Synthesis and anti-influenza a virus activity of 2,2-dialkylamantadines and related compounds

Article abstract of DOI:10.1021/ml300279b

The synthesis of several 2,2-dialkyladamantyl-1-Amines through the combination of a Ritter reaction with a Wagner-Meerwein rearrangement from noradamantane alcohols is reported. Several of the novel amines displayed low micromolar activities against sever

Full text of DOI:10.1021/ml300279b

Letter
pubs.acs.org/acsmedchemlett
Synthesis and Anti-influenza A Virus Activity of 2,2-
Dialkylamantadines and Related Compounds
Eva Torres,† Roser Fernandez,† Stephanie Miquet,† Merce Font-Bardia,§,∥ Evelien Vanderlinden,‡
́
́
̀
Lieve Naesens,*,‡ and Santiago Vazquez*,†
́
†Laboratori de Química Farmaceu
̀ ̀
tica (Unitat Associada al CSIC), Facultat de Farmacia, and Institute of Biomedicine (IBUB),
Universitat de Barcelona, Av. Diagonal 643, Barcelona E-08028, Spain
‡Rega Institute for Medical Research, University of Leuven, 3000 Leuven, Belgium
§Crystallografia, Mineralogia i Dipos
̀
its Minerals, Universitat de Barcelona, Martí i Franques
̀
s/n, Barcelona E-08028, Spain
∥Unitat de Difraccio
́
de RX, Centre Científic i Tecnologic de la Universitat de Barcelona (CCiTUB), Universitat de Barcelona, Sole i
̀
́
Sabarís 1-3, Barcelona E-08028, Spain
S
* Supporting Information
ABSTRACT: The synthesis of several 2,2-dialkyladamantyl-1-amines through the
combination of a Ritter reaction with a Wagner−Meerwein rearrangement from
noradamantane alcohols is reported. Several of the novel amines displayed low micromolar
activities against several H1N1 influenza virus strains, including the amantadine-resistant
A/PuertoRico/8/34 strain. Most of the compounds did not show cytotoxicity for MDCK
cells.
KEYWORDS: amantadine, influenza, M2 channel, Ritter reaction, Wagner−Meerwein rearrangement
nfluenza A virus is a global cause of significant morbidity and
mortality, which is related to its easy transmission and ability
protein], only a few were active against amantadine-resistant
strains.
I
to escape from existing immunity. The possible introduction
into the human population of emerging swine and highly
pathogenic avian influenza viruses is considered a global health
threat.1 Currently available drugs for the treatment of influenza
virus infections comprise the M2 ion channel blockers
amantadine and rimantadine (Chart 1)2,3 and the neuramini-
To the best of our knowledge, 2,2-dialkylamantadines have
not been synthesized so far. To explore whether the addition of
two alkyl groups can be beneficial for the antiviral activity of
amantadine, we decided to synthesize and evaluate the antiviral
activity of a series of 2,2-dialkylamantadines starting from the
known 3-noradamantanecarboxylic acid, taking advantage of a
unique combination of the Ritter reaction with the Wagner−
Meerwein rearrangement.
Chart 1. Structures of Amantadine and Rimantadine
In the study reported here, we found that several 2,2-
dialkylamantadines display low micromolar activity against
several influenza virus strains. Interestingly, this activity was
markedly subtype-dependent. While most of the compounds
were active against A/H1N1 strains, whether carrying an
amantadine-sensitive M2 channel or a mutated, amantadine-
resistant M2 channel, neither of the compounds was active
against influenza A/H3N2 strains.
In 1993, Stoelting and Shiner reported that the solvolysis of
several 1-(3-noradamantyl)ethyl sulfonates led to 2-methyl-1-
adamantanol, in excellent yields. In a similar way, 2-(3-
noradamantyl)propan-2-ol (1) led to 2,2,-dimethyl-1-adaman-
tanol (2) in high yield (Scheme 1).11 Presumably, the large
release of strain promotes the observed Wagner−Meerwein
rearrangement.
dase inhibitors oseltamivir and zanamivir.4 However, most of
the currently circulating influenza strains are resistant to the M2
ion channel blockers, and resistance to the neuraminidase
inhibitors (in particular oseltamivir) is also on rise.5−9
Accordingly, novel anti-influenza virus drugs are urgently
needed.
