Novel anti-Alzheimer phenol-lipoyl hybrids: Synthesis, physico-chemical characterization, and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2019.111880

To date, drugs that hit a single target are inadequate for the treatment of neurodegenerative diseases, such as Alzheimer's or Parkinson's diseases. The development of multitarget ligands, able to interact with the different pathways involved in the progession of these disorders, represents a great challenge for medicinal chemists. In this context, we report here the synthesis and biological evaluation of phenol-lipoyl hybrids (SV1-13), obtained via a linking strategy, to take advantage of the synergistic effect due to the antioxidant portions and anti-amyloid properties of the single constituents present in the hybrid molecule. Biological results showed that SV5 and SV10 possessed the best protective activity against Aβ_1-42 induced neurotoxicity in differentiated SH-SY5Y cells. SV9 and SV10 showed remarkable antioxidant properties due to their ability to counteract the damage caused by H₂O₂ in SHSY-5Y-treated cells. Hovewer, SV5, showing moderate antioxidant and good neuroprotective activities, resulted the best candidate for further experiments since it also resulted stable both simulated and plasma fluids.

Full text of DOI:10.1016/j.ejmech.2019.111880

Journal Pre-proof
Novel anti-Alzheimer phenol-lipoyl hybrids: Synthesis, physico-chemical
characterization, and biological evaluation
Aikaterini Pagoni, Lisa Marinelli, Antonio Di Stefano, Michele Ciulla, Hasan Turkez,
Adil Mardinoglu, Stamatia Vassiliou, Ivana Cacciatore
PII:
S0223-5234(19)31032-3
DOI:
https://doi.org/10.1016/j.ejmech.2019.111880
EJMECH 111880
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 October 2019
Revised Date: 8 November 2019
Accepted Date: 11 November 2019
Please cite this article as: A. Pagoni, L. Marinelli, A. Di Stefano, M. Ciulla, H. Turkez, A. Mardinoglu,
S. Vassiliou, I. Cacciatore, Novel anti-Alzheimer phenol-lipoyl hybrids: Synthesis, physico-chemical
characterization, and biological evaluation, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.111880.
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Novel anti-Alzheimer phenol-lipoyl hybrids:
synthesis, physico-chemical characterization,
and biological evaluation
Aikaterini Pagoni,a Lisa Marinelli, b Antonio Di Stefano, b Michele Ciulla, b Hasan Turkez,c
Adil Mardinoglud,e Stamatia Vassiliou,**,a and Ivana Cacciatore*, b
aLaboratory of Organic Chemistry, Department of Chemistry, National and Kapodistrian
b
University of Athens, Athens, Greece; Department of Pharmacy, “G. D’Annunzio”
c
University of Chieti-Pescara, 66100 Chieti Scalo (CH), ITALY; Department of Molecular
Biology and Genetics, Faculty of Science, Erzurum Technical University, Erzurum,
TURKEY; dScience for Life Laboratory, KTH, Royal Institute of Technology, Stockholm, SE-
e
17121, SWEDEN; Centre for Host-Microbiome Interactions, Faculty of Dentistry, Oral &
Craniofacial Sciences, King’s College London, London, SE1 9RT, UNITED KINGDOM.
KEYWORDS
Alzheimer’s disease, Hybrid, Lipoic acid, Polyphenol.
ABBREVIATIONS
DMAP,
4-Dimethylaminopyridine;
EDC.HCl,
N-(3-dimethylaminopropyl)-N′-
ethylcarbodiimide hydrochloride; HOBt, 1-Hydroxybenzotriazole; TEA, triethylamine; TFA,
trifluoroacetic acid.
1

ABSTRACT
To date, drugs that hit a single target are inadequate for the treatment of neurodegenerative
diseases, such as Alzheimer’s or Parkinson’s diseases. The development of multitarget
ligands, able to interact with the different pathways involved in the progession of these
disorders, represents a great challenge for medicinal chemists.
In this context, we report here the synthesis and biological evaluation of phenol-lipoyl
hybrids (SV1-13), obtained via a linking strategy, to take advantage of the synergistic effect
due to the antioxidant portions and anti-amyloid properties of the single constituents present
in the hybrid molecule. Biological results showed that SV5 and SV10 possessed the best
protective activity against Aβ1-42 induced neurotoxicity in differentiated SH-SY5Y cells. SV9
and SV10 showed remarkable antioxidant properties due to their ability to counteract the
damage caused by H2O2 in SHSY-5Y-treated cells. Hovewer, SV5, showing moderate
antioxidant and good neuroprotective activities, resulted the best candidate for further
experiments since it also resulted stable both simulated and plasma fluids.
2

INTRODUCTION
Alzheimer’s Disease (AD) is a progressive neurodegenerative disorder characterized by
several pathological hallmarks: deposition of Aβ oligomers, neurofibrillary tangles, and
hyperphosphorylated tau proteins which lead to loss of memory and cognitive impairment [1,
2]. The etiology of AD is not yet clear, but it is certain that the simultaneous presence of
oxidative stress, metal dishomeostasis, neuroinflammation, and proteins accumulation
contribute to the development of the disorder [3]. To date, the current pharmacological
treatment (Acetylcholinesterase inhibitors and N-metyl-D-aspartate antagonist) is only
symptomatic so there is no effective cure to reduce or block the progression of AD [4].
Recently, natural compounds gained much attention for their intrinsic therapeutic potential
[5]. In this context, phenolic acids, such as caffeic or ferulic acids, are strong antioxidants
able to scavenge reactive oxygen species (ROS) thus reducing oxidative damage that
characterizes AD [6-10]. Notably, like other polyphenols, they can interfere with the
deposition of the amyloid peptide destabilizing the β-sheet conformation in in vitro models of
AD [11-13].
Although lipoic acid (LA) and its reduced form (dihydrolipoic acid, DHLA) are structurally
different from polyphenols, they can act as multifunctional antioxidant against ROS and
chelator for metal ions such as zinc or copper involved in the pathogenesis of AD [14].
Moreover, they increase the level of reduced glutathione, downregulate the inflammatory
process, scavenge lipid peroxidation products, and increase acetylcholinesterase production
by activation of choline acetyltransferase [15].
Small molecules targeting Aβ and ROS concurrently could be effective in the prevention
and/or treatment of AD. In our previous papers we reported the design and synthesis of
different lipoyl hybrids as multifunctional agents for management of neurodegenerative
diseases [16-17]. Hybrid molecules can be obtained by linking two or more pharmacophoric
3

portions into a single compound able to interact with multiple targets [18]. Moreover, hybrids
are endowed with better drug-like properties such as low molecular weight that allow them to
easily pass the blood brain barrier, better water and fat solubility both to guarantee good oral
absorption and distribution to the target sites.
Recently, hybrid compounds combining polyphenols and lipoic acid have been reported in
the literature acting as selective butyrylcholinesterase (BuChE) inhibitors, an enzyme which,
along with acetylcholinesterase (AChE), has an important role in the development and
progression of AD [19-22].
Based on these data, our study describes the development of phenol-lipoyl hybrids (SV1-13)
via a linking strategy (Figure 1). The phenol-lipoyl hybrids differ by a) the phenolic acid and
b) the linkage between the two units; since they incorporate antioxidant and anti-amyloid
portions, SV1-13 could provide a synergistic effect against the neurodegenerative process in
patients affected by AD. The polyamines were chosen as linkers for two main reasons: a)
they were recently proposed as universal template for the design of multitarget-directed
ligands for the managemnet of multifactorial neurodegenerative diseases, such as
Alzheimer’s disease [23]; b) pharmacological evidences reported that polyamines exerts
beneficial effects on learning and memory in the therapeutic approach of cognitive
pathologies [24].
Thus, the synthesis and in vitro capabilities of SV1-13 against neurotoxicity and apoptosis
induced by Aβ1-42 in SHSY-5Y neuroblastoma cells were analysed. The antioxidant
properties of SV hybrids were also investigated considering the presence of different
antioxidant portions in each hybrid. Finally, SV1-13 were also subjected to stability studies in
simulated gastric, intestinal, and plasma fluids.
4

