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Synthesis of some insect juvenile hormone analogues from thujone Leyten, Wayne J. 1984

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SYNTHESIS OF SOME INSECT JUVENILE HORMONE ANALOGUES FROM THUJONE by WAYNE J . LEYTEN B.Sc. (Hons.). University of B r i t i s h Columbia, 1 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Chemistry) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA November, 1984 • Wayne J . Leyten, 1984 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date DE-6 (3/81) i i -ABSTRACT Treatment of cedar leaf o i l with aqueous potassium permanganate resulted in the oxidative ring opening of thujone (XXIV) to y i e l d the c r y s t a l l i n e a-thujaketonic acid (XXV). 7 0 This material, because of i t s a v a i l a b i l i t y and interesting structure, represented an a t t r a c t i v e starting material for the synthesis of analogues of insect juvenile hormone. Therefore, to achieve t h i s aim, a-thujaketonic acid (XXV) was converted to form two products (or 'half-molecules') which were then coupled together and transformed to the analogues. In the i n i t i a l study Grignard treatment of (XXV) produced an intermediate t e r t i a r y alcohol (LVIII) which cyclized spontaneously to a lactone (XXVI). The l a t t e r compound resisted further transformation and t h i s approach was abandoned. On the other hand, (XXV) was refluxed in water to give B-thujaketonic acid (XXIX). This ring-opened acid was hydrogenated to give the ketoacid (XXX) which reacted with excess methylmagnesiurn iodide to y i e l d the alcohol acid (XXXI). In the last reaction of t h i s sequence some carboxylic acid was found to be converted to an alcohol ketone. This product (XXXII) was apparently formed via attack of the excess Grignard reagent (on to the salt of the acid) to y i e l d the ketone from the acid. This alcohol ketone (XXXII) was reduced to a diol (XXXIII) and the l a t t e r converted to an acetate derivative (XXIV) 1n order to investigate i t s structure. Next, the desired alcohol acid (XXXI) was pyrolyzed to give the o l e f i n acids (XXVII) and (XXVIII) which were separated via s i l v e r n i t r a t e impregnated s i l i c a gel column chromatography. The required intermediate (XXVII) afforded one of the necessary products or 'half molecules'. In order to improve the overall y i e l d of the required synthon (XXVII), a-thujaketonic acid was quantitatively converted to the methylene derivative (LIX) via reaction with two equivalents of methyl triphenyphosphorane. 5 9 Pyrolytic ring opening of the cyclopropane ring system in (LIX) afforded a good y i e l d of the dienoic acid (XXXV). Reduction of the desired acid (XXXV) with potassium in l i q u i d ammonia gave an 85% y i e l d of the key intermediate (XXVII). The second required intermediate was synthesized via the e s t e r i f i c a t i o n of B-thujaketonic acid (XXIX) with diazomethane in ether. This yielded the ketoester (XXXVII) which was used in the following coupling reaction sequence. Thus the key intermediate (XXVII) was treated with lithium diisopropylamide (LDA) in tetrahydrofuran (THF) to form the carboxylic acid dianion which was reacted with the keto ester (XXXVII) in THF to give a mixture of B-hydroxy carboxylic acids (XXXIX) and (XL). This product mixture was dissolved in dry pyridine and excess benzene-sulfonyl chloride was added 1n order to achieve the required c y c l i z a t i o n to the expected o l e f i n i c B-lactones (XLI) and (XLII). Epoxidation of this mixture with metachloroperbenzoic acid (MCPBA) in methylene chloride yielded the corresponding epoxy B-lactones (XLIII) and (XLIV). The f i n a l step in the synthetic strategy was to u t i l i z e the pyrolytic decomposition of the intermediate 8-lactone function to the central double bond inherent in the juvenile hormone systems. Therefore, the - iv -epoxy lactones (XLIII) and (XLIV) were subjected to such pyrolytic conditions but only decomposition products resulted. For this reason further studies with (XLIII) and (XLIV) were abandoned. The o l e f i n acid (XXVII) was reacted with mercuric acetate in dry alcohol and the mercury intermediate thus derived was converted with sodium borohydride in methanol or ethanol to y i e l d respectively the methoxy (IL) or the ethoxy acids (XLVIII). The ethoxy acid (XLVIII) was reacted with LDA in THF to give the expected carboxylic acid dianion, the l a t t e r upon addition of the ketoester (XXXVII) yielded a mixture of the 8-hydroxycarboxylic acids (L) and ( L I ) . This mixture was lactonized with benzenesulfonyl chloride in pyridine as described above to y i e l d the ethoxy 8-lactones (L11) and (LI 11). These were pyrolyzed and separated to give the juvenile hormone analogues (LIV) and (LV). Analogous investigations on the methoxy acid (IL) yielded the analogous B-hydroxy carboxylic acids (LVI) and (LVII). Time constraints precluded the conversion of these to the hormone analogues. - V -TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS v LIST OF TABLES v i i i LIST OF FIGURES i x LIST OF SCHEMES xi ABBREVIATIONS x i i ACKNOWLEDGEMENT xi i i DEDICATION xiv 1. INTRODUCTION 1 1.1 General Background 1 1.2 Insect Hormonal Control 2 1.3 Hormonal Control of Insect Metamorphosis 3 1.4 Hormonal Control of Insect Populations 7 1.5 Structure-Activity of Aryl Terpenes as Juvenile Hormone Mimics 13 1.6 Specific Juvenile Hormone Analogues 31 1.7 Anti-Juvenile Hormones 32 1.8 Environmental/Economic Aspects 34 2. SCOPE OF PRESENT WORK 36 3. DISCUSSION 38 3.1 Analysis of Cedar Leaf Oil 38 3.2 a-Thujaketonic Acid 48 3.3 Synthetic Objectives 49 v i -Page 3.4 The Coupling Reaction 50 3.5 The Synthesis of Left Hand Halves 55 3.6 Preparation of the Right Hand Half 63 3.7 The Coupling Reaction 63 3.8 Preparation of 8-Lactones and Attempted Preparation of Anlogues 65 3.9 Preparation of Hormone Analogues 69 3.10 Preliminary Investigations Toward the Synthesis of Other Juvenile Hormone Analogues 72 4. EXPERIMENTAL 74 3-Isopropyl-3,4-cyclopropyl-6-dimethyl-tetrahydro-l,2-pyrone (XXVI) 75 3-Isopropyl-6-methyl-5- and 6-heptenoic acids (XXVII) and (XXVIII) 78 3-Isopropyl-6-keto-heptanoic acid (XXX) 78 6- Hydroxy-3-isopropyl-6-methylheptanoic acid (XXXI) 85 7- Hydroxy-4-isopropyl-7-methyl-2-octanone (XXXII) 88 7- Hydroxy-4-isopropyl-7-methyl-2,7-octanediol (XXXIII) 88 2- Acetoxy-7-methyl-4-isopropyl-7-octanol (XXXIV) 93 3- Isopropyl-6-methyl-2-(E)-5-heptadienoic acid (XXXV) 93 3-Isopropyl-6-methyl-3,5-heptadienoic acid (XXXVI) 98 8- Hydroxycarboxylic acids (XXXIX) and (XL) 98 The (3-Lactones (XLI) and (XLII) 101 The Epoxy 8-lactones (XLIII) and (XLIV) 104 The p-Hydroxycarboxylic acid (XLV) 104 The 8-lactone (XLVI) 107 - vi i -Page 6-Ethoxy-3-isopropyl-6-methylheptanoic acid (XLVIII) 107 6-Methoxy-3-isopropyl-6-methyl heptanoic acid (IL) 110 The Ethoxy B-hydroxy carboxylic acids (L) and (LI) 110 The Ethoxy B-lactones (LI I) and (LI 11) 110 The Hormone Analogues (LIV) and (LV) 117 REFERENCES 120 - vi i i -LIST OF TABLES Table Page 1 Juvenile Hormone A c t i v i t y of Various Compounds with Terpenoid Backbones on J_. mol i t o r 8 2 Effect of Modifying Methyl Juvenate Unsaturation on Juvenile Hormone A c t i v i t y in T_. mol i t o r 10 3 Effect on Juvenile Hormone A c t i v i t y in T. molitor of Modification of the Epoxide end of Methyl Juvenate 11 4 Juvenile Hormone A c t i v i t y on Rhodnius prolixus 12 5 Lis t of Compounds Tested for Juvenile Hormone A c t i v i t y . I S 6 A c t i v i t y Ratings (AR) of the Juvenile Hormone Mimics Tested 2f 7 Dimensions in Angstroms of Compounds for Some Juvenile Hormone Analogues 26 8 Average Composition of the Oil of the Leaves of Thuja piicata (Donn.) 40 9 Occurrence of Terpenes in the Leaf Oils of North American Conifers of the Family Cupressacaeae and th e i r Chemosystematic Value 42 10 Percent Thujone in Samples of Cedar Grown in Different Environments 44 11 Structural Comparison of Juvenile Hormone with Possible Analogues Available from a-Thujaketonic Acid.. 54 - ix -LIST OF FIGURES Figure Page 1 Endocrine Regulations of Life Cycle of Yellow Mealworm 6 2 Comparison of Electronegative Centers of Juvenile Hormone (I) with Ecdysone (II) 9 3 Dimensional Symbols and Cartesian Orientation for Mimics 23 4 Hypothetical Receptor Site 25 5 Schematic Drawing of Location of Foliage Picked from 5-year-old Thuja plicata 39 6 Chromatogram of the Leaf Oil of Thuja pi i c a t a as obtained on a 6 f t . x 1/4 i n . O.D. Polyethylene Glycol (PEG 1540) Column at 100° and 60 ml Helium per min 41 7 Typical GLC Chromatogram of Cedar Leaf Oil 4?; 8 100 MHz. N.M.R. Spectrum of (XXVI) 76 9 I.R. Spectrum of (XXVI) 77 10 100 MHz. N.M.R. Spectrum of (XXX) 83 11 I.R. Spectrum of (XXX) 84 12 100 MHz. N.M.R. Spectrum of (XXXV) 96 13 I.R. Spectrum of (XXXV) 97 14 270 MHz. N.M.R. Spectrum of (XXVII) 79 15 I.R. Spectrum of (XXVII) 80 16 100 MHz. N.M.R. Spectrum of (XXVIII) 81 17 I.R. Spectrum of (XXVIII) 82 18 100 MHz. N.M.R. Spectrum of (XXXII) 89 19 I.R. Spectrum of (XXXII) 90 20 100 MHz. N.M.R. Spectrum of (XXXIII) 91 - X -Figure Page 21 I.R. Spectrum of (XXXIII) 92 22 100 MHz. N.M.R. Spectrum of (XXXIV) 94 23 I.R. Spectrum of (XXXIV) 95 24 100 MHz. N.M.R. Spectrum of (XXXI) 86 25 I.R. Spectrum of (XXXI) 87 26 100 MHz - N.M.R. Spectrum of (XXXIX) and (XL) 99 27 I.R. Spectrum of (XXXIX) and (XL) 100 28 100 MHz. N.M.R. Spectrum of (XLI) and (XLII) 102 29 I.R. Spectrum of (XLI) and (XLII) 103 30 100 MHz. N.M.R. Spectrum of (XLIII) and (XLIV) 105 31 I.R. Spectrum of (XLIII) and (XLIV) 106 32 100 MHz. N.M.R. Spectrum of (IL) I l l 33 I.R. Spectrum of (IL) 112 34 100 MHz N.M.R. Spectrum of (XLVIII) 108 35 I.R. Spectrum of (XLVIII) 109 36 100 MHz. N.M.R. Spectrum of (L) and (LI) 113 37 I.R. Spectrum of (L) and (LI) 114 38 100 MHz. N.M.R. Spectrum of (LI I) and (LI 11) 115 39 I.R. Spectrum of (LII) and (LIII) 116 40 400 MHz. N.M.R. Spectrum of (LIV) and (LV) 118 41 I.R. Spectrum of (LIV) and (LV) 119 - xi -LIST OF SCHEMES Schemes Page 1 Major Degradation Products of RO-103108 after Exposure to Air and Sunlight in Polluted Water for 4 Weeks 15 2 Elaboration of a-Thujaketonic Acid into Synthetic 'Left-Hand' Halves 51 3 Elaboration of a a-Thujaketonic Acid into Synthetic 'Right-Hand' Halves 52 4 The Coupling Reaction 53 5 Elaboration of a-Thujaketonic Acid to a 'Left-Hand' Half XXVII 5£ 6 Elaboration of a-Thujaketonic Acid (via new route) 58 - xi i -ABBREVIATIONS AND FOOTNOTES The following abbreviations are used in this work: THF tetrahydrofuran ether diethyl ether mmoles minimoles TLC thin-layer chromatography LDA lithium diisopropylamide CClu carbon tetrachloride HC1 aqueous hydrochloric acid MCPBA meta chloroperbenzoic acid HMPA hexamethyl phosphoric triamide GLC gas l i q u i d chromatography N.M.R. lH nuclear magnetic resonance GC-MS gas chromatography coupled to a mass spectrometer I.R. infra red HRMS high resolution mass spectrum B.C. B r i t i s h Columbia Sask. Saskatchewan JH Juvenile hormone JHA Juvenile hormone analogue CA Corpora a l l a t a QSAR Quantitative structure-activity relationships BD Beginning dark portion of cedarleaf o i l fraction EL End l i g h t portion of cedarleaf o i l fraction FOOTNOTES dried the solution was dried over anhydrous sodium sulphate and f i l t e r e d extracted the solution was successively extracted with portions of the solvent at least three times xi i i ACKNOWLEDGEMENTS I wish to express my appreciation to Dr. James P. Kutney for his help and encouragement throughout t h i s work. Also I am very grateful to Dr. Brian R. Worth for invaluable advice and teaching. F i n a l l y I wish to thank Drs. M. Singh, J. B. Paine ( I I I ) , and H. Jacobs for their time, knowledge and kindness. - xiv DEDICATION I dedicate this work to my parents, for t h e i r work and dreams have made a l l t h i s possible. Also I am eternally grateful to Diana for her be! i e f . - 1 -1. INTRODUCTION 1.1 6eneral Background Human beings have been on earth somewhere between one and two mill i o n years, a time span that most of us have d i f f i c u l t y in grasping. Try now to visua l i z e 250 mi l l i o n years, the period for which insects are known to have t h r i v e d . 1 Insects are the most successful animal l i f e form on this planet. It i s l i t t l e wonder, then, that man must fight insects to ensure his su r v i v a l . Besides th e i r being in direct competition with man for agricultural products, insects are pests and disease vectors. Insecticides have been used to control insects since antiquity, but only since the 1940's have chemical pesticides provided effective control. Nowadays there i s great cause for concern about the environmental effects of conventional pesticides such as DDT. Conventional biocides have side effects: magnification in the food chain, deleterious effects on non-target organisms, r e l a t i v e l y high acute t o x i c i t y to man and (occasionally) carcinogenic, mutagenic or teratogenic properties. Many insect species have rapidly and perversely developed immunity to many previously used pesticides. 