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[72] Synthesis of acetylated derivatives of polyamines
420
ANALOGS AND DERIVATIVES
[72_]
Synthesis of CNCH2CH2NH(CH2)4NHCH2CH2CN" 2HCI. s This was prepared from 1,4-diaminobutane and acrylonitrile by a modification of the methods of Schultz m and of Jackson and Rosenthal21 Technical acrylonitrile (51 ml; 770 mmol), used without further purification, was added over a 2-min period to a mixture of 30 ml (about 300 mmol) of technical 1,4-diaminobutane (Aldrich) and 300 ml of 97-98% ethanol at room temperature. After 72 hr at room temperature, the solution was cooled to 0°, and cold 6 N HC1 (approximately 100 ml) was added until the pH was approximately 1-2. A heavy white precipitate formed immediately; after several hours at 0°, the precipitate was collected by filtration and washed with cold 95% ethanol. The precipitate was then dissolved in 150 ml of hot H20 and recrystallized by the addition of 500 ml of hot absolute ethanol; on cooling at - l 0 °, crystals of the desired nitrile formed. The crystals were collected by filtration, and dried under vacuum; yield, 45 g (170 mmol); mp 225-227 ° (capillary; decomposition). Additional material could be obtained from the mother liquors after evaporation. These samples were then fractionally recrystallized from ethanol-water. Synthesis of HOOCCH2CH2NH(CH2)4NHCH2CH2COOH • 2HCl. The above dinitrile, 29 g (1 l0 mmol), and 6 N HC1, 150 ml, were refluxed for 6 hr. The mixture was then evaporated to dryness in a flash evaporator. This material was then purified by fractional crystallization from ethanol-water; mp 221-222 ° (capillary, decomposition). l0 H. P. Schultz, J. Am. Chem. Soc. 70, 2666 (1948). n E. L. Jackson and S. M. Rosenthal, J. Org. Chem. 25, 1055 (1960).
[72] S y n t h e s i s o f A c e t y l a t e d D e r i v a t i v e s o f P o l y a m i n e s
By HERBERT TABOR and CELIA WHITE TABOR Monoacetyl-l,3-diaminopropane Monohydrochloride 1,2 Monoacetyl-l,3-diaminopropane monohydrochloride is used as a starting material for the synthesis of N-monoacetylspermidine (see below). The synthesis of monoacetylputrescine hydrochloride was described in this series, Vol. 17B [256]. Monoacetylcadaverine hydrochloride can also be prepared by a comparable procedure. We found, however, that the protocol listed did not completely r e m o v e the unacetylated cadaverine in the 2-propanol step. Therefore, the extract was chromatographed on D o w e x 50-X2, and eluted with 0.4 N HCI. (The column size for a synthesis from 24.9 g of
METHODS IN ENZYMOLOGY,VOL. 94
Copyright © 1983by AcademicPress, Inc. AII rights of reproduction in any form reserved. ISBN 0-12-181994-9
[72]
SYNTHESIS OF ACETYLATED POLYAMINE DERIVATIVES
421
1,3-Diaminopropane (86.1 g, 1.16 mol) is cooled in ice and added to 200 ml of glacial acetic acid with magnetic stirring and cooling. Then 95 ml of acetic anhydride (1 mol) are added dropwise with magnetic stirring over a period of 1 hr; during this time the temperature is maintained at 5073°. The mixture is stored for 3 days at room temperature and then evaporated to dryness in v a c u o . The residual oil is dissolved in 250 ml of 5 N HC1, and the solution is again evaporated to dryness. The residue is refluxed with 300 ml of 2-propanol, and filtered to remove diaminopropane dihydrochloride. Monoacetyl-l,3-diaminopropane monohydrochloride crystallizes upon cooling. Additional crystalline material can be obtained upon concentration of the mother liquor. The crystals can be recrystallized from a large volume of hot 2-propanol. The yield is 60.6 g (34%). N1-Monoacetylspermidine 3
