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Bioconjugate Chem. 1990, 7, 96-99
LETTERS Synthesis of 1 - (Aminooxy ) -4- [ (3-nitro- 2-pyr idyl) dithi01butane and 1- (Aminooxy)- 4 4 (3-nitro-2-pyridyl)dithio]but-2-ene, Novel Heterobifunctional Cross-Linking Reagents l-(Aminooxy)-4-[ (3-nitro-2-pyridyl)dithiolbutane hydrochloride (1) and the related unsaturated compound 2 are stable alkoxylamino cross-linking reagents which react readily with ketones to give the corresponding alkoximes and which couple readily with thiolated proteins in aqueous media to provide stable protein conjugates.
The most commonly employed procedures for attaching drugs to proteins involve thiolation of the protein using heterobifunctional cross-linking reagents such as SPDP [N-succinimidyl3-(2-pyridyldithio)propionate] (I, 2) and subsequent coupling with a nucleophilic moiety of the drug. In the case of antibodies, procedures have been developed which call for periodate oxidation of the carbohydrate region of the antibody, subsequent reaction of the exposed aldehyde moieties with an amine, and reduction of the resultant imine with borohydride to yield stable alkylamino functionalized proteins (3). As part of a program concerned with targeting of monoclonal antibody (MAb)-drug combinations for chemotherapeutic applications, we sought alternatives to currently employed strategies (2). Toward this end, the alkoxylamino c o m p o u n d l-(aminooxy)-4-[(3-nitro-2pyridy1)dithiolbutane hydrochloride (1, Chart I) was prepared (vide supra) and found to react readily with the ketone moiety of adriamycin to produce a stable alkoxime ( 4 ) . This drug-linker combination readily coupled with thiolated antibody ( I ) in neutral phosphate buffer solution to produce a disulfide-linked drug-antibody conjugate (Scheme I) which was found to be exceptionally stable, requiring exposure to strongly acidic media (aqueous phosphoric acid, ca. pH 2) to release the adriamycin
1
Scheme I
2
1
L
O
H
MeOH
R
Adriamycin
N oS -'s/ R
&OH
OZN
MAb-SH
R= N'O-SsMAb
RL
O
H
HO
Scheme I1
(4).
In addition, 1 was found to react with periodatetreated antibodies in aqueous solution (Scheme 11)to produce the alkoximino-modified proteins (vide infra) not requiring further modification to improve their stability (Kaneko, T., and Webb, R., unpublished results). The preparation of 1 proceeds as follows. The alkylation of the sodium salt of ethyl N-hydroxyacetimidate (3; NaH/THF, Chart 11) with l,4-dibromobutane gave bromide 4 (5)after fractional distillation to remove starting 3. Displacement of bromide 4 with potassium thioacetate in EtOH to give acetylthio compound 5 was followed by removal of the acetate (K,CO,/MeOH) and the acetimidate (HCl/EtOH/H,O) to yield 1-(aminooxy)-4mercaptobutane hydrochloride (6). Introduction of the (3-nitro-2-pyridy1)dithio group was accomplished by treatment of 6 in methanol with (methoxycarbony1)sulfenyl chloride (6) to give crystalline l-(aminooxy)-4-[(methoxycarbony1)dithiolbutane hydrochloride (7), which was stirred in methanol with 3-nitro-2-mercaptopyridine (7) to yield the desired alkylamino dithio compound 1. Compound 1 could be recrystallized from a mixture of meth-
