Design, synthesis, and biological evaluation of tetrahydroisoquinolines derivatives as novel, selective PDE4 inhibitors for antipsoriasis treatment

Article abstract of DOI:10.1016/j.ejmech.2020.113004

Psoriasis is a kind of chronic inflammatory skin disorder, while the long-term use of conventional therapies for this disease are limited by severe adverse effects. Novel small molecules associated with new therapeutic mechanisms are greatly needed. It is known that phosphodiesterase 4 (PDE4) plays a central role in regulating inflammatory responses through hydrolyzing intracellular cyclic adenosine monophosphate (cAMP), making PDE4 to be an important target for the treatment of inflammatory diseases (e.g. psoriasis). In our previous work, we identified a series of novel PDE4 inhibitors with a tetrahydroisoquinoline scaffold through structure-based drug design, among which compound 1 showed moderate inhibition activity against PDE4. In this study, a series of novel tetrahydroisoquinoline derivatives were developed based on the crystal structure of PDE4D in complex with compound 1. Anti-inflammatory effects of these compounds were evaluated, and compound 36, with high safety, permeability and selectivity, exhibited significant inhibitory potency against the enzymatic activity of PDE4D and the TNF-α release from the LPS-stimulated RAW 264.7 and hPBMCs. Moreover, an in vivo study demonstrated that a topical administration of 36 achieved more significant efficacy than calcipotriol to improve the features of psoriasis-like skin inflammation. Overall, our study provides a basis for further development of tetrahydroisoquinoline-based PDE4 inhibitors against psoriasis.

Full text of DOI:10.1016/j.ejmech.2020.113004

Journal Pre-proof
Design, Synthesis, and Biological evaluation of Tetrahydroisoquinolines Derivatives
as Novel, Selective PDE4 Inhibitors for Antipsoriasis Treatment
Rui Zhang, Heng Li, Xianglei Zhang, Jian Li, Haixia Su, Qiukai Lu, Guangyu Dong,
Huixia Dou, Chen Fan, Zhanni Gu, Qianwen Mu, Wei Tang, Yechun Xu, Hong Liu
PII:
S0223-5234(20)30976-4
DOI:
https://doi.org/10.1016/j.ejmech.2020.113004
EJMECH 113004
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 September 2020
Revised Date: 3 November 2020
Accepted Date: 5 November 2020
Please cite this article as: R. Zhang, H. Li, X. Zhang, J. Li, H. Su, Q. Lu, G. Dong, H. Dou, C. Fan, Z. Gu,
Q. Mu, W. Tang, Y. Xu, H. Liu, Design, Synthesis, and Biological evaluation of Tetrahydroisoquinolines
Derivatives as Novel, Selective PDE4 Inhibitors for Antipsoriasis Treatment, European Journal of
Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.113004.
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Graphical Abstract

Title:
Design, Synthesis, and Biological evaluation of
Tetrahydroisoquinolines Derivatives as Novel, Selective
PDE4 Inhibitors for Antipsoriasis Treatment

Graphical Abstract

Highlights:
‹
‹
‹
51 tetrahydroisoquinolines derivatives as novel PDE4 inhibitors were designed,
synthesized, and evaluated.
The structure-activity relationship (SAR) of this novel series of compounds was
demonstrated.
The optimum compound 36 exhibited high safety, potency (PDE4D IC = 0.36
5
0
μM, TNF-α IC = 6.76 μM), permeability and selectivity in vitro.
5
0
‹
‹
The crystal structure of PDE4D in complex with compound 36 was solved.
Compound 36 exhibited superior therapeutic efficacy compared with calcipotriol
against the IMQ-induced murine psoriasis-like skin inflammation.

Design, Synthesis, and Biological evaluation of
Tetrahydroisoquinolines Derivatives as Novel, Selective
PDE4 Inhibitors for Antipsoriasis Treatment
a,b,#
b,c,#
b,d,#
a
b,d
b,c
Rui Zhang , Heng Li , Xianglei Zhang , Jian Li , Haixia Su , Qiukai Lu ,
a
b
,d
c
a
b,d
,b,c
Guangyu Dong , Huixia Dou , Chen Fan , Zhanni Gu , Qianwen Mu , Wei Tang* ,
,
b,d
,
a,b
Yechun Xu* and Hong Liu*
a
State Key Laboratory of Drug Research and CAS Key Laboratory of Receptor
Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555
Zu Chong Zhi Road, Shanghai, 201203, China.
b
University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing 100049,
China.
c
Laboratory of Anti-inflammation and Immunopharmacology, Shanghai Institute of
Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai
2
01203, China.
d
State Key Laboratory of Drug Research, Drug Discovery and Design Center,
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road,
Shanghai 201203, China.
#
These authors contributed equally to this study.
Corresponding Authors
*
W. Tang: Phone, +86-21-50806820; E-mail, tangwei@simm.ac.cn.
Y. Xu: Phone, +86-21-50801267; E-mail, ycxu@simm.ac.cn.
*
*
H. Liu: Phone, +86-21-50807042; E-mail, hliu@simm.ac.cn.

