[Agr. Biol. Chem., Vol. 29, No. 3, p. 243•`248, 1965]

Saligenin Cyclic Phosphoramidates and Phosphoramidothionates

as Pesticides

By Morifusa ETO, Ken KOBAYASHI,_??_ Takeshi KATO,_??_ Kenichi KOJIMA*

and Yasuyoshi OSHIMA

Department of Agricultural Chemistry, Kyushu University, Fukuoka and *Institute for Agricultural Chemicals, Toa Noyaku Co., Odawara

Received October 5, 1964

Several saligenin cyclic phosphoramidates and phosphoramidothionates were synthesized and their pesticidal activities against insects, mites and nematodes were examined. 2-

Methylamino-4H-1,3,2-benzodioxaphosphorin-2-oxide and its thiono analog had high acti vities. They were also effective as systemic insecticides. When the size or number of substituent on nitrogen increased, the pesticidal activities decreased.

Our investigations on saligenin cyclic phos EXPERIMENTALS phorus esters have started with the finding of Syntheses. the biologically active metabolite of tri-o-tolyl All cyclic phosphoramidates and phosphoramido phosphate.1,2) It has been demonstrated that thionates were synthesized from saligenin and ap methyl phosphate (Salioxon) and methyl phos propriate phosphoramidic dichloride or phosphor amidothioic dichloride by the action of proper phorothionate (Salithion) derivatives in this dehydrogen chloride agent. The reaction of saligenin series have high insecticidal activities.3,4) As with reactive dichloride as monoalkylphosphoramidic some known systemic insecticides" such as dichloride and some others was proceeded by the Schradan (octamethylpyrophosphoramide) and action of tertiary amine such as pyridine (Procedures Mipafox (N, N'-diisopropylphosphorodiamidic Ap and Ap') or triethylamine (Procedure At) in u fluoride) have phosphoramide linkage, it ap cooled condition. Liquid dichloride was usually added peared that systemic insecticidal properties dropwise to the mixture of saligenin and the base would be expected from amidate derivatives in chloroform (Procedures Ap and At). Solid dichlo in this series. The investigations described ride was, however, dissolved in cold chloroform with in this paper are concerned with syntheses saligenin, then the amine was added dropwise to the and pesticidal activities including systemic chilled mixture (Procedure Ap'). When the dichloride is less reactive as almost all phosphoramidothioic and activity of saligenin cyclic phosphoramidates diethyl phosphoramidic dichlorides, heating the mix and phosphoramidothionates. ture of reactants in toluene in the presence of anhydrous potassium carbonate and copper powder was useful (Procedure B). The procedures are illust _??_ Research fellow from Toa Noyaku Co., Odawara. _??_ Present address. Mikasa Chemical Co., Amagi. rated by the following examples. Phosphoramidic 1) J. E. Casida, M. Eto and R. L. Baron, Nature, 191, 1396 dichlorides and phosphoramidothioic dichlorides were (1961). 2) M. Eto, J. E. Casida and T. Eto, Biochem. Pharniacol., prepared according to Michaelis' procedure.6) 11, 337 (1962). 2-Methylamino-4H-1, 3, 2-benzodioxaphosphorin-2- 3) M. Eto, Y. Kinoshita, T. Kato and Y. Oshima, Nature, 200, 171 (1963). oxide (K-19) (Procedure At). To a mixture of 4) M. Eto, Y. Kinoshita, T. Kato and Y. Oshima, This saligenin, 9.2g, triethylamine, 15g, and 90ml chloro- Journal, 27, 789 (1963). 5) P. A. Giang, J. Assoc. Offic. Agr. Chem., 37, 642 (1954). 6) A. Michaelis, Ann., 326, 129 (1903). 244 Morifusa ETO, Ken KOBAYASHI, Takeshi KATO, Kenichi KOJIMA and Yasuyoshi OSHIMA

form was dropwise added 12g methylphosphoramidic borer, Chile supressalis Walker, were topically treated dichloride with stirring and cooling in an ice bath. as previously reported.7.8)

After the completion of addition,, the reaction mix Pot tests for rice stem-borer were performed by ture was kept overnight at room temperature, and spraying 20ml of 0.025% emulsion to 3 young rice

then washed sequentially with ice water, dilute hydro plants cultured in a pot. The spray was applied at chloric acid, aqueous sodium bicarbonate solution distance of 1m with a power sprayer at 5lbs/in2. and water. The solvent was removed in vacuo after Fifteen first instar larvae were made the plants infest drying over anhydrous sodium sulfate. Crude crystals, seven days before the spray. In some experiments,

