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Mutagenesis vol. 18 no. 4 pp. 321-329, July 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press

Structure–activity relationship of oxadiazoles and allylic structures in the Ames test: an industry screening approach

Wolfgang Muster1, Silvio Albertini and Elmar Gocke

Pharma Research Non-clinical Development, Drug Safety, F. Hoffmann-La Roche Ltd, CH-4070 Basel, Switzerland


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results and discussion
 Conclusion
 Appendix 1. Detailed chemical...
 References
 
In recent years genotoxicity testing has become more and more important in the process of early screening for potential development compounds. In the case that a pharmacologically interesting structure is found to be positive in an in vitro mutagenicity test a straightforward approach starts by sorting out what substructure is responsible for the activity observed in the test. The Ames test is a rapid, convenient test system which has been effectively used in structure–activity relationship studies for mutagenicity, since it can rapidly establish differences in the mutagenic action of isomers and chemical analogs. The lead compound with a benzodiazepine-like structure and close analogs exhibited weak, but unequivocal positive effects in the Ames test (strains TA1535 and TA 100) after metabolic activation by rat liver homogenate fraction (S9). To identify substances within this class of compounds devoid of mutagenic liability an extensive structure–activity investigation was undertaken. More than 50 compounds were tested in the two critical bacterial strains, using a standard plate incorporation and a preincubation modification. It quickly became evident that the benzodiazepine structure was not involved. First hints that the allyl side chain were responsible for the Ames activity had to be refined in a more complex, but clear-cut structure–activity relationship during the course of the experiments. It was shown that all compounds with an allyl side chain, independent of the heterocycle, but surprisingly also all compounds with a specific arrangement of the heteroatoms in the oxadiazole ring, showed positive effects in at least one strain. Based on these investigations it was possible to select pharmacologically active structures without mutagenic liability.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results and discussion
 Conclusion
 Appendix 1. Detailed chemical...
 References
 
Drug-induced anxiolysis, sedation and amnesia are essential for successful therapeutic or diagnostic interventions. For most patients, invasive medical and surgical procedures are associated with some degree of acute anxiety. Even if the intervention itself is not painful, patient anxiety may complicate the procedure for the physician. Moreover, memories of an unpleasant procedure may make the patient reluctant to submit to repetitive examinations.

The optimal combination of sedation, anxiolysis and amnesia is most likely brought about by a benzodiazepine receptor (BZR)-mediated mechanism. Ligands with high intrinsic activity at the BZR (full agonists) have the highest chance of displaying this desirable profile (Hunkeler et al., 1981Go; Leonard, 1999Go; Teuber et al., 1999Go). Midazolam, which became available in the 1980s, has increasingly become the drug of choice for short interventions in ambulatory patients (Reves et al., 1985Go; Kanto, 1985Go; Dundee et al., 1984Go). Midazolam displays the safety and efficacy profiles of a BZR full agonist, while exhibiting a slower onset of action and recovery as well as a more variable inter-individual efficacy than is desirable for the majority of procedures currently performed under conscious sedation. Therefore, an i.v. sedative with shorter onset and duration of action and less variable pharmacological action is needed in short procedures.

Several compounds were synthesized and in the course of their safety evaluation tested in the Salmonella/mammalian microsome assay (Ames test).

The lead compound Ro 47–3555 (8-chloro-7-fluoro-1-(4-diallylaminomethyl-1,2,4-oxadiazol-2-yl)-12,12a-dihydro-9H, 11H-azeto[2,1-c]imidazo[1,5-a][1,4]benzodiazepin-9-one) and close analogs exhibited weak but unequivocal positive effects in tester strains TA1535 and TA100 after metabolic activation by a rat liver homogenate.

The Salmonella/mammalian microsome assay (Ames test) (Ames et al., 1973, 1975; McCann et al., 1975Go; Maron and Ames, 1983Go) is the most widely used test system for mutagenicity studies. This rapid, convenient and inexpensive test system has been effectively used in structure–activity relationship studies, since it can differentiate between isomers and chemical analogs (Shahin, 1987, 1989, 1993).

The combination of test data and an understanding of structural aspects of mutagenicity offer a basis for avoiding human exposure to hazardous chemicals (Miller and Miller, 1977Go).

To identify substances within this class of compounds devoid of mutagenic liability an extensive structure–activity investigation was undertaken.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results and discussion
 Conclusion
 Appendix 1. Detailed chemical...
 References
 
Chemicals
Structures and identification numbers of all 53 tested compounds are shown in Appendix 1.

Allylamine (CAS no. 107–11–9) and diallylamine (CAS no. 124–02–7) were obtained from Aldrich Chemical (Milwaukee, WI). Glyceraldehyde (CAS no. 56–82–6), glycidol (CAS no. 556–52–5) and acrolein (CAS no. 107–02–8) were obtained from Fluka Chemie GmbH (Buchs, Switzerland).

All other test compounds were synthesized in house. Chemical structures were confirmed by IR, NMR and mass spectroscopy. They were pure according to DC analysis and showed <0.3% deviation from the calculated microanalysis values.

