Acylguanidine derivatives of zanamivir and oseltamivir: Potential orally available prodrugs against influenza viruses

Article abstract of DOI:10.1016/j.ejmech.2018.05.030

Zanamivir (ZA) and guanidino-oseltamivir carboxylic acid (GOC) are very potent inhibitors against influenza neuraminidase (NA). The guanidinium moiety plays an important role in NA binding; however, its polar cationic nature also hinders the use of ZA and GOC from oral administration. In this study, we investigated the use of ZA and GOC acylguanidine derivatives as possible orally available prodrugs. The acylguanidine derivatives were prepared by coupling with either n-octanoic acid or (S)-naproxen. The lipophilic acyl substituents were verified to improve cell permeability, and may also improve the bioavailability of acylguanidine compounds. In comparison, the acylguanidines bearing linear octanoyl chain showed better NA inhibitory activity and higher hydrolysis rate than the corresponding derivatives having bulky branched naproxen moiety. Our molecular docking experiments revealed that the straight octanoyl chain could extend to the 150-cavity and 430-cavity of NA to gain extra hydrophobic interactions. Mice receiving the ZA octanoylguanidine derivative survived from influenza infection better than those treated with ZA, whereas the GOC octanoylguanidine derivative could be orally administrated to treat mice with efficacy equal to oseltamivir. Our present study demonstrates that incorporation of appropriate lipophilic acyl substituents to the polar guanidine group of ZA and GOC is a feasible approach to develop oral drugs for influenza therapy.

Full text of DOI:10.1016/j.ejmech.2018.05.030

Accepted Manuscript
Acylguanidine derivatives of zanamivir and oseltamivir: Potential orally available
prodrugs against influenza viruses
Peng-Hao Hsu, Din-Chi Chiu, Kuan-Lin Wu, Pei-Shan Lee, Jia-Tsrong Jan, Yih-
Shyun E. Cheng, Keng-Chang Tsai, Ting-Jen Cheng, Jim-Min Fang
PII:
S0223-5234(18)30443-4
10.1016/j.ejmech.2018.05.030
EJMECH 10441
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 January 2018
Revised Date: 5 May 2018
Accepted Date: 20 May 2018
Please cite this article as: P.-H. Hsu, D.-C. Chiu, K.-L. Wu, P.-S. Lee, J.-T. Jan, Y.-S.E. Cheng, K.-C.
Tsai, T.-J. Cheng, J.-M. Fang, Acylguanidine derivatives of zanamivir and oseltamivir: Potential orally
available prodrugs against influenza viruses, European Journal of Medicinal Chemistry (2018), doi:
10.1016/j.ejmech.2018.05.030.
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ACCEPTED MANUSCRIPT
Graphical abstract

ACCEPTED MANUSCRIPT
Acylguanidine Derivatives of Zanamivir and Oseltamivir: Potential Orally Available
Prodrugs against Influenza Viruses
Peng-Hao Hsu,a Din-Chi Chiu,a Kuan-Lin Wu,a Pei-Shan Lee,a Jia-Tsrong Jan,b Yih-Shyun E.
Cheng,b Keng-Chang Tsai,c,d Ting-Jen Cheng,b and Jim-Min Fanga,b,*
aDepartment of Chemistry, National Taiwan University, Taipei 106, Taiwan
bThe Genomics Research Center, Academia Sinica, Taipei 115, Taiwan
cNational Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei 112,
Taiwan
dThe Ph.D. Program for Medical Biotechnology, College of Medical Science and Technology,
Taipei Medical University, Taipei 110, 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:
Zanamivir (ZA) and guanidino-oseltamivir carboxylic acid (GOC) are very potent inhibitors
against influenza neuraminidase (NA). The guanidinium moiety plays an important role in NA
binding; however, its polar cationic nature also hinders the use of ZA and GOC from oral
administration. In this study, we investigated the use of ZA and GOC acylguanidine
derivatives as possible orally available prodrugs. The acylguanidine derivatives were prepared
by coupling with either n-octanoic acid or (S)-naproxen. The lipophilic acyl substituents were
verified to improve cell permeability, and may also improve the bioavailability of
acylguanidine compounds. In comparison, the acylguanidines bearing linear octanoyl chain
showed better NA inhibitory activity and higher hydrolysis rate than the corresponding
derivatives having bulky branched naproxen moiety. Our molecular docking experiments
revealed that the straight octanoyl chain could extend to the 150-cavity and 430-cavity of NA
to gain extra hydrophobic interactions. Mice receiving the ZA octanoylguanidine derivative
survived from influenza infection better than those treated with ZA, whereas the GOC
octanoylguanidine derivative could be orally administrated to treat mice with efficacy equal to
oseltamivir. Our present study demonstrates that incorporation of appropriate lipophilic acyl
substituents to the polar guanidine group of ZA and GOC is a feasible approach to develop
oral drugs for influenza therapy.
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Keywords: Acylguanidine, Zanamivir, Oseltamivir, Neuraminidase inhibitor, Influenza.
Research highlights
• ZA and GOC acylguanidines are potent influenza neuraminidase (NA) inhibitors.
• Alkyl chain of acylguanidine improves lipophilicity and fits NA 150 & 143-cavities.
• ZA and GOC acylguanidines as prodrugs undergo hydrolysis in physiological conditions.
• ZA octanoylguanidine is better than ZA to treat influenza-infected mice in low dosage.
• GOC octanoylguanidine treats mice well by oral administration against influenza.
Abbreviations:
Arg, arginine; Boc, tert-butoxycarbonyl; (Boc)2O, di(tert-butyl) dicarbonate; CC50, half
maximal cytotoxicity concentration; CPE, cytopathic effect; DIEA, diisopropyl ethylamine;
DMSO, dimethylsulfoxide; EC50, half maximal effective concentration; ESI, electrospray
ionization; Gln, glutamine; GOC, guanidino-oseltamivir carboxylic acid; HA, hemagglutinin,
HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; IC50,
half maximal inhibitiory concentration; LC−MS, liquid chromatography−mass spectrometry;
LD50, median lethal dose; MDCK, Madin–Darby canine kidney; MUNANA,
2-(4-methylumbelliferyl)-α-D-N-acetyl-neuraminic acid; NA, neuraminidase; OC, oseltamivir
carboxylic acid; OS, oseltamivir; PBS, phosphate buffered saline; PDB, Protein Data Bank;
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PE, peramivir; Pro, proline; Ser, serine; TFA, trifluoroacetic acid; THF, tetrahydrofuran; Thr;
threonine; TPG, tamiphosphor guanidine; Val, valine; ZA, zanamivir.
