770 Joumal of Medicinal Chemistry, 1971, Vol. 14, No. 8 hydrazine.HC1, 250 mg of NaOAc, and 1.0 ml of AcOH and refluxed for 40 min. The mixt was poured over a large amount of ice water and the ppt formed was collected by filtration. Recrystn from MeOH yielded 900 mg of 6, mp 154-157'. Receptor Binding Studies,-Uteri from 10 immature rabbits weigbing less than 2 kg were removed, minced, and washed several times a i t h buffer t o rid the tissue of blood. Uteri were then homogenized in 0.4 vol of buffer (0.01 M Tris-HC1 buffer, p H 8.0, contg 0.001 Jf EDTA and 0.25 M sucrose) a t 4". The homogenates were first centrifuged a t 12,0008 for 30 min, followed by 273,0008 for 1.0 hr. Reaction mixts consisting of ( a ) 0.2 ml of buffer (0.01 X Tris-HC1, p H 8.0, contg 0.001 M E D T S , 0.25 M sucrose, and 25,000 cpm of progesterone-tlml), ( b ) nonradioactive compds reported in this paper a t a concn of 100 ng/ml, and (c) 5 0 ~ of 1 uterine cytosol were incubated a t 4' for 16 hr. After incubation bound us. unbound steroids were sepd as earlier reported, and the amt of bound progesterone-t was determined.
Acknowledgment.--We wish to thank Dr. I. Scheer and Dr. J. DaVanzo for their encouragement and interest. Thanks are also due to hfrs. E. Kaffitz, Miss C. DeDella, and Mrs. B. Fuller for their technical assistance.
P-Lactam Antimicrobial Agents W h i c h Possess A n t i f u n g a l Activity WILLIAMJ. GOTTSTEIN, ANS H. EACHIJS, PETER F. MISCO,LEE C. CHENEY,* MARTINMISIEK,AND KENNETH E. PRICE
Research Division, Bristol Laboratories, Division of Bristol-Myers Company, Syracuse, iVew York 13601 Received February 12, 1911
Although the cell walls of bacteria and fungi differ widely in their mucopeptide and mucopolysaccharide arrangement, we thought that it would be interesting to prepare some /3-lactam-containing semisynthetic compounds and test them against certain fungi. Unexpectedly, we have found that a few of these antibiotics inhibit the growth of some pathogenic fungi in vitro.
I1 (15),R'= CeHSOCH(CHJC0; R"=COSK 111(16), R' = C,H,OCH,CO; R" = CHO
Chemistry.-The compounds listed in Tables I and I1 were prepared by two procedures unless otherwise noted. All of the thiocarbamoylmercaptomethylcephalosporanic acids were prepared by direct acylation of 7-aminocephalosporanic acid (7-ACA) with the Scarboxymethyl dithiocarbamate-carboxylic mixed anhydride (method A). The carbamoyImercaptomethy1cephalosporins were prepared from the S-carboxymethyl N,N-disubstituted dithiocarbamates and 7-ACA
NOTES
in the presence of Izand S a 1 (method B).It2 N-Benyzlthiocarbamoylmercaptoacetamidocephalosporanic acid (I) (14) was prepared from sodium 7-(2-bromoacetamido)cephalosporanate3 and potassium N-benzyldithiocarbamate. Potassium 6- [ (-)-a-phenoxypropionamido]thiopenicillanate (11) (15) and 6-phenox) acetamidopenicillanal (111) \!ere prepared h y published procedures. Antifungal Activity.