Journal of Medicinal Chemistry, 1971, Vol. 14, N o . 6 463
NOTES
TABLE I11 POTENTIATING
EFFECTO F 2,4-DIAMINOPYRIMIDINES I N COMBINATION WITH SULFISOXAZOLE (SI) AGAINST BACTERI.4L INFECTIONS I N MICE 7
SI Organism
Streptococcus pyogenes Diplococcus pneumoniae Staphylococcus aureus E. coli Klebsiella pneumoniae Proteus vulgaris Pseudomonas aeruginosa Salmonella typhosa Salmonella schottmuelleri a Pyrimidine dose, 50 mg/kg,
Strain
+ sa=
potentiation (-fold)
4 6301 Smith 257
2.1 >5.0 2.0
KA 190 B P58a
2.3 1.1
CDso, mg/kg per os SI 3ca potentiation (-fold)
+
3.8 2.1 4.0b 5.7b 3.1 2.6b 1.4 11.0 5.4
+
+
SI Sda potentiation (-fold)
SI 8ea potentiation (-fold)
6.2
1.7 3.8 >11.0 >8.9 4.1 9.2 0.8 4.0 1.4
2.6 2.1 >3.5 4.7 1.7 11.1 >1.2
2.5 2.5
0.6
1.9
except 10 mgjkg.
After evapn of EtzO, the residue was fractioned under vacuum. 3,4,5-Trichlorobenzyl alcohol distilled a t 155-170" (11 mm) (log, 25%), solidified in the receiver, and melted a t 111-112'. 3,4,5Trichloro-2'-cyanodihydrocinnamaldehydedimethyl acetal followed a t 195-208' (11 mm) (20 g, 35%) and crystd upon standing, mp 85-86'. 2,4-Diamino-5-(3,4,5-trichlorobenzyl)pyrimidine (3d).-3,4,5Trichloro-2 '-cyanodihydrocinnamaldehyde dimethyl acetal (15 g, 0.04 mole) was refluxed with methanolic guanidine (100 ml, 1 M ) for 2 hr and subsequently the solvent was distilled from an oil bath a t 140'. The remaining solid was slurried with H20 filtered by suction and purified via the acetate. The base melted a t 285-286'. The compd formed a monohydrate, which was dehydrated upon drying a t 100". Biological Resuk1l--The in vivo antibacterial activities of 3a-e were tested in mice infected with 100-1000 MLD's of representative Gram-positive and Gram-negative bacteria and treated by oral administration of the respective substances. Compd 3b protected 50y0 of the animals infected with Staphylococcus aureus Smith, Escherichia coli 257, Klebsiella pneumoniae KA, Proteus vulgaris 190, and Salmonella typhosa P58a a t doses of 140, 841, 698, 19, and 268 mg/kg, respectively, but was inactive a t doses of 1000 to 2000 mg/kg against Streptococcus pyogenes 4, Diplococcus pneumoniae 6301, Pseudomonas aeruginosa B, and Salmonella schottmuelleri. Compound 3a protected 507, of the animals infected with S. typhosa P58a at a dose of 177 mg/kg but was inactive a t 500-1000 mg/kg against the other organisms tested. No protective effect was detected when 3c-e were tested a t doses of 250-500, 50, and 100 mg/kg, respectively, against any of the 9 bacterial infections. When the compds were tested in vivo a t a fixed concn orally of 50 mg/kg (except that 3b was administered a t 10 mg/kg against S. aureus Smith, E. coli 257, and P . vulgaris 190) in combination with graded doses of sulfisoxazoleagainst the bacterial infections, various degrees of potentiation of sulfisoxazole were observed. There was a two-fold or greater increase in the activity of sulfisoxazole against S . pyogenes 4 in combination with 3a-d (2.1-, 3.8-, 2.6-, and 6.2-foldI respectively); against D . pneumoniae 6301 in combination with 3b,c,e (2.1-, 2.1-, and 3.8-fo1dl respectively); against S. aureus 209 in combination with 3a-e (>5.0-, 4.0-, >3.5-, 2 . 6 , and >ll.O-fold, respectively); against E. coli 257 in combination with 3a-e (2.0-, 5.7-, 4.7-, 2.5-, and >8.9-fold, respectively); against K . pneumoniae KA in combination with 3b and 3e (3.1-, and 4.1-fo1dl respectively); against P . vulgaris 190 in combination with 3a,b,c,e (2.3-, 2.6-, 11.1-, and 9.2-fold, respectively); in combination with 3b and 3e against S.typhosa P58a (11.0- and 4.0-fo1dl respectively) and in combination with 3b against S. schottmuelleri (5.4-fold). No potentiation of sulfisoxazole was observed with any compound against P. aeruginosa. These results are summarized in Table 111.
