Synthesis of camphecene derivatives using click chemistry methodology and study of their antiviral activity

Article abstract of DOI:10.1016/j.bmcl.2017.03.051

A series of seventeen tetrazole derivatives of 1,7,7-trimethyl-[2.2.1]bicycloheptane were synthesized using click chemistry methodology and characterized by spectral data. Studies of cytotoxicity and in vitro antiviral activity against influenza virus A/Puerto Rico/8/34 (H1N1) in MDCK cells of the compounds obtained were performed. The structure-activity relationship analysis suggests that to possess virus-inhibiting activity, the compounds of this group should bear oxygen atom with a short linker (C2-C4), either as a hydroxyl group (18, 19, 29), keto-group (21) or as a part of a heterocycle (24). These compounds demonstrated low cytotoxicity along with high anti-viral activity.

Full text of DOI:10.1016/j.bmcl.2017.03.051

Accepted Manuscript
Synthesis of Camphecene Derivatives using Click Chemistry Methodology and
Study of their Antiviral Activity
Oleg I. Artyushin, Elena V. Sharova, Natalya M. Vinogradova, Galina K.
Genkina, Aleksandra A. Moiseeva, Zinaida.S. Klemenkova, Iana R.
Orshanskaya, Anna A. Shtro, Renata A. Kadyrova, Vladimir V. Zarubaev, Olga
I. Yarovaya, Nariman F. Salakhutdinov, Valery K. Brel
PII:
S0960-894X(17)30301-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.03.051
BMCL 24803
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
31 January 2017
21 March 2017
21 March 2017
Please cite this article as: Artyushin, O.I., Sharova, E.V., Vinogradova, N.M., Genkina, G.K., Moiseeva, A.A.,
Klemenkova, Zinaida.S., Orshanskaya, I.R., Shtro, A.A., Kadyrova, R.A., Zarubaev, V.V., Yarovaya, O.I.,
Salakhutdinov, N.F., Brel, V.K., Synthesis of Camphecene Derivatives using Click Chemistry Methodology and
Study of their Antiviral Activity, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/
j.bmcl.2017.03.051
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Synthesis of Camphecene Derivatives using Click Chemistry Methodology
and Study of their Antiviral Activity
Oleg I. Artyushina, Elena V. Sharovaa, Natalya M. Vinogradovaa, Galina K. Genkinaa,
Aleksandra A. Moiseevaa, Zinaida. S. Klemenkovaa, Iana R. Orshanskayab, Anna A. Shtrob,
Renata A. Kadyrovab, Vladimir V. Zarubaevb, Оlga I. Yarovayac,d,
Nariman F. Salakhutdinovc,d, Valery K. Brela,*
a A.N. Nesmeyanov Institute of Organoelement Compound of Russian Academy of Sciences, 28,
Vavilova St., GSP-1, V-334, 119991 Moscow, Russian Federation
bDepartment of Chemotherapy, Influenza Research Institute, 15/17 Prof. Popova St.,
197376 St. Petersburg, Russian Federation
cNovosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian Academy of
Sciences, Lavrentjev Avenue 9, 630090, Novosibirsk, Russian Federation
dNovosibirsk State University, Pirogova St. 2, 630090, Novosibirsk, Russian Federation
Keywords: Acetylenes, Antivirals, Azides, Camphor, Click chemistry, Imine derivatives,
Influenza, Triazoles.
ABSTRACT
A series of seventeen tetrazole derivatives of 1,7,7-trimethyl-[2.2.1]bicycloheptane were
synthesized using click chemistry methodology and characterized by spectral data. Studies of
cytotoxitity and in vitro antiviral activity against influenza virus A/Puerto Rico/8/34 (H1N1) in
MDCK cells of the compounds obtained were performed. The structure-activity relationship
analysis suggests that to possess virus-inhibiting activity, the compounds of this group should
bear oxygen atom with short linker (C2-C4), either as a hydroxyl group (18, 19, 29), keto-group
(21) or as a part of heterocycle (24). These compounds demonstrated low cytotoxicity along with
high anti-viral activity.
Influenza viruses A and B cause seasonal epidemics, and changing the antigenic subtype of the
virus results in pandemics spreading rapidly from one country to another and covering all
regions of the world. Influenza A virus presents an additional risk factor for the emergence of
zoonotic diseases as well as occurrence of strains causing pandemics due to the possibility of
changing the host. Presently, there are two classes of chemical compounds that are
internationally recognized as etiotropic anti-influenza drugs, namely, inhibitors of M2 viral
protein (adamantane derivatives) and viral neuraminidase NA inhibitors (oseltamivir, peramivir,
* Corresponding author. Tel.: +74991356373
E-mail address: v_brel@mail.ru (Valery. K. Brel)

