Synthesis and evaluation of phenylisoserine derivatives for the SARS-CoV 3CL protease inhibitor

Article abstract of DOI:10.1016/j.bmcl.2017.04.056

Synthesis and evaluation of new scaffold phenylisoserine derivatives connected with the essential functional groups against SARS CoV 3CL protease are described. The phenylisoserine backbone was found by simulation on GOLD software and the structure activi

Full text of DOI:10.1016/j.bmcl.2017.04.056

Bioorganic & Medicinal Chemistry Letters 27 (2017) 2746–2751
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of phenylisoserine derivatives for the SARS-
CoV 3CL protease inhibitor
a
a
a
a
Hiroyuki Konno a, , Takumi Onuma , Ikumi Nitanai , Masaki Wakabayashi , Shigekazu Yano ,
⇑
Kenta Teruya b, Kenichi Akaji c,
⇑
a Department of Biological Engineering, Graduate School of Science and Engineering, Yamagata University, Yonezawa, Yamagata 992-8510, Japan
b Department of Neurochemistry, Tohoku University Graduate School of Medicine, Aoba-ku, Sendai 980-8575, Japan
c Department of Medicinal Chemistry, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607-8414, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis and evaluation of new scaffold phenylisoserine derivatives connected with the essential func-
tional groups against SARS CoV 3CL protease are described. The phenylisoserine backbone was found by
simulation on GOLD software and the structure activity relationship study of phenylisoserine derivatives
Received 30 March 2017
Revised 15 April 2017
Accepted 18 April 2017
Available online 20 April 2017
gave SK80 with an IC50 value of 43
l
M against SARS CoV 3CL R188I mutant protease.
Ó 2017 Elsevier Ltd. All rights reserved.
Keywords:
SARS CoV
SARS 3CL protease
Phenylisoserine derivative
Docking simulation
Cinnamoyl
Severe acute respiratory syndrome (SARS) is a contagious respi-
ratory disease in humans which is caused by the SARS coronavirus
(SARS-CoV).1–3 The key enzyme in the processing of polyproteins
pp1a and pp1ab, translated by the viral RNA genome of SARS-
CoV is called a 3C-like protease (3CL protease). Due to its func-
tional importance in the viral life cycle, SARS-CoV 3CL protease is
considered to be an attractive target for a drug for SARS therapy.
For this reason, a variety of 3CL protease inhibitors have been
reported in the literature over the past decade.4–11 They are non-
peptidyl compounds derived from natural products and/or sub-
strate-based peptidemimetics. However, no effective therapeutic
drug or vaccine has been developed to date, although many candi-
dates targeting SARS 3CL protease have been identified. In a
research program on SARS 3CL protease, we found for the first time
that mature SARS 3CL protease is subject to degradation at the
188Arg/189Gln site. R188I mutant protease with high activity
and stability was prepared.10 Although our group additionally
found that tetrapeptide aldehyde Ac-Thr-Val-Cha-His-H (1)
showed high inhibitory activity with an IC50 value of 98 nM against
SARS 3CL R188I mutant protease in 2008,12 there are generally
Next, we designed and synthesized serine derivatives focusing on
the P1, P2 and P4 sites of tetrapeptide aldehyde (1) by the combi-
nation of docking simulation and a structure activity relationship
study. As for the results, SK23 (2) was given as a candidate com-
pound against SARS 3CL R188I mutant protease.13 In this case,
the inhibitory activity of SK23 (2) was much lower than the pep-
tide aldehyde inhibitor (1). However, SK23 (2) has no thiol capture
unit and achieved a marked structural transformation from pep-
tidyl compounds based on a substrate mimic strategy. Investiga-
tion of a scaffold with three essential functional groups is needed
to develop small molecular inhibitors using a serine scaffold for
non-peptidyl compounds (Fig. 1).
Scaffold hopping techniques have been widely applied by
medicinal chemists to discover equipotent compounds with novel
backbones that have improved properties.14 Scaffold hopping into
four major categories, namely heterocycles replacements, ring
opening or closure, peptidemimetics and topology-based hopping,
has been classified. In the technique for SARS 3CL protease inhibi-
tors, we are interested in a peptidomimetics approach based on the
scaffold hopping strategy. We herein report the design, synthesis
and evaluation of a new scaffold, a phenylisoserine (PIS) derivative,
for SARS-CoV 3CL protease inhibitors.
enzymatic digestions of peptide chains and a-proton racemization.
We performed a variety of molecular mechanics calculations
with SPARTAN from Wavefunction and docking simulations of pro-
tein interactions using GOLD from CCDC for the 1,2-hydroxyamine
⇑
Corresponding author.
E-mail addresses: konno@yz.yamagata-u.ac.jp (H. Konno), akaji@mb.kyoto-phu.
ac.jp (K. Akaji).
http://dx.doi.org/10.1016/j.bmcl.2017.04.056
0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.

