Discovery of furyl/thienyl β-carboline derivatives as potent and selective PDE5 inhibitors with excellent vasorelaxant effect

Article abstract of DOI:10.1016/j.ejmech.2018.09.028

Based on our previous studies and predictive docking results, furans and thiophenes were introduced to the privileged tetrahydro-β-carboline scaffold to generate more potent and selective PDE5 inhibitors. A total of 66 novel furyl/thienyl tetrahydro-β-carboline derivatives were designed, synthesized and evaluated for PDE5 inhibition. Tetrahydro-β-carboline-piperazinedione 19f and tetrahydro-β-carboline-hydantoin 26b with optimized pendant 5-ethylfuran/5-ethylthiophene were identified as the most potent PDE5 inhibitors, and showed high selectivity towards PDE5 versus other PDE isozymes, especially PDE6 and PDE11. Further vasorelaxant activity assessments revealed that these PDE5 inhibitors also exhibited significant angiectasis on the norepinephrine-precontracted 3^rd-order mesenteric arteries (110–150 μm) via NO–sGC–cGMP pathway, implying their further application for the treatment of vascular diseases.

Full text of DOI:10.1016/j.ejmech.2018.09.028

Accepted Manuscript
Discovery of furyl/thienyl β-carboline derivatives as potent and selective PDE5
inhibitors with excellent vasorelaxant effect
Hongbo Zheng, Yifeng Wu, Bin Sun, Chuanle Cheng, Yanan Qiao, Yuehua Jiang,
Shengtian Zhao, Zhiyu Xie, Jing Tan, Hongxiang Lou
PII:
S0223-5234(18)30800-6
10.1016/j.ejmech.2018.09.028
EJMECH 10739
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 June 2018
Revised Date: 23 August 2018
Accepted Date: 10 September 2018
Please cite this article as: H. Zheng, Y. Wu, B. Sun, C. Cheng, Y. Qiao, Y. Jiang, S. Zhao, Z. Xie, J.
Tan, H. Lou, Discovery of furyl/thienyl β-carboline derivatives as potent and selective PDE5 inhibitors
with excellent vasorelaxant effect, European Journal of Medicinal Chemistry (2018), doi: https://
doi.org/10.1016/j.ejmech.2018.09.028.
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Eur. J. Med. Chem.
ACCEPTED MANUSCRIPT
Discovery of Furyl/Thienyl β-Carboline Derivatives as Potent and Selective
PDE5 Inhibitors with Excellent Vasorelaxant Effect.
Hongbo Zheng,a Yifeng Wu,a Bin Sun,a,b Chuanle Cheng,a Yanan Qiao,a Yuehua Jiang,c Shengtian Zhao,c
Zhiyu Xie,a Jing Tan,a Hongxiang Loua *
aDepartment of Natural Products Chemistry, Key Laboratory of Natural Products & Chemical Biology,
Ministry of Education, School of Pharmaceutical Sciences, Shandong University, Jinan, 250012, China.
bNational Glycoengineering Research Center, Shandong University, Jinan, 250012, China.
cCentral Laboratory, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan
250014, China.
Abstract
Based on our previous studies and predictive docking results, furans and thiophenes were introduced
to the privileged tetrahydro-β-carboline scaffold to generate more potent and selective PDE5
inhibitors. A total of 66 novel furyl/thienyl tetrahydro-β-carboline derivatives were designed,
synthesized and evaluated for PDE5 inhibition. Tetrahydro-β-carboline-piperazinedione 19f and
tetrahydro-β-carboline-hydantoin 26b with optimized pendant 5-ethylfuran/5-ethylthiophene were
identified as the most potent PDE5 inhibitors, and showed high selectivity towards PDE5 versus
other PDE isozymes, especially PDE6 and PDE11. Further vasorelaxant activity assessments
revealed that these PDE5 inhibitors also exhibited significant angiectasis on the
norepinephrine-precontracted 3rd-order mesenteric arteries (110–150 µm) via NO–sGC–cGMP
pathway, implying their further application for the treatment of vascular diseases.
Keywords: β-Carboline derivatives; PDE5 inhibitors; Vasorelaxant effect; Structure-activity
relationship.

Graphic Abstract
ACCEPTED MANUSCRIPT

1. Introduction
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The ubiquitous secondary messengers cyclic adenosine monophosphate (cAMP) and cyclic
guanosine monophosphate (cGMP) play important roles in regulating many biological processes and
mediating responses to diverse cellular stimulations. [1] cGMP is also a well-known substrate in
regulating the relaxation of blood vessels. [2] Hydrolysis of either cAMP or cGMP is dependent on
phosphodiesterases (PDEs), a superfamily coded by 21 identified genes and have been divided into
11 subtypes (PDE1–11). [3]
Phosphodiesterase 5 (PDE5) is an enzyme specific catalyzing the hydrolysis of cGMP and is
widely expressed in various tissues, such as platelets, vascular smooth muscle, brain, lung, heart,
kidney, and corpus cavernosum. The PDE5 inhibitors have been applied for decades to treat male
erectile dysfunction (MED) and pulmonary arterial hypertension (PAH), for example,
FDA-approved drugs, sildenafil, vardenafil, tadalafil and avanafil (Fig.1). Recently, the PDE5
inhibitors were also proved to be effective for cognitive improvement in the treatment of
neurodegenerative diseases (e.g. Alzheimer’s disease, AD), possibly due to their vasorelaxant effects.
[4-6] As reported, inhibition of PDE5 could result elevation of cGMP level and further promote
vasodilation via NO–sGC–cGMP pathway, especially in penile, pulmonary and cerebral vasculatures.
[6, 7] In addition, many PDE5 inhibitors are also being investigated as therapeutics for cancer, stroke,
Raynaud’s disease and overactive bladder. [8-11] Therefore, searching for more potent and selective
PDE5 inhibitors is still much of importance.
Figure 1. Examples of commercial PDE5 inhibitors. (PDE5 potency and selectivity were listed as literature reports
[12-15])

[Figure 1 here]
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Tadalafil (Cialis®), the 3rd FDA-approved PDE5 inhibitor, was found to be both highly potent
on PDE5 enzyme and selective versus other phosphodiesterases, especially for PDE6 (up to
1000-fold). As can be seen from the structures in Fig.1, tadalafil is structurally distinctive with the
tetrahydro-β-carboline pharmacophore. Moreover, many β-carboline-derived molecules, such as
compounds 1–4 shown in Fig.2, were also reported with significant PDE5 potency, and high
selectivity for other PDE subtypes. [16-18] β-Carboline analogues have been well-documented
privileged structures for developing potent and selective PDE5 inhibitors.
Figure 2. Examples of β-carboline analogues 1–4. [16-18]
[Figure 2 here]
Recently, we reported the discovery of series of furyl/thienyl tetrahydro-β-carbolines and
pyrroloquinolones as PDE5 inhibitors, based on the natural β-carboline alkaloid perlolyrine (5a). [19,
20] Compounds 6–7 with pendant furan/thiophene exhibited excellent PDE5 inhibitory activity but a
relative low selectivity against PDE6 and PDE11. Meanwhile, we also noticed that the furyl/thienyl
moieties were becoming more popular in the structures of recent discovered PDE5 inhibitors (Fig. 3,
compound 8–13). [5, 21-23] To our best known, very few α-furyl/thienyl β-carboline analogues
have been investigated for the PDE5 activity, as well as their structure-activity relationships,
druggabilitiy et al. Therefore, introducing the furans and thiophenes to the privileged β-carbolines
scaffolds will provide a small molecular library of novel β-carboline derivatives for further PDE5
inhibitory activity investigation. This promising approach may lead to the discovery of novel
β-carboline analogues with high PDE5 inhibitory activity, well PDE isozymes selectivity and good
vasorelaxant activity.
In the present manuscript, several privileged β-carboline-derived scaffolds (Series A-G, Fig.4)
were selected to introduce pendant furans or thiophenes for PDE5 inhibitors discovery. The

designed compounds were synthesized and then tested for PDE5 potency and PDE subtypes
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selectivity. In addition, these compounds also underwent further in vitro vasorelaxant activity
assessment to evaluate their therapeutic potential for the treatment of vascular diseases.
Figure 3. Furyl and thienyl PDE5 inhibitors 5–13. [5, 21-23]
[Figure 3 here]
2. Results and discussion
2.1. Design and Chemistry
According to PDE5/tadalafil complexes and several docking modeling of β-carbolines in PDE5
catalytic site [24, 25], the unambiguous binding mode that comprise major interaction types with
PDE5 enzyme could be observed to explain the high potency of β-carboline scaffold. There is one
key H-bond between the indole core nitrogen atom and Gln817, while adequate hydrophobic
contacts with Q pocket residues can also be detected, including interactions between: a) immutable
indole fragment and Q pocket residues Phe 820; b) alternative α-substituents and Q2 pocket formed
by Ala 779, Leu 804, Ile 813 and Met 816. On the other hand, the structural advantages of
β-carbolines for their PDE subtypes selectivity were also been explained by molecular docking. [26,
27] For the purpose of discovering novel chemical entities as more potent and selective PDE5
inhibitors, several privileged β-carboline-derived scaffolds (series A-G, as shown in Fig. 4) were
selected to introduce our previously reported furan/thiophene moieties as α-substituents, because
the furan/thiophene fragments could occupy Q2 pocket well which is possibly due to their small

size and relatively higher lipophilicity (eg. clog p = 2.816 and 2.346 for 5-ethylthiophene and
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5-ethylfuran, respectively). [20, 21]
Figure 4. Design of diverse β-carboline-derived PDE5 inhibitors.
[Figure 4 here]
Our hypotheses were also confirmed by predictive binding mode of β-carbolines in the catalytic
pocket of PDE5. As given in Fig. 5, the docked poses of several representative compounds were
listed and their key interactions with PDE5 were recognized as similar pattern: i) all the β-carboline
analogues exhibit crucial monodentate H–bonding interaction with residue Gln817; ii) the
α-substituted furyl and thienyl rings are principally involved in hydrophobic interaction with the Q2
pocket residues; iii) these derivatives could make π-π stacking interactions with Phe820. With these
essential interactions, the β-carboline derivatives could tightly bind to the PDE5 enzyme and may
showed high PDE5 potency. Furthermore, considering the differences between PDE subtypes, we
also proposed that introducing furans or thiophenes as α-substituents may lead to improved
selectivity for PDE5 versus other PDEs. On the basis of the above molecular design, we then began
the systematic synthesis of the target compounds.