Compounds were dissolved in either ultrapure deionized water or dimethyl sulfoxide (DMSO) and tested up to either bacteriotoxic or precipitating concentrations.

Ames test
Tests were performed as described by Maron and Ames (1983Go). The Salmonella tester strains were obtained from B.N. Ames (University of California, Berkely, CA). Overnight cultures (20 ml) were inoculated from frozen aliquots containing 10% DMSO. Strain TA102 was grown with 0.3–0.5 µg tetracycline/ml NB to ensure an adequate copy number of the pAQ1 plasmid (Levin et al., 1984Go; Albertini and Gocke, 1988Go).

All experiments were at least run in duplicate. Reproducibility within and between experiments was very high and did not contradict the classification of the test results.

A positive result is defined as a reproducible, dose-related increase in the number of his+ revertants. The increase should reach a doubling of the number of spontaneous revertants [2- to 2.9-fold, weakly positive (+); 3- to 4.9-fold, positive (++); ≥5-fold, strongly positive (+++)]. A 1.5- to 1.9-fold increase in the mutant frequency is classified as ‘borderline’ (w+), which is especially relevant in tester strain TA100, which has a higher spontaneous mutant frequency. A reproducible, dose-related increase in strain TA100 above 1.5-fold that of the negative control leads to a positive classification, whereas in TA1535 a w+ finding has to be accompanied by a stronger response in another test modification.

Rat liver S9
Rat liver S9 was prepared according to Ames et al. (1975Go). Liver enzymes were induced with either Aroclor 1254 (used in V79/HPRT tests) or phenobarbital (Siegfried, Switzerland; diluted in pyrogen-free bi-distilled water) and ß-naphthoflavone (SERVA, Heidelberg, Germany; suspended in corn oil) following the method of Matsushima et al. (1976Go). The protein contents determined according to Lowry et al. (1951Go) were in the range 30–40 mg/ml. The preparations were shown to be sterile.


   Results and discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
Conclusion
 Appendix 1. Detailed chemical...
 References
 
A series of midazolam follow-up candidate compounds was tested using seven tester strains (Salmonella typhimurium TA1535, TA1537, TA97, TA98, TA100 and TA102 and Escherichia coli WP2 uvrA).

One of the lead compounds, an imidazo-diazepinone derivative, namely Ro 47–3555, showed weak but unequivocal positive effects in the Ames test (strains TA1535 and TA100) after metabolic activation by an exogenous rat liver homogenate fraction (Table I).


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  		Table I.. Mutagenicity evaluation of Ro 47–3555 in the bacterial mutagenicity assay (Ames test) (values are means ± SD of four plates)
 
Subsequently, to elucidate the genotoxicity of Ro 47–3555 the full standard genotoxicity test battery, except a second in vivo test, was performed (Table II).


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  		Table II.. Genotoxicity test results for Ro 47–3555
 
The weak but unequivocally positive finding in the Ames test was considered as a potential liability for the development of a sedative and anxiolytic compound, despite the absence of genotoxic activity in the other four tests of the battery. Although an isolated positive Ames result is normally considered to be of no significant regulatory impact, such activity is avoided if possible to be on the safe side during further drug development.

Therefore, an extensive structure–activity relationship investigation was undertaken with the goal of identifying substances within this class of compounds devoid of mutagenic activity.

Compounds were tested using Salmonella typhimurium TA1535 and TA100 in the presence and absence of S9 using two different methods: a standard plate incorporation assay and a preincubation modification, known to be more sensitive for several classes of compounds. The responsiveness of the tester strains was demonstrated by using appropriate positive controls. The results are summarized in Table III.


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  		Table III.. Summary table of all Ames data, sorted according to positive results (see Appendix for detailed chemical structures)
 
Allyl component
Initially our efforts concentrated on the two allyl side chains attached to the heterocycle via a methylene group. Pairwise comparison of several structures led to the hypothesis that the allyl groups were responsible for the mutagenic activity in the Ames test (see Table IV). The role of the allyl groups becomes clear on stepwise elimination. The secondary amine with one allyl group (Ro 47–3539) showed decreased mutagenic activity in both strains (compared with Ro 48–3848), and the primary amine without allyl side chains (Ro 47–3406) was clearly negative.


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  		Table IV.. Pairwise comparison of several tested structures
 
For substances containing allyl or allylic side chains a direct mechanism of action involving an allylic cation which then reacts with nucleophilic centers of nucleic acid bases has been discussed in the literature (SN1 mechanism). Directly acting allyl compounds need a good leaving group at the {gamma}-carbon to facilitate the formation of an allylic cation (Eder et al., 1980Go). The N-allylic side groups as present in our test compounds cannot be expected to act directly because the secondary (-NHR) and tertiary (-NRR) groups are chemically very poor leaving groups. A metabolic epoxidation of the double bond in olefinic structures as shown in the case of vinyl chloride has been regarded as the common principle leading to their mutagenic and carcinogenic activities (Lijinsky and Andrews, 1980Go). Since the mutagenic activity was predominantly observed with metabolic activation, an epoxidation mechanism seems to be more likely than the SN1 mechanism via an allylic cation.

