Synthesis of benzopolycyclic cage amines: NMDA receptor antagonist, trypanocidal and antiviral activities

Article abstract of DOI:10.1016/j.bmc.2011.11.050

The synthesis of several 6,7,8,9,10,11-hexahydro-9-methyl-5,7:9,11- dimethano-5H-benzocyclononen-7-amines is reported. Several of them display low micromolar NMDA receptor antagonist and/or trypanocidal activities. Two compounds are endowed with micromola

Full text of DOI:10.1016/j.bmc.2011.11.050

Bioorganic & Medicinal Chemistry 20 (2012) 942–948
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of benzopolycyclic cage amines: NMDA receptor antagonist,
trypanocidal and antiviral activities
Eva Torres a, María D. Duque a, Marta López-Querol b, Martin C. Taylor c, Lieve Naesens d, Chunlong Ma e,
Lawrence H. Pinto e, Francesc X. Sureda b, John M. Kelly c, Santiago Vázquez a,
⇑
a Laboratori de Química Farmacèutica (Unitat Associada al CSIC), Facultat de Farmàcia and Institute of Biomedicine (IBUB), Universitat de Barcelona,
Av. Diagonal, 643, Barcelona E-08028, Spain
b Unitat de Farmacologia, Facultat de Medicina i Ciències de la Salut, Universitat Rovira i Virgili, C./St. Llorenç 21, Reus E-43201, Spain
c London School of Hygiene and Tropical Medicine, Department of Infectious and Tropical Diseases, Keppel Street, London WC1E 7HT, United Kingdom
d Rega Institute for Medical Research, Katolieke Universiteit Leuven, 3000 Leuven, Belgium
e Department of Neurobiology and Physiology, Northwestern University, Evanston, IL 60208-3500, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of several 6,7,8,9,10,11-hexahydro-9-methyl-5,7:9,11-dimethano-5H-benzocyclononen-7-
amines is reported. Several of them display low micromolar NMDA receptor antagonist and/or trypano-
cidal activities. Two compounds are endowed with micromolar anti vesicular stomatitis virus activity,
while only one compound shows micromolar anti-influenza activity. The anti-influenza activity of this
compound does not seem to be mediated by blocking of the M2 protein.
Received 6 August 2011
Revised 21 November 2011
Accepted 23 November 2011
Available online 2 December 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
NMDA
Trypanosoma
Amantadine
Antivirals
Cage compounds
1. Introduction
amantadine but less potent than memantine.4 Moreover, several
of these compoundsdisplayed an anti-influenza activity very similar
Amantadine and its dimethyl derivative memantine, are two
low-affinity, uncompetitive NMDAR antagonists, clinically used
to treat Parkinson’s and Alzheimer’s diseases, respectively.1 Aman-
tadine and its analogue rimantadine also target the M2 ion-chan-
nel protein of the influenza A virus, having prophylactic and
therapeutic activities in influenza A virus infections. However,
many influenza virus strains have developed resistance to amanta-
dine and rimantadine.2 Finally, amantadine, rimantadine and re-
lated polycyclic amines possess trypanocidal properties (Fig. 1).3
Considering the interesting pharmacological profile of amanta-
dine, rimantadine and memantine, these compounds are ideal
leads in the development of new agents to treat excitotoxic
NMDAR channel mediated neurodegenerative diseases, influenza
infections or African trypanosomiasis.
to that of amantadine.5 We also found that oxapolycyclic amines
such as 3-alkyl(2-oxadamantyl)-1-amines and 1,2,3,5,6,7-hexahy-
dro-1,5:3,7-dimethano-4-benzoxonin-3-amines of general struc-
tures 1 and 2, respectively, were also active as NMDA receptor
antagonists. While these compounds lacked anti-influenza activity,
several of them displayed trypanocidal activity (Fig. 2).6
Although several 1,2,3,5,6,7-hexahydro-1,5:3,7-dimethano-4-
benzoxonin-3-amines, 2, displayed low micromolar activity as
NMDA receptor antagonists, the presence of an hemiaminal subunit
in these compounds may raise serious concerns about their stability
and thus of their further development as therapeutical agents. In
fact, it is known that NGP1-01, a micromolar NMDA receptor antag-
onist containing an hemiaminal unit,7 decomposes easily in aque-
ous medium.8
Recently, we started a research project directed to the synthesis
and pharmacological evaluation of new analogs of amantadine,
memantine and rimantadine. So far, we have synthesized ring-con-
tracted analogs of these drugs and found that they showed NMDA
receptor antagonism, many of them being more potent than
NH2
R
NH2
R
R = H, amantadine
R = Me, memantine
rimantadine
⇑
Corresponding author. Tel.: +34 934024533; fax: +34 934035941.
E-mail address: svazquez@ub.edu (S. Vázquez).
Fig. 1. Structures of amantadine, memantine and rimantadine.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.11.050

E. Torres et al. / Bioorg. Med. Chem. 20 (2012) 942–948
943
was obtained by reaction of 3 with methyl chloroformate followed
by LiAlH4-mediated reduction of the intermediate carbamate, in
28% overall yield. Alkylation of 3 with propargyl bromide led to a
mixture of 9 and 10 that were separated by column chromatogra-
phy. Finally, alkylation of 3 with 1,5-dibromopentane gave the
piperidine derivative 12 in 35% yield (Scheme 1).
R3
R3
O
O
R1
R1
N
R2
N
R2
1
2
10
1
4
CH3
9
7
11
13
12
2
3
2.2. NMDA receptor antagonist activity
8
3
4a 5
H
N
H
6
To evaluate if the synthesized compounds were able to antago-
nize NMDA receptors, we have measured its effect on the increase
in intracellular calcium evoked by glutamate or NMDA (both
Fig. 2. General structures of previously studied polycycles 1 and 2 and the new
amine 3.
100 lM, in the presence of 10 lM of glycine) on rat cultured cere-
bellar granule neurons.12 Inspection of the results shown in Table 1
reveals that piperidine derivative 12 had a value of IC50 (NMDA)
very similar to that of amantadine, while compounds 3, 8, 9 and
11 had values of IC50 (NMDA) that were in the micromolar order,
lower than amantadine but higher than memantine (Table 1).
Benzyl derivatives 6 and 7 were devoid of activity, in agreement
with our previous results with derivatives of general structures 1
and 2,6 but in striking contrast with NGP1-01, a low micromolar
In this Letter we wish to report the synthesis and pharmacolog-
ical evaluation of several benzopolycyclic amines derived from 3
(Fig. 2). Several of these derivatives, that are devoid of the hemiami-
nal unit, show low micromolar NMDA receptor antagonist activity,
although with higher IC50 than the previously described oxa-ana-
logues. Interestingly, several of the new derivatives are endowed
with low micromolar trypanocidal activity while only one com-
pound has anti-influenza activity.
