key: cord-0912869-4wcsz4is
authors: Camps, Pelayo; Duque, María D.; Vázquez, Santiago; Naesens, Lieve; Clercq, Erik De; Sureda, Francesc X.; López-Querol, Marta; Camins, Antoni; Pallàs, Mercè; Prathalingam, S. Radhika; Kelly, John M.; Romero, Vanessa; Ivorra, Dolores; Cortés, Diego
title: Synthesis and pharmacological evaluation of several ring-contracted amantadine analogs
date: 2008-12-01
journal: Bioorg Med Chem
DOI: 10.1016/j.bmc.2008.10.028
sha: a1030b14be6fdd295b18de21525a37f0a0561444
doc_id: 912869
cord_uid: 4wcsz4is

The synthesis of several (3-noradamantyl)amines, [(3-noradamantyl)methyl]amines, (3,7-dimethyl-1-bisnoradamantyl)amines, and [(3,7-dimethyl-1-bisnoradamantyl)methyl]amines is reported. They were evaluated against a wide range of viruses and one of them inhibited the cytopathicity of influenza A virus at a concentration similar to that of amantadine. Several of the new polycyclic amines show an interesting activity as NMDA receptor antagonists. A rimantadine analogue displayed significant trypanocidal activity. Moreover, to further characterize the pharmacology of these compounds, their effects on dopamine uptake were also assessed.

1-Adamantylamine (amantadine) and (a-methyl-1-adamantyl)methylamine (rimantadine) have prophylactic and therapeutic activity in influenza A virus infections. 1 Related adamantane derivatives also show antiviral activity. 2 Adamantane derivatives are inexpensive, but resistance against the drugs develops readily and treatment is frequently complicated by central nervous system (CNS) side-effects. In fact, amantadine and its 3,5-dimethyl analogue memantine are NMDA receptor antagonists and are approved for the treatment of Parkinson's and Alzheimer's disease, respectively. 3 Thus, the design of new amantadine-related antiinfluenza agents without CNS side-effects is a highly desirable goal. Amantadine, rimantadine, memantine, and related polycyclic amines also possess trypanocidal activity (Fig. 1 ). 4 Biological activity has also been found in other polycyclic cage amines. For example, compounds 1À4 have anti-influenza activ-ity, 5 5 is a MAO-B inhibitor, 6 and 6 is a NMDA receptor antagonist (Fig. 2) . 7 For more than 20 years two of us (P.C. and S.V.) have worked on a project aimed at exploring the structure and the reactivity of noradamantane 8 and bisnoradamantane derivatives. 9 Up to now, our work on this topic has been done from a purely synthetic point of view. For example, we have developed several general entries to these skeletons. However, systematic studies directed towards the synthesis of biologically active noradamantane and bisnoradamantane derivatives have not yet been carried out (Fig. 3) It is well known in medicinal chemistry that when drugs contain cyclic systems, it is generally worth synthesizing analogues where the ring is opened, expanded or contracted by one unit, because these analogues show similar activity to the parent compound. 11 Tricyclo[3.3.1.0 3, 7 ] non-3-ylamine [(3-noradamantyl)amine] and tricyclo[3.3.0.0 3,7 ]oct-1-ylamine [ (1-bisnoradamantyl) amine] may be viewed as ring contracted analogs of amantadine, featuring a skeleton with one and two carbon less than the model, respectively. For this reason, in this paper, we describe the preparation of a series of (3-noradamantyl)amines, (1-bisnoradamantyl) amines and related compounds as well as the results of their antiviral, trypanocidal, NMDA receptor antagonist, and dopamine reuptake inhibitory activities.

Starting from the known amine 7, 10, 12 we have prepared noradamantane amines 8À12 using classical methods in amine chemistry. Thus, reductive alkylation of 7 with several aromatic aldehydes afforded secondary amines 8a-f in moderate to high yields. Dimethylated derivative 10 was prepared as previously described in the literature. 13 Monomethyl derivative 12 was synthesized from 8a by reductive alkylation followed by catalytic debenzylation in good overall yield. Finally, piperidine derivative 11 was prepared by alkylation of primary amine 7 with 1,5-dibromopentane in 51% yield (Scheme 1).

Starting from the known amide 13, 14 a series of 3-(noradamantyl)methylamines were synthesized. Thus, reduction of amide 13 with LiAlH 4 followed by acidic work-up led to the hydrochloride of amine 14 in 90% yield. Reductive alkylation of 14 with benzaldehyde and NaCNBH 3 in methanol gave 15 in 77% yield. Reductive methylation of 15 followed by catalytic hydrogenation led to 19 in high yield. Reductive methylation of 14 with formic acid and formaldehyde furnished dimethyl derivative 17 in 68% yield. Finally, reaction of 14 with 1H-pyrazol-1-carboxamidine led to guanidine 18 in 84% yield (Scheme 2).

On the other hand, starting from the known acid 20, 9g we have prepared amines 21À23, 25À28, and 30À36 using classical methods in amine chemistry. Although the synthesis of amine 21 from acid 20 was very low yielding using the classical Schmid's or Curtius' reactions (14% and 24% yield, respectively), application of the Yamada's modification of the Curtius reaction allowed us to obtain and fully characterize the hydrochloride of amine 21 in 73% yield.

From this amine, reductive alkylation with benzaldehyde led to 22a in 60% yield. Similarly, reductive alkylation with 2-thiophenecarbaldehyde led to 22b in 69% yield. Reductive methylation of 22a followed by catalytic debenzylation furnished secondary amine 28 in high yield. Dimethylated derivative 26 was obtained in 83% yield by treating amine 21 with excess of formic acid and formaldehyde. Finally, dibenzylated compound 27 was prepared by double alkylation of 21 with benzyl chloride in 73% yield (Scheme 3). Moreover, reaction of 20 with methyllithium gave ketone 24 in low yield. Reaction of 24 with hydroxylamine followed by reduction of the obtained oxime with LiAlH 4 gave amine 25, a compound that can be viewed as a ring contracted analog of the antiviral rimantadine (Scheme 3).

