- Email: [email protected]
Preparation and properties of some compounds of biological interest
I47
NOTES
Preparation
and properties
of some compounds
of biological
interest
In connection with work on the identification of unknown urinary metabolites, h a number of new compounds were synthesized. This communication describes the preparation and chromatographic characteristics of thirteen such compounds.
,C(
Prepavatio2z of materials Catechol monosulfate.
One drop of chlorosulfonic acid was stirred vigorously with 0.1 g of catechol, dissolved in 2 ml of benzene, for 5 min at o”. 5 ml of ice water were added and the mixture extracted three times with 10 ml portions of ether to remove unreacted catechol. The aqueous layer was chromatographed in BuOH-AcOH-E-I,0 (4: I : I). Two bands were identified by spraying a strip with diazotized sulfanilic acid (DSA). The upper band was catechol monosulfate, and the lower was probably catechol disulfate. The area corresponding to catechol monosulfate was cut out and eluted with water. Cateclzol mono$&os$hate. The method of GENVRESSE~was modified as follows: Catechol (1.1 g, 0.01 mole) was heated with 1.43 g of P,O, (0.01 mole) in ~aczco (15 mm Hg) at 120--130~ for z h, then distilled at 200’ to remove excess catechol. The residue was dissolved in water, and chromatographed in BuOH-AcOW-H,O (4: I : I). The area containing catechol monophosphate was cut out and elhted with water. q-Gzcaltidi~zoimidazolc. Guanine was heated with 4 N hydrochloric acid for 2 h at 160~ in a sealed tubes. The reaction mixture, after chromatographic separation in isopropanol-concentrated ammonium hydroxide-water (8 : I : I), gave two DSApositive spots, one yellow and the other red, changing to mauve. The yellow spot was xanthine and the other was guanidinoimidazole, which was confirmed by a positive pentacyanoaquaferriate (PCF) reaction. 4-Gzca7zidi7zo-5-imidazoZecarboxamide. 4-Amino-5-imidazolecarboxamide (AIC) was heated with cyanamide in a boiling water bath for.1 h. The unreacted AIC was removed by chromatographing the mixture in isopropanol-ammonium hydroxidewater (S : I : I). 4-Guanidino-g-imidazolecarboxamide gave a positive PCF test in addition to a. positive DSA. Methyl q-aminoimidaaole-pcarboxylale. This was prepared by the catalytic reduction of methyl 4-nitroimidazole-5-carboxylate (California Biochemicals) with Ha in the presence of 5 o/o Pd on charcoal. 4-Amimoimidazole and +amino-pimidazolecarboxylic acid. These compounds were prepared according to the method of RABINOW~TZ~. 4-Formamido-g-imidazolecarboxamide. SHAW%~ method was used for the prep- . aration. 4-Ureido-5-imidazolecarboxylic acid. This was a gift from Dr. J. RABINOWITZof the National Institute of Health, Bethesda, Maryland. Glycocyamidine. An aqueous solution (pH 2) of glycocyamine was boiled for 5 min. The product was isolated by paper chromatography in isopropanol-ammonium hydroxide-water (8 : I : I). PCF was used as the spray reagent. ~?4W.~~~ Tawocyamine. This was prepared from taurine and freshly prepared S-ethyl;:;,.. ‘>-thiourea hydrobromide in ammoniacal medium according to MORRISONet aZ.b.The final product was purified by crystallization from water and ethanol (I : IO). J. Chrotnaiog.,
20
(1965)
x47-149
OF SYNTHESIZED
STANDARDS
-
P P -
d
-
b -
-
-
-
-
OR
R -
YGn
B
-
-
Gy Gy -
Agf
P
PY -
-
Y
-
11
-
-
Bn
-GY -
-
GyB B
vpM -
B Gy -
vpBn Y -
B Gy -
vpB
P P
R -
R
P GY
P
PCF
-
-
0
-
RBn _ MP R R
P--
P
P
R
Bn Bn RBn R
1Vin. DQC DQC- Hg- AM. base DPC
PGY -
-
MV GyB Y+OR Gy
-
R+P OR B
R R OR
DlNA Elir.
OY RP+V
Y
DSA
-
-
Gy
R
GY
-
-
67
23 33 14 II
46
39
25 3s 24 22
39
4s 54 So
4
82 39 62
.-
5
the solved
5S
53 56 55 54
57
60 7S 45 44
Sr
5s
5S
53 54 52 40
67
DC -
79 DC 52
St 70
jj 231 -
so 53 $I
3
63 73 DC** 5s
3s
65 3;
2
DC 12
5s 26
42
3s 46 25
B.&I. I
RF x IOO b
l U.V.: b = weak blue; d = weakly defined, slightly darker than background; P = purple, darker than background. Colors: The following colors were as previously defined’: B = blue; Bn = brown; Gy = gray; GyB = gray blue; 11 = mauve; MP = mauve purple; MV = mauve violet; 0 = orange; OR = orange red; OY = orange yellow; P = purple; R = red; RBn = red brown; RP = red purple; V = violet; Y = yellow; YGn = yellow green. Adjectives: p = pale; vp = very pale; - indicates no reaction. l * DC = compound decomposed in the solvent.
