Monoterpenoid-based inhibitors of filoviruses targeting the glycoprotein-mediated entry process

European Journal of Medicinal Chemistry 207 (2020) 1 12726

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European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Research paper

Monoterpenoid-based inhibitors of ﬁloviruses targeting the

glycoprotein-mediated entry process

,

Anastasiya S. Sokolova ^{a *}, Olga I. Yarovaya ^{a b}, Anastasiya V. Zybkina ^c,

Ekaterina D. Mordvinova ^a^c, Nadezhda S. Shcherbakova ^c, Anna V. Zaykovskaya ^c,

,

Dmitriy S. Baev ^a, Tatyana G. Tolstikova ^a^b, Dmitriy N. Shcherbakov ^c, Oleg V. Pyankov ^c,

,

Rinat A. Maksyutov ^c, Nariman F. Salakhutdinov ^a

^aN.N.Vorozhtsov Novosibirsk Institute of Organic Chemistry SB RAS, 630090, Novosibirsk, Lavrent’ev Av., 9, Russia

^bNovosibirsk State University, 630090, Novosibirsk, Pirogova St., 1, Russia

^cState Research Center of Virology and Biotechnology VECTOR, Rospotrebnadzor, 630559, Koltsovo, Novosibirsk Region, Russia

a r t i c l e i n f o

a b s t r a c t

Article history:

In this study, we screened a large library of (þ)-camphor and (ꢀ)-borneol derivatives to assess their

ﬁlovirus entry inhibition activities using pseudotype systems. Structure-activity relationship studies

revealed several compounds exhibiting submicromolar IC₅₀values. These compounds were evaluated for

their effect against natural Ebola virus (EBOV) and Marburg virus. Compound 3b (As-358) exhibited the

Received 28 May 2020

Received in revised form

21 July 2020

Accepted 3 August 2020

Available online 20 August 2020

good antiviral potency (IC₅₀¼ 3.7 M, SI ¼ 118) against Marburg virus, while the hydrochloride salt of

m

this compound 3b·HCl had a strong inhibitory effect against Ebola virus (IC₅₀¼ 9.1 M, SI ¼ 31) and good

m

in vivo safety (LD₅₀> 1000 mg/kg). The results of molecular docking and in vitro mutagenesis analyses

suggest that the synthesized compounds bind to the active binding site of EBOV glycoprotein similar to

the known inhibitor toremifene.

Keywords:

Borneol

Camphor

Ebola virus

Marburg virus

Glycoprotein

Mutagenesis study

1. Introduction

declared a new outbreak of Ebola virus disease in North Kivu

Province. The latest numbers provided by the WHO regarding the

Filoviruses belong to the Filoviridae family and can cause severe

hemorrhagic fever in humans and nonhuman primates, with a

mortality rate of up to 90%. Two human pathogenic genera of this

virus family have been identiﬁed: Ebolavirus and Marburgvirus. The

genus Ebolavirus has six species, among which the prototypic virus

Zaire ebolavirus [now known as Ebola virus (EBOV)], Sudan (SUDV),

Bundibugyo (BDBV), Reston (RESTV), Taï Forest (TAFV) and Bombali

virus (BOMV). Bombali virus is a new species that was identiﬁed in

Sierra Leon and Angolan free-tailed bats [1]. The Ebola virus was

ﬁrst identiﬁed in 1976 during outbreaks in the Sudan and Congo [2],

since which time several outbreaks have occurred. The largest

outbreak in West African 2013e16 resulted in more than 28,000

infected cases and over 11,000 deaths. On August 1, 2018, the

Ministry of Health of the Democratic Republic of the Congo (DRC)

Ebola outbreak in DRC as of March 16, 2020 included 3310

conﬁrmed cases and 2264 deaths [3]. Human pathogenic ﬁloviruses

are believed to exclusively occur in regions of Africa, although

ﬁlovirus-speciﬁc antibodies have been detected in animals in

Singapore [4] and China [5]. The results of these serological studies

suggest that ﬁloviruses could also be widely distributed in Asia.

To date, no antiviral therapeutic for Ebola virus infections has

been licensed, with treatment primarily focusing on supportive

measures. A number of experimental therapies were tested during

the latest Ebola virus epidemic, including monoclonal antibodies

(ZMapp, mAb 114 and REGN-EB3) and the small-molecule inhibitor

remdesivir (GS-5734), although evidence for the efﬁcacy of these

therapies is still lacking [6]. Thus, the continuous re-emergence of

Ebola virus disease (EVD) outbreaks in Africa coupled with the

potential risk of the expansion of epidemics in other continents, as

well as the possibility of the use EBOV as bioweapon and a lack of

approved therapeutics to treat EVD makes the development of

effective anti-EBOV therapeutics one of the top public health

* Corresponding author.

E-mail address: asokolova@nioch.nsc.ru (A.S. Sokolova).

https://doi.org/10.1016/j.ejmech.2020.1 12726

2

A.S. Sokolova et al. / European Journal of Medicinal Chemistry 207 (2020) 112726

priorities.

library camphor and borneol derivatives in a Marburg glycoprotein-

The RNA genome of EBOV harbors seven genes that encode at

least ten proteins: nucleoprotein (NP); viral proteins (VP24, VP30,

VP35, and VP40); glycoprotein (GP); soluble GP (sGP); small sGP;

mediated VSV pseudotype system, which resulted in the identiﬁ-

cation of N-heterocyclic borneol derivatives that block the entry

step of Marburg virus [19]. Compounds 3b (As-358) and 4b con-

taining a piperidine and 4-methylpiperidine moieties exhibited the

D-

peptide; and polymerase (L) [7]. EBOV glycoprotein (EBOV GP) is

the key protein that mediates viral entry into host cells and pro-

vides a potential target for the discovery anti-EBOV agents, because

inhibition of this glycoprotein can block the propagation of the

virus at an early stage, minimizing the chance for the virus to

evolve. The entry process includes ﬁve steps: 1) viral attachment to

the cell surface via interactions between GP and host cell receptors;

2) viral uptake into host cells by macropinocytosis; 3) GP cleavage

by cathepsins B and L; 4) GP interaction with the host protein

Niemann-Pick C1 (NPC1), which is necessary for the virus to bind

the endosomal membrane [8]; and 5) the release of viral particles.

Each of these steps, as well as the cellular functions associated with

these steps, are potential targets for the discovery anti-ﬁlovirus

agents. Recently, a large number of small-molecule entry in-

hibitors have been identiﬁed, several of them illustrated in Fig. 1

and including inhibitors of cell surface attachment

(MLS000534476), macropinocytosis inhibitor (MLS000394177) and

lysosomal cysteine cathepsin inhibitors, such as R11P [9,10]. Also an

inhibitor of two-pore Ca^2þchannel 2 (TPC2), alkaloid tetrandrine,

showed partial protective activity in vivo in a lethal mouse model

[1 1].

highest virus-speciﬁc activities, with IC₅₀values of 9 and 4

mM,

respectively (Fig. 3). Preliminary structure-activity relationship

(SAR) studies revealed that crucial structural fragment included a

1,7,7-trimethylbicyclo[2.2.1]heptan scaffold and heterocyclic ring,

such as piperidine, 4-methylpiperidine, 1-methylpiperazine, 1-

ethylpiperazine. Moreover, showed that the combination of this

saturated N-heterocycle and 1,7,7-trimethylbicyclo[2.2.1]heptan

scaffold was favorable for antiviral activity against orthopoxvirus

infection [20].

In this study, we expanded our SAR studies of borneol esters

with the aim of identifying other potent small molecule ﬁlovirus

inhibitors (Fig. 4). We synthesized a series of (ꢀ)-borneol esters to

assess the effects of different N-containing heterocycles as well as

the effects of spacer length and the chiral conﬁguration of 1,7,7-

trimethylbicyclo[2.2.1]heptan scaffold on ﬁlovirus inhibition. A

series of (þ)-camphor amides were synthesized to evaluate the

effect of the replacement of the ester group by an amide on anti-

ﬁlovirus activity. In addition, we conducted molecular docking

and site-directed mutagenesis analyses to identify a possible

binding site of the synthesized agents.

Several FDA-approved drugs inhibit the EBOV entry process by

destabilizing the GP. The most prominent of these compounds

include the selective estrogen receptor modulator toremifene

(Fig. 1); the painkiller ibuprofen; the antipsychotic drugs benz-

tropine and thioridazine; and the antidepressants paroxetine and

sertraline [12]. High-resolution complexes of GP-toremifene and

GP-ibuprofen were obtained and show that they bind in a cavity

between the attachment (GP1) and fusion (GP2) subunits (Fig. 2).

Thus, targeting the entry process is a rapidly developing strategy

for the discovery of antivirals against ﬁloviruses.

Recently, we identiﬁed camphor and borneol derivatives as

effective inhibitors of inﬂuenza virus replication [13e15]. Among

these compounds, (þ)-1,7,7-trimethylbicyclo[2.2.1] heptan-2-

ylidene-aminoethanol (camphecene) is one of the most effective

[15,16]. Through fusion inhibition assays, camphecene was shown

to inhibit the surface glycoprotein hemagglutinin (HA) activities of

inﬂuenza A and B viruses [1 7]. In addition, we identiﬁed hetero-

cyclic derivatives of (ꢀ)-borneol as potent inhibitors of inﬂuenza A

viruses. Similar to ﬁlovirus GP, the inﬂuenza virus protein HA binds

sialic acid on host cells and is responsible for membrane fusion.

Moreover, inﬂuenza virus and ﬁlovirus class I fusion proteins have

similar pre- and postfusion forms [18]. Therefore, we screened large

2. Results

2.1. Chemistry

The syntheses of compounds 2-7a-c and 8a-b were previously

reported [14,20]. Using similar methods, we synthesized com-

pounds 3-4d, 8c and 9e14a-c as illustrated in Scheme 1. Acylation

of (ꢀ)-borneol with chloroacetyl chloride or the chlorine-

containing chloride 1a-c generated esters 2a-d with good yields.

The desired products were produced from interactions between the

key intermediates 2a-d with the corresponding amines.

Being a nitrogenous compound, salt formation was envisaged as

one of the approaches to enhance solubility of synthesized com-

pounds, therefore, HCl salts of compounds 3b and 4b (3b·HCl and

4b·HCl) were generated.

To assess the effect of the ester group on antiviral activity, a

series of amides containing heterocycles and linkers of different

lengths were synthesized. Target amides were generated using

commercially available (þ)-camphor. The syntheses of amides

18e20a-b and 21b were previously reported [20]. The new amides

were synthesized using a similar method as depicted in Scheme 2.

Fig. 1. Compounds inhibiting the ﬁlovirus entry process.

A.S. Sokolova et al. / European Journal of Medicinal Chemistry 207 (2020) 112726

3

Fig. 2. Overall structure of EBOV GP-toremifene (left). A schematic representation of the GP-toremifene complex (right), showing GP1 (green), GP2 (blue), and the mucin-like

domain (pink). An enlarged fragment of an inhibitor-binding site complex (center). (For interpretation of the references to color in this ﬁgure legend, the reader is referred to

the Web version of this article.)

Fig. 3. Structures of antiviral agents based on (þ)-camphor and (ꢀ)-borneol.

Fig. 4. The strategy used for structure-activity relationship studies.

In the ﬁrst stage, camphor oxime was prepared, and its subsequent

reduction with NaBH₄in the presence of NiCl₂resulted in the (1R,

2R)-bornyl amine 16. The reaction between amine 16 and chlor-

oacetyl chloride or the chlorine-containing compounds 1a-b led to

key intermediates 17a-c. At the ﬁnal stage, the chlorine-containing

amides 17a-c were reacted with morpholine, 4-methylpiperidine,

1-methylpiperazine or 1-ethylpiperazine to obtain the target sub-

stances 18e21a-c.

derivatives were generated. The synthesis of two enantiomers of

isoborneol derivatives is shown in Scheme 3. (þ)-Camphor NaBH₄

was reduced to give

a 10:1 exo:endo mixture of isomers

[(þ)-isoborneol and (þ)-borneol, respectively]. Subsequent chro-

matographic separation led to the desired exo isomer, namely,

(þ)-isoborneol, and (ꢀ)-isoborneol was prepared in a similar

manner from commercially available (ꢀ)-camphor. From the pre-

pared (þ)- and (ꢀ)-isoborneols, the target compounds (1R,2R,4R)-

23e24 and (1S,2S,4S)-23e24 were similarly prepared as shown in

Scheme 1.

To study the effect of the chiral conﬁguration of borneol hydroxy

groups on antiviral activity, a group of (þ)- and (ꢀ)-isoborneol

4

A.S. Sokolova et al. / European Journal of Medicinal Chemistry 207 (2020) 112726

Scheme 1. Reagents and conditions: (i) corresponding chloroacetyl chloride, Et₃N, CH₂Cl₂, r.t.; (ii) the corresponding amine, Et₃N, CH₂Cl₂, r.t.; (iii) diethylamine or triethylamine,

K₂CO₃, CH₃CN, r.t.-reﬂux.

Scheme 2. Reagents and conditions: (i) NH₂OH$HCl, AcONa, and EtOHeH₂O (8:2), reﬂux; (ii) NiCl₂, NaBH₄, and MeOH, ꢀ30 ^ꢁC.

Scheme 3. Reagents and conditions: (i) NaBH₄and MeOH, ꢀ30 ^ꢁC; (ii) chloroacetyl chloride, Et₃N, and CH₂Cl₂, r.t.; (iii) piperidine or 4-methylpiperidine, Et₃N, and CH₂Cl₂, r.t.

2.2. Evaluation of in vitro antiviral activity using pseudotype

viruses and SAR study

can only be performed in laboratories with BSL-4 containment. To

overcome this issue, in the ﬁrst stage, the activity of synthesized

compounds was study using a “surrogate” system, which allowed

for entry inhibitors to be identiﬁed under BSL-2 containment. This

system included vesicular stomatitis virus (VSV) particles

Considering the high lethality of EBOV and the lack of prophy-

lactic and therapeutic treatments, studies using the authentic virus

A.S. Sokolova et al. / European Journal of Medicinal Chemistry 207 (2020) 112726

5

pseudotyped with EBOV GP. The potency against Ebola pseudotype

viruses (rVSV- G-EboV-GP) of all synthesized compounds and

2.3. In vitro antiviral activity against EBOV and MARV

D

cytotoxicity are displayed in Tables 1e3. The sertraline was used as

a reference drug, since there are data conﬁrming the efﬁcient

binding of this drug to GP EBOV [12]. To determine the speciﬁc

As previously mentioned, we identiﬁed compounds 3b and 4b

as perspective inhibitors of the entry step of Marburg virus. Further

analysis of the in vitro antiviral activities of these derivatives using

inhibitory activity of the compounds toward rVSV-

antiviral activities against viral particles pseudotyped G VSV (rVSV-

G-G) were determined. Using this approach, compounds that

similarly inhibit both viruses will likely affect cellular mechanisms

common to the entry of both viruses.

First, we examined the effect of different heterocyclic or dia-

lkylamino groups and linker lengths on antiviral activity (Table 1).

