Thiophene-based water-soluble fullerene derivatives as highly potent antiherpetic pharmaceuticals

Article abstract of DOI:10.1039/d0ob01826k

Here we report the Friedel-Crafts arylation of chlorofullerenes C60Cl6 and C70Cl8 with thiophene-based methyl esters. While C60Cl6 formed expected Cs-C60R5Cl products, C70Cl8 demonstrated a tendency for both substitution of chlorine atoms and addition of an extra thiophene unit, thus forming Cs-C70R8 and C1-C70R9H compounds. The synthesized water-soluble C60 and C70 fullerene derivatives with thiophene-based addends demonstrated high activity against a broad range of viruses, including human immunodeficiency virus, influenza virus, cytomegalovirus, and herpes simplex virus. The record activity of C70 fullerene derivatives against herpes simplex virus together with low toxicity in mice makes them promising candidates for the development of novel non-nucleoside antiherpetic drugs.

Full text of DOI:10.1039/d0ob01826k

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Kushch and P. Troshin, Org. Biomol. Chem., 2020, DOI: 10.1039/D0OB01826K.
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DOI: 10.1039/D0OB01826K
ARTICLE
Thiophene-based water-soluble fullerene derivatives as highly
potent antiherpetic pharmaceuticals†
Olga A. Kraevaya,a,b* Alexander S. Peregudov,c Natalia E. Fedorova,d Regina R. Klimova,d Ivan A.
Godovikov,c Denis V. Mishchenko,b Alexander F. Shestakov,b,e Dominique Schols,f Alla A. Kushch,d
and Pavel A. Troshina,b
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Here we report Friedel-Crafts arylation of chlorofullerenes C60Cl6 and C70Cl8 with thiophene-based methyl esters. While
C60Cl6 formed expected Cs-C60R5Cl products, C70Cl8 demonstrated a tendency to both substitution of chlorine atoms and
addition of extra thiophene unit, thus forming Cs-C70R8 and C1-C70R9H compounds. The synthesized water-soluble C60 and
C70 fullerene derivatives with thiophene-based addends demonstrated high activity against a broad range of viruses,
including human immunodeficiency virus, influenza virus, cytomegalovirus, and herpes simplex virus. Record activity of C70
fullerene derivatives against herpes simplex virus together with low toxicity in mice make them promising candidates for
the development of novel non-nucleoside antiherpetic drugs.
based on the acyclic guanosine analogues, such as acyclovir
and ganciclovir.10 Unfortunately, long-term treatment with
nucleoside analogues usually leads to the development of drug
resistance. For example, several clinical surveys reported a
Introduction
The unique structure and electronic properties of the
fullerenes lead to their massive application in organic and
hybrid electronics,1 supramolecular chemistry,2 and medicine.3
Outstanding antioxidant, antitumor, neuroprotective and
antiviral activity of the water-soluble fullerene derivatives
makes them one of the most perspective platforms for
designing novel drugs.3 Although these unique compounds
demonstrated significant antiviral activity against a wide range
of viruses, such as human immunodeficiency virus (HIV),4
influenza,5 Zika, Dengue,6 hepatitis C,7 and Ebola8 viruses, their
anti-herpetic activity is still not thoroughly and systematically
investigated.
Herpesviral infections are prevalent in the human population
and caused mainly by herpes simplex virus (HSV) and
cytomegalovirus (HCMV). Although the majority of herpesviral
infections are mild and asymptomatic, infection in the fetal
and perinatal periods can be fatal or cause severe birth
defects.9 First-line treatment of the herpesviral infections is
rate of acyclovir-resistant HSV isolates of up to 36%.10,11
Therefore, the development of novel types of antiherpetic
drugs with different mechanisms of action is highly important.
Mixtures of fullerenols C60(OH)12-14, C60(OH)18-24, C60(OH)30-38
were reported to inhibit HSV in the concentrations of 46-123
µM, which is comparable or less effective than acyclovir
(EC50=48 µM).12 Polycarboxylic fullerene derivatives with
aromatic addends demonstrated the HSV half-inhibiting
concentrations of 0.2-10 µM and selectivity indexes SI
(SI=CC50/EC50) up to 1962, which illustrates their much higher
potency as compared to acyclovir.13 Water-soluble fullerene
derivatives with the residues of amino acids demonstrated
pronounced activity against HCMV with the SI values up to
5000, which makes them five times more selective than
ganciclovir (SI=1000).14 Though the mechanism of the
antiherpetic activity of the fullerene derivatives is still not
completely understood, their antioxidative activity is believed
to play a role in the inhibition of cytomegalovirus.14
While screening the antiherpetic activity of a broad range of
water-soluble fullerene derivatives, we discovered an
outstanding anti-HSV activity of the thiophene-based C60 and,
especially, C70 derivatives. Although our previous work
described the synthesis of Cs-C60R5Cl and Cs-C70R8 fullerene
derivatives, where R is the residue of the thiophene-based
carboxylic acid,15 here we report synthesis of previously
unknown compounds Cs-C60R5X (X = H, Me, Et) and C1-C70R9H
(Scheme 1) to reveal the structure-activity relationships.
