1,2,3-Triazole-containing derivatives of rupestonic acid: Click-chemical synthesis and antiviral activities against influenza viruses

Article abstract of DOI:10.1016/j.ejmech.2014.02.029

Two series of rupestonic acid derivatives, (1-substituted-1H-1,2,3-triazol- 4-yl)methyl 2-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen- 5-yl)acrylate and N-(1-substituted-1H-1,2,3-triazol-4-yl)methyl 2-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl) acrylamide were easily and efficiently synthesized via click chemistry. These compounds were tested for their in vitro activities against various strains of influenza A virus (H1N1, oseltamivir resistant H1N1, H3N2) and influenza B virus. The results showed that nine compounds were active against the H1N1 strain of influenza A virus and among them the best one 14a, was as active as the reference drugs, Oseltamivir and Ribavirin. Some of them were also active on the Oseltamivir resistant H1N1 strain. In regards to influenza B virus, twenty-one compounds over thirty were active and seven of them 7b, 8b, 9b, 10a, 11b, 12b, 13b showed better activity than Ribavirin. The structure-activity relationship of these compounds is discussed on the basis of each type of the viruses studied. Furthermore, four best representative compounds 7b, 10a, 12b and 14a were evaluated in a plaque assay experiment using MDCK cells and RBV as control compound and the results showed that 7b, 10a and 12b were better than RBV in inhibiting plaque formation, in good accordance with their anti-influenza B activities.

Full text of DOI:10.1016/j.ejmech.2014.02.029

Accepted Manuscript
1,2,3-Triazole-containing derivatives of rupestonic acid: Click-chemical synthesis and
antiviral activities against influenza viruses
Yao-Wu He, Chang-Zhi Dong, Jiang-Yu Zhao, Lin-Lin Ma, Yu-Huan Li, Haji Akber
Aisa
PII:
S0223-5234(14)00149-4
10.1016/j.ejmech.2014.02.029
EJMECH 6746
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 April 2013
Revised Date: 31 January 2014
Accepted Date: 10 February 2014
Please cite this article as: Y.-W. He, C.-Z. Dong, J.-Y. Zhao, L.-L. Ma, Y.-H. Li , H.A. Aisa,
1,2,3-Triazole-containing derivatives of rupestonic acid: Click-chemical synthesis and antiviral
activities against influenza viruses, European Journal of Medicinal Chemistry (2014), doi: 10.1016/
j.ejmech.2014.02.029.
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ACCEPTED MANUSCRIPT
Highlights:
•
•
•
Triazole derivatives of rupestonic acid (RA) prepared using click chemistry;
Some of them more active against Influenza A (H1N1) and B viruses than RA;
Best compounds 4 times more efficient than Ribavirin against Influenza B virus.

ACCEPTED MANUSCRIPT
1
,2,3-Triazole-containing derivatives of rupestonic acid: Click-chemical synthesis and
antiviral activities against influenza viruses
1, 2
2,3,*
1
4
4
Yao-Wu He , Chang-Zhi Dong , Jiang-Yu Zhao , Lin-Lin MA , LI Yu-Huan , Haji Akber
1
,*
Aisa
Key Laboratory of Plant Resources and Chemistry in Arid Regions, Xinjiang Technical Institute
of Physics and Chemistry, Chinese Academy of Sciences, Beijing South Road 40–1, Urumqi, Xinjiang,
1
8
30011, P. R. China
2
Université Paris Diderot, Sorbonne Paris Cité, ITODYS, UMR 7086 CNRS, 15 rue J-A de Baïf,
7
5205 Paris Cedex 13, France.
3
School of Light Industry and Chemical Engineering, Guangdong University of Technology,
Guangzhou, 510006, P. R. China.
4
Institute of Medicinal Biotechnology, Chinese Academy of Medical Science and Peking Union
Medical College, Beijing, 100050, P. R. China
Corresponding authors. Tel. : +33 1 57 27 88 81, fax : +33 1 57 27 72 63, E-mail address:
dong@univ-paris-diderot.fr (C. Z. Dong); Tel. : +86 991 368 06 35, fax : +86 991 383 89 57,
haji@ms.xjb.ac.cn (H. A. Aisa)
1

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Abstract: Two series of rupestonic acid derivatives, (1-substituted-1H-1,2,3-triazol-4-yl)methyl
2
-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylate
N-(1-substituted-1H-1,2,3-triazol-4-yl)methyl
-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylamide were easily and
and
2
efficiently synthesized via click chemistry. These compounds were tested for their in vitro activities
against various strains of influenza A virus (H1N1, oseltamivir resistant H1N1, H3N2) and influenza B
virus. The results showed that nine compounds were active against the H1N1 strain of influenza
A
virus and among them the best one 14a, was as active as the reference drugs, Oseltamivir and Ribavirin.
Some of them were also active on the Oseltamivir resistant H1N1 strain. In regards to influenza B virus,
twenty-one compounds over thirty were active and seven of them 7b, 8b, 9b, 10a, 11b, 12b, 13b
showed better activity than Ribavirin. The structure-activity relationship of these compounds is
discussed on the basis of each type of the viruses studied. Furthermore, four best representative
compounds 7b, 10a, 12b and 14a were evaluated in a plaque assay experiment using MDCK cells and
RBV as control compound and the results showed that 7b, 10a and 12b were better than RBV in
inhibiting plaque formation, in good accordance with their anti-influenza B activities.
Keywords: Rupestonic acid; 1,2,3-triazole; click chemistry; influenza virus.
2

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1
.
Introduction
Influenza is a serious public health problem that causes severe illnesses and deaths for higher risk
populations [1]. It is an acute viral infection that spreads easily from person to person and anybody in
any age group. An epidemic can take an economic toll through reduction of workforce productivity,
and strain health services. Annual epidemics of influenza result in about 3 to 5 million cases of severe
illness, and up to 500 000 deaths worldwide [2]. The influenza is caused by influenza viruses which can
be classified into three types, A, B and C [3]. Type A influenza viruses are further divided into
subtypes according to different combinations of virus surface proteins. Among many subtypes of
influenza A viruses, currently H1N1 and H3N2 subtypes are circulating in human beings. The subtype
H7N9 has been reported since a few weeks to have infected humans in China, but no evidence has been
found for its capacity to be vehiculated from man to man. Influenza B viruses are normally found only
in humans. Although influenza type B viruses can cause human epidemics, they have not caused
pandemics. Type C influenza viruses are much less frequent than A and B [1].
To date, two types of small molecular drugs are available for the treatment of influenza.
Adamantine (amantadine and remantadine) are effective against the type A viruses and the inhibitors of
influenza neuraminidase, such as oseltamivir and zanamivir, are used for the treatment of both types A
and B viruses [4]. However, the amantidine is strictly limited actually because of rapid emergence of
the drug resistance and its CNS side effects [5]. Meanwhile, the oseltamivir resistant viruses have been
reported as well [6]. Therefore, it is necessary to develop novel and effective drugs to overcome the
limitations of the existing antiviral agents.
Natural products represented and will represent an important source of lead compounds for drug
discovery [7]. Artemisia rupestris L is a well-known medicinal plant in Xinjiang, China and has been
traditionally used for detoxification, anti-hypersusceptibility and protecting liver, also as antitumor,
antibacterial and antiviral agents [8]. It is the main ingredient in Compound Yizhihao Granule (Fufang
Yizhihao Keli, No. Z20026711), which is prescribed to treat colds in China since more than 10 years.
2
-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylic acid (rupestonic acid,
R.A.), isolated from Artemisia rupestris L [9], is a guaiane type sesquiterpene with multifunctional
groups. It shows anti-influenza virus activities and low toxicity (IC50 = 25.8 ꢀM and TC50 = 4653 ꢀM
against influenza H3N2 virus) [10]. In order to improve its efficacy, our research group has long been
3

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involved in synthesizing and testing rupestonic acid derivatives against influenza A and B viruses. The
rupestonic acid derivatives, bearing a 1,3,4-triazoles [10] or isoxazole moiety [11] we developed
recently, exhibit a good in vitro inhibitory activity against influenza A and B viruses. Unfortunately,
the high toxicity associated has limited their further development. Hence, it remains a challenge for us
to design and synthesize new derivatives with higher efficiency and less toxicity against influenza
virus.
1
,2,3-Triazole has been frequently used in drugs with a large spectrum of therapeutic properties,
such as anti-allergic, antifungal, antibacterial and antiviral (against HIV or influenza virus) activities
12-17]. Recent innovations in click chemistry have made 1,4-disubstituted-1,2,3-triazoles easily
[
accessible from alkynes and azides, through Huisgen 1,3-dipolar cycloaddition [18]. This copper (I)
catalyzed reaction is mild and very efficient, requiring no protecting groups and no purification in
7
many cases with the reaction rate accelerated up to 10 times. This has put it in a class of its own and
enabled many novel applications [19], in particular in the synthesis of drug-like molecules to accelerate
drug discovery process [20].
Starting from rupestonic acid, we report in this work the synthesis of its 1,2,3-triazole-containing
derivatives and their in vitro biological activities against influenza viruses. The structure-activity
relationship of these compounds is discussed on the basis of each type of the viruses studied.
Furthermore, in a plaque assay experiment three representative compounds were shown to be better
inhibitors than RBV, in good accordance with their anti-influenza B activities.
2
.
Results and discussion
.1. Chemistry
The synthetic route for the title compounds is shown in Scheme 1. Rupestonic acid was isolated
2
from raw roots of A. rupestris L. in a global yield of about 0.1%. It reacted with 3-bromoprop-1-yne or
prop-2-yn-1-amine to afford the compounds a or b containing a terminal alkyne function. In parallel,
substituted benzyl and alkyl azides (1-15) were prepared according to the experimental procedures
-
1
described in the literature [21] and characterized by the absorption at 2100 cm in the IR spectra,
corresponding to the azide function. They were used without further purification. The compounds a
and b were then submitted to the copper-catalyzed [3+2] cycloaddition with the azides (1-15) in the
presence of CuSO and sodium ascorbate in a non-homogeneous mixture of solvents (CH Cl /H O, 1:1,
4
2
2
2
4

ACCEPTED MANUSCRIPT
v/v) to give the rupestonic acid derivatives 1-15a and 1-15b in moderate to good yields (35% to 74%).
1
One aromatic singlet was observed in the corresponding H NMR spectra (δ about 7.50 ppm) and
can be attributed to the proton H-5’ of the triazole ring of all the final products. Besides of the NMR
data, the identity of all the rupestonic acid derivatives was further confirmed by elemental analysis, as
well as HRMS for the majority of the compounds.
Scheme 1. Synthesis of 1,2,3-triazole-containing derivatives of rupestonic acid. Reagents and
conditions: i) NaN , DMSO; ii) K CO , DMF; iii) EDCI, HOBt, DCM; iv) CuSO /Na-Ascorbate,
3
2
3
4
DCM/H O
2
2
.2. Antiviral activities against influenza viruses
The antiviral activities of the synthesized compounds against influenza A virus of the subtypes
FM/1/47/H1N1, oseltamivir resistant tianjinjinnan/15/2009/H1N1, hanfang/359/95/H3N2, as well as
influenza B virus of the subtype jifang/13/97 were evaluated by cytopathic effects (CPE) using MDCK
cells with oseltamivir and Ribavirin (RBV) as control drugs. The results are summarized in Table 1.
The cytotoxicity of each compound was determined and is reported in Table 1 as well. Since one
substituent to the triazole ring had not the same influence on the toxicity in the ester series as in the
amide series, the highest concentrations tested were not the same in both series for a given substituent.
When tested against influenza A virus, there were nine compounds (2a, 6a, 14a, 15a, 3b, 4b, 5b,
6
b and 14b) active against the strain A/FM/1/47/H1N1. The most active compound, 14a (IC = 2.82
50
µg/mL) is as active as the control drugs, Oseltamivir (IC50 = 2.81 µg/mL) and RBV (IC = 1.92
50
µg/mL). Unfortunately, its selectivity index is less interesting, due to its higher cytotoxicity than the
control drugs. Curiously, all compounds containing a triazole ring substituted by a halogenated (F or Cl)
benzyl group are inactive, no matter what is the nature or the number of the halogens. As for the others,
one substituent seems not to influence the antiviral activity in the same manner in the ester series as in
the amide one. The compound 2a in the ester series containing a 3-methylbenzyl substituent is active,
while the compound 2b in the amide series containing the same substituent is not. This is also the case
for the couples 3a/3b-5a/5b.
Interestingly, when tested against Oseltamivir-resistant influenza A virus (the strain of
5

ACCEPTED MANUSCRIPT
Tianjinjinnan/15/2009/H1N1), seven of the new rupestonic acid derivatives (2a, 6a, 7a, 15a, 13b, 14b
and 15b) were revealed active and the compounds 2a and 14b demonstrated the best activity (IC50
9.7 µg/mL), only about three times less active than RBV, the unique reference drug. But their
selectivity index is much worse than that of RBV, due to their relatively high cytotoxicity.
=
1
On the contrary to rupestonic acid, the new derivatives seem quite inefficient against the H3N2
subtype of influenza A virus, since only two of them (7a, 13b) display moderate potency against the
H3N2 strain, hanfang/359/95/H3N2, compared to the control compounds.
In brief, these new compounds are much more efficient against the H1N1 subtypes of influenza
A virus (H1N1, Oseltamivir resistant H1N1) than the H3N2 subtype. Their selectivity index is in
general moderate compared to the reference drugs, due to their high cytotoxicity and modest activity.
However, the results were much more encouraging when the new compounds were tested against
influenza B virus. More than two thirds of them are active (Table 1) and seven of them (10a, 7b, 8b,
9
b, 11b, 12b, 13b) possess a higher potency than RBV, more than four times better for the best ones.
In contrary to the results observed with influenza A viruses, the majority of the compounds bearing a
halogen atom or two showed interesting activity against influenza B virus. In addition, it seems that the
position of the halogen on the phenyl group has an influence on the antiviral activity. Introduction of a
chlorine atom to the position 2 (7a, 7b) or position 4 (9a, 9b) of the phenyl ring confers a better
potency than to the position 3 (8a, 8b). Note that the amide derivatives are generally more active
against the type B virus than the ester ones. On the other hand, the presence of a deactivating group on
the phenyl ring as F and Cl induces a beneficial effect to the activity, while an activating group as
methyl or methoxyl, no matter where it is attached to the phenyl ring, is detrimental to the activity.
Nevertheless, there are some exceptions to these general trends: 1) the compound 11a, 13a and 10b
were observed inactive although they possess one or more halogens on the phenyl ring, and 2) 2a in
which is found a methyl group at the position 2 of the phenyl ring is as active as 7a in which is found a
chlorine atom at the same position.
In brief, this SAR study seems indicating that an electron-withdrawing group on the phenyl ring in
particular at the positions 3 or 4 would increase the antiviral activity against influenza B virus.
Four representative compounds 10a, 14a, 7b and 12b were evaluated in a plaque assay experiment
using MDCK cells and Ribavirin (RBV) as control drug. The results (Table 2) demonstrated that the
6

ACCEPTED MANUSCRIPT
compounds 10a, 7b and 12b with good anti-influenza B activities were better inhibitors than RBV and
the compound 14a with good anti-influenza A activities, but lower anti-influenza B activities was as
good as the control drug. This result is consistent with the anti-viral activities of these compounds.
Insert Table 1 here
Insert Table 2 here
3
.
Conclusion
In this work, we have synthesized two series of rupestonic acid derivatives,
(
1-substituted-1H-1,2,3-triazol-4-yl)methyl esters and N-(1-substituted-1H-1,2,3-triazol-4-yl)methyl
amides. Their antiviral activity has been evaluated against the influenza A and B viruses. Certain
compounds display interesting potencies against influenza A viruses and the best one is as active as the
reference drugs to kill the H1N1 subtype virus. Unfortunately, the selectivity index of these compounds
is in general moderate compared to that of the controls. When tested against influenza B virus, the
results are really exciting, as seven of the new compounds are more active than RBV, the only
reference drug in this study. Although the selectivity index of these compounds remains to be a little
worse than RBV, it may be acceptable taking into account their better performance than the control
(
more than four times better). The SAR study did not give any clear correlation between the substituent
and the activity when they were evaluated against influenza A viruses. However, a deactivating group
on the phenyl ring seems beneficial to the antiviral activity against the type B virus. The results
obtained in this work will help us to adjust our research focus in this field and the best compounds in
this work are actually under investigation for their pharmacological properties.
4
.
Experimental Procedures and Characterization
.1. General Information
All chemicals and solvents were purchased from Aldrich, Sodipro and VMR. IR spectra were
4
1
13
recorded on Perkin Elmer Spectrum 100 FT-IR. H NMR and C NMR spectra were recorded on
Bruker AVANCE III 400 NMR. Results are presented as, chemical shift δ in ppm, multiplicity, J
values in Hertz (Hz), number of protons, proton’s position. Multiplicities are shown as the
7

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abbreviations: s (singlet), brs (broad singlet), d (doublet), t (triplet), m (multiplet). Elemental analyses
were performed for C, H and N by the Service de Microanalyse, ICSN-CNRS and were within ± 0.4%
of theoretical values. Mass spectrometry was performed using a QSTAR Elite LC/MS/MS system from
Applied Biosystems/MDS Sciex (Concord, ON, Canada) equipped with an electrospray ionization (ESI)
ion source. The reactions were monitored by TLC on Merck silica gel 60 F254 plates with UV and
iodine vapor detection. Commercial solvents in reagent grade were used without any treatment.
4
.2.
-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylic acid (Rupestonic
acid)
Raw roots of A. rupestris L. (2 kg) were extracted three times with 95% ethanol. The extracts
Preparation
of
2
were combined and evaporated to dryness under reduced pressure. The residue was dissolved in water,
then the solution was extracted three times with ethyl acetate, and the combined ethyl acetate layers
were extracted three times with 5% NaHCO . The NaHCO fractions were combined and neutralized to
3
3
pH 2 with conc. HCl, then extracted with ethyl acetate. After evaporation of the solvent to dryness, the
crude sample (400 g) was purified by silica gel chromatography (petroleum ether /EtOAc, 5:1 to 2:1,
1
v/v) to furnish the title compound (2.3 g) as colorless crystals; m.p. 131-132 °C; H NMR (400 MHz,
CDCl ) δ 6.37 (s, 1H, CH =C), 5.73 (s, 1H, CH =C), 3.18 (m, 1H, CHCH CO), 2.89 (m, 2H,
3
2
2
2
CHCCO H, CH C=C), 2.61 (dd, J = 18.0, 6.0 Hz, 1H, CH CO), 2.46 (dd, J = 19.2, 12.0 Hz, 1H,
2
2
2
CH C=C), 2.13 (m, 1H, CHCH ), 2.06 (d, J = 18.9 Hz, 1H, CH CO), 1.87 – 1.80 (m, 2H, CH CHCH ),
2
3
2
2
3
1
3
1
.80 – 1.66 (m, 2H, CH CH CHCH ), 1.64 (s, 3H, CH C=C), 0.66 (d, J = 7.1 Hz, 3H, CH CH);
C
2
2
3
3
3
NMR (100 MHz, CDCl ) δ 208.73, 174.93, 171.56, 145.86, 138.01, 125.55, 46.10, 41.44, 38.49, 37.85,
3
-
1
3
6.70, 35.42, 31.72, 12.23, 8.11 [10]; IR (v/cm ): 2952, 2923, 2897, 1712, 1660, 1617.
.3. General procedure for the synthesis of alkyl and aryl azides (1-15)
The title compounds were prepared according to the method previously reported [21]. Briefly,
4
alkyl or benzyl bromides or chlorides (5 mmol) and sodium azide (6 mmol) in DMSO (10 mL) were
stirred overnight. The reaction mixture was diluted with cold water (10 mL) and extracted with diethyl
ether. The combined organic layers were washed successively with water, brine and dried over
anhydrous sodium sulfate. The solvent was removed by rotary evaporator to produce liquid azides. The
azides (50-85% yields) were used in the following reaction without further purification.
8

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4
.4.
Synthesis
of
prop-2-ynyl
2
-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylate (a)
To a solution of rupestonic acid (1.24 g, 5.0 mmol) in dry DMF (10 mL) were added successively
anhydrous potassium carbonate (1.04 g, 7.5 mmol) and 3-bromoprop-1-yne (0.88 g, 7.5 mmol). The
resultant mixture was stirred at room temperature overnight and the reaction was stopped by addition of
water (20 mL). It was then extracted with diethyl ether and the organic layer was washed successively
with water, brine and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced
pressure and the crude product was purified by silica gel (60–120 mesh) column chromatography using
1
hexane–EtOAc, (1:1, v/v) as eluent to give the title compound as white oil (1.28 g, 90% yield); H
NMR (400 MHz, CDCl ) δ 6.29 (s, 1H, CH =C), 5.70 (s, 1H, CH =C), 4.78 (d, J = 2.5 Hz, 2H, CH O),
3
2
2
2
3
.20 – 3.17 (m, 1H, CHCH CO), 2.96 – 2.90 (m, 1H, CHCCO ), 2.86 – 2.80 (m, 1H, CH C=C), 2.59
2 2 2
(
ddd, J = 18.8, 6.6, 1.2Hz, 1H, CH CO), 2.49 (t, J = 2.4 Hz, 1H, CH), 2.46 (ddd, J = 19.4, 12.4, 1.2 Hz,
2
1
H, CH C=C), 2.17-2.10 (m, 1H, CHCH ), 2.04 (dt, J = 18.8, 1.6 Hz, 1H, CH CO), 1.89 – 1.74 (m, 3H,
2 3 2
CH CHCH , CH CH CHCH ), 1.68 – 1.60 (m, 4H, CH C=C, CH CH CHCH ), 0.66 (d, J = 7.1 Hz, 3H,
2
3
2
2
3
3
2
2
3
13
CH CH); C NMR (101 MHz, CDCl ) δ 208.21, 174.10, 166.09, 145.76, 138.01, 124.45, 75.10, 52.49,
3
3
-
1
4
1
5.99, 41.50, 38.35, 38.21, 36.70, 35.46, 31.78, 12.25, 8.13; IR (v/cm ): 3267, 2957, 2920, 2875, 1717,
+
688, 1627; HRMS (ESI) calcd for C H O (M+H) : 287.1647, found: 287.1655; Anal. Calcd. for
18
23
3
C H O ·0.22 CHCl : C, 70.00; H, 7.16; Found: C, 69.66; H, 7.17.
18
22
3
3
4
.5.
Synthesis
of
N-(prop-2-ynyl)
2
-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylamide (b)
To a stirred solution of rupestonic acid (1.24 g, 5.0 mmol) in dry DCM (10 mL) at 0°C were
added successively EDCI (1.15 g, 6.0 mmol), HOBt▪H O (0.92 g, 6.0 mmol),
2
N,N-diisopropylethylamine (1.0 mL, 6.0 mmol) and prop-2-yn-1-amine (0.41g, 7.5 mmol). After stirred
at room temperature overnight, the reaction mixture was diluted with DCM (10 mL), washed
successively with saturated NaHCO solution and water (three times). The organic layer was then dried
3
over anhydrous sodium sulfate and the solvent evaporated under reduced pressure. The crude product
was purified by silica gel (60–120 mesh) column chromatography using hexane–EtOAc (1:1, v/v) as
1
eluent to give the title compound as yellow oil (1.16 g, 81% yield); H NMR (400 MHz, CDCl ) δ 6.10
3
(
s, 1H, NH), 5.55 (s, 1H, CH =C), 5.37 (s, 1H, CH =C), 4.11 (dd, J = 5.3, 2.6 Hz, 2H, CH NH), 3.21 –
2 2 2
9

ACCEPTED MANUSCRIPT
3
1
.14 (m, 1H, CHCH CO), 2.96-2.90 (m, 1H, CHCCONH), 2.89-2.83 (m, 1H, CH C=C), 2.58 (ddd, J =
2
2
8.8, 6.6, 1.2 Hz, 1H, CH CO), 2.56 (t, J = 2.6 Hz, CH), 2.46 (ddd, J = 19.5, 12.2, 1.3 Hz, 1H,
2
CH C=C), 2.18 – 2.06 (m, 1H, CHCH ), 2.03 (dt, J = 18.8, 1.4 Hz, 1H, CH CO), 1.84-1.74 (m, 3H,
2
3
2
CH CHCH , CH CH CHCH ), 1.66-1.55 (m, 4H, CH C=C, CH CH CHCH ), 0.64 (d, J = 7.1 Hz, 3H,
2
3
2
2
3
3
2
2
3
13
CH CH); C NMR (101 MHz, CDCl ) δ 208.14, 174.18, 168.55, 151.21, 137.81, 115.55, 79.32, 71.84,
3
3
-
1
4
1
5.85, 41.38, 38.65, 38.12, 36.56, 35.33, 31.26, 29.47, 12.08, 7.99; IR (v/cm ): 3298, 2958, 2920, 1734,
+
683, 1659, 1619, 1519; HRMS (ESI) calcd for C H NO (M+H) : 286.1807, found: 286.1807; Anal.
1
8
24
2
Calcd. for C H NO ·0.25H O: C, 74.58; H, 8.17; N, 4.83; Found: C, 74.42; H, 8.18; N, 4.60.
18
22
3
2
4
.6.
-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylate
N-(1-substituted-1H-1,2,3-triazol-4-yl)methyl
-(5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylamide (1-15b)
Prop-2-ynyl 2-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylate or
N-(prop-2-ynyl) 2-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylamide
1.0 eq.) and alkyl or aryl azide (2.0 eq.) was dissolved in DCM/H O (1:1). To this solution, CuSO4 (1
General procedure for the synthesis of (1-substituted-1H-1,2,3-triazol-4-yl)methyl
2
(1-15a) and
2
(
2
eq.) and sodium ascorbate (2 eq.) were added. The reaction mixture was stirred at room temperature
overnight. After completion of the reaction, the product was extracted with DCM. The organic extract
was washed with water and brine. The solvent was removed under reduced pressure to afford crude
product, which was purified by column chromatography on silica gel using either cyclohexane/ ethyl
acetate (3:1 to 2:1, v/v) as eluent to furnish the compounds (1-15a), or MeOH/DCM (100:1 to 50:1, v/v)
as eluent to provide the compounds (1-15b).
4
.6.1. (1-(2-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl
2
-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylate (1a): yellow oil
1
(
70 % yield); H NMR (400 MHz, CDCl ) δ 7.42 (s, 1H, H-het), 7.31 – 7.14 (m, 4H, H-Ph), 6.22 (s, 1H,
3
CH =C), 5.63 (s, 1H, CH =C), 5.53 (s, 2H, NCH Ph), 5.28 (s, 2H, CH O), 3.22–3.07 (m, 1H,
2
2
2
2
CHCH CO), 2.88 (t, J = 11.0 Hz, 1H, CHCCO ), 2.78 (d, J = 19.6 Hz, 1H, CH C=C), 2.58 (dd, J =
2
2
2
1
2
4
8.8, 6.5 Hz, 1H, CH CO), 2.47 – 2.33 (m, 1H, CH C=C), 2.27 (s, 3H, CH -Ph), 2.12 (m, 1H, CHCH ),
2 2 3 3
.03 (d, J = 18.8 Hz, 1H, CH CO), 1.82 – 1.73 (m, 3H, CH CHCH , CH CH CHCH ), 1.71 – 1.54 (m,
2
2
3
2
2
3
1
3
H, CH C=C, CH CH CHCH ), 0.64 (d, J = 7.1 Hz, 3H, CH CH); C NMR (101 MHz, CDCl ) δ
3
2
2
3
3
3
1
0

ACCEPTED MANUSCRIPT
2
08.22, 174.20, 166.71, 145.94, 143.05, 137.97, 137.06, 132.35, 131.24, 129.62, 129.43, 126.85,
-
1
1
24.17, 123.62, 58.09, 52.59, 45.80, 41.33, 38.22, 36.68, 35.45, 31.77, 19.10, 12.23, 8.12; IR (v/cm ):
+
2
954, 2922, 2853, 1689, 1626, 1457; HRMS (ESI) calcd for C H N O (M+H) : 434.2444, found:
2
6
32
3
3
4
34.2440; Anal. Calcd. for C H N O ·1.5H O ·0.45 C H : C, 69.16; H,7.97; N, 8.43; Found: C, 69.35;
26 31 3 3 2 6 12
H, 7.72; N, 8.04.
4
.6.2.
(1-(3-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl
2
-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylate (2a): yellow oil
1
(
72.6% yield); H NMR (400 MHz, CDCl ) δ 7.53 (s, 1H, H-het), 7.27 – 7.06 (m, 4H, H-Ph), 6.22 (s,
3
1
H, CH =C), 5.63 (s, 1H, CH =C), 5.47 (s, 2H, NCH Ph), 5.28 (s, 2H, CH O), 3.18 – 3.13 (m, 1H,
2 2 2 2
CHCH CO), 2.88 (t, J = 11.2 Hz, 1H, CHCCO ), 2.78 (d, J = 19.0 Hz, 1H, CH C=C), 2.57 (ddd, J =
2
2
2
1
1
1
8.8, 6.6, 1.1 Hz, 1H, CH CO), 2.46 – 2.36 (m, 1H, CH C=C), 2.33 (s, 3H, CH -Ph), 2.14 – 2.08 (m,
2 2 3
H, CHCH ), 2.03 (dt, J = 18.8, 1.4 Hz, 1H, CH CO), 1.80-1.71(m, 3H, CH CHCH , CH CH CHCH ),
3
2
2
3
2
2
3
1
3
.64-1.56 (m, 4H, CH C=C, CH CH CHCH ), 0.64 (d, J = 7.1 Hz, 3H, CH CH); C NMR (101 MHz,
3
2
2
3
3
CDCl ) δ 208.38, 174.43, 166.72, 145.91, 143.18, 139.16, 137.94, 134.35, 129.75, 129.16, 128.98,
3
1
25.31, 124.19, 123.80, 58.08, 54.40, 45.98, 41.45, 38.20, 36.64, 35.41, 31.76, 21.43, 12.22, 8.10; IR
-
1
(
v/cm ): 3142, 2922, 1690, 1627, 1440; Anal. Calcd. for C H N O ·0.42 CHCl : C, 65.61; H, 6.55; N,
26 31 3 3 3
8
.69; Found: C, 65.21; H, 6.62; N, 8.46
.6.3.
4
(1-(4-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl
2
-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylate (3a): yellow oil
1
(
74.2% yield); H NMR (400 MHz, CDCl ) δ 7.51 (s, 1H, H-het), 7.17 (s, 4H, H-Ph), 6.22 (s, 1H,
3
CH =C), 5.62 (s, 1H, CH =C), 5.47 (s, 2H, NCH Ph), 5.28 (s, 2H, CH O), 3.21 – 3.10 (m, 1H,
2
2
2
2
CHCH CO), 2.88 (m, 1H, CHCCO ), 2.78 (d, J = 19.5 Hz, 1H, CH C=C), 2.57 (ddd, J = 18.7, 6.6, 1.0
2
2
2
Hz, 1H, CH CO), 2.41 (dd, J = 19.0, 12.0 Hz, 1H, CH C=C), 2.34 (s, 3H, CH -Ph), 2.11 (m, 1H,
2
2
3
CHCH ), 2.03 (d, J = 18.8 Hz, 1H, CH CO), 1.80 – 1.76 (m, 3H, CH CHCH , CH CH CHCH ),
3
2
2
3
2
2
3
1
3
1
.64-1.76 (m, 4H, CH C=C, CH CH CHCH ), 0.64 (d, J = 7.1 Hz, 3H, CH CH); C NMR (101 MHz,
3 2 2 3 3
CDCl ) δ 208.19, 174.19, 166.58, 145.86, 143.17, 138.97, 137.94, 131.44, 129.93, 128.30, 124.14,
3
-
1
1
2
23.67, 58.09, 54.19, 45.95, 41.47, 38.21, 36.66, 35.43, 31.75, 21.27, 12.22, 8.09; IR (v/cm ):2953,
+
923, 2853, 1691, 1627, 1458; HRMS (ESI) calcd for C H N O (M+H) : 434.2444, found: 434.2445;
2
6
32
3
3
1
1

ACCEPTED MANUSCRIPT
Anal. Calcd. for C H N O ·0.5 H O·0.5C H : C, 71.87; H, 7.90; N, 8.67; Found: C, 72.12; H, 7.76; N,
26
31
3
3
2
6
12
8
.42
4
.6.4
(1-benzyl-1H-1,2,3-triazol-4-yl)methyl
2
-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylate(4a): yellow oil
1
(
73.4% yield); H NMR (400 MHz, CDCl ) δ 7.54 (s, 1H, H-het), 7.42 – 7.25 (m, 5H, H-Ph), 6.22 (s,
3
1
H, CH =C), 5.63 (s, 1H, CH =C), 5.52 (s, 2H, NCH Ph), 5.28 (s, 2H, CH O), 3.22 – 3.08 (m, 1H,
2 2 2 2
CHCH CO), 2.97 – 2.83 (m, 1H, CHCCO ), 2.78 (dd, J = 19.5, 1.0 Hz, 1H, CH C=C), 2.57 (ddd, J =
2
2
2
1
1
8.7, 6.6, 1.1 Hz, 1H, CH CO), 2.48 – 2.33 (m, 1H, CH C=C), 2.21–2.07 (m, 1H, CHCH ), 2.01 (d, J =
2 2 3
8.9 Hz, 1H, CH CO), 1.81 – 1.69 (m, 3H, CH CHCH , CH CH CHCH ), 1.65 – 1.54 (m, 4H,
2
2
3
2
2
3
1
3
CH C=C, CH CH CHCH ), 0.64 (d, J = 7.1 Hz, 3H, CH CH); C NMR (101 MHz, CDCl ) δ 208.22,
3
2
2
3
3
3
1
4
1
74.23, 166.73, 145.99, 138.03, 134.48, 129.36, 129.09, 128.23, 124.13, 123.78, 58.13, 54.43, 45.99,
-
1
1.50, 38.39, 38.10, 36.70, 35.46, 31.79, 12.25, 8.14; IR (v/cm ): 2955, 2923, 2854, 1687, 1627, 1497,
+
455; HRMS (ESI) calcd for C H N O (M+H) : 420.2287, found: 420.2281; Anal. Calcd. for
25
30
3
3
C H N O ·0.3CHCl ·0.35C H : C, 67.88; H, 6.97; N, 8.67; Found: C, 67.74; H, 7.23; N, 8.38.
25
29
3
3
3
6
12
4
.6.5 (1-(3-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl
2
-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylate (5a): yellow oil
1
(
65% yield); H NMR (400 MHz, CDCl ) δ 7.54 (s, 1H, H-het), 7.31 – 7.27, 6.98 - 6.79 (m, 4H,
3
H-Ph), 6.23 (s, 1H, CH =C), 5.64 (s, 1H, CH =Cb), 5.49 (s, 2H, NCH Ph), 5.29 (s, 2H, CH O), 3.78 (s,
2
2
2
2
3
1
H, OCH ), 3.16 – 3.18 (m, 1H, CHCH CO), 2.89 (t, J = 11.6 Hz, 1H, CHCCO ), 2.79 (d, J = 20.3 Hz,
3 2 2
H, CH C=C), 2.58 (dd, J = 18.8, 6.6 Hz, 1H, CH CO), 2.42 (dd, J = 17.8, 9.3 Hz, 1H, CH C=C), 2.12
2
2
2
(
m, 1H, CHCH ), 2.04 (d, J = 20.2 Hz, 1H, CH CO), 1.89 – 1.67 (m, 3H, CH CHCH ,
3
2
2
3
1
3
CH CH CHCH ), 1.63 (m, 4H, CH C=C, CH CH CHCH ), 0.65 (d, J = 7.1 Hz, 3H, CH CH);
C
2
2
3
3
2
2
3
3
NMR (101 MHz, CDCl ) δ 208.34, 174.37, 166.72, 160.28, 145.92, 143.26, 137.95, 135.89, 130.38,
3
1
24.20, 123.83, 120.42, 114.35, 113.98, 58.09, 55.44, 54.33, 45.99, 41.47, 38.37, 38.06, 36.66, 35.43,
-
1
+
3
1.77, 12.23, 8.11; IR (v/cm ): 2958, 2923, 1689, 1627; HRMS (ESI) calcd for C H N O (M+H) :
26 32 3 4
4
50.2393, found: 450.2338; Anal. Calcd. for C H N O ·0.25 CHCl : C, 65.74; H, 6.57; N, 8.77; Found:
2
6
31
3
4
3
C, 65.85; H, 6.75; N, 8.54
1
2

ACCEPTED MANUSCRIPT
4
.6.6. (1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl
2
-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylate (6a): yellow oil
1
(
67.2% yield); H NMR (400 MHz, CDCl ) δ 7.50 (s, 1H, H-het), 7.23 (d, J = 8.8 Hz, 2H, H-Ph),
3
6
.89 (d, J = 8.7 Hz, 2H, H-Ph), 6.22 (s, 1H, CH =C), 5.63 (s, 1H, CH =C), 5.45 (s, 2H, NCH Ph), 5.28
2 2 2
(
s, 2H, CH O), 3.80 (s, 3H, OCH ), 3.23 – 3.12 (m, 1H, CHCH CO), 2.91-2.86 (m, 1H, CHCCO ),
2 3 2 2
2
1
1
=
.79 (dd, J = 19.5, 1.0 Hz, 1H, CH C=C), 2.58 (ddd, J = 18.7, 6.6, 1.1 Hz, 1H, CH CO), 2.49 – 2.35 (m,
2
2
H, CH C=C), 2.17 – 2.08 (m, 1H, CHCH ), 2.08 – 1.99 (dt, J = 18.8, 1.2 Hz , 1H, CH CO), 1.81 –
2
3
2
.70 (m, 3H, CH CHCH , CH CH CHCH ), 1.67 – 1.58 (m, 4H, CH C=C, CH CH CHCH ), 0.64 (d, J
2
3
2
2
3
3
2
2
3
13
7.1 Hz, 3H, CH CH); C NMR (101 MHz, CDCl ) δ 208.28, 174.27, 166.76, 160.20, 145.98, 143.18,
3
3
1
37.99, 129.89, 126.44, 124.17, 123.56, 114.69, 58.14, 55.50, 53.98, 46.01, 41.50, 38.24, 36.70, 35.46,
-
1
3
1.80, 29.85, 12.25, 8.13; IR (v/cm ): 2921, 1698, 1611, 1513; Anal. Calcd. for C H N O ·0.55
26 31 3 4
CHCl ·0.40 C H : C, 63.32; H, 6.68; N, 7.66; Found: C, 63.14; H, 6.88; N, 7.36.
3
6
12
4
.6.7. (1-(2-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl
2
-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylate (7a): yellow oil
1
(
66% yield); H NMR (400 MHz, CDCl ) δ 7.64 (s, 1H, H-het), 7.43 (dd, J = 7.9, 1.3 Hz, 1H,
3
H-Ph), 7.34 – 7.21 (m, 3H, H-Ph), 6.23 (s, 1H, CH =C), 5.66 (s, 2H, NCH Ph), 5.63 (s, 1H, CH =C),
2
2
2
5
0
1
3
3
1
3
.30 (s, 2H, CH O), 3.17 – 3.15 (m, 1H, CHCH CO), 2.90 – 2.88 (m, 1H, CHCCO ), 2.79 (dd, J = 19.5,
2 2 2
.9 Hz, 1H, CH C=C), 2.58 (ddd, J = 18.7, 6.6, 1.1 Hz, 1H, CH CO), 2.42 (ddd, J = 19.8, 12.4, 1 Hz,
2
2
H, CH C=C), 2.16 – 2.10 (m, 1H, CHCH ), 2.03 (dt, J = 18.8, 1.4 Hz , 1H, CH CO), 1.81 – 1.66 (m,
2
3
2
H, CH CHCH , CH CH CHCH ), 1.65 – 1.57 (m, 4H, CH C=C, CH CH CHCH ), 0.64 (d, J = 7.1 Hz,
2
3
2
2
3
3
2
2
3
13
H, CH CH); C NMR (101 MHz, CDCl ) δ 208.31, 174.30, 166.74, 145.97, 143.17, 137.99, 133.76,
3
3
32.33, 130.69, 130.57, 130.16, 127.79, 124.20, 124.15, 58.10, 51.66, 46.01, 41.50, 38.38, 38.14, 36.70,
-
1
5.46, 31.80, 12.25, 8.13; IR (v/cm ): 2922, 2852, 1699, 1624, 1447; HRMS (ESI) calcd for
+
C H N O Cl (M+H) : 454.1897, found: 454.1851; Anal. Calcd. for C H ClN O ·0.25 CHCl ·0.35
25
29
3
3
25 28
3
3
3
C H : C, 63.97; H, 6.38; N, 8.19; Found: C, 64.16; H, 6.60; N, 7.96.
6
12
4
.6.8. (1-(3-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl
2
-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylate (8a): yellow oil
1
(
47% yield); H NMR (400 MHz, CDCl ) δ 7.57 (s, 1H, H-het), 7.36 – 7.28 (m, 2H, H-Ph), 7.25
3
(
dd, J = 2.3, 1.8 Hz, 1H, H-Ph), 7.15 (dt, J = 6.9, 1.7 Hz, 1H, H-Ph), 6.24 (s, 1H, CH =C), 5.64 (s, 1H,
2
1
3

ACCEPTED MANUSCRIPT
CH =C), 5.50 (s, 2H, NCH Ph), 5.30 (s, 2H, CH O), 3.17 – 3.15 (m, 1H, CHCH CO), 2.92 – 2.86 (m,
2
2
2
2
1
2
H, CHCCO ), 2.79 (dd, J = 19.5, 1.0 Hz, 1H, CH C=C), 2.58 (ddd, J = 18.7, 6.6, 1.1 Hz, 1H, CH CO),
2 2 2
.42 (ddd, J = 19.3, 12.3, 1.2 Hz, 1H, CH C=C), 2.14 – 2.10 (m, 1H, CHCH ), 2.03 (dt, J = 18.8, 1.4
2
3
Hz, 1H, CH CO), 1.81 – 1.70 (m, 3H, CH CHCH , CH CH CHCH ), 1.65 – 1.58 (m, 4H, CH C=C,
2
2
3
2
2
3
3
1
3
CH CH CHCH ), 0.64 (d, J = 7.1 Hz, 3H, CH CH); C NMR (101 MHz, CDCl ) δ 208.28, 174.24,
2
2
3
3
3
1
5
1
6
66.76, 145.93, 143.53, 137.99, 136.44, 135.26, 130.61, 129.27, 128.28, 126.27, 124.26, 123.91, 58.06,
-
1
3.68, 46.00, 41.49, 38.24, 36.69, 35.45, 31.81, 27.04, 12.24, 8.13; IR (v/cm ): 2922, 2852, 1693, 1626,
435; Anal. Calcd. for C H ClN O ·0.25 CHCl ·0.35 C H : C, 63.97; H, 6.38; N, 8.19; Found: C,
25
28
3
3
3
6
12
4.18; H, 6.61; N, 8.09.
.6.9.
4
(1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl
2
-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylate (9a): yellow oil
1
(
53.5% yield); H NMR (400 MHz, CDCl ) δ 7.54 (s, 1H, H-het), 7.35 (d, J = 11.0 Hz, 2H, H-Ph), 7.21
3
(
d, J = 8.6 Hz, 2H, H-Ph), 6.23 (s, 1H, CH =C), 5.62 (s, 1H, CH =C), 5.49 (s, 2H, NCH Ph), 5.29 (s,
2
2
2
2
1
2
H, CH O), 3.17-3.16 (m, 1H, CHCH CO), 2.89 (dd, J = 16.2, 5.6 Hz, 1H, CHCCO ), 2.81 – 2.76 (m,
2 2 2
H, CH C=C), 2.58 (ddd, J = 18.7, 6.6, 1.1 Hz, 1H, CH CO), 2.46 – 2.37 (m, 1H, CH C=C), 2.18 –
2
2
2
.08 (m, 1H, CHCH ), 2.04 (m, J = 18.9 Hz, 1H, CH CO), 1.80 – 1.69 (m, 3H, CH CHCH ,
3
2
2
3
CH CH CHCH ), 1.65 – 1.55 (m, 4H, CH C=C, CH CH CHCH ), 0.65 (d, J = 7.1 Hz, 3H, CH CH);
2
2
3
3
2
2
3
3
13
C NMR (101 MHz, CDCl ) δ 208.19, 174.17, 166.70, 161.95, 159.48, 145.94, 137.96, 131.22, 130.82,
3
1
1
25.01, 124.08, 121.80, 116.13, 115.92, 58.07, 47.93, 45.96, 41.48, 38.36, 38.10, 36.59, 35.44, 31.76,
-
1
2.23, 8.10; IR (v/cm ): 2953, 2923, 2853, 1690, 1628, 1607, 1511; HRMS (ESI) calcd for
+
C H ClN O (M+H) : 454.1897, found: 454.1893; Anal. Calcd. for C H ClN O ·0.20 CHCl ·0.60
25
29
3
3
25 28
3
3
3
C H : C, 65.44; H, 6.75; N, 7.95; Found: C, 65.50; H, 6.82; N, 7.86.
6
12
4
.6.10.
(1-(2-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl
2
-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylate (10a): yellow oil
1
(
45.7% yield); H NMR (400 MHz, CDCl ) δ 7.63 (s, 1H, H-het), 7.43 – 7.05 (m, 4H, H-Ph), , 6.23 (s,
3
1
H, CH =C), 5.63 (s, 1H, CH =C), 5.58 (s, 2H, NCH Ph), 5.29 (d, J = 1.7 Hz, 2H, CH O), 3.17 – 3.16
2 2 2 2
(
m, 1H, CHCH CO), 2.89 (t, J = 11.1 Hz, 1H, CHCCO ), 2.79 (d, J = 19.5 Hz, 1H, CH C=C), 2.58 (dd,
2 2 2
J = 18.2, 7.2 Hz, 1H, CH CO), 2.42 (dd, J = 19.5, 12.2 Hz, 1H, CH C=C), 2.12 (m, 1H, CHCH ), 2.03
2
2
3
(
d, J = 18.8 Hz, 1H, CH CO), 1.82 – 1.68 (m, 3H, CH CHCH , CH CH CHCH ), 1.65 – 1.55 (m, 4H,
2 2 3 2 2 3
1
4

ACCEPTED MANUSCRIPT
1
3
CH C=C, CH CH CHCH ), 0.64 (dd, J = 7.1, 2.0 Hz, 3H, CH CH); C NMR (101 MHz, CDCl ) δ
3
2
2
3
3
3
2
1
3
4
8
08.19, 174.17, 166.70, 161.95, 159.48, 145.94, 137.96, 131.21, 130.83, 125.01, 124.18, 123.97,
-
1
21.80, 116.02, 58.07, 47.93, 45.96, 41.48, 38.36, 38.10, 36.59, 35.44, 31.76, 12.23, 8.10; IR (v/cm ):
+
143, 2957, 2922, 2854, 1711, 1688, 1628, 1589, 1493; HRMS (ESI) calcd for C H N O F (M+H) :
2
5
29
3
3
38.2193, found: 438.2187; Anal. Calcd. for C H FN O ·0.6 H O·0.25 C H : C, 67.81; H, 6.91; N,
2
5
28
3
3
2
6
12
.95; Found: C, 68.04; H, 7.13; N, 8.57.
.6.11.
4
(1-(3-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl
2
-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylate (11a): yellow oil
1
(
60.1% yield); H NMR (400 MHz, CDCl ) δ 7.57 (s, 1H, H-het), 7.35 (m, 1H, H-Ph), 7.08 – 6.93 (m,
3
3
–
H, H-Ph), 6.24 (s, 1H, CH =C), 5.64 (s, 1H, CH =C), 5.52 (s, 2H, NCH Ph), 5.30 (s, 2H, CH O), 3.19
2 2 2 2
3.13 (m, 1H, CHCH CO), 2.92 – 2.86 (m, 1H, CHCCO ), 2.77 (d, J = 19.5 Hz, 1H, CH C=C), 2.58
2
2
2
(
ddd, J = 18.7, 6.6, 1.0 Hz, 1H, CH CO), 2.49 – 2.32 (m, 1H, CH C=C), 2.15 – 2.09 (m, 1H, CHCH ),
2 2 3
2
1
.02 (dt, J = 18.8, 1.2 Hz , 1H, CH CO), 1.81 – 1.69 (m, 3H, CH CHCH , CH CH CHCH ), 1.66 –
2 2 3 2 2 3
1
3
.55 (m, 4H, CH C=C, CH CH CHCH ), 0.64 (d, J = 7.1 Hz, 3H, CH CH5); C NMR (101 MHz,
3
2
2
3
3
CDCl ) δ 208.15, 174.12, 166.62, 164.27, 161.80, 145.76, 143.37, 137.85, 130.84, 124.11, 123.69,
3
-
1
1
3
4
6
15.94, 115.03, 57.91, 53.50, 45.86, 41.34, 38.23, 37.97, 36.54, 35.30, 31.66, 12.09, 7.97; IR (v/cm ):
+
142, 2959, 2922, 1712, 1687, 1628, 1592, 1489, 1452; HRMS (ESI) calcd for C H N O F (M+H) :
2
5
29
3
3
38.2193, found: 438.2179; Anal. Calcd. for C H FN O ·H O: C, 65.92; H, 6.64; N, 9.22; Found: C,
2
5
28
3
3
2
5.88; H, 6.34; N, 9.25..
.6.12.
4
(1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl
2
-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylate (12a): yellow oil
1
(
57.2% yield); H NMR (400 MHz, CDCl ) δ 7.53 (s, 1H, H-het), 7.28 – 7.06 (m, 4H, H-Ph), 6.23 (s,
3
1
H, CH =C), 5.64 (s, 1H, CH =C), 5.49 (s, 2H, NCH Ph), 5.29 (s, 2H, CH O), 3.17 – 3.16 (m, 1H,
2 2 2 2
CHCH CO), 2.89 (dd, J = 15.8, 5.5 Hz, 1H, CHCCO ), 2.83 – 2.73 (m, 1H, CH C=C), 2.58 (ddd, J =
2
2
2
1
8.7, 6.6, 1.1 Hz, 1H, CH CO)., 2.49 – 2.35 (m, 1H, CH C=C), 2.15 – 2.10 (m, 1H, CHCH ), 2.04 (d, J
2 2 3
=
18.9 Hz, 1H, CH CO), 1.83-1.76 (m, 3H, CH CHCH , CH CH CHCH ), 1.65 – 1.57 (m, 4H,
2 2 3 2 2 3
1
3
CH C=C, CH CH CHCH ), 0.64 (d, J = 7.1 Hz, 3H, CH CH); C NMR (101 MHz, CDCl ) δ 208.19,
3
2
2
3
3
3
1
5
74.12, 166.74, 164.30, 161.83, 130.39, 130.36, 130.20, 130.12, 124.21, 123.71, 116.44, 116.22, 58.07,
-
1
3.64, 45.98, 41.47, 38.36, 38.09, 36.68, 35.44, 31.78, 12.23, 8.12; IR (v/cm ): 3140, 2923, 2853, 1690,
1
5

ACCEPTED MANUSCRIPT
+
1
628, 1607, 1511; HRMS (ESI) calcd for C H N O F (M+H) : 438.2193, found: 438.2194; Anal.
2
5
29
3
3
Calcd. for C H FN O ·0.30 CHCl ·0.60 C H : C, 66.24; H, 6.84; N, 8.02; Found: C, 66.06; H,6.86; N,
25
28
3
3
3
6
12
7
.90.
4
.6.13.
(1-(3,4-dichlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl
2
-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylate (13a): yellow oil
1
(
(
76.6% yield); H NMR (400 MHz, CDCl ) δ 7.58 (s, 1H, H-het), 7.45 (d, J = 8.2 Hz, 1H, H-Ph), 7.36
3
d, J = 2.1 Hz, 1H, H-Ph), 7.11 (dd, J = 8.3, 2.1 Hz, 1H, H-Ph), 6.24 (s, 1H, CH =C), 5.65 (s, 1H,
2
CH =C), 5.48 (s, 2H, NCH Ph), 5.30 (s, 2H, CH O), 3.21 – 3.11 (m, 1H, CHCH CO), 2.97-2.86 (m,
2
2
2
2
1
2
1
H, CHCCO ), 2.79 (dd, J = 19.4, 0.9 Hz, 1H, CH C=C), 2.58 (ddd, J = 18.7, 6.6, 1.1 Hz, 1H, CH CO),
2 2 2
.42 (ddd, J = 19.5, 12.4, 0.9 Hz, 1H, CH C=C), 2.22 – 2.07 (m, 1H, CHCH 0), 2.04 (d, J = 18.8 Hz ,
2
3
H, CH CO), 1.83 – 1.72 (m, 3H, CH CHCH , CH CH CHCH ), 1.66 – 1.55 (m, 4H, CH C=C,
2
2
3
2
2
3
3
1
3
CH CH CHCH ), 0.64 (d, J = 7.1 Hz, 3H, CH CH); C NMR (101 MHz, CDCl ) δ 208.19, 174.10,
2
2
3
3
3
1
66.75, 145.90, 143.67, 138.00, 134.63, 133.56, 133.48, 131.32, 130.09, 127.38, 124.29, 123.91, 58.02,
-
1
5
3.07, 45.98, 41.48, 38.35, 38.08, 36.68, 35.44, 31.82, 12.24, 8.13; IR (v/cm ): 2954, 2923, 2853, 1688,
1
627, 1462; Anal. Calcd. for C H Cl N O ·0.25 CHCl ·0.30 C H : C, 59.74; H, 5.72; N, 7.73; Found:
25
27
2
3
3
3
6
12
C, 59.82; H, 5.90; N, 7.53.
4
.6.14.
(1-(cyclohex-2-enyl)-1H-1,2,3-triazol-4-yl)methyl
2
-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylate (14a): yellow oil
1
(
39% yield); H NMR (400 MHz, CDCl ) δ 7.67 (s, 1H, H-het), 6.25 (s, 1H, CH =C), 6.15 (m, 1H,
3
2
CH=CH), 5.77 (m, 1H, CH=CH), 5.64 (s, 1H, CH =C), 5.28 (m, 3H, CH O, CHN), 3.18 – 3.17 (m, 1H,
2
2
CHCH CO), 2.91 (m, 1H, CHCCO ), 2.81 (m, J = 19.3 Hz, 1H, 1H, CH C=C), 2.58 (ddd, J = 18.4, 6.2,
2
2
2
0
1
7
1
.8 Hz, 1H, CH CO), 2.45-2.37 (m, 1H, CH C=C), 2.22 -2.10 (m, 1H, CHCH ), 2.03 (d, J = 18.8 Hz,
2 2 3
H, CH CO), 1.99 – 1.57 (m, 13H, CH CHCH , CH CH CHCH , CH C=C, CH CH CH ), 0.65 (d, J =
2
2
3
2
2
3
3
2
2
2
13
.1 Hz, 3H, CH CH); C NMR (101 MHz, CDCl ) δ 208.24, 174.28, 166.79, 146.00, 137.95, 134.26,
3
3
24.14, 123.95, 58.23, 56.18, 46.00, 41.48, 38.42, 38.10, 36.68, 35.45, 31.75, 24.70, 19.13, 12.24, 8.11;
-
1
+
IR (v/cm ): 2954, 2921, 2852, 1690, 1627, 1456; HRMS (ESI) calcd for C H N O (M+H) :
2
4
32
3
3
4
10.2443, found: 410.2431; Anal. Calcd. for C H N O ·0.6 H O·0.33C H : C, 69.65; H, 8.14; N, 9.37;
2
4
31
3
3
2
6
12
Found: C, 69.54; H, 7.92; N, 9.30.
1
6

ACCEPTED MANUSCRIPT
4
.6.15.
(1-cyclohexyl-1H-1,2,3-triazol-4-yl)methyl
2
-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylate (15a): yellow oil
1
(
64.8% yield); H NMR (400 MHz, CDCl ) δ 7.61 (s, 1H, H-het), 6.25 (s, 1H, CH =C), 5.64 (s, 1H,
3
2
CH =C), 5.30 (s, J = 2H, CH O), 4.43 (m, 1H, CHN), 3.24 – 3.11 (m, 1H, CHCH CO), 2.93 – 2.88 (m,
2
2
2
1
2
2
H, CHCCO ), 2.81 – 2.78 (m, CH C=C), 2.58 (ddd, J = 18.7, 6.6, 1.0 Hz, 1H, CH CO), 2.42 (ddd, J =
2 2 2
0.0, 12.4 0.8 Hz, 1H, CH C=C), 2.23 –2.17 (m, 2H, NCHCH ), 2.15 – 2.07 (m, 1H, CHCH ), 2.05 –
2
2
3
.01 (m, J = 18.8, 1.2 Hz, 1H, CH CO), 1.95 – 1.88 (m, 2H, NCHCH ), 1.81 – 1.59 (m, 13H,
2
2
1
3
CH CHCH , CH CH CHCH , CH C=C, CH CH CH CH CH ), 0.64 (d, J = 7.1 Hz, 3H, CH CH);
C
2
3
2
2
3
3
2
2
2
2
2
3
NMR (101 MHz, CDCl ) δ 208.24, 174.28, 166.80, 146.00, 137.94, 124.12, 60.37, 58.25, 45.99, 41.47,
3
-
1
3
1
8.42, 38.07, 36.67, 35.44, 33.66, 31.75, 25.25, 25.21, 12.23, 8.11; IR (v/cm ): 3142, 2924, 2855, 1691,
+
628, 1450; HRMS (ESI) calcd for C H N O (M+H) : 412.2600, found: 412.2599; Anal. Calcd. for
24
34
3
3
C H N O ·0.3 CHCl ·0.30 C H : C, 66.30; H, 7.87; N, 8.89; Found: C, 66.08; H,7.92; N, 8.61.
24
33
3
3
3
6
12
4
.6.16.
N-(1-(2-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl
2
-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylamide (1b): yellow oil
1
(
52% yield); H NMR (400 MHz, CDCl ) δ 7.38 (s, 1H, H-het), 7.32 – 7.13 (m, 4H, H-Ph), 6.67 (br,
3
1
H, NH), 5.55 (s, 1H, CH =C), 5.51 (s, 2H, NCH Ph), 5.34 (d, J = 0.8 Hz, 1H, CH =C), 4.54 (s, 2H,
2 2 2
CH NHCO), 3.19-3.13 (m, 1H, CHCH CO), 2.93 – 2.85 (m, 1H, CHCCO ), 2.82 (d, J = 19.6 Hz, 1H,
2
2
2
CH C=C), 2.57 (ddd, J = 18.7, 6.6, 1.0 Hz, 1H, CH CO), 2.43 (dd, J = 19.3, 12.3 Hz, 1H, CH C=C),
2
2
2
2
1
=
.28 (s, 3H, CH -Ph), 2.15 – 2.07 (m, 1H, CHCH ), 2.07 – 1.98 (dt, J = 18, 1 Hz, 1H, CH CO), 1.83 –
3
3
2
.70 (m, 3H, CH CHCH , CH CH CHCH ), 1.67 – 1.51 (m, 4H, CH C=C, CH CH CHCH ), 0.65 (d, J
2
3
2
2
3
3
2
2
3
13
7.2 Hz, 3H, CH CH); C NMR (101 MHz, CDCl ) δ 208.29, 174.41, 169.08, 151.34, 137.92, 137.04,
3
3
1
3
32.32, 131.25, 129.65, 129.45, 126.87, 115.71, 60.53, 52.67, 45.99, 41.52, 38.71, 38.29, 36.71, 35.48,
-
1
1.38, 19.12, 12.22, 8.13; IR (v/cm ): 3330, 2923, 1687, 1622; HRMS (ESI) calcd for C H N O
2
2
6
33
4
+
(
M+H) : 433.2603, found: 433.2603; Anal. Calcd. for C H N O ·0.9 CH Cl ·0.2 CH OH: C, 63.13; H,
26 32 4 2 2 2 3
6
.77; N, 10.87; Found: C, 63.26; H,6.67; N, 10.72.
4
.6.17.
N-(1-(3-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl
2
-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylamide (2b): yellow oil
1
(
59.4% yield); H NMR (400 MHz, CDCl ) δ 7.47 (s, 1H, H-het), 7.2 (t, J = 7.4 Hz, 1H, H-Ph), 7.16 (d,
3
1
7

ACCEPTED MANUSCRIPT
J = 7.6 Hz, 1H, H-Ph), 7.08 – 7.04 (m, 2H, H-Ph), 6.72 (t, J = 4.8 Hz, 1H, NH), 5.55 (s, 1H, CH =C),
2
5
.45 (s, 2H, NCH Ph), 5.33 (d, 1H, CH =C), 4.53 (d, J = 5.6 Hz, 2H, CH NHCO), 3.17 – 3.13 (m, 1H,
2 2 2
CHCH CO), 2.94 – 2.86 (m, 1H, CHCCO ), 2.83 (d, J = 19.6 Hz, 1H, CH C=C), 2.56 (ddd, J = 18.7,
2
2
2
6
–
.6, 0.9 Hz, 1H, CH CO), 2.43 (ddd, J = 19.8, 12.2, 0.8 Hz, 1H, CH C=C), 2.33 (s, 3H, CH -Ph), 2.14
2 2 3
2.07 (m, 1H, CHCH ), 2.02 (d, J = 19.0 Hz, 1H, CH CO), 1.82 – 1.69 (m, 3H, CH CHCH ,
3
2
2
3
CH CH CHCH ), 1.65 – 1.53 (m, 4H, CH C=C, CH CH CHCH ), 0.63 (d, J = 7.1 Hz, 3H, CH CH);
2
2
3
3
2
2
3
3
13
C NMR (101 MHz, CDCl ) δ 208.27, 174.40, 169.09, 151.32, 144.77, 139.17, 137.90, 134.35, 129.77,
3
1
2
29.17, 129.00, 125.33, 122.19, 115.71, 54.45, 45.97, 41.50, 38.67, 38.30, 36.69, 35.46, 35.22, 31.39,
-
1
1.45, 12.20, 8.11; IR (v/cm ): 3330, 3142, 2956, 2921, 2873, 1684, 1658, 1618; Anal. Calcd. for
C H N O ·0.5 CH Cl ·0.3 CH OH: C, 66.40; H, 7.11; N, 11.56; Found: C, 66.13; H,7.12; N, 11.46.
26
32
4
2
2
2
3
4
.6.18.
N-(1-(4-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl
2
-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylamide (3b): yellow oil
1
(
34.8% yield); H NMR (400 MHz, CDCl ) δ 7.45 (s, 1H, H-het), 7.16 (s, 4H, H-Ph), 6.69 (br, 1H,
3
NH), 5.55 (s, 1H, CH =C), 5.45 (s, 2H, NCH Ph), 5.33 (d, J = 0.6 Hz, 1H, CH =C), 4.52 (d, J = 5.5 Hz,
2
2
2
2
1
H, CH NHCO), 3.18 – 3.13 (m, 1H, CHCH CO), 2.94 – 2.85 (m, 1H, CHCCO ), 2.82 (d, J = 19.9 Hz,
2 2 2
H, CH C=C), 2.57 (ddd, J = 18.7, 6.6, 0.9 Hz, 1H, CH CO), 2.42 (ddd, J = 19.5, 11.7, 0.8 Hz, 1H,
2
2
CH C=C), 2.34 (s, 3H, CH -Ph), 2.16 – 2.07 (m, 1H, CHCH ), 2.06 – 1.98 (m, 1H, CH CO), 1.82-1.71
2
3
3
2
(
m, 3H, CH CHCH , CH CH CHCH ), 1.69 – 1.50 (m, 3H, CH C=C, CH CH CHCH ), 0.64 (t, J = 7.3
2
3
2
2
3
3
2
2
3
13
Hz, 3H, CH CH); C NMR (101 MHz, CDCl ) δ 208.28, 174.42, 169.09, 151.32, 139.02, 137.90,
3
3
1
2
31.41, 129.96, 128.33, 122.11, 115.71, 60.52, 54.27, 45.97, 41.51, 38.49, 36.69, 35.47, 35.19, 31.38,
-
1
1.29, 12.20, 8.11; IR (v/cm ): 3331, 2956, 2923, 2855, 1683, 1658, 1617, 1516; HRMS (ESI) calcd
+
for C H N O (M+H) : 433.2604, found: 433.2584; Anal. Calcd. for C H N O ·0.2 CH Cl ·0.4
26
33
4
2
26 32
4
2
2
2
CH OH: C, 69.08; H, 7.41; N, 12.12; Found: C, 69.41; H,7.49; N, 11.97.
3
4
.6.19.
N-(1-benzyl-1H-1,2,3-triazol-4-yl)methyl
2
-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylamide (4b): yellow oil
1
(
63.4% yield); H NMR (400 MHz, CDCl ) δ 7.49 (s, 1H, H-het), 7.36 – 7.23 (m, 5H, H-Ph), 6.82 (br,
3
1
H, NH), 5.56 (s, 1H, CH =C), 5.48 (s, 2H, NCH Ph), 5.33 (s, 1H, CH =C), 4.52 (d, J = 4.9 Hz, 2H,
2 2 2
CH NHCO), 3.18 – 3.12 (m, 1H, CHCH CO), 2.93 – 2.76 (m, 2H, CHCCO , CH C=C), 2.56 (dd, J =
2
2
2
2
1
8.8, 6.6 Hz, 1H, CH CO), 2.42 (dd, J = 19.3, 12.0 Hz, 1H, CH C=C), 2.17 – 2.05 (1H, CHCH ), 2.01
2 2 3
1
8

ACCEPTED MANUSCRIPT
(
d, J = 18.8 Hz, 1H, CH CO), 1.82 – 1.71 (m, 3H, CH CHCH , CH CH CHCH ), 1.65 – 1.47 (m, 4H,
2
2
3
2
2
3
1
3
CH C=C, CH CH CHCH ), 0.62 (d, J = 7.1 Hz, 3H, CH CH); C NMR (101 MHz, CDCl ) δ 208.34,
3
2
2
3
3
3
1
1
2
74.53, 169.11, 151.24, 137.87, 134.47, 129.28, 129.00, 128.95, 128.38, 128.33, 128.24, 122.31,
-
1
15.75, 54.41, 45.97, 41.49, 38.63, 38.29, 36.67, 35.44, 35.17, 31.36, 12.19, 8.10; IR (v/cm ): 3330,
+
957, 2922, 1683, 1658, 1621; HRMS (ESI) calcd for C H N O (M+H) : 419.2447, found: 419.2448;
2
5
31
4
2
Anal. Calcd. for C H N O ·0.4 CDCl ·0.2 CH Ph: C, 66.33; H, 6.57; N, 11.55; Found: C, 66.45;
25
30
4
2
3
3
H,6.32; N, 11.42.
.6.20.
4
N-(1-(3-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl
2
-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylamide (5b): yellow oil
1
(
54.9% yield); H NMR (400 MHz, CDCl ) δ 7.48 (s, 1H, H-het), 7.28 – 6.79 (m, 4H, H-Ph), 6.63 (t, J
3
=
4.3 Hz, 1H, NH), 5.55 (s, 1H, CH =C), 5.47 (s, 2H, NCH Ph), 5.34 (d, J = 0.9 Hz, 1H, CH =C), 4.55
2 2 2
(
d, J = 5.6 Hz, 2H, CH NHCO), 3.78 (s, 3H, OCH ), 3.19 – 3.13 (m, 1H, CHCH CO), 2.94-2.87 (m,
2 3 2
1
H, CHCCO ), 2.83 (d, J = 19.9 Hz, 1H, CH C=C), 2.57 (ddd, J = 18.7, 6.6, 1.1 Hz, 1H, CH CO), 2.43
2 2 2
(
dd, J = 19.0, 12.8 Hz, 1H, CH C=C), 2.14 – 2.08 (m, 1H, CHCH ), 2.07 – 1.99 (m, 1H, CH CO), 1.82
2 3 2
–
1.72 (m, 3H, , CH CHCH , CH CH CHCH ), 1.64 – 1.57 (m, 4H, CH C=C, CH CH CHCH ), 0.64
2 3 2 2 3 3 2 2 3
13
(
d, J = 7.1 Hz, 3H, CH CH); C NMR (101 MHz, CDCl ) δ 208.34, 174.45, 169.11, 160.28, 151.35,
3 3
1
3
37.93, 135.90, 130.41, 120.44, 115.73, 114.33, 114.06, 55.46, 54.39, 46.00, 41.52, 38.69, 38.31, 36.70,
-
1
5.48, 35.22, 31.41, 12.22, 8.13; IR (v/cm ): 3331, 2957, 2922, 2854, 1683, 1659, 1614; HRMS (ESI)
+
calcd for C H N O (M+H) : 449.2553, found: 449.2555; Anal. Calcd. for C H N O ·0.1
26
33
4
3
26 32
4
3
CH Cl ·0.4 CH OH: C, 67.72; H, 7.26; N, 11.93; Found: C, 68.02; H,7.33; N, 11.60.
2
2
3
4
.6.21
N-(1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl
2
-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylamide (6b): yellow oil
1
(
62.1% yield); H NMR (400 MHz, CDCl ) δ 7.44 (s, 1H, H-het), 7.21 (dt, J = 8.8, 2.6 Hz, 2H, H-Ph),
3
6
.88 (dt, J = 8.8, 2.6 Hz, 2H, H-Ph), 6.76 (t, J = 5.2 Hz, 1H, NH), 5.55 (s, 1H, CH =C), 5.42 (s, 2H,
2
NCH Ph), 5.32 (d, J = 0.8 Hz, 1H, CH =C), 4.51 (d, J = 5.6 Hz, 2H, CH NHCO), 3.79 (s, 3H, OCH ),
2
2
2
3
3
2
2
.27 – 3.13 (m, 1H, CHCH CO), 2.93 – 2.86 (m, 1H, CHCCO ), 2.82 (d, J = 20.2 Hz, 1H, CH C=C),
2 2 2
.56 (ddd, J = 18.8, 6.6, 1.2 Hz, 1H, CH CO), 2.46 (ddd, J = 19.5, 12.2, 1.3 Hz, 1H, CH C=C), 2.14 –
2
2
.06 (m, 1H, CHCH ), 2.02 (dt, J = 18.8, 1.4 Hz, 1H, CH CO), 1.82 – 1.70 (m, 3H, CH CHCH ,
3
2
2
3
CH CH CHCH ), 1.63 – 1.51 (m, 4H, CH C=C, CH CH CHCH ), 0.63 (d, J = 7.1 Hz, 3H, CH CH);
2
2
3
3
2
2
3
3
1
9

ACCEPTED MANUSCRIPT
13
C NMR (101 MHz, CDCl ) δ 208.26, 174.42, 169.08, 160.14, 151.29, 144.74, 137.87, 129.83, 126.44,
3
1
21.96, 115.69, 114.64, 55.46, 53.96, 45.96, 41.49, 38.65, 38.28, 36.67, 35.45, 35.21, 31.36, 12.19,
-
1
8
.10; IR (v/cm ): 3323, 3136, 2956, 2922, 1683, 1657, 1613; Anal. Calcd. for C H N O ·0.35 CH Cl :
26 32 4 3 2 2
C, 66.15; H, 6.90; N, 11.71; Found: C, 66.02; H,7.02; N, 11.98.
4
.6.22.
N-(1-(2-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl
2
-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylamide (7b): yellow oil
1
(
66% yield); H NMR (400 MHz, CDCl ) δ 7.87 (s, 1H, H-het), 7.44 – 7.20 (m, 4H, H-Ph), 6.75 (br,
3
1
3
H, NH), 5.66 (s, 2H, NCH Ph), 5.56 (s, 1H, CH =C), 5.34 (s, 1H, CH =C), 4.62 (br, 2H, CH NHCO),
2 2 2 2
.19 – 3.13 (m, 1H, CHCH CO), 2.91 (t, J = 11.0 Hz, 1H, CHCCO ), 2.83 (d, J = 19.5 Hz, 1H,
2
2
CH C=C), 2.57 (dd, J = 18.8, 6.4 Hz, 1H, CH CO), 2.43 (dd, J = 19.3, 11.8 Hz, 1H, CH C=C), 2.15 –
2
2
2
2
.07 (m, 1H, CHCH ), 2.02 (d, J = 19.4 Hz, 1H, CH CO), 1.82 – 1.71 (m, 3H, CH CHCH ,
3 2 2 3
CH CH CHCH ), 1.65 – 1.53 (m, 4H, CH C=C, CH CH CHCH ), 0.63 (d, J = 7.1 Hz, 3H, CH CH);
2
2
3
3
2
2
3
3
13
C NMR (101 MHz, CDCl ) δ 208.26, 174.38, 169.07, 151.34, 137.93, 133.85, 132.22, 130.69, 130.60,
3
1
30.13, 127.72, 115.87, 53.53, 52.36, 46.03, 41.51, 38.75, 38.30, 36.74, 35.47, 31.38, 12.22, 8.13; IR
-
1
(
v/cm ): 3328, 3142, 2962, 2921, 2874, 1682, 1658, 1619; HRMS (ESI) calcd for C H ClN O
25 30 4 2
+
(
M+H) : 453.2057, found: 453.2036; Anal. Calcd. for C H ClN O ·0.35 CH Cl : C, 63.05; H, 6.20; N,
25 29 4 2 2 2
1
1.61; Found: C, 62.86; H,6.25; N, 11.56.
.6.23.
4
N-(1-(3-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl
2
-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylamide (8b): yellow oil
1
(
57.8% yield); H NMR (400 MHz, CDCl ) δ 7.53 (s, 1H, H-het), 7.36 – 7.12 (m, 4H, H-Ph), 6.68 (br,
3
1
H, NH), 5.56 (s, 1H, CH =C), 5.47 (s, 2H, NCH Ph), 5.34 (s, 1H, CH =C), 4.56 (d, J = 5.2 Hz, 2H,
2 2 2
CH NHCO), 3.18 – 3.13 (m, 1H, CHCH CO), 2.96 – 2.77 (m, 2H, CHCCO , CH C=C), 2.57 (ddd, J =
2
2
2
2
1
8.7, 6.6, 1.0 Hz, 1H, CH CO), 2.43 (dd, J = 18.7, 12.1 Hz, 1H, CH C=C), 2.15 – 2.07 (m, 1H,
2 2
CHCH ), 2.02 (d, J = 19.2 Hz, 1H, CH CO), 1.82 – 1.71 (m, 3H, CH CHCH , CH CH CHCH ), 1.64 –
3
2
2
3
2
2
3
1
3
1
.52 (m, 4H, CH C=C, CH CH CHCH ), 0.63 (d, J = 7.1 Hz, 3H, CH CH); C NMR (101 MHz,
3 2 2 3 3
CDCl ) δ 208.26, 174.34, 169.15, 151.30, 137.93, 136.40, 135.24, 130.60, 129.27, 128.29, 126.28,
3
-
1
1
3
6
15.80, 53.76, 45.98, 41.51, 38.69, 38.30, 36.70, 35.47, 35.19, 31.41, 12.21, 8.13; IR (v/cm ): 3320,
137, 3054, 2957, 2922, 1683, 1657, 1619; Anal. Calcd. for C H ClN O ·0.45 CH Cl ·0.2 CH OH: C,
2
5
29
4
2
2
2
3
1.88; H, 6.22; N, 11.26; Found: C, 61.72; H,6.18; N, 11.23.
2
0

ACCEPTED MANUSCRIPT
4
.6.24.
N-(1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl
2
-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylamide (9b): yellow oil
1
(
54.5% yield); H NMR (400 MHz, CDCl ) δ 7.49 (s, 1H, H-het), 7.37 – 7.32 (m, 2H, H-Ph), 7.23 –
3
7
1
1
.18 (m, 2H, H-Ph), 6.63 (br, 1H, NH), 5.55 (s, 1H, CH =C), 5.47 (s, 2H, NCH Ph), 5.34 (d, J = 0.9 Hz,
2 2
H, CH =C), 4.54 (d, J = 5.6 Hz, 2H, CH NHCO), 3.19 – 3.13 (m, 1H, CHCH CO), 2.94 – 2.86 (m,
2
2
2
H, CHCCO ), 2.83 (d, J = 19.6 Hz, 1H, CH C=C), 2.57 (ddd, J = 18.8, 6.6, 1.1 Hz, 1H, CH CO), 2.43
2
2
2
(
ddd, J = 18.5, 12.3, 0.8 Hz, 1H, CH C=C), 2.16 – 2.09 (m, 1H, CHCH ), 2.05 (dt, J =18.8, 2.4Hz, 1H,
2 3
CH CO), 1.82 – 1.70 (m, 3H, CH CHCH , CH CH CHCH ), 1.66 – 1.55 (m, 4H, CH C=C,
2
2
3
2
2
3
3
1
3
CH CH CHCH ), 0.64 (d, J = 7.1 Hz, 3H, CH CH); C NMR (101 MHz, CDCl ) δ 208.28, 174.33,
2
2
3
3
3
1
3
69.15, 151.32, 137.94, 135.17, 132.97, 129.59, 129.55, 122.24, 115.76, 53.71, 45.99, 41.52, 38.70,
-
1
8.29, 36.71, 35.48, 35.19, 31.41, 12.22, 8.14; IR (v/cm ): 3330, 2961, 2922, 1682, 1657, 1618; Anal.
Calcd. for C H ClN O ·0.2 CH Cl : C, 64.38; H, 6.31; N, 11.92; Found: C, 64.39; H,6.41; N, 11.80.
25
29
4
2
2
2
4
.6.25.
N-(1-(2-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl
2
-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylamide (10b): yellow oil
1
(
59.8% yield); H NMR (400 MHz, CDCl ) δ 7.56 (s, 1H, H-het), 7.41 – 7.08 (m, 4H, H-Ph), 6.65 (br,
3
1
H, NH), 5.56 (s, 2H, NCH Ph), 5.55 (s, H, CH =C), 5.34 (d, J = 0.9 Hz, 1H, CH =C), 4.53 (d, J = 5.6
2 2 2
Hz, 2H, CH NHCO), 3.19 – 3.13 (m, 1H, CHCH CO), 2.94 – 2.87 (m, 1H, CHCCO ), 2.83 (d, J = 19.6
2
2
2
Hz, 1H, CH C=C), 2.57 (ddd, J = 18.7, 6.6, 1.1 Hz, 1H, CH CO), 2.43 (dd, J = 19.0, 11.8 Hz, 1H,
2
2
CH C=C), 2.15 – 2.07 (m, 1H, CHCH ), 2.03 (dt, J = 19.2, 1.2 Hz, 1H, CH CO), 1.83 – 1.72 (m, 3H ,
2
3
2
CH CHCH , CH CH CHCH ), 1.67 – 1.53 (m, 4H, CH C=C, CH CH CHCH ), 0.64 (t, J = 7.1 Hz, 3H,
2
3
2
2
3
3
2
2
3
13
CH CH); C NMR (101 MHz, CDCl ) δ 208.31, 174.40, 169.11, 161.98, 151.37, 137.94, 131.26,
3
3
1
1
4
1
30.83, 125.02, 116.19, 115.98, 115.71, 48.02, 45.99, 41.53, 38.72, 38.30, 36.71, 35.49, 35.18, 31.39,
-
1
+
2.22, 8.13; IR (v/cm ): 3315, 2921, 1682, 1618; HRMS (ESI) calcd for C H N O F (M+H) :
2
5
30
4
2
37.2354, found: 437.2351; Anal. Calcd. for C H FN O ·0.9 CH Cl ·0.5 CH OH: C, 59.92; H, 6.25; N,
2
5
29
4
2
2
2
3
0.59; Found: C, 59.84; H,6.03; N, 10.55.
.6.26.
4
N-(1-(3-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl
2
-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylamide (11b): yellow oil
1
(
58.8% yield); H NMR (400 MHz, CDCl ) δ 7.52 (s, 1H, H-het), 7.34 (m, 1H, H-Ph), 7.09 – 6.87 (m,
3
3
H, H-Ph), 6.70 (br, 1H, NH), 5.55 (s, 1H, CH =C), 5.49 (s, 2H, NCH Ph), 5.34 (d, J = 0.9 Hz, 1H,
2 2
2
1

ACCEPTED MANUSCRIPT
CH =C), 4.55 (d, J = 5.5 Hz, 2H, CH NHCO), 3.18 – 3.13 (m, 1H, CHCH CO), 2.94 – 2.78 (m, 2H,
2
2
2
CHCCO , CH C=C), 2.58 (ddd, J = 18.8, 6.6, 1.2 Hz, 1H, CH CO), 2.46 (ddd, J = 19.5, 12.2, 1.3 Hz,
2
2
2
1
3
3
1
3
H, CH C=C), 2.15 – 2.07 (m, 1H, CHCH ), 2.02 (dt, J = 19.2, 1.4 Hz, 1H, CH CO), 1.82 – 1.71 (m,
2 3 2
H, CH CHCH , CH CH CHCH ), 1.64 – 1.50 (m, 4H, CH C=C, CH CH CHCH ), 0.63 (d, J = 7.1 Hz,
2
3
2
2
3
3
2
2
3
13
H, CH CH); C NMR (101 MHz, CDCl ) δ 208.27, 174.23, 166.75, 164.40, 161.93, 145.92, 143.50,
3
3
37.98, 136.86, 131.98, 124.26, 123.71, 116.08, 115.17, 58.06, 53.71, 45.99, 41.48, 38.37, 38.09, 36.68,
-
1
5.44, 31.79, 12.23, 8.12; IR (v/cm ): 3315, 3138, 3056, 2958, 2922, 1683, 1658, 1618; Anal. Calcd.
for C H FN O ·0.55 CH Cl ·0.3 CH OH: C, 62.97; H, 6.41; N, 11.37; Found: C, 62.82; H, 6.39; N,
25
29
4
2
2
2
3
1
1.20.
4
.6.27.
N-(1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methyl
2
-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylamide (12b): yellow oil
1
(
61.7% yield); H NMR (400 MHz, CDCl ) δ 7.50 (s, 1H, H-het), 7.26 – 7.20 (m, 2H, H-Ph), 7.03 (m,
3
2
H, H-Ph), 6.89 (br, 1H, NH), 5.55 (s, 1H, CH =C), 5.45 (s, 2H, NCH Ph), 5.32 (s, 1H, CH =C), 4.51
2 2 2
(
d, J = 5.5 Hz, 2H, CH NHCO), 3.19 – 3.08 (m, 1H, CHCH CO), 2.92 – 2.85 (m, 1H, CHCCO ), 2.81
2 2 2
(
d, J = 19.6 Hz, 1H, CH C=C), 2.54 (ddd, J = 18.7, 6.6, 1.0 Hz, 1H, CH CO), 2.41 (ddd, J = 19.6, 12.2,
2 2
0
1
=
.6 Hz, 1H, CH C=C), 2.16 – 2.04 (m, 1H, CHCH ), 2.00 (dt, J = 19.2, 1.2 Hz, 1H, CH CO), 1.79 –
2
3
2
.70 (m, 3H, CH CHCH , CH CH CHCH ), 1.64 – 1.47 (m, 4H, CH C=C, CH CH CHCH ), 0.62 (t, J
2
3
2
2
3
3
2
2
3
13
7.3 Hz, 3H, CH CH); C NMR (101 MHz, CDCl ) δ 208.28, 174.34, 169.14, 164.31, 161.84, 151.35,
3
3
1
3
37.94, 130.21, 130.13, 116.45, 116.23, 115.73, 53.71, 46.00, 41.51, 38.71, 38.29, 36.71, 35.47, 35.22,
-
1
1.40, 12.21, 8.13; IR (v/cm ): 3321, 2957, 2922, 1683, 1657, 1618; Anal. Calcd. for
C H FN O ·0.85 CH Cl ·0.3 CH OH: C, 60.57; H, 6.21; N, 10.81; Found: C, 60.78; H, 6.05; N, 10.65.
25
29
4
2
2
2
3
4
.6.28.
N-(1-(3,4-dichlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl
2
-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylamide (13b): yellow oil
1
(
(
66.7% yield); H NMR (400 MHz, CDCl ) δ 7.55 (s, 1H, H-het), 7.43 (d, J = 8.2 Hz, 1H, H-Ph), 7.34
3
d, J = 2.1 Hz, 1H, H-Ph, 7.10 (dd, J = 8.3, 2.1 Hz, 1H, H-Ph), 6.82 (t, J = 5.3 Hz, 1H, NH), 5.56 (s, 1H,
CH =C), 5.44 (s, 2H, NCH Ph), 5.34 (d, J = 0.8 Hz, 1H, CH =C), 4.54 (d, J = 5.7 Hz, 2H, CH NHCO),
2
2
2
2
3
2
2
.17 – 3.12 (m, 1H, CHCH CO), 2.93 – 2.86 (m, 1H, CHCCO ), 2.82 (d, J = 19.6 Hz, 1H, CH C=C),
2 2 2
.55 (ddd, J = 18.7, 6.6, 1.0 Hz, 1H, CH CO), 2.42 (ddd, J = 19.8, 12.5, 0.8 Hz, 1H, CH C=C), 2.14 –
2
2
.06 (m, 1H, CHCH ), 2.01 (dt, J =18.8, 1.2 Hz, 1H, CH CO), 1.84 – 1.67 (m, 3H, CH CHCH ,
3
2
2
3
2
2

ACCEPTED MANUSCRIPT
CH CH CHCH ), 1.64 – 1.51 (m, 4H, CH C=C, CH CH CHCH ), 0.62 (d, J = 7.1 Hz, 3H, CH CH);
2
2
3
3
2
2
3
3
13
C NMR (101 MHz, CDCl ) δ 208.25, 174.37, 169.16, 151.20, 145.34, 137.88, 134.65, 133.49, 133.40,
3
1
31.25, 130.07, 127.36, 122.46, 115.82, 60.50, 53.05, 45.96, 41.48, 38.63, 38.27, 36.66, 35.30, 31.39,
-
1
1
2.18, 8.10; IR (v/cm ): 3317, 2957, 2922, 1683, 1658, 1618; Anal. Calcd. for C H Cl N O ·1.1
25 28 2 4 2
CH Cl ·0.3 CH OH: C, 53.67; H, 5.36; N, 9.49; Found: C, 53.76; H, 5.15; N, 9.37.
2
2
3
4
.6.29.
N-(1-(cyclohex-2-enyl)-1H-1,2,3-triazol-4-yl)methyl
2
-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylamide (14b): yellow oil
1
(
63.1% yield); H NMR (400 MHz, CDCl ) δ 7.57 (s, 1H, H-het), 6.72 (brs, 1H, NH), 6.14 (m, 1H,
3
CH=CH), 5.76 (m, 1H, CH=CH), 5.56 (s, 1H, , CH =C), 5.34 (s, 1H, CH =C), 5.23 (m, 1H, CHN),
2
2
4
.56 (d, J = 5.6 Hz, 2H, CH NHCO), 3.23 – 3.14 (m, 1H, CHCH CO), 2.92 (t, J = 11.7 Hz, 1H,
2 2
CHCCO ), 2.84 (d, J = 19.0 Hz, 1H, CH C=C), 2.57 (dd, J = 19.2, 6.0 Hz, 1H, CH CO), 2.44 (dd, J =
2
2
2
1
9.6, 12.2 Hz, 1H, CH C=C), 2.18 – 2.15 (m, 1H, CHCH ), 2.06 – 1.89 (m, 1H, CH CO), 1.86 – 1.53
2 3 2
1
3
(
m, 13H, CH CHCH , CH CH CHCH , CH C=C, CH CH CH ), 0.64 (d, J = 7.1 Hz, 3H, CH CH); C
2 3 2 2 3 3 2 2 2 3
NMR (101 MHz, CDCl ) δ 208.36, 174.52, 169.11, 151.37, 137.91, 134.20, 123.96, 120.97, 115.65,
3
5
6.21, 46.00, 41.52, 38.72, 38.30, 36.71, 35.48, 35.26, 31.40, 30.69, 24.70, 19.18, 12.22, 8.13; IR
-
1
(
v/cm ): 3335, 2924, 2872, 1683, 1658, 1620; Anal. Calcd. for C H N O ·0.7 CH Cl ·0.7 CH OH: C,
24 32 4 2 2 2 3
6
2.18; H, 7.44; N, 11.42; Found: C, 62.16; H,7.18; N, 11.12
.6.30. N-(1-cyclohexyl-1H-1,2,3-triazol-4-yl)methyl
4
2
-((5R,8S,8aS)-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)acrylamide (15b): yellow oil
1
(
53.0% yield); H NMR (400 MHz, CDCl ) δ 7.55 (s, 1H, H-het), 6.75 (brs, 1H, NH), 5.57 (s, 1H,
3
CH =C), 5.34 (s, 1H, CH =C), 4.56 (d, J = 5.6 Hz, 2H, CH NHCO), 4.41 (m, 1H, CHN), 3.20 – 3.15
2
2
2
(
m, 1H, CHCH CO), 2.92 (t, J = 11.7 Hz, 1H, CHCCO ), 2.88 – 2.78 (m, 1H, CH C=C), 2.57 (ddd, J =
2 2 2
1
2
1
8.7, 6.6, 1.1 Hz, 1H, CH CO), 2.49 – 2.37 (m, 1H, CH C=C), 2.18-2.15 (m, 2H, NCHCH ), 2.14 –
2 2 2
.09 (m, 1H, CHCH ), 2.07 – 1.98 (m, 1H, CH CO), 1.96 – 1.88 (m, 2H, NCHCH ), 1.84 – 1.21 (m,
3
2
2
3H, CH CHCH , CH CH CHCH , CH C=C, CH CH CH CH CH ), 0.64 (t, J = 6.0 Hz, 3H, CH CH);
2
3
2
2
3
3
2
2
2
2
2
3
13
C NMR (101 MHz, CDCl ) δ 208.26, 174.43, 169.11, 151.36, 137.89, 115.68, 60.45, 53.55, 45.99,
3
-
1
4
1.51, 38.69, 38.31, 36.71, 35.48, 35.27, 33.65, 31.39, 25.27, 25.22, 12.20, 8.12; IR (v/cm ): 3336,
3
137, 2929, 2858, 1688, 1659, 1620; Anal. Calcd. for C H N O ·0.35 CH Cl ·0.1 CH OH: C, 66.20;
2
4
34
4
2
2
2
3
H, 7.98; N, 12.63; Found: C, 66.04; H,8.02; N, 12.62
2
3

ACCEPTED MANUSCRIPT
4
4
.7. Activity screening
.7.1. The procedure for the anti-influenza virus assay
Each compound was dissolved in DMSO at an initial concentration of 20 mg/mL and then diluted
three fold successively to obtain 8 different concentrations as stock solutions for the following
experiments. Madin-Darby canine kidney (MDCK) cells were seeded in 96-well trays and cultured at
3
7 °C in a humidified CO incubator (95% air, 5%CO ) for 24 h. Then, the cells were infected with
2 2
influenza A virus. All of infected tissue culture plates (96 wells) were incubated at 37 °C for 2 h, and
then the medium was removed. Subsequently, different concentrations of drug maintain solutions were
added to the wells (one per well), and the plates were incubated again for 40 h at 37 °C. Then, the
inhibition of the virus-induced cytopathic effect (CPE) for each sample was recorded relative to the cell
control and the virus control. The 50% cell-inhibitory concentration (IC ) values of active compounds
5
0
were calculated accordingly. The commercial drugs ribavirin (RBV) and oseltamivir as Positive
contrast
4
.7.2. The procedure for cytotoxicity assay
MDCK cells were seeded in 96-well trays, each well 25000 cells and cultured at 37 °C in a
humidified CO incubator (95% air, 5% CO ) for 24 h. 3 times dilution compound was added cell
2
2
monolayer, and continue to cultivate 48 hours, then observe the cytopathic record results. The 50%
cytotoxic concentration (TC ) values of active compounds were calculated by the methods of Reed &
50
Muench
.7.3. The procedure for the anti-influenza B virus plaque inhibition assay
4
Monolayers of MDCK cells in twelve well plate were washed with PBS and infected with
mL/well of certain dilution of the virus in the maintenance medium without trypsin. After 2 h
1
incubation at 37 °C in 5% CO , the virus was removed and washed plate with PBS for 3 times ,then
2
added 1mL/well of 1.5% agarose overlay (1 ml) containing Eagle minimal essential medium, trypsin (2
ꢀ
g/ml), and different concentration drug or compounds. Plates were incubated at 37°C in 5% CO for
2
future 3-4 days to allow plaque formation. Whereafter, agar was removed and plaques were stained
with crystal violet (0.5%) and counted. The percentage of plaque inhibition relative to the infected
control (no drug) plates was determined for each drug concentration, and the 50% inhibitory
concentration (IC ) was calculated by the Reed & Muench methods.
50
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4

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Acknowledgments
This work was financially supported by the Funds for International Cooperation and Exchange of the
National Natural Science Foundation of China (No. 31110103908) and the National Basic Research
Program of China (No. 2012CB723502).
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Scheme 1. Synthesis of 1,2,3-triazole-containing derivatives of rupestonic acid. Reagents and
conditions: i) NaN , DMSO; ii) K CO , DMF; iii) EDCI, HOBt, DCM; iv) CuSO /Na-Ascorbate,
3
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3
4
DCM/H O.
2
Table 1. Structure and inhibitory activity of compounds against influenza A and B viruses
Table 2. Inhibition of the plaque formation by four best representative compounds using MDCK cells
and RBV as control drug.
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