Synthesis of a s-methoprene derivative "s-methobutene" with enhanced bioactivity against dipteran insects

Abstract

Mosquitoes and mosquito-borne illness remain to significantly impact public health and human well-being nowadays. Mitigation by pesticides against larvae and adults has played a critical role in mosquito management. Larviciding is more cost effective than adulticiding in mosquito control operations. However, mosquito larvicides are historically low for numerous reasons. Currently larvicides with microbial and insect growth regulators (IGRs) account for most of the available products. Screening of new active ingredients (Als) or improvement of existing Als is desired to augment the arsenals for mosquito control. S-methoprene has been one of the main targets for research and development. The efficacy and safety of S-methoprene has been well documented since the late 1960s, and numerous products have been commercialized to combat pests of economic importance.

Description

TITLE Synthesis of a S-methoprene Derivative "S-methobutene" with Enhanced Bioactivity Against Dipteran Insects. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. Provisional Patent Application No. 63 / 366,003 filed on June 7, 2022 and titled “Synthesis of a S-methoprene derivative "S-methobutene" with enhanced bioactivity against Dipteran insects.” The entire disclosure of the prior application is considered to be part of the disclosure of the accompanying application and is hereby incorporated by reference. BACKGROUND OF THE INVENTION 1. Field of the Invention

[0002] This invention relates to the use of biopesticides for the control of dipteran insects, in particular, the synthesis and bioactivity of a new S-methoprene derivative called S-methobutene for the control of dipteran insects. S-Methobutene shows improved juvenile hormone-like activity over S-methoprene as well as improved performance parameters against ultraviolet irradiation, hydrolysis, and degradation by microbial activities over S-methoprene. 2. Description of the Related Art

[0003] Mosquitoes and mosquito-borne diseases remain one of the significant public health burdens which adversely impact social and economic development and productivity, particularlyin tropical and subtropical regions worldwide (Sutherst 2004, Dahmana and Mediannikov 2020, Chala et al.2021). Generally, integrated mosquito control is one of the most effective measures in mitigation of mosquito-related public health and well-being concerns. Strategic applications of environmentally compatible pesticides against aquatic immature stages and air-borne adult stage have been playing critical roles upon the evolution of public health pesticides from conventional to biorational options (Connelly and Borchert 20202). In mosquito control, larviciding aiming larvae that dwell in aquatic environments is more achieving and cost-effective than adulticiding targeting adults that disperse easily and distribute broadly in the cryptic environmental niches (Antonia-Nkondjio et al.2018, Derua et al.2019). Plus, larviciding appears to face less pesticide resistance challenges due to limited exposures to larvicides with diverse modes of action, as compared with adulticiding where adult mosquitoes are subjected primarily to pyrethroids that are applied to control not only mosquitoes (Su 2022), but also the pests of urban and household importance at much larger scales. However, availability of environmentally friendly larvicides is historically low for numerous reasons, for instance strict regulations, high research and development cost, narrow market arena, and risk of resistance development. Currently, microbials, insect growth regulators (IGRs), botanicals and a few other miscellaneous active ingredients are the only resources for larvicide development.

[0004] Among the IGRs ranging from juvenile hormone analogs (JHAs) (Henrick 2007, Webb et al.2012, Su 2018), chitin synthesis inhibitors (CSIs) (Bellini et al.2009) and molting hormone agonists (MHAs) (Beckage et al.2004), tremendous research and development effort has been made to methoprene, one of the JHAs that has achieved the significant success ever since it was synthesized in late 1960s (Lowe et al.1975). The early chemical synthesis led to thebirth of mixture of inactive R- and active S- chiral forms, ensued by the improved technology which made the S-methoprene with high purity available (Henrick 2007, Su 2018). Methoprene was recognized as a soft pesticide that regulates the growth process of the target species instead of killing them directly, thus a word “biorational” was coined for this compound and its related ones such as hydroprene and kinoprene (Djerassi et al.1974, Menn and Henrick 1981). Methoprene was registered as a chemical pesticide by the United States Environmental Protection Agency (US EPA) in 1975. This registration was revised to biopesticide in 1982 since methoprene possesses a similar molecular structure and an identical function to the natural juvenile hormone III (JH III) in mosquitoes (US EPA 2023a, b). To date, the efficacy and safety against a broad spectrum of pest species in public health, urban, livestock and stored product protection have been well documented. The low effective concentrations and doses, low risk of resistance development, and lack of cross resistance to the pesticides with different mode of action, even the one with same mode of action such as pyriproxyfen, have made methoprene an irreplaceable option in sustainable vector and pest management (Su et al.2018, 2019, 2021, Su 2022). However, the methoprene molecule has its weakness such as vulnerability to ultraviolet irradiation, hydrolysis, and degradation by microbial activities (Silberhorn 2005), which has created the challenges in formulation development, particularly in the cases where extended efficacy is desired. Sunlight and micro-organisms break down methoprene rapidly in soil, water, and on plants. In soil, about half of the original amount is gone within 10-14 days. In water, it takes 1-28 days for methoprene residue to break down by half, depending on the availability of sunlight. When methoprene is formulated in a briquette, pellet, or granule, the release is slowed. Methoprene’s full breakdown when released as a briquette has been reported up to 18 months.

