Boronate, trifluoroborate, sulfone, sulfinate and sulfonate congeners of oseltamivir carboxylic acid: Synthesis and anti-influenza activity

Article abstract of DOI:10.1016/j.ejmech.2018.12.027

Tamiflu readily undergoes endogenous hydrolysis to give oseltamivir carboxylic acid (OC) as the active anti-influenza agent to inhibit the viral neuraminidase (NA). GOC is derived from OC by replacing the 5-amino group with a guanidino group. In this study, OC and GOC congeners with the carboxylic acid bioisosteres of boronic acid, trifluoroborate, sulfone, sulfinic acid, sulfonic acid and sulfonate ester were first synthesized, starting with conversion of OC to a Barton ester, followed by halodecarboxylation to give the iodocyclohexene, which served as a pivotal intermediate for palladium-catalyzed coupling reactions with appropriate diboron and thiol reagents. The enzymatic and cell-based assays indicated that the GOC congeners consistently displayed better NA inhibition and anti-influenza activity than the corresponding OC congeners. The GOC sulfonic acid congener (7a) was the most potent anti-influenza agent, showing EC₅₀ = 2.2 nM against the wild-type H1N1 virus, presumably because the sulfonic acid 7a was more lipophilic than GOC and exerted stronger interactions on the three arginine residues (R118, R292 and R371) in the NA active site. Although the trifluoroborates, sulfones and sulfonate esters did not have acidic proton, they still exhibited appreciable NA inhibitory activity, indicating that the polarized B?F and S→O bonds still made sufficient interactions with the tri-arginine motif.

Full text of DOI:10.1016/j.ejmech.2018.12.027

Accepted Manuscript
Boronate, trifluoroborate, sulfone, sulfinate and sulfonate congeners of oseltamivir
carboxylic acid: Synthesis and Anti-influenza activity
Bei-Tao Hong, Yih-Shyun E. Cheng, Ting-Jen Cheng, Jim-Min Fang
PII:
S0223-5234(18)31064-X
DOI:
https://doi.org/10.1016/j.ejmech.2018.12.027
EJMECH 10960
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 July 2018
Revised Date: 25 October 2018
Accepted Date: 12 December 2018
Please cite this article as: B.-T. Hong, Y.-S.E. Cheng, T.-J. Cheng, J.-M. Fang, Boronate, trifluoroborate,
sulfone, sulfinate and sulfonate congeners of oseltamivir carboxylic acid: Synthesis and Anti-
influenza activity, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2018.12.027.
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ACCEPTED MANUSCRIPT
Boronate, Trifluoroborate, Sulfone, Sulfinate and Sulfonate Congeners of Oseltamivir
Carboxylic Acid: Synthesis and Anti-influenza Activity
Bei-Tao Honga, Yih-Shyun E. Chengb, Ting-Jen Chengb, and Jim-Min Fanga,b,*
a Department of Chemistry, National Taiwan University, Taipei 106, Taiwan
b The Genomics Research Center, Academia Sinica, Taipei 115, Taiwan
* Corresponding author. J.-M.F.: Department of Chemistry, National Taiwan University,
Taipei, 106, Taiwan. E-mail address: jmfang@ntu.edu.tw.
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Abstract:
Tamiflu readily undergoes endogenous hydrolysis to give oseltamivir carboxylic acid (OC) as
the active anti-influenza agent to inhibit the viral neuraminidase (NA). GOC is derived from
OC by replacing the 5-amino group with a guanidino group. In this study, OC and GOC
congeners with the carboxylic acid bioisosteres of boronic acid, trifluoroborate, sulfone,
sulfinic acid, sulfonic acid and sulfonate ester were first synthesized, starting with conversion
of OC to a Barton ester, followed by halodecarboxylation to give the iodocyclohexene, which
served as a pivotal intermediate for palladium-catalyzed coupling reactions with appropriate
diboron and thiol reagents. The enzymatic and cell-based assays indicated that the GOC
congeners consistently displayed better NA inhibition and anti-influenza activity than the
corresponding OC congeners. The GOC sulfonic acid congener (7a) was the most potent
anti-influenza agent, showing EC50 = 2.2 nM against the wild-type H1N1 virus, presumably
because the sulfonic acid 7a was more lipophilic than GOC and exerted stronger interactions
on the three arginine residues (R118, R292 and R371) in the NA active site. Although the
trifluoroborates, sulfones and sulfonate esters did not have acidic proton, they still exhibited
appreciable NA inhibitory activity, indicating that the polarized B−F and S→O bonds still
made sufficient interactions with the tri-arginine motif.
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Keywords:
Influenza; Neuraminidase inhibitor; Oseltamivir; Bioisosteres; Sulfonic acid; Boronic acid;
Trifluoroborate; Sulfone; Sulfinic acid.
Research highlights
• First synthesis of boronate and sulfonate related OC and GOC bioisosteres 2a−9b.
• OC derived iodocyclohexene 16a is a key intermediate for syntheses of bioisosteres.
• Acidity and lipophilicity of bioisosteres are important to anti-influenza activity.
• GOC congeners show better NA inhibition and anti-influenza activity than OC congeners.
• GOC sulfonic acid is a potent inhibitor against H1N1 virus (EC50 = 2.2 nM).
