Synthetic Analogues of Aminoadamantane as Influenza Viral Inhibitors—In Vitro, in Silico and QSAR Studies

Article abstract of DOI:10.3390/molecules25173989

A series of nineteen amino acid analogues of amantadine (Amt) and rimantadine (Rim) were synthesized and their antiviral activity was evaluated against influenza virus A (H3N2). Among these analogues, the conjugation of rimantadine with glycine illustrated high antiviral activity combined with low cytotoxicity. Moreover, this compound presented a profoundly high stability after in vitro incubation in human plasma for 24 h. Its thermal stability was established using differential and gravimetric thermal analysis. The crystal structure of glycyl-rimantadine revealed that it crystallizes in the orthorhombic Pbca space group. The structure–activity relationship for this class of compounds was established, with CoMFA (Comparative Molecular Field Analysis) 3D-Quantitative Structure Activity Relationships (3D-QSAR) studies predicting the activities of synthetic molecules. In addition, molecular docking studies were conducted, revealing the structural requirements for the activity of the synthetic molecules.

Full text of DOI:10.3390/molecules25173989

molecules
Article
Synthetic Analogues of Aminoadamantane as
Influenza Viral Inhibitors—In Vitro, In Silico and
QSAR Studies
Radoslav Chayrov 1, Nikolaos A. Parisis 2 , Maria V. Chatziathanasiadou 2, Eleni Vrontaki 3,4
Kalliopi Moschovou 3, Georgia Melagraki 5, Hristina Sbirkova-Dimitrova 6, Boris Shivachev 6,
,
Michaela Schmidtke 7 , Yavor Mitrev 8, Martin Sticha 9 , Thomas Mavromoustakos 3
,
Andreas G. Tzakos 2,* and Ivanka Stankova 1,
*
1
Department of Chemistry, South-West University “Neofit Rilski”, 2700 Blagoevgrad, Bulgaria;
rchayrov@swu.bg
Section of Organic Chemistry and Biochemistry, Department of Chemistry, University of Ioannina,
45110 Ioannina, Greece; nikparis@gmail.com (N.A.P.); m.chatziathanasiadou@gmail.com (M.V.C.)
Division of Organic Chemistry, Department of Chemistry, National and Kapodistri‘an University of Athens,
15771 Zografou, Greece; evrontaki@pharm.uoa.gr (E.V.); kmoschovou@chem.uoa.gr (K.M.);
tmavrom@chem.uoa.gr (T.M.)
2
3
4
5
6
7
8
9
Division of Pharmaceutical Chemistry, Department of Pharmacy,
National and Kapodistrian University of Athens, 15771 Zografou, Greece
Division of Physical Sciences and Applications, Department of Military Sciences, Hellenic Military Academy,
Vari-Koropi Avenue, 16672 Vári, Greece; georgiamelagraki@gmail.com
Institute of Mineralogy and Crystallography “Acad. Ivan Kostov”, Bulgarian Academy of Sciences,
1113 Sofia, Bulgaria; sbirkova@mail.bg (H.S.-D.); bls@clmc.bas.bg (B.S.)
Department of Virology and Antiviral Therapy, Friedrich Schiller University, 207745 Jena, Germany;
michaela.schmidtke@med.uni-jena.de
Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Science,
1113 Sofia, Bulgaria; yavor@orgchm.bas.bg
Department of Analytical Chemistry, Charles University, 11636 Prague 1, Czech Republic;
martin.sticha@natur.cuni.cz
*
Correspondence: atzakos@uoi.gr (A.G.T.); ivastankova@abv.bg (I.S.); Tel./Fax: +359-73-88-55-16 (I.S.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Accepted: 31 August 2020; Published: 1 September 2020
Abstract: A series of nineteen amino acid analogues of amantadine (Amt) and rimantadine (Rim) were
synthesized and their antiviral activity was evaluated against influenza virus A (H3N2). Among these
analogues, the conjugation of rimantadine with glycine illustrated high antiviral activity combined
with low cytotoxicity. Moreover, this compound presented a profoundly high stability after in vitro
incubation in human plasma for 24 h. Its thermal stability was established using differential and
gravimetric thermal analysis. The crystal structure of glycyl-rimantadine revealed that it crystallizes
in the orthorhombic Pbca space group. The structure–activity relationship for this class of compounds
was established, with CoMFA (Comparative Molecular Field Analysis) 3D-Quantitative Structure
Activity Relationships (3D-QSAR) studies predicting the activities of synthetic molecules. In addition,
molecular docking studies were conducted, revealing the structural requirements for the activity of
the synthetic molecules.
Keywords: adamantane derivatives; amino acids; 3D-QSAR; molecular docking; X-Ray crystallography;
plasma stability; mass spectrometry; LC–MS/MS.
Molecules 2020, 25, 3989; doi:10.3390/molecules25173989
www.mdpi.com/journal/molecules

Molecules 2020, 25, 3989
2 of 21
1. Introduction
The synthesis of a series of adamantane derivatives led to the discovery of several drugs that are
currently used in clinical practice, such as antivirals, antidiabetics, antimicrobials, anti-inflammatories
and CNS-acting compounds [1].
Amantadine (Amt) was discovered in the 60s and it was the first adamantane derivative used in
medical practice exhibiting antiviral activity against influenza A strains. Rimantadine (Rim) has been
also used since the start of the 1980s to treat and prevent influenza A infection. Both drugs mechanism
of action is associated with the proton conduction channel (M2) of influenza A virus. The lack of the
M2 ion channel in influenza B virus renders these two drugs ineffective against this virus. There is
an increasing number of resistant influenza strains every year as a result of the spontaneous mutations
in the viral genome. This fact has led the researchers to the quest for new antiviral drugs that can
overcome the resistant virus strains.
One approach that could enhance the antiviral activity of this class of compounds is the
incorporation of additional active functional groups in the drug’s scaffold that can disrupt proton
transport. Sources of such active functional groups can be amino acids and peptides incorporated into
amantadine and rimantadine, using peptide synthesis techniques.
In addition to being the building blocks of proteins and peptides, amino acids serve as precursors
of many kinds of small molecules that have important and diverse biological roles. Shibnev et al. [2]
studied antiviral activity against influenza A subtypes A (H1N1) and A (H3N2) of various amino
acid derivatives of adamantane. It is established that some of the synthesized compounds can inhibit
influenza A virus strains resistant to rimantadine. The authors suggest that adamantane carbocycle as a
membranotropic carrier is capable of transporting functional groups containing in amino acid to the M2
protein of influenza A/H5N1 virus, thus causing inhibition of influenza A/H5N1 virus replication [
3,4].
Hong et al. [ ] reported that many oseltamivir carboxylate (A) (neuraminidase inhibitor, NA)
5
analogs have been synthesized (i.e., with lipophilic alkyl and aryl groups other than the 3-pentyl group,
different amido groups at the C-4 position or relocating the C–C double bond in the cyclohexene scaffold)
bearing anti-influenza activity. However, such structural modifications have not led to enhanced
anti-influenza activity with respect to oseltamivir carboxylate (A). In contrast, the replacement of the
amino group at the C-5 position in guanidated oseltamivir carboxylate (B) by a more basic guanidino
group enhanced the inhibitory activity against NA (Figure 1) [6].
O
O
HO
OH
O
O
O
O
OH
HO
O
H
OH
HN
HN
HN
HN
NH2
NH
NH2
HN
NH2
NH
O
O
O
(C)
) guanidated oseltamivir carboxylate
(B)
(A)
Figure 1. Chemical structures of: (
and (C) zanamivir.
A
) oseltamivir carboxylate; (
B
Guanidation has been successfully used in the modification of another class of influenza drugs,
the neuraminidase inhibitors—zanamivir (C) [ ]. In this frame, derivatization of the synthesized
7
compounds with guanidine could yield even more promising results.
In addition, docking combined with the 3D-Quantitative Structure Activity Relationships
(3D-QSAR) results proposes that next generation potential M2 inhibitors should include lipophilic
adamantane combined with polar groups, e.g., guanidino group [8].
The aim of this study was the synthesis of amino acid derivatives of amantadine and rimantadine
and the investigation of their antiviral activity in vitro against influenza viruses. Some analogues
were also guanidated in the amino group. X-ray crystallography was achieved for the analogue with
the lowest half maximal Inhibitory Concentration (IC50). A plasma stability assay was conducted