ACCEPTED MANUSCRIPT
Figure 5. Crystal structure of PDE5A in complex with Tadalafil (upper-left, cyan); Predictive binding mode of
series A-G compounds within PDE5 catalytic site, PDB code 1xoz [25]. (upper-middle, series A-B compounds 19f
(marine), 26b (paleyellow); upper-right, series C compounds 30a (purple), 30b (vermeil)); down-left, series D
compounds 6 (navy), 35c (orange); down-middle, series E compounds 36h (blue), 40a (yellow); down-right, series
F-G compounds 41 (aquamarine), 37a (meganta).
[Figure 5 here]
The general synthetic procedures for the β-carboline derivatives were illustrated in scheme 1–4.
Enantiopure D–tryptophan 14 was employed to construct the tetrahydro-β-carboline scaffold via
Pictet-Splenger reaction. The intermediate 15 then underwent esterification, acylation and
cyclization to form the piperazinedione structure 18. The condition for oxidative coupling of 18 with
diverse potassium trifluoroborate derivatives was also established by employing excess TEMPO (4
eq.) as oxidant (scheme 1, and scheme S1 Supporting Information), and under such oxidative
condition, the diastereoisomers 19a–f, 20a–f were prepared in acceptable yields. Alternatively, the
L–tryptophan (L)-14 was used as starting material, and compounds 21c,f, 22c,f were afforded
according to similar synthetic procedure of compounds 19a–f, 20a–f. Next, starting from
(D)-ester-14, the 3-substituted thiophenes derivatives 20g–h, 21g–h were synthesized according to
previously reported methods. [21]
Tetrahydro-β-carboline (3S)-16 was also applied to afford hydantoin derivatives 25-Et and
25-Bu by treatment with ethyl/butyl isocyanate in refluxing in butanone (Scheme 2). Unexpectedly,

following oxidative C–H functionalization exhibited high diastereo-selectivity, and only
ACCEPTED MANUSCRIPT
trans-isomers 26a–c,e were isolated (dr > 20 : 1). Accordingly, a series of 1,3-disubstituted
tetrahydro-β-carboline 23-Deri (as mixture of cis-and trans-isomers) were prepared via a modified
Pictet-Spengler reaction, from both D– and L- tryptophan (3R and 3S, respectively). These
intermediates were then subjected to cyclization with ethyl/butyl isocyanates to form the hydantoin
ring, under the condition of refluxing in butanone. All the desired enantiomers and diastereoisomers
26a–e, 27b–e, 28b, 28c, 28e, 29b, 29c, 29e were provided as illustrated in scheme 1. [17] Changing
solvent and temperature led to the acyclic tetrahydro-β-carboline products 30a,b.
Similarly, starting from the commercial available tryptamine 31, tetrahydro-β-carboline
substrates 32A–D bearing heteroaromatic biaryls or acyls were synthesized smoothly as our
previous reports. [20, 28] These compounds were subsequently submitted to the TEMPO-mediated
C–H functionalization with corresponding potassium trifluoroborate derivatives, and the
α-furayl/thienyl β-carbolines 33a–g, 34a,b, 35a–c were prepared in good to excellent yield (Scheme
3). After hydrolysis with NaOH, compound 35c was also obtained.
Finally, most β-carboline compounds were submitted to DDQ oxidation for further modification.
Interestingly, diverse β-carboline substrates may lead to structurally distinctive products in the
presence of DDQ oxidative condition. As shown in scheme 4, when starting from the N-bipyridine
substrates 33a–g, 6, only 2,3-disubstitued indoles 36a–h can be obtained. However, once replaced
the bipyridine with pyrimidine, for example 34a,b, carbolinones 37a,b then became the major
products, along with the ring-opening products 38a,b. In addition, compounds 19f, 22f, 26c, 29c
also underwent this condition smoothly to yield the indole derivatives 39a,b, 40a,b, and, moreover,
fully aromatic β-carboline product 41 were obtained as well. Unfortunately, furyl derivatives 19c,
21c, 26b and 29b decomposed quickly in the presence of DDQ and no corresponding products could
be obtained.
The determination of absolute configurations is mainly relying on NMR analysis. Furthermore,
the X-ray single crystallography structures of compounds 19c, 19f, 21c, 26b–c, 27c, 28c, 29c were
also displayed in Fig. 6.

Scheme 1. Preparation of series A β-carbolines derivitives , tetrahydro-β-carboline-piperazinediones,.a
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O
O
O
O
OMe
NMe
O
OR
(R)
OH
Cl
a
c
e
d
N
N
NH
NH2
HCl
O
N
N
N
N
H
H
H
H
15 R = H
16 R = Me
b
17
18
14
O
O
19a, 20a X = O, R1 = 5-H, 9%, dr = 1 : 1.8
12aR
12aR
NMe
O
NMe
19b, 20b X = O, R1 = 5-Me, 11%,dr = 1 : 2.0
19c, 20c X = O, R1 = 5-Et, 12% dr = 1 : 1.9
19d, 20d X = S, R1 = 5-Br, 20%, dr = 1 : 3.3
19e, 20e X = S, R1 = 5-Me, 25%,dr = 1 : 2.8
19f, 20f X = S, R1 = 5-Et, 22%, dr = 1 : 2.6
5-substituented
furans/thiophenes
N
N
6R
6S
O
N
N
H
H
X
X
19g, 20g X = S, R1 = 3-Br, 51%, dr = 1 : 1.8
19h, 20h X = S, R1 = 3-Me, 69%, dr = 1 : 2.3
3-substituented
thiophenes
19a-h (cis)
R1
R1
20a-h (trans)
O
O
O
Series A compounds 19-22
12aS
12aS
6R
NMe
NMe
(S)
OH
a-e
N
N
NH2
HCl
6S
O
O
N
N
H
N
H
H
X
X
21c, 22c X = O, R1 = Et, 13%, dr = 1 : 1.9
21f, 22f X = S, R1 = Et, 21%, dr = 1 : 2.5
R1
R1
22c,f (trans)
(3S)
-14
21c,f (cis)
O
COOMe
Cl
COOMe
COOMe
NH2
(R)
OH
b
N
f
c
d
NH
X
NH2
HCl
O
N
H
R1
N
N
H
N
H
H
X
R1
23-S-Br R1 = Br
24-S-Br R1 = Br
(D)-ester-14
14
23-S-Me R1 = Me
24-S-Me R1 = Me
aReaction conditions: a) HCHO, NaOH, H2O, 105 ºC; b) SO2Cl2, MeOH, 0 ºC then reflux; c) chloroacetyl chloride,
Et3N, CH2Cl2, r.t.; d) methylamine, MeOH, 70 ºC; e) appropriate potassium trifluoroborate derivatives, TEMPO (4
eq.), CH2Cl2/CHCl3, r.t; f) aldehydes, CHCl3, TFA, r.t.;
[Scheme 1 here]
Scheme 2. Preparation of series B and series C β-carbolines derivitives, tetrahydro-β-carboline-hydantoins and
2,3-diacyl-β-carbolines.a
aReaction conditions: g) isocyanates, butanone, reflux; h) appropriate potassium trifluoroborate derivatives,
TEMPO (3 eq.), CH2Cl, r.t.; i) ethyl isocyanates, DIEA, CH2Cl2, r.t.
bOnly trans-configuration isomers can be isolated.
cOnly 5S,11aS-isomer 26a can be isolated.
[Scheme 2 here]

Scheme 3. Preparation of series D β-carbolines derivitives , α-furyl/thienyl-tetrahydro-β-carbolines. a
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aReaction conditions: a) appropriate potassium trifluoroborate derivatives, TEMPO (1 eq.), CH2Cl2, r.t.; b) MeOH,
NaOH, reflux;
[Scheme 3 here]
Scheme 4. Preparation of series E-G β-carbolines derivatives via DDQ oxidation, 2,3-substituted-indoles,
β-carboline-3-carboxamide and β-carboline-4-ones.a
36a
36b
36c
36d
36e
36f
A
, X = O, R1 = 5-Me, 36%
A
, X = O, R1 = 5-Et, 41%
R2
R2
R2
R2
R2
R2
R2
R2
=
=
=
=
=
=
=
=
N
N
N
HN
O
N
N
A
, X + R1 = benzuofuran-2-yl, 49%
a
A
, X + R1 = (5-OMe)benzuofuran-2-yl, 37%
N
H
X
A
, X = S, R1 = 5-Me, 63%
N
A
, X = S, R1 = 5-Pr, 48%
H
X
R1
36g
36h
A
A
, X = S, R1 = 4,5-Me, 38%
, X = S, R1 = 5-Et, 51%
33a-g,6
36a-h
R1
Series E compounds 36,38,39
O
OMe
OMe
OMe
OMe
OMe
OMe
N
N
N
HN
N
a
N
N
O
N
H
N
X
X
H
N
H
X
Et
Et
34a
34b
38a
38b
X = O
X = S
X = O, 22%
X = S, 20%
37a
37b
X = O, 48%
X = S, 32%
Et
Series F compounds 37
O
N
O
O
n = 1, R = Me
n = 0, R = Et
R
O
N
NEt
a
NHEt
N
n = 0, 1
HN
HN
O
N
O
O
O
N
H
N
H
N
O
N
H
S
H
S
S
S
Et
Et
Et
Et
41
31%
39a R
39b S
19f
22f
40a R
40b S
29d
26d
-isomers, 47% from
-isomers, 42% from
-isomers, 53% from
-isomers, 50% from
19f, 22f (n = 1, R = Me)
26d, 29d (n = 0, R = Et)
Series G compounds 41
aReaction conditions: a) DDQ, THF/H2O, -78 ºC, then 25 ºC.
[Scheme 4 here]