Abbreviations:
Boc, tert-butoxycarbonyl; (BPin)2, bis(pinacolato)diboron; CCK, cholecystokinin; cLogD,
calculated distribution coefficient; CPE, cytopathic effect; dba, dibenzylideneacetone; DMAP,
(4-dimethylamino)pyridine; DMF, N,N-dimethylformamide; GABA, γ-aminobutyric acid;
GOC,
guanidino
oseltamivir
carboxylic
acid;
HBTU,
MDCK,
2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate;
Madin−Darby canine kidney; MUNANA, 2'-(4-methylumbelliferyl)-α-D-N-acetylneuraminic
acid; NA, neuraminidase; OC, oseltamivir carboxylic acid; SD, standard deviation; TCID50,
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50% cell culture infectious dose; Tf, trifluoromethanesulfonyl; TFA, trifluoroacetic acid; THF,
tetrahydrofuran; Xantphos, 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene.
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1. Introduction
Influenza is a highly contagious airborne disease, which has become a persistent global
public health issue; effective drugs are needed for treatment of influenza infection. Influenza
neuraminidase (NA), a glycoprotein on the surface of the virus, is responsible for the
enzymatic cleavage of the terminal sialic acid residue from the sialo-receptors on host cells to
facilitate the release of progeny viruses for propagation and infectivity. NA is a good target
for development of anti-influenza drug because NA is located on the virus surface, and its
active site is conserved across various influenza viral strains. Several anti-influenza drugs,
such as zanamivir, [1, 2] oseltamivir, [3, 4] peramivir [5, 6] and laninamivir octanoate, [7, 8]
have been developed as NA targeting agents. Tamiflu (the phosphate salt of oseltamivir) is an
oral prodrug, which is hydrolyzed by endogenous esterase to oseltamivir carboxylic acid (OC,
1a in Fig. 1) as the active NA inhibitor. The cyclohexene core structure of OC is designed to
mimic the oxocarbenium-like intermediate in the NA catalyzed hydrolysis of the sialoside. [9,
10] The carboxyl group of OC exhibits strong electrostatic interactions with the three arginine
residues (R118, R292 and R371) in the S1 site of influenza NA. [1, 11] In addition, OC has a
3-pentoxy substituent at C-3, in lieu of the glycerol side chain in sialoside, to attain
hydrophobic interactions with NA. GOC (1b), [3] the guanidino analog of OC, has a more
basic guanidino group at C-5 to enhance the electrostatic interactions with the acidic residues
(E119, D151 and E227) in the S2 site of NA. However, GOC and its ethyl ester have not been
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developed for therapeutic use due to their poor pharmacokinetic properties.
Fig. 1. Chemical structures of oseltamivir carboxylic acid (1a, OC), guanidino OC (1b, GOC),
and their carboxyl bioisosteres (2a−13b).
Bioisosteres are substituents through rational modifications to mimic the structure of an
active ingredient with similar chemical, physical, electronic, conformational and biological
properties. [12, 13] As an approach to explore new anti-influenza drugs, we have previously
developed the OC and GOC congeners by replacing the carboxylic acid with bioisosteres, [14]
including phosphonic acid (10a/10b), [15−18] phosphonate monoester (11a/11b), [16, 18, 19]
hydroxamate (12a/12b) [20] and acyl sulfonamide (13a/13b) [20]. Compounds 10a
(tamiphosphor) and 10b are more potent NA inhibitors than their carboxyl congeners OC and
GOC, because the phosphonate group exhibits stronger electronic interactions with the
tri-arginine (R118, R292 and R371) motif in NA. [15] Phosphonate monoalkyl esters 11a and
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11b also exhibit high NA inhibitory activities because these compounds still contain a
negatively charged group to render sufficient electrostatic interactions with the three arginine
residues in the NA active site. [18, 19] The hydroxamate (12a/12b) and acyl sulfonamide
(13a/13b) bioisosteres are less potent than OC and GOC, but still showed the IC50 values in
nanomolar range against the NA of wild-type influenza virus (H1N1). [20]
Compared with carboxylic acid (pKa ≈ 4.5), boronic acid is a weaker acid (pKa ≈ 10.0)
whereas sulfinic acid ((pKa ≈ 2.0) and sulfonic acid (pKa ≈ −0.5) are stronger acids. Use of
boronic acid and sulfonic acid as the bioisosteres of carboxylic acid has been demonstrated in
drug development. For example, bortezomib (velcadeTM) is a synthetic boronic acid drug used
as protease inhibitor in cancer treatment. [21] In protein degradation, a specific peptide bond
(−CONH−) of planar structure is attacked by a nucleophilic amino acid (e.g. serine) to form
an intermediate of tetrahedral structure. Boron atom has an empty p orbital to accommodate
nucleophile (e.g. water, alcohol, amine and thiol), thus becoming a negatively charged
tetrahedral structure to mimic the intermediate during enzymatic degradation of peptide bond.
The trifluoroborate salt with three electronegative fluorine atoms also exists in a tetrahedral
structure to act as protease inhibitor. [22] In comparison with boronic acid and boronate ester,
the corresponding trifluoroborate salts are usually easier to purify and preserve. In some cases,
trifluoroborate salts are even more potent than boronic acid in inhibition of serine proteases.
[22]
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In an antagonist of cholecystokinin (CCK) receptor the carboxylic acid is replaced by
sulfonic acid to give a dipeptoid hormone, which is more active and selective to CCK receptor.
[23] As a surrogate of γ-aminobutyric acid (GABA), 3-aminopropylsulfonic acid also
improves the activity to agonize GABAA receptor. [24] Some sulfo-sialic acid analogues are
reported to have good inhibitory activity against influenza and bacterial neuraminidases. [25]
Bren, Miertus and coworkers have applied molecular dynamics simulations in conjunction
with a linear interaction energy method to predict that sulfonate 7a, a putative compound in
their publication, should be a very strong binder to N1 subtype neuraminidase. [26] The
negatively charged sulfonate group of 7a is considered to have great hydrogen bonding
capacity to gain strong electrostatic interactions with the three arginine residues in the S1 site
of NA.
