FORSCHUNG 141 (,HIMIA 47 (1993) Nr." (Mai)
Chim;a 47 (/993) 141-/47 © Neue SchIVeizerische Chemische Gesellscllllft /SSN 0009-4293
Benzodiazepines, the Story of the Antagonist Flumazenil and of the Partial Agonist Bretazenil
Walter Hunkeler: Born 1938 in Altishofen. Walter Hunkeler* Lucerne. 1955-58 apprenticeship as laborato- ry assistentatS;egfr;edAG inZofingen. 1960- 63 study of chemistry at the Institute of Tech- nology of the State of Berne. Burgdorf. and Abstract. The story offlumazenil and bretazenil is a typical example of a serendipitious 1965-69 at the University of Berne. Doctoral drug discovery. In 1979 benzodiazepine antagonists were unknown. No one was thesis under the direction of Prof. H. Sclwlteg- looking for them, but they were discovered nevertheless. Ro 15-1788 was selected for ger. Postdoctoral research at the ETH-Zurich with Prof. A. Eschenmoser. Since 1974 at F. clinical trial. Today 'this compound has the generic name f1umazenil, the trade name Hoffmann-La Roche AG in Basel. Anexate, and is registered in 70 countries. Itis the first specific benzodiazepine receptor antagonist for clinical use. Minor structural modifications of benzodiazepine antago- nists led to partial agonists, compounds with powerful anxiolytic and anticonvulsant properties. Compared with full agonists they have markedly reduced sedative and alcohol-potentiating effects. Bretazenil is in clinical trial; it exhibits promising anxio- other stems from the triazolobenzo-di- lytic activity especially in patients with panic attacks. In open clinical studies it has azepines, which were synthesized and in- shown remarkable antipsychotic activity. Structure-activity relationships and spin-offs vestigated at about the same time at Take- are discussed. da Chemical Industries in Japan and at the Upjohn Company in the United States [4][5]. These are more potent than previ- 1. The Chemical Pedigree of know this, when they started synthesizing ous benzodiazepines. FlumazeniJ (20) simpler analogues of anthramycin, since Alprazolam (4), to take one example, the existence of the BZR had not yet been is more potent in terms of anticonvulsant Chlordiazepoxide (1, Librium) is the established. A numberoftheir compounds and anxiolytic effects. The question that first anxiolytic with the structure of a showed some anticonvulsant and anti-con- now arose was whether annelation by a benzodiazepine. It was synthesized in 1955 flict activity in animal tests. Thus, 5 was tri azo I0 ring would also work in a different and submitted for testing by Leo H. Stern- selected as a candidate for anxiolytic eval- series, e.g., with the Lederle anthramycin bach in 1957 at F. Hofjinann-La Roche, uation in a limited clinical trial in man, but derivative? Would this result in a more Nutley, USA [1]. The time elapsing be- it failed to produce sufficient anxiolytic potent substance? Compound 6 was syn- tween the first pharmacological testing activity. Their results were published [3]. thesized to answer these questions: and introduction on the market was only This is one branch of the pedigree; the In December 1978, the two imida- two and a half years. In 1963, diazepam (2, Valium), the best known and best investi- gated benzodiazepine reached the market place. At this time, when benzodiazepines \ 0 YN'N were a major focus of attention in Nutley, ,~i:-" o~o one group there, Leimgruber, Batcho, and "y Schenker was working on antibiotics. They ~ /, ~ /, ~y;) CIM;0 " isolated anthramycin (3) and elucidated 0 0 its structure [2]. To their great surprise, Chlordiazepoxide Diazepam they discovered that it, too, was a 1,4- benzodiazepine. 0 0 Anthramycin (3) was later found to H3C N ,... , (' 0••.•.•••.••.• have no affinity for the benzodiazepine y " A receptor (BZR). Chemists working at Led- ~ I ~ rt"{) N erie at that time were not in a position to "1 CC;H CIM; 0 "'~~, : I ".6 ~ 0 0 CONH,o ~ ::~" 0 ~ *Correspondence: Dr. W. Hunkeler
