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Spot test for 3-pyridinols
MICROCIIEMICAL
JOURNrZL
11,
Spot
237-242
Test
(1966)
for
3-Pyridinols
HARVEY W. YUROW, LINWOOD B. MORTON, AND SAMUEL SASS Chemical
Research
Division, Chemical Research and Development US Army Edgewood Arsenal, Maryland Received
December
Laboratories,
21, 1965
INTRODUCTION At present there are no satisfactory calorimetric methods to distinguish among the hydroxypyridines (8). The 3-pyridinols, which behave as phenols compared to the keto-like Z- and 4-pyridinols, give a positive test with phenolic reagents such as the Folin-Denis phosphotungstomolybdate mixture (9). However, positive results are also obtained with this reagent and 2-pyridinols substituted in the 6-position by groups such as hydroxy, methyl, methoxy, amino, etc. (7). In this report, a new, sensitive spot-test technique is described which appears to be capable of distinguishing 3-pyridinols, either unsubstituted in the 2-position or having groups labile to bromine such as hydroxymethyl in the 2-position, from phenols and 2- or 4-pyridinols, and 2-pyridinols substituted in the 6-position. In the test, the compound in acid solution is treated with a slight excess of bromine water followed by addition of sodium arsenite to remove excess bromine. Brief heating of the solution gives an orange color which changes to deep blue when brought to a pH of 5 to 6 with buffer. The blue color can be formed directly on addition of buffer without heating, but is then somewhat less intense. MATERIALS AND METHODS Reagents
Sulfuric acid, 1.O X Bromine water, saturated, approximately Sodium arsenite, 4% aqueous solution Tripotassium phosphate, 1.O IM Ammonium hydroxide, 15 N
0.2 M
The compounds tested were those commercially available, with the exceptions of 6-amino-2-pyridinol, prepared from 2,6-diaminopyridine (12) ; and 3-hydroxyquinoline, prepared from 3-aminoquinoline (6). 237
238
HARVEY
W.
YUROW
ET
AL.
2 k 5 - I
u I
I I I I I I I
I”Z~IIII
I
I I I I
I I I
SPOT
TEST
FOR
239
3-PYRIDINOLS
Procedures Solution test. A drop of the aqueoustest solution, approximately neutral, is treated in a test tube with one drop of 1.0 N sulfuric acid and one drop of saturated bromine water, followed after 3 minutes by one drop of 47% sodium arsenite. The solution is heated in a boiling water bath for 5 minutes (orange color), cooled rapidly to room temperature and mixed with one drop of 1.0 M tripotassium phosphate. A blue color, gradually changing to pink, indicates a positive test. Filter paper test. The approximately neutral, aqueous test solution is spotted on filter paper such as Whatman No. 50 (13) and the moist spot exposed to saturated bromine water in a wide-mouth jar for 15 seconds. The paper is heated in an oven for 2 minutes at 110°C. It is removed and exposed to ammonia fumes for 1 minute on each side. A blue or cyan spot indicates a positive test. RESULTS
Qualitative
Tests
Sensitivity limits were determined for a number of 3-pyridinol compounds under spot-test conditions in solution, and as spots on filter paper. These results are shown in Table 1 along with test results for a number of related compounds.Results for mixtures of 3-pyridinol and possibleinterferencesare given in Table 2. TABLE DETECTION
OF 3-PYRIDINOL
Mixture
(5 ug) + 2-pyridinol
3-Pyridinol
(5 pg) f
3-Pyridinol (50 l-e) 3-Pyridinol
a Before
OF OTHER
(0.30 ml)
3-Pyridinol
3-Pyridinol
2
IN THE PRESENCE
COMPOUNDS
Resulta (50 ug)
+
light
blue +
very
(5 ug) f 6-amino-2-pyridinol
Deep blue ppt., colorless soln. blue ppt., light blue soln.
+
deep
(5 pg)
+ phenol
Cloudy soln.
light
blue
(5 pg)
+ pyridine
Test for Quantitative
(50 ug)
orange
Pale orange soln., black ppt. pale blue soln., green ppt.
and after
2,6-pyridinediol
Light
(50 ug) (50 pg)
the addition
of tripotassium
Light
light orange
orange
soln.