a. Skolkovo Institute of Science and Technology, Nobel St. 3, Moscow, 143026,
Russia. E-mail: okraevaya@inbox.ru.
b. IPCP RAS, Semenov Prospect 1, Chernogolovka, 142432, Russia.
c. INEOS RAS, Vavylova St. 28, B-334, Moscow, 119991, Russia.
d. Honored Academician N.F.Gamaleya National Research Centre for Epidemiology
and Microbiology of the Ministry of Health of the Russian Federation, Gamaleya
st. 18, 123098, Moscow, Russia.
e. Faculty of Fundamental Physics & Chemical Engineering, Moscow Lomonosov
State University, GSP 1,1-51 Leninskie Gory, Moscow 119991, Russia.
f. Rega Institute for Medical Research, KU Leuven, Herestraat 49, 3000 Leuven,
Belgium.
†
Electronic
Supplementary
Information
(ESI)
available.
See
DOI: 10.1039/x0xx00000x
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antiviral activity of compounds dramatically.13 To reveal the
OH
View Article Online
O
X
influence of this modification on the anDtOivI:i1r0a.l10a3c9t/iDv0itOyBo01f8t2h6eK
thiophene-based fullerene derivatives, we synthesized
compounds 1b,c,d, 2b, and 3b using the methods, reported
earlier (Scheme 3).13,17
HO
X
OH
S
previous work
O
Cl
O
S
Cl
Cl
X
S
OH
S
Cl
Cl
X
Cl
Cl
X
S
O
HO
O
S
Friedel-Crafts
reaction conditions
X
O
S
HO
X
S
O
Cl
S
O
HO
X
OH
O
X
R
R
R
S
OCH3
OH
Limited solubility of the
acidic form of Cs-C70R8
prevents its further
S
S
S
X
O
R
R
R
Cs-C70R8
this work
S
S
S
S
S
S
P(OX)3
PhCH3
110oC, 30 min
Friedel-Crafts
reaction conditions
PPh3, H2O
PhCH3
H
Cl
X
R
R
R
S
S
S
confersion to C1-C70R9H
H3CO
r.t., 10 min
OCH3
X
OCH3
O
X
O
X
O
X
S
S
H3CO
X
H3CO
S
S
S
OCH3
H
OCH3
R
R
R
S
O
O
O
S
O
S
R
R
R
X
X
S
S
X = Me (c), Et (d)
1b: 94%
2b: 89%
3b: 91%
1: R=CH2COOCH3
2: R=CH2CH2COOCH3
3: R=CH2CH2CH2COOCH3
X
X
S
S
1c: 94%
1d: 90%
H3CO
H3CO
O
O
S
S
X
X
OCH3
OCH3
S
S
O
O
X
X
S
S
O
O
S
S
O
O
Scheme 3. Synthesis of the fullerene derivatives C60R5X (X=H, Me, Et).
H3CO
H3CO
X
X
OCH3
OCH3
O
O
X
X
OCH3
OCH3
C1-C70R9H
Cs-C70R8
Typically, Friedel-Crafts reactions of chlorofullerene C70Cl8 with
phenyl-substituted esters18 and thiophene-based acids15 lead
to the formation of pure Cs-C70R8 fullerene derivatives.
Surprisingly, the reaction of chlorofullerene C70Cl8 with
thiophene-based esters resulted in the formation of two major
products: expected Cs-C70R8 derivative and untypical C1-
symmetrical C70R9H. Interestingly, while reaction with 3-
thienylpropanoic and 4-thienylbutanoic acid esters lead to the
formation of both Cs-C70R8 and C1-C70R9H fullerene derivatives,
2-thienylacetic acid reacted selectively forming Cs-C70R8.
Most probably, after the substitution of all eight chlorine
atoms on the fullerene cage, reactive 7,8-double bond (atom
numbering following R. Taylor19) on the C70 cage is attacked by
other thiophene ester molecule, leading to the formation of
C1-C70R9H. Limited solubility and the tendency for aggregation
of compound 4a in solution, observed visually and also
confirmed by the peak broadening in 1H NMR spectra,
prevents its interaction with the excess of the thiophene-
based ester. The low solubility of Cs-C70R8, where R is the
residue of the thiophene-based carboxylic acid, in 1,2-
dichlorobenzene is also believed to be the driving force for the
selective formation of pure Cs-C70R8 fullerene derivatives in the
reactions of C70Cl8 with thiophene-based carboxylic acids
(Scheme 4).15
Scheme 1. Thiophene-based C70 fullerene derivatives.