F. Hoffmann-La Roche. Ltd. Anthramycin Alprazolam PRPN ]5/235 CH-4002 Basel FORSCHUNG 142 CHIW,\ ~71IQQJ) Nr. SIMail zoesters 7 and 8 were synthesized and rine, had with 6.4 nM an even greater Table I. Billdillg Activities OfCol/1f1ol/luls submitted for testing together with the affinity for the BZR than diazepam. As a SYllthesi~ed 0/1 the Way to Fluma-;,ellil (20) triazolo compound 6. All turned out to be consequence of this preliminary result, inactive in the usual tests for benzodi- compounds without the CI-atom in the omp und Inhibition f 'II'lhu/cpam azepmes. phenyl ring were synthesized. Thus, the blllding i/ll'ifrrJ, IC'Ill' in nM Luckily for us, the benzodiazepine re- first three compounds in this new series ceptor (BZR) had been discovered the already indicated that structure-activity 9 > 1000 year before [6][7]. Usingthe3H-diazepam relationship were different from those de- binding assay it became possible to deter- termined for the older benzodiazepines, 10 > 1000 mine the affinity of a compound for the such as 2. The fact that 7 and 8, with an BZR in vitro. The three compounds were affinity for the BZR, failed to show BZ- 11 > IO{){) tested in this assay, and the triazolo deriv- like activity was not very discouraging ati ve 6 that we had favored proved to have initially, because for metabolic or phar- 12 ;> 1000 no affinity for the BZR. However, the macokinetic reasons in vitro activity is imidazo-ester 8, with a CI-atom in the often not accompanied by in vivo activity. 13 u same position as in diazepam, had an IC50 Therefore, synthetic work in the series of of 62 nM. Compound 7, without the chlo- imidazobenzodiazepinones continued. 14 . .0
0 0 0 Rapid hydrolysisoftheester7, the lead (' /'.... compound, to the inactive acid 9 was a N!J OH ~'~ Ii ° possible explanation for inactivity of 7 in vivo. The amide 10 was also inactive ill 0;,6~ I: 12 13 14 very high affinity for the BZR. In spite of this, the compounds displayed no or only borderline activity in I'il'o. The possibility Scheme J that these compounds might be antago- nists was discussed. Our suspicions were CI,CCH(OH), H NH H,NOH. HCI H AcOH, H,O, increased, when further testing revealed I~ Na,SO., H,O DN~/OH"-': ""N H,SO, H,SO, 0 D ' 0 I that the lead compound 7 also bound to the F ~ I ~ 95% F 89% F~ ~ 80% receptor in vivo, demonstrating that the 0 16 15 17 target, the BZR in the brain, was reached. In early September 1979 a rat was sedated o 1. POCI" CHCI, with diazepam (2), after which it received p-CH,·C.H,N(CH,), H 0 an injection of7. Even before the injection 2 2. CNCH2C02Et CH,NHCH,C0DMF, AcOH H BC;N)I "-': KOtBu,DMF was finished the rat got up and walked off. ~ New tests were immediately devised. In 75% F N\ 78% ,~r~ each situation the effects of diazepam o o Ro 15·1788 and other benzodiazepines were reversed 18 19 flumazenil 20 ANEXATE by 7. ,. NaH, CIPO(OEt), 2 CH2(CO,EI), ~ HC(OC,Hslo 2. Optimization: Flumazenil (20) (Scheme 1) HY'COOEt 1. NaNO" AcOH ~j-COOEt The lead compound 7 was not a pure N =) NaOEt,E!OH 2.H"Pd ()"-) antagonist. Some minor agonistic activity e.g. FBC; N was found, in aconflict test in rats. We \ F~N\ o o wanted a pure antagonist for therapeutic applications and as a scientific tool. In 21 22 23 October 1979 Ro /5-1788, f1umazenil (20) FORSCHUNG 143 CHIMIA ~71Iqq.l) Nr. ~ (Mail was synthesized, and, in January 1980, selected for clinical trial. 