Taking into account that amantadine was initially licensed in
the United States in 1966, it is not surprising that many
hundreds of derivatives have been synthesized and tested,
including 1-substituted adamantanes, 2-substituted adaman-
tanes, azaspiroadamantanes, aminospiroadamantanes, 1,2-annu-
lated adamantane derivatives, and 2-azaadamantanes.10 Inter-
estingly, while several of them proved to be active against
amantadine-sensitive strains [containing the wild-type (wt) M2
In the most familiar form of the Ritter reaction, an alcohol is
treated with strong acid to generate a carbenium ion. The
Received: September 10, 2012
Accepted: October 22, 2012
Published: October 22, 2012
© 2012 American Chemical Society
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al. had described that, while the acetamides resulting from the
Ritter reaction of tertiary alcohols were very difficult to
hydrolyze, the corresponding chloroacetamides underwent easy
cleavage to the corresponding tert-alkylamines using thiourea.19
Thus, the reaction of tertiary alcohol 3a with chloroacetoni-
trile using the standard conditions developed by Jirgensons et
al. led to the expected rearranged chloroacetamide 6a in 89%
yield. Cleavage of the chloroacetyl group with thiourea
furnished 2,2-dimethylamantadine, 7a, in 78% yield (Scheme
3).
Scheme 1. Rearrangement of Noradamantyl Alcohol 1 to
Adamantanol 2
nitrile then reacts with it to produce a nitrilium ion whose
hydrolysis affords an amide product.
Thus, we envisaged that submitting 1-(3-noradamantyl)-1,1-
dialkylmethanols (3) to a Ritter reaction may led to N-(2,2-
dialkyl-1-adamantyl)amides (6), through the Wagner−Meer-
wein rearrangement of the initially generated carbenium ion.
Hydrolysis of these amides should lead to the expected 2,2-
dialkylamantadine derivatives (7) (Scheme 2). Interestingly,
After successfully proving the viability of our approach, we
synthesized a series of tertiary alcohols from ester 8. The
alcohols were subsequently transformed into the corresponding
2,2-dialkylamantadine derivatives in high yields (Scheme 3).
Remarkably, while the reaction of ester 8 with methyllithium,
ethyllithium, and propylmagnesium chloride led to the
expected tertiary noradamantyl alcohols in good yields, addition
of an excess of isopropylmagnesium bromide to 8 led to a
secondary alcohol, 10. Similarly, the reaction of 8 with
pentamethylenebis(magnesium bromide) did not furnish 13,
but 1-(3-noradamantyl)-5-hexen-1-ol, 9, in 93% yield. The
reductive behavior of organometallic reagents on steric
encumbered ketones is well documented, and these alcohols
may arise from the reduction by a magnesium species of the
initially obtained alkyl noradamantyl ketones.20 We finally
succeeded in the synthesis of the cyclohexanol 13 using the
known iodide 12 as the starting material.21 The reaction of 12
with t-butyllithium followed by addition of cyclohexanone led
to the expected alcohol in 56% yield. Alcohol 13 smoothly
underwent the Ritter reaction to the chloroacetamide 14, which
was cleaved with thiourea to amine 15 (Scheme 3). Of note,
from 10, we directly obtained the rearranged amine 11 using an
excess of urea in trifluoroacetic acid at 115 °C in 43% yield.
This one-step procedure for the synthesis of the amine from
the alcohol seems to be general, as the application of this
Scheme 2. Attempted Synthesis of 2,2-Dialkylamantadines 7
from Noradamanyl Alcohols 3
although the combination of a Ritter reaction with a Wagner−
Meerwein rearrangement has been used for mechanistic
studies,12,13 its combination for synthetic purposes has been
very limited.14−17
First, we studied the suitability of our approach using alcohol
3a,11 obtained in 91% yield from known ester 8.18 Jirgensons et
a
Scheme 3. Synthesis of Amantadine Analogues through Ritter Reaction and Wagner−Meerwein Rearrangement
a
Reagents and conditions: (i) For 3a: CH3Li, Et2O, reflux, overnight, 91% yield; for 3b: CH3CH2Li, Et2O, reflux, overnight, 56% yield; for 3c:
CH3CH2CH2MgCl, THF, reflux, overnight, 89% yield; for 10: (CH3)2CHMgBr, Et2O, reflux, overnight, 75% yield; for 17: CH3Li, Et2O, reflux,
overnight, 51% yield. (ii) ClCH2CN, AcOH, H2SO4, rt, overnight, 89% for 6a; 89.5% for 6b; 91% for 6c; 38% for 14; and 93% from 17. (iii)
Thiourea, ethanol, AcOH, reflux, overnight, 78% for 7a; 64% for 7b; 95% for 7c; 62% for 15; and 74% for 18. (iv) BrMg(CH2)5MgBr, THF, reflux,
24 h, 93% yield. (v) Urea, CF3CO2H, 115 °C, overnight, 43%. (vi) t-BuLi, Et2O, pentane, −78 °C to room temperature, 56% yield.