The most promising compounds demonstrated high efficiency
of HSV inhibition (EC50=0.04 µM) together with low
cytotoxicity (CC50=1690 µM) leading to the outstanding
selectivity indexes exceeding 40 000. Record anti-HSV activity
and low acute toxicity in mice make them promising
candidates for the development of novel antiherpetic drugs.
Results and discussion
Synthesis of the fullerene derivatives
The experimental procedure for the synthesis of the
thiophene-based fullerene derivatives was quite simple.
Reaction conditions were similar to those reported
previously,15 except thiophene-based methyl esters were used
instead of thiophene-based acids. Typically, chlorofullerene
C60Cl6 or C70Cl8 was dissolved in o-dichlorobenzene together
with an excess of thiophene-based ester (25 eq.) (ESI, Fig. S1)
and SnCl4 (cat.) under stirring at 70oC in inert atmosphere for
30-60 min. Reaction mixtures were analysed using analytical
HPLC (ESI, Fig. S2). Flash column chromatography and
preparative HPLC were used to purify the samples.
The products derived from C60Cl6 have Cs-symmetrical
structures similar to those reported before for the adducts
with thiophene16 and thiophene-based acids15 and contain five
organic addends and one chlorine atom attached to the
fullerene cage (Scheme 2). Compounds 1-3(a) were
synthesized with ester-protected carboxylic groups to perform
their further functionalization as described below.
R
R
R
R
R
S
H
S
S
S
S
R
R
S
S
S
OCH3
S
S
R
S
R
S
O
n
R
SnCl4, 1,2-DCB
80oC, 30 min
R
S
S
R
S
R
S
S
S
this work
R
Cl
Cl
Cl
R
R
(a)
(b)
R
Cl
Cl
Cl
Cl
4: R=CH2COOCH3
5: R=CH2CH2COOCH3
6: R=CH2CH2CH2COOCH3
4a: 81%
5a: 55%, 5b: 43%
6a: 40%, 6b: 32%
R
S
R
S
Cl
R
previous work
S
Cl
Cl
R
R
S
S
S
OH
S
S
R
R
Cl
Cl
Cl
S
O
n
1a: R=CH2COOCH3, 51%
R
S
S
2a: R=CH2CH2COOCH3, 47%
3a: R=CH2CH2CH2COOCH3, 36%
SnCl4, 1,2-DCB
80oC, 30 min
R
SnCl4, 1,2-DCB
80oC, 30 min
S
R
S
Cl
Cl
S
S
R
R
R
(a)
R
A4: R=CH2COOH, 95%
A5: R=CH2CH2COOH, 95%
A6: R=CH2CH2CH2COOH, 95%
Scheme 2. Synthesis if the thiophene-based C60 fullerene derivatives C60R5Cl.
Substitution of the chlorine atom in the arylated fullerene
derivatives C60R5Cl by hydrogen or alkyl group can change the
Scheme 4. Synthesis of the thiophene-based C70 fullerene derivatives.
2 | J. Name., 2012, 00, 1-3
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The observed substitution and addition reactions on the ortho-positioned bulky -OOtBu and –CF3 groupsV.ieDwuAertictleoOntlhinee
fullerene C70 cage emphasize the importance of the solubility- small size of a hydrogen atom, steric repDuOlsIi:o1n0.1b0e3t9w/De0eOnBo01r8th26oK-
driven effects in the fullerene chemistry and provide the positioned substituents in C70R9H compounds 5-6(b) is low,
access to the water-soluble fullerene derivatives with the which leads to more typical addition across 7,8-double bond as
untypical arrangement of the addends on the fullerene cage.
Characterization and structure determination
shown on Fig. 2(B). UV/vis spectra of compounds 5-6(b) show
strong similarity with the spectra of the previously reported
fullerene derivatives with the same addition pattern, such as
C70(CF3)8Cl2,27 C70(CF3)8(CN)2,28 and C70(CF3)8[C(PhOMe)2],31
(ESI, Fig. S58). Dynamics of the HPLC profiles for the reactions
of C70Cl8 with thiophene-based esters (ESI, Fig. S59) confirmed
in situ conversion of Cs-C70R8 to untypical fullerene derivatives
C1-C70R9H, thus proving that eight of nine addends in the
structure of C1-C70R9H have the same positions as in Cs-C70R8.
Quantum chemical calculations were performed to simulate 1H
and 13C NMR spectra of isomers B-D (Fig. 2). Comparison of
theoretical and experimental NMR spectra undoubtedly
confirmed type B structure for C1-C70R9H compounds. For C
and D isomers, the predicted signals of sp3-hybridized C atoms
of the fullerene cage were strongly displaced as compared to
the experimental chemical shifts (ESI, Figs. S60-61).