The SAR results showed that a morpholine ring is not favorable for

inhibiting activity, although the derivatives 5a-c displayed low

D

G-EboV-GP,

rVSV-DG-EboV-GP viruses showed that they also effectively inhibit

the entry step of Ebola virus. Thus, agents 3b and 4b and their

hydrochloride salts 3b·HCl and 4b·HCl were assessed for their

antiviral activity against the authentic ﬁloviruses EBOV (strain

Zaire) and MARV (strain Popp). Anti-EBOV and anti-MARV assays

were conducted at SRC VB Vector in a maximum containment fa-

cility (BSL-4). EBOV and MARV were obtained from the State

Collection of Viral Infections and Rickettsioses Agents of SRC VB

Vector.

D

toxicity (CC₅₀

¼

210e326

m

M). The piperidine and 4-

As shown in Table 4, agents 3b, 4b, 3b·HCl and 4b·HCl were

more active against MARV than EBOV. Derivative 3b was observed

to be very potent toward MARV, with micromolar activity

methylpiperidine derivatives 3-4b-d, which had a long linker

(n ꢂ 2), displayed potent inhibition efﬁciency, with high SI values of

162e956. The hydrochloride salts 3b·HCl and 4b·HCl showed

(IC₅₀¼ 3.7

compound 4b had a micromolar IC₅₀value 2.2

higher degree of toxicity (CC₅₀¼ 69 M) compared to the agent 3b.

Interestingly, the conversion of compound 4b into the hydrochlo-

M) while

m

M) and low toxicity (CC₅₀¼ 443

mM). In contrast,

M and displayed a

promising antiviral activity (IC₅₀¼ 0.8 and 0.4

were signiﬁcantly more toxic (CC₅₀¼ 87.0 and 69.8

tively) compared to the free bases 3b and 4b (CC₅₀¼ 289.7 and

279.7 M, respectively). Almost all of the 4-substituted piperazine

mM, respectively) but

m

mM, respec-

m

ride salt 4b·HCl led to decreased toxicity (СС₅₀¼ 144

m

derivatives displayed good antiviral potency, with the exception of

the ethyl piperazine-1-carboxylate derivatives 8a-c, which dis-

played weak inhibition. The derivatives 6-7b-c and 9b-c, which

harbor alkyl or hydroxyethylene groups in the piperazine ring with

linker lengths of n ꢂ 2, were signiﬁcantly more active, exhibiting

retaining the same micromolar IC₅₀value. The salt 3b·HCl, which

contained a piperedin ring, was the most effective inhibitor of

EBOV. Despite the fact that piperazine derivatives 6b and 7b

showed potency against Ebola pseudotype viruses, antiviral activity

against the authentic ﬁloviruses did not conﬁrm. The morpholine

derivative 5b showed low activity in the p-EBOV and p-MARV assay

IC₅₀values between 0.1 and 1.8

mM and CC₅₀values ranging from

99.2 to 282

m

M. The piperazine derivative 10c, containing 2-

(IC₅₀¼ 8.5 and 10

mM, respectively) and was not against the

aminoethyl substitution at the nitrogen atom, showed low IC₅₀

values and high toxicity, while compound 10a had a CC₅₀value of

authentic ﬁloviruses. These ﬁndings demonstrate that the pseu-

dotyped viruses bearing ﬁlovirus glycoproteins are a reasonable

predictor of activity against authentic ﬁloviruses.

247

group at the nitrogen atom signiﬁcantly increased the toxicity of

compounds 11a-c, which had CC₅₀values ranging from 30 to 59 M.

mM and an SI value of 114. Interestingly, a lipophilic benzyl

m

2.4. Molecular docking studies

In addition, the azepane derivatives 12a and 12c demonstrated high

toxicity, whereas compound 12b, with linker length of n ¼ 2,

We performed molecular docking studies of synthesized com-

pounds in the active binding site of known inhibitors using an XP

(extra precision) docking algorithm implemented in the Glide pro-

gram (Schrӧdinger Suite 2016e1). Models (PDB IDs: 6F6S, 6F6N,

6F6I, and 6F5U) representing the envelope glycoprotein of the

Ebola virus (Zaire ebolavirus, strain Mayinga-76) were selected in

combination with pharmacologically signiﬁcant inhibitors (reso-

lution of 2.07 Å). Features of spatial orientation and noncovalent

interactions of the synthesized ligands were compared with the

data obtained for known inhibitors. Toremifene, sertraline, benz-

tropine, bepridil, paroxetine were selected as known inhibitors. An

analysis of the binding site of EBOV GP with cocrystallized in-

hibitors revealed the presence of a central hydrophobic region (A), a

deep hydrophobic pocket (B), and a hydrophilic groove (C) formed

by several charged amino acid residues (Fig. 5A). Known inhibitors

are located in the central region by the primary parts of the mol-

ecules and possess hydrophobic chains positioned in a deep pocket

B. Benztropine, bepridil, paroxetine, sertraline were characterized

by interactions with amino acid residues 517 and 548 in this region.

For torimephene, an additional structural feature included the

presence of a “tail” containing polar groups interacting with

charged residues of the hydrophilic furrow, particularly with res-

idue 522 (Fig. 5B).

exhibited a high CC₅₀value of 182 mM and moderate activity, with

an IC₅₀value of 5.1 mM. Derivatives with N,N-dialkylamine sym-

metric groups did not show appreciable antiviral activity, with the

exception of (1S,2S,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl 4-

(diethylamino)butanoate (14c) displayed high anti-rVSV-

GP activity, with an IC₅₀value of 0.3 M, and low toxicity

(CC₅₀¼ 165 M). These results indicated that the piperidine, 4-

DG-EboV-

m

methylpiperidine, and 4-alkyl or -hydroxyalkyl substituted piper-

azine ring may be more favorable than morpholine, azepane or 4-

benzyl substituted piperazine groups. In this study, we conﬁrmed

that linker length greatly affects toxicity. The piperidine, 4-

methylpiperidine, N-ethyl piperazine and azepane derivatives

were the least toxic, with linker lengths of n ¼ 2, compared to

compounds where n ¼ 1 or 3,4.

Next, we examined the effect of the chiral conﬁguration of the

1,7,7-trimethylbicyclo[2.2.1]heptan moiety on antiviral activity

(Table 2). In general, comparing compounds with the same sub-

stituents 3e4 a and (1R,2R,4R)-, (1S,2S,4S)-23e24, the (þ)- and

(ꢀ)-isoborneol derivatives (1R,2R,4R)-23e24 and (1S,2S,4S)-23e24

showed lower values of IC₅₀than (ꢀ)-borneol derivatives 3a and

4a. However, (þ)- and (ꢀ)-isoborneol derivatives were more toxic

than analogues in the (ꢀ)-borneol series.

In the (þ)-camphor amide series of compounds 18e21a-c, all

The synthesized compounds demonstrate a “hybrid” type of

binding, where the hydrophobic 1,7,7-trimethylbicyclo [2.2.1]

heptan fragment occupies the central region of the binding site,

interacting with leucines 515 and 558, methionine 548, alanine 101,

tyrosine 517. An aliphatic tail with a nitrogen-containing hetero-

cycle is located in a hydrophilic groove, forming electrostatic in-

teractions with Glu 100 and Asp522 (Fig. 6).

examined compounds possessed good to excellent antiviral activ-

ities, with IC₅₀values between 0.6 and 27 mM, and ﬁve of these

compounds had SI values greater than 100 (Table 3). The MTT test

results showed an increase in toxicity with increasing linker length

for compounds 17e19a-c, whereas 4-methylpiperidine derivatives

exhibited decreased toxicity with increasing linker length.

6

A.S. Sokolova et al. / European Journal of Medicinal Chemistry 207 (2020) 112726

Table 1

Antiviral activities of the synthesized (ꢀ)-borneol derivatives.

Compound

n

X/R

CC₅₀

(

m

M)^a

IC50 (

mM)

SIEboV-GP^d

Ebind^e

EboV-GP^b

VSV-G^c

3a

3b

3c

3d

3b·HCl

4a

4b

4c

4d

n ¼ 1

n ¼ 2

n ¼ 3

n ¼ 4

n ¼ 2

n ¼ 1

n ¼ 2

n ¼ 3

n ¼ 4

n ¼ 2

n ¼ 1

n ¼ 2

n ¼ 3

CH₂

141.4 14.1

289.7 22.6

74.8 6.1

100.9 12.1

87.0 7.8

15.7 0.8

1.7 0.1

0.3 0.01

0.6 0.05

0.8 0.06

8.5 1.1

0.3 0.04

9.3 1.1

0.2 0.02

0.4 0.03

24.9 1.1

8.5 0.7

6.5 0.5

122.5 13.5

93.0 9.3

50.1 4.5

105.4 8.4

89.4 6.3

35.0 2.4

289.7 19.7

276.4 13.5

83.5 7.3

9

ꢀ3.65

ꢀ4.14

ꢀ3.63

ꢀ4.48

ꢀ4.14

ꢀ3.43

ꢀ3.80

ꢀ4.02

ꢀ4.70

ꢀ3.80

ꢀ3.07

ꢀ4.28

ꢀ4.49

170

230

162

112

8

956

18

408

171

13

CH₂

CHCH₃

O

71.6 6.4

279.7 28.3

171.1 12.8

73.0 3.1

4b·HCl

5a

5b

69.8 5.6

71.5 5.4

326.9 45.8

304.7 19.5

210.1 14.3

290.7 26.5

319.8 30.1

287.7 14.4

O

36

33

5c

6a

6b

6c

7a

7b

7c

8a

8b

8c

n ¼ 1

n ¼ 2

n ¼ 3

n ¼ 1

n ¼ 2

n ¼ 3

n ¼ 1

n ¼ 2

n ¼ 3

CH₃

C₂H₅

312.5 30.6

282.0 25.7

99.2 8.8

129.7 11.5

263.6 24.3

130.8 10.5

227.0 13.6

109.7 9.5

60.4 4.7

6.8 0.8

0.3 0.03

0.6 0.04

1.6 0.1

0.1 0.01

19.9 2.1

28.3 1.9

2.6 0.2

484.7 48.0

173.2 15.7

97.3 8.7

263.6 21.9

80.5 5.9

165.3 11.4

112.9 8.9

NT

46

ꢀ3.53

ꢀ4.89

ꢀ5.00

ꢀ3.30

ꢀ4.85

ꢀ4.77

ꢀ3.70

ꢀ3.99

ꢀ4.83

870

160

80

170

880

11

4

23

49.1 3.8

9a

9b

9c

10a

10c

n ¼ 1

n ¼ 2

n ¼ 3

n ¼ 1

n ¼ 3

295.9 32.8

271.8 32.6

224.1 28.0

247.3 23.0

71.1 5.7

13.9 1.0

1.8 0.1

0.3 0.02

2.2 0.2

0.6 0.1

394.2 38.2

258.9 25.9

192.0 19.2

24.1 2.4

21

ꢀ4.94

ꢀ5.33

ꢀ6.07

ꢀ5.51

ꢀ6.77

153

658

114

125

50.4 5.1

11a

11b

11c

n ¼ 1

n ¼ 2

n ¼ 3

59.4 5.6

39.0 3.7

30.1 2.0

1.3 0.1

0.3 0.03

39.8 3.4

27.0 2.4

26.0 2.3

44

150

100

ꢀ3.20

ꢀ4.69

ꢀ5.83

12a

12b

12c

n ¼ 1

n ¼ 2

n ¼ 3

71.6 7.9

182.5 16.2

62.2 5.7

1.0 0.1

5.1 0.4

0.2 0.02

NT

70

36

250

ꢀ3.01

ꢀ2.99

ꢀ4.21

191.2 11.1

NT

13a

13b

13c

14a

14b

14c

Sertraline

n ¼ 1

n ¼ 2

n ¼ 3

n ¼ 1

n ¼ 2

n ¼ 3

CH₃

C₂H₅

388.6 38.1

505.2 34.9

100.6 7.0

344.0 33.0

326.9 31.7

165.8 12.8

408 35.9

250.7 22.3

30.0 2.6

1.5 0.1

522.6 63.2

NT

560.9 66.7

411.4 48.9

195.4 23.1

NT

2

ꢀ3.58

ꢀ3.21

ꢀ3.53

ꢀ3.09

ꢀ2.55

ꢀ3.05

ꢀ8.24

17

67

61

26

490

582

5.6 0.5

12.4 1.1

0.3 0.03

0.7 0.07

A.S. Sokolova et al. / European Journal of Medicinal Chemistry 207 (2020) 112726

7

The data represent the mean SD from three independent experiments.

NT not tested.

a

CC₅₀: the median cytotoxic dose, i.e. the concentration causing 50% cell death.

b

c

IC₅₀(EboV-GP): the concentration of a compound required to inhibit rVSV-

DG-EboV-GP infection of HEK293T cells by 50%.

IC₅₀(VSV-G): the concentration of a compound required to inhibit rVSV-

DG-G infection of HEK293T cells by 50%.

d

e

SI_EBOV-GP¼ CC₅₀/IC₅₀(EboV-GP).

E_bind: binding energy, kcal/mol (the value is not genuine binding energy but rather an estimated docking score).

the GP2 subunit. The antiviral activity of the mutated EboV-GP

particles was compared with the wild-type WT-EboV-GP. The

mutant pseudotyped VSV particles were tested against the

following selected compounds: 4b, 3b and 7b, which were tested

on natural ﬁlovirus; 4c and 6c, with long linkers; and two

(þ)-camphor amides, 18c and 21a.

Table 2

Antiviral activities of the (þ)- and (ꢀ)-isoborneol derivatives against rVSV-

DG-

EboV-GP virus.

Compound

R

CC₅₀

(

m

M)^a

IC_{50 EboV-GP}

SI^c

E_bind

b

d

In the antiviral assay, the IC₅₀values of agent 3b toward Y517A-

EboV-GP and M548A-EboV-GP were 3.7 and 47.1 mM, respectively

(Table 5). A 20-fold increase in the IC₅₀value for M548A-EboV-GP

was compared to that of the WT-EboV-GP was observed. In

contrast, a similar ability of 3b to inhibit WT-EboV-GP and mutant

(1R,2R,4R)-23

(1R,2R,4R)-24

H

CH₃

80.5 9.9

34.1 4.0

2.9 0.3

3.1 0.3

28

11

ꢀ3.99

ꢀ4.36

D522A-EboV-GP was observed (IC₅₀1.7 and 2.2 mM respectively).

Similar results were observed for compound 4b, where a greater

than 30-fold increase in the IC₅₀value for M548A-EboV-GP was

detected compared to that observed for WT-EboV-GP. The inhibi-

tory activity of derivatives 4c and 6c toward all mutant pseudo-

typed viral particles and WT-EboV-GP were different. For agent 4с,

a greater than 20-fold decrease in the IC₅₀value toward the mutant

Y517A-EboV-GP and D522A-EboV-GP pseudotyped viral particles

(1S,2S,4S)-23

(1S,2S,4S)-24

H

CH₃

89.5 8.0

34.1 2.7

3.6 0.3

4.4 0.4

25

8

ꢀ3.72

ꢀ4.36

a

CC₅₀: the median cytotoxic dose, i.e. the concentration causing 50% cell death.

