New triterpene and antibacterial labdenoic acid derivatives from Moldenhawera nutans

Abstracts

A new triterpene derivative (22beta-hydroxylupeol) was isolated from the MeOH extract of stems of Moldenhawera nutans (Leguminosae) together with labdenoic acid derivatives of common occurrence in this species. From the labd-8(17)-en-15-oic acid were prepared simple derivatives, which exhibited in vitro weak activity against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella cholerasuis, and Vibrio parahaemolyticus.

Moldenhawera nutans; Leguminosae; antibacterial labdene derivatives; 22beta-hydroxylupeol

Um novo triterpeno (22beta-hidroxilupeol) foi isolado do extrato em metanol do caule de Moldenhawera nutans (Leguminosae) além de diterpenos derivados do ácido labdenóico de ocorrência comum nesta espécie. A partir do ácido labd-8(17)-en-15-óico foram preparados derivados com atividade in vitro discreta frente à Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella cholerasuis e Vibrio parahaemolyticus.

SHORT REPORT

New triterpene and antibacterial labdenoic acid derivatives from Moldenhawera nutans

Juceni P. David^I,*; Jailton Ferrari^II; Jorge M. David^II; Alaíse G. Guimarães^I; Fernanda W. de M. Lima^I; Geórgia L. S. de Souza^I

^IFaculdade de Farmácia, Universidade Federal da Bahia, 40170-290 Salvador-BA, Brazil

^IIInstituto de Química, Universidade Federal da Bahia, 40170-290 Salvador-BA, Brazil

ABSTRACT

A new triterpene derivative (22b-hydroxylupeol) was isolated from the MeOH extract of stems of Moldenhawera nutans (Leguminosae) together with labdenoic acid derivatives of common occurrence in this species. From the labd-8(17)-en-15-oic acid were prepared simple derivatives, which exhibited in vitro weak activity against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella cholerasuis, and Vibrio parahaemolyticus.

Keywords:Moldenhawera nutans, Leguminosae, antibacterial labdene derivatives, 22b-hydroxylupeol

RESUMO

Um novo triterpeno (22b-hidroxilupeol) foi isolado do extrato em metanol do caule de Moldenhawera nutans (Leguminosae) além de diterpenos derivados do ácido labdenóico de ocorrência comum nesta espécie. A partir do ácido labd-8(17)-en-15-óico foram preparados derivados com atividade in vitro discreta frente à Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Salmonella cholerasuis e Vibrio parahaemolyticus.

Introduction

Genus Moldenhawera Schrard. (Leguminosae: Caesalpinoideae), endemic to Northeast Brazil, is represented by approximately ten species.¹ Previous phytochemical study of M. nutans resulted in the isolation of four known labdene diterpenes besides a new bis-diterpene named moldenin.² The present work describes the results of the fractionation of the hexane phase obtained from the MeOH extract of M. nutans. Besides the known diterpenes previously isolated, it was also obtained the 3-oxo-labd-8(17)-en-15-oic acid (1) as a methyl derivative (1a), and three triterpenes, lupeol, betulin, and the new lupane derivative (2). The labd-8(17)-en-15-oic acid (3) was the predominant compound in this extract. From this compound, derivatives (4-8) were prepared and some of them were submitted to in vitro antibacterial assays.

Results and Discussion

The structural elucidation of 1a (Figure 1) was based on MS, IR, and NMR data analyses. Comparison of NMR data of the methyl ester derivative with moldenin,² methyl ent-3-oxo-labd-8(17)-en-15-oate³ and literature data⁴ allowed establishing the labdanic structure. The normal series of this compound was confirmed by positive optical rotation.

