Neuroprotective effects of mercaptoethylleonurine and mercaptoethylguanidine analogs on hydrogen peroxide-induced apoptosis in human neuronal SH-SY5Y cells

Article abstract of DOI:10.1016/j.bmcl.2013.01.038

A series of mercaptoethylleonurine and mercaptoethylguanidine derivatives were designed and synthesized. Their neuroprotective effects toward H ₂O₂-induced apoptosis were investigated in human SH-SY5Y cells. The results from these studies identified several potent compounds, with compound 8k emerging as the most effective. Further investigation demonstrated that 8k reduced H₂O₂-induced activation of mitochondrial apoptosis by inhibiting the expression of Bax and elevating the expression of Bcl-2. Moreover, the molecular mechanism underlying the observed neuroprotective effects of 8k was exerted via the Akt and JNK pathways. Compound 8k can be a lead compound for further discovery of neuroprotective medicine.

Full text of DOI:10.1016/j.bmcl.2013.01.038

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1793–1796
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Neuroprotective effects of mercaptoethylleonurine
and mercaptoethylguanidine analogs on hydrogen peroxide-induced
apoptosis in human neuronal SH-SY5Y cells
c
a
a,d,
Zhao-Lin Que a, , Wen-Jiang Zhou b, , Jun Chang a, , Xin-Hua Liu , Jian-Ming Yu , Xun Sun
⇑
⇑
a Department of Natural Products Chemistry, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China
b Animal Center, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China
c Department of Pharmacology, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China
d Key Lab of Smart Drug Delivery, Ministry of Education & PLA, Fudan University, 826 Zhangheng Road, Shanghai 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A
series of mercaptoethylleonurine and mercaptoethylguanidine derivatives were designed and
Received 20 November 2012
Revised 30 December 2012
Accepted 12 January 2013
Available online 23 January 2013
synthesized. Their neuroprotective effects toward H2O2-induced apoptosis were investigated in human
SH-SY5Y cells. The results from these studies identified several potent compounds, with compound 8k
emerging as the most effective. Further investigation demonstrated that 8k reduced H2O2-induced
activation of mitochondrial apoptosis by inhibiting the expression of Bax and elevating the expression
of Bcl-2. Moreover, the molecular mechanism underlying the observed neuroprotective effects of 8k
was exerted via the Akt and JNK pathways. Compound 8k can be a lead compound for further discovery
of neuroprotective medicine.
Keywords:
Mercaptoethylleonurine
Mercaptoethylguanidine
Neuroprotective effect
SH-SY5Y cell
Ó 2013 Elsevier Ltd. All rights reserved.
Parkinson’s disease (PD), the second most common neurode-
generative disorder after Alzheimer’s disease, is mainly character-
ized by the loss of dopaminergic neurons in the substantial nigra
pars compacta.1,2 Although the underlying mechanisms in the neu-
rodegenerative processes of PD remain unknown, many studies
have suggested the involvement of oxidative stress and mitochon-
drial dysfunction in the activation of an apoptotic cascade that ulti-
mately results in the loss of dopaminergic neurons.3,4 Oxidative
stress caused by excessive reactive oxygen species (ROS) produc-
tion has been shown to lead to cellular dysfunction, culminating
in cell death.5 The most common ROS are oxygen radicals, such
as superoxide and hydroxyl radicals, and non-free radicals, such
as hydrogen peroxide. All of these reactive oxygen species are gen-
erated in various redox processes in the human body. The genera-
tion of ROS in normal cells is tightly regulated by biological
antioxidants and antioxidant enzymes. These ROS cause oxidative
damage to molecules such as carbohydrates, proteins, lipids, and
DNA. Many antioxidant compounds can prevent these oxidative-
stress-related disorders by scavenging ROS.6 Hydrogen peroxide
induces apoptosis in a variety of cells, including neuronal cells,
and is a precursor of highly reactive free radicals.7
Many naturally occurring compounds have been utilized as
leads in drug discovery, and efforts also have been made to search
compounds with neuroprotective effects.8–10 Leonurine (Fig. 1), a
cardioprotective drug candidate, was reported to exhibit cardio-
protective effects both in vivo and in vitro,8,9 and its preclinical
study is on-going. Moreover, Shi et al. recently reported that leon-
urine significantly reduced 6-hydroxydopamine (6-OHDA)-in-
duced cell death in dopaminergic SH-SY5Y cells and attenuated
apomorphine-elicited rotational behavior in 6-OHDA-lesioned
rats.11 Structurally, the guanidine moiety in leonurine may play a
very important role in its activity. Similarly, mercaptoethylguani-
dine (MEG), another scavenger bearing a guanidine unit, can react
with peroxynitrite and protect against peroxynitrite-induced
oxidative damage.12 MEG was also reported to have neuroprotec-
tive,13 cardioprotective,14–16 and radioprotective effects.17
Although both leonurine and MEG have interesting neuroprotec-
tive effects, their structure–activity relationships (SAR) have not
been extensively explored.
