Camphor-based symmetric diimines as inhibitors of influenza virus reproduction

Article abstract of DOI:10.1016/j.bmc.2014.02.038

Influenza is a continuing world-wide public health problem that causes significant morbidity and mortality during seasonal epidemics and sporadic pandemics. The purpose of the study was synthesis and investigation of antiviral activity of camphor-based symmetric diimines and diamines. A set of C2-symmetric nitrogen-containing camphor derivatives have been synthesized. The antiviral activity of these compounds was studied against rimantadine- and amantadine-resistant influenza virus A/California/7/09 (H1N1)pdm09 in MDCK cells. The highest efficacy in virus inhibiting was shown for compounds 2a-e with cage moieties bound by aliphatic linkers. The therapeutic index (selectivity index) for 2b exceeded that for reference compounds amantadine, deitiforin and rimantadine almost 10-fold. As shown by structure-activity analysis, the length of the linker has a dramatic effect on the toxicity of compounds. Compound 2e with -C₁₂H₂₄- linker exhibited the lowest toxicity (CTD₅₀ = 2216 μM). Derivatives of camphor, therefore, can be considered as prospective antiinfluenza compounds active against influenza viruses resistant to adamantane-based drugs.

Full text of DOI:10.1016/j.bmc.2014.02.038

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Camphor-based symmetric diimines as inhibitors of influenza virus
reproduction
Anastasiya S. Sokolova a,b, Olga I. Yarovaya a,b, Dina V. Korchagina a, Vladimir V. Zarubaev c,
,
⇑
Tatiana S. Tretiak c, Pavel M. Anfimov c, Oleg I. Kiselev c, Nariman F. Salakhutdinov a
a Novosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian Academy of Sciences, Lavrentjev Avenue 9, 630090 Novosibirsk, Russia
b Novosibirsk State University, Pirogova St. 2, 630090 Novosibirsk, Russia
c Department of Chemotherapy, Influenza Research Institute, 15/17 Prof. Popova St., 197376 St. Petersburg, Russia
a r t i c l e i n f o
a b s t r a c t
Article history:
Influenza is a continuing world-wide public health problem that causes significant morbidity and mor-
tality during seasonal epidemics and sporadic pandemics. The purpose of the study was synthesis and
investigation of antiviral activity of camphor-based symmetric diimines and diamines. A set of C2-sym-
metric nitrogen-containing camphor derivatives have been synthesized. The antiviral activity of these
compounds was studied against rimantadine- and amantadine-resistant influenza virus A/California/7/
09 (H1N1)pdm09 in MDCK cells. The highest efficacy in virus inhibiting was shown for compounds
2a–e with cage moieties bound by aliphatic linkers. The therapeutic index (selectivity index) for 2b
exceeded that for reference compounds amantadine, deitiforin and rimantadine almost 10-fold. As shown
by structure–activity analysis, the length of the linker has a dramatic effect on the toxicity of compounds.
Received 3 October 2013
Revised 14 February 2014
Accepted 18 February 2014
Available online xxxx
Keywords:
Camphor
Diimine derivatives
Antivirals
Influenza
Compound 2e with –C12H24– linker exhibited the lowest toxicity (CTD50 = 2216 lM). Derivatives of cam-
phor, therefore, can be considered as prospective antiinfluenza compounds active against influenza
viruses resistant to adamantane-based drugs.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
Due to specific genome organization (in particular, segmented
nature and lack of the mechanism of correction of replication er-
Development of new drugs for treatment and prophylaxis of
viral infections is one of the most important directions of modern
pharmacology and medicinal chemistry. Outbreaks of avian influ-
enza H5N1 (1997–2006) followed by pandemic of ‘swine flu’
A(H1N1)pdm09, as well as the recent introduction of avian virus
of H7N9 subtype to the human population make it necessary to re-
vise the problem of searching for and developing novel antivirals.1
As demonstrated by practice-based research, natural com-
pounds are the most promising source for the development of
drugs. Approximately half of novel drugs approved for clinical
use in USA are the naturally derived compounds and their modified
derivatives.2 There are currently few antivirals against influenza
infection, for most of which fast emergence of drug resistance
has been documented.3 For this reason, creation of novel effective
antivirals with the alternative mechanism and broad spectrum of
activity is one of the most important goals in the field of treatment
and prophylaxis of influenza.
rors) and short-term life cycle, influenza virus demonstrates high
genetic variability and high rate of mutations. As a result, its
antigenic structure is highly variable due to selective pressure
of host immune response. In addition, the presence of specific
antivirals also serves as a selective factor resulting in fast emer-
gence of drug-resistant virus strains. Taken together, these
processes lead to the selection of viral variants that are capable
both of escaping the immune inactivation by neutralizing anti-
bodies and of overcoming the suppressive action of antiviral
drugs.
Three chemical classes of compounds are currently used as anti-
influenza drugs. They are targeted against different stages of viral
life cycle in infected cells. Among them, the most commonly used
compounds include neuraminidase inhibitors zanamivir
(RelenzaÒ), oseltamivir (TamifluÒ), peramivir (RapiactaÒ), and
laninamivir (InavirÒ).4 They interfere with the activity of viral
neuraminidase, which plays an essential role in the release of prog-
eny virus particles from the surface of a host cell. Ribavirin and
favipiravir are antivirals of broad range of activity, which exhibit
⇑
Corresponding author. Tel.: +7 812 499 1527; fax: +7 812 499 1500.
E-mail addresses: ooo@nioch.nsc.ru (O.I. Yarovaya), zarubaev@influenza.spb.ru
a
suppressive effect against almost all RNA-genome human
viruses.5 Meanwhile, being a nucleoside analog, ribavirin possesses
(V.V. Zarubaev).
http://dx.doi.org/10.1016/j.bmc.2014.02.038
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Sokolova, A. S.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.02.038

2
A. S. Sokolova et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
numerous side effects, including the reduction of haemoglobin le-
vel, neutropenia, pulmonary edema, etc.6
Adamantane derivatives, which were first discovered as specific
anti-influenza drugs with the direct mechanism of activity are rep-
2. Results and discussion
2.1. Chemistry
resented by two compounds: Rimantadine (a-methyl-1-adaman-
Starting from the known terpene camphor 1, we have pre-
pared new N,N0-bis(isobornyl)diimine 2a–g and diamine 3b,c,e,f,g
and 4b,c using the conventional methods of amine chemistry.
Camphor is an inexpensive natural compound, one of the first
plant metabolites isolated in the chemically pure form. For a long
time, the technology for isolating this compound from camphor
tree wood has been known in China in Japan. The analeptic prop-
erties of camphor have been known since the 18th century.
Today, it is used to treat acute and chronic heart failure, respira-
tory depression, soporific and narcotic poisoning, as well as an
agent for topical use.24 A typical feature of a camphor molecule
is its cage-hydrocarbon (rigid) structure that is similar to a cer-
tain extent to that of adamantane. Having the nearly spherical
structure, adamantane derivatives, amantadine and rimantadine,
exhibit high penetrability through the cell membrane and, hence,
high antiviral activity.
tylamine hydrochloride) and amantadine (1-aminoadamantane).7
These compounds block virally encoded protein M2 that acts as a
proton channel required for acidification of the virion core, and
thus prevent hemagglutinin cleavage and further fusion of mem-
branes of the viral envelope and lysosomal vacuole necessary for
virus decapsidation.8,9
Despite high initial efficacy of adamantanes as anti-influenza
drugs, drug-resistant strains can be easily selected both in vitro
and in patients receiving adamantane-based therapy.10 Adaman-
tane resistance among circulating influenza A viruses increased
rapidly worldwide starting from 2003 to 2004. The percentage of
adamantane-resistant influenza A virus isolates increased from
0.4% in 1994–1995 to 92% in 2005–2006 influenza season.11 The
resistance is mainly conferred by amino acid substitutions in M2
protein L26F, V27A, S31N and G34E.12
Being widely used cage structures, adamantanes were used for
the rational design of a large class of antiviral compounds.13 In par-
ticular, attempts were undertaken to overcome the adamantane-
resistance of influenza virus. Among them, aminospiroadaman-
tanes, although inactive against the mutant S31N, were revealed
to be a submicromolar inhibitor of the clinically important mutant
Aliphatic diimines (Schiff bases) 2a–e were obtained according
to the procedure in Ref. 25 using azeotropic distillation of water
and purified by column chromatography. Diimines 2f,g were ob-
tained by the method described previously26 using tetraethylxysi-
lane as a dehydrating agent. In the latter case, we isolated
iminoamines 5f,g along with C2-symmetric camphor diimine. Ali-
phatic diimines 2b,c,e are reduced in a regio- and stereo selective
manner,27 yielding symmetric exo-, exo-diamines 3b,c,e. N-Methyl-
ation with dimethyl sulfate of amines 3b,c give tertiary amines
4b,c.