Finally, reduction of the amide 29, easily available from acid 20, with LiAlH 4 followed by acidic work-up gave the hydrochloride of amine 30 in 70% overall yield. Following a similar sequence of the previously used with amine 21, amines 31, 32, 33, and 36 were obtained in high yields. Piperidine derivative 34 was obtained by alkylation of amine 30 with 1,5-dibromopentane in 53% yield. Finally, reaction of 30 with 1H-pyrazol-1-carboxamidine led to guanidine 35 in 90% yield (Scheme 4).

The structure of all new compounds was confirmed by elemental analysis or accurate mass measurement, IR, 1 H NMR, 13 C NMR, and mass spectral data.

The tsetse fly-transmitted protozoan parasite Trypanosoma brucei is the causative agent of Human African Trypanosomiasis (HAT). After a major upsurge of the disease in the late 1990s throughout many parts of sub-Saharan Africa, annual infections have now fallen to 70,000 as a result of major surveillance and treatment programmes. 15 However, over 60 million people remain at risk, and in some areas death rates exceed those from HIV/AIDS and malaria. The drugs currently available to treat HAT require administration under medical supervision and are characterized by limited effi-cacy, toxicity, and resistance. For example, the arsenical drug melarsoprol, which is used to treat late stage disease, can result in a reactive encephalopathy which kills up to 10% of patients. In the absence of treatment, HAT is invariably fatal and new drugs are therefore urgently required. Recently, it was reported that the anti-influenza virus drug rimantadine was active in vitro against bloodstream form T. brucei, and that other aminoadamantane derivatives had enhanced activity. 4, 16 To extend these observations, we have tested several new bisnoradamantanes and related compounds for activity against bloodstream form T. brucei.

The noradamantylamines and [(3-noradamantyl)methyl]amines described in this paper and the birnoradamantyl derivatives 21, 22a, 23, 27, 28, 32, 34, and 36 were found to have no significant activity against cultured bloodstream from T. brucei at concentrations up to 5 lg mL À1 . Compounds 26, 30, and 31 showed transient effect on growth at 5 lg mL À1 , but cells grew to normal density. Rimantadine analog 25 was the most active of the compounds tested and we established its IC 50 (6.02 ± 0.36 lM) and IC 90 (9.48 ± 2.64 lM) values. Amine 25 was found to be slightly more active than rimantadine (IC 50 = 7.04 ± 0.12 lM; IC 90 = 13.97 ± 1.68 lM) and at least 20 times more active than amantadine (IC 50 > 130 lM).

NMDA receptor antagonists are highly interesting compounds since these receptors have been involved in several neurodegenerative disorders. In fact, memantine is widely used in therapeutics to slow down the progression of Alzheimer's disease. 17 The activity of the different new polycyclic compounds was assayed on cerebellar granule neurons loaded with the calcium-sensitive probe Fura-2. 18 Addition of glutamate or NMDA (100 lM) in the presence of glycine (10 lM) produced a robust and stable increase in intracellular calcium that was challenged with cumulative additions of the compounds to be tested. Although all the noradamantyl derivatives and several of the bisnoradamantyl compounds were able to inhibit calcium entry through NMDA receptors, none of the compounds was more potent than memantine against glutamate-or NMDA-induced calcium increase in cerebellar granule neurons (Table 1 ).

In general, the bisnoradamantane derivatives are more potent as NMDA receptor antagonists than the noradamantane amines. For example, amine 21 is 4 times more potent as NMDA receptor antagonist than 3-noradamantylamine, 7, and the guanidine derivative 35 is 3.5 times more potent than its corresponding noradamantyl analog, 18.

Bisnoradamantylamines were usually more active than their corresponding (bisnoradamantyl)methylamine analogs as exemplified by the pairs 21/30, 26/33, and 28/36, and alkyl substitution causes a reduction in the potency (e.g., series 21/28/26 or 30/36/ 33). The guanidine derivative 35 was the more potent compound, being 10 times more potent than amantadine and 5 times less potent than memantine. Attempts to synthesize a guanidine derivative from 21 were not successful.

None of the synthesized compounds was found to have antiviral activity against the enveloped DNA viruses herpes simplex virus (type 1 or type 2) or vaccinia virus; the enveloped RNA viruses fe-line coronavirus, parainfluenza-3 virus, respiratory syncytial virus, vesicular stomatitis virus, sindbis virus, or Punta Toro virus; or the non-enveloped RNA viruses Coxsackievirus B4 and Reovirus-1. In the influenza virus assays, only compounds 7 and 14, two primary amines, displayed reasonable activity against the influenza A/ H1N1 and A/H3N2 subtypes, secondary and tertiary amines were not active ( Table 2 ). The antiviral data obtained by microscopy were confirmed by a colorimetric cell viability assay (data not shown). The highest selectivity was noted with compound 7 tested against the A/H3N2 subtype. As anticipated, all compounds proved to be inactive against influenza B virus, which is known to be insensitive to amantadine and rimantadine.

It is known that amantadine increases extracellular dopamine levels by antagonism of the NMDA receptor, 19 although the exact mechanism has not been fully elucidated. As several of our new amines showed NMDA receptor antagonist activity with IC 50 similar or even lower than amantadine, we have determined their effect on [ 3 H]dopamine uptake in rat striatal synaptosomes (Table   3 ). At the concentration tested (100 lM), several of the compounds were able to inhibit [ 3 H]dopamine uptake in some manner, show- ing similar values of inhibition than amantadine or memantine. However, it seems that no correlation exists with their potency as antagonists at the NMDA receptor. Probably, other mechanisms are being involved in the regulation of dopamine release, like different activities at D 2 receptors or through inhibition at the dopamine transporter.

In summary, we have synthesized and fully characterized several (3-noradamantyl)amines, (3-noradamantyl)methylamines, (3-bisnoradamantyl)amines, and (3-bisnoradamantyl)methylamines. Although these compounds were less potent than memantine against NMDA-induced calcium increase in cerebellar granule neurons, several compounds were more potent than amantadine, the bisnoradamantane amines being more potent than the corresponding noradamantane amines. Interestingly, none of those compounds showed antiviral activity, while compound 14, that displayed reasonable activity against the influenza A/H1N1 and A/H3N2 subtypes, showed no NMDA receptor antagonist activity. Moreover, none of the compounds were significantly more potent, at the tested concentration, than amantadine or memantine as inhibitors of the dopamine uptake.

