Development and Characterization of a Fluorescent Probe for GLS1 and the Application for High-Throughput Screening of Allosteric Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.9b01035

Glutaminase (GLS1) is a cancer energy metabolism protein which plays a predominant role in cell growth and proliferation. Because of its major involvement in malignant tumor, small-molecule GLS1 inhibitors are urgently needed to assess its therapeutic potential and for probing their underlying biology function. Recent studies showed that targeting the allosteric binding site represented a promising strategy for identifying potent and selective GLS1 inhibitors. Herein, we present the synthesis of two fluorescent probes targeting the allosteric binding site of GLS1 and their usage as mechanistic tools in multiple applicable assay platform. The fluorescence polarization (FP)-based binding assay enables easy, fast, and reliable screen of allosteric inhibitors from our in-house compound library obtained through click chemistry method. The obtained compound C147 (named as CPU-L1) has been proved to be more potent and with greater solubility than the control compound CB839, which could serve as promising leads for further optimization as novel GLS1 inhibitors.

Full text of DOI:10.1021/acs.jmedchem.9b01035

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Article
Development and Characterization of a Fluorescent Probe for GLS1 and
the Application for High-Throughput Screening of Allosteric Inhibitors
Xi Xu, Zijian Kuang, Jie Han, Ying Meng, Lei Li, Hongyu Luan, Pengfei
Xu, Jubo Wang, Cheng Luo, Hong Ding, Zhiyu Li, and Jinlei Bian
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01035 • Publication Date (Web): 11 Oct 2019
Downloaded from pubs.acs.org on October 12, 2019
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Development and Characterization of a Fluorescent Probe for GLS1
and the Application for High-Throughput Screening of Allosteric
Inhibitors
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Xi Xu, Zijian Kuang, Jie Han, Ying Meng, Lei Li, Hongyu Luan, Pengfei Xu, Jubo Wang,
‡
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,†
,†
Cheng Luo, Hong Ding, Zhiyu Li,* Jinlei Bian*
†
Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing
210009, P.R.China
‡
CAS Key Laboratory of Receptor Research, State Key Laboratory of Drug Research, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai
201203, China
*Corresponding author. Tel.: +86 13951678592 (Z. Li); +86 15151865295 (J. Bian)
E-mail address: zhiyuli@cpu.edu.cn (Z. Li); bianjl@cpu.edu.cn (J. Bian)
ABSTRACT:
Glutaminase (GLS1) is a cancer energy metabolism protein which plays a predominant role in
cell growth and proliferation. Due to its major involvement in malignant tumor, small-molecule
GLS1 inhibitors are urgently needed to assess its therapeutic potential and for probing their
underlying biology function. Recent studies showed that targeting the allosteric binding site
represented a promising strategy for identifying potent and selective GLS1 inhibitors. Herein, we
present the synthesis of two fluorescent probes targeting the allosteric binding site of GLS1 and
their usage as mechanistic tools in multiple applicable assay platform. The fluorescence
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polarization (FP)-based binding assay enables easy, fast and reliable screen of allosteric inhibitors
from our in-house compound library obtained through click chemistry method. The obtained
compound C147 (named as CPU-L1) has been proved to be more potent and with greater
solubility than the control compound CB839, which could serve as promising leads for further
optimization as novel GLS1 inhibitors.
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Keywords: glutaminase, fluorescence polarization, high-throughput screening, click chemistry
INTRODUCTION
Warburg effect, which was first proposed nearly a century ago, is the observation that even
under conditions of sufficient oxygen, cancer cells are likely to convert glucose to lactic acid by
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glycolysis. Cancer cells reprogram extensively novel cellular energy metabolism, including
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glycolysis and glutaminolysis instead of pyruvate oxidation. Glutaminolysis serves as a
significant role in many energy-generating and biosynthesis process for the growth and
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proliferation of cancer cells. Glutamine is hydrolyzed by glutaminase (GLS) into glutamate,
which is further oxidatively deaminated into α-ketoglutarate (α-KG) by glutamate dehydrogenase.
Then, α-KG enters the tricarboxylic acid (TCA) cycle and involves in the production of
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nucleotides, ATP, certain amino acids, lipids and glutathione in mitochondria. Glutaminase, as the
first key enzyme in glutamine metabolism, has become a potential therapeutic target for the
development of anti-cancer drug.
In mammalian cells, there are two paralogous GLS genes, GLS1 (or GLS) and GLS2. GLS1
encodes two alternatively spliced isozymes: kidney glutaminase (KGA) and glutaminase C (GAC).
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GLS2 also encodes two isozymes: liver glutaminase (LGA) and glutaminase B. While the
relationship of GLS2 and cancer is not certain, it has been intensively studied that GLS1 is linked
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to the progression of many malignant tumors. More importantly, the expression of GLS1
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appears to be highly upregulated and activated in some cancers that need much energy supply and
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nitrogen-rich substances. In addition, some growing cancer cells exhibited “glutamine addiction”,
in which the proliferation of cancer cells are highly dependent on glutamine, with rapid dying after
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glutamine depletion. Therefore, the development of inhibitors targeting GLS1 represents a novel
and promising orientation for glutamine-addicted cancer therapeutics.
Currently, GLS1 inhibitors could be divided into two categories, including active site inhibitors
and allosteric site inhibitors. The active site inhibitors 6-diazo-5-oxo-L-norleucine (DON),
azaserine, and acivicin (Figure 1A), which showed similar chemical structures with glutamine,
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exhibited robust anti-cancer effects in vitro and in vivo by inhibiting glutaminase.
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these glutamine mimetic have serious toxicity and side effects due to the low selectivity.
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Therefore, the clinical development of active site GLS1 inhibitors are limited.
Figure 1. Representative chemical structures of GLS1 inhibitors. (A) active site GLS1 inhibitors; (B)
allosteric site GLS1 inhibitors.
Bis-2-(5-phenylacetamido-1,2,4-thiadiazol)ethyl sulfide (BPTES), the first reported allosteric
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inhibitors, showed potent and specific GLS1 inhibitory activity. Based on the scaffold of BPTES,
a great many of derivatives of it were designed and synthesized for the purposed of improving the
drug-like properties and GLS1 inhibitory activity. Several representative compounds are listed in
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Figure 1B.
There is only one GLS1 allosteric inhibitor (CB839) in clinical trials currently.
Therefore, it is urgent to discover some potent GLS1 allosteric inhibitors with novel structures to
provide more drug options for clinical researches.
It is significant to develop efficacious assay technology platforms for discovering GLS1
inhibitors due to the potential mechanism of compound action and compound-mediated assay
interference. The most common method employed to evaluate the GLS1 inhibitors was the
enzymatic-based assay, which was hydrolysis of glutamine to glutamate under the catalysis of
GLS1. The available enzymatic-based assay has quite a few deficiencies. Firstly, the activity of
GLS1 can be quantitatively reflected by measuring the absorbance of NADH/NADPH at 340 nm
in glutamine hydrolysis assay. The disadvantage of the method is the relatively short and
unspecific wavelengths at which many small molecule compounds absorb. Secondly, the amount
of GLS1 protein required for glutamine hydrolysis assay is relatively large. Thirdly, the glutamine
hydrolysis assay involves two steps, which are cumbersome and unsuitable for high-throughput
screening (HTS). Moreover, many other reagents are required for glutamine hydrolysis assay,
such as: glutamate dehydrogenase (GDH), NAD/NADH and ADP. These additional reagents could
reduce the stability of the experiment and increase the cost. Thus, an easy, fast and reliable binding
assay for HTS of allosteric GLS1 inhibitors were urgent to be developed, which could be used to
combine with the glutamine hydrolysis assays to ensure the precise assessment of GLS1 inhibitors
and provide useful information for further optimization. Recently, fluorescence-based techniques
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have undergone a huge development for studying biochemical process and employed for drug
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discovery.