As a continuing study to get a better picture for the relationship between the oseltamivir
carboxylic acid and bioisosteres, we designed and synthesized new OC and GOC congeners
(Fig. 1), including boronates 2a/2b, trifluoroborates 3a/3b, sulfones 4a−5b, sulfinic acids
6a/6b, sulfonic acids 7a/7b as well as sulfonate esters 8a/8b and 9a/9b.
2. Results and discussions
2.1. Chemical synthesis
Scheme 1 shows the synthesis of OC and GOC boronic acids (2a/2b) and trifluoroborates
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ACCEPTED MANUSCRIPT
(3a/3b). The tert-butoxycarbonyl (Boc) protected oseltamivir (14) was prepared by the
previously reported procedure. [16, 19, 27] After saponification, the carboxylic acid product
was reacted with 3-hydroxy-4-methyl-2(3H)-thiazolethione in the presence of HBTU, DMAP
and Et3N to give a Barton ester, which underwent photolysis in the presence of CF3CH2I to
give the decarboxylative halogenation product 16a. [16, 27−30] After testing several
palladium catalysts, Pd(PPh3)4 was chosen for the Pd-catalyzed coupling reaction between the
iodocyclohexene 16a and bis(pinacolato)diboron to furnish the boronate ester 17a. [31]
On the other hand, the Boc protecting group in 16a was removed by trifluoroacetic acid
(TFA),
and
the
amine
intermediate
was
treated
with
1,3-di-Boc-2-(trifluoromethanesulfonyl)guanidine to give the guanidino derivative 16b,
which was subjected to the palladium-catalyzed boronation to afford compound 17b.
Attempts to hydrolyze boronate ester 17a (or 17b) in various conditions (e.g. using 1−3 M
HCl solutions in Et2O or 1,4-dioxane, BBr3 in CH2Cl2, NaIO4−HCl in THF−H2O) failed to
give the desired boronic acid 2a (or 2b). Alternatively, the boronate esters 17a and 17b were
first treated with KHF2 to give the trifluoroborate intermediates 18a and 18b, [22, 32−34]
which were then successfully converted to the corresponding boronic acids 2a and 2b in 1 M
aqueous HCl solution by concomitant removal of the Boc group. Treatment of 2a and 2b with
KHF2 afforded the trifluoroborates 3a and 3b, respectively.
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Scheme 1. Synthesis of boronic acids 2a/2b and trifluoroborates 3a/3b
O
O
O
I
b
e
OR
AcHN
AcHN
NHBoc
NHZ
14
15
R = C2H5
R = H
16a
16b
Z = Boc
Z = (BocNH)C=NBoc
a
c, d
K+
F
B F
O
f
O
O
B
O
F
AcHN
AcHN
NHZ
NHZ
18a 18b
/
17a 17b
/
K+
F
OH
f
g
O
B F
O
B
OH
F
AcHN
AcHN
NHY
3a 3b
NHY
2a 2b
/
/
a
b
series: Y = H, Z = Boc
series: Y = (H2N)C=NH, Z = (BocNH)C=NBoc
Reagents and conditions: (a)
1
M
KOH(aq)
,
MeOH, rt,
2
h; (b)
3-hydroxy-4-methyl-2(3H)-thiazolethione, HBTU, Et3N, DMAP (cat.), CH2Cl2, rt, 3 h; then
CF3CH2I, CH2Cl2, hν, 30 min; (c) CF3COOH, CH2Cl2, rt, 3 h; (d) (BocNH)(TfNH)C=NBoc,
o
NEt3, CH2Cl2, rt, 10 h; (e) (BPin)2, Pd(PPh3)4, KOAc, DMF, 90 C, 4 h; (f) KHF2, H2O,
MeOH, rt, 10 h; (g) 1 M HCl in H2O, rt, 8 h. Yields: 15 (90% from oseltamivir), 16a (54%),
16b (72%), 2a (48% from 16a), 2b (38% from 16b), 3a (72%), 3b (67%). Boc =
tert-butoxycarbonyl, (BPin)2 = bis(pinacolato)diboron, DMAP = (4-dimethylamino)pyridine,
HBTU = 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, rt =
room temperature, Tf = trifluoromethanesulfonyl.
Using iodocyclohexene 16a as the pivotal compound, OC and GOC sulfones (4a/4b and
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5a/5b), sulfinic acids (6a/6b) and sulfonic acids (7a/7b) were synthesized. By the catalysis of
Pd2(dba)3 and xantphos, [35] the iodine atom in 16a was readily substituted by
2-(4-pyridyl)ethylthiolate to give the thioether 19, which was then oxidized by
m-chloroperbenzoic acid (m-CPBA) to give the sulfone product 20 (Scheme 2). Treatment of
sulfone 20 with an alkylating agent CH3I in the presence of a strong base t-BuOK produced
methylsulfone 21. The reaction possibly proceeded with a β-elimination that was promoted by
prior methylation of the pyridine moiety, giving the intermediates of sulfinate ion B and
1-methyl-4-ethenylpyridium ion C. [36] The subsequent transfer of methyl group from the
intermediate C to sulfinate B would yield the observed product 21. On the other hand,
removal of the Boc protecting group from 20 yielded compound 4a. By the procedure similar
to that for conversion of 2a to 2b, guanidination of 4a and 5a afforded 4b and 5b,
respectively.
