Pest controlling compositions

A pest-controlling composition which comprises: PA0 (A) a first compound capable of substantially inhibiting a phosphodiesterase enzyme (PDE) of said pest; and PA0 (B) a second compound having pest-dontrolling activity towards said pest, selected from the group consisting of PA1 (1) a substantial octopamine agonist toward an octopamine receptor present in said pest; PA1 (2) a compound directly and substantially stimulating the enzyme, adenylate cyclase; and PA1 (3) a cyclic adenosine monophosphate (cAMP) analogue.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to pest controlling compositions formed by 
mixing a first compound having pest controlling activity together with a 
second compound capable of inhibiting a phosphodiesterase enzyme of the 
pest. The invention also relates to methods of controlling pests by 
treatment with the aforementioned compositions. 
2. Description of the Background Art 
Despite the recent development and great promise of such advanced pest 
controlling compositions as chemical sterilants, pheromones or 
ecologically-based insect control strategies, it is doubtless that, at 
present, the use of chemical pesticides still plays a predominant role. 
The use of insecticides often represents the difference between profitable 
crop production for farmers and no marketable crop at all, and the value 
of insecticides in controlling human and animal diseases has been 
dramatic. 
Therefore, in parallel to the aforementioned newer technologies for pest 
control, there has been active research and investigation into the 
detailed biochemical modes of action of existing known chemical 
pesticides. Thus, for example, Nathanson et al., Molecular Pharmacology 
20:68-75 (1981) presented evidence indicating that the formamidine 
pesticides chlordimeform (CDM) and N-demethylchlordimeform (DCDM) may 
affect octopaminergic neurotransmission. CDM and DCDM have been reported 
to mimic the effects of octopamine in stimulating light emission in the 
firefly lantern (Hollingworth, R. M. et al., Science, 208:74-76 (1980)), 
and in effecting nerve-evoked muscle responses in the locust leg (Evans, 
P.D., Nature, 287:60-62 (1980)). Nathanson et al., supra, found that DCDM, 
which is the probable in vivo metabolite of CDM, is about six-fold more 
potent than octopamine itself as a partial agonist of light organ 
octopamine-stimulated adenylate cyclase. Stimulation by the formamidines 
resulted in increased formation of the intracellular messenger, cyclic 
AMP. This stimulation was blocked by cyproheptadine, clozapine, 
fluphenazine and phentolamine compounds, also known to block the 
octopamine receptor. Nathanson et al. concluded that DCDM is the most 
potent octopaminergic compound described. 
Similar results were observed by Hollingworth et al. (reported in the 
Scientific Papers of the Institute of Organic and Physical Chemistry of 
Wroclaw Technical University, No. 22, Conference 7 (1980)). These authors 
demonstrated that certain formamidines act on octopamine receptors to 
induce the synthesis of cyclic AMP, and that this response is blocked by 
both phentolamine and cyproheptadine, which are known to act as 
octopaminergic antagonists in insects. The authors also suggested that 
these formamidines are potent stimulators of the octopamine sensitive 
adenylate cyclases in the thoracic ganglia of Periplaneta americana, and 
in the ventral nerve cord and fat body of M. sexta. The authors suggest 
that the stimulation of octopamine receptors underlies a number of toxic 
responses seen with formamidines on insects. 
It should be noted that the presence of an insect adenylate cyclase enzyme 
which is sensitive to octopamine as a "neuro transmitter" has been known 
for some time (Nathanson et al, Science, 180:308-310 (1973) (cockroach); 
Nathanson, ibid: 203: 65-68 (1979) (firefly); Evans, J., Neurochem, 
30:1015-1022 (1978) (cockroach)). 
The study of cyclic AMP (cAMP) as a "second messenger" has led to the 
accepted model that a hormone or neurotransmitter binds at a cell-membrane 
bound receptor, which activates adenylate cyclase to a form capable of 
converting ATP in the cytoplasm of the cell into cAMP. cAMP then relays 
the signal brought by the hormone or neurotransmitter from the membrane to 
the interior of the cell. Agonists of the hormone or neurotransmitter are, 
by definition, capable of eliciting the same response (see, for example, 
Nathanson and Greengard, Scientific American, 237:108-119 (1977)). Once 
formed inside the cell, cyclic AMP presumably binds to a protein kinase 
which is then capable of phosphorylating appropriate proteins, etc. 
Given the continuous need for increased selectivity and effectiveness in 
pest control agents, it became desirable that the greater understanding of 
the biochemical mode of action of the formamidines be utilizable in some 
manner to improve their effectiveness, and to lead to a general rational 
formulation of pest control agents. 
SUMMARY OF THE INVENTION 
The present invention arose out of the initial observations by the inventor 
and others that the mode of action of certain formamidine pesticides was 
through their octopaminergic agonist activity on octopamine receptors 
present in the pest, and that these pest control agents were acting 
through generation of cAMP as a "second messenger." The inventor then 
observed that the effectiveness of any octopaminergic agonist pest control 
agent could be greatly enhanced when the quantity and half-life of 
generated cAMP was regulated by inhibiting insect phosphodiesterase 
enzymes, which are capable of hydrolyzing cAMP. Thus, addition of 
phosphodiesterase inhibitors to octopaminergic agonist pest control agents 
increases the action and effectiveness of these types of agents. Large 
amounts of experimental data have confirmed the generality of this 
invention. 
Thus, in one embodiment, the present invention provides a pest controlling 
composition which comprises: 
(A) a first compound capable of inhibiting a phosphodiesterase enzyme of 
said pest; and 
(B) a second compound having pest-controlling activity towards said pest, 
selected from the group consisting of 
(1) an octopamine agonist toward an octopamine receptor present in said 
pest; 
(2) a compound directly stimulating the enzyme adenylate cyclase; and 
(3) a cyclic adenosine monophosphate (cAMP) analogue. 
These compositions are synergistic, i.e., the combination of the first 
compound (A) and the second compound (B) results in the correlated action 
of both compounds which, together, have greater total effect than the sum 
of their individual effects. 
The synergism observed in the compositions of the present invention should 
be distinguished from the more classical insecticide synergism. Thus, for 
example, it is known that pyrethrin insecticides, when used alone, have 
reversible action due to the detoxication effect by microsomal insect 
oxidases. Since the detoxication enzymes are inhibited by a number of 
compounds, especially those of the methylenedioxyphenyl structure, these 
compounds (called "synergists"), when used at various ratios, activate the 
pyrethrins by about 2 to 30 times. (See, for example, Encyclopedia of 
Chemical Technology, 3d Edition, Vol. 13, pages 424-425.) 
Microsomal oxidase inhibitors inhibit enzymes which are directly involved 
in the destruction of insecticides. In the present invention, on the other 
hand, the phosphodiesterase inhibitors do not act on enzymes involved in 
the destruction of the octopamine agonist, but inhibit the hydrolysis of 
cyclic AMP acting as "secondary messenger." 
FIG. 21 indicates the three types of pest control agents having pest 
control activity useful as compounds (B). These are either octopamine 
agonists (B1), direct adenylate cyclase enzyme stimulators (B2), or cyclic 
AMP analogues (B3). Octopamine agonists act by binding to a receptor which 
activates adenylate cyclase which, in turn, produces secondary messenger 
cyclic AMP. Enzyme stimulators also act through the production of cyclic 
AMP, but do so by interacting directly with adenylate cyclase, bypassing 
the receptor. Once octopamine agonists or enzyme stimulators lead to the 
production of cyclic AMP, the cyclic AMP can either be hydrolyzed by the 
action of phosphodiesterase enzymes (PDE) or bind to a cyclic AMP receptor 
generating hormonal-type activity. The third type of pest control 
compound, the cyclic AMP analogue (B3), can either be hydrolyzed by the 
action of PDE or bind to a cyclic AMP receptor generating hormonal-like 
activity. 
With all three types of pest control agents, the addition of PDE inhibitors 
blocks or decreases the competing hydrolyses of the cyclic AMP or cyclic 
AMP analogue, increasing hormonal-like and pest control activity. 
In another embodiment of the invention, there is provided a method for 
controlling pests by treating said pests with a composition as hereinabove 
in an amount effective to provide pest control, by either pesticidal or 
pestistatic activity.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The terms "pest controlling" or "pest controlling activity," used 
throughout the specification and claims, are meant to include any 
pesticidal (killing) or pestistatic (preventing the host plant from being 
eaten, or inhibiting, maiming or generally interfering) activities of a 
composition against a given pest at any stage in its life cycle. Thus, 
these terms not only include killing, but also include such activities as 
the production of behavioral abnormalities (e.g., tremor, incoordination, 
hyperactivity, anorexia, leaf walk-off behavior) which interfere with such 
activities such as but not limited to eating, molting, hatching, mobility 
or plant attachment. The terms also include activities of chemisterilants 
which produce sterility in insects by preventing the production of ova or 
sperm, by causing death of sperm or ova, or by producing severe injury to 
the genetic material of sperm or ova, so that the larvae that are produced 
do not develop into mature progeny. 
The terms also include repellants, which are substances that protect 
animals, plants or products from insect attack by making food or living 
conditions unattractive or offensive. These may be poisonous, mildly 
toxic, or non-poisonous. 
The terms also include attractants, food lures, sex pheromones, aggregation 
pheromones, and the like. Any compound (B) which has such "pest 
controlling activity" as defined and which is (i) an octopamine agonist 
toward an octopamine receptor present in the pest, (ii) a direct 
stimulator of pest adenylate cyclase, or (iii) a cyclic AMP analogue, is 
included in the present invention. 