Abstract
Psoriasis is a kind of chronic inflammatory skin disorder, while the long-term use
of conventional therapies for this disease are limited by severe adverse effects. Novel
small molecules associated with new therapeutic mechanisms are greatly needed. It is
known that phosphodiesterase 4 (PDE4) plays a central role in regulating
inflammatory
responses through hydrolyzing intracellular cyclic adenosine
monophosphate (cAMP), making PDE4 to be an important target for the treatment of
inflammatory diseases (e.g. psoriasis). In our previous work, we identified a series of
novel PDE4 inhibitors with a tetrahydroisoquinoline scaffold through structure-based
drug design, among which compound 1 showed moderate inhibition activity against
PDE4. In this study, a series of novel tetrahydroisoquinoline derivatives were
developed based on the crystal structure of PDE4D in complex with compound 1.
Anti-inflammatory effects of these compounds were evaluated, and compound 36,
with high safety, permeability and selectivity, exhibited significant inhibitory potency
against the enzymatic activity of PDE4D and the TNF-α release from the
LPS-stimulated RAW 264.7 and hPBMCs. Moreover, an in vivo study demonstrated
that a topical administration of 36 achieved more significant efficacy than calcipotriol
to improve the features of psoriasis-like skin inflammation. Overall, our study
provides a basis for further development of tetrahydroisoquinoline-based PDE4
inhibitors against psoriasis.
Keywords: PDE4 inhibitors, tetrahydroisoquinoline, antipsoriasis

1
. Introduction
Psoriasis is a kind of chronic inflammatory skin disorder, the pathogenesis of
which is complicated and often linked with multiple factors such as autoimmune
activation, genetic susceptibility and environment [1]. It is generally accepted that the
disturbed interaction between innate and adaptive immune systems is the main cause
of psoriasis, which is closely related to the abnormal activation of keratinocytes,
dendritic cells, macrophages, and T cells [2-4]. Conventional treatments of psoriasis
consist of topical ointments (corticosteroids, emollients, vitamin D, etc.) and systemic
therapies (methotrexate, ciclosporin, acitretin, etc.) [5, 6].
However, the drug-drug interactions and severe adverse effects limit the
long-term use of these drugs. Moreover, phototherapy and photochemotherapy are
also considered as effective strategies for psoriasis patients, however, they are
time-consuming and can only be used for short-term prevention of disease
deterioration. Recently, biological agents, including infliximab, adalimumab and
secukinumab, have been emerged in clinical as efficacious options for psoriasis
treatment [7, 8]. Unfortunately, high cost, poor compliance and tolerance limited their
wide applications. Compared with the macromolecular antibodies, small molecules
possess several advantages [9, 10]. Apart from being cost-friendly and convenient for
administration, small molecules can enter cells and selectively inhibit the
inflammatory signaling pathways, thereby providing alternative therapeutic strategies
for patients who respond inadequately to conventional treatments or targeted
biological agents.
Phosphodiesterase 4 (PDE4), known as one of the 11 members of the PDE
super-family, is a cyclic adenosine monophosphate (cAMP) specific hydrolase, which
is widely expressed in numerous cells, including keratinocytes, endothelial cells,
hematopoietic cells, and nerve cells [11]. PDE4 plays a central role in inflammatory
responses through the regulation of intracellular cAMP levels and downstream protein
kinase A pathways [12], which makes PDE4 to be a primary target for the treatment of
inflammatory diseases like psoriasis [13-17]. Apremilast, approved in 2014 by FDA

and in 2015 by the European Commission, is the first oral PDE4 inhibitor used for
treating psoriasis and psoriatic arthritis (PsA), which inspires us to seek a new PDE4
inhibitor with higher safety and potency for anti-psoriasis treatment [18-20].
Berberine (BBR) is a kind of tetrahydroisoquinoline alkaloid with a proven
medicinal history in Chinese medicinal systems [21]. Clinical investigations on BBR
have demonstrated its broad spectrum of pharmacological effects, including
anti-inflammatory effects [22, 23]. Previously, we have built a chemical library
comprising intermediates, precursors and multiple analogues of berberine for
discovery of lead compounds. Based on this library, we obtained a series of novel
PDE4 inhibitors with tetrahydroisoquinoline skeleton utilizing the structure-based
drug design strategy, among which compound 1 showed high inhibitory activity
against PDE4D (IC50 = 0.14 μM) [24, 25]. In this study, a series of novel
tetrahydroisoquinoline derivatives were designed and synthesized on the basis of the
crystal structure of PDE4D-1. Two commonly used inflammatory cell models, mouse
monocyte/macrophage leukemia cell line (RAW 264.7) and human peripheral blood
mononuclear cells (hPBMCs), were used for in vitro anti-inflammatory activity
evaluation. The structure-activity relationship (SAR) of these new compounds was
explored and compound 36 was evaluated in the imiquimod-induced murine psoriasis
model.
2
. Results and discussion
2
.1. Design
To seek for a more potent PDE4 inhibitor, we got a deep insight into the binding
mode of compound 1 with PDE4D (Figure 1A, B). Based on our previous study, the
carbonyl group on the tetrahydroisoquinoline core of compound 1 oriented toward the
2
+
metal binding (MB) site but had no obvious interaction with the two metal ions (Zn
2
+
and Mg ) (Figure 1B, PDB code: 6IMO) [24, 26]. Thus, we replaced the formamide
group with larger groups to give compounds 4–20 (Figure 1C), expecting that the
extended groups could form stronger interactions with the two ions or surrounding

residues. In addition, 6,7-dimethyoxyl groups of 1 inserted into a hydrophobic pocket
(Q-pocket) and both oxygen atoms formed hydrogen bonds (H-bonds) with Q369
(Figure 1B), playing an indispensable role in binding of 1 to PDE4D. We noticed that
the cavity of the Q2 pocket (formed by Q369 and F372) was larger than that of the Q1
formed by N321 and I336) and it was not fully occupied by the 7-methyoxyl
(
substituent [26]. Thus, we introduced ethyoxyl or benzyloxyl groups and synthesized
compounds 21-28 (Figure 1C), in order to occupy the Q2 pocket while retain the
H-bond interaction. Finally, we turned our attention to the indole group. Even though
the crystal structure of the PDE4D-1 complex demonstrated that the terminal indole
ring was flexible and only formed few hydrophobic interactions with M273 and M357,
acting as a hydrophobic clamp (HC, Figure 1B), these interactions were essential for
it to maintain the PDE4 inhibitory activity. Therefore, we devoted more attention to
evaluating the influence of the “tail” on the binding affinity of the compounds with
PDE4D (Figure 1C), including the introduction of heterocycles (compounds 29-30),
the change of the linker between the tetrahydroisoquinoline core and the indole ring
(compounds 31-32), and the substituents on the indole ring (compounds 33-52).
Figure 1. Complex structure of compound 1 with PDE4D catalytic domain. (A)
Binding pose of compound 1 in the PDE4D catalytic pocket. Compound 1 and
PDE4D are shown as cyan sticks and yellow surface, respectively. (B) Detailed
interactions between compound 1 and PDE4D generated by Ligplot. Two H-bonds
formed between conserved Q369 and catechol group of compound 1 are shown as
black dash lines. (C) Schematic diagram for design of compounds 2-52 based on the
analysis of the crystal structure of compound 1 in complex with PDE4D.

