A Novel Parkinson's Disease Drug Candidate with Potent Anti-neuroinflammatory Effects through the Src Signaling Pathway

Article abstract of DOI:10.1021/acs.jmedchem.6b00976

Numerous drug treatments are available for Parkinson's disease (PD), an age-related neurodegenerative disease, but most cause serious side effects. Therefore, novel therapeutic strategies that halt disease progression and allow for long-term administration are urgently needed. Neuroinflammation critically contributes to the pathogenesis of PD. Here, we report the discovery and optimization of phloroglucinol derivatives, a novel class of anti-neuroinflammatory compounds. Structural modifications of the hit compound 3-methyl-1-(2,4,6-trihydroxyphenyl)butan-1-one produced 43 derivatives, including a preclinical candidate (compound 21), that exhibited potent in vitro anti-neuroinflammatory effects, good blood-brain barrier penetration, and desirable safety margins in mice at a median lethal dose (LD₅₀) >5000 mg/kg. Its in vivo efficacy was demonstrated in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- and MPTP/probenecid (prob)-induced subacute and chronic PD models, respectively, and α-synuclein transgenic mice. Mechanistic studies revealed neuroinflammation inhibition by targeting Src/phosphatase and tensin homologue deleted on chromosome 10 (PTEN)/Akt signaling might be promising. We highlighted the potential usefulness of phloroglucinol derivatives in PD treatment.

Full text of DOI:10.1021/acs.jmedchem.6b00976

Subscriber access provided by Syracuse University Libraries
Article
A novel Parkinson’s disease drug candidate with potent anti-
neuroinflammatory effects through the Src signaling pathway
Yadan Wang, Xiuqi Bao, Song Xu, Wenwen Yu, Shengnan Cao,
Jinping Hu, Yan Li, Xiaoliang Wang, Dan Zhang, and Shi-Shan Yu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00976 • Publication Date (Web): 12 Sep 2016
Downloaded from http://pubs.acs.org on September 12, 2016
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 63
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
12
13
A novel Parkinson’s disease drug candidate with
potent anti-neuroinflammatory effects through the
Src signaling pathway
14
15
16
17
18
19
20
2
2
‡
‡
Yadan Wang, Xiuqi Bao, Song Xu, Wenwen Yu, Shengnan Cao, Jinping Hu, Yan Li, Xiaoliang
23
24
Wang, Dan Zhang* and Shishan Yu*
25
26
27
28
29
30
31
3
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of
Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College,
Beijing 100050, China.
3
34
35
36
37
38
39
40
41
42
4
4
45
46
47
48
49
50
51
52
53
5
5
56
57
58
59
60
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 63
1
2
3
4
5
6
7
8
9
1
1
12
13
14
15
16
17
18
19
20
2
ABSTRACT: Numerous drug treatments are available for Parkinson’s disease (PD), an age-
related neurodegenerative disease, but most cause serious side effects. Therefore, novel
therapeutic strategies that halt disease progression and allow for long-term administration are
urgently needed. Neuroinflammation critically contributes to the pathogenesis of PD. Here, we
report the discovery and optimization of phloroglucinol derivatives, a novel class of anti-
neuroinflammatory compounds. Structural modifications of the hit compound 3-methyl-1-(2,4,6-
trihydroxyphenyl)butan-1-one produced 43 derivatives, including a preclinical candidate
(compound 21) that exhibited potent in vitro anti-neuroinflammatory effects, good blood-brain
barrier penetration, and desirable safety margins in mice at a median lethal dose (LD ) > 5000
2
23
5
0
24
25
26
27
28
29
30
31
3
3
34
35
mg/kg. Its in vivo efficacy was demonstrated in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP)- and MPTP/probenecid (prob)-induced sub-acute and chronic PD models, respectively,
and α-synuclein transgenic mice. Mechanistic studies revealed neuroinflammation inhibition by
targeting Src/phosphatase and tensin homolog deleted on chromosome 10 (PTEN)/Akt signaling.
We highlighted the potential usefulness of phloroglucinol derivatives in PD treatment.
36
37
38
39
40
41
42
4
4
45
46
47
48
49
50
51
52
53
5
5
56
57
58
59
60
ACS Paragon Plus Environment

Page 3 of 63
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
INTRODUCTION
Parkinson’s disease (PD) is an age-related, progressive neurodegenerative disorder characterized
by the loss of dopaminergic neurons in the substantia nigra pars compacta, which decreases the
1
1
1
interstitial dopamine level and leads to bradykinesia, rigidity, and tremor. Therefore, current
12
1
14
treatment focuses on dopamine replacement, the inhibition of dopamine degradation using
15
16
catechol-O-methyl-transferase (COMT) or monoamine oxidase type B (MAO-B) inhibitors,
17
18
19
20
2
2
23
2
-5
dopamine reuptake, and direct dopamine receptor agonists. The available PD medications offer
valuable symptomatic relief, but they are often associated with significant and intolerable side
effects. Furthermore, importantly, these drugs cannot slow PD progression. Therefore, the
24
25
6
development of broader and more fundamental therapeutic approaches to PD is critical. The
26
2
28
“neuroprotective therapy” concept has emerged as a potential treatment for PD and may
2
30
7,8
effectively slow down or stop the disease progression.
31
3
Neuroinflammation, which is well recognized as a key pathophysiological event contributing
3
34
35
9,10
to progressive nigral dopaminergic neuron loss in PD,
may be another target for
36
37
38
39
40
41
42
neuroprotective therapy. Microglia and astrocytes are major mediators of neuroinflammation in
PD. Several reports have demonstrated the activation of microglial and astroglial cells near
1
1–13
damaged or dying dopaminergic neurons in the substantia nigra.
Positron emission
4
4
tomography (PET) imaging of PD patients shows increased microglial activation, and elevated
45
4
47
levels of proinflammatory mediators are evident in the substantia nigra during postmortem
4
49
14,15
examinations.
Epidemiological studies have demonstrated that nonsteroidal anti-
50
51
1
6
inflammatory drugs (NSAIDs) confer a decreased risk of PD. Therefore, preventative and
52
53
5
curative treatments for PD have focused on discovering active substances that attenuate
5
56
57
58
59
60
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 63
1
2
3
4
5
6
7
8
9
1
1
12
13
14
15
16
17
18
19
20
2
excessive glial activation and protect dopaminergic neurons. Several agents that target
1
7
inflammatory pathways are currently under investigation.
In this article, we describe the discovery and optimization of a novel anti-PD series of
phloroglucinol compounds with significant potential anti-neuroinflammatory effects and, more
importantly, the drugability investigation that enabled the selection of a preclinical drug
candidate with potent in vitro and in vivo anti-neuroinflammatory activities, desirable
pharmacokinetic properties, and extremely low toxicity. Its underlying mechanisms were also
investigated, and the results support investigating this novel agent as a potential PD therapy
further.
2
23
24
25
26
RESULTS AND DISCUSSION
27
28
29
30
31
3
3
34
35
Discovery of the hit compound. 3-Methyl-1-(2,4,6-trihydroxyphenyl)butan-1-one (1), a
known phloroglucinol derivative, was previously isolated by our research group from Lysidice
rhodostegiaroots. It was identified as the initial hit compound by screening for biological
activities, which revealed a moderate inhibitory effect on nitric oxide (NO) production in
lipopolysaccharide (LPS)-induced BV2 cells, with a half-maximal inhibitory concentration (IC )
36
37
38
5
0
39
4
41
42
4
4
45
of 50.6 μM. Moreover, this compound showed certain “lead-like” properties, including low
molecular weight, and a combination of polar and nonpolar moieties. Furthermore, it provided
multiple diversification points for robust optimization.
46
47
48
49
50
51
52
53
5
5
5
Figure 1. The structure of the hit compound.
57
58
59
60
ACS Paragon Plus Environment

