Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest Control Downloaded from pubs.acs.org by COLUMBIA UNIV on 12/05/18. For personal use only.
Chapter 8
A Review of Aminothiazoline Chemistry Barbara Wedel,*,1 Wolfgang von Deyn,2 Sebastian Soergel,2 Matthias Pohlman,1 Douglas Anspaugh,1 Ramani Kandasamy,1 Fae Malone,1 Daniel Houtz,1 Nancy Rankl,1 John Dorsch,1 Lynn Stam,1 Brecht London,1 Ronan le Vezouet,2 Christopher Koradin,2 and Markus Kordes2 1BASF
Corp., 26 Davis Drive, Research Triangle Park, North Carolina 27709-3528, United States 2BASF SE, B009, Ludwigshafen, Delaware 67056, United States *E-mail:
[email protected].
Aminothiazoline (ATZ) chemistry was developed starting from a random in vivo screening hit with moderate activity in aphids. A series of compounds was synthesized to increase potency and breadth of spectrum towards other piercing sucking insects. ATZs showed no cross-resistance to chloronicotinyl insecticides (CNIs) and pyrethroids and controlled multi-resistant whitefly species. The main symptoms in aphids were wandering behavior along with fast feeding cessation, the speed of control was slow. As to the mode of action, based on structural similarity of the original screening hit with a known octopaminergic agonist we closer examined the possibility of ATZs signaling through insect octopamine receptors. Although in vitro activity in the micromolar range could be observed with ATZs on some octopamine receptors, there was no clear in vivo-in vitro correlation suggesting a possibly new mode of action for this chemistry. Initial Tox- and Ecotox profiles were very positive, but late stage toxicity testing raised concern that together with a lack of systemicity and limited spectrum prompted us to stop further activity on ATZs.
© 2017 American Chemical Society
Discovery and Overview of Aminothiazolines In this book chapter aminothiazoline chemistry is reviewed, which provides excellent control of piercing-sucking insects. The initial discovery, structure-activity relationship, mode of action work and biological spectrum will be covered in the following sections. The chemistry was developed from a random screening hit, the N-(1,2-diphenylethyl)aminooxazoline derivatives shown in Figure 1, which showed moderate activity on cotton aphid (Aphis gossypii) and green peach aphid (Myzus persicae). Chemical variation of the scaffold resulted in increased activity on aphids and expansion of the spectrum to whiteflies. Further structure optimization led to the lead analog shown on the right in Figure 1, which showed excellent activity on aphids and whiteflies and further expansion of the spectrum to thrips and hoppers. Using a scaffold hopping approach, Lambert et al. recently reported insecticidal activity for structurally related thioureas and isothioureas, that controlled Myzus persicae and Bemisia tabaci in laboratory bioassays (1).
Figure 1. Development of aminothiazoline chemistry from the original Aminooxazoline screening hit into a screening hit cluster with heteroatom (X), prodrug group (PG) and R-group variation on both phenyl rings (R1-R6). The lead Aminothoiazoline analog with an extended pest spectrum is shown on the right side.
The mode of action appears to be novel and will be discussed in more detail below. The route of exposure was mainly contact with limited oral activity. While the speed of aphid control was slow, feeding cessation was fast, which is an important factor in virus transmission. No cross-resistance to conventional insecticide classes such as CNIs and pyrethroids was observed. Translaminar plant movement could be seen for several aminothiazoline analogs, but no appreciable systemicity could be detected. 140
The regulatory profile of the lead aminothiazoline analog was very favorable: acute toxicity in rats was low (LD50 > 500 mg/kg) with no observed eye or skin irritation and a negative Ames test (non-mutagenic). With regards to ecotoxicology, there were no major concerns for aquatic organisms (acute fish, Daphnia and Chironomus) and low acute toxicity for wildlife (quail) and bees. Environmental fate studies showed a high organic carbon-water partition co-efficient and therefore no leaching risk. Soil degradation was moderate to slow and the compound was rapidly degraded in air.
Figure 2. Summary of structure-activity relationship of aminothiazoline chemistry. Heterocycle substitutions (1.) including prodrug groups (PG), A-ring (2.) and B-ring substitutions (3.) providing best in vivo activity are depicted.