+
blue
light
+
phosphate.
Application
Studies were made on the quantitation of the bromine method for 3pyridinols. The absorbancy index (1 gm per liter) for the isolated colored substance from 3-pyridinol (see Discussion) in pH 6.5 phosphate buffer
240
HARVEY
W.
YUROW
ET
AL.
at 610 rnp was 37; and in 0.5 N sulfuric acid, was 50 at 470 ml& with the latter being relatively more stable. An attempt to make the spot-test for 3-pyridinol quantitative was unsuccessful because of nonlinearity in the range of 20-100 pg per milliliter initial concentration. DISCUSSION
Isolation
and Properties
of the Colored Pyvidinol Substance
The reaction of a slight excess of bromine water with aqueous 3-pyridinol is reported by Den Hertog et al. to give 2,4,6-tribromo-3-pyridinol as a white precipitate in 50% yield (5), and by Fisher and Renouf to give a dibromo 3-pyridinol hydrobromide (3). Observations in our laboratory indicate t,hat when bromine water is added slowly to aqueous 3-pyridinol as the free base, a deep blue color is first formed which turns to orange and is masked as more bromine water (3.1 M total) is added. If the mixture is allowed to stand for about 0.5 hour at room temperature, or is heated for several minutes at 60°C it changes from pale yellow to colorless, to deep orange. The colored pyridinol product was isolated by treating a 1% solution of 3-pyridinol in 0.1 N sulfuric acid with a slight excess (3.1 N) of saturated bromine water followed by boiling for 5 minutes. A bright green solid (15% yield based upon initial weight of 3-pyridinol) was deposited, collected by filtration, and dried in air. The substance, which decomposes above 300°C without melting, gave an elemental analysis of C = 37.3%, H = 3.4%, N = 9.0%, and Br = 15.3%. It was slightly soluble in water and such organic solvents as acetone, methanol, etc., but dissolved readily in dimethyl formamide or dimethyl sulfoxide to give a deep orange solution; in dilute sodium hydroxide, to give a deep blue solution; and in concentrated sulfuric acid, to give an intense magenta solution. The orange color of the substance in dilute sulfuric acid is bleached by ferric ion, by hydrosulfite, and by bromine water; but is returned on exposure to air for the latter two. The color is reversibly changed to blue at pH 4. The substance is also reduced by titanium trichloride in methanol to a brick-red color; and in aqueous solution, to an orange precipitate characteristic of o-quinones (14). The infrared spectrum as a potassium bromide pellet indicates strongly conjugated carbonyl groups and phenolic groups. Nature of the Isolated Substance A clue to the identity of the green substance from 3-pyridinol (and similar but not necessarily identical products which can also be formed
SPOT
TEST
FOR
3-PYRIDINOLS
241
by the aforementioned hydroxymethyl 3-pyridinols), is found in a comparison of the chemical and physical properties described above with work reported in the literature for azaquinones. Boyer and Kruger prepared 6-hydroxy-1,2-azaquinone as the purple quinhydrone by oxidation of 3amino-2-pyridinol with potassium bromate in acid solution at 3”-5°C (I), while Schickh et al. formed 1,2-azaquinone by the oxidation of 2,3-pyridinediol with manganese dioxide and sulfuric acid (11). These compounds are characterized by relatively high melting points, a blue color in basic solution, and a red color in concentrated sulfuric acid, and have the general structure:
Mechanism of Formation The mechanism of the conversion of 3-pyridinol to the assumed azaquinone is still uncertain. However, one may speculate that in addition to attack on 3-pyridinol by molecular bromine to give the 2,4,6-tribromo derivative as the major product (3)) a side reaction may occur with 3pyridinol and hypobromous acid to give hydroxylation to a pyridinediol followed by oxidation to an azaquinone, analogous to the results of Boyer and Kruger. The fact that 2-hydroxymethyl-3-pyridinol and 2,6-bis(hydroxymethyl)-3-pyridinol react similarly to 3-pyridinol in giving the blue color is not unexpected, since a number of ortho-substituted phenols such as saligenin or salicylic acid readily lose the ortho group when treated with bromine water (10). The odor of formaldehyde was detected when 2hydroxymethyl-3-pyridinol was treated with bromine water and heated. A degree of differentiation between 3-pyridinol and its hydroxymethyl derivatives is possible by treatment with one drop, each, of ferric ammonium sulfate (0.5% in lo-” N sulfuric acid) and 3% hydrogen peroxide; the first-named compound giving a much more intense violet color, which may be the result of diol formation (2) followed by complexation with ferric ion (4). The test is also given by 6-amino-2-pyridinol. SUMMARY A sensitive spot tion products, on tive Folin-Denis arsenite followed
test to distinguish between 3-pyridinol and certain of its substituthe one hand, and various phenols or 2-pyridinols giving a positest involves treatment with bromine water; then excess sodium by brief heating and basification. The blue color formed has spot-
242
HARVEY
W.