2.1. The Synthesis of Flumazenil (20) Starting with 4-fluoroaniline (15) the isatin 17 is synthesized via the Sandmeyer synthesis; isatin is then oxidized with per- acetic acid to the isatoic anhydride 18. Reaction with sarcosine in DMF leads to 24 25 the benzodiazepine-2,5-dione 19. This is converted to the iminochloride by reac- o tion with POCI3. In the key step the imida- zoester is built up by reaction with depro- rfll 0.•...•...... tonated ethyl isocyanoacetate [8]. Since ethyl isocyanoacetate is not very stable, an CIn;N~I :::,... N alternative synthesis based on the synthe- \ sis of midazolam was developed for large o scale-production. Tnthis synthesis diethyl- 26 27 malonate is used. The diester 21 is then transformed to the monoester 22 hy deethoxycarbonylation. Nitrosation and catalytic reduction lead to the amino com- Table 2. Influence of Substitution of the Phenyl Ring 011 the Pharmacological Acti\'ity pound 23. The final carbon atom is intro- Compounu Inhlbillon or H-UI 2.2. Indications for Flumazenil (20) ? Flumazenil (20) is used when the ef- fects of benzodiazepines are no longer desired. For example: - To reverse the effect ofbenzodiazepine overdose. - To reverse sedation in patients who have been given benzodiazepines to diazepam partial flumazenil agonist agonist antagonist help them tolerate an unpleasant med- ical examination such as bronchosco- py. - To terminate anaesthesia induced and tion at C(8) is optimal only for the older concentrated on the synthesis of com- maintained by benzodiazepines. benzodiazepines, like diazepam, but not pounds with chlorine in position seven. - As a diagnostic help in cases of coma for the series of the antagonists. Surpris- of unknown origin; if the patient does ingly, the fluoro-substituted flumazenil is not wake up after being given 2 mg of more potent than the unsubstituted com- 4. Serendipity Again. Partial Agonists flumazenil i. V., benzodiazepine over- pound 14. Therefore, we wondered dose can be ruled out. It is evident that whether this might in fact be the optimum If there are agonists and antagonists flumazenil also works in the case of position. In orderto clarify this we synthe- for a particular receptor, there should also intoxication with zopiclone or zolpi- sized all possible CI-substituted isomers be ligands in the range between the two, or dem, since these two hypnotics that do 24-27. in pharmacological terms, with an inter- not have the structure of a benzodi- We found a major deviation from the mediate intrinsic efficacy. On the one hand, azepine nevertheless bind to the BZR. situation with classical benzodiazepines we had agonists like diazepam (2) and on in that the isomer substituted at position the other hand, antagonists like flumaze- seven (24) was 40 times more potent in a nil (20). What would a partial agonist look 3. The Aromatic Substitution test for antagonism than the one substitut- like? Frankly we had no idea. ed in position eight (25). The isomers The methyl, ethyl, and isopropyl esters At the very beginning of this pro- substituted at C(9) (26) or at C(] 0) (27) are all powerful antagonists. However, gramme it became clear that CI-substitu- were inactive (Table 2). Consequently, we with the propyl ester the potency drops FORSCHUNG 144 (,HIMI,\ 471199.11 NI'.' IMail tenfold. The hexyl ester still has an excel- lent affinity for the BZR ill vitro, but is virtually inactive illl'il'O. The length of the ester is obviously restricted