Long-chain triazolyl acids as inhibitors of osteoclastogenesis

Article abstract of DOI:10.1016/j.bmc.2013.05.013

Saturated fatty acids (e.g., palmitic acid) are known to moderately inhibit the development of osteoclasts in vitro. In pursuit of more effective inhibitors of osteoclastogenesis we explored two new classes of palmitic acid analogues containing either an ether or triazolyl group at various positions along the chain. The compounds were evaluated for their ability to inhibit the formation of osteoclasts in primary mouse bone marrow cultures. The oxyacids were generally prepared by condensation of the appropriate alkyl halides and diols, followed by Jones oxidation. The triazolyl acids were prepared by copper-catalysed click chemistry between alkyl azides and acetylenic acids, or with the appropriately-protected azides and alkynes, followed by deprotection and oxidation. The oxyacids were little more effective than palmitic acid, but the triazolyl analogues were much more effective osteoclastogenesis inhibitors, especially when the triazole was distant from the acid unit.

Full text of DOI:10.1016/j.bmc.2013.05.013

Accepted Manuscript
Long-chain Triazolyl Acids as Inhibitors of Osteoclastogenesis
Andrew J. Marshall, Jian-Ming Lin, Andrew Grey, Ian R. Reid, Jillian Cornish,
William A. Denny
PII:
S0968-0896(13)00438-0
DOI:
http://dx.doi.org/10.1016/j.bmc.2013.05.013
BMC 10839
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
5 April 2013
2 May 2013
8 May 2013
Please cite this article as: Marshall, A.J., Lin, J-M., Grey, A., Reid, I.R., Cornish, J., Denny, W.A., Long-chain
Triazolyl Acids as Inhibitors of Osteoclastogenesis, Bioorganic & Medicinal Chemistry (2013), doi: http://
dx.doi.org/10.1016/j.bmc.2013.05.013
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Graphical Abstractr

Long-chain Triazolyl Acids as Inhibitors of Osteoclastogenesis
Andrew J. Marshall,a Jian-Ming Lin,b Andrew Grey,b Ian R. Reid,b Jillian Cornish,b
and William A. Dennya,*
aAuckland Cancer Society Research Centre, School of Medical Sciences and
bDepartment of Medicine, The University of Auckland, Private Bag 92019, Auckland
1142, New Zealand
Abstract
Saturated fatty acids (e.g., palmitic acid) are known to moderately inhibit the
development of osteoclasts in vitro. In pursuit of more effective inhibitors of
osteoclastogenesis we explored two new classes of palmitic acid analogues containing
either an ether or triazolyl group at various positions along the chain. The compounds
were evaluated for their ability to inhibit the formation of osteoclasts in primary
mouse bone marrow cultures. The oxyacids were generally prepared by condensation
of the appropriate alkyl halides and diols, followed by Jones oxidation. The triazolyl
acids were prepared by copper-catalysed click chemistry between alkyl azides and
acetylenic acids, or with the appropriately-protected azides and alkynes, followed by
deprotection and oxidation. The oxyacids were little more effective than palmitic acid,
but the triazolyl analogues were much more effective osteoclastogenesis inhibitors,
especially when the triazole was distant from the acid unit.
Graphical abstract
1

Keywords
Click chemistry, osteoclastogenesis, osteoporosis, oxyacids, triazolyl acids
Abbreviations
HMPA, hexamethylphosphoramide; OPG, osteoprotegerin; TBTA, tris[(1-benzyl-1H-
1,2,3-triazol-4-yl)methyl]amine; TEMPO, (2,2,6,6-tetramethylpiperidin-1-yl)oxy;
TMAI, tetramethylammonium iodide
1. Introduction
Osteoporosis is a disorder of skeletal fragility consequent upon decreased quantity
and quality of bone. It is an important public health problem: among those > 50 years
old, 50% of women and 20% of men will experience at least one osteoporotic fracture
during their remaining lifetime.1 The annual health care costs associated with
osteoporotic fractures are estimated to exceed US$50bn in the USA and Europe.1 As
osteoporotic fractures occur most frequently in older people, the ageing of the world
population is likely to worsen the scope and scale of the problem.
2

Bone remodelling occurs throughout life, coordinated by the interplay of osteocytes,
bone-resorbing osteoclasts and bone-forming osteoblasts, and regulated by local and
systemic factors. In skeletal homeostasis, the components of bone remodelling,
osteoclastic bone resorption and osteoblast-mediated bone formation, are balanced.
Bone loss, which is a pivotal contributor to skeletal fragility, is the result of an
imbalance in the relative activity of osteoblasts and osteoclasts. Over time, the bone
loss accumulates, leading to a decrease in the mechanical strength of bone.
Inhibition of osteoclastic bone resorption is a proven strategy for reducing the risk of
osteoporotic fractures.2 Among currently available therapies for osteoporosis, all but
one work by primarily inhibiting osteoclastic bone resorption. Among the anti-
resorptive therapies, aminobisphosphonates dominate.3 Several of these
pyrophosphate analogues have prolonged skeletal half-lives, accumulate in bone
with continuing use, and have a very slow offset of action. They are effective in
reducing fracture risk, but there are uncertainties about their long-term safety.4,5
Consequently, development of non-bisphosphonate inhibitors of bone resorption for
treatment of osteoporosis is ongoing.6
We have recently established7 that C14-18 saturated fatty acids inhibit the
development of osteoclasts in vitro, with palmitic acid (1) showing the greatest effect.
It was considered likely that this effect is mediated by binding of the fatty acids to the
cell surface receptor GPR120, with the known8 GPR120 receptor agonist GW9508 (2)
also exhibiting osteoclastogenic activity in these assays.7
3

However, therapeutic exploitation of this observation is complicated by the modest
potency, rapid metabolism and relative insolubility of the natural long-chain fatty
acids, which likely limits them reaching the bone environment in sufficient
concentration for long enough to exert a substantial therapeutic effect in vivo. With
this in mind, we explore here two novel classes of palmitic-length fatty acids,
modified by ether (compounds 3-10) or triazole (compounds 11-16) units, for their
ability to inhibit osteoclast development in vitro.
2. Results and Discussion
2.1. Chemistry
Oxyacid 3 was synthesised by Williamson ether synthesis using sodium hydride and
tetrahydrofuran as the base, in low yield, due to competing addition of the alkoxide
ion of 16 to the carbonyl group of 17 (Scheme 1A).9 Subsequent analogues 4-9 were
prepared by displacement of alkyl halides 18a-f with the requisite diols 19a-f to give a
separable mixture of the desired alcohols 20a-f in moderate yield from unreacted diols
and bis alkylated products (not isolated). Subsequent Jones oxidation of alcohols
20a-f afforded the corresponding carboxylic acids 4-9 (Scheme 1B). The trioxa
analogue 10 was prepared by straightforward coupling of 2-(2-
methoxyethoxy)ethanol (21) and 8-iodooctanoic acid 22 at –78 oC (Scheme 1C).
The triazole acid 11 was prepared by a copper-catalysed “click chemistry”10 between
1-azidoundecane (24) and 3-butyn-1-ol (25) to give alcohol 26, which underwent
4