Aminothiazoline Chemistry Key structure-in vivo activity relationships are summarized in Figure 2. 5-ring heterocycles showed best activity with thiazolines showing higher activity than oxazolines or imidazolines (Figure 2). While heterocyclic substitutions on the B-ring were tolerated, but showed weakened activity, heterocyclic A-ring substitutions generally lost biological activity with the exception of thiophene. Figure 3 shows the preferred synthesis route for aminothiazolines: Commercially available 2,3-dimethylbenzaldehyde was treated with lithium bis(trimethylsilyl)amide to form a silyl-imine. A Grignard reagent – prepared from the respective chloride – was added afterwards yielding the diphenylethylamine. With the addition of thiophosgene an isothiocyanate was produced in very good yields. Ethanolamine was added to form a thiourea compound which was subsequently dehydrated under various conditions to form the lead aminothiazoline. 141
Figure 3. Synthesis route for the lead aminothiazoline analog. The synthesis route was very robust and reproducible from small mg to kg amount scale.
Aminothiazoline Mode of Action Octopamine is an essential neurotransmitter, neuromodulator and neurohormone in the insect nervous sytem involved in coordination of locomotor behavior, modulation of sensory responses, learning and memory as reviewed elsewhere (2, 3). Octopamine and tyramine receptors are interesting targets for the development of Insecticides and octopamine receptor agonists such as amitraz (AM) and chlordimeform (CDM) have long been know to have insecticidal/acaricidal properties (4, 5). Both formamidines are thought to be pro-insecticides, which are metabolized within the insect to the active metabolites BTS27271 and demethylchlordimeform (DCDM), respectively (Figure 4). Searching for new classes of octopamine agonists, Jennings et al. discovered that some 2-aminooxazolines identified in an in vitro approach were aphicidal and acaricidal, and exhibited symptomology characteristic for octopaminergic agonists (6). The phenylaminooxazoline AC-6, one of the most potent compounds in the series, showed structural similarity to our initial screening hit, which had also been an aminooxazoline (Figure 4). This prompted us to investigate octopamine receptor activation as a putative mode of action for our aminothiazoline chemistry. We decided to develop a cell line stably expressing the Drosophila melanogaster octopamine receptor Oa2 (Dm-Oa2), which is also known in literature as OctR or Octβ1R and corresponds to CG6919. Insect neurohormone GPCRs and octopamine receptors in particular have been reviewed elsewhere (7, 8). A classification system based on structural similarities and signaling properties with vertebrate adrenergic receptors was proposed in 2005 by Evans 142
and Maqueira (9). They suggested to distinguish between α-adrenergic-like octopamine receptors (OctαRs), β-adrenergic-like octopamine receptors (OctβRs) and tyraminergic receptors. Based on literature evidence, Dm-Oa2 activation causes elevation of cAMP levels when expressed in HEK293 cells (10). We stably expressed Dm-Oa2 in a CHO-Gα16 background thus forcing the coupling of the GPCR to increases in cytosolic calcium levels via stimulation of PLC-β as shown in Figure 5.
Figure 4. Structures of the initial in vivo screening hit, the octopaminergic agonist AC-6 described in(6), amitraz and its active metabolite BTS27271 and chlordimeform and its active metabolite DCDM. BTS27271 is also known in literature as DPMF (N-(2,4-dimethylphenyl)-N‘-methylformamidine).
We pharmacologically characterized the Dm-Oa2/CHO-Gα16 cell line as summarized in Table 1, using the parental CHO-Gα16 line as a control. The natural ligand octopamine activated Dm-Oa2 with an EC50 of 13.6 nM. Agonists and antagonists of mammalian GPCRs described in literature to affect insect octopamine receptors were also tested. Mianserin and cyproheptadine acted as antagonists of Oa2 with an IC50 of 31.2 nM and 50.8 nM, respectively. Naphazoline, tolazoline and clonidine had agonist activity on Dm-Oa2 with a rank order of potency of naphazoline (5.7 nM) > clonidine (75.1 nM) > tolazoline (217.5 nM). As to be expected, the pro-insecticide forms of the two formamidines amitraz (EC50 = 176 nM) and CDM (EC50 = 13600 nM) were less potent on Dm-Oa2 than the respective active metabolites BTS27271 and DCDM (EC50 of 12 nM and 16 nM, respectively; see Table 1). 143
Figure 5. Dm-Oa2 was stably expressed in a CHO-Ga16 background linking GPCR activation to increases in intracellular Ca2+ levels via activation of PLC and activation of the ER-residing IP3 receptor. The increased calcium release can be measured with fluorescent calcium indicator dyes such as Fluo-4.
