Novel 3-(2-adamantyl)pyrrolidines with potent activity against influenza A virus - Identification of aminoadamantane derivatives bearing two pharmacophoric amine groups

Article abstract of DOI:10.1016/S0960-894X(01)00388-2

The 3-(2-adamantyl)pyrrolidines 8a-g, 14 were synthesized and evaluated for activity against influenza A virus. The parent N-H compound 14 was several times more active than amantadine against H₂N₂ and H₃N₂ influenza A virus. The combined use of NMR spectroscopy and computational chemistry showed that the conformation around the pyrrolidine-adamantyl carbon-carbon bond is trans and the pyrrolidine heterocycle has an envelope conformation with C-2 out of the plane of the other ring atoms. N-Dialkylaminoethyl substitution of compound 14 resulted in the potent diamine analogues 8e,f,g. Interestingly, their lactam amine precursors were also active. Compounds 8e,f,g are the first adamantane derivatives, bearing two amine groups, reported to be active against influenza A virus.

Full text of DOI:10.1016/S0960-894X(01)00388-2

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2137–2142
Novel 3-(2-Adamantyl)pyrrolidines with Potent Activity Against
Influenza A Virus—Identification of Aminoadamantane
Derivatives Bearing Two Pharmacophoric Amine Groups
George Stamatiou,a Antonios Kolocouris,a Nicolas Kolocouris,a George Fytas,a
George B. Foscolos,a,* Johan Neytsb and Erik De Clercqb
aSchool of Pharmacy, Department of Pharmaceutical Chemistry, Universiy of Athens, Panepistimioupoli-Zografou,
GR-15771 Athens, Greece
bRega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium
Received 3 February 2001; accepted 22 May 2001
Abstract—The 3-(2-adamantyl)pyrrolidines 8a–g, 14 were synthesized and evaluated for activity against influenza A virus. The
parent N–H compound 14 was several times more active than amantadine against H2N2 and H3N2 influenza A virus. The combined
use of NMR spectroscopy and computational chemistry showed that the conformation around the pyrrolidine–adamantyl carbon–
carbon bond is trans and the pyrrolidine heterocycle has an envelope conformation with C-2 out of the plane of the other ring
atoms. N-Dialkylaminoethyl substitution of compound 14 resulted in the potent diamine analogues 8e,f,g. Interestingly, their lac-
tam amine precursors were also active. Compounds 8e,f,g are the first adamantane derivatives, bearing two amine groups, reported
to be active against influenza A virus. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
to secondary bacterial pneumonia or primary viral
Influenza A is a major respiratory tract disease affecting
millions of people each year. Influenza A is character-
ized by the abrupt onset of constitutional and respira-
tory signs and symptoms (e.g., fever, myalgia, headache,
severe malaise, nonproductive cough, sore throat and
rhinitis).1 However, in some persons the infection can
cause, for example pulmonary or cardiac disease or lead
pneumonia. Epidemics of influenza 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.2,3 Influenza A viruses have the ability
to undergo changes by the mechanisms of antigenic drift
and shift and new evolving strains can be a serious
threat to the human population.4 Thus, pandemic
*Corresponding author. Fax: +30-1-727-4747; e-mail: ankol@pharm.uoa.gr
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00388-2