The reduction of diimines 2f,g by NaBH4 in the presence of NiCl2
yielded the corresponding aromatic secondary amines 3f,g. How-
ever, the degree of conversion in this case is low; the yield of dia-
mines was 34–46%. The attempts to select conditions for reducing
compounds 2f,g were unsuccessful. Many well-established meth-
ods failed to give the clean reaction, including the use of complex
metal hydrides (i.e. NaBH4, NaBH3CN, NaBH(OAc)3, LiAlH4,
Zn(BH4)2) in a number of solvents. The stability of compounds
2f,g against reduction can probably be attributed to their rigid aro-
matic structure and the presence of conjugation in the molecule
(Scheme 1).
V27A (IC50 = 0.31
mutant L26F (IC50 = 5.6
l
M) and also showed to be active against the
l
M).14
Bananins, which have been shown to possess an inhibitory
activity against SARS coronavirus, are the closest compounds to
classical adamantanes.15 More widely, cage structures appeared
promising core structures for the development of novel antivirals.
Among them, Deitiforin (2-(10-aminoethyl)bicyclo[2.2.1]heptane)
is one of the most interesting drugs based on natural cage com-
pounds, bornanes (Fig. 1).16 In this regard, it is important to note
that this compound is in fact of natural origin and that borneol
and isoborneol have been widely known in folk medicine as anti-
inflammatory compounds.17
Natural compounds, including monoterpenoids, are a promis-
ing source of new antiviral compounds.18,19 Oxygen-containing
monoterpenoid with a para-menthane framework showed a sig-
nificant activity against the influenza virus,20 nitrogen contain-
ing compounds based on the hydrochlorides of 1- and
2-adamantylamines and monoterpene aldehydes demonstrated
antiviral activity against the influenza virus higher than that
of rimantadine and amantadine.21 Meanwhile, it is known that
Schiff bases are among the most important groups of biomole-
cules. These compounds have been found to reveal both remark-
The endo position of H1 proton in compounds 3b,c,e,f,g is sup-
ported by the values of the constants of spin–spin coupling be-
tween it and protons H2n and H2k (8.8 Hz and 7.5 Hz,
respectively). In case of the exo position of H1 atom, its constant
of spin–spin coupling with H2k proton would be equal to ꢀ12–
13 Hz. The absence of long-range spin–spin proton coupling be-
tween protons H5k and H1 also argues in favor of the fact that
the H1 proton occupies the exo position. Compounds 2b–g,
3b,c,e,f,g, 4b,c and 5f,g are new and have not been previously de-
scribed in the literature. Symmetric camphor diimine and diamine
based on commercially available (1R)-camphor with ethane-1,2-
diamine and propane-1,3-diamine were previously used as a chiral
bidentate ligands.28
able biological activities and
a variety of valuable practical
applications.22,23 The aim of this work was to synthesize new
nitrogen containing compounds, including Schiff bases and
amines, based on camphor and to study their antiviral activity
against rimantadine-resistant influenza virus A/California/07/09
(H1N1)pdm09.
2.2. Study of biological activity. Antiviral activity
The obtained compounds 2a–g, 3b,c,g, 4b,c and 5f,g have been
studied as potential antiviral agents. Adamantane- and bornane-
based derivatives were used as reference compounds due to their
close similarity to the compounds under investigation in having ri-
gid cage fragments in their structures.
NH2 HCl
NH2
It should be noted that the initial camphor molecule does not
possess a virus-inhibiting activity. Meanwhile, the study revealed
a high inhibiting effect of symmetric compounds with aliphatic
linkers between two camphor residues (compounds 2a–e) against
NH2
Deitiforin
HCl
Amantadine
Rimantadine
Figure 1. Anti-influenza drugs.
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A. S. Sokolova et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
3
6
9
2
4
5
2`
a
b
c
8
7
1`
1
N
N
H
N
N
H
6`
R
O
R
10
1
3 b,c,e
2 a-e
d
N
N
R
14
14`
4 b,c
16
12
15
16`
11`
15`
11
14
+
NH2
X
N
X
N
N
13
5 f,g
2 f,g
c
R- a) -(CH2)3-, b) -(CH2)6-,
c) -(CH2)7-, d) -(CH2)8-, e) -(CH2)12-,
-X- f) -CH2-, g) -O-
X
N
N
H
H
3 f,g
Scheme 1. Reagents and conditions: (a) NH2-R-NH2 (0.50 equiv), BF3ꢁEt2O (1–5 mol %), PhMe, reflux (Dean–Stark), 12–48 h; (b) NH2-Ph-X-Ph-NH2 (0.50 equiv), SiOEt4
(1 equiv), H2SO4 (5 mol %) reflux; (c) NiCl2 (4 equiv), NaBH4 (20 equiv), MeOH, –30 °C to rt; (d) (CH3O)2SO2 (10 equiv), K2CO3 (1.0 equiv), MeOH, rt, 12 h.
influenza virus A/California/07/09 (H1N1)pdm09, mostly due to a
decrease in toxicity. Compounds 2a,b,c,e were shown to possess
the highest antiviral activity (Table 1). Diimine 2b has a selectivity
index exceeding that for reference compounds 9-fold and more.
During the course of the study, three types of modifications
were applied to these compounds. As shown by structure–activity
analysis, compounds bearing an amino group lost their efficacy in
suppression of virus replication. With few exceptions, these com-
pounds had much higher toxicity (low CTD50 values), resulting in
lowering of their SIs. Indeed, ED50 values for diamines 3b and 3c
are 0.77 and 3.2 lM, respectively, the values that could be recog-
nized as a high antiviral activity. Nevertheless, their toxicity ap-
peared to be high and, as a consequence, SI’s are low. Second, the
introduction of aromatic moieties into linker also resulted in lack
of virus suppression. Indeed, symmetrical diimines 2f,g connected
by aromatic linkers, amino-imines 5f,g and diamine 3g did not ex-
hibit high antiviral activity. The combination of these two modifi-
cations did not restore the antiviral activity (compounds 3f,g).
Based on the results of the study, two issues should be dis-
cussed. First, among the compounds 2a–2e, with one exception
(2e), the strong dependence of anti-viral activity of the linker
length can be observed. One can note that optimal number of car-
bon atoms in the linker is 6 (2b). The activity of compounds de-
creased when this number both increased (2c, 2d) and decreased
(2a). This led to speculation that there are two sites of interaction
of terminal camphor moieties with viral target, and that they dem-
onstrate optimal activity when connected with the spacer of
appropriate length.
Second, it should be noted that the described experiments were
conducted using rimantadine-resistant strain of influenza virus.
The resistance is due to amino acid substitution S31N (GenBank
accession number AGI54797.1) typical of resistant strains. There-
fore, despite structure similarity of substances under investigation
with the known anti-influenza drugs amantadine and rimantadine,
as well as with other M2 cage inhibitors, the mechanism of activity
of symmetric camphor-based diimines should be different. Ini-
tially, adamantane derivatives were shown to interact with amino
acids within the M2 proton channel.30 Recently, another adaman-
tane-binding site was discovered in M2.31,32 The mutations confer-
ring amantadine- and rimantadine-resistance are located in
transmembrane domain of M2 facing inside the channel. S31N is
Table 1
Antiviral activity of camphor-based compounds 1–5 against influenza virus A/
California/7/09 (H1N1)pdm09 in MDCK cells
a
b
Compound
CTD50
,
lM
ED50
,
lM
SIc
1
2a
2b
2c
2d
2e
2f
2g
3289.5 216.0
283.6 25.4
1346.6 126.1
1256.3 114.2
390.1 35.9
2216.2 214.8
>1072.9
>1067.0
5.4 0.4
10.4 1.0
1644.7 144.4
9.3 0.8
2
30
89
55
16
49
3
3
7
3
15.1 1.1
22.8 2.3
24.2 2.1
45.0 3.9
351.4 32.3
373.4 35.1
0.77 0.1
3.2 0.2
3b
3c
3e
14.4 1.5
4.4 0.3
3
3f
3g
4b
4c
5f
5g
>638.2
>635.5
110.3 9.1
697.6 72.2
30.1 2.9
153.2 11.9
207.6 19.5
110.1 10.4
40.4 3.8
15.0 1.2
4.2 0.0
4
3
1
17
2
32.9 2.7
8
Rimantadine
Amantadine
Deitiforin
Ribavirind
335.2 26.8
284.1 21.4
1266.2 81.5
>2000.0
67.0 4.9
64.2 4.7
208.6 15.4
24.6
5
4
6
>81.0
a
CTD50—cytotoxic concentration; the concentration resulting in death of 50% of
cells.
b
ED50—50% effective concentration; the concentration leading to 50% inhibition
of virus replication.
c
SI—selectivity index, ratio CTD50/ED50
Data are taken from Ref. 29.
.
d
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4
A. S. Sokolova et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
the most widely distributed mutation among drug-resistant
strains. Due to spatial limitations, few modifications and side
groups can be introduced into adamantane derivative without
lacking its ability to fit into proton channel. Interaction with exter-
nal amino acids suggests more wide range of chemical modifica-
tions resulting in high virus-inhibiting activity.
