NOTES
November 1967
1173
SCHEME I
Si( CHzCHCOSH,),
I
KHz.HC1 19
Sz(CHzCHCONH2)z
I
HSCHZCHCONHS
C~H~CH~SCHZCHCOXIIZ ,
I I
NHn.IIC1 1
NHCOCH3 20
NHCOCHI
11
.1 HSCHzCHCONIiz
I
XHCOCHS 2
alcoholic hydrogen chloride. Reduction of 19 with sodium in liquid ammonia gave a desired intermediate, L-2-amino-3-mercaptopropionamidehydrochloride (1)) of 1 gave in excellent yield. Selective N-a~etylation'-~ 2 (Scheme I). Intermediate 1 xyas also prepared by sodium-liquid ammonia debenzylation of L-2-amino-3-benzylthiopropionamide (8) and purified through its insoluble mercuric mercaptide. Earlier workersg had obtained 1 in solution by reduction of L-iY,i\;'-bis(benzy1oxycarbony1)cystine diamide or L-S-benzyl-S-(benzyloxycarbony1)cysteine amide, but isolated it only in the form of its oxidation product, L-cystine diamide. Two other routes to 2 involved acetylation of intermediates 19 and 8, followed by reduction of the resulting acetylated compounds (20 and 11, respectively). Four analogs of 2 mere synthesized (Table I). Compounds 4 and 5 were prepared by reduction of the corresponding acylated cystine diamides (21 and 23, respectively) . L-2-Formamido-3-mercapt o-S-phenylpropionamide (6) was readily prepared by removallo of the S-diphenylmethyl blocking group from L-3-diphenylmethylthio-2-formamido-S-phenylpropionamide (17). Compound 17 was obtained in excellent yield from L-S-diphenylmethyl-?-formylcysteinelo and aniline by the n',K'-dicyclohexylcarbodiimide method. Applying the carbodiimide method to the condensation of NAC with aniline produced 7. These findings are similar to the results reported by Sheehan and Hessll where K-carbobenzoxyserine was found to react in a like manner. Mucolytic Data.-The activity of three compounds in reducing the viscosity of a mucoproteinlz solution is shown in Table 11. XAC is included as reference material. Substantially greater mucolytic activity is demon5tr:~tedby the 2-acylamino-3-mercaptopropiorlamides (1-5) u t each time period. Two less soluble compounds (6 and 7) do not show good activity. Oxidative Stability.-In addition to the good mucolytic I)ropertics esliihitccl by severd of these products, :I greater resistance to autoxidation was demonstrated. ( 1 1 ) J C Sheehan and G P. Hess, J A m Chem Soc , 7 7 , 1067 (1955) (12) i. L Sheffner. A n n . N. Y . A c d . Scz., 106, 298 (1963).
The results in Table I11 show that thiol group oxidation in 2 is minor in comparison to that in XAC and Lcysteine. Acute Toxicities.-The acute intravenous toxicities for L-2-acetamido-3-mercaptopropionamide (2) and NA4Cxvere conducted with groups of ten male albino mice13 weighing 18-25 g and with groups of ten male albino ratsI4 weighing 120-165 g. Solutions of 2 in distilled water at pH 5 and in K-saline, adjusted to pH 6.5-7, were administered to mice and rats, respectively. Sodium salt solutions of KAC in distilled water at pH 7 were used in both species. Injections in mice were made at a rate of 0.3 ml/min with volumes of solution that ranged from 10 to 30 ml/kg and in rats at 0.1 ml/min with a volume of 10 ml/kg. Toxic side effects of each compound indicated a generalized depression of the central nervous system. Deaths occurred within 24 hr after treatment and apparently resulted from respiratory failure. The median lethal doses (LD,,) in mice were determined to be 2820 (2611-3046) and 3500 mg/kg (3420-4220) for 2 and SAC, respectively. In rats the LDjo doses were 1870 mg/kg (1655-2113) for 2 and 2330 mg/kg (2473-2625) for SAC. Experimental Sectionlc Examples of Preparative Methods. A. ~-3,3'-Dithiobis(2aminopropionamide) Dihydrochloride or L-Cystine Diamide Dihydrochloride (19).-~-Cystine dimethyl ester dihydrochloridelo (34.1 g, 0.1 mole) was added with stirring to 300 ml of liquid S H 3 . T h e KHs wax allowed to evaporate mid the residue xas warmed (50') in t~ucim. The residual crude solid ( 2 7 . 5 g ) w:is slurried with warm JleOH. il trace of iiisoluble material was collected (discarded) and the filtrate was acidified with alcoholic HCI to give 24 g (77 5)of 19. ( 1 8 ) Swiss \\'&ster strain of mire ubtained from 1,aboratory Supply Corn],any, Iridiana~olir,Inrl. (14) \Viatar strain of rat8 ubtained from llarlan Indualries, Cuniberland, Ind. (15) Median lethal doses were calculated according t o t h e method of T. J. Litchfield and F. Kilroxon, J . P h n m n c o l . E&. T i i e r u p . , 96, Y Y (1949). (16) \\'e a r e grateful t u Alesars. ,John G. Scliinidt, Clareiice Kennedy. a n d Charles A I . Combs for t h e analytical and instrumental data. T h e infrared spectra of all described compounds were consistent n i t h tlie assiqned structures. I n general. deionized water, a s well as a n atmosphere of N ? was used when irorking Trith SH compounds.