N~-Acetylspermidine is synthesized by treatment of N-monoacetyl1,3-diaminopropane with 4-bromobutyronitrile, followed by catalytic reduction. 4 To 1.45 g of N-acetyl-l,3-diaminopropane hydrochloride are added 45 ml of absolute ethanol and 1.5 g of K 2 C O 3 . A solution of 1.47 g of 4-bromobutyronitrile in 20 ml of absolute ethanol is added dropwise with magnetic stirring. After I hr at room temperature, the mixture is refluxed for 12 hr. After the solution is cooled, 100 ml of absolute ethanol, 0.7 ml of concentrated H 2 8 0 4 , and 300 mg of PtO2 are added; hydrogenation is carried out for 2½ hr at room temperature at atmospheric pressure. The catalyst is removed by filtration; the solution is diluted with 900 ml of water and passed through a Dowex 50-X2 column (H + form; 2 × 20 cm). The column is washed with water and 250 ml of 0.5 N HC1. The acetylspermidine is then eluted with 500 ml of 1 N HCI; the solution is evaporated to dryness in v a c u o , and N~-monoacetylspermidine dihydrochloride 5 is crystallized from 2-propanol (yield 590 mg). cadaverine was 5 x 13.5 cm). The fractions with monoacetylcadaverine were identified by chromatography on paper with a solvent containing l-butanol, 2; acetic acid, 1; pyridine, 0.5; water, 1 (Rf for cadaverine is 0.43; R I for monoacetylcadaverine is 0.71). These fractions were dried, then recrystallized from alcohol-ether. 3 We have used the following numbering system as discussed in this series, Vol. 17B [256]: 1
2
3
4
5
6
7
8
NH2CH2CH2CH:NHCH2CH2CH2CH2NHv 4 H. Tabor and C. W. Tabor, J. Biol. Chem. 250, 2648 (1975). This method of synthesis of Nl-monoacetylspermidine is superior to the less specific procedure that we described in this series, Vol. 17B [256]. The latter procedure sometimes results in mixtures of the N Iand the NS-monoacetyl derivatives. 5 A method for the unambiguous synthesis of NS-monoacetylspermidine dihydrochloride was presented in this series, Vol. 17B [256].
422
ANALOGS AND DERIVATIVES
[73]
Diacetyl-1,6-Diaminohexane 6
1,6-Diaminohexane(320 ml) is cooled and mixed with 500 ml of cooled glacial acetic acid. Then 720 ml of acetic anhydride are added dropwise with stirring. The mixture is stored overnight at room temperature and finally heated for 2 hr at 60°. Water (500 ml) is added, and the mixture is evaporated to dryness on a steam bath to a volume of less than 500 ml. After cooling, crystals of diacetyl-l,6-diaminohexane appear; they are collected by filtration. Additional crystals can be obtained from the mother liquor (yield 260 g). The crystals can be recrystallized from hot water or hot acetone (mp 125-126°). A similar method can be used to prepare diacetylputrescine. 6 Diacetyl-1,6-diaminohexane and diacetyl-1,4-diaminobutane are often referred to as hexamethylene bisacetamide and tetramethylene bisacetamide, respectively. These compounds are of special interest, as they are capable of inducing hemoglobin formation in Friend erythroleukemia cells 7 and of inducing differentiation of neuroblastoma cells. 8 They also induce procollagen formation in malignant mesenchymal cells. 9 7 R. C. Reuben, R. C. Wife, R. Breslow, R. A. Rifkind, and P. A. Marks, Proc. Natl. Acad. Sci. U.S.A. 73, 862 (1976). 8 C. Palfrey, Y. Kimhi, U. Z. Littauer, R. C. Reuben, and P. A. Marks, Biochem. Biophys. Res. Commun. 76, 937 (1977). 9 A. S. Rabson, R. Stern, T. S. Tralka, J. Costa, and J. Wilczek, Proc. Natl. Acad. Sci. U.S.A. 74, 5060 (1977).
[73] a - P u t r e s c i n y l t h y m i n e By A. M. B. KROPINSKI,K. L. MALTMAN,and R. A. J. WARREN
The hypermodified pyrimidine t~-putrescinylthymine replaces half the thymine residues in the DNA of bacteriophage 4~W-14.1,2 O
H
H N ~ C . . c..CH2 --N --CH z --CH 2 --CH z - C H z --NH 2 J
lJ
o%C-.N/CH H
i A. M. B. Kropinski and R. A. J. Warren, J. Gen. Virol. 6, 85 (1970). z A. M. B. Kropinski, R. J. Bose, and R. A. J. Warren, Biochemistry 12, 151 (1973).
