High-throughput synthesis of azide libraries suitable for direct "click" chemistry and in situ screening

Article abstract of DOI:10.1039/b902338k

A key challenge in current drug discovery is the development of high-throughput (HT) amenable chemical reactions that allow rapid synthesis of diverse chemical libraries of enzyme inhibitors. The Cu(I)-catalyzed, 1,3-dipolar cycloaddition between an azide and an alkyne, better known as "click chemistry", is one such method that has received the most attention in recent years. Despite its popularity, there is still a lack of robust and efficient chemical strategies that give access to diverse libraries of azide-containing building blocks (key components in click chemistry). We report herein a highly robust and efficient strategy for high-throughput synthesis of a 325-member azide library. The method is highlighted by its simplicity and product purity. The utility of the library is demonstrated with the subsequent "click" synthesis of the corresponding bidentate inhibitors against PTP1B.

Full text of DOI:10.1039/b902338k

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
High-throughput synthesis of azide libraries suitable for direct “click”
chemistry and in situ screening†
Rajavel Srinivasan,a Lay Pheng Tan,b Hao Wu,a Peng-Yu Yang,a,c Karunakaran A. Kalesha and
Shao Q. Yao*a,b,c
Received 4th February 2009, Accepted 10th February 2009
First published as an Advance Article on the web 11th March 2009
DOI: 10.1039/b902338k
A key challenge in current drug discovery is the development of high-throughput (HT) amenable
chemical reactions that allow rapid synthesis of diverse chemical libraries of enzyme inhibitors. The
Cu(I)-catalyzed, 1,3-dipolar cycloaddition between an azide and an alkyne, better known as “click
chemistry”, is one such method that has received the most attention in recent years. Despite its
popularity, there is still a lack of robust and efficient chemical strategies that give access to diverse
libraries of azide-containing building blocks (key components in click chemistry). We report herein a
highly robust and efficient strategy for high-throughput synthesis of a 325-member azide library. The
method is highlighted by its simplicity and product purity. The utility of the library is demonstrated
with the subsequent “click” synthesis of the corresponding bidentate inhibitors against PTP1B.
Introduction
The field of high-throughput enzymology (or “Catalomics” as
we call it) necessitates the development of highly efficient and
robust synthetic methods that enable rapid generation of en-
zyme probes and inhibitors.1 The Cu(I)-catalyzed, 1,3-dipolar
cycloaddition between an azide and an alkyne, better known
as “click chemistry”, is one such method that has received the
most attention in recent years.2 The salient features of this
reaction include its high efficiency, chemoselectivity, modularity
and biocompatibility, which allow direct in situ screening of
the products formed without the need of tedious purification
steps.3 Consequently, many research groups have successfully
adopted this platform for the high-throughput screening and
discovery of inhibitors against numerous enzymes, including
HIV-1 protease,4a,b sulfotransferases,4c fucosyltransferases,4d ma-
trix metalloproteases,4e acetylcholinesterase,4f and others.4g–i For
example, we recently used click chemistry to discover a potent,
cell-permeable inhibitor against PTP1B by assembling together
an alkyne-containing isoxazole “warhead” (a bioisostere of phos-
photyrosine) and an azide-modified aryl group that binds to
the secondary binding site in the enzyme (Fig. 1).4g PTP1B has
previously been identified as a key target for diseases such as
diabetes, obesity and cancer, and our inhibitor design was based
on the elegant work reported by Zhang et al and researchers from
Abbott laboratories.5
Fig. 1 PTP1B inhibitor assembled by “click chemistry”.4g
screening methods),4 though advantageous, remains to be truly
high-throughput due to the lack of a robust and efficient chemical
strategy that gives access to diverse libraries of azide-containing
building blocks. The standard azidation method calls for the use of
NaN3 (a highly toxic chemical) which, while efficient, necessitates
the compounds to be individually synthesized and purified using
solution phase chemistry, therefore is time-consuming and low-
throughput.4 Recently, improved methods for azide synthesis have
shown some compatibility with click chemistry.6 For example,
Fokin et al reported the in situ generation of azides from alkyl
and aryl halides followed by direct “click” chemistry.6a Other
methods include the in situ generation of azides from alcohols,6b
the direct conversion of amines to azides by employing the
diazotransfer reaction using TfN3 or imidazole-1-sulfonyl azide
hydrochloride,6c–e the use of TPP/DDQ/n-Bu4NN3 reagents to
convert alcohols directly to azides,6f and the microwave-assisted
azidation of tosylates.6g,h All of them, again due to the use of NaN3
(or equivalents), are not suitable for direct in situ screening with
enzymes, unless additional purification steps (either before or after
click chemistry) are carried out. Herein, we report a truly high-
throughput amenable, solid-phase strategy for the synthesis of
high-quality azides which, without purification, could be used for
“click assembly” and direct in situ screening of enzyme inhibitors
(Scheme 1, top). With this new method, the whole process
When compared with other existing drug discovery approaches,
our method (as well as other published “click”-based enzyme
aDepartment of Chemistry, National University of Singapore, 3 Science
Drive 3, Singapore 117543. E-mail: chmyaosq@nus.edu.sg; Fax: (+65)
67791691; Tel: (+65) 68741683
bDepartment of Biological Sciences, National University of Singapore,
Singapore 117543
cNUS MedChem Program of the Office of Life Sciences, National University
of Singapore, Singapore 117543
† Electronic supplementary information (ESI) available: Detailed experi-
mental procedures and results. See DOI: 10.1039/b902338k
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Scheme 1 (top) Workflow of our strategy. (bottom) Traceless solid-phase synthesis of azide libraries.
of inhibitor discovery can be now completed in less than one
week.
of as-is starting materials (i.e. same as solution-phase methods) as
building blocks and is easily amenable to high-throughput synthe-
sis (i.e. IRORI(tm) split-and-pool sorting technology8) of a large
number of azides. We routinely make several hundreds of azides
in milligram scales in 2–3 days; (2) it is highly robust, capable of
making a variety of azides from different functionalized building
blocks (i.e. amines, acids, sulfonyl chloride, and chloroformates)
with suitable linkers; (3) it gives rise to products with extremely
high purity, thus allowing them to be used directly for “click”
assembly followed by in situ enzyme screening.