bR
anol, ethyl acetate, and ether and was stable when stored a t room temperature for several months. Unsaturated compound 2 was prepared in a similar fashion, i.e. by alkylation of 3 (NaH/THF) with 1,4-dibromo-(E)-but-2ene to yield bromide 8 (Chart 11); subsequent thioacetate displacement (KSAc/EtOH) giving 9, deprotection (K,CO,/MeOH then aqueous HCl/EtOH) to thiol 10 and disulfide formation [ (methoxycarbony1)sulfenyl chloride/ 3-nitro-2-mercaptopyridine/MeOH] yielding 2. Both 1 and 2 could be reacted directly with carbonyl compounds or used as their corresponding free bases by first stirring with a slight excess of triethylamine in methanol. Experimental Procedures. All solvents and reagents were employed as received, with the following exceptions: Aldrich anhydrous-grade tetrahydrofuran and anhydrous-grade hexane were used for reactions employing
1043-1802/90/2901-0096$02.50/0 0 1990 American Chemical Society
Bioconjugate Chem., Vol. 1, No. 2, 1990 97
Letters
Chart I1 Et0
HCI H,N-OR
161.79, 161.67 (CH,C(OEt)=N), 73.09, 72.49 (NOCH,), 61.93,61.86 (CH,CH,O), 33.41,30.88, 29.59, 27.52,25.51, 14.27, 13.40; IR (neat) 2980, 2941,1647 (s), 1377 (s), 1306 (s), 1263, 1252, 1119, 1076, 978 cm-l; MS (DCI/ methane) m / e 240 MH+ 2, 18), 238 (MH', 19), 158 (MH' - Br, 60); exact mass calcd for C,H,,NO,Br 237.0364, found 237.0358. Anal. (C,H,,NO,Br) C, H, N. Ethyl N-[[4- (Acetylt hio)butylloxy lacetimidate (5). A solution of ethyl N-[(4-bromobutyl)oxy]acetimidate (4, 34.00 g, 0.143 mol) in absolute ethanol (300 mL) was treated with potassium thioacetate (20.00 g, 0.175 mol) and the resulting yellow suspension heated a t reflux for 1h. Thin-layer chromatographic analysis of the reaction mixture revealed the absence of the starting bromide (R, 0.6 in A) and the presence of the product thioacetate ( R f 0.5 in A). The mixture was cooled, filtered, and concentrated in vacuo, and the slurry was partitioned between ethyl acetate and water. The combined ethyl acetate layers were washed with saturated aqueous NaHCO, solution, water, and brine and then dried over MgSO,, filtered, and concentrated in vacuo. The oil remaining was distilled to yield 30 g (90%) of ethyl-N[ [4-(acetylthio)butyl]oxy]acetimidate( 5 ) as a clear, yellow, foul-smelling liquid: bp 105-108 "C (2 mmHg); 'H NMR (CDCl,) 6 3.90 (9, J = 6 Hz, 2 H, OCH,CH,), 3.79 (t, J = 6 Hz, 2 H, NOCH,), 2.81 (t,J = 6 Hz, 2 H, CH,SAc), 2.22 (s, 3 H, CH,COS), 1.81 (s, 3 H, CH,C(OEt)=N), 1.58 (br m, 4 H , CH,CH,), 1.16 (t, J = 6 Hz, 3 H , OCH,CH,); 13C NMR (CDC1,) 6 196.45 (SC=O), 162.74 and 162.66 (CH,C(OEt)=N), 73.55 and 73.05 (CH,O), 62.35 and 62.32 (CH,CH,O), 30.73, 29.12, 28.81, 28.61, 28.23, 26.45, 25.74, 14.48, 13.66; IR (neat) 2990,2945,2875, 1700 (s, C=O), 1650,1380,1305 (s), 630 cm-'; MS (DCI/ methane) m / e (relative abundance) 234 (MH+,100);exact mass calcd