oxidation using TEMPO, NaOCl/NaClO2 in phosphate buffer,11 to 11 (Scheme 2).
Acids 12 and 13 were prepared similarly but more directly, from azides 29, 30 and the
acetylene acids 31 and 32.
The remaining triazole acids 14-16 were prepared from the bromoalcohols 33a-c
which were protected as the THP ethers (34a-c), then reacted with n-butyllithium and
trimethylsilylacetylene to give silyl ethers 35a-c.12 These were deblocked with TBAF
to give the acetylenes 3a-c. Reaction of these with azides 38a-c (from bromides 37a-
c) gave the desired triazoles 39a-c. These were deprotected with TFA and the
resulting alcohols 40a-c were oxidised to acids 14-16.
2.2. Structure-activity relationships
The compounds were evaluated for their ability to inhibit the formation of osteoclasts
in primary mouse bone marrow cultures, prepared as previously described.13,14 Table
1 gives comparative results at a compound concentration of 10 µg/mL, with activity
determined as the % inhibition of cell differentiation growth over untreated cultures.
The positive control compounds used were osteoprotegerin (OPG) (at 0.01 µg/mL) (in
each assay) and occasionally GW9058 (2) (at 25 µg/mL). These control compound
concentrations were chosen to give a mid-range response (about 35% inhibition).
The mono ether acids 3-10 were prepared as the structurally-closest analogues to 1,
retaining the 15-atom chain off the carboxylic acid unit and replacing one CH2 unit by
O at various positions along the chain. This provides a significant drop in clogP (by
2.47 units, from 7.21 to 4.74; ChemDraw Ultra, version 11.01), while retaining much
5

of the flexibility of the alkyl chain. This was considered desirable since it was
reported7 that unsaturation in C18 fatty acids decreased their anti-osteoclastogenic
activity. While compound 3 was not active in this assay, compounds 4, 5 and 9
showed dose-response (Figure 1A,B) and modest but repeatable activity, comparable
to that of 1 itself, showing that such substitutions were at least viable. Compounds 6-8
were inactive, as was the triether analogue 10.
Compounds 11-16 explore the similar insertion of a triazole unit, counting this as a 2-
atom insertion and maintaining a 15-atom chain off the carboxylic acid unit. With this
modification we sought to provide aromatic character and rigidity to the backbone
while also improving solubility. This change does provide an even larger reduction in
clogP than does an oxygen atom (by 3.6 units) over 1. All of the triazole analogues
showed good dose-responses (Figures 1C-E) and substantially better activity than
either 1 (Figure 1F) or the GPR120 receptor agonist 2 (Figure 1G), with the activity
broadly increasing as the distance between the triazole and acid units was increased
(Figure 2).
2.3. Conclusions
The data above indicate that the anti-osteoclastogenic activity of natural long chain
saturated fatty acids is changed very little by the introduction of an ether moiety
anywhere in the chain, but is improved markedly by the introduction of an aromatic
triazole group, especially when placed distant from the acid unit. In a primary rodent
culture system commonly used to evaluate osteoclast development, the most potent of
the triazolyl analogues were comparable in efficacy to the GPR120 agonist GW9508,
6

and osteoprotegerin, the endogenous inhibitor of osteoclastogenesis. In clinical
practice, agents that decrease osteoclastic bone resorption by 50-80% are effective in
reducing the risk of fragility fractures.2 Further characterization of the skeletal effects
of these compounds is justified.
3. Experimental Section
3.1. Chemistry
1H NMR spectra were recorded on a Bruker Avance 400 spectrometer; chemical shifts
are reported in δ using SiMe4 as the internal standard when measured in CDCl3, and
the residual DMSO as internal standard when measured in d6-DMSO. Low resolution
mass spectra were recorded on a Thermo Finnigan MSQ single quadrupole mass
spectrometer using atmospheric pressure chemical ionisation (APCI). High resolution
mass spectra were obtained on a Bruker microTOF-QII mass spectrometer using
electrospray ionisation (ESI). Analyses were carried out in The Campbell
Microanalytical Laboratory, University of Otago, Dunedin, New Zealand. Melting
points were determined on an Electrothermal 2300 Melting Point Apparatus. Silica
gel chromatography was performed using 200-320 mesh silica gel obtained from APS
Finechem Ltd. Yields have not been optimised.
3.1.1. (Tridecyloxy)acetic acid (3) (Scheme 1A). Tridecanol (16) (500 mg, 2.4
mmol) was added to iodoacetic acid (17) (669 mg, 3.6 mmol) and NaH (130 mg, 5.4
mmol) in THF (10 mL) at -70 °C and then stirred for 16 h at room temperature.9 The
reaction was then basified with 2M NaOH and the resulting precipitate was collect by
7

filtration, dissolved in MeOH (50 mL) and acidified to pH 3 with 2M HCl. The
reaction was concentrated to remove MeOH and the solution was extraction twice
with EtOAc and dried over Na2SO4. The product was purified by flash column
chromatography; Eluting with EtOAc/CH2Cl2 (10:90) gave foreruns and then elution
with neat EtOAc then gave 3 (50 mg, 8%) as a white solid; mp 51-53 oC (hexanes).
1H NMR [DMSO] δ 12.53 (br s, 1H), 3.95 (s, 2H), 3.41 (t, J = 6.6 Hz, 2H), 1.48
(quin, J = 6.8 Hz, 2H), 1.34-1.19 (m, 20H), 0.85 (t, J = 6.7 Hz, 3H). LCMS (APCI-)
257.7 (M-H, 100%). Anal. Calcd for C15H30O3: C, 69.72; H, 11.70. Found C, 69.86;
H, 11.80.
3.1.2. 3-(Dodecyloxy)propanoic acid (4) (Scheme 1B). NaH (315 mg, 13.1 mmol)
was added in one portion to 1,3-propanediol (19a) (1 g, 13.1 mmol) and TBAI (16
mg, 3 mol%) in THF (10 mL) at 0 °C for 30 min. 1-Bromododecane (18a) (2.94 g,
11.8 mmol) was then added and the reaction was refluxed overnight, then cooled,
neutralised with sat. NH4Cl and extracted into EtOAc, washed with brine and dried
with MgSO4.The product was purified by flash column chromatography; CH2Cl2 gave
foreruns, and then elution with EtOAc/CH2Cl2 (5:95) gave 3-(dodecyloxy)propan-1-ol
(20a) (1.06 g, 31%) as a colourless oil. 1H NMR [DMSO]  4.36 (t, J = 5.2 Hz, 1H),
3.43 (q, J = 6.4 Hz, 2H), 3.38 (t, J = 6.5 Hz, 2H), 3.31 (t, J = 6.5 Hz, 2H), 1.62 (quin,
J = 6.4 Hz, 2H), 1.46 (quin, J = 6.4 Hz, 2H), 1.24 (s, 18H), 0.85 (t, J = 6.8 Hz, 3H).
LCMS (APCI+) 245.9 (MH+, 100%).
CrO3 (88 mg, 0.88 mmol) in H2SO4 (0.5 mL) / H2O (1.5 mL) was added dropwise to a
solution of 20a (108 mg, 0.44 mmol) in acetone (2 mL) until an orange colour
persisted. The reaction was quenched over isopropanol, and the resulting precipitate
8