IC_{50 EboV-GP}: the concentration of a compound required to inhibit rVSV-DG-

(IC₅₀0.4

mM) was measured compared to that observed for WT-

b

EboV-GP (IC₅₀9.3

mM). For the derivative 6c, a greater than 20-

EboV-GP infection of HEK293T cells by 50%,

m

M.

c

fold increase in the IC₅₀value was noted for all mutant pseudo-

typed viral particles compared with that observed for WT-EboV-GP.

The inhibitory activities of agent 7b toward the mutant pseudo-

typed viral particles were slightly different from the antiviral ac-

tivity against WT-EboV-GP. The IC₅₀values of amide 18b against

WT-EboV-GP and mutant D522A-EboV-GP were almost the same

SI_EBOV-GP¼ CC₅₀/IC₅₀(EboV-GP).

d

E_bind: binding energy, kcal/mol (the value is not genuine binding energy but

rather an estimated docking score).

2.5. Site-directed mutagenesis

(IC₅₀22.1 and 17.7 mM, respectively), while the IC₅₀values against

WT-EboV-GP and mutants Y517A-EboV-GP, M548A-EboV-GP

signiﬁcantly different. For the derivative 21a there is a signiﬁcant

difference was only observed for the mutation Y517A.

Based on the molecular docking results we made 3 rVSV pseu-

dotyped with mutant GP proteins carrying single alanine sub-

stitutions at the 517, 522 and 548 amino acids positions of chain B of

Table 3

Antiviral activities of the (þ)-camphor amides.

M)^a

IC₅₀

(m

M)

SI_EboV-GP

E_bind

d

e

Compound

n

Y

CC₅₀

(

m

EboV-GP^b

VSV-G^c

18a

18b

18c

19a

19b

19c

20a

20b

20c

21a

21b

21c

n ¼ 1

n ¼ 2

n ¼ 3

n ¼ 1

n ¼ 2

n ¼ 3

n ¼ 1

n ¼ 2

n ¼ 3

n ¼ 1

n ¼ 2

n ¼ 3

O

838.1 71.2

312.5 37.8

171.8 21.1

853.3 101.5

485.6 55.4

474.7 51.7

660.2 71.3

564.9 49.7

268.2 17.9

78.0 6.9

5.7 0.7

22.1 2.2

9.4 0.7

13.3 1.1

2.6 0.3

2.5 0.3

8.5 0.8

0.6 0.04

2.1 0.14

27.0 1.6

2.6 0.1

3.4 0.3

838.1 74.6

147

14

18

ꢀ3.95

ꢀ3.61

ꢀ4.76

ꢀ3.81

ꢀ5.89

ꢀ5.29

ꢀ4.67

ꢀ5.15

ꢀ5.76

ꢀ4.65

ꢀ3.70

ꢀ4.56

312.5 21.9

171.8 10.3

853.3 42.7

485.6 19.4

474.7 21.4

660.2 30.4

564.9 49.7

268.2 23.6

78.0 6.9

NeCH₃

NeC₂H₅

CHCH₃

64

187

191

78

908

129

2.9

36

93.6 10.0

141.6 14.3

93.6 7.3

141.6 12.7

41

a

CC₅₀: the median cytotoxic dose, i.e. the concentration causing 50% cell death.

IC₅₀(EboV-GP): the concentration of a compound required to inhibit rVSV-

IC₅₀(VSV-G): the concentration of a compound required to inhibit rVSV-

b

c

D

G-EboV-GP infection of HEK293T cells by 50%.

DG-G infection of HEK293T cells by 50%.

d

e

SI_EBOV-GP¼ CC₅₀/IC₅₀(EboV-GP).

Ebind: binding energy, kcal/mol (the value is not genuine binding energy but rather an estimated docking score).

8

A.S. Sokolova et al. / European Journal of Medicinal Chemistry 207 (2020) 112726

Table 4

Inhibitory activities of synthesized derivatives against EBOV and MARV.

Compound

CC₅₀

(m

M)^a

IC₅₀

(

m

M)^bMARV

SI MARV^c

IC₅₀

(

m

M)^dEBOV

SI EBOV^e

3b

3b·HCl

4b

4b·HCl

5b

6b

442.9 82.9

279.1 38.7

69.3 5.2

144.0 33.2

122.0 25.6

61.4 38.5

19.2 11.8

3.7 1.7

18.1 9.3

2.2 1.4

5.7 3.4

NA

17.8 4.1

2.43 0.14

118

15

32

47.5 8.2

9.1 2.1

54.3 14.8

64.7 11.2

NA

3.73 0.62

9

31

1.3

2.2

25

7b

1

Sertralineⁱ

NA: not active.

The data represent the mean SD from three independent experiments.

a

СC₅₀: 50% cytotoxic concentration (Vero cells).

IC₅₀: the concentration of a compound required to inhibit MARV (strain Popp) infection of Vero cells by 50%.

SI MARV: is the selectivity index, which is the CC₅₀/IC₅₀MARV ratio.

IC₅₀: the concentration of a compound required to inhibit EBOV (strain Zaire) infection of Vero cells by 50%.

SI EBOV: is the CC₅₀/IC₅₀EBOV ratio.

IC₅₀values (Vero E6 cells) are taken from Johansen et al. [21].

b

c

d

e

i

Fig. 5. А: The inhibitor binding site pockets. B: Superposition the of pharmacologically signiﬁcant inhibitors in binding site of EBOV GP. Toremifene, paroxetine, sertraline and

bepridil are shown as yellow, orange, pink and green, respectively. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the Web version of this

article.)

2.6. Acute toxicity studies

activity. Notably, the most promising anti-EBOV agent (3b·HCl)

demonstrated good in vivo safety. In addition, the in silico absorp-

tion, distribution, metabolism and excretion (ADME) study results

for compounds 3-7b, 9b-c, 18b and 20b are shown in Table 1S. The

calculated parameters are in good agreement with the Lipinski [22]

and Ghose [23] rules. For all new compounds, logP does not exceed

5, which indicates good lipophilicity. The most active compounds

are characterized by a large scatter of logP values in the range

1.3e4.7. An increase in the linker length directly affects the increase

in the logP value in the series of compounds that are identical in

structure of the substituent (See SI).

The safety of the most active compound (3b·HCl) against EBOV

was evaluated in outbred ICR mice. Groups of mice were adminis-

tered either a single dose (1.08, 0.44, 0.27, and 0.14 g/kg) or the

vehicle control by gavage in the form of a suspension. The survival

of the mice was monitored and recorded over 7 days. No animal

deaths were recorded during in the (3b·HCl)-treated mice over the

entire observation period. Thus, the results of this study indicated

that compound (3b·HCl) is not toxic to adult mice with intragastric

administration (LD₅₀> 1000 mg/kg).

Considering that the synthesized derivatives selectively inhibi-

ted rVSV-DG-EboV-GP but did not show activity against VSV-G

3. Discussion

indicate that the likely target of these compounds is GP EBOV.

The viral glycoprotein GP is consists of two subunits, the receptor

attachment glycoprotein 1 (GP₁) and membrane fusion glycopro-

tein 2 (GP₂). GP₁is responsible for cell surface recognition/attach-

ment and viral uptake into host cells, containing two exterior

domains that are responsible for host cell binding and a receptor

binding domain (RBD) that interacts with endosomal receptor

Niemann-Pick C1 (NPC1). GP₂is a class I viral fusion protein that

catalyzes the fusion of the virus with the host membrane. Previ-

ously, a large number of Food and Drug Administration (FDA)-

approved drugs were shown to be active against Ebola virus

infection. It was shown the anticancer drug toremifene and the

painkiller ibuprofen bind in a cavity between the attachment (GP₁)

In this study, we describe the preparation and antiviral activities

of N-heterocyclic monoterpenoids derivatives containing an 1,7,7-

trimethylbicyclo[2.2.1]heptan scaffold. All synthesized com-

pounds were screened in a cell-based assay for their anti-EBOV

activity using a VSV pseudotype by GP EBOV. Almost all of the

synthesized compounds showed inhibitory potency against rVSV-

D

G-EboV-GP, ranging from the micromolar range (IC₅₀¼ 30

nanomolar range (IC₅₀¼ 0.1 M). Moreover, 36 compounds were

identiﬁed as having low cytotoxicity (CC₅₀˃ 100 M) and high

mM) to

m

selectivity index values. The in vitro study results against bona ﬁde

EBOV and MARV for the most promising derivatives (3b, 3b·HCl, 4b

and 4b·HCl) demonstrated their excellent to moderate antiviral

A.S. Sokolova et al. / European Journal of Medicinal Chemistry 207 (2020) 112726

9

Fig. 6. Noncovalent interactions of synthesized agents with amino acid residues of the binding site GP EBOV. 3b (A); 4b (B); 4c (C); 9c (D); 9b (E); 10a (F); 18b (G); 21a (H); 10c (I).

Interactions are shown by dashed lines (green, hydrogen bonds; orange, electrostatic interactions), while hydrophobic interactions are not indicated. (For interpretation of the

references to color in this ﬁgure legend, the reader is referred to the Web version of this article.)

Table 5

In vitro alanine mutagenesis of the EboV-GP residues Y517, D522 and M548.

Compound

IC₅₀(mM)

WT-EboV-GP^a

Y517A-EboV-GP^b

D522A-EboV-GP^c

M548A-EboV-GP^d

3b

4b

4с

1.7 0.2

0.3 0.03

9.3 0.8

0.6 0.1

1.6 0.2

22.1 2.7

27.0 1.9

3.7 0.4

2.2 0.3

4.9 0.5

47.1 6.1

112.2 14.7

1.2 0.2

12.2 1.4

1.0 0.1

48.1 4,8

0.4 0.03

18.3 2.2

1.8 0.1

173.1 11.8

1.7 0.2

0.4 0.04

18.3 1.9

0.5 0.04

17.7 1.4

11.6 0.8

6с

7b

18b

21a

65.2 5.8

44.2 3.4

a

IC₅₀(WT-EboV-GP): the concentration of a compound required to inhibit rVSV-

D

G-EboV-GP infection by 50%.

IC₅₀(Y517A-EboV-GP): the concentration of a compound required to inhibit rVSV- G-Н517A-EboV-GP infection by 50%.

IC₅₀(D522A-EboV-GP): the concentration of a compound required to inhibit rVSV- G-D522A-EboV-GP infection by 50%.

G-M548A-EboV-GP infection by 50%.

b

c

D

d

IC₅₀(M548A-EboV-GP): the concentration of a compound required to inhibit rVSV-

and fusion (GP₂) subunits at the entrance to a large tunnel that links

with equivalent tunnels from the other monomers of the trimer at

the 3-fold axis. These drugs belong to various pharmacological

groups and have different chemical structures but bind in the same

cavity on the EBOV GP. Therefore, we performed molecular docking

analyses of synthesized compounds in the active binding site of

known inhibitors, such as benztropine, bepridil, paroxetine, ser-

traline (Figs. 6 and 7).

additional hydrogen bond through a hydroxyl group with the

charged residue Glu100 (Fig. 6). Glu100 and Thr519 also participate

in the interaction with charged nitrogen in the piperidine or

piperazine rings, which is probably one of the reasons why the

hydrochloride salt 3b·HCl more active against EBOV than the free

base 3b. Overall, the binding interaction mode of a synthesized

ligand with EBOV GP could identify key amino acids, including

Glu100, Asp522, Tyr 517, Leu 515, and Met 548, which are important

for the of binding in the active site. These results suggest that the

synthesized compounds bind EBOV GP similarly to toremifene.

To validate these results, we performed an in vitro mutagenesis

study of the glycoprotein residues involved in the binding site. To

this end, the amino acids M548A, Y517A (two key residues

responsible for hydrophobic interaction of all tested compounds)

and D522A were all mutated to alanine. Then, the afﬁnities of the

The in silico docking results of synthesized ligands in the binding

site EBOV GP (i.e., calculated E_bind) provided a structural basis to

interpret the experimental in vitro data. The derivatives 9b, 9с, 10

and 10с with hydroxyl or amino groups display high antiviral ac-

tivity against rVSV- G-EboV-GP and exhibited high afﬁnities in in

а

D

silico simulations. This result can be explained by additional in-

teractions between the functional groups of ligands with the amino

acid Asp522. Notably, the binding mode of compound 9b has an

rVSV-DG-EboV-GP mutants toward compounds 3b, 4b-c, 6c, 7b,

10

A.S. Sokolova et al. / European Journal of Medicinal Chemistry 207 (2020) 112726

groups to three makes it possible for the agents to interact with

amino acid D522. As shown in Fig. 8, in contrast to derivative 6b

and 6с (n ¼ 2, 3), derivative 6a (n ¼ 1) had no contacts with residue

522 and showed the lowest antiviral activity (IC_50EboV-GP¼ 6.8

m

M)

compared to 6b-c (IC_50EboV-GP¼ 0.3 and 0.6

mM, respectively). In

general, for (ꢀ)-borneol derivatives, both the computational and

in vitro experimental results indicated that an increase in the

aliphatic carbon chain length (n ¼ 1e3) increased the antiviral

potency (Table 1).

(þ)-Camphor-based amides are probably located in the binding

site as shown above. As depicted in Table 5, for agents 18b and 21a,

the greatest IC₅₀value change was observed for mutants Y517A-

EboV-GP and M548A-EboV-GP, which was consistent with the in

silico trend, which do not involve interactions with D522 (Fig. 5).

The residues lining the binding site described above are highly

conserved among ﬁloviruses, with the exception Marburg virus,

suggesting that the antiviral activity these compounds against

MARV will require additional research. As shown in Table 4, the

anti-MARV activities of agents 3b and 4b were considerable higher

than that observed against EBOV. Additional studies are required to

localize the binding site on the MARV GP for the synthesized

derivatives.

Fig. 7. In silico docking results of compound 3b. Regions of EBOV GP are marked by

color: receptor binding domain (RBD), green; glycan cap, red; fusion peptide, rose; N-

terminal heptad repeat (NHR), blue; and C-terminal heptad repeat (CHR), yellow. (For

interpretation of the references to color in this ﬁgure legend, the reader is referred to

the Web version of this article.)

4. Conclusion

In this study, we describe

a

series of (þ)-camphor and

18c and 20a were evaluated in vitro (Table 5). As shown in Table 5,

agent 4b exhibited a greater change in IC₅₀values for mutants

carrying mutations at positions 517 and 548 than at position 522

compared with that observed for the wild-type viral particles.

