Eur J Med Chem (I 997) 32,27-38 0 Elsevier, Paris 27 1-Oxa-3,8-diazaspiro[4S]decan-2-one derivatives with a potent inhibitory effect on neural Ca-uptake and protecting action against TET-induced brain edema and memory and learning deficits? E Tdthl, B Kiss*, A Gere, E K&-p&, J Tiirley, 6 Pdosi, A Kis-Varga, M Padczai, S Szabd, D Gro6, I Laszlovszky, E Lapis, K Csomor, L Szporny Chemical Works of Gedeon Richter Ltd, PO Box 27, H-1475 Budapest, Hungary (Received 31 October 1995; accepted 10 July 1996) Summary - A series of novel I-oxa-3,8-diazaspiro[4.5]decan-2-one derivatives 8-71 were synthesized. Several representatives were examined for their in vitro inhibitory action on 4sCa-uptake into cerebrocortical synaptosomes depolarized by potassium and veratrine and on triethyltin-induced brain edema. Of the compounds displaying most potent inhibitory action on veratrine-induced ‘Wa-uptake into cerebrocortical synaptosomes and outstanding protection against triethyltin chloride (TET) induced brain edema in rats, four were tested for their antihypoxic action and prevention of learning and memory deficits elicited by various agents (eg, electroshock, diazepam, scopolamine, carbon dioxide and normobaric hypoxia). In some of these tests the four compounds showed remarkable protecting/restoring activity. It is assumed that the beneficial effects of these compounds in brain edema formation are probably related to their actions on intracellular Ca*+- and Na+-movements. These cellular effects may also play role in their antiamnesic actions, but other mechanisms may also be involved. On the basis of results obtained in the tests used, the pharmacological profile of the novel 1-oxa-3,8-diazaspiro[4.5]decan-2-one derivatives seems to differ from that of known Ca 2+-antagonists such as flunarizine or nimodipine and Na+-channel blocker, phenytoin. Out of the four most active compounds tested, one (44) was selected for further investigation and this compound is currently under preclinical development with the code name of RGH-2716 or TDN-345. 1-oxa-3,8-diazaspiro[4S]decan-2-one I neural CaZ+ -uptake inhibition Introduction Brain injury of either ischemic or traumatic type rapidly causes disturbances in cellular energy production, profoundly alters many processes related ion homeostasis (eg, it greatly increases intracellular Ca?+ concentration and Na+-influx) and leads to enhanced water permeability (ie, edema) [l] and thus may produce functional consequences manifested in deterioration of cognitive performance (ie, various deficits in learning and memory functions). Compounds interfering with one or more components of the above pathological cellular events or their sequence could have therapeutical potential as neuroprotective agents. l-Oxa-3,8-diazaspiro[4,5]decan-2-ones substituted at different positions have been described as compounds possessing various pharmacological effects, *Correspondence and reprints tThis paper is dedicated to the memory of the first author, E T&h, who died after submission of the manuscript. / TET edema / antiamnesic action eg, bronchodilatory, adrenolytic, antihistamine [2, 31, antihypertensive [4] and tachykinin NK, receptor antagonistic [5] activity. We have previously found that 4-methyleneor 4-hydroxy-4-methyl-l-oxadiazaspiro[4S]decan-2-ones remarkably inhibited “5Ca uptake into depolarized cerebrocortical synaptosomes and they possessed antihypoxic action in various experimental situations [6]. In order to obtain more potent neural 4sCa uptake inhibitory compounds with beneficial actions on brain metabolism and with memory enhancing or restoring potency, further modifications of these structures were carried out that resulted in novel compounds having potent inhibitory action on depolarization-induced 4jCa uptake into rat cerebrocortical synaptosomes. Some representatives of these compounds (eg, 12, 44, 54, 61) offered complete protection against triethyl-tin chloride (TET) induced brain edema in rats and they showed prevention of learning and memory functions in mice against deleterious. effects of different interventions (eg, administration of diazepam, scopolamine, inhalation of carbon dioxide or hypobaric hypoxia) known to cause learning and memory deficits. 28 Chemistry The novel 1-oxa-3,8-diazaspiro[4.5]decan-2-ones described in this paper can be obtained by several different ways (eg, schemes l-3). The structures and physical data of the compounds are listed in table I. Method 1 (A,B) (scheme 1) involved the condensation of 4-methylene-1-oxa-3,8-diazaspiro[4.5]decan2-one derivatives 2 [6] with a phenylalkane derivative of formula 1, wherein Y is a leaving group (eg, a mesyl, tosyl group or a halogen (chlorine or bromine)) in the presence of a base (eg, inorganic or tertiary organic bases may be used) in an inert organic solvent at reflux, optionally in the presence of an alkali metal iodide catalyst. This gave compound(s) 3 which, upon treatment with aqueous mineral and/or organic acids, could be hydrated to the corresponding 4-hydroxy-4methyl-1-oxa-3,8-diazaspiro[4.5]decan-2-one derivatives 4. The phenylalkane derivatives 1 were either commercially available or synthesized by standard methods 5 Scheme 2. Method 6 2. Reagents: a) CH,ONa/toluene; 50 “C; 65588%. r2, 71. Method 2 (scheme 2) involved the reaction of 4-ethynyl-4-piperidinol derivatives 5 with the appropriate isocyanates performed with CH,ONa in toluene or K,CO, in DMF or CH,COOK in 2-picoline, which afforded the corresponding 4-carbamoyloxy-4-ethynylpiperidine derivatives 6. Under the basic reaction conditions compounds 6 were cyclized intramolecularly (without isolation) to the corresponding l-oxa3,8-diazaspiro[4.5]decan-2-one compound 3. The ethynylcarbinols 5 were prepared by ethynylation of the