Heterocyclic aromatic ring
Further investigations showed that the allyl side chains were the major critical substructures, but their removal was not sufficient to fully get rid of the mutagenic activity. The aromatic heterocycle also caused a weak mutagenic activity, even in the absence of allylic side chains, when the heteroatoms had a specific arrangement. It is remarkable that only the position in the heterocycle is decisive for the observed effects. The 1,2,4-oxadiazol-3-CH2-N-derivatives (O-N arrangement) without allylic side chains were all found to be positive in tester strain TA100 using the preincubation version. If the amino side chain is attached to the 5 position, i.e. 1,2,4-oxadiazol-5-CH2-N-derivatives (N-O arrangement), all tested structures without an allyl group were negative. It might be hypothesized that 1,2,4-oxadiazoles in the O-N arrangement are more prone to ring opening than the corresponding 1,2,4-oxadiazoles in the N-O arrangement. Some metabolism studies of 1,2,4-oxadiazole rings have demonstrated that reductive or hydrative cleavages can occur (Gyarmati et al., 1976Go). Yabuki et al. (1993Go) described a novel metabolite with a different ring-opening pattern leading to a cyanamide. Cyanamide itself tested positive in the Ames test and the cyanamide substructure is regarded as a structural alert by Ashby and Tennant (1988, 1991). Such a mechanism might be an explanation for the observed mutagenic activity of the O-N arrangement of 1,2,4-oxadiazole compounds without allyl substituents.

Furthermore, there seems to be an interaction between these aminoallyl side chains and the aromatic heterocycle. By comparing the structures of Ro 47–1706 and Ro 48–1086 it becomes obvious that increasing the chain length between the aromatic heterocycle and the aminoallyl side chain from C1 to C2 reduces the mutagenic activity. Further evidence for such an interaction between the subgroups was observed by comparing the effects for allyl-containing compounds with an O-N or N-O arrangement in the heterocyclic ring. Generally the structures with an O-N arrangement showed higher mutagenic activity than with the N-O arrangement. It might be argued that there is an influence of the aromatic system on the allyl groups which facilitates the activation step. There are a variety of different mechanisms ({pi}-electron interactions, N-oxide formation in the heterocycle, influence on enzymatic epoxide formation, etc.) which might be hypothesized, but there are no experimental results available which allow preference to be given to one theory over another.


   Conclusion
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusion
Appendix 1. Detailed chemical...
 References
 
In conclusion, it appears that the mutagenic activity based on the allyl side chain and its amplification by the aromatic heterocycle seems not to be mechanistically directly related to the effects observed for non-allylic compounds with the O-N arrangement. It can be assumed that the complex structure–activity relations are a consequence of the two different but probably interacting mechanisms (Figure 1).



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  		Fig. 1. Structure–activity relationship (SAR) of 1,2,4-oxadiazol-CH2-N-allyl derivatives in the Ames test. The complex structure–activity relationship (for detailed results see Tables III and V) can be summarized as follows: (i) all compounds with an allyl group(s) adjacent to an oxadiazole ring were mutagenic in at least one strain; (ii) all compounds with the oxadiazole ring in the ‘O-N’ arrangement showed at least weak mutagenic effects in strain TA100, preincubation version, independent of the side chain used; (iii) compounds containing allyl group(s) in the side chain and O-N arrangement in the aromatic heterocycle are more strongly positive than N-O arranged heteroaromatics; (iv) longer chains, i.e. a C2 bridge, between the allyl groups and the oxadiazole ring reduces the mutagenic effect; (v) replacement of the oxadiazole ring by other 5-membered heterocycles leads to a negative outcome, as long as no allyl groups are attached; (vi) no influence of the basic imidazo-diazepinone structure could be observed, with broad variations possible without any influence on the mutagenic activity.

 
In the end, two structures were selected for further development based on these structure–activity investigations which turned out to be free of any genotoxic liability in the complete standard genotoxicity test battery and showed promising pharmacological activities.


   Acknowledgements
 
We wish to thank Dr W.Hunkeler, Dr H.Stadler, Dr U.Widmer and Dr B.Büttelmann for synthesis work and Dr U.Widmer for very helpful discussions about the chemical aspects of this manuscript. Furthermore we are indebted to Ms C.Kieffer, Ms S.Marget-Muller and Ms J.Muller-Freudenreich for their excellent technical assistance.


   Appendix 1. Detailed chemical structures of test compounds
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusion
 Appendix 1. Detailed chemical...
References
 



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  		Table V.. Summary table of the obtained structure-activity relationship
 

   Notes
 
1To whom correspondence should be addressed. Tel: +41 61 68 81580; Fax: +41 61 68 88418; Email: wolfgang.muster{at}roche.com


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusion
 Appendix 1. Detailed chemical...
 References
 

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Received on March 25, 2002; accepted on March 31, 2003.


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