Discovery of DC_H31 as potential mutant IDH1 inhibitor through NADPH-based high throughput screening

Article abstract of DOI:10.1016/j.bmc.2019.05.040

IDH1 mutations are early events in the development of IDH-mutant gliomas and leukemias and are associated with various regulation of molecular process. Mutations of active site in IDH1 could lead to high levels of 2-HG and the suppression of cellular differentiation, while these changes can be reversed by molecule inhibitors target mutant IDH1. Here, through in-house developed enzymatic assay-based high throughput screening platform, we discovered DC_H31 as a novel IDH1-R132H/C inhibitor, with the IC₅₀ value of 0.41 μmol/L and 2.7 μmol/L respectively. In addition, saturable SPR binding assay indicated that DC_H31 bound to IDH1-R132H/C due to specific interaction. Further computational docking studies and structure-activity relationship (SAR) suggest that DC_H31 could occupy the allosteric pocket between the two monomers of IDH1-R132H homodimer, which accounts for its inhibitory ability. And it is possible to conclude that DC_H31 acts via an allosteric mechanism of inhibition. At the cellular level, DC_H31 could inhibit cell proliferation, promote cell differentiation and reduce the production of 2-HG with a dose-dependent manner in HT1080 cells. Taken together, DC_H31 is a potent selective inhibitor of IDH1-R132H/C both in vitro and in vivo, which can promote the development of more potent pan-inhibitors against IDH1-R132H/C through further structural decoration and provide a new insight for the pharmacological treatment of gliomas.

Full text of DOI:10.1016/j.bmc.2019.05.040

Accepted Manuscript
Discovery of DC_H31 as Potential mutant IDH1 Inhibitor through NADPH-
based High Throughput Screening
Zhe Duan, Jingqiu Liu, Liping Niu, Jun Wang, Mingqian Feng, Hua Chen,
Cheng Luo
PII:
S0968-0896(19)30606-6
https://doi.org/10.1016/j.bmc.2019.05.040
BMC 14927
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
12 April 2019
24 May 2019
27 May 2019
Please cite this article as: Duan, Z., Liu, J., Niu, L., Wang, J., Feng, M., Chen, H., Luo, C., Discovery of DC_H31
as Potential mutant IDH1 Inhibitor through NADPH-based High Throughput Screening, Bioorganic & Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.05.040
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Discovery of DC_H31 as Potential mutant IDH1 Inhibitor
through NADPH-based High Throughput Screening
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a
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a*
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Zhe Duan , Jingqiu Liu , Liping Niu , Jun Wang , Mingqian Feng , Hua Chen , Cheng Luo
a
College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070,
China
b
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy
of Sciences, Shanghai 201203 P. R. China
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Key Laboratory of Chemical Biology of Hebei Province, College of Chemistry and Environmental
Science, Hebei University, Bao ding 071002, China
These authors contributed equally: Zhe Duan and Jingqiu Liu
*Corresponding authors
fengmingqian@mail.hzau.edu.cn
Cheng Luo: cluo@simm.ac.cn
Hua Chen: hua-todd@163.com
Abstract:
IDH1 mutations are early events in the development of IDH-mutant gliomas and leukemias
and are associated with various regulation of molecular process. Mutations of active site in IDH1
could lead to high levels of 2-HG and the suppression of cellular differentiation, while these
changes can be reversed by molecule inhibitors target mutant IDH1. Here, through in-house
developed enzymatic assay-based high throughput screening platform, we discovered DC_H31 as
a novel IDH1-R132H/C inhibitor, with the IC value of 0.41 μmol/L and 2.7 μmol/L respectively.
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In addition, saturable SPR binding assay indicated that DC_H31 bound to IDH1-R132H/C due to
specific interaction. Further computational docking studies and structure-activity relationship
(SAR) suggest that DC_H31 could occupy the allosteric pocket between the two monomers of
IDH1-R132H homodimer, which accounts for its inhibitory ability. And it is possible to conclude

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that DC_H31 acts via an allosteric mechanism of inhibition. At the cellular level, DC_H31 could
inhibit cell proliferation, promote cell differentiation and reduce the production of 2-HG with a
dose-dependent manner in HT1080 cells. Taken together, DC_H31 is a potent selective inhibitor
of IDH1-R132H/C both in vitro and in vivo, which can promote the development of more potent
pan-inhibitors against IDH1-R132H/C through further structural decoration and provide a new
insight for the pharmacological treatment of gliomas.
Keywords: IDH1 mutation; high-throughput Screening; pan-inhibitor; 2-HG; gliomas
Graphical abstract:
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Isocitrate dehydrogenase 1 (IDH1) is a homodimeric metabolic enzyme that participates in
lipid metabolism and glycolytic pathway and catalyzes the oxidative decarboxylation of isocitrate to
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generate a-ketoglutarate (α-KG) and CO2, accompanied with consuming NADP+ to produce
[1]
NADPH . Whole-genome sequencing revealed IDH1 mutations were frequent in low-grade
gliomas (70-80%) and acute myeloid leukemia (AML) (10-15%)[2-4]. The most prevalent
heterozygous mutated residues in IDH1 are located in the enzyme active cavity corresponding to
amino acid residue 132[5] that replaces an active-site arginine residue with inactive missense mutation
histidine and cystein, accounting to 92%. These mutations render IDH1-R132H/C a neomorphic
activity that reduce α-ketoglutarate (α-KG) to the oncometabolite 2-hydroxyglutarate (2-HG) with
[6]
concomitant reduction of NADPH , and finally cause reduction of cellular concentration of α-KG
[7]
and the accumulation of the D-2HG . α-KG is a substrate of prolyl hydroxylase domain proteins
(PHD) which could lead to hydroxylation and degradation of hypoxia inducible factor (HIF). The
high level 2-HG may inhibit ten-eleven translocation 2 (TET2), PHD and histone demethylases,
which could induce changes in the cell methylome and epigenetic profiles, resulting in blockade
of cell differentiation and cell proliferation[8, 9]. Previous studies also demonstrated that the
accumulation of 2-HG might be utilized diagnostically in oncology clinics [5, 10-15]. Therefore, due
to the pivotal role of IDH1 mutations in tumorigenesis makes IDH1-R132H/C becoming a
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promising novel therapeutic target . [16]
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IDH1 mutations are therapeutically advantageous for inhibiting IDH1-mutant gliomas[17, 18]
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There are several types of drugs targeting IDH1 mutations, such as metabolism inhibitors,
demethylating agents, and vaccines [5, 19-23]. Small molecule inhibitors targeting mutant IDH1
would lead to reduction of 2-HG and regulate related metabolic pathways. Among the small
molecule inhibitors, Ivossidenib (AG-120) is a specific and progressing IDH1-R132H/C inhibitor
that restores normal cellular process to AML patients and approved by FDA in 2018 [24, 25].
Nevertheless, there is still urgent need to develop novel pan-inhibitors for IDH1-R132H/C for
treatment of gliomas.
In the current study, a new mutation–specific inhibitor, named DC_H31, was identified by a
high-throughput screening. DC_H31 can reduce the production of 2-HG in HT1080 cells. The
identification of this lead molecule may help us deep understand the mechanism of the action and
develop better drugs of the class with improved therapeutic effects.