Scheme 2. Synthesis of sulfones 4a/4b and 5a/5b
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Reagents and conditions: (a) HS(CH2)2Py, Pd2(dba)3, xantphos, i-Pr2NEt, DMF, 90 oC, 4 h; (b)
o
m-ClC6H4CO3H, CH2Cl2, 0 C, 1 h; (c) MeI, t-BuOK, Me2CO, H2O, rt, 10 h; (d) CF3CO2H,
CH2Cl2, rt, 3 h. (e) (BocNH)(TfNH)C=NBoc, NEt3, CH2Cl2, rt, 10 h; Yields: 20 (62% from
16a), 4a (37%), 4b (32%), 5a (67% from 20), 5b (28%). dba = dibenzylideneacetone, DMF =
N,N-dimethylformamide, xantphos = 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene.
The sulfone compound 23a was obtained by the Pd-catalyzed coupling reaction of 16a
with 2-ethylhexyl 3-sulfanylpropanoate, followed by oxidation of the thioether intermediate
22 with m-CPBA (Scheme 3). The β-elimination reaction of 23a occurred on treatment with
t-BuOK to give the sulfinic acid 24a, which was converted to the desired OC sulfinate
bioisostere 6a by removal of the Boc protecting group. Alternatively, oxidation of 24a with
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m-CPBA afforded the sulfonic acid 25a, and the subsequent removal of Boc group gave the
OC sulfonic acid 7a. By the procedure similar to that for 6a and 7a, the Boc protecting groups
of guanidine analogs 23b and 25b were removed to afford GOC sulfinic acid 6b and GOC
sulfonic acid 7b.
Scheme 3. Synthesis of sulfinic acids 6a/6b and sulfonic acids 7a/7b
Reagents and conditions: (a) HS(CH2)2CO2R, Pd2(dba)3, xantphos, i-Pr2NEt, DMF, 90 oC, 4 h;
(b) m-ClC6H4CO3H, CH2Cl2, 0 oC, 1 h; (c) CF3CO2H, CH2Cl2, rt, 3 h; (d)
(BocNH)(TfNH)C=NBoc, NEt3, CH2Cl2, rt, 10 h; (e) t-BuOK, Me2CO, H2O, rt, 10 h. Yields:
23a (71% from 16a), 23b (72%), 6a (61% from 23a), 6b (48% from 23b), 7a (47% from 23a),
7b (31% from 23b).
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The alkylation reactions of sulfonic acid 25a with 1-iodobutane and
1-iodo-3-phenylpropane were carried out by using Ag2O as the promoter to give the sulfonate
esters 26 and 27, respectively (Scheme 4). After removal of the Boc protecting group, the
desired OC sulfonate esters 8a and 9a were obtained. The corresponding GOC sulfonate
esters 8b and 9b were then prepared by guanidination of 8a and 9a, respectively.
Scheme 4. Synthesis of sulfonate esters 8a–9b
Reagents and conditions: (a) Ag2O, MeCN, 80 oC, 3 h; then n-BuI or Ph(CH2)3I, 80 oC, 10 h;
(b) CF3CO2H, CH2Cl2, rt, 3 h; (c) (BocNH)(TfNH)C=NBoc, NEt3, CH2Cl2, rt, 10 h; Yields:
26 (38%), 27 (45%), 28 (52%), 29 (65%), 8a (29%), 8b (60%), 9a (25%), 9b (58%).
2.2. Neuraminidase inhibition and anti-influenza activity
Table 1 shows the NA inhibition (IC50) and anti-influenza activity (EC50) against
A/WSN/33 (H1N1) virus. The NA inhibitory activity was measured by using
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2-(4-methylumbelliferyl)-α-
D
-N-acetyl-neuraminic acid (MUNANA) as the fluorogenic
substrate. The anti-influenza activity was evaluated by the cytopathic effect of Madin-Darby
canine kidney (MDCK) cells under viral infection. In general, GOC (1b) and other b-series
compounds bearing a guanidino group at the C-5 position displayed better NA inhibition and
anti-influenza activity than the corresponding OC (1a) and a-series compounds having a C-5
amino group. This result was attributable to the stronger interactions generated by the more
basic guanidino group with the acidic residues (E119, D151 and E227) in NA active site.
In comparison with OC, the NA inhibitory activity of boronic acid 2a dramatically
decreased by 400 fold with a boronate group replacing the carboxyl group. Boronate 2a was
also 60-fold less effective than OC to protect MDCK cells from the infection of H1N1 viruses.
However, by introduction of a C-5 guanidino substituent, compound 2b regained the NA
inhibition and anti-influenza activities, showing the IC50 and EC50 values of 28 and 580 nM,
respectively. Furthermore, the trifluoroborate 3b, with IC50 = 20 nM and EC50 = 270 nM,
were more potent than boronate 2b. This result indicated that the fluorine atoms having high
electronegativity could render appreciable electrostatic interactions with the tri-arginine motif
in the S1 site of NA. Since trifluoroborate compound tends to be more stable and easier in
isolation than the corresponding boronate compound, it is worthwhile to exploit
trifluoroborate as a general carboxyl bioisostere in drug discovery.
Because boron atom has an empty p orbital that may provide opportunity for covalent
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bond formation. For this concern, we also incubated the boronate and trifluoroborate
compounds (2a, 2b, 3a and 3b) respectively with NA in various lengths of time (0, 0.5, 1, 2, 8,
16, 24, 42 and 48 h) to monitor the possibility of covalent modifications, which would result
in irreversible inhibition and time-dependent shift in IC50 values. The NA inhibitory activities
were measured, and the resulted IC50 values showed no shift with incubation time. Therefore,
the inhibition of NA by compounds 2a, 2b, 3a and 3b may not proceed with an irreversible
process caused by covalent bond formation.