It is worth noting that compound 1 possesses a chiral carbon, and our previous
study has revealed that the (S)-enantiomer is the dominant configuration with higher
PDE4 inhibitory activity. Thus, we only investigated the influence of configuration on
part of the compounds, while for the others, only (S)-enantiomers were synthesized
and evaluated. Altogether, 51 new tetrahydroisoquinoline derivatives were
synthesized and evaluated as novel PDE4 inhibitors.
2
.2. Synthesis of tetrahydroisoquinoline derivatives
The synthetic routes of compounds 1-28 are depicted in Scheme 1. Condensation
of 3,4-disubstutituted phenylethylamines (1a-c) and 2-(1H-indol-2-yl)-acetic acid
with HATU afforded the corresponding amide intermediates (3a-c). 3a-c were then
treated with POCl to afford the dihydroisoquinoline derivatives (4a-c). Racemic
3
tetrahydroisoquinolines intermediates 5a-c were obtained by reduction of 4a-c with
NaBH . While asymmetric reduction with Noyori catalyst gave the S- (6a-b) and R-
4
(7a) isomers respectively. Finally, 5a-c, 6a-b and 7a were reacted with HCOOEt,
anhydride, carboxylic acid, halogenide or acyl chloride to afford the target products.
a
Scheme 1. Synthetic Methods of Compounds 1-28

a
Reagents and conditions: (a) HATU, TEA, DCM, rt, 85%; (b) POCl , CH CN, Ar,
3
3
8
0 °C, 90%; (c) MeOH, NaBH , 90%; (d) HCOOEt, TEA, 65 °C, or (RCO) O, TEA,
4 2
DCM or RX, K CO , CH CN, 80 °C or RCOCl, DIPEA, DCM or RCOOH, HATU,
2
3
3
TEA, DCM, 80-88%; (e) La(OTf) , AgSbF , (R,R)-Noyori catalyst, HCOONa,
3
6
MeOH/H O (v/v) = 1:2, Ar, rt; (f) La(OTf) , AgSbF , (S,S)-Noyori catalyst,
2
3
6
HCOONa, MeOH/H O (v/v) = 1:2, Ar, rt.
2
The synthetic routes of compounds 29-32 are outlined in Scheme 2.
Condensation of 3,4-dimethoxyphenylethylamine with indicated carboxylic acids
gave the amide intermediates 9a-c. The following synthetic steps were similar to that
of compound 1.
a
Scheme 2. Synthetic Methods of Compounds 29-32

a
Reagents and conditions: (a) HATU, TEA, DCM, 85%; (b) POCl , CH CN, Ar, 80 °C,
3
3
9
0%; (c) MeOH, NaBH , 90%; (d) HCOOEt, TEA, 65 °C, 80-88%; (e) La(OTf) ,
4 3
AgSbF , (R,R)-Noyori catalyst, HCOONa, MeOH/H O (v/v) = 1:2, Ar, rt.
6
2
Compounds 33-41, 43-51 were synthesized using the same methods with
compounds 29-32, only by changing the starting materials (Scheme 3). For
compounds 42 and 52, a benzene ring was introduced through Suziki-coupling [27] to
give the amide intermediates 18a-b. The following steps are showed in Scheme 4.
a
Scheme 3. Synthetic Methods of Compounds 33-41, 43-51

a
Reagents and conditions: (a) HATU, TEA, DCM, 85%; (b) POCl , CH CN, Ar, 80 °C,
3
3
9
0%; (c) MeOH, NaBH , 90%; (d) HCOOEt, TEA 65 °C, 80-88%; (e) La(OTf) ,
4 3
AgSbF , (R,R)-Noyori catalyst, HCOONa, MeOH/H O (v/v) = 1:2, Ar, rt.
6
2
a
Scheme 4. Synthetic Methods of Compounds 42, 52
a
Reagents and conditions: (a) HATU, TEA, DCM, 85%; (b) Phenylboronic acid,
Pd(dbpf)Cl , K CO , CH CN/H O (v/v 1:1), Ar, 60 °C, 90%; (c) POCl , CH CN, Ar,
2
2
3
3
2
3
3
8
0 °C, 90%; (d) La(OTf) , AgSbF , (R,R)-Noyori catalyst, HCOONa, MeOH/H O
3 6 2
(v/v) = 1:2, Ar, rt; (e) HCOOEt, TEA, 65 °C, 80-88%.
2
.3. Structure−activity relationships of tetrahydroisoquinoline derivatives
Inhibitory activities against PDE4D of all the synthesized compounds mentioned
above were determined using a scintillation proximity assay (SPA) for a preliminary
SAR study [28] Meanwhile, TNF-α, known as an important pro-inflammatory