was collected by filtration and eluted through a pad of silica gel with EtOAc/CH2Cl2
(1:9) gave 4 (36 mg, 30%) as a white solid: mp 53-55 °C (hexanes). 1H NMR
[DMSO]  12.14 (s, 1H), 3.54 (t, J = 6.3 Hz, 2H), 3.34 (t, J = 6.6 Hz, 2H), 2.41 (t, J
= 6.3 Hz, 2H), 1.45 (quin, J = 6.6 Hz, 2H), 1.24 (s, 18H), 0.85 (t, J = 6.8 Hz, 3H).
LCMS (APCI-) 257.7 (M-H+, 100%). Anal. Calcd for C15H30O3: C, 69.72; H, 11.70.
Found C, 69.83; H, 11.97.
3.1.3. 5-(Decyloxy)pentanoic acid (5). Pentane-1,5-diol (19b) (1.0 g, 9.6 mmol) was
similarly treated with NaH/TBAI, followed by 1-bromodecane (18b) (1.9 g, 8.6
mmol). Workup as above and purification by flash column chromatography; eluting
with neat CH2Cl2, gave foreruns, and then elution with EtOAc/CH2Cl2 (5:95) gave 5-
1
(decyloxy)pentan-1-ol (20b) (500 mg, 25%) as a colourless oil. H NMR [CDCl3] 
4.32 (t, J = 5.2 Hz, 1H), 3.37 (dd, J = 6.5, 1.1 Hz, 2H), 3.32 (m, 4H), 1.53-1.35 (m,
6H), 1.34-1.20 (m, 16H), 0.85 (t, J = 6.7 Hz, 3H). LCMS (APCI+) 245.9 (100%).
Similar treatment of 20b (100 mg, 0.43 mol) with CrO3 in H2SO4 / H2O and
purification by flash column chromatography; eluting with EtOAc/CH2Cl2 (0.1:99)
gave foreruns, and then elution with EtOAc/CH2Cl2 (10:90) gave 5 (31 mg, 29%) as a
colourless semi-solid: mp 38-40 °C (hexanes). 1H NMR [DMSO]  12.02 (s, 1H),
3.36-3.28 (m, 4H), 2.21 (t, J = 7.1 Hz, 2H), 1.59-1.41 (m, 6H), 1.31-1.19 (m, 14H),
0.85 (t, J = 6.8 Hz, 3H). LCMS (APCI-) 257.7 (M-H+, 100%). HRMS (ESI+) Calcd
for C15H31O3: 259.2268; found (MH+) 259.2266.
3.1.4. 6-(Nonyloxy)hexanoic acid (6). Hexane-1,6-diol (19c) (1.57 g, 7.6 mmol) was
similarly treated with NaH/TBAI, followed by 1-bromononane (18c) (1.57 g, 7.6
9

mmol). Workup as above and purification by flash column chromatography; eluting
with EtOAc/CH2Cl2 (1:99), gave foreruns, and then elution with EtOAc/CH2Cl2 (1:9)
gave 6-(nonyloxy)hexan-1-ol (20c) (378 mg, 20%) as a colourless oil. 1H NMR
[CDCl3]  3.63 (t, J = 6.6 Hz, 2H), 3.39 (td, J =6.6 Hz, 4.0 Hz, 4H), 1.69-1.50 (m,
7H), 1.44-1.34 (m, 4H), 1.34-1.20 (m, 12H), 0.88 (t, J = 6.78 Hz, 3H). LCMS
(APCI+) 245.9 (MH+, 100%).
Similar treatment of 20c (270 mg, 1.1 mmol) with CrO3 in H2SO4 / H2O and
purification by flash column chromatography; eluting with EtOAc/CH2Cl2 (10:90),
gave foreruns, and then further elution with EtOAc/CH2Cl2 (10:90) gave 6 (110 mg,
39%) as a colourless oil. 1H NMR [DMSO]  11.97 (s, 1H), 3.31 (t, J = 6.5 Hz, 4H),
2.18 (t, J = 7.4 Hz, 2H), 1.56-1.41 (m, 6H), 1.35-1.17 (m, 14H), 0.85 (t, J = 6.8 Hz,
3H). LCMS (APCI-) 257.7 (M-H+, 100%). HRMS (ESI+) Calcd for C15H31NaO3:
281.2087; (MH+Na)+ 281.2088.
3.1.5. 9-(Hexyloxy)nonanoic acid (7). Nonane-1,9-diol (19d) (2.08 g, 13.0 mmol)
was similarly treated with NaH/TBAI, followed by 1-bromohexane (18d) (1.93 g,
11.7 mmol). Workup as above and purification by flash column chromatography with
EtOAc/CH2Cl2 (5:95) gave 8-(heptyloxy)octan-1-ol (20d) (200 mg, 7%) as a
1
colourless oil. H NMR [CDCl3]  3.62 (t, J = 6.6 Hz, 2H), 3.39 (t, 6.8 Hz, 4H), 1.71
(br s, 1H), 1.56 (quin, 6.7 Hz, 6H), 1.39-1.32 (m, 16H), 0.89 (t, J = 6.8Hz, 3H).
LCMS (APCI+) 245.9 (MH+, 100%).
Similar treatment of 20d (150 mg, 0.61 mmol) with CrO3 in H2SO4 / H2O and
purification by flash column chromatography, eluting with CH2Cl2 gave foreruns, and
10

then elution with EtOAc/CH2Cl2 (1:9) gave 7 (30 mg, 19%) as a colourless oil. 1H
NMR [DMSO] 11.98 (s, 1H), 3.31 (t, J = 6.5 Hz, 4H), 2.18 (t, J = 7.4 Hz, 2H), 1.46
(quin, J = 6.8 Hz, 6H), 1.33-1.19 (m, 14H), 0.86 (t, J = 6.9 Hz, 3H). LCMS (APCI-)
257.7 (M-H+, 100%). HRMS (ESI+) Calcd for C15H31O3: 259.2268; found (MH+)
259.2267.
3.1.6. 10-(Pentyloxy)decanoic acid (8). Decane-1,10-diol (19e) (2.27 g, 13.0 mmol)
was similarly treated with NaH/TBAI, followed by 1-bromopentane (18e) (1.96 g,
13.0 mmol). Workup as above and purification by flash column chromatography with
neat CH2Cl2 gave foreruns, then elution with EtOAc/CH2Cl2 (5:95) gave 10-
1
(pentyloxy)decan-1-ol (20e) 1.0 g, 32%) as a colourless oil. H NMR [CDCl3]  3.62
(t, J = 6.6 Hz, 2H), 3.39 (t, 6.8 Hz, 4H), 1.65-1.50 (m, 7H), 1.40-1.23 (m, 16H), 0.89
(t, J = 6.8 Hz, 3H). LCMS (APCI+) 245.9 (MH+, 100%).
Similar treatment of 20e (500 mg, 2.0 mmol) with CrO3 in H2SO4 / H2O and
purification by flash column chromatography; eluting with CH2Cl2 gave foreruns, and
then elution with EtOAc/CH2Cl2 (1:9) gave 8 (333 mg, 65%) as a colourless semi-
solid. 1H NMR [DMSO] 11.95 (s, 1H), 3.34-3.29 (m, 4H), 2.18 (t, J = 7.4 Hz, 2H),
1.56-1.37 (m, 6H), 1.35-1.16 (m, 14H), 0.86 (t, J = 7.0 Hz, 3H). LCMS (APCI-)
257.7 (M-H+, 100%). HRMS (ESI+) Calcd for C15H31O3: 259.2268; found (MH+)
259.2268.
3.1.7. 12-Propoxydodecanoic acid (9). Dodecane-1,10-diol (19f) (2.63 g, 13 mmol)
was similarly treated with NaH /TBAI followed by 1-bromopropane (18f) (1.93 g,
11.7 mmol) with overnight reflux. Workup as above and purification by flash column
11

chromatography; eluting with neat CH2Cl2 gave foreruns, then elution with
EtOAc/CH2Cl2 (5:95) gave 12-(pentyloxy)decan-1-ol (20f) (1.0 g, 32%) as a
1
colourless oil. H NMR [DMSO]  4.30 (t, J = 5.2 Hz, 1H), 3.37 (td J = 6.5, 5.3 Hz,
2H), 3.3-3.2 (m, 2H), 3.28 (t, J = 6.6 Hz, 2H), 1.56-1.34 (m, 16H), 1.54-1.33 (m, 6H),
0.85 (t, J = 7.4 Hz, 3H). LCMS (APCI+) 245.9 (MH+, 100%).
Similar treatment of 20f (149 mg, 0.59 mmol) with CrO3 in H2SO4 / H2O and
purification by flash column chromatography; eluting with CH2Cl2 gave foreruns, and
then elution with EtOAc/CH2Cl2 (1:9) gave 9 (72 mg, 47%) as a colourless oil. 1H
NMR [DMSO] 11.95 (s, 1H), 3.34-3.29 (m, 6H), 2.18 (t, J = 7.4 Hz, 2H), 1.56-1.37
(m, 6H), 1.35-1.16 (m, 12H), 0.86 (t, J = 7.0 Hz, 3H). LCMS (APCI-) 257.7 (M-H+,
100%). HRMS (ESI+) Calcd for C15H31NaO3: 281.2087; (MH+Na)+ 281.2084.
3.1.8. 8-[2-(2-Methoxyethoxy)ethoxy]octanoic acid (10) (Scheme 1C. NaH (600
mg, 24 mmol was added to a stirred solution of 8-iodooctanoic acid (22) (2.24 g, 8.16
mmol) in THF (30 mL) at -78 °C for 20 mins, followed by the addition of 2-(2-
methoxyethoxy)ethanol (21) (500 mg, 4.16 mmol). The reaction was stirred at room
temperature overnight, cooled to 0 °C and acidified to pH 3 with 1 M HCl. The
solution was extracted with EtOAc x3, washed with brine and dried over Na2SO4. The
crude yellow oil was purified by flash column chromatography; elution with neat
CH2Cl2 gave foreruns, then further elution with EtOAc/CH2Cl2 (15:75) gave 10 (140
mg, 13%) as a yellow oil; 1H NMR [DMSO]  11.95 (s, 1H), 3.53-3.40 (m, 8H), 3.36
(t, J = 6.6 Hz, 2H), 3.24-3.22 (m, 3H), 2.18 (t, J = 7.4 Hz, 2H), 1.55-1.41 (m, 4H),
1.53-1.42 (m, 6H). LCMS (APCI-) 261.6 (M-H+, 100%). HRMS (ESI+) Calcd for
C13H27O5: 263.1853; (MH+) 263.1861.
12

3.1.9. (1-Undecyl-1H-1,2,3-triazol-4-yl)acetic acid (11) (Scheme 2). 1-
Bromoundecane (23) (1.5 g, 6.3 mmol) and NaN3 (0.61 g, 9.5 mmol) in DMSO were
heated in a microwave chamber at 100 °C for 30 min. The reaction mixture was
cooled in an ice bath and diluted with water, extracted into Et2O, washed with brine
and dried over Na2SO4 to give crude 1-azidoundecane (24) as a tan oil (0.93 g, 74%),
which was used directly in the next step without further characterisation.
CuSO4.5H2O (44.9 mg, 5 mol%), TBTA (191 mg, 10 mol%) and sodium ascorbate
(107 mg, 15 mol%) were added to a solution of 24 (930 mg, 4.7 mmol) and 3-butyn-
1-ol (25) (253 mg, 3.6 mmol) in CH2Cl2/DMF/water (5 mL: 5 mL: 5 mL).[10] The
reaction mixture sealed under nitrogen and heated to 50 °C overnight, then diluted
with EtOAc, washed 4 x with brine, dried over Na2SO4 and concentrated to dryness to
give crude 2-(1-undecyl-1H-1,2,3-triazol-4-yl)ethanol (26) as a white solid (600 mg,
68%), which was used directly in the next step without further purification. 1H NMR
[DMSO]  7.83 (s, 1H), 4.67 (t, J = 5.3 Hz, 1H), 4.27 (t, J = 7.1 Hz, 2H), 3.60 (td, J
= 7.0, 5.3 Hz, 2H), 2.75 (t, J = 6.9 Hz, 2H), 1.77 (quin, J = 7.1 Hz, 2H), 1.32-1.15
(m, 16H), 0.87 (t, J = 6.7 Hz, 3H). LCMS (APCI+) 268.7 (MH+, 100%).
A mixture of 26 (200 mg, 0.78 mmol), TEMPO (8 mg, 7 mol%), NaOCl (50 µL, as a
0.27 molL-1 solution, 2.0 mol%) and NaClO2 (203 mg, 2.2 mmol) in acetonitrile (4
mL) and phosphate buffer (0.67M NaH2PO4 and 0.67M Na2HPO4, 4 mL) and the
reaction was heated at 60 °C overnight.11 The resulting precipitate was collected by
filtration, washed with water and hexanes to give 11 (100 mg, 44%) as a white solid;
mp 100-102 °C (hexanes). 1H NMR [DMSO]  12.44 (br s, 1H), 7.95 (s, 1H), 4.31 (t,
13