High purity of the isolated thiophene-based fullerene
derivatives was revealed by HPLC, and composition was
confirmed by ESI mass spectra. Molecular structures of all
synthesized fullerene derivatives were additionally proved by
1
1
1D (1H, 13C) and 2D (1H-1H COSY, H-13C HSQC, H-13C HMBC)
correlation NMR spectroscopy (ESI, Figs. S3-57). UV/vis spectra
1
were used to confirm the addition pattern. H and 13C NMR
spectra of compound 5b are shown in Figure 1 as an example.
a
(D) C1-C70R10
(R=CF3)
(C) C2-C70R10
(R=OOtBu)
(B) Cs-C70R10
R=H, Cl, Ph, this work, etc.
(A) Cs-C70R8
Figure 2. Schlegel diagrams for the different families of C70R10 fullerene
derivatives.
b
Compounds 1-6 comprised from five to nine esterified
carboxylic groups, which were deprotected by acidic
hydrolysis. The corresponding polycarboxylic acids A1-A6 were
obtained with nearly quantitative yields. Spectroscopic data
for compounds A1a, A4a, A5a, A6a has been reported in the
previous work.15 Structures and composition of the
synthesized acids A1(b-d), A2(a,b), A3(a,b), A5b, A6b were
confirmed by 1D and 2D NMR spectra. Water-soluble
potassium salts K1-K6 were prepared by treatment of the acids
with a stoichiometric amount of aqueous K2CO3.
Biological activity
Antiviral properties of thiophene-based water-soluble
fullerene derivatives were systematically explored in vitro
against HIV-1 (NL4.3, BaL), HIV-2 (ROD), Influenza A (H1N1,
H3N2) and B, HCMV and HSV (Table 1, ESI, Tables S1-S4).
Compound K5b demonstrated pronounced activity against
HIV-1/NL4.3 with EC50 value of less than 1 µM. Water-soluble
fullerene derivative K2b effectively inhibited Influenza A H3N2
virus in the concentration of 0.7 µM. Compounds K3b and K5b
selectively inhibited HCMV in the concentration of ~3 µM in
prophylactic and virulicide schemes, respectively.
Figure 1. 1H (a) and 13C (b) NMR spectra of compound 5b
While the presence of σs symmetry plane in the molecules of
the fullerene derivatives Cs-C60R5X (X=Cl, H, Me, Et) and Cs-
C70R8 undoubtedly confirms their structure, C1-symmetrical
structures of compounds C70R9H (5-6(b)) were predicted using
quantum chemical calculations and proved by absorbance
spectroscopy data and dynamic HPLC experiments.
Within a couple of specific exceptions, most of all C70
derivatives bearing ten substituents, including C70H10,20
C70Cl10,21 C70Br10,22 C70Me10,23 C70Ph10,24 C70Ph9(OH),25
While most of the synthesised compounds demonstrated
pronounced, but not record activities against HIV-1, HIV-2,
influenza A and B and HCMV, their activities against HSV were
outstanding (Table 1).
C70Ph8(OH)2,26 C70(CF3)8Cl2,27 C70(CF3)8(CN)2 have the Cs-
28
symmetrical “equatorial-belt” structure, shown on Fig. 2(B).
The exceptions are represented by C2-C70(OOtBu)1029 (Fig. 2(C))
30
and C1-C70(CF3)10 (Fig. 2(D)) derivatives, where untypical
addition patterns are explained by notable steric repulsion of
This journal is © The Royal Society of Chemistry 20xx
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outstanding anti-HSV activity of Cs-C70R8 fullerene derivatives,
exceeding antiviral activity of acyclovir and other widely used
antiherpetic drugs; their low acute toxicity and high availability
makes them promising candidates for the development of
novel non-nucleoside anti-herpetic pharmaceuticals.
R
S
View Article Online
DOI: 10.1039/D0OB01826K
R
R
R
S
R
R
H
S
S
S
S
R
R
R
R
R
S
S
S
S
S
S
X
R
S
R
R
S
S
S
S
R
S
R
S
S
S
R
S
R
R
R
R
(a)
(b)
R
X=Cl (a), H (b), Me (c), Et (d)
K4: R=CH2COOK*
K5: R=CH2CH2COOK*
K6: R=CH2CH2CH2COOK*
*K1a, K4a, K5a, K6a were reported in ref. 16
Experimental
Synthesis of С60 fullerene derivatives 1-3(a)
K1: R=CH2COOK*
K2: R=CH2CH2COOK
K3: R=CH2CH2CH2COOK
Figure 3. Structures of thiophene-based water-soluble fullerene derivatives.
A solution of chlorofullerene C60Cl6 (500 mg, 0.536 mmol, 1
eq.) in 70 mL of 1,2-dichlorobenzene was degassed, and the
reaction system was filled with argon. Then methyl ester of the
thiophene-based carboxylic acid (10.7 mmol, 20 eq.) and SnCl4
(50 μL) were added to the reaction mixture. The reaction
mixture was heated to 70-80° C and stirred at this temperature
for 30-60 minutes. Afterwards, it was cooled to the room
temperature, diluted with toluene (200 mL), and poured on
the top of the silica gel column. The target product was eluted
using toluene/methanol mixtures (97-90% : 3-10% v/v). The
obtained solutions were concentrated at the rotary
evaporator, and then preparative HPLC (C18 column) was used
to purify the compounds. Fullerene derivatives 1-3(a) were
obtained as bright red-orange powders.