cytokine in the development of inflammation and autoimmune diseases, is mainly
produced in monocytes or macrophages. Mouse monocyte/macrophage leukemia cell
line (RAW 264.7) is one of the most commonly used inflammatory cell models, which
can release a variety of inflammatory factors such as TNF-α after induction and
activation by lipopolysaccharide (LPS). Therefore, the inhibition of TNF-α secretion
in LPS-stimulated RAW 264.7 cells was measured for further SAR analysis. In
addition, their CC values toward RAW 264.7 were also measured as preliminary
5
0
safety evaluation indicators.
First, the racemic compound 2 and the (R)-enantiomer 3 were synthesized to
testify the influence of configuration on PDE4D inhibitory activity. As expected, both
2
and 3 showed lower inhibitory activity compared with 1 (1, IC = 0.14 μM; 2, IC
50 50
=
0.39 μM; 3, IC = 1.81 μM; Table 1). Then compounds 4-20 were synthesized by
50
replacing the formamide with different amide or alkylamine. Only the compounds
with acetamide showed PDE4D inhibitory activity comparable with that of 1 (Table 1).
Furthermore, the (S)-enantiomer (5, IC = 0.89 μM) exhibited a slightly lower IC
5
0
50
value relative to that of the racemate (4, IC = 1.13 μM). However, the inhibition rate
5
0
of TNF-α production in RAW 264.7 cells of 4 and 5 (IC > 50 μM) was not desirable.
5
0
The analogues 21-28 with 7-ethyoxyl or -benzyloxyl substituents were then prepared
in order to to fully fill the Q2 pocket of PDE4D (Table 2). Even though compounds
2
1-23 (21, IC = 0.075 μM; 22, IC = 0.042 μM; 23, IC = 0.17 μM) displayed
50 50 50
enhanced PDE4D inhibitory compared with the 7-methyoxyl substituted analogues (2,
and 5), their inhibitory activities against the TNF-α secretion in RAW 264.7 cells
1
were unexpectedly poor. We speculated that these compounds may degraded rapidly
after entering cells, leading to their poor activity at cellular level. Compound 28 with
a 7-benzyloxyl substituent showed moderate bioactivity at both enzymatic and
cellular levels, with IC values of 3.57 μM and 36.7 μM respectively. Nevertheless,
5
0
its cytotoxicity was relatively high (CC = 53.4 μM), so it was not suitable for a
5
0
further study. On the other hand, none of compounds 25-27 exhibited satisfactory
inhibitory activity against PDE4D. Collectively, formamide or acetamide may be
favorable for improving the binding affinity of the compounds with the MB site of

PDE4D, while 7-methyoxyl or 7-ethyoxyl is necessary for keeping the potency of the
derivatives.
Table 1. Inhibitory Activities of Compounds 1-20 against PDE4D and TNF-α
Secretion in the LPS-stimulated RAW264.7 Cells
RAW264.7
TNF-α IC50
PDE4D
RAW264.7
1
Compds
R
Configuration
IC (μM)
CC (μM)
5
0
50
(μM)
1
2
3
4
5
6
7
8
9
S
Rac
R
0.14 ± 0.03
0.39 ± 0.05
1.81 ± 0.18
1.13 ± 0.21
0.89 ± 0.11
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
Rac
S
a
Rac
S
> 1 (-8.2%)
> 1 (-4.4%)
> 1 (-1.8%)
> 1 (-3.5%)
> 1 (34.7%)
> 1 (42.7%)
> 1 (28.0%)
Rac
S
1
0
Rac
Rac
S
11
1
1
2
3
Rac
> 1 (10.4%)
> 50
> 50

1
4
5
Rac
Rac
> 1 (5.0%)
> 1 (2.8%)
> 50
> 50
> 50
> 50
1
1
1
1
1
2
6
7
8
9
0
S
Rac
S
> 1 (6.1%)
> 1 (3.3%)
> 50
> 50
> 50
> 50
> 50
> 50
-
> 1 (10.2%)
> 1 (-14.7%)
> 1 (-18.9%)
b
Rac
S
-
-
-
a
b
PDE4D inhibition rates at 1 μM were presented in brackets.
Not measured
Table 2. Inhibitory Activities of Compounds 21-28 against PDE4D and TNF-α
Secretion in the LPS-stimulated RAW264.7 Cells
RAW264.7
PDE4D IC50
RAW264.7
CC (μM)
1
2
Compds
R
R
Configuration
TNF-α
(μM)
5
0
IC (μM)
5
0
21
22
23
24
Et
Et
Et
Et
Rac
S
0.075 ± 0.006
0.042 ± 0.012
0.17 ± 0.02
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
S
S
2.40 ± 0.17

a
25
26
27
28
S
S
> 1 (22.9%)
> 50
> 50
> 50
36.7
> 50
> 50
> 50
53.4
Et
Et
> 1 (19.4%)
> 1 (20.3%)
3.57 ± 0.07
S
Et
Bn
Rac
a
PDE4D inhibition rates at 1 μM were presented in brackets.
Subsequently, we paid our attention to the tail modification (Table 3). We
attempted to replace the indole ring with a benzene or thiophene ring. Unfortunately,
the potency of the resulting compounds toward PDE4D almost disappeared. The
PDE4D inhibition rate of 29 and 30 at 1 μM were only 6.3 % and 7.3 %, respectively.
The inhibitory activity of analogues (31, 32) with a linker between the
tetrahydroisoquinoline core and 2-position of indole also decreased drastically. Thus,
we retained the original linker and focused on investigating different substitutions on
the indole ring. Based on the aforementioned SAR study of the tetrahydroisoquinoline
core, we kept the formamide group and synthesized two series of derivatives,
compounds 33-42 with the 7-methyoxyl and compounds 43-52 with the 7-ethyoxyl.
Compounds 33-37 with a halogen-substituted indole all showed moderate to good
potency toward PDE4D. The derivatives with the 6-chloroindole (34, IC = 0.61 μM;
5
0
3
5, IC = 0.55 μM) showed comparable inhibitory activity against PDE4D compared
50
with the ones with the 4-chloroindole (33, IC = 0.36 μM) or 7-chloroindole (36, IC
50
5
0
=
0.36 μM). Both compounds 33 and 36 also exhibited moderate inhibitory activity in
cells (33, IC = 34.9 μM, 36, IC = 14.5 μM). By contrast, introduction of 5-bromo
5
0
50
gave the less potent derivative (37, IC = 1.03 μM). Even though 37 showed better
5
0
inhibitory activity against TNF-α release in the RAW264.7 cell than that of 33, its
cytotoxicity was unfortunately higher (CC = 38.0 μM). Subsequently, we changed
5
0
the bromine to other groups, like methyl (38), methoxyl (39), or phenyl (42). Among
these derivatives, 38 with the 5-methyl group displayed the most potent inhibitory
activity against PDE4D, with an IC value of 0.30 μM, while its inhibition rate of
5
0