J = 7.1 Hz, 2H), 3.65-3.52 (m, 2H), 1.78 (quin, J = 7.2 Hz, 2H), 1.35-1.15 (m, 16H),
0.85 (t, J = 6.7 Hz, 3H). HRMS (ESI+) Calcd for C15H27N3O2: 282.2176; found
(MH+) 282.2173. Anal. Calcd for C15H27N3O2.0.3H2O: C, 62.82; H, 9.70, N, 14.65.
Found C, 62.75; H, 9.84; N, 14.58.
3.1.10. 3-(1-Decyl-1H-1,2,3-triazol-4-yl)propanoic acid (12). Similar reaction of 1-
bromodecane (27) (2.0 g, 9.6 mmol) and NaN3 (0.94 g, 14.4 mmol) and workup of the
product gave 1-azidodecane (29) as a tan oil (1.62 g, 86%) that was used directly in
the next step. CuSO4.5H2O (74 mg, 5 mol%), TBTA (220 mg, 7 mol%) and sodium
ascorbate (176 mg, 15 mol%) were added to a solution of 27 (1.30 g, 6 mmol) and 5-
hexynoic acid (31) (723 mg, 5.9 mmol) in THF/water (15 mL/ 5 mL). The reaction
mixture sealed under nitrogen and heated to 50 °C overnight, then concentrated under
reduced pressure to a volume of ~5 mL and diluted with water. The resulting
precipitate was collected by filtration, taken up in MeOH and dried over Na2SO4 and
concentrated to dryness to give 12 (500 mg, 35%) as a white solid. 1H NMR [DMSO]
 12.09 (s, 1H), 7.85 (s, 1H), 4.27 (t, J = 7.0 Hz, 2H), 2.61 (t, J = 7.5 Hz, 2H), 2.31-
2.15 (m, 2H), 1.85-1.70 (m, 4H), 1.35-1.15 (m, 12H), 0.85 (t, J = 6.7 Hz, 3H). LCMS
(APCI+) 282.7 (MH+, 100%). Anal. Calcd for C15H27N3O2: C, 64.02; H, 9.67, N,
14.93. Found C, 64.24; H, 9.7; N, 15.05.
3.1.11. 4-(1-Nonyl-1H-1,2,3-triazol-4-yl)butanoic acid (13). Similar reaction of 1-
bromononane (28) (2.0 g, 9.6 mmol) and NaN3 (0.94 g, 14.4 mmol) gave crude 1-
azidononane (30) (1.62 g, 86%), which was used directly in the next step. Crude 30
(1.30 g, 6 mmol) was reacted with 4-butynoic acid (31) (723 mg, 5.9 mmol) as above,
and the resulting precipitate was collected by filtration, taken up in MeOH and dried
14

over Na2SO4 and concentrated to dryness to give 13 (1.3g, 78%) as a white solid; mp
1
77-78 °C (hexanes). H NMR [DMSO]  12.09 (s, 1H), 7.85 (s, 1H), 4.27 (t, J = 7.0
Hz, 2H), 2.61 (t, J = 7.5 Hz, 2H), 2.31-2.15 (m, 2H), 1.85-1.70 (m, 4H), 1.35-1.15
(m, 12H), 0.85 (t, J = 6.7 Hz, 3H). LCMS (APCI+) 282.7 (MH+, 100%). Anal. Calcd
for C15H27N3O2: C, 64.02; H, 9.67, N, 14.93. Found C, 64.24; H, 9.7; N, 15.05.
3.1.12. 6-(1-Heptyl-1H-1,2,3-triazol-4-yl)hexanoic acid (14) (Scheme 3). 3,4-
Dihydro-2H-pyran (1.40 g, 16.0 mmol) was added to stirred solution of 6-bromo-1-
hexanol (33a) (2.0 g, 11.0 mmol) and camphorsulfonic acid (116 mg, 5 mol%) in
Et2O at room temperature overnight. The reaction was basified with sat. K2CO3 and
extracted into Et2O, washed with brine and dried over Na2SO4. Purification by flash
column chromatography; neat CH2Cl2 gave 2-((6-bromohexyl)oxy)tetrahydro-2H-
pyran (34a) (2.70 g, 95%) as a colourless oil. 1H NMR [DMSO]  4.55-4.50 (m, 1H),
3.72 (ddd, J = 11.2, 8.1, 3.1 Hz, 1H), 3.61 (td, J = 9.7, 6.7 Hz, 1H), 3.52 (t, J = 6.7
Hz, 2H), 3.46-3.37 (m, 1H), 3.35-3.28 (m, 1H), 1.84-1.76 (m, 2H), 1.73-1.64 (m, 1H),
1.64-1.54 (m, 1H), 1.54-1.28 (m, 10H). HRMS (ESI+) Calcd for C11H21BrNaO2:
287.0617; (MNa+) 287.0613
n-Butyllithium (2.07 mL of a 2.0 M solution in cyclohexanes, 4.1 mmol) was added
dropwise to a solution of trimethylsilylacetylene (0.60 mL, as a solution in THF, 4.1
mmol) in THF (8 mL) at -60 °C, then the reaction was allowed to warm to 0 °C for 30
min.12 The reaction was then cooled to -20 °C, HMPA (2 mL) was added dropwise
followed by 34a (1.0 g, 3.8 mmol). The reaction was stirred for 5 h at 0 °C and room
temperature for an additional 14 h, then quenched with sat. NH4Cl and extracted into
EtOAc, washed with brine and dried over Na2SO4. The crude oil was filtered through
15

