Discovery of a new class of valosine containing protein (VCP/P97) inhibitors for the treatment of non-small cell lung cancer

Article abstract of DOI:10.1016/j.bmc.2018.12.036

Valosine containing protein (VCP/p97) is a member of the AAA ATPase family involved in several essential cellular functions and plays an important role in the ubiquitin-mediated degradation of misfolded proteins. P97 has a significant role in maintaining the cellular protein homeostasis for tumor cell growth and survival and has been found overexpressed in many tumor types. No new molecule entities based on p97 target were approved in clinic. Herein, a series of novel pyrimidine structures as p97 inhibitors were designed and synthesized. After enzymatic evaluations, structure-activity relationships (SAR) were discussed in detailed. Among the screened compounds, derivative 35 showed excellent enzymatic inhibitory activity (IC₅₀, 36 nM). The cellular inhibition results showed that compound 35 had good antiproliferative activity against the non-small cell lung cancer A549 cells (IC₅₀, 1.61 μM). Liver microsome stability showed that the half-life of compound 35 in human liver microsome was 42.3 min, which was more stable than the control CB-5083 (25.8 min). The in vivo pharmacokinetic results showed that the elimination phase half-lives of compound 35 were 4.57 h for ig and 3.64 h for iv, respectively and the oral bioavailability was only 4.5%. These results indicated that compound 35 could be effective for intravenous treatment of non-small cell lung cancer.

Full text of DOI:10.1016/j.bmc.2018.12.036

Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of a new class of valosine containing protein (VCP/P97) inhibitors
for the treatment of non-small cell lung cancer
a
a
a
b
c
b
d
c
Xueyuan Wang , Enhe Bai , Hui Zhou , Sijia Sha , Hang Miao , Yanru Qin , Zhaogang Liu ,
c
a
b,⁎
c
a,⁎
Jia Wang , Haoyang Zhang , Meng Lei , Jia Liu , Ou Hai , Yongqiang Zhu
a
b
c
d
College of Life Science, Nanjing Normal University, No. 1 Wenyuan Road, Nanjing 210037, PR China
College of Science, Nanjing Forestry University, No. 159 Longpan Road, Nanjing 210037, PR China
Jiangsu Chia Tai Fenghai Pharmaceutical Co. Ltd., No. 9 Weidi Road, Nanjing 210046, PR China
School of Bio-engineering, Qilu University of Technology (Shandong Academy of Sciences), No. 3501 Daxue Rd. Changqing District, Jinan 250353, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Valosine containing protein (VCP/p97) is a member of the AAA ATPase family involved in several essential
cellular functions and plays an important role in the ubiquitin-mediated degradation of misfolded proteins. P97
has a significant role in maintaining the cellular protein homeostasis for tumor cell growth and survival and has
been found overexpressed in many tumor types. No new molecule entities based on p97 target were approved in
clinic. Herein, a series of novel pyrimidine structures as p97 inhibitors were designed and synthesized. After
enzymatic evaluations, structure-activity relationships (SAR) were discussed in detailed. Among the screened
compounds, derivative 35 showed excellent enzymatic inhibitory activity (IC50, 36 nM). The cellular inhibition
results showed that compound 35 had good antiproliferative activity against the non-small cell lung cancer A549
cells (IC50, 1.61 μM). Liver microsome stability showed that the half-life of compound 35 in human liver mi-
crosome was 42.3 min, which was more stable than the control CB-5083 (25.8 min). The in vivo pharmacoki-
netic results showed that the elimination phase half-lives of compound 35 were 4.57 h for ig and 3.64 h for iv,
respectively and the oral bioavailability was only 4.5%. These results indicated that compound 35 could be
effective for intravenous treatment of non-small cell lung cancer.
Valosine containing protein
Non-small cell lung cancer
Structure-activity relationships
Pyrimidine derivative
Pharmacokinetic
1
. Introduction
Valosin-containing protein (VCP/p97) is a member of the AAA
off rate of ADP.1 The D2 domain is responsible for the major ATPase
activity of p97 under physiological conditions. The D2 ATPase region
has been displayed to have both a higher K for ATP and a faster hy-
m
1
0
(
ATPase associated with various cellular activities) ATPase family. P97
drolysis of ATP to ADP. The N-terminal domain binds various cofac-
tors that interact with a variety of substrate proteins. Many studies have
revealed p97′s role in promoting ERAD in cooperation with the ubi-
quitin-proteasome system (UPS). P97 serves as a force-generating ma-
chine to remove misfolded poly-ubiquitinated proteins from the ER into
promotes several biological processes, including ubiquitin-dependent
protein degradation, endoplasmic reticulum-associated degradation
(
ERAD), nuclear membrane fusion after completion of mitosis, Golgi
reassembly, activation of transcription factors and autophagy, where it
supplies the mechanical force required for extracting proteins by ATP
the cytosol and then transports them to the proteasome for degrada-
1
–4
11,12
hydrolysis.
As most AAA-ATPases, p97 structurally adopts a ring-
tion
.
shaped homohexamer structure comprising six identical 90 kDa sub-
units arranged in a ring, with each protomer containing three domains:
P97 plays an important role in maintaining the cellular protein
homeostasis for tumor cell growth and survival, which was found to be
5
–10
two ATPase domains (D1 and D2) and one N-terminal domain.
The
overexpressed in several malignancies including non-small cell lung
cancer and associated with malignancy.1
3–18
For instance, it had been
D1 domain has low basal hydrolytic activity, due in part to a very low
Abbreviations: VCP, valosine containing protein; AAA, ATPases associated with various cellular activities; ER, endoplasmic reticulum; ERAD, endoplasmic re-
ticulum-associated degradation; SAR, structure-activity relationship; UPS, ubiquitin-proteasome system; IC50, half-maximum inhibitory concentration; AUC, area
under the curve; T1/2, halftime; SD, Sprague-Dawley; PK, pharmacokinetics; F, absolute bioavailability in %; HPLC, high performance liquid chromatography; HRMS,
high-resolution mass spectra; ESI, electrospray source; DCM, dichloromethane; DMSO, dimethyl sulfoxide; THF, tetrahydrofuran; LDA, lithium diisopropylamide
⁎
Corresponding authors.
E-mail addresses: hk-lm@163.com (M. Lei), zhyqscu@hotmail.com (Y. Zhu).
https://doi.org/10.1016/j.bmc.2018.12.036
Received 23 November 2018; Received in revised form 19 December 2018; Accepted 27 December 2018
0968-0896/ © 2018 Elsevier Ltd. All rights reserved.
Please cite this article as: Zhu, Y., Bioorganic & Medicinal Chemistry, https://doi.org/10.1016/j.bmc.2018.12.036

X. Wang et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
Fig. 1. The p97 inhibitors reported in the literatures.
shown that RNA interference or overexpression of ATPase deficient
protein in tumor cell lines could disrupt the function of p97 to cause cell
62%). Then Boc-protecting group was introduced to the N atom of in-
dole ring of 11 and the nitro group was reduced to amino group by Fe to
give amine 12 (yield 71%). Finally, amine 12 reacted with different
1
9,20
death.
Besides, gene knockout of p97 in mice was found to be
21
2
embryonic lethal, while mutations of the p97 gene were thought to be
R Cl and deprotected the Boc groups to yield 13a-13j (yields 75%-
2
2
associated with the neurodegenerative diseases. So in recent years,
p97 had drawn great attention as a potential drug target for developing
small molecules for cancer therapy. Up to now, a number of p97 in-
85%).
The synthesis of target molecules CB-5083 and 17–20 was sum-
marized in Scheme 2. Intermediate 5c was coupled with cyanoindole 9
2
3–30
hibitors had been reported (Fig. 1).
Among them, compound CB-
in the presence of Pd
2
(dba)
3
as the catalyst and Cs
2
CO as base to get
3
5
083 was the first selective p97 inhibitor with the requisite pharma-
the compound 16 (yield 74%). Intermediate 16 reacted with acet-
aldehyde oxime in the presence of palladium acetate and triphenyl-
phosphine to convert the nitrile group into the primary carboxamide to
yield target molecule CB-5083 (yield 70%). And then CB-5083 reacted
with Lawesson's reagent to change the O atom into S one to give mo-
lecule 17 (yield 81%). Intermediate 5c reacted with commercially
3
1
cological properties that showed promising preclinical activities. Be-
sides, two phase I clinical trials (NCT02243917 and NCT02223598) of
CB-5083 had been completed in 2017. However, due to the toxicities of
CB-5083, the clinical trial was discontinued. So there is a necessity to
develop new compounds to meet the unmet medical need. Herein, we
described the design, synthesis and structure-activity relationships
available N-(1H-indol-4-yl)acetamide in the presence of Pd
2
(dba) , X-
3
(
SAR) of a series of novel p97 inhibitors. Among the inhibitors, com-
Phos and cesium carbonate to give target molecule 18 (yield 72%).
Similar reaction between 5c and 3-aminobenzonitrile produced the
target molecule 19 (yield 66%).
pound 35 exhibited good in vitro activities and microsomal stabilities.
Furthermore, compound 35 showed good pharmacokinetic results,
which indicated that compound 35 might be effective for the treatment
of non-small cell lung cancer.
The synthesis of target molecules 20–25 was illustrated in Scheme
3. Cyanoindole 9 was coupled with intermediates 5a, 5b, 5d-5f, re-
spectively and then the nitrile groups were hydrolyzed to the primary
carboxamides to give target molecules 20–24 (yields 60%-72%). The
target molecule 25 was prepared from commercially available 2,4-di-
chloro pyrimidine 14, which firstly reacted with benzylamine to form
compound 15 (yield 85%). Intermediate 15 was coupled with 9 and
then the nitrile substituent was converted into the carboxamide to get
target molecule 25 (yield 60%). Similar substitution reactions were
performed between 5c and indole derivatives 13a-13j to give the target
molecules 26–31 and 35–38 (yields 60%–78%) and intermediate 13c
reacted with 5d, 5f and 5g to produce target molecules 32–34 (yields
64%-74%) as showed in Scheme 4.
2
. Results and discussion
2.1. Chemistry
The synthesis of key intermediates 5a-5g, 9 and 13a-13j was out-
lined in Scheme 1. Compounds 1a-1c reacted with dimethyl carbonate
in the presence of NaH to produce methyl esters 2a-2c (yields 29%-
3
5%). And then the carbonyl groups of intermediates 2a-2c were am-
monified by ammonium acetate to give amines 3a-3c (yields 83%-
0%). Subsequently, compounds 3a-3c reacted with 2,2,2-tri-
9
chloroacetyl isocyanate and ammonia to form 2,4-diol pyrimidyl het-
erocyclic rings 4a-4c, which reacted with POCl
3
and then were am-
2.2. Biological evaluation
1
monified by different amines R NH
2
to produce key intermediates 5a-
5
g (yields 19%-26%). 1H-indole-4-carbonitrile 6 reacted with benze-
The kinase inhibitory activities of the target compounds were
evaluated via ADP-Glo assay (Promega) against purified human p97
enzyme. Cell-based assay included a 72 h Cell Counting Kit-8 (CCK8)
viability assay. Metabolic stabilities were investigated in five liver mi-
crosomes, mouse, rat, dog, monkey and human. Absolute bioavail-
ability (F %) was determined by the pharmacokinetic assessment of
areas under the plasma concentration versus time curves following iv
and ig administrations.
nesulfonyl chloride to afford N-protected intermediate 7 with the yield
of 75%. Intermediate 7 reacted with lithium diisopropylamide at
−
40 °C followed by methyl iodide to yield indole derivative 8 (yield
6
9%). Then the N-terminal protected group benzenesulfonyl was re-
moved with sodium hydroxide solution to form 2-methyl-4-cyano in-
dole 9 (yield 90%). 3-Nitroaniline 10 condensed with acetone under
strongly basic conditions to give 2-methyl-4-nitro indole 11 (yield
2

