248 Journal of Medicinal Chemistry, 1971, Val. 14, N o . 3
Benzothiazolines as Antituberculous Agents P. J. PALMER,* R . B. TRIQG, A N D J. V. WARRINGTON Twyford Laboratories Ltd., L m d m , England Received September I,1970
2-Arylbenzothiazoles have been shown by previous workers’ to have antimicrobial properties, and in particular t o be active both in vityo and in vivo against Mycobacteiium tuberculosis. It was therefore of interest t o prepare a series of benzothiazolines containing aryl or heterocyclic groups in the 2 position, and to examine their biological properties. Three types of benzothiazoline (1-111) were synthesized by condensation of an aldehyde or ketone with the appropriately substituted o-aminobenzenethiol or its hydrochloride in EtOH. Reaction normally occurred readily either a t room temp or on brief warming. Compounds of type I are readity oxidized t o the corresponding benzotliiazole, and the physical data quoted in the literature for a number of benzothiazolines \vould suggest cont:imiriation I\ ith the oxidation product. I n general, \\-e liuve found that benzothiazolines in which the 2 substituent is electron attracting are considerably more stable th:w those ivith electron-donating groups. The ]titter :ire often converted into the corresponding benzothiazolc simply by crystallization or by treatment with m i n e d acid. The assignment of the betizothiazoline structure I to the products obtained \vas supported2 by S H stretching bands at 3100-3390 cm-’ in their ir spectra and a band (E 3000-10,000) a t 315 rt 5 mp in the uv. I n contrast, the corresponding benzothiazoles showed strong bands (E 20,000-25,000) in the region 300-350 mp. Purity of products was further confirmed by tlc on silica (fluorescent) ; under uv light, benzothiazoles fluoresced bright blue, whereas the berizotliiazolines gave quenched spots. I n several cases, benzothiazoliiies were oxidized to the corresponding benzothiazole using commercial 311~02.
I
111
11
I\‘
The pyridylbenzothiazolines (I; It = 2-pyridyl) suffered ring opening3 when attempts were made to Kmethylate them with J I e I in methanolic NaOH solution; the product 11-as the azometliine (IV; R = x-pyridyl). Acylation of (I; R = 2-pyridyl) with AcnO and with BzCl in pyridine gave (11; R1 = Ac, R2 = 2-pyridyl) and (11; R1 = Bz, R2 = 2-pyridyl). These could not be satisfactorily reduced to N-alliylated benzothiazolines with LAH, though a similar compound (11; R1 = Ac, It? = p-diethylaminoethoxyphenyl) gave the corre(1) (a) \V. Logemann, S. Galimberti, G . Tosolini, I . de Carneri, and G . Coypi, Fnrmaco Ed. S c i . , 16, 795 (1961). (b) R . Prescott a n d J. 1M. \Vebb, Anlibiot. Chemolher., 8, 33 (1958). (c) 11. D. Cossey, R . N . Gartside, and F . F. Stephens, Arzneim.-Fordr., 16, 33 (1966). (2) F. J. Goetz, J . Heterocycl. Chem., 6 , 509 (1968). (3) C/. F. J. Goetz, ibid., 4 , 80 (19137).