for CloHlgNO,S 233.1086, found 233.1084. Anal. (C1&1gNO,S) C, H, N, S. l-(Aminooxy)-4-mercaptobutane Hydrochloride (6). A solution of ethyl N-[ [4-(acetylthio)butyl]oxy]acetimidate (5,35.00 g, 0.150 mol) in absolute methanol (100 mL) was thoroughly degassed with nitrogen and treated with anhydrous K,CO, (20.00 g, 0.150 mol), and the resulting yellow suspension was stirred vigorously for 12 h. Thinlayer chromatographic analysis of the reaction mixture indicated the absence of the starting thioacetate 5 (R, 0.5 in A) and presence of the product thiol (Rf.0.65 in A). The suspension was filtered and concentrated in vacuo to a yellow slurry, which was dissolved in ethanol and treated with concentrated aqueous HCl(30 mL), and the resulting mixture was heated on a steam bath for 1 h. The volatiles were removed in vacuo, and the residue was azeotroped with 4 successive volumes of absolute ethanol. The volatile amine was isolated as its hydrochloride by dissolution of the solid in aqueous NaHCO,, extraction with diethyl ether, and treatment of the ether solution slowly dropwise with HC1 in diethyl ether (1.0 M) until no further solid had formed. The solid was collected by filtration, washed with hexane, and recrystallized from ethanol-hexane to yield 12.0 g (51%) of 1-(aminooxy)-4-mercaptobutane hydrochloride (6) as a white solid: mp 134 "C dec; 'H NMR (CD,OD) 6 5.08 (br s, 6 H, exch, NH,, SH, HCl), 3.85 (t,J = 6 Hz, 2 H, NOCH,), 2.30 (t, J = 6 Hz, 2 H, CH,S), 1.54 (m, complex, 2 H, OCH,CH,), 1.46 (m, complex, 2 H, CH,CH,S); 13CNMR (CD,OD) 6 76.39 (NOCH,), 31.37 (CH,S), 27.70 (OCH,CH,), 24.99 and 24.85 (CH,CH,S); IR (KBr) 3450 (br), 2980 (s), 2790, 1502, 1025 cm-l; MS (DCI) m / e (relative abundance) 122 (MH+, 45), 104 (MH+ - NH,, 12), 75 (MH+ - N,H,O, P); exact mass calcd for C,H,,NOS
+
3:R=H 4 : R = (CH,),Br 5 : R = (CH,),SAc
6 R = (CHd4SH
7 : R = (CH2)4SSQCH3 10 : R = CH&H=CHCH,SH
8 : R = CH,CH=CHCH,Br 9 : R = CH,CH=CHCHZSAc
NaH; ethyl N-hydroxyacetimidate was obtained from Aldrich, and distilled from CaH, under nitrogen prior to use; (methoxycarbony1)sulfenyl chloride was obtained from Fluka or prepared from (chlorocarbony1)sulfenyl chloride (obtained from Aldrich) according to the procedure of Field and Ravichandran (6) and was distilled under argon prior to use; Sigma absolute methanol was employed as received after degassing with nitrogen. Thin-layer chromatography was performed on EM Science silica gel 60 F,,, using the following solvent systems: system A, 3:l hexane/ethyl acetate; system B, 2:l hexane/ethyl acetate; system C: 1:l ethyl acetate/hexane. Proton and carbon-13 NMR spectra were obtained on a Varian 300 spectrometer at 300 and 75 MHz, respectively. Mass spectra and exact mass measurements were performed by the Analytical Research Department, Bristol-MyersSquibb, Wallingford, CT. Elemental analyses were performed by Oneida Research Services, Whitesboro, NY. Values reported were within 0.4% of the theoretical values unless otherwise noted. Ethyl