1 This build up of tolerance has been ascribed to the short generation time of many major pests; insects quickly adapt and evolve defenses against applied chemicals. These problems have a l l spurred interest in the development of new methods for controlling insect pests. These methods need to be: highly selective to the target organism, very effective in controlling - 2 -the problem, and r e l a t i v e l y innocuous to the environment. The main areas investigated have been: insect pheremones, pyrethroids, and insect hormonal control. Pheremones are chemicals used by insects to communicate with others members of the same species. Many pheremones have recently been is o l a t e d , i d e n t i f i e d , and synthesized. These compounds hold promise in disrupting insect mating, but problems remain in formulating and u t i l i z i n g these materials. Pyrethroids are analogues of chrysanthemic acid and the other natural pyrethrins. These chemicals act as rapid and powerful insect poisons. They generally exhibit low t o x i c i t y to mammals and are useful for the consumer insecticide market. Insect hormonal control involves regulating insect populations through manipulation of th e i r neuro-endocrine systems. This method holds considerable promise for the future. 1.2 Insect Hormonal Control I t has been known since Roman times that more had to be known about the l i f e cycle of insects 1n order to control them.1 In 1934, S i r Vincent Wigglesworth, in a b e a u t i f u l , s i m p l i s t i c , c l a s s i c paper, showed2 that the molting and metamorphosis of Rhodnius prolixus nymphs were 3 regulated by hormones. In 1956, Carroll Williams at Harvard discovered a rich source of a so-called 'juvenile hormone' (JH) in the adult males of Hyalophora cecropia. It was then that the p o s s i b i l i t y of hormonal control of insect populations was f i r s t entertained. In 1967, H. Roller - 3 -et al made a major breakthrough. After a 100,000-fold pur i f i c a t i o n of the above H. cecropia extract, they were able to determine the structure of t h i s JH. It was found to be methyl trans, trans, cis-10,ll-epoxy-7-ethyl-3,11-dimethyl-2,6-tridecadienoate ( I ) . Since that time interest in the f i e l d of insect chemistry has grown exponentially. It i s the subject of books, conferences, symposia, and thousands of s c i e n t i f i c publications. 5 1.3 Hormonal Control of Insect Metamorphosis At puberty, human beings release hormones. This release causes them to mature and assume adult characteristics. In insects, the exact opposite is true - a hormone which was present a l l during youth is removed. The removal of the hormone causes the insect to mature. 6 Hormonal control over the development and maturation of the insect can be considered to be regulated by three main groups of hormones.6 The neurosecretory c e l l s (residing in the protocerebrum part 7 of the insect brain) release 'brain hormones.' These are believed to be polypeptides. They activate the prothoracic glands, which, in turn, (I) - 4 -secrete one or more closely related steroids, the ecdysones. 7 Only (III) is physiologically active. It is believed that the ecdysones induce molting in a l l arthropods. 1 0' 9 Molting is the process by which an insect grows. During molting the hard exoskeleton (or cuticle) a-ecdysone (II) B-ecdysone (III) s p l i t s and the insect emerges to grow into i t s new c u t i c l e . The kind of new c u t i c l e which i s secreted (by the epidermal c e l l s ) at each molt i s affected by the third group of insect hormones--the JH's. Since 1967 f o u r 1 1 natural JH's have been discovered (IV-VI), as well as the original (1), which i s known as JH-1. ( I V ) JH-2 (VI) JH-4 JH i s secreted by the corpora a l l a t a (CA). If any JH i s present during the insect molt (as i s the case in immature larvae) then the insect remains the same - a larva remains a larva. The new cutic l e w i l l be the same as the old one - simply larger. Once the larva has completed i t s larval l i f e and reaches a certain s i z e , i t is ready for metamorphosis (Fig. 1). The level of JH drops in i t ' s body. In the absence of JH the molting hormone causes the insect to molt to either the pupal or adult form (depending on species). In the adult insect JH plays a great number of roles: regulating diapause, controlling ovarian development, affecting egg maturation, controlling the development of internal organs, 1 0 etc. - 6 -£5 JUVEN\U^ Ho^rnoNE Fig. 1. Endocrine Regulations of Life Cycle of Yellow Mealworm. - 7 -1.4 Hormonal Control of Insect Populations Any a b i l i t y to disrupt maturation and metamorphosis of insects would lead to an e f f e c t i v e , permanent method of control. It is known that JH i s present during certain times of the insect l i f e cycle, while i t is absent during other times. If JH was applied while the insect's own level of JH was low, then population control might be achieved. This i s found to be true in the laboratory. JH's affect v i r t u a l l y a l l insects upon which they have been t e s t e d . 1 3 Conversely, none of the compounds tested were found to have any effect when applied to young insect l a r v a e . 1 3 To be e f f e c t i v e , JH must be applied when the insect's own level of JH is low. At that point, endogenous JH interferes dramatically with maturation. The development of the midgut, central nervous system, and glands are a l l a f f e c t e d . 7 * 1 4 JH application results in the insect molting to a larval-pupal or larval-adultoid intermediate. These creatures appear to be mixtures of body parts fron both stages of the l i f e cycle. A l t e r n a t i v e l y , JH may cause the insect to molt to produce giant larvae or giant pupae.7 No matter in what fashion the JH application manifests i t s e l f , the insects usually die or become innocuous. The use of natural JH's as insect control agents has some drawbacks. There is a lack of species s p e c i f i c i t y . They are unstable in the environment and are expensive. To overcome these drawbacks, a number of Juvenile Hormone Analogues (JHA's) were synthesized (Table 1) by Jacobson and co-workers. 1 0 These JHA's have the terpenoid backbones l u and some exhibit high a c t i v i t y . Structure-activity relationships - 8 -Table 1 Juvenile Hormone A c t i v i t y of Various Compounds with Terpenoid Backbones on T. molitor u Compound Ac t i v i t y (ug) a 1. 2. 4. 6 . 7. 8. 9. 10. n • ^ ^ ^ ^ ^ (JH-4) 0.03 3.0 3.0 3.0 0.1 1.0 1.0 0.1 (JH-3) 0.03 (JH-1) 0.001 0.001 aDose required to give a c t i v i t y rating 1.0. - 9 -(Table 1,2,3) suggest the essential nature of the o l e f i n i c and expoxide moieties. White et a l . 1 5 (Table 4) presented a hypothesis to explain these relationships. In Fig. 2, one can notice that the electronegative Fig. 2 - Comparison of Electronegative Centers of Juvenile Hormone (I) with Ecdysone ( I I ) . centers of these two classes of insect hormones generally coincide. Many JHA's can be made to f i t this skeleton. Whites' results suggest the trans-ol efins posses more a c t i v i t y than the cis-forms. Some JHA's show higher a c t i v i t y than the natural JH's. This a c t i v i t y may he due to the JHA's f a c i l i t y for c u t i c l e penetration, i t s ' metabolic s t a b i l i t y , i t ' s a b i l i t y to interefere with natural JH metabolism, conformational si mi 1 a r i t y to JH, etc. The JHA's can have quite a variety of structures. The extent of research in this area i s so extensive that some modelling of receptor-- 10 -Table 2 Effect of Modifying Methyl Juvenate Unsaturatipn on Juvenile Hormone A c t i v i t y in T. molitor Compound Acti vi ty (pg) a Methyl 1aurate > 10 Methyl 10,11-epoxyundecanoate > 10 Methyl 10,11-epoxy-11-methyl - tridecanoate > 10 Methyl 10,11-epoxy-ll-methyl -dodecanoate > 10 Methyl 3,7,11-trimethyldodecanoate > 10 Methyl 10,1l-epoxy-3,7,11-trimethyl -dodecanoate > 10 Methyl 10 ,ll-epoxy-3,7,11-trimethyl-2-dodecanoate > 10 Methyl 10,1l-epoxy-3,7,11-trimethyl-6-dodecanoate > 10 Methyl farnesate 10,11-epoxide 0, .031 Cecropia JH (mixed isomers) 0.01 aDose required to give a c t i v i t y rating 1.0. - 11 -s i t e geometry i s now possible. Thus I would l i k e to include a short review on some recent structure-activity relationships of some JHA's. 1 6 These compounds are quite similar to those described later in this t h e s i s , and have been the result of thorough studies. Table 3 Effect on Juvenile Hormone A c t i v i t y in J_. mol i tor of Modification of the Epoxide end of Methyl Juvenate 4 Compound A c t i v i t y (ug) a aDose required to give a c t i v i t y rating 1.0. - 12 -Table 4 Juvenile Hormone A c t i v i t y on Rhodnius prolixus Compound (mixed isomers except Dose where otherwise stated) ^ a |gb Cecropia JH 1 aDose in pg required to give a score of 10, i. e . half-juvenalised; DDose in pg required to give a score of 19, i. e . complete supernumerary larva. - 13 -1.5 A Structure-Activity Study of Aryl Terpenes as Juvenile  Hormone Mimics Of the hundreds of chemicals that have been investigated as to their juvenile hormone mimicking properties, probably the largest single group of these is the aryl acyclic monoterpenes. A number of these have high orders of a c t i v i t y in many different insect species. Examples: ( V I I ) (E)-6,7-epoxy-3,7-dimethyl-l-[3,4-(methylenedioxy)-phenoxy]-2-octene Code: R0-20360 Hoffmann-La Roche Synthesized by: W.S. Bowers 1 7 A c t i v i t y : This is highly active against the horn f l y and stable f l y . ( V I I I ) - 14 -(E)-6,7-epoxy-3,7-dimethyl-l-(p-ethylphenoxy)-2-octene Code: RO-20458 Stauffer Chemical Corp. Synthesized by: F.M. P a l l o s 1 8 A c t i v i t y : This material i s highly active against the stable f l y ; also, t h i s compound shows considerable promise as a mosquito l a r v i c i d a l agent. Based on promising p i l o t f i e l d tests which were conducted in Europe in collaboration with Hoffmann-La Roche, the World Health Organization has entered into o f f i c i a l mosquito control t r i a l s in Indonesia, India and Nigeria. ( I X ) 6,7-epoxy-l(p-ethylphenoxy)-3-ethyl-7-methyl-non ane Code: R0-103108 1 9 Synthesized by: M. Suchy A c t i v i t y : This compound gave good control of natural populations of summerfruit t o r t r i x moth and scale insects. Studies on this compound have contributed a knowledge of how applied JHA's are decomposed in the environment, as shown in the following scheme. - 15 -OH OH Scheme 1 Major Degradation Products of RO-103108 after Exposure to Air and Sunlight in Polluted Water for 4 weeks. 1 6 (D) ( X ) (E,2)-6,7-epoxy-3,7-dimethyl-oct-2-enyl-6-ethyl-3-pyridyl ether - 16 -and ( X I ) (E,Z) 7-ethoxy-3,7-dimethyl-oct-2-enyl-6-ethyl-3-pyridyl ether Code: HS-104 Synthesized by: H. S o l i i 2 0 A c t i v i t y : These are both highly active against: Tenebrio molitor, Ga l l e r i a mellonella and Culex pi pi ens (mosquito). (E) (XIV) - 17 -A new class of aryl alkenyl ethers with juvenile hormone a c t i v i t y containing a dichlorovinyl group at the end of the aliphatic chain was synthesized by: P. P i c c a r d i 2 1 A c t i v i t y : Active against Tenebrio molitor and P i e r i s brassicae; the oral t o x i c i t i e s to rats are low. The acute oral LD50 being generally >2000 mg/kg. (F) (XV) (XVI) O (XVI I) A series of N-alkyl carbamate derivatives of geranyl phenyl ethers was synthesized by: B.D. Hammock 22 - 18 -A c t i v i t y : These compounds exhibit low juvenile hormone-like a c t i v i t y , but are very potent inhibitors of Musca  domestica juvenile hormone esterase. Biological Assay Large numbers of analogues of the following type, (Compounds 1-23, Table 5) have been bioassayed. Results of these studies indicate that the maximum juvenile hormone-like response was obtained when the benzene ring was para substituted and also when the terminal double bond in the component R was epoxidized, or contains alkoxy groups. The question i s : Why do these compounds have so high a juvenile hormone a c t i v i t y , and what kind of factors decide i t ? Quantitative structure-activity relationships (QSAR) have been successful in predicting substituent effect on biological response in many series of pharmaceuticals. 2 3" 2 5 Recent advances in QSAR have allowed the s t a t i s t i c a l assessment of the contribution of s t e r i c , electronic, and l i p o p h i l i c parameters on biological a c t i v i t y . - 19 -4. 5. 6. 7. 8. 9. 10. 11. 12. 14. 