8.05g, m.p. 86•Ž, were recrystallized from benzene the larvae were made infest three, five or eight days to yield pure crystals, m.p. 87•Ž. after the spray in order to examine the residual activity. Mortality accounts were done three days 2-n-Propylamino-4H-1, 3, 2-benzodioxaphosphorin- 2-oxide (K-41) (Procedure Ap'). To a mixture, chilled after the treatments. All tests were triplicated. at -4•Ž, of 12.3g saligenin, 16.2g propylphosphor Systemic activities. Systemic insecticidal activities

amidic dichloride and 100ml chloroform, 16g pyridine against green rice leafhopper were examined by using was dropped with stirring. The mixture was stirred 10 adult females (susceptible Odawara strain) on 3

for four hours at room temperature after the addition young rice plants of which roots were soaked in 20ml of pyridine. After washing and drying by usual emulsion diluted with tap water. They were kept in a small net cage at 25•Ž and mortality account manner, the solvent was removed in vacuo. The residue was distilled in vacuo at 135•`140•Ž (0.5 was made after twenty-four hours. The tests were triplicated. mmHg) to yield 8g of liquid. Systemic activity test against rice stem-borer was 2-Dimethylamino-4H-1, 3, 2-benzodioxaphosphorin- carried out as follows: Seven rice plants in a pot 2-sulfide (K-36) (Procedure B). Saligenin, 12.3g, were infested with 10 to 12 first instar larvae four was dissolved in 100ml of toluene by heating. Thirty days before application of insecticide. Acetone solu grams of finely powdered anhydrous potassium carbo tion of chemicals was dropped to water covering soil nate and copper powder, 0.5g, were added thereto, in the pot. Twelve grams chemicals an acre were and the mixture was added dropwise with dimethyl applied. Mortality was counted seven days after the phosphoroamidothioic dichloride, 18g, while being treatment. The test was duplicated. stirred and kept at 60•Ž. After one hour, the tem Acaricidal activity. Primary leaf of kidney bean perature of the reaction mixture was raised to 70 •` 80•Ž, and the mixture stirred for fifteen hours at plants was dipped for ten seconds in aqueous emul sion of test chemicals. The leaf when dry was in that temperature and filtered. The filtrate was washed fested with about 50 adults of Tetranychus telarius. and dryed by ordinary manner. The solvent was They were held for three days before making mor removed in vacuo and the residue distilled in vacuo tality account. at 118•`122•Ž (0.2mmHg) to yield 6.2g of liquid. Nematocidal activity. Rhabditis sp. was suspended Pesticidal Tests. in an aqueous solution of chemicals and kept at 25•Ž Insecticidal activities. Four days old females of for twenty-four hours. The tests were duplicated. oriental house flies, Musca domestica vicina Macquart

Takatsugi strain, were topically treated with the RESULTS acetone solution of test chemicals and kept at 25•Ž. Saligenin cyclic phosphoramidates and their The mortality account was made after twenty-four thiono analogs were synthesized by the phos hours. The tests were duplicated. phorylation of saligenin with appropriate Knock down time was measured by a film method.

A sheet of filter paper was placed on the bottom of phosphoramidic dichlorides and their thiono analogs respectively in the presence of suitable a beaker (diameter 9cm). Acetone solution contain ing 100ƒÊg of test chemicals was poured on the filter de-hydrogen chloride reagent such as tertiary

paper and after the evaporation of the solvent, 2ml amine or potassium carbonate. Their synthetic

of sugared milk was spread on the paper. Oriental procedures, yields, physical and analytical house flies of Sapporo strain were used. 7) S. Kitakata, A. Shiino and K. Kojima, Botyu-Kagaku, Adult females of green rice leafhopper, Nephotettix 28, 29 (1963). 8) K. Kojima, T. Ishizuka and S. Kitakata, ibid., 28, 55 bipuntatus cincticeps Uhler, and larvae of rice stem (1963). Saligenin Cyclic Phosphoramidates and Phosphoramidothionates as Pesticides 245

TABLE I. PHYSICAL AND ANALYTICAL DATA OF SALIGENIN CYCLIC PHOSPHORAMIDATES AND PHOSPHORAMIDOTHI.ONATES(I) PRODUCED BY THE REACTION

* uncorrected temperature. t Tertiary amine (A) or potassium carbonate (B) was used as de-hydrogen chloride agent. For detail, see the ex perimental part.

TABLE 11. TOXICITIES TO INSECTS AND MOUSE OF (II) IN COMPARISONWITH KNOWN INSECTICIDES

* Crude sample was used.