To demonstrate our strategy, a 325-member azide library was
synthesized as shown in Scheme 1 (bottom). For comparison, 9
azides were synthesized using standard solution-phase method.4g
Depending on the choice of building blocks and linkers, either
route A or B was taken. In route A, 40 different commercially
Results and discussion
Chemistry
As shown in Scheme 1, our strategy made use of the commercially
available PL-FMP resin on which an immobilized 4-formyl-3-
methoxyphenoxy moiety efficiently captured a variety of primary
amines via reductive amination and converted them to the cor-
responding secondary amines which were subsequently acylated.7
Following TFA cleavage, the desired products were obtained in
uniformly high quality. There are numerous advantages in this
method: (1) it is a traceless solid-phase strategy, allowing the use
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Table 1 IC50 of selected “hits” against PTP1B
available aromatic amine building blocks were treated with the
PL-FMP aldehyde resin, 7, in the presence of Na(OAc)3BH/2%
glacial acetic acid, to give the corresponding secondary amines,
8. The reductive amination reaction proceeded smoothly with a
variety of aromatic amines bearing different substituents such as
-F, -Cl, -OR, -SCH3, -CO2R and -R. Benzyl, naphthyl and anthra-
cenyl amines too underwent reductive amination smoothly. Subse-
quently, treatment with azidoalkynoyl chlorides, 5, furnishing the
N-acylated products, 9. Other coupling conditions attempted (e.g.
DIC/EDC/HATU/PyBOP) failed to give the desired products
in sufficient purity. In route B, we first carried out reductive
amination with azide-functionalized amine linkers 6 to give 10.
Subsequently, the resulting resins were treated with different
commercially available building blocks, including acid chlorides,
aryl acids, sulfonyl chlorides and chloroformates, giving 11, 12, 13
and 14 respectively. We found that coupling conditions using acid
chlorides, sulfonyl chlorides and chloroformates (giving 11, 13 &
14 respectively) were excellent as expected. With aryl acid building
blocks, however, the PyBOP/HOAt coupling was the method of
choice, providing complete coupling to give 12. Other coupling
conditions were tried but failed to give satisfactory results. We
also found building blocks bearing different functionalities such
as -F, -Cl, -Br, -R, -OH, -OCH3, -CF3, -CN, -OH, -NO2 and -vinyl
were tolerated. Finally, cleavage of the resins with optimized TFA
cocktails gave the desired products, which, upon concentration
in vacuo, analysis and characterizations by LC-MS/NMR, showed
uniformly high purity (> 95% in most cases). In total, 198 amide-
bearing azides (i.e. A1-2C to D4-6C, E1-2C to G10-2C, H1-2C to
H1-6C), 115 different sulfonamide-containing azides (i.e. I1-2C to
J11-6C), and 3 carbamate-derivatized azides (i.e. L1-2C to L1-6C)
were successfully prepared. Stock solutions of these azides were
prepared in uniform concentration with DMSO in 96-deep well
plates and used directly, without any purification, for subsequent
in situ click assembly followed by enzymatic screening.
Inhibitor
IC50 (mM)
Inhibitor
IC50 (mM)
A5-2C-W2
A6-2C-W2
B7-2C-W2
B8-2C-W2
634
159
55
B9-2C-W2
B10-2C-W2
B11-2C-W2
C8-2C-W2
34.0
148
11.1
24.0
70.8
Fig. 2 Structure of eight representative hits.
Inhibitor screening
To make potential cell-permeable, bidentate inhibitors against
PTP1B, we subsequently “click”-assembled the above 325-
member azide library with the alkyne-modified isoxazole warhead,
1, as shown in Scheme 1. The choice of this warhead was
guided by our previous results and computational modeling.4g
The synthesis of the warhead is essentially the same as previously
reported. Optimizations of the click chemistry conditions were
essential in order for the products to be generated in quantitative
yield. With CuSO4/sodium ascorbate catalysts and H2O/t-BuOH
as cosolvents, we were able to carry out the reactions in high
throughput and small scale (100 mL/reaction volume) using 384-
deep well plates. At the end of the reaction, (~2 days), products
were evaporated in vacuo, redissolved in DMSO, and directly
characterized (by LCMS and NMR); most were found to be of
sufficient purity (> 95% excluding excess starting materials in some
cases; see ESI) and used for direct in situ enzymatic screening. The
inhibition potency of the 325-member “click” products against
PTP1B was determined using a standard fluorescence microplate-
based assay as previously reported.4g From the preliminary
results, eight representative “hits” were identified (Fig. 2). They
were further purified, characterized and their IC50 values were
determined (Table 1). Of particular interest is B11-2C-W2, which
has an IC50 of 11.1 mM, nearly as potent as the best “click”
inhibitor previously identified (shown in Fig. 1). This thus con-
firms the utility of our azide library and its potential applications
with other click chemistry-based strategies. We are currently
testing the in vivo activity of this new target compound and its
analogs.
Conclusions
In conclusion, we have developed a solid-phase strategy for the
synthesis of high-quality azides which could be used for direct
“click assembly” and in situ screening of enzyme inhibitors. With
this new method, the whole process of inhibitor discovery, starting
from the synthesis of building blocks to the identification of
enzyme inhibitors, could now be completed in less than one week.
The entire operation was performed in 96/384-well plates without
any purification, therefore is truly high-throughput amenable. We
foresee azide libraries synthesized using this approach will be
equally useful for other classes of enzymes. Our present approach
thus serves as a powerful chemical tool in click chemistry and in
the emerging field of “Catalomics”.1
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General protocol for cleavage and release of azides (A1-2C to
D4-6C). Each dried resin was treated with a 1.5 ml solution
containing TFA (50%), DCM (45%) and water (5%) and the
mixture was shaken for 4 hours and transferred into 3 different 96-
well plates and concentrated in vacuo to afford the azides below.
Representative yield varied from 50% to 80%. The azides were
re-dissolved in DMSO (1 mL) to give a stock solution (50 mM
assuming 50% yield).
Experimental
Material and methods
All chemicals were purchased from commercial vendors and used
without further purification, unless otherwise noted. H NMR
1
and 13C NMR spectra were recorded on a Bruker model Avance
300 MHz or DPX-300 MHz or DPX-500 MHz NMR spectrom-
eter. Chemical shifts are reported in parts per million referenced
with respect to residual solvent (CHCl3 = 7.26 ppm and DMSO-
d6 = 2.50 ppm) or from internal standard tetramethylsilane
(TMS = 0.00 ppm). The following abbreviations were used in
reporting spectra: s = singlet, d = doublet, t = triplet, q = quartet,
m = multiplet, dd = doublet of doublets. HPLC grade solvents
were used for all the reactions. Shimadzu LCMS-IT-TOF system
equipped with auto-sampler (cat. no. LCMS-2010EV) was used
for the analysis of the azide and the 1,2,3-triazole library. Sciclone
ALH 3000 Liquid Handler Workstation (Caliper Life Sciences)
and Multi-drop Combi dispenser (Thermo Scientific) were used
to dispense and transfer reagents, solvents and reactants during the
library synthesis. 384-deep well polypropylene plates (Genetix. cat.
no. X7020) were used as reaction plates for the library synthesis.
