Novel PDE5 inhibitors derived from rutaecarpine for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.bmcl.2020.127097

A series of novel rutaecarpine derivatives were synthesized and subjected to pharmacological evaluation as PDE5 inhibitors. The structure–activity relationships were discussed and their binding conformation and simultaneous interaction mode were further clarified by the molecular docking studies. Among the 25 analogues, compound 8i exhibited most potent PDE5 inhibition with IC₅₀ values about 0.086 μM. Moreover, it also produced good effects against scopolamine-induced cognitive impairment in vivo. These results might bring significant instruction for further development of potential PDE5 inhibitors derived from rutaecarpine as a good candidate drug for the treatment of Alzheimer's disease.

Full text of DOI:10.1016/j.bmcl.2020.127097

Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel PDE5 inhibitors derived from rutaecarpine for the treatment of
Alzheimer’s disease
Xian-Feng Huanga, Yan-Hua Donga, Jin-Hui Wangb, Heng-Ming Kea, Guo-Qiang Songa,
⁎
De-Feng Xua,
a School of Pharmaceutical Engineering and Life Sciences & Advanced Catalysis and Green Manufacturing Collaborative Innovation Center, Changzhou University,
Changzhou, Jiangsu 213164, PR China
b Wenzhou No. 3 Clinical Institute Affiliated to Wenzhou Medical University, Wenzhou People’s Hospital, Wenzhou, Zhejiang 325000, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of novel rutaecarpine derivatives were synthesized and subjected to pharmacological evaluation as
PDE5 inhibitors. The structure–activity relationships were discussed and their binding conformation and si-
multaneous interaction mode were further clarified by the molecular docking studies. Among the 25 analogues,
compound 8i exhibited most potent PDE5 inhibition with IC50 values about 0.086 μM. Moreover, it also pro-
duced good effects against scopolamine-induced cognitive impairment in vivo. These results might bring sig-
nificant instruction for further development of potential PDE5 inhibitors derived from rutaecarpine as a good
candidate drug for the treatment of Alzheimer’s disease.
PDE5 inhibitors
Rutaecarpine
Alzheimer’s disease
Alzheimer’s disease (AD), being a long-term and incurable neuro-
degenerative brain disorder, poses a major health problem worldwide.1
As the most common form of dementia, AD is characterized by ag-
gregation of amyloid β peptides (Aβ) into fibrillar plaques that lead to
memory loss.2,3 Currently, many hypotheses, including low levels of
acetylcholine, the deposition of Aβ plaques and the aggregation of tau
protein, etc., have been proposed to explain the pathophysiology of
AD.4 However, the pathogenic mechanism of AD is still not fully un-
derstood. Consequently, there is no ideal drugs for the treatment of AD,
and only a few anti-AD drugs such as acetylcholine esterase inhibitors
(donepezil, rivastigmine, and galantamine), NMDA (N-methyl-D-aspar-
tate) receptor antagonists (memantine) have been used clinically,
which only achieve limited treatment effects accompanied by a lot of
side effects in AD patients.5 Thus, the search for AD drugs remains an
urgent issue in the pharmaceutical community.
cavernosum. PDE5 inhibitors are used to regulate many biological
processes.12 In fact, several PDE5 inhibitors have been approved as
therapeutics for the treatment of various diseases. Currently, various
chemical structures of PDE5 inhibitors including cGMP-based deriva-
tives, β-carboline-derived molecules, quinoline derivatives, iso-
quinolinone derivatives and pyridopyrazinone derivatives have been
reported.13–17 The most well-known drugs are sildenafil, vardenafil,
tadalafil and avanafil, which were approved by the FDA for the treat-
ment of male erectile dysfunction (Viagra) and pulmonary hypertension
(Revatio).18 However, an upregulation of PDE5 expression was also
found in the brains of mild AD patients, and recent studies have de-
monstrated that several PDE5 inhibitors possess a potential therapeutic
effect on AD by reversing cognitive impairment and improving learning
and memory.19–23
Rutaecarpine [Fig. 1], a major quinazolinocarboline alkaloid iso-
lated from Evodia rutaecarpa, exerts extensive biological and pharma-
cological activities such as vasodilation, anti-inflammation and neuro-
Phosphodiesterases (PDEs), being responsible for the hydrolysis of
two second messengers, cyclic AMP (cAMP) and cyclic GMP (cGMP),
can affect neuronal cell survival, and they may play a vital part in
neurodegenerative diseases, such as AD, Parkinson’s disease and
Huntington’s disease, etc.6 It has been reported that different inhibitors
of PDE subtypes such as PDE3, PDE4, PDE5 and PDE9 showed sig-
nificant memory-enhancing effects in vitro and in vivo.7–11 Of all the
PDE subtypes, PDE5 specifically hydrolyzes cGMP, which is widely
distributed in vascular smooth muscle cells, lungs, platelets, and corpus
protectiveeffects.24,25
Considering
quinoline
structures
have
demonstrated to be potent PDE5 inhibitors, we decided to use rutae-
carpine to develop PDE5 inhibitors for the treatment of AD. In this
study, a series of novel PDE5 inhibitors based on rutaecarpine were
synthesized, and tested with PDE5 inhibitory activities and memory
and cognitive function in vivo.