Figure 1. Design strategy towards phenolic acid – lipoic acid hybrids (SV1-13).
RESULTS AND DISCUSSION
Synthesis of SV1-13
Phenol-lipoyl hybrids (SV1-13) were synthesized following a sequence of reactions depicted
in Schemes 1-4. Several Boc-aminoalcohols and Boc-diamines 2a-g, prepared as previously
described [25-28], were used as linkers (Schemes 1-4). The 3,4-Dimethoxyphenylacetic acid
(1) was coupled to the suitable Boc-aminoalcohols (2a, 2c, or 2f) or Boc-diamines (2b, 2d, or
2e) in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDC.HCl) and catalytic amount of 4-dimetylaminopyridine (DMAP) to obtain ester
derivatives 3a, 3c, and 3f or 1-hydroxybenzotriazole (HOBt) to afford amide derivatives 3b,
3d, and 3e, respectively. Mild TFA-mediated removal of the Boc group followed by coupling
with (±) α-lipoic acid resulted in hybrids 4a-b, 4e-f, and SV1-2. Removal of di-methoxy
5

.
groups with boron trifluoride dimethylsulfide complex (BF3 SMe2) provided lipoyl hybrids
SV7-10 and SV12-13 in good yields.
Scheme 1. Synthesis of SV1-2, 8-10, 12-13
Reagents and reaction conditions: i) 2a, 2c, or 2f, EDC.HCl, 10% DMAP, CH2Cl2, 0oC→ rt, 18 h, or 2b, 2d, 2e,
or 2g, ΗΟΒt, CH2Cl2, 0°C→ rt, 18 h; ii) 5%TFA/CH2Cl2, 0°C→ rt, 1.5 h; iii) EDC.HCl, lipoic acid, TEA, ΗΟΒt,
.
CH2Cl2, 0.5% cysteine, 0°C→ rt, 18 h; iv) BF3 SMe2, CH2Cl2, 0°C→ rt, 22 h.
For the synthesis of SV4 (Scheme 2), caffeic acid 5 was transformed to the corresponding
dimethoxy derivative 6 using dimethylsulfate [29]. Standard acylation reaction using the
linker 2e in the presence of EDC.HCl gave the derivative 7e. Boc group deprotection with
5%TFA/CH2Cl2 followed by coupling with lipoic acid furnished SV4 in good yield.
Scheme 2. Synthesis of SV4
6

Reagents and reaction conditions: i) Dimethyl sulfate, NaOH, H2O, rt, 18 h; ii) 2e, EDC.HCl, ΗΟΒt, CH2Cl2,
0°C→ rt, 18 h; iii) 5%TFA/CH2Cl2, 0°C→ rt, 1.5 h; iv) EDC.HCl, lipoic acid, TEA, ΗΟΒt, CH2Cl2, 0.5%
cysteine, 0°C→ rt, 18 h.
In a similar manner, starting from ferulic acid 8, hybrids SV5-6 were obtained in satisfactory
yields (Scheme 3). Following the same synthetic procedure, SV3 and SV11 were synthetized
as reported in Scheme 4.
Scheme 3. Synthesis of SV5-6
Reagents and reaction conditions: i) 2b or 2d, EDC.HCl, ΗΟΒt, CH2Cl2, 0°C→ rt, 18 h; ii) 5%TFA/CH2Cl2,
0°C→ rt, 1.5 h; iii) EDC.HCl, lipoic acid, TEA, ΗΟΒt, CH2Cl2, 0.5% cysteine, 0°C→ rt, 18 h.
Scheme 4. Synthesis of SV3 and SV11
7

Reagents and reaction conditions: i) EDC.HCl, ΗΟΒt, CH2Cl2, 0°C→ rt, 18 h; ii) 5%TFA/CH2Cl2, 0°C→ rt, 1.5
.
h; iii) EDC.HCl, lipoic acid, TEA, ΗΟΒt, CH2Cl2, 0.5% cysteine, 0°C→ rt, 18 h; iv) BF3 SMe2, CH2Cl2, 0°C→ rt,
22 h.
1
The structure of all synthesized compounds was confirmed by H-, 13C-NMR and Mass
spectrometry. The purity of the new hybrid compounds was verified by HPLC analysis and
was found > 95% (Supporting Information).
Physico-chemical characterization of SV1-13
Physico-chemical properties of all synthetized SV1-13 were analyzed by HPLC. Results
showed that all hybrids possessed a poor water solubility even if derivatives with free
catechol groups resulted slightly water-soluble compared to compounds with the protected
catechol ring (Table 1). Moreover, poor water-soluble hybrids SV1-5 showed LogP values
higher than 3 suggesting that these compounds are quite lipophilic while hybrids SV7-9,
showing LogP values lower than 2, could be better absorbed by gastrointestinal tract [30].
8

Table 1. Physicochemical properties of SV 1-13.
Compound
SV1
Water Solubility (mg/mL)a
0.110 (± 0.001)
0.042 (± 0.002)
0.010 (± 0.003)
-
Classificationb
cLogPc
3.63
2.73
3.95
3.33
3.19
2.92
1.59
1.29
1.81
2.95
2.49
2.71
2.43
VSS
PI
SV2
SV 3
PI
SV 4
PI
SV 5
0.049 (± 0.002)
0.023 (± 0.001)
2.688 (± 0.026)
0.081 (± 0.001)
0.235 (± 0.010)
-
PI
SV 6
PI
SV 7
SS
PI
SV 8
SV 9
VSS
PI
SV 10
SV 11
SV 12
SV 13
2.484 (± 0.017)
0.933 (± 0.087)
0.736 (± 0.002)
SS
VSS
VSS
aValues are means of three experiments, standard deviation is given in parentheses; bFS (Freely Soluble), S
(Soluble), SPS (Sparingly Soluble), SS (Slightly Soluble), VSS (Very Slightly Soluble), PI (Practically
Insoluble); cValues are calculated with ACD LogP software package, version 4.55.
Stability studies performed in simulated gastric fluid (SGF) at pH 1.2 in presence of pepsin
revealed that SV2 and SV5-7 are the most stable compounds (Table 2). Similar results were
obtained in simulated intestinal fluid (SIF) at pH 6.8 suggesting that these hybrids, containing
two amide bonds and a double bond (SV5-6), are hardly attackable by proteolytic enzymes
(Table 2). Plasma stability studies confirmed these data (Table 3). On the other hand, hybrids
SV1, SV10-13 were not very stable at pH 1.2 and underwent to immediate hydrolysis in SIF
at pH 6.8 (Table 2). Probably, the introduction of an ester bond between the catechol residue
and the linker is detrimental for the stability of the compounds. In human plasma the same
hybrids showed a half-life less than 1 h suggesting that they do not possess physico-chemical
properties suitable for further studies (Table 3). The structure-activity relationships
highlighted the larger effect of the amide bond on the stability of the hybrids than the ester
junction.
9