The question of whether a given compound (B) is an octopamine agonist (B1) 
can be readily answered by measuring adenylate cyclase activity of the 
octopamine-sensitive adenylate cyclase present in broken cell preparations 
of the firefly light organ. Generally, the broken cell preparations are 
prepared according to the method described in a paper by Nathanson et al. 
(Molecular Pharmacology 20:68-75 (1981), which is herein incorporated by 
reference. Specimens of Photinus pyralis are prepared by opening their 
tail sections, cleaning them, removing the light organs, and homogenizing 
the cyclase-containing fraction. Adenylate cyclase activity is measured in 
appropriate buffer-containing ATP and the compound to be tested. If 
necessary, the compounds (B) to be tested are initially solubilized and 
appropriate solvent controls are run in parallel. The enzyme reaction is 
initiated by addition of ATP, stopped by heating, and centrifuged. Cyclic 
AMP can be measured by any test which indicates the presence thereof, 
preferably by the protein binding assay of Brown et al. (Advances in 
Cyclic Nucleotide Research 2:25-40 (1972)). Normally, the solution mixture 
contains a phosphodiesterase inhibitor such as theophylline, so as to 
provide linear measurements with respect to time and enzyme concentration. 
The determination of the constant K.sub.a, which is the concentration of 
agonist B1 necessary for halfmaximal activation of cyclase activity, is 
carried out by measuring cyclase activity in the preparation, and plotting 
the activity (above control activity) versus the semilogarithm of the 
particular agonist concentration. This is done for a series of increasing 
concentrations until maximal activity (Vmax) is reached. K.sub.a.sup.B is 
then calculated from the graph as the agonist concentration required for 
one-half of Vmax. K.sub.a.sup.B is compared with the constant 
(K.sub.a.sup.oct) determined in an analogous way using .+-. p-octopamine 
as the agonist. The ratio K.sub.a.sup.oct /K.sub.a.sup.B is then an 
indication of whether the compound (B1) is better (ratio greater than 1) 
or worse (ratio smaller than 1) than (.+-. )-p-octopamine. Maximal 
activation of enzyme activity as a percentage of maximal activation seen 
in the presence of (.+-.) p-octopamine can be denoted as % Vmax. 
Generally, an octopamine agonist having a K.sub.a.sup.oct /K.sub.a.sup.B 
ratio greater than 0.05, preferably 0.05 to 1000, most preferably 0.1 to 
1000, as measured by the firefly lantern test, is used. Also, generally, 
octopamine agonists having Vmax anywhere between 5 and upwards of 100%, 
preferably between 10 and upwards of 100%, of the Vmax of 
(.+-.)-p-octopamine can be used. The values of Vmax for any desired 
octopamine agonist are not as important as the values of the ratio of K's. 
As long as the K.sub.a.sup.oct /K.sub.a.sup.B ratio falls within the 
stated range, the Vmax values can vary widely. 
In addition to the above method employing the firefly light organ, 
octopamine-sensitive adenylate cyclase can also be measured in tissue 
preparations from the nerve cord of any desired particular insect pest, 
using a modification of the method appearing in Nathanson et al. (Science 
180:308-310 (1973)) herein incorporated by reference. In this modification 
(which is not necessary if the firefly light organ is used), dopamine (10 
micromolar) and serotonin (10 micromolar) are added to all (including 
control) assay tubes. This is done in order to be sure that the tested 
compounds (B1) are affecting only octopamine receptors (known to be 
present in all insect nerve cords) and not dopamine or serotonin 
receptors. Otherwise, the procedure is identical to that described above. 
Among the preferred octopamine agonists B1 are those belonging to the 
families of the phenylethylamines (I): 
##STR1## 
Cyclic Amindines (II): 
##STR2## 
where X is N--R.sup.11, O, CH.sub.2, or S, and n may be 1 or 2; such as 
2-(phenylimino) imidazolidines (X.dbd.NH, n=1); 2-(phenylimino) 
pyrrolidines (X.dbd.CH.sub.2, n=1); 2-(phenylimino) oxazolidines (X=0, 
n=1); 2 (phenylimino) thiazolidines (X.dbd.S, n=1) and 2-(phenylimino) 
thiazines (X.dbd.S, n=2). See, e.g., the compounds in DeJong et al, Europ. 
J. Pharm. 69:175-188 (1981); 
2-benzylimidazolines (III): 
##STR3## 
Formamidines (IV): 
##STR4## 
R.sup.3, R.sup.4, R.sup.11, R.sup.16 and R.sup.17 stand for hydrogen, 
lower alkyl or lower alkyl substituted by hydroxy or lower (C.sub.1 
-C.sub.6) alkoxy; R.sup.12 stands for hyrogen or hydroxyl. R.sup.1 and 
R.sup.2 are the same or different and selected from the group consisting 
of hydrogen, hydroxy and lower (C.sub.1 -C.sub.6) alkyl; R.sup.5, R.sup.6, 
R.sup.7, R.sup.8, R.sup.9, R.sup.10, R.sup.13, R.sup.14, and R.sup.15 are 
the same or different and selected from the group consisting of hydrogen, 
hydroxy, fluorine, chlorine, bromine, iodine, nitro, lower (C.sub.1 
-C.sub.6) alkyl, lower (C.sub.1 -C.sub.6) alkoxy, lower haloalkyl, amino, 
mono lower alkylamino, di-lower alkyl amino, hydroxy-substituted lower 
alkyl and lower acylamino. 
Also, in compounds of formulae (II) or (III) above R.sup.8, R.sup.9 or 
R.sup.8, R.sup.10 together may form a six membered phenyl, pyridine, 
diazine, or cyclohexyl ring fused to the noted phenyl ring. For example, 
systems of formulae V and VI can also be used: 
##STR5## 
where R.sup.10, R.sup.11, R.sup.12 and n are as defined previously. 
Specific compounds useful as octopamine agonists (B1) include 
phenylethylamines of the formula (VII): 
##STR6## 
where R.sup.5 is OH and R.sup.6 is CH.sub.3, C.sub.2 H.sub.5, i-C.sub.3 
H.sub.7, C.sub.6 H.sub.11, NH.sub.3, F, Cl, Br I, NHSO.sub.2 CH.sub.3, OH, 
H or OCH.sub.3 ; or where R.sup.6 is OH and R.sup.5 i-C.sub.3 H.sub.7, 
CH.sub.3, C.sub.2 H.sub.5, C.sub.6 H.sub.11, NH.sub.3, Cl, Br, I, NH 
SO.sub.2 CH.sub.3, OCH.sub.3 or H. 
Other specific compounds B1 include cyclic amidines of the formula (VIII): 
##STR7## 
where R is phenyl; o-tolyl; 2,6 dimethylphenyl; 2,3 (cyclohexyl) phenyl; 
2,6-diethylphenyl; 2,6-difluorophenyl; 2-chlorophenyl; 2,6-dichlorophenyl; 
3-chlorophenyl; 2,5-dichlorophenyl; 3,5-dichlorophenyl; 
5-bromoquinoxaline; 2-methyl,3-bromophenyl; 2-chloro,3-methylphenyl, 
2-chloro,4-methylphenyl; 3-fluoro,6-methyl-phenyl; 2,6-dichloro, 
4-hydroxyphenyl; 3,4-dihydroxyphenyl; or 4-chlorophenyl. 
Other specific compounds B1 include cyclic amidines of the formula (IX): 
##STR8## 
where R' is phenyl, o-tolyl, 2,6-dimethylphenyl, 2-chlorophenyl, 
2,6-dichlorophenyl, 4-chlorophenyl, or 4-methoxyphenyl. 
Other specific compounds B1 include cyclid amidines of the formula (X): 
##STR9## 
where R" is H, 2-CH.sub.3, 2-6-diCH.sub.3, 4-CH.sub.3, 4-Cl or 2,6-diCl. 
Other specific compounds B1 include cyclic amidines of the formula (XI): 
##STR10## 
where R'" is 2,6-dimethyl; 2,6-diethyl; 2,6-dichloro; 2,4,6-trimethyl; 
2,4-dichloro; 2,4-dimethyl; 2-chloro-4-methyl; 4-chloro-2-methyl; 
4-chloro; 2-chloro; 2-methyl or 4-methyl; where n is 1 or 2. 
Other specific compounds B1 include 2-benzylimidazoline of the formula 
(XII): 
##STR11## 
where R"" is phenyl, o-tolyl, 2,6-dichlorophenyl, 4-CH.sub.3 O phenyl, 2,3 
naphthyl (naphazoline), 2,6-dimethyl, 4-.sup.t Butylphenyl 
(xylometazoline), 2,6-dimethyl, 3-hydroxy, 4-.sup.t Butylphenyl 
(oxymetazoline), or 
##STR12## 
(tetrahydrozoline). 
The syntheses and preparation of phenylethylamines of formula (I) is 
described, for example, in LeClerc et al., J. Med. Chem. 23:738-744 
(1980). The syntheses and preparation of cyclic amidines of formulas (II) 
or (III) is described, for example, in Rouot, et al., Journal of Medicinal 
Chemistry, Vol. 19, 1049 (1976); Oxley et al, J. Chem Soc., 497 (1947), 
Faust et al., J. Org. Chem. 26:4044 (1961); Van der Stelt et al., Arzneim. 
Forsch 15:1251 (1965) or Jen et al., J. Med. Chem 15:727 (1972) and ibid, 
18:90 (1975). The syntheses and preparation of formamidines of formula 
(IV) can be found, for example, in Chang et al, J. Agric. Food Chem. 
25:493-501 (1977). 
