- Email: [email protected]
Synthesis and activity towards Alzheimer's disease in vitro: Tacrine, phenolic acid and ligustrazine hybrids
Accepted Manuscript Synthesis and activity towards Alzheimer's disease in vitro: Tacrine, phenolic acid and ligustrazine hybrids Guoliang Li, Ge Hong, Xinyu Li, Yan Zhang, Zengping Xu, Lina Mao, Xizeng Feng, Tianjun Liu PII:
S0223-5234(18)30028-X
DOI:
10.1016/j.ejmech.2018.01.028
Reference:
EJMECH 10105
To appear in:
European Journal of Medicinal Chemistry
Received Date: 1 December 2017 Revised Date:
2 January 2018
Accepted Date: 8 January 2018
Please cite this article as: G. Li, G. Hong, X. Li, Y. Zhang, Z. Xu, L. Mao, X. Feng, T. Liu, Synthesis and activity towards Alzheimer's disease in vitro: Tacrine, phenolic acid and ligustrazine hybrids, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.01.028. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Synthesis and Activity towards Alzheimer’s Disease in Vitro: Tacrine, Phenolic acid and Ligustrazine Hybrids Guoliang Li a, Ge Hong a, Xinyu Li b, Yan Zhang a, Zengping Xu a, Lina Mao a, Xizeng Feng b, Tianjun Liu a, * a
Tianjin Key Laboratory of Biomedical Materials, Institute of Biomedical Engineering, Chinese Academy of Medical Sciences
& Peking Union Medical College, Tianjin 300192, China b
State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Bioactive Materials, Ministry of Education, College
of Life Sciences, Nankai University, Tianjin 300071, China
RI PT
*Corresponding author: Tianjun Liu, Email: [email protected]
Abstract
A series of novel tacrine-phenolic acid dihybrids and tacrine-phenolic acid-ligustrazine trihybrids were synthesized, characterized and screened as novel potential anti-Alzheimer drug candidates. These compounds showed potent inhibition activity towards cholinesterases (ChEs), among of them, 9i was the most potent one towards acetylcholinesterase (eeAChE, IC50
SC
= 3.9 nM; hAChE, IC50 = 65.2 nM). 9i could also effectively block β-amyloid (Aβ) self-aggregation with an inhibition ratio of 47% at 20 µM. In addition, its strong anti-oxidation activity could protect PC12 cells from CoCl2-damage in the experimental condition while no neurotoxicity. Furthermore, its hepatotoxicity was lower than tacrine in vitro and in vivo. Kinetic and molecular modeling studies revealed that 9i worked in a mixed-type way, could interact simultaneously with catalytic active site
M AN U
(CAS) and peripheral anionic site (PAS) of AChE. Therefore, 9i was a promising multifunctional candidate for the treatment of AD.
Keywords: Tacrine; Alzheimer’s disease; Cholinesterase inhibitors; Aβ aggregation; Neuroprotection 1. Introduction
Alzheimer’s disease (AD), characterized by cognitive and memory impairment, is an age-related, progressive, and irreversible neurodegenerative disorder [1]. Today, over 46 million people suffer this dementia worldwide, and would increase to 131.5 million by 2050 [2]. However prevention and treatment of AD still remained elusive to date. The pathogenesis of AD is
TE D
complex and related to the abnormality and dysfunction of multi-systems, several factors, including low levels of acetylcholine, β-amyloid deposits, τ-protein aggregation, oxidative stress and neurological disorder, play crucial roles in the AD patients [3-6]. Current treatment of AD focuses on increasing cholinergic neurotransmission in the brain by inhibiting ChEs with medicines like donepezil, rivastigmine, (-)-huprine A and galantamine (Figure 1). Clinical results demonstrated inhibition of ChEs could only improve symptoms, have no profound disease-modifying effects [7]. While multi-target direct ligands (MTDLs), which
EP
combined two or more distinct pharmacological moieties, could work simultaneously on more than one pathological target, thereby resulted in potent therapeutic effectiveness. Fragments in MTDLs could synergistically contribute to the overall activity profile of the ensemble system by acting on the targets involved in disease pathogenesis. So MTDLs have been developed as
AC C
potential agents for the treatment of AD [8-10]. Ligustrazine (2,3,5,6-tetramethylpyrazine, TMP), the main bioactive alkaloid derived from the traditional Chinese medicine
Rhizoma Chuanxiong (Ligusticum chuanxiong Hort.) (Figure 1) [11], had multiple pharmacological activities, including anti-oxidation, anti-inflammatory, thrombolysis, hepato-protection, vascular protection and neuroprotection, it could effectively protect the nervous system from oxidation stress and inflammatory [12-18]. Phenolic acids like caffeic acid, salvianolic acid, p-coumaric acid, ferulic acid, etc., as anti-oxidative ingredients, they showed potent neuroprotective and hepato-protective effects (Figure 1) [19-25]. Conjugation ligustrazine with phenolic acids could achieve much better neuroprotective activity than the single component [26-28]. Inspired by these results, here we would use either phenolic acids or phenolic-ligustrazine hybrids to modify tacrine, the aim was to obtain the novel multifunctional ChEs inhibitors which had combined activities like ChEs inhibition, neuroprotection and hepato-protection. This would be beneficial for the treatment of AD. Tacrine was the first approved ChEs inhibitor by FDA to treat AD, although its hepatotoxicity blocked its use in clinic, to find novel anti-AD drugs based on tacrine was still of interest [29-33]. Over the past years, many multifunctional tacrine hybrids had been developed by conjugating tacrine with antioxidants [34], metal chelating ligands [35], neuroprotective agents [36], NO
ACCEPTED MANUSCRIPT releasing moieties [37], or the inhibitory ligands to cholinesterase [38], β-secretase [39], β-amyloid [40] and monoamine oxidases [41], etc. Typical examples included tacrine-lophine hybrids [42], tetracyclic tacrine scaffolds [43-46], heterobivalent tacrine derivatives [47], tacrine-melatonin hybrids [48-50], tacrine-8-hydroxyquinoline hybrids [51], tacrine-chromene hybrids [52], tacrine-coumarin hybrids [40, 53], tacrine homodimers [54, 55], tacrine-NO donor group [56], tacrine-lipoic acid hybrids [57], tacrine-selegiline hybrids [41], tacrine-carbazole hybrids [58], tacrine-dihrydropyridine hybrids (tacripyrines) [59], bis-tacrine hybrids bearing peptide moiety [39], tacrine-ferulic acid scaffolds [60, 61], tacrine-ferulic acid-NO donor trihybrids [37], tacrine-caffeic acid hybrids [62], tacrine-trolox hybrids [34], tacrine-resveratrol hybrids [36], tacrine-propargylamine
RI PT
hybrids [63], tacrine–ebselen hybrids [64], tacrine-silibinin codrug [65], tacrine-1,2,3-triazole hybrids [66], cystamine-tacrine dimer [67], tacrine-tianeptine hybrids [68], pyrano-tacrine hybrids [69], tacrine-multialkoxy benezene hybrids [8, 70], quinone-tacrine hybrids [71], tacrine-(β-carboline) hybrids [72], tacrine-bifendate conjugates [73]. The results reported above proved the effectiveness of the MTDLs strategy. Inspired by these MTDLs approaches, we designed a series of novel tacrine hybrids based upon tacrine, phenoic acids and TMP.
SC
AChE features two distinct binding sites: the catalytic active site (CAS), located at the bottom of the gorge, was the binding site for both substrates and inhibitors; while the peripheral anionic site (PAS), situated at the entrance of the gorge, was the binding site of enzyme inhibitors; a mid-gorge recognition site between CAS and PAS was also identified [29]. Previous studies have clearly highlighted the superiority of dual site inhibitors for AChE, such as bis(7)-tacrine, was able to bind both CAS and
M AN U
PAS concomitant [54, 74]. This rationalized design of novel chemical entities towards AChE by MTDLs strategy. Both the crystal structure of AChE and mixed type docking characteristics of tacrine hybrids reported previously implied that the flexible conformation with alkylene chain as linker would be better [29]. Here tacrine and phenolic acids conjugated by 2, 3, 4, 6 and 8 methylene groups were randomly designed, their synthesis and pharmacological evaluation was reported in this study, including their AChE and butyrocholinesterase (BuChE) inhibition activity, kinetics of enzyme inhibition, inhibition of self-mediated Aβ
AC C
EP
TE D
aggregation, antioxidant activity, as well as neuroprotection study in vitro and hepatotoxicity assay in vitro and in vivo.
Figure 1. Chemical structures of tacrine, donepezil, rivastigmine, (-)-huprine A, galantamine, TMP, p-coumaric acid, sinapic acid, ferulic acid, caffeic acid, salvianolic acid and tacrine derivatives. 2. Results and discussion 2.1. Chemistry Scheme 1. Synthesis of intermediates 3a−3e.
ACCEPTED MANUSCRIPT Reagents and conditions: (a) POCl3, reflux, 3 h; (b) α,ω-diamino-n-alkane, pentanol, 180 °C, 18 h.
SC
RI PT
Scheme 2. Synthesis of intermediates 7a−7c.
Reagents and conditions: (c) anhydrous DMF, Bn-Br, NaHCO3, r.t., 12 h; (d) anhydrous DMF, TMP-Br, K2CO3, 85 °C, 2 h; (e) C2H5OH, KOH, 60 °C, 40 min; (f) THF, H2, Pd/C, r.t., 12 h.
M AN U
Scheme 3. Synthesis of compounds 9a−9j, 10a−10o, 11a−11e and 12a−12e. O R3 R2
OH
g
N
O O
OH
O R1
i
10a R1 = H, R3 = H, n = 2 10b R1 = H, R3 = H, n = 3 10c R1 = H, R3 = H, n = 4 10d R1 = H, R3 = H, n = 6 10e R1 = H, R3 = H, n = 8 10f R1 = OMe, R3 = OMe, n = 2 10g R1 = OMe, R3 = OMe, n = 3 10h R1 = OMe, R3 = OMe, n = 4 10i R1 = OMe, R3 = OMe, n = 6 10j R1 = OMe, R3 = OMe, n = 8
N
O
N nH
O
N
R1
N N
N
N
3a-e
OH
O R1
N
7a R1 = H, R3 = OMe
10k R1 = H, R3 = OMe, n = 2 10l R1 = H, R3 = OMe, n = 3 10m R1 = H, R3 = OMe, n = 4 10n R1 = H, R3 = OMe, n = 6 10o R1 = H, R3 = OMe, n = 8
N
O
N
N N
N
N
i
O O
HN OH
i
OH
O
3a-e 7c
N
N nH
O OH
O R1
N N N
12a n = 2, 12b n = 3, 12c n = 4, 12d n = 6, 12e n = 8
11a R1 = H, n = 2, 11b R1 = H, n = 3 11c R1 = H, n = 4, 11d R1 = H, n = 6 11e R1 = H, n = 8
EP
7b R1 = H
HN
3a-e
R1
N
O
N
N
O
9a R1 = H, R2 = OH, R3 = H, n = 2 9b R1 = H, R2 = OH, R3 = H, n = 3 9c R1 = H, R2 = OH, R3 = H, n = 4 9d R1 = H, R2 = OH, R3 = H, n = 6 9e R1 = H, R2 = OH, R3 = H, n = 8 9f R1 = OMe, R2 = OH, R3 = OMe, n = 2 9g R1 = OMe, R2 = OH, R3 = OMe, n = 3 9h R1 = OMe, R2 = OH, R3 = OMe, n = 4 9i R1 = OMe, R2 = OH, R3 = OMe, n = 6 9j R1 = OMe, R2 = OH, R3 = OMe, n = 8
R3
R3
N nH
h
R1
N
8a R1 = H, R2 = OH, R3 = H 8b R1 = OMe, R2 = OH, R3 = OMe
N N
HN
R2
3a-e
R1
O
O
R3
N nH
HN
TE D
O C
Reagents and conditions: (g) anhydrous DMF, EDCI/DMAP, r.t., 12 h; (h) anhydrous DMF, TMP-Br, K2CO3, 60 °C, 6 h; (i) anhydrous CH2Cl2, EDCI/HOBT, r.t., 12 h.
AC C
The target compounds were the conjugation of tacrine, phenolic acids and ligustrazine. Compound 2, 9-chloro-1,2,3,4-tetrahydroacridine as the starting compound, was synthesized from reaction of anthranilic acid with cyclohexanone. Then 2 reacted with the corresponding α,ω-diamino-n-alkane to yield the series amines 3a−3e (Scheme 1). In the tandem reaction, due to the self-coupling of two phenolic acids moieties, direct reaction of tacrine with phenolic acids like ferulic acid, caffeic acid as well as salvianolic acid was lower in yields. Therefore, the economic way was phenolic acid firstly coupling with TMP. However in the coupling reaction of phenolic acids 4a−4c with TMP-Br, carboxylic group would cost more TMP-Br, so in practice the corresponding benzyl ester 5a−5c instead, was reacted with TMP-Br to give compounds 6a−6c. Treated 6a−6c by KOH or H2, Pd/C offered intermediates 7a−7c. The procedure is outlined in Scheme 2. Synthesis of the target compounds were depicted in Scheme 3. Reaction of the series amines 3a−3e with the acids 8a−8b in presence of EDCI and DMAP in anhydrous DMF at room temperature yielded the series 9a−9j. The series 9a−9j reacted with intermediate TMP-Br to produce the target compounds 10a−10j. Reaction of intermediates 7a−7c with the series amines 3a−3e in presence of EDCI and HOBT in anhydrous CH2Cl2 yielded series 10k−10o, 11a−11e and 12a−12e. Totally 35 new target compounds were synthesized, their chemical structure characterized by 1H NMR, 13C NMR and high resolution mass spectra
ACCEPTED MANUSCRIPT
RI PT
(HRMS), was right, their purity was over 95% measured by HPLC.
Figure 2. The purity of representative compounds 9i (A) and 10i (B) analyzed by HPLC (Kromasil C18 column, eluted by acetonitrile/water (15/85-95/5) containing 0.1% formic acid at a flow rate of 1 mL/min). 2.2. Cholinesterase inhibition assay in vitro
The inhibition potency towards AChE and BuChE for each hybrid was assayed by the Ellman’s method with slightly
SC
modifications [75]. The IC50 values were summarized in Table 1. The tested 35 compounds (9a–12e) all showed AChE and BuChE inhibitory activities. As control, tacrine had potent inhibition potency (IC50 of AChE = 70.7 nM, IC50 of BuChE = 7.2 nM) in our experiment, however as it was modified by the phenolic acids, like sinapic acid or p-coumaric acid, the potency changed with the linker as well as the auxiliary, some of them were more potent than tacrine. The inhibition activity of sinapic acid
M AN U
dihybrids 9f–9j for AChE was slightly better than that of p-coumaric acid dihybrids 9a–9e, and the activity roughly increased with the electron density in the cinnamic acid moieties [70], changed in a tendency of 4-OH, 2,3-di(OCH3) > 4-OH, 3-OCH3 > 4-OH > 3, 4-di(OH) at the same chain length (here in order to extract the common rule, we compared our data with others’ reported previously by L. Fang [60] and X. Chao [62]). Among these compounds, compound 9i (IC50 = 3.9 nM) exhibited the best AChE inhibitory activity. In the trihybrid system of tacrine conjugation with sinapic acid or p-coumaric acid and ligustrazine, i.e., 10a-10j, they showed irregular change in inhibition activity toward either AChE or BuChE, and compared with their precursor dihybrids, no obvious improvement in inhibition activity. The contribution of ligustrazine moiety was complicated in
TE D
these system, these results could be possibly ascribed to suitable docking positions of the ligustrazine unit at the AChE gorge [76]. The trihybrids 10a, 10f, and 10i showed good inhibitory activities toward AChE, in which compound 10i (IC50 of AChE = 2.6 nM) was the best. As for BuChE, compared with tacrine, the trihybrids 10a–10j showed relatively weak inhibitory activities, except for compounds 10b (IC50 = 5.1 nM), 10c (IC50 = 2.3 nM).
The contribution of ligustrazine moiety was complicated in the trihybrid system of tacrine, ferulic acid or caffeic acid and
EP
ligustrazine due to the spacer length, the bulk difference as well as the space in docking target. Compared to tacrine-ferulic acid dihybrids (IC50 = 1.2 eq, IC50 = 1.4 eq), the inhibition activity of tacrine–ferulic acid–ligustrazine trihybrids 10k (IC50 = 2.1 eq) and 10l (IC50 = 5.8 eq) for AChE was stronger than that of the corresponding tacrine dihybrids with same carbon chain length (2
AC C
and 3). While 10m (IC50 = 0.38 eq) showed lower inhibition activity than tacrine-ferulic acid dihybrid (IC50 = 5.9 eq) with four-carbon chain length. In addition, the AChE inhibition activity changed little between the tacrine–caffeic acid dihybrids (IC50 = 0.5 eq, IC50 = 0.5 eq) and tacrine–caffeic acid–ligustrazine 11a (IC50 = 0.65 eq) and 11d (IC50 = 0.5 eq) with same carbon chain length (2 and 6). When the carbon chain length was three, the trihybrid 11b (IC50 = 5.2 eq) exhibited potent AChE inhibition activity relative to that of the corresponding dihybrid (IC50 = 0.1 eq) (here we compared our data with others’ reported previously by L. Fang [60] and X. Chao [62], and eq was the abbreviation of tacrine equivalent). Among trihybrids like tacrine–ferulic acid–ligustrazine (10k–10o) and tacrine–caffeic acid–ligustrazine (11a–11e), they had the similar molecular structure. At the same linker one ligustrazine moiety on the benzene ring usually has a higher ChEs (both AChE and BuChE) inhibitory activity than that of two ligustrazine moieties, such as 10k > 11a, 10n > 11d, 10o > 11e. However, this regularity did not work in the case of tacrine–salvianolic acid–ligustrazine trihybrids, 12d (IC50 = 1.3 nM), 12e (IC50 = 3.8 nM), which exhibited surprising BuChE inhibitory activity. The possible reason was that BuChE could accommodate bulkier inhibitors than AChE [76]. Results from 11a–12e suggested that at the same linker, trans olefinic bond could usually enhance the AChE inhibition activity, such as 11a > 12a, 11b > 12b, 11c > 12c, 11d > 12d. Except 12e > 11e, a different bonding
ACCEPTED MANUSCRIPT conformation may be given at longer linker. The linker length notably influenced the inhibition ability to BuChE, chains containing 6 carbon atom (9i, 10i, 10n, 12d) and 8 carbon atoms (9j, 10j, 11e, 12e) generally showed potent inhibition activities in our study. So the linker length and the bulk of ligustrazine had a certain impact on the inhibition activity. Generally, for the system reported here, their inhibition potency towards AChE and BuChE was dependent on the integral contribution of the linker length, the electronic density in conjugation partner as well as the steric effect. Table 1. Inhibition of AChE and BuChE (IC50 Values). IC50 (nM)a Sturcture
IC50 (nM)a Compd.
n AChEb
BuChEc
Sturcture
n AChEb
BuChEc
RI PT
Compd.
2.6±0.7
28.6±6.5
8
144.9±11.3
16.6±3.7
2
33.1±5.0
502.4±57.1
3
12.2±0.3
22.6±5.6
4
188.0±19.9
32.4±4.5
6
14.7±1.9
28.3±3.3
8
304.1±17.9
173.8±24.6
2
108.5±15.9
﹥1000
11b
3
13.7±4.0
199.1±6.0
9.3±3.9
11c
4
138.7±18.6
110.9±8.7
44.9±7.1
588.8±73.8
11d
6
136.2±26.6
68.1±3.2
3
205.1±9.7
5.2±0.3
11e
8
﹥1000
61.8±9.5
10c
4
333.7±45.0
2.3±0.6
12a
2
﹥1000
816.2±66.0
10d
6
303.7±31.9
147.0±5.1
12b
3
﹥1000
164.5±7.7
10e
8
263.5±26.8
228.1±54.3
12c
4
﹥1000
﹥1000
10f
2
65.3±7.5
612.7±60.7
12d
6
270.3±53.5
1.3±0.1
3
115.7±3.3
﹥1000
12e
8
65.1±5.6
3.8±0.4
4
178.9±26.6
﹥1000
Tacrine
70.7±3.9
7.2±0.2
109.5±15.4
302.2±8.1
10i
9b
3
127.5±7.0
43.3±2.7
10j
9c
4
138.9±15.5
29.8±2.5
10k
9d
6
44.6±9.6
24.1±0.5
10l
9e
8
522.0±21.1
278.5±5.0
10m
9f
2
100.5±7.3
﹥1000
10n
9g
3
25.8±0.7
329.8±5.8
10o
9h
4
64.4±3.7
240.9±7.1
11a
9i
6
3.9±0.8
24.3±3.1
9j
8
32.5±1.5
10a
2
10b
TE D
M AN U
2
10g 10h
SC
6
9a
Results were the mean of three independent experiments (n = 3) ± SD.
b
AChE from electric eel, Type VI-S, EC 3.1.1.7.
c
BuChE form equine serum, EC 3.1.1.8.
EP
a
In the following step, selected compounds 9i and 10i were also evaluated as inhibitors of human AChE (hAChE) and human BuChE (hBuChE), results (Table 2) showed that 9i (IC50 = 65.2 nM) was a potent inhibitor towards hAChE, nearly 2-fold of that
AC C
tacrine (IC50 = 116.8 nM), but its inhibition activity towards hBuChE (IC50 = 48.9 nM) was relatively lower than tacrine (IC50 = 34.4 nM). Compound 10i (IC50 of hAChE = 108.4 nM, IC50 of hBuChE = 577.0 nM) exhibited lower activity than tacrine towards either hAChE or hBuChE.
Table 2. Inhibition of hAChE and hBuChE (IC50 Values). IC50 (nM)a
Compd.
hAChEb
hBuChEc
9i
65.2±13.2
48.8±19.5
10i
108.4 ±25.5
577.0±37.5
Tacrine
116.8±15.9
34.4±4.1
a
Results were the mean of three independent experiments (n = 3) ± SD.
b
hAChE from human erythrocytes, EC 3.1.1.7.
c
hBuChE form human serum, EC 3.1.1.8.
2.3. Prediction of ADME property and blood-brain barrier permeability.
ACCEPTED MANUSCRIPT The ADME (absorption, distribution, metabolism and excretion) properties of all of the target compounds 9a−12e were predicted
using
MOE
2008.10,
their
BBB
permeation
and
HIA
were
predicted
using
admetSAR
server
(http://lmmd.ecust.edu.cn:8000/ predict/) (Table 3) [77]. It can be seen that not all tested compounds met the criteria of lipinski’s rule of five. According to this prediction, tacrine–p-coumaric acid hybrids showed higher probability of BBB permeation than that of sinapic acid hybrids, while less difference among HIA data. Compounds 9a−9e, 9h−9j, 10a−10e, 10m−10o, 11c−11e (9i included) could be absorbed by human intestinal and were able to penetrate brain; therefore, they were predicted that could be
RI PT
oral administrated as CNS-active compounds. Table 3. The ADME properties and BBB permeability of compounds 9a−12e. Comp.