suitably substituted 4-piperidone derivatives as described elsewhere [8]. The isocyanates were commercially available. In Method 3 (A,B) (scheme 3), the reaction of the appropriate 1,3-dioxolan-2-ones 7 with primary Scheme 3. Method 3A. Reagents: a) an excess of R3-NH2 as solvent, or xylene; 60-89%. Method 3B. Reagents: b) 4-methylbenzenesulphonic acid, xylene, reflux; 8X-96%. amines provided the 4-hydroxy-4-methyl-I-oxa-3,8diazaspiro[4.5]decane derivatives 4. Subsequent dehydration of the carbinol 4 to give 3 was accomplished by refluxing and stirring a mixture of 4 and 4-methylbenzenesulphonic acid catalyst in xylene under a water separator. The dioxolanes 7 were obtained by acid catalysed cyclization of the 4-carbamoyloxy-4ethynylpiperidine derivatives of the formula 6, eg, with dry hydrogen chloride gas, and subsequent hydrolysis of the intermediate 2-imino- 1,3-dioxolane hydrochlorides as described elsewhere [8]. The appropriate amines were commercially available. Pharmacology 3 4 Scheme 1. Method IA. Reagents: a) 4-methyl-2-pentanone, K,COI, KI, reflux; 32-89%. Method IB. Reagents: b) HCOOH/3 M HCl; 5 “C; 94-99%. Thirty members of the substituted I-oxa-3,8-diazaspiro[4.5]decan-2-ones 8-71 falling into three major structural groups were tested for their inhibitory effects on Wa-uptake into cerebrocortical synapto- 29 Table I. Physical data of I-oxa-3,8-diazaspiro[4S]decan-2-one Compound RI RI R3 8 H 4-a 4-F H 4-F H 4-F 4-F H 4-Cl 4-F H 4-F 4-F 4-F 4-F H 4-Cl 4-F 4-F 4-F 4-F 4-Cl 4-F 4-F 4-F 4-F H 4-a 4-F 4-F 4-F 4-F H 4-Cl 4-F 4-F 4-F 4-F 4-F 4-F 4-F H H H H 4-F-Phenyl H H 4-F-Phenyl H H H Phenyl 4-F-Phenyl 4-F-Phenyl H 4-F-Phenyl H H H 4-F-Phenyl H 4-F-Phenyl H H 4-F-Phenyl H 4-F-Phenyl H H H 4-F-Phenyl H 4-F-Phenyl H H H 4-F-Phenyl H 4-F-Phenyl 4-F-Phenyl 4-F-Phenyl 4-F-Phenyl CH, CH, CH, CK CH, CH, CK CK C&L C,Hs C,Hs GHj GK 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 CZHj GHs GH, n-Q& n-C,H, n-C& n-C,H, n-C,H, r2-C3H, i-C,H, X,H, X,H, i-C3H, X,H, n&H, rX,H, n-C,H, n-C4H, n-C,H, n-C,H, t-C,H, t-C,H, t-C,H, t-C,H, t-C,H, t&H, n-C& n-WL n-C& R4 derivatives. R-5 =CHZ =CH, =CH, =CH, =CH, OH CH, OH CH, OH CH, =CH, =CH, =CH, =CH, =CH, =CH, OH CH, OH CH, =CH> =CH, =CH, =CH, OH CH? OH CH, =CH, =CH, =CH, OH CH, OH CH, =CH, =CH, =CH, =CH, OH CH, OH CH, =CH, =CH, =CH, =CH, OH CH, OH CH, =CH, =CH, OH CH, IZ MP (“Cl Formula 1 1 1 2 3 1 1 3 1 1 1 2 2 3 1 3 1 1 1 3 1 3 1 1 3 1 3 1 1 1 3 1 3 1 1 1 3 1 3 3 3 3 119-120 118-119 74-75 35-36 90-9 1 184-185 > 300 220-223” 121-122 106-107 83-84 93-94 152-154 ill-112 > 300 23 l-235” 97-98 82-83 78-79 107-10s 246-248” 134-136” 102-103 103-104 118-l 19 > 300 25 l-253” 70-7 1 86-87 91-92 94-95 > 300” 218-221” 106-107 104-105 93-94 90-92 286-288a 2 1 8-220a 121-122 106-107 107-109 C,,H&Q C,,H2,C1N,02 C,,H,,FN,O, C ,&N~O~ GA&N@~ C JLJW~ C,,H,,FN,Oq-HCl C,,H,,,F,NZO,-HCl C ,JW~O~ C ,BH,,CINZOl C ,&JWO~ CzsH,,,NzQ CZ,H,,F,N,Oz.maleate G&&NO~ C,,H,,FN,O,.HCl C,,H,ZF2N20,.HCl ‘XL&Q C,,H&lN,O, C,,H,sFN@~ G,H,JW,Q C,,H,,FN,O,.HCI G7H,,F,N,0,.HC1 C,,H,,ClN,O, GWWQ C,,H,,W:O, C,,H,,FN,O,.HCl C,,H,,F,N,O,.HCl G&,&OI CZOH&lNZOZ ‘%H#N@~ C28H33FZNZO> C,,H,,FN,O,.HCl C,,H,,F,N,O,-HCl G&&O, GJWWO, Cx&FN,O, G,H,P,N,O~ C,,,H2,FN,0,.HCl C,,H,,F,N,O,.HCl Ci,H,,,F,N,O,.maleate CyH4hF2NZOZ.maleate C,,H,,F,N,O,.HCl Method 79 81 91 68 76 98 97 78 85 87 92 58 55 88 98 96 77 81 86 89 82 97 77 88 74 60 96 78 75 96 80 89 87 83 68 76 85 98 95 92 82 88 IA 1A 3B 2 1A 1B 1B 3A 1A 1A 3B 1A 1A 2 1B 1B 2 2 1A 1A 3A 1B 2 1A 1A 3A 1B 2 2 3B 2 3A 3A 1A 2 2 1A 1B 1B 3B 1A 3A 30 Table I. Continued. - Compound RI R2 R3 50 51 4-F-Phenyl H H H 4-F-Phenyl H H 4-F-Phenyl H H H 4-F-Phenyl H 4-F-Phenyl H 4-F-Phenyl H 4-F-Phenyl H 4-F-Phenyl H 4-F-Phenyl n-C ,“H,, Cyclohexyl Cyclohexyl Cyclohexyl Cyclohexyl Cyclohexyl Cyclohexyl Cyclohexyl Phenyl Phenyl Phenyl Phenyl Phenyl Phenyl Benzyl Benzyl Benzyl Benzyl 1-Naphthyl 1-Naphthyl 1-Naphthyl I-Naphthyl 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 4-F H 4-Cl 4-F 4-F 4-Cl 4-F 4-F H 4-Cl 4-F 4-F 4-F 4-F 4-F 4-F 4-F 4-F 4-F 4-F 4-F 4-F RS n CH, =CH2 =CH, =CH2 =CH, OH CH, OH CH, OH CH, =CH2 =CH, =CH, =CH, OH CH, OH CH, =CH, =CH, OH CH, OH CH, =CH> =CH, OH CH, OH CH, 3 1 1 1 3 1 1 3 1 1 1 3 1 3 1 3 1 3 1 3 1 3 R4 OH MP PC) 109-111 152-1.53 134-135 125-126 122-123 310-315” >315 258-260” 137-138 134-135 146-147 125-126 > 300 274-276” 100-101 81-82 290-295” 177-179a 160-161 127-128 288-290” 180-182” Formula C,,H,,F,N,O,.HCl GJL&O~ C2,H,,C1N,02 C,ZH,,FN,O: C,,,bP,N,Q C,,H,,ClN,03-HCl C,,H,,FN,O,.HCl G,H,,W~Q-HCl G,WW, Cs2H,,C1N,02 CZIH,,FN201 G,Hx,W@~ C,,H,,FN,O,.HCl C,,H,,F,N,OI.HCl GJWN@~ C, ,WW,O, C,,H,,FN,OR.HCI C,,H,,F2N,0,.HCl G,H,sFN,Q GJWJ’W~ C,,H,,FNZO,.HCl C,,H,,W,Q~HCl Yield ( 56) Method 97 75 92 83 81 94 87 99 72 85 78 81 97 97 32 88 98 82 65 78 97 94 1B 2 3B 2 1A 1B 3A IB 1A 2 1A 2 1B 1B 1A 3B 1B 3A 2 IA 1B 1B “Decomposed. somes depolarized by 60 mM K+ or 20 PM veratrine. Further, 13 representatives of the three groups were also examined for their protective effect against the formation of brain edema in rats intoxicated with subchronic administration of TET. The most active compounds in these tests (12, 44, 54, 61) were examined for antihypoxic actions (in hypobaric hypoxia) using SH rats and in various experimental situations where deficits in learning and memory processes were produced by various agents such as diazepam, scopolamine, carbon dioxide, electroshock in mice or by normobaric hypoxia in SH rats. The four selected compounds were more active in most of these tests than flunarizine, nimodipine (Ca2+-antagonists) and phenytoin (Na+-channel blocker) that were used as reference substances. Results and discussion Based on the general formula given in table I the most characteristic representatives of 