15. Table 5 L i s t of Compounds Tested for Juvenile Hormone A c t i v i t y 1 6 R-O-R - 0 - < 0 > P r O - @ > -13. R - O - ® - 7 " ^ R - 0 olo 16 17. 18. 19. 20. 21. 22. Ps~o-eH a-^-23. frV S ~ ® r - / 24. 25. 26. 27. 9, O •oc«.a - 20 -This type of analysis has been recently extended to the entomological area by Metcalf and Yu, 2 5> 2 6 but p a r t i c u l a r l y to a study 2 7 of aryl acyclic monoterpene ethers by Matthew J. Zabik. ( X V I I I ) These JH mimics were based on variations on the structure of 6,7-epoxy geranyl-4-ethyl-phenyl ether (XVIII). Representative classes consist of (see Table 6): a) 6,7-epoxy geranyl cyclohexyl ethers b) 6,7-epoxy geranyl aryl ethers with different substituents c) miscellaneous ethers The mimics were assayed on laboratory reared adult prediapause cereal leaf beetles Onlema melanopus. Evaluation of juvenile hormone a c t i v i t y has been described by N i l l e s . Juvenile hormone analogues (JHA's) were applied 50 or 100 mg per insect in acetone on the ventral side of the abdomen. Either females alone (designated F) were treated, or both males and females (designated MF) were treated. Blank runs in which insects were treated only with acetone consistently gave zero oviposition and insects entered normal diapause toward the end of the test period. - 21 -Table 6 A c t i v i t y Ratinqs (AR) of the Juvenile Hormone Mimics Tested 1 6 # Structure Dosage A.R w9 M.F. F 100 24 16 50 1 4 100 114 29 50 50 19 100 24 0 50 29 0 100 0 3 50 3 0 100 0 36 50 9 10 100 34 28 50 18 22 100 53 16 50 36 7 100 13 5 50 32 2 100 2 0 50 0 0 100 2 0 50 0 0 100 0 0 50 2 0 100 23 35 50 40 36 100 62 20 50 1 2 100 12 0 50 0 0 100 38 6 50 0 5 Dosage Structure pg A. M.F. R. F 16. 100 50 0 15 5 0 17. 100 50 126 25 27 64 18. 100 50 2 1 0 0 19. 100 50 5 16 2 0 20. 100 50 5 16 27 0 21. 100 50 26 0 6 0 22. 100 50 21 0 3 1 23. 100 50 0 10 0 0 24. 100 50 2.1 11 2.0 18 25. 100 50 3 10 0 0 26. 100 50 0 0 0 0 27. 100 50 87 38 30 13 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. R = - 22 -Thus an a c t i v i t y rating (AR) for a candidate mimic should be d i r e c t l y proportional to the total number of eggs l a i d and the molar quantity of the mimic applied. It should be inversely proportional to the number of days following treatment on which oviposition f i r s t occurs. Accordingly we 1 6 define the a c t i v i t y rating (AR) as an equation: AR - ™ D-V T - i s the total number of eggs l a i d by the test colony for 14 days inclusive following topical application of the mimic. G - i s the granmetric factor relative to JH. D - i s the number of days elapsed, following treatment on which oviposition f i r s t occurs. V - i s the v i t a l i t y of test colony after 14 days expressed as a decimal f r a c t i o n . As mentioned, advances in quantitative structure a c t i v i t y relationship (QSAR) have allowed the s t a t i s t i c a l assessment of contribution of s t e r i c , electronic and l i p o p h i l i c parameters on biological a c t i v i t y . 1 6 Assumpti on: In this study 1 6 a term W i s employed (see Fig. 3), empirically defined by the following equation: W = (C - 2.08) + (B - 2.75) (W - 6.29) + (Ta - 2.00) - 2 3 -Y Fig. 3 - Dimensional Symbols and Cartesian Orienation for Mimics The parameters are defined as follows: L i s the distance from the oxygen nucleus to the furthest extent of the van der Waals' radius of the pure substituent. B is the distance from the oxygen nucleus to the centre of the ring . C i s the maximum length of the pure substituent from the C-4 nucleus to the van der Waals' radius of the terminal hydrogen of the substituent. W' i s the width of the group inclusive of the van der Waals' r a d i i . - 24 -A is the distance in the XZ plane perpendicular to the axis joining C-l and C-4 and measured to the van der Waals1 radius of substituent on C-2 or C-6. T, is the distance above the plane defined by C - l , C-3 and C-S. a is the distance below this plane. T i s the thickness of the group. A l l measurments are made with respect to the x, y and z axes with the plane of the benzene ring parallel to the xz coordinate plane. In the case of compound with a cyclohexane ring, the plane defined by C - l , 3 and 5 was placed parallel to the XZ plane. 2 3 TT the 1 i pophi 1 i c i t y parameters are from the study by Hansch. 2 3 a value(s) were used from the compilation of Hansch. J equal to the sum of the four AR values for each mimic. J Q i s the value for mimic 4, the arbitrary standard (see Table 5). In the table below are collected values for the mentioned terms for a few mimics. Qualitative examination of tables reveals several s t e r i c effects on JH response which lead to a picture of a hypothetical receptor si t e for the JH mimics as shown in Figure 4: - 25 -Fig. 4 - Hypothetical Receptor Site In drawing a hypothetical receptor site with c r i t i c a l dimensions, part A of the receptor may, or may not, be d i r e c t l y connected to part B of the receptor s i t e . The shape of this hypothetical receptor site is purely a rationale for the experimental JH a c t i v i t i e s . There is the unsubstantiated assumption that the epoxy oxygen is involved in binding that end of the mimic to the receptor. In fa c t , the epoxy mimics are more JH active than the olefins from which they are derived. A second assumption is that there is a "pouch" at P which holds the para substituent and "locks i n " the aryl end of the mimic. Table 7 1 6 Dimensions in Angstroms of Compounds for Some Juvenile Hormone Analogues If log L W A B C T . T I O I T O b a J / J o 4. x l v ^ J ^ v ^ o ^ ^ 0 , 0 0 6 ' 2 2 6 * 2 9 3 * 1 4 2 " 7 5 2 * 0 8 1 , 0 0 l ' 0 0 0 , 0 0 0 , 0 0 °*° 0 2. - ^ y ^ J ^ S c > i ^ ^ 1 , 5 4 9 8 * 8 8 6 ' 6 8 3 * 3 4 2 , 7 4 3 , 8 3 3 , 2 5 2 , 2 9 1 , 0 4 1 , 5 7 6* ^^^O^y^o--^^' 1 , 2 3 8 ' A 5 6 , 2 9 3 ' 1 4 2 , 7 5 4 * 3 6 3 , 1 5 K 7 2 3 ' 1 0 l ' 0 2 ~ ° * 1 5 9. ^ j ^ / ^ J ^ ^ o - ^ ^ " -0.482 7.21 7.68 4.54 2.75 3.07 2.23 1.72 0.469 0.99 -0.30 U * / ^ J t y ^ ^ O ) - 0 , 1 2 4 7 ' 2 1 9 " 0 7 4 , 5 4 2 , 7 5 3 * 0 7 2 , 2 3 1 , 7 2 0 , 2 8 3 1 , 2 9 + 0 ' 7 5 12. x\^0t^s0_^5y_} l*4 8 7 9 ' 2 6 7-7 8 4 ' 6 4 2 ' 7 5 5 ' 1 2 4 ' 6 4 K 7 2 4*23 l ' 5 5 -°*13 I 7 ' 1.605 7.85 6.29 .14 3.37 3.07 2.23 1.72 2.24 - 27 -Conclusions: i b 1. Biologial a c t i v i t y i s in direct proportion to C and B and inversely proportional to W and Ta (see Fig. 3). 2. Increasing Ta (see Fig. 3) beyond 2.3A causes a sharp drop in a c t i v i t y (see Table 6). This is apparently due to the bulky groups of the following examples being too thick to f i t into the opening at P (see Fig. 4). 3. Increasing the length of the para substitutent increases a c t i v i t y l i n e a r l y in the series (see Table 6): Differentiation here between a steric effect and a l i p o p h i l i c effect i s d i f f i c u l t ; probably both are operational. - 28 -4. The decrease in a c t i v i t y for: could be ascribed to the folding of the n-butyl group resulting in an increase in W or Ta. 5. The decrease in a c t i v i t y for: and can be rationalized by the fact that phenyl group and t o l y l group are too bulky to f i t the pouch. 6. Mimics: and - 29 -give poor response, since the hydrogen and methyl groups respec-t i v e l y are too short to lock in the aryl end of the substrate. The cyclohexyl mimics: give poor response, since the axial ethyl group w i l l not lock in to the pouch and s t i l l allow the remainder of the substrate to f i t the receptor. Mimic: which has only a para-methyl group can f i t this pouch since the intervening methylene between the oxygen and the ring pushes the methyl group into the pouch. (2.4 times greater than) - 30 -9. This thioether possesses lower biological a c t i v i t y than geranyl phenyl ether: This could be due either to loss of coordinate covalent bonding due to the decreased electron density of sulfur versus oxygen, or alte r n a t i v e l y , i t may be due to the change in the bond angle. The difference between the average C-O-C bond angle and the average C-S-C bond angle i s about 6 ° . This would result in moving the terminal hydrogen of the para-ethyl group about 0.8A in the Z dire c t i o n . This may be enough to make a proper f i t at P d i f f i c u l t . 1 0 . The effect of the o l e f i n i c bond on a c t i v i t y i s revealed by comparing compounds: The saturated JHA should have shown an increase in a c t i v i t y on the basis of 1 i p o p h i l i c i t y since saturating the double bond should increase TT by about 0.45. Tr(CH 2CH 2CH 3) = 1.55 TT(CH 2CH = C H 2 ) = 1.10 The decrease in a c t i v i t y could be due to steric factors (changing two sp 2 to sp 3) and perhaps is due less to possible bonding of the olef i n moiety to the receptor. - 31 -11. Those mimics with a W value above 7.5A show decreased JH a c t i v i t y . From this reason i t i s concluded that the receptor slot cannot be much wider than shown in Figure 4. Mimics: give low a c t i v i t i e s since they are too wide to f i t the s l o t . 12. Final caution: This analysis and experimental results are only applicable to leaf beetles due to the well-known variations in structure-activity responses shown by different species of insects. 1.6 Specific JHA's Species selective JHA's are rare but not unknown. Juvabione or paper factor (XIX) was f i r s t isolated from balsam f i r wood in 1967. It possesses a high degree of JH a c t i v i t y against Pyrrhocoris apterus 2 9 bugs. Dehydrojuvabione (XX) also possesses this a c t i v i t y . - 32 -Tests conducted on members of the families Hemiptera, Lepidoptera, Coleoptera, Di ptera, and Orthopotera showed that these compounds were only effective on Hemiptera pyrrhocoridae species. 1.7 Anti-JH's• The narrow developmental period in the insect l i f e cycle during which excess JH can effect a lethal derangement has proven a drawback to the development of these hormones as widely successful pesticides. Perhaps the most serious deficiency in this method of insect control i s the fact that the immature and adult insects are unaffected by an excess of JH, and are able to produce their damage f i . e . plant destruction, disease transmission, e t c . ] 3 0 It i s known from cl a s s i c a l insect endocrinology that surgical removal of the corpora a l l a t a (CA) has a variety of effects. These effects include precocious metamorphosis 3 1 and the onset of s t e r i l i t y in adult female i n s e c t s . 3 2 * 3 3 Some insects are unable to produce pheremones34 or enter into diapause. 3 5 Other insects in the absence of JH are forced to complete thei r metamorphosis to adults, regardless of environmental c o n d i t i o n s . 3 6 ' 3 7 Application of JH-III to diapausing beetles terminates th e i r diapause. 3 8 Since JH i s necessary for insect development throughout the immature insect phases, for development of the ovaries, for pheremone production, and for larval dispause, a means of stopping JH production (or a c t i v i t y ) would be a more effective insecticide than the application 3 0 of juvenile hormones themselves. - 33 -In 1976, W.S. Bowers et al . discovered two a n t i a l l a t o t r o p i c [or anti-JH] compounds in the common bedding pi ant Agerstiurn  houstontonianum. The extract of this plant, when applied to second instar Oncopeltus fasciatus nymphs causes then to molt to apparently normal third and fourth instar larvae, and then to tiny adults, omitting 3 9 the f i f t h nymphal stage. P u r i f i c a t i o n of the extract, followed by structural ellucidation shows the active materials to be: 7-methoxy-2,-2-dimethylchromene (XXI) and 6,7-dimethoxy-2,2-dimethychromene (XXII). These materials were named precocene I and precocene I I , r e s p e c t i v e l y . 3 9 Using pure materials, i t was found that (XXII) was about tenfold more active than (XXI). The precocenes cause precocious metamorphosis and prevent ovarian development in female insects. This proved that the corpora a l l a t a (CA) i s inactive since i t i s known that the CA is the source of the gonadotropic (Juvenile) hormone.32 Precocene treatment of adult insects (soon after their emergence) prevents ovarian development. 3 9 Treatment of normal Colorado potato beetles with precocene II causes many of them (XXI) (XXII) - 34 -to enter a diapause state. It is known that the onset of diapause in these and other Coleoptera is dependent on the cessation of JH s e c r e t i o n . 3 5 * 3 8 Other effects of the precocenes include: ovicidal a c t i v i t y , 3 9 reversal of applied JH e f f e c t s , 3 9 i n h i b i t i o n of JH synthesis in v i t r o , 4 0 , i n h i b i t i o n of CA development 4 1 and atrophy of the CA in Locustra m i g r a t o r i a . 