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Synthesis of certain derivatives of thiols*,†
Scientific Edition
J O U R N A L O F THE AMERICAN PHARMACEUTICAL ASSOCIATION JUSTIN
L. POWERS, EDITOR,WASHINGTON, D. C.
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VOLUMEXXXVIII
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NUMBER 7 CONSECUTIVE No. 14
JULY, I949
Synthesis of Certain Derivatives of Thiols*J By JOHN W. BOENIGK,$ JOHN E. CHRISTIAN, Seven aromatic thiols have been used to prepare two series of sulfides. Thiols used were o-thiophenol, o-thiocresol, m-thiocresol, p-thiocresol, o-chlorobenzyl thiol, 2,4-dichlorobenzyl thiol, and 3,4-dichlorobenzyl thiol. A series was prepared of 3-diethylaminopropyl derivatives and 3:diethylamino2-propanol derivatives. The compounds are analogous to Benadryl and Pyribenzamine type compounds with sulfur replacing the oxygen and nitrogen, respectively. The general structure of the molecule differs from these antihistamine drugs in that only one aryl group is present instead of the diaryl groups of Benadryl and Pyribenzamine. The aryl structure was varied to show the influence of substituent groups, in order that some of the thiols used would contain chlorine on the ring; others would contain methyl groups. HE PURPOSE of
the research project was to synthesize sulfur compounds analogous to Benadryl and Pyribenzamine type compounds with sulfur replacing the oxygen and nitrogen linkage, respectively. The general structure of
L. JENKINS
the molecule differs from these antihistamine drugs in that only one aryl group is present instead of the diaryl groups of Benadryl and Pyribenzamine. The aryl structure was varied to show the influence of substituent groups, in order that some of the thiols used would contain chlorine on the ring; others would contain methyl groups. It was planned to prepare two general groups of thiol derivatives, one in which the substituent was 3-diethylaminopropyl, and the other in which the substituent was 3-diethylamino-2propanol. The diethylaminopropyl substituent has been shown to have neurotropic activity (1) and the sulfur linkage to have musculotropic activity (2), the combination of the side chain and the sulfur may have desirable antispasmodic or antihistamine activity. The purpose of the second side chain was to test the influence of such a secondary alcohol. METHOD OF PREPARATION
*
Received April 4, 1949. from the Research Laboratories, Purdqe University School of Pharmacy Lafayette Ind. Presented to the'Scientific Section, A.'Pa. A,, Jarksonville meeting, April, 1949. t The work reported in this paper was made possible through a grant from the American Foundation for Pharmaceutical Education. $ Present address: The University of Texas, School of Pharmacy, Austin, Tcx.
a n d GLENN
The thiols used in the research project were either commercially available or were synthesized by either reduction of the corresponding disulfide or reaction of an appropriate benzyl chloride with thiourea (3). (See Tables I and 11.)
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AMERICANPHARMACEUTICAL ASSOCIATION
JOURNAL OF THE
The 3-diethylaminopropyl dei ivatives were prepared according t o the following sequence of reactions :
+ + +
KOH + Ar-S-K f H20 1. Ai-SH 2. Ar-S-K Cl-CHZ-CHZ-CHZN(C2Hs)?-HC1 KOH Ar-ELCHzCHz-CHz-N(CzH5)z 2KC1+ HzO 3. Ar-S-CHz-CHz-CHz-N(C?Hs)z HCI Ar-S-CHe-CH2-CH2-N(C&)Hz-Cl -.f
+
+
-.f
The 3-diethylamino-2-propanolderivatives were prepared as follows : 1. Ar-SH
2. Ar-SNa
+ NaOH Ar-SNa + He0 Hz H Hz + C-C-C-N-(C~H,)Z + NdOH -.f
\/ 0
-*
Ar-S-CH%-CH-CHZ-N(
CzH&
1
+ NaOH
OH The compounds listed in T a b k I11 were all prepared by the same method. The thiol (0.1 mole) was dissolved in 50 cc. methyl alcohol and a solution
of KOH 14.4 Gm. (0.2 mole plus 10% excess) in 50 cc. methyl alcohol added. This mixture was refluxed one-half hour. The 1-diethylamino-3-chloropropane HC1 (18.6 Gm.) (0.1 mole) was dissolved in 25 cc. methyl alcohol and added slowly t o the refluxing mixture. The refluxing w+s continued for four hours, the solution cooled and filtered through a Biichner funnel. The methyl alcohol was removed by distillation at atmospheric pressure and the residual oil washed with water and dissolved in benzene. The benzene solution was dried with anhydrous Na2S04, the benzene removed by vacuum distillation, and the residual oil distilled under reduced pressure. The 1-diethylamino-3-chloropropanewas prepared by two methods (8, 9). Method A.-Diethylamine 117 Gm. (1.6 mole) was added slowly t o trimethylene chlorobromide 126 Gm. (0.8 mole) while the reaction mixture was maintained a t 30”. The mixture was allowed t o react a t 35-40” for three hours, and stand overnight. The salt formed (diethylamine HBr) was removed by washing with 80 cc cold water. The residual oil was added t o 500 cc. cold 2 N HCl and the unreacted trimethylene chlorobromide removed with ether. The acid solution was made alkaline with K2C03,
TABLE I.-DISULFIDES PREPARED Ohtd.”