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Figure1. The diagram of NADPH-based high throughput screening assay. A) The flowchart of biochemical
assay-based high throughput screening for the discovery of DC_H31. B) The Z’ factor determination of
established biochemical assays. C) Proportional fluorescence signal at different NADPH concentrations. D) The
IC50 determination of AGI-5198 against IDH1-R132H measured by biochemical assays. (Each point represents the
mean ±SD of three replicates)
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In our study, a new reliable and robust NADPH-based HTS assay targeting IDH1-R132H/C
was developed to identify mutant-specific inhibitor. IDH1-R132H/C catalyze the convert of α-KG
to 2-HG mediated with NADPH consumption with a concomitant decrease in fluorescence at 460
nm, therefore inhibition of IDH1-R132H/C enzyme activity by small molecule was evaluated by a
fluorescent assays of NADPH depletion. In the assay, the remaining NADPH, which is inversely
proportional to IDH1-R132H/C activity, was measured at the endpoint of the reaction by
fluorescent reader (λex = 355nm,λem = 460nm) (Fig. 1A), and the fluorescence signal is
proportional to the NADPH concentration(Fig. 1C). The Z’ factor of the biochemical assay was
0.76 and S/B ratio was 7.6 demonstrated the confidence of this NADPH fluorescence-based
biochemical assay(Fig. 1B). The feasibility of this HTS method was further verified by testing the
inhibitory activities of the positive compound AGI-5198[17, 18], the IC50 value for the compound
through this method was 82 nmol/L(Fig. 1D), which was in line with previous reports. All these
data demonstrate that the assay is reliable and robust and can be used for molecular screening
target mutant IDH1 and wide type (WT) IDH1. Compared to existing mutant IDH1-mediated
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NADPH consumption of a diaphorase/resazurin-based detection assay[26], the assay saved
diaphorase converts resazurin into resorufin steps, needed less time and simplified screening
process for drug discovery of mutant IDH1.
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Figure2. The results of high throughput screening and preliminary hit validation. A) Activity of 71
compounds screened from primary HTS in biochemical assays in WT IDH1 (x axis) versus that in IDH1-R132C (y
axis). The selected 6 compounds are shown in red. B) IC50 value of 6 compounds screened from secondary round
screening. C) The IC50 determination of DC_H31 against WT IDH1, IDH1-R132H and IDH1-R132C measured by
biochemical assays. (Each point represents the mean ±SD of three replicates. D) The chemical structure of
DC_H31.
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A library containing 25,000 compounds with diverse structures was screened by this
NADPH-based HTS assay using DMSO as a negative control and non-enzymatic reaction as a
positive control to indicate an inhibition. Through primary HTS with a single concentration (10
μmol/L), 71 compounds which inhibitory activity more than 80% were selected and confirmed in
an IDH1-R132C and WT IDH1 assay, and we obtained 6 compounds screened from secondary

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round screening, as shown in Figure 2A. After that, all of these 6 compounds were identified by a
selection criteria that inhibition as varies with a serious of concentrations between 50 μmol/L to
0.068 μmol/L (Fig. 2B). Finally, DC_H31 was identified as a potential inhibitor with an IC value
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of 0.41 μmol/L for IDH1-R132H and 2.7 μmol/L for IDH1-R132C, but inhibited WT IDH1
minimally (Fig. 2C and Fig.2D).
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Figure3. DC_H31 binds to IDH1-R132H/C but not WT IDH1 by Surface Plasmon Resonance measurements.
A) DC_H31 binds to IDH1-R132H with the KD is 3.8 μmol/L. B) DC_H31 binds to IDH1-R132C with the KD is
0.72 μmol/L. C) DC_H31 could not observably bind to WT IDH1 under the same detection conditions with IDH1-
R132H/C.
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To further investigating the interaction of compound DC_H31 and IDH1-R132H/C, Surface

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Plasmon Resonance (SPR), an optical technique for hit validation [27], was utilized to measure the
interactions between the newly discovered DC_H31 towards IDH1-R132H/C and WT IDH1.The
data were fit in both kinetic and equilibrium modes. Compound DC_H31 could directly bind to
IDH1-R132H and IDH1-R132C with an equilibrium dissociation constant KD of 3.8 μmol/L and
0.72 μmol/L (Figs. 3A and 3B), while the KD value for WT IDH1 was noticeably higher, over 50
μmol/L (Fig. 3C). The result is similar to the IDH1-R132H/C IC50 and WT IDH1 IC50 value,
suggesting that DC_H31 is a potential selective inhibitor to bind IDH1-R132H/C directly.
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Figure4. Putative binding mode between DC_H31 and IDH1-R132H. A) Overview of the allosteric binding
pocket between the two monomers. H132 is shown as gray sticks. B) Detailed view of the allosteric binding pocket
for DC_H31 (green). Hydrogen bonds (yellow) are indicated by yellow dotted line. C) Schematic diagram
showing predicted interactions between IDH1-R132H and DC_H31. DC_H31 is colored in purple. Hydrophobic
interactions are plotted in red arcs. D) Overlay of one monomer of the IDH1-R132H (gray) bound to DC_H31
(green) and the WT IDH1 (wheat). Regulatory segment in IDH1-R132H and WT IDH1 is depicted in yellow and
lavender respectively.
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The selective and effective inhibitory activity of DC_H31 against IDH1-R132H/C prompted

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us to disclose the molecular mechanism of the inhibitory activity of DC_H31. Molecular docking
study was employed to reveal the binding mode and structural details of its interactions with
mutant IDH1. The crystal structures of IDH1-R132H (PDB ID: 5LGE)[28] were selected and a
putative binding mode was generated by Glide program with XP mode of the maestro. The results
suggested that DC_H31 occupied the allosteric pocket between the two monomers far away from
the mutation site with 12.6 Å. (Fig. 4A.). The binding site is also not located at the active site,
Tyr139, because of highly polarity as defined by the amino acids lining the site [29]. The allosteric
pocket lined on three sides by Tyr285, Trp124, Met259, Trp267, and the remaining side was
formed by residues in a regulatory segment such as Ser280, Gln277[30]( Fig.4B.). An assumption
was proposed that DC_H31 inhibits the IDH1-R132H enzyme function through an allosteric
mechanism of inhibition.
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OH
R1
R3
R1
R1 Br
a
R1 MgBr
b
R2
c
R2
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CH-H-X
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Scheme 1. Synthesis of the compounds CH-H-X. Conditions and reagents: (a) R Br, Mg, I , THF, 30 C; (b) 3,5-
o
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di-tert-butyl-4-hydroxybenzaldehyde or 3,5-di-tert-butylbenzaldehyde, 30 C, two steps: 65-70% yields; (c)1)
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2 2
MsCl, CH Cl , rt; 2) R XH (X = N; O; S), DMF,80-90 C, two steps: 50-70% yields.
We next synthesized a series of derivatives(Scheme.1) and evaluated the structure-activity
relationships (SAR) of DC_H31 and its derivatives based on the results of biochemistry assay to
validate the scaffold authenticity of DC_H31. DC_H31 formed two hydrogen bond to the carbonyl
group of Gln277 and hydroxyl group Ser280 respectively. The hydroxyl in the scaffold of
DC_H31 formed a hydrogen bond to the carbonyl group of Gln277, which may clearly explain the
different inhibitory activity between DC_H31 and CH-H-16 or CH-H-17. Inhibitory activity
totally disappeared when the hydroxyl was moved from DC_H31. Another hydrogen bond was
observed between nitrogen atom of morpholine group and Ser280. Obviously, the hydrogen bond
is vital to the inhibitory activity, which accounts for the huge difference in inhibitory activity
between CH-H-3, CH-H-4 and DC_H31. However, the oxygen atom at the morpholine group had
less importance, and the activity was no much change when the oxygen atom was replaced by a
nitrogenous group (CH-H-2). However, the activity gradually decreased when the nitrogenous

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group was linked with various lengths of chains because of the steric hindrance, such as
compounds CH-H-5 to CH-H-11.
Besides polar interactions as mentioned above, a highly hydrophobic environment
surrounded by the allosteric pocket may contribute to stabilize the DC_H31 conformation in the
pocket. In addition, it should be noticed that the pyridine group of the molecular forms three edge-
to-face interaction with Trp124, Tyr285, Trp267 in three different directions respectively and is
stabilized by powerful hydrophobic forces around the pocket (Fig. 4C). In addition, any chemical
modifications on this group could reduce the inhibitory activity. It is clear that the pyridine
group plays a crucial role to the improvement in inhibitory activity, which clarifies the
molecular mechanism for better inhibitory activity of DC_H31 compared to its analogues (CH-
H12, CH-H13, and CH-H14).
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Table 1.Structures and biochemical IC (μmol/L) data for DC_H31 and its analogues.
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Biochemical IC (μmol/L)
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Compounds.
R1
R2
R3
IDH1-
IDH1-
R132C
WT
R132H
IDH1
OH
N
CH-H-1
CH-H-2
CH-H-3
CH-H-4
CH-H-5
N
O
0
.41
2.7
4.9
43.7
~50
>50
>50
6.4
OH
OH
OH
OH
N
N
N
N
N
N
3
.0
S
>
>
50
50
>50
>50
4.8
O
N
N
1
2