X. Wang et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
a a
1
Scheme 1. Synthesis of key intermediates 5a-5g, 9 and 13a-13j. Reagents and conditions: (a) NaH, (MeO)
2
CO, THF, 45 °C; (b) NH
4
OAc, MeOH, rt; (c) Cl
3
CCONCO,
MeCN, rt; (d) NH
3
, MeOH, 70 °C; (e) POCl
3
, 120 °C; (f) MeCN, R NH
2
rt; (g) PhSO
2
Cl, NaH, THF, rt; (h) MeI, LDA, THF, −40 °C; (i) NaOH, MeOH, H
2
O, 40 °C; (j)
2
Acetone, t-BuOK, DMSO; (k) (Boc)
2
O, Et
3
N, DCM, rt; (l) Fe, NH
4
Cl, EtOH, 60 °C; (m) R Cl, Et
3
N, rt; (n) TFA, DCM, rt.
2
.3. Discussion
heterocycle (morpholinyl 21), aliphatic ring (cyclopropyl 22) led to the
complete loss of p97 activities. In addition, the replacement of pyran
ring (CB-5083) with oxepane (23) showed potent p97 activity (IC50
82.8 nM). However, when the pyran ring (CB-5083) was replaced by
oxecane (24, 119.7 nM) or was removed (25, 119.7 nM), all failed to
increase the p97 potencies.
Enzymatic. The enzymatic results of compounds 17–25 and CB-5083
were showed in Table 1. While the O atom (compound CB-5083, IC50
2
1
7.0 nM) on the formamide was substituted by S one (17, IC50
92.6 nM), it led to the activity decrease by more than seven folds.
2
When the 4-indol-formamide group of CB-5083 was changed to acetyl
substituted 4-indol-amine and 2-methyl was removed at the same time,
the activity of compound 18 (IC50 922.1 nM) was greatly reduced.
Replacement of indol ring with benzyl one resulted in an inactive
compound 19. Subsequently, the replacement of the phenyl ring on
benzylamino group by aromatic heterocycle (pyridyl 20), aliphatic
Attention was next focused on the modification of the R groups of
1
2-methylindole and R groups of pyrimidin-4-amine in CB-5083 as il-
lustrated in Table 2. When the formamide of CB-5083 was changed to
acetyl substituted 4-indol-amine, the activity of compound 26
(129.4 nM) was reduced greatly. The introduction of 2-chloroacetyl
group at the R2 position (27, 308.1 nM) also failed to increase the p97
3

X. Wang et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
a a
Scheme 2. Synthesis of the target molecules CB-5083 and 17–19. Reagents and conditions: (a) Cs
2
CO
3
, Pd
2
3
(dba) , X-Phos, dioxane, 100 °C; (b) Pd(OAc)
2
, Ph P,
3
MeC = NOH, EtOH, H
2
O, 90 °C; (c) Lawesson’s reagent, THF, 80 °C; (d) MeCN, BnNH , rt.
2
with different sulfamide groups to give compounds 35–38 (Table 3).
Compared with acrylamide substituent (28, IC50 58.3 nM), methane-
sulfonamide (35, 36.4 nM) and cyclopropyl (37, 34.6 nM) substituents
showed better activities. However, benzenesulfonamide (36, 134.9 nM)
and trifluoromethanesulfonamide groups (38, 100.9 nM) resulted in
decreased potencies in a certain extent.
Cellular. The compounds with the IC50 values of enzymatic inhibi-
tion less than 0.1 μM were further evaluated their potential antitumor
effects in A549 cells. The biological results were listed in Table 4. These
compounds showed significant inhibitory potency against A549 cell
lines with the IC50 values less than 5 μM. Especially compound 35 in-
hibited the cell proliferation at 1 μM level, which was nearly as active as
CB-5083. Therefore, compound 35 was used for further evaluation
Microsome stabilities. The metabolic stabilities of the promising
compound 35 were determined with various species of liver micro-
somes, such as human, mouse, rat, dog and monkey. And the compound
CB-5083 was selected as the standard. The half-life (T1/2) and intrinsic
clearance (CLint) parameters were used to evaluate their metabolic
stabilities. It could give a good indication of the in vivo hepatic clear-
ance when the overall clearance mechanism was metabolic and when
oxidative metabolism dominates (i.e., CLmetabolic ≫ CLrenal + CLbiliary
+
a a
Scheme 3. Synthesis of the target molecules 20–25. Reagents and conditions:
3
2,33
CLother.
The data were showed in Table 5. The results revealed that
(
a) Cs
2
CO
3
,
Pd
2
(dba)
3
,
X-Phos, dioxane, 100 °C; (b) Pd(OAc)
, rt.
2
,
3
Ph P,
both compounds 35 and CB-5083 displayed good metabolic stabilities
in human, mouse and dog species. While for the rat and money liver
microsomes, the two compounds were metabolized too rapidly. The
half-life of compound 35 in human liver microsome was 42.3 min,
which was more stable than the control CB-5083 (25.8 min).
MeC = NOH, EtOH, H
2
O, 90 °C; (c) MeCN, BnNH
2
potency. Conversely, vinyl group at the R2 position (28, 58.3 nM) sig-
nificantly increased the activity. Furthermore, compared with com-
2
pounds 26–28, larger sterically hindered groups (29–31) at R position
Pharmacokinetic. With these encouraging in vitro data, candidate 35
was further investigated by profiling ig and iv PK in male Sprague-
Dawley (SD) rats. The results were illustrated in Table 6. It indicated
that the elimination phase half-lives of compound 35 were 4.57 h for ig
and 3.64 h for iv, respectively. The oral bioavailability of compound 35
was only 4.2%, which was not suitable for developing an oral drug.
However, the data of Cmax, T1/2 and AUC reflected that compound 35
led to the decreased activities, which indicated that smaller groups at
2
the R position were more favorable for the improvement of activity.
Since the introduction of vinyl substituent to compound 28 showed
1
good activity, we changed the benzylamine moiety at the R position
into pyridyl (32), cyclopropyl (33) and naphthyl (34) groups. However,
the biological results indicated that the generated compounds were
inactive. Subsequently, we replaced the acrylamide of compound 28
4

X. Wang et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
Scheme 4. Synthesis of the target molecules 26–38. a aReagents and conditions: (a) Cs
(dba) , X-Phos, dioxane, 100 °C.
2
CO
3
, Pd
2
3
could be a good candidate for iv injection. Nowadays, the in vivo ef-
ficacy of this compound was being carried out to evaluate the drug-
availability.
in vivo efficacy of this compound was being performed to evaluate the
drug-availability.
4
. Experimental section
3
. Conclusion
4.1. General methods
A novel series of p97 inhibitors were designed and synthesized. The
structure activity relationship (SAR) was discussed in detail and the
results demonstrated that the benzylamine linked to the pyrimidine
structure and the pyran ring were necessary for maintaining good ac-
tivities. Besides, hydrophilic and smaller groups in the indole structure
showed better cellular activities. From the optimized results, compound
Unless otherwise indicated, chemicals, solvents and reagents were
purchased from commercial suppliers and they were used without any
purification. Absolutely anhydrous solvents (CH
2
Cl , THF, DMF, etc.)
2
were purchased from Energy packaged under nitrogen in Sure/Seal
bottles. All reactions involving air or moisture-sensitive reagents were
performed under an argon atmosphere. All reactions were detected by
thin layer chromatography on silica gel 60 plate coated with 0.25 mm
layer and spotted with UV light or iodine. All final products were purified
to > 95% purity. The purity of the final products was determined by
HPLC (Thermo) on an Agilent Poroshell 120 EC-C18 column
(50 mm × 4.6 mm, 2.7 μm) with 0.1% FA/ACN (gradient eluted pro-
gram: 0–5 min 90/10–5/95 v/v; 5–11.9 min 5/95 v/ v; 11.9–12.1 min 5/
95–90/10 v/v; 12.1–15 min 90/10 v/ v) at 0.3 mL/min flow rate and
3
5 was screened and showed nanomolar level in the inhibition of p97
activity. Further cellular assay indicated that compound 35 could in-
hibit the proliferation of non-small cell lung cancer lines A549 with the
IC50 value of 1.61 μM. Candidate 35 exhibited good liver microsomal
stabilities in mice, dog and human. For human liver microsome, the
half-life of compound 35 was 42.3 min, which was more stable than the
control CB-5083 (25.8 min). The in vivo pharmacokinetic results
showed that the elimination phase half-lives of compound 35 were
1
13
4
.57 h for ig and 3.64 h for iv, respectively. However, the oral bioa-
254 nm detector wavelength. H and C spectra were acquired in CDCl ,
3
vailability was only 4.5%, which needs to be improved in the next
work. These results showed that compound 35 might be an effective
candidate for intravenous treatment of non-small cell lung cancer. The
DMSO‑d
6
or CD OD at room temperature on a Bruker Avance 400
3
spectrometer with chemical shift (δ, ppm) reported relative to TMS as an
internal standard. High-resolution mass spectra (HRMS) were recorded
on a ZAB-HS instrument using an electrospray source (ESI).
5

X. Wang et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
Table 1
P97 inhibitory activities of CB-5083 and 17–25.a
Compd.
n
1
X
R1
Ar
IC50 (nM)
192.6
1
7
O
1
8
1
O
922.1
1
2
9
0
1
1
O
O
NA
NAb
21
22
23
24
25
1
1
2
3
O
NAb
O
NAb
CH
2
2
82.8
119.7
160.9
CH
CB-5083
1
O
27.0
a
b
All experiments were repeated three times.
NA: No activity.
6