1-oxa-3&diazaspiro- ]4S]decan-2-ones included in this report were selected for pharmacological experiments. These compounds were divided into three major groups (groups 1,2 and 3) according to the number of IZ (table I). The effects of these selected compounds on the WZa-uptake into cerebrocortical synaptosomes depolarized by different depolarizing agents (ie, by 60 mM K+ or 20 FM veratrine) are summarized in tables IIIV. Flunarizine (Caz+-antagonist of diphenylpiperazine type), nimodipine (dihydropyridine type Ca*+-antagonist) and phenytoin (a prototype Na+-channel blocker) were also tested for comparison. The examples given in group 1 (table II), where 12 = 1 and Rt = F and R* = H in all cases, did not or only slightly inhibited 45Ca-uptake induced by potassium depolarization whereas their action on veratrineinduced QCa-uptake was somewhat more expressed especially when R3 was a bulky, aromatic group (eg, I-naphthyl, compound 68). Replacement of methylene group at positions R4 and Rs with R4 = OH and Rs = CH, in these analogues resulted in a fairly potent inhibitor of veratrine-induced Wa-uptake with IC,,, of 1.34 PM (compound 70). Reference compounds, flu- 31 II. Effects of compoundsbelonging to group 1 and reference compoundson the in vitro synaptosomal“Taz+-uptake stimulated with 60 mM K+ or 20 pM veratrine. Table Compound 10 37 64 68 14 39 66 70 Flunarizine Nimodipine Phenytoin R’ R2 Rd R3 4-F 4-F 4-F 4-F 4-F 4-F 4-F 4-F CR n-C,H, Benzyl 1-Naphthyl CH, n-C,H, Benzyl LNaphthyl Rj =CH, =CH, =CH2 =CHZ OH CH, OH CH, OH CH, OH CH, X&uptake inhibition (IC,J’ K+stimulation Veratrine stimulation >7 30 77 30 7 30 >7 30 >7 30 77 30 40.2 48.3 22.6 208 =300 7 30 19.0 16.0 9.0 > 30 7 30 4.7 I .34 1.3 4.7 24b “IC,, values are given in pM; hpercentageinhibition at 50 pM. Table III. Effects of compoundsbelonging to group 2 on the in vitro synaptosomal+Wal+-uptake stimulatedwith 60 mM K+ or 20 yM veratrine R1 cl Cb+C&-CH,R2’ Compound 11 19 20 RI H H 4-F R2 H Phenyl 4-F-Phenyl R3 R4 CK C,J& W& Rj =CH, =CH, =CH, 4jCa-uptake inhibition (IC,,)” K+stimulation Veratrine stimulation 100 80.2 18.1 7 30 1.75 0.97 7 “IC,,, values are given in PM. nimodipine and phenytoin also displayed moderate or no inhibition in QCa-uptake induced by potassium depolarization while the actions of flunarizine and nimodipine, but not that of phenytoin, in the inhibition of veratrine-induced Wa-uptake were comparable to those of most active analogues in this group. In group 2 (table III) where n = 2, introduction of R2 = phenyl and especially that of R? = 4-F-phenyl narizine, (compound 20) greatly improved the ability to inhibit veratrine-induced Wa-uptake inhibitory activity (IC,, = 0.97 FM) even though it had a relatively small alkyl substituent at the R3 position. Compound 20 was even active in inhibition of potassium depolarization induced Wa-uptake (I& = 18.1 FM). These results clearly indicate that at least three structural elements, ie, length of alkyl chain between the diazaspiro[4S]decan-2-one and phenyl moieties, 32 Table IV. Effects of compounds in vitro synaptosomal dTa’+-uptake KC or 20 pM veratrine. belonging to group 3 on the stimulated with 60 mM Fm OH-OH*--CH2-w--N Or” 37 R4 KY RS’R3 F Cornjound R3 R4 R.7 “sea-uptake inhibition (G”Y K+Veratrine stimulation stimulation 12 21 27 32 38 44 54 61 69 15 23 29 34 40 46 57 63 67 71 CH, GH, n-C,H, i-C,H, I&H, t-C,H, Cyclohexyl Phenyl 1-Naphthyl CK GHs n-W% i-C,H, n&H, t-C,H, Cyclohexyl Phenyl Benzyl 1-Naphthyl =CH? =CH, =CH, =CH, =CH, =CH, =CH, =CH, =CH, OH CH, OH CH, OH CH, OH CH, OH CH, OH CH, OH CH, OH CK OH CH, OH CH, 17.7 6.7 8.2 9.8 5.8 5.8 5.3 2.1 32.6 11.3 9.9 24.4 7.4 6.1 18.5 31.4 40.4 15.2 15.2 1.60 0.30 0.37 1.34 0.32 0.36 0.14 0.26 0.38 0.84 0.87 0.7 1 0.93 1.66 0.69 0.59 0.93 0.71 0.23 “IC,,, values are given in PM. substitutions on diazaspiro[45]decan-2-one part and R2-substitution(s) could be important to obtain compounds with potent Wa-uptake inhibitory activity. Indeed, in group 3 (table IV) where length of alkyl chain was extended to 4 (ie, n = 3), RI = F and R2 = 4-fluorophenyl in all examples, several potent inhibitors of synaptosomal Wa-uptake were found (IC,, values varied from 0.23 and 1.66 pM for veratrineinduced uptake and for 2.1 and 40.4 PM for potassium-induced uptake, respectively). Compounds with R4R5 = methylene showed somewhat higher inhibitory activity in case of potassium depolarization compared to those where R4 = OH and Rs = CH,, but no such a tendency could be observed in case of veratrine depolarization. Similarly, no clear-cut relationship could be seen between the quality and bulkiness of substitutions at position of R3 and the WZa-uptake inhibitory activity of the compounds. Three members of compounds belonging to group 1 were examined for their protecting action against edema formation induced by TET intoxication. Flunarizine, nimodipine and phenytoin were again used as reference compounds. These results are depicted in table V. Out of these compounds only 70, which possesses a 1-naphthyl substitution at position R3, produced protection of 55%. This might correspond well to the inhibitory activity seen in the in vitro Ca2+uptake experiments. On the other hand, however, compounds from group 2 (19, 20) with IC,, values in micromolar range for veratrine-induced uptake of Ca2+ (0.97 and 1.75 PM, respectively) did not show any protecting activity against TET-induced alterations. The poor absorption of the compounds might explain the lack of effect but the toxicity observed with 20 apparently contradicts to this assumption. However, all compounds selected from group 3 as potent inhibitors of in vitro synaptosomal Ca2+ uptake proved to be very effective against TET-induced water content increase in rat brain; the compounds tested offered practically