2570 A
3500 il
Color reaciiom to digerellt spray reagents’
CHARACTERISTICS
Catechol monosulfate CXechol monophosphate 4-Guanidinoimidazole 4-Guanidino-5-imidazolecarboxamide Methyl 4-aminoimidazole5-carboxylate 4-Aminoimidazole 4-Amino-5-imidazolecarboxylic acid 4-Formamido-5-imidazolecarboxamide q-Ureido-5-imidazolecarboxylic acid Glycocyamidine Taurocyamine Homotaurocyamine q-Methoxy-3-hydroxymandelic acid
CHROMATOGRAPHIC
TABLE I
NOTES
I49
Homotazwocyamine. This was prepared as in the method above; homotaurine being used as the starting material instead of taurine. \. q-Metkoxy-3-laydyoxyrvtavtdelic acid. The method of CORSON et al.0 for the syni* thesis of mandelic acid was modified as follows : 1.5 g (0.03 moles) of sodium cyanide, 5 ml of water, and 4.5 g (0.03 moles) of isovanillin were placed in a 50 ml beaker and stirred, while 8.5 ml of a saturated solution of sodium bisulfite and g g of ice were added over a period of IO min. The layer of 4-methoxy-3-hydroxymandelonitrile which appeared was separated using a separatory funnel. The crude nitrile was at once transferred to an evaporating dish and 4.3 ml of concentrated hydrochloric acid was added. The hydrolysis was allowed to proceed in the cold for about 12 h, after which the mixture was heated on a steam bath to drive off the water and excess hy.clrochloric acid. The final product was dissolved in ethanol.
Each material was chromatographed unidimensionally in qz-butanol-acetic acid-water (4 : I : I) (solvent I), isopropanol-ammonia-water (8 : I : I) (solvent 2)) methanol-+butanol-benzene-water (z : I :I :I) (solvent 3)) isobutyric acid-o.5 N NH,OH (IO: 6) (solvent 4), and +butanol-pyridine-water (I :I :I) (solvent 5). Each RF value given in Table I is the average of at least three descending runs on Whatman No. I: paper on different days. Color reactions under ultraviolet light and with the various spray reagents are also recorded in Table I and are those appearing after chromatograms were run in solvent no. 3. Eleven spray reagents were used as described previously’. They were: diazotized sulfanilic acid (DSA), diazotized +nitroaniline (DNA), Ehrlich’s reagent (pdimethylaminobenzaldehyde in ethanol and I-ICI.) (Ehr.), ammoniacal silver nitrate (Ag*), Bratton-Marshall (nitrous acid, then coupling component) (B.M.) , ninhydrin (Nin.), 2,6-dichloroquinonechlorimide (DQC), saturated boras sprayed over D QC (DQC base), mercury-diphenyl carbazone (Hg-DPC), anisidine (An.), and pentacyanoaquaferriate reagent (PCF). The color description was correlated and recorded as described previously’. The chromatographic data obtained are summarized in the table. This research was supported by Grant No. H-5664 from the National Institute of Health, Rethesda, Maryland. K&nsmeqs Laboratory of Newological The Un&ersity of British Cohmbia, I M. P. GENVRESSE, Coopt. Rend., z G. HUNTBR AND J. A. NELSON, 3
4 5
6
**,$ 7 .:i
Research, Vancower
127 (1898)
(Canada)
N. P. SEN P. L. MCGEER
523.
Res., Igl3 (x941) 2gG. J, RABINOWITZ, J. Biol. Cl&em., 218 (1956) 175. E. S~IAW, J. Biol. Cltem., 18s (1950) 439. J, I?. MORRISON, A. H. ENNOR AND D. E. GRIFFITIIS,Biochem. J., 65 (1958) 447. B. B. CORSON, R. A. DODGE, S. A. HARRIS AND J. S. YEAW, in HI. GILMAN AND A. H. QLATT (Editors), Organic Sym!hesis, CoElective VoZzbme I, John Wiley and Sons, Lonclon, 1941, p. 336. E. G. MCGEIZR, M. C. ROBERTSON AND I?.L. MCGEER, Can. J. Biochem. Playsiol., 39 (196x) 605.
’ Received
April
r&h,
1965
Cm.J.
