Development of novel proteasome inhibitors based on phthalazinone scaffold

Article abstract of DOI:10.1016/j.bmcl.2016.04.067

In this study we designed a series of proteasome inhibitors using pyridazinone as initial scaffold, and extended the structure with rational design by computer aided drug design (CADD). Two different synthetic routes were explored and the biological evaluation of the phthalazinone derivatives was investigated. Most importantly, electron positive triphenylphosphine group was first introduced in the structure of proteasome inhibitors and potent inhibition was achieved. As 6c was the most potent inhibitor of proteasome, we examined the structure-activity relationship (SAR) of 6c analogs.

Full text of DOI:10.1016/j.bmcl.2016.04.067

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of novel proteasome inhibitors based on phthalazinone
scaffold
⇑
⇑
Lingfei Yang, Wei Wang, Qi Sun, Fengrong Xu, Yan Niu, Chao Wang, Lei Liang , Ping Xu
Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, China
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study we designed a series of proteasome inhibitors using pyridazinone as initial scaffold, and
extended the structure with rational design by computer aided drug design (CADD). Two different syn-
thetic routes were explored and the biological evaluation of the phthalazinone derivatives was investi-
gated. Most importantly, electron positive triphenylphosphine group was first introduced in the
structure of proteasome inhibitors and potent inhibition was achieved. As 6c was the most potent inhi-
bitor of proteasome, we examined the structure–activity relationship (SAR) of 6c analogs.
Ó 2016 Elsevier Ltd. All rights reserved.
Received 12 January 2016
Revised 11 April 2016
Accepted 23 April 2016
Available online xxxx
Keywords:
Proteasome inhibitors
Phthalazinone
Triphenylphosphine
Drug design
Structure–activity relationship
The ubiquitin proteasome pathway (UPP) is essential for intra-
cellular protein degradation in eukaryotic cells and is involved in
many vital cellular processes such as signal transduction, apopto-
sis, cell cycling and antigen processing for appropriate immune
responses.1 The 26S proteasome is known as a large multicatalytic
protein complex, consisting of two 19S regulatory particles and
one 20S catalytic core. The active sites of the 20S proteasome are
located on the b1, b2 and b5 subunits with caspase-like (C-L), tryp-
sin-like (T-L), and chymotrypsin-like (ChT-L) activities, respec-
tively. The proteasome is a validated drug target for treatment of
cancer, and three proteasome inhibitors bortezomib, carfilzomib,
and marizomib have recently been approved by FDA for the treat-
ment of multiple myeloma. Besides, several second-generation
proteasome inhibitors are currently in clinical trials.2,3
In terms of the molecular structure or scaffold, current protea-
some inhibitors are mainly based on peptides and peptidomimet-
ics, which mimic the substrates binding to threonine of b5
subunit. For instance, we previously reported a series of furan-
based proteasome b5 subunit inhibitors, which are simply di- or
tri-peptide derivatives.4,5 However, many novel proteasome inhi-
bitors have been developed in the past two decades and various
small-molecule proteasome inhibitors with different scaffolds
have been investigated.6–12
When the C4 and C5 of pyridazinone are conjugated with a ben-
zene ring, the conjugation is extended and the new structure is
p
formed as phthalazinone. Phthalazinone is another important
pharmacophore contained in several drugs including Olaparib
(Fig. 1a), the first poly ADP-ribose polymerase (PARP) inhibitor
approved to treat advanced ovarian cancer in women with defec-
tive BRCA genes.30 However, neither pyridazinone, nor phthalazi-
none has ever been reported in designing proteasome inhibitors.
Our group is devoted to discover proteasome inhibitors with
novel scaffolds. In this study, we designed a series of proteasome
inhibitors using pyridazinone as initial scaffold and extended the
structure through rational design. Two different synthetic routes
were explored and the biological evaluation of the phthalazinone
derivatives was investigated. Most importantly, positively charged
triphenylphosphine group was first introduced into a proteasome
inhibitor and potent activity has been achieved.