Characterizations of selected azides
Except for the azides cleaved from 9b resin (with 3C linker; data not
shown), almost all azides synthesized from the above protocols are
of high purity (90–95%; see ESI Part 2 for detailed structures, ID
and characterizations). Representative compounds were further
characterizations, without any purification, by LCMS, 1H and 13
NMR, as shown below and in ESI.
C
(A2-2C). 2-Azido-N-(3-fluoro-phenyl)-acetamide. 1H-NMR
(500 MHz, DMSO-d6) d 10.3 (s, 1H), 7.58 (d, J = 8 Hz, 1H),
7.39–7.29 (m, 1H), 7.09 (d, J = 50 Hz, 1H), 6.92 (m, 1H), 4.06 (s,
2H). 13C-NMR (125 MHz, DMSO-d6) d 167.1, 163.6, 131.0, 140.1,
115.5, 110.6, 106.6, 51.8. ESI-MS(TOF): m/z 195.060 [M + H]+.
(B1-2C). 2-Azido-N-(2-phenoxy-phenyl)-acetamide.
1H-
Solid-phase synthesis of azides
NMR (500 MHz, DMSO-d6) d 9.71 (s, 1H), 7.39 (t, J = 8 Hz,
3H), 7.16–7.11 (m, 3H), 7.00 (d, J = 8 Hz, 2H), 6.89–6.87 (m,
1H), 4.06 (s, 2H). 13C-NMR (125 MHz, DMSO-d6) d 156.5, 129.9,
128.9, 125.3, 123.6, 123.5, 118.6, 118.5, 51.1. ESI-MS(TOF): m/z
269.098 [M + H]+.
Chemical synthesis of the alkyne warhead, 1, is described in
the ESI. The construction of the azide library was achieved by
R
IRORIꢀ split-and-pool directed sorting technology.8 Full details
of the synthesis are described in the ESI. Final products were
released from the solid phase by our optimized TFA cleavage
protocol (below). For complete structural information, ID and
full characterizations of all azides, see Part 2 (in the ESI).
Representative procedures for the solid-phase synthesis of resins 8
and 9, followed TFA cleavage to give A1-2C to D4-6C (as shown
in Scheme 1) are given below.
(B2-2C). 2-Azido-N-(3,4-dimethoxy-phenyl)-acetamide. 1H-
NMR (500 MHz, DMSO-d6) d 9.36 (s, 1H), 7.71 (s, 1H), 6.97
(d, J = 8.8 Hz, 1H), 6.66 (m, 1H), 4.14 (s, 2H), 3.79 (s, 3H), 3.69
(s, 3H). 13C-NMR (125 MHz, DMSO-d6) d 166.9, 153.4, 143.9,
127.8, 112.4, 109.0, 108.49, 56.7, 55.8, 51.8. ESI-MS(TOF): m/z
237.091 [M + H]+.
Synthesis of the reductive aminated resin (8). PL-FMP resin
(200 ¥ 150 mg, 0.9 mmol/g) was taken in 200 MacroKanTM
reactors each containing a RF tag. The resin was swelled in 1,2-
dichloroethane (250 mL) for about two hours after which the
solvent was decanted. The 200 microreactors were then distributed
equally into 40 different bottles of capacity 50 mL containing 2%
acetic acid in 1,2-dichloroethane (30 mL). Amines (A1–D4) (5 eq)
were added to the bottles, so that each bottle contains a unique
amine. The reaction mixture was then incubated for about 3 hours
after which sodium triacetoxyborohydride (6 eq) was added. After
shaking for another 8 hours the solution was decanted and the
reactors were combined and washed with DCM (200 mL ¥ 5),
MeOH (200 mL ¥ 2) and THF (200 mL ¥ 3) and dried to afford
the resin 8.
(B5-2C). 2-Azido-N-(4-pentyloxy-phenyl)-acetamide.
1H-
NMR (500 MHz, DMSO-d6) d 10.06 (s, 1H), 6.96 (s, 2H), 4.00 (s,
2H), 3.74 (s, 6H), 3.62 (s, 3H). 13C-NMR (125 MHz, DMSO-d6) d
166.0, 152.7, 134.4, 133.6, 97.0, 60.05, 55.6, 51.2. ESI-MS(TOF):
m/z 267.100 [M + H]+.
(B12-2C). 2-Azido-N-(4-pentyl-phenyl)-acetamide. 1H-NMR
(500 MHz, DMSO-d6) d 10.10 (s, 1H), 7.28 (s, 1H), 7.22 (t, J =
8.2 Hz, 1H), 7.11 (d, J = 8.2 Hz, 1H), 6.66 (d, J = 8 Hz, 1H), 4.02
(s, 2H), 3.73 (s, 3H). 13C-NMR (125 MHz, DMSO-d6) d 166.8,
160.0, 140.0, 130.1, 112.0, 109.6, 105.5, 55.4, 51.7. ESI-MS(TOF):
m/z 207.080 [M + H]+.
(C4-2C). 2-Azido-N-(3,4-dimethyl-phenyl)-acetamide. 1H-
NMR (500 MHz, DMSO-d6) d 9.96 (s, 1H), 7.36 (s, 1H), 7.29 (d,
J = 8.2 Hz, 1H), 7.06 (d, J = 8.2 Hz, 1H), 3.99 (s, 2H), 2.19 (s,
3H), 2.16 (s, 3H). 13C-NMR (125 MHz, DMSO-d6) d 165.8, 136.3,
136.0, 131.4, 129.6, 120.4, 116.7, 51.2, 19.5, 18.7. ESI-MS(TOF):
m/z 205.101 [M + H]+.
Synthesis of the N-acylated resin (9a–e). The 200 reductive
aminated resins were separated into 5 sets (40 different reductive
aminated resins ¥ 5 sets). To the first set, DCM (100 mL), DIEA
(10 eq) and acid chloride, 5a-e (5 eq) were added and the reaction
mixture was shaken for about 8 hours after which the solution
was decanted and the resin was washed with DCM (100 mL ¥ 3),
MeOH (100 mL ¥ 2) and THF (100 mL ¥ 3) and dried to afford
resin 9a-e.