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Figure 6. Single crystal structure of compounds 19c, 19f, 21c, 26b–c, 27c, 28c, 29c.
[Figure 6 here]

2.2. PDE5 Inhibitory Activity and PDE Subtypes Selectivity
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All the derivatives were evaluated for in vitro inhibitory activity against recombinant human
PDE5 according to our previously reported procedures. [20] Firstly, these compounds were tested
the percentage of inhibition at 100 nM (Results were displayed in Supporting Iinformation, table
S1-S6). For compounds with >50% inhibition and several other representative structures 34a, 35c,
41, the concentration-response tests were performed to determined their IC50 values (table 1–2).
Moreover, the most potent compounds 19c–f, 21f, 26a–e, 28b, 29b,c,e and 37a,b were also tested
inhibitory activity against a panel of other PDEs (PDE1–4,6,11, 19f, 26c further tested PDE7-10) for
deciding their selectivity profiles. Tadalafil was employed as a positive control.
As shown in table 1, piperazinedione analogues with pendant α-furyl/thienyl substituents
produced several candidates with excellent PDE5 inhibitory activities. Exploration of the
structure-activity relationships (SAR) around the heterocycles was also carried out. Based on the
biological evaluation, stereochemical state of C-6 and C-12a is crucial important: almost all the (6S,
12aR) isomers 19b–h (entry 1–7) exhibited potent inhibition against PDE5 enzyme, but the rest
enantiomers and diastereoisomers provided only 35–40% inhibition (Table S1, Supporting
Information) except for compounds 20h, 21c, 22c,f (entry 8–11, IC50 =19.9–97.5 nM). By
comparing the introduced heteroaromatic systems in C-6 position, we also concluded that
thiophenes were more suitable than furans for the tetrahydro-β-carboline-piperazinedione scaffold
(entry 1–2, 9). Among these thienyl derivatives, compound 19d–f bearing 5-substituted thiophenes
(entry 3–5) exhibited good PDE5 inhibitory activities, while compounds 20g–h with 3-substitued
thiophenes were less active (entry 6–7). Compound 20f with 5-ethylthiophene showed the highest
potency with an IC50 of 3.87 nM, and noticeably, its enantiomer 21f and diastereoisomer 22f also
exhibited high PDE5 potency (entry 10–11), implying 5-ethylthienyl moiety is well appropriate
choice for developing novel potent PDE5 inhibitors.
We then expanded piperazinedione ring to a five-membered hydantoin structure. Again, PDE5
inhibitory activities of these β-carboline derivatives 26–29 were also highly influenced by the
α-substituents. Compounds 26b,c with pendant 5-ethylfuran or 5-ethylthiophene were the most
potent PDE5 inhibitors, whereas shorter chain or different substituted position on the heteroaromatic

systems would decrease their PDE5 potency (26a–d, entry 12–15). These results provided hydantoin
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26b with a 5-ethylfuran substituent at C-5 to be the most potent PDE5 inhibitor, IC50 = 2.92 nM.
Subsequent SAR analysis revealed that stereochemical configuration of C-5 was also an important
determinant, as all (5S)-isomers 26b,c, 29b,c (entry 13–14, 20–21) could strongly inhibit PDE5
(IC50 < 10 nM) and their respective (5R)-diastereomers 27b,c, 28b,c (entry 16–19) only
demonstrated high to moderate PDE5 potency. Additionally, increasing the length of N4-alkyl chain
on the terminal ring led to a 10-fold decrease in potency (26e, 29e, entry 22–23), and a possible
interpretation attributed to the absence of the hydrophobic interaction with Ile665 (H-loop).
Unfortunately, according to the results shown in Table 2 and Table S4-S6 (Supporting
Informating), all N-biaryl/N-carbamoyl tetrahydro-β-carbolines 30a,b, 33a–g, 35a–c displayed only
15–47% inhibition at 100 nM, and the best representative compounds 34a, 35c with optimized
5-ethylfuan/5-ethylthienyl moiety were tested with moderate PDE5 potency (entry 24–25). We
subsequently introduced a carbonyl group at the C-4 position, and as expected, the PDE5 inhibitory
activity of β-carboline-4-ones 37a,b was significantly improved (entry 26–27). The improved PDE5
potency for 37a,b is mainly due to the C4-carbonyl group could increase the acidity of the indole
nitrogen which are benefit for the formation of H-bond with Gln 817 of PDE5 enzyme. However,
after eliminating hydrogens of the pyridine ring, compound 41 (entry 28) exhibited a huge decrease
in PDE5 inhibition, comparing to previously described potent β-carboline PDE5 inhibitors.
Regarding the 2,3-disubstituted indole analogues 36a–h, 38a,b, 39a,b, 40a,b, no compound was
determined with PDE5 inhibitory activity, even after replacing various α-substituents and N-aryls.
These results indicated that the binding affinity for these indole derivatives was much weaker than
the corresponding tetrahydro-β-carbolines, and proposed the necessity of keeping intact
tetrahydro-β-carboline scaffold for further development of novel PDE5 inhibitor.

Table 1. PDE5 potency for compound 19b, 20b–h, 21f and 22c,f. a
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Entry
Compd
X
R1
IC50 for PDE5 (nM)a
Tetrahydro-β-carboline-piperazinedione：
1
2
19b
19c
19d
19e
19f
19g
19h
20h
21c
21f
22f
O
O
S
S
S
S
S
–
5-Me
5-Et
5-Br
5-Me
5-Et
3-Br
3-Me
–
36.9 ± 7.26
12.7 ± 0.85
7.20 ± 0.52
8.68 ± 0.19
3.87 ± 0.61
80.2 ± 1.74
50.2 ± 6.50
97.5 ± 22.1
56.4 ± 6.89
19.9 ± 3.91
61.0 ± 8.44
3
4
5
6
7
8
9
O
S
–
–
10
11
–
–
Tetrahydro-β-carboline-hydantoin, R = Et,
12
13
14
15
16
17
18
19
20
21
26a
26b
26c
26d
27b
27c
28b
28c
29b
29c
O
O
S
5-Me
5-Et
5-Et
3-Me
5-Et
5-Et
5-Et
5-Et
5-Et
5-Et
10.5 ± 0.42
2.92 ± 0.17
5.24 ± 1.34
21.4 ± 0.78
81.2 ± 8.36
383 ± 4.50
27.6 ± 2.08
155 ± 33.4
8.44 ± 1.65
7.02 ± 0.51
S
O
S
O
S
O
S
R = Bu,
22
26e
29e
S
S
–
5-Et
5-Et
–
30.3 ± 4.95
36.4 ± 5.13
4.67 ± 0.32
23
Tadalafilb
aIC50 was the mean of three tests. b IC50 value for tadalafil was experimental result.
[Table 1 here]

Table 2. PDE5 potency for compoundA34Ca,C35EcP, 3T7Ea,Db aMndA41N. USCRIPT
Entry
24
Compd
34a
IC50 for PDE5 (nM)a
177 ± 20.4
25
35c
235 ± 49.8
26
37a
52.5 ± 7.02
27
37b
41
39.7 ± 6.38
28
227 ± 14.5
aIC50 was the mean of three tests.
[Table 2 here]
Finally, the isozyme selectivity of compounds 19c–f, 21f, 26a–e, 28b, 29b,c,e and 37a,b were
determined, upon comparison of their potency for PDE5 inhibition with their potency for inhibition
of PDE1A, PDE2A, PDE3B, PDE4A, PDE6C, PDE7A, PDE8A1, PDE9A2, PDE 10A1 and
PDE11A. As summarized in table 3, these compounds were all inactive for PDE1–4 at the
concentrations of 20 µM or 10 µM, showing excellent selectivity to PDE5 versus such PDEs. The
most potent compound 19f and 26b exhibited >1 000-fold selectivity to PDE5 versus PDE7–10. The
IC50 values of these compounds for PDE6 and PDE11 were also calculated by testing a range of 8
concentrations (0.33–1 000 nM), and their inhibitory potency against PDE6 and PDE11 was
significantly influenced by various α-furyl/thienyl moieties and distinctive tetrahydro-β-carboline
scaffolds. Tetrahydro-β-carboline-piperazinedione 19c with pendant 5-ethylfuran exhibited high
PDE6 selectivity but only poor selective for PDE11, whereas thienyl derivatives 19d–f including
enantiomer 21f showed improved selective for PDE11 (entry 29–33). The most potent compound
19f, exhibited > 258- and 70- fold selectivity for PDE6 and PDE11, respectively. However,
tetrahydro-β-carboline-hydantoin 26a–e, 28b displayed moderate PDE6 selectivity and high PDE11
selectivity (entry 34–39), while compound 26b showed respective 43- and > 342- fold selectivity
over PDE6 and PDE11. Conversely, their diastereomer 29b,c,e with (5S,11aR)-configuration