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1. Introduction
Influenza is a long-standing problem that has caused seasonal epidemics and occassional
pandemics to claim a great number of lives. Influenza viruses are classified into A, B and C
groups according to their nucleoproteins and matrix proteins. The most virulent group A
viruses are further divided into various subtypes depending on their hemagglutinin (HA) and
neuraminidase (NA) on the membrane of viruses. Influenza virus proliferate itself by invading
host cells to produce its progeny, in which HA is responsible for specific binding of influenza
virus to the sialoprotein on the membrane of host cell, whereas NA is responsible for cleaving
the terminal sialic acid from the surface glycoprotein of host cell to release progeny virus.
Because different subtypes of NA still keep relatively conservative active sites, [1] it is
advantageous to design NA inhibitors as effective anti-influenza drugs. [2–5] The NA
inhibitors zanamivir (1, ZA), [6, 7] oseltamivir (2a, OS) [8–10] and peramivir (3, PE) [11–13]
are currently used in therapies of influenza infection. TamifluTM, the phosphate salt of OS, is
manufactured as an orally available prodrug, which is hydrolyzed by endogenous esterase to
release the corresponding carboxylic acid (2b, OC) as the active gradient. Emergence of the
OS-resistant influenza viruses, such as the H1N1 H275Y strain, is a serious concern in clinic
treatment. ZA and PE are not treated as oral drugs, mainly because they carry the hydrophilic
charged carboxyl and guanidino groups, rendering low bioavailability and fast renal
elimination. Although GOC (4), the guanidino analog of OC, shows higher NA inhibitory
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activity, [14] incorporation of the guanidino group greatly reduces its bioavailability (< 5%),
[15] Therefore, GOC and its ethyl ester derivative have not been developed for therapeutic
use in spite of their preferable NA inhibitory activities.
Derivatization of ZA and GOC, including modification of the guanidino group, has been
explored to improve their usefulness in treatment of influenza. [5, 16–19] For example, the
ZA derivatives having an extended (piperazinocarbonyl)propyl substituent at the internal
N-position of the guanidino group was found to exhibit a potent anti-influenza activity against
H1N1 virus with EC50 = 0.77 µM. [20] This ZA derivative renders extra interactions in the
150-cavity [3] of group-1 neuraminidases as the guanidino group is decorated, and the
hydrophobic alkyl substituent may also improve pharmacokinetics properties. In another study,
[21] the guanidino group of ZA is modified with appropriate acyl substituents at the external
N-position to attain extra bindings in the 150-cavity of H1N1 virus. Furthermore, a library of
1,2,3-triazole-containing acylguanidine derivatives of ZA is constructed to seek for potent NA
inhibitors. [22] Recently, some GOC acylguanidines are found to possess higher activities
than OC against wild-type H1N1 and OS-resistant H259Y viruses. [23]
From a different angle of view, acylguanidine compounds are liable to hydrolysis in
acidic or basic conditions, [24–26] and even by bioactivation. [27] Therefore, the
acylguanidine derivatives of ZA and GOC may act as prodrugs if they can be hydrolyzed in
body to give the parental guanidine drugs. In such case, modification with hydrophobic acyl
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substituent may enhance the drug lipophilicity for better membrane permeability and oral
administration. [19] To illustrate this concept, a thrombin inhibitor containing guanidino
group has been prepared as the acylguanidine derivatives to uphold membrane permeability
and drug absorptivity. [27] In one of our previous studies, [28] we also observed that
acylguanidine 6 effectively protected Madin–Darby canine kidney (MDCK) cells from the
infection of H1N1 viruses with EC50 of 0.8 nM, similar to tamiphosphor guanidine (5, EC50 =
3.8 nM), even 6 has poor inhibitory activity against NA (IC50 > 100 µM). This result suggests
the possible conversion of acylguanidine 6 to the parental drug 5 in the cell-based assay.
Although some ZA and GOC acylguanidine derivatives have been revealed to possess
good NA inhibitory activity, [21–23] further experiments are required to elucidate whether
they can act as prodrugs or not. In this study, we prepared the ZA and GOC acylguanidine
derivatives 7a, 7b, 8a and 8b (Fig. 1), and then investigated their anti-influenza activities and
susceptibility to hydrolysis in different conditions. Due to steric effect, compounds 7b and 8b
carrying an α-naphthylpropanoyl group are predicted to be less sensitive to hydrolysis than
their counterparts 7a and 8a bearing n-octanoyl group. [27]
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Fig. 1. Chemical structures of influenza neuraminidase inhibitors and acylguanidine
derivatives.
2. Results and discussions
2.1. Chemical synthesis
Mono-Boc protected S-methylisothiourea (10) was prepared, [23, 29] and reacted with
n-octanoic acid and (S)-naproxen, respectively, in the presence of HBTU and DIEA to give
the corresponding acylisothiourea compounds 12a and 12b (Scheme 1). Using sialic acid as
the starting material, compound 13 was prepared according to the previously reported method.
[30] Using HgCl2 as an activator, the coupling reactions of amine 13 with 12a and 12b were
carried out to give 14a and 14b in 78% and 89% yields, respectively. Compounds 14a and
14b were treated trifluoroacetic acid to remove the Boc protecting group, and the ethyl ester
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was subsequently saponified at room temperature for 30 min to give the desired products 7a
and 7b, respectively, without hydrolysis of the acylguanidine group.
Scheme 1. Synthesis of acylguanidine derivatives 7a, 7b, 8a and 8b. Reagents and conditions:
(a) (Boc)2O, NaHCO3, THF/H2O (1:1), rt, 14 h; (b) HBTU, DIEA, CH2Cl2, rt, 14 h; (c) HgCl2,
Et3N, CH2Cl2, rt, 6 h; (d) TFA, CH2Cl2, rt, 1 h; (e) KOH, CH3CN/H2O (1:1), rt, 30 min. Boc =
tert-butoxycarbonyl, (Boc)2O = di(tert-butyl) dicarbonate, DIEA = diisopropyl ethylamine,
HBTU = 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, TFA =
trifluoroacetic acid, THF = tetrahydrofuran.
For preparation of the GOC acylguanidines, Tamiflu capsule was treated with (Boc)2O in
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the presence of NaHCO3 to afford the Boc-protecting oseltamivir 15. [31, 32] The Boc group
was removed, and the intermediate aminium salt underwent the Hg(II)-promoted coupling
reactions with 12a and 12b to afford compounds 16a and 16b, respectively. The desired
acylguanidines 8a and 8b were obtained from 16a and 16b, respectively, after removal of the
Boc group and saponification of the ester.
2.2. Neuraminidase inhibition and cell-based assay
With ZA and GOC acylguanidines 7a–8b in hand, we then evaluated their NA inhibition
and anti-influenza activities (Table 1). The ZA acylguanidines 7a and 7b showed the NA
inhibitory activities against wild-type (WSN H1N1) virus and the H275Y mutant with IC50
values of 180 and 290 nM, respectively, inferior to ZA by approximately 160 fold.