Based on our previous results, we proposed that three impor-
tant positions of the pyridazinone scaffold could be modified and
the stepwise extension strategy was shown in Figure 1b. In order
to occupy the small S2 pocket of the proteasome b5 site, we intro-
duced a benzene ring conjugated to the C4 and C5 positions of
pyridazinone yielding phthalazinone. Then we introduced a N,N-
diethylaniline group targeting S1 pocket, and a bromobutyl group
extending to S3 pocket. Thus the compound 5a was first designed
and the corresponding docking model showed that 5a was able to
fully occupy all three pockets. Unfortunately, compound 5a did not
show any observable proteasome inhibition (Table 1). We
speculated that perhaps the S3 pocket may have low affinity to
Pyridazinone has been proved as a superior pharmacophore for
building a broad range of chemicals with versatile functions.13–29
⇑
Corresponding authors. Tel.: +86 8280 1505.
E-mail address: leiliang@bjmu.edu.cn (L. Liang).
http://dx.doi.org/10.1016/j.bmcl.2016.04.067
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Yang, L.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.04.067

2
L. Yang et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
yphenylboronicacid. Compound 4 can be obtained by copper(I) cat-
a
alyzed C-N bond coupling reaction between 3 and various aromatic
halides. Next, compound 5 was easily obtained by reflux of 4 and
1,4-dibromobutane in acetonitrile. Finally, 6 was prepared by
nucleophilic substitution of 5 by different nucleophiles such as
triphenylphosphine. The total yields were ranging from 18% to 30%.
In another case, different aliphatic groups were introduced to
the 2-NH of phthalazinone. Thus the synthetic route was altered.
6
7
O
5
4
5
4
8
N
NH
N
3
6
O
1
O
N
1
NH
2
O
N
3
NH
2
F
N
O
pyridazinone
phthalazinone
Olaparib
b
Table 1
HO
O
Structure and proteasome ChT-L inhibitory activity by enzyme assaysa
HO
O
N
NH
N
N
NH
O
O
N
N
R2
R1
O
O
HO
O
R2
N
R1
N
N
R1
n
Compd
5a
R1
R2
Inhib. [%]b
NIc
IC50
NDd
[lM]
Figure 1. (a) Phthalazinone structure in drug. (b) The design of phthalazinone
derivatives by stepwise extension of the pyridazinone scaffold.
N
N
–Br
6a
–PPh+3
99.1 0.1
10.25 0.45
17.63 0.56
9.88 0.21
14.23 0.15
S
–PPh+3
–PPh+3
–PPh+3
93.2 0.3
99.5 0.6
94.3 2.0
the bromobutyl chain. Thus, we considered to substitute it with
triphenylphosphine group, which is much more bulky, to bind to
the wider S3 pocket. As we expected, the biochemical evaluation
result showed that the new compound 6a exhibited significant
enhancement of proteasome inhibition with a single modification
(Fig. 2).
6b
6c
6d
NH
6e
–PPh+3
98.7 0.2
13.76 0.22
To further optimize our design, we synthesized a series phtha-
lazinone 6a derivatives, and evaluated their proteasome inhibition
activity. As shown in Figure 3, we designed 7a, 7b and 7c by elim-
inating the extended moieties of phthalazinone scaffold. As we
expected, when the phthalazinone scaffold was replaced by phenyl
or pyridazinone, no activity has been observed, which proved the
phthalazinone structure is irreplaceable. Moreover, 7c showed no
activity that confirmed the necessary modification of phenol group
by alkyl chain. Furthermore, we investigated the substituted posi-
tion and length of alkyl chain. When the alkyl was linked to the
meta-phenol of 7d, the activity diminished due to the altered trend
of alkyl chain toward the S3 pocket. In the case of 7e, the length of
alkyl chain was prolonged while the activity maintained.