H. Konno et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 2746–2751
2747
role to interact with the mutant protease. This result strongly sup-
ports those of Bai’s15 and our13 information (Scheme 1).
As depicted in Scheme 1, the PIS derivative (4), designed from a
computer-assisted docking simulation, was synthesized as the tar-
get molecule. Additionally, a structure activity relationship study
of the PIS derivative (4) in terms of functionalities to interact with
the corresponding P10 pocket was attempted. Functionalities of the
P10 position (A) of 5 were designed and synthesized to evaluate its
inhibitory activity against the R188I mutant (4). In other words, a
cyclohexyl ring was essential for functionality of the P1 position on
the PIS scaffold in the preliminary studies. Inhibitory activities of
the synthesized PIS derivatives in absence of the cyclohexyl ring
at the P2 position showed extremely weak activity against SARS
3CL R188I mutant protease (IC50 > 1 mM). In addition, cinnamoyl
functionality of the P4 position was effective to maintain the inhi-
bitory activity (Scheme 1).
Fig. 1. Tetrapeptide aldehyde (1) and SK23 (2) for the SARS-CoV 3CL protease
inhibitor.
compounds in-house. In an attempt to give good coverage of the
substrate-recognition pocket of SARS 3CL R188I mutant protease
(PDB code 3AW1), we found that the isoserine backbone had rea-
sonable interactions with the mutant protease to give an isoserine
derivative (3), which can be considered to replace the amine group
The designed (2R,3S)-PIS derivative (4) was synthesized as
shown in Scheme 2. At three step sequence for the protection of
active functionalities with amine, carboxylic acid and hydroxy
groups of (2R,3S)-PIS, gave the protected PIS derivative (6) in 64%
yield over 3 steps. After the saponification of the methyl ester of
6, coupling of the resultant carboxylic acid with cyclohexylamine
by DMT-MM16/DIPEA gave the PIS amide (7) in 54% yield over 2
steps. Deprotection of the Cbz group of 7 by hydrogenolysis and
coupling between the resultant amino group and cinnamic acid
with DMT-MM/DIPEA successfully proceeded to afford the cin-
namoyl derivative (8) in satisfactory yield. TFA-assisted deprotec-
tion of the MOM group gave the alcohol (9) in 76% yield. Finally,
treatment of the alcohol (9) with BzCl/NaI/DIPEA in CH2Cl2 gave
the target compound (4) in 80% yield without any problems. On
the other hand, the P10 moiety of the hydroxy group of 9 was intro-
duced by coupling with the five cinnamic acid derivatives prepared
by us in the previous literature13 to afford 10–14 in lower yields.
Thus, a variety of the coupling reagents was employed between
the PIS derivative (9) with a large steric hindrance and cinnamic
acid, it was difficult to improve the chemical yields of the corre-
sponding ester (10). As a result, we selected DMT-MM/DIPEA or
HATU17,18/HOAt/DIPEA in CH2Cl2 as the coupling conditions to give
the desired materials (10)-(14). Despite the poor chemical yields, it
was available for the evaluation of inhibitory activities against
SARS-CoV R188I mutant protease. We recognized the chemical
structures for synthetic compounds clearly by 1H and 13C NMRs,
IR and MS spectra (Scheme 2).
at the a-position and a hydroxy group at the b-position of SK23 (2).
Using the docking simulation of the (R)-isomer (3) with SARS 3CL
protease, there was a hydrophobic space on the S2 pocket of the
R188I mutant protease with the (R)-isomer (3). In the comparison
with several hydrophobic functionalities, alkane, cycloalkane and
aromatic rings, the phenyl group was fitted on the S2 pocket of
the R188I mutant protease to obtain a (2R,3S)-PIS derivative (4).
The docking simulations of the other three stereoisomers of
(2R,3S)-4 hardly interacted with the R188I mutant protease and
therefore the (2R,3S)-isomer (4) as a candidate for this research
was selected. The (2R,3S)-PIS derivative (4) has the following char-
acteristics: (a) The substrates by nature have involved small amino
acids (Ser, Ala or Gly) at the P10 position of SARS 3CL protease.
However, it may be preferable to adopt the phenyl group, which
was optimized as the serine-type inhibitor SK23 (2).13 (b) As with
SK23 (2), the cyclohexyl ring may have the essential functionality
of the P1 position, as well as stabilizing interactions of other posi-
tions. (c) The substrates at the P2 position are Leu, Val, Phe and Met
with the hydrophobic side chains and therefore the S2 pocket is
rather hydrophobic in nature. Consequently, the phenyl ring makes
good contact with target regions. This is interesting in the results of
docking simulations. The S2 pocket of the R188I mutant protease
barely allowed the phenyl functionality of the serine-type inhibi-
tor. (d) Although the S4 pocket prefers hydrophobic side chains
(Ala, Val or Pro), aromatic derivatives on the synthetic substrates
are acceptable. Especially, cinnamoyl residues play an important
Scheme 1. Molecular design from serine to PIS template.

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H. Konno et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 2746–2751
Scheme 2. Synthesis of the designed PIS compound (4) and its derivatives (10)–(14).
To evaluate the importance of the carbonyl group at the P10
position, the benzyl-type derivative (16) was prepared. Bn-protec-
tion of the hydroxy group of Cbz-PIS-OMe gave Cbz-PIS(Bn)-OMe
in 52% yield. The saponification of methyl ester followed by the
introduction of cyclohexylamine by DMT/MM was subjected to
afford 15 in moderate yield. After deprotection of the Cbz group
by hydrogenolysis, coupling with cinnamic acid was carried out
to give the designed benzyl-type derivative (16) in 47% yield
(Scheme 3).
As depicted in Scheme 4, the PIS phenylpropanoate (18) was
prepared from 7 using the synthetic protocols as mentioned above.
To investigate the importance of the flexibility of the cinnamoyl
group at the P10 position, phenylpropanoate was introduced.
Deprotection of the MOM group of 7 with TFA and coupling of
the resultant hydroxyl group with cinnamic acid gave 17 in 9%
Scheme 4. Synthesis of the PIS derivative (18).
yield. Removal of the Cbz group and coupling of cinnamic acid
were carried out to give the PIS phenylpropanoate (18). In these
cases, coupling reactions between the PIS derivatives with a large
steric hindrance and cinnamic acid prove difficult to give the
desired compounds in high yields (Scheme 4).
The inhibitory activity against SARS 3CL R188I mutant protease
was determined by the previous procedure using a synthetic
decapeptide with the S01 cleavage sequence as a substrate. Syn-
thetic PIS derivatives with modification at the P10 position were
evaluated as IC50 values shown in Table 1. The PIS derivative (4;
SK80) designed by docking simulations exhibited an IC50 value of
43
lM (entry 1). The serine type inhibitor SK23 (2), which has
same functionalities, showed an IC50 value of 30
l
M and therefore
we expected that the active site of the 3CL R188I mutant protease
fitted with these ligands via similar binding modes. To investigate
Scheme 3. Synthesis of the benzyl-type derivative (16).