Page 5 of 63
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
12
13
14
15
16
17
18
19
20
2
2
23
24
25
26
27
28
29
30
31
3
3
34
35
36
37
Hit-to-lead medicinal chemistry optimization. With careful analysis, it was supposed
that the three phenolic hydroxyl group contained in the hit compound might bring some
negative effects as follows: 1) The polar of the molecule is strong, which may affect the
absorption; 2) Glucuronidation of multi-hydroxyl groups may shorten the acting time of
the compound within the body; 3) The stability of the compound is poor since the
phenolic hydroxyl group is sensitive to chemical environment. Besides, the hit
compound was not potent enough and its activity should be improved markedly.
Therefore, structural modifications were performed to increase the anti-neuroinflammatory
activities of the hit compound as well as improve its physicochemical properties. According to
the basic principles of medicinal chemistry, the following strategies were used to achieve these
purposes: 1) The structural skeleton of the hit compound was retained, and minimal
modifications were made to adjust the lipid-water partition coefficients and/or improve its
stability, such as changing the number of hydroxyl groups, methylating the hydroxyl groups at
different positions, and altering the length of the sidechain. 2) Substantial changes were made
by introducing various common nitrogen-containing activity-modifying pharmacophores into
the molecule, such as amide, aminoketone, amino, propanolamine and furoxan units, to enhance
the pharmacological effects. In the latter case, the number and methylation of hydroxyl groups,
as well as the length of the sidechain can be modified simultaneously. As the hit compound
was simple, slight change might lead to wide variation in the structure. Consequently, 43
phloroglucinol derivatives belonging six series (2–8, 9–10, 11–17, 18–25, 26–43, and 44) were
synthesized, 30 of which were new (indicated by *).
38
39
40
41
42
4
4
45
46
47
48
49
50
51
52
53
5
5
56
Compound 2 was synthesized from 1 by methylating the hydroxyl group at the ortho position
of the carbonyl with Me SO after protecting the other two with methoxymethyl (MOM) groups
2
4
57
58
59
60
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 63
1
2
3
4
5
6
7
8
9
(
Scheme 1), and 3−8 were prepared via Friedel–Crafts reactions, with BF −Et O as a catalyst
3
2
1
8
(Scheme 2).
a
Scheme 1. Synthesis of compound 2.
1
1
12
13
14
15
16
1
a
Reagents and conditions: (a) CH OCH Cl, DIPEA, CH Cl , 0 °C, 30 min; (b) Me SO ,
3
2
2
2
2
4
1
19
K CO , acetone, reflux, 5 h; (c) 2N HCl, reflux, 1 h.
2 3
20
2
2
23
a
Scheme 2. Synthesis of compounds 3−8.
24
25
26
27
28
29
30
31
3
3
34
35
36
37
38
a
Reagents and conditions: (a) isovaleryl chloride, BF −Et O, 80 °C, 2 h; (b) valeryl
3
2
39
40
chloride, BF −Et O, 80 °C, 2 h; (c) acetyl chloride, BF −Et O, 80 °C, 2 h.
3
2
3
2
41
42
4
4
Amides 9 and 10 were synthesized as follows: 2,4,6-trihydroxybenzoic acid was reacted with
45
46
oxalyl chloride to give acyl chloride, which reacted with amines to yield the corresponding
47
48
49
amides (Scheme 3).
5
51
a
Scheme 3. Synthesis of compounds 9 and 10
.
52
53
5
5
56
57
58
59
60
ACS Paragon Plus Environment

Page 7 of 63
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
12
13
a
Reagents and conditions: (a) oxalyl chloride, DMSO, CH Cl , rt, 1 h; (b) isopropylamine,
2
2
THF, rt, 1 h; (c) butylamine, THF, rt, 1 h.
14
15
16
17
18
19
20
2
2
The aminoketone analogs 11−17 were prepared in two steps: acylation of 1,3,5-
trimethoxybenzene or phloroglucinol with an omega-chloro alkyl nitril through Houben−Hoesch
reactions catalyzed by HCl gas and introduction of amino groups by replacing the chloro group
23
2
1
9
with various amines in the presence of K CO (Schemes 4 and 5). The order of the two steps
2
3
25
26
27
28
29
30
31
3
3
could be reversed for the synthesis of 11−14; however, the amines must first react with 4-
chlorobutanenitrile for the synthesis of 15−17 because the hydroxyl groups of phloroglucinol
were unstable in the alkaline environment created by K CO .
2
3
a
Scheme 4. Synthesis of compounds 11−14.
34
35
36
37
38
39
40
41
42
4
4
45
46
47
48
49
5
a
Reagents and conditions: (a) chloroacetonitrile, HCl gas, PhCl, rt, 6 h; (b) H O, reflux, 1
2
5
52
53
5
h; (c) piperidine, K CO , acetone, reflux, 24 h; (d) morpholine, K CO , acetone, reflux,
2
3
2
3
2
4 h; (e) 1-methylpiperazine, K CO , acetone, reflux, 24 h; (f) 1-ethylpiperazine, K CO ,
2 3 2 3
acetone, reflux, 24 h.
5
56
5
58
a
Scheme 5. Synthesis of compounds 15−17.
59
60
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 63
1
2
3
4
5
6
7
8
9
1
1
12
13
14
15
16
a
Reagents and conditions: (a) piperidine, K CO , NaI, acetonitrile, rt, 20 h; (b) N-
2
3
17
18
19
2
methylcyclohexanamine, K CO , NaI, acetonitrile, rt, 20 h; (c) 1-(3-
2
3
chlorophenyl)piperazine, K CO , NaI, acetonitrile, rt, 20 h; (d) (i) phloroglucinol, HCl
2
3
gas, PhNO , rt, 12 h; (ii) H O, reflux, 1 h.
2
2
2
2
23
2
Compounds 18−25 were obtained from 2,4,6-trimethoxybenzaldehyde by adding amines or
25
26
27
alkyl (methyl or ethyl) ester of the amino acid hydrochloride salts, followed by treatment with
28
29
NaBH CN in anhydrous MeOH (pH～6, Scheme 6) and, then, hydrolysis of the ester, if
3
30
31
3
2
0
necessary.
3
34
a
Scheme 6. Synthesis of compounds 18−25.
35
36
37
38
39
40
41
42
4
4
45
46
47
48
49
50
51
52
53
5
5
56
57
58
59
60
ACS Paragon Plus Environment