METHODS IN ENZYMOLOGY, VOL. 94
Copyright © 1983by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181994-9
ANALOGS AND DERIVATIVES
[72_]
Synthesis of CNCH2CH2NH(CH2)4NHCH2CH2CN" 2HCI. s This was prepared from 1,4-diaminobutane and acrylonitrile by a modification of the methods of Schultz m and of Jackson and Rosenthal21 Technical acrylonitrile (51 ml; 770 mmol), used without further purification, was added over a 2-min period to a mixture of 30 ml (about 300 mmol) of technical 1,4-diaminobutane (Aldrich) and 300 ml of 97-98% ethanol at room temperature. After 72 hr at room temperature, the solution was cooled to 0°, and cold 6 N HC1 (approximately 100 ml) was added until the pH was approximately 1-2. A heavy white precipitate formed immediately; after several hours at 0°, the precipitate was collected by filtration and washed with cold 95% ethanol. The precipitate was then dissolved in 150 ml of hot H20 and recrystallized by the addition of 500 ml of hot absolute ethanol; on cooling at - l 0 °, crystals of the desired nitrile formed. The crystals were collected by filtration, and dried under vacuum; yield, 45 g (170 mmol); mp 225-227 ° (capillary; decomposition). Additional material could be obtained from the mother liquors after evaporation. These samples were then fractionally recrystallized from ethanol-water. Synthesis of HOOCCH2CH2NH(CH2)4NHCH2CH2COOH • 2HCl. The above dinitrile, 29 g (1 l0 mmol), and 6 N HC1, 150 ml, were refluxed for 6 hr. The mixture was then evaporated to dryness in a flash evaporator. This material was then purified by fractional crystallization from ethanol-water; mp 221-222 ° (capillary, decomposition). l0 H. P. Schultz, J. Am. Chem. Soc. 70, 2666 (1948). n E. L. Jackson and S. M. Rosenthal, J. Org. Chem. 25, 1055 (1960).
[72] S y n t h e s i s o f A c e t y l a t e d D e r i v a t i v e s o f P o l y a m i n e s
By HERBERT TABOR and CELIA WHITE TABOR Monoacetyl-l,3-diaminopropane Monohydrochloride 1,2 Monoacetyl-l,3-diaminopropane monohydrochloride is used as a starting material for the synthesis of N-monoacetylspermidine (see below). The synthesis of monoacetylputrescine hydrochloride was described in this series, Vol. 17B [256]. Monoacetylcadaverine hydrochloride can also be prepared by a comparable procedure. We found, however, that the protocol listed did not completely r e m o v e the unacetylated cadaverine in the 2-propanol step. Therefore, the extract was chromatographed on D o w e x 50-X2, and eluted with 0.4 N HCI. (The column size for a synthesis from 24.9 g of
METHODS IN ENZYMOLOGY,VOL. 94
Copyright © 1983by AcademicPress, Inc. AII rights of reproduction in any form reserved. ISBN 0-12-181994-9
[72]
SYNTHESIS OF ACETYLATED POLYAMINE DERIVATIVES
421
1,3-Diaminopropane (86.1 g, 1.16 mol) is cooled in ice and added to 200 ml of glacial acetic acid with magnetic stirring and cooling. Then 95 ml of acetic anhydride (1 mol) are added dropwise with magnetic stirring over a period of 1 hr; during this time the temperature is maintained at 5073°. The mixture is stored for 3 days at room temperature and then evaporated to dryness in v a c u o . The residual oil is dissolved in 250 ml of 5 N HC1, and the solution is again evaporated to dryness. The residue is refluxed with 300 ml of 2-propanol, and filtered to remove diaminopropane dihydrochloride. Monoacetyl-l,3-diaminopropane monohydrochloride crystallizes upon cooling. Additional crystalline material can be obtained upon concentration of the mother liquor. The crystals can be recrystallized from a large volume of hot 2-propanol. The yield is 60.6 g (34%). N1-Monoacetylspermidine 3