2,2’-Dichloro-dibcnzyl disulfide 2,2’,4,4’-Tetrachloro-dibenzyl disulfide 3,3’,4,4’-Tetrachloro-dibenzyl disulfide
M.P,OC.
88-89 7 1-72 91-92
Lit.
Obtd.
89-90 (4) 74-75 (4) 94-95 (4)
75 70 72
yo Yield
Lit.
96 84 89
Uncorrected. Corrected. The disulfides were prepared by the procedure described by Christian el at. (4): 1. 2 Z’-Dichloro-dibenzyl disulfide: o-Chlorobenzyl chloride (30 Gm.) was dissolved in 150 cc. refluxing ethyl alcohol. To the hot alcoholic solution a warm alcoholic solution of NazSz, prepared by dissolving 21.8 Gm. of Nad39HrO and 2.9 Gm. of sulfur in 210 cc. of hot ethyl alcohol, was added slowly. The refluxing was continued for 4 hr., the mixture filtered through a Biichner funnel, and the filtrate allowed t o cool. The precipitate was recrystallized from ethyl alcohol. 2. 2 2’ 4 4’-Tetrachloro-dibenzyl disulfide and 3,3’ 4 4’-tetrachloro-dibenzyl disulfide were prepared b y the same procedure &hi the corresponding dichlorobenzyl chloride t3b Gm.), Na&SHzO (17.9 Gm.) and sulfur (2.4 Gm.). a
TABLE II.-THIOLS PREPARED
o-Chlorobenzyl thiol 2.4-Dichlorobenzyl thiol 3,4-Dichlorobenzyl thiol
Method Used
Yield, %
A B A B A B
Quant. 73 Quant. 68 Quant. .78
B. P.,
C./Mm.
120-121/25 100-104/13 151-152/29 133-135/16 170-171 /31 139-141713
Refractive Index, O C.
3 5-Dinitrobenzoate Esters M. P., C.“
1.5650/28 1.5658/28 1.5993/29 1.5992/29 1.6000/29 i.6012’/29
112.5-113.5 112.5-113.5 110.5-111.5 110.5-111.5 97.0- 98.0 97.0- 98.C
Uncorrected. The thiols were prepared either by (A) reduction of corresponding disulfides using zinc and hydrochloric acid, or (B)reaction of appropriate henzyl chloride with tluourea (3). Both methods were used for all three thiols. Melhod A-Reduction of Disul$de.-A modification of t h e procedure b y Allen, et ol. (5) was used. The appropriate disulfide (50-60 Gm.) was dissolved in 200 cc. of glacial acetic acid. T h e solution was heated t o boiling and 30 Gm. of zinc dust and 90 cc. of concentrated hydrochloric acid were added in divided portions so as t o maintain a rapid evolution of hydrogen at all times. When all of the zinc was dissolved, a sample was removed and diluted with water. If the diluted sample formed a clear solution with sodium hydroxide solution, the reaction was discontinued, and the warm solution filtered through a Bdchner funnel. If the diluted sample did not form a clear solution, additional amounts of zinc a n d hydrochloric acid were added until i t did so. The filtrate was diluted with twice its volume of distilled water, and the thiol layer which separated, removed by means of a separatory funnel and the aqueous portion extracted with ether. The ether was evaporated on a w.ater bath and the residue combined with the crude thiol and dried with anhydrous sodium sulfate. The dried thiol was distilled under reduced pressure. Melhod B-Thiourea Method.-A modification described b y Urquhart, d al. (3) was used. A mixture of o-chlorobenzyl chloride 79.3 Gm. (0.5 mole), thiourea 38 Gm. (0.5 mole), and ethyl alcohol 250 cc. was refluxed on a steam bath for 12 hr. A solution of NaOH 30 Gm. (0.75 mole) in 300 cc. of water was added and the mixture refluxed an additional 4 hr. The thiol layer was separated’and the aqueous layer was acidified with dilute shfuric acid (7 cc. conc. HzSOd t o 50 cc. water), and extracted with 75 cc. benzene. The benzene extract and the crude thiol were combined and washed with water, then dried with anhydrous sodium sulfate. The benzene was removed by evaporation, and the thiol distilled under reduced pressure. The 3,5-dinitrobenzoate esters of the thiols were prepared b y a method given b y Wertheim (12). I n a test tube were placed 2.3 Gm. (0.01 mole) of 3,5-dinitro-benzoyl chloride, (0.015 mole) of the thiol, and 4 drops of pyridine. The test tube was heated until fumes of HCI ceased to appear (about 10 min.). In certain cases i t was necessary to heat t h e test tube in a steam bath for a n additional 10 min. A few drops of water were added to the mixture, then an excess ofpyridine, and the mass stirred vigorously until i t solidified. The reaction mixturc was then filtered and dried on a porous plate. m t e r i a l was recrystallized from dilute ethyl alcohol.