Page 9 of 63
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
12
13
14
15
16
17
18
19
20
2
2
23
24
25
26
2
a
Reagents and conditions: (a) isopropylamine, NaBH CN, MeOH, HOAc, argon, rt, 3 h;
3
28
29
3
(
b) L-GlyOEt-HCl, NaBH CN, MeOH, argon, rt, 3 h; (c) L-AlaOMe-HCl, NaBH CN,
3
3
MeOH, argon, rt, 3 h; (d) L-SerOMe-HCl, NaBH CN, MeOH, argon, rt, 3 h; (e) L-
3
3
3
3
3
Asp(OMe) -HCl, NaBH CN, MeOH, argon, rt, 3 h; (f) L-Glu(OMe) -HCl, NaBH CN,
2
3
2
3
MeOH, argon, rt, 3 h; (g) L-PheOMe-HCl, NaBH CN, MeOH, argon, rt, 3 h; (h) L-
3
HisOMe-HCl, NaBH CN, MeOH, argon, rt, 3 h; (i) KOH, EtOH-H O (1:1), rt, 2 h,
3
2
+
Amberlite 120 H Resin.
35
36
37
38
39
40
41
42
4
4
45
46
47
48
49
50
51
The synthesis of racemic aryloxypropanolamine hydrochloride salts 26−31 is outlined in
Scheme 7. Condensations of 2-(chloromethyl)oxirane with 3, 5, and 1a, the O-benzyl-protected
2
1
product of 1, which was achieved through treatment with NaH in anhydrous DMF, provided
the epoxides 3b, 5b and 1b, respectively. These subsequently reacted with various amines,
resulting in the corresponding aryloxypropanolamines. The hydrochloride salts were obtained
through treatment with ethereal HCl, followed by a deprotection step, if necessary.
52
53
a
Scheme 7. Synthesis of compounds 26−31.
5
5
56
57
58
59
60
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 63
1
2
3
4
5
6
7
8
9
1
1
12
13
14
15
16
17
18
19
20
2
2
23
24
2
a
Reagents and conditions: (a) benzyl p-toluenesulfonate, K CO , acetone, reflux, 3 h; (b)
2
3
26
27
28
2
-(chloromethyl)oxirane, NaH, DMF, N , 90 °C, 2 h; (c) (i) isopropylamine, 70 °C, 3 h;
2
(
ii) ethereal HCl, 10 min; (d) (i) tert-butylamine, 70 °C, 3 h; (ii) ethereal HCl, 10 min; (e)
2
30
H , 3 atm, Pd–C, MeOH, HCl, rt, 12 h.
2
31
3
3
34
The synthesis of chiral aryloxypropanolamine hydrochloride salts 32−43 is shown in Scheme
35
36
8
. Condensation of (R)-or (S)-4-(chloromethyl)-2,2-dimethyl-1,3-dioxolane with 3, 5, and 1a,
37
38
39
40
41
42
4
followed by hydrolysis of 1,3-dioxolane to diols, p-toluenesulfonylation of the primary alcohol-
OH and amination with various amines resulted in the corresponding chiral
aryloxypropanolamine, which had the following configuration.
4
4
a
Scheme 8. Synthesis of compounds 32−43.
46
47
48
49
50
51
52
53
5
5
56
57
58
59
60
ACS Paragon Plus Environment

Page 11 of 63
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
12
13
14
15
16
17
18
19
20
2
2
23
24
25
26
27
28
29
30
31
3
3
34
35
3
37
38
a
Reagents and conditions: (a) (S)-4-(chloromethyl)-2,2-dimethyl-1,3-dioxolane, NaH,
DMF, N , 90 °C, 24 h; (b) (R)-4-(chloromethyl)-2,2-dimethyl-1,3-dioxolane, NaH, DMF,
2
3
4
41
42
N , 90 °C, 24 h; (c) 4% H SO , THF, rt, 8 h; (d) TsCl, pyridine, CH Cl , rt, 48 h; (e) (i)
isopropylamine, 70 °C, 3 h; (ii) ethereal HCl, 10 min; (f) (i) tert-butylamine, 70 °C, 3 h;
2
2
4
2
2
(
ii) ethereal HCl, 10 min; (g) H , 3 atm, Pd–C, MeOH, HCl, rt, 12 h.
2
4
4
45
46
Compound 44 was synthesized according to the sequence shown in Scheme 9. The reaction of
47
48
2
2
cinnamyl alcoholand NaNO in HOAc resulted in 4-hydroxymethyl-3-phenyl furoxan (44a),
2
49
5
51
52
53
which was converted into 4-chloromethyl-3-phenyl furoxan (44b) through treatment with SOCl2.
Condensation of the resulting chloride with 2a in the presence of K CO , followed by
2
3
5
5
deprotection using 2N HCl, provided the desired product 44.
56
57
58
a
Scheme 9. Synthesis of compound 44.
59
60
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 63
1
2
3
4
5
6
7
8
9
1
1
12
13
14
15
16
1
a
Reagents and conditions: (a) NaNO , HOAc, H O, rt, 24 h; (b) SOCl , pyridine, CH Cl ,
2
2
2
2
2
18
19
rt, 3 h; (c) K CO , KI, acetone, reflux, 5 h; (d) 2N HCl, reflux, 1 h.
2
3
20
2
2
23
24
25
26
27
28
29
30
31
3
3
34
35
36
37
38
39
40
41
42
4
4
45
All synthesized phloroglucinol derivatives were tested for anti-inflammatory activity using the
same method as for the hit compound screen (Table 1). The activity profile exhibited varied
results for each analogue, and their structure-activity relationships were discussed by structural
series. Series 1: Compared with the hit compound 1, the results of 2−6 showed that the numbers
and positions of the phenolic hydroxyl groups and their methyl ethers greatly influenced the
inhibitory activity against NO production in the LPS-induced BV2 cells. It seemed that reducing
the number of hydroxyl groups to two (5) or one (6) might cause negative impact on the anti-
inflammatory activity. While methylation of one (2) or all the three (4) rather than two (3)
hydroxyl groups markedly increase the activity. Moreover, the results of 7 and 8 suggested that
the length of the sidechain were also important for the activity, and valeryl would be more
46
47
48
49
50
51
52
53
appropriate than isovaleryl and isobutyryl groups. Series 2 and 3: Introducing an amide (9 and 10)
or an aminoketone (11−17) group seemed to make little sense to improve the activity. However,
among the aminoketone analogues, 11−14 with substitution of methoxyl in place of hydroxyl
groups and shorter length of the sidechain were slightly more effective. Series 4: Compounds
5
5
56
1
9−25, which incorporated amino acid substituents, exhibited significantly different activities.
57
58
59
60
ACS Paragon Plus Environment