X. Wang et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
Table 2
Table 3
P97 inhibitory activities of compounds 26–34.a
P97 inhibitory activities of compounds 32–35.a
Compd.
R2
IC50 (nM)
36.4
3
5
Compd.
R1
R2
IC50 (nM)
129.4
26
27
28
29
3
6
134.9
308.1
58.3
3
3
7
8
34.6
447.2
100.9
3
0
387.1
a
All experiments were repeated three times.
Table 4
3
3
1
2
448.7
Cell viabilities of representative compounds
against A549 cancer cell lines.a
Compd
5104.5
IC50 (μM)
2
2
3
3
3
8
5
7
2.6
4.6
1.6
2.1
1.3
3
3
4
NAb
NAb
CB-5083
3
a
All experiments were repeated at least
three times.
Table 5
Stabilities of compounds 35 and CB-5083 in liver microsomes of various spe-
a
b
All experiments were repeated three times.
NA: No activity.
cies.a
Liver microsomes 35
CB-5083
4
.2. Chemistry
.2.1. N-benzyl-2-chloro-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-4-amine
T
1/2 (min) CLint (μL/min/
T
1/2 (min) CLint (μL/min/
kg)
kg)
4
(
5c)
To a 0 °C tetrahydro-4H- pyran-4-one (1c) (10 g, 100 mmol) in THF
300 mL) was added NaH (6 g, 250 mmol). The mixture was stirred for
Human
Mouse
Rat
42.3
47.8
16.8
36.3
12.3
29.5
25.8
141.4
17.0
36.5
14.7
48.4
114.2
148.7
55.0
38.6
146.5
54.7
(
Dog
3
0 min, and dimethyl carbonate (23 g, 250 mmol) was then added and
Monkey
152.3
127.4
stirred for an additional 30 min. The resulting mixture was then stirred
at 45 °C for 12 h and then poured into a 0 °C HCl solution (0.4 N,
a
All experiments were repeated at least three times.
3
00 mL). The aqueous phase was separated and extracted with ethyl
acetate (100 mL × 3); the combined organic layers were washed with
mixture was concentrated in vacuo, and diluted with water (50 mL) and
extracted with DCM (50 mL × 3). The separated organic layer was
water (100 mL) and brine (100 mL), dried over Na
2
SO , filtered and
4
evaporated in vacuo. The residue was purified by column chromato-
graphy (petroleum ether/EtOAc = 25:1) to give methyl-4-oxote-
dried over Na
2
SO and concentrated in vacuo. The resulting crude
4
methyl 4-amino-5,6-dihydro-2H-pyran-3-carboxylate 3c was then dis-
solved in acetonitrile (50 mL), and 2,2,2-trichloroacetyl isocyanate
(7.2 g, 38.46 mmol) was added. The resulting mixture was stirred for
30 min, and the precipitated solids were collected and dissolved in a
solution of ammonia in methanol (10 mL, 7 N). Then the resulting
trahydro-2H-pyran-3- carboxylate (2c) (5.5 g, yield 35%, purity 98%)
+
as a colorless liquid. MS (ESI) m/z 159.1 [M+H]
.
2
c (5.5 g, 34.97 mmol) and ammonium acetate (8.1 g, 104.9 mmol)
in methanol (100 mL) were stirred at room temperature overnight. The
7

X. Wang et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
Table 6
Single-dose pharmacokinetic parameters of compound 35 following iv and oral in SD rats.a
PK parameters
C
max (ng/mL)
T
max (h)
T
1/2 (h)
AUC0-t (ng·h/mL)
AUC0-inf (ng·h/mL)
MRT(h)
F(%)
4.2
iv (5 mg/kg)
ig (30 mg/kg)
3376.3
190.9
0.05
2.67
3.64
4.57
6947
1770
7422
2564
4.51
7.08
a
iv and ig dose formulations: solution in PEG400/ Castor oil/EtOH/water (40:10:5:45, v/v/v/v). n = 3 animals per study.
mixture was heated at 70 °C for 1 h. The reaction was cooled down and
(9) (2.6 g, yield 90%, purity 98%) as a yellow solid. MS (ESI) m/z 157.3
+ 1
the precipitated solids were collected and dried to afford the diol (4c)
[M+H]
1H, Ph), 7.13 (m, 1H, Ph), 6.30 (m, 1H, 3-H of indole), 2.44 (s, 3H,
CH ).
;
H NMR (400 MHz, DMSO‑d ) δ 7.61 (m, 1H, Ph), 7.43 (m,
6
(
3.8 g, yield 65%, purity 97%) as a white solid. MS (ESI) m/z 169.2 [M
+
+
H]
.
3
A solution of 4c (3.8 g, 22.7 mmol) in POCl (20 mL) was refluxed
3
and stirred for 3 h. After being cooled to room temperature, the mixture
4
.2.3. N-(2-methyl-1H-indol-4-yl)acetamide (13a)
was concentrated in vacuo. The residue was diluted with water
At room temperature, to solution of 3-nitroaniline (1 g,
a
(
100 mL) and extracted with DCM (50 mL × 3). The combined organic
7
.25 mmol) in DMSO (20 mL) was added acetone (0.8 g, 14.5 mmol).
layers were washed with brine (50 mL), dried over Na
2
SO , filtered and
4
The mixture was stirred for 5 min, and t-BuOK (1.2 g, 10.87 mmol) was
concentrated in vacuo. The residue was purified by column chromato-
then added. The reaction mixture was stirred at room temperature for
graphy (petroleum ether/EtOAc = 25:1) to give intermediate 2,4-di-
2
4 h and then was added water (50 mL) and the pH was adjusted to 4
chloro-7,8-dihydro-5H-pyrano[4,3-d]pyrimidine (1.4 g, yield 32%,
+
and extracted with DCM (50 mL × 3); the combined organic layers
purity 97%). MS (ESI) m/z 203.4 [M+H]
.
were washed with water (50 mL) and brine (50 mL), dried over Na
2
4
SO ,
To
a solution of 2,4-dichloro-7,8-dihydro-5H-pyrano[4,3-d]pyr-
filtered and evaporated in vacuo. The residue was purified by column
chromatography (petroleum ether/EtOAc = 10:1) to give 2-methyl-4-
imidine (1.4 g, 6.83 mmol) in acetonitrile (50 mL) was added phe-
nylmethanamine (2.2 g, 20.5 mmol) and triethylamine (2.1 g,
nitro-1H-indole (11) (0.8 g, yield 62%, purity 98%) as yellow solid. MS
2
0.5 mmol). The resulting solution was then stirred at room tempera-
−
(
(
ESI) m/z 175.4 [M−H]
.
ture for 12 h and concentrated in vacuo. The residue was purified by
column chromatography (petroleum ether/EtOAc = 5:1) to afford
compound 5c (1.6 g, yield 86%, purity 98%). MS (ESI) m/z 276.6
At room temperature to a solution of 11 (0.8 g, 4.48 mmol) in DCM
30 mL) were added (Boc) O (1.5 g, 6.72 mmol) and DMAP (109 mg,
2
+
1
0.89 mmol). Then the mixture was stirred for 3 h. The reaction was
washed with water (50 mL) and brine (50 mL). The separated organic
layer was concentrated in vacuo to give the crude tert-butyl 2-methyl-4-
nitro-1H-indole-1-carboxylate (1.1 g, yield 91%, purity 95%) as yellow
solid, which was used in the next step without further purification. MS
[
M + H]
;
H NMR (400 MHz, DMSO‑d
6
) δ 7.39–7.18 (m, 5H, Ph),
Ph), 3.88 (t,
), 2.62 (t, J = 5.0 Hz, 2H, OCH CH ).
4
.55 (d, J = 5.9 Hz, 2H, OCH
2
), 4.44 (s, 2H, CH
2
J = 5.6 Hz, 2H, OCH
2
CH
2
2
2
−
1
4
.2.2. 2-methyl-1H-indole-4-carbonitrile (9).
(ESI) m/z 275.3 [M−H]
J = 8.3 Hz, 1H, Ph), 8.14 (dd, J
(t, J = 8.2 Hz, 1H, Ph), 7.09 (s, 1H, 3-H of indole), 2.64 (s, 3H, CH
1.65 (s, 9H, CH ).
,
H NMR (400 MHz, DMSO‑d
= 0.8 Hz, J = 8.2 Hz, 1H, Ph), 7.44
),
6
) δ 8.47 (d,
To a solution of 1H-indole-4-carbonitrile (6) (5 g, 35.17 mmol) in
1
2
THF (100 mL) was added NaH (1.3 g, 52.75 mmol) at 0 °C. The mixture
was stirred for 5 min and benzenesulfonyl chloride (6.5 g, 42.21 mmol)
was then added. The reaction was allowed to room temperature and
stirred for an additional 30 min and then poured into a precooled satu-
3
3
At room temperature, to a solution of tert-butyl 2-methyl-4-nitro-
1H-indole-1-carboxylate (1.1 g, 4.05 mmol) in ethanol (40 mL) was
added Fe (1.1 g, 20.26 mmol) and saturated ammonium chloride solu-
tion (8 mL). Then the mixture was stirred at 60 °C for 3 h. The resulting
solution was filtered and concentrated in vacuo and then the resulting
solid was diluted with water (50 mL) and extracted with DCM
(50 mL × 3); the combined organic layers were washed with water
rated aqueous NH Cl solution (300 mL). The aqueous phase was sepa-
4
rated and extracted with ethyl acetate (100 mL × 3); the combined or-
ganic layers were washed with water (100 mL) and brine (100 mL), dried
over Na
2
SO , filtered and evaporated in vacuo. The residue was re-
4
crystallized (EtOAc) to give the 1-(phenylsulfonyl)-1H-indole-4-carboni-
+
trile (7) (7.4 g, yield 75%, purity 98%). MS (ESI) m/z 283.1 [M+H] .
(50 mL) and brine (50 mL), dried over Na
2
SO , filtered and evaporated
4
At −40 °C, to the solution of 7 (7.4 g, 26.38 mmol) in THF (100 mL)
was slowly added LDA (2.0 M in THF, 26.4 mL, 52.8 mmol). The mix-
ture was stirred for an additional 1 h and then MeI (7.5 g, 52.76 mmol)
was added. The resulting mixture was then allowed to warm to room
temperature and stirred for an extra 12 h. The mixture was poured into
in vacuo. The residue was purified by column chromatography (pet-
roleum ether/EtOAc = 10:1) to give tert-butyl 4-amino-2-methyl-1H-
indole-1-carboxylate (12) as yellow solid (0.8 g, yield 78%, purity
+
98%). MS (ESI) m/z 247.2 [M+H]
.
At room temperature, to a solution of 12 (0.8 g, 3.17 mmol) in DCM
a precooled saturated aqueous NH
4
Cl solution (200 mL) and extracted
(30 mL) were added acetyl chloride (298 mg, 3.8 mmol) and Et N
3
with ethyl acetate (100 mL × 3); the combined organic layers were
(0.96 g, 9.51 mmol). Then the mixture was stirred for 3 h. The reaction
solution was concentrated in vacuo to give the crude tert-butyl 4-
acetamido-2-methyl-1H-indole-1-carboxylate (813 mg, yield 89%,
washed with water (100 mL) and brine (100 mL), dried over Na
2
SO ,
4
filtered and evaporated in vacuo. The residue was purified by column
chromatography (petroleum ether/EtOAc = 20:1) to give 2-methyl-1-
purity 95%), which was used in the next step without further pur-
+
(
phenylsulfonyl)-1H-indole-4-carbonitrile (8) (5.4 g, yield 69%, purity
ification. MS (ESI) m/z 289.5 [M+H]
.
+
9
1
8%) as a white solid. MS (ESI) m/z 297.4 [M+H]
.
To a 0 °C solution of tert-butyl 4-acetamido-2-methyl-1H-indole-1-
carboxylate (813 mg, 2.82 mmol) in DCM (15 mL) was added tri-
fluoroacetic acid (3.2 g, 28.2 mmol). Then the reaction was stirred at
room temperature for an additional 3 h and evaporated in vacuo. The
residue was purified by column chromatography (petroleum ether/
EtOAc = 3:1) to give N-(2-methyl-1H-indol-4-yl)acetamide (13a) as
brown solid (477 mg, yield 90%, purity 98%). MS (ESI) m/z 189.0 [M
At room temperature, to a solution of intermediate (8) (5.4 g,
8.19 mmol) in ethanol (100 mL) was added aqueous sodium hydroxide
solution (4 M, 18.2 mL, 72.76 mmol). Then the mixture was stirred at
4
0 °C for 12 h. The resulting solution was concentrated in vacuo and
diluted with water (50 mL) and extracted with ethyl acetate
(
(
50 mL × 3); the combined organic layers were washed with water
+
1
50 mL) and brine (50 mL), dried over Na
2
SO
4
, filtered and evaporated
+H] ; H NMR (400 MHz, DMSO‑d ) δ 7.50 (m, 1H, Ph), 6.99 (m, 1H,
6
in vacuo. The residue was purified by column chromatography (pet-
roleum ether/EtOAc = 20:1) to give 2-methyl-1H-indole-4-carbonitrile
Ph), 6.89 (t, J = 7.9 Hz, 1H, Ph), 6.37 (s, 1H, 3-H of indole), 2.37 (s,
3H, CH ), 2.11 (s, 3H, CH ).
3
3
8

X. Wang et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
4
.2.4. 1-(4-(benzylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-2-yl)-
-methyl-1H-indole-4-carbonitrile (16)
H of indole), 7.43–7.37 (m, 2H, Ph), 7.33 (dd, J
2H, Ph), 7.25–7.18 (m, 1H, Ph), 7.05 (t, J = 8.1 Hz, 1H, Ph), 6.91 (d,
J = 3.7 Hz, 1H, 3-H of indole), 4.72 (d, J = 5.8 Hz, 2H, OCH ), 4.55 (s,
2H, CH Ph), 3.96 (t, J = 5.6 Hz, 2H, OCH CH ), 2.74 (t, J = 5.6 Hz, 2H,
OCH CH
1
= 6.9 Hz, J
2
= 8.4 Hz,
2
At room temperature, to the solution of N-benzyl-2-chloro-7,8-di-
2
hydro-5H-pyrano [4,3-d]pyrimidin-4-amine (5c) (200 mg, 0.725 mmol)
and 2-methyl-1H-indole-4-carbonitrile (9) (113 mg, 0.725 mmol) in
2
2
2
1
3
2
2
), 2.13 (s, 3H, CH
3
). C NMR (100 MHz, DMSO‑d ) δ 168.56,
6
1
,4-dioxane (20 mL) was added cesium carbonate (354 mg, 1.09 mmol),
158.91, 158.54, 154.81, 139.78, 135.28, 130.85, 128.36, 126.93,
Pd
2
(dba) (100 mg, 0.11 mmol) and X-Phos (52 mg, 0.11 mmol). The
3
126.75, 124.86, 123.04, 113.27, 111.92, 107.04, 102.98, 64.00, 62.76,
mixture was degassed and refilled with nitrogen three times. The re-
sulting mixture was stirred at 105 °C for 12 h and cooled to room
temperature. The volatiles were evaporated in vacuo, and the resulting
residue was dissolved in DCM (50 mL), washed with water (50 mL) and
43.92, 31.20, 23.89. m.p.: 88–90 °C. HRMS calcd for C24
H
23
N
5
O [M
2
+
+Na] 436.1743, found 436.1747.
4
.2.8. 3-((4-(benzylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-2-yl)
brine (30 mL), dried over Na
2
4
SO , filtered, and evaporated in vacuo.
amino) benzamide (19)
The residue was purified by column chromatography (petroleum ether/
Compound 19 was synthesized from 5c and 3-aminobenzonitrile
EtOAc = 4:1) to give intermediate 16 (212 mg, yield 74%, purity 98%).
1
+
1
according to the procedure for preparing CB-5083. 66% yield. H NMR
MS (ESI) m/z 396.2 [M+H] ; H NMR (400 MHz, DMSO‑d ) δ 7.92 (d,
6
(
400 MHz, DMSO‑d ) δ 7.83–7.74 (m, 1H, Ph), 7.40–7.11 (m, 8H, Ph),
6
J = 8.4 Hz, 1H, Ph), 7.52 (dd, J
1
= 0.9 Hz, J
.37–7.23 (m, 5H, Ph), 7.02 (m, 1H, Ph), 6.53 (s, 1H, 3-H of indole),
.63 (d, J = 5.9 Hz, 2H, OCH ), 4.61 (s, 2H, CH Ph) , 3.98 (t,
CH ), 2.76 (t, J = 5.7 Hz, 2H, OCH CH ),
).
2
= 7.5 Hz, 1H, Ph),
4
.64 (d, J = 5.9 Hz, 2H, OCH
J = 5.6 Hz, 2H, OCH CH ), 2.56 (t, J = 5.7 Hz, 2H, OCH
) δ 174.59, 168.53, 158.18, 140.20, 134.87,
34.84, 128.20, 128.00, 127.09, 126.57, 120.95, 119.09, 117.99,
2
), 4.46 (s, 2H, CH
2
Ph), 3.89 (t,
7
4
13
2
2
6
2
CH
2
).
C
2
2
NMR (100 MHz, DMSO‑d
J = 5.6 Hz, 2H, OCH
2
3
2
2
2
1
1
1
.31–1.20 (m, 3H, CH
02.53, 64.06, 62.89, 43.49, 31.38. m.p.: 224–226 °C. HRMS calcd for
C
21
H
21
N
5
O
2
[M+Na]+ 398.1587, found 398.1591.
4
.2.5. 1-(4-(benzylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-2-yl)-
-methyl-1H-indole-4-carboxamide (CB-5083)
2
At room temperature, to the solution of the 16 (262 mg,
4
.2.9. 1-(4-(benzylamino)-6,7,8,9-tetrahydro-5H-cyclohepta[d]pyrimidin-
0
.663 mmol) in ethanol (12 mL) was added PPh (34 mg, 0.13 mmol),
3
2
-yl)-2- methyl-1H-indole-4-carboxamide (20)
Pd(OAc)
2
(29.2 mg, 0.13 mmol), acetaldehyde oxime (35.4 mg,
Compound 20 was synthesized from 5a and 9 according to the
0
2
.60 mmol) and water (1.5 mL). The resulting mixture was refluxed for
h, cooled down to room temperature and concentrated in vacuo. The
1
procedure for preparing CB-5083. 65% yield. H NMR (400 MHz,
DMSO‑d
6
) δ 7.87–7.83 (m, 1H, Ph), 7.64–7.60 (m, 1H, Ph), 7.58–7.54
residue was purified by flash chromatography (petroleum ether/
(
m, 1H, Ph), 7.34–7.22 (m, 5H, Ph), 6.82 (s, 1H, 3-H of indole), 4.63 (d,
1
EtOAc = 2:1) to afford CB-5083 (191 mg, yield 70%, purity 99%).
H
J = 5.8 Hz, 2H, CH
2
Ph), 2.89–2.74 (m, 4H, CH ), 2.47 (s, 3H, CH ),
2
3
NMR (400 MHz, CD
Ph), 7.48–7.44 (m, 1H, Ph), 7.36–7.28 (m, 5H, Ph), 6.78 (t, J = 1.0 Hz,
H, 3-H of indole), 4.71 (s, 2H, OCH ), 4.64 (s, 2H, CH Ph), 4.05 (t,
J = 5.7 Hz, 2H, OCH CH ), 2.82 (m, 2H, OCH CH ), 2.45 (s, 3H, CH ).
3
OD) δ 7.75–7.70 (m, 1H, Ph), 7.59–7.52 (m, 1H,
1
3
1
.90–1.83 (m, 2H, CH
2
), 1.69–1.58 (m, 4H, CH ). C NMR (100 MHz,
2
DMSO‑d
6
) δ 171.16, 169.48, 160.67, 154.52, 140.03, 138.97, 137.73,
1
2
2
1
1
6
4
28.90, 128.70, 128.58, 127.64, 127.60, 127.35, 124.09, 117.34,
2
2
2
2
3
13.58, 104.46, 45.75, 38.16, 26.51, 25.74, 25.46, 16.30. m.p.:
1
3
C NMR (100 MHz, CD OD) δ 174.08, 160.60, 156.40, 140.56, 138.89,
3
2–64 °C. HRMS calcd for C26
26.2256.
H
27
N
5
O [M + H]+ 426.2288, found
1
1
33.80, 133.08, 129.99, 128.59, 129.58, 127.87, 125.74, 125.25,
21.96, 117.40, 109.80, 105.42, 65.67, 64.08, 45.19, 31.78, 15.56.
+
m.p.: 71–73 °C. HRMS calcd for C24
found 436.1747.
H
23
N
5
O
2
[M+Na] 436.1743,
4
.2.10. 1-(4-(benzylamino)-5,6,7,8,9,10-hexahydrocycloocta[d]
pyrimidin-2-yl)-2- methyl-1H-indole-4-carboxamide (21)
4
.2.6. 1-(4-(benzylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-2-yl)-
Compound 21 was synthesized from 5b and 9 according to the
2
-methyl-1H -indole-4-carbothioamide (17)
1
procedure for preparing CB-5083. 60% yield. H NMR (400 MHz,
At room temperature, to the solution of CB-5083 (100 mg,
.24 mmol) in THF (20 mL) were added Lawesson's reagent (146.7 mg,
.36 mmol). The resulting solution was then refluxed for overnight and
DMSO‑d ) δ 7.84–7.77 (m, 1H, Ph), 7.63–7.59 (m, 1H, Ph), 7.57–7.53
6
0
0
(
m, 1H, Ph), 7.45–7.14 (m, 5H, Ph), 6.83 (d, J = 4.5 Hz, 1H, 3-H of
indole), 4.63 (d, J = 5.9 Hz, 2H, CH Ph), 2.85–2.74 (m, 4H, CH ), 2.46
s, 3H, CH ), 1.79–1.63 (m, 4H, CH ), 1.51–1.36 (m, 4H, CH
) δ 169.89, 169.53, 160.76, 140.24, 138.37,
36.98, 136.89, 132.10, 131.58, 131.48, 128.87, 128.76, 128.38,
26.34, 119.04, 118.79, 113.25, 111.39, 100.47, 43.83, 34.02, 31.19,
2
2
2
concentrated in vacuo. The residue was dissolved in DCM (50 mL),
13
(
3
2
).
C
washed with water (50 mL) and brine (30 mL), dried over Na
2
SO , fil-
4
NMR (100 MHz, DMSO‑d
6
tered, and evaporated in vacuo. The residue was purified by column
1
1
2
chromatography (petroleum ether/EtOAc = 2:1) to give 17 (83 mg,
1
yield 81%, purity 98%). H NMR (400 MHz, DMSO‑d
6
) δ 9.85 (s, 1H,
9.08, 25.97, 25.81, 13.49. m.p.: 50–52 °C. HRMS calcd for C27
H
29
5
N O
NH), 9.31 (s, 1H, NH), 7.78 (d, J = 8.3 Hz, 1H, Ph), 7.36 (d, J = 7.5 Hz,
H, Ph), 7.34–7.19 (m, 5H, Ph), 6.94–6.89 (m, 1H, Ph), 6.69 (s, 1H, 3-H
of indole), 4.64 (d, J = 5.8 Hz, 2H, OCH ), 4.61 (s, 2H, CH Ph), 3.99 (t,
), 2.77 (t, J = 5.4 Hz, 2H, OCH CH ), 2.47 (s,
M+Na]+ 462.2264, found 462.2266.
[
1
2
2
J = 5.6 Hz, 2H, OCH
2
CH
2
2
2
4.2.11. 2-methyl-1-(4-((pyridin-4-ylmethyl)amino)-7,8-dihydro-5H-
1
3
3
1
1
3
4
H, CH
3
). C NMR (100 MHz, DMSO‑d
6
) δ 201.42, 159.09, 158.75,
pyrano[4,3-d] pyrimidin-2-yl)-1H-indole-4-carboxamide (22)
54.67, 139.70, 138.00, 136.49, 132.91, 128.42, 126.71, 126.54,
25.00, 120.68, 120.04, 115.22, 108.34, 104.44, 63.97, 62.74, 43.65,
Compound 22 was synthesized from 5d and 9 according to the
1
procedure for preparing CB-5083. 72% yield. H NMR (400 MHz,
0.72, 15.66. m.p.: 72–74 °C. HRMS calcd for C24
H
23
N
5
OS [M + H]+
DMSO‑d ) δ 8.61 (d, J = 5.9 Hz, 2H, Py), 7.97–7.86 (m, 1H, Ph), 7.52
6
30.1696, found 430.1687.
(d, J = 5.1 Hz, 2H, Ph), 7.45 (d, J = 7.4 Hz, 1H, Py), 7.40–7.36 (m, 1H,
Py), 6.83 (d, J = 4.5 Hz, 1H, 3-H of indole), 4.71 (d, J = 5.7 Hz, 2H,
4
.2.7. N-(1-(4-(benzylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-2-
CH
J = 5.7 Hz, 2H, OCH
(100 MHz, DMSO‑d ) δ 169.51, 159.35, 158.74, 154.66, 149.53,
2
Py), 4.65 (s, 2H, OCH
2
), 4.00 (t, J = 5.6 Hz, 2H, OCH
2
CH ), 2.78 (t,
2
13
yl)-1H-indol −4-yl)acetamide (18)
2
CH
2
), 2.18 (t, J = 7.3 Hz, 3H, CH ). C NMR
3
Compound 18 was synthesized from 5c and N-(1H-indol-4-yl)acet-
6
1
amide according to the procedure for preparing 16. 72% yield. H NMR
138.33, 136.88, 127.00, 125.31, 121.80, 120.60, 115.88, 108.52,
(
400 MHz, DMSO‑d ) δ 9.68 (s, 1H, NH), 8.25 (d, J = 8.4 Hz, 1H, Ph),
6
105.23, 64.01, 62.71, 42.92, 30.73, 15.59. m.p.: 76–78 °C. HRMS calcd
8
.10 (d, J = 3.7 Hz, 1H, Ph), 7.65 (dd, J
1
= 4.1 Hz, J
2
= 7.2 Hz, 1H, 2-
for C23
H
22
N
6
O
2
[M+Na]+ 437.1696, found 437.1727.
9

X. Wang et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
4
.2.12. 2-methyl-1-(4-((2-morpholinoethyl)amino)-7,8-dihydro-5H-
139.68, 136.97, 136.39, 128.66, 128.42, 126.74, 126.59, 120.95,
pyrano[4,3-d] pyrimidin-2-yl)-1H-indole-4-carboxamide (23).
113.20, 110.48, 108.21, 102.05, 63.97, 62.75, 43.62, 43.52, 30.66,
+
Compound 23 was synthesized from 5e and 9 according to the
15.75. m.p.: 64–66 °C. HRMS calcd for
462.1691, found 462.1734.
C
25
H
24ClN
5
O
2
[M+H]
1
procedure for preparing CB-5083. 70% yield. H NMR (400 MHz,
DMSO‑d
6
) δ 8.22 (d, J = 8.2 Hz, 1H, Ph), 7.51 (dd, J = 0.9 Hz,
1
J
2
= 7.5 Hz, 1H, Ph), 7.12 (dd, J
.92–6.89 (m, 1H, 3-H of indole), 4.47 (s, 2H, CH
H, CH ), 3.55 (t, J = 4.7 Hz, 6H, CH ), 2.74 (t, J = 5.7 Hz, 2H, CH
.65 (d, J = 1.1 Hz, 2H, CH ), 2.40 (s, 4H, CH
) δ 169.52, 158.89, 158.79, 154.81, 138.39,
1
= 7.4 Hz, J
2
= 8.3 Hz, 1H, Ph),
), 3.95 (t, J = 5.6 Hz,
),
4.2.17. N-(1-(4-(benzylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-
6
2
2
2
2-yl)-2- methyl-1H-indol-4-yl)acrylamide (28)
2
2
2
Compound 28 was synthesized from 5c and 13c according to the
1
3
1
2
2
), 1.75 (s, 3H, CH
3
).
C
procedure for preparing 16. 64% yield. H NMR (400 MHz, DMSO‑d
6
) δ
NMR (100 MHz, DMSO‑d
6
9.74 (s, 1H), 7.64 (d, J = 2.4 Hz, 1H, Ph), 7.52 (d, J = 8.3 Hz, 1H, Ph),
1
6
37.02, 127.11, 125.39, 120.67, 116.24, 108.09, 105.28, 66.28, 63.98,
7.38–7.21 (m, 5H, Ph), 6.87 (t, J = 8.1 Hz, 1H, Ph), 6.72–6.62 (m, 1H,
2.67, 55.01, 53.41, 31.38, 14.05. m.p.: 58–60 °C. HRMS calcd for
COCHCH
2
), 6.60 (s, 1H, 3-H of indole), 6.26 (dd,
J
1
= 2.1 Hz,
= 10.2 Hz, 1H
), 4.60 (s, 2H, CH Ph), 3.98
), 2.75 (t, J = 5.7 Hz, 2H, OCH CH ), 2.46
+
C
23
H
28
N
6
O
3
[M+Na] 459.2115, found 459.2118.
J
2
= 17.0 Hz, 1H COCHCH
COCHCH ), 4.65 (d, J = 5.8 Hz, 2H, OCH
(t, J = 5.6 Hz, 2H, OCH CH
2
), 5.74 (dd, J
1
2
= 2.1 Hz, J
2
2
2
4
.2.13. 1-(4-((cyclopropylmethyl)amino)-7,8-dihydro-5H-pyrano[4,3-d]
2
2
2
2
1
3
pyrimidin-2-yl) −2-methyl-1H-indole-4-carboxamide (24)
(s, 3H, CH
3
). C NMR (100 MHz, DMSO‑d ) δ 163.24, 159.03, 158.73,
6
Compound 24 was synthesized from 5f and 9 according to the
154.80, 139.70, 136.94, 136.13, 132.03, 129.22, 128.39, 126.70,
1
procedure for preparing CB-5083. 65% yield. H NMR (400 MHz,
126.58, 121.57, 113.08, 110.11, 108.12, 102.22, 66.37, 63.97, 43.59,
+
DMSO‑d
6
) δ 7.77 (s, 1H, Ph), 7.51 (dd, J
1
= 0.9 Hz, J
2
= 7.5 Hz, 1H,
30.70, 15.77. m.p.: 36–38 °C. HRMS calcd for C26H25
440.2081, found 440.2084.
N
5
2
O [M+H]
Ph), 7.45–7.34 (m, 1H, Ph), 6.91 (t, J = 1.0 Hz, 1H, 3-H of indole), 4.50
(
s, 2H, OCH
2
), 3.96 (t, J = 5.6 Hz, 2H, OCH
Ph), 2.74 (t, J = 5.7 Hz, 2H, OCH CH
), 0.88–0.81 (m, 1H, CH), 0.46–0.40 (m, 2H, CH
2
CH
), 2.65 (d, J = 1.0 Hz,
), 0.25–0.20
) δ 171.56, 169.54, 158.80,
2
), 3.30 (t, J = 6.2 Hz,
2
3
H, CH
H, CH
2
3
2
2
4.2.18. N-(1-(4-(benzylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-
2
2-yl)-2-methyl −1H-indol-4-yl)cinnamamide (29)
1
3
(
m, 2H, CH
2
). C NMR (100 MHz, DMSO‑d
6
Compound 29 was synthesized from 5c and 13d according to the
1
1
1
47.16, 138.37, 137.06, 127.10, 125.38, 120.62, 116.24, 107.91,
procedure for preparing 16. 75% yield. H NMR (400 MHz, DMSO‑d
6
) δ
05.24, 63.98, 62.80, 55.00, 31.21, 14.05, 10.91, 3.49. m.p.: 50–52 °C.
9.76 (s, 1H, NH), 7.68–7.64 (m, 1H, Ph), 7.63 (s, 1H, Ph), 7.53 (d,
J = 8.3 Hz, 1H, CH), 7.50–7.22 (m, 10H, Ph), 7.13 (d, J = 15.7 Hz, 1H,
CH), 6.89 (t, J = 8.1 Hz, 1H, Ph), 6.67 (s, 1H, 3-H of indole), 4.66 (d,
+
HRMS calcd for C21
H
23
N
5
O
2
[M+Na] 400.1743, found 400.1747.
4
.2.14. 1-(4-(benzylamino)pyrimidin-2-yl)-2-methyl-1H-indole-4-
J = 5.9 Hz, 2H, OCH
2
), 4.60 (s, 2H, CH
), 2.76 (t, J = 5.7 Hz, 2H, OCH
2
2
Ph), 3.98 (t, J = 5.7 Hz, 2H,
CH ), 2.49–2.45 (s, 3H, CH ).
carboxamide (25)
OCH CH
2
2
2
3
1
3
Compound 25 was prepared from commercially available 2,4-di-
chloro pyrimidine 14, which firstly reacted with benzylamine to form
C NMR (100 MHz, DMSO‑d ) δ 159.05, 158.75, 154.83, 139.91,
6
139.72, 136.95, 136.13, 135.00, 129.09, 128.41, 127.75, 126.73,
compound 15 and then coupled with 9 via the similar procedure for