laninamivir and zanamivir)1. M2 Inhibitors are presently not recommended for the treatment of
influenza because most of circulating virus strains are resistant to adamantine derivatives2. Many
examples of appearance of viral strains resistant also to NA inhibitors including viruses of
pandemic or potentially pandemic H1N1pdm09, H5N1, H7N9 subtypes, are known3. One cannot
give up on the economic dimension accompanying the annual influenza epidemic. Therefore,
development of novel agents possessing high activity against the influenza virus is one of the
vital tasks in medicinal chemistry. Previously we have shown that the product of reaction
between camphor and aminoethanol, referred by us to as camphecene, has a wide range of
antiviral activity along with a low toxicity4 (Fig. 1). It was shown that the inhibiting activity of
camphecene is based on the inhibition of viral hemagglutinin (HA) and is therefore the most
prominent in the early stages of virus replication5. This agent is a top compound in our library of
the camphor-based compounds6. Natural bicyclic camphor framework is similar to that of
deitiforin which is an antiviral drug applied in Russia earlier. A wide spectrum of antiviral
activity in the camphor amino-derivatives was found as early as in the 90s7. Authors8 suggest
that aminocamphor derivatives presents the M2 ion channel blockers involved in viral
replication. The present study was directed on the synthesis of camphecene using the click
chemistry methodology. Most attention was concentrated on the synthesis of compounds bearing
both previously identified pharmacophoric moiety and a substituted triazole ring9.
HCl
NH2
NH2
OH
N
NH2
HCl
Camphecene
Rimantadine
Amantadine
Deitiforin
CTD50 (µM) 2500,
ED50 (µM) 5,
SI 500
CTD50 (µM) 335,
ED50 (µM) 67,
SI 5
CTD50 (µM) 1260,
ED50 (µM) 206,
SI 6
CTD50 (µM) 280,
ED50 (µM) 70,
SI 4
Fig 1. Structural formula of anti-influenza drugs and antiviral activity of camphor derivatives
against influenza A(H1N1) pdm09 virus. (CTD50 - the median cytotoxic dose; EC50 - the 50%
effective concentration; SI - the selectivity index)
In search for novel camphor-based compounds having a pronounced antiviral activity, we
decided to use the click chemistry methodology. This choice was not by coincidence. Reported
in literature there are a great number of successful applications of catalytic cycloaddition
reactions to design biologically active compounds. In particular, [2+3] cycloaddition reaction of
azides to acetylenes was used to construct organic conjugates displaying antiviral activity10. In
some cases, introduction of the 1,2,3-triazole ring results in a rise of antiviral activity10b-d
.

Therefore, we decided to use click reactions in our study to produce conjugates of 1,7,7-
trimethyl-[2.2.1]bicycloheptane with various pharmacophoric groups. This methodology
suggests the formation of various biologically active conjugates in reaction of [2+3]
cycloaddition of azides to acetylenes11. In this study, we have maintained the camphecene
scaffold and linked 1,2,3-triazole as a headgroup. The design strategy is shown in Fig. 2. It was
also important for us to identify the influence of linker length and type of R (R') in the 1,2,3-
triazole ring.
Scaffold
1,2,3-triazole ring
N
N
R
N
N
previos work
this work
Linker
OH
O
N
N
N
(+) Camphor
Camphecene
O
N
R1
N
Scaffold
1,2,3-triazole ring
Fig 2. Design strategy for triazole-containing compounds
Based on the above, we have synthesized acetylene 2 and azide 4 building blocks (Scheme 1).
Camphecene propargyl ether 2 was obtained in one step by the Williamson reaction12 using
camphecene alkoxylate obtained in situ by reaction of 1 and sodium hydride in toluene. After a
work-up procedure of the reaction mixture with propargyl bromide at room temperature followed
by isolation of the target compound by column chromatography, hitherto unknown acetylene 2
was obtained in 58% yield. The second azide bulding block was obtained in two steps. At the
first step, reaction of camphecene and methyl sulfonyl chloride gave mesylate 3 in nearly
quantitative yield requiring no further purification. Analytically pure sample of the compound 3
was obtained through the column chromatography on silica gel using a СНСl3/СН3ОН mixture
as an eluent. Nucleophilic substitution of the methylsulfonate group for the azide one was
conducted under standard conditions13 using acetonitrile as a solvent. After purification on the
silica gel, azide 4 was obtained in 96% yield.