to C,. If this is true, the tert-butyl esters (the dlmethyle- thyl esters) should, by rights, also be po- tent antagonists. So we synthesized the tert-butyl ester 28 with optimum aromatic substitution and found that it is one of the most potent antagonists -at least ten times more potent than flumazenil (20). We 28 29 30 immediately synthesized both tetracyclic analogues but the results were disappoint- ing: both were completely inactive in the most important test for antagonism (Table 3). However, in the receptor-binding test they were just as potent as the tricyclic compound 28. Could they be agonists? Compound 29 was found to lack agonistic activity against penty lenetetrazole-i n- duced seizures in mice. In spite of this, the compound was examined in the conflict test in rats: this time it proved to be consid- erably more potent than diazepam (2). The 31 32 anti-pentylenetetrazole test was repeated bretazenil in rats, and the compound was 75 times Ro 16-6028 Ro 18-2598 more potent than diazepam (2) in the same species. Apparently we were dealing with a difference between animal species. As it turned out, this class of compounds also works in humans. 0 N Had we discovered nothing more than 0" )- a new but more potent diazepam'? No, it if was the first partial agonist acting at the :: r ~ -:N BZR. In some tests, e.g. in the horizontal QS)' y(t<~~ N wire test, 29 antagonized the effect of the N 1 \ full agonist diazepam (2) (Fig. 1). H Sr 0 In this test the animals are lifted by the tail and allowed to grasp a horizon- 33 34 35 tally strung wire with their forepaws and are then released. Untreated control ani- mals heave themselves onto the wire with at least one hindpaw. Diazepam (2) impairs this performance (right side). Flumazenil (20) given 15 min after di- azepam (2) restores control behavior (left 1 q--== side) [10]. 1~ ::::-.. N ~ N (f;\ (f;\ 4.1. Bretazellil (31) CI 0 CI 0 Bretazenil (31), the bromo analogue of 39 40 41 29 was selected for clinical development [II]. It produces anticonflict effects, in- dicative of anxiolytic activity in man, at much lower doses and over a much wider dose range than diazepam. It is also a very potent anticonvulsant. Apart from these 0 0 0 effects, the following additional advan- tages were revealed by animal experi- L(0~ tro~ments: - strongly reduced sedative activity ~ N ~ N - virtually no disturbance of motor con- ~rif' 3 y; N \ ~ CI 0 1231 0 \ 0 trol - no development of tolerance 24 54 55 - no development of physical depend- ence FORSCHUNG 145 - greatly reduced potentiation of the sed- ative effects of alcohol For a thorough discussion of the con- cept of partial agonism see [12]. Bretazenil (31) is a chiral compound. Its configuration is (S). The (R)-enanti- omer 32 has 10,000 times less affinity for the BZR and was not acti ve in the pharma- cological tests at the doses shown (Table 4). Incidentally, it costs less to synthesize the pure (S)-compound than the racemic compound, because natural (S)-proline is cheaper than racemic proline. 5.1,2,4-0xadiazoles As esters are metabolically unstable, their duration of action is relatively short. Two hydrogen-bond acceptors appear to be optimal, since the ketones and ethers corresponding to the esters are considera- bly less potent. We were looking for an alternative. In the case of j3-carbolines (33) it was possible to replace the ester by various heterocycles, e.g. by 1,2,4-oxadi- azoles 34 [13]. In the case of the benzodi- azepines, this concept also worked well. The pharmacological profile was shifted towards more agonism. Soon after our patent application