Also, it should be noted that applicant has filed on even date a commonly 
assigned patent application entitled "Pest Controlling Agents" having Ser. 
No. 605,847, now U.S. Pat. No. 4,678,775 which discloses a number of 
formamidine, cyclic amidine and phenylethylamine octopamine agonists as 
pest-controlling agents. The full disclosure of this copending patent 
application is herein incorporated by reference. 
Other examples of specific compounds (B1) which are octopamine agonists are 
listed in the Tables in accompanying examples. 
The second type of pest controlling compounds (B2) useable in the present 
compositions are direct stimulators of the pest enzyme adenylate cyclase. 
These compounds bypass the receptor, and interact with one or another of 
the associated catalytic or regulatory subunits of adenylate cyclase, 
thereby stimulating the formation of cyclic AMP. Suitable compounds can be 
determined from assay of pest adenylate cyclase as described above. 
Generally, a compound (at a concentration of less than 1 millimolar) 
causing a stimulation of adenylate cyclase of at least 10% that due to a 
Vmax concentration of (.+-.)-p-octopamine is preferred. A direct 
stimulator of adenylate cyclase can be distinguished from an octopamine 
agonist in that the stimulatory activity of the former (but not the 
latter) at a concentration causing half-maximal activation of the enzyme, 
is not significantly reduced by the addition of known octopamine receptor 
antagonists, such as phentolamine or cyproheptadine, used at a 
concentration of 100 micromolar. 
Among preferred direct enzyme stimulators are those belonging to the 
diterpenes, (XIII), forskolin (XIV) and its derivatives: 
##STR13## 
R.sup.18-23 stand for hydrogen, hydroxyl, oxy, keto, lower alkyl, lower 
alkene, lower alkoxy, carboxy and carboxyamino. 
In forskolin R.sup.18 .dbd.R.sup.19 .dbd.R.sup.21 .dbd.OH; R.sup.20 
.dbd.OCOMe; R.sup.22 .dbd.0; and R.sup.23 .dbd.CH.dbd.CH.sub.2. 
Certain bacterial-derived toxins, such as cholera toxin can also be used. 
The preparation of forskolin derivatives is described in Seamon, K. and 
Daly, J. W, J. Med. Chem. 26:436-439 (1983). The structure and action of 
cholera toxin are described in Van Heyningen, S., Biosci. Repts. 2:135-146 
(1982). 
The third type of pest controlling compounds (B3) useable in the present 
compositions are cyclic adenosine monophosphate analogues. These are 
compounds which havecyclic AMP activity, and are capable of binding to the 
appropriate pest protein kinase to activate the same. The potency of a 
particular cyclic AMP analogue can be determined from the calculated 
K.sub.a and Vmax of the analogue for activating cyclic AMP-dependent 
protein kinase found in insect nerve cord or firefly lantern, using the 
method described by Nathanson in Cyclic AMP: A Possible Role in Insect 
Nervous System Function, Ph.D. Thesis, Yale Univ., 1973, pp. 81-82, herein 
incorporated by reference. The K.sub.a and Vmax for the analogue can be 
compared, in the same tissue, with the K.sub.a and Vmax for cyclic AMP, 
itself, in stimulating protein kinase. 
Generally, a cyclic AMP analogue with a K.sub.a.sup.B /K.sub.a (cyclic AMP) 
ratio greater than 0.01, preferably about 0.01 to 100 or more, most 
preferably 0.05 to 100 or more is used. Also, generally, cyclic AMP 
analogues having a Vmax anywhere between 5 and upwards of 100% of the Vmax 
for cyclic AMP can be used. 
A number of biologically active cyclic AMP analogues have been synthesized. 
See Revankar and Robins, in Handbook of Experimental Pharmacology, 58/I 
(ed. J. Nathanson, J. Kebabian) pp. 17-151 (Spring-Verlag, N.Y.) 1982. 
Among the preferred ones are 6-n-butylamino-8-benzylthio-cyclic AMP; 
8-p-chlorophenylthio-cyclic AMP; 8-chloro-cyclic AMP; 8-bromo-cyclic AMP; 
N.sup.6 -monobutyryl or N.sup.6,2'-0-dibutyryl cyclic AMP; 7-deaza-cyclic 
AMP; and 1-deaza-cyclic AMP. 
The compound (A) is one capable of inhibiting a phosphodiesterase enzyme of 
the pest being controlled. The inhibition is such that it should prevent 
or greatly decrease the hydrolysis of endogenous cAMP produced by 
activation of adenylate cyclase. Alternatively, the phospodiesterase 
inhibitor should be capable of inhibiting the hydrolysis of the cyclic 
adenosine monophosphate analogue. The inhibition of phosphodiesterase may 
be either through a competitive or non-competitive mode. Further, the 
phosphodiesterase should be that of the particular pest being controlled, 
but may generally also be the phosphodiesterase present in the broken cell 
preparations described previously, obtained from the firefly lantern. 
Thus, the testing of any particular PDE inhibitor can be carried out on 
isolated pest PDE's or specifically on firefly lantern PDE. 
The ability of any compound (A) to inhibit phosphodiesterase (PDE) activity 
in broken cell preparations of firefly lantern or in pest tissues can be 
determined either (1) by measuring the decrease in rate of hydrolysis of 
an added amount of cyclic AMP by PDE (see Methods Section of Nathanson et 
al., Mol. Pharmacol. 12:390-398 (1975)), or (2) by measuring the rate of 
accumulation of one of the breakdown products of cyclic AMP, such as 
5'-AMP or adenosine (see method of Filburn et al., Anal. Biochem. 
52:505-516 (1973)). Both of these are herein incorporated by reference. 
Generally, any compound capable of maximally inhibiting PDE activity by at 
least 50% (V.sub.max -inhibition) and preferably by at least 80% is 
preferred. Also, in terms of the concentration of the compound required 
for such inhibition, this can be quantitated by determining the IC 
.sub.50-inhibition, i.e., the concentration of the compound required to 
cause 50% of the maximal inhibitioncaused by the compound at any 
concentration. Generally, any compound with an IC .sub.50-inhibition for 
PDE of less than 10 mM and preferably less than 2.5 mM is preferred. 
Of particular interest are purine derivatives, such as caffeine, 
theophylline, xanthine, methylxanthine, isobutylmethylxanthine (IBMX), and 
lower alkyl or substitution homologues or analogues thereof. See, e.g. 
Kramer, et al., Biochem, 16:3316 (1977); Garst et al., J. Med. Chem., 
19:499 (1976); Amer et al., J. Pharm. Sci., 64:1 (1975); or Beavo et al., 
Mol. Pharm., 6:597 (1970). For the purposes of this invention, halide, 
hydroxy, keto, lower alkoxy, lower straight alkyl, lower branched alkyl, 
amino, lower alkylamino, lower halo alkyl, fluorine, chlorine, bromine, 
iodo, nitro, mercapto, alkene-oxy, cyano, alkyl-cyano, phenyl, benzyl, 
substituted benzyl, or the like substituents on any of the aforementioned 
compounds are equivalent if they do not interfere with the inhibitory 
activity of the PDE inhibitor, and do not substantially block the 
agonistic activity of the octopaminergic agonist. 
Of interest are also the phosphodiesterase inhibitors described by 
Rojakovick et al., (Pesticide Biochemistry and Physiology 610-19 (1976)) 
which belong to the family of quinoxaline dithiols. These compounds, 
denoted as oxythioquinox, SAS 2185, 1948, 2501, 2061, 2551 or 2079, are 
those of the formula (XV): 
##STR14## 
where R.sup.24 can be hydrogen, lower alkyl, lower alkoxy or 
trifluoromethyl, R.sup.25 and R.sup.26 can be the same or different and 
selected from the group of H, COOR.sup.27, where R.sup.27 is lower alkyl; 
or both R.sup.25 and R.sup.26 taken together may form a group of the 
formula --CO--, bridging both S atoms. It should be noted that Rojakovick 
et al., found these compounds to be phosphodiesterase inhibitors, as 
determined by cockroach brain adenylate cyclase and PDE in vitro. However, 
the authors concluded that there was no direct relationship of the PDE 
inhibition activity to their mode of toxic action since, on the basis of 
broad distribution of PDE in the animal kingdom, it appeared unlikely to 
them that PDE inhibition was a direct cause of their selective pest 
controlling activity. 
Another family of PDE inhibitors are the benzylisoquinoline derivatives, 
such as papaverine (See, for example, U.S. Pat. No. 3,978,213 to Lapinet 
et al., which relates to the cosmetic use of mixtures of cyclic AMP and 
phosphodiesterase inhibitors; or Amer et al., supra, p.17). 
Another family of PDE inhibitors are the substituted pyrrolidones, such as 
4-(3-cyclopentyloxy-4-methylphenyl)-2-pyrrolidine (ZK 62711). See Schwabe 
et al., Mol. Pharmacol. 12:900-910, 1976. 
Another family of PDE inhibitors are the 
4-(3,4-dialkoxybenzyl)-2-imidazolidinones, such as 
(4-(3-butoxy-4-methoxbenzyl)-2-imidazolidinone (Ro 20-1724). See Sheppard 
et al., Biochem. J. 120:20P (1970). 
Another family of PDE inhibitors are the benzodiazepine derivatives, such 
as diazepam. See Dalton et al., Proc. Soc. Exp. Bio. Med. 145:407-10 
(1974). 
Another family of PDE inhibitors are the tricyclic agents, such as the 
phenothiazines. See Honda et al., Biochim. Biophys. Acta 161:267 (1968). 
