Bryostatins

Abstract: Antineoplastic agents designated bryostatins (4, 5, 6, 7 and 8) have been isolated from the marine animals Bugula neritina L. and Amathia convoluta (Bryozoan phylum). These and analo­ gues thereof are novel compounds.

Description: This invention relates to bryostatins which are antibiotics isolated from the marine bryozoans Bugula neratina and Amathia convoluta . EP-A-0109811 describes the antineoplastic agents designated bryostatin 1, bryostatin 2 and bryostatin 3 which have been isolated from Bugula neratina . This organism and its location are fully described in EP-A-0109811, as are methods for the isolation and purification of bryostatins. Amathia convoluta occurs in small shrub-like clusters, 10-30 mm tall, where each individual animal of the greyish-brown colony is approximately 0.1 mm in size. See R. Woolcott et al , "Biology of Bryozoans" (Academic Press, N.Y., 1981) and J.S. Rylands, Bryozoans, (Hutchinson and Co., Ltd., London, 1970). In general, A . convoluta resembles the bryozoan Bugula neritina L., and both occur together in certain areas of the north-eastern Gulf of Mexico. Novel compounds according to the present invention are of formula I wherein R is -COCH₂CH₂CH₃ or -COCH₃, and R₁ is -COCH₂CH(CH₃)₂, -COCH₂CH₂CH₃ or -COCH₃. The invention includes also the acylates of these compounds. Particular novel compounds according to the invention are five bryostatins which are defined (with respect to the given formula) as follows: Bryostatin 4: R is COCH₂CH₂CH₃, R₁ is COCH₂CH(CH₃)₂ Bryostatin 5: R is COCH₃, R₁ is COCH₂CH(CH₃)₂ Bryostatin 6: R is COCH₃, R₁ is COCH₂CH₂CH₃ Bryostatin 7: R and R₁ are each COCH₃ Bryostatin 8: R and R₁ are each -COCH₂CH₂CH₃. In particular, Bryostatin 4 has the following formula (I-4), as determined by ¹³C-NMR: The novel compounds of the given formula have free hydroxyl groups. Various acyl esters thereof can be prepared by methods known per se . Acyl derivatives according to the invention can be formed with any of the acids generically or specifically disclosed in EP-A-0109811 at from page 6 line 7 to page 8 line 30. A variety of methods can be used to isolate and purify the bryostatins from samples of B . neritina and A . convoluta , including solvent extraction, partition chromatography, silica gel chromatography, liquid-liquid distribution in a Craig apparatus, absorption on resins, and crystallisation from solvents. These procedures are also all described in EP-A-0109811. All solvents employed for chromatography were redistilled. Column chromatography was performed using either Sephadex® LH-20, from Pharmacia Fine Chemicals AB, Uppsala, Sweden, or silica gel (70-230 mesh) from E. Merck, Darmstadt, Germany. HPLC columns (9.4 x 500 nm) were Partisil®-10 M-9-ODS-2 (C-18 reversed phase) and Partisil®-10 M-9 columns from Whatman, Inc., Clifton, New Jersey, USA. An Altex HPLC with System Controller Model 420 and Model 110A pumps effected the final separations. Preparative and Reversed Phase (KC-18) LTC Plates were provided by Whatman, and Silica gel GF Uniplates were supplied by Analtech, Inc., Newark, Delaware, USA. Plates were visualised by UV light and/or detected by anisaldehyde-acetic acid-sulfuric acid spray (plates heated to about 150 C for 5-10 minutes). Fractions were collected with either Gilson FC-80 (microfractionator) or FC-220 (race-track fractionator) equipment, and a Gilson UV monitor Model HM was used for chromatographic fractionations. Melting points are uncorrected and were observed with a Kofler-type melting point apparatus. Ultra-violet spectra were recorded with a Hewlett-Packard 8540A UV/VIS spectrometer. Optical rotations were recorded using a Perkin-Elmer Model 214 polarimeter. Mass spectra were recorded with a MAT-312 mass spectrometer, and infra-red spectra with a Nicolet FTIR Model MX-1 spectrometer. NMR spectra were obtained with Bruker WH-90, Bruker WH-400 and Varian XL-100 instruments; deuteriochloroform was employed as