(C7-2C). 2-Azido-N-(4-ethyl-phenyl)-acetamide. 1H-NMR
(500 MHz, DMSO-d6) d 10.0 (s, 1H), 7.47 (d, J = 8.8 Hz, 2H),
7.15 (d, J = 8.2 Hz, 2H), 4.00 (s, 2H), 2.55 (m, 2H), 1.15 (t, J =
7.6 Hz, 3H). 13C-NMR (125 MHz, DMSO-d6) d 166.4, 139.5,
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136.6, 128.5, 119.8, 51.7, 28.0, 16.1. ESI-MS(TOF): m/z 205.103
(B8-4C). 4-Azido-N -(4-trifluoromethoxy-phenyl)-butyr-
amide. 1H-NMR (500 MHz, DMSO-d6) d 10.09 (s, 1H), 7.66–7.64
(d, J = 8.85 Hz, 2H), 7.25–7.23 (d, J = 8.2 Hz, 2H), 3.36–3.33
(t, J = 6.6, 2H), 2.38–2.35 (m, 2H), 1.83–1.78 (m, 2H). 13C-NMR
(125 MHz, DMSO-d6) d 170.49, 143.34, 138.37, 121.47, 120.30,
119.09, 50.24, 33.16, 24.15.
[M + H]+.
(C10-2C). 3-(2-Azido-acetylamino)-benzoic acid ethyl ester.
1H-NMR (500 MHz, DMSO-d6) d 10.36 (s, 1H), 8.24 (s, 1H), 7.85
(d, J = 8.2 Hz, 1H), 4.32 (m, J = 7 Hz, 2H), 4.07 (s, 2H), 1.32 (t,
J = 7 Hz, 3H). 13C-NMR (125 MHz, DMSO-d6) d 166.6, 165.4,
138.7, 130.4, 129.3, 124.2, 123.6, 119.6, 60.8, 51.2, 14.1.
(B11-4C). 4-Azido-N-(4-phenoxy-phenyl)-butyramide. 1H-
NMR (500 MHz, DMSO-d6) d 9.92 (s, 1H), 7.57–7.56 (m,
2H), 7.32–7.29 (m, 2H), 7.05–7.02 (m, 1H), 6.94–6.90 (m, 4H),
3.36–3.33 (t, J = 6.92 Hz, 2H), 2.37–2.34 (t, J = 7.25 Hz, 2H),
1.83–1.78 (m, 2H). 13C-NMR (125 MHz, DMSO-d6) d 170.04,
157.38, 151.47, 135.13, 129.85, 122.83, 120.70, 119.37, 117.72,
50.26, 33.10, 24.26.
(C11-2C). 2-Azido-N-(4-heptyl-phenyl)-acetamide. 1H-NMR
(500 MHz, DMSO-d6) d 10.03 (s, 1H), 7.46 (d, J = 8.8 Hz, 2H),
7.12 (d, J = 8.8 Hz, 2H), 4.01 (s, 2H), 2.51 (t, 2H), 1.52 (m, 2H),
1.24 (m, 8H), 0.85 (t, J = 7 Hz, 3H). 13C-NMR (125 MHz, DMSO-
d6) d 165.9, 137.6, 136.0, 128.4, 119.2, 51.2, 34.4, 31.1, 30.9, 28.5,
28.4, 22.0, 13.8. ESI-MS(TOF): m/z 275.179 [M + H]+.
(B12-4C). 4-Azido-N-(4-methoxy-phenyl)-butyramide. 1H-
NMR (500 MHz, DMSO-d6) d 9.87 (s, 1H), 3.67 (s, 3H),
3.36–3.329 (m, 2H), 2.36–2.33 (t, J = 7.25 Hz, 2H), 1.82–1.77 (m,
2H). 13C-NMR (125 MHz, DMSO-d6) d 170.31, 159.44, 140.34,
129.36, 111.32, 108.42, 104.86, 54.86, 50.25, 33.23, 24.18.
(D1-2C). N-Anthracen-1-yl-2-azido-acetamide.
1H-NMR
(500 MHz, DMSO-d6) d 10.40 (s, 1H), 8.48 (d, J = 13.8 Hz, 3H),
8.06 (t, J = 9.4 Hz, 3H), 7.56 (d, J = 9.4 Hz, 1H), 7.47 (m, 2H),
4.14 (s, 2H). 13C-NMR (125 MHz, DMSO-d6) d 167.1, 135.8,
132.2, 131.5, 129.3, 128.5, 128.1, 126.2, 125.6, 125.5, 121.2, 116.7,
114.5, 51.9. ESI-MS(TOF): m/z 277.099 [M + H]+.
(C6-4C). 4-Azido-N-(4-tert-butyl-phenyl)-butyramide. 1H-
NMR (500 MHz, DMSO-d6) d 9.80 (s, 1H), 7.45–7.44 (d, J =
8.8 Hz, 2H), 7.25–7.24 (d, J = 8.2 Hz, 2H), 3.35–3.32 (t, J =
6.62 Hz, 2H), 2.35–2.32 (t, J = 7.57 Hz, 2H), 1.82–1.76 (m, 2H),
1.2 (s, 9H). 13C-NMR (125 MHz, DMSO-d6) d 170.00, 145.26,
136.58, 125.14, 118.84, 50.27, 33.89, 33.10, 31.13, 24.28.
(A1-4C). 4-Azido-N-(4-fluoro-phenyl)-butyramide. 1H-NMR
(300 MHz, DMSO-d6) d 9.94 (s, 1H), 7.57–7.54 (m, 2H), 7.09–7.06
(m, 2H), 3.35–3.33 (t, J = 6.62 Hz, 2H), 2.36–2.33 (m, 2H), 1.81–
1.78 (m, 2H). 13C-NMR (75 MHz, DMSO-d6) d 170.12, 158.75,
156.84, 135.56, 120.76(d), 115.20(d), 50.24, 33.07, 24.19.
(C7-4C). 4-Azido-N-(4-ethyl-phenyl)-butyramide. 1H-NMR
(300 MHz, CDCl3) d 7.36–7.33 (d, J = 8.7 Hz, 2H), 6.85–6.82
(d, J = 8.7 Hz, 2H), 4.03–3.96 (m, 2H), 3.42–3.38 (t, J = 6.27 Hz,
2H), 2.47–2.43 (t, J = 7.14 Hz, 2H), 2.01–1.97 (t, J = 6.79 Hz,
2H), 1.41–1.37 (t, J = 6.79 Hz, 3H). 13C-NMR (75 MHz, CDCl3)
d 170.83, 142, 130, 122.30, 114.76, 63.68, 50.64, 33.91.
(A6-4C). 4-Azido-N-(3-fluoro-4-methyl-phenyl)-butyramide.