Acylguanidines 7a and 7b were nontoxic to MDCK cells (CC50 > 100 µM), but efficiently
protected MDCK cells from WSN virus infection with EC50 values of 90 and 140 nM,
respectively. As the EC50 values were even lower than their IC50 values, we speculated that
acylguanidines 7a (ZA-oct) and 7b (ZA-nap) might be hydrolyzed, at least in parts, to yield
the parental ZA. The GOC acylguanidines 8a and 8b also displayed remarkable cellular
protection against WSN virus with EC50 values of 1 and 2 nM, respectively, even though they
are inferior NA inhibitors than GOC. This result might be also accounted for the increased
lipophilicity of 8a and 8b to facilitate cell absorption.
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Table 1. Neuraminidase inhibition (IC50), anti-influenza activity (EC50) and calculated
lipophilicity.
WSN
EC50c (nM)
H275Y
EC50c (nM)
Lipophilicity
Compounda
IC50b (nM)
1 ± 0e
IC50b (nM)
2 ± 0e
cLogPd cLogDd
1 (ZA)
2b (OC)
27 ± 11
16 ± 4
7 ± 4
150 ± 20
26000 ± 11000
2100 ± 900
480 ± 30
–4.13
0.43
–5.77
–1.84
–1.55
–2.92
–2.63
1.30
1 ± 0e
370 ± 100
2 ± 1
4 (GOC)
1 ± 0e
0.09
7a (ZA-oct)
7b (ZA-nap)
8a (GOC-oct)
8b (GOC-nap)
180 ± 50
200 ± 50
8 ± 1
90 ± 15
140 ± 10
1 ± 0e
290 ± 78
260 ± 70
18 ± 5
–1.27
–0.99
2.95
900 ± 210
6600 ± 2600
19000 ± 8000
53 ± 9
2 ± 1
190 ± 50
3.23
1.59
a All the test compounds are nontoxic to MDCK cells (CC50 > 100 µM).
b
IC50 value is the half maximal inhibitory concentration against the neuraminidase of
influenza virus A/WSN/1933 (H1N1) or the H275Y mutant.
c
EC50 value is half maximal effective concentration for protection of MDCK cytopathic
effects due to influenza virus infection.
d Octanol−water partition coefficient calculated using MarvinSketch 17.29
e
The values were between 0.1 to 0.4, which were round-up in order to standardize all the
numbers to the same standard deviation significant digits.
Similar to the previous studies, [21–23] acylguanidine derivatives (e.g. 7a−8b) generally
displayed weaker NA inhibitory activities than their parental anti-influenza agents (Table 1).
Some anti-influenza activity could be reverted by hydrolysis of acylguanidine to the parental
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guanidine as inferred from the cell-based assay. The EC50 values in Table 1 might just reflect
the combined effect of the acylguanidine and its hydrolysis product. It was interesting to note
that GOC-oct 8a still had sufficient binding strength with the neuraminidase of WSN virus
(IC50 = 7.9 nM) and exhibited high protective effect on MDCK cells (EC50 = 0.5 nM) against
the infection of WSN virus, presumably a consequence of enhanced permeability of the
acylguanidine combined with the partial hydrolysis to more active guanidine 4 (GOC). In
contrast, the binding strength of 8a with H275Y mutant was weaker, and no better
anti-influenza activity than GOC was observed. Following the similar trend, the anti-influenza
activity of GOC-nap 8b against wild-type and H275Y mutant was correlated to its binding
strength with NA.
2.3. Cell permeability study
To validate the assumption that the lipophilic acyl group could bolster up cell permeation,
we measured the cell permeability of 4 and 8a. We first identified the characteristic fragments
at m/z = 180 for guanidine 4 and at m/z = 365 for acylguanidine 8a (Fig. S1). The calibration
curves for concentrations of 4 and 8a were established, respectively, on the basis of the ion
currents (expressed as integral area) of the characteristic fragments in LC−MS spectra (Fig.
S2). Thus MDCK cells were incubated with 100 µM of 4 or 8a for 48 h, and the cell lysates
were prepared for the quantification of 4 and 8a in the cells by LC−MS analyses. The
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experiments showed that the cell lysate of the cells treated with 8a contained 1225 nM of 8a
and 130.4 nM of the hydrolysis product 4, equivalent to a sum of 1355.4 nM 8a entering the
MDCK cells after a 48-hour incubation (Fig. S3). In comparison, the parallel experiment
showed that the cell lysates of the cells treated with 4 only contained 47.6 nM of 4. Thus,
acylguanidine 8a has better cell permeability than guanidine 4 by 28 fold.
2.4. Stability of acylguanidine compounds in buffer and plasma
According to our cell permeability study, approximately 10% of 8a was hydrolyzed at 48
h of incubation to release more active NA inhibitor 4. To have a deeper insight into the
attributes of acylguanidines 7a–8b, we also examined their hydrolytic behaviors in different
media (Table 2). These compounds were stable in acidic media; no appreciable degradation
occurred after incubation for 96 h at pH 1.3 or 4.1 (entries 1 and 2). The acylguanidine
derivatives were slowly hydrolyzed in neural phosphate buffer, whereas the hydrolytic
degradation of octanoylguanidine compounds 7a and 8a accelerated in basic conditions
(entries 5 and 6). For unknown reason, the naproxen-derived acylguanidine 8b could sustain
longer in basic borate or glycine buffers (t = 76.7−81.4 h) than that in neutral condition (t =
½
½
64.9 h). The stability experiments also revealed that octanoylguanidines (7a and 8a) were
more easily hydrolyzed than the naproxen-derived acylguanidines (7b and 8b). This result is
in agreement with the previous finding that shows acylguanidine with α-substituent will slow
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down the hydrolysis rate. [27] The similar trend was observed in hydrolysis of acylguanidines
in rat plasma and rabbit serum (entry 7). Compound 7a in rat plasma was hydrolyzed with a
half-life at 29.5 h, compared with 67.7 h of 7b. The LC–MS analysis showed a signal at m/z
333 corresponding to the protonated molecule ion [M + H] of ZA, indicating that ZA was the
hydrolysis product of 7a (Fig. S4). Both 8a and 8b were rather reluctant to hydrolysis in
rabbit serum; thus 55% of 8a and 76% of 8b remained after incubation for 96 h at 37 oC.