Page 13 of 63
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
12
13
14
15
16
17
18
19
20
2
Weak inhibition of NO release was observed for compound 19, which had a Gly substituent,
whereas 21−23, contained Ser, Asp, and Glu substituents, respectively, showed high inhibition
levels, suggesting that sidechain substituents with a certain polarity and electrical properties
might be necessary for activity. Furthermore, 24 and 25, with Phe and His substituents,
respectively, are mostly inactive, indicating that aromatic functional groups in the sidechain are
not desirable. Series 5: Compounds 26−43 differed from the hit compound by an additional
propanolamine unit at 2’-hydroxyl position, most of which showed moderate or marginal anti-
neuroinflammatory activity except for compound 34 (IC 11.2 μM). The absolute configuration
5
0
2
23
24
25
26
27
28
29
of the chiral carbon in propanolamine unit had certain influence on the activity, but no clear
regularity could be concluded. Series 6: Introduction a furoxan substituent by formation of ether
resulted in significant improvement in the activity, which may provide an effective way to get
more potent anti-neuroinflammatory product. But unfortunately, only one compound was
synthesized for a variety of reasons.
30
31
3
3
34
35
36
37
38
39
40
In a word, the structure activity relationships of the phloroglucinol derivatives are very
complicated. Both minimal and substantial modification may influence the anti-
neuroinflammatory effect. Finally, compounds 2, 21, 23, and 44, which had IC values of 5.8,
5
0
41
42
6
.6, 8.4, and 8.5 μM, respectively, were selected as target compounds for further investigations.
4
4
It is noteworthy that except for 2, the other three proposed compounds are remarkably different
45
4
47
from the hit compound in their structures, which may lead to a different mode of action.
48
49
Table 1. Inhibitory activities of compounds 1−44 against LPS-induced NO release in
50
51
BV2 cells.
52
53
5
a
Compound
IC50 (μM)
Compound
IC (μM)
50
5
56
5
1
2
50.7
24
25
—
—
5.8
58
59
60
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 63
1
2
3
4
5
6
7
8
9
1
1
12
13
14
15
16
17
18
19
20
2
2
23
2
25
2
27
28
29
30
31
3
3
34
35
36
3
3
4
5
6
7
8
9
51.5
11.1
113.4
90.7
11.2
194.7
92.6
37.1
24.9
32.4
95.3
22.7
182.2
54.8
26
27
84.8
75.0
—
28
29
137.5
74.0
21.7
138.4
41.3
11.2
—
30
31
32
1
0
1
2
3
33
1
1
1
34
35
36
25.6
40.9
53.8
28.2
—
14
15
37
38
1
1
1
1
2
6
7
8
9
0
39
b
—
40
—
—
41
—
42
24.1
37.3
8.5
39.1
6.6
43
21
22
44
38
3
40
41
c
14.4
8.4
Curcumin
2.2
2
3
42
4
4
a
b
half-maximal inhibitory concentration. inhibition rate was less than 50% at the
c
maximal concentration of 0.01 M. positive control.
45
46
47
48
49
50
51
52
53
5
5
56
57
58
In vitro efficacy of lead compounds in primary cultures. The four promising compounds
were further examined for anti-neuroinflammatory effects in primary mixed mesencephalic
neuronal/glial cultures. The microglia were activated by LPS stimulation, as indicated by the
over-expression of membrane Iba-1, a marker of microglia activation, and they showed increased
production of IL-1β and TNF-α. The neuroinflammatory response was greatly inhibited by
59
60
ACS Paragon Plus Environment

Page 15 of 63
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
12
13
compounds 2, 21, 23, and 44 at 10 μM (Figure 2A‒C). The LPS-induced neuroinflammation also
induced dopaminergic neurotoxicity, as reflected by the reduced expression of tyrosine
hydroxylase (TH), which was rescued by these compounds (Figure 2D). These data suggest that
the four compounds potentially suppressed neuroinflammation and protected dopaminergic
neurons.
14
15
16
17
18
19
20
2
2
23
24
25
26
27
28
29
30
31
3
3
34
35
36
37
38
39
Figure 2. The effects of compounds 2, 21, 23, and 44 on neuroinflammation and TH
40
41
42
4
4
45
46
47
expression in primary mixed midbrain neuronal/glial cultures stimulated with LPS.
Control group: primary mixed mesencephalic neuronal/glial cultures with no treatment; LPS
group: primary mixed neuronal/glial cultures stimulated with LPS (100 ng/mL); Other groups:
48
49
1
0 μM of 2, 21, 23 and 44 incubated with cells for 1 hour before LPS challenge, and then
50
51
52
53
5
5
56
57
incubated for 5 h to detect cytokines, 24 h to measure Iba-1 expression and 7 days to measure
TH expression. (A) Effects of the indicated compounds on Iba-1 expression in microglia (n = 4).
A representative image is shown. Inhibitory effects of the indicated compounds on (B) IL-1β
and (C) TNF-α production in medium measured by ELISA assay (n = 6). (D) Effects of the
58
59
60
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 63
1
2
3
4
5
6
7
8
9
indicated compounds on TH expression measured by Western blot (n = 4). A representative
immunoblot image is shown. Data are the mean ± standard deviation (SD),*P < 0.05 and
#
##
*
*P < 0.01 versus control cells; P < 0.05 and P < 0.01 versus LPS-treated cells.
1
1
Pharmacokinetic properties and safety profiles. The in vivo preliminary
12
1
14
15
pharmacokinetic properties of these four lead compounds were investigated in mice
following the intragastric administration of each compound at a dose of 50 mg/kg (Table 2).
16
17
18
All compounds were detectable in the plasma 5 min after administration. However, only
2
19
2
2
2
23
and 21 efficiently passed through the blood-brain barrier, whereas the concentrations of 23
and 44 in the brain were low (just above the limit of detection) and undetected, respectively.
24
25
Therefore, compounds 2 and 21 were selected for further safety studies.
26
2
28
29
30
The results of in vitro toxicity study indicated that neither of the two compounds had
selectivity for hERG. In the in vivo toxicity study, both showed satisfactory maximum tolerated
31
3
doses after being orally administered to mice, with median lethal dose (LD ) values of 2000
50
3
34
35
36
37
38
39
40
41
and >5000 mg/kg for compounds 2 and 21, respectively. Nevertheless, the 14-day toxicity study
showed that compound 2 exhibited hepatotoxicity and nephrotoxicity at a higher dose (1000
mg/kg), whereas compound 21 had no detectable adverse effects. Considering the long-
term administration requirements for PD medications, compound 21 was chosen instead of
compound 2 for further in vivo efficacy studies.
42
4
4
45
46
47
Table 2. Pharmacokinetic properties of the lead compounds.
48
49
Blood
Brain
50
51
5
53
5
5
56
57
Compound
AUC0-∞
b
Cmax
AUC0-∞
Cmax
a
t
1/2 (h)
c
t1/2 (h)
(μg∙[L·h])
(μg/L)
(μg∙[L·h])
(μg/L)
6
42.2
10.8 ±
1361.6 ±
561.1
1981.2 ±
346.7
4668.0 ±
1624.4
2
d
6.4 ± 0.7
1.1 ± 0.3
3.4 ± 0.6
1.5 ± 0.4
2
1
5733.0 ±
2590.0 ±
497.1 ±
192.0 ±
58
59
60
ACS Paragon Plus Environment