The preparation of a series of rutaecarpine derivatives 6a-6e, 7a-7j
⁎ Corresponding author.
E-mail address: markxu@cczu.edu.cn (D.-F. Xu).
https://doi.org/10.1016/j.bmcl.2020.127097
Received 3 February 2020; Received in revised form 4 March 2020; Accepted 4 March 2020
0960-894X/©2020ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:Xian-FengHuang,etal.,Bioorganic&MedicinalChemistryLetters,https://doi.org/10.1016/j.bmcl.2020.127097

X.-F. Huang, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Table 1
In vitro PDE5 inhibitory activity of rutaecarpine derivatives.
Compounds
R1
R2
R3
PDE5A (μM)a
CLogP
6a
H
H
H
1.23
0.13
0.09
3.20
4.09
4.62
5.54
5.14
3.15
4.12
4.65
5.58
5.18
3.13
4.13
4.66
5.59
5.19
2.54
3.66
4.18
5.11
4.71
1.87
3.06
3.59
4.52
4.12
6b
H
H
CH2CH3
1.12
6c
H
H
(CH2)2CH3
(CH2)2CH(CH3)2
(CH2)3CH3
H
1.7
0.15
Fig. 1. The structure of rutaecarpine.
6d
H
H
0.61
0.07
0.11
6e
H
H
1.12
and 8a-8j was accomplished using the general methods outlined in
Scheme 1..26,27 The reaction of anthranilamide (1) with 5-chlorovaleryl
chloride in CH2Cl2 at ice water furnished the corresponding acid amide
2, which reacted with two equivalents of potassium tert-butoxide in
THF led to two steps of cyclisation and afford 3 and 4, respectively. The
mackinazolinone 4 reacted with in situ generated diazonium salts of
substituted benzenamines at −5 to 5 °C and gave the corresponding
hydrazone 5. The Fischer-indole reaction of hydrazone 5 in refluxing
glacial acetic acid yielded compounds 6a, 7a, and 7f, which was re-
acted with alkyl bromide in DMF to afford 6b-6e, 7b-7e and 7g-7f,
respectively. Compounds 8a-8j were obtained by the further de-
methylation of 7a-7j using BBr3.
7a
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
CH3O
OH
H
22.05
11.63
12.12
6.17
3.52
7b
H
CH2CH3
2.02
1.97
7c
H
(CH2)2CH3
(CH2)2CH(CH3)2
(CH2)3CH3
H
7d
H
0.77
7e
H
9.19
1.05
7f
CH3O
CH3O
CH3O
CH3O
CH3O
H
45.62
47.32
19.76
18.23
20.63
1.36
5.84
7g
CH2CH3
4.49
3.12
2.59
3.76
7h
(CH2)2CH3
(CH2)2CH(CH3)2
(CH2)3CH3
H
7i
7j
8a
0.18
8b
OH
H
CH2CH3
0.97
0.07
0.15
0.02
0.07
0.08
0.11
0.05
8c
OH
H
(CH2)2CH3
(CH2)2CH(CH3)2
(CH2)3CH3
H
1.33
8d
OH
H
0.11
8e
OH
H
0.63
Rutaecarpine and its derivatives (25 compounds) were evaluated for
the PDE5 inhibitory activities. The inhibitory activities were presented
as IC50 (μM) and the results were summarized in Table 1. The well
known PDE5 inhibitor tadalafil was used as positive reference. Rutae-
carpine in this study exhibited PDE5 inhibition with IC50 value about
1.23 μM, manifesting it is a promising candidate as PDE5 inhibitors.