As shown in Table 1, the lead ATZ analog did not exhibit high potency on Dm-Oa2 with an EC50 only in the micromolar range. We also tested the lead ATZ in antagonist mode challenging the receptor with 100nM Octopamine, but could not observe any inhibitory effect. We were wondering whether species differences might be responsible for the lack of activity of the lead on Dm-Oa2 and therefore also cloned and stably expressed the corresponding aphid receptor Mp-Oa2 (Myzus persicae) in the same CHO-Gα16 background as the Drosophila receptor Dm-Oa2. However, in both cell lines the lead ATZ analog was 850-fold (Dm-Oa2) and 80fold less potent (Mp-Oa2) than the active form of amitraz, BTS27271. We tested an entire series of ATZ derivatives on the two Oa2-receptor expressing cell lines, which exhibited a range of potencies, but no clear in vitro - in vivo correlation was observed (data not shown). 144
Table 1. Pharmacological Characterization of the Dm-Oa2/CHO-Gα16 Cell Line. Agonist or Antagonist Activity of Modulators of Mammalian GPCRs, Formamidines, Octopamine and the Lead ATZ Compound Were Determined.
We next decided to establish an octopamine receptor panel. We included the β-adrenergic-like octopamine receptors (Dm-Oa2, Mp-Oa2, Dm-Octβ2R Tc-Octβ3R, the α-adrenergic-like octopamine receptor Dm-oamb, and the tyraminergic receptor Dm-Tyr, also referred to as Oct-TyrR1, a Type 1 receptor (Table 2). The receptors were cloned from Drosophila melanogaster (Dm), Myzus persicae (Mp) or Tribolium confusum (Tc) as indicated and stably expressed in a CHO-Gα16 background as described above. As shown in Table 2, octopamine exhibited highest potency on Tc-Octβ3R with an EC50 of 2 nM, while Dm-TyrR showed clear preference for tyramine over octopamine (EC50 of 165 nM; tyramine). In the cell lines tested (Dm-Oa2, Mp-Oa2 and Dm-OAMB), formamidine pro-insecticides showed lower potency than their corresponding active forms (Table 2). Interestingly, Kita et al. recently reported that the active amitraz metabolite DPMF (BTS27271) was more potent in increasing intracellular cAMP levels signaling through b-adrenergic-like octopamine receptors (EC50 = 79.6 pM) than elevating intracellular Ca2+ levels signaling through α-adrenergic-like octopamine receptors EC50 = 1.17 nM (11). Since we forced coupling through Gα16 and therefore a calcium readout in our assay panel, 145
we may not have been able to see this differential activation in our system. The above mentioned phenylaminooxazoline AC-6 that showed structural similarity with our initial in vivo screening hit, exhibited nM potency on Dm-Oa2 and Mp-Oa2. However, our ATZ lead analog exhibited only µM potency on Dm-Oa2, Mp-Oa2, Tc-Octβ3R and Dm-oamb while being inactive on Dm-Octβ2R and Dm-TyrR. There was no antagonist activity for the ATZ lead on any of tested receptors when challenged with 100nM octopamine or 250nM tyramine, respectively.
Table 2. Agonist Potencies of Octopamine, Formamidines, AC-6 and the ATZ lead on an Octopamine Receptor Panel. An Asterisk* Indicates That an EC50 Could Not Be Determined since No Detectable Increase in Intracellular Calcium Could Be Detected.