4 2 More work needs to be done in this area. The co-evolution of plants and insects has been going on for millions of years, and we humans have just begun to investigate the mechanisms by which plants protect themselves. This newest area of the so-called 'fourth genera-tions' insecticides holds much promise for new insect control agents. 1.8 Environmental/Economic Aspects Most of the JHA's tested exhibit low t o x i c i t y towards non-arthropods. 1 + 3 - 1 + 8 Acute t o x i c i t y tests show some JHA's to be 1 4 e s s e n t i a l l y non-toxic. Mice fed 35 ug. of 2- C labelled JH had 4 4 excreted 35% of the radiation in 24 hrs - the rest was retained. Long term chronic studies have, generally, not been done. Most JHA's show adverse effects on wasps, ladybeetles and other beneficial insect p r e d a t o r s . 4 9 - 5 2 Spot application coupled with careful control can minimize these adverse effects. Effects on birds, mammals and other non-target organisms are thus minimized whilst the v i t a l problems of insect control are solved. The success of JHA research can be i l l u s t r a t e d by the licensing of Altosid (XXIII) (by the Environmental Protection Agency) in 1975. - 35 -This compound is applied at the rate of 3-4 ounces per acre in an aerial spray and gives essentially 100% control of floodwater mosquitoes. 5 3' 5 4 It is also used to control f l i e s in manure and to increase s i l k production from silkworms. 3 0 Since 1975, other JHA's have met the stringent licensing requirements of the United States 3 0 , 5 5 } 5 6 (XXIII) - 36 -2. SCOPE OF PRESENT WORK The previous discussion has provided a brief summary of some recent work in the various areas of insect chemistry. Thus, i t i s clear that certain chemical structural features provide biological a c t i v i t y of the juvenile hormone type. Additional research i s needed in this area to provide a better understanding of structure-activity relationships, and hence to develop useful applications of chemistry in the f i e l d of insect control. Keeping these objectives in mind, i t was decided to embark on a synthetic program leading to novel analogues of juvenile hormones, and then to evaluate their potential usefulness. It seemed attractive to u t i l i z e , as starting materials, some organic compounds which are presently regarded as waste byproducts in the forest industry. It was f e l t that successful chemistry performed on these wastes would stimulate useful applications for them. In B r i t i s h Columbia, three species of commercially important trees have been studied with regard to their chemical composition. 5 7' 5 8 These are the Western red cedar, Douglas f i r and the Western hemlock. Current forest practices generate large volumes of waste material, which i s generally disposed of by dumping or burning. These materials are thus a f i r e and pollution hazard. Our research group has been p a r t i c u l a r l y interested in the Western red cedar (Thuja plicata Donn.). The main component in the leaves and branches is the monoterpene thujone (XXIV). - 37 -(XXIV) Thujone can be obtained by an economically feasible steam d i s t i l l a t i o n and fractionation process, and constitutes about 85% of the steam d i s t i l l e d cedar leaf o i l . Our research group has done a lot of 5 9 6 3 work " in converting thujone into commercially viable products. We are in close l i a i s o n with Alan Moss and Associates, in Kelowna B.C., as part of a larger research program funded by the B.C. Science Council, and begun in 1979. Alan Moss' group has made considerable progress in evaluating various methods of extracting the cedar leaf o i l from trees grown under different conditions. Their study involves the overall economics of the steam disti11ation/fractionation process in forested areas, u t i l i z i n g portable steam d i s t i l l a t i o n apparatus. Due to the coordinated nature of the research program, i t was decided to study the y i e l d and the quality of o i l obtained from cedar trees grown in different areas, and under different conditions of age, a l t i t u d e , r a i n f a l l , etc. Thus a study was done u t i l i z i n g gas chromatography as an analytical method to determine o i l composition. Concomitantly, a synthetic program was undertaken to produce some novel JHA's u t i l i z i n g thujone as the sole carbon source. - 38 -3. DISCUSSION 3.1 Analysis of Cedar Leaf Oil Western red cedar Thuja plicata (Donn.) is a rich source of thujone. The steam-volatile o i l derived from red cedar has been previously examined by von Rudloff 6 4» 6 5 and the components ide n t i f i e d by means of GC-MS, I.R. spectroscopy, and preparative GLC followed by coinjection with authentic samples (See Table 8). L i t t l e difference was found by this author between Western red cedar and the Eastern white cedar Thuja o c c i d e n t a l s (_L.) insofar as monoterpenoid content i s concerned. 6 6 von R u d l o f f 6 5 examined steam v o l a t i l e o i l s from a mature tree grown near Prince Rupert, B.C., and a young tree grown near Saskatoon Sask. He found l i t t l e qualitative or quantitative difference between their o i l s derived from the trees as a whole (see Table 8). He then investigated the o i l derived from specific portions of the young tree. These results are tabulated below; a typical chromatogram is shown in Figure 6. Samples 1 - 1 0 were picked in January while samples A, B, C were picked in mid March. Samples 1 - 4 were older leaves (approx. 1 - 2 years old). As shown in Figure 5, they were derived from various parts of the tree. The o i l from these l e a f l e t s were found to be similar in composition, but a d i s t i n c t l y lower amount of hydrocarbons i s character-i s t i c of the younger leaves. The recorded values were outside the range of experimental error and were consistent in this trend. No other characteristic difference was found. The samples A, B, and C were - 39 -a l l low in hydrocarbon content and this was attributed to the return of the growing season before this branch was collected. Fig. 5 - Schematic Drawing of Location of Foliage Picked from 5-year-old Thuja plicata Samples 1 to 10 were picked during the second and third week of January 1962; samples A to C were collected on March 12, 1962. von Rudloff's o i l preparation method consisted of steam d i s t i l l a t i o n followed by: ether extraction of the l i q u i d , bicarbonate wash, drying, and ether evaporation. As such, his preparation removed free acids and non-ether-soluble compounds from the samples. His T a h l e 8 A v e r a g e C o m p o s i t i o n o f t h e O i l o f t h e L e a v e s o f T h u j a p i i c a t a L e a f O i l Sample Compound P r i n c e ( P e a k N o . ) * RRTt R u p e r t S a s k a t o o n 1 2 3 4 5 6 7 8 9 10 A R C ( a ) H y d r o c a r b o n s I . d - a - P i n e n e 0 . 2 9 2 . 0 1 . 5 0 . 8 0 . 6 0 . 4 0 . 8 t r . t r . t r . 0 . 3 0 . 1 t r . 0 . 3 0 . 3 0 . 3 2 . (Camphene) 0 . 4 1 0 . 4 0 . 2 t r . t r . t r . t r . - - - - - - 0 . 2 0 . 3 t r . 3 . d - S a b i nene 0 . 5 9 6 . 4 6 . 3 6 . 5 a 5 . 2 3 . 5 1 . 3 1 2 . 2 2 . 7 2 2 . 2 1 . 5 2 1 . 5 4 . ( C a r - 4 - e n e ) 0 . 7 8 2 . 8 2 . 8 1 . 5 2 1 . 9 2 . 5 0 . 5 0 . 3 0 . 5 1 . 8 0 . 6 0 . 5 0 . 6 0 . 8 0 . 8 5 . d - l i m o n e n e 1 . 0 0 0 . 5 0 . 3 0 . 4 0 . 6 0 . 1 0 . 2 t r . t r . 0 . 1 t r . 0 . 1 0 . 2 t r . t r . t r . 6 . ( l , 8 , C i n e o l e ) 1 . 0 8 0 . 1 t r . 7 . 8 . ( f - T e r p i n e n e ) ( p - C y m e n e ) 1 . 3 0 1 . 5 5 0 . 1 0 . 2 t r . 0 . 1 0 . 8 0 . 8 0 . 2 0 . 5 t r . t r . 0 . 3 t r . 0 . 2 0 . 2 0 . 2 . t r . t r 9 . ( T e r p i n o l e n e ) 1 . 6 5 0 . 4 0 . 2 (b) O x y g e n a t e d 1 0 . 1 - T h u j o n e 0 . 6 3 7 6 . 0 7 7 . 5 79 77 82 82 8 5 . 5 86 86 84 86 85 86 88 87 11 . d - I s o t h u j o n e 0 . 6 8 7 . 5 7 . 8 8 7 8 7 8 . 5 9 8 8 7 7 9 7 . 5 8 . 5 1 2 . ( A r o m - e s t e r ) 1 . 9 1 . 9 1 . 5 0 . 5 1 1 2 . 5 1 . 5 1 0 . 8 2 . 5 1 I 1 . 3 0 . 5 0 . 5 1 3 . d - T e r p i n e n - 4 - o l 1 . 3 9 1 .7 1 . 2 3 1 1 1 . 5 I 1 . 5 0 . 8 2 . 4 3 0 . 5 0 . 5 0 . 6 Age o f b r a n c h l e t s ( y e a r s ) : mi xed mi xed 4 - 5 3-4 3-4 3 1-2 1 1-2 1 2 1 3 - 4 2 - 3 1-2 *Naines i n p a r e n t h e s e s r e f e r to compounds i d e n t i f i e d by r e t e n t i o n t i m e s o n l y . t R e l a t i v e r e t e n t i o n t i m e s on t h e a d i p a t e (b) o x y g e n a t e d t e r p e n e s at 1 1 0 ° and 100 p o l y e s t e r c o l u m n : ( a ) ml h e l i u m p e r m i n u t e . h y d r o c a r b o n s at 6 0 ° and 150 ml h e l i u m p e r m i n u t e . + + C o m p o s i t i o n g i v e s a % o f t o t a l i n s a m p l e . - 41 -findings d i f f e r from those of other (generally e a r l i e r ) worke r s b b ~ b B in that he found no 3-pinene, cor-3-ene, a-phell andrene, thujyl alcohol, thujyl acetate, borneol , bornyl acetate, fenchone, or camphor. Other methods of o i l isolation were used by these other investigators ( i . e . solvent extraction of foliage) and this may explain the incongruities. An alternate explanation may be that von Rudloff's GLC method did not elute these compounds. Our study has reinvestigated the composition of the steam-volatile o i l ; peaks eluted after thujone have been found to be (reproducibly) present in a l l samples. Time (min.) Fig. 6 - Chromatogram of the Leaf Oil of Thuja plicata as Obtained on a 6 f t . x 1/4 i n . 0.0. Polyethylene Glycol (PEG 1540) Column at 100° and 60 ml Helium per min. 1. a-pinene; 2. camphene (+ a-fenchene); 3. sabinene; 4. (?) car-4-ene; 5. limonene; 6. 1,8-cineole; 7. y-terpinene; 8. p-cymene; 9. terpinolene; 10. thujone; 11. isothujone; 12. (?) aromatic ester; 13. terpinen-4-ol. This study deals with leaf o i l samples derived by various methods from trees grown in different climatic conditions. Within each - 42 -T a b l e 9 O c c u r r e n c e o f T e r p e n e s i n t h e L e a f O i l s o f N o r t h A m e r i c a n C o n i f e r s o f t h e F a m i l y C u p r e s s a c a e a e and t h e i r C h e m o s y s t e m a t i c V a l u e 5 7 P e a k * N o . Chemae c y p e r i s t J u n i p e r u s T h u j a S a n t e n e 1 - - -T r i c y c l e n e 2 - -/+0 -F e n c h e n e 3 + - +d a - T h u j e n e 4 - +1D +d a - P i n e n e 5 ++d + + Camphene 6 t - l + n t B - P i nene 7 + t/+ri + S a b i n e n e 8 t t/++D +d M y r c e n e 9 + +d t 3 - C a r e n e 10 •n+d + t a - P h e l 1 a n d r e n e 11 t t -a - T e r p i nene 12 t t/+D + p-Cymene 13 t t/+D + L i m o n e n e 14 ++d +/++d + B - P h e l 1 a n d r e n e 15 + t/+d t c i s - O c i m e n e 16 - t -t r a n s - O c i m e n e 17 - - -• y - T e r p i n e n e 18 t t/+D t T e r p i no! ene 19 + + + l : 8 - C i n e o l e 20 - - t F e n c h o n e 21 - - ++D L i n a l o o l 22 - + -T h u j o n e 22a - t ++D F e n c h o l 23 - - -I s o t h u j o n e 23a - t ++D C a m p h o t e n i c a l d e h y d e 24 - -It -Camphor 25 - -/++0 +d T e r p i n e n 4 - o l 28 t -/++D +d a - T e r p i n e o l 29 t -/+ri t C i t r o n e l l o l 30 t +d -C a r v o n e 32a - -B o r n y l a c e t a t e 33 t -/+D + M y r t e n y l a c e t a t e + + 33a - -/++D -a - T e r p i n y l a c e t a t e 34a - t t G e r a n y l a c e t a t e 35 - t t S a f r o l e 36a - -/+D -M e t h y l E u g e n o l 38a - -/+n -E u g e n o l 39 - t/+D -C i 6 H y d r o c a r b o n s 41 +D + + C 1 6 O x y g e n a t e d 42 +D + /++D + U n i d e n t i f i e d $ 43 - - +D M e t h y l c i t r o n e l l a t e 44 - +D -U n i d e n t i f i e d 4 4 a - -/+D -U n i d e n t i f i e d 45 - t/+D -E l emol 46 - + /++D -E l e m y l a c e t a t e 47 - t/+D -Eudesmol i s o m e r s 48 - + d -a - C y p e r o n e 49 - +d -A c e t a t e II 50 - t/++D -U n i d e n t i f i e d 51 - t/+D -K e y . t - t r a c e (up t o 0 . 3 % ) ; + = 0 . 5 - 10%; + + = above 10% 0 - m a j o r ; d - m i n o r d i a g n o s t i c v a l u e . * I n s e q u e n c e o f e l u t i o n f r o m n o n - p o l a r GLC c o l u m n s : a l s o i n F 1 0 , 1 2 , 1 3 . t A l a s k a C e d a r ( C . n o o t k a t e n s i s ) o n l y . + + S o m e J u n i p e r o i l s c o n t a i n a l s o s m a l l amonts o f m y r t e n o l m y r t e n a l ( j A r o m a t i c - 43 -geographical area different parts of the foliage were used as biomass to produce the o i l . It is hoped that by analyzing the sundry samples a correlation (between thujone content and growth conditions of the tree) can be made with the aim of optimizing the economic f e a s i b i l i t y of cedar leaf o i l production. As an economic f e a s i b i l i t y study, our work has sacrific e d s e n s i t i v i t y for resolution. Hence the minor (< .2%) monoterpenoid components, being of only chemosystematic interest, were not investigated. The results of our study are tabulated in Table 10 together with the data submitted by Alan Moss & Assoc. The % thujone peak i s followed by the length of the GLC run in minutes. A l l samples were kept in the dark, dried by means of 4A molecular sieves for 15 minutes and injected by means of a Hamilton mi c r o l i t e r syringe. Injection volume was 0.5 u l . A l l samples were injected at least twice to check reproduci-b i l i t y . Samples were injected neat to preclude any solvent mediated a r t i f a c t production and to check on the elution of low-boiling components. A typical chromatogram, with instrumental conditions, is shown in Fig. 7. A l l runs were done on the same GLC column (a 6 feet x 1/4" i . d . stainless steel column packed with 5% FFAP on Chromosorb HP/W). Discussion Batches 1 - 3 came from the twigs, leaves, and branches of 40 year old trees. Batch 4 came from the wood of a 20 year old tree. Batches 5 - 7 came from the twigs, leaves, and branches of 20 year old - 44 -T a b l e 10 W e i g h t % T h u j o n e i n Wood Y i e l d % % T h u j o n e o f Sample ( a v e r a g e d ) B a t c h D a t e o f C o l 1 e c t i on G r e e n Dry Run A Run B G r e e n Dry 1 12/11/79 0 . 