Table 2. Stability tests of acylguanidine compounds.
t
1/2 (h)a
entry
Media
7ab
> 96c
> 96c
39.2
56.8
26.1
NDd
29.5
7bb
8ab
8bb
> 96c
NDe
64.9
69.7
76.7
81.4
> 96c
1
2
3
4
5
6
7
HCl/KCl solution (50 mM, pH 1.3)
acetate buffer (50 mM, pH 4.1)
phosphate buffer (50 mM, pH 7.4)
phosphate buffer (10 mM, pH 7.4)
borate buffer (50 mM, pH 9.1)
glycine buffer (50 mM, pH 9.2)
animal plasmaf
NDd
NDd
NDd
67.2
NDd
NDd
67.7
> 96c
NDe
NDe
NDe
18.1
22.4
≈ 96g
a Half-life was determined by HPLC analysis of the remaining acylguanidine compound. The
calculation was based on pseudo-first order rate law.
b Three duplicate experiments were performed for 7a and 7b, whereas single measurement
was taken for 8a and 8b.
c More than 75% of the indicated compound remained after incubation for 96 h at 37 oC.
d ND: Not determined. The hydrolysis tests were not performed.
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e ND: Not determined. The hydrolysis tests were not performed due to incomplete dissolution
of the substrates.
f Experiments of 7a and 7b was performed in rat plasma, whereas that of 8a and 8b was
conducted in rabbit serum.
g Only 55% of 8a remained after incubation for 96 h at 37 oC.
2.5. Molecular modeling
To address the distinction of IC50 values between a- and b-series acylguanidines, we
conducted molecular docking experiments on the GOC acylguanidines 8a and 8b to compare
their binding modes in the open-form NA of H1N1 virus (Fig. 2). In addition to the key
interactions of the C1-carboxy, C3-pentoxy, C4-acetamido and C5-gaunidino groups in the
NA active site, [33] the carbonyl group of the acylguanidine moiety also generates a hydrogen
bond with Arg-118. The similar hydrogen-bond interaction is also shown in the binding
modes of other GOC and ZA acylguanidines. [21–23] The straight hydrocarbon chain of 8a
extends to the 150-cavity [3] and 430-cavity [34] to gain hydrophobic interactions with
Val-116, Gln-136, Ser-145 and Thr-439. In contrast, the bulky naproxen moiety of 8b cannot
access the 150-cavity, and would rather points away from the NA pocket. Moreover, the
hydrophobic interactions induced by the naphthyl ring with Arg-430 and Pro-431 in the
430-cavity are significantly less robust than those possessed by compound 8a. Taken together,
the molecular docking experiments are in agreement with the results of NA inhibition assays,
i.e. 7a and 8a bearing a linear octanoyl substituent at the guanidine moiety can fit the
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open-form NA to attain better inhibitory activity than 7b and 8b.
Fig. 2. Schematic representations of molecular modeling of compounds 8a (a) and 8b (b) in
the open-form neuraminidase (N1 subtype, PDB code: 2HU0).
2.6. Mice experiments
ZA requires a dose of 400 µg/kg/day (1.2 µmol/kg/day) by intranasal administrations
twice (with half of the dosage at each administration) for treatment of influenza infected
patients. Taking 10 times metabolic rate of mouse, the mice experiment was first conducted
using the dosage of 12 µmol/kg/day by intranasal instillation. Phosphate buffered saline (PBS)
was utilized as negative control. Fig. 3a shows that the acylguanidine 7a (ZA-oct) was as
effective as ZA in protection of mice (100% survival) from the infection of H1N1 virus, but
acylguanidine 7b (ZA-nap) was somewhat inferior with 70% survival rate at day 21. When
mice were treated with a low dosage of 2.4 µmol/kg/day, compound 7a showed somewhat
better efficacy than ZA (Fig. 3b). The in vitro stability tests suggested that the half-life of
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compound 7a is approximately 30 h (Table 2). Compared to the reported half-life of
zanamivir of 2−5 h [35], it is possible that the introduction of lipophilic group helps the
stability of 7a. Also, the cell permeability tests implied that higher amount of compound with
lipophilic group is available to the cells compared to the parental compound. Therefore, the
observed virus challenge results are ascribed to higher availabilities and stabilities, combined
to the antiviral effects of the ZA derivative and ZA. This mechanism is similar to that
proposed for laninamivir octanoate, which is a long-acting NA inhibitor. [36]
Fig. 3. Survival rates of virus-infected mice on treatment with ZA acylguanidines. The
administered dosage of each compound was 12 µmol/kg/day (a) and 2.4 µmol/kg/day (b).
Compounds were administered by intranasal instillation and continued twice daily for 5 days.
BALB/c mice were intranasally challenged with 10 LD50 of A/WSN/1933 (H1N1) viruses.
The animal experiments were conducted with 10 mice per group. ***P<0.0001.
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Compared to cLogP of 1.16 for OS, GOC acylguanidine 8a is predicted to have a suitable
lipophilicity (cLogP = 2.95) for oral administration in mice experiment. OS requires a dose of
3 mg/kg/day (9.6 µmol/kg/day) for influenza treatment. Fig. 4 shows that 8a and OS have
equivalent efficacy by oral administration at 48 µmol/kg/day dosage to protect mice from the
infection of WSN virus. This experiment suggests the possible development of GOC
acylguanidine as an orally available drug in influenza therapy.
Fig. 4. Survival rates of virus-infected mice on treatment with GOC acylguanidine 8a.
Compounds were administered by oral gavage and continued twice daily for 5 days. The
administered dosage of each compound was 48 µmol/kg/day. BALB/c mice were intranasally
challenged with 10 LD50 of A/WSN/1933 (H1N1) viruses. The animal experiments were
conducted with 10 mice per group. ***P<0.0001. **P<0.001.
3. Conclusion
In this study, we designed and synthesized the ZA and GOC acylguanidine derivatives 7a,
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7b, 8a and 8b. Similar to the previous studies, [21–23] these acylguanidine derivatives
generally displayed weaker NA inhibitory activities than their parental anti-influenza agents
(ZA or GOC, Table 1). Nonetheless, the GOC octanoylguanidine derivative 8a still possess
high inhibitory activity with IC50 of 8 nM and EC50 of 1 nM against wild-type H1N1 virus. By
conducting cell permeability experiments, we ensured that 8a is 28-fold more readily uptaken
by MDCK cells compared to the parental agent, and approximately 10% of 8a can be
hydrolyzed to produce 4. Our experiments also indicated that the acylguanidines were prone
to hydrolysis in neutral and basic buffer solutions as well as in animal plasma at physiological
condition, leading to liberation of the parental anti-influenza agents ZA and GOC (Table 2).
The hydrolysis of acylguanidines 7a and 8a was appreciably faster than their counterparts 7b
and 8b. Taken together, the ZA and GOC acylguanidine derivatives are not only active to NA
but also act further as prodrugs by hydrolysis in cell to generate the parental guanidines (ZA
and GOC). Incorporation of appropriate lipophilic acyl substituents to the polar guanidine
group of ZA and GOC can improve the cell permeability and render the possible oral
administration for treatment of influenza.