Another family are various purine-ribose derivatives, including puromycin 
and derivatives of cyclic nucleotides (other than cyclic AMP or active 
cyclic AMP analogues). See Amer et al., J. Pharm. Sci. 64:1-37 (1975) 
Table VI. 
Anther PDE inhibitor is 
SQ20009:(1-ethyl-4-isopropylidenehydrazino-14-pyrazolo(3,4)pyridine-5-carb 
oxylate ethyl ester. See Beer et al., Science 176:428 (1972). 
In general, any compound which inhibits PDE as described above and which, 
at the same concentration, does not substantially block the activity of 
the octopaminergic agonist in stimulating octopamine-sensitive adenylate 
cyclase (as measured above), can be used. 
The PDE inhibitor may be present alone or in combination with other active 
or non-active compounds. For example, it is known that tea leaves and 
coffee beans contain caffeine. Kaplan et al., S.A. Med. T. 48:510 (1974). 
Kola nuts also contain caffeine (J. Food Sci., 38:911 (1973). Thus ground 
tea leaves, or kola nuts, when combined with any of the compounds (B) are 
covered by the present invention. 
The molecular inhibition of PDE in vitro by a PDE inhibitor correlates with 
the molecular inhibition of the enzyme in vivo. However, it may be that a 
compound which is an excellent PDE inhibitor in vitro does not show good 
in vivo synergistic activity. Other factors, such as possible metabolism, 
transport or absorption of the compound may influence its overall 
effectiveness. One of skill in the art, however, can by a simple 
preliminary trial on the desired pest ascertain quite quickly and 
routinely whether a chosen agent is useful in vivo. 
The % ratio by weight of compound (A) to compound (B) (octopamine agonist, 
direct enzyme stimulator, or cyclic adenosine monophosphate analogue), can 
be varied from 0.001% to 99.99%, preferably 10% to 90%. Preferably, the 
ratio is adjusted so as to effect maximal pesticidal or pestistatic effect 
in the combination. 
The pest controlling compositions of the present invention can be 
formulated as dusts, water dispersions, emulsions, and solutions. They may 
comprise accessory agents such as dust carriers, solvents, emulsifiers, 
wetting and dispersing agents, stickers, deodorants and masking agents 
(see for example, Encyclopedia of Chemical Technology, Vol. 13, page 416 
et seq.). 
Dusts generally will contain low concentration, 0.1-20%, of the compound 
(B), although ground preparations may be used and diluted. Carriers 
commonly include organic flours, sulfur, silicon oxides, lime, gypsum, 
talc, pyrophyllite, bentonites, kaolins, attapulgite, and volcanic ash. 
Selection of the carrier can be made on the basis of compatibility with 
the desired pest control composition (including pH, moisture content, and 
stability), particle size, abrasiveness, absorbability, density, 
wettability, and cost. The mixture of the composition of the invention and 
diluent is made by a variety of simple operations such as milling, solvent 
impregnations, fusing and grinding. Particle sizes usually range from 
0.5-4.0 microns in diameter. 
Wettable powders can be prepared by blending the mixture of the invention 
in high concentrations, usually from 15-95%, with a dust carrier such as 
bentonite which wets and suspends properly in water. 1 to 2% of a 
surface-active agent is usually added to improve the wetting and 
suspendibility of the powder. 
The pest-controlling composition can also be used in granules, which are 
pelleted mixtures of the composition, usually at 2.5-10%, and a dust 
carrier, e.g., adsorptive clay, bentonite or diatomaceous earth, and 
commonly within particle sizes of 250 to 590 microns. Granules can be 
prepared by impregnations of the carrier with a solution or slurry of the 
composition and can be used principally for mosquito larvae treatment or 
soil applications. 
The composition can also be applied in the form of an emulsion, which 
comprises a solution of the composition in water immiscible organic 
solvents, commonly at 15-50%, with a few percent of surface active agent 
to promote emulsification, wetting, and spreading. The choice of solvent 
is predicated upon solubility, safety to plants and animals, volatility, 
flammability, compatibility, odor and cost. The most commonly used 
solvents are kerosene, xylenes, and related petroleum factions, 
methylisobutylketone and amyl acetate. Water emulsion sprays from such 
emulsive concentrates can be used for plant protection and for household 
insect control. 
The composition can also be mixed with baits, usually comprising 1-5% of 
composition with a carrier especially attractive to insects. Carriers 
include sugar for house flies, protein hydrolysate for fruit flies, bran 
for grasshoppers, and honey, chocolate or peanut butter for ants. 
The composition can be included in slow release formulations which 
incorporate non-persistent compounds, insect growth regulators and sex 
pheromones in a variety of granular microencapsulated and hollow fiber 
preparations. 
The pest controlling compositions of the present invention will be applied 
depending on the properties of the particular pest controlling compound, 
the habits of the pest to be controlled and the site of the application to 
be made. It can be applied by spraying, dusting or fumigation. 
Doses of the combined weight of the two active ingredients may typically 
vary between 0.001-100 lbs/acre, preferably between 0.001-5 lbs/acre. 
Sprays are the most common means of application and generally will involve 
the use of water as the principal carrier, although volatile oils can also 
be used. The pest-control compositions of the invention can be used in 
dilute sprays (e.g., 0.001-10%) or in concentrate sprays in which the 
composition is contained at 10-98%, and the amount of carrier to be 
applied is quite reduced. The use of concentrate and ultra low volume 
sprays will allow the use of atomizing nozzles producing droplets of 30 to 
80 microns in diameter. Spraying can be carried out by airplane or 
helicopter. 
Aerosols can also be used to apply the pest controlling compositions. These 
are particularly preferred as space sprays for application to enclosures, 
particularly against flying insects. Aerosols are applied by liquified gas 
dispersion or bomb but can be generated on a larger scale by rotary 
atomizers or twin fluid atomizers. 
A simple means of pest control composition dispersal is by dusting. The 
pest controlling composition is applied by introducing a finely divided 
carrier with particles typically of 0.5-3 microns in diameter into a 
moving air stream. 
Any octopamine-receptor containing pest is treatable by the formulation of 
the present invention. These pests include all invertebrate pests, 
including, but not limited to, round worms (e.g., hookworm, trichina, 
ascaris); flatworms (e.g., liver flukes and tapeworms); jointed worms 
(e.g., leeches); molluscs (e.g., parasitic snails); and arthropods 
(insects, spiders, centipedes, millipedes, crustaceans (e.g., barnacles)). 
In particular, included among the arthropods are ticks; mites (both plant 
and animal); lepidoptera (butterflies and moths and their larvae); 
hemiptera (bugs); homoptera (aphids, scales); and coleoptera (beetles). 
Also included are spiders; anoplura (lice); diptera (flies and 
mosquitoes); trichoptera; orthoptera (e.g., roaches); odonta; thysanura 
(e.g., silverfish); collembola (e.g., fleas); dermaptera (earwigs); 
isoptera (termites); ephemerids (mayflies); plecoptera; mallophaga (biting 
lice); thysanoptera; siphonaptera (fleas); dictyoptera (roaches); 
psocoptera (e.g., book lice); and certain hymenoptera (e.g., those whose 
larva feed on leaves). 
EXAMPLES 
Having now generally described this invention, the same will become better 
understood by reference to certain specific examples which are included 
herein for purposes of illustration only and are not intended to be 
limiting unless otherwise specified. 
IN VITRO METHODS 
I. Determination of Octopamine Agonist Activity 
A. Firefly Light Organ 
Specimens of Photinus pyralis were collected in summer, frozen on dry ice, 
and stored at -90.degree.. Under these conditions, octopamine sensitive 
enzyme activity remains stable for six months or longer. For each 
experiment, a number of insects were thawed and maintained at 4.degree. C. 
Tail sections were opened through a dorsal midline incision and the 
abdominal cavity was cleaned out of all gut, fat reproductive organs, and 
ganglia. The light organs were then removed from the ventral cuticle, 
cleaned of any adhering nonlantern tissue, and homogenized (10 mg/ml) in 6 
mM Tris-maleate buffer (pH 7.4). To prepare a P.sub.2 fraction, the 
homogenate was diluted to a volume of 30 ml in 6 mM Tris-maleate and 
centrifuged at 120,000.times.g for 20 minutes. The supernatant was 
discarded, and the pellet was resuspended by homogenization in 30 ml of 
buffer and again centrifuged at 120,000.times.g for 20 minutes. The 
resulting pellet (P.sub.2 fraction) was resuspended in a volume of 6 mM 
Tris-maleate equivalent to the starting amount and maintained at 0.degree. 
until it was used. Alternatively, the homogenate may be used directly 
without preparing P.sub.2 fraction. 