NOTES
sponding N-Et compound in reasonable yield bj- this procedure. The required K-methylated compounds (11; R1 = N e , R, = z-pyridyl) were prepared by condensation of the appropriate pyridine aldehyde nitli omethylaminothiophenol. This compound 11 a5 p e pared easily and in high yield b! LAH reduction of bib(2-formamidophenyl) disulfide, rather than by the more tedious literature4 procedure. For the synthesis of benzene ring substituted betizothiazolines, pure samples of the approprintel! sub-t 1tuted o-aminothiophenols were required Thew \\ere best obtained, in some cases, by LAH reductio11 of the corresponding di- or trisulphide, followed by distills‘1 t‘1011 of the free thiol under reduced pressure in S 2 ‘1* t 1110sphere. Biological Activity.-The benzothiazolines I\ ere tested 277 z d r o against a range of organisms including Grampositive and Gram-negative bacteria, dermatophytes, and Entamoeba histolytica. A number of compounds shou ed activity against Gram-positive bacteria slid against dermatophytes, but this \ \ a s not of L: d3ciently high order t o n arrant furthcr stud).. I//uitw activity against A I . tube)culosis (H37Rv) I\ as found for most of the bt~nzothiazolin~s (see Table I), and becauw of their mse of preparation and relative sta pyridyl compounds (16-18) \\-ere taken as a further ptructurc-:ictivity study. S-Substit creased activit! (23-27) but introduction of :I 2-1’11 group (30) increased activity as, also, in gener:il, did benzene ring substitutioti (34, 36, 37, 39-43, 45, 47-49). Greatest activity \\as found for the 6-methoxy conipound (48), and this and severiil others were tested for Only slight activity I\ as found for anv of the compounds. Experimental Section6 General Methods for Preparing Benzothiazolines. Method A. -The aldehyde, dissolved in an equal vol of MeOH or Et,OIl was added t80 an eqllimolar quantity of o-amiiiobenze~iethiol (oABT) iii the same solvent ( 5 ml/g). The react,ion mixt wab mairitaiiied wider the conditions specified when the product had either crystd or was pptd by the careful addn of H& (see Table I). Method B.-Equimolar quantities of the X-substituted o-ABT and the appropriak aldehyde were heat,ed together for 5 mill at 150°, the reaction mixt was cooled and triturated with hexane, and the solid was collected and crystd (see Table 11). Method C.-o-ABT.HCl (0.04 mole) in EtOH (20 ml) was treated wit>h the appropriate pyridyl ketone (0.04 mole) i 1 1 Et,OH (10 ml), warming briefly if necessary t,o effect sohi. The reactioii mixt was maintained under the conditions specified and evapd to dryness under reduced pressure, the residue was dissolvedin HzO, the soln was basified with dil NH40H, and the product was isolated by EtzOextn (see Table 11). Method D.-The appropriate pyridine carboxaldehyde (0.1 mole) was added to the substitut,ed o-ABT.HC1 (0.01 mole) in EtOH (10 ml) and the resulting crystals were collected. In some cases, EttO (ca. 15 ml) was added and the mixt’ was stored at 0’ for 1 hr before collect,ingthe product (see Table 111). 2-(5-Nitro-2-furyl)benzothiazoline.-~-Nitrof~1rfural (2.2 g) in Et20 (100 ml) w a s added dropwise to a stirred solii of o-ABT (4) A. I . Kiprianov and Z.N. Pazenko, Z h . Obsheh. K h i m . , 1 9 , 1 5 2 3 (1949): Chem. Abstr., 44, 3488 (1950). (5) I n vivo screening \vas done using a slight modification t o the method of G . P. Toumans and A. S. Youmans, Amer. Rev. Tuberc.. 64, 541 (1951). (6) Where analyses are indicated only b y symbols of t h e elements, analytical results obtained for those elements were within +0.4% of t h e theoretical values. Melting points were determined with a Rciclli apparaton using open capillary tubes. I r spectra mere recorded for Nujol mulls on a Perkin-Elmer 237 spectrophotometer. ‘cv spectra were determined for EtOH solutions.