0 8 _ 9 3 . 9 ( 3 8 ) 9 2 . 8 ( 4 6 ) 0 . 0 7 5 -2 II 0 . 0 7 - 8 7 . 2 ( 3 5 ) 8 8 . 0 ( 7 5 ) 0 . 0 6 1 -3 13/11/79 0 . 0 8 - 8 4 . 6 ( 3 3 ) 8 1 . 2 ( 8 5 ) 0 . 0 5 6 -14/11/79 0 . 1 9 - 8 4 . 6 ( 3 3 ) 8 6 . 4 ( 3 7 ) 0 . 1 6 -5 16/11/79 0 . 2 6 - 9 2 . 1 ( 3 1 ) 9 1 . 5 ( 3 4 ) 0 . 2 4 -6+7 2 2 / 1 1 / 7 9 0 . 1 9 - 9 1 . 3 ( 3 0 ) 9 0 . 6 ( 5 7 ) 0 . 1 7 -8BD 2 3 / 6 / 8 0 0 . 5 5 1 . 1 8 8 5 . 7 ( 3 4 ) 8 7 . 3 ( 3 5 ) 0 . 4 8 1 . 0 2 8EL 2 3 / 6 / 8 0 0 . 5 5 1 . 1 8 8 5 . 5 ( 3 4 ) 8 5 . 2 ( 3 7 ) 0 . 4 7 1 .01 980 2 4 / 6 0 . 7 3 1 . 5 6 8 4 . 6 ( 3 7 ) 8 4 . 8 ( 1 2 5 ) 0 . 6 2 1 . 3 2 9EL " 0 73 1 . 5 6 8 2 . 1 ( 3 5 ) 8 4 . 8 ( 1 2 5 ) 0 . 6 2 1 . 3 0 10BD " 0 . 7 0 1 . 4 9 8 4 . 1 ( 3 3 ) 8 5 . 0 ( 3 8 ) 0 . 5 9 1 . 2 6 10EL n 0 . 7 0 1 . 4 9 8 0 . 7 ( 3 3 ) 8 0 . 0 ( 3 5 ) 0 . 5 6 1 . 2 0 11BD II 0 . 6 4 1 . 3 7 8 3 . 3 ( 4 8 ) 8 2 . 1 (62) 0 . 5 2 1 . 1 3 11EL 2 5 / 6 / 8 0 0 . 6 4 1 . 3 7 8 4 . 0 ( 1 5 0 ) 8 5 . 0 ( 5 0 ) 0 . 5 4 1 . 1 6 120 it 0 . 7 3 1 . 5 6 8 1 . 7 ( 5 0 ) 16J ( 4 0 ) 0 . 5 8 1 . 2 3 12L " 0 . 7 3 1 . 5 6 8 4 . 1 ( 4 4 ) 8 6 . 3 ( 3 5 ) 0 . 6 2 1 . 3 3 13BD 2 6 / 6 / 8 0 0 . 7 0 1 . 4 9 7 8 . 8 ( 4 5 ) 8 1 . 8 ( 3 8 ) 0 . 5 6 1 . 2 0 13EL ii 0 . 7 0 1 . 4 9 7 1 . 6 ( 4 0 ) 7 2 . 1 ( 3 1 ) 0 . 5 0 1 . 2 0 14BD " 0 . 5 4 1 . 1 4 7 4 . 4 ( 5 6 ) 8 3 . 0 ( 3 2 ) 0 . 4 2 0 . 9 0 14EL 0 . 5 4 1 . 1 4 7 8 . 8 ( 4 5 ) 8 1 . 0 ( 3 6 ) 0 . 4 3 0 . 9 1 15BD 2 7 / 6 / 8 0 0 . 6 9 1 . 4 6 7 7 . 4 ( 4 8 ) 8 1 . 3 ( 3 4 ) 0 . 5 4 1 . 1 4 15EL 0 . 6 9 1 . 4 6 7 7 . 4 ( 3 3 ) 7 2 . 2 ( 5 5 ) 0 . 5 2 1 . 0 9 16BD " 0 . 6 4 1 . 3 6 7 2 . 3 ( 3 8 ) 7 8 . 5 (32) 0 . 4 8 1 . 0 3 16EL " 0 . 6 4 1 . 3 6 7 8 . 7 (31) 7 7 . 5 ( 3 4 ) 0 . 5 0 1 . 0 6 17BD 0 . 6 9 1 . 4 8 7 8 . 5 ( 3 8 ) 7 5 . 8 ( 5 8 ) 0 . 5 3 1 . 1 4 17EL " 0 . 6 9 1 . 4 8 7 9 . 0 ( 3 4 ) 8 0 . 6 ( 3 2 ) 0 . 5 5 1 . 1 8 18EL 0 . 7 0 1 . 4 8 8 1 . 7 ( 3 3 ) 8 2 . 7 ( 3 3 ) 0 . 5 8 1 . 2 2 19BD 1/7/80 0 . 5 4 1 . 1 5 8 5 . 6 ( 3 2 ) 8 3 . 7 ( 3 2 ) 0 . 4 6 0 . 9 7 19EL n 0 . 5 4 1 . 1 5 7 3 . 1 ( 3 5 ) 7 5 . 4 ( 3 5 ) 0 . 4 0 0 . 8 5 20BD n 0 . 7 5 1 . 6 0 7 9 . 1 ( 4 9 ) 8 2 . 0 ( 3 3 ) 0 . 6 0 1 . 2 9 20EL " 0 . 7 5 1 . 6 0 8 0 . 3 ( 3 8 ) 8 0 . 5 ( 4 1 ) 0 . 6 0 1 . 2 9 n i x 2 / 7 / 8 0 0 . 4 1 0 . 8 7 7 1 . 6 ( 4 2 ) 8 0 . 7 ( 3 4 ) 0 . 5 2 % 1.12% mi x t o . t o t o 8 2 . 6 ( 3 3 ) 7 9 . 7 ( 3 3 ) mix 8 / 7 / 8 0 0 . 8 0 1 . 6 9 8 1 . 1 ( 3 3 ) 8 0 . 1 ( 3 4 ) n i x 7 8 . 4 ( 3 8 ) 8 0 . 2 ( 3 3 ) a v e r a g e 0 . 6 6 1 . 4 1 7 9 . 3% - 4 5 -4. ee E 10 l . 04 7 . 5 5 B . 3 0 9 . 28 1 3 . 03 1 4 . 6 0 1 7 . 3 9 21 . 6 9 2 3 . 22 2 4 . 2 9 15:18 2t>. Ib 2 7 . 8 6 §1:1* 2 9 . 1 7 3 0 . 2 1 31 . 6 0 S30PB4 TEMPI 250 1 00 168 IT1ME1 2 0 . 80 1 RR7E 10. 00 TEHP2 258 170 ITIME2 3 8 . 00 1HJ TEMP 400 250 249 FID TEMP 400 300 299 RUX TEMP 400 0 92 CHT SPIl 0 . 50 . ZERO 10. 0 ATTN 2 t 12 'F ID SGHL SLP SENS 0 . 00 1 . 1 5 IRREfi R E J 50000 FLOW fi 2 0 . 8 20 . 2 FLOW B 0 19 . 8 HP RUN • 218 fiPR/29/81 TIME 11 :47 :28 ID: 1 - 1 7 8 - 2 0 0 fiREfi V. RT flRER ftREfi X 0.51 £ 6 7 6 0 0.503 0 . 7 6 2 5 1 8 0 0 1 .691 1 .04 5 5 8 5 0 0 4. 147 1.17 160300 1 .287 1 . 47 119100 0. 697 1.79 71 12B 0.536 1 .99 167788 1.414 4 . 0 0 10560800 7 9 . 6 9 2 7 . 5 5 5 4 5 4 0 0 .411 6 .30 5 4 7 4 0 0.412 9 .28 192000 1.446 2 1 . 6 9 6 0 3 6 0 0. 455 2 3 . 2 2 117306 0.684 2 4 . 29 0 2 1 0 0 0.618 2 5 . 28 109906 0. 628 2 5 . 68 6 6 5 4 0 0.516 2 6 . 16 74728 0 .563 2 7 . 8 6 1 17488 0. 884 2 7 . 76 6 2 4 4 0 0 . 4 7 0 2e. 21 131400 0. 990 2 9 . 17 71460 0. 538 30 . 21 9 2 6 4 0 8.69ft F i g . 7 - T y p i c a l GLC c h r o n a t o g r a n of certa ' - l e a f o i l . - 46 -trees. Batches 5 - 2 0 were prepared from foliage < 1 cm in diameter while batches 1 - 3 came from foliage < 3.8 cm in diameter. F i n a l l y , batches 1 - 7 were prepared from cedar grown in a r e l a t i v e l y dry area while a l l other o i l s came from cedars grown in a wet be l t , and of approximate age of 5- 15 years. As Table 10 shows, the wet belt cedars averaged a consistently higher (at least 2x) concentration of thujone as a % of wet weight of cedar, while the dry belt cedars had a consistently higher % thujone as a % of total o i l weight. It seems that dry-grown cedars give less o i l containing more thujone or, a l t e r n a t i v e l y , the age of the trees nay be the factor, rather than the climatic condition, von Rudloff found l i t t l e difference (see Table 8) in o i l composition relative to age of the tree as previously mentioned. The "mix" at the bottom of Table 10 refers to o i l samples from various batches mixed together. Four of these were taken at random and % thujone determined. The averaged % thujone in the o i l was multiplied by the average % y i e l d of o i l (both wet wood and dry wood as biomass) to y i e l d a % thujone in weight of wood used. These figures should be "ballpark figures" as to what can be expected for a commercial operation centered in the area in which these wet belt cedars grow. Thus 1000 kg. of wet cedar foilage (grown in the wet belt) should yi e l d around 520 g. of cedar leaf o i l . This figure does not include experimental uncertainty. The minor peaks eluted before thujone compare well with the minor components of von Rudloffs' results in Table 8. These often do not show - 47 -up in the results of the run since they are present in only trace amounts. Overall, the two chromatograms (Figs. 6 and 7) are similar and the peaks 1 - 9 of von Rudloff can probably be di r e c t l y compared to the peaks (eluted before thujone) in Fig. 7 since the GLC column stationary phases are r e l a t i v e l y s i m i l a r , hence order of elution should be the same. The compounds eluted after thujone are not inconsequential as a percent of total material, von Rudloff did not observe these, very probably as a result of using an isothermal run at 100°C. My method used (see Fig. 7) an i n i t i a l temp, of 100°C for Time 1 followed by a rise (Rate = 10°C/min) to (Temp 2) 170°C followed by a further time (Time 2) at this new temperature. These conditions could elute materials (not seen at 100°C isothermal) by d r a s t i c a l l y increasing their v o l a t i l i t y . Thus, some of the compounds detected by other workers 6 9' 6 1 4 6 6 such as kaurene, rimuene, and cupressene found by Alpin and Cambie may be the compounds with higher retention times seen in this study. No eff o r t was made to isolate these compounds or to identify their structure due to their low occurrence, yet their presence in a l l the o i l samples i s interesting. GC-MS of these samples should r e c t i f y the ambiguity by giving a HRMS of the peaks. In closing, l e t me mention that thujone was not resolved from isothujone by this program. The l a t t e r can be seen as a shoulder on the thujone peak and hence % thujone should be read as % of (thujone plus  i sothujone mi xed). For our chemical purposes they are the same compound since one can be epimerized to the other. - 48 -3.2 a-Thujaketonic Acid Since a-thujaketonic acid, a thujone derived compound, was the central compound in our synthesis i t seemed interesting to highlight i t s preparation and spectral data. The treatment of cedar leaf o i l with aqueous potassium permanganate i s a known r e a c t i o n . 7 0 These conditions result in an oxidative cleavage of thujone (XXIV) to a-thujaketonic acid (XXV). (XXIV) (XXV) The spectral data of this acid (XXV) were identical to that of previously published r e s u l t s . 1 1 4 Due to the central role that this material occupied in regards to the synthetic investigation i t s ' N.M.R. spectrum was recorded and analyzed for future reference. 1 4 The spectrum contained a three proton singlet at 62.22 which could be attributed to the methyl group adjacent to the ketonic carbonyl while at 60.94 occurred a six proton doublet (J = 6Hz) which was assigned to the methyl protons of the iso-propyl group. The four l i n e resonance centered at - 49 -52.49 was assigned to the methylene protons adjacent to the carboxylate group. These two protons are adjacent to a chiral centre and thus gave rise to the observed AB pattern (J = 17 Hz.). Centered at 61.91 was a one proton doublet of doublets which was assigned to the proton on the cyclopropane adjacent to the ketonic carbonyl. This proton along with the methylene protons of the cyclopropane ring formed an ABX system. One of the cyclopropane methylene protons gave rise to the doublet of doublets centered at 60.88, the other cyclopropyl proton and the methine proton of the isopropyl group then accounted for the multiplet which spanned the region 61.5 to 61.1. 3.3 Synthetic Objectives Previous work in thujone c h e m i s t r y 5 9 * 6 3 have given rise to the possible analogues via Wittig olefination of the ketonic carbonyl of o-thujaketonic acid (XXV). It was decided to continue in this area by making suitable intermediates ( i . e . , appropriate units of the f i n a l JHA) - 50 -e n t i r e l y from thujone and then to develop suitable methods of coupling these, via olefination processes, to complete this convergent synthesis. The work described in this section of the thesis was aimed at: (1) the transformation of thujone into a number of 'left-hand halves' (see Scheme 2) to act as nucleophiles in the coupling reaction, (2) the preparation of a-thujaketonic acid analogues into a number of 'right-hand halves' to act as electrophiles in the coupling reaction, (see Scheme 3) (3) the development of methods to couple these two halves together, (Scheme 4), and (4) further transformation of these products into suitable JHA's. If this was successful i t should be possible to produce hormone analogues of the type shown in Table 11. 3.4 The Coupling Reaction After investigating the possible pathways for coupling the two synthetic halves together, the 6-lactone route was decided upon for the following reasons. F i r s t l y , B-lactones are easily prepared from B-hydroxy carboyxlic a c i d s , 8 4 - 8 7 , 7 8 , 7 9 v i a the formation of mixed ^ anhydrides. These B-hydroxy acids are often readily obtainable via the nucleophilic attack of a carboxylic acid dianion onto an aldehydic or ketonic carbonyl system. 8 4~ 8 7» 7 8» 7 9 Secondly, these carboxylate and ketonic groups are present in thujone-derived compounds (as seen in Scheme 2, 3) hence no functional group transformations would be necessary. Thirdly, the use of the B-lactone route leads to no ambiguity about the position of the central double bond, since B-lactones pyrolyze to alkenes without any isomerization of the double Scheme 2 - Elaboration of a-Thujaketonic Acid into Synthetic 'Left-Hand' Halves. Scheme 3 - Elaboration of a-Thujaketonic Acid to Synthetic 'Right-Hand' Halves. - 53 -V f U& H T - H r \ N D HFALF^ O 1) L "DrX 4/ (S,.leGLU\v.) C H — COO o ^ — o X U c H 3 R' J H 5>v fc\^-V\p^ <k VNPAT. C O O H OVA * 5 ° C 7 C H C H 3 Scheme 4 - The Coupling Reaction. - 54 -Table 11. Structural Comparison of Juvenile Hormone with Possible Analogues Available from a-Thujaketonic Acid. R = Methyl, Ethyl R' = Methyl, Ethyl A l l compounds also available with (Z)-isomer of central o l e f i n . - 55 -bond. ° ' B» y Lastly, the use of this method obviates any need to shorten the carbon chain by one carbon (this chain shortening is necessary to arrive at a JHA of the same chain length as the natural JHs). The elimination of carbon dioxide in the pyrolysis step achieves t h i s necessity in a very desirable manner. 