exhibited better PDE6 selectivity but less selective for PDE11 (entry 40–42). These data also
ACCEPTED MANUSCRIPT
indicate that the configuration is determinant for regulating the binding affinity with PDE6 and
PDE11 enzymes. It is notable that compound 26a with characteristic 5-methylfuran ring lead to
strong inhibition against PDE11 enzyme with the IC50 value of 4 nM. In addition, carboline-4-ones
37a,b only displayed 4–12 fold selectivity for PDE6 or PDE11 (entry 43–44).
The stability during PDE assays for representative compound 19c,f, 26b,c, 30a, 36h, 37a, 41
were also determined (Fig. S9–S18 in Supportin Inforamtion). According to the results, these
compounds underwent the PDE assays smoothly without significant impurities obtained.
Table 3. Selectivity profiles for the most potent compounds 19c–f, 21f, 26a–e, 28b, 29b,c,e and 37a,b. a
PDE5
IC50 (nM)
PDE1-3
IC50 (nM)
PDE4
IC50 (nM)
PDE6
IC50 (nM)
Selectiveity Indexb
(PDE6/PDE5)
PDE7-10
PDE11
IC50 (nM)
Selectiveity Index
(PDE11/PDE5)
Entry Compd
IC50 (nM)
c
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
19c
19d
19e
19f
12.7
7.20
8.68
3.87
19.9
10.5
2.92
5.24
21.4
30.3
27.6
8.44
7.02
36.4
52.5
39.7
4.67
> 20 000
> 20 000
> 20 000
> 20 000
> 20 000
> 20 000
> 20 000
> 20 000
> 20 000
> 20 000
> 20 000
> 20 000
> 20 000
> 20 000
–
> 10 000
> 10 000
> 10 000
> 10 000
> 10 000
> 10 000
> 10 000
> 10 000
> 10 000
> 10 000
> 10 000
> 10 000
> 10 000
> 10 000
–
> 1 000
> 79
–
119
9
30
136
19
–
219
> 1 000
> 1 000
> 1 000
62
> 115
> 258
> 50
6
–
173
20
> 4 000
272
70
21f
–
> 1 000
4
> 50
0.4
> 342
> 190
7
26a
26b
26c
26d
26e
28b
29b
29c
29e
37a
37b
–
127
43
> 3 000
> 1 000
> 1 000
155
56
11
–
13
0.6
–
107
4
–
> 1 000
802
> 33
29
10
0.4
–
> 1 000
> 1 000
> 1 000
288
> 118
> 142
> 27
5
–
99
12
–
169
24
–
386
10
–
–
634
12
–
–
253
6
153
4
Tadalafild
> 10 000
> 10 000
> 10 000
5 100
> 10 000
47
10
aIC50 was the mean of three replicates. Isoforms for PDE subtypes are PDE1A, PDE2A, PDE3B, PDE4A, PDE6C,
PDE7A, PDE8A1, PDE9A2, PDE 10A1 and PDE11A.
bSelectivity Index = PDE-X(IC50)/PDE-5(IC50).
cNot determined.
dFor tadalafil, IC50 values for PDE5 and PDE11 was experimental results, IC50 values for PDE1–4,6-10 were listed
as literature data. [14]
[Table 3 here]
2.3. Vasodilatory Activity
PDE5 inhibitors could potentiate the intrinsic NO-sGC-cGMP signaling through inhibition of
cGMP hydrolysis, inducing further vasodilation and leading to corresponding therapeutic effects.

Therefore, the most potent PDE5 inhibitors 19c–f, 26a–c and 29b–c (PDE5 IC50 = 2.92–12.7 nM),
ACCEPTED MANUSCRIPT
were investigated the in vitro vasorelaxant activity using a pressure myograph. [29-31] As shown in
table 4, all tested compounds exhibited significantly vasodilatory effect on the isolated rat 3rd-order
mesenteric arteries (110–150 µm, pre-contracted by 20 µM norepinephrine). Obviously, their
vasodilatory activities were in accordance with the PDE5 potency. We found that all α-thiophene
β-carbolines 19d–f, 26c, 29c as well as one single furyl analogue 26b exhibited excellent
vasorelaxant activities, while the remaining compounds 19c, 26a, 29b with pendant furans only
displayed moderate efficacy. The above data indicate that these potent PDE5 inhibitors have
immense therapeutic potential for vascular diseases.
Additionally, representative real-time vasorelaxant course and contraction-concentration
response were displayed in Fig.7 and Fig.S1 (Supporting Information). The isolated arteries were
firstly pre-contracted by adding 20 µM norepinephrine (Left column), and then PDE5 inhibitors
were added with increased dosages. These compounds could inhibit PDE5 enzyme, elevate cGMP
level, and lead to significant vasodilation via NO-sGC-cGMP signaling. At the end of each
experiment, vessel was washed with Ca2+-free solution for entire dilation (Right column). It is
obviously that the diameter of contract arteries gradually increased as PDE5 inhibitors accumulation
dose-dependently.
Table 4. Vasorelaxant activity of compounds 19c–f, 26a–c and 29b,c.a
Compound
EC50 (nM)
19c
19d
19e
19f
30
26a
26b
26c
29b
29c
Tadalafil
725
55
71
191
63
58
225
103
78
aCalculated from 7 concentrations (0.01–10 000 nM), n = 6.
[Table 4 here]

ACCEPTED MANUSCRIPT
Figure 7. PDE5 inhibitors 19d–e, 26b,c, 29c induced vasorelaxant effect on mesenteric resistance arteries. Arteries
(110–150 µm) were constricted by 20 µM norepinephrine, and added tested compounds. At the end, vessels were
washed with Ca2+-free solution for entire dilation.
[Figure 7 here]
2.4. Molecular Modeling Analysis
According to the proposed binding mode of compounds 19f and 26b with PDE5 enzyme,
reasonable interactions can be found to support our experimental results, explaining their high PDE5
potency and selectivity. In the hypothetical protein-ligand complex structure (Fig.8), compounds 19f
and 26b exhibit similar docking poses and both fit nicely in the catalytic domain of PDE5 enzyme.
The key interaction of 19f and 26b with PDE5 enzyme is the hydrogen bonding with Gln817, which
considered crucial for reducing cGMP affinity. The indole ring in both compounds also showed π–π
stacking with the hydrophobic residue Phe820. Additionally, the pendant 5-ethylfuran and

5-ethylthiophene of 19f and 26b were all buried deeply within central hydrophobic cavity of PDE5
ACCEPTED MANUSCRIPT
enzyme, which are benefit for their high PDE5 potencies.
Figure 8. Proposed molecular modeling of compounds 19f (cyan) and 26b (pink) with PDE5: (Left) Overall view;
(middle) molecular surface of the binding pockets for 19f and 26b with PDE5; and (right) proposed binding model
of 19f and 26b in the tadalafil bonding site; distances were both 1.9 Å. PDB code 1xoz [23].
[Figure 8 here]
3. Conclusion
Inspired by natural product perlolyrine 5a and our previous efforts, we designed and
constructed a small library of tetrahydro-β-carboline derivatives with characteristic pendant
furan/thiophene moieties for seeking more potent and structurally distinctive PDE5 inhibitors. By
employing oxidative conditions under the presence of TEMPO or DDQ, a total of 66
tetrahydro-β-carboline analogues were prepared. Further PDE assays and SAR analysis provided
compounds 19f and 26b with optimized 5-ethylfuryl or 5-ethylthienyl moiety as the most potent and
selective PDE5 inhibitors. Furthermore, the high PDE5 potency of compounds 19c–f, 26a–c and
29b–c also lead to significant vasorelaxant effects against norepinephrine-precontracted resistance
artery, which implies their considerable therapeutic potential for the treatment of vascular diseases.
In addition, the present study indicated that the optimized 5-ethylfuran/5-ethylthiophene substituents
were privileged substructures to occupy the Q2 pocket of PDE5 enzyme based on the docking results.
We also expect these valuable fragments could be further employed for the discovery of more potent
and selective PDE5 inhibitors.

4. Experiment Section
ACCEPTED MANUSCRIPT
4.1. General materials and methods.
Melting points were determined with an X-6 melting point apparatus (Beijing TECHInstrument Co. Ltd.).
NMR spectra were obtained using a Bruker Avance AVIII-400 spectrometer operating at 400 (1H) or 100 (13C)
MHz. HR-ESI-MS was conducted with an LTQ Orbitrap XL instrument. Single crystals were examined on a
Bruker D8 VENTURE diffractometer. HPLC was performed with an Agilent 1200 system equipped with a
G1311A isopump, a G1322A degasser, and a G1315D DAD detector using an Eclipse XDB-C18 (150 mm × 4.6
mm × 5 µm). Column chromatography was conducted on silica gel (200–300 mesh) or performed on a Teledyne
ISCO CombiFlash Rf Automated Flash Chromatography System with RediSep Rf disposable flash columns. All
solvents used were of analytical grade. Precoated silica gel GF254 plates (Qingdao Haiyang Chemical Co., Ltd.)
were used for thin-layer chromatography. All reactions were conducted under a nitrogen atmosphere with dry
solvents under anhydrous conditions. The purities of all biologically evaluated compounds were established as ≥
95% by HPLC analysis with UV detection. The outdiameter of mesenteric artery was measured by DMT-112PP
Pressure Myograph System (Danish Myo Technology A/S).
4.2. General synthetic procedures.
The general procedures for the synthesis of diverse α-furyl/thienyl tetrahyro-β-carbolines analogues were
described with typical examples as following. The analytical data, HPLC assays, X-ray crystallographic analysis
for individual compounds can be found in the supporting information.
Methyl (R)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylate (16). A mixture of (D)-tryptophan
(2.04 g, 10 mmol, 1.1 eq.), NaOH (440 mg, 11 mol, 1.1 eq.), and H2O (30 mL) was stirred, and then formalin (37%,
860 µL, 11 mol, 1.1 eq.) was added. The mixture was stirred for 2 h at room temperature and subsequently refluxed
for another 3 h. The reaction was cooled and neutralized with glacial acetic acid to pH = 5. After filtration, the
1
precipitate was collected and dried in vacuo to yield product 15 (4.5 g, 66%). mp. >280 ºC. H NMR (400 MHz,
DMSO-d6) δ 10.92 (s, 1H), 7.44 (d, J = 7.9 Hz, 1H), 7.33 (d, J = 8.0 Hz, 1H), 7.07 (t, J = 7.5 Hz, 1H), 6.99 (t, J =
7.5 Hz, 1H), 4.26 – 4.11 (m, 2H), 3.60 (dd, J = 10.2, 4.9 Hz, 2H), 3.17 (s, 5H), 3.14 – 3.10 (m, 1H), 2.81 (dd, J =
15.8, 10.2 Hz, 1H). 13C NMR (101 MHz, DMSO) δ 170.5, 136.7, 126.8, 126.2, 122.2, 119.5, 118.4, 111.8, 105.0,
54.3, 40.6, 22.4. ESI-MS m/z 215 [M - H]-, [α]20 = +96.60
°, c = 0.25 in 5% AcOH/MeOH. Compound 15 (868
D
mg, 4 mmol, 1 eq.) was then dissolved in 16 mL anhydrous MeOH, SO2Cl2 (416 µL, 6 mmol, 1.5 eq.) was add at
0 °C. After stirred at room temperature for 2 h, the mixture was reflux for another 15 h, and then the mixture was
adjusted to pH > 9 with an aqueous solution of NaHCO3. The reaction was then extracted with EtOAc (3 × 50 mL)
and the organic phase was concentrated in vacuo, compound 16 was obtained as a light yellow solid (800 mg,
86%). mp. 147-149 ºC. 1H NMR (400 MHz, DMSO-d6) δ 10.71 (s, 1H), 7.37 (d, J = 7.7 Hz, 1H), 7.26 (d, J = 8.0

