Discovery of Potent, Selective, and Direct Acid Sphingomyelinase Inhibitors with Antidepressant Activity

Article abstract of DOI:10.1021/acs.jmedchem.9b00739

Recent studies on sphingolipids suggest that acid sphingomyelinase (ASM), which plays a central role in the pathogenesis of major depression, is emerging to be a novel target for developing antidepressants. Herein we first described the design, synthesis,

Full text of DOI:10.1021/acs.jmedchem.9b00739

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Article
Discovery of Potent, Selective, and Direct Acid
Sphingomyelinase Inhibitors with Antidepressant Activity
Kan Yang, Jinying Yu, Keyi Nong, Youzhi Wang, Ao Niu, Wenlu Chen, Jibin Dong, and Jinxin Wang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00739 • Publication Date (Web): 16 Jan 2020
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Discovery of Potent, Selective, and Direct Acid
Sphingomyelinase Inhibitors with
Antidepressant Activity
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Kan Yang1,2, Jinying Yu1, Keyi Nong1,Youzhi Wang1, Ao Niu1, Wenlu Chen1, Jibin
Dong3*, Jinxin Wang1*
1Jiangsu Key Laboratory of Drug Design and Optimization, Department of Medicinal
Chemistry, School of Pharmacy, China Pharmaceutical University, Nanjing 210009,
China
2Key Laboratory of Pharmaceutical Quality Control of Hebei Province, College of
Pharmaceutical Sciences, Hebei University, Baoding 071002, China
3Department of Biochemistry, School of Pharmacy, Fudan University, Shanghai
201203, China
Abstract
Recent studies on sphingolipids suggest that acid sphingomyelinase (ASM) which
plays a central role in the pathogenesis of major depression, is emerging to be a novel
target for developing antidepressants. Herein we firstly described the design,
synthesis, and biological evaluation of hydroxamic acid-based direct inhibitors of
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ASM with the effort of validating their antidepressant effects in vivo. As a result, a
series of novel ASM inhibitors were developed using a structure-based approach. Our
studies demonstrated that the administration of 21b improved depression-like
behaviors of rats. Importantly, this positive result was relevant to the inhibition of
ASM and the increasing neurogenesis in hippocampus. To the best of our knowledge,
this is the first time that direct inhibitors of ASM were developed to support the
possibility of ASM as a potential therapeutic target for depression.
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Introduction
Major depression disorder (MDD) is a severe, chronic and life-threatening illness,
which is regarded as a significant cause of global disability and mortality.1 It usually
takes several weeks for the currently used antidepressants to achieve remarkable
effectiveness. However, adverse effects of clinical antidepressants occur faster, such as
sexual dysfunction, nausea/vomiting, and sleep disruption.2 Moreover, 12 % – 20 % of
MDD patients have developed drug resistance.3 The mechanism of MDD is
complicated.4 Previously, the lack of monoaminergic neurotransmitters was the focus
of pathophysiological concepts. Most antidepressant drugs were found regulating
extracellular concentration of serotonin and norepinephrine. However, the activity of
some antidepressants did not correlate with their monoaminergic effect. With the
growing understanding of the mechanism of depression, the reduction in
neurogenesis, particularly in the hippocampus, has been raised as a central factor of
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MDD.5-7 Thus, the recent advances that ASM plays a central role in neurogenesis and
may be a noteworthy target for the development of novel anti-depressants has dawn
our attention. 8-10
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ASM is a zinc-dependent enzyme that acts as a lysosomal or secretory hydrolase,
catalyzing the degradation of sphingomyelin (SM) to phosphorylcholine and ceramide
(Cers). The released Cers molecules in the cell membrane or mitochondrial
membrane can spontaneously form Cers-enriched "lipid platforms",11 which serve to
trap and cluster receptor molecules and to initiate stress and apoptotic signals.
Additionally, Cers
can directly regulating a series of apoptotic signals, such as
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protein kinase C isoforms
and cathepsin D.13 Thus, Cers have been regarded as
pivotal factors in the regulation of apoptosis.14-16
Clinical studies demonstrated that depressed patients displayed significantly higher
levels of ASM activity and Cers concentration in their cultivated blood cells or plasma
compared to the control groups.17, 18 Furthermore, splicing events of ASM were found
less occurring in MDD patients, which led to higher expression of ASM.19 Another
direct and convincing evidence is that most of the antidepressants are functional
inhibitors of ASM, which detaches ASM from the inner lysosomal membranes. This
will result in the proteolytic degradation of ASM.20, 21 In 2013, a study from Gulbins's
22
group revealed that the effects of antidepressant drugs might be mediated by the
ASM-Cers system through a mechanism of Cers negatively related neuronal
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proliferation, maturation, and survival. Administration of amitriptyline and
fluoxetine with the therapeutic concentrations not only reduced hippocampus ASM
activity and Cers concentrations, but also increased hippocampus neurogenesis. 22 In
addition, it was found that the inhibition of ASM could prevent the stress-induced
P38 activation, thereby increasing neurogenesis and reducing depression-like
symptoms.23 A recent study revealed that free-choice alcohol drinking reduced
depression-like behavior in depressed animals through the normalization of brain
ASM activity.24 All together, these studies demonstrated that ASM is a promising drug
target to develop new antidepressant agents (Figure 1).