Herein, by considering the common structural features of
leonurine and MEG, we designed and synthesized a series of new
mercaptoethylleonurine (MEL) analogs (1a–h) by incorporating a
mercaptoethyl group and changing the chain length (R1 group) to
find new chemical entities with better neuroprotective activity
than leonurine and MEG. The in vivo biological activities could
depend on many parameters, including bioavailability. Given that
increasing aqueous solubility generally results in enhanced
⇑
Corresponding authors. Tel./fax: +86 21 51980003.
E-mail addresses: jchang@fudan.edu.cn (J. Chang), sunxunf@shmu.edu.cn
(X. Sun).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.01.038

1794
Z.-L. Que et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1793–1796
OH
MeO
OMe
NH
NH2
R2
NH
SH
NH
R1
O
O
SH
X
N
H
N
NH
Leonurine
R1
H
SH
NH
O
O
N
H
N
H
H2N
N
H
Mercaptoethylleonurine (MEL)
analogs (1a-h)
Mercaptoethylguanidine (MEG)
analogs (8a-k)
X = OH, NH2
Mercaptoethylguanidine (MEG)
Figure 1. Design strategy.
X = OH, R1 = (CH2)2
X = OH,
X = OH,
R
R
1 = CH2CH(CH3)
1 = CH(CH3)CH2
8a
8g
8h
X = OH, R1 = (CH2)3
8b
NH
NH
R1
8c X = OH, R1 = (CH2)4
8d X = OH, R1 = (CH2)5
8e X = OH, R1 = (CH2)6
8i X = OH, R1 = CH2CH(CH2CH3)
8j X = NH2, R1 = (CH2)2
R1
X
b
N
a
SH.CS2
SH
S
+
X
N
H
N
H
X
N
H
N
H
R1 NH2
8k X = NH2, R1 = (CH2)3
NH2
4a-k
X = OH, R1
=
8a-k
8f
2
3a-k
OH
c
OBn
MeO
OMe
MeO
OMe
R1
NBoc
N
R1
d
e
NH
R1
SH
NBoc
N
N
HO
SH
OBn
SH
Boc Boc
O
O
N
N
H
O
O
N
H
MeO
OMe
Boc Boc
5b-f
7a-e
1a-e
1a R1 = (CH2)3
1b R1 = (CH2)4
7a R1 = (CH2)3
5b
R
1 = (CH2)3
5c R1 = (CH2)4
7b R1 = (CH2)4
R1 = (CH2)5
1c
1d
O
OH
R1 = (CH2)5
R1 = (CH2)6
=
R
R
1 = (CH2)5
1 = (CH2)6
=
5d
5e
7c
7d
6a
d
R
1 = (CH2)6
5f R1
=
1e R1
7e R1
R2
R2
NBoc
N
(CH2)4
f
NH
R1
SH
NBoc
N
N
(CH2)4
HO
SH
Boc Boc
SH
O
O
N
H
N
O
O
R2
H
N
Boc Boc
1f-h
5c
7f-h
7f R2 = 3,5-diMeO
1f R1 = (CH2)4, R2 = 3,5-diMeO
O
OH
R
2 = 3,4,5-triMeO
R2 = 4-MeO
1g
1h
R
R
1 = (CH2)4, R2 = 3,4,5-triMeO
1 = (CH2)4, R2 = 4-MeO
7g
7h
6b-c
Scheme 1. Reagents and conditions: (a) (i) HBr, MeCN, H2O, 110 °C, 1–2 h; (ii) CS2, H2O, 0 °C, 12 h; (b) HCl, MeOH, 40–50 °C, 2–3 h; (c) Na2CO3, (Boc)2O, dioxane:H2O = 1.5:1,
rt, 24–72 h; (d) EDC, DMAP, CH2Cl2, 0 °C, 3 h; then rt, 24–72 h; (e) (i) HCOOH, rt, 24–28 h; (ii) HCl, MeOH, rt, 2–3 h; (f) HCOOH, rt, 24–28 h.