YUROW
ET
AL.
test sensitivities of the order of 5 pg/O.25 ml in a test tube; or 1 pg/cm2 on filter paper. Although the structure of the colored species has not been definitely established, it appears, on the basis of chemical and physical evidence, to be an azaquinone, REFERENCES
2. 3.
4. 5.
6.
7.
8. 9. 10. 11. 12.
13. 14.
BOYER, J. H., AND KRUGER, S., Azaquinones. I. Oxidation of certain hydroxyand aminopyridones. J. Am. Chem. Sot. 79, 3552-3554 (1957). CHWALA, H., AND PAILER, M., Uber die Oxydation von Phenolen mit H,O, bei Anwesenheit von FeSO,. J. Pratt. Chem. 152, 45-48 (1939). DEN HERTOG, H. J., SCHEP~N, F. R., DE BRVYN, J., AND THYSSE, G. J. E., Bromo-derivatives of 3-hydroxypyridine. Rec. Truv. Chim. 69, 1281-1288 (1950). DEN HERTOG, H. J., WIBAIJT, J. P., SCHEZMAN, F. R., AND VAN DER WAL, A. A., The dihydroxy and diethoxy pyridines. Rec. Trav. Chim. 69, 700-710 (1950). FISHER, O., AND RENOUF, E., Einige Derivative des Chinolins und Pyridine. Chem. Ber. 17, 755-764 (1884) ; Zur Kenntniss des Oxypyridins aus Pyridinsulfotiure, Chem. Ber. 17, 1896-1899 (1884). HEY, W. H., AND WILLIAMS, J. M., New Therapeutic Agents of the Quinoline Series, Part VII. 2-, 3- and 4-Pyridylquinolines ; 4-Pyridylquinaldiones ; and 2-Pyridyllepidines. J. Chem. Sot., 1960, 1678-1683. KLEIPOOL, R. J. C., AND WIB.~UT, J. P., Note on the colour reaction of derivatives of hydroxypyridines with the folin and Denis reagent. Rec. Trav. Chim. 69, 59-60 (1950). KLINGSBERG, E., Heterocyclic compounds-pyridine and derivatives, Part 3, p. 631, Wiley (Interscience), New York, 1962. KUHN, R., AND WENDT, B., Uber den Oxydativen Abbau des Adermins. Chem. Bev. 72,305-309 (1939). RUDERMAN, I. W., Bromination of phenols and phenol alcohols. 2nd. Eng. Chem., Anal. Ed., 18, 753-759 (1946). SCHICKH, 0. N., BINZ, A., AND SCHULZ, A., Derivative des 3-Amino-pyridine. Chem. Ber. 69, 2593-2605 (1936). SEIDE, 0. A., AND TITOV, A. J., Zur Frage der Struktur von P-Amino-pyridin und 2,6-Diamino-pyridin. Hydrolyze der Amino Gruppe in 2-Amino-pyridin, 2,6Diamino-pyridin, 2-Amino-6-oxy-pyridin und 2-oxy-4-methyl-7-amino napbthyridin-( 1,s). Chem. Ber. 69, 1884-1893 (1936). WEST, P. W., AND HAMILTON, W. C., A study of the effect of media upon spot test reactions. Mikrochemie ver. Mikrochim. Acta 38, 100-113 (1951). WEYGAND, F., AND CSENDES, E., Eine Nachweismethode fur Endiole und Enole der 1.3 Diketone. Chem. Ber. 85, 45-57 (1952).