Table 2. Stability of SV 1-13 in simulated gastric fluid at pH 1.2 (SGF) and in intestinal fluid at pH 6.8 (SIF) with Pepsin and Pancreatin,
respectively.
Time
point
(min)
0
SV1
SV2
SV3
SV4
SV5
SV6
SV7 SV8 SV9
SV10 SV11 SV12 SV13
-
-
-
-
-
-
-
-
-
-
-
-
-
15.23
(±
2.90
(±
5.60
(±
4.67
(±
0.36
(±
0.25
(±
0.63 10.37 15.27
(± (± (±
45.21
(±
12.34
(±
35.42
72.4
15
30
(± 0.76)
(± 0.64)
0.53)
35.89
(±
0.03)
1.89
(±
0.34)
7.35
(±
0.19)
8.23
(±
0.01)
0.43
(±
0.01)
1.28
(±
0.02) 0.94) 0.14)
0.99 37.63 47.45
0.84)
63.52
(±
0.88)
46.72
(±
SGF
79.35
86.54
(±
(±
(±
(± 0.40)
(± 0.63)
0.75)
60.98
(±
0.56)
4.03
(±
0.24)
30.76
(±
0.53)
22.16
(±
0.02)
1.78
(±
0.06)
3.45
(±
0.01) 0.74) 0.15)
2.74 57.89 65.48
0.54)
78.90
(±
0.71)
67.34
(±
100
100
60
0
(±
(±
(±
(± 0.05)
(± 0.50)
0.53)
0.38)
-
0.82)
-
0.99)
-
0.76)
-
0.54)
-
0.15) 0.65) 0.15)
0.75)
-
0.82)
-
-
-
-
0.45
(±
12.01
(±
3
0.54
(±
2.52
(±
1.89 1.50 5.60
(± (± (±
3.87
(±
2.50
(±
15
(±
0.07)
0.73
(±
0.86)
21.06
(±
0.89)
3.64
(±
0.01)
0.86
(±
0.62)
3.01
(±
0.03) 0.07) 0.36)
2.17 20.44 30.40
0.19)
45.89
(±
0.08)
35.40
(±
30
60
(±
(±
(±
0.21)
0.90
(±
0.95)
0.27)
0.05)
0.21)
0.04) 0.87) 0.17)
0.75)
0.49)
Immediate
hydrolysis
Immediate
hydrolysis
Immediate
hydrolysis
SIF
27.20
11.79
1.04
5.20
4.02 42.70 56.30
60.78
54.62
(±0.50) (±0.23) (±0.45) (±0.20) (±0.30)(±0.05)(±0.09)
(±0.42)
(±0.60)
0.01)
1.32
(±
31.38
(±
22.21
(±
2.50
(±
4.15
(±
3.78 63.00 70.00
(± (± (±
89.78
(±
70.45
(±
120
180
0.09)
2.09
(±
0.50)
40.30
(±
0.81)
34.75
(±
0.09)
3.46
(±
0.28)
4.80
(±
0.17) 0.86) 0.16)
4.32 88.91 90.20
0.92)
100
(±
0.84)
100
(±
(±
(±
(±
0.11)
0.75)
0.86)
0.04)
0.37)
0.20) 0.86) 0.66)
0.08)
0.47)
a Values are means of three experiments; standard deviation is given in parentheses. b unstable RD% > 5%, stable RD% < 5%.
Table 3. Human plasma stability of SV 1-13.
SV1
SV2
SV3
SV4
SV5
SV6
SV7
SV8
SV9
SV10
SV11
SV12 SV13
10

0.62
(±
17.23
(±
36.44
(±
72.37
(±
57.44
(±
23.14
(±
1.49
(±
9.21
(±
0.03
(±
2.16
(±
0.75
(±
1.13
(±
> 12
(±)
t
½
(h) a
Human
plasma
0.12)
1.146
(±
4.84)
0.042
(±
2.88)
0.019
(±
1.46)
0.010
(±
1.82)
0.012
(±
7.21)
0.031
(±
0.36)
0.479
(±
0.05)
0.08
(±
0.01)
20.56
(±
0.02)
0.32
(±
0.01)
0.93
(±
0.05)
0.613
(±
kobs (h-
-
1
a
)
0.224)
0.012) 0.002) 0.001) 0.001) 0.010) 0.117) 0.001) 2.631) 0.004) 0.003) 0.025)
a Values are means of three experiments; standard deviation is given in parentheses.
Biological studies
Effect of SV1-13 on neurotoxicity induced by Aβ1-42 in SH-SY5Y cells.
In the current study, we aimed to test the protective effects of SV1-13 on Aβ-induced
differentiated SH-SY5Y cell death. First, as a preliminary study, the toxicity of SV1-13 on
SH-SY5Y cells was investigated in a wide range of concentrations (1-250 mg/L). Results
showed that they did not cause any significant cytotoxicity compared to control cells (Fihure
1S). Afterwards, we examined the protective effects of SV1-13 on viability of SHSY-5Y
cells exposed to Aβ1-42, a pathogenic inducer of AD. Based on our previous study [31], the
viability of differentiated SH-SY5Y cells decreased with increasing concentration of Aβ1-42
,
and the 50% decrease in cell proliferation was determined when treated with 20ꢀµM of Aβ1–
42. Therefore, this concentration was used in all subsequent experiments.
The viability of differentiated SH-SY5Y cells was determined using 3-(4,5-dimethylthiazol-
2-yl)-2,5- diphenyltetrazolium bromide (MTT). The cell viability was elevated in a
concentration-dependent manner when the cells, exposed to Aβ1-42, were treated with SV
hybrids. Similar results were obtained using lactate dehydrogenase (LDH). Figure 2 shows
that the increased cytotoxicity in the cells exposed to Aβ1-42 was decreased after treatment
with SV hybrids. These results indicated that these compounds have exhibited different
protection levels on cellular toxicity caused by Aβ1-42. Based on these data, SV2, SV3, SV5,
SV9, and SV10 were chosen for further biological experiments due to their higher potential
11

protective activity against Aβ1-42 induced neurotoxicity in differentiated SH-SY5Y cells
(Figure 2).
Figure 2. The neuroprotective effects of SV derivatives against Aβ1–42 at a concentration of 20 µM was
examined by MTT and LDH assay. Values are shown as the percentage of cell viability and represented as
mean ± SEM of five independent experiments. **p = 0.001; *p = 0.05; n.s. p > 0.05.
Effect of SV2, SV3, SV5, SV9, and SV10 on Aβ1-42 -induced apoptosis in SH-SY5Y cells
Because of their neuroprotective activity on Aβ1-42-induced cell death, SV2, SV3, SV5, SV9,
and SV10 were chosen for further studies. To examine their effect on the apoptotic cell death
of SH-SY5Y cells, Hoechst 33258 staining was performed to reveal DNA fragmentation or
chromatin condensation. The blue stained cells, after exposure to Aβ1-42 with or without SV
hybrids for 48 h, were reported in Figure 3. The cells in the control group (Panel A) have
regular shape while exposure to Aβ1-42 significantly affect their morphology including the
presence of condensed chromatin (seen as a bright blue) and fragmented nuclear DNA
12