[0005] As a synthetic biopesticide, S-methoprene has been utilized to combat arthropods of agricultural, urban, medical, and veterinary importance due to its high efficacy, low risk of resistance development and friendly environmental profile. Numerous products based on S-methoprene have been developed and commercialized since the 1970s. However, S-methoprene continues to be susceptible to hydrolysis, photolysis and microbial activity which leads to the short longevity under field conditions.

[0006] Therefore, there exists a need for a derivative of methoprene that (i) is capable of juvenile hormone-like activity, (ii) has the same or improved efficacy, stability and safety against a broad spectrum of pest species in public health, urban, livestock, and stored product protection, (iii) is effective at low concentrations and doses, (iv) has low risk of resistance development, and (v) lacks cross resistance to the pesticides with different mode of action. BRIEF SUMMARY OF THE INVENTION

[0007] Upon the advancement of chemical synthesis technology, the stability and bioactivity improvement of S-methoprene through non-insect growth regulation molecular domain modification was achieved: a derivative of S-methoprene, named S-methobutene in the present disclosure, was successfully synthesized. In addition to S-methoprene, S-methobutene also has significant chemical similarities to sibling compounds S-hydroprene as well as S-kinoprene. S-methoprene, S-hydroprene, and S-kinoprene were registered as biopesticide in 1975, 1988 and 1997, respectively by the US EPA. Between S-methoprene and S-methobutene, the only difference is located at -O-C(CH3)3structure found at the end of the S-methobutene molecular structure. In S-methoprene, the structure found at the end of its molecular structure is-O-CH(CH3)2. This is shown in FIG.1.

[0008] The S-methobutene derivative demonstrates significant improvement over S-methoprene both in chemical stability and bioactivity against dipteran insects such as the yellow fever mosquito Aedes aegypti, the southern California malaria mosquito Anopheles hermsi and the southern house mosquito Culex quinquefasciatus (Diptera: Culicidae).

[0009] Neither this summary nor the following detailed description defines or limits the invention. The invention is defined by the claims. BRIEF DESCRIPTION OF DRAWINGS

[0010] The present invention will become more fully understood from the detailed description and accompanying drawings and tables. Other systems, methods, features, and advantages of the invention will be or will become apparent to one with skill in the art upon examination of the following figures and detailed description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the invention, and be protected by the accompanying claims. In the drawings, like reference numerals may designate like parts throughout the different views, wherein:

[0011] FIG.1 contains a table showing the chemical similarities of S-methobutene and three closely related juvenile hormone analogs: S-methoprene, S-hydroprene, and S-kinoprene.

[0012] FIG.2 shows the purity of trans-S-methobutene (98.841%) and impurity cis-S-methobutene (1.159%) by Gas chromatography (GC).

[0013] FIG.3 shows the hydrogen profile in S-methobutene by Nuclear Magnetic Resonance.

[0014] FIG.4 shows the carbon profile in S-methobutene by Nuclear Magnetic Resonance.

[0015] FIG.5 shows the chemical identification of S-methobutene by mass spectrometry (MS): chemical formula C20H36O3with exact mass of 324.2664.

[0016] FIG.6 shows a table containing the water quality profile of tap water by Riverside Public Utilities, Riverside, California, USA (2022).

[0017] FIG.7 shows the average weekly minimum and maximum room temperatures during the aging testing.

[0018] FIG.8 shows the average weekly minimum and maximum relative humidity during the aging testing.

[0019] FIG.9 shows a table displaying the comparative bioactivity of technical S-methoprene and S-methobutene in laboratory bioassays against Aedes aegypti, Anopheles hermsi, and Culex quinquefasciatus (Diptera: Culicidae)

[0020] FIG.10 shows the proportion of stage-specific mortality in Aedes aegypti, Anopheles hermsi, and Culex quinquefasciatus in response to treatments by S-methoprene and S-methobutene.

[0021] FIG.11 shows a set of charts comparing the activity of technical S-methoprene and S-methobutene when the aqueous dilutions were aged for up to 120 days at 2.5 ppb against the southern house mosquito Culex quinquefasciatus (Diptera: Culicidae).

