Azapropellanes with anti-influenza a virus activity

Article abstract of DOI:10.1021/ml500108s

The synthesis of several [4,4,3], [4,3,3], and [3,3,3]azapropellanes is reported. Several of the novel amines displayed low-micromolar activities against an amantadine-resistant H1N1 strain, but they did not show activity against an amantadine-sensitive H3N2 strain. None of the tested compounds inhibit the influenza A/M2 proton channel function. Most of the compounds did not show cytotoxicity for MDCK cells.

Full text of DOI:10.1021/ml500108s

Letter
pubs.acs.org/acsmedchemlett
Azapropellanes with Anti-Influenza A Virus Activity
Eva Torres,† Rosana Leiva,† Sabrina Gazzarrini,‡ Matías Rey-Carrizo,† Marta Frigole-Vivas,†
́
Anna Moroni,‡ Lieve Naesens,§ and Santiago Vazquez*,†
́
†Laboratori de Química Farmaceu
Universitat de Barcelona, Av. Diagonal 643, Barcelona E-08028, Spain
̀
tica (Unitat Associada al CSIC), Facultat de Farmac
̀
ia, and Institute of Biomedicine (IBUB),
‡Department of Biosciences and National Research Council (CNR) Biophysics Institute (IBF), University of Milan, Via Celoria 26,
20133 Milan, Italy
§Rega Institute for Medical Research, KU Leuven, 3000 Leuven, Belgium
S
* Supporting Information
ABSTRACT: The synthesis of several [4,4,3], [4,3,3], and [3,3,3]azapropellanes is reported. Several
of the novel amines displayed low-micromolar activities against an amantadine-resistant H1N1 strain,
but they did not show activity against an amantadine-sensitive H3N2 strain. None of the tested
compounds inhibit the influenza A/M2 proton channel function. Most of the compounds did not
show cytotoxicity for MDCK cells.
KEYWORDS: Amantadine, influenza, M2 channel, hemagglutinin, propellane
uman influenza A virus, a member of the Orthomyxovir-
idae, causes significant morbidity and mortality, partic-
ularly in infants, elderly people, and those suffering from
previous pathology or immunodeficiency.1 Its capacity to cause
widespread epidemics is related to its fast droplet transmission
and ability to escape from existing immunity.
Scheme 1. Simplification of Diene 1 to Tetracyclo 2 and to
[4,4,3]-, [4,3,3]-, and [3,3,3]-Azapropellanes of General
Structures I, II, and III
H
The life cycle of influenza viruses is well documented and
most viral proteins are regarded as potential therapeutic
targets.2−6 However, presently available drugs for the treatment
of influenza virus infections only comprise the M2 ion channel
blockers amantadine (Amt) and rimantadine (Chart 1),7,8 and
the neuraminidase inhibitors oseltamivir, zanamivir, peramivir,
and laninamivir.5 Taking into account that most of the
currently circulating influenza strains are resistant to the M2
ion channel blockers and as resistance to the neuraminidase
inhibitors (in particular oseltamivir) is also rising,9,10 novel anti-
influenza virus drugs, preferably with a novel mechanism of
action, are urgently needed.