126.59, 122.63, 121.69, 120.46, 108.14, 102.12, 63.99, 62.76, 43.60,
1
+
preparing CB-5083. 60% yield. H NMR (400 MHz, CDCl
3
) δ 8.06 (d,
31.21, 15.83. m.p.: 89–91 °C. HRMS calcd for C32
538.2213, found 538.2216.
H
29
N
5
O [M+Na]
2
J = 6.0 Hz, 1H, CH), 7.77 (s, 1H, Ph), 7.62–7.53 (m, 2H, Ph), 7.52–7.44
(
m, 2H, Ph), 7.44–7.34 (m, 2H, Ph), 6.94 (d, J = 9.7 Hz, 1H, Ph), 6.74
s, 1H, CH), 6.43 (s, 1H, 3-H of indole), 4.58 (s, 2H, CH Ph), 2.45 (s,
(
2
4.2.19. (E)-N-(1-(4-(benzylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-
1
3
3
1
1
5
3
H, CH
3
). C NMR (100 MHz, CD
3
OD) δ 174.03, 164.97, 158.42,
2-yl)-2- methyl-1H-indol-4-yl)-4-(dimethylamino)but-2-enamide (30)
55.51, 140.36, 138.87, 133.81, 130.05, 129.92, 129.65, 128.72,
Compound 30 was synthesized from 5c and 13e according to the
1
28.10, 125.82, 122.14, 122.01, 117.59, 105.81, 45.14, 14.47. m.p.:
procedure for preparing 16. 60% yield. H NMR (400 MHz, DMSO‑d
6
) δ
+
8–60 °C. HRMS calcd for C21
80.1485.
H
19
N
5
O [M+Na] 380.1481, found
9.67 (s, 1H, NH), 7.72–7.60 (m, 1H, Ph), 7.52 (d, J = 8.3 Hz, 1H, Ph),
7.43–7.28 (m, 5H, Ph), 6.96–6.83 (m, 1H, Ph), 6.79–6.68 (m, 1H, CH),
6
.62 (s, 1H, CH), 6.60–6.50 (m, 1H, 3-H of indole), 4.65 (d, J = 5.9 Hz,
4
.2.15. N-(1-(4-(benzylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-
2H, OCH
3.17 (d, J = 6.0 Hz, 2H, CH
2
), 4.59 (s, 2H, CH
2
Ph), 3.98 (t, J = 5.6 Hz, 2H, OCH
CH ), 2.46
) δ
2
CH
2
),
2
-yl)-2- methyl-1H-indol-4-yl)acetamide (26)
2
), 2.75 (t, J = 5.6 Hz, 2H, OCH
2
2
1
3
Compound 26 was synthesized from 5c and 13a according to the
(s, 3H, CH
3
), 2.26 (s, 6H, CH
3
). C NMR (100 MHz, DMSO‑d
6
1
procedure for preparing 16. 69% yield. H NMR (400 MHz, DMSO‑d
6
) δ
174.66, 163.21, 159.05, 158.76, 154.85, 139.74, 136.97, 136.07,
7
.72 (d, J = 8.3 Hz, 1H, Ph), 7.56 (d, J = 7.8 Hz, 1H, Ph), 7.28–7.17
m, 5H, Ph), 6.95 (t, J = 8.0 Hz, 1H, Ph, 6.18 (s, 1H, 3-H of indole),
.62 (d, J = 5.4 Hz, 2H, OCH ), 4.46 (s, 2H, CH Ph), 3.96 (t,
CH ), 2.79 (t, J = 5.8 Hz, 2H, OCH CH ), 2.14 (s,
128.43, 126.74, 126.61, 121.61, 112.93, 110.55, 110.02, 108.14,
(
102.30, 64.01, 62.80, 59.50, 48.68, 43.62, 31.40, 13.51. m.p.:
+
4
2
2
60–62 °C. HRMS calcd for C29
519.2483.
H
32
N
6
O
2
[M+Na] 519.2478, found
J = 5.6 Hz, 2H, OCH
2
2
2
2
1
3
6
1
1
8
4
H, CH
3
). C NMR (100 MHz, CDCl ) δ 168.54, 159.98, 158.78,
3
55.70, 138.45, 137.57, 128.92, 127.61, 122.41, 113.84, 111.03,
4.2.20. N-(1-(4-(benzylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-
07.28, 101.04, 64.99, 62.77, 45.13, 31.36, 24.66, 14.30. m.p.:
2-yl)-2-methyl-1H-indol-4-yl)-2-(pyrrolidin-1-yl)acetamide (31)
+
7–89 °C. HRMS calcd for C25
50.1902.
H
25
N
5
O
2
[M+Na] 450.1900, found
Compound 31 was synthesized from 5c and 13f according to the
1
procedure for preparing 16. 72% yield. H NMR (400 MHz, DMSO‑d
6
) δ
7
.56 (d, J = 7.7 Hz, 1H, Ph), 7.51 (d, J = 8.3 Hz, 1H, Ph), 7.37–7.20
(m, 5H, Ph), 6.87 (t, J = 8.0 Hz, 1H, Ph), 6.34 (m, 1H, 3-H of indole),
4.64 (d, J = 5.9 Hz, 2H, OCH ), 4.59 (s, 2H, CH Ph), 3.98 (t,
J = 5.6 Hz, 2H, OCH CH ), 3.31 (s, 2H, COCH ), 2.75 (t, J = 5.6 Hz,
2H, OCH CH ), 2.65 (m, 4H, NCH CH ), 2.45 (s, 3H, CH ), 1.78 (m, 4H,
NCH CH ). C NMR (100 MHz, DMSO‑d ) δ 168.87, 159.46, 159.14,
4
.2.16. N-(1-(4-(benzylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-
2
-yl)-2- methyl-1H-indol-4-yl)-2-chloroacetamide (27)
2
2
Compound 27 was synthesized from 5c and 13b according to the
2
2
2
1
procedure for preparing 16. 73% yield. H NMR (400 MHz, DMSO‑d
6
) δ
2
2
2
2
2
3
1
3
7
.53 (s, 1H, Ph), 7.51 (s, 1H, Ph), 7.37–7.21 (m, 5H, Ph), 6.88 (t,
J = 8.1 Hz, 1H, Ph), 6.55 (s, 1H, 3-H of indole), 4.65 (d, J = 5.9 Hz, 2H,
OCH ), 4.59 (s, 2H, CH Ph), 4.37 (s, 2H, COCH ), 3.98 (t, J = 5.6 Hz,
H, OCH CH ), 2.75 (t, J = 5.7 Hz, 2H, OCH CH ), 2.46 (s, 3H, CH ).
C NMR (100 MHz, DMSO‑d ) δ 164.80, 158.97, 158.76, 154.70,
2
6
155.20, 140.12, 137.29, 136.94, 129.23, 128.82, 127.13, 127.00,
122.08, 120.91, 112.89, 110.39, 108.58, 101.56, 64.39, 63.16, 59.51,
54.23, 44.03, 31.12, 23.99, 16.17. m.p.: 59–61 °C. HRMS calcd for
2
2
2
2
2
2
2
2
3
1
3
[M+H]+ 497.2659, found 497.2697.
6
C
29
H
32
N
O
6 2
10

X. Wang et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
4
.2.21. N-(2-methyl-1-(4-((pyridin-4-ylmethyl)amino)-7,8-dihydro-5H-
4.2.25. N-(1-(4-(benzylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-
pyrano[4,3-d] pyrimidin-2-yl)-1H-indol-4-yl)acrylamide (32)
2-yl)-2-methyl −1H-indol-4-yl)benzenesulfonamide (36)
Compound 32 was synthesized from 5d and 13c according to the
Compound 36 was synthesized from 5c and 13h according to the
1
1
procedure for preparing 16. 78% yield. H NMR (400 MHz, DMSO‑d
6
) δ
procedure for preparing 16. 74% yield. H NMR (400 MHz, CDCl
3
) δ
9
.73 (s, 1H, NH), 8.51 (d, J = 4.8 Hz, 2H, Py), 7.77–7.58 (m, 2H, Py),
.41 (d, J = 8.4 Hz, 1H, Ph), 7.32 (d, J = 5.0 Hz, 2H, Ph), 6.83 (t,
), 6.26 (d,
), 5.74 (d, J = 10.3 Hz, 1H, COCHCH ),
.65 (s, 1H, CH), 4.63 (s, 2H, CH Ph), 4.62–4.42 (m, 2H, OCH ), 3.99
), 2.76 (s, 2H, OCH CH ), 2.39 (s, 3H,
7.68–7.44 (m, 5H, Ph), 7.41–7.34 (m, 5H, Ph), 6.99–6.94 (m, 2H, Ph),
7
6.60 (s, 1H, 3-H of indole), 6.14 (t, J = 1.0 Hz, 1H, Ph), 4.72 (d,
J = 8.1 Hz, 1H, 3-H of indole), 6.58 (s, 1H, COCHCH
2
J = 5.3 Hz, 2H, OCH
2
), 4.56 (d, J = 5.4 Hz, 2H, CH Ph), 4.06–4.00 (m,
2
13
J = 16.9 Hz, 1H, COCHCH
2
2
2H, OCH
2
CH
2
), 2.89–2.83 (m, 2H, OCH
2
CH
2
), 2.52 (s, 3H, CH
3
).
C
4
2
2
NMR (100 MHz, DMSO‑d ) δ 158.99, 158.71, 154.66, 140.20, 139.66,
6
(
t, J = 5.8 Hz, 2H, OCH
2
CH
2
2
2
138.84, 137.04, 134.65, 132.69, 129.51, 129.11, 128.41, 128.23,
127.63, 126.72, 126.53, 121.57, 114.64, 110.99, 108.17, 102.24,
63.95, 62.73, 43.56, 31.21, 15.75. m.p.: 84–86 °C. HRMS calcd for
1
3
CH
3
). C NMR (100 MHz, DMSO‑d ) δ 163.26, 158.60, 151.99, 146.17,
6
1
1
1
4
36.86, 136.55, 136.04, 131.99, 129.30, 126.62, 122.81, 121.48,
20.79, 113.15, 102.35, 66.40, 64.00, 43.14, 32.95, 15.65. m.p.:
C
29
H
27
N
5
O
3
S [M+Na]+ 548.1726, found 548.1731.
+
12–114 °C. HRMS calcd for C26
63.1928.
H
25
N
5
O
2
[M+Na] 463.1900, found
4.2.26. N-(1-(4-(benzylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-
2
-yl)-2-methyl-1H-indol-4-yl)cyclopropanesulfonamide (37)
Compound 37 was synthesized from 5c and 13i according to the
1
4
.2.22. N-(1-(4-((cyclopropylmethyl)amino)-7,8-dihydro-5H-pyrano[4,3-
procedure for preparing 16. 64% yield. H NMR (400 MHz, CDCl
3
) δ
d]pyrimidin- 2-yl)-2-methyl-1H-indol-4-yl)acrylamide (33)
7.38–7.31 (m, 5H, Ph), 7.22 (d, J = 7.7 Hz, 1H, Ph), 7.06 (t, J = 8.0 Hz,
1H, Ph), 6.42 (s, 1H, 3-H of indole), 6.41 (d, J = 1.1 Hz, 1H, Ph), 4.76
Compound 33 was synthesized from 5f and 13c according to the
1
procedure for preparing 16. 68% yield. H NMR (400 MHz, DMSO‑d
6
) δ
(d, J = 5.4 Hz, 2H, OCH
2
), 4.58 (d, J = 1.4 Hz, 2H, CH
2
Ph), 4.07 (t,
CH ), 2.62 (d,
), 2.43 (m, 1H, CH), 1.15–1.12 (m, 2H, CH ),
) δ 159.04,
9
.77 (s, 1H, NH), 7.86 (d, J = 8.3 Hz, 1H, Ph), 7.71 (d, J = 7.7 Hz, 1H,
Ph), 7.39 (d, J = 8.7 Hz, 1H, Ph), 7.13–6.96 (m, 1H, COCH), 6.76–6.60
m, 1H, 3-H of indole), 6.27 (dd, J = 2.1 Hz, J = 17.0 Hz, 1H,
COCHCH ), 5.80–5.71 (m, 1H, COCHCH ), 4.49 (s, 2H, OCH ), 3.95 (t,
J = 5.7 Hz, 2H, OCH CH ), 3.29 (d, J = 6.1 Hz, 2H, CH ), 2.76–2.69
), 2.63 (dd, J = 1.0 Hz, J = 4.5 Hz, 3H, CH ),
), 0.25–0.19 (m, 2H,
J = 5.6 Hz, 2H, OCH
J = 1.0 Hz, 3H, CH
2
CH
2
), 2.94–2.87 (m, 2H, OCH
2
2
3
2
1
3
(
1
2
0.89–0.85 (m, 2H, CH
2
). C NMR (100 MHz, DMSO‑d
6
2
2
2
158.75, 154.78, 139.69, 137.13, 136.68, 128.41, 128.22, 126.71,
126.54, 123.69, 121.68, 115.80, 111.11, 108.17, 102.54, 63.98, 62.76,
2
2
2
(
m, 2H, OCH
2
CH
2
1
2
2
3
43.59, 30.71, 15.86, 5.15. m.p.: 83–85 °C. HRMS calcd for C26
[M+Na]+ 512.1726, found 512.1727.
H
27
N
5 3
O S
1
.14–1.06 (m, 1H, CH), 0.45–0.39 (m, 2H, CH
1
3
CH
2
). C NMR (100 MHz, DMSO‑d ) δ 163.29, 158.82, 158.73, 154.87,
6
1
1
37.07, 136.14, 132.06, 129.38, 126.63, 121.65, 120.83, 113.10,
4.2.27. N-(1-(4-(benzylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-
10.18, 107.83, 102.25, 63.97, 62.80, 55.00, 31.21, 16.11, 10.93, 3.49.
2-yl)-2- methyl-1H-indol-4-yl)-1,1,1-trifluoromethanesulfonamide (38)
+
m.p.: 68–70 °C. HRMS calcd for C23
found 426.1916.
H
25
N
5
O
2
[M+Na] 426.1900,
Compound 38 was synthesized from 5c and 13j according to the
1
procedure for preparing 16. 66% yield. H NMR (400 MHz, CDCl
3
) δ
7
.36–7.27 (m, 5H, Ph), 7.09–7.02 (m, 2H, Ph), 6.92 (t, J = 8.0 Hz, 1H,
Ph), 6.38 (s, 1H, 3-H of indole), 4.66 (s, 2H, OCH
2
), 4.53 (s, 2H,
4
.2.23. N-(2-methyl-1-(4-((naphthalen-1-ylmethyl)amino)-7,8-dihydro-
CH
2
Ph), 4.00 (t, J = 5.6 Hz, 2H, OCH
2
CH ), 2.81 (t, J = 5.7 Hz, 2H,
2
1
3
5
H-pyrano [4,3-d]pyrimidin-2-yl)-1H-indol-4-yl)acrylamide (34).
OCH
2
CH
2
), 2.46 (s, 3H, CH
3
). C NMR (100 MHz, DMSO‑d ) δ 163.26,
6
Compound 34 was synthesized from 5g and 13c according to the
159.35, 153.71, 139.51, 136.53, 136.29, 128.32, 128.21, 126.59,
1
procedure for preparing 16. 73% yield. H NMR (400 MHz, DMSO‑d
6
) δ
126.42, 122.35, 120.59, 117.37, 111.16, 108.25, 103.89, 63.45, 62.80,
9
.67 (s, 1H, NH), 8.17–8.10 (m, 1H, Ph), 8.02–7.96 (m, 1H, Ph), 7.85
d, J = 7.9 Hz, 1H, Ph), 7.76–7.68 (m, 1H, Ph), 7.58–7.53 (m, 2H, Ph),
.50–7.34 (m, 4H, Ph), 6.52 (d, J = 4.0 Hz, 1H, COCHCH ), 6.51–6.45
m, 1H, 3-H of indole), 6.27–6.19 (m, 1H, COCHCH
43.25, 31.41, 14.75. m.p.: 50–52 °C. HRMS calcd for C24
H
22
3
F N
5
O S [M
3
+
(
+Na] 540.1287, found 540.1290.
7
2
(
2
), 5.74–5.69 (m,
4.3. Biological assay
1
4
2
1
1
1
8
4
H, COCHCH
2
), 5.12 (d, J = 5.5 Hz, 2H, CH
2
Ph), 4.66 (s, 2H, OCH
CH
2
2
),
),
.03–3.96 (m, 2H, OCH
2
CH ), 2.77 (t, J = 5.7 Hz, 2H, OCH
2
2
4.3.1. Kinase inhibition assay
1
3
.39–2.34 (m, 3H, CH
3
). C NMR (100 MHz, DMSO‑d
6
) δ 163.20,
The ATPase assay was performed according to the following pro-
tocol: compounds were diluted in DMSO with a 3-fold 10-point serial
dilution starting at 10 μM. The tenth concentration point was solvent
control group (no drug). The assay was done in a 384-well plate with
each row as a single dilution series with duplicate of each compound
concentration point. In 4 μL total volume, 60 μg/mL p97 hexameric
enzyme and 100 μM ATP were added to start the reaction. The plate
was sealed and incubated at 25 °C for 60 min after mixing thoroughly in
an orbital shaker. ADP Glo reagents 1 and 2 were added according to
the manufacturer’s protocol (Promega, Madison, WI). The luminescence
was measured by CLARIO Star Plate Reader as the end point of the
reaction. The program Graphpad Prism 6 was used to fit nonlinear
curve and calculate IC50 of each compounds.31
59.12, 158.83, 154.88, 136.85, 134.55, 133.36, 132.00, 130.75,
28.64, 127.24, 126.23, 125.86, 125.55, 123.38, 123.15, 121.26,
20.69, 108.25, 102.29, 64.02, 62.84, 55.00, 31.21, 14.05. m.p.:
+
2–84 °C. HRMS calcd for C30
H
27
N
5
O
2
[M+H] 490.2237, found
90.2242.
4
.2.24. N-(1-(4-(benzylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-
2
-yl)-2-methyl −1H-indol-4-yl)methanesulfonamide (35)
Compound 35 was synthesized from 5c and 13g according to the
1
procedure for preparing 16 in 76% yield. H NMR (400 MHz, CDCl
3
) δ
7
1
.37–7.33 (m, 5H, Ph), 7.20 (d, J = 7.6 Hz, 1H, Ph), 7.08 (t, J = 8.0 Hz,
H, Ph), 6.44 (s, 1H, 3-H of indole), 6.36 (s, 1H, Ph), 4.76 (d,
), 4.58 (s, 2H, CH Ph), 4.08 (t, J = 5.6 Hz, 2H,
), 2.95 (s, 3H, CH ), 2.92 (t, J = 5.7 Hz, 2H, OCH CH ), 2.62
J = 5.3 Hz, 2H, OCH
2
2
4.3.2. Cell culture and inhibition of cell proliferation
OCH
2
CH
2
3
2
2
A549 cell lines were cultured according to ATCC guidelines. Cells
were plated (2000 cells/well) in a volume of 90 μL/well of complete
1
3
(
s, 3H, CH
3
). C NMR (100 MHz, DMSO‑d ) δ 159.06, 158.75, 154.75,
6
1
1
1
4
39.70, 137.19, 136.83, 128.42, 128.20, 126.72, 126.54, 123.35,
media in 96-well cell culture plates and cultured at 37 °C with 5% CO
2
21.79, 115.41, 111.17, 108.23, 102.38, 63.98, 62.75, 43.60, 30.71,
for 24 h. Inhibitors were dissolved in DMSO (less than 0.1%) and tested
in duplicate utilizing 3-fold serial dilutions with the highest con-
centration at 10 μM. Inhibitors were incubated with cells at 37 °C with
5.82. m.p.: 111–113 °C. HRMS calcd for C24
64.1750, found 464.1720.
H
25
N
5
O
3
S [M+H]+
11