TNF-α production was not satisfactory. Although compound 42 bearing a
-phenylindole showed lower inhibitory activity at the enzymatic level, its ability to
5
reduce the production of TNF-α was comparable to that of 36. Meanwhile, the CC50
value of 42 was also low. Analogue 40 with 2,5-disubsitituent on the indole ring
maintained the PDE4D inhibitory activity yet displayed weak TNF-α inhibition in
RAW264.7 cells. When the nitrogen of the indole ring was blocked by a methyl group,
the resulting compound 41 exhibited significant decreased ability in inhibiting
PDE4D. Among compounds 43-47, 43 with a 4-chloroindole and 46 with a
7
-chloroindole showed moderate inhibitory activity at both the enzymatic and cellular
levels, (43, PDE4D IC = 0.81 μM, TNF-α IC = 37.5 μM; 46, PDE4D IC = 0.63
5
0
50
50
μM, TNF-α IC50 = 14.8 μM), but both of them were less potent than their
7
-methyoxyl counterparts (33 and 36). Replacement of the 5-bromo (47) with a
-methyl (48), 5-methoxyl (49) or 5-phenyl (52) resulted in less potent inhibitors, of
5
which the PDE4D IC values were more than 1 μM. Compound 50 (IC = 0.63 μM)
5
0
50
with a 2-methyl-5-methoxyl exhibited the PDE4D inhibitory activity more potent than
that of 49 but less potent than that of 40 (IC = 0.61 μM).
5
0
Table 3. Inhibitory Activities of Compounds 29-52 against PDE4D and TNF-α
Secretion in the LPS-stimulated RAW264.7 Cells
RAW264.7
PDE4D IC50
RAW264.7
CC (μM)
2
3
Compds
R
R
Configuration
TNF-α
(μM)
5
0
IC (μM)
5
0
a
b
29
30
31
Me
Me
Me
S
S
> 1 (6.3%)
-
-
> 1 (7.3%)
-
-
Rac
> 1 (-24.9 %)
> 50
> 50

3
3
2
3
Me
Me
S
S
> 1 (-13.9%)
0.36 ± 0.04
> 50
34.9
> 50
> 50
Cl
N
H
3
4
5
Me
Me
Rac
S
0.61 ± 0.03
0.55 ± 0.04
> 50
> 50
> 50
> 50
3
3
6
Me
S
0.36 ± 0.02
14.5
> 50
37
38
39
Me
Me
Me
S
S
S
1.03 ± 0.10
0.30 ± 0.04
1.16 ± 0.19
29.2
> 50
> 50
38.0
> 50
> 50
4
4
0
1
Me
Me
S
0.61 ± 0.04
> 1 (23.5%)
> 50
> 50
> 50
> 50
Rac
4
4
2
3
Me
Et
S
S
> 1 (41.5%)
0.81 ± 0.07
13.8
37.5
16.2
> 50

4
4
5
Et
Et
Rac
S
> 1 (11.7%)
> 1 (21.7%)
-
-
-
-
4
4
6
Et
Et
S
0.63 ± 0.08
14.8
45.5
47
48
49
S
S
S
1.41 ± 0.21
> 1 (42.8%)
> 1 (30.7%)
> 50
> 50
> 50
> 50
> 50
> 50
Et
Et
5
0
1
S
S
0.63 ± 0.01
> 1 (25.3%)
> 50
> 50
Et
Et
5
-
-
5
2
S
> 1 (12.3%)
-
-
Et
a
b
PDE4D inhibition rates at 1 μM were presented in brackets.
Not measured
2
.4. Inhibition of TNF-α production from hPBMC by the selected compounds
It is known that inhibition of PDE4 would elevate intracellular cAMP levels,
which would subsequently reduce the production of TNF-α from human peripheral
blood mononuclear cells (hPBMCs) [29]. Consequently, an enzyme-linked
immunosorbent assay (ELISA) was used to measure the inhibition of TNF-α

production in the LPS-stimulated hPBMCs, for the compounds with moderate to good
inhibitory activity at both the enzymatic and cellular levels.
As shown in Table 4, most of the derivatives exhibited low inhibitory potency on
the TNF-α release in hPBMCs, with the inhibition rate lower than 25% at 10 μM.
Fortunately, compounds 23 and 36 performed more potently than compound 1 (IC =
5
0
3
3.34 μM), with IC values of 7.18 and 6.76 μM, respectively. Moreover, the results
50
of the Caco-2 permeability assay revealed that compound 36 possesses perfect
cell-penetrating ability. Its efflux ratio was about 3-fold lower than that of compound
1
(Table 5), which is in accord with its high TNF-α inhibitory activity. Taking a
consideration of the PDE4D enzymatic inhibitory activity and the inhibitory activity
of the TNF-α release in both RAW 264.7 cells and hPBMCs, we eventually chose
compound 36 for a further study of crystal structure determination, selectivity and in
vivo efficacy.
Table 4. Inhibitory Activities of Selected Compounds against the TNF-α Release
from the LPS-stimulated hPBMCs
O
R2
N
O
R1
R3
hPBMCs TNF-α
IC (μM)
1
2
3
Compds
R
R
R
5
0
1
Me
Et
33.34 ± 2.91
7.18 ± 2.17
2
3
8
a
2
Bn
> 1 (-6.2 %)

