Heterocyclic rimantadine analogues with antiviral activity

Article abstract of DOI:10.1016/j.bmc.2003.09.024

2-(1-Adamantyl)pyrrolidines 6, 7, 2-(1-adamantyl)piperidines 10, 12a-c, 15a,b and 2-(1-adamantyl)hexahydroazepines 19, 21, 22 were synthesized and tested for their antiviral activity against influenza A, B viruses and the human immunodeficiency virus type

Full text of DOI:10.1016/j.bmc.2003.09.024

Bioorganic & Medicinal Chemistry 11 (2003) 5485–5492
Heterocyclic Rimantadine Analogues with Antiviral Activity
George Stamatiou,a George B. Foscolos,a,* George Fytas,a,* Antonios Kolocouris,a
Nicolas Kolocouris,a Christophe Pannecouque,b Myriam Witvrouw,b Elizaveta Padalko,b
Johan Neytsb and Erik De Clercqb
aDepartment of Pharmacy, Division of Pharmaceutical Chemistry, University of Athens, Panepistimioupoli-Zografou,
GR-15771 Athens, Greece
bRega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
Received 18 February 2003; accepted 15 September 2003
Abstract—2-(1-Adamantyl)pyrrolidines 6, 7, 2-(1-adamantyl)piperidines 10, 12a–c, 15a,b and 2-(1-adamantyl)hexahydroazepines
19, 21, 22 were synthesized and tested for their antiviral activity against influenza A, B viruses and the human immunodeficiency
virus type 1 (HIV-1) and type 2 (HIV-2). The synthetic procedure followed for the preparation of the parent piperidine 10 repre-
sents a general method for the synthesis of 2-alkyl- or cycloalkyl-substituted piperidine alkaloids. Parent aminoadamantanes 6, 10
and 19 contain the 1-aminoethyl pharmacophore group of rimantadine drug 2, extended into a saturated nitrogen heterocycle:
pyrrolidine, piperidine and hexahydroazepine, respectively. The ring size effect in anti-influenza A activity was investigated. Riman-
tadine analogues 6 and 10 were, respectively, 6- and 4-fold more active than the drug Rimantadine 2, whereas the hexahydroazepine
derivative 19 was inactive. Thus, enlargement from a 5-(pyrrolidine)- or 6-(piperidine)- to a 7-(hexahydroazepine)- membered hetero-
cyclic ring dramatically reduced the anti-influenza virus A activity. Substitution of piperidine 10 with a dialkyaminoethyl group led to
the active compounds 15a and 15b: compound 15a was active against influenza A virus whereas both 15a and 15b were active against
HIV-1.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
Amantadine 1 and Rimantadine 2 are anti-influenza
virus A drugs that inhibit virus replication at micro-
molar concentrations.5,6 Many aminoadamantane car-
bocycles and heterocycles with activity against influenza
A virus were synthesized in our laboratory during the
past 8 years.6,7 The antiviral activity of synthetic 2-
(1-adamantyl)pyrrolidines 6, 7, 2-(1-adamantyl)piper-
idines 10, 12a–c, 15a,b and 2-(1-adamantyl) azepines 19,
21, 22 is described in the present paper.
Millions of people each year become infected with the
influenza A virus. Influenza A is characterized by the
abrupt onset of constitutional and respiratory signs and
symptoms (e.g., fever, myalgia, headache, severe
malaise, nonproductive cough, sore throat and rhini-
tis).1 In some persons the infection can cause pulmonary
or cardiac disease or lead to secondary bacterial pneu-
monia or primary viral pmeumonia. Epidemics of influ-
enza occur during the winter months nearly every year
and are responsible for an average of approximately
20,000 deaths per year in the United States.2 Pandemic
lethal influenza A viruses appeared in 1918 (‘Spanish’
flu), 1957 (‘Asian’ flu) and 1968 (‘Hong Kong’ flu).3
Given that influenza shifts occur every 20–30 years and
that a new lethal variant appeared in Hong Kong in
1997,4 the danger of future influenza A pandemics is
real and new more effective drugs are needed.
*Corresponding authors. Tel.: +30-210-727-4808/4810; fax: +30-210-
7274747; e-mail: ankol@pharm.uoa.gr
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2003.09.024

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G. Stamatiou et al. / Bioorg. Med. Chem. 11 (2003) 5485–5492
Parent molecules 6, 10, 19 contain the 1-aminoethyl
pharmacophore group of drug Rimantadine 2 extended
into a pyrrolidine, piperidine or hexahydroazepine het-
erocycle. Thus, we sought mainly to compare the anti-
influenza A virus potency of these structurally modified
rimantadines with the activity of Rimantadine 2. The
effect of N-dialkylaminoethyl group substitution was
also investigated since it appeared to be important for
activity against influenza A virus and HIV-1, as was
shown previously in preliminary reports.7c,e
procedures used for the preparation of the corresponding
piperidine analogues 10, 12a and 15a (Scheme 3).
Antiviral activity evaluation
The potency of the new aminoadamantane heterocycles
6, 7, 10, 12a–c, 15a,b, 19 and 21, 22, was examined in
vitro against influenza A (H2N2) and B viruses, and was
compared to the activity of Amantadine 1, Rimantadine
2 and Ribavirin (Table 1). The compounds were also
evaluated against HIV-1 and -2; AZT, Nevirapine (BI-
RG587) and Ritonavir (ABT538) were used as controls
(Table 1). The sources of viruses, and methods and
antiviral assays used were as previously reported.13ꢀ18
Results and Discussion
Chemistry
From the MIC50 values shown in Table 1, it is obvious
that 6, 10 and, to a lesser extent, 15a were potent anti-
influenza virus A compounds. Compounds 6 and 10
The synthetic protocol used for the synthesis of pyrroli-
dines was based on the dry distillation of N-acylpyrro-
lidinone 4 in the presence of CaO, as has already been
described elsewhere in detail.7b From this reaction, 2-(1-
adamantyl)-1-pyrroline 5 was obtained which was easily
converted to the parent pyrrolidine 6 (Scheme 1).
Reductive methylation of pyrrolidine 6 with CH2O/
8
NaCNBH3 afforded the N-methyl derivative 7.