With respect to symptomology, chlordimeform-treated aphids showed hyperactivity while ATZ treated aphids exhibited mostly extensive wandering behavior. Our compiled data suggest a novel mode of action for ATZ, different from existing octopamine receptor agonists. Given that there is micromolar activity on some of the octopamine receptors one might speculate that ATZs might act on another insect GPCR. 146
Aminothiazoline Biology As mentioned above the original aminooxazoline screening hit shown in Figure 1 exhibited only moderate activity on the tested aphids (Aphis gossypii and Myzus persicae). With chemical variation, compounds with increased activity on aphids and widened spectrum to whiteflies could be identified. Further structure optimization yielded the ATZ lead analog, which showed excellent activity on aphids in the range of commercial standards, also controlled whiteflies including the difficult to manage Q biotype and additionally expanded the spectrum to thrips and hoppers as summarized in Table 3.
Table 3. Efficacy and Residual Activity of the Lead ATZ analog.
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Summary and Conclusions ATZ chemistry was developed from a random in vivo screening hit and shows preference for controlling piercing-sucking insects. Although the speed of control was slow, feeding cessation was fast with aphids exhibiting a wandering behavior. In vitro activity on octopamine receptors was observed for several ATZ analogs, but there was no clear in vivo-in vitro correlation suggesting a possibly new mode of action for this chemistry. Attractive features of the chemistry were its lack of cross-resistance to CNIs and pyrethroids and very positive initial Tox- and Ecotox profiles. However late stage toxicity testing raised some concerns that together with a lack of systemicity and somewhat limited spectrum prompted us to stop activity on this chemistry.
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Lambert, W. T.; Goldsmith, M. E.; Sparks, T. C. Insecticidal activity of novel thioureas and isothioureas. Pest Manage. Sci. 2017, 73, 743–751. 2. Roeder, T. Tyramine and octopamine: ruling behavior and metabolism. Ann. Rev. Entomol. 2005, 50, 447–477. 3. Roeder, T. Octopamine in invertebrates. Prog. Neurobiol. 1999, 59, 533–61. 4. Hirashima, A. Tyramine and octopamine receptors as a source of biorational insecticides. In Biorational Control of Arthropod Pests: Application and Resistance Management; Ishaaya, I., Horowitz, A. R., Eds.; Springer Netherlands: Dordrecht, 2009; pp 83−109. 5. Hollingworth, R. M. Chemistry, biological activity, and uses of formamidine pesticides. Environ. Health Perspect. 1976, 14, 57–69. 6. Jennings, K. R.; Kuhn, D. G.; Kukel, C. F.; Trotto, S. H.; Whitney, W. K. A biorationally synthesized octopaminergic insecticide: 2-(4-chloro-otoluidino)-2-oxazoline. Pestic. Biochem. Physiol. 1988, 30, 190–197. 7. Hauser, F.; Cazzamali, G.; Williamson, M.; Blenau, W.; Grimmelikhuijzen, C. J. A review of neurohormone GPCRs present in the fruitfly Drosophila melanogaster and the honey bee Apis mellifera. Prog. Neurobiol. 2006, 80, 1–19. 8. Ohta, H.; Ozoe, Y. Chapter two - molecular signalling, pharmacology, and physiology of octopamine and tyramine receptors as potential insect pest control targets. In Advances in Insect Physiology; Ephraim, C., Ed.; Academic Press, 2014; Vol. 46, pp 73−166. 9. Evans, P. D.; Maqueira, B. Insect octopamine receptors: a new classification scheme based on studies of cloned Drosophila G-protein coupled receptors. Invert. Neurosci. 2005, 5, 111–118. 10. Balfanz, S.; Strunker, T.; Frings, S.; Baumann, A. A family of octopamine receptors that specifically induce cyclic AMP production or Ca2+ release in Drosophila melanogaster. J. Neurochem. 2005, 93, 440–451. 11. Kita, T.; Hayashi, T.; Ohtani, T.; Takao, H.; Takasu, H.; Liu, G.; Ohta, H.; Ozoe, F.; Ozoe, Y. Amitraz and its metabolite differentially activate α- and βadrenergic-like octopamine receptors. Pest Manage. Sci. 2017, 73, 984–990. 148