bioavailabilities in vivo, we also designed and synthesized 8a–k in
an attempt to improve bioavailability by increasing the hydrophilic
character of the molecules.
found that the benzyl group could be only easily removed in 4 N
HCl in MeOH. Analogs 1f–h were obtained in good yields in a sim-
ilar manner as 1a–e. The MEG derivatives were prepared from 4a–
k using hydrochloric acid in methanol at 40 °C, followed by HPLC
purification to provide compounds 8a–k in 29–50% yield.19
Eight MEL analogs (1a–h) and 11 MEG analogs (8a–k) (Table 1)
were evaluated for their neuroprotective activity toward H2O2-in-
duced apoptosis in human SH-SY5Y cells.20 The cytotoxic potential
The synthesis of our target compounds started from 2-amino-2-
thiazoline (2), a commercially available starting material, as illus-
trated in Scheme 1. The treatment of 2-amino-2-thiazoline (2) with
amino alcohols (3a–3i) or diamines (3j–3k) in the presence of 40%
hydrobromic acid followed by the addition of carbon disulfide
afforded guanidine derivatives (4a–k).18 Guanidines 4b–f were
converted to their corresponding tri-Boc substituted intermediates
5b–f using excess Boc2O and sodium carbonate as a base in 49–66%
yield. The free hydroxyl groups in 5b–f were coupled with 4-ben-
zyloxy-3,5-dimethoxy-benzoic acid (6a) to afford esters 7a–e in
36–48% yield in the presence of N-ethyl-N0-(3-dimethylaminopro-
pyl) carbodiimide hydrochloride (EDC) and 4-dimethylaminopyri-
dine (DMAP). After subsequently removing the Boc and benzyl
protecting groups on 7a–e with formic acid and hydrochloric acid,
new MEL analogs (1a–e) were synthesized in desirable yields. It is
worth mentioning that the debenzylation step was problematic.
After numerous attempts of failure (using various palladium cata-
lytic hydrogenations; ferric chloride; Li-naphthalene; etc.), it was
of 1a–h and 8a–k has been performed at 5–100 lM concentration
by MTT assay and cytotoxic effect of them was not observed at the
dosage used in this study. Among analogs 1a–h, only 1a exhibited
greater neuroprotective activity than leonurine relative to the po-
sitive control N-acetyl-cysteine (NAC) at 5 mM. Increasing the side
chain length (R1 substitution) from three carbons (1a) to four, five,
and six carbons (1b–d, respectively), did not improve their neuro-
protective activity. Although compound 1d had a similar activity to
1a at low concentrations (5
lower at higher concentrations (25
l
M), the activities were dramatically
M and 50 M). Replacing the
l
l
straight side chain with cyclohexyl linkage also decreased the
activity of 1e relative to 1a. Compared with leonurine, compound
1b, which contained a mercaptoethyl group on the guanidine of

Z.-L. Que et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1793–1796
1795
Table 1
Neuroprotective activity of compounds 1a–1h, and 8a–8k on H2O2-induced SH-SY5Y cells
Figure 2. Compound 8k modulated H2O2-induced Bax and Bcl-2 protein changes in SH-SY5Y cells. Cells were treated with 8k (5, 25, 50
l
M) or NAC (5 mM) for 4 h and then
stimulated by H2O2 (100 lM) for another 12 h before western blotting. (A) Bar graph showing the quantitative analysis of Bcl-2 expression with GAPDH as the loading control.
(B) Bar graph showing the quantitative analysis of Bax expression with GAPDH as the loading control. Data are mean SEM of results from at least three independent
experiments, each performed in duplicate, #P <0.05 versus control cells; &P <0.05 versus H2O2-stimulated cells.
leonurine, also exhibited only moderate activity. Further modifica-
tion of the substitution patterns on the phenyl ring (1f–h) did not
produce any promising analogs. Interestingly, after removing the
phenyl ring, some analogs in the free hydroxyl analog series
(8a–i), such as 8b and 8d, retained good activity at all three doses.
Further SAR development included the replacement of the hydro-
xyl group with an amino group to yield compound 8k, a bioisostere
of 8b. Surprisingly, compound 8k was the most potent analog both
series. Based on its excellent neuroprotective activity, 8k was
chosen for further study.