2138
G. Stamatiou et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2137–2142
influenza A viruses appeared in 1918 (‘Spanish’ H1N1),
1957 (‘Asian’ H2N2) and 1968 (‘Hong Kong’ H3N2).
Given that influenza shifts occur every 20–30 years and
that a new lethal variant appeared in Hong Kong in
1997, the danger of future influenza A pandemics is real.
The parent N–H pyrrolidine 14 was synthesized using
the N-trimethylsilyl 2-pyrrolidinone 9 as starting mate-
rial (Scheme 2). Lithiation of lactam 9 and subsequent
reaction with 2-adamantanone afforded alcohol 10.
Compound 10 was treated with thionylchloride and
ethanolic potassium hydroxide solution to give methy-
lenelactam 12. Catalytic hydrogenation and subsequent
LiAlH4 reduction resulted in the pyrrolidine 14.8
Amantadine 1 and rimantadine 2 are anti-influenza A
drugs that inhibit virus replication at micromolar con-
centrations.5,6 Many aminoadamantanes active against
influenza A virus were synthesized in our laboratory
during the past 5 years.6,7 Of these compounds, the
most potent ones bear an amino group substitution at
the C-2 position of the adamantane nucleus. In order to
further examine the stereoelectronic requirements for
optimal activity we report here the synthesis of the 3-(2-
adamantyl)pyrrolidines 14 and 8a–g and their anti-
influenza A virus activity evaluation. The biological
activity of the lactam amine precursors of diamines 8e
and 8f was also examined.
The preparation of the parent pyrrolidine 14 through
demethylation of N-methyl derivative 8a failed to suc-
ceed. Although the reaction of N-methylpyrrolidine 8a
with 2,2,2-trichloroethyl chloroformate gave the corre-
sponding carbamate, treatment of the latter with Zn/
AcOH failed to produce the N–H derivative as was
expected from literature data.9 Instead the N-methyl
derivative 8a was identified to be the reaction product.
The above described routes can be considered as general
for the 3-substitution of g-lactams and pyrrolidines with
cycloalkyl groups.
Results and Discussion
Chemistry
Antiviral activity evaluation
The cytotoxicity and activity of the new aminoada-
mantane heterocycles 7e,f, 8a–g and 14 were examined
against influenza A virus strains H2N2 and H3N2
according to previously reported methods (Table 1).10
The synthetic route followed for the preparation of the
novel compounds is illustrated in Scheme 1. N-Alkyl 2-
pyrrolidinones 4a–g were prepared through N-alkyl-
ation of 2-pyrrolidinone 3 and used as starting materials
for the preparation of the target compounds 8a–g.
Alcohols 5a–g were synthesized by the reaction of 2-
oxo-3-pyrrolidinyl lithium with 2-adamantanone. Sub-
sequent dehydration and catalytic hydrogenation of the
indermediate methylene lactams 6a–g afforded the 3-(2-
adamantyl)-2-pyrrolidinones 7a–g. Reduction of lactams
7a–g with LiAlH4 yielded compounds 8a–g.
From the MIC50 and MCC50 values presented in Table
1, it appears that, except for compounds 8b–d, all the
others were active against influenza virus A strains.
Whereas N-alkylation of the parent amine 14 caused a
dramatic reduction in antiviral activity, N-dialkylami-
noethyl substitution led to active analogues. The activ-
ity of compounds 14, 8e,g was 2–7 times higher than
that of amantadine, while compounds 14 and 8e had a
Scheme 1. Reagents: (a) NaH, RX, DMF or benzene, 60–70 ꢀC(23–58%); (b) LDA, ꢁ70 ꢀC; (c) adamantanone, THF, ꢁ80 ꢀCand then H +/H2O,
0 ꢀC(73–95%); (d) TsOH, benzene, reflux (79–96%); (e) H , PtO2, ethanol, 40 lb/in2, rt quant; (f) LiAlH4, THF, 20–25 h reflux (72–89%).
2