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Figure 1 Supposed mechanism of acid sphingomyelinase in the development of
depression. ASM, acid sphingomyelinase
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Inhibition of ASM could promote neurogenesis and reduce depression-like
behavior. However, until now, direct inhibitors of ASM are few,25 including
analogues of SM,26 bisphosphonates,27 phosphatidylinositol-3,5-bisphosphates
derivatives,28, 29 benzopyrone derivatives,30 and xanthones (Figure 2).31, 32 Therefore,
in this work, we developed novel direct inhibitors of ASM to disclose the role of ASM
in MDD and to verify the effectiveness of ASM as an anti-depression target.
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OH
HO
O-
OH
C12H25
O
O
O
O
O
P
S
N+
P
C12H25
O
O
PO3H2
NH2
PO3H2
HO
O
H
HO
OH
HN
N
O
P
O
OH
S
OH
ARC39 (IC50 = 20 nM)
AD2765 (Ki = 70 M)
1
(IC50 = 0.44 M)
O
H2N
O
O
O
OH
O
Br
O
O
O
OH
O
O
C10H21
N
H
HO
OH
(IC50 = 14.1 M)
O
a-Mangostin
WJYK50 (IC50 = 0.48 M)
2 (IC50 = 3.1 M)
Figure 2 Representative structures of acid sphingomyelinase inhibitors.
Previously, our research group have identified a family of direct ASM inhibitors
using a ligand-based pharmacophore model and confirmed a hydroxamic acid group
as an essential Zn2+ bonding group (ZBG), which led to the discovery of WJYK50
(IC50 = 0.48 μM, Figure 2).33 In our previous pharmacokinetics study, WJYK50
showed some deficiencies In this work, a structure-based design approach was
performed for the first time to discover novel hydroxamic acid-based ASM direct
inhibitors. As a result, a series of new compounds were identified as effective and
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selective ASM inhibitors, among which compound 21b was assessed with in vivo
behavioral tests, enzyme inhibitory activity test, and neurogenesis studies.
Collectively, the promising and innovative results obtained from this study further
validated the possibility of targeting ASM to prevent and treat major depression.
Results and discussion
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Design of novel ASM inhibitors
In 2016, three research groups resolved the crystal structures of ASM.34-36 These
breakthroughs revealed a large ligand binding cavity defined by certain residues in
ASM protein and provided possibilities for structure-based drug design. Firstly,
molecular docking simulation was carried out to gain insight into the detail binding
mode of WJYK50 in ASM (PDB code: 5fi9). The hydroxamic acid group of WJYK50
inserted into the di-zinc catalytic center, whereas its ten-carbon lipid tail sat in a
relatively featureless shallow groove in a linear mode (Figure 3A). This binding
conformation was very similar to that of the known inhibitor ARC39. Both the alkyl
chain of ARC39 and WJYK50 did not locate to the position of SM fatty-acid chains,
which sat along a hydrophobic track that extended from the edge of the active site to
the saposin domain.34 The polar hydroxamic acid head of WJYK50 competitively
occupied the catalytic site where the choline headgroup of SM located to. Moreover,
this group made hydrogen bond contacts with residues His208, Asp276 and His280.
His280 has been regarded as the key residue for protonation of the ester oxygen of
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substrate SM (Figure 3B).35 Reducing the flexibility of WJYK50 by replacing flexible
alkyl chain with rigid rings might improve π-π or p-π interactions with ASM. Thus, the
single benzene of WJYK50 was replaced by a (benzyloxy)benzene scaffold (Figure 3C).
Substitutions of the two benzene rings were also explored. This led to the design of
compounds 9a – 9i and 12a – 12j (Table 1 and Table 2).
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Figure 3 Docking pose of WJYK50 in acid sphingomyelinase and optimizing strategy.
Hydrogen bonds are shown as green lines and metal contacts in yellow.
Enzyme inhibitory activity screening and structure−activity relationship studies of
compounds 9a – 9i and 12a – 12j.