These results agree with those of the molecular docking studies,

where for agent 4b, there is no contact with 522, while this ligand

has interactions with Y517 and M548 (Fig. 5). Moreover, the results

of the molecular docking and mutagenesis analyses explain the

dependence of antiviral activity on the length of the aliphatic “tail”,

which is probably due to the necessary length for the interaction of

the nitrogen atom with amino acid residue 522. An in vitro study

using mutant pseudotyped VSV particles of agents 4c and 6c also

conﬁrmed this hypothesis. As shown in Table 5, these agents

demonstrated considerable changes in IC₅₀values in inhibiting the

Y517A-EboV-GP, M548A-EboV-GP and D522A-EboV-GP virus par-

ticles. Thus, an increase in the linker length from two methylene

(ꢀ)-borneol derivatives as potent ﬁlovirus enter inhibitors. The

relationships between the structural properties of the synthesized

ligands and their estimated afﬁnities for EBOV GP using pseudotype

viruses and in silico approach can be summarized as follows. (1)

(ꢀ)-Borneol derivatives that contain a 4-N-substituted piperazine/

piperidine heterocycle were the most potent against rVSV-DG-

EboV-GP. (2) The longer aliphatic linker in both series (ꢀ)-borneol

ester and (þ)-camphor amide is associated with lower binding

energies for EBOV GP and higher toxicity. (3) The conﬁguration the

1,7,7-trimethylbicyclo[2.2.1]heptan moiety is important, and high

activity and toxicity were discovered for (ꢀ)-borneol derivatives.

The agents 3b (As-358), 4b and their hydrochloride salts 3b·HCl and

4b·HCl showed excellent antiviral activities using a “surrogate”

system, which conﬁrmed their inhibitory activity and in relation

toward authentic ﬁlovirus. Based on the molecular docking and

in vitro mutagenesis results, we suggest that the possible mecha-

nism of action of the synthesized compounds is binding to the

active site of EBOV glycoprotein, similar to that of the known in-

hibitor toremifene.

5. Experimental section

5.1. General

Reagents and solvents were purchased from commercial sup-

pliers and used as received. Dry solvents were obtained according

to standard procedures. GC MS was performed using a 7820A gas

chromatograph (Agilent Technologies); a ﬂame-ionization detec-

tor; a HP-5 capillary column (0.25 mm ꢃ 30 m 0.25

mm); and He as

carrier gas (ﬂow rate 2 mL/min, ﬂow division 99:1). Optical rotation

was assessed using a polAAr 3005 spectrometer with a CHCl₃so-

lution. ¹H and ¹³C NMR spectra were recorded on Bruker spec-

trometers, including an AV-300 instrument at 300.13 MHz (¹H) and

75.47 MHz (¹³C), an AV-400 instrument at 400.13 MHz (¹H) and

100.61 MHz (¹³C), and a DRX-500 instrument at 500.13 MHz (¹H)

and 125.76 MHz (¹³C) in CDCl₃; chemical shifts

d were reported in

ppm relative to residual CHCl₃[d(CHCl₃) 7.24, d(CDCl₃) 76.90 ppm],

J in Hz. The structure of the products was determined by analyzing

the ¹H and ¹³C NMR spectra. HR-MS analyses were performed using

Fig. 8. Superposition of the (ꢀ)-borneol derivatives 6a, 6b and 6c are shown as yellow,

orange and green, respectively. (For interpretation of the references to color in this

ﬁgure legend, the reader is referred to the Web version of this article.)

A.S. Sokolova et al. / European Journal of Medicinal Chemistry 207 (2020) 112726

11

a DFS Thermo Scientiﬁc spectrometer in a full scan mode (0e500

m/z, 70 eV electron impact ionization, direct sample administra-

H-18e, H-19e, H-20), 1.78e1.94 (3H, m, H-5endo, H-17a, H-16a),

2.23e2.35 (5H, m, H-2exo, H-12, H-15), 2.79e2.85 (2H, m, H-17e, H-

tion). Elemental analysis was performed using

3000 C,H,N,S-analyzer. Column chromatography was performed on

silica gel (60e200 m, Macherey-Nagel). The purity of the target

a

Euro EA

16e), 4.81e4.86 (1H, m, H-1exo). ¹³C NMR (

d, ppm): 173.47 s (C-11),

79.16 d (C-1), 58.27 t (C-15), 53.62 t (C-16, C-17), 48.26 s (C-6),

47.31 s (C-7), 44.42 d (C-3), 36.38 t (C-2), 34.13 t (C-12), 33.90 t (C-

18, C-19), 30.39 d (C-20), 27.58 t (C-4), 26.66 t (C-5), 26.15 t (C-14),

22.84 t (C-13), 21.46 q (Me-21), 19.24 q (Me-9), 18.38 q (Me-10),

m

compounds was determined by gas chromatography. All of the

target compounds reported in this study had a purity of ꢂ95%.

13.05

335.2819).

q

(Me-8). HR-MS: 335.2817 (M^þ, C₂₁H₃₇O₂N₁; calcd

5.2. Chemistry

5.2.1. Syntheses of esters 3e7 a-c, 8 a-b and amides 18e20 a-b, 21c

Syntheses and structural characterization of the (ꢀ)-borneol

ester 3e8 a-b [14] and the ester 3-7c as well as the (þ)-camphor

amides 18e20 a-b, 21b [20] were described in our previous

publication.

5.2.4. Synthesis of the hydrochloride salts 3b·HCl and 4b·HCl

Excess hydrochloric acid was prepared by dissolving gaseous

hydrogen chloride in Et₂O, which was added to solutions of com-

pounds 3b or 4b in hexane. The resulting precipitate was collected

by ﬁltration, washed with hexane (10 mL), and then dried to yield

3b·HCl and 4b·HCl, respectively.

5.2.2. Synthesis of (1S,2R,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-

2-yl 5-chloropentanoate 2d

1-(3-Oxo-3-((1S,2R,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-

yloxy)propyl)piperidinium chloride 3b·HCl.

To a solution of 5-chloropentanoic acid in dry CH₂Cl₂, excess

oxalyl chloride and N,N-dimethylformamide (one drop) were

added. Then, the mixture was stirred at room temperature for 3 h,

after which the excess oxalyl chloride was removed using a rotary

evaporator. The resulting 5-chloropentanoyl chloride was imme-

diately used in a subsequent reaction.

Yield 91%; white powder. ¹H NMR (

d, ppm, J/Hz): 0.75 (3H, с, Мe-

10), 0.81 (3H, с,

М

e-8), 0.83 (3H, с,

М

e-9), 0.89 (1H, dd, ²J ¼ 13.7,

J_{2endo, 1exo}¼ 3.5, H-2endo), 1.12e1.29 (2H, m, H-4endo, H-5exo),

1.32e1.44 (1H, m, H-17a), 1.60e1.64 (1H, m, H-3), 1.64e1.74 (1H, m,

H-4exo), 1.75e1.89 (4H, m, H-5endo, H-17e, H-15a, H-16a),

2.14e2.32 (3H, m, H-2exo, H-15e, Н-17e), 2.57e2.69 (2H, m, Н-11),

3.02e3.14 (2H, m, H-13а, H-14а), 3.17e3.26 (2H, m, H-13e, H-14e),

5-Chloropentanoyl chloride (5 mmol) and Et₃N (5 mmol) was

added to a solution of (ꢀ)-borneol (4.5 mmol) in CH₂Cl₂at 0e5 ^ꢁC,

after which the mixture was stirred at room temperature for 24 h.

The reaction mixture was washed with brine and extracted with

CH₂Cl₂. Then, the combined organic phase was dried over anhy-

drous Na₂SO₄and the solvent was removed. The crude product was

then puriﬁed by ﬂash CC (silica gel, eluent: hexane-ethyl acetate).

3.42e3.51 (2H, m, H-13), 4.79e4.85 (1H, m, H-1exo). ¹³C NMR (

d,

ppm): 170.37 s (C-11), 81.24 d (C-1), 53.35 and 53.29 t (C-14, C-15),

52.17 t (C-13), 48.56 s (C-6), 47.66 s (C-7), 44.54 d (C-3), 36.41 t (C-

2), 28.92 t (C-4), 27.72 t (C-5), 26.76 t (C-12), 22.36 t (C-16, C-17),

21.79 t (C-18), 19.44 q (Me-9), 18.58 q (Me-10), 13.33 q (Me-8). Anal.

Calcd for C₁₈H₃₂ClNO₂, C, 65.53; H, 9.78; Cl, 10.75; N, 4.25; O, 9.70.

Found, %: C, 65.58; H, 9.68; Cl, 10.64; N, 4.30. M.p. ¼ 245.1 ^ꢁC.

4-Methyl-1-(3-oxo-3-((1S,2R,4S)-1,7,7-trimethylbicyclo[2.2.1]

heptan-2-yloxy)propyl)piperidinium chloride 4b·HCl.

Yield 58%; pale yellow oil. ¹H NMR (

d, ppm, J/Hz): 0.79 (3H, s, Me-9),

0.84 (3H, s, Me-10), 0.87 (3H, s, Me-8), 0.92 (1H, dd, ²J ¼ 13.7, J_2endo,

¼ 3.4, H-2endo), 1.15e1.32 (2H, m, H-4endo, H-5exo), 1.60e1.94

1exo

(7H, m, H-3, H-4exo, H-5endo, H-13, H-14), 2.28e2.37 (3H, m, H-12,

H-2exo), 3.49e3.58 (2H, m, H-15), and 4.83e4.88 (1H, m, H-1exo).

Yield 85%; white powder. ¹H NMR (

10), 0.81 (3H, с, e-8), 0.83 (3H, с,

d, ppm, J/Hz): 0.75 (3H, с, Мe-

М

e-9), 0.89 (1H, dd, ²J ¼ 14.2,

J_{2endo, 1exo}¼ 3.4, H-2endo), 0.98 (3H, d, J ¼ 5.7 Hz, Me-19), 1.13e1.28

(2H, m, H-4endo, H-5exo), 1.49e2.01 (8H, m, H-3, H-4exo, H-5endo,

H-16a, H-17a, H-16e, H-17e, H-18), 2.22e2.32 (1H, m, H-2exo),

2.57e2.69 (2H, m, Н-11), 3.04e3.11 (2H, m, H-13а, H-14а),

3.17e3.25 (2H, m, H-13e, H-14e), 3.41e3.49 (2H, m, H-13),

5.2.3. General synthetic procedure for compounds 3-4d

A

mixture of chloride 2d (2.5 mmol), piperidine/4-

methylpiperidine (3 mmol), Et₃N (3 mmol) and 10 mL CH₂Cl₂

were reﬂuxed for 8 h. After completion, the mixture was washed

with brine and extracted with CH₂Cl₂. The combined organic phase

was dried over Na₂SO₄, and the solvent was removed in vacuo. The

crude product was then puriﬁed by silica gel CC (eluent: hexane-

ethyl acetate).

4.80e4.85 (1H, m, H-1exo). ¹³C NMR (

d, ppm): 170.38 s (C-11),

81.22 d (C-1), 53.23 and 53.17 t (C-14, C-15), 52.27 t (C-13), 48.55 s

(C-6), 47.65 s (C-7), 44.52 d (C-3), 36.41 t (C-2), 30.58 t (C-16, C-17),

29.09 d (C-18), 28.95 t (C-4), 27.71 t (C-5), 26.75 t (C-11), 20.72 t

(Me-19), 19.44 q (Me-9), 18.58 q (Me-10), 13.33 q (Me-8). Anal.

Calcd for C₁₉H₃₄ClNO₂, C, 66.35; H, 9.96; Cl, 10.31; N, 4.07; O, 9.30.

Found, %: C, 66.61; H, 11.09; Cl, 10.74; N, 4.09. M.p. ¼ 231.9 ^ꢁC.

(1S,2R,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl

5-(piper-

idin-1-yl)pentanoate 3d.

Yield 47%; pale yellow oil. ¹H NMR (

d, ppm, J/Hz): 0.79 (3H, s,

Me-10), 0.84 (3H, s, Me-8), 0.87 (3H, s, Me-9), 0.91 (1H, dd, ²J ¼ 12.9,

J_{2endo, 1exo}¼ 4.5, H-2endo), 1.14e1.31 (2H, m, H-4endo, H-5exo),

1.34e1.43 (2H, m, H-20), 1.45e1.76 (11H, m, H-3, H-4exo, H-5endo,

H-13, H-14, H-18, H-19), 1.86e1.94 (1H, m, H-5endo), 2.23e2.41

(9H, m, H-2exo, H-12, H-15, H-16, H-17), 4.82e4.87 (1H, m, H-

5.2.5. General procedures A: syntheses of esters 8c, 9-12a-c

A1: A solution of (1S,2R,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-

2-yl 2-chloroacetate (2a; 1 equiv), Et₃N (1 equiv) and the requisite

secondary amine (1.5 equiv) in CH₂Cl₂was stirred at room tem-

perature for 12 h. The resulting mixture was diluted with brine and

extracted twice with CH₂Cl₂. Then, the combined organic layer was

dried with anhydrous Na₂SO₄and evaporated. The crude residues

were puriﬁed via silica gel column chromatography (hexane-ethyl

acetate eluent).

1exo). ¹³C NMR (

d, ppm): 173.83 s (C-11), 79.50 d (C-1), 58.96 t (C-

15), 54.49 t (C-16, C-17), 48.59 s (C-6), 47.64 s (C-7), 44.75 d (C-3),

36.70 t (C-2), 34.46 t (C-12), 27.91 t (C-4), 26.98 t (C-5), 26.31 t (C-

14), 25.85 t (C-18, C-19), 24.34 t (C-20), 23.17 t (C-13), 19.57 q (Me-

9), 18.70 q (Me-10), 13.38 q (Me-8). HR-MS: 321.2663 (M^þ,

C

₂₀H₃₅O₂N₁; calcd 321.2662).

(1S,2R,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl

A2: A solution of (1S,2R,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-

2-yl 3-chloropropanoate (2b; 1 equiv), Et₃N (1 equiv) and the

requisite secondary amine (1.5 equiv) in CH₂Cl₂was reﬂuxed for

12e24 h. The resulting mixture was diluted with brine and

extracted twice with CH₂Cl₂. Then, the combined organic layer was

dried with anhydrous Na₂SO₄and evaporated. The crude residues

were puriﬁed via silica gel column chromatography (hexane-ethyl

5-(4-

methylpiperidin-1-yl)pentanoate 4d.

Yield 51%; pale yellow oil. ¹H NMR (

d

, ppm, J/Hz)): 0.78 (3H, s,

Me-10), 0.83 (3H, s, Me-8), 0.86 (3H, s, Me-9), 0.87 (3H, d, J ¼ 6.6 Hz,

Me-21), 0.91 (1H, m, H-2endo), 1.12e1.31 (6H, m, H-4endo, H-5exo,

H-18a, H-19a, H-14), 1.46e1.74 (8H, m, H-3, H-4exo, H-5endo, H-13,

12

A.S. Sokolova et al. / European Journal of Medicinal Chemistry 207 (2020) 112726

acetate eluent).

352.2716 (M^þ, C₂₀H₃₆O₃N₂; calcd 352.2721).

A3: A solution of (1S,2R,4S)-1,7,7-trimethylbicyclo[2.2.1]-hep-

tan-2-yl 4-chlorobutanoate (2c; 1 equiv), Et₃N (1 equiv), DBU (one

drop) and the requisite secondary amine (1.5 equiv) in CH₃CN was

reﬂuxed for 18e24 h. The reaction mixture was concentrated under

reduced pressure, diluted with brine and extracted twice with

CH₂Cl₂. Then, the combined organic layer was dried with anhy-

drous Na₂SO₄and evaporated. The crude residues were puriﬁed via

silica gel column chromatography (hexane-ethylacetate eluent).