3.5 The Synthesis of Left Hand Halves The f i r s t synthetic scheme was envisaged as shown in Scheme 5. After methyl ation of the ketone, an acid catalyzed ring opening of the cyclopropylcarbinol to the dienoic a c i d 6 0 was planned, and th i s product would subsequently be transformed to the key intermediate. Grignard reaction of a-thujaketonic acid (XXV) with methyl magnesium iodide gave, after workup, only a small amount of acidic mate rial. This proved to be unreacted starting material. Isolation of the neutral products of the reaction produced a c r y s t a l l i n e material (XXVI) in high y i e l d , apparently formed via spontaneous cyclizations of the alcohol (LVIII). (XXVI) - 56 -Scheme 5 - Elaboration of a-Thujaketonic Acid to a Left-Hand Hal (XXVII). - 57 -This lactone proved intractable to a l l attempted methods of ring opening-both of the lactone moiety or of the cyclopropane ring. Strongly acidic or basic conditions were tried as well as pyrolysis and hydrogenolysis; these yielded only tarry decomposition products or unreacted starting material. Thus, this scheme was abandoned in favour of another route (Scheme 6). The a-thujaketonic acid (XXV) was pyrolyzed to the known B-thujaketonic acid ( X X I X ) . 1 4 ' 5 9 This ring-opened material was hydrogenated at one atmosphere pressure over 5% palladium on charcoal to give the racemic saturated keto-acid (XXX). Qualitatively the N.M.R. spectrum of this compounded proved quite similar to that of the starting material. The v i n y l i c proton of 6-thujaketonic acid was absent and the former a l l y l i c protons were shifted to higher f i e l d (61.2 - 1.7) where they appeared as an unresolved multiplet. Grignard reaction of (XXX) with excess methyl magnesium iodide gave the alcohol acid (XXXI), as well as considerable amounts of neutral material. This neutral product showed a three proton singlet at 62.16 in the N.M.R. and proved to be compound (XXXII). This appears to be analogous to the formation of ketones from carboxylic acids via the use of alkyl lithium reagents. ( X X X I I ) - 58 -COOH ( X X V " ) (xxxi) Pd/C COOH 2> 2) Stpo*aJt COOH C H j , COOH (xxv u) (xxvui) Scheme 6 - Elaboration of a-Thujaketonic Acid (via new route). - 59 -The proposed structure of (XXXII) was further substantiated by the following conversions. Borohydride reduction of (XXXII) in methanol gave the diol (XXXIII). The N.M.R. spectrum of (XXXIII) showed a one proton multiplet at 63.90; acetylation of the l a t t e r with acetic anhydride in pyridine gave the monoacetate (XXXIV). The N.M.R. spectrum of (XXXIV) was similar to that of (XXXIII) with these exceptions: (XXXIV) contained a three proton singlet at 62.05 - attributed to the methyl of the acetate group - and i t also contained a one proton multiplet at 64.90 as compared to the one proton multiplet at 63.90 in (XXXIII). The use of 2.0 equivalents of methyl magnesium iodide in the Grignard reaction gave a good y i e l d of the desired acid (XXXI) with no ketone byproduct (XXXII) being isolated. (XXXIII) (XXXIV) Pyrolysis of (XXXI) at 125°C for one hour followed by d i s t i l l a t i o n in vacuo gave the desired al kenes (XXVII) and (XXVIII) in good y i e l d ; attempted dehydration of (XXXI) via f e r r i c chloride on 71 s i l i c a was unsuccessful. - 60 -Examination of the dehydration product via 270 MHz N.M.R. showed two o l e f i n i c protons: a singlet at 64.73, and a t r i p l e t (J = 6Hz) at 65.13. The former represents the v i n y l i c methylene of (XXVIII) while the l a t t e r represents the single o l e f i n i c proton on (XXVII). The integration of these two resonances showed that the ratio of the products (XXVII)/(XXVIII) was 4:1. (XXVIII) - 61 -Attempts to separate (XXVII) and (XXVIII) by exhaustive r e c r y s t a l l i z a t i o n of the dicyclohexylammonium salts f a i l e d , as did attempts at TLC and HPLC. Separation was achieved via column chromatography on s i l i c a gel impregnated with 12.5% (by weight) s i l v e r n i t r a t e , in the absence of l i g h t . 7 2 Analytical separation on HPLC was achieved by u t i l i z i n g a s i l v e r nitrate impregnated p-porasil column. At this time, an interesting transformation was unearthed. Heating of the known14 methylene derivative of ct-thujaketonic acid (LIX) for 5-6 hours at 140-145°C gave, as a c r y s t a l l i n e material, the dienoic acid (XXXV). This appears to be a [1,5] sigmatropic s h i f t with the cyclopropane ring acting as an alkene extending conjugation. Compound (XXXV) gave a very good first-order N.M.R. spectrum. At 65.71 there was a one proton singlet ( C 2-H), and at 65.09 there was a one proton t r i p l e t (J = 6Hz.), attributed to the other vinyl hydrogen, analogously to compound (XXVII). A two proton doublet (J = 6Hz.) centered at 63.39 was assigned to the d i - a l l y l i c protons, while the a l l y l i c methine gave a septet (J = 6Hz.) at 62.41. A six proton singlet COOH (LIX) (XXXV) - 62 -at 61.68 was assigned to the vinyl methyl groups while the isopropyl methyl resonances appeared as a doublet (J = 6Hz.) at 61.02 and 61.10. Heating of (XXXV) causes further isomerization to (XXXVI). As th i s reaction proceeds, the vinyl resonances of (XXXV) (in i t s N.M.R. spectrum) disappear as a two proton singlet at 66.10 appears. This transformation i s of interest as ol e f i n migration tends to be towards carbonyl systems, rather than away from them. (XXXVI) Conditions for the reduction of the C 2 - C 3 o l e f i n of (XXXV) to form the key intermediate (XXVII) were investigated. Attempted 73 reduction with 3% sodium amalgam in various solvents were unsuccess-f u l , as were Clemmensen reduction c o n d i t i o n s . 7 4 Hydrogenation was not selective enough. Attempted reduction with potassium intercalated in graphite was also u n s u c c e s s f u l . 7 5 " 7 7 Reduction was achieved (in over 85% yield) via potassium in l i q u i d ammonia followed by quenching with dry iso-propanol, 8 2 Compound (XXVII) prepared in this way was identical to that prepared e a r l i e r ( i . e . (XXXI) + (XXVII) + (XXVIII)). - 63 -3.6 Preparation of the Right Hand Half The right hand half used was prepared by esterifying 6-thujaketonic acid (XXIX) with diazomethane in ether. A quantitative y i e l d of a material identical to that of a published sample 1 4 was obtained. This material (XXXVII) was used in the coupling reaction. Attempts to produce the isomeric (Z)-B-thujaketonic methyl ester (XXXVIII) were abandoned due to the low y i e l d reported 1 4 and confirmed by our experiments. (XXXVII) (XXXVIII) 3.7 The Coupling Reaction The f i r s t attempts were made using compounds (XXVII) and (XXXVII) as seen below (see Scheme 4): - 64 -2> (XXXVX) CVH(^  (XL) Thus (XXVII) was reacted with 2.1 equivalents of lithium diisopropylamide in dry THF under nitrogen at -78°C, followed by warming of the mixture to 50°C for four hours. Next, the acid dianion intermediate was cooled to -78°C, and (XXXVII) was added (via syringe) as a solution in dry THF. After t h i r t y minutes, the reaction mixture was poured into ice water and extracted with ether. The ether was dried, f i l t e r e d , and evaporated to yield unreacted (XXXVII). The - 65 -aqueous phase was cooled to 0°C in an ice bath and carefully a c i d i f i e d (1 M HC1) to pH 5.0. This solution was extracted exhaustively with ether and the combined ethereal extracts were washed with brine, dried (Na2S0L,), f i l t e r e d , and evaporated in vacuo to give the crude B-hydroxy carboxylic acids (XXXIX) and (XL). This mixture was kept at 5°C to preclude any p o s s i b i l i t y of dehydration to form the unsaturated acid. Analysis of the crude mixture by N.M.R. showed the presence of two o l e f i n i c resonances, a singlet at 65.70 (proton adjacent to ester carbonyl), and a t r i p l e t (J = 6Hz.) at 65.12. These resonances had integrations in the ratio of 3:4, which implied a ratio of 75% B-hydroxy acids (XXXIX) and (XL) to 25% unreacted starting acid (XXVII). 3.8 Preparation of g-lactones and Attempted Preparation of Analogues The crude B-hydroxy acids (XXXIX) and (XL) were dried via azeotropic d i s t i l l a t i o n (with ethyl acetate), in vacuo, at 0°C. This dried material was dissolved in dry, cold pyridine.and three equivalents of dry, d i s t i l l e d benzenesul fonyl chloride were added, at 0°C. This mixture was stired for 48 hours at 5°C. A l l subsequent steps were performed at 5°C maximum temperature (in the cold room). The purple solution was poured into ice-water and extracted with ether. The organic layers were combined, washed with saturated bicarbonate solution, saturated copper sulphate (to remove pyridine), and brine, dried (Na2S0i4), f i l t e r e d , and evaporated in vacuo (at 0°C) to give the crude B-lactones (XLI), and (XLII). The crude mixture was analyzed by infra-red spectroscopy and the characteristic B-lactone absorbance at - 66 -1805 cm - 1 was very strong. The crude 3-lactones were dissolved in dry methylene chloride and 1.01 equivalents of metachloroperbenzoic acid (previously purified by washing with a pH 7.10 buffer and drying to constant weight) were added. After s t i r r i n g for 24 hours at 5°C, the mixture was poured into ice-water and extracted with ether. The combined organic layers were washed with saturated bicarbonate and brine, dried (Na 2S0 4), f i l t e r e d , and evaporated in vacuo (at 0°C) to give the crude epoxy lactones (XL 111) and XLIV). This residue was chromatographed on s i l i c a gel to give homogenous materials (by TLC). (XLI Tl) and (XLIV) - 67 -These 'homogenous' epoxylactones s t i l l showed the characteristic 8-lactone absorbance at 1805 cm - 1 in the I.R. These materials were pyrolyzed at 110°C for one hour in 2-methyoxyethanol. Only tarry decomposition products were seen. Repetition of this reaction sequence several times gave identical results; q u a l i t a t i v e l y , the 6-lactones and epoxy 3-1actones appeared to be formed in reasonable y i e l d . The 3-1 actones gave a parent M* in the mass spectrum and showed the expected infra-red spectrum. The epoxidized material was more polar than the o l e f i n 3-lactones (by TLC). Due to the l a b i l i t y and i n s t a b i l i t y of these compounds complete characterization was d i f f i c u l t . The pyrolysis step to remove carbon dioxide seemed to cause the whole molecule to decompose. It was decided to try a parallel set of reactions to test for any technical d i f f i c u l t i e s . Thus, the carboxylic acid dianion of the key intermediate (XXVII) was quenched wth D20. Examination of this product (at 270 MHz) by N.M.R. showed that the integration of the methylene protons adjacent to the carboxylic acid (at 62.25) corresponds to about 1.2 protons. Examination of a pure sample of (XXVII) at (270 MHz) shows (at 62.25), a well resolved AB quartet s p l i t into doublets by the adjacent methine (J = 14, 6 Hz). The integration (of pure XXVII) for these resonances i s about two protons. Thus the extent of anion formation is 80%. - 68 -S3l^eauw.L0A D (XXVII) (XXVII) 80% D Next, the dianion of (XXVII) was quenched with hexadeuteroacetone to examine the r e a c t i v i t y of the dianion to a ketonic electrophile very similar to that used ( i . e . XXXVII) in the coupling reaction. This reaction was run under identical conditions to those of the coupling reaction. After workup the product (XLV) by examined. Its N.M.R. spectrum was very similar to that of the starting acid (XXVII) except that the resonance at 62.25 was shifted to 62.50. This i s attributed to the deshielding effect of the neighbouring carbinol. This material (XLV) gave a parent mass of 248.2255 (calculated 248.3100) in the mass spectrum and appears to be pure. It was formed in 67% y i e l d . COOH C O O H LOF\/THP C P 3 OH (XXVII) (XLV) - 69 -O (XLVI) Lactonization as described above produced the 6-lactone (XLVI). With questions of technical competence muted, further attempts were made on the synthetic pathway. The yield of B-hydroxy carboxylic acids (XXXIX) and (XL) was, at best, 75%, even i f HMPA was added to the dianion coupling reaction m i x t u r e . 6 0 > 7 8 . 7 9 Since the lactonization and pyrolysis reactions worked on d 6-acetone, i t was decided that the epoxide moiety was i n t e r f e r i n g . Since the epoxide end of the natural JH is a known site of metabolism, 8 0 i t was decided to pursue this synthesis with some alkoxy derivatives to make the alkoxy JHA's (see Scheme 2, and compound (XXIII)). 