complete protection. This effect appeared in all compounds regardless of their substitution at positions Rx, R4 or Rs. Moreover, these compounds not only protected against edema formation but the gross neurological deficits (ie, the greatly reduced righting and grasping reflex, reduction in motor activity) and body weight loss caused by TET administration also completely or almost completely disappeared. In case of compound 44 the above effects were greatly dose-dependent between 5 and 100 ymol/kg and already 5 ymol/kg produced a protection of 33% against TET-induced edema. The protecting activity in edema experiments also correlated with the change in brain Na+ content. Namely, a 44.5% decrease was seen in brain Na+ content already after administration of 5 pmol/kg (= 2.3 mg/kg) 44 compared to rats treated with TET, whereas higher doses (ie, 10, 25, 50 and 100 pmol/kg) resulted in a complete restoration in Na+ content (data not shown), TET intoxication, like ischemia, is known to cause several cerebral metabolic changes in the brain accompanied with profound behavioural effects in addition to cerebral edema formation and increase in Na+-content [ 17, 181. Therefore, it is reasonable to assume that the beneficial effects of this particular group of 1-oxa-3,8-diazaspiro[4.5]decan-2-ones found in TET-edema experiments might be related mainly to their inhibitory effects on transmembrane Na+-flux and/or Cal+-movements but their protective actions on intermediary metabolism of nerve cell may also contribute to the anti-edema effects. Regarding the inhibitory effects on Na+- and Ca2+-movements, the assumption is largely supported with the notion that out of reference substances phenytoin (an Na+-channel blocker) and flunarizine, that is also known to directly 33 Table V. Effects of various I-oxa-3,8-diazaspiro[4S]decan-2-ones TET-induced brain edema in rats. Group with different substitutions and reference compounds on R” R5 Inhibition (7~) CH, CK CH, 10.1 4.0 5.53 Compound” RI R2 R3 :t 70 4-F 4-F 4-F H H H CH, n-C,H, 1-Naphthyl :; 20 H H 4-F H Phenyl 4-F-Phenyl CK GK GK =CH, =CH, =CH2 8.6 7.1 1.9” 4-F 4-F 4-F 4-F 4-F 4-F 4-F 4-F-Phenyl 4-F-Phenyl 4-F-Phenyl 4-F-Phenyl 4-F-Phenyl 4-F-Phenyl 4-F-Phenyl CH, n-C,H, =CH2 106.3b 92.3b 108.6b 113.7b 95.0b 85.9b 94.3b Group 1 Group 2 Group 3 t; 44 2’: :z t-C,H, Cyclohexyl Phenyl 1-Naphthyl t-C,H, OH :i =CH2 =CH, =CH, =CH, =CHZ OH CH, Flunarizine Nimodipine Phenytoin 67.6b -20.2 79.8b “All compounds were administered orally in a dose of 0.1 mmol/kg twice a day for 5 days, 1 and 6 h after 2.5 mg/kg TET; bsignificantly different from TET + vehicle-treated group, P < 0.00 1; ctoxic at the dose investigated. interfere with Na+-channels [19], produced almost 80 and 67% protection, respectively, against TET-induced brain edema whereas nimodipine had no protective activity and rather slightly enhanced TET-induced edema and other neurological symptoms. Compounds found to be most effective in TET experiments either in prevention of brain edema or neurological deficits (ie, 12, 44, 54 and 61) were selected for further studies. The relatively low acute toxicity of these compounds (oral LD,, > 1000 mg/kg in all four cases) also contributed to their selection. Table VI shows the protecting actions of the selected and reference compounds against hypobaric hypoxia-induced lethality in SH rats. Out of the four, two (ie, compounds 12 and 44) showed antihypoxic actions manifested in the increase of survival rate and these actions were apparently dose-dependent in the dose range studied. In this test only nimodipine proved to be active and no significant protection was seen in case of flunarizine and phenytoin. Out of the four compounds, three demonstrated significant antiamnesic effects in the diazepaminduced anterograde amnesia model in mice (table VII). In case of 12 and 61 these effects were already apparent in doses as low as 0.1 mg/kg. Neither of the reference compounds showed protecting activity against diazepam-induced anterograde amnesia, rather a tendency to further deteriorate was observed. The antiamnesic actions of the four selected compounds Table VI. Effects of test compounds 12, 44, 54 and 61, and reference compounds on hypobaric hypoxia-induced lethality in SH-rats. Compound Dose” (w/kg) Protected animals I%) 12 10 50 20 7Ob 44 5 10 50 10 20 60” 54 10 50 10 20 61 10 50 0 0 Flunarizine 10 50 20 40 Nimodipine 5 10 30 60” Phenytoin 10 50 0 20 “Compounds were given orally 1 h before hypoxia challenge; bsignificantly different from control, P < 0.05. 34 Table VII. Antiamnesic effects of compounds 12, 44, 54 and 61 and reference compounds against diazepam-induced grade amnesia. Treatment Dose” (mg/kg) Control - DIAZ + vehicle 3 DIAZ + 12 Retention htencp time (s) (mean f SEM) Prevention (9%) antero- Retention time > 200 sb (70) - 60 60 f 14.6 - 0 3 + 0.1 3 + 10.0 226 f 31.5’ 188 + 31.5 123’ 95’ 60 50 DIAZ + 44 3 + 0.1 3 + 10.0 168 * 35.4 217 f 35.7’ 80 116’ 40 70 DIAZ + 54 3 + 0.1 3 + 10.0 105 + 36.1 I84 -c 30.8 33 92 22 33 DIAZ + 61 3 + 0.1 3 + 10.0 230 f 34.4’ 294 + 5.8 126” 173” 80 100 Control - I86 f 38.2 - 60 DIAZ + vehicle 3 76.7 f 27.4 - 0 DIAZ + flunarizine 3 +O.I 3 + 10.0 43.7 + 27.4 46.4 + 14.8 -30 -28 10 0 DIAZ + nimodipine 3 + 0.1 3 + 10.0 97.1 f 35.7 17.1 f 3.4 19 -54 20 0 DIAZ + phenytoin 3 + 0.1 3 + 10.0 25.3 + 4.7 67.3 -c 26.9 -47 -9 0 10 195 k41.0 “Diazepam (DIAZ) was administered ip whereas reference and test compounds were given orally (for other details see Experimental protocols); bnumber of animals (given in percentage) “significantly different from DIAZ + vehicle group. P < 0.05. were further studied in scopolamine-induced anterograde amnesia, electroshock and carbon dioxide induced retrograde amnesia models. Neither of them