September, 1963
ANTIRADIATION COMPOUNDS.I V
509
Antiradiation Compounds. IV. Trithiocarbonates of p-Mercaptoethylguanidinesl
Department of Clwnistry, illassachusetts College of Phamnclcy, Boston, Afassachusetts
Received Dccember I O , 1962 p-~\I(,rcaptoctliylguanidineand S-substituted derivatives, prepared in aqucous solution by rcarraiigc~iiwntof the corresponding p-aminoethylisothiuronium compounds, gave insoluble adducts Rith carbon disulhde. Both infrared and ultraviolet absorption spectral characteristics as well as chemical evidence showed the adducts to bc trithiocarbonates rather than dithiocarbamates or cycllc structures. N,S-Diacyl derivatives of p-inercaptoetliylguanidine were prepared from the trithiocarbonate by reaction with acj 1 chlorides. Several of the tritliiocarbonates, as well as one of the diacyl derivatives, furnished significant protection to mice against an otherwise lethal dose of X-irradiation.
Conversion of xercaptan bases to compounds with reactive sulfur-containing functional groups has produced effective radiation-protective agents for animals. This has been demonstrated in the case of 2-mercaptoet hylami ne (NEA) and 2-mercaptoe t h ylguanidiiie (RIEG) by coiiversioii to Buiite salts,2thioph~sphates,~ and acyl t h i ~ e s t e r s . ~The t'rithiocarbonates of AIEA and AIEG have now been prepared and found to have appreciable radiation-protective ability in mice. The trithiocarbonate zwitterion of RlEA was previously believed t'o be a dithiocarbamic acid, 4 , 5 but evidence for the trithiocarbonate structure has been
ethaiiethiol (IY), n liicli caii bc only a trithiocaiboiiatc, absorbed a t 1032 cin. -l. Furthermore, no absorptioii near 2550 ciii.-l, due to a inercapto group, n-as evident with any of these compounds. 8
11
+NH&EIzCHZSCS111
8 t
11
(C2Ht)K;HCH&HzSCS
IV
Evidence from comparison of tlie ultraviolet absorption spectra of dithiocarbamates and trithiocarbonates also favored a trithiocarbonate structure (see Table I). The trithiocarbonate zwitterion of 2-diethylamiiioshorn-ed trithiocarbonate absorption peaks8 ethaiiethiol Reaction of carbon disulfide with an aqueous solution at 226 mp (log emax 4.07) and 303 mp (log emax 4.18), of 2-mercaptoethylguaiiidine, prepared from the alkaline rearrangement of 2-(2-aninoethyl)-2-thiopseudo- and the trithiocarbonate zwitterion of RlEA showed very similar absorption a t 226 mp (log emax 3.96) and urea dihydrobromide (XET), gave immediate precipita304 mp (log emax 4.13). The potassium salts of ethyl tion of an insoluble yellow adduct. The product is and hexyl trithiocarbonates, however, absorbed a t 300 most likely the trithiocarbonat'e zwitterion, as has mp (log emax 3.46) and 333 nip (log emax 3.10) and a t 302 been found from the corresponding reactions with MEA nip (log elnxx 3.61) and 333 mp (log emax 3-24), respecand 2-diethylamiiioethaiiethi01.~ However, since a tively. The carbon disulfide adduct of AIEG showed dithiocarbamate (I) or trit'hiocarboiiate (11) structure, absorption in the ultraviolet resembling that of the both theoret'ically possiblc from this reaction, give the alkyl tritliiocarbonates when observed in neutral solsanic clemeiit'al analysis, it was necessary to distinguish vent (GHsOH), with peaks a t 304 mp (log emax 3.36) bctiveen t'he possible structures. and 353 mp (log emax 2.43). When observed in alkaline S "2 ethanol, however, the 1\IEGCS2adduct revealed ultraI/ I1 violet absorption similar to that of the MEA and diethyl -SCSCH~CH~NWCXH~ I 11 MEA zwitterions, with peaks at 225 mp (log emax 4.00) and 303 nip (log emax 4.11). The iY-alkylated JIEG triExaniiiiatioii of tlic iiifi,arcd absorption spectra of t'hc thiocarbonates showed closely similar absorption in thc RlEG adduct showed definite trithiocarbonate abultraviolet. sorption' a t 1038 em.