[0022] FIG.12 shows a set of charts comparing the activity of technical S-methoprene and S-methobutene when the aqueous dilutions were aged for up to 120 days at 10 ppb against the southern house mosquito Culex quinquefasciatus (Diptera: Culicidae).

[0023] FIG.13 shows a set of charts comparing the activity of technical S-methoprene and S-methobutene when the aqueous dilutions were aged for up to 120 days at 100 ppb against the southern house mosquito Culex quinquefasciatus (Diptera: Culicidae).

[0024] FIG.14 shows the molecular structure of S-methobutene. DEFINITIONS

[0025] The following acronyms have already been used or will be used in this disclosure: AI - Active Ingredient. CI - Confidence Interval. CSI - Chitin Synthesis Inhibitor. HPGC - High Performance Gas Chromatography. HPLC - High-Performance Liquid Chromatography. HRMS - High-Resolution Mass Spectrometry. IE - Inhibition Of Emergence. IGR - Insect Growth Regulator. JH III - Juvenile Hormone III. JHA - Juvenile Hormone Analog. MHA - Molting Hormone Agonist. MS - Mass Spectrometry. NMR - Nuclear Magnetic Resonance. PIP - Plant Incorporated Protectant. SE% - Standard Errors.TLC - Thin Layer Chromatography US EPA - United States Environmental Protection Agency. UTC - Untreated Control. DETAILED DESCRIPTION OF THE INVENTION

[0026] The compounds of methobutene are useful for the control of arthropods, particularly insects. The compounds are applied using suitable carrier substances, either liquid or solid carriers, such as water, emulsifiers, diatomaceous earth, gypsum, activated carbon , silica, talc, natural and synthetic resins, and the like. Generally, compositions for application will contain up to about 75% of the active compound and more usually less than 25%, sufficient composition should be applied to provide at parts per billion scale of the active compound per insect. Typical insects controlled by the present invention are Diptera, such as mosquitos, midges, houseflies; Hemiptera, such as Pyrrhocoridae and Miridae; Lepidoptera, such as Pyralidae, Noctuidae and Gelechiidae; and Coleoptera, such as Tenebrionidae, Chrysomelidae and Dermestidaen just name a few, for example, Pyrrhocoris apterus, Lygus hesperus, Aphids, Tenebrio molitor, Triboleum confusm, Diabrotica undecimpunctata, Dermestes maculatus, Alfalfa weevil, potato tuber moth, Aedes aegypti and Musca domestica. The effectiveness of methobutene to control insects is attributed to the property to mimic the activity of juvenile hormone as demonstrated herein. While the methods of applying and carriers for conventional insecticides are usually adaptable to the practical use of methobutene, the mechanism of action of methobutene is unlike that of conventional insecticides. Whereas conventional insecticides are dependent upon direct knock-down effect, toxicity effect or paralyzing effect, methobutene achieves control by reasonof its ability to inhibit metamorphosis, inhibit reproduction due to abnormal development, break diapause at an unfavorable time, or act as a direct insecticide, particularly at the embryo stage and larvae stage at the higher doses. Treatment of insects in accordance with the present invention can be achieved via ingestion of the active compound in the normal food of the insect and by topical application (cuticle absorption), that is — by contact of the epidermis of the insect as by spraying the insect and habitat of the insect or exposure to vapors of the active compound which penetrate into the insect.

[0027] This disclosure will start with a discussion of the synthesis of S-methobutene and its chemical properties. The disclosure will then discuss testing methods and the results obtained when comparing the bioactivity of S-methoprene and S-methobutene against the following mosquitoes: the yellow fever mosquito Aedes aegypti, the southern California malaria mosquito Anopheles hermsi and the southern house mosquito Culex quinquefasciatus. Finally, the disclosure will conclude with a discussion of the test results. 1. Synthesis of S-Methobutene

[0028] In a 100-mL three-necked flasks, successively adding (2E, 4E, 7S) -11-methoxy - 3, 7, 11 – trimethyl l - 2, 4 -dodecarenoic acid (2.69g, 10 mmol) and anhydrous dichloromethane (20 mL) under nitrogen protection and mixing evenly. After mixing evenly, acetyl chloride (7.84 g, 7.13 ml, 100 mmol) was slowly added dropwise at 0℃ and the resultant mixture was stirred for 0.5 hours and then moved to room temperature to react for 2 hours. Then tert-butanol (1.1 g, 1.37 mL, 15 mmol) was added to the mixture and the reaction was allowed to continue for another 2 hours at room temperature [using TLC (thin layer chromatography) to monitor thereaction]. After the mixture was fully converted, saturated sodium bicarbonate solution (20 mL) was slowly added to quench the reaction, followed by extraction with ether for three times, each time for 10 mL. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 40 / 1) to obtain the target products as a colorless oily liquid (2.96 g, 91% yield). With additional processing, S-Methobutene may also be converted into a dry form such as powder or granules. The purity of trans-S-methobutene reached 98.841% and impurity cis-S-methobutene was as little as 1.159% by Gas chromatography (GC) (Figure 2). The hydrogen and carbon profile by NMR and chemical identity by mass spectrometry (MS) for S-methobutene led to a formula of C20H36O3with molecular weight 324.2664 (FIGs 3 to 5).