N-[(4-Bromobutyl)oxy]acetimidate(4). A 3-L three-necked round-bottomed flask equipped with mechanical stirrer, addition funnel, and gas inlet, was ovendried, flushed with nitrogen, and allowed to cool under nitrogen. It was then charged with NaH (20.40 g, 0.509 mol, 60% dispersion in oil). The NaH was washed twice with hexane, covered with tetrahydrofuran (1.6 L), and, with vigorous stirring, ethyl N-hydroxyacetimidate (3,47.74 g, 0.463 mol) was added rapidly dropwise. Gas evolution was noted upon addition. After the addition was complete, the grey suspension was heated a t reflux for 18 h. T h e suspension was t h e n cooled a n d 1,4dibromobutane (100.00 g, 0.463 mol) was added rapidly dropwise. After the addition was complete, the resulting suspension was refluxed for 2 days under nitrogen. Thin-layer chromatographic analysis of the reaction mixture indicated the presence of the starting acetimidate (R, 0.4 in A) and the presence of a less polar product (R, 0.6 in A). The mixture was cooled and treated with methanol (50 mL) followed by saturated aqueous NH,C1(200 mL). The tetrahydrofuran was removed in vacuo, and the slurry that remained was partitioned between ethyl acetate and water. The combined ethyl acetate layers were washed with brine, dried over MgSO,, filtered, and concentrated in vacuo to an oil which was distilled under vacuum to yield recovered ethyl N-hydroxyacetimidate [20 g, 1970, bp 35-40 "C (2 mmHg)], a mixed fraction (ca. 1 2 g), and 60 g (52%) of ethyl N-[(4-bromobutyl)oxylacetimidate (4)as a colorless oil: bp 75-77 "C (2 mmHg); 'H NMR (CDCl,) 6 3.88 (9, J = 7 Hz, 2 H, OCH,CH,), 3.80 (t, J = 6 Hz, 2 H, NOCH,), 3.33 (t, J = 6 Hz, 2 H, CH,Br), 1.93 (m, 1H, OCH,CH,), 1.84 (m, 1H, OCH,CH,), 1.80 (s, 3 H, CH,(EtO)=N), 1.65 (m, 2 H, CH,CH,Br), 1.15 (t, J = 7 Hz, 3 H, OCH,CH,); 13C NMR (CDC1,) 6
98
Bioconjugate Chem., Vol. 1, No. 2, 1990
122.0640, found 122.0638. Anal. (C,H,,NOS.HCl) C, H, N, S. In separate runs, t h e intermediate ethyl N-[(4mercaptobutyl)oxy]acetimidate could be isolated after treatment with K,CO, in MeOH by partitioning of the residue between ethyl acetate and water. The combined ethyl acetate layers were washed with brine, dried over K,CO,, and concentrated in vacuo to a light yellow liquid which was distilled to give ethyl N-[(4-mercaptobuty1)oxylacetimidate as a clear, yellow, foul-smelling liquid: bp 65-67 "C (5 mmHg); 'H NMR (CDCI,) 6 4.00 (9, J = 6 Hz, 2 H, OCH,CH,), 3.90 (t, J = 6 Hz, 2 H , OCH,CH,), 2.57 (q, J = 7 Hz, 2 H, CH,S), 1.92 (s, 3 H, CH,C(OEt)=N), 1.72 (m, 4 H, OCH,CH,CH,CH,), 1.35 (t, J = 7 Hz, 1 H, SH), 1.27 (t,J = 6 Hz, 3 H, OCH,CH,); I3C NMR (CDC1,) 6 162.75 (CH,C(OEt)=N), 73.14 (OCH,CH,), 62.39, 30.93 (CH,SH), 27.86 (CH,CH,SH), 24.68 (OCH,CH,), 14.51 (OCHJH,), 13.72; MS (DCI/ methane) m / e (relative abundance) 192 (MH+, P). 