33
36
37
43
46
> 1 (23.3 %)
6.76 ± 2.08
> 1 (16.7 %)
> 1 (22.8 %)
> 1 (21.5 %)
Me
Me
Me
Et
Et
a
TNF-α inhibition rates at 1 μM were presented in brackets.
Table 5. Caco-2 Permeability of Compounds 1 and 36
Mean
Compds Flow Direction Recovery
%)
Mean
Fabs
(%)
a
Mean P
(
Efflux Permeability
app
b
-
6
c
d
10 cm/s)
Ratio
class
(
apical to basal
basal to apical
apical to basal
basal to apical
100
90
14.80
21.73
34.58
15.08
1
100
1.47
high
high
108
85
3
6
100
0.44
a
The Papp values in the A to B or B to A were calculated by the following formula:
Papp = (V /(area × time)) × ([drug]acceptor/[drug]initial donor) where, V = the volume in
A
A
the acceptor well (in this assay: 0.3 or 1.0 mL), area = the surface area of the
2
membrane (in this assay: 0.33 cm ), time = the total transport time in seconds (in this
b
c
assay: 5400 s). F : Human absorbed fraction. Efflux ratio (B to A/A to B ratio) =
abs
d
Papp (B to A)/Papp (A to B). Permeability class: high, F % > 70%; moderate, 30% <
abs
Fabs % < 70%; poor: Fabs % < 30%.

2
.5. Crystal structures of PDE4D in complex with compound 36 and apremilast
We solved the crystal structures of the catalytic domain of PDE4D in complex
with compound 36 and apremilast (Figure 2). The structure of the PDE4D-36
complex (PDB code: 7CBJ) showed that the binding conformation of compound 36 in
the ligand binding pocket of PDE4D was similar to that of compound 1. The catechol
group formed two H-bonds with the conserved glutamine (Q369) and the π-π stacking
interactions between F372 and tetrahydroisoquinoline scaffold were maintained too.
While in compared with compound 1, the installation of a chlorine atom at the
7
-position of the indole ring led this ring to flip away and bury into a new
hydrophobic pocket formed by residues M273, H276, L319, and I376. As a result, the
indole ring of compound 36 was stabilized, as the density map of it was complete.
However, the inhibitory activity of compound 36 against PDE4D was weaker than
those of compound 1 and apremilast (IC values of 0.14 µM and 0.077 µM for the
5
0
two compounds, respectively). It is consisted with our previous finding that
interactions between the indole ring and hydrophobic clamp was important to improve
the inhibitory activity. In structurally, the binding mode of apremilast with PDE4D
(PDB code: 7CBQ) was quite different from that of compound 36. Except the
H-bonds with Q369, a direct H-bond with H160 in the M-pocket and a water
molecule mediated H-bond with P356 in the H-pocket were formed (Figure 2D), and
the latter may account for the high inhibitory activity of apremilast. In contrast, the
carbonyl group of compounds 1 and 36 oriented to the solvent accessible region and
made little interactions with residues within the M- and H-pockets.

Figure 2. Crystal structures of PDE4D in complex with compounds 1, 36 and
apremilast. (A) Superimposed binding modes of three compounds with PDE4D.
Compounds 1, 36 and apremilast are shown as cyan, yellow and pink sticks,
respectively. The PDE4D catalytic domain is shown as gray surface. (B-D) Detailed
interactions of 1 (B), 36 (C) or apremilast (D) with PDE4D, respectively. H-bonds are
shown as grey dash lines.
2
.6. Selectivity of compound 36 against other PDE isoforms
Selectivity of compound 36 against other PDE isoforms was examined before the
in vivo study. As shown in Tables 6 and S2, compound 36 displayed > 13-fold
selectivity versus PDE1-3,6, and > 55-fold selectivity versus PDE5,7-9,11, indicating
its high selectivity for PDE4.

Table 6. Selectivity of Compound 36 to 11 PDE Enzymes
PDEs
PDE1A
PDE2A
PDE3A
PDE4A
PDE5A
IC (µM)
Selectivity index
5
0
a
a
a
a
a
> 5
> 5
> 5
> 13
> 13
> 13
-
0.36
> 20
> 5
> 55
> 13
> 55
> 55
> 55
-
a
a
PDE6C
PDE7A
PDE8A
PDE9A
> 20
> 20
> 20
76.5%
> 20
a
a
a
a
b
PDE10A
PDE11A
> 55
a
b
Commercial recombinant human full-length PDEs (BPS Bioscience, Inc.).
PDE10A inhibition rate at 5 μM
2
.7. Compound 36 attenuated IMQ-induced murine psoriasis-like skin
inflammation.
Encouraged by the enzymatic inhibitory activity and anti-inflammatory effect of
compound 36, pharmacological effects of compound 36 were further evaluated in the
following in vivo investigations. Since PDE4 are widely expressed in numerous cells
[
11], oral administration of PDE4 inhibitors may cause some side effects, the most
common ones of which are nausea and diarrhea [20]. Besides, compound 36 displayed
poor pharmacokinetics properties after p.o. administration in rats, with the
bioavailability of only 0.75% (Table S3). Given the preferable transdermal absorption
characteristics (Table S4), compound 36 with a topical treatment strategy was
conducted in the imiquimod (IMQ)-induced murine psoriasis model, which could
extensively mimic the clinical manifestations of psoriasis patients [30, 31]. During
consecutive application of the IMQ cream on BALB/c mice for 7 days, obvious loss
of body weight was observed, which could be largely reversed upon the topical