Adenylate cyclase activation by test compounds was measured in test tubes 
containing (in 0.3 ml) 80 mM Tris-maleate, pH 7.4; 10 mM theophylline; 8 
mM MgCl.sub.2 ; 0.1 mM GTP; 0.5 mM ethylene glycol bis (beta-aminoethyl 
ether)-N,N,N'N'-tetraacetic acid; 2 mM ATP; 0.06 ml of P.sub.2 fraction; 
and the various compounds to be tested. Prior experiments had determined 
that, under these conditions, octopamine-sensitive adenylate cyclase 
activity is optimized. Test compounds were initially solubilized (prior to 
aqueous dilution) in water or (if soluble) in 50% (v/v) methanol. If 
insoluble in 50% methanol, compounds may be dissolved initially in 100% 
methanol, or 100% dimethylsulfoxide, or 100% polyethylene glycol. Final 
solvent concentration after dilution can be kept as high as 15% for 
methanol and DSMO and as high as 20% for polyethylene glycol. Appropriate 
solvent controls were run in parallel. The enzyme reaction (5 minutes at 
30.degree.) was initiated by addition of ATP, stopped by heating to 
90.degree. for 2 minutes, and then centrifuged at 1000.times.g for 15 
minutes to remove insoluble material. Cyclic AMP in the supernatant was 
measured by protein-binding assay, according to the method of Brown et 
al., Adv. Cyclic Nucleotide Res. 2:25-40 (1970). Under the above assay 
conditions, enzyme activity is linear with respect to time and enzyme 
concentration, and phosphodiesterase activity is nearly completely 
inhibited. Previous experiments had shown that the cyclic AMP produced in 
this reaction cochromatographs on Dowex AG-50X.RTM. with authentic cyclic 
AMP. Protein concentration was determined by the method of Lowry et al., 
Journal of Biological Chemistry 193:265-275 (1951). 
B. Other Pest Tissues 
In those cases in which other pest tissues are used to measure octopamine 
agonist activity, the procedure is identical to that above, except that 
the tissue to be used is the brain, segmental ganglia, or the entire nerve 
cord of the insect pest, with or without the brain. The tissue is 
homogenized (usually 15 mg/ml) as above in 6 mM Tris maleate, pH 7.4. 
Assay conditions are identical to those described above except that 
dopamine (10 micromolar) and serotonin (10 micromolar) are added to all 
(including control) assay tubes when testing compounds which may affect 
receptors other than octopamine receptors. This is done to cancel out the 
effects of dopamine and serotonin receptors which are usually present in 
nerve cord. It assures that the compound (B) tested is affecting only 
octopamine receptors (known to be present in all insect nerve cords). 
II. Determination of Adenylate Cyclase Stimulating Activity 
As detailed previously, the adenylate cyclase assay, either in the firefly 
or in other insect pest, is also used to identify a direct stimulator of 
adenylate cyclase. 
III. Determination of Cyclic AMP-Dependent Protein Kinase Activity (for 
Determination of Activity of Cyclic AMP Analolgues 
A P.sub.2 pellet is prepared as described above from either firefly lantern 
or insect pest nerve tissue. In this assay, the pellet is used both as a 
source of protein kinase and as the substrate which is phosphorylated. The 
assay mixture (total volume 0.2 ml) contains: 10 micromoles sodium 
glycerol phosphate buffer, pH 7.4; 1 millimicromole gamma-.sup.32 P-ATP, 
approx. 10.sup.6 cpm; 2 micromoles MgCl.sub.2 ; 2 micromoles NaF; 0.4 
micromoles theophylline; 0.06 micromoles EGTA; 10-100 micrograms protein 
of P.sub.2 pellet; .+-. various concentrations of cyclic AMP or the cyclic 
AMP analogue to be tested (typically to give a final concentration of from 
10.sup.-9 -10.sup.-4 M). 
The reaction is initiated by the addition of tissue and the incubation is 
for 5 minutes at 30.degree. C. The reaction is terminated by the addition 
of 4 ml of 7.5% trichloroacetic acid (TCA). 0.2 ml of 0.63% bovine serum 
albumin is added, the mixture is centrifuged at low speed, and the 
supernatant is discarded. The precipitate is dissolved in 0.1 ml of 1N 
NaOH and the TCA precipitation repeated 4 more times. The protein-bound 
.sup.32 P is then redissolved in NaOH and counted in a scintillation 
spectrometer. The amount of increase in cpm over control tubes incubated 
in the absence of cyclic AMP or cyclic AMP analogue represents 
cyclic-AMP-dependent protein kinase activity. Activity constants are 
calculated as described previously. 
IV. Determination of PDE Inhibitory Activity 
In this assay (modified slightly from Filburn and Karn (Analyt. Biochem. 
52: 505-516 (1973)), the rate of hydrolysis of labeled cyclic AMP to 
5'-AMP by PDE is measured by converting the breakdown product (5'-AMP) to 
adenosine, which can be separated and measured by alumina chromatography. 
Specifically, tissue from either the firefly lantern or pest is 
homogenized (10-20 mg/ml) in 6 mM Tris-maleate buffer, pH 7.4. The 
hydrolysis reaction is run in test tubes containing (in 100 microliters): 
80 mM Tris maleate, pH 7.4; 6 mM MgSO.sub.4 ; 10 pmoles tritiated cyclic 
AMP (2.times.10.sup.4 to 2.times.10.sup.5 cpm, depending upon the activity 
of the enzyme); 20 microliters of tissue homogenate; and various 
concentrations of the compound to be tested. In those cases in which the 
compound is insoluble in aqueous solution at pH 7.4, pH can be varied 
between pH 6.5 and 9.0 for optimization of solubility. In addition, the 
test compound can be initially solubilized in either 100% methanol or DMSO 
and diluted to a final concentration of 10% methanol or DMSO in the assay 
(in which case solvent controls are run in parallel). 
The reaction is started by the addition of homogenate, run for 4 min at 
37.degree. C., and terminated by boiling for 90 sec. The 5'AMP formed is 
then converted to adenosine by the addition of 20 microliters of an 
aqueous solution of 0.5 Units/ml of 5'-nucleotidase (Sigma, Grade IV), 
vortexed, and incubated for 30 minutes at 37.degree. C. The second 
reaction is stopped by addition of 0.4 ml of 0.1N ammonium acetate, pH 
4.0. The entire sample is then applied to a 0.5.times.8 cm column 
containing neutral alumina prewashed with 20 ml of 0.1N ammonium acetate, 
pH 4.0. The void volume is discarded and the column is then eluted with 2 
ml of 0.1N ammonium acetate, pH 4.0, the eluent collected and counted by 
liquid scintillation spectrometry. Activity is cpms above that due to a 
blank which was initially incubated for 0 seconds in the first reaction. 
IN VIVO METHODS 
To test the effects of PDE inhibition on the pesticidal and pestistatic 
effects of some of the disclosed compounds, the effects on the feeding 
behavior of tobacco hornworms (Manducca sexta) were investigated. This 
species is one of the several types of insects particularly susceptible to 
octopamine type insecticides. The ease of rearing this species from eggs 
in the laboratory and the ability to maintain them on artificial media, 
makes it possible to test compounds on large numbers of larvae of the same 
age. 
For testing, single tomato leaves were placed in a closed container, with 
stems hydrated by means of a small, 3 ml waterfilled bottle. Compounds, 
dissolved in water or methanol, were sprayed on the tomato leaves with an 
ultra fine atomizer. Six, 3-day-old larvae were placed on each leaf, 
allowed to feed for 24-108 hours, and then the percentage of leaf 
remaining was determined by planimetry, weight, or "blind" visual 
observation. An active compound or an active synergist was one which 
resulted in an increase in percentage of leaf remaining, compared with 
control. 
In some cases, test agents were tested for ovacidal activity by dipping 
groups of 10-50 Manducca eggs in drug solutions for 60 seconds and then 
determining the percentage of eggs which produced viable larvae. A 
compound or synergist with active ovacidal activity was one which 
decreased the percentage of eggs hatched, relative to control. 
EXAMPLE 1 
Phenylethylamines as Octopamine Agonists 
Table 1 shows the structure/activity relationships of phenylethylamines 
interacting with octopamine sensitive adenylate cyclase of the firefly 
(Photinus pyralis). 
