SOTES
N a y 1969 highest dose tested of 100 mg,kg. Additional in vivo data can be found in Table 11. TABLE I1 Approx single sc doseQ mg/kg-S. aureus UC-76 H-228
--EDso, S. aureus Penicillin
Resistance index in mice'
1 so 80 Penicillin G 16 35 1.6 Osacillin 2,4,6-Trimet hyl70 450 6.4 vycloheptatrienevart)onyl penicillin Standard mouse protection tests [X. IV. Fisher, 31. C. Manning, I,.A. Gagliardi, 11.It. Gaetz, and A. L. IMandson, .Lntibiot. =Inn., 2 W (1959-1960)] involving single subcutaneous dose therapy (0.5 nil) concurrent with lethal intraperitoneal mucinized challenges with either S. aweus UC-76 (sensitive) or H-228 (resistant); groups of t,en mice were used. I n vivo resistance index obtained hy comparing data from the two S . aweus st,rains. (I
529
attributed2 to a "steric effect" exerted by the bulky group(s) attached to the acyl portion. Thus, in some penicillins3 containing ortho-substituted aromatic acyl groups, such as methicillin, diphenicillin, nafcillin, etc., the cleavage of the lactam ring by penicillinaseproducing bacteria is considerably retarded or even prevented, presumably due to the inability of the active sites of the enzyme to achieve the prerequisite fit with the specfic groups on the peiiicilliii. K e wish to describe the preparation of a series of novel seniisyiithetic peiiicilliiis 1 , derived from bell ziiorcaradieiiccarbox~lic acids (la,7b-dihydro-lH-cyclopropa [ n ] n n p l i t h n l c i i c - l - ~ ~ ~ ~ r ~acids). , ~ ~ x ~ l i cFrom con-
Experimental Section Tlc was carried out on silica gel HF plates in the solvent system 1 BuOH-H20--IcOH (80: SO: 2 ) with 6-aminopenicillanic acid (Aldrich Chemical Co.) as standard. The spots were detected siderations of molecular models of these derivatives, it under uv light and with the alkaline SH20H-FeC13 spray. was anticipated that the unique spacial arrangement4 General Procedure for the Preparation of Penicillins.-The particular cycloheptatrienecarboxylic acid was refluxed in Et20 of the acyl group in the vicinity of the lactam ring in the presence of excess SOC12 for 2-3 hr. Evaporation of the mould confer some interesting antibacterial properties, solution and removal of excess reagent gave the corresponding particularly against penicillinase-producing strains of acid chloride which was examined by ir spectroscopy for the S . aureus. absence of C=O peaks. The preparation of the penicillins followed the procedure described below. The majority of the beiiziiorcaradieiiecarboxylic acids -1solution containing 0.39 g (0.002 mole) of 2,4,6-trimethylrequired for this study were prepared by published cycloheptatrienecarhonyl chlorideBin 20 ml of CHsC12 was added procedures. The penicillins were prepared by the acid dropwise to a stirred solution of 6-aminopenicillanic acid (0.42 g, chloride method and were isolated in the form of 0.002 mole) and Et3S (0.7 ml) in 7 nil of CHtC12 at 0". The amorphous potassium salts. Their homogeneity was solution was stirred for 2 hr after the addition was completed and then allowed to warm to room temperature. Evaporation established qualitatively by tlc and ir spectral data, and t o dryness folloived by addition of Ne&O (20 ml) and filtration quantitatively by a uv spectral assay and colorimetric afforded a pale yellow solution which was concentrated to dryness. procedures. The residue was dissolved in 15 ml of H20, the solution was Results of the antibacterial tests5 are listed in Tables covered with 30 ml of EtO.Ic, and the pH was adjusted t o 2.2 with 10: aqueous H2S04 a t 0 '. The organic phase was separated. I and 11. It can be seen that, in general, the derivatives washed rapidly (HTO), dried (XaeSO,), and filtered. -1solution exhibit in vitro antibacterial activities against penicilof SOYc potassium 2-ethylhexanoate in BuOH was added t o the linase-producing strains, which are somewhat superior filtrate and the lvhole was concentrated to a small volume to penicillin G, but none approach the levels shown by (ca. 5 ml). Et20 was then added t o the neutral solution until oxacillin. The most promising compound appeared to precipitation was complete. ;\fter st'anding overnight a t 0 the precipitated K salt of the penicillin was filtered, washed h c the unsubstituted penicillin 1, which has a favorable (EtrO), and dried (P205);yield, 0.3 g of an almost colorless solid. ratio of activities us. the two X.aureus strains. I t is of Tlc revealed the presence of only one spot. dnal. ( C ~ ~ H E K S ~ O ~ . interest to note that minor variations in the nature and 2Hz0) C, 50.08; H, 4.90; N, 6.23; S, 7.12. Found: C, 50.62; positions of substituents in the substituted compound H, 4.60; S,5.93; S,6.72. ')
Acknowledgment.-The authors thailk Dr. C. L. Heifetz of these laboratories for carrying out the biological test's.