[0029] FIG.14 shows the molecular structure of S-Methobutene. 2. Testing Methods and The Results Obtained

[0030] As for the test materials, technical S-methoprene (purity 96.69%, Lot# SC-L20201123) and S-methobutene (purity 99.00%, Lot# 20210108) were synthesized in the chemistry laboratory at the East China Normal University (Shanghai, China) and provided by Synergetica International, Inc. (Marlboro, NJ, USA). For both test materials, the hydrogen and carbon profiles, chemical identities and chemical formulas were determined by NMR and HRMS, and purity was measured by HPGC. The comparative chemical profiles of S-methoprene and S-methobutene are shown in FIG.1. As for the mosquitoes, the yellow fever mosquito Aedes aegypti, was supplied by Benzon Research (Carlisle, PA, USA) from its long-term susceptible colony. The southern California malaria mosquito Anopheles hermsi Barr& Guptavanij originated from field-collected larvae and host-seeking female adults from southern California. The southern house mosquito Culex quinquefasciatus was from a long-term in-house susceptible colony. The late 4th instar larvae with good biofitness, that were about to pupate, were used in laboratory bioassays (Noguchi and Ohtaki 1974).

[0031] Concurrent laboratory bioassays were conducted to determine the comparative inhibition of emergence (% IE) by S-methoprene and S-methobutene against three test species. To prepare the bioassay treatments, previously published details (Su et al.2021) were followed with minor modifications. The stock solution of S-methoprene was made by adding 10.3 μL of sample to 9,989.7 μL of HPLC grade acetone leading 0.1%, 10-fold serial dilutions were made by adding 1 mL of the upper concentration to 9 mL of the same diluent to generate the lower concentrations to 0.00000001% (0.1 ppb). The dilutions of S-methobutene were prepared in the same way from 10.1 μL technical materials due to its higher AI level. Twenty-five late 4thinstar larvae were transferred to 100 mL tap water as provided by the local supplier (Riverside Public Utilities, CA, USA) (see FIG.6) that was held in 6-OZ Styrofoam (polystyrene) cups. The straight tap water was used purposely to test the tolerance of the S-methoprene and S-methobutene to various chemical factors that were associated with the tap water. The appropriate amount of dilutions from each product was added to each treatment cup by controlling the added amount within 100-1000 μL, where the added amount was negligible for its impact on the change of the final volume and intended methoprene concentration. The concentration range was 0.00025 to 5 ppb, a broad span to cover the variabilities in bioactivity between pesticide samples and susceptibility among test species. A small piece (approximately 100 mg) of rabbit pellets was added to each bioassay cup to support them until pupation, withoutcausing over-eutrophication when larval feeding slowed down upon pupation. The bioassay cups were covered by a clear plastic dome with a screened top, where emerged adults were confined, and the cups were held in an insectary measuring 3.0 × 2.0 × 2.5 m. The conditions were maintained at temperature 27.8-29.4°C, photoperiod L: D = 16 h: 8 h (one T-8 Clear LED light tube -18W, 2200 lumens, 5000 K Daylight) (Sun Lake Lighting, Pasadena, CA). The bioassay cups were placed on a rack approximately 1.50 m from the light source that was installed on the ceiling. Results were read when all treated individuals emerged as adults or died prior to emergence. Only free exuviae were counted and considered as successful emergence. Mortality was read as dead larvae, dead pupae, or incompletely emerged adults with wings and / or legs attached to the exuviae.