In the present study, we report the first small-molecule fluorescent probe based on
affinity, which can be used for the fluorescence polarization (FP) method to quantitatively
ascertain in vitro binding affinity of allosteric inhibitors targeting GLS1. We have optimized the
experimental conditions and demonstrated the feasibility of conducting the method for HTS of
GLS1 inhibitors. Take advantage of this fluorescent probe, we discovered several novel GLS1
inhibitors through screening our in-house compound library which was obtained through click
chemistry method.
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RESULTS AND DISCUSSION
Designation of fluorescent-labeled CB839 probes.
It has robustly been demonstrated that CB839 is a potent, selective and orally bioavailable
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GLS1 inhibitor.
CB839 exhibits extremely strong allosteric binding mechanism and
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slow-on/slow-off kinetic behavior with GLS1. In this work, the cocrystal structure of CB839
bound to the allosteric site of hKGA (PDB ID: 5JYO) was deeply analyzed to guide the design of
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small molecule probes. The structure revealed that the nitrogen atoms in thiadiazole and
pyridazinyl group of CB839 could form hydrogen bonds with the protein backbone amide groups
of the Phe322 and Leu323. The pyridazinyl and acetyl groups made hydrogen bonds with Tyr394,
Lys320 and Asn324. The thiadiazole group was also involved in a water-mediated interaction with
Asp327. However, the trifluoromethoxyphenyl group in the end of CB839 was not engaged in any
interactions with GLS1 and orientated towards the solvent site (Figure 2A and 2B). Thus, the
fragment I was selected as the recognition group (Figure 2C). For the fluorophores, we selected
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the fluorescein isothiocyanate (FITC) and 7-nitro-1,2,3-benzoxadiazole (NBD), which have been
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widely used in many probes.
In addition, different linkers with varying composition and length
were evaluated as survey of the potential chemical space. We conceived that a fluorescent tag, i.e.
FITC and NBD, conjugated via a flexible linker to CB839 will not only afford a fluorescent probe
but also recapitulate the binding performance of the parent ligand CB839 (Figure 2C). In this
paper, we just reported the best two molecules probe 1 and probe 2 among our designed probes.
Data of other molecules are not shown here.
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Figure 2. (A) Cocrystal structure of CB839 (yellow) bound to the allosteric site of GLS1 (PDB: 5JYO).
(B) The 2D diagram of binding mode of CB839 in complex with GLS1 (PDB: 5JYO). (C) The
principle of affinity-based fluorescent probe and the structures of fluorophores used in this paper.
Synthesis of two fluorescent probes.
Scheme 1 illustrates the synthesis of probe 1. 4-Pentynoic acid (3) was reacted with
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thiosemicarbazide in the presence of phosphorus oxychloride to give aminothiadiazole 4.
Intermediate 5 was obtained by coupling 4 with 2-pyridine acetic acid. Under the catalysis of
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(beta-4)-platinum and CuI, 5 was reacted with 6-iodopyridazin-3-amine by a C-C coupling
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reaction to give intermediate 6, which was further hydrogenated to intermediate 7 by Raney-Ni
and hydrogen. 7 was converted to intermediate 8 by treating with 1,5-pentanedioic acid.
Subsequent reaction with N-boc-ethylenediamine followed by hydrolysis gave intermediate 10.
Finally, compound 10 was condensed with FITC in the presence of TEA afforded the desired
probe 1.
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Scheme 1. Synthesis of probe 1
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Reagents and conditions: (a) thiosemicarbazide, POCl , 80 C, 4 h, 80%; (b) 2-pyridine acetic acid
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hydrochloride, HATU, DIPEA, DMF, rt, 2 h, 92%; (c) 6-iodopyridazin-3-amine, (beta-4)-platinum, CuI,
o
TEA, DMA, 60 C, 3.5 h, 68%; (d) Raney-Ni, H , MeOH, rt, 12 h, 97%; (e) 1,5-pentanedioic acid,
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DMTMM, DMF, 35 C, 12 h, 45%; (f) N-boc-ethylenediamine, HATU, DIPEA, DMF, rt, 2 h, 69%; (g)
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trifluoroacetic acid, DCM, rt, 1.5 h, 72%; (h) FITC, TEA, THF, 35 C, 2 h, 31%.
The synthesis of target probe 2 was exhibited in Scheme 2. Starting from commercially
available 4-chloro-7-nitro-1,2,3-benzoxadiazole (11), it was treated with N-boc-ethylenediamine
and trifluoroacetic acid to give intermediate 13. Reaction of 13 with intermediate 8 in the presence
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of HATU and DIPEA afforded the desired probe 2.
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Scheme 2. Synthesis of probe 2
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Reagents and conditions: (a) N-boc-ethylenediamine, TEA, DMF, DCM, rt, 1 h, 76%; (b)
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trifluoroacetic acid, DCM, rt, 10 h, 81%; (c) 8, HATU, DIPEA, DMF, 35 C, 1 h, 52%.
Characterization and Screening of Potential Probes for GLS1.
Absorption and fluorescence spectra of probe 1 and probe 2 were measured to investigate the
spectroscopic properties of the ligand and how these were affected by the environment. Figure S1
showed that different solvents had a certain impact on the spectroscopic properties. Probe 1 had a
maximum UV absorption at 481 nm, an excitation wavelength at 486 nm, an emission wavelength
at 529 nm with a Stokes shift of 43 nm and a considerable high fluorescence quantum yield (Ф) of
88.27% in PBS (Table 1 and Figure S1). The wavelength difference and fluorescence quantum
yield were suitable for further biological evaluation in vitro.
Table 1. Absorption and Fluorescence Properties of probe 1 and 2 in PBS.
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probe
λmax [nm]
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λex [nm]
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λem [nm]
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SS [nm]
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The absorption spectrum of probe 2 was almost similar in four solvents with a maximum UV
absorption at 470 nm (Figure S2). However, the excitation and emission spectrum of probe 2 were
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different in various solvents. The increase in polarity of the environment (from 60% dioxane in
PBS to aqueous buffer) induced an increase in the fluorescence intensity. Probe 2 had an
excitation wavelength at 475 nm, an emission wavelength at 546 nm, with a Stokes shift of 71 nm
and a quantum yield of 9.86% in PBS. Because the fluorescence quantum yield of probe 1 is
significantly higher than that of probe 2, probe 1 was selected to be mainly evaluated in following
experiments.
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Fluorescent Properties of Probe 1 combined with GLS1 and Nonspecific Protein (BSA). For
further determination of the affinity of the probe 1, it was incubated with a series of concentrations
of GLS1 protein. As showed in Figure S3A, the fluorescence intensity was gradually enhanced
with increased GLS1 protein concentrations. When incubated with 1 μM GLS1 protein, the
fluorescence intensity was 2-fold higher than that of the blank group. In order to study the
selectivity of probe 1 for GLS1, bovine-serum albumin (BSA) was employed to evaluate. Though
the probe 1 presented a slightly higher fluorescence response when treated with BSA than blank
group, it was much lower than the probe 1 treating with GLS1 (Figure S3B and Figure S3C). The
results indicated that probe 1 exhibited potent fluorescence selectivity with GLS1 and had very
weak nonspecific binding with BSA.