The molecular docking experiments revealed that the straight octanoyl chain of 8a could
extend to the 150-cavity and 430-cavity of NA to gain extra hydrophobic interactions. In
comparison, the GOC acylguanidine 8b showed a weaker affinity to NA by 7 fold because the
bulky branched naproxen moiety could not be incorporated into the 150-cavity. Following the
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similar trend, the ZA octanoyl derivative 7a also showed better NA inhibitory activity than the
naproxen derivative 7b. By intranasal administration, the octanoylguanidine derivative 7a
(ZA-oct) secured higher mice survival than ZA in treatment with H1N1 influenza, but 7b
(ZA-nap) was less effective (Fig. 3). By oral gavage, mice receiving 8a (GOC-oct) showed
comparable survival rate to those treated with OS (Fig. 4).
5. Experimental section
5.1. General part
All the reagents and solvents were reagent grade and were used without further
purification unless otherwise indicated. All solvents were ACS grade unless indicated
otherwise. CH2Cl2 was distilled from CaH2. All non-aqueous reactions were conducted in
oven-dried glassware under a slight positive pressure of argon or nitrogen unless otherwise
noted. Reactions were magnetically stirred and monitored by thin-layer chromatography
(TLC) on silica gel using phosphomolybdic acid as visualizing agents. Silica gel (Merck,
0.040–0.063 mm) and LiChroprep RP-18 (Merck, 0.040–0.063 mm) were used for column
chromatography. Melting points were recorded on a Yanaco or Electrothermal MEL-TEMP
1101D apparatus and are not corrected. Optical rotations were measured on digital
polarimeter of Japan JASCO Co. DIP-1000. [α]D values are given in units of 10–1 deg cm2 g–1.
Infrared (IR) spectra were recorded on a Thermo Nicolet iS5 FT-IR spectrometer. Nuclear
magnetic resonance (NMR) spectra were obtained on Bruker DPX-400 (400 MHz) or Bruker
AVIII-400 (400 MHz) spectrometer. Chemical shifts (δ) are given in parts per million (ppm)
relative to internal standards: CHCl3 (δH = 7.24), CDCl3 (δC = 77.0 for the central line of
triplet), HDO (δH = 4.81), CH3OD (δH = 3.31), CD3OD (δC = 49.0), (CH3)2SO (δH = 2.50) and
20

ACCEPTED MANUSCRIPT
(CD3)2SO (δC = 39.5). The splitting patterns are reported as s (singlet), d (doublet), t (triplet),
q (quartet), quin (quintet), m (multiplet), dd (double of doublets) and br (broad). Coupling
constants (J) are given in Hz. The ESI−MS experiments for compound characterization were
conducted on a Bruker Daltonics BioTOF III high-resolution mass spectrometer.
High-performance liquid chromatography (HPLC) was performed on Agilent 1100 Series
instrument equipped with a degasser, a Quat pump, and a UV detector. LC–MS experiments
for compound characterization were performed on Agilent 1200 Series instrument and Agilent
6320 Ion Trap LC–MS System.
5.2. Material and methods
Influenza virus A/WSN/1933 (H1N1) was obtained from Dr. Shin-Ru Shih at Chang Gung
University in Taiwan, and influenza virus A/Udorn/1972 (H3N2) was from Dr. Jia-Tsrong Jan
(The Genomics Research Center, Academia Sinica). WSN 274Y was selected with Tamiflu
from influenza virus A/WSN/1933 (H1N1) in our lab. All Viruses were cultured in the
allantoic cavities of 10-day-old embryonated chicken 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% fetal bovine serum (GibcoBRL) and
penicillin-streptomycin (GibcoBRL) at 37 oC under 5% CO2.
5.3. Synthesis and characterization of compounds
5.3.1. Tert-Butyl [amino(methylthio)methylene]carbamate (10). [29]
To a solution of S-methylisothiourea hemisulfate salt (9) (1085 mg, 7.80 mmol) and
NaHCO3 (655 mg, 7.80 mmol) in H2O (15 mL) and THF (15 mL) was added di(tert-butyl)
dicarbonate (1283 mg, 5.88 mmol) dropwise. The mixture was stirred at room temperature
21

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under argon for 14 h, and then concentrated under reduced pressure. The residue was
partitioned between EtOAc and H2O. The organic phase was dried over MgSO4, filtered, and
purified by silica gel chromatography (EtOAc/hexane = 1:4) to afford the desired product 10
(1104 mg, 99%). C7H14N2O2S; 1H NMR (CDCl3, 400 MHz) δ 2.39 (3 H, s), 1.45 (9 H, s); 13C
NMR (CDCl3, 100 MHz) δ 172.3, 160.4, 79.1, 27.5, 12.7; HRMS (ESI) calcd for C7H15N2O2S:
191.0854, found: m/z 191.0848 [M + H]+.
5.3.2. Tert-Butyl [(methylthio)(octanamido)methylene]carbamate (12a)
To a solution of octanoic acid (158 µL, 1.00 mmol) in anhydrous CH2Cl2 (5 mL) was
added HBTU (417 mg, 1.10 mmol), DIEA (192 µL, 1.10 mmol) and compound 10 (209 mg,
1.19 mmol). The mixture was stirred at room temperature under argon for 14 h, and then
concentrated under reduced pressure. The residue was diluted with EtOAc and washed with 1
M HCl(aq), saturated NaHCO3(aq) and brine. The organic phase was dried over MgSO4, filtered,
and purified by silica gel chromatography (EtOAc/hexane = 1:19) to afford the desired
o
product 12a (169 mg, 71% yield). C15H28N2O3S; white solid; mp 68.0–69.2 C; TLC
(EtOAc/hexane = 1:9) Rf = 0.45; IR υmax (neat) 2930, 1748, 1567 cm–1; 1H NMR (CDCl3, 400
MHz, containing two tautomers (3:1)) δ 12.41 (0.75 H, s), 12.12 (0.25 H, s), 2.50–2.29 (5 H,
m), 1.71–1.58 (2 H, m), 1.48 (9 H, s), 1.37–1.19 (8 H, m), 0.84 (3 H, t, J = 5.4 Hz); 13C NMR
(CDCl3, 100 MHz) δ 171.1, 170.9, 160.6, 80.6, 77.2, 37.0, 31.3, 28.7, 27.7 (3 ×), 24.3, 22.3,
14.1, 13.7; HRMS (ESI) calcd for C15H29N2O3S: 317.1899 found: m/z 317.1897 [M + H]+.
5.3.3.
