The GABA Antagonist Î³-Benzene Hexachloride and its

Chapter 3

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The GABA Antagonist γ-Benzene Hexachloride and its Polychlorinated Cyclohexane Analogs Keiji Tanaka,*,1 Takaaki Sakamoto,1 Takaaki Iwai,1 Kou Kuroda,1 Karin Nagasaki,1 Yoshihisa Ozoe,2 Miki Akamatsu,3 and Kazuhiko Matsuda1 1Faculty

of Agriculture, Kindai University, 3327-204 Nakamachi, Narashi, Nara, Japan 2Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu-cho, Matsue, Shimane 690-8504, Japan 3Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan *E-mail: [email protected].

GABA receptor Cl- ion channel complex (GABA-gated chloride channel) is a scientifically and practically important target site for various pharmaceutical and animal health drugs, and insecticides. γ-Benzene hexachloride (γ-BHC, lindane, one of eight BHC isomers) is well known to be a noncompetitive antagonist of the GABA-gated chloride channel. However why only γ-BHC, out of eight BHC isomers is insecticidal, remains unclear. In the early 1970s a number of γ-BHC analogs were synthesized, in which some chlorine atoms were replaced by other substitutes such as hydrogen, halogens other than chlorine and alkoxy groups, etc. Among these analogs, γ-BHC was the most insecticidal against the housefly and the German cockroach. Recently, we have focused on the polychlorinated γ-BHC analogs having four to eight chlorine (Cl) atoms on the cyclohexane ring and studied not only their insecticidal but also GABA antagonist activities to elucidate the molecular requirements and the role of Cl atom(s) for γ-BHC activity.

© 2017 American Chemical Society Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Introduction γ-Benzene hexachloride (γ-BHC (I); also known as lindane) is a polychlorinated chemical, which was extensively used for crop production after the Second World War and is now regarded as one of the legacy insecticides. γ-BHC (I) is insecticidal against many species of insects and shows potent nerve excitation in insects, fish, and mammals exposed to it (1–5). The γ-BHC-mediated nerve excitation was shown to be the result of the inhibitory action on the γ-aminobutyric acid (GABA)-gated Cl- channel (6, 7). BHC is the generic name of eight stereoisomers of very simple compounds having the empirical formula C6H6Cl6 (Figure 1); these stereoisomers are also called by various other names, including 1,2,3,4,5,6-hexachlorocyclohexane, HCH, and HCCH. γ-BHC (I) is one of the BHC stereoisomers and is the only one among the eight to have high insecticidal activity. Other isomers are slightly insecticidal or almost inactive against insects. α-BHC (III), whose structure is the most similar to that of γ-BHC (I), causes γ-BHC-like nerve excitation in insects and is known to be the GABA antagonist interacting with the picrotoxinin receptor (GABA receptor, i.e., GABA-gated Cl- channel) (8). However its insecticidal activity is 1/1000 of that of γ-BHC (I) (2). Why only γ-BHC (I) is insecticidal and which structural arrangements are responsible for its insecticidal activity remain unclear (4). To address this question and elucidate the structural arrangements responsible for the insecticidal activity of γ-BHC (I), various γ-BHC analogs in which some of the chlorine atoms of γ-BHC (I) were replaced by other substituents were synthesized and the structure-insecticidal activity relationship of these γ-BHC analogs was studied based on the the Hansch-Fujita approach (9). γ-BHC (I) itself was one of the highly active compounds studied. It was obvious from the findings that the activity of γ-BHC (I) could be the result of critical structural and steric requirements and/or hydrophobicity of chlorine atoms attached to the cyclohexane ring, although the exact underlying mechanism remains unknown. To address the question, and to more precisely evaluate the structural and steric requirements associated with the insecticidal and GABA antagonistic activities of γ-BHC, we focused not only on γ-BHC (I), but also α-BHC (III), which is less insecticidal than γ-BHC (I), but induces the nerve excitation similar to γ-BHC to insects and mammals (1, 2). We synthesized various stereoisomers of tetra-chlorocyclohexanes (TetraCl), penta-one (PentaCl), hexa-one (BHC), hepta-one (HeptaCl), and octa-one (OctaCl), from (36/45)tetrachlorocyclohex-1-ene (10–12) or by the photochlorination of benzene, mono- and (o, m or p)-dichlorobenzene and/or additional photochlorination of γ-BHC (I) (13–16), in which the fundamental molecular structure of γ-BHC (I) (three vicinal Cl atoms in the axial and the other three in the equatorial conformations) or α-BHC (III) (two vicinal Cl atoms in the axial and the other four in the equatorial conformations) was retained. Some of the chemicals such as (45/36)- and (145/6)-TetraCl (XIV and XII, respectively) and (12345/36)-HeptaCl (XV) were newly synthesized from (36/45)-tetrachlorocyclohex-1-ene or isolated from the additional photochlorination mixture of γ-BHC (I) for this study (12, 16). Further, the 42 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