MWa
HBDa
HBAa
LogPa
Rotora
ASAa (Å2)
ASA-Pa (Å2)
BBB permeation (±) and probabilityb
HIAb
9a
387
3
5
4.01
7
697.03
153.53
BBB+ 0.8789
0.9699
9b
401
3
5
4.45
8
739.98
143.90
BBB+ 0.9005
9c
415
3
5
4.89
9
762.92
143.42
BBB+ 0.9366
9d
443
3
5
5.78
11
818.24
138.99
BBB+ 0.9366
9e
471
3
5
6.66
13
868.80
134.22
9f
447
3
7
3.99
9
733.44
192.34
9g
461
3
7
4.43
10
814.61
195.09
9h
475
3
7
4.87
11
838.14
194.52
BBB+ 0.5000
0.9554
9i
503
3
7
5.76
13
884.86
193.35
BBB+ 0.5000
0.9554
9j
531
3
7
6.64
15
958.90
186.72
BBB+ 0.5000
0.9554
10a
521
2
7
4.53
10
888.92
126.12
BBB+ 0.5745
0.9851
10b
535
2
7
4.97
11
943.25
118.09
BBB+ 0.5599
0.9874
10c
549
2
7
5.41
12
989.80
120.95
BBB+ 0.6943
0.9939
10d
577
2
7
6.29
14
1009.95
114.32
BBB+ 0.6943
0.9939
10e
605
2
7
7.18
16
1064.77
110.52
BBB+ 0.6943
0.9939
10f
581
2
9
4.01
12
949.61
186.25
BBB- 0.7967
0.9236
10g
595
2
9
4.45
13
1022.45
181.84
BBB- 0.7867
0.9345
10h
609
2
9
4.90
14
1035.15
180.76
BBB- 0.6594
0.9678
10i
637
2
9
5.78
16
1097.94
172.70
BBB- 0.6594
0.9678
10j
665
2
9
6.66
18
1085.11
175.91
BBB- 0.6594
0.9678
10k
551
2
10l
565
2
10m
579
2
0.9907
SC
0.9907
BBB+ 0.9366
0.9907
BBB- 0.7251
0.8645
BBB- 0.6523
0.9105
M AN U
TE D
EP
0.9808
4.27
11
937.98
167.48
BBB- 0.6321
0.9705
8
4.71
12
982.16
143.48
BBB- 0.6420
0.9748
8
5.15
13
955.21
145.76
BBB+ 0.5194
0.9879
AC C
8
10n
607
2
8
6.04
15
1041.88
138.56
BBB+ 0.5194
0.9879
10o
635
2
8
6.92
17
1115.12
141.03
BBB+ 0.5194
0.9879
11a
671
2
10
4.52
13
1072.9
148.48
BBB- 0.6229
0.9840
11b
685
2
10
4.96
14
1079.8
134.55
BBB- 0.6276
0.9864
11c
699
2
10
5.41
15
1182.7
140.11
BBB+ 0.5214
0.9935
11d
727
2
10
6.29
17
1193.7
142.08
BBB+ 0.5214
0.9935
11e
756
2
10
7.17
19
1268.2
135.88
BBB+ 0.5214
0.9935
12a
689
3
11
3.39
14
1105.94
139.23
BBB- 0.9320
0.6597
12b
703
3
11
3.83
15
1050.16
111.51
BBB- 0.9619
0.5223
12c
717
3
11
4.28
16
1029.06
124.95
BBB- 0.9320
0.6597
12d
745
3
11
5.16
18
1226.38
137.65
BBB- 0.9320
0.6597
12e
774
3
11
6.04
20
1252.38
158.21
BBB- 0.9320
0.6597
ACCEPTED MANUSCRIPT a
MW (molecular weight), HBD (number of H-bond donors), HBA (number of H-bond acceptors), LogP (log octanol/water
partition coefficient), Rotor (number of rotatable bonds), ASA (water accessible surface area) and ASA-P (total polar surface area) were predicted using MOE 2008.10. b
The BBB (blood-brain barrier) permeability and HIA (probability of human intestinal absorption) were predicted using
admetSAR server (http://lmmd.ecust.edu.cn:8000/ predict/). 2.4. Inhibition of Aβ42 self-aggregation Compounds 9i and 10i, because of potent AChE inhibition activities in our screening compounds, their potential inhibition
RI PT
activities towards Aβ42 aggregation were further examined using thioflavin T (ThT) fluorescence method [78]. Hybrid 9i could efficiently inhibit the self-mediated Aβ42 aggregation, and its activity was dose-dependent with a good S shape curve, less potencies at lower concentrations, higher potencies at higher concentrations (20, 100 µM) compared with tacrine (Figure 3), inhibition ratio 47% at 20 µM, 48% at 100 µM, following a plateau stage. Compound 10i displayed less potent inhibition activity
M AN U
SC
for Aβ42 self-aggregation.
Figure 3. Inhibition of Aβ42 self-aggregation curves for 9i (solid square), 10i (solid triangle) and tacrine (solid circle) determined by the ThT assay.
2.5. Inhibition of Aβ42 fibril formation monitored by atomic force microscope (AFM)
Aβ42 aggregation behavior in the absence and presence of compounds 9i, 10i and tacrine, were monitored using AFM. As
TE D
shown in Figure 4, Aβ42 alone easily aggregated into dense, thick and large fibrils at concentration of 0.1 mM, while amorphous deposits with some well-defined Aβ42 fibrils formed in the presence of tacrine (0.1 mM). No obvious Aβ42 fibril was observed in the presence of 9i (0.1 mM) and 10i (0.1 mM), and more patchy aggregations occurred in 10i group. The small dots were the crystal of 10i extracted due to the poor water solubility, other than Aβ42 aggregation. These AFM experimental results were in agreement with the results of ThT studies (Figure 3), which revealed that compound 9i could efficiently inhibit the Aβ42 fibrils
AC C
EP
formation.
Figure 4. AFM image of Aβ42 aggregation (CAβ = 0.1 mM) recorded in the absence (A) and presence of tacrine (0.1 mM, B), 9i (0.1 mM, C) 10i (0.1 mM, D) incubated at 37 °C for 4 days, respectively. 2.6. Antioxidant activity in vitro 9i and 10i, the most potent two compounds screened in AChE inhibition study, their antioxidant activities were evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay with trolox as reference compound (Table 4) [34]. Results showed that 9i had very potent peroxyl radical scavenging capacity. However, compound 10i, with phenolic hydroxyl group covered by a TMP moiety, exhibited a poor antioxidant activity with IC50 over 1000 µM.
ACCEPTED MANUSCRIPT Table 4. Inhibitory activities in DPPH assay.
a
Compd.
IC50 (µM)a
9i
85.8±3.5
10i
﹥1000
Trolox
52.9±1.3
Results were the mean of three independent experiments (n = 3) ± SD.
2.7. Neuroprotection
RI PT
Neurotoxicity of compound 9i, 10i on NGF-differentiated PC12 cells were determined at 1.25, 2.5, 5 and 10 µM with tacrine as control, shown in Figure 5 (A). The toxicity of tacrine increased with concentration, while compound 9i exhibited no neurotoxicity to NGF-differentiated PC12 cells at the experimental concentration range of 10 µM (131%). Unfortunately, 10i exhibited significant neurotoxicity, larger than tacrine. Neuroprotection effect was studied on the NGF-differentiated PC12 cells treated with CoCl2 [79, 80], with trolox as reference, shown in Figure 5 (B). The viability of CoCl2-damaged PC12 cell was only
SC
51.2%, and increased to 101.3% pretreated by 10 µM trolox, while it increased to 74.5%, 92.4%, 99.5% and 106.8% respectively, pretreated by compound 9i at 1.25, 2.5, 5 and 10 µM, the cell viability increased in a dose dependent manner. This indicated that 9i could protect PC12 cells from CoCl2 damage. Compound 10i exhibited neuroprotective effect at 1.25 µM (73.3%); its cytotoxicity increased with increasing concentrations, the cell viabilities were 37.7%, 15%, 11.2% at 2.5, 5 and 10 µM
EP
TE D
might come from radical scavenging action.
M AN U
respectively. This result was positive-correlated with their antioxidant assay, which indicated that the neuroprotective action of 9i
Figure 5. Neurotoxicity (A) and neuroprotection (B) of 9i and 10i on normal and CoCl2-damaged PC12 cells. Data were expressed as mean ± SD (n = 3). ** p < 0.01: A, compared with NGF group; B, compared with CoCl2 group.
AC C
2.8. Hepatotoxicity study in vitro and in vivo
The goal of the present work was to find novel candidate with potent anti-AD activity and lower toxicity, and the conjugate
reported here were the derivatives of tacrine, a withdraw medicine due to obvious hepatotoxicity in clinic. Compound 9i had potent activity in ChEs inhibition and block Aβ42 self-aggregation had been proved in vitro, so the toxicity study, especially the hepatotoxicity becomes very important for its further study as a candidate. Its hepatotoxicity in vitro was analyzed via MTT assay of HepG2 cell line (Figure 6) [44], which was incubated with 9i of 1.25, 2.5, 5 and 10 µM respectively for 24 h. A concentration-dependent decrease in the cell viability was observed for tacrine and 9i, whereas the cell viability of 9i (98.4%, 96.3%, 94.9% and 81.8%) was higher than that of tacrine (92.5%, 90.9%, 89.8% and 77.6%) at the same condition. This suggested 9i had a lower hepatotoxicity profile with respect to tacrine. Its hepatotoxicity in vivo was assayed with adult male ICR mice as model [37], orally administrated compound 9i at dose of 12.82 µmol/100 g b wt. Samples taken at 8 h, 24 h and 36 h were subjected to hepatotoxicity assay respectively. Results (Table 5) indicated that tacrine had higher hepatotoxicity with higher ALT and AST values at each time point, while compound 9i had lower hepatotoxicity than tacrine from 8 h to 36 h, both ALT and AST values were very approached the control group except relative higher AST value at 8 h. Considering its potent ChEs
ACCEPTED MANUSCRIPT
RI PT
inhibition activity and lower hepatotoxicity than tacrine, 9i was a promising anti-AD candidate.
Figure 6. In vitro hepatotoxicity of 9i on HepG2 cell line. Data were expressed as mean ± SD (n = 3). * p < 0.05, ** p < 0.01,
SC
compared with control group.
Table 5. ALT and AST values at different time points measured after ICR mice oral administration of tacrine and 9i. ALT(U/L)
AST(U/L)
Groups 8h
24 h
36 h
8h
24 h
36 h
Control
40.9±2.2
37.3±2.9
27.8±1.8
25.5±1.6
20.8±2.2
Tacrine
45.3±2.0*
55.7±2.5**
35.1±1.7**
38.8±2.1**
28.3±1.4**
21.9±1.6**
9i
43.2±3.4
40.6±2.1
29.7±2.7
34.3±1.8**
21.7±2.7
19.9±1.8
M AN U
18.5±1.7
Values were expressed as mean ± SD (n = 8. t-test, compared to control of the same time after administration, * p ≤ 0.05, ** p ≤ 0.01). 2.9. Kinetic characterization of AChE inhibition
Because of the best total performance including cholinesterases inhibition, inhibition of Aβ42 aggregation, antioxidant
TE D
activity and neuroprotection as well as hepatotoxicity study, 9i was selected for a kinetic study to gain information on the mechanism of inhibition. Kinetic characterization of 9i inhibition AChE was elucidated from the Lineweaver-Burk reciprocal plots. Both slopes (decreased Vmax) and intercepts (higher Km) increased with increasing concentrations of 9i (Figure 7),
AC C
EP
implied 9i was a mixed-type inhibitor.
Figure 7. Lineweaver-Burk plots resulting from subvelocity curves of AChE activity at different substrate concentrations (0.05~0.5 mM) in the absence and presence of 2.5, 5, 10 nM 9i. 2.10. Molecular modeling study
To further study the interaction mode of 9i with ChEs, molecular docking study was performed using software package MOE 2008.10 [72]. The X-ray crystal structure of the TcAChE complex with bis(7)-tacrine (PDB code: 2CKM) was applied to build the starting model of AChE. Tacrine moiety of 9i was located into the CAS of TcAChE, via aromatic π–π stacking interactions with the phenyl ring from Phe 330 (3.66 Å) and the indole ring from Trp 84 (3.45 Å), respectively. The sinapic acid moiety occupied the PAS of the enzyme, and bound with residues Ser 286, Asp 285 via hydrophobic interactions (Figure 8, A1 and A2). In addition, the interaction mechanism of 9i with hAChE in complex with donepezil (PDB code: 4EY7) was also carried out [53]. As shown in Figure 8 (A3) and (A4), sinapic acid moiety was located into PAS of hAChE through aromatic π–π
ACCEPTED MANUSCRIPT stacking interaction with Trp 286 (3.43 Å). Tacrine moiety occupied the CAS and stacked against the indole ring of Trp 86 (4.18 Å). In middle gorge, a hydrophobic interactions with the residues Tyr124 (2.68 Å) was observed, which could enhance the interaction with hAChE. These results indicated that compound 9i could bind to the CAS and PAS of AChE in interaction model of both TcAChE and hAChE. Since equine serum BuChE, whose crystal structure has not been reported, is homology with human BuChE, so, the hBuChE (PDB code: 1P0I) was used in the docking study. A π–π stacking interaction was found between tacrine and Trp 82 (3.41 Å), and two hydrogen bonds were observed between the hydroxyl group and Ser 198 (2.95 Å), methoxy group and His 438 (3.23
RI PT
Å) (Figure 8, B1 and B2). These docking models could also give some clues to other dihybrids or trihybrids interaction with
M AN U
SC
ChEs in this report.
Figure 8. 3D, 2D docking model of compound 9i with TcAChE (A1, A2), hAChE (A3, A4) and hBuChE (B1, B2). These figures were depicted using the ligand interactions applied in MOE. Atom colors: gray–carbon atoms, dark blue–nitrogen atoms, red– oxygen atoms.
TE D
3. Conclusion
In conclusion, a series of novel tacrine-phenolic acid dihybrids and tacrine-phenolic acid-ligustrazine trihybrids were designed and synthesized as multifunctional ChEs inhibitor for the treatment of AD. Most of them could effectively inhibit ChEs in vitro. Compound 9i (IC50 of eeAChE = 3.9 nM, IC50 of hAChE = 65.2 nM; IC50 of eqBuChE = 24.3 nM, IC50 of hBuChE = 48.8 nM) was the most potent AChE inhibitor (18 or 2-fold activity of tacrine), but less potent towards BuChE than tacrine. 9i
EP
could also block Aβ42 self-aggregation with an inhibition ratio of 47% at 20 µM. Its strong anti-oxidation activity could protect PC12 cells from CoCl2-damage in the experimental condition while no neurotoxicity. Hepatotoxicity assays in vitro and in vivo indicated that 9i had no obvious hepatotoxicity, was safer than tacrine. Kinetic and molecular modeling study revealed that 9i
AC C
was a mixed-type AChE inhibitor, could interact simultaneously with CAS and PAS of AChE. Summary above, the new hybrid 9i, with characteristics of potent AChE inhibition, blocking Aβ42 self-aggregation, neuroprotection and low hepatotoxicity, was a promising anti-AD drug candidate. 4. Experimental section 4.1. Chemistry 1
H NMR spectra and 13C NMR were acquired on a Bruker AVANCE III (400MHz) or VARIAN INOVA (500MHz) and
referenced to tetramethylsilane (TMS). Chemical shifts were reported in ppm (δ) using the residue solvent line as the internal standard. High resolution mass spectra (HRMS) were recorded on Agilent 6520 Q-TOF LC/MS and Varian 7.0T FTMS (MALDI). Melting points are uncorrected and were measured using a digital melting point apparatus (Shenguang WRS-1B, shanghai, China). Flash column chromatography was performed with silica gel (200–300 mesh). Reactions were followed by thin-layer chromatography (TLC) on silica gel GF254 plates (Qingdao Haiyang Chemical Plant, Qingdao, China), and the spots were visualized by modified bismuth potassium iodide or using a UV lamp (λ = 254 nm). The purity of the synthesized compounds was over 95% analyzed by HPLC
(Waters
2695 Alliance system), with Kromasil C18 column eluted by
ACCEPTED MANUSCRIPT acetonitrile/water (15/85-95/5) containing 0.1% formic acid at a flow rate of 1 mL/min. Compounds 3a−3e were prepared following previously published methods [58, 71]. 4.2. Preparation of intermediates 7a−7c Compound TMP-Br was prepared according to our previously reported method [27]. Compound 6a−6c was gained according to the method described by Kojima and Tranchimand [81, 82]. An aqueous solution of KOH (24.90 mmol) was added to a solution of intermediates (6a and 6b, 7.01 mmol) in ethanol. The mixture was stirred for 40 min at 60 °C. Then the pH of mixture was adjusted to 3 with 10% hydrochloric acid to afford crude compounds, filtered, and purified by recrystallization from
solid, yield 89%. HRMS (ESI) m/z 449.2184 [M+H]+, calcd. for [C25H29N4O4]+, 449.2189.
RI PT
ethyl acetate. 7a, white solid, yield 83%. HRMS (ESI) m/z 329.1498 [M+H]+, calcd. for [C18H21N2O4]+, 329.1501. 7b, white
To a solution of 6c (7.01 mmol) in methanol (20 mL), was added Pd/C 10% (0.01 g). Then the suspension was stirred under hydrogen atmosphere at room temperature for 12 h. The mixture was filtered, concentrated under vacuum and purified by silica
HRMS (ESI) m/z 467.2293 [M+H]+, calcd. for [C25H31N4O5]+, 467.2294. 4.3. General procedure I for the preparation of compounds 9a−9j
SC
gel chromatography (petroleum ether: acetone = 4:1) to give corresponding target compound 7c. 7c, light yellow solid, yield 84%,
To a stirred solution of cinnamic acids (4.14 mmol), EDCI (4.97 mmol), DMAP (1.24 mmol) in anhydrous DMF (10 mL) was added intermediate 3a−3e. The reaction mixture was stirred at room temperature for 12 h. It was concentrated to dryness and
M AN U
redissolved in CH2Cl2 (30 mL), washed successively with water (2 × 30 mL) and brine (30 mL), dried over sodium sulfate, concentrated under vacuum and purified by silica gel chromatography (CH2Cl2: MeOH = 20:1 ) to give compound 9a−9j. (E)-3-(4-hydroxyphenyl)-N-(2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)acrylamide (9a) Compound 9a was prepared according to the general procedure I, light yellow solid, yield 63%, mp > 210 °C. 1H NMR (400 MHz, DMSO) δ 9.94 (s, 1H, -OH), 8.62 (t, J = 5.7 Hz, 1H, -CONH), 8.46 (d, J = 8.7 Hz, 1H), 8.01 (br, 1H, -NH), 7.93 (d, J = 8.2 Hz, 1H), 7.81 - 7.77 (m, 1H), 7.52 - 7.49 (m, 1H), 7.35 - 7.30 (m, 3H), 6.78 - 6.76 (m, 2H), 6.39 (d, J = 15.7 Hz, 1H), 3.98 3.95 (m, 2H), 3.54 - 3.49 (m, 2H), 2.94 (br, 2H), 2.64 (br, 2H), 1.77 (br, 4H). 13C NMR (101 MHz, DMSO) δ 166.97, 159.08,
TE D
155.89, 150.50, 139.38, 137.84, 132.44, 129.22, 129.22, 125.57, 125.18, 124.95, 119.01, 117.80, 115.75, 115.75, 115.49, 111.21, 48.22, 27.83, 23.78, 21.42, 20.21. HRMS (ESI) m/z 388.2025 [M+H]+, calcd. for [C24H26N3O2]+, 388.2025. (E)-3-(4-hydroxyphenyl)-N-(3-((1,2,3,4-tetrahydroacridin-9-yl)amino)propyl)acrylamide (9b) Compound 9b was prepared according to the general procedure I, light yellow oil, yield 65%. 1H NMR (400 MHz, DMSO) δ 9.90 (br, 1H, -OH), 8.34 (d, J = 8.6 Hz, 1H), 8.29 (br, 1H, -CONH), 7.87 (d, J = 8.4 Hz, 1H), 7.75 - 7.71 (m, 1H), 7.50 - 7.46 (m,
EP
1H), 7.39 -7.37 (m, 2H), 7.32 (d, J = 15.7 Hz, 1H), 7.20 (br, 1H, -NH), 6.81 (d, J = 8.5 Hz, 2H), 6.42 (d, J = 15.7 Hz, 1H), 3.78 3.74 (m, 2H), 3.28 - 3.24 (m, 2H), 2.96 (br, 2H), 2.71 (br, 2H), 1.87 - 1.81 (br, 6H). 13C NMR (101 MHz, DMSO) δ 165.79, 158.89, 153.85, 152.91, 138.71, 130.96, 129.09, 129.09, 125.76, 124.42, 124.26, 121.97, 118.41, 117.01, 115.71, 115.71, 115.71,
AC C
112.64, 44.76, 35.88, 30.33, 29.64, 24.31, 21.83, 20.89. HRMS (ESI) m/z 402.2182 [M+H]+, calcd. for [C25H28N3O2]+, 402.2182. (E)-3-(4-hydroxyphenyl)-N-(4-((1,2,3,4-tetrahydroacridin-9-yl)amino)butyl)acrylamide (9c) Compound 9c was prepared according to the general procedure I, light yellow oil, yield 69%. 1H NMR (400 MHz, DMSO) δ 9.94 (s, 1H, -OH), 8.43 (d, J = 8.6 Hz, 1H), 8.15 - 8.13 (m, 1H, -CONH), 7.98 (d, J = 8.0 Hz, 1H), 7.84 - 7.80 (m, 2H), 7.57 7.53 (m, 1H), 7.35 (d, J = 8.6 Hz, 2H), 7.28 (d, J = 15.7 Hz, 1H), 6.80 (d, J = 8.6 Hz, 2H), 6.42 (d, J = 15.7 Hz, 1H), 3.90 - 3.85 (m, 2H), 3.20 - 3.15 (m, 2H), 3.00 (br, 2H), 2.67 (br, 2H), 1.81 (br, 4H), 1.78 - 1.73 (m, 2H), 1.56 - 1.49 (m, 2H). 13C NMR (101 MHz, DMSO) δ 165.33, 158.83, 155.55, 150.61, 138.37, 137.84, 132.39, 128.99, 128.99, 125.80, 124.96, 124.96, 119.10, 118.72, 115.70, 115.55, 115.55, 111.07, 46.82, 38.05, 27.88, 27.36, 26.24, 23.96, 21.42, 20.20. HRMS (ESI) m/z 416.2341 [M+H]+, calcd. for [C26H30N3O2]+, 416.2338. (E)-3-(4-hydroxyphenyl)-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)acrylamide (9d) Compound 9d was prepared according to the general procedure I, light yellow oil, yield 66%. 1H NMR (400 MHz, DMSO) δ 9.94 (s, 1H, -OH), 8.39 (d, J = 8.6 Hz, 1H), 8.10 - 8.08 (m, 1H, -CONH), 7.98 (d, J = 8.2 Hz, 1H), 7.83 - 7.78 (m, 2H), 7.56 7.52 (m, 1H), 7.34 (d, J = 8.6 Hz, 2H), 7.28 (d, J = 15.7 Hz, 1H), 6.79 (d, J = 8.6 Hz, 2H), 6.45 (d, J = 15.7 Hz, 1H), 3.84 - 3.79
ACCEPTED MANUSCRIPT (m, 2H), 3.14 - 3.12 (m, 2H), 3.00 (br, 2H), 2.65 (br, 2H), 1.80 (br, 4H), 1.73 - 1.70 (m, 2H), 1.45 - 1.42 (m, 2H), 1.33 - 1.32 (m, 4H). 13C NMR (101 MHz, DMSO) δ 165.28, 158.80, 155.41, 150.64, 138.28, 138.00, 132.29, 128.96, 128.96, 125.84, 124.92, 124.87, 119.24, 118.85, 115.69, 115.69, 115.56, 111.08, 47.04, 38.37, 29.77, 29.02, 27.95, 25.97, 25.70, 23.94, 21.43, 20.25. HRMS (ESI) m/z 444.2652 [M+H]+, calcd. for [C28H34N3O2]+, 444.2651. (E)-3-(4-hydroxyphenyl)-N-(8-((1,2,3,4-tetrahydroacridin-9-yl)amino)octyl)acrylamide (9e) Compound 9e was prepared according to the general procedure I, light yellow oil, yield 70%. 1H NMR (500 MHz, CD3OD) δ 8.29 (d, J = 8.7 Hz, 1H), 7.77 - 7.75 (m, 1H), 7.71 - 7.70 (m, 1H), 7.52 - 7.49 (m, 1H), 7.33 (d, J = 15.7 Hz, 1H), 7.27 (d, J = 8.7
RI PT
Hz, 2H), 6.68 (d, J = 8.6 Hz, 2H), 6.35 (d, J = 15.7 Hz, 1H), 3.87 - 3.84 (m, 2H), 3.23 - 3.20 (m, 2H), 2.94 - 2.92 (m, 2H), 2.61 2.59 (m, 2H), 1.90 - 1.86 (m, 4H), 1.80 - 1.74 (m, 2H), 1.51 - 1.49 (m, 2H), 1.39 - 1.31 (m, 8H). 13C NMR (101 MHz, DMSO) δ 165.22, 158.79, 155.52, 150.65, 138.28, 137.98, 132.39, 128.98, 128.98, 125.86, 124.95, 124.90, 119.22, 118.86, 115.69, 115.69, 115.57, 111.10, 47.16, 38.52, 30.72, 29.73, 29.11, 28.50, 27.93, 26.28, 25.91, 23.92, 21.42, 20.26. HRMS (ESI) m/z 472.2968 [M+H]+, calcd. for [C30H38N3O2]+, 472.2964. Due to rapid proton exchange with MeOH in CD3OD, the proton NH and phenol
SC
OH missed in the 1H NMR spectrum.