Page 17 of 63
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
6
35.7
420.2
72.2
27.2
3
195.1 ±
1645.0 ±
233.4
e
23
1.1 ± 0.2
≈ LOD
–
–
≈ LOD
891.1
4
4
37.6 ± 18.0 1.2 ± 0.7 26.5 ± 8.4
ND
ND
a
b
c
d
1
1
12
The area under the curve from 0 h to infinity. Half-life. Maximum concentration. The
values are the mean ± SD (n = 6). Limit of detection
e
13
14
15
16
17
18
19
20
2
2
23
24
25
26
27
28
29
30
In vivo efficacy of compound 21 in PD mouse models. Compound 21 was successively
evaluated in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- and MPTP/probenecid (prob)-
induced sub-acute and chronic PD mouse models, respectively, and in α-synuclein (syn) transgenic
mice. Obvious dopaminergic neuron dysfunction and neuroinflammatory responses were observed in
the three different PD models. Compound 21 greatly improved the motor behavior dysfunction of the
mice by increasing their staying time on the rod in the rotarod tests and their performance scores in the
pole tests. Furthermore, it increased the dopamine levels in the striatum, reversed the decline in TH-
positive neurons in the substantia nigra, and decreased the productions of proinflammatory molecules,
such as IL-1β and TNF-α, at doses of 10 and 20 mg/kg after short- (MPTP, 10-day) or long-term
31
3
3
34
35
36
37
(MPTP/prob, 7-week and α-syn transgenic mice, 10-week) administration (Figures 3‒5). The in vivo
38
3
40
study demonstrated the potent neuroprotective effects of compound 21 on dopaminergic neurons,
41
42
which were closely associated with its suppression of neuroinflammation in the brain.
4
4
45
46
47
48
49
50
51
52
53
5
5
56
57
58
59
60
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 63
1
2
3
4
5
6
7
8
9
1
1
12
13
14
15
16
17
18
19
20
2
2
23
24
25
26
27
28
29
30
31
3
3
34
35
36
37
38
39
40
41
42
Figure 3. The effect of compound 21 on the MPTP-induced sub-acute PD mouse model.
Control group: mice were treated with 0.5% sodium carboxymethylcellulose (CMC-Na);
MPTP group: mice were injected with MPTP hydrochloride (30 mg/kg, i.p.) for 5 consecutive
days; Other groups: mice were administered different doses of compound 21 (5, 10, 15 mg/kg)
4
4
45
46
and L-DOPA (20 mg/kg) 30 min before each MPTP injection, and continued administered for
47
48
49
50
51
52
53
5
5
56
57
58
the next 7 days. Compound 21 improved motor behavior of mice, as measured by (A) rotarod
test and (B) pole test (n = 15). (C) The effect of compound 21 on dopamine level (n = 8). (D)
The effect of compound 21 on the TH-positive staining neurons. Representative sections of
substantia nigra from 5 mice are shown. Inhibitory effect of compound 21 on (E) IL-1β and (F)
TNF-α production in the mouse midbrain measured by ELISA assay on the 12th day after the
59
60
ACS Paragon Plus Environment

Page 19 of 63
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
administration of compound 21 ( n = 6). Data are the mean ± SD; *P < 0.05 and **P < 0.01
#
##
versus control mice; P < 0.05 and P < 0.01 versus MPTP injected mice.
1
1
12
13
14
15
16
17
18
19
20
2
2
23
24
25
26
27
28
29
30
31
3
3
34
35
36
37
38
39
40
41
42
4
4
45
46
47
48
Figure 4. The effect of compound 21 on the MPTP/prob-induced chronic PD mouse model.
Control group: mice were treated with 0.5% CMC-Na; MPTP/prob group: mice were
administered 10 doses of MPTP hydrochloride (25 mg/kg, subcutaneously, s.c.) in combination
with probenecid (250 mg/kg, i.p.) on a 5-week schedule; Other groups: mice showing
behavioural dysfunction after the last MPTP/prob injection were administered different doses of
49
50
51
52
53
5
compound 21 (5, 10, 15 mg/kg) and L-DOPA (20 mg/kg) once a day for 7 weeks. Compound 21
5
56
improved motor behavior of mice, as measured by (A) rotarod test and (B) pole test (n = 15). (C)
57
58
59
60
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 63
1
2
3
4
5
6
7
8
9
The effect of compound 21 on dopamine level (n = 8). (D) The effect of compound 21 on the
TH-positive staining neurons. Representative sections of substantia nigra from 5 mice are shown.
Inhibitory effect of compound 21 on (E) IL-1β and (F) TNF-α production in the mouse midbrain
measured by ELISA assay (n = 6). Data are the mean ± SD; *P < 0.05 and **P < 0.01 versus
1
1
12
1
14
#
##
control mice; P < 0.05 and P < 0.01 versus MPTP/prob-treated mice.
15
16
17
18
19
20
2
2
23
24
25
26
27
28
29
30
31
3
3
34
35
36
37
38
39
40
41
42
4
4
45
46
47
48
49
50
51
52
53
Figure 5. The effect of compound 21 on α-syn transgenic mice. WT group: mice were
treated with 0.5% CMC-Na; α-Syn group: α-Syn (A53T) transgenic mice; Other groups: mice
were administered different doses of compound 21 (5, 10, 15 mg/kg) and L-DOPA (20 mg/kg)
5
5
once a day for 12 weeks. Compound 21 improved motor behavior of mice, as measured by (A)
56
57
58
rotarod test and (B) pole test (n = 15). (C) The effect of compound 21 on dopamine level (n = 8).
59
60
ACS Paragon Plus Environment