-_
Synthetic Penicillins Derived from Benznorcaradienecarboxylic Acids
caused appreciable variations in i n vitro activity, particularly against Streptococcus pyoye?zes (15 and 16). Compound 1 mas well tolerated in mice at the highest dose tested of 100 mg kg. Additional data on i n vivo tests using 1 are provided in Table 11, together with comparative data on penicillin G and oxacillin. Experimental Section
STEPIIES
H.\SESSI.\X~ . I S D GUSTHER
SCHUTZE
Research Laboratories, Parke, Davis and Company, . 4 i z n ATbor, .Ilichigan .$&'lo6 Received :\-owember 6,1968
The eiihaiiced antibacterial activity of certain commercially available penicillins against penicillinaseproducirig strains of Staphylococcus aureus has been (1) To \%hornall correspondence should be addressed a t the Departmelit of ChemlstiJ, Uiiiveisity of SZontreal, Alontreal, Quebec, Canada.
Melting points are uncorrected. Tlc were developed in the solvent system BuOH-H?O-.IcOH (SO: 80: 2) using silica gel HF plates and spots were detected by uv visualization and the alkaline SHzOH-FeC13 spray. la,2,3,7b-Tetrahydro-2,3-dichloro-lH-cyclopropa[a]naphthaIene-1-carboxylic Acid ( 1 3 b - A solution containing 1.86 g (0.01 mole) of la,7b-dihydro-1H-cyclopropa[a]naphthalene-l(2) R.Knox, Nature, 192, 492 (1961). (3) For a review see, J. 0. Klein and SI. Finland, N e u Engl. J . M e d . . 269, 1019, 1074, 1129 (1963). ( 4 ) R. Huisgen and G . Juppe, Chem. Ber., 94.2332 (1961). ( 5 ) 31.W. Fisher, 11.C . Nanning, L. A. Gagliardi, hl. R. G aetz, and A . I.. Erlandson, ilntibiot. A n n . , 293 (1959-1960).
0.1
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./
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0 .I
23.0
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30.0
0.2
0.4
12.5
0.4
-I
I .ti
12.3
0.4
i
:3. I
25.0
0.S
j
0.4
12.5
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diniethyl-lH-c~ycluprop:t[a]naphtl~ale~~ecarhosylic acid6 (0.635 g, rsriios?;lic acid' in 30 ml of CHC1, was saturated with C12 at, 0 '. 3 mmoles) in 75 ml of AIeOH was hydrogenated (5% Pd-C) at After stirring at room temperature for 30 min, the solvent and 3.5 kg/cm2. After 24 hr, the catalyst was filtered and the fi1trat)e excess C12 were removed under redu(aed pressure leaving a white was evaporated to give the product' as a while solid. Recrystal$()lid ( 2 g). Iiecrystallization ( ~ 1 ~gave 0 ~ t h e) pure product, lizatioii (AIeOH) gave the pure product (0.13 g), 11111 168-171". i 1 1 ~151-132°. .LTICLI. (CirH,oCl?O?)C1. . I ? I I J ~ . (C1.1HiaOn)C, 13. la,2,3,7b-Tetraliydro-2,6-dimethyl-1H-cyclopropaja]napl~tl~a.. lene-1-carboxylic Acid (5).---1 solution of 13,71,-dihydlo-",0((;) R. Huisgen and G . J u y i w , Tetrahedron, 15, 7 (1961).