solvent, with tetramethylsilane as internal standard. The extraction and solvent-partitioning of B . neritina is described in detail in Example 1 and Chart IV of EP-A-0109811. A similar procedure has been used for A . convoluta . Thus, 235 kg (wet weight) A . convoluta were initially reduced to a 61 g carbon tetrachloride PS active fraction as outlined in the Chart, Part 1. The gel permeation-partition type chromatographic separation employing Sephadex HL-20 with 2:3 methylene chloride-methanol as solvent caused a further rapid concentration in fraction D of the major antineoplastic constituents. Useful further separation and concentration were achieved by repeating this step as shown in Part 2 of the Chart. Fraction H seemed to contain the most potent antineoplastic constituents and was further separated by careful fractionation on silica gel to yield active fraction M. The silica gel chromatographic separation with changes in solvent was further expanded to produce active fraction N. The relatively small weight of N (37.5 ng) allowed partial separation by preparative layer chromatography, to yield active fraction O. Even the 18.2 mg fraction O from some 245 kg of animal was still a challenging mixture that required extensive reverse phase and normal phase HPLC procedures for final separation. By this means, bryostatins 4 and 6 were isolated in 1.6 mg and 0.6 mg yields, respectively. Meanwhile, the promising Sephadex® LH-20 fractions G and I were combined as outlined in the Chart, Part 3, and subjected to the same type of fractionation as just summarised for obtaining bryostatins 4 and 6. However, the active fraction P corresponding to fraction M of the preceding separation was considerably more complicated, and required the introduction of a very careful partition chromatographic step utilising the LH-20 gel. The resulting PS in vivo very active fraction Q was separable into the principal active antineoplastic constituent of A . convoluta , utilising the techniques developed for separating fractions M-0. Additional quantities of bryostatins 4 (1a, 6.0 mg) and 6 (1b, 5.7 mg) were isolated accompanied by bryostatin 5 (lc, 3.1 mg) and a new bryostatin designated 8 (2, 4.2 mg). The combined total yields of bryostatins 4 and 6 amounted to 3.1 x 10⁻⁶% and 2.6 x 10⁻⁶%, bryostatin 5, 1.3 x 10⁻⁶% and bryostatin 8, 1.7 x 10⁻⁶%. Results of present PS in vivo antineoplastic evaluations (cf. Separation Scheme Part 3) of bryostatins 4-6 suggests that bryostatin 8 will eventually be found to exhibit similar remarkably high levels. A parallel study of Bugula neritina antineoplastic components also led to the assignment of the structures indicated here for bryostatins 4 and 6. Recognition of bryostatin 8 as a member of this unique series of macrocyclic lactones first arose from viewing the characteristic reddish-purple color produced upon heating a thin-layer chromatogram of the substance sprayed with anisaldehyde reagent. Detailed interpretation of the 400 MHz ¹H-NMR (Table 1) and ¹³C-NMR (Table 2) indicated that bryostatin 8 retained the basic bryopyran ring system and substitution pattern of bryostatin 1. The principal differences seemed to be at C-7 and C-20 where the acetate and (E,E)-octa-2,4-dienoate esters appeared to be absent. Application of the solution phase secondary ion mass spectrometric techniques to bryostatin 8 that we developed for routine detection of molecular ions proved rewarding. A solfolane solution containing sodium iodide gave a molecular ion complex at 903 [M+Na]⁺ and an important fragment ion at 815 [M+Na-88]⁺. Elimination of only 88 mass units led to the assumption that bryostatin 8 was esterified at C-7 and C-20 with butyrate substituents. As part of the structural elucidation of bryostatin 2 it was found that bryostatin 1 could be selectively hydrolyzed using 1% hydrochloric acid in aqueous-methanol. The reaction led to bryostatin 2 as major product and suggested that the steric hindrance around C-20 