Peramivir conjugates as orally available agents against influenza H275Y mutant

Article abstract of DOI:10.1016/j.ejmech.2017.12.072

Peramivir is an efficacious neuraminidase (NA) inhibitor for treatment of influenza by intravenous administration. However, the efficacy of peramivir toward the H275Y mutant is appreciably reduced. To address this drawback, conjugation of peramivir with c

Full text of DOI:10.1016/j.ejmech.2017.12.072

Accepted Manuscript
Peramivir conjugates as orally available agents against influenza H275Y mutant
Peng-Cheng Wang, Din-Chi Chiu, Jia-Tsrong Jan, Wen-I. Huang, Yin-Chen Tseng,
Ting-Ting Li, Ting-Jen Cheng, Keng-Chang Tsai, Jim-Min Fang
PII:
S0223-5234(17)31103-0
10.1016/j.ejmech.2017.12.072
EJMECH 10050
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 18 October 2017
Revised Date: 15 December 2017
Accepted Date: 20 December 2017
Please cite this article as: P.-C. Wang, D.-C. Chiu, J.-T. Jan, W.-I. Huang, Y.-C. Tseng, T.-T. Li, T.-J.
Cheng, K.-C. Tsai, J.-M. Fang, Peramivir conjugates as orally available agents against influenza H275Y
mutant, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2017.12.072.
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ACCEPTED MANUSCRIPT
Peramivir Conjugates as Orally Available Agents against Influenza H275Y Mutant
a,e
a,e
b
b
b
Peng-Cheng Wang, Din-Chi Chiu, Jia-Tsrong Jan, Wen-I Huang, Yin-Chen Tseng,
b
b
c,d
a,b,
Ting-Ting Li, Ting-Jen Cheng, Keng-Chang Tsai, and Jim-Min Fang *
a
Department of Chemistry, National Taiwan University, Taipei 106, Taiwan
b
The Genomics Research Center, Academia Sinica, Taipei 115, Taiwan
c
National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei 112,
Taiwan
d
The Ph.D. Program for Medical Biotechnology, College of Medical Science and Technology,
Taipei Medical University, Taipei 110, Taiwan
e
P.-C. W and D.-C. C contributed equally to this work.
_
*
_________________________
Corresponding author. J.-M.F.: Department of Chemistry, National Taiwan University,
Taipei, 106, Taiwan. Tel.: (8862)33661663; fax: (8862)2367812. E-mail address:
jmfang@ntu.edu.tw.

ACCEPTED MANUSCRIPT
Abstract:
Peramivir is an efficacious neuraminidase (NA) inhibitor for treatment of influenza by
intravenous administration. However, the efficacy of peramivir toward the H275Y mutant is
appreciably reduced. To address this drawback, conjugation of peramivir with caffeic acid is
devised in this study to enhance the binding affinity with neuraminidases. The C2-OH group
of peramivir is elaborated to link with caffeate derivatives, giving the desired conjugates 8
and 9 that possess potent NA inhibitory activity against both wild-type and H275Y viruses
with the IC values in nanomolar range. The molecular modeling reveals that the caffeate
5
0
moiety of conjugate 9 prefers to reside in the 295-cavity of H275Y neuraminidase, thus
providing additional hydrogen bonds and hydrophobic interactions to compensate the reduced
binding affinity of the peramivir moiety due to Glu-276 dislocation in H275Y mutant. In
comparison with peramivir, the lipophilicity of conjugates 8 and 9 also increases by
incorporation of the caffeate moiety. Thus, conjugates 8 and 9 offer better effect to protect
MDCK cells from infection of H275Y virus with low EC value (~17 nM). Administration of
50
conjugates 8 or 9 by oral gavage is effective in treatment of mice that are infected by lethal
dose of wild-type or H275Y influenza viruses. Considering drug metabolism, since the ester
linkage in conjugate 8 is susceptible to hydrolysis in plasma, conjugate 9 with robust amide
linkage may be a better candidate for development into orally available anti-influenza drug
that is also active to mutant viruses.

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Keywords:
Peramivir, caffeic acid, conjugate, neuraminidase, inhibitor, influenza, drug resistance.
Research highlights
•
•
•
•
•
Peramivir-caffeate conjugates are potent inhibitors of influenza neuraminidases.
The 295-cavity in neuraminidase is found for binding of the caffeate moiety.
The conjugates at 17 nM protect MDCK cells from infection of H275Y mutant.
The conjugates are orally available for treatment of influenza-infected mice.
Compared with oseltamivir, the conjugates greatly improve survival rate of mice.
Abbreviations:
Ala, alanine; All, allyl; Asn, asparagine; Asp, aspartic acid; CDI, 1,1′-carbonyl diimidazole;
DIBAL-H, diisobutylaluminium hydride; DMAP, 4-dimethylaminopyridine, EDCI,
N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide; ESI, Electrospray ionization; Glu,
glutamic acid; His, histidine; 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 ;
PE, peramivir; TFA, trifluoroacetic acid; THF, tetrahydrofuran; Trp, tryptophan; Tyr, tyrosine;
ZA, zanamivir.

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1
. Introduction
Influenza is an acute respiratory disease with great virulence. Three anti-influenza agents,
zanamivir (ZA, 1), oseltamivir (OS, 2) and peramivir (PE, 4) that target the viral
neuraminidase (NA) are currently used in clinic by distinct administrations. Oseltamivir is an
orally available prodrug that is readily hydrolyzed in body to generate the corresponding
carboxylic acid (OC, 3) as the active NA inhibitor. Though these neuraminidase inhibitors are
efficacious anti-influenza drugs, therapies are sometimes strikingly found to be ineffective. In
particular, the pandemic H5N1 avian influenza in 2003 has resulted in a fatality rate over 80%
[
1, 2]. In addition to the drug resistance, extravagant inflammatory reactions induced by the
cross-species infection not only greatly complicate clinical treatment but also cause increased
death toll.
To reduce the risk of drug-resistance in influenza treatment, the combination
chemotherapy consisting of two or more drugs for targeting different viral proteins or host
immune response has been applied to provide additive or synergistic antiviral activities [3–5].
However, using multi-component drug-cocktails may have drawback in complex
pharmacokinetics and unpredictable drug-drug interaction [6]. In an alternative approach,
conjugate inhibitors are designed to incorporate multiple drugs by covalent linkage [7, 8].
Such approach is especially valuable in controlling over complex disease systems and
soothing drug resistance by targeting multiple proteins with a conjugate compound to offer