H. Konno et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 2746–2751
2749
Table 1
Inhibitory activities of phenylisoserine derivatives.
c
Entry
1
Compound
A
ClogPa
6.12
tPSAb
84.5
IC50
4
43
SK80
2
3
16
11
6.25
5.99
67.4
NI
104.7
>500
4
5
9
14
H
4.17
5.95
78.4
112.2
>500
250
6
7
8
13
6.66
6.65
6.20
103.0
84.5
67.4
125
85
10
17d
75
9
18
12
6.69
6.57
84.5
93.7
65
65
10
a
b
c
ClogP was calculated by ChemBio3D Ultra 12.0 (PerkinElmer).
tPSA was calculated by ChemBio3D Ultra 12.0 (PerkinElmer).
lM.
d
The amino group of 17 was protected by the Cbz group.
the importance of the benzoyl carbonyl group at the P10 position,
the benzyl ether (16) was evaluated. As a result, 16 showed no
inhibitory activity (entry 2). In addition, the PIS derivative with a
phenolic hydroxyl group (11) and the synthetic intermediate with
a hydroxyl group (9) also showed lower inhibitory activities
The binding mode of the SK80 (4) to SARS 3CL protease pre-
dicted by docking simulation using GOLD software is exhibited in
Fig. 2. Based on these results, we decided to synthesize and evalu-
ate inhibitory activity against SARS 3CL mutant protease. It appears
to be a reasonable binding mode by the surface mode of the pro-
tease with SK80 (4) shown in Fig. 2A. The functionalities at the
P10, P1, P2 and P4 sites of SK80 (4) were located on the S10, S1,
S2 and S4 pockets with hydrophobic spaces, respectively. As
depicted in Fig. 2B, two amide groups of SK80 (4) were involved
in hydrogen bonding interactions with His164 and Gln189. These
interactions of SK80 (4) with the R188I mutant protease were
found to be identical to those of SK23 (2) with the protease. This
suggests that carbonyl of the benzoyl group of the inhibitor (4)
interacts with the active site of Cys145 and His41 of SARS 3CL pro-
tease. Interestingly, the distances of hydrogen and oxygen between
the two amide groups of SK80 (4) as the docking simulation exhib-
ited 2.1 and 3.1 Å, respectively. These values are reasonable to esti-
mate the intramolecular hydrogen bonding networks. Therefore,
the fixed eight-membered cyclic structure in the scaffold motif of
SK80 (4) is proposed to locate the active site of SARS 3CL mutant
protease by induced fit (Fig. 2C). In comparison with the stable
conformation of SK80 (4) in absence of the SARS 3CL protease, a
molecular mechanics calculation for conformational analysis by
SPARTAN’14 was attempted (Fig. 2D). The distances of intramolec-
ular hydrogen bonds between the corresponding two amide
groups of 4 showed 2.0 and 5.3 Å, respectively. This result suggests
that the conformation of 4 by the intramolecular hydrogen bond of
two amide groups is partly fixed to increase the inhibitory activi-
ties against the SARS 3CL mutant protease. We conclude that the
two amide groups of SK80 (4) interact to form inter- and intra-
(IC50 > 500 lM) (entries 3 and 4). These results were expected from
the docking simulations and structure activity relationship study
of serine type inhibitors in our previous report. Next, the PIS
derivatives with modified cinnamoyl groups were evaluated. The
IC50 values of 14 and 13 were 250 and 125 lM, respectively
(entries 5 and 6). The inhibitors containing 3,5- and/or 4-methoxy
groups on a phenyl ring exhibited moderate activities. In contrast,
cinnamate derivatives 10, 17 and the phenylpropanoate derivative
18 increased the inhibitory activities to show the IC50 values of 65–
85 lM (entries 7, 8, 9). The CH2O of Cbz group of 17 and the double
bond of cinnamoyl group of 10 played a similar role and conse-
quently both the phenyl rings interact with the S4 pocket to show
approximately same activities. The inhibitor containing a 3-meth-
oxy group on a phenyl ring (12) had 65 lM as an IC50 value (entry
10). These results suggested that a planar aromatic ring and its
hydrophobic functionality were essential factors to produce a rea-
sonable 3CL protease inhibitor. Although the methoxy groups of
the phenyl rings had increased infrequently inhibitory activities
against SARS 3CL protease in our previous studies, there is hardly
critical influence. As with SK23 (2), the benzoyl group at P10 posi-
tion was optimized to give the inhibitor 4. To show reasonable IC50
values against SARS 3CL protease, it is important to satisfy both
conditions with around 6 for ClogP values and 65–90 for tPSA. A
similar tendency can also be seen in the structure activity relation-
ship study of serine-type inhibitors (Table 1).

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H. Konno et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 2746–2751
Fig. 2. Docking simulation of selected inhibitors bound to SARS 3CL protease (PDB code 3AW1) using GOLD from CCDC. Molecular graphic image shown using PyMOL from
Schrödinger; oxygen (red) and nitrogen (blue) of inhibitors; Cys145 (red) of SARS 3CL protease. (A) Surface mode with 4, (B) model of interaction with 4, (C) Conformational
analysis of 4. (D) Conformational analysis of 4 by SPARTAN’14.
molecular hydrogen bondings and the binding of the SARS 3CL
mutant protease and SK80 (4) using induced fit are shown (Fig. 2).
Since any protease inhibitor needs to have an orthogonal rela-
tionship between anti-virus activity and microbial activities or
cytotoxicity, antibacterial and antifungal activities and cytotoxicity
for SK80 (4) were investigated. The antifungal activities of SK80 (4)
against Saccharomyces cerevisiae strain X2180-1A and Candida vis-
wanathii NBRC10321 were evaluated by our assay protocols.19 In
addition, we tested the bacterial activities of SK80 (4) against
E. coli NBRC3972 and Pseudomonas putida NBRC14164 for gram-
negative and Micrococcus luteus NBRC13867, Bacillus subtilis
NBRC111470 and Staphylococcus epidermidis NBRC12993 for
gram-positive. As a result, there were no inhibitory activities for
any screened microbials (MICs > 800 mg/mL). The cytotoxicity of
SK80 (4) was performed against Hela cells by a standard MTT assay
Acknowledgements
This work was supported in part by an Adaptable and Seamless
Technology transfer Program (A-STEP) through target driven R&D,
Japan Science and Technology Agency.
A. Supplementary data
Supplementary data (additional experimental procedures, and
analytical data for synthetic compounds. This material is available
free of charge.) associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.bmcl.2017.04.056.
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