was responsible for this very helpful selectivity. When bryostatin 8 was allowed to react with 1% hydrochloric acid in aqueous methanol for 1 day at room temperature the major product was presumed to be C-20 butyrate ester if accompanied by the isomeric 1g on the basis of mass spectral and proton magnetic resonance studies. A solution phase secondary ion mass spectrum of the hydrolysis product exhibited a molecular ion complex at 833 [M+Na]⁺ and a quite revealing fragment ion at 745 [M+Na-88]⁺. Thus loss of one butyl ester by selective hydrolysis and the second by mass spectral promoted cleavage provided compelling support for the C-7, C-20 dibutyrate structure 2 for bryostatin 8. Non-branching of the butyl group was ascertained by the NMR studies of bryostatins 4, 6 and 8. When structures for the principal antineoplastic constituents of A. convoluta were established a possible relationship between this animal and the related Bugula neritina came under scrutiny. Voucher animal specimens from all prior collections along with 100 plus kilogram amounts of A. convoluta available from the 1981 and more recent (1982) recollections were closely examined. Amounts ranging from <0.2% to approximately 3% dry weight of Bugula neritina were found growing from the A. convoluta in a parasitic or epiphytic-like manner. The 1981 recollection described herein was found to have approximately 2.5% by weight of attached Bugula neritina . From our experience with the isolation and characterization of bryostatins 4-6 from B. neritina collected in the same general area as A. convoluta , the yields of these bryostatins seem to be two to four times greater than would be expected from A. convoluta containing some 2.5% of B. neritina . But this proportion was too close to safely conclude that they were produced by A. convoluta . On the other hand bryostatin 8 was isolated from A. convoluta in a quantity 50 times greater than would be expected from the companion B. neritina . Thus, bryostatin 8 seems to be a genuine constituent of A. convoluta and/or both animals have a relationship where the bryostatins may be transferred and concentrated by the A. convoluta . Alternatively, if the real source of the bryostatins resides in a common food source such as a dinoflagellate these relationships may be even more complex. The isolation and purification methods chosen can be monitored at each step by performing in vitro and/or in vivo antitumor tests as described by R.I. Geran, N.H. Greenberg, M.M. MacDonald, A.M. Schumacher and B.S. Abbot in Cancer Chemother . Rep . Part 3, Vol. 3 (2): 1-103 (1972); and by Schmidt, J.M.; Pettit, G.R. in Experientia , 34: 659-660 (1978). Such tests include the determination of the concentration of active material required to inhibit the growth of tumor cells in culture, (e.g., the concentration required to inhibit growth by 50 % or the ED₅₀), and of the dose of active material required to prolong the life of mice bearing transplanted tumours. Bryostatin 4 inhibits the PS cell line with an ED₅₀ of 1 to 0.1 ng/kg, and produces a 62% increase in life extension at 46 µg/kg. Bryostatin 5 has an ED₅₀ in the PS cell line of 1.3 to 0.26 ng/ml and an in vivo life extension of 88% at 185 µg/kg. Bryostatin 6 has an ED₅₀ of 3.0 ng/ml and an in vivo life extension of 82% at 185 µg/kg. Bryostatin 7 has an ED₅₀ in the PS cell line of 0.026 ng/ml, and an in vivo life extension of 77% at 92 µg/kg. Bryostatin 8 has an ED₅₀ of 1.3 ng/ml in the murine P₃₈₈ lymphocytic leukemia assay (PS cell line). The novel bryostatins are antineoplastic. For this purpose, they may be administered to humans or animals. Illustrative diseases for treatment, dosage levels, body concentrations and dosage forms are described in detail in EP-A-0109811 at from page 18 line 24 to page 21 line 31. Further, illustrative compositions are given in EP-A-0109811 at from page 21 line 35 to page 25 line 33. The following Examples illustrate the preparation of products