Amantadine displays both anti-influenza activity and NMDA receptor antagonism. As selectivity is usually highly desirable in drugs, the amines herein reported open the way for the design of new aminopolycyclic compounds with selective anti-influenza or NMDA receptor antagonist activity.

Interestingly, amine 25, that is 2.5 times more potent than amantadine as NMDA receptor antagonist, also displayed trypanocidal activity, being slightly more active than rimantadine and at least 21 times more active than amantadine.

Guanidine 35 is a polycyclic cage compound with selective NMDA receptor antagonist activity (IC 50 = 7.1 lM) without antiviral and trypanocidal activities. The synthesis and pharmacological evaluation of more polycyclic cage amines is in progress to reach more potent and selective derivatives.

Melting points were determined in open capillary tubes. Unless otherwise stated, NMR spectra were recorded in CD 3 OD in the following spectrometers: 1 H NMR (500 MHz), 13 C NMR (100.6 MHz). Chemical shifts (d) are reported in ppm related to internal tetramethylsilane (TMS). Assignments given for the NMR spectra are based on DEPT, COSY 1 H/ 1 H, and HETCOR 1 H/ 13 C (HSQC and HMBC sequences for one bond and long range 1 H/ 13 C heterocorrelations, respectively) and NOESY experiments for selected compounds. For the MS and GC/MS analyses the sample was introduced directly or through a gas chromatograph. For GC/MS analyses a 30-m column [5% diphenyl-95% dimethylpolysiloxane, conditions: 10 psi, initial temperature: 35°C (2 min), then heating at a range of 8°C/min till 300°C, then isothermic at 300°C] was used. The electron impact (70 eV) or chemical ionization (CH 4 ) techniques were used. Only significant ions are given: those with higher relative ratio, except for the ions with higher m/z values. Accurate mass measurements were obtained using ESI technic. Absorption values in the IR spectra (KBr) are given as wave-numbers (cm À1 ). Column chromatography was performed on silica gel 60 Å (35-70 mesh). For the thin-layer chromatography (TLC) aluminum-backed sheets with silica gel 60 F 254 were used and spots were visualized with UV light and/or 1% aqueous solutions of KMnO 4 .

To a solution of 7ÁHCl (600 mg, 3.46 mmol) in MeOH (10 mL), NaBH 3 CN (95%, 445 mg, 6.72 mmol), AcOH (0.3 mL), and benzaldehyde (0.5 mL, 4.92 mmol) were added and the mixture was stirred at room temperature for 2 h. An additional portion of NaBH 3 CN (95%, 220 mg, 3.33 mmol) and benzaldehyde (0.25 mL, 2.46 mmol) were added, the mixture was stirred at room temperature overnight and concentrated to dryness. Water (20 mL) was added to the residue, the suspension was basified with 1 N NaOH and was extracted with EtOAc (3Â 10 mL). The combined organic extracts were washed with brine (2Â 10 mL), dried with anhyd Na 2 SO 4 , filtered, and concentrated in vacuo. The residue was taken in EtOAc and the amine 8a was precipitated as its hydrochloride (839 mg, 92% yield) by adding an excess of Et 2 OÁHCl. The analytical sample of 8aÁHCl was obtained by crystallization from MeOH, mp >300°C (dec). IR: 2942 IR: , 2757 IR: , 2656 IR: , 2600 IR: , 2428 IR: , 2363 IR: , 2340 IR: , 1458 IR: , 1425 IR: , 1331 From 7ÁHCl (173 mg, 1.00 mmol), NaBH 3 CN (95%, 142 mg, 2.15 mmol), AcOH (0.3 mL), and 4-methoxybenzaldehyde (0.18 mL, 1.48 mmol) in MeOH (7 mL) and following the above procedure, 8bÁHCl was obtained (233 mg, 79% yield). The analytical sample of 8bÁHCl was obtained by crystallization from 2-propanol, mp >300°C (dec). IR: 2926 IR: , 2794 IR: , 2751 IR: , 2716 IR: , 2678 IR: , 2592 IR: , 2578 IR: , 2443 IR: , 1613 IR: , 1588 IR: , 1514 IR: , 1460 IR: , 1440 IR: , 1333 IR: , 1306 IR: , 1272 IR: , 1248 IR: , 1178 IR: , 1040 , 8.20; N, 4.74; Cl, 12.59. Found: C, 68.69; H, 8.19; N, 4.70; Cl, 12.73. 4.1.4 . N-(2-Methoxybenzyl)(tricyclo[3.3.1.0 3,7 ]non-3-yl)amine hydrochloride (8cÁHCl)

From 7ÁHCl (173 mg, 1.00 mmol), NaBH 3 CN (95%, 142 mg, 2.15 mmol), AcOH (0.3 mL), and 2-methoxybenzaldehyde (0.18 mL, 1.46 mmol) in MeOH (7 mL) and following the procedure described for 8a, 8cÁHCl was obtained (128 mg, 44% yield). The analytical sample of 8cÁHCl was obtained by crystallization from 2-propanol, mp 262À263°C. IR: 2956 IR: , 2921 IR: , 2717 IR: , 2678 IR: , 2582 IR: , 2433 IR: , 1605 IR: , 1584 IR: , 1496 IR: , 1466 IR: , 1456 IR: , 1446 IR: , 1435 IR: , 1332 IR: , 1292 IR: , 1253 IR: , 1121 IR: , 1050 IR: , 1031 , 8.23; N, 4.77; Cl, 12.07. Found: C, 69.21; H, 8.29; N, 4.80; Cl, 12.32. 4.1.5 