Molecules 2020, 25, 3989
3 of 21
for this analogue in human plasma, utilizing liquid chromatography coupled with tandem mass
spectrometry (LC–MS/MS). Molecular docking combined with the 3D-QSAR study revealed the
molecular requirements for drug activities.
2. Results and Discussion
2.1. Synthesis of the Compounds
The target compounds 2 (a–j), 3a, 3e and 3g–h were prepared from Boc-amino acids 1 (a–j) and
amantadine or rimantadine using the coupling reagent TBTU (2-(1H-Benzotriazole-1-yl)-1,
1,3,3-tetramethylaminium tetrafluoroborate) in the presence of DMAP 4-(Dimethylamino)pyridine [
9
].
◦
Boc-protected groups were removed in CH2Cl2/TFA at 0 C and lead to
4
(
a–
j)
, 5a, 5e and 5g–h
(Scheme 1).
O
O
Boc-HN
CH3
H2N
NH
CH3
NH
R"'
CH
R"'
CH
R',R''
i
ii
+ R'''COOH
2 (a-j)
1 (a-j)
4 (a-j)
O
O
Boc-HN
H2N
NH
NH
R"'
R"'
(5a, 5e, 5g-h)
(3a, 3e, 3g-h)
Scheme 1. Synthesis of amino acid derivatives of adamantane. R0 = NH2—amantadine;
R00 = NH2CH(CH3)—rimantadine; R000COOH = Boc-Ala (1a); Boc-Gly (1b); Boc-Ile (1c); Boc-Leu
(
1d); Boc-Phe (1e); Boc-(4-F)-D-Phe (1f); Boc-(4-F)-Phe (1g); Boc-Val (1h); Boc-β-Ala (1i); Boc-Tyr (1j);
(i) TBTU/DMAP and (ii) TFA.
The derivatives, Ala-Rimantadine (4a), β-Ala-Rimantadine (4i), Tyr-Rimantadine (4j),
Ala-Amantadine (5a) and Val-Amantadine (5h), with the lowest cytotoxicity, were guanidated.
Guanidated analogs (6a 6i 7a and 7h) were obtained by treating the corresponding aminoadamantane
,
–j,
(4a, 4i–j, 5a and 5h) with 1H-pyrazole-1-carboxamidine·HCl in 2 mL acetonitrile (Scheme 2) [10].
CH3
NH
O
(4 a, 4 i-j)
NH
N
H2N
NH
N
CH
.
HCI
R"'
NH
H2N
(1H-pyrazole-1-carboxamidine.HCI)
NH
(6 a, 6 i-j)
O
H2N
NH
NH
(5a, 5h)
R"'
(7a, 7h)
Scheme 2. Guanidation of adamantane analogues.

Molecules 2020, 25, 3989
4 of 21
Boc-(4-F)-Phe-OH was used to modify amantadine since (4-F)-phenylalanine has proven to
antiviral activity [11]. Thus, we synthesized and compared the activity between the three amino acids
with a similar structure, such as phenylalanine, (4-F)-phenylalanine and tyrosine.
2.2. Biological Studies
To improve the oral bioavailability of the antiviral drug acyclovir in 1992, Beauchamp L.M. et al.,
synthesized eighteen amino acid esters of acyclovir. Among them, L-valyl ester (Valacyclovir) was
proved to be a very effective prodrug. Orally introduced in the organism, it possesses 3–4 times higher
bioavailability than the acyclovir. Valacyclovir is stable in water–acid solutions, it is swiftly absorbed
in vivo at the intestine and subsequently metabolized to acyclovir. According to the literature, the valine
derivative is non-toxic, which is also confirmed by our new data for the amantadine amide. The success
of valine derivatives against certain viruses, probably due to the amphiphilicity of valine motivated us
to couple the structurally similar amino acids leucine and isoleucine with adamantanes [12–14].
Their amphiphilic role could play a major role not only in the increased solubility of the new
derivatives but also in their facilitated transportation through the cellular and viral membranes [15].
Alanine, one of the most important amino acids, is metabolized in the liver to glucose. This process,
known as the glucose-alanine cycle, is reversible and represents one of the main mechanisms for the
biochemical synthesis of glucose in the body. Studying the work by Kim et al. [16], we decided to link
alanine and amantadine via an amide bond. Alanine exhibits more pronounced hydrophilic properties
over the previously described derivatives, which are important for increased bioavailability.
In addition, the structurally similar highly hydrophilic amino acid glycine is very suitable for
ameliorating the hydrophobicity of amantadine and can thus, lead to higher bioavailability. This could
also be the case for β-alanine. Nonetheless, the use of this amino acid in the synthesis could not be
beneficial since it adopts a linear structure and its amino group resembles polyamine. M2 proteins
have a specific binding site for polyamines, which is different from the amantadine binding site [17].
Taking into consideration the significance of the nature of the inhibitors in the active sites,
we decided to use the guanidino group to modify part of the newly obtained compounds [18].
We chose to modify some of the synthesized derivatives, which showed poor antiviral activity and low
cytotoxicity and at the same time to study their biological activity in vitro (Table 1).
Table 1. Cytotoxicity concentration, high-nontoxic concentration and inhibition concentration of
investigated compounds.
CC50 (µM)
MDCK Cells
HNTC (µM)
MDCK Cells
IC50 (µM)
Against A/Hong Kong/68
Compound
Ala-Rim (4a)
Gly-Rim (4b)
Ile-Rim (4c)
Leu-Rim (4d)
Phe-Rim (4e)
>100
>100
58.35
>100
21.93
19.91
15.46
70.33
>100
>100
>100
89.4
83.61
>100
>100
>100
>100
>100
>100
>100
>100
14.65
13.89
5.33
11.66
4.75
6.23
4.37
10.2
20.78
9.97
1.53
0.11
n/a
0.75
n/a
n/a
n/a
n/a
15.72
0.52
1.41
0.75
0.81
1.32
41.9
n/a
8.19
18.37
17.13
0.39
0.002
(4-F)-D-Phe-Rim (4f)
(4-F)-Phe-Rim (4g)
Val-Rim (4h)
β-Ala-Rim (4i)
Tyr-Rim (4j)
Ala-Am (5a)
Phe-Am (5e)
(4-F)-Phe-Am (5g)
Val-Am (5h)
Gua-Ala-Rim (6a)
Gua-β-Ala-Rim (6i)
Gua-Tyr-Rim (6j)
Gua-Ala-Am (7a)
Gua-Val-Am (7h)
Amantadine
>100
15.1
15.35
>100
>100
>100
36.69
>100
>100
>100
>100
Oseltamivir

Molecules 2020, 25, 3989
5 of 21
As shown in Table 1 the highest antiviral activity of the rimantadine analogs was clearly
demonstrated by glycyl-rimantadine (4b). Compounds 4d and 4j (Leu and Tyr analogues) also
displayed antiviral activity but at higher concentrations than 4b and amantadine. One should note
that the IC50 of 4b is about 3.5 times lower than that of amantadine, though with comparable 50%
cytotoxic concentration (CC50) and thus potentially allowing the use of higher concentration during
cell treatment. Probably 4b small structure allows it to easily fit the M2-ion channel. The branched
with aliphatic amino acids rimantadine analogues (Val, Leu, Ile, Ala) do not show antiviral activity.
Increasing the volume of the molecule with aromatic amino acids (Phe, 4-F-Phe, Tyr) leads to a decrease
or complete disappearance of the antiviral activity. It is important to stress that there is no difference in
activity between the R and S isomers 4f and 4g and that the different conformational structure of
(4i) greatly decreases the activity.
β-Ala
From the amantadine analogs (5a
–
h) only the phenylalanine and 4-F-phenylalanine (5e
,
5g
)
displayed antiviral activity, although at higher concentrations than amantadine. Probably the aromatic
ring, as a larger and more rigid structure, does not fit as well in the M2 ion channel.
Modification of amino acid analogs of rimantadine and amantadine with larger and more basic
guanidine functional group leads to decreased antiviral activity in contrast to guanidated neuraminidase
inhibitors (zanamivir).
2.3. 3D-QSAR Studies
The statistical results of the obtained 3D-QSAR comparative molecular field analysis (CoMFA)
model for the biologically evaluated molecules are listed in Table 2.
Table 2. Statistical parameters of the comparative molecular field analysis (CoMFA) model.
CoMFA Model
ONC
4
Q2
0.847
0.127
0.954
0.070
123.383
SEP
R2
SEE
F value
Field contribution
Steric: Electrostatic
R2 test
0.694:0.306
0.867
An optimal number of components (ONC) of four corresponds to the lowest standard error of
prediction (SEP) of 0.127 and the highest cross-validated Q2 of 0.847. A non-cross-validated R2 of 0.954,
a standard error of estimates (SEE) of 0.070 and the degree of statistical confidence F value of 123.383
show an excellent statistical correlation between the predicted and the actual values of inhibition
(Table 2). The CoMFA analysis shows that the relative field contributions were 69.4 and 30.6% for the
steric and electrostatic field, respectively. The external validation gave a value of R2 test equal to 0.867
upholding an excellent correlation between the predicted and actual values of inhibition.
The results of the CoMFA method were also graphically represented by field contour maps.
In these maps the areas around the compound that interacts favorably or unfavorably with the possible
receptor are visualized. Therefore, the contour maps are used to identify the structural features relevant
to the biological activity of derivatives and their obtained information can be utilized for further design
of amino adamantane analogs as blockers of the M2 wild type ion channel.
In Figure 2, the CoMFA fields around compound 29 an active compound from the training set are
displayed. The steric interactions are represented by green and yellow contour maps, where bulky
groups near the green regions increase the inhibitory activity but they cause the opposite effect near
the yellow ones. As it can be observed, the green regions are more than yellow ones for the steric field
in the contour map of the CoMFA model (Figure 2a). The large green regions around the aromatic