(selectivity index) for 2b exceeded that for reference compounds
amantadine, deitiforin and rimantadine almost 10-fold. As shown
by structure–activity analysis, the length of the linker has a
dramatic effect on the toxicity of compounds. Compound 2e
with
–C12H24
–
linker
exhibited
the
lowest
toxicity
(CTD50 = 2216
lM).
In order to address these issues, we performed a computer sim-
ulation of interaction of the most active compound 2b with M2
proton channel of influenza virus (Fig. 2).
According to the model, 2b binds to two adjacent sites in M2 in
the area of amino acids 44–55. Each camphor moiety is coordi-
nated with charged (Asp44 and Arg45) and aromatic (Phe48,
Tyr52 and Phe55) amino acids. No hydrogen bonds have been
established between 2b and M2. Their interaction can be provided
by hydrophobic contacts with Phe48, Ile51 and Phe55.
The results obtained suggest that structure containing two
imine groups linked by the aliphatic chain is the key element re-
quired for antiviral activity of the studied compounds. This can
be illustrated by comparing of three compounds, diimines 2b,c,e
and their derivatives diamines 3b,c,e. Despite low effective con-
centrations of 3b,c,e their toxicity is much higher than that for 2.
This results in lower SI values. In summary, symmetric diimines
2a–e should be considered as a highly promising class of com-
pounds effective against influenza.
It should be mentioned that despite docking experiment de-
scribes the interaction of 2b with specific amino acids of M2, in a
framework of our study no direct evidence of such binding was ob-
tained. For this reason, direct experiments should be performed to
confirm this hypothesis using biophysical (conductance or binding
testing) or virological (selection of resistant mutants and analysis
of amino acid changes) methods.
As follows from the results obtained, symmetric dimeric deriv-
atives of camphor with linker of specific length are more active
than those with different spacer. This might be due to existence
of two binding sites in the viral target. Combination of two cam-
phor moieties in one molecule might increase the strength of bind-
ing of the compound with target by using two binding sites instead
of one. Regarding the M2 protein, this is illustrated on the model
presented, where two camphor moieties bind to two adjacent sites
(amino acids 44–55) of neighboring polypeptide chains of M2. One
can suppose, for instance, that such type of binding could restrict
the ability of cytoplasmic domain of M2 to open the proton chan-
nel, or stabilizes its closed state. Nevertheless, once again, direct
M2-inhibiting activity of 2b should be further demonstrated by
specific tests.
4. Experimental section
4.1. General chemical methods
Reagents and solvents were purchased from commercial suppli-
ers and used as received. Dry solvents were obtained according to
the standard procedures. GC: 7820A gas chromatograph (Agilent
Tech., USA); flame-ionization detector; HP-5 capillary column, He
as carrier gas (flow rate 2 ml/min, flow division 99:1). Optical rota-
tion: polAAr 3005 spectrometer; CHCl3 soln. 1H and 13C NMR spec-
tra: Bruker DRX-500 apparatus at 500.13 MHz (1H) and
125.76 MHz (13C) in CDCl3; chemical shifts d in ppm relative to
residual CHCl3 [d(H) 7.24, d(C) 76.90 ppm], J in Hz. The structure
of the products was determined by analyzing the 1H and 13C
NMR spectra, assignments on a routine basis by a combination of
1D and 2D experiments (COSY, COLOC, HSCQ, HMBC). HR-MS:
DFS Thermo Scientific spectrometer in
a full scan mode
(15–500 m/z, 70 eV electron impact ionization, direct sample
administration). Spectral and analytical studies were carried out
at the Collective Chemical Service Center of the Siberian Branch
of the Russian Academy of Sciences. Column chromatography
(CC) was performed on silica gel (60–200 l, Macherey-Nagel). The
purity of the target compounds was determined by gas chromatog-
raphy methods. All the target compounds reported in this paper
have a purity of at least 95%.
3. Conclusion
In the present study, we have synthesized a set of C2-sym-
metric compounds with two camphor moieties (1,7,7-trim-
ethylbicyclo[2.2.1]heptan) and two imine (amine) groups bound
by linkers of various size and rigidity. The antiviral activity of
these compounds was studied against influenza virus A/Califor-
nia/7/09 (H1N1)pdm09 in MDCK cells. The highest efficacy in
virus inhibiting was shown for compounds 2a–e with cage
moieties bound by aliphatic linkers. The therapeutic index
The following reagents were used in this study: (1R)-(+)
camphor (Alfa Aesar 98%, ½a D25
ꢂ
45.5 (CHCl3, c 0.8)), ee 99, pro-
pane-1,3-diamine (Aldrich 98%), hexane-1,6-diamine (Aldrich
98%), heptane-1,7-diamine (Acros 98%), octane-1,8-diamine (Acros
98%), dodecane-1,12-diamine (ABCR 98%), 4,40-methylenedianiline
(ABCR 97%) 4,40-oxydianiline (ABCR 97%).
Figure 2. Model of interaction of 2b (marked in magenta) with M2 proton channel of influenza virus. (A) General view (outer domain of M2 is on the top and labeled blue), (B)
close view from the inner side of M2.
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A. S. Sokolova et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5
0
The enumeration of atoms in compounds is given in order to as-
sign the signals in the NMR spectra and does not coincide with the
enumeration of atoms in the IUPAC name (see Supplementary).
The specific rotation is given in (deg ml)⁄(g dm)ꢃ1; concentrations
2J 16.8 Hz), 1.79 ddddd (H4exo, H4 exo
,
2J 12.2, J4exo,5exo 12.2,
0
J4exo,5endo 4.3, J4exo,3 4.3, J4exo,2exo 3.2 Hz), 1.87 dd (H3, H3 , J3,4exo
0
4.3, J3,2exo 4.3 Hz), 2.28 ddd (H2exo, H2 exo, 2J 16.8, J2exo,3 4.3, J2exo,4exo
0
3.2 Hz), 3.12 dt (H11a, H11a
,
2J 12.1, J11a,12 7.3 Hz), 3.17 dt (H11b
,
0
0
of solutions, in (g) (100 ml)ꢃ1
.
H11 b
,
2J 12.1, J11b,12 7.3 Hz). 13C NMR (d, ppm): 180.97 s (C1, C1 ),
0
0
0
0
35.21 t (C2, C2 ), 43.73 d (C3, C3 ), 27.38 t (C4, C4 ), 32.10 t (C5, C5 ),
0
0
0
4.2. General synthetic procedure for diimin 2a–e (method a)
53.23 s (C6, C6 ), 46.67 s (C7, C7 ), 18.84 q (C8, C8 ), 19.40 q (C9,
0
0
0
0
C9 ), 11.32 q (C10, C10 ), 52.26 t (C11, C11 ), 30.39 t (C12, C12 ), 27.40
0
To a solution of (1R)-(+)-camphor 1 (3 g, 19.7 mmol, 1.0 equiv)
and diamine (9.8 mmol, 0.5 equiv) in toluene (60 ml) 0.15 ml
BF3ꢁEt2O in 5 ml toluene were added. The solution was heated at
reflux with a Deane-Stark trap condenser until no further water ap-
peared. The combined organic layers were washed two times with
brine, dried (Na2SO4) and evaporated to dryness. The reaction solu-
tion was then concentrated and crude product purified by flash sil-
ica gel column chromatography (hexane-ethyl acetate eluent) to
obtain the desired diimine 2a–e.
t (C13, C13 ), 29.28 t (C14). ½a D25
ꢂ
20.7 (CHCl3, c = 0.66). HRMS: calcd
for C27H46N2: 398.3653, found: 398.3656.