The synthetic route followed for the preparation of
piperidines 10, 12a–c and 15a,b is illustrated in Scheme 2.
Tertiary alcohol 8 was synthesized from ester 7 and 1,4-
bis(bromomagnesium)butane.9 The reaction of tertiary
alcohol 8 with trichloroacetic acid or concd sulfuric acid
— the last giving the best results — and sodium azide
led to tetrahydropyridine 9, the formation of which was
accomplished by cyclopentane ring expansion of the
intermediate alkylazide via nitrenium ion.10 Reduction
of imine 9 with NaBH4 gave 2-(1-adamantyl)piperidine
10. The above described sequence, based on the reaction
between 1,4-bis(bromomagnesium)butane and an ester
function to form 1-substituted-1-cyclopentanols, can be
considered as general route for the synthesis of 2-alkyl-
or cycloalkyl-substituted piperidine alkaloid analogues.
Scheme 1. Reagents and conditions: (a) 1-AdCOCl, THF, reflux 7 h,
(94%); (b) CaO, ꢀ (35%); (c) NaBH4, MeOH–AcOH (3:1), ꢀ30 ꢁC,
and then rt 4 h (83%); (d) 37% CH2O, CH3CN, 10 min, then
NaCNBH3, 30 min, AcOH, 45 min (83%).
Piperidine 10 was suitably N-acylated to afford amides
11a,c,d, which were converted to piperidines 12a–c by
reduction with LiAlH4. Alternatively N-methylpiper-
idine 12a was obtained through NaBH4 reduction of
quaternary salt 11b.11 N-Bromoacetylation of parent
piperidine 10 resulted in bromoacetamide 13, which
after treatment with the appropriate secondary amines
gave dialkylaminoacetamides 14a,b. These compounds
were converted to the corresponding diamines 15a,b by
means of LiAlH4.
For the synthesis of the hexahydroazepine derivatives
19 and 21, 22, 1-adamantyl lithium was needed. This
reagent was prepared from 1-bromoadamantane 16,
using the procedure of Kraus and Siclovan.12 1-Ada-
mantyl lithium was then reacted with cyclohexanone to
afford the tertiary alcohol 17.12 Tetrahydroazepine 18
was obtained from the tertiary alcohol 17 through
cyclohexane ring expansion of the intermediate alkyl-
azide. Parent hexahydroazepine 19 and its N-substituted
derivatives 21, 22 were obtained according to the
Scheme 2. Reagents and conditions: (a) BrMg(CH2)4MgBr/THF and
then NH4Cl, H2O (67%); (b) NaN3/trichloroacetic acid, CHCl3, 0 ꢁC,
29 h and then concd H2SO4, CHCl3, 0 ꢁC, 0.5 h (method A, 51%) or
NaN3/concd H2SO4, CHCl3, 0 ꢁC 1.5 h (method B, 73%); (c) NaBH4,
MeOH, 1 h, 0 ꢁC, and then rt 20 h (92%); (d) RCOCl, Et3N, ether or
THF, 0 ꢁC, and then rt 24 h (66–91%) for 11a,c,d or BrCH2COCl/
K2CO3, CHCl3/H2O, 0 ꢁC, and then rt for 2.5 h (52%) for 13 (e) di-
ethylamine or piperidine, benzene, 30 min, 0 ꢁC, and then 24 h rt (76–
86%); (f) LiAlH4, THF, reflux (70–89%); (g) MeI, reflux, 3 h (60%);
(h) NaBH4, CHCl3/MeOH 2:1, 0 ꢁC, 1 h, and then rt 24 h (87%).

G. Stamatiou et al. / Bioorg. Med. Chem. 11 (2003) 5485–5492
5487
Table 1. Anti-influenza virus A, B and anti-HIV activity and cytotoxicity of heterocyclic Rimantadine analogues — pyrrolidines 6, 7, piperidines
10, 12a–c, 15a,b and hexahydroazepines 19, 21, 22a — in MDCKb and MT-4 cellsb
Compd
Influenza
HIV
EC50c (mM)
MTCd (mM)
EC50e (mM)
CC50f (mM)
b
A H2N2
B
HIV-1
HIV-2
6
7
10
12a
12b
12c
15a
15b
19
21
22
2.5
>196
3.3
>978
244
522
51.3
>561
186
>39
>840
45
13.9
22.1
—
>1035
>196
>978
>928
>162
>724
>616
>561
>186
>35
>840
—
1035
196
978
928
162
145
616
561
—
—
—
—
>403
>415
>265
>694
>276
>54
>101
>281
>6.6
—
—
—
0.002
>75.1
0.14
—
—
403
413
263
63
275
53
101
281
6.6
—
—
—
36.8
75.1
>14.2
>403
>415
75.2
>64
40.8
3.6
>101
>281
>66
—
186
39
840
Amantadine, 1
Rimantadine, 2
Ribavirin
AZT
Nevirapine
Ritonavir
>1333
>1160
>710
—
—
—
—
—
—
0.001
0.05
0.06
19.9
—
—
—
—
—
aAminoadamantanes 6, 7, 10, 12a–c, 15a,b, 19, 21, 22 were tested as hydrochlorides or fumarate salts.
bAbbreviations and strains used: MDCK, Madin–Darby canine kidney cells, human epithelial cells; influenza A H2N2 (A2 Japan/305/57); MT-4
represents a human T-4 lymphocytic cell line.
cConcentration required to reduce virus-induced CPE in MDCK cells by 50%, as determined by the MTS method.
dMinimal toxic concentration or concentration that causes microscopically detectable toxicity in virus infected cultures.
e50% Effective concentration, or concentration which inhibited virus-induced cytopathogenicity by 50%.
f50% Cytotoxic concentration, or concentration which inhibited MT-4 cell growth by 50%, as compared with control cultures. All data represent
mean values for at least two separate experiments.
inactive compound. It appears that enlargement from a
five-, six- to a seven-membered ring detrimentally
reduced the activity against influenza A virus. This pro-
vides a novel observation regarding the SAR in ami-
noadamantane series.
N-Alkylation of parent amines 6 and 10 caused a dra-
matic reduction in anti-influenza virus A potency.