Molecules 2020, 25, 3989
6 of 21
group of the amino adamantane scaffold indicate that bulky groups at o- and m- positions increase the
activity, whereas the substitution of bulky groups at p-position of phenyl group decreased the activity.
The electrostatic interactions are represented by blue and red contour maps, where electropositive
groups were favored near the blue regions, whereas electronegative groups were favored near the red
regions, therefore, electropositive groups were favored at m-position and electronegative groups were
favored at p-position of phenyl (Figure 2b). Based on the 3D-QSAR CoMFA model, a suggested scaffold
of a compound that could be designed and synthesized with potent inhibition against influenza A is
depicted in Scheme 3.
Figure 2. CoMFA StDev*Coeff contour maps around the active compound 29: (a) for steric field (green:
bulky groups are favored; yellow: bulky groups are disfavored) and ( ) for electrostatic field (blue:
b
electropositive groups are favored; red: electronegative groups are favored).
Scheme 3. Suggested scaffold based on the 3D-QSAR CoMFA model.
The Enalos Model Acceptability Criteria Konstanz Information Miner, (KNIME) node applied
to the data shows that the model passed Tropsha’s recommended tests for predictive ability and
the results are depicted in Table 3. Using this predictive and validated 3D-QSAR CoMFA model,
the predicted inhibitory values of synthesized and evaluated Am analogs from the present study
were calculated. The biological activity values (IC50) of compounds were compared relatively with
the predicted ones (p% inhibition). The active compounds against A/Hong Kong/68 (IC50 < 30
µM)
were predicted as active (p% > 0.6), and the only inactive one with IC50 = 59.71 M was predicted
µ
as inactive (p% = 0.413, Table 4). The correlation plot of the experimental inhibition values versus
predicted ones for the 3D-QSAR CoMFA model is shown in Figure 3. It is noticeable that the studied

Molecules 2020, 25, 3989
7 of 21
synthesized analogs are S enantiomers. R enantiomers were also designed, in order for their inhibition
to be predicted.
Table 3. Enalos Model Acceptability Criteria KNIME node screenshots for the CoMFA model.
Criterion
Assessment
Result
R2
= 0.862
pred
R2
> 0.6
> 0.5
PASS
PASS
PASS
PASS
PASS
PASS
PASS
pred
R2
R2
= 0.867
test
test
(R2 − R20)/R2 < 0.1
(R2 − R020)/R2 < 0.1
|(R2 − R20)|/R2< 0.3
0.85 < k < 1.15
(R2 − R20)/R2 = 0.015
(R2 – R020)/R2 = 0.0
|(R2
−
R20)|/R2 = 0.013
k = 1.028
0.85 < k0 < 1.15
k0 = 0932
Table 4. The predictions of synthesized studied analogs based on obtained 3D-QSAR CoMFA model.
ID
Predicted Values CoMFA p(%) IC50 (µM)
Active *
Prediction (Active)
1
0.820
0.736
0.713
0.745
0.735
0.645
0.668
0.645
−0.057
0.39
1.41
18.37
0.75
0.81
1.32
17.13
3.93
59.71
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
39(S)
40(S)
41(S)
42(S)
43(S)
44(S)
45(S)
46(S)
* Active compound only if IC50 < 30 µM.
Figure 3. Correlation plot of the experimental inhibition values versus predicted ones for the 3D-QSAR
CoMFA model.
For continuous QSAR, criteria followed in developing activity/property predictors are:
(i) correlation coefficient R between the predicted and actual values; (ii) coefficients of determination
2
(predicted versus observed activities R20, and observed versus predicted activities R0 for regressions
0
through the origin) and (iii) slopes k and k0 of regression lines through the origin.
2.4. Molecular Docking Study of the Glycyl-Rimantadine
Glycyl-rimantadine (4b) showed the lowest IC50 (0.11 µM; Figure 4). For this reason, it was sought
interest, to dock it inside the M2TM of Influenza A (Figure 5(B1)). The M2 transmembrane (TM) domain
forms a homotetrameric pore and acts as an ion channel by transporting selectively H+ from low pH

Molecules 2020, 25, 3989
8 of 21
conditions of the endosome into the viral interior [19]. The co-crystallized rimantadine in the M2TM of
Influenza A was superimposed with the docked rimantadine using the grid-based ligand docking
with energetics (GLIDE) algorithm. The small root mean square deviation (RMSD) (<0.1) of this
superposition demonstrates that the use of GLIDE for molecular docking reproduces crystallographic
experimental data for structurally similar molecules. In the present molecular docking procedure
Rim and glycyl-rimantadine (4b) were used as flexible ligands to be docked to the rigid receptor, and
XP (extra precision) method was selected in order to calculate the GLIDE score (Figure 5(A1,B1)).
The docking calculations showed that glycyl-rimantadine (4b) had a lower XP GScore (
−
7.61 kcal mol−1
)
value than rimantadine’s value (
−
7.14 kcal mol−1), so the binding and the inhibition it causes was
expected to be of similar magnitude. The analysis of M2TM–rimantadine interactions (Figure 5(A2))
revealed that the adamantane moiety was oriented to the N-terminus of the virus, while the ammonium
group was directed to C-terminus (His37). Furthermore, the adamantane moiety was bounded in
a hydrophobic pocket consisting of the residues Val37, Ala30 and Ser31. Specifically, the hydroxyl
of Ser31 forms a hydrogen bond to a carbonyl of Val27. It is noteworthy that the bulky adamantane
moiety is responsible for shifting the bound water molecules near the top of the pore. As a consequence,
the complex is stabilized through hydrophobic interactions (van der Waals) and blocks proton entry
into the pore. The ammonium group is adjacent to the four Ala30 carbonyl-associated water molecules.
Actually, the ammonium group does not form hydrogen bonds with facing carbonyl groups but it
mimics hydrogen that interacts with water networks connected directly to the proton gating residue
His37. We examined the intermolecular interactions developed between M2TM and glycyl-rimantadine
(4b) (Figure 5(B2)). Two important observations were described. First, both molecules rimantadine and
glycyl-rimantadine (4b) had their adamantane segment located in the hydrophobic pocket surrounded
by the residues Val27, Ala30 and Ser31. Secondly, glycyl-rimantadine (4b) adopts an additional
hydrogen bond compared to rimantadine with a water molecule, which in a consequence is hydrogen
bonding with the key amino acid His37 responsible for the opening of the M2TM channel.
Figure 4. The two-dimensional (2D) and three-dimensional (3D) structures of rimantadine and
glycyl-rimantadine (4b).

Molecules 2020, 25, 3989
9 of 21
Figure 5. (A1). The 3D docked pose of M2TM–rimantadine in the crystallized M2TM (PDB code:
6BOC) with the presence of water molecules (red color). Hydrogen bonds are depicted as yellow
dashes. (A2). The 2D docked pose of M2TM–rimantadine interactions within 5Å. (B1). The 3D docked
pose of M2TM–glycyl-rimantadine in the crystallized M2TM (PDB code: 6BOC) with the presence of
water molecules (red color). Hydrogen bonds are depicted as yellow dashes. (B2). The 2D docked pose
of M2TM–glycyl-rimantadine interactions within 5Å.
2.5. In Vitro Human Plasma Stability of Glycyl-Rimantadine (4b)
The evaluation of the stability of glycyl-rimantadine (4b) in human plasma was accomplished
by setting up the appropriate LC–MS/MS protocol. In order to choose the optimal chromatographic
conditions (mobile phase and ratio, column type, flow rate, etc.) for achieving a high resolution and
a short runtime, several tests were conducted. A mobile phase composed of formic acid 0.1% in water
(A) and acetonitrile (B) along with a reversed-phase C18 column Kinetex (Phenomenex) met those
criteria. A gradient elution was chosen for the analysis, at a constant flow rate of 0.25 mL min−1 with
the following profile: initial phase (B) concentration of 5% until 0.3 min, then increased to 80% within
1.2 min, kept constant for 1 min at 80% and then reduced to 5% at 2.6 min, until the end of the run at
4 min. The temperature in the auto-sampler and the column oven was maintained at 15 ◦C and 40 ◦C,
respectively. The sample injection volume was set to 5 µL.
Protein precipitation with acetonitrile produced clean chromatograms on blank plasma samples
and was therefore chosen as the sample clean-up method. The retention times for the glycyl-rimantadine
(4b) and Ala-rimantadine (4a) (internal standard, IS) were 2.60 min and 2.65 min respectively and