The activation of phosphatidylinositol 3-kinase (PI3 K) is known
to promote cell survival and prevent apoptosis.21,22 The anti-apop-
totic effects of PI3 K are mediated by its downstream target Akt,
which may regulate the expression of several apoptosis-related
genes, such as Bcl-2/Bax proteins, which mediate apoptosis.23,24
Therefore, the elucidation of the molecular mechanism involved
in this effect might provide novel insight into the process of
neurodegeneration and suggest the potential utility of MEL and
MEG analogs for protection against neurodegenerative diseases
caused by oxidative stress and apoptosis.
In this study, H2O2 stimulation significantly inhibited the
expression of Bcl-2 and increased Bax expression in SH-SY5Y cells
(P <0.05). In the cells treated with compound 8k and NAC, the ef-
fects of H2O2 on the expression of Bcl-2 and Bax were evidently re-
versed (Fig. 2). Additionally, the effects of 8k were dose-
dependent. As shown in Figure 3, compound 8k can attenuate
the H2O2-induced phosphorylation of JNK and Akt in SH-SY5Y cells
but does not affect the phosphorylation of p38 and ERK relative to
the H2O2-stimulated cells.
In summary, eight MEL analogs (1a–h) and eleven MEG analogs
(8a–k) were designed and synthesized. Analogs 1a, 8b, 8d, 8j, and
8k showed neuroprotective activity against H2O2-induced apopto-
sis in human SH-SY5Y cells, with 8k exhibiting the highest neuro-
protective activity. Further studies showed that compound 8k can

1796
Z.-L. Que et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1793–1796
2. Schapira, A. H.; Bezard, E.; Brotchie, J.; Calon, F.; Collingridge, G. L.; Ferger, B.;
Hengerer, B.; Hirsch, E.; Jenner, P.; Le Novere, N.; Obeso, J. A.; Schwarzschild, M.
A.; Spampinato, U.; Davidai, G. Nat. Rev. Drug Disc. 2006, 5, 845.
3. Mattson, M. P. Natl. Rev. Mol. Cell Biol. 2000, 1, 120.
4. Brown, J. M.; Yamamoto, B. K. Pharmacol. Ther. 2003, 99, 45.
5. Fleury, C.; Mignotte, B.; Vayssiere, J. L. Biochemie 2002, 84, 131.
6. Slemmer, J. E.; Shacka, J. J.; Sweeney, M. I.; Weber, J. T. Curr. Med. Chem. 2008,
15, 404.
7. Sherer, T. B.; Betarbet, R.; Stout, A. K.; Lund, S.; Baptista, M.; Panov, A. V.;
Cookson, M. R.; Greenamyre, J. T. J. Neurosci. 2002, 22, 7006.
8. Liu, X. H.; Xin, H.; Hou, A. J.; Zhu, Y. Z. Clin. Exp. Pharmacol. 2009, 36, 696.
9. Loh, K. P.; Qi, J.; Tan, B. K.; Liu, X. H.; Wei, B. G.; Zhu, Y. Z. Stroke 2010, 41, 2661.
10. Kang, S. S.; Lee, J. Y.; Choi, Y. K.; Kim, G. S.; Han, B. H. Bioorg. Med. Chem. Lett.
2004, 14, 2261.
11. Shi, X. R.; Hong, Z. Y.; Liu, H. R.; Zhang, Y. C.; Zhu, Y. Z. Neurochem. Int. 2011, 58,
851.
12. Szabo, C.; Ferrer-Sueta, G.; Zingarelli, B.; Southan, G. J.; Salzman, A. L.; Radi, R. J.
Biol. Chem. 1997, 272, 9030.
13. Moochhala, S. M.; Lu, J.; Xing, M. C.; Anuar, F.; Ng, K. C.; Yang, K. L.; Whiteman,
M.; Atan, S. J. Trauma 2005, 59, 448.
14. Szabo, A.; Hake, P.; Salzman, A. L.; Szabo, C. Crit. Care Med. 1999, 27, 1343.
15. Zingarelli, B.; Ischiropoulos, H.; Salzman, A. L.; Szabo, C. Eur. J. Pharmacol. 1997,
338, 55.