The HREIMS of 2 exhibited a molecular ion signal at m/z 442.3829, indicating the molecular formula C₃₀H₅₀O₂ (requires 442.3811). The ¹H NMR data (Experimental section) showed characteristic signals of lupane triterpene, seven methyl groups, one isopropenyl (d 4.61, 4.71, and 1.70) and two signals of oxymethine hydrogens (d 3.68 and 3.20). The ¹³C NMR spectra (BB and DEPT 135º) displayed 30 signals and confirmed the data above through the resonances displayed at d 19.3, 109.8, 150.3, as well as at d 78.0 and 79.7 for the isopropenyl and two oxymethine groups, respectively. The presence of an additional oxymethine signal indicated that 2 is a hydroxylated lupeol derivative. The localization of the hydroxyl group at C-22 of the lupane framework was proposed by comparison with ¹³C NMR data (Table 1) of 16-hydroxylupeol⁵ and the correlations observed in the long range HETCOR of H-22 (d 3.68) and C-28 (d 12.2), C-19 (d 45.9) and C-18 (d 45.1) as well as by the carbon shifts observed in the diacetyl derivative (2a) (Table 1). Thus, the NMR spectra of 2a showed a shielding effect at C-21 (Dd = -3.5 ppm), which is indicative that C-22 bears a hydroxyl group. The insignificant effect observed at C-19 in the NMR spectra of 2a together with the coupling constants observed in the ¹H NMR suggested that the hydroxyl group is in equatorial position. The ¹HNMR nOe difference spectra of this compound permitted to corroborate the proposition. When the H-22 was irradiated it was possible to assign increments in Ha-21 (25%), H-29 (3%) and, H-19 (10%). These findings are indicative that all the affected protons were in the same plane. The fragmentation pattern observed in the MS of 2 was also indicative of a hydroxyl group in C-22 of the cyclopentyl ring, especially by mass fragments of 2 at m/z 374 and 2a at m/z 458 (Figure 2).

Since labd-8(17)-en-15-oic acid (3) showed antibacterial activity (Table 2), some of its simple chemical derivatives were prepared and also evaluated. Derivative 3 was refluxed with MeOH/HCl to obtain the isomeric methyl esters 4a and 5. Next, 4a was submitted to allylic oxidation by t-butyl chromate⁶ and furnished 6a. It was also prepared the epoxy derivatives 7a and 8a from 4a by reaction with MCPBA. The derivatives 7a and 8a are new and they were characterized by spectrometric analyses data. Compounds 1a, 2, 4a, and 6a-8a were submitted to antibacterial evaluation and none of them was active; while the acid derivatives (1, 4, 6-8) obtained after saponification, together with 9, a labdane previously obtained from this plant, showed moderate antibacterial activity at 90 µg per disc (Table 2). These results indicate the need of the presence of an acid group for the antibacterial activity and also that structural features contribute to the inhibition area as well.

Experimental

General procedures

The ¹H and ¹³C NMR, DEPT, COSY, and HETEROCOSY (J 140 and 9 Hz) spectra were obtained on a Varian Gemini 2000 instrument employing CDCl₃ as both solvent and reference. The FTIR spectrum was recorded on a JASCO spectrophotometer Mod. Valor III. MS was recorded on a Micromass Autospec spectrometer (HRMS) and an HP model 5973 spectrometer (EIMS). Melting points were measured on a Microquímica MIAPF 301 apparatus and are uncorrected. Column chromatography was carried out on silica gel 60 and, silica gel TLC was used to monitor the fraction employing iodine fumes, Liberman-Buchard spray reagent, and UV light (254/366 nm).

Plant material

The plant material of M. nutans were collected at sandy soil of Reserva do Parque da Lagoa do Abaeté, Salvador, BA, Brazil in the spring of 1997 and identified by Prof. Maria L. S. Guedes of Herbarium Alexandre Leal Costa, where a voucher (#029057) is deposited.

Extraction and isolation

The powdered stem (4 kg) of Moldenhawera nutans was extracted with MeOH. The methanol extract (153 g) was partitioned with hexane, furnishing 73.2 g of extract. The hexane phase was purified through CC over silica gel with mixtures of hexane/EtOAc as eluents. The fraction eluted with 5% of EtOAc furnished the labd-8(17)-en-15-oic acid (3, 42 g). All the fractions eluted from the main CC (7.3 g) with hexane:EtOAc (8:2) were submitted to methylation using an ether solution of N-methyl-N-nitroso-p-toluenesulfonamide (Diazald^®). The product of this reaction was then subjected to silica gel CC and from the fraction eluted with hexane:EtOAc (9:1) afforded 39.9 mg of 1a (methyl 3-oxo-labd-8(17)-en-15-oate), lupeol (10.5 mg), and 22b-hydroxylupeol (2, 70.3 mg). The fraction eluted with hexane:EtOAc led to the isolation of 25.4 mg of betulin⁷ and methyl 3-hydroxy-labd-8(17)-en-15-oate (9a, 102 mg).