Page 21 of 63
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(
D) The effect of compound 21 on the TH-positive staining neurons. Representative sections of
substantia nigra from 5 mice are shown. Inhibitory effect of compound 21 on (E) IL-1β and (F)
TNF-α production in the mouse midbrain measured by ELISA assay (n = 6). Data are the mean ±
1
1
#
##
SD; *P < 0.05 and **P < 0.01 versus control mice; P < 0.05 and P < 0.01 versus α-syn
12
1
14
transgenic mice.
15
16
17
18
19
20
2
2
23
24
25
26
27
28
29
30
31
3
3
34
35
36
37
38
39
40
41
42
4
4
Mechanistic studies of neuroprotective effects. To elucidate the molecular target of
compound 21 in neuroinflammation, we performed isobaric tags for relative and absolute
quantification (iTRAQ)-labelling proteomic studies in a MPTP/prob-induced chronic PD mouse
model. We quantified 3508 proteins and applied a threshold of >1.5-fold to identify proteins that
were differentially expressed, and 132 met the criteria (Supporting Information, Proteomics
Data). Among these proteins, we selected those that were reportedly associated with
neuroinflammation to validate the proteomic quantization. Finally, twenty proteins were
analyzed, and eight were consistent with the proteomic analysis, as described in Figure 6. The
Western blot analysis confirmed phosphatase and tensin homolog deleted on chromosome 10
(PTEN); Akt and NO synthase (NOS) were the most affected by compound 21. Akt is reportedly
2
3,24
the downstream effector of PTEN, and its phosphorylation may induce NOS activation.
PTEN counteracts the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway and controls
45
4
47
2
3,25
inflammatory responses.
The present results indicated that the PTEN/Akt pathway and
48
49
50
51
52
53
5
downstream NOS might be involved in the anti-inflammatory activity of compound 21.
PTEN function is primarily regulated by phosphorylation through specific kinases, such as Src-
tyrosine kinase, glycogen synthase kinase-3 (GSK3)-β, casein kinase 2 (CK2), liver kinase B1
5
56
(LKB1), Rho guanosine triphosphatases (GTPases) activating protein 21 (ARHGAP21), and Rho-
57
58
59
60
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 63
1
2
3
4
5
6
7
8
9
1
1
12
13
associated protein kinase 1 (ROCK1), which is frequently associated with functionally inactive
26,27
PTEN.
Among these kinases, the expression levels of GSK3β, ARHGAP21, ROCK1 and Src
were affected by compound 21 in the proteomic quantization. The four proteins were then validated,
and Src-tyrosine kinase was the most obviously altered by compound 21 (Supporting Information,
Figures S1 and S2). Src-tyrosine kinase is a major modulator in the proximal intracellular signaling
14
15
16
28,29
pathways in innate cells and activates monocytes and macrophages.
Inhibition of Src reportedly
17
18
19
20
2
2
23
3
0
suppresses neuroinflammation and protects mice from ischemia-induced brain damage. Our
present results revealed that in addition to PTEN and Akt phosphorylation, Src activation was also
inhibited by compound 21 (Figures 6A‒C). Src primarily phosphorylates PTEN at Tyr240,
24
25
26
27
31
inactivating it and phosphorylating Akt. We knocked down the Src gene in primary microglia and
midbrain mixed neuronal/glial cultures and stimulated the cells with LPS, 1-methyl-4-
28
2
30
+
phenylpyridinium ion (MPP ), or α-synuclein. We found that the phosphorylation of PTEN and Akt
31
3
3
34
35
36
37
38
39
40
41
42
4
4
decreased (Figure 6D), the inflammatory response was reduced, and the injury of dopaminergic
neurons was alleviated (Figure 6E). Most importantly, we found that the suppression of
neuroinflammation (Figures 6E and F) and the neuroprotective activity of compound 21 were
mostly abrogated when Src was silenced (Figures 6G‒I). The above data prove that Src tyrosine
kinase was the main target of compound 21 in its neuroinflammation regulation and associated
neuroprotection.
45
46
47
48
49
50
51
52
53
5
5
56
57
58
59
60
ACS Paragon Plus Environment

Page 23 of 63
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
1
1
12
13
14
15
16
17
18
19
20
2
2
23
24
25
26
27
28
29
30
31
3
3
34
35
36
37
38
39
40
41
42
4
4
45
46
47
48
Figure 6. Compound 21 suppressed neuroinflammation by inhibiting Src signaling.
49
50
51
52
53
5
5
C
ontrol group: primary microglia with no treatment; LPS group: primary microglia
stimulated with LPS (100 ng/mL); LPS + 21 group: the microglia was incubated with compound
1 (10 μM) for 1 hour followed by LPS challenge; Negative control group: primary microglia
2
56
57
58
or mixed mesencephalic neuronal/glial cultures were tranfected with empty vector;
59
60
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 63
1
2
3
4
5
6
7
8
9
Negative control + LPS group: primary microglia or mixed mesencephalic neuronal/glial
cultures were tranfected with empty vector followed by LPS challenge; Src siRNA group:
microglia or mixed mesencephalic neuronal/glial cultures were tranfected with Src siRNA;
1
1
+
Src siRNA + LPS/MPP /α-Syn group: primary microglia or mixed mesencephalic
12
1
14
15
+
neuronal/glial cultures were tranfected with Src siRNA, LPS (100 ng/mL), MPP (30 μM), or
+
α-syn (250 nM) was added 4 h later; Src siRNA + LPS/MPP /α-Syn +21 group: primary
16
17
18
microglia or mixed neuronal/glial cultures were tranfected with Src siRNA, compound 21 (10
19
2
2
+
μM) was added 1 h before LPS, MPP or α-Syn challenge. The effects of compound 21 on (
A)
2
23
24
25
26
2
28
p-PTEN, (
regulation of Src on PTEN and Akt in primary microglia cultures (n = 4). The effects of
compound 21 on ( ) IL-1β and ( ) TNF-α production in LPS-stimulated primary
B) p-Akt and (C) p-Src expression in primary microglia cultures. (D) The
E
F
29
30
microglia cultures (n = 6). The effects of compound 21 on TH expression in (
G
) LPS-, (
H)
31
3
+
MPP - and (
I
) α-syn-stimulated primary mixed mesencephalic neuronal/glial cultures (n =
3
34
35
4
). A representative immunoblot images are shown. The results are shown as the mean ±
3
37
38
39
#
##
+
SD; **P < 0.01 versus control cells; P < 0.05 and P < 0.01 versus LPS-, MPP - or α-
syn- treated cells; ΔP < 0.05 versus Src siRNA-treated cells.
40
41
42
4
4
CONCLUSIONS
45
4
47
48
49
50
51
52
53
In this paper, we reported the discovery and optimization of a novel class of anti-
neuroinflammatory compounds: phloroglucinol derivatives. The structure-activity
relationships of this series were complex, and changes in either the phenolic hydroxyl
groups or the sidechain of the hit compound significantly influenced the activities. Note
that the introduction of various nitrogen-containing pharmacophores into the hit
5
5
56
57
58
59
60
ACS Paragon Plus Environment