-Results of the tests are iumniarized in Table I. As can be seen, the muat activc compound is sodium (i~-benzyIdithiocarbani~~~lacetamido)cephalosporanate (I) (Table I, 14). It i.: noteworthy that this cephalosporin showed tIvo- to fourfold greater antifungal activity than the antifungal acid 176from which it was derived. I t is also of interest that the penicillin aldehyde I11 (16) which is virtually without activity zn vitro aganist bacteria, showed antifungal effects. Compound 14 was tested in an esperimental systemic Cryptococcus weofol'maris infection of mice but was found to case n o prolongation of survival time. Thus, the probability exists that, although these compounds are active t n vitro, they are not present in an active form in animal tissues at high enough concentrations to give protection against systemic fungal infections. Experimental Section7 Sodium 7-~i~,~~r-Dimethyldithiocarbamoylacetamido)cepha~osporanate (Table I, 1) (Method A).-To a soln of 1.8 g (0.01 mole) of S-carboxymethyl N,X-dimeth>-ldithiocarbamate aiid 1 g (0.01 mole) of EtaS in 50 ml of T H F at 0" was added 1.2 g (0.01 mole) of isovaleryl chloride. The niixt \vas stirred for 10 min and a s o h of 2.8 g (0.01 mole) of T-AC.4 and 1.1 g (0.11 mole) of Et3N in 25 ml of H20 was added all at oiice. The s o h was stirred for 0.5 hr and the T H F was evapd under reduced pressure a t 30' (15 mm). The aq residue was acidified with 1 : 1 H,POI and extd with EtOAc. The exts were washed wit'h H2O and dried (Xa;lSO,). The soln was treated with sodium 2-ethylhexanoate and the white cryst salt was collected and recrystd three times from aq n-BuOH to give '2.3 g of product (see Table 11). Sodium 7-(~~~-Benzyldithiocarbamoylacetamido)cepha~osporanate (14).-To a soln of 1 l . i g (0.03mole) of sodium 7-(2-bromoacetamido)cephalosporanate3 and 2.3 g of SaHC03 in 200 ml of H 2 0 a t 10" was added, with vigorous stirring, 6.6 g (0.03 mole) of potassium .Y-benzyldithiocarbamate.6 The s o h was stirred in the cold for 1 hr as the p H Fas kept around 7 with 2 A-NaHC03 soln. The s o h was washed with Et,O, layered with 100 ml of EtOAc, and acidified t o pH 4 with 425;; H3POr. The org layer was sepd, and the aq phase was again estd with EtOBc. The exts were washed with HzO, dried (Xa2S04), and evapd to an oil under 15 mm pressure a t 35'. The residue, after trituration with dry EtzO was collected by filtration and was dried over (1) W.J. Gottstein and .\. H . Eachus, C. S. Patent 3,351,141 (1568); Chem. Abstr., 69, 66992 (1568). (2) W.J. Gottstein, A. H. Eachus, and L. C. Cheney, J . Ore. Chem., 5 6 , 1653 (1570). (3) L. B. Crast, J r . , South .ifrican Patent 6i/07,783 (1966); Chem. Abstr., 7 0 , 68389 (1569). (4) W.J. Gottstein, R . B. Babel, I,. B. Crast, Jr., J. 1\I. Essery, R . K . Fraser, J. C . Godfrey. C . T. Holdrege, W. F. Minor, AI. E. S e u b e r t , C. .\. Panetta, and L. C . Cheney, J . .?fed. Chem., 8, 754 (1965). ( 5 ) W.J. Gottstein, G. E . Bocian, L. B. Crast, Jr., K . Uadabo, J . hI. Essery, J. C . Godfrey, and L. C . Cheney, J . Org. Chem., 31, 1922 (1966). ( 6 ) A. Rieche, J. Hilgetag. D. Martin, and I . Kreyzi, Arch. Pharni. ( W e i n heim), 886, 310 (1963). (7) Melting points were determined on a Fisher-Johns apparatus, and are uncorrected. I r apectra were recorded on a Beokman I R 9 spectrometer, nmr spectra on a Varian A-60 spectrometer a i a sweep x i d t h of 500 cps using D20 a s a solvent. A11 spectra ivere consistent with structure. Klrere analyses are indicated only b y symbols of t h e elements, analytical results obtained for those elements were xithin ~ 0 . 4 7of ~t h e theoretical values. T h e authors wish t o thank M r , R . M , Downing and M r . D . F. Whitehead for the microanalytical and spectral d a t a , respectively.