Acknowledgment.-The microanalyses were obtained by Dr. F. Scheidl and his associates of our Microanalytical Laboratory. The nmr spectra were obtained by Dr. T. Williams of our Physical Chemistry (11) T h e i n vivo test methodologies may be found in E . Grunberg and Ag. Chemother., 1966, 430 (1967).
W.F. DeLorenzo, Antimicrob.
+
SI 8b" potentiation (-fold)
Department. We gratefully acknowledge the technical assistance of Mr. Sam Gruenman.
Seeds as Sources of L - D o p a ' E. l)AXENBICHLER,* CECIL H. VANETTEN, E. ANN HALLINAN, FONTAINT. It. EARLE,
RhLVIN
Northern Utilization Rcsearch and Development Division, Agricultural Research Service, U . S . Department of Agriculture, Peoria, Illinois 61604 A N D ARTHUR
s. BARCLAY
Crops Research Divasion, Agricultural Research Service,
U . S.Department of .Igriculture, Beltsville, Maryland 20705 Received October 1 , 1970
The L isomer of dopa [3-(3,4-dihydroxyphenyl)alanine] is being used for symptomatic relief of Parkinson's disease.2 I t is presently obtained by synthesis or by processing fish flours3 A patent has been issued4 for its preparation from velvet bean seed. Since the isolation of dopa from V i c i a f a b a in 1913,j the compound has been reported in plant parts of species of the legumes B a p tisia, L u p i n u s , M u c u n a (including Stizolobium) , and V i c i a at levels up to 1.9%.436-8 The compound has also been reported in the Euphorbiaceae as 1.7y0of the fresh weight of the latex of Euphorbia lathyrusg and in the latex from Euphorbia clendroides.l0 I n the course of a survey in which amino acids in seed meals were determined by ion-exchange chromatography of acid hydrolysates, an unidentified peak eluting after leucine1l?l2was observed. The elution position of (1) Presented a t Division of Medicinal Chemistry, 160th National Meeting of t h e American Chemical Society, Chicago, Ill., Sept 1970. (2) J. E. Randal, Todag's Health, 48, 34 (1970). (3) Chem. Eng. News, 48, 43 (Jan 26, 1970). (4) Don V. Wysong, D o i r Chemical Co., U. S. Patent 3,253,023 (1966); Chem. Abstr., 66, 5529a (1966). ( 5 ) M. Guggenheim, 2. Fh&d. Chem., 88, 276 (1913); Chem. Abstr., 8 , 1128 (1913). (6) G.Just, J. Kagan, and T. J . Mabry, personal communication, 1970. (7) hf. Damadoran and R . Rasaswams, Biochem. J . , 31, 2149 (1937). (8) T. Yoshida, Tohoku J . E z p . M e d . , 48, 27 (1945). (9) I. Lisa, Flora ( J e n a ) , 161, 351 (1961); Chem. Absfr., 67, 3786e (1961). (10) M . .4dinolfi. Rend. Accad. Sci. Fis. Mat., N a p l e s , 31, 335 (1964); C h e m . Absfr.. 64, 3961g (1964). (11) C . H. VanEtten, R . W.Miller, I. A . Wolf?, and Q . Jones, J . Agr. Food Chem., 11, 399 (1963). (12) C . H. VanEtten, W ,F. Kirolek, J. E. Peters, and .I S. ,Barclay, ibid., 16, 1077 (1967).
