Design, synthesis, and evaluation of carboxyl-modified oseltamivir derivatives with improved lipophilicity as neuraminidase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2018.09.014

In this study, a series of carboxyl-modified oseltamivir analogs with improved lipophilicity were designed and synthesized, and their inhibitory activities against neuraminidase from influenza A virus H5N1 subtype were evaluated. The results demonstrated that compound 5m exhibited potent inhibitory activity (IC₅₀ = 1.30 ± 0.23 μM), and it targeted the recently discovered 430-cavity. Compound 5m (Log D = ?0.12) is more lipophilic than oseltamivir carboxylate (Log D = ?1.69) at pH 7.4, which is potentially propitious to improved membrane permeability and oral drug absorption. Meanwhile, 5m showed high stability in human liver microsomes. The findings of this study can be valuable in identifying neuraminidase inhibitors with optimal lipophilicity and in the exploration of 430-cavity.

Full text of DOI:10.1016/j.bmcl.2018.09.014

Accepted Manuscript
Design, synthesis, and evaluation of carboxyl-modified Oseltamivir derivatives
with improved lipophilicity as neuraminidase inhibitors
Boyu Wang, Kuanglei Wang, Peipei Meng, Yaping Hu, Fei Yang, Kemin Liu,
Zaiqiang Lei, Binfeng Chen, Yongshou Tian
PII:
S0960-894X(18)30745-5
https://doi.org/10.1016/j.bmcl.2018.09.014
BMCL 26035
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
7 May 2018
16 July 2018
11 September 2018
Please cite this article as: Wang, B., Wang, K., Meng, P., Hu, Y., Yang, F., Liu, K., Lei, Z., Chen, B., Tian, Y.,
Design, synthesis, and evaluation of carboxyl-modified Oseltamivir derivatives with improved lipophilicity as
neuraminidase inhibitors, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.
2018.09.014
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Design, synthesis, and evaluation of carboxyl-modified
Oseltamivir derivatives with improved lipophilicity as
neuraminidase inhibitors
Boyu Wanga·Kuanglei Wangb, c·Peipei Menga·Yaping Hua·Fei Yanga·Kemin
Liua·Zaiqiang Leia· Binfeng Chena·Yongshou Tiana*
a
Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of Education,
Shenyang Pharmaceutical University, Benxi, Liaoning 117004, China
b Wuyi University, Jiangmen, Guangdong 529020, China
c International Healthcare Innovation Institute (Jiangmen), Jiangmen, Guangdong, 529080, China
✉Yong-Shou Tian*
102030207@syphu.deu.cn
Bo-yu Wang
375156282@qq.com
Kuang-lei Wang
wangkuangl@163.com
Pei-pei Meng
mpp18741422207@163.com
Ya-ping Hu
444209585@qq.com
Fei Yang
825233646@qq.com
Ke-min Liu
1573738278@qq.com
Zai-qiang Lei
lei116051220@qq.com
Bin-feng Chen
757220347@qq.com
1

Abstract In this study, a series of carboxyl-modified oseltamivir analogs with
improved lipophilicity were designed and synthesized, and their inhibitory activities
against neuraminidase from influenza A virus H5N1 subtype were evaluated. The
results demonstrated that compound 5m exhibited potent inhibitory activity (IC50
=
1.30 ± 0.23 μM), and it targeted the recently discovered 430-cavity. Compound 5m
(Log D = -0.12) is more lipophilic than oseltamivir carboxylate (Log D = -1.69) at pH
7.4, which is potentially propitious to improved membrane permeability and oral drug
absorption. Meanwhile, 5m showed high stability in human liver microsomes. The
findings of this study can be valuable in identifying neuraminidase inhibitors with
optimal lipophilicity and in the exploration of 430-cavity.
Keywords Influenza virus · Neuraminidase inhibitors · Oseltamivir derivatives
1. Neuraminidase and inhibitors
Influenza is a highly contagious disease, and influenza A and B viruses cause
seasonal epidemics. Novel influenza strains that emerge periodically could cause an
influenza pandemic. In the past centuries, influenza epidemics have caused numerous
human deaths.1-4 Vaccines and drugs are available for the prevention and treatment of
influenza, respectively. Vaccines are effective only for influenza caused by a matched
virus. In cases where vaccines are ineffective, anti-influenza drugs are administered.
There are two types of anti-influenza drugs, namely Matrix-2 (M2) proton channel
inhibitors and neuraminidase (NA) inhibitors. M2 proton channel inhibitors5-6 are no
2