JOURNrZL
11,
Spot
237-242
Test
(1966)
for
3-Pyridinols
HARVEY W. YUROW, LINWOOD B. MORTON, AND SAMUEL SASS Chemical
Research
Division, Chemical Research and Development US Army Edgewood Arsenal, Maryland Received
December
Laboratories,
21, 1965
INTRODUCTION At present there are no satisfactory calorimetric methods to distinguish among the hydroxypyridines (8). The 3-pyridinols, which behave as phenols compared to the keto-like Z- and 4-pyridinols, give a positive test with phenolic reagents such as the Folin-Denis phosphotungstomolybdate mixture (9). However, positive results are also obtained with this reagent and 2-pyridinols substituted in the 6-position by groups such as hydroxy, methyl, methoxy, amino, etc. (7). In this report, a new, sensitive spot-test technique is described which appears to be capable of distinguishing 3-pyridinols, either unsubstituted in the 2-position or having groups labile to bromine such as hydroxymethyl in the 2-position, from phenols and 2- or 4-pyridinols, and 2-pyridinols substituted in the 6-position. In the test, the compound in acid solution is treated with a slight excess of bromine water followed by addition of sodium arsenite to remove excess bromine. Brief heating of the solution gives an orange color which changes to deep blue when brought to a pH of 5 to 6 with buffer. The blue color can be formed directly on addition of buffer without heating, but is then somewhat less intense. MATERIALS AND METHODS Reagents
Sulfuric acid, 1.O X Bromine water, saturated, approximately Sodium arsenite, 4% aqueous solution Tripotassium phosphate, 1.O IM Ammonium hydroxide, 15 N
0.2 M
The compounds tested were those commercially available, with the exceptions of 6-amino-2-pyridinol, prepared from 2,6-diaminopyridine (12) ; and 3-hydroxyquinoline, prepared from 3-aminoquinoline (6). 237
238
HARVEY
W.
YUROW
ET
AL.
2 k 5 - I
u I
I I I I I I I
I”Z~IIII
I
I I I I
I I I
SPOT
TEST
FOR
239
3-PYRIDINOLS
Procedures Solution test. A drop of the aqueoustest solution, approximately neutral, is treated in a test tube with one drop of 1.0 N sulfuric acid and one drop of saturated bromine water, followed after 3 minutes by one drop of 47% sodium arsenite. The solution is heated in a boiling water bath for 5 minutes (orange color), cooled rapidly to room temperature and mixed with one drop of 1.0 M tripotassium phosphate. A blue color, gradually changing to pink, indicates a positive test. Filter paper test. The approximately neutral, aqueous test solution is spotted on filter paper such as Whatman No. 50 (13) and the moist spot exposed to saturated bromine water in a wide-mouth jar for 15 seconds. The paper is heated in an oven for 2 minutes at 110°C. It is removed and exposed to ammonia fumes for 1 minute on each side. A blue or cyan spot indicates a positive test. RESULTS
Qualitative
Tests
Sensitivity limits were determined for a number of 3-pyridinol compounds under spot-test conditions in solution, and as spots on filter paper. These results are shown in Table 1 along with test results for a number of related compounds.Results for mixtures of 3-pyridinol and possibleinterferencesare given in Table 2. TABLE DETECTION
OF 3-PYRIDINOL
Mixture
(5 ug) + 2-pyridinol
3-Pyridinol
(5 pg) f
3-Pyridinol (50 l-e) 3-Pyridinol
a Before
OF OTHER
(0.30 ml)
3-Pyridinol
3-Pyridinol
2
IN THE PRESENCE
COMPOUNDS
Resulta (50 ug)
+
light
blue +
very
(5 ug) f 6-amino-2-pyridinol
Deep blue ppt., colorless soln. blue ppt., light blue soln.
+
deep
(5 pg)
+ phenol
Cloudy soln.
light
blue
(5 pg)
+ pyridine
Test for Quantitative
(50 ug)
orange
Pale orange soln., black ppt. pale blue soln., green ppt.
and after
2,6-pyridinediol
Light
(50 ug) (50 pg)
the addition
of tripotassium
Light
light orange
orange
soln.