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Enzymatic assays of the target compounds against ASM and neutral
sphingomyelinase (NSM) were performed by evaluating their abilities of inhibiting
the production of 7-nitro-2-1,3-benzoxadiazol (NBD)-labeled ceramide. Results were
shown in Table 1 and Table 2. These compounds varied with the type and size of
substituent group on the ring B as well as the R2 group at C-4 position of ring A.
Overall, most compounds with a biphenyl scaffold showed low micromole IC50
values, among which compound 9f was found to be the most active compound. In
addition, the fact that compound 9f was far more potent than 9g identified the
chlorine substituent on the terminal benzene as a key element for ASM inhibition.
Same phenomenon was observed between 9c and 9b. Replacement of the terminal
benzene by pyridine (9d and 9h) led to great decrease of inhibitory potency. The
introduction of C6H13 alkyl also resulted in low micromole ASM inhibition, like 9e.
Compounds 12b – 12e and 12j almost lost potency. However, compound 12f and 12g
were more active than 12a – 12e. Because of the presence of methylene in R1 group,
12f and 12g showed a little more flexibility than 12b – 12e. This result indicated that
the direct connection of three aromatic rings was unfavorable for their inhibitory
activity against ASM, which implied that these structures shouldn’t be strongly rigid.
Furthermore, bulky substitutes were not suitable for ring B due to the weak activity
of 9a – 9h when comparing with 9i and 12a – 12j. Furthermore, these active
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compounds were tested their activity against neutral sphingomyelinase (NSM) to
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demonstrate their selectivity. As a result, none of these compounds inhibited NSM at
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a concentration of 100 μM.
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Table 1. Structures of 9a – 9i and their inhibitory activity against ASM and NSM.
R1
O
B
O
OH
N
A
H
R2
IC50 (μM)
ASM a NSM b
Compound
R1
R2
c
9a
C6H13
Phenyl
H
H
H
H
2.11 ± 0.30
19.61 ± 2.98
8.96 ± 2.03
52.3 ± 4.98
-
9b
-
9c
4-Cl-Phenyl
Pyridin-3-yl
C6H13
nt d
nt
-
9d
9e
OMe 1.96 ± 0.23
9f
4-Cl-Phenyl OMe 0.34 ± 0.05
Phenyl OMe 3.63 ± 0.34
Pyridin-3-yl OMe 4.97 ± 1.01
-
9g
9h
-
-
9i
Styryl
H
>100
nt
nt
WJYK50
0.48 ± 0.10
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b
c
aASM, acid sphingomyelinase. NSM, neutral sphingomyelinase. -, inactive at 100
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μM. dnt, not tested. Data are expressed as means ± SD of 3 experiments.
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Table 2. Structures of 12a – 12j and their inhibitory activity against ASM and NSM.
O
N
R1
N
O
B
O
OH
N
A
H
R2
IC50 (μM)
ASM a
Compound
R1
R2
NSM b
nt c
nt
12a
12b
12c
12d
12e
12f
Ethyl
H
H
H
H
H
H
H
34.72 ± 3.25
>100
Phenyl
4-Br-Phenyl
2-F-Phenyl
3-F-Phenyl
Benzyl
>100
nt
>100
nt
>100
nt
7.83 ± 1.90
1.73 ± 0.20
nt
d
12g
12h
Phenethyl
Ethyl
-
OMe 8.22 ± 1.30
nt
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12i
12j
Phenyl
OMe 15.91 ± 1.32
nt
nt
4-Br-Phenyl OMe
>100
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c
d
aASM, acid sphingomyelinase. NSM, neutral sphingomyelinase. nt, not tested. -,
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inactive at 100 μM. Data are expressed as means ± SD of 3 experiments.
Design of compounds 15a – 15e, 18, and 21a – 21b
Most of the compounds were quite hydrophobic and insoluble in physiological
saline due to their three benzene rings, such as 9a – 9c, 9e – 9g. Thus, we decided to
replace ring A or ring B by a heterocyclic ring, which might improve the drug
likeness. Moreover, it was found that the imidazole of His457 lies under the ring A of
9f by docking 9f into ASM (Figure 4). Therefore, if ring A was optimized to a smaller
heterocycle to make the molecular going deep into the catalytic pocket, the
compounds might form Pi interactions with His457. These facts led to the design of
compounds 15a – 15e, 18, and 21a – 21b.
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Figure 4 Docking poses of 9f identified in acid sphingomyelinase. Hydrogen bonds are
shown as green lines, metal contacts in yellow, and Pi contacts in blue.