Tert-Butyl
[[2-(6-methoxynaphthalen-2-yl)propanamido](methylthio)methylene]carbamate (12b)
To a solution of (S)-naproxen (11b) (115 mg, 0.50 mmol) in anhydrous CH2Cl2 (5 mL)
was added HBTU (247 mg, 0.65mmol), DIEA (130 µL, 0.75mmol), and compound 10 (114
mg, 0.6 mmol). The mixture was stirred at room temperature under argon for 14 h, and then
22

ACCEPTED MANUSCRIPT
concentrated under reduced pressure. The residue was diluted with EtOAc and washed with 1
M HCl(aq), saturated NaHCO3(aq), and brine. The organic phase was dried over MgSO4, filtered,
and purified by silica gel chromatography (EtOAc/hexane = 1:9) to afford the desired product
12b (169 mg, 84%, containing tautomers (11:9)). C21H26N2O4S; colorless syrup; TLC
(EtOAc/hexane = 1:9) Rf = 0.27; [α]24 –18.4 (c = 1.0, CH2Cl2); IR υmax (neat) 2933, 1747,
D
1557 cm–1; 1H NMR (CDCl3, 400 MHz) δ 12.31 (0.55 H, s), 12.19 (0.45 H, s), 7.69 (3 H, m),
7.38 (1 H, br), 7.15–7.07 (2 H, m), 4.02–3.90 (1 H, br), 3.87 (3 H, s), 2.30 (3 H, s), 1.62 (3 H,
d, J = 6.8 Hz), 1.37 (9 H, br); 13C NMR (CDCl3, 100 MHz) δ 186.3, 172.1, 171.0, 170.2,
169.4, 160.2, 157.4, 157.2, 150.3, 150.1, 136.4, 133.8, 133.6, 133.2, 128.9, 128.6, 127.4,
126.4, 126.2, 125.9, 125.3, 118.,8 118.4, 105.2, 82.7, 82.3, 80.5, 77.2, 53.7, 50.9, 47.6, 27.4,
17.5, 17.0, 14.1, 13.9; HRMS (ESI) calcd for C21H27N2O4S: 403.1692, found: m/z 403.1700
[M + H]+.
5.3.4.
Methyl
5-acetamido-6-(1,2,3-triacetoxypropyl)-4-[2-(tert-butoxycarbonyl)]-3-octanoyl]guanidino-4,5
-dihydro-6H-pyran-2-carboxylate (14a)
Amine 13 was prepared from sialic acid according to the previously reported method. [30]
To a solution of amine 13 (138 mg, 0.32 mmol) in CH2Cl2 (5 mL) were added compound 12a
(100 mg, 0.32 mmol), HgCl2 (95 mg, 0.35 mmol), and Et3N (49 µL, 0.35 mmol). The mixture
was stirred at room temperature under argon for 6 h, filtered through Celite, and concentrated
under reduced pressure. The residue was diluted with EtOAc and washed with 1 M HCl(aq)
,
saturated NaHCO3(aq), and brine. The organic phase was dried over MgSO4, filtered, and
purified by silica gel chromatography (EtOAc/hexane = 1:1 to 3:2) to afford the coupling
product 14a (172 mg, 78%). C32H50N4O13; white foam; TLC (EtOAc/hexane = 3:2) Rf = 0.45;
[α]24D +58.6 (c = 1.0, CH2Cl2); IR υmax (neat) 3269, 2930, 1746, 1657 cm–1; 1H NMR (CDCl3,
400 MHz, two tautomers (17:3)) δ 12.21 (0.15 H, s), 12.19 (0.85 H, s), 9.12 (0.85 H, d, J =
23

ACCEPTED MANUSCRIPT
8.4 Hz), 8.48 (0.15 H, d, J = 8.4 Hz), 6.00 (0.85 H, d, J = 8.8 Hz), 5.91 (0.15 H, d, J = 2.4 Hz),
5.86 (0.85 H, d, J = 2.4 Hz), 5.76 (0.15 H, d, J = 9.2 Hz), 5.46–5.38 (1 H, m), 5.33–5.25 (1 H,
m), 5.20–5.05 (1 H, m), 4.71–4.60 (1 H, m), 4.36–4.20 (2 H, m), 4.20–4.10 (1 H, m), 3.84–
3.73 (3 H, m), 2.41–2.30 (2 H, m), 2.13–2.00 (9 H, m), 1.88–1.80 (3 H, m), 1.67–1.57 (2 H,
m), 1.51–1.41 (9 H, m), 1.35–1.19 (8 H, m), 0.85 (3 H, t, J = 6.8 Hz); 13C NMR (CDCl3, 100
MHz, two tautomers (17:3)) δ 187.6, 174.4, 170.6, 170.5, 170.2, 170.1, 170.0, 169.89, 169.86,
162.9, 161.4, 156.6, 156.1, 152.5, 144.4, 144.3, 109.8, 109.7, 83.2, 79.3, 77.1, 71.4, 67.7, 62.0,
52.0, 49.3, 49.1, 46.8, 46.7, 41.1, 37.5, 31.4, 31.2, 29.0, 28.8, 28.52, 28.46, 27.8, 27.6, 25.3,
24.2, 22.6, 22.3, 22.2, 20.5, 20.4, 13.72, 13.68; HRMS (ESI) calcd for C32H51N4O13: 699.3453,
found: m/z 699.3453 [M + H]+.
5.3.5.
Methyl
5-acetamido-6-(1,2,3-triacetoxypropyl)-4-[2-(tert-butoxycarbonyl)-3-(2-(6-methoxynaphthale
n-2-yl)propanoyl)guanidino-4,5-dihydro-6H-pyran-2-carboxylate (14b)
According to the procedure similar to that for compound 14a, the reaction of amine 13
(180 mg, 0.42 mmol) in CH2Cl2 (5 mL) with compound 12b (169 mg, 0.42 mmol) in the
presence of HgCl2 (125 mg, 0.46 mmol) and Et3N (64 µL, 0.46 mmol) afforded the coupling
product 14b (293 mg, 89%). C38H48N4O14; white foam; TLC (EtOAc/hexane = 3:2) Rf = 0.26;
[α]24D +49.0 (c = 1.0, CH2Cl2); IR υmax (neat) 3280, 2933, 1748, 1606 cm–1; 1H NMR (CDCl3,
400 MHz, two tautomers (3:2)) δ 12.24 (0.6 H, s), 11.97 (0.4 H, s), 8.98 (0.6 H, d, J = 8.4 Hz),
8.39 (0.4 H, d, J = 7.2 Hz), 7.77–7.62 (3 H, m), 7.46 (0.4 H, d, J = 8.4 Hz), 7.33 (0.6 H, d, J =
8.4 Hz), 7.18–7.07 (2 H, m), 6.12 (0.6 H, d, J = 8.8 Hz), 5.87–5.78 (0.4 H, m), 5.42–5.37 (0.6
H, m), 5.30–5.23 (1 H, m), 5.23–5.18 (0.4 H, m), 5.09–4.97 (1 H, m), 4.69–4.59 (1 H, m),
4.29–4.05 (3 H, m), 3.95–3.82 (3 H, m), 3.82–3.66 (4 H, m), 2.18–1.97 (9 H, m), 1.89–1.78 (3
13
H, m), 1.62–1.52 (3 H, m), 1.43–1.28 (9 H, m); C NMR (CDCl3, 100 MHz, two tautomers
(3:2)) δ 187.6, 175.2, 170.6, 170.2, 170.09, 170.06, 169.8, 169.6, 169.5, 162.5, 161.3, 161.2,
24

ACCEPTED MANUSCRIPT
157.4, 157.1, 156.5, 156.1, 152.1, 144.0, 143.9, 137.2, 133.6, 133.5, 133.1, 128.9, 128.7,
128.6, 128.4, 127.3, 126.6, 126.3, 126.2, 125.6, 125.4, 118.7, 118.6, 109.8, 105.3, 105.1, 83.2,
79.0, 77.2, 76.8, 71.3, 71.0, 71.0, 67.6, 67.3, 61.9, 54.74, 54.69, 51.82, 51.76, 50.7, 49.2, 47.8,
46.3, 46.2, 27.5, 27.3, 22.5, 22.1, 20.4, 20.3, 17.8, 16.8; HRMS (ESI) calcd for C38H49N4O14:
785.3245, found: m/z 782.3236 [M + H]+.