From 7ÁHCl (173 mg, 1.00 mmol), NaBH 3 CN (95%, 142 mg, 2.15 mmol), AcOH (0.3 mL), and 3-methoxybenzaldehyde (0.18 mL, 1.45 mmol) in MeOH (7 mL) and following the procedure described for 8a, 8dÁHCl was obtained (228 mg, 78% yield). The analytical sample of 8dÁHCl was obtained by crystallization from 2-propanol, mp >257°C (dec). IR: 3002, 2923 IR: 3002, , 2873 IR: 3002, , 2792 IR: 3002, , 2734 IR: 3002, , 2708 IR: 3002, , 2678 IR: 3002, , 2585 IR: 3002, , 2446 IR: 3002, , 1606 IR: 3002, , 1599 IR: 3002, , 1588 IR: 3002, , 1494 IR: 3002, , 1460 IR: 3002, , 1437 IR: 3002, , 1331 IR: 3002, , 1306 IR: 3002, , 1272 IR: 3002, , 1256 IR: 3002, , 1174 IR: 3002, , 1035 

From 7ÁHCl (150 mg, 0.86 mmol), NaBH 3 CN (95%, 125 mg, 1.89 mmol), AcOH (0.3 mL), and 4-fluorobenzaldehyde (0.14 mL, 1.31 mmol) in MeOH (8 mL) and following the procedure described for 8a, 8eÁHCl was obtained (186 mg, 77% yield). The analytical sample of 8eÁHCl was obtained by crystallization from ethyl acetate, mp >300°C (dec). IR: 2947 IR: , 2920 IR: , 2797 IR: , 2754 IR: , 2580 IR: , 2446 IR: , 1604 IR: , 1590 IR: , 1514 IR: , 1457 IR: , 1440 IR: , 1429 IR: , 1334 IR: , 1232 IR: , 1163 IR: , 1127 7.53; N, 4.81; Cl, 13.38; F, 6.52. Found: C, 66.01; H, 7.67; N, 4.83; Cl, 13.31 ; F, 6.34.

From 7ÁHCl (150 mg, 0.86 mmol), NaBH 3 CN (95%, 125 mg, 1.89 mmol), AcOH (0.3 mL), and 2-thiophenecarbaldehyde (0.12 mL, 1.37 mmol) in MeOH (8 mL) and following the procedure described for 8a, 8fÁHCl was obtained (145 mg, 66% yield). The analytical sample of 8fÁHCl was obtained by crystallization from EtOAc, mp 278°C (dec). IR: 2939 IR: 2917 IR: , 2790 IR: , 2747 IR: , 2442 IR: , 1589 IR: , 1457 IR: , 1440 IR: , 1429 IR: , 1334 IR: , 1256 IR: , 1194 IR: , 1126 , 695 cm À1 . To a solution of 8aÁHCl (395 mg, 1.5 mmol) in acetonitrile (10 mL), formaldehyde (1.18 mL, 37% wt. in water solution, 15 mmol), and NaBH 3 CN (95%, 238 mg, 4.28 mmol) were added. The mixture was stirred for 30 min at room temperature, AcOH (0.3 mL) was added and the mixture was stirred at room temperature for 2 h. An additional portion of NaBH 3 CN (95%, 283 mg, 4.28 mmol) was added and the mixture was further stirred at room temperature for 2 h. The mixture was concentrated to dryness, 2 N NaOH (20 mL) was added and the suspension was extracted with CH 2 Cl 2 (3Â 20 mL). The combined organic phases were washed with H 2 O (2Â 20 mL), dried with anhyd Na 2 SO 4 , filtered, and concentrated in vacuo. The residue was taken in EtOAc and the amine 9 was precipitated as its hydrochloride (327 mg, 91% yield) by adding an excess of Et 2 OÁHCl. The analytical sample of 9ÁHCl was obtained by crystallization from 2-propanol, mp 258À259°C (dec) . IR: 3042, 2927 IR: 3042, , 2872 IR: 3042, , 2850 IR: 3042, , 2608 IR: 3042, , 2553 IR: 3042, , 2520 IR: 3042, , 2487 IR: 3042, , 2472 IR: 3042, , 2391 IR: 3042, , 1498 IR: 3042, , 1458 IR: 3042, , 1405 IR: 3042, , 1332 (17), 91 (96). Anal. Calcd for C 17 H 23 NÁHCl (277.84): C, 73.49; H, 8.71; N, 5.04; Cl, 12.76. Found: C, 73.61; H, 8.74; N, 5.04; Cl, 7 ] non-3-yl)piperidine hydrochloride (11ÁHCl)

To a solution of 7ÁHCl (173 mg, 1.00 mmol) in DMF (2.5 mL), anhyd Et 3 N (0.4 mL, 2.9 mmol) was added and the suspension was stirred at room temperature for 2 h. 1,5-Dibromopentane (0.17 mL, 1.2 mmol) was added and the mixture was heated at 60°C for 26 h. To the cold mixture, water (15 mL) was added and the solution was washed with EtOAc (3Â 10 mL). The aqueous phase was basified with 2 N NaOH (5 mL) and extracted with ethyl acetate (3Â10 mL). The combined organic extracts were washed with water (3Â 10 mL), dried with anhyd Na 2 SO 4 , filtered and excess of Et 2 OÁHCl was added. The solution was concentrated in vacuo to dryness to give 11ÁHCl (117 mg, 48% yield). The analytical sample of 11ÁHCl was obtained by crystallization from MeOH/Et 2 O, mp >264°C (dec). IR: 2941 IR: , 2930 IR: , 2871 IR: , 2851 IR: , 2634 IR: , 2530 IR: , 2451 IR: , 2401 IR: , 1471 IR: , 1455 IR: , 1351 IR: , 1333 IR: , 1187 IR: , 1124 IR: , 1016 , 708 cm À1 . 1 H NMR 1.54 (dm, J = 13.0 Hz, 1H, 4 0 -H ax ), 1.62 (dquint, J = 13.3 Hz, 1H, (14) 10.00; N, 5.79. Found: C, 69.60; H, 10.0; N, 7 ] non-3-yl)amine hydrochloride (12ÁHCl)

