&Hapten Synthesis | Hot Paper |

Development of Tetramethylenedisulfotetramine (TETS) HaptenLibrary: Synthesis, Electrophysiological Studies, and ImmuneResponse in Rabbits.

Bogdan Barnych,[a] Natalia Vasylieva,[a] Tom Joseph,[a] Susan Hulsizer,[b] Hai M. Nguyen,[c]

Tomas Cajka,[d] Isaac Pessah,[b] Heike Wulff,[c] Shirley J. Gee,[a] and Bruce D. Hammock*[a]

Abstract: There is a need for fast detection methods for the

banned rodenticide tetramethylenedisulfotetramine (TETS),a highly potent blocker of the g-aminobutyric acid (GABAA)

receptors. General synthetic approach toward two groups of

analogues was developed. Screening of the resulting libraryof compounds by FLIPR or whole-cell voltage-clamp re-

vealed that, despite the structural differences, some of the

TETS analogues retained GABAA receptor inhibition; howev-er, their potency was an order of magnitude lower. Antibod-

ies raised in rabbits against some of the TETS analogues con-

jugated to protein recognized free TETS and will be used forthe development of an immunoassay for TETS.

Introduction

TETS (tetramethylenedisulfotetramine, tetramine) is a highlylethal neurotoxic rodenticide. It is a non-competitive channel

blocker of the g-aminobutyric acid (GABAA) receptors that in-duces excessive excitation of the adult central nervous system

(CNS). The LD50 in laboratory animals is 0.1 mg kg@1, and 7–

10 mg is considered to be a lethal dose for humans.[1] Despitebeing banned worldwide, it is still available on the black

market in China and other countries because of its ease ofmanufacture, profitability, and effectiveness as a rodenticide.[2]

The illegal use of TETS has led to multiple accidental humanpoisoning cases. Additionally, being water soluble, odorless,

and tasteless, it has been often used in intentional poisonings

and thus, is considered as a potential threat agent.[3] Morethan three thousand poisonings between 2000 and 2012 inChina have been associated with TETS.[3b]

China implemented multiple regulatory enforcement meas-ures, which had a positive impact on the frequency of such

events, but failed to completely remove TETS-containing prod-ucts from the open market because of the lack of technical

means to test for highly toxic rodenticides such as TETS.[2]

Access to TETS in China and its illegal export to other countries

necessitates better methods for its detection.[2] Current meth-

ods for the detection of TETS are mainly GC-MS based andthus require a laboratory setting, are laborious, and expensive,

limiting their use. Immunoanalytical methods, on the otherhand, are widely used for the detection of small molecules in

different matrices[4] and have the advantage of being cheap,portable, and high throughput. However, they require conjuga-tion of the small-molecule analyte to a larger protein to gener-

ate an appropriate immune response and raise analyte-selec-tive IgG antibodies.

Tetramine has a unique chemical structure including a rigidcage and multiple heteroatoms that may provide recognition

points for antibodies. However, TETS lacks reactive functionalgroups that could be easily functionalized and used as the at-

tachment points for the preparation of the immunizing and

coating antigens. Therefore, haptens have to be synthesizedde novo, not by modification of the target analyte (TETS) or its

precursors as it is typically done for other analytes.[5] An addi-tional challenge is to develop a synthetic route that would ex-

clude production of free TETS as a by-product. These are prob-ably the main reasons for the lack of an immunoassay for TETS

to date. Interestingly, one monoclonal antibody (mAb) devel-

oped against cyclodiene pesticides, such as aldrin, was shownto cross-react with TETS.[6] However, due to significant structur-

al differences between cyclodienes and TETS, the affinity of themonoclonal antibody (mAb) to TETS was low (IC50 = 3 mm or

0.72 mg mL@1) and not suitable for analytical use. Thus, the de-velopment of structurally close TETS analogues possessing

[a] Dr. B. Barnych, Dr. N. Vasylieva, T. Joseph, S. J. Gee, Prof. B. D. HammockDepartment of Entomology and Nematology, andUCD Comprehensive Cancer CenterUniversity of California Davis, Davis, California 95616 (United States)E-mail : bdhammock@ucdavis.edu

[b] S. Hulsizer, Prof. I. PessahDepartment of Molecular Biosciences, School of Veterinary MedicineUniversity of California Davis, Davis, California 95616 (United States)

[c] Dr. H. M. Nguyen, Prof. H. WulffDepartment of Pharmacology, School of Medicine,University of California Davis, Davis, California 95616 (United States)

[d] Dr. T. CajkaUC Davis Genome Center-Metabolomics,University of California Davis, Davis, California 95616 (United States)

Supporting Information, including full experimental details, and the ORCIDidentification number for the author of this article can be found underhttps ://doi.org/10.1002/chem.201700783.