longer recommended owing to severe side effects and development of drug resistance.
7
NA inhibitors (NAIs) are the mainstay of treatment for influenza.8-9 NA, a viral
surface glycoprotein, can facilitate the release of nascent viruses from host cells by
cleaving the glycosidic bond formed between sialic acid and galactose.10 By inhibiting
NA, NAIs prevent the influenza virus from infecting normal cells, thus preventing
their propagation and achieving success in the treatment of influenza.
Fig. 1 Interaction of S1-S5 subsites of neuraminidase active site with oseltamivir carboxylate
Fig. 2 Active ingredients of four neuraminidase inhibitors
The active site of NA is empirically divided into five subsites (S1-S5) (Fig. 1).
S1 containing three basic amino acids (Arg118, Arg292, and Arg371) and S2
comprising three acidic amino acids (Glu119, Asp151, and Glu227) could form ionic
bonds with the acidic and basic fragments of ligands, respectively, which facilitate
potent inhibition. Therefore, the clear majority of potent NAIs, including the active
3

ingredients of four drugs (Fig. 2), carried both acidic and basic moieties. Compounds
with a zwitterionic structure coupled with low molecular weight generally have poor
lipophilicity, which results in poor bioavailability of drugs. Thus, zanamivir hydrate
(1, Fig. 2)11 and laninamivir octanoate (5, Fig. 2) were approved for inhalation and
peramivir hydrate (4, Fig. 2) was used for injection. Oseltamivir (OS) (2, Fig. 2)
phosphate, a prodrug of oseltamivir carboxylate (OC) (3, Fig. 2), is the only oral
agent with suitable lipophilicity and good bioavailability.12
Recently, 371- and 430-cavity located near S1 were found (Fig. 3), suggesting
new opportunities for NAI design.13-15 The 430-cavity is
a
relatively
positively-charged pocket consisting of R430-T439. The 371-cavity comprising R371,
W403, I427, P431, and K432 is a hydrophobic cavity. Several NAIs interacting with
371- or 430-cavity have been reported. Hong et al. grafted an indole ring fragment on
the carboxyl group of oseltamivir to obtain the highly active compound A (Fig. 4)
(A/WSN/33 H1N1 IC50= 6.4 nM, OC: IC50= 1.78 nM), and the elongated groups at
the C-1-position were projected toward the 371-cavity region. In addition, they
observed additional interactions.16 Recently, Ju et al. modified the carboxyl group of
oseltamivir to obtain compound B (Fig. 4), which exhibits good inhibitory effects
with IC50 value of 0.088 μM (A/chicken/China/1220/2012 H5N1 OC: IC50= 0.023
μM).17 Feng et al. modified the carboxyl group of zanamivir and obtained compound
C (A /Indonesia/5/2005 H5N1 IC50= 0.001 μM, OC: IC50= 0.012 μM) (Fig. 4), and
the elongated groups at the C-1-position were projected toward the 430-loop region.
Moreover, they mentioned that aryl group facilitates interaction with 430-cavity.18
4

Currently, only a few compounds acting on the new active cavities near the S1
region have been reported, and the activity of compound A is slightly weaker than
that of the control drug. Compound C showed potent activity. Owing to its high
polarity, it is highly soluble in water, leading to poor drug absorption. Because
430-cavity is relatively positively charged, it can interact with phenyl or hydrophobic
substituents. The 371-cavity can interact with hydrophobic substituents. Therefore,
the addition of lipophilic substituents with new active cavities to the listed NAIs can
lead to additional interactions and increase the inhibitory effects on the virus. It can
also improve the lipophilicity of compounds as well as the absorption of drugs.
Therefore, it is necessary to develop potent NAIs acting on the new active sites near
the S1 region.
Fig. 3 Structure of compound OC and the active cavities in H5N1 (PDB code: 2HU4)
5

Fig. 4 Structure of compounds A, B, and C
2. Synthesis route of target compounds
Intrigued by the newly discovered cavities near S1, two types of C-1-modified
OC analogs with improved lipophilicity were designed (Fig. 5). One was a lipophilic
substituent with aromatic and/or alkyl groups to produce additional hydrophobic and
π-π interactions. The other type contained a carboxyl group to retain electrostatic
interactions with Arg118, Arg292, and Arg371.
Fig. 5 Modification of oseltamivir at the carboxylic moiety by adding amido groups
Two kinds of oseltamivir derivatives were synthesized via the process outlined in
Scheme 1. The amino group of oseltamivir was protected by t-Boc to yield compound
1. Hydrolysis of compound 1 with NaOH yielded compound 2. Compound 2 was
6