OH
OH
OH
OH
OH
OH
OH
N
N
N
N
N
N
CH-H-6
CH-H-7
CH-H-8
CH-H-9
CH-H-10
CH-H-11
CH-H-12
N
N
N
N
>
50
>50
9
>50
12.79
31
>50
>
>
50
50
18
>50
7
N
N
N
N
>50
3
N
N
N
4
.76
N
>
>
50
50
9
8.5
N
N
O
O
>50
>50
F
OH
OH
N
CH-H-13
CH-H-14
>
50
>50
4.7
>50
0.8
CH3
H C
3
N
N
N
O
O
2
0
N
N
H
H
CH-H-15
CH-H-16
>
>
50
50
>50
>50
>50
>50
N
N
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Note: The IC50 values for IDH1-R132H and IDH1-R132C and WT IDH1 are the mean of three determinations
performed as described method.
When the IDH1-R132H:DC_H31 conformation was overlaid to the WT IDH1 structure
(PDB: 1T09) [31], we found the α10 regulatory segment (seg-2), a partially α-helix structure, in
WT IDH1 conformation blocked the homologous allosteric site which was bound by DC_H31 in
IDH1-R132H (Fig. 4D). In the WT IDH1 structures, Arg132 formed an ionic interaction with
Asn271 of the seg-2, whereas the ionic interaction did not produce because of the mutation of Arg

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to His in IDH1-R132H [6, 31]. Destabilization of seg-2 due to the lacking of the R132:N271
interaction may afford DC_H31 have access to the allosteric site of IDH1-R132H achieving
mutant selectivity. Taken together, mutant selectivity for IDH1 is achieved by the intrinsic lability
of regulatory segment in IDH1-R132H compared to WT IDH1. However, we need further
confirmation to verify that the long distance between H132 to the allosteric pocket is the reason
for the pan-inhibitory activity.
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Figure5. DC_H31 decreases intracellular 2-HG production and affects cell proliferation and differentiation.
A) DC_H31 inhibits intracellular 2-HG production in HT1080 cells within 48h. B) And C) DC_31 inhibits
HT1080 cell proliferation but have weak effect on U87-MG. D) qRT-PCR results of Sox2 and GFAP expression in
HT1080 cells treatment with DC_H31 after 6 days. E) DC_H31 increases expression of GFAP and promotes
HT1080 cell differentiation in the presence of DC_H31 for 6 days. (Error bar are mean ±S.D. for three replicates)
As HT1080 cells produced the high level of 2-HG due to the neomorphic enzyme function of
the missense mutation in IDH1, to further demonstrate the inhibition of IDH1 mutation in cells by
DC_H31, the 2-HG assay was carried out with 0.625 μmol/L, 1.25 μmol/L, 2.5 μmol/L, 5 μmol/L
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DC_H31 . As shown in the figure 5A, 2-HG levels declined to half of DMSO control when
DC_H31 concentration up to 1.25 μmol/L, and measurements of 2-HG production in a culture
medium of HT1080 cells demonstrated dose-dependent inhibition after 48h of treatment.
As mentioned above, inhibiting the mutation could affect cells proliferation. HT1080 cell line
harboring IDH1-R132C mutation and U-87 MG cell line, a WT IDH1 cell, were chosen for the

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cell proliferation assay to evaluate the cellular activity of DC_H31. As shown in figure 5B and 5C,
inhibition of HT1080 cell proliferation was observed in a dose-dependent manner after 72h
treatment with a range of concentrations of DC_H31. The compound could inhibit HT1080 cells
growth with the IC value of 5 μmol/L while the minimal effect on the proliferation of U-87 MG
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cells was seen with the IC value of 48.7 μmol/L. Noticeably, HT1080 cells were more sensitive
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to inhibition by DC_H31 than U-87 MG cells, at the nearly 10-fold difference between the two
cell lines.
Furthermore, in order to determine whether DC_H31 could induce differentiation in HT1080
cells, SRY-box2 (SOX2) and glial fibrillary acidic protein (GFAP) which are used to indicate the
degree of differentiation of HT1080[32] were selected to determine the inhibition of DC_H31
against the transcription of IDH1-R132C downstream genes. After the treatment with 1 μmol/L
DC_H31 or DMSO for 6 days, quantitative fluorescence real-time PCR (qRT-PCR) was used to
measure the transcription of the two genes in HT1080 cells. As shown in Fig. 5D, DC_H31 could
inhibit the transcription of SOX2 genes and upregulate the transcription of GFAP genes.
Meanwhile, western blot assay was performed to evaluate the alteration of the protein GFAP. As
shown in Fig. 5E, treatment with 0.5 μmol/L DC_H31 induced the expression of GFAP compared
to DMSO control, confirming the inhibitory activity of DC_H31 with on-target behavior.
IDH1–R132H/C are highly attractive targets for the treatment of gliomas and AML.
Although many IDH inhibitors have been discovered since the first report in 2012, there is still
pressing need to develop novel pan-inhibitors for IDH1-R132H/C. In this study, we identified a
small molecular, DC_H31, that could potentially inhibit both IDH1-R132H and IDH1-R132C
with an IC50 value of 0.41 μmol/L and 2.7 μmol/L respectively. At the cellular level, DC_H31
effectively inhibited the proliferation of HT1080 cells and was capable of reducing 2-HG levels in
the HT1080 cell line. In addition, the transcription of mutant IDH1 downstream genes was altered
by DC_H31, and the protein abundance of GFAP also increased which validated inhibitory
activity of this compound. Molecular docking studies revealed the potential binding mechanism of
DC_H31 with IDH1-R132H and facilitated to discovery better mutant IDH1 inhibitors. Overall,
these results demonstrate that DC_H31 deserves further structure optimization as a pan-inhibitor
of IDH1-R132H and IDH1-R132C for their potential use in the chemotherapeutics of patients with
IDH1-mutant gliomas and AML.
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Acknowledgements
Funding: This work was funded by Large-scale Protein Preparation System at the National
Facility for Protein Science in Shanghai (NFPS), Zhanjiang Lab, and China for providing
technical support and assistance in data collection and analysis. We gratefully acknowledge
financial support from the National Natural Science Foundation of China (21472208, 81625022,
and 81430084 to C.L.); K.C. Wong Education Foundation to C.L., Chinese Academy of Sciences
(XDA12020353 and XDA12050401) and China Postdoctoral Science Foundation (2017M621571
to L.Y.).
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Author Contributions
Cheng Luo, Mingqian Feng, Zhe Duan and Jingqiu Liu designed the study, Zhe Duan and
Jingqiu Liu performed the assays, Hua Chen and Liping Niu instructed the chemical synthesis
including DC_H31 and the derivatives, Jun Wang performed molecular docking analysis, Zhe
Duan and Jingqiu Liu analyzed data wrote the manuscript with input from all of the authors.