1- (Aminooxy)-4-[ (methoxycarbonyl)dithio]butane Hydrochloride (7). A solution of (methoxycarbony1)sulfenyl chloride (5 mL, 0.061 mol) in dry, degassed MeOH was cooled to 0 "C and treated with 1-(aminooxy)4-mercaptobutane hydrochloride (6,g.O g, 0.045 mol), and the resulting yellow solution was stirred a t 0 "C for 1 h (HCl gas evolution was noted). The yellow solution was then thoroughly degassed with N,, and the solvents were removed in vacuo. The white solid obtained was recrystallized from methanol/CH,Cl,/hexane to yield (in three crops) 5.4 g (75% ) of l-(aminooxy)-4-[ (methoxycarbonyl)dithio]butane hydrochloride (7) as sharp-melting, white flakes: mp 107-108 "C; 'H NMR (CD,OD) 6 4.6 (br s, exch, 6 H, NH,, HCl), 3.82 (t, J = 6 Hz, 2 H, NOCH,), 3.63 (s, 3 H, OCH,), 2.59 (t, J = 6 Hz, 2 H, CH,S), 1.57 (m, 4 H, OCH,CH,CH,CH,S); 13CNMR (CD,OD) 6 172.10 (SCOOCH,), 76.28 (NOCH,), 56.45 (SCH,), 39.78 (OCH,CH,), 27.52 (CH,CH,), 25.97 (COOCH,); IR (CH,OH) 3433 (br), 1737 (s, C=O), 1195, 1142 (s) cm-'; MS (DCI/methane) m / e (relative abundance) 212 (MH+, 22), 179 (MH+ - CH,OH, P); exact mass calcd for C,H,,NO,S, 211.0337, f o u n d 211.0335. Anal. (C,H13N03S,~HCl)C, H, N, S, C1. 1-(Aminooxy)-4-[(3-nitro-2-pyridyl)dithiolbutane Hydrochloride (1). A solution of l-(aminooxy)-4[ (methoxycarbonyl)dithio] butane hydrochloride (7, 1.50 g, 0.00949 mol) in dry, degassed methanol (30 mL) was treated with 3-nitro-2-mercaptopyridine (1.48 g, 0.00949 mol) and the resulting yellow solution was stirred under N, a t room temperature. After 12 h, thin-layer chromatographic analysis of the reaction mixture revealed the absence of the starting material 7 (R, 0.5 in B) and presence of the product pyridine 1 (R, 0.4 in B). The mixture was filtered to remove unreacted 3-nitro-2-mercaptopyridine, and the product was precipitated by the addition of ethyl acetate and finally ether to yield 1.3 g (50%) of 1-(aminooxy)-4-[ (3-nitro-2-pyridyl)dithiolbutane hydrochloride ( 1 ) as a yellow solid. An analytical sample was obtained by recrystallization from MeOH/ethyl acetate/ ether to give 1 as a white, crystalline solid: mp 134-135 "C; 'H NMR (CD,OD) 6 8.85 (dd, J = 1.5, 4.5 Hz, 1 H, ArH), 8.59 (dd, J = 1.5, 8 Hz, 1 H, ArH), 7.49 (dd, J = 4.5, 8 Hz, 1 H, ArH), 4.88 (br s, exch, 6 H), 4.05 (t,J = 6 Hz, 2 H, CH,ON), 2.91 (t, J = 6 Hz, 2 H, CH,SS), 1.83 (m, 4 H, OCH,CH,CH,CH,S); 13CNMR (CD,OD) 6 158.25 (Ar), 155.71 (Ar), 144.96 (Ar), 135.83 (Ar), 123.28 (Ar), 76.31 (OCH,CH,), 38.91 (CH,SS), 27.78 (CH,CH,SS), 26.20 (OCH,CH,); IR (KBr) 3434 (br), 2946,1584, 1558,1512, 1402, 1398 (s), 1342 (s), 744 cm-'; MS (DCI) m / e (relative abundance) 278 (MH+ + 2, lo), 276 (MH+, P); exact
Webb and Kaneko
mass calcd for C,Hl,N,03S, 276.0477, found 276.0473. Anal. (C,H,,N,O,S,~HCl) C, H, N, S, C1. Ethyl N-[(4-Bromo-(E)-but-2-enyl)oxy]acetimidate (8). A 3-L three-necked round-bottomed flask equipped with mechanical stirrer, addition funnel, and gas inlet was oven dried, flushed with nitrogen, and allowed to cool under nitrogen. It was then charged with NaH (18.80 g, 0.470 mol, 60% dispersion in oil). The NaH was washed twice with hexane and covered with tetrahydrofuran (1 L), and with vigorous stirring, ethyl N-hydroxyacetimidate (3,43.82 g, 0.420 mol) wad added rapidly dropwise. Gas evolution was noted upon addition. After the addition was complete, the grey suspension was heated at reflux for 18 h. The suspension was then cooled and 1,4-dibromo-2-butene (100.00 g, 0.470 mol) was added rapidly dropwise. After the addition was complete, the resulting suspension was refluxed for 2 days under nitrogen. Thin-layer chromatographic analysis of the reaction mixture indicated the presence of the starting acetimidate (R, 0.4 in A) and the presence of a less polar product (Rf0.7 in A). The mixture was cooled and treated with methanol (50 mL) followed by saturated aqueous NH,C1 (200 mL). The tetrahydrofuran was removed in vacuo, and the slurry that remained was partitioned between ethyl acetate and water. The combined ethyl acetate layers were washed with brine, dried over MgSO,, filtered, and concentrated in vacuo to an oil which was distilled under vacuum to yield recovered ethyl N-hydroxyacetimidate (3,10.0 g, 23%) and 64.5 g (65%) of (8) as ethyl N-[ (4-bromo-(E)-but-2-enyl)oxy]acetimidate a colorless oil: bp 70-72 "C (2 mmHg); 'H NMR (CDC1,) 6 5.89 (m, 2 H, OCH,CH=CHCH,), 4.37 (m, 2 H, NOCH,), 3.97 (9, J = 6 Hz, 2 H, OCH,CH,) overlapping 3.93 (m, 2 H, CH,Br), 1.90 (s, 3 H, CH,(OEt)=N), 1.23 (t, J = 6 Hz, 3 H , OCH,CH,); 13C NMR (CDC1,) 6 163.21 (CH,C (OEt)=N) , 132.15 (OCH,CH=C H ) , 129.38 (BrCH,CH=CH), 73.11 (CH,CH,OC(CH,)=N), 62.53 (OCH,), 32.30 (BrCH,), 14.51 (OCH,CH,), 13.85 (CH,C(OEt)=N); IR (neat) 2981,1647 (s), 1378 (s),1307 (s), 1207, 1119, 1095, 1062, 1027 (s), 969 cm-'; MS (DCI) m / e (relative abundance) 238 (MH+ + 2,75), 236 (MH+, 80), 156 (MH+ - HBr, P); exact mass calcd for CsH,,N0,Br 235.0208, found 235.0203. Anal. (C,H,,NO,Br) C, H, N. Ethyl N-[[4-( Acetylthio)-(E)-but-2-enyl]oxy]acetimidate (9). A solution of ethyl N-[(4-bromo-(E)-but2-eny1)oxylacetimidate (8, 32.4 g, 0.137 mol) in absolute ethanol (200 mL) was treated with potassium thioacetate (16.0 g, 0.140 mol) and the resulting yellow suspension was heated at reflux for 1 h. Thin-layer chromatographic analysis of the reaction mixture revealed the absence of the starting bromide 8 (R, 0.7 in A) and the presence of the more polar product thioacetate 9 (R, 0.5 in A). The mixture was cooled, filtered, and concentrated in vacuo, and the slurry was partitioned between ethyl acetate and water. The combined ethyl acetate layers were washed with saturated aqueous NaHCO, solution, water, and brine and then dried over MgSO,, filtered, and concentrated in vacuo. The oil remaining was chromatographed over SiO, eluting with 9:l hexane/ ethyl acetate to yield 30 g (90%) of ethyl N-[[4-(acetylthio)-(E)-but-2-enyl]oxy]acetimidate(9) as a clear, yellow, foul-smelling liquid: 'H NMR (CDC1,) 6 5.75 (dt, J = 6, 15 Hz, 1 H, CH=CHCH,SAc), 5.61 (dt, J = 7, 15 Hz, 1 H , OCH,CH=CH), 2.80 (d, J = 7 Hz, 2 H , OCH,CH=CH), 3.93 (9, J = 7 Hz, 2 H, CHSCHZOC(CH,)=N), 3.47 (d, J = 6 Hz, 2 H, CH,SAc), 2.26 (s, 3 H, SCOCH,), 1.85 (s, 3 H, CH,C(OEt)=N), 1.18 (t, J = 7 Hz, 3 H, OCH,CH,); 13C NMR (CDC1,) 6 194.88