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active functional groups will facilitate development of a cheapimmunoanalytical method for its detection, and may be useful

for regulatory and enforcement agencies charged with envi-ronmental, agricultural, and homeland security. Additionally,

such analogues could be used for the development of photo-affinity labels allowing identification of the TETS binding site,

in-depth study of the mechanisms of its toxicity and evaluationof treatment options.

Therefore, in this work we developed a synthetic route to

generate a library of TETS-like compounds. The potency ofthese compounds as excito-toxicants were assessed in bioas-

says with primary cultures of mouse hippocampal neurons andcultured cells expressing human GABAA receptors, and com-

pared directly to TETS. The most promising analogues wereconjugated to the carrier protein and injected in rabbits to

produce polyclonal antibodies.

Results and Discussion

We designed four types of analogues with different degrees of

similarity to TETS (Scheme 1). Although overall surface comple-

mentarity is considered to be an important determinant for an-tigen recognition, specific interactions like electrostatic and hy-

drogen bonding are frequently more critical determinants of

antibody affinity.[7] It is therefore generally accepted thata good hapten will preserve the distinctive functional groupsas well as the overall antigen structure.[8] Additionally, toensure that distinctive functional groups remain well-exposed

and available for interaction with the antibody, the spacer armshould be as remote from them as possible.[8c] Following theseconsiderations, TETS analogue 1 having a linker arm attached

to one of the methylene bridges should be an ideal hapten be-cause it preserves all the structural features of the parent com-

pound like the adamantane structure and both sulfamide func-tions. Theoretically, its synthesis would involve co-condensa-

tion of formaldehyde and aldehyde with sulfamide resulting in

formation of the mixture of TETS-like compounds including an-alogue 1 and TETS. Clearly, this approach would suffer from

drawbacks such as poor yields and complicated chromato-graphic separation of the desired product. Most importantly,

the possibility of formation of analogue 1 or its stability isdoubtful. At least, under standard reaction conditions, previous

publications show that replacement of formaldehyde by morebulky aldehydes in the condensation reaction with sulfamide

precludes the formation of the tricyclic core.[9] Thus eitherstructural or functional group modifications were required

during the process of hapten design.It was shown previously that monoalkyl or monoaryl sulfa-

mides could be engaged in a similar reaction with formalde-hyde as a parent nonsubstituted sulfamide giving TETS-like

compounds 4 lacking one methylene bridge (Scheme 1).[9, 10]

Although synthetically very attractive this approach wouldresult in haptens having two linker units that may cause com-plications during conjugation or negatively influence TETS rec-ognition. This problem could be overcome by using asymmet-rically substituted analogues 4 (Me = R1 ¼6 R2). Unfortunately,condensation of equimolar amounts of N-methylsulfamide and

methyl 6-(sulfamoylamino)hexanoate with polyformaldehyde

not only gave poor yields of asymmetric analogue 4 b (Table 1)but also its separation from the symmetrical analogues was

complicated.

Scheme 1. Design of compounds with functional determinants similar toTETS.