b
O
OH
N
S
N
O
O
3
a
1
c
O
N
N3
N
2
4
f
f
N3R1
R
e
d
N
25-28
N
5-14
O
N
N
N
33
N
N
N
N
N
R1
O
N
R
N
N
29-32
15-24
OH
R
Si
OH
OH
5, 15
6, 16
7, 17
9, 19
8, 18
O
O
O
H
N
O
O
O
P
P
N
O
O
N
H
O
P
10, 20
11, 21
O
O
O
O
14, 24
12, 22
13, 23
OH
R1
O
O
P
O
O
O
28,32
25, 29
26,30
27,31
Scheme 1. Reagents and conditions: (a) 1. NaH, Toluene, 2. BrCH2C≡CH, (b) CH3SO2Cl,
Et3N, -10°C, (c) NaN3, CH3CN, 60°C, (d) R≡CH (1.0 equiv.), Cu+, t-BuOH/H2O, 24 h, (e) N3R1
(1.0 equiv), Cu+, t-BuOH/H2O, 24 h, (f) 4, 2, (1:1), Cu+, t-BuOH/H2O, 24 h.
Structure of the compounds 2 and 4 were unambiguously confirmed by the NMR and IR
spectroscopy techniques. In the 1Н NMR spectrum of compound 2, among the signals
characteristic of the starting camphecene, a triplet at 2.41 ppm with is observed with the spin-
spin coupling constant JHH =4.0 Hz evidencing the presence of the acetylenic proton. In the IR

spectrum an absorption band at 2112 cm-1 (νC≡C) is observed14. For compound 4 the highly
intensive absorption band at ν (N3) 2099 cm-1 in the IR spectrum is the most important
characteristic for organic azides15.
At the next step of our study, the azide building block 4 was reacted with acetylenes, and
the acetylenic building block was reacted with organic azides, correspondingly. It should be
noted that synthesis of triazoles by the cycloaddition reaction is well studied. In particular,
organic azides were established to react readily with non-symmetrical acetylenes to give adducts
as a mixture of two geometric isomers16. Formation of isomers decreases the synthetic value of
this reaction since isolation of individual isomer presents a laborious task. In 2001, Sharpless
demonstrated that copper-catalyzed reaction between organic azides and acetylenes results in a
regioselective addition to give a single isomer17. Presently, various modifications of click
reactions are known18. We have chosen the most simple and well-characterized variant, namely,
carrying out the reaction in a t-BuOH-water mixture, with catalysis of the Cu(I) ions generated in
situ from CuSO4 and sodium ascorbate19. With this procedure and the azide building block 4 in
hand, we performed its cycloaddition to a number of acetylenes. As acetylenic substrates the
compounds of various structure having a triple bond were chosen. Commercially available
acetylenes 5-10 were used. Compounds 12, 13 were obtained according to the previously
reported procedures20, 21
.
Reaction of azide 4 and acetylenes 5-14 under catalytic conditions was carried out at
20°C for 24 hours (monitoring by TLC). As the reaction proceeds, the ν (C≡C) and ν (N3)
absorption bands at 2100 cm-1 are gradually decreased while an absorption band in the range of
1300-1000 cm-1 appears testifying the formation of the 1,2,3-triazole ring. An addition evidence
of the triazole ring formation is the NMR spectroscopy data. In the 13C NMR spectra of
compounds 15-24 signals of the carbon atoms of opposite polarity are observed at 120 and 140
ppm, characteristic of the 1,2,3-triazole ring22. Compounds 15-24 were isolated by column
chromatography. Using acetylenic building block 2 in the catalytic reactions with organic azides
25-28, we have also obtained the triazole-containing derivatives of 29-32. Commercially
available azides 26, 27, and azides 2823 2524 obtained according to the previously reported
procedures were used. The reaction conditions were similar to those used to obtain compounds
15-24.
At a final step of the synthetic part of our study, we performed a catalytic cycloaddition
reaction between acetylenic 2 and azidic 4 building blocks. Azide 2 reacted with acetylene 4
under catalytic conditions for 24 hours at 20°C (monitored by TLC). After a standard work-up
and purification adduct 33 was isolated in 75% yield as a single isomer.