[0032] To compare the bioactivity of S-methoprene with S-methobutene upon aging, the aqueous suspensions of both were made according to the aging schedule of 0 (freshly prepared), 7, 14, 30, 60, 90 and 120 days, and assayed on the same day (see below). The stock solution was made by adding 10.3 μL of technical S-methoprene to 989.7 μL of pure acetone leading to 1%, 10-fold serial dilutions were then made by adding 100 μL of the upper concentration to 900 μL acetone to reach the lower concentration to 0.0001% (1000 ppb). The amount of 10.1 μL of S-methobutene was used to make the same dilutions due to its higher AI level. The appropriate aliquot of dilutions in acetone was added to 500 mL tap water with the same origin as previously described to reach the final concentration of 2.5, 10 and 100 ppb, where added amount ranged 50 – 125 μL with negligible increase of total volume and impact on final concentration. Each aqueous preparation was held in a 500-mL clear glass bottle, then secured tightly by a black phenolic screw cap with polypropylene polycone liner and stored at room conditions. Three (3)replicates were made for each concentration, and for each age interval of 0, 7, 14, 30, 60, 90 and 120 days. UTC, i.e. tap water, only in the same amount was prepared at each aging interval with three (3) replicates. The storage room was 3.0 x 3.0 x 2.75 m. The photoperiod was L: D = 16h: 8h by one T-8 Clear LED light tube (18W, 2200 lumens, 5000 K Daylight) (Same supplier as previously described). The bottles that contained diluted aqueous samples were placed on the floor and approximately 2.75 m from the light source that was installed on the ceiling. The weekly average minimum and maximum room temperatures and relative humidity (RH) were monitored by a Thermo-Hygrometer (Fisher Scientific, Waltham, MA): average minimum and maximum room temperatures were 22.0 and 24.9°C, and the average minimum and maximum RH ranged 33.8 to 40.3% during the 120-day aging process are shown in FIGs.7 and 8 respectively.

[0033] All bioassays on the aged samples (0-120 days) were conducted concurrently along with untreated controls against the southern house mosquito Culex quinquefasciatus. The remaining aged samples after setting the bioassays were stored at 4°C as backups in case any assay needs to be repeated. In bioassay, 25 late 4th instar larvae were transferred to 100 mL of previously aged aqueous preparation that was held in 6-OZ Styrofoam cups. Three (3) replicates were made for each concentration and untreated control. A small piece (approximately 100 mg) of rabbit pellets was added to each bioassay cup to support them until pupation. The bioassay cups were covered by a clear plastic dome with a screened top and were held in the same conditions as previously described. The progress of the assay was observed, and emerged adults were confined by the plastic dome. Results were read when all treated individuals emerged as adults or died prior to emergence. Only free exuviae were counted and considered as successfulemergence.

[0034] Concentration–response data with approximately 5-95% IE in laboratory bioassays on the three species were corrected by Abbott formula (Abbott 1925) in case the total mortality in UTC was greater than 5% but less than 20%, then analyzed using POLO Plus (Robertson et al. 2006) to calculate the concentrations causing 10%, 50%, and 90% IE (respectively IE10, IE50, and IE90) and their 95% CIs. In cases where the total mortality in UTC reached 20% or greater, the test was discarded and repeated. Significant differences in IE levels were indicated by separate 95% CIs (Su et al.2021). The proportion of stage-specific mortality (%) was calculated by 100 × (total number of dead at a given stage of larvae, pupae, or incomplete emergence / total number of dead individuals) across all test concentrations.

[0035] In aged aqueous preparations, the IE% was calculated by 100 × (individuals that failed to emerge to adults / total late instar larvae assayed), where data correction was conducted if mortality in UTC was greater than 5% but less than 20% by Abbott formula (Abbott 1925). In cases where the total mortality in UTC reached 20% or greater, the test was discarded and repeated. The SE% were calculated after data correction: 100 × sqrt of p × (1-p) / N, where p was IE% in decimal format and N was total individuals observed. At each concentration of 2.5, 10 and 100 ppb, the trend of activity loss as indicated by decreased IE% during aging from 0 to 120 days were drawn by 2nd order polynomial curve fitting. The significance in comparative activity between S-methoprene and S-methobutene at each aging interval was analyzed by Chi-square test at χ2= 3.84, p = 0.05 or χ2= 6.63, p = 0.01 when df = 1 (Stangroom 2022). The correlation and regressions between IE% and length of aging were established. The strength of correlations between IE% and length of aging was indicated by the significant levels of correlationcoefficient r and p values (one-tail, df = n-2) between two test materials. The pace of activity losses upon aging was indicated by the separated 95% CIs of the slope values (Mann 2017). The greater absolute value of the slope in the regression equation, i.e., the steeper regression line, implied faster activity loss in contrast to the smaller slope that was associated with a flatter line.

[0036] The dose-response correlations and regressions were established for both S-methoprene and S-methobutene against three test species where correlation coefficient (r2) ranged 0.86 to 0.99 within approximately 5-95% IE. The IE10, IE50and IE90and their 95% CIs were generated mostly within the actual test concentration range. Against all test species, S-methobutene was significantly more active than S-methoprene as indicated by separate 95% CI ranges (p < 0.05). The same significance indication was also held true for different species, where Aedes aegypti and Anopheles hermsi were significantly more susceptible than Culex quinquefasciatus to both test materials (see FIG.9). S-methoprene and S-methobutene exhibited the same mode of action, where the mortality presented as dead larvae occasionally, dead pupae predominantly, and incompletely emerged adults sometimes with wings and / or legs attached to the exuviae (see FIG.10). While pupal mortality was the majority, more larval mortality tended to occur at higher concentrations while incomplete adult emergence was generally more prevalent at the lower concentrations.