(E)-3-(4-hydroxy-3,5-dimethoxyphenyl)-N-(2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)acrylamide (9f) Compound 9f was prepared according to the general procedure I, white solid, yield 66%, mp > 210 °C. 1H NMR (500 MHz, DMSO) δ 8.86 (s, 1H, -OH), 8.57 (t, J = 5.9 Hz, 1H, -CONH), 8.50 (d, J = 8.7 Hz, 1H), 8.06 (br, 1H, -NH), 7.95 (dd, J = 8.5 Hz,
M AN U
0.9 Hz, 1H), 7.86 - 7.83 (m, 1H), 7.57 - 7.54 (m, 1H), 7.35 (d, J = 15.7 Hz, 1H), 6.84 (s, 2H), 6.47 (d, J = 15.7 Hz, 1H), 4.03 4.00 (m, 2H), 3.78 (s, 6H), 3.57 - 3.54 (m, 2H), 2.99 - 2.97 (m, 2H), 2.70 - 2.68 (m, 2H), 1.82 - 1.80 (m, 4H). 13C NMR (101 MHz, DMSO) δ 166.89, 155.96, 150.54, 148.04, 148.04, 139.93, 137.86, 137.56, 132.49, 125.18, 125.00, 124.95, 119.02, 118.54, 115.50, 111.23, 105.43, 105.43, 55.98, 55.98, 48.29, 27.83, 23.75, 21.42, 20.21. HRMS (ESI) m/z 448.2240 [M+H]+, calcd. for [C26H30N3O4]+, 448.2236.
(E)-3-(4-hydroxy-3,5-dimethoxyphenyl)-N-(3-((1,2,3,4-tetrahydroacridin-9-yl)amino)propyl)acrylamide (9g) Compound 9g was prepared according to the general procedure I, light yellow solid, yield 59%, mp > 210 °C. 1H NMR (400
TE D
MHz, DMSO) δ 8.82 (br, 1H, -OH), 8.15 (d, J = 8.3 Hz, 1H), 8.05 - 8.02 (m, 1H, -CONH), 7.72 - 7.70 (m, 1H), 7.53 - 7.50 (m, 1H), 7.36 - 7.34 (m, 1H), 7.35 (d, J = 15.7 Hz, 1H), 6.85 (s, 2H), 6.46 (d, J = 15.7 Hz, 1H), 5.57 (t, J = 6.3 Hz, 1H, -NH), 3.79 (s, 6H), 3.46 -3.41 (m, 2H), 3.26 - 3.21 (m, 2H), 2.92 - 2.89 (m, 2H), 2.75 - 2.73 (m, 2H), 1.82 - 1.80 (m, 4H), 1.76 - 1.69 (m, 2H). 13
C NMR (101 MHz, DMSO) δ 165.62, 157.93, 150.09, 148.05, 148.05, 146.85, 139.27, 137.33, 128.28, 127.81, 125.24, 123.27,
122.83, 120.28, 119.24, 115.99, 105.27, 105.27, 55.94, 55.94, 45.15, 36.16, 33.47, 30.77, 25.11, 22.73, 22.39. HRMS (ESI) m/z
EP
462.2397 [M+H]+, calcd. for [C27H32N3O4]+, 462.2393.
(E)-3-(4-hydroxy-3,5-dimethoxyphenyl)-N-(4-((1,2,3,4-tetrahydroacridin-9-yl)amino)butyl)acrylamide (9h) Compound 9h was prepared according to the general procedure I, light yellow oil, yield 66%. 1H NMR (400 MHz, DMSO) δ
AC C
8.79 (br, 1H, -OH), 8.13 (d, J = 8.4 Hz, 1H), 7.94 - 7.91 (m, 1H, -CONH), 7.70 (d, J = 8.4 Hz, 1H), 7.54 - 7.50 (m, 1H), 7.36 7.34 (m, 1H), 7.30 (d, J = 15.8 Hz, 1H), 6.83 (s, 2H), 6.44 (d, J = 15.7 Hz, 1H), 5.49 (br, 1H, -NH), 3.78 (s, 6H), 3.44 - 3.40 (m, 2H), 3.17 - 3.13 (m, 2H), 2.91 - 2.89 (m, 2H), 2.73 - 2.70 (m, 2H), 1.81- 1.76 (m, 4H), 1.61 - 1.56 (m, 2H), 1.51 - 1.46 (m, 2H). 13
C NMR (101 MHz, DMSO) δ 165.19, 157.63, 150.42, 148.05, 148.05, 146.52, 139.01, 137.26, 127.98, 127.89, 125.30, 123.26,
123.05, 120.08, 119.45, 115.70, 105.21, 105.21, 55.94, 55.94, 47.65, 38.36, 33.26, 28.09, 26.59, 25.00, 22.67, 22.31. HRMS (ESI) m/z 476.2549 [M+H]+, calcd. for [C28H34N3O4]+, 476.2549. (E)-3-(4-hydroxy-3,5-dimethoxyphenyl)-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)acrylamide (9i) Compound 9i was prepared according to the general procedure I, light yellow oil, yield 64%. 1H NMR (400 MHz, DMSO) δ 8.74 (br, 1H, -OH), 8.17 (d, J = 8.4 Hz, 1H), 7.92 - 7.89 (m, 1H, -CONH), 7.75 - 7.73 (m, 1H), 7.60 - 7.56 (m, 1H), 7.40- 7.36 (m, 1H), 7.30 (d, J = 15.6 Hz, 1H), 6.83 (s, 2H), 6.46 (d, J = 15.7 Hz, 1H), 5.90 (br, 1H, -NH), 3.78 (s, 6H), 3.52 - 3.47 (m, 2H), 3.15 - 3.10 (m, 2H), 2.91 - 2.90 (m, 2H), 2.71 - 2.68 (m, 2H), 1.81 - 1.80 (m, 4H), 1.61 - 1.57 (m, 2H), 1.43 - 1.40 (m, 2H), 1.30 - 1.29 (m, 4H). 13C NMR (101 MHz, DMSO) δ 165.13, 156.24, 151.46, 148.03, 148.03, 145.08, 138.91, 137.23, 128.83, 126.22, 125.31, 123.54, 123.43, 119.53, 119.19, 114.78, 105.19, 105.19, 55.93, 55.93, 47.72, 38.45, 32.23, 30.37, 29.08, 26.12, 25.95, 24.76,
ACCEPTED MANUSCRIPT 22.41, 21.93. HRMS (ESI) m/z 504.2867 [M+H]+, calcd. for [C30H38N3O4]+, 504.2862. (E)-3-(4-hydroxy-3,5-dimethoxyphenyl)-N-(8-((1,2,3,4-tetrahydroacridin-9-yl)amino)octyl)acrylamide (9j) Compound 9j was prepared according to the general procedure I, light yellow oil, yield 60%. 1H NMR (400 MHz, DMSO) δ 8.75 (br, 1H, -OH), 8.27 (d, J = 8.6 Hz, 1H), 7.97 - 7.94 (m, 1H, -CONH), 7.84 (d, J = 8.4 Hz, 1H), 7.71 - 7.67 (m, 1H), 7.47 - 7.43 (m, 1H), 7.30 (d, J = 15.6 Hz, 1H), 6.83 (s, 2H), 6.75 (br, 1H, -NH), 6.49 (d, J = 15.7 Hz, 1H), 3.78 (s, 6H), 3.66 - 3.61 (m, 2H), 3.15 - 3.10 (m, 2H), 2.95 (br, 2H), 2.67 (br, 2H), 1.81 (br, 4H), 1.66 - 1.61 (m, 2H), 1.40 - 1.39 (m, 2H), 1.29 - 1.24 (m, 8H). 13C NMR (101 MHz, DMSO) δ 165.15, 153.66, 153.35, 148.04, 148.04, 141.72, 138.88, 137.24, 130.47, 125.34, 124.15, 123.00,
21.96, 21.18. HRMS (ESI) m/z 532.3180 [M+H]+, calcd. for [C32H42N3O4]+, 532.3175. 4.4. General procedure II for the preparation of compounds 10a−10j
RI PT
123.00, 119.59, 117.52, 113.07, 105.20, 105.20, 55.95, 55.95, 47.50, 38.56, 30.27, 30.09, 29.11, 28.56, 28.56, 26.31, 26.06, 24.37,
To a solution of 9a−9j (0.516 mmol) in anhydrous DMF (10 mL) was added K2CO3 (0.774 mmol) and TMP-Br (0.619 mmol). The reaction mixture was stirred for 6 h at 60°C. It was filtered, concentrated and redissolved in CH2Cl2 (30 mL).Then it
SC
was washed with water (2 × 30 mL) and brine (30 mL), dried over sodium sulfate, concentrated under vacuum and purified by silica gel chromatography (CH2Cl2: MeOH=30:1) to afford corresponding target compound 10a−10j.
(E)-N-(2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)-3-(4-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)acrylamide (10a)
M AN U
Compound 10a was prepared according to the general procedure II, light yellow solid, yield 79%, mp > 210 °C. 1H NMR (400 MHz, DMSO) δ 8.66 - 8.63 (m, 1H, -CONH), 8.51 (d, J = 8.7 Hz, 1H), 8.02 (br, 1H, -NH), 7.96 - 7.94 (m, 1H), 7.87 - 7.83 (m, 1H), 7.59 - 7.55 (m, 1H), 7.51 (d, J = 8.8 Hz, 2H), 7.41 (d, J = 15.8 Hz, 1H), 7.07 (d, J = 8.8 Hz, 2H), 6.50 (d, J = 15.8 Hz, 1H), 5.18 (s, 2H), 4.03 - 4.00 (m, 2H), 3.59 - 3.54 (m, 2H), 2.99 (br, 2H), 2.70 (br, 2H), 2.48 (s, 3H), 2.45 (s, 3H), 2.44 (s, 3H), 1.83 (br, 4H). 13C NMR (101 MHz, DMSO) δ 166.72, 159.48, 155.92, 150.98, 150.59, 149.24, 148.23, 145.08, 138.85, 137.91, 132.50, 129.13, 129.13, 127.65, 125.19, 124.99, 119.19, 119.10, 115.54, 115.15, 115.15, 111.31, 69.38, 48.17, 27.90, 23.79, 21.44, 21.20, 20.91, 20.25, 20.10. HRMS (ESI) m/z 522.2871 [M+H]+, calcd. for [C32H36N5O2]+, 522.2869.
TE D
(E)-N-(3-((1,2,3,4-tetrahydroacridin-9-yl)amino)propyl)-3-(4-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)acrylamide (10b)
Compound 10b was prepared according to the general procedure II, light yellow oil, yield 79%. 1H NMR (400 MHz, DMSO) δ 8.41 - 8.38 (m, 1H), 8.37 (br, 1H, -CONH), 7.95 - 7.92 (m, 1H), 7.92 - 7.90 (m, 1H), 7.78 - 7.74 (m, 1H), 7.54 - 7.52 (m, 1H, -NH), 7.50 - 7.48 (m, 2H), 7.36 (d, J = 15.8 Hz, 1H), 7.06 (d, J = 8.7 Hz, 2H), 6.51 (d, J = 15.8 Hz, 1H), 5.18 (s, 2H), 3.85 - 3.80
EP
(m, 2H), 3.29 - 3.24 (m, 2H), 2.97 (br, 2H), 2.70 (br, 2H), 2.48 (s, 3H), 2.44 (br, 6H), 1.90 - 1.87 (m, 2H), 1.80 (br, 4H). 13C NMR (101 MHz, DMSO) δ 165.59, 159.34, 154.60, 151.82, 150.97, 149.24, 148.22, 145.10, 139.40, 138.14, 131.58, 128.98, 128.98, 127.82, 124.65, 124.53, 120.72, 119.80, 116.34, 115.12, 115.12, 111.96, 69.38, 44.68, 35.86, 30.21, 28.85, 24.16, 21.65, 21.18,
AC C
20.90, 20.58, 20.09. HRMS (ESI) m/z 536.3030 [M+H]+, calcd. for [C33H38N5O2]+, 536.3026. (E)-N-(4-((1,2,3,4-tetrahydroacridin-9-yl)amino)butyl)-3-(4-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)acrylamide (10c)
Compound 10c was prepared according to the general procedure II, light yellow oil, yield 82%. 1H NMR (400 MHz, DMSO) δ 8.41 (d, J = 15.7 Hz, 1H), 8.14 - 8.11 (m, 1H, -CONH), 7.88 - 7.86 (m, 1H), 7.86 - 7.83 (m, 1H), 7.81 - 7.79 (m, 1H, -NH), 7.58 7.54 (m, 1H), 7.47 (d, J = 8.7 Hz, 2H), 7.33 (d, J = 15.7 Hz, 1H), 7.05 (d, J = 15.7 Hz, 2H), 6.51 (d, J = 15.8 Hz, 1H), 5.17 (s, 2H), 3.91 - 3.86 (m, 2H), 3.22 - 3.17 (m, 2H), 2.98 (br, 2H), 2.66 (br, 2H), 2.47 (s, 3H), 2.43 (br, 6H), 1.81 (br, 4H), 1.77 - 1.74 (m, 2H), 1.58 - 1.51 (m, 2H). 13C NMR (101 MHz, DMSO) δ 165.16, 159.28, 155.67, 150.96, 150.45, 149.24, 148.23, 145.12, 137.88, 137.69, 132.60, 128.92, 128.92, 127.89, 125.04, 125.04, 120.07, 118.92, 115.44, 115.11, 115.11, 111.12, 69.39, 45.62, 35.63, 27.86, 27.36, 26.24, 23.92, 21.37, 21.18, 20.90, 20.19, 20.09. HRMS (ESI) m/z 550.3190 [M+H]+, calcd. for [C34H40N5O2]+, 550.3182. (E)-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)-3-(4-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)acrylamide (10d)
ACCEPTED MANUSCRIPT Compound 10d was prepared according to the general procedure II, light yellow oil, yield 81%. 1H NMR (400 MHz, DMSO) δ 8.40 (d, J = 8.6 Hz, 1H), 8.08 - 8.06 (m, 1H, -CONH), 7.94 (d, J = 8.3 Hz, 1H), 7.85 - 7.81 (m, 1H), 7.75 (br, 1H, -NH), 7.58 7.54 (m, 1H), 7.48 (d, J = 8.7 Hz, 2H), 7.33 (d, J = 15.7 Hz, 1H), 7.06 (d, J = 8.7 Hz, 2H), 6.50 (d, J = 15.8 Hz, 1H), 5.18 (s, 2H), 3.87 - 3.82 (m, 2H), 3.16 - 3.12 (m, 2H), 3.00 (br, 2H), 2.66 (br, 2H), 2.48 (s, 3H), 2.45 (s, 3H), 2.44 (s, 3H), 1.82 (br, 4H), 1.74 1.71 (m, 2H), 1.46 - 1.43 (m, 2H), 1.34 (br, 4H). 13C NMR (101 MHz, DMSO) δ 165.04, 159.26, 155.50, 150.98, 150.67, 149.25, 148.23, 145.13, 138.02, 137.80, 132.41, 128.91, 128.91, 127.94, 124.94, 124.94, 120.20, 119.26, 115.58, 115.12, 115.12, 111.16, 69.38, 47.11, 38.40, 29.76, 28.99, 27.98, 25.97, 25.70, 23.90, 21.42, 21.19, 20.90, 20.27, 20.10. HRMS (ESI) m/z 578.3500
RI PT
[M+H]+, calcd. for [C36H44N5O2]+, 578.3495.
(E)-N-(8-((1,2,3,4-tetrahydroacridin-9-yl)amino)octyl)-3-(4-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)acrylamide (10e)
Compound 10e was prepared according to the general procedure II, light yellow oil, yield 85%. 1H NMR (400 MHz, DMSO) δ 8.33 (d, J = 8.5 Hz, 1H), 8.07 - 8.05 (m, 1H, -CONH), 7.90 (d, J = 8.1 Hz, 1H), 7.78 - 7.74 (m, 1H), 7.52 - 7.51 (m, 1H), 7.48 (d,
SC
J = 8.7 Hz, 2H), 7.33 (d, J = 15.7 Hz, 1H), 7.29 (br, 1H, -NH), 7.05 (d, J = 8.7 Hz, 2H), 6.51 (d, J = 15.8 Hz, 1H), 5.17 (s, 2H), 3.76 - 3.72 (m, 2H), 3.15 - 3.10 (m, 2H), 2.98 (br, 2H), 2.66 (br, 2H), 2.48 (s, 3H), 2.44 (s, 3H), 2.44 (s, 3H), 1.81 (br, 4H), 1.70 1.64 (m, 2H), 1.41 - 1.40 (m, 2H), 1.31 - 1.24 (m, 8H). 13C NMR (101 MHz, DMSO) δ 165.03, 159.25, 154.53, 152.09, 150.98, 149.26, 148.24, 145.13, 139.61, 137.79, 131.52, 128.91, 128.91, 127.97, 124.57, 120.98, 120.96, 120.23, 116.46, 115.11, 115.11,
M AN U
112.01, 69.39, 47.33, 38.57, 29.90, 29.10, 29.02, 28.53, 28.49, 26.30, 25.98, 24.12, 21.67, 21.19, 20.90, 20.69, 20.10. HRMS (ESI) m/z 606.3813 [M+H]+, calcd. for [C38H48N5O2]+, 606.3808.
(E)-3-(3,5-dimethoxy-4-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-N-(2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl) acrylamide (10f)
Compound 10f was prepared according to the general procedure II, light yellow solid, yield 74% mp 203.8-204.7 °C. 1H NMR (400 MHz, DMSO) δ 8.58 - 8.55 (m, 1H, -CONH), 8.51 (d, J = 8.7 Hz, 1H), 8.01 (br, 1H, -NH), 7.92 - 7.85 (m, 2H), 7.61 - 7.56 (m, 1H), 7.40 (d, J = 15.7 Hz, 1H), 6.89 (s, 2H), 6.59 (d, J = 15.7 Hz, 1H), 4.99 (s, 2H), 4.04 - 4.03 (m, 2H), 3.77 (br, 6H), 3.59-
TE D
3.58 (m, 2H), 2.99 (br, 2H), 2.74 - 2.71 (m, 2H), 2.59 (s, 3H), 2.44 (s, 3H), 2.40 (s, 3H), 1.85 (br, 4H). 13C NMR (101 MHz, DMSO) δ 166.55, 156.03, 153.29, 153.29, 150.60, 150.04, 150.04, 147.68, 145.83, 139.39, 137.90, 137.21, 132.67, 130.65, 125.26, 125.05, 120.92, 119.08, 115.51, 111.35, 104.96, 104.96, 73.53, 55.87, 55.87, 48.23, 27.91, 23.76, 21.43, 21.20, 20.83, 20.28, 20.02. HRMS (ESI) m/z 582.3078 [M+H]+, calcd. for [C34H40N5O4]+, 582.3080. (E)-3-(3,5-dimethoxy-4-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-N-(3-((1,2,3,4-tetrahydroacridin-9-yl)amino)propyl
EP
)acrylamide (10g)
Compound 10g was prepared according to the general procedure II, light yellow oil, yield 80%. 1H NMR (400 MHz, DMSO) δ 8.35 (d, J = 8.6 Hz, 1H), 8.29 (t, J = 5.0 Hz, 1H, -CONH), 7.86 (d, J =8.1 Hz, 1H), 7.77 - 7.73 (m, 1H), 7.52 - 7.48 (m, 1H), 7.36
AC C
(d, J = 15.7 Hz, 1H), 7.21 (br, 1H, -NH), 6.88 (s, 2H), 6.56 (d, J = 15.8 Hz, 1H), 4.99 (s, 2H), 3.79 - 3.77 (m, 6H), 3.33 - 3.30 (m, 2H), 3.29 - 3.26 (m, 2H), 2.97 (s, 2H), 2.74 - 2.72 (m, 2H), 2.59 (s, 3H), 2.44 (s, 3H), 2.40 (s, 3H), 1.91 - 1.88 (m, 2H), 1.86 1.83 (m, 4H). 13C NMR (101 MHz, DMSO) δ 165.39, 153.28, 153.28, 153.28, 150.58, 150.04, 147.67, 145.85, 138.71, 138.71, 137.06, 131.13, 130.84, 124.51, 124.36, 121.50, 121.50, 118.86, 116.98, 112.64, 104.84, 104.84, 73.52, 55.85, 55.85, 44.84, 35.98, 30.25, 29.59, 24.28, 21.82, 21.20, 20.89, 20.83, 20.02. HRMS (ESI) m/z 596.3235 [M+H]+, calcd. for [C35H42N5O4]+, 596.3237.