(observed as blue spots) (Panel B). On the contrary, fragmentation and condensation of DNA
were decreased when the cells exposed to Aβ1-42 plus SV hybrids, particularly with SV2
(Panel C), SV5 (Panel E), and SV10 (Panel G).
Figure 3. SH-SY5Y cells stained with Hoechst 33258 showing the the antiapoptotic effects of SV derivatives
against Aβ1–42 (20 µM). A) Control, B) Aβ1–42, C) Aβ1–42 + SV2, D) Aβ1–42 + SV3, E) Aβ1–42 + SV5, F) Aβ1–42
SV9, G) Aβ1–42 + SV10; Original magnification, × 200.
+
Furthermore, the effect of SV2, SV3, SV5, SV9, and SV10 on apoptotic cell death caused by
Aβ1-42 in differentiated SH-SY5Y cells was assessed by Annexin V-FITC and propidium
iodide (PI) double staining (Figure 4). Cells were incubated with Aβ1-42 with or without
hybrids for 48 h, and then analyzed by flow cytometry. As shown in Figure 4, Aβ1-42 caused
an increase in cell death (apoptosis 34.46%, necrosis 6.99%) in SH-SY5Y cells. On the other
hand, SV hybrids provided protection of differentiated SH-SY5Y cells against Aβ1-42 induced
cell death.
13

A
β 1-42
A 1-42 + SV3
β
Control
A 1-42 + SV2
β
A 1-42 + SV5
β
A 1-42 + SV10
β
A 1-42 + SV9
β
Annexin-V
Figure 4. Representative images of flow cytometry evaluation of cell apoptosis after treatment with SV
derivatives against Aβ1-42 (20 µM) induced toxicity in SH-SY5Y cells. The apoptotic rate was determined by
annexin V/PI staining. The lower left quadrant I represents viable cells (Annexin V−/PI −); the lower right
quadrant II represents the cells in the early stage of apoptosis (Annexin V+/PI −); the upper left quadrant III
represents the cells in the late stage of apoptosis (Annexin V+/PI +); and the upper right quadrant IV represents
necrotic cells (Annexin V−/PI +).
Effect of SV2, SV3, SV5, SV9, and SV10 on gene expression profile in SH-SY5Y cells
exposed to Aβ1-42
To further explore the mechanism underlying the protective effects of SV hybrids on Aβ1-42
-
induced neurotoxicity of differentiated SH-SY5Y cells, the mRNA levels of 12 different AD
related genes were analyzed. Cells were treated with Aβ1-42 with or without SV hybrids and
changes in the gene expression levels of treated cells compared to control group were shown
in Figure 5. It was observed that Aβ1-42 in SH-SY5Y cell line led to an increase in the gene
expressions of APOE, NCTSN, APH1α, PSENEN, APP, TNF-α, EGFR, MAPT, BDNF,
BACE1 and a decrease in the gene expressions of PSEN and ADAM10. On the other hand, SV
14

hybrids caused different ameliorative changes in the gene expression profile of cells exposed
to Aβ1-42. Among all tested SV hybrids, SV5 and SV10 showed the best activity against Aβ1-42
induced neurotoxicity in SH-SY5Y cells.
3,5
3
2,5
2
3
2
3,5
3
2,5
2
1
1,5
1
0,5
0
-0,5
-1
1,5
1
0
-1
-2
-3
0,5
0
-0,5
-1
-1,5
Treatment with Aβ1–42
+ SV2
Treatment with Aβ1–42
Treatment with Aβ1–42
+
SV3
4
3
3
5
4
3
2
2
1
2
1
1
0
0
0
-1
-2
-3
-4
-1
-2
-3
-1
-2
-3
-4
Treatment with Aβ1–42
+ SV5
Treatment with Aβ1–42
+ SV9
Treatment with Aβ1–42
+
SV10
Figure 5. Gene expression profile in differentiated SH-SY5Y cells treated with SV2, SV3, SV5, SV9, and SV10
against Aβ1-42 (20 µM). Up-regulation and down-regulation of each gene are represented as a fold-change
relative to untreated cells.
Cytotoxic effects of SV2, SV3, SV5, SV9, and SV10 on human whole blood cells
Human whole blood cells were treated with SV2, SV3, SV5, SV9, and SV10 and tested to
evaluate their potential cytotoxic effects. A wide range of hybrids concentrations were
screened and incubated with the blood cells for 24 h. The cytotoxicity of each hybrid was
determined using both MTT and LDH release assays. The positive control, that was treated
with Triton-X and H2O2 (50 µM), showed the lowest cell viability rates (< 20% and < 50%,
respectively) as compared to untreated (control (-)) cells. On the contrary, after 24 h of
treatments, the cultures showed excellent viability even up to the concentration of 400 µM of
SV hybrids. Although the minimum cell viability rates were observed after treatment with
400 µM of SV3, the determined cell viability rates were still higher than 70%. Likewise, it
was observed in LDH release assay that a 24 h treatment of blood cells to SV2, SV3, SV5,
15

SV9, and SV10 concentrations led no decrease of the number of viable cells, whereas there
was a slight reduction in cell viability at the concentration of 400 µM of SV3 (data not
shown). For all that, this reduction did not reflect cytotoxic potential since the observed
values were above 75%. Eventually, the cytotoxicity analysis revealed that SV2, SV3, SV5,
SV9, and SV10 hybrids have non-cytotoxic nature on human blood cells.
Antioxidant properties of SV2, SV3, SV5, SV9, and SV10
SV2, SV3, SV5, SV9, and SV10 were also evaluated for their potential antioxidant activity
using TAC assay (Figure 6). Significant differences were observed in the supporting capacity
of SV hybrids. Maximum % increases of TAC levels decreased in the following order: SV10
(32.3%) > SV9 (26.5 %) > SV5 (6.7 %) > SV2 (4.2 %) > SV3 (2.0 %) as compared to control
(-) level. Ascorbic acid (AA) increased the TAC level in 10.7%.
16

Figure 6. The increases of TAC levels (%) as compared to level of control (-) group after treatment with
different concentrations (0-400 mg/L) of SV2, SV3, SV5, SV9, and SV10 for 72 h in whole blood cultures.
(AA: Ascorbic acid).
Taking account of cell viability rates (> 95%) and results obatained by TAC assay, two
effective concentrations were selected as 50 and 100 µM for further cyto-protectivity
evaluations. So, the blood cells were simultaneously incubated with 50 or 100 µM of selected
SV hybrids and H2O2 to reveal whether these derivatives conferred cytoprotection against
oxidative damages of cells. Blood cells treated with H2O2 caused 56.9% and 52.2% decreases
in cell viability when compared to control (-) rates (MTT and LDH release assays),
respectively. However, SV2, SV3, SV5, SV9, and SV10 led to improvement of the cell
viability. A clear concentration-dependent cyto-protection was monitored, and the cell
17

viability was increased between the ranges of 41.6 and 86.8% by SV hybrids indicating 0.5
and 42.0% recovery from H2O2-induced cell death in MTT analysis. Similarly, the cell
viability was increased between the ranges of 48.2 and 89.3% by SV hybrids indicating 0.4%
and 41.5% recovery from H2O2-induced cell death in LDH release analysis (Figure 7 and
Table 1S).
Figure 7. Protective effect of SV2, SV3, SV5, SV9, and SV10 on human blood cells against H2O2 induced cell
damage as analyzed by MTT and LDH release assays. Different letters in the same column indicate statistically
significant differences (P < 0.05) from each other, as analyzed by Dunnett's multiple comparison test.
Figure 8 reflects the observed TAC levels in cultures after incubation with selected SV
hybrids and H2O2, simultaneously. The TAC levels significantly (p < 0.05) decreased when
blood cells were treated with H2O2. In fact, the exposure to H2O2 caused 83.3% decrease in
TAC level as compared to untreated cultures. By contrast with, SV hybrids provided
antioxidative restoration and minimized these decreases in clear compound type and
concentration manners. Nominately, 50 µM and 100 µM SV3 exhibited the weakest (12.5%
and 29.2%) but SV9 and SV10 provided the strongest protection (reaching to 79.2%) against
H2O2-induced oxidative insults.
18