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Enzyme inhibitory activity screening and structure−activity relationship studies of
15a –15e, 18, and 21a – 21b
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Compound 15a – 15e showed decreased inhibitory activity on ASM when
comparing with 9f and 9g, which indicated that hydrophobic benzene was more
beneficial than heterocycle at the position of ring B. In accordance with 9f, attempts
to substitute 15a with a chlorine moiety at either the C-3 (15c) or C-4 (15d) on the
biphenyl ring induced higher potency than 15a. Placement of a fluorine at C-2 didn’t
affect the potency because of the similar IC50 values of 15a and 15b. When ring A was
changed to isoxazole, compounds showed comparable activity to 9g and 9f. The
biphenyl group without any substituent (21a) produced moderate ASM inhibition.
Compounds 18 and 21b exhibited higher potency than 21a. Screening of active
compounds against NSM found that all the compounds did not inhibit NSM at 100
μM. Finally, compound 21b was chosen for further studies.
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Table 3. Structures of 15a – 15e and their inhibitory activity against ASM and NSM.
O
N
O
R1
O
O
OH
N
N
H
IC50 (μM)
Compound
R1
ASM a
NSM b
nt c
15a
15b
15c
15d
15e
H
36.45 ± 1.69
21.60 ± 1.55
9.22 ± 0.36
21.61 ± 2.13
2-F
nt
d
3-Cl
4-Cl
-
-
3,4- dimethoxy 13.78 ± 0.89
nt
b
c
d
aASM, acid sphingomyelinase. NSM, neutral sphingomyelinase. nt, not tested. -,
inactive at 100 μM. Data are expressed as means ± SD of 3 experiments.
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Table 4. Structures of 18, 21a, 21b and their inhibitory activity against ASM and NSM.
O
O
R1
N
O
HN OH
IC50 (μM)
NSM b
Compound
R1
ASM a
c
18
C10H21
0.36 ± 0.06
0.88 ± 0.10
-
21a
-
Cl
21b
0.32 ± 0.03
-
b
c
aASM, acid sphingomyelinase. NSM, neutral sphingomyelinase. -, inactive at 100
μM. dnt, not tested. Data are expressed as means ± SD of 3 experiments.
Membrane penetration and brain exposure
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Before in vivo anti-depressant test, we evaluated the membrane penetration and
brain exposure of compound 21b. Human microvascular endothelial hCMEC/D3 cells
were used for membrane transmittance study. As shown in Figure 5, the uptake of
21b by hCMEC/D3 cells gradually increased from 0.5 h to 2 h, suggesting that 21b
effectively penetrated the cell membrane. However, the uptake of 21b was decreased
at 4 h. This was probably because that cell proliferation led to decreased drug intake
at per unit protein. Another possibility was that the cells expelled the drug. By
calculating the ratio of the intracellular concentration to the original dose
concentration, it was found that the uptake rate of 21b gradually increased over time,
further proving 21b could successfully penetrate through the cell membrane.
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Figure 5 Cellular uptake of 21b in hCMEC/D3 cells. Values are expressed as mean ±
SD from 3 independent experiments.
Then, in vivo brain exposure was studied. SD rats were administrated with 21b at
an intraperitoneal dose of 48 mg/kg. Concentrations of 21b in plasma and brain were
quantified by LC-MS/MS. From table 5, it could be found that the drug content in
brain tissue was much higher than that in plasma, and this trend was more obvious
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over time. This result strongly proved that 21b penetrated the BBB very well. Besides,
the concentrations in brain and plasma were all obviously decreased at 4 h, most
probably due to the metabolism.
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Table 5. Plasma and brain concentration of 21b in SD rats.
Brain Conc. Plasma Conc.
Time point
Brain/Plasma Conc. ratio
(ng·g-1)
2122
(ng·mL-1)
709
0.5 h
2 h
2.94
6.49
9.55
4756
762
4 h
1298
144
Values are expressed as mean from 3 independent experiments.
In Vivo Behavioral Studies
Cytotoxicity evaluation using the MTT method supported that 21b was safe to
NIH3T3 cells (IC50 =161 ± 33.46 μM) (Figure S4). A functional inhibitor amitriptyline
was chosen as positive control and did not showed any cytotoxicity.
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Reserpine (4 mg/Kg) was used to induce depression of rats by intraperitoneal
injection (ip) daily. Rats were administered compounds 21b (6, 12, and 24 mg/Kg, ip)
or amitriptyline (12 mg/Kg, ip), a tricyclic antidepressant, as well as a functional
inhibitor of ASM. Results from sugar water preference test proved that 21b improved
sugar consumption in a dose dependent manner after two days tap-water drinking
and following 20 h water depletion and fasting (Figure 6A). In the open-field test, the
total time in center of rats were decreased (Figure 6B) and times passing the grid
(Figure 4C), frequency of upright (Figure 6D), and frequency of grooming (Figure 6E)
were all increased by treating the depressed rats with amitriptyline and 21b,
respectively. In both experiments, the high dose of 21b showed comparable effects to
that of amitriptyline, supporting the notion that inhibiting ASM will generate
beneficial effect on depression.