4.2.4. N1E,N8E-N1,N8-bis((1R,2R,4R)-1,7,7-
trimethylbicyclo[2.2.1]heptan-2-ylidene)octane-1,8-diamine
(2d)
Yield: 61%. Colourless oil; 1H NMR (d, ppm, J/Hz): 0.69 s (C9H3,
0
0
0
C9 H3), 0.86 s (C8H3, C8 H3), 0.91 s (C10H3, C10 H3), 1.13 ddd (H4endo
,
,
0
H4 endo, 2J 12.3, J4endo,5endo 9.3, J4endo,5exo 4.2 Hz), 1.19–1.30 m (2H13
0
0
0
2H13 , 2H14, 2H14 ), 1.29 ddd (H5endo, H5 endo
,
2J 13.0, J5endo,4endo 9.3,
0
0
4.2.1. N1E,N3E-N1,N3-bis((1R,2R,4R)-1,7,7-
trimethylbicyclo[2.2.1]heptan-2-ylidene)propane-1,3-diamine
(2a)
J5endo,4exo 4.4 Hz), 1.47–1.56 m (2H12, 2H12 ), 1.59 ddd (H5exo, H5 exo
0
2J 13.0, J5exo,4exo 12.2, J5exo,4endo 4.2 Hz), 1.77 d (H2endo, H2 endo
,
2J
0
0
16.7 Hz), 1.74-1.82 m (H4exo, H4 exo), 1.86 dd (H3, H3 , J3,2exo 4.4,
0
Yield: 74%. Colourless oil; 1H NMR (d, ppm, J/Hz): 0.68 s (C9H3,
J3,4exo 4.4 Hz), 2.27 ddd (H2exo, H2 exo
,
2J 16.7, J2exo,3 4.4, J2exo,4exo
0
0
0
0
C9 H3), 0.86 s (C8H3, C8 H3), 0.90 s (C10H3, C10 H3), 1.12 ddd (H4endo
,
,
,
,
3.2 Hz), 3.12 dt (H11a, H11 a
,
2J 12.1, J11a,12 7.3 Hz), 3.16 dt (H11b
,
0
0
0
H4 endo
,
2J 12.3, J4endo,5endo 9.3, J4endo 5exo
,
4.2 Hz), 1.28 ddd (H5endo
H11 b
,
2J 12.1, J11b,12 7.3 Hz). 13C NMR (d, ppm): 181.00 s (C1, C1 ),
0
0
0
0
0
H5 endo,2J 13.0, J5endo,4endo 9.3, J5endo,4exo 4.4 Hz), 1.59 ddd (H5exo
35.19 t (C2, C2 ), 43.71 d (C3, C3 ), 27.35 t (C4, C4 ), 32.07 t (C5, C5 ),
0
0
0
0
H5 exo 2J 13.0, J5exo,4exo 12.2, J5exo,4endo 4.2 Hz),), 1.77 d (H2endo
53.23 s (C6, C6 ), 46.66 s (C7, C7 ), 19.38 q (C8, C8 ), 18.81 q (C9,
0 0 0 0
0
0
H2 endo
,
2J 16.9 Hz), 1.73–1.83 m (H4exo, H4 exo, 2H12), 1.86 dd (H3,
C9 ), 11.29 q (C10, C10 ), 52.22 t (C11, C11 ), 30.37 t (C12, C12 ), 27.33
0
0
0
0
H3 J3,2exo = J3,4exo = 4.4 Hz), 2.27 ddd (H2exo, H2 exo
,
2J 16.9, J2exo,3
t (C13, C13 ), 29.32 t (C14, C14 ). ½a D25
ꢃ32.0 (CHCl3, c = 0.92). HRMS:
ꢂ
0
4.4, J2exo,4exo 3.3 Hz), 3.17 dt (H11a, H11 a
,
2J 12.2, J11a,12 7.3 Hz),
calcd for C28H48N2: 412.3812, found: 412.3813.
0
3.22 dt (H11b, H11 b
,
2J 12.2, J11b,12 7.3 Hz). 13C NMR (d, ppm):
0
0
0
181.43 s (C1, C1 ), 35.18 t (C2, C2 ), 43.71 d (C3, C3 ), 27.34 t (C4,
4.2.5. N1E,N12E-N1,N12-bis((1R,2R,4R)-1,7,7-
trimethylbicyclo[2.2.1]heptan-2-ylidene)dodecane-1,12-
diamine (2e)
0
0
0
0
C4 ), 32.06 t (C5, C5 ), 53.27 s (C6, C6 ), 46.69 s (C7, C7 ), 18.81 q
0
0
0
0
(C8, C8 ), 19.40 q (C9, C9 ), 11.28 q (C10, C10 ), 49.98 t (C11, C11 ),
31.31
(C12).
ꢃ31 (CHCl3, c = 0.72). HRMS: calcd for
t
½
a 2D2
ꢂ
Yield: 49%. Colourless oil; 1H NMR (d, ppm, J/Hz): 0.71 s (C9H3,
0
0
0
C
23H38N2: 342.3030, found: 342.3029. This compound was previ-
C9 H3), 0.88 s (C8H3, C8 H3), 0.92 s (C10H3, C10 H3), 1.15 ddd (H4endo
,
0
ously obtained.33 Spectral date agree with those specified in
literature.
H4 endo
,
2J 12.2, J4endo,
9.3, J4endo,
4.2 Hz), 1.18–1.28 m
5exo
0
05endo
0
0
(2H13, 2H13 , 2H14, 2H14 , 2H15, 2H15 , 2H16, 2H16 ), 1.30 ddd (H5endo
,
0
H5 endo
,
2J 12.8, J5endo,
9.3, J5endo,
4.3 Hz), 1.49–1.57 m
4endo
4exo
0
0
4.2.2. N1E,N6E-N1,N6-bis((1R,2R,4R)-1,7,7-
trimethylbicyclo[2.2.1]heptan-2-ylidene)hexane-1,6-diamine
(2b)
(2H12, 2H12 ), 1.61 ddd (H5exo, H5 exo
,
2J 12.8, J5exo,4exo 12.2, J5exo,
0
4.2 Hz), 1.78 d (H2endo, H2 endo
,
2J 16.8 Hz), 1.76-1.84 m
4endo
0
0
(H4exo, H4 exo), 1.88 dd (H3, H3 , J3,2exo 4.4, J3,4exo 4.4 Hz), 2.28 ddd
0
Yield: 70%. Colourless oil; 1H NMR (d, ppm, J/Hz): 0.70 s (C9H3,
(H2exo, H2 exo
,
2J 16.8, J2exo,3 4.4, J2exo,
3.2 Hz), 3.13 dt (H11a
,
4exo
0
0
0
0
0
C9 H3), 0.87 s (C8H3, C8 H3), 0.91 s (C10H3, C10 H3), 1.14 ddd (H4endo
,
,
,
,
H11 a
,
2J 12.1, J11a,12 7.3 Hz), 3.17 dt (H11b, H11 b
,
2J 12.1, J11b,12
0
0
0
H4 endo, 2J 12.2, J4endo,5endo 9.3, J4endo 5exo
,
4.1 Hz), 1.24–1.32 m (2H13
7.3 Hz). 13C NMR (d, ppm): 180.98 s (C1, C1 ), 35.23 t (C2, C2 ),
0
0
0
0
0
0
2H13 , H5endo, H5 endo), 1.50–1.57 m (2H12, 2H12 ), 1.60 ddd (H5exo
43.75 d (C3, C3 ), 27.40 t (C4, C4 ), 32.11 t (C5, C5 ), 53.25 s (C6,
0
0
0
0
0
H5 exo
,
2J 13.0, J5exo,4exo 12.2, J5exo,4endo 4.1 Hz), 1.77 d (H2endo
C6 ), 46.68 s (C7, C7 ), 18.85 q (C8, C8 ), 19.41 q (C9, C9 ), 11.33 q
0 0 0 0
0
0
H2 endo
,
2J 16.8 Hz), 1.79 ddddd (H4exo, H4 exo 2J 12.2, J4exo,5exo 12.2,
(C10, C10 ), 52.28 t (C11, C11 ), 30.42 t (C12, C12 ), 27.41 t (C13, C13 ),
0
0
0
0
J4exo,5endo = J4exo,3 = 4.3, J4exo,2exo 3.2 Hz), 1.87 dd (H3, H3 , J3,2exo
=
29.38 t, 29.46 t, 29.49 t (C14, C14 ; C15, C15 ; C16, C16 ). ½a 2D5
ꢃ32.7
ꢂ
0
J3,4exo = 4.3 Hz), 2.27 ddd (H2exo, H2 exo
,
2J 16.8, J2exo,3 4.3, J2exo,4exo
(CHCl3, c = 0.8). HRMS: calcd for C32H56N2: 468.4435, found:
0
3.2 Hz), 3.12 dt (H11a, H11 a
,
2J 12.0, J11a,12 7.3 Hz), 3.17 dt (H11b
,
468.4435.
0
0
H11 b
,
2J 12.0, J11b,12 7.3 Hz). 13C NMR (d, ppm): 180.99 s (C1, C1 ),
0
0
0
0
35.21 t (C2, C2 ), 43.71 d (C3, C3 ), 27.37 t (C4, C4 ), 32.09 t (C5, C5 ),
4.3. General method for synthesis of compounds 2f-g (method
b)
0
0
0
53.23 s (C6, C6 ), 46.66 s (C7, C7 ), 18.83 q (C8, C8 ), 19.40 q (C9,
0
0
0
0
C9 ), 11.31 q (C10, C10 ), 52.19 t (C11, C11 ), 30.44 t (C12, C12 ), 27.31
0
t (C13, C13 ). ½a 2D2
ꢂ
18 (CHCl3, c = 1.0). HRMS: calcd for C26H44N2:
384.3503, found: 384.3499.