464
Journal of Medicinal Chemisfry,1971, Vol. 14, N o . 6
this peak was reported13 and later confirmed in our laboratory to be identical n-ith that of authentic L-dopa. Based on the appearance of this peak, seed from four species of legumes, Baptisia leucantha, Dolzchos lablab. Mucuna deei-inqiana, and Vicia faba,were the only ones of 379 species analyzed that contained dopa. By calculations from the peak area, the seed from M . deeiinqiana contained 3.0yo and the remaining three species leqs than 0.5yc. Current interest i n the compound prompted us t o search for other, possibly richer, sources. TIC\vas used as the primary tool in screening the seed extracts for detection of dopa. In the nianrier described, dopa could be detected in the extracts of seeds containing amounts of the compound in excess of about 0.p5~c. uv absorption procedure was used for quantitative measurements. I t proved to be a rapid and reliable mearii of ehtimstirig the compound in seed of the Jlucuna species;. ,I total of 724 species from 447 genera of 135 families have heen w-eened b j t h e tlc procedure. About 5OyL of t he genera arid 75ycof the speciei I\ ere in 7 of the 13,j f:imilici. These 7 fwmiliei and the number of genera J\ itliin th(>mthat v e r e examined are iti follovs. L c quminosae, lST,; Boraqinaceae, 29; Euphoi.biaceae, '26 Co.i,iposztae, 2'2; Labiatoe, 0 ; Grainineae, S; arid Lauraecae, 7. Yearly all the remaining families were represented by 1-3 speciec from each One large family, ilic C'i uczfci ae, 11a i not trstcd hecnuic a large number of rrucifcr speciei 11ere previously analyzed for amino arid dopa I\ a- not found on recxnmiriation of the tl:1 t :i The legume family received the greatest attention iri our wrvey xi ith 332 \pecies because (1) the first isolation of L-dopa \\as from 1'. faba.j and ('2) 1-3y0 amount- of t h e compound were found in seed of ionie qpecies of J l u ~ u n a'. ~ S o n e of the seed from 30 ipecics of T'zcza examined coiitaincd enough dopn t o give a pobitive test by the method used. One of 3 accession, of 1'. faba was also arinlyzetl by t h e more sensitive ion-exchange amino :rcitl arialyzer and contained leqi than 0.1% dopa. Sesame seed (Sesanium intlicunz) reportedly coritniiis wnie dopa * Seed from sebame, from Roqerza longi$om (both from the family Petlaliaceae), arid from '2 species of t hc cloiely related famil! J l a i fynzaceae, I\ hich is wmct ime. included a i a part of the Petlalzaceae, did not coiit:tiii ciiough dopa i o be detected. Examination of s ~ e dfrom 3'2 ipccic.. of L'upAo~bia gave negative results, ;ilthough the compouiid i i rcported in the latex from 2 ipeciei 1'' Tlic. cur1 ent 1) examined 724 speciei, \\hen conibirietl x\ it11 tlic ~reviou.specie\ ex:~niined1)) :imino wid analT v\,li ' ? , 1 4 reprewit :L total of 106'2 different species froin 160 ylarit f:iinilie+ I l o p a iqolated i n 2.:37c 1ield from '11. r/ee/wgianagave :HI i i \pectrum in good agreement u i t h that from the icfLlrciicr material, and i t 5 rotation, [a111-S.37" (c 2.0, 1 S HC'1). compared favorably iiith the rotation, [a111 - \ 41'. reported 111 the patent O n l , ~ 111 seed of the genu5 3lucuna \\ere amounts f o u l i t 1 :il)ovc O..jc; (Table 1) The large amounts of the conipouiid fouiid in t hc Jlut una $ample6 and the failure
NOTES
to find corresponding amounts in other species suggested that members of this genus are the best seed sources. T.mL>: I 1lOP.l CONTI.:NT O F 1ILTCUNS.L sPI:CII:S
Species
l>opa,
.TI. aterrinin 31. aterrima 116.aff. atcrrinia d l . aff. atcrriina .If. aff. aterriina J I . aff. rlcoingiana .If. aff, tleeringiantr .If. holtonii .I/. 1ircn.s
ip.