treatment of compound 36 (Figure 3A). Moreover, as presented in Figure 3B, C,
severe skin lesions occurred on the back skins following the IMQ application,
including scales, thickness and erythema. Therapeutically, compound 36 significantly
attenuated the experimental symptoms in a dose-dependent manner during the
treatment, which was comparable to the calcipotriol-treated group (Figure 3B, C).
Collectively, these results demonstrated that compound 36 exhibited the therapeutic
potential to improve the features of psoriasis-like skin inflammation.
Figure 3. Topical treatment of compound 36 dose-dependently ameliorated the
experimental murine psoriatic inflammation. (A) Body weights were monitored
daily as the percentage of body weight at day 0. (B) The represent phenotypic pictures
of the back skins in each group. (C) The Psoriasis Area and Severity Index (PASI)
scores, including scales, thickness, and erythema. Data were shown as mean ± SEM;
n = 5 mice per group; * p < 0.05, ** p < 0.01, and *** p < 0.001, compared with the
vehicle group.
3
. Conclusions
Small molecules with minimal side effects and high potency for anti-psoriasis
treatment are greatly needed. Based on the previously reported compound 1 with
moderate PDE4D inhibitory activity, a series of novel tetrahydroisoquinoline
derivatives were designed and synthesized. Set out from the crystal structure of the
PDE4D-1 complex, we investigated the structure−activity relationship on substituents

supposed to interact with the metal binding site, Q2 pocket and hydrophobic clamp of
the enzyme, respectively. The structure-guided optimization resulted in 12 novel
PDE4D inhibitors (2, 5, 21-23, 33, 36, 38, 40, 43, 46, and 50) with IC values in the
5
0
sub-micromolar range. Among them, compound 36 was found to be a safe, permeable
and selective PDE4 inhibitor with enhanced inhibition of the TNF-α secretion in the
LPS-stimulated RAW 264.7 and hPBMCs. The crystal structure of compound 36 in
complex with PDE4D was solved and compared with those of PDE4D-1 and
PDE4D-Apremilast. Moreover, topical administration of 36 exhibited superior
therapeutic efficacy compared with calcipotriol, the first-line medication for psoriasis,
against the IMQ-induced murine psoriasis-like skin inflammation. Overall, our study
lays a solid foundation for further development of small-molecular PDE4 inhibitors
for effective anti-psoriasis treatment.
4
4
4
. Experimental sections
.1. Chemistry
.1.1. Materials and methods. All of the reagents (chemicals) were purchased from
commercial chemical reagent company and directly used without further purification.
Analytical thin layer chromatography (TLC) was performed on HSGF 254. All
products were characterized by their NMR, LR-MS and HR-MS. The purity of the
target compounds was determined by HPLC (Agilent Eclipse XDB-C18, 5 μm, 4.6
mm × 150 mm, 30 °C, UV 214/230/254/280 nm, flow rate = 1.0 mL/min) with
aqueous CH CN or CH OH (40−70%) for 20 min, and the peak areas were calculated
3
3
at 230 nm.
4
.1.2. Synthesis of compounds 1-52
S)-1-((1H-indol-3-yl)methyl)-6,7-dimethoxy-3,4-dihydroisoquinoline-2(1
H)-carbaldehyde (1). A solution of 6a (300 mg, 0.93 mmol) and trimethylamine
TEA, 140 μL, 1.02 mmol) in ethyl formate (5.0 mL) was kept stirring for 6 h at
5 °C. The mixture was then diluted with EA. After being washed with saturated
NH4Cl and NaCl, the organic layer, the organic layer was dried over Na SO The
(
(
5
2
4.

solvent was removed under vacuum. The residue was purified by column
chromatography (PE/EA v: v = 1:1) to give compound 1 (245 mg, 75%) as a white
o
1
solid. HPLC purity: 98.66 %. m.p.: 88-90 C. H NMR (500 MHz, DMSO-d ) δ 10.94,
6
1
0.86 (2 × s, 1H), 8.08, 7.48 (2 × s, 1H), 7.68, 7.57 (2 × d, J = 7.9 Hz, 1H), 7.35, 7.33
2 × d, J = 8.2 Hz, 1H), 7.18 – 6.94 (m, 3H), 6.91, 6.67 (2 × s, 1H), 6.73, 6.44 (2 × s,
H), 5.45, 4.82 (2 × dd, J = 10.4, 4.3 Hz, 1H), 4.16, 3.69 (2 × ddd, J = 13.0, 6.2, 2.5
Hz, 1H), 3.73, 3.70 (2 × s, 3H), 3.73, 3.49 (2 × s, 3H), 3.43 – 3.37, 3.27 – 3.04 (m,
(
1
1
3
3
1
1
1
H), 2.82 – 2.59 (m, 2H). C NMR (126 MHz, DMSO-d ) δ 161.4, 160.6, 147.7,
6
47.4, 147.2, 146.8, 136.2, 136.1, 128.6, 127.8, 127.6, 127.1, 125.5, 125.4, 124.4,
23.8, 120.9, 120.8, 118.6, 118.4, 118.3, 112.0, 111.9, 111.4, 111.3, 110.9, 110.7,
10.6, 110.4, 56.5, 55.6, 55.5, 55.4, 55.2, 50.1, 33.0, 32.4, 31.4, 28.7, 27.2. ESI-MS
+
+
+
m/z 351.0 [M+H] . HR-MS: (ESI, m/z) calcd for C H N O [M+H] 351.1703,
2
1
23
2
3
found 351.1704.
-((1H-indol-3-yl)methyl)-6,7-dimethoxy-3,4-dihydroisoquinoline-2(1H)-carba
ldehyde (2). White solid. This compound was prepared using a similar method of
1
o
1
compound 1, by replacing 6a with 5a. HPLC purity: 99.17 %. m.p.: 93-95 C. H
NMR (400 MHz, DMSO-d ) δ 10.92, 10.83 (2 × s, 1H), 8.08, 7.46 (2 × s, 1H), 7.69,
6
7
.58 (2 × d, J = 7.8 Hz, 1H), 7.39 – 6.94 (m, 4H), 6.92, 6.67 (2 × s, 1H), 6.73, 6.45 (2
s, 1H), 5.45, 4.81 (2 × dd, J = 10.4, 4.2 Hz, 1H), 4.22 – 4.11, 3.69 – 3.65 (2 × m,
H), 3.73, 3.70 (2 × s, 3H), 3.73, 3.49 (2 × s, 3H), 3.42 – 3.03 (m, 3H), 2.80 – 2.61 (m,
×
1
2
1
1
5
1
3
H). C NMR (126 MHz, CDCl ) δ 161.6, 161.5, 148.3, 147.8, 147.7, 147.3, 136.4,
3
36.1, 127.9, 127.9, 127.8, 126.8, 126.1, 125.2, 123.6, 123.0, 122.4, 122.1, 119.8,
19.6, 119.1, 118.1, 112.0, 111.6, 111.4, 111.2, 111.0, 110.6, 110.0, 57.4, 56.1, 56.0,
+
5.9, 55.6, 51.2, 40.8, 34.0, 33.3, 31.8, 29.2, 27.8. ESI-MS m/z 351.1 [M+H] .
+
+
HR-MS: (ESI, m/z) calcd for C H N O [M+H] 351.1703, found 351.1697.
2
1
23
2
3
(
R)-1-((1H-indol-3-yl)methyl)-6,7-dimethoxy-3,4-dihydroisoquinoline-2(1
H)-carbaldehyde (3). White solid. This compound was prepared using a similar
method of compound 1, by replacing 6a with 7a. HPLC purity: 99.46 %. m.p.:
o
1
1
59-161 C. H NMR (600 MHz, DMSO-d ) δ 10.89, 10.88 (2 × s, 1H), 8.08, 7.46 (2
6
×
s, 1H), 7.70, 7.57 (2 × d, J = 7.9 Hz, 1H), 7.34, 7.33 (d, J = 8.1 Hz, 1H), 7.13 – 6.92