Figure 8. The effects of concomitant treatments of SV2, SV3, SV5, SV9, or SV10 and H2O2 on TAC levels (as
mmol/L) of cultured human blood cells. Bars with different letters are considered statistically different (P <
0.05), as analyzed by Dunnett's multiple comparison test.
In summary, we reported the synthesis and biological characterization of 13 novel hybrids
containing phenols and lipoic acid as novel multifunctional antioxidants for the development
of potential anti-Alzheimer agents. Taking together all the obtained data, the present study
demonstrated that SV2, SV3, SV5, SV9, and SV10 derivatives exhibited non-cytotoxic and
cyto-protective actions. The in vitro assays showed that most of the hybrids had significant
neuroprotective activity against H2O2-induced oxidative stress in SHSY-5Y cells. Notably,
19

SV9 and SV10 showed the highest antioxidant properties but they resulted unstable,
especially SV10, in simulated fluids and human plasma. In fact, stability studies in simulated
and plasma fluids confirmed that the introduction of ester bond - between phenolic portion
and the linker (SV10) - compared to the amide linkage is detrimental for the stability of the
synthetized hybrids.
The best activity against neurotoxicity induced by Aβ1-42 in SHSY-5Y-treated cells was
exhibited by SV5 and SV10; the presence of ferulic and 3,4-dihydoxyphenilacetic acids as
phenolic portions, respectively in SV5 and SV10, represent important structural motifs for a
strong free radical scavenging character and anti-amyloidogenic properties. Hydroxyl groups
of SV hybrids could form non-covalent interactions with the β-sheet structures interfering
with their assembling.
In conclusion, the moderate antioxidant properties of SV5 (compared to SV9 and SV10) but
its good potential as neuroprotective compound against Aβ1-42 and its high stability in
simulated fluids and human plasma (hal-life > 72 h), render this hybrid a potential
multifunctional candidate for further studies to the development of new anti-Alzheimer
agents.
EXPERIMENTAL PART
General. All used reagents were purchased at the highest quality from commercial suppliers
and were used without further purification. Reactions were monitored by thin-layer
chromatography (TLC) carried out on 0.25 mm silica gel plates (silica gel 60F254) and
components were visualized by UV light absorbance. Purification of compounds by column
1
chromatography was carried out on silica gel (70-230 mesh) with the indicated solvents. H
and 13C spectra were recorded on a Varian VXR-300 spectrometer (Varian Medical Systems, Inc.,
1
13
Palo Alto, CA, USA). H and C spectra are referenced according to the residual peak of the
20

solvent based on literature data. 13C spectra are fully proton decoupled. ESI mass spectral
analyses were performed on a mass spectrometer (Thermo Finnigan), using direct sample
injection. Negative or positive ion ESI spectra were acquired by adjusting the needle and
cone voltages accordingly. The spectroscopy and analytical data were obtained in the
Laboratory of Organic Chemistry of the University of Athens. Analyses indicated by the
symbols of the elements were within ± 0.4% of the theoretical values.
General esterification procedure (Method A)
To a stirred solution of the appropriate carboxylic acid (1.00 mmol), Boc-protected
aminoalcohol (1.00 mmol) and DMAP (0.10 mmol) in CH2Cl2 (5 ml) at 0oC, EDC†HCl (1.00
mmol) was added. After stirred at room temperature overnight, the mixture was diluted with
CH2Cl2, washed with citric acid 5%, H2O, NaHCO3 5%, H2O and brine. The organic phase
was dried over Na2SO4, filtered, and concentrated to obtain yellow oil, which was further
purified by column chromatography on silica gel using appropriate mixture of CH2Cl2/MeOH
or AcOEt/Petroleum ether.
2-((tert-butoxycarbonyl)amino)ethyl 2-(3,4-dimethoxyphenyl)acetate (3a)
Following the general method A, compound 3a was obtained as thicky oil in 77% yield. Rf
1
(CH2Cl2/MeOH 9.8:0.2) = 0.29. H NMR (200 MHz, CDCl3) δ 6.77 (s, 3H), 4.80 (br s, 1H),
4.10 (t, J = 5.4 Hz, 2H), 3.82 (dd, J = 3.6, 2.1 Hz, 6H), 3.53 (s, 2H), 3.32 (d, J = 5.4 Hz, 2H),
13
1.40 (s, 9H). C NMR (50 MHz, CDCl3) δ 171.5, 155. 6, 148.7, 148.0, 126.1, 121.2, 112.2,
111.05, 79.3, 63.7, 55.6, 40.5, 39.4, 28.1.
3-((tert-butoxycarbonyl)amino)propyl 2-(3,4-dimethoxyphenyl)acetate, 3c
Following the general method A, compound 3c was obtained as thicky oil in 75% yield. Rf
1
(CH2Cl2/MeOH 9.8:0.2) = 0.38. H NMR (200 MHz, CDCl3) δ 6.69 (s, 3H), 4.93 (br s, 1H),
4.02 (t, J = 6.2 Hz, 2H), 3.72 (d, J = 4.1 Hz, 6H), 3.43 (s, 2H), 3.02 (q, J = 6.4 Hz, 2H), 1.67
21

13
(p, J = 6.4 Hz, 2H), 1.31 (s, 9H). C NMR (50 MHz, CDCl3) δ 171.4, 155.5, 148.5, 147.7,
126.0, 120.9, 112.01, 110.8, 78.5, 61.8, 55.3, 40.3, 36.8, 28.6, 27.9.
5-((tert-butoxycarbonyl)amino)pentyl 2-(3,4-dimethoxyphenyl)acetate (3f)
Following the general method A, compound 3f was obtained as thicky oil in 83% yield. Rf
(CH2Cl2/MeOH 9.8:0.2) = 0.45. 1H NMR (200 MHz, CDCl3) δ 6.68 (d, J = 2.9 Hz, 3H), 4.80
(br s, 1H), 3.93 (td, J = 6.2, 3.8 Hz, 2H), 3.71 (t, J = 3.9 Hz, 6H), 3.41 (d, J = 3.8 Hz, 2H),
2.95 (d, J = 5.9 Hz, 2H), 1.48 (dd, J = 15.7, 8.9 Hz, 2H), 1.36 – 1.11 (m, 13H). 13C NMR (50
MHz, CDCl3) δ 171.4, 155.6, 148.3, 147.6, 126.1, 120.9, 111.9, 110.7, 78.3, 64.2, 55.3, 40.4,
39.9, 29.2, 27.9, 27.8, 22.7.
2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethyl
(E)-3-(3,4-dimethoxyphenyl)acrylate
(10g)
Following the general method A, compound 10g was obtained as thicky oil in 57% yield. Rf
(AcOEt/P.E. 1:1) = 0.60. 1H NMR (200 MHz, CDCl3) δ 7.43 (dd, J = 15.9, 2.4 Hz, 1H), 6.93
– 6.79 (m, 2H), 6.64 (dd, J = 8.0, 2.3 Hz, 1H), 6.14 (dd, J = 15.9, 2.3 Hz, 1H), 5.02 (br s,
1H), 4.12 (d, J = 2.7 Hz, 2H), 3.68 (d, J = 2.1 Hz, 6H), 3.52 (s, 2H), 3.40 – 3.30 (m, 2H),
3.12 (d, J = 3.6 Hz, 2H), 1.22 (d, J = 2.1 Hz, 9H). 13C NMR (50 MHz, CDCl3) δ 166.5, 155.5,
150.7, 148.7, 144.6, 126.8, 122.3, 114.9, 110.5, 109.2, 78.5, 69.7, 68.5, 62.9, 55.4, 55.3, 39.8,
27.9.
2-(2-((tert-butoxycarbonyl)amino)ethoxy)ethyl 2-(3,4-dimethoxyphenyl)acetate (11g)
Following the general method A, compound 11g was obtained as thicky oil in 74% yield. Rf
(CH2Cl2/MeOH 9.8:0.2) = 0.35. 1H NMR (200 MHz, CDCl3) δ 6.70 (t, J = 2.7 Hz, 3H), 4.98
(br s, J = 2.7 Hz, 1H), 4.15 – 4.04 (m, 2H), 3.72 (t, J = 3.6 Hz, 6H), 3.50 (dt, J = 7.4, 4.0 Hz,
4H), 3.40 – 3.32 (m, 2H), 3.21 – 3.07 (m, 2H), 1.32 (d, J = 3.3 Hz, 9H). 13C NMR (50 MHz,
CDCl3) δ 171.2, 155.5, 148.5, 147.7, 125.9, 121.0, 112.1, 110.8, 78.7, 69.6, 68.3, 63.3, 55.4,
40.2, 39.8, 27.9.
22