Page 25 of 63
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
compound enhanced the potency more efficiently than minor modifications, such as
changing the number of hydroxyl groups, methylating the hydroxyl groups at different
positions, and altering the length of the sidechain. These results might provide a new
strategy for the structural modification of other similar compounds.
1
1
12
1
14
15
16
17
18
19
20
2
2
23
24
25
26
27
Our study provided evidence supporting compound 21 as a potential candidate for PD
treatment because of its potent in vitro and in vivo anti-neuroinflammatory activities, novel
mechanism, impressive penetration of the blood-brain barrier, and low toxicity. Moreover, our
results yielded further proof that neuroinflammation suppression might become an effective
disease-modifying therapy that could delay or slow the progression of PD and thereby open up a
new strategy for the future treatment of PD patients. We believe that further structural
modification based on compound 21 and its exact mechanisms would enable the development of
more potent agents used for PD. And these studies are currently underway in our laboratory.
28
29
30
31
3
3
EXPERIMENTAL SECTION
34
35
36
Chemistry. Reagents and solvents were of commercial quality and used without further
37
38
39
40
41
42
4
4
45
46
47
48
49
50
51
52
1
13
purification. H and C NMR spectra were acquired on an INOVA-500, Mecury-400 or Mecury-
300 spectrometer. HRESIMS data were obtained on an Agilent 6520 Accurate-Mass-Q-TOF
LC/MS spectrometer. ESIMS data were recorded on an Agilent 1100 Series LC/MSD Trap/SL
spectrometer. Optical rotations were measured on a JASCO P-2000 automatic polarimeter. TLC
was conducted with glass precoated with silica gel GF254 (Qingdao Marine Chemical Inc.,
China) to monitor the progression of the reactions. Silica gel (200–300 mesh, Qingdao Marine
Chemical Inc., China) were used for column chromatography (CC). The purity of the test
compounds was determined by HPLC analysis using an Agilent 1100 series instrument equipped
with a DAD detector and a YMC-Pack ODS column (100 mm × 4.6 mm, 5 μm) at 210 nm, and
53
5
5
56
57
58
59
60
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 63
1
2
3
4
5
6
7
8
9
was generally ≥ 95%, if not denoted. Preparative HPLC was performed on a Shimadzu LC-6AD
instrument with a SPD-20A detector using a YMC-Pack ODS-A column (250 × 50 mm, 5 μm).
1
-(2,4-Dihydroxy-6-methoxyphenyl)-3-methylbutan-1-one (2). To a stirred suspension of 1
1
1
(
2.10 g, 10.0 mmol) in anhydrous CH Cl (50 mL) was added DIPEA (2.71 g, 21.0 mmol) and
2
2
12
1
14
the reaction mixture was stirred for 15 min at 0 °C, and then CH OCH Cl (2.00 g, 25.0 mmol) in
3 2
15
16
CH Cl (20 mL) was added dropwise. The mixture was stirred for 1 h at room temperature and
2
2
17
18
19
20
subsequently poured into cold water (20 mL), and extracted with chloroform (3 × 50 mL). The
combined organic layers were dried over Na SO and concentrated in vacuo. The residue
2
4
2
2
23
24
25
obtained on column purification (silica, petroleum ether/EtOAc, 15:1 as eluent) afforded 2a as a
colorless oil (1.40 g, 47%).
26
2
28
29
30
To a solution of 2a (0.30 g, 1.0 mmol) in anhydrous acetone (20 mL) was added anhydrous
K CO (0.27 g, 2.0 mmol), and the reaction mixture was refluxed for 15 min, followed by
2
3
31
3
addition of Me SO (100 µL, 2.0 mmol). After refluxing for an additional 5 h, the solvent was
2
4
3
34
35
36
37
removed in vacuo. Water (20 mL) was then added to the residue, and the aqueous layer was
extracted with EtOAc (3 × 25 mL). The combined organic layers were dried over Na SO and
2
4
38
3
40
evaporated in vacuo. The residue obtained on column purification (silica, petroleum ether/EtOAc,
41
42
3
0:1 as eluent) afforded 2b (0.25 g, 60%) as a yellow oil.
4
4
To a solution of 2b (0.25g, 0.8 mmol) in MeOH (10 mL) was added 2N HCl (2 mL), and the
45
4
47
48
49
reaction mixture was refluxed for 1 h. Then the solvent was removed in vacuo, and the resulting
residue was dissolved in EtOAc (50 mL) and washed with water. The organic layers were dried
50
5
over Na SO and concentrated in vacuo. The residue obtained on column purification (silica,
2
4
52
53
5
petroleum ether/EtOAc, 5:1 as eluent) afforded 2 as a white solid (0.18 g, 83%, mp 122–123°C ).
5
56
1
H NMR (500 MHz, DMSO-d ) δ 13.59 (br s, OH-2′, OH-4′), 5.94 (1H, s, H-5′), 5.85 (1H, s, H-
6
57
58
59
60
ACS Paragon Plus Environment