[0037] FIG.11 shows the comparative activity of technical S-methoprene and S-methobutene when the aqueous dilutions were aged for up to 120 days at 2.5 ppb against the southern house mosquito Culex quinquefasciatus (Diptera: Culicidae). The upper chart shows that the significant difference in IE% between S-methoprene and S-methobutene was indicated by Chi-square test at p < 0.05 (χ2= 4.34-4.76) or p < 0.01 (χ2= 8.31-12.93) on each aginginterval with exception of day 120. The middle and bottom charts show the correlation and regression between IE% and length of aging were established. The correlations were highly significant and were stronger in S-methobutene (r2= 0.936, p < 0.001) than in S-methoprene (r2= 0.787, p < 0.005). The insignificant difference in slope values of the regression equation was indicated by overlapping 95% CIs (p > 0.05).

[0038] FIG.12 shows the comparative activity of technical S-methoprene and S-methobutene when the aqueous dilutions were aged for up to 120 days at 10 ppb against the southern house mosquito Culex quinquefasciatus (Diptera: Culicidae). The upper chart shows that the significant difference in IE% between S-methoprene and S-methobutene was indicated by Chi-square test at p < 0.05 (χ2= 3.85-5.51) or p < 0.01 (χ2= 11.31-17.64) on each aging interval. The middle and bottom charts show the correlation and regression between IE% and length of aging were established. The correlations were highly significant and were stronger in S-methobutene (r2= 0.957, p < 0.0005) than in S-methoprene (r2= 0.818, p < 0.005). The insignificant difference in slope values of the regression equation was indicated by overlapping 95% CIs (p > 0.05).

[0039] FIG.13 shows the comparative activity of technical S-methoprene and S-methobutene when the aqueous dilutions were aged for up to 120 days at 100 ppb against the southern house mosquito Culex quinquefasciatus (Diptera: Culicidae). The upper chart shows that the significant difference in IE% between S-methoprene and S-methobutene was indicated by Chi-square test at p < 0.01 (χ2= 10.17-30.88) on each aging interval with exceptions on day 0 and 7. The middle and bottom charts show the correlation and regression between IE% and length of aging were established. The correlations were highly significant and were stronger inS-methobutene (r2= 0.932, p < 0.0005) than in S-methoprene (r2= 0.831, p < 0.0025). The significant difference in slope values of the regression equation was indicated by separate 95% CIs (p < 0.05).

[0040] At 2.5 ppb, the difference in IE% between S-methoprene and S-methobutene was significant on days 0, 60 and 90 (χ2= 4.34-4.76, p < 0.05), but highly significant on days 7, 15 and 30 (χ2= 8.31-12.93, p < 0.01). This trend stayed on day 120 but did not reach a significant level (see the upper chart in FIG.11). The same but stronger trend prevailed at 10 ppb, where the IE% in S-methobutene was significantly higher on days 0 and 120 (χ2= 3.85-5.51, p < 0.05), but highly significant on days 7 through 90 (χ2= 11.31-17.64, p < 0.01) than those in S-methoprene (see the upper chart in FIG.12). The significant differences in IE% between S-methoprene and S-methobutene were further amplified at 100 ppb where high significance was observed on days 15 through 120 (χ2= 10.17-30.88, p < 0.01). This difference in performance on days 0 and 7 was however masked by the overwhelmingly high activity (100% IE) of both test materials. Overall, the activity decline was more obvious in S-methoprene than in S-methobutene during days 7-30 at 2.5 and 10 ppb, and days 14-30 at 100 ppb (see the upper chart in FIG.13).

[0041] A significant negative correlation and regression was established between the IE% and aging intervals, where the r2ranged 0.787-0.831 (p < 0.0025-0.005) for S-methoprene, and 0.932-0.957 (p < 0.0005) for S-methobutene. The lower r2in S-methoprene was caused by the rapid decline in activity on earlier days (7-30 days). At 2.5 ppb, the activity of S-methoprene and S-methobutene declined in the similar pace as indicated by the overlapped 95% CIs of the slope values: -3.040 to 1.885 in S-methoprene and -0.476 to -0.698 in S-methobutene. This trend remained at 10 ppb, where the 95% CIs of slope values were -2.263 to 1.067 in S-methopreneand -1.991 to -0.741 in S-methobutene. At the highest concentration 100 ppb, however, S-methoprene lost its activity at significantly faster pace than did S-methobutene, the 95% CIs of the slope values were well separated in two test materials: -0.667 to -0.651 for the former and -0.314 to -0.322 for the latter (see the middle and lower charts in FIG.11 through 13). 3. Discussion of Results