Camphor (15 mmol) and the diamine (7.5 mmol) were com-
bined and treated with one drop of coned H2SO4. Si(OEt)4
(15 mmol) was added and the mixture was placed into a flask
equipped with a still head. The solution was heated at 160 °C under
argon. The distillate (EtOH) was discarded and the residue was dis-
solved in Et2O (50 mL) and washed with saturated NaHCO3 solu-
tion and H2O (25 mL each). The Et2O solution was dried (Na2SO4)
and solvent removed under reduced pressure. The residue was dis-
solved in 10 mL of 95% EtOH and treated with 2 mL of 1 M KOH in
EtOH. The solution was stirred for 15-20 min and filtered; the pre-
cipitate was washed with Et2O. The filtrate was washed with H2O
(220 mL) and dried (Na2SO4). The solvent was removed under
4.2.3. N1E,N7E-N1,N7-bis((1R,2R,4R)-1,7,7-
trimethylbicyclo[2.2.1]heptan-2-ylidene)heptane-1,7-diamine
(2c)
Yield: 70%. Colourless oil; 1H NMR (d, ppm, J/Hz): 0.70 s (C9H3,
0
0
0
C9 H3), 0.87 s (C8H3, C8 H3), 0.91 s (C10H3, C10 H3), 1.14 ddd (H4endo
,
,
0
H4 endo, 2J 12.2, J4endo,5endo 9.3, J4endo 5exo
,
4.2 Hz), 1.20–1.29 m (2H13
0
0
2H13 , 2H14), 1.30 ddd (H5endo, H5 endo
,
2J 12.8, J5endo,4endo 9.3,
0
0
J5endo,4exo 4.3 Hz), 1.49–1.56 m (2H12, 2H12 ), 1.60 ddd (H5exo, H5 exo
0
2J 12.8, J5exo,4exo 12.2, J5exo,4endo 4.2 Hz), 1.77 d (H2endo, H2 endo
,
Please cite this article in press as: Sokolova, A. S.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.02.038

6
A. S. Sokolova et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
reduced pressure and purified by flash silica gel column chroma-
tography (hexane-ethyl acetate eluent) to obtain the desired 2f,g
and 5f,g.
1.71-1.78 m (H5exo), 1.75 d (H2endo
H3), 2.20 ddd (H2exo 2J 17.7, J2exo,3 4.4, J2exo,4exo 3.2 Hz), 3.53 br s
(NH2), 6.63 d (H19, H21, J19,18 (21,22) 8.8 Hz), 6.65 d (H12, H16, J12,13
, ,
2J 17.7 Hz), 1.82-1.90 m (H4exo
,
8.8 Hz), 6.82 d (H18, H22, J18,19
8.8 Hz, 6.85 d (H13
,
(16,15)
(22,21)
4.3.1. (E)-N-((1R,2R,4R)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-
ylidene)-4-(4-((E)-((1R,2R,4R)-1,7,7-
H15, J13,12
8.8 Hz). 13C NMR (d, ppm): 184.89 s (C1), 36.18 t
(15,16)
(C2), 43.71 d (C3), 27.32 t (C4), 31.93 t (C5), 53.85 s (C6), 46.97 s
(C7), 18.90 q (C8), 19.44 q (C9), 11.12 q (C10), 146.79 s (C11),
120.47 d (C12, C16), 118.18 d (C13, C15), 154.21 s (C14), 149.54 s
trimethylbicyclo[2.2.1]heptan-2-ylidene)amino)benzyl)aniline
(2f)
Yield: 42%. Yellowish amorphous solid; mp 129–130 °C; 1H
(C17), 120.17 d (C18, C22), 116.07 d (C19, C21), 142.04 s (C20). ½a 3D1
ꢂ
0
0
NMR (d, ppm, J/Hz): 0.84 s (C9H3, C9 H3), 0.95 s (C8H3, C8 H3), 1.06
28.2 (CHCl3, c = 0.56). HRMS: calcd for C22H426O1N2: 334.2040,
0
0
s (C10H3, C10 H3), 1.18–1.26 m (H4endo, H4 endo), 1.49 ddd (H5endo
,
,
found: 334.2036.
0
H5 endo
,
,
2J 13.0, J5endo,4endo 9.5, J5endo,4exo 4.3 Hz), 1.73 d (H2endo
0
0
H2 endo
2J 17.7 Hz), 1.74 ddd (H5exo, H5 exo
,
2J 13.0, J5exo,4exo 12.2,
4.4. General synthetic procedure for reduction of diimine 2a-g
(method c)
0
0
J5exo,4endo 4.2 Hz), 1.81-1.89 m (H4exo, H4 exo), 1.87 dd (H3, H3 ,
0
J3,2exo 4.3, J3,4exo 4.3 Hz), 2.19 ddd (H2exo, H2 exo
,
2J 17.7, J2exo,3 4.3,
0
0
J2exo,4exo 3.2 Hz), 3.88
s
(2H17), 6.63
d
(H12
,
H12
,
H16
,
H16
,
Camphor diimine (10 mmol) and NiCl2ꢁ6H20 (40 mmol) were
taken in methanol (100 ml) and cooled to –30 °C. NaBH4
(200 mmol) was added in portions to this mixture from a solid
addition flask under stirring at ꢃ30 °C over a period of 1 h. The
reaction mixture was further stirred for 1 h at ꢃ30 °C and for 4 h
at room temperature. 3 N NaOH solution (15 ml) and ether
(100 ml) were added, and the contents were filtered. The aqueous
and organic layers of the filtrate were separated. The organic layer
was washed with saturated NaCl solution (2 ꢄ 15 ml), dried over
anhydrous magnesium sulfate. The solvent was removed under re-
duced pressure and crude product purified by flash silica gel col-
umn chromatography to obtain the desired 3b,c,e,f,g.
0
0
J12,13(16,15) 8.2 Hz), 7.05 d (H13, H13 , H15, H15 , J13,12(15,16) 8.2 Hz).
0
0
13C NMR (d, ppm): 184.43 s (C1, C1 ), 36.14 t (C2, C2 ), 43.71 d (C3,
0
0
0
0
C3 ), 27.34 t (C4, C4 ), 31.97 t (C5, C5 ), 53.81 s (C6, C6 ), 46.99 s (C7,
0
0
0
0
C7 ), 18.93 q (C8, C8 ), 19.45 q (C9, C9 ), 11.12 q (C10, C10 ), 150.01 s
0
0
0
(C11, C11 ), 119.37 d (C12, C12 ), 129.24 d (C13, C13 ), 135.86 s (C14
,
0
0
0
C14 ), 129.24 d (C15, C15 ), 119.37 d (C16, C16 ), 40.58 t (C17). ½a 3D0
ꢂ
29.0 (CHCl3, c = 0.6). HRMS: calcd for C33H42N2: 466.3347, found:
466.3343.
4.3.2. (E)-4-(4-aminobenzyl)-N-((1R,2R,4R)-1,7,7-
trimethylbicyclo[2.2.1]heptan-2-ylidene)aniline (5f)
Yield: 14%. Yellowish amorphous solid; mp 130–132 °C; 1H
NMR (d, ppm, J/Hz): 0.84 s (C9H3), 0.95 s (C8H3), 1.07 s (C10H3),
4.4.1. N1,N6-bis((1R,2R,4R)-1,7,7-trimethylbicyclo[2.2.1]heptan-
2-yl)hexane-1,6-diamine (3b)
1.19–1.26 m (H4endo), 1.50 ddd (H5endo
,
2J 12.8, J5endo,4endo 9.5,
J5endo,4exo 4.3 Hz), 1.73 d (H2endo
,
2J 17.7 Hz), 1.73–1.78 m (H5exo),
Yield: 92%. Colourless oil; 1H NMR (d, ppm, J/Hz): 0.77 s (C9H3,
0
0
0
1.82–1.90 m (H4exo, H3), 2.18 ddd (H2exo, 2J 17.7, J2exo,3 4.3, J2exo,4exo
3.2 Hz), 3.53 br s (NH2), 3.81 s (2H17), 6.60 d (H20, H22, J20,19 (22,23)
8.2 Hz), 6.63 d (H12, H16, J12,13 (16,15) 8.2 Hz), 6.95 d (H19, H23, J19,20
8.2 Hz), 7.05 d (H13, H15, J13,12 8.2 Hz). 13C NMR (d, ppm): 184.40 s
(C1), 36.12 t (C2), 43.68 d (C3), 27.31 t (C4), 31.94 t (C5), 53.77 s
(C6), 46.96 s (C7), 18.90 q (C8), 19.42 q (C9), 11.11 q (C10), 149.91
s (C11), 119.34 d (C12, C16), 129.13 d (C13, C15), 136.22 s (C14),
40.32 t (C17), 131.48 s (C18), 129.56 d (C19, C23), 115.11 d
C9 H3), 0.83 s (C10H3, C10 H3), 0.98 s (C8H3, C8 H3), 0.99–1.07 m
0
0
0
(H4endo, H4 endo, H5endo, H5 endo), 1.25–1.32 m (2H13, 2H13 ), 1.35–
0
0
0
1.44 m (2H12, 2H12 ), 1.44–1.55 m (H5exo, H5 exo, 2H2, 2H2 ), 1.60–
0
0
0
1.69 m (H3, H3 , H4exo, H4 exo), 2.40 dt (H11a, H11 a
,
2J 11.3, J11a,12
,
0
7.0 Hz), 2.46 dd (H1endo, H1 endo, J1endo,2endo 7.0, J1endo,2exo 6.2 Hz),
0
2.50 dt (H11b, H11 b
,
2J 11.3, J11b,12 7.2 Hz). 13C NMR (d, ppm):
0
0
0
66.84 d (C1, C1 ), 39.06 t (C2, C2 ), 45.18 d (C3, C3 ), 27.28 t (C4,
0
0
0
0
C4 ), 36.89 t (C5, C5 ), 48.20 s (C6, C6 ), 46.51 s (C7, C7 ), 20.41 q
0
0
0
0
(C20,C22), 144.25 s (C21). ½a 3D0
ꢂ
18.0 (CHCl3, c = 0.6). HRMS: calcd
for C23H28N2: 332.2244, found: 332.2247.