Journal of Medicinal Chemistry, 1971, Vol. 14, N o . S 249
NOTES
TABLE I PREPARED BY METHOD A
NO. 1 2 3 4 5
R PI1 m-Cyanophenyl p-Nitrophenyl Piperonyl v-CsHdOCHrCHzNEtz
Solvent EtOH EtOH MeOH MeOH EtOH
Time. min 30 60 30 960 30 10
Temp, QC 25 25 25 25
71
25
90
138-140
MezCO-hexane
48d
61-62 128-130 46.6-47.5 115.51 169.5-172'
CsHrhexane EtOH MeOH C6Hshexane EtOH-HzO
oh
EtOH
7
30 78 EtOH MeOH 15 25 2 MeOH 65 25 960 EtOH See Experimental Section EtOH 30 25 See Experimental Section EtOH 960 25 MeOH 30 25 EtOH 5 60 EtOH 5 60 EtOH 5 60 MeOH 15 25 EtOH 2 25 See Experimental Section EtOH 2 78
9 10 11 12 13 14 15 16 17 18 19 20 21 22
Benzyl Ferrooenyl Undeoenyl 2-Benzothiazolyl 2-Renzimidazolyl 2-Fury1 5-Nitro-2-fury1 2-Pyrrolyl 2-Quinolyl 2-Pyridyl 3-Pyridyl 4-Pyridyl 2-Pyridyl N-oxide 4-Hydroxy-3-pyridyl 6-Methyl-2-pyridyl N-Methyl-2-pyridinium iodide
Cryst solvent EtzO-hexane EtOH-HzO MeOH
MP, OC 76-77" 92-94 119.5-121 55-56 63-64
6 8
Yield,
% 52 40 63
43
79 51
84' 53 99
oil*
75 55 96 73 73 57
121-122 83. 5-85 107.5-1W 99-101 89-9 1 103-104 179-180 226.5-228' 93.5-94.5 134,5-136
43 92 65
85b
e
CeHshexane
CaHshexane CsHchexane CsHs-hexane MezCO MeiCO MezCO EtOH-hexane j
CsHs-hexane EtOH-EtrO
Formula CiaHiiNS CidHioNzS CirHioNnOzS CiiHiiNOzS CisHzsNiOS
Analysis C, H , N C, H, N, S C , H , N, S C
C, H, N
MIC' 200 6.25 > 200 200 12.5
>200 50
CicHirNS ClrHisFeNS CiiHzsNS CI~HIONBZ CirHiiNaS .O :5HzOP CIIHPNOS CiiHaNzOrS CiiHiNiS CisHizNzS CIZHIQNZS CIZHIONZS CizHioNrS CIZHI~NZOS CizHiiClNzOS CiaHizNzS CirHirINzS
0.8 100 50 25 3.1 6.3 0.6 1.6 2.2 4.4 6.25 6.25 1.6 3.1
Reaction mixt allowed to warm to 25" during reaction period. Lit. [M. Claasz, Chem. Ber., 49, 1141 (1916)l gives mp 108-109'. Compd unstable; further characterized by oxidn to known [M. T. Bogert and A. Stull, J. Amer. Chem. SOC.,47, 3078 (1925)l 2-piperonylbenzothiazole. After chromatog on Si02 using hexane-CsHs (1:9), e After chromatog on Si02 using CEHE. Resolidifies and remelts a t 280-300'; has a lower melting form, mp 93-94', identical spectroscopically with higher mp form. Resolidifies and remelts a t 270-280'; HzO of cryst confirmed by ir spec. Bp 128-138' (0.01 mm); compd unstable; further characterized by oxidn to Free base unstable; prepd using o-aminothiophenol-HC1; isolated as HC1 salt which was knownc 2-furylbenzothiazole. N: calcd 10.6; found 10.0. MIC values washed with hot EtOH. Pptd with EtzO; product deteriorates rapidly on storage; quoted in Tables 1-111 are in pg/ml and were detd by serial diln in Proskauer and Beck medium. Isoniazid had MIC 0.04-0.08.
'
'
TABLE I1
R1 I
No.