G. Stamatiou et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2137–2142
2139
Scheme 2. Reagents: (a) NaH, Me3SiCl, benzene, rt, 12 h (67%); (b) LDA, ꢁ70 ꢀC; (c) adamantanone, THF, ꢁ80 ꢀCand then H +/H2O, 0 ꢀC
(95%); (d) SOCl2, CHCl3, reflux, 3 h; (e) CH3ONa/CH3OH, reflux (53% from 10); (f) H2, PtO2, EtOH, 40 lb/in2 (quant); (g) LiAlH4, THF, 15 h
reflux (68%).
Table 1. Anti-influenza A virus activity and cytotoxicity of amino-
adamantane derivatives 7e,f, 8a–g and 14a in MDCK cellsb
pounds 8e,g bearing two amine groups is interesting
since these compounds are the first examples of active
aminoadamantanes bearing two amine groups. The
specific activity of compounds 8e,g can be exerted by
three pharmocophores, that is adamantane and two
amine groups. It is interesting to note that the lactam
amines 7e,f were also active anti-influenza A compounds,
albeit less potent than the diamines 8e–g.
d
Compound
MIC50c (mM)
MCC50
(mM)
Influenza virus
b
Influenza virus
b
A H2N2
A H3N2
7e
7f
8a
8b
8c
8d
8e
14.7
11.1
18.8
>29.7
>28.2
>26.9
0.38
8.9
1.7
0.60
2.6
614.8
14.7
11.1
18.8
>29.7
>28.2
>26.9
9.4
>14.9
0.35
5.0
122.4
92.0
ꢃ156.4
148.2
140.9
134.3
78.7
74.5
14.6
124.4
Conformational analysis
Recently, we have been involved in the investigation of
the conformational properties of potent aminoada-
mantanes with the future goal a deeper understanding
of their SAR relationship. In this work a combination of
computational chemistry and NMR spectroscopy was
used in order to explore the conformational preferences12
of the parent pyrrolidine 14 in its protonated form.13
8f
8g
14
Amantadine
Ribavirin
12.8
122.9
>533.3
ꢃ1024.6
All data represent mean values for at least two separate experiments.
aAminoadamantanes 7e,f 8a–g and 14 were tested as hydrochlorides or
fumarate salts.
The molecular dynamics simulation and grid scan
search analysis around the C3–C20 bond revealed many
low energy conformers for the protonated form of
compound 14.13 The conformers obtained differed in
the pyrrolidine conformation,14 adamantyl orientation
and C3–C20 bond conformations. Thus, the pyrrolidine
heterocycle can adopt an envelope conformation with
any atom out of the plane of the four other atoms,
the 2-adamantyl group can be axial or equatorial, while
the conformation around the C3–C20 bond can be trans
or gauche.
bAbbreviations and strains used: MDCK, Madin–Darby canine kid-
ney cells, human epithelial cells; influenza A H2N2 (A2 Japan/305/57),
influenza A H3N2 (X31).
cMinimum inhibitory concentration or concentration required to
reduce virus-induced cytopathogenicity by 50%.
dMinimum cytotoxic concentration or concentration required to cause
a microscopically detectable alteration of normal cell morphology.
selectivity index ꢂ 210. In the past, it has been reported
that N-aminoethyl substitution of amantadine resulted
in inactive analogues.11 Thus, the potency of com-

2140
G. Stamatiou et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2137–2142
However, conformers A–D with an envelope E(2) pyr-
rolidine conformation, that is with the C-2 atom out of
the plane of the other atoms, are most favored. The
conformational descriptors of these conformers are
summarized in Table 2. Conformers A, B and C with a
2-adamantyl group in the equatorial position have
trans, gauche-(ꢁ) and gauche-(+) orientation around
the C3–C20 bond, while in conformer D, the 2-ada-
mantyl group is axial and the C3–C20 bond conforma-
tion is trans (Fig. 1). The molecular mechanics
calculations suggest A to be the lowest in energy con-
former.14a All the remaining conformers are higher in
energy by more than 3.3 kcal molꢁ1 (Table 2) and are
considered unpopulated at room temperature according
to equation ÁGꢀ=ꢁRTlnK.14b AM1 and MNDO cal-
culations produce almost completely flat structures and
failed to give significant information as has already been
pointed out in recent studies on pyrrolidine struc-
ture.14a,15 The E(2) structure of pyrrolidine is consistent
with the triplet (J=10.4 Hz) at 2.61 ppm assigned at the
H-2 axial proton which must be trans to H-3 proton and
the NOE correlation between H-2 and H-5 axial pro-
tons. Experimental information for the conformation
around H3–C3–C20–H20 dihedral angle can be extracted
from the adamantane H-20 signal. However, this was
not resolved for protonated 14 but appeared as a
doublet (Jꢂ10.7 Hz) at 1.41 ppm in the proton NMR
spectrum of the free amine form. Thus, the conforma-
tion by rotation around the C3–C20 bond must be anti
for compound 14.
The anti conformation by rotation around C3–C20 bond
can be explained on the basis of the more generalized
situation found in 1,1,2,2-tetrasubstituted ethanes. The
last molecules can adopt two low energy conformations,
that is, the anti-staggered conformation E with two
gauche interactions and the gauche-staggered conforma-
tion F or its enantiomer with three gauche interactions
(Fig. 2). In fact, when the four substituents are methyl
groups, as in 2,3-dimethylbutane, the two conformations
Table 2. Low energy conformers of parent pyrrolidine 14. Relative energies (kcal molꢁ1) were calculated using the MM+ force field14
Compound
Conformer
Conformer descriptors
Relative energy
Conformation
around C3–C20 bond
2–Ada group
orientation
Pyrrolidine
conformation
14
A
B
C
D
trans
gauche(ꢁ)
gauche(+)
trans
eqb
eq
eq
axd
E(2)c
E(2)
E(2)
E(2)
0.00
5.50
4.61
3.32
aAd, adamantyl.
bEquatorial.
cEnvelope with C-2 out of plane.
dAxial.
Figure 1. Low energy conformations A–D of the compound 14 as resulting from molecular mechanics calculations.