[0042] Due to its benign environmental and non-target profile, synthetic S-methoprene is considered as a biochemical and received favorite registration status as a biopesticide along with microbial pesticides and PIP by US EPA (2023a). Furthermore, Code of Federal Regulations of the United States of America recognized S-methoprene as organic pesticide and exemption from requirement of a tolerance (CFR §180.1033) (Code of Federal Regulation 2023). However, as an unsaturated hydrocarbon with two double bonds, methoprene degrades rapidly in sunlight when in water and on inert surfaces. It metabolizes rapidly in soil under both aerobic and anaerobic conditions. The major degradation product is carbon dioxide. Because of its rapid degradation, methoprene does not persist for long periods in soil and is unlikely to contaminate groundwater (Silberhorn 2005). While short persistence in the environment due to its photolysis, hydrolysis and microbial degradation can be desired, it also creates a challenge in formulating residual products for the habitats where persistent control is needed. Considerable effort has been made to preserve the technical S-methoprene in formulation matrix to achieve long residual efficacy, which lead to a few long-lasting formulations on the market for labor and cost saving applications.

[0043] Previously, a racemic mixture, or racemate RS-tert-butyl methoprene was attemptedand its bioactivity was analyzed against limited target species of interest. However, the bioactive S-chirality of tert-butyl methoprene (i.e., S-methobutene in this disclosure) never became available due to limitations of earlier chemical synthesis technologies (Henrick 1982). In current studies, S-tert-butyl methoprene was successfully synthesized in the laboratory by replacing the isopropyl group [-CH(CH3)2] with a tert-butyl group [-C(CH3)3] and was named S-methobutene for the first time. Against three test species, S-methobutene consistently outperformed S-methoprene as indicated by lower IE10, IE50and IE90each with separate 95% CIs between the two test materials. Further studies are needed to elucidate whether S-methobutene does exert higher IE activity intrinsically than S-methoprene, or if the former is more stable hence higher stability than the latter in the environment. Nevertheless, the tert-butyl substituent is chemically bulky and often used in kinetic stabilization. Because a neighboring C-H bond donates some of its electron density into a carbocation’s empty p-orbital and interacts with the carbocation, the tert-butyl carbocation is more stable than the isopropyl carbonation since the tert-butyl carbocation has three alkyl groups / 9 hyperconjugation C-H bonds as compared with two alkyl groups / 6 hyperconjugation C-H bonds in isopropyl carbonation (Doubtnut 2023, Wikipedia 2023). The bioassay was conducted using tap water where test materials were subjected to the existing profile of water quality parameters including the physical, chemical, and microbial measurements (Riverside Public Utilities 2022, see also FIG.6). Among these measurements were low number of coliforms, high pH (8.2), chlorine (0.61 ppm), and other various organic and inorganic materials, which could have challenged the stability of S-methoprene and S-methobutene during the week-long bioassay process. Obviously, the S-methobutene withstood the challenges associated with the tap water and resulted in significantly lower IE10, IE50and IE90than did S-methoprene.

[0044] The observed stage-specific inhibition of emergence in both test materials represented the typical mortality pattern in JHAs that have been published previously (Su et al.2108, 2019, 2021). The derivative S-methobutene induced a similar mortality pattern to that in S-methoprene. Most mortality occurred at pupal stage when the pupating larvae (old 4thinstars) completed larval growth and proceeded to pupation when internal JH III was at the lowest level and showed the highest susceptibility to external JHAs. At the lower concentrations, some larvae successfully pupated, and adult emergence was attempted but aborted with wings or legs attached to the pupal exuviae. On the other hand, some larvae died prior to pupation due the exposure to higher concentrations of test materials as external JHAs, which overwhelmed the declining internal JH III.