5.3.6.
5-Acetamido-4-(3-octanoylguanidino)-6-(1,2,3-trihydroxypropyl)-4,5-dihydro-6H-pyran-2-ca
rboxylic acid (7a)
To a solution of compound 14a (58 mg, 0.083 mmol) in CH2Cl2 (1 mL) was added TFA
(1 mL). The mixture was stirred at room temperature for 1 h, and then concentrated under
reduced pressure. The residue was dissolved in CH3CN (1 mL), and then added 1 M KOH(aq)
(1 mL). The mixture was stirred at room temperature for 30 min, acidified by adding TFA,
and concentrated under reduced pressure. The residue was purified by reversed-phase
chromatography (RP-18; elution with gradients of MeOH in H2O (0−60%)) to afford the
desired compound 7a (29 mg, 76%). The purity of 7a was 97.2% as shown by HPLC on an
HC-C18 column (Agilent, 4.6 × 250 mm, 5 µm particle size), tR = 8.5 min [MeOH/H2O = 2:3
to 7:3 for 20 min then 7:3 to 100% H2O for 20 min] at a flow rate of 1.0 mL/min. C20H34N4O8;
white solid (hygroscopic); [α]24 +70.3 (c = 1.0, DMSO); IR υmax (KBr) 3398, 2929, 1696,
D
1
1637 cm–1; H NMR (CD3OD/CF3CO2D = 2:1, 400 MHz) δ 5.92 (1 H, d, J = 2.8 Hz), 4.72–
4.62 (1 H, m), 4.51 (1 H, d, J = 8.8 Hz), 4.40–4.30 (1 H, m), 3.92–3.80 (2 H, m), 3.78–3.69 (2
H, m), 2.43 (2 H, t, J = 7.2 Hz), 2.00 (3 H, s), 1.68–1.55 (2 H, m), 1.37–1.18 (8 H, m), 0.83 (3
H, t, J = 6.4 Hz); 13C NMR (CD3OD/CF3CO2D = 2:1, 100 MHz) δ 176.9, 174.4, 164.6, 154.9,
146.3, 106.3, 76.7, 70.7, 69.6, 63.8, 50.4, 37.2, 31.9, 29.12, 29.07, 24.5, 22.7, 21.8, 13.3;
HRMS (ESI) calcd for C20H35N4O8: 459.2455, found: m/z 459.2449 [M + H]+.
25

ACCEPTED MANUSCRIPT
5.3.7.
5-Acetamido-4-(3-(2-(6-methoxynaphthalen-2-yl)propanoyl)guanidino-6-(1,2,3-trihydroxypro
pyl)-4,5-dihydro-6H-pyran-2-carboxylic acid (7b)
According to the procedure similar to that for compound 7a, compound 14b (293 mg,
0.38 mmol) was treated with TFA in CH2Cl2 to remove the Boc group and subsequently
saponified with 1 M KOH(aq) in CH3CN to give the desired compound 7b (189 mg, 93%). The
purity of 7b was > 99% as shown by HPLC on an HC-C18 column (Agilent, 4.6 × 250 mm, 5
µm particle size), tR = 7.8 min [MeOH/H2O = 2:3 to 7:3 for 20 min then 7:3 to 100% H2O for
20 min] at a flow rate of 1.0 mL/min. C26H32N4O9; white solid (hygroscopic); [α]24D +28.9 (c
1
= 1.0, DMSO); IR υmax (KBr) 3415, 2938, 1691, 1605 cm–1; H NMR (CD3OD/CF3CO2D =
2:1, 400 MHz) δ 7.69–7.60 (3 H, m), 7.37–7.25 (2 H, m), 7.10–7.03 (1 H, m), 5.86 (1 H, s),
4.62–4.55 (1 H, m), 4.50–4.42 (1 H, m), 4.32–4.25 (1 H, m), 3.95–3.78 (6 H, m), 3.73–3.66 (2
H, m), 1.92 (3 H, s), 1.47 (3 H, d, J = 7.2 Hz); 13C NMR (CD3OD/CF3CO2D = 2:1, 100 MHz)
δ 177.8, 174.3, 164.5, 158.6, 155.1, 146.3, 134.8, 134.7, 134.6, 129.6, 129.5, 128.0, 126.9,
125.9, 119.5, 106.1, 105.9, 76.7, 70.7, 69.6, 63.8, 54.9, 50.3, 21.8, 17.6; HRMS (ESI) calcd
for C26H33N4O9: 545.2248, found: m/z 545.2249 [M + H]+.
5.3.8.