3.9 Preparation of Hormone Analogues Oxymercuration of (XXVIII) with mercuric acetate in anhydrous ethanol, followed by demercuration with sodium borohydride in aqueous caustic soda gave the ethoxy acid (XLVIII) in 80% y i e l d . Upon N.M.R. examination (XLVIII) showed the complete disappearance of any vinyl resonances, while a two proton AB quartet (J = 8Hz.) (the methylene of the ethoxy group) appeared at 63.35. \ - 70 -COOH fto COOH (XXVIII) (XLVIII) Replacement of (XXVII) with (XLVIII) in the coupling reaction (as described above) scheme gave, after the usual work up, a 70% y i e l d of the mixed ethoxy 8-hydroxy carboxylic acids (L) and (LI). The N.M.R. spectrum of this product shows the 65.68 singlet (one hydrogen in intensity) of the proton adjacent to the ester carbonyl, a three proton singlet at 63.66, attributed to the ester methyl group, and a two proton multiplet assigned to the methylene of the ethoxy moiety. This crude material was lactonized with benzene sulfonyl chloride in pyridine; i t showed the 3-lactone absorbance at 1805 cm - 1 in the infra-red spectrum. After TLC p u r i f i c a t i o n , a homogenous material was examined by N.M.R. It showed a spectrum very similar to that of the B-hydroxy acids. The spectra of (L11) and (L111) show the appearance of two singlets at 61.52 and 61.63. These were assigned to the methyl groups B-to the lactone in the expected isomers (see wavy lines in LII and L I I I ) . After pyrolysis of t h i s mixture of lactones (LII) and (L111) and TLC p u r i f i c a t i o n , a homogenous product was obtained. The N.M.R. of this - 71 -material was very similar to that of the above 3-lactones; the differences include the appearance of a broad doublet (J = 9 Hz) centered at 64.90. This broad one proton resonance was assigned to the proton on the internal double bond. Two singlets at 61.64 and 61.77 (intensity 3H) were assigned to the vinyl methyl groups of the internal double bond. Since the two resonances are seen, this product appeared to be a mixture of the two juvenile hormone analogues (LIV) and (LV). The overall yield of this hormone mixture, from the ethoxy acid (XLVIII), was 56%. C O O C H 3 (XLVIII) (L) and (LI) 3 (LID and (LIII) - 72 -(LII) and (LIII) (LIV) and (LV) 3.10 Preliminary Investigations Toward the Synthesis o f Other JHA Oxymercuration of (XXVIII) with methanol solvent gave the methoxy acid ( I L ) , analogously to the ethoxy acid (XLVIII), in 80% y i e l d . The N.M.R. spectrum of (IL) showed no vinyl l i e resonances but a singlet at 63.18 (3H in i n t e n s i t y ) , attributed to the methoxy methyl was seen. The methoxy acid was subjected to the usual dianion coupling reaction conditions and workup, and (XXXVII) was used as the electrophile. A mixture of B-hydroxycarboxylic acids (LVI) and (LVII) was obtained in 70% y i e l d . These gave a N.M.R. spectrum very similar to that of the ethoxy acids (L) and (LI) with the other differences being attributed to the difference between the methoxy group vs. the ethoxy group. Unfortunately time constraints prevented us from characterizing - 73 -t h i s compound carrying t h i s material through to the hormone stage. With the ethoxy hormone experience in hand this was thought to present no d i f f i c u l t y , however. (xxvm) LTDftjTHF C O O H - 74 -4. EXPERIMENTAL The a-thujaketonic acid (XXV) used was prepared from cedar leaf o i l by the method of Thompson.81 The cedar leaf o i l was supplied by MacMillan Bloedel Research Ltd., and Alan Moss and Assoc., and generally consisted of approximately 88% thujone and 12% terpenoid impurity. Products were generally characterized by the boiling point (BP), melting point (mp), infrared spectrum (I.R.), proton magnetic resonance spectrum (N.M.R.), low resolution mass spectrum (LRMS), high resolution mass spectrum (HRMS), and elemental analysis. Except for the a-, and B-thujaketonic acids, and the lactone (XXVI) no physical or 8 3 spectral data for any of the compounds prepared had been published. Boiling points were determined during d i s t i l l a t i o n . Melting points were uncorrected. Infrared spectra were recorded on the Perkin Elmer Model 710 spectrophotometer. Polystyrene was used as the calibrant and the samples were run as 3% solutions in chloroform with a path length of 0.1 mm between salt plates. The position of the absorption was recorded in wavenumbers (cm - 1). Proton magnetic resonance spectra were usually obtained on the Varian XL-100 model. Spectral data were occasionally obtained on the 270 MHz or the Bruker 400 MHz instruments and reference to these i s made below. The solvent was deuterochloroform and tetramethylsi 1ane was used as the internal standard. Mass spectra were run on the Varian/Mat model CH4B or AEI MS-902 spectrometers. Electron energy was generally 70 eV. Analytical GLC separations were performed on the Hewlett-Packard 5830A gas - 75 -chromatograph. A l l samples were homogenous by GLC before analysis. Column chromatography u t i l i z e d Merck s i l i c a gel 60 (70-230 mesh). Thin layer chromatography u t i l i z e d Merck s i l i c a gel GF 2 5 l 4. Combustion analyses obtained were performed by Mr. P. Borda. 3-Isopropyl-3,4-cyclopropyl-6,6-dimethyl-tetrahydro-l,2-pyrone (XXVI) To magnesium turnings (0.750 g., 0.031 moles) in dry ether, (40 ml), under nitrogen, was added methyl iodide (1.9 ml, 0.033 moles), with s t i r r i n g . After a l l the magnesium had dissolved, the Grignard reagent was cooled to 0°C (ice bath). Dry a-thujaketonic acid (1.15 g., 0. 0062 moles) was added as a solution in dry THF (20 ml), via syringe, over ten minutes. After s t i r r i n g for one hour at 0°C, the solution was warmed to 25°C and st i r r e d for 24 hours. The mixture was poured into ice-water and extracted with ether. The combined organic layers were washed with brine, dried, and evaporated in vacuo to yi e l d a s o l i d . Recrystal1ization gave 0.745 g (66%) of (XXVI) as green c r y s t a l s , mp (hexane/ether): 21.5 - 23°C. N.M.R. (Fig. 8): 62.58 (2H, AB quartet (J = 19 Hz),-0-C0-CH2), 61.38 (3H, singl e t , C H 3-C-O), 61.42 (3H, s i n g l e t , CH3-C-0), 60.96 (7H, multipiet ,-CH(CH 3) 2), 60.48(2H, doublet (J = 6 Hz), cyclopropyl CH 2), 61.20 (IH, m u l t i p l e t , cyclopropyl methine). 1. R. (Fig. 9): 1725 cm"1 (C = 0), 1300 cm"1, 1150 cm"1 (C-0). HRMS: M+ 182.130, (calculated for C n H 1 8 0 2 : 182.262), (M-CH 3) + 167.11. Analysis: Calculated for C n H 1 8 0 2 : C, 72.53; H, 9.89; found: C, 72.46; H, 10.00. Fig. 8 - 100 MHz. N.M.R. Spectrum of (XXVI). Fig. 9 - I.R. Spectrum of (XXVI). - 78 -3-Isopropy1-6-methy1-5- and 6-heptenoic acids (XXVII) and (XXVIII) The alcohol acid (XXXI) (2.91 g., 0.0144 moles), was slowly heated to 140°C and the pressure was then slowly reduced. D i s t i l l a t i o n (122°C at 1 torr) gave 2.6 g of an o i l . The l a t t e r was chromatographed in the dark on a column containing 50 g of 12.5% s i l v e r n i t r a t e impregnated s i l i c a gel. Elution with chloroform/methanol/acetic acid gave 1.14 g of the acid (XXVII) (71%) and 0.36 g of the acid (XXVIII) (18%). The trisubstituted olefin acid (XXVII) gave: N.M.R. (at 270 MHz) (Fig. 14): 62.40 (AB quartet s p l i t into doublets (J = 17,7 Hz), CH2-C00), 65.48 (1H, t r i p l e t (J = 7 Hz), C = CH), 61.80 (3H, s i n g l e t , vinyl methyl), 61.70 (3H, sing l e t , vinyl methyl), 60.95 (6H, two doublets (J = 6 Hz), (CH 3) 2C-) I.R. (Fig. 15): 1710 cm"1 (C = 0), 1645 cm - 1 (C = C). HRMS: M+ 184.1463, (calculated for CnH 2 00 2: 184.2778). Analysis: Calculated for C n H 2 0 0 2 : C, 71.69; H, 10.94; found: C, 71.52; H, 10.96. The disubstituted o l e f i n acid (XXVIII) gave: N.M.R. (Fig. 16): 64.65 (2H, singlet, CH2 = C), 61.66 (3H, sing l e t , vinyl methyl), 60.88 (6H, two doublets (J = € Hz) , (CH 3) 2C-), 68.10 (1H, broad singlet (D20 exchangeable), COOH), 62.30 (2H, AB quartet, CH2-C00). I.R. (Fig. 17): 1710 cm"1 (C =0), 1646 cm"1 (C = C). HRMS: M+ 184.1462, (calculated for CnH 2 00 2: 184.2778). Analysis: calculated for CnH 2 00 2: C, 71.69; H, 10.94; found: C, 71.52; H, 10.96. 3-Isopropy1-6-keto-heptanoic acid (XXX) 6-Thujaketonic acid (XXIX) (1.64 g, 0.009 moles) was dissolved in ethanol (30 ml) and 5% palladium on carbon (0.17 g) was added. The sti r r e d mixture was hydrogenated at one atmosphere pressure un t i l 1S0O > 106b BOO i 1 • F1g. 14 - 270 MHz. N.M.R. Spectrum of (XXVII). Fig. 15 - I.R. Spectrum of (XXVII). so MOONS 7.0 • 0 WKVTNUMM* fcw'l F1g. 17 - I.R. Spectrum of (XXVIII). F 1 g . 10 - 100 MHz. N.M.R. Spectrum of (XXX). VWWlNUMBIllCM 'I WAVINUMM* fcfi'. ' Fig. 11 - I.R. Spectrum of (XXX). - 85 -hydrogen uptake ceased (ca. 7 hours). The slurry was f i l t e r e d and evaporated in vacuo to y i e l d an o i l which was d i s t i l l e d (125°C at 1 torr) to give (XXX) (1.65 g, 100%) as on o i l . N.M.R. (Fig. 10): 610.60 (1H, broad singlet (D20 exchangeable), COOH), 62.40, (2H, multiplet, CH2C00), 62.20 (2H, multiplet, CH2C0), 62.07 (3H, sin g l e t , CH3C0), 60.80 (6H, two doublets (J =1 Hz), (CH 3) 2-C). I.R. (Fig. 11): 1710 cm"1 (C = 0). HRMS: M+ 186.126, (calculated for C 1 0H 1 80 3: 186.250), (M -CH 3) + 171.102, (M - H 20) + 168.115. Analysis: Calculated for C i 0H i 8 0 3: C, 64.47; H, 9.74; found: C, 64.27; H, 9.71. 6-Hydroxy-3-isopropy1-6-methyiheptanoic acid (XXXI) To magnesium turnings (3.28 g, 0.135 moles) in dry ether (160 ml) under nitrogen, was added methyl iodide (8.5 ml, 0.136 moles) via syringe, with dry ether (60 ml). The mixture was stir r e d to dissolve a l l the magnesium and cooled to 0°C. The keto-acid (XXX) (8.35 g, 0.47 moles), was added as a solution in dry THF (200 ml). The mixture was warmed to 22°C and stir r e d for 24 hours. The products were poured into water and extracted with ether. The organic layers were combined and labelled NEUTRAL FRACTION. The aqueous phase was cooled to 0°C (ice-bath) and aci d i f i e d to pH 4.0 (1M HC1). The solution was extracted with ether; the combined organic phases were washed with brine, dried, and evaporated in vacuo. The residue was d i s t i l l e d (121°C at 1 torr) to give (XXXI) (8.34 g, 90%) as an o i l . If 2.0 equivalents of Grignard reagent were used, the yi e l d of (XXXI) was 90%; no neutral material was isolated. N.M.R. (Fig. 24): 60.85 (6H, two doublets (J = 1 Hz), (CH 3) 2C), 61.18 (6H, sin g l e t , (CH 3) 2C-0), 62.25 (2H, multiplet, CH2C00), 66.32 (2H, broad singlet ( D 2 O exchangeable) OH and COOH). I.R. F1g. 25 - I.R. Spectrum of (XXXI). - 88 -(Fig. 25): 1710 cm"1 (C = 0), 3400 cm"1 (OH), 910 cm - 1. HRMS: M+ 202.1588, (calculated for C 1 1 H 2 2 O 2 : 202.2930), (M-H 20) + 184.1468. Analysis: Calculated for C n H 2 2 0 3 : C, 65.31; H, 10.96; found: C, 65.52; H, 1 0 . 7 9 . 7-Hydroxy-4-isopropyl-7-methyl-2-octanone (XXXII) The portion labelled NEUTRAL FRACTION in the synthesis of (XXXI) was dried and evaporated to give a residue which was d i s t i l l e d (132°C at 15 torr) to give (XXXII) (0.617 g, 7%) as an o i l . N.M.R. (Fig. 18): 62.15 ( 3 H , singlet, - C0CH3), 61.20 ( 6 H , s i n g l e t , (CH 3) 2C0), 62.30 (2H, multiplet, CH2C00), 61.60 (IH, singlet (D 20-exchangeable), OH), 60.88 ( 6 H , two doublets (J = 1 Hz), (CH 3) 2C-) I.R. Fig. 19: 1710 cm"1 (C = 0), 3500 cm - 1 (OH), HRMS: (M - CH 3) + 185.1543, (calculated for C 1 2H 2 40 2-CH 3: 185.2857). Analysis: Calculated for Ci 2H 2 1 +0 2: C, 7 1 . 9 5 ; H, 12.08; found: C, 71.62; H, 12.08. 7-Hydroxy-4-isopropyl-7-methyl-2 ,7-octanediol (XXXIII) The keto-acid (XXXII) (100 mg, 0.5 mmoles), was dissolved in dry methanol (10 ml) and cooled to 0°C. Sodium borohydride (220 mg, 6 mmoles) was added and the mixture stirred for ten hours at 22°C. The products were poured into water and extracted with ether. The organic phase was washed with brine, dried, and evaporated in vacuo to give an o i l which d i s t i l l e d (115°C at 0.5 torr) to give 80 mg of (XXXIII) (80%). N.M.R. (Fig. 20): 63.85 (IH, multiplet, CH-0H), 61.50 (2H, broad singlet (D20 exchangeable), OH's), 5U20 ( 9 H , s i n g l e t , (CH 3) 2-C0 and CH3-C0), 60.88 ( 6 H , multiplet, (CH 3) 2-C). I.R. (Fig. 21): 3400 F1g. 18 - 100 MHz. N.M.R. Spectrum of (XXXII). Fig. 19 - I.R. Spectrum of (XXXII). Fig. 20 - 100 MHz. N.M.R. Spectrum of (XXXIII). F1g. 21 - I.R. Spectrum of (XXXIII). - 93 -cm"1 (OH). HRMS: (M-CH3)+ 187.1698, (calculated for C 1 2H 2 60 2-CH 3: 187.3015). 2- Acetoxy-7-methyl-4-isopropyl-7-octanol (XXXIV) The diol (XXXIII) (80 mg, 0.4 mmoles) was dissolved in 1.5 ml dry pyridine, and 1 ml acetic anhydride was added. The solution was stirred for 18 hours, and poured into water. After one hour, the mixture was extracted with ether; the combined organic layers were washed with saturated bicarbonate and brine, dried, and evaporated in vacuo to yi e l d a residue. This material d i s t i l l e d (118°C at 0.5 torr) to give 80 mg. of (XXXIV) (80%) as an o i l . N.M.R. (Fig. 22): 60.84 (6H, two doublets (J = 7Hz.), (CH 3) 2-C), 62.00 (2H, singlet, CH3-C00), 64.92 (IH, multiplet, CH-0C0), 61.65 (IH, broad singlet (D20 exchangeable), OH), 61.16 (9H, multiplet,(CH 3) 2-C0 and CH3-C-00C) I.R. (Fig. 23): 1710 cm"1 (C = 0), 3400 cm"1(OH). HRMS: (M-CH 3) + 229.1796, (calculated for Ci 4H 2803-CH 3: 229.3387). 