insecticidal and GABA antagonistic activities of these compounds were studied in terms of their structures and the orientation of Cl atoms on their cyclohexane rings.

Figure 1. Conformations of BHC isomers. Also shown under their conformations are the same structures derived from a “planar” cyclohexane ring. These planar structures make it easy to visualize the orientation (up or down) of Cl atoms on their cyclohexane rings.

Materials and Methods Chemicals The configurations of the chemicals in this chapter are indicated by fractional notations, whereby numerals in the numerator denote a Cl atom above the plane of the ring whilst numerals in the denominator denote that below the plane. Short vertical lines in the structural formulas represent Cl atoms ( See Tables 1 and 2). Various stereoisomers of TetraCl, PentaCl, BHC, HeptaCl, and OctaCl were used in this study. The stereoisomers of PentaCl (X and XI) and TetraCl (XIII) were synthesized by the substitution of hydrogen atom(s) for one or two Cl atoms of γ-BHC (I) according to previously reported methods (10, 11). The 43 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


stereoisomers of TetraCl (XIV and XII) were newly synthesized for this study (12). Stereoisomers of HeptaCl (XV and XVI) and OctaCl (XVII, XVIII, and XIX) were obtained by the photochlorination of monochlorobenzene or o- or m-dichlorobenzene or the additional photochlorination of γ-BHC (I) in CCl4 saturated with Cl2 under sunlight or UV (AS ONE-Handy-UV lamp, SLUV-4, at wavelength of 365 nm) (13–16). Each of these stereoisomers was isolated from the reaction mixture by silica gel column chromatography and their chemical structures were elucidated and assigned by proton magnetic resonance (PMR, ULTRASHIELDTM 400 PLUS, Bruker and JEOL JNM-ECZ600R). As some of the products interconverted between two possible conformers, the PMR spectra of these compounds were recorded at low temperatures (-80°C) to confirm their structures (16).

Evaluation of Activities Insecticidal Activity Insecticidal activity was evaluated using four-to-five-day-old female adult houseflies (Musca domestica, Takatsuki strain, obtained from Earth Chemical Co., Ltd., and kept with cube sugars, skim milk and water at 25°C room temperature and 60% humidity with a 12:12 h light:dark photoperiod). The houseflies were held at 25°C after topical application of acetone solution (0.5 μ1) containing various amounts of the chemicals to the thorax and the mortality was recorded in 24 hrs. The median lethal dose (LD50), which was analyzed from the data of two or three replicated experiments by standard probit analysis (17, 18). Synergized LD50 values were determined by the application of 0.5 µl of 5% piperonyl butoxide in acetone, 1 hr prior to the application of the chemicals. Control data were obtained by the application of acetone (0.5 μ1). Time-poisoning symptom and mortality relations of German cockroach (Blattella germanica, obtained from Chemical Ecology Lab, Kyoto University and kept with dog foods and water in the insect rearing room (25°C room temperature and 60% humidity, a 12:12 h light:dark photoperiod)) against γ-BHC (I) and (12345/36)-HeptaCl (XV) were examined by a surface contact method, in which the inner surfaces of glass beakers (300 ml) were coated with a fixed amount of γ-BHC (I) and (12345/36)-HeptaCl (XV) (0.15 mg/beaker and 0.16 mg/beaker, respectively) with acetone. Control data were obtained by using acetone only. Male cockroaches in batches of 5 were exposed to the toxicant in duplicates and their poisoning symptoms were recorded over the time. (See Table 3)