Table 1. Synthesis of TETS analogues

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Next, we studied whether compounds having two sulfamidefunctions connected through a variable-length linker undergo

intramolecular condensation with formaldehyde to give tricy-clic TETS-like compounds. To answer this question, disulfa-

mides 8 had to be synthesized. Among multiple synthetic pro-cedures available[11] we chose the method described by Masui

et al.[12] because of its reported water tolerance and high prod-uct yields. Thus, reaction of the CSI (chlorosulfonyl isocyanate)with tert-butanol followed by the addition of the triethylamine

and primary amine provided the Boc-protected (Boc = tert-bu-toxycarbonyl) sulfamides 8 in good to excellent yields (details

in the Supporting Information). The next steps called for cleav-age of the Boc protecting group followed by condensationwith formaldehyde. Considering that acidic media is necessaryfor both reactions, these two steps were tested in a one-pot

procedure. Furthermore, because the bicyclic TETS analogue4 a is known, this condensation reaction was first tested withtert-butyl (N-methylsulfamoyl)carbamate 8 k and produced the

compound having identical physicochemical characteristics tothe previously reported 4 a in 73 % yield. Next, this reaction

was tested with a simple di-Boc-protected disulfamide derivedfrom easily available ethylene diamine. Reaction of di-tert-butyl

(((2-(hydrosulfonylamino)ethyl)amino)sulfonyl)dicarbamate with

dimethoxymethane in trifluoroacetic acid gave the TETS ana-logue 2 a as a racemic mixture in 92 % yield (Table 1). To inves-

tigate the scope of this transformation and optimize reactionconditions a variety of differently substituted 1,2-diamines

were tested. Considering that the choice of commercially avail-able functionalized diamines is limited, the substrate scope

was first tested with most commonly available nonfunctional-

ized diamines followed by functionalized ones, which were ob-tained by multistep synthesis as described in the Supporting

Information. Presence of aliphatic or carboxylate substituentson the ethylene bridge proved to be tolerable, however, in this

case, the products were obtained as a mixture of two diaste-reomers (Table 1, 2 b–d). Although flash column chromatogra-

phy has been tested as a purification technique, use of this

technique for purification was complicated owing to relativelylow polarity and very poor solubility of 2 in non-polar solvents.

The major products were obtained in pure or almost pureform by recrystallization from methanol. Interestingly, introduc-

tion of a benzyl group on the ethylene bridge not only dra-matically deteriorated the reaction yield but also resulted in

formation of product 2 e with an unexpected structure. Re-

placement of the benzyl group by p-nitrobenzyl resulted in for-mation of a mixture of at least 2 products, which, after recrys-

tallization, gave pure 2 f in 30 % yield. Comparison of the NMRspectra of crude product, 2 f and 2 e revealed that chemical

shifts of the minor product from the mixture were very similarto those of 2 e and thus it likely had the same structure. Ab-

sence of the TETS-like product in the case of disulfamide 8 ecan be explained by intramolecular sulfamidoalkylation of thearomatic ring resulting in the formation of benzo-annelated

side-products similar to a previous report.[9]

Disulfamide derived from (1R,2R)-cyclohexane-1,2-diamine

also reacted with dimethoxymethane, resulting in formation ofTETS analogue 2 g in 55 % yield as a single diastereoisomer.

(1R,2S)-Cyclohexane-1,2-diamine, on the other hand, failed togive the desired tricyclic product probably because of consid-erable van der Waals repulsive forces between the tricycliccore and the axial hydrogens of the cyclohexane moiety, whichrender this compound unstable. The only product that was ob-tained in only 5.2 % yield was the disulfamide 2 h structurally

related to 2 e, but missing -CH2OCH2- bridge. Its structure wasconfirmed by spectroscopic methods and X-ray crystallography.Replacement of the ethylene bridge by phenylene was well-

tolerated (Table 1, 2 i and j).Studies of NOE and COSY spectra of analogues 2 revealed

the following interesting features. A NOE effect was observedbetween spatially close pairs of hydrogens of the methylenebridges H1a-H2b and H1b-H3b, whereas spin–spin correlations inthe COSY spectra were observed only for distant protons of

the same methylene groups H1b-H2a and H1a-H3a (Figure 1).

Modeling of the analogues 2 revealed that the systems H@C@N@C@H for which these spin-spin correlations were observed

are almost flat and thus they could be attributed to long-

range “W”-type correlations.[13] A NOE effect was also observedbetween spatially close protons of methylene and ethylene

bridges H2a-H6a and H3a-H5b, and therefore could be used toconfirm the relative stereochemistry in these analogues.