Hz, 1H), 7.08 – 6.97 (m, 1H), 6.93 (t, J = 7.3 Hz, 1H), 4.09 – 3.84 (m, 2H), 3.73 (dd, J = 8.9, 4.7 Hz, 1H), 2.93 (dd,
ACCEPTED MANUSCRIPT
J = 15.1, 4.7 Hz, 1H), 2.85 (s, 1H), 2.74 (dd, J = 14.9, 8.7 Hz, 1H). 13C NMR (101 MHz, DMSO) δ 174.1, 136.3,
133.9, 127.4, 120.9, 118.8, 117.7, 111.4, 106.0, 55.7, 52.1, 41.9, 25.5. ESI-MS m/z 231 [M + H]+, [α]20D = -69.50
°,
c = 1.0 in chloroform.
(R)-2-methyl-2,3,6,7,12,12a-hexahydropyrazino[1',2':1,6]pyrido[3,4-b]indole-1,4-dione (18). chloroacetyl
chloride (381 µL, 4.8 mmol, 1.1 eq.) and distilled triethylamine (1.2 mL, 8.8 mmol, 2 eq.) were added to the
solution of compound 16 (1 g, 4.4 mmol, 1 eq.) in 40 mL anhydrous dichloromethane, and the reaction mixture
was stirred at room temperature for 2 h. The reaction was quenched by ice-water and extracted with
dichloromethane (3 × 20 mL). The organic extracts were concentrated in vacuo, and the crude product was then
purified by silica gel column chromatography (20%–50% EtOAc/Hexane) to yield compounds 17 (1.05 g, 81%).
mp. 177-179 ºC. 1H NMR (400 MHz, DMSO-d6, rotamer seen) δ 10.94 (d, J = 27.6 Hz, 1H), 7.45 (dd, J = 7.9, 4.2
Hz, 1H), 7.31 (dd, J = 8.1, 2.2 Hz, 1H), 7.06 (td, J = 8.1, 7.6, 3.3 Hz, 1H), 6.98 (t, J = 7.5 Hz, 1H), 5.73 – 5.64 (m,
0.49H), 5.25 (d, J = 5.9 Hz, 0.51H), 5.04 (d, J = 17.2 Hz, 0.50H), 4.94 (d, J = 15.5 Hz, 0.52H), 4.81 – 4.63 (m, 2H),
4.54 (d, J = 13.6 Hz, 0.52H), 4.28 (d, J = 16.9 Hz, 0.52H), 3.57 (d, J = 4.0 Hz, 3H), 3.42 – 3.28 (m, 1H), 3.11 (dd,
J = 15.7, 6.1 Hz, 0.55H), 2.97 (dd, J = 15.9, 6.4 Hz, 0.51H). 13C NMR (101 MHz, DMSO, rotamer seen) δ 171.4,
171.3, 167.4, 167.3, 136.8, 136.6, 129.9, 129.5, 126.6, 126.5, 121.7, 121.6, 119.2, 119.1, 118.2, 118.2, 111.6, 111.6,
105.0, 104.5, 55.4, 53.1, 52.9, 51.3, 43.2, 42.9, 41.5, 23.8, 23.0. ESI-MS m/z 307 [M + H]+ [α]20 = -90.80
°, c =
D
0.5 in chloroform. Intermediate 17 (500 mg,1.4 mmol, 1 eq.) was then reflux with methylamine (alcoholic solution,
30% w/w, 1.1 mL, 7 mmol, 5 eq.) in 5 mL MeOH for 3 h. The mixture was evaporated in vacuo to yield compound
18 as orange solid (350 mg, 91%). mp. 267-269 ºC. 1H NMR (400 MHz, DMSO-d6) δ 10.96 (s, 1H), 7.43 (d, J =
7.7 Hz, 1H), 7.33 (d, J = 8.0 Hz, 1H), 7.07 (t, J = 6.9 Hz, 1H), 6.98 (t, J = 6.9 Hz, 1H), 5.37 (dd, J = 16.5, 1.4 Hz,
1H), 4.32 (dd, J = 11.7, 4.2 Hz, 1H), 4.19 (dd, J = 17.3, 3.6 Hz, 2H), 4.02 (d, J = 17.6 Hz, 1H), 3.20 (dd, J = 15.2,
4.3 Hz, 1H), 2.90 (s, 3H), 2.88 – 2.78 (m, 1H). 13C NMR (101 MHz, DMSO) δ 165.5, 163.3, 136.4, 130.3, 126.8,
121.5, 119.2, 118.1, 111.6, 106.1, 56.4, 51.2, 33.1, 27.0. ESI-MS m/z 270 [M + H]+, [α]20D = +151.40
°, c = 0.5 in
chloroform.
(6S,12aR)- and (6R,12aR)-6-(5-Ethylfuran-2-yl)-2-methyl-2,3,6,7,12,12a-hexahydropyrazino[1',2':1,6]
pyrido[3,4-b]indole-1,4-dione (19c and 20c). Compound 18 (135 mg, 0.5 mmol, 1 eq.) and potassium
5-ethylfuran-2-yl)trifluoroborate (327 mg, 1.5 mmol, 3 eq.) was dissolved in 20 mL CH2Cl2/CHCl3 (anhydrous, v/v
= 1 : 1), and this solution was added 2,2,6,6-tetramethyl-piperidine N-oxide salt (T+BF4−) (500 mg, 2 mmol, 4 eq.)
in 4 portions, the mixture was stirred for 2 h at room temperature before quenched by adding saturated NaHCO3
solution at 0 °C. The organic phase was separated and the aqueous layer was extracted with dichloromethane (3 ×
15 mL). The combined organic phase was dried, concentrated and purified by using a Flash Chromatography
System (0%–8% MeOH/CH2Cl2) to give compound 19c and 20c (8 mg and 15 mg, 12%). (6S,12aR)-isomer (19c)

Colorless crystal, mp. 221-223 ºC. tR = 3.368 min. 1H NMR (400 MHz, DMSO-d6) δ 11.20 (s, 1H), 7.58 (d, J = 7.8
ACCEPTED MANUSCRIPT
Hz, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.08 (t, J = 7.6 Hz, 1H), 7.01 (t, J = 7.1 Hz, 1H), 6.36 (s, 1H), 6.05 (d, J = 3.1
Hz, 1H), 5.91 (d, J = 3.1 Hz, 1H), 4.32 (dd, J = 11.8, 4.4 Hz, 1H), 4.20 (dd, J = 17.1, 1.5 Hz, 1H), 3.96 (d, J = 17.0
Hz, 1H), 3.50 (dd, J = 15.4, 4.6 Hz, 1H), 2.93 (s, 4H), 2.45 (q, J = 7.5 Hz, 2H), 1.05 (t, J = 7.5 Hz, 3H). 13C NMR
(101 MHz, DMSO) δ 167.3, 167.2, 156.6, 151.8, 136.4, 131.8, 126.1, 121.7, 119.4, 118.6, 112.0, 107.2, 106.7,
105.2, 55.8, 52.0, 49.0, 33.4, 22.9, 21.1, 12.1. ESI-MS m/z 364 [M + H]+, HR-EI-MS: m/z 366.1257 [M + H]+,
+
(calcd for C21H22N3O3 , 364.1656). [α]20 = +32.00°, c = 0.5 in chloroform. (6R,12aR)-isomer (20c) Colorless
D
crystal, mp. 230-232 ºC. 1H NMR (400 MHz, DMSO-d6) δ 11.11 (s, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.32 (d, J = 8.1
Hz, 1H), 7.11 (t, J = 7.0 Hz, 1H), 7.01 (t, J = 7.4 Hz, 1H), 6.85 (s, 1H), 6.10 (d, J = 3.2 Hz, 1H), 6.05 (d, J = 3.1
Hz, 1H), 4.37 – 4.17 (m, 2H), 4.07 (d, J = 17.8 Hz, 1H), 3.21 (td, J = 17.4, 16.4, 4.8 Hz, 1H), 2.97 (dd, J = 14.7,
12.6 Hz, 1H), 2.88 (s, 3H), 2.58 (q, J = 7.6 Hz, 2H), 1.13 (t, J = 7.5 Hz, 3H). 13C NMR (101 MHz, DMSO) δ 165.2,
163.1, 158.2, 149.8, 136.6, 129.5, 126.3, 122.2, 119.3, 118.6, 111.8, 110.9, 107.5, 105.5, 53.3, 51.1, 46.2, 33.1,
+
26.9, 21.2, 12.3. ESI-MS m/z 364 [M + H]+, HR-EI-MS: m/z 366.1258 [M + H]+, (calcd for C21H22N3O3 ,
364.1656). [α]20D = +214.60°, c = 0.5 in chloroform.
(6S,12aR)- and (6R,12aR)-2-Methyl-6-(3-methylthiophen-2-yl)-2,3,6,7,12,12a-hexahydropyrazino[1',2':
1,6]pyrido[3,4-b]indole-1,4-dione (19h and 20h). D-Tryptophan methyl ester (2 g, 9 mmol, 1 eq.) and
2-formyl-3-methylthiophene (1.1 mL, 10 mmol, 1.1 eq.) was dissolved in 75 mL anhydrous CHCl3, and the
mixture was added 1 mL TFA dropwise. The reaction was quenched by adding 50 mL water and the organic phase
was separated. After dried and concentrated, the crude product was purified by silica gel column chromatography
(0%–5% MeOH/CH2Cl2) to yield 23-S-Me as a mixture of cis and trans isomers (1.2 g, 41%). The two
diastereomers were further separated by prepared TLC (Hexane EtOAc 1).
:
=
5
:
(1S,3R)-methyl-1-(3-methylfuran-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylate (23-S-Me-1).
White solid, mp. 78-80 ºC. 1H NMR (400 MHz, Acetone-d6) δ 9.60 (s, 1H), 7.54 – 7.40 (m, 1H), 7.33 – 7.18 (m,
2H), 7.02 (pd, J = 7.1, 1.5 Hz, 2H), 6.85 (d, J = 5.1 Hz, 1H), 5.70 (t, J = 2.3 Hz, 1H), 3.98 (dd, J = 11.2, 4.1 Hz,
1H), 3.78 (s, 3H) 3.13 (ddd, J = 14.9, 4.2, 1.9 Hz, 1H), 2.95 – 2.90 (m, 1H), 2.31 (s, 3H). 13C NMR (101 MHz,
Acetone) δ 172.7, 138.4, 136.8, 135.4, 135.1, 129.8, 127.2, 124.0, 121.1, 118.8, 117.8, 111.1, 107.0, 56.9, 51.8,
51.4, 25.4, 13.1. ESI-MS m/z 327, [α]20
= +21.60°, c = 1.0 in chloroform. (1R,3R)-methyl
D
1-(3-methylfuran-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3- carboxylate (23-S-Me-2). White solid,
mp. 73-75o ºC. 1H NMR (400 MHz, Acetone-d6) δ 9.75 (s, 1H), 7.49 (dd, J = 7.5, 1.4 Hz, 1H), 7.28 (dd, J = 7.9,
1.2 Hz, 1H), 7.20 (d, J = 5.0 Hz, 1H), 7.09 – 6.99 (m, 2H), 6.86 (d, J = 5.1 Hz, 1H), 5.82 (d, J = 1.6 Hz, 1H), 4.10
(dd, J = 6.8, 5.2 Hz, 1H), 3.69 (s, 3H), 3.17 (ddd, J = 15.2, 5.3, 1.3 Hz, 1H), 3.02 (ddd, J = 15.2, 6.8, 1.6 Hz, 1H),
2.33 (s, 3H). ESI-MS m/z 327, [α]20 = +40.40
°, c = 0.5 in chloroform. Then 23-S-Me (500 mg, 1.5 mmol, 1eq.,
D
mixture of 23-S-Me-1 and 23-S-Me-2) was stirred with distilled triethylamine (0.42 mL, 3.0 mmol, 2 eq.) and

chloroacetyl chloride (130 µL, 1.6 AmCmColE, P1.T1 EeDq.)MtoAyNieUldSmCiRxtuIPreT24-S-Me (472 mg, 78%). The two
diastereomers
were
purified
by
CombiFlash
(20%–60%
EtOAc/Hexane).
(1S,3R)-methyl-2-(2-chloroacetyl)-1-(3-methylthiophen-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-ca
1
rboxylate (24-S-Me-1). White solid, mp. 187-189 ºC. H NMR (400 MHz, DMSO-d6, rotamer seen) δ 10.95 (s,
1H), 7.53 (d, J = 7.8 Hz, 1H), 7.30 (d, J = 8.1 Hz, 2H), 7.10 (t, J = 7.5 Hz, 1H), 7.02 (t, J = 7.4 Hz, 1H), 6.98 (s,
1H), 6.79 (d, J = 5.4 Hz, 1H), 5.25 (d, J = 7.9 Hz, 1H), 4.83 (d, J = 14.4 Hz, 1H), 4.48 (d, J = 13.4 Hz, 1H), 3.51 (d,
J = 16.1 Hz, 1H), 3.19 (s, 3H), 3.12 (dd, J = 16.3, 7.9 Hz, 1H), 2.02 (s, 3H). 13C NMR (101 MHz, DMSO, rotamer
seen) δ 170.9, 166.8, 136.8, 136.7, 136.4, 131.4, 130.7, 126.2, 125.3, 122.1, 119.2, 118.7, 111.8, 105.7, 52.8, 52.5,
46.7, 43.6, 21.4, 14.3. ESI-MS m/z 403, [α]20 = -59.2
°, c = 1.0 in chloroform. (1R,3R)-methyl
D
2-(2-chloroacetyl)-1-(3-methylthiophen-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylate
(24-S-Me-2). White solid, mp. 96-98 ºC. 1H NMR (400 MHz, DMSO-d6, rotamer seen) δ 10.81 (s, 1H), 7.50 (d, J
= 7.8 Hz, 1H), 7.39 – 6.91 (m, 4H), 6.74 (s, 1H), 6.63 – 6.15 (m, 1H), 5.31 (s, 1H), 4.69 (d, J = 14.2 Hz, 1H), 4.49
– 4.13 (m, 1H), 3.68 – 3.38 (m, 4H), 3.29 – 3.14 (m, 1H), 2.46 (s, 3H). 13C NMR (101 MHz, DMSO, rotamer seen)
δ 172.0, 168.1, 136.9, 135.0, 130.5, 126.2, 123.5, 122.0, 119.4, 118.6, 112.0, 104.3, 57.1, 53.1, 50.6, 43.7, 24.0,
14.4. ESI-MS m/z 403, [α]20 = +17.20
°, c = 0.5 in chloroform. Finally, 24-S-Me (472 mg, 1.2 mmol, 1eq.,
D
mixture of 24-S-Me-1 and 24-S-Me-2) was directly used to reflux with methylamine (alcoholic solution, 30% w/w,
0.95 mL, 6 mmol, 5 eq.) in 5 mL MeOH, the reaction was concentrated and the crude product was purified on a
Flash Chromatography System (0%–8% MeOH/CH2Cl2) to give compound 19h and 20h (98 mg and 191 mg, 69%).
1
(6S,12aR)-isomer (19h) Pale yellow crystal, mp. 288-289 ºC. H NMR (400 MHz, DMSO-d6) δ 10.99 (s, 1H),
7.58 (d, J = 7.5 Hz, 1H), 7.31 (d, J = 7.7 Hz, 1H), 7.16 (d, J = 5.1 Hz, 1H), 7.08 (t, J = 7.3 Hz, 1H), 7.01 (t, J = 7.3
Hz, 1H), 6.70 (d, J = 5.0 Hz, 1H), 6.50 (d, J = 6.2 Hz, 1H), 4.40 (dd, J = 11.8, 4.5 Hz, 1H), 4.17 (dd, J = 17.3, 1.6
Hz, 1H), 3.94 (d, J = 17.1 Hz, 1H), 3.57 (dd, J = 16.1, 4.8 Hz, 1H), 3.04 (dd, J = 15.7, 11.6 Hz, 1H), 2.91 (s, 3H),
2.47 (s, 3H). 13C NMR (101 MHz, DMSO) δ 167.3, 167.0, 140.8, 136.8, 134.8, 134.6, 130.0, 126.2, 123.8, 121.9,
119.4, 118.7, 111.9, 105.2, 56.0, 51.9, 49.8, 33.3, 23.6, 14.4. ESI-MS m/z 366 [M + H]+, HR-EI-MS: m/z 366.1271
[M + H]+, (calcd for C20H20N3O2S+, 366.1271). [α]20 = +83.40°, c = 0.5 in chloroform. (6R,12aR)-isomer (20h)
D
Colorless crystal, mp. 237-238 ºC. 1H NMR (400 MHz, DMSO-d6) δ 11.13 (s, 1H), 7.51 (d, J = 7.8 Hz, 1H), 7.39
(d, J = 5.1 Hz, 1H), 7.31 (d, J = 8.2 Hz, 1H), 7.17 – 7.08 (m, 2H), 7.02 (t, J = 7.5 Hz, 1H), 6.95 (d, J = 5.1 Hz, 1H),
4.37 (dd, J = 11.9, 4.3 Hz, 1H), 4.26 (d, J = 17.8 Hz, 1H), 4.06 (d, J = 17.7 Hz, 1H), 3.27 (dd, J = 15.5, 4.3 Hz,
1H), 3.01 (dd, J = 14.9, 12.3 Hz, 1H), 2.86 (s, 3H), 2.33 (s, 3H). 13C NMR (101 MHz, DMSO) δ 165.1, 163.0,
136.7, 136.6, 136.1, 132.0, 131.0, 126.2, 125.5, 122.3, 119.4, 118.7, 111.9, 107.1, 52.9, 51.0, 45.7, 33.0, 27.0, 14.3.
ESI-MS m/z 366 [M + H]+, HR-EI-MS: m/z 366.1272 [M + H]+, (calcd for C20H20N3O2S+, 366.1271). [α]20
+211.00°, c = 0.5 in chloroform.
=
D
(5S,11aS)-2-ethyl-5-(5-ethylfuran-2-yl)-5,6,11,11a-tetrahydro-1H-imidazo[1',5':1,6]pyrido[3,4-b]indole-