Fluorescence Polarization (FP) assay.
Then, probe 1 was employed as a fluorescent tracer to develop a competition binding FP assay.
It was well known that the change of mP (ΔmP) reflects the interaction between the
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fluorescent-labeled compound (in the case probe 1) and the protein (GLS1). To develop a
powerful and steady fluorescence polarization assay, the binding affinity of the probe to the
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protein should be high and the binding range from maximum mP at saturation to minimal mP with
no protein should be relatively large.
Figure 3A indicated that probe 1 and probe 2 exhibited obvious concentration-dependent FP
response signal in assay buffer-1 with 10 mM Tris pH = 8.5 and 150 mM NaCl. The ΔmP value of
probe 1 between maximum mP and minimal mP was about 120, which was in a reasonable assay
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window. Compared with probe 1, probe 2 exhibited a lower assay window with ΔmP value being
85. To investigate whether the assay buffer with different pH had an effect on ΔmP value, FP
response signal of probe 1 and probe 2 were examined in assay buffer-2 with 10 mM Tris pH = 7.4
and 150 mM NaCl and assay buffer-3 with 10 mM Tris pH = 6.0 and 150 mM NaCl. Experimental
results (Figure S4) indicated probe 1 and probe 2 showed almost similar FP response signal in
various pH assay buffer, respectively. Combined with the spectroscopic properties, probe 1 was
employed for further HTS of novel GLS1 inhibitors by FP assay.
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Figure 3. (A) Determination of the optimal concentration of free probe 1 and probe 2 for fluorescence
polarization assay by varying the concentration of the free probes without GLS1 protein in assay
buffer-1 with 10 mM Tris pH = 8.5 and 150 mM NaCl. (B) The structure of probe 1. (C) Binding of the
probe 1 (25 nM) to various concentrations of GLS1 protein. These assay buffer constants with 10 mM
Tris pH = 8.5 and 150 mM NaCl. (D) SPR-based binding assay of probe 1 with GLS1. All data are
mean values from three separate experiments.
1
1
1
1
1
1
1
1
1
1
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To avoid the use of high amounts of probe but also to eliminate possible inconsistencies in
concentration measurements, the concentration of probe 1 set as 25 nM appeared optimal for a
suitable and reproducible FP effects. We next performed a saturation binding experiment by
titrating a dilution series of GLS1 protein to probe 1. FP signals were expressed as the change in
millipolarization ΔmP, where ΔmP is the mP of the GLS1 mixture minus the mP of probe 1 only
37
in assay buffer. FP response signal (ΔmP) was gradually increased with the increased GLS1
protein concentration (Figure 3C). Moreover, it has been demonstrated that there was no
discernible binding affinity between FITC and GLS1, thus confirming specificity of the probe 1—
GLS1 interaction. Concentration of GLS1 was plotted versus ΔmP, and the data were fit to the
38
following equation (1) using KaleidaGraph (v4.1.1, Synergy Software) to determine the K of
d
probe 1:
n
n
38
ΔmP = [Pmin + Pmax × (x/K ) ]/[1+(x/K ) ])
(1)
d
d
where Pmin and Pmax are the minimum and maximum observed ΔmP values, respectively, x is the
GLS1 concentration, and n is the Hill coefficient of the binding curve. The K of probe 1 with
d
GLS1 protein was calculated to be 0.45 μM (Figure 3C). Given the prominent potency of probe 1,
surface plasmon resonance (SPR) based binding assay was performed to quantitatively determine
the binding affinity between probe 1 and GLS1. The results demonstrated that the interactions
between probe 1 and GLS1 was dose-dependent and the equilibrium dissociation constant (K ) is
d
0.61 μM (Figure 3D). The SPR measurement was consistent with FP assay. The dimethyl
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sulfoxide (DMSO) tolerance in GLS1 FP assay was also evaluated. Different concentrations of
DMSO was added into assay mixture. As shown in Figure S5, the assay window is reasonably
stable in the presence of DMSO at least up to 8%. In addition, our GLS1 FP assay exhibited
excellent signal-to-noise ratio (S/N) and an acceptable screening window coefficient (denoted
Z’-factor). The Z’ factor represents a characteristic parameter for the quality of the assay itself and
is a measure of the reproducibility in the difference in signal between free and bound tracer
controls across a large number of assay wells. Z’ factor with more than 0.5 is considered to be
0
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acceptable for a good HTS assay. In our GLS1 FP assay, the Z’ factor is 0.96, indicating that the
assay conditions are suitable for HTS (Figure S6).
Figure 4. (A) Competition Binding effect of known allosteric site GLS1 inhibitors (BPTES and
CB839) against probe 1/GLS1 interaction. (B) SPR-based binding assay of BPTES with GLS1. (C)
SPR-based binding assay of CB839 with GLS1.
To evaluate whether the FP assay results correlate with a relevant biological parameter and the
robustness in vitro screening for allosteric GLS1 inhibitors, we examined the binding affinities of
known GLS1 inhibitors BPTES and CB839 through FP assay using probe 1. Figure 4A showed
the results of BPTES and CB839 with IC50 values at 2.9 μM and 0.11 μM, respectively, which
were in good agreement with the values (BPTES K = 4.3 μM and CB839 K = 92 nM)
d
d
determined by SPR (Figure 4B and 4C). These results supported the accuracy of FP assay to
screen small-molecule compounds targeting the allosteric site of GLS1.
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Figure 5. (A) The interactions of BPTES in the allosteric binding site of GLS1 (PDB: 3UO9). (B) The
design of GLS1 inhibitors library by click chemistry.
GLS1 Inhibitors Library Generation by Click Chemistry.
To show the feasibility of the FP assay method to screen quantities of candidate ligands for
binding to GLS1, we performed a screen of our in-house made GLS1 inhibitors library. According
to structure-activity relationship (SAR) study and crystal structures analysis, one of the two phenyl
4
,28
groups of BPTES could be replaced due to the poor interaction with GLS1 (Figure 5A). Thus,
our in-house GLS1 inhibitors library were designed using a biosoteric replacement of the phenyl
by 1,2,3-triazole core to improve the activity and drug-like property of BPTES (Figure 5B). A
200-member derivatives of BPTES were rapidly and efficient synthesized using “click chemistry”
(Figure S7 and S8). This library was used for primary screening by assuming a nearly 100% yield
of the click reaction. These compounds were then evaluated at 10 μM in duplicates in the FP assay
against GLS1. At 10 μM, all pure alkynes, azides and catalyst showed no or low disruption effect
of probe 1—GLS1 binding interaction with a range from 0% to 6%. In the primary screening, 36
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members were considered as “hit” compounds with inhibition of >30% of the fluorescent probe 1
(25 nM) binding to the GLS1. Then, these compounds were selected for further synthesis (Scheme
S1, S2 and S3) and purification.
0
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Figure 6. (A) Comparison of FP and enzyme-activity assay results from triazole-containing compounds
against GLS1. These compounds were fully purified and characterized. And all target compounds had ≥
9
5% purity. (B) Thermal shift profiles of GLS1 inhibitors interacting with GLS1. The melting
temperature shifts compared with apo GLS1 are depicted in the insertion. (C) SPR-based binding assay
of C90 with GLS1. (D) SPR-based binding assay of C147 with GLS1.