Next, we tested if one of the methylene bridges in TETScould be replaced by propylene or if it could be completelydeleted. For this purpose, we synthesized di-Boc-protected di-

sulfamides starting from 1,3-diaminopropane, 1,8-diamino-naphthalene, and hydrazine, which were then reacted with di-methoxymethane in trifluoroacetic acid (Table 1, 3 a, 3 b, 7). Allthree reactions resulted in complex reaction mixtures showing

no sign of the desired product by NMR spectroscopy or massspectrometry. For 3 a and b, this result compares well with lit-

erature data showing that, unlike ethylenediamine, 1,3-diami-nopropane does not give a tricyclic condensation product withformaldehyde presumably because of steric factors.[14] Howev-

er, it is likely that faster alternative condensation reactionscompared to the formation of the eight-membered ring, re-

quired for construction of 3 a, b skeleton, are responsible forabsence of the desired product.

We also studied approaches to synthesize TETS analogues 5and 6, which possess a lower degree of similarity to the parentmolecule, but still have an adamantane-like structure and one

of the two sulfamide functions preserved. Hapten 5 was veryattractive from the synthetic point of view because precur-

sors—bispidine derivatives—have been described and areeasily accessible, thus leaving solely the feasibility of sulfamide

Figure 1. Significant NOE and spin-spin correlations observed for 2.

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bridge formation uncertain. Thus, after the intermediates 10and 11 were synthesized (Scheme 2) according to a literature

procedure,[15] the possibility of introduction of a sulfamidebridge was studied. For this transformation, we first tested the

reaction conditions employing sulfuryl chloride as sulfurylation

reagent and triethylamine or pyridine as a base. In both cases,reactions resulted in complex mixtures as judged by NMR

spectra. To overcome this problem, a range of alternative sul-furylation reagents were tested, but none were successful at

introducing the sulfamide bridge in bispidine derivatives 10and 11.

In parallel to our studies toward the TETS analogue 5, we

also explored approaches aiming at synthesis of TETS ana-logues 6. Among the multitude of possible radicals R in the

TETS analogue with general formula 6 (Scheme 1), we chosehydroxymethylene (HOCH2-) and amino groups because of theavailability of precursors pentaerythritol and Tris base. Synthe-

sis of compound 6 a commenced by preparation of the knowntriamine 16 (Scheme 3).[16] Briefly, pentaerythritol was first con-

verted into monobenzyl derivative 15 via orthoester protec-tion, alkylation, and deprotection sequence. Tosylation of deriv-

ative 15 followed by nucleophilic substitution with azide ionand palladium-catalyzed hydrogenation gave triamine 16.

Transformation of 16 into the adamantane-like sulfamide 17was achieved in 24–45 % yield by its treatment with an equi-

molar amount of catechol sulfate under reflux followed by ad-dition of formaldehyde. Alternatively, refluxing triamine 16with sulfamide in pyridine followed by the addition of formal-dehyde also gave the tricyclic sulfamide 17, but in lower yield.The next step was benzyl deprotection using hydrogen andpalladium on carbon. However, despite all efforts, the hydroge-

nation either did not proceed or was not chemoselective asconcomitant hydrogenative cleavage of the C@N bond at themethylene bridge was occurring.

Elimination of the deprotection step was envisioned toavoid this problem and to considerably shorten the synthetic

pathway. Thus, the unprotected triamino alcohol 18, ananalogue of 16, was prepared in two steps from commercial

pentaerythritol tribromide by nucleophilic substitution with

azide ion followed by palladium-catalyzed hydrogenation(Scheme 4).[17] The resulting triamine 18 was transformed into

TETS analogue 6 a by treatment with catechol sulfate followedby addition of formaldehyde. The yield of 6 a (8.4–15 %) was

considerably lower than the yield for 17. This might be attrib-

uted to poor solubility of the reaction intermediates obtainedfrom 18 in dioxane (a gummy substance was observed in the

reaction mixture) leading to higher amounts of polymericsulfamides.