was filed [14] an appli- cation by acompetitor was published [15]. We found the cyclopropyl-substituted Fig. ]. The horizontal wire test I,2,4-oxadiazoles very interesting and, to our relief, they were not the object of a claim by our competitor. Their duration of Table 3. A Small Structural Difference with all Important Change in Pharmaceutical Effect action is longer than that of the corre- amp und 28 29 311 sponding methyl derivatives. The reason for this might be that the formation of Binding radicals, intermediates in the pathway of .. 0 ':L .3 cytochrome-P4S0-catalyzed metabolism, nlag ni 111 0.2 100 inac:t 50lnacl is much more difficult in the case of cyclo- propane than an alkane like 2,2-dimethyl- Ag nism 100 in:1C:1 0.29 O. , propane [16]. HW ntagonisl11 O.()O~ 2.0 30 sl;I'1 Binding: Inhibition of3H-diazepam binding in vitro, rats. IC ()' nM. Scheme 2 S Antagonism: Antagonism of diazepam protection \'s. pentylenetetrazole, mice, EDso in mg/kg, p.o. Agonism: Prevention of pentylenetetrazole-induced convulsions, rats, EDso in mg/kg, p.o. HWT: Antagonism of diazepam in the horizontal wire test, rats, EDso in mg/kg, lUi. a Table 4. Comparison of the Two Enantiol11ers 31 (Illd 32 ompounu 31 32 ~r~CI 0 24 36 Binding 2.2 _5000 gonism 0.07 100 ina t II\R ntill!onism 0.. o ina t I~R ont1. I Test () inacl ::,.. N 0.02 \ YCI ; CI a a ~t Binding: Inhibition of 'W-Diazepam binding in vitro, rats, IC ' nM,p.o. 37 36 so Agonism: Prevention of pentylenetetrazole-induced convulsions, rats, EDso in mg/kg, p.o. HWT: Antagonism of diazepam in the horizontal wire test, rats, EDso in mg/kg, p.o. Conflict test: Fixed ratio 1 conflict, rats, FSD in mglkg, p.o. FORSCHUNG 146 CHI~lIA 47 (1993\ Nr, S (M.i) Scheme 3 6. The Rational Approach Finally? No, But Still More Serendipity. o Alkynes r~00~ We were convinced that high affinity for the BZR can be achieved best if the I) substituent on the imidazolo ring has two F ~ N 'C'H, hydrogen-bridge acceptors. Esters and ~ o 1,2,4-oxadiazoles are typical examples. 52 Replacement of these cornerstones by alkynes is, therefore. not logical at all. Nevertheles we went ahead. Was it the pure joy of chemistry in action? The ester 24 is hydrolyzed to the acid, 51 which is decarboxylated to 36. Iodine is introduced regiospecifically at C(3) with iodine in DMF, CaC01 as base (-.t37), which reacts smoothly 'with a variety of 53 alkynes in DMF with triethylamine as base and bis(triphenylphosphine)palla- dium(II) dichloride and copper([) iodide as catalysts to give compounds of type 38 in yields ranging from 60 to 90% [17]. Table 5. Pharma('(}logica/ Results (!f the A/kyne Deri\,(llil'es 39-41 The pharmacological results were ex- iting (Table 5). Not only did this new ompollnd Inhibition (Jf ntag.(lOl~m of diazepam Prevention of alldi geni' series of compounds have a high affinity 'H-Illimal 'nil pr te II n \'.\. penly- tOIll on\u!'lon in for the BZR, they also displayed a wide hllldlll • ill 1'/11'0. lenetctrHl.Oh:. mice. DBA/ll mi e. p.o .. variety of pharmacological profiles. 39 is I su.l1\l 11./1,. £: D51.1 111 mglkg E/)su in mg/kg a partial inverse agonist, 40 an antagonist with very low intrinsic activity and 41 a partial agonist. 39 1.6 0.24 100 ina 't 7. Structure-Activity Relationship ~o 1.1 0.13 49 The structure-activity relationship in 41 50maCI 0.9 this series of benzodiazepines is very in- 0 I":: ~"\; i{O~i{~ ~.