(E)-3-(3,5-dimethoxy-4-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-N-(4-((1,2,3,4-tetrahydroacridin-9-yl)amino)butyl) acrylamide (10h) Compound 10h was prepared according to the general procedure II, light yellow oil, yield 81%. 1H NMR (400 MHz, DMSO) δ 8.32 (d, J = 8.6 Hz, 1H), 8.16 (br, 1H, -CONH), 7.87 (d, J = 8.5 Hz, 1H), 7.73 - 7.69 (m, 1H), 7.49 - 7.46 (m, 1H), 7.34 (d, J =15.7 Hz, 1H), 7.01 (br, 1H, -NH), 6.86 (s, 2H), 6.61 (d, J = 15.7 Hz, 1H), 4.97 (s, 2H), 3.75 (br, 6H), 3.73 - 3.71 (m, 2H), 3.21 3.17 (m, 2H), 2.96 (br, 2H), 2.68 (br, 2H), 2.58 (s, 3H), 2.43 (s, 3H), 2.39 (s, 3H), 1.81 (br, 4H), 1.75 - 1.68 (m, 2H), 1.56 - 1.49 (m, 2H). 13C NMR (101 MHz, DMSO) δ 164.97, 153.78, 153.29, 153.29, 153.11, 150.60, 150.07, 147.69, 145.88, 141.00, 138.41,
ACCEPTED MANUSCRIPT 136.98, 130.97, 130.89, 124.41, 124.34, 122.23, 121.86, 117.15, 112.72, 104.78, 104.78, 73.53, 55.85, 55.85, 47.14, 38.29, 29.82, 27.65, 26.34, 24.37, 21.90, 21.21, 20.99, 20.85, 20.04. HRMS (ESI) m/z 610.3389 [M+H]+, calcd. for [C36H44N5O4]+, 610.3393. (E)-3-(3,5-dimethoxy-4-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl) acrylamide (10i) Compound 10i was prepared according to the general procedure II, light yellow oil, yield 75%. 1H NMR (400 MHz, DMSO) δ 8.23 (d, J = 8.5 Hz, 1H), 8.03 (t, J = 5.5 Hz, 1H, -CONH), 7.78 (d, J = 8.2 Hz, 1H), 7.66 - 7.62 (m, 1H), 7.44 - 7.41 (m, 1H), 7.33 (d, J = 15.7 Hz, 1H), 6.87 (s, 2H), 6.58 (d, J = 15.7 Hz, 1H), 6.38 (br, 1H, -NH), 4.98 (s, 2H), 3.80 - 3.75 (m, 6H), 3.59 - 3.57 (m,
RI PT
2H), 3.17 - 3.12 (m, 2H), 2.94 - 2.92 (m, 2H) 2.70 - 2.68 (m, 2H), 2.59 (s, 3H), 2.43 (s, 3H), 2.39 (s, 3H), 1.81 (br, 4H), 1.64 1.61 (m, 2H), 1.44 - 1.41 (m, 2H), 1.31 (br, 4H). 13C NMR (101 MHz, DMSO) δ 164.83, 154.83, 153.27, 153.27, 152.51, 150.57, 150.05, 147.67, 145.86, 138.34, 138.34, 136.95, 130.97, 129.76, 124.50, 123.91, 123.84, 121.90, 118.28, 113.87, 104.76, 104.76, 73.52, 55.83, 55.83, 47.58, 38.50, 31.18, 30.24, 29.03, 26.10, 25.91, 24.58, 22.18, 21.53, 21.19, 20.83, 20.02. HRMS (ESI) m/z 638.3705 [M+H]+, calcd. for [C38H48N5O4]+, 638.3706.
SC
(E)-3-(3,5-dimethoxy-4-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-N-(8-((1,2,3,4-tetrahydroacridin-9-yl)amino)octyl) acrylamide (10j)
Compound 10j was prepared according to the general procedure II, light yellow oil, yield 83%. 1H NMR (400 MHz, DMSO) δ 8.27 (d, J = 8.6 Hz, 1H), 8.01 (t, J = 5.4 Hz, 1H, -CONH), 7.81 (d, J = 8.4 Hz, 1H), 7.73 - 7.69 (m, 1H), 7.49 - 7.45 (m, 1H), 7.34
M AN U
(d, J = 15.7 Hz, 1H), 6.87 (s, 2H), 6.79 (br, 1H, -NH), 6.58 (d, J = 15.7 Hz, 1H), 4.98 (s, 2H), 3.75 (br, 6H), 3.66 - 3.65 (m, 2H), 3.14 - 3.11 (m, 2H), 2.95 (br, 2H), 2.67 (br, 2H), 2.59 (s, 3H), 2.44 (s, 3H), 2.40 (s, 3H), 1.82 (br, 4H), 1.67 - 1.63 (m, 2H), 1.41 (br, 2H), 1.25 (br, 8H). 13C NMR (101 MHz, DMSO) δ 164.81, 153.48, 153.27, 153.27, 153.27, 150.58, 150.05, 147.68, 145.87, 138.34, 138.34, 136.95, 130.98, 124.22, 124.22, 122.83, 121.92, 117.44, 113.00, 109.64, 104.76, 104.76, 73.52, 55.83, 55.83, 47.51, 38.61, 30.77, 30.08, 29.07, 28.58, 28.58, 26.31, 26.07, 24.36, 21.94, 21.20, 21.16, 20.84, 20.02. HRMS (ESI) m/z 666.4018 [M+H]+, calcd. for [C40H52N5O4]+, 666.4019.
4.5. General procedure III for the preparation of compounds 10k−10o, 11a−11e and 12a−12e
TE D
To a stirred solution of 7a−7c (0.428 mmol), EDCI (0.514 mmol), HOBT (0.428 mmol) in anhydrous CH2Cl2 (10 mL) was added intermediate 3a−3e, the reaction mixture was stirred at room temperature for 12 h. It was diluted with CH2Cl2 (20 mL), washed successively with water (2 × 30 mL) and brine (30 mL), dried over sodium sulfate, concentrated under vacuum and purified by silica gel chromatography (CHCl3: MeOH = 20:1 ) to give compound 10k−10o, 11a−11e and 12a−12e. (E)-3-(3-methoxy-4-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-N-(2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)acry
EP
lamide (10k)
Compound 10k was prepared according to the general procedure III, white solid, yield 56%, mp > 210 °C. 1H NMR (500 MHz, DMSO+CD3OD) δ 8.43 (d, J = 8.7 Hz, 1H), 7.81 - 7.78 (m, 1H), 7.75 - 7.73 (m, 1H), 7.55 - 7.51 (m, 1H), 7.38 (d, J = 15.7 Hz,
AC C
1H), 7.11 (d, J = 1.5 Hz, 1H), 7.09 - 7.07 (m, 1H), 7.07 - 7.06 (m, 1H), 6.43 (d, J = 15.7 Hz, 1H), 5.12 (s, 2H), 4.05 - 4.03 (m, 2H), 3.75 (s, 3H), 3.60 - 3.57 (m, 2H), 2.93 - 2.90 (m, 2H), 2.68 - 2.66 (m, 2H), 2.47 (s, 3H), 2.42 (s, 3H), 2.42 (s, 3H), 1.85 1.82 (m, 4H). 13C NMR (101 MHz, DMSO+MeOD) δ 168.43, 157.15, 151.89, 151.25, 150.46, 150.41, 150.25, 149.15, 146.14, 140.75, 138.91, 133.27, 129.05, 125.91, 125.69, 122.15, 119.74, 119.46, 116.42, 114.58, 112.34, 111.28, 70.98, 56.01, 49.66, 46.78, 28.70, 24.40, 22.22, 21.32, 21.04, 21.04, 20.29. HRMS (ESI) m/z 552.2971 [M+H]+, calcd. for [C33H38N5O3]+, 552.2975. Due to rapid proton exchange with MeOH in CD3OD, the proton NH missed in the 1H NMR spectrum. (E)-3-(3-methoxy-4-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-N-(3-((1,2,3,4-tetrahydroacridin-9-yl)amino)propyl)ac rylamide (10l) Compound 10l was prepared according to the general procedure III, light yellow solid, yield 58%, mp 150.7-151.4 °C. 1H NMR (500 MHz, DMSO) δ 8.42 (d, J = 8.6 Hz, 1H), 8.38 - 8.36 (m, 1H, -CONH), 7.94 - 7.93 (m, 2H), 7.85 - 7.82 (m, 1H), 7.56 - 7.53 (m, 1H), 7.34 (d, J = 15.7 Hz, 1H), 7.16 (d, J = 1.7 Hz, 1H), 7.14 (d, J = 8.4 Hz, 1H), 7.10 (dd, J = 8.4 Hz, 1.7 Hz, 1H), 6.53 (d, J = 15.8 Hz, 1H), 5.15 (s, 2H), 3.91 - 3.89 (m, 2H), 3.77 (s, 3H), 3.29 - 3.26 (m, 2H), 2.99 (br, 2H), 2.69 (br, 2H), 2.48 (s, 3H), 2.45 (s, 3H), 2.44 (s, 3H), 1.94 - 1.89 (m, 2H), 1.81 (br, 4H). 13C NMR (101 MHz, DMSO) δ 165.61, 155.62, 150.95, 150.58,
ACCEPTED MANUSCRIPT 149.44, 149.29, 149.00, 148.15, 145.19, 138.51, 137.88, 132.41, 128.32, 124.95, 124.89, 121.02, 120.03, 119.15, 115.53, 113.79, 111.18, 110.52, 70.11, 55.53, 44.64, 35.81, 30.09, 27.89, 23.90, 21.41, 21.16, 20.86, 20.20, 20.05. HRMS (ESI) m/z 566.3128 [M+H]+, calcd. for [C34H40N5O3]+, 566.3131. (E)-3-(3-methoxy-4-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-N-(4-((1,2,3,4-tetrahydroacridin-9-yl)amino)butyl)acr ylamide (10m) Compound 10m was prepared according to the general procedure III, light yellow oil, yield 57%. 1H NMR (500 MHz, DMSO) δ 8.42 (d, J = 8.9 Hz, 1H), 8.16 - 8.13 (m, 1H, -CONH), 7.95 - 7.91 (m, 1H), 7.85 - 7.82 (m, 2H), 7.57 - 7.54 (m, 1H), 7.32 (d, J =
RI PT
15.7 Hz, 1H), 7.14 (d, J = 1.7 Hz, 1H), 7.13 (d, J = 8.6 Hz, 1H), 7.09 - 7.07 (m, 1H), 6.54 - 6.49 (m, 1H), 5.15 (s, 2H), 3.89 3.85 (m, 2H), 3.77 (s, 3H), 3.21 - 3.17 (m, 2H), 2.99 (br, 2H), 2.66 (br, 2H), 2.48 (s, 3H), 2.45 (s, 3H), 2.44 (s, 3H), 1.82 (br, 4H), 1.79 - 1.75 (m, 2H), 1.56 - 1.50 (m, 2H). 13C NMR (101 MHz, DMSO) δ 165.11, 155.54, 150.95, 150.67, 149.45, 149.29, 148.92, 148.15, 145.20, 138.19, 137.90, 132.39, 128.42, 124.95, 124.95, 120.98, 120.38, 119.17, 115.58, 113.78, 111.14, 110.40, 70.11, 55.51, 46.85, 38.11, 27.92, 27.36, 26.21, 23.94, 21.41, 21.17, 20.86, 20.21, 20.05. HRMS (ESI) m/z 580.3285 [M+H]+, calcd. for
SC
[C35H42N5O3]+, 580.3288.
(E)-3-(3-methoxy-4-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)acr ylamide (10n)
Compound 10n was prepared according to the general procedure III, light yellow oil, yield 55%. 1H NMR (500 MHz, DMSO) δ
M AN U
8.40 (d, J = 8.7 Hz, 1H), 8.08 - 8.06 (m, 1H, -CONH), 7.94 - 7.93 (m, 1H), 7.85 - 7.82 (m, 1H), 7.78 (br, 1H, -NH), 7.58 - 7.54 (m, 1H), 7.32 (d, J = 15.7 Hz, 1H), 7.15 (d, J = 1.7 Hz, 1H), 7.12 (d, J = 8.4 Hz, 1H), 7.07 (dd, J = 8.4 Hz, 1.7 Hz, 1H), 6.53 (d, J = 15.7 Hz, 1H), 5.14 (s, 2H), 3.86 - 3.82 (m, 2H), 3.76 (s, 3H), 3.16 - 3.13 (m, 2H), 2.99 (br, 2H), 2.65 (br, 2H), 2.48 (s, 3H), 2.45 (s, 3H), 2.44 (s, 3H), 1.82 (br, 4H), 1.75 - 1.69 (m, 2H), 1.46 - 1.41 (m, 2H), 1.36 - 1.30 (m, 4H). 13C NMR (101 MHz, DMSO) δ 165.01, 155.52, 150.94, 150.64, 149.44, 149.29, 148.89, 148.14, 145.20, 138.10, 137.95, 132.41, 128.47, 124.93, 124.93, 120.97, 120.49, 119.21, 115.55, 113.79, 111.13, 110.39, 70.11, 55.50, 47.10, 38.39, 29.74, 28.96, 27.94, 25.94, 25.68, 23.87, 21.39, 21.16, 20.86, 20.24, 20.05. HRMS (ESI) m/z 608.3598 [M+H]+, calcd. for [C37H46N5O3]+, 608.3601.
TE D
(E)-3-(3-methoxy-4-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-N-(8-((1,2,3,4-tetrahydroacridin-9-yl)amino)octyl)acry lamide (10o)
Compound 10o was prepared according to the general procedure III, light yellow oil, yield 53%. 1H NMR (500 MHz, DMSO) δ 8.11 (d, J = 8.5 Hz, 1H), 7.98 - 7.96 (m, 1H, -CONH), 7.70 - 7.69 (m, 1H), 7.53 - 7.50 (m, 1H), 7.34 - 7.33 (m, 1H), 7.33 (d, J = 15.7 Hz, 1H), 7.16 (d, J = 1.7 Hz, 1H), 7.12 (d, J = 8.4 Hz, 1H), 7.08 (dd, J = 8.4 Hz, 1.7 Hz, 1H), 6.50 (d, J = 15.7 Hz, 1H), 5.53
EP
(br, 1H, -NH), 5.15 (s, 2H), 3.77 (s, 3H), 3.42 - 3.38 (m, 2H), 3.14 - 3.10 (m, 2H), 2.91 - 2.88 (m, 2H), 2.70 - 2.68 (m, 2H), 2.48 (s, 3H), 2.45 (s, 3H), 2.44 (s, 3H), 1.82 - 1.78 (m, 4H), 1.57 - 1.51 (m, 2H), 1.41 - 1.38 (m, 2H), 1.28 - 1.22 (m, 8H). 13C NMR (101 MHz, DMSO) δ 164.94, 157.45, 150.94, 150.54, 149.45, 149.30, 148.88, 148.14, 146.40, 145.21, 138.14, 128.48, 127.97,
AC C
127.75, 123.16, 123.06, 120.98, 120.46, 119.97, 115.52, 113.78, 110.37, 70.11, 55.48, 47.86, 38.55, 33.17, 30.47, 29.05, 28.61, 28.57, 26.27, 26.17, 24.91, 22.61, 22.28, 21.16, 20.85, 20.04. HRMS (ESI) m/z 636.3909 [M+H]+, calcd. for [C39H50N5O3]+, 636.3914.
(E)-3-(3,4-bis((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-N-(2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)acrylamide (11a)
Compound 11a was prepared according to the general procedure III, white solid, yield 72%, mp 112.1-113.0 °C. 1H NMR (500 MHz, DMSO) δ 8.64 - 8.62 (m, 1H, -CONH), 8.51 (d, J = 8.8 Hz, 1H), 8.01 (br, 1H, -NH), 7.94 - 7.93 (m, 1H), 7.87 - 7.84 (m, 1H), 7.58 - 7.55 (m, 1H), 7.41 (m, 2H), 7.16 -7.15 (m, 2H), 6.53 (d, J = 15.8 Hz, 1H), 5.16 (s, 4H), 4.13 - 4.02 (m, 2H), 3.59 3.56 (m, 2H), 2.99 (br, 2H), 2.70 (br, 2H), 2.43 (br, 6H), 2.42 (br, 6H), 2.41 (br, 3H), 2.41 (br, 3H), 1.83 - 1.82 (m, 4H). 13C NMR (101 MHz, DMSO) δ 166.72, 155.89, 150.93, 150.93, 150.68, 149.59, 149.46, 149.40, 148.17, 148.12, 148.09, 145.19, 145.13, 139.10, 138.04, 132.49, 128.14, 125.18, 124.97, 121.92, 119.60, 119.22, 115.59, 114.20, 113.13, 111.37, 70.29, 70.17, 48.25, 45.42, 27.97, 23.77, 21.44, 21.16, 21.16, 20.84, 20.83, 20.28, 19.99, 19.99. HRMS (ESI) m/z 672.3659 [M+H]+, calcd. for [C40H46N7O3]+, 672.3662.
ACCEPTED MANUSCRIPT (E)-3-(3,4-bis((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-N-(3-((1,2,3,4-tetrahydroacridin-9-yl)amino)propyl)acrylami de (11b) Compound 11b was prepared according to the general procedure III, light yellow solid, yield 71%, mp 68.7-69.5 °C. 1H NMR (500 MHz, DMSO) δ 8.43 - 8.41 (m, 1H), 8.37 (br, 1H, -CONH), 7.94 - 7.91 (m, 1H), 7.89 (br, 1H, -NH), 7.84 - 7.81 (m, 1H), 7.56 - 7.53 (m, 1H), 7.38 (d, J = 1.4 Hz, 1H), 7.34 (d, J = 15.7 Hz, 1H), 7.17 - 7.16 (m, 1H), 7.14 - 7.12 (m, 1H) 6.53 (dd, J = 15.8 Hz, 3.3 Hz, 1H), 5.16 (s, 4H), 3.92 - 3.88 (m, 2H), 3.30 - 3.27 (m, 2H), 2.99 (br, 2H), 2.69 (br, 2H), 2.43 (br, 12H), 2.41 (br, 6H), 1.95 - 1.89 (m, 2H), 1.82 (br, 4H). 13C NMR (101 MHz, DMSO) δ 165.60, 155.50, 150.89, 150.89, 150.72, 149.45, 149.45,
RI PT
149.38, 148.18, 148.09, 148.07, 145.20, 145.14, 138.39, 138.06, 132.31, 128.33, 124.92, 124.85, 121.72, 120.20, 119.33, 115.63, 114.23, 113.11, 111.27, 70.32, 70.19, 44.66, 35.90, 30.11, 28.00, 23.94, 21.45, 21.15, 20.83, 20.81, 20.25, 20.08, 19.98, 19.98. HRMS (ESI) m/z 686.3815 [M+H]+, calcd. for [C41H48N7O3]+, 686.3819.
(E)-3-(3,4-15bis((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-N-(4-((1,2,3,4-tetrahydroacridin-9-yl)amino)butyl)acrylam ide (11c)
SC
Compound 11c was prepared according to the general procedure III, light yellow solid, yield 85%, mp 67.5-68.4 °C. 1H NMR (500 MHz, DMSO) δ 8.41 (d, J = 8.6 Hz, 1H), 8.16 - 8.14 (m, 1H, -CONH), 7.92 (d, J = 8.6 Hz, 1H), 7.84 - 7.81 (m, 1H), 7.75 (br, 1H, -NH), 7.57 - 7.54 (m, 1H), 7.36 (d, J = 1.2 Hz, 1H), 7.31 (d, J = 15.7 Hz, 1H), 7.16 - 7.15 (m, 1H), 7.12 -7.11 (m, 1H), 6.52 (d, J = 15.8 Hz, 1H), 5.15 (s, 4H), 3.89 - 3.85 (m, 2H), 3.21 - 3.19 (m, 2H), 2.99 (br, 2H), 2.67 (br, 2H), 2.43 (br, 12H), 2.41
M AN U
(br, 6H), 1.82 (br, 4H), 1.77 - 1.73 (m, 2H), 1.56 - 1.50 (m, 2H). 13C NMR (101 MHz, DMSO) δ 165.09, 155.44, 150.90, 150.90, 150.86, 149.45, 149.38, 149.38, 148.17, 148.09, 148.07, 145.20, 145.15, 138.09, 138.09, 132.32, 128.42, 124.92, 124.92, 121.66, 120.52, 119.40, 115.70, 114.25, 113.03, 111.28, 70.31, 70.19, 46.90, 38.14, 28.07, 27.38, 26.24, 23.95, 21.44, 21.15, 21.15, 20.83, 20.81, 20.28, 19.98, 19.98. HRMS (ESI) m/z 700.3975 [M+H]+, calcd. for [C42H50N7O3]+, 700.3975. (E)-3-(3,4-bis((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)acrylamid e (11d)
Compound 11d was prepared according to the general procedure III, light yellow oil, yield 79%. 1H NMR (500 MHz, DMSO) δ
TE D
8.40 (d, J = 8.7 Hz, 1H), 8.11 - 8.08 (m, 1H, -CONH), 7.93 (d, J = 8.6 Hz, 1H), 7.84 - 7.81 (m, 1H), 7.77 (br, 1H, -NH), 7.57 7.54 (m, 1H), 7.37 (d, J = 1.5 Hz, 1H), 7.32 (d, J = 15.7 Hz, 1H), 7.16 - 7.14 (m, 1H), 7.12 - 7.10 (m, 1H), 6.54 (d, J = 15.8 Hz, 1H), 5.15 (s, 4H), 3.86 - 3.81 (m, 2H), 3.15 - 3.13 (m, 2H), 2.99 (br, 2H), 2.66 (br, 2H), 2.42 (br, 9H), 2.42 (br, 3H), 2.41 (br, 6H), 1.82 (br, 4H), 1.75 - 1.70 (m, 2H), 1.47 - 1.42 (m, 2H), 1.37 - 1.31 (m, 4H). 13C NMR (101 MHz, DMSO) δ 165.01, 155.49, 150.89, 150.89, 150.70, 149.45, 149.38, 149.34, 148.17, 148.09, 148.07, 145.20, 145.15, 138.02, 137.98, 132.38, 128.47, 124.91,
EP
124.91, 121.65, 120.66, 119.28, 115.58, 114.24, 113.00, 111.17, 70.31, 70.19, 47.12, 38.46, 29.76, 28.96, 27.98, 25.94, 25.69, 23.88, 21.41, 21.14, 21.14, 20.82, 20.81, 20.26, 19.97, 19.97. HRMS (ESI) m/z 728.4287 [M+H]+, calcd. for [C44H54N7O3]+, 728.4288.