Methyl 3-oxo-labd-8(17)-en-15-oate (1a)

Pale yellow oil. 33.4º (c 0.1, CHCl₃), IR(film) n_max/cm^-1: 3080, 2950, 1737, 1706, 1643, 1385, 1160, 1008, 890; C₂₁H₃₄O₃ (Found: C, 75.1; H, 10.5%. Requires: C, 75.4; H, 10.2%), EIMS: 70 eV (rel. int.) m/z: 334 [M⁺] (18), 319 (4), 303 (7), 233 (51), 220 (15), 205 (48), 177 (16), 163 (22), 137 (34), 135 (100), 123 (68), 109 (68); ¹H NMR (300 MHz, CDCl₃): d 4.88 (sl, 1H, H-17a), 4.55 (sl, 1H, H-17b), 3.64 (s, 3H, OCH₃), 1.08 (s, 3H, Me-19), 1.01 (s, 3H, Me-18), 0.93 (d, J 6.6 Hz, 3H, Me-16), 0.85 (s, 3H, Me-20); ¹³C NMR (75 MHz, CDCl₃): d 35.3 (C-1), 34.4 (C-2), 216.2 (C-3), 47.4 (C-4), 54.8 (C-5), 24.8 (C-6), 37.6 (C-7), 146.9 (C-8), 55.6 (C-9), 39.0 (C-10), 21.1 (C-11), 37.3 (C-12), 30.5 (C-13), 41.5 (C-14), 173.2 (C-15), 21.4 (C-16), 107.2 (C-17), 19.3 (C-18), 25.6 (C-19), 13.8 (C-20), 51.0 (OCH₃).

22b-Hydroxylupeol (2)

White amorphous powder; mp 158-160 ºC. 71.2º (c 0.1, CHCl₃); IR(film) n_max/cm^-1: 3401, 2934, 2868, 1643, 1455, 1385, 880; HREIMS: m/z 442.3829 (C₃₀H₅₀O₂ requires 442.3811), EIMS: 70 eV (rel. int.) m/z: 442 [M⁺] (12), 427 (4), 374 (100), 291 (6), 273 (57), 247 (14), 234 (12), 219 (8), 207 (23), 189 (31), 175 (14), 161 (13), 147 (16), 135 (35), 121 (24), 107 (26); ¹H NMR (300 MHz, CDCl₃): d 4.71 (m, 1H, H-29b), 4.61 (m, 1H, H-29a), 3.68 (t, J 8.8 Hz, 1H, H-22), 3.20 (dd, J 10.7 and 5.6 Hz, 1H, H-3), 2.46 (m, 1H, H-19), 1.70 (s, 3H, Me-30), 1.60 (m, 1H, H-5), 1.06 (s, 3H, Me-23), 0.98 (s, 3H, Me-24), 0.94 (s, 3H, Me-26), 0.85 and 0.84 (s, 3H each, Me-28 or Me-27), 0.79 (s, 3H, Me-25); ¹³C NMR (75 MHz, CDCl₃): see Table 1.

Preparation of derivatives

Acetylation of 22b-hydroxylupeol (2)

Compound 2 (15.0 mg) was added to a solution of pyridine (0.5 mL) and acetic anhydride (2.0 mL) and the mixture was left at room temperature for 24 h. Cold H₂O was added and the diacetyl derivative (2a, 14.3 mg) was extracted with CHCl₃.

3b,22b-Diacetoxylup-20(29)-ene (2a)

Oil, EIMS: 70 eV (rel. int.) m/z: 526 [M⁺] (35), 466 (42), 458 (50), 416 (46), 398 (10), 289 (21), 276 (39), 249 (19), 216 (38), 201 (50), 189 (95), 161 (27), 135 (58), 107 (42); ¹H NMR (300 MHz, CDCl₃): d 4.70 (sl, 1H, H-29b), 4.60 (sl, 1H, H-29a), 4.65 (t, J 8.9 Hz, 1H, H-22), 4.46 (dd, J 10.9 and 5.8 Hz, 1H, H-3), 2.48 (m, 1H, H-19), 2.04 (s, 3H ), 2.03 (s, 3H), 1.69 (sl, 3H, Me-30), 1.03 (s, 3H, Me-23), 0.92 (s, 3H, Me-24 and Me-26), 0.85, 0.84 and 0.83 (s, 3H each, Me-28 or Me-27 or Me-25); ¹³C NMR (75 MHz, CDCl₃): see Table 1.