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Figure 6 Anti-depressant effect of compound 21b. Amitriptyline was used as 12
mg/Kg. A, sugar water preference test. B-D, open-field test. Values are expressed as
mean ± SD from 8 independent experiments. *, P value compared to control group
(**p<0.01, ***p<0.001, ****p<0.0001). #, P value compared the model group (# p<0.05, #
# p<0.01, # # # p<0.001, # # # # p<0.0001).
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In vivo ASM activity and neurogenesis
To test whether 21b inhibited ASM in the brain of rats, we separated the cerebral
cortex and hippocampus and tested the relative enzyme activity of ASM. As a result,
in both the cortex and hippocampus, activity of ASM was obvious increased without
treatment of drugs (about 53 % and 73 %, respectively), while this increase in
hippocampus was higher than in cortex (Figure 7). Rats administered with 21b at a
dose of 24 mg/Kg demonstrated a reduction of the cortical and hippocampal ASM
activity by approximately 27 % and 33 % respectively, comparing to the model group.
Figure 7 Relative acid sphingomyelinase activity in cerebral cortex and hippocampus.
Amitriptyline was used as 12 mg/Kg. The activity of the normal group was regarded as
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100 %, and the Y-axis reflected the relative percentage of each group to the normal
group. Values are expressed as mean ± SD from 5 independent experiments. *, P value
compared to control group (*p<0.05, **p<0.01, ****p<0.0001). #, P value compared the
model group (# p<0.05, # # p<0.01).
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Meanwhile, to substantiate the role of compound 21b on neurogenesis, cell
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proliferation in hippocampus was detected using the EDU method. Treatment of
wildtype rats with reserpine resulted in decrease of neurogenesis in the hippocampus
(Figure 8), consistent with previously reported data. 22 ASM inhibitor 21b prevented
the inhibitory effect of reserpine on neurogenesis and restored neurogenesis in the
brain of depressed rats in a dose dependent manner. Additionally, while amitriptyline
exhibited higher anti-depression effect than 21b, its influence on neurogenesis was
less than 21b. The preferable in vivo effects of 21b was closely consistent with the
previous reports on biological studies in the anti-depression potency, ASM activity
and neurogenesis. The above findings further confirmed ASM as an effective target
for depression treatment.
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Figure 8 Neurogenesis in hippocampus. The dose of amitriptyline was 12 mg/Kg.
Values are expressed as mean ± SD from 5 independent experiments, ***p<0.005
compared to control group, # # p<0.01 compared the model group.
Pure enzyme inhibition of active compounds
We complementally tested the active compounds 9a, 9c, 9f, 9h, 12f, 12g, 18, 21a,
and 21b with recombinant human ASM (95% > purity). The results were shown in
Table 6. The lead compound, WJYK50, showed a slightly higher IC50 value (0.89 μM)
than the previous one. Compound 9c (0.36 μM) was found more active than others.
And 9f had an IC50 value of 8.07 μM. It was less active than 9c. This assay led us to
ponder that the methoxy group at C-4 position of WJYK50 should be deleted.
Molecule docking strongly indicated that the hydroxamic acid of 9c binding to ZN702
in a classical bidentate mode (Figure S7). Moreover, the hydroxy and amino groups of
hydroxamic acid also combined with the other Zn2+ (ZN701) respectively. Compounds
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18, 21a, and 21b were found unsatisfactory activity in the pure enzyme inhibition
assay. As decreased NBD-labeled ceramide generation was detected in the pure
enzyme screening assay, our designed compounds can be determined to inhibit ASM
in the direct manner.
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Table 6. Inhibitory activity against recombinant human ASM.
Pure ASM a
Pure ASM a
Compound
Compound
(IC50, μM)
(IC50, μM)
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WJYK50 0.89 ± 0.28
12f
12g
18
4.93 ± 1.21
4.25 ± 0.86
2.20 ± 0.36
4.09 ± 0.48
3.37 ± 0.82
9a
9c
9f
3.38 ±0.37
0.36 ± 0.11
8.07 ± 0.55
3.83 ± 0.69
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21a
21b
9h
aPure ASM, recombinant human ASM (purity >95%) was used. Data are expressed as
means ± SD of 3 experiments.
Synthesis of the compounds
We began the synthesis from two commercially available materials, 6a and 6b.