.,i 0 4,:; 4.4
4.6 4.7 4,s 4. 6.7 .>. 2 : ,1
4.4 In defatted, air-dried seed meal. .I/. sp.
5
Origin
LIexico Florida Colombia Costa Rice Nigeria Louisiana Rhodesia Guatemala Florida Georgia Japan
The genus Jlzicuna contains an estimated': 160 species of scandent herbs or shrubs distributed in both the S e w and Old World tropics arid subtropics. Taxonomically, the species of Mucuna have been divided into 3 or 4 sections or subgenera. The only economically important species of Mucuna are the velvet beans belonging to the section Xfizolobiunz, a group considered by Fome taxonomists to constitute a separate genus. Velvet beans arc viny or bush-like annual herbs that are cultivated in various warm regions of the world for fodder, green manure, arid for their edible seeds. With the exception of 14. Iioltonii and 111. urens, both of I\ hich are n ild tropical lianas, the Mucuna species included in the present survey are cultivated velvet beans. -4lthough percentages of dopa are highest in M. holtonii and 21. ui'eizs, these species, because of their wild habit, have little to recommend then1 as potential seed sources of the compound. The variability in dopa content exhibited in our limited sampling of the cultivated velvet beans suggests that high-yielding strains might be developed through breeding arid selection. Experimental Section A 500-mg sample of finely ground, defatted seed meal was heated on a steam bath with 5 ml of 0.1 iV HC1 for 5 min. After coolilig, the mixt was shaken vigorously with 10 ml of EtOH arid centrifuged. For tlc, 1 nil of the ext was concd to 0.5 ml, and a 5- to 10-pl portion was spotted 011 a 20 x 20 cm plate of silica gel G'G (0.25-mm layer). The air-dried plate was developed with i-PrO€I-EtOAc-HzO-AcOIJ (20: 19: 10: l), and the solvent froiit was allowed to migrate 12.5 cm above the point, of sample application. When the plate was sprayed with ninhydrin reagent (0.5CGin n-BuOH-MezCO 1: 1) arid heated 10 mill at 110", dopa (R, 0.55) gave a characteristic gray-blue spot. Two spots of known dopa were applied to each plate as reference material for comparison of color and migration. If dopa was detected in aii extract by tlc, the amt present was estimated by measuring the uv absorption a t 283 nm. A11 ext, prepd as described above, was decanted into a 300-in1 volumetric flask and a11 additional 25 ml of Et0I-I added to the residue meal. After vigorous shaking, the sample was again centrifuged and the alcohol wash was decanted iiito the volunletric flask with the initial ext and made t o vol with EtOH. The added Et011 usually caused flirther pptn, so a portion of the s o h was filtered for measurement of uv absorption. A calibration curve prepd with known dopa was a straight h i e up to 100 pg of dopa/"l, the highest concn measured. Since (15) J. Hutcl~inson,"The Genera of Ploivering Planta, Dicotyledons 1," The Clarendon Press, Oxford, 1961, PI) 433-431. (16) The mention of firm names or trade products does not imply that they are endorsed or recommended l ~ ytile Department of Agriculttlre over o t h e r firms or similar prodllcts not mentioned.
Journal of Medicinal Chemistry, 1971, Vol. 14, No. 6 465
NEW COMPOCNDS exts from seed meals may be expected to exhibit nonspecific absorption in the uv, it was considered likely that a correction would be necessary. Minima on either side of the Am., 283 peak occurred near 255 and 305 nm. To provide the desired correction, absorbances a t 255 and 305 nm were averaged and subtracted from the absorbance a t the maximum to give a net value. From our measurements a net absorbance of 1.000 was equiv to 65 pglml. Confidence in the uv absorption as a rapid means of quantitation was gained by comparison of the value (6.7Yc) obtained for M , holtonii with that calcd for M . holtonii when detd on the ionexchange analyzer (6.2%). Dopa was isolated from M . deeringiana by the patented process.' The dopa used for reference and calibration measurements
was from Mann Research Laboratories. Uv measurements were made with a Beckman Model DK-2a recording spectrophotometer. The names of the 135 families, 447 genera, and 724 species examined in the present work are available from the authors on request.