The influenza virus enters its target cells by receptor-
mediated endocytosis, which is followed by acid-induced fusion
of the viral and endosomal membranes. This fusion event is
mediated by a conformational change of the influenza
hemagglutinin (HA) protein, triggered by the low pH in the
endosomal lumen.11 The endosomal acidic pH also activates
the membrane-spanning A/M2 protein, which acts as a proton
channel to conduct protons into the virion interior. The
decrease in intravirion pH results in uncoating of the viral
ribonucleoprotein, which is transferred, through the fusion
pore, to the cytoplasm.12
Since this endosomal escape of the virus critically depends on
the activity of the M2 proton channel and the HA protein,
dually acting agents which combine blockade of the M2
channel with an inhibitory effect on HA refolding, appear highly
attractive. This dual approach could also, at least in theory,
increase the barrier for selecting Amt resistance.13
Optimized Amt analogues might be able to exert this dual
pharmacological effect, provided that both the M2 and HA
inhibition occur at similar and clinically relevant compound
Chart 1. Structures of Amantadine and Rimantadine
Received: March 13, 2014
Accepted: May 2, 2014
Published: May 2, 2014
© 2014 American Chemical Society
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a
Scheme 2. Synthesis of Novel [4,4,3]- and [4,3,3]-Azapropellanes
a
Reagents and conditions: (i) NaAlH2(OCH2CH2OCH3)2, toluene, reflux, overnight, 46% yield for 4; 71% for 7; 40% yield for 11; 82% yield for 13;
74% yield for 18. (ii) H2, Pd/C, methanol, 1 atm, rt, 93% yield. (iii) 5 N NaOH, reflux, overnight, then conc HCl, 81% yield. (iv) urea, 220 °C, 30
min, 80% yield. (v) 1H-pyrazole-1-carboxamidine hydrochloride, triethylamine, acetonitrile, reflux, 6 h, 49% yield. (vi) LiHMDS, anhydrous THF,
−78 °C. (vii) 3-Chloro-2-(chloromethyl)-1-propene, anhydrous THF, −78 °C to room temperature, overnight, 70% overall yield. (viii) CAN, H2O,
acetonitrile, rt, 2 h. (ix) ethanol, aq NH4OH, reflux, 2 h, 70% overall yield.
concentrations. For comparison, the M2 blocking effect of Amt
is achieved at micromolar concentrations, whereas its effect on
HA refolding requires much higher concentrations (i.e., in the
range of 100 μM). These high Amt concentrations cannot be
achieved in vivo with the standard Amt drug regimens, since the
reported plasma concentration values for Amt are in the range
of 2 μM.14 Hence, to potentially exploit the effect of Amt
analogues on HA refolding, novel derivatives with a more
potent activity (i.e., IC50 values in the micromolar range) would
be required.
It is well-known in medicinal chemistry that for drugs
containing cyclic systems, it is generally worth synthesizing
analogues where the ring is opened, expanded or contracted by
one unit. These analogues show similar activity to the parent
compound and sometimes display improved pharmacokinetic
profile. Also, it is of interest to simplify a polycyclic active
compound by removing some of the rings.15 Previously we
synthesized and evaluated a series of ring-contracted, ring-
expanded, and ring-rearranged analogues of Amt and
rimantadine. Although we found several compounds with
very interesting activities as M2 channel blockers or modifiers
of the HA refolding, no dual agents were found.16−20
expected, amine 1 was active in a cytopathic effect (CPE) assay
(EC50 = 14 μM) against the Amt-sensitive A/HK/7/87 strain,
an H3N2 virus with a wt-type A/M2 channel. Interestingly,
amine 1 was also active (EC50 = 16 μM) against the A/PR/8/
34 strain, an H1N1 virus carrying two characteristic Amt
resistance mutations in its M2 protein (S31N and V27T). A
detailed study of the mechanism of action of this and several
related analogues suggested that their target was the H1 type
HA, through an effect on its refolding.19 Thus, this compound
may be considered a lead for dually acting compounds. Of note,
we have also reported that a simplified analogue of diene 1, 3-
azatetracyclo[5.2.1.15,8.01,5]undecane, 2, displayed a better
inhibition of the wt A/M2 channel (IC50 = 11.7 μM) than 1
(IC50 = 33.5 μM) and was slightly more potent in the CPE
assay (EC50 = 14.0 μM for 1; EC50 = 7.9 μM for 2) against the
Amt-sensitive A/HK/7/87 strain. In sharp contrast with diene
1, which was also active against the A/PR/8/34 strain,
azaundecane 2 was inactive against this Amt-resistant strain
(Scheme 1).20
In the present work, we report on the synthesis and antiviral
activity of further ring-simplified analogues of 1. We found that
several of these novel polycyclic amines, that have considerable
higher conformational freedom than 1 and 2, display low
micromolar activity against the Amt-resistant A/PR/8/34 strain
(A/H1N1). However, this activity was markedly subtype-
Recently, we have reported that diene 1 displayed similar
IC50 values for wild-type (wt) A/M2 to Amt, although it was
inactive against the Amt-resistant S31N form of A/M2.17 As
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a
Scheme 3. Synthesis of Novel [3,3,3]Azapropellanes
a
Reagents and conditions: (i) NaAlH2(OCH2CH2OCH3)2, toluene, reflux, overnight, 75% yield for 20; 80% yield for 24; 93% yield for 31. (ii) H2,
Pd/C, methanol, 1 atm, rt, 90% yield for 21; 95% yield for 26; 87% yield for 27. (iii) 1H-Pyrazole-1-carboxamidine hydrochloride, triethylamine,
acetonitrile, reflux, 6 h, 18% yield for 22; 75% yield for 25. (iv) LiHMDS, anhydrous THF, −78 °C. (v) 1,3-Dichloropropane, anhydrous THF, −78
°C to room temperature, overnight, 72% overall yield. (vi) CAN, H2O, acetonitrile, rt, 2 h. (vii) ethanol, aq NH4OH, reflux, 2 h, 83% overall yield.