May 1969
531
NOTES
TABLE I1 Penicillin
Penicillin G Oxacillin Compound 1
Acute single sc dosea EDN,mg/kg S. aureua S. aweus UC-76 H-22Sb
1 16 20
80 25 110
Researches in the Field of Antiviral Compounds. Mannich Bases of 3-hydroxy coumarin
Resistance indexC
80 1.6 5.5
Standard mouse protection tests, involving single-dose therapy concurrent with lethal intraperitoneal challenges. Groups of ten mice were used. For details, see ref 5. S. aureus H-228 refers to a penicillinase-producing strain. I n vivo resistance index was obtained by comparing data from the two S. aureus strains.
GIAN31. CINGOLANI, FULVIO GUALTIERI,AND
3-Azido-la,2,3,7b-tetrahydro-2-hydroxy-lH-cyclopropa [a]-
Acknowledgment.-The authors wish to thank Dr. C. L. Heifetz of these laboratories for carrying out the biological tests. (7) F. R. Batchelor, F. P. Doyle, J. H. C. Nayler, and G. N. Robinson, Nature, 183, 257 (1959).
PIGINI~
Instztute of Pharmaceutzcal and Organzc Chemzstry, Unzverszty of Camerzno, Camerzno, Italy
(I
naphthalene-1-carboxylic Acid (14).-A solution of 2,3-epoxyla,2,3,7b-tetrahydro-lH-cyclopropa [alnaphthalene-1-carboxyl ethyl ester6 (2.3 g, 0.01 mole) in 40 ml of methyl Cellosolve containing 1.3 g of SaN3, 0.5 g of SH&l, and 2 drops of H t 0 was refluxed for 4 hr. After cooling and evaporating to dryness, the residue was extracted (EtzO) t o give 0.9 g of a pale yellow liquid, exhibiting strong Kz and OH absorption bands in the ir spectrum. A portion of the ester (0.18 g ) was dissolved in MeOH containing 1OyoS a O H and the solution was allowed to stir at 0" for 1 hr. The MeOH was removed under reduced pressure without heating, the residue was dissolved in H20 and acidified, and the solution was extracted (Et20). Processing the extracts in the usual way afforded the product as an amorphous white powder, mp 115120". Anal. (C12H11SaOa) C, H, S. 3-Azido-la,2,3,7b-tetrahyclro-2-methoxy-lH-cyc~opropa[a]naphthalene-1-carboxylic Acid (15).-To a refluxing solution of the preceding compound (2.73 g, 0.01 mole) in 30 ml of ?\le1 was added 15 g (0.06 mole) of Ag20, in portions. The mixture was stirred under reflux overnight and filtered, and the filtrate was evaporated to dryness (yield 2.5 g). -4portion of the oily residue (2.1 g ) in 15 ml of RIeOH was added for 20 ml of cold NeOH containing 0.56 g of SaOH. After stirring for 30 min a t 0", the solution was concentrated at low temperature, the residue was acidified with dilute HC1, and the solution was extracted (EtsO). Processing the Et20 extracts afforded the product as a pale yellow oil which was used as such in further steps; ir (liquid film), 2100 cm-' (azide). General Procedure for the Preparation of Penicillins.-The particular acid was converted t o the acid chloride by refluxing in CsHs containing a slight excess of SOcl2 during 2-3 hr. The acid chlorides obtained by removing the solvent and drying the residue over KOH pellets zn vacuo had the expected spectral properties and were used as such. A typical preparation is described in detail in the case of 1. A solution of la,7b-dihydro-1H-cyclopropa[a]naphthalene-lcarbonyl chloride4 (0.4 g, 0.2 mmole) in 15 ml of CH2C12 was added dropwise to a stirring solution of 