SCIENTIFIC EDITION
359
TABLE III.-DIETHYLAMINOPROPYL SULFIDE HYDROCHLORIDES Thiol Used
B. P., C./Mm. Amine
o-Chbrobenzyl thiol 2,4-Dichlorobenzyl thiol 3,4-Dichlorobenzyl thiol ThioDhenol o-Th:ocresol m-Thiocresol D-Thiocresol
Yield Amine, Gm.5
190-193/12
20
165-168/4 169-172/3 141-143/6 150-15315 168-173/13 155-158/7
M. P., C b Amine HC1
(80%)
--Nitrogenb--Calcd. Found
r - C I c as HCICalcd. Found
83-84
4.54
4.46
11.49
11.45
22.5 (73%)
116-117
4.08
4.02
10.31
10.29
24.9 (81%) 18.1 (81%) 16 9 (71%) 20 8 (88%) 19 4 (81%)
75-76 61-63 126-127 65-66 97-98
4.08 6.26 5 11 5 90 5 11
4.03 5.90d 4 93 5 41d 5 08
10.31 13.67 12 95 12 95 12.95
10.24 13.15 12.98 12 52 12 90
Recrystallizing solvent: ethyl acetate. * Chlorine Nitrogen determined by a semimicro Kjeldahl method (7). determined by the method described by Blicke and Zienty (6).
a
d Amine hydrochlorides were very hygroscopic, N analysis made on free base.
the oil separated, and the aqueous layer extracted with ether. The combined ether extracts and oil were dried with anhydrous KzC03. After evaporation of the ether, the residual oil was distilled under reduced pressure. The yield obtained was 59 Gm. (49% theoretical), the boiling point was 68-70 O a t 20 mm. Method B.-A solution of y-diethylaminopropanol 78.5 Gm. (0.6 mole) in 130 cc. of chloroform was added slowly with cooling t o a solution of thionyl chloride 145 Gm. (1.21 mole) in 600 cc. of chloroform. The mixture was refluxed for three hours, after which the solvent and excess thiouyl chloride were removed by distillation. The residue was treated with 150 cc. of a 40% solution of NaOH and extracted with 1000 cc. of ether. The ether solution was dried over anhydrous Na2S04 and the ether evaporated. The residue distilled as a colorless oil 62-64' a t 19 mm., yield 79.3 Gm. (88%theoretical). Gilman and Shirley (9) reported a yield of 730/, theoretical, boiling point 73-75' at 20 mm. l-Diethylamino-2,3-epoxypropanewas also prepared by two methods (10, 11). Method A.-A mixture of epichlorohydrin (46.3 Gm.), diethylamine (30.0 Gm.) and water (1.0 Gm.) was well mixed while maintaining the temperature a t 28-30 O for four hours. The resultant oil was shaken with 50 cc. 20% K~COX, then with 60 cc. of a 35% NaOH solution for one hour. The oil was then shaken with 50 cc. of a 50% KOH solution. The resultant oil after separation from the KOH solution was distilled under reduced pressure. The yield obtained was 35.8 Gm. (55.4% theoretical). The boiling point of the product was 40-50" a t 8 mm. Method B.-A mixture of epichlorohydrin (111.2 Gm.), diethylamine (86.4 Gm.), and 3.6 C h . water was stirred vigorously for six hours maintain-
ing the temperature of the reaction mixture at 2830". The reaction mixture was then cooled t o 20" and a solution of NaOH (50 Gm. in 91 cc. H20) was added slowly, meanwhile maintaining vigorous stirring. The reaction mixture was stirred for a n additional forty minutes and poured into 200 cc. water. The top layer was separated and the aqueous layer extracted with ether. The ether extracts plus the oil were dried over KOH pellets. The ether layer was separated from the KOH and the ether removed by evaporation. The residual oil distilled a t 53-56' at 17 mm., the yield being 75 Gm. (50% of theoretical). Gilman reported a 62% yield and the boiling point as 62-65" at 20 mm. The compounds listed in Table IV were prepared as fOllOW5: The sodium hydroxide 8.8 Gm. (0.2 mole plus 10% excess) was dissolved in 50 cc. of water and the thiol (0.2 mole) added t o the NaOH solution. The l-diethylamino-2,3-epoxypropane 25.8 Gm. (0.2 mole) was dissolved in 50 cc. of water, and added slowly with vigorous stirring t o the thiolNaOH solution. The mixture was stirred for an additional four hours, and the resultant oily product after being washed with water, was dissolved in 50 cc. of benzene. The benzene solution was dried with anhydrous sodium sulfate and the benzene removed by evaporation under reduced pressure. The residual oil was distilled under reduced pressure. All attempts t o convert these amines t o crystalline hydrochlorides failed.
SUMMARY
1. Seven diethylaminopropyl and seven diethylaminohydroxypropyl derivatives of thiols have been prepared and characterized.
TABLE IV.-1-DIETHYLAMINO-2-HYDROXY-PROPYLSULFIDES Thiol Used
o-Chlorabenzyl thiol 2,4-Dichlorobenzyl thiol 3,4-Dichlorobenzyl thiol Thiophenol o-Thiocresol m-Thiocresol p-Thiocresol
B. P.,
O
C./Mm.
190-195/4 210-215/4 210-215/4 168-171/4 154-157/1 168-172/3 168-172/3
Yield Amine, Gm.
17 42.8 44.3 41.3 35.6 32.0 35.0
(60%)b (66%) (69%) (86%) (65%) (60%) (64%)
Nitrogen determined by semimicro Kjeldahl method (7). b Made using 0.1 molar quantities, others made using 0.2 molar quantities.