AC C
(E)-3-(3,4-bis((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-N-(8-((1,2,3,4-tetrahydroacridin-9-yl)amino)octyl)acrylamide (11e)
Compound 11e was prepared according to the general procedure III, light yellow oil, yield 81%. 1H NMR (500 MHz, DMSO) δ 8.21 - 8.20 (m, 1H), 8.02 - 8.00 (m, 1H, -CONH), 7.76 (d, J = 8.5 Hz, 1H), 7.64 - 7.61 (m, 1H), 7.43 - 7.39 (m, 1H), 7.37 (d, J = 1.6 Hz, 1H), 7.32 (d, J = 15.7 Hz, 1H), 7.16 - 7.14 (m, 1H), 7.13 - 7.11 (m, 1H), 6.52 (d, J = 15.6 Hz, 1H), 6.29 (br, 1H, -NH), 5.16 (s, 2H), 5.15 (s, 2H), 3.57 - 3.54 (m, 2H), 3.14 - 3.11 (m, 2H), 2.93 - 2.91 (m, 2H), 2.69 - 2.67 (m, 2H), 2.43 (br, 9H), 2.42 (br, 9H) 1.82 - 1.79 (m, 4H), 1.64 - 1.58 (m, 2H), 1.43 - 1.38 (m, 2H), 1.30 - 1.24 (m, 8H). 13C NMR (101 MHz, DMSO) δ 164.96, 155.35, 152.14, 150.90, 150.90, 149.46, 149.40, 149.34, 148.18, 148.10, 148.08, 145.22, 145.17, 143.78, 138.02, 138.02, 129.40, 128.48, 125.10, 123.72, 123.65, 121.68, 120.63, 118.59, 114.23, 112.97, 70.29, 70.19, 47.68, 38.62, 31.53, 30.25, 29.06, 28.57, 28.57, 26.28, 26.10, 24.59, 22.24, 21.66, 21.15, 21.15, 20.83, 20.81, 19.98, 19.98. HRMS (ESI) m/z 756.4598 [M+H]+, calcd. for [C46H58N7O3]+, 756.4601. 3-(3,4-bis((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-2-hydroxy-N-(2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)pro panamide (12a)
ACCEPTED MANUSCRIPT Compound 12a was prepared according to the general procedure III, light yellow oil, yield 82%. 1H NMR (500 MHz, DMSO) δ 8.44 (d, J = 8.8 Hz, 1H), 8.35 (t, J = 5.9 Hz, 1H, -CONH), 7.93 - 7.91 (m, 1H), 7.84 - 7.81 (m, 2H), 7.55 - 7.52 (m, 1H), 7.01 (d, J = 1.7 Hz, 1H), 6.92 (d, J = 8.2 Hz, 1H), 6.71 (dd, J = 8.1 Hz, 1.5 Hz, 1H), 5.00 - 4.98 (m, 4H), 4.12 - 4.10 (m, 1H), 3.91 - 3.88 (m, 2H), 3.54 - 3.41 (m, 2H) 3.39 - 3.35 (m, 3H), 2.98 (br, 2H), 2.65 - 2.60 (m, 2H), 2.41 (br, 6H), 2.40 (s, 3H), 2.39 (s, 3H), 2.36 (s, 3H), 2.36 (s, 3H), 1.82 (br, 4H). 13C NMR (101 MHz, DMSO) δ 174.86, 155.70, 150.89, 150.72, 150.71, 150.66, 149.42, 149.37, 148.02, 148.02, 147.82, 146.73, 145.44, 145.22, 138.04, 132.39, 131.60, 125.23, 124.88, 122.47, 119.25, 115.52, 114.04,
(ESI) m/z 690.3768 [M+H]+, calcd. for [C40H48N7O4]+, 690.3768.
RI PT
111.22, 72.02, 70.43, 70.37, 48.38, 45.35, 38.80, 28.05, 23.63, 21.45, 21.20, 21.19, 20.87, 20.83, 20.31, 19.99, 19.99. HRMS
3-(3,4-bis((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-2-hydroxy-N-(3-((1,2,3,4-tetrahydroacridin-9-yl)amino)propyl)pr opanamide (12b)
Compound 12b was prepared according to the general procedure III, light yellow oil, yield 81%. 1H NMR (500 MHz, CD3OD) δ 8.16 (d, J = 8.6 Hz, 1H), 7.70 - 7.67 (m, 2H), 7.42 - 7.40 (m, 1H), 7.05 (d, J = 1.8 Hz, 1H), 6.93 (d, J = 8.2 Hz, 1H), 6.84 (dd, J =
SC
8.2 Hz, 1.8 Hz, 1H), 5.04 (d, J = 1.2 Hz, 2H), 4.96 (br, 2H), 4.29 - 4.27 (m, 1H), 3.64 - 3.59 (m, 1H), 3.51 - 3.46 (m, 1H), 3.43 3.38 (m, 1H), 3.32 - 3.30 (m, 1H), 3.23- 3.17 (m, 1H), 3.01 - 2.98 (m, 1H), 2.92 - 2.90 (m, 2H), 2.86 - 2.82 (m, 1H), 2.66 - 2.65 (m, 2H), 2.43 (s, 3H), 2.42 (s, 3H), 2.40 (s, 3H), 2.40 (s, 6H), 2.35 (s, 3H), 1.85 - 1.84 (m, 4H), 1.82 - 1.78 (m, 2H). 13C NMR (101 MHz, MeOD) δ 176.74, 156.03, 154.19, 152.53, 152.46, 151.15, 151.13, 150.16, 150.07, 149.66, 148.76, 147.40, 147.33,
M AN U
142.42, 133.00, 132.45, 125.83, 125.53, 124.51, 122.80, 118.37, 118.06, 116.06, 114.21, 73.64, 71.97, 71.92, 45.90, 41.23, 36.82, 31.76, 30.99, 25.38, 23.29, 22.38, 21.36, 21.34, 21.17, 21.15, 20.43, 20.40. HRMS (ESI) m/z 704.3925 [M+H]+, calcd. for [C41H50N7O4]+, 704.3924.
3-(3,4-bis((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-2-hydroxy-N-(4-((1,2,3,4-tetrahydroacridin-9-yl)amino)butyl)pro panamide (12c)
Compound 12c was prepared according to the general procedure III, light yellow oil, yield 87%. 1H NMR (500 MHz, CD3OD) δ 7.92 (d, J = 8.6 Hz, 1H), 7.58 (d, J = 8.4 Hz, 1H), 7.49 - 7.46 (m, 1H), 7.05 - 7.02 (m, 1H), 7.00 (d, J = 1.7 Hz, 1H), 6.91 (d, J =
TE D
8.3 Hz, 1H), 6.77 (dd, J = 8.2 Hz, 1.7 Hz, 1H), 5.01 (s, 2H), 4.93 (s, 2H), 4.21 - 4.18 (m, 1H), 3.59 - 3.49 (m, 2H), 3.24 - 3.21 (m, 2H), 2.98 – 2.94 (m, 1H), 2.93 - 2.89 (m, 1H), 2.86 - 2.80 (m, 3H), 2.54 - 2.51 (m, 2H), 2.36 (s, 3H), 2.31 (s, 3H), 2.28 (s, 3H), 2.27 (s, 3H), 2.20 (s, 3H), 2.18 (s, 3H), 1.79 - 1.75 (m, 2H), 1.74 - 1.68 (m, 2H), 1.43 - 1.38 (m, 2H), 1.37 - 1.28 (m, 2H). 13C NMR (101 MHz, MeOD) δ 176.00, 155.58, 154.98, 152.55, 152.49, 151.20, 151.15, 150.20, 150.10, 149.55, 148.63, 147.29, 147.29, 143.09, 132.72, 131.96, 125.38, 125.25, 124.56, 123.32, 118.75, 117.55, 115.56, 114.40, 73.56, 71.85, 71.70, 49.09,
EP
41.18, 39.41, 31.44, 29.11, 27.84, 25.46, 23.41, 22.58, 21.30, 21.30, 21.07, 21.03, 20.42, 20.38. HRMS (ESI) m/z 718.4078 [M+H]+, calcd. for [C42H52N7O4]+, 718.4081.
3-(3,4-bis((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-2-hydroxy-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)pro
AC C
panamide (12d)
Compound 12d was prepared according to the general procedure III, light yellow oil, yield 85%. 1H NMR (500 MHz, DMSO) δ 8.31 (d, J = 8.7 Hz, 1H), 7.90 - 7.88 (m, 1H), 7.79 - 7.76 (m, 1H, -CONH), 7.71 - 7.68 (m, 1H), 7.55 (br, 1H, -NH), 7.50 - 7.47 (m, 1H), 7.02 (d, J = 1.6 Hz, 1H), 6.98 (d, J = 8.3 Hz, 1H), 6.74 (dd, J = 8.2 Hz, 1.5 Hz, 1H), 5.05 (s, 2H), 5.02 (s, 2H), 4.05 4.03 (m, 1H), 3.79 - 3.76 (m, 2H), 3.08 - 3.04 (m, 1H), 3.01 - 2.97 (m, 4H), 2.67 - 2.63 (m, 2H) 2.61 (br, 2H), 2.40 (s, 3H), 2.39 (s, 6H), 2.38 (s, 3H), 2.37 (s, 3H), 2.36 (s, 3H), 1.79 (br, 4H), 1.69 - 1.66 (m, 2H), 1.33 - 1.30 (m, 4H), 1.19 - 1.18 (m, 2H). 13C NMR (101 MHz, DMSO) δ 173.19, 155.02, 151.25, 150.79, 150.76, 150.68, 149.32, 149.32, 148.14, 148.03, 147.68, 146.55, 145.47, 145.43, 139.07, 131.93, 131.27, 124.81, 124.45, 122.19, 120.21, 115.74, 114.05, 111.45, 72.17, 70.46, 70.29, 47.31, 38.32, 37.93, 29.88, 29.00, 28.46, 25.85, 25.76, 23.96, 21.53, 21.45, 21.19, 20.85, 20.46, 20.12, 20.03, 20.01. HRMS (ESI) m/z 746.4388 [M+H]+, calcd. for [C44H56N7O4]+, 746.4394. 3-(3,4-bis((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-2-hydroxy-N-(8-((1,2,3,4-tetrahydroacridin-9-yl)amino)octyl)pro panamide (12e) Compound 12e was prepared according to the general procedure III, light yellow oil, yield 85%. 1H NMR (500 MHz, CD3OD) δ
ACCEPTED MANUSCRIPT 8.02 (d, J = 8.4 Hz, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.54 - 7.51 (m, 1H), 7.30 - 7.27 (m, 1H), 6.99 (d, J = 1.7 Hz, 1H), 6.93 (d, J = 8.3 Hz, 1H), 6.76 (dd, J = 8.2 Hz, 1.7 Hz, 1H), 5.04 (s, 2H), 5.00 (s, 2H), 4.17 - 4.15 (m, 1H), 3.58 - 3.54 (m, 2H), 3.16 - 3.10 (m, 1H), 3.03 - 2.98 (m, 1H), 2.97 - 2.93 (m, 1H), 2.91 -2.90 (m, 3H), 2.80 - 2.75 (m, 1H), 2.63 - 2.62 (m, 2H), 2.40 (s, 3H), 2.37 (br, 9H), 2.35 (s, 3H), 2.34 (s, 3H), 1.86 - 1.84 (m, 4H), 1.64 - 1.58 (m, 2H), 1.31 - 1.26 (m, 10H). 13C NMR (101 MHz, MeOD) δ 175.99, 156.63, 154.67, 152.53, 152.49, 151.23, 151.23, 150.21, 150.13, 149.76, 148.76, 147.54, 147.50, 145.23, 133.02, 131.10, 125.36, 125.17, 125.03, 124.34, 119.79, 117.94, 116.10, 115.31, 73.73, 72.01, 71.82, 49.53, 41.25, 39.86, 32.60, 32.02, 30.38, 30.17, 30.17, 27.74, 27.65, 25.69, 23.73, 23.09, 21.31, 21.29, 21.13, 21.10, 20.44, 20.41. HRMS (ESI) m/z 774.4708 [M+H]+,
RI PT
calcd. for [C46H60N7O4]+, 774.4707. 4.6. Biological activity 4.6.1. Cholinesterase inhibition assay in vitro
The inhibitory activity was measured by Ellman’s assay with slightly modifications [70]. Acetylcholinesterase (AChE from electric eel and human erythrocytes, EC 3.1.1.7.), Butyrylcholinesterase (BuChE from equine serum and human serum, EC
SC
3.1.1.8.), 5,5'-dithiobis(2-nitrobenzoic acid), acetylthiocholine (ATC), and butyrylthiocholine (BTC) iodides were purchased from Sigma-Aldrich. Enzyme solutions of eeAChE and eqBuChE, hAChE and hBuChE were prepared by dissolving the corresponding enzymes in Tris-HC1-pH 8.0 containing 0.1% w/v bovine serum albumin to give 0.22 and 0.3, 0.05 and 0.024 units/mL, respectively, each in 5 mL aliquots. DTNB (0.015 M) and ATC/BTC iodide (0.03 M) solutions were prepared in
concentrations (normally 10-5~10−11 M).
M AN U
1×PBS. Stock solutions of the test compounds were prepared in DMSO and diluted with PBS to final concentration at least five
For measurement, 50 µL of the respective enzyme was added in a 96-well plate containing 20 µL of PBS and 20 µL of test compound solution, incubated for 5 min at 37 °C. Then, 100 µL of DTNB were added and incubated for 5 min at 37 °C in the dark. Finally, 10 µL of the substrate solution (ATC/BTC) was added and incubated for 3 min at 37 °C. The absorbance of each well was measured at 415 nm using a microplate reader. For the reference value, 20 µL of PBS replaced the test compound solution. For determining the blank value, additionally 100 µL of Tris-HC1 replaced the enzyme solution. Results are expressed
TE D
as the mean ± SD at least three different experiments. The inhibition curve was obtained by plotting percentage enzyme activity (100% for the reference) versus logarithm of test compound concentration. 4.6.2. Inhibition of Aβ42 self-aggregation
Inhibition of Aβ42 self-aggregation was measured using a Thioflavin T-binding assay. HFIP pretreated Aβ42 samples (Sigma-Aldrich) were resolubilized with PBS (pH = 7.4) to give a 40 µM solution. And ThT solution was prepared in PBS at a
EP
concentration of 10 µM. Then, 10 µL of 40 µM Aβ42 solution was added to each well containing 10 µL of different concentrations of test compounds (0.0064, 0.032, 0.16, 0.8, 4, 20, 100 µM). After incubated for 24h, the samples were diluted to a final volume of 200 µL with ThT solution. Fluorescence was measured on a Varioskan Flash spectral scanning multimode
AC C
reader (Thermo, Varioskan Flash 3001, USA) with excitation and emission wavelengths at 435 nm and 485 nm, respectively. 4.6.3. Atomic force microscope assay For the inhibition of Aβ42 aggregation experiment, 45 µL PBS was added in a 96-well plate containing 2.5 µL of 2 mM/L
(DMSO) Aβ42 stock solution and 2.5 µL (2 mM/L DMSO) of test compound solution, shaken for 12 hours. Then, the mixed solution was incubated for 4 days at 37 °C. The final concentration of Aβ42 and the compounds were 0.1 mM. For the control, 2.5 µL of DMSO replaced the test compound solution. Aliquots (5 µL) of the samples diluted 100-fold with PBS were dropped to the mica plate surface (about 0.5 cm2), naturally dried at ambient temperature. The aggregation state of Aβ42 were examined with atomic force microscope (Veeco Multi Mode 8/B0021, Veeco German) equipped with antimony doped silicon of conical shape (Veeco). The Aβ42 aggregation was scanned in intelligent mode and the force constant was monitored at 3 N/m. 4.6.4. Antioxidant activity in vitro The antioxidant activities of compounds 9i and 10i were evaluated by DPPH free-radical scavenging assay according to the method of Lee et al. with slightly modifications [34, 83]. Briefly, 95 mL of DPPH radical solution (300 mM) was added in a 96-well plate containing 5 mL of different concentrations of test compound dissolved in MeOH, and incubated for 30 min at
ACCEPTED MANUSCRIPT 37 °C in the dark. The absorbance of each well was measured at 517 nm using a microplate reader (Thermo, Varioskan Flash 3001, USA). 4.6.5. Cell viability assay on the PC12 cells CoCl2 was cytotoxic towards PC12 cells by production of reactive oxygen species (ROS) [79, 80]. Trolox, a strong antioxidant, which can effectively prevent oxidative neuronal death, was used as a reference compound at 10 µM. The PC12 cell line was purchased from Institute of Materia of Chinese Academy of Medical Science. Cells were cultured in RPMI 1640 medium supplemented with 5% (v/v) fetal bovine serum (FBS), 10% (v/v) heat inactivated horse serum and 100 U/mL
RI PT
penicillin-streptomycin (Thermo Technologies, USA) and incubated at 37 °C in a humidified atmosphere of 5% CO2. PC12 cells, maintained with serum-free media for 14 h, were placed on 96-well dishes pre-coated with poly-L-lysine at 7 × 104 cells/mL, differentiated by treated with NGF (50 ng/mL). Thereafter, the differentiated PC12 cells were pretreated with different concentrations of test compound (1.25, 2.5, 5 and 10 mM) for 36 h, respectively; then it was exposed to CoCl2 for 12 h. Cell viability was measured by using the MTT assay and the absorption was measured by a well plate reader at 490 nm (Thermo
SC
Multiskan GO, USA). For the neurotoxicity, test compounds was incubated for 48 h and measured by MTT assay. Cell viability was expressed as a percentage of the control. 4.6.6. Hepatotoxicity study in vitro
HepG2 cells (human hepatocellular liver carcinoma cell line purchased from Institute of Materia of Chinese Academy of
M AN U
Medical Science), were grown in DMEM supplemented with 10% FBS and 100 U/mL of penicillin-streptomycin (Thermo Technologies, USA) at 37 °C in a humidified atmosphere containing 5% CO2. For the experiments, cells (6 × 103 cells/well) were seeded in 96-well plate in complete medium. After 24 h, the medium was removed, and cells were exposed to the increasing concentrations of compound 9i or tacrine (1.25, 2.5, 5 and 10 µM) in DMEM for further 24 h. Cell survival was measured through MTT assay. 4.6.7. Hepatotoxicity study in vivo
Adult male ICR mice (weighing 22−25g) from Beijing HFK Bio-Technology.co., LTD (Beijing, China) were used to
TE D
perform experiments. Mice were housed in standard controlled conditions (22 ± 2 °C, 55 ± 10% humidity) with a reversed 12h light/dark cycle. Food and water were available ad libitum in the home cage. Tacrine hydrochloride was dissolved in CMC-Na solution (0.5 g CMC-Na in 100 mL of distilled water) and 3 mg/100 g b wt, corresponding to 12.82 µmol/100 g b wt, was given orally. 9i was dissolved in CMC-Na solution, and the equimolar dose corresponding to tacrine was administered. Heparinized serum was obtained 8, 24, and 36 h after dosing from the retro bulbar plexus to determine aspartate aminotransferase (AST) and
EP
alanine aminotransferase (ALT) activity, two indicators of a liver damage, using routine methods. All animal protocols were approved by the Animal Care and Use Committee of PUMC. 4.6.8. Kinetic characterization of AChE inhibition
AC C
The kinetic characterization of AChE was performed in the same manner with four different concentrations (0, 2.5, 5, 10 nm) of compound 9i. Lineweaver-Burk reciprocal plots were constructed by plotting 3/velocity against 1/ [substrate] at varying concentrations of the substrate acetylthiocholine iodide (0.05~0.5 mM). Data analysis was performed with Graph Pad Prism 5 software (San Diego, CA, USA). 4.6.9. Docking study
Molecular docking study was performed using Molecular Operating Environment (MOE) software version 2008.10
(Chemical Computing Group, Montreal, Canada). The X-ray crystal structure of TcAChE complexed with bis(7)-tacrine (PDB code: 2CKM), hAChE in complex with donepezil (PDB code: 4EY7) and hBuChE (PDB code: 1P0I) were obtained from the Protein Data Base (PDB). All water molecules in PDB files were removed and hydrogen atoms were subsequently added to the protein. Compound 9i was constructed using the builder interface of the MOE program and energy minimized using MMFF94x force field. Then 9i was docked into the active site of the protein by the ‘Triangle Matcher’ method. The Dock scoring was done using ASE scoring function and the Force field was selected as the refinement method. The retained best poses were analyzed using the MOE’s pose viewer utility.
ACCEPTED MANUSCRIPT Competing interests The authors declare that they have no competing interests. Acknowledgments We thank the Tianjin special support program for talent development, the key technologies R & D program of Tianjin (12ZCDZSY11900) and the Medical and Health Science and Technology Innovation Project of Chinese Academy of Medical Science (2017-I2M-3-021) for financial support of this study. We also thank Chinese Academy of Medical Sciences Peking
Authors’ contributions
RI PT
Union Medical College Information technology Center for their help in molecular modeling study.
Tianjun Liu and Guoliang Li designed the study; GuoLiang Li, Ge Hong, Xinxu Li, Zengping Xu, Lina Mao and Yan Zhang performed experiments; Guoliang Li analyzed data with the help of Tianjun Liu and Xizeng Feng; Tianjun Liu and Guoliang Li wrote the paper. All authors read and approved the final manuscript. References
SC
[1] J. Mendiola-Precoma, L.C. Berumen, K. Padilla, G. Garcia-Alcocer, Therapies for prevention and treatment of Alzheimer's disease, BioMed. Res. Int. 2016 (2016) 2589276.
[2] M. Prince, A. Wimo, M. Guerchet, G.C. Ali, Y.T. Wu, M. Prina, World Alzheimer Report 2015. The global impact of dementia. An analysis of prevalence, incidence, cost and trends, London: Alzheimer's Disease International, 2015.
M AN U
[3] Z. Wang, Y. Wang, W. Li, F. Mao, Y. Sun, L. Huang, X. Li, Design, synthesis, and evaluation of multitarget-directed selenium-containing clioquinol derivatives for the treatment of Alzheimer's disease, ACS Chem. Neurosci. 5 (2014) 952–962. [4] M.G. Savelieff, S. Lee, Y. Liu, M.H. Lim, Untangling amyloid-beta, tau, and metals in Alzheimer's disease, ACS Chem. Biol. 8 (2013) 856-865.
[5] S.S. Choi, S.R. Lee, S.U. Kim, H.J. Lee, Alzheimer's disease and stem cell therapy, Exp. Neurobiol. 23 (2014) 45–52. [6] C. Rochais, C. Lecoutey, F. Gaven, P. Giannoni, K. Hamidouche, D. Hedou, E. Dubost, D. Genest, S. Yahiaoui, T. Freret, V. Bouet, F. Dauphin, J. Sopkova de Oliveira Santos, C. Ballandonne, S. Corvaisier, A. Malzert-Freon, R. Legay, M. Boulouard, S.
TE D
Claeysen, P. Dallemagne, Novel multitarget-directed ligands (MTDLs) with acetylcholinesterase (AChE) inhibitory and serotonergic subtype 4 receptor (5-HT4R) agonist activities as potential agents against Alzheimer's disease: the design of donecopride, J. Med. Chem. 58 (2015) 3172–3187.
[7] M. Citron, Alzheimer's disease: strategies for disease modification, Nat. Rev. Drug. Discov. 9 (2010) 387–398. [8] C. Zhang, Q.Y. Du, L.D. Chen, W.H. Wu, S.Y. Liao, L.H. Yu, X.T. Liang, Design, synthesis and evaluation of novel
200–209.