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Letter
procedure to the alcohol 6c led to the corresponding amine 7c
in 31% yield. However, the yield for the one-step procedure
was much lower than the overall yield obtained using the
cloroacetonitrile method.
derivative 22 in low yield. Reductive methylation of secondary
amine 21 with formaldehyde and NaCNBH3 led, in 80% yield,
to the tertiary amine 23, which on catalytic hydrogenation
quantitatively furnished secondary amine 24. Similarly, N,N-
dimethylderivatives of amines 7b, 7c, 11, and 18 were also
synthesized, in high yields, by reductive alkylation with
formaldehyde and NaCNBH3. Finally, secondary amine 26
was synthesized by treatment of 7b with methyl chloroformiate
followed by reduction with LiAlH4 in 92% overall yield
(Scheme 4).
Interestingly, our approach was also successfully imple-
mented in the ring expansion of a bisnoradamantane derivative
to the corresponding noradamantane amine. Thus, reaction of
known ester 16 with excess of methyllithium followed by the
application of Jirgensons' conditions to the alcohol 17 led to
the noradamantane derivative 18 in good yield (Scheme 3).22
To gain further insight into the structure−activity relation-
ship SAR, a small series of N-alkylated and N,N-dialkylated
derivatives were synthesized from adamantylamine 7a, using
standard amine chemistry (Scheme 4). Thus, from 7a,
reductive alkylation with formaldehyde, acetaldehyde, or
benzaldehyde and NaCNBH3 led to amines 19a, 20, and 21,
respectively, in high yield. Several attempts to synthesize the
N,N-diethylderivative of 7a, either from 7a or from 20, met
with failure, probably as a consequence of the steric hindrance.
Alkylation of 7a with 1,5-dibromopentane led to piperidine
The structure of all new compounds was confirmed by
1
elemental analysis and/or accurate mass measurement, IR, H
NMR, 13C NMR, and mass spectral data. The amines were fully
characterized as their corresponding hydrochloride or tartrate
salts. Moreover, the structure of chloroacetamide 14 was
established by X-ray crystallography.23
Antiviral cell culture assays were performed to determine the
antiviral activity of all of the synthesized compounds against a
broad panel of DNA and RNA viruses. None of the compounds
displayed activity against the enveloped DNA viruses herpes
simplex virus or vaccinia virus; the enveloped RNA viruses
feline coronavirus, parainfluenza-3 virus, respiratory syncytial
virus, vesicular stomatitis virus, sindbis virus, or Punta Toro
virus, or the nonenveloped RNA viruses Coxsackievirus B4 and
Reovirus-1.
Scheme 4. Synthesis of N- and N,N-Substituted 2,2-
a
Dialkylamantadines and Related Compounds
In influenza virus-infected Madin−Darby canine kidney
(MDCK) cells, several compounds displayed low micromolar
activity against the influenza A/H1N1 subtype, but no
compound was active against the influenza A/H3N2 subtype
(Table 1). The antiviral data obtained by microscopic
inspection of the viral cytopathic effect (CPE) at day 3
postinfection were confirmed by a colorimetric cell viability
assay.24 As anticipated, all compounds proved to be inactive
against influenza B virus, which is known to be insensitive to
amantadine and rimantadine.
Analysis of the data in Table 1 revealed the following trends.
First, with the single exception of the dipropyl derivative 7c, all
of the primary amines were more potent than amantadine and
rimantadine against the A/PR/8/34 strain of influenza A/
H1N1. Although the more potent compound was the
spiroderivative 15 (EC50 = 1.1 μM), the highest selectivity
(i.e., ratio of cytotoxic to antiviral concentration) was noted
with compound 7b (EC50 = 2.0 μM; ratio of CC50 to EC50
>
50). Second, while the introduction of one or two methyl
groups into the primary amine was beneficial (i.e., 7c vs 19c) or
practically neutral with regard to antiviral activity (i.e., 7b vs
19b or 26, 18 vs 25, and 11 vs 19d), the introduction of larger
groups was clearly deleterious for the activity, leading largely to
inactive, cytotoxic compounds.