RI
Rr
2-Pyridyl Method
Time, Temp, hr OC
Yield,
%
MP, 'C
Cryst solvent
Salts
MIC
a 12.5 62 61-62 EtOH-HZ0 Me H 2 B b 50 65 98-99 E tOH-Hz0 Me H 3 B 25 121.5-123 EtOH-Hz0 56 Me H 4 B C Et H 2 B Quant Oil d 25 4 B Et H 62. 5-65d EtOH-Hz0 72 6.3 96.5-98 2 H Me c 20 0 73 MelCO >200 H Et 87 Me&O 76-78 2 C 0 . 5 25 0.6 H Ph c 20 0 71 118-119 2 MeZCO 12.5 H 2-Pyridyl 87 139-140 MeOH 2 C 0 . 5 25 1.6 44 Hf Me MeOH-E tzO 2 196-199 c 0 . 2 25 C: calcd, 68.4; found 68.85; HCl salt, mp 179-182' dec (EtOH-EtzO) ClaH&lNaS (C, H I N). HCl salt, mp 174.5-176.5' (EtOH). C13HlsClN2S (C, H I N). Characterized as picrate, mp 149-150" dec (EtOH), C Z O H I ~ N ~(C, O ~HSI N). Bp 146-152' (0.015 mm); picrate, mp 146-8' (EtOH), CzoH17Na07S (C, HI N). e N: calcd, 12.3; found, 11.7. 15-Me group in benzene ring; kolated and characterized as HCl salt. 23 24 25 26 27 28 29 30 31 32 (1
(2.0 g) in Et20 (100 ml) a t -60' under Ne. The soln was allowed to warm to 25' overnight, the bulk of the solvent removed in uucuo a t 25' and the residue triturated with petr ether (see Table I). 2-(6-Methyl-2-pyridyl)benzothiazolineand -benzothiazole.A soln of 6-methylpyridine-2-carboxaldehyde(0.97 g) in EtOH (1 ml) was added to o-ABT (1.0 g) in EtOH (5 ml). The resulting soln was allowed to stand for 1 hr a t 25', refluxed for 2 min, and cooled to O", to give pale yellow crystals(W%) of 2(6-methyl-2Amax 313 pyridy1)benzothiazole: mp 148-149' (C&-hexane): C, HI N. On further standm r ( e 22,100). Anal. (CI~HIONZS) ing, the filtrate deposited the required benzothiazoline (65%) (see Table I). Condensation of o-ABT 'HC1 with tkmethylpyridine-
2-carboxaldehyde in EtOH for 1 hr a t 25' gave, by pptn with Et20, 2-(6-methyl-2-pyridyl)benzothiazoline~HCl,mp 184-185' (EtOH-EtnO). Anal. ( C I ~ H I ~ C ~ N C,ZHI S )N, S. 2 4 2-Benzimidazolyl)benzothiazoline.-Benzimidazole-~carboxaldehyde (0.5 g) was dissolved in the min vol of 2 N HC1 and the soln was dild with EtOH (10 ml) and treated with a soln of o-ABT. HC1 (0.55 g) in EtOH (5 ml). The soln was refluxed for 2 min, cooled, basified with ",OH, and dild with HzO. The resulting solid was crystd to give the required compd (see Table I). 3-Acetyl-2-(2-pyridyl)benzothiazoline.-AczO (8 ml) was added to a soln of 16 (4.1 g) in pyridine (8 ml) and the soln w&skept a t 25' for 18 hr. Working up in the usual way gave an oil (4.5 9). A sample for analysis was distd a t 0.015 mm. Anal. (CIC
250 Journal of Medicinal Chemistry, 1971, Vol. 14, N o . 3
TABLE I11 PREPARED BY METHOD D
NO.
X
z-Pyndyl
Yield, %
5-Me 2 89 6-Me 3 81 5-Me 4 42 5-C1 2 92 3-C1 3 79 5-CI 4 94 5-CFa 2 82 5-CF3 3 88 5-CF3 4 14 5-NO, 2 83 5-NOz 3 91 6-CI 2 77 6-Cl 3 90 6-CI 4 88 6-Me0 2 85 6-Me0 3 96 6-Me0 4 92 a Free base. Resolidifies and remelts 232". found, 50.0. 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49
MP, ' C
Cryst solvent
EtOH-EtzO EtOH-Et20 EtOH-Et@ E tOH-E t 2 0 MeOII-E t 2 0 hleOH-E t20 MeOH-Et?O MeOH-Et20 EtOH-E t 2 0 EtOH-Et20 EtOH EtOH-Et20 E tOH-E tz0 MeOH E tOH-E t20 EtOH-Et20 E tOH-E tz0 C1: calcd, 24.9; found, 23.8.