General amidation procedure (Method B)
To a stirred solution of the appropriate carboxylic acid (1.00 mmol), Boc-protected diamine
(1.00 mmol) and HOBt (1.00 mmol) in CH2Cl2 (5 ml) at 0oC, EDC†HCl (1.00 mmol) was
added. After stirred at room temperature overnight, the mixture was diluted with CH2Cl2,
washed with citric acid 5%, H2O, NaHCO3 5%, H2O and brine. The organic phase was dried
over Na2SO4, filtered, and concentrated to obtain yellow oil, which was further purified by
column chromatography on silica gel using appropriate mixture of CH2Cl2/MeOH.
tert-butyl (2-(2-(3,4-dimethoxyphenyl)acetamido)ethyl)carbamate (3b)
Following the general method B, compound 3b was obtained as white solid in 83% yield. Rf
1
(CH2Cl2/MeOH 9.5:0.5) = 0.61. H NMR (200 MHz, CDCl3) δ 6.76 (s, 3H), 6.36 (br s, 1H),
13
5.12 (br s, 1H), 3.82 (s, 6H), 3.44 (s, 2H), 3.26 (s, 2H), 3.17 (s, 2H), 1.38 (s, 9H). C NMR
(50 MHz, CDCl3) δ 172.0, 156.5, 148.9, 148.0, 127.1, 121.3, 112.2, 111.2, 79.3, 55.7, 43.1,
40.4, 40.1, 28.2.
tert-butyl (3-(2-(3,4-dimethoxyphenyl)acetamido)propyl)carbamate (3d)
Following the general method B, compound 3d was obtained as thicky oil in 85% yield. Rf
(CH2Cl2/MeOH 9.5:0.5) = 0.46. 1H NMR (200 MHz, CDCl3) δ 6.65 (t, J = 4.4 Hz, 4H), 5.30
(d, J = 5.0 Hz, 1H), 3.73 – 3.61 (m, 2H), 3.30 (d, J = 4.9 Hz, 6H), 3.15 – 3.00 (m, 2H), 2.91
13
(d, J = 5.4 Hz, 2H), 1.39 (d, J = 5.3 Hz, 2H), 1.24 (d, J = 5.0 Hz, 9H). C NMR (50 MHz,
CDCl3) δ 171.5, 156.1, 148.5, 147.5, 127.3, 120.9, 111.9, 110.9, 78.4, 55.3, 42.7, 36.5, 35.6,
29.5, 27.9.
tert-butyl (4-(2-(3,4-dimethoxyphenyl)acetamido)butyl)carbamate (3e)
Following the general method B, compound 3e was obtained as white solid in 89% yield. Rf
(CH2Cl2/MeOH 9.5:0.5) = 0.43. 1H NMR (200 MHz, CDCl3) δ 6.69 (s, 3H), 6.17 (br s, 1H),
4.87 (br s, 1H), 3.73 (s, 6H), 3.36 (s, 2H), 3.09 (d, J = 5.4 Hz, 2H), 2.95 (d, J = 5.2 Hz, 2H),
23

1.31 (s, 13H). 13C NMR (50 MHz, CDCl3) δ 171.1, 155.8, 148.7, 147.7, 127.3, 121.1, 112.0,
111.1, 78.6, 55.5, 55.5, 42.8, 39.7, 38.9, 28.0, 27.1, 26.2.
tert-butyl (E)-(4-(3-(3,4-dimethoxyphenyl)acrylamido)butyl)carbamate (7e)
Following the general method B, compound 7e was obtained as white solid in 82% yield. Rf
1
(CH2Cl2/MeOH 9.5:0.5) = 0.41. H NMR (200 MHz, CDCl3) δ 7.44 (d, J = 15.6 Hz, 1H),
7.01 (br s, 1H), 6.95 – 6.83 (m, 2H), 6.66 (d, J = 8.1 Hz, 1H), 6.36 (d, J = 15.6 Hz, 1H), 5.01
(br s, 1H), 3.72 (d, J = 8.6 Hz, 6H), 3.28 (d, J = 5.4 Hz, 2H), 3.02 (d, J = 5.7 Hz, 2H), 1.46 (s,
13
4H), 1.32 (s, 9H). C NMR (50 MHz, CDCl3) δ 166.2, 156.0, 150.0, 148.6, 139. 8, 127.6,
121.4, 118.9, 110.7, 109.4, 78.7, 55.5, 55.4, 39.8, 39.0, 28.1, 27.3, 26.4.
tert-butyl (E)-(2-(3-(4-hydroxy-3-methoxyphenyl)acrylamido)ethyl)carbamate (9b)
Following the general method B, compound 9b was obtained as white solid in 76% yield. Rf
1
(CH2Cl2/MeOH 9.5:0.5) = 0.33. H NMR (200 MHz, CD3OD-d4) δ 7.42 (d, J = 15.7 Hz,
1H), 6.96 (dd, J = 11.5, 3.2 Hz, 2H), 6.75 (dd, J = 8.0, 3.8 Hz, 1H), 6.39 (d, J = 15.7 Hz, 1H),
4.94 (br s, 3H), 3.80 – 3.71 (m, 3H), 3.41 – 3.33 (m, 1H), 3.26 – 3.14 (m, 3H), 1.37 (s, 9H).
13C NMR (50 MHz, CD3OD-d4) δ 169.2, 158.3, 149.6, 149.0, 142.0, 127.9, 123.0, 118.5,
116.3, 111.3, 80.0, 56.2, 40.9, 40.5, 28.7.
tert-butyl (E)-(3-(3-(4-hydroxy-3-methoxyphenyl)acrylamido)propyl)carbamate (9d)
Following the general method B, compound 9d was obtained as white solid in 69% yield. Rf
1
(CH2Cl2/MeOH 9.5:0.5) = 0.39. H NMR (200 MHz, CDCl3) δ 7.48 (d, J = 15.5 Hz, 1H),
7.02 (br s, 1H), 6.95 – 6.79 (m, 3H), 6.31 (d, J = 15.6 Hz, 1H), 5.33 (br s, 1H), 3.75 (s, 3H),
13
3.37 (d, J = 5.2 Hz, 2H), 3.14 (d, J = 5.2 Hz, 2H), 1.63 (s, 2H), 1.40 (s, 9H). C NMR (50
MHz, CDCl3) δ 166.9, 156.7, 147.6, 147.0, 140.7, 126.9, 121.9, 118.0, 114.9, 109.7, 79.2,
55.6, 36.9, 36.0, 29.9, 28.2.
General procedure for the lipoyl amides synthesis
24

Boc-protected compound (1 mmol) was suspended in CH2Cl2 (10 mL), and then
trifluoroacetic acid (0.5 mL) was added at 0ºC. After stirring at room temperature for 1.5 h,
the volatiles were removed under reduced pressure to obtain the crude product which was
dissolved in CH2Cl2 (7 mL) and directly coupled with lipoic acid (1 mmol) at 0ºC, in the
presence of Et3N (1.10 mmol), EDC†HCl (1.2 mmol), HOBt (1.00 mmol) and 0.5% cysteine
to avoid polymerization. After stirred at room temperature overnight, in the dark, the reaction
mixture was taken up with CH2Cl2 and washed with HCl 0.5 N, water, 5% NaHCO3, water,
and brine. The organic phase was dried over Na2SO4, filtered, and concentrated to obtain the
crude product, which was further purified by column chromatography on silica gel
(CH2Cl2/MeOH).
2-(5-(1,2-dithiolan-3-yl)pentanamido)ethyl 2-(3,4-dimethoxyphenyl)acetate (4a)
Following the general method, compound 4a was obtained as thick oil in 59% yield. Rf
(CH2Cl2/MeOH 9.5:0.5) = 0.56. 1H NMR (200 MHz, CDCl3) δ 6.76 (d, J = 1.5 Hz, 3H), 5.92
(br s, 1H), 4.12 (t, J = 5.4 Hz, 2H), 3.82 – 3.76 (m, 6H), 3.55 – 3.37 (m, 5H), 3.18 – 2.96 (m,
2H), 2.39 (td, J = 12.4, 6.4 Hz, 1H), 2.07 (t, J = 7.1 Hz, 2H), 1.93 – 1.74 (m, 1H), 1.60 (ddd,
13
J = 13.9, 9.1, 2.2 Hz, 4H), 1.38 (dd, J = 14.2, 6.1 Hz, 2H). C NMR (50 MHz, CDCl3) δ
172.7, 171.6, 148.6, 147.9, 126.0, 121.1, 112.1, 110.9, 63.2, 56.2, 55.6, 40.5, 40.0, 38.3, 38.2,
36.0, 34.3, 28.6, 25.0.
N-(2-(2-(3,4-dimethoxyphenyl)acetamido)ethyl)-5-(1,2-dithiolan-3-yl)pentanamide (4b)
Following the general method, compound 4b was obtained as white solid in 45% yield. Rf
1
(CH2Cl2/MeOH 9:1) = 0.55. H NMR (200 MHz, CDCl3) δ 6.83 (br s, 1H), 6.79 – 6.66 (m,
4H), 3.78 (t, J = 1.4 Hz, 6H), 3.54 – 3.42 (m, 1H), 3.39 (s, 2H), 3.24 (s, 4H), 3.16 – 2.94 (m,
2H), 2.46 – 2.28 (m, 1H), 2.06 (t, J = 7.2 Hz, 2H), 1.82 (dq, J = 13.7, 6.9 Hz, 1H), 1.65 –
1.44 (m, 4H), 1.43 – 1.25 (m, 2H). 13C NMR (50 MHz, CDCl3) δ 173.6, 172.4, 148.7, 147.8,
25

127.1, 121.1, 112.2, 111.1, 56.2, 55.6, 55.6, 42.8, 40.0, 39.6, 39.5, 38.2, 35 9, 34.3, 28.6,
25.1.
N-(4-(2-(3,4-dimethoxyphenyl)acetamido)butyl)-5-(1,2-dithiolan-3-yl)pentanamide (4e)
Following the general method, compound 4e was obtained as white solid in 40% yield. Rf
(CH2Cl2/MeOH 9.5:0.5) = 0.45. 1H NMR (200 MHz, CDCl3) δ 6.82 – 6.69 (m, 3H), 6.29 (br
s, 1H), 6.11 (br s, 1H), 3.80 (d, J = 3.5 Hz, 6H), 3.52 (dd, J = 13.7, 6.9 Hz, 1H), 3.43 (s, 2H),
3.21 – 2.97 (m, 6H), 2.39 (td, J = 12.4, 6.4 Hz, 1H), 2.11 (t, J = 7.3 Hz, 2H), 1.83 (dq, J =
13.8, 6.9 Hz, 1H), 1.69 – 1.50 (m, 4H), 1.41 (t, J = 6.4 Hz, 6H). 13C NMR (50 MHz, CDCl3)
δ 172.9, 171.4, 148.9, 147.9, 127.3, 121.3, 112.2, 111.2, 56.3, 55.7, 43.0, 40.0, 39.0, 38.8,
38.3, 36.1, 34.4, 28.7, 26.7, 26.5, 25.2.
5-(5-(1,2-dithiolan-3-yl)pentanamido)pentyl 2-(3,4-dimethoxyphenyl)acetate (4f)
Following the general method, compound 4f was obtained as white solid in 70% yield. Rf
(CH2Cl2/MeOH 9.5:0.5) = 0.71. 1H NMR (200 MHz, CDCl3) δ 6.80 – 6.73 (m, 3H), 5.76 (br
s, 1H), 4.04 (t, J = 6.5 Hz, 2H), 3.85 – 3.78 (m, 6H), 3.60 – 3.45 (m, 3H), 3.24 – 3.00 (m,
4H), 2.41 (td, J = 12.4, 6.4 Hz, 1H), 2.12 (t, J = 7.2 Hz, 2H), 1.85 (dq, J = 13.8, 6.9 Hz, 1H),
13
1.58 (dt, J = 14.0, 6.7 Hz, 6H), 1.49 – 1.20 (m, 6H). C NMR (50 MHz, CDCl3) δ 172.6,
171.7, 148.6, 147.8, 126.3, 121.2, 112.2, 110.9, 64.4, 56.2, 55.7, 55.7, 40.7, 40.0, 39.1, 38.3,
36.2, 34.4, 29.0, 28.7, 28.0, 25.2, 23.0.
2-(2-(5-(1,2-dithiolan-3-yl)pentanamido)ethoxy)ethyl
(12g)
2-(3,4-dimethoxyphenyl)acetate
Following the general method, compound 12g was obtained as thick oil in 72% yield. Rf
(CH2Cl2/MeOH 9.5:0.5) = 0.62. 1H NMR (200 MHz, CDCl3) δ 6.79 (d, J = 1.9 Hz, 3H), 5.92
(br s, 1H), 4.23 (dd, J = 5.5, 3.8 Hz, 2H), 3.83 (dd, J = 4.1, 2.0 Hz, 6H), 3.66 – 3.60 (m, 2H),
3.57 (d, J = 1.9 Hz, 2H), 3.49 (t, J = 5.6 Hz, 3H), 3.43 – 3.32 (m, 2H), 3.20 – 2.99 (m, 2H),
2.42 (td, J = 12.4, 6.4 Hz, 1H), 2.13 (t, J = 7.2 Hz, 2H), 1.96 – 1.77 (m, 1H), 1.73 – 1.53 (m,
26