Page 27 of 63
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
3
′), 3.80 (3H, s, OCH ), 2.77 (2H, d, J = 6.5 Hz, H-2), 2.07 (1H, m, H-3), 0.89 (6H, d, J = 6.5
3
13
Hz, CH -4, CH -5); C NMR (125 MHz, DMSO-d ) δ 204.0, 165.7, 164.4, 162.4, 104.2, 95.3,
3
3
6
+
9
2
0.9, 55.3, 51.9, 24.5, 22.2 (2C); HRESIMS m/z = 225.1126 [M + H] (calcd for C H O ,
12 17 4
1
1
25.1121).
12
1
14
15
General Procedure for Acylation by Friedel–Crafts Reaction for Compounds 3−8 (GP1). The
phenolic precursor (10.0 mmol) was dissolved in BF –Et O (20 mL), and an amount of 1 equiv
3
2
16
17
18
19
20
of acyl chloride was added, and the reaction mixture was stirred at 80 °C for 2 h. After cooling,
the reaction mixture was poured into ice-cooled 5% KOAc solution (50 mL), and extracted with
2
2
23
EtOAc (3 × 30 mL). The combined organic layers were washed with saturated NaHCO solution
3
24
2
and dried over Na SO and concentrated in vacuo. The resulting product was obtained by column
2
4
26
2
28
29
30
chromatography.
-(2-Hydroxy-4,6-dimethoxyphenyl)-3-methylbutan-1-one (3). This compound was prepared
1
31
3
from 3,5-dimethoxyphenol and isovaleryl chloride by means of GP1 as a white solid (1.70 g,
3
34
35
1
7
1%, mp 54–55°C ). Eluent for column chromatography: petroleum ether/EtOAc (30:1); H
36
37
NMR (500 MHz, DMSO-d ) δ 13.59 (1H, s, OH-2′), 6.09 (1H, s, H-5′), 6.07 (1H, s, H-3′), 3.84
6
38
3
40
41
(3H, s, OCH -4′), 3.79 (3H, s, OCH -6′ ), 2.78 (2H, d, J = 6.5 Hz, H-2), 2.08 (1H, m, H-3), 0.91
3
3
+
(
6H, d, J = 6.5 Hz, CH -4, CH -5); ESIMS m/z = 237 [M + H] .
3 3
42
4
4
1
-(2,4,6-Trimethoxyphenyl)-3-methylbutan-1-one (4). This compound was prepared from
45
4
47
1,3,5-trimethoxybenzene and isovaleryl chloride by means of GP1 as a colorless oil (1.23 g,
4
49
1
4
9%). Eluent for column chromatography: petroleum ether/EtOAc (30:1); H NMR (500 MHz,
50
5
DMSO-d ) δ 6.24 (2H, s, H-3′, H-5′), 3.79 (3H, s, OCH -4′), 3.72 (6H, s, OCH -2′, OCH -6′),
6
3
3
3
52
53
5
2
.48 (2H, d, J = 7.0 Hz, H-2), 2.03 (1H, m, H-3), 0.87 (6H, d, J = 7.0 Hz, CH -4, CH -5); ESIMS
3 3
5
56
+
m/z = 275 [M + Na] .
57
58
59
60
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 63
1
2
3
4
5
6
7
8
9
1
-(2-Hydroxy-4-methoxyphenyl)-3-methylbutan-1-one (5). This compound was prepared from
3
-methoxyphenol and isovaleryl chloride by means of GP1 as a colorless oil (1.95 g, 94%).
1
Eluent for column chromatography: petroleum ether/EtOAc (25:1); H NMR (500 MHz, DMSO-
1
1
d6) δ 12.81 (br s, OH- 2′), 7.82 (1H, d, J = 8.5 Hz, H-6′), 6.47 (1H, dd, J = 8.5, 2.0 Hz, H-5′),
12
1
14
15
6.44 (1H, d, J = 2.0 Hz, H-3′), 3.79 (3H, s, OCH ), 2.79 (2H, d, J = 6.5 Hz, H-2), 2.12 (1H, m,
3
+
H-3), 0.91 (6H, d, J = 6.5 Hz, CH -4, CH -5); ESIMS m/z = 209 [M + H] .
3
3
16
17
18
1
-(4-Hydroxyphenyl)-3-methylbutan-1-one (6). This compound was prepared from phenol and
19
2
2
2
23
isovaleryl chloride by means of GP1 as a white solid (1.00 g, 56%, mp 90–92°C ). Eluent for
1
column chromatography: petroleum ether/EtOAc (20:1); H NMR (500 MHz, DMSO-d ) δ 7.80
6
24
25
(2H, d, J = 8.0 Hz, H-2′, 6′), 6.81 (2H, d, J = 8.0 Hz, H-3′, 5′), 2.73 (2H, d, J = 6.5 Hz, H-2), 2.10
26
2
28
+
(1H, m, H-3), 0.90 (6H, d, J = 6.5 Hz, CH -4, CH -5); ESIMS m/z = 179 [M + H] .
3
3
29
30
1
-(2,4,6-Trihydroxyphenyl)pentan-1-one (7). This compound was prepared from
31
3
phloroglucinol and valeryl chloride by means of GP1 as a white solid (1.19 g, 57%, mp 130–
3
34
35
1
1
31°C ). Eluent for column chromatography: CH Cl /MeOH (30:1); H NMR (500 MHz,
2
2
36
37
DMSO-d ) δ 12.21 (2H, s, OH-2′, OH-6′), 10.30 (1H, s, OH-4′), 5.78 (2H, s, H-3′, H-5′), 2.96
6
38
3
40
41
(2H, t, J = 7.5 Hz, H-2), 1.55 (2H, m, H-3), 1.31 (2H, m, H-4), 0.87 (3H, t, J = 7.5 Hz, CH -5);
3
+
ESIMS m/z = 211 [M + H] .
42
4
4
1
-(2,4,6-Trihydroxyphenyl)ethanone (8). This compound was prepared from phloroglucinol
45
4
47
and acetyl chloride by means of GP1 as a white solid (0.70 g, 42%, mp 224°C ). Eluent for
4
49
1
column chromatography: CH Cl /MeOH (30:1); H NMR (500 MHz, DMSO-d ) δ 12.21 (br s,
2
2
6
50
5
OH-2′, OH-6′), 10.30 (br s, OH-4′), 5.78 (2H, s, H-3′, H-5′), 2.53 (3H, s, CH -2); ESIMS m/z =
3
52
53
5
+
1
69 [M + H] .
5
56
57
58
59
60
ACS Paragon Plus Environment

Page 29 of 63
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
General Procedure for Amidation for Compounds 9 and 10 (GP2). To a stirred suspension of
2
,4,6-trihydroxybenzoic acid (0.34 g, 2.0 mmol) and DMF (0.1 mL) in anhydrous CH Cl (30
2 2
mL) was added dropwise oxalyl chloride (0.51 g, 4.0 mmol) in CH Cl (10 mL). The reaction
2
2
1
1
12
13
14
15
16
17
18
19
20
2
mixture was stirred at room temperature for 1h and then evaporated in vacuo to give a clear oil.
This crude oil was dissolved in anhydrous THF (5 mL), and amine (5.0 mmol) in THF (5 mL)
was added dropwise in an ice-bath. After stirring at room temperature for 1h, the mixture was
filtered and the filtrate was concentrated in vacuo to dryness. The resulting product was obtained
by column chromatography.
2
23
24
25
N-Isopropyl-2,4,6-trihydroxybenzamide (9). This compound was prepared using
isopropylamine by means of GP2 as a white solid (0.31 g, 80%, mp 100–102°C ). Eluent for
26
2
28
1
column chromatography: CH Cl /MeOH (25:1); H NMR (500 MHz, DMSO-d ) δ 8.42 (1H, d,
2
2
6
29
30
J = 7.5 Hz, NH), 5.77 (2H, s, H-3′, H- 5′), 4.03 (1H, m, H-3), 1.15 (6H, d, J = 6.0 Hz, CH -4,
3
31
3
+
CH -5); ESIMS m/z = 212 [M + H] .
3
3
34
35
36
37
N-Butyl-2,4,6-trihydroxybenzamide (10). This compound was prepared using butylamine by
means of GP2 as a white solid (0.32 g, 73%, mp 73–75°C ). Eluent for column chromatography:
38
3
40
1
CH Cl /MeOH (25:1); H NMR (500 MHz, DMSO-d ) δ 12.62 (2H, s, OH-2′, OH-6′), 9.87 (1H,
2
2
6
41
42
s, OH-4′), 8.60 (1H, t, J = 5.5 Hz, NH), 5.77 (2H, s, H-3′, H-5′), 3.26 (2H, dd, J = 13.0, 6.5 Hz,
4
4
H-3), 1.47 (1H, m, H-4), 1.30 (1H, m, H-5), 0.89 (3H, t, J = 7.0 Hz, CH -6); LC-ESI-MS (m/z):
3
45
4
47
+
ESIMS m/z = 226 [M + H] .
48
49
50
51
52
53
5
5
General Procedure for Introduction of Aminoketone Unit for Compounds 11−14 (GP3). To a
stirred solution of 1,3,5-trimethoxybenzene (2.00 g, 12.0 mmol) in chlorobenzene (50 mL) was
added chloroacetonitrile (0.90 g, 12.0 mmol), and the reaction mixture was stirred at room
temperature with HCl gas bubbled in for 6 h, followed by filtration. A solution of the precipitate
56
57
58
59
60
ACS Paragon Plus Environment