The following results showed that the introduction of substituted group
such as methoxy and hydroxyl on phenyl ring (R1 and R2) of rutae-
carpine could significantly influence the inhibitory activity of the de-
rivatives. According to the screening data, we found that the OH group
substitution can increase their inhibitory activity. Especially, compound
8i showed an IC50 of 86 nM. On the contrary, compounds with CH3O
groups remarkably decreased the PDE5 inhibitory activities. For ex-
ample, compounds 7f-7j with two CH3O groups only exhibited weak
activities (IC50 values: 18.23–47.32 μM). The influence of substituent
group on N atom (R3) on PDE5 inhibitory activity was also evaluated. It
seems that these compounds with N-butyl substituents and isopentenyl
compounds exhibited more potent PDE5 inhibitory activities than other
compounds with small substituent group. In brief, the bulk of R3 sub-
stituents deeply influenced PDE5 inhibitory activities. Large R sub-
stituents were favorable for the activity, while small R substituents
were adverse. The most potent compound 8i was also evaluated for the
PDE5 inhibitory selectivity over other PDEs (PDE2, 4, 5, 9 and 10)
[Table 2]. The compound displayed > 5000-fold selectivity with
IC50 > 500 μM against PDE2, 4, 9, and inhibited moderately PDE6 and
10 with IC50 values of 43.8 and 32.1 μM, respectively. It was noticed
8f
OH
OH
OH
OH
OH
OH
0.79
8g
OH
CH2CH3
0.97
8h
OH
(CH2)2CH3
(CH2)2CH(CH3)2
(CH2)3CH3
0.33
8i
OH
0.086
0.26
0.009
0.03
0.0001
8j
OH
Tadalafil
Donepezil
0.005
> 100
a
Results are expressed as the mean of at least three experiments.
that compound 8i showed a very excellent selectivity over PDE6
(> 500-fold), while sildenafil has only 10-fold selectivity over PDE6,
which is the main reason for its side effect of visual disturbance.28
Furthermore, most of the derivatives have appropriate values of CLogP
(2.0–5.0), indicating favorable lipophilicity that may allow blood
brain barrier penetration.
To explore the interaction mode of the optimal compound 8i with
PDE5, molecular docking simulation was performed using discovery
studio 2017 software based on the crystal structure of hPDE5A com-
plexed tadalafil (PDB ID: 1UDU). The predicted binding mode of
compound 8i within the active site pockets of hPDE5A is presented in
Fig. 2. In general, compound 8i contacts with Gln817, Phe820, Met816,
Gln775, Tyr 612, Leu804 and Phe786 of hPDE5A via three hydrogen
bonds and hydrophobic interactions. In detail, the indole fragment of 8i
interacts with the phenyl ring of Phe820 through face-to-face π-π
stacking interactions and the OH of indole fragment interacts with
Gln817, Gln775 and Tyr 612 via hydrogen bond interactions. Moreover,
Scheme 1. Synthesis of rutaecarpine derivatives.
Reagents and conditions: (a) N(C2H5)3, 5-chlor-
ovaleryl chloride, CH2Cl2, 0 °C to r.t., 2 h; (b)
potassium tert-butoxide, THF, 0 °C, 2 h; (c) po-
tassium tert-butoxide, THF, r.t., 5 h; (d) amines,
NaNO2, 20% HCl, CH3COOH, 0 °C; (e) ZnCl2,
CH3COOH, reflux; (f) R3Br, K2CO3, DMF,
37–80 °C; (g) BBr3, CH2Cl2, 0 °C to r.t.
2

X.-F. Huang, et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Table 2
PDE selectivity of compounds 8i.
Compounds
PDE IC50 (μM)a
PDE5
0.086
PDE2
PDE4
PDE6
43.8
PDE9
PDE10
32.1
8i
0.007
> 500
> 500
2.7
> 500
2.9
a
Results are expressed as the mean of at least three experiments.
into the cavity toward the hydrophobic Met816, Leu804 and Phe786,
which is different with compound 8i. Overall, the above docking stu-
dies provided an explanation for enzyme assay results that both sub-
stitution on aromatic ring and side chain on N atom could evidently
influence the inhibitory activity of these derivatives.
To evaluate the in vivo effect of compound 8i on cognitive im-
provement, the passageway water maze experiment was performed on a
scopolamine (Scop)-induced cognitive deficit mouse model with done-
pezil (Don) as a positive control (Ctrl). As shown in Fig. 3, the Scop
group (model group) exhibited a longer escape latency and more fre-
quent errors than the Ctrl group (***p < 0.001). The groups treated
with 5 and 10 mg/kg compound 8i demonstrated shorter escape la-
tencies and less frequent errors than the Scop group [F(4, 40) = 9.65,
#p < 0.05; F(4, 40) = 13.69, ##p < 0.01], confirming that the
compound could relieve the Scop-induced learning and memory deficits
at dosages of 5 and 10 mg/kg, which was comparable to donepezil.
In conclusion, a series of novel rutaecarpine derivatives with ex-
cellent PDE5 inhibitory activities were designed and synthesized, the
SARs of the 25 compounds were explicit and provided some insights
into the structural modification of effective PDE5 inhibitors based on
rutaecarpine. Of all the rutaecarpine derivatives, compounds 8i showed
high inhibitory activities with the IC50 values of 86 nM, and good se-
lectivity over PDEs 2, 4, 6, 9 and 10. Furthermore, The in vivo study
suggested that compound 8i could improve cognitive dysfunction in an
AD mouse model. Our study provided a basis for the rational design of
novel rutaecarpine derivatives as PDE5 inhibitors, and subsequent ef-
forts on further optimization of this structural class would lead to more
potent and selective PDE5 inhibitors as a good candidate drug for the
treatment of AD.