dependent, as none of the compounds were active against the
Amt-sensitive influenza A/HK/7/87 strain, an H3N2 virus.
Seven compounds, including the most potent amine, 24, were
evaluated as M2 channel blockers, but were unable to inhibit
the activity of the wt and the V27A mutant, thus suggesting that
they interact with a different target in the virus.
We envisaged that the simplification of the hexacyclic amine
1 may lead to three different propellane scaffolds, I, II, and III
(Scheme 1). Propellanes are defined as tricyclic compounds in
which the three rings have a common carbon−carbon bond.21
Of note, the use of propellane scaffolds in the field of medicinal
chemistry has been rather limited,22 and, in fact, to the best of
our knowledge, the use of [4,3,3] and [3,3,3]azapropellanes is
unprecedented, and only two works have been published in
connection with the [4,4,3]azapropellane scaffold.23,24
The synthesis of azapropellanes related to the general
structure I was straightforward. Thus, reduction of known
imide 3 led to diene 4 in 46% yield.23,24 The catalytic
hydrogenation of 4 led to the [4,4,3]azapropellane 5 in 93%
yield. In a similar fashion, starting from the known imide 6,23
the azapropellane 7 was isolated in 71% yield.24 Finally,
hydrolysis of the known anhydride 8 led to diacid 9 in 81%
yield. Reaction of 9 with melting urea furnished imide 10 that
was subsequently reduced to the diene 11 (Scheme 2).
Three compounds related to the general structure II were
synthesized. Reduction of the known imide 12 furnished 13 in
82% yield. Reaction of 13 with 1H-pyrazole-1-carboxamidine
led to the guanidine 14 in 49% yield. For the synthesis of the
amine 18 featuring a methylidene group we started from the
known N-(p-methoxybenzyl)imide 15. Alkylation of 15 using
lithium hexamethyldisilazane (LiHMDS) and 3-chloro-2-
(chloromethyl)-1-propene furnished imide 16 in 70% yield.
Removal of the protecting group was accomplished with cerium
ammonium nitrate (CAN) in 70% yield. Reduction of the imide
gave propellane 18 in 74% yield (Scheme 2).
Finally, seven compounds containing the [3,3,3]-
azapropellane scaffold were synthesized (Scheme 3). Reduction
of the known imide 19 with sodium bis(2-methoxyethoxy)-
aluminum hydride furnished 20 in 75% yield. The hydro-
genolysis of the benzyl protecting group furnished the
[3,3,3]azapropellane 21 in 90% yield. Finally, reaction of 21
with 1H-pyrazole-1-carboxamidine led to the guanidine 22 in
very low yield. Similarly, reduction of imide 23, previously
synthesized by our group, gave diene 24 in 80% yield. The
guanidine 25 was synthesized following the general procedure
in 75% yield. The catalytic hydrogenation of 24 and 25 gave the
fully saturated compounds 27 and 26, respectively, as mixtures
of stereoisomers, in very high yields.
For the synthesis of 31 we started from the imide 28,
previously synthesized by our group. Reaction of 27 with
LiHMDS and 1,3-dichloropropane gave imide 29 in 72% yield.