[3H]4′-Ethynyl-4-n-propylbicycloorthobenzoate ([3H]EBOB) Binding Assay For displacement assays, [3H]EBOB was incubated with membranes prepared from housefly heads according to the procedures described by Deng et al. (19), Cole et al. (20), and Ju et al. (21). The incubation mixtures consisted of 0.5 nM [3H]EBOB and various concentrations of a test chemical; these were added to 44 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.

the membrane preparation (0.2 mg protein) in assay buffer (1 ml). The mixtures were shaken at 60 rpm and incubated for 70 min. at 22°C and were then filtered using Whatman GF/B glass-fiber filters followed by two rinses with 5 ml of chilled assay buffer. The membranes were collected on glass filters and the bound radioactivity was determined using a liquid scintillation counter. Specific binding was calculated as the difference between total [3H]EBOB bound with 0.5 nM [3H]EBOB and nonspecific 3H bound on the addition of α-endosulfan (1 µM). Each experiment was repeated at least three times to account for the variability of the tissue and the technique.


Fluorometric Imaging Plate Reader (FLIPR) Membrane Potential (FMP) Assay Antagonistic action on Drosophila GABA receptor (ac-splice variant), expressed on Drosophila Mel-2 cells, was characterized using the FMP assay kit (Molecular Devices Corporation, Sunnyvale, CA, USA) according to the procedure described by Nakao et al. (22). In the assay, change in membrane potential was quantified based on the change in the fluorescence of the FMP dye. The experiments were performed three times in duplicate for each chemical.

Results Insecticidal Activities γ-BHC (I) was the most insecticidal of the chemicals that were tested in this study (Tables 1 and 2, Figure 2). With regard to the relationship between the structures of the tested chemicals and their insecticidal activity, there seems to be some structural requirement of the chlorine atom and the orientation of the substituted atoms on the cyclohexane ring. (245/36)-PentaCl (X) and (1245/6)PentaCl(XI) were less active than γ-BHC (I) but more active thanα-BHC (III) . (245/36)-PentaCl (X) was the compound with the substitution of five chlorine atoms that were structurally similar to those of γ-BHC (I) and α-BHC (III) on the cyclohexane ring. This finding clearly indicates that a Cl atom on the 1- or 3position of the cyclohexane ring is necessary for insecticidal activity. The activities of all TetraCl isomers tested ( i.e., (45/36)-TetraCl (XIV); (145/6)-TetraCl (XII) and (1245/)-TetraCl (XIII)) were inferior to those of the PentaCl isomers (X and XI) (Table 1). Among the TetraCl isomers tested, only (145/6)-TetraCl (XII) showed a slight insecticidal activity (LD50, 2.69 μg/housefly, an average weight of house flies was 16.7mg/housefly) and the others are almost inactive (mortality was 0% at 10 μg/housefly).

45 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 2. GABA antagonist ([3H]EBOB binding inhibition) activities of α-BHC (III) and γ-BHC (I). a) Each data are from three determinations.

Among the HeptaCl and OctaCl isomers tested in this study, (12345/36)HeptaCl (XV), (1245/136)-HeptaCl (XVI) and (12456/136)-OctaCl (XVII), which had an additional one or two chlorine atoms substituted on the cyclohexane ring of γ-BHC (I), were insecticidal but the others were not (Table 2). (12345/36)-HeptaCl (XV) having geminal chlorine atoms at the 3-position of cyclohexane ring, a hybrid structural isomer of γ-BHC (I) and θ-BHC (VIII), was more insecticidal than (1245/136)-HeptaCl (XVI) having geminal chlorine atoms on the 1-position of cyclohexane ring, that isomer of γ-BHC (I) and α-BHC (III). This finding indicates that the additional chlorine atom substitution on 1-position of γ-BHC (I) is not favorable to its insecticidal activity, and resulted in a less activity. Certainly (12456/136)-OctaCl (XVII), a hybrid structural isomer of (12345/36)-HeptaCl (XV) and (1245/136)-HeptaCl (XVI), was less active than (12345/36)-HeptaCl (XV). The intoxication symptoms observed in German cockroaches treated with (12345/36)-HeptaCl (XV) were fairly identical to those associated with γ-BHC (I) intoxication except in terms of the time of onset: the treatment first resulted in depression (calm and sedate) symptoms, followed by excitingly running around, turning upside down, and convulsion (Table 3). The symptoms of (12345/36)-HeptaCl (XV) intoxication manifested slower than those of γ-BHC (I) intoxication. The differences between intoxication with these two compounds may be attributable to their physicochemical properties such as the hydrophobicities (lipophilicities). 46 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Table 1. Insecticidal Activities against Housefly (Topical Application LD50) and GABA Antagonist Activities (Evaluated by [3H]EBOB Binding and FMP Assays) of α-BHC (III), γ-BHC (I) and Isomers of TetraCl and PentaCl