G. Stamatiou et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2137–2142
2141
around C3–C20 rotor and an E(2) envelope conformation
were identified for this series of compounds.
The fact that aminoadamantanes with a second amine
group, that is, compounds 8e and 8g, have potent anti-
viral activity is unprecedented and surmises the presence
of a corresponding binding site in the M2 transmem-
brane pore. Dialkylaminoethyl substitution of 1-ada-
mantanamine reportedly led to inactive compounds.11
Yet, N-dialkylaminoethyl substitution on 3-(2-ada-
mantyl)pyrrolidines generates potent anti-influenza A
virus activity.
It was also shown that lactam amine precursors of di-
amines are less active compounds. These findings sug-
gest that certain stereoelectronic requirements must be
fulfilled for aminoadamantanes to exert M2 ion channel
blocking activity and demontrate anti-influenza A virus
potency.5
Figure 2. Newman structures to describe the conformational behavior
of 1,1,2,2-tetrasubstituted ethanes.
Acknowledgements
This research activity was supported by a research grant
from the University of Athens, Greece. We thank Dr. T.
Mavromoustakos for some useful discussions at early
stages of this work.
are about equal in energy,16 whereas for larger sub-
stituents, only the gauche conformations F and enan-
tiomer are populated.17
The anti conformation E is less stable than the gauche F
for large substituents because one of the favored ways
of relieving strain at tertiary atoms such as H–CR1R2R3
is by opening up of bond angles R–C–R. If this occurs
in conformation E, gauche-substituents are forced
nearer each other with no obvious possibility of relief,
see structure G. If this occurs in conformation F,
gauche-substituents are moved apart somewhat and
steric interaction may be further eased by rotation
about the central bond, see structure H. A contrasting
example of a 1,1,2,2-tetrasubstituted bond is provided
by the bond from the ring to the 2-adamantyl sub-
stituent in compound 14. As pointed out above, the
vicinal coupling constant of 10.4 Hz across this bond
shows that it strongly prefers to adopt the trans con-
formation. Here bond angle opening is resisted by the
adamantyl structure and the gauche conformers, calcu-
lated to be more than 4.6 kcal molꢁ1 higher in energy,
are unpopulated.
References and Notes
1. Cox, N. J.; Subarao, K. Lancet 1999, 354, 1277.
2. Simonsen, L.; Schonberger, L. B.; Stroup, D. F.; Arden, N.
H.; Cox, N. J. The impact of influenza on mortality in the
USA. In Options for the Control of Influenza III; Brown, L. E.,
Hampson, A. W., Webster, R. G., Eds.; Amsterdam: Elsevier
Science, 1996; pp 26–33.
3. Lui, K.-J.; Kendal, A. P. Am. J. Public Health 1987, 77,
712.
4. (a) Taubenberger, J. K.; Reid, A. H.; Fanning, T. G. Vir-
ology 2000, 274, 241. (b) Taubenberger, J. K.; Reid, A. H.;
Kraftt, A. E.; Bijwaard, K. E.; Fanning, T. G. Science 1997,
275, 1793.
5. (a) 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. (d) Duff, K. C.; Gilchrist, P. J.; Saxena, A. M.;
Bradshaw, J. P. Virology 1992, 188, 14.
N-Alkyl derivatives of the parent amine have the same
pyrrolidine conformation and an equatorial N-sub-
stituent orientation. It seems that the dramatic decrease
of activity observed for N-alkyl derivatives can be
attributed simply to steric reasons.
6. Wolff, M. E., Ed. In Burger’s Medicinal Chemistry; John
Wiley & Sons: New York, 1998; Part II, pp 590–591.
7. (a) Kolocouris, A. PhD Thesis (1995), available from
National Documentation Center, Vas. Constantinou 48,
Athens, Greece. (b) Kolocouris, N.; Foscolos, G. B.; Kolo-
couris, A.; Marakos, P.; Pouli, N.; Fytas, G.; Ikeda, S.; De
Clercq, E. J. Med. Chem. 1994, 37, 2896. (c) Kolocouris, N.;
Kolocouris, A.; Foscolos, G. B.; Fytas, G.; Neyts, J.; Padalko,
E.; Balzarini, J.; Snoeck, R.; Andrei, G.; De Clercq, E. J. Med.
Chem. 1996, 39, 3307. (d) Kolocouris, A.; Tatarides, D.; Fytas,
G.; Foscolos, G.; Mavromoustakos, T.; Kolocouris, N.
Bioorg. Med. Chem. Lett. 1999, 9, 3465.
Conclusion
This work reports the synthesis of a new series of potent
aminoadamantanes. Pyrrolidines 14, 8a,e,g were found
active against H2N2 and H3N2 strains of influenza virus
A. Through a combination of NMR spectroscopy and
molecular mechanics calculations an anti conformation
1
8. The H and 13CNMR spectra of free pyrrolidine 14 was
1
assigned using COSY and CHCORR spectroscopy. H NMR
(CDCl3, 200 MHz): d 1.21–1.37 (m, 1H, 4-H), 1.38–1.44 (d,

2142
G. Stamatiou et al. / Bioorg. Med. Chem. Lett. 11 (2001) 2137–2142
1H, 20-H), 1.48–1.99 (15H, adamantane–H, 4-H), 2.30–2.47
(m, 2H, 2, 3-H), 2.90–3.08 (m, 2H, 5-H), 3.10–3.20 (dd, 1H, 2-
H), 4.03 (bs, 1H, N–H); 13C NMR (CDCl3, 50 MHz) d 27.7,
28.1 (50,70-C), 30.6 (4-C), 31.0, 31.3 (10,30-C), 31.8 (40,90-C),
38.3 (60-C), 39.1, 39.2 (80,100-C), 40.5 (3-C), 46.2 (5-C), 49.5
(20-C), 51.0 (2-C). Spectra and elemental analysis of all the
synthesized compounds were in accord with their structure.
9. Isidor, J. L.; Carlson, R. M. J. Org. Chem. 1973, 38, 554.
10. Shigeta, S.; Konno, K.; Yokota, T.; Nakamura, K.; De
Clercq, E. Antimicrob. Agents Chemother. 1998, 32, 906.
11. Aldrich, P. E.; Hermann, E. C.; Meier, W. E.; Paulshock,
M.; Prichard, W. W.; Snyder, J. A.; Watts, J. A. J. Med.
Chem. 1971, 14, 535.
14. (a) Molecular mechanics were performed using the MM+
force field provided by Hyperchem. This force field is an
extension of the MM2 force field. Molecular mechanics calcu-
lations give the most consistent results with experimental data
for pyrrolidine conformers, see ref 15. An initial structure was
constructed and minimized using conjugate gradient and
Newton–Raphson algorithms and an energy gradient toler-
˚
ance of 0.001 kcal molꢁ1
A
ꢁ1. This structure was then sub-
jected to grid scan search analysis using 10ꢀ steps. (b)
According to equation ÁGꢀ=ꢁRTlnK the relative population
D/A is 0.4:99.6 at 298 K (see Table 2).
15. (a) Pfafferott, G.; Oberhammer, H.; Boggs, J. E.; Cami-
nati, W. J. Am. Chem. Soc. 1985, 107, 2305. (b) Dobado, J. A.;
Molina Molina, J.; Rodriguez Espinosa, M. J. Mol. Struct.
1994, 303, 205.
12. Howard, A. E.; Kollman, P. A. J. Med. Chem. 1988, 31,
1669.
13. (a) Solutions of protonated compound 14 in DMSO-d6
solution (fumarate salt) or CDCl3 solution (TFA-d1 salt) were
studied. The 1H and 2D COSY, HMQC and NOESYPH
NMR spectra were run on a Bruker DRX 400 machine. (b)
Aminoadamantanes have been considered to act biologically
through their protonated form; see ref 5.
16. Anderson, J. E. In The Chemistry of Alkanes and
Cycloalkanes; Patai, S., Rappoport, Z. Eds.; Wiley: New
York, 1992; Chapter 3, III, G.
17. (a) Lunazzi, L.; Macciantelli, D.; Bernardi, B.; Ingold,
K. U. J. Am. Chem. Soc. 1977, 99, 4573. (b) Ritter, W.; Hull,
W.; Cantow, H. J. Tetrahedron Lett. 1978, 30, 933.