Jietacins, azoxy antibiotics with potent nematocidal activity: Design, synthesis, and biological evaluation against parasitic nematodes

Article abstract of DOI:10.1016/j.ejmech.2017.12.031

Jietacins, an azoxy antibiotic class of chemicals, were isolated from the culture broth of Streptomyces sp. KP-197. They have a unique structural motif, including a vinyl azoxy group and a long acyclic aliphatic chain, which is usually branched but non-branched in the case of jietacin C. During a drug discovery program, we found that jietacins display potent anthelmintic activity against parasitic nematodes and that jietacin A has a moderate or low acute toxicity (LD₅₀ > 300 mg/kg) and no mutagenic potential in a mini Ames screen. This suggests that jietacins have potential for drug discovery research. In order to create a novel anthelmintic agent, we performed design, synthesis, and biological evaluation of jietacin derivatives against parasitic nematodes. Of these derivatives, we found that a fully synthesized simplified derivative exhibited better anthelmintic activity against three parasitic nematodes than natural jietacins. In addition, it had a better efficacy in vivo through oral administration against a mouse nematode. This indicated that the azoxy motif could prove useful as a template for anthelmintic discovery, possibly creating a class of anthelmintic with novel skeletons, a potential new mode of action, and providing further insight for rational drug design.

Full text of DOI:10.1016/j.ejmech.2017.12.031

Accepted Manuscript
Jietacins, azoxy antibiotics with potent nematocidal activity: Design, synthesis, and
biological evaluation against parasitic nematodes
Akihiro Sugawara, Masahiko Kubo, Tomoyasu Hirose, Kyoichi Yahagi, Noriaki
Tsunoda, Yoshihiko Noguchi, Takuji Nakashima, Yoko Takahashi, Claudia Welz,
Dennis Mueller, Christina Mertens, Johannes Koebberling, Satoshi Ōmura, Toshiaki
Sunazuka
PII:
S0223-5234(17)31037-1
10.1016/j.ejmech.2017.12.031
EJMECH 10009
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 October 2017
Revised Date: 25 November 2017
Accepted Date: 9 December 2017
Please cite this article as: A. Sugawara, M. Kubo, T. Hirose, K. Yahagi, N. Tsunoda, Y. Noguchi, T.
Nakashima, Y. Takahashi, C. Welz, D. Mueller, C. Mertens, J. Koebberling, S. Ōmura, T. Sunazuka,
Jietacins, azoxy antibiotics with potent nematocidal activity: Design, synthesis, and biological
evaluation against parasitic nematodes, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2017.12.031.
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ACCEPTED MANUSCRIPT
Jietacins, azoxy antibiotics with potent nematocidal activity: Design, synthesis, and
biological evaluation against parasitic nematodes
a,d
a
a
a
Akihiro Sugawara , Masahiko Kubo , Tomoyasu Hirose , Kyoichi Yahagi , Noriaki
a
a
a
a
b
Tsunoda , Yoshihiko Noguchi , Takuji Nakashima , Yoko Takahashi , Claudia Welz ,
b
c
b
a
Dennis Mueller , Christina Mertens , Johannes Koebberling , Satoshi Ōmura *,
a
Toshiaki Sunazuka **
a
Kitasato Institute for Life Sciences and Graduate School of Infection Control
Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-8641,
Japan.
b
Bayer AG Drug Discovery Pharmaceuticals, Wuppertal, Berlin, Germany.
c
Bayer Animal Health GmbH, Monheim, Germany.
Key words, jietacin, vinyl azoxy, nematocidal, anthelmintic, natural product, late-stage
diversification
Present address
d
Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3, Aza-Aoba,
Aramaki, Aoba-ku, Sendai 980-8578, Japan
Corresponding authors
Professor Toshiaki Sunazuka
Kitasato Institute for Life Sciences and Graduate School of Infection Control Sciences,
Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-8641, Japan.
Phone & Fax; +81-3-5791-6340
Email; sunazuka@lisci.kitasato-u.ac.jp
Distinguished Professor Emeritus Satoshi Ōmura
Phone; +81-3-5791-6101
Email; omuras@insti.kitasato-u.ac.jp
1

ACCEPTED MANUSCRIPT
Authors’ contribution is as follows:
A.S., S.Ō., and T.S. designed the research;
A.S., C.W., S.Ō., and T.S. wrote the paper;
A.S., M.K., T.H., K.Y., N.T. and Y.N. synthesized jietacin derivatives and related
substrates;
C.W. and C.M. performed the evaluation of anthelmintic activity;
D.M. performed the toxicological analysis;
T.N. and Y.T. isolated natural products;
J.K., S.Ō and T.S. provided scientific direction and established collaboration.
Email:
Akihiro Sugawara; sugawara@lisci.kitasato-u.ac.jp
Masahiko Kubo; masahiko882@gmail.com
Tomoyasu Hirose; thirose@lisci.kitasato-u.ac.jp
Kyoichi Yahagi; kyahagi01@gmail.com
Noriaki Tsunoda; tsunoda-n@microbiopharm.com
Yoshihiko Noguchi; ynoguchi@lisci.kitasato-u.ac.jp
Takuji Nakashima; takuji@lisci.kitasato-u.ac.jp
Yoko Takahashi; ytakaha@lisci.kitasato-u.ac.jp
Claudia Welz; claudia.welz@bayer.com
Dennis Mueller; dennis.mueller1@bayer.com
Christina Mertens; christina.mertens@bayer.com
Johannes Koebberling; johannes.koebberling@bayer.com
Satoshi Ōmura; omuras@insti.kitasato-u.ac.jp
Toshiaki Sunazuka; sunazuka@lisci.kitasato-u.ac.jp
Highlights
•
•
Jietacin A, an azoxy antibiotic, was shown to have potent anthelmintic activity.
A simplified jietacin derivative exhibits better anthelmintic activity than that of jietacin
A.
•
•
•
Structure-activity relationships of azoxy antibiotics were revealed.
A simplified derivative showed anthelmintic activity via oral administration.
Azoxy motif was found to be a useful platform for drug discovery.
2

ACCEPTED MANUSCRIPT
Financial interest
The authors declare no competing financial interest.
Abbreviations
AcOH, acetic acid; Cbz, carboxybenzyl; (COCl) , oxalyl chloride; DCM,
2
Dichloromethane; DMSO, Dimethyl sulfoxide; DMF, Dimethylformamide; Et N,
3
Triethylamine; LD50, lethal dose 50; NaN , sodium azide; NaBH CN, Sodium
3
3
cyanoborohydride; TBHP, tert-butyl hydroperoxide; TBS, tert-butyldimethylsilyl;
TBTA, Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amin)
Graphical abstract
3

ACCEPTED MANUSCRIPT
Abstract
Jietacins, an azoxy antibiotic class of chemicals, were isolated from the culture broth of
Streptomyces sp. KP-197. They have a unique structural motif, including a vinyl azoxy
group and a long acyclic aliphatic chain, which is usually branched but non-branched in
the case of jietacin C. During a drug discovery program, we found that jietacins display
potent anthelmintic activity against parasitic nematodes and that jietacin A has a
moderate or low acute toxicity (LD > 300 mg/kg) and no mutagenic potential in a
5
0
mini Ames screen. This suggests that jietacins have potential for drug discovery
research. In order to create a novel anthelmintic agent, we performed design, synthesis,
and biological evaluation of jietacin derivatives against parasitic nematodes. Of these
derivatives, we found that a fully synthesized simplified derivative exhibited better
anthelmintic activity against three parasitic nematodes than natural jietacins. In addition,
it had a better efficacy in vivo through oral administration against a mouse nematode.
This indicated that the azoxy motif could prove useful as a template for anthelmintic
discovery, possibly creating a class of anthelmintic with novel skeletons, a potential
new mode of action, and providing further insight for rational drug design.
4

ACCEPTED MANUSCRIPT
1
. Introduction
1
.1 Helminths and anthelmintics
Helminths are parasitic worms that have caused major and widespread disease in
both humans and animals for centuries [1]. The two major phyla of helminths include
nematodes and platyhelminths. The World Health Organization reported that the number
of people infected with soil-transmitted helminths surpasses a billion people worldwide
[
2]. Such infections are a major cause of long-term illness, leading to productivity loss
and poverty, and may be fatal. Likewise, helminth infections in animals are also of
grave concern, since they lead to economic losses, animal welfare issues, and impact on
human food supplies.
Historically, helminth infections have been treated with several classes of broad
spectrum anthelmintics [3], such as benzimidazoles [4], probenzimidazoles [5],
salicylanilides and substituted phenols [6], imidazothiazoles [7], tetrahydropyrimidines
[
8], organophosphates [9], macrocyclic lactones [10-16]. These compounds display
variable modes of action and impact, while options for vaccination against helminth
parasites remain severely limited. Unfortunately, reports of drug-resistant nematodes
have been steadily increasing for currently available classes of anthelmintics [17-19].
Although novel classes of anthelmintic agents, such as octadepsipeptides [20, 21],
aminoacetonitriles [22, 23], and spiroindoles [24] have been approved in order to help
overcome drug-resistance, anthelmintic agents with a novel skeleton, which may have a
new mode of action, are still urgently needed for use in both human and animal health.
1
.2 Jietacins, azoxy antibiotics
The Kitasato research team has a long history of searching for anthelmintics from
microbial metabolites originating in natural sources. In fact, a collaboration between the
Merck, Sharpe and Dohme research laboratories and the Kitasato research group
discovered the avermectins, the world’s first ‘endectocides’, the compounds quickly
becoming widely used as antiparasitic agents in veterinary medicine [10]. In 1987,
during screening for nematocidal agents from natural sources, the azoxy antibiotics
5

ACCEPTED MANUSCRIPT
jietacin A (1) and B (2) were isolated from the culture broth of Streptomyces sp. KP-197
(Fig. 2) [25, 26]. More recently, in 2014, we discovered more novel jietacin analogs
(jietacin C (3) and D (4)), together with different components of the aliphatic side chain,
from the same culture broth (Fig. 1) [27].
Aliphatic long chain
Vinyl azoxy
O
N
N
Jietacin A (1)
O
O
N
N
O
Jietacin B (2)
O
N
N
O
Jietacin C (3)
O
N
N
O
Jietacin D (4)
Properties of Jietacin A
-
-
-
-
Nematocidal activity
No antibacterial activity
Moderate or low acute toxicity LD50 >300 mg/kg (rat)
No mutagenic potential (mini Ames test; negative)
Fig. 1. Structure of azoxy antibiotics (jietacin A-D) and some bioactive properties of
jietacin A.
While there are some other natural products bearing an azoxy moiety, such as
elaiomycin [28-30], valanimycin [31], maniwamycins [32], LL-BH 872α [33], MH-071,
MH-072 [34], and pyrinadine A [35], the jietacins contain a unique structural motif, a
vinyl azoxy group and long acyclic aliphatic chains.
In terms of biological activity, we previously reported that 1 and 2 showed 10-times
greater nematocidal activity against the pine wood nematode Bursaphelenchus
xylophilus (LD100 = 0.25 ppm) than avermectin B1a (LD100 = 2.5 ppm), the main
component of the commercially available antiparasitic agent [36]. Given the attractive
6

ACCEPTED MANUSCRIPT
nematocidal activity and structural features of jietacins, a drug discovery program was
started to identify a potential jietacin-derived candidate as a new anthelmintic. Initially,
we further investigated the biological properties of 1 in order to identify a suitable
candidate lead compound. As a result, we found that 1 showed potent activity in an
assay using Nippostrongylus braziliensis (up to 100% inhibition of AChE (acetylcholine
esterase) secretion at 10 and 1 ppm dosage) [37]. This is comparable to emodepside, a
commercially available antiparasitic agent. The acute oral toxicity of 1 in female rats
was absent, with no adverse clinical signs and no lethality at a dosage of 300 mg/kg.
The LD is therefore considered to be >300 mg/kg. No evidence for mutagenic impact
5
0
was found in a mini-Ames test with three different Salmonella typhimurium mutant
strains. In contrast to the anthelmintic activity, no antibacterial activity of natural 1
against 27 strains was observed [38].
Given the anthelmintic activity in vitro of 1 and the fact that it did not show any
significant toxicity in our assays, we decided to utilize this natural product as a lead
compound for chemical optimization and clarification of structure-activity relationships
(SARs).
Herein, we report the synthesis of novel jietacin derivatives, which showed greater
anthelmintic activity against several nematodes than the parent jietacin A, insights into
their SARs, and evaluation of biological activity in vitro and in vivo.
2
. Results and discussion
2
.1 Initial SAR maps based on natural jietacins
We previously reported on SAR concerned with activity against the pine wood
nematode, Bursaphelenchus xylophilus [36], and showed that 1) changing a ketone to an
acetal or an alcohol did not lead to a significant reduction in activity, suggesting a
ketone does not play an important role and that 2) the vinyl azoxy group was necessary
for anthelmintic activity at low concentration.
To further investigate SAR, we evaluated in vitro activity of four natural products
(1-4), plus one derivative, against gastro-intestinal nematodes and compared the results
with those for emodepside and ivermectin (IVM), both commercially available
mainstream drugs. Tests were carried out using larvae of Haemonchus contortus and
7

ACCEPTED MANUSCRIPT
Cooperia curticei, both parasites of sheep, and adults of the rat nematode
Nippostrongylus braziliensis. Anthelmintic activity for 1-4 ((R)-4 is not a natural
product [27]) is summarized in table 1. Of significance, 1-4 and (R)-4 showed 100%
efficacy (inhibition of AChE secretion) in N. braziliensis at 1 ppm, the same as
emodepside (100% efficacy at 10 and 1 ppm) and ivermectin. Lower concentrations
were not tested. Against third-stage larvae of H. contortus and C. curticei, jietacins
showed only low or moderate efficacy with one exception: 3, showed 100% efficacy at
4
ppm against H. contortus, which was again comparable to emodepside and ivermectin.
(R)-4 had no efficacy against the larvae.
Overall, we found that, with regard to 2, 3, and (R/S)-4, possession of a
non-branched side chain (e.g. 3), caused better anthelmintic activity compared with
possession of a branched side chain (e.g. 2 and (R/S)-4), even though they have the
same carbon length.
Consequently we found that changing functional groups of the side chains plays a
crucial role for generating anthelmintic activity, although the vinyl azoxy group is
necessary for the activity based on our previous work [36]. To complete these SARs
with respect to side chains, testing of more derivatives will be required.
Table 1
In vitro anthelmintic activity of jietacin A-D, ivermectin, and emodepside.
H. contortus
C. curticei
(efficacy %)
4
N. braziliensis
1
1
2
(
efficacy %)
4
(efficacy %)
2
0
0.8
ppm
0
20
0.8
ppm
0-80
60-80
0
10
ppm
100
100
100
100
100
100
100
1
Compound
ppm
ppm
ppm
ppm
ppm
100
100
100
100
100
100
100
Emodepside
Ivermectin
80-100
80-100
0
80-100
90-100 90-100
80-100 40-80
90
60
40
60
40
0
90
60
0
Jietacin A (1)
Jietacin B (2)
Jietacin C (3)
Jietacin D (4)
R)-Jietacin D ((R)-4)
0
0
0
0
0
0
0
60
0
100
40
100
40
0
40
0
0
0
(
0
0
0
1
Efficacy in larval assay: decrease of motility, 100% efficacy means that all larvae were immotile, 0%
efficacy means that all larvae were motile. Results for jietacins are presented as the mean of duplicate
8

ACCEPTED MANUSCRIPT
tests. In case of the reference compounds emodepside and ivermectin, compounds were tested more often
and in at least two batches, each test performed in duplicate. Therefore, a range of the results is indicated.
The range observed for emodepside in C. curticei at 0.8 ppm is considered to be due to the impact of
potential variability in concentration of the highly lipophilic compound at a sensitive point in the
dose-response curve. Lower concentrations of emodepside (0.16 ppm, 0.032 ppm) have no efficacy in the
respective assay (data not shown).
2
Efficacy in Nippostrongylus assay: decrease of acetylcholine esterase activity compared to negative
control. Anthelmintic activity was categorized using the following scale: 100: >84%-100% = full activity
(
complete AChE inhibition compared to negative control); 84: >60%-84% = good activity; 60:>35%-60%
=
weak activity, and 35: 0%-35% = no activity.
2
.2 Strategy for side-chain modification
Based on our initial SAR maps, our new strategy focused on synthesis of various
side-chain derivatives, which were 1) alkyl, aryl groups with triazole linkage via click
reaction, 2) acyl derivatives with ketone linkage via Grignard reagents, and 3) aliphatic
alkyl chains with no linkage (Fig. 2). To accomplish this strategy, we decided to utilize
two approaches, late-stage diversification [39-41] and function-oriented synthesis [42,
4
3].
Late-stage
diversification
N
N
N
R
N3
N
N
N
O
N
O
8
Triazole derivatives 5
O
O
R
H
O
N
N
N
R
N
OMe
N
O
O
9
(ref. 27)
Acyl derivatives 6
H N
OH
2
N
Cbz
N
N
10
n
O
Function-oriented synthesis
Alkyl-chain derivatives 7
Fig. 2. Strategy; three derivative targets.
9

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2
.3 Synthesis
Although the challenging task for this synthesis is to easily construct a vinyl azoxy
moiety, we previously developed an effective construction of such a moiety through a
4
-step synthesis [27].
Based on our design, we began with synthesis of triazole derivatives utilizing our
synthetic route (Scheme 1). We envisioned that various functional groups can be
introduced by late-stage copper-catalyzed triazole reaction with azides and alkynes, in
one-step, at the final reaction. Swern oxidation of mono-TBS alcohol 11 [44] gave the
corresponding aldehyde (not shown), followed immediately by reductive hydrazination
with Cbz hydrazine 10, to give the corresponding hydrazine 12 in 82% yield over 2
steps. The azo moiety (13) was formed in 82% yield by air oxidation of the
corresponding hydrazine, after removal of the Cbz group of 12. Regioselective
oxidation [45, 46] of 13 gave azoxy 14 (in 72% yield) followed by removal of TBS
group to afford 15 in quantative yield. To construct the vinyl group, bis-mesylation of
1
5 gave the corresponding mesylated compound, followed by chemoselective
β-elimination using the difference of acidity of the two protons to give 16 in 82% yield.
Finally, substitution reaction with NaN led to the triazole precursor 8 in 85% yield.
3
With the azide in hand, a late-stage diversification in the presence of a vinyl azoxy
moiety utilizing copper catalyzed triazole reaction [47-54] with 16-type acetylenes
readily led to the corresponding triazole 5a-q bearing aromatic, aliphatic, or hydrophilic
groups in 35-97% yields [55].
1
0

ACCEPTED MANUSCRIPT
Scheme 1. Synthesis of triazole derivatives 5a-q. Reagents and conditions;
a) DMSO, (COCl) , Et N, DCM, –78 ˚C, then 10, AcOH, NaBH CN, CHCl , 0 ˚C, 82%
2
3
3
3
over 2 steps, b) H , Pd(OH) , MeOH, then air [O], rt 82%, c) TBHP, VO(acac) , DCM,
2
2
2
0
8
8
9
˚C, 72%, d) AcOH, H O, THF, rt, quant., e) MsCl, Et N, DCM, rt, then DBU, 35 ˚C,
2 3
1%, f) NaN , DMF, 50 ˚C, 85%, g) R-N , Cu(MeCN) PF , TBTA [56], MeOH, rt, (5a;
3
3
4
6
5%, 5b; 96%, 5c; 89%, 5d; 84%, 5e; 91%, 5f; 93%, 5g; 83%, 5h; 91%, 5i; 84%, 5j;
1%, 5k; 97%, 5l; 96%, 5m; 51%, 5n; 35%, 5o; 73%, 5p; 89%, 5q; 61%).
Based on the results with triazole derivatives (vide infra), we focused on making
acyl analogs with ketone linkage via Grignard reagents in the presence of LaCl •2LiCl
3
[
57] as an additive, previously developed through our total synthesis process (Scheme
). We devised a “late-stage diversification” strategy which accelerated easy production
of alkyl chain analogs with only one-step from 9. Since SARs with respect to triazole
2
11

ACCEPTED MANUSCRIPT
analogs indicated that an aliphatic alkyl group showed better activity than others, we
introduced some alkyl groups via Grignard reagents in the presence of LaCl •2LiCl. In
3
fact, treatment of the Weinreb amide 9 [27], which we reported previously, with various
Grignard reagents in the presence of LaCl •2LiCl, gave 6a-g in 47-84% yields.
3
6
steps
(ref. 27)
O
N
N
N
O
OMe
9
common key intermediate
Late-stage
diversification
a
O
N
R
N
O
6a-f
6
6
6
6
6
6
a; R=Me (84%)
b; R=Et (58%)
c; R=n-Bu (53%)
d; R=Pentadecyl (53%)
e; R=Isopentyl (60%)
f; R=Phenylethyl (47%)
Scheme 2. Synthesis of 6a-f. Reagents and conditions; a) R-MgBr, LaCl •2LiCl, THF
3
(6a; 84%, 6b; 58%, 6c; 53%, 6d; 53%, 6e; 60%, 6f; 47%)
We next envisioned that function-oriented synthesis might be utilized; i.e. total steps
will theoretically be reduced from 7 to 4 by deletion of a carbonyl group, because we
assumed, based on SARs, that a ketone does not play an important role in anthelmintic
activity. In order to demonstrate this, we designed a ketone-deficient alkyl-chain
derivative 7b, based on 6c. In the same fashion, we designed 7a and 7c to reconfirm the
impact of side-chain carbon length. In fact, 7a-c were synthesized from the aldehydes
1
7a-c (n=4,10,16) (Scheme 3). Likewise, use of the regioisomer of azoxy moiety 21,
obtained after oxidation of azo, led to the vinyl azoxy 22 in 73 % yield [58-60].
1
2

ACCEPTED MANUSCRIPT
A) Synthesis of alkyl chain derivatives 7a-c with different lengths.
a
b
H
O
N
OH
N
OH
N
N
n
n
n
Cbz
1
1
1
7a (n=4)
7b (n=10)
7c (n=16)
18a (n=4) : 77%
18b (n=10): 81%
18c (n=16) : 2 steps 66%
19a (n=4) : 85%
19b (n=10): 72%
19c (n=16) : 82%
c
d
N
OH
N
N
O
N
O
n
n
2
2
2
0a (n=4): 55%
0b (n=10): 72% (21: 22%)
0c (n=16): 50%
7a (n=4) : 91%
7b (n=10): 71%
7c (n=16) : 77%
B) Synthesis of regioisomer 22 of azoxy moiety 7b
O
N
d
O
OH
N
N
N
1
10
10
2
22
Scheme 3. A) Synthesis of alkyl chain derivatives 7a-c. B) Synthesis of 22, regioisomer
of azoxy moiety 7b. Reagents and conditions; a) 10, AcOH, NaBH CN, CHCl , 0 ˚C,
3
3
(18a; 77%, 18b; 81%, 18c; 66% (2 steps)), b) H , Pd(OH) , MeOH, then air [O], rt,
2 2
(
19a; 85%, 19b; 72%, 19c; 82%), c) TBHP, VO(acac) , DCM, 0 ˚C, (20a; 55%, 20b;
2
7
2%, 20c; 50%), d) MsCl, Et N, DCM, rt, then DBU, 35 ˚C, (7a; 91%, 7b; 71%, 7c;
3
7
7%, 22; 73%)
2
.4 Biological evaluation
In vitro anthelmintic activity of triazoles 5a-q are summarized in Table 2. In order to
investigate the impact of the triazole moiety on anthelmintic activity, we designed
triazole derivative 5a to possess the same carbon pendant (15-carbon chain), as 1.
Biological evaluation indicated that both 1 and 5a had similar properties (100% efficacy
at 1 ppm against N. braziliensis), suggesting that the triazole moiety might be replaced
by a ketone group. We, therefore, focused on making triazole derivatives to introduce
various functional groups for better understanding of SARs. Introduction of alcohol
(5b), nitrile (5c), bromo (5e), and acetate (5l) groups on the alkyl chain led to reduced
efficacy in the N. braziliensis assay at 1 ppm. Likewise, activity of other alkyl chain
1
3

ACCEPTED MANUSCRIPT
derivatives (5d, 5f, 5g, 5h, 5i, 5j, and 5k) and a silyl derivative (5m) did not satisfy our
criteria (<100% efficacy at 1 ppm against N. braziliensis). No n-butyl group (5n) on the
triazole moiety led to loss of activity (35% efficacy at 1 ppm against N. braziliensis).
Although a phenyl derivative (5o) showed activity (100% at 1 ppm against N.
braziliensis), it was not effective against either H. contortus or C. curticei, while other
two aromatic derivatives (5p, 5q) had no effect whatsoever. In case of compound 5e, it
should be noted that in the N. braziliensis assay it had no activity (35%) at 10 ppm, but
weak activity (60%) was found at 1 ppm [61]. As a result, no derivatives with stronger
activity than 5a were obtained. Especially, introduction of a hydrophilic functional
group on the triazole moiety clearly decreased activity at 1 ppm against N. braziliensis.
We thus concluded that efficacy would be affected by changing the aliphatic chain at a
side chain.
In terms of biological activities of acyl derivatives, Weinreb amide 9, a common key
intermediate, lost anthelmintic activity (35% efficacy at 1 ppm N. braziliensis) (Table 2).
Although the alkyl group bearing an appropriate carbon length (methyl 6a, ethyl 6b,
n-butyl 6c, isopentyl 6e) showed good anthelmintic activity, pentadexyl 6d completely
lost activity against N. braziliensis, presumably due to having a lengthy alkyl chain.
Although the phenylethyl 6f showed good activity against H. contortus and N.
braziliensis, activity of 6f against C. curticei was weak. Consequently, we concluded
that anthelmintic activity of the n-butyl 6c demonstrated the best balance (80% efficacy
at 4 ppm against H. contortus, 100% efficacy at 20 ppm against C. curticei, 100%
efficacy at 1 ppm against N. braziliensis).
Next, we evaluated biological activities of alkyl-chain derivatives (Table 2).
Although 7a-c indicated 100% efficacy at 1 ppm dosage against N. braziliensis, almost
no activity of 7a (C6) and 7c (C16) analogs against H. contortus or C. curticei was
observed. Pleasingly, 7b (bearing a C12 aliphatic chain) exhibited stronger anthelmintic
activity against all three nematodes than the other compounds, indicating that
appropriate moderate carbon length is necessary to bestow anthelmintic characteristics.
Our attention next turned to regioisomers of the azoxy moiety. Biological evaluation
revealed that 22, a regioisomer of 7b, had significantly reduced anthelmintic activity,
suggesting the direction of the azoxy moiety plays an important role in bestowing
anthelmintic activity and that it may be recognized by a target protein.
1
4

ACCEPTED MANUSCRIPT
Table 2
In vitro anthelmintic activity of derivatives 5a-q, 6a-6f, 7a-7c, 9, and 22.
H. contortus
C. curticei
N. braziliensis
1
1
2
(
efficacy %)
(efficacy %)
(efficacy)
2
ppm
0
4
ppm
0.8
ppm
20
ppm
4
ppm
0.8
ppm
10
ppm
1
ppm
Compound
5
a
100
40
0
100
40
40
100
100
0
0
0
0
0
0
0
0
0
0
5
5
5
5
5
5
b
c
d
e
f
0
0
0
0
0
0
0
0
60
84
60
35
60
60
60
84
0
6
8
0
0
90
90
90
0
0
100
35
40
40
0
0
0
100
100
g
100
0
100
80
40
0
0
0
0
60
60
100
40
40
60
80
0
0
0
5
5
5
5
5
5
5
5
h
i
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
100
100
100
84
60
84
35
35
35
60
35
100
6
0
0
40
40
0
j
0
0
k
l
0
0
0
84
0
40
0
m
n
o
100
84
4
0
0
0
0
0
0
100
0
80
0
0
5
5
p
q
0
0
0
0
100
84
35
60
0
0
0
9
6
6
6
6
6
6
7
7
7
2
0
0
0
40
0
0
0
0
0
0
0
0
0
0
0
0
0
0
84
100
100
100
0
35
100
100
100
0
a
b
c
d
e
f
100
100
100
0
60
60
80
0
90
100
100
0
40
0
0
0
0
0
100
100
40
100
0
0
0
100
90
0
0
100
100
100
100
100
84
100
100
100
100
100
35
60
0
0
0
a
b
c
2
0
0
100
0
60
0
100
0
80
0
40
0
0
0
0
1
Efficacy in larval assay: decrease of motility, 100% efficacy means that all larvae were immotile, 0%
efficacy means that all larvae were motile
2
Efficacy in Nippostrongylus assay: decrease of acetylcholine esterase activity compared to negative
control. Anthelmintic activity was categorized using the following scale: 100: >84%-100% = full activity
(
=
complete AChE inhibition compared to negative control); 84: >60%-84% = good activity; 60:>35%-60%
weak activity, and 35: 0%-35% = no activity.
1
5

ACCEPTED MANUSCRIPT
2
.5 SAR maps
The SAR of jietacins and their derivatives are summarized; 1) No carbonyl group
is needed (7b vs 6c); 2) the vinyl azoxy moiety participates in displaying stronger
inhibition (as shown in previous work) [36]; 3) chain length significantly influences
anthelmintic activity. Although shorter (7a; C6) and longer (7c; C16) carbon length
decreased activity, moderate chain length (7b; C9-14) exhibited good anthelmintic
activity against H. contortus, and C. curticei; 4) non-branched chains tend to correlate
with greater potency compared to branched; 5) hydrophobicity in a side chain is
preferable, e.g. attachment of hetero atoms in a side chain decreased activity (triazole
derivatives 5a-q); 7) Regioisomer of azoxy moiety, including a vinyl group, plays an
important role in nematocidal impact (7b vs 22).
2
.6 In vivo assay in mice
Based on the promising in vitro results, we subsequently tested selected compounds
for their anthelmintic activity in vivo using a mouse model. Mice were infected with
Heligmosomoides polygyrus, a gastrointestinal nematode in mice. Mice were treated
orally once or daily on four consecutive days. As shown in Table 3, jietacin A had full
efficacy (100%) at 100 mg/kg after 4-day treatment (entry 2). A reduced amount of
jietacin A had no efficacy (<50%) at 25 mg/kg on 1 day (entry 3), additionally, no
efficacy was observed at 10 mg/kg on 1 day (entry 4). Notably, the derivative 7b had
full efficacy (100%) at 25 mg/kg following 4-day treatment (entry 6). Although this
result is not favourably comparable to emodepside (full efficacy, 10 mg/kg at 4-day
regimen (entry 1)), this is the first example, to the best of our knowledge, that a
compound having an azoxy moiety exhibited 100% anthelmintic efficacy in vivo,
thereby indicating azoxy compounds have the possibility of becoming good lead
candidates in future. While 7b fully efficacious at a dosage of 25 mg/kg for four days, at
a high dosage (100 mg/kg using a 4-day regimen, entry 5) animals showed strong
adverse effects and were euthanized for animal welfare reasons after the third treatment.
When the dosage was decreased (25 mg/kg on 1 day), efficacy was only 73% (entry 7),
and a dosage of 10 mg/kg on a 1-day regimen had no or little effect (<50%, entry 8).
Other derivatives bearing a triazole moiety, 5a, 5g, 5i, and 5o had mostly no efficacy
(<50%, entry 9, 10, and 12), or 70% efficacy (entry 11) at 100 mg/kg with a 4-day
1
6

ACCEPTED MANUSCRIPT
regimen, indicating that a triazole moiety may be a significant element for anthelmintic
activity.
Table 3
Emodepside and jietacins against Heligmosomoides polygyru in vivo.
entry
Dosage (mg/kg) Treatment days
Formulation
Efficacy
100%
100%
<50%
<50%
toxic
1
2
3
4
5
6
7
8
9
Emodepside
10
100
25
4
4
1
1
4
4
1
1
4
4
4
4
A
B
B
B
A
B
B
B
A
A
A
A
Jietacin A
Jietacin A
Jietacin A
10
7b
7b
7b
7b
5a
5g
5i
100
25
100%
73%
25
10
<50%
<50%
<50%
70%
100
100
100
100
1
1
1
0
1
2
5o
<50%
Formulation A: 25% Cremophor EL, 75% water.
Formulation B: 10% Transcutol, 10% Cremophor EL, 80% physiol. NaCl solution (0.9%).
3
. Conclusion
Our results confirm that jietacins possess potent anthelmintic activity against
parasitic nematodes. Jietacin A is known to have moderate or low acute toxicity (LD >
5
0
3
00 mg/kg) and shows no mutagenic potential in a mini Ames screen, significant
indication of the promising properties of this compound for future drug development. In
addition, this study revealed not only design, synthesis, and biological evaluation of
jietacin derivatives against pathogenic nematodes, but also clarification of their SARs.
Of the derivatives evaluated, we found that a simplified derivative, 7b, exhibited better
anthelmintic activity in vitro than natural jietacin and had better efficacy in vivo
following oral administration against a mouse nematode, but lower tolerability. Further
1
7

ACCEPTED MANUSCRIPT
optimization of the compounds is therefore necessary to identify the best drug candidate
with a sufficient therapeutic potential. Nonetheless, to the best of our knowledge, this is
the first example of compounds with an azoxy moiety exhibiting 100% anthelmintic
efficacy in vivo. Therefore, our results indicate that an azoxy motif may be a useful
template for anthelmintic drug discovery. This has the promise of creating a class of
anthelmintics with novel skeletons, a potentially new mode of action, and an insight for
rational drug design. Although the purpose of this study was to develop veterinary
medicines, we believe that the results provide a useful insight for drug development,
with respect to azoxy antibiotics, for both human and animal health.
4
. Experimental section
4
.1. Chemistry
Pre-coated silica gel plates with a fluorescent indicator (Merck 60 F254, Cat.-No.
.05744.0001, Merck KGaA, Darmstadt, Germany) were used for analytical and
1
preparative thin layer chromatography. Flash column chromatography was carried out
with Kanto Chemical silica gel (Silica gel 60N, spherical neutral, 40-50 µm, Cat.-No.
3
7563-84, Kanto Chemical Co., Inc., Tokyo Japan) or Merck silica gel 230-400 mesh
1
ASTM (60N, 40-63 µm, Cat.-No. 109385). H NMR spectra were recorded at 500 MHz
1
3
and C NMR spectra were recorded at 125 MHz on JEOL ECA-500 (500 MHz, JEOL
Ltd., Tokyo, Japan). The chemical shifts are expressed in ppm downfield from internal
1
13
solvent peaks CDCl (7.26 ppm, H NMR), CDCl (77.16 ppm, C NMR), CD OD
3
3
3
1
13
(
3.31 ppm, H NMR), CD OD (49.0 ppm, C NMR), and coupling constant (J values)
3
are given in Hertz. The coupling patterns are expressed by s (singlet), d (doublet), dd
double doublet), ddd (double double doublet), t (triplet), dt (double triplet), q (quartet),
(
dq (double quartet), m (multiplet), br (broadened), app (appearance). All infrared
spectra were measured using a Horiba FT-210 spectrometer (Horiba Ltd., Kyoto, Japan).
High- and low-resolution mass spectra were measured on a JEOL JMS-700 MStation
and JEOL JMS-T100LP. Melting points were measured with a Yanaco Micro Melting
Point System MP-500P (Anatec Yanaco Corporation, Kyoto, Japan).
4
.1.1. General procedure for azo formation
1
8

ACCEPTED MANUSCRIPT
1
0% Pd(OH) (2.0 equiv.) was suspended in MeOH (0.1 M) at 0 ˚C under N
2
2
atmosphere. To the resulting suspension was added a solution of Cbz-hydrazine (1.0
equiv.) in MeOH (0.5 M). The solution was subjected to an atmosphere of hydrogen and
stirred at room temperature for 2 h. After the starting material was consumed, the
mixture was filtered through a pad of Celite and washed with a mixture of MeOH and
CHCl (1/10, v/v) The filtrate was concentrated in vacuo. The crude product was
3
.
immediately oxidized at room temperature by exposure to air until the hydrazine was
consumed. The crude product was purified by flash column chromatography to give the
azo compound.
4
.1.2 General procedure for azoxy formation
To a solution of azo compound (1.0 equiv.) and VO(acac) (0.1 equiv.) in DCM (0.1
2
M) was added TBHP (5.5 M in decane, 1.2 equiv.) at 0 ˚C under N atmosphere. After
2
stirring at the same temperature until the starting material was consumed, the resulting
mixture was quenched with saturated aqueous Na S O , and extracted with CHCl . The
2
2
3
3
organic layer was washed with brine, dried over Na SO , filtered, and concentrated. The
2
4
residue was purified by flash column chromatography to give the azoxy compound and
its regioisomer.
4
.1.3. General procedure for triazole reaction
To a solution of azide 8 in MeOH (0.2 M) at room temperature were added
acetylenes
(1.2
equiv.),
tetrakis(acetonitrile)copper(I)
hexafluorophosphate
(
(
Cu(MeCN) PF , 0.05 equiv.), and tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine
4
6
TBTA, 0.05 equiv.). The reaction mixture was stirred at room temperature until the
starting material was consumed. After the reaction mixture was concentrated in vacuo,
the crude product was purified by silica gel column chromatography
(
hexane/EtOAc=5/1 to CHCl /MeOH=10/1) to afford the desired triazole compounds
3
5
a-q.
4
.1.4. General procedure for Grignard reaction
To a solution of LaCl ・2LiCl (4.0 equiv.) in THF (2.0 M) was added drop-wise
3
Grignard reagent (4.0 equiv.). After stirring for 30 min at 0 ℃, a mixture of Grignard
1
9

ACCEPTED MANUSCRIPT
reagent and LaCl ・2LiCl was added to a solution of Weinreb amide (1.0 equiv.) in THF
3
(0.5M) at –40 ℃ in another flask and the mixture was stirred for 10 min. The reaction
mixture was then quenched with saturated aqueous NH Cl, extracted with EtOAc. The
4
organic layers were dried over Na SO , filtered, and concentrated in vacuo. The residue
2
4
was purified by preparative PLC on silica gel to afford a ketone analog.
4
.1.5. General procedure for reductive hydrazination
To a solution of 10 (1.0 equiv.) in MeOH (0.1 M) was added drop-wise aldehyde
(1.0 equiv.) at 0 ˚C. After stirring at room temperature for 30 min, to the mixture was
added AcOH (2.0 equiv.) and NaBH CN (6.0 equiv.) at 0 ˚C. The mixture was allowed
3
to warm to room temperature and stirred until all starting material was consumed. The
resulting mixture was diluted with CHCl and washed with pH 7.2 phosphate buffer,
3
and H O, brine, dried over Na SO , filtered, and concentrated in vacuo. The residue was
2
2
4
purified by flash column chromatography to give a corresponding Cbz-hydrazine.
4
.1.6. General procedure for construction of vinyl azoxy group
To a solution of azoxy ethylalcohol (1.0 equiv.) in DCM (0.1 M) were added Et N
3
(2.0 equiv.) and MsCl (1.2 equiv.) at room temperature. After stirring for 10 min, the
mixture was added to DBU (2.0 equiv.) and stirred at 35 ˚C for 3 min. The resulting
mixture was diluted with EtOAc and washed with saturated aqueous NH Cl and H O,
4
2
brine, dried over Na SO , filtered, and concentrated. The residue was purified by
2
4
preparative TLC on silica gel to give the vinyl azoxy compound.
4
.1.7. 8-[(tert-Butyldimethylsilyl)oxy]octan-1-ol (11)
To a solution of 1,8-octadiol (10.0 g, 68.4 mmol) in MeOH (340 mL) were added
Et N (47.8 mL, 342 mmol), TBSCl (9.79 g, 65.0 mmol) and DMAP (0.418 g, 3.42
3
mmol) and the mixture was stirred at 0 ˚C under nitrogen atmosphere. After stirring for
1
h, the reaction mixture was added to CHCl (600 mL) washed with saturated aqueous
3
NaHCO (900 mL), saturated aqueous NH Cl (900 mL, x2) and brine (900 mL). The
3
4
organic layer was dried with Na SO , filtered and concentrated in vacuo. The crude
2
4
product was purified by column chromatography on silica gel (hexane/EtOAc=15/1 to
5
/1) to afford 11 (9.06 g, 51%) as a colorless oil.
-1
IR (Diamond Prism) ν (cm ) : 3349, 2931, 2858, 1471, 1387, 1362, 1255, 1099, 1059,
2
0

ACCEPTED MANUSCRIPT
1
8
37, 775, 665; H NMR (500 MHz, CDCl ) δ (ppm) : 3.63 (t, J = 6.6 Hz, 2H), 3.59 (t, J
3
=
6.6 Hz, 2H), 1.58-1.46 (complex m, 4H), 1.35-1.30 (complex m, 8H), 0.88 (s, 9H),
1
3
0
.04 (s, 6H); C NMR (125 MHz, CDCl ) δ (ppm) : 63.3, 62.9, 32.8, 32.7, 29.4 (2C),
3
+
2
5.9 (3C), 25.69, 25.66, 18.3, -5.3 (2C); HRMS (FAB, NBA) m/z : 261.2250 [M+H] ,
calcd for C H O Si : 261.2242.
1
4
33
2
4
.1.8. Benzyl 2-{8-[(tert-butyldimethylsilyl)oxy]octyl}-1-(2-hydroxyethyl)-
hydrazine-1-carboxylate (12)
To a solution of DMSO (4.46 mL, 62.8 mmol) in DCM (96.0 mL) was added oxalyl
chloride (2.65 mL, 100 mmol) drop-wise using a syringe at –78 ˚C under nitrogen
atmosphere. After stirring for 30 min, to the reaction mixture was added 11 (5.03 g, 19.3
mmol) drop-wise using a syringe, the mixture being stirred for an additional 30 min at –
7
8 ˚C, before Et N (16.1 mL, 377 mmol) was added. After stirring for 10 min at –78 ˚C,
3
the reaction mixture was warmed to 0 ˚C and stirred for 1 h, then washed with saturated
aqueous NH Cl (90 mL), 10% aqueous NaHSO (90 mL, x2), saturated aqueous
4
3
NaHCO (90 mL), brine (90 mL), dried with Na SO , filtered and concentrated in vacuo.
3
2
4
The crude product was taken on to the next step without purification. To a stirred
solution of 10 (4.87 g, 23.2 mmol) in CHCl (190 mL) at 0 ˚C was added the crude
3
aldehyde, the reaction mixture subsequently being warmed to room temperature and
stirred. After stirring for 45 min, the reaction mixture was cooled to 0 ˚C, and AcOH
(2.4 mL, 38.6 mmol) was added. After stirring for 10 min, the mixture was added to
NaBH CN (7.28 g, 377 mmol) at 0 ˚C and stirred for 10 min at room temperature. The
3
reaction mixture was washed with phosphate buffer solution in water (pH 7.2, 190 mL),
brine (190 mL), dried with Na SO , filtered and concentrated in vacuo. The crude
2
4
product was purified by column chromatography on silica gel (hexane/EtOAc=5/1 to
3
/1) to afford 12 (7.19 g, 82%) as a colorless oil.
-1
IR (Diamond Prism) ν (cm ) : 3995, 2931, 2854, 1697, 1458, 1404, 1350, 1250, 1095,
1
8
33, 771, 694; H NMR (500 MHz, CDCl ) δ (ppm) : 7.36 (m, 5H), 5.16 (s, 2H), 3.80
3
(m, 2H), 3.62 (t, J = 4.9 Hz, 2H), 3.59 (t, J = 6.9 Hz, 2H), 2.88 (m, 2H), 1.51-1.45
1
3
(
complex m, 4H), 1.28 (complex m, 8H), 0.89 (s, 9H), 0.04 (s, 6H); C NMR (125
MHz, CDCl ) δ (ppm) : 156.5, 135.9, 128.2 (2C), 127.9, 127.7 (2C), 67.3, 62.9, 61.3,
3
4
9.9, 32.5, 29.2, 29.0, 28.9 27.5, 26.8, 25.7 (3C), 25.4, 18.1, –5.5 (2C); HRMS (FAB,
+
NBA) m/z : 451.3144 [M+H] , calcd for C H N O Si : 453.3149.
2
4
45
2
4
2
1

ACCEPTED MANUSCRIPT
4
.1.9. (E)-2-({8-[(tert-Butyldimethylsilyl)oxy]octyl}diazenyl)ethan-1-ol (13)
1
2 (8.80 g, 19.4 mmol) was converted to 13 (5.04 g, 82%) which appeared as a
yellow oil, according to the general procedure for azo formation.
-1
IR (Diamond Prism) ν (cm ) : 3316, 3224, 2931, 2854, 1511, 1462, 1427, 1334, 1257,
1
1
203, 1057, 972, 872, 833, 764,717, 617; H NMR (500 MHz, CDCl ) δ (ppm) : 4.04
3
(m, 2H), 3.99 (m, 2H), 3.81 (t, J = 6.8 Hz, 2H), 3.59 (t, J = 6.9 Hz, 2H), 1.79 (dt, J =
1
4.4, 6.9 Hz, 2H), 1.50 (m, 2H), 1.36-1.31 (complex m, 8H), 0.89 (s, 9H), 0.04 (s, 6H);
1
3
C NMR (125 MHz, CDCl ) δ (ppm) : 69.9, 69.6, 63.3, 60.3, 32.8, 29.3, 29.2, 27.5,
3
+
2
7.2, 26.0 (3C), 25.7, 18.3, –5.3 (2C); HRMS (FAB, NBA) m/z : 317.2617 [M+H] ,
calcd for C H N O Si : 317.2624
1
6
37
2
2
4
.1.10. (Z)-2-{8-[(tert-Butyldimethylsilyl)oxy]octyl}-1-(2-hydroxyethyl)diazene 1-oxide
(14)
1
3 (1.00 g, 3.16 mmol) was converted to 14 (751 mg, 72%), appearing as a yellow
oil, according to the general procedure for azoxy formation. IR (Diamond Prism) ν
-1
1
(
cm ) : 3396, 2931, 2854, 1504, 1466, 1419, 1311, 1250, 1195, 1095, 833, 771, 663; H
NMR (500 MHz, CDCl ) δ (ppm) : 4.31 (m, 2H), 4.04 (m, 2H), 3.59 (t, J = 6.9 Hz, 2H),
3
3
.45 (t, J = 6.9 Hz, 2H), 1.71 (dt, J = 14.4, 6.9 Hz, 2H), 1.50 (m, 2H), 1.41-1.31
13
(
complex m, 8H), 0.89 (s, 9H), 0.04 (s, 6H); C NMR (125 MHz, CDCl ) δ (ppm) :
3
7
0.4, 63.3, 59.5, 52.1, 32.8, 29.3 (2C), 27.7, 27.0, 26.0 (3C), 25.7, 18.4, –5.3 (2C);
+
HRMS (FAB, NBA) m/z : 333.2569 [M+H] , calcd for C H N O Si : 333.2573.
1
6
37
2
3
4
.1.11. (Z)-1-(2-Hydroxyethyl)-2-(8-hydroxyoctyl)diazene 1-oxide (15)
To a stirred solution of 14 (5.88 g, 17.7 mmol) in THF (51 mL) at 0 ˚C were added
water (51 mL) and AcOH (76 mL), the mixture then being warmed to room temperature.
After stirring for 2 h, the reaction mixture was quenched with saturated aqueous
NaHCO (180 mL) and extracted with EtOAc (150 mL, x2). The combined organic
3
layers were washed with brine (300 mL), dried with Na SO , filtered and concentrated
2
4
in vacuo. The crude product was purified by column chromatography on silica gel
(hexane/EtOAc=1/2) to afford 15 (3.86 g, quant) as a colorless solid.
-1
mp 47.5 ˚C; IR (Diamond Prism) ν (cm ): 3314, 3224, 2931, 2854, 1512, 1469, 1427,
1
1
336, 1257, 1203, 1057, 972, 872, 833, 764, 717, 617; H NMR (500 MHz, CDCl ) δ
3
2
2

ACCEPTED MANUSCRIPT
(ppm) : 4.31 (m, 2H), 4.05 (m, 2H), 3.64 (dt, J = 5.4, 1.2 Hz, 2H), 3.45 (t, J = 6.9 Hz,
2
H), 1.71 (dt, J = 14.4, 6.9 Hz, 2H), 1.55 (dt, J = 13.8, 6.9 Hz, 2H) 1.41-1.32 (complex
1
3
m, 8H); C NMR (125 MHz, CDCl ) δ (ppm) : 70.5, 62.8, 59.3, 52.1, 32.6, 29.2, 29.1,
3
+
2
7.6, 26.9, 25.6; HRMS (FAB, NBA) m/z : 219.1713 [M+H] , calcd for C H N O :
1
0
23
2
3
2
19.1709.
4
.1.12. (Z)-2-{8-[(Methylsulfonyl)oxy]octyl}-1-vinyldiazene 1-oxide (16)
To a solution of 15 (0.502 g, 2.30 mmol) in DCM (23 mL) were added Et N (0.950
3
mL, 6.89 mmol) and MsCl (0.430 mL, 5.51 mmol). After stirring for 5 min, the
corresponding bis-mesylate was observed on TLC. DBU (1.03 mL, 6.89 mmol) was
added and the mixture was stirred at 35 ˚C for 3 min. The reaction mixture was washed
with saturated aqueous NH Cl (20 mL x2), brine (30 mL), dried with Na SO , filtered
4
2
4
and concentrated in vacuo. The crude product was purified by column chromatography
on silica gel (hexane/EtOAc=2/1) to provide product 16 (0.447 g, 70%) as a colorless
solid.
-1
mp 52.1 ˚C; IR (Diamond Prism) ν (cm ) : 3124, 3039, 2931, 2854, 2190, 1466, 1419,
1
1
7
327, 1265, 1184, 1041, 941, 825, 733, 656; H NMR (500 MHz, CDCl ) δ (ppm) :
3
.10 (dd, J = 15.0, 8.0 Hz, 1H), 6.42 (d, J = 15.0 Hz, 1H), 5.50 (d, J = 7.5 Hz, 1H), 4.22
(t, J = 6.9 Hz, 2H), 3.53 (t, J = 6.9 Hz, 2H), 3.00 (s, 3H), 1.76-1.73 (complex m, 4H),
1
3
1
5
.42-1.35 (complex m, 8H); C NMR (125 MHz, CDCl ) δ (ppm) : 143.4, 115.3, 70.1,
3
2.5, 37.3, 29.1 (2C), 28.9, 27.6, 27.0, 25.3; HRMS (FAB, NBA) m/z : 279.1383
+
[
M+H] , calcd for C H N O S : 279.1379.
11
23
2
4
4
.1.13. (Z)-2-(8-Azidooctyl)-1-vinyldiazene 1-oxide (8)
To a solution of 16 (1.52 g, 5.46 mmol) in DMF (56 mL) was added NaN (0.526 g,
3
8
.19 mmol) at 50 ˚C. After stirring for 1.5 h, water (50 mL) was added to the reaction
mixture which was then extracted with a mixture of hexane/EtOAc = 1/1 (25 mL/25 mL,
v/v). The organic layer was separated and the aqueous layer was extracted with a
mixture of hexane/EtOAc=1/1 (25 mL/25 mL, v/v). The combined organic layer were
washed with brine (150 mL), dried with Na SO , filtered and concentrated in vacuo.
2
4
The crude product was purified by flash column chromatography on silica gel
(hexane/EtOAc=2/1) to afford 8 (1.05 g, 85%) as a yellow oil.
-1
IR (Diamond Prism) ν (cm ) : 3112, 3089, 2931, 2858, 2096, 1732, 1506, 1473, 1419,
2
3

ACCEPTED MANUSCRIPT
1
1
373, 1348, 1311, 1265, 1052, 953, 773, 723, 654; H NMR (500 MHz, CD OD) δ
3
(ppm) : 7.21 (dd, J = 15.0, 7.5 Hz, 1H), 6.35 (d, J = 15.0 Hz, 1H), 5.57 (d, J = 7.5 Hz,
1
7
H), 3.52 (t, J = 6.9 Hz, 2H), 3.28 (t, J= 6.9 Hz, 2H), 1.76 (m, 2H), 1.59 (dt, J = 14.9,
1
3
.5 Hz, 2H), 1.45-1.38 (complex m, 8H); C NMR (125 MHz, CD OD) δ (ppm) : 144.9,
3
116.0, 53.6, 52.5, 30.4 (2C), 30.0, 29.0, 28.3, 27.9; HRMS (FAB, NBA) m/z : 226.1671
+
[
M+H] , calcd for C H N O : 226.1668.
10 20 5
4
.1.14. (Z)-2-[8-(4-Butyl-1H-1,2,3-triazol-1-yl)octyl]-1-vinyldiazene 1-oxide (5a)
According to the general procedure for synthesis of triazole analogs, 8 (80.9 mg,
.359 mmol) with 1-hexyne (50 µL, 0.431 mmol) was converted to 5a (93.7 mg, 85%)
0
as a colorless solid.
-1
mp 62.2 ˚C; IR (Diamond Prism) ν (cm ) : 3214, 3078, 2924, 2854, 1643, 1550,
1
1
466,1411, 1342, 1265, 1157,1057, 957, 749, 779, 725, 656; H NMR (500 MHz,
CDCl ) δ (ppm) : 7.24 (s, 1H), 7.09 (dd, J = 14.9, 7.5 Hz, 1H), 6.41 (d, J = 14.9 Hz, 1H),
3
5
7
2
.50 (d, J = 7.5 Hz, 1H), 4.30 (t, J = 7.5 Hz, 2H), 3.53 (t, J = 7.5 Hz, 2H), 2.71 (d, J =
.5 Hz, 2H), 1.88 (dt, J = 14.9, 7.5 Hz, 2H), 1.74 (dt, J = 14.9, 7.5 Hz, 2H), 1.65 (m,
1
3
H), 1.43-1.29 (complex m, 10H), 0.93 (t, J = 7.5 Hz, 3H); C NMR (125 MHz,
CDCl ) δ (ppm) : 148.3, 143.4, 120.3, 115.2, 52.4, 50.1, 31.5, 30.3, 29.0, 28.8, 27.6,
3
+
2
6.9, 26.4, 25.3, 22.3, 13.8; HRMS (ESI) m/z : 330.2269 [M+Na] , calcd for
C H N NaO : 330.2270
16
29
5
4
.1.15.
-oxide (5b)
(Z)-2-{8-[4-(4-Hydroxybutyl)-1H-1,2,3-triazol-1-yl]octyl}-1-vinyldiazene
1
According to the general procedure for synthesis of triazole analogs, 8 (80.2 mg,
.356 mmol) with 5-hexyne-1-ol (50 µL, 0.427 mmol) was converted to 5b (93.1 mg,
1%) as a colorless solid.
0
8
-1
mp 69.9 ˚C; IR (Diamond Prism) ν (cm ) : 3448, 3402, 3116, 3062, 2924, 2854, 1550,
1
1
465, 1342, 1255, 1211, 1165, 1049, 997, 949, 864, 787, 648; H NMR (500 MHz,
CDCl ) δ (ppm) : 7.28 (s, 1H), 7.09 (dd, J = 14.9, 7.5 Hz, 1H), 6.41 (d, J = 14.9 Hz, 1H),
3
5
7
1
.50 (d, J = 7.5 Hz, 1H), 4.31 (t, J = 7.5 Hz, 2H), 3.68 (t, J = 6.9 Hz, 2H), 3.52 (t, J =
.5 Hz, 2H), 2.76 (t, J = 7.5 Hz, 2H), 1.98 (brs, 1H), 1.88 (dt, J = 14.9, 7.5 Hz, 2H),
13
.81-1.73 (complex m 4H), 1.64 (m, 2H), 1.43-1.34 (complex m, 8H); C NMR (125
MHz, CDCl ) δ (ppm) : 147.8, 143.2, 120.4, 115.2, 61.9, 52.3, 50.0, 32.0, 30.1, 28.9,
3
2
4

ACCEPTED MANUSCRIPT
+
2
8.7, 27.4, 26.8, 26.2, 25.5, 25.1; HRMS (ESI) m/z : 346.2221 [M+Na] , calcd for
C H N NaO : 346.2219
16
29
5
2
4
.1.16. (Z)-2-{8-[4-(4-Cyanobutyl)-1H-1,2,3-triazol-1-yl]octyl}-1-vinyldiazene 1-oxide
5c)
According to the general procedure for synthesis of triazole analogs, 8 (80.9 mg,
.359 mmol) with 6-heptynenitrile (50 µL, 0.431 mmol) was converted to 5c (106.7 mg,
9%) as a colorless solid.
(
0
8
-1
mp 50.5 ˚C; IR (Diamond Prism) ν (cm ) : 3109, 3070, 2924, 2854, 2244, 2209, 2185,
1
2
013, 1643, 1550, 1465, 1408, 1319, 1273, 1219, 1157, 1049, 964, 841, 771, 656; H
NMR (500 MHz, CDCl )δ(ppm) : 7.29 (s, 1H), 7.09 (dd, J = 14.9, 7.5 Hz, 1H), 6.41 (d,
3
J = 14.9 Hz, 1H), 5.50 (d, J = 7.5 Hz, 1H), 4.31 (t, J = 7.2 Hz, 2H), 3.53 (t, J = 6.9 Hz,
2
1
H), 2.77 (t, J = 7.2Hz, 2H), 2.38 (t, J = 6.9 Hz, 2H), 1.87 (m, 4H), 1.74 (m, 4H),
1
3
.42-1.29 (complex m, 8H); C NMR (125 MHz, CDCl ) δ (ppm) : 146.6, 143.2, 120.4,
3
119.3, 115.0, 52.2, 49.9 , 30.0, 28.7, 28.6, 28.1, 27.4, 26.7, 26.1, 24.49, 24.45, 16,6;
+
HRMS (ESI) m/z : 355.2230 [M+Na] , calcd for C H N NaO : 355.2222.
17
28
6
4
.1.17. (Z)-2-[8-(4-Isobutyl-1H-1,2,3-triazol-1-yl)octyl]-1-vinyldiazene 1-oxide (5d)
According to the general procedure for synthesis of triazole analogs, 8 (80.5 mg,
.355 mmol) with 4-methyl-1-pentyne (50 µL, 0.426 mmol) was converted to 5d (92.0
0
mg, 84%) as a colorless solid.
-1
mp 49.9 ˚C; IR (Diamond Prism) ν (cm ) : 3116, 3062, 2924, 2854, 1550, 1466, 1365,
1
1
327, 1273, 1219, 1157, 1049, 957, 856, 771, 717, 663; H NMR (500 MHz, CDCl ) δ
3
(ppm) : 7.29 (s, 1H), 7.11 (dd, J = 14.9, 8.0 Hz, 1H), 6.44 (d, J = 14.9 Hz, 1H), 5.51 (d,
J = 8.0 Hz, 1H), 4.33 (t, J = 7.5 Hz, 2H), 3.56 (t, J = 7.5 Hz, 2H), 2.61 (d, J = 6.9 Hz,
2
H), 1.98 (m, 1H), 1.91 (dt, J = 14.9, 7.5 Hz, 2H), 1.76 (dt, J = 14.9, 7.5 Hz, 2H),
1
3
1
.45-1.35 (complex m, 8H), 0.96 (d, J = 6.9 Hz, 6H); C NMR (125 MHz, CDCl ) δ
3
(ppm) : 147.1, 143.5, 120.9, 115.3, 52.5, 50.1, 34.8, 30.3, 29.1, 28.9, 28.7, 27.6, 27.0,
+
2
6.4, 22.3 (2C); HRMS (ESI) m/z : 330.2275 [M+Na] , calcd for C H N NaO :
1
6
29
5
3
30.2270
4
.1.18. (Z)-2-{8-[4-(2-Bromoethyl)-1H-1,2,3-triazol-1-yl]octyl}-1-vinyldiazene 1-oxide
(5e)
2
5

ACCEPTED MANUSCRIPT
According to the general procedure for synthesis of triazole analogs, 8 (80.8 mg,
.358 mmol) with 1-hexyne (40 µL, 0.433 mmol) was converted to 5e (116.8 mg, 91%)
0
as a colorless solid.
-1
mp 55.3 ˚C; IR (Diamond Prism) ν (cm ) : 3162, 3124, 2924, 2854, 1550, 1466, 1419,
1
1
319, 1257, 1219, 1142, 1057, 957, 883, 764, 725, 671; H NMR (500 MHz, CDCl ) δ
3
(ppm) : 7.43 (s, 1H), 7.09 (dd, J = 14.9, 7.5 Hz, 1H), 6.41 (d, J = 14.9 Hz, 1H), 5.49 (d,
J = 7.5 Hz, 1H), 4.33 (t, J = 7.5 Hz, 2H), 3.65 (t, J= 6.9 Hz, 2H), 3.53 (t, J = 7.5 Hz,
2
2
1
3
H), 3.30 (t, J = 6.9 Hz, 2H), 1.90 (dt, J = 14.9, 7.5 Hz, 2H), 1.74 (dt, J = 14.9, 7.5 Hz,
1
3
H), 1.43-1.31 (complex m, 8H); C NMR (125 MHz, CDCl ) δ (ppm) : 144.5, 143.3,
3
21.4, 115.2, 52.3, 50.1, 31.6, 30.1, 29.3, 28.9, 28.7, 27.5, 26.8, 26.2; HRMS (ESI) m/z :
+
80.1059 [M+Na] , calcd for C H N BrNaO : 380.1062
1
4
24
5
4
.1.19. (Z)-2-[8-(4-Isopropyl-1H-1,2,3-triazol-1-yl)octyl]-1-vinyldiazene 1-oxide (5f)
According to the general procedure for synthesis of triazole analogs, 8 (80.8 mg,
.359 mmol) with 3-methyl-1-butyne (40 µL, 0.431 mmol) was converted to 5f (97.8
0
mg, 93%) as a colorless solid.
-1
mp 51.3 ˚C; IR (Diamond Prism) ν (cm ) : 3109, 3062, 2924, 2854, 1550, 1466, 1365,
1
1
327, 1265, 1211, 1173, 1103, 1049, 949, 849, 764, 656; H NMR (500 MHz, CDCl ) δ
3
(ppm) : 7.22 (s, 1H), 7.09 (dd, J = 14.9, 8.0 Hz, 1H), 6.40 (d, J = 14.9 Hz, 1H), 5.48 (d,
J = 8.0 Hz, 1H), 4.29 (t, J = 7.5 Hz, 2H), 3.52 (t, J = 6.9 Hz, 2H), 3.08 (m, 1H), 1.88 (dt,
J = 14.4, 7.2 Hz, 2H), 1.74 (dt, J = 14.6, 7.3 Hz, 2H), 1.43-1.29 (complex m, 8H), 1.30
1
3
(
d, J = 7.0 Hz, 6H),; C NMR (125 MHz, CDCl ) δ (ppm) : 154.3, 143.3, 118.8, 115.1,
3
5
3
2.3, 49.9, 30.1, 28.9, 28.7, 27.5, 26.8, 26.3, 25.7, 22.4 (2C); HRMS (ESI) m/z :
+
16.2123 [M+Na] , calcd for C H N NaO : 316.2113
1
5
27
5
4
.1.20. (Z)-2-{8-[4-(Prop-1-en-2-yl)-1H-1,2,3-triazol-1-yl]octyl}-1-vinyldiazene 1-oxide
(
5g)
According to the general procedure for synthesis of triazole analogs, 8 (80.0 mg,
.355 mmol) with 2-methyl-1-butene-1-yne (86.2 mg, 0.426 mmol) was converted to 5g
85.0 mg, 83%) as a colorless amorphous.
0
(
-1
IR (Diamond Prism) ν (cm ) : 3008, 2931, 2854, 1643, 1466, 1373, 1319, 1219, 1111,
1
1
049, 949, 895, 748, 663; H NMR (500 MHz, CDCl ) δ (ppm) : 7.46 (s, 1H), 7.03 (dd,
3
J = 14.9, 7.5 Hz, 1H), 6.34 (d, J = 14.9 Hz, 1H), 5.63 (s, 1H), 5.43 (d, J = 7.5 Hz, 1H),
2
6

ACCEPTED MANUSCRIPT
5
1
.01 (m, 1H), 4.26 (t, J = 7.5 Hz, 2H), 3.46 (t, J= 6.9 Hz, 2H), 2.07 (s, 3H), 1.83 (dt, J =
1
3
4.4, 7.2 Hz, 2H), 1.66 (dt, J = 14.8, 7.4 Hz, 2H), 1.35-1.25 (complex m, 8H); C NMR
(
125 MHz, CDCl ) δ (ppm) : 148.4, 143.2, 133.5, 119.3, 115.1, 112.0, 52.3, 50.0, 30.1,
3
+
2
8.9, 28.7, 27.4, 26.8, 26.2, 20.5; HRMS (ESI) m/z : 314.1962 [M+Na] , calcd for
C H N NaO : 314.1957
15
25
5
4
.1.21. (Z)-2-{8-[4-(tert-Butyl)-1H-1,2,3-triazol-1-yl]octyl}-1-vinyldiazene 1-oxide (5h)
According to the general procedure for synthesis of triazole analogs, 8 (80.6 mg,
.358 mmol) with 3,3-dimethyl-1-butyne (50 µL, 0.430 mmol) was converted to 5h
0
(99.5 mg, 91%) as a colorless amorphous.
-1
IR (Diamond Prism) ν (cm ) : 3124, 3070, 2931, 2854, 1643, 1542, 1466, 1365, 1275,
1
1
218, 1126, 1049, 949, 841, 764, 717, 656; H NMR (500 MHz, CDCl ) δ (ppm) : 7.22
3
(s, 1H), 7.10 (dd, J = 14.9, 7.5 Hz, 1H), 6.41 (d, J = 14.9 Hz, 1H), 5.50 (d, J = 7.5 Hz,
1
H), 4.29 (t, J = 7.5 Hz, 2H), 3.53 (t, J = 6.9 Hz, 2H), 1.88 (dt, J = 14.4, 7.2 Hz, 2H),
13
1
.75 (dt, J = 14.8, 7.4 Hz, 2H), 1.44-1.32 (complex m, 8H), 1.35 (s, 9H); C NMR (125
MHz, CDCl ) δ (ppm) : 157.3, 143.3, 118.1, 115.1, 52.2, 49.9, 30.5, 30.2 (3C), 30.1,
3
+
2
8.8, 28.7, 27.4, 26.8, 26.3; HRMS (ESI) m/z : 330.2271 [M+Na] , calcd for
C H N NaO : 330.2270
16
29
5
4
.1.22. (Z)-2-{8-[4-(2-Hydroxypropan-2-yl)-1H-1,2,3-triazol-1-yl]octyl}-1-vinyl-
diazene 1-oxide (5i)
According to the general procedure for synthesis of triazole analogs, 8 (82.8 mg,
0
.368 mmol) with 2-methy-3-butyne-2-ol (40 µL, 0.441 mmol) was converted to 5i
(103.6 mg, 95%) as a colorless solid.
-1
mp 71.0 ˚C; IR (Diamond Prism) ν (cm ) : 3309, 3124, 2970, 2924, 2854, 1543, 1466,
1
1
365, 1265, 1211, 1192, 1134, 1049, 964, 489, 764, 656; H NMR (500 MHz, CDCl ) δ
3
(ppm) : 7.42 (s, 1H), 7.08 (dd, J = 14.9, 7.5 Hz, 1H), 6.39 (d, J = 14.9 Hz, 1H), 5.48 (d,
J = 7.5 Hz, 1H), 4.30 (t, J = 7.5 Hz, 2H), 3.51 (t, J= 6.9 Hz, 2H), 2.80 (s, 1H), 1.87 (dt,
J = 13.8, 6.9 Hz, 2H), 1.72 (dt, J = 15.0, 7.5 Hz, 2H), 1.61 (s, 6H), 1.41-1.30 (complex
1
3
m, 8H); C NMR (125 MHz, CDCl ) δ (ppm) : 155.5, 143.3, 118.9, 115.3, 68.3, 52.3,
3
5
0.1, 30.3 (2C), 30.1, 28.9, 28.7, 27.5, 26.8, 26.3; HRMS (ESI) m/z : 332.2064
+
[
M+Na] , calcd for C H N NaO : 332.2062
1
5
27
5
2
2
7

ACCEPTED MANUSCRIPT
4
.1.23. (Z)-2-[8-(4-Cyclopropyl-1H-1,2,3-triazol-1-yl)octyl]-1-vinyldiazene 1-oxide (5j)
According to the general procedure for synthesis of triazole analogs, 8 (82.2 mg,
.365 mmol) with cyclopropylacetylene (40 µL, 0.438 mmol) was converted to 5j (97
0
mg, 91%) as a colorless solid.
-1
mp 66.1 ˚C; IR (Diamond Prism) ν (cm ) : 3132, 3100, 2924, 2854, 2021, 1558, 1466,
1
1
412, 1327, 1265, 1211, 1157, 1049, 957, 895, 802, 764, 656; H NMR (500 MHz,
CDCl ) δ (ppm) : 7.20 (s, 1H), 7.09 (dd, J = 14.9, 8.0 Hz, 1H), 6.41 (d, J = 14.9 Hz,
3
1
1
8
H,), 5.50 (d, J = 8.0 Hz, 1H), 4.27 (t, J = 7.5 Hz, 2H), 3.53 (t, J = 7.5 Hz, 2H), 1.94 (m,
H), 1.86 (dt, J = 14.9, 7.5 Hz, 2H), 1.74 (dt, J = 7.5, 14.9, 2H), 1.43-1.30 (complex m,
1
3
H), 0.94 (m, 2H), 0.83 (m, 2H); C NMR (125 MHz, CDCl ) δ (ppm) : 150.1, 143.4,
3
119.4, 115.3, 52.5, 50.2, 30.3, 29.0, 28.9, 27.6, 27.0, 26.4, 7.7 (2C), 6.7; HRMS (ESI)
+
m/z : 314.1956 [M+Na] , calcd for C H N NaO : 314.1957
1
5
25
5
4
.1.24. (Z)-2-{8-[4-(1-Hydroxycyclohexyl)-1H-1,2,3-triazol-1-yl]octyl}-1-vinyldiazene
-oxide (5k)
1
According to the general procedure for synthesis of triazole analogs, 8 (80.0 mg,
.355 mmol) with 1-Ethynyl-1-cyclohexanol (53.2 mg, 0.427 mmol) was converted to
k (124.0 mg, 97%) as a colorless solid.
0
5
-1
mp 88.4 ˚C; IR (Diamond Prism) ν (cm ) : 3224, 3101, 3055, 2931, 2854, 1466, 1415,
1
1
358, 1319, 1257, 1173, 1049, 949, 902, 849, 764, 656; H NMR (500 MHz, CDCl ) δ
3
(
ppm) : 7.42 (s, 1H), 7.09 (dd, J = 14.9, 7.5 Hz, 1H), 6.41 (d, J = 15.5 Hz, 1H), 5.49 (d,
J = 7.5 Hz, 1H), 4.32 (t, J = 7.5 Hz, 2H), 3.53 (t, J = 6.8 Hz, 2H), 2.13 (brs, 1H),
2
1
.00-1.86 (complex m, 7H), 1.79-1.70 (complex m, 4H), 1.65-1.52 (complex m, 3H),
1
3
.43-1.32 (complex m, 8H); C NMR (125 MHz, CDCl ) δ (ppm) : 155.5, 143.3, 119.3,
3
115.3, 69.4, 52.4, 50.1, 38.0 (2C), 30.1, 28.9, 28.7, 27.5, 26.8, 26.3, 25.3, 21.9 (2C);
+
HRMS (ESI) m/z : 372.2377 [M+Na] , calcd for C H N NaO : 372.2375.
18
31
5
2
4
.1.25. (Z)-2-{8-[4-(Acetoxymethyl)-1H-1,2,3-triazol-1-yl]octyl}-1-vinyldiazene 1-oxide
(5l)
According to the general procedure for synthesis of triazole analogs, 8 (82.4 mg,
.366 mmol) with propargyl acetate (40 µL, 0.439 mmol) was converted to 5l (114.1 mg,
7%) as a yellow solid.
0
9
-1
mp 56.0 ˚C; IR (Diamond Prism) ν (cm ) : 3124, 2924, 2013, 1736, 1466, 1365, 1227,
2
8

ACCEPTED MANUSCRIPT
1
1
149, 1034, 957, 849, 787, 656; H NMR (500 MHz, CDCl ) δ (ppm) : 7.58 (s, 1H),
3
7
.10 (dd, J = 14.9, 7.5 Hz, 1H), 6.41 (d, J = 14.9 Hz, 1H), 5.50 (d, J = 7.5 Hz, 1H), 5.21
(s, 2H), 4.34 (t, J = 7.5 Hz, 2H), 3.53 (t, J = 6.9 Hz, 2H), 2.07 (s, 3H), 1.90 (dt, J = 14.9,
1
3
7
.5 Hz, 2H), 1.75 (dt, J = 14.9, 7.5 Hz, 2 H), 1.42-1.31 (complex m, 8H); C NMR
(
125 MHz, CDCl ) δ (ppm) : 170.6, 143.2, 142.5, 123.3, 115.1, 57.5, 52.2, 50.1, 30.0,
3
+
2
8.8, 28.6, 27.4, 26.7, 26.2, 20.6; HRMS (ESI) m/z : 346.1860 [M+Na] , calcd for
C H N NaO : 346.1855
15
25
5
3
4
.1.26. (Z)-2-{8-[4-(Trimethylsilyl)-1H-1,2,3-triazol-1-yl]octyl}-1-vinyldiazene 1-oxide
(5m) and (Z)-2-[8-(1H-1,2,3-triazol-1-yl)octyl]-1-vinyldiazene 1-oxide (5n)
According to the general procedure for synthesis of triazole analogs, 8 (83.1 mg,
0
.368 mmol) with trimethylsilylacethylene (60 µL, 0.442 mmol) was converted to 5m
(30.9 mg, 51%) and 5n (32.0 mg, 35%) as a colorless solid.
-1
5
m; mp 55.6 ˚C; IR (Diamond Prism) ν (cm ) : 3093, 2931, 2854, 1643, 1466, 1365,
1
1
327, 1250, 1196, 1126, 1049, 949, 841, 756, 656; H NMR (500 MHz, CDCl ) δ
3
(ppm) : 7.48 (s, 1H), 7.08 (dd, J = 14.9, 7.5 Hz, 1H), 6.40 (d, J = 14.9 Hz, 1H), 5.48 (d,
J = 7.5 Hz, 1H), 4.35 (t, J = 7.4 Hz, 2H), 3.51 (t, J = 6.9 Hz, 2H), 1.89 (dt, J = 13.7, 6.9
1
3
Hz, 2H), 1.73 (dt, J = 14.8, 7.4 Hz, 2H), 1.42-1.33 (complex m, 8H), 0.30 (s, 9H); C
NMR (125 MHz, CDCl ) δ (ppm) : 146.3, 143.3, 128.6, 115.2, 52.4, 49.6, 30.3, 28.9,
3
+
2
8.8, 27.6, 26.9, 26.4, –1.2 (3C); HRMS (ESI) m/z : 346.2042 [M+Na] , calcd for
C H N NaOSi: 346.2039
15
29
5
-1
5
n; mp 54.2 ˚C; IR (Diamond Prism) ν (cm ) : 3111, 2952, 2927, 2856, 2102, 1722,
1
1
487, 1468, 1325, 1271, 1217, 1122, 1084, 1049, 960, 949, 827, 761, 656; H NMR
(
500 MHz, CDCl ) δ (ppm) : 7.71 (s, 1H), 7.53 (s, 1H), 7.10 (dd, J = 14.9, 7.5 Hz, 1H),
3
6
.42 (d, J = 14.9 Hz, 1H), 5.50 (d, J = 7.5 Hz, 1H), 4.39 (t, J = 7.5 Hz, 2H), 3.53 (t, J =
.9 Hz, 2H), 1.92 (dt, J = 14.4, 7.2 Hz, 2H), 1.75 (dt, J = 14.8, 7.4 Hz, 2H), 1.44-1.35
6
1
3
(
complex m, 8H); C NMR (125 MHz, CDCl ) δ (ppm) : 143.3, 133.7, 123.1, 115.3,
3
5
2.4, 50.1, 30.2, 29.0, 28.8, 27.6, 26.9, 26.3; HRMS (ESI) m/z : 274.1647
+
[
M+Na] calcd for C H N NaO : 274.1644.
1
2
21
5
4
.1.27. (Z)-2-(8-(4-Phenyl-1H-1,2,3-triazol-1-yl)octyl)-1-vinyldiazene 1-oxide (5o)
According to the general procedure for synthesis of triazole analogs, 8 (82.8 mg,
.368 mmol) with ethynyl benzene (50 µL, 0.442 mmol) was converted to 5o (88.0 mg,
0
2
9