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First Potent Inhibitors of Mutant IDH1 That Lower Tumor 2-HG in Vivo. ACS MED CHEM LETT
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Yan H, Parsons DW, Jin G, McLendon R, Rasheed BA, Yuan W, et al.. IDH1 and IDH2
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associated IDH1 mutations produce 2-hydroxyglutarate. NATURE 2010.
Dinardo CD, Propert KJ, Loren AW, Elisabeth P, Zhuoxin S, Levine RL, et al.. Serum 2-
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hydroxyglutarate levels predict isocitrate dehydrogenase mutations and clinical outcome in acute
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enantiomer of 2-hydroxyglutarate linked to EGLN activation. NATURE 2012; 483 (7390): 484-88.
Xu W, Yang H, Liu Y, Yang Y, Wang P, Kim SH, et al.. Oncometabolite 2-hydroxyglutarate is a
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10 Stefan G, Cairns RA, Minden MD, Driggers EM, Bittinger MA, Hyun Gyung J, et al.. Cancer-
associated metabolite 2-hydroxyglutarate accumulates in acute myelogenous leukemia with isocitrate
dehydrogenase 1 and 2 mutations.. J EXP MED 2010; 207 (2): 339-44.
11 Fathi AT, Hossein S, Borger DR, Ballen KK, Amrein PC, Attar EC, et al.. Prospective serial
evaluation of 2-hydroxyglutarate, during treatment of newly diagnosed acute myeloid leukemia, to
assess disease activity and therapeutic response. BLOOD 2012; 120 (23): 4649-52.
12 Leopold S, David C, Jochen M, Claus-Dieter L, Christine-Maria H, Heike P, et al.. Increased
levels of 2-hydroxyglutarate in AML patients with IDH1-R132H and IDH2-R140Q mutations. EUR J
HAEMATOL 2010; 85 (5): 457-59.
13 Maxime J, Elena M, Véronique S, Jean-Baptiste M, Aline R, Cyril Q, et al.. Serum 2-
hydroxyglutarate production in IDH1- and IDH2-mutated de novo acute myeloid leukemia: a study by
the Acute Leukemia French Association group. J CLIN ONCOL 2014; 32 (4): 297-305.
14 Borger DR, Lipika G, Thomas Y, Poon RT, Marek A, Vikram D, et al.. Circulating
oncometabolite 2-hydroxyglutarate is a potential surrogate biomarker in patients with isocitrate
dehydrogenase-mutant intrahepatic cholangiocarcinoma. CLIN CANCER RES 2014; 20 (7): 1884-90.
15 Wiseman DH, Small HF, Wilks DP, Waddell ID, Dennis MW, Ogilvie DJ, et al.. Elevated plasma
2-hydroxyglutarate in acute myeloid leukaemia: association with the IDH1 SNP rs11554137 and severe

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renal impairment. BRIT J HAEMATOL 2014; 166 (1): 145-48.
16 Hodges TR, Choi BD, Bigner DD, Hai Y, Sampson JH. Isocitrate dehydrogenase 1: what it means
to the neurosurgeon: a review. J NEUROSURG 2013; 118 (6): 1176-80.
17 Dan R, Janeta PM, Nicolaos P, Sevin T, Christian G, Carl C, et al.. An inhibitor of mutant IDH1
delays growth and promotes differentiation of glioma cells. SCIENCE 2013; 340 (6132): 626-30.
18 Fang W, Jeremy T, Byron DL, Virginie PL, Stefanie S, Erica H, et al.. Targeted inhibition of
mutant IDH2 in leukemia cells induces cellular differentiation. SCIENCE 2013; 340 (6132): 622-26.
19 Theresa S, Lukas B, Stefan P, Felix S, Benedikt W, Jasmin Q, et al.. A vaccine targeting mutant
IDH1 induces antitumour immunity. NATURE 2014; 512 (7514): 324-27.
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20 Adnan E, Ippolito JE, Lynne C, Jan C, Jayne M, David PW. A high-throughput fluorimetric assay
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24 Popovicimuller J, Lemieux RM, Artin E, Saunders JO, Salituro FG, Travins J, et al.. Discovery of
AG-120 (Ivosidenib): A First-in-Class Mutant IDH1 Inhibitor for the Treatment of IDH1 Mutant
Cancers. ACS MED CHEM LETT 2018; 9 (4): 300-05.
25 Theresa S, Lukas B, Stefan P, Felix S, Benedikt W, Jasmin Q, et al.. A vaccine targeting mutant
IDH1 induces antitumour immunity. NATURE 2014; 512 (7514): 324-27.
26 Okoye-Okafor UC, Bartholdy B, Cartier J, Gao EN, Pietrak B, Rendina AR, et al.. New IDH1
mutant inhibitors for treatment of acute myeloid leukemia. NAT CHEM BIOL 2015; 11 (11): 878-86.
27 Myszka DG. KINETIC ANALYSIS OF MACROMOLECULAR INTERACTIONS USING
SURFACE PLASMON RESONANCE BIOSENSORS. Curr Opin Biotechnol 1997; 8 (1): 50-57.
28 Pusch S, Krausert S, Fischer V, Balss J, Ott M, Schrimpf D, et al.. Pan-mutant IDH1 inhibitor
BAY 1436032 for effective treatment of IDH1 mutant astrocytoma in vivo. ACTA NEUROPATHOL
2017; 133 (4): 629-44.
29 Levell JR, Caferro T, Chenail G, Dix I, Dooley J, Firestone B, et al.. Optimization of 3-
Pyrimidin-4-yl-oxazolidin-2-ones as Allosteric and Mutant Specific Inhibitors of IDH1. ACS MED
CHEM LETT 2017; 8 (2): 151.
30 Yang, Chen, Zhong, Yingjie, Peng, Zheng, et al.. Molecular mechanisms of ＂off-on switch＂
of activities of human IDH1 by tumor-associated mutation R132H. CELL RES 2010; 20 (11): 1188-
200.
31 Xiang X, Jingyue Z, Zhen X, Baozhen P, Qiuhua H, Eddy A, et al.. Structures of human cytosolic
NADP-dependent isocitrate dehydrogenase reveal a novel self-regulatory mechanism of activity. J
BIOL CHEM 2004; 279 (32): 33946.
32 Hsieh JK, Hong CS, Manjila S, Cohen ML, Lo S, Rogers L, et al.. An IDH1-mutated primary
gliosarcoma: case report. J NEUROSURG 2016; 126 (2): 1-05.
33 Zhang JH, Chung TDY, Oldenburg KR. A Simple Statistical Parameter for Use in Evaluation and
Validation of High Throughput Screening Assays. J BIOMOL SCREEN 1999; 4 (2): 67-73.

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34 Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, et al..
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PHARMACOL SIN 2018; 39 (9).
a high-throughput screening assay. ACTA

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Supplementary Materials for
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3
Discovery of DC_H31 as Potential mutant IDH1 Inhibitor
through NADPH-based High Throughput Screening
a
b
c
b
a*
c*
b*
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Zhe Duan , Jingqiu Liu , Liping Niu , Jun Wang , Mingqian Feng , Hua Chen , Cheng Luo
a
College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070,
China
b
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy
of Sciences, Shanghai 201203 P. R. China
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c
Key Laboratory of Chemical Biology of Hebei Province, College of Chemistry and Environmental
Science, Hebei University, Bao ding 071002, China
These authors contributed equally: Zhe Duan and Jingqiu Liu
*Corresponding authors
fengmingqian@mail.hzau.edu.cn
Cheng Luo: cluo@simm.ac.cn
Hua Chen: hua-todd@163.com

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Materials and methods
Protein expression and purification
Proteins were over expressed in bacterial systems and purified by affinity chromatography.
The WT IDH1 insert was amplified by standard PCR procedure and ligated to the pET28a vector
using CloneExpress One Step Cloning Kit (Vazyme). The correct construct was verified by
Sanger sequencing. Based on the WT IDH1 plasmid, the IDH1-R132H/C was obtained using the
site-directed mutagenesis kit .
The plasmids were transformed into BL21 cells for over expressing the fusion protein with an
N-terminal 6×His-tag. (Both competent cell purchased from TRANSGEN BIOTECH). The
transformed BL21 cells were cultured in LB media containing 50 μg/ml kanamycin, 0.4 mmol/L
isotropy-β-D-thiogalactoside at 16°C for 13-15 hours, then collected and sonicated in pre-cooled
lysis buffer (20 mmol/L Tris-HCl pH 7.5, 150 mmol/L NaCl, 1 mmol/L β-ME and 20 mmol/L
imidazole). Cell debris were centrifuged at 18,000 rpm for 1 hours at 4℃. The supernatant was
loaded on 5 ml HisTrap HP, Superdex 75 10/300 column (GE Healthcare). Purified proteins were
stored at −80°C.
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Compound library
A compound library containing 2, 5000 compounds of diverse structures was purchased from
the SPECS Company (SPECS_SC_10mg_ Dec2016). AGI-5198, serving as a positive control [17]
,
was purchased from MCE. The purity of all compounds is greater than 95% (LCMS). All
compounds were dissolved in Dimethyl sulfoxide (DMSO) and stored in 4 °C for a long time.