Molecules 2020, 25, 3989
10 of 21
the total analysis cycle time was 4 min. Tandem mass spectrometry was used for the detection of
the compounds in the positive electrospray ionization (ESI) mode. The tuning parameters (cone
gas flow, heated probe temperature, cone temperature, etc.) were optimized by direct infusion
of a solution containing glycyl-rimantadine (4b) and IS at a concentration of 75 ng mL−1 to have
a consistent response.
The stability profile of glycyl-rimantadine (4b) in human plasma after 24 h incubation at 37 ◦C is
presented in Figure 6. Glycyl-rimantadine (4b) was characterized by profoundly high stability in human
plasma, since after 6 h the degradation was insignificant (97.5%
±
4.6%). After 12 h approximately
91% ± 3.1% of the conjugate was still present in plasma, while at 24 h of incubation the initial amount
was only reduced to 83.7% ± 3.7%.
Figure 6. Stability of glycyl-rimantadine (4b) after incubation in human plasma at 37 ◦C for 24 h. The %
remaining amount of glycyl-rimantadine (4b) is the estimated concentration after 0, 2, 4, 6, 12 and 24 h
in respect to the initial concentration of the conjugate. Each data point represents the average of three
replicates ± SD.
2.6. Elucidation of the Structure of Glycyl-Rimantadine (4b)
Single crystal X-ray study showed that the compound 4b crystallizes in a centrosymmetric manner
(space group Pbca) as a glycyl-rimantadine trifluoroacetate salt. A summary of the most important
crystal data and refinement indicators is provided in Table S1. The asymmetric unit of the studied
compound contains one molecule glycyl-rimantadine, one molecule of trifluoroacetic acid (TFA) and
one solvent water molecules (Figure 7). The bond lengths and angles of the glycyl-rimantadine
molecule are shown in Table S2. The X-ray refinement reveals the formation of a zwitterionic
TFA−–NH3+-glycyl-rimantadine structure. In support of this fact, the C=O and C–O bond lengths
in the TFA– were almost identical (1.231 and 1.242 Å). The crystal structure revealed the presence of
several intermolecular hydrogen bonds (Table 5). In the crystal structure, the molecules were oriented
head (glycyl) to tail (rimantadine moiety). The bulky rimantadine prevented the formation of hydrogen
bonding while the glycyl moiety, TFA and water were all involved in intermolecular interactions.
Thus, three-dimensional packing of the structure was governed by the molecular features (hydrophilic
and hydrophobic parts) and hydrogen bonding interactions (Figure 8).

Molecules 2020, 25, 3989
11 of 21
Figure 7. Oak Ridge Thermal Ellipsoid Plot (ORTEP) diagram of the glycyl-rimantadine (4b) molecule
present in the asymmetric unit along with employed numbering scheme; adenosine diphosphates
(ADPs) are at the 50% displacement probability level while hydrogen atoms are shown as spheres with
arbitrary radii.
Table 5. Hydrogen bonds geometry (Å, ◦) for glycyl-rimantadine (4b).
D -
H . . .
A
D-H . . . A (◦)
d(D-H) (Å)
d(H . . . A) (Å)
d(D . . . A) (Å)
N1 -
N2 -
N2 -
N2 -
C14 -
O4 -
O4 -
O4 -
N2 -
C11 -
H1
H2A
H2B
H2B
H14A
H4C
H4C
H4D
H2C
H11
O11
O2
0.89 (3)
0.98 (3)
0.89 (3)
0.89 (3)
0.95 (3)
0.95 (4)
0.95 (4)
0.86 (4)
1.00 (3)
1.01 (2)
2.25 (3)
1.78 (3)
2.17 (3)
2.64 (3)
2.59 (3)
1.88 (4)
2.53 (3)
2.00 (4)
1.87 (4)
2.35 (2)
3.135 (2)
2.744 (2)
2.922 (3)
3.193 (3)
3.285 (5)
2.811 (3)
3.152 (6)
2.855 (3)
2.847 (3)
2.861 (3)
173 (2)
169 (2)
141 (2)
121 (2)
130.5 (2)
166 (3)
123 (2)
175 (4)
163 (3)
110.0 (1)
O32
O43
F254
O25
F265
O36
O4
O1
Figure 8. Three-dimensional packing and hydrogen bonding systems in the crystal structure of
Glycyl-rimantadine (4b): a view along the a and b axes.

Molecules 2020, 25, 3989
12 of 21
2.7. Thermal Analysis and Stability of Glycyl-Rimantadine (4b)
The thermal behavior of the glycyl-rimantadine (4b) compound was investigated from 20 to
250 ◦C using differential thermal analysis (DTA)—thermogravimetric (TG) analyses (Figure 9).
◦
DTA measurement showed two endothermic effects: the first was in the range of 40–70 C and
◦
the second in the range of 150–170 C. The first endo effect was accompanied by 4% weight loss,
which corresponded to a loss of one water molecule and is in agreement with the crystal structure.
The second DTA effect was not associated with weight loss and corresponded to the melting of the
compound. After the melting, a pronounced weight loss was initiated due to the volatility. During the
cooling stage, an endo effect was observed at around 90 ◦C.
Figure 9. Differential thermal analysis (DTA)-TG curves for glycyl-rimantadine.
3. Materials and Methods
3.1. Chemicals and Reagents
Unless otherwise stated, the starting materials, reagents and solvents were commercially obtained
and used as supplied without further purification. Amantadine and rimantadine as hydrochloride salts
were from Sigma-Aldrich (St. Louis, Missouri, USA) and the amino acids were purchased from Bachem
(Bubendorf, Switzerland). Analytical thin-layer chromatography (TLC) was run on Merck silica gel
60 F-254, with detection by UV light (254 nm). The TLC chromatograms were displayed by ninhydrin
solution and Sakaguchi I/II reagents for guanidated derivatives. The reaction was observed by TLC
Silicagel 60 F254 (Merck, Darmstadt, Germany) by the following mobile phases: CHCl3/CH3OH (95:5);
CHCl3/CH3OH/CH3COOH (95:5:3) and nBuOH/CH3COOH/H2O (3:1:1).
1H and 13C spectra were recorded on Bruker Avance II+ spectrometer (14.09 T magnet, Billerica,
Massachusetts, USA) operating at 600.11 MHz 1H frequencies, equipped with a 5 mm BBO probe
with z-gradient coil. The temperature was maintained at 293 K, using Bruker B-VT 3000 (v1.9, Bruker,
Billerica, Massachusetts, USA) temperature unit with airflow of 535 L h−1. All chemical shifts were
reported in parts per million (ppm), referenced against tetramethylsylane (TMS, 0.00 ppm) or using
the residual solvent signal (7.27 ppm for CDCl3 of 2.5 ppm for DMSO).
Electrospray mass spectrometry (ESI-MS) experiments were acquired on Bruker Compact
QTOF-MS (Bruker Daltonics, Billerica, Massachusetts, USA) and controlled by the Compass Control
software (v1.9, Bruker, Billerica, Massachusetts, USA). The data analysis was performed and the

