November 1965 NOTES 877

primary or secondary amine was heated on an oil bath a t 130- 140' for 3 hr. with constant stirring. The mixture was then cooled and 20 g. of a 50% aqueous NaOH solution was added. The mixture was extracted with 200 ml. of ether and the extract was dried overnight. The dried extract was then distilled to yield the desirpd product.

Spasmolytic Activity.-Anticholinergic activity was deter- mined on isolated guinea pig ileum suspended in Locke-Ringer's solution in a water bath a t 37". Acetylcholine ( 1 5 0 p.p.m.) was used as the spasmogenic agent. The compounds being tested were added to the medium 30 sec. before the addition of acetylcholine, and the inhibition of the acetylcholine-induced contractioii u-as measured by comparison with control values.

The procediire used to determine the inhibition of BaClz-in- duced contractions was the same, except that the final concen- tration of BaClz was 1:10,000 and rabbit ileum was the test tissue. The results are shown in Table 11.

Acknowledgment.-The authors wish to express their thanks to Dr. Beiler and his staff at the Research Laboratories of the National Drug Company for the pharmacological evaluation.

Amino Acid Analogs. I. Analogs of the Glutamic Acid-Proline Interconversion. 111. Substituted 2-Acetarnido-4-benzoylbutyric

Acids and 5-Phenylprolines

HERMAN GERSHOS,' ALFRED SCaLA, AXD RAULO PBR11EQIAAI

Pjister Chemical W o r k s , Inc., Ridgejield, S e w Jersey

Received June 7 , 1065

In a previous paper,2 a rationale for our interest in the preparation of proline and glutamic acid analogs was presented. I n that work, the syntheses of 2-acet- amido-4benzoylbutyric acid and 5-phenylproline were reported, and the methods were thought to be suitable for the preparation of aromatic substitution products xyhich were desired for biological study.

In this study, 10 glutamic acid and 10 proline analogs and the necessary intermediates3 will be described.

The P-chloropropiophenones (I) were prepared by acylation of the appropriate benzene derivative with p- chloropropionyl chloride by means of aluminum chlo- ride in carbon disulfide, according to Allen, Cressman, arid BelL4 The chloro ketones mere used without fur- ther purification in the condensation with ethyl acet- amidomalonate using sodium ethoxide in anhydrous ethyl alcohol as the condensing medium. Upon hy- drolysis of the acetaniidomalonates (11) x-ith alkali, malonic acids (111) were obtained which were de- carboxylated t o the acetylated glutamic acid analogs (IV). Acid hydrolysis of I1 yielded 2-(substituted pheiiyl)-l-pyrroline-5-carboxylic acids (V). Subse- quent hydrogenation of the pyrrolines (T') over Adams' ratalyqt at 3-4 atni. of hydrogen yielded the coi-re-

(1) T o al iorn re,juests for reprints should he made:

(2) H. Gershon and A. Scala, J . O w . Chem.. 26, 2347 (1561). ( 3 ) Three of the intermediates prepared in this study, 2-(4-chlorophenyl)-,

2-(4-methoxyphen).l)-, and 2-(4-aminophen3.1)-l-p).rroline-j-carhox3.lic acid, were claimed in a recent patent [F. Leonard, British Patent 577,097 (1564)l bu t were not characterized. The method of synthesis described in the patent is analogous to tha t reported by Gershon and Scala2 for the preparation of 2-phenyl-l-pyrroline-5-carboxylic acid.

(4) C. F. H . Allen, H. \T. J. Cressman, and A. C. Bell, Can . J . K e s . , 8, 440 (1933).

I b y c e Thompson Insti tute for Plant Research, Yonkers, N. I-. 10701.

SCHEME I

I

COOCzH5 E R ~ ~ C O C H ~ C H ~ C N H C O C H ~ - I I

- COOCzH5

I1 v H,-PtO, I NaOH I

COOH R~-&-$&F R l d C O C H 2 C H 2 C N I HCOCH3

I H COOH

VI I11

1-CO2

--, - - ~ b, R, = Ci; R z = H e , R, = Br; R2 = H

e, R1 = CBHB; Rz = H d, Ri = CH,;

f. RY = CHqO: R? = H

Rz = H

g; R; = CGHSO; l?z = H

i, R1 = F; R2 = F j , R, = C1; Rz = C1

IIh-IT-h, R, = CHBCONH; Vh, TTh, R1 = Rl

Rz = H SHz'HCl; R, = H

sporiding proline analogs (VI). These reactions are summarized in Scheme I, and the physical and analyti- cal data on compounds of types 11-VI are listed in Tables I-V, respectively.

The genus Leuconostoc is composed of streptococcus- like bacteria that secrete large quantities of gum. These organisms which are prevalent in sugar refineries interfere with the processing of cane sugar.5 Leuconostoc inesenteroides P-60 is a species that has been used as an assay organism for 18 amino acids6 Among these are glutamic acid and proline.

To learn whether these glutamic acid and proline analogs possess diff ererices in biological activity from the unsubstituted analogs and to learn whether such compounds could be useful in the cane sugar industry,

( 5 ) .I, T. Henrici and E. J. Ordal, "The Biology of Bacteria." D. C. Heath

(6) B. F. Steele, H. E. Sauberlich, XI, S. Reynolds. and C. ..2. Ijaumann, and Co.. Boston, XIass., 1948, p. 416.

J . B i d . Chem., 177, 533 (1949).

RL I COOC > H

I

S O T E S 878

TABLE IT'

Rl&

I'CTED ~-PHESYL-~-ITRROLIXE-~-CARBOXYLIC ACID HYDROCHLORIDES (1.)

Yield. 31. y . ,a __-_ Calcd., <h---- ,- I:oitn,l, b;

v K, H 2 54 " C . dec. Formula C H L- c H s a F H 83.0 108-110 C11H1,C1F1702 54.22 4.55 5.75 5 4 . 5 4 4.03 5 : 3 0 I, C1 H -'' II .8 107-109 C11H11CI2N02 50.79 4 , 2 6 5.39 50.75 4.32 3 . 4 2 C Br H 5 2 . 5 192-194 CIIHl1BrCl?r'O2 43.37 3.64 4.60 43.40 3.65 4.64 d CHJ H 91.5 70 Cl?HiJClS02 60.13 5,8!) 5.84 60.02 5.68 5.52 e CsH; H 89.8 90 CiiHisClS 0 2 67.66 5 .34 4.64 67.98 5 .30 4.23 f CHZO H 9 7 . 7 110 CnH1AClSOa 56.36 .5,52 5.48 56.62 5.81 5.2s g CsHjO H 86.8 123 CiiHi6ClY03 64.25 ,5.08 4.41 64.42 5.35 4.16 h NHZ.HC1 H 80.0 253 CllH14C12N202 47.67 5.09 10.11 47.65 5.12 10.20 i F F 92 .5 112-1 14 Cl1HlOC1F2NO2 50.49 3.85 5 . 3 5 30.71 3 . 9 7 4 , 0 5 J C1 C1 85.6 172-174 C~IH~,CI~NO, 44.85 3.42 4.76 4 5 . 2 2 3.76 4 . 5 0

a Aiialyticd samples; crystallized froiii niethyl alcohol-ether iiiistures.

VI

a 1) r d e f r h

.l

1

Ri F CI Br CHs C6H5 CHI0 C6HjO NH?.HCI F C1

R? H H H H H H H H F e1

TABLE V SUBS'TITUTED ~-PHESYLPROLINE HYDROCHLORIUBS (\.I)

R l -&CO~~c l H

Yield, 3I .p . ,Q -----Calr~:i.. [ V - 470 " C . Formula C H

92.5 140 CiiH13CIFNO2 5 3 . 78 4 . 0 2 80.0 150-152 CiiHiaCl,N02 50.40 3 OO 92.0 93 C I I H ~ B B ~ C I X O ~ 43.09 4.27 93.0 109-11 1 C ~ Y H I ~ C ~ S O ~ 50 63 6 .67 96 .5 190 CiiHi8ClSO2 67.21 5.9T 84.8 17i-179 C I ~ H I ~ C ~ N O , 5.593 6.26 96.8 188-190 CiiH,&1NO3 6L85 5.67 88.8 137 CliHi6Cldi20? 47.32 5 . 78 84.0 175-1 77 C11H12C1F2N02 50.11 4.59 70. 0 203-205 CllHl?Cl,NO, 44,55 4 , OS

-___ S

5 .70 5 . 3 4 4 . 5 7 5.80 4.61 5 .44 4.38

10,04 j 31 4.72

---- l o u n i i . I,i--

c, H

53 s i 4 .90 . iO . 2 2 5 , Oi 43.01 4.12 59.71 6 S 5 66.94 3 . 6 3 5,568 6 . 2 4 63.61 T,.:i6 47.28 , i . i t 5 50.20 4.41 44.50 3.97

r2iialytical samples: crystalliLed from rnethyl alcohol-ether mixtures

TABLE VI h T I B A C T E R I A L AC'l.1 O F SELECTED 2-AC'ETAh.IIDO-4-

BEKZOYLBUTYRIC ACIDS ASD ~-PHESYLPROLINES AGAISST

('onipouiid, but the low level of activity of the test rom- Iioutids would nialce them useless for the purpose of the study undertalren.

All of these compounds were screened by the Caricw Chemotherapy Kational Service Center (SIH) agaiiist at least three 1nou.w tumors, Sarcoiiia 180, Carcinoma 75.5 or Ehrlich ascit>es, and leukemia L1210. These

L. / r U 3 S e n k t ' O i t k S P-60 I N SIhlLTL.4'l.ED C A S E S U G A R JUICE --Grout11 a t levels testeda--

Compd. 11 1 R2 O.lYo 0.5y0 1.0%

I NHCOCH,

lief. 2 H H + + I Ya F H + + 1I.e CaHa H + 1T.f CHJO H + IT'i F F +

- - -

R2

lief. 2 H H + + \'Id CH3 H + \7e CeH5 H + \.If CH,O H + \'I j C1 CI +

- - - -

(i + = growth, - = roniplete illhibition.

data are cotitairled in Table VI1

Experimental Section

Chemical.' p-Acetamido-p-chloropropiophenone (Ih).-A mixture of 137 g. (1.01 moles) of acetanilide, 210 g. (1.58 moles)

- of anhydrous AICI,, and 400 nil. of dry carbon disulfide w'as pre- - pared. p-Chloropropionyl chloride (100 g., 0.79 mole) was added - dropwise with vigorous agitatioii, diiriiiq 2 hr., :xiid the teni-

peratiiw of the reacntioii was maintained at ahinit 20" by means of a water bath. Upon completion of additioii of the p-chloro- propioiil-l chloride, agitatioii was c,oiitiuiied overnight. The prodiict mas drowiied in a sliirrl- of 500 g. of ire in 200 ml. of coilcentrated HC!, and the solid ni:tterial that formed was re- nioved by filtration, washed t l io ro i i~ l~ ly with water and dried at 05" iiiider vaciiIim. The yield of prodiict was 173 g. (97.5ci ), n1.p. 12S-140° der. An aiialyticd sample was rrystallized from n misture of acetoile mid ethyl nlcoliol, ni.p. 134-1 57" der.

- -

- - - - - ( i ) This work was completed several years ago, and melting points \%ere

then taken in a Hershberg melting point apparatus and are uncorrected. The synthetic procedures are general.

880

F COOH

II1:t l I Ib 1IIC IIId IIIe I I Ig I I Ih 1111 I I I j

w I NHCOCH,

I \.g CbH ,o H I\*h CH3CONH H n - I F F I\-j ('I ('1

R2 I ,HC1

I311 C'I H

3

CTI, InC ha

6.i 1"; 125

2 3 50 511

I 2,5 12.3 2 3

. iOO ,311)

125 125 500 125 500 300 125 250 500

500

300 500 500

,500 300 600 ,500

300 500 500 500

500

100 100 ,500

2.50

500 125

T (

(3 3 .jO 76 6h ! I O 58 6 3 tiJ

105 % J 62

1 h'3 106 $16 84 i!l 84

124 166 121

h i

'30 7'3 95

7% 117 102 78

71 Y 0 7 2 h ,5

5ti

:3 7 123 72

IO,!

102 !)ti

\ 'ri I I I B Lg

t i3 li

)

2 3 11 -5 ci

200 11

4(NI 4 I ( I

12,j

4.50 112 .AX1 4.50 100 22.5 4.50

430

4.3 I 4.50 400 -200 400 450 450 400 400 90

400 :350 4.50

:L50

80

173 173 22.5 112 200 112

T ( '

h4 47

$7 102

9.3 6S 70 61 7 2 7 1

52

44 64

131 105 109 64 93

B"

7% titi 44

104 60 92 64 3 2 60 04

44 X h

-- I I

50 78

124

44 SO 49

10:2

47

- - i .)

-----I, 12 10 CT I,,')

lnir

c,O 47

2 0 20 4 .i

400 23

201) 200

- , I (J

! I l l

430 12,j 3 5 0 4.50 100 225 450 430 450

500 450 200

400 4.50 1'3 400

! )O 400 350 430

5 0 0

00

:350

225

400 I I2

Kovember 1965 XOTES 88 1

Compd. RI RZ

T'f CHIO H v g C6H5O H Vh NH2.HCl H

Vi F F V j C1 C1

TABLE \.I1 (Continued) --S180------ , Ca775 or EA-

NTLP T/C: NTL? T/C.C mg./kg. % m d k g . x

500 68 350 C 90 125 98 112 C 116 250 50 112 C 68

125 99 88 C 108 125 78 100 C 59

61

Ref. 2 VIa YIt, VIc S'Id J-Ie T'If

T'Ih T'Ii YIj

T'Ig

H H F H c1 H Br H CHI H C6H6 H CHBO H C6Hj0 H NHe.HC1 H F F c1 CI

500 250 125 500 250 125 500 500 375 500 100

100 78 66 99 75 75

102 72 80 62 89

450 200 112 350 100 56

225 225 320 200

80

C C C E C C C C C C C

58 95 82 82

100 93

137 108 55 7 0 63

L121+-- NTL? T/C,C

mg .! kg . %

350 102 112 85 225 87

88 103 100 103

225 200 112 350 100 112 450 110 320

72 91 29 73 04 96 88 04 06

400 89 80 104

a We are indebted to Dr. Howard W. Bond, Cancer Cheniotherapy National Service Center, National Institutes of Health, Bethesda The details of the screening procedures can be found in Cancer Chemotherapy Rept.,

E = Ehrlichascites, C = Carcinoma 755. 14, ?*Id., for making these data available to us. 1,42 (1959). * XTL = maximum nontoxic level. T/C = treated tumor/control tumor.

Anal . Calcd. for ClIH~~ClNO?: C, 58.54; H, 5.32; N, 6.28. Found: C, 58.63; H, 5.80; N , 6.00.

Ethyl 2-Acetamido-4-(3-p-acetamidobenzoyl)-2-carbethoxy- butyrate (IIh).-To a mixture of 76 g. (0.35 mole) of ethyl acetamidomalonate, 79 g. (0.35 mole) of Ih, and 150 ml. of an- hydrous ethyl alcohol kept a t 20" was added a solution of 9 g. (0.39 g.-atom) of sodium in 150 ml. of anhydrous ethyl alcohol, dropwise with agitation. Agitation was continued overnight a t room temperature. A41cohol was removed by flash evaporation, and the residiie was extracted with 500 ml. of methylene chloride and washed free of salts with two 100-ml. portions of water. hlethylene chloride was flash evaporated, and the residue was crystallized from isopropyl alcohol and yielded 58 g. of product, m.p. 149-158", 39.8% yield. il l1 analytical sample was crystal- lized from isopropyl alcohol, m.p. 150-152'.

Acid (IIIh).-IIh (20.5 g., 0.05 mole) was suspended in a solution prepared from XaOH (10 g., 0.25 mole) dissolved in a mixture of 90 ml. of water and 40 ml. of methyl alcohol. After standing overnight a t 40", the hydrolysate was acidified with concentrated HC1. The precipitate was filtered, washed free of chloride, and dried at 50' iinder vaciium. The yield of malonic acid was 13.5 g. (77.1%), m.p. 137-140" dec. Recrystallization from methyl alcohol did not change the melting point.

~~-2-Acetamido-4-( p-acetamidobenzoy1)butyric acid ( IVh) was obtained by heating 12 g. (0.034 mole) of I I Ih in 250 ml. of water for 2 hr. under reflux. Upon cooling, 5.5 g. (52.2y6) of product was obtained, m.p. 155-166'. An analytical sample was prepared by crystallizatioii from aqueons methyl alcohol, m.p. 165-168".

2 4 p-Aminopheny1)-1 -pyrroline-5-carboxylic Acid Dihydrochlo- ride (Vh).-IIh (22 g., 0.054 mole) was heated under reflux with 150 ml. of concentrated HC1 overnight. The solution was evaporated to dryness under vacuum, and the residue was dis- solved in water, decolorized with charcoal, and evaporated again. The product was then dissulved in methyl alcohol and allowed to stand in the freezer for several days when 12 g. (SOTc) of com- polind was ohtain3d which melted at 240-242' dec. An analyti- cal sample was obtained by crystallization from a mixture of met,liyl alcohol and ether, m.p. 252' der,.

5 4 p-Aminopheny1)proline Dihydrochloride (VIh).-Compound Vh (14.7 g., 0.053 mole) was dissolved in 150 ml. of methyl alcohol and was hydrogenated in a Parr hydrogenator in the presence of 50 mg. of platinum oxide under 3-4 atni. When the theoretical uptake of hydrogen was observed, the catalyst was removed by filtratioii and the solvent was evaporated under a

Acetamido [ 3 4 p - acetamidophenyl)-3-oxopropyl] malonic

stream of air. The product (13 g., 88.8%) obtained melted at 131-137'. For analysis, a sample was recrystallized from a mixture of methyl alcohol and ether, m.p. 137".

Microbiological Assay.-Aqueous medium ( 100 ml.) contained sucrose (10 g.); glucose (1.0 g.); yeast extract, Difco (1.0 g.); peptone, Difco (0.3 g.); beef extract, Difco (0.2 9 . ) ; and inorganic salts (0.3 g.). The salt mixture was composed of K ~ H P O I (150 mg.), ("&SO4 (150 mg.), sodium citrate (149 mg.), CaCL (25 mg.), hIgS04.7Hz0 (25 mg.), FeNH4S04 (0.5 mg.), and Zn- SOd.7H20 (0.5 mg.). Solution8 of the test compounds were made in water and the pH was adjusted to 7 with ",OH. The levels of compound were set so that, 1 ml. of solution when diluted t'o 10 ml. would yield final concentrations of 0.1, 0.5, and 1.070, respectively. A soliltion (1 ml.) of test, compound, made sterile by filtration throiigh a Seitz filter, was added a,septically to 9 ml. of medium, also made sterile by filtration through a Seitz filter. Inoculation with I,. mesenteroides P-60 was effected by addit,ion of 2 loopfds of an 18-hr. culture of the organism in Eugon broth (BBL). After incubation for 24 hr. at 37", the culture tubes were examined for turbidity in a Klett-Summerson colorimeter using a No. 66 filter. The data recorded indicated 100% inhibi- tion as compared to gron-th.

Reduction of Steroidal Enamines with Potassium Borohydride

ARVIK P. SHROFF

Ortho Research Foundation, Karitnn, .\-ew Jerseg

Received J u n e 23, 1965

de Winters, et al., and have reported that the removal of the oxygen function at C-3 of A4-3- keto steroids resulted in compounds with anabolic or progestational activity. Kind and Dorfmanj showed

(1) 3'1. S. de Winters, C. W. Siegmann, and S. A. Szpilfogel, Chem.

(2) N. E. Borglin, Acta Endocrinol., Supp l . , 58 (1960). ( 3 ) 0. Halpern, J. .\. Edwards, and J. A. Zderic, Chem. I n d . (London),

1571 (1962). (4) K. Irmscher. H. G . Kraf t , and K. Bruckner, J . .Wed. Chem., 7, 348

(1964).

Ind . (London), 905 (1959).