1H-NMR (300 MHz, DMSO-d6) d 10.03 (s, 1H), 7.55–7.52
(m, 1H), 7.18–7.17 (m, 2H), 3.40–3.37 (t, J = 6.625 Hz,
2H), 2.51–2.50 (m, 2H), 2.16 (s, 3H), 1.86–1.81 (q, 2H). 13C-
NMR (75 MHz, DMSO-d6) d 170.37, 161.12, 159.21, 138.54(d),
131.25(d), 118.18(d), 114.53, 105.80, 5.21, 33.13, 24.52, 13.54.
(A12-5C). 5-Azido-pentanoic acid (2-ethoxy-phenyl)-amide.
1H-NMR (500 MHz, DMSO-d6) d 8.87 (s, 1H), 7.85 (m, 1H),
7.00–6.97 (m, 2H), 6.86–6.83 (m, 1H), 4.06–3.99 (m, 2H), 3.34–
3.32 (m, 2H), 2.52–2.37 (m, 2H), 1.58 (m, 4H), 1.32–1.21 (m,
3H). 13C-NMR (125 MHz, DMSO-d6) d 170.87, 148.98, 127.45,
124.33, 122.37, 120.07, 112.17, 63.82, 50.38, 44.75, 35.40, 27.74,
22.39, 14.52. ESI-MS(TOF): m/z 263.148 [M + H]+.
(A8-4C). 4-Azido-N-(4-chloro-phenyl)-butyramide.
1H-
NMR (500 MHz, DMSO-d6) d 10.02 (s, 1H), 7.58–7.56 (d,
J = 8.85 Hz, 2H), 7.30–7.28 (d, J = 8.8 Hz, 2H), 3.35–3.33 (t,
J = 6.95 Hz, 2H), 2.37 (m, 2H), 1.82–1.77 (m, 2H). 13C-NMR
(125 MHz, DMSO-d6) d 170.40, 138.09, 128.48, 126.49, 120.52,
50.22, 33.16, 24.12.
(A11-4C). 4-Azido-N-(4-chloro-2-methoxy-phenyl)-butyra-
mide. 1H-NMR (500 MHz, DMSO-d6) d 9.29 (s, 1H), 8.10 (s, 1H),
7.10–7.08 (m, 1H), 7.05–7.03 (m, 1H), 3.83 (s, 3H), 3.39–3.36 (t,
J = 6.9 Hz, 2H), 2.54–2.49 (m, 2H), 1.86–1.80 (m, 2H). 13C-NMR
(125 MHz, DMSO-d6) d 170.94, 148.05, 128.58, 123.74, 123.29,
120.84, 112.85, 112.35, 55.96, 50.21, 32.96, 24.26.
(B3-5C). 5-Azido-pentanoic acid (4-ethoxy-phenyl)-amide.
1H-NMR (300 MHz, DMSO-d6) d 9.68 (s, 1H), 7.44–7.41 (d,
J = 15 Hz, 2H), 6.81–6.78 (d, J = 15.05 Hz, 2H), 3.95–3.88 (m,
2H), 3.33–3.28 (d, J = 10.67 Hz, 2H), 2.27–2.23 (t, J = 11.5 Hz,
2H), 1.58–1.52 (m, 4H), 1.27–1.20 (m, 3H). 13C-NMR (75 MHz,
DMSO-d6) d 170.27, 154.24, 132.33, 120.55, 114.26, 63.01, 50.38,
35.55, 27.84, 22.43, 14.64. ESI-MS(TOF): m/z 263.148 [M + H]+.
(B4-4C). 4-Azido-N-(2-methoxy-phenyl)-butyramide.
1H-
NMR (500 MHz, DMSO-d6) d 9.06 (s, 1H), 7.88–7.86 (m, 1H),
7.03–6.97 (m, 2H), 6.86–6.83 (t, 1H), 3.8 (s, 3H), 3.35–3.32 (t, J =
6.92 Hz, 2H), 2.46–2.41 (m, 2H), 1.80–1.77 (m, 2H). 13C-NMR
(125 MHz, DMSO-d6) d 170.41, 149.64, 127.23, 124.26, 122.13,
120.09, 111.06, 55.55, 50.25, 32.94, 24.38.
(B4-5C). 5-Azido-pentanoic acid (2-methoxy-phenyl)-amide.
1H-NMR (300 MHz, DMSO-d6) d 8.99 (s, 1H), 7.89–7.87 (d,
J = 12.85, 1H), 7.04–6.96 (m, 2H), 6.87–6.81 (m, 1H), 3.77 (s,
3H), 3.33–3.29 (t, J = 10.55 Hz, 2H), 2.39–2.35 (t, J = 11.1 Hz,
2H), 1.61–1.51 (m, 4H). 13C-NMR (75 MHz, DMSO-d6) d 170.99,
149.60, 127.30, 124.20, 122.06, 120.10, 111.04, 55.55, 50.35, 35.35,
27.77, 22.37. ESI-MS(TOF): m/z 249.133 [M + H]+.
(B7-4C). 4-Azido-N-(4-methylsulfanyl-phenyl)-butyramide.
1H-NMR (500 MHz, DMSO-d6) d 9.89 (s, 1H), 7.51–7.50 (m,
2H), 7.17–1.16 (m, 2H), 3.35–3.32 (t, J = 6.95 Hz, 3H), 2.38 (m,
3H), 2.36–2.33 (t, 2H), 1.82–1.77 (q, 2H). 13C-NMR (125 MHz,
DMSO-d6) d 170.14, 136.73, 131.42, 127.13, 119.71, 50.26, 33.14,
24.20, 15.57.
(B6-5C). 5-Azido-pentanoic acid (3-isopropoxy-phenyl)-
1
amide. H-NMR (500 MHz, DMSO-d6) d 9.78 (s, 1H), 7.24 (s,
1H), 7.13–7.08 (m, 2H), 6.54–6.51 (m, 1H), 4.51–4.43 (m, 1H), 3.3
(t, 2H), 2.30–2.26 (t, J = 11.65 Hz, 2H), 1.63–1.50 (m, 4H), 1.21
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(s, 3H), 1.23 (s, 3H). 13C-NMR (125 MHz, DMSO-d6) d 170.85,
157.64, 140.42, 129.34, 111.11, 110.12, 106.52, 69.06, 50.38, 35.74,
27.81, 22.23, 21.79. ESI-MS(TOF): m/z 277.163 [M + H]+.
2H), 3.29 (t, J = 6.74 Hz, 2H), 2.34 (t, J = 7.32 Hz, 2H), 1.52
(m, 4H), 1.32 (m, 2H); 13C-NMR (75 MHz, DMSO-d6) d 171.46,
149.31, 127.93, 124.64, 122.67, 120.47, 112.59, 64.22, 50.93, 36.31,
28.42, 26.12, 25.12, 14.93. ESI-MS(TOF): m/z 277.157 [M + H]+.
(B10-5C). 5-Azido-pentanoic acid (3,4-dimethoxy-phenyl)-
1
(B3-6C). 6-Azido-hexanoic acid (4-ethoxy-phenyl)-amide.
1H- NMR (300 MHz, DMSO-d6) d 9.64 (s, 1H), 7.42 (d, J =
9.03 Hz, 2H), 6.79 (d, J = 8.88 Hz, 2H), 3.92 (q, J = 7.08 Hz,
2H,), 3.28 (t, J = 6.82 Hz, 2H), 2.22 (t, J = 7.39 Hz, 2H), 1.52
(m, 4H), 1.26 (m, 2H); 13C-NMR (75 MHz, DMSO-d6) d 170.86,
154.60, 132.79, 120.92, 114.66, 63.41, 50.90, 36.45, 28.41, 26.20,
25.07, 15.04. ESI-MS(TOF): m/z [M + H]+ = 277.156.
amide. H-NMR (300 MHz, DMSO-d6) d 9.7 (s, 1H), 7.06–7.03
(d, J = 8.55 Hz, 1H), 6.83–6.80 (d, J = 8.52 Hz, 1H), 3.7 (s,
6H), 3.33–3.29 (t, J = 6.24 Hz, 2H), 2.28–2.24 (t, J = 6.73 Hz,
2H), 1.60–1.50 (m, 4H). 13C-NMR (75 MHz, DMSO-d6) d 170.39,
148.50, 144.63, 132.96, 112.08, 110.94, 104.33, 55.71, 55.30, 50.40,
38.63, 35.65, 27.86, 22.32. ESI-MS(TOF): m/z 279.143 [M + H]+.
(C2-5C). 5-Azido-pentanoic
acid
(5-chloro-2-phenoxy-
1
(B4-6C). 6-Azido-hexanoic acid (2-methoxy-phenyl)-amide.
1H-NMR (300 MHz, DMSO-d6) d 8.97 (s, 1H), 7.88 (d, J =
7.71 Hz, 1H), 6.99 (m, 2H), 6.84 (m, 1H), 3.78 (s, 3H), 3.28 (t,
J= 6.82 Hz, 2H), 2.34 (t, J = 7.23 Hz, 2H), 1.55 (m, 4H), 1.34
(m, 2H); 13C-NMR (75 MHz, DMSO-d6) d 171.59, 149.96, 127.77,
124.53, 122.40, 120.51, 111.44, 55.97, 50.94, 36.24, 28.41, 26.16,
25.12. ESI-MS(TOF): m/z [M + H]+ = 263.142.
phenyl)-amide. H-NMR (300 MHz, DMSO-d6) d 9.56 (s, 1H),
8.10 (s, 1H), 7.37–7.32 (m, 2H), 7.10–7.06 (m, 2H), 6.97–6.94 (d,
J = 13.95 Hz, 2H), 6.87–6.84 (d, J = 14.5 Hz, 1H), 3.24 (t, 2H),
2.36–2.31 (t, J = 11.5 Hz, 2H), 1.52–1.41 (m, 4H). 13C-NMR
(75 MHz, DMSO-d6) d 171.67, 156.34, 145.81, 131.15, 129.89,
127.13, 124.11, 123.59, 122.71, 120.09, 118.30. ESI-MS(TOF):
m/z 345.110 [M + H]+.
(C5-5C). 5-Azido-pentanoic acid (3-ethyl-phenyl)-amide. 1H-
NMR (500 MHz, DMSO-d6) d 9.77 (s, 1H), 7.40–7.31 (m, 2H),
7.16–7.07 (m, 1H), 6.83–6.77 (m, 1H), 3.33–3.27 (m, 2H), 2.52
(m, 2H), 2.30–2.22 (m, 2H), 1.55 (m, 4H), 1.14–1.05 (m, 3H).13C-
NMR (125 MHz, DMSO-d6) d 170.77, 144.12, 139.24, 128.47,
122.44, 118.39, 116.49, 50.39, 35.70, 28.23, 27.83, 22.29, 15.46.
ESI-MS(TOF): m/z 247.153 [M + H]+.
(B5-6C). 6-Azido-hexanoic acid (3,4,5-trimethoxy-phenyl)-
1
amide. H-NMR (300 MHz, DMSO-d6) d 9.74 (s, 1H), 6.94 (s,
2H), 3.68 (s, 6H), 3.58 (s, 3H), 3.29 (t, J = 6.73 Hz, 2H), 2.23 (t,
J = 7.23 Hz, 2H), 1.53 (m, 4H), 1.31 (m, 2H); 13C-NMR (75 MHz,
DMSO-d6) d 171.29, 153.04, 135.86, 133.56, 97.13, 60.45, 56.02,
50.90, 36.65, 28.43, 26.19, 24.98. ESI-MS(TOF): m/z 323.160
[M + H]+.
(B10-6C). 6-Azido-hexanoic acid (3,4-dimethoxy-phenyl)-
amide. H-NMR (300 MHz, DMSO-d6) d 9.67 (s, 1H), 7.26 (s,
(C9-5C). 4-(5-Azido-pentanoylamino)-benzoic acid ethyl es-
ter. H-NMR (500 MHz, DMSO-d6) d 10.19 (s, 1H), 7.87–7.84
1
1
1H), 7.03 (d, J = 8.55 Hz, 1H), 6.82 (d, J = 8.70 Hz, 1H), 3.68
(s, 6H), 3.29 (t, J = 6.73 Hz, 2H), 2.23 (t, J = 7.30 Hz, 2H), 1.53
(m, 4H), 1.31 (m, 2H); 13C-NMR (75 MHz, DMSO-d6) d 170.96,
148.88, 144.97, 133.40, 112.49, 111.28, 104.69, 56.12, 55.69, 50.90,
36.54, 28.42, 26.20, 25.03. ESI-MS: m/z 293.153 [M + H]+.
(d, J = 12.35 Hz, 2H), 7.70–7.67 (d, J = 13.6 Hz, 2H), 4.26–
4.22 (m, 2H), 3.33–3.29 (t, J = 10 Hz, 2H), 2.36–2.32 (t, J =
11.1 Hz, 2H), 1.61–1.51 (m, 4H), 1.28–1.22 (m, 3H). 13C-NMR
(125 MHz, DMSO-d6) d 171.48, 165.29, 143.56, 130.14, 123.95,
118.31, 60.32, 50.38, 35.78, 27.79, 22.10, 14.15. ESI-MS(TOF):
m/z 291.144 [M + H]+.
(B11-6C). 6-Azido-hexanoic acid (4-phenoxy-phenyl)-amide.
1H-NMR (300 MHz, DMSO-d6) d 9.84 (s, 1H), 7.55 (d, J =
8.88 Hz, 2H), 7.31 (t, J = 7.56 Hz, 2H), 7.05 (t, J = 7.39 Hz,
1H), 6.92 (m, 4H), 3.28 (t, J = 6.80 Hz, 2H), 2.26 (t, J = 7.32 Hz,
2H), 1.54 (m, 4H), 1.34 (m, 2H); 13C-NMR (75 MHz, DMSO-d6)
d 171.22, 157.82, 151.79, 135.66, 130.28, 123.23, 121.04, 119.81,
118.11, 50.90, 36.51, 28.41, 26.18, 25.03. ESI-MS(TOF): m/z
325.159 [M + H]+.
(A1-6C). 6-Azido-hexanoic acid (4-fluoro-phenyl)-amide. 1H-
NMR (300 MHz, DMSO-d6) d 9.87 (s, 1H), 7.55 (m, 2H), 7.07
(m, 2H), 3.28 (t, J = 6.74 Hz, 2H), 2.25 (t, J = 7.23 Hz, 2H), 1.51
(m, 4H), 1.31 (m, 2H); 13C-NMR (75 MHz, DMSO-d6) d 171.31,
159.74, 136.06, 121.13, 115.37, 50.89, 36.49, 28.40, 26.18, 24.97.
ESI-MS(TOF): m/z 251.124 [M + H]+.
(A8-6C). 6-Azido-hexanoic acid (4-chloro-phenyl)-amide. 1H-
NMR (300 MHz, DMSO-d6) d 9.95 (s, 1H), 7.56 (d, J = 8.85 Hz,
2H), 7.30 (d, J = 8.88 Hz, 2H), 3.28 (t, J = 6.82 Hz, 2H), 2.27 (t,
J = 7.30 Hz, 2H), 1.51 (m, 4H), 1.33 (m, 2H); 13C-NMR (75 MHz,
DMSO-d6) d 171.59, 138.62, 128.89, 126.81, 120.89, 50.88, 36.57,
28.39, 26.15, 24.90. ESI-MS(TOF): m/z 267.091 [M + H]+.
Click chemistry
Alkyne 1 and a total of 325 azides (see ESI for complete details)
were subject to Cu(I) catalyzed 1,3-dipolar cycloaddition which
resulted in a 325 member library. The library was assembled in a
384 well plate. The conditions used for click chemistry were first
optimized (ESI).
Alkyne solution (25 mM, 10 mL) was dispensed into each well of
the 384-well plate using a Multi-drop Combi dispenser (Thermo
Scientific) followed by the transfer of azide solutions (50 mM,
7 mL) from the 96-well storage plate to the first quadrant of the
384-well reaction plate using a Sciclone ALH 3000 Liquid Handler
Workstation (Caliper Life Sciences). Similarly other azides were
transferred from the 96-well storage plates to the rest of the three
quadrants of the 384-reation plate. t-BuOH (43 mL) and catalyst
mix (40 mL; containing a mixture of CuSO4 solution (25 mM, 2 mL)
(A11-6C). 6-Azido-hexanoic acid (4-chloro-2-methoxy-
1
phenyl)-amide. H-NMR (300 MHz, DMSO-d6) d 9.15 (s, 1H),
8.06 (s, 1H), 7.10–6.95 (m, 2H), 3.79 (s, 3H), 3.28 (t, J = 6.80 Hz,
2H), 2.37 (t, J = 7.32 Hz, 2H), 1.52 (m, 4H), 1.31 (m, 2H);
13C-NMR (75 MHz, DMSO-d6) d 172.10, 148.40, 129.09, 124.14,
123.54, 121.11, 112.76, 56.38, 50.91, 36.19, 28.39, 26.11, 24.99.
ESI-MS(TOF): m/z 297.104 [M + H]+.
(A12-6C). 6-Azido-hexanoic acid (2-ethoxy-phenyl)-amide.
1H-NMR (300 MHz, DMSO-d6) d 8.81 (s, 1H), 7.85 (d, 1H, J =
7.74 Hz), 7.03–6.95 (m, 2H), 6.86–6.79 (m, 1H), 4.03 (q, J = 6.9 Hz,
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and Sodium ascorbate (50 mM, 5 mL) in water (33 mL)) were added
to each well in the reaction plate using the bulk liquid dispenser.
The 384-well plate was carefully sealed using a silicone based cap-
mat and shaken for 2 days. The solvent was dried in vacuo and
the resulting residue was redissolved in DMSO (100 mL/well).
The ‘click’ products were analyzed by LC-MS to determine the
quality and identity (see Part 3 of the ESI for complete data).
The concentration of the product in each well was estimated to
be 2.5 mM based on the assumption that all alkynes were reacted
quantitatively. The compounds were stored in -20 ◦C. Selected
products were further scaled-up, purified (by RP-HPLC) and fully
characterized (by NMR). Subsequent biological screenings were
carried out first with the direct “click” product (for preliminary
screening), then with the HPLC-purified products (for detailed
kinetic studies).
6H), 5.33 (s, 2H), 5.47 (s, 2H), 6.89–6.91 (d, J= 8.70 Hz, 1H),
7.02–7.05 (m, 3H), 7.29 (s, 1H), 7.45–7.50 (dd, J1 = 2.31 Hz, J2 =
11.34 Hz, 1H), 7.94–7.97 (m, 1H), 8.32 (s, 1H), 10.35 (s, 1H);
ESI-MS (IT-TOF) m/z = [M + H]+ 498.1454.
(B11-2C-W2). 5-(4-Fluoro-2-((1-(2-oxo-2-(4-phenoxyphenyl-
amino)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)isoxazole-
3-carboxylic acid. 1H NMR (300 MHz, DMSO-d6) d 5.36 (s, 2H),
5.47 (s, 2H), 6.95–7.13 (m, 7H), 7.33–39 (m, 2H), 7.45–7.50 (m,
3H), 7.54–7.58 (m, 2H), 7.97 (m, 1H), 8.33 (s, 1H), 10.51 (s, 1H);
ESI-MS (IT-TOF) m/z 530.1579 [M + H]+.
(C8-2C-W2). 5-(2-((1-(2-(4-Cyclohexylphenylamino)-2-oxo-
ethyl)-1H-1,2,3-triazol-4-yl)methoxy)-4-fluorophenyl)isoxazole-
1
3-carboxylic acid. H-NMR (300 MHz, DMSO-d6) d 0.82–0.87
(m, 4H), 1.22–1.32 (m, 5H), 1.48–1.58 (m, 2H), 5.34 (s, 2H), 5.47
(s, 2H), 6.98–7.01 (m, 1H), 7.05 (s, 1H), 7.14 (d, J = 8.37 Hz, 2H),
7.45–7.50 (m, 3H), 7.94–7.97 (m, 1H), 8.32 (s, 1H), 10.39 (s, 1H);
ESI-MS (IT-TOF) m/z 508.2006 [M + H]+.
Characterizations of selected “click” inhibitors
Detailed synthesis and characterizations of all compounds and
intermediates are reported in the ESI. Shown below are charac-
terizations of selected inhibitors.
Biological screenings
The PTP1B activity was determined by measuring the rate of
hydrolysis of a commercially available, fluorogenic substrate, 6,8-
difluoro-4-methylumbelliferyl phosphate (DIFMUP) (Invitrogen,
USA).
(A5-2C-W2). 5-(4-Fluoro-2-((1-(2-(3-fluorobenzylamino)-2-
oxoethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)isoxazole-3-
carboxylic acid. 1H-NMR (300 MHz, DMSO-d6) d 4.34 (m, 2H),
5.23 (s, 2H), 5.45 (s, 2H), 6.97–7.01 (m, 1H), 7.03 (s, 1H), 7.08–7.13
(m, 2H), 7.33–7.41 (m, 1H), 7.44–7.48 (dd, J1 = 11.34 Hz, J2 =
2.31 Hz, 1H), 7.93–7.99 (m, 1H), 8.28 (s, 1H), 8.88 (t, J = 5.76 Hz,
1H); ESI-MS (IT-TOF) m/z 470.1288 [M + H]+.
The inhibition of phosphatases were screened using black
polypropylene flat-bottom 384-well microtiter plates (Greiner,
Germany) in a total reaction volume of 25 mL/well, monitored
with a Tecan F200 fluorescence plate reader (Tecan, Germany),
using the exciting and emission filters recommended by the
vendor. In the preliminary screening with unpurified “click”
products, A single-concentration assay format was adopted, in
which a uniform concentration of inhibitors (ranging between
25–3 mM) was used, for quick identification of potential inhibitors
as previously described.4g Controls were done with 1,3-dipolar
ligation reactions carried out without either the alkyne or the
azide component; they showed no inhibition against PTP1B (up to
260 mM inhibitor concentration). From the preliminary screening
results, eight representative “click” products were identified, and
accordingly scaled-up and further purified (by RP-HPLC) before
being investigated by the measurement of their IC50 values against
PTP1B. The IC50 values were obtained using dose-dependent
reactions by varying the concentrations of the inhibitor, under
the same enzyme and substrate concentration. Briefly, a two-
fold dilution series of an inhibitor, from approximately 400 mM
to 3.125 mM (final concentrations) was prepared. The reaction
condition is as shown below.
(A6-2C-W2). 5-(4-Fluoro-2-((1-(2-(3-fluoro-4-methylphenyl-
amino)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-iso-
xazole-3-carboxylic acid. 1H-NMR (300 MHz, DMSO-d6) d 2.17
(s, 3H), 5.36 (s, 2H), 5.47 (s, 2H), 6.98–7.01 (m, 1H), 7.03 (s, 1H),
7.17–7.24 (m, 2H), 7.45–7.52 (m, 2H), 7.96 (m, 1H), 8.33 (s, 1H),
∑
10.59 (s, 1H); ESI-MS (IT-TOF) m/z 470.1295 [M + H]+ .
(B7-2C-W2). 5-(4-Fluoro-2-((1-(2-(4-(methylthio)phenyl-
amino)-2-oxoethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-
1
isoxazole-3-carboxylic acid. H-NMR (300 MHz, DMSO-d6) d
2.45 (s, 3H), 5.35 (s, 2H), 5.46 (s, 2H), 6.98–7.01 (m, 1H), 7.03 (s,
1H), 7.24 (d, J = 8.70 Hz, 2H), 7.49–7.55 (m, 3H), 7.93–7.99 (m,
1H), 8.33 (s, 1H), 10.48 (s, 1H); ESI-MS (IT-TOF) m/z 484.1053
∑
[M + H]+ .
(B8-2C-W2). 5-(4-Fluoro-2-((1-(2-oxo-2-(4-(trifluorometho-
xy)phenylamino)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-
1
isoxazole-3-carboxylic acid. H-NMR (300 MHz, MeOH-d4) d
5.39 (s, 2H), 5.44 (s, 2H), 6.88–6.95 (m, 1H), 7.05 (s, 1H), 7.23–
7.25 (m, 2H), 7.28–7.29 (m,1H), 7.66–7.69 (m, 2H), 7.95–8.00 (m,
1H), 8.26 (s, 1H); ESI-MS (IT-TOF) m/z 522.1054 [M + H]+.
PTB 1B (2.4 mg/mL) = 5 mL
DIFMUP (20 mM) = 5 mL
Inhibitor (varied) = 5 mL
(B9-2C-W2). 5-(4-Fluoro-2-((1-(2-(4-methoxyphenylamino)-
Assay Buffer (1X) = 10 mL
2-oxoethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)isoxazole-3-
[Assay buffer = 25 mM HEPES, 150 mM NaCl and 0.1 mg/mL
BSA, pH = 7.5].
1
carboxylic acid. H-NMR (300 MHz, DMSO-d6) d 3.75 (s, 3H),
5.33 (s, 2H), 5.46 (s, 2H), 6.88–6.91 (d, J = 8.88 Hz, 2H), 7.01–7.09
(m, 2H), 7.45–7.49 (m, 3H), 7.93–7.99 (m, 1H), 8.32 (s, 1H), 10.34
(s, 1H); ESI-MS (IT-TOF) m/z 468.1424 [M + H]+.
The enzymatic reactions were allowed to incubate at room
temperature for 30 minutes before being initiated by the addition of
DIFMUP. The enzymatic reactions were immediately monitored
at lex = 355 nm and lem = 460 nm for a period of 15 minutes.
The IC50 was calculated by fitting the fluorescence outputs
obtained using the Graphpad Prism software v.4.03 (GraphPad,
(B10-2C-W2). 5-(2-((1-(2-(3,4-Dimethoxyphenylamino)-2-
oxoethyl)-1H-1,2,3-triazol-4-yl)methoxy)-4-fluorophenyl)isoxa-
zole-3-carboxylic acid. 1H-NMR (300 MHz, DMSO-d6) d 3.71 (s,
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San Diego). Each IC50 plots were generated by 6 to 8 data points.
Results are shown in Table 1 and ESI.
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Acknowledgements
Funding support was provided by the Ministry of Education
(R143-000-280-112) and the Agency for Science, Technology and
Research (A*Star) of Singapore (R154-000-274-305).
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