Molecules 2020, 25, 3989
13 of 21
monoisotopic mass values were calculated using Data analysis software (v4.4, Bruker, Billerica,
Massachusetts, USA). The analyses were conducted in the positive ion mode at a scan range from
m/z 50 to 1000 and nitrogen was used as nebulizer gas at a pressure of 4 psi and flow of 3 L min−1
for the dry gas. The capillary voltage and temperature were set at 4500 V and 220 ◦C, respectively.
An external calibration for mass accuracy was carried out by using sodium formate as calibration
solution. The precursor ion of each compound was selected, and ESI–MS/MS analysis was performed
by collision-induced dissociation (CID); nitrogen was the collision gas, and the collision energy
varied from 5 to 40 eV. MSn experiments were conducted on an ion trap instrument Esquire 3000
(Billerica, Massachusetts, USA) and controlled by the Esquire Control software (v5.3.11, Bruker, Billerica,
Massachusetts, USA). ESI-MS data were collected in positive-ion mode at a scan range from m/z 50 to
500. In all ESI-MS measurements, the nebulizer gas pressure was 124.1 kPa at a flow rate of 5 L min−1
;
the desolvation temperature was 300 ◦C and capillary voltage was adjusted to 4000 V. The sample
solutions were delivered to nebulizer by a syringe pump Cole Parmer (Chicago, Illinois, USA) at a flow
rate 3 µL min−1
.
Formic acid (98%, LC–MS grade) and dimethyl sulfoxide (DMSO, 99.7%) were obtained from
≥
Honeywell Fluka (Charlotte, North Carolina, USA) and Thermo Scientific (Waltham, Massachusetts,
USA), respectively. LC–MS grade acetonitrile and water were purchased from Thermo Fisher Scientific.
Membrane filters (0.2
µm pore size and 47 mm diameter, Waltham, Massachusetts, USA and Minisart
RC 4 syringe filters (0.2
µm pore size) were purchased from Sartorius (Göttingen, Germany Drug-free,
pooled human plasma from healthy donors was a kind offer from the Blood Donation Center of the
University Hospital of Ioannina (Ioannina, Greece).
General Procedure for Synthesis of the 2(a–j), 3a, 3e and 3g–h.
The Boc-AA (1(a–j) (3 mmol), DIPEA (3.1 mmol) were added to a solution of TBTU (3 mmol)
in CH2Cl2 (15 mL). After stirred the mixture was treated with amantadine or rimantadine (3 mmol)
with DMAP (3 mmol). This mixture was stirred at RT for 3 h, and then evaporated to dryness.
After evaporation the residue was purified by flash chromatography (ethyl acetate:n-hexan, 1:1) as
the eluent.
3.1.1. General Procedure for Boc-Group Deprotection of the 4(a–j), 5a, 5e and 5g–h
One equiv. of 2(a–j), 3a, 3e and 3g–h was dissolved in 10-fold excess of trifuoroacetic acid (TFA) at
0 ◦C. The reaction mixture was stirred until Boc group running out (the chromatographic control was
carried out in systems chloroform-methanol 95:5 ratio). The solvent was evaporated and the residue
was dissolved in 10 mL methanol. Until the pH reached approximately 8, 25% ammonia solution was
added. The solvent was evaporated under vacuum. Obtained crystals was dissolved in ethyl acetate
and washed with water 3
removed. The yield on each compound was various, but it was within 65–82%.
×
25 mL. The organic layer dried on anhydrous Na2SO4 and solvent was
3.1.2. Preparative Method of the 6a, 6i, 6j, 7a and 7h
In 2 mL acetonitrile, 0.67 mmol of 4a, 4i, 4j, 5a and 5h was dissolved. Of 1H-pyrazole-1-
carboxamidine hydrochloride 1.0 mmol and 2.0 mmol TEA were added to the solution. The reaction
continued for 48 h at room temperature. After that the solvent was evaporated under vacuum.
The residue was dissolved in 25 mL chloroform and washed few times with 5% sodium hydrogensulfate
(pH 3). The organic layer was dried with sodium sulfate anhydrous. The solvent was evaporated
under vacuum and residue was crystalized by methanol/diethyl ether. After 12 h at 4 ◦C the mixture
was filtered and dried at least 24 h in a desiccator, filled with calcium dichloride.
3.2. Cytotoxicity
The synthesized compounds were tested for antiviral activity against the influenza virus A/H3N2,
strain Hong Kong 68.

Molecules 2020, 25, 3989
14 of 21
In order to identify new potential antiviral drugs, small amounts of extracts or compounds have to
be examined for cytotoxicity and antiviral activity in primary screening using a rapid, easy, inexpensive
and highly standardized test system. The cytotoxic and the antiviral effects were quantified using
a crystal violet uptake assay allowing automated handling of large numbers of candidate agents.
To ensure comparable results with plaque reduction assays, the 50% plaque inhibitory concentrations
of amantadine and oseltamivir were used to standardize the anti-influenza virus A tests. Madin-Darby
Kanine Kidney Cells (MDCK) cells were seeded at 2
microtiter plates (Falcon 3075). The cytotoxicity of the test compounds was determined on 2-day-old
confluent MDCK cell monolayers grown in the internal 60 wells (5% CO2, 37 ◦C). After removal of
×
104 cells per well in 96-well flat-bottomed
the growth medium, two-fold dilutions of the compounds in 100 µL test medium were added and
incubated at 37 ◦C in a 5% CO2 atmosphere for 72 h. Cells in six wells without compound treatment
served as basic controls. The Dynex Immuno Assay System (DIAS, Guernsey, UK) developed for
automated ELISA techniques was applied to wash gently, stain, measure and analyze the viability
of the cell monolayers in cytotoxic, as well as antiviral assays. The staining procedure undertaken
with crystal violet was based on the principles described by Nain et al. for the determination of the
cytotoxic activity of TNF-
washed three times with 300 mL physiological sodium chloride solution to remove dead cells. Secondly,
the cells were fixed and stained in one step with 50 L of 0.03% crystal violet (w/v) in 20% methanol
for 10 min. After six further automated washings with 300 L of water, the stained monolayers were
treated for 20 min with 100 L lysis buffer (0.8979 g of sodium citrate and 1.25 mL of 1 N HCl in 98.05 mL
α [20]. At first, the supernatant was aspirated and the cell monolayers were
µ
µ
µ
of 47.5% ethanol) to elute the crystal violet. Then the optical density of individual wells was quantified
spectrophotometrically at 540/630 nm and analyzed with the DIAS. Cell viability was evaluated as
the percentage of the mean value of optical density, resulting from the six cell controls, which was set
100%. The 50% cytotoxic concentrations (CC50) were calculated from the mean dose–response curves
of three assays each with two parallels.
3.3. Antiviral Activity
To exclude non-specific antiviral activities of test compounds, their cytotoxic effects have to be
determined not only on confluent cell monolayers but also on proliferating cell cultures in the primary
antiviral screening. The cytotoxicity and the growth inhibition of compounds were quantified using an
automated crystal violet staining procedure and the electronic cell analyzer system CASY 1, respectively,
on day 3 after compound application. The 50% cytotoxic and growth inhibition concentrations were
calculated from the mean dose–response curve (3.9 through 1000 µ
g mL−1; dilution factor 2) of three
separate experiments each with two parallels in the indicated cell lines.
3.4. 3D-QSAR Model Development
For the development of the 3D-QSAR model, 38 molecules were used with experimental inhibitory
data obtained from the literature [21,22]. The selected structures of amino adamantane analogs were
built using ChemBioDraw Ultra 14.0 [23] and their SMILE strings were imported in the project table
of Maestro10.2, the graphical user interface (GUI) by Schrödinger [24]. 2D chemical structures were
converted to 3D using LigPrep3.4, [25] in which all the hydrogen atoms were added, ionization states
were generated at user-defined pH 7, and the molecules were geometrically refined. All the structures
were subsequently submitted in full structure optimization, using the minimization procedure of
MacroModel 10.8 [26]. For the minimization, water was selected as the solvent and a standard molecular
mechanics energy function (OPLS_20057 force field) with the Polak–Ribiere conjugated gradient method
(PRCG, 1000 iterations with gradient 0.01 kcal mol−1 Å−1) was applied [27]. The dataset was divided
into two sets, training and test set, followed by the module previously described [28]. Therefore,
the training set consisted of 28 molecules and the test one consisted of the remaining 10 molecules.
The experimental data of 38 adamantane analogs expressed as the percent inhibition (%), ranging from
0.7 to 91, were converted into (×10−2) values and are defined in Supplementary Table S1.