~ N \ CI 0 CI 0 ({(tvCI 0 CI 0 24 28 42 43 0 A 9 0 n i s v m i~i~*CI 0 CI 0 iiCI 0 i:S~CI 0 44 29 45 46 i-t'it~ ii~0 a 0 a 0 CI 0 i1~CI 0 47 48 49 50 Fig. 2. Structure-activ- Agonism ity relationship FORSCHUNG 147 CHIMIA 47 11l)<).1 1Nr.' (Mail teresting. It is fascinating how the slight- barbiturates and ethanol, in the afore-men- [12] W. Haefely, 'Transmitter Amino Acid est modification affects the pharmacolog- tioned horizontal wire test. Receptors: Structures, Transduction, and ical profile. In contrast to flumazenil (20) Ro ] 5- Models for Drug Development', Eds. E.A. FiX. 2 shows twelve compounds: the 45/3 was active against some effects of Barnard and E. Costa, Thieme. New York, 1991, p. 91. ethyl, tert-butyl and cyclopropylmethyl some doses of phenobarbitone and etha- [131 G. Neef. U. Eder, R. Schmiechen. A. Huth, esters as well as the cyclopropyl-oxadia- nol. These results were presented as post- D. Rathz, D. Seidel mann, W. Kehr, D. zoles. In the top row are the tricyclic ers at a meeting of the British Pharmaco- Palenschat, e.T. Braestrup. J.A. Christen- compounds, followed by the tetracyclic logical Society in Southampton [27]. Ap- son, M. EngelsLoft, Eur. Pat. 0054507 as- compounds with the five-membered ring proximately one year later the lay press signed to Schering AG. Berlin. Priority: and those with the four-membered ring. made up a big story out of this compound 17.12.80. The agonistic component increases pro- as a possible alcohol antagonist or sober- [14] W. Hunkeler, E. Kyburz, Eur. Pat. 0150040 assigned to F. H(~fflll(/nn-L(/ Roche AG, gressively from top to bottom and from ing-up pill. The development of such a pill Basel. Priority: 19.01.84. left to right. 24 is not a pure antagonist, as was never considered by us for many [151 e.T. Braestrup, V.A. Christensen. M. En- it also has proconvulsant properties. its reasons, the clinical most important one gelstoft. F. Waetjen, Eur. Pal. 0109921 neighbors, the tricyclic lert-butyl ester and that the toxicity of ethanol is not reduced assigned to Schering AG, Berlin. Date of the tetracyclic ethyl ester are pure antago- by Ro ]5-45]3 [28]. Nevertheless, it re- publication: 30.05.84. nists. By combining the two structural mains an interesting tool in pharmacology [16] G.Boche, H.M. Wlaborsky, 'The Chemi- stry of the Cyclopropyl Group', Ed. Z. elements we obtain the partial agonist 29, [29][30]. Rappoport,John Wiley & Sons, New York. the chloro analogue of bretazeniJ (31). 1987, p. 707. One step down and one to the right we [17] W. HunkeleI', E. Kyburz. M. Meier, Eur. have 49 which was, under the code number Conclusion Pat. 0285837 assigned LaF. H(~trll/(//1II-La Ro 17-1812, for a short time in clinical Roche AG. Basel. trial r 181. It is definitely more agonistic The benzodiazepines have been around [18] B. Saletu, J. Gruenberger, L. Linzmayer. than 29. Compound 50 at bottom right is a for more than 30 years, however, their Meth. Find. Exp. Gin. Pharlllacol. 1986.8. 373. full agonist. It is unable to antagonize medicinal chemistry and pharmacology [19] M.e. Potier, L. Prado de Carvalho, R.H. diazepam in the horizontal wire test, as it have never been more exciting than today. Dodd, c.L. Borwn, J. RossieI', L(I(' Sci. produces an agonistic effect itself in this To end with the author's credo: Creativity 1988,43, 1287. test. and fun are inseparable. [20] A. Persson, E. Ehrin. L. Farde, P. Mindus. G. Sedwal1, Int. J. Nellmsci. 1986,31.223. [21] Y. Samson, S. Pappata, P. Hantraye. J.e. The development of the antagonists and par- Baron, M. Maziere, Electroenceplllliogra- 8. Spin-offs tial agonists was the result of exciting teamwork. phy C/in. Neumphysiol. 