--Nitrogen'Calcd.
Found
4.85 4.34 4.34 5.85 5.50 5.50 5.50
4.56 4.23 4.14 5.79 5.32 5.37 5.28
Refractive Index (/" C.) Amine
1.5435/30 1.5522/27 1.5515/30 1.5412/25 1.5408/30 1.5376/30 1.5371/30
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JOURNAL OF THE
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2* Sufficient quantities Of these have been prepared for pharmacological study. REFERENCES ( 1 ) Halpern, B. N., Arch. intern. pharmacodynamic, 59, 149 (1938). (2) Burnter, R. R., and Cusic, J. W., J . A m . Chem. Soc., 65, 262(1943). (3) Urquhart, G;,G., Gates, J. W., Jr., and Connor. R . , “Organic Syntheses, John Wiley and Sons, Inc.. New York, Vol. 21 1941 p. 36. (4) ’ChriGian J. E. Jenkins G. L. Keagle, L. C., and Crum, J. A,, TH& JO&AL, 35,’328(1946).
( 5 ) All:& C. P. H., and MacKay, D. D., “Organic Syntheses, John Wiley and Sons, Inc.. New York. Coll., vol.11, 1943, P. 580. ( 6 ) Blicke, F. F., and Zienty, F. B., J. A m . Chem. SOC., 61,776(1939). (7) “The Hengar Technique for the Kjeldahl Procedure,” Hengar Company, Philadelphia, Pa. ( 8 ) Breslow D. S. Walker H. G. Yost R. S., and Hauser. C. R., j . A m . khem. So;., 67, 14?4(194$. (9) Gilman, H., and Shirley D. A., ibid., 66, 888(1944). (10) Eisleb, O., Substituted ‘ 1,3-diamino-2-propanols to Winthrop Chemical Company, New York, N. Y., U. S. Patent No. 1,790,042, Jan. 27, 1931; Chcm. Abstr.. 25, 1259 (1931). (11) Gilman, H., cf al., J . A m . Chem. Soc., 68, 1291(1946). (12) Wertheirn. E., ibid.. 51, 3661(1929).
Assay of Heparin. 111. Measurement of the End Point by Physical Means. Influence of Surface on Clotting*J By R. H. K. FOSTER In the assay of heparin using beef or sheep plasma the degree of clotting has been estimated by visual inspection. This estimate, visual density, has been correlated with light transmission as measured photoelectrically and with the weights of dried clots. Good agreement was obtained. At times the surface of thoroughly cleaned and stored Pyrex tubes interfered with the aa’on of heparin in some unknown manner so that very irregular end-point responses were obtained. Concentration-response clotting curves obtained using freshly cleaned .Pyrex glass tubes were similar to those obtained using siliconecoated glass tubes or cellulose nitrate tubes. method previously described (I) the end point was considered t o be that amount of heparin which would just permit the formation of a 50 per cent clot. Since a 50 per cent clot is seldom obtained in any tube, the concentration of heparin corresponding to a 50 per cent clot must be calculated by interpolation from a clotting curve. The degree of clotting in the various tubes has always been estimated visually. I t is the purpose of this paper to correlate visually estimated degrees of opacity with opacity measured photoelectrically and with the weights of dried clots. In the course of this work some very marked irregularities were observed when clotting took place in Pyrex glass tubes but were completely eliminated when clotting was allowed to proceed in silicone-coated Pyrex tubes or in celN THE ASSAY
I
*
Received Nov. 10, 1948, from the Department of Pharmacology, St. Louis University School of Medicine, St. Louis, Mo. Supported in part by a grant-in-aid from the U. S. Public Health Service t The heparin used in this investigation was kindly supplied by Hoffmann-LaRoche, Inc., Nutley, N . J.
lulose nitrate tubes. A preliminary report of this investigation has been made elsewhere (2). MATERIALS, CRITERIA, AND PROCEDURES Opacity determined by inspection is termed visual density. This, representing the degree of coagulation, was estimated in terms of an arbitrary scale, 0 to 4+, with several intermediate values (1). The transmission of light, here measured photoelectrically, is a reciprocal function of opacity, the latter reflecting the degree of coagulation. Results are reported in terms of per cent transmission of light since this simplified the plotting of curves. However, for convenience, the term optical density is employed. Since optical density is -log 1/10the light transmission scale has been inverted to make these curves parallel to those for visual density and clot weight. The instrument accounts for light absorbed (or scattered) by the plasma as well as by the clot itself in contrast to the eye which discounts the plasma color when inspecting for visual density. In reporting the weights of dried clots it is not presymed that the weights represent pure fibrin. Highspeed centrifuging was employed to compact the fibrin clots but it is more than probable that the clots still contained some plasma and/or serum which when dried would add to the weight of the dried fibrin. Certain characteristics of the clot such as size and number of fibrin fibrils, their specific gravity, and their “malleability” must influence the ease of packing during centrifugalization and hence entrained material. It is probable that different concentrations of heparin alter these factors to such an extent that the proportionate amount of packing varies and the ratio of the amount of fibrin present to the entrained impurities is not strictly constant. Nevertheless, it is assumed that the clot weights as determined represent reasonable parallelism to the “true” weight of the fibrin.