6-aminopenicillanic acid' (0.42 g, 2 mmoles) and Et3S (0.7 ml, 0.46 mmole) in CHCl? (7 ml) at 0". The mixture was stirred a t 0 " for 2 hr and warmed to room temperature, and the solvent was removed under reduced pressure. bIe2CO (20 ml) was added, the insoluble material was filtered, the filtrate was concentrated, and the residue was dissolved in H20 (15 ml). The solution was covered with 20 ml of EtOAc and cooled and the pH was adjusted to 2.2 with cold aqueous H2S04. The organic layer was separated, washed quickly (HlO), dried (Sa2804), and heated with 1 ml of a 507, solution of potassium 2-ethylhexanoate in BuOH. The solution was concentrated a t low temperature to about 5 ml and heated with EtzO until precipitation was complete. After standing at 5" for several hours, the precipitate was filtered, washed well (EtlO), and dried; yield 0.2 g of an off-white solid. The product exhibited a single spot on tlc. Anal. Calcd for C ? G H ~ ~ O ~ X ? S K . HC, ? O55.00; : H, 4.35; Ti, 6.37; 8, 7.26. Found: C, 55.58; H, 4.54; X, 6.90; S, 7.31.
hI.4RI.4
Recezved October 16, 1968
The synthesis of some 2,4-dioxo-3-hydroxyiminochromane derivatives showing good antiviral activity has been dealt with in a previous note;2these compounds, however, developed a high degree of cytotoxicity. I n order to lower their toxicity we have started synthetic work with the aim of modifying the active molecule while preserving the cyclic a-dicarbonyl structure. The present note deals with the preparation of several 4-N, N-dialkylaminomethyl-3-hydroxycoumarins obtained through the Mannich reaction. 3-Hydroxycoumarin was treated with formaldehyde and various primary and secondary amines so that products containing an a-dicarbonyl as well as a basic group were obtained; the latter is reported to be present in many antiviral compound^.^ The synthetic steps leading to these compounds are described in the Experimental Section; the synthesized compounds are listed in Table I. Compounds 3, 4, 7, and 8 are quite unstable Rlannich bases and give, when boiled in EtOH, 4,4'-methylenebis(3-hydroxy~oumarin)~ thus showing that the aminomethyl group replaces position 4. All the bases react with AczO as reported in the literature for analogous Mannich bases;: in fact 12 is obtained by substitution of the aminomethyl group by an acetoxymethyl group and simultaneous acetylation of the 3-hydroxyl group. A by-product [4,4'-methylenebis(3-acetoxycoumarin) (13)] precipitates, its amount depending on the relative instability to heat of the starting base.
1-11
12
I n order to confirm its structure, 13 mas also synthesized by reaction of 4,4'-methylenebis(3-hydroxycoumarin) with Ac20. The structure of 12 has been (1) hl. Pigini thanks the National Research Council of Italy for a fellowship; (2) &I. Giannella and F. Gualtieri, Farmaco, Ed. Sei., 23, 1104 (1968). (3) (a) R. C. Fort, Jr., a n d P. Von R . Schleyer, Chem. Rev., 64. 277 (1964). (b) A. HollinsheadandP. K. Smith,J. Pharmacol. Ezp. Ther., 123.54 (1968); ( 0 ) B. Melander, Antibiot. Chemotherapy, 10, 34 (1960). (4) K. N. Trivedi and S. Sethna, J . Org. Chem., 25, 1817 (1960). ( 5 ) H. A. Bruson and C. W. MacRlullen, J . d m e r . Chem. Soc., 63, 270
C.
(194 1).