produced significant protecting activity in scopolamineinduced anterograde amnesia model. However, 51 showed significant prevention at the dose of 0.1 mg/kg in carbon dioxide induced retrograde amnesia and 61 had the same action at the dose of 10 mg/kg in electroshock-induced retrograde amnesia model (data not shown). Table VIII shows the results obtained with the four selected and three reference compounds in hypoxia-induced memory disturbances. In the dose tested (ie, 10 mg/kg) only 54 and 61 gave significant protecting activity and no such an action was found in case of reference compounds. The actions of the compounds in TET-induced brain edema may well be related to their inhibitory effects on veratrine-induced alterations in cerebrocortical synaptosomes. Veratrine is known to prevent inactivation of Na+-channels, a phenomenon leading not entering into the black compartment of the box in 200 s; Table VIII. Effects of compounds 12, 44, 54 and 61, and reference compounds against normobaric hypoxia-induced memory impairment in SH rats. Compound 12 44 54 61 10 10 IO 10 0 24 84h 74h Flunarizine Nimodipine Phenytoin IO IO IO 0 0 0 “Compounds were given orally 1 h before normobaric hypoxia challenge; bsignificantly different from control + vehicle: P < 0.05. 35 to greatly increased Na+-influx and, as a consequence, to the depolarization of intracellular organelles, such as mitochondria and endoplasmic reticulum, and thus causing a massive release of Ca2+ from these sites. The potent and preferential inhibitory effects of some of the I-oxa-3,8-diazaspiro[4S]decan-2-one derivatives described in this paper lends support to the hypothesis that they interact with membrane Na+-channels and, by preventing stimulus evoked Na+-influx, they inhibit intracellular Ca2+-release rather than transmembrane Ca2+-flux. Although no such data are available at present their direct effects on intracellular Cal+-release cannot be ruled out. Prevention by these compounds against intracellular Na+-increase could be directly demonstrated in TET experiments. Although the above cellular mechanism (ie, inhibition of Na+-influx and intracellular Ca2+-release) may partly contribute to the antihypoxic and antiamnesic actions of the four compounds found in various models, detailed investigations (not shown here) indicate the participation of other mechanism(s), too. Regarding the likely interactions of the four selected compounds with neuronal Na+- and Ca2+-movements and their related (or unrelated) pharmacological (eg, antihypoxic or antiamnesic) actions, data presented here indicate that their (biochemical and pharmacological) profile may be somewhat different from those of known Ca2+- and Na+-channel blockers (ie, flunarizine, nimodipine and phenytoin) included in our experiments as reference substances. According to our data obtained so far the pharmacological actions are possibly unrelated to direct involvement of cholinergic system as neither of the four compounds possessed effects on either M, or M, cholinergic receptors (data not shown). In vitro binding experiments demonstrated, however, moderate interactions between the two compounds 44 and 61 and a,-adrenergic receptors (IC,, = 180 and 289 nM) while all the four compound showed slight interaction with S-2 serotoninergic receptors (IC,,, = 118, 204, 195 and 194 nM, respectively) and with dopaminergic D2 (IC,, = 89, 60, 11.5 and 9 1 nM, respectively) receptors. In addition, 44 had an IC,,, of 230 nM in the in vitro D, dopaminergic binding assay. In vivo experiments confirmed the slight accelerating effects of 44 on brain serotoninergic and dopaminergic systems but these effects were manifested only in doses higher than those offering antiamnesic and antiedema actions. Despite the mild in vitro interactions with a,adrenergic receptors no significant cardiovascular effects (eg, alterations in mean arterial blood pressure, heart rate, cerebral and femoral blood flow) in anesthetised dogs were found with the four compounds (data not demonstrated here). Although they interacted with dopaminergic and serotoninergic receptors no behavioural changes related to these monoaminergic systems were seen at pharmacological doses. Moreover, no hypothermizing effects of the compounds that could, at least partly, explain the antihypoxic effects of the compounds were observed. Experimental protocols Chemistry Melting points were determined on Btlchi 5 IO apparatus and are uncorrected. IR spectra were obtained with a Nicolet 20DXC FTIR spectrophotometer. tH-NMR and *K-NMR spectra were recorded on Varian VXR-300 and UNITY plus 500 NMR spectrometer using tetramethylsilane as an internal standard. The chemical shifts marked ‘a’ correspond to the numerical middle of the relevant fluorine-coupled t3C multiplet. Elemental analysis (C, H, N and halogen) were in agreement with calculated values (within & 0.4%). Method IA. 8-/4,4-Bis(4-~uorap~envljhurvl]-3-(1, l-dimethyleth~l)-4-methylene-l-oxa-3,K-diazasp~ro~4.5]decnn-Z-one 44 A mixture of 3-( 1,l -dimethylethyl)-4-methylene1-oxa-3,8diazaspiro[4S]decan-2-one (22.43 i, 0.10 mol). 1,I’-(4-chlorobutvlidene)bis(4-fluorobenzene) (30.88 e. 0.11 mol). anhvdrous potassium ‘carbonate ( 15.2 g, d. 11 molr and potassium -iodide (1.82 g, 0.011 mol) in 4-methyl-2-pentanone (224 mL) was vigorously stirred and refluxed for 18-20 h and monitored by TLC. After cooling, water (50 mL) was added to the reaction mixture, the organic phase was separated. washed with saturated aqueous sodium chloride solution, dried over anhydrous magnesium sulphate and evaporated under reduced pressure. Consecutive crystallization from isopropyl ether and isopropano1 gave the title compound in 85.0% yield, mp 9 l-93 “C. IR (cm-t) 1756 (CO), 1641 (C=C), 1230 (C-O-C), 1219 (ArF), 1600 (Ar-skeletal), 834 (Ar-H). t H-NMR (500 MHz, CDC&, 30 “C, 6 [ppm]): 1.44 (2H, m, NCHCH,CH,CH), 1.58 (9H, s, fBu(CH,)), 1.76 (2H, m, H,-6,10), 1.80 (2H. m. H:,6,10), 2.00 (2H. m, NCH&H,CH#ZH), 2.29 (2H, m, H,-7.9), 2.34 (2H, m, NCH2CH2CH2CH), 2.72 (2H, m, H,-7,9), 3.87 (IH, t, NCH&H,CH,CH), 4.09 and 4.46 (2H, d and d, =CH?), 6.96 (4H, m, fluoro-phenyl H-3.5), 7.15 (4H, m, fluorophenyl H-2,6). tsC-NMR (125 MHz, CDCI,, 30 “C, F [ppm]): 25.5 (NCH,CH,CH$H), 28.3 (tBu(CH,)), 33.9 (NCH,CH,CH,CH), 36.8 (C-6,10), 49.2 (C-7,9), 49.8 (NCHZCH2CH2CH), 57.0 (tBu(C)). 