TABLE 1 
__________________________________________________________________________ 
Structure-activity Relationships of Phenylethylamines 
Interacting with Octopamine-sensitive Adenylate Cyclase 
Compound 
##STR15## (% OCT)Vmax 
##STR16## 
__________________________________________________________________________ 
.beta.-phenylethylamine 
-- H H H.sub.2 
13 .+-. 2 
0.03 
(+)-amphetamine 
-- H CH.sub.3 
H.sub.2 
N.A. -- 
(.+-.)-phenylethanolamine 
-- OH H H.sub.2 
45 .+-. 1 
0.11 
(-hydroxyphenyl 
ethylamine 
(.+-.)-norephedrine 
-- OH CH.sub.3 
H.sub.2 
8 .+-. 1 
&lt;0.09 
(phenylpropanolamine) 
(.+-.)-o-octopamine 
2-OH OH H H.sub.2 
23 .+-. 1 
0.05 
(.+-.)-m-octopamine 
3-OH OH H H.sub.2 
9 .+-. 1 
0.1 
(norphenylephrine) 
(-)-phenylephrine 
3-OH OH H HCH.sub.3 
17 .+-. 1 
&lt;0.2 
tyramine 4-OH H H H.sub. 2 
57 .+-. 1 
0.15 
(.+-.)-p-hydroxyamphet- 
4-OH H CH.sub.3 
H.sub.2 
8 .+-. 3 
&lt;0.05 
amine 
(.+-.)-p-octopamine 
4-OH OH H H.sub.2 
81 .+-. 1 
0.06 
(.+-.)-p-octopamine 
4-OH OH H H.sub.2 
100 .+-. 1 
1.0 
(-)-p-octopamine 
4-OH OH H H.sub.2 
101 .+-. 2 
1.3 
(.+-.)-Nmethyloctopamine 
4-OH OH H HCH.sub.3 
108 .+-. 10 
1.8 
(synephrine) 
(.+-.)-.alpha.-methyloctopamine 
4-OH OH CH.sub.3 
H.sub.2 
44 .+-. 2 
0.05 
(p-hydroxynorephedrine) 
(.+-.)-N,Ndimethyl- 
4-OH OH H (CH.sub.3).sub.2 
43 .+-. 4 
0.9 
octopamine 
(.+-.)-.alpha.-methylsynephrine 
4-OH OH CH.sub.3 
HCH.sub.3 
7 .+-. 2 
&lt;0.01 
(p-hydroxyephedrine) 
p-hydroxymandelic 
4-OH OH OHO.sup.F 
-- N.A.* -- 
acid 
isoxsuprine 4-OH OH CH.sub.3 
1** N.A. -- 
p-methoxphenylethyl- 
4-OCH.sub.3 
H H H.sub.2 
6 .+-. 3 
0.10 
amine 
(p-methoxytyramine) 
p-fluoro-phenyl- 
4-F OH H.sub.2 
H.sub.2 
72 .+-. 8 
0.11 
ethanolamine 
2,4-dichlorophenyl- 
2-Cl,4-Cl OH H H.sub.2 
14 .+-. 1 
0.67 
ethanolamine 
m-chloro-octopamine 
3-Cl,4-OH OH H H.sub.2 
77 .+-. 1 
0.28 
dopamine 3-OH,4-OH H H H.sub.2 
4 .+-. 1 
0.26 
Nmethyldopamine 
3-OH,4-OH H H HCH.sub.3 
11 .+-. 1 
0.19 
(-)-nonrepinephrine 
3-OH,4-OH OH H H.sub.2 
72 .+-. 1 
0.13 
(-)-epinephrine 
3-OH,4-OH OH H HCH.sub.3 
75 .+-. 2 
0.20 
(-)-isoproterenol 
3-OH,4-OH OH H HCH(CH.sub.3).sub.2 
19 .+-. 1 
&lt;0.03 
(.+-.)-normetanephrine 
3-OCH.sub.3,4-OH 
OH H H.sub.2 
68 .+-. 2 
0.07 
salbutamol 3-CH.sub.2OH,4-OH 
OH H HC(CH.sub.3).sub.3 
N.A. -- 
zinterol 3-NHSO.sub.2 CH.sub.3,4-OH 
OH H 2** N.A. -- 
__________________________________________________________________________ 
*No Activity 
##STR17## 
- 
##STR18## 
- 
##STR19## 
- 
It can be seen that in most instances the half-maximal activation constant 
K.sub.a for the desired pest controlling compound ranges between less than 
0.01 of (.+-.)-p-octopamine to greater than (.+-.)-p-octopamine, whereas 
the Vmax ranges from less than 4 to 108% of Vmax of octopamine. Examples 
of compounds satisfying the criteria of most preferred octopamine agonists 
(K.sub.a.sup.oct /Ka.sup.B &gt;0.1; Vmax&gt;10%) are p-octopamine, 
N-methyl-octopamine, p-fluoro- phenylethanolamine, and 
2,4-dichlorophenylethanolamine. As will be shown later, the pesticidal 
activity of all four of these examples is synergized by combination with a 
compound having phosphodiesterase inhibitory activity. Note here that 
m-octopamine has much less activity than the active positional isomer, 
p-octopamine. As will be shown below, and confirming the in vitro/in vivo 
correlation, m-octopamine also has much less pesticidal activity than 
p-octopamine, and the activity shows much less synergism. 
EXAMPLE 2 
Clonidines as Octopamine Agonists 
A number of clonidine analogues were investigated as octopamine agonists in 
the octopamine receptor of the firefly lantern. Inhibitory properties were 
also tested. Table 2 shows the results of these experiments. 
TABLE 2 
__________________________________________________________________________ 
OCTOPAMINE RECEPTOR ACTIVITY OF THE CLONIDINES 
##STR20## (% OCT)Vmax 
##STR21## 
(.mu.m).sup.K.sbsp.i 
__________________________________________________________________________ 
NC 10 
-- N.A. N.A. 190 
NC 12 
4-Br 34 3.0 -- 
NC 8 2-Cl, 4-Cl 47 3.7 -- 
NC 7 2-CH.sub.3, 4-Cl 68 9.8 -- 
NC 9 2-CH.sub.3, 4-CH.sub.3 
78 5.1 -- 
NC 2 2-Cl, 5-Cl 9 1.1 23 
NC 6 2-Br, 6-Br 35 2.1 -- 
clonidine 
2-Cl, 6-Cl 35 0.95 20 
NC 4 2-CH.sub.3, 6-CH.sub.3 
80 0.41 -- 
NC 5 2-CH.sub.2 CH.sub.3, 6-CH.sub.2 CH.sub.3 
97 19.0 -- 
NC 3 2-Cl, 4-Cl, 5-Cl 80 2.8 -- 
NC 11 
2-Cl, 4-Cl, 6-Cl 60 1.9 -- 
NC 13 
2-CH.sub.3, 4-CH.sub.3, 6-CH.sub.3 
112 4.3 -- 
NC 14 
2-Cl, 4NH.sub.2, 6-Cl 
29 0.7 440 
NC 15 
2-Cl, 4-N(CH.sub.3).sub.2, 6-Cl 
38 1.9 -- 
NC 16 
2-Cl, 4-NCH.sub.3 (CH.sub.2 CH.sub.2 Cl), 6-Cl 
6 4.8 10 
NC 17 
2-Cl, 4-CH.sub.2 NCH.sub.3 (CH.sub.2 CH.sub.2 Cl), 
N.A. N.A. 250 
__________________________________________________________________________ 
Among the findings resulting from this data is the fact that several 
clonidine derivatives (some having low mammalian potency) are extremely 
potent octopamine agonists. For example, NC 5 is almost 20 times more 
potent than octopamine and has an equivalent Vmax. This makes this 
compound the most potent octopamine agonist yet discovered. Several other 
compounds are also more potent than octopamine. From the compounds shown 
in Table 2, two examples (NC5 and NC7) which satisfy the criteria as most 
preferred octopamine agonists will be shown below to be pesticides, and to 
have their pesticidal activity markedly synergized by phosphodiesterase 
inhibition. 
EXAMPLE 3 
Formamidines as Octopamine Agonists 
FIG. 1 shows the agonist activity of three formamidine compounds using the 
firefly lantern octopamine receptor. This Figure indicates that 
mono-demethylchlordimeform (DCDM) (Ka ratio=6; V.sub.max =76%) and 
di-demethylchlordimeform (DDCDM) (Ka ratio=4; V.sub.max =68%) have greater 
potency than octopamine. Of interest, as will be shown later, is the fact 
that the relative potency of mono demethylchlordimeform (DCDM) and 
didemethylchlordimeform (DDCDM) as per octopamine agonist activity 
parallel their pesticidal activity in vivo. Thus, against the octopamine 
receptor, DCDM is 50% more potent than DDCDM; similarly, as will be shown 
below, DCDM is more potent in inhibiting feeding behavior of tobacco 
hornworms. Also shown in FIG. 1 is the fact that chlordimeform (CDM), 
which is converted in insects to DCDM, has a Ka ratio of 0.5 and a 
V.sub.max of about 10%. As will be shown later, PDE inhibition markedly 
increases the pesticidal activity of all three formamidines. 
FIG. 2 shows the effect of three other formamidines on octopamine-activated 
adenylate cyclase. 
EXAMPLE 4 
Demonstration of Octopamine Agonist Activity Measured in Tissue from an 
Insect Pest 
In order to show that adenylate cyclase activation in tissue from an insect 
pest can also be used to define octopamine agonists, members of three 
chemical groups (phenylethanolamines, phenyliminoimidazolidines, and 
formamidines) were tested for their ability to activate adenylate cyclase 
in broken cell preparations of insect nerve cord. FIG. 3 shows that 
octopamine, NC7 (see Table 2), and DDCDM were potent activators of 
adenylate cyclase in nerve cord of tobacco hornworm. All three of these 
compounds fulfilled the criteria of being most preferred octopamine 
agonists. As will be described below, all of these compounds have 
pesticidal activity, and their antifeeding effects are greatly enhanced by 
PDE inhibition. 
EXAMPLE 5 
A Direct Stimulator of Adenylate Cyclase in the Firefly Lantern and in Pest 
Tissue 
FIG. 4 shows the effect of forskolin in directly activating adenylate 
cyclase in firefly lantern. FIG. 5 also shows the effect of forskolin in 
directly activating adenylate cyclase activity in broken cell perparations 
from the nerve cord of tobacco hornworm larvae. In both tissues, forskolin 
fulfills the criteria of a preferred enzyme activator. As will be shown 
below, forskolin has activity as a pesticide and this activity is 
synergized by phosphodiesterase inhibition. 
EXAMPLE 6 
Comparison of PDE Inhibitors on the Phosphodiesterase Enzyme from the 
Firefly Lantern 
As described above, compounds (A) useful as synergists are those which can 
inhibit phosphodiesterase enzyme activity with a V.sub.max -inhibition 
more than 50% and an IC.sub.50 -inhibition of less than 10 mM, preferably 
less than 2.5 mM. FIG. 6 shows the effects of three methylxanthines, IBMX, 
theophylline, and caffeine, active as phosphodiesterase inhibitors against 
the Firefly phosphodiesterase enzyme. Although all three compounds fulfill 
the criteria as active synergists, the results show that the IC.sub.50 for 
IBMX is less than those for the other two compounds, indicating that IBMX 
is a more potent inhibitor of the enzyme. 
As will be shown below, all three compounds are able to synergize the 
pesticidal activity of Group B compounds. 
To further demonstrate the generality of the findings as related to PDE 
inhibition, another compound was defined which was structurally unrelated 
to the methylxanthines but which was an effective phosphodiesterase 
inhibitor. FIG. 7 shows that puromycin inhibited PDE activity with an 
IC.sub.50 -inhibition of less than 2 mM and V.sub.max greater than 70%. As 
will be shown below, puromycin is also able to act as a pesticide 
synergist. 