Preparation of derivatives of labd-8(17)-en-15-oic acid (3)

Compound 3 (1.0 g) was refluxed with 36 mL of an acidic methanolic solution (HCl 0.048 N) under stirring for 4.5 h. Sequentially, water was added and the solution was extracted with CHCl₃. The product was submitted to Si gel with AgNO₃ CC and eluted with hexane:EtOAc (9:1). This procedure furnished 489.4 mg of methyl labd-8-en-15-oate⁸ (4a, 49%) and 210 mg of methyl labd-7-en-15-oate⁹ (5a, 21%). Methyl labd-8-en-15-oate (4a, 480 mg) was diluted to 11 mL of CCl₄ and sequentially added with 3.5 mL of acetic acid, 2.0 mL of acetic anhydride, and 4.5 mL of solution of terc-butyl chromate freshly prepared by established procedures.⁶ The system was refluxed for 90 minutes followed by the addition of 21 mL of an oxalic acid (5 %) aqueous solution. The mixture was stirred until two layers were produced and next it was extracted with CHCl₃. The organic phase was recovered and washed with a 10% solution of Na₂CO₃ and yielded 243.1 mg of methyl 7-oxo-labd-8-en-15-oate¹⁰ (6a, 50%). In parallel, to a solution of 100 mg of 4a in 4 mL of CH₂Cl₂ was added another solution of 80 mg MCPBA in 4 mL of dichloromethane and the mixture was stirred for 30 minutes. Next, purification of the mixture reaction product through CC over Al₂O₃ with hexane/EtOAc (7:3) as an eluent, furnished compounds 7a (24.3 mg) and 8a (15.8 mg).

Methyl labd-8-en-15-oate (4a)

Oil. 60.2º (c 0.8, CHCl₃); IR(film) n_max/cm^-1: 2947, 2926, 1741, 1643, 1459, 1154, 887; C₂₁H₃₆O₂, EIMS: 70 eV (rel. int.) m/z: 320 [M⁺] (13), 305 (24), 264 (5), 196 (10), 191 (100), 177 (13), 163 (15), 149 (28), 135 (33), 121 (53), 109 (47); ¹H NMR (300 MHz, CDCl₃): d 3.67 (s, 3H, OCH₃), 1.54 (s, 3H, Me-17), 0.97 (d, J 6.4 Hz, 3H, Me-16), 0.87 (s, 3H, Me-19), 0.83 (s, 3H, Me-18) and 0.79 (s, 3H, Me-20); ¹³C NMR (75 MHz, CDCl₃): d 33.6 (C-1), 19.1 (C-2), 39.5 (C-3), 33.3 (C-4), 51.8 (C-5), 25.4 (C-6), 36.9 (C-7), 125.4 (C-8), 140.4 (C-9), 38.9 (C-10), 19,0 (C-11), 41.4 (C-12), 31.5 (C-13), 37.3 (C-14), 173.6 (C-15), 19.0 (C-16), 20.1 (C-17), 21.7 (C-18), 33.3 (C-19), 19.6 (C-20), 51.9 (OCH₃).

Methyl labd-7-en-15-oate (5a)

Oil. 30.2º (c 0.2, CHCl₃), C₂₁H₃₆O₂, EIMS: 70 eV (rel. int.) m/z: 320 [M⁺] (9), 305 (15), 196 (12), 191 (52), 177 (6), 163 (10), 149 (15), 135 (20), 122 (100), 109 (84); ¹H NMR (300 MHz, CDCl₃): d 5.39 (sl, 1H, H-7), 3.66 (s, 3H, OCH₃), 1.68 (s, 3H, Me-17), 0.97 (d, J 6.5 Hz, 3H, Me-16), 0.84 (s, 3H, Me-19), 0.81 (s, 3H, Me-18) and 0.78 (s, 3H, Me-20); ¹³C NMR (75 MHz, CDCl₃): d 39.2 (C-1), 19.0 (C-2), 42.2 (C-3), 32.7 (C-4), 54.7 (C-5), 23.8 (C-6), 122.1 (C-7), 135.1 (C-8), 49.8 (C-9), 36.6 (C-10), 25.3 (C-11), 37.1 (C-12), 30.8 (C-13), 41.7 (C-14), 172.9 (C-15), 18.5 (C-16), 22.2 (C-17), 21.7 (C-18), 33.1 (C-19), 13.5 (C-20), 51.7 (OCH₃).