However, N-dimethylaminoethyl substitution of the
piperidine derivative 10 led to the active analogue 15a.
This result has also been observed in a previous work
concerning a different class of aminoadamantane het-
erocycles.7e The specific activity of compound 15a could
be attributed to three pharmocophore groups, that is,
adamantane and two amine groups.
Scheme 3. Reagents and conditions: (a) (i) Li/THF, sonication, (ii)
cyclohexanone, THF, 0 ꢁC 5 h, and then MeOH–H2O 1:1 (72%); (b)
NaN3/concd. H2SO4, CHCl3, 0 ꢁC 1.5 h (30%) (c) NaBH4, MeOH,
0 ꢁC 1 h, and then rt 2 h (73%); (d) RCOCl, Et3N, ether or THF, rt 24
h (84–94%) for 21a or BrCH2COCl/K2CO3, CHCl3/H2O, 0 ꢁC 3 h, rt
for 30 min (86%) for 21b (e) piperidine, benzene, 30 min 0 ꢁC, 24 h rt
(90%); (f) LiAlH4, THF, reflux (73% for 22 and 96% for 23).
All compounds were inactive against influenza B virus,
which is in accordance with their putative mode action,
that is their interaction with influenza A M2 protein
which is absent from influenza B virion.
were, respectively, 18- and 14-fold more potent than
Amantadine 1, and 9 and 7 times more potent than
Ribavirin, whereas 15a proved equipotent to Amanta-
dine 1 and was 2.3 times less potent than Ribavirin.
N-H, N-methyl and N-benzyl piperidines 10, 12a and
12c proved to be inactive against HIV-1, whereas the N-
cyclopropylmethyl derivative 12b showed an EC50 of
75.2 mM. N-Substitution by a dialkylaminoethyl group
further improved the activity. Indeed, compounds 15a
and 15b showed an EC50 of 40.8 and 3.6 mM, respec-
tively. Interestingly, replacement of the dimethylamino
moiety in 15a by a piperidino group in 15b led to a
substantial increase in potency. The active compounds
12b, 15a, 16b showed selectivity against HIV-1. The
hexahydroazepines 19 and 21, 22 were found inactive
against HIV.
Compounds 6, 10 and 19 contain a 1-aminoethyl phar-
macophore group of Rimantadine 2 extended into a
saturated nitrogen heterocycle: pyrrolidine, piperidine
and hexahydroazepine, respectively. Pyrrolidine 6 was
somewhat more potent than the piperidine analogue 10,
but both the heterocyclic Rimantadine analogues 6 and
10 were, respectively, 6- and 4-fold more potent, and
than Rimantadine 2. The hexahydroazepine 19 was an

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Aminoadamantane derivatives have not primarily been
pursued as inhibitors of HIV replication. This fact
makes the activity of 12b and 15a and 15b against HIV-
1 even more intriguing; although their potency is con-
siderably lower compared to that of the control com-
pounds Azidothymidine, Nevirapine and Ritonavir. The
new aminoadamantane derivatives may be considered
as the starting point for the development of new anti-
HIV-1 agents. In analogy to the mechanism of anti-
influenza A virus activity exhibited by adamantana-
mines,5 the new compounds can be surmised to interact
with an early step (i.e., fusion/uncoating) of the HIV
replicative cycle. Since conversion of 10 to 15b results in
a marked shift from anti-influenza to anti-HIV activity,
studies are currently underway to obtain further insight
in the structure–activity relationships of this series of
compounds.
MSL 400 spectrometers, respectively, using CDCl3 as
solvent and TMS as internal standard. Carbon multi-
plicities were established by DEPT experiments. The 2D
NMR techniques (HMQC and COSY) were used for
the elucidation of the structures of some derivatives.
Microanalyses were carried out by the Service Central
de Microanalyse (CNRS) France, and the results
obtained had a maximum deviation of ꢂ0.4% from the
theoretical value.
1-(1-Adamantyl)cyclopentanol 8 was synthesized from
the reaction of 1,4-bis(bromomagnesium) butane with
ethyl 1-adamantanecarboxylate in 67% yield.8 1-(1-
Adamantyl)cyclohexanol 17 was synthesized from the
reaction of 1-adamantyl lithium with cyclohexanone in
72% yield.12 2-(1-Adamantyl)pyrrolidines 6, 7 were
synthesized according a previous publication.7b
3,4,5,6 - Tetrahydro - 2 - (tricyclo[3.3.1.13,7]dec - 1 - yl)pyri-
dine, 9. Method A. A mixture of trichloroacetic acid
(37.5 g, 0.230 mol) and NaN3 (4.57 g, 0.070 mol) in
chloroform (110 mL) was stirred for 15 min at 0 ꢁC. To
the resulting suspension was added in one portion ada-
mantylcyclopentanol 8 (5 g, 22.7 mmol) and stirring was
continued for 5 h at 0 ꢁC. The mixture was allowed to
stand for 24 h at 0 ꢁC and then poured into a mixture of
ice and 26% aqueous ammonia solution. The organic
layer was separated and the aqueous layer was extracted
with chloroform. The combined organic extracts were
washed three times with water, dried (Na2SO4) and
concentrated under vacuum at ambient temperature to
a volume of ꢃ80 mL. To the resulting solution con-
centrated sulfuric acid (7.5 mL) was added dropwise at
0 ꢁC and the mixture was stirred for 30 min and then
poured into a mixture of ice and 26% aqueous ammo-
nia solution. The organic layer was separated and the
aqueous layer was extracted with chloroform. The
combined organic extracts were washed with water and
evaporated almost to dryness. The residue was dissolved
in ether and extracted with 5% hydrochloric acid solu-
tion. The acidic aqueous phase was washed with ether
and then made alkaline with solid Na2CO3. The result-
ing oil was extracted with ether and the organic extracts
were dried and evaporated to dryness. The oily residue
was solidified on cooling and was filtered through neu-
tral aluminum oxide (100–125 mesh) with ether as the
eluent to afford 2.50 g of piperideine 9: yield 51%.