Synthesis of TETS analogue 6 b started from Tris base, whichwas first transformed into trichloride followed by the azidation

and protection of the amine function by a Boc group to give

intermediate triazide (Scheme 5).[18] Palladium-catalyzed hydro-genation of this triazide gave monoprotected tetraamine 19[19]

that was reacted with catechol sulfate in refluxing dioxane fol-lowed by reaction with formaldehyde at RT. Treatment of the

resulting intermediate 20 with trifluoroacetic acid liberated thefree TETS analogue 6 b in quantitative yield. All products and

intermediates were characterized by NMR and other spectro-scopic methods. Additionally, the identities of compounds 2 e,

Scheme 2. Synthesis of bispidine derivatives 10–11 and tentatives of theirsulfurylation. (TfO = triflate.)

Scheme 3. First synthetic approach toward hapten 6 a. (pTSA = p-toluenesul-fonic acid; TsCl = tosyl chloride; Pyr = pyridine.)

Scheme 4. Synthesis of the hapten 6 a.

Scheme 5. Synthesis of the hapten 6 b. (TFA = trifluoroacetic acid.)

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2 g–j, 4 a, 17, and 20 were proven by X-ray crystallography(see the Supporting Information).

Development of immune response in rabbits

Analogues 2 d, 2 j, 2 k (nitro group reduced to amine in 2 f),

6 a, and 6 b were selected from our library for animal immuni-zation. These compounds represented the diversity of the syn-

thesized library and possessed functional groups that could beused for chemically linking to the carrier protein. Thyroglobulin

was chosen as a carrier protein for immunization because ofits high immunogenicity and ease of use. Bovine serum albu-

min and conalbumin were used as carrier proteins in the prep-

aration of coating antigens. For the conjugation 6 a and 6 bwere first reacted with succinic anhydride to give correspond-

ing monoester and monoamide, respectively. The resultingmonosuccinates of 6, as well as haptens 2 d, 2 j and 2 c (2 cwas only used for making coating antigen) were then directlyconjugated to the carrier protein through a carboxylic acid

functionality using the standard activated-ester method.

Amine 2 k was conjugated to the carrier protein by usingeither diazotization or glutaraldehyde methods (see the Sup-

porting Information).[20]

After immunization of rabbits, serum from the final bleedwas analyzed in a competitive ELISA format with TETS(5 mg L@1). The aim of this experiment was to study if synthetic

analogues used for immunization could elicit an immune re-sponse and result in antibodies that recognized TETS. Figure 2demonstrates that sera from rabbits immunized with haptens

2 j, 2 k, and 2 d recognized the corresponding haptens and itsbinding was, to some extent, inhibited by TETS. By contrast,

even though sera obtained from rabbits immunized with hapt-ens 6 b and 6 a still recognized the corresponding immunizing

haptens, their binding was not altered by TETS. These data

suggest that either haptens 6 b and 6 a are fairly distinct fromthe TETS structure and thus sera do not recognize the analyte

or the affinity of the developed antibodies is much greater forthe haptens and thus the concentration of TETS tested is not

high enough to produce a visible inhibition effect. Here wepresent promising preliminary data for immunoassay develop-

ment from rabbit sera immunized with haptens 2. Further eval-uation of developed antibodies against TETS is a subject of

a separate study.[21]

TETS analogues as tools for biological applications

The history of terror acts involving chemical agents[22] has

raised concern about banned substances with high toxicity, in-cluding TETS. For instance, within the U.S. Department of

Health and Human Services, the NIH is making a significanteffort to pursue the development of new and improved medi-

cal countermeasures designed to prevent, diagnose, and/ortreat the pathology caused by TETS.[23] To develop a successful

treatment approach for the poisoning, it is important to under-stand the mechanism of action of the agent. So far, TETS hasonly been identified to block the GABA receptor chloride chan-

nel (GABAAR), but the binding site remains unidentified. Knowl-edge of structure–activity relationship of TETS and analogues

may prove to be useful in this regard, for example by helpingdesign photoaffinity probes.