Compounds 6a or 6b were substituted by alpha,4-dibromotoluene, yielding
compounds 7a or 7b, respectively. Compounds 7a or 7b were further converted to 8a
– 8i through Heck or Suzuki reactions. The Heck products 8a and 8b were reduced to
get their corresponding saturated alkyl intermediates. Compounds 6a or 6b were
respectively substituted by 4-cyanobenyl chloride to yield 10a or 10b. 10a or 10b
were then reacted with hydroxylamine hydrochloride and NaHCO3 in ethanol to
yield their amidoximes, which were then respectively reacted with carboxylic acids
using EDCI and DMAP to afford 1,2,4-oxadiazole products 11a – 11j. In the last step,
compounds 8a – 8i or 11a – 11j were allowed to hydrolyze to give the corresponding
carboxylic acids, which were following activated by thionyl chloride and converted
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to the hydroxamic acid (9a – 9i) by adding them to a solution of hydroxylamine base.
The hydroxylamine solution was freshly prepared from hydroxylamine hydrochloride
with NaOH in tetrahydrofuran and water.
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Br
R1
R1
O
O
O
O
O
O
OH
HO
R2
O
b
a
c
O
N
O
O
H
R2
, R2 = H
, R2 = OMe
R2
R2
8a-8i
9a-9i
7a
7b
6a
6b
, R2 = H
, R2 = OMe
a
O
O
N
N
R1
R1
NC
N
O
N
O
O
O
c
d
O
O
OH
O
O
N
H
R2
R2
R2
12a-12j
11a-11j
10a
10b
, R2 = H
, R2 = OMe
o
Scheme 1. Reagents and conditions:(a) RBr, K2CO3, KI, acetone, 60 C; (b) Heck: i:
o
1-Hexene or styrene, Et3N, Pd(PPH3)2Cl2, DMF, 120 C, or Suzuki: Aromatic boric
o
o
acid, CsCO3, Pd(PPh3)4, dioxane, 100 C; (c) i: NaOH, MeOH, H2O, 100 C, ii: SOCl2,
o
DMF, dichloromethane, 40 C, iii: NaOH, NH2OH•HCl, THF, H2O, rt; (d) i:
o
NH2OH•HCl, NaHCO3, EtOH, 80 C, ii: Carboxylic acid, EDCI, DMAP, DMF, 0 – 120
oC.
Commercially available material 6b was substituted by chloroacetonitrile to yield
compound 13. Under the condition of hydroxylamine hydrochloride and NaHCO3 in
ethanol, 13 was converted to an active intermediate, which was combined with
carboxylic acid and cyclizing at a high temperature to produce compounds 14.
Compound 16 was produced from dimethyl acetylenedicarboxylate and
hydroxycarbamide. Compounds 15a – 15e, 17, 29, 20a, 20b were synthesized
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following the same procedure mentioned above. Intermediates 17 or 20 were
converted to the title compounds 18 or 21 through a solution of potassium
hydroxylamine.
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O
O
N
O
O
N
O
O
R1
R1
O
O
OH
HO
O
O
O
NC
O
O
a
b
c
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H
14a-14e
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15a-15e
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C10H21
O
HO
C10H21
O
O
O
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O
e
OH
b
d
MeOOC
COOMe
+
H2N
N
H
N
N
N
O
O
O
HN OH
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Br
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HO
N
O
O
b
f
O
e
O
O
O
N
O
O
O
O
N
O
N
O
O
O
HN OH
19
20a-20b
21a-21b
16
o
Scheme 2. Reagents and conditions:(a) Chloroacetonitrile, K2CO3, KI, acetone, 60 C;
(b) i: NH2OH•HCl, NaHCO3, EtOH, 80 oC, ii: carboxylic acid, EDCI, DMAP, DMF, 0 –
120 C; (c) i: NaOH, MeOH, H2O, 100 C, ii: SOCl2, DMF, dichloromethane, 40 oC, iii:
o
o
o
NaOH, NH2OH•HCl, THF, H2O, rt; (d) DBU, MeOH, 0 C – rt, then 10% HCl; (e)
NH2OK in MeOH, MeOH, rt; (f) Aromatic boric acid, CsCO3, Pd(PPh3)4, dioxane, 100
oC.
Conclusions
Overall, we disclosed the design, synthesis, and biological screening of a series of
direct and selective ASM inhibitors. Through two rounds of optimization, compounds
9a, 9c, 9f, 9h, 12f, 12g, 18, 21a and 21b were identified as effective ASM direct
inhibitors. Among those compounds, 21b showed very low cytotoxicity in vitro.