Methyl 7-oxo-labd-8-en-15-oate (6a)

Oil. 41.1º (c 1.0, CHCl₃); IR(film) n_max/cm^-1: 2953, 1736, 1661, 1605, 1461, 1437, 1156, 1080, 1007; C₂₁H₃₄O₃, EIMS: 70 eV (rel. int.) m/z: 334 [M⁺] (30), 303 (7), 233 (52), 220 (15), 205 (44), 177 (10), 163 (10), 149 (13), 135 (100), 123 (48), 109 (56); ¹H NMR (300 MHz, CDCl₃): d 3.56 (s, 3H, OCH₃), 1.62 (s, 3H, Me-17), 0.90 (d, J 6.4 Hz, 3H, Me-16), 0.96 (s, 3H, Me-19), 0.80 (s, 3H, Me-18) and 0.76 (s, 3H, Me-20); ¹³C NMR (75 MHz, CDCl₃): d 35.7^* (C-1), 18.5 (C-2), 41.2^# (C-3), 33.0 (C-4), 50.1 (C-5), 35.2^* (C-6), 200.0 (C-7), 129.7 (C-8), 168.0 (C-9), 40.8 (C-10), 26.9 (C-11), 35.1 (C-12), 31.3 (C-13), 41.0^# (C-14), 173.1 (C-15), 19.4 (C-16), 11.2 (C-17), 21.2 (C-18), 32.4 (C-19), 18.1 (C-20), 51.3 (OCH₃), ^*,# values may be interchangeable.

Methyl 8b,9b-epoxy-labd-8-en-15-oate (7a)

Oil. 38.2º (c 0.8, CHCl₃), C₂₁H₃₆O₃, EIMS: 70 eV (rel. int.) m/z: 336 [M⁺] (14), 321 (10), 305 (7), 278 (15), 264 (20), 253 (25), 251 (23), 235 (20), 207 (100), 177 (18), 163 (25), 149 (58), 125 (57), 121 (35), 109 (47); ¹H NMR (300 MHz, CDCl₃): d 3.53 (s, 3H, OCH₃), 1.13 (s, 3H, Me-17), 0.89 (d, J 6.4 Hz, 3H, Me-16), 0.96, 0.78 (s, 3H, Me-19 and Me-18) and 0.76 (s, 3H, Me-20); ¹³C NMR (75 MHz, CDCl₃): d 33.2 (C-1), 17.2 (C-2), 41.5 (C-3), 32.8 (C-4), 42.3 (C-5), 23.9 (C-6), 43.5 (C-7), 62.2 (C-8), 72.2 (C-9), 38.5 (C-10), 18.4 (C-11), 41.4 (C-12), 31.0 (C-13), 35.1 (C-14), 173.5 (C-15), 19.6 (C-16), 21.2 (C-17), 21.9 (C-18), 33.5 (C-19), 17.2 (C-20), 51.3 (OCH₃).

Methyl 8a,9a-epoxy-labd-8-en-15-oate (8a)

Oil. C₂₁H₃₆O₃. 42.2º (c 0.6, CHCl₃), EIMS: 70 eV (rel. int.) m/z: 336 [M⁺] (15), 321 (13), 305 (6), 278 (15), 264 (23), 253 (26), 251 (26), 235 (18), 207 (100), 177 (18), 163 (22), 149 (62), 125 (59), 121 (36), 109 (52); ¹H NMR (300 MHz, CDCl₃): d 3.53 (s, 3H, OCH₃), 1.24 (s, 3H, Me-17), 0.90 (d, J 6.4 Hz, 3H, Me-16), 1.00, 0.80 (s, 3H, Me-19 and Me-18) and 0.74 (s, 3H, Me-20); ¹³C NMR (75MHz, CDCl₃): d 33.4 (C-1), 16.7 (C-2), 41.4 (C-3), 33.6 (C-4), 53.8 (C-5), 29.2 (C-6), 29.0 (C-7), 64.7 (C-8), 72.5 (C-9), 38.7 (C-10), 19.7 (C-11), 41.4 (C-12), 31.4 (C-13), 37.0 (C-14), 173.5 (C-15), 19.5 (C-16), 21.4 (C-17), 21.9 (C-18), 33.2 (C-19), 16.5 (C-20), 51.3 (OCH₃).