J O U R N A L O F THE AMERICAN PHARMACEUTICAL ASSOCIATION JUSTIN
L. POWERS, EDITOR,WASHINGTON, D. C.
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VOLUMEXXXVIII
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NUMBER 7 CONSECUTIVE No. 14
JULY, I949
Synthesis of Certain Derivatives of Thiols*J By JOHN W. BOENIGK,$ JOHN E. CHRISTIAN, Seven aromatic thiols have been used to prepare two series of sulfides. Thiols used were o-thiophenol, o-thiocresol, m-thiocresol, p-thiocresol, o-chlorobenzyl thiol, 2,4-dichlorobenzyl thiol, and 3,4-dichlorobenzyl thiol. A series was prepared of 3-diethylaminopropyl derivatives and 3:diethylamino2-propanol derivatives. The compounds are analogous to Benadryl and Pyribenzamine type compounds with sulfur replacing the oxygen and nitrogen, respectively. The general structure of the molecule differs from these antihistamine drugs in that only one aryl group is present instead of the diaryl groups of Benadryl and Pyribenzamine. The aryl structure was varied to show the influence of substituent groups, in order that some of the thiols used would contain chlorine on the ring; others would contain methyl groups. HE PURPOSE of
the research project was to synthesize sulfur compounds analogous to Benadryl and Pyribenzamine type compounds with sulfur replacing the oxygen and nitrogen linkage, respectively. The general structure of
L. JENKINS
the molecule differs from these antihistamine drugs in that only one aryl group is present instead of the diaryl groups of Benadryl and Pyribenzamine. The aryl structure was varied to show the influence of substituent groups, in order that some of the thiols used would contain chlorine on the ring; others would contain methyl groups. It was planned to prepare two general groups of thiol derivatives, one in which the substituent was 3-diethylaminopropyl, and the other in which the substituent was 3-diethylamino-2propanol. The diethylaminopropyl substituent has been shown to have neurotropic activity (1) and the sulfur linkage to have musculotropic activity (2), the combination of the side chain and the sulfur may have desirable antispasmodic or antihistamine activity. The purpose of the second side chain was to test the influence of such a secondary alcohol. METHOD OF PREPARATION
*
Received April 4, 1949. from the Research Laboratories, Purdqe University School of Pharmacy Lafayette Ind. Presented to the'Scientific Section, A.'Pa. A,, Jarksonville meeting, April, 1949. t The work reported in this paper was made possible through a grant from the American Foundation for Pharmaceutical Education. $ Present address: The University of Texas, School of Pharmacy, Austin, Tcx.
a n d GLENN
The thiols used in the research project were either commercially available or were synthesized by either reduction of the corresponding disulfide or reaction of an appropriate benzyl chloride with thiourea (3). (See Tables I and 11.)
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AMERICANPHARMACEUTICAL ASSOCIATION
JOURNAL OF THE
The 3-diethylaminopropyl dei ivatives were prepared according t o the following sequence of reactions :
+ + +
KOH + Ar-S-K f H20 1. Ai-SH 2. Ar-S-K Cl-CHZ-CHZ-CHZN(C2Hs)?-HC1 KOH Ar-ELCHzCHz-CHz-N(CzH5)z 2KC1+ HzO 3. Ar-S-CHz-CHz-CHz-N(C?Hs)z HCI Ar-S-CHe-CH2-CH2-N(C&)Hz-Cl -.f
+
+
-.f
The 3-diethylamino-2-propanolderivatives were prepared as follows : 1. Ar-SH
2. Ar-SNa
+ NaOH Ar-SNa + He0 Hz H Hz + C-C-C-N-(C~H,)Z + NdOH -.f
\/ 0
-*
Ar-S-CH%-CH-CHZ-N(
CzH&
1
+ NaOH
OH The compounds listed in T a b k I11 were all prepared by the same method. The thiol (0.1 mole) was dissolved in 50 cc. methyl alcohol and a solution
of KOH 14.4 Gm. (0.2 mole plus 10% excess) in 50 cc. methyl alcohol added. This mixture was refluxed one-half hour. The 1-diethylamino-3-chloropropane HC1 (18.6 Gm.) (0.1 mole) was dissolved in 25 cc. methyl alcohol and added slowly t o the refluxing mixture. The refluxing w+s continued for four hours, the solution cooled and filtered through a Biichner funnel. The methyl alcohol was removed by distillation at atmospheric pressure and the residual oil washed with water and dissolved in benzene. The benzene solution was dried with anhydrous Na2S04, the benzene removed by vacuum distillation, and the residual oil distilled under reduced pressure. The 1-diethylamino-3-chloropropanewas prepared by two methods (8, 9). Method A.-Diethylamine 117 Gm. (1.6 mole) was added slowly t o trimethylene chlorobromide 126 Gm. (0.8 mole) while the reaction mixture was maintained a t 30”. The mixture was allowed t o react a t 35-40” for three hours, and stand overnight. The salt formed (diethylamine HBr) was removed by washing with 80 cc cold water. The residual oil was added t o 500 cc. cold 2 N HCl and the unreacted trimethylene chlorobromide removed with ether. The acid solution was made alkaline with K2C03,
TABLE I.-DISULFIDES PREPARED Ohtd.”