Conclusion
The major aim of this study was to examine the anti-
influenza A virus activity of heterocyclic Rimantadine 2
analogues 6, 10 and 19. The effect of ring size was
investigated. Pyrrolidine and piperidine analogues 6 and
10 were more potent in vitro, by 6- and 4-fold, respec-
tively, than the drug Rimantadine 2, whereas hexahy-
droazepine 19 was inactive. Extension from a five- or
six- to a seven-membered ring detrimentally reduced the
potency against influenza A virus.
Substitution of piperidine 10 with a dialkyaminoethyl
group led to compounds 15a and b, with compound 15a
showing modest activity against both influenza A virus
and HIV-1, and 15b being markedly active against HIV-1.
As it has been proposed by molecular modelling studies,
amantadine 1 blocks the M2 channel activity because its
two pharmacophoric groups, that is, adamantane and
amine groups, are complementary in shape, hydro-
phobicity and polarity with the luminal space between
M2 Leu26 and His37.5c The anti-influenza A virus
activity of compound 15a may be determined by three
pharmocophore groups, the adamantane and the two
amine groups. Since this result has also been observed
previously with a different class of compounds,7e it will
be of interest to verify in the future whether this effect is
general for aminoadamantanes.
Aminoadamantanes 15a and, in particular, 15b have
interesting activity against HIV-1 and can possibly be
considered as lead compounds for the development of a
new class of anti-HIV-1 agents.
Method B. To a vigorously stirred mixture of ada-
mantylcyclopentanol 8 (880 mg, 4.0 mmol) and NaN3
(1.4 g, 21.0 mmol) in chloroform (30 mL) was added
dropwise concentrated sulfuric acid (4 mL) under ice
cooling. The mixture was stirred for 1.5 h and poured
into a mixture of ice and 26% aqueous ammonia solu-
tion. The organic layer was separated and the aqueous
layer was extracted with chloroform. The combined
organic extracts were washed many times with water
and evaporated almost to dryness. The residue was dis-
solved in ether and extracted with 5% hydrochloric acid
solution. The acidic aqueous phase was washed with
ether and then made alkaline with solid Na2CO3. The
resulting oil was extracted with ether and the organic
Experimental
Chemistry
Melting points were determined using a Buchi capillary
apparatus and are uncorrected. IR spectra were recor-
ded on a Perkin-Elmer 833 spectrometer.1H and 13C
NMR spectra were recorded on a Bruker AC200 and

G. Stamatiou et al. / Bioorg. Med. Chem. 11 (2003) 5485–5492
5489
extracts were dried (Na2SO4) and evaporated to dry-
ness. The oily residue was heated at 30–35 ꢁC under
vacuum (1 mm Hg) and the yellow solid was chroma-
tographed on neutral aluminum oxide (100–125 mesh)
with ether as the eluent to afford 630 mg of piperideine
9: yield 73%; mp 34–36 ꢁC (n-pentane); IR (Nujol): n
cooling. The inorganic precipitate was filtered off and
washed with THF, and the filtrate was concentrated
under vacuum. The residue was dissolved in ether and
extracted with HCl 5%. The aqueous layer was made
alkaline with solid Na2CO3 and the solid formed was
extracted with ether. The combined ether extracts were
dried (Na2SO4) and the solvent was evaporated. The
resulting residue was chromatographed on silica gel
with ether as an eluent to give the piperidine 12a (710
mg, 89%).
(C¼N) 1651 cmꢀ1
;
1H NMR (CDCl3, 200 MHz) d
(ppm) 1.44–1.70 (m, 16H, 2,4,6,8,9,10-adamantane H,
4,5-tetrahydropyridine H), 1.96 (s, 3H, 3,5,7-ada-
mantane H), 2.06–2,12 (t, 2H, 3-tetrahydropyridine H),
3.50–3.60 (m, 2H, 6-tetrahydropyridine H); 13C NMR
(CDCl3, 50 MHz) d (ppm) 19.6 (4-tetrahydropyridine
C), 21.9 (5-tetrahydropyridine C), 23.5 (3-tetra-
hydropyridine C), 28.4 (3,5,7-adamantane C), 36.8
(4,6,10-adamantane C), 39.6 (2,8,9-adamantane C), 41.3
(1-adamantane C), 49.1 (6-tetrahydropyridine C), 177.0
(2-tetrahydropyridine C). Picrate: mp 177–179 ꢁC
(MeOH) Anal. (C21H26N4O7) C, H.
Method B. A suspension of tetrahydropyridine 9 (510
mg, 2.30 mmol) and CH3I (12 mL) was refluxed for 3 h.
The reaction mixture was ice-cooled and the pre-
cipitated crystalline salt 11b was filtered off, washed
with ether and dried: yield 500 mg (60%); 162–164 ꢁC
(EtOH–Et2O); anal. (C16H26IN) C, H.
To a stirred solution of tetrahydropyridinium iodide
11b (1.08 g, 3.0 mmol) in chloroform–methanol 2:1 (20
mL) was added portionwise under ice cooling NaBH4
(500 mg, 13.0 mmol) and stirring was continued for 24 h
at ambient temperature. The solvent was evaporated
under vacuum at room temperature and water was
added into the residue with vigorous stirring. The aqu-
eous mixture was extracted with chloroform, and the
combined organic phases were washed with water and
dried (Na2SO4). After evaporation of the solvent, the
residue was dissolved in ether and the ethereal solution
was extracted many times with HCl 5%. The acidic
aqueous phase was made alkaline with solid Na2CO3,
extracted with ether and the organic solution was dried
(Na2SO4). After evaporation of the solvent the residue
was chromatographed to afford crystalline piperidine
12a: (610 mg, 87%); mp 54–55 ꢁC (ether–n-pentane); 1H
NMR (CDCl3, 200 MHz) d (ppm) 1.31–1.84 (complex
m, 18H, 2,4,6,8,9,10-adamantane H, 3,4,5-piperidine
H), 1.92 (s, 3H, 3,5,7-adamantane H), 2.09 (dd, J=2.7,
10.8 Hz, 1H, 2-piperidine H), 2.41 (s, 3H, CH3), 2.70–
2.96 (m, 2H, 6-piperidine H); 13C NMR (CDCl3,
50 MHz) d (ppm) 17.71 (3-piperidine C), 19.05 (5-piper-
idine C), 24.53 (4-piperidine C), 28.68 (3,5,7-ada-
mantane C), 36.32 (1-adamantane C), 37.22 (4,6,10-
adamantane C), 39.17 (CH3), 39.75 (2,8,9-adamantane
C), 55.75 (6-piperidine C), 70.80 (2-piperidine C).