N OTEH
1175
--~ __ Found. LZ---
,_-_
____
C
H
N
S(SH)
C
H
N
23.00 37.02
5 79
.i.6.i 6.44
6 86 6 86 .i40 5 92 6 71
(21.1) (20.3) (20.3) 18.20 (18.76)
23.23 37.32
40.89 40.89 ,53,3.5 . J J , 44
41.10 40.79 53 , -50 .5.5 . 27 57.03
6.96 6.70 5.46 6.13 6.70
37.12 5 7 . 12 64.94 3.62 .i6 . 7 2 6 7 . 10 70.74 21.16 23.15
92 6 39 6 39 .i78 6 81 6 80 6 38 .i 68 3 92 3 18
17.89 17.2i 17. 27 1.;. 90 15.90 12.49 11.76 13.32 11.36 11.75 11.10 11.10 8.91 10. ;i2 9.43 9.78 7.18 13.72 18.01
5 3 . 34 57.24 57.03 64.89 3.66 56.99 66.92 70.72 41.10 23. 4:3
.5,84 6.48 6.46 5.8.5 6.97 6,90 6.37 .i. 7 3 6.22 5.14
17..i3 17.06 17.09 1,;. 78 15.88 12.38 11.30 13.32 11.30 11.67 10.90 10.78 8.70 10.is7 9.16 9 . 69 7.13 13.60 17.74
37. 2.5
3 6.;
17.38
3 7 . 55
5.90
17.68
41.12
6 34
15.99
18. so
40.87
6.29
13.71
18.01
- 107.39 (4)
33.80
4 96
12.54
14.86
53.84
4.87
12.34
14.10
-248.:;
41.12
6 33
41.14
6.40
-. _-. l l
cll
.X. 44
6 21
;i
13 . 23 12.99 13.45 12.71 10.20 10 82
S(SH)
(20.6) (20.4) (20.8) 18.29 (18.0) 14.83 12.73 13.55 12.82 10.21
+- 12.28 14.51 ( 1 ) (1) 0.0 (1) - 1 4 , 6 3( 2 ) -28.23 ( 3 ) +17.11 ( 4 ) -43.82 ( 2 ) +24.3 (5) +1.13(2) - 30.98 ( 6 ) 0.0 ( 2 ) -77:$.4 ( 2 ) -l:Ltj!j([i)
10.98
-1.2 (4) 0.0 ( 4 ) 16.8Y (4) -21.8 ( 3 ) - 1!%,9.5( 7 )
+
-124.49(3)
(8)
-76. 75 ( 3 )
($5)0.3, H 2 0 ; (6) 2, EtOH: ( 7 ) 1, 1 S HCl; (8) 1, DAIF e Racemic form. f 1 .Y HC1 was emdoved as the reaction solvent. 0 Benmy1 chloride was Iised as the acylating agent. * Carried out at atmospheric presbure. The rescduk mass, remaining after the solvent (XIeOH-SHj) had evaporated, was suspended in XIeOH and treated with an equivalent of anhydrous NaOAc. 1 The autoxidation product. %
for 10 min and then cooling, the white product was collected, washed with EtOAc, and dried; yield 10.5 g (83%). F.-After treating 200 g (0.765 mole) of S-benzyl-L-cysteine methyl ester hydrochloride with 2.1 1. of NeOH saturated with SH,, the mixture was concentrated to dryness and treated with ,500 ml of HpO, 42 g (0.51 mole) of anhydrous NaOAc, and 86.5 g (0.846 mole) of AcZO. The reaction temperature rose to 50" atid the product precipitated. After stirring the mixture with 400 ml of additional H10, the crude solid was collected, washed with HsO, and dried; yield 170 g (87%). The DL form (12)was obtained as the first, crop by solution of the crude solid in 1.2 1. of warm EtOH, followed by dilution with 150 ml of HpO; yield X ( 4 5 ) . Compound 11 was obtained on cooling- the filtrate: yield 105 g (54cmall volume, diluted with EtOAr-heptalie, ailti stored a t 0'. The separated solid was collected aiid triturated with ; i c ~ t ~ ~ r c giving 3.3 g (555:) of 15. In a later preparatioii the gumni.~reactiuti pI'(Jdll('i was i riturated with acetone to give comparable product i l l i:
