N-benzylpiperidinol derivatives as novel USP7 inhibitors: Structure–activity relationships and X-ray crystallographic studies

Article abstract of DOI:10.1016/j.ejmech.2020.112279

USP7 as a deubiquitinase plays important roles in regulating the stability of some oncoproteins including MDM2 and DNMT1, and thus represents a potential anticancer target. Through comparative analysis of USP7 co-crystal structures in complex with the rep

Full text of DOI:10.1016/j.ejmech.2020.112279

Journal Pre-proof
N-benzylpiperidinol derivatives as novel USP7 inhibitors: Structure–activity
relationships and X-ray crystallographic studies
Minglei Li, Shengjie Liu, Hui Chen, Xinyu Zhou, Jin Zhou, Shuxi Zhou, Haoliang Yuan,
Qing-Long Xu, Jun Liu, Keguang Cheng, Hongbin Sun, Yue Wang, Caiping Chen,
Xiaoan Wen
PII:
S0223-5234(20)30248-8
DOI:
https://doi.org/10.1016/j.ejmech.2020.112279
EJMECH 112279
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 January 2020
Revised Date: 25 March 2020
Accepted Date: 25 March 2020
Please cite this article as: M. Li, S. Liu, H. Chen, X. Zhou, J. Zhou, S. Zhou, H. Yuan, Q.-L. Xu, J. Liu,
K. Cheng, H. Sun, Y. Wang, C. Chen, X. Wen, N-benzylpiperidinol derivatives as novel USP7 inhibitors:
Structure–activity relationships and X-ray crystallographic studies, European Journal of Medicinal
Chemistry (2020), doi: https://doi.org/10.1016/j.ejmech.2020.112279.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier Masson SAS.

N-benzylpiperidinol derivatives as novel USP7 inhibitors: structure–activity
relationships and X-ray crystallographic studies
a,
a,
a,
a
a
a
†
†
Minglei Li , Shengjie Liu †, Hui Chen , Xinyu Zhou , Jin Zhou , Shuxi Zhou ,
a
b
Haoliang Yuan a, Qing-Long Xu a, Jun Liu , Keguang Cheng , Hongbin Sun a, Yue
Wang c,*, Caiping Chen a,*, Xiaoan Wen a,*
aJiangsu Key Laboratory of Drug Discovery for Metabolic Disease, State Key
Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009,
China
b State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal
Resources, School of Chemistry and Pharmacy of Guangxi Normal University, Guilin
541004, China
c School of Science, China Pharmaceutical University, Nanjing 211198, China
† These authors contributed equally to this article.
*
Corresponding authors: Tel/fax: +86-25-83271050. E-mail: wxagj@126.com (X. A.
Wen); caiping.chen@cpu.edu.cn (C. P. Chen); zwy_1115@126.com (Y. Wang)
Graphical abstract
Highlights
ꢀ Fifty-five piperidinol derivatives were synthesized and biologically evaluated.
ꢀ Compound L55 exhibited potent USP7 inhibitory activity (IC50 = 40.8 nM).

ꢀ X-ray crystallographic studies revealed a new pose for L55 to bind to USP7.
ꢀ L55 downregulated MDM2 and DNMT1 and upregulated p53 and p21 in RS4;11
cells.
ꢀ LNCaP (IC50 = 29.6 nM) and RS4;11 (IC50 = 41.6 nM) were highly sensitive to
L55.
Abstract
USP7 as a deubiquitinase plays important roles in regulating the stability of some
oncoproteins including MDM2 and DNMT1, and thus represents a potential
anticancer target. Through comparative analysis of USP7 co-crystal structures in
complex with the reported piperidinol inhibitors, we noticed that the USP7 Phe409
sub-site might have good adaptability to the ligands. Based on this observation, 55
N-aromatic and N-benzyl piperidinol derivatives were designed, synthesized and
biologically evaluated, among which compound L55 was identified as a highly
selective and potent USP7 inhibitor (IC50 = 40.8 nM, KD = 78.3 nM). X-ray
crystallographic studies revealed that L55 bound to USP7 with a new pose that was
very different from the previously reported inhibitors. The results of cellular assays
showed that L55 had strong antitumor activity against LNCaP (IC50 = 29.6 nM) and
RS4;11 (IC50 = 41.6 nM) cells, probably through inducing cell death and restricting
G0/G1 and S phases. Moreover, L55 dose-dependently reduced the protein levels of
MDM2 and DNMT1 and increased the protein levels of p53 and p21. These findings
could have valuable implications for designing novel structural classes of USP7
inhibitors.
Key words: USP7, piperidine, structure-activity relationship, crystallographic study,
deubiquitinase
1. INTRODUCTION
The ubiquitin–proteasome system (UPS) is critical to maintain protein homeostasis

in the human body. With ubiquitin (a highly conserved 76-amino-acid polypeptide)
1
linked to or removed from target proteins , the fates of these proteins can be
regulated in many aspects, such as cellular localization, physiological functions and
2
protein stability . Not surprisingly, abnormal modulation of the UPS is closely
3
4
5
correlated to various diseases (e.g., cancer , infection , and neurodegeneration ).
Therefore, pharmacological interference of UPS represents a promising therapeutic
approach to diseases such as cancer, as evidenced by the approval of the proteasome
inhibitor bortezomib for the treatment of multiple myeloma and mantle cell
lymphoma 6. As an important component of the UPS, deubiquitinating enzymes play a
key role in maintaining the normal functions of the UPS 7-10. Ubiquitin-specific
proteases (USP) constitute the largest family of deubiquitinating enzymes, among
which ubiquitin-specific protease 7 (USP7, also known as HAUSP) has been
extensively investigated as a potential target for cancer treatment. Notably, USP7
deubiquitinates and stabilizes MDM2, leading to decreased stability of the tumor
suppressor p53 11, 12. Inhibition of USP7 has been shown to increase p53 stability, thus
promoting cancer cell apoptosis 12-14. In addition, USP7 deubiquitinates many other
16
proteins, including DNMT1 15, N-Myc
and Tip60 17, 18, among others. Thus,
inhibition of USP7 can also induce anti-tumor effects in a p53-independent manner 19.
Early efforts by other research groups identified several classes of USP7 inhibitors,
including HBX41108, HBX19818 and P5091 (Fig. 1) 20-22, but the potency and
selectivity of theses inhibitors were poor. Recently, a series of piperidinol derivatives
(Fig. 1: FT671, FT827, CP4, CP5) were identified as potent and highly selective
USP7 inhibitors 23-26, and facile semi-synthetic methods were established to produce
key probe reagents for exploring deubiquitinase activity 27, 28. X-ray crystallographic
studies revealed that the piperidinol and pyrimidone subunits in FT671 and CP5 were
two key pharmacophores with important hydrogen bond interactions with USP7
Asp295, Val296, Gln297 and Phe409, respectively 24, 26. In particular, we noticed that
the Phe409 sub-site of USP7 showed a great adaption to the ligands, which could be
used for the design of new inhibitors.

O
Cl
N
O
O2N
S
O
Cl
N
N
CN
CN
N
N
H
S
Cl
Cl
HBX19818
P5091
HBX41108
O
N
N
F
OH
O
OH
N
N
N
N
N
N
N
O
HN
N
N
N
S
O
O
F
F
O
FT671
FT827
F
O
N
O
N
OH
OH
N
N
N
N
N
N
N
N
O
O
CF3
CP5
CP4
NH2
NH2
Fig. 1. Structures of some USP7 inhibitors.
As a part of our program to develop novel USP7 inhibitors for cancer therapy 16, 29,
30, we worked on the structural modifications of the piperidinol class of USP7
inhibitors, with a particular focus on the USP7 Phe409 sub-site. Thus, a series of
N-aromatic and N-benzyl piperidinol derivatives were designed, synthesized and
biologically evaluated. Among these compounds, compound L55 was found to
potently inhibit USP7 (IC50 = 40.8 nM, KD = 78.3 nM). Moreover, L55 showed strong
antiproliferative activity in the cancer cell lines LNCaP (IC50 = 29.6 nM) and RS4;11
(IC50 = 41.6 nM). More importantly, X-ray crystallographic studies revealed that L55
bound to USP7 with a new pose that was very different from the previously reported
inhibitors.
2. RESULTS AND DISCUSSION
2.1 Design of target compounds
Considering that the USP7 Phe409 sub-site exhibited great adaptability to the
ligands, we examined the potency outcome of replacing the flexible long chain of
FT671 with a heteroaromatic group, and thus, a series of N-heteroaromatic
piperidinol derivatives (Fig. 2: L1-L13) were designed. It was expected that the