With the results above, we summarized the first round SAR
information. It is confirmed that both the phthalazinone and posi-
tively charged triphenylphosphine group are irreplaceable. The
substituted position of alkyl on the phenol group is also crucial
for the proteasome inhibition activity but the length is not
important.
In the second round optimization, a series of phthalazinone
derivatives were designed and synthesized with some common
features including (1) retention of the phthalazinone and triph-
enylphosphine groups; (2) modification of 2-NH of phthalazinone
by various aromatic and aliphatic groups; (3) fixation of –OH on
the para-position of the phenyl group; (4) installation of the alkyl
on the –OH group is n-butyl.
Depending on the different N-substitution of the phthalazinone,
two synthetic routes were carried out.33–37 In one case, the aro-
matic group was introduced to the 2-NH of phthalazinone by cop-
per(I) catalyzed C–N bond coupling reaction. As shown in
Scheme 1, compound 6 can be synthesized efficiently by 5 steps.
The starting material 2,3-dihydrophthalazine-1,4-dione 1 was
refluxed with POBr3 for 24 h. After cooling, the white precipitation
can be filtered and subsequently refluxed in acetic acid for 2 h.
After these two steps, a white product 4-bromophthalazin-1(2H)-
one 2 can be filtered out for next step without purification. Suzuki
coupling reaction was employed to prepare 3 with 4-hydrox-
N
N
N
6f
–P(Cy)+3
98.5 1.6
8.6 0.4
13.34 0.08
ND
N
6g
N
N
6h
6i
5.8 0.6
ND
ND
N
N
10.3 0.9
N
N
N
N
6j
–PPh2
19.6 2.1
5.1 2.0
8.0 1.3
13.2 2.6
ND
ND
ND
ND
O
N
6k
6l
N
6m
N
8a
8b
8c
8d
–Me
–Et
n-Propyl
n-Butyl
–PPh+3
–PPh+3
–PPh+3
–PPh+3
NI
NI
ND
ND
ND
ND
12.1 1.4
35.8 1.1
8e
8f
–PPh+3
–PPh+3
52.4 0.4
48.2 2.8
26.05 0.11
23.05 0.27
8g
8h
–PPh+3
–PPh+3
70.5 0.7
21.54 0.09
16.14 0.29
84.9 0.5
80.9 2.4
8i
–PPh+3
—
11.86 0.17
MG-132e
—
37.0 3.2 nM
a
Values represent the mean SD of three independent experiments, each based
on four biological replicates.
b
Percent inhibition at 25
NI: no inhibition.
l .
g mLÀ1
c
d
e
ND: not determined.
N-(Benzyloxycarbonyl)-leucinyl-leucinyl-leucinal: a potent proteasome inhi-
bitor, used as positive control.
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L. Yang et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
S2
S2
O
O
O
O
N
N
N N
S2
Br
PPh3
S3
S3
N
N
S1
S3
S1
S1
5a
6a
Figure 2. Docking of compound 5a in proteasome b5 site and the further optimization of the terminal group. Gold 3.0 was used to dock the inhibitors into the b5 subunit of
the 20S yeast proteasome, the structure of which was obtained from the RCSB Protein Data Bank (PDB ID: 3SHJ).9 The structure of the human 20S proteasome is quite similar
to that of the yeast 20S proteasome, and the chymotrypsin active site is highly conserved.31 Before docking, the protein and ligands were prepared in Discovery Studio 2.5.32
O
O
N
N
HO
O
O
N
N
Ph3P
Ph3P
N
N
7a NI
7b NI
7c NI
O
O
O
N
N
N N
O
Ph3P
Ph3P
N
N
7d NI
7e 90.3 + 0.4
Figure 3. Structures and proteasome ChT-L inhibitory activity of compounds 7a–e. Values represent the mean SD of three independent experiments, each based on four
biological replicates. Percent inhibition at 25
control.