197 161-170 261-268 202-203 196-197 225-254 163- 164 148-157 137-141 126 5-128' 187-188' 186-187 192-193 128 5-131" 194-195 194-196 183"
Analysis
C, H, N, CI C, H, N, C1 C, H, N, CI C, H, N ; C1. C, H, N, CI C, H, N,Cl H, N, CI; Cd C, H, N, CI H, N , CI; Ce c, H, N c, H, N C, H, N, CI C, H, N , C1 C, H, N, C1 C, H, N, Cl C, H, N, C1 C, H, CI found, 48.4. e C
MIC
3.1 3.1
30 0.8 1.6
t2,5 1.6 3.1 1.0 1.25 0.4 3 1 1. o 6 3 1.9 0.16 1.6 calcd, 49.0;
H12NzOS)C, H, N. The compd gave a hydrochloride, mp 177was refluxed 30 min, cooled, neutralized with NHAOH, and dild 183' (-MeOH-EtZO). Anal. ( C ~ ~ H ~ ~ C ~C, N H, Z ON. S) with H20 to give a solid (1.87 9): mp 169.5-170.5" (CBHI3-Benzoyi-2-( 2-pyridyl)benzothiazoline.-To a s o h of 16 hexane); Xmar 333 mp ( e 27,600). Anal. (ClaHloNzS) C, I€, N. (2.14 g) in Et20 (50 ml) was added a soln of BzCl (1.41 g) in 2-Amino-4-methylbenzenethiol.-3-Nitro-4-chlorotol~1e1le Et20 (13 ml). The mixt was stirred at 25" for 1.5 hr and filtered (51.3 g) was added during 20 min to a stirred solti of sodium and the filtrate treated with EtZO-HCl t o give a solid (1.47 g), polysulfide prepd from S (Fi7.6 g), NazS.9Hz0 (216 g), and 1320 mp 125-137" dec. Conversion into the free base gave the compd: (200 ml) at 70'. The reaction niixt was stirred at 90-95" for mp 110-112" (MeOH): vmax 1672 cm-l. Anal. ( C I ~ H ~ ~ N Z O S2) hr, EtOH (100 ml) added, and heating continued for a further H, N; C : calcd, 71.7; found 71.1. 1 hr before removing the solvent by steam distn. The residue Attempted N-Methylation of 2-(Pyridyl)benzothiazolines.was cooled to 5" and the solid (A; 11.3 g), mp 132-134", collected. A s o h of 17 (1.5 g) in MeOH (15 ml) was treated with 40yG The filtrate was acidified (50% AcOH) and Et@ extd to give a NaOH soln (0.7 mi; 1 equiv). To the red s o h was added Me1 residue (30 g) of S and an EtzO-sol solid (B; 19.0 g), mp 96-130'. (0.44 ml; 1 equiv) and after 30 min the reaction mixt was poured Crystn of A from MeOH gave bis(2-amino-4-methylphenyl) into HZO and the product was isolated by Et20 extn to give IV ( R trisulfide, mp 135-136'. Anal. (ClrHleNtS3) C, H, S; K : = 3-pyridyl): mp 67-68.5' (hexane); Xmax 206.3, 253, 268 (sh), calcd, 9.1; fo&d 9.8. and 367.5 (br) ( e 25,400, 21,100, 18,700, and 4200, resp). Anal. A soln of either A or B (5.52 n ) in a mixt of T H F (40 ml) arid (ClaH12NzS) C, H, N. The same compd was obtained when Et20 (30 ml) was added to LAH-(2.5 g) in Et20 (50 ml), the mixt equimolar amts of o-methylthioaniline and pyridine-3-carboxalwas refluxed for 1hr, cooled, and decompd with H20 and dil HCl. dehyde were heated for 30 min in EtOH. The Et20 layer was dried and treated with HC1-Et20 to give Similar reaction of Me1 with 16 and 18 gave I V (R = 2-pyrithe title compound as its hydrochloride (5.9 g), mp 187-189". dyl), mp 87-88' (hexane). Anal. (ClaHlzNzS) C , H, N and The free base had mp 42-44', bp 92-93' (2 mm). Ana'. ((37(IV; R = 4-pyridyl) mp 84.Fj-85.5' (hexane). Anal. ((313HgNS) C, H, N. Oxidn of the amine in 50% HC1 with .?OYc HitN2S) C, H, N. FeC13 soln gave bis(2-amino-4-methylphenyl)disulfide, mp 723-Ethyl-2-p-( 6-diethylaminoethoxy )pheny1benzothiazoline.73' (EtOH). Anal. (CIJII~NZSZ) C, H, N , S. Compd 5 (3.28 g) in Et20 (15 ml) contg AczO (1.41 ml) was left 2-Amino-4-chlorobenzenethiol.