(C8, C8 ), 20.50 q (C9, C9 ), 12.05 q (C10, C10 ), 48.78 t (C11, C11 ),
0
0
30.38 t (C12, C12 ), 27.34 t (C13, C13 ). ½a D28
ꢂ
55 (CHCl3 c = 0.72). HRMS:
calcd for C26H48N2: 388.3808, found: 388.3812.
4.3.3. (E)-N-((1R,2R,4R)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-
ylidene)-4-(4-((E)-((1R,2R,4R)-1,7,7-
trimethylbicyclo[2.2.1]heptan-2-
4.4.2. N1,N7-bis((1R,2R,4R)-1,7,7-trimethylbicyclo[2.2.1]heptan-
2-yl)heptane-1,7-diamine (3c)
ylidene)amino)phenoxy)aniline (2g)
Yield: 87%. Colourless oil; 1H NMR (d, ppm, J/Hz): 0.77 s (C9H3,
0
0
0
Yield: 34%. White amorphous solid; mp 164–165 °C; 1H NMR (d,
C9 H3), 0.84 s (C10H3, C10 H3), 0.98 s (C8H3, C8 H3), 0.99–1.07 m
0
0
0
0
0
ppm, J/Hz): 0.83 s (C9H3, C9 H3), 0.95 s (C8H3, C8 H3), 1.06 s (C10H3,
(H4endo, H4 endo, H5endo, H5 endo), 1.23–1.31 m (2H13, 2H13 , 2H14),
0 0 0
0
0
0
C10 H3), 1.18–1.26 m (H4endo, H4 endo), 1.49 ddd (H5endo, H5 endo
,
,
2J
2J
2J
1.35–1.43 m (2H12, 2H12 ), 1.43–1.56 m (H5exo, H5 exo, 2H2, 2H2 ),
0 0 0
0
13.0, J5endo,4endo 9.4, J5endo,4exo 4.3 Hz), 1.75 ddd (H5exo, H5 exo
1.61–1.70 m (H3, H3 , H4exo, H4 exo), 2.40 dt (H11a, H11 a
,
2J 11.3,
0
0
13.0, J5exo,4exo 12.2, J5exo,4endo 4.2 Hz), 1.76 d (H2endo, H2 endo
,
J11a,12 7.0 Hz), 2.47 dd (H1endo, H1 endo, J1endo 2endo
,
7.0, J1endo,2exo
0
0
0
0
17.7 Hz), 1.82-1.90 m (H4exo, H4 exo, H3, H3 ), 2.21 ddd (H2exo, H2 exo
,
6.2 Hz), 2.50 dt (H11b, H11 b
,
2J 11.3, J11b,12 7.2 Hz). 13C NMR (d,
0
0
0
0
0
2J 17.7, J2exo,3 4.4, J2exo,4exo 3.2 Hz), 6.67 d (H12, H12 , H16, H16 , J12,13
ppm): 66.85 d (C1, C1 ), 39.07 t (C2, C2 ), 45.18 d (C3, C3 ), 27.28 t
0
0
0
0
0
0
8.8 Hz), 6.89 d (H13, H13 , H15, H15 , J13,12 8.8 Hz). 13C NMR (d,
(C4, C4 ), 36.90 t (C5, C5 ), 48.21 s (C6, C6 ), 46.52 s (C7, C7 ), 20.41
(16,15)
0
0
0
0
0
0
0
ppm): 184.94 s (C1, C1 ), 36.17 t (C2, C2 ), 43.70 d (C3, C3 ), 27.31 t (C4,
q (C8, C8 ), 20.50 q (C9, C9 ), 12.06 q (C10, C10 ), 48.84 t (C11, C11 ),
0 0
0
0
0
0
C4 ), 31.93 t (C5, C5 ), 53.86 s (C6, C6 ), 46.98 s (C7, C7 ), 18.89 q (C8,
30.37 t (C12, C12 ), 27.38 t (C13, C13 ), 29.42 t (C14). ½a 2D8
ꢂ
75.1 (CHCl3,
0
0
0
0
C8 ), 19.43 q (C9, C9 ), 11.10 q (C10, C10 ), 147.29 s (C11, C11 ), 120.53 d
c = 0.66). HRMS: calcd for C27H50N2: 402.3965, found: 402.3960.
0
0
0
(C12, C12 ), 118.98 d (C13, C13 ), 153.36 s (C14, C14 ), 118.98 d (C15
,
0
0
C15 ), 120.53 d (C16, C16 ). ½a D31
ꢂ
30.3 (CHCl3, c = 0.56). HRMS: calcd
4.4.3. N1,N12-bis((1R,2R,4R)-1,7,7-
trimethylbicyclo[2.2.1]heptan-2-yl)dodecane-1,12-diamine (3e)
for C32H40N2: 468.3135, found: 468.3138.
Yield: 82%. Colourless oil; 1H NMR (d, ppm, J/Hz): 0.78 s (C9H3,
0
0
0
4.3.4. (E)-4-(4-aminophenoxy)-N-((1R,2R,4R)-1,7,7-
C9 H3), 0.84 s (C10H3, C10 H3), 0.99 s (C8H3, C8 H3), 0.99-1.07 m
0
0
0
trimethylbicyclo[2.2.1]heptan-2-ylidene)aniline (5g)
Yield: 11%. White amorphous solid; mp 118–120 °C; 1H NMR (d,
ppm, J/Hz): 0.83 s (C9H3), 0.95 s (C8H3), 1.06 s (C10H3), 1.18–1.26 m
(H4endo, H4 endo, H5endo, H5 endo), 1.21–1.30 m (2H13, 2H13 , 2H14
,
0 0 0 0
2H14 , 2H15, 2H15 , 2H16, 2H16 ), 1.36–1.43 m (2H12, 2H12 ), 1.43–
0
0
1.54 m (H5exo, H5 exo), 1.49–1.58 m (2H2, 2H2 ), 1.61–1.64 m (H3,
0
0
0
(H4endo), 1.49 ddd (H5endo
,
2J 13.0, J5endo,4endo 9.4, J5endo,4exo 4.4 Hz),
H3 ), 1.63–1.71 m (H4exo, H4 exo), 2.41 dt (H11a, H11 a 2J 11.3, J11a,12
,
Please cite this article in press as: Sokolova, A. S.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.02.038

A. S. Sokolova et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
7
0
0
7.0 Hz), 2.47 dd (H1endo, H1 endo, J1endo,2endo 7.0, J1endo,2exo 6.2 Hz),
6.0, J2exo,3 4.3, J2exo,4exo 3.2 Hz), 2.14 s (C14H3, C14 H3), 2.15 dd
0
0
2.51 dt (H11b, H11 b
,
2J 11.3, J11b,12 7.3 Hz). 13C NMR (d, ppm):
(H1endo, H1 endo, J1endo,2endo 8.5, J1endo,2exo 6.0 Hz), 2.18 ddd (H11a
,
0
0
0
0
0
66.87 d (C1, C1 ), 39.08 t (C2, C2 ), 45.20 d (C3, C3 ), 27.29 t (C4,
H11 a
,
2J 12.8, J11a,12a 9.8, J11a,12b 5.4 Hz), 2.39 ddd (H11b H11 b
,
2J
,
0
0
0
0
C4 ), 36.92 t (C5, C5 ), 48.22 s (C6, C6 ), 46.53 s (C7, C7 ), 20.42 q
12.8, J11b,12b 10.0, J11b,12a 5.4 Hz). 13C NMR (d, ppm): 73.23 d (C1,
0
0
0
0
0
0
0
0
(C8, C8 ), 20.51 q (C9, C9 ), 12.06 q (C10, C10 ), 48.87 t (C11, C11 ),
C1 ), 34.93 t (C2, C2 ), 44.91 d (C3, C3 ), 27.31 t (C4, C4 ), 37.22 t (C5,
0
0
0
0
0
0
30.41 t (C12, C12 ), 27.41 t (C13, C13 ), 29.52 t, 29.50 t, 29.49 t (C14
,
C5 ), 49.38 s (C6, C6 ), 46.94 s (C7, C7 ), 19.61 q (C8, C8 ), 20.72 q
0 0 0 0
0
0
0
C14 ; C15, C15 ; C16, C16 ). ½a 2D5
ꢂ
ꢃ88.6 (CHCl3, c = 1). HRMS: calcd
(C9, C9 ), 14.39 q (C10, C10 ), 57.33 t (C11, C11 ), 26.94 t (C12, C12 ),
0
0
for C32H60N2: 472.4751, found: 472.4752.
27.47 t (C13, C13 ), 40.80 q (C14, C14 ). ½a D28
ꢃ85.6 (CHCl3 c = 0.5).
ꢂ
HRMS: calcd for C28H52N2: 416.4127, found: 416.4125.