reacted with different amines to afford compounds 3a-n. The t-Boc group of
compounds 3a-n was then hydrolyzed in the presence of trifluoroacetic acid (TFA) to
yield compounds 4a-n. The hydrolysis of compounds 4g-n with NaOH yielded the
target compounds 5g-n. The complete data are provided in supplemental material.
Scheme 1. Reagents and conditions: a (Boc)2O, Et3N, rt; b NaOH, CH3OH:H2O (5:1), rt; c HATU,
DIPEA, CH2Cl2, R-NH2, rt; d TFA, rt; e NaOH, CH3OH:H2O (5:1), rt.
3. Biological activity of the target compounds
The compounds were tested for their abilities to inhibit NA from H5N1 subtype
7

using OC as a positive control (see Supplementary Data for details). First, we tested
the inhibition rates of compounds 4a-h and 5g-n at concentrations of 1 and 10 μM.
Then, the IC50 values of compounds 5g-k and 5m-n with good inhibition rates were
assayed. The results are presented in Tables 1 and 2, respectively.
As shown in Table 1, the inhibitory effects of compounds 4a-d were less potent
than that of OC. This may be due that compounds 4a-d lost the electrostatic
interactions with S1 in the absence of carboxyl fragment. Compounds 4e-h exhibited
better inhibitory activities than compounds 4a-d. The additional ester moieties of
compounds 4e-h acting as hydrogen bond acceptors might form additional H-bond
interactions with the three basic residues (Arg118, Arg292, and Arg371), resulting in
improved inhibitory activities. Compounds 5g-h, obtained by changing the ester
fragments of compounds 4g-h to the corresponding carboxyl fragments, showed
general improvement in inhibitory activities. This can be attributed to the ability of
carboxyl fragment to form electrostatic interactions with basic amino acids. The
improvement in inhibitory activities could also be attributed to the enhanced capacity
to generate hydrogen bonds both as an acceptor and a donor. Meanwhile, substituents
other than carboxyl fragments also played a vital role in the inhibitory activities, as
shown by compounds 5g-n. Compounds 5g and 5l, containing one benzene ring,
showed dissatisfactory inhibitory activities, and compound 5g exhibited the most
powerful activity with inhibition rates of 21.1 and 80.4% at concentrations of 1 and 10
μM, respectively. The biological activities of compounds 5h-k and 5m-n improved to
some extent owing to the absence of phenyl groups, with the inhibition rates at 10 μM
8

exceeding 80.4%. It is worth noting that the activities of 4f and 4g containing phenyl
groups were significantly higher than those of 4e and 4h, which is contradictory to the
results obtained from the activity data of 5g-n. We considered that the carboxyl group,
on the one hand, formed strong ionic and hydrogen bond interactions with the basic
amino acids Arg292, Arg371, and Arg118 in the S1 region. However, the anchoring of
carboxyl group to S1 region limits the binding of rigid groups such as phenyl group to
NA, resulting in unsatisfactory effects for compounds 5g and 5l containing phenyl
group. The IC50 values of compounds 5m and 5n, which showed the most potent
activities among compounds 4a-h and 5g-n, were 1.30 and 4.18 μM, respectively. The
t-butyl fragment in compound 5m favored potent inhibition, because t-butyl formed
new interactions with NA. Fragments larger or smaller than the optimum t-butyl
fragment led to unfavorable biological activities. Although compound 5m exhibited
weaker inhibitory activity than OC, its lipophilicity was likely to be higher, which is
propitious to improved membrane permeability, oral bioavailability, and even
therapeutic effect.
Coincidentally, compound B (against A/chicken/China/1220/2012 H5N1, IC50
=0.088 μM, OC: IC50= 0.023 μM) found by Zhu et al.18 has the same structure as a
compound in our earlier patent application,19 and this compound was identified as
compound 5n in this paper (against A/Anhui/2005 H5N1, IC50 =4.18±0.33 μM, OC:
IC50= 0.21±0.02 μM). However, in our study, a new compound 5m (against
A/Anhui/2005 H5N1, IC50 =1.30±0.23 μM) showed stronger inhibitory activity than
5n. It can be suggested that compound 5m has better activity than compound B found
9

by Zhu et al. To some extent, the discovery of compound 5m contributed to the
discovery of carboxyl-modified potent NAIs.
Table 1
Structures and in vitro inhibition rates[a] of compounds against neuraminidase from H5N1 subtype
Compound
R
@ 1 μM
@ 10 μM
4a
N.D.[b]
21.4%
4b
4c
4d
13.5%
24.7%
5.0%
19.3%
29.6%
55.3%
4e
23.6%
50.5%
4f
4g
4h
26.5%
24.9%
13.1%
56.7%
63.2%
41.7%
5g
21.1%
80.4%
5h
5i
38.9%
27.8%
N.D.
84.4%
81.1%
85.5%
85.7%
8.3%
5j
5k
5l
N.D.
N.D.
5m
58.3%
91.0%
10