-Reaction of 2,5-dichlororiiovernight a t 25'. The oily product with Et20-HC1 gave a solid trobenzene (57.5 g) with sodium polysulfide as above gave, after (1.8 g), mp 162-164" (EtOH-EtzO). Conversion into the free adjusting the pH of the reaction mixt to 7 with AcOH, a solid base and redn with LAH (1 g) in Et20(100 ml) gave the compd (75.9 g). This was dissolved in hot EtOH (500 ml), the soln as an oil (1.06 g) which gave a citrate, mp 100-102' (EtOHfiltered, and the filtrate concd to give successive crops of solid: O ~NS;) C : calcd, 59.1; found, EtzO). Anal. ( C Z ~ H ~ ~ N ZH, A, mp 129-134" (9.95 9); B, mp 110-139' (4.4 9); C, mp 13458.6. 138' (2.53 g); and a final crop D, (31 g of gummy solid). Crystn 2 4 2-Pyridyl)-5-methylbenzothiazole.-Pyridine-2-carboxaldeof C (EtOH-MeCO) gave bis(2-amino-4-chlorophenyl) trisulhyde (1.07 g) and 2-aminc-4methylbenzenethiol(l.39g) were reC, H, N, S. fide, mp 142.5-143.5". Anal. (C12H1&1~N~S3) fluxed in PhNOz (10 ml) for 15 min, the solvent was removed by Redn of D (5 g ) with LAH (2 g ) in Et20 (200 ml) using the steam distn, the residue was extd into CeHe, and the CeHe soln was procedure described above gave the required compd, mp 44-46', filtered through a column of alumina. Elution with CeHe gave bp 102' (1.8 mm). Anal. (CeH&lNS) C, H, N, c1, S. a solid (0.66 g), mp 150-154O, raised by crystn (EtOH) to 2-Amino-5-chlorobenzenethiol.-6-Chloro-l,3,2-benzothi152-155'. Anal. (ClsHloN8)C, H, N. azathionium chlorides (140 g) was added portionwise to Hz0 2-(2-Pyridyl)-5-chiorobenzothiazole.-Compd 36 ( 1.0 g) was (2 1.) and after 2 hr, EtzO (1 1.) was added and the mixt was stirred converted into the free base, and to a soln of this in Me2C0 for 0.5 hr longer. The Et20 layer was sepd, the HzOlayer was extd (20 ml) was added a slight excess of Jones' chromic acid ~ o l n . ~ with EtzO (2 x 1 l.), and the Et20 exts were combined, washed, Concn to small bulk and isolation of the product in the usual and dried to afford a purple solid (99 g), mp 112-114" dec. This way gave a solid (400 mg) which, after several crystns from solid in MeOH (1 1.) was added to a stirred soln of NaOH (100 g) EtOH, gave the benzothiazole, mp 170-172'. Anal. (C12H7in HzO (1 1.) a t 10-15°, the mixt allowed to warm to 2 5 O , the p H ClNzS) C, H, N. adjusted to 7 with 50% AcOH, and the product extd into Etz.0. 2 4 3-Indolyl)benzothiazole.-o-ABT ( 1.64 g) and indole-3-carAddn of EtZO-HCl gave the thiolaHC1 (67.5 g), mp 210-212'. boxaldehyde (2.0 g) in EtOH (20 ml) contg 2 drops of concd HC1 The free base had mp 76-79O (C8He-hexane), bp 85-86" (0.2 mm). -___ (7) A . Bowers, T. G . Halsall, E. R . H . Jones, and A . J. Lemin, J . Chem. Soc., 2548 (1953).
(8) K . J. Farrington and W.K. Warburton, Aust. J . Chem., 8, 545 (1955).
