23

4-OH 3,5-I2

4-Cl

A1

2110 210-

j

49

C16H14ClI2NOj

S

-7

+22

+56h

-8

-24

4-OH

4-Cl

B

218-. 2200

j

l2

C16H16ClN03

0

+6

+8

-14

+7

2

4-Cl

H

A

223224O

53

C16H16C1N02

S

+I&

+glh

-19

-4lh

26

4-Cl

4-Cl

A

214217'

4s

Cl6HlsC12N'?z

0

+66

+56h

-50

-nh

j

525 Table I. (continued). %

Activitv, No

Prepn. methoda

Y

X

Ser~,m

IIDI.

Serum

changee

tot.21

Sel-Ul?l triglyceride

Body wt. gain

mP, OC

Yield,b % 30

Cl7HlaC1N"z

S

-8

+21

-37h

-7

82

C16H16C1N03

S

-10

+4

0

+6

formula=

Dietd

cholesterol

cholesterol

&7

4-CH3

4-Cl

A

222224'

28

4-Cl

4-OH

A

221224'

2

4-Cl

4-OC6H5

A

208210°

72

C22H20C1N03m

S

+6

-13h

-3lh

-7

z!

4-Cl

4-C6H5

A

223224O

33

C22H20C1N02"

S

-1

-7"

-7

-7

31

H

4-Cl

A

222224'

38

C16”16C1N%

S

+6ah

+49h

-6Th

-2oh

L

j

“The letter refers to the general methods described in the text. bNo e,Yorts were made to optimize yields. CA11compounds were analysed for C, H, N and halogen (Cl or Bf), if present, within *0.4% of theory unless otherwise stated. dOxoid (0) or semi-synthetic (S). For rats maintained on Oxoid, HDL cholesterol was measured using serum samples pooled from 4 rats so that no statistical analysis is possible. eAll the compounds were screened orally at 100 mg/kg for 7 d. The ‘A change for each parameter given is that compared with an appropriate control value. fThe oroduct was dissolved in dilute NaOH solution and -urecioitated bv acidification with dilute HCI. ^ gLit. Galue is 215-220°C [7]. Vignificantly different from control, p ~0.05. iC: calcd. : 72.46; found: 71.94. jMelts with decomposition. kRecryst. from aq. CH30H. ‘The product was isolated by bringing the reaction mixture to pH 4 with cont. HCl. mH: calcd.: 5.28; found: 5.80. “H: calcd. : 5.51 ; found: 6.17. OCompounds l-23 inclusive were prepared from L-amino acids. Compounds 24-30 inclusive were prepared from DL-amino acids. Compound 31 was prepared from a D-amino acid.

Chemistry

Compounds in Tables I-V were prepared from various amino acids 1 and aldehydes 2 by in situ reduction of the corresponding Schiff’s base with sodium cyanoborohydride (Scheme 1, method A) [7]. Alternatively, an ester 5 was prepared by catalytic reduction of the Schiff’s base formed from 4 and 2 and hydrolysed to give the desired product 3 [7] (Scheme 1, method B). Compound 40 was prepared by reaction of a mixture of phenylalanine methyl ester and 2-(4-chlorophenyl)-ethyl-p-toluene sulphonate [8] with METHOD

A

R3

2

R3

'

R -f-(CH21nCU2H

+

1 RCHO

NaBH3CN > d-b

IcH21mNH2

(cH21mNHCH2Rl

(21

III METHOD

-ICH21nc02~

I31

T

B

NoOH/

R3

Et OH

R3

I

H2IPd-C “‘-;

-fCH2)&02R liH2lm 141

Scheme 1.

NH2

*

(21

anhydrous K,C03 followed by saponification of the ester (method C). Compound 55 was prepared by catalytic reduction of a mixture of histidine monohydrochloride and 4-chlorobenzaldehyde (method D) [9]. The 2-pyridylglycine derivative 69 was prepared by hydrolysis of the ester obtained by condensing ethyl a-bromo-2-pyridyl acetate [lo] with 4-chlorobenzylamine (method E). A modification of this method was used to prepare compound 67 [Ill; methyl a-bromophenylacetate was condensed with 4-chloroaniline in the presence of anhydrous K,CO, in refluxing acetone and the resulting ester was then hydrolysed to give the desired product. The a-methyl derivative 73 was obtained via alkylation [12] of the Schiff’s base formed from 4chlorobenzaldehyde and phenylalanine methyl ester followed by base hydrolysis (method F). N-Methylation of 2 (BRL 26314) with formic acid and formaldehyde gave 75 (method G). Finally ethyl-3-oxo-4-phenylbutyrate was condensed with 4-chlorobenzylamine in the presence of iodine to give 3-(6chlorobenzyl)amino-4-phenylbut-2-enoate which was hydrogenated. Hydrolysis -of the ester gave 76 in 28% overall yield (method H).