Table 2. Insecticidal Activities against Housefly (Topical Application LD50) and GABA Antagonist Activities (Evaluated by [3H]EBOB Binding and FMP Assays) of Various Isomers of BHC, HeptaCl and OctaCl

Table 3. Insecticidal Activity and Poisoning Symptom: Film Contact Method Using German Cockroaches (Blattella germanica)



[3H]EBOB Binding Assay for the Evaluation of in Vitro GABA Antagonistic Activity The [3H]EBOB binding assay to evaluate the in vitro GABA antagonistic activities of BHC isomers and γ-BHC analogs showed that γ-BHC (I) had the most potent in vitro GABA antagonistic activity among the chemicals tested, as shown in Tables 1 and 2. α-BHC (III) also had some GABA antagonistic activity, but it was weaker than that of γ-BHC (I). Other BHC isomers (β (II), δ (IV), and ε (V)) were not active at the concentration of 10 μM. These GABA antagonistic activities evaluated by [3H]EBOB binding assay shown in Table 2 are in agreement with the corresponding insecticidal activities against the housefly. The GABA antagonistic activities of all TetraCl isomers tested (i.e., (45/36)-TetraCl (XIV), (145/6)-TetraCl (XII) and (1245/)-TetraCl (XIII)) were inferior to those of the PentaCl isomers (X and XI) (Table 1). Among the HeptaCl and OctaCl isomers, (12345/36)-HeptaCl (XV) had potent GABA antagonistic activity, but (1245/136)-HeptaCl (XVI) was less active than the (12345/36)-isomer (XV). The OctaCl isomers (XVII, XVIII, and XIX) were not active at 10 μM (Table 2).

FMP Assay With regard to the evaluation of the in vitro GABA antagonistic activity, the FMP assay was roughly one order less sensitive than the [3H]EBOB binding assay (Table 1 and 2). However, the activities of γ-BHC analogs evaluated by this method were highly comparable to the corresponding activities evaluated by the [3H]EBOB binding assay. Among γ-BHC (I) and its analogs, γ-BHC (I) was most active, followed by (12345/36)-HeptaCl (XV) and (245/36)-PentaCl (X) (Tables 1, 2 and Figure 3, 4).

Figure 3. FLIPR membrane potential (FMP) assay with Drosophila melanogaster GABA receptor: GABA antagonist activities of BHC isomers. Individual data are means ± SD of two experiments, each experiment involving three determinations. 49 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Figure 4. FLIPR membrane potential (FMP) assay with Drosophila melanogaster GABA receptor: GABA antagonist activity of α-BHC (III), γ-BHC (I) and isomers of HeptaCl and OctaCl. Individual data are means ± SD of two experiments, each experiment involving three determinations.