EP
tacrine-multialkoxybenzene hybrids as multi-targeted compounds against Alzheimer's disease, Eur. J. Med. Chem. 116 (2016)
[9] A. Cavalli, M.L. Bolognesi, A. Minarini, M. Rosini, V. Tumiatti, M. Recanatini, C. Melchiorre, Multi-target-directed ligands
AC C
to combat neurodegenerative diseases, J. Med. Chem. 51 (2008) 347–372. [10] R. Leon, A.G. Garcia, J. Marco-Contelles, Recent advances in the multitarget-directed ligands approach for the treatment of Alzheimer's disease, Med. Res. Rev. 33 (2013) 139–189. [11] K. Xu, P. Wang, X. Xu, F. Chu, J. Lin, Y. Zhang, H. Lei, An overview on structural modifications of ligustrazine and biological evaluation of its synthetic derivatives, Res. Chem. Intermediat. (2013) doi:10.1007/s11164–013–1281–2. [12] M. Kim, S.O. Kim, M. Lee, J.H. Lee, W.S. Jung, S.K. Moon, Y.S. Kim, K.H. Cho, C.N. Ko, E.H. Lee, Tetramethylpyrazine, a natural alkaloid, attenuates pro-inflammatory mediators induced by amyloid beta and interferon-gamma in rat brain microglia, Eur. J. Pharmacol. 740 (2014) 504–511. [13] T.K. Kao, Y.C. Ou, J.S. Kuo, W.Y. Chen, S.L. Liao, C.W. Wu, C.J. Chen, N.N. Ling, Y.H. Zhang, W.H. Peng, Neuroprotection by tetramethylpyrazine against ischemic brain injury in rats, Neurochem. Int. 48 (2006) 166–176. [14] C. Zhang, S.Z. Wang, P.P. Zuo, X. Cui, J. Cai, Protective effect of tetramethylpyrazine on learning and memory function in D-galactose-lesioned mice, Chin. Med. Sci. J. 19 (2004) 180–184. [15] H. Chen, G. Li, P. Zhan, X. Liu, Ligustrazine derivatives. Part 5: design, synthesis and biological evaluation of novel
ACCEPTED MANUSCRIPT ligustrazinyloxy-cinnamic acid derivatives as potent cardiovascular agents, Eur. J. Med. Chem. 46 (2011) 5609–5615. [16] T. Zhang, J. Gu, L. Wu, N. Li, Y. Sun, P. Yu, Y. Wang, G. Zhang, Z. Zhang, Neuroprotective and axonal outgrowth-promoting effects of tetramethylpyrazine nitrone in chronic cerebral hypoperfusion rats and primary hippocampal neurons exposed to hypoxia, Neuropharmacology 118 (2017) 137–147. [17] C. Lu, J. Zhang, X. Shi, S. Miao, L. Bi, S. Zhang, Q. Yang, X. Zhou, M. Zhang, Y. Xie, Q. Miao, S. Wang, Neuroprotective effects of tetramethylpyrazine against dopaminergic neuron injury in a rat model of Parkinson's disease induced by MPTP, Int. J. Biol. Sci. 10 (2014) 350–357.
RI PT
[18] E.C. So, K.L. Wong, T.C. Huang, S.C. Tasi, C.F. Liu, Tetramethylpyrazine protects mice against thioacetamide-induced acute hepatotoxicity, J. Biomed. Sci. 9 (2002) 410–414.
[19] Y. Wang, X. Zhang, C. Xu, G. Zhang, Z. Zhang, P. Yu, L. Shan, Y. Sun, Y. Wang, Synthesis and biological evaluation of danshensu and tetramethylpyrazine conjugates as cardioprotective agents, Chem. Pharm. Bull. (Tokyo) 65 (2017) 381–388.
[20] P. Picone, D. Nuzzo, M. Di Carlo, Ferulic acid: a natural antioxidant against oxidative stress induced by oligomeric A-beta
SC
on sea urchin embryo, Biol. Bull. 224 (2013) 18–28.
[21] Y. Huang, M. Jin, R. Pi, J. Zhang, M. Chen, Y. Ouyang, A. Liu, X. Chao, P. Liu, J. Liu, C. Ramassamy, J. Qin, Protective effects of caffeic acid and caffeic acid phenethyl ester against acrolein-induced neurotoxicity in HT22 mouse hippocampal cells, Neurosci. Lett. 535 (2013) 146–151.
M AN U
[22] A. Abdel-Moneim, A.I. Yousef, S.M. Abd El-Twab, E.S. Abdel Reheim, M.B. Ashour, Gallic acid and p-coumaric acid attenuate type 2 diabetes-induced neurodegeneration in rats, Metab. Brain Dis. 32 (2017) 1279–1286. [23] F.N. Ekinci Akdemir, M. Albayrak, M. Calik, Y. Bayir, I. Gulcin, The protective effects of p-coumaric acid on acute liver and kidney damages induced by cisplatin, Biomedicines 5 (2017) pii: E18.
[24] S.Y. Park, G. Ahn, J.H. Um, E.J. Han, C.B. Ahn, N.Y. Yoon, J.Y. Je, Hepatoprotective effect of chitosan-caffeic acid conjugate against ethanol-treated mice, Exp. Toxicol. Pathol. 69 (2017) 618–624.
[25] W. Qu, H. Huang, K. Li, C. Qin, Danshensu-mediated protective effect against hepatic fibrosis induced by carbon
TE D
tetrachloride in rats, Pathol. Biol. (Paris) 62 (2014) 348–353.
[26] P. Wang, H. Zhang, F. Chu, X. Xu, J. Lin, C. Chen, G. Li, Y. Cheng, L. Wang, Q. Li, Y. Zhang, H. Lei, Synthesis and protective effect of new ligustrazine-benzoic acid derivatives against CoCl2-induced neurotoxicity in differentiated PC12 cells, Molecules 18 (2013) 13027–13042.
[27] G. Li, X. Xu, K. Xu, F. Chu, J. Song, S. Zhou, B. Xu, Y. Gong, H. Zhang, Y. Zhang, P. Wang, H. Lei, Ligustrazinyl amides: a
EP
novel class of ligustrazine-phenolic acid derivatives with neuroprotective effects, Chem. Cent. J. 9 (2015) 9. [28] G. Li, Y. Tian, Y. Zhang, Y. Hong, Y. Hao, C. Chen, P. Wang, H. Lei, A novel ligustrazine derivative T-VA prevents neurotoxicity in differentiated PC12 cells and protects the brain against ischemia injury in MCAO rats, Int. J. Mol. Sci. 16 (2015)
AC C
21759–21774.
[29] L. Ismaili, B. Refouvelet, M. Benchekroun, S. Brogi, M. Brindisi, S. Gemma, G. Campiani, S. Filipic, D. Agbaba, G. Esteban, M. Unzeta, K. Nikolic, S. Butini, J. Marco-Contelles, Multitarget compounds bearing tacrine- and donepezil-like structural and functional motifs for the potential treatment of Alzheimer's disease, Prog. Neurobiol. 151 (2017) 4–34. [30] N. Guzior, A. Wieckowska, D. Panek, B. Malawska, Recent development of multifunctional agents as potential drug candidates for the treatment of Alzheimer's disease, Curr. Med. Chem. 22 (2015) 373–404. [31] B. Sameem, M. Saeedi, M. Mahdavi, A. Shafiee, A review on tacrine-based scaffolds as multi-target drugs (MTDLs) for Alzheimer's disease, Eur. J. Med. Chem. 128 (2017) 332–345. [32] S. Hamulakova, L. Janovec, M. Hrabinova, P. Kristian, K. Kuca, M. Banasova, J. Imrich, Synthesis, design and biological evaluation of novel highly potent tacrine congeners for the treatment of Alzheimer's disease, Eur. J. Med. Chem. 55 (2012) 23– 31. [33] E.K. Reddy, C. Remya, K. Mantosh, A.M. Sajith, R.V. Omkumar, C. Sadasivan, S. Anwar, Novel tacrine derivatives exhibiting improved acetylcholinesterase inhibition: design, synthesis and biological evaluation, Eur. J. Med. Chem. 139 (2017)
ACCEPTED MANUSCRIPT 367–377. [34] S.S. Xie, J.S. Lan, X.B. Wang, N. Jiang, G. Dong, Z.R. Li, K.D. Wang, P.P. Guo, L.Y. Kong, Multifunctional tacrine-trolox hybrids for the treatment of Alzheimer's disease with cholinergic, antioxidant, neuroprotective and hepatoprotective properties, Eur. J. Med. Chem. 93 (2015) 42–50. [35] S.S. Xie, X.B. Wang, J.Y. Li, L. Yang, L.Y. Kong, Design, synthesis and evaluation of novel tacrine-coumarin hybrids as multifunctional cholinesterase inhibitors against Alzheimer's disease, Eur. J. Med. Chem. 64 (2013) 540–553. [36] J. Jerabek, E. Uliassi, L. Guidotti, J. Korabecny, O. Soukup, V. Sepsova, M. Hrabinova, K. Kuca, M. Bartolini, L.E.
RI PT
Pena-Altamira, S. Petralla, B. Monti, M. Roberti, M.L. Bolognesi, Tacrine-resveratrol fused hybrids as multi-target-directed ligands against Alzheimer's disease, Eur. J. Med. Chem. 127 (2017) 250–262.
[37] Y. Chen, J. Sun, L. Fang, M. Liu, S. Peng, H. Liao, J. Lehmann, Y. Zhang, Tacrine-ferulic acid-nitric oxide (NO) donor trihybrids as potent, multifunctional acetyl- and butyrylcholinesterase inhibitors, J. Med. Chem. 55 (2012) 4309–4321.
[38] P. Camps, X. Formosa, C. Galdeano, T. Gomez, D. Munoz-Torrero, M. Scarpellini, E. Viayna, A. Badia, M.V. Clos, A.
SC
Camins, M. Pallas, M. Bartolini, F. Mancini, V. Andrisano, J. Estelrich, M. Lizondo, A. Bidon-Chanal, F.J. Luque, Novel donepezil-based inhibitors of acetyl- and butyrylcholinesterase and acetylcholinesterase-induced beta-amyloid aggregation, J. Med. Chem. 51 (2008) 3588–3598.
[39] S. Butini, M. Brindisi, S. Brogi, S. Maramai, E. Guarino, A. Panico, A. Saxena, V. Chauhan, R. Colombo, L. Verga, E. De
M AN U
Lorenzi, M. Bartolini, V. Andrisano, E. Novellino, G. Campiani, S. Gemma, Multifunctional cholinesterase and amyloid Beta fibrillization modulators. Synthesis and biological investigation, ACS Med. Chem. Lett. 4 (2013) 1178–1182. [40] Q. Sun, D.Y. Peng, S.G. Yang, X.L. Zhu, W.C. Yang, G.F. Yang, Syntheses of coumarin-tacrine hybrids as dual-site acetylcholinesterase inhibitors and their activity against butylcholinesterase, abeta aggregation, and beta-secretase, Bioorg. Med. Chem. 22 (2014) 4784–4791.
[41] C. Lu, Q. Zhou, J. Yan, Z. Du, L. Huang, X. Li, A novel series of tacrine-selegiline hybrids with cholinesterase and monoamine oxidase inhibition activities for the treatment of Alzheimer's disease, Eur. J. Med. Chem. 62 (2013) 745–753.
TE D
[42] J.S. da Costa, J.P. Lopes, D. Russowsky, C.L. Petzhold, A.C. Borges, M.A. Ceschi, E. Konrath, C. Batassini, P.S. Lunardi, C.A. Goncalves, Synthesis of tacrine-lophine hybrids via one-pot four component reaction and biological evaluation as acetyland butyrylcholinesterase inhibitors, Eur. J. Med. Chem. 62 (2013) 556–563. [43] M. Khoobi, F. Ghanoni, H. Nadri, A. Moradi, M. Pirali Hamedani, F. Homayouni Moghadam, S. Emami, M. Vosooghi, R. Zadmard, A. Foroumadi, A. Shafiee, New tetracyclic tacrine analogs containing pyrano[2,3-c]pyrazole: efficient synthesis,
EP
biological assessment and docking simulation study, Eur. J. Med. Chem. 89 (2015) 296–303. [44] L. Pourabdi, M. Khoobi, H. Nadri, A. Moradi, F.H. Moghadam, S. Emami, M.M. Mojtahedi, I. Haririan, H. Forootanfar, A. Ameri, A. Foroumadi, A. Shafiee, Synthesis and structure-activity relationship study of tacrine-based pyrano[2,3-c]pyrazoles
AC C
targeting AChE/BuChE and 15-LOX, Eur. J. Med. Chem. 123 (2016) 298–308. [45] M. Eghtedari, Y. Sarrafi, H. Nadri, M. Mahdavi, A. Moradi, F. Homayouni Moghadam, S. Emami, L. Firoozpour, A. Asadipour, O. Sabzevari, A. Foroumadi, New tacrine-derived AChE/BuChE inhibitors: Synthesis and biological evaluation of 5-amino-2-phenyl-4H-pyrano[2,3-b]quinoline-3-carboxylates, Eur. J. Med. Chem. 128 (2017) 237–246. [46] L. Jalili-Baleh, H. Nadri, A. Moradi, S.N.A. Bukhari, M. Shakibaie, M. Jafari, M. Golshani, F. Homayouni Moghadam, L. Firoozpour, A. Asadipour, S. Emami, M. Khoobi, A. Foroumadi, New racemic annulated pyrazolo[1,2-b]phthalazines as tacrine-like AChE inhibitors with potential use in Alzheimer's disease, Eur. J. Med. Chem. 139 (2017) 280–289. [47] W. Luo, Y.P. Li, Y. He, S.L. Huang, D. Li, L.Q. Gu, Z.S. Huang, Synthesis and evaluation of heterobivalent tacrine derivatives as potential multi-functional anti-Alzheimer agents, Eur. J. Med. Chem. 46 (2011) 2609–2616. [48] R.J. Reiter, D.X. Tan, J.C. Mayo, R.M. Sainz, J. Leon, Z. Czarnocki, Melatonin as an antioxidant: biochemical mechanisms and pathophysiological implications in humans, Acta biochim. Pol. 50 (2003) 1129–1146. [49] M.I. Rodriguez-Franco, M.I. Fernandez-Bachiller, C. Perez, B. Hernandez-Ledesma, B. Bartolome, Novel tacrine-melatonin hybrids as dual-acting drugs for Alzheimer disease, with improved acetylcholinesterase inhibitory and antioxidant properties, J.
ACCEPTED MANUSCRIPT Med. Chem. 49 (2006) 459–462. [50] M.I. Fernandez-Bachiller, C. Perez, N.E. Campillo, J.A. Paez, G.C. Gonzalez-Munoz, P. Usan, E. Garcia-Palomero, M.G. Lopez, M. Villarroya, A.G. Garcia, A. Martinez, M.I. Rodriguez-Franco, Tacrine-melatonin hybrids as multifunctional agents for Alzheimer's disease, with cholinergic, antioxidant, and neuroprotective properties, ChemMedChem. 4 (2009) 828–841. [51] M.I. Fernandez-Bachiller, C. Perez, G.C. Gonzalez-Munoz, S. Conde, M.G. Lopez, M. Villarroya, A.G. Garcia, M.I. Rodriguez-Franco, Novel tacrine-8-hydroxyquinoline hybrids as multifunctional agents for the treatment of Alzheimer's disease, with neuroprotective, cholinergic, antioxidant, and copper-complexing properties, J. Med. Chem. 53 (2010) 4927–4937.
RI PT
[52] M.I. Fernandez-Bachiller, C. Perez, L. Monjas, J. Rademann, M.I. Rodriguez-Franco, New tacrine-4-oxo-4H-chromene hybrids as multifunctional agents for the treatment of Alzheimer's disease, with cholinergic, antioxidant, and beta-amyloid-reducing properties, J. Med. Chem. 55 (2012) 1303–1317.
[53] S.S. Xie, X. Wang, N. Jiang, W. Yu, K.D. Wang, J.S. Lan, Z.R. Li, L.Y. Kong, Multi-target tacrine-coumarin hybrids: cholinesterase and monoamine oxidase B inhibition properties against Alzheimer's disease, Eur. J. Med. Chem. 95 (2015) 153–
SC
165.
[54] M.K. Hu, L.J. Wu, G. Hsiao, M.H. Yen, Homodimeric tacrine congeners as acetylcholinesterase inhibitors, J. Med. Chem. 45 (2002) 2277–2282.
[55] J.M. Roldan-Pena, D. Alejandre-Ramos, O. Lopez, I. Maya, I. Lagunes, J.M. Padron, L.E. Pena-Altamira, M. Bartolini, B.
M AN U
Monti, M.L. Bolognesi, J.G. Fernandez-Bolanos, New tacrine dimers with antioxidant linkers as dual drugs: Anti-Alzheimer's and antiproliferative agents, Eur. J. Med. Chem. 138 (2017) 761–773.
[56] L. Fang, D. Appenroth, M. Decker, M. Kiehntopf, C. Roegler, T. Deufel, C. Fleck, S. Peng, Y. Zhang, J. Lehmann, Synthesis and biological evaluation of NO-donor-tacrine hybrids as hepatoprotective anti-Alzheimer drug candidates, J. Med. Chem. 51 (2008) 713–716.
[57] M. Rosini, V. Andrisano, M. Bartolini, M.L. Bolognesi, P. Hrelia, A. Minarini, A. Tarozzi, C. Melchiorre, Rational approach to discover multipotent anti-Alzheimer drugs, J. Med. Chem. 48 (2005) 360–363.
TE D
[58] S. Thiratmatrakul, C. Yenjai, P. Waiwut, O. Vajragupta, P. Reubroycharoen, M. Tohda, C. Boonyarat, Synthesis, biological evaluation and molecular modeling study of novel tacrine-carbazole hybrids as potential multifunctional agents for the treatment of Alzheimer's disease, Eur. J. Med. Chem. 75 (2014) 21–30.
[59] J. Marco-Contelles, R. Leon, C. de Los Rios, A. Guglietta, J. Terencio, M.G. Lopez, A.G. Garcia, M. Villarroya, Novel multipotent tacrine-dihydropyridine hybrids with improved acetylcholinesterase inhibitory and neuroprotective activities as
EP
potential drugs for the treatment of Alzheimer's disease, J. Med. Chem. 49 (2006) 7607–7610. [60] L. Fang, B. Kraus, J. Lehmann, J. Heilmann, Y. Zhang, M. Decker, Design and synthesis of tacrine-ferulic acid hybrids as multi-potent anti-Alzheimer drug candidates, Bioorg. Med. Chem. Lett. 18 (2008) 2905–2909.
AC C
[61] Y. Fu, Y. Mu, H. Lei, P. Wang, X. Li, Q. Leng, L. Han, X. Qu, Z. Wang, X. Huang, Design, synthesis and evaluation of novel tacrine-ferulic acid hybrids as multifunctional drug candidates against Alzheimer's disease, Molecules 21 (2016) pii: E1338. [62] X. Chao, X. He, Y. Yang, X. Zhou, M. Jin, S. Liu, Z. Cheng, P. Liu, Y. Wang, J. Yu, Y. Tan, Y. Huang, J. Qin, S. Rapposelli, R. Pi, Design, synthesis and pharmacological evaluation of novel tacrine-caffeic acid hybrids as multi-targeted compounds against Alzheimer's disease, Bioorg. Med. Chem. Lett. 22 (2012) 6498–6502. [63] F. Mao, J. Li, H. Wei, L. Huang, X. Li, Tacrine-propargylamine derivatives with improved acetylcholinesterase inhibitory activity and lower hepatotoxicity as a potential lead compound for the treatment of Alzheimer's disease, J. Enzyme Inhib. Med. Chem. 30 (2015) 995–1001. [64] F. Mao, J. Chen, Q. Zhou, Z. Luo, L. Huang, X. Li, Novel tacrine-ebselen hybrids with improved cholinesterase inhibitory, hydrogen peroxide and peroxynitrite scavenging activity, Bioorg. Med. Chem. Lett. 23 (2013) 6737–6742. [65] X. Chen, K. Zenger, A. Lupp, B. Kling, J. Heilmann, C. Fleck, B. Kraus, M. Decker, Tacrine-silibinin codrug shows neuroand hepatoprotective effects in vitro and pro-cognitive and hepatoprotective effects in vivo, J. Med. Chem. 55 (2012) 5231–5242. [66] Z. Najafi, M. Mahdavi, M. Saeedi, E. Karimpour-Razkenari, R. Asatouri, F. Vafadarnejad, F.H. Moghadam, M. Khanavi, M.
ACCEPTED MANUSCRIPT Sharifzadeh, T. Akbarzadeh, Novel tacrine-1,2,3-triazole hybrids: In vitro, in vivo biological evaluation and docking study of cholinesterase inhibitors, Eur. J. Med. Chem. 125 (2017) 1200–1212. [67] A. Minarini, A. Milelli, V. Tumiatti, M. Rosini, E. Simoni, M.L. Bolognesi, V. Andrisano, M. Bartolini, E. Motori, C. Angeloni, S. Hrelia, Cystamine-tacrine dimer: a new multi-target-directed ligand as potential therapeutic agent for Alzheimer's disease treatment, Neuropharmacology 62 (2012) 997–1003. [68] M.A. Ceschi, J.S. da Costa, J.P.B. Lopes, V.S. Camara, L.F. Campo, A.C.A. Borges, C.A.S. Goncalves, D.F. de Souza, E.L. Konrath, A.L.M. Karl, I.A. Guedes, L.E. Dardenne, Novel series of tacrine-tianeptine hybrids: synthesis, cholinesterase
RI PT
inhibitory activity, S100B secretion and a molecular modeling approach, Eur. J. Med. Chem. 121 (2016) 758–772.
[69] N. Garcia-Font, H. Hayour, A. Belfaitah, J. Pedraz, I. Moraleda, I. Iriepa, A. Bouraiou, M. Chioua, J. Marco-Contelles, M.J. Oset-Gasque, Potent anticholinesterasic and neuroprotective pyranotacrines as inhibitors of beta-amyloid aggregation, oxidative stress and tau-phosphorylation for Alzheimer's disease, Eur. J. Med. Chem. 118 (2016) 178–192.
[70] W. Luo, Y.P. Li, Y. He, S.L. Huang, J.H. Tan, T.M. Ou, D. Li, L.Q. Gu, Z.S. Huang, Design, synthesis and evaluation of
SC
novel tacrine-multialkoxybenzene hybrids as dual inhibitors for cholinesterases and amyloid beta aggregation, Bioorg. Med. Chem. 19 (2011) 763–770.
[71] E. Nepovimova, E. Uliassi, J. Korabecny, L.E. Pena-Altamira, S. Samez, A. Pesaresi, G.E. Garcia, M. Bartolini, V. Andrisano, C. Bergamini, R. Fato, D. Lamba, M. Roberti, K. Kuca, B. Monti, M.L. Bolognesi, Multitarget drug design strategy:
Med. Chem. 57 (2014) 8576–8589.
M AN U
quinone-tacrine hybrids designed to block amyloid-beta aggregation and to exert anticholinesterase and antioxidant effects, J.
[72] J.S. Lan, S.S. Xie, S.Y. Li, L.F. Pan, X.B. Wang, L.Y. Kong, Design, synthesis and evaluation of novel tacrine-(beta-carboline) hybrids as multifunctional agents for the treatment of Alzheimer's disease, Bioorg. Med. Chem. 22 (2014) 6089–6104.
[73] J. Cen, H. Guo, C. Hong, J. Lv, Y. Yang, T. Wang, D. Fang, W. Luo, Development of tacrine-bifendate conjugates with improved cholinesterase inhibitory and pro-cognitive efficacy and reduced hepatotoxicity, Eur. J. Med. Chem. 144 (2017) 128–
TE D
136.
[74] E.H. Rydberg, B. Brumshtein, H.M. Greenblatt, D.M. Wong, D. Shaya, L.D. Williams, P.R. Carlier, Y.P. Pang, I. Silman, J.L. Sussman, Complexes of alkylene-linked tacrine dimers with Torpedo californica acetylcholinesterase: binding of bis5-tacrine produces a dramatic rearrangement in the active-site gorge, J. Med. Chem. 49 (2006) 5491–5500. [75] G.L. Ellman, K.D. Courtney, V. Andres, Jr., R.M. Feather-Stone, A new and rapid colorimetric determination of
EP
acetylcholinesterase activity, Biochem. Pharmacol. 7 (1961) 88–95. [76] X. Zha, D. Lamba, L. Zhang, Y. Lou, C. Xu, D. Kang, L. Chen, Y. Xu, L. Zhang, A. De Simone, S. Samez, A. Pesaresi, J. Stojan, M.G. Lopez, J. Egea, V. Andrisano, M. Bartolini, Novel tacrine-benzofuran hybrids as potent multitarget-directed ligands
AC C
for the treatment of Alzheimer's disease: design, synthesis, biological evaluation, and X-ray crystallography, J. Med. Chem. 59 (2016) 114–131.