Table 1. Neuraminidase inhibition (IC50) and anti-influenza activity (EC50) against A/WSN/33
(H1N1) virusa
Compound IC50b (nM) EC50c (nM) CC50d (µM) cLogDe pKa
e
1a (OC)
1.8 ± 0.6
1.0 ± 0.2
28 ± 4
14 ± 4
NDf
NDf
NDf
NDf
NDf
NDf
NDf
> 100
−1.84
−1.55
−1.75
−2.48
NAh
4.4
4.6
1b (GOC)
730 ± 200 1700 ± 380
28 ± 2 580 ± 130
3400 ± 600 860 ± 730
10.0
10.0
NAg
NAg
NAg
NAg
2a
2b
3a
3b
4a
4b
20 ± 6
270 ± 80
1500 ± 790
100 ± 60
NAh
330 ± 40
9.8 ± 4.5
−0.93
−2.56
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180 ± 10
2.4 ± 0.2
22 ± 3
1200 ± 250
46 ± 13
48 ± 13
7.8 ± 2.5
14 ± 3
NDf
> 100
> 100
> 100
> 100
> 100
NDf
−1.88
−3.51
−2.20
−1.09
−1.44
−1.15
−0.85
−1.47
1.55
NAg
NAg
2.0
5a
5b
6a
6b
7a
7b
8a
8b
9a
9b
2.2 ± 0.1
1.8 ± 0.3
1.3 ± 0.1
27 ± 5
2.0
−0.5
−0.5
NAg
NAg
NAg
NAg
2.2 ± 0.3
190 ± 100
31 ± 4
3.3 ± 0.7
45 ± 13
2.0 ± 0.1
> 100
NDf
280 ± 30
24 ± 8
> 100
−0.33
a A/WSN/33 is a wild-type human H1N1 virus.
b A fluorescent substrate, 2-(4-methylumbelliferyl)-α-
D
-N-acetylneuraminic acid (MUNANA),
was used to determine the IC50 values that are compound concentrations causing 50%
inhibition of influenza neuraminidase. Data are shown as mean ± SD of three experiments.
The anti-influenza activities against influenza virus were measured as EC50 values that are
the compound concentrations for 50% protection of MDCK cytopathic effects due to the
infection by influenza virus. Data are shown as mean ± SD of three experiments.
c
d The compound was nontoxic to human 293T cells (CC50 > 100 µM).
e
The octanol−PBS distribution coefficient (cLogD) and acidity (pKa) are calculated values
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using MarvinSketch.
f ND: not determined (no cytotoxicity test was performed).
g NA: not available (cannot be calculated).
Based on molecular dynamics simulations, Bren and coworkers have proposed that the
strength of electrostatic interactions provided by carboxylate ion and its bioisosteres might be
correlated to their acidity. [26] The acidity in decreasing order is sulfonic acid (pKa = −0.5),
sulfinic acid (pKa = 2.0), carboxylic acid (pKa ≈ 4.5) and boronic acid (pKa = 10.0). The
sulfonic acid 7a (EC50 = 14 nM) exhibited a higher anti-influenza activity than OC (EC50 = 28
nM), even they showed a similar NA inhibitory activity. Interestingly, compound 7a was
predicted to be more lipophilic than OC by comparing their distribution coefficients (cLogD
in Table 1). Thus, compound 7a might be more permeable to attain better effect in protecting
cells from viral infection. Among the examined compounds, the GOC sulfonic acid
bioisostere 7b was the most potent anti-influenza agent with EC50 = 2.2 nM. The sulfinic
acids 6a and 6b still possessed good NA inhibition and anti-influenza activities. The GOC
sulfone congeners (4b & 5b) as well as sulfonate esters (8b & 9b) also inhibited influenza
viral NA with IC50 in nanomolar range, indicating that the polarized S→O bonds could also
provide sufficient electrostatic interactions with the tri-arginine motif. To our knowledge,
there is no endogenous enzyme in mammal cells accountable for the hydrolysis of sulfonate
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ester to sulfonic acid; thus sulfonate esters (e.g. 8b & 9b) may not act as the prodrugs of
sulfonic acid (7b).
Lipophilicity is an important factor for the physicochemical properties and
pharmacokinetic behavior of drugs. The partition of an ionic compound between octanol and
PBS buffer (pH = 7.4) is represented as the distribution coefficient (LogD). Interestingly,
Table 1 shows that compounds 1b (GOC), 6b and 7b bearing a C5-guanidino group are
predicted to be more lipophilic, with higher cLogD, than their corresponding compounds 1a
(OC), 6a and 7a. This result is in agreement with our previous observation in the phosphonate
congeners 10a/10b and 11a/11b. [18] The increased lipophilic property of 1b, 6b, 7b, 10b and
11b could be attributable to their zwitterionic structures, [37, 38] in which the C-5
guanidinium ion could pair with the carboxylate, sulfinate, sulfonate and phosphonate ions in
either intra- or intermolecular manner. It is noted that Huryn, Ballatore and coworkers have
previously found that phenylpropionic acid (LogD = −0.49) is more lipophilic than the
corresponding sulfinic acid (LogD = −1.30) and sulfonic acid (LogD = −1.45) congeners. [39]
In our case, compounds 1a/1b, 6a/6b and 7a/7b may comprise zwitterionic structures in the
presence of positively charged amino or guanidino substituents, which are absent in
phenylpropionic acid. Since sulfonic acid is a stronger acid than carboxylic acid, congeners
7a/7b will form the sulfonate−aminium/sulfonate−guanidinium ion-pairs more effectively
than OC/GOC, thus ascribing to the higher lipophilicity of 7a/7b.