A solution of 9ÁHCl (275 mg, 0.99 mmol) and 5% Pd/C (50% in water, 10 mg) in absolute EtOH (25 mL) was hydrogenated at 1 atm for 24 h. The suspension was filtered, the residue was washed with EtOH, and the organic layer was concentrated in vacuo to give a solid. Crystallization from MeOH/Et 2 O gave 12ÁHCl (165 mg, 89% yield), mp 165À166°C. IR: 2947 IR: , 2922 IR: , 2870 IR: , 2856 IR: , 2763 IR: , 2728 IR: , 2692 IR: , 2587 IR: , 2516 IR: , 2458 IR: , 1466 IR: , 1420 IR: , 1348 IR: , 1332 IR: , 1311 IR: , 1258 IR: , 1160 IR: , 1111 IR: , 1094 IR: , 1060 IR: , 1018 , 9.71; N, 7.23. Found: C, 62.13; H, 9.63; N, 7.20. 4.1.11. [(Tricyclo[3.3.1.0 3, 7 ] non-3-yl)methyl]amine hydrochloride (14ÁHCl)

To a cold (0°C) solution of 13 (600 mg, 3.64 mmol) in anhyd THF (50 mL), LiAlH 4 (443 mg, 11.1 mmol) was added and the suspension was heated under reflux for 15 h. The suspension was cooled (ice bath), carefully basified with 10 N NaOH (10 mL), and stirred for 1 h at room temperature. The precipitate was filtered and washed with CH 2 Cl 2 (3Â 25 mL). The combined filtrate and washings were dried with anhyd Na 2 SO 4 , filtered, and excess of Et 2 OÁHCl was added. The solution was concentrated in vacuo to give a solid that was crystallized from MeOH/Et 2 O to give 14ÁHCl (613 mg, 90% yield), mp >300°C (dec). IR: 3019, 2926 , 2867 , 1597 , 1499 , 1384 , 1337 , 1116 10 H 17 NÁHCl (187.71): C, 63.99; H, 9.66; N, 7.46; Cl, 18.89. Found: C, 64.07; H, 9.66; N, 7.43; Cl, 7 ] non-3-yl)methyl]amine hydrochloride (15ÁHCl)

From 14ÁHCl (500 mg, 2.67 mmol), MeOH (10 mL), NaBH 3 CN (95%, 380 mg, 5.74 mmol), AcOH (0.3 mL), benzaldehyde (0.4 mL, 3.92 mmol), an additional portion of NaBH 3 CN (95%, 190 mg, 2.87 mmol), and benzaldehyde (0.2 mL, 1.96 mmol) and following the procedure described for 8a, 15ÁHCl was obtained (567 mg, 77% yield). The analytical sample of 15ÁHCl was obtained by crystallization from EtOAc, mp >277°C (dec). IR: 2931 IR: , 2865 IR: , 2781 IR: , 1588 IR: , 1446 , 8.65; N, 4.94; Cl, 13.77. Found: C, 72.03; H, 8.56; N, 4.85, Cl, 7 ] non-3-yl)methyl]amine hydrochloride (16ÁHCl)

From 15ÁHCl (465 mg, 1.67 mmol), acetonitrile (10 mL), formaldehyde (1.31 mL, 37% wt. in water solution, 16.7 mmol), and two portions of NaBH 3 CN (95%, 314 mg, 4.75 mmol) and following the procedure described for 9, the amine 16 was obtained (411 mg, 96% yield). Its hydrochloride was obtained by adding an excess of Et 2 OÁHCl to a solution of the amine in EtOAc, followed by concentration in vacuo to dryness. The analytical sample of 16ÁHCl was obtained by crystallization from EtOAc, mp 251À252°C. IR: 3042, 2924 IR: 3042, , 2865 IR: 3042, , 2698 IR: 3042, , 2641 IR: 3042, , 2550 IR: 3042, , 2530 IR: 3042, , 1458 IR: 3042, , 1423 IR: 3042, , 1337 IR: 3042, , 1085 2.94 (s, 3 H, 3.32 (d, J = 13.5 Hz, 1H, CH a N), 3.46 (d, J = 13.5 Hz, 1H, CH b N), 4.37 (d, J = 12.2 Hz, 1H , CH a C 6 H 5 ), 4.43 (d, J = 12.2 Hz, 1H, CH b C 6 H 5 ), 3H, -H], 7.59 [m, 2H, (17) 18 H 25 NÁHCl (291.86): C, 74.07; H, 8.98; N, 4.80; Cl, 12.15. Found: C, 74.15; H, 8.96; N, 4.81; Cl, 12.43. 4.1.14. N, 7 ] non-3-yl)methyl]amine hydrochloride (17ÁHCl)

To a cold (0°C) solution of 14 (128 mg, 0.68 mmol) in Et 2 O (5 mL), formaldehyde (1.38 mL, 37% wt. in water solution, 17.6 mmol) and, dropwise, formic acid (1.17 mL, 30.5 mmol) were added and the mixture was stirred at 80°C for 10 h. To the cold mixture Et 2 O (15 mL) was added, 5 N NaOH (5 mL) was added dropwise and the suspension was stirred at room temperature for 15 min. The organic layer was separated and the aqueous phase was extracted with Et 2 O (4Â 10 mL). The combined organic phases were dried with anhyd Na 2 SO 4 , filtered, and an excess of Et 2 OÁHCl was added. Concentration in vacuo gave 17ÁHCl (100 mg, 68% yield). The analytical sample of 17ÁHCl was obtained by crystallization from MeOH/Et 2 O, mp >260°C (dec). IR (KBr): 2930 , 2915 , 2868 , 2847 , 2681 , 2570 , 2468 , 2360 , 1471 , 1413 , 1338 , 1306 , 1225 , 1062 (7), 79 (8), 77 (7), 58 ([CH 2 @N(CH 3 ) 2 ] + , 100). Anal. Calcd for C 12 H 21 NÁ1.05HClÁ0. 25H 2 O (222.09): C, 64.90; H, 10.23; N, 6.31; Cl, 16.76. Found: C, 64.77; H, 10.42; N, 6.32; Cl, 7 ] non-3-yl)methyl]guanidine hydrochloride (18ÁHCl)