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Furthermore, SAR studies reflected that the biphenyl scaffold with a terminal
chlorine substituent was necessary for ASM inhibitors. It’s worth to note that
compound 21b could permeate the cell membrane well and successfully crossed the
blood-brain barrier and distributed in brain tissue. Treatment of depressed rats with
21b demonstrated favorable antidepressant-like effects in the sugar water preference
test and open-field test. Furthermore, this result was in close correlation with the
decreased brain ASM activity and restoring neurogenesis in hippocampus by
administration of 21b. Previously, beneficial effects on neurogenesis of the
antidepressants have been attributed to their functional inhibition on the ASM/Cers
system.8 In this work, it is the first time to validate the effects on neurogenesis and
depression by mean of direct inhibition of ASM.
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Altogether, intervention in the ASM/Cers system by small molecule direct
inhibitor was applied to provide sufficient evidences in support of ASM as a promising
antidepressant target. In addition, this research could offer practical guidance for
developing more potent direct ASM inhibitors and serve as a starting point to launch
an MDD drug discovery program based this potential target.
Experimental Section
Molecular docking
The docking study was performed with CCDC GOLD 5.3.0 software. The co-crystal
structures of ASM with an inhibitor (ARC39) (PDB code: 5fi9) was chosen as the
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receptor.33 Small molecules were drawn in ChemDraw and the initial lowest energy
conformations were calculated in Chem3D. Protein was preliminary prepared in DS
3.0 with adding hydrogens, deleting glycosyls and water and conducting the “Clean
Protein” protocol. The prepared protein was then imported into GOLD and edited
with extracting ligand. The binding site was defined at the position of the original
included ligand ARC39. Goldscore_P450_csd was chosen as the configuration
template and GoldScore was chosen as scoring function. Other parameters were kept
default. Docking poses were loaded into Gold for a visualization of ligand-receptor
interactions and analysis.
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Chemistry
General: Starting materials and reagents were obtained from commercially available
sources with high–grade purity. Melting points were determined on a MEL-TEMP Ⅱ
1
apparatus (Laboratory Devices, MA, USA). H NMR and 13C NMR spectra were
recorded on a Bruker AV-300 MHz instrument (Bruker Biospin AG, Switzerland) in
CDCl3 or DMSO-d6 with TMS as the internal standard. MS were obtained on Waters
Q-Tof microTM (Waters, MA, USA) with the ESI (+, -) protonation interface. High–
resolution mass spectra (HRMS) were recorded on Q-Tof Premier hybrid mass
spectrometer with electron spray ionization (ESI). HPLC analysis was performed on a
Shimadzu SPD-20A/20AV HPLC system (Shimadzu, Tokyo, Japan). The column used
was a C18 column (5 μ micron, 250 × 4.60 mm) at a temperature of 30 °C and a flow
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rate of 1.0 mL/min. The purity of title compounds was higher than 95 %, which was
assessed at 254 nm using methanol /water (0.1% AcOH) (80:20). All title compounds
were examined for PAINS and passed. Anhydrous reactions were carried out in dried
glassware under a nitrogen atmosphere. The boiling range for petroleum ether was 60
– 90 °C.
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General procedure for the preparation of compounds 7a and 7b.
To a solution of the substrate (6a or 6b, 1 g, 1 equiv.) in acetone was added
potassium carbonate (3 equiv.), potassium iodide (0.1 equiv.), and 4-bromobenzyl
bromide (1.2 equiv.). The mixture was heated to reflux for 4 h. After the reaction was
finished, the mixture was filtered and the filtrate was concentrated and purified by
chromatography on silica gel to afford the target products.
General procedure for the preparation of compounds 8a – 8i.
Heck procedure: A mixture of the substrate (7a or 7b, 100 mg, 1 equiv.), alkene (3
equiv.), trans-dichlorobis(triphenyl-phosphine)Palladium(II) (0.05 equiv.), and
triethylamine (3 equiv.) in N,N-dimethylformamide (10 mL) was heated to 120 °C for
6 h under N2 atmosphere. When the reaction was finished, the mixture was diluted
with water and extracted by ethyl acetate. The organic phase was washed three times
by saline and dried over Na2SO4. The solution was concentrated and purified by
chromatography on silica gel to afford the target products.
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Suzuki procedure: A mixture of the substrate (7a or 7b, 100 mg), aromatic boracic
acid (1.5 equiv.), and tetrakis(triphenylphosphine)palladium(0) (0.05 equiv.), and
cesium carbonate (2 equiv.) in 1,4-dioxane (10 mL) was heated to 100 °C for 4 h under
N2 atmosphere. When the reaction was finished, the mixture was filtered and the
solid was washed by dichloromethane. The filtrate was concentrated and purified by
chromatography on silica gel to afford the target products.