1987,67,28. I thank my colleagues for their invaluable contri- [22J H. Carmann. W. HunkeleI'. Eur. Pal. butions. Special thanks go to Marc Meier and Flumazenil (20) is widely used as a 0353754 assigned to F. H(~lJin(//111-LaRo- Beatrix Stauffer who, in my laboratory, synthe- tool in eNS pharmacology. If an effect che AG, Basel. sized all new benzodiazepines mentioned in this cannot be antagonized with tlumazenil it [23] H.F. Beer, P.A. Bltiuenstein. P.H. Hasler. paper and well over 1000 additional compounds B. Delaloye, G. Riccabona, I. Bangerl, W. is not mediated by the BZR. 3H-Flumaze- in connection with this project. nil is a very good ligand for in vitro and in HunkeleI', E.P. Bonetti, L. Pieri, J.G. Ri- vivo studies [19] because the nonspecific chards, P.A. Schubiger ..J. Nile/. Med. 1990, 31, 1007. binding is low. [24] S.W. Woods. J.P. Seibyl, A.W. Goddard, IIC-Flumazenil is an excellent ligand H.M. Dey, S.S. Zoghbi. M. Germine, R.M. for positron emission tomography (PET), Received: March 17,1993 Baldwin, E.O. Smith, D.S. Charney, G.R. which makes it possible to show the pres- Heminger P.B. Hoffer, R.B. Innis, Psych- iar. Res.: Nel/mill/aging 1992,45,67. ence and the distribution of the BZR in [I] L.R. Sternbach, Angew. Chem. 1971,83, [25] B. Kaegi, Schwei::.. Arch. Tierheilk. 1990, man [20][21]. 70. 132,251. Both labelled compounds are synthe- [2] W. Leimgruber, A.D. Batcho, F. Schenker, [26] H. Mohler, W. Sieghart, J.G. Richards, W. 1. Am. Chem. Soc. 1965, 87, 5793. sized from 51 either by alkylation with HunkeleI', Eur. J. PIll/I'll/am/. 1984. 102. [3] W.B. Wright, Jr., H.J. Brabander, E.N. C3H,I or with IICH3T (Scheme 3). 191. Greenblatt, J.P. Day, R.A. Hardy, Jr., J. 123I-Iomazenil (54) is used as ligand [27] E.P. Bonetti, W.P. Burkard, M. Gabl, H. Med. Chem.1978,21, 1087. for the BZR in single photon emission Mohler, Brit. J. PllI/rllll/col. 1985,86, 463P. [4] K. Meguro, Y. Kuwada, Tetrahedron Lett. [28] DJ. Nutt, R.G. Lister, D. Rusche, E.P. computed tomography (SPECT). 54 is also 1970,4039. Bonetti, R.E. Reese. R. Rufener, El/r. J. used as a brain imaging agent in humans [5] J.B. Hester, Jr., DJ. Duchamp, C.G. Chi- Pharmacal. 1988, 151. 356. [22-24]. It is on the market in Europe. chester, Tetrahedron Lett. 1971, 1609. [29] E.P. Bonetti, W.P. Burkard, M. Gabl, W. [6] H. Mohler, T. Okada, Science 1977, 198, Sarmazenil (24) is licensed out as a HunkeleI', H.P. Lorez, J.R. Martin, H. 849. benzodiazepine antagonist for use in vet- Mohler. W. Osterrieder, L. Pieri, P. Polc, [7] R.F. Squires, C. Braestrup, Nature (Lon- eri nary medici ne after anaesthesi a induced J.G. Richards, R. Schaffner, R. Scher- don) 1977,266, 732. with a benzodiazepine [25]. schlicht, P. Schoch, W.E. Haefely, Phar- [8] A. Walser, US Pat. 4118386 (October macal. Biochem. Behl/\'. 1989, 3 I, 733. The azido derivative 55 was synthe- I978) assigned to Ho.fj'mann-La Roche Inc., [30] E.R. Korpi, C. Kleingoor, H. Kettcnmann, sized as a photoaffinity label for the BZR. Nutley, NJ, USA. P. Seeburg, Nature (Lolldoll) 1993,361. [9] W. HunkeleI', H. Mohler, L. Pieri, P. Polc, Under UV-irradiation it binds covalently 356. to the receptor [26]. The 3H-labelled de- E.P. Bonetti, R. Cumin, R. Schaffner, W. rivative is commercially available as 3H Haefely, Nature (London) 1981,290,514. Ro /5-45]3. Yet, Ro ]5-4513 is better [] 0] E.P. Bonetti, L. Pieri, R. Cumin, R. Schaff- ner, E.R. Gamzu, R.K.M. MillieI', W. Hae- known for other reasons: When its phar- fely, Psychopharmacology 1982, 78,8. macological profile was elaborated it was [II] L. Pieri, W. HunkeleI', R. Jauch, W.A. also tested for possible interaction with Merz, G. Roncari, U. Timm, Drugs Future the effects of other CNS depressants, like 1988, 13, 730.