+
blue
light
+
phosphate.
Application
Studies were made on the quantitation of the bromine method for 3pyridinols. The absorbancy index (1 gm per liter) for the isolated colored substance from 3-pyridinol (see Discussion) in pH 6.5 phosphate buffer
240
HARVEY
W.
YUROW
ET
AL.
at 610 rnp was 37; and in 0.5 N sulfuric acid, was 50 at 470 ml& with the latter being relatively more stable. An attempt to make the spot-test for 3-pyridinol quantitative was unsuccessful because of nonlinearity in the range of 20-100 pg per milliliter initial concentration. DISCUSSION
Isolation
and Properties
of the Colored Pyvidinol Substance
The reaction of a slight excess of bromine water with aqueous 3-pyridinol is reported by Den Hertog et al. to give 2,4,6-tribromo-3-pyridinol as a white precipitate in 50% yield (5), and by Fisher and Renouf to give a dibromo 3-pyridinol hydrobromide (3). Observations in our laboratory indicate t,hat when bromine water is added slowly to aqueous 3-pyridinol as the free base, a deep blue color is first formed which turns to orange and is masked as more bromine water (3.1 M total) is added. If the mixture is allowed to stand for about 0.5 hour at room temperature, or is heated for several minutes at 60°C it changes from pale yellow to colorless, to deep orange. The colored pyridinol product was isolated by treating a 1% solution of 3-pyridinol in 0.1 N sulfuric acid with a slight excess (3.1 N) of saturated bromine water followed by boiling for 5 minutes. A bright green solid (15% yield based upon initial weight of 3-pyridinol) was deposited, collected by filtration, and dried in air. The substance, which decomposes above 300°C without melting, gave an elemental analysis of C = 37.3%, H = 3.4%, N = 9.0%, and Br = 15.3%. It was slightly soluble in water and such organic solvents as acetone, methanol, etc., but dissolved readily in dimethyl formamide or dimethyl sulfoxide to give a deep orange solution; in dilute sodium hydroxide, to give a deep blue solution; and in concentrated sulfuric acid, to give an intense magenta solution. The orange color of the substance in dilute sulfuric acid is bleached by ferric ion, by hydrosulfite, and by bromine water; but is returned on exposure to air for the latter two. The color is reversibly changed to blue at pH 4. The substance is also reduced by titanium trichloride in methanol to a brick-red color; and in aqueous solution, to an orange precipitate characteristic of o-quinones (14). The infrared spectrum as a potassium bromide pellet indicates strongly conjugated carbonyl groups and phenolic groups. Nature of the Isolated Substance A clue to the identity of the green substance from 3-pyridinol (and similar but not necessarily identical products which can also be formed
SPOT
TEST
FOR
3-PYRIDINOLS
241
by the aforementioned hydroxymethyl 3-pyridinols), is found in a comparison of the chemical and physical properties described above with work reported in the literature for azaquinones. Boyer and Kruger prepared 6-hydroxy-1,2-azaquinone as the purple quinhydrone by oxidation of 3amino-2-pyridinol with potassium bromate in acid solution at 3”-5°C (I), while Schickh et al. formed 1,2-azaquinone by the oxidation of 2,3-pyridinediol with manganese dioxide and sulfuric acid (11). These compounds are characterized by relatively high melting points, a blue color in basic solution, and a red color in concentrated sulfuric acid, and have the general structure:
Mechanism of Formation The mechanism of the conversion of 3-pyridinol to the assumed azaquinone is still uncertain. However, one may speculate that in addition to attack on 3-pyridinol by molecular bromine to give the 2,4,6-tribromo derivative as the major product (3)) a side reaction may occur with 3pyridinol and hypobromous acid to give hydroxylation to a pyridinediol followed by oxidation to an azaquinone, analogous to the results of Boyer and Kruger. The fact that 2-hydroxymethyl-3-pyridinol and 2,6-bis(hydroxymethyl)-3-pyridinol react similarly to 3-pyridinol in giving the blue color is not unexpected, since a number of ortho-substituted phenols such as saligenin or salicylic acid readily lose the ortho group when treated with bromine water (10). The odor of formaldehyde was detected when 2hydroxymethyl-3-pyridinol was treated with bromine water and heated. A degree of differentiation between 3-pyridinol and its hydroxymethyl derivatives is possible by treatment with one drop, each, of ferric ammonium sulfate (0.5% in lo-” N sulfuric acid) and 3% hydrogen peroxide; the first-named compound giving a much more intense violet color, which may be the result of diol formation (2) followed by complexation with ferric ion (4). The test is also given by 6-amino-2-pyridinol. SUMMARY A sensitive spot tion products, on tive Folin-Denis arsenite followed
test to distinguish between 3-pyridinol and certain of its substituthe one hand, and various phenols or 2-pyridinols giving a positest involves treatment with bromine water; then excess sodium by brief heating and basification. The blue color formed has spot-
242
HARVEY
W.