16. Ozturk, M.; Ozler, M.; Kurt, Y. G.; Ozturk, B.; Uysal, B.; Ersoz, N.; Yasar, M.;
Demirbas, S.; Kurt, B.; Acikel, C.; Oztas, Y.; Arpaci, F.; Topal, T.; Ozet, A.;
Ataergin, S.; Kuzhan, O.; Oter, S.; Korkmaz, A. J. Pineal Res. 2011, 50, 89.
17. Mandrugin, A. A.; Fedoseev, V. M.; Dubova, O. V.; Rodunin, A. A.;
Konstantinova, M. M.; Dontsova, G. V. Radiats. Biol. Radioekol. 1999, 39, 254.
18. Mandrugin, A. A.; Fedsoseev, V. M.; Rodunin, A. A.; Semenko, M. N. Chem.
Heterocycl. Compd. 2001, 37, 360.
19. Representative experiment for the preparation of 1-(3-aminopropyl)-3-(2-
mercaptoethyl)guanidine (8k): 2-Amino-2-thiazoline (2, 306 mg, 3.0 mmol)
was dissolved in acetonitrile–water (5 mL, 3:2), and 40% hydrobromic acid
(0.6 mL) was added. After the mixture was stirred for 5 min, compound 3k
(12.0 mmol) was added. The solution was heated in a sealed vessel at 110 °C
for 1–2 h. After cooling to 0 °C, water (5 mL) and carbon disulfide (0.5 mL) were
added with stirring, and the mixture was kept at 0 °C for 12 h. The yellow solid
formed was filtered, washed with ethanol and ether, and dried to give
compounds 4k as a yellowish solid in 45% yield, which will be used directly in
the next step. Compound 4k was dissolved in MeOH (5 mL), and 4 N HCl in
MeOH (5 mL) was added. The solution was heated to 40–50 °C and stirred until
all of the yellow solid was dissolved (2–3 h). Next, the reaction mixture was
concentrated and the residue was purified by HPLC to give compound 8k as a
yellowish oil (42%). 1H NMR (CD3OD, 400 MHz) d ppm: 3.51 (2H, d, J = 6.26 Hz),
3.37 (4H, m), 2.91 (2H, d, J = 6.46 Hz), 1.96 (2H, m); 13C NMR (CD3OD,
100 MHz) d ppm: 153.22, 43.73, 38.39, 23.00, 19.71; C6H16N4S: ESIMS m/z
160.1 [MꢀNH3+H]+; HRMS m/z calcd 160.0864 [MꢀNH3+H]+, found 160.0888.
20. Zhang, H. A.; Gao, M.; Zhang, L.; Zhao, Y.; Shi, L. L.; Chen, B. N.; Wang, Y. H.;
Wang, S. B.; Du, G. H. Biochem. Biophys. Res. Commun. 2012, 421, 479.
21. Burke, R. E. Pharmacol. Ther. 2007, 114, 261.
Figure 3. Compound 8k attenuated H2O2-induced phosphorylation of Akt and JNK
in SH-SY5Y cells. Cells were pretreated with 50
model groups were stimulated with H2O2 (100
l
M 8k for 4 h. Next, the cells in
lM) for the indicated periods and
then collected. The phosphorylation of Akt, JNK, ERK, and p38 was detected by
Western blot using the sum of Akt, JNK, ERK, and p38 as the loading control. Data
are results from at least three independent experiments, each performed in
duplicate.
reduce the H2O2-induced activation of mitochondrial apoptosis by
inhibiting the expression of Bax and elevating the expression of
Bcl-2. Moreover, the molecular mechanism underlying the ob-
served neuroprotective effects of 8k was more probable via the
Akt and JNK pathways. This finding may provide insight for our fu-
ture design of leonurine analogs with optimal neuroprotective
activities.
Acknowledgments
Financial support by the National Natural Science Foundation of
China (No: 81172920), Shanghai Municipal Committee of Science
and Technology (No: 10431903100), and National Basic Research
Program of China (973 Program, No: 2010CB912603) are
acknowledged.
22. Timmons, S.; Coakley, M. F.; Moloney, A. M. Neurosci. Lett. 2009, 467, 30.
23. Wu, Y.; Shang, Y.; Sun, S.; Liu, R. Eur. J. Pharmacol. 2007, 564, 47.
24. Zhang, D.; Zhang, J. J.; Liu, G. T. Neuropharmacology 2007, 52, 423.
References and notes
1. de Lau, L. M.; Breteler, M. M. Lancet Neurol. 2006, 5, 525.