2,2’-Dichloro-dibcnzyl disulfide 2,2’,4,4’-Tetrachloro-dibenzyl disulfide 3,3’,4,4’-Tetrachloro-dibenzyl disulfide
M.P,OC.
88-89 7 1-72 91-92
Lit.
Obtd.
89-90 (4) 74-75 (4) 94-95 (4)
75 70 72
yo Yield
Lit.
96 84 89
Uncorrected. Corrected. The disulfides were prepared by the procedure described by Christian el at. (4): 1. 2 Z’-Dichloro-dibenzyl disulfide: o-Chlorobenzyl chloride (30 Gm.) was dissolved in 150 cc. refluxing ethyl alcohol. To the hot alcoholic solution a warm alcoholic solution of NazSz, prepared by dissolving 21.8 Gm. of Nad39HrO and 2.9 Gm. of sulfur in 210 cc. of hot ethyl alcohol, was added slowly. The refluxing was continued for 4 hr., the mixture filtered through a Biichner funnel, and the filtrate allowed t o cool. The precipitate was recrystallized from ethyl alcohol. 2. 2 2’ 4 4’-Tetrachloro-dibenzyl disulfide and 3,3’ 4 4’-tetrachloro-dibenzyl disulfide were prepared b y the same procedure &hi the corresponding dichlorobenzyl chloride t3b Gm.), Na&SHzO (17.9 Gm.) and sulfur (2.4 Gm.). a
TABLE II.-THIOLS PREPARED
o-Chlorobenzyl thiol 2.4-Dichlorobenzyl thiol 3,4-Dichlorobenzyl thiol
Method Used
Yield, %
A B A B A B
Quant. 73 Quant. 68 Quant. .78
B. P.,
C./Mm.
120-121/25 100-104/13 151-152/29 133-135/16 170-171 /31 139-141713
Refractive Index, O C.
3 5-Dinitrobenzoate Esters M. P., C.“
1.5650/28 1.5658/28 1.5993/29 1.5992/29 1.6000/29 i.6012’/29
112.5-113.5 112.5-113.5 110.5-111.5 110.5-111.5 97.0- 98.0 97.0- 98.C
Uncorrected. The thiols were prepared either by (A) reduction of corresponding disulfides using zinc and hydrochloric acid, or (B)reaction of appropriate henzyl chloride with tluourea (3). Both methods were used for all three thiols. Melhod A-Reduction of Disul$de.-A modification of t h e procedure b y Allen, et ol. (5) was used. The appropriate disulfide (50-60 Gm.) was dissolved in 200 cc. of glacial acetic acid. T h e solution was heated t o boiling and 30 Gm. of zinc dust and 90 cc. of concentrated hydrochloric acid were added in divided portions so as t o maintain a rapid evolution of hydrogen at all times. When all of the zinc was dissolved, a sample was removed and diluted with water. If the diluted sample formed a clear solution with sodium hydroxide solution, the reaction was discontinued, and the warm solution filtered through a Bdchner funnel. If the diluted sample did not form a clear solution, additional amounts of zinc a n d hydrochloric acid were added until i t did so. The filtrate was diluted with twice its volume of distilled water, and the thiol layer which separated, removed by means of a separatory funnel and the aqueous portion extracted with ether. The ether was evaporated on a w.ater bath and the residue combined with the crude thiol and dried with anhydrous sodium sulfate. The dried thiol was distilled under reduced pressure. Melhod B-Thiourea Method.-A modification described b y Urquhart, d al. (3) was used. A mixture of o-chlorobenzyl chloride 79.3 Gm. (0.5 mole), thiourea 38 Gm. (0.5 mole), and ethyl alcohol 250 cc. was refluxed on a steam bath for 12 hr. A solution of NaOH 30 Gm. (0.75 mole) in 300 cc. of water was added and the mixture refluxed an additional 4 hr. The thiol layer was separated’and the aqueous layer was acidified with dilute shfuric acid (7 cc. conc. HzSOd t o 50 cc. water), and extracted with 75 cc. benzene. The benzene extract and the crude thiol were combined and washed with water, then dried with anhydrous sodium sulfate. The benzene was removed by evaporation, and the thiol distilled under reduced pressure. The 3,5-dinitrobenzoate esters of the thiols were prepared b y a method given b y Wertheim (12). I n a test tube were placed 2.3 Gm. (0.01 mole) of 3,5-dinitro-benzoyl chloride, (0.015 mole) of the thiol, and 4 drops of pyridine. The test tube was heated until fumes of HCI ceased to appear (about 10 min.). In certain cases i t was necessary to heat t h e test tube in a steam bath for a n additional 10 min. A few drops of water were added to the mixture, then an excess ofpyridine, and the mass stirred vigorously until i t solidified. The reaction mixturc was then filtered and dried on a porous plate. m t e r i a l was recrystallized from dilute ethyl alcohol.