’
J. Chromatog., 20 ( 196.5)
147-149
NOTES
Preparation
and properties
of some compounds
of biological
interest
In connection with work on the identification of unknown urinary metabolites, h a number of new compounds were synthesized. This communication describes the preparation and chromatographic characteristics of thirteen such compounds.
,C(
Prepavatio2z of materials Catechol monosulfate.
One drop of chlorosulfonic acid was stirred vigorously with 0.1 g of catechol, dissolved in 2 ml of benzene, for 5 min at o”. 5 ml of ice water were added and the mixture extracted three times with 10 ml portions of ether to remove unreacted catechol. The aqueous layer was chromatographed in BuOH-AcOH-E-I,0 (4: I : I). Two bands were identified by spraying a strip with diazotized sulfanilic acid (DSA). The upper band was catechol monosulfate, and the lower was probably catechol disulfate. The area corresponding to catechol monosulfate was cut out and eluted with water. Cateclzol mono$&os$hate. The method of GENVRESSE~was modified as follows: Catechol (1.1 g, 0.01 mole) was heated with 1.43 g of P,O, (0.01 mole) in ~aczco (15 mm Hg) at 120--130~ for z h, then distilled at 200’ to remove excess catechol. The residue was dissolved in water, and chromatographed in BuOH-AcOW-H,O (4: I : I). The area containing catechol monophosphate was cut out and elhted with water. q-Gzcaltidi~zoimidazolc. Guanine was heated with 4 N hydrochloric acid for 2 h at 160~ in a sealed tubes. The reaction mixture, after chromatographic separation in isopropanol-concentrated ammonium hydroxide-water (8 : I : I), gave two DSApositive spots, one yellow and the other red, changing to mauve. The yellow spot was xanthine and the other was guanidinoimidazole, which was confirmed by a positive pentacyanoaquaferriate (PCF) reaction. 4-Gzca7zidi7zo-5-imidazoZecarboxamide. 4-Amino-5-imidazolecarboxamide (AIC) was heated with cyanamide in a boiling water bath for.1 h. The unreacted AIC was removed by chromatographing the mixture in isopropanol-ammonium hydroxidewater (S : I : I). 4-Guanidino-g-imidazolecarboxamide gave a positive PCF test in addition to a. positive DSA. Methyl q-aminoimidaaole-pcarboxylale. This was prepared by the catalytic reduction of methyl 4-nitroimidazole-5-carboxylate (California Biochemicals) with Ha in the presence of 5 o/o Pd on charcoal. 4-Amimoimidazole and +amino-pimidazolecarboxylic acid. These compounds were prepared according to the method of RABINOW~TZ~. 4-Formamido-g-imidazolecarboxamide. SHAW%~ method was used for the prep- . aration. 4-Ureido-5-imidazolecarboxylic acid. This was a gift from Dr. J. RABINOWITZof the National Institute of Health, Bethesda, Maryland. Glycocyamidine. An aqueous solution (pH 2) of glycocyamine was boiled for 5 min. The product was isolated by paper chromatography in isopropanol-ammonium hydroxide-water (8 : I : I). PCF was used as the spray reagent. ~?4W.~~~ Tawocyamine. This was prepared from taurine and freshly prepared S-ethyl;:;,.. ‘>-thiourea hydrobromide in ammoniacal medium according to MORRISONet aZ.b.The final product was purified by crystallization from water and ethanol (I : IO). J. Chrotnaiog.,
20
(1965)
x47-149
OF SYNTHESIZED
STANDARDS
-
P P -
d
-
b -
-
-
-
-
OR
R -
YGn
B
-
-
Gy Gy -
Agf
P
PY -
-
Y
-
11
-
-
Bn
-GY -
-
GyB B
vpM -
B Gy -
vpBn Y -
B Gy -
vpB
P P
R -
R
P GY
P
PCF
-
-
0
-
RBn _ MP R R
P--
P
P
R
Bn Bn RBn R
1Vin. DQC DQC- Hg- AM. base DPC
PGY -
-
MV GyB Y+OR Gy
-
R+P OR B
R R OR
DlNA Elir.
OY RP+V
Y
DSA
-
-
Gy
R
GY
-
-
67
23 33 14 II
46
39
25 3s 24 22
39
4s 54 So
4
82 39 62
.-
5
the solved
5S
53 56 55 54
57
60 7S 45 44
Sr
5s
5S
53 54 52 40
67
DC -
79 DC 52
St 70
jj 231 -
so 53 $I
3
63 73 DC** 5s
3s
65 3;
2
DC 12
5s 26
42
3s 46 25
B.&I. I
RF x IOO b
l U.V.: b = weak blue; d = weakly defined, slightly darker than background; P = purple, darker than background. Colors: The following colors were as previously defined’: B = blue; Bn = brown; Gy = gray; GyB = gray blue; 11 = mauve; MP = mauve purple; MV = mauve violet; 0 = orange; OR = orange red; OY = orange yellow; P = purple; R = red; RBn = red brown; RP = red purple; V = violet; Y = yellow; YGn = yellow green. Adjectives: p = pale; vp = very pale; - indicates no reaction. l * DC = compound decomposed in the solvent.