heteroatoms could make hydrogen bond interactions with USP7 Try 465, and the
heteroaromatic rings could mimic the bending pose of the long chain of FT671.
Fig. 2. Design of the target compounds.
In addition, we noticed that although the piperidinol amide group of FT827
hydrogen bonded with USP7 Try465, it also formed a dihedral angle of nearly 90
degrees with its attached phenyl group 23. Thus, we considered deleting the amide
oxygen atom and the outer phenyl group of the diphenyl so that the ligands could be
easily adjusted to the optimal binding poses. In this regard, a series of N-benzyl
piperidinol derivatives were designed (Fig. 2: L14-L53). Furthermore, upon finding
2-chloro-4-methoxycarbonylbenzyl as one of the preferred groups, compounds L54
and L55 were designed, as the (4-aminomethyl)phenyl group and the
pyrazolo[4,3-d]pyrimidine skeleton were reported to add more hydrogen bond
interactions with USP7 24.
2.2 Synthesis of target compounds
The synthetic route to L1-L49 is depicted in Scheme 1. The key intermediate 5 was
synthesized according to previously reported procedure 23. In the presence of
triethylamine,
4-fluorophenylhydrazine
hydrochloride
and
2-(ethoxymethylene)malonoitrile were cyclized under heating to form pyrozole 1,
which was hydrolyzed to afford amide 2. Reaction of 1 with triethyl
methanetricarboxylate in acetic anhydride under reflux afforded pyrimidone 3,
followed by a nucleophilic substitution reaction occurring with tert-butyl
1-oxa-6-azaspiro[2.5]octane-6-carboxylate in the presence of Cs2CO3 to give
piperidinol 4. Compound 4 was Boc-deprotected by TFA and finally reacted with
diverse halides to offer the target compounds L1-L49. Ester L49 was hydrolyzed in

50% hydroxylamine aqueous solution to give carboxylic acid L50, which was
amidated with different amines to provide L51-L53.
Scheme 1. Synthesis of L1-L49 Reagents and conditions: (a) TEA, EtOH, reflux, 10
.
h, 66%; (b) con. H2SO4, r.t., 75%; (c) triethyl orthoformate, acetic anhydride, reflux,
53%; (d) tert-butyl 1-oxa-6-azaspiro[2.5]octane-6-carb-oxylate, Cs2CO3, DMF, 80 ℃,
33%; (e) TFA, DCM, r.t.; (f) K2CO3, DMF, r.t. or 120 ℃, 16-86%; (g) 50% NH2OH
in H2O, 90 ℃, 73%; (h) for L51 or L53: oxalyl chloride, DMF (cat.), DCM; then
ammonia methanol solution or 50% NH2OH in H2O, r.t., 30% or 31%; for L52: 27%
methylamine in EtOH, HATU, DIPEA, DMF, r.t., 40%.
The synthesis of L54 and L55 is outlined in Scheme 2. The intermediate 11 was
prepared from commercially available methyl 4-nitro-1H-pyrazole-3-carboxylate over
six steps: methylation, reduction, cyclization, bromination, aminolysis cleavage of
24
epoxy
and
Boc-deprotection
.
Treatment
of
11
with
methyl
4-(bromomethyl)-3-chlorobenzoate in the presence of potassium carbonate gave
compound 12, which reacted with boronate 15 or 16 under Suzuki coupling
conditions to afford 17 or L54. Boc-deprotection of 17 in trifluoroacetic acid gave
compound L55 as the trifluoroacetate form.

Scheme 2. Synthesis of L54 and L55. Reagents and conditions: (a) K2CO3, MeI,
acetone, 70 , 27%; (b) Pd/C, H2, MeOH, r.t., 97%; (c) formamidine acetate, DIPEA,
n-BuOH, 110 °C, 94%; (d) Br2, AcOH, 95 °C, 36%; (e) tert-butyl
1-oxa-6-azaspiro[2.5]octane-6-carboxylate, Cs2CO3, DMF, 80 °C, 33%; (f) TFA,
DCM, r.t.; (g) methyl 4-(bromomethyl)-3-chlorobenzoate, K2CO3, DMF, r.t., 32%; (h)
for 13: (Boc)2O, N2, DIPEA, CHCl3, r.t., 92%; for 14: acetyl chloride, TEA, dry DCM,
76%; (i) AcOK, Bis(pinacolato)diboron, Pd(dppf)Cl2, DMSO, Ar, 80 °C, 16-95%; (j)
Pd(PPh3)4, K2CO3, DMF, Ar, 110 °C, 60-65%; (k) TFA, DCM, r.t., 79%.
2.3 USP7 inhibitory activity and SAR analysis of the target compounds
The inhibitory activity of all the target compounds against full-length USP7 was
30
evaluated using a Ub-Rho assay with the trifluoroacetic acid salt of CP4 as the
positive control (its synthetic details are included in Supporting Information). A
preliminary screen was performed at a compound concentration of 10 ꢀM or 1 ꢀM.
For those compounds with inhibition ≥50%, IC50 values were further determined.
As shown in Table 1, all N-aromatic compounds had poor or no activity at 10 µM,
with the exception of halides L5-L7, which had 32-43% inhibition against USP7.

Table 1. USP7 inhibitory activity of L1-L13
Cpd
R
Inhibition (%)
NA
Cpd
R
Inhibition (%)
NA
L1
L8
NA
NA
21.9±7.8
NA
L2
L3
L4
L5
L9
15.5±2.2
19.6±4.4
32.1±1.0
L10
L11
L12
NA
43.3±5.2
39.3±6.4
NA
L6
L13
(trifluoroacetic
acid salt)
92.4±6.8 a
L7
CP4
a Determined at a compound concentration of 1 µM. NA: not active.
As shown in Table 2, N-benzyl piperidinol compound L14 exhibited poor activity,
and no improvement was observed on aza-benzyl compounds L15-17. Notably,
introduction of NO2, CN, halides and COOMe to the ortho (L20, L23, L25, L28 and
L30) and para (L22, L24, L27, L29 and L31) positions of the phenyl group of the
N-benzyl fragments generally led to a large increase in activity. In particular, ortho
chloride L25, ortho bromide L28 and para ester L37 had IC50 values of 3.4, 3.1 and
4.5 µM, respectively. In contrast, ortho ester L35 was not active, and compounds
L32-L34, with electron-donating groups CH3 and CF3, had poor activity, indicating a
strong preference of small and electron-withdrawn groups for the ortho and para
positions.
The above findings prompted us to investigate ortho and para di-substituted
compounds. To our delight, combinations of ortho-halides and para-COOMe

dramatically improved the activity. Compounds L43 (IC50 = 0.8 µM), L44 (IC50 = 0.8
µM) and L45 (IC50 = 0.9 µM) bearing both ortho-halides and para-COOMe were
more potent than compounds L23 (IC50 = 9.8 µM), L25 (IC50 = 3.4 µM) and L28
(IC50 = 3.1 µM) bearing only ortho-halides. Notably, very small structural changes on
methyl ester L44 caused sharp decrease in potency since acid L50 and amides L51-53
were almost not active at 1 µM.
Table 2. USP7 inhibitory activity of L14-L53
Inhibition
(%)
IC50
Inhibition
(%)
IC50
Cpd
R
Cpd
R
(µM)
(µM)
L14
24.9±7.1
ND
ND
ND
ND
L35
NA
ND
L15
L16
L17
NA
L36
L37
L38
NA
ND
20.7±5.5
14.3±6.6
65.2±20.7
28.6±5.8
4.5±0.6
ND
L18
L19
NA
NA
ND
ND
L39
L40
17.1±7.1
75.3±0.7
ND
1.6±0.4
L20
L21
L22
40.5±5.3
21.4±4.5
53.7±3.8
ND
ND
L41
L42
L43
66.5±18.3
72.7±2.9
61.1±3.3 a
3.3±0.9
1.9±0.6
0.8±0.3
9.0±4.5

Inhibition
(%)
IC50
Inhibition
(%)
IC50
Cpd
L23
L24
L25
R
Cpd
L44
L45
L46
R
(µM)
(µM)
59.6±4.5
30.9±7.8
82.4±1.5
9.8±1.2
ND
65.9±3.7 a
55.7±6.7 a
41.2±5.5
0.8±0.2
0.9±0.2
ND
3.4±0.4
L26
23.9±6.9
ND
L47
62.9±18.6 11.4±1.2
L27
L28
60.6±21.5 8.0±0.9
68.6±10.3 3.1±0.4
L48
L49
43.5±3.9
ND
ND
42.7±5.1 a
L29
L30
L31
L32
51.9±3.0
6.7±1.8
L50
L51
L52
L53
17.8±8.1 a
NA a
ND
ND
ND
ND
59.4±13.2 8.1±2.7
18.2±5.7
34.9±3.8
ND
ND
NA a
NA a
L33
L34
31.8±5.2
23.1±5.6
ND
ND
(trifluoroacetic
acid salt)
0.0197±0
.0072
CP4
92.4±6.8 a
a Determined at a compound concentration 1 µM; NA: not active; ND: not determined.
As shown in Table 3, replacing the pyrazolo[3,4-d]pyrimidone fragment with the
pyrazolo[4,3-d]pyrimidone fragment significantly increased the potency, consistent
24
with a previous report . The intermediate 12 (IC50 = 156.6 nM) was 4 times more
potent than L44. Substitution of the bromine atom of 12 with 4-aminomethylphenyl
group further improved the potency by approximately 4-fold, giving rise to the most
potent compound in this study, L55 (IC50 = 40.8 nM). Boc-protection or acetylation of
the benzylamino group of L55 caused a significant reduction of potency (compound
17 IC50 = 208.8 nM; L54 IC50 = 121.3 nM).

Table 3. USP7 inhibitory activity of 12, 17, L54 and L55
Cpd
R3
Br
IC50 (nM)
12
156.6±9.5
17
208.8±21.9
L54
121.3±0.56
L55
CP4
40.8±4.4
19.7±7.2
(trifluoroacetic acid salt)
(trifluoroacetic acid salt)
The deubiquitinase inhibitory selectivity of compound
L55 was further evaluated. As shown in Table S4 (see Supporting Information),
similar to the control compound CP4 (trifluoroacetic acid salt), compound L55 did
not inhibit a panel of the tested deubiquitinases UCH-L1, UCH-L5, USP2, USP25,
USP28 and USP11 at 20 µM, indicating its excellent inhibitory selectivity for USP7.
2.4 Binding affinity of L55 to USP7
The binding affinity of L55 to USP7 was determined using Biolayer Interferometry
(BLI) technology. As shown in Fig. 3, L55 bound to the USP7 catalytic domain with
high affinity (KD = 78.3 nM), which was comparable to the positive control CP4
(trifluoroacetic acid salt, KD = 41.6 nM) and consistent with the IC50 values.

Fig. 3. Binding profiles for the interaction of CP4 (A) and L55 (B) with the USP7
catalytic domain (determined by BLI).
2.5 X-ray crystallographic study of USP7 co-crystals in complex with L55
To reveal the crucial interactions between compound L55 and USP7, we cultivated
co-crystals of USP7 catalytic domain protein with L55 and determined their structure
by an X-ray diffraction method. A co-crystal structure at 2.26 Å resolution was
successfully obtained. The X-ray crystallographic studies showed that similar to CP5
24, L55 bound to USP7 in the ubiquitin C-terminal binding cleft between the palm and
thumb areas (Fig. 4A and Fig. 4B). The rigid skeletons of L55 and CP5 had very
similar poses and interactions with USP7 (Fig. 4C and Fig. 4D). Their
(4-aminomethyl)phenyl groups both stretched out to the USP7 surface and made
hydrogen bond interactions with Gln351. Their pyrazolopyrimidone groups were both
buried in USP7 and made multiple hydrogen bond interactions with Gln297, Arg408
and Phe409.

Fig. 4. Comparison of the co-crystal structures of L55 (A, C) and CP5 (B, D) binding
to the USP7 catalytic domain.
Unexpectedly, in the USP7 complex with L55, Phe409 showed a large upshift, thus
adding good pi-pi interactions to the pyrazole ring of L55 (Fig. 4C). Moreover, L55
was very different from CP5 in the pose of the piperidinol group (Fig. 4D), although
their hydroxyl groups both made hydrogen bond interactions with Asp295 and Val296.
The piperidinol ring of L55 stood up and was nearly parallel to the pyrimidone ring,
probably due to the lack of a hydrogen bond interaction with Tyr465, while the
piperidinol ring of CP5 laid flat in the USP7 pocket. Additionally, unlike the flexible
chain in CP5, the benzyl group of L55 did not bend inward but stretched outside,
which was probably the cause of the Phe409 shift. The 2-chloro and
4-methoxycarbonyl groups on the benzyl of L55 were very close to the binding
pocket boundary, thus not allowing for replacement by large groups. Together, this
new co-crystal structure would assist in the design of novel USP7 inhibitors.

2.6 In vitro anticancer effect and action mechanism of L55
We next performed the cell viability assay for compound L55 against a panel of
cancer cell lines including LNCaP, RS4;11, HCT116, NB4, K562 and Huh-7 cells.
RS4;11 (lymphoblastic leukemia cell line) and LNCaP (prostate cancer cell line) were
previously reported to be highly sensitive to CP4 24. As expected, we found that
among the test cell lines, RS4;11 and LNCaP were the most sensitive to L55 and CP4.
As shown in Table 4, L55 exhibited nanomolar levels of inhibitory activity on LNCaP
and RS4;11 and was slightly less potent than CP4. Conversely, L55 showed very
weak inhibitory activity on HCT116, NB4, K562 and Huh-7 cells (see Supporting
Information Fig. S3).
Table 4. Inhibitory activity of L55 and CP4 on cancer cell growth
LNCaP
RS4;11
HCT116
IC50/µM
NB4
K562
Huh-7
Cpd
IC50/nM
IC50/nM
IC50/µM
IC50/µM
IC50/µM
29.6±2.1
20.1±2.6
41.6±4.1
6.9±2.2
32.8±6.2
>50
5.0±0.5
>50
27.9±2.8
24.1±1.9
3.6±0.4
L55
CP4
10.7±1.6
To understand the anti-cancer mechanism of L55, we assessed its effects on cell
death and the cell cycle in RS4;11 cells. The Annexin-V/PI assay revealed that the
ratios of dying cells (Annexin V positive/PI positive, representing late apoptosis or
necrosis) increased significantly with the L55 treatment time (Fig. 5A). Flow
cytometry analysis of the cell cycle distribution revealed a time-dependent increase in
the subG (apoptotic cell) population, further supporting the cytotoxic effect of L55.
Moreover, upon L55 treatment, the proportions of G2/M cells were clearly reduced,
while the proportions of G0/G1 and S cells were not apparently altered, suggesting
that L55 might arrest cell progression by restricting the G0/G1 and S phases (Fig.
5B).

Fig. 5. Effects of L55 on cell death and cell cycle distribution. (A) Flow cytometric
analysis of cell death of RS4;11 cells treated with L55 (1 µM) for 0 h, 24 h, 48 h or 72
h. (B) The cell cycles of RS4;11 cells were analyzed via flow cytometry after
treatment with L55 (1 µM) for 0 h, 24 h, 48 h or 72 h.
We next assessed the effects of compound L55 on downstream pathway
components of USP7 signaling in RS4;11 cells. As shown in Fig. 6, the protein levels
of MDM2 were dose-dependently reduced, while the protein levels of p53 and p21
were increased by L55 treatment. This result was consistent with the finding that L55
induced cell death and cell cycle arrest. In addition, another USP7 substrate, DNMT1
(DNA methyltransferase 1), was also downregulated by L55 in
a
concentration-dependent manner. Interestingly, L55 also induced a decrease in the
protein level of USP7.

Fig. 6. Effects of L55 on the protein levels of USP7, DNMT1, MDM2, p53 and p21
in RS4;11 cells. Western blotting analysis of RS4;11 cells treated with L55, CP4 (1
µM) or DMSO for 24 h.
3. CONCLUSIONS
In this study, 55 piperidinol derivatives were designed, synthesized and biologically
evaluated to find new USP7 inhibitors. The results of the USP7 inhibitory assay
showed that N-benzyl piperidinol derivatives exhibited potent USP7 inhibitory
activity, among which compound L55 was identified as a highly selective and potent
USP7 inhibitor (USP7 IC50 = 40.8 nM, KD = 78.3 nM). L55 showed strong in vitro
antitumor activity against LNCaP (IC50 = 29.6 nM) and RS4;11 (IC50 = 41.6 nM) cells.
Mechanism studies demonstrated that L55 caused marked cell death and arrested cell
progression through restricting G0/G1 and S phases in RS4;11 cells. Moreover, L55
dose-dependently reduced the protein levels of oncoproteins MDM2 and DNMT1 and
increased the protein levels of tumor suppressors p53 and p21. Interestingly, L55 also
reduced USP7 protein levels in RS4;11 cells. More importantly, the co-crystal
structure of USP7 in complex with L55 revealed that L55 bound to USP7 at the
previously reported site but with a new pose that was very different from the
previously reported inhibitors. This co-crystal structure of USP7 in complex with L55
would assist in the future design of novel structural classes of USP7 inhibitors.

4. Experimental section
4.1 Chemistry
General information General procedures of the synthesis route are described as
below. Commercially available reagents and solvents were used without further
purification. Analytical thin-layer chromatography (TLC) was performed on silica gel
60 F254 plates (Qingdao Ocean Chemical Company, China). Column
chromatography was carried out on silica gel (200-300 mesh, Qingdao Ocean
1
Chemical Company, China). H and 13C NMR spectra were recorded on an
ACF*300Q Bruker spectrometer with Me4Si as the internal reference. Proton
coupling patterns were described as singlet (s), doublet (d), doublet of doublets (dd),
triplet (t), triplet of doublets (td), quartet (q), multiplet (m), and broad (br). Low and
high-resolution mass spectra (LRMS and HRMS) were obtained in electrospray
ionization mode. The mass analyzer type used for the HRMS measurements was TOF.
The melting point (m. p.) was measured on a microscopic melting point apparatus. All
compounds underwent UPLC to determine purity immediately before processing the
Ub-Rho assays. A Waters Acquity UPLC system comprising the Quaternary Solvent
manager, Sample Manager-FTN, PDA Detector were employed. The UPLC
conditions were as follows: ACQUITY UPLC® BEH C18 column, 2.1×50 mm, 1.7
ꢀm; detection wavelength: 254 nm.
4.1.1
1-(4-fluorophenyl)-5-((4-hydroxy-1-(pyridin-2-yl)piperidin-4-yl)methyl)-1,5-dihydro-
4H-pyrazolo[3,4-d]pyrimidin-4-one (L1)
A mixture of 5 (88 mg, 0.2 mmol) 23, 2-bromopyridine (21 µL, 0.22 mmol) and
K2CO3 (102 mg, 0.74 mmol) in DMF (2 mL) was stirred at 120 °C for 20 h. After
cooling to room temperature, the mixture was diluted with water (10 mL), extracted
with ethyl acetate (10 mL × 3), and the organic layer was washed with water (10
mL×3) and brine (10 mL), dried over sodium sulfate, filtered and concentrated. The
residue was purified by column chromatography (silica gel, petroleum ether : ethyl
acetate = 1 : 1) to give compound L1 (37 mg, 34% yield) as a white solid. m.p.

1
204-206 °C. H NMR (300 MHz, d6-DMSO) δ 8.42 – 8.32 (m, 2H), 8.12 – 8.04 (m,
3H), 7.54 – 7.37 (m, 3H), 6.86 – 6.79 (m, 1H), 6.61 – 6.53 (m, 1H), 4.90 (s, 1H), 4.05
(s, 2H), 4.00 – 3.90 (m, 2H), 3.26 – 3.16 (m, 2H), 1.64 – 1.53 (m, 2H), 1.48 – 1.39 (m,
2H).ESI-MS: m/z 443.1 [M+Na]+. LC tR: 0.910 min, purity 97.24%.
4.1.2
1-(4-fluorophenyl)-5-((4-hydroxy-1-(pyrimidin-2-yl)piperidin-4-yl)methyl)-1,5-dihyd
ro-4H-pyrazolo[3,4-d]pyrimidin-4-one (L2)
Compound L2 was prepared according to the procedure described for compound L1.
L2: white solid, 34% yield. m.p. 204-206 °C. 1H NMR (300 MHz, d6-DMSO) δ 8.42
– 8.30 (m, 4H), 8.15 – 8.00 (m, 2H), 7.51 – 7.35 (m, 2H), 6.62 – 6.54 (m, 1H), 4.96 (s,
1H), 4.37 – 4.26 (m, 2H), 4.06 (s, 2H), 3.29 – 3.23 (m, 2H), 1.64 – 1.37 (m, 4H).
ESI-MS: m/z 422.2 [M+H]+. LC tR: 0.889 min, purity 97.78%.
4.1.3
1-(4-fluorophenyl)-5-((4-hydroxy-1-(quinolin-2-yl)piperidin-4-yl)methyl)-1,5-dihydr
o-4H-pyrazolo[3,4-d]pyrimidin-4-one (L3)
Compound L3 was prepared according to the procedure described for compound L1.
L3: white solid, 39% yield. m.p. 203-205 °C. 1H NMR (300 MHz, d6-DMSO) δ 8.38
(s, 1H), 8.37 (s, 1H), 8.14 – 8.05 (m, 2H), 8.04 – 7.97 (m, 1H), 7.72 – 7.65 (m, 1H),
7.59 – 7.49 (m, 2H), 7.50 – 7.38 (m, 2H), 7.30 – 7.15 (m, 2H), 4.96 (s, 1H), 4.31 –
4.14 (m, 2H), 4.06 (s, 2H), 3.46 – 3.33 (m, 2H), 1.75 – 1.56 (m, 2H), 1.56 – 1.41 (m,
2H). ESI-MS: m/z 471.2 [M+H]+. LC tR: 2.336 min, purity 95.85%.
4.1.4
1-(4-fluorophenyl)-5-((1-(3-fluoropyridin-2-yl)-4-hydroxypiperidin-4-yl)methyl)-1,5-
dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (L4)
Compound L4 was prepared according to the procedure described for compound L1.
L4: white solid, 39% yield. m.p. 165-167 °C. 1H NMR (300 MHz, d6-DMSO) δ 8.45
– 8.30 (m, 2H), 8.16 – 7.93 (m, 3H), 7.54 – 7.33 (m, 3H), 6.91 – 6.77 (m, 1H), 4.91 (s,

1H), 4.06 (s, 2H), 3.80 – 3.63 (m, 2H), 3.27 – 3.15 (m, 2H), 1.77 – 1.62 (m, 2H), 1.55
– 1.43 (m, 2H). ESI-MS: m/z 439.1 [M+H]+. LC tR: 0.597 min, purity 97.42%.
4.1.5
5-((1-(3-chloropyridin-2-yl)-4-hydroxypiperidin-4-yl)methyl)-1-(4-fluorophenyl)-1,5-
dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (L5)
Compound L5 was prepared according to the procedure described for compound L1.
L5: white solid, 36% yield. m.p. 175-177 °C. 1H NMR (300 MHz, d6-DMSO) δ 8.46
– 8.31 (m, 2H), 8.24 – 8.15 (m, 1H), 8.15 – 8.01 (m, 2H), 7.81 – 7.71 (m, 1H), 7.51 –
7.36 (m, 2H), 7.03 – 6.89 (m, 1H), 4.87 (s, 1H), 4.08 (s, 2H), 3.57 – 3.40 (m, 2H),
3.22 – 3.04 (m, 2H), 1.86 – 1.66 (m, 2H), 1.60 – 1.44 (m, 2H). ESI-MS: m/z 455.1
[M+H]+. LC tR: 1.620 min, purity 98.47%.
4.1.6
5-((1-(3-bromopyridin-2-yl)-4-hydroxypiperidin-4-yl)methyl)-1-(4-fluorophenyl)-1,5-
dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (L6)
Compound L6 was prepared according to the procedure described for compound L1.
L6: white solid, 48% yield. m.p. 171-173 °C. 1H NMR (300 MHz, d6-DMSO) δ 8.38
(s, 1H), 8.37 (s, 1H), 8.27 – 8.18 (m, 1H), 8.14 – 8.02 (m, 2H), 7.98 – 7.89 (m, 1H),
7.48 – 7.38 (m, 2H), 6.95 – 6.84 (m, 1H), 4.87 (s, 1H), 4.10 (s, 2H), 3.51 – 3.38 (m,
2H), 3.16 – 3.00 (m, 2H), 1.86 – 1.65 (m, 2H), 1.62 – 1.48 (m, 2H). ESI-MS: m/z
455.1 [M+Na]+. LC tR: 0.900 min, purity 97.56%.
4.1.7
1-(4-fluorophenyl)-5-((4-hydroxy-1-(3-iodopyridin-2-yl)piperidin-4-yl)methyl)-1,5-di
hydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (L7)
Compound L7 was prepared according to the procedure described for compound L1.
L7: white solid, 55% yield. m.p. 215-217 °C. 1H NMR (300 MHz, d6-DMSO) δ 8.41
– 8.35 (m, 2H), 8.29 – 8.22 (m, 1H), 8.19 – 8.12 (m, 1H), 8.12 – 8.04 (m, 2H), 7.50 –
7.36 (m, 2H), 6.81 – 6.71 (m, 1H), 4.86 (s, 1H), 4.10 (s, 2H), 3.41 – 3.31 (m, 2H),

3.14 – 2.98 (m, 2H), 1.88 – 1.68 (m, 2H), 1.64 – 1.45 (m, 2H). ESI-MS: m/z 547.2
[M+H]+. LC tR: 2.157 min, purity 98.52%.
4.1.8
2-(4-((1-(4-fluorophenyl)-4-oxo-1,4-dihydro-5H-pyrazolo[3,4-d]pyrimidin-5-yl)meth
yl)-4-hydroxypiperidin-1-yl)nicotinonitrile (L8)
Compound L8 was prepared according to the procedure described for compound L1.
L8: white solid, 48% yield. m.p. 185-187 °C. 1H NMR (300 MHz, d6-DMSO) δ 8.44
– 8.32 (m, 3H), 8.14 – 7.98 (m, 3H), 7.50 – 7.38 (m, 2H), 6.93 – 6.84 (m, 1H), 4.98 (s,
1H), 4.09 (s, 2H), 4.04 – 3.91 (m, 2H), 3.42 – 3.33 (m, 2H), 1.82 – 1.65 (m, 2H), 1.61
– 1.44 (m, 2H). ESI-MS: m/z 468.1 [M+Na]+. LC tR: 1.037 min, purity 98.23%.
4.1.9
1-(4-fluorophenyl)-5-((4-hydroxy-1-(6-(trifluoromethyl)pyridin-2-yl)piperidin-4-yl)m
ethyl)-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (L9)
Compound L9 was prepared according to the procedure described for compound L1.
L9: white solid, 40% yield. m.p. 233-235 °C. 1H NMR (300 MHz, d6-DMSO) δ 8.43
– 8.32 (m, 2H), 8.14 – 8.01 (m, 2H), 7.79 – 7.65 (m, 1H), 7.53 – 7.38 (m, 2H), 7.20 –
7.07 (m, 1H), 7.04 – 6.93 (m, 1H), 4.97 (s, 1H), 4.06 (s, 2H), 4.05 – 3.96 (m, 2H),
3.29 – 3.18 (m, 2H), 1.70 – 1.56 (m, 2H), 1.53 – 1.38 (m, 2H). ESI-MS: m/z 511.1
[M+Na]+. LC tR: 2.734 min, purity 96.26%.
4.1.10
2-(4-((1-(4-fluorophenyl)-4-oxo-1,4-dihydro-5H-pyrazolo[3,4-d]pyrimidin-5-yl)meth
yl)-4-hydroxypiperidin-1-yl)nicotinaldehyde (L10)
Compound L10 was prepared according to the procedure described for compound L1.
L10: white solid, 49% yield. m.p. 172-175 °C. 1H NMR (300 MHz, d6-DMSO) δ 9.92
(s, 1H), 8.44 – 8.33 (m, 3H), 8.14 – 7.97 (m, 3H), 7.51 – 7.32 (m, 2H), 7.05 – 6.89 (m,
1H), 4.95 (s, 1H), 4.09 (s, 2H), 3.70 – 3.51 (m, 2H), 3.43 – 3.32 (m, 2H), 1.88 – 1.68
(m, 2H), 1.57 – 1.38 (m, 2H). ESI-MS: m/z 471.1 [M+Na]+. LC tR: 0.912 min, purity
96.41%.

4.1.11
6-(4-((1-(4-fluorophenyl)-4-oxo-1,4-dihydro-5H-pyrazolo[3,4-d]pyrimidin-5-yl)meth
yl)-4-hydroxypiperidin-1-yl)picolinaldehyde (L11)
Compound L11 was prepared according to the procedure described for compound L1.
L11: white solid, 19% yield. m.p. 170-172 °C. 1H NMR (300 MHz, d6-DMSO) δ 9.79
(s, 1H), 8.38 (s, 1H), 8.37 (s, 1H), 8.15 – 8.02 (m, 2H), 7.79 – 7.65 (m, 1H), 7.49 –
7.37 (m, 2H), 7.22 – 7.09 (m, 2H), 4.97 (s, 1H), 4.19 – 4.00 (m, 4H), 3.42 – 3.31 (m,
2H), 1.71 – 1.54 (m, 2H), 1.54 – 1.36 (m, 2H). ESI-MS: m/z 471.2 [M+Na]+. LC tR:
0.445 min, purity 95.78%.
4.1.12
1-(4-fluorophenyl)-5-((4-hydroxy-1-(thiazol-2-yl)piperidin-4-yl)methyl)-1,5-dihydro-
4H-pyrazolo[3,4-d]pyrimidin-4-one (L12)
A mixture of 5 (221 mg, 0.5 mmol), 2-bromothiazole (82 mg, 0.5 mmol) and K2CO3
(138 mg, 1 mmol) in DMF (2 mL) was stirred at 90 °C for 67 h. After cooling to room
temperature, the mixture was diluted with water (10 mL), extracted with ethyl acetate
(10 mL × 3), and the organic layer was washed with water (10 mL×3) and brine (10
mL), dried over sodium sulfate, filtered and concentrated. The residue was purified by
column chromatography (silica gel, DCM: CH3OH= 1: 1) to give compound L12 (34
1
mg, 16% yield) as a white solid. m.p. 210-212 °C. H NMR (300 MHz, d6-DMSO) δ
8.36 (d, J = 2.6 Hz, 2H), 8.18 – 7.99 (m, 2H), 7.42 (t, J = 8.8 Hz, 2H), 7.13 (d, J = 3.6
Hz, 1H), 6.80 (d, J = 3.6 Hz, 1H), 5.00 (s, 1H), 4.06 (s, 2H), 3.77 – 3.59 (m, 2H), 3.28
(t, J = 10.8 Hz, 2H), 1.68 (t, J = 10.2 Hz, 2H), 1.60 – 1.43 (m, 2H). ESI-MS: m/z
449.2 [M+Na]+. LC tR: 0.856 min, purity 97.06%.
4.1.13
Ethyl
2-(4-((1-(4-fluorophenyl)-4-oxo-1,4-dihydro-5H-pyrazolo[3,4-d]pyrimidin-5-yl)meth
yl)-4-hydroxypiperidin-1-yl)thiazole-4-carboxylate (L13)
Compound L13 was prepared according to the procedure described for compound
1
L12. L13: white solid, 18% yield. m.p. 184-186 °C. H NMR (300 MHz, d6-DMSO)

δ 8.35 (d, J = 1.8 Hz, 2H), 8.06 (dd, J = 8.7, 4.8 Hz, 2H), 7.65 (s, 1H), 7.41 (t, J = 8.7
Hz, 2H), 5.01 (s, 1H), 4.21 (q, J = 7.0 Hz, 2H), 4.06 (s, 2H), 3.68 (d, J = 12.6 Hz, 2H),
1.68 (t, J = 10.2 Hz, 2H), 1.58 – 1.41 (m, 2H), 1.25 (t, J = 7.0 Hz, 3H). ESI-MS: m/z
521.2 [M+Na]+. LC tR: 2.779 min, purity 97.84%.
4.1.14
5-((1-benzyl-4-hydroxypiperidin-4-yl)methyl)-1-(4-fluorophenyl)-1,5-dihydro-4H-pyr
azolo[3,4-d]pyrimidin-4-one (L14)
A mixture of 5 (88 mg, 0.2 mmol), benzyl bromide (36 µL, 0.3 mmol) and K2CO3
(102 mg, 0.74 mmol) in DMF (4 mL) was stirred at ambient temperature for 2 h. The
mixture was diluted with water (9 mL), extracted with ethyl acetate (6 mL × 3), and
the organic layer was washed with water (10 mL×3) and brine (10 mL), dried over
sodium sulfate, filtered and concentrated. The residue was purified by column
chromatography (silica gel, DCM:methol = 20:1) to give compound L14 (27 mg, 31%
1
yield) as a light yellow solid. m.p. 178-180 °C. H NMR (300 MHz, d6-DMSO) δ
8.42 – 8.24 (m, 2H), 8.18 – 7.99 (m, 2H), 7.54 – 7.08 (m, 7H), 4.61 (s, 1H), 4.03 (s,
2H), 3.44 (s, 2H), 2.49 – 2.37 (m, 2H), 2.39 – 2.17 (m, 2H), 1.75 – 1.54 (m, 2H), 1.51
– 1.32 (m, 2H). ESI-MS: m/z 434.2 [M+H]+. LC tR: 1.572 min, purity >99%.
4.1.15
1-(4-fluorophenyl)-5-((4-hydroxy-1-(pyridin-2-ylmethyl)piperidin-4-yl)methyl)-1,5-d
ihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (L15)
Compound L15 was prepared according to the procedure described for compound
1
L14. L15: light yellow solid, 31% yield. m.p. 124-126 °C. H NMR (300 MHz,
d6-DMSO) δ 8.54 – 8.40 (m, 1H), 8.40 – 8.28 (m, 2H), 8.16 – 7.95 (m, 2H), 7.85 –
7.64 (m, 1H), 7.52 – 7.32 (m, 3H), 7.28 – 7.15 (m, 1H), 4.66 (s, 1H), 4.01 (s, 2H),
3.57 (s, 2H), 2.64 – 2.50 (m, 2H), 2.44 – 2.24 (m, 2H), 1.72 – 1.51 (m, 2H), 1.47 –
1.28 (m, 2H). ESI-MS: m/z 435.2 [M+H]+. LC tR: 0.647 min, purity 98.94%.
4.1.16
1-(4-fluorophenyl)-5-((4-hydroxy-1-(pyridin-3-ylmethyl)piperidin-4-yl)methyl)-1,5-d

ihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (L16)
Compound L16 was prepared according to the procedure described for compound
L14. L16: white solid, 54% yield. m.p. 198-200 °C. 1H NMR (300 MHz, d6-DMSO) δ
8.54 – 8.40 (m, 2H), 8.37 – 8.27 (m, 2H), 8.14 – 7.98 (m, 2H), 7.77 – 7.62 (m, 1H),
7.51 – 7.22 (m, 3H), 4.66 (s, 1H), 4.00 (s, 2H), 3.47 (s, 2H), 2.60 – 2.49 (m, 2H), 2.38
– 2.19 (m, 2H), 1.70 – 1.49 (m, 2H), 1.48 – 1.28 (m, 2H). ESI-MS: m/z 435.2 [M+H]+.
LC tR: 0.904 min, purity >99%.
4.1.17
1-(4-fluorophenyl)-5-((4-hydroxy-1-(pyridin-4-ylmethyl)piperidin-4-yl)methyl)-1,5-d
ihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (L17)
Compound L17 was prepared according to the procedure described for compound
1
L14. L17: light yellow solid, 61% yield. m.p. 244-246 °C. H NMR (300 MHz,
d6-DMSO) δ 8.59 – 8.43 (m, 2H), 8.42 – 8.28 (m, 2H), 8.17 – 8.00 (m, 2H), 7.50 –
7.37 (m, 2H), 7.37 – 7.26 (m, 2H), 4.69 (s, 1H), 4.03 (s, 2H), 3.50 (s, 2H), 3.31 (s,
2H), 2.57 – 2.51 (m, 2H), 2.40 – 2.22 (m, 2H), 1.73 – 1.54 (m, 2H), 1.50 – 1.32 (m,
2H). ESI-MS: m/z 435.2 [M+H]+. LC tR: 0.912 min, purity 97.69%.
4.1.18
Methyl
2-(2-chlorophenyl)-2-(4-((1-(4-fluorophenyl)-4-oxo-1,4-dihydro-5H-pyrazolo[3,4-d]p
yrimidin-5-yl)methyl)-4-hydroxypiperidin-1-yl)acetate (L18)
A mixture of 5 (221 mg, 0.5 mmol), methyl 2-bromo-2-(2-chlorophenyl)acetate (131
mg, 0.5 mmol) and K2CO3 (138 mg, 1 mmol) in DMF (4 mL) was stirred at ambient
temperature for 2 h. The mixture was diluted with water (9 mL), extracted with ethyl
acetate (6 mL × 3), and the organic layer was washed with water (10 mL×3) and brine
(10 mL), dried over sodium sulfate, filtered and concentrated. The residue was
purified by column chromatography (silica gel, DCM : CH3OH = 20 : 1) to give
compound L18 (150 mg, yield 57%) as a white solid. 1H NMR (300 MHz, d6-DMSO)
δ 8.34 (d, J = 5.5 Hz, 2H), 8.07 (dd, J = 9.0, 4.9 Hz, 2H), 7.58 (d, J = 7.3 Hz, 1H),
7.50 – 7.28 (m, 5H), 4.70 (s, 1H), 4.58 (s, 1H), 4.02 (s, 2H), 3.62 (s, 3H), 2.67 – 2.57

(m, 1H), 2.48 – 2.35 (m, 3H), 1.72 – 1.50 (m, 2H), 1.50 – 1.32 (m, 2H). ESI-MS: m/z
548.2 [M+Na]+. m.p. 168-170 °C. LC tR: 5.308 min, purity 98.88%.
4.1.19
5-((1-benzhydryl-4-hydroxypiperidin-4-yl)methyl)-1-(4-fluorophenyl)-1,5-dihydro-4
H-pyrazolo[3,4-d]pyrimidin-4-one (L19)
Compound L19 was prepared according to the procedure described for compound
1
L14. L19: white solid, 35% yield. m.p. 220-222 °C. H NMR (300 MHz, d6-DMSO)
δ 8.31 (s, 2H), 8.04 (s, 2H), 7.38 (s, 6H), 7.32 – 7.20 (m, 4H), 7.20 – 7.04 (m, 2H),
4.63 (s, 1H), 4.30 (s, 1H), 4.01 (s, 2H), 2.47 – 2.37 (m, 2H), 2.32 – 2.03 (m, 2H), 1.79
– 1.51 (m, 2H), 1.50 – 1.26 (m, 2H). ESI-MS: m/z 532.3 [M+Na]+. LC tR: 1.083 min,
purity 96.31%.
4.1.20
1-(4-fluorophenyl)-5-((4-hydroxy-1-(2-nitrobenzyl)piperidin-4-yl)methyl)-1,5-dihydr
o-4H-pyrazolo[3,4-d]pyrimidin-4-one (L20)
Compound L20 was prepared according to the procedure described for compound
1
L14. L20: light yellow solid, 40% yield. m.p. 180-182 °C. H NMR (300 MHz,
d6-DMSO) δ 8.35 (s, 1H), 8.33 (s, 1H), 8.13 – 8.00 (m, 2H), 7.88 – 7.79 (m, 1H), 7.70
– 7.57 (m, 2H), 7.57 – 7.36 (m, 3H), 4.67 (s, 1H), 4.00 (s, 2H), 3.71 (s, 2H), 2.47 –
2.21 (m, 4H), 1.64 – 1.44 (m, 2H), 1.42 – 1.27 (m, 2H). ESI-MS: m/z 479.2 [M+H]+.
LC tR: 1.524 min, purity 97.51%.
4.1.21
1-(4-fluorophenyl)-5-((4-hydroxy-1-(3-nitrobenzyl)piperidin-4-yl)methyl)-1,5-dihydr
o-4H-pyrazolo[3,4-d]pyrimidin-4-one (L21)
Compound L21 was prepared according to the procedure described for compound
1
L14. L21: white solid, 44% yield. m.p. 90-92 °C. H NMR (300 MHz, d6-DMSO) δ
8.34 (s, 2H), 8.22 – 8.02 (m, 4H), 7.76 (d, J = 7.4 Hz, 1H), 7.62 (t, J = 7.8 Hz, 1H),
7.42 (t, J = 8.7 Hz, 2H), 4.68 (s, 1H), 4.03 (s, 2H), 3.61 (s, 2H), 2.60 – 2.51 (m, 2H),
2.44 – 2.23 (m, 2H), 1.74 – 1.54 (m, 2H), 1.50 – 1.36 (m, 2H). ESI-MS: m/z 479.2