SCIENTIFIC EDITION
359
TABLE III.-DIETHYLAMINOPROPYL SULFIDE HYDROCHLORIDES Thiol Used
B. P., C./Mm. Amine
o-Chbrobenzyl thiol 2,4-Dichlorobenzyl thiol 3,4-Dichlorobenzyl thiol ThioDhenol o-Th:ocresol m-Thiocresol D-Thiocresol
Yield Amine, Gm.5
190-193/12
20
165-168/4 169-172/3 141-143/6 150-15315 168-173/13 155-158/7
M. P., C b Amine HC1
(80%)
--Nitrogenb--Calcd. Found
r - C I c as HCICalcd. Found
83-84
4.54
4.46
11.49
11.45
22.5 (73%)
116-117
4.08
4.02
10.31
10.29
24.9 (81%) 18.1 (81%) 16 9 (71%) 20 8 (88%) 19 4 (81%)
75-76 61-63 126-127 65-66 97-98
4.08 6.26 5 11 5 90 5 11
4.03 5.90d 4 93 5 41d 5 08
10.31 13.67 12 95 12 95 12.95
10.24 13.15 12.98 12 52 12 90
Recrystallizing solvent: ethyl acetate. * Chlorine Nitrogen determined by a semimicro Kjeldahl method (7). determined by the method described by Blicke and Zienty (6).
a
d Amine hydrochlorides were very hygroscopic, N analysis made on free base.
the oil separated, and the aqueous layer extracted with ether. The combined ether extracts and oil were dried with anhydrous KzC03. After evaporation of the ether, the residual oil was distilled under reduced pressure. The yield obtained was 59 Gm. (49% theoretical), the boiling point was 68-70 O a t 20 mm. Method B.-A solution of y-diethylaminopropanol 78.5 Gm. (0.6 mole) in 130 cc. of chloroform was added slowly with cooling t o a solution of thionyl chloride 145 Gm. (1.21 mole) in 600 cc. of chloroform. The mixture was refluxed for three hours, after which the solvent and excess thiouyl chloride were removed by distillation. The residue was treated with 150 cc. of a 40% solution of NaOH and extracted with 1000 cc. of ether. The ether solution was dried over anhydrous Na2S04 and the ether evaporated. The residue distilled as a colorless oil 62-64' a t 19 mm., yield 79.3 Gm. (88%theoretical). Gilman and Shirley (9) reported a yield of 730/, theoretical, boiling point 73-75' at 20 mm. l-Diethylamino-2,3-epoxypropanewas also prepared by two methods (10, 11). Method A.-A mixture of epichlorohydrin (46.3 Gm.), diethylamine (30.0 Gm.) and water (1.0 Gm.) was well mixed while maintaining the temperature a t 28-30 O for four hours. The resultant oil was shaken with 50 cc. 20% K~COX, then with 60 cc. of a 35% NaOH solution for one hour. The oil was then shaken with 50 cc. of a 50% KOH solution. The resultant oil after separation from the KOH solution was distilled under reduced pressure. The yield obtained was 35.8 Gm. (55.4% theoretical). The boiling point of the product was 40-50" a t 8 mm. Method B.-A mixture of epichlorohydrin (111.2 Gm.), diethylamine (86.4 Gm.), and 3.6 C h . water was stirred vigorously for six hours maintain-
ing the temperature of the reaction mixture at 2830". The reaction mixture was then cooled t o 20" and a solution of NaOH (50 Gm. in 91 cc. H20) was added slowly, meanwhile maintaining vigorous stirring. The reaction mixture was stirred for a n additional forty minutes and poured into 200 cc. water. The top layer was separated and the aqueous layer extracted with ether. The ether extracts plus the oil were dried over KOH pellets. The ether layer was separated from the KOH and the ether removed by evaporation. The residual oil distilled a t 53-56' at 17 mm., the yield being 75 Gm. (50% of theoretical). Gilman reported a 62% yield and the boiling point as 62-65" at 20 mm. The compounds listed in Table IV were prepared as fOllOW5: The sodium hydroxide 8.8 Gm. (0.2 mole plus 10% excess) was dissolved in 50 cc. of water and the thiol (0.2 mole) added t o the NaOH solution. The l-diethylamino-2,3-epoxypropane 25.8 Gm. (0.2 mole) was dissolved in 50 cc. of water, and added slowly with vigorous stirring t o the thiolNaOH solution. The mixture was stirred for an additional four hours, and the resultant oily product after being washed with water, was dissolved in 50 cc. of benzene. The benzene solution was dried with anhydrous sodium sulfate and the benzene removed by evaporation under reduced pressure. The residual oil was distilled under reduced pressure. All attempts t o convert these amines t o crystalline hydrochlorides failed.
SUMMARY
1. Seven diethylaminopropyl and seven diethylaminohydroxypropyl derivatives of thiols have been prepared and characterized.
TABLE IV.-1-DIETHYLAMINO-2-HYDROXY-PROPYLSULFIDES Thiol Used
o-Chlorabenzyl thiol 2,4-Dichlorobenzyl thiol 3,4-Dichlorobenzyl thiol Thiophenol o-Thiocresol m-Thiocresol p-Thiocresol
B. P.,
O
C./Mm.
190-195/4 210-215/4 210-215/4 168-171/4 154-157/1 168-172/3 168-172/3
Yield Amine, Gm.
17 42.8 44.3 41.3 35.6 32.0 35.0
(60%)b (66%) (69%) (86%) (65%) (60%) (64%)
Nitrogen determined by semimicro Kjeldahl method (7). b Made using 0.1 molar quantities, others made using 0.2 molar quantities.