58.4 (NCH,CH,CH,CH), 79.1 (C-5) 85.2 (=CH?), 115.3 (fluoro-phenyl C-3.5)“, 129.1 (fluorophenyl C-2,6)“, 140.5 (fluoro-phenyl C-l)“, 149.7 (C-4), 154.5 (C-2). 161.4 (fluoro-phenyl C-4)“. Compound 44 is currently under development with code numbers of RGH-2716 and TDN-345 in Chemical Works of Gedeon Richter Ltd and Takeda Chemical Industries Ltd, respectively. Method I B. 8-[4,4-Bis(4-fluorophenyl)hut~l~-3-c?;clohex~l-4hydra,~~-4-metlzyl-/-nxa-3,8-dia~aspir~~[4.S]d~can-2-one~HCl 57 To a stirred solution of 8-[4,4-bis-(4-fluorophenyl)butyl]-3cyclohexyl-4-methylene-l-oxa-3,8-diazaspiro[4.5]decan-2-one (4.9 g, 0.01 mol) in 10 mL formic acid 50 mL of 1 M hydrochloric acid was added and the reaction mixture was then stirred for additional 30 min at 5 “C. The crystals precipitated were filtered, washed with water and dried to afford the hydrochloride in 98.5% yield; decomposes at 260 “C. 36 IR (cm-t) 3220 (OH), 2750-2200 (+NH), 1735 (CO), 1232 (AI--F), 1133 (C-O-C), 1607 (Ar-skeletal), 833 (Ar-H). IH-NMR (500 MHz, DMSO-d,, 30 “C, 6 [ppm]: 1.05 and 1.57 (2H, m, cyclohexyl H-4), 1.24 and 1.73-(2H, m, cyclohexyl H-3. interchangeable). 1.26 and 1.73 (2H. m. cvclohexvl H-5). interchangeabli), 1.31(3H, s, 4-CH,),‘l.59 (2H: m, NCH,CH;: CH,CH), 1.60 and 1.90 (2H, m, cyclohexyl H-2), 1.68 and 2.03 (2H, m, cyclohexyl H-6), 1.71 and 2.25 (2H, m, H-10) 1.99 and 2.14 (2H, m, C-6), 2.05 (2H, m, NCH,CH,CH&H), 2.92 and 3.43 (2H, m, H-7), 2.95 and 3.34 (2H, m, C-9), 3.07 (lH, m, cyclohexyl H-l), 3.10 (2H, m, NCH2CHZCH1CH), 4.01 (H, t, NCH,CH,CH,CIE), 6.19 (lH, s, OH), 7.11 (4H, m, fluorophenyl H-3,5), 7.35 (4H, m, fluoro-phenyl H-2,6), 10.80 (lH, br, HCl). 13C-NMR (125 MHz, DMSO-d,, 30 “C, 6 [ppm]: 20.5 (4-CH,), 21.8 (NCH,CH,CH,CH), 24.9 (cyclohexyl C-4), 25.5 and 25.6 (cyclohexyl C-3,5), 26.1 (C-6), 29.0 (C-IO), 29.8 (cyclohexyl C-6), 30.0 (cyclohexyl C-2) 32.0 (NCH,CH,C& CH), 47.9 (C-9), 48.2 (C-7), 48.2 (NCH&H$H,CH), 50.9 (cyclohexyl C-l), 55.3 (NCH,CH2CH2CH), 79.8 (C-5), 89.3 (C-4), 115.1 (fluoro-phenyl C-3,5)a, 129.2 (fluoro-phenyl C-2,6)a, 140.6 (fluoro-phenyl C-l)a, 153.5 (C-2), 160.6 (fluorophenyl C-4)a. Method 2. 8-[4,4-Bis(4-Jluorophenyl)butyl]-4-methylene-3phenyl-l-oxa-3,8-diazaspiro[4.5]decan-2-one 61 To a’ stirred suspension- of I-[4,4-bis(4-fluorophenyl)butyl]-4ethvnvl-4-nioeridinol (7.38 g. 0.02 mol) and sodium methoxide (0.22 g, O.bd4 mol) in‘30 rn’, toluene, a solution of phenyl isocyanate (2.62 g, 0.022 mol) in 8 mL toluene was added at 50 “C under argon. After stirring for 1 h at the same temperature it was cooled, washed with water, dried (anhydrous magnesium sulphate) and concentrated under reduced pressure. The residue was crystallized from ethanol to give the title compound in 81% yield, mp 125-126 “C. IR (cm-t) 1771, 1765 (CO), 1687, 1647 (C=C), 1231 (COC), 1225, 1217 (Ar-F), 1601 (Ar- skeletal), 825, 769, 698 (Ar-H). tH-NMR (300 MHz, CDCI,, 24 “C, 6 [ppm]): 1.47 (2H, m, NCH&H$ZH$H), 1.99 (4H, m, H-6,10), 2.01 (2H, m, NCH,CH,CH,CH), 2.38 (2H, m, H,-7,9), 2.43 (2H, m, NCH,CH,CH;CH), 2.82 (2H, ‘m, H,-7,9), 3.88 (H, t, NCH&H&H&X), 4.07 and 4.16 (2H. d and d. = CH,). 6.97 (4H. m. fluoro-uhenvl H-3,5), 7.17 (4H, m, fluoroLphen;l’H-2,6), 7.33 (2H, m, bhen$l H-2,6), 7.39 (IH, m, phenyl H-4), 7.47 (2H, m, phenyl H-3,5). t3C-NMR (75 MHz, CDCI,, 24 “C, 6 [ppm]): 25.4 (NCH$XCHCH), 33.7 (NCH,CH,CH,CH), 36.8 (C-6,10), 49.0 (C-7,9), 49.7 (NCH,CHaCH&‘H), 58.4 (NCH,CH,CH,CH), 82.0 (C-5) 82.0 (=CH,), 115.2 (fluoro-phenyl C-3,5)a, 127.0 (phenyl C2,6), 128.3 (phenyl C-4), 129.0 (fluoro-phenyl C-2,6)a, 129.5 (phenyl C-3,5), 133.8 (phenyl C-l), 140.4 (fluoro-phenyl C-l)a, 150.5 (C-4), 154.4 (C-2), 161.3 (fluoro-phenyl C-4)a. Method 3A. 3-Butyl-8-[2-(4-fluorophenyl)ethyl]-4-hydroxy-4methyl-I-oxa-3,8-diazaspiro[4S]decan-2-one HC139 Twenty-five millimoles (7.2 g) of 8-[2-(4-fluorophenyl)ethyl]4-methylene- 1,3-dioxa-8-azaspiro[4.5]decan-2-one was dissolved in 36 mL of n-butylamine. When the reaction had subsided and returned to 24-25 “C the reaction mixture was stirred at room temperature for 20 h, then evaporated under reduced pressure. The resulting solid was recrystallized from benzene, and melted at 155-156 “C. The vield was 89%. The melting point of the HCl salt is greater than 300 “C. IR (cm-t) 3280 (OHY, 1730 (CO), 1232 (Ar-F), 1120 (C-O0. 1598 (Ar-skeletal). 832 (Ar-H). tH-NMR (500 MHz. DMSO-d,, 30 “C, 6 [pim]): 0.90 (3H, t, NCH,CH,CH,CH,); 1.29 (2H, m, NCH2CH2CH2CH3), 1.34 (3H, s, 4-CH,), 1.51 (2H, m, NCH,CH,CH,CH,), 1.80 (lH, m, H,-lo), 2.07 (IH, m, ~I, H,-6), 2.21 (IH, m, H,-6) 2.31 (lH, m, H,-lo), 3.06 (lH, m, H,-7), 3.07 (1 H, m, H,-9), 3.11 (2H, m, -CH,CH,N), 3.12 (2H, m, NCH,CH,CH&H,), 3.31 (2H, m, -CH,CHZN), 3.53 (lH, m, H,-9), 3.63 (lH, m, H,-7), 6.26 (lH, s, OH), 7.17 (2H, m, fluoro-phenyl H-3,5), 7.33 (2H, m, fluoro-phenyl H-2,6), 11.3 (lH, br, HCl). tsC-NMR (125 MHz, DMSO-d,, 30 “C, 6 [ppm]): 13.6 (NCH,CH,CH,CH,), 19.5 (NCH,CH,CH,CH,), 20.4 (4.CH,), 26.1 (C-6), 28.5 (-CH2CH2N), 29.2 (C-IO), 3 1.1 (NCH,C’H,CH,CH,), 39.2 (NC’H2CH2CH2CH3), 47.9 (C-9), 48.2 (C-7), 56.0 (-CH,CH,N), 80.4 (C-5) 88.5 (C-4), 115.3 (fluoro-phenyl C-3,5)a, 130.4 (fluoro-phenyl C-2,6)a, 133.2 (fluoro-phenyl C-l)a, 155.4 (C-2), 161.0 (fluoro-phenyl C-4)a. Method 