NOTES
Journal of Medicinal Chemistry, 1971, Vol. 14, N o . 3 251
Anal. (CsHeCINS) C, H, C1, N, S. The thiol readily oxidized in air to give bis(2-amino-3-chlorophe1iyl) disulfide, mp 109-110' (MeOH). Anal. ( C ~ ~ H ~ O C C, ~ ZH, N ~N,S C1; ~ ) S: calcd 20.2; found, 20.8%.
Gly-Gly Ale ester (3). Hydrolysis of 3 with 1 N KaOH in MeOH gave the free acid, Z-O-tert-Bu-Tyr-y-tertBu-Glu-Gly-Gly (4), which was coupled to pentachloro2-Amino-5-methylbenzenethiol.-2-Ami1io-~met~hylbe1izothi- phenol using l-cyclohex~l-3-(2-morpholinoethyl)carazole (52 g), KOH (160 g), and HzO (320 ml) were refluxed 8 hr bodiimide methyl p-toluenesulfonate to obtain the under Nz. The s o h was cooled and the p H adjust>edt,o 7 with tetrapeptide active ester, Z-O-lert-Bu-Tyr-y-tert-BuSOYG AcOH to afford a solid (44 9). Cleavage of this with LA11 Glu-Gly-Gly pentachlorophenyl ester (5). The caras before gave the thiol, mp 68-70', bp 91" ( 2 mm). ,4nal. bobenzoxy protecting group was removed by catalytic (CgHiNS) C, H, N, S. Oxidation with FeC13 gave the correspoiidiiig disulfide, nip 88" (EtOH). Anal. (CIIHIGN~SI) C, hydrogenolysis in the presence of dry HC1 to give the H , N, S. polymerizing unit 0-tert-Bu-Tyr- y-tert-Bu-Glu-Gly-Gly 2-Amino-4-trifluoromethylbenzenethiol.-Red1i of bis(2pentachlorophenyl ester HC1 (6). The polymerization amino-4-t rifluoromethylphenyl) disulfides (4.5 g) with LAH as was performed a t a reagent concentration of 100 above gave the conipd, bp 77-78' (2 mm), n 2 5 1.533'2. ~ Ilespite mmoles/l. in the presence of a performed monomer, several attempts, the compd failed to analyze satisfactorily, possibly due to its high F content. Anal. Calcd for CTHeF,NS; since this has been shown to produce linear high mol C, 43.6; H , 3.1; N, 7.25; S, 16.6; F, 29..7. Found: C, 44.7; wt polypeptides.'P2+* Following this established proH,3.8;N,7.1;S, 13.8,5;F,29.0. cedure, the insoluble polymer, poly (0-tert-Bu-Tyr- y-terto-Methylaminobenzenethiol.-Bis(2-formamidopheiiyl) diBu-Glu-Gly-G1y)Glg methyl ester \vas prepared, from sulfidelo (5.0 g ) in Et20 (130 ml) was added to LAH (5.0 g ) i n Et20 (50 ml) and the solii was refluxed 1 hr, cooled, aiid decompd which the protecting tert-Bu groups were removed by under N P with 2 N HC1. The EtzO layer was washed, dried, aiid the use of 90% F3CC02H to yield poly(Tyr-Glu-Glydistd to give the thiol (4.18 g), bp 127-128" (18 mm). G1y)Gly methyl ester (1). After extensive dialysis, Acknowledgment.-The authors thank A h . W. Iafffor the biological data. through calibrated columns of Sephadex G-1009 and Corning CPG 10-240 glass granules. By this means (9) Sandoz Ltd., Ketlierlands Application 6,512,671: Chem. Abslr.. 61, 8929 (1966). the mol wt of the polypeptide was found to be at least (10) M. L. Tomlinson, J. Chem. Soc., 1607 (1936). 1 x 105. Immunochemistry.