--Nitrogen'Calcd.
Found
4.85 4.34 4.34 5.85 5.50 5.50 5.50
4.56 4.23 4.14 5.79 5.32 5.37 5.28
Refractive Index (/" C.) Amine
1.5435/30 1.5522/27 1.5515/30 1.5412/25 1.5408/30 1.5376/30 1.5371/30
360
JOURNAL OF THE
AMERICAN PHARMACEUTICAL ASSOCIATION
2* Sufficient quantities Of these have been prepared for pharmacological study. REFERENCES ( 1 ) Halpern, B. N., Arch. intern. pharmacodynamic, 59, 149 (1938). (2) Burnter, R. R., and Cusic, J. W., J . A m . Chem. Soc., 65, 262(1943). (3) Urquhart, G;,G., Gates, J. W., Jr., and Connor. R . , “Organic Syntheses, John Wiley and Sons, Inc.. New York, Vol. 21 1941 p. 36. (4) ’ChriGian J. E. Jenkins G. L. Keagle, L. C., and Crum, J. A,, TH& JO&AL, 35,’328(1946).
( 5 ) All:& C. P. H., and MacKay, D. D., “Organic Syntheses, John Wiley and Sons, Inc.. New York. Coll., vol.11, 1943, P. 580. ( 6 ) Blicke, F. F., and Zienty, F. B., J. A m . Chem. SOC., 61,776(1939). (7) “The Hengar Technique for the Kjeldahl Procedure,” Hengar Company, Philadelphia, Pa. ( 8 ) Breslow D. S. Walker H. G. Yost R. S., and Hauser. C. R., j . A m . khem. So;., 67, 14?4(194$. (9) Gilman, H., and Shirley D. A., ibid., 66, 888(1944). (10) Eisleb, O., Substituted ‘ 1,3-diamino-2-propanols to Winthrop Chemical Company, New York, N. Y., U. S. Patent No. 1,790,042, Jan. 27, 1931; Chcm. Abstr.. 25, 1259 (1931). (11) Gilman, H., cf al., J . A m . Chem. Soc., 68, 1291(1946). (12) Wertheirn. E., ibid.. 51, 3661(1929).
Assay of Heparin. 111. Measurement of the End Point by Physical Means. Influence of Surface on Clotting*J By R. H. K. FOSTER In the assay of heparin using beef or sheep plasma the degree of clotting has been estimated by visual inspection. This estimate, visual density, has been correlated with light transmission as measured photoelectrically and with the weights of dried clots. Good agreement was obtained. At times the surface of thoroughly cleaned and stored Pyrex tubes interfered with the aa’on of heparin in some unknown manner so that very irregular end-point responses were obtained. Concentration-response clotting curves obtained using freshly cleaned .Pyrex glass tubes were similar to those obtained using siliconecoated glass tubes or cellulose nitrate tubes. method previously described (I) the end point was considered t o be that amount of heparin which would just permit the formation of a 50 per cent clot. Since a 50 per cent clot is seldom obtained in any tube, the concentration of heparin corresponding to a 50 per cent clot must be calculated by interpolation from a clotting curve. The degree of clotting in the various tubes has always been estimated visually. I t is the purpose of this paper to correlate visually estimated degrees of opacity with opacity measured photoelectrically and with the weights of dried clots. In the course of this work some very marked irregularities were observed when clotting took place in Pyrex glass tubes but were completely eliminated when clotting was allowed to proceed in silicone-coated Pyrex tubes or in celN THE ASSAY
I
*
Received Nov. 10, 1948, from the Department of Pharmacology, St. Louis University School of Medicine, St. Louis, Mo. Supported in part by a grant-in-aid from the U. S. Public Health Service t The heparin used in this investigation was kindly supplied by Hoffmann-LaRoche, Inc., Nutley, N . J.
lulose nitrate tubes. A preliminary report of this investigation has been made elsewhere (2). MATERIALS, CRITERIA, AND PROCEDURES Opacity determined by inspection is termed visual density. This, representing the degree of coagulation, was estimated in terms of an arbitrary scale, 0 to 4+, with several intermediate values (1). The transmission of light, here measured photoelectrically, is a reciprocal function of opacity, the latter reflecting the degree of coagulation. Results are reported in terms of per cent transmission of light since this simplified the plotting of curves. However, for convenience, the term optical density is employed. Since optical density is -log 1/10the light transmission scale has been inverted to make these curves parallel to those for visual density and clot weight. The instrument accounts for light absorbed (or scattered) by the plasma as well as by the clot itself in contrast to the eye which discounts the plasma color when inspecting for visual density. In reporting the weights of dried clots it is not presymed that the weights represent pure fibrin. Highspeed centrifuging was employed to compact the fibrin clots but it is more than probable that the clots still contained some plasma and/or serum which when dried would add to the weight of the dried fibrin. Certain characteristics of the clot such as size and number of fibrin fibrils, their specific gravity, and their “malleability” must influence the ease of packing during centrifugalization and hence entrained material. It is probable that different concentrations of heparin alter these factors to such an extent that the proportionate amount of packing varies and the ratio of the amount of fibrin present to the entrained impurities is not strictly constant. Nevertheless, it is assumed that the clot weights as determined represent reasonable parallelism to the “true” weight of the fibrin.