[77] F. Cheng, W. Li, Y. Zhou, J. Shen, Z. Wu, G. Liu, P.W. Lee, Y. Tang, admetSAR: a comprehensive source and free tool for assessment of chemical ADMET properties, J. Chem Inf. Model. 52 (2012) 3099–3105. [78] N.A. Vyas, S.S. Bhat, A.S. Kumbhar, U.B. Sonawane, V. Jani, R.R. Joshi, S.N. Ramteke, P.P. Kulkarni, B. Joshi, Ruthenium(II) polypyridyl complex as inhibitor of acetylcholinesterase and Abeta aggregation, Eur. J. Med. Chem. 75 (2014) 375–381. [79] J.X. Chen, T. Zhao, D.X. Huang, Protective effects of edaravone against cobalt chloride-induced apoptosis in PC12 cells, Neurosci. Bull. 25 (2009) 67–74. [80] J. Hu, T.Z. Zhao, W.H. Chu, C.X. Luo, W.H. Tang, L. Yi, H. Feng, Protective effects of 20-hydroxyecdysone on CoCl(2)-induced cell injury in PC12 cells, J. Cell. Biochem. 111 (2010) 1512–1521. [81] S. Tranchimand, T. Tron, C. Gaudin, G. Iacazioa, First chemical synthesis of three natural depsides involved in flavonol catabolism and related to quercetinase catalysis, Synth. Commun. 36 (2006) 587–597.
ACCEPTED MANUSCRIPT [82] T. Kojima, N. Hirasa, D. Noguchi, T. Ishizuka, S. Miyazaki, Y. Shiota, K. Yoshizawa, S. Fukuzumi, Synthesis and characterization of ruthenium(II)-pyridylamine complexes with catechol pendants as metal binding sites, Inorg. Chem. 49 (2010) 3737–3745. [83] S.K. Lee, Z.H. Mbwambo, H. Chung, L. Luyengi, E.J. Gamez, R.G. Mehta, A.D. Kinghorn, J.M. Pezzuto, Evaluation of the
AC C
EP
TE D
M AN U
SC
RI PT
antioxidant potential of natural products, Comb. Chem. High Throughput Screen. 1 (1998) 35–46.
ACCEPTED MANUSCRIPT
Highlights 1. 35 novel tacrine-phenolic acid dihybrids and tacrine-phenolic acid-ligustrazine trihybrids were synthesized and screened for anti-AD purpose.
RI PT
2. 9i was the most potent AChE inhibitor (eeAChE, IC50 = 3.9 nM; hAChE, IC50 = 65.2 nM). 3. 9i could effectively block Aβ42 self-aggregation with a ratio of 47% at 20 µM. 4. 9i had potent neuroprotection towards PC12 cells while no neurotoxicity.
AC C
EP
TE D
M AN U
SC
5. 9i showed lower hepatotoxicity than tacrine.
S0223-5234(18)30028-X
DOI:
10.1016/j.ejmech.2018.01.028
Reference:
EJMECH 10105
To appear in:
European Journal of Medicinal Chemistry
Received Date: 1 December 2017 Revised Date:
2 January 2018
Accepted Date: 8 January 2018
Please cite this article as: G. Li, G. Hong, X. Li, Y. Zhang, Z. Xu, L. Mao, X. Feng, T. Liu, Synthesis and activity towards Alzheimer's disease in vitro: Tacrine, phenolic acid and ligustrazine hybrids, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.01.028. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Synthesis and Activity towards Alzheimer’s Disease in Vitro: Tacrine, Phenolic acid and Ligustrazine Hybrids Guoliang Li a, Ge Hong a, Xinyu Li b, Yan Zhang a, Zengping Xu a, Lina Mao a, Xizeng Feng b, Tianjun Liu a, * a
Tianjin Key Laboratory of Biomedical Materials, Institute of Biomedical Engineering, Chinese Academy of Medical Sciences
& Peking Union Medical College, Tianjin 300192, China b
State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Bioactive Materials, Ministry of Education, College
of Life Sciences, Nankai University, Tianjin 300071, China
RI PT
*Corresponding author: Tianjun Liu, Email: [email protected]
Abstract
A series of novel tacrine-phenolic acid dihybrids and tacrine-phenolic acid-ligustrazine trihybrids were synthesized, characterized and screened as novel potential anti-Alzheimer drug candidates. These compounds showed potent inhibition activity towards cholinesterases (ChEs), among of them, 9i was the most potent one towards acetylcholinesterase (eeAChE, IC50
SC
= 3.9 nM; hAChE, IC50 = 65.2 nM). 9i could also effectively block β-amyloid (Aβ) self-aggregation with an inhibition ratio of 47% at 20 µM. In addition, its strong anti-oxidation activity could protect PC12 cells from CoCl2-damage in the experimental condition while no neurotoxicity. Furthermore, its hepatotoxicity was lower than tacrine in vitro and in vivo. Kinetic and molecular modeling studies revealed that 9i worked in a mixed-type way, could interact simultaneously with catalytic active site
M AN U
(CAS) and peripheral anionic site (PAS) of AChE. Therefore, 9i was a promising multifunctional candidate for the treatment of AD.
Keywords: Tacrine; Alzheimer’s disease; Cholinesterase inhibitors; Aβ aggregation; Neuroprotection 1. Introduction
Alzheimer’s disease (AD), characterized by cognitive and memory impairment, is an age-related, progressive, and irreversible neurodegenerative disorder [1]. Today, over 46 million people suffer this dementia worldwide, and would increase to 131.5 million by 2050 [2]. However prevention and treatment of AD still remained elusive to date. The pathogenesis of AD is
TE D
complex and related to the abnormality and dysfunction of multi-systems, several factors, including low levels of acetylcholine, β-amyloid deposits, τ-protein aggregation, oxidative stress and neurological disorder, play crucial roles in the AD patients [3-6]. Current treatment of AD focuses on increasing cholinergic neurotransmission in the brain by inhibiting ChEs with medicines like donepezil, rivastigmine, (-)-huprine A and galantamine (Figure 1). Clinical results demonstrated inhibition of ChEs could only improve symptoms, have no profound disease-modifying effects [7]. While multi-target direct ligands (MTDLs), which
EP
combined two or more distinct pharmacological moieties, could work simultaneously on more than one pathological target, thereby resulted in potent therapeutic effectiveness. Fragments in MTDLs could synergistically contribute to the overall activity profile of the ensemble system by acting on the targets involved in disease pathogenesis. So MTDLs have been developed as
AC C
potential agents for the treatment of AD [8-10]. Ligustrazine (2,3,5,6-tetramethylpyrazine, TMP), the main bioactive alkaloid derived from the traditional Chinese medicine
Rhizoma Chuanxiong (Ligusticum chuanxiong Hort.) (Figure 1) [11], had multiple pharmacological activities, including anti-oxidation, anti-inflammatory, thrombolysis, hepato-protection, vascular protection and neuroprotection, it could effectively protect the nervous system from oxidation stress and inflammatory [12-18]. Phenolic acids like caffeic acid, salvianolic acid, p-coumaric acid, ferulic acid, etc., as anti-oxidative ingredients, they showed potent neuroprotective and hepato-protective effects (Figure 1) [19-25]. Conjugation ligustrazine with phenolic acids could achieve much better neuroprotective activity than the single component [26-28]. Inspired by these results, here we would use either phenolic acids or phenolic-ligustrazine hybrids to modify tacrine, the aim was to obtain the novel multifunctional ChEs inhibitors which had combined activities like ChEs inhibition, neuroprotection and hepato-protection. This would be beneficial for the treatment of AD. Tacrine was the first approved ChEs inhibitor by FDA to treat AD, although its hepatotoxicity blocked its use in clinic, to find novel anti-AD drugs based on tacrine was still of interest [29-33]. Over the past years, many multifunctional tacrine hybrids had been developed by conjugating tacrine with antioxidants [34], metal chelating ligands [35], neuroprotective agents [36], NO
ACCEPTED MANUSCRIPT releasing moieties [37], or the inhibitory ligands to cholinesterase [38], β-secretase [39], β-amyloid [40] and monoamine oxidases [41], etc. Typical examples included tacrine-lophine hybrids [42], tetracyclic tacrine scaffolds [43-46], heterobivalent tacrine derivatives [47], tacrine-melatonin hybrids [48-50], tacrine-8-hydroxyquinoline hybrids [51], tacrine-chromene hybrids [52], tacrine-coumarin hybrids [40, 53], tacrine homodimers [54, 55], tacrine-NO donor group [56], tacrine-lipoic acid hybrids [57], tacrine-selegiline hybrids [41], tacrine-carbazole hybrids [58], tacrine-dihrydropyridine hybrids (tacripyrines) [59], bis-tacrine hybrids bearing peptide moiety [39], tacrine-ferulic acid scaffolds [60, 61], tacrine-ferulic acid-NO donor trihybrids [37], tacrine-caffeic acid hybrids [62], tacrine-trolox hybrids [34], tacrine-resveratrol hybrids [36], tacrine-propargylamine
RI PT
hybrids [63], tacrine–ebselen hybrids [64], tacrine-silibinin codrug [65], tacrine-1,2,3-triazole hybrids [66], cystamine-tacrine dimer [67], tacrine-tianeptine hybrids [68], pyrano-tacrine hybrids [69], tacrine-multialkoxy benezene hybrids [8, 70], quinone-tacrine hybrids [71], tacrine-(β-carboline) hybrids [72], tacrine-bifendate conjugates [73]. The results reported above proved the effectiveness of the MTDLs strategy. Inspired by these MTDLs approaches, we designed a series of novel tacrine hybrids based upon tacrine, phenoic acids and TMP.
SC
AChE features two distinct binding sites: the catalytic active site (CAS), located at the bottom of the gorge, was the binding site for both substrates and inhibitors; while the peripheral anionic site (PAS), situated at the entrance of the gorge, was the binding site of enzyme inhibitors; a mid-gorge recognition site between CAS and PAS was also identified [29]. Previous studies have clearly highlighted the superiority of dual site inhibitors for AChE, such as bis(7)-tacrine, was able to bind both CAS and
M AN U
PAS concomitant [54, 74]. This rationalized design of novel chemical entities towards AChE by MTDLs strategy. Both the crystal structure of AChE and mixed type docking characteristics of tacrine hybrids reported previously implied that the flexible conformation with alkylene chain as linker would be better [29]. Here tacrine and phenolic acids conjugated by 2, 3, 4, 6 and 8 methylene groups were randomly designed, their synthesis and pharmacological evaluation was reported in this study, including their AChE and butyrocholinesterase (BuChE) inhibition activity, kinetics of enzyme inhibition, inhibition of self-mediated Aβ
AC C
EP
TE D
aggregation, antioxidant activity, as well as neuroprotection study in vitro and hepatotoxicity assay in vitro and in vivo.
Figure 1. Chemical structures of tacrine, donepezil, rivastigmine, (-)-huprine A, galantamine, TMP, p-coumaric acid, sinapic acid, ferulic acid, caffeic acid, salvianolic acid and tacrine derivatives. 2. Results and discussion 2.1. Chemistry Scheme 1. Synthesis of intermediates 3a−3e.
ACCEPTED MANUSCRIPT Reagents and conditions: (a) POCl3, reflux, 3 h; (b) α,ω-diamino-n-alkane, pentanol, 180 °C, 18 h.
SC
RI PT
Scheme 2. Synthesis of intermediates 7a−7c.
Reagents and conditions: (c) anhydrous DMF, Bn-Br, NaHCO3, r.t., 12 h; (d) anhydrous DMF, TMP-Br, K2CO3, 85 °C, 2 h; (e) C2H5OH, KOH, 60 °C, 40 min; (f) THF, H2, Pd/C, r.t., 12 h.
M AN U
Scheme 3. Synthesis of compounds 9a−9j, 10a−10o, 11a−11e and 12a−12e. O R3 R2
OH
g
N
O O
OH
O R1
i
10a R1 = H, R3 = H, n = 2 10b R1 = H, R3 = H, n = 3 10c R1 = H, R3 = H, n = 4 10d R1 = H, R3 = H, n = 6 10e R1 = H, R3 = H, n = 8 10f R1 = OMe, R3 = OMe, n = 2 10g R1 = OMe, R3 = OMe, n = 3 10h R1 = OMe, R3 = OMe, n = 4 10i R1 = OMe, R3 = OMe, n = 6 10j R1 = OMe, R3 = OMe, n = 8
N
O
N nH
O
N
R1
N N
N
N
3a-e
OH
O R1
N
7a R1 = H, R3 = OMe
10k R1 = H, R3 = OMe, n = 2 10l R1 = H, R3 = OMe, n = 3 10m R1 = H, R3 = OMe, n = 4 10n R1 = H, R3 = OMe, n = 6 10o R1 = H, R3 = OMe, n = 8
N
O
N
N N
N
N
i
O O
HN OH
i
OH
O
3a-e 7c
N
N nH
O OH
O R1
N N N
12a n = 2, 12b n = 3, 12c n = 4, 12d n = 6, 12e n = 8
11a R1 = H, n = 2, 11b R1 = H, n = 3 11c R1 = H, n = 4, 11d R1 = H, n = 6 11e R1 = H, n = 8
EP
7b R1 = H
HN
3a-e
R1
N
O
N
N
O
9a R1 = H, R2 = OH, R3 = H, n = 2 9b R1 = H, R2 = OH, R3 = H, n = 3 9c R1 = H, R2 = OH, R3 = H, n = 4 9d R1 = H, R2 = OH, R3 = H, n = 6 9e R1 = H, R2 = OH, R3 = H, n = 8 9f R1 = OMe, R2 = OH, R3 = OMe, n = 2 9g R1 = OMe, R2 = OH, R3 = OMe, n = 3 9h R1 = OMe, R2 = OH, R3 = OMe, n = 4 9i R1 = OMe, R2 = OH, R3 = OMe, n = 6 9j R1 = OMe, R2 = OH, R3 = OMe, n = 8
R3
R3
N nH
h
R1
N
8a R1 = H, R2 = OH, R3 = H 8b R1 = OMe, R2 = OH, R3 = OMe
N N
HN
R2
3a-e
R1
O
O
R3
N nH
HN
TE D
O C
Reagents and conditions: (g) anhydrous DMF, EDCI/DMAP, r.t., 12 h; (h) anhydrous DMF, TMP-Br, K2CO3, 60 °C, 6 h; (i) anhydrous CH2Cl2, EDCI/HOBT, r.t., 12 h.
AC C
The target compounds were the conjugation of tacrine, phenolic acids and ligustrazine. Compound 2, 9-chloro-1,2,3,4-tetrahydroacridine as the starting compound, was synthesized from reaction of anthranilic acid with cyclohexanone. Then 2 reacted with the corresponding α,ω-diamino-n-alkane to yield the series amines 3a−3e (Scheme 1). In the tandem reaction, due to the self-coupling of two phenolic acids moieties, direct reaction of tacrine with phenolic acids like ferulic acid, caffeic acid as well as salvianolic acid was lower in yields. Therefore, the economic way was phenolic acid firstly coupling with TMP. However in the coupling reaction of phenolic acids 4a−4c with TMP-Br, carboxylic group would cost more TMP-Br, so in practice the corresponding benzyl ester 5a−5c instead, was reacted with TMP-Br to give compounds 6a−6c. Treated 6a−6c by KOH or H2, Pd/C offered intermediates 7a−7c. The procedure is outlined in Scheme 2. Synthesis of the target compounds were depicted in Scheme 3. Reaction of the series amines 3a−3e with the acids 8a−8b in presence of EDCI and DMAP in anhydrous DMF at room temperature yielded the series 9a−9j. The series 9a−9j reacted with intermediate TMP-Br to produce the target compounds 10a−10j. Reaction of intermediates 7a−7c with the series amines 3a−3e in presence of EDCI and HOBT in anhydrous CH2Cl2 yielded series 10k−10o, 11a−11e and 12a−12e. Totally 35 new target compounds were synthesized, their chemical structure characterized by 1H NMR, 13C NMR and high resolution mass spectra
ACCEPTED MANUSCRIPT
RI PT
(HRMS), was right, their purity was over 95% measured by HPLC.
Figure 2. The purity of representative compounds 9i (A) and 10i (B) analyzed by HPLC (Kromasil C18 column, eluted by acetonitrile/water (15/85-95/5) containing 0.1% formic acid at a flow rate of 1 mL/min). 2.2. Cholinesterase inhibition assay in vitro
The inhibition potency towards AChE and BuChE for each hybrid was assayed by the Ellman’s method with slightly
SC
modifications [75]. The IC50 values were summarized in Table 1. The tested 35 compounds (9a–12e) all showed AChE and BuChE inhibitory activities. As control, tacrine had potent inhibition potency (IC50 of AChE = 70.7 nM, IC50 of BuChE = 7.2 nM) in our experiment, however as it was modified by the phenolic acids, like sinapic acid or p-coumaric acid, the potency changed with the linker as well as the auxiliary, some of them were more potent than tacrine. The inhibition activity of sinapic acid
M AN U
dihybrids 9f–9j for AChE was slightly better than that of p-coumaric acid dihybrids 9a–9e, and the activity roughly increased with the electron density in the cinnamic acid moieties [70], changed in a tendency of 4-OH, 2,3-di(OCH3) > 4-OH, 3-OCH3 > 4-OH > 3, 4-di(OH) at the same chain length (here in order to extract the common rule, we compared our data with others’ reported previously by L. Fang [60] and X. Chao [62]). Among these compounds, compound 9i (IC50 = 3.9 nM) exhibited the best AChE inhibitory activity. In the trihybrid system of tacrine conjugation with sinapic acid or p-coumaric acid and ligustrazine, i.e., 10a-10j, they showed irregular change in inhibition activity toward either AChE or BuChE, and compared with their precursor dihybrids, no obvious improvement in inhibition activity. The contribution of ligustrazine moiety was complicated in
TE D
these system, these results could be possibly ascribed to suitable docking positions of the ligustrazine unit at the AChE gorge [76]. The trihybrids 10a, 10f, and 10i showed good inhibitory activities toward AChE, in which compound 10i (IC50 of AChE = 2.6 nM) was the best. As for BuChE, compared with tacrine, the trihybrids 10a–10j showed relatively weak inhibitory activities, except for compounds 10b (IC50 = 5.1 nM), 10c (IC50 = 2.3 nM).
The contribution of ligustrazine moiety was complicated in the trihybrid system of tacrine, ferulic acid or caffeic acid and
EP
ligustrazine due to the spacer length, the bulk difference as well as the space in docking target. Compared to tacrine-ferulic acid dihybrids (IC50 = 1.2 eq, IC50 = 1.4 eq), the inhibition activity of tacrine–ferulic acid–ligustrazine trihybrids 10k (IC50 = 2.1 eq) and 10l (IC50 = 5.8 eq) for AChE was stronger than that of the corresponding tacrine dihybrids with same carbon chain length (2
AC C
and 3). While 10m (IC50 = 0.38 eq) showed lower inhibition activity than tacrine-ferulic acid dihybrid (IC50 = 5.9 eq) with four-carbon chain length. In addition, the AChE inhibition activity changed little between the tacrine–caffeic acid dihybrids (IC50 = 0.5 eq, IC50 = 0.5 eq) and tacrine–caffeic acid–ligustrazine 11a (IC50 = 0.65 eq) and 11d (IC50 = 0.5 eq) with same carbon chain length (2 and 6). When the carbon chain length was three, the trihybrid 11b (IC50 = 5.2 eq) exhibited potent AChE inhibition activity relative to that of the corresponding dihybrid (IC50 = 0.1 eq) (here we compared our data with others’ reported previously by L. Fang [60] and X. Chao [62], and eq was the abbreviation of tacrine equivalent). Among trihybrids like tacrine–ferulic acid–ligustrazine (10k–10o) and tacrine–caffeic acid–ligustrazine (11a–11e), they had the similar molecular structure. At the same linker one ligustrazine moiety on the benzene ring usually has a higher ChEs (both AChE and BuChE) inhibitory activity than that of two ligustrazine moieties, such as 10k > 11a, 10n > 11d, 10o > 11e. However, this regularity did not work in the case of tacrine–salvianolic acid–ligustrazine trihybrids, 12d (IC50 = 1.3 nM), 12e (IC50 = 3.8 nM), which exhibited surprising BuChE inhibitory activity. The possible reason was that BuChE could accommodate bulkier inhibitors than AChE [76]. Results from 11a–12e suggested that at the same linker, trans olefinic bond could usually enhance the AChE inhibition activity, such as 11a > 12a, 11b > 12b, 11c > 12c, 11d > 12d. Except 12e > 11e, a different bonding
ACCEPTED MANUSCRIPT conformation may be given at longer linker. The linker length notably influenced the inhibition ability to BuChE, chains containing 6 carbon atom (9i, 10i, 10n, 12d) and 8 carbon atoms (9j, 10j, 11e, 12e) generally showed potent inhibition activities in our study. So the linker length and the bulk of ligustrazine had a certain impact on the inhibition activity. Generally, for the system reported here, their inhibition potency towards AChE and BuChE was dependent on the integral contribution of the linker length, the electronic density in conjugation partner as well as the steric effect. Table 1. Inhibition of AChE and BuChE (IC50 Values). IC50 (nM)a Sturcture
IC50 (nM)a Compd.
n AChEb
BuChEc
Sturcture
n AChEb
BuChEc
RI PT
Compd.
2.6±0.7
28.6±6.5
8
144.9±11.3
16.6±3.7
2
33.1±5.0
502.4±57.1
3
12.2±0.3
22.6±5.6
4
188.0±19.9
32.4±4.5
6
14.7±1.9
28.3±3.3
8
304.1±17.9
173.8±24.6
2
108.5±15.9
﹥1000
11b
3
13.7±4.0
199.1±6.0
9.3±3.9
11c
4
138.7±18.6
110.9±8.7
44.9±7.1
588.8±73.8
11d
6
136.2±26.6
68.1±3.2
3
205.1±9.7
5.2±0.3
11e
8
﹥1000
61.8±9.5
10c
4
333.7±45.0
2.3±0.6
12a
2
﹥1000
816.2±66.0
10d
6
303.7±31.9
147.0±5.1
12b
3
﹥1000
164.5±7.7
10e
8
263.5±26.8
228.1±54.3
12c
4
﹥1000
﹥1000
10f
2
65.3±7.5
612.7±60.7
12d
6
270.3±53.5
1.3±0.1
3
115.7±3.3
﹥1000
12e
8
65.1±5.6
3.8±0.4
4
178.9±26.6
﹥1000
Tacrine
70.7±3.9
7.2±0.2
109.5±15.4
302.2±8.1
10i
9b
3
127.5±7.0
43.3±2.7
10j
9c
4
138.9±15.5
29.8±2.5
10k
9d
6
44.6±9.6
24.1±0.5
10l
9e
8
522.0±21.1
278.5±5.0
10m
9f
2
100.5±7.3
﹥1000
10n
9g
3
25.8±0.7
329.8±5.8
10o
9h
4
64.4±3.7
240.9±7.1
11a
9i
6
3.9±0.8
24.3±3.1
9j
8
32.5±1.5
10a
2
10b
TE D
M AN U
2
10g 10h
SC
6
9a
Results were the mean of three independent experiments (n = 3) ± SD.
b
AChE from electric eel, Type VI-S, EC 3.1.1.7.
c
BuChE form equine serum, EC 3.1.1.8.