Hydrolyses of the ester derivatives

All the methyl ester derivatives were submitted to hydrolysis by adding 1.5 mL of a solution of 10 mg NaOH in H₂O/EtOH (1:1) to 10 mg of each compound for 30 minutes. After this time, the reaction medium was saturated with 3 mL of brine and the produced ppted was acidified with 4 mL of an aqueous solution of H₂SO₄ and extracted with CHCl₃. The organic phase was washed and dried over NaSO₄, yielding acid compounds.

Antibacterial assays

This assay was performed by disc diffusion method using the established protocol.¹¹ The antibacterial activity using paper disk with f 9 mm was determined through the microorganism growth inhibition halo for Staphylococcus aureus (ATCC 10708), Pseudomonas aeruginosa (ATTCC 15442), Escherichia coli (ATCC 112229), Salmonella cholerasuis (ATCC 10708), and Vibrio parahaemolyticus (ATCC 17802) under the action of test substances (90 mg). Penicillin (10 mg), tetracycline (30 mg) and chloramphenicol (30 mg) were used as positive controls.

Supplementary Information

Supplementary data of 1a, 2, 2a, 4a and 6a as ¹³C and ¹H NMR spectra are available free of charge at http://jbcs.sbq.org.br, as PDF file.

Acknowledgments

The authors are grateful to Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq (Brazil), FAPESB and Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for fellowship support and grants. We are in debt to Dr. Ignacio Lopez of Universidad de Extremadura, Badajoz, Spain for HREIMS.

Received: February 23, 2007

Web Release Date: December 12, 2007

Supplementary Information

• 1. Queiroz, L. P.; Lewis, G. P.; Allkin, R.; Kew Bull. 1999, 54, 817.
• 2. David, J. P.; David, J. M.; Yang, S-W.; Cordell, G. A.; Phytochemistry 1999, 50, 443.
• 3. Branco, A.; Pinto, A. C.; Braz Filho, R.; An. Acad. Bras. Cienc. 2004, 76, 505.
• 4. Wehrli, F. W.; Nishida, T. In Fortschritte der Chemie Organischer Naturstoffe/Progress in the Chemistry of Organic Natural Products; Herz, W.; Grisebach, H.; Kirby, G. W., eds.; Springer-Verlag: New York, 1979; v. 36, p. 1.
• 5. Wenkert, E.; Baddeley, G. V.; Burfitt, I. R.; Moreno, L. N.; Org. Magn. Reson. 1978, 11, 337.
• 6. Pinto, A. C.; Pereira, A. L.; Kelecom, A.; Porreca, L. M.; Ribeiro, N. M.; Barnes, R. A.; Chem. Pharm. Bull. 1988, 36, 4689.
• 7. Mahato, S. B.; Kundu, A. P.; Phytochemistry 1994, 37, 1517.
• 8. Weyerstahl, P.; Marschall, H.; Weirauch, M.; Thefeld, K.; Surburg, H.; Flavor Fragrance J. 1998, 13, 295.
• 9. Pinto, A. C.; Braga, W. F.; Rezende, C. M.; Garrido, F. M. S.; Veiga Jr.; V. F.; Bergter, L.; Patitucci, M. L.; Antunes, O. A. C.; J. Braz. Chem. Soc 2000, 11, 355.
• 10. De Pascual, J. T.; Bellido, I. S.; Basabe, P.; Marcos, I. S.; Ruano, I. F.; Urones, J. G.; Phytochemistry 1982, 21, 899.
• 11. Bauer, A. W.; Kirby, M. M.; Sherris, J. C.; Turck, M.; Am. J. Clin. Pathol. 1966, 45, 493.

Publication Dates

• Publication in this collection
  12 Feb 2008
• Date of issue
  2007

History

• Accepted
  23 Feb 2007
• Received
  23 Feb 2007