[0045] During the 120-day aging process, the outperformance by S-methobutene over S-methoprene at 2.5, 10 and 100 ppb could be attributable at least in part to its ability to withstand the hydrolysis, photolysis, and microbial degradation, unquantified though, during aging process as well as an approximately week-long bioassay process. The differences of activity between two test materials minimized upon aging to 120 days at the lower concentrations of 2.5 and 10 ppb, this situation was expected to occur as well at the highest concentration of 100 ppb upon further aging beyond 120 days. The slower activity decline during early days (days 7-21) resulted in stronger correlations between IE% and length of aging in S-methobutene than in S-methoprene. The difference in pace of activity loss was further indicated by the significantly flatter regression line between IE% and length of aging in S-methobutene than in S-methoprene at 100 ppb, which were also believed to be at least partiallyrelated to the enhanced molecular stability due to the tert-butyl group in S-methobutene as explained previously (Doubtnut 2023, Wikipedia 2023). As compared with the adverse factors related to methoprene degradation in week-long bioassays, most of those factors such as time of aging, exposures to various inorganic and organic materials, and microbial reproduction could have been intensified during 120-day aging. The exception might be chlorine which could have broken down gradually during the aging process. Of course, the fate of degradation of other inorganic and organic materials that originated from the tap water and prevailed in the aqueous suspensions for aging was unknown, nevertheless both S-methoprene and S-methobutene were exposed to the identical conditions, and their activity loss during aging was measured in the same way, therefore the differences in their IE activity during aging was validated.

[0046] The following is an example of a water-dispersible powder formulation in accordance with the present invention: Hi Sil 233 73.5% Igepon-T-77 1.0% Defoamer 0.5% Methobutene 25.0%

[0047] Hi Sil is a trademark of PPG Industries. lgepon T-77 is an anionic wetting agent of GAF Corporation. Defoamer is soap flakes but other defoamers can be used.

[0048] The following is an example of an emulsive formulation in accordance with the present invention: Solvent 14% Atlox 3403F 1%Atlox 3404F 3% Methobutene 82%

[0049] Solvent is xylene although other solvents can be used. Atlox is a trademark of Atlas Chemical Industries, Inc. the emulsive is diluted in water and applied. A deactivator, such as a tertiary amine, can be added to above formulation, usually in the amount of about 1% depending on shelf-life desired.

[0050] Other delivery formulations included ultra-low volume spray concentrate (5-25%), granules (1-4.25%), pellets (1-4.25%), briquettes (8.2%), water soluble pouches (4.25%), etc.

[0051] In summary, the new derivative S-methobutene was significantly more active than S-methoprene and exhibited a similar mode of action against different mosquito species tested to S-methoprene and other JHAs that was published previously. During a 120-day aging process, S-methobutene also consistently outperformed S-methoprene at various test concentrations against Culex quinquefasciatus and lost its activity at a significantly slower pace than did S-methoprene at 100 ppb. The enhanced bioactivity in S-methobutene could be intrinsic, and / or the improved stability due to its bulky tert-butyl group in contrast to the isopropyl group in S-methoprene. As compared with existing S-methoprene products, lower dose, longer efficacy, and more cost-effective applications of S-methobutene products are desired in future mosquito control operations.

[0052] Having now fully described the invention, it will be appreciated by those skilled in the art that the invention can be performed within a wide range of parameters within what is claimed without departing from the spirit and scope of the invention.REFERENCES CITED Abbott, W. S.1925. A method of computing the effectiveness of an insecticide. J. Econ. Entomol.18: 265–267. Antonio-Nkondjio, C., N. N. Sandjo, P. Awono-Ambene, and C. S. Wondji.2018. Implementing a larviciding efficacy or effectiveness control intervention against malaria vectors: key parameters for success. Parasites Vectors 11: 57 Beckage, N. E., K. M. Marion, W. E. Walton, M. C. Wirth, and F. F. Tan.2004. Comparative larvicidal toxicities of three ecdysone agonists on the mosquitoes Aedes aegypti, Culex quinquefasciatus, and Anopheles gambiae. Arch. Insect Biochem. Physiol.57: 111–122. Bellini, R., A. Alessandro, C. Marco, C. Roberta, D. Luciano, M. Maurizio, P. Roberto, V. Rodolfo, C. Giancarlo, and L. 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Claims

CLAIMS What is claimed is:

1. A compound of the following formula: whe3.

2. A method for the manufacture of the composition of claim 1 in liquid form comprising: adding (2E, 4E, 7S)-11-methoxy-3, 7, 11–trimethyl l-2, 4-dodecarenoic acid and anhydrous dichloromethane under nitrogen protection to create a mixture; adding acetyl chloride to said mixture; allowing said mixture to react; adding tert-butanol to said mixture; allowing said mixture to react; adding saturated sodium bicarbonate solution to said mixture; extracting by ether.

3. A method for the manufacture of the composition of claim 2 in dry form further comprising the step of drying said mixture using anhydrous sodium sulfate.

4. A composition for controlling insects which comprises as an active ingredient an insecticidally effective amount of the compound according to claim 1 and aninert carrier or diluent.

5. A method for controlling insects selected from the group consisting of Hemiptera, Lepidoptera, Coleoptera, Diptera, Dictyoptera, Orthoptera, Hemoptera, Hymenoptera and Aphaniptera comprising applying an insecticidally effective amount of a compound according to claim 1 to said insects or the locus where the insect pests propagate.

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