Acknowledgments.-We thank Alrs. Gertrude Rose for technical assistance and Mr. J. F. Cavins for estimation measurements of dopa with the amino acid analyzer.
New Compounds 2-(5-Nitro-2-thienyl)cinchoninic Acids I. L.4LEZARI1*F. GHABGHARAN, AND R. MAGHSOUDI Department of Chemistry, Faculty of Pharmacy, University of Tehran, Tehran, I r a n
The antibacterial activities of 2-(5-nitro-2-furyl)cinchoninic acid and derivatives have been reported.' I n a search for more potent antibacterial compounds, we have been preparing a series of their S analogs, 2-(5nitro-2-thieny1)cinchoninic acids.
R, Preliminary in vitro tests of the compounds prepared, against Pseudomonas aeruginosa, Proteus vulgaris, Salmonella typhosa, and Staphylococcus album did not show significant activity.
Yield, No.
Rl
Rz
R3
%
Mp, "C deo
2105 280 250 299 4 F 261 5 c1 293 6 C1 H 250 95 7 Br H 262 74 8 Br H 222 H 90 9 CHI H NO2 78 308 H 10 CHI 242 H 82 11 H CHa 282 NO2 91 12 H CHa a All compds were analyzed for C, H, and the anal. results were satisfactory. All compds were subjected to nmr and ir spectroscopy. The spectroscopic data were as expected. * Lit. [P. Schaefer, K. S.Kulkarni, li. Costin, J. Higgins, and L. M. Honig, J. Heterocycl. Chem., 7, 607 (1970)l gives mp 209-211". 1 2 3
H H F
H H H H H
H NO2 H NO2 H NO2 H NO,
80 63 79 84 73 85
Acknowledgments-The authors gratefully acknowledge the constant encouragement of Professor A. Zargari of Tehran University.
Experimental Section2 2 4 2-Thieny1)cinchoninic Acids.-A mixture of 0.02-mole quantities of an appropriate isat,in and 2-acetylthiophene in 15 ml of aq 2OYc KOH and 15 ml of EtOH was heated under reflux for 12 hr. The reaction mixt was cooled and acidified with dil HCl and the resulting yellow ppt was removed by filtration and crystd from AcOH (See Table I). 2-(5-Nitro-2-thienyl)cinchoninicAcids.-To a cold s o h of 0.01 mole of 2-(2-thienyl)cinchoninic acid in 15 ml of concd &SO', 3 ml of a mixt of concd H2S04 and concd HNOl (1:l) was added with vigorous stirring. After 1 hr, 200 g of crushed ice was added t o the reaction mixt and the resulting ppt was filtered and crystd from AcOH. The positions of the NO2 groups were confirmed by nmr spectroscopy (DMSO). (See Table I.) (1) Homer A. nurch, J. .Wed. Chem., 11, 535 (1969). (2) Melting points were taken on a Kofler hot stage microscope and were uncorrected. The ir spectra were determined with a Leitr Model I11 spectrograph. Nmr spectra were obtained on a Varian A60.4 instrument.
Analogs of Albizziin DALER. SARGENT A N D CHARLES G. SKINNER' Departmrnt qf Chemistry, A'orth Texas State University, Denton, Texas 76?203 Received October 22, 1070
The experimental and clinicrl use of asparaginase as as antitumor agent' has led to a renewed interest in the synthesis of analogs of asparagine. Albieziin, L-2amino-3-ureidopropionic acid,:! contains an SH group (1) J. D.Broome, T r a n s . A'. Y . Acad. Sci., SO, 690 (1968). (2) A. Kjaer. P. 0. Larsen, and R . Gmelin, Ezperientza, 16, 253 (1959); Chrm. Abstr., 64, 17263f (1960).