X. Wang et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
5
% CO
2
. After 72 h treatment, Cell Counting Kit-8 (CCK-8) was added to
8. Peters JM, Harris JR, Lustig A, et al. Ubiquitous soluble Mg2+-ATPase complex: a
structural study. J Mol Biol. 1992;223(2):557–571.
the plates to measure cell viabilities. Absorbance at 450 nm was mea-
sured and using GraphPad software to fit sigmoidal curve to determine
IC50 value.
9
.
Rouiller I, Butel VM, Latterich M, et al. A major conformational change in p97 AAA
ATPase upon ATP binding. Mol Cell. 2000;6(6):1485–1490.
10. Wang Q, Song C, Li CC. Molecular perspectives on p97-VCP: progress in under-
standing its structure and diverse biological functions. J Struct Biol.
2
004;146(1–2):44–57.
4.3.3. In vitro liver microsome stabilities
1
1. Raasi S, Wolf DH. Ubiquitin receptors and ERAD: a network of pathways to the
The liver microsomal incubations consisted of PBS (pH 7.4) con-
proteasome. Semin Cell Dev Biol. 2007;18(6):780–791.
taining 1 μM compounds 35 and CB-5083, 40 mM NADP, 80 mM G6P,
12. Wolf DH, Stolz A. The Cdc48 machine in endoplasmic reticulum associated protein
degradation. Biochim Biophys Acta. 2012;1823(1):117–124.
1
00 U/mL G6PDH, 120 mM MgCl and 0.5 mg/mL liver microsomes
2
1
3. Zhu WW, Kang L, Gao YP, et al. Expression level of valosin containing protein is
associated with prognosis of primary orbital MALT lymphoma. Asian Pac J Cancer
Prev. 2013;14(11):6439–6443.
(
mouse, rat, dog, monkey and human, respectively). The reaction
mixtures were pre-incubated for 3 min at 37 °C before the addition of
corresponding liver microsomes, then terminated at 0, 5, 10, 20, 30,
1
4. Tsujimoto Y, Tomita Y, Hoshida Y, et al. Elevated expression of valosin-containing
protein (p97) is associated with poor prognosis of prostate cancer. Clin Cancer Res.
6
0 min by adding equal volume of ice-cold acetonitrile. The final con-
2
004;10(9):3007–3012.
centration of organic solvents was < 0.1% in all incubations. The
samples were centrifuged at 4000 rpm for 20 min at 4 °C and then
analyzed by liquid chromatography-mass spectrometry (ABSciex
API4000).
15. Yamamoto S, Tomita Y, Hoshida Y, et al. Expression level of valosin-containing
protein (p97) is correlated with progression and prognosis of non-small-cell lung
carcinoma. Ann Surg Oncol. 2004;11(7):697–704.
1
1
1
6. Yamamoto S, Tomita Y, Hoshida Y, et al. Increased expression of valosin-containing
protein (p97) is associated with lymph node metastasis and prognosis of pancreatic
ductal adenocarcinoma. Ann Surg Oncol. 2004;11(2):165–172.
7. Yamamoto S, Tomita Y, Hoshida Y, et al. Expression level of valosin-containing
protein (VCP) as a prognostic marker for gingival squamous cell carcinoma. Ann
Oncol. 2004;15(9):1432–1438.
4.3.4. In vivo pharmacokinetics
Male Sprague-Dawley rats (200 g) were administrated with the test
8. Nadeau ME, Rico C, Tsoi M, et al. Pharmacological targeting of valosin containing
protein (VCP) induces DNA damage and selectively kills canine lymphoma cells. BMC
Cancer. 2015;15:479.
compounds intravenously (iv) at 5 mg/kg or orally (ig) at 30 mg/kg.
Compound 35 was dissolved in mixture of 40% PEG400, 10% Castor
oil, 5% EtOH and 45% buffered saline for tail-vein or oral administra-
tion. Blood samples (0.1 mL) were then obtained via orbital sinus
puncture at 2 min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h and 24 h time
points and collected into heparinized tubes. Heparinized blood samples
collected for PK analyses were centrifuged at 4000 rpm for 10 min at
19. Wojcik C, Yano M, DeMartino GN. RNA interference of valosin-containing protein
(VCP/p97) reveals multiple cellular roles linked to ubiquitin/proteasome-dependent
proteolysis. J Cell Sci. 2004;117(Pt 2):281–292.
2
0. Kobayashi T, Tanaka K, Inoue K, et al. Functional ATPase activity of p97/valosin-
containing protein (VCP) is required for the quality control of endoplasmic reticulum
in neuronally differentiated mammalian PC12 cells. J Biol Chem.
2
002;277(49):47358–47365.
4
°C. LC/MS/MS analysis of compound 35 was performed under opti-
2
1. Muller JM, Deinhardt K, Rosewell I, et al. Targeted deletion of p97 (VCP/CDC48) in
mouse results in early embryonic lethality. Biochem Biophys Res Commun.
2007;354(2):459–465.
mized conditions to obtain the best sensitivity and selectivity of the
analyte in selected reaction monitoring mode (SRM). Plasma con-
centration-time data were analyzed by a non-compartment model using
the software Kinetica 5.1.
2
2
2. Kakizuka A. Roles of VCP in human neurodegenerative disorders. Biochem Soc Trans.
2
008;36:105–108.
3. Bursavich MG, Parker DP, Willardsen JA, et al. 2-Anilino-4-aryl-1,3-thiazole in-
hibitors of valosin-containing protein (VCP or p97). Bioorg Med Chem Lett.
2
010;20(5):1677–1679.
Acknowledgments
2
4. Chou TF, Bulfer SL, Weihl CC, et al. Specific inhibition of p97/VCP ATPase and
kinetic analysis demonstrate interaction between D1 and D2 ATPase domains. J Mol
Biol. 2014;426(15):2886–2899.
This work was supported by the following grants: National Natural
Science Foundation of China (21877061), Natural Science Foundation
of Jiangsu Province (BK20171448), National and Local Joint
Engineering Research Center of Biomedical Functional Materials and
Priority Academic Program Development of Jiangsu Higher Education
Institutions (PAPD).
2
2
5. Magnaghi P, D'Alessio R, Valsasina B, et al. Covalent and allosteric inhibitors of the
ATPase VCP/p97 induce cancer cell death. Nat Chem Biol. 2013;9(9):548–556.
6. Polucci P, Magnaghi P, Angiolini M. Alkylsulfanyl-1,2,4-triazoles, Alkylsulfanyl-
1,2,4-triazoles, a new class of allosteric valosine containing protein inhibitors.
Synthesis and structure-activity relationships. J Med Chem. 2013;56(2):437–450.
7. Cervi G, Magnaghi P, Asa D, et al. Discovery of 2-(cyclohexylmethylamino)pyr-
imidines as a new class of reversible valosine containing protein inhibitors. J Med
Chem. 2014;57(24):10443–10454.
2
References
28. Chou TF, Brown SJ, Minond D, et al. Reversible inhibitor of p97, DBeQ, impairs both
ubiquitin-dependent and autophagic protein clearance pathways. Proc Natl Acad Sci
USA. 2011;108(12):4834–4839.
1
2
3
.
.
.
Meyer H, Bug M, Bremer S. Emerging functions of the VCP/p97 AAA-ATPase in the
ubiquitin system. Nat Cell Biol. 2012;14(2):117–123.
2
3
3
3
3
9. Chou TF, Li K, Frankowski KJ, et al. Structure-activity relationship study reveals
ML240 and ML241 as potent and selective inhibitors of p97 ATPase. ChemMedChem.
Chou TF, Deshaies RJ. Development of p97 AAA ATPase inhibitors. Autophagy.
2
013;8(2):297–312.
2
014;7(9):1091–1092.
0. Zhou HJ, Wang J, Yao B, et al. Discovery of a first-in-class, potent, selective, and
Dargemont C, Ossareh-Nazari B. Cdc48/p97, a key actor in the interplay between
orally bioavailable inhibitor of the p97 AAA ATPase (CB-5083). J Med Chem.
autophagy and ubiquitin/proteasome catabolic pathways. Biochim Biophys Acta.
2
015;58(24):9480–9497.
2
012;1823(1):138–144.
1. Anderson DJ, Le Moigne R, Djakovic S, et al. Targeting the AAA ATPase p97 as an
4
.
Orme CM, Bogan JS. The ubiquitin regulatory X (UBX) domain-containing protein
TUG regulates the p97 ATPase and resides at the endoplasmic reticulum-golgi in-
termediate compartment. J Biol Chem. 2012;287(9):6679–6692.
approach to treat cancer through disruption of protein homeostasis. Cancer Cell.
2
015;28(5):653–665.
2. Obach RS. Prediction of human clearance of twenty-nine drugs from hepatic mi-
crosomal intrinsic clearance data: an examination of in vitro half-life approach and
nonspecific binding to microsomes. Drug Metab Dispos. 1999;27(11):1350–1359.
3. Baranczewski P, Stanczak A, Sundberg K, et al. Introduction to in vitro estimation of
metabolic stability and drug interactions of new chemical entities in drug discovery
and development. Pharmacol Rep. 2006;58(4):453–472.
5
6
.
.
Brunger AT, DeLaBarre B, Davies JM, et al. X-ray structure determination at low
resolution. Acta Crystallogr D Biol Crystallogr. 2009;65(Pt 2):128–133.
Dreveny I, Kondo H, Uchiyama K, et al. Structural basis of the interaction between
the AAA ATPase p97/VCP and its adaptor protein p47. EMBO J.
2
004;23(5):1030–1039.
7
.
Huyton T, Pye VE, Briggs LC, et al. The crystal structure of murine p97/VCP at 3.6 Å.
J Struct Biol. 2003;144(3):337–348.
12