3B. 3-Butyl-S-[2-{4-Jluorophenyl)ethyl]-4-methyleneI-oxa-3,8-diazaspiro-[4.5]decan-2-one 37 A mixture containing 3-butyl-8-[2-(4-fluorophenyl)ethyl]-4hydroxy-4-methyl-l-oxa-3,8-diazaspiro[4.5]decan-2-one (7.3 g, 0.02 mol) and 4-methylbenzene sulphonic acid monohydrate (1.5 g, 0.008 mol) in xylene (72 mL) was boiled under stirring while the water formed in the reaction was azeotropically distilled off. The reaction was monitored by TLC. After termination of the reaction the mixture was cooled, treated with NaHC03 (sat aq sol), and the organic phase was washed with water, dried over anhydrous magnesium sulphate, and evaporated to dryness under reduced pressure. Crystallization from diisopropyl ether provided the product, mp 91-92 “C, yield 96%. IR (cm-t) 2809, 2792, 2771 (N-CH,), 1757 (C=O), 1677 (C=C), 1222 (Ar-F), 1134 (COC), 827 (=CH,), 1599 (Ar-skeletal), 808 (Ar-H). tH-NMR (500 MHz, CDCI,, 30 “C, 6 [ppm]): 0.95 (3H, t, NCH,CH,CH,CH,), 1.35 (2H, m, NCHZCH2CH2CH,), 1.59 (2H, m, NCH,CH,CH,CH3), 1.85 (2H, m, H,-6,10), 1.92 (2H, m, H,-6,10), 2.46 (2H, m, H,-7,9), 2.63 (2H, m, -CH,CH,N), 2.78 (2H, m, -CH,CH,N), 2.90 (2H, m, H,-7,9), 3.45 (2H, m, NCH,CH2CH,CH,), 4.03 and 4.12 (2H, d and d, =CH,), 6.97 (2H, m, fluoro-phenyl H-3,5), 7.15 (2H, m, fluorophenyl H-2,6). tsC-NMR (125 MHz, CDCl,, 30 “C, 6 [ppm]): 13.7 (NCH,CH2CH,CH,), 19.9 (NCH,CH,CH&H,), 28.4 (NCH,CH,CH,CH,), 32.9 (-CH,CH,N), 36.9 (C-6,10), 41.2 (NCH,CH,CH,CH,), 49.1 (C-7,9), 60.4 (-CH,CH,N), 80.0 (=CH,), 81.4 (C-5), 115.1 (lluoro-phenyl C-3,5)a, 130.0 (fluoro-phenyl C-2,6)a, 135.8 (fluoro-phenyl C-l)a, 149.8 (C4) 155.5 (C-2), 161.4 (fluoro-phenyl C-4)a. Pharmacology General procedures In diazepam-, scopolamine-, electroshock- or carbon dioxideinduced amnesia tests, male NMRI mice (Charles-River, Hungary) weighing 24-26 g were used after fasting of 16 h, while in the hypobaric hypoxia tests selectively bred, male, Wistarderived spontaneously hypertensive rats (from our own breeding colony) weighing 200-220 g were used. In the TET-induced brain edema studies male, Hannover-Wistar rats weighing 180-200 g from our conventional breeding colony were used. Rats of the same strain were used for the in vitro 4sCa-uptake experiments. Drugs were suspended in 1% Tween-80 and given orally in a volume of 1.0 mL/lOO g body weight (for mice) or 0.5 mL/lOO g body weight (for rats), respectively. TET was obtained from Merck-Schuchardt (FRG), phenytoin (free acid) and flunarizine dihydrochloride were from Sigma. Diazepam and nimodipine were synthesized in our Chemical Departments. JsCaCl, was purchased from Amersham. All other reagents were obtained from commercial sources and were of analytical grade. 37 Student’s r-test (for TET experiments) and Mann-Whitney U-test (for amnesia experiments) was used for statistical comparisons. $Waz+-uptake into rat cerebrocortical synaptosomes [9] Rats were killed by cervical dislocation. The brains were removed and the cerebral cortices were dissected rapidly. The cortices were weighted and homogenised in 10 volumes of icecold 0.32 M sucrose solution. The homogenates was centrifuged at 1000 g for 10 min at 4 “C. The supernatant was recentrifuueed at 12 000 P for 20 min at 4 “C. The nellet obtained (P2 fracGon) was suspended in 0.32 M sucrose solution. The synaptosomal fraction (20 mg protein/ml) were used immediately. The incubation solution contained 112 mM NaCI, 5 mM KCl, 1.3 mM MgC12, 1.2 mM CaCI,, 1.2 mM NaH,PO,, 10 mM glucose and 20 mM Tris base. This solution was bubbled with 95% O2 and 5% CO, until its pH reached 7.4. Crude P2 fraction (1 mg protein) and test agents were added into tubes and preincubated at 37 “C for 20 min. When the depolarisation was induced by potassium 4sCa-uptake was initiated by addition of 50 uL 4sCaC1, (2.8 kBa) in 1.2 M KCl. Basal (unstimulated) 4sCa-uptake was initiat:d by addition of 50 iL JsCaCl, in 1.2 M NaCl. The final volume of the incubation mixture was 1 mL and the incubation time was 20 s. The uptake was terminated bv adding: 5 mL stoonine solution (120 mM NaCl. 5 mM KCl, 5 -mM EGTA, 20 rnLM T~s, pH 7.4) which was followed by rapid filtration through Whatman GF/C glass fiber filters. The filters were washed twice with 5 mL washing solution (132 mM NaCI, 5 mM KCl, 1.3 mM MgCl,, 1.2 mM CaCI,, 20 mM Tris, pH 7.4). When veratrine was used as depolarising agent 4sCa-uptake was initiated bv addition of 50 uL 4sCaC1, in 400 uM veratrine solution. The basal (unstimulatkd) ‘Ya-uptake was initiated by addition of 50 pL 45CaC12 in distilled water. Incubation, termination and filtration of samples was identical with that of described for K+-induced 4sCa-uptake. The filters were then placed into vials and dried at 40 “C for about 1 h. Radioactivity was determined by liquid scintillation spectrometry. I&” values (concentrations giving 50% inhibition) were calculated by using the data of two or three independent experiments from individual curves consisting of at least four different concentrations of the test compounds. Probit analysis was used for the calculation of ICXo values. TET-induced edema in rat brain /IO] Rats were intoxicated by daily oral administration of 2.5 mg/kg TET. The test comuounds were eiven orallv in a dose of 100 pmol/kg 1 h after TET treatmgnt and 6 h’thereafter. This treatment protocol was repeated on five consecutive days and animals were decapitated 2 h after the last treatment with test compounds. The brains were rapidly removed, rinsed in saline, blotted on filter paper and their wet weights were immediately measured. Brain samples were dried for at least 48 h at 100 “C and the dried residues were re-weighed. The difference between the wet and dry weight represents the brain water content which is expressed as a percentage of wet weight. Hypobaric