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3. Conclusion
In this study, we first synthesized the boronate, trifluoroborate, sulfone, sulfinate and
sulfonate congeners of oseltamivir carboxylic acid and the guanidino derivatives. The
synthesis started with elaboration of oseltamivir carboxylic acid (OC) to a Barton ester,
followed by Hunsdiecker−Barton halodecarboxylation to give the iodocyclohexene 16a,
which served as the pivotal intermediate for the palladium-catalyzed coupling reactions with
appropriate diboron and thiol reagents. The synthesized boronate, trifluoroborate, sulfone,
sulfinate and sulfonate congeners of OC and its guanidino analogs were subject to influenza
virus neuraminidase inhibition and cell-based anti-influenza assays. The GOC congeners (b
series with C5-guanidino group) consistently showed better NA inhibition and anti-influenza
activity than the corresponding OC congeners (a series with C5-amino group). Along with the
interactions of the C5-amino (or guanidino), C-4 acetamide, and C3-pentoxy substituents in
the S2–S5 sites of NA, the electrostatic interactions of carboxylate ion (or its bioisostere) with
the three arginine residues (R118, R292 and R371) in the S1 site played an essential role in
NA inhibition. The sulfonic acids 7a and 7b display high NA inhibitory activity, and 7b is the
most potent anti-influenza agent (EC50 = 2.2 nM) among the examined compounds. The order
of NA inhibitory activity for sulfonic acids (7a/7b) > sulfinic acids (6a/6b) > boronic acids
(2a/2b) seems to correlate with their strength of acidity; thus the sulfonic acid can make the
20

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strongest interactions with the tri-arginine motif in the S1 site of NA. However, the acidity
may not be the sole factor to be accounted for the affinity toward NA and the ability in virus
suppression. For example, the NA inhibitory activity of the sulfinic acid congener 6a (IC50 =
22 nM) is inferior to OC (IC50 = 1.8 nM), even though sulfinic acid (pKa = 2.0) is a stronger
acid than carboxylic acid (pKa = 4.5). In another aspect, the sulfinic acid congener 6b displays
the anti-influenza activity comparable to GOC in the cell-based assay; even 6b has lower NA
inhibitory activity. This result is presumably related to the higher lipophilicity of 6b for
enhanced cell permeability. In agreement with this rationale, OC and the sulfonic acid
congener 7a have a similar NA inhibitory activity, but 7a is more lipophilic to show a higher
anti-influenza activity. Our study also indicates that trifluoroborate (3b), sulfones
(4a/4b/5a/5b) and sulfonate esters (8a/8b/9a/9b) exhibit good NA inhibitory activity with
IC50 values in nanomolar range, because the electronegative fluorine and oxygen atoms on the
polarized B–F and S→O bonds can also make sufficient electrostatic interactions with the
tri-arginine motif.
4. Experimental section
4.1. General part
All the reagents were commercially available and used without further purification unless
indicated otherwise. All solvents were anhydrous grade unless indicated otherwise. Reactions
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were magnetically stirred and monitored by thin-layer chromatography on silica gel. Flash
chromatography was performed on silica gel (40–63 µm particle size) and LiChroprep RP-18
(40–63 µm particle size). Yields were reported for spectroscopically pure compounds. Melting
1
points were recorded on a Yanaco melting point apparatus. H and 13C NMR spectra were
recorded on Varian Unity Plus-400 (400 MHz) spectrometer. 19F NMR spectra were recorded
on Varian Unity Plus-400 (400 MHz) spectrometer. Chemical shifts (δ) are given in parts per
million (ppm) relative to residual signals of solvents as the internal standards: CHCl3 (δH =
7.24), CDCl3 (δC = 77.0 for the central line of triplet), CD2HOD (δH = 3.31), CD3OD (δC =
49.0), DHO (δH = 4.81), (CH3)2SO (δH = 2.50) and (CD3)2SO (δC = 39.5). Coupling constants
(J) are given in hertz (Hz) and the splitting patterns are reported as s (singlet), d (doublet), t
(triplet), q (quartet), quint (quintet), m (multiplet), br (broad), and dd (doublet of doublets). IR
spectra were recorded on Varian 640 or Nicolet Magna 500-II. Optical rotations were
recorded on digital polarimeter of Japan JASCO Co. DIP-1000. [α]D values were given in
units of 10–1 deg cm2g–1. ESI mass spectra were recorded on a high-resolution mass
spectrometer.
4.2. Materials
Influenza A/WSN/1933 (H1N1) was from Dr. Shin-Ru Shih at Chang Gung University in
Taiwan. All viruses were cultured in the allantoic cavities of 10-day-old embryonated chicken
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eggs for 72 h, and purified by sucrose gradient centrifugation. Madin–Darby canine kidney
(MDCK) cells were obtained from American Type Culture Collection (Manassas, Va.), and
were grown in DMEM (Dulbecco’s modified Eagle medium, GibcoBRL) containing 10%
o
fetal bovine serum (GibcoBRL) and penicillin-streptomycin (GibcoBRL) at 37 C under 5%
CO2.
4.3. Determination of influenza virus TCID50
The TCID50 (50% tissue culture infectious dose) was determined by serial dilution of the
influenza virus stock solution onto 100 µL MDCK cells at 1 × 105 cells/mL in 96-well
microplates. The infected cells were incubated at 37 oC under 5.0% CO2 for 48 h and added to
each well with 100 µL of CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay
o
reagent (Promega). After the incubation at 37 C for 15 min, absorbance at 490 nm was read
on a plate reader. Influenza virus TCID50 was determined using Reed–Müench method. [40,
41]
4.4. Determination of neuraminidase activity by a fluorescent assay
The NA activity was measured by using diluted allantoic fluid harvested from influenza
virus infected embryonated eggs or by recombinant neuraminidase proteins. A fluorometric
assay was used to determine the NA activity with the fluorogenic substrate
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2-(4-methylumbelliferyl)-α-
D
-N-acetylneuraminic
acid
(MUNANA;
Sigma).