(m, 3H), 6.92, 6.67 (2 × s, 1H), 6.73, 6.45 (2 × s, 1H), 5.46 – 5.44, 4.83 – 4.80 (2 × m,
1
3
H), 4.19 – 4.14, 3.71 – 3.65 (2 × m, 1H), 3.73, 3.70 (2 × s, 3H), 3.73, 3.49 (2 × s,
1
3
H), 3.45 – 3.35, 3.29 – 3.04 (m, 3H), 2.84 – 2.61 (2 × m, 2H). C NMR (151 MHz,
DMSO-d ) δ 161.4, 160.6, 147.7, 147.4, 147.2, 146.8, 136.2, 136.1, 128.5, 127.8,
6
1
1
5
27.6, 127.1, 125.5, 125.3, 124.4, 123.8, 120.9, 120.8, 118.6, 118.5, 118.4, 118.3,
12.0, 111.8, 111.4, 111.3, 110.8, 110.7, 110.6, 110.4, 56.5, 55.6, 55.5, 55.4, 55.1,
+
4.9, 50.1, 33.0, 32.4, 31.4, 28.7, 27.2. ESI-MS m/z 351.1 [M+H] . HR-MS: (ESI,
+
+
m/z) calcd for C H N O [M+H] 351.1703, found 351.1707.
2
1
23
2
3
1
-(1-((1H-indol-3-yl)methyl)-6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)
-
yl)ethan-1-one (4). To a solution of 5a (150 mg, 0.47 mmol) and N,
N-Diisopropylethylamine (DIPEA, 230 μL, 1.40 mmol) in DCM (5 mL) was slowly
o
added acetyl chloride (40 μL, 0.56 mmol) at 0 C. The mixture was then stirred at
room temperature for 2 h. After being washed with saturated NH Cl and brine, the
4
solvent was dried over Na SO and concentrated under vacuum. The residue was
2
4
purified by column chromatography (PE/EA v: v = 1:1) to afford compound 4 as a
o
1
white solid (150 mg, 89%). HPLC purity: 99.64 %. m.p.: 155-157 C. H NMR (400
MHz, CDCl ) δ 8.89, 8.59 (2 × s, 1H), 7.64, 7.59 (2 × d, J = 7.7 Hz, 1H), 7.38, 7.31 (2
3
×
m, 1H), 7.22 – 7.04 (m, 2H), 6.87, 6.83 (2 × d, J = 2.4 Hz, 1H), 6.64, 6.20 (2 × s,
H), 6.58 (s, 1H), 5.83 – 5.81, 4.94 – 4.91 (2 × m, 1H), 4.82 – 4.79, 3.24 – 3.17 (2 ×
m, 2H), 3.87, 3.83 (2 × s, 3H), 3.80, 3.46 (2 × s, 3H), 3.67 – 3.63, 3.51 – 3.47 (2 × m,
1
1
1
1
1
5
H), 3.34 – 3.27 (2 × m, 1H), 2.93 – 2.79 (2 × m, 1H), 2.74 – 2.64 (2 × m, 1H), 2.15,
1
3
.53 (2 × s, 3H). C NMR (126 MHz, CDCl ) δ 170.2, 169.6, 148.1, 147.6, 147.3,
3
47.0, 136.3, 136.2, 129.1, 128.4, 128.0, 127.2, 126.6, 125.6, 123.7, 123.1, 122.1,
21.8, 119.7, 119.4, 119.2, 117.9, 112.3, 111.7, 111.6, 111.4, 111.1, 110.9, 110.2,
7.9, 56.1, 56.0, 55.9, 55.6, 53.1, 41.7, 34.9, 32.7, 32.0, 28.7, 28.0, 22.2, 21.1.
+
+
+
ESI-MS m/z 365.1 [M+H] . HR-MS: (ESI, m/z) calcd for C H N O [M+H]
2
2
25
2
3
3
65.1860, found 365.1863.
S)-1-(1-((1H-indol-3-yl)methyl)-6,7-dimethoxy-3,4-dihydroisoquinolin-2(
H)-yl)ethan-1-one (5). White solid. This compound was prepared using a
(
1
similar method of compound 4, by replacing 5a with 6a. HPLC purity: 99.83 %.