Effects of TETS and TETS analogues on Ca2 ++ oscillations inprimary cultured hippocampal neurons

For initial screening for neuroactive analogues, we used

a FLIPR bioassay detecting Ca2 + oscillations in cultured hippo-campal neurons. Cultured hippocampal neurons (13–28 days in

vitro (DIV)) display a balance of glutamatergic (excitatory) and

GABAergic (inhibitory) signaling, which results in spontaneoussynchronous Ca2 + oscillations of approximately 10 second du-

ration. Application of GABAA receptor (GABAAR) antagonistssuch as picrotoxin, bicuculin, and TETS, result in an increase in

the amplitude of intracellular calcium peaks as revealedthrough a Ca2 + indicator, such as Fluo-4. Therefore, the FLIPR

assay allows sensitive detection and high-throughput screen-

ing of neuroactive compounds. Recordings of individual somasreveal that these oscillations are synchronous across a field of

view, making them appropriate to study with lower spatial res-olution, as the average signal from a large portion of a well in

a 96-well plate. As with all primary neuronal cultures, there areculture-to-culture variations in the precise balance of types of

neurons, but with so many wells from one culture, the com-plete dose-response curve for TETS can be obtained from one

plate in one run with 5 duplicate wells with FLIPR.[24]

Addition of vehicle (0.03 % dimethyl sulfoxide [DMSO]) hadno significant effect on properties of the synchronous Ca2 + os-

cillations (SCO). By contrast, TETS at 10 mm significantly in-creased the amplitude and decreased the frequency of the

SCOs.[24] Eight analogues of TETS were tested at 30 mm, andcompared to TETS (Figure 3). Analog 4 a produced a similar

effect to TETS, with an increased SCO amplitude and lower

spike frequency. The traces from this analogue were indistin-guishable from TETS (Figure S1 in the Supporting Information).

None of the other analogues tested had a significant differencein the SCO amplitude from vehicle. The TETS analogue 4 a was

tested at 3 concentrations: 3, 10, and 30 mm and compared toTETS at concentrations from 0.06 mm to 20 mm (Figure 4). The

Figure 2. Inhibition of antibody binding on homologous coating antigen inthe presence of TETS at 5 mg L@1. Each bar represents serum from individualanimal.

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EC50 value for 4 a was found to be 7.15 mm (95 % confidence in-

terval (CI): 3.13–16.35 mm). Our use of TETS in this study was tocompare on each plate, the amplitude increase of each ana-logue with a maximal TETS response. An earlier study used

a wider range of TETS concentrations on identically preparedcultures with the same procedures as this study, in the samelab, found the EC50 of TETS on amplitude of SCO to be 1.8 mm(95 % CI: 1.12 to 2.80 mm).[24] Our study used a narrower rangeof TETS, and indicated a lower EC50 of 0.5 mm, but did not in-clude measurements at the lower concentrations of TETS

needed to state the EC50 with confidence. Using both values,our conclusion is that TETS is at least 4 times more potentthan the most effective TETS analogue tested (4 a), whereas

the maximal response of the two is the same (efficacy).

Inhibition of GABAA currents by TETS and its analogues

TETS causes neuronal hyperexcitability by competitively bind-

ing to GABAA receptors and reducing the hyperpolarizing chlo-ride currents. To evaluate further neuromodulating properties

and to determine the potency of the TETS analogues in inhibit-ing GABA-induced currents, the effect of the TETS analogues

on currents produced by cultured cells expressing a1b2g2LGABAA receptors were measured using whole-cell voltage-

clamp and compared against current inhibition by TETS. De-spite being a low-throughput technique, manual patch-clamp-ing directly measures the activity of the compound on GABAARcurrent. Additionally, in contrast to cultured hippocampal neu-

rons, we controlled the receptor subunits being expressed inour heterologous cells and thus the exact identity of the

GABAA receptors being tested is always known. In this report,we chose the most abundant subunit combination in themammalian CNS (a1b2g2L) to determine the potency of TETS

and its analogues. In contrast to TETS (IC50 = 7.9:2.6 mm, n =

12), TETS analogue 4 a, identified as the most active analoguein the SCO assays, only exhibited modest activity (IC50 = 48.0:13.2 mm, n = 10) (Figure 5). Two additional analogues, 2 a and

2 c, which also showed some activity in the FLIPR assays, weredetermined to have comparable inhibitory effect on GABA-in-

duced currents as 4 a at the 50 mm test concentration (4 a43.9:2.1 %, n = 3; 2 a 59.8:20.5 %, n = 5; 2 c 59.4:12.6 %,n = 3). None of the tested analogues were more potent than