1,3(2H)-dione (26b) Intermediate 16 A(30C0CmEgP, 1T.3EmDmMol,A1eNq.U) wSaCsRdiIsPsoTlved in 5 mL butanone and reflux with
ethyl isocyanate (265 µL, 2.1 mmol, 1.6 eq.) for 8 h. The reaction was concentrated in vacuo, and the crude residue
1
was further purified (20%–50% EtOAc/Hexane) to yield compound 25-Et (68 mg, 77%). mp. 186-187 ºC. H
NMR (400 MHz, Acetone-d6) δ 10.16 (s, 1H), 7.51 (d, J = 7.8 Hz, 1H), 7.39 (d, J = 8.0 Hz, 1H), 7.20 – 6.98 (m,
2H), 5.03 (dd, J = 16.2, 1.8 Hz, 1H), 4.43 (dt, J = 16.3, 1.8 Hz, 1H), 4.32 (ddd, J = 11.1, 5.5, 2.5 Hz, 1H), 3.54 (q,
J = 7.2 Hz, 2H), 3.29 (ddd, J = 14.9, 5.5, 1.5 Hz, 1H), 2.75 (ddd, J = 15.1, 10.9, 2.1 Hz, 1H), 1.18 (t, J = 7.2 Hz,
3H). 13C NMR (101 MHz, Acetone) δ 172.4, 154.8, 136.9, 129.7, 126.8, 121.6, 119.1, 117.8, 111.1, 105.5, 54.9,
37.3, 33.0, 22.8, 12.9. m/z 270 [M + H]+, [α]20 = +27.80
°, c = 0.5 in chloroform Compound 25-Et (54 mg, 0.2
D
mmol, 1eq.) and potassium (5-ethylfuran-2-yl)trifluoroborate (140 mg, 0.6 mmol, 3 eq.) was dissolved in a mixed
solution of 12 mL CH2Cl2/CHCl3 (anhydrous, v/v = 1 : 1). The mixture was added T+BF4− (150 mg, 0.6 mmol, 3
eq.) in 3 portions, and subsequently stirred for 2 h at room temperature. The reaction was quenched by adding
saturated NaHCO3 solution at 0 °C. After extracted with dichloromethane (3 × 20 mL) and concentration, the
residue was purified by silica gel column chromatography (10%–40% EtOAc/Hexane) to yield 26b (23 mg, 32%).
Colorless crystal, mp. 150-152 ºC. 1H NMR (400 MHz, DMSO-d6) δ 11.04 (s, 1H), 7.52 (d, J = 7.8 Hz, 1H), 7.33
(d, J = 8.0 Hz, 1H), 7.11 (t, J = 7.5 Hz, 1H), 7.02 (t, J = 7.4 Hz, 1H), 6.24 (t, J = 2.6 Hz, 2H), 6.05 (d, J = 3.1 Hz,
1H), 4.49 (dd, J = 10.8, 5.7 Hz, 1H), 3.47 (q, J = 7.2 Hz, 2H), 3.33 (dd, J = 15.2, 5.8 Hz, 1H), 2.80 (ddd, J = 15.2,
10.9, 1.8 Hz, 1H), 2.56 (q, J = 7.6 Hz, 2H), 1.17 – 1.07 (m, 6H). 13C NMR (101 MHz, DMSO) δ 172.8, 157.8,
154.4, 150.5, 137.0, 130.0, 126.2, 122.2, 119.3, 118.7, 111.9, 109.2, 106.2, 105.5, 53.5, 45.8, 33.5, 23.1, 21.2, 13.7,
+
12.2. ESI-MS m/z 364 [M + H]+, HR-EI-MS: m/z 364.1657 [M + H]+, (calcd for C21H22N3O3 , 364.1656). [α]20
=
D
-206.80°, c = 0.5 in chloroform.
(5S,11aS)- and (5R,11aS)- 2-Ethyl-5-(5-ethylthiophen-2-yl)-5,6,11,11a-tetrahydro-1H-imidazo[1',5':1,6]
pyrido[3,4-b]indole-1,3(2H)-dione (26c and 27c). Intermediate (3S)-methyl 1-(5-ethylthiophen-2-yl)-2,3,4,9-
tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylate (3S)-23-Deri-c (cis/trans mixture, 170 mg, 0.5 mmol, 1 eq.) was
dissolved in 5 mL butanone and reflux with ethyl isocyanate (63 µL, 0.8 mmol, 1.6 eq.) for 8 h. The mixture was
evaporated in vacuo and purified on the Flash Chromatography System (0%–60% EtOAc/Hexane) to give
1
compound 26c and 27c (90 and 42 mg, 69%). (5S,11aS)-isomer (26c). Colorless crystal, mp. 218-219 ºC. H
NMR (400 MHz, DMSO-d6) δ 11.09 (s, 1H), 7.53 (d, J = 7.8 Hz, 1H), 7.32 (d, J = 8.0 Hz, 1H), 7.11 (t, J = 7.6 Hz,
1H), 7.02 (t, J = 7.4 Hz, 1H), 6.88 (d, J = 3.5 Hz, 1H), 6.74 (d, J = 3.4 Hz, 1H), 6.42 (s, 1H), 4.45 (dd, J = 10.9, 5.6
Hz, 1H), 3.46 (q, J = 7.1 Hz, 2H), 3.34 (dd, J = 15.5, 6.1 Hz, 1H), 2.87 – 2.67 (m, 3H), 1.19 (t, J = 7.5 Hz, 3H),
1.12 (t, J = 7.2 Hz, 3H). 13C NMR (101 MHz, DMSO) δ 172.5, 154.3, 147.7, 140.5, 137.0, 131.5, 126.8, 126.1,
123.8, 122.3, 119.3, 118.8, 111.9, 106.2, 52.9, 47.3, 33.5, 23.3, 22.9, 16.2, 13.8. ESI-MS m/z 380 [M + H]+,
HR-EI-MS: m/z 380.1428 [M + H]+, (calcd for C21H22N3O2S+, 380.1427). [α]20D = -243.40°, c = 0.5 in chloroform.
1
(5R,11aS)-isomer (27c). Pale yellow crystal, mp. 233-235 ºC. H NMR (400 MHz, DMSO-d6) δ 10.85 (s, 1H),

7.54 (d, J = 7.7 Hz, 1H), 7.28 (d, J = 8A.0 CHzC, E1HP)T, 7E.1D5 –M7.A04N(mU,S2HC)R, 7I.P00T(t, J = 7.4 Hz, 1H), 6.67 (d, J = 3.5 Hz,
1H), 6.18 (s, 1H), 4.50 (dd, J = 11.5, 4.2 Hz, 1H), 3.51 – 3.23 (m, 3H), 2.82 (ddd, J = 14.0, 11.6, 1.8 Hz, 1H), 2.69
(q, J = 7.6 Hz, 2H), 1.14 (t, J = 7.5 Hz, 3H), 1.09 (t, J = 7.2 Hz, 3H). 13C NMR (101 MHz, DMSO) δ 171.6, 154.5,
146.9, 141.9, 137.0, 134.9, 126.7, 126.3, 123.0, 122.0, 119.3, 118.8, 111.9, 105.5, 58.4, 51.9, 33.2, 23.3, 22.3, 15.9,
13.8. ESI-MS m/z 380 [M + H]+, HR-EI-MS: m/z 380.1427 [M + H]+, (calcd for C21H22N3O2S+, 380.1427). [α]20
D
= -76.00°, c = 0.5 in chloroform.
Methyl (1S,3R)-2-(ethylcarbamoyl)-1-(5-ethylfuran-2-yl)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b] indole-3-
carboxylate (30a). 23-Deri-b (cis/trans mixture, 32 mg, 0.1 mmol, 1 eq.) was dissolved in 3 mL anhydrous
CH2Cl2 and added distilled DIEA (20 µL, 0.11mmol, 1.1 eq.), ethyl isocyanate (10 µL, 1.3 mmol, 1.3 eq.). The
mixture was stirred for 3 h at room temperature. After workup, the resulting solid was purified (20%–50%
EtOAc/Hexane) to yield compound 30a (5 mg, 13%). Yellow crystal, mp. 93-95 ºC. 1H NMR (400 MHz, DMSO-d6) δ
10.92 (s, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.30 (d, J = 8.1 Hz, 1H), 7.09 (t, J = 7.4 Hz, 1H), 7.00 (t, J = 7.4 Hz, 1H), 6.79 (t,
J = 5.3 Hz, 1H), 6.50 (s, 1H), 5.95 (d, J = 3.0 Hz, 1H), 5.68 (d, J = 3.1 Hz, 1H), 5.39 (d, J = 6.6 Hz, 1H), 3.37 (s, 1H),
3.28 – 3.07 (m, 5H), 2.82 (dd, J = 15.7, 6.8 Hz, 1H), 2.57 (q, J = 7.6 Hz, 2H), 1.15 (t, J = 7.5 Hz, 3H), 1.09 (t, J = 7.1 Hz,
3H). 13C NMR (101 MHz, DMSO) δ 172.3, 157.2, 157.1, 151.5, 136.7, 129.7, 126.5, 121.8, 118.9, 118.5, 111.7, 109.8,
107.0, 105.0, 52.3, 50.2, 47.0, 36.0, 21.6, 21.3, 15.9, 12.5. ESI-MS m/z 396 [M + H]+, HR-EI-MS: m/z 396.1913 [M +
+
H]+, (calcd for C22H26N3O4 , 396.1918). [α]20D = +63.80°, c = 0.5 in chloroform.
(E)-3-(3-Bromophenyl)-1-(1-(5-ethylthiophen-2-yl)-1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)prop-2
-en-1-one (35a). (E)-3-(3-bromophenyl)-1-(1,3,4,9-tetrahydro-2H-pyrido[3,4-b]indol-2-yl)prop-2-en-1-one 32c
(43 mg, 0.1 mmol, 1eq.) was prepared and dissolved in
2 mL anhydrous CH2Cl2, potassium
(5-thiophen-2-yl)trifluoroborate (44 mg, 0.2 mmol, 2 eq.) and T+BF4− (24 mg, 0.1 mmol, 1 eq.) was added in
sequence. The reaction completed within 10 min, and subsequently quenched by saturated NaHCO3. After further
extraction with dichloromethane (3 × 15 mL), concentration and purification (0%–40% EtOAc/Hexane), product
35a was obtained (43 mg, 89%). Yellow crystal, mp. 105-107 ºC. 1H NMR (400 MHz, DMSO-d6) δ 11.09 (s, 1H),
8.12 (q, J = 3.6, 2.7 Hz, 1H), 7.75 (d, J = 7.9 Hz, 1H), 7.61 – 7.55 (m, 2H), 7.51 – 7.44 (m, 2H), 7.39 – 7.34 (m,
1H), 7.32 (d, J = 8.0 Hz, 1H), 7.10 (t, J = 7.3 Hz, 1H), 7.01 (t, J = 7.4 Hz, 1H), 6.97 (s, 1H), 6.71 – 6.62 (m, 2H),
4.61 – 4.47 (m, 1H), 3.43 (ddd, J = 13.8, 10.0, 6.0 Hz, 1H), 2.92 – 2.82 (m, 2H), 2.75 (q, J = 7.6 Hz, 3H), 1.19 (t, J
= 7.4 Hz, 4H). 13C NMR (101 MHz, DMSO) δ 164.8, 147.5, 141.4, 140.7, 138.0, 136.6, 132.7, 132.2, 131.3, 130.6,
128.2, 127.0, 126.5, 123.3, 122.8, 121.9, 120.4, 119.1, 118.5, 111.8, 108.4, 48.5, 23.3, 22.6, 16.3. ESI-MS m/z 491
[M + H]+, HR-EI-MS: m/z 491.0793 [M + H]+, (calcd for C26H24BrN2OS+, 491.0787).
2-(5-(3,4-Dimethoxyphenyl)pyrimidin-2-yl)-1-(5-ethylfuran-2-yl)-1,2,3,9-tetrahydro-4H-pyrido[3,4-b]ind
ol-4-one (37a) and (3-(2-((5-(3,4-Dimethoxyphenyl)pyrimidin-2-yl)amino)ethyl)-1H-indol-2-yl)(5-ethylfuran-
2-yl)methanone (38a) The well-mixed powder of 34a (104 mg, 0.21 mmol, 1eq.) and DDQ (98 mg, 0.43 mmol, 2