After obtaining these compounds, the enzymatic assay was employed to evaluate the GLS1
inhibitory activity and the results were consistent with the FP assay (Figure 6A), which further
confirmed the validity and stability of the FP method using probe 1. In general, compounds
C81-C91 and C147-C153 at 10 µM displayed inhibition >50% of the probe 1 (25 nM) binding to
the GLS1. And these compounds (C81-C91, C147-C153) are more potent than compounds
C61-C78 and BPTES. Next, the half maximal inhibitory concentration (IC ) against GLS1 of
5
0
C81-C91 and C147-C153 were further tested by FP and enzymatic assay (Table 2). Compared
with BPTES, all of these compounds exhibited more potent GLS1 inhibitory activity. Among
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them, compound C147 showed the highest inhibitory potency against GLS1 with an IC value of
5
0
0.041 μM by FP assay and 0.027 μM by enzyme-activity assay, respectively, which were more
potent than that of CB839 with an IC50 value of 0.11 μM by FP assay and 0.038 μM by
enzyme-activity assay, respectively. Representative compounds C90 and C147 were selected to
validate the binding interactions with GLS1 by protein thermal shift (PTS) assay and surface
plasmon resonance (SPR) analysis. BPTES and CB839 were selected as positive references.
Thermal shift assay (Figure 6B) showed that C90 and C147 gave rise to considerable stabilization
1
1
1
1
1
1
1
1
1
1
2
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0
1
2
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4
5
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8
9
0
1
2
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4
5
6
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9
0
1
2
3
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9
0
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5
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0
1
2
3
4
5
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7
8
9
0
o
o
of GLS1 protein with △T value being 7.7 C and 16.5 C, respectively. SPR analysis indicated a
m
dissociation constant (K ) of 0.35 μM and 0.068 μM (Figure 6C and 6D). In addition, the
d
antiproliferative activity in vitro of C81-C91 and C147-C153 were tested on HCT116 and
1
9,26
MDA-MB-436 cells, which were reported to exhibit glutamine-dependent profiles.
Compound
C147 exhibited similar antiproliferative activity against HCT116 and MDA-MB-436 compared
with CB839 (HCT116 IC : 0.15 vs 0.06 μM; MDA-MB-436 IC : 0.18 vs 0.52 μM, respectively).
50
50
Table 2. GLS1 Inhibitory Activities and in vitro Antiproliferative Activities.
Antiproliferative activity
a
GLS1 IC (μM)
5
0
a
IC (μM)
5
0
b
Comp .
Ar
Ph
FP
Enzyme-activity
0.90 ± 0.26
0.84 ± 0.31
2.50 ± 0.31
1.11 ± 0.14
1.25 ± 0.09
0.98 ± 0.10
HCT116
4.78 ± 0.58
5.14 ± 1.93
1.05 ± 0.44
1.09 ± 0.65
3.41 ± 0.28
1.85 ± 0.19
MDA-MB-436
1.16 ± 0.87
2.95 ± 0.57
10.67 ± 0.79
0.35 ± 0.12
1.14 ± 0.49
4.06 ± 0.42
C81
C82
C83
C84
C85
C86
0.92 ± 0.11
0.93 ± 0.09
2.75 ± 0.24
1.22 ± 0.30
1.37 ± 0.16
1.08 ± 0.20
4’-CN-Ph
4’-NO -Ph
2
4’-F-Ph
4’-Cl-Ph
4’-Br-Ph
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C87
C88
4’-CH
3
-Ph
-Ph
0.56 ± 0.08
0.42 ± 0.21
0.51 ± 0.35
1.57 ± 0.73
0.56 ± 0.29
1.79 ± 0.58
5.49 ± 0.71
4’-OCH
0.76 ± 0.10
0.85 ± 0.05
3
3
’,4’-2OCH3-
Ph
C89
C90
C91
0.50 ± 0.29
0.28 ± 0.06
0.52 ± 0.09
0.45 ± 0.31
1.01 ± 0.62
0.74 ± 0.38
0.80 ± 0.28
0.43 ± 0.35
0.76 ± 0.13
0
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2’-CH3-
0.50 ± 0.13
0.78 ± 0.03
4
6
’-CH -Ph
3
2
’-CH3-
’-CH -Ph
3
C147
C148
4’-CH
3
-Ph
-Ph
0.041 ± 0.02
0.19 ± 0.11
0.027 ± 0.01
0.15 ± 0.02
0.15 ± 0.03
0.23 ± 0.10
0.18 ± 0.06
0.63 ± 0.15
4’-OCH
3
3’,4’-2OCH3-
C149
C150
C151
0.40 ± 0.08
0.21 ± 0.13
0.46 ± 0.18
0.33 ± 0.05
0.11 ± 0.10
0.32 ± 0.09
0.38 ± 0.05
0.72 ± 0.17
0.27 ± 0.05
0.40 ± 0.19
0.20 ± 0.09
0.34 ± 0.21
Ph
2
’-CH3-
4’-CH -Ph
3
2
’-CH3-
6
’-CH -Ph
3
C152
C153
4’-OCF
3
-Ph
0.39 ± 0.21
0.073 ± 0.03
2.90 ± 0.11
0.11 ± 0.08
0.30 ± 0.11
0.044 ± 0.02
3.69 ± 0.26
0.25 ± 0.10
0.18 ± 0.05
1.02 ± 0.13
0.06 ± 0.02
0.31 ± 0.19
0.21 ± 0.08
2.40 ± 0.79
0.52 ± 0.20
4’-CF -Ph
3
BPTES
CB839
0.038 ± 0.01
a
b
All data are mean values from triplicate experiments. These compounds were fully purified and
characterized. And all target compounds had ≥ 95% purity.
In order to confirm whether C147 (Figure 7B) could block the hydrolysis of glutamine by
inhibiting GLS1, its inhibition of cellular glutamate production was examined with HCT116 cells.
Experimental results (Figure 7A) indicated that C147 could inhibit the glutamate production in a
dose-dependent manner in HCT116, similar with CB839. Moreover, it is well known that reactive
oxygen species (ROS) is a key regulator of cancer cell growth, proliferation and glutamine
3
,34
metabolism plays a vital role for ROS homeostasis. The intracellular ROS level was monitored
using a ROS-sensitive fluorogenic dye (2’,7’-dichlorodihydrofluorescein diacetate, DCFH-DA) by
2
6
fluorescence microscopy and flow cytometry. As shown in Figure 7C and 7D, C147 could
potently increase the ROS levels in a dose-dependent manner in HCT116, which was similar with
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that of CB839.
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Figure 7. (A) Effect of compound C147 and CB839 on diminishing the cellular glutamate levels in
HCT116 cell. (B) The chemical structure of C147. (C) Fluorescence microscopic images of
intracellular ROS production by DCFH-DA staining (green) in HCT116 cells (D) Quantitatively
analysis of ROS generation by the flow cytometry.
In addition, the preliminary physicochemical properties of the representative compound C147
was also determined. Lipid-water distribution coefficient (LogD) and intrinsic aqueous solubility
(Table S1) of compound C147, BPTES and CB839 were determined by the LC-MS based
32
shake-flask method. Experimental results showed that C147 displayed potent aqueous solubility
for 20.8 µg/mL, nearly 9 times better than CB839 and the LogD of C147 was similar with that of
CB839 (1.29 vs 1.90). These results demonstrated that introduction of triazole could indeed
improve the aqueous solubility. Further optimization and mechanism studies of compound C147
are in progress in our lab.