Ethyl 4-(4-oxo-4-((1S,2R,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-

2-yloxy)butyl)piperazine-1-carboxylate 8c (procedure A3). Yield 37%;

(1S,2R,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl

2-(4-(2-

aminoethyl)piperazin-1-yl)acetate 10a (procedure A1). Yield 25%;

brown oil. ¹H NMR (

d

, ppm, J/Hz): 0.76 (3H, s, Me-10), 0.80 (3H, s,

Me-8), 0.84 (3H, s, Me-9), 0.91 (1H, dd, ²J ¼ 14.2, J_{2endo, 1exo}¼ 3.2, H-

2endo), 1.11e1.29 (2H, m, H-4endo, H-5exo), 1.59e1.73 (2H, m, H-

4exo, H-3), 1.80e1.88 (1H, m, H-5endo), 2.08 (2H, br. s, NH₂),

2.25e2.34 (1H, m, H-2exo), 2.37 (2H, t, J ¼ 6.0, H-17), 2.39e2.62

(8H, br. s, H-13, H-14, H-15, H-16), 2.72 (2H, t, J ¼ 6.0, H-18), 3.17

(2H, s, H-12), 4.87 (1H, m, H-1exo). ¹³C NMR (

d, ppm): 170.55 s (C-

11), 80.07 d (C-1), 60.92 t (C-17), 59.36 t (C-12), 52.91 and 52.88 t

(C-13, C-14, C-15, C-16), 48.65 s (C-6), 47.69 s (C-7), 44.72 d (C-3),

38.66 t (C-18), 36.66 t (C-2), 27.89 t (C-4), 26.98 t (C-5), 19.58 q (Me-

9), 18.71 q (Me-8), 13.44 q (Me-10). HR-MS: 323.2554 (M^þ,

₁₈H₃₃O₂N₃; calcd 323.2567).

(1S,2R,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl

pale yellow oil. ¹H NMR (

d, ppm, J/Hz): 0.79 (3H, s, Me-10), 0.83 (3H,

s, Me-8), 0.87 (3H, s, Me-9), 0.91 (1H, dd, ²J ¼ 14.2, J_{2endo, 1exo}¼ 3.2,

H-2endo), 1.14e1.30 (5H, m, H-4endo, H-5exo, Me-21), 1.61e1.65

(1H, m, H-3), 1.65e1.74 (1H, m, H-4exo), 1.78 (2H, quint, J ¼ 7.2, H-

13), 1.84e1.92 (1H, m, H-5endo), 2.25e2.42 (9H, m, H-2exo, H-12,

H-14, H-15, H-16), 3.39e3.47 (4H, br. s, H-17, H-18), 4.09 (2H, q,

C

4-(4-(2-

aminoethyl)piperazin-1-yl)butanoate 10c (procedure A3). Yield

J ¼ 7.05, H-20), 4.85 (1H, m, H-1exo). ¹³C NMR (

d

, ppm): 173.48 s (C-

35%; brown oil. ¹H NMR (

d, ppm, J/Hz): 0.78 (3H, s, Me-10), 0.83 (3H,

11), 155.31 s (C-19), 79.78 d (C-1), 61.26 t (C-20), 57.44 t (C-14),

52.64 t (C-15, C-16), 48.64 s (C-6), 47.68 s (C-7), 44.79 d (C-3),

43.25 t (C-17, C-18), 36.72 t (C-2), 32.19 t (C-12), 27.94 t (C-4), 27.02 t

(C-5), 21.73 t (C-13),19.57 q (Me-9),18.70 q (Me-8),14.53 q (Me-21),

s, Me-8), 0.86 (3H, s, Me-9), 0.91 (1H, dd, ²J ¼ 14.2, J_{2endo, 1exo}¼ 3.2,

H-2endo),1.13e1.30 (2H, m, H-4endo, H-5exo),1.61e1.66 (1H, m, H-

3), 1.66e1.94 (4H, m, H-4exo, H-13, H-5endo), 1.96e2.04 (2H, br. s,

NH₂), 2.26e2.56 (15H, m, H-2exo, H-12, H-14, H-15, H-16, H-17, H-

13.41

380.2670).

(1S,2S,4S)-1,7,7-Trimethylbicyclo[2.2.1]heptan-2-yl

hydroxyethyl)piperazin-1-yl)acetate 9a (procedure A1). Yield 36%;

pale yellow oil. ¹H NMR (

, ppm, J/Hz): 0.76 (3H, s, Me-10), 0.80 (3H,

q

(Me-10). HR-MS: 380.2666 (M^þ, C₂₁H₃₆O₄N₂; calcd

18, H-19), 4.84 (1H, m, H-1exo). ¹³C NMR (

d, ppm): 173.67 s (C-11),

79.66 d (C-1), 57.48 t (C-14), 56.12 t (C-19), 52.88 and 52.62 t (C-15,

C-16, C-17, C-18), 48.60 s (C-6), 47.65 s (C-7), 44.71 d (C-3), 36.69 t

(C-2), 34.29 t (C-20), 32.39 t (C-12), 27.91 t (C-4), 26.97 t (C-5),

22.08 t (C-13),19.56 q (Me-9),18.69 q (Me-8),13.42 q (Me-10). Anal.

Calcd for C₂₀H₃₇O₂N₃, C, 68.33; H, 10.61; N,11.95. Found, %: C, 68.63;

H, 10.07; N, 11.53.

2-(4-(2-

d

s, Me-8), 0.83 (3H, s, Me-9), 0.91 (1H, dd, ²J ¼ 14.2, J_{2endo, 1exo}¼ 3.2,

H-2endo),1.11e1.28 (2H, m, H-4endo, H-5exo),1.59e1.73 (2H, m, H-

4exo, H-3), 1.78e1.88 (1H, m, H-5endo), 2.23e2.35 (1H, m, H-2exo),

2.43e2.67 (10H, br. s, H-13, H-14, H-15, H-16, H-17), 2.94 (1H, br. s,

OH), 3.17 (2H, s, H-12), 3.54 (2H, t, J ¼ 5.4, H-18), 4.87 (1H, m, H-

(1S,2R,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl

2-(4-

benzylpiperazin-1-yl)acetate 11a (procedure A1). Yield 42%; yellow

oil. ¹H NMR (

d, ppm, J/Hz): 0.80 (3H, s, Me-10), 0.84 (3H, s, Me-8),

1exo). ¹³C NMR (

d

, ppm): 170.34 s (C-11), 79.96 d (C-1), 59.09 t (C-

0.87 (3H, s, Me-9), 0.95 (1H, dd, ²J ¼ 13.7, J_{2endo, 1exo}¼ 3.3, H-

2endo), 1.14e1.31 (2H, m, H-4endo, H-5exo), 1.63e1.66 (1H,m, H-3),

1.68e1.76 (1H, m, H-4exo), 1.83e1.92 (1H, m, H-5endo), 2.27e2.37

(1H, m, H-2exo), 2.46e2.64 (8H, br. s, H-13, H-14, H-15, H-16), 3.20

(2H, s, H-12), 3.50 (2H, s, H-17), 4.91 (1H, m, H-1exo), 7.19e7.24 (1H,

18), 59.05 t (C-12), 57.47 t (C-17), 52.62 and 52.45 t (C-13, C-14, C-

15, C-16), 48.50 s (C-6), 47.54 s (C-7), 44.55 d (C-3), 36.52 t (C-2),

27.75 t (C-4), 26.84 t (C-5), 19.45 q (Me-9), 18.58 q (Me-8), 13.32 q

(Me-10). HR-MS: 324.2413 (M^þ, C₁₈H₃₂O₃N₂; calcd 324.2407).

(1S,2R,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl

3-(4-(2-

m, H-21), 7.27e7.30 (4H, m, H-19, H-20, H-22, H-23). ¹³C NMR (

d,

hydroxyethyl)piperazin-1-yl)propanoate 9b (procedure A2). Yield

ppm): 170.56 s (C-11), 138.00 s (C-18), 129.04 d (C-19, C-23),

128.06 d (C-20, C-22), 126.88 d (C-21), 80.02 d (C-1), 62.83 t (C-17),

59.36 t (C-12), 52.86 and 52.73 t (C-13, C-14, C-15, C-16), 48.63 s (C-

6), 47.67 s (C-7), 44.69 d (C-3), 36.64 t (C-2), 27.87 t (C-4), 26.96 t (C-

5), 19.57 q (Me-9), 18.71 q (Me-8), 13.43 q (Me-10). HR-MS:

370.2614 (M^þ, C₂₃H₃₄O₂N₂; calcd 370.2615).

45%; pale yellow oil. ¹H NMR (

d, ppm, J/Hz): 0.78 (3H, s, Me-10),

0.82 (3H, s, Me-8), 0.85 (3H, s, Me-9), 0.93 (1H, dd, ²J ¼ 14.2,

J_{2endo, 1exo}¼ 3.2, H-2endo), 1.12e1.29 (2H, m, H-4endo, H-5exo),

1.60e1.64 (1H, m, H-3), 1.65e1.75 (1H, m, H-4exo), 1.81e1.91 (1H,

m, H-5endo), 2.24e2.34 (1H, m, H-2exo), 2.38e2.72 (14H, br. s, H-

12, H-13, H-14, H-15, H-16, H-17, H-18), 3.28 (1H, br. s, OH),

(1S,2R,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl

benzylpiperazin-1-yl)propanoate 11b (procedure A2). Yield 45%;

pale yellow oil. ¹H NMR (

, ppm, J/Hz): 0.80 (3H, s, Me-10), 0.84 (3H,

3-(4-

3.57e3.63 (2H, m, H-19), 4.85 (1H, m, H-1exo). ¹³C NMR (

d, ppm):

172.47 s (C-11), 79.71 d (C-1), 59.20 t (C-19), 57.35 t (C-18), 53.38 t

(C-13), 52.63 and 52.30 t (C-14, C-15, C-16, C-17), 48.59 s (C-6),

47.59 s (C-7), 44.64 d (C-3), 36.47 t (C-2), 32.54 t (C-12), 27.85 t (C-

4), 26.92 t (C-5), 19.49 q (Me-9), 18.63 q (Me-8), 13.32 q (Me-10).

Anal. Calcd for C₁₉H₂₄O₃N₂, C, 67.42; H, 10.12; N, 8.28. Found, %: C,

67.04; H, 9.88; N, 8.21.

d

s, Me-8), 0.88 (3H, s, Me-9), 0.95 (1H, dd, ²J ¼ 13.7, J_{2endo, 1exo}¼ 3.3,

H-2endo), 1.15e1.31 (2H, m, H-4endo, H-5exo), 1.62e1.67 (1H,m, H-

3), 1.66e1.76 (1H, m, H-4exo), 1.85e1.94 (1H, m, H-5endo),

2.26e2.36 (1H, m, H-2exo), 2.37e2.54 (10H, br. s, H-12, H-14, H-15,

H-16), 2.65e2.69 (2H, m, H-13), 3.48 (2H, s, H-18), 4.86 (1H, m, H-

1exo), 7.19e7.23 (1H, m, H-22), 7.26e7.30 (4H, m, H-20, H-21, H-23,

(1S,2R,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl

hydroxyethyl)piperazin-1-yl)butanoate 9c (procedure A3). Yield

27%; pale yellow oil. ¹H NMR (

, ppm, J/Hz): 0.77 (3H, s, Me-10),

4-(4-(2-

H-24). ¹³C NMR (

d, ppm): 172.74 s (C-11), 137.96 s (C-19), 129.08 d

d

(C-20, C-24), 128.07 d (C-21, C-23), 126.89 d (C-22), 79.70 d (C-1),

62.92 t (C-18), 53.60 t (C-13), 52.91 and 52.75 t (C-14, C-15, C-16, C-

17), 48.66 s (C-6), 47.66 s (C-7), 44.73 d (C-3), 36.54 t (C-2), 32.69 t

(C-12), 27.91 t (C-4), 26.99 t (C-5), 19.58 q (Me-9), 18.71 q (Me-8),

0.82 (3H, s, Me-8), 0.85 (3H, s, Me-9), 0.90 (1H, dd, ²J ¼ 14.2,

J_{2endo, 1exo}¼ 3.2, H-2endo), 1.10e1.30 (2H, m, H-4endo, H-5exo),

1.59e1.64 (1H, m, H-3), 1.66e1.93 (4H, m, H-4exo, H-13, H-5endo),

2.27e2.36 (5H, m, H-2exo, H-12, H-14), 2.36e2.55 (10H, br. s, H-15,

H-16, H-17, H-18, H-19), 2.82 (1H, br. s, OH), 3.56 (2H, t, J ¼ 5.4, H-

13.38

384.2771).

(1S,2R,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl

benzylpiperazin-1-yl)butanoate 11c (procedure A3). Yield 31%; yel-

q

(Me-10). HR-MS: 384.2767 (M^þ, C₂₄H₃₆O₂N₂; calcd

20), 4.83 (1H, m, H-1exo). ¹³C NMR (

d, ppm): 173.58 s (C-11), 79.57 d

4-(4-

(C-1), 59.18 t (C-20), 57.59 t (C-14), 57.46 t (C-19), 52.96 and 52.71 t

(C-15, C-16, C-17, C-18), 48.57 s (C-6), 47.60 s (C-7), 44.71 d (C-3),

36.66 t (C-2), 32.38 t (C-12), 27.88 t (C-4), 26.95 t (C-5), 22.10 t (C-

13), 19.52 q (Me-9), 18.65 q (Me-8), 13.36 q (Me-10). HR-MS:

low oil. ¹H NMR (

d, ppm, J/Hz): 0.79 (3H, s, Me-10), 0.84 (3H, s, Me-

8), 0.87 (3H, s, Me-9), 0.92 (1H, dd, ²J ¼ 13.7, J_{2endo, 1exo}¼ 3.3, H-

2endo), 1.15e1.30 (2H, m, H-4endo, H-5exo), 1.62e1.66 (1H,m, H-3),

A.S. Sokolova et al. / European Journal of Medicinal Chemistry 207 (2020) 112726

13

1.66e1.75 (1H, m, H-4exo), 1.79 (2H, quint, J ¼ 7.4, H-13), 1.85e1.94

(1H, m, H-5endo), 2.26e2.37 (5H, m, H-2exo, H-12, H-14),

2.38e2.56 (8H, br. s, H-15, H-16, H-17, H-18), 3.48 (2H, s, H-19), 4.85

(1H, m, H-1exo), 7.19e7.23 (1H, m, H-23), 7.25e7.29 (4H, m, H-21,

(2H, m, H-4endo, H-5exo), 1.61e1.74 (2H, m, H-3, H-4exo),

1.84e1.91 (1H, m, H-5endo), 2.32 (6H, s, M ꢀ 13, Me-14), 2.29e2.33

(1H, m, H-2exo), 3.15 (2H, s, H-12), 4.91 (1H, m, H-1exo). ¹³C NMR (

d,

ppm): 170.86 s (C-11), 79.88 d (C-1), 60.28 t (C-12), 48.61 s (C-6),

47.65 s (C-7), 45.00 q (Me-13, Me-14), 44.68 d (C-3), 36.61 t (C-2),

27.85 t (C-4), 26.98 t (C-5), 19.55 q (Me-9), 18.68 q (Me-8), 13.37 q

(Me-10). HR-MS: 239.1884 (M^þ, C₁₄H₂₅O₂N₁; calcd 239.1880).