TETS (69.4:1.0 %, n = 4) on our receptors of choice.Thus, we have successfully synthesized and identified ana-

logues of TETS that are active on GABAA receptors. Despitebeing several fold less potent than TETS in our electrophysio-

logical studies, these analogues retained the functional groups

required for binding and blocking a1b2g2L GABAA receptors.Additionally, although TETS is a known GABAA receptor inhibi-

tor, its exact binding site and selectivity for the various GABAA

receptor subtypes have not been investigated. Thus, further

testing of these analogues and TETS on additional GABAA re-ceptor subtypes will provide the information on their exact po-

Figure 3. Screening for neuroactive TETS analogues with the FLIPR bioassayin cultured hippocampal neurons. The SCO amplitude after the addition ofTETS at 10 mm or TETS analogues at 30 mm, compared to the addition of0.03 % DMSO vehicle control. The mean amplitude for each well after theaddition is compared to the mean amplitude for that well before the addi-tion, then normalized to the vehicle response. n = 7 or 8 for TETS and ana-logues; n = 12 for vehicle. ANOVA comparison with vehicle control, *** indi-cates p<0.0001, all other differences were not significant from vehicle. Barsindicate standard error of the mean (SEM).

Figure 4. TETS analogue 4 a has lower potency but similar efficacy to TETS.n = 4 for each group, bars indicate SEM.

Figure 5. Effect of TETS and its analogues on GABA-induced currents. Top:Dose-response association curves showing percentage of the a1b2g2L cur-rent blocked by increasing concentrations of either TETS or its analogue 4 a.Bottom: Percentage of current blocked by TETS and its analogues at testconcentration of 50 mm. Percentage blocked for TETS is 69.4:1.0 % (n = 4),4 a is 43.9:2.1 % (n = 3), 2 a is 59.8:20.5 % (n = 5), and 2 c is 59.4:12.6 %(n = 3). Error bars indicate SD.

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tency and selectivity on the GABAA receptors. The fact that an-alogues and TETS exhibit a different potency ranking in the

SCO and the patch-clamp experiments is probably caused bythe fundamentally different nature of the two assays, which in

one case uses Ca2 + signaling as a downstream effect of GABAA

receptor blockade, whereas direct measuring of the blockadeof GABA-induced chloride currents occurs in the other. Anotherpossibility could be that the TETS analogues exhibit differentialselectivity for different GABAA receptor subtypes present in thehippocampal neurons used for the SCO experiments.

Conclusions

In summary, we have developed a general synthetic approachtoward two classes of tricyclic sulfamides structurally related to

the neurotoxic TETS molecule. Bioactivity of some of the syn-

thesized compounds was evaluated by studying their effectson synchronous calcium oscillations in cultured hippocampal

neurons and on receptor currents in cells expressing a1b2g2LGABAAR and the results were compared to those of the parent

TETS compound. Although none of the tested TETS analogueswas as potent as TETS, the potency of some was only an order

of magnitude lower on a1b2g2L GABAAR compared to TETS. In

view of a recent burst of research interest in the sulfamidepharmacophore for the development of new medicines for

a broad spectrum of pharmacological targets[25] the tricyclicsulfamides described here appear to be useful building blocks

for the construction of new drug candidates. Six TETS ana-logues were used for conjugation to the carrier protein and

rabbit immunization. Preliminary data suggest that the pro-

duced antibodies recognized TETS with sensitivities higherthan 5 mg mL@1. Use of these sera for the development of the

first immunoassay for sensitive detection and quantification ofTETS will be published in due course.

Acknowledgements

This work was supported by the CounterACT Program, Nation-al Institutes of Health Office of the Director, and the National

Institute of Neurological Disorders and Stroke, Grant NumberU54 NS079202; and NIEHS, Superfund Research Program, P42

ES004699.

Conflict of interest

The authors declare no conflict of interest.

Keywords: antibodies · cage convulsants · GABA ·neurotoxicity · poly-heterocycles · tetramine

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Manuscript received: February 17, 2017

Revised manuscript received: April 8, 2017

Accepted manuscript online: April 14, 2017

Version of record online: May 30, 2017

Chem. Eur. J. 2017, 23, 8466 – 8472 www.chemeurj.org T 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim8472

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