> R2-+H21&02R

Biological results and discussion lcH21rn~~~~2~’ I51

The compounds were tested in male rats for their effects on serum total cholesterol, HDL cholesterol, and tri-

526 Table

II. 3-Aryl-2-[(substituted)methyl]

aminopropionic

acids.’ /\ X

D-

CH2 fH C02H NH CH2 It1 Activity,

% Changee 1

NO

X

32

H

Rl 1-naphthyl

Prepn. Methoda

Yieldb %

mp, OC

formulaC

Dietd

Serum HDL cholesterol

s:;s cholesterol

SerWll triglyceride

Body wt. gain

A

ZOZ202.s”

24

C20HlW2g

S

+ah

+d

-ah

210211oi

4o

‘3oHlF2

S

+3oh

+17h

-57h

-10

247248’

46

C24H20CLN02

S

+4

-2

+5

+12

33

H

2-naphthyl

A

24

H

IO-C1,9-anthracenyl

A

f

-2

2

H

1-adamantyl

Ak

224225’

10

C20H27NC2

0

-4

-2

-16

5!!

H

CH2-4-ClC6H4-

C

275’

14

C17HlsClN02

0

-17

-2

-4

-40

41

4-Cl

2-pyridyl

Aj

211212oi

I2

C15H15C1N202

s

+v+h

+7sh

-3oh

-17

-42

4-Cl

L-pyridyl

A.’

210212O

32

C15H15ClN202

S

0

-8

-21

-14

43

4-Cl

P-fury1

A

22S229O

31

C14’h4ClNO3

S

+w+h

+6lh

-47h

-2oh

44

4-a

5-Cl,Z-thienyl

A

220221°

24

C14H13C12N02S

S

+3lh

+lh

-42h

+4

45

4-Cl

222223oi

4S

%HlsClN02

S

-10

+10

-14

+4

c

CH L CHC6Hlj

A

-7

a-‘See corresponding footnotes to Table I. fRecrystallised from CaHsOH. gC: calcd. : 78.66; found: 78.10. iSignificantly different from control, JJ ~0.05. iMelts with decomposition. jThe product was isolated by bringing the reaction mixture to pH 6 with cont. HCl. kThe product was dissolved in dilute NaOH solution and precipitated by acidification with dilute HCl. inclusive from DL-precursors. iCompounds 3240 inclusive were prepared from L-precursors ; compounds 4145

glycerides as described under Experimental protocols. For historical reasons, some compounds were tested using a stock diet, whilst compounds prepared more recently were tested using a semi-synthetic diet devised to give a basal lipoprotein pattern of some physiological relevance [13]. Valuable information can be obtained for the various classes of lipid-modulating agents by comparing the metabolic responses in the two diets but for the present series the effect on HDL cholesterol metabolism and on triglyceride concentration was similar in either diet [5]. Liver weights were monitored routinely and the concentration of liver lipids measured only when significant effect on serum lipoproteins was observed. As the weight and lipid content of the liver rarely changed by more than lo%, the results are not shown for the primary evaluation. The activities of 3-aryl-2[(aryl)methyl]aminopropionic acids are shown in Table I. The majority of these compounds (l-21) are N-[(aryl)methyl]phenylalanine analogues differing only in the substituent Y in the aryl group. In this

series it was found that 3-, 4, or 3,4-disubstitution with halogen (Cl, Br) or CF, (compounds 2, 3, 13, 14 and 18) gave the greatest HDL elevation. In contrast, compounds bearing an o&o-substituent of this type (16, 17, 20 and 21) were essentially inactive. Compounds with other Y groups, even electron-withdrawing groups, such as methylsulphonyl (compound ll), were either inactive or had a small, nonsignificant effect on HDL. If o-substituted compounds (in which steric factors may be paramount) are ignored, all those compounds in the series 1-21 with good activity had aromatic substituents (Y) falling in the 3-0, +n quadrant of a two-dimensional Craig plot [14, 151. Introduction of a substituent (X) into the other aromatic ring whilst maintaining Y as 4-Cl led to loss of activity except when X was also 4-Cl (compound 26). Compound 25 was also very active. (X=Cl; Y=H) Most of the compounds in Table I were prepared from L-starting materials by a reductive process (method A) believed to involve minimal racemisation. However, absolute

527 Table III. 3-Substituted-2-[(aryl)methyl]aminopropionic

acids.j RZCH2 $H C02H NH CH2

/ -Q

erepn. NO.

Y

R2

-46

H

3-indolyl

methoda B

mP, OC

\ -

Y

Activity,

Yieldb

23724@f

a-%ee corresponding footnotes to Table I. fMelts with decomposition. Qignificantly different from control, p <0.05. Wontrol value was unusually low. icontains 1.5 mol of Hz0 of crystallisation. jcompounds 46-48 were prepared from L-precursors.

% 43

Serum formulaC

Died

cholesterol

C18”18N202

0

-29

‘h&7ClN20z

0

+55

All other compounds

stereochemistry appears to be unimportant factor in determining activity since the D-compound 31 was very potent (compare the L-isomer 2) as were the DL-compounds 25 and 26 noted above. Table II lists compounds in which various groups R1 were introduced into the side chain in place of substituted phenyl. In this series, an aromatic group R1 (e.g., naphthyl as in 32 and 33) or a heteroaromatic group (as in 41 and 43) appeared to be essential, albeit insufficient, for activity. It is noteworthy that 40, with an extended side chain, was inactive. Table III lists analogues in which the other end of the molecule was varied: that is, compounds in which an aryl or heteroaryl group R2 was introduced whilst retaining for the most part, side chains giving best results in the derivatives listed in Table I. Most of the compounds in Table III were active, the I-naphthyl derivative 49 being a notable exception. In Table IV, compounds 59-64 (prepared from Lstarting materials) and 68-72 (racemic) were prepared to examine the activity of N-4-chlorobenzyl derivatives of amino acids other than aryl- or heteroaryl-substituted alanines. The most active compounds in this series were

HDL

Sf?YXm rota1 cholesterol +12 R +2og

% Changee S~tY.Illl triglyceride

wt.

Body gain

-4

+5

-368

+2

were prepared from m-precursors.

the substituted glycine derivatives 59,69 and 70. Introduction of a 4-OH substituent into the phenyl ring in 60 led to loss of activity as in the phenylalanine analogue 24. Compounds in which the group R2 did not contain an aromatic or heteroaromatic group were all inactive and in one case (compound 71) extremely toxic. Table V lists a number of miscellaneous derivatives, prepared to evaluate the effect of inserting an a-methyl group (74), and N-methyl group (76) or making various alterations to the spacing of the nitrogen atom relative to the carboxy group. None of the compounds in this Table was of interest. As regards biological effects other than HDL elevation, it was notable that some compounds that markedly raised HDL also decreased body weight (bw) gain, but the two effects are not necessarily associated, e.g., compounds 2, 47 and 57, had little effect on growth. Compounds that markedly raised HDL also tended to elevate total cholesterol, a result that is not surprising, since HDL accounts for a large proportion of the total circulating cholesterol in rats. In addition, the elevation in HDL was often, but not always, associated with a decrease in triglycerides.

528 Table IV. Various disubstituted

amino acidsr R2

7"

CO2H

NH-R1

R1

NO.

R2

Prepll. methoda

59

4-C1

C6H4CH2

C6H5

A

60

4-a

C6H4CH2

4-H0C6H4

Ag

C6H5CH2SCH2

Ah

61

4-Cl

C6H4CH2

62

4-Cl

C6H4CH2

-63

4-a

CgHqCH*

(CH2)2SCH2

A

C"3

196198'

34

245'

+125f

+42f

-34f

- 24f

29

Cl5Hl4ClN03

0

-12

+6

-19

+4

C17Hl8ClNO2S

0

+61

+52f

-35f

-22f

Cl7HlgClN202S

S

+4

+4

-2Sf

+18

CloHl2ClN'+

0

+4

-7

+4

+6

79

ZlO2130i

32

ClgH24N202j'k

0

+11

-12f

-2lf

-8

0

-10

-8

+4

+6

4-Cl

C6H4CH2

C&(CH2)2

B

69

4-Cl

C6H4CH2

2-pyridyl

E

4-c1.

C6H4CH2

CH3S(CH2)2

S

B

68

C6H4CH2

C15"14ClN02

-4

166 16s"m*n

4-C1

29

-5

E

11

Body gain

-4

C6H5

E-C4Hg

"t.

-5

B

C6H4CH2

triglyceride

0

(I-dodecyljCH2

4-Cl

total cholesterol

CloHlzClNO3

192197O

11

Serum

HDL cholesterol

33

(3-indolyllCH2

4-(3,5-di-CH3)isoxazolyl

Serum

Dietd

Ag

(3-indolyljCR2

70

SelYlm

% Changee

21822ooi

(2-norbornyl)CH2

C6H4

formula=

%

36

65

4-C1

Yieldb

209210° 217219O ZOl202O

4-C1

67

HOCH2

B

'C

64

66

C6HqCH2

(4-pyridylj-

mP>

Activity,

236237' 118120°

B

2002flz" 216218'

Ag

219221a<

Ag

3g 4o

C23%6N202

1

0

+3

-8

0

+62

+6Sf

Cu+Hl3CW02

S

+64f

+103f

-34

CIOWLV~~

S

+121f

+9

-54f

59

C13HlsClN'+

0

46

C12Hl6ClN02S

0

78 6o 9

Cl4Hl2ClNO2 Cl7H2-~ClN02~

See footnote -5

+1

+4 -11

-1 -56f 0 -47f

s +5

-3

&-%ee corresponding footnotes to Table I. ‘Significantly different from control, p ~0.05. gThe product was isolated by bringing the reaction mixture to pH 4 with cont. HCI. hThe product was dissolved in dilute NaOH solution and precipitated by acidification with dilute HCI. iMelts with decomposition. jH: calcd. : 7.93 ; found: 8.44. kContains 1 mol Hz0 of crystallisation. iC: calcd.: 74.15; found: 73.61. mRecrystallised from CHsOH. “Lit. mp: 175OC [ll]. OBased on a-bromophenylacetic acid. PH: calcd. : 6.64 ; found : 5.97. qRequires: C: 57.05; H: 5.13; N: 9.51; Cl: 12.03. Found: C: 56.62; H: 5.62; N: 9.05; Cl: 11.10. *Compounds 59-66 were prepared from L-; compounds 67-72 from DL-starting materials. SCompound was withdrawn because of toxic effects.

In contrast to the lack of effect of diet on lipoprotein modulation by the substituted amino acids, the response of standard compounds is very different (Table VI). In the semi-synthetic diet HDL is significantly increased by bezafibrate and fenofibrate and modestly increased by cholestyramine but significantly decreased by clofibrate and probucol. Each of ,these changes is similar to the usual clinical response and substantiates the use of this diet as a model for evaluating the possible clinical effects of novel compounds. In general, rats fed on a stock diet provide a poorer prediction of the clinical response: probucol and cholestyramine are essentially inactive and the fibrates all tend to decrease HDL.

On further evaluation in hyperlipidaemic rats, a dosedependent decrease in VLDL cholesterol and total triglycerides and an increase in HDL cholesterol was observed (Table VII) for compounds 2, 47 and 59. There was no consistent effect on liver lipids but a deleterious action on liver function was seen for compound 59. Compounds 2 and 47 were subsequently compared in a rabbit model of atherosclerosis [16]. After dosing for 28 days, compound 47 induced a smaller rise in serum HDL cholesterol than 2 (+41x vs +84x) and this was associated with a smaller effect on the severity of the aortic sudanophilic lesions (-17 % vs -53 %) and a smaller decrease in abdominal arterial intimal-medial thickening (-24 % vs -45 %).

529 Table V. Various di-, tri- or tetrasubstituted

amino acids.”

k” 7 -(CH2),

R* -

(CH2jm

C02H NR1. R4 Activity,

NO.

R1

R2

Serum HDL cholesterol

Serum total cholesterol

Serum triglyceride

R3

R4

m

n

myt:ehpondb

:;

11

0

0

F

2702720

448

Cl7HlSClN02

0

-23

-9

0

0

A

268272O

21

CllHl4ClN02

0

-15

0

-13

18

Cl,H18ClN02 S

-17h

-3

-4

C17HlSClN02

-12

12

4-Cl

C,jH4CH2 CgH5CH2CH3

E

4-Cl

CgH4CH2

CH3

Yield,c %

% Change=

CH3H

Ei

4-Cl

C6H4CH2 C6H5CH2

H

CH3

o 0

G

1871X8"

E

4-Cl

C6H4CH2 C$5CH2

H

H

0

1

H

1992000

82

4-Cl

C,jH4CH2

H

H

0

1

Aj

1900

28

CgH5

formud

ni.etf

S

Table VI. Comparison

of standard

compounds

0

Body gain +3

+36h -7

+38h

-37h

-23h

-4

-23h

-2

+6

“Starting material was racemic unless otherwise noted. WSee corresponding footnotes to Table I. gBased on the corresponding Schiff’s base (Method F). “Significantly different from control, JJ ~0.05. ‘Starting material was L-configuration. jThe product was dissolved in dilute NaOH solution and precipitated by acidification kH: calcd. : 5.56; found : 5.07. rC: calcd. : 67.25 ; found : 65.26; H: calcd. : 5.93 ; found : 5.39. “H: calcd. : 5.57; found: 5.05. *Control value was unusually low. oRecrystallised from CHsOH. PAmino acid prepared according to the method of Secrist and Logue [19].

wt.

with dilute HCI.

using the 2 diets.

, % change Stock HDL cholesterol Clofibrate

-19

-30a

(100

mg/kg)

-3P

-23

Fenofibrate

(100

mg/kg)

-36a

-15

(250

Cholestyramine

“Significantly

mg/kg)

Total cholesterol

Bezafibrate

Probucol

(100

mg/kg) (2,000

mg/kg)

compared

with

appropriate

Diet Total triglyceride

=

control Semi-synthetic Total cholesterol

HDL cholesterol

Diet Total triglyceride

-14

-3oa

+10

-50=

-16

-29 a

-42a

+43

-44a

+70.a

+18

-35=

.=

-1

+3

-30=

-lga

-11

-12

+5

+3

+9

+15

-32a

-19

different from control, p ~0.05.

In more detailed biochemical studies [.5], compound 2 was found to promote reverse cholesterol transport and to increase the biliary excretion of bile acids in rats and one consequence appears to be a work-induced hypertrophy of the biliary ducts. The toxicological significance of this effect is not clear and further studies would be needed if clinical progression of compounds of this type were to be considered.

Experimental protocols Biological methods Male Sprague-Dawley rats (SO-100 g) were obtained from A. Tuck and Son, Battlesbridge, U.K., and received food and water ad libitum. The rats were fed either on a stock laboratory (Oxoid) diet or on a semi-synthetic diet, as described previously [13], for 7 days and were then allocated to experimental groups of 8 animals so that the mean

530 Table VII. Direct comparison

of compounds

2 (BRL 26314), 47 and 59 at two dietary levels (semi-synthetic

T Body wt. gain

Control

Serum ,iver wt. body wt.

4355.6

5.9to.3

HDL, t- chol 1

LDL chola

40

25

55

24

58

24

Compound (mg/kg body wt./day) 2. (5(-J) 2

(100)

44k3.9

5.9+0.4

48~2.1

6.2tO.4

42. (50)

4552.3

5.6?0.3

51

22

47

38t2.0

5.8i0.5

53

18

(100)

59

(so)b

4Ot2.8

59

(1OO)C

17t3.6

d

6.920.4

79

29

6.4~0.5

76

30

&Pooled samples. b2/8 rats showed green tinting of serum. Q/8 rats showed green tinting of serum. %ignificantly different from control, p <0.05.

body weight (bw) of each group was the same. The compounds to be tested were added to the diet at a level of 0.1% (w/w) in the primary evaluation, this is equivalent to a daily dose of approximately 100 mg/kg bw. The experimental diets were given for 7 days, an untreated control group being included in each experiment. Serum total cholesterol and triglycerides were measured using a Technicon Autoanalyser [5] and lipoproteins were measured by the chemical precipitation methods described previously [13]. Liver lipid was determined on CHCls-CHsOH extracts of pooled liver samples [17]. The HDL assay chosen for the present studies principally measures the subfraction HDLz in rat plasma. This subfraction accounts for approximately 80% of HDL cholesterol in rats but other components of rat HDL, HDLI and HDLc, may be partly co-isolated with LDL [5], so that the interpretation of changes in that fraction is difficult. For this reason, the discussion of lipoprotein changes in the primary evaluation is confined to HDLz.

Melting points were uncorrected and measured on a Kofler hot-stage apparatus. The spectra of all new compounds were consistent with the assigned structures. Infra-red spectra were measured on a PerkinElmer 197 instrument and proton NMR spectra were obtained on a Varian EM-390 90 MHz machine. Mass spectra were run on a VG7070F mass spectrometer. Each analytical sample was homogeneous. Optical rotations were measured on non-racemic compounds and were of solutions, concentration = 1 in 1 :l 6 N HCl/acetic acid. Column chromatography on silica used Merck Kieselgel 60. The a-amino acids used were either available commercially or were synthesised by reaction of the appropriate alkyl halide with diethyl acetamidomalonate in the presence of sodium ethoxide followed by hydrolysis with refluxing 4648% aqueous HBr [18]. Method A [7] L-2- (4-ChCorobenzyC)amino-3-phenylpropionic [N-(4-ChCorobenzyC)-~~phenylalanine].

acid

2

(BRL

26314).

A mixture of L-phenylalanine (40 g, 0.24 mol) and 4-chlorobenzaldehyde (33.8 g, 0.24 mol) in CHaOH (600 ml) was stirred at room temperature for 1 h and then sodium cyanoborohydride (15.2 g, 0.24 mol) was added in one portion. The reaction mixture was stirred at room temperature for 7 h and then allowed to stand overnight. The precipitate was filtered off, washed well with CHaOH and Hz0 and dried in vacua giving 39.8 g of 2 as a colourless microcrystalline solid (57 % yield) ; vrnBX(KBr) 1620 cm-l ; 4 (TFA-dl): 3.45 (2H, m); 4.45 (3H, m) and 7.4 (9H, m).

lipid

(mg/lOO

IVLDL chola

ml)

Liver

Total chol

Total t riglycerid

lipid

Chol

diet).

(mg/g)

Triglyceride I

33

9Rf4.5

374236

4.8

9.7

27

105f6.7

9.7

169t16"

6.1

17

99T3.8

141+7.7d

5.8

7.7

31

104_+6.4

168c16d

6.4

10.5

22

93t2.8

23

135f7.6’

16

121f6.0’

Method B [7] 2- (I-Chlorobenzyl) benzyC)tryptophan).

a

f 1

150112d

5.8

9.0

135+154

4.6

10.4

103f13d

4.2

6.8

-

amino-3-

(3GndoCyC)propionic

acid

47.

[N- (4-Chloro-

A mixture of L-tryptophan methyl ester (4.2 g, 0.019 mol) and 4-chlorobenzaldehyde (2.48 g, 0.017 mol) in benzene (100 ml) was refluxed for 5 min using a Dean and Stark apparatus. The reaction mixture was cooled and evaporated to dryness to give the crude Schiff’s base (8 g). This was hydrogenated at afmospheric pressure in CHsOH (100 ml) with platinum oxide catalyst (0.2 g) until 1 eq. of hydrogen had been absorbed. The catalyst was filtered off and the solvent evaporated to give N-(4-chlorobenzyl)tryptophan methyl ester (6 g) as a yellow oil which crystallised on standing. The methyl ester was hydrolysed by heating under reflux with aqueousmethanolic NaOH (1.5 g of NaOH in 100 ml of 1 :l HzO/CH~OH) for 2 h. The reaction mixture was cooled, the CHaOH evaporated and the solution diluted to 100 ml with HzO. Extraction of this solution with CHC13 (which was discarded) followed by acidification of the aqueous solution with 2 M HCl gave a precipitate which was filtered off, washed with Hz0 and dried in vacua to give 47 (2.6 g, 41 ‘A overall yield) as a white solid; Y,,, (KBr) 3485, 1585 and 1575 cm-l; mass spectrum M+ 328; 6 (DMSO-ds): 3.1 (2H, m); 3.4 (lH, m); 3.88 (2H, d, J = 3 Hz); 6.0 (2H, br s, D20 exchangeable); 7.25 (9H, m) and 10.82 (IH, s, D20 exchangeable). Method C ~-2-[2-(4-ChCorophenyl)ethyC]amino-3-phenyCpropionic

acid 40. A solution of L-phenylalanine methyl, ester (6.7 g, 0.037 mol) and 2-(4-chlorophenyl)ethyl-p-toluene sulphonate [8] (11.6 g, 0.37 mol) in toluene (150 ml) was treated with anhydrous K2C03 (5 g) and stirred under reflux for 4 h. The mixture was poured into Hz0 (300 ml) and the organic layer was washed with 2 M HCl, Hz0 and dried over anhydrous MgS04. Evaporation of the organic solvent gave a brown oil which deposited unchanged 2-(4-chlorophenyl)ethyl-p-toluene sulphonate on cooling from a solution in hot MeOH. The mother liquors were evaporated to give an oil (2 g); vrnax (film) 1740 cm-l; 6 (CDCL): 1.46 (lH, s, DzO exchangeable); ca. 2.66 (4H, m); 2.87 (2H, d, J = 7 Hz) ; 3.46 (lH, t, J = 7 Hz); 3.60 (3H, s) and ca. 7.10 (9H, m). This oil was refluxed for 2 h in aqueous CH30H (50 ml) containing NaOH (0.53 g). The mixture was cooled, acidified with 2 M HCl and the colourless product was collected and triturated with CH30H to give 40 (1.6 g, 14% yield); vmax (KBr) 1570 cm-l; 6 (TFA-dl): 3.20 (2H, t, J = 8 Hz) ; ca. 3.5 (4H, m) ; 4.47 (1 H, dd, J = 6, 9 Hz) ; and ca. 7.20 (9H, m). Method

D [9] (4XhCorobenzyC) ChCorobenzyC)-DL-histidine].

DL-2-

amino-3-

(4-imidazoCyC)propionic

acid

55.

pN- (4-

A solution of DL-histidine mono hydrochloride (7 g, 0.03 mol) in Ha0 (30 ml) was added to 4-chlorobenzal-

531 dehyde (17.6 g, 0.13 mol) in CzHsOH (80 ml) containing N, N-dimethylbenzylamine (20 ml) and the mixture was hydrogenated at 2 atm pressure in the presence of 10% Pd-C (5 g) for 7 h at room temperature. The solution was filtered, evaporated and the residue was dissolved in the minimum amount of H20. Addition of acetone with cooling gave a colourless solid which afforded pure 55 (1.6 g) as the mono hydrate, after recrystallisation from CzH50H and finally from Ha0 (13 % yield) ; rmax (KBr) 3400, 3335, 1650, 1630 and 1550 cm-l; 6 (TFA-dl): 3.70 (2H, d, J = 6 Hz); 4.47 (3H, m); 7.50 (5H, s); and 8.60 (lH, s). Method E DL-2- /4-Chlorobenzvlamino

I -2- (2-nvridvl) acetic acid 69. Ethyl abromo-2-pyridylacetate (14103 g, o.o58-mol) (prepared by bromination of ethyl 2-pyridylacetate [lo] was dissolved in tetrahydrofuran (THF) (150 ml) and anhydrous KaC03 (20.7 g, 0.15 mol) was added. A solution of 4-chlorobenzylamine (12.21 g, 0.086 mol) in THF (150 ml) was added dropwise to the stirred suspension over a period of 15 min. The mixture was stirred at ambient temperature overnight and then filtered. The filtrate was evaporated and the residue was chromatographed on silica gel eluting with CHaOH-CHzClz mixtures of increasing polarity (to 4%, v/v, CH~OH/CH~CIZ). The ester was isolated as an oil (12.17 g, 69.5% yield); vrnBX (film) 1735 cm-l; 6 (CDCla) : 1.20 (3H, t, J = 7 Hz); 2.99 (lH, br s, DzO exchangeable) ; 3.76 (2H, d, J
yield) ; vmax (KBr) 1610 cm-l; 6 (DMSO-do): 2.20 (3H, s); 2.92 (lH, dd, J = 3, 8 Hz); 3.50 (lH, t, J = 8 Hz); 3.51 (lH, d, J = 14 Hz); 3.75 (lH, d, J = 14 Hz); ca. 7.10 (4H, m); and 7.22 (5H, s). Method H DL-3-(I-Chlorobenzyl)amino-4-phenylbutyric

acid 76. Ethyl-3-0x0-4phenylbutyrate (6.6 g, 0.032 mol) and 4-chlorobenzylamine (4.53 g, 0.032 mol) were dissolved in benzene (50 ml) containing a trace of 1~. The reaction mixture was allowed to stand at room temperature overnight and then the solvent was removed in vacua and the product was chromatographed to give ethyl 3-(4-chlorobenzyl)amibo-4-phenylbut-2-enoate (7.6 g); 6 (DMSO-de): 1.13 (3H, t, J = 7.5 Hz); 3.56 (2H, s); 3.96 (2H, q, J = 7.5 Hz); 4.33 (lH, s); 4.40 (2H, d, J = 6 Hz); ca. 7.30 (9H, m); and 8.86 (lH, br t, J = 6 Hz, DzO exchangeable). This material was hydrogenated at atmospheric pressure using PtOa catalyst (0.2 g) in CzHjOH (80 ml) as solvent until hydrogen uptake had ceased. The solution was filtered, the solvent removed in vacua and the product chromatographed eluting with 2 ‘A CHsOH in CHaCla to give oL-ethyl-3-(4-chlorobenzyl)amino-4-phenylbutyrate (3.6 g); vmBX(film) 1730 cm-l ; mass spectrum (NH3 C.I.) MH+ 332 ; 6 (CDCL) : 1.20 (3H, t, J = 8 Hz); 1.70 (lH, br s, Da0 exchangeable); 2.35 (2H, d, J = 6 Hz); cu. 2.75 (2H, m); cu. 3.16 (lH, m); 3.70 (2H, s) ; 4.06 (2H, q. J = 8 Hz) ; and ca. 7.15 (9H, m). The ester was hydrolysed by refluxing with NaOH (0.8 g) in HzO/CZHZOH (4:1, 150 ml). After 4 h, the reaction mixture was cooled, diluted with Hz0 and extracted with (C2H5)20. The aqueous layer was acidified (pH 4) with dilute HCl and the white precipitate was filtered, washed with CzHsOH and dried in vacua to give 76 (2.7 g, 28 % overall yield); vmax (KBr) 1620, cu. 1575 cm-l; 6 (TFA-dl): ca. 3.15 (4H, m); 3.96 (lH, m); 4.40 (2H, s); and ca. 7.30 (9H, m).

Acknowledgments The authors wish to thank Mr. G. Baker, Miss H. A. Birch, Mrs. R. I. E. Carter and Mr. G. Bond for their skilled technical assistance.

Method F [12] 2-(4-Chlorobenzyl)amino-2-methyl-3-phenylpropionic

acid 73. A solution of methyl-Z[N-(4-chlorobenzylidene)]-amino-3-phenylpropionate (see method B) (5 g, 0.016 mol) in dry THF (20 ml) was added dropwise to a stirred solution of KOQHa (1.8 g, 0.016 mol) in dry THF (50 ml) under NB at -78OC. After 30 min, KI (0.2 g) was added, followed by CHaI (2.3 g, 0.016 mol) dropwise and the solution stirred overnight at ambient temperature. Saturated aqueous NH&l was added, followed by (Cd&)20 and the ethereal layer separated, dried (anhydrous NaaS04) and evaporated to give methyl 2-[N-(4-chlorobenzylidene)]amino-2methyl-3-phenylpropionate (5.3 g, 0.016 mol) as an orange oil. This crude product was dissolved in CHaOH (40 ml) and NaBHaCN (2 g, 0.03 mol) was added portionwise over 30 min and the solution stirred at room temperature overnight. Acidification with cont. HCl and dilution with Hz0 (150 ml) gave a colourless precipitate which was recrystallised from CzHbOH to give methyl 2-(4-chlorobenzyl)amino2-methyl-3-phenylpropionate (4. 8g); vmax (KBr) 1745 cm-l; 6 (TFAdi): 1.97 (3H, s); 3.47 (2H, s); 3.97 (3H, s); 4.21 (lH, d, J = 12 Hz); 4.47 (1H. d. J = 12 Hz): and cu. 7.3 (9H. m). Hvdrolvsis was carried out with KOH (2 g) in refluxing CHadHLH& (4-3, v/v! 35 ml) for 5 h. Acidification with 2 M HCl gave a colourless solid which was filtered, washed with CHaOH, triturated with hot Hz0 and dried giving 73 (2.2 g, 44% yield); vmsx (KBr) 1608 and 1575 cm-l ; (TFAdi): 2.00 (3H, s); 3.36 (lH, d, J = 15 Hz); 3.60 (lH, d, J = 15 Hz); 4.40 (2H, br s); and cu. 7.30 (9H, m). Meihod G L-2-N-(4-Chlorobenzyl), [N- (CChlorobenzyl),

N-methylamino-3-phenylpvopionic N-methyl-L-phenylalanine].

acid

75.

N-(4-Chlorobenzyl)L-phenvlalanine 2 (10 g, 0.034 mol) was heated on a steam bath for 3 h with formic acid (6% ml) and 37 % aqueous formaldehyde solution (20 ml). The reaction mixture was cooled and poured into H20. A white precipitate formed which was filtered, washed with Hz0 and CHaOH and recrystallised from CHaOH to give 75 (1.92 g, 18%

References 1 Lipid Research Clinics (1984) J. Am. Med. Assoc. 251, 351-364 2 Fears R. (1984) Drugs Today 20, 257-294 3 Sircar I., Hoefle M. & Maxwell R. E. (1983) J. Med. Chem. 26, 1020-1027 Eisenberg S. (1984) J. Liuid Res. 25, 1017-1058 Fears R, Rush W.‘R., Walker P. & Ferres H. (1984)IBiochem. Pharmacol. 33, 209-217 U.K. Patent No. 1576007 published 1.10.80 Takemura S., Terauchi H., Kowata K., Nakano K., Okumura Y., Li K. & Inamori Y. (1978) J. Pharm. Sot. Jpn. 98, 869-879 8 Schadt F. L. III, Lancelot C. J. & Schleyer P. vR. (1978) J. Am. Chem. Sot. 100, 228-246 9 Reinhold V. N., Ishikawa Y. & Melville D. B. (1968) J. Med. Chem. 11, 258-260 10 Edwards 0. E., Chaput M., Clarke F. H. & Singh T. (1954) Can. J. Chem. 32. 785-791 11 Bhatt S. B. & Parikh A. R. (1976) Curr. Sci. 45, 547 12 Stork G.. Leona A. Y. W. & Touzin A. M. (1976) \ , J. Ora. Chem. 41, 34911349313 Rush W. R. & Fears R. (1982) Biochem. Pharmacol. 31, 24232426 14 Craig P. N. (19 ) in: Buwer’s Medicinal Chemistrv 4th edn.. Part-I, Ch. ‘8, pp. 343 15 Craig P. N. (1971) J. Med. Chem. 14, 680-684 16 Fears R., Esmail A., Walker P., Rush W. R. & Ferres H. (1984) Biochem. Pharmacol. 33, 219-228 17 Folch J., Lees M. & Sloane Stanley G. H. (1957) J. Biol. Chem. 226, 497-509 18 Albertson N. F. &Archer S. (1945) J. Am. Chem. Sot. 67,308-310 19 Secrist J. A, III & Logue M. W. (1972) J. Org. Chem. 37, 335-336