YUROW
ET
AL.
test sensitivities of the order of 5 pg/O.25 ml in a test tube; or 1 pg/cm2 on filter paper. Although the structure of the colored species has not been definitely established, it appears, on the basis of chemical and physical evidence, to be an azaquinone, REFERENCES
2. 3.
4. 5.
6.
7.
8. 9. 10. 11. 12.
13. 14.
BOYER, J. H., AND KRUGER, S., Azaquinones. I. Oxidation of certain hydroxyand aminopyridones. J. Am. Chem. Sot. 79, 3552-3554 (1957). CHWALA, H., AND PAILER, M., Uber die Oxydation von Phenolen mit H,O, bei Anwesenheit von FeSO,. J. Pratt. Chem. 152, 45-48 (1939). DEN HERTOG, H. J., SCHEP~N, F. R., DE BRVYN, J., AND THYSSE, G. J. E., Bromo-derivatives of 3-hydroxypyridine. Rec. Truv. Chim. 69, 1281-1288 (1950). DEN HERTOG, H. J., WIBAIJT, J. P., SCHEZMAN, F. R., AND VAN DER WAL, A. A., The dihydroxy and diethoxy pyridines. Rec. Trav. Chim. 69, 700-710 (1950). FISHER, O., AND RENOUF, E., Einige Derivative des Chinolins und Pyridine. Chem. Ber. 17, 755-764 (1884) ; Zur Kenntniss des Oxypyridins aus Pyridinsulfotiure, Chem. Ber. 17, 1896-1899 (1884). HEY, W. H., AND WILLIAMS, J. M., New Therapeutic Agents of the Quinoline Series, Part VII. 2-, 3- and 4-Pyridylquinolines ; 4-Pyridylquinaldiones ; and 2-Pyridyllepidines. J. Chem. Sot., 1960, 1678-1683. KLEIPOOL, R. J. C., AND WIB.~UT, J. P., Note on the colour reaction of derivatives of hydroxypyridines with the folin and Denis reagent. Rec. Trav. Chim. 69, 59-60 (1950). KLINGSBERG, E., Heterocyclic compounds-pyridine and derivatives, Part 3, p. 631, Wiley (Interscience), New York, 1962. KUHN, R., AND WENDT, B., Uber den Oxydativen Abbau des Adermins. Chem. Bev. 72,305-309 (1939). RUDERMAN, I. W., Bromination of phenols and phenol alcohols. 2nd. Eng. Chem., Anal. Ed., 18, 753-759 (1946). SCHICKH, 0. N., BINZ, A., AND SCHULZ, A., Derivative des 3-Amino-pyridine. Chem. Ber. 69, 2593-2605 (1936). SEIDE, 0. A., AND TITOV, A. J., Zur Frage der Struktur von P-Amino-pyridin und 2,6-Diamino-pyridin. Hydrolyze der Amino Gruppe in 2-Amino-pyridin, 2,6Diamino-pyridin, 2-Amino-6-oxy-pyridin und 2-oxy-4-methyl-7-amino napbthyridin-( 1,s). Chem. Ber. 69, 1884-1893 (1936). WEST, P. W., AND HAMILTON, W. C., A study of the effect of media upon spot test reactions. Mikrochemie ver. Mikrochim. Acta 38, 100-113 (1951). WEYGAND, F., AND CSENDES, E., Eine Nachweismethode fur Endiole und Enole der 1.3 Diketone. Chem. Ber. 85, 45-57 (1952).