Numbers in parentheses are mortality percentage at 10ƒÊg dose per female house fly. 246 Morifusa ETO, Ken KOBAYASHI, Takeshi KATO, Kenichi KOJIMA and Yasuyoshi OSHIMA data are shown in Table I. Their infrared desirable properties as insecticides. It killed spectra examined in chloroform solution 100% larvae infesting rice plants by spraying showed characteristic absorption bands as 20ml a pot of 0.025% emulsion. Its oxo sociated with P-O-C (alkyl), P-O-C (aryl), and analog (K-19) killed 78.3%. The homologous N-H (in N-mono-substituted derivatives) in compounds carrying bigger substituent(s) than the regions 1020cm-1, 1250cm-1 and 3400cm-1 methyl group showed only weak activity. respectively. Ethyl parathion killed the insect, 97.2%, by Insecticidal activities against oriental house the same treatment. The residual activity on fly, Musca domestica vicina Macquart Takatsuki rice plants of K-35 against the insect larvae strain, rice stem-borer, Chilo supressalis Walker, was slightly less than Parathion. Sixty-two and green rice leafhopper, Nephotettix bipuntatus percent larvae invading rice plants which had cincticeps Uhler, and toxicity for mouse are been sprayed with K-35 before five days were shown in Table II. Saligenin cyclic methyl killed, while 77% killed by the same treatment phosphoramidate (K-19) and its thiono analog with Parathion. Systemic insecticidal activity (K-35) are most insecticidal for all tested against rice stem-borer in rice plant stem insect species and are comparable to Parathion is shown in Table III. The methylphos and Malathion. When the length of alkyl phoramidothionate, K-35, is more effective group increases, the insecticidal activities re than ƒÁ-BHC, which is now widely used in markably decrease. Aromatic derivative (K- this country for the control of the insect. 10) is almost non-toxic to insects. Similar The systemic activity of some saligenin relationship between the size of substituent cyclic phosphoramidates against green rice and insecticidal activity has been observed in leafhopper on rice plants was also demon the series of saligenin cyclic phosphates.4) strated (Table IV). The activity of K-19 Dialkylphosphoramidates (K-20 and 23) are was higher than Schradan and slightly lower less toxic to the insects than corresponding mono-substituted derivatives (K-19 and 22). TABLE III. SYSTEMIC ACTIVITY OF I AGAINST RICE Insecticidal activities against house fly of thiono STEM-BORDER INFESTING RICE PLANTS analogs (K-35, 36, 37 and 38) are almost equal to or greater than those observed in the cor responding oxo analogs (K-19, 20, 22 and 23). Particularly, diethyl derivative (K-38) is much more active than its corresponding oxo analog

(K-23). Against green rice leafhopper, how ever, thiono analogs including Salithion4) are inferior to their oxo analogs (K-19 and Salioxon). Much higher insecticidal activity TABLE IV. SYSTEMIC ACTIVITY AGAINST GREEN of these cyclic phosphoramidates against the RICE LEAFHOPPER ON RICE PLANTS WHOSE house fly of Sapporo strain was observed. ROOTS WERE SOAKED IN AN AQUEOUS EMUL The rapid effect to knock down house fly SION OF THE CHEMICALS appeared to be an outstanding property of K-19. The values of LT,, in min/1.6ƒÊg/cm2 were 9.1, 19.1 and 49.9 for K-19, Salioxon and Parathion respectively. In the following pot tests against rice stem borer, it was observed that saligenin cyclic methylphosphoramidothionate (K-35) has some Saligenin Cyclic Phosphoramidates and Phosphoramidothionates as Pesticides 247