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High-throughput screening with enzyme activity assay
Mutant IDH1 catalyzed the conversion of α-KG to 2-HG accompanied by NADPH
consumption, and the inhibition of IDH1-R132H/C enzyme activity by small molecule was
evaluated by a fluorescent assays of NADPH depletion to identify lead compounds. In the assay,
the remaining NADPH, which is inversely proportional to IDH1-R132H/C activity, was measured
at the endpoint of the reaction by a fluorescent reader (BMG POLARstar omega, λex = 355nm,
λem = 460nm).
Compounds were prepared in 100% DMSO as 10mM stock and diluted 1:1000 into the final
reaction of the first screen. The positive control drug was also prepared as 10 mmol/L stock in
DMSO. The enzyme reactions were performed in 384-well plates at room temperature containing
1X assay buffer (20 mmol/L Tris, pH 7.5, 150 mmol/L NaCl, 5 mmol/L MgCl2), 10 µmol/L
NADPH, 2.5 mmol/L αKG and 100nmol/L IDH1-R132H enzyme or 20 nmol/L IDH1-R132C
enzyme, and plates were incubated at room temperature for 40 minutes in a final volume of 50 µl.
The wild type enzyme reactions were also performed in 384-well plate under the same conditions
but contain 10µmol/L NADP+, 5mmol/L isocitrate and 60 nmol/L IDH1 enzyme.
This assay was also used to determine DC_H31 which was prepared with a dose-response
curve starting with 50 μmol/L in seven dilution steps in IDH1-R132H/C test and starting with 150
μmol/L in five dilution steps in wild type IDH1 assay. The relative fluorescence versus inhibitor
concentration was proportional.
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Z′ factor and S/B calculation
The Z’ factor, as a parameter of the high-throughput screen assay [33], was calculated to

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evaluate the reliability of the test methods as the below formula:
Z′=1-(3*(δp+δn) /|μn−μp|)
In this formula, μp, δp, μn and δn were used for the means and standard deviations of the
positive (p) and negative (n) controls respectively. The reaction adding DMSO was employed as
negative control and lacking of mutation protein in the action was positive control. The S/B value
was the result of the mean of the negative controls over the mean of the positive controls in the
biochemical reactions.
Surface Plasmon Resonance (SPR)-based binding assays
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SPR is an efficient method for measuring interactions between small molecule and protein [27]
In this study, we applied SPR to measure the interactions between the newly discovered DC_H31
towards IDH1-R132H/C and WT IDH1. The SPR-based binding assay was performed with HBS
buffer (10 mmol/L HEPES pH 7.4, 150 mmol/L NaCl, and 0.1% (v/v) DMSO) on GE Healthcare
Biacore T200 instrument at 25 °C. IDH1-R132H, IDH1-R132C and WT IDH1 were diluted in 10
mmol/L sodium acetate, and covalently immobilized on the CM5 sensor chip respectively by the
standard amine-coupling procedure. The test compound DC_H31 was serially diluted to a
concentration range between 25 μmol/L to 0.78 μmol/L with HBS buffer in IDH1-R132H/C test,
or between 50 μmol/L to 1.56 μmol/L in WT IDH1 test, and injected into the chip at 30 μL/min
rate. The combination step was set at 120 s, following 150 s dissociation step. The KD value of
DC_H31 was calculated by the state model of Biacore T200 evaluation software.
.
Cell culture and viability assays

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The sarcoma cell line HT1080, featuring an endogenous IDH1-R132C mutation, and likely
glioblastoma cell line U-87 MG were both purchased from American Type Culture Collection
(ATCC) and maintained in DMEM medium with 10% fetal bovine serum supplement and 1%
penicillin and streptomycin antibiotic (Gibco® Invitrogen, Carlsbad, USA) under standard culture
conditions (37 °C, 5% CO2).
For cell viability assay, the two cell lines were seeded into 96-well plates. After attachment to
the culture plate, the cells were treated with a range of concentrations of compounds for 72 h in
triplicate, and the same concentration of DMSO was employed as negative control. The fraction of
viable cells was estimated through Cell Titer-Glo luminescent assays using multilabel reader
(Envision, PerkinElmer). All data were analyzed by GraphPad Prism 5.0 software and fitted to
nonlinear curve to calculate IC value.
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Western Blot
HT1080 cells were cultured in 6-well plate at the appropriate density and treated with
indicated concentrations of DC_H31 or DMSO control for 6 days after cell attachment. Treated
HT1080 cells was collected and lysed by cell lysis buffer. Then, protein samples quantified by
BCA protein determination method [34] were separated by 12% SDS-polyacrylamide gradient gel
and transferred to nitrocellulose membranes. The membranes were blocked in 5% (w/v) nonfat
milk for 1h and then incubated with anti-GFAP and anti-GAPDH primary antibodies at 4 ℃
overnight. Subsequently, after washed by TBST three times, the membranes were incubated with
HRP-conjugated anti-mouse or anti-rabbit secondary antibody for 2h and detected in
ChemiScope3400 imaging system using ECL substrate.

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Quantitative RT-PCR
For the q-PCR analysis assay, HT1080 cells were also cultured in 6-well plates and treated
with 1 μmol/L DC_H31 or DMSO control for 6 days. Treated cells then were collected and lysed
for RNA extraction using the Trizol Reagent kit. After that, previous RNA was reverse
transcripted for Real-time PCR analysis on the Quant Studio 6 Flex Real-Time PCR system using
the SYBR Green Real-Time PCR Master Mix. Data were visualized with the expression value for
SOX2 gene and GFAP gene in the DMSO control or treated samples.
Measurement of 2-HG
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The level of 2-Hydroxyglutarate(2-HG) was quantified in HT1080 cells in the absence or
presence of DC_H31 compound by LC/MS. Firstly, HT1080 cells were seeded into a 6-well plate.
After attaching to plate, cells received DC_H31 with various concentrations and received the same
concentration of DMSO as a negative control in complete medium for 48 h. Cell culture medium
was then harvested for 2-HG extraction and measured by LC/MS using DIONEX UltiMate 3000
and Thermo Fisher Scientific TSQ Quantiva. A Calibration curve was done by deterring a
standard 2-HG dissolved in 80:20 methanol: water. LC was performed on Waters XSELECT HSS
T3analytical column. The metabolite 2-HG and the standard 2-HG was monitored by the transition
of 147.0 m/z > 101.1 m/z and 51.2 m/z using a DP −35.0, collision energy −16.0, and CXP −9.0.
Data were acquired and analyzed with Thermo Xcalibur3.0.63. The peak areas of the metabolite
2-HG were normalized by the peak area of the standard 2-HG. The value of 2-HG concentration
was normalized to the DMSO negative control and expressed as percentages of negative control
activity [6]
.