a pad of silica with CH2Cl2 and concentrated to dryness to give (by 1HNMR) a 2:1
mixture of trimethyl-(8-((tetrahydro-2H-pyran-2-yl)oxy)oct-1-yn-1-yl)silane (35a)
and 2-(oct-7-yn-1-yloxy)tetrahydro-2H-pyran (36a) (1.0 g, 98%). This (1.0 g, 3.8
mmol) was taken up in THF (10 mL) and stirred with TBAF (5.6 mL as a 1 molL-1
solution in THF, 5.6 mmol) at room temperature for 48 h. The reaction was diluted
with EtOAc, and washed with sat. NaHCO3, brine, dried over Na2SO4 and
concentrated to dryness to give only 36a (740 mg, 91%) as a colourless oil. 1H NMR
[DMSO]  4.53 (t, J = 3.5 Hz, 1H), 3.72 (ddd, J = 11.2, 8.0, 3.1 Hz, 1H), 3.66-3.55
(m, 1H), 3.47-3.37 (m, 1H), 3.36-3.24 (m, 1H), 2.73 (t, J = 2.6 Hz, 1H), 2.14 (td, J =
6.9, 2.7 Hz, 2H), 1.80-1.20 (m, 14H). HRMS (ESI+) Calcd for C13H22NaO2: 233.1512;
(MNa+) 233.1517.
1-Bromoheptane (37a) (470 mg, 2.6 mmol) and NaN3 (0.51 g, 7.9 mmol) in DMSO
were heated in a microwave chamber at 100 °C for 1 h. The reaction mixture was
cooled in an ice bath and diluted with water, extracted into Et2O, washed with brine
and dried over Na2SO4 to give 1-azidoheptane (38a) as a tan oil (293 mg, 80%). The
material was not characterised further and was used directly in the next step.
CuSO4.5H2O (22 mg, 5 mol%), TBTA (65 mg, 7 mol%) and sodium ascorbate (52
mg, 15 mol%) were added to a solution of 36a (273 mg, 1.93 mmol) and 38a (330
mg, 1.76 mmol) in THF/water (15 mL: 5 mL). The reaction mixture was sealed under
nitrogen and heated to 50 °C overnight, diluted with Et2O, washed with water, brine
and dried over Na2SO4 and concentrated to dryness. Purification by flash column
chromatography; eluting with CH2Cl2 gave foreruns, further elution with
EtOAc/CH2Cl2 (1:9) gave 1-heptyl-4-(6-((tetrahydro-2H-pyran-2-yl)oxy)hexyl)-1H-
16

1
1,2,3-triazole (39a) (280 mg, 45%) as a colourless oil. H NMR [DMSO]  7.82 (s,
1H), 4.52 (t, J = 3.5 Hz, 1H), 4.27 (t, J = 7.1 Hz, 2H), 3.71 (ddd, J = 11.2, 8.0, 3.0
Hz, 1H), 3.59 (td, J = 9.6, 6.7 Hz, 1H), 3.46-3.36 (m, 1H), 3.34-3.26 (m, 1H), 2.58 (t,
J = 7.5 Hz, 2H), 1.82-1.66 (m, 3H), 1.66-1.53 (m, 3H), 1.54-1.38 (m, 6H), 1.38-1.11
(m, 12H), 0.84 (t, J = 7.0 Hz, 3H). LCMS (APCI+) 352.9 (MH+, 20%).
A mixture of 39a (250 mg, 0.71 mmol) and trifluoroacetic acid (1 mL) in CH2Cl2 (5
mL) was stirred for 48 h. The reaction was cooled to 0 °C and neutralised with sat.
K2CO3, and extracted into EtOAc, washed with water, brine, dried with Na2SO4 and
concentrated to give 6-(1-heptyl-1H-1,2,3-triazol-4-yl)hexan-1-ol (40a) as a
1
colourless oil (140 mg, 74%). H NMR [DMSO]  1H NMR 7.83 (s, 1H), 4.37 (t, J =
6.6 Hz, 2H), 4.27 (t, J = 7.1 Hz, 2H), 2.10 (t, J = 7.5 Hz, 2H), 1.83-1.72 (m, 2H),
1.72-1.63 (m, 2H), 1.64-1.52 (m, 2H), 1.43-1.14 (m, 13H), 0.84 (t, J = 7.0 Hz, 3H).
LCMS (APCI+) 268.9 (MH+, 100%).
A mixture of 40a (140 mg, 0.53 mmol), TEMPO (6 mg, 7 mol%), NaOCl (33 µL, as a
0.27 molL-1 solution, 2.0 mol%) and NaClO2 (203 mg, 2.2 mmol) in acetonitrile (4
mL) and phosphate buffer (0.67M NaH2PO4 and 0.67M Na2HPO4, 4 mL) were stirred
at 50 °C for 2 days. The reaction was diluted with brine, and extracted into EtOAc,
dried over Na2SO4 to give an oil that crystallised on standing to give 14 (74 mg, 50%)
1
as a white solid; mp 68-70 °C (hexanes). H NMR [DMSO]  11.99 (s, 1H), 7.83 (s,
1H), 4.27 (t, J = 7.1 Hz, 2H), 2.58 (t, J = 7.5 Hz, 2H), 2.18 (t, J = 7.3 Hz, 2H), 1.83-
1.70 (m, 2H), 1.51-45 (m, 4H), 1.35-1.13 (m, 10H), 0.85 (t, J = 7.0 Hz, 3H). LCMS
(APCI+) 282.7 (MH+, 100%). Anal. Calcd for C15H27N3O2: C, 64.02; H, 9.67, N,
14.93. Found C, 64.21; H, 9.75; N, 15.12.
17

3.1.13. 8-(1-Pentyl-1H-1,2,3-triazol-4-yl)octanoic acid (15). Similar reaction of 8-
bromooctan-1-ol (33b) (2.0 g, 9.6.0 mmol) with 3,4-dihydro-2H-pyran (1.20 g, 9.6
mmol) and purification by flash column chromatography with neat CH2Cl2 gave 2-
((8-bromooctyl)oxy)tetrahydro-2H-pyran (34b) (2.60 g, 95%) as a colourless oil. 1H
NMR [DMSO]  4.52 (t, J = 3.5 Hz, 1H), 3.72 (ddd, J = 11.1, 8.0, 3.0 Hz, 1H), 3.60
(td, J = 9.6, 6.7 Hz, 1H), 3.52 (t, J = 6.7 Hz, 2H), 3.46-3.37 (m, 1H), 3.35-3.25 (m,
1H), 1.85-1.74 (m, 2H), 1.75-1.65 (m, 1H), 1.64-1.55 (m, 1H), 1.55-1.20 (m, 14H).
HRMS (ESI+) Calcd for C13H25BrNaO2: 315.0930; (MNa+) 315.0932.
Similar reaction of 34b (0.60 g, 2.04) with trimethylsilylacetylene and filtration of the
crude product through a pad of silica with CH2Cl2 gave a mixture (571 mg, 97%) (2:1
by 1HNMR) of trimethyl(10-((tetrahydro-2H-pyran-2-yl)oxy)dec-1-yn-1-yl)silane
(35b) and 2-(dec-9-yn-1-yloxy)tetrahydro-2H-pyran (36b). Treatment of this as
above with TBAF (4.0 mL as a 1 molL-1 solution in THF, 4.0 mmol) at room
temperature for 48 h gave only 36b (370 mg, 92%) as a colourless oil. 1H NMR
[DMSO]  4.52 (t, J = 3.5 Hz, 1H), 3.72 (ddd, J = 11.1, 8.0, 3.0 Hz, 1H), 3.60 (td, J
= 9.6, 6.7 Hz, 1H), 3.45-3.37 (m, 1H), 3.36-3.26 (m, 1H), 2.72 (t, J = 2.6 Hz, 1H),
2.14 (td, J = 7.0, 2.7 Hz, 2H), 1.76-1.65 (m, 1H), 1.54-1.38 (m, 8H), 1.38-1.21 (m,
9H). HRMS (ESI+) Calcd for C15H26NaO2: 261.1825; (MNa+) 261.1830.
Crude 1-azidopentane (38b) was prepared as above from 1-bromopentane (37b) (450
mg, 3.0 mmol) as a tan oil (355 mg, 85% yield) and was used directly in the next step.
CuSO4.5H2O (24.6 mg, 5 mol%), TBTA (73.2 mg, 7 mol%) and sodium ascorbate
(58.6 mg, 15 mol%) were added to a solution of 1-azidopentane (38b) (417 mg, 4
18

mmol) and 2-(dec-9-yn-1-yloxy)tetrahydro-2H-pyran (36b) (470 mg, 5.9 mmol) in
THF/water (15 mL: 5 mL). The reaction mixture sealed under nitrogen and heated to
50 °C overnight, then concentrated under reduced pressure to a volume of ~5 mL and
diluted with water. The resulting precipitate was collected by filtration, taken up in
MeOH and dried over Na2SO4 and concentrated to dryness to give 1-pentyl-4-(8-
((tetrahydro-2H-pyran-2-yl)oxy)octyl)-1H-1,2,3-triazole (39b) as a green solid (144
mg, 20.8%) that was used crude in the subsequent step. LCMS (APCI+) 352.9 (MH+,
20%).
A mixture of 39b (144 mg, 0.41 mmol) and trifluoroacetic acid (1 mL) was stirred in
CH2Cl2 (5 mL) for 48 h. The reaction was cooled to 0 °C and neutralised with sat.
K2CO3, and extracted into EtOAc, washed with water, brine, dried with Na2SO4 and
concentrated to give 8-(1-pentyl-1H-1,2,3-triazol-4-yl)octan-1-ol as a colourless oil
1
(40b) (110 mg, 95%) as a colourless oil. H NMR [DMSO]  7.83 (s, 1H), 4.31 (t, J
= 5.2 Hz, 1H), 4.27 (t, J = 7.1, 2H), 3.37 (td, J = 6.5, 5.2 Hz, 2H), 2.59 (t, J = 7.5
Hz, 2H), 1.78 (quin, J = 7.3 Hz, 2H), 1.62-1.51 (m, 2H), 1.44-1.33 (m, 2H), 1.33-1.12
(m, 12H), 0.90 (t, J = 7.2 Hz, 3H). LCMS (APCI+) 268.7 (MH+, 100%).
A mixture of 8-(1-pentyl-1H-1,2,3-triazol-4-yl)octan-1-ol (40b) (110 mg, 0.41 mmol),
TEMPO (4.5 mg, 7 mol%), NaOCl (26 µL, as a 0.27 molL-1 solution, 2.0 mol%) and
NaClO2 (148 mg, 1.6 mmol) in acetonitrile (4 mL) and phosphate buffer (4 mL) were
stirred at 50 °C for 2 days. The reaction was diluted with brine, and extracted into
EtOAc, dried over Na2SO4 to give an oil that crystallised on standing to give 15 (49
1
mg, 42%) as a white solid; mp 62-68 °C (hexanes). H NMR [DMSO]  11.73 (s,
1H), 7.82 (s, 1H), 4.27 (t, J = 7.1 Hz, 2H), 2.58 (t, J = 7.5 Hz, 2H), 2.18 (t, J = 7.3
19

Hz, 2H), 1.78 (quin, J = 7.2 Hz, 2H), 1.62-1.51 (m, 2H), 1.51-1.38 (m, 2H), 1.36-1.12
(m, 10H), 0.84 (t, J = 7.2 Hz, 3H). LCMS (APCI+) 282.7 (MH+, 100%). Anal. Calcd
for C15H27N3O2: C, 64.02; H, 9.67, N, 14.93. Found C, 63.79; H, 9.69; N, 15.07.
3.1.14. 11-(1-Ethyl-1H-1,2,3-triazol-4-yl)undecanoic acid (16). Similar reaction of
11-bromo-1-undecanol (33c) (4.0 g, 16.0 mmol) with 3,4-dihydro-2H-pyran (1.47 g,
17.5 mmol) and purification of the product by flash column chromatography in neat
CH2Cl2 gave 2-((11-bromoundecyl)oxy)tetrahydro-2H-pyran (34c) (5 g, 95%) as a
1
colourless oil. H NMR [DMSO]  4.52 (t, J = 3.5 Hz, 1H), 3.72 (ddd, J = 11.2, 8.0,
3.1 Hz, 1H), 3.60 (td, J = 9.6, 6.7 Hz, 1H), 3.52 (t, J = 6.7 Hz, 2H), 3.46-3.37 (m,
1H), 3.35-3.27 (m, 1H), 1.83-1.74 (m, 2H), 1.73-1.64 (m, 1H), 1.64-1.54 (m, 1H),
1.52-1.21 (m, 20H). HRMS (ESI+) Calcd for C16H31BrNaO2: 357.1400; (MNa+)
357.1393.
Similar reaction of 34c (1.0 g, 3.0 mol) and filtration of the crude through a pad of
silica with CH2Cl2 gave a mixture (0.65 g, 65%) (65:35 by 1HNMR) of trimethyl(13-
((tetrahydro-2H-pyran-2-yl)oxy)tridec-1-yn-1-yl)silane (35c) and 2-(tridec-12-yn-1-
yloxy)tetrahydro-2H-pyran (36c). Treatment of this as above with TBAF (3.7 mL as a
1 molL-1 solution in THF, 3.7 mmol) at room temperature for 48 h gave only 36c
(0.53 g, 63%) as a colourless oil. 1H NMR [DMSO]  4.54-4.50 (m, 1H), 3.72 (ddd, J
= 11.1, 8.0, 3.0 Hz, 1H), 3.59 (td, J = 9.6, 6.7 Hz, 1H), 3.45-3.37 (m, 1H), 3.35-3.26
(m, 1H), 2.72 (t, J = 2.6 Hz, 1H), 2.13 (td, J = 7.0, 2.7 Hz, 2H), 1.79-1.64 (m, 1H),
1.63-1.55 (m, 1H), 1.54-1.38 (m, 8H), 1.38-1.18 (m, 14H). HRMS (ESI+) Calcd for
C18H32NaO2: 303.2295; (MNa+) 303.2299.
20

Ethyl iodide (37c) (2.76 g, 17.8 mmol) and NaN3 (230 mg, 17.4 mmol) was stirred at
40 °C in a sealed tube for 1 hour. The reaction was filtered and the crude 2-
azidoethane (38c) was reacted as above with 36c (300 mg, 1.0 mmol) at 40 °C for 1
week. The product was purified by flash column chromatography with neat CH2Cl2 to
give unreacted alkyne, and further elution with EtOAc/CH2Cl2 (10:90) gave 1-ethyl-
4-(11-((tetrahydro-2H-pyran-2-yl)oxy)undecyl)-1H-1,2,3-triazole (39c) (110 mg,
29%as a colourless oil (110 mg, 29%). 1H NMR [DMSO]  7.83 (s, 1H), 4.54-4.50
(m, 1H), 4.31 (q, J = 7.3 Hz, 2H), 3.72 (ddd, J = 11.2, 8.0, 3.0 Hz, 1H), 3.59 (td, J =
9.6, 6.7 Hz, 1H), 3.45-3.37 (m, 1H), 3.35-3.26 (m, 1H), 2.58 (t, J = 7.6 Hz, 2H), 1.77-
1.64 (m, 2H), 1.63-1.53 (m, 2H), 1.52-1.36 (m, 9H), 1.34-1.20 (m, 14H). LCMS
(APCI+) 352.9 (MH+, 20%).
Similar reaction of 39c (110 mg, 0.31 mmol) with trifluoroacetic acid for 7 days gave
11-(1-ethyl-1H-1,2,3-triazol-4-yl)undecan-1-ol (40c) (70 mg, 84%) as a colourless oil.
1H NMR [DMSO]  7.83 (s, 1H), 4.37 (t, J = 6.6 Hz, 2H), 4.31 (q, J = 7.3 Hz, 2H),
2.58 (t, J = 7.6 Hz, 2H), 1.68 (quin, J = 6.8 Hz, 2H), 1.62-1.52 (m, 2H), 1.40 (t, J =
7.3 Hz, 4H), 1.35-1.20 (m, 14H). LCMS (APCI+) 268.9 (MH+, 20%).
A mixture of 40c (70 mg, 0.26 mmol) with TEMPO (3.5 mg, 7 mol%), NaOCl (18
µL, as a 0.27 molL-1 solution, 2.0 mol%) NaClO2 (72.9 mg, 0.79 mmol) as above at
1
50 °C for 2 days gave 16 (20 mg, 25%) as a white solid; mp 61-63°C (hexanes). H
NMR [DMSO]  11.95 (s, 1H), 7.83 (s, 1H), 4.31 (q, J = 7.3 Hz, 2H), 2.58 (t, J = 7.6
Hz, 2H), 2.18 (t, J = 7.4 Hz, 2H), 1.62-1.52 (m, 2H), 1.51-1.43 (m, 2H), 1.40 (t, J =
7.3 Hz, 3H), 1.32-1.21 (m, 12H). LCMS (APCI+) 282.7 (MH+, 100%). Anal. Calcd
for C15H27N3O2: C, 64.02; H, 9.67, N, 14.93. Found C, 63.80; H, 9.60; N, 14.99.
21

3.2. Biology
3.2.1. Reagents, animals and cell culture conditions
Cell culture medium and fetal bovine serum were purchased from Invitrogen NZ
(Auckland, New Zealand), and the media used contained 100 units/mL of penicillin
and 100 µg/mL streptomycin, which were from Life Technologies. Cells were
cultured in an incubator with humidified atmosphere and 5% CO2 at 37°C. All the
animal work had the approval of the University of Auckland animal ethical
committee.
3.2.2. Osteoclastogenesis assay (murine bone marrow)
Osteoclast development was determined as previously described.14 In brief, femora
and tibiae of 4-6 week old male CD-1 mice were aseptically removed and dissected
free of adhering tissues. Bone marrow cells were collected by flushing the marrow
cavity with α-minimum essential medium (αMEM) using a syringe with a 23G
needle. After incubation for 2 hours in αMEM containing 10% fetal bovine serum
(Invitrogen NZ), non-adherent cells were collected and seeded in a 48-well plate at 5
x 105 cells/0.5 mL/well (day 0). The osteoclastogenic factor 1,25-dihydroxyvitamin
D3 (10-8 M final, Merck) was added on days 0, 2 and 4 while the test compounds
were added on days 2 and 4. The cells were fed 0.5 mL fresh medium on day 2 and
0.5 mL medium was replaced on day 4. On day 7, cells were fixed and stained for the
22

osteoclast marker tartrate-resistant acid phosphatase (TRAP), using the acid
phosphatase kit (Sigma) according to the manufacturer’s instruction. TRAP positive
cells with three or more nuclei were counted as osteoclasts.
Acknowledgements
The authors acknowledge Drs Shannon Black and Lydia Liew for NMR spectroscopy
services and Wilson Sun for technical assistance, and the Biopharma Thematic
Research Initiative, University of Auckland, for financial support of this work.
References
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Sambrook, P.; Cooper, C. Lancet 2006, 357, 2010-2018.
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Cornish, J.; MacGibbon, A.; Lin, J-M.; Watson, M.; Callon, K.E.; Tong, P. C.;
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13.
Itoh, Y.; Kawamata. Y.; Harada, M.; Kobayashi, M.; Fujii, R.; Fukusumi, S.;
Ogi, K.; Hosoya, M.; Tanaka, Y.; Uejima, H.; Tanaka, H.; Maruyama, M.;
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Cornish, J, Callon, K. E.; Bava, U.; Kamona, S. A.; Cooper, G. J.; Reid I. R.
Bone 2001, 29, 162-168.
25

Table 1. Activity of palmitic acid analogues on osteoclast formation in mouse bone
marrow cultures
No
n
X
m
% of controla
73.8±7.2
28.2±2.5
100
1 (palmitic)
12
CH2
1
2 (GW9058)b
12
11
9
O
O
O
O
O
O
O
1
2
3
80.8±5.2
65.4±12.8
100
4
4
5
8
5
6
5
8
100
7
4
9
100
8
2
11
73.2±12.8
100
9
10
11
12
13
14
15
16
OPGc
10 triazole
1
2
3
5
7
38.8±10.3
26.7±4.5
22.2±6.4
21.5±0.9
33.8±7.4
16.9±4.6
32.6±1.6
9
8
6
4
1
triazole
triazole
triazole
triazole
triazole 10
26

Footnotes for Table 1
aPercentage activity of test compounds in osteoclastogenesis, assessed in a murine
bone marrow culture treated with 1,25-dihydroxyvitaminD3 for 7 days (throughout the
whole culture period) (see Experimental Section for details). Test compounds were
added at 10 µg/mL (37 µM). bGW9058 was dosed at 25 µg/mL (72 µM). cOPG was
dosed at 0.01 µg/mL.
27

Scheme 1. Synthesis of oxyacids 3-9
Reagents and conditions: (i) NaH, THF, -70 to 20 oC, 16 h; (ii) NaH, TBAI, THF, 0
°C, 30 min, then bromoalkane, reflux, 16 h.
28