To a cold (0°C) solution of 14ÁHCl (150 mg, 0.8 mmol) in acetonitrile (2.5 mL), anhyd Et 3 N (0.2 mL, 1.45 mmol), and 1H-pyrazol-1carboxamide hydrochloride (140 mg, 0.94 mmol) were added. The suspension was heated at 70°C for 6 h and cooled at 0°C for 24 h. The precipitate was filtered and washed with cold acetonitrile (2Â 5 mL) to give 18ÁHCl (129 mg, 70% yield). The above product was taken in EtOAc, excess Et 2 OÁHCl was added, and the solvent was eliminated in vacuo. The analytical sample of 18ÁHCl was obtained by crystallization from MeOH/Et 2 O, mp 298À299°C. IR (KBr): 3357, 3262, 3169, 2926 , 2861 , 1664 , 1623 , 1578 , 1356 , 1094 , 57.51; H, 8.77; N, 18.29; Cl, 15.43. Found: C, 57.65; H, 8.88; N, 18.23; Cl, 7 ] non-3-yl)methyl]amine hydrochloride (19ÁHCl) From 16ÁHCl (267 mg, 0.91 mmol), 5% Pd/C (50% in water, 10 mg), and absolute EtOH (30 mL) and following the procedure described for 12, 19ÁHCl (143 mg, 78% yield) was obtained after crystallization from MeOH/Et 2 O, mp >224°C (dec). IR: 2931 IR: , 2866 IR: , 2770 IR: , 2435 IR: , 1668 IR: , 1611 IR: , 1460 IR: , 1430 IR: , 1398 IR: , 1338 IR: , 1302 IR: , 1154 IR: , 1028 To a solution of acid 20 (389 mg, 2.16 mmol) in toluene (6.5 mL), Et 3 N (0.4 mL, 2.9 mmol), and diphenylphosphorylazide (875 mg, 3.18 mmol) were added and the mixture was heated under reflux for 3 h. The cold (ice-bath) solution was washed with cold 1 N HCl (10Â 5 mL). Then, 6 N HCl (9 mL) was added to the organic solution and the mixture was heated under reflux for 24 h.

The organic layer was separated, the aqueous layer was washed with Et 2 O (3Â 5 mL) and the water was removed in a freeze-dryer giving 21ÁHCl as a white solid (296 mg, 73% yield). The analytical sample was obtained by crystallization from water, mp 210À211°C. IR: 2959 IR: , 2945 IR: , 2891 IR: , 2863 IR: , 2765 IR: , 2685 IR: , 2579 IR: , 1602 IR: , 1493 IR: , 1480 IR: , 1460 IR: , 1308 IR: , 1285 IR: , 1163 , 9.67; N, 7.46; Cl, 18.89. Found: C, 63.71; H, 9.70; N, 7.48; Cl, 7 ] oct-1-yl)amine hydrochloride (22aÁHCl)

To a solution of 21ÁHCl (770 mg, 4.11 mmol) in MeOH (15 mL), NaBH 3 CN (95%, 585 mg, 8.86 mmol), AcOH (0.3 mL), and benzaldehyde (653 mg, 6.16 mmol) were added, and the mixture was stirred at room temperature for 18 h. The solution was concentrated to dryness, water (30 mL) was added to the residue and the mixture was extracted with Et 2 O (3Â 25 mL). The combined organic extracts were washed with 2 N NaOH (3Â 25 mL) and brine (2Â 25 mL), dried (anhyd Na 2 SO 4 ) and concentrated in vacuo. The residue was subjected to column chromatography (silica gel; hexane/ EtOAc, 97/3) to give amine 22a (594 mg, 60% yield). An analytical sample of 22aÁHCl was obtained by adding an excess of Et 2 OÁHCl to a solution of 22a in EtOAc and filtration of the formed precipitate, mp >258°C (dec). IR: 2957 IR: , 2934 IR: , 2917 IR: , 2911 IR: , 2759 IR: , 2739 IR: , 2732 IR: , 2725 IR: , 2626 IR: , 2619 IR: , 2578 IR: , 2423 IR: , 1459 IR: , 1452 IR: , 1308 IR: , 1160 IR: , 1028 , 693 cm À1 . 1 H NMR 1.23 (s, 6H, C3 (7) , 8.71; N, 5.04; Cl, 12.76. Found: C, 73.83; H, 8.78; N, 5.02; Cl, 12.92. 4.1.19 . N-(2-Thenyl)-3,7-dimethyl(tricyclo[3.3.0.0 3,7 ]oct-1-yl)amine hydrochloride (22bÁHCl)

From 21ÁHCl (187.7 mg, 1.00 mmol), NaBH 3 CN (95%, 198 mg, 3 mmol), acetic acid (0.3 mL), and 2-thiophenecarbaldehyde (0.15 mL, 1.65 mmol) in methanol (10 mL) and following the above procedure, 22bÁHCl (196 mg, 69% yield) was obtained. The analytical sample of 22bÁHCl was obtained by crystallization from MeOH/ Et 2 O, mp >255°C (dec). IR: 3062, 2952 IR: 3062, , 2919 IR: 3062, , 2887 IR: 3062, , 2727 IR: 3062, , 2689 IR: 3062, , 2549 IR: 3062, , 2451 IR: 3062, , 1583 IR: 3062, , 1477 IR: 3062, , 1440 IR: 3062, , 1375 IR: 3062, , 1307 IR: 3062, , 1280 IR: 3062, , 1247 IR: 3062, , 1159 IR: 3062, , 1068 IR: 3062, , 1007 , 701 cm À1 . 1 H NMR 1.23 (s, 6 H, C3 (7) 13 C NMR 16.4 [CH 3 , C3(7)-CH 3 ], 43.5 (CH 2 , CH 2 N), 43.6 (CH, C5), 47.8 [C, C3 (7)], 53.6 [CH 2 , C4 (6)], 54.9 [CH 2 , C2(8)], 68.0 (C, C1), 128.7 (CH, Ar-C4), 129.2 (CH, Ar-C5), 4.1.23. N, 7 ] oct-1-yl)amine hydrochloride (27ÁHCl)