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Binding mode analysis
Molecular docking experiments were performed to analyze the possible binding modes of
DC_H31 using the molecular modeling software Maestro (Schrödinger, LLC: New York, NY,
2009) [35]. Firstly, the structure of DC_H31 was drawn using Chem3D and prepared by LigPrep
and Advanced Conformational Search to generate different protonation states and conformations
with the OPLS3 force field. Then the crystal structure of IDH1-R132H selected from the protein
data bank (PDB code: 5LGE) was optimized as molecular docking receptor through the Protein
preparation Wizard module in Maestro with a pH value of 7.0. In addition, a 20 Å × 20 Å × 20 Å
receptor grid box centered at the BAY 1436032 binding center was generated in the Receptor Grid
Generation Module. Other parameters were set as default. Finally, Glide extra-precision (XP)
mode of induced-fit docking was used to dock the prepared ligand molecular into the defined
binding site with standard protocols. After Glide docking, the binding mode of the ligand was
chosen according to the score.
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Chemistry
Column chromatography was carried out on flash silica gel (300-400 mesh). TLC analysis was
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conducted on silica gel plates (Silica G UV254). Melting points were measured in an open
capillary on an SGW X-4 melting point apparatus and were uncorrected. NMR spectra were
recorded at 400 MHz and 600 MHz for 1H on a Bruker instrument. Chemical shifts (δ values) and
coupling constants, (J values) were given in ppm and hertz, respectively, using TMS (1HNMR)
solvent as internal standard. The High Resolution Mass Spectra (HRMS) were carried out on an
FTICR-MS (Ionspec 7.0T) mass spectrometer with electrospray ionization (ESI). Element analysis
was performed using a Heraeus (CHNO, rapid) elemental analyzer. The silica gel (300-400 mesh)
for flash column chromatography was from Qingdao Marine Chemical (China).
General procedure for the synthesis of compounds 2

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A mixture of magnesium (Mg, 769 mg, 32 mmol), iodine (50 mg, 0.2 mmol) in THF (8 mL)
was added slowly 6 mL of a solution of 2-bromopyridine (1.2 mL, 12.8 mmol) in THF. After the
dropwise addition finished, the reaction mixture was heated at 30 °C for 2 hours. Then, 3,5-di-tert-
butyl-4-hydroxybenzaldehyde (592 mg, 2.5mmol) was added dropwise. Their action mixture was
stirred at 30°C for 24 h. After completion of the reaction by TLC, 20% ammonium chloride
solution was added to quench the reaction. The mixture was extracted with dichloromethane (50
mL×3). The organic layer was washed with brine and dried over Na SO . The solid was filtered
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off, and the filtrate was concentrated under reduced pressure to give 2,6-di-tert-butyl-4-
(hydroxy(pyridin-2-yl) methyl) phenol2 ( 498 mg, yield 63%).
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General procedure for the synthesis of compounds CH-H-X
Methane sulfonyl chloride (58 μL, 0.7 mmol) and triethylamine (238 μL) was added to a
solution of compound 2 (180 mg, 0.6 mmol) in dichloromethane (5 mL) and stirred at room
temperature for 1 h. After completion of the reaction by TLC, ice water was added to quench the
reaction. The mixture was extracted with dichloromethane (20 mL×3). The organic layer was
washed with brine and dried over Na SO . The solid was filtered off, and the filtrate was
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concentrated under reduced pressure to give the methylsulfonylated product (210 mg, yield 93%).
To a solution of the methylsulfonylated product (210mg, 0.6 mmol) in DMF (5 mL) was added
K CO (111 mg, 0.8 mmol), morpholine (93 μL, 1.0 mmol). The mixture was stirred at 90 °C for 2
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h. After completion of the reaction by TLC, The mixture was extracted with dichloromethane (20
mL×3). The organic layer was washed with brine and dried over Na SO . The solid was filtered
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off, and the filtrate was concentrated under reduced pressure. The residues were separated by
silica gel column chromatography (V petroleum ether: V ethyl acetate = 8:1) to give CH-H-1 (108
mg, yield 53%). Under the same conditions, compounds CH-H-2 to CH-H-16 were obtained.
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2,6-di-tert-butyl-4-(morpholino(pyridin-2-yl)methyl)phenol (CH-H-1):
o
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Light yellow solid, yield 53%, m.p. 142.3-145.8 C; H NMR (600 MHz, CDCl )
3
δ(ppm): 8.51 (d, J = 4.2 Hz, 1H), 7.62 (td, J = 7.8, 1.8 Hz, 1H), 7.57 (d, J = 7.8 Hz,
1H), 7.24 (s, 2H), 7.11 – 7.09 (m, 1H), 5.11 (s, 1H), 4.30 (s, 1H), 3.75 – 3.68 (m, 4H),
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2.42 (s, 2H), 2.33 (s, 2H), 1.40 (s, 18H); C NMR (150 MHz, CDCl ) δ(ppm): 162.5,
3

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153.0, 149.1, 136.5, 135.8, 130.8, 125.0, 122.2, 121.9, 78.5, 67.1, 52.6, 34.4, 30.4;
+
HRMS (ESI): Calcd for C H N O Na ([M+Na] ): 405.2518, Found: 405.2526; Anal.
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Calcd for C H N O : C, 75.35; H, 8.96; N, 7.32. Found: C, 75.43; H, 8.95; N, 7.36.
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2,6-di-tert-butyl-4-((4-methylpiperazin-1-yl)(pyridin-2-yl)methyl)phenol (CH-H-2):
o
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White solid, yield 70%, m.p. 134.2-136.5 C; H NMR (600 MHz, CDCl ) δ(ppm):
3
8.49 (d, J = 4.8 Hz, 1H), 7.60 (td, J = 8.4, 1.8 Hz, 1H), 7.55 (d, J = 7.8 Hz, 1H), 7.23
(s, 2H), 7.08 – 7.06 (m, 1H), 5.08 (s, 1H), 4.33 (s, 1H), 2.45 – 2.67 (m, 11H), 1.40 (s,
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18H); C NMR (150 MHz, CDCl ) δ(ppm): 162.9, 152.8,149.0,136.4,135.7, 131.3,
3
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125.0, 122.2, 121.7, 77.9, 55.4, 51.8, 46.0, 34.3, 30.4; HRMS (ESI): Calcd for
+
C H N ONa ([M+Na] ): 418.2834, Found: 418.2839; Anal. Calcd for C H N O: C,
2
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25 37
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75.91; H, 9.43; N, 10.62. Found: C, 75.98; H, 9.36; N, 10.58.
2,6-di-tert-butyl-4-((cyclohexylthio)(pyridin-2-yl)methyl)phenol (CH-H-3):
o
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1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Light yellow solid, yield 52%, m.p. 101.3-102.6 C; H NMR (600 MHz, CDCl )
3
δ(ppm): 8.53 (d, J = 4.2 Hz, 1H), 7.64 (td, J =7.8, 1.8 Hz, 1H), 7.58 (d, J = 7.8 Hz,
1H), 7.26 (s, 2H), 7.13 – 7.11 (m, 1H), 5.29 (s, 1H), 5.12 (s, 1H), 2.49 – 2.46 (m, 1H),
1.96 (d, J = 11.4 Hz, 1H), 1.83 (d, J = 11.4 Hz, 1H), 1.72 – 1.70 (m, 2H), 1.53 – 1.48
1
3
(m, 1H), 1.40 (s, 18H), 1.36-1.30 (m, 2H), 1.30 – 1.16 (m, 3H); C NMR (150 MHz,
CDCl ) δ(ppm): 162.5, 152.9, 148.9, 136.7, 135.8, 131.2, 125.0, 122.6, 121.7, 54.7,
3
+
43.7, 34.4, 33.4, 30.3, 25.9, 25.8; HRMS (ESI): Calcd for C H NOSNa ([M+Na] ):
2
6
37
434.2493, Found: 434.2487; Anal. Calcd for C H NOS: C, 75.86; H, 9.06; N, 3.40.
2
6
37
Found: C, 75.92; H, 9.16; N, 3.45.
2,6-di-tert-butyl-4-((cyclohexyloxy)(pyridin-2-yl)methyl)phenol (CH-H-4):
o
1
Yellow solid, yield 67%, m.p. 112.1-113.0 C; H NMR (600 MHz, CDCl ) δ(ppm):
3
8.52 (d, J = 4.6 Hz, 1H), 7.68 (t, J = 7.8 Hz, 1H), 7.59 (d, J = 7.8 Hz, 1H), 7.16–7.13
(m, 3H), 5.60 (s, 1H), 5.10 (s, 1H), 3.38-3.34 (m, 1H), 2.02 (d, J = 11.4 Hz, 1H), 1.89
(d, J = 11.4 Hz, 1H), 1.77–1.70 (m, 2H), 1.50–1.43 (m, 3H), 1.39 (s, 18H), 1.23 –
1
3
1.86 (m, 3H); C NMR (150 MHz, CDCl ) δ(ppm): 163.2, 153.1, 148.7, 136.6, 135.5,
3