hypoxia induced lethality in SH rats [II] The method described by Nakanishi et al [ll] was adapted to rats. One hour after the oral administration of the test compounds spontaneously hypertensive (SH) rats (ten per dose group) were placed (two at a time) into a desiccator of 6 L. The barometric pressure is then decreased to 22.66 kPa (170 mmHg) in 20 s. The survival time (ie, interval between reaching the low pressure and the last chest movement) of the animals was determined. By definition, those animals were considered as protected that survived 30% longer than the mean survival time of the control (ie, vehicle-treated) group. The mean survival time of vehicle-treated animals was 249 f 28.8 s (n = 10). Anterograde amnesia induced by diazepam or scopolamine /12, 131 For the measurement of diazepam/scopolamine-induced anterograde amnesia an eight-channel computer-controlled stepthrough passive avoidance apparatus (consisting of dark and light compartments separated by a guillotine door) was used. Exploration trial. Mice were pre-selected before learning experiment. Animals that did not enter the dark compartment in 30 s during the exploration trial were excluded from further study. Acquisition trial. Next day pre-selected animals (ten in each group) were placed in the lit compartment and when they crossed the door between the dark and light compartments with all four limbs within 30 s they received a foot shock (1 mA for 3 s) through the bottom stainless-steel grid. Latency time to enter the dark compartment was registered. Twenty-four hours after acquisition animals Retention trial. were placed again in the light compartment and the latency time to enter the dark compartment was registered up to 300 s. Test compounds were given orally 1 h prior to acquisition trial and 30 min or 1 h later the mice received diazepam (3 mg/kg, ip) or scopolamine (3 mg/kg, ip), respectively. Carbon dioxide- and electroconvulsive shock-induced retrograde amnesia [14, 151 The method was essentially same as those used for diazepam/scopolamine-induced anterograde amnesia but amnesia was induced in retrograde fashion either by carbon dioxide or electroshock (ECS). In the former case immediately after the acquisition trial mice were placed in plastic boxes (20 x 20 x 20 cm) flushed with carbon dioxide. Twenty seconds later animals were taken by artificial respiration and from the boxes, reanimated returned to their home cages. Test compounds were administered 23 h after the acquisition trial and retention was tested 1 h later. In case of ECS-induced amnesia, I h after acquisition trial mice received auricular ECS (20 mA for 0.2 s) and the test compounds were given orally 1 h later and retention was tested 24 h after the acquisition. Hypoxia-induced memory impairment [ 161 SH rats (six in each group) were trained in microprocessorcontrolled shuttle boxes to-develop two-way conditioned avoidance resnonse (CAR). Animals performed dailv 30 cvcles consisting’ of a 15 s intercycle interval, a 15 s -conditToned stimulus (periodical light, 1 Hz frequency) and a 10 s footshock (0.8 mA) as unconditioned stimulus in each day for 3 days. The animals had to change their compartment while the light was on otherwise they received a footshock. On the 4th day they were treated with the test compounds and 60 min later they were placed into the shuttle box where hypoxic conditions were maintained by perfusion of air and nitrogen (200 L/mm/ box) in a ratio giving 6% O2 content in the inspired air. Following 20 min equilibration period the animals performed 30 cycles. The number of conditioned avoidance responses was recorded automatically by a computerised program and group means were calculated. In each experiment two control groups were used (placebo and placebo + hypoxia). 38 Compounds were administered orally to mice (ten animals per dose group) and the deaths were recorded for 5 days. They were tested in at least five different doses up to 1000 mg/kg. References I 2 3 4 5 6 Siesjii BK, Katsura K, Palmark K, Smith. MS (1992) In: Phrrrmucolo~~ oj Ce&rrr/ Ichernra (Krieglstein JJ. Oberpichler-Swenk H. eds) Wissenschaftlithe Verlaggesellschaft mbH, Stuttgart. 5 I I-525 Regmer G. Canwart R, Le Douarec JC, Laubie M, Duhault J (1969) Chin7 Ther 4, IXS~IYJ Rognonl F, Marchmi F, Piacenza G, Paracchini S (IY78) Boll Chirn Frrw I I7,39740 I Canmn JM, Clark RD. Kluge AF. Nelson JT. Srrosbrrg AM, Unger SH. Michel AD. Whiting RL (I 9X I) J n/led Chem 24, I32O- I328 Smith PW, Cooper AWJ, Bell R, Bersford lJM, Gore PM, McElroy AB, Pritchard JM, Saez V, Taylor NR (1995) J Med Chrm 3X.3772-3779 Gedeon Richter Ltd (1991) EP 412 820; Chem Abstr (199 I) 115, 87X0 t 7 S~ndelar K. Rajhner .M, Ccrvena I ! I9731 Co//e<! Cxclz CIwn Comrnun 38, 3879.3901 X Gedeon Richter Ltd (1991) EP414 421; C/wrnAb.rrr(l991) 114, 247260 j Y Wu PH. Phillis JN, Thierry DLJ (IY82) ./.~&/nclwnl 39. 700-70X IO LinnCe P. Quiniou M, Godin C. Le Polles JB (1984) Ann Phrrnn Fr 42, 431442 I I Nakanishi M, Yaauda H. Tsumagari T (1973) I# Sci I3,464-467 I2 Broekamp CL, Pichon ML, Lloyd K (1984) P.~~cho/~ha~nlrrc~~~~~~~ X3, l22Z I25 I3 Groir D, P&loai 8. S~porny L (1987) Drug Dev Res I I, 29-36 14 lshihara Y. Yukimasa H, Miyamoto M, Goto G (1992) Chrn~ Pharmad Bull 40,1177-II85 15 Gro6 D, P.%losl 6, Szporny L (1989) In: Phurmacolog~ rfCerdm/ Iwhcmio (Krieglstem JJ, ed) Wissenschaftliche Verlaggesellschnft mbH, Stuttgart, 299%305 I6 GroCl D. P&w 8, Szporny L (1988) Drug Drv RPS 15.75-85 17 McMillan DE, Wenger GR (1985) Pharmoco/ Rer- 37. 365-379 IX Young AR, Gotti B, Legrain Y, MacKew.ie ET, Niowicki JP (19X2) In: Cerrhrirl H>apoxio in Parhogmrsis of Migrzinc (Clifford RF. ed 1, Pitman Books Ltd. London. 155-175 I9 Taylor CP, Meldrum BS ( 1995) Twnd,s Pharrnu~vl Sci 16, 309-3 I6