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Methyl (E)-3-((4-(hex-1-en-1-yl)benzyl)oxy)benzoate (8a)
Oil (65 mg, 63.8 %), 1H-NMR (300 MHz, CDCl3): δ 7.70-7.67 (m, 2H), 7.46-7.34 (m,
5H), 7.26-7.17 (m, 2H), 6.44-6.22 (m, 0.5H), 5.59-5.46 (m, 0.5H), 5.13-5.10 (m, 2H),
3.94 (s, 3H), 2.28-2.00 (m, 2H), 1.73-1.28 (m, 4H), 1.02-0.88 (m, 3H); ESI-MS m/z:
325.21 [M + H] +.
Methyl 3-([1,1'-biphenyl]-4-ylmethoxy)benzoate (8b)
White solid (73 mg, 73.0 %), M.P. 84-86 °C, 1H-NMR (300 MHz, CDCl3): δ 7.68-7.65
(m, 2H), 7.60-7.57 (m, 2H), 7.53-7.51 (m, 4H), 7.43-7.31 (m, 4H), 7.21-7.17 (m, 1H),
5.15 (s, 2H), 3.92 (s, 3H); ESI-MS m/z: 319.1 [M + H] +.
Methyl 3-((4'-chloro-[1,1'-biphenyl]-4-yl)methoxy)benzoate (8c)
1
White solid (80 mg, 72.3 %), M.P. 118-120 °C, H-NMR (300 MHz, CDCl3): δ
7.70-7.53 (m, 7H), 7.45-7.33 (m, 3H), 7.28 (m, 1H), 7.23-7.20 (m, 1H), 5.17 (s, 2H),
3.94 (s, 3H), 388 (s, 3H); ESI-MS m/z: 375.1 [M + Na] +.
Methyl 3-((4-(pyridin-3-yl)benzyl)oxy)benzoate (8d)
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White solid (63 mg, 62.8 %), M.P. 106-107 °C, 1H-NMR (300 MHz, CDCl3): δ 8.86 (m,
1H), 8.61-8.60 (m, 1H), 7.90-7.87 (m, 1H), 7.68-7.55 (m, 6H), 7.40-7.34 (m, 2H),
7.21-7.18 (m, 1H), 5.17 (s, 2H), 3.92 (s, 3H); ESI-MS m/z: 320.1 [M + H]+.
Methyl (E)-3-((4-(hex-1-en-1-yl)benzyl)oxy)-4-methoxybenzoate (8e)
Oil (65 mg, 64.4 %), 1H-NMR (300 MHz, CDCl3): δ 7.70-7.67 (m, 2H), 7.46-7.34 (m,
5H), 7.26-7.17 (m, 2H), 6.44-6.22 (m, 0.5H), 5.59-5.46 (m, 0.5H), 5.13-5.10 (m, 2H),
3.94 (s, 3H), 2.28-2.00 (m, 2H), 1.73-1.28 (m, 4H), 1.02-0.88 (m, 3H); ESI-MS m/z:
325.21 [M + H] +.
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Methyl 3-((4'-chloro-[1,1'-biphenyl]-4-yl)methoxy)-4-methoxybenzoate (8f)
1
White solid (88 mg, 81.2 %), M.P. 140-142 °C, H-NMR (300 MHz, CDCl3): δ
7.72-7.68 (m, 1H), 7.64-7.63 (m, 1H), 7.56-7.50 (m, 6H), 7.42-7.39 (m, 2H), 6.93-6.91
(m, 1H), 5.22 (s, 2H), 3.95 (s, 3H), 388 (s, 3H); ESI-MS m/z: 383.1 [M + H] +.
Methyl 3-([1,1'-biphenyl]-4-ylmethoxy)-4-methoxybenzoate (8g)
1
White solid (78 mg, 78.2 %), M.P. 118-120 °C, H-NMR (300 MHz, CDCl3): δ
7.74-7.70 (m, 1H), 7.67-7.55 (m, 7H), 7.49-7.44 (m, 2H), 7.39-7.34 (m, 1H), 6.95-6.93
(m, 1H), 5.24 (s, 2H), 3.97 (s, 3H), 3.90 (s, 3H); ESI-MS m/z: 371.1 [M + Na] +.
Methyl 4-methoxy-3-((4-(pyridin-3-yl)benzyl)oxy)benzoate (8h)
White solid (75 mg, 75.0 %), M.P. 106-107 °C, 1H-NMR (300 MHz, CDCl3): δ 8.87 (m,
1H), 8.62-8.60 (m, 1H), 7.91-7.87 (m, 1H), 7.74-7.70 (m, 2H), 7.65-6.63 (m, 1H),
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