5n
57.6%
66.7%
91.4%
91.0%
OC
[a]: Inhibition rates of compounds against NA from A/Anhui/2005 H5N1 subtype at this concentration.
[b]: Not Detected.
Table 2
IC50 of compounds against neuraminidase from H5N1 subtype
Compound
IC50 (μM)
21.89±1.1
24.44±1.0
46.44±0.7
4.18±0.33
Compound
IC50 (μM)
6.55±2.8
5g
5i
5h
5j
30.74±2.1
1.30±0.23
0.21±0.02
5k
5n
5m
OC
4. Lipophilicity of compound 5m
Compounds with appropriate lipophilicity can readily traverse a membrane and
then distribute to their sites of action, which is conducive to improving bioavailability
and therapeutic effects. Most of the potent NAIs have poor bioavailability owing to
poor lipophilicity and membrane permeability. Based on the above consideration, we
adopted octanol/phosphate-buffered saline (PBS) distribution coefficient (Log D) to
explore the lipophilicity of compound 5m (see Supplementary Data for details). As
shown in Table 3, compound 5m showed the highest lipophilicity at approximately
pH 6.8. Compound 5m (Log D = -0.1159) was approximately fifteen times more
lipophilic than OC (Log D = -1.69) at pH 7.4, which led to potentially improved
membrane permeability, oral bioavailability, and therapeutic effect.
11

Table 3
Distribution coefficients of compound 5m between octanol and PBS buffer
pH
5.8
D
Log D
-2.5
0.0028
0.8998
0.6812
0.7658
0.6183
6.8
-0.0458
-0.1667
-0.1159
-0.2087
-1.69
7
7.4
7.6
7.4 (OC)
5. Molecular docking
To understand the interactions between compound 5m and NA further, molecular
docking was performed using MAESTRO. Images depicting the proposed binding
modes were generated by Discovery Studio 2016 Client. The resolved NA (PDB code:
2HU4) from H5N1 subtype was downloaded from RCSB protein data bank (PDB).
As shown in Figure 6, the binding model of compound 5m and NA was similar
to that of OC and NA. Compared with the binding model of OC and NA, the OC part
of 5m retained C3-(pentan-3-yloxy) to form hydrophobic interactions with Trp178
and IIe222, and retained C5-NH2 to form H-bond interactions with Asp151 and
Glu119. Although the hydrogen bond interactions between C4-acetamido of OC and
Arg152 did not occur in the new compound 5m, acetamido of 5m formed new
hydrogen bond interactions with Glu227. In 5m, the same structure as OC maintains
the original interactions between OC and NA. However, the two binding models do
12

not coincide completely; the carboxyl group at C1 site of OC have three ionic bonds
and two hydrogen bonds with Arg292, Arg371, and Arg118. Compared with OC, the
carboxyl group of 5m forms only one ionic bond interaction and one hydrogen bond
interaction with Arg118 and Arg371, because the carboxyl group of 5m is pushed to
the deeper region of NA. Although the carboxyl group of 5m forms a new π-cation
interaction with Tyr347, the loss of several interactions was observed in 5m when
compared with the interactions between OC and NA. The t-butyl group of 5m was
projected toward the newly discovered 430-cavity and formed new hydrophobic
interactions with Val149 and Pro431. The new hydrophobic interactions make up for
the lost interactions between the carboxyl group of OC and NA to some extent.
Therefore, 5m shows good activity, and may have beneficial effects in vivo owing to
the introduction of lipophilic group.
13

Fig. 6 A and B: Docking modes of compounds OC and 5m with neuraminidase (PDB code:
2HU4), respectively. C: Compound 5m (blue) and OC (yellow) are superposed within NA (PDB
code: 2HU4). This image was obtained using Discovery Studio 2016 Client.
6. Metabolic stability assay in vitro
Table 4
Human liver microsomal stability of compound 5m
Compounds
5m
T1/2 (min)
>145
14.5
Remaining (T = 60 min)
84.1%
5.2%
2.4%
0.1%
Testosterone
Diclofenac
Propafenone
11.6
6.1
Liver weight: 20 g/kg for humans.
Metabolic stability of drugs can affect pharmacodynamic properties such as oral
efficacy and duration of action. Generally, the degree to which a compound is
metabolized within the liver is expressed as the metabolic half-time (T1/2) in vitro.
Compound 5m with the most potent inhibitory activity was selected to perform
in vitro metabolic stability study in human liver microsomes. The control compounds
14