H-22, H-24, H-25). ¹³C NMR (

d, ppm): 173.67 s (C-11), 137.96 s (C-

20), 129.10 d (C-20, C-24), 128.05 d (C-21, C-23), 126.88 d (C-22),

79.60 d (C-1), 62.95 t (C-18), 57.60 t (C-14), 53.02 and 52.92 t (C-15,

C-16, C-17, C-18), 48.62 s (C-6), 47.65 s (C-7), 44.77 d (C-3), 36.70 t

(C-2), 32.52 t (C-12), 27.92 t (C-4), 27.00 t (C-5), 22.20 t (C-13), 19.57

q (Me-9), 18.71 q (Me-8), 13.41 q (Me-10). HR-MS: 398.2930 (M^þ,

(1S,2R,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl

2-(dieth-

ylamino)acetate 14a. A solution of 2a (4.3 mmol) and CH₃CN

(10 mL) was treated with excess of diethylamine (6 mmol) and

K₂CO₃(7 mmol) was added. The mixture left for the night at room

temperature. The precipitate was ﬁltered off and the solvent was

removed at reduced pressure. The crude product was puriﬁed by

column chromatography silica gel eluent hexane/EtOAc 100/0 / 0/

C

₂₅H₃₈O₂N₂; calcd 398.2928).

(1S,2R,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl 2-(azepan-1-

yl)acetate 12a (procedure A1). Yield 51%; yellow oil. ¹H NMR (

d,

ppm, J/Hz): 0.80 (3H, s, Me-10), 0.85 (3H, s, Me-8), 0.87 (3H, s, Me-

9), 0.95 (1H, dd, ²J ¼ 13.7, J_{2endo, 1exo}¼ 3.3, H-2endo), 1.15e1.34 (2H,

m, H-4endo, H-5exo), 1.54e1.77 (12H, m, H-4endo, H-5exo, H-3, H-

4exo, H-14, H-15, H-16, H-17), 1.87e1.95 (1H, m, H-5endo),

2.29e2.39 (1H, m, H-2exo), 2.78 (4H, t, J ¼ 5.2, H-13, H-18), 3.40

100. Yield 65%; pale yellow oil. ¹H NMR (

d, ppm, J/Hz): 0.78 (3H, s,

Me-10), 0.82 (3H, s, Me-8), 0.85 (3H, s, Me-9), 0.92 (1H, dd, ²J ¼ 13.7,

J_2endo,

¼ 3.3, H-2endo), 1.03 (6H, t, J ¼ 7.2, Me-15, Me-16),

1exo

1.14e1.29 (2H, m, H-4endo, H-5exo), 1.61e1.75 (2H, m, H-3, H-

4exo), 1.85e1.92 (1H, m, H-5endo), 2.27e2.35 (1H, m, H-2exo), 2.63

(4H, q, J ¼ 7.2, H-13, H-14), 3.31 (2H, s, H-12), 4.88 (1H, m, H-1exo).

(2H, s, H-12), 4.90 (1H, m, H-1exo). ¹³C NMR (

d, ppm): 171.69 s (C-

11), 80.01 d (C-1), 59.79 t (C-12), 54.93 t (C-13, C-18), 48.60 s (C-6),

47.67 s (C-7), 44.72 d (C-3), 36.71 t (C-2), 28.44 t (C-14, C-17), 27.89 t

(C-4), 27.07 t (C-5), 26.87 t (C-15, C-16),19.58 q (Me-9),18.70 q (Me-

8), 13.43 q (Me-10). HR-MS: 293.2348 (M^þ, C₁₈H₃₁O₂N₁; calcd

293.2349).

¹³C NMR (

d, ppm): 171.72 s (C-11), 79.78 d (C-1), 53.85 t (C-12),

48.55 s (C-6), 47.61 s (C-7), 47.54 t (C-13, C-14), 44.68 d (C-3), 36.68 t

(C-2), 27.86 t (C-4), 27.04 t (C-5), 19.54 q (Me-9), 18.67 q (Me-8),

13.37 q (Me-10), 12.35 q (M ꢀ 15, Me-16). HR-MS: 267.2196 (M^þ,

(1S,2R,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl 3-(azepan-1-

C₁₆H₂₉O₂N₁; calcd 267.2193).

yl)propanoate 12b (procedure A2). Yield 47%; pale yellow oil. ¹H

NMR (

d

, ppm, J/Hz): 0.80 (3H, s, Me-10), 0.84 (3H, s, Me-8), 0.87 (3H,

5.2.7. General synthetic procedures for the preparation of 13 b,c and

14 b,c

s, Me-9), 0.95 (1H, dd, ²J ¼ 13.7, J_2endo,

¼ 3.3, H-2endo),

1exo

1.14e1.30 (2H, m, H-4endo, H-5exo), 1.51e1.76 (12H, m, H-4endo,

H-5exo, H-3, H-4exo, H-15, H-16, H-17, H-18), 1.87e1.96 (1H, m, H-

5endo), 2.26e2.37 (1H, m, H-2exo), 2.47 (2H, t, J ¼ 7.2, H-12),

2.59e2.65 (4H, m, H-14, H-19), 2.83 (2H, t, J ¼ 7.2, H-13), 4.85 (1H,

A solution of 2b or 2c (2.8 mmol) and CH₃CN (7 mL) was treated

with an excess of dimethylamine (1.5 mL 40% wt.) solution in

ethanol (for the synthesis compounds 13b,c) or diethylamine (for

the synthesis compounds 14b,c), and K₂CO₃(7 mmol) was added.

The mixture was reﬂuxed for 12, after which the precipitate was

ﬁltered off and the solvent was removed at reduced pressure. Then,

CH₂Cl₂was added to the resulting mixture, after which the solution

was diluted with brine and extracted twice with CH₂Cl₂. The

combined organic layer was dried with anhydrous Na₂SO₄and

evaporated. The crude residues were puriﬁed via silica gel column

chromatography (hexane-ethylacetate eluent).

m, H-1exo). ¹³C NMR (

d, ppm): 172.68 s (C-11), 79.33 d (C-1), 54.71 t

(C-14, C-19), 53.36 t (C-13), 48.30 s (C-6), 47.33 s (C-7), 44.43 d (C-

3), 36.26 t (C-2), 32.84 t (C-12), 27.63 t (C-15, C-18), 27.57 t (C-4),

26.68 t (C-5), 26.48 t (C-16, C-17), 19.25 q (Me-9), 18.39 q (Me-8),

13.03

307.2506).

(1S,2R,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl 4-(azepan-1-

yl)butanoate 12c (procedure A3). Yield 45%; yellow oil. ¹H NMR (

q

(Me-10). HR-MS: 307.2507 (M^þ, C₁₉H₃₃O₂N₁; calcd

d

,

(1S,2R,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl 3-(dimethyla-

ppm, J/Hz): 0.79 (3H, s, Me-10), 0.84 (3H, s, Me-8), 0.87 (3H, s, Me-

9), 0.92 (1H, dd, ²J ¼ 13.7, J_{2endo, 1exo}¼ 3.3, H-2endo), 1.15e1.30 (2H,

m, H-4endo, H-5exo), 1.51e1.66 (11H, m, H-4endo, H-5exo, H-3, H-

16, H-17, H-18, H-19), 1.66e1.81 (3H, m, H-4exo, H-13), 1.87e1.95

(1H, m, H-5endo), 2.26e2.37 (3H, m, H-2exo, H-12), 2.48 (2H, t,

J ¼ 7.4, H-14), 2.58e2.64 (4H, m, H-15, H-20), 4.85 (1H, m, H-1exo).

mino)propanoate 13b. Yield 53%; pale yellow oil. ¹H NMR (

d, ppm, J/

Hz): 0.78 (3H, s, Me-10), 0.82 (3H, s, Me-8), 0.86 (3H, s, Me-9), 0.93

(1H, dd, ²J ¼ 13.7, J_{2endo, 1exo}¼ 3.3, H-2endo), 1.14e1.30 (2H, m, H-

4endo, H-5exo), 1.61e1.75 (2H, m, H-3, H-4exo), 1.85e1.94 (1H, m,

H-5endo), 2.21 (6H, s, M ꢀ 14, Me-15), 2.25e2.35 (1H, m, H-2exo),

2.42e2.61 (4H, AB, J ¼ 7.1, H-12, H-13), 4.85 (1H, m, H-1exo). ¹³C

¹³C NMR (

d

, ppm): 173.22 s (C-11), 80.01 d (C-1), 56.71 t (C-14),

NMR (d, ppm): 172.63 s (C-11), 79.75 d (C-1), 54.78 t (C-13), 48.65 s

54.71 t (C-15, C-20), 48.61 s (C-6), 47.67 s (C-7), 44.73 d (C-3),

36.65 t (C-2), 31.69 t (C-12), 27.88 t (C-4), 26.95 t (C-5), 26.84 t (C-

16, C-19), 25.69 t (C-17, C-18), 21.14 t (C-13), 19.54 q (Me-9), 18.67 q

(Me-8), 13.39 q (Me-10). HR-MS: 321.2659 (M^þ, C₂₀H₃₅O₂N₁; calcd

321.2662).

(C-6), 47.66 s (C-7), 45.05 q (Me-14, Me-15), 44.82 d (C-3), 36.61 t

(C-2), 33.17 t (C-12), 27.90 t (C-4), 27.03 t (C-5),19.56 q (Me-9),18.69

q (Me-8), 13.29 q (Me-10). HR-MS: 253.2033 (M^þ, C₁₅H₂₇O₂N; calcd

253.2036).

(1S,2R,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl 4-(dimethyla-

mino)butanoate 13c. Yield 47%; pale yellow oil. ¹H NMR (

d, ppm, J/

5.2.6. Preparation of derivatives 13a and 14a

Hz): 0.79 (3H, s, Me-10), 0.83 (3H, s, Me-8), 0.86 (3H, s, Me-9), 0.91

(1H, dd, ²J ¼ 13.7, J_{2endo, 1exo}¼ 3.3, H-2endo), 1.14e1.30 (2H, m, H-

4endo, H-5exo), 1.60e1.75 (2H, m, H-3, H-4exo), 1.77e1.93 (3H, m,

H-5endo, H-13), 2.28 (6H, s, M ꢀ 15, Me-16), 2.29e2.40 (5H, m, H-

(1S,2R,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl 2-(dimethyla-

mino)acetate 13a. A solution of (1S,2R,4S)-1,7,7-trimethylbicyclo

[2.2.1]heptan-2-yl 2-chloroacetate 2a (6.5 mmol) and CH₃CN

(10 mL) was treated with excess of dimethylamine (2.5 mL, 40% wt.)

solution in ethanol and K₂CO₃(7 mmol) was added. The mixture

left for the night at room temperature. The precipitate was ﬁltered

off and the solvent was removed at reduced pressure. The crude

product was puriﬁed by column chromatography silica gel eluent

hexane/EtOAc 100/0 / 0/100. Yield 71%; pale yellow oil. ¹H NMR

2exo, H-12, H-14), 4.84 (1H, m, H-1exo). ¹³C NMR (

d, ppm): 173.68 s

(C-11), 79.59 d (C-1), 58.70 t (C-14), 48.55 s (C-6), 47.61 s (C-7),

45.20 q (Me-15, Me-16), 44.69 d (C-3), 36.63 t (C-2), 32.26 t (C-12),

27.87 t (C-4), 26.92 t (C-5), 22.82 t (C-13), 19.54 q (Me-9), 18.67 q

(Me-8), 13.36 q (Me-10). HR-MS: 267.1197 (M^þ, C₁₆H₂₉NO₂; calcd

267.2193).

(d

, ppm, J/Hz): 0.79 (3H, s, Me-10), 0.83 (3H, s, Me-8), 0.86 (3H, s,

(1S,2R,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl

3-(dieth-

, ppm,

Me-9), 0.93 (1H, dd, ²J ¼ 13.7, J_{2endo, 1exo}¼ 3.3, H-2endo), 1.15e1.30

ylamino)propanoate 14b. Yield 58%; pale yellow oil. ¹H NMR (

d

14

A.S. Sokolova et al. / European Journal of Medicinal Chemistry 207 (2020) 112726

J/Hz): 0.79 (3H, s, Me-10), 0.83 (3H, s, Me-8), 0.86 (3H, s, Me-9), 0.93

(1H, dd, ²J ¼ 13.7, J_{2endo, 1exo}¼ 3.3, H-2endo), 0.99 (6H, t, J ¼ 7.2, Me-

16, Me-17), 1.15e1.29 (2H, m, H-4endo, H-5exo), 1.61e1.64 (1H, m,

H-3), 1.65e1.73 (1H, m, H-4exo), 1.86e1.92 (1H, m, H-5endo), 2.21

(6H, s, M ꢀ 14, Me-15), 2.26e2.34 (1H, m, H-2exo), 2.39e2.44 (2H,

m, H-12), 2.48 (4H, q, J ¼ 7.2, H-14, H-15), 2.74e2.80 (2H, m, H-13),

J ¼ 4.3, 8.6, H-1exo), 5.55 (1H, NeH).

5.2.8. General procedure for syntheses of amides 18-21c

A solution comprising 17c (1.5 mmol) in acetonitrile (15 ml), the

corresponding amine (2 mmol), Et₃N (2 mmol) and one drop of

DBU were reﬂuxed for 12 h. After completion, the mixture was

concentrated in a vacuum. Brine and CHCl₃were added to the

residue, and then the mixture was extracted twice with CHCl₃.

Then, the combined organic phase was dried over Na₂SO₄, and the

solvent was removed in vacuo. The crude product was puriﬁed by

silica gel CC (eluent: hexane-ethyl acetate).

4.84 (1H, m, H-1exo). ¹³C NMR (

d, ppm): 173.08 s (C-11), 79.59 d (C-

1), 48.57 s (C-6), 48.23 t (C-13), 47.61 s (C-7), 46.51 t (C-14, C-15),

44.71 d (C-3), 36.56 t (C-2), 32.35 t (C-12), 27.86 t (C-4), 26.96 t (C-

5), 19.54 q (Me-9), 18.68 q (Me-8), 13.33 q (Me-10), 11.70 q (Me-16,

Me-17). HR-MS: 281.2348 (M^þ, C₁₇H₃₁O₂N₁; calcd 281.2349).