EP
a
In the following step, selected compounds 9i and 10i were also evaluated as inhibitors of human AChE (hAChE) and human BuChE (hBuChE), results (Table 2) showed that 9i (IC50 = 65.2 nM) was a potent inhibitor towards hAChE, nearly 2-fold of that
AC C
tacrine (IC50 = 116.8 nM), but its inhibition activity towards hBuChE (IC50 = 48.9 nM) was relatively lower than tacrine (IC50 = 34.4 nM). Compound 10i (IC50 of hAChE = 108.4 nM, IC50 of hBuChE = 577.0 nM) exhibited lower activity than tacrine towards either hAChE or hBuChE.
Table 2. Inhibition of hAChE and hBuChE (IC50 Values). IC50 (nM)a
Compd.
hAChEb
hBuChEc
9i
65.2±13.2
48.8±19.5
10i
108.4 ±25.5
577.0±37.5
Tacrine
116.8±15.9
34.4±4.1
a
Results were the mean of three independent experiments (n = 3) ± SD.
b
hAChE from human erythrocytes, EC 3.1.1.7.
c
hBuChE form human serum, EC 3.1.1.8.
2.3. Prediction of ADME property and blood-brain barrier permeability.
ACCEPTED MANUSCRIPT The ADME (absorption, distribution, metabolism and excretion) properties of all of the target compounds 9a−12e were predicted
using
MOE
2008.10,
their
BBB
permeation
and
HIA
were
predicted
using
admetSAR
server
(http://lmmd.ecust.edu.cn:8000/ predict/) (Table 3) [77]. It can be seen that not all tested compounds met the criteria of lipinski’s rule of five. According to this prediction, tacrine–p-coumaric acid hybrids showed higher probability of BBB permeation than that of sinapic acid hybrids, while less difference among HIA data. Compounds 9a−9e, 9h−9j, 10a−10e, 10m−10o, 11c−11e (9i included) could be absorbed by human intestinal and were able to penetrate brain; therefore, they were predicted that could be
RI PT
oral administrated as CNS-active compounds. Table 3. The ADME properties and BBB permeability of compounds 9a−12e. Comp.
MWa
HBDa
HBAa
LogPa
Rotora
ASAa (Å2)
ASA-Pa (Å2)
BBB permeation (±) and probabilityb
HIAb
9a
387
3
5
4.01
7
697.03
153.53
BBB+ 0.8789
0.9699
9b
401
3
5
4.45
8
739.98
143.90
BBB+ 0.9005
9c
415
3
5
4.89
9
762.92
143.42
BBB+ 0.9366
9d
443
3
5
5.78
11
818.24
138.99
BBB+ 0.9366
9e
471
3
5
6.66
13
868.80
134.22
9f
447
3
7
3.99
9
733.44
192.34
9g
461
3
7
4.43
10
814.61
195.09
9h
475
3
7
4.87
11
838.14
194.52
BBB+ 0.5000
0.9554
9i
503
3
7
5.76
13
884.86
193.35
BBB+ 0.5000
0.9554
9j
531
3
7
6.64
15
958.90
186.72
BBB+ 0.5000
0.9554
10a
521
2
7
4.53
10
888.92
126.12
BBB+ 0.5745
0.9851
10b
535
2
7
4.97
11
943.25
118.09
BBB+ 0.5599
0.9874
10c
549
2
7
5.41
12
989.80
120.95
BBB+ 0.6943
0.9939
10d
577
2
7
6.29
14
1009.95
114.32
BBB+ 0.6943
0.9939
10e
605
2
7
7.18
16
1064.77
110.52
BBB+ 0.6943
0.9939
10f
581
2
9
4.01
12
949.61
186.25
BBB- 0.7967
0.9236
10g
595
2
9
4.45
13
1022.45
181.84
BBB- 0.7867
0.9345
10h
609
2
9
4.90
14
1035.15
180.76
BBB- 0.6594
0.9678
10i
637
2
9
5.78
16
1097.94
172.70
BBB- 0.6594
0.9678
10j
665
2
9
6.66
18
1085.11
175.91
BBB- 0.6594
0.9678
10k
551
2
10l
565
2
10m
579
2
0.9907
SC
0.9907
BBB+ 0.9366
0.9907
BBB- 0.7251
0.8645
BBB- 0.6523
0.9105
M AN U
TE D
EP
0.9808
4.27
11
937.98
167.48
BBB- 0.6321
0.9705
8
4.71
12
982.16
143.48
BBB- 0.6420
0.9748
8
5.15
13
955.21
145.76
BBB+ 0.5194
0.9879
AC C
8
10n
607
2
8
6.04
15
1041.88
138.56
BBB+ 0.5194
0.9879
10o
635
2
8
6.92
17
1115.12
141.03
BBB+ 0.5194
0.9879
11a
671
2
10
4.52
13
1072.9
148.48
BBB- 0.6229
0.9840
11b
685
2
10
4.96
14
1079.8
134.55
BBB- 0.6276
0.9864
11c
699
2
10
5.41
15
1182.7
140.11
BBB+ 0.5214
0.9935
11d
727
2
10
6.29
17
1193.7
142.08
BBB+ 0.5214
0.9935
11e
756
2
10
7.17
19
1268.2
135.88
BBB+ 0.5214
0.9935
12a
689
3
11
3.39
14
1105.94
139.23
BBB- 0.9320
0.6597
12b
703
3
11
3.83
15
1050.16
111.51
BBB- 0.9619
0.5223
12c
717
3
11
4.28
16
1029.06
124.95
BBB- 0.9320
0.6597
12d
745
3
11
5.16
18
1226.38
137.65
BBB- 0.9320
0.6597
12e
774
3
11
6.04
20
1252.38
158.21
BBB- 0.9320
0.6597
ACCEPTED MANUSCRIPT a
MW (molecular weight), HBD (number of H-bond donors), HBA (number of H-bond acceptors), LogP (log octanol/water
partition coefficient), Rotor (number of rotatable bonds), ASA (water accessible surface area) and ASA-P (total polar surface area) were predicted using MOE 2008.10. b
The BBB (blood-brain barrier) permeability and HIA (probability of human intestinal absorption) were predicted using
admetSAR server (http://lmmd.ecust.edu.cn:8000/ predict/). 2.4. Inhibition of Aβ42 self-aggregation Compounds 9i and 10i, because of potent AChE inhibition activities in our screening compounds, their potential inhibition
RI PT
activities towards Aβ42 aggregation were further examined using thioflavin T (ThT) fluorescence method [78]. Hybrid 9i could efficiently inhibit the self-mediated Aβ42 aggregation, and its activity was dose-dependent with a good S shape curve, less potencies at lower concentrations, higher potencies at higher concentrations (20, 100 µM) compared with tacrine (Figure 3), inhibition ratio 47% at 20 µM, 48% at 100 µM, following a plateau stage. Compound 10i displayed less potent inhibition activity
M AN U
SC
for Aβ42 self-aggregation.
Figure 3. Inhibition of Aβ42 self-aggregation curves for 9i (solid square), 10i (solid triangle) and tacrine (solid circle) determined by the ThT assay.
2.5. Inhibition of Aβ42 fibril formation monitored by atomic force microscope (AFM)
Aβ42 aggregation behavior in the absence and presence of compounds 9i, 10i and tacrine, were monitored using AFM. As
TE D
shown in Figure 4, Aβ42 alone easily aggregated into dense, thick and large fibrils at concentration of 0.1 mM, while amorphous deposits with some well-defined Aβ42 fibrils formed in the presence of tacrine (0.1 mM). No obvious Aβ42 fibril was observed in the presence of 9i (0.1 mM) and 10i (0.1 mM), and more patchy aggregations occurred in 10i group. The small dots were the crystal of 10i extracted due to the poor water solubility, other than Aβ42 aggregation. These AFM experimental results were in agreement with the results of ThT studies (Figure 3), which revealed that compound 9i could efficiently inhibit the Aβ42 fibrils
AC C
EP
formation.
Figure 4. AFM image of Aβ42 aggregation (CAβ = 0.1 mM) recorded in the absence (A) and presence of tacrine (0.1 mM, B), 9i (0.1 mM, C) 10i (0.1 mM, D) incubated at 37 °C for 4 days, respectively. 2.6. Antioxidant activity in vitro 9i and 10i, the most potent two compounds screened in AChE inhibition study, their antioxidant activities were evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay with trolox as reference compound (Table 4) [34]. Results showed that 9i had very potent peroxyl radical scavenging capacity. However, compound 10i, with phenolic hydroxyl group covered by a TMP moiety, exhibited a poor antioxidant activity with IC50 over 1000 µM.
ACCEPTED MANUSCRIPT Table 4. Inhibitory activities in DPPH assay.
a
Compd.
IC50 (µM)a
9i
85.8±3.5
10i
﹥1000
Trolox
52.9±1.3
Results were the mean of three independent experiments (n = 3) ± SD.
2.7. Neuroprotection
RI PT
Neurotoxicity of compound 9i, 10i on NGF-differentiated PC12 cells were determined at 1.25, 2.5, 5 and 10 µM with tacrine as control, shown in Figure 5 (A). The toxicity of tacrine increased with concentration, while compound 9i exhibited no neurotoxicity to NGF-differentiated PC12 cells at the experimental concentration range of 10 µM (131%). Unfortunately, 10i exhibited significant neurotoxicity, larger than tacrine. Neuroprotection effect was studied on the NGF-differentiated PC12 cells treated with CoCl2 [79, 80], with trolox as reference, shown in Figure 5 (B). The viability of CoCl2-damaged PC12 cell was only
SC
51.2%, and increased to 101.3% pretreated by 10 µM trolox, while it increased to 74.5%, 92.4%, 99.5% and 106.8% respectively, pretreated by compound 9i at 1.25, 2.5, 5 and 10 µM, the cell viability increased in a dose dependent manner. This indicated that 9i could protect PC12 cells from CoCl2 damage. Compound 10i exhibited neuroprotective effect at 1.25 µM (73.3%); its cytotoxicity increased with increasing concentrations, the cell viabilities were 37.7%, 15%, 11.2% at 2.5, 5 and 10 µM
EP
TE D
might come from radical scavenging action.
M AN U
respectively. This result was positive-correlated with their antioxidant assay, which indicated that the neuroprotective action of 9i
Figure 5. Neurotoxicity (A) and neuroprotection (B) of 9i and 10i on normal and CoCl2-damaged PC12 cells. Data were expressed as mean ± SD (n = 3). ** p < 0.01: A, compared with NGF group; B, compared with CoCl2 group.
AC C
2.8. Hepatotoxicity study in vitro and in vivo
The goal of the present work was to find novel candidate with potent anti-AD activity and lower toxicity, and the conjugate
reported here were the derivatives of tacrine, a withdraw medicine due to obvious hepatotoxicity in clinic. Compound 9i had potent activity in ChEs inhibition and block Aβ42 self-aggregation had been proved in vitro, so the toxicity study, especially the hepatotoxicity becomes very important for its further study as a candidate. Its hepatotoxicity in vitro was analyzed via MTT assay of HepG2 cell line (Figure 6) [44], which was incubated with 9i of 1.25, 2.5, 5 and 10 µM respectively for 24 h. A concentration-dependent decrease in the cell viability was observed for tacrine and 9i, whereas the cell viability of 9i (98.4%, 96.3%, 94.9% and 81.8%) was higher than that of tacrine (92.5%, 90.9%, 89.8% and 77.6%) at the same condition. This suggested 9i had a lower hepatotoxicity profile with respect to tacrine. Its hepatotoxicity in vivo was assayed with adult male ICR mice as model [37], orally administrated compound 9i at dose of 12.82 µmol/100 g b wt. Samples taken at 8 h, 24 h and 36 h were subjected to hepatotoxicity assay respectively. Results (Table 5) indicated that tacrine had higher hepatotoxicity with higher ALT and AST values at each time point, while compound 9i had lower hepatotoxicity than tacrine from 8 h to 36 h, both ALT and AST values were very approached the control group except relative higher AST value at 8 h. Considering its potent ChEs
ACCEPTED MANUSCRIPT
RI PT
inhibition activity and lower hepatotoxicity than tacrine, 9i was a promising anti-AD candidate.
Figure 6. In vitro hepatotoxicity of 9i on HepG2 cell line. Data were expressed as mean ± SD (n = 3). * p < 0.05, ** p < 0.01,
SC
compared with control group.
Table 5. ALT and AST values at different time points measured after ICR mice oral administration of tacrine and 9i. ALT(U/L)
AST(U/L)
Groups 8h
24 h
36 h
8h
24 h
36 h
Control
40.9±2.2
37.3±2.9
27.8±1.8
25.5±1.6
20.8±2.2
Tacrine
45.3±2.0*
55.7±2.5**
35.1±1.7**
38.8±2.1**
28.3±1.4**
21.9±1.6**
9i
43.2±3.4
40.6±2.1
29.7±2.7
34.3±1.8**
21.7±2.7
19.9±1.8
M AN U
18.5±1.7
Values were expressed as mean ± SD (n = 8. t-test, compared to control of the same time after administration, * p ≤ 0.05, ** p ≤ 0.01). 2.9. Kinetic characterization of AChE inhibition
Because of the best total performance including cholinesterases inhibition, inhibition of Aβ42 aggregation, antioxidant
TE D
activity and neuroprotection as well as hepatotoxicity study, 9i was selected for a kinetic study to gain information on the mechanism of inhibition. Kinetic characterization of 9i inhibition AChE was elucidated from the Lineweaver-Burk reciprocal plots. Both slopes (decreased Vmax) and intercepts (higher Km) increased with increasing concentrations of 9i (Figure 7),
AC C
EP
implied 9i was a mixed-type inhibitor.
Figure 7. Lineweaver-Burk plots resulting from subvelocity curves of AChE activity at different substrate concentrations (0.05~0.5 mM) in the absence and presence of 2.5, 5, 10 nM 9i. 2.10. Molecular modeling study
To further study the interaction mode of 9i with ChEs, molecular docking study was performed using software package MOE 2008.10 [72]. The X-ray crystal structure of the TcAChE complex with bis(7)-tacrine (PDB code: 2CKM) was applied to build the starting model of AChE. Tacrine moiety of 9i was located into the CAS of TcAChE, via aromatic π–π stacking interactions with the phenyl ring from Phe 330 (3.66 Å) and the indole ring from Trp 84 (3.45 Å), respectively. The sinapic acid moiety occupied the PAS of the enzyme, and bound with residues Ser 286, Asp 285 via hydrophobic interactions (Figure 8, A1 and A2). In addition, the interaction mechanism of 9i with hAChE in complex with donepezil (PDB code: 4EY7) was also carried out [53]. As shown in Figure 8 (A3) and (A4), sinapic acid moiety was located into PAS of hAChE through aromatic π–π
ACCEPTED MANUSCRIPT stacking interaction with Trp 286 (3.43 Å). Tacrine moiety occupied the CAS and stacked against the indole ring of Trp 86 (4.18 Å). In middle gorge, a hydrophobic interactions with the residues Tyr124 (2.68 Å) was observed, which could enhance the interaction with hAChE. These results indicated that compound 9i could bind to the CAS and PAS of AChE in interaction model of both TcAChE and hAChE. Since equine serum BuChE, whose crystal structure has not been reported, is homology with human BuChE, so, the hBuChE (PDB code: 1P0I) was used in the docking study. A π–π stacking interaction was found between tacrine and Trp 82 (3.41 Å), and two hydrogen bonds were observed between the hydroxyl group and Ser 198 (2.95 Å), methoxy group and His 438 (3.23
RI PT
Å) (Figure 8, B1 and B2). These docking models could also give some clues to other dihybrids or trihybrids interaction with
M AN U
SC
ChEs in this report.
Figure 8. 3D, 2D docking model of compound 9i with TcAChE (A1, A2), hAChE (A3, A4) and hBuChE (B1, B2). These figures were depicted using the ligand interactions applied in MOE. Atom colors: gray–carbon atoms, dark blue–nitrogen atoms, red– oxygen atoms.
TE D
3. Conclusion
In conclusion, a series of novel tacrine-phenolic acid dihybrids and tacrine-phenolic acid-ligustrazine trihybrids were designed and synthesized as multifunctional ChEs inhibitor for the treatment of AD. Most of them could effectively inhibit ChEs in vitro. Compound 9i (IC50 of eeAChE = 3.9 nM, IC50 of hAChE = 65.2 nM; IC50 of eqBuChE = 24.3 nM, IC50 of hBuChE = 48.8 nM) was the most potent AChE inhibitor (18 or 2-fold activity of tacrine), but less potent towards BuChE than tacrine. 9i
EP
could also block Aβ42 self-aggregation with an inhibition ratio of 47% at 20 µM. Its strong anti-oxidation activity could protect PC12 cells from CoCl2-damage in the experimental condition while no neurotoxicity. Hepatotoxicity assays in vitro and in vivo indicated that 9i had no obvious hepatotoxicity, was safer than tacrine. Kinetic and molecular modeling study revealed that 9i
AC C
was a mixed-type AChE inhibitor, could interact simultaneously with CAS and PAS of AChE. Summary above, the new hybrid 9i, with characteristics of potent AChE inhibition, blocking Aβ42 self-aggregation, neuroprotection and low hepatotoxicity, was a promising anti-AD drug candidate. 4. Experimental section 4.1. Chemistry 1
H NMR spectra and 13C NMR were acquired on a Bruker AVANCE III (400MHz) or VARIAN INOVA (500MHz) and
referenced to tetramethylsilane (TMS). Chemical shifts were reported in ppm (δ) using the residue solvent line as the internal standard. High resolution mass spectra (HRMS) were recorded on Agilent 6520 Q-TOF LC/MS and Varian 7.0T FTMS (MALDI). Melting points are uncorrected and were measured using a digital melting point apparatus (Shenguang WRS-1B, shanghai, China). Flash column chromatography was performed with silica gel (200–300 mesh). Reactions were followed by thin-layer chromatography (TLC) on silica gel GF254 plates (Qingdao Haiyang Chemical Plant, Qingdao, China), and the spots were visualized by modified bismuth potassium iodide or using a UV lamp (λ = 254 nm). The purity of the synthesized compounds was over 95% analyzed by HPLC
(Waters
2695 Alliance system), with Kromasil C18 column eluted by
ACCEPTED MANUSCRIPT acetonitrile/water (15/85-95/5) containing 0.1% formic acid at a flow rate of 1 mL/min. Compounds 3a−3e were prepared following previously published methods [58, 71]. 4.2. Preparation of intermediates 7a−7c Compound TMP-Br was prepared according to our previously reported method [27]. Compound 6a−6c was gained according to the method described by Kojima and Tranchimand [81, 82]. An aqueous solution of KOH (24.90 mmol) was added to a solution of intermediates (6a and 6b, 7.01 mmol) in ethanol. The mixture was stirred for 40 min at 60 °C. Then the pH of mixture was adjusted to 3 with 10% hydrochloric acid to afford crude compounds, filtered, and purified by recrystallization from
solid, yield 89%. HRMS (ESI) m/z 449.2184 [M+H]+, calcd. for [C25H29N4O4]+, 449.2189.
RI PT
ethyl acetate. 7a, white solid, yield 83%. HRMS (ESI) m/z 329.1498 [M+H]+, calcd. for [C18H21N2O4]+, 329.1501. 7b, white
To a solution of 6c (7.01 mmol) in methanol (20 mL), was added Pd/C 10% (0.01 g). Then the suspension was stirred under hydrogen atmosphere at room temperature for 12 h. The mixture was filtered, concentrated under vacuum and purified by silica
HRMS (ESI) m/z 467.2293 [M+H]+, calcd. for [C25H31N4O5]+, 467.2294. 4.3. General procedure I for the preparation of compounds 9a−9j
SC
gel chromatography (petroleum ether: acetone = 4:1) to give corresponding target compound 7c. 7c, light yellow solid, yield 84%,
To a stirred solution of cinnamic acids (4.14 mmol), EDCI (4.97 mmol), DMAP (1.24 mmol) in anhydrous DMF (10 mL) was added intermediate 3a−3e. The reaction mixture was stirred at room temperature for 12 h. It was concentrated to dryness and
M AN U
redissolved in CH2Cl2 (30 mL), washed successively with water (2 × 30 mL) and brine (30 mL), dried over sodium sulfate, concentrated under vacuum and purified by silica gel chromatography (CH2Cl2: MeOH = 20:1 ) to give compound 9a−9j. (E)-3-(4-hydroxyphenyl)-N-(2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)acrylamide (9a) Compound 9a was prepared according to the general procedure I, light yellow solid, yield 63%, mp > 210 °C. 1H NMR (400 MHz, DMSO) δ 9.94 (s, 1H, -OH), 8.62 (t, J = 5.7 Hz, 1H, -CONH), 8.46 (d, J = 8.7 Hz, 1H), 8.01 (br, 1H, -NH), 7.93 (d, J = 8.2 Hz, 1H), 7.81 - 7.77 (m, 1H), 7.52 - 7.49 (m, 1H), 7.35 - 7.30 (m, 3H), 6.78 - 6.76 (m, 2H), 6.39 (d, J = 15.7 Hz, 1H), 3.98 3.95 (m, 2H), 3.54 - 3.49 (m, 2H), 2.94 (br, 2H), 2.64 (br, 2H), 1.77 (br, 4H). 13C NMR (101 MHz, DMSO) δ 166.97, 159.08,
TE D
155.89, 150.50, 139.38, 137.84, 132.44, 129.22, 129.22, 125.57, 125.18, 124.95, 119.01, 117.80, 115.75, 115.75, 115.49, 111.21, 48.22, 27.83, 23.78, 21.42, 20.21. HRMS (ESI) m/z 388.2025 [M+H]+, calcd. for [C24H26N3O2]+, 388.2025. (E)-3-(4-hydroxyphenyl)-N-(3-((1,2,3,4-tetrahydroacridin-9-yl)amino)propyl)acrylamide (9b) Compound 9b was prepared according to the general procedure I, light yellow oil, yield 65%. 1H NMR (400 MHz, DMSO) δ 9.90 (br, 1H, -OH), 8.34 (d, J = 8.6 Hz, 1H), 8.29 (br, 1H, -CONH), 7.87 (d, J = 8.4 Hz, 1H), 7.75 - 7.71 (m, 1H), 7.50 - 7.46 (m,
EP
1H), 7.39 -7.37 (m, 2H), 7.32 (d, J = 15.7 Hz, 1H), 7.20 (br, 1H, -NH), 6.81 (d, J = 8.5 Hz, 2H), 6.42 (d, J = 15.7 Hz, 1H), 3.78 3.74 (m, 2H), 3.28 - 3.24 (m, 2H), 2.96 (br, 2H), 2.71 (br, 2H), 1.87 - 1.81 (br, 6H). 13C NMR (101 MHz, DMSO) δ 165.79, 158.89, 153.85, 152.91, 138.71, 130.96, 129.09, 129.09, 125.76, 124.42, 124.26, 121.97, 118.41, 117.01, 115.71, 115.71, 115.71,
AC C
112.64, 44.76, 35.88, 30.33, 29.64, 24.31, 21.83, 20.89. HRMS (ESI) m/z 402.2182 [M+H]+, calcd. for [C25H28N3O2]+, 402.2182. (E)-3-(4-hydroxyphenyl)-N-(4-((1,2,3,4-tetrahydroacridin-9-yl)amino)butyl)acrylamide (9c) Compound 9c was prepared according to the general procedure I, light yