A rapid synthesis of lavendustin-mimetic small molecules by click fragment assembly

Article abstract of DOI:10.1016/j.bmcl.2010.05.014

Lavendustin-mimetic small molecules modifying the linker -CH ₂-NH- with an 1,2,3-triazole ring have been synthesized via a click chemistry. Two pharmacophoric fragments of lavendustin were varied to investigate chemical space and the auxophoric -CH₂-NH- was altered to an 1,2,3-triazole for rapid click conjugation. The small molecules were evaluated against HCT116 colon cancer and CCRF-CEM leukemia cell lines. Among 28 analogues, 3-phenylpropyl ester 26b inhibited CCRF-CEM leukemia cell growth with GI₅₀ value of 0.9 μM.

Full text of DOI:10.1016/j.bmcl.2010.05.014

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3930–3935
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A rapid synthesis of lavendustin-mimetic small molecules by click
fragment assembly
*
Jieun Yoon, Jae-Sang Ryu
Center for Cell Signaling & Drug Discovery Research, College of Pharmacy and Division of Life & Pharmaceutical Sciences, Ewha Womans University, 11-1 Daehyun-Dong,
Seodaemun-Gu, Seoul 120-750, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Lavendustin-mimetic small molecules modifying the linker –CH2–NH– with an 1,2,3-triazole ring have
been synthesized via a click chemistry. Two pharmacophoric fragments of lavendustin were varied to
investigate chemical space and the auxophoric –CH2–NH– was altered to an 1,2,3-triazole for rapid click
conjugation. The small molecules were evaluated against HCT116 colon cancer and CCRF-CEM leukemia
cell lines. Among 28 analogues, 3-phenylpropyl ester 26b inhibited CCRF-CEM leukemia cell growth with
Received 30 March 2010
Revised 3 May 2010
Accepted 6 May 2010
Available online 12 May 2010
GI50 value of 0.9 lM.
Keywords:
Lavendustin
Click chemistry
Library
Ó 2010 Elsevier Ltd. All rights reserved.
Triazole
Anticancer
Due to the growing interest in small molecules to help under-
stand biological processes, the demand for the generation of
small-molecule libraries inspired by bioactive natural products
and their metabolites has increased significantly. In this context,
a rapid access to natural product-like small molecules has become
crucial for systematically dissecting the complex protein functions
and for discovering drug leads. Chemistry tailored to produce mol-
ecules rapidly and reliably by connecting small units is click chem-
protein-tyrosine kinase (PTK) in cell-free extract.4 However, due
to their high polarity and poor cell permeability, they significantly
lost their cellular activity in intact cell line.4 The further research
on improving cell penetration and antiproliferative activity led to
the discovery of novel structures including compound 4 and its
derivatives,5 which were found to inhibit not only the nonreceptor
PTK Syk and the receptor PTK EGFR but also tubulin
polymerization.
istry,1 which is
a
chemical philosophy introduced by Barry
Lavendustin C (3) consists of two fragments, salicylic acid and
hydroxybenzene, which are responsible for biological activity and
also found in lavendustin A (1) and B (2). However, such polar frag-
ments are also considered to decrease cell permeability. Therefore,
when devising a rapid route to lavendustin-mimetic small mole-
cules, the followings should be considered: (1) the modification
of pharmacophoric fragments to improve cell permeability; (2)
the change of the auxophoric –CH2–NH– with a linkage to facilitate
the assembly of fragments. In this study, we synthesized a series of
lavendustin-mimetic small molecules replacing the –CH2–NH–
with an 1,2,3-triazole ring. The salicylic acid fragment and the
hydroxybenzene fragment were varied structurally to explore sub-
tle chemical space and linked with a rigid lipophilic triazole
(Fig. 2). This bioisosteric triazole replacement would increase
hydrophobicity and make these molecules suitable for rapid paral-
lel synthesis. Particularly, click chemistry would allow the substit-
uents of the 1,2,3-triazole ring to be easily varied through the use
of different azides or alkynes. Similar bioisosteric triazole replace-
ments are often found in the literatures. Recently, Genazzani and
co-workers modified the –CH@CH– of natural product resveratrol
K. Sharpless. The Cu(I)-catalyzed Huisgen [3+2] cycloaddition,2
the representative click chemistry, has been used in various appli-
cations such as drug discovery and chemical biology due to high
chemical yield, high selectivity, wide scope, atom economy, and
simple purification. Especially, fragment-based rapid assembly
using click chemistry and in situ screening are emerging as a ver-
satile tool to identify novel inhibitors quickly against a number of
biological targets.3 The resulting 1,4-disubstituted 1,2,3-triazoles
are similar to amide bonds in terms of distance and planarity,
and often used as a scaffold in the discovery of enzyme inhibitors.
Recently, potential pharmaceuticals based on 1,2,3-triazoles have
been developed exponentially.
Lavendustin A (1) (Fig. 1), a metabolite of Streptomyces griseo-
lavendus, was first isolated by Onoda in 1989.4 This natural metab-
olite and its synthetic partial structure 3 were reported to inhibit
* Corresponding author. Tel.: +82 2 3277 3008; fax: +82 2 3277 2851.
E-mail address: ryuj@ewha.ac.kr (J.-S. Ryu).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.014

J. Yoon, J.-S. Ryu / Bioorg. Med. Chem. Lett. 20 (2010) 3930–3935
3931
HO
N
HO
N
OCH3
OCH3
OCH3
OCH3
OCH3
CBr4, Ph3P
n-BuLi, THF
O
OH
O
H
H
CH2Cl2
0 ºC, 0.5h
-78 oC→ rt
HO
HO
HO
HO
O
O
OCH3
OH
OH
Br
Br
6 (99%)
7 (85%)
5
OTBS
Lavendustin B (2)
HO
OH
OTBS
Lavendustin A (1)
TBSCl
imidazole
CBr4, Ph3P
CH2Cl2
HO
H
DMF
rt, 5 h
0 ºC, 2 h
F
O
OH
O
OH
OTBS
OH
O
OTBS
Br
Br
NH
NH
HO
HO
N
H
9 (83%)
10 (91%)
8
HO
OTBS
OH
TBAF
THF
Lavendustin C (3)
4
n-BuLi, THF
Figure 1. Structures of lavendustin analogues.
-78 ºC → rt
rt, 2 h
OTBS
OH
11 (84%)
12 (90%)
Hydroxybenzene fragment
HO
OCH3
OH
Y
O
OH
OH
O
NH
N
HO
HO
N
N
X
OCH3
OH
triazole linker
HO
7
12
13
Lavendustin C (3)
Salicylate fragment
OCH3
OCH3
Figure 2. Fragment-based click approach for lavendustin-mimetic small molecule
synthesis.
OCH3
OCH3
16
14
15
with 1,2,3-triazole and identified a potent antiproliferative agent
Scheme 1. Structure and synthesis of alkyne building blocks.
3b
showing low nanomolar IC50
.
We used six alkynes and five azides for the rapid assembly of
salicylate fragments and hydroxybenzene fragments of lavendu-
stin-mimetic small molecules. Alkyne 7 and 12 were readily pre-
pared under Corey–Fuchs conditions6 from the aldehyde 5 and 8,
respectively (Scheme 1). Commercially available alkynes 13–16
were also used to investigate the role of –OH in the hydroxyben-
zene fragment of lavendustin C (3). Five different azide building
blocks bearing salicylamide or alkylsalicylate were synthesized as
lipophilic surrogates of the salicylate fragment (Scheme 2). Synthe-
sis of the azide 23 commenced from commercially available
5-aminosalicylic acid (17). After esterification, a treatment of the
diazonium salt, formed by the addition of aqueous solution of
NaNO2 to a HCl solution of 22, with NaN3 led to an azide 23. How-
ever, the same condition did not provide azide 18. After changing
HCl to H2SO4,7 we were able to obtain azide 18 in 99% yield, which
was coupled with amines and alcohols in the presence of 1,
10-carbonyldiimidazole.
With alkyne and azide building blocks in hand, we performed a
small screening of catalytic conditions for azide 18, 23 and alkyne
13, 16 to find the optimal conditions for click reaction (Table 1). In
this simulation, azide 23 bearing methylester afforded the corre-
sponding triazoles in almost quantitative yields. However, azide
18 bearing salicylic acid did not react with alkynes even in the
presence of 20 mol % of catalyst. Interestingly, when we used
20 mol % of catalyst, we observed the line broadening effect in 1H
NMR of the crude reaction mixture, presumably caused by the
bidentate complex formation of Cu and salicylic acid.8 Therefore,
the salicylic acid analogues 28a–28f were synthesized from hydro-
lysis of the corresponding methylsalicylate analogues 27a–27f
after click reaction of azide 23 and alkyne 7, 12–16 (Scheme 3).
Library synthesis was well proceeded in 24 conical tubes. After
four days, the resulting triazoles were precipitated, separated by
centrifugation, and washed with H2O and ether. The conical tube
reactions really facilitated work-up processes such as solid separa-
tion by centrifugation and solvent removal by GeneVac™ without
sample transfer. All precipitated compounds were submitted to
HPLC and 1H NMR analysis to verify their purity and authenticity.
Indeed, 23 out of 24 were confirmed as the expected product. Only
25b was not yielded under standard conditions (Table 2). To ensure
the complete removal of a trace of Cu, which is a potentially bioac-
tive metal, all crude products were further purified through a short
pad of silica gel before bioassay. Hydrolysis of methyl ester ana-
logues 27a–27f provided the salicylic acid analogues 28a and
28c–28f except 28b. We eventually synthesized the 28-membered
lavendustin-mimetic small molecules with minimum side prod-
ucts, as judged by HPLC and 1H NMR characterizations. The yields
of the synthesized compounds are shown in Table 2. The yields of
the salicylic acid analogues 28a–28f are for two steps; click reac-
tion and hydrolysis.
The 28-membered library was next screened for antiprolifera-
tive activity against HCT116 colon cancer and CCRF-CEM leukemia
cell lines.9 For easy comparison, the potency was expressed as %
inhibition at 10 lM concentration of the reaction products
(Table 2). GI50 values for selected compounds are listed in Table
3 compared to the anticancer drug, doxorubicin, as a positive con-
trol. Among the 28 small molecules, two ester (26b and 27b) ana-
logues and seven salicylamide (24a, 24c, 24d, 24f, 25a, 25e, and

3932
J. Yoon, J.-S. Ryu / Bioorg. Med. Chem. Lett. 20 (2010) 3930–3935
F
O
a. CDI, THF, reflux, 3 h
b. H2N
N3
N
H
OH
F
TEA , reflux, 23 h
19 (62%)
a. H2SO4, H2O
NaNO2, 0 ºC
1.5 h
O
O
N3
O
a. CDI, CH3CN, reflux, 3.5 h
H2N
OH
OH
OH
N3
N
H
CF3
b.
b. NaN3, H2O
0 ºC→ rt
13 h
OH
OH
H2N
CF3
18 (99%)
17
20 (54%)
TEA, reflux, 13.5 h
CH3OH, H2SO4
80 ºC, 18 h
O
a. CDI, THF, reflux, 3 h
N3
O
HO
b.
OH
21 (91%)
a. c-HCl, NaNO2,
H2O, 0 ºC
10 min
O
O
TEA , reflux, 22 h
N3
H2N
OCH3
OH
23 (60%)
OCH3
b. NaN3, H2O,
0 ºC, 40 min
OH
22 (87%)
F
O
O
O
O
O
N3
N3
N3
N3
N3
OH
O
OCH3
OH
N
H
CF3
N
H
OH
18
OH
OH
OH
19
20
21
23
Scheme 2. Structure and synthesis of azide building blocks.
Table 1
among a series of compounds against CCRF-CEM leukemia cell
lines and exhibited the GI50 value of 0.9 M. Interestingly, 4-fluor-
Screening of loadings of catalyst and reducing agent
l
ophenylethyl amide 24b, a rigid triazole analogue of 4, was less ac-
Entries Azide Alkyne CuSO4 (mol %) Na ascorbate (mol %) Yield (%)
tive (15% inhibition of CCRF-CEM and 18% inhibition of HCT116 at
1
2
3
4
5
6
7
8
23
23
23
23
18
18
18
18
13
16
13
16
13
16
13
16
2
2
20
20
2
2
20
20
10
10
100
100
10
10
100
100
99
77
99
99
0a
10
and 9.2
Previously, it was reported that lavendustin C potently inhibited
EGF-R tyrosine kinase in A431 cell-free extract with 0.012 g/mL
IC50
4 However, lack of cellular activity was observed in the HaCaT
keratinocyte proliferation assay (IC50 >100
M).12 It was attributed
l
M) than 4, which showed GI50 values of 0.29
lM (CCRF-CEM)
l
M (HCT116).11
l
0a
0b
0b
.
l
to insufficient cell penetration caused by the polar functional
groups, in particular the carboxylic moiety.12 Not surprisingly, all
the salicylic acid analogues 28a, 28c–28f exhibited no cellular
activity presumably due to the high polarity and poor cell perme-
ability of salicylic acid. This result is consistent with the cell-based
assay of natural lavendustins4 and supports the library design that
the salicylic acid fragment should be replaced by a lipophilic
surrogate.
In conclusion, we have developed synthetic protocol for the
rapid assembly of 28-membered lavendustin-mimetic small
molecules using ‘click chemistry’: Cu(I)-catalyzed 1,3-dipolar
cycloaddition between alkynes and azides, which afforded
triazoles in good yields after minimum work-up processes. Fur-
ther silica gel filtration completely removed a trace of Cu and
afforded pure compounds suitable for biological evaluation
against cancer cell lines. In antiproliferative screening, 26b
showed cytotoxic activity on the CCRF-CEM leukemia cell line
a
No reaction.
No reaction, the line broadening in 1H NMR was observed.
b
Y
N
CuSO4 (20 mol%)
Na. Ascorbate
O
O
N3
N
Y
X
N
+
X
t
-BuOH-H2O
rt, 4 d
HO
HO
19-21, 23
7, 12-16
24a-24f (X = 4-F-Ph(CH2)2NH-)
25a-25f (X = 3-CF3-PhNH-)
26a-26f (X = Ph(CH2)3-)
27a-27f (X = CH3O-)
X = NHR, OR, OH
Y = OCH3, OH
1N NaOH, CH3OH
80 ºC, 0.5h
28a-28f (X = HO-)
Scheme 3. Click assembly for lavendustin-mimetic small molecules.
with GI50 value of 0.9 lM. This compound could be a good can-
didate for further design of cell-permeable antiproliferative
agents.
25f) analogues showed moderate potency with GI50 values of 0.9–
23
M. Particularly, 3-phenylpropyl ester 26b10 was the best
l

Table 2
Chemical yields and cell growth inhibition of lavendustin-mimetic small molecules synthesized by ‘click chemistry’ (28a–28f yields for two steps)
OCH3
OH
OCH3
OCH3
OH
OCH3
OCH3
16
OCH3
OH
14
13
24c, 57
7
12
24b, 41
15
24e, 60
F
Yield (%)
24a, 70
24d, 80
24f, 75
O
% Inhibition (HCT116)a
44
35
18
15
0
35
62
21
23
11
43
35
% Inhibition (CCRF-CEM)a
N3
N
H
OH
19
Yield (%)
25a, 29
46
15
25b, 0
—
—
25c, 99
25
13
25d, 21
9
12
25e, 24
61
65
25f, 45
62
73
O
% Inhibition (HCT116)a
N3
% Inhibition (CCRF-CEM)a
N
CF3
H
OH
20
O
Yield (%)
26a, 76
13
5
26b, 34
39
81
26c, 72
11
9
26d, 81
12
5
26e, 69
7
4
26f, 76
19
8
% Inhibition (HCT116)a
N3
N3
N3
% Inhibition (CCRF-CEM)a
O
OH
21
O
Yield (%)
27a, 84
6
3
27b, 29
15
52
27c, 77
6
5
27d, 85
6
16
27e, 74
8
6
27f, 94
3
3
% Inhibition (HCT116)a
% Inhibition (CCRF-CEM)a
OCH3
OH
23
Yield (%)
28a, 74b
0
9
28b, 0b
—
—
28c, 82b
5
3
28d, 64b
8
9
28e, 78b
4
9
28f, 76b
2
9
O
% Inhibition (HCT116)a
% Inhibition (CCRF-CEM)a
OH
OH
18
Inhibition at 10
a
lM.
b
Yields for two steps; click reaction and hydrolysis.

3934
J. Yoon, J.-S. Ryu / Bioorg. Med. Chem. Lett. 20 (2010) 3930–3935
Table 3
GI50 of selected lavendustin-mimetic small molecules
Compounds
GI50 (lM)
HCT116
CCRF-CEM
OCH3
H3CO
F
O
13.57 0.96
22.87 0.67
N
24a
24c
N
N
N
H
HO
OH
F
O
>100
22.78 0.04
N
N
N
N
H
HO
OCH3
F
24d
12.50 0.77
>100
O
N
N
N
N
N
H
HO
H3CO
OCH3
F
16.10 1.54
>100
O
24f
N
N
N
N
H
HO
OCH3
H3CO
O
25a
12.90 0.79
>100
N
N
F3C
N
H
HO
OCH3
OCH3
O
25e
25f
7.92 0.67
10.45 0.37
N
N
N
F3C
N
H
HO
H3CO
4.37 0.39
6.41 0.50
O
N
N
N
F3C
N
H
HO
OH
HO
O
26b
27b
20.40 2.79
0.93 0.03
N
N
N
O
HO
OH
HO
O
>100
13.90 1.66
0.02 0.003
N
N
N
H3CO
HO
Doxorubicin
0.40 0.21

J. Yoon, J.-S. Ryu / Bioorg. Med. Chem. Lett. 20 (2010) 3930–3935
3935
7. Xiong, Y.; Bernardi, D.; Bratton, S.; Ward, M. D.; Battaglia, E.; Finel, M.; Drake, R.
R.; Radominska-Pandya, A. Biochemistry 2006, 45, 2322.
Acknowledgments
8. Hanic, F.; Michalov, J. Acta Crystallogr. 1960, 13, 299.
This work was supported by the Korea Research Foundation
Grant funded by the Korean Government (MOEHRD) (KRF-2007-
313-E00645). J.Y. was supported by the Brain Korea 21 project.
Authors thank Hwayoung Yun and Dr. Seung-Mann Paek for mass
spectroscopy.
9. Cytotoxic activity of the synthesized compounds against human cancer cell
lines was investigated using the MTT assay. HCT116 (Human colorectal
carcinoma:KCLB) were supplied from the KCLB/ATCC and grown in RPMI
1640/DMEM (Gibco BRL) supplemented with 10% (V/V) heat inactivated Fetal
Bovine Serum (FBS) and maintained at 37 °C in a humidified atmosphere with
5% CO2. The cells (5 ꢀ 104 cells/ml) were seeded into 96-well plate. Various
concentrations of samples was added to each well in duplicate, then incubated
at 37 °C with 5% CO2 for two days such that time cells are in the exponential
phase of growth at the time of sample addition. Add 15
(Promrga, Cell Titer 96) to each well. Incubate the plate at 37 °C for up to 4 h in
humidified, 5% CO2 atmosphere. After incubation, add 100 of the
Solubilization Solution/Stop Mix (Promrga, Cell Titer 96) to each well. Allow
the plate to stand overnight in sealed container with humidified
atmosphere at room temperature to completely solubilize the formazan
crystals. The optical density was measured using microplate reader
(Versamax, Molecular Devices) with a 570 nm wavelength and the anticancer
effective concentration was expressed as a GI50
lL of the Dye Solution
Supplementary data
a
lL
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.05.014.
a
a
a
References and notes
.
10. Yield: 34%. TLC: Rf 0.27 (EtOAc/Hex = 2:3). 1H NMR (400 MHz, acetone-d6): d
10.96 (br s, 1H), 9.66 (br s, 1H), 8.92 (s, 1H), 8.35 (d, 1H, J = 3.2 Hz), 8.10 (dd,
1H, J = 8.8 Hz, 3.2 Hz), 7.92 (br s, 1H), 7.39 (d, 1H, J = 3.2 Hz), 7.29–7.26 (m, 4H),
7.23 (d, 2H, J = 8.8 Hz), 7.13 (m, 1H), 6.86 (d, 1H, J = 8.8 Hz), 6.77 (dd, 1H,
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