EXAMPLE 7 
Comparison of PDE Inhibitors on the Phosphodiesterase Enzyme from Insect 
Pest Tissue 
To confirm the use of the firefly light organ as a general tissue by which 
to define useful compounds, other experiments were run using nerve tissue 
from an insect pest. FIG. 8 shows the effect of IBMX and theophylline on 
PDE activity in a broken cell preparation from tobacco hornworm larvae. As 
can be seen, the pattern of inhibition in the hornworm is quite similar to 
that in the firefly light organ. As will be shown below, when tested 
against living tobacco hornworm larvae, both IBMX and theophylline were 
able to synergize the pesticidal activity of Group B compounds. 
To further confirm that phosphodiesterase inhibitory activity is the 
criterion for determining the synergistic potential of a compound, a 
weakly active methylxanthine analogue was studied. FIG. 8 shows that 
8-phenyl-theophylline failed to inhibit phosphodiesterase activity by more 
than 30%, and therefore fell outside of the criteria defined above for a 
PDE inhibitor. As will be described below, 8-phenyl-theophylline had very 
little activity as a pesticide synergist. 
EXAMPLE 8 
Demonstration of Increased Cyclic AMP Content in Pest Nerve Cord Caused by 
a Combination of a Phosphodiesterase Inhibitor and an Octopamine Agonist 
As described earlier and as shown in Scheme I, the pesticidal activity of 
octopamine agonists is mediated through the increased formation of cyclic 
AMP within the cells of the insect pest. In order to directly demonstrate 
that a PDE inhibitor, as defined above, can augment the increase in cyclic 
AMP caused by an octopamine agonist (as defined above) in an insect pest, 
further experiments were run using intact nerve cords of tobacco 
hornworms. These intact nerve cords were incubated under physiological 
conditions in the presence of octopamine, NC7, or DDCDM, first in the 
absence of a PDE inhibitor and then in the presence of 0.1 mM IBMX. After 
5 minutes, the intact nerve cord was quickly treated to release all cyclic 
AMP which was present within the tissue. 
Table 3 compares the levels of cyclic AMP within nerve tissue under 
different treatments. As can be seen, with all three octopamine agonists 
(octopamine, NC7, and DDCDM), addition of IBMX markedly increased the 
level of cyclic AMP within the nerve tissue. 
TABLE 3 
______________________________________ 
Effect of PDE Inhibition of Increasing Cyclic AMP 
in Insect Pest Tissue 
Cyclic AMP Content 
(Fold-increase over Control) 
Compound -IBMX +0.1 mM IBMX 
______________________________________ 
Octopamine 0.9 .+-. 0.3 
6.4 .+-. 1.0 
NC 7 2.2 .+-. 0.2 
13.9 .+-. 3.0 
DDCDM 5.4 .+-. 3.8 
11.1 .+-. 2.7 
______________________________________ 
Also of considerable interest was the fact that, consistent with the 
invention, the level of cyclic AMP within the tissue paralleled pesticidal 
activity. For example, in the absence of the synergist, octopamine caused 
little elevation of cyclic AMP, and likewise had little pesticidal 
activity (see FIGS. 9, 10 below). In the presence of IBMX, however, 
octopamine considerably elevated both cyclic AMP content and pesticidal 
activity. 
EXAMPLE 9 
In Vivo Tests of Compositions Containing A PDE Inhibitor and Octopamines 
This and the following series of examples demonstrate, in vivo, the effects 
of PDE inhibitors on increasing pesticidal (pestistatic) or ovacidal 
activity of compounds of Type B (see Scheme I). FIG. 9 shows the percent 
leaf remaining at 72 hours following spraying of a series of matched 
tomato leaves with various concentrations of (.+-.)-p-octopamine or 
(.+-.)-p-octopamine in the presence of 0.1% IBMX. Comparison of the 
results shows that the addition of IBMX to (.+-.)-p-octopamine greatly 
increases the percent leaf remaining, thus acting as a synergist. 
Of interest, and further confirming the invention are the results shown in 
FIG. 10. Here (.+-.)-m-octopamine, an octopamine analogue which falls 
short of the in vitro criteria of a most preferred octopamine agonist, was 
tested in the absence and presence of IBMX. Although IBMX had a very small 
inhibitory effect by itself, combination with m-octopamine failed to 
increase pesticidal activity. 
EXAMPLE 10 
In Vivo Tests of Compositions Containing a PDE Inhibitor and Formamidines 
A. Leaf Test 
Table 4 below shows the results obtained upon testing tomato leaves in the 
presence of CDM, DCDM, and DDCDM, with or without the phosphodiesterase 
inhibitor IBMX. The data indicates clearly that addition of IBMX at 0.1 
g/100 ml to the mixture markedly synergized the ability of each of the 
formamidines to inhibit consumption of the leaf. 
For clarity of demonstration of this effect, FIG. 11 shows 4 leaves 72 
hours after treatment with either vehicle alone (upper left); 0.1% DDCDM 
(upper right); 0.1% IBMX (lower left); or a combination of 0.1% DDCDM and 
0.1% IBMX (lower right). As can be seen, the tobacco hornworms have almost 
entirely consumed the leaves treated with vehicle or IBMX and have eaten 
substantial amounts of the leaf treated with DDCDM. The leaf treated with 
the combination of IBMX and DDCDM, however, is virtually untouched. 
TABLE 4 
______________________________________ 
% Leaf Remaining 
Agonist No PDE PDE Inhibitor 
(gm/100 ml) Inhibitor (0.1 g/100 ml) 
______________________________________ 
CDM C* &lt;5 IBMX 15 
10.sup.-4 
&lt;5 50 
10.sup.-3 
&lt;5 65 
10.sup.-2 
&lt;5 85 
10.sup.-1 
70 90 
1 90 90 
DCDM C* &lt;5 IBMX 20 
10.sup.-4 
&lt;5 75 
10.sup.-3 
45 90 
10.sup.-2 
95 98 
DDCDM C* 5 IBMX 22 
10.sup.-3 
5 20 
10.sup.-2 
7 85 
10.sup.-1 
30 98 
1 100 100 
______________________________________ 
*Control = no Agonist 
B. Ovacidal Properties 
Table 5 shows the ovacidal properties of DCDM and CDM in the presence and 
absence of IBMX as a phosphodiesterase inhibitor. 
TABLE 5 
______________________________________ 
% Living Larvae 
Agonist No PDE PDE Inhibitor 
(gm/100 ml) Inhibitor (0.1 g/100 ml) 
______________________________________ 
DCDM C* 80 IBMX 90 
10.sup.-2 
50 10 
10.sup.-1 
50 3 
1 2 2 
CDM C* 80 IBMX 90 
10.sup.-2 
60 10 
10.sup.-1 
25 10 
1 2 2 
______________________________________ 
*Control = no agonist 
The results indicate that the addition of IBMX produced a substantial 
decrease in % living larvae and, therefore, an increase in ovacidal 
activity. This was particularly apparent at lower concentrations of the 
agonist; in other words, the PDE inhibitor shifted the dose-response curve 
to the left and increased the potency of the agonist as an ovacidal agent. 
C. Dose-Dependent Effects of Phosphodiesterase Inhibitors 
The synergistic effects of PDE inhibitors are apparent at certain, but not 
all, concentrations of the PDE inhibitor. If the concentration is too low 
then no substantial synergism occurs. At intermediate concentrations, 
synergism will occur. At higher concentrations the PDE inhibitor, itself, 
will inhibit insect feeding. In other words, the PDE inhibitor, at certain 
concentrations, has pest-controlling activity. This is due to the fact 
that, even in the absence of an octopamine agonist, the adenyltte cyclase 
enzyme in the animal slowly produces cyclic AMP which, normally, is easily 
broken down by the PDE present in the tissue. However, if the PDE is 
inhibited to a great enough degree, this cyclic AMP will accumulate and 
act to disrupt the insect's feeding. For example, in the absence of a PDE 
inhibitor, the cyclic AMP content of hornworm nerve cord incubated for 5 
minutes in vitro was found to be 8.9.+-.1.6 pmoles/mg. In the presence of 
100 micromoles/liter of IBMX, cyclic AMP content increased to 21.4.+-.0.8 
pmoles/mg. 
FIG. 12 shows the dose-dependent effect of IBMX on hornworm feeding in the 
absence or presence of a fixed concentration (0.1%) of DDCDM. As can be 
seen, at low concentrations (less than 0.001 gm/100 ml) IBMX had no effect 
by itself and did not increase the activity of DDCDM. At intermediate 
concentrations (0.01 to 0.3 gm/100 ml), IBMX acted as a synergist of 
DDCDM. At concentrations of 0.4-5 gm/b 100 ml, IBMX itself acted to 
inhibit feeding; i.e., it had the properties of a primary pesticide. 
Further data demonstrating that PDE inhibition is the essential property of 
a compound predicating anti-feeding activity is shown in FIG. 13. This 
graph shows the relationship, for three compounds, between the dose 
sprayed on tomato leaves and the ability to inhibit feeding of tobacco 
hornworm larvae. As can be seen, IBMX is more potent than theophylline, 
and 8-phenyl-theophylline shows the least activity, being unable to 
inhibit feeding more than 35%, even at a high dose. The relationship 
between the antifeeding activities of these three compounds is remarkably 
similar to their relative PDE inhibitory abilities shown in FIG. 8. 