The antiviral effect of three compounds displaying good
selectivities, 7b (a primary amine), 19b (a tertiary amine), and
26 (a secondary amine), was further evaluated against a broader
panel of influenza A/H1N1 and A/H3N2 viruses, including two
amantadine sensitive A/H1N1 strains (A/Ned/378/05 and A/
FM/1/47) and X-31, a chimeric strain containing the H3 and
N2 proteins of A/Aichi/2/68 and the other proteins (including
M2) from the amantadine-resistant A/PR/8/34 strain. As
shown in Table 2, the three compounds behave similarly,
showing activity against the three A/H1N1 strains and being
inactive against the three A/H3N2 strains.
a
Reagents and conditions: (i) H2CO, NaBH3CN, methanol, room
temperature, overnight; 84% for 19a; 31% for 19b; 83% for 19c; 68%
for 19d; 73% for 23; and 96% for 25. (ii) CH3CHO, NaBH3CN,
methanol, rt, overnight, 84% yield. (iii) C6H5CHO, NaBH3CN,
methanol, room temperature, overnight, 83% yield. (iv) 1,5-
Dibromopentane, NaI, Et3N, anh. DMF, 60 °C, 26 h, 22% yield. (v)
H2, Pd/C, ethanol, 38 atm, 100 °C, 24 h, 80% yield. (vi) ClCO2CH3,
Et3N, Et2O, room temperature, overnight. (vii) LiAlH4, THF, reflux,
20 h, 62% yield overall.
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 is sufficient to render the
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ACS Medicinal Chemistry Letters
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a
Table 1. Antiviral Activity in Influenza Virus-Infected MDCK Cells
b
antiviral EC50 (μM)
influenza A/H1N1
CPE MTS
15
influenza A/H3N2
CPE MTS
influenza B
cytotoxicity (μM)
c
d
compd
CPE
>100
MTS
>100
MCC
CC50
7a
7b
7c
11
15
18
3
10
3
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
167 33
≥100
77 20
>100
2.0 0.0
>100
1.7
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
8.0 1.7
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>50
>100
>100
6.0 4.0
5.3 2.1
6.0 4.0
≥100
2.9 1.4
8.6 3.5
4.7 1.1
>100
5.8
1.2 0.1
0.9 0.2
4.5 2.4
4.8 0.1
>100
>100
1.1 0.1
4.6 2.4
4.0 0.0
>100
>100
>100
19a
>100
110 90
68 26
20
>100
200
4
0
85
3
21
>100
>100
>100
1.1
0.8
0.7
22
>100
>100
>100
4
23
>100
>100
>100
4
24
6.0 1.0
4.0 2.5
4.0 2.5
6.0 1.9
5.8 2.1
5.1 3.2
53 11
63 18
7.9 0.6
10.0
9.6
0
>100
110 90
≥100
≥100
≥100
≥100
>100
70 28
>100
>100
>100
>100
≥100
>100
>100
≥20
19b
>100
26
>100
19c
6.7 0.8
5.7 2.4
6.0 1.2
>100
25
>100
19d
>100
amantadine
rimantadine
ribavirin
3.4 1.7
0.17 0.08
7.7 1.2
>100
>100
8.1 3.4
500
≥100
>100
8.2 1.4
7.0 0.4
a
b
MDCK: Madin−Darby canine kidney cells. Virus strains: A/PR/8/34 (A/H1N1), A/HK/7/87 (A/H3N2), and B/HK/5/72. The EC50
represents the 50% effective concentration, or compound concentration producing 50% inhibition of virus replication, as determined by microscopic
c
scoring of the CPE, or by the MTS cell viability test. MCC: minimum cytotoxic concentration or compound concentration producing minimal
d
alterations in cell morphology. CC50: 50% cytotoxic concentration, as determined by the MTS cell viability test. Values shown are the mean SEM
of 2−5 determinations.
Table 2. Activity against a Broader Panel of Influenza A/H1N1 and A/H3N2 Viruses
a
antiviral activity (EC50 in μM)
A/H1N1 subtype
A/H3N2 subtype
A/Ishikawa/7/82
b
compd
A/PR/8/34
A/Ned/378/05 A/FM/1/47
A/HK/7/87
A/X-31
>100
cytotoxicity (MCC in μM)
7b
1.2 0.5
0.70 0.39
5.0 2.5
53 11
4.7 1.3
23
7.0 0.0
43 12
9.3 0.3
5.1 2.4
1.9 1.1
>100
>100
>100
>100
100
19b
26
3
>100
>100
≥100
100
8.7 0.3
2.0 0.0
0.50 0.22
1.5 0.8
1.7 0.5
7.6 1.0
>100
>100
amantadine
3.4 1.7
0.17 0.08
11
8
61 12
7.0 1.7
0.11 0.05
0.16 0.05
8.8 0.2
500
rimantadine
oseltamivir carboxylate
zanamivir
63 18
0.45 0.15
3.0 1.0
8.7 3.8
8.8 0.3
≥100
>100
>200
≥100
21
9
16
5.0 1.6
10
2
29
5
6.0 1.3
8.7 0.7
1.2 0.9
8.6 0.4
ribavirin
1
a
The EC50 represents the compound concentration producing 50% inhibition of virus replication, as determined by microscopic scoring of the CPE.