(1S,2R,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl

4-(dieth-

, ppm,

4-Morpholino-N-((1R,2R,4R)-1,7,7-trimethylbicyclo[2.2.1]heptan-

ylamino)butanoate 14c. Yield 42%; pale yellow oil. ¹H NMR (

d

2-yl)butanamide 18c. Yield: 46%, yellow oil. ¹H NMR (

d, ppm, J/Hz):

J/Hz): 0.79 (3H, s, Me-10), 0.83 (3H, s, Me-8), 0.86 (3H, s, Me-9), 0.91

(1H, dd, ²J ¼ 13.7, J_{2endo, 1exo}¼ 3.3, H-2endo), 1.01 (6H, t, J ¼ 7.2, Me-

17, Me-18), 1.13e1.32 (2H, m, H-4endo, H-5exo), 1.61e1.82 (6H, m,

H-4endo, H-5exo, H-3, H-4exo, H-13), 1.83e1.95 (1H, m, H-5endo),

2.25e2.37 (3H, m, H-2exo, H-12), 2.41e2.58 (6H, m, H-14, H-15, H-

0.79 (6H, s, Me-10, Me-9), 0.86 (3H, s, Me-8), 1.07e1.15 (1H, m, H-

4endo), 1.19e1.28 (1H, m, H-5endo), 1.44e1.56 (2H, m, H-5exo, H-

2endo), 1.60e1.71 (2H, m, H-4exo, H-3), 1.75e1.85 (3H, m, H-2exo,

H-13), 2.16 (2H, t, J ¼ 7.3, H-12), 2.32 (2H, t, J ¼ 7.2, H-14), 2.35e2.41

(2H, br. s, H-15, H-16), 3.64e3.68 (4H, m, H-17, H-18), 3.87 (1H, dt,

16), 4.85 (1H, m, H-1exo). ¹³C NMR (

d

, ppm): 173.75 s (C-11), 79.64 d

J ¼ 4.9, 9.2, H-1exo), 5.44 (1H, NeH).¹³C NMR (

d, ppm): 171.6 s (C-

(C-1), 51.86 t (C-14), 48.61 s (C-6), 47.66 s (C-7), 46.79 t (C-15, C-16),

44.79 d (C-3), 36.69 t (C-2), 32.37 t (C-12), 27.92 t (C-4), 27.00 t (C-

5), 22.21 t (C-13), 19.57 q (Me-9), 18.70 q (Me-8), 13.37 q (Me-10),

11.40 q (Me-17, Me-18). HR-MS: 295.2513 (M^þ, C₁₈H₃₃NO₂; calcd

295.2506).

11), 66.8 t (C-17, C-18), 57.5 t (C-14), 56.4 d (C-1), 53.5 t (C-15, C-16),

48.3 s (C-6), 46.9 s (C-7), 44.8 d (C-3), 39.0 t (C-2), 35.8 t (C-5), 34.3 t

(C-12), 26.8 t (C-4), 22.3 t (C-13), 20.2 q, 20.1 q (Me-8, Me-9), 11.6 q

(Me-10). HRMS: calcd for C₁₈H₃₂O₂N₂: 308.2458; found: 308.2451.

4-(4-Methylpiperazin-1-yl)-N-((1R,2R,4R)-1,7,7-trimethylbicyclo

[2.2.1]heptan-2-yl)butanamide 19c. Yield: 28%, brown oil. ¹H NMR

2-(4-Ethylpiperazin-1-yl)-N-((1R,2R,4R)-1,7,7-trimethylbicyclo

[2.2.1]heptan-2-yl)acetamide 21a.

A

solution of 2-chloro-N-

(d, ppm, J/Hz): 0.78 (6H, s, Me-10, Me-9), 0.85 (3H, s, Me-8),

((1R,2R,4R)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl)acetamide

(17a; 1 equiv), Et₃N (1 equiv) and the 1-ethylpiperazine (1.5 equiv)

in CH₂Cl₂was reﬂuxed for 12 h. After completion, the mixture was

washed with brine and extracted twice with CHCl₃. Then, the

combined organic phase was dried over Na₂SO₄, and the solvent

was removed in vacuo. The crude product was puriﬁed by silica gel

column chromatography (eluent: hexane/ethyl acetate). Yield: 35%,

1.07e1.14 (1H, m, H-4endo), 1.20e1.28 (1H, m, H-5endo),

1.45e1.55 (2H, m, H-5exo, H-2endo), 1.60e1.70 (2H, m, H-4exo, H-

3), 1.74e1.84 (3H, m, H-2exo, H-13), 2.15 (2H, t, J ¼ 6.8, H-12), 2.26

(3H, s, H-19), 2.10e2.53 (10H, m, H-14, H-15, H-16, H-17, H-18), 3.85

(1H, dt, J ¼ 4.3, 8.6, H-1exo), 5.56 (1H, NeH).¹³C NMR (

d, ppm): 171.

7 s (C-11), 57.0 t (C-14), 56.4 d (C-1), 54.8 t (C-15, C-16), 52.7 t (C-17,

C-18), 48.3 s (C-6), 46.9 s (C-7), 45.8 q (Me-19), 44.7 d (C-3), 38.9 t

(C-2), 35.7 t (C-5), 34.3 t (C-12), 26.8 t (C-4), 22.5 t (C-13), 20. 2 q,

20.1 q (Me-8, Me-9), 11.6 q (Me-10). HR-MS: 321.2775 (M^þ,

yellow oil. ¹H NMR (

d, ppm, J/Hz): 0.77 (3H, s, Me-10), 0.80 (3H, s,

Me-9), 0.89 (3H, s, Me-8), 1.05 (3H, t, J ¼ 7.2, Me-18), 1.07e1.15 (1H,

m, H-4endo), 1.21e1.30 (1H, m, H-5endo), 1.47e1.56 (2H, m, H-

5exo, H-2endo), 1.69e1.72 (2H, m, H-4exo, H-3), 1.81 (1H, dd,

J ¼ 9.0, 13.2, H-2exo), 2.37 (2H, q, J ¼ 7.2, H-17), 2.42e2.63 (8H, br. s,

H-13, H-14, H-15, H-16), 2.94 (2H, AB-d, J ¼ 4.2, H-12), 3.83 (1H, dt,

C₁₉H₃₅O₁N₃; calcd 321.2775).

4-(4-Methylpiperidin-1-yl)-N-((1R,2R,4R)-1,7,7-trimethylbicyclo

[2.2.1]heptan-2-yl)butanamide 20c. Yield: 36%, yellow oil. ¹H NMR

(d, ppm, J/Hz): 0.79 (6H, s, Me-10, Me-9), 0.86 (3H, s, Me-8), 0.88

J ¼ 4.3, 8.6, H-1exo), 7.36 (1H, NeH).¹³C NMR (

d

, ppm): 168. 9 s (C-

(3H, d, J ¼ 6.5, Me-20), 1.08e1.30 (5H, m, H-4endo, H-5endo, H-18a,

H-19, H-19a), 1.45e1.72 (6H, m, H-5exo, H-2endo, H-4exo, H-3, H-

17e, H-18e), 1.75e1.92 (5H, m, H-2exo, H-15a, H-16a, H-13), 2.15

(2H, t, J ¼ 6.8, H-12), 2.28e2.36 (2H, m, H-14), 2.78e2.88 (2H, m, H-

11), 61.2 t (C-12), 55.7 d (C-1), 53.3 t (C-15, C-16), 52.9 t (C-13, C-14),

52.1 t (C-17), 48.3 s (C-6), 46.9 s (C-7), 44.8 d (C-3), 39.1 t (C-2),

35.6 t (C-5), 26.9 t (C-4), 20. 0 q, 20.1 q (Me-8, Me-9), 11.9 q (Me-10),

11.8 q (Me-18). HR-MS: 307.2614 (M^þ, C₁₈H₃₃O₁N₃; calcd 307.2618).

4-Chloro-N-((1R,2R,4R)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl)

14e, H-15e), 3.87 (1H, dt, J ¼ 4.9, 9.2, H-1exo), 5.57 (1H, NeH).¹³

C

NMR (d, ppm): 171.9 s (C-11), 57.5 t (C-14), 56.4 d (C-1), 53.9 t (C-15,

butanamide

17c.

Excess

oxalyl

chloride

and

N,N-

C-16), 48.4 s (C-6), 46.9 s (C-7), 44.8 d (C-3), 39.0 t (C-2), 35.9 t (C-5),

34.6 t (C-18, C-17), 34.1 (C-12), 30.7 d (C-19), 26.9 t (C-4), 22.9 t (C-

13), 21.7 q (Me-20), 20. 2 q, 20.1 q (Me-8, Me-9), 11.6 q (Me-10). HR-

MS: 320.2819 (M^þ, C₂₀H₃₆O₁N₂; calcd 320.2822).

dimethylformamide (one drop) were added to a solution of 4-

chlorobutanoic acid in dry CH₂Cl₂. The mixture was stirred at

room temperature for 4 h in an Ar atmosphere, after which the

excess oxalyl chloride was removed using a rotary evaporator. The

resulting 4-chlorobutanoyl chloride was immediately used in a

subsequent reaction.

4-(4-Ethylpiperazin-1-yl)-N-((1R,2R,4R)-1,7,7-trimethylbicyclo

[2.2.1]heptan-2-yl)butanamide 21c. Yield: 32%, yellow oil. ¹H NMR

(d, ppm, J/Hz): 0.79 (6H, s, Me-10, Me-9), 0.86 (3H, s, Me-8), 1.05

4-Chlorobutanoyl chloride (10 mmol) in CH₂Cl₂(5 ml) and Et₃N

(10 mmol) to a solution of (þ)-isobornyl amine (8 mmol) in CH₂Cl₂

was added the at 0e5 ^ꢁC, and the mixture was stirred at room

temperature for 24 h in an atmosphere of Ar. The reaction mixture

was washed with brine and extracted with CH₂Cl₂. Then, the

combined organic phase was dried over anhydrous Na₂SO₄and the

solvent was removed. The resulting amide was used in a subse-

quent reaction without puriﬁcation. Yield 62%; yellow solid. ¹H

(3H, t, J ¼ 7.1, Me-20),1.08e1.15 (1H, m, H-4endo),1.21e1.29 (1H, m,

H-5endo), 1.45e1.56 (2H, m, H-5exo, H-2endo), 1.61e1.71 (2H, m,

H-4exo, H-3),1.74e1.86 (3H, m, H-2exo, H-13), 2.15 (2H, t, J ¼ 6.8, H-

12), 2.28e2.58 (12H, m, H-14, H-15, H-16, H-17, H-18, H-19), 3.87

(1H, dt, J ¼ 4.3, 8.6, H-1exo), 5.48 (1H, NeH).¹³C NMR (

d, ppm): 171.

8 s (C-11), 56.9 t (C-14), 56.4 d (C-1), 52.8 t (C-15, C-16), 52.6 t (C-17,

C-18), 52.2 t (C-19), 48.3 s (C-6), 46.9 s (C-7), 44.7 d (C-3), 39.0 t (C-

2), 35.7 t (C-5), 34.5 t (C-12), 26.8 t (C-4), 22.7 t (C-13), 20. 2 q, 20.1 q

(Me-8, Me-9),11.8 q (Me-20),11.6 q (Me-10). HR-MS: 335.2934 (M^þ,

NMR (d, ppm, J/Hz): 0.78 (3H, s, Me-10), 0.79 (3H, s, Me-9), 0.86 (3H,

s, Me-8), 1.06e1.14 (1H, m, H-4endo), 1.18e1.27 (1H, m, H-5endo),

1.47e1.56 (2H, m, H-5exo, H-2endo), 1.60e1.71 (2H, m, H-4exo, H-

3), 1.80 (1H, dd, J ¼ 9.0, 13.2, H-2exo), 2.02e2.07 (2H, m, H-13),

2.28e2.33 (2H, m, H-12), 3.51e3.61 (2H, m, H-14), (3.86 (1H, dt,

C₂₀H₃₇O₁N₃; calcd 335.2931).

5.2.9. Synthesis of (þ)- and (ꢀ)-isoborneols

(þ)-Camphor (3.00 g, 20 mmol) or (ꢀ)-camphor (3.00 g,

A.S. Sokolova et al. / European Journal of Medicinal Chemistry 207 (2020) 112726

15

20 mmol) was dissolved in methanol (20 mL), and sodium boro-

hydride (3.5 g, 92 mmol) was added in portions. The solution was

cooled to ꢀ30 ^ꢁC and stirred for 3 h and then for 4 h at room

temperature. The reaction mixture was then diluted with 2 M HCl

and extracted with diethyl ether. Subsequently, the combined

organic extracts were washed with brine (20 mL), dried with

anhydrous Na₂SO₄and evaporated to yield the white solid products

(þ)-isoborneol (14 mmol, 71%) and (ꢀ)-isoborneol (15 mmol, 75%),

the ¹H NMR spectrum for which showed it to consisted of two di-

astereomers in an approximately 9:1 ratio. The diastereomers were

separated by silica gel column chromatography (eluent: hexane/

ethyl acetate). NMR spectra for (þ)-isoborneol and (ꢀ)-isoborneol

agreed with those presented in the literature [24]. (þ)-Isoborneol

yellow oil. ¹H NMR (

d, ppm, J/Hz): 0.80 (6H, s, Me-8, Me-9), 0.88

(3H, d, J ¼ 5.8, Me-18), 0.94 (3H, s, Me-8), 1.00e1.16 (2H, m, H-

4endo, H-5endo), 1.21e1.34 (3H, m, H-15a, H-17, H-16a), 1.46e1.82

(7H, m, H-15e, H-16e, H-3, H-4exo, H-5exo, H-2endo, H-2exo),

2.07e2.16 (2H, m, H-13a, H-14a), 2.30 (1H, m, H-2exo), 2.82e2.91

(2H, m, H-13e, H-14e), 3.13 (2H, s, H-12), 4.69 (1H, m, H-1exo). ¹³

C

NMR (d, ppm): 170.19 s (C-11), 80.87 d (C-1), 59.91 t (C-12), 53.54

and 53.50 t (C-13, C-14), 48.55 s (C-6), 46.79 s (C-7), 44.86 d (C-3),

38.65 t (C-2), 34.05 t (C-15, C-16), 33.54 t (C-5), 30.20 d (C-17),

26.87 t (C-4), 21.76 q (Me-18), 19.97 q and 19.79 q (Me-8, Me-9),

11.42 q (Me-10). ½a _D²⁵

¼ -38.8 (c 0.5, CHCl₃). HR-MS: 293.2345

ꢄ

(M^þ, C₁₈H₃₁NO₂; calcd 293.2349).

(1S,2S,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl 2-(piperidin-

½

a ²_D⁶

ꢄ

¼ -20 (c 0.5, CHCl₃), mp ¼ 203.0e204.9. (ꢀ)-Isoborneol

¼ 23.3 (c 0.3, CHCl₃), mp ¼ 197.8.

1-yl)acetate (1S,2S,4S)-23. ¹H and ¹³C NMR specters and HRMS

ꢄ

coincide with compound (1R,2R,4R)-23. ½a _D²²

ꢄ

¼ 31 (c 0.4, CHCl₃).

2-(4-

(1S,2S,4S)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl

methylpiperidin-1-yl)acetate (1S,2S,4S)-24. ¹H and ¹³C NMR spec-

5.2.10. Synthesis of (1R,2R,4R)- and (1S,2S,4S)-1,7,7-

trimethylbicyclo[2.2.1]heptan-2-yl 2-chloroacetates [(1R,2R,4R)-22

and (1S,2S,4S)-22)]

ters and HRMS coincide with compound (1R,2R,4R)-24. ½a _D²²

¼ 38

ꢄ

(c 0.4, CHCl₃).