Hydrochloride: mp>260 ꢁC (EtOH–Et2O); Picrate: mp
159–161 ꢁC (MeOH), anal. (C22H30N4O7) C, H.7
2-(Tricyclo[3.3.1.13,7]dec-1-yl)piperidine, 10. To a stirred
solution of tetrahydropyridine 9 (2.68 g, 12.3 mol) in
methanol (50 mL) NaBH4 (2.0 g, 53.0 mmol) was added
in small portions under ice cooling. The reaction mix-
ture was stirred for 20 h at ambient temperature and the
solvent was removed under reduced pressure. The resi-
due was treated with HCl (10%) and the aqueous mix-
ture was washed with ether and then made alkaline with
solid Na2CO3. The oil formed was extracted with ether,
the combined organic extracts were dried and evaporated
to give piperidine 10 as a white solid: yield 2.51 g (93%);
mp 73–74 ꢁC (ether–n-pentane); 1H NMR (CDCl3,
200 MHz)
d (ppm) 1.04–1.80 (complex m, 20H,
2,4,6,8,9,10-adamantane H, 2,3,4,5-piperidine H, NH),
1.91 (s, 3H, 3,5,7-adamantane H), 2.46–2.58 (m, 1H, 6-
piperidine H), 3.06–3.11 (m, 1H, 6-piperidine H); 13C
NMR (CDCl3, 50 MHz) d (ppm) 25.40 (3-piperidine C),
25.50 (5-piperidine C), 26.70 (4-piperidine C), 28.50
(3,5,7-adamantane C), 35.10 (1-adamantane C), 37.30
(4,6,10-adamantane C), 38.70 (2,8,9-adamantane C),
48.0 (6-piperidine C), 66.90 (2-piperidine C). Hydro-
chloride: mp>300 ꢁC (EtOH–Et2O); Picrate: mp 221–
223 ꢁC (MeOH–Et2O) anal. (C21H28N4O7) C, H.
1-Methyl-2-(tricyclo[3.3.1.13,7]dec-1-yl)piperidine, 12a.
Method A. Ethyl chloroformate (1.09 g, 10.0 mmol) in
dry ether (20 mL) was added dropwise under ice cooling
to a stirred solution of the piperidine 10 (1.30 g, 5.93
mmol) and triethylamine (1.95 g, 19.3 mmol) in dry
ether (20 mL). The mixture was stirred at room tem-
perature for 24 h and water was added. The organic
phase was separated and the aqueous phase was extrac-
ted with ether. The combined organic extracts were
washed with water, HCl (2%), water and dried. The
solvent was evaporated in vacuo to afford carbamate
11a as an oil (1.33 g, 77%); IR (film) 1700 cmꢀ1, which
was used without further purification for the preparation
of derivative 12a.
1-Cyclopropylmethyl-2-(tricyclo[3.3.1.13,7]dec-1-yl)piper-
idine, 12b. Cyclopropanecarbonyl chloride (2.68 g, 25.6
mmol) in dry THF (10 mL) was added dropwise under
ice cooling to a stirred solution of the piperidine 10
(1.70 g, 7.76 mmol) and triethylamine (4.04 g, 39.9
mmol) in dry THF (40 mL). The mixture was stirred at
75 ꢁC for 2 h and the precipitated triethylamine hydro-
chloride was filtered off and washed with THF. The fil-
trate was evaporated under vacuum and the residue was
dissolved in ether. The organic solution was washed
with water, HCl 5%, NaOH 5%, water and dried
(Na2SO4). The solvent was evaporated in vacuo and the
oil residue was chromatographed on aluminum oxide
using ether as an eluent to afford amide 11c as an oil
To a stirred suspension of LiAlH4 (1.40 g, 36.9 mmol) in
dry THF (40 mL) was added dropwise a solution of the
carbamate 11a (1.0 g, 3.40 mmol) in dry THF (20 mL).
The reaction mixture was refluxed for 20 h and then
hydrolyzed with water and NaOH 20% under ice
(2.04 g, 91%); IR (Film) 1637 cmꢀ1
.

5490
G. Stamatiou et al. / Bioorg. Med. Chem. 11 (2003) 5485–5492
Compound 12b was prepared by the LiAlH4 (1.98 g,
52.2 mmol) reduction (9 h) of N-cyclopropanecarbonyl
derivative 11c (1.50 g, 5.22 mmol) in dry THF (40 mL),
using the procedure followed for the preparation of 12a
(method A): yield 70% (oil); 1H NMR (CDCl3,
200 MHz) d (ppm) 0.04–0.51 (m, 4H, 2,3-cyclopropane
H), 0.75–0.94 (m, 1H, 1-cyclopropane H), 1.23–1.75
(complex m, 18H, 2,4,6,8,9,10-adamantane H, 3,4,5-
piperidine H), 1.92 (s, 3H, 3,5,7-adamantane H), 2.04–
2.11 (m, 1H, 2-piperidine H), 2.38–2.62 (m, 2H,
C3H5CH2N), 2.77–3.10 (m, 2H, 6-piperidine H); 13C
NMR (CDCl3, 50 MHz) d (ppm) 3.68, 3.86 (2,3-cyclo-
propane C), 10.52 (1-cyclopropane C), 18.26, 18.65,
22.13 (3,4,5-piperidine C), 28.69 (3,5,7-adamantane C),
36.90 (1-adamantane C), 37.32 (4,6,10-adamantane C),
39.68 (2,8,9-adamantane C), 46.60 (6-piperidine C),
57.93 (C3H5CH2N), 70.37 (2-piperidine C). Hydrochlor-
ide: mp 219–221 ꢁC (EtOH–Et2O); Picrate: mp 152–
154 ꢁC (MeOH); anal. (C25H34N4O7) C, H.