Deprotection of the imide with CAN led to 30 in 83% yield,
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ACS Medicinal Chemistry Letters
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a
Table 1. Antiviral Activity in Influenza Virus-Infected MDCK Cells and Inhibitory Effect on A/M2 wt Proton Channel
b
Function
c
antiviral EC50 (μM)
inhibitory effect on A/M2 wt proton channel
d
influenza A/H1N1
influenza A/H3N2
CPE MTS
function
cytotoxicity
e
f
compd
117,19
CPE
16
MTS
Inh by 100 μM for 2 min (%)
IC50 (μM)
MCC (μM)
CC50 (μM)
14
82.5 0.8
93.9 0.2
ND
33.5
11.7
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
16.0
>100
≥59
>100
100
220
>100
38
>100
30
7.9
7.2
4
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
54
>100
>100
>100
>100
>100
>100
>100
>100
100
5
5.9
13
>100
ND
7
30
14
>100
19.1 1.8
ND
11
7
5.6
>100
13
70
56
>100
ND
14
>100
45
>100
43
>100
ND
18
>100
14.9 3.9
47.1 2.3
ND
21
>100
>100
<0.8
>100
2.0
>100
>100
<0.8
>100
1.6
>100
22
>100
24
>100
29.3 2.3
ND
>100
16
>100
20
25
>100
26
>100
8.8 2.2
58.7 2.9
29.9 1.1
91.0 2.1
>100
>100
>100
500
>20
27
24
20
>100
>100
>100
>100
>100
31
>100
53 11
63 18
>100
>100
Amantadine18,20
Rimantadine18
3.4 1.7
0.17 0.08
≥100
a
b
c
MDCK: Madin−Darby canine kidney cells. See text and Supporting Information for details. Compounds 7, 18, 21, 24, 26, 27, and 31 were also
d
tested in the V27A mutant and 21, 24, and 27 also in the S31N mutant. None of them significantly inhibit these channels. Virus strains: A/PR/8/34
(A/H1N1); A/HK/7/87 (A/H3N2). The EC50 represents the 50% effective concentration, or compound concentration producing 50% inhibition
e
of virus replication, as determined by microscopic scoring of the CPE, or by the MTS cell viability test. MCC: minimum cytotoxic concentration, or
f
compound concentration producing minimal 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. ND: not determined.
whose reduction with sodium bis(2-methoxyethyoxy)aluminum
hydride furnished 31 in 93% yield (Scheme 3).
Interestingly, going from diene 4 to its tetramethyl derivative
11 also increased the activity (EC50 = 38 μM for 4 vs EC50 = 7
μM for 11). Unfortunately, several attempts to carry out the
reduction of 11 either with catalytic hydrogenation or diimide
were unsuccessful, likely due to steric hindrance. Second, ring
contraction from the [4,4,3]azapropellane to the [4,3,3]-
azapropellane did not improve the activity (compare 7 vs
13), while the introduction of a methylidene group in the five-
membered ring slightly increased the activity (compare 13 vs
18). Third, the smallest member of this series, [3,3,3]-
azapropellane 21 was inactive as was its monomethylidene
derivative 31. However, introduction of a second methylidene
group, as in 24, led to an active compound (EC50 < 0.8 μM),
nearly 2 orders of magnitude more potent than Amt and
rimantadine. Finally, with the only exception of compound 26,
that was more potent than amine 27, the introduction of a
guanidine group was clearly deleterious for the activity, leading
largely to inactive compounds.
It is well-known that the target of Amt and rimantadine is the
influenza A virus M2 channel protein and that a single S31N
mutation in M2 is sufficient to render the virus resistant to both
drugs.26−28 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.
The influenza A/PR/8/34 strain that we used in our CPE
assay has an M2 protein carrying two substitutions associated
with amantadine resistance (i.e., S31N and V27T). In order to
assess if the target of the novel amines was the M2 protein, the
inhibitory activity of seven compounds (7, 18, 21, 24, 26, 27
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.
Antiviral cell culture assays were performed to determine the
antiviral activity of all 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.