4.4.4. (1R,2R,4R)-1,7,7-trimethyl-N-(4-(4-((1R,2R,4R)-1,7,7-
trimethylbicyclo[2.2.1]heptan-2-
4.5.2. N1,N7-dimethyl-N1,N7-bis((1R,2R,4R)-1,7,7-
ylamino)benzyl)phenyl)bicyclo[2.2.1]heptan-2-amine (3f)
trimethylbicyclo[2.2.1]heptan-2-yl)heptane-1,7-diamine (4c)
Yield: 46%. Yellowish amorphous solid; mp 134 °C; 1H NMR (d,
Yield: 44%. Colourless oil; 1H NMR (d, ppm, J/Hz): 0.77 s (C9H3,
0
0
0
0
0
ppm, J/Hz): 0.86 s (C9H3, C9 H3), 0.92 s (C10H3, C10 H3), 1.02 s
C9 H30 ), 0.91 s (C8H3, C8 H3), 0.95 s (C10H3, C10 H3), 0.98–1.05 m
0
0
0
0
0
0
(C8H3, C8 H3), 1.13–1.30 m (H4endo, H4 endo, H5endo, H5 endo), 1.56-
(H4endo, H4 endo, H5endo, H5 endo), 1.17–1.31 m (2H13, 2H13 , 2H14),
0
0
0
0
0
1.81 m (H2exo, H2 exo, H3, H3 , H4exo, H4 exo, H5exo, H5 exo), 1.87 dd
1.37 dd (H2endo, H2 endo
,
2J 12.4, J2endo 1endo
,
8.6 Hz), 1.32–1.44 m
0
0
0
0
0
(H2endo, H2 endo
,
2J 12.7, J2endo,1endo 8.3 Hz), 3.24 dd (H1endo, H1 endo
,
(H5exo, H5 exo, 2H12, 2H12 ), 1.57 dd (H3, H3 , J3,2exo = J3,4exo = 4.3 Hz),
0
0
J1endo,2endo 8.3, J1endo,2exo 4.6 Hz), 3.62 br s (2NH), 3.75 s (2H17),
1.60–1.68 m (H4exo
,
H4 exo), 1.85 dddd (H2exo
,
H2 exo
,
2J 12.4,
0
0
0
6.49 d (H12, H12 , H16, H16 , J12,13
8.2 Hz), 6.96 d (H13, H13
,
J2exo,1endo 6.1, J2exo,3 4.3, J2exo,4exo 3.2 Hz), 2.14 s (C15H3, C15H30 ),
(16,15)
0
0
0
H15, H15 , J13,12 8.2 Hz). 13C NMR (d, ppm): 61.60 d (C1, C1 ), 40.69 t
2.15 dd (H1endo, H1 endo, J1endo,2exo 8.6, J1endo,2exo 6.1 Hz), 2.18 ddd
20
30
40
50
110a
(C2, C ), 45.07 d (C3, C ), 27.28 t (C4, C ), 36.63 t (C5, C ), 48.58
(H11a, H
,
2J 12.8, J11a,12a 9.8, J11a,12b 5.3 Hz), 2.40 ddd (H11b
,
0
0
0
0
0
s (C6, C6 ), 47.02 s (C7, C7 ), 20.35 q (C8, C8 ), 20.35 q (C9, C9 ),
H11 b
,
2J 12.8, J11b,12b 10.2, J11b,12a 5.4 Hz). 13C NMR (d, ppm): 73.21
0
0
0
0
0
0
0
12.15 q (C10, C10 ), 146.10 s (C11, C11 ), 112.54 d (C12, C12 ), 129.34
d (C1, C1 ), 34.92 t (C2, C2 ), 44.92 d (C3, C3 ), 27.32 t (C4, C4 ),
0
0
0
0
0
0
d (C13, C13 ), 130.00 s (C14, C14 ), 129.34 d (C15, C15 ), 112.54 d
37.22 t (C5, C5 ), 49.37 s (C6, C6 ), 46.94 s (C7, C7 ), 19.61 q (C8,
0 0 0 0
0
(C16, C16 ), 39.89 t (C17). ½a 3D1
ꢂ
25.0 (CHCl3, c = 0.6). HRMS: calcd
C8 ), 20.73 q (C9, C9 ), 14.37 q (C10, C10 ), 57.35 t (C11, C11 ), 26.86 t
0 0 0 0
for C33H46N2: 470.3649, found: 470.3656.
(C12, C12 ), 27.44 t (C13, C13 ), 29.66 t (C14, C14 ), 40.82 q (C15, C15 ).
85.6 (CHCl3, c = 0.5). HRMS: calcd for C29H54N2: 430.4283,
½ ꢂ
a 2D8
4.4.5. (1R,2R,4R)-1,7,7-trimethyl-N-(4-(4-((1R,2R,4R)-1,7,7-
trimethylbicyclo[2.2.1]heptan-2-
found: 430.4282.
ylamino)phenoxy)phenyl)bicyclo[2.2.1]heptan-2-amine (3g)
4.6. Viruses and cells
Yield: 34%. White amorphous solid; mp 123–125 °C; 1H NMR (d,
0
0
ppm, J/Hz): 0.85 s (C9H3, C9 H3), 0.92 s (C10H3, C10 H3), 1.02 s (C8H3,
Influenza virus A/California/07/09 (H1N1)pdm09 from the col-
lection of viruses of the Research Institute of Influenza (St. Peters-
burg, Russia) was used. The virus was cultivated in 10-12-day-old
chicken embryos for 48 h at +37 °C. MDCK cells (ATCC CCL 34) in a
minimal essential medium (MEM, PAA, Austria, Cat.# E15-825)
were seeded on 96-well plates (Orange Scientific no. 5530100)
and incubated at 37 °C in 5% CO2 until a confluent monolayer
0
0
0
C8 H3), 1.13–1.20 m (H4endo, H4 endo), 1.24 ddd (H5endo, H5 endo
,
,
2J
2J
0
13.0, J5endo,4endo 9.3, J5endo,4exo 4.2 Hz), 1.61 ddd (H5exo, H5 exo
0
13.0, J5exo,4exo 12.2, J5exo,4endo 4.2 Hz), 1.64–1.80 m (H2exo, H2 exo
,
0
0
0
H3, H3 , H4exo, H4 exo), 1.86 dd (H2endo, H2 endo
,
2J 12.8, J2endo,1endo
0
8.3 Hz), 3.21 dd (H1endo, H1 endo, J1endo,2endo 8.3, J1endo,2exo 4.7 Hz),
0
0
3.60 br s (2NH), 6.50 d (H12, H12 , H16, H16 , J12,13
8.8 Hz),
(16,15)
0
0
6.80 dd (H13, H13 , H15, H15 , J13,12 8.8 Hz). 13C NMR (d, ppm): 62.14
formed. To cultivate the virus, 5% albumin, trypsin (1
16 mM HEPES (pH 7.6) were added.
lg/ml), and
0
0
0
0
d (C1, C1 ), 40.65 t (C2, C2 ), 45.11 d (C3, C3 ), 27.29 t (C4, C4 ),
0
0
0
36.68 t (C5, C5 ), 48.65 s (C6, C6 ), 47.04 s (C7, C7 ), 20.38 q (C8,
0
0
0
0
C8 ), 20.36 q (C9, C9 ), 12.17 q (C10, C10 ), 143.73 s (C11, C11 ),
4.7. Toxicity studies
0
0
0
113.42 d (C12, C12 ), 119.31 d (C13, C13 ), 149.33 s (C14, C14 ),
0
0
119.31 d (C15, C15 ), 113.42 d (C16, C16 ). ½a 3D0
ꢂ
33.0 (CHCl3, c = 0.6).
HRMS: calcd for C32H44O1N2: 472.3450, found: 472.3448.
Microtetrazoliumtest(MTT)wasusedtostudycytotoxicityofthe
compounds (Mossman, 1983). Briefly, series of two-fold dilutions of
each compound (1000–4 lg/ml) in MEM were prepared. MDCK cells
4.5. General method for synthesis of compounds 4b,c (method
d)
were incubated for 48 h at 37 °C in 5% CO2 in the presence of the dis-
solved substances. The degree of destruction of the cell monolayer
was then evaluated in the microtetrazolium test (MTT). The cells
were washed twice with saline, and a solution of 3-(4,5-dimethyl-
thiazolyl-2)-2,5-diphenyltetrazolium bromide (ICN Biochemicals
Camphor diamine (1.5 mmol), K2CO3 (1.5 mmol), and dimethyl
sulfate (15 mmol) were mixed in methanol. Reaction mixture was
stirring at room temperature during 12 h. Than 10 ml 5% NaOH and
saturated NaCl solution was added. The aqueous layer was ex-
tracted with CH2Cl2 (3 ꢄ 10 ml). The combined organic phases
were dried over Na2SO4. The solvent was evaporated to dryness
and crude product purified by flash silica gel column chromatogra-
phy to obtain the desired 4b and 4c.