2570 A
3500 il
Color reaciiom to digerellt spray reagents’
CHARACTERISTICS
Catechol monosulfate CXechol monophosphate 4-Guanidinoimidazole 4-Guanidino-5-imidazolecarboxamide Methyl 4-aminoimidazole5-carboxylate 4-Aminoimidazole 4-Amino-5-imidazolecarboxylic acid 4-Formamido-5-imidazolecarboxamide q-Ureido-5-imidazolecarboxylic acid Glycocyamidine Taurocyamine Homotaurocyamine q-Methoxy-3-hydroxymandelic acid
CHROMATOGRAPHIC
TABLE I
NOTES
I49
Homotazwocyamine. This was prepared as in the method above; homotaurine being used as the starting material instead of taurine. \. q-Metkoxy-3-laydyoxyrvtavtdelic acid. The method of CORSON et al.0 for the syni* thesis of mandelic acid was modified as follows : 1.5 g (0.03 moles) of sodium cyanide, 5 ml of water, and 4.5 g (0.03 moles) of isovanillin were placed in a 50 ml beaker and stirred, while 8.5 ml of a saturated solution of sodium bisulfite and g g of ice were added over a period of IO min. The layer of 4-methoxy-3-hydroxymandelonitrile which appeared was separated using a separatory funnel. The crude nitrile was at once transferred to an evaporating dish and 4.3 ml of concentrated hydrochloric acid was added. The hydrolysis was allowed to proceed in the cold for about 12 h, after which the mixture was heated on a steam bath to drive off the water and excess hy.clrochloric acid. The final product was dissolved in ethanol.
Each material was chromatographed unidimensionally in qz-butanol-acetic acid-water (4 : I : I) (solvent I), isopropanol-ammonia-water (8 : I : I) (solvent 2)) methanol-+butanol-benzene-water (z : I :I :I) (solvent 3)) isobutyric acid-o.5 N NH,OH (IO: 6) (solvent 4), and +butanol-pyridine-water (I :I :I) (solvent 5). Each RF value given in Table I is the average of at least three descending runs on Whatman No. I: paper on different days. Color reactions under ultraviolet light and with the various spray reagents are also recorded in Table I and are those appearing after chromatograms were run in solvent no. 3. Eleven spray reagents were used as described previously’. They were: diazotized sulfanilic acid (DSA), diazotized +nitroaniline (DNA), Ehrlich’s reagent (pdimethylaminobenzaldehyde in ethanol and I-ICI.) (Ehr.), ammoniacal silver nitrate (Ag*), Bratton-Marshall (nitrous acid, then coupling component) (B.M.) , ninhydrin (Nin.), 2,6-dichloroquinonechlorimide (DQC), saturated boras sprayed over D QC (DQC base), mercury-diphenyl carbazone (Hg-DPC), anisidine (An.), and pentacyanoaquaferriate reagent (PCF). The color description was correlated and recorded as described previously’. The chromatographic data obtained are summarized in the table. This research was supported by Grant No. H-5664 from the National Institute of Health, Rethesda, Maryland. K&nsmeqs Laboratory of Newological The Un&ersity of British Cohmbia, I M. P. GENVRESSE, Coopt. Rend., z G. HUNTBR AND J. A. NELSON, 3
4 5
6
**,$ 7 .:i
Research, Vancower
127 (1898)
(Canada)
N. P. SEN P. L. MCGEER
523.
Res., Igl3 (x941) 2gG. J, RABINOWITZ, J. Biol. Cl&em., 218 (1956) 175. E. S~IAW, J. Biol. Cltem., 18s (1950) 439. J, I?. MORRISON, A. H. ENNOR AND D. E. GRIFFITIIS,Biochem. J., 65 (1958) 447. B. B. CORSON, R. A. DODGE, S. A. HARRIS AND J. S. YEAW, in HI. GILMAN AND A. H. QLATT (Editors), Organic Sym!hesis, CoElective VoZzbme I, John Wiley and Sons, Lonclon, 1941, p. 336. E. G. MCGEIZR, M. C. ROBERTSON AND I?.L. MCGEER, Can. J. Biochem. Playsiol., 39 (196x) 605.
’ Received
April
r&h,
1965
Cm.J.
’
J. Chromatog., 20 ( 196.5)
147-149