The most effective compounds K5a and K6a combined low
cytotoxicity and high antiviral activity resulting in the high
selectivity indexes (SI=CC50/EC50) of about 40000. These SI
values represent the best results reported so far for fullerene
derivatives and exceed by several times SI of the common drug
acyclovir. It should be emphasized that fullerene derivative
K6a works in all treatment schemes: microbicidal,
prophylactic, virulicide, and therapeutic. Compounds K5a and
K6a have similar structures with eight symmetrically attached
addends, which differ only by one CH2 group.
Acute toxicity in mice was investigated for the most promising
compound K5a (ESI, Fig. S62). Semi-lethal dose LD50 was
determined as 483 ± 27 mg/kg. Lower acute toxicity of K5a as
compared to the conventional drug aspirin (LD50=167 mg/kg)
suggests its safety for biomedical application.
Synthesis of С60 fullerene derivatives 1-3(b)
Replacement of Cl in С60R5Cl with H was achieved using
Ph3P/H2O treatment reported previously.17
Table 1. Antiviral properties of the thiophene-based water-soluble fullerene
derivatives
Synthesis of С60 fullerene derivatives 1(c, d)
Substitution of Cl in С60R5Cl with X was achieved using P(OX)3
treatment reported earlier.13
Synthesis of С70 fullerene derivatives 4-6(a, b)
a
b
СС50,
µM
EC50,
µM
Virus strain
HSV
Compound
SI
K5ac
K6ac
K3bd
K5be
K5b
K3a
1692
1600
672
0.0419
0.04
2.96
3.02
0.70
2.31
1.31
40380
40000
227
141
>142
>43
A dispersion of chlorofullerene C70Cl8 (500 mg, 0.445 mmol, 1
eq.) in 70 mL of 1,2-dichlorobenzene was degassed, and the
reaction system was filled with argon. Then methyl ester of the
thiophene-based carboxylic acid (14.2 mmol, 32 eq.) and SnCl4
(50 μL) were added to the reaction mixture. The reaction
mixture was heated to 70-80° C and stirred at this temperature
until HPLC profile demonstrated conversion to the required
product(s). Afterwards, it was cooled to the room
temperature, diluted with toluene (200 mL), and poured on
the top of the silica gel column. The target product was eluted
using toluene/methanol mixture (85% : 15% v/v). The obtained
solution was concentrated at the rotary evaporator, and then
preparative HPLC (C18 column) was used to separate
compounds 5-6(a, b). Compounds 4-6(a) were obtained as
brownish-orange powders. Compounds 5-6(b) appeared as
yellow powders.
HCMV
426
HIV-1 NL4.3
HIV-1 BaL
HIV-2 ROD
Influenza
A/H3N2
> 100
> 100
> 100
K3b
>76
K2b
50.6
0.7
72
aCC50 - 50% cytotoxic concentration; bEC50 - 50% effective concentration, or
c
concentration producing 50% inhibition of virus-induced cytopathic effect; – in
microbicidal scheme; d – in prophylactic scheme; e – in virulicide scheme
Conclusions
We have thoroughly studied the reactivity of chlorofullerenes
C60Cl6 and C70Cl8 with thiophene-based esters under Friedel-
Crafts arylation conditions. While C60Cl6 formed expected Cs-
C60R5Cl derivatives, chlorofullerene C70Cl8 demonstrated a
tendency to both substitution of chlorine atoms and addition
of extra thiophene molecule to the 7,8-double bond, forming
Cs-C70R8 and C1-C70R9H. C70 fullerene derivatives with nine
organic addends and a hydrogen atom attached to the cage
were synthesized and characterized for the first time.
General procedure for the synthesis of polycarboxylic acids A(1-
6)(a-d)
Fullerene derivative (1-6)(a-d) (0.05 mmol), toluene (30 mL),
acetic acid (30 mL) and HCl (5 mL) were introduced into a two-
necked round bottom 100 mL flask equipped with a stirring
bar, condenser and thermometer. The mixture was stirred at
65-70oC for 5 days and concentrated using a rotary evaporator
to afford a powder, which was washed with acetonitrile and
dried in air.
Thiophene-based
water-soluble
fullerene
derivatives
demonstrated high activity against a wide range of viruses,
including HIV, Influenza virus, HCMV, and HSV. The
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General procedure for the synthesis of potassium salts K(1-6)(a-d)
animals were kept under standard vivarium condiVtiieownAsrtiwcleitOhnl1in2e
h lighting regime and free access to wateDrOaIn: d10f.1o0o3d9/.D0OB01826K
The test compound was administered to animals once
intraperitoneally in the form of an aqueous solution in the
dose range of 200-800 mg/kg (body weight). Observation of
animals was carried out continuously for the first 30 minutes
after administration of the substance, then with an interval of
1 hour for 4 hours, then every day once a day. The total follow-
up period was 30 days.