l
g mLÀ1. NI: no inhibition. N-(Benzyloxycarbonyl)-leucinyl-leucinyl-leucinal: a potent proteasome inhibitor, used as positive
ii
iii
i
HO
O
HO
O
Br
O
O
O
N
N
N
NH
HN NH
N NH
R1
4
3
1
2
iv
v
O
O
O
O
N
N
N
N
Br
R1
R2
R1
R1 = Aryl
6
5
Scheme 1. Synthesis of compound 6. Reagents and conditions: (i) step 1: 1,2-dichloroethane, POBr3, reflux for 24 h, cooling and filtration; step 2: acetic acid, reflux for 2 h,
cooling and filtration; (ii) 4-hydroxyphenylboronicacid, Pd(PPh3)4, Na2CO3, DMF, H2O, 120 °C, 48 h; (iii) 4-bromo-N,N-diethylaniline, CuI, Cs2CO3, DMF, 120 °C, 36 h; (iv) 1,4-
dibromobutane, K2CO3, CH3CN, reflux for 18 h; (v) R (R = PPh3 or other nucleophiles), CH3CN, reflux for 24 h.
As outlined in Scheme 2, the final products can be synthesized in 5
steps with total yields from 20% to 37%. Compound 2 was prepared
in the similar procedure as depicted in Scheme 1. In order to pre-
pare 9, compound 2 was mixed with various alkyl halides and
sodium hydride in DMF, the reaction was accomplished under
room temperature for 24 h. Then Suzuki coupling reaction was uti-
lized to construct 10. Finally, 8 was obtained via the same proce-
dure as described in Scheme 1.
After the second round synthesis, totally 23 phthalazinone
derivatives were obtained for biochemical evaluation. As shown
in Table 1, we investigated the effect of N-substituted group. Var-
ious aromatic groups including N,N-diethylamino 6a, 2-thiophene
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4
L. Yang et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
iii
ii
i
Br
O
Br
O
HO
O
O
O
N
N
R
HN NH
N
NH
N N
R
1
2
9
10
iv
v
O
O
O
O
N
N
R
N
N
R
Br
Ph3P
R = -Alkyl
11
8
Scheme 2. Synthesis of compound 8. Reagents and conditions: (i) step 1: 1,2-dichloroethane, POBr3, reflux for 24 h, cooling and filtration; step 2: acetic acid, reflux for 2 h,
cooling and filtration; (ii) NaH, alkyl halide, DMF, 0 °C – rt, 24 h; (iii) 4-hydroxyphenylboronicacid, Pd(PPh3)4, Na2CO3, DMF, H2O, 120 °C, 48 h; (iv) 1,4-dibromobutane, K2CO3,
CH3CN, reflux for 18 h; (v) PPh3, CH3CN, reflux for 24 h.
6b, phenyl 6c, p-methylaminophenyl 6d, and naphthyl 6e, were
Acknowledgments
introduced and obvious activity were observed. The best result
was achieved by 6c which showed IC50 less than 10
exhibited considerable proteasome activity with IC50 10
l
M. 6a also
M. To
This work was supported by the National Natural Science Foun-
dation of China (21202003/21172012), the National Basic Research
Program of China (2012CB518000) and the Specialized Research
Fund for the Doctoral Program of Higher Education of China
(20120001110010).
l
investigate whether the PPh3 could be replaced by other positively
charged groups, we synthesized several compounds with different
positively charged groups including P(Cy)3 6f, pyridine 6g, methyl
imidazole 6h and triethylamine 6i. However, only 6f showed com-
petitive activity probably due to the similar structure with triph-
enylphosphine. 6g, 6h and 6i showed poor inhibition activity
which indicating that positive charge might not be the only essen-
tial factor, the size of terminal group also plays important role. Fur-
thermore, we investigated electroneutral terminal groups such as
bromine 5a, diphenylphosphine 6j, morpholine 6k, 6-H-pyridine
6l, 4-H-pyrrole 6m, all of them exhibited negligible inhibition
activity.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.04.
067.
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