1
Despite the rather complex H and 13C NMR spectra for this compound one may
unambiguously confirm that each group of atoms shows a single group of signals and in the
triazole ring only one signal of the C-H protons is observed (i.e. there is only one singlet in the
20
1H NMR spectrum). Compound 33 is optically active and has [α]D -82.9. In the IR spectrum
two νC=N bands are observed, and the 13C NMR spectrum shows two sets of signals of the
camphor framework. There are no signals of the starting azide and acetylene in spectra of the
final compounds.
The obtained compounds 2, 4, 14-24 and 29-33 have been studied as potential antiviral
agents. Adamantane- and bornane-based derivatives were used as reference compounds due to
their similarity to the compounds under investigation in having rigid cage fragments in their
structures.
Table 1. Antiviral activity of camphor-based compounds 2, 4, 15-24, 29-33 against influenza
virus A/Puerto Rico/ in MDCK cells.
Compound
CTD50a, µM
ED50b, µM
SIc
971 52
>1364
303 19
32 2
>429,2
455 50
56 7
2
3
2
4
5
15
>31
1
16
>1087
>1034,5
>987
616 73
55 6
2
17
19
31
2
18
32 5
19
195 16
865 61
92 8
>100
20
25 4
34
2
21
>58,4
314 40
45 5
22
>555
2
23
>836
18
19
2
24
>938
50 7
29
138 11
204 13
>641
63 8
30
31
24 3
8
299 32
98 11
2
32
117 8
344 20
266 19
1113 73
1
33
Rimantadine
Amantadine
Deitiforin
72
60
6
8
5
4
181 22
6

a CTD50 - cytotoxic concentration; the concentration affording 50% death of cells.
b ED50 - effective concentration; the concentration affording 50% inhibition of virus replication.
c SI - selectivity index, ratio CTD50/ ED50
As can be seen from the data obtained, most part of the compounds are not toxic for cells.
Trimethylsilyl derivative 16 showed the highest toxicity. In terms of anti-influenza activity, it
should be noted that no modifications performed gave a rise of activity compared to the initial
compound camphecene. The highest antiviral activity along with a low toxicity was revealed for
compounds 19 and 21. The selectivity index of these compounds is several times higher than that
for a reference drug. The structure-activity relationship analysis suggests that to possess virus-
inhibiting activity, the compounds of this group should bear oxygen atom with a short linker
(C2-C4), either as a hydroxyl group 18, 19, 29, keto-group 21 or as a part of heterocycle 24.
These compounds demonstrated low cytotoxicity with high antiviral activity (low values of
IC50). Phosphorus- or silicon-containing compounds as well as compounds with aliphatic,
benzene, tert-butyl or nitrogen substituents showed no selectivity to influenza virus. This
suggests that in addition to camphor-based imine moiety, other groups contribute to the binding
of these ligands to a specific viral target.
In our study, the model influenza virus A/Puerto Rico/8/34 (H1N1) was used. Given that
influenza viruses of types A and B, as well as subtypes H1N1 and H3N2 substantially differ in
their structure, particularly in the case of hemagglutinin, it is important to determine whether
triazole-modified camphor derivatives are effective against viruses H3N2 and B, too, or their
spectrum of activity is restricted by the H1N1 virus only. Aisa et al.9 have demonstrated that the
compounds from the chemical library of aminoalkyl rupestonates, being active against H1N1
virus, were not active against H3N2 and B viruses (for instance, for the most active compound
SI’s are 188, 5 and less than 1, respectively). This issue regarding triazole derivatives of
camphecene should be the subject of our further studies.
Acknowledgments
This work has been partially financially supported by Russian Scientific Foundation,
grant No. 15-03-00663
Supplementary data
Supplementary data associated with this article can be found, in the online version, at
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Influenza virus
A/H1N1 Puerto Rico
N
N
[CuI],
R
CH
N3
N
N
R
N
R = n-C4H9, (CH2)nOH (n=1,2), CH2NHCH3, CH2CH[P(O)(OEt)2], etc.
click
chemistry
OH
N
Camphecene
N
N
[CuI],
R1N3
N
R1
O
OCH2C
CH
N
N
R1 = CH2CH2OH, Ph, CH2C(O)OC(CH3)3, CH2(CH2)2CH2P(O)(OEt)2, etc.
25.