3- Isopropy1-6-wethy1-2-(E)-5-heptadienoic acid (XXXV) The known methylene derivative of a-thujaketonic acid (60g, 0.3 moles) was prepared by the l i t e r a t u r e 1 4 method (in 100% yi e l d vs 90%lk), and was refluxed, under nitrogen, under reduced pressure (140°C at 3-4 t o r r ) , for six hours. After cooling the residue s o l i d i f i e d and was recrystal1ized (to constant mp) to yield (XXXV) (42 g, 70%). mp (hexane/ether): 65°C. N.M.R. (Fig. 12): 65.72 (IH, single t , C2-H) , 65.10 (IH, t r i p l e t (J = 7 Hz.), CH = C), 63.40 (2H, doublet (J = 7 Hz.), a l l y l i c methylene), 62.43 (IH, septet (J = 7 Hz.), isopropyl methine), -1—1—I I. I I I I I I I I I I I I I I I I I I t I I I I. I I I I I I I I I I I I I I • I I I • • I I I I I I I I . I I I • I I I I I I I • I I • • I J i—I I I i i i I i i I i I i i — i — i I i i — I I—I I I i _ J I I i I I ' i i I i i • i I i i i i ' ' i i i L_i I u _ i I i i i i l i i i l l ~ i — i — i — | — i — i — i — i — | — i — i — i — I — | — i — i — i — r - | — r — i — i — I — [ — ' I — I — I I | — I — i — i — i — | — i — i — I — r — | — i — i — i — i — | — i i i —1— | — i — r — i — r — | — i — i — I — r — ^ — i — i — i — i — | I i — r — i — | — i— i— i — i—p— i — i — i— i—p F 1 g . 22 - 100 MHz. N.M.R. Spectrum of (XXXIV). Fig. 23 - I.R. Spectrum of (XXXIV). V Fig. 12 - 100 MHz. N.M.R. Spectrum of (XXXV). F1g. 13 - I.R. Spectrum of (XXXV). - 98 -61.70 (6H, s i n g l e t , vinyl methyls), 61.08 (6H, doublet (J = 7 Hz), isopropyl methyls). I.R. (Fig. 13): 1690 cm - 1 (C = 0), 1633 cm"1 (C = C). HRMS: M+ 182.1309, (calculated for C n H 1 8 0 2 : 182.2620), (M - C 3 H 7 ) + 139.0760. Analysis: calculated for C n H i 8 0 2 : C, 72.49; H, 9.95; found: C, 72.20; H, 9.98. 3-Isopropy1-6-Methy1-3,5-heptadienoic acid (XXXVI) The acid (XXXV), (100 mg, 0.55 mmoles), was heated at 140-145°C (at 5 torr pressure), under nitrogen, for 7 hours to give an o i l . This residue was purified by T.L.C. to give (XXXVI) (60 mg, 60%) as an o i l . N.M.R.: 66.05 (2H, singlet, C = CH - CH = C), 61.80 (3H, s i n g l e t , CH 3 -C = C), 61.75 (3H, s i n g l e t , CH3 - C = C), 61.05 (6H, two doublets (J = 7 Hz), (CH 3) 2-C), 62.70 (2H, multiplet, CH2-C00), 62.35 (1H, multiplet, isopropyl methine). I.R.: 1710 cm'1 (COOH), 1635 cm"1 (C = C). HRMS: M+ 182.1314, (calculated for C n H i 8 0 2 : 182.2620). g-Hydroxycarboxylic acids (XXXIX) and XL) To dry, d i s t i l l e d diisopropylamine (0.922 g, 9.11 mmoles) in dry tetrahydrofuran (6.3 ml), under dry nitrogen at -78°C was added, via syringe, 5.3 ml. of 1.70 21 n-butyl lithium in hexane. The solution was stirred for one hour at -78°C, and then raised to -23°C (freezing CC1 4). The olefin acid (XXVII) (0.763 g; 4.14 mmoles), was added, via syringe, as a solution in dry THF (1 ml). After s t i r r i n g for one hour at -23°C and then for four hours at 50°C, the solution was cooled to -78°C where the keto-ester (XXXVII) (0.821 g, 4.14 mmoles) was added, via syringe, as a solution in dry THF (1 ml). After warming to room F1g. 27 - I.R. Spectrum of (XXXIX) and (XL). - 101 -temperature and s t i r r i n g one hour, the mixture was poured into ice water and extracted with ether. The ether layer was dried and evaporated to yi e l d 0.25 g of unreacted (XXXVII). The aqueous phase was cooled to 0°C (ice bath), a c i d i f i e d to pH 5.0, extracted with ether, washed with brine, dried and evaporated in vacuo to yi e l d the B-hydroxycarboxylic acids (XXXIX) and (XL) (1.33 g, 70%). [This material also contains about 0.23 g of unreacted (XXVII)]. N.M.R. (Fig. 26): 65.70 (1H, sing l e t , C 2-H), 65.12 (1H, t r i p l e t (J = 7 Hz.), C = CH), 63.64 (3H, sin g l e t , C00CH3), 61.64 (3H, s i n g l e t , CH3 - C = C), 61.72 (3H, s i n g l e t , CH3 - C = C). I.R. (Fig. 27): 1710 cm"1 (C = 0), 1635 cm"1 (C = C), 3500 cm"1 (OH). LMRS M+ 382; 364,349. The g-Lactones (XLI) and (XLII) The mixture of B-hydroxy acids (XXXIX) and (XL) (1.0 g, 0.25 mmoles), was dissolved in dry pyridine (5 ml) and cooled to 0°C. Benzene sulfonyl chloride (0.2 g, 1.1 mmoles) was added and the mixture was st i r r e d for 48 hours at 5°C. A l l subsequent operations were performed at 5°C (cold-room) to preclude any decarboxylation. The products were poured into water and extracted with ether. The combined organic phases were washed with saturated sodium bicarbonate and brine, dried, and evaporated in vacuo to yiel d the B-lactones (XLI) and (XLII) (0.75 g, 75%). N.M.R. (Fig. 28): 65.77 (1H, s i n g l e t , C 2-H), 65.12 (1H, t r i p l e t (J = 6 Hz.), C = CH), 63.70 (3H, sing l e t , C00CH3), 61.60 (3H, si n g l e t , CH3 - C = C), 61.70 (3H, s i n g l e t , CH3 - C = C), 60.85 (6H, multip l e t , (CH 3) 2C-), 61.05 (6H, multiplet, (CH 3) 2-C - C = C). I.R. (Fig. 29): 1805 cm"1 (B-lactone), 1706 cm"1 (C = 0), 1635 cm"1 (C = C). LRMS: M+ 364; 346,337. y 1 1 1 1 1 I 1 1 1 i i 1 I 1 1 I 1 1 1 L _ y ii 1 1 1 i 1 1 u 1 1 1 L _ l 1 1 1 1 1 1 1 1 1 I 1 1 1 1 l 1 1 1 1 1 1 l l 1 1^1 i t i t i i i l l . 1 i i i 1 i . . i 1 i I _ i : I 1 1 i I . i I 1 | 1 1 1 1 1 ' • 1  1 1 1 1  1 1 1 1 1 1 1 1 1 I I i _ i [ i i 1 i i i t i A . -| .— — 1 • '— j 1 1 1 r>J| 1 . 1 1—| 1 1 1 ' 1 1 1 1 1 1 1 1 1 1 1 1 1 ' ' 1 1 1 1 1 1 1—T 1 1 1— Fig. 28 - 100 MHz. N.M.R. Spectrum of (XLI) and (XLII). Fig. 29 - I.R. Spectrum of (XLL) and (XLII). - 104 -The Epoxy g-lactones (XLIII) and (XLIV) The $-lactones (XLI) and (XLII) (198 mg, 0.5 mmole) were dried via azeotropic d i s t i l l a t i o n with ethyl acetate in vacuo at 5°C. A l l the following operations were carried out at 5°C (cold room). These compounds were dissolved in dry CH2C12 (2 ml) and metachloroperbenzoic acid (128 mg, 0.75 mmoles) was added along with 10 mg sodium bicarbonate. The solution was stirred for 48 hours, poured into water and extracted with CH 2C1 2. The organic layer was successively washed with saturated sodium b i s u l f i t e , saturated sodium bicarbonate, and brine, dried, and evaporated in vacuo to yi e l d the epoxy lactones. (XLIII) and (XLIV), (151 mg, 80%). N.M.R. (Fig. 30): 65.70 (1H, sin g l e t , C 2-H), 63.70 (3H, s i n g l e t , C00CH3), 61.22 (3H, si n g l e t , CH3-C-C), 61.27 (3H, singlet, CH3-C-C), 60.90 (fiH, multiplet, (CH 3) 2-C-), 61.02 (3H, doublet (J = 3 Hz.), a l l y l i c methyl), 61.10 (3H, doublet (J = 3 Hz), a l l y l i c methyl). I.R. (Fig. 31): 1805 cm"1 (6 lactone), 1712 cm"1 (C00CH3), 1640 cm"1 (C = C), 1185 cm"1 (C-C?). LRMS: (M-C0 2-CH 3) + 320. The g-Hydroxycarboxylic acid (XLV) The dianion of (XXVII) (550 mg, 3 mmoles) was formed in the usual way (See Experimental, compounds (XXXIX) and (XL)) and alkylated with hexadeuteroacetone (0.25 ml). The reaction mixture was worked up in the usual way (see above) to yi e l d 500 mg (67%) of (XLV). N.M.R.: 65.20 (1H, t r i p l e t (J = 6Hz), C = C), 61.75 (3H, s i n g l e t , CH3C = C), 61.70 (3H, singlet, CH3-C = C), 60.90 (6H, multiplet, (CH 3) 2-C), 66.05 (1H, broad singlet (D 20 exchangeable) OH), I.R.: 1710 cm"! (C = 0), 1650 cm-1 F1g. 31 - I.R. Spectrum of (XLIII) and (XLIV). - 107 -(C =C), 3500 cm - 1 (OH). HRMS: M+ 248.2255, (calculated for C 1 4H 2 0D 50 3:• 248.2100). The 3-Lactone (XLVI) The $-hydroxy carboxylic acid (XLV) (500 mg., 2 mmoles), was lactonized (as described above under compounds (XLI) and (XLII)) and worked up (as above) to yi e l d the 3-lactone (XLVI) (411 mg, 89%). N.M.R.: 65.10 (1H, sing l e t , C = CH), 62.40 (1H, multiplet, CH-COO), 61.62 (3H, s i n g l e t , CH3-C = C), 61.70 (3H, s i n g l e t , CH3-C = C), 60.90 (6H, multiplet, (CH 3) 2-C). I.R.: 1805 cm"1 (C = 0), 1650 cm"1 (C = C). HRMS: M+ 230.2154, (calculated for Cll+H18D6.02: 230.2950). 6-Ethoxy-3-isopropyl-6-roethy1heptanoic acid (XLVIII) The olefi n acid (XXVIII) or (XXVII), (9000 mg., 4.9 m-noles), was dissolved in dry ethanol (20 ml) under nitrogen. Mercuric acetate (1700 mg, 5.3 mmoles) was added, and the mixture was stir r e d for 4 hours. Excess sodium borohydride in 3_M sodium hydroxide was added and the mixture was stir r e d overnight. The solution was aci d i f i e d to pH 2.0 with 2M_ HCL and extracted with ether. The combined organic layers were washed with brine, dried and evaporated to give a residue which was d i s t i l l e d (140°C at 3 torr) to give (XLVIII) (958 mg, 85%). N.M.R. (Fig 34): 63.38 (2H, quartet (J = 8Hz), -CH2-0-C), 62.25 (2H, multiplet, CH2-C00), 61.16 (6H, s i n g l e t , (CH 3) 2 C-0), 60.87 (6H, two doublets (J =7Hz), (CH 3) 2-C). I.R.: (Fig 35) 1710 cm"1 (C = 0). LRMS: M+ 230, 215. Analysis: calculated for C 1 3H 2 60 3: C, 62.57; H, 11.38; found: C, 62.36; H, 11.51. • F1g. 34 - 100 MHz N.M.R. Spectrum of (XLVIII). Fig. 35 - I.R. Spectrum of (XLVIII). - 110 -6-Nethoxy-3-isopropy1-6-methy1heptanoic acid (IL) The o l e f i n acid (XXVIII) (or XXVII) (641 mg., 3.5 mmoles), was dissolved in dry methanol (25 ml) under nitrogen. Mercuric acetate was added (1.2 g, 3.75 mmoles) and the mixture was sti r r e d four hours. Excess sodium borohydride in 3M_ NaOH was added and the product was sti r r e d overnight. The mixture was acidfied to pH 2.0 with 3_M HC1, and extracted with ether. The combined ethereal layers were washed with brine, dried and evaporated in vacuo to give a residue which d i s t i l l e d (135°C at 3 torr) to give (IL) (700 mg, 92%) as an o i l . N.M.R. (Fig 32): 63.18 (3H, s i n g l e t , -0-CH3), 62.27 (2H, multiplet, CH2-C000), 61.14 (6H, s i n g l e t , (CH 3) 2-C). I.R. (Fig. 33): 1710 cm"1 (C = 0). LRMS: M+ 216, 201. Analysis: calculated for C 1 2H 2 40 3: C, 66.57; H, 11.18; found: C, 66.77;H, 11.38. The Ethoxy 3-hydroxycarboxylic acids (L) and (LI) The ethoxy acid (XLVIII) (560 mg, 2.0 mmoles) was subjected to the usual dianion forming conditions (See Experimental (XXXVIII) and (XXXIX)) and alkylated with B-thujaketonic acid methyl ester (XXXVII) (400 mg, 2 mmoles) in the usual way. After the regular work-up was isolated 642 mg of (L) and (LI) (75%), N.M.R. (Fig. 36): 65.71 (1H, singlet, C 2-H), 63.71 (3H, sing l e t , C00CH3), 63.40 (2H, multiplet, CH2-0-C). I.R. (Fig. 37): 1710 cm"1 (C = 0), 1640 cm"1 (C = C), 1180 cm"1, 3500 cm"1 (OH). LRMS: (M-H 20) + 410, 395. The Ethoxy g-1actones (LII) and (LIII) The crude B-hydroxy acids (LI) and (L) (110 mg, 0.25 mmoles) were lactonized as described above (See Experimental (XLI) and (XLII) to Fig. 33 - I.R. Spectrum of (IL). F1g. 36 - 100 MHz. N.M.R. Spectrum of (L) and (LI). F1g. 37 - I.R. Spectrum of (L) and (LI). ! • • I I • ' • i I I ' I I I I I I I I I I ,1 I I I I I I I I I I I I I I l_J L_J—I I I—I—I—I—I—U_l—I—I—1—I—L_l—I—l_l—L_J—I—I J I I I L _ l I I I I I • I I I I ' l l I I I I I I 1 I I I 1 I I I I I I I I I I I l_l I I I I I 1 I I 1 1 1 1—I 1 1 1 1 1 1 1 1 1 ' 1 1 — 1 — i — r — i — | — i — i — i — i — | — i — i — i — i — | — i — i — i — i — | — i — i — i — i — | — r — i — i — i — | — i — I — 1 — i — | — i — i — i — I — | — r — 1 — i — i — | — i — i — - i — | — i — i — i — i — | — i — i — i — ' — | — ' — ' 1 ' r -' ' — 1 — r - | — i — 1 — ' — • | • ' 9 Fig. 38 - 100 MHz. N.M.R. Spectrum of (LII) and ( L I U ) . MtCtOMS 3"00 J500 1000 1800 l « 0 0 MOO ITOO IOOO W A V I N O M M t l C M - ' ) W A V T K I U M B r * t " M 'I Flq. 39 - I.R. Spectrum of (LII) and ( L I I I ) . - 117 -give, after the usual workup, the lactones (85 mg., 80%), (LII) and (LI 11) as an o i l . N.M.R. (Fig. 38): 65.74 (IH, si n g l e t , C 2-H), 63.73 (3H, s i n g l e t , C00CH3), 63.39 (2H, multiplet, CH2-0-C) I.R. (Fig. 39): 1810 cm - 1 (C = 0), 1710 cm"1 (C00CH3), 1640 cm - 1 (C = C). LRMS: M+ 410, (M-CH 3) + 395. The Hormone Analogues (LIV) and (LV) The B-hydroxy acids (L) and (LI) were olefin-lactonized (112 mg. 0.25 mmole) via dissolving in 3 mL dry pyridine, adding dry benzenesulfonyl chloride (0.07 ml, 0.5 mmoles) and heating to 45-55°C for 6 hours. The mixture was poured into saturated bicarbonate solution and st i r r e d overnight. The solution was extracted with ether; the organic layer was washed with brine, dried, and evaporated in vacuo to give the JHA's (LV) and (LIV) as an o i l (88 mg, 93%). This material was purified by TLC to give 51 mg of a homogenous o i l . N.M.R. (Fig. 40) (400 MHz): 65.66 (IH, si n g l e t , C 2-H), 64.92 (IH, multiplet, C = CH), 63.69 (3H, si n g l e t , C00CH3), 63.34 (2H, quartet (J =6Hz), CH2-C-0-C), 61.80 (IH, si n g l e t , (E)-isomer vinyl methyl), 60.85 (6H, multiplet, a l l y l i c isopropyl methyl groups). I.R. (Fig 41): 1710 cm"1 (C = 0), 1640 cm"1 (C = C), 1165 cm"1, 910 cm"1. HRMS: M+ 366.3109, (calculated for C23H42O3: 366.5830). Analysis: calculated for C23H42O3; C, 75.36; H, 11.55; found: C, 75.36; H, 11.51. 1000 500 F1g. 41 - I.R. Spectrum of (LIV) and (LV). - 120 -REFERENCES 1. G. W. Ware, The Pesticide Rook, p. 27, W. H. Freeman and Comp., San Francisco, 1978. 2. V.B. Wigglesworth, Quart J . Microscop. S c i . , 77, 1919 (1934). 3. C. M. Williams, Nature, 178, 212 (1956). 4. H. Roller, K. H. Dahm C. C. Sweeley, and B. M. Trost, Angew. Chemie  Intern. Ed. Engl., 6, 179 (1969). 5. See, for example, The Juvenile Hormones, edited by L. I. Gi l b e r t , Plenum Press Inc., New York, 1975. 6. C. E. 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