Table 1
IC50
(lM) values for 6,7,8,9,10,11-hexahydro-5,7:9,11-dimethano-5H-benzocyclo-
nonen-7-amines as NMDA antagonistsa,b
2. Results and discussion
2.1. Synthesis
Compound
Glutamate (100
l
M)
NMDA (100 lM)
3
8
9
11
22 3.9
455 216
>500
87 28
>500
13.6 3.4
19.4 3.3
27.5 13.2
11.8 3.1
101 25
92 29
Inspired by the work of Bishop et al., we prepared amine 3 from
known diene 4.9 Thus, Ritter-type transannular cyclization with
chloroacetonitrile in acidic medium,10 and subsequent cleavage of
the chloroacetyl group in the resulting chloroacetamide 5 with thio-
urea led to amine 3 in medium overall yield.11 Starting from amine 3
we have prepared several secondary and tertiary amines using clas-
sical methods in amine chemistry. Briefly, reductive alkylation of 3
with benzaldehyde and NaBH3CN led to 6 in 59% while reductive
alkylation of 3 and 6 with formaldehyde and NaBH3CN furnished
7 and 11 in 55% and 75% yield, respectively. Hydrogenolysis of
amine 7 led to secondary amine 8 in 74% yield. Alternatively, 8
12
Amantadine
Memantine
358 130
55 12
1.5 0.1
a
Functional data were obtained from primary cultures of cerebellar granule
neurons using the method described in Section 4.2. by measuring the intracellular
calcium concentration. Cells were challenged with glutamate (2nd column) or
NMDA (3rd column) as indicated. Data shown are means SEM of at least three
separate experiments carried out on three different batches of cultured cells.
b
Compounds 6, 7, and 10 were found to have low potency as glutamate (IC50
>500
lM) and NMDA receptor antagonists (IC50 >200 lM).
CH3
CH3
a
b
NH
O
NH2
4
Cl
5
3
CH3
NH
CH3
CH3
d
c
e
CH3
CH3
3
N
H
N
CH2C6H5
CH2C6H5
7
6
8
f
CH3
CH3
N
g
d
+
N
H
9
10
CH3
CH3
N
CH3
N
CH3
12
11
Scheme 1. Synthesis of benzopolycyclic amines from known diene 4. Reagents and conditions: (a) chloroacetonitrile, acetic acid, conc. H2SO4, 0 °C to rt, overnight, 71%; (b)
acetic acid, thiourea, ethanol, reflux, overnight, 62%; (c) benzaldehyde, NaBH3CN, AcOH, MeOH, rt, 18 h, 68%; (d) CH2O, NaBH3CN, AcOH, acetonitrile (for 7) or methanol (for
11), rt, 2 h, 55% for 7, 75% for 11; (e) H2, Pd/C, 1 atm, 90 °C, 24 h, 74%; (f) propargyl bromide, K2CO3, NaI, acetonitrile, reflux, 18 h, 17% of 9 and 48% of 10; (g) 1,5-
dibromopentane, Et3N, DMF, 60 °C, 26 h, 35%.

944
E. Torres et al. / Bioorg. Med. Chem. 20 (2012) 942–948
NMDA receptor antagonist that features a benzyl group in its
structure.7
In our previous work with amines of general structure 2 we
found that the NMDA receptor antagonist activity increased in
going from the primary amine to the tertiary lower alkyl amine
with upwards of 60 million people at risk. A process of antigenic
variation prevents elimination of the parasite by the immune sys-
tem, and the prospects of developing a vaccine are thought to be
remote. New drugs against HAT are therefore a major priority.
Drugs currently available against HAT are characterized by prob-
lems including toxicity, limited efficacy, cost and the need to
administer under medical supervision.
The anti-influenza virus drug rimantadine has been shown to be
active in vitro against bloodstream form T. brucei and other ada-
mantane derivatives have even greater activity.3,4,13 Moreover,
very recently, we found that several derivatives of general struc-
tures 1 and 2 showed significant activity against bloodstream form
T. brucei.6 We therefore investigated if the newly synthesized poly-
cyclic cage amines reported here also displayed significant activity
against this parasite.
(R1@R2@R3@H; IC50 = 35
R1@R2@CH3, R3@H; IC50 = 6
proved in going from 3 to 8 and 11.
l
M; R1@R3@H, R2@CH3; IC50 = 6
lM;
lM). However, the activity did not im-
We had previously found that the replacement of the methylene
unit of C-2 in amantadine by an oxygen atom, as in compound 1
(R1@R2@R3@H), led to less potent NMDA receptor antagonists.6a
Taking into account the good activity of several of our oxapolycyclic
amines of general structure 2, we expected that the replacement of
the oxygen atom on these derivatives by a methylene unit, as in
the newer derivatives herein reported, would lead to more potent
NMDA receptor antagonists. In fact, amine 3 (IC50 = 13.6
much more potent than the previously described amine
(R1@R2@H, R3@CH3; IC50 = 98 M). However, this trend is not
general, and, for example, amine 11 is 3 times less potent
(IC50 = 12 M) than tertiary amine 2 (R1@R2@R3@CH3; IC50 = 4 M).
Worthy of note, the IC50 of compound 8 as NMDA receptor
antagonist is more than 20 times lower than its IC50 as glutamate
receptor antagonist, in a similar manner than has been found for
memantine.
l
M) is
A total of 7 compounds were tested. Of these, 11 displayed little
or no inhibitory activity against cultured bloodstream form T. brucei
2
l
at 5 l lM). However, compound 10 inhibited para-
g mLÀ1 (15–20
site growth, at a level comparable to memantine. More interest-
ingly, compounds 3, 6, 7, 8 and 9 significantly inhibited parasite
growth, at levels lower than rimantadine and memantine (Table
2). The IC90 of the more active compound, 9, was more than 4 times
lower than that of rimantadine. Inspection of the results shown in
Table 2 reveals that secondary amines 6, 8 and 9 were more potent
than tertiary amines 7, 10 and 11.
l
l
2.3. Trypanocidal activity
2.4. Antiviral activity
Human African trypanosomiasis (HAT) is caused by infection
with protozoan parasites of the Trypanosoma brucei species com-
plex. The disease is endemic in many parts of sub-Saharan Africa,
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,
sindbis virus or Punta Toro virus; or the non-enveloped RNA
viruses Coxsackievirus B4 and Reovirus-1.
Table 2
IC50 and IC90
(lM) values for oxapolycyclic cage amines that display inhibitory
activity against cultured bloodstream form T. bruceia
Compound 10 was active against vesicular stomatitis virus
(VSV) in HeLa cells, with an EC50 (50% antiviral effective concentra-
Compound
IC50
(
l
M)
IC90 (lM)
3
6
7
8
9
10
4.80 0.41
3.00 0.32
5.25 0.99
4.46 0.54
1.99 0.46
13.44 0.56
7.04 0.27
13.75 1.26
9.60 0.30
6.55 0.32
10.85 0.59
6.66 0.18
3.14 0.10
21.61 0.44
13.97 1.68
21.43 0.55
tion) of 4
concentration) of 25. Another compound, 12, was only weakly ac-
tive (EC50 = 54 M). The antiviral data obtained by microscopy
lM and a selectivity index (ratio of cytotoxic to antiviral
l
were confirmed by a colorimetric cell viability assay. However,
both compounds were not active against VSV in HEL cells, which
suggest that the anti-VSV activity may be cell type-dependent.
In the influenza virus assays, all compounds proved to be inac-
tive against the A/H3N2 subtype and influenza B virus. Interest-
ingly, benzyl derivative 6 displayed reasonable activity against
Rimantadine
Memantine
a
Compounds were tested for in vitro activity against bloodstream form T. brucei
at a range of concentrations and IC50 and IC90 values established (Section 4.5). Data
are the mean of triplicate experiments SEM. Values for rimantadine and
memantine are shown for comparison.
the influenza A/H1N1 subtype (antiviral EC50 = 10 lM).
Addition of the benzene ring to
the adamantane nucleusis compatible
with anti-trypanosoma and NMD Areceptor
antagonist activities but detrimental
for anti-influenza activity.
CH3
Introduction of this methylene unit instead
of the oxygen atom previously studied,
improves the anti-trypanosoma activity.
R
N
R'
A benzyl substituent is compatible
with anti-trypanosoma activity but
detrimental for NMDA antagonism.
NMDA receptor antagonist activity is
better when R=R'=H and R=R'=CH3.
CH3
CH3
CH3
N
CH3
N
H
11
9
Fig. 3. Main features of the SAR of the new benzopolycyclic family of compounds, including 11, the most potent compound as NMDA receptor antagonist
(IC50 = 11.8 3.1 M), and 9, the most potent compound as trypanocidal (IC50 = 1.99 0.46 M; IC90 = 3.14 0.10 M).
l
l
l

E. Torres et al. / Bioorg. Med. Chem. 20 (2012) 942–948
945
It is well-known that the target of amantadine and rimantadine
is the influenza A virus M2 channel protein and that a single S31N
mutation in M2 renders the virus resistant to both drugs.2,14 As most
of the currently circulating subtypes of influenza A virus, A/H3N2
and A/H1N1, carry the S31N mutation in M2, there is an urgent need
for the development of novel anti-influenza drugs that are effective
against the most common amantadine-resistant mutants.15
The influenza A/PuertoRico/8/34 strain used for testing the activ-
ity of amine 6 has an M2 channel carrying two substituions associ-
ated with amantadine resistance, that is S31N and V27T. In order to
assess if the target of 6 was the M2 protein, the inhibitory activity of
6 was tested on A/M2 channels expressed in Xenopus oocytes using
the two-electrode voltage clamps (TEV) technique. Amine 6, at
4.1.2. N-(6,7,8,9,10,11-Hexahydro-9-methyl-5,7:9,11-dimethano
-5H-benzocyclononen-7-yl)chloroacetamide, 5
A suspension of diene 4 (5.3 g, 25.2 mmol), chloroacetonitrile
(6.5 mL) and acetic acid (17 mL) was cooled to 0 °C and concen-
trated H2SO4 (8.2 mL, 151.2 mmol) was added dropwise
(T < 10 °C). The mixture was allowed to reach room temperature
and was stirred overnight. The suspension was added to ice
(120 g) and a yellow solid precipitated. The precipitate was filtered
and solved in dichloromethane (100 mL). The organic phase was
dried with anhyd Na2SO4, filtered and concentrated in vacuo to give
5 as a yellow solid (5.44 g, 71% yield). The analytical sample of 5 was
obtained by crystallization from CH2Cl2, mp 166À168 °C. IR 3320,
3301, 3062, 2949, 2910, 2861, 1659, 1651, 1540, 1492, 1451,
1431, 1361, 1328, 1312, 1210, 1145, 1091, 1009, 971, 943, 765,
100 lM, was unable to inhibit the activity of the wild-type (aman-
tadine-sensitive) and the S31N and the V27A (amantadine-resis-
tant) mutants, thus suggesting that 6 interacts with a different
target in the virus. Neither 3 nor 11 were able to inhibit the activity
of the wild-type and the S31N and the V27A mutants.
747, 694, 651, 610, 507, 478 cmÀ1 1H NMR (CDCl3) 0.94 (s, 3H,
.
CH3), 1.56 [d, J = 13.5 Hz, 2H, 10(13)-Hb], 1.67 [ddd, J = 13.5 Hz,
J’ = 6.0 Hz, J’’ = 2.0 Hz, 2H, 10(13)-Ha], 1.85 (s, 2H, 8-H2), 2.09 [d,
J = 13 Hz, 2H, 6(12)-Hb], 2.17 [ddd, J = 13 Hz, J’ = 6.0 Hz, J’’ = 2.0 Hz,
2H, 6(12)-Ha], 3.09 (tt, J = 6.0 Hz, J’ = 2.0 Hz, 2H, 5(11)-H], 3.93 (s,
2H, CH2Cl), 6.31 (br s, 1H, NH), 7.04–7.10 [complex signal, 4H,
1(4)-H and 2(3)-H]. 13C NMR (CDCl3) 32.1 (CH3, C9-CH3), 33.6 (C,
C9), 38.7 [CH2, C6(12)], 40.9 [CH, C5(11)], 41.0 [CH2, C10(13)],
42.9 (CH2, CH2Cl), 46.9 (CH2, C8), 54.9 (C, C7), 126.4 [CH, C2(3)],
128.0 [CH, C1(4)], 146.0 [C, C4a(C11a)], 164.5 (C, CO). MS (EI), m/z
(%): 303 (MÁ+, 31), 268 (18), 211 [(C16H19)+, 29], 210 (100), 195
(28), 182 (13), 181 (15), 167 (11), 156 (15), 155 (82), 154 (20), 143
(14), 142 (13), 141 (20), 129 (19), 128 (23), 115 (17). Anal. Calcd.
for C18H22ClNO (303.83): C 71.16, H 7.30, N 4.61, Cl 11.67. Found
C 71.07, H 7.28, N 4.50, Cl 11.44.
3. Conclusions
In summary, we have synthesized and fully characterized sev-
eral benzopolycyclic amines. Several of the derivatives were more
potent than amantadine against NMDA-induced calcium increase
in cerebellar granule neurons, although they were less potent than
memantine. Several amines displayed a significant level of trypan-
ocidal activity, being up to four times more potent than rimanta-
dine. Figure 3 summarizes the main features of the SAR of these
new compounds, including the most potent compounds.
Regarding antiviral activity, compound 10 displayed anti-VSV
activity in HeLa cells, but not in HEL cells, suggesting that its anti-
viral activity can be cell type-dependent. Finally, benzyl derivative
6, that is not active as NMDA receptor antagonist, is a low micromo-
lar inhibitor of the influenza A/H1N1 subtype. Interestingly, this
anti-viral activity is not mediated by M2 channel blocking.
The synthesis and pharmacological evaluation of more polycy-
clic cage amines are in progress.
4.1.3. 6,7,8,9,10,11-Hexahydro-9-methyl-5,7:9,11-dimethano-5H
-benzocyclononen-7-amine hydrochloride, 3ÁHCl
A mixture of chloroacetamide 5 (7.21 g, 23.7 mml), thiourea
(2.16 g, 28.44 mmol), acetic acid (8.7 mL) in ethanol (44 mL) was
stirred under reflux for 18 h. To the cold solution water (250 mL),
and 10 N NaOH (80 mL) were added. The base aqueous solution
was extracted with EtOAc (3 Â 300 mL). The combined organic
extracts were dried with anhyd Na2SO4, filtered and concentrated
in vacuo to give a brown oily residue (7.19 g). The residue was ta-
ken in EtOAc and the amine 3 was precipitated as its hydrochloride
by adding an excess of Et2OÁHCl (3.87 g, 62% yield). The analytical
sample was obtained by crystallization from 2-propanol, mp
287À288 °C. IR 3499, 2985, 2944, 2907, 2860, 2717, 2680, 2622,
2581, 2548, 2061, 1606, 1509, 1495, 1454, 1362, 757, 578,
4. Experimental
4.1. Chemistry
4.1.1. General
Melting points were determined in open capillary tubes. Unless
otherwise stated, NMR spectra were recorded in CD3OD in the fol-
lowing spectrometers: 1H NMR (500 MHz), 13C NMR (100.6 MHz).
Chemical shits (d) are reported in ppm related to internal tetra-
methylsilane (TMS). Assignments given for the NMR spectra are
based on DEPT, COSY 1H/1H, HETCOR 1H/13C (HSQC and HMBC se-
quences for one bond and long range 1H/13C 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-meter
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 (CH4) techniques
were used. Only significant ions are given: those with higher rela-
tive 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 (cmÀ1).
Only the more intense bands are given. 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
F254 were used and spots were visualized with UV light and/or
1% aqueous solution of KMnO4.
471 cmÀ1 1H NMR 0.87 (s, 3H, CH3), 1.38 [d, J = 13.5 Hz, 2H,
.
10(13)-Hb], 1.54 [dd, J = 13.5 Hz, J’ = 6.0 Hz, 2H, 10(13)-Ha], 1.58
(s, 2H, 8-H2), 1.73 [d, J = 13.0 Hz, 2H, 6(12)-Hb], 1.96 [dd,
J = 13.0 Hz, J’ = 6.0 Hz, 2H, 6(12)-Ha], 3.09 [t, J = 6.0 Hz, 2H, 5(11)-
H], 7.05 (s, 4H, Ar-H), 8.31 (br s, 3H, NH3). 13C NMR 32.3 (CH3,
C9-CH3), 34.7 (C, C9), 39.3 [CH2, C6(12)], 41.6 [CH2, C10(13)],
41.7 [CH, C5(11)], 47.2 (CH2, C8), 55.5 (C, C7), 128.0 [CH, C2(3)],
129.2 [CH, C1(4)], 146.4 [C, C4a(C11a)]. MS (EI), m/z (%): 227
(MÁ+, 100), 212 (28), 185 (65), 171 (12), 170 (52), 157 (15), 156
(58), 155 (32), 153 (11), 144 (21), 143 (15), 141 (17), 130 (12),
129 (18), 128 (25), 115 (22), 108 (22), 94 (27). HRMS-ESI+ m/z
[M+H]+ calcd for C16H22N+H: 228.1746, found: 228.1743.
4.1.4. N-Benzyl-6,7,8,9,10,11-hexahydro-9-methyl-5,7:9,11-dime
thano-5H-benzocyclononen-7-amine hydrochloride, 6ÁHCl
To a solution of amine 3ÁHCl (200 mg, 0.76 mmol) in MeOH
(10 mL), NaBH3CN (104 mg, 1.65 mmol), AcOH (0.25 mL) and
benzaldehyde (80.4 mg, 0.76 mmol) were added and the mixture
was stirred at room temperature for 18 h and concentrated in va-
cuo to dryness. Water (10 mL) was added to the residue, and the
mixture was extracted with Et2O (4 Â 15 mL). The combined or-
ganic extracts were washed with 2 N NaOH (3 Â 25 mL), brine

946
E. Torres et al. / Bioorg. Med. Chem. 20 (2012) 942–948
(2 Â 25 mL), dried with anhyd Na2SO4, filtered and concentrated in
vacuo. The residue was taken in EtOAc and the amine 6 was precip-
itated as its hydrochloride (159 mg, 68% yield) by adding an excess
of Et2OÁHCl. The analytical sample was obtained by crystallization
from methanol/Et2O, mp 263À264 °C. IR 3410, 2948, 2912, 2840,
2733, 2407, 1494, 1455, 1364, 1347, 1307, 1217, 1127, 992, 943,
for C24H30NClÁ0.3H2O (373.37): C 77.21, H 8.26, N 3.75, Cl 9.50.
Found C 76.96, H 8.21, N 3.67, Cl 9.89.
4.1.6. N-Methyl-6,7,8,9,10,11-hexahydro-9-methyl-5,7:9,11-dim
ethano-5H-benzocyclononen-7-amine hydrochloride, 8ÁHCl
A suspension of 7ÁHCl (184 mg, 0.50 mmol) and 5% Pd/C (50% in
water, 50 mg) in absolute EtOH (25 mL) was hydrogenated at 1 atm
and 90 °C for 24 h. The suspension was filtered, the residue was
washed with EtOH and to the combined organic filtrates an excess
of Et2OÁHCl was added. Evaporation of the solvents from the filtrate
in vacuo followed by crystallization of the residue from methanol/
Et2O gave 8ÁHCl (102.8 mg, 74% yield) as a white solid, mp >225
dec. IR 3419, 2910, 2846, 2692, 2133, 2094, 2061, 1867, 1447,
755, 696, 567, 501 cmÀ1 1H NMR (CDCl3) 0.89 (s, 3H, CH3), 1.50
.
[d, J = 13.5 Hz, 2H, 10(13)-Hb], 1.66 [dd, J = 13.5 Hz, J’ = 6.0 Hz, 2H,
10(13)-Ha], 1.92 [d, J = 12.5 Hz, 2H, 6(12)-Hb], 2.00 (s, 2H, 8-H2),
2.24 [dd, J = 12.5 Hz, J’ = 6.0 Hz, 2H, 6(12)-Ha], 3.08 [m, 2H, 5(11)-
H], 3.80 (br s, 2H, CH2C6H5), 7.00 [m, 2H, 1(4)-Ar-H], 7.08 [m, 2H,
2(3)-Ar-H], 7.20 (t, J = 7.5 Hz, 1, Ar-Hpara), 7.27 (t, J = 7.5 Hz, 2H,
Ar-Hmeta), 7.61 (d, J = 7.5 Hz, 2H, Ar-Horto), 9.61 (br s, 2H, NH2).
13C NMR 32.3 (CH3, C9-CH3), 35.0 (C, C9), 37.0 [CH2, C6(12)], 41.7
[CH, C5(11)], 41.8 [CH2, C10(13)], 45.0 (CH2, CH2C6H5), 45.4 (CH2,
C8), 62.0 (C, C7), 128.0 [CH, C2(3)], 129.2 [CH, C1(4)], 130.3 (CH,
Ar-Cmeta), 130.5 (CH, Ar-Cpara), 131.2 (CH, Ar-Cortho), 133.1 (C, Ar-
1367, 1215, 1172, 1126, 1072, 1007, 957, 773, 574, 509 cmÀ1 1H
.
NMR 1.00 (s, 3H, C9-CH3), 1.56 [d, J = 13.5 Hz, 2H, 10(13)-Hb], 1.70
(s, 2H, 8-H2), 1.73 [dd, J = 13.5 Hz, J’ = 6.0 Hz, 2H, 10(13)-Ha], 1.90
[d, J = 12.5 Hz, 2H, 6(12)-Hb], 2.07 [dd, J = 12.5 Hz, J’ = 6.5 Hz, 2H,
6(12)-Ha], 2.61 (s, 3H, N-CH3), 3.24 [m, 2H, 5(11)-H], 7.10 (s, 4H,
Ar-H). 13C NMR 25.6 (CH3, CH3-N), 32.3 (CH3, C9-CH3), 34.9 (C, C9),
36.8 [CH2, C6(12)], 41.5 [CH, C5(11)], 41.7 [CH2, C10(13)], 45.3
(CH2, C8), 60.3 (C, C7), 128.0 [CH, C2(3)], 129.2 [CH, C1(4)], 146.3
[C, C4a(C11a)]. MS (EI), m/z (%): 241 (MÁ+, 100), 226 (35), 200 (12),
199 (72), 198 (12) 184 (40), 171 (14), 170 (47), 169 (13), 158 (14),
156 (11), 155 (30), 153 (11), 144 (11), 143 (12), 141 (23), 129 (22),
128 (28), 122 (21), 115 (27), 108 (29), 91 (10), 71 (28), 57 (10), 56
(15). HRMS-ESI+ m/z [M+H]+ calcd for C17H23N+H: 242.1903, found:
242.1902. Anal. Calcd. for C17H23NÁ1.6HCl (299.71): C 68.13, H 8.27,
N 4.67. Found C 68.11, H 8.20, N 4.27.
C
ipso), 146.4 [C, C4a(C11a)]. MS (EI), m/z (%): 317 (MÁ+, 100), 302
(23), 276 (15), 275 (64), 260 (19), 246 (23), 211 [(C16H19)+, 17],
184 (21), 169 (11), 155 (28), 147 (16), 146 (14), 143 (18), 141
(18), 129 (18), 128 (19), 115 (15), 91 (82). HRMS-ESI+ m/z
[M+H]+ calcd for C23H28N+H: 318.2216, found: 318.2215. Anal.
Calcd. for C23H27NÁ1.1HClÁ0.3H2O (362.99): C 76.11, H 7.97, N
3.86, Cl 10.74. Found C 76.42, H 7.65, N 3.62, Cl 10.36.
4.1.5. N-Benzyl-N-methyl-6,7,8,9,10,11-hexahydro-9-methyl-5,
7:9,11-dimethano-5H-benzocyclononen-7-amine
hydrochloride, 7ÁHCl
To a solution of 6ÁHCl (327 mg, 0.96 mmol) in acetonitrile
(10 mL), formaldehyde (0.75 mL, 37% wt. in water solution,
9.60 mmol) and NaBH3CN (180 mg, 2.88 mmol) were added. The
mixture was stirred for 30 min at room temperature, acetic acid
(0.5 mL) was added and the mixture was stirred at room tempera-
ture for 2 h. An additional portion of NaBH3CN (180 mg, 2.88 mmol)
was added and the mixture was further stirred at room temperature
for 2 h. The mixture was concentrated to dryness, 2 N NaOH (10 mL)
was added and the suspension was extracted with CH2Cl2
(3 Â 15 mL). The combined organic phases were washed with H2O
(2 Â 10 mL), dried with anhyd Na2SO4, filtered and concentrated
in vacuo to give 7 as an oil. Its hydrochloride was obtained by adding
an excess of Et2OÁHCl to a solution of the amine in EtOAc, followed
by filtration of the white solid precipitate (194 mg, 55% yield). The
analytical sample was obtained by crystallization from methanol/
Et2O, mp 250À251 °C. IR 3411, 3016, 2963, 2943, 2914, 2865,
2844, 2461, 2375, 1492, 1452, 1421, 1384, 1308, 1277, 1264,
1214, 1177, 1156, 1121, 1101,1052, 1005, 973, 950, 892, 869, 756,
4.1.7. N-Propargyl-6,7,8,9,10,11-hexahydro-9-methyl-5,7:9,11-di
methano-5H-benzocyclononen-7-amine hydrochloride, 9ÁHCl,
and N,N-dipropargyl-6,7,8,9,10,11-hexahydro-9-methyl-5,7:9,
11-dimethano-5H-benzocyclononen-7-amine hydrochloride,
10ÁHCl
A suspension of 3ÁHCl (400 mg, 1.52 mmol), K2CO3 (246 mg,
1.78 mmol), propargyl bromide (0.22 mL, 80% solution in toluene,
1.98 mmol) and NaI (21 mg, 0.14 mmol) in acetonitrile (15 mL)
was heated under reflux for 18 h. After concentration to dryness
in vacuo, the residue was taken in CH2Cl2 (20 mL) and the solution
was washed with water (3 Â 10 mL). The organic layer was dried
with anhyd Na2SO4, filtered and concentrated in vacuo to give a res-
idue which was subjected to column chromatography (hexane/
EtOAc mixtures) affording 10 (hexane/EtOAc 99/1, 220 mg, 48%
yield) and 9 (hexane/EtOAc 97/3, 70 mg, 17% yield). The hydrochlo-
rides of 9 and 10 were obtained by adding an excess of Et2OÁHCl to a
solution of the corresponding amine in EtOAc, followed by filtration
of the formed precipitate. The analytical samples were obtained by
crystallization from methanol/Et2O. 9ÁHCl: mp 175–176 °C (metha-
nol/Et2O). IR 3487, 3221, 2908, 2446, 2366, 2125, 1628, 1451, 850,
746, 698, 573, 515 cmÀ1 1H NMR (CDCl3) 1.00 (s, 3H, CH3), 1.59
.
[d, J = 13.5 Hz, 2H, 10(13)-Hb], 1.73 [ddm, J = 13.5 Hz, J’ = 6.0 Hz,
2H, 10(13)-Ha], 2.01 (d, J = 12 Hz, 1H, 6-Hb or 12-Hb), 2.06 (complex
signal, 2H, 8-Ha and 12-Hb or 6-Hb), 2.25 (d, J = 12.0 Hz, 1H, 8-Hb),
2.38 (d, J = 5.0 Hz, 3H, N–CH3), 2.55 (m, 1H) and 2.65 (m, 1H) (6-
Ha and 12-Ha), 3.25 (t, J = 6.0 Hz, 1H) and 3.27 (t, J = 6.0 Hz, 1H)
(5H and 11-H), 3.65 (dd, J = 12.5 Hz, J’ = 9.0 Hz, 1H, CH2-C6H5),
4.66 (dd, J = 12.5 Hz, J’ = 3.5 Hz, 1H, CH2-C6H5), 7.06 [m, 2H, 1(4)-
Ar-H], 7.11 [m, 2H, 2(3)-Ar-H], 7.33-7.39 (complex signal, 3H, Ar-
761, 548 cmÀ1 1H NMR 1.00 (s, 3H, C9-CH3), 1.56 [d, J = 13.5 Hz,
.
2H, 10(13)-Hb], 1.71 (s, 2H, 8-H2), 1.73 [ddd, J = 13.5 Hz, J’ = 6.0 Hz,
J’ = 2.0 Hz, 2H, 10(13)-Ha], 1.93 [d, J = 12.5 Hz, 2H, 6(12)-Hb], 2.08
[dm, J = 12.5 Hz, 2H, 6(12)-Ha], 3.20 (t, J = 2.5 Hz, 1H, CH2C„CH),
3.23 [tm, J’ = 6.0 Hz, 2H, 5(11)-H], 3.94 (d, J = 2.5 Hz, 2H, CH2C„CH),
7.10 (s, 4H, Ar-H). 13C NMR 30.5 (CH2, CH2C„CH), 32.3 (CH3, C9-
CH3), 34.9 (C, C9), 37.0 [CH2, C6(12)], 41.5 [CH, C5(11)], 41.6 [CH2,
C10(13)], 45.3 (CH2, C8), 61.7 (C, C7), 75.5 (CH, CH2C„CH), 78.7
(C, CH2C „CH), 128.0 [CH, C2(3)], 129.2 [CH, C1(4)], 146.2 [C,
C4a(C11a)]. HRMS-ESI+ m/z [M+H]+ calcd for C19H23N+H:
266.1903, found: 266.1898. Anal. Calcd for C19H23NÁ1.5HCl
(320.09): C 71.30, H 7.71, N 4.38. Found C 71.56, H 7.73, N 4.15.
10ÁHCl: mp 199–200 °C (methanol/Et2O). IR 3305, 3239, 3194,
2951, 2907, 2346, 2118, 1493, 1474, 1455, 1435, 1380, 1355,
1308, 1212, 1166, 1039, 1007, 959, 884, 845, 755, 691, 639,
Hmeta and Arpara), 7.69 (dd, J = 7.5 Hz, J = 2.0 Hz, 2H, Ar-Hortho), 11.9
(br s, 2H, NH2). 13C NMR (CDCl3) 31.7 (CH3, CH3-N), 31.9 (CH3, C9-
CH3), 33.1 (CH2) and 33.5 (CH2) (C6 and C12), 34.5 (C, C9), 40.1
(CH2), and 40.28 (CH2) (C10 and C13), 40.31 [CH, C5(11)], 43.2
(CH2, C8), 52.5 (CH2, CH2C6H5), 67.9 (C, C7), 127.0 (CH, Ar-Cmeta),
128.0 [CH, C2(3)], 129.0 [CH, C1(4)], 129.2 (C, Ar-Cipso), 129.7 (CH,
Ar-Cpara), 131.7 (CH, Ar-Cortho), 144.89 (C), and 144.92 (C) (C4a and
C11a). MS (EI), m/z (%): 331 (MÁ+, 100), 316 (33), 290 (18), 289
(77), 288 (12), 274 (12), 260 (10), 246 (11), 240 (10), 211
[(C16H19)+, 11], 198 (22), 169 (10), 161 (10), 160 (16), 155 (30),
143 (18), 141 (18), 129 (18), 128 (18), 115 (13), 91 (61). Anal. Calcd.
.
546 cmÀ1 1H NMR 1.03 (s, 3H, C9-CH3), 1.54 [d, J = 13.5 Hz, 2H,
10(13)-Hb], 1.73 [dd, J = 13.5 Hz, J’ = 6.0 Hz, 2H, 10(13)-Ha], 1.90 (s,

E. Torres et al. / Bioorg. Med. Chem. 20 (2012) 942–948
947
2H, 8-H2), 2.13 [d, J = 12.0 Hz, 2H, 6(12)-Hb], 2.30 [dd, J = 12.0 Hz,
J’ = 6.5 Hz, 2H, 6(12)-Ha], 3.27 [t, J’ = 6.0 Hz, 2H, 5(11)-H], 4.32 (br
s, 4H, CH2C„CH), 7.10 (s, 4H, Ar-H). The signal corresponding to
the terminal „CH was not observed. 13C NMR 32.5 (CH3, C9-CH3),
35.9 (C, C9), 36.0 [CH2, C6(12)], 38.4 (CH2, CH2C„CH), 41.4 [CH2,
C10(13)], 41.9 [CH, C5(11)], 45.0 (CH2, C8), 71.7 (C, C7), 74.8 (CH,
CH2C„CH), 80.9 (C, CH2C „CH), 128.1 [CH, C2(3)], 129.2 [CH,
C1(4)], 146.3 [C, C4a(C11a)]. MS (EI), m/z (%): 303 (MÁ+, 87), 302
(35), 288 (28), 260 (38), 246 (19), 232 (10), 218 (12), 211 (25), 206
(27), 169 (21), 167 (11), 165 (11), 156 (15), 155 (77), 153 (15),
144 (11), 143 (42), 142 (13), 141 (40), 133 (63), 132 (100), 130
(18), 129 (46), 128 (35), 127 (11), 118 (11), 117 (16), 115 (30), 91
(15). Anal. Calcd for C22H26ClN (339.91): C 77.74, H 7.71, N 4.12,
Cl 10.43. Found C 77.63, H 7.61, N 3.94, Cl 10.06.
C9-CH3), 1.52 (br s, 1H, piperidine 4-Hax), 1.55 [d, J = 13.5 Hz, 2H,
10(13)-Hb], 1.73 [dd, J = 12.0 Hz, J’ = 6.5 Hz, 2H, 10(13)-Ha], 1.83 (s,
2H, 8-H2), 1.83–1.98 [complex signal, 5H, piperidine 4-Heq, 3(5)-
Hax and 3(5)-Heq], 2.04 [d, J = 12.5 Hz, 2H, 6(12)-Hb], 2.18 [dd,
J = 12.5 Hz, J’ = 6.5 Hz, 2H, 6(12)-Ha], 2.92 [br s, 2H, piperidine
2(6)-Hax], 3.27 [tm, J’ = 6.5 Hz, 2H, 5(11)-H], 3.68 [br s, 2H, piperidine
2(6)-Heq], 7.10 (s, 4H, Ar-H). 13C NMR 23.2 (CH2, piperidine C4), 25.0
[CH2, piperidine C3(5)], 32.5 (CH3, C9-CH3), 34.9 [CH2, C6(12)], 35.6
(C, C9), 41.5 [CH2, C10(13)], 41.8 [CH, C5(11)], 44.3 (CH2, C8), 47.7
[CH2, piperidine C2(6)], 68.8 (C, C7), 128.0 [CH, C2(3)], 129.2 [CH,
C1(4)], 146.4 [C, C4a(C11a)]. Anal. Calcd for. C21H30NClÁ0.5H2O
(340.93): C 73.98, H 9.16, N 4.11. Found C 73.79, H 8.93, N 3.91.
4.2. NMDA receptor antagonist activity
4.1.8. N,N-Dimethyl-6,7,8,9,10,11-hexahydro-9-methyl-5,7:9,11-
dimethano-5H-benzocyclononen-7-amine hydrochloride,
11ÁHCl
The functional assay of antagonist activity at NMDA receptors
was performed using primary cultures of cerebellar granule neu-
rons, that were prepared according to established protocols.12 Cells
To a solution of 3ÁHCl (300 mg, 1.14 mmol) in MeOH (10 mL),
NaBH3CN (205 mg, 3.27 mmol), AcOH (0.3 mL) and formaldehyde
(0.26 mL, 37% wt. in water solution, 3.48 mmol) were added and
the mixture was stirred at room temperature for 8 h. An additional
portion of NaBH3CN (205 mg, 3.27 mmol) and formaldehyde
(0.26 mL, 37% wt. in water solution, 3.48 mmol) were added, the
mixture was stirred at room temperature for 18 h and then it
was concentrated in vacuo to dryness. Water (20 mL) was added
to the residue, the suspension was basified with 1 N NaOH
(10 mL) and was extracted with EtOAc (4 Â 15 mL). The combined
organic extracts were washed with brine (2 Â 10 mL), dried with
anhyd Na2SO4, filtered and concentrated in vacuo to give 11 as a
yellow oil. Its hydrochloride was obtained by adding an excess of
Et2OÁHCl to a solution of the amine in EtOAc, followed by filtration
of the white solid precipitate (249 mg, 75% yield), mp 226À227 °C.
IR 3468, 3411, 3244, 3063, 3022, 2945, 2917, 2892, 2865, 2846,
2670, 2462, 2071, 1635, 1492, 1458, 1449, 1409, 1369, 1309,
were grown on 10 mm poly-
used for the experiments after 7-14 days in vitro. Cells were loaded
with 6 M Fura-2 AM (Invitrogen-Molecular Probes) for 45 min.
L-lysine coated glass cover slips, and
l
Afterwards, the coverslip was mounted on a quartz cuvette con-
taining 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. Anal-
ysis from each sample was recorded real-time during 1200 s. After
stimulation with NMDA or glutamate (100
lM, in the presence of
10 M glycine), increasing cumulative concentrations of the com-
l
pound 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.
4.3. Antiviral evaluation
1215, 1176, 1155, 1125, 1096, 1051, 980, 903, 756, 584 cmÀ1
.
1H
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].16 The compounds’ inhibitory effect on virus replication as
well as their cytotoxicity were monitored by microscopical exam-
ination, and confirmed by the colorimetric MTS cell viability assay.
NMR (CDCl3) 0.97 (s, 3H, C9-CH3), 1.56 [d, J = 13.5 Hz, 2H, 10(13)-
Hb], 1.66 [dd, J = 13.5 Hz, J’ = 6.0 Hz, 2H, 10(13)-Ha], 1.88 (s, 2H,
8-H2), 1.92 [d, J = 12.0 Hz, 2H, 6(12)-Hb], 2.17 (br s, 1 H, NH),
2.25 [dd, J = 12.0 Hz, J’ = 6.0 Hz, 2H, 6(12)-Ha], 2.69 (s, 6H, N-CH3),
3.20 [t, J = 6.0 Hz, 2H, 5(11)-H], 7.04 [m, 2H, 1(4)-Ar-H], 7.10 [m,
2H, 2(3)-Ar-H]. 13C NMR (CDCl3) 31.8 (CH3, C9-CH3), 33.2 [CH2,
C6(12)], 34.3 (C, C9), 36.4 (CH3, CH3-N), 40.0 [CH2, C10(13)], 40.1
[CH, C5(11)], 43.6 (CH2, C8), 65.3 (C, C7), 127.1 [CH, C2(3)], 128.2
[CH, C1(4)], 144.8 [C, C4a(C11a)]. MS (EI), m/z (%): 255 (MÁ+, 100),
240 (39), 214 (15), 213 (88), 212 (17), 198 (29), 185 (11), 184
(22), 170 (13), 169 (10), 155 (35), 153 (10), 143 (12), 141 (22),
136 (12), 129 (17), 128 (23), 122 (22), 115 (20), 85 (24). Anal.
Calcd. for C18H26NClÁ1.3H2O (315.28): C 68.57, H 9.14, N 4.44, Cl
11.24. Found C 68.29, H 8.84, N 4.52, Cl 11.65.
4.4. Plasmid, mRNA synthesis, and microinjection of oocytes.
Two-electrode voltage clamp analysis
The cDNA encoding the influenza A/Udorn/72 (A/M2) was in-
serted into pGEM3 vector for the expression on oocyte plasma
membrane. A/M2 S31N and A/M2 V27A mutants were generated
by QuikChange site-directed mutagenesis kit (Agilent Technolo-
gies). The synthesis of mRNA and microinjection of oocytes have
been described previously.17
Macroscopic membrane current was recorded 48–72 h after
injection as described previously.18 The tested compounds were
applied at pH 5.5 at various concentrations when the inward cur-
rent reaches maximum. The compounds were applied for 2 min,
and residual membrane current was compared with the membrane
current before the application of compounds. Membrane currents
were analyzed with pCLAMP 10.0 software package (Axon Instru-
ments, Sunnyvale, CA).
4.1.9. N-(6,7,8,9,10,11-hexahydro-9-methyl-5,7:9,11-dimethano
-5H-benzocyclononen-7-yl)piperidine hydrochloride, 12ÁHCl
To a solution of 3ÁHCl (288 mg, 1.09 mmol) in DMF (4.0 mL), an-
hyd Et3N (0.45 mL, 3.27 mmol) was added and the suspension was
stirred at room temperature for 2 h. 1,5-Dibromopentane (0.18 mL,
1.3 mmol) was added and the mixture was heated at 60 °C for
26 h. To the cold mixture, water (15 mL) and 2 N NaOH (10 mL) were
added and the solution was extracted with EtOAc (6 Â 15 mL). The
combined organic extracts were washed with water (3 Â 15 mL),
dried with anhyd Na2SO4 and filtered. Excess of Et2OÁHCl was added
and the solution was concentrated in vacuo to dryness to give 12ÁHCl
(125 mg, 35%yield). Theanalytical sampleof 12ÁHClwas obtainedby
crystallization from methanol/Et2O, mp 280 °C (sub). IR 3427, 3023,
2951, 2924, 2900, 2858, 2661, 2530, 2014, 1941, 1455, 1374, 1297,
4.5. T. brucei culturing and drug testing
Cultures of bloodstream form T. brucei (strain 427) were main-
tained at 37 °C in modified Iscove’s medium (pH 7.4).19 Trypanoci-
dal activity was assessed by growing parasites for 48 h in the
1218, 1122, 1094, 973, 752, 551 cmÀ1 1H NMR 1.02 (s, 3H,
.

948
E. Torres et al. / Bioorg. Med. Chem. 20 (2012) 942–948
Abbruscato, T. J.; Bickel, U.; Klein, J.; van der Schyf, C. J. Neurosci. Lett. 2005, 383,
49; (c) Grobler, E.; Grobler, A.; van der Schyf, C. J.; Malan, S. F. Bioorg. Med.
Chem. 2006, 14, 1176; (d) Kiewert, C.; Hartmann, J.; Stoll, J.; Thekkumkara, T. J.;
Geldenhuys, W. J.; Klein, J. Neurochem. Res. 2006, 31, 395; (e) Geldenhuys, W. J.;
Malan, S. F.; Bloomquist, J. R.; van der Schyf, C. J. Bioorg. Med. Chem. 2007, 15,
1525; (f) Hao, J.; Mdzinarishvili, A.; Abbruscato, T. J.; Klein, J.; Geldenhuys, W.
J.; van der Schyf, C. J.; Bickel, U. Brain Res. 2008, 1196, 113.
presence of various drug concentrations and determining the levels
which inhibited growth by 50% (IC50) and 90% (IC90). In the case of
untreated cultures (volume 4 mL), cell densities increased from
0.25 Â 105 to 1 Â 106 cells mLÀ1 over this period. Experiments
were performed in triplicate. Cell densities at each drug concentra-
tion were determined using a hemocytometer (Weber Scientific
International Ltd), and drug sensitivity was expressed as a percent-
age of growth of control cells.
8. (a) du Preez, J. L.; Lotter, A. P.; Guillory, J. K. Pharmazie 1996, 51, 223; (b) du
Preez, J. L.; Lotter, A. P. Drug Dev. Ind. Pharm. 1996, 22, 1249.
9. (a) Bishop, R.; Landers, A. E. Aust. J. Chem. 1979, 32, 2675; (b) Amini; Bishop, R.
Aust. J. Chem. 1983, 36, 2465; (c) Amini; Bishop, R.; Burgess, G.; Craig, D. C.;
Dance, I. G.; Scudder, M. L. Aust. J. Chem. 1989, 42, 1919.
9c
Acknowledgments
10. The Ritter reaction of diene
4 with acetonitrile was described in Ref.
However, in our hands, the hydrolysis of this acetamide with aq. HCl gave
amine in low yield. Only under very drastic basic conditions (NaOH,
3
F.X.S. and S.V. gratefully acknowledge financial support from
Ministerio de Educación y Ciencia (S.V.: Project CTQ2008-03768;
F.X.S.: Project SAF 2006-13092-C02-01) and Comissionat per a Uni-
versitats i Recerca (S.V.: Project 2005-SGR-00180, F.X.S.: Project
2009-SGR-853). J.M.K. was funded by the Wellcome Trust (Grant
number 084175). E.T. and M.D.D. thank the Ministerio de Educación
(FPU Program).
diethyleneglycol at 210°C for 24 h) 3 was obtained in high yield (89%).
11. Jirgensons, A.; Kauss, V.; Kalvinsh, I.; Gold, M. R. Synthesis 2000, 1709.
12. Canudas, A. M.; Pubill, D.; Sureda, F. X.; Verdaguer, E.; Camps, P.; Muñoz-
Torrero, D.; Jiménez, A.; Camins, A.; Pallàs, M. Exp. Neurol. 2003, 180, 123.
13. (a) Papanastasiou, I.; Tsotinis, A.; Kolocouris, N.; Prathalingam, S. R.; Kelly, J. M.
J. Med. Chem. 2008, 51, 1496; (b) Zoidis, G.; Tsotinis, A.; Kolocouris, N.; Kelly, J.
M.; Prathalingam, S. R.; Naesens, L.; De Clercq, E. Org. Biomol. Chem. 2008, 6,
3177; (c) Papanastasiou, I.; Tsotinis, A.; Zoidis, G.; Kolocouris, N.; Prathalingam,
S. R.; Kelly, J. M. ChemMedChem 2009, 4, 1059; (d) Zoidis, G.; Kolocouris, N.;
Kelly, J. M.; Prathalingam, S. R.; Naesens, L.; De Clercq, E. Eur. J. Med. Chem.
2010, 45, 5022; (e) Fytas, C.; Zoidis, G.; Tzoutzas, N.; Taylor, M. C.; Fytas, G.;
Kelly, J. M. J. Med. Chem. 2011, 54, 5250.
References and notes
14. (a) Cady, S. D.; Schmidt-Rohr, K.; Wang, J.; Soto, C. S.; DeGrado, W. F. Nature
2010, 463, 689; (b) Acharya, R.; Carnevale, V.; Fiorin, G.; Levine, B. G.;
Polishchuk, A. L.; Balannik, V.; Samish, I.; Lamb, R. A.; Pinto, L. H. Proc. Natl.
Acad. Sci. U.S.A. 2010, 107, 15075; (c) Wang, J.; Qui, J. X.; Soto, C.; DeGrado, W. F.
Curr. Opin. Struct. Biol. 2011, 21, 68.
15. (a) Deyde, V. M.; Xu, X.; Bright, R. A.; Shaw, M.; Smith, C. B.; Zhang, Y.; Shu, Y.;
Gubareva, L. V.; Cox, N. J.; Klimov, A. I. J. Infect. Dis. 2007, 196, 249; (b) Furuse,
Y.; Suzuki, A.; Kamigaki, T.; Oshitani, H. Virol. J. 2009, 6, 67; (c) Astrahan, P.;
Arkin, I. T. Biochim. Biophys. Acta, Biomembr. 2011, 1808, 547; (d) Balannik, V.;
Carnevale, V.; Fiorin, G.; Levine, B. G.; Lamb, R. A.; Klein, M. L.; DeGrado, W. F.;
Pinto, L. H. Biochemistry 2010, 49, 696.
16. (a) Setaki, D.; Tataridis, D.; Stamatiou, G.; Kolocouris, A.; Foscolos, G. B.; Fytas, G.;
Kolocouris, N.; Padalko, E.; Neyts, J.; De Clercq, E. Bioorg. Chem. 2006, 34, 248; (b)
Naesens, L.; Vanderlinden, E.; Roth, E.; Jeko, J.; Andrei, G.; Snoeck, R.;
Pannecouque, C.; Illyes, E.; Batta, G.; Herczegh, P.; Sztaricskai, F. Antiviral Res.
2009, 82, 89.
17. Ma, C.; Soto, C. S.; Ohigashi, Y.; Taylor, A.; Bournas, V.; Glawe, B.; Udo, M. K.;
DeGrado, W. F.; Lamb, R. A.; Pinto, L. H. J. Biol. Chem. 2008, 283, 15921.
18. Balannik, V.; Wang, J.; Ohigashi, Y.; Jing, X.; Magavern, E.; Lamb, R. A.; DeGrado,
W. F.; Pinto, L. H. Biochemistry 2009, 48, 11872.
1. (a) Danysz, W.; Parsons, C. G.; Kornhuber, J.; Schmidt, W. J.; Quack, G. Neurosci.
Biobehav. Rev. 1997, 21, 455; (b) Parsons, C. G.; Danysz, W.; Quack, G.
Neuropharmacology 1999, 38, 735; (c) Parsons, C. G.; Stöffler, A.; Danysz, W.
Neuropharmacology 2007, 53, 699; (d) Gilling, K. E.; Jatzke, C.; Hechenberger,
M.; Parsons, C. G. Neuropharmacology 2009, 56, 866.
2. Hay, A. J.; Wolstenholme, A. J.; Skehel, J. J.; Smith, M. H. EMBO J. 1985, 4, 3021;
(b) Hay, A. J. Semin. Virol. 1992, 3, 21; (c) Pinto, L. H.; Holsinger, L. J.; Lamb, R. A.
Cell 1992, 69, 517.
3. (a) Kelly, J. M.; Miles, M. A.; Skinner, A. C. Antimicrob. Agents Chemother. 1999,
43, 985; (b) Kelly, J. M.; Quack, G.; Miles, M. M. Antimicrob. Agents Chemother.
2001, 45, 1360.
4. Camps, P.; Duque, M. D.; Vázquez, S.; Naesens, L.; De Clercq, E.; Sureda, F. S.;
López-Querol, M.; Camins, A.; Pallàs, M.; Prathalingam, S. R.; Kelly, J. M.;
Romero, V.; Ivorra, D.; Cortés, D. Bioorg. Med. Chem. 2008, 16, 9925.
5. Duque, M. D.; Ma, C.; Torres, E.; Wang, J.; Naesens, L.; Juárez-Jiménez, J.; Camps,
P.; Luque, F. J.; DeGrado, W. F.; Lamb, R. A.; Pinto, L. H.; Vázquez, S. J. Med. Chem.
2011, 54, 2646.
6. (a) Duque, M. D.; Camps, P.; Profire, L.; Montaner, S.; Vázquez, S.; Sureda, F. S.;
Mallol, J.; López-Querol, M.; Naesens, L.; De Clercq, E.; Prathalingam, S. R.;
Kelly, J. M. Bioorg. Med. Chem. 2009, 17, 3198; (b) Duque, M. D.; Camps, P.;
Torres, E.; Valverde, E.; Sureda, F. S.; López-Querol, M.; Camins, A.;
Prathalingam, S. R.; Kelly, J. M.; Vázquez, S. Bioorg. Med. Chem. 2010, 18, 46.
7. (a) Geldenhuys, W. J.; Malan, S. F.; Bloomquist, J. R.; Marchand, A. P.; van der
Schyf, C. J. Med. Res. Rev. 2005, 25, 21; (b) Mdzinarishvili, A.; Geldenhuys, W. J.;
19. Hirumi, H.; Hirumi, K. J. Parasitol. 1989, 75, 985.