The optimal dose of PDE inhibitor as a primary pesticide can be derived 
from graphs such as FIG. 13. The optimal synergistic concentration of a 
PDE inhibitor can be derived from graphs such as shown in FIG. 12. In 
general, optimal synergistic dose will vary, depending upon various 
factors such as the concentration of primary agonist, the method of 
application, and the species of pest treated. 
D. Other PDE Inhibitors 
The in vivo leaf tests were also carried out for DDCDM alone, caffeine 
alone, and mixtures of caffeine plus DDCDM. Results are shown in FIG. 14, 
which depicts results in a different format from dose-response graphs. 
FIG. 14 is a time-course experiment showing the progressive eating and 
diminution of leaf size caused by larvae on leaves treated as shown. 
The results indicate that mixtures of caffeine with DDCDM are much more 
powerful in inhibiting leaf feeding than caffeine alone or DDCDM alone. 
This effect becomes more apparent as time progresses. 
Experiments were also carried out with mixtures of theophylline and DDCDM. 
These results are indicated in Table 6 below: 
TABLE 6 
______________________________________ 
% Leaf Remaining 
(increase over 
Agonist control) 
______________________________________ 
Control 0 
DDCDM 0.1 g/100 ml 0 
1% Theophylline 11 
1% Theophylline + DDCDM 
61 
0.1 gm/100 ml 
______________________________________ 
The results indicate that, whereas DDCDM alone or theophylline alone allow 
only a small amount of leaf to remain (0-11% more than control), the 
mixture of both allows much more of the leaf to remain. 
8-Phenyl-theophylline, a compound structurally related to methylxanthine 
was tested next. FIG. 13 shows that 8-phenyl-theophylline, by itself, had 
little antifeeding activity. As shown in FIG. 15, results of in vivo 
testing revealed that, compared with 0.1% IBMX, 0.1% 8-phenyl-theophylline 
had much less activity as a synergist of 0.1% DDCDM. These results would 
have been unexpected on the basis of chemical structure alone, but were 
predicted by the in vitro PDE assay procedure, as shown in FIG. 8, which 
indicates that 8-phenyl-theophylline has little activity as a PDE 
inhibitor. These results with 8-phenyl-theophylline further confirm the in 
vitro/in vivo correlation of the invention. 
Experiments were also carried out with the structurally-unrelated PDE 
inhibitor, puromycin (see FIG. 7), in mixtures with the primary pest 
compound, DDCDM. These results, which are shown in FIG. 16, demonstrate 
that puromycin was able to synergize the pesticidal activity of DDCDM. 
This result further confirms the generality of the findings with PDE 
inhibition. 
EXAMPLE 11 
In Vivo Leaf Test of Compositions Containing a PDE Inhibitor and 
Phenylethanolamines 
Table 7 shows the results obtained for mixtures comprising various 
phenylethanolamines in the presence or absence of IBMX as a PDE inhibitor. 
TABLE 7 
______________________________________ 
% Leaf Remaining 
Agonist No PDE PDE Inhibitor 
(gm/100 ml) Inhibitor (0.1 g/100 ml) 
______________________________________ 
4-F--phenylethanol- 
C* &lt;5 IBMX 25 
amine 
10.sup.-3 
&lt;5 50 
10.sup.-2 
&lt;5 80 
10.sup.-1 
&lt;5 98 
0.3 25 95 
2,4-dichlorophenyl- 
C* &lt;5 IBMX 20 
ethanolamine 
10.sup.-2 
&lt;5 30 
10.sup.-1 
&lt;5 50 
1 10 80 
Synephrine C* &lt;5 IBMX 42 
10.sup.-2 
&lt;5 42 
10.sup.-1 
&lt;5 55 
1 30 90 
______________________________________ 
*Control = No Agonist 
The results show that in all instances, addition of IBMX markedly enhanced 
the ability of the phenylethanolamines to inhibit the leaf feeding 
activity of the pest. 
EXAMPLE 12 
In Vivo Leaf Tests of Compositions Containing a PDE Inhibitor and 
Phenyliminoimidazolidines (Clonidines). 
Mixtures of various clondine derivatives with IMBX were prepared and tested 
in the aforementioned in vivo tests. Table 8 shows the results. 
TABLE 8 
______________________________________ 
% Leaf Remaining 
Agonist No PDE PDE Inhibitor 
(gm/100 ml) Inhibitor 
(0.1 g/100 ml) 
______________________________________ 
NC 7.sup.(1) 
C* 25 IBMX 30 
10.sup.-3 23 48 
10.sup.-2 30 85 
10.sup.-1 55 95 
3 .times. 10.sup.-1 
80 100 
clonidine C* 5 IBMX 35 
10.sup.-2 5 55 
10.sup.-1 20 55 
1 65 63 
NC 5.sup.(1) 
C* 10 IBMX 18 
10.sup.-3 10 17 
10.sup.-2 15 30 
10.sup.-1 30 45 
3 .times. 10.sup.-1 
40 70 
NC 10.sup.(1) 
C* 10 IBMX 15 
10.sup.-3 5 12 
10.sup.-2 5 15 
10.sup.-1 5 13 
1 5 15 
______________________________________ 
.sup.(1) See Table 2, supra 
*Control = No agonist 
The data indicate that, with NC7, clonidine, and NC5, addition of IBMX 
results in substantial increases in the potency of the pest controlling 
compound. It will be noted that these three pest controlling compounds, on 
the basis of in vitro testing (Table 2), all satisfied the criteria of 
being most preferred compounds of type B. Of interest, and further 
confirming the invention, was the fact that the structural analogue, NC10, 
was inactive in vivo and was not synergized by the PDE inhibitor. Thus, as 
can be seen in Table 8, the addition of various concentrations of NC 10 to 
the PDE inhibitor, IBMX, did not increase antifeeding activity over that 
of the PDE inhibitor alone. This unexpected result was correctly predicted 
by the in vitro assay data shown in Table 2, where NC10 had no activity. 
EXAMPLE 13 
In Vivo Leaf Test of a Composition Containing a PDE Inhibitor and a Direct 
Enzyme Stimulator 
As described previously, the diterpene forskolin is an example of a 
compound which directly stimulates adenylate cyclase, thereby producing 
cyclic AMP. This example shows that inhibition of PDE enhances the 
antifeeding activity of forskolin on tobacco hornworm larvae. FIG. 17 
shows that forskolin, alone, has antifeeding activity. FIG. 17 also shows 
that this antifeeding activity is enhanced in the presence of (0.1%) of 
the PDE inhibitor IBMX. 
EXAMPLE 14 
In Vivo Leaf Test of a Composition Containing a PDE Inhibitor and a cAMP 
Analogue 
Mixtures containing IBMX 0.1% and 2 cyclic AMP analogues were tested by the 
leaf test. The results are shown in FIG. 18 for p-Cl-phenylthio cyclic 
AMP, and FIG. 19 for n-butylaminobenzylthio cyclic AMP. As can be seen, 
both compounds, at a concentration of 1%, also showed some antifeeding 
activity in the absence of IBMX. 
It has been previously shown, above, that PDE inhibition can increase the 
concentration of cyclic AMP in insect tissue. To further confirm the 
invention this example demonstrates directly that PDE inhibition can also 
increase the levels of cyclic AMP analogues, such as butyl-benzylthio 
cyclic AMP, in insect pest tissue. In order to do this, a fixed amount of 
butyl-benzylthio cyclic AMP was incubated in vitro for 4 hours in the 
presence of a tissue homogenate from tobacco hornworm nerve cord. In some 
tubes, IBMX (0.1 mM) was added to inhibit PDE. After the incubation, the 
amount of butyl-benzylthio cyclic AMP remaining was measured by protein 
binding assay (Brown et al., supra), using a standard curve based upon 
butyl-benzylthio cyclic AMP. FIG. 20 shows that addition of IBMX more than 
doubled the amount of butyl-benzylthio cyclic AMP present after 4 hours. 
EXAMPLE 15 
Determination of Levels of PDE Inhibitor Compounds in Insects In Vivo 
In order to supply further evidence that PDE compounds of type A were 
exerting their effects through inhibition of PDE, it was determined that 
these compounds, when applied to the tomato leaves, a) were able to be 
absorbed by the tobacco hornworms, and b) that the concentration of 
compound within the insect corresponded approximately to the concentration 
necessary to cause substantial inhibition of PDE activity in vitro. 
To determine this, tobacco hornworms were allowed to feed on tomato leaves 
treated with a 1% spray of theophylline. This concentration was chosen 
since it is an amount which, by itself, inhibits feeding in the tobacco 
hornworm (see FIG. 13). After 72 hours, worms (alive or dead) were 
removed, quickly rinsed of any compound adhering to their outside cuticle, 
and then homogenized. The homogenate was centrifuged at 2000.times.g for 5 
minutes and the supernatant was assayed for theophylline concentration by 
a standard immunoenzymatic procedure (Emit-aad.RTM. Theophylline Assay, 
Syva Company, Palo Alto, Calif.). By this procedure, the internal 
concentration within the insects was determined to be 4.0 mM. From FIGS. 
6,7 or 8 it will be seen that this concentration of theophylline is 
sufficient to cause at least 70% inhibition of PDE enzyme activity in 
vitro. The correspondence between in vitro and in vivo results is 
remarkably close and serves to further support the invention. 
Having now fully described this invention it will be understood by those of 
skill in the art that the same can be performed within a wide and 
equivalent range of compositions, parameters, structures, modes of 
application, pests, formulations, and ranges without effecting the scope 
of the invention or any embodiment thereof.