of the invention. Example 1 Isolation and Purification of Bryostatin 4 The methylene chloride phase of a methylene chloride-methanol-water extract of Bugula neritina (109 kg wet wt., from the Gulf of Mexico) was successively partitioned in 9:1 to 4:1 v/v methanol-water between hexane and carbon tetrachloride. The overall separations were guided by bioassay (PS system). The carbon tetrachloride fraction was subjected to an extensive series of gel permeation (Sephadex® LH-20, 2:3 v/v methylene chloride-methanol), silica gel (methylene chloride-methanol) partition (Sephadex® LH-20, hexane-methylene chloride-methanol 10:10:1), Prep TL (hexane-ethyl acetate), reverse phase (C-18, methanol-water) and normal phase (partisil® 10, ethyl acetate-heptane-methanol-water) HPL chromatographic procedures. Recrystallization from methylene chloride-methanol afforded 44.5 mg (4.1 x 10⁻⁵ yield) of bryostatin 4 as an amorphous powder melting at 198-200° C; FAB⁴ MS m/z 894 (M⁺ for C₄₆H₇₀O₁₇); [α]²⁷ D +93.6°; (c=0.032, CH₃OH); UV (CH₃OH) λ max (ε, 36,500); IR (KBr) 3470, 2980-2945, 1740, 1725, 1660-1645, 1440, 1390, 1370, 1290, 1240, 1170, 1100, 1080, 1050, and 1000 cm⁻¹; and ¹³C NMR as entered (tentative assignments) on Formula I-4. Detailed interpretation of the 400 MHz NMR spectrum of bryostatin 4 uncovered a series of signals that very closely corresponded to the high resolution proton assignments for the bryopyran ring system of bryostatins 13 a and 2.3 b . In addition, the side-chain protons of the esters at C-7 and C-20 characteristic of bryostatins 1-3 were absent. However, the ¹³C NMR signals at δ 172.03 and δ 172.22 suggested the presence of new ester substitution at C-7 and C-20. Such a conclusion was further strengthened by the C-7 carbon resonance at δ 72.46 and the C-20 carbon resonance at δ 79.13. Similarly, in the ¹H-NMR spectrum of bryostatin 1, the C-7 proton appeared at δ 5.10 (m) and the C-20 proton at δ 5.167 as a sharp singlet. Both signals were very characteristic of the bryostatin 1 molecular geometry. Because of substantial difficulties in recognizing the molecular ion in mass spectra of bryostatins, by a variety of methods, a new series of techniques based on solution phase secondary ion mass spectrometry was developed. These new procedures utilizing an alkali metal iodide, silver tetrafluoroborate or thallium tetrafluoroborate in sulfolane provided, with a great variety of substances including the bryostatins, the facile and routine detection of molecular ions. Application of these new mass spectral methods to further determining the structure of bryostatin 4 proved decisive. With sodium iodide in sulfolane, bryostatin 4 exhibited m/z 917 [M+Na]⁺ with the parent peak at 899 [M+Na-18]⁺, 829 [M+Na-88]⁺ and 815 [M+Na-102]⁺. With silver tetrafluoroborate was found m/z 1001 and 1003 [M+Ag107 and Ag109]⁺, 983 and 985 [M+Ag-18]⁺, 913 and 915 [M+Ag-88]⁺ and 899 and 901 [M+Ag-102]⁺. The loss of 88 mass units suggested elimination of a butyrate ester and water while the loss of 102 indicated elimination of a valerate group and water. Confirmation for these assumptions was obtained by assignment of the remaining ¹H- and ¹³C-NMR signals. By this means the butyrate group was found to be normal and the valerate ester to be iso. A study of the selective acid-catalyzed hydrolysis of bryostatin 1 (1a) to bryostatin 2 (1b) with hydrochloric acid was used as the basis for assigning positions to the butyrate and isovalerate esters. The relative steric compression of C-20 compared to C-7 seemed to favor hydrolysis at the less hindered position (C-7) and bryostatin 1 was found convertable to bryostatin 2 in reasonable yield. Selective hydrolysis of bryostatin 4 (1 mg) with hydrochloric acid (1% in aqueous methanol, 24 hour at room temperature) yielded bryostatin 4a (0.72 mg, FAB MS m/z 833 [M+Na]⁺ for C₄₁H₆₂O₁₆) and as a minor product (100 µg) bryostatin 4b (FAB MS m/z 847 [M+Na]⁺ for C₄₂H₆₄O₁₆). From these results the butyrate ester