MCC: minimum cytotoxic concentration or compound concentration producing minimal alterations in cell morphology. See ref 24 for a
b
description of the virus strains. Values shown are the mean SEM of 2−5 determinations.
virus resistant to both drugs.25−29 As most of the currently
circulating strains of influenza A virus of the A/H3N2 or A/
H1N1 subtype 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.7,30 The influenza A/PuertoRico/8/34 strain
that we used has an M2 protein carrying two substitutions
associated with amantadine resistance (i.e., S31N and V27T),
and this strain was particularly sensitive to our new amines.
From the results in Table 2, it appears that the target of these
new amantadine analogues is not the M2 protein, as the
compounds displayed antiviral activity against all of the studied
A/H1N1 strains, regardless of whether they carried a wt,
amantadine-sensitive, or a mutated, amantadine-resistant M2
protein. Moreover, the new derivatives are inactive against all
A/H3N2 strains studied, also regardless of carrying a wt or a
mutated M2 protein.
To sum up, we have synthesized, fully characterized, and
evaluated a series of novel 2,2-disubstituted amantadine
analogues. Six compounds (7b, 18, 19a, 19b, 19d, and 26)
were endowed with EC50 values up to 1 order of magnitude
lower than that of amantadine, while not being cytotoxic. Three
of the new amines displayed low micromolar activities against
several A/H1N1 strains, including the amantadine-resistant A/
PR/8/34 strain, while being inactive against A/H3N2 strains.
The mechanism of action of these compounds is currently
under study.
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ACS Medicinal Chemistry Letters
Letter
(9) Nguyen, H. T.; Fry, A. M.; Gubareva, L. V. Neuraminidase
inhibitor resistance in influenza viruses and laboratory testing
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ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures for the synthesis and characterization
of novel compounds, a structural figure with probability
ellipsoids and the CIF file from the X-ray of compound 14,
antiviral assays in influenza virus-infected MDCK cells, and
cytotoxicity assays. This material is available free of charge via
the Internet at http://pubs.acs.org.
(10) Duque, M. D.; Torres, E.; Valverde, E.; Barniol, M.; Guardiola,
S.; Rey, M.; Vaz
́
quez, S. Inhibitors of the M2 channel of influenza A
virus. In Recent Advances in Pharmaceutical Sciences; Munoz-Torrero,
D., Ed.; Transworld Research Network: Kerala (India), 2011; Chapter
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̃
(11) Stoelting, D. T.; Shiner, V. J., Jr. Solvolysis of 1-(3-
Noradamantyl)ethyl Sulfonates. J. Am. Chem. Soc. 1993, 115, 1695−
1705.
AUTHOR INFORMATION
Corresponding Author
*Tel: +32 16337345. Fax: +32 16337340. E-mail: lieve.
naesens@rega.kuleuven.be (L.N.). Tel: +34 934024533. Fax:
+34 934035941. E-mail: svazquez@ub.edu (S.V.).
■
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́
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Author Contributions
The manuscript was written through contributions of all
authors.
Funding
This work was funded by the Spanish Ministerio de Ciencia e
́
Innovacion (FPU Fellowship to E.T., Grant CTQ2011-22433
to S.V.), the Fonds voor Wetenschappelijk Onderzoek
Vlaanderen (FWO No. 9.0188.07 to L.N.), and the
Geconcerteerde Onderzoeksacties (GOA/10/014 to L.N.).
(17) Kovalskaya, S. S.; Kozlov, N. G.; Dikusar, E. A. Transformations
of 2-(phenylethynyl)isocamphanol under acid catalysis. Russ. J. Org.
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Notes
(18) Moss, R. A.; Sauers, R. R.; Sheridan, R. S.; Tian, J.; Zuev, P. S.
Carbon tunneling in the ring expansion of noradamantylchlorocar-
bene. J. Am. Chem. Soc. 2004, 126, 10196−10197.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
E.V. and L.N. acknowledge the technical assistance from L.
Persoons and W. van Dam.
(19) Jirgensons, A.; Kauss, V.; Kalvinsh, I.; Gold, M. R. A practical
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ABBREVIATIONS
■
CPE, cytopathic effect; MDCK, Madin−Darby canine kidney;
MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphen-
yl)-2-(4-sulfophenyl)-2H-tetrazolium; wt, wild-type
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graphic data for this paper. These data can be obtained free of from the
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif.
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