Fullerene-based polycarboxylic acid (0.05 mmol), distilled
water (20 mL) and stoichiometric amount of anhydrous
potassium carbonate (A(1-3)(a-d): 17.3 mg 0.125 mmol; A(4-
6)(a): 27.6 mg 0.2 mmol; A(5-6)(b): 31.1 mg 0.225 mmol) were
placed into a single-necked flask and the reaction mixture was
stirred until complete dissolution. Then the solution was
filtered through a PES syringe filter (pore size 0.45 μm) and
freeze-dried to afford a powder of water-soluble salt with
quantitative yield.
To determine the toxic doses, the toxicity curve was
constructed, and the LD16, LD50, LD84, LD100 parameters were
calculated using the probit analysis method.
Quantum chemical calculations
Theoretical calculations were performed using PBE density
functional method32 using the PRIRODA program package33 at
the Joint Supercomputer Center of the Russian Academy of
Sciences.
For geometry optimization SBK pseudopotential32 was used
and extended basis set H [5s 1p / 3s 1p] C, O, S [5s 5p 2d / 3s
3p 2d] was used for valence shells. Magnetic shielding
constants were calculated using Λ2 basis H [8s 4p 2d / 3s 2p
1d] C, O [12s 8p 4d 2f/ 4s 3p 2d 1f] S [18s 13p 5d 2f/5s 4p 2d
1f]34 of cc-pVTZ quality at PBE/SBK optimized geometry.
Antiviral activity assays
It was shown that with intraperitoneal administration of
aqueous solutions of K5a in doses up to 300 mg/kg, the death
of animals was not observed throughout the entire
observation time (30 days). Animals begin to die after
administration of the test compound in doses of 400 to 700
mg/kg. In the surviving animals, no clinical manifestations of
toxicity and changes in behavioral reactions were observed
until the end of the experiment.
Thus, studies of the acute toxicity of K5a with intraperitoneal
administration of its aqueous solutions showed that a semi-
lethal dose of LD50 = 483 ± 27 mg/kg, which allows it to be
Activities of the fullerene derivatives against Herpes Simplex classified as toxicity class III, i.e. compound K5a is moderately
virus type 1 (HSV1), human cytomegalovirus (HCMV), influenza toxic in accordance with GOST 12.1.007–76. Toxic doses were
viruses (Influenza A/H1N1 A/Ned/378/05, Influenza A/H3N2 also calculated: LD16 = 412, LD84 = 566 and LD100 = 608 mg/kg.
A/HK/7/87, and Influenza
B
B/Ned/537/05), human
immunodeficiency virus (HIV-1, NL4.3WT and BaL; HIV-2, ROD)
were determined using standard in vitro assays (ESI, Tables S1-
S4).
Conflicts of interest
There are no conflicts to declare.
HSV-sensitive Vero cells and HCMV-sensitive human embryo
lung fibroblasts (HELF) were used to evaluate the cytotoxicity
and antiviral activity of compounds. Antiviral activity was
evaluated by monitoring the ability of the compound to inhibit
a plaque formation in the cells infected with corresponding
viruses. Selectivity indices (SI) were calculated as CC50/EC50
(CC50 - concentration of the compound reducing the viability of
the cells by 50% within 1 (HSV1) or 3 (HCMV) days after its
addition to the culture medium, EC50 - concentration of
compound providing the 50% inhibition of the plaque-forming
activity). Full description of the procedures (cytotoxicity and
antiviral assays) was provided previously.13
MDCK (Madin Darby canine kidney cells) were used to
evaluate the cytotoxicity and antiviral activity of the
compounds against influenza viruses. Details were provided
previously.18
TZM-bl cell line was used to evaluate the cytotoxicity and
antiviral activity of the compounds against HIV.35
Acknowledgements
This work was supported by the Russian Science Foundation
(project 19-13-00411).
Notes and references
1
D. M. Guldi, B. M. Illescas, C. M. Atienza, M. Wielopolskia
and N. Martín, Chem. Soc. Rev., 2009, 38, 1587-1597; C.-Z. Li,
H.-L. Yip and A. K.-Y. Jen, J. Mater. Chem., 2012, 22, 4161-
4177; E. Castro, O. Fernandez-Delgado, F. Arslan, G. Zavala,
T. Yang, S. Seetharaman, F. D'Souza and L. Echegoyen, New
J. Chem., 2018, 42, 14551-14558.
F. Diederich and M. Gómez-López, Chem. Soc. Rev., 1999,
28, 263-277; D. Bonifazi, O. Engerc and F. Diederich, Chem.
Soc. Rev., 2007, 36, 390-414; D. Sun, F. S. Tham, C. A. Reed, L.
Chaker and P. D. W. Boyd, J. Am. Chem. Soc., 2002, 124, 23,
6604-6612; C. Fuertes-Espinosa, C. Garcia-Simon, M. Pujals,
M. Garcia-Borras, L. Gomez, T. Parella, J. Juanhuix, I. Imaz, D.
Maspoch, M. Costas and X. Ribas, Chem, 2020, 6, 1–18.
E. Castro, A. H. Garcia, G. Zavala and L. Echegoyen, J. Mater.
Chem. B, 2017, 5, 6523-6535; R. Bakry, R. M. Vallant, M.
Najam-ul-Haq, M. Rainer, Z. Szabo, C. W. Huck and G. K.
Bonn, Int. J. Nanomedicine, 2007, 2, 4, 639–649.
2
Determination of the acute toxicity of compound K5a in mice
All manipulations with the animals were performed in
accordance with the solutions of the Commission on Bioethics
of the Institute of Problems of Chemical Physics, Russian
Academy of Sciences (IPCP RAS) and Russian state veterinary
service (approval 250 №0038716). Acute toxicity studies were
conducted under ISO 10993-11-2011 standard.36 Female BDF1
laboratory mice with the weight of 20-22 g were used. The
3
4
E. Castro, Z. S. Martinez, C.-S. Seong, A. Cabrera-Espinoza, M.
Ruiz, A. Hernandez Garcia, F. Valdez, M. Llano and L.
Echegoyen, J. Med. Chem., 2016, 59, 10963–10973; Z. S.
Martinez, E. Castro, C.-S. Seong, M. R. Cerón, L. Echegoyen
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
Please do not adjust margins

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Organic & Biomolecular Chemistry
Page 6 of 6
ARTICLE
Journal Name
and M. Llano, Antimicr. Agents Chemother., 2016, 60, 5731– 23 H. Al-Matar, A. K. A. Sada, A. G. Avent, R. Taylor and X. W.
View Article Online
5741.
S. Tollas, I. Bereczki, A. Borbás, G. Batta, E. Vanderlinden, L. 24 A. G. Avent, P. R. Birkett, A. D. Darwish, H. W. Kroto, R.
Naesens and P. Herczegh, Bioorg. Med. Chem. Lett., 2014, Taylor and D. R. M. Walton, Tetrahedron, 1996, 52, 5235.
24, 2420–2423; M. Shoji, E. Takahashi, D. Hatakeyama, Y. 25 P. R. Birkett, A. G. Avent, A. D. Darwish, H. W. Kroto, R.
Wei, J. Chem. Soc., Perkin Trans. 2, 2002, 1251.
DOI: 10.1039/D0OB01826K
5
Iwai, Y. Morita, R. Shirayama, N. Echigo, H. Kido, S.
Nakamura, T. Mashino, T. Okutani and T. Kuzuhara, PLOS
ONE, 2013, 8, 66337.
J. Ramos-Soriano, J. J. Reina, B. M. Illescas, N. de la Cruz, L.
Rodríguez-Perez, F. Lasala, J. Rojo, R. Delgado and N. Martín,
J. Am. Chem. Soc., 2019, 141, 15403−15412.
H. Kataoka, T. Ohe, K. Takahashi, S. Nakamura and T.
Mashino, Bioorg. Med. Chem. Lett., 2016, 26, 4565–4567; S.
Xiao, Q. Wang, F. Yu, Y. Peng, M. Yang, M. Sollogoub, P. 28 T. T. Clikeman, I. V. Kuvychko, N. B. Shustova, Y.-S. Chen, A.
Taylor and D. R. M. Walton, Chem. Commun., 1996, 1231-
1232.
26 A. G. Avent, P. R. Birkett, A. D. Darwish, H. W. Kroto, R.
Taylor and D. R. M. Walton, J. Chem. Soc., Perkin Trans. 2,
2001, 68-72.
27 A. A. Goryunkov, N. A. Samokhvalova, P. A. Khavrel, N. M.
Belov, V. Yu. Markov, L. N. Sidorov and S. I. Troyanov, New J.
Chem., 2011, 35, 32-35.
6
7
Sinay, Y. Zhang, L. Zhang and D. Zhou, Bioorg. Med. Chem.,
2012, 20, 5616–5622.
B. M. Illescas, J. Rojo, R. Delgado and N. Martín, J. Am. Chem. 29 Z. Xiao, F. D. Wang, S. H. Huang, L. B. Gan, J. Zhou, G. Yuan,
Soc., 2017, 139, 17, 6018–6025. A. Muñoz, D. Sigwalt, B. M. M. J. Lu and J. Q. Pan, J. Org. Chem., 2005, 70, 2060.
Illescas, J. Luczkowiak, L. Rodríguez-Pérez, I. Nierengarten, 30 I. E. Kareev, I. V. Kuvychko, A. A. Popov, S. F. Lebedkin, S. M.
M. Holler, J.-S. Remy, K. Buffet, S. P. incent, J. Rojo, R.
Delgado, J.-F. Nierengarten and N. Martín, Nature Chemistry,
2016, 8, 1, 50–57.