N~-Acetylspermidine is synthesized by treatment of N-monoacetyl1,3-diaminopropane with 4-bromobutyronitrile, followed by catalytic reduction. 4 To 1.45 g of N-acetyl-l,3-diaminopropane hydrochloride are added 45 ml of absolute ethanol and 1.5 g of K 2 C O 3 . A solution of 1.47 g of 4-bromobutyronitrile in 20 ml of absolute ethanol is added dropwise with magnetic stirring. After I hr at room temperature, the mixture is refluxed for 12 hr. After the solution is cooled, 100 ml of absolute ethanol, 0.7 ml of concentrated H 2 8 0 4 , and 300 mg of PtO2 are added; hydrogenation is carried out for 2½ hr at room temperature at atmospheric pressure. The catalyst is removed by filtration; the solution is diluted with 900 ml of water and passed through a Dowex 50-X2 column (H + form; 2 × 20 cm). The column is washed with water and 250 ml of 0.5 N HC1. The acetylspermidine is then eluted with 500 ml of 1 N HCI; the solution is evaporated to dryness in v a c u o , and N~-monoacetylspermidine dihydrochloride 5 is crystallized from 2-propanol (yield 590 mg). cadaverine was 5 x 13.5 cm). The fractions with monoacetylcadaverine were identified by chromatography on paper with a solvent containing l-butanol, 2; acetic acid, 1; pyridine, 0.5; water, 1 (Rf for cadaverine is 0.43; R I for monoacetylcadaverine is 0.71). These fractions were dried, then recrystallized from alcohol-ether. 3 We have used the following numbering system as discussed in this series, Vol. 17B [256]: 1
2
3
4
5
6
7
8
NH2CH2CH2CH:NHCH2CH2CH2CH2NHv 4 H. Tabor and C. W. Tabor, J. Biol. Chem. 250, 2648 (1975). This method of synthesis of Nl-monoacetylspermidine is superior to the less specific procedure that we described in this series, Vol. 17B [256]. The latter procedure sometimes results in mixtures of the N Iand the NS-monoacetyl derivatives. 5 A method for the unambiguous synthesis of NS-monoacetylspermidine dihydrochloride was presented in this series, Vol. 17B [256].
422
ANALOGS AND DERIVATIVES
[73]
Diacetyl-1,6-Diaminohexane 6
1,6-Diaminohexane(320 ml) is cooled and mixed with 500 ml of cooled glacial acetic acid. Then 720 ml of acetic anhydride are added dropwise with stirring. The mixture is stored overnight at room temperature and finally heated for 2 hr at 60°. Water (500 ml) is added, and the mixture is evaporated to dryness on a steam bath to a volume of less than 500 ml. After cooling, crystals of diacetyl-l,6-diaminohexane appear; they are collected by filtration. Additional crystals can be obtained from the mother liquor (yield 260 g). The crystals can be recrystallized from hot water or hot acetone (mp 125-126°). A similar method can be used to prepare diacetylputrescine. 6 Diacetyl-1,6-diaminohexane and diacetyl-1,4-diaminobutane are often referred to as hexamethylene bisacetamide and tetramethylene bisacetamide, respectively. These compounds are of special interest, as they are capable of inducing hemoglobin formation in Friend erythroleukemia cells 7 and of inducing differentiation of neuroblastoma cells. 8 They also induce procollagen formation in malignant mesenchymal cells. 9 7 R. C. Reuben, R. C. Wife, R. Breslow, R. A. Rifkind, and P. A. Marks, Proc. Natl. Acad. Sci. U.S.A. 73, 862 (1976). 8 C. Palfrey, Y. Kimhi, U. Z. Littauer, R. C. Reuben, and P. A. Marks, Biochem. Biophys. Res. Commun. 76, 937 (1977). 9 A. S. Rabson, R. Stern, T. S. Tralka, J. Costa, and J. Wilczek, Proc. Natl. Acad. Sci. U.S.A. 74, 5060 (1977).
[73] a - P u t r e s c i n y l t h y m i n e By A. M. B. KROPINSKI,K. L. MALTMAN,and R. A. J. WARREN
The hypermodified pyrimidine t~-putrescinylthymine replaces half the thymine residues in the DNA of bacteriophage 4~W-14.1,2 O
H
H N ~ C . . c..CH2 --N --CH z --CH 2 --CH z - C H z --NH 2 J
lJ
o%C-.N/CH H
i A. M. B. Kropinski and R. A. J. Warren, J. Gen. Virol. 6, 85 (1970). z A. M. B. Kropinski, R. J. Bose, and R. A. J. Warren, Biochemistry 12, 151 (1973).
METHODS IN ENZYMOLOGY, VOL. 94
Copyright © 1983by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181994-9