Letters
(SCOCH,), 162.36 (CH,C(OEt)=N), 129.89 (CH=CH), 128.03 (CH=CH), 73.23 (CH,CH,OC(CH,)=N), 62.09 (NOCH,), 30.86 (CH,SCO), 30.35 (CH,COS), 14.30 (CH,CH,O), 13.56 (CH,C(OEt)=N); IR (neat) 2981,2933, 2866, 1695 (s, C=O), 1646 (9, C=N), 1378 (s), 1306 (s, NOCH,), 1105, 1024, 969, 627 cm-l; MS (DCI) m / e (relative abundance) 234 (MH+ + 2, 8), 232 (MH+, P), 190 (MH+- COCH,, lo), 156 (MH+- SCOCH,, 12), 129 (MH+ - CH,C(OEt)=N, 38); exact mass calcd for Cl,Hl,NO,S 231.0920, found 231.0928. Anal. (CloHlgNO,S) C, H, N, S. l-(Aminooxy)-4-mercapto-(E)-but-2-ene Hydrochloride (10). A solution of ethyl N - [[4-(acetylthio)-(E)-but2-enylloxylacetimidate (9, 18.50 g, 0.0799 mol) in absolute methanol (100 mL) was thoroughly degassed with nitrogen and treated with anhydrous K,CO, (20.00 g, 0.144 mol), and the resulting yellow suspension was stirred vigorously for 2 h. The suspension was filtered and concentrated in vacuo to a yellow slurry which was dissolved in ethanol (100 mL) and treated with concentrated aqueous HC1 (10 mL), and the resulting mixture was heated on a steam bath for 1 h. The yellow solution was concentrated in vacuo, and the residue was azeotroped with 4 successive volumes of absolute ethanol. The slurry remaining was dissolved in water and washed with ether (3 X 50 mL), and the ether layers were discarded. The solution was then basified with 2 N NaOH to pH 10 and extracted exhaustively with diethyl ether. The combined ether layers were dried over MgSO,, filtered, and treated slowly dropwise with HC1 in diethyl ether (1.0 M) until no more solid had formed. The solid was collected by filtration, washed with hexane, dried in vacuo and recrystallized from ethanol-hexane to yield 9.0 g (60%) of l-(aminooxy)-4-mercapto-(E)-but-2-ene hydrochloride (10) as a white solid: mp 134-136 OC dec; 'H NMR (CD,OD) 6 5.77 (m, 5 lines, 1 H, CH=CH), 5.54 (m, 5 lines, 1 H, CH=CH), 4.63 (br s, 6 H, exch, NH,, SH, HCl), 4.31 (d, J = 6 Hz, 2 H, NOCH,), 3.15 (d, J = 6 Hz, 2 H, CH,S); I3C NMR (CD,OD) 6 136.42 (NOCH,CH= CH), 127.02 (CH=CHCH,S), 76.43 (OCH,), 41.35 (CH,S); IR (KBr) 2953,2665,1402,970 cm-l; MS (DCI) m / e (relative abundance) 118 (MH+ - 2, P); exact mass calcd for C , H , , N O S 1 2 0 . 0 4 8 3 , f o u n d 120.0485. A n a l . (C4HllNOS*HC1)C, H, N, C1. 1-(Aminooxy)-4-[(3-nitro-2-pyridyl)dithio]-(E)but-2-ene Hydrochloride (2). A solution of 1-(aminooxy)-4-mercapto-(E)-but-2-ene hydrochloride (10,2.00 g, 0.0088 mol) in dry, degassed (N,) methanol (20 mL) was cooled to 0 "C under N, and treated with (methoxycarbony1)sulfenyl chloride (0.72 mL, 0.0088 mol). The resulting light yellow solution was stirred for 15 min and then degassed with N, and concentrated in vacuo. The slurry remaining was dissolved in methanol (20 mL) and treated in one batch with 3-nitro-2-mercaptopyidine (1.40 g, 0.0088 mol). The resulting suspension was stirred under N, at room temperature for 2 h, a t which time thin-layer chromatographic analysis of the reaction mixture revealed the absence of 3-nitro-2-mercaptopyridine ( R , 0.1 in C) and the presence of the product ( R , 0.2 in C). The solution