eq.) was cooled to -78 °C, and added a solution of THF/H2O (8 mL, v/v = 9 : 1 mL) dropwise. The resulting
ACCEPTED MANUSCRIPT
mixture was stirred at -78 °C for 1 h, and then warmed up to room temperature for stirring another hour. The
reaction was finally quenched with saturated NaHCO3 and extracted with dichloromethane (3 × 15 mL). Organic
phase was dried, concentrated and purified (0%–65% EtOAc/Hexane) to yield compound 37a (51 mg, 48%) and
38a (23 mg, 22%). Compound 37a. Brown crystal, mp. 133-134 ºC. 1H NMR (400 MHz, Chloroform-d) δ 9.59 (s,
1H), 8.49 (s, 2H), 8.26 – 8.10 (m, 1H), 7.57 (s, 1H), 7.38 – 7.27 (m, 1H), 7.26 – 7.18 (m, 2H), 6.93 (dd, J = 8.2,
2.1 Hz, 1H), 6.91 – 6.78 (m, 2H), 6.57 (d, J = 3.6 Hz, 1H), 6.44 (d, J = 3.5 Hz, 1H), 5.37 (d, J = 18.3 Hz, 1H), 3.94
(d, J = 18.2 Hz, 1H), 3.83 (d, J = 6.4 Hz, 6H), 2.61 (q, J = 7.5 Hz, 2H), 1.11 (t, J = 7.5 Hz, 3H). 13C NMR (101
MHz, CDCl3) δ 189.4, 159.2, 155.6, 149.5, 149.2, 148.8, 148.5, 136.5, 136.1, 128.1, 126.7, 124.4, 124.1, 123.0,
122.7, 121.8, 118.5, 112.1, 111.8, 111.7, 109.3, 56.0, 49.3, 48.8, 23.4, 15.7. ESI-MS m/z 511 [M + H]+, HR-EI-MS:
m/z 511.1790 [M + H]+, (calcd for C29H27N4O3S+, 511.1798). Compound 38a.Yellow crystal, mp. 160-162 ºC. 1H
NMR (400 MHz, DMSO-d6) δ 11.60 (s, 1H), 8.60 (s, 2H), 7.85 (d, J = 8.1 Hz, 1H), 7.71 (d, J = 3.8 Hz, 1H), 7.49
(d, J = 8.3 Hz, 1H), 7.34 – 7.28 (m, 1H), 7.20 (d, J = 2.1 Hz, 1H), 7.16 – 7.10 (m, 2H), 7.08 (d, J = 3.7 Hz, 1H),
7.00 (d, J = 8.3 Hz, 1H), 3.84 (s, 3H), 3.78 (s, 3H), 3.62 – 3.54 (m, 2H), 3.29 (t, J = 7.3 Hz, 2H), 2.90 (t, J = 7.5 Hz,
2H), 1.29 (t, J = 7.5 Hz, 3H). 13C NMR (101 MHz, DMSO) δ 179.6, 161.7, 157.2, 149.7, 148.5, 141.7, 137.2,
135.7, 131.8, 128.5, 128.1, 126.3, 125.4, 122.4, 121.1, 120.9, 120.3, 117.8, 113.2, 112.8, 109.7, 56.0, 56.0, 42.6,
25.0, 23.8, 16.1. ESI-MS m/z 513 [M + H]+, HR-EI-MS: m/z 513.1959 [M + H]+, (calcd for C29H29N4O3S+,
513.1955).
4.3. Bioassay.
4.3.1 PDE assays.
These assays were carried out as described previously. [32] All of the enzymatic reactions were conducted at
25°C for 60 min. The 50 µL reaction mixture contains 40 mM MOPS at pH 7.5, 0.5 mM EDTA, 15 mM MgCl2,
0.15 mg/mL BSA, 1 mM DTT, 0.05% ProClin 200, 15 ng/mL PDE5 (or other PDE isoforms) and 100 nM
FAM-cyclic-3’,5-GMP (or FAM-cyclic-3’,5’-AMP for different PDE isoforms). The compounds were diluted in 10%
DMSO, and 5 µL of the dilution was added to a 50 µL reaction for a final concentration of 1% DMSO in all
reactions. The reaction mixture was incubated at 25 °C for 1 h. Then, 100 µL of diluted binding agent was added to
each well and incubated at 25 °C for 1 h with slow shaking. The fluorescence polarization of the sample was read
using an excitation filter of 360 nm and an emission filter of 480 nm. The IC50 values were calculated using
nonlinear regression with a normalized dose-response fit using Prism 6 (GraphPad software, San Diego, CA). The
PDEs employed included PDE1A (BPS Bioscience Inc. #60010), PDE2A (BPS Bioscience Inc. #60021), PDE3B
(BPS Bioscience Inc. #60031), PDE4A1A (BPS Bioscience Inc. #60040), PDE5A (BPS Bioscience Inc. #60050),
PDE6C (BPS Bioscience Inc. #60060), PDE7A (BPS Bioscience Inc. #60070) PDE8A1 (BPS Bioscience Inc.
#60080), PDE9A2 (BPS Bioscience Inc. #60090), PDE 10A1 (BPS Bioscience Inc. #60099) and PDE11A (BPS

Bioscience Inc. #60110).
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4.3.2. Blood vessel myography
The vasorelaxant activity of compound 19c–f, 26a–c and 29b,c were determined by according to literature
reported protocols.[29-31] Wistar rats (250–280 g), male and female, were sacrificed by cervical dislocation. The
abdominal cavity was opened and the mesenteric tissue was dissected out quickly. 3rd mesenteric arteries (110–150
µm) were isolated with the aid of stereo-dissection microscope, and the isolated artery was soon placed in ice-cold
PSS solution (composition in mmol/L: NaCl 144, KCl 5.8, MgCl2 1.2, CaCl2 2.5, HEPES 5 and glucose 11.1, pH
7.4), pre-equilibrated with 95% O2/5% CO2 for 15 min. Isolated mesenteric artery was cut into about 3–4 mm
length rings without branching. The vessel was carefully transferred to the chamber glass cannulas and well
mounted. The vessel equilibrated for 35 min at a transmural pressure sequence, from 10 mmHg to 60 mmHg (10
mmHg pressure raised every 5 min). The outer diameter was measured by Pressure Myograph System
(DMT-112PP, Danish Myo Technology A/S). Each vessel was checked for well responses to the addition of K–PSS
solution (120 mM KCl) and acetylcholine (10-5 µM) before agonist and antagonist stimulation. Then vessel then
contracted with phenylephrine (20 µM), and tested compounds were continuous added carefully. Ca2+-free PSS
solution was finally applied for maximal dilation. The vasorelaxant effect was determined as a percentage of the
response calculated using the following formula: Response (%) = (Record diameter (µm) - Minimized diameter
(µm)) / (Maximal diameter (µm) - Minimized diameter (µm)) × 100%.
All the EC50 values were calculated using nonlinear regression with a normalized dose-response fit using Prism
6 (GraphPad software, San Diego, CA).
4.3.3. Docking.
Docking studies were performed using SYBYL-X and the GOLD program as described [33], employing the
crystal structure of PDE5 in a complex with sildenafil downloaded from the PDB (PDB code 1xoz).
Acknowledgement
This work was financially supported by the National Natural Science Foundation of China (81630093), Focus
on Research and Development Plan in Shandong Province (2017CXGC1403), Focus on research and development
plan in Shandong province (2018GSF118213) and the National Science and Technology Project of China (No.
2018ZX09301042-002)
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