-'. The corresponding N,N,E'Moreover, the characteristic ultraviolet absorption8 trimethyl derivative of RIEG, where dithiocarbamate of dithiocarbamates at 240-260 mp, 290-310 mp, and formation would be less likely, also showed this ab340-360 mp was not in evidence with the carbon disulsorption a t 1026 em. -l. The trithiocarbonat'e zwitfide adducts of MEG and its alkyl derivatives. As seen terion of MEA (111) showed this peak a t 1018 from Table I, dithiocarbamate zwitterions showed aband the carbon disulfide adduct of 2-diethylaminosorption peaks a t these wave lengths, as did the dithio(1) This project was carried o u t under a contract (D.-\-49-193-hlD-2029) carbamate of the S-methyl ether of >TEA (Y). I n this with tlie Office of tlie Surgeon Genpral, U. S. Army Xedical Research and Development Command. connection, the S-heptyl ether of K,K'-dicyclohexyl (2) B. Holmberg and 73. Sorbo, Nature, 188,832 (1959): A. Kalusayner, MEG gave a reaction with carbon disulfide in the usual P. Czerniak, and E. D. Borginann, Radiation Res., 14, 23 (1961). manner to give an insoluble adduct (T'I) that showed (3) €3. Sorbo. Arch. Bzochem. Biophys., 98, 342 (1962); D. P. Jacobus, 0. B. Ramsay, R. E. SImlding, and D. C. Dittmer, Abstracts, 141st Kational characteristic dithiocarbamate peaks in the ultraviolet Meeting, American Chemical Society, March, 1962, p. 30K. a t 243 mp (log emnx 4.42) and 288 mp (log elnax 2.77). (4) W. 0. Foye, R. N. Diivall, and J . hIickles, J . Pharm. Sci., 61, 168 +
(1962). ( 5 ) W.0. Foye and J. ;\Iickles, J . M e d . I'harm. Chem., 5, 826 (1962). (6) W. 0. Foye, J. R. hlarsliall, and J. Mickles, J . I'harm. Sci., 63, 406 (1963). (7) J. I. Jones, W. Kynaston, and J. L. Hales, J . C h e m . S o c . , 614 (1857).
(8) J. A . Barltrop, P If.€I.Lses, and 32. Calvln, J . Am. Chem. Soc., 76, 4348 (1954); 11. J. Janssen, Rec. f r a t . c h m . 79, 454, 464, 1068 (1960) 1' Rfason. E. C Holdsworth, and R . Emmott, J C h e m So? , 292 (1953). (9) H. P. Koch, ?bid , 401 (1949).
s
3 ;!I 3 3; 2 s2
30 I 3,i2
:: t'H,SCHLCIl,?rrHCh NH:
I'
h
I/
o u t l)y t hci twliai 101 of 1 , \ '-die\-clohcxyl AIFX; ii\-(11 o1)ioiiiide. .Uthougli a hydrohalide of ATE:(; o i i t dc.rivativtls had uot previously beeii isolated, t 1ii\ compound was sufficiently insoluble in aqueou+ iiicdia to permit its isolation, after rearrangement of the corresponding AET derivative with aqueous itiiuii01iia Reaction of the >LEG derivative n ith carboii disulfidt.. however. remo\.ed the hydiogeii bromide, gix irig t h e trithiocarhonate ZIT itterioii. Furthei*more,iwictioii 01 S,S-diacyl l I E ( ; derivatives v itli carbon disulhdc 111 alkaline media gavc no insoluble adduct until 1,ot 11 acyl groups ~vcroIcmoved during hydrolysis. The S,S-diacyl derivatives of N E G ~ t e r epiepaietl t)) utilizing the trithiocarbonate, which could be isolated from aqueous solutioii. Preyiously, l I E G had beeii isolated oiily as tlw flavianate.ll Heating the trithiocarboiiatc \T ith acyl chlorides having chain Itwgth Yix 0 1 1 1 1 0 1 ~cai-bons ~ gave solid S.S-diacpl ckriva + of cmhoii diwlfido f'liphjcal (wiIstLtiit\ \
1NC6HII
3 20 2 li!)
XHCsHii ' 1
C,HI,SCH,CH~JHCX(CsHii )CS'-C,H~,SCH,CH,THCNHC~HI I r-1
Chemical evidence in favor of trithiocarboiiate ratliei than dithiocarbamate formation was found in the ~xwtioiiof the disulfide of 1IEG with carboii disulfidr. T h e degree of alkalinity iwjuired for a reaction to tak(> place also cleared the disulfide linkage, giving thr same 1 I E G trithiocarbonate zwitterion as before. 111 this coiiriectioii, no dithiocarbamates of the guanidiiie group have previously appeared in the literature, with tlir cxceptioii of aiiiiuoguanidine dithiocarha11iates. l o The inarked tciideiicy for zwitterion formation i i i t IIO prcpai 2 tioii of t h ( w trithiocarhoiiates was hi.ought
79, 10) I 1
I I'!\
.mil
I
L~liiliit
Jliit14i
I ' , i t t n t 7 ~ 7 , X i i( I ' l X
i t h 3 (lOj7l
September, 1963
ANTIRADIATION COMPOUNDS.IV
PHYSICAL PROPERTIES OF M.p., OC.
Yield,
Compound
N,S-Dihexanoyl MEG 'HC1 S,S-Diheptanoyl MEG .HCI N,S-Dioctanoyl MEG.HC1 S,S-Dimyristoyl MEG.HC1 N,N'-Dicyclohexyl MEG.HBr
83-86 87-89 94-95 107-109 209-210
26 22 54 45 49
%
THE
511
TABLEI1 8-MERCAPTOETHYLGVANIDISE DERIVATIVES
Formula
F C a r b o n , %,-Hydrogen, 70Calcd. Found Calcd. Found
c l i i t r o g e n , %Calcd. Found
51.13 8 . 5 3 8 . 4 1 11.95 54.07 8.96 9.06 11.07 56.16 9.33 9 . 4 8 10.31 64.16 10.77 10.40 7.30 49,27 8 . 2 4 7.31 11.54
CljH30ClX302S 51.21 C17H,4CINa02S 53.75 Ci9H3sClN30zS 55.95 C31H62ClhT302S64.64 C15HS0BrN3S 49.45
-Sulfur, %-Calcd. Found
c
11.68 11.31 10.20 7.16 11.21
9.10 8.43 7.85 5.56 8.79
9.52 8.96 8.25 6.09 8.76
21.85 20.08 26.77 16.65 11.52 20.55
49.29 45.93 45.71 40.51 26.74 45.93
49.00 45.46 46.55 40.21 26.30 45.98
S
/I
TRITHIOCARBONATES, RSCS-
a
140- 142 159-161 131-132 116-1 18 156-159 133-135
74 71 21 58 56 80
24.60 28.71 22 86 35.44 53.48 28.71
25.00 28.71 23.00 35.37 53.60 29.09
4.64 5.26 4.76 6.33 8.08 5.26
4.80 5.15 4.67 6 53 8.17 5.53
21.51 20.10 26.67 17.72 11.70 20.10
15c-152
80
35.74 35.49
5.53
5 . 6 5 17.87 17.50 40.85 40.89
142-144
67
30.10 30.94
5.47
5.72 17.55 17.98 40.19 40.71
Anal. Calcd. HzO: 7.10. Found: 6.32 (by loss of weight).
of the compounds prepared are listed in Table 11. Antiradiation Properties.-Tests for the ability of some of the compounds discussed to protect mice against a lethal dose of X-irradiation (800 r.) have been carried out a t the Walter Reed Army Institute of Research under the direction of Dr. D. P. Jacobus. Tests were carried out as previously i n d i ~ a t e d ,and ~ the trithiocarbonate of AIEG was described as giving good protection, comparable to that from hIEG itself, in the dose range of 350-750 mg./kg. The trithiocarbonates of the Y-amino derivative (S'-amino-2-guanidinoethyltrithiocarbonate zwitterion) and the X-methyl derivative also gave good protection, as well as did the trithiocarbonate of 3-niercaptopropylguanidine (NPG). The trithiocarbonates of the dicyclohexyl and imidazole derivatives gave only slight protection, whereas the trithiocarbonate of the trimethyl derivative gave no protection. The hydrobromide of dicyclohexyl AIEG also gave no protection, but the latter two compounds were too toxic for appreciable doses to be employed (doses of less than 50 mg./kg. were used). It appears, therefore, that two or three alkyl groups on the guanidine nitrogens seriously diminish or remove radioprotective ability. One of the 3,s-diacyl derivatives, the dioctanoyl, gave fair protection in the 150-350 mg./kg. dose range. I n addition, more detailed data for the protective ability of XIEG trithiocarbonate (GET) us. 800 and 900 r. in mice is presented in Table 111. These results were obtained through the courtesy of Dr. Richard I. H. Wang of Roswell Park Memorial Institute, who plans to publish a more complete study elsewhere. The protective ability of the compound is obscured somewhat in these results by the toxicity of the propylene glycol solutions employed: the LD,, of GET in propylene glycol is about 0.1 ml. of a 4% solution by the intraperitoneal route. Experimental12 2-Guanidinoethyltrithiocarbonate Zwitterion.-S-(2-Aminoethyl)-isothiuronium bromide hydrobromide (5.6 g., 0.02 mole)
TABLEI11 RADIOPROTECTIVE ABILITYOF 2-GUAXIDISOETHYLTRITHIOCARBONATE(GET) I N
hf ICE' %
Compound; amount administeredb
Dose, r.
XO.of mice used
No. of mice died
mortality (30 days)
900 20 11 55 0.1 ml. 3% GET 900 20 6 30 .1 ml. 2% GET 900 20 0 0 . 3 ml. 0.8% AET' 900 20 20 100 Control 800 20 11 55 0.1 ml. 3% GET 800 20 0 0 .1 ml. 27, GET 800 20 0 0 .3 ml. 0.8% AET" 800 20 16 80 .1 ml. propylene glycol 800 20 17 85 Control a Determined a t Roswell Park Memorial Institute by Dr. R. I. H. Wang. b In propylene glycol; administered 15 min prior t o radiation exposure. c In saline solution. (Matheson Coleman and Bell) was dissolved in 15 ml. of water and treated with 3 ml. of concentrated ammonium hydroxide. The resulting solution of 2-mercaptoethylguanidine was added dropwise with stirring to 4 ml. of carbon disulfide surrounded by an ice bath. A bright yellow precipitate was isolated and was washed wit,h water and ethanol and was air dried. The yield was 2.9 g. (747,) of product which melted a t 140-142". Trithiocarbonates of the S-alkyl MEG derivatives were prepared in similar fashion. N,S-Diacyl-2-mercaptoethylguanidine Hydrochlorides.-The acid chloride (15 ml.) and 3.5 g. (0.018 mole) of 2-guanidinoethyltrithiocarbonate zwitterion were heated on a steam bath for 1 hr. after the mixture became homogeneous. The solution was cooled, and 200 ml. of ether was added. The resulting solution was refrigerated, and the crystals which appeared were filtered and washed with ether. In the case of the N,S-dimyristoyl derivative, the product could be recrystallized from absolute alcohol. In general, the compounds were slightly soluble in water, very soluble in alcohol, and gave negative tests for a sulfhydryl group with dilute iodine solution. 2-Guanidinoethyl Disulfide Dihydr0bromide.'~-S-(2-Amino(12) Meking points were taken on either a Fisher-Johns or hIel-Temp block. Analyses for carbon, hydrogen, and nitrogen were done by Weiler and Strauss, Oxford, England. (13) The disulfide of MEG has been tested for radioprotective ability by E. E. Schwartz a n d E. Shapiro, Radiation Res., 13, 768 (19601, b u t n o synthetic procedure was described.