-Two rabbits I\ ere immunized v i t h poly(Tyr-Glu-Gly-G1y)Gly methyl estcr (1) using Linear Polypeptides of a Known Sequence the same protocol as that previously d c ~ c r i b e d . ~To of Amino Acids. Synthesis and aliquots of thc sera obtained from each rabbit v a s added Immunochemical Properties of up to 10,000pg of thc synthetic polypeptide 1. KO Poly (L-tyrosyl-L-glutamylglycylg1ycyl)glycine precipitin reaction was observed. The possibility that the polypeptide 1 could be ft Methyl Ester hapten for the antigen poly(Tyr-Glu-Ala-G1y)Gly-II4C Et was also investigated. Incremental amounts BRIANJ. JOHNSON*A N D ELWOODG. Tiwsrc of the polypeptide poly(Tyr-Glu-Gly-G1y)GlyN e 1 Department of Chemistry, l'ujts University, were added to aliquots of antisera to poly(Tyr-GluMedjord, Massachusetts 06165 Ala-Gly)Gly-l-14C Et containing a k n o 11~ amount of the antigen. No inhibition of the precipitin reaction lieceived August 13, 1970 was observed. Conclusions.-Replacement of the alanyl moicty in A recent investigation of the immunochemical proppoly(Tyr-Glu-Ala-Gly)Gly-l-~4C Et by thc sterically Et ester1r2has erties of p~ly(Try-Glu-Gly-Gly)Gly-l-~~C smaller and optically in:tct ive glycyl residuc cauqed a demonstrated that the polypeptide is antigenic, elicitloss in antigenicity. Thus, although the nlanyl rcsidue ing antibodies in rabbit^.^ is believed pot to be part of the active site of the antiIt has been shown that the alanyl residue is not part gen4 this residue does confer antigenicity t o the moleof the active site of this a ~ i t i g e n . ~However, it was cule. This is most probably achievcd by the alanyl unknown if the alanyl residue played any role in the residue maintaining such a favorable conformation t hnt antigenicity of the polypeptide. To investigate this the antigenic determin:mte are made available. The latter point it was considered that substitution of the conformation of the polypeptide poly(Tyr-Glu-Glyalanyl residue with the sterically smaller and optically G1y)Gly Me is thought t o be very different since this inactive glycyl residue may affect the immunochemical polymer does not inhibit the precipitin reaction beproperties of the molecule. T o this end we wish to tween the antigen and its antisera. report the synthesis and immunochemical properties of poly (Tyr-Glu-Gly-G1y)glycine ;\le ester (1). Experimental Section Chemistry.-Z-y-tert-butylglutamic acid pentachlorophenyl ester was coupled to Gly-Gly i\Ie.HC15 to hlelting points were taken with a Mel-Temp apparatus aiid yield Z-y-tert-Bu-Glu-Gly-Gly Me ester. Catalytic are uncorrected. Optical rotations were taken with a Carl Zeiss precision polarimeter. hydrogenolysis of the fully protected tripeptide in the r-tertBu-Glu-Gly-Gly Me .HCl (2).-To a mixtlire of 10.0 g presence of dry HCl produced y-terl-Bu-Glu-Gly-Gly (0.055 mole) of Gly-Gly Me.HC1 in 300 ml of CHZC12 was added Ale.HC1 (2). This material was coupled to Z-O-tert5.55 g (0.055 mole) of EtaN aiid 29.25 g (0.05 mole) of Z-7-tert Bu-tyrosine pentachlorophenyl ester' to give the fully butylglutamic acid pentachlorophenyl ester. The mixture was protected tetrapeptide, Z-0-tert-Bu-Tyr-y-tert-Bu-Glustirred overnight a t room temp, then coricd zn vacuo. The solid (1) B. J. Johnson a n d E. G. Trask, J. Chem. SOC.C, 2644 (1Q69). (2) B. J. Johnson, J . Pharm. Sei., in press. (3) 13. J. Johnson and E. G . Trask, abid., 69, 724 (1970). (4) B. J. Johnson, abad., in press. ( 5 ) H.Brookmann and H. Musso, Chem. Ber., 87, 587 (1954).
residue was dissolved in EtOAc and washed with 10% citric acid (6) B. J. Johnson and D. S. Rea, Can. J . Chem., 48, 2509 (1970). (7) B. J. Johnson, J . Chem. SOC.C, 1412 (1969).
(8) B. J. Johnson, ibid., C, 3008 (1968). (9) P.Andrew, Biochem. J . , 91, 222 (1964).