was assigned to C-20 and the isovalerate ester to C-7. Example 2 Bryostatins 4, 5, 6, and 7 and Acylated Derivatives Collection and Extraction : The recollection of Bugula neritina from the Eastern Gulf of Mexico (Alligator Harbor, Franklin County, Florida) in July 1982, afforded 50 kg wet weight of marine animal which was preserved in 2-propanol. The extraction and solvent partitioning sequences described above provided 37.6 g of bryostatin-enriched methylene chloride extract (PS ED₅₀, 0.058 µg/ml). The extract was subjected to a series of column chromatographic separations which gave bryostatin 4 and later, bryostatins 5-7. Bryostatin 5 From a succession of silica gel column chromatographies and gel permeation (Sephadex® LH-20) chromatography, an active fraction (0.38 g) was isolated as a white amorphous powder with PS ED₅₀ = 1.6 x 10⁻³ µg/ml and which contained bryostatins 4-7 by TLC (7:3 n-hexane:acetone and 2:3 n-hexane :ethyl acetate). By using dry column (2 x 60 cm) silica gel chromatographic methods with n-hexane : acetone (5:1 → 1:1) and n-hexane : ethyl acetate (2:1 → 1:2), six fractions were selected for further purification. The first fraction contained bryostatin 4. The second, third and fifth fractions were subjected to HPLC using Partisil®-10 M-9-ODS-2 (C-18 reversed phase) with a 1:1 → 9:1 methanol :water gradient (flow rate of 2 ml/min). The second fraction (15 mg) yielded pure bryostatin 5 (14.1 mg), melting at 169-172° C (needles from CH₂Cl₂-CH₃OH); FAB MS m/z 866 for C₄₄H₆₆O₁₇:[α]²⁷ D +106.92 (c=0.028, CH₃OH); uv (CH₃OH) λ max 226 nm (ε 36,300); and ir (KBr) 3465, 2980-2935, 1740, 1725, 1660-1640, 1440, 1385, 1370, 1290, 1230, 1165, 1100, 1080, 1060, and 1000 cm⁻¹. Bryostatin 6 The third fraction (65 mg) was subjected to the same HPLC final purification to yield bryostatin 6, 61.9 mg, mp 172-175° C (needles from CH₂Cl₂-CH₃OH); FAB MS m/Z 824 for C₄₁H₆₀O₁₇:[α]²⁷ D +39.92 (c = 0.05, CH₃OH); uv (CH₃OH) λ max 228 nm (ε 35,350); ir (KBr) 3460, 2980-2950, 1740, 1720, 1660-1650, 1440, 1380, 1370, 1280, 1240, 1160, 1100, 1080, 1060, and 100 cm⁻¹. Acid-Catalyzed Hydrolysis of Bryostatin 5 A specimen of bryostatin 5 (3b, 1 mg) was subjected to hydrolysis in 0.5 ml of 1% hydrochloric acid for 24 hour at room temperature. The product, obtained by extraction with methylene chloride, washing with water and drying, was separated by HPLC reversed phase (C-18) column chromatography with methanol:water (from 1:1 to 9:1) to give the C-20 acetate 3e (300 µg) and the C-7 isovalerate 3f (100 µg). The C-20 acetate was obtained as an amorphous powder from aqueous methanol melting at 153-156° C; FAB MS:m/z 805 [M+Na]⁺ for C₃₉H₅₈O₁₆, 787 [M+Na-18]⁺; ir (KBr):3475, 2980-2950, 1740, 1720, 1660-1638, 1435, 1380-1370, 1290, 1240, 1160, 1100, 1090, 1075, 1048, 1005 and 870 cm⁻¹. The C-7 iso-valerate was obtained as amorphous solid from aqueous methanol melting at 147-152° C; FAB MS:mz 847 [M+Na]⁺ for C₄₂H₆₄O₁₆, 829 [M+Na-18]⁺ and 745 [M+Na-102°]⁺; ir (KBr):3470, 3435, 2975-2950, 1738, 1720, 1660-1635, 1440, 1390-1370, 1290, 1240, 1160, 1100, 1075, 1050, 1000, and 870 cm⁻¹. The product was found to be the same as the compound from bryostatin 4 by similar acid hydrolysis. Acid-Catalyzed Hydrolysis of Bryostatin 6 A specimen of bryostatin 6 (1 mg) was hydrolyzed using the same method as described for bryostatin 5. HPLC of the crude product in the same manner as described above provided the C-20 acetate (350 µg), identical to that obtained from bryostatin 5, and the C-7 butyrate (150 µg). The C-7 butyrate was obtained as an amorphous solid melting at 66-170° C; FAB MS:m/z 833 [M+Na]⁺ for C₄₁H₆₂O₁₆, 815 [M+Na-18]⁺ and 745 [M+Na-88]⁺; IR (KBr): 3475, 3430, 2975-2945, 1736, 1720, 1640, 1620, 1440, 1390, 1095, 1080, 1040, and 870 cm⁻¹. Acid-Catalyzed Hydrolysis of Bryostatin 7 A sample of bryostatin 7 (1 mg) was hydrolyzed and the crude product purified by HPLC as outlined for bryostatin 5. The major product was the 20-acetate (370 µg) which was found to be the same product obtained from bryostatin 5. Acetylation of Bryostatin 4 A sample of bryostatin 4 (9.0 mg) was treated with acetic anhydride (0.14 ml) and pyridine (0.2 ml) at room temperature for 4 hours. The mixture was poured into ice-water and extracted with methylene chloride. The extract was washed with dil. hydrochloride acid and water, concentrated and dried under reduced pressure. The crude product (9.5 mg) was purified by HPLC using the same procedure as described for bryostatin 5 above. Pure bryostatin 4 acetate 3h (8.0 mg, 88.9% yield) was obtained as colorless needles from CH₂Cl₂-CH₃OH and melted at 159-162° C:[α]²⁷ D +58.71 (c=0.034, CH₃OH); uv (CH₃OH); λ max 227 nm (ε 36,600); ir (KBr) 3460, 2985-2935, 1745, 1730, 1660-1640, 1440, 1380, 1370, 1290, 1240, 1165, 1100, 1050, and 1000 cm⁻¹. Tables I-IV show the ¹H NMR and ¹³C NMR of bryostatins 5-7. Example 3 Separation of the gel permeation fractions G and I was even more challenging. Both fractions were combined and the 27.56 g combined fraction (Chart B Part 3) was first separated as summarized for fractions H→M to yield PS active fraction P (979 mg). At this stage it was found best to utilize a parition chromatographic procedure employing Sephadex LH-20 and the solvent system n-hexane-methylene chloride-methanol (10:10:1). A 3 cm x 120 cm sized column was used and 12 ml fractions totalling 240 ml were collected. The resulting most active fraction (Q, 266 mg) was further fractionated as noted above for fraction M→N. The remaining separation steps were accomplished by the same general procedures employed with fraction 0 (Chart B Part 2). In order of elution appearance from the HPLC silica gel, were obtained bryostatin 4 (6.0 mg), bryostatin 5 (3.1 mg), bryostatin 6 (5.7 mg) and bryostatin 8 (4.2 mg). Presumably these substances are derived from the association of Amathia convoluta with Bugula neritina . However, bryostatin 8 appears to be a genuine constituent of Amathia convoluta and is characterized as follows. Bryostatin 8 Bryostatin 8 was brought to analytical purity by rechromatography of the original 4.2 mg on a HPLC reverse phase C-18 column with methanol-water (from 50:50 to 90:10) as eluent. The resulting 2.8 mg of bryostatin 8 was obtained as a colorless amorphous powder melting at 170-173° C: [α]²⁷ 2D + 49.9 (c, 0.04, CH₃OH); uv λCH₃OH max 266 (ε 37,500); ir ν KBr max 3551, 3475, 3415, 2975-2950, 1742, 1721, 1640, 1616, 1449, 1380, 1245, 1225, 1165, 1095, 1075, 1050 and 870 cm⁻¹; MS: 903 [M+Na]⁺. The 400 MHz proton, carbon-13 and carbon magnetic resonance data has been displayed in Tables V and VI in comparison with bryostatin 1. Acid-Catalyzed Hydrolysis of Bryostatin 8 to Mono-Butyrate Esters An 0.8 mg specimen of bryostatin 8 was hydrolyzed (24 hours at room temperature) in 0.2 ml of 1% hydrochloric acid in methanol. The products (0.5 mg) were isolated by HPLC reverse phase column (C-18) chromatography with methanol-water (from 50:50 to 90:10). Results of the solution phase secondary ion mass and high resolution ¹H-NMR (400 MHz) spectral studies of the hydrolysis product have been recorded in the discussion section.

Claims: A compound of formula wherein R is -COCH₂CH₂CH₃ or -COCH₃, and R₁ is -COCH₂CH(CH₃)₂, -COCH₂CH₂CH₃ or -COCH₃, or an acylate thereof. The compound of claim 1 wherein R is -COCH₂CH₂CH₃ and R₁ is -COCH₂CH(CH₃)₂. The compound of claim 1 wherein R is -COCH₃ and R₁ is -COCH₂CH(CH₃)₂. The compound of claim 1 wherein R is -COCH₃ and R₁ is -COCH₂CH₂CH₃. The compound of claim 1 wherein R and R₁ are each -COCH₃. The compound of claim 1 wherein R and R₁ are each -COCH₂CH₂CH₃.

Inventors: HERALD CHERRY L, KAMANO YOSKIAKI, PETTIT GEORGE ROBERT, HERALD, CHERRY L., KAMANO, YOSKIAKI, PETTIT, GEORGE ROBERT
Applicants: UNIV ARIZONA, ARIZONA BOARD OF REGENTS, ARIZONA STATE UNIVERSITY
Application year: 1985
Main classification: C07D493
IPC codes: B01J20, G01N30, A61K35, C07D493, A61K31, A61P31, A61P35, C07D323