The
fluorescence of the released 4-methylumbelliferone was measured in Envision plate reader
(Perkin-Elmer, Wellesley, MA) by using excitation and emission wavelength of 365 and 460
nm, respectively. Neuraminidase activity was determined at 200 µM of MUNANA (for
diluted allantoic fluid) or at 5 µM of MUNANA (for recombinant influenza neuraminidases).
Enzyme activity was expressed as the fluorescence increase during 15 min incubation at room
temperature.
4.5. Determination of IC50 of neuraminidase inhibitors
Neuraminidase inhibition was determined by mixing inhibitor (0.02–83000 nM) and NA
for 10 min at room temperature followed by the addition of substrate. The IC50 values were
determined from the dose–response curves by plotting the percent inhibition of NA activity
versus inhibitor concentrations using Graph Pad Prism 4.
4.6. Determination of EC50 and CC50 of neuraminidase inhibitors
The anti-influenza activities of neuraminidase inhibitors were measured by the EC50
values, which were the concentrations of NA inhibitor for 50% protection of the influenza
virus infection-mediated cytopathic effects (CPE). Fifty microliters of diluted influenza virus
at 100 TCID50 was mixed with equal volumes of NA inhibitors at varied concentrations. The
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mixtures were used to infect 100 µL of MDCK cells at 1 × 105 cells/mL in 96-wells. After 48
o
h of incubation at 37 C under 5.0% CO2, the cytopathic effects were determined with
CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay reagent as described above.
The EC50 values were determined by fitting the curve of percent CPE versus the
concentrations of NA inhibitor using Graph Pad Prism 4. The CC50 values (50% cytotoxic
concentrations) of NA inhibitor to MDCK cells were determined by the procedures similar to
the EC50 determination but without virus infection.
4.7. Synthesis and characterization of compounds
4.7.1. Compound characterization
New compounds were characterized by their physical and spectroscopic properties (mp,
1
13
11
TLC, [α], IR, ESI−MS, H, C and B NMR). Purity of synthetic compounds was assessed
to be ≥95% by HPLC analysis on an HC-C18 Agilent column (4.6 × 250 mm, 5 µm particle
size) with the indicated eluent at a flow rate of 1 mL/min and detection at 254 nm wavelength.
4.7.2.
4-Acetamido-5-(tert-butoxycarbonyl)amino-3-(1-ethylpropoxy)-1-cyclohexenecarboxylic acid
(15) [19]
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To a mixture of oseltamivir (as the phosphate salt, 0.75 g, 1.83 mmol) and NaHCO3 (0.77
g, 9.14 mmol) in THF/H2O (10 mL/10 mL) was added di-tert-butyl dicarbonate [(Boc)2O]
(0.52 g, 2.38 mmol). The mixture was stirred for 2 h at room temperature, and concentrated
under reduced pressure. The residue was diluted with CH2Cl2, and washed with water. The
aqueous phase was extracted with CH2Cl2 (3 ×). The combined organic phase was dried over
MgSO4, filtered, and concentrated under reduced pressure to give 14 (0.82 g), the Boc
derivative of oseltamivir, which was used in the next step without further purification.
The above-prepared Boc derivative 14 (0.82 g) was stirred with KOH (3.5 mL, 1.0 M) in
MeOH (6.6 mL, 0.3 M) at room temperature for 2 h, and then neutralized with Dowex 50W×8.
The mixture was filtered, washed with MeOH, and the filtrate was concentrated under
reduced pressure to give acid 15 (0.63 g, 90% yield from oseltamivir). C19H32N2O6; white
solid; mp 217–219 oC; TLC (EtOAc/hexane, 2:1) Rf = 0.10; [α]D23 –109.6 (c = 1, MeOH); IR
1
(film) 3442, 3321, 2936, 1690, 1656, 1537, 1294, 1052, 1013 cm–1; H NMR (400 MHz,
CD3OD) δ 6.79 (1 H, s), 4.11 (1 H, d, J = 8.4 Hz), 3.88–3.83 (1 H, m), 3.75–3.68 (1 H, m),
3.41 (1 H, t, J = 5.6 Hz), 2.71–2.66 (1 H, m), 2.28–2.19 (1 H, m), 1.96 (3 H, s), 1.56–1.49 ( 4
H, m), 1.44 (9 H, s), 0.90–0.85 (6 H, m); 13C NMR (100 MHz, CDCl3) δ 169.5, 168.1, 155.3,
135.0, 131.6, 81.0, 77.7, 75.4, 54.5, 49.3, 31.0, 28.4 (3 ×), 26.0, 25.4, 23.0, 9.6, 9.2; HRMS
calcd for C19H33N2O6: 385.2339, found: m/z 385.2337 [M + H]+.
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4.7.3. 4-Acetamido-5-(tert-butoxycarbonyl)amino-3-(1-ethylpropoxy)-1-iodo-1-cyclohexene
(16a) [19]
To a solution of carboxylic acid 15 (0.25 g, 0.65 mmol) in anhydrous CH2Cl2 (8 mL)
were added Et3N (0.14 mL, 0.98 mmol), HBTU (0.37 g, 0.98 mmol), and DMAP (catalytic
amount). The mixture was stirred at room temperature for 30 min, and 3-hydroxy-4-methyl
thiazolethione (0.13 g, 0.86 mmol) was added. The mixture was stirred for another 3 h at
room temperature, and concentrated under reduced pressure. The residue was purified by
flash chromatography on a silica gel column (EtOAc/hexane, 1:1 to 2:1) to afford an
intermediate Barton ester (0.3 g, 90% yield).