[M+H]+. LC tR: 1.484 min, purity 94.43%.
4.1.22
1-(4-fluorophenyl)-5-((4-hydroxy-1-(4-nitrobenzyl)piperidin-4-yl)methyl)-1,5-dihydr
o-4H-pyrazolo[3,4-d]pyrimidin-4-one (L22)
Compound L22 was prepared according to the procedure described for compound
L14. L22: 51% yield as white solid. m.p. 214-216 °C. 1H NMR (300 MHz, d6-DMSO)
δ 8.35 (d, J = 3.5 Hz, 2H), 8.18 (d, J = 8.4 Hz, 2H), 8.07 (dd, J = 8.8, 4.9 Hz, 2H),
7.59 (d, J = 8.4 Hz, 2H), 7.42 (t, J = 8.8 Hz, 2H), 4.69 (s, 1H), 4.03 (s, 2H), 3.60 (s,
2H), 2.57 – 2.51 (m, 2H), 2.33 (t, J = 10.2 Hz, 2H), 1.63 (t, J = 10.1 Hz, 2H), 1.41 (d,
J = 12.9 Hz, 2H). ESI-MS: m/z 479.2 [M+H]+. LC tR:1.444 min, purity 93.09%.
4.1.23
5-((1-(2-fluorobenzyl)-4-hydroxypiperidin-4-yl)methyl)-1-(4-fluorophenyl)-1,5-dihyd
ro-4H-pyrazolo[3,4-d]pyrimidin-4-one (L23)
Compound L23 was prepared according to the procedure described for compound
L14. L23: white solid, 53% yield. m.p. 174-176 °C. 1H NMR (300 MHz, d6-DMSO) δ
8.35 (s, 1H), 8.33 (s, 1H), 8.11 – 8.01 (m, 2H), 7.48 – 7.36 (m, 3H), 7.35 – 7.25 (m,
1H), 7.20 – 7.09 (m, 2H), 4.65 (s, 1H), 4.01 (s, 2H), 3.58 – 3.45 (m, 2H), 2.39 – 2.25
(m, 2H), 1.65 – 1.54 (m, 2H), 1.41 – 1.33 (m, 2H). ESI-MS: m/z 452.2 [M+H]+. LC tR:
1.216 min, purity 97.49%.
4.1.24
5-((1-(4-fluorobenzyl)-4-hydroxypiperidin-4-yl)methyl)-1-(4-fluorophenyl)-1,5-dihyd
ro-4H-pyrazolo[3,4-d]pyrimidin-4-one (L24)
Compound L24 was prepared according to the procedure described for compound
L14. L24: white solid, 58% yield. m.p. 206-208 °C. 1H NMR (300 MHz, d6-DMSO) δ
8.35 (s, 1H), 8.34 (s, 1H), 8.13 – 8.01 (m, 2H), 7.50 – 7.37 (m, 2H), 7.38 – 7.24 (m,
2H), 7.19 – 7.06 (m, 2H), 4.66 (s, 1H), 4.02 (s, 2H), 3.44 (s, 2H), 2.49 – 2.44 (m, 2H),
2.33 – 2.19 (m, 2H), 1.71 – 1.50 (m, 2H), 1.48 – 1.30 (m, 2H). ESI-MS: m/z 452.3
[M+H]+. LC tR: 0.933 min, purity 96.77%.

4.1.25
5-((1-(2-chlorobenzyl)-4-hydroxypiperidin-4-yl)methyl)-1-(4-fluorophenyl)-1,5-dihyd
ro-4H-pyrazolo[3,4-d]pyrimidin-4-one (L25)
Compound L25 was prepared according to the procedure described for compound
1
L14. L25: white solid, 48% yield. m.p. 218-220 °C. H NMR (300 MHz, d6-DMSO)
δ 8.36 (s, 1H), 8.34 (s, 1H), 8.14 – 8.00 (m, 2H), 7.54 – 7.37 (m, 4H), 7.37 – 7.20 (m,
2H), 4.69 (s, 1H), 4.03 (s, 2H), 3.55 (s, 2H), 2.62 – 2.52 (m, 2H), 2.43 – 2.30 (m, 2H),
1.69 – 1.54 (m, 2H), 1.47 – 1.34 (m, 2H). ESI-MS: m/z 468.2 [M+H]+. LC tR: 1.411
min, purity 96.24%.
4.1.26
5-((1-(3-chlorobenzyl)-4-hydroxypiperidin-4-yl)methyl)-1-(4-fluorophenyl)-1,5-dihyd
ro-4H-pyrazolo[3,4-d]pyrimidin-4-one (L26)
Compound L26 was prepared according to the procedure described for compound
L14. L26: white solid, 65% yield. m.p. 194-196 °C. 1H NMR (300 MHz, d6-DMSO) δ
8.35 (s, 1H), 8.34 (s, 1H), 8.13 – 7.99 (m, 2H), 7.50 – 7.21 (m, 6H), 4.66 (s, 1H), 4.02
(s, 2H), 3.47 (s, 2H), 2.49 – 2.45 (m, 2H), 2.36 – 2.22 (m, 2H), 1.68 – 1.53 (m, 2H),
1.46 – 1.33 (m, 2H). ESI-MS: m/z 468.1[M+H]+. LC tR: 1.139 min, purity 95.44%.
4.1.27
5-((1-(4-chlorobenzyl)-4-hydroxypiperidin-4-yl)methyl)-1-(4-fluorophenyl)-1,5-dihyd
ro-4H-pyrazolo[3,4-d]pyrimidin-4-one (L27)
Compound L27 was prepared according to the procedure described for compound
L14. L27: white solid, 39% yield. m.p. 224-226 °C. 1H NMR (300 MHz, d6-DMSO) δ
8.47 – 8.25 (m, 2H), 8.18 – 7.95 (m, 2H), 7.68 – 7.20 (m, 6H), 4.67 (s, 1H), 4.03 (s,
2H), 3.44 (s, 2H), 2.49 – 2.41 (m, 2H), 2.36 – 2.16 (m, 2H), 1.72 – 1.52 (m, 2H), 1.47
– 1.31 (m, 2H). ESI-MS: m/z 468.2 [M+H]+. LC tR: 2.271 min, purity >99%.
4.1.28
5-((1-(2-bromobenzyl)-4-hydroxypiperidin-4-yl)methyl)-1-(4-fluorophenyl)-1,5-dihy
dro-4H-pyrazolo[3,4-d]pyrimidin-4-one (L28)