In influenza virus-infected 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 CPE at day 3
post infection were confirmed by a colorimetric cell viability
assay.25 All compounds proved to be inactive against influenza
B virus (results not shown). Only two compounds, guanidines
22 and 25 were cytotoxic for the MDCK cells.
Analysis of the data in Table 1 revealed the following trends.
First, within the series of [4,4,3]azapropellanes, progressive
saturation of the diene 4 to alkene 7 and to the fully saturated 5
increased the antiviral activity against the A/PR/8/34 strain
(EC50 = 38, 30, and 5.9 μM for 4, 7 and 5, respectively). This
trend had been previously seen on going from diene 1 (EC50
=
16 μM) to its fully saturated derivative (EC50 = 2.0 μM).25
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ACS Medicinal Chemistry Letters
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and 31) was tested on A/M2 channels expressed in Xenopus
oocytes using the two-electrode voltage clamps (TEV)
technique. None of them, at 100 μM, was able to significantly
inhibit the activity of the wt, the V27A (Amt-resistant) or the
S31N (Amt-resistant) mutants, indicating that for the
compounds that showed antiviral activity in the CPE assay
the viral target is not the M2 protein.
ABBREVIATIONS
■
Amt, amantadine; Bn, benzyl; CAN, cerium ammonium nitrate;
CPE, cytopathic effect; HA, hemagglutinin; MDCK, Madin−
Darby canine kidney; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium;
PMB, p-methoxybenzyl; wt, wild-type
After combining the results that we previously reported for
diene 1,17,19 those of its simpler analogue 2,20 and those of the
azapropellane disclosed herein, it seems that keeping a
conformationally rigid scaffold is more suitable for targeting
the M2 channel.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Procedures for the synthesis and characterization of novel
compounds. Antiviral and cytotoxicity assays. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
(15) Patrick, G. L. An Introduction to Medicinal Chemistry; Oxford
University Press: Oxford, 2005; pp 204−205 and 626−629.
(16) Camps, P.; Duque, M. D.; Vaz
E.; Sureda, F. S.; Lo
Prathalingam, S. R.; Kelly, J. M.; Romero, V.; Ivorra, D.; Cortes
Synthesis and pharmacological evaluation of several ring-contracted
amantadine analogs. Bioorg. Med. Chem. 2008, 16, 9925−9936.
(17) Duque, M. D.; Ma, C.; Torres, E.; Wang, J.; Naesens, L.; Juarez-
́
Corresponding Author
*Tel: +34 934024533. Fax: +34 934035941. E-mail: svazquez@
ub.edu.
́
quez, S.; Naesens, L.; De Clercq,
pez-Querol, M.; Camins, A.; Pallas, M.;
, D.
́
̀
́
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 Generalitat de Catalunya (FI Fellowship to R.L.),
and the Geconcerteerde Onderzoeksacties (GOA/10/014 to
L.N.). M.R.-C. acknowledges a predoctoral grant from the
Government of Andorra (ATCR2012/2013-00XX-AND).
Jimen
Pinto, L. H.; Vaz
inhibitors of the M2 ion channel of influenza A virus. J. Med. Chem.
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́
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dez, R.; Miquet, S.; Font-
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Notes
Bardia, M.; Naesens, L.; Vaz
́
The authors declare no competing financial interest.
activity of 2,2-dialkylamantadines and related compounds. ACS Med.
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(19) Torres, E.; Duque, M. D.; Vanderlinden, E.; Ma, C.; Pinto, L.
ACKNOWLEDGMENTS
■
L.N. acknowledges the technical assistance from W. van Dam.
́
H.; Camps, P.; Froeyen, M.; Vazquez, S.; Naesens, L. Role of the viral
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ACS Medicinal Chemistry Letters
Letter
hemagglutinin in the anti-influenza virus activity of newly synthesized
polycyclic amine compounds. Antiviral Res. 2013, 99, 281−291.
(20) Rey-Carrizo, M.; Torres, E.; Ma, C.; Barniol-Xicota, M.; Wang,
J.; Wu, Y.; Naesens, L.; DeGrado, W. F.; Lamb, R. A.; Pinto, L. H.;
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