A mixture of 21ÁHCl (178 mg, 0.95 mmol), K 2 CO 3 (1.03 g, 7.5 mmol), benzyl chloride (0.29 mL, 2.5 mmol) and NaI (100 mg, 0.67 mmol) in acetonitrile (10 mL) was heated under reflux for 24 h. The mixture was concentrated in vacuo and EtOAc (30 mL) was added to the residue. The organic solution was washed with water (2Â 20 mL), dried (anhy Na 2 SO 4 ) and concentrated in vacuo. The residue was subjected to column chromatography (silica gel; hexane/EtOAc, 99:1) to give amine 27 (130 mg, 41% yield). An analytical sample of 27ÁHCl was obtained by adding an excess of Et 2 OÁHCl to a solution of 27 in EtOAc and filtration of the formed precipitate, mp 191À192°C. IR: 3034, 2959, 2920, 2887, 2864,

The antiviral activity of the compounds was determined in established cell culture assays using a selection of DNA and RNA viruses, including three subtypes of influenza virus [A/Puerto Rico/8/34 (H1N1); A/Hong Kong/7/87 (H3N2) and B/Hong Kong/ 5/72]. 21 The compounds' inhibitory effect on virus replication as well as their cytotoxicity were monitored by microscopical examination, and confirmed by the colorimetric MTS cell viability assay.

Female Wistar rats (200-250 g) were used throughout. Briefly, rats were killed by decapitation and the striatum was dissected and homogenized in 10 volumes (w/v) of 0.32 M sucrose using a Potter-Elvejhem. The resulting crude synaptosomal preparation was centrifuged at 1000g for 10 min. The supernatant was stored and the pellet was resuspended in 10 volumes of 0.32 M sucrose and recentrifuged. The two supernatants were combined and the mixture centrifuged at 16,000g for 30 min. The resultant pellet was suspended in 10 volumes of ice-cold Krebs medium. Protein concentrations were determined using the Bradford protein assay.

[ 3 H]Dopamine uptake was evaluated on aliquots of the synaptosomal preparation. After a 10 min preincubation at 37°C in Krebs buffer containing 10 lM pargyline (to block metabolism of dopamine by monoamine oxidase), [ 3 H]dopamine (47 Ci/mmole, Amersham) was added to a final 0.5 nM concentration. Ten minute incubations were stopped by dilution into ice-cold Krebs medium. Samples were filtered rapidly through Grade 30 fiberglass filters (Schleicher & Schuell) using a Brandel cell harvester (model M-24, Biochemical Research and Development Laboratories, Inc.). Filters were washed twice with 3 mL cold Krebs medium and dried. Non-specific [ 3 H]DA uptake was determined in duplicate samples in the presence of 10 lM nomifensine (dopamine uptake inhibitor). Filters were placed into scintillation mixture (Optiphase 'Hisafe' 2, Perkin-Elmer) and radioactivity was determined by scintillation spectrometry.

MS (EI), m/z (%): 247 (M Å+ , 2)

)amine hydrochloride (23ÁHCl) From a solution of 22aÁHCl (635 mg, 2.29 mmol), acetonitrile (15 mL), formaldehyde (1.81 mL, 37% wt. in water solution, 23 mmol), two portions of NaBH 3 CN (95%, 455 mg, 6.88 mmol) and following the procedure described for 9, the amine 23 (516 mg, 88.5% yield) was obtained. An analytical sample of 23ÁHCl was obtained by adding an excess of Et 2 OÁHCl to a solution of 23 in EtOAc followed by filtration of the obtained precipitate

MS (EI), m/z (%): 255 (M Å+ , 3)

This salt was added to anhyd Et 2 O (20 mL) and the resulting suspension was cooled to 0°C. Methyllithium (18.8 mL, 1.6 M in Et 2 O, 30 mmol) was added dropwise and the suspension was heated under reflux for 18 h. To the cold (ice-bath) mixture, water (15 mL) was added dropwise, and the mixture was further stirred for 15 min. The organic layer was separated and the aqueous phase was extracted with Et 2 O (3Â 15 mL). The combined organic phases were dried (anhyd Na 2 SO 4 ) and concentrated in vacuo at room temperature to give ketone 24 (382 mg, 43% yield; 57% yield based on unrecovered starting material). The aqueous layer was made acidic and extracted with CH 2 Cl 2 (4Â 10 mL). The combined organic phases were dried (anhyd Na 2 SO 4 ), and concentrated in vacuo to give starting acid 20 (223 mg). IR: 2956

MS (EI), m/z (%): 178 (M Å+ , 3)

Accurate mass measurement (ESI + ) calcd for

To a solution of ketone 24 (168 mg, 0.94 mmol) in ethanol (1 mL), hydroxylamine hydrochloride (103 mg, 1.49 mmol), water (0.1 mL), and powdered NaOH (190 mg, 4.75 mmol) were added and the mixture was heated under reflux for 5 min. The cold solution (ice-bath) was added to a cold solution (ice bath) of concd HCl (0.64 mmol, 7.72 mmol) and water (3.5 mL). The obtained precipitate was filtered, washed with cold water (2Â 2 mL) and dried in vacuo over P 4 O 10 to give the title oxime (135 mg, 74% yield) that was used without further purification in the next step

1062 cm À1 . 1 H NMR 1.192 (s, 3H) and 1.195 (s, 3H) [C3-CH 3 and C7-CH 3

oct-1-yl)amine hydrochloride (26ÁHCl) From amine 21 (151 mg, 1.0 mmol) in Et 2 O (5 mL), formaldehyde (1.8 mL, 37% wt. in water solution, 22.8 mmol), and formic acid (1.5 mL, 39 mmol) and following the procedure described for 17, the amine 26 was obtained as its hydrochloride. The analytical sample of 26ÁHCl (180 mg, 83.5% yield) was obtained by crystallization from MeOH/Et 2 O, mp 173À174°C. IR 2999

75.4 (C, C1). MS (EI), m/z (%): 179 (M Å+ , 2)