Molecules 2020, 25, 3989
15 of 21
3.5. CoMFA Analysis
The CoMFA calculations were performed using SYBYL 8.0 software provided by TRIPOS [29].
Atomic charges of molecules were calculated using the Gasteiger–Hückel method, which is
a combination of two other charge computational methods: the Gasteiger–Marsili [30] method
that calculates the
σ component of the atomic charge, and the Hückel [27] method that calculates the
π
component of the atomic charge. The total charge is the sum of the charges calculated by the two
methods. A rigid-body atom-by-atom superimposition of one molecule onto another was performed
using the amino adamantane core (11 atoms) as the common substructure, and the alignment of
compounds was applied based on amantadine (Figure 10).
Figure 10. Dataset alignment superimposition of training set used for the 3D-QSAR analysis based on
the amino amantadine skeleton.
The CoMFA fields are generated by creating a grid around the molecule and calculating the steric
and electrostatic potentials at each point on the grid using a + 1 charged probe atom of a hybridized
sp3 carbon [31
,32]. The steric and electrostatic field energies were calculated using the Lennard-Jones
and the Coulomb potentials, respectively, with a 1/r2 distance-dependent dielectric constant in all
intersections of regularly spaced (0.2 nm) grid. The truncation for both the steric and electrostatic
energies was set to 30 kcal mol−1. This indicates that any steric or electrostatic field value that exceeds
this value will be replaced with 30 kcal mol−1, thus makes a plateau of the fields close to the center of
any atom.
3.6. Partial Least Square (PLS) Analysis
The partial least square (PLS) analysis [33,34] applied in SYBYL 8.0 was used to derive the
3D-QSAR model and linearly correlate the CoMFA fields with experimental values. The CoMFA fields
were used as independent variables and the inhibitory values as dependent variables. PLS analysis was
performed in two steps. In the first step, the cross-validation analysis performed using leave-one-out
(LOO) method was applied to the training set of the dataset in order to determine (Q2), SEP and ONC.
In the LOO method, one compound is omitted from the training set and the model is derived involving
the rest of the molecules. Then using this model, the activity of the molecule that was removed can be
predicted [32]. The model with the lowest SEP and as possible highest Q2 was selected for the next
step of the analysis. The value of Q2 is calculated by the following equation:
ꢀ
ꢁ
P
2
2
Yobs − Ypred
Yobs − Ytrain
Q2 = 1 −
(1)
ꢀ
ꢁ
P
where Yobs and Ypred indicate observed and predicted activity values, respectively, and Ytrain indicates
the mean of observed activity values of the training set. A model is considered acceptable when the
value of Q2 exceeds 0.5 [35,36].

Molecules 2020, 25, 3989
16 of 21
In the second step, using the ONC obtained from cross-validation analysis, a non-cross-validated
method was applied and the non-cross-validated R2 (conventional) was calculated according to the
following equation:
P
2
(Yobs − Ycalc
)
2
R2 = 1 −
(2)
ꢀ
ꢁ
P
Yobs − Y
In addition, the statistical significance of the model is described by the standard error of estimate
(SEE) and the probability value (F-value). To speed up the analysis and reduce noise, a minimum
column filter value
σ
(2.00 kcal mol−1) was used. The application of non-cross-validated method led to
the build of the CoMFA model.
3.7. 3D-QSAR CoMFA Model Validation
The predictive capability of the 3D-QSAR CoMFA model was evaluated using the test set [37].
The predictive correlation (R2test) is calculated using the following equation:
ꢀ
ꢁ
P
2
Ypred(test) − Ytest
R2test = 1 −
(3)
ꢀ
ꢁ
P
2
Ytest − Ytrain
where Ypred(test) and Ytest indicate predicted and observed activity values of the test set, respectively.
For a predictive QSAR model, the value of the R2
should be more than 0.5.
test
To assess the predictive power of the produced 3D-QSAR model, the Enalos Model Acceptability
Criteria KNIME node was additionally used [38] including all proposed validation tests [39].
3.8. Molecular Docking of the Glycyl-Rimantadine (4b) in the Binding Site of Rimantadine Bound to the
Influenza A M2 Transmembrane Domain
Molecular docking is one of the most preferable methods in drug discovery design because of
its ability to predict the conformation of small molecule ligands within the protein binding site with
accuracy. In addition, the most likely binding conformation and the corresponding intermolecular
interactions can be determined.
The ligands (rimantadine and glycyl-rimantadine) were built using ChemBioDraw Ultra, [22
and their SMILES used in order to create 3D-structure using Maestro 10.2 Graphical User Interface
(GUI) [23]. Then, the two ligands were minimized by means of Maestro MacroModel 10.8, [25
]
]
where all hydrogens were added and molecules were subjected to complete structure minimization.
For the minimization, water was chosen as a solvent, OPLS_2005 [26] force field and the algorithm
Polak-Ribiere (PRCG, convergence value 0.01 kcal mol−1
Å
−1) were used. Subsequently, the molecules
were checked to have the right pH (7.4) using LigPrep 2.5. [40].
The 3D-structure of Influenza A M2TM (open conformation)–rimantadine (PDB code: 6BOC)
served as a model structure for the docking calculations. The “Protein Prep. Wizard” application from
Schrödinger’s Maestro software was used, which is a collection of tools that prepares the 3D-structure
of proteins [40–43].
Using the “Preprocess and analyze structure” tool, the sequence of the bonds was checked,
all hydrogen atoms were added, all disulfide bonds were checked, and all water molecules greater
than 5 Å from the active site of the protein were deleted. Epik 2.3 was used in order to predict the
ionization of amino acids [40–43].
Finally, using the “Impref” application and the OPLS_2005 force field optimized, the positions of
the enzyme hydrogen atoms by keeping all other atoms immobilized [23].
For the molecular docking was used Schrödinger’s GLIDE (grid-based ligand docking with
energetics) algorithm [44]. The binding region was defined by a 12 Å
coincides with that of the ligand and selected the binding of ligands similar in size to the crystallized
ligand. The other parameters used were the program default. The precision level XP (XP extra
×
12 Å
×
12 Å grid whose center

Molecules 2020, 25, 3989
17 of 21
precision) and XP GScore were used for docking and grading, respectively. The poses representing the
lowest value of the scoring function (XP GScore) were further analyzed to identify which of the ligand
conformations most likely describe the correct binding mode.
3.9. LC–MS/MS Plasma Stability Assay
3.9.1. LC–MS/MS Conditions
The separation of glycyl-rimantadine (4b) and Ala-rimantadine (4a) (internal standard, IS) was
performed on an ultra-high-performance liquid chromatography (UHPLC) system (Bruker Daltonics,
Germany), utilizing an RP Kinetex C18 column 100 mm
×
2.1 mm, 2.6 µm, with a proguard column
2.1 mm (Phenomenex, USA). The mobile phase was composed of formic acid 0.1% in water (A) and
acetonitrile (B). Gradient elution was chosen for the analysis, at a constant flow rate of 0.25 mL min−1
and the following gradient profile: initial phase (B) concentration of 5% until 0.3 min, then increased to
80% within 1.2 min, kept constant for 1 min at 80% and then reduced to 5% at 2.6 min, until the end of
the run at 4 min. The temperature in the autosampler and the column oven was maintained at 15 ◦C
and 40 ◦C, respectively. The sample injection volume was set to 5 µL.
The EVOQ Elite ER (Bruker Daltonics, Germany) triple quadrupole mass spectrometer was
operated in positive ionization electrospray mode (ESI) in multiple reaction monitoring (MRM) for
the detection of glycyl-rimantadine (4b) and IS. The optimal MRM transitions for monitoring the
analytes were determined and set as follows: m/z 237.1
→
163.2, 237.1
→
75.5 and 237.1
→
81.4 for
glycyl-rimantadine and 251.1 163.2, 251.1 89.4 and 251.1
→
→
→
180.2 for the IS. The optimum ESI
parameters were determined and set as follows: spray voltage at 4500 V; heated probe gas flow at
50 units; heated probe temperature at 150 ◦C; cone gas flow at 20 units; cone temperature at 300 ◦C
and nebulizer gas flow at 50 units. The control of the LC and the MS, as well as the data acquisition,
was performed using the MSWS software, version 8.2.1 (Bruker Datonics, Germany).
3.9.2. Stock, Working, Calibration Solutions and Extraction Procedure
Stock solutions (1 mg mL−1 and 100
µ
g mL−1) and working solutions for the glycyl-rimantadine
(4b) and the IS were prepared in DMSO. Five working solutions with concentrations equal to 4, 10, 20,
30 and 40 µ
g mL−1 were used in order to prepare the final calibration standards (10, 25, 50, 75 and
100 ng mL−1) for the analyte. The final concentration of the IS was 50 ng mL−1. Protein precipitation
was applied to extract the analyte from plasma. Specifically, for each calibrator 5 µL of the respective
working solution were spiked in 90
for plasma precipitation and 5 L of the IS working solution (20
vortexing and centrifugation for 10 min at 10,000 g were employed to separate and collect the liquid
supernatant containing the analyte and the IS. The supernatant was filtered with 0.2 m syringe filters
and 100 L were transferred in LC–MS vial containing 300 L water (LC–MS grade). Finally, 5 L were
µL of plasma. Then, 400
µ
L of ice-cold acetonitrile were used
µ
µ
g mL−1) were added. Vigorous
×
µ
µ
µ
µ
injected into the LC–MS system. The calibration standards for the conjugate were freshly prepared on
the experimental days by spiking the appropriate amounts of working solutions into the blank human
plasma. All stock and working solutions were stored at −20 ◦C prior to analysis.
3.9.3. In Vitro Plasma Stability
The in vitro plasma stability of glycyl-rimantadine (4b) was conducted by LC–MS/MS. In 90
µ
L of
blank human plasma, 5 µL of the respective working solution (30 µ
for 0, 2, 4, 6, 12 and 24 h in a thermoshaker at 37 ◦C. At the end of the incubation time the samples
were processed as described above. All samples were tested in triplicates. The glycyl-rimantadine
g mL−1) were added and incubated
(4b) concentration was calculated via the standard calibration curve that was designed by plotting the
area of the conjugate versus the IS area against the nominal concentration of the calibration standards.
The plot of the remaining percentage of the parent compound against time was designed for the
glycyl-rimantadine analogue through GraphPad Prism 8.0.1 (Bruker, Billerica, Massachusetts, USA).

Molecules 2020, 25, 3989
18 of 21
3.10. Thermal Analyses
DTA and TG thermal analysis were carried out simultaneously in a thermal analyzer Stanton
◦
Redcroft STA780 at the following conditions: heating rate of 5 C min–1, dry argon as carrier gas
(30 mL min–1) and sample weight of 7 mg in corundum crucible.
3.11. Crystal Structure Analysis
3.11.1. Sample Crystallization
In order to obtain crystals with better quality 50 mg of the dry powder (microcrystals) of
glycyl-rimantadine trifluoroacetate (C16H27N2O4F3) were dissolved in ethanol, methanol and acetone
(3 mL) at room temperature. Large crystals (0.3
×
0.2
×
0.15 mm3) suitable for single crystal X-ray
studies grew by slow evaporation within 2–3 days from all solutions, however the diffraction quality
was significantly better for the crystals obtained from methanol solution. Crystals were collected
directly from the mother liquors, e.g., from the three solvents used.
3.11.2. Data Collection and Crystal Structure Refinement
Selected crystal was mounted on a glass capillary and all data were collected at room temperature
(290 K) on an Oxford Diffraction SupernovaDual four cycle diffractometer, equipped with Atlas CCD
detector using Mo-Kα radiation (λ = 0.71013 Å) from the micro-focus source. The determination of
unit cell parameters, data integration, scaling and absorption corrections were carried out using the
CrysalisPro. [45]. The phases were obtained by direct methods with ShelxS-2018/3. The refinement
of the structure involved several cycles of refinement using full-matrix least-squares on F2 with
the ShelxL-2018 package [46,47]. Initially non-hydrogen atoms (C, O, N and F) were located and
positioned from difference Fourier map. After that, all hydrogen atoms were located and positioned
in the resulting difference Fourier map. The non-hydrogen atoms were refined anisotropically and
hydrogen atoms were refined using the riding model. The drawings of the asymmetric unit and
three-dimensional representations of glycyl-rimantadine (4b) were carried out using ORTEP [48
]
and Mercury programs [49]. The crystallographic data (coordinates and structure factors) have been
verified using check CIF/PLATON [50].
4. Conclusions
The antiviral activity in vitro against the influenza virus A/H3N2 strain Hong Kong/68 of new
analogs of amantadine and rimantadine conjugated with amino acids was investigated.
The results revealed that the highest antiviral activity combined with low cytotoxicity was
demonstrated by the rimantadine derivative with the structurally simplest amino acid-glycine. IC50 of
the conjugate was about 3.5 lower than amantadine. Moreover, glycyl-rimantadine (4b) shows a high
stability profile after incubation in human plasma for 24 h. Docking studies disclosed that rimantadine
and glycyl-rimantadine (4b) had their adamantane segment located in the same hydrophobic pocket.
Furthermore, glycyl-rimantadine adopted an additional hydrogen bond compared to rimantadine
with a water molecule, which in a consequence was hydrogen bonding with the key amino acid His37.
In addition, the various aliphatic and aromatic amino acids as substitutes in the rimantadine
molecule had no significant effect on antiviral activity. The corresponding amantadine analogs were
considerably less effective and only phenylalanyl-amantadine (5e) and (4-F)-phenylalanyl-amantadine
(5g) exhibited activity although lower than that of the amantadine in the same concentration. The results
indicate that electron-acceptor substituent (4-F)-phenylalanine did not change the activity. Probably
the aromatic ring, as a larger structure, did not fit in the M2 ion channel.
QSAR studies on adamantane analogues revealed that the CoMFA model could predict the
activities of the synthetic molecules.

Molecules 2020, 25, 3989
19 of 21
Amantadine and rimantadine analogues conjugated with guanidine show low toxicity but they
also exhibited lower activity. Unlike guanidated oseltamivir analogues [ ] the guanidation of the
6
rimantadine and amantadine analogues did not lead to increasing of antiviral activity.
Supplementary Materials: The following are available online. The purity and the molecular structure of the
synthesized compounds was confirmed with 1H and 13C NMR spectroscopy.
Author Contributions: I.S. and A.G.T. conceived the project and prepared manuscript. R.C., N.A.P., M.V.C., E.V.,
K.M., G.M., Y.M., M.S. (Martin Sticha) and H.S.-D. performed and analyzed experiments. B.S., M.S. (Michaela
Schmidtke) and T.M. interpreted the data and co-wrote the manuscript. All authors have read and agreed to the
published version of the manuscript.
Funding: This research was funded by South-West University (Project RPY-A3/19).
Acknowledgments: Special thanks to A. Kolocouris for his fruitful discussions and comments on the 3D-QSAR part.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
References
1.
Spilovska, K.; Zemek, F.; Korabecny, J.; Nepovimova, E.; Soukup, O.; Windisch, M.; Kuca, K. Adamantane-a
lead structure for drugs in clinical practice. Curr. Med. Chem. 2016, 23, 3245–3266. [CrossRef] [PubMed]
Shibnev, V.A.; Garaev, T.M.; Finogenova, M.P.; Shevchenko, E.S.; Burtseva, E.I. New adamantane
derivatives can overcome resistance of influenza A(H1N1)pdm2009 and A(H3N2) viruses to remantadine.
Bull. Exp. Biol. Med. 2012, 153, 233–235. [CrossRef]
2.
3.
4.
5.
Deryabin, P.G.; Garaev, T.M.; Finogenova, M.P.; Botikov, A.G.; Shibnev, V.A. Amino acid derivatives of
adamantane carbocycle are capable of inhibiting replication of highly virulent avian influenza A/H5N1 Virus.
Bull. Exp. Biol. Med. 2014, 157, 62–65. [CrossRef] [PubMed]
Shibnev, V.A.; Garaev, T.M.; Deryabin, P.G.; Finogenova, M.P.; Botikov, A.G.; Mishin, D.V. New carbocyclic
amino acid derivatives inhibit infection caused by highly pathogenic influenza A virus strain (H5N1).
Bull. Exp. Biol. Med. 2016, 161, 284–287. [CrossRef] [PubMed]
Hong, B.T.; Chen, C.L.; Fang, J.M.; Tsai, K.C.; Wang, S.Y.; Huang, W.I.; Cheng, Y.E.; Wong, C.H. Oseltamivir
hydroxamate and acyl sulfonamide derivatives as influenza neuraminidase inhibitors. Bioorg. Med. Chem.
2014, 22, 6647–6654. [CrossRef]
6.
7.
8.
9.
Jackson, R.J.; Cooper, K.L.; Tappenden, P.; Rees, A.; Simpson, E.L.; Read, R.C.; Nicholson, K.G. Oseltamivir,
zanamivir and amantadine in the prevention of influenza: A systematic review. J. Infect. 2011, 62, 14–25.
FDA Approves Using Relenza to Prevent Flu, March 29, 2006. Available online: www.cidrap.umn.edu
(accessed on 1 September 2020).
Gaiday, A.V.; Levandovskiy, I.A.; Byler, K.G.; Shubina, T.E. Mechanism of influenza A M2 ion-channel
inhibition: A docking and QSAR study. Comput. Sci. ICCS 2008, 5102, 360–368. [CrossRef]
Knorr, R.; Trzeciak, A.; Annwarth, W.; Gillessen, D. New coupling reagents in peptide chemistry.
Tetrahedron Lett. 1989, 30, 1927–1930. [CrossRef]
10. Bernatowicz, M.; Wu, Y.; Matsueda, G. 1H-Pyrazole-1-carboxamidine hydrochloride an attractive reagent for
guanylation of amines and its application to peptide synthesis. J. Org. Chem. 1992, 57, 2497–2502. [CrossRef]
11. Schnell, J.R.; Chou, J.J. Structure and mechanism of the M2 proton channel of influenza A virus. Nature 2008
451, 591–595. [CrossRef]
,
12. Beauchamp, L.M.; Orr, G.F.; Demiranda, P.; Burnette, T.; Krenitsky, T.A. Amino-acid ester prodrugs of
acyclovir. Antivir. Chem. Chemother. 1992, 3, 157–164. [CrossRef]
13. Han, H.; de Vrueh, R.L.; Rhie, J.K.; Covitz, K.M.; Smith, P.L.; Lee, C.P.; Oh, D.M.; Sadee, W.; Amidon, G.L.
50-Amino acid esters of antiviral nucleosides, acyclovir, and AZT are absorbed by the intestinal PEPT1
peptide transporter. Pharm. Res. 1998, 15, 1154–1159. [CrossRef] [PubMed]
14. Shen, W.; Kim, J.S.; Mitchell, S.; Kish, P.; Kijek, P.; Hilfinger, J. 50-O-D-valyl ara A, a potential prodrug for
improving oral bioavailability of the antiviral agent vidarabine. Nucleos. Nucleot. Nucl. Acids 2009, 28, 43–55.
[CrossRef]