1
2
132.4, 124.0, 122.0, 121.0, 81.9, 75.6, 34.3, 32.8, 32.3, 30.3, 25.8, 24.2; HRMS (ESI):
+
Calcd for C H NO Na ([M+Na] ): 418.2722, Found: 418.2718; Anal. Calcd for
2
6
37
2
3
4
5
C H NO : C, 78.94; H, 9.43; N, 3.54. Found: C, 78.89; H, 9.50; N, 3.62.
26 37 2
2,6-di-tert-butyl-4-((4-ethylpiperazin-1-yl)(pyridin-2-yl)methyl)phenol (CH-H-5):
o
1
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
White solid, yield 57%, m.p. 129.5-130.5 C; H NMR (600 MHz, CDCl ) δ(ppm):
3
8.49 (d, J = 4.8 Hz, 1H), 7.60 (t, J = 7.2 Hz, 1H), 7.55 (d, J = 7.8 Hz, 1H), 7.22 (s,
2H), 7.07( t, J = 6.0 Hz, 1H), 5.07 (s, 1H), 4.35 (s, 1H), 2.50 – 2.41 (m, 10H), 1.39 (s,
1
3
18H), 1.07 (t, J = 7.2 Hz, 3H); C NMR (150 MHz, CDCl ) δ(ppm): 162.8, 152.9,
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
149.0, 136.4, 135.7, 131.3, 125.1, 122.22, 121.7, 77.8, 53.0, 52.3, 51.6, 34.3, 30.4,
+
11.9; HRMS (ESI): Calcd for C H N ONa ([M+Na] ): 432.2991, Found: 432.2993;
2
6
39
3
Anal. Calcd for C H N O: C, 76.24; H, 9.60; N, 10.26. Found: C, 76.31; H, 9.56; N,
2
6
39
3
10.42.
2,6-di-tert-butyl-4-((4-phenylpiperazin-1-yl)(pyridin-2-yl)methyl)phenol (CH-H-6):
o
1
White solid, yield 52%, m.p. 101.2-102.1 C; H NMR (400 MHz, CDCl ) δ(ppm):
3
8.53 (d, J = 4.8 Hz, 1H), 7.65 – 7.59 (m, 2H), 7.27 (s, 2H), 7.23(d, J = 8.8 Hz, 2H )
7.12 – 7.08 (m, 1H), 6.90 (d, J = 8.0 Hz, 2H), 6.84 (t, J = 7.2 Hz, 1H ), 5.12 (s, 1H),
4.38 (s, 1H), 3.24 – 3.16 (m, 4H), 2.62 – 2.59 (m, 2H), 2.52 – 2.48 (m, 2H), 1.40 (s,
1
3
1H); C NMR (100 MHz, CDCl ) δ(ppm): 162.8, 153.0, 151.4, 149.0, 136.6, 135.8,
3
131.2, 129.1, 125.0, 122.2, 121.9, 77.9, 51.9, 49.2, 34.4, 30.4; HRMS (ESI): Calcd for
+
C H N ONa ([M+Na] ): 480.2991, Found: 480.2995; Anal. Calcd for C H N O: C,
3
0
39
3
30 39
3
78.73; H, 8.59; N, 9.18. Found: C, 78.64; H, 8.72; N, 9.31.
2,6-di-tert-butyl-4-((4-butylpiperazin-1-yl)(pyridin-2-yl)methyl)phenol (CH-H-7):
o
1
Light yellow solid, yield 63%, m.p. 57.8-59.2 C; H NMR (400 MHz, CDCl )
3
δ(ppm): 8.49 (d, J = 4.8 Hz, 1H), 7.61-7.59 (m, 1H), 7.56 (d, J = 7.8 Hz, 1H), 7.22 (s,
2H), 7.06 (t, J =6.0 Hz, 1H), 5.07 (s, 1H), 4.34 (s, 1H), 2.46 (s, 4H), 2.39 – 2.31 (m,
4H), 1.45 (t, J = 7.8Hz, 2H), 1.40 (s, 18H), 1.33 – 1.25 (m, 4H), 0.90 (t, J = 7.2 Hz,
1
3
3H); C NMR (100 MHz, CDCl ) δ(ppm): 162.9, 152.8, 149.0, 136.4, 135.6, 131.4,
3

1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
125.0, 122.2, 121.7, 77.9, 58.5, 53.5, 51.8, 34.3, 30.4, 29.0, 20.8, 14.0; HRMS (ESI):
+
Calcd for C H N ONa ([M+Na] ): 460.3303, Found: 460.3309; Anal. Calcd for
2
8
43
3
C H N O: C, 76.84; H, 9.90; N, 9.60. Found: C, 76.92; H, 9.81; N, 9.73.
2
8
43
3
2,6-di-tert-butyl-4-((4-(cyclopropylmethyl)piperazin-1-yl)(pyridin-2
yl)methyl)phenol (CH-H-8):
o
1
Light yellow solid, yield 49%, m.p. 48.2-50.3 C; H NMR (400 MHz, CDCl )
3
δ(ppm): 8.45 (d, J = 3.2Hz, 1H), 7.57 (t, J = 5.2 Hz, 1H), 7.52 (d, J = 5.2 Hz, 1H),
7.17 (s, 2H), 7.04 (t, J = 4.0 Hz, 1H), 5.04 (s, 1H), 4.30 (s, 1H), 2.59 – 2.18 (m, 10H),
1.42 (s, 18H), 0.84 – 0.79 (m, 1H), 0.44 (d, J = 5.2 Hz, 2H), 0.04 (d, J = 4.8 Hz, 2H);
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
1
3
C NMR (100 MHz, CDCl ) δ(ppm): 162.9, 152.8, 148.9, 136.4, 135.7, 131.3, 125.0,
3
122.2, 121.6, 77.9, 63.8, 53.5, 51.7, 34.3, 30.4, 8.3, 3.9, 3.8; HRMS (ESI): Calcd for
+
C H N ONa ([M+Na] ): 458.3147, Found: 458.3141; Anal. Calcd for C H N O: C,
2
8
41
3
28 41
3
77.20; H, 9.49; N, 9.65. Found: C, 77.08; H, 9.54; N, 9.66.
2,6-di-tert-butyl-4-((4-cyclohexylpiperazin-1-yl)(pyridin-2-yl)methyl)phenol (CH-
H-9):
o
1
Light yellow solid, yield 50%, m.p. 129.2-130.8 C; H NMR (400 MHz, CDCl )
3
δ(ppm): 8.49 (d, J = 4.2 Hz, 1H), 7.60 – 7.58 (m, 1H), 7.55 (d, J = 7.8Hz,1H), 7.21 (s,
1H), 7.06 (t, J = 6.0 Hz, 2H), 5.07(s, 1H), 4.32 (s, 1H), 2.59 (s, 4H), 2.45 (s, 2H), 2.37
(s, 2H), 2.22 – 2.18 (m, 1H), 1.88 (d, J = 10.2 Hz, 2H), 1.76 (d, J = 12.0 Hz, 2H), 1.60
1
3
(d, J = 12.6 Hz, 1H), 1.39 (s, 18H), 1.26 – 1.18 (m, 2H); C NMR (100 MHz, CDCl )
3
δ(ppm): 162.9, 152.8, 149.0, 136.4, 135.6, 131.4, 1251, 122.2, 121.6, 77.9, 63.5, 52.3,
+
49.1, 34.3, 30.4, 29.0, 26.3, 25.9; HRMS (ESI): Calcd for C H N ONa ([M+Na] ):
3
0
45
3
486.3460, Found: 486.3464; Anal. Calcd for C H N O: C, 77.71; H, 9.78; N, 9.06.
3
0
45
3
Found: C, 77.67; H, 9.84; N, 9.11.
2,6-di-tert-butyl-4-((4-isopropylpiperazin-1-yl)(pyridin-2-yl)methyl)phenol (CH-H-
10):
o
1
Light yellow solid, yield 67%, m.p. 137.6-138.5 C; H NMR (400 MHz, CDCl )
3
δ(ppm): 8.54 (d, J = 4.2 Hz, 1H), 7.68 (td, J = 7.8, 1.2 Hz, 1H), 7.41 (d, J = 7.8 Hz,