Jaumal of Medicinal Chemistry, 1971, Vol. 14, No. 8 771
NOTES
TABLE J I n Vitro ANTIFUNGAL ACTIVITY”
I
R or compd
Compd
T . mentaorophutd8
Lowest level of inhibition C. neoformans
M . canis
H . capsdatum
1 2
(CH3)zNC(S)SCH2 (CzH5)zNC(S)SCH2
250 250
>250 >250
63 250
63 31
3
CNWSCH,
250
250
>250
31
>250
s2jo
63
16
>25u
>250
>250
250
250
250
250
31
>250
>250
>250
16
7
C~HSCHZN(CH,)C(S)SCH~
8
(CH3),NCOSCH,
18
63
63
>250
9
(CzHJzNCOSCH,
31
31
63
125
10
CNCOSCH,
>250
>250
>250
125
11
CNCOSCH2
250
>250
>250
125
12
CsHsN(CHa)COCHt
125
>250
>250
250
126
>250
>250
250
13
o”NcOSCH,
W
8 8 1.6 250 16 25 13 50 63 8 32 2 4 A. Rieche, J. Hilgetag, D. Martin, and I. Kreyzi, Arch. Pharm. (Wein4
14 I 15 I1 16 I11 17b CeH5CHzNHC(S)SCH&OH a Agar dilution tests; minimum inhibitory concn, pg/ml. heim), 296,310 (1963).
13 62
TABLE I1 Compd
1
2 3
4
Method of prepn
A A A A A A
5 6 7
A
8
B
Solvent
Aq n-BuOH EtOAc Aq n-BuOH Aq n-BuOH Aq n-BuOH Aq MezCO Amorphous from EtOAc E t 0Ac-Et20
Deoompn point, OC
Yield, %
210 195-199 182- 185 250 192 160 >210
60
95-96
40
162-165 9 B Aq n-BuOH DMF-Me2CO 158-159 10 B 210-212 11 B Aq n-BuOH Aq n-BuOH 185-187 12 B 169-1 70 13 B Aq n-BuOH Anal. results were within 0.470 of the theoretical values. P205 a t 15 mm to give 8 g of product. The acid was dissolved in EtOAc and treated with solid sodium 2-ethylhexanoate. Crystals were collected by filtration, washed with hot EtOAc, a t 15 mm, yield, 4 g (2670) of a and dried overnight over P z O ~ light yellow cryst product. Recrystn from MezCO-EtOH gave an anal. sample, mp 185’ dec. Anal. (Cz0H~oN3NaO~S~) C, H, N. The semisynthetic compds were tested for activity against Trichophyton mentagrophytes, Microsporum canis, Cryptococcus neoformans, and Histoplasma capsdatum by a twofold serial diln method. The compds were dissolved in DMSO, brought to
24 32
51 58 42 10
20 45 37
25 40
vol mth sterile Ha0 (1: 9) a t 1 mg/ml, and serially dild twofold in appropriate liq media. These medicated dilns were dispensed in 2-ml amts into sterile, capped, 1.56-cm diameter test tubes prior to inoculation. The tests for activity against T. mentagrophytes and M. canis were carried out in Sabouraud’s dextrose broth (Difco), C. neoformans in antibiotic medium KO. 3 (Difco), and H. capsulatum in Salvin’s Y-P medium.8 Stock suspensions of the test cultures were prepd as follows and then used a t the rate of 0.05 ml of inoculum per 2 ml of
(e b.9. S a v i n , J . Bacteh’d., 64, 655 (1947).
772 Journal of Medicinal Chemistry, 1971, Vol. 14, N o . 8
NOTES
medicated broth: T . mentayrophytes and :\I. canis were grown on Sabouraud's dextrose agar slants at 28' for 7-10 days. Surface growth from an agar slant culture was taken up into 5 ml of sterile 1320, homogenized, and dild 300-fold with rterile H 2 0 . C. neoformans was grown on Sabouraud's dextrose agar slant.3 a t 22" for 2 days. The surface growth from an agar slant culture was taken up into ,j ml of sterile H20, homogenized, and dild 23-fold with sterile H,O. H . capsulation was grown in Salvin'.+ Y-P semifluid medium at 22" for 7 day.