Discussion γ-BHC exerts its insecticidal activity by binding to the insect GABA receptor in the insect nervous system. The GABA receptor (i.e., the GABA-gated Clchannel) is a member of the Cys-loop ligand-gated ion channel (LGIC) family in the nervous system of insects and mammals (23). Insects that are resistant to γ-BHC and cyclodiene is the result of a single point mutation (alanine to serine) in the ion channel lining of the transmembrane 2 (TM2) region (24, 25). This point mutation confers target-site insensitivity and reveals hypothetically the primary binding site in the channel pore. γ-BHC (I) could bind inside the channel pore in the TM2 region, which is a part of the GABA receptor. To examine the location of the binding site in the pore, the docking of γ-BHC into the homomeric β3 GABA receptor constructed on the basis of the crystal structure revealed by Miller and Aricescu was attempted (4, 26, 27). As a result, we were able to confirm that γ-BHC is docked on the cytoplasmic side within the channel (Figure 5). Among the BHC isomers (Figure 1 and Table 2), γ-BHC (I) showed the most highly insecticidal activity against houseflies, followed by α-BHC (III) (25.6 μg/housefly) (Figure 2 and Table 2). γ-BHC (I) and α-BHC (III) induced the excitation and resulted in killing of houseflies and German cockroaches. The intoxication symptom of German cockroaches applied with (12345/36)-HeptaCl (XV) was also quite identical with that of γ-BHC (I) except the onset times of these symptoms (Table 3). (12345/36)-HeptaCl (XV) took more time to show the onset of these symptoms than γ-BHC (I). The difference between those two compounds must depend on their physicochemical properties such as their hydrophobicities (i.e., lipophilicities, of which numerical values are expressed as partition coefficients of the drugs between water and a relatively nonpolar 50 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


organic solvent such as 1-octanol). The passive penetration of drugs to their site of action in insects and mammals has been considered to be dependent on their physicochemical properties (28, 29).

Figure 5. Docking of γ-BHC into thecrystal structure of a human homomeric β3 GABAA receptor revealed by Miller and Aricescu (26, 27).

Other BHC isomers were neither excitatory nor insecticidal at the highest dosage tested, and although δ-BHC (IV) showed weak insecticidal activity (LD50, 10.2 μg/housefly), it was a depressant on the CNS and did not cause an excitatory effect. As many researchers have pointed out (1, 30–33), δ-BHC (IV) might interact allosterically with sites other than the γ-BHC binding site on the GABA receptor and act as a depressant in houseflies. Regarding the molecular requirements for activity, especially structural and steric requirements of chlorine atoms on the cyclohexane ring, this study clearly shows that 6- (and/or 3-) chlorine atom substituted at three vicinal chlorine atoms in the axial conformation and three in the equatorial conformation are necessary for potent GABA antagonistic activity. The change in the orientation of one chlorine atom from axial (γ-BHC (I)) to equatorial (α-BHC(III)) at the 1-position on the cyclohexane ring drastically decreased the insecticidal and GABA antagonistic activities. The intermediate compound of γ-BHC (I) and α-BHC (III), i.e., (245/ 36)-PentaCl (X) was less insecticidal than γ-BHC (I) but more active than αBHC (III). The GABA antagonistic activity of this compound (X) is also at an intermediate level between those of γ-BHC (I) and α-BHC (III). The equatorial chlorine atom at the 1-position on the cyclohexane ring does not seem favorable and causes steric hindrance for binding on the inside pore of the chloride ion channel of the GABA receptor. 51 Gross et al.; Advances in Agrochemicals: Ion Channels and G Protein-Coupled Receptors (GPCRs) as Targets for Pest ... ACS Symposium Series; American Chemical Society: Washington, DC, 2017.


Conclusion From the structure-insecticidal and GABA antagonistic activities of γ-BHC (I), its analogs and its related compounds tested in this and previous studies, γ-BHC (I) was confirmed to be the most insecticidal compound with the potent GABA antagonistic activity, which could have the most suitable molecular form to fit inside the pore of the chloride ion channel of the GABA receptor. Undoubtedly, γ-BHC (I) and α-BHC (III) could be regarded as GABA antagonists; other BHC isomers (β(II)-, δ(IV)-, ε(V)-, and η(VII)-isomers) except ζ(VI)- and θ(VIII)-isomers cannot be regarded as GABA antagonists. It is unknown whether ζ-BHC (VI) and θ-BHC (VIII) have GABA antagonistic activity or not, because these two isomers have not been available. However judging from the GABA antagonistic activity of (1245/6)-PentaCl (XI) and (12345/36)-HeptaCl (XV), as shown in Tables 1-3, these isomers are likely active GABA antagonists, because they have four essential chlorine atoms, i.e., axaeye ( ζ-isomer (VI): aeaeae and θ-isomer (VI): aeaeee or aaaeae ), similar to γ-BHC (I) (aaaeee)

Acknowledgments We thank Dr. Toshifumi Nakao (Mitsui Chemicals Agro.) for the FMP assay.

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The GABA Antagonist Î³-Benzene Hexachloride and its

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