Ethyl
4-acetamido-5-(2-(tert-butoxycarbonyl)-3-octanoyl)guanidino-3-(pentyl-3-oxy)cyclohex-1-en
e-1-carboxylate (16a)
To a suspension of Tamiflu capsule (985 mg, 2.40 mmol) and NaHCO3 (1007 mg, 11.99
mmol) in THF/H2O (10 mL/10 mL) was added (Boc)2O (720 µL, 3.13 mmol). The mixture
was stirred at room temperature for 5 h. The organic layer was collected, and the aqueous
layer was washed with EtOAc. All organic phase was combined, dried over MgSO4, filtered,
and purified by silica gel chromatography (EtOAc/hexane = 3:7 to 4:6) to afford the
Boc-protecting oseltamivir 15 (907 mg, 92%). [31]
26

ACCEPTED MANUSCRIPT
To a solution of compound 15 (128 mg, 0.31 mmol) in CH2Cl2 (2 mL) was added TFA (2
mL). The mixture was stirred at room temperature for 1 h, and then concentrated under
reduced pressure to give the corresponding amine. The amine product was dissolved in
CH2Cl2 (4 mL), and compound 12a (98 mg, 0.31 mmol), HgCl2 (92 mg, 0.34 mmol), and
Et3N (216 µL, 1.55 mmol) were added. The mixture was stirred at room temperature under
argon for 6 h, filtered through Celite, and concentrated under reduced pressure. The residue
was diluted with EtOAc and washed with 1 M HCl(aq), saturated NaHCO3(aq), and brine. The
organic phase was dried over MgSO4, filtered, and purified by silica gel chromatography
(EtOAc/hexane = 2:3 to 1:1) to afford the coupling product 16a (141 mg, 77%). C30H52N4O7;
white foam; TLC (EtOAc/hexane = 1:1) Rf = 0.48; [α]24 –65.1 (c = 1.0, CH2Cl2); IR υmax
D
1
(neat) 3280, 2929, 1722, 1614 cm–1; H NMR (CDCl3, 400 MHz, two tautomers (4:1)) δ
12.18 (0.8 H, s), 12.12 (0.2 H, s), 9.19 (0.8 H, d, J = 8.4 Hz), 8.65 (0.2 H, d, J = 7.6 Hz), 6.75
(0.2 H, s), 6.71 (0.8 H, s), 6.42–6.31 (1 H, m), 4.39–4.25 (1 H, m), 4.14–3.89 (4 H, m), 3.27
(1 H, quin, J = 5.6 Hz), 2.71 (1 H, dd, J = 17.6, 5.2 Hz), 2.32–2.19 (3 H, m), 1.82 (0.6 H, s),
1.80 (2.4 H, s), 1.61–1.48 (2 H, m), 1.48–1.31 (13 H, m), 1.27–1.10 (11 H, m), 0.84–0.69 (9
H, m); 13C NMR (CDCl3, 100 MHz, two tautomers (4:1)) δ 187.6, 174.3, 170.2, 170.1, 165.8,
165.7, 163.2, 156.4, 155.9, 152.5, 137.6, 137.1, 128.8, 128.5, 82.9, 82.6, 82.2, 79.4, 75.3, 75.2,
60.69, 60.66, 53.6, 53.0, 47.9, 41.2, 37.6, 31.5, 31.3, 30.0, 29.5, 29.1, 29.0, 28.62, 28.59, 28.0,
27.9, 27.7, 25.88, 25.85, 25.5, 24.3, 23.0, 22.9, 22.4, 22.3, 13.9, 13.82, 13.77, 9.4, 9.3, 9.14,
9.08; HRMS (ESI) calcd for C30H53N4O7: 581.3914, found: m/z 581.3912 [M + H]+.
5.3.9.
Ethyl
4-acetamido-5-(2-(tert-butoxycarbonyl)-
3-(2-(6-methoxynaphthalen-2-yl)propanoyl)guanidino-3-(pentyl-3-oxy)cyclohex-1-ene-1-carb
oxylate (16b)
According to the procedure similar to that for compound 16a, compound 15 (157 mg,
0.38 mmol) was treated with TFA to give the corresponding amine, which reacted with 12a
27

ACCEPTED MANUSCRIPT
(151 mg, 0.38 mmol) in the presence of HgCl2 (114 mg, 0.42 mmol) and Et3N (265 µL, 1.90
mmol) to afford the coupling product 16b (225 mg, 89%). C36H50N4O8; white foam; TLC
(EtOAc/hexane = 1:1) Rf = 0.40; [α]24 –101.1 (c = 1.0, CH2Cl2); IR υmax (neat) 3287, 2933,
D
1722, 1606 cm–1; 1H NMR (CDCl3, 400 MHz, two tautomers (3:2)) δ 12.28 (0.4 H, s), 12.02
(0.6 H, s), 9.09 (0.4 H, d, J = 8.4 Hz), 8.52 (0.6 H, d, J = 8.0 Hz), 7.67–7.57 (3 H, m), 7.41
(0.6 H, d, J = 9.6 Hz), 7.29 (0.4 H, d, J = 9.6 Hz), 7.07–6.98 (2 H, m), 6.70 (0.4 H, s), 6.67
(0.6 H, s), 6.34 (0.4 H, d, J = 8.8 Hz), 5.37 (0.6 H, d, J = 9.2 Hz), 4.31–4.01 (3.4 H, m), 3.95–
3.85 (1 H, m), 3.82–3.69 (4 H, m), 3.51–3.46 (0.6 H, m), 3.28–3.22 (0.4 H, m), 2.98–2.92 (0.6
H, m), 2.72–2.62 (1 H, m), 2.32–2.22 (1 H, m), 1.80 (1.2 H, s), 1.53 (3 H, d, J = 7.2 Hz),
1.45–1.33 (9.4 H, m), 1.30–1.13 (8.4 H, s), 0.84–0.73 (4.2 H, m), 0.63 (1.8 H, t, J = 7.6 Hz);
13C NMR (CDCl3, 100 MHz, two tautomers (3:2)) δ 187.8, 175.1, 170.1, 169.9, 165.5, 162.9,
157.5, 157.2, 156.7, 156.1, 152.3, 137.6, 137.5, 137.3, 133.8, 133.7, 133.0, 129.0, 128.8,
128.7, 128.6, 128.4, 127.4, 126.8, 126.3, 126.2, 125.64, 125.55, 118.8, 118.7, 105.3, 83.1,
82.6, 82.1, 79.2, 76.1, 75.1, 60.6, 60.5, 54.93, 54.85, 53.9, 53.3, 51.0, 48.2, 48.1, 47.8, 29.7,
27.8, 27.6, 25.8, 25.6, 25.5, 25.3, 22.8, 22.5, 17.8, 17.0, 13.9, 9.2, 9.0, 8.8; HRMS (ESI) calcd
for C36H51N4O8: 667.3707, found: m/z 667.3702 [M + H]+.
5.3.10. 4-Acetamido-3-(pentyl-3-oxy)-5-(3-octanoylguanidino)cyclohex-1-ene-1-carboxylic
acid (8a)
To a solution of compound 16a (47 mg, 0.081 mmol) in CH2Cl2 (1 mL) was added TFA
(1 mL). The mixture was stirred at room temperature for 1 h, and then concentrated under
reduced pressure. The residue was dissolved in CH3CN (1 mL) and then added 1 M KOH(aq)
(1 mL). The mixture was stirred at room temperature for 30 min, acidified by adding TFA,
and concentrated under reduced pressure. The residue was purified by reversed-phase
chromatography (RP-18; elution with gradients of MeOH in H2O (0−75%)) to afford the
desired compound 8a (22 mg, 60%). The purity of 8a was > 99% as shown by HPLC on an
28