Fig. 2. Binding mode of compounds 8i (red) and 7a (blue) within the active
pocket of PDE5A (PDB ID: 1UDU). Hydrogen bonds are represented by dashed
lines (green).
quinazolin-4(3H)-one of 8i were also found to form hydrophobic in-
teractions with the phenyl ring of Phe786. The side chain on N atom of
8i occupies the hydrophobic pocket of hPDE5A, forming hydrophobic
interactions with Met816, Leu804 and Phe786. By contrast, compound
7a, exhibiting weak PDE5A inhibitory activity, was accommodated into
the active pocket with a slightly different behaviour. Although its in-
dole fragment also interacts with the phenyl ring of Phe820 through a
face-to-face π-π stacking, no hydrogen bonds were observed in this
mode. On the other hand, its methoxy substituted at the indole ring fits
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Fig. 3. Effects of compound 8i (5 and 10 mg/kg) on scopolamine-induced cognitive deficits in mice. Donepezil (10 mg/kg) as a reference. (A) The escape latency of
mice in the passageway water maze. (B) The number of errors of mice in the passageway water maze. Data are shown a s the mean
group, #p < 0.05 and ##p < 0.05 vs Scop group (one-way ANOVA followed by post hoc Dunnett’s test).
SEM, ***p < 0.001 vs control
3

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Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxxx
Acknowledgment
8. Zhu L, Yang JY, Xue X, et al. Mech Ageing Dev. 2015;150:34.
9. Gomez L, Massari ME, Vickers T, et al. J Med Chem. 2017;60:2037.
10. Park SH, Kim JH, Bae SS, et al. Biochem Biophys Res Commun. 2011;408:602.
11. García-Osta A, Cuadrado-Tejedor M, García-Barroso C, Oyarzábal J, Franco R. ACS
Chem Neurosci. 2012;3:832.
This study was supported by the Postgraduate Research & Practice
Innovation Program of Jiangsu Province (SJCX19_0631).
12. Andersson KE. Br J Pharmacol. 2018;175:2554.
13. Yu G, Mason H, Wu X, et al. J Med Chem. 2003;46:457.
Appendix A. Supplementary data
14. Yu G, Mason HJ, Wu X, et al. J Med Chem. 2001;44:1025.
15. Watanabe N, Adachi H, Takase Y, et al. J. Med. Chem. 2000;43:2523.
16. Ukita T, Nakamura Y, Kubo A, et al. J Med Chem. 2001;44:2204.
17. Ukita T, Nakamura Y, Kubo A, et al. Bioorg Med Chem Lett. 2003;13:2341.
18. Bruzziches R, Francomano D, Gareri P, Lenzi A, Aversa A. Expert Opin Pharmacother.
2013;14:1333.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bmcl.2020.127097.
19. Prickaerts J, Steinbusch HWM, Smits JFM, Vente JD. Eur J Pharmacol. 1997;337:125.
20. Boccia MM, Blake MG, Krawczyk MC, Baratti CM. Behav Brain Res. 2011;220:319.
21. Hosseini-Sharifabad A, Ghahremani MH, Sabzevari O, Naghdi N, Abdollahi M.
Pharmacol Biochem Behav. 2012;101:311.
References
1. Wimo A, Jonsson L, Bond J, Prince M, Winblad B. Alzheimers Dement.
2013;9(1–11):e3.
22. Al-Amin MM, Hasan SM, Alam T, et al. Eur J Pharmacol. 2014;745:84.
23. Bi Y, Stoy P, Adam L, et al. Bioorg Med Chem Lett. 2004;14:1577.
24. Choi YH, Shin EM, Kim YS, Cai XF, Lee JJ, Kim HP. Arch Pharm Res. 2006;29:293.
25. Hu CP, Xiao L, Deng HW, Li YJ. Planta Med. 2002;68:705.
26. Charlotte LS, Steven VL. Synthesis. 2017;49:135.
2. Blennow K, de Leon MJ, Zetterberg H. Lancet. 2006;368:387.
3. Masliah E. Histol Histopathol. 1995;10:509.
4. Wilson RS, Segawa E, Boyle PA, Anagnos SE, Hizel LP, Bennett DA. Psychol Aging.
2012;27:1008.
5. Loveman E, Green C, Kirby J, et al. Health Technol Assess. 2006;10:1.
6. Bollen E, Prickaerts J. IUBMB Life. 2012;64:965.
7. Reneerkens OA, Rutten K, Steinbusch HW, Blokland A, Prickaerts J.
Psychopharmacology. 2009;202:419.
27. Santosh BM, Narshinha PA. Tetrahedron. 2004;60:3417.
28. Marmor MF, Kessler R. Surv. Ophthalmol. 1999;44:153.
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