A solution containing the intermediate Barton ester (0.3 g, 0.58 mmol) and trifluoroethyl
iodide (2 mL, 1.5 mmol) in anhydrous CH2Cl2 (0.3 M, 2 mL) was irradiated by 500 W lamp
at room temperature for 0.5 h. The mixture was concentrated under reduced pressure, and
purified by flash chromatography on a silica gel column (EtOAc/hexane, 1:3 to 1:1) to afford
compound 16a (164 mg, 0.35 mmol, 54% overall yield). C18H31IN2O4; pale yellow solid, mp
161–163 oC; TLC (EtOAc/hexane, 1:1) Rf = 0.5; 1H NMR (400 MHz, CDCl3) δ 6.31 (1 H, s),
5.61 (1 H, d, J = 16.4 Hz), 5.26 (1 H, d, J = 8.8 Hz), 4.08 (1 H, q, J = 9.6 Hz), 3.84–3.75 (2 H,
m), 3.28 (1 H, t, J = 5.6 Hz), 2.86 (1 H, dd, J = 4.4, 5.8 Hz), 2.60 (1 H, dd, J = 1.7, 4.08 Hz),
1.98 (3 H, s), 1.54–1.41 (13 H, m), 0.90–0.85 (6 H, m); 13C NMR (100 MHz, CDCl3) δ 170.7,
155.8, 138.0, 94.5, 82.0, 79.6, 77.5, 53.3, 50.5, 45.1, 28.4 (3 ×), 26.2, 25.8, 23.4, 9.7, 9.4;
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HRMS calcd for C18H32IN2O4: 467.1407, found: m/z 467.1403 [M + H]+.
4.7.4.
(3R,4R,5S)-4-Acetamido-5-[N2,N3-bis(tert-butoxycarbonyl)guanidino]-3-(1-ethylpropoxy)-1-i
odocyclohexene (16b)
Trifluoroacetic acid (TFA, 0.03 mL, 0.4 mmol) was added to a solution of the iodo
compound 16a (300 mg, 0.32 mmol) in anhydrous CH2Cl2 (5 mL). The mixture was stirred at
room temperature for 3 h, and then concentrated under reduced pressure to give an amine
product
as
brown
oil.
The
amine
compound,
1,3-di-Boc-2-(trifluoromethanesulfonyl)guanidine (156 mg, 0.4 mmol) and NEt3 (0.05 mL,
0.4 mmol) were dissolved in CH2Cl2 (5 mL). The mixture was stirred at room temperature for
10 h, concentrated under reduced pressure, and purified by silica gel column chromatography
(EtOAc/hexane = 2:1) to give the guanidination product 16b (140 mg, 72% yield).
21
C24H41IN4O6; colorless oil; [α]D –18.0 (c = 1, CH2Cl2); IR (film) 3417, 2979, 2881, 2366,
1734, 1637, 1373, 1233, 1152 cm–1; 1H NMR (400 MHz, CDCl3) δ 8.72 (1 H, d, J = 8.0 Hz),
6.28 (1 H, s), 6.21 (1 H, d, J = 8.8 Hz), 4.39 (1 H, quint, J = 8.0 Hz), 4.13–4.05 (1 H, m), 3.79
(1 H, br), 3.24 (1 H, quint, J = 5.6 Hz), 2.90 (1 H, dd, J = 18.0, 5.6 Hz), 2.70–2.63 (1 H, m),
1.90 (3 H, s), 1.45 (22 H, br), 0.87–0.80 (6 H, m). 13C NMR (100 MHz, CDCl3) δ 170.7,
163.0, 156.4, 152.4, 137.8, 94.5, 83.4, 82.7, 79.7, 52.5, 48.9, 43.9, 28.2 (3 ×), 27.9 (3 ×), 26.0,
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25.7 (2 ×), 23.1, 9.6, 9.3; HRMS calcd for C24H42IN4O6: 609.2141, found: m/z 609.2149 [M +
H]+.
4.7.5. (3R,4R,5S)-4-Acetamido-5-amino-3-(1-ethylpropoxy)-1-cyclohexeneboronic acid (2a)
Tetrakis(triphenylphosphine)palladium(0) (6 mg, 5 µmol) was added to a solution of iodo
compound 16a (47 mg, 0.1 mmol), KOAc (29 mg, 0.3 mmol), and bis(pinacolato)diboron (51
mg, 0.2 mmol) in anhydrous DMF (1 mL). The solution was heated to 90 oC and stirred for 4
h. The mixture was filtered through a pad of Celite, and the filtrate was concentrated under
reduced pressure to give a crude boronate ester 17a as yellow oil. A solution of 17a and
potassium hydrogenfluoride (KHF2, 31 mg, 0.4 mmol) in MeOH (2 mL) and H2O (1 mL) was
stirred for 10 h at room temperature, and then concentrated under reduced pressure to give
white solids, which were dissolved in MeOH (5 mL) and filtered. The filtrate was
concentrated under reduced pressure to give a crude trifluoroborate product 18a as white
solids. Compound 18a was dissolved in 1 M HCl (2 mL), stirred for 8 h at room temperature,
and concentrated under reduced pressure. The residue was purified on a reversed-phase RP-18
column (MeOH/H2O = 1:9 to 1:4) to give the desired product of boronic acid 2a (14 mg, 48%
yield from 16a). The purity of product 16a was > 99% as shown by HPLC on an HC-C18
column (Agilent, 4.6 × 250 mm, 5 µm particle size), tR = 10.6 min (MeOH/H2O = 3:7).
23
C13H25BN2O4; hygroscopic solid; [α]D –57.5 (c = 1, MeOH); IR (film) 3434, 2792, 2156,
29