13
4H), 1.49 – 1.32 (m, 2H). C NMR (50 MHz, CDCl3) δ 172.6, 171.7, 148.7, 148.0, 126.1,
121.2, 112.2, 111.0, 69.6, 68.7, 63.5, 56.3, 55.7, 40.6, 40.1, 38.9, 38.3, 36.1, 34.5, 28.7, 25.2.
34.3, 28.5, 28.4, 25.1.
3-(5-(1,2-dithiolan-3-yl)pentanamido)propyl 2-(3,4-dimethoxyphenyl)acetate (SV1)
Following the general method, SV1 was obtained as white solid in 61% yield. Rf
(CH2Cl2/MeOH 9.5:0.5) = 0.67. 1H NMR (200 MHz, CDCl3) δ 6.75 (d, J = 5.6 Hz, 3H), 6.09
(t, J = 5.6 Hz, 1H), 4.07 (t, J = 6.2 Hz, 2H), 3.78 (d, J = 3.1 Hz, 6H), 3.55 – 3.39 (m, 3H),
3.18 (q, J = 6.4 Hz, 2H), 3.03 (dt, J = 6.5, 4.9 Hz, 2H), 2.36 (td, J = 12.4, 6.1 Hz, 1H), 2.06 (t,
J = 7.3 Hz, 2H), 1.89 – 1.68 (m, 3H), 1.67 – 1.48 (m, 4H), 1.37 (dd, J = 13.9, 7.1 Hz, 2H).
13C NMR (50 MHz, CDCl3) δ 172.6, 171.7, 148.5, 147.7, 126.0, 121.0, 112.0, 110.9, 62.0,
56.1, 55.5, 40.5, 39.9, 38.1, 36.0, 35.7, 34.3, 28.5, 28.4, 25.1. MS (ESI) m/z calculated for
-
C21H30NO4S2 [M-H]- 440.1, found 440.0. Anal. Calcd for C21H31NO5S2: C, 57.12; H, 7.08;
N, 3.17; O, 18.12; S, 14.52. Found: C, 57.20; H, 7.15; N, 3.09; O, 18.04; S, 14.55.
N-(3-(2-(3,4-dimethoxyphenyl)acetamido)propyl)-5-(1,2-dithiolan-3-yl)pentanamide
(SV2)
Following the general method, SV2 was obtained as off-white solid in 42% yield. Rf
1
(CH2Cl2/MeOH 9.5:0.5) = 0.43. H NMR (200 MHz, CDCl3) δ 6.81 – 6.57 (m, 5H), 3.76 –
3.70 (m, 6H), 3.51 – 3.32 (m, 3H), 3.20 – 2.90 (m, 6H), 2.34 (td, J = 12.4, 6.4 Hz, 1H), 2.08
(t, J = 7.2 Hz, 2H), 1.78 (td, J = 13.7, 7.0 Hz, 1H), 1.65 – 1.23 (m, 8H). 13C NMR (50 MHz,
CDCl3) δ 173.1, 171.8, 148.6, 147.7, 127.2, 121.0, 112.0, 111.1, 56.1, 55.5, 55.5, 42.9, 39.9,
+
38.1, 36.0, 35.7, 35.3, 34.2, 29.2, 28.5, 25.1. MS (ESI) m/z calculated for C21H33N2O4S2
[M+H]+ 441.2, found 441.2. Anal. Calcd for C21H31N2O4S2: C, 57.37; H, 7.11; N, 6.37; O,
14.56; S, 14.59. Found: C, 57,35; H, 7,17; N, 6,41; O, 14,52; S, 14,55.
2-(2-(5-(1,2-dithiolan-3-yl)pentanamido)ethoxy)ethyl
dimethoxyphenyl)acrylate (SV3)
(E)-3-(3,4-
27

Following the general method, compound SV3 was obtained as yellowish solid in 70% yield.
Rf (CH2Cl2/MeOH 9.5:0.5) = 0.63. 1H NMR (200 MHz, CDCl3) δ 7.46 (d, J = 15.9 Hz, 1H),
6.97 – 6.81 (m, 2H), 6.69 (d, J = 8.2 Hz, 1H), 6.27 – 6.06 (m, 2H), 4.23 – 4.09 (m, 2H), 3.71
(t, J = 2.4 Hz, 6H), 3.60 – 3.51 (m, 2H), 3.41 (dd, J = 10.2, 5.4 Hz, 2H), 3.28 (dd, J = 11.7,
6.9 Hz, 3H), 3.02 – 2.82 (m, 2H), 2.23 (dt, J = 18.9, 6.4 Hz, 1H), 2.02 (t, J = 7.4 Hz, 2H),
1.67 (dt, J = 19.7, 6.9 Hz, 1H), 1.45 (dd, J = 10.3, 4.9 Hz, 4H), 1.26 (dd, J = 8.2, 5.8 Hz, 2H).
13C NMR (50 MHz, CDCl3) δ 172.4, 166.6, 150.7, 148.6, 144.7, 126.6, 122.3, 114.7, 110.6,
109.1, 769.2, 68.5, 62.8, 55.9, 55.4, 55.4, 39.7, 38.6, 37.9, 35.7, 34.1, 28.4, 24.9. MS (ESI)
-
m/z calculated for C23H34NO6S2 [M+H]+ 484.2, found 484.2. Anal. Calcd for C23H33NO6S2:
C, 57.12; H, 6.88; N, 2.90; O, 19.85; S, 13.26. Found: C, 57,11; H, 6.88; N, 2,87; O, 19.95; S,
13.19.
(E)-N-(4-(3-(3,4-dimethoxyphenyl)acrylamido)butyl)-5-(1,2-dithiolan-3-yl)pentanamide
(SV4)
Following the general method, compound SV4 was obtained as yellowish solid in 65% yield.
Rf (CH2Cl2/MeOH 9.5:0.5) = 0.29. 1H NMR (200 MHz, CDCl3) δ 7.56 (d, J = 15.5 Hz, 1H),
7.14 – 6.98 (m, 2H), 6.84 (d, J = 8.2 Hz, 1H), 6.43 – 6.29 (m, 2H), 6.21 (br s, 1H), 3.89 (s,
6H), 3.62 – 3.48 (m, 1H), 3.41 (d, J = 5.4 Hz, 2H), 3.30 (d, J = 5.7 Hz, 2H), 3.13 (dd, J =
12.9, 6.1 Hz, 2H), 2.54 – 2.31 (m, 1H), 2.21 (t, J = 7.3 Hz, 2H), 1.88 (td, J = 13.8, 7.0 Hz,
1H), 1.73 – 1.57 (m, 7H), 1.50 (d, J = 8.3 Hz, 3H). 13C NMR (50 MHz, CDCl3) δ 173.2,
166.5, 150.5, 149.0, 140.7, 127.7, 121.9, 118.5, 111.0, 109.6, 56.4, 55.9, 55.8, 40.2, 39.2,
+
39.1, 38.4, 36.3, 34.6, 28.9, 26.9, 26.8, 25.4. MS (ESI) m/z calculated for C23H35N2O4S2
[M+H]+ 467.2, found 467.2. Anal. Calcd for C23H34N2O4S2: C, 59.20; H, 7.34; N, 6.00; O,
13.71; S, 13.74. Found: C, 59.18; H, 7.35; N, 6.02; O, 13.75; S, 13,70.
(E)-5-(1,2-dithiolan-3-yl)-N-(3-(3-(4-hydroxy-3-
methoxyphenyl)acrylamido)propyl)pentanamide (SV5)
28