were testosterone, diclofenac, and propafenone.20-23 The data are shown in Table 4
(see Supplementary Data for details). After incubation with human liver microsomes
for 1 h, 5m (84.1%) was detected. The T1/2 was more than 145 min, which indicated
that 5m showed good stability in human liver microsomes.
In summary, we designed 16 oseltamivir analogs by modification at the C-1
position to make use of the newly discovered cavities. Compound 5m exhibited a
slightly weaker inhibitory activity than OC. Molecular modeling showed that the
introduced t-butyl moiety of 5m interacted with NA and was projected toward the
430-loop region. Compound 5m showed high metabolic stability in human liver
microsomes. Notably, the lipophilicity of compound 5m was improved, which
favored membrane permeability and oral drug absorption. To a certain extent, we
achieved beneficial anti-NA activity and lipophilicity, which will be of value in the
rational design of new types of NAIs with potent inhibitory activity under the
precondition of optimal Log D.
Acknowledgements This work was financially supported by Foundation of Shenyang
Science and Technology Bureau (NO. F13-196-9-00).
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing interests.
Abbreviation list
NA, neuraminidase; OS, oseltamivir; OC, oseltamivir carboxylate; TFA, trifluor-
15

oacetic acid; HATU, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyr-
idinium 3-oxid hexafluorophosphate; DIPEA, N,N-diisopropylethylamine; PDB,
Protein Data Bank
References
[1] Dawood FS, Jain S, Finelli L, et al. N. Engl. J. Med. 2009; 360: 2616-2650.
[2] Lewis DB. Rev. Med. 2006; 57: 139.
[3] Taubenberger JK, Morens DM. Emerg. Inf. Dis. 2006; 12: 15-22.
[4] Yu H, Cowling BJ, Feng L, et al. Lancet. 2013; 382: 138-145.
[5] Davie WL, Grunert RR, Haff RF, et al. Science. 1964; 144: 862.
[6] Schnell JR, Chou JJ. Nature. 2008; 451: 591.
[7] Pielak RM, Schnell JR, Chou JJ. Proc. Natl. Acad. Sci. U. S. A. 2009; 106:
7379-7384.
[8] De Clercq E. Nat. Rev. Drug Discovery. 2006; 5: 1015−1025.
[9] Schmidt AC. Drugs. 2004; 64: 2031−2046.
[10] Gong J, Xu W, Zhang J. Curr. Med. Chem. 2007; 14: 113.
[11] Dunn CJ, Goa KL. Drugs. 1999; 58: 761-784.
[12] Lew W, Chen X, Kim CU. Curr. Med. Chem. 2000; 7: 663-672.
[13] Rommie E, David DL. J. AM. CHEM. SOC. 2007; 129: 7764-7765.
[14] Rommie E, Cheng XL. J. AM. CHEM. SOC. 2009; 131: 4702-4709.
[15] Cheng LS, Rommie E, Xu D, et al. J. Med. Chem. 2008; 51: 3878-3894.
[16] Hong BT, Chen CL, Fang JM, et al. Bioorganic & Medicinal Chemistry. 2014; 22:
6647-6654.
[17] Ju H, Zhang J, Sun Z, et al. European Journal of Medicinal Chemistry. 2018; 146:
220-231.
[18] Feng E, Shin W, Zhu X, et al. Journal of Medicinal Chemistry. 2013; 56:
671-684.
[19] Tian YS, Wang KL, Wang By, et al. 2018; CN 108101804A.
[20] Shi D, Yang J, Yang D, et al. J. Pharmacol. Exp. Ther. 2006; 319: 1477-1484.
16

[21] Sweeny DJ, Lynch G, Bidgood AM, et.al. Drug Metab. Dispos. 2000; 28:
37-741.
[22] Sławiński J, Szafrański K, Pogorzelska A, et al. Eur. J. Med. Chem. 2017; 132:
236-248.
[23] Zhao Y, Bai L, Liu L, et al. J. Med. Chem. 2017; 60: 3887-3901.
17

18

Highlights
• A series of carboxyl-modified oseltamivir analogs with improved lipophilicity were
designed and synthesized.
• Inhibitory activities of compounds against neuraminidase from influenza A virus
H5N1 subtype were evaluated.
•The molecular docking studies of 5m was performed.
• Compound 5m targeted the recently discovered 430-cavity.
• Lipophilicity and metabolic stability of compound 5m were evaluated.
19