Chloroacetyl chloride (2.4 ml, 0.03 mol) was added to a mixture

of (þ)-isoborneol or (ꢀ)-isoborneol (3.00 g, 0.02 mol) and Et₃N

(2.8 ml, 0.02 mol) in 20 ml dry CH₂Cl₂at 15e18^ꢁС, after which the

mixture was stirred at room temperature (with slight heating for

(ꢀ)-isoborneol) for 12 h. The reaction mixture was washed with

brine and extracted with CH₂Cl₂. Then, the combined organic phase

was dried over anhydrous Na₂SO₄and the solvent was removed.

The resulting product was used in a further reaction without

puriﬁcation.

5.3. Molecular modeling

Molecular modeling was carried out in the Schrodinger Maestro

visualization environment using applications from the Schrodinger

Small Molecule Drug Discovery Suite 2016-1 package. Three-

dimensional structures of the derivatives were empirically ob-

tained in the LigPrep application using the OPLS3 force ﬁeld [25].

For calculations, the XRD models (PDB IDs 6F5U, 6F6N, and 6F6I) of

EBOV GP inhibited by various drugs from the Protein Data Bank

were chosen [12]. To model a possible mechanism of inhibition for

the selected target, molecular docking of new compounds was

performed at the binding site of EBOV GP using Glide [26]. The

search area for docking was selected according to the size of known

inhibitors. Docking was performed in comparison with known in-

hibitors. The three-dimensional structures of inhibitors were ob-

tained in the PubChem database and prepared in the LigPrep

application. Noncovalent interactions of molecules in the binding

site were visualized using Biovia Discovery Studio Visualizer and

Schrodinger Maestro.

(1R,2R,4R)-22. Yield: 55%, pale yellow oil. ¹H NMR (

d, ppm, J/

Hz): 0.82 (3H, s), 0.83 (3H, s), 0.85e0.88 (1H, m), 0.95 (3H, s),

1.00e1.17 (2H, m), 1.49e1.59 (1H, m), 1.60e1.70 (1H, m), 1.71e1.81

(2H, m), 4.00 (2H, s), 4.70e4.75 (1H, m). HR-MS: 230.1066 (M^þ,

C

₁₂H₁₉O³₂⁵Cl₁; calcd 230.1068). ½a ²_D⁵

ꢄ

¼ -68.5 (c 0.9, CHCl₃).

(1S,2S,4S)-22. Yield: 43%, colorless oil. ¹H NMR (

d

, ppm, J/Hz):

0.82 (3H, s), 0.84 (3H, s), 0.85e0.88 (1H, m), 0.95 (3H, s), 1.00e1.16

(2H, m), 1.50e1.59 (1H, m), 1.63e1.70 (1H, m), 1.71e1.81 (2H, m),

4.00 (2H, s), 4.70e4.74 (1H, m). HR-MS: 230.1066 (M^þ,

C

₁₂H₁₉O³₂⁵Cl₁; calcd 230.1068).

5.2.11. General procedure for the nucleophilic substitution

(1R,2R,4R)-22 and (1S,2S,4S)-22 with piperidin and 4-

methylpiperidin

5.4. Biological assays

The requisite amine (1.1 mmol) piperidin or 4-methylpiperidin

was added to a solution of (1R,2R,4R)-22 or (1S,2S,4S)-22 (0.2 g,

0.9 mmol) and Et₃N (0.1 ml, 0.9 mmol) in CH₂Cl₂(5 ml), and the

mixture was stirred at room temperature for 12 h. The reaction

mixture was washed with brine and extracted with CH₂Cl₂. Then,

the combined organic phase was dried over anhydrous Na₂SO₄and

the solvent was removed. The crude products were puriﬁed by

column chromatography on silica gel using a gradient of EtOAc

(1e15%) in hexane.

5.4.1. Cell lines

HEK293FT and HEK293T cells were maintained in DMEM

(Invitrogen) containing 10% fetal bovine serum (FBS; Gibco),

0.6 mg/mL

L-glutamine (Invitrogen) and 50

mg/ml gentamicin.

5.4.2. Production of rVSV-

GP, D522A-EboV-GP, and M548A-EboV-GP)

To generate Ebola glycoprotein (or variants with amino acid

substitutions) pseudotyped VSVs containing the ﬁreﬂy luciferase

gene, 293FT cells grown in a T75 were transfected with a pGPE

plasmid (pGPE-Y517A, pGPE-D522A, or pGPE-M548A) using the

CaCl₂method (23 mg of plasmid per cell monolayer in a T75 culture

vial). After 16 h the culture medium was replaced, and a suspension

of VSV of the ﬁreﬂy luciferase gene pseudotyped by surface VSV

glycoprotein G (rVSV-DG-G) was added to the cells [27]. After 6 h,

D

G pseudotyped EboV-GP (Y517A-EboV-

(1R,2R,4R)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl 2-(piperidin-

1-yl)acetate (1R,2R,4R)-23. ¹H NMR (

d, ppm, J/Hz): 0.79 and 0.80

(6H, s, Me-8, Me-9), 0.93 (3H, s, Me-8), 1.99e1.15 (2H, m, H-4endo,

H-5endo), 1.34e1.81 (11H, m, H-15, H-16, H-17, H-3, H-4exo, H-

5exo, H-2endo, H-2exo), 2.43e2.51 (4H, m, H-13, H-14), 3.11 (2H, s,

H-12), 4.68 (1H, m, H-1exo). ¹³C NMR (

d, ppm): 169.82 s (C-11),

80.54 d (C-1), 59.94 t (S-12), 53.73 t (C-13, C-14), 48.22 s (C-6),

47.46 s (C-7), 44.55 d (C-3), 38.33 t (C-2), 33.21 t (C-5), 26.54 t (C-4),

25.39 t (C-15, C-17), 23.47 t (C-16), 19.64 q and 19.46 q (Me-8 and

the cells were washed, and the medium was exchanged with fresh

medium. Pseudoviruses were harvested after 48 h by ﬁltering the

culture medium through a 0.45-mm ﬁlter after centrifugation to

Me-9), 11.08 q (Me-10). ½a _D²²

¼ -46.1 (c 0.4, CHCl₃). HR-MS:

ꢄ

remove cell debris. Pseudoviruses were stored at ꢀ80 ^ꢁC, and their

functional activity was determined using a HEK293T cell culture,

with the luminescence level recorded using a Stat Fax 4400

luminometer.

279.2191 (M^þ, C₁₇H₂₉O₂N₁; calcd 279.2193).

(1R,2R,4R)-1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl

2-(4-

methylpiperidin-1-yl)acetate (1R,2R,4R)-24. Yield: 48%, pale

16

A.S. Sokolova et al. / European Journal of Medicinal Chemistry 207 (2020) 112726

5.4.3. Plasmids

on a Model 680 Microplate (Bio-Rad) Readerat 535 nm and plotted

against concentration of the compounds. Each concentration was

tested in three parallels. The 50% cytotoxic dose (CC₅₀) 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.

The plasmid pGPE containing the Ebola virus surface glycopro-

tein gene of the Mayinga strain was obtained by inserting the

synthesized gene into the vector pcDNA3.1. Prior to synthesis,

codon optimization of the GP gene was performed.

To construct the plasmids pGPE-Y517A, pGPE-D522A and pGPE-

M548A, site-directed mutagenesis was performed. To this end, the

primers Ebo-F and Ebo-R were designed that ﬂank the GPE gene

sequence. Then, for each of the amino acid substitutions, the cor-

responding pairs of primers containing nucleotide substitutions

introducing the desired amino acid change (517-F, 517-R, 522-F,

522-R, 548-F and 548-R) were designed. PCR was performed with

primer pairs Ebo-F, 517-R (522-R, 548-R) and Ebo-R, 517-F (522-F,

548-F). PCR products were puriﬁed, and ampliﬁed annealing was

performed using the primers Ebo-F and Ebo-R. The resulting

product was inserted into the original plasmid pGPE.

5.4.6. Ebola and Marburg Virus Infections under BSL-4 conditions

EBOV (strain Zaire) and MARV (strain Popp) were obtained from

the State Collection of Viral Infections and Rickettsioses Agents of

SRC VB Vector suspended in culture supernatant. Titer EBOV was

4.5 0.75 lgTCD₅₀/ml, titer MARV was 5.3 0.63 lgTCD₅₀/ml. Anti-

EBOV and anti-MARV assays were conducted at SRC VB Vector in a

maximum containment facility (BSL-4). Vero cell culture (African

green monkey kidney cell line) were seeded into 96-well plates and

were grown into conﬂuent monolayers. Series of 3-fold dilutions of

each compound (previously dissolved in 100% DMSO) in DMEM

medium were prepared starting of 300

centrations were prepared of each inhibitors. The compounds were

added of 100 L/well. Viruses were used at a multiplicity of infec-

tion of 0.01 (equivalent to a dose of 100 TCID₅₀per well). For the

treatment assay, 100 L of diluted virus (100 TCID₅₀) was added. For

cytotoxicity assay to each well was added 100 L DMEM medium.

mg/ml. Six decreasing con-

Oligonucleotide name

Nucleotide sequence

Ebo-F

Ebo-R

517-R

517-F

522-F

522-R

548-F

548-R

5⁰-ttttgctagccgccaccatgggcgttacaggaatattgcagtt-3⁰

5⁰-ttttttcggccgaaactaaaagacaaatttgcatatac-3⁰

5⁰-cctgagtagtccaggcatgtaaattagggttgcatttgg-3⁰

5⁰-ccaaatgcaaccctaatttacatgcctggactactcagg-3⁰

5⁰-atttacattactggactactcagcttgaaggtgctgcaatcg-3⁰

5⁰-ccgattgcagcaccttcaagctgagtagtccagtaatgtaaat-3⁰

5⁰-gggaatttacatagaggggctcgcgcacaatcaagatggttta-3⁰

5⁰-taaaccatcttgattgtgcgcgagcccctctatgtaaattccc-3⁰

m

After incubation at 37 ^ꢁC and 5% CO₂for 10 days, neutral red was

then added. The absorbance was measured at 490 nm using a

microplate reader (Thermo Scientiﬁc Multiskan FC). The 50%

cytotoxicity concentration (CC₅₀) and 50% inhibitory concentration

(IC₅₀) was calculated using SOFTmax PRO 4.0 with the 4-parameter

analysis method. Each inhibitor was analyzed at least 3 times.

5.4.4. Pseudotype virus entry inhibition assay

Viral entry inhibition assays were performed using HEK293T

cells. Cells were seeded in 96-well plates (100 ml at a density of

10⁵cells per ml) one day before analysis. The following day, po-

tential inhibitor compounds were titrated in 96-well round-bottom

5.4.7. Acute toxicity

ICR mice weighing 22e25 g were obtained from the animal fa-

cility of SRC VB Vector, Rospotrebnadzor and maintained under

standard conditions with free access to food and water. All exper-

iments were performed in accordance with the ‘‘European

Convention for the Protection of Vertebrate Animals Used for

Experimental and other Scientiﬁc Purposes’’, 2010, and were

approved by the Ethic Committee of the N.N. Vorozhtsov Institute

of Organic Chemistry SB RAS (protocol No. 10/2019). The mice were

weighed and randomly divided into groups (2 mice/group). Test

compounds were administered by gavage (0.1 mL/10 g) at various

doses, whereas the control groups received only solvent. Because of

the lower water solubility of the compounds, a solution of DMSO in

water (10%) was used as the solvent.

plates at a dilution of 1:4. Next, 10-ml aliquots of pseudoviruses

(10⁵PFU) were added to each well, and the mixtures were incu-

bated for 1 h in a CO₂incubator at 37 ^ꢁC under an atmosphere with

5% CO₂. After incubation, an aliquot of each mixture was added to a

monolayer of cells. As a negative control, cells were treated with the

same volume of medium as that used for the pseudovirus com-

pound mixture was added to cells. After incubating in a CO₂incu-

bator for 48 h, the luminescence level was measured using a Stat

Fax 4400 plate luminometer. To this end, the medium was carefully

removed from the wells, the cells were washed with 100

PBS and then 25 l of Luciferase Cell Culture Lysis Buffer (Promega)

was added. After 5 min, 50 l of Luciferase Assay Reagent (Promega)

ml/well of

m

was added. The percentage of inhibition was evaluated by deter-

mining the degree of decrease in luminescence in the wells with

the compounds relative to the control wells (cells with the virus

without compounds). All test compounds were dissolved in

dimethylsulfoxide (DMSO) at a concentration of 10 mg/ml and used

Funding sources

This work was supported by a grant from the Russian Science

Foundation 19-73-00125.

at a ﬁnal concentration of 375 to 0.02 mg/ml.

Declaration of competing interest

5.4.5. Cytotoxicity assays

MTT reduction was used to study cytotoxicity of the com-

pounds.[28] Brieﬂy, series of two-fold dilutions of each compound

The authors declare that they have no known competing

ﬁnancial interests or personal relationships that could have

appeared to inﬂuence the work reported in this paper.

(15.6e1000

HEK293T cells (100

and incubated for 48 h at 37 ^ꢁC in 5% CO₂. Than 20

m

M) in 10% DMEM were prepared in 96-well plates.

l at a density of 10⁵cells per ml) were added

l (1/10 vol) of a

m

solution of 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium

bromide (Sigma) (5 mg/ml) in phosphate-buffered saline was

added to each well. After 2 h incubation, the solution was removed

Acknowledgment

The authors would like to acknowledge the Multi-Access

Chemical Research Center SB RAS for spectral and analytical

measurements.

from wells and DMSO (100

ml per well) was added to dissolve the

formazan crystals. The optical density of cells was then measured

A.S. Sokolova et al. / European Journal of Medicinal Chemistry 207 (2020) 112726

17

borneol derivatives as potent inhibitors of the in ﬂ uenza A virus, Med. Chem.

Commun 8 (2017) 960e963.

Appendix A. Supplementary data

[15] A.S. Sokolova, O.I. Yarovaya, D.S. Baev, A.V. Shernyukov, A. Shtro,

V.V. Zarubaev, N.F. Salakhutdinov, Aliphatic and alicyclic camphor imines as

effective inhibitors of in ﬂ uenza virus H1N1, Eur. J. Med. Chem. 127 (2017)

661e670.

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2020.112726.

[16] A.S. Sokolova, O.I. Yarovaya, A.V. Shernyukov, Y.V. Gatilov, Y.V. Razumova,

V.V. Zarubaev, T.S. Tretiak, O.I. Kiselev, N.F. Salakhutdinov, Discovery of a new

class of antiviral compounds: camphor imine derivatives, Eur. J. Med. Chem.

105 (2015) 263e273.

[17] V.V. Zarubaev, A.V. Garshinina, T.S. Tretiak, V.A. Fedorova, A.A. Shtro,

A.S. Sokolova, O.I. Yarovaya, N.F. Salakhutdinov, Broad range of inhibiting

action of novel camphor-based compound with anti-hemagglutinin activity

against in ﬂ uenza viruses in vitro and in vivo, Antivir. Res. 120 (2015)

126e133.

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Monoterpenoid-based inhibitors of filoviruses targeting the glycoprotein-mediated entry process

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