Molecules 2020, 25, 3989
20 of 21
15. Kim, C.U.; Lew, W.; Williams, M.A.; Wu, H.; Zhang, L.; Chen, X.; Escarpe, P.A.; Mendel, D.B.; Laver, W.G.;
Stevens, R.C. Structure-activity relationship studies of novel carbocyclic influenza neuraminidase inhibitors.
J. Med. Chem. 1998, 41, 2451–2460. [CrossRef] [PubMed]
16. Kim, S.H.; Tangallapally, R.; Kim, I.C.; Trovato, R.; Parker, D.; Patton, J.S.; Reeves, L.; Bradford, C.;
Wettstein, D.; Baichwal, V.; et al. Discovery of an L-alanine ester prodrug of the Hsp90 inhibitor, MPC-3100.
Bioorg. Med. Chem. Lett. 2015, 25, 5254–5257. [CrossRef] [PubMed]
17. Lin, T.I.; Heider, H.; Schroeder, C. Different modes of inhibition by adamantane amine derivatives and
natural polyamines of the functionally reconstituted influenza virus M2 proton channel protein. J. Gen. Virol.
1997, 78, 767–774. [CrossRef]
18. Leonov, H.; Astrahan, P.; Krugliak, M.; Arkin, I.T. How do aminoadamantanes block the influenza M2
channel, and how does resistance develop? J. Am. Chem. Soc. 2011, 133, 9903–9911. [CrossRef]
19. Lamb, R.A.; Zebedee, S.L.; Richardson, C.D. Influenza virus M2 protein is an integral membrane protein
expressed on the infected-cell surface. Cell 1985, 40, 627–633. [CrossRef]
20. Nain, M.; Hinder, F.; Gong, J.H.; Schmidt, A.; Bender, A.; Sprenger, H.; Gemsa, D. Tumor necrosis factor-alpha
production of influenza A virus-infected macrophages and potentiating effect of lipopolysaccharides.
J. Immunol. 1990, 145, 1921–1928.
21. Li, F.; Ma, C.; Hu, Y.; Wang, Y.; Wang, J. Discovery of potent antivirals against amantadine-resistant Influenza
A viruses by targeting the M2-S31N proton channel. Acs Infect. Dis. 2016, 2, 726–733. [CrossRef]
22. Wang, J.; Ma, C.; Wang, J.; Jo, H.; Canturk, B.; Fiorin, G.; Pinto, L.H.; Lamb, R.A.; Klein, M.L.; DeGrado, W.F.
Discovery of novel dual inhibitors of the wild-type and the most prevalent drug-resistant mutant, S31N, of
the M2 proton channel from influenza A virus. J. Med. Chem. 2013, 56, 2804–2812. [CrossRef] [PubMed]
23. ChemBioDraw Ultra, version 14.0.; PerkinElmer. 2014. Available online: https://www.perkinelmer.com
(accessed on 1 September 2020).
24. Maestro, version 10.2.; Schroedinger, LLC: New York, NY, USA, 2015.
25. LigPrep, version 3.4.; Schroedinger, LLC: New York, NY, USA, 2015.
26. MacroModel, version 10.8.; Schroedinger, LLC: New York, NY, USA, 2015.
27. Kaminski, G.A.; Friesner, R.A.; Tirado-Rives, J.; Jorgensen, W.L. Evaluation and reparametrization of the
OPLS-AA force field for proteins via comparison with accurate quantum chemical calculations on peptides.
J. Phys. Chem. B 2001, 105, 6474–6487. [CrossRef]
28. Vrontaki, E.; Melagraki, G.; Mavromoustakos, T.; Afantitis, A. Searching for anthranilic acid-based thumb
pocket 2 HCV NS5B polymerase inhibitors through a combination of molecular docking, 3D-QSAR and
virtual screening. J. Enzym. Inhib. Med. Chem. 2016, 31, 38–52. [CrossRef] [PubMed]
29. Tripos Associates. SYBYL QSAR and COMFA, version 8.0.; Tripos Associates: St. Louis, MO, USA, 2007.
30. Gasteiger, J.; Marsili, M. Iterative partial equalization of orbital electronegativity—a rapid access to atomic
charges. Tetrahedron 1980, 36, 3219–3228. [CrossRef]
31. Purcell, P.; Singer, A. A brief review and table of semiempirical parameters used in the Hueckel molecular
orbital method. J. Chem. Eng. Data 1967, 12, 235–246. [CrossRef]
32. Clark, M.; Cramer, R.D.; Vanopdenbosch, N. Validation of the general-purpose Tripos 5.2 force-field.
J. Comput. Chem. 1989, 10, 982–1012. [CrossRef]
33. Cramer, R.D.; Patterson, D.E.; Bunce, J.D. Comparative molecular-field analysis (CoMFA).1. Effect of shape
on binding of steroids to carrier proteins. J. Am. Chem. Soc. 1988, 110, 5959–5967. [CrossRef]
34. Frank, I.; Feikema, J.; Constantine, N.; Kowalski, B. Prediction of product quality from spectral data using
the partial least-squares method. J. Chem. Inf. Comput. Sci. 1984, 24, 20–24. [CrossRef]
35. Rannar, S.; Lindgren, F.; Geladi, P.; Wold, S. A Pls kernel algorithm for data sets with many variables and
fewer objects. 1. theory and algorithm. J. Chemometr. 1994, 8, 111–125. [CrossRef]
36. Cramer, R.D.; Bunce, J.D.; Patterson, D.E.; Frank, I.E. Cross-validation, bootstrapping, and partial least-squares
compared with multiple-regression in conventional QSAR studies. Quant. Struct-Act. Rel. 1988, 7, 18–25.
[CrossRef]
37. Hawkins, D.M.; Basak, S.C.; Mills, D. Assessing model fit by cross-validation. J. Chem. Inf. Comput. Sci. 2003
,
43, 579–586. [CrossRef] [PubMed]
38. Tropsha, A. Best practices for QSAR model development, validation, and exploitation. Mol. Inform. 2010, 29,
476–488. [CrossRef] [PubMed]

Molecules 2020, 25, 3989
21 of 21
39. Melagraki, G.; Afantitis, A. Enalos KNIME nodes: Exploring corrosion inhibition of steel in acidic medium.
Chemometr. Intell. Lab. 2013, 123, 9–14. [CrossRef]
40. Golbraikh, A.; Tropsha, A. Beware of q2! J. Mol. Graph. Model. 2002, 20, 269–276. [CrossRef]
41. LigPrep, version 2.5.; Schroedinger, LLC: New York, NY, USA, 2012.
42. Protein Preparation Wizard, Epik version 2.3.; Schroedinger, LLC: New York, NY, USA, 2012.
43. Impact, version 5.8.; Schroedinger, LLC: New York, NY, USA, 2012.
44. Prime, version 3.1.; Schroedinger, LLC: New York, NY, USA, 2012.
45. CrysAlis PRO version 171.36.20; Agilent Technologies, Ltd.: Yarnton, UK, 2011.
46. Sheldrick, G.M. A short history of SHELX. Acta Crystallogr. A 2008, 64, 112–122. [CrossRef] [PubMed]
47. Sheldrick, G.M. Crystal structure refinement with SHELXL. Acta Crystallogr. C Struct. Chem. 2015, 71, 3–8.
[CrossRef] [PubMed]
48. Farrugia, L.J. WinGX and ORTEP for Windows: An update. J. Appl. Crystallogr. 2012, 45, 849–854. [CrossRef]
49. Macrae, C.F.; Bruno, I.J.; Chisholm, J.A.; Edgington, P.R.; McCabe, P.; Pidcock, E.; Rodriguez-Monge, L.;
Taylor, R.; van de Streek, J.; Wood, P.A. Mercury CSD 2.0-new features for the visualization and investigation
of crystal structures. J. Appl. Crystallogr. 2008, 41, 466–470. [CrossRef]
50. Spek, A.L. Structure validation in chemical crystallography. Acta Crystallogr. D 2009, 65, 148–155. [CrossRef]
Sample Availability: Samples of the compounds are not available.
©
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).