Inc., Aurora, Ohio) (0.5 lg/ml) in saline was added to the wells. After
1 h incubation, the wells were washed and the formazan residue dis-
solved in DMSO (0.1 ml per well). The optical density of cells was
then measured on a Victor 2 1440 multifunctional reader (Perkin El-
mer, Finland) at 535 nm and plotted against concentration of the
compounds. Each concentration was tested in three parallels. The
50% cytotoxic dose (CTD50) of each compound (i.e., the compound
concentration that causes the death of 50% cells in a culture, or
decreasing the optical density twice as compared to the control
wells) was calculated from the data obtained.
4.5.1. N1,N6-dimethyl-N1,N6-bis((1R,2R,4R)-1,7,7-
trimethylbicyclo[2.2.1]heptan-2-yl) hexane-1,6-diamine (4b)
Yield: 52%. Colourless oil; 1H NMR (d, ppm, J/Hz): 0.77 s (C9H3,
0
0
0
C9 H3), 0.92 s (C8H3, C8 H3), 0.95 s (C10H3, C10 H3), 0.98–1.06 m
0
0
0
(H4endo, H4 endo, H5endo, H5 endo), 1.18–1.26 m (2H13, 2H13 ), 1.37 dd
4.8. Determination of the antiviral activity
0
(H2endo, H2 endo
,
2J 12.6, J2endo 1endo 8.8 Hz), 1.31–1.50 m (H5exo
0
,
,
0
0
H5 exo, 2H12, 2H12 ), 1.57 dd (H3, H3 , J3,2exo = J3,4exo 4.3 Hz), 1.59–
The compounds in appropriate concentrations were incubated
with MDCK cells for 1 h at 37 °C. The cell culture was then infected
0
0
1.69 m (H4exo, H4 exo), 1.85 dddd (H2exo, H2 exo
,
2J 12.6, J2exo,1endo
Please cite this article in press as: Sokolova, A. S.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.02.038

8
A. S. Sokolova et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
with 10-fold dilutions of the virus (10ꢃ1 to 10ꢃ6). The plates were
incubated for 48 h at 37 °C in the presence of 5% CO2. The infection
activity of the virus was evaluated in the hemagglutination reac-
6. Torriani, F. J.; Rodriguez-Torres, M.; Rockstroh, J. K.; Lissen, E.; Gonzalez-Garcia,
J.; Lazzarin, A.; Carosi, G.; Sasadeusz, J.; Katlama, C.; Montaner, J.; Sette, H.;
Passe, S.; De Pamphilis, J.; Duff, F.; Schrenk, U. M.; Dieterich, D. T. N. Eng. J. Med.
2004, 351, 438.
7. Davies, W. L.; Grunert, R. R.; Haff, R. F.; McGahen, J. W.; Neumayer, E. M.;
Paulshock, M.; Watts, J. C.; Wood, T. R.; Hermann, E. C.; Hoffmann, C. E. Science
1964, 144, 862.
8. Scholtissek, C.; Quack, G.; Klenk, H. D.; Webster, R. G. Antiviral Res. 1998, 37, 83.
9. Cady, S. D.; Schmidt-Rohr, K.; Wang, J.; Soto, C. S.; DeGrado, W. F.; Hong, M. H.
Nature 2010, 463, 689.
10. Belshe, R. B.; Burk, B.; Newman, F.; Cerruti, R. L.; Sim, I. S. J. Infect. Dis. 1989,
159, 430.
11. Antiviral Agents for the Treatment and Chemoprophylaxis of Influenza.
Recommendations and Reports 2011, 60, 1. http://www.cdc.gov/mmwr/
preview/mmwrhtml/rr6001a1.htm).
12. Abed, Y.; Goyette, N.; Boivin, G. Antimicrob. Agents Chemother. 2005, 49, 556.
13. Zoidis, G.; Kolocouris, N.; Naesens, L.; Clercq, E. D. Bioorg. Med. Chem. 2009, 17,
1534.
14. DeGrado, W.F.; Wang, J. (University of Pennsylvania), 2011, WO 2011/022191.
15. Tanner, J. A.; Zheng, B. J.; Zhou, J.; Watt, R. M.; Jiang, J. Q.; Wong, K. L.; Lin, Y. P.;
Lu, L. Y.; He, M. L.; Kung, H. F.; Kesel, A. J. Chem. Biol. 2005, 12, 303.
16. Tandura, S. N.; Zarubaev, V. V.; Anfimov, P. M.; Kiselev, O. I. Antibiot. Khimioter.
2013, 58, 36.
tion with chicken erythrocytes.
A virus-containing solution
(100 l) was placed in the wells of a round-bottom plate. An equal
l
amount of a 1% suspension of chicken erythrocytes in saline was
added. The reaction was evaluated after 60 min incubation at room
temperature. Each concentration of the compounds was tested in
three parallels. A virus titer was considered as reciprocal to deci-
mal logarithm of maximum dilution that caused complete aggluti-
nation of erythrocytes and was expressed in logarithms of the 50%
experimental infection dose (log10 EID50). The antiviral activity of
the compounds was estimated by the decrease in the virus titer
as compared to the control. The 50% effective dose (ED50) of the
drug, i.e., the concentration at which the virus production de-
creased by a factor of two (a virus titer per 0.3 log10 EID50) and
the selectivity index (the ratio between CTD50 and ED50) were cal-
culated from the data obtained.
17. Xiong, Z. Y.; Xiao, F. M.; Xu, X.; Wu, Y. F.; Jiang, X. M. Zhongguo Zhong Yao Za Zhi
2013, 38, 786.
18. Jassim, S. A. A.; Naji, M. A. J. Appl. Microbiol. 2003, 95, 412.
19. Astani, A.; Reichling, J.; Schnitzler, P. Phytother. Res. 2010, 24(5), 673–679.
20. Ardashov, O. V.; Zarubaev, V. V.; Shtro, A. A.; Korchagina, D. V.; Volcho, K. P.;
Salakhutdinov, N. F.; Kiselev, O. I. Lett. Drug Des. Discov. 2011, 8, 375.
21. Teplov, G. V.; Suslov, E. V.; Zarubaev, V. V.; Shtro, A. A.; Karpinskaya, L. A.;
Rogachev, A. D.; Korchagina, D. V.; Volcho, K. P.; Salakhutdinov, N. F.; Kiselev,
O. I. Lett. Drug Des. Discov. 2013, 10, 477.
4.9. Computer modeling
The molecular docking for modeling the interaction between
compounds under investigation and influenza virus M2 protein
(protein database code 2LOJ) was done by Hex online server
(http://hexserver.loria.fr/).
22. Sztanke, K.; Maziarka, A.; Osinka, A.; Sztanke, M. Bioorg. Med. Chem. 2013, 21,
3648.
23. Bhat, M. A.; Al-Omar, M. A. Med. Chem. Res. 2013, 22, 4522.
24. Mann, J. C.; Hobbs, J. B.; Banthorpe, D. V.; Harborne, J. B. Natural Products: Their
Chemistry And Biological Significance; Longman Scientific & Technical: Harlow,
Essex, England, 1994.
25. Caselli, A.; Giovenzana, G. B.; Palmisano, G.; Sisti, M.; Pilati, T. Tetrahedron:
Asymmetry 2003, 14, 1451.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.02.038.
26. Love, B.; Ren, J. J. Org. Chem. 1993, 58, 5556.
27. Periasamy, M.; Devasagayaraj, A.; Satyanarayana, N.; Narayana, C. Synth.
Commun. 1989, 19, 565.
References
28. Sanjeevakumar, N.; Periasamy, M. Tetrahedron: Asymmetry 1842, 2009, 20.
29. Sokolov, D. N.; Zarubaev, V. V.; Shtro, A. A.; Polovinka, M. P.; Luzina, O. A.;
Komarova, N. I.; Salakhutdinov, N. F.; Kiselev, O. I. Bioorg. Med. Chem. Lett. 2012,
22, 7060.
30. Pinto, L. H.; Lamb, R. A. Trends Microbiol. 1995, 3, 271.
31. Schnell, J. R.; Chou, J. J. Nature 2008, 451, 591.
1. Nicoll, A.; Brown, C.; Karcher, F.; Penttinen, P.; Hegermann-Lindencrone, M.;
Villanueva, S.; Ciotti, M.; Jean-Gilles, L.; Rehmeta, S.; Nguyen-Van-Tamd, J. Bull.
World Health Organ. 2012, 90, 311.
2. De Clercq, E. In Advances in Antiviral Drug Design; Elsevier, 2007; Vol. 5,
3. Hauge, S. H.; Dudman, S.; Borgen, K.; Lackenby, A.; Hungnes, O. Infect. Dis. 2009,
15, 155.
32. Pielak, R. M.; Schnell, J. R.; Chou, J. J. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 7379.
33. Raza, Z.; Dakovic, S.; Vinkovic, V.; Dunjic, V. Croat. Chem. Acta 1996, 69, 1545.
4. Ison, M. G. Influenza Other Respir. Viruses 2013, 7, 7.
5. Lee, S. M.; Yen, H. L. Antiviral Res. 2012, 96, 391.
Please cite this article in press as: Sokolova, A. S.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.02.038