Bioconjugate Chem., Vol. 1, No. 2, 1990 99
was filtered to remove a small amount of precipitate, and the filtrate was diluted with ethyl acetate and ether. The resulting light yellow solid was filtered, washed with ether, and dried in vacuo to yield 1.34 g (56%) of yellow, solid material. An analytical sample was obtained by recrystallization from methanol/ethyl acetate/ether to yield 1-(aminooxy)-4-[ (3-nitro-2-pyridyl)dithiol-(E)-but-2ene hydrochloride (2) as light yellow flakes: mp 135 OC (sharp); 'H NMR (CD,OD) 6 8.62 (dd, J = 1.5, 5 Hz, 1 H, ArH), 8.36 (dd, J = 1.5, 8.5 Hz, 1 H, ArH), 7.26 (dd, J = 5, 8.5 Hz, 1 H, ArH), 5.77 (m, complex (5 lines), 1H, CH=CH), 5.40 (dt, J = 7, 12 Hz, 1 H, CH=CH), 4.19 (d, J = 6.5 Hz, 2 H, OCH,), 3.32 (d, J = 7 Hz, 2 H, CH,S); I3C NMR (CD,OD) 6 160.49 (Ar), 158.23 (Ar), 138.36 (CH= CH), 138.21 (Ar), 129.89 (CH=CH), 78.78 (OCH,), 43.11 (CH,S); IR (KBr) 3433,2942,1585,1558,1521,1396,1342, 746 cm-'; MS (DCI/methane) m / e (relative abundance) 274 (MH+ + 1,95), 241 (MH+ - NH,OH, P); exact mass calcd for CgHllN,O,S, 274.0320, found 274.0317. Anal. (CgHllN,O,SyHC1) C, H, N, S. LITERATURE CITED (1) Carlsson, J., Drevin, H., and Axen, R. (1978) Protein Thiolation and Reversible Protein-Protein Conjugation. Biochem. J . 173 723-737. (2) Upeslacis, J., and Hinman, L. (1988) Chemical Modification of Antibodies for Cancer Chemotherapy. Annu. Rep. Med. Chem. 23,151-160. (3) Rodwell, J., and McKearn, T. (1987) Antibody Conjugates for the Delivery of Compounds to Target Sites. US Patent 4,671,958. (4) Braslawsky, G., Fitzgerald, K., Edson, M., Daues, A., Kaneko, T., Webb, R., Knipe, J., and Greenfield, R. (1989) Internalization as a Requirement for MAb-Directed Cell Killing by ADM-Immunoconjugates. Presented at the 80th Annual Meeting of the American Association for Cancer Research, Abstract #4494. (5) K h o m u t o v , A. R., a n d K h o m u t o v , R. M. (1986) Aminooxythiothreitol-New Agent for Introducing Reactive Thiol Groups Into Nucleic Acids. Bioorg. Khim. 12 (6),845847. Bromide 4 is reported in this paper, but without experimental details. (6) Field, L., and Ravichandran, R. (1979) Organic Disulfides and Related Substances. 42. Synthesis and Properties of Some Tertiary Disulfides, Especially Involving Penicillamine. J . Org. Chem. 44 (15),2624-2629. (7) Surrey, A. R., and Lindwall, H. G. (1940) The reaction of 2-Chloro-5-nitropyridine and Thiourea. J. Am. Chem. SOC. 62,1697-1698.
Robert R. Webb II**+and Takushi Kaneko$ Department o f Antitumor Chemistry, Bristol-Myers-Squibb, Pharmaceutical Research and Development Division, 5 Research Parkway, Wallingford, Connecticut 06492- 7660 February 12, 1990 Current address: Medicinal and Biomolecular Chemistry Department, Genentech Inc., 460 Pt. San Bruno Blvd., South San Francisco, CA 94038. Current address: Pfizer Central Research, Groton, CT 06430.