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CONCLUSIONS
In this study, using the potent small-molecule GLS1 inhibitor CB839 as the structure template,
we have synthesized two small-molecule probes 1 and 2 with FITC and NBD fluorescent group,
respectively. Probe 1 exhibited desirable spectroscopic properties, such as a high quantum yield in
PBS environments, high potency, selectivity and on-target action, which was successfully used to
establish a reliable robust and customizable FP-based in vitro HTS for allosteric inhibitors of
GLS1.
0
1
2
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0
In addition, a compound library of GLS1 inhibitors containing triazole group was rapidly
constructed by click chemistry. The inhibitory activity of these compounds against GLS1 were
cross-screened by FP and enzyme activity assay. It has been demonstrated that the introduction of
a suitable triazole moiety in the appropriate position of BPTES indeed could increase the GLS1
inhibitory activity and improve aqueous solubility. In summary, we propose that our probe and FP
assay reported in this work are versatile and valuable tools for the in vitro evaluation of GLS1
characteristics, which will prove useful for the identification and profiling of new drug candidates
targeting the allosteric binding site. In addition, the GLS1 potent inhibitor C147 which was
discovered through the high throughput screening method could be effective lead compound for
further evaluation.
EXPERIMENTAL SECTION
General Chemistry Methods.
All reagents and solvents were from commercial sources and, unless otherwise noted, were used
without further purification. All reactions were monitored by thin-layer chromatography (TLC) on
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0.25 mm silica gel plates with fluorescent indicator (GF ) and visualized under UV light. With
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1
13
tetramethylsilane (TMS) as internal standard, the H and C NMR spectra were recorded on
o
Bruker AV-300 (300 and 75 MHz) apparatus at 25 C. Samples were prepared as solutions in
1
1
1
1
1
1
1
1
1
1
2
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0
deuterated solvent. EI-MS was collected on shimadzu GCMS-2010 instruments. HR-MS spectral
data was obtained on Agilent technologies 6520 Accurate-Mass Q-TOF LC/MS instruments. All
target compounds were purified via silica gel (60 Å, 70-230 mesh) column chromatography.
Melting points were measured by XT-4 melting point apparatus. Purity of all target compounds
was ≥ 95%, as determined by HPLC (SHIMADZU Labsolutions) analysis on the Aglilent C18
column (4.6 × 150 mm, 5 μm) using gradient elution (Mobile Phase: A Phase = 0.1% formic acid
in H O, B Phase = MeOH) at a flow rate of 0.5 mL/min.
2
Synthetic Procedures.
5-(But-3-yn-1-yl)-1,3,4-thiadiazol-2-amine (4)
To a solution of compound 3 (1.50 g, 0.015 mol) in POCl was added thiosemicarbazide (1.39 g,
3
0.015 mol). The reaction mixture was stirred at 80 °C for 4 h. After cooling to the room
temperature, the mixture was basified with NaOH to pH 9 and extracted with ethyl acetate. The
reaction mixture was partitioned between ethyl acetate (15 mL) and water (20 mL). The water
layer was extracted with ethyl acetate (15 mL × 3). The organic layer was combined, washed with
saturated brine, dried over anhydrous Na SO and concentrated in vacuo to afford 4 as a tawny
2
4
1
solid, yield 88%. H NMR (300 MHz, DMSO-d ): δ 6.99 (s, 2H), 2.95 (t, J = 7.0 Hz, 2H), 2.83 (d,
6
J = 2.4 Hz, 1H), 2.50 (dd, J = 7.1, 4.6 Hz, 2H) ppm. HRMS (ESI): m/z, calcd for C H N S [M +
6
8
3
+
H] , 154.0433; found: 154.0434.
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
3
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3
3
4
4
4
4
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4
4
4
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5
5
5
5
5
5
5
5
5
5
6
N-(5-(but-3-yn-1-yl)-1,3,4-thiadiazol-2-yl)-2-(pyridin-2-yl)acetamide (5)
To a solution of compound 4 (3.00 g, 0.022 mol) in DMF was added 2-pyridine acetic acid
hydrochloride (3.74 g, 0.022 mmol), HATU (8.80 g, 0.039 mmol) and DIPEA (7.50 g, 0.059
mmol). The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was
diluted in 50 mL water and was extracted with ethyl acetate (30 mL × 3). The organic layer was
0
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4
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6
7
8
9
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9
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8
9
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2
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6
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8
9
0
1
dried over sodium sulfate, filtered and concentrated in vacuo to afford 5, yield 92%. H NMR (300
MHz, DMSO-d ): δ 12.71 (s, 1H), 8.50 (d, J = 4.8 Hz, 1H), 7.78 (td, J = 7.7, 1.7 Hz, 1H), 7.40 (d,
6
J = 7.7 Hz, 1H), 7.29 (dd, J = 6.7, 5.0 Hz, 1H), 4.02 (s, 2H), 3.16 (t, J = 7.0 Hz, 2H), 2.88 (t, J =
2.6 Hz, 1H), 2.62 (td, J = 7.0, 2.6 Hz, 2H) ppm. HRMS (ESI): m/z, calcd for C H N OS [M +
13 13 4
+
H] , 273.0805; found: 273.0807.
N-(5-(4-(6-aminopyridazin-3-yl)but-3-yn-1-yl)-1,3,4-thiadiazol-2-yl)-2-(pyridin-2-yl)acetami-
de (6)
To a solution of compound 5 (2.00 g, 7.40 mmol) in DMA was added 6-iodopyridazin-3-amine
(1.35 g, 6.11 mmol), CuI (0.12 g, 0.61 mmol), TEA (3.10 g, 30.65 mmol) and (beta-4)-platinum
(0.71 g, 0.61 mmol). The reaction mixture was stirred at room temperature for 3.5 h under
nitrogen atmosphere. After cooling to the room temperature, the reaction mixture was then diluted
in 30 mL isopropyl ether. After filtration, the reddish brown oily substance was collected, when
add some water, we can get some gray solid. The residue was purified by column chromatography
1
to afford the titled compound as a gray solid, yield 68%. H NMR (300 MHz, DMSO-d ): δ 12.69
6
(s, 1H), 8.49 (d, J = 4.0 Hz, 1H), 7.76 (t, J = 7.1 Hz, 1H), 7.39 (d, J = 7.8 Hz, 1H), 7.33-7.17 (m,
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2H), 6.69 (d, J = 9.1 Hz, 1H), 6.63 (s, 2H), 4.01 (s, 2H), 3.31-3.23 (m, 2H), 2.90 (t, J = 6.8 Hz, 2H)
+
ppm. HRMS (ESI): m/z, calcd for C H N OS [M + H] , 366.1132; found: 366.1135.
17
16
7
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
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3
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3
3
3
3
3
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3
4
4
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4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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2
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4
5
6
7
8
9
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6
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8
9
0
N-(5-(4-(6-aminopyridazin-3-yl)butyl)-1,3,4-thiadiazol-2-yl)-2-(pyridin-2-yl)acetamide (7)
To a solution of compound 6 (1.00 g, 2.74 mmol) in methanol was added Raney Nickel (2 ml).
The reaction mixture was stirred at room temperature for 12 h under hydrogen. The solution was
filtered to remove the catalyst. The filtrate was concentrated to afford 7 as a yellow solid, yield
1
9
7%. H NMR (300 MHz, DMSO-d ): δ 12.67 (s, 1H), 8.50 (d, J = 4.9 Hz, 1H), 7.77 (td, J = 7.7,
6
1.8 Hz, 1H), 7.40 (d, J = 7.8 Hz, 1H), 7.29 (dd, J = 6.9, 5.3 Hz, 1H), 7.19 (d, J = 9.0 Hz, 1H), 6.76
(d, J = 9.1 Hz, 1H), 6.28 (s, 2H), 4.01 (s, 2H), 3.00 (t, J = 6.9 Hz, 2H), 2.70 (t, J = 6.9 Hz, 2H),
+
1.70 (s, 4H) ppm. HRMS (ESI): m/z, calcd for C H N OS [M + H] , 370.1445; found: 370.1451.
1
7
20
7
5-Oxo-5-((6-(4-(5-(2-(pyridin-2-yl)acetamido)-1,3,4-thiadiazol-2-yl)butyl)pyridazin-3-yl)ami-
no)pentanoic acid (8)
To a solution of compound 7 (0.50 g, 1.35 mmol) in DMF was added 1,5-pentanedioic acid (0.27
g, 2.01 mmol) and DMTMM (0.56 g, 2.03 mmol). The reaction mixture was stirred at 45 °C for 12
h. The reaction mixture was partitioned between DCM: MEOH = 10:1 (15 mL) and water (30 mL).
The water layer was extracted with DCM/MEOH = 10:1 (15 mL × 3). The organic layer was
combined, washed with saturated brine (10 mL × 3), dried over sodium sulfate, filtered and
concentrated. The crude product was purified by column chromatography to afford the titled
1
compound as a yellow solid, yield 45%. H NMR (300 MHz, DMSO-d ): δ 10.99 (s, 1H), 8.58 (s,
6
1H), 8.24 (d, J = 8.9 Hz, 1H), 7.92 (d, J = 8.1 Hz, 1H), 7.55 (dd, J = 15.2, 8.5 Hz, 2H), 7.43 (s,
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1H), 4.09 (s, 2H), 3.02 (s, 2H), 2.89 (s, 2H), 2.45 (d, J = 6.4 Hz, 2H), 2.26 (d, J = 7.0 Hz, 2H),
+
1
4
.85-1.70 (m, 6H) ppm. HRMS (ESI): m/z, calcd for C H N O S [M + H] , 484.1761; found:
2
2
26
7
4
84.1761.
0
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9
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Tert-butyl(2-(5-oxo-5-((6-(4-(5-(2-(pyridin-2-yl)acetamido)-1,3,4-thiadiazol-2-yl)butyl)pyrid-
azin-3-yl)amino)pentanamido)ethyl)carbamate (9)
To a solution of compound 8 (0.42 g, 0.86 mmol) in DMF was added N-boc-ethylenediamine
(0.16 g, 1.05 mmol), HATU (0.67 mg, 1.77 mmol) and DIPEA (0.56 g, 4.36 mmol). The reaction
mixture was stirred at room temperature for 2 h. The reaction mixture was partitioned between
DCM/MEOH = 10:1 (15 mL) and water (30 mL). The water layer was extracted with
DCM/MEOH = 10:1 (10 mL × 3). The organic layer was combined, washed with saturated NaCl
solution (10 mL × 3), dried over sodium sulfate, filtered and concentrated. The residue was
purified by column chromatography to afford the titled compound as a faint yellow solid, yield
1
6
9%. H NMR (300 MHz, DMSO-d ): δ 10.99 (s, 1H), 8.64 (d, J = 4.3 Hz, 1H), 8.25 (d, J = 9.1
6
Hz, 1H), 8.04 (d, J = 8.0 Hz, 1H), 7.81 (s, 1H), 7.67-7.49 (m, 3H), 6.76 (s, 1H), 4.15 (s, 2H),
3.10-2.83 (m, 8H), 2.41 (s, 2H), 2.14-2.03 (m, 2H), 1.85-1.68 (m, 6H), 1.35 (s, 9H) ppm. HRMS
+
(
ESI): m/z, calcd for C H N O S [M + H] , 626.2868; found: 626.2868.
29
40
9
5
1
5
N -(2-aminoethyl)-N -(6-(4-(5-(2-(pyridin-2-yl)acetamido)-1,3,4-thiadiazol-2-yl)butyl)pyrida-
zin-3-yl)glutaramide (10)
To a solution of compound 9 (0.22 g, 0.35 mmol) in DCM was added trifluoroacetic acid (5 mL).
The reaction mixture was stirred at room temperature for 1.5 h under nitrogen. The mixture was
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basified with NaHCO to pH 8. After filtration, the solid was purified by column chromatography
3
1
to afford the titled compound as a yellow solid, yield 72%. H NMR (300 MHz, DMSO-d ): δ
6
1
7
2
0.99 (s, 1H), 8.61 (d, J = 5.4 Hz, 1H), 8.23 (d, J = 9.2 Hz, 1H), 8.02 (dd, J = 13.7, 6.6 Hz, 2H),
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
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8
9
0
1
2
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5
6
7
8
9
0
1
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7
8
9
0
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7
8
9
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9
0
.58 (d, J = 9.0 Hz, 2H), 7.53-7.46 (m, 1H), 4.13 (s, 2H), 3.27 (dd, J = 12.0, 6.3 Hz, 2H), 3.01 (s,
H), 2.92-2.80 (m, 4H), 2.44 (t, J = 7.3 Hz, 2H), 2.15 (t, J = 7.6 Hz, 2H), 1.87-1.78 (m, 2H), 1.73
+
(s, 4H), 1.22 (s, 2H) ppm. HRMS (ESI): m/z, calcd for C H N O S [M + H] , 526.2343; found:
2
4
32
9
3
526.2364.
1
N -(2-(3-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]-6-yl)thioureido)ethyl)-
5
N -(6-(4-(5-(2-(pyridin-2-yl)acetamido)-1,3,4-thiadiazol-2-yl)butyl)pyridazin-3-yl)glutarami-
de (probe 1)
To a solution of compound 10 (40 mg, 0.076 mmol) in THF was added FITC (20 mg, 0.051 mmol)
and TEA (20 mg, 0.20 mmol). The reaction mixture was stirred at 35 ℃ for 2 h. The reaction
mixture was concentrated in vacuum. The luminous yellow solid can be obtained by preparing
1
high performance liquid phase, yield 35%. mp 179.2-180.1 °C. H NMR (300 MHz, MeOD-d ): δ
4
8.69 (s, 1H), 8.39 (d, J = 8.9 Hz, 1H), 8.22 (d, J = 7.5 Hz, 1H), 8.13 (s, 1H), 7.80-7.65 (m, 4H),
7.18 (d, J = 7.9 Hz, 1H), 6.74 (d, J = 12.1 Hz, 4H), 6.58 (d, J = 8.8 Hz, 2H), 3.74 (dd, J = 13.1, 6.5
Hz, 6H), 3.05 (s, 2H), 2.95 (s, 2H), 2.53 (s, 2H), 2.31 (d, J = 6.9 Hz, 2H), 2.00 (s, 2H), 1.82 (s, 4H)
1
3
ppm. C NMR (75 MHz, DMSO-d ): δ 181.14, 172.97, 172.64, 168.97, 168.73, 164.46, 159.96,
6
159.88, 158.54, 154.77, 154.55, 152.33, 148.82, 147.14, 142.08, 138.19, 130.10, 129.50, 128.64,
126.78, 125.12, 123.02, 119.15, 113.03, 110.14, 102.68, 83.23, 54.59, 48.87, 44.02, 38.15, 35.80,
34.99, 34.65, 32.39, 29.48, 29.15, 29.03, 28.72, 27.30, 23.23, 21.32, 18.76, 17.15, 14.41 ppm.
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6
+
HRMS (ESI): m/z, calcd for C H N O S [M + H] , 915.2701; found: 915.2662. HPLC: t =
45
43 10
8
2
R
13.038 min, 97.5% purity.
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Tert-butyl (2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethyl)carbamate (12)
To a solution of compound 11 (0.50 g, 2.51 mmol) in DMF was added N-boc-ethylenediamine
(0.40 g, 2.51 mmol), TEA (0.28 g, 2.77 mmol) in DCM. The reaction mixture was stirred at room
temperature for 1 h. The reaction mixture was partitioned between ethyl acetate (10 mL) and water
(
30 mL). The water layer was extracted with ethyl acetate (10 mL × 3). The organic layer was
combined, washed with saturated brine (10 mL × 3), dried over sodium sulfate, filtered and
1
concentrated, yield 76%. H NMR (400 MHz, CDCl ): δ 8.49 (d, J = 8.6 Hz, 1H), 7.69 (s, 1H),
3
6.18 (d, J = 8.6 Hz, 1H), 5.12 (s, 1H), 3.63 (s, 4H), 1.48 (s, 9H) ppm. HRMS (ESI): m/z, calcd for
+
C H N O [M + H] , 324.1302; found: 324.1292.
1
3
18
5
5
1
N -(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)ethane-1,2-diamine (13)
To a solution of compound 12 (0.20 g, 0.62 mmol) in DCM was added 2, 2, 2-trifluoroacetic acid
(5 mL). The reaction mixture was stirred at room temperature for 4 h under nitrogen. The mixture
was basified with NaHCO to pH 7. After filtration, the solid was purified by column
3
1
chromatography to afford the titled compound as a black solid, yield 81%. H NMR (300 MHz,
DMSO-d ): δ 8.58 (d, J = 8.8 Hz, 1H), 8.28 (s, 1H), 6.50 (d, J = 8.9 Hz, 1H), 3.78 (s, 2H), 3.18 (t,
6
-
J = 6.0 Hz, 2H), 1.32 (d, J = 35.7 Hz, 1H) ppm. HRMS (ESI): m/z, calcd for C H N O [M - H] ,
8
8
5
3
222.0633; found: 222.0638.
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1
5
N -(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethyl)-N -(6-(4-(5-(2-(pyridin-2-yl)aceta-
mido)-1,3,4-thiadiazol-2-yl)butyl)pyridazin-3-yl)glutaramide (Probe 2)
To a solution of compound 8 (20 mg, 0.041 mmol) in DMF was added compound 13 (12 mg,
1
1
1
1
1
1
1
1
1
1
2
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3
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4
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4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
6
7
8
9
0
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5
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8
9
0
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4
5
6
7
8
9
0
0
.052 mmol), HATU (31 mg, 0.082 mmol) and DIPEA (31 mg, 0.24 mmol). The reaction mixture
was stirred at 35 ℃ for 1 h. The reaction mixture was added some water, we can obtain some gray
solid. The solid was purified by column chromatography to afford the titled compound as a yellow
1
solid, yield 52%. m.p. 137.9-139.8 °C. H NMR (300 MHz, DMSO-d ): δ 12.71 (s, 1H), 10.99 (s,
6
1
H), 9.47 (s, 1H), 8.53 (d, J = 9.8 Hz, 2H), 8.26-8.04 (m, 2H), 7.81 (d, J = 7.1 Hz, 1H), 7.55 (d, J
=
8.8 Hz, 1H), 7.46-7.26 (m, 2H), 6.44 (d, J = 9.4 Hz, 1H), 4.01 (s, 2H), 3.16 (s, 3H), 3.01 (s, 2H),
2
.88 (s, 2H), 2.73 (s, 1H), 2.40 (s, 2H), 1.99 (d, J = 5.7 Hz, 2H), 1.75 (d, J = 15.5 Hz, 6H) ppm.
1
3
C NMR (101 MHz, DMSO-d ): δ 172.93, 172.87, 168.97, 164.47, 159.93, 158.56, 155.36,
6
1
9
54.52, 149.57, 145.84, 144.93, 144.55, 138.42, 137.23, 128.52, 124.64, 122.67, 121.35, 118.97,
9.63, 44.54, 43.55, 37.67, 35.74, 34.99, 34.70, 29.05, 29.00, 28.73, 21.25 ppm. HRMS (ESI):
+
m/z, calcd for C H N O S [M + H] , 689.2361; found: 689.2371. HPLC: t = 11.910 min, 95.0%
30 33 12
6
R
purity.
Ethyl 5-(5-amino-1,3,4-thiadiazol-2-yl)pentanoate (15)
To a solution of compound 14 (10 g, 57.40 mmol) in POCl (30 mL) was added thiosemicarbazide
3
(
5.23 g, 57.40 mmol). The reaction mixture was stirred at 85 °C for 4 h. After cooling to the room
temperature, the viscous oil was poured onto ∼100 g ice. The filtrate was basified using solid
NaOH until pH 10. The solid that precipitated out of solution was filtered off and washed several
times with H O to get desired compound 15 as a white solid, yield 42%. 15 was ready for the next
2
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5
6
+
step without the further purification. HRMS (ESI): m/z, calcd for C H N O S [M + H] ,
9
16
3
2
230.0958; found: 230.0962.
0
1
2
3
4
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8
9
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Ethyl 5-(5-(2-phenylacetamido)-1,3,4-thiadiazol-2-yl)pentanoate (16)
The compound 15 (2.00 g, 8.73 mmol) was dissolved in THF (20 mL) and followed by addition of
triethylamine (1.3 mL, 9.62 mmol) and dropwise addition of phenylacetyl chloride (1.35 g, 8.73
mmol). The reaction mixture was stirred at room temperature for 24 h. The solvent was removed
under vacuum. The crude product was recrystallized using water to give compound 16 as a white
solid, yield 92%. 16 was ready for the next step without the further purification. HRMS (ESI): m/z,
+
calcd for C H N O S [M + H] , 348.1376; found: 348.1371.
17
22
3
3
5
-(5-(2-Phenylacetamido)-1,3,4-thiadiazol-2-yl)pentanoic acid (17)
The compound 16 (2.00 g) was dissolved in 4 N NaOH (15 mL) and MeOH (10 mL) followed by
stirring for 3 h at room temperature. The solvent was removed under reduced pressure and the
basic solution was acidified with HCl. The precipitate was filtered off to obtain compound 17 as a
white solid, yield 90%. 17 was ready for the next step without the further purification. HRMS
+
(
ESI): m/z, calcd for C H N O S [M + H] , 320.1063; found: 320.1064.
15
18
3
3
N-(5-(4-(5-amino-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazol-2-yl)-2-phenylacetamide (18)
Compound 17 (2.00 g, 6.20 mmol) and thiosemicarbazide (0.62 g, 6.80 mmol) were dissolved in
POCl (10 mL) and heated to 85 °C for 4 h. The reaction was cooled and the black viscous oil was
3
poured onto ice. The solution was basified using NaOH to pH 10. The precipitate was filtered off
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to afford the titled compound 18 as a black solid, yield 43%. 18 was ready for the next step
+
without the further purification. HRMS (ESI): m/z, calcd for C H N OS [M + H] , 375.1056;
1
6
19
6
2
found: 375.1056.
1
1
1
1
1
1
1
1
1
1
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2
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4
4
4
4
4
4
5
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5
5
5
5
5
5
5
5
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0
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-Chloro-N-(5-(4-(5-(2-phenylacetamido)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazol-2-yl)a-
cetamide (19)
To a solution of compound 18 (3.00 g, 8.02 mmol) in DMF was added TEA (2.43 g, 24.06 mmol)
and dropwise addition of chloroacetyl chloride (1.80 g, 16.04 mmol). The reaction mixture was
stirred at room temperature for 12 h. The reaction was cooled and the reaction mixture was poured
onto water. The precipitate was filtered off to obtain white solid, yield 84%. 19 was ready for the
+
next step without the further purification. HRMS (ESI): m/z, calcd for C H ClN O S [M + H] ,
1
8
20
6
2 2
451.0772; found: 451.0785.
2
-Azido-N-(5-(4-(5-(2-phenylacetamido)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazol-2-yl)ac-
etamide (A4)
To a solution of compound 19 (3.50 g, 7.78 mmol) in DMF was added NaN (1.52 g, 23.30 mmol).
3
The reaction mixture was stirred at room temperature for 12 h. The reaction mixture was then
diluted in water ether. After the water layer was extracted with DCM (30 mL × 3). Finally the
organic layer was dried over sodium sulfate, filtered and concentrated. And the crude product was
purified by column chromatography to afford the titled compound as a white solid, yield 90%.
+
HRMS (ESI): m/z, calcd for C H N O S [M + H] , 458.1176; found: 458.1148.
18
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2-(4-(2-Hydroxyethyl)-1H-1,2,3-triazol-1-yl)-N-(5-(4-(5-(2-phenylacetamido)-1,3,4-thiadiazol
-2-yl)butyl)-1,3,4-thiadiazol-2-yl)acetamide (C61)
To a solution of compound A4 (0.20 g, 0.44 mmol) in DMF was added CuI (16.70 mg, 0.088
mmol in water) and 3-butyn-1-ol B1 (77.05 mg, 1.10 mmol). The reaction mixture was stirred in
microwave 300 W at 100 °C for 0.5 h. The reaction mixture was then diluted in water. The
precipitate was filtered off to obtain white solid. The residue was purified by column
chromatography (DCM/MeOH = 20:1) to afford the titled compound C61 as a white solid, yield
0
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2
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0
o
1
4
5
5%. m.p. 208-210 C. H NMR (300 MHz, DMSO-d ): δ 12.66 (s, 2H), 7.90 (s, 1H), 7.31 (s, 5H),
6
.43 (s, 2H), 4.69 (s, 1H), 3.79 (s, 2H), 3.64 (s, 2H), 3.00 (s, 4H), 2.79 (s, 2H), 1.74 (s, 4H) ppm.
13
C NMR (75 MHz, DMSO-d ): δ 169.80, 165.77, 164.73, 164.28, 158.62, 144.89, 135.11, 129.71,
6
128.89, 127.34, 124.56, 60.79, 51.81, 42.01, 31.61, 29.56, 29.47, 29.14, 28.90, 28.84, 28.68, 22.54,
+
4
.68 ppm. HRMS (ESI): m/z, calcd for C H N O S [M + H] , 528.1595; found: 528.1577.
2
2
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HPLC: t = 21.625 min, 97.8% purity.
R
2-(4-(2-Hydroxypropan-2-yl)-1H-1,2,3-triazol-1-yl)-N-(5-(4-(5-(2-phenylacetamido)-1,3,4-thi-
adiazol-2-yl)butyl)-1,3,4-thiadiazol-2-yl)acetamide (C62)
Compound C62 was prepared from A4 as described for the preparation of C61 except
o
1
2
-methyl-3-butyn-2-ol B2 was used in place of B1: white solid (39% yield). m.p. 210-212 C. H
NMR (300 MHz, DMSO-d ): δ 12.93 (s, 1H), 12.67 (s, 1H), 7.92 (s, 1H), 7.32 (s, 5H), 5.44 (s,
6
13
2
H), 5.15 (s, 1H), 3.80 (s, 2H), 3.01 (s, 4H), 1.75 (s, 4H), 1.48 (s, 6H) ppm. C NMR (75 MHz,
DMSO-d ): δ 169.84, 165.68, 164.80, 164.36, 158.72, 156.30, 135.09, 129.72, 128.91, 127.37,
6
1
22.66, 67.54, 51.83, 42.02, 31.16, 28.84, 28.68 ppm. HRMS (ESI): m/z, calcd for C H N O S
23 28 9 3 2
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[M + H] , 542.1751; found: 542.1740. HPLC: t = 22.069 min, 96.8% purity.
R
2
-(4-(2-Hydroxybutan-2-yl)-1H-1,2,3-triazol-1-yl)-N-(5-(4-(5-(2-phenylacetamido)-1,3,4-thia-
1
1
1
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diazol-2-yl)butyl)-1,3,4-thiadiazol-2-yl)acetamide (C63)
Compound C63 was prepared from A4 as described for the preparation of C61 except
o
1
3-methylpent-1-yn-3-ol B3 was used in place of B1: white solid (40% yield). m.p. 220-223 C. H
NMR (300 MHz, DMSO-d ): δ 12.93 (s, 1H), 12.65 (s, 1H), 7.90 (s, 1H), 7.32 (s, 5H), 5.45 (s,
6
1
3
2
H), 5.00 (s, 1H), 3.80 (s, 2H), 3.02 (s, 4H), 1.75 (s, 6H), 1.44 (s, 3H), 0.76 (s, 3H) ppm.
C
NMR (75 MHz, DMSO-d ): δ 169.81, 165.76, 164.27, 158.61, 155.25, 135.12, 129.72, 128.90,
6
1
27.35, 123.49, 70.24, 51.84, 42.02, 35.97, 29.46, 28.86, 28.70, 8.77 ppm. HRMS (ESI): m/z,
+
calcd for C H N O S [M + H] , 556.1908; found: 556.1891. HPLC: t = 22.457 min, 97.9%
24
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2
R
purity.
3
-(1-(2-Oxo-2-((5-(4-(5-(2-phenylacetamido)-1,3,4-thiadiazol-2-yl)butyl)-1,3,4-thiadiazol-2-
yl)amino)ethyl)-1H-1,2,3-triazol-4-yl)propanoic acid (C64)
Compound C64 was prepared from A4 as described for the preparation of C61 except
o
1
4
-pentynoicacid B4 was used in place of B1: white solid (35% yield). m.p. 220-224 C. H NMR
(
300 MHz, DMSO-d ): δ 12.96-12.86 (m, 1H), 12.64 (s, 2H), 7.89 (s, 1H), 7.32 (s, 5H), 5.43 (s,
6
1
3
2
H), 3.79 (s, 2H), 3.00 (s, 6H), 2.58 (d, J = 7.3 Hz, 2H), 1.75 (s, 4H) ppm. C NMR (75 MHz,
DMSO-d ): δ 169.81, 164.18, 160.65, 139.27, 135.11, 131.28, 129.72, 128.89, 127.35, 61.07,
6
52.35, 42.02, 31.67, 28.84, 28.72, 14.63 ppm. HRMS (ESI): m/z, calcd for C H N O S [M +
2
3
26
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4 2
+
H] , 556.1544; found: 556.1521. HPLC: t = 19.619 min, 98.9% purity.
R
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