A. Popov, O. V. Boltalina, S. H. Strauss, Chem. Eur. J., 2013,
19, 5070-5080.
8
9
Miller, O. P. Anderson, S. H. Strauss and O. V. Boltalina,
Angew. Chem., Int. Ed., 2005, 44, 7984.
31 A. V. Rybalchenko, M. G. Apenova, O. O. Semivrazhskaya, N.
M. Belov, V. Yu. Markov, S. I. Troyanov, I. N. Ioffe, N. S.
Lukonina, L. N. Sidorov, T. V. Magdesieva and A. A.
Goryunkov, Electrochim. Acta, 2016, 191, 980-986.
32 J. P. Perdew, K. Burke and M. Ernzerhof, Phys. Rev. Lett.,
1996, 77, 3865-3868.
W. J. Muller, C. A. Jones and D. M. Koelle, Curr. Immunol.
Rev., 2010, 6, 1, 38–55.
10 Y.-C. Jiang, H. Feng, Y.-C. Lin and X.-R. Guo, Int. J. Oral Sci.,
2016, 8, 1, 1–6.
11 J. C. Pottage and H. A. Kessler, Infect. Agents Dis., 1995, 4, 3,
115-24.
33 D. N. Laikov, Chem. Phys. Lett., 1997, 281, 151-156.
12 M. Yu. Eropkin, E. Yu. Melenevskaya, K. V. Nasonova, T. S. 34 D. N. Laikov, Chem. Phys. Lett., 2005, 416, 116-120.
Bryazzhikova, E. M. Eropkina, D. M. Danilenko and O. I. 35 O. A. Troshina, P. A. Troshin, A. S. Peregudov, V. I.
Kiselev, Pharmaceutical Chemistry Journal, 2013, 47, 2, 87-
91;
Kozlovskiy, J. Balzarini and R. N. Lyubovskaya, Org. Biomol.
Chem., 2007, 5, 2783.
13 N. E. Fedorova, R. R. Klimova, Yu. A. Tulenev, E. V. Chichev, A. 36 A. N. Mironov, Preclinical Drug Research Guide, 2012, 944.
B. Kornev, P. A. Troshin and A. A. Kushch, Mendeleev
Commun., 2012, 22, 254–256; O. A. Kraevaya, A. S.
Peregudov, S. I. Troyanov, I. Godovikov, N. E. Fedorova, R. R.
Klimova, V. A. Sergeeva, L. V. Kameneva, E. S. Ershova, V. M.
Martynenko, S. Claes, A. A. Kushch, S. V. Kostyuk, D. Schols,
A. F. Shestakov and P.l A. Troshin, Org. Biomol. Chem., 2019,
17, 7155-7160.
14 R. A. Kotel´nikova, I. I. Faingol´d, D. A. Poletaeva, D. V.
Mishchenko, V. S. Romanova, V. N. Shtol´ko, G. N. Bogdanov,
A. Yu. Rybkin, E. S. Frog, A. V. Smolina, A. A. Kushch, N. E.
Fedorova and A. I. Kotel´nikov, Russian Chemical Bulletin,
International Edition, 2011, 60, 6, 1172—1176.
15 O. A. Kraevaya, A. S. Peregudov, I. A. Godovikov, E. V.
Shchurik, V. M. Martynenko, A. F. Shestakov, J. Balzarini, D.
Schols and P. A. Troshin, Chem. Commun., 2020, 56, 1179-
1182.
16 P. R. Birkett, A. G. Avent, A. D. Darwish, I. Hahn, H. W. Kroto,
G. J. Langley, J. O’Loughlin, R. Taylor and D. R. M. Walton, J.
Chem. Soc., Perkin Trans. 2, 1997, 1121-1125.
17 A. G. Avent, P. R. Birkett, J. D. Crane, A. D. Darwish, G. J.
Langley, H. W. Kroto, R. Taylor and D. R. M. Walton, J. Chem.
Soc., Chem. Commun., 1994, 1463-1464.
18 A. B. Kornev, A. S. Peregudov, V. M. Martynenko, J. Balzarini,
B. Hoorelbeke and P. A. Troshin, Chem. Commun., 2011, 47,
8298–8300.
19 E. W. Godly and R. Taylor, Fullerene Sci. Technol., 1997, 5,
1667-1708.
20 H. P. Spielmann, B. R. Weedon and M. S. Meier, J. Org.
Chem., 2000, 65, 2755.
21 P. R. Birkett, A. G. Avent, A. D. Darwish, H.W. Kroto, R. Taylor
and D. R. M. Walton, J. Chem. Soc., Chem. Commun., 1995,
683.
22 S. I. Troyanov, A. A. Popov, N. I. Denisenko, O. V. Boltalina, L.
N. Sidorov and E. Kemniz, Angew. Chem., Int. Ed., 2003, 42,
2395.
6 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
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