Ar-4-H). 13 C NMR 16

Anal. Calcd for C 24 H 29 NÁ1

Accurate mass measurement (ESI + ) calcd for

yl)amine hydrochloride (28ÁHCl) A mixture of 23ÁHCl (390 mg, 1.33 mmol) and 10% Pd/C (50% in water, 10 mg) in absolute EtOH (80 mL) was hydrogenated at 38 atm and 100°C for 24 h. The suspension was filtered, the residue was washed with EtOH and the organic layer was concentrated in vacuo to give a solid. Crystallization from MeOH/Et 2 O gave 28ÁHCl (240 mg, 89% yield). An analytical sample of 28ÁHCl was obtained by crystallization from THF, mp 167À168°C. IR (KBr)

85 (s, mobile H). 13 C NMR (75.4 MHz) 16.5 [CH

The oily yellow residue (1.16 g) was dissolved in CHCl 3 , the solution was cooled to 0°C and NH 4 OH (60 mL, 25% aqueous solution) was added dropwise. After stirring for 15 h at room temperature, the suspension was extracted with CH 2 Cl 2 (4Â 30 mL). The aqueous layer was made acidic with concd HCl and extracted with EtOAc (3Â 30 mL). The combined EtOAc extracts were dried (anhyd Na 2 SO 4 ) and concentrated in vacuo to give the starting acid 20 (438 mg). The combined CH 2 Cl 2 extracts were washed with brine (2Â 30 mL), dried (anhyd Na 2 SO 4 ), and concentrated in vacuo to give amide 29 (572 mg, 51% yield, 84% yield taking into account the recovered starting acid). An analytical sample of 29 was obtained by crystallization from EtOAc

MS (EI), m/z (%): 179 (M Å+ , 4)

LiAlH 4 (388 mg, 9.72 mmol) was added and the suspension was heated under reflux for 15 h. The suspension was cooled (ice bath), carefully basified with 10 N NaOH (5 mL) and stirred for 1 h at room temperature. The precipitate was filtered and washed with CH 2 Cl 2 (3Â 25 mL). The combined filtrate and washings were dried (anhyd Na 2 SO 4 ) and excess of Et 2 OÁHCl was added. The solution was concentrated in vacuo to give a solid that was crystallized from MeOH/Et 2 O to give 30ÁHCl (534 mg, 83% yield)

Anal. Calcd for C 11 H 19 NÁHClÁ0

AcOH (0.3 mL), and benzaldehyde (395 mg, 3.68 mmol) and following the procedure described for 22a, 31ÁHCl (489 mg, 68% yield) was obtained

Anal. Calcd for C 18 H 25 NÁHClÁ0

260 mg, 70.5% yield) was obtained. Its hydrochloride was obtained by adding an excess of Et 2 OÁHCl to a solution of the amine in EtOAc followed by concentration to dryness in vacuo. The analytical sample of 32ÁHCl was obtained by crystallization from EtOAc, mp 215À216°C

MS (EI), m/z (%): 269 (M Å+

Anal. Calcd for C 19 H 27 NÁHCl (305.89): C, 74

mL, 37% wt. in water solution, 12.7 mmol) and formic acid (0.85 mL, 22 mmol) and following the procedure described for 17, 33ÁHCl was obtained. The analytical sample of 33ÁHCl (68 mg, 61% yield) was obtained by crystallization from THF

Accurate mass measurement (ESI + ) calcd for

The analytical sample of 34ÁHCl was obtained by crystallization from 2-propanol

61.5 (CH 2 , CH 2 N). MS (EI), m/z (%):233 (M Å+ , 9

Found: C, 71

mg, 77% yield) was obtained. The analytical sample of 35ÁHCl was obtained by crystallization from MeOH/Et 2 O, mp 214À215°C

3,7 ]oct-1-yl)methyl]amine hydrochloride (36ÁHCl) From 32ÁHCl (200 mg, 0.65 mmol), 10% Pd/C (50% in water, 10 mg) and absolute ethanol (25 mL) and following the procedure described for 28, 36ÁHCl (120 mg, 85% yield) was obtained after crystallization from MeOH/Et 2 O, mp >255°C

27 (s, 2H, CH 2 N), 4.86 (s, mobile H). 13 C NMR 16

k) Camps, P.; Fernández

We found a patent that claimed antiviral activity for some bisnor-and noradamantane derivatives, but activity details are not given. See, Hoover

An Introduction to Medicinal Chemistry

Financial support from Ministerio de Educación y Ciencia (P.C. 

Cultures of bloodstream form T. brucei (strain 427) were maintained at 37°C in modified Iscove's medium (pH 7.4) . 20 Trypanocidal activity was assessed by growing parasites for 48 h in the presence of various drug concentrations and determining the levels which inhibited growth by 50% (IC 50 ) and 90% (IC 90 ). In the case of untreated cultures (volume 4 mL), cell densities increased from 0.25 Â 10 5 to 1 Â 10 6 cells mL À1 over this period. Experiments were performed in triplicate. Cell densities at each drug concentration were determined using a hemocytometer (Weber Scientific International Ltd), and drug sensitivity was expressed as a percentage of growth of control cells.

The functional assay of antagonist activity at NMDA receptors was performed using primary cultures of cerebellar granule neurons, which were prepared according to established protocols. 18 Cells were grown on 10 mm poly-L-lysine coated glass cover slips, and used for the experiments after 7-14 days in vitro. Cells were loaded with 6 lM Fura-2 AM (Invitrogen-Molecular Probes) for 45 min. Afterwards, the coverslip was mounted on a quartz cuvette containing a Locke-Hepes buffer using a special holder. Measurements were performed using a PerkinElmer LS-50B fluorometer equipped with a fast-filter accessory, under mild agitation and at 37°C. Analysis from each sample was recorded real-time during 1200 s. After stimulation with NMDA or glutamate (100 lM, in the presence of 10 lM glycine), increasing cumulative concentrations of the compound to be tested were added. The percentages of inhibition at every tested concentration were analyzed using a non-linear regression curve fitting (variable slope) by using the software GraphPad Prism 4.0.