Compound L28 was prepared according to the procedure described for compound
L14. L28: white solid, 41% yield. m.p. 214-216 °C. 1H NMR (300 MHz, d6-DMSO) δ
8.46 – 8.24 (m, 2H), 8.19 – 7.94 (m, 2H), 7.65 – 7.53 (m, 1H), 7.53 – 7.26 (m, 4H),
7.27 – 7.10 (m, 1H), 4.68 (s, 1H), 4.03 (s, 2H), 3.52 (s, 2H), 2.61 – 2.53 (m, 2H), 2.45
– 2.27 (m, 2H), 1.75 – 1.52 (m, 2H), 1.49 – 1.31 (m, 2H). ESI-MS: m/z 512.2 [M+H]+.
LC tR: 2.580 min, purity 97.52%.
4.1.29
5-((1-(4-bromobenzyl)-4-hydroxypiperidin-4-yl)methyl)-1-(4-fluorophenyl)-1,5-dihy
dro-4H-pyrazolo[3,4-d]pyrimidin-4-one (L29)
Compound L29 was prepared according to the procedure described for compound
L14. L29: white solid, 72% yield. m.p. 208-210 °C. 1H NMR (300 MHz, d6-DMSO) δ
8.35 (s, 1H), 8.33 (s, 1H), 8.13 – 8.01 (m, 2H), 7.54 – 7.37 (m, 4H), 7.31 – 7.21 (m,
2H), 4.65 (s, 1H), 4.02 (s, 2H), 3.43 (s, 2H), 2.49 – 2.42 (m, 2H), 2.34 – 2.19 (m, 2H),
1.65 – 1.51 (m, 2H), 1.44 – 1.35 (m, 2H). ESI-MS: m/z 512.2 [M+H]+. LC tR: 1.202
min, purity 95.07%.
4.1.30
2-((4-((1-(4-fluorophenyl)-4-oxo-1,4-dihydro-5H-pyrazolo[3,4-d]pyrimidin-5-yl)meth
yl)-4-hydroxypiperidin-1-yl)methyl)benzonitrile (L30)
Compound L30 was prepared according to the procedure described for compound
L14. L30: white solid, 45% yield. m.p. 184-186 °C. 1H NMR (300 MHz, d6-DMSO) δ
8.44 – 8.26 (m, 2H), 8.14 – 7.99 (m, 2H), 7.84 – 7.75 (m, 1H), 7.73 – 7.62 (m, 1H),
7.62 – 7.53 (m, 1H), 7.51 – 7.33 (m, 3H), 4.72 (s, 1H), 4.02 (s, 2H), 3.65 (s, 2H), 2.62
– 2.52 (m, 2H), 2.45 – 2.29 (m, 2H), 1.68 – 1.50 (m, 2H), 1.49 – 1.33 (m, 2H).
ESI-MS: m/z 459.2 [M+H]+. LC tR: 1.211 min, purity 97.71%.
4.1.31
4-((4-((1-(4-fluorophenyl)-4-oxo-1,4-dihydro-5H-pyrazolo[3,4-d]pyrimidin-5-yl)meth
yl)-4-hydroxypiperidin-1-yl)methyl)benzonitrile (L31)
Compound L31 was prepared according to the procedure described for compound

L14. L31: white solid, 22% yield. m.p. 204-206 °C. 1H NMR (300 MHz, d6-DMSO) δ
8.36 (s, 1H), 8.34 (s, 1H), 8.15 – 8.01 (m, 2H), 7.85 – 7.70 (m, 2H), 7.58 – 7.34 (m,
4H), 4.68 (s, 1H), 4.02 (s, 2H), 3.55 (s, 2H), 2.64 – 2.52 (m, 2H), 2.40 – 2.23 (m, 2H),
1.69 – 1.51 (m, 2H), 1.50 – 1.31 (m, 2H). ESI-MS: m/z 459.2 [M+H]+. LC tR: 1.141
min, purity 96.61%.
4.1.32
1-(4-fluorophenyl)-5-((4-hydroxy-1-(4-(trifluoromethyl)benzyl)piperidin-4-yl)methyl)
-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (L32)
Compound L32 was prepared according to the procedure described for compound L1.
L32; white solid, 36% yield. m.p. 177-179 °C. 1H NMR (300 MHz, d6-DMSO) δ 8.35
(s, 1H), 8.34 (s, 1H), 8.14 – 8.01 (m, 2H), 7.74 – 7.62 (m, 2H), 7.58 – 7.48 (m, 2H),
7.48 – 7.35 (m, 2H), 4.67 (s, 1H), 4.03 (s, 2H), 3.56 (s, 2H), 2.49 – 2.44 (m, 2H), 2.42
– 2.17 (m, 2H), 1.72 – 1.51 (m, 2H), 1.51 – 1.32 (m, 2H). ESI-MS: m/z 502.2 [M+H]+.
LC tR: 1.153 min, purity 96.15%.
4.1.33
1-(4-fluorophenyl)-5-((4-hydroxy-1-(2-methylbenzyl)piperidin-4-yl)methyl)-1,5-dihy
dro-4H-pyrazolo[3,4-d]pyrimidin-4-one (L33)
Compound L33 was prepared according to the procedure described for compound
1
L14. L33: white solid, 32% yield. m.p. 204-206 °C. H NMR (300 MHz, d6-DMSO)
δ 8.36 (s, 2H), 8.07 (dd, J = 8.8, 4.8 Hz, 2H), 7.43 (t, J = 8.7 Hz, 2H), 7.17 (d, J =
24.0 Hz, 4H), 4.68 (s, 1H), 4.03 (s, 2H), 3.40 (s, 2H), 3.32 (s, 3H), 2.31 (s, 4H), 1.56
(d, J = 8.8 Hz, 2H), 1.39 (d, J = 10.4 Hz, 2H). ESI-MS: m/z 448.2 [M+H]+. LC tR:
2.364 min, purity 97.34%.
4.1.34
1-(4-fluorophenyl)-5-((4-hydroxy-1-(4-methylbenzyl)piperidin-4-yl)methyl)-1,5-dihy
dro-4H-pyrazolo[3,4-d]pyrimidin-4-one (L34)
Compound L34 was prepared according to the procedure described for compound

L14. L34: white solid, 60% yield. m.p. 200-202 °C. 1H NMR (300 MHz, d6-DMSO) δ
8.35 (s, 1H), 8.34 (s, 1H), 8.15 – 7.99 (m, 2H), 7.74 – 7.65 (m, 1H), 7.49 – 7.34 (m,
2H), 7.24 – 7.03 (m, 3H), 4.64 (s, 1H), 4.01 (s, 2H), 3.41 (s, 2H), 2.49 – 2.42 (m, 2H),
2.38 – 2.12 (m, 5H), 1.65 – 1.54 (m, 2H), 1.43 – 1.35 (m, 2H). ESI-MS: m/z 448.2
[M+H]+. LC tR: 1.420 min, purity 97.13%.
4.1.35
Methyl
2-((4-((1-(4-fluorophenyl)-4-oxo-1,4-dihydro-5H-pyrazolo[3,4-d]pyrimidin-5-yl)meth
yl)-4-hydroxypiperidin-1-yl)methyl)benzoate (L35)
Compound L35 was prepared according to the procedure described for compound
1
L14. L35: white solid, 57% yield. m.p. 180-182 °C. H NMR (300 MHz, d6-DMSO)
δ 8.32 (d, J = 5.4 Hz, 2H), 8.05 (dd, J = 8.8, 4.9 Hz, 2H), 7.55 (d, J = 7.4 Hz, 1H),
7.49 – 7.26 (m, 5H), 4.65 (s, 1H), 3.98 (s, 2H), 3.77 (s, 3H), 3.62 (s, 2H), 2.43 – 2.31
(m, 2H), 2.25 (t, J = 9.8 Hz, 2H), 1.50 (t, J = 9.8 Hz, 2H), 1.40 – 1.26 (m, 2H).
ESI-MS: m/z 492.2 [M+H]+. LC tR: 1.330 min, purity 96.44%.
4.1.36
Methyl
3-((4-((1-(4-fluorophenyl)-4-oxo-1,4-dihydro-5H-pyrazolo[3,4-d]pyrimidin-5-yl)meth
yl)-4-hydroxypiperidin-1-yl)methyl)benzoate (L36)
Compound L36 was prepared according to the procedure described for compound
1
L14. L36: white solid, 63% yield. m.p. 158-160 °C. H NMR (300 MHz, d6-DMSO)
δ 8.33 (d, J = 3.5 Hz, 2H), 8.05 (dd, J = 8.8, 4.9 Hz, 2H), 7.88 (s, 1H), 7.82 (d, J = 7.4
Hz, 1H), 7.55 (d, J = 7.2 Hz, 1H), 7.50 – 7.32 (m, 3H), 4.65 (s, 1H), 4.01 (s, 2H), 3.84
(s, 3H), 3.51 (s, 2H), 2.49 (s, 2H), 2.30 (d, J = 9.8 Hz, 2H), 1.57 (d, J = 9.6 Hz, 2H),
1.47 – 1.28 (m, 2H). ESI-MS: m/z 492.3 [M+H]+. LC tR: 1.146 min, purity 97.29%.
4.1.37
Methyl
4-((4-((1-(4-fluorophenyl)-4-oxo-1,4-dihydro-5H-pyrazolo[3,4-d]pyrimidin-5-yl)meth
yl)-4-hydroxypiperidin-1-yl)methyl)benzoate (L37)
Compound L37 was prepared according to the procedure described for compound
1
L14. L24: white solid, 57% yield. m.p. 186-188 °C. H NMR (300 MHz, d6-DMSO)