than Thiment (O, O-diethyl S-ethylthiomethyl analogs11) have been investigated. These ex phosphorodithioate ). perimental results showed that N-ethylphos The acaricidal activity of K-19 was also phoramidate and its thiono analog were most observed. It killed 100% of adult Tetranychus active against insects and isopropylamidates. telarius on a bean leaf dipped into 0.01% were next. In contrast with these results, emulsion for ten seconds. monomethyl derivatives were most active in Saligenin cyclic phosphoramidates also the present series of saligenin cyclic phos showed nematocidal activity (Table V). They phoramidates and their thiono analogs. When are generally more toxic to Rhabditis than the size of alkyl group increased, the activity a well known nematocide D-D (mixture of markedly decreased as similarly observed in 1, 3-dichloropropene and 1, 2-dichloropropane). a previous study on the series of phosphate Mono-methylphosphoramidate (K-19) was the analogs.4) most effective. The correlation between the Some organophosphorus esters containing structure and the activity was similar to that sulfide linkage (e. g. Thimet), carboxyamide in the toxicity for house fly. group (e. g. Dimethoate: O, O-dimethyl S-(N - methylcarbamoylmethyl) phosphorodithioate) TABLE V. NEMATOCIDALACTIVITY OF I To Rhabditis or phosphoramide group (e. g. Schradan) are known as systemic insecticides. It was demon strated that saligenin cyclic phosphorus esters could also be endowed with systemic insecti cidal activity by the introduction of alkylamino group on phosphorus atom. Several organic phosphates have been re cently investigated for nematocidal uses. Diethyl 2, 4-dichlorophenyl phosphorothionate (VC-13-Nemacide)12) and diethyl pyrazinyl phosphorothionate (Cynem)13) recently became commercially available nematocides. In the series of saligenin cyclic phosphates, the nematocidal activity of 2-ethoxy-4 H-1, 3, 2- Numbers in parentheses are mortality percentage at in dicated concentration. benzodioxaphosphorin-2-oxide was reported in a previous paper.14) It killed 70% Rhabditis DISCUSSION at 200 p.p.m. The present analogous phos Although great numbers of organophosphates phoramidates were much more active as and their sulfur analogs have been evaluated mentioned above. 2-Methylamino-4H-1, 3, 2- as insecticides, relatively little has been pub benzodioxaphosphorin-2-oxide (K-19) killed lished on the evaluation of phosphoramidates 100% the nematodes at a concentration less and phosphoramidothionates in spite of that than 10 p.p.m. 0-2-chloro-4-tert-butylphenyl O-methyl methyl- It has been observed 4, 15) that the aryl deri phosphoramidate (Ruelene) and its thiono vatives of saligenin cyclic phosphates remarka- analog (Narlene) are known as antihelmintics.9) 11) E. H. Blair, K. C. Kauer and E. E. Kenaga, J. Agr. The correlation between insecticidal activities Food Chem., 11, 237 (1963). and chemical structure of O-2, 4, 5-trichloro 12) G. Schrader, Die Entwicklung neuer insektizider Phosphorsaure-Ester" Verlag Chemie, 1963, p. 209. phenyl phosphoramidates10) and their thiono 13) R. E. Mostinger, Plant Disease Reporter, 45, 335 (1961). 14) M. Eto, T. Eto and Y. Oshima, This Journal, 26, 630 9) J. F. Landram and R. J. Shaver, J. Parasitology (Sup 1962). plement), 45, 55 (1959); J. F. Landram, ibid., 45, 56 (1959). 15) M. Eto, K. Hanada, Y. Namazu and Y. Oshima, ibid., 10) T. R. Fukuto, R. L. Metcalf, M. Y. Winton and R. B. _??_ 27, 723 (1963). March, J. Econ. Entomol., 56, 808 (1963). 248 Morifusa ETO, Ken KOBAYASHI, Takeshi LATO, Kenichi KOJIMA and Yasuyoshi OSHIMA

TABLE VI. EFFECTS OF SUBSTITUENT (R) ON derivative. Dimethylphosphoramidate is CHEMICAL AND BIOLOGICAL ACTIVITIES OF ranked between them. Their insecticidal SALIGENIN CYCLIC PHOSPHORAMIDATES activities are in this order. The effects of the substituents on biological activities may be attributed to steric interaction between the molecule and the active centre of the target enzyme. The mono-methylphosphoramidate (K-19) has high anticholinesterase activity in vitro (Table VI). This suggests that a metabolic activation in vivo, e.g., to an N-oxide16) or similar derivatives17) as has been shown to Khyd: hydrolysis rate constant in m/15 phosphate buffer, occur with Schradan may not necessarily occur pH 7.7 at 25•Ž. I50ChE: molar concentration to inhibit 50% housefly cholin with K-19. esterase. * No detectable hydrolysis occurred . Salioxon and Salithion which have been discovered as high insecticidal compounds in bly differ from analogous alkyl derivatives the series of saligenin cyclic phosphorus esters in their chemical and biological properties. are rather unstable. The presented phos This is also the case for the present series of phoramidates were more stable as expected: saligenin cyclic phosphoramidates. Hydrolysis K-19 was ten times more stable than Salioxon rate constants, I50 values for house fly and twice than Salithion. cholinesterase and LD50 values for house fly of three representatives are shown as an ex Acknowledgements. This work was partially ample in Table VI. Phenylphosphoramidate supported by the grant from Asahi News is most reactive and about twice reactive Paper Co., Ltd. and also by Sumitomo Chem. than mono-methyl derivative. Dimethyiphos Co., Ltd. Skilled technical assistance of Miss phoramidate is quite stable. Mono-methyl Hirai and Miss Nagata is acknowledged. phosphoramidate shows high inhibitory activity 16) J. E. Casida, T. C. Allen and M. A. Stahmann, J. Biol. to house fly cholinesterase, whereas phenyl Chem., 210, 607 (1954). 17) R. D. O'Brien, "Toxic Phosphorus Esters", Academic phosphoramidate is a weak inhibitor, less Press, 1960, p. 66. active than one hundredth of mono-methyl