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multifunction.
In our previous studies, this strategy has been utilized to develop better anti-influenza
drugs. For example, compounds 6 and 7 (Fig. 1), which are prepared by conjugation of
zanamivir with caffeic acid (5), not only increases the lipophilicity but also improves overall
efficacy in protection of Madin-Darby canine kidney (MDCK) cells against influenza
infection [9, 10]. Furthermore, the conjugate compounds 6 and 7 exhibit superior effect in
treatment of mice that are lethally infected by human or avian influenza viruses. Nonetheless,
compounds 6 (cLog P = −1.63) and 7 (cLog P = −2.36) with low lipophilicity are still not
ideal for oral administration. In comparison, peramivir (cLog P = 0.43) is more lipophilic than
zanamivir (cLog P = −4.13). We thus investigated whether peramivir conjugates 8 (cLog P =
1
.65) and 9 (cLog P = 0.69) with appropriate lipophilicity can be developed into oral drugs?
According to the crystal structure of peramivir-NA complex [11, 12], the C2-OH group is
feasible to conjugation because it does not directly interact with the amino acid residues in the
NA active site. In this study, the peramivir derivatives 10, 11 and 12 were also prepared to
compare their anti-influenza activities.

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Fig. 1. Chemical structures of anti-influenza drugs and the conjugate derivatives.
2
. Results and discussions
2
.1. Chemical Synthesis
The appropriate caffeic acid derivatives 16a and 16b for conjugation with peramivir
were first synthesized (Scheme 1). The alkylation reaction of caffeic acid (5) with excess allyl
bromide in the presence of K CO gave a per-allylation product 13, which was then subjected
2
3
to saponification to afford carboxylic acid 14. The coupling reactions of acid 14 with
N-Boc-3-aminopropanol and N-Boc-1,3-propanediamine were promoted by EDCI and DMAP
to afford ester 15a and amide 15b, respectively. Removal of the Boc protecting groups in 15a
and 15b with trifluoroacetic acid formed the aminium salts 16a and 16b, respectively.
Alternatively, allyl ester 13 was reduced with diisobutylaluminium hydride to yield alcohol 17.

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The alkylation reaction of 17 with 3-azidopropyl methanesulfonate was carried out to furnish
ether 18, which underwent Staudinger reduction to give amine 19 in an excellent yield.
On the other hand, the peramivir derivative 20 was prepared according to the previously
reported method [13]. The hydroxyl group was activated with 1,1′-carbonyldiimidazole (CDI)
to give an imidazolide intermediate 21. Without further purification, imidazolide 21 was
treated with aminium salt 16a in the presence of Et N to give the intermediate carbamate,
3
which was successfully converted to the desired peramivir-caffeate conjugate 8 by a sequence
of reactions, including saponification with 1 M KOH, deallylation with Pd(PPh ) and
3
4
removal of the Boc groups with TFA. It was noted that the methyl ester was selectively
hydrolyzed without disrupting the linkage of caffeate ester. Starting with the coupling reaction
of imidazolide 21 with 16b, the peramivir-caffeate conjugate 9 with amide linkage was
synthesized by a procedure similar to that for 8.
The peramivir conjugate 10 having an ether linkage was also synthesized by a similar
approach. Imidazolide 21 was treated with amine 19 to furnish compound 22. After
saponification of the methyl ester, the allylic groups on the phenyl ring were selectively
removed by the catalysis of Pd(PPh ) at room temperature. Since the cinnamyl ether was
3
4
highly sensitive to acidic conditions, the C=C bond was reduced by the Pd-catalyzed
hydrogenation prior to using TFA for removal of the Boc groups.

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Scheme 1. Synthetic pathway of conjugate products 8–12. Reagents and reaction conditions: (a) allyl bromide,
o
K CO , acetone, reflux, 23 h, 61%; (b) 1 M NaOH, MeOH, 60 C, 4 h, 81%; (c) EDCI, DMAP, CH Cl , rt, 5 h,
2
3
2
2
o
1
5a (53%), 15b (71%); (d) TFA, CH Cl , rt, 1−2 h; (e) DIBAL-H, toluene, –78 to 0 C, 2 h, 89%; (f) NaH, THF,
2 2
rt, 25 h, 48%; (g) PPh , THF/H O = 9:1, rt, 13 h, 99%; (h) CDI, THF, rt, 22 h; (i) Et N, MeCN, rt, 4–22 h, 22
3
2
3
(
62% from 20); (j) KOH, THF, rt, 1–1.5 h; (k) Pd(PPh ) , morpholine, THF, rt, 3 h; (l) TFA, CH Cl , rt, 14–22 h,
3 4 2 2
8
1
(30% from 20), 9 (19% from 20), 10 (32% from 22), 12 (59%); (m) H (1 atm), Pd/C, MeOH, rt, 9 h; (n) H O,
2 2
o
00 C, 1.5 h, 84%. All = allyl, CDI = 1,1′-carbonyl diimidazole; DIBAL-H = diisobutylaluminium hydride;
DMAP = 4-(dimethylamino)pyridine; EDCI = N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide; TFA =
trifluoroacetic acid; THF = tetrahydrofuran.

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To understand the contribution of caffeic residue to anti-influenza activity as well as the
possible enzyme-catalyzed hydrolysis of the ester and amide linkages, we also synthesized the
analogous compounds 11 and 12, which were presumed to be the hydrolytic products of the
peramivir conjugates 8 and 9. Noticeably, deprotection of the Boc groups of 23 was
performed in boiling water, [14] instead of using TFA that could lead to a side product by
acylation of the primary alcohol.
2
.2. Enzymatic and cell-based assays
Table 1 shows the neuraminidase inhibition and anti-influenza activity of the peramivir
conjugate compounds in comparison with OC (3) and PE (4). The NA inhibitory activity was
measured by employing 2-(4-methylumbelliferyl)-α- -N-acetyl-neuraminic acid (MUNANA)
D
as the fluorogenic substrate. The anti-influenza activity was evaluated by the cytopathic effect
of Madin-Darby canine kidney (MDCK) cells under viral infection.
The peramivir conjugates 8−12 were potent NA inhibitors with the IC50 values in
nanomolar range against the wild-type H1N1 virus. The cell-based assay indicated that
compounds 8−10 possessed higher anti-influenza activities than OC and PE. The clinically
relevant H275Y mutant is well known for its severe resistance to oseltamivir and peramivir
[
15, 16]. Replacement of the histidine residue at the 275-position of wild-type H1N1 virus by
tyrosine will push the neighboring Glu-276 away, and cause a large dislocation of the side

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chain (i.e. pentoxy in OC and pentyl in PE), thus decreasing the hydrophobic interactions in
the NA active site. To our delight, the peramivir conjugates 8, 9 and 10 were very potent NA
inhibitors against wild-type H1N1 virus (IC = 0.44−1.05 nM), and still active to the H275Y
5
0
mutant with low IC values (10.5−15.3 nM). Of importance, the conjugates 8 and 9 exhibited
5
0
appreciable protection effect of MDCK cells against H275Y mutant infection with EC of
5
0
1
5.5−18.1 nM, outperforming PE and OC by 40−200 fold.
Table 1
Neuraminidase inhibition, anti-influenza activity, partition and distribution coefficients of the
peramivir conjugate compounds.
a
b
c
wild-type virus
H275Y mutant
MDCK
f
f
cmpd
cLog P
cLog D
d
e
d
e
c
IC (nM)
EC (nM)
IC (nM)
50
EC (nM)
CC (nM)
5
0
50
50
50
5
3
4
8
9
0.99 ± 0.40
16.6 ± 4.0
272 ± 64
23.1 ± 9.3
10.5 ± 2.2
10.9 ± 1.7
15.3 ± 2.2
35.4 ± 7.3
34.3 ± 3.0
3520 ± 1260
643 ± 164
15.5 ± 3.5
18.1 ± 2.3
71.6 ± 5.5
1010 ± 478
405 ± 47
>10
−0.48
0.43
−2.12
−1.84
0.00
5
0.48 ± 0.08 6.68 ± 4.82
1.05 ± 0.67 1.68 ± 0.46
0.44 ± 0.10 1.56 ± 0.02
0.67 ± 0.05 3.50 ± 1.73
0.64 ± 0.07 2.22 ± 0.25
0.94 ± 0.04 2.58 ± 1.48
>10
5
>10
1.65
5
>10
0.69
−0.96
−0.07
−2.42
−2.57
5
1
0
>10
1.72
5
11
>10
−0.78
−0.88
5
1
2
>10
a
b
c
A/WSN/1933 (H1N1) influenza virus.
H275Y mutant of A/WSN/1933 virus.
All of the test compounds exhibit 50% cytotoxic concentration (CC ) higher than 100 µM against
50

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Madin-Darby canine kidney (MDCK) cells.
d
e
f
Half maximal inhibitory concentration against influenza neuraminidase.
Concentration of NA inhibitor for 50% protection of the cytopathic effects due to infection of influenza virus.
The calculated partition coefficient (cLog P) and distribution coefficient (cLog D) were obtained using
MarvinSketch (https://www.reaxys.com/reaxys/js/sre_5_3_1_05/child_java.jsp).
The conjugates 8 and 9 were further selected to test their activities against various
neuraminidase subtypes (Table 2). Both compounds showed comparable activities to PE (4).
Taking lipophilicity into consideration, medication involving 9 would be more effective due
to the improved bioavailability. We also noted that compounds 4, 8 and 9 all showed
relatively lower activity, in sub-micromolar IC , against the E119A and R292K mutants of
5
0
N2 and N9 viruses in comparison with the H275Y mutant of N1 viruses. We speculated that
mutation of the E119 or R292 ionic residue would mitigate the electrostatic interactions with
inhibitor in the NA active site.
Table 2
Inhibitory activity of PE conjugates 8 and 9 against various influenza NAs.
IC (nM)
5
0
a
neuraminidase
strain
4
8
9
b
H1N1
wild-type
H275Y
0.4 ± 0.1
3.6 ± 1.0
0.9 ± 0.1
8.6 ± 3.8
1.8 ± 0.6
5.3 ± 0.6

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b
b
H5N1
H3N2
wild-type
H275Y
0.3 ± 0.4
18.4 ± 3.9
2.0 ± 1.4
4.0 ± 0.1
73 ± 2
3.6 ± 0.1
10 ± 1
wild-type
E119A
27.2 ± 9.0
590 ± 263
426 ± 155
4.1 ± 0.5
11 ± 3
451 ± 138
146 ± 109
16.7 ± 5.9
92.5 ± 14.0
564 ± 208
640 ± 247
11.2 ± 2.1
166 ± 10
R292K
b
H7N9
wild-type
R292K
c
ND
a
b
c
Influenza recombinant neuraminidases were prepared from HEK293 cells, and the purified enzymes were used
for this study.
Influenza viruses: A/WSN/1933 (H1N1), A/Vietnam/1194/2004 (H5N1), A/Brisbane/10/2007 (H3N2), and
A/Shanghai/01/2014 (H7N9).
Not determined.
2
.3. Molecular modeling
Referring to our previous research [17, 18], the long extended caffeic moiety may locate
in a pockect like 150, 371 or 430-cavity, and thus enhance the inhibitory activity against
influenza neuraminidase. As a consequence, we employed a molecular modeling experiment
for compound 9 in H275Y neuraminidase to reveal the hidden facts of inhibitor-protein
interactions. Out of our expectation, the 3,4-dihydroxycinammyl group prefers to reside in a
cavity surrounded by the 247, 295 and 347 loops, namely 295-cavity that is not previously
reported (Fig. 2A). In this cavity, compound 9 utilizes the C=O group of the carbamate moiety
to form a hydrogen bond with Tyr-347, and the para-OH group on the phenyl ring to form

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another hydrogen bond with Trp-295 (Fig. 2B). In addition, the aryl ring of 9 provides
hydrophobic interactions with Ala-346 and Asn-247. By this disposition of compound 9, the
N–H of the carbamate group also shares a hydrogen bond with Asp-151 in 150-cavity. Due to
the structural similarity, compounds 8 and 10 are considered to apply the similar binding
mode with H275Y neuraminidase. All these interacitons may compensate the loss of binding
affinity of peramivir in H275Y due to the Glu-276 translocation that is caused by replacement
of His-275 with a larger size of tyrosine [15, 19]. Thus, the extended caffeate side chain
contributes to the outstanding potency of the peramivir conjugates.
Fig. 2. Schematic representations of the docking experiment of compound 9 with H275Y
mutant (N1 neuraminidase, PDB code: 3CL0) in a view of (A) van der Waals surface to
identify the binding pockets; (B) amino acid residues to display inhibitor-protein interactions.
2
.4. Animal experiments
MarvinSketch was utilized to predict the partition coefficients (cLog P) and distribution
coefficients (cLog D). As shown in Table 1, compounds 8, 9 and 10 are predicted to have

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adequate lipophilicity for oral administration. To find out the most auspicious candidate for
development into orally available anti-influenza drug, we thus performed the in vivo
experiments by oral administration of the test compound to the mice infected by WSN or
H275Y viruses (Fig. 3). Oseltamivir (2) or peramivir (4) were used as the positive control,
while phosphate buffered saline (PBS) was used as the negative control. Oseltamivir is an oral
drug requiring a dose of 3 mg/kg/day (i.e. 481 µmol/50 kg/day) for treatment of influenza
infected patients. Taking 10 times metabolic rate of mouse, the equivalent dose for a mouse
weighing 20 g is 1.92 µmol/day. In a preliminary experiment (Fig. 3A), mice was challenged
with WSN viruses and then treated with test compound at a dose of 0.96 µmol/day for
consecutive 5 days. While the peramivir derivative 10 only provided partially protection of
mice with 50% survival at day 14, the peramivir conjugate 9 secured 100% survival of mice,
indicating the superior effect by conjugation of a caffeate moiety to peramivir. Furthermore,
the peramivir-caffeate conjugates 8 and 9 at a reduced dose of 0.72 µmol/day shows better
protection activity in mice survival than oseltamivir at a dose of 0.96 µmol/day (Fig. 3B).
As the H275Y mutant is resistant to oseltamivir, only 20% of the H275Y infected mice
survived at day 10 on treatment with oseltamivir (Fig. 3C). In comparison, all the peramivir
derivatives 8, 9 and 10 rendered significantly better mice survival than oseltamivir.
Particularly, 90% of the mice receiving treatment of compound 9 survived at day 14. Inferring
from the time-course in recording the body weights of the mice challenged with H275Y

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viruses (Fig. 3D), compounds 8, 9 and 10 also helped mice to recover their weights sooner
than oseltamivir. In addition, the weight loss of mice treated with peramivir derivatives were
significantly smaller compared with the control groups. These observations indicated that the
faster rehabilitation was enabled to abate illness severity.
Fig. 3. Using peramivir conjugates for treatment of the influenza virus infected mice. BALB/c
mice were intranasally challenged with 10 mouse LD of viruses. The experiments were
5
0
conducted with 10 mice per group. (A) Survival rate of mice challenged by A/WSN/1933
H1N1) viruses and treated with the test compound at a dose of 0.96 µmol/20 g/day. (B)
(
Survival rate of mice challenged by WSN viruses and treated with OS (0.96 µmol/20 g/day)
or peramivir conjugate at a reduced dose (0.72 µmol/20 g/day). Survival rate (C) and average
body weight (D) of mice challenged by WSN/H275Y viruses and treated with the test
****
***
**
*
compound at a dose of 0.96 µmol/20 g/day.
.05.
P < 0.0001.
P < 0.001. P < 0.01. P <
0

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2
.5. Stability study
As shown in Table 1, the peramivir ester conjugate 8 and amide conjugate 9 possess
similar lipophilicity and anti-influenza activity. However, compounds 8 and 9 express
somewhat distinct efficacies in the mice experiments. The discrepancy may originate from the
tendency on cleavage of cinnamyl ester and amide through enzymatic process. To support our
speculation, we treated compounds 8 and 9 in rabbit serum, and examined the possible
degradative products 11 and 12 by HPLC and ESI−MS analyses (Fig. 4). According to the
HPLC diagram (Fig. 4A), alcohol 11 was generated from ester 8 on incubation in rabbit serum
o
at 37 C for 24 h, and the content of 11 was estimated to be 26% by the ratio of integral area
affiliated with the molar absorptivity determined at 214 nm wavelength (Fig. 4B). This ratio is
well correlated to that in the mass spectrum (Fig. 4C). In a sharp contrast, the peramivir amide
conjugate 9 was robust in rabbit serum, and only minute amount of the possible degradative
product 12 could be detected in the ESI−MS spectrum (Fig. 4D). Even ester 8 and amide 9
possess similar activity against H275Y virus with close EC values (15.5 and 18.1 nM),
50
survival of the H275Y infected mice on receiving treatment with 8 was less than that treated
with 9 (Fig. 3C). This phenomenon could be attributable to a partial degradation of ester 8 to
alcohol 11, which is much less active to H275Y mutant (EC ≈ 1 µM).
5
0

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Fig. 4. (A) HPLC diagram of peramivir ester conjugate 8 after incubation with rabbit serum at
o
3
7 C for 24 h. (B) Calibration curve of integral area versus concentration of 8 and 11 to
determine their molar absorptivity at a wavelength of 214 nm. (C) and (D) ESI−MS
characterizations of the degradative products 11 and 12 from the hydrolysis of ester 8 and
amide 9 in rabbit serum for 24 h incubation, respectively.
3
. Conclusion
The efficacy of peramivir toward H275Y influenza virus is significantly reduced by a less

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favorable interaction of the 3-pentyl side chain with the translocated Glu-276 in the mutant
neuraminidase. To solve the problem of drug-resistance, we elaborate peramivir by
conjugation with caffeic acid, which provides additional interactions in the H275Y
neuraminidase to compensate the loss of binding affinity. The structure of peramivir contains
a C2-OH that does not directly interact with the NA active site, granting us an opportunity of
derivatization. The C2-OH in a peramivir derivative 20 is first activated as the imidazolide,
which is readily connected with appropriate caffeate derivatives to afford peramivir-caffeate
conjugates such as compounds 8 and 9 with ester or amide linkage (Scheme 1).
The enzymatic and cell-based assays indicate that conjugates 8 and 9 are potent NA
inhibitors against the wild-type H1N1 virus and H275Y mutant with the IC and EC values
50
50
in nanomolar range. Interestingly, the conjugates 8 and 9 offer higher activity than the
parental peramivir by 40 fold to protect MDCK cells from infection of H275Y mutant by
incorporation of the caffeate moiety (Table 1). Moreover, conjugates 8 and 9 with adequate
lipophilicity facilitate oral administration for treatment of mice that are infected by lethal dose
of wild-type or H275Y influenza viruses (Fig. 3). Since the ester linkage in conjugate 8 is
vulnerable to hydrolysis in plasma, conjugate 9 with robust amide linkage is a more
promising candidate for further development into an orally available and expedient
anti-influenza drug that is still active to mutant viruses.

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4
. Experimental section
4
.1. General
All the reagents were commercially available and used without further purification
unless indicated otherwise. All solvents were anhydrous grade unless indicated otherwise.
Reactions 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.
1
13
Melting points were recorded on a Yanaco melting point apparatus. H and C NMR spectra
were recorded on Varian Unity Plus-400 (400 MHz) or Bruker AVIII FT-NMR (500 MHz)
spectrometers. Chemical shifts were given in δ values relative to tetramethylsilane (TMS);
coupling constants J were given in hertz (Hz). Internal standards were CHCl (δ = 7.24 ppm),
3
H
1
CD OH (δ = 3.31 ppm) or HDO (δ = 4.81 ppm) for H-NMR spectra; and CDCl (δ = 77.0
3
H
H
3
C
ppm for the central line of triplet) or CD OD (δ = 49.2 ppm for the central line of septet) was
3
C
1
3
for C-NMR spectra. The splitting patterns are reported as s (singlet), d (doublet), t (triplet), q
(quartet), quin (quintet), m (multiplet), dd (double of doublets), ddd (double of doublets of
doublets), ddt (doublet of doublet of triplet), dt (double of triplet) and td (triple of doublet).
Infrared (IR) spectra were recorded on Nicolet Magna 500−II spectrometer. Optical rotations
were recorded on digital polarimeter of Japan JASCO P-2000. [α] values were given in units
D
–
1
2
–1
of 10 deg cm g . Electrospray ionization (ESI) mass spectra were recorded on a

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high-resolution mass spectrometer.
4
.2. Material
Influenza A/WSN/1933 (H1N1) virus was obtained from Dr. Shin-Ru at Chang Gung
University in Taiwan. 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
o
(
GibcoBRL) at 37 C under 5% CO .
2
The oseltamivir-resistance A/WSN H275Y mutant was created using a 12-plasmid
system that was based on cotransfection of mammalian cells with 8 plasmids encoding virion
sense RNA under the control of a human PolI promoter and 4 plasmids encoding mRNA
encoding the RNP complex (PB1, PB2, PA and nucleoprotein gene products) under the
control of a PolII promoter. An H275Y mutation was introduced in the NA gene, and the
sequence was confirmed to generate the A/WSN H275Y virus.
4
.3. Synthetic procedures and characterization of compounds
Oseltamivir (2) [20, 21], oseltamivir carboxylic acid (3) [20, 21] and peramivir (4) [22]

ACCEPTED MANUSCRIPT
were prepared according to the previously reported procedures. Purity of synthetic
compounds was assessed to be ≥ 95% by HPLC analysis.
4
.3.1.
(1S,2S,3R,4R)-3-[(S)-1-Acetamido-2-ethylbutyl]-2-[[3-((E)-3-(3,4-dihydroxyphenyl)acryloylo
xy)propyl]carbamoyloxy]-4-guanidinocyclopentane-1-carboxylic acid (8)
According to the known procedure [10], the alkylation reaction of caffeic acid with allyl
bromide (4 equiv) in the presence of K CO gave a per-allylation product 13 (61% yield),
2
3
which was subjected to saponification to afford carboxylic acid 14 (81% yield). The coupling
reaction of acid 14 with tert-butyl (3-hydroxypropyl)carbamate [23] was performed in the
presence
of
-(dimethylamino)pyridine (DMAP) to give ester 15a (53% yield). To a solution of carbamate
5a (317 mg, 0.76 mmol) in CH Cl (2 mL) was added TFA (2 mL). The mixture was stirred
N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide
(EDCI)
and
4
1
2
2
at room temperature for 2 h, and concentrated under reduced pressure to give the
corresponding amine 16a (as the TFA salt).
The peramivir derivative 20 was prepared according to the previously reported
method.[13] To a solution of alcohol 20 (349 mg, 0.64 mmol) in anhydrous THF (2.3 mL) was
added 1,1′-carbonyldiimidazole (CDI) (313 mg, 1.93 mmol). The mixture was stirred at room
temperature for 22 h, then partitioned between EtOAc and water. The organic layer was

ACCEPTED MANUSCRIPT
washed with brine, dried over MgSO and filtered. The filtrate was concentrated under
4
reduced pressure to give imidazolide 21. A solution of the intermediate imidazolide 21 (404
mg, 0.63 mmol) and Et N (0.18 mL, 1.29 mmol) in CH CN (11 mL) was added to another
3
3
solution of the above-prepared aminium salt 16a in CH CN (2 mL). The mixture was stirred
3
at room temperature for 8 h, then concentrated under reduced pressure. The residue was
partitioned between CH Cl and brine, and the organic layer was dried over MgSO , filtered,
2
2
4
and concentrated under reduced pressure. The residue was purified by flash chromatography
silica gel, EtOAc/hexane 1:3 to 1:1 gradients) to afford a carbamate product (410 mg, 72%
yield based on compound 20). C H N O ; foam; TLC (EtOAc/hexane = 1:1) R = 0.21; [α]
(
4
5
67
5
13
f
2
D
5
−1 1
−
51.2 (c = 1.0, CHCl ); IR νmax (neat) 3322, 2966, 1716, 1650, 1507, 1258, 1025 cm ; H
3
NMR (400 MHz, CDCl ) δ 11.41 (1 H, s), 8.56 (1 H, br), 8.24 (1 H, d, J = 10.4 Hz), 7.53 (1 H,
3
d, J = 16.0 Hz), 7.03−7.01 (2 H, m), 6.81 (1 H, d, J = 8.8 Hz), 6.22 (1 H, d, J = 15.6 Hz),
6
.07−6.00 (2 H, m), 5.79 (1 H, t, J = 6.0 Hz), 5.40 (1 H, ddd, J = 7.2, 3.2, 1.6 Hz), 5.35 (1 H,
ddd, J = 7.2, 3.2, 1.6 Hz), 5.26−5.22 (2 H, m), 5.03 (1 H, dd, J = 6.8, 2.8 Hz), 4.60−4.57 (4 H,
m), 4.48 (1 H, br), 4.24−4.08 (3 H, m), 3.68 (3 H, s), 3.21 (2 H, q, J = 6.8 Hz), 2.92 (1 H, td, J
=
8.4, 3.2 Hz), 2.42 (1 H, ddd, J = 13.2, 8.0, 8.0 Hz), 2.26 (1 H, t, J = 7.2 Hz), 2.04 (3 H, s),
1
1
1
.89−1.82 (2 H, m), 1.79−1.72 (1 H, m), 1.45 (9 H, s), 1.43 (9 H, s), 1.40−1.24 (3 H, m),
1
3
.23−1.01 (2 H, m) 0.81−0.74 (6 H, m); C NMR (100 MHz, CDCl ) δ 173.9, 169.9, 167.1,
3
62.5, 156.5, 155.6, 152.9, 150.4, 148.4, 144.5, 132.9, 132.8, 127.5, 122.6, 117.8 (2 ×), 115.7,

ACCEPTED MANUSCRIPT
113.2, 112.4, 83.8, 79.9, 78.5, 69.8, 69.6, 61.6, 52.2, 51.9, 50.3, 49.2, 48.0, 44.7, 37.6, 34.9,
2
9.0, 28.1 (3 ×), 27.9 (3 ×), 23.9, 22.8, 21.7, 11.3, 11.0; ESI–HRMS calcd for C H N O :
4
5
68
5
13
+
8
86.4814, found: m/z 886.4813 [M + H] .
To a solution of the as-synthesized carbamate (80 mg, 0.09 mmol) in THF (2 mL) was
added 1 M KOH (2 mL, 2 mmol). The mixture was stirred at room temperature for 1 h,
neutralized with Dowex 50W×8 (acid form) and filtered. The filtrate was concentrated under
reduced pressure to afford the corresponding carboxylic acid. To the above-prepared
carboxylic acid (51 mg, 0.06 mmol) in anhydrous THF (1 mL) were added Pd(PPh ) (10 mg,
3
4
0
.009 mmol) and morpholine (0.1 mL, 1.1 mmol). The mixture was stirred at room
temperature for 3 h, then concentrated under reduced pressure. The residue was partitioned
between EtOAc and 1 M HCl. The organic layer was washed with brine, dried over MgSO4,
concentrated under reduced pressure. The residue was purified by flash chromatography
(
silica gel, MeOH/CH Cl 1:25 to 1:16 gradients) to give the corresponding
2 2
3
0
,4-dihydroxyphenyl compound. The above prepared 3,4-dihydroxyphenyl compound (32 mg,
.04 mmol) was dissolved in CH Cl (1 mL), and TFA (0.06 mL, 0.78 mmol) was added to
2
2
o
the solution at 0 C. The mixture was stirred at room temperature for 14 h, and concentrated
under reduced pressure. The residue was purified on a reversed-phase RP-18 column
(
MeOH/H O = 1:1) to afford the guanidine compound 8 (22 mg, 41% yield for 2 steps). The
2
purity of product 8 was 98.6% as shown by HPLC on an HC-C18 column (Merck, 4.6 × 100

ACCEPTED MANUSCRIPT
mm, 2 µm particle size), t = 7.0 min [MeOH/(0.1% TFA aqueous solution) = 9:11 to 1:1] at a
R
o
25
flow rate of 1.0 mL/min. C H N O ; yellow solid, mp 207–209 C; [α]
D
−34.8 (c = 1.0,
2
8
41
5
9
−1 1
MeOH); IR νmax (neat) 3342, 2966, 1663, 1528, 1258, 1176, 1164 cm ; H NMR (400 MHz,
CD OD) δ 7.54 (1 H, d, J = 15.6 Hz), 7.06 (1 H, s), 6.93 (1 H, d, J = 7.6 Hz), 6.77 (1 H, d, J =
3
8
.4 Hz), 6.26 (1 H, d, J = 16.0 Hz), 5.33 (1 H, d, J = 4.4 Hz), 4.47 (1 H, d, J = 11.2 Hz), 4.20
2 H, t, J = 6.0 Hz), 3.95−3.91 (1 H, m), 3.29−3.19 (2 H, m), 2.78−2.63 (3 H, m), 1.99 (3 H, s),
.92−1.87 (2 H, m), 1.74−1.67 (2 H, m), 1.45−1.43 (1 H, m), 1.26−1.20 (1 H, m), 1.13−1.04
(
1
1
3
(
2 H, m), 1.00−0.93 (6 H, m); C NMR (100 MHz, CD OD) δ 181.8, 173.8, 169.4, 158.0,
3
1
4
5
57.9, 149.8, 147.2, 147.0, 127.8, 123.2, 116.6, 115.2, 115.1, 79.9, 63.2, 57.4, 53.1, 51.2, 50.4,
5.9, 38.9, 37.5, 30.4, 23.8, 23.0, 22.4, 13.3, 13.1; ESI–HRMS calcd for C H N O :
28
42
5
9
+
92.2983, found: m/z 592.2973[M + H] .
4
.3.2.
(1S,2S,3R,4R)-3-[(S)-1-Acetamido-2-ethylbutyl]-2-[[3-((E)-3-(3,4-dihydroxyphenyl)acrylamid
o)propyl]carbamoyloxy]-4-guanidinocyclopentane-1-carboxylic acid (9)
The coupling reaction of acid 14 with tert-butyl (3-aminopropyl)carbamate was
performed in the presence of EDCI and DMAP to give amide 15b (71% yield). To a solution
of carbamate 15b (317 mg, 0.76 mmol) in CH Cl (2 mL) was added TFA (2 mL). The
2
2
mixture was stirred at room temperature for 1 h, and concentrated under reduced pressure to

ACCEPTED MANUSCRIPT
give the corresponding amine 16b (as the TFA salt).
To a CH CN solution (2 mL) of the above-prepared aminium salt 16b (327 mg, 0.76
3
mmol) and Et N (0.18 mL, 1.29 mmol) was added another CH CN solution (11 mL) of
3
3
imidazolide 21 (404 mg, 0.63 mmol) that was prepared from acid 20 and CDI (313 mg, 1.93
mmol). The mixture was stirred at room temperature for 8 h, then concentrated under reduced
pressure. The residue was partitioned between CH Cl and brine. The organic layer was dried
2
2
over MgSO , filtered, and concentrated under reduced pressure. The residue was purified by
4
flash chromatography (silica gel, EtOAc/hexane 1:2 to 3:2 gradients) to afford the
corresponding carbamate (347mg, 62% yield based on compound 20). C H N O ; foam;
4
5
68
6
12
25
D
TLC (EtOAc); R = 0.22; [α]
−72.6 (c = 1.0, CHCl ); IR ν
(neat) 3301, 2929, 1728,
max
f
3
−1
1
1
654, 1373, 1258, 1140 cm ; H NMR (400 MHz, CDCl ) δ 11.42 (1 H, s), 8.57 (1 H, d, J =
3
8
.0 Hz), 8.25 (1 H, d, J = 9.6 Hz), 7.47 (1 H, d, J = 15.6 Hz), 7.02−7.00 (2 H, m), 6.80 (2 H, d,
J = 8.0 Hz), 6.29 (1 H, d, J = 15.6 Hz), 6.09−5.98 (2 H, m), 5.81−5.80 (1 H, m), 5.39 (2 H, dd,
J = 17.2, 5.2 Hz), 5.25 (2 H, d, J = 10.4 Hz), 5.11 (1 H, dd, J = 6.8, 2.8 Hz), 4.59 (2 H, s),
4
.58 (2 H, s), 4.49 (1 H, quin, J = 8.4 Hz), 4.12 (1 H, t, J = 8.4 Hz), 3.70 (3 H, s), 3.40−3.38
2 H, m), 3.28−3.21 (1 H, m), 3.17−3.13 (1 H, m), 2.92−2.88 (1 H, m), 2.48−2.40 (1 H, m),
.29−2.25 (2 H, m), 2.08 (3 H, s), 1.83−1.70 (3 H, m), 1.48 (9 H, s), 1.45 (9 H, s), 1.37−1.25
(
2
1
3
(
3 H, m), 1.19−1.12 (1 H, m), 0.84−0.77 (6 H, m); C NMR (100 MHz, CDCl ) δ 173.9,
3
1
70.0, 166.3, 162.6, 157.1, 155.7, 153.0, 149.8, 148.4, 140.1, 133.1, 133.0, 128.3, 121.9,

ACCEPTED MANUSCRIPT
119.2, 117.8 (2 ×), 113.5, 112.6, 83.9, 80.0, 78.6, 69.9, 69.7, 52.3, 51.8, 50.4, 49.3, 48.0, 44.8,
3
7.6, 36.0, 34.8, 29.4, 28.2 (3 ×), 27.9 (3 ×), 23.9, 22.8, 21.8, 11.4, 11.0; ESI–HRMS calcd for
+
C H N O : 885.4973, found: m/z 885.4968 [M + H] .
45
69
6
12
By the procedure similar to that for compound 8, the as-synthesized carbamate (241 mg,
.27 mmol) was subjected to saponification in 1 M KOH, followed by removal of the allyl
groups with Pd(PPh ) and then removal of the Boc groups with TFA to afford the guanidine
0
3
4
compound 9 (48 mg, 30% yield). The purity of product 9 was 95.0% as shown by HPLC on
an HC-C18 column (Merck, 4.6 × 100 mm, 2 µm particle size), t = 2.9 min [MeOH/(0.1%
R
TFA aqueous solution) = 9:11] at a flow rate of 1.0 mL/min. C H N O ; pale yellow solid,
2
8
42
6
8
o
25
D
mp 220–222 C; [α]
−48.2 (c = 1.0, MeOH); IR νmax (neat) 3318, 1654, 1552, 1389, 1270,
−1 1
1
115 cm ; H NMR (400 MHz, CD OD) δ 7.39 (1 H, d, J = 16.0 Hz), 7.01 (1 H, d, J = 2.4
3
Hz), 6.90 (1 H, dd, J = 8.4, 2.0 Hz), 6.76 (1 H, d, J = 8.0 Hz), 6.36 (1 H, d, J = 16.0 Hz), 5.34
(
1 H, d, J = 4.4 Hz), 4.48 (1 H, d, J = 10.4 Hz), 3.97−3.91 (1 H, m), 3.35−3.33 (2 H, m), 3.17
2 H, t, J = 6.8 Hz), 2.76−2.60 (3 H, m), 1.98 (3 H, s), 1.76−1.66 (4 H, m), 1.47−1.42 (1 H, m),
(
1
.24−1.22 (1 H, m), 1.12−1.03 (2 H, m), 0.98 (3 H, t, J = 6.8 Hz), 0.93 (3 H, t, J = 6.8 Hz);
1
3
C NMR (100 MHz, CD OD) δ 181.9, 173.7, 169.6, 158.1, 158.0, 148.9, 146.9, 142.4, 128.4,
3
1
22.3, 118.5, 116.6, 115.2, 79.9, 57.4, 53.0, 51.3, 50.2, 45.9, 39.4, 38.0, 37.6, 31.1, 23.8, 23.0,
+
2
2.4, 13.3, 13.1; ESI–HRMS calcd for C H N O : 591.3142, found: m/z 591.3140 [M + H] .
2
8
43
6
8

ACCEPTED MANUSCRIPT
4
.3.3.
(1S,2S,3R,4R)-3-[(S)-1-Acetamido-2-ethylbutyl]-2-[[3-((E)-3-(3,4-bis(allyloxy)phenyl)propox
y)propyl]carbamoyloxy]-4-guanidinocyclopentane-1-carboxylic acid (10)
A solution of ester 13 (2.00 g, 6.66 mmol) in anhydrous toluene (20 mL) was stirred and
o
cooled to −78 C. DIBAL-H (16.64 mmol, 13.87 mL of 1.2 M solution in toluene) was added
o
o
dropwise via syringe pump. The mixture was stirred at –78 C for 1 h and 0 C for another 1 h.
o
The reaction was quenched with water at 0 C, and the mixture was filtered through a pad of
Celite. The filtrate was partitioned between EtOAc and water. The organic layer was washed
with brine, dried over MgSO , and concentrated under reduced pressure. The residue was
4
purified by flash chromatography (silica gel, EtOAc/hexane 1:4 to 1:3 gradients) to afford
1
.46 g (89% yield) of (E)-3-[3,4-bis(allyloxy)phenyl]prop-2-en-1-ol (17). C H O ; white
15 18 3
o
solid, mp 37−38 C; TLC (EtOAc/hexane = 1:1) R = 0.41; IR ν
(neat) 3354, 2921, 1650,
max
f
−1 1
1
516, 1269, 1139, 1021 cm ; H NMR (400 MHz, CDCl ) δ 6.94 (1 H, d, J = 1.2 Hz), 6.88 (1
3
H, dd, J = 8.0, 1.6 Hz), 6.81 (1 H, d, J = 8.4 Hz), 6.50 (1 H , d, J = 16.0 Hz), 6.23−6.16 (1 H,
m), 6.12−6.01 (2 H, m), 5.43−5.37 (2 H, m), 5.29−5.24 (2 H, m), 4.61−4.58 (4 H, m),
1
3
4
1
.28−4.27 (2 H, m), 1.57 (1 H, br); C NMR (100 MHz, CDCl ) δ 148.1, 148.0, 133.1, 133.0,
3
30.4, 129.9, 126.5, 119.7, 117.4 (2 ×), 113.7, 111.7, 69.8, 69.7, 63.4; ESI–HRMS calcd for
+
C H O Na: 269.1154, found: m/z 269.1164 [M + Na] .
15
18
3
To a solution of NaH (650 mg, 16.25 mmol, 60% suspension in mineral oil) in anhydrous

ACCEPTED MANUSCRIPT
THF (2 mL) under an atmosphere of nitrogen was added dropwise a solution of alcohol 17
o
(
1.00 g, 4.06 mmol) in anhydrous THF (18 mL) at 0 C. The mixture was stirred at room
temperature for 30 min, and then a solution of 3-azidopropyl methanesulfonate (1.09 g, 6.09
mmol) in THF (10 mL) was added dropwise. The mixture was stirred at room temperature for
o
2
5 h, quenched with water at 0 C, and concentrated under reduced pressure. The residue was
partitioned between EtOAc and water. The organic layer was washed with brine, dried over
MgSO , and concentrated under reduced pressure. The residue was purified by flash
4
chromatography (silica gel, EtOAc/hexane 1:18 to 1:15 gradients) to afford 642 mg (48%
yield)
C H N O ; yellow oil; TLC (EtOAc/hexane = 1:3) R = 0.57; IR ν (neat) 3379, 2925,
max
of
(E)-1,2-bis(allyloxy)-4-[3-(3-azidopropoxy)prop-1-en-1-yl]benzene
(18).
18
23
3
3
f
−1 1
1
683, 1597, 1516, 1270, 1136 cm ; H NMR (400 MHz, CDCl ) δ 6.95 (1 H, d, J = 1.6 Hz),
3
6
.89 (1 H, dd, J = 8.4, 2.0 Hz), 6.81 (1 H, d, J = 8.4 Hz), 6.49 (1 H, d, J = 15.6 Hz), 6.14−6.01
(3 H, m), 5.43−5.37 (2 H, m), 5.28−5.24 (2 H, m), 4.60 (4 H, m), 4.10 (2 H, d, J = 6.4 Hz),
1
3
3
.54 (2 H, t, J = 6.0 Hz), 3.41 (2 H, t, J = 6.4 Hz), 1.87 (2 H, quin, J = 6.0 Hz); C NMR (100
MHz, CDCl ) δ 148.3, 148.2, 133.3, 133.2, 132.3, 129.9, 123.8, 119.9, 117.5 (2 ×), 113.8,
3
111.8, 71.7, 70.0, 69.9, 66.8, 48.6, 29.4.
To a solution of azide 18 (608 mg, 1.85 mmol) in water (0.67 mL) and THF (6 mL) was
added PPh (969 mg, 3.69 mmol). The mixture was stirred for 13 h at room temperature,
3
concentrated under reduced pressure, and purified by flash chromatography (silica gel,

ACCEPTED MANUSCRIPT
MeOH/CH Cl2 1:40 to 1:10 gradients) to afford 554 mg (99% yield) of
2
(
E)-3-[(3-(3,4-bis(allyloxy)phenyl)allyl)oxy]propan-1-amine (19). C H NO ; yellow oil;
18 25 3
TLC (MeOH/CH Cl = 1:5) R = 0.09; IR ν (neat) 3330, 2925, 1654, 1511, 1270, 1123
max
2
2
f
−1 1
cm ; H NMR (400 MHz, CDCl ) δ 6.94 (1 H, d, J = 2.0 Hz), 6.87 (2 H, dd, J = 8.8, 2.0 Hz),
3
6
.46 (1 H, d, J = 15.6 Hz), 6.13−5.99 (3 H, m), 5.40 (1 H, dd, J = 7.2, 1.2 Hz), 5.36 (1 H, dd,
J = 7.2, 1.2 Hz), 5.25 (1 H, d, J = 1.2 Hz), 5.23 (1 H, d, J = 1.6 Hz), 4.59−4.55 (4 H, m), 4.07
2 H, d, J = 6.0 Hz), 3.53 (2 H, t, J = 6.0 Hz), 3.38 (2 H, br), 2.89 (2 H, t, J = 6.8 Hz), 1.80 (2
(
1
3
H, quin, J = 6.4 Hz); C NMR (100 MHz, CDCl ) δ 148.5, 148.4,133.4, 133.3, 132.3, 130.0,
3
1
24.0, 120.0, 117.6 (2 ×), 113.8, 111.8, 71.7, 69.9, 69.8, 68.2, 39.3, 31.7; ESI–HRMS calcd
+
for C H NO : 304.1913, found: m/z 304.1910 [M + H]
1
8
26
3
To a solution of alcohol 20 (349 mg, 0.64 mmol) in anhydrous THF (2.3 mL) was added
CDI (313 mg, 1.93 mmol). The mixture was stirred at room temperature for 22 h, and
partitioned between EtOAc and water. The organic layer was washed with brine, dried over
MgSO , filtered, and concentrated under reduced pressure to give the corresponding
4
imidazolide 21. To a solution of amine 19 (231 mg, 0.76 mmol) and Et N (0.18 mL, 1.29
3
mmol) in CH CN (2 mL) was added another solution of imidazolide 21 (404 mg, 0.63 mmol)
3
in CH CN (11 mL). The mixture was stirred at room temperature for 4 h, concentrated under
3
reduced pressure, and partitioned between CH Cl and brine. The organic layer was dried over
2
2
MgSO , filtered, and concentrated under reduced pressure. The residue was purified by flash
4