To a stirred solution of bromoacetamide 13 (750 mg,
2.20 mmol) in dry benzene (10 mL) was added dropwise
under ice cooling a solution of Me2NH (300 mg, 6.60
mmol) in the same solvent (8 mL). The mixture was
stirred for 30 min at 0 ꢁC and 24 h at ambient tempera-
ture. The solvent was evaporated in vacuo and water
was added to the residue. The aqueous mixture was
extracted with ether and the organic extracts were
washed with water and extracted with HCl (5%). The
acidic aqueous solution was washed with ether and was
made alkaline with solid Na2CO3. The mixture was
extracted many times with ether and the combined
organic extracts were dried (Na2SO4) and evaporated
under vacuum. The solid residue was chromatographed
on silica gel with a mixture of ether–n-hexane 2:1 as an
eluent to afford compound 14a; yield 580 mg (86%); mp
91–93 ꢁC (ether–n-pentane); IR (Nujol) 1629 cmꢀ1
;
Picrate: mp 178–180 ꢁC (MeOH–Et2O); anal.
(C25H35N5O8) C, H.
1-Phenylmethyl-2-(tricyclo[3.3.1.13,7]dec-1-yl)piperidine,
12c. Amide 11d was prepared by the procedure used
for 11c, by the reaction of benzoyl chloride (2.81 g, 20.0
mmol) with piperidine 10 (1.77 g, 8,08 mmol) in the
presence of triethylamine (3.30 g, 32.6 mmol): yield
84%; mp 123–125 ꢁC (ether–n-pentane); anal.
(C22H29NO) C, H.
Diamine 15a was prepared by the LiAlH4 (1.12 g, 29.6
mmol) reduction (10 h) of dimethylaminoacetamide 14a
(900 mg, 2.96 mmol) in dry THF (45 mL), using the
procedure followed for the preparation of 12a. The pro-
duct was purified by column chromatography on silica
1
gel using ether as an eluent: yield 78% (oil); H NMR
(CDCl3, 200 MHz) d (ppm) 1.40–1.75 (complex m, 18H,
2,4,6,8,9,10-adamantane H, 3,4,5-piperidine H), 1.93 (s,
3H, 3,5,7-adamantane H), 2.04–2.10 (m, 1H, 2-piper-
idine H), 2.25 (s, 6H, 2ÂCH3), 2.41–2.94 (complex m,
6H, NCH2CH2N, 6-piperidine H); 13C NMR (CDCl3,
50 MHz) d (ppm) 18.40, 19.0, 22.20 (3,4,5-piperidine C),
28.70 (3,5,7-adamantane C), 37.30 (4,6,10-adamantane
C), 38.70 (1-adamantane C), 39.90 (2,8,9-adamantane
C), 45.90 (CH3), 48.10 (NCH2CH2NMe2), 50.80
(NCH2CH2NMe2), 58.70 (6-piperidine C), 71.40
(2-piperidine C). Fumarate: mp 167–170 ꢁC dec (EtOH–
Et2O); anal. (C23H38N2O4) C, H.
Compound 12c was prepared by the LiAlH4 (1.75 g,
46.1 mmol) reduction (20 h) of N-benzoylcarbonyl
derivative 11d (1.40 g, 4.33 mmol) in dry THF (45 mL),
using the procedure followed for the preparation of 12b:
yield 83%; mp 77–78 ꢁC (n-pentane); H NMR (CDCl3,
1
200 MHz)
d (ppm) 1.42–1.83 (complex m, 18H,
2,4,6,8,9,10-adamantane H, 3,4,5-piperidine H), 1.97 (s,
3H, 3,5,7-adamantane H), 2.15–2.26 (m, 1H, 2-piper-
idine H), 2.42–2.76 (m, 2H, 6-piperidine H), 3.69–4.0
(m, 2H, CH2C6H5), 7.22–7.45 (m, 5H, C6H5); 13C NMR
(CDCl3, 50 MHz) d (ppm) 17.80, 19.10, 21.80 (3,4,5-
piperidine C), 28.70 (3,5,7-adamantane C), 37.40
(4,6,10-adamantane C), 38.10 (1-adamantane C), 39.70
(2,8,9-adamantane C), 44.60 (CH2C6H5), 57.6 (6-piper-
idine C), 70.80 (2-piperidine C), 126.50, 128.10, 128.50,
141.10 (C6H5). Hydrochloride: mp 121–123 ꢁC dec
(EtOH–Et2O). Anal. (C22H31N) C, H.
1-[2-(1-Piperidino)ethyl]-2-(tricyclo[3.3.1.13,7]dec-1-yl)pi-
peridine, 15b. Compound 14b was prepared by the pro-
cedure used for 14a, by the reaction of bromacetamide
13 (1.30 g, 3.82 mmol) with piperidine (800 mg, 9.40
mmol) in dry benzene (20 mL). The solid residue was
chromatographed on silica gel with ether–n-hexane 2:1
as an eluent: yield 1.0 g (76%); mp 36–38 ꢁC (ether–n-
pentane); IR (film) 1642 cmꢀ1; Picrate: mp 195–197 ꢁC
(MeOH); anal. (C28H39N5O8) C, H.
1-[2-(Dimethylamino)ethyl]-2-(tricyclo[3.3.1.13,7]dec-1-
yl)piperidine, 15a. To a vigorously stirred mixture of
adamantylpiperidine 10 (1.70 g, 7.76 mmol) and K2CO3
(850 mg, 6.10 mmol) in dichloromethane (10 mL)/water
(10 mL) was added dropwise under ice cooling a solu-
tion of bromoacetyl chloride (1.23 g, 7.81 mmol) in di-
chloromethane (5 mL). The mixture was stirred for 3 h
at 0 ꢁC and 30 min at room temperature. Water was
added, the organic phase was separated and the aqu-
eous was extracted with dichloromethane. The com-
bined organic extracts were evaporated and the oily
residue was dissolved in ether. The ether solution was
washed successively with water, Na2CO3 (5%), water,
HCl (5%), brine and dried (Na2SO4). Evaporation of
the solvent afford crystalline bromoamide 13; yield 2.21
g (84%); mp 61–63 ꢁC (ether–n-pentane); IR (Nujol)
1648 cmꢀ1; anal. (C17H26BrNO) C, H.
The solid diamine 15b was prepared by the LiAlH4 (450
mg, 11.6 mmol) reduction (12 h) of piperidinocetamide
14b (450 mg, 11.6 mmol) in dry THF (40 mL), using the
procedure followed for the preparation of 15a: yield 730
mg (76%); mp 46–47 ꢁC (ether–n-pentane); 1H NMR
(CDCl3, 200 MHz) d (ppm) 1.34–1.67 (complex m, 24H,
2,4,6,8,9,10-adamantane H, 3,4,5-piperidine H, 30,40,50-
piperidine H), 1.86–2.01 (m, 4H, 2-piperidine H, 3,5,7-
adamantane H), 2.31–2.46 (m, 6H, 6-piperidine H, 20,60-
piperidine H), 2.52–2.88 (m, 4H, NCH2CH2N) ; 13C
NMR (CDCl3, 50 MHz) d (ppm) 18.40, 19.0, 22.20
(3,4,5-piperidine C), 24.30 (40-piperidine C), 25.90 (30,50-
piperidine C), 28.60 (3,5,7-adamantane C), 37.30
(4,6,10-adamantane C), 39.90 (2,8,9-adamantane C),

G. Stamatiou et al. / Bioorg. Med. Chem. 11 (2003) 5485–5492
5491
40.10 (1-adamantane C), 47.60 (NCH2CH2N), 50.50
(NCH2CH2N), 55.1 (20,60-piperidine C), 58.90 (6-piper-
idine C), 71.20 (2-piperidine C). Fumarate: mp 193–
195 ꢁC (EtOH–Et2O); Bipicrate: mp 209–211 ꢁC (MeOH);
anal. (C34H44N8O14) C, H.
(CDCl3, 50 MHz) d (ppm) 24.39, 26.81, 27.93, 30.16
(3,4,5,6-azepine C), 28.71 (3,5,7-adamantane C), 36.81
(1-adamantane C), 37.45 (4,6,10-adamantane C), 39.28
(2,8,9-adamantane C), 45.31 (N-CH3), 52.01 (7-azepine
C), 73.67 (2-azepine C). Hydrochloride mp 206–208 ꢁC
(EtOH–Et2O); Picrate: mp 199–201 ꢁC (MeOH); anal.
(C23H32N4O7) C, H.
4,5,6,7-Tetrahydro-2-(tricyclo[3.3.1.13,7]dec-1-yl)-3H-
azepine, 18. Compound 18 was prepared by the proce-
dure used for the tetrahydropyridine analogue 9
(method B), by treating a chloroform solution (30 mL)
of 1-adamantylcyclohexanol 17 (1.0 g, 4.30 mmol) with
sodium azide (1.50 g, 23.0 mmol) and concd H2SO4 (4
mL): yield 300 mg (30%); mp 47–49 ꢁC (n-pentane); IR
1-[2-(1-Piperidine)ethyl]-2-(tricyclo[3.3.1.13,7]dec-1-yl)-
hexahydro-1H-azepine, 22. The solid bromacetylated
hexahydroazepine 20b was prepared by the procedure
used for 13, by treating hexahydroazepine 19 (900 g,
3.90 mmol) in dichloromethane (18 mL) — water (10
mL) with bromacetyl chloride (680 mg, 4.30 mmol) in di-
chloromethane (10 mL) in the presence of K2CO3 (600 mg,
4.30 mmol) in water (4 mL): yield 1.21 g (86%); mp 85–
87 ꢁC (ether–n-pentane); IR (Nujol) 1635 cmꢀ1; anal.
(C18H28BrNO) C, H.
(Nujol) 1655 cmꢀ1 1H NMR (CDCl3, 200 MHz) d
;
(ppm) 1.23–1.37 (m, 2H, 4-azepine H), 1.38–1.50 (m,
2H, 6-azepine H), 1.52–1.77 (m, 14H, 2,4,6,8,9,10-ada-
mantane H, 5-azepine H), 1.96 (br s, 3H, 3,5,7-ada-
mantane H), 2.32 (ꢃt, 2H, J=ꢃ8 Hz, 3-azepine H),
3.53 (ꢃt, 2H, J=ꢃ6 Hz, 1H, 7-azepine H); 13C NMR
(CDCl3, 50 MHz) d (ppm) 23.98 (4-azepine C), 26.20 (6-
azepine C), 27.07 (3-azepine C), 28.15 (3,5,7-ada-
mantane C), 31.33 (5-azepine C), 36.72 (4,6,10-ada-
mantane C), 38.97 (2,8,9-adamantane C), 42.17 (1-
adamantane C), 51.44 (7-azepine C), 183.55 (2-azepine
C). Picrate: mp 161–163 ꢁC (MeOH–Et2O); anal.
(C22H28N4O7) C, H.
Treating a solution of bromacetylated derivative 20b
(1.0 g, 2.80 mmol) in dry benzene (8 mL) with piperidine
(720 mg, 8.50 mmol) in dry benzene (10 mL) using the
same procedure described for compound 14b afforded
910 mg of acylated azepine 20c: yield 90%; mp 136–
138 ꢁC (n-pentane); IR (Nujol) 1646 cmꢀ1; anal.
(C23H38N2O) C, H.
Hexahydro-2-(tricyclo[3.3.1.13,7]dec-1-yl)-1H-azepine,
19. Compound 19 was prepared by the procedure used
for the piperidine analogue 10, by treating a methanol
solution (20 mL) of tetrahydroazepine 18 (1.0 g, 4.33
mmol) with NaBH4 (730 mg, 19.3 mmol) for 1 h at 0 ꢁC
and 2 h at ambient temperature: yield 740 mg (73%);
mp 65–67 ꢁC (n-pentane); IR (Nujol) 3365 cmꢀ1; ‘p1H
NMR (CDCl3, 200 MHz) d (ppm) 1.23–1.74 (m, 21H,
2,4,6,8,9,10-adamantane H, 1,3,4,5,6-azepine H), 1.96
(dd, 1H, J=2.7, 10.1 Hz, 1H, 2-azepine H), 1.91 (br
s, 3H, 3,5,7-adamantane H), 2.52–2.61 (m, 1H, 7-
azepine H), 2.91–3.0 (m, 1H, 7-azepine H); 13C NMR
(CDCl3, 50 MHz) d (ppm) 26.70, 27.10, 28.90, 31.60
(3,4,5,6-azepine C), 28.50 (3,5,7-adamantane C), 36.60
(1-adamantane C), 37.30 (4,6,10-adamantane C),
38.70 (2,8,9-adamantane C), 49.0 (7-azepine C), 67.9
(2-azepine C). Hydrochloride mp >280 ꢁC (EtOH–
Et2O); Picrate: mp 172–174 ꢁC (MeOH–Et2O); anal.
(C22H30N4O7) C, H.
Piperidinoethyl hexahydroazepine 22 was prepared by
the LiAlH4 (370 mg, 9.80 mmol) reduction of piper-
idinoacetyl hexahydroazepine 20c (700 mg, 1.95 mmol)
in dry THF (30 mL), using the procedure followed for
the preparation of 15b. The residue was chromato-
graphed on silica gel with ether as an eluent to afford
solid compound 22: yield 620 mg (92%); mp 49 ꢁC; H
1
NMR (CDCl3, 200 MHz) d (ppm) 1.23–2.03 (complex
m, 30H, adamantane H, 2,3,4,5,6-azepine H, 3,4,5-
piperidine H), 2.35–2.71 (m, 10H, NCH2CH2N, 7-aze-
pine H, 2,6-piperidine-H); 13C NMR (CDCl3, 50 MHz) d
(ppm) 24.46 (4-piperidine C), 26.04 (3,5-piperidine C),
23.84, 26.28, 27.74, 26.69 (3,4,5,6-azepine C), 28.75
(3,5,7-adamantane C), 36.81 (1-adamantane C), 37.48
(4,6,10-adamantane C), 38.88, 39.44 (2,8,9-adamantane
C), 47.51, 50.47 (NCH2CH2N), 55.35 (2,6-piperidine C),
58.46 (7-azepine C), 74.49 (2-azepine C). Monofumarate:
mp 209–211 ꢁC (EtOH–Et2O); anal. (C27H44N2O4) C,
H.
Hexahydro-1-methyl-2-(tricyclo[3.3.1.13,7]dec-1-yl)-1H-
azepine, 21. The oily carbamate ester 20a was prepared
by the procedure used for 11a (method A), by the reac-
tion of ethyl chloroformate (760 mg, 7.0 mmol) with
hexahydroazepine 19 (990 mg, 4.25 mmol) in the pre-
sence of triethylamine (1.40 g, 13.8 mmol) in dry ether
Biological evaluation
Reference compounds. Dextran sulfate with an average
molecular weight of 5000 was purchased from Sigma
(St. Louis, MO). AZT (azdothymidine) was synthesized
as previously described.14 Nevirapine (BI-RG587) was
obtained from Boehringer Ingelheim (Ridgefield, CN).
Ritonavir (ABT538) was kindly provided by J. M. Leo-
nard, Abbott Laboratories (Abbott Park, IL, USA).
(15 mL): yield 1.01 g (78%); IR (film) 1697 cmꢀ1
.
The solid azepine 21 was prepared by the LiAlH4 (1.20
g, 31.6 mmol) reduction of carbamate ester 20a (960 mg,
3.15 mmol) in dry THF (60 mL), using the procedure
followed for the preparation of 12a (method A): yield
570 mg (73%); mp 56–58 ꢁC (n-pentane); 1H NMR
(CDCl3, 200 MHz) d (ppm) 1.12–1.92 (complex m, 24H,
adamantane H, 2,3,4,5,6-azepine H), 2.52 (s, 3H, N-
CH3), 2.69–2.93 (m, 2H, 7-azepine H); 13C NMR
Cells and viruses. The MT-4 cells15 were grown in
humidified atmosphere with 5% CO2 and maintained in
RPMI 1640 medium supplemented with 10% heat-
inactivated fetal calf serum, 2 mM l-glutamine, 0.1%
sodium bicarbonate and 20 mg/mL of gentamicin. The
origin of HIV-1(IIIB) and HIV-2 (ROD) virus stocks

5492
G. Stamatiou et al. / Bioorg. Med. Chem. 11 (2003) 5485–5492
has been described previously.16 HIV-1 and HIV-
2(ROD) stocks were obtained from the culture super-
natant of HIV-1- or HIV-2-infected MT-4 cells, respec-
tively. Influenza A viruses (H2N2) and B were kindly
provided by Dr. K. Andries (Janssen Pharmaceutica,
Beerse, Belgium) and stocks were prepared on MDCK
cells.
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Antiviral activity and cytotoxicity assays. The inhibitory
effects of the rimantadine derivatives on HIV-1 strain
IIIB and HIV-2 strain ROD replication were monitored
by measuring the viability of MT-4 cells 5 days after
infection. Cytotoxicity of the compounds was deter-
mined in parallel by measuring the viability of mock-
infected cells on day 5. The number of viable cells was
quantified semi-automatically by a tetrazolium-based
colorimetric method using 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium (MTT), as described by Pau-
wels et al.17,18 Anti-influenza virus activity was assessed
in 96-well plates on 1-day confluent MDCK cells. Cells
were infected with 100CCID50 virus/well. After 1 h of
virus absorption virus was removed and serial dilutions
of the test compounds were added after which cultures
were further incubated at 35 ꢁC (5% CO2) for 4–5 days.
Cytopathic effects (and, in parallel, cytotoxic effects in
non-infected cultures) were read using the MTS-method
(Promega).
Acknowledgements
This research activity was supported by a research grant
from the University of Athens, Greece. We thank ISEP/
FORTIS, the European Commission (QLRT-2000-
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30291 and the Rene Descartes Prize (Grant HPAW-CT-
´
2002-90001) for financial support. We are grateful to
Cindy Heens and Kristien Erven for excellent technical
assistance.
14. Barre
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Chamaret, S.; Gruest, J.; Dauguet, C.; Axler-Blin, C.; Ve
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zinet-
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Brun, F.; Rouzioux, C.; Rozenbaum, W.; Montagnier, L.
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