1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
1H), 7.20 (s, 2H), 7.09 (dd, J = 6.6, 5.4 Hz, 1H ), 4.36 (s, 1H), 3.40-3.35 (m, 1H),
3.16 – 3.08 (m, 4H), 2.69 (s, 2H), 2.62 (s, 2H), 1.36 (s, 18H), 1.24 (d, J = 6.0 Hz, 6H);
1
3
C NMR (100 MHz, CDCl ) δ(ppm): 161.3, 153.3, 149.1, 137.1, 136.1, 130.1, 125.0,
3
122.6, 122.2, 76.5, 56.0, 48.9, 47.2, 34.3, 48.9, 47.2, 34.3, 30.3, 16.8, 16.7; HRMS
+
(ESI): Calcd for C H N ONa ([M+Na] ): 446.3147, Found: 446.3143; Anal. Calcd
2
7
41
3
for C H N O: C, 76.55; H, 9.76; N, 9.92. Found: C, 76.58; H, 9.80; N, 9.85.
2
7
41
3
2,6-di-tert-butyl-4-((4-(tert-butyl)piperazin-1-yl)(pyridin-2-yl)methyl)phenol (CH-
H-11):
o
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
Light yellow solid, yield 68%, m.p. 123.1-126.4 C; H NMR (400 MHz, CDCl )
3
δ(ppm): 8.49 (d, J = 3.2 Hz, 1H), 7.61 – 7.56 (m, 2H), 7.20 (s, 2H), 7.07 (t, J = 4.0 Hz,
1
3
1H), 5.06 (s, 1H), 4.31 (s, 1H), 2.60 – 2.38 (m, 8H), 1.39 (s, 18H), 1.05 (s, 9H); C
NMR (100 MHz, CDCl ) δ(ppm): 163.0, 152.8, 149.0, 136.4, 135.6, 131.3, 125.2,
3
122.2, 121.6, 78.0, 52.6, 45.8, 34.3, 30.4, 25.8; HRMS (ESI): Calcd for C H N ONa
2
8
43
3
+
([M+Na] ): 460.3303, Found: 460.3307; Anal. Calcd for C H N O: C, 76.84; H,
2
8
43
3
9.90; N, 9.60. Found: C, 76.88; H, 9.96; N, 9.68.
2,6-di-tert-butyl-4-((3-fluorophenyl)(morpholino)methyl)phenol (CH-H-12):
o
1
Light yellow solid, yield 63%, m.p. 120.1-121.1 C; H NMR (600 MHz, CDCl )
3
δ(ppm): 7.24 – 7.15 (m, 5H), 6.85 (t, J = 8.4 Hz, 1H), 5.09 (s, 1H), 4.10 (s, 1H), 3.73
1
3
– 3.68 (m, 4H), 2.33 (s, 4H), 1.41 (s, 18H); C NMR (150 MHz, CDCl ) δ(ppm):
3
1
2
163.0 (d, J =243.9), 152.8, 146.0, 135.8, 131.8, 129.8, 124.5, 123.5, 114.5 (d, J
CF
CF
3
=21.3 Hz), 113.6 (d, J = 21.5 Hz), 76.2, 67.2, 52.5, 34.3, 30.4; HRMS (ESI): Calcd
CF
+
for C H FNO Na ([M+Na] ): 422.2471, Found: 422.2475; Anal. Calcd for
2
5
34
2
C H FNO : C, 75.15; H, 8.58; N, 4.76. Found: C, 75.23; H, 8.65; N, 4.74.
2
5
34
2
2,6-di-tert-butyl-4-((6-methylpyridin-2-yl)(morpholino)methyl)phenol (CH-H-13):
o
1
Light yellow solid, yield 48%, m.p. 68.5-69.3 C; H NMR (400 MHz, CDCl )
3
δ(ppm): 7.50 (t, J = 7.6 Hz, 1H), 7.41 (d, J = 7.6 Hz, 1H), 7.28 (s, 2H), 6.94 (d, J =

1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7.6 Hz, 1H), 5.09 (s, 1H), 4.29 (s, 1H), 3.73 – 3.66 (m, 4H), 2.50 (s, 3H), 2.42–
1
3
2.40(m, 2H), 2.33-2.30 (m, 2H), 1.40 (s, 18H); C NMR (100 MHz, CDCl ) δ(ppm):
3
161.9, 157.5, 152.9, 136.7, 135.7, 131.1, 125.1, 121.4, 118.9, 78.5, 67.2, 52.6, 34.4,
+
30.4; HRMS (ESI): Calcd for C H N O Na ([M+Na] ): 419.2674, Found: 419.2679;
2
5
36
2
2
Anal. Calcd for C H N O : C, 75.72; H, 9.15; N, 7.06. Found: C, 75.84; H, 9.19; N,
2
5
36
2
2
7.13.
2,6-di-tert-butyl-4-((3-methylpyridin-2-yl)(morpholino)methyl)phenol (CH-H-14):
o
1
Light yellow solid, yield 56%, m.p. 95.3-96.6 C; H NMR (400 MHz, CDCl )
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
δ(ppm): 8.51 (d, J = 2.8Hz, 1H), 7.33 (d, J = 7.2 Hz, 1H), 7.27 (d, J = 13.2 Hz, 2H),
6.97 – 6.97 (m, 1H), 5.08 (s, 1H), 4.41 (s, 1H), 3.74 (d, J = 13.6 Hz, 4H), 2.42 (s, H),
1
3
2.37 (s, 7H) 1.38 (s, 18H); C NMR (100 MHz, CDCl ) δ(ppm): 158.9, 153.0, 147.1,
3
138.4, 135.5, 131.7, 129.4, 125.9, 121.4, 74.2, 66.9, 52.9, 34.3, 30.4, 29.6, 19.3;
+
HRMS (ESI): Calcd for C H N O Na ([M+Na] ): 419.2674, Found: 419.2672; Anal.
2
5
36
2
2
Calcd for C H N O : C, 75.72; H, 9.15; N, 7.06. Found: C, 75.77; H, 9.18; N, 7.07.
2
5
36
2
2
4-((3,5-di-tert-butylphenyl)(pyridin-2-yl)methyl)morpholine (CH-H-15):
o
1
Light yellow solid, yield 73%, m.p. 93.6-94.5 C; H NMR (600 MHz, CDCl )
3
δ(ppm): 8.51 (d, J = 4.2 Hz, 1H), 7.61 (td, J = 8.4, 1.8 Hz, 1H), 7.57 (d, J = 7.8 Hz,
1H), 7.33 (d, J = 1.8 Hz, 2H), 7.25 (d, J = 1.8 Hz, 2H), 7.09–7.07 (m, 1H), 4.38 (s,
1
3
1H), 3.76–3.69 (m, 4H), 2.44 – 2.34 (m, 2H), 1.29 (s, 18H); C NMR (150 MHz,
CDCl ) δ(ppm): 162.1, 150.8, 149.1, 139.5, 136.5, 122.6, 122.3, 121.9, 121.2, 79.0,
3
+
67.1, 52.7, 34.8, 31.5; HRMS (ESI): Calcd for C H N ONa ([M+Na] ): 389.2569,
2
4
34
2
Found: 389.2573; Anal. Calcd for C H N O: C, 78.64; H, 9.35; N, 7.64. Found: C,
2
4
34
2
78.66; H, 9.42; N, 7.67.
1-((3,5-di-tert-butylphenyl)(pyridin-2-yl)methyl)-4-methylpiperazine (CH-H-16):
o
1
Light yellow solid, yield 78%, m.p. 97.8-98.1 C; H NMR (400 MHz, CDCl )
3
δ(ppm): 8.48 (s, 1H), 7.54 (dd, J = 14.4, 7.2 Hz, 2H), 7.31 (s, 2H), 7.23 (s, 1H), 7.04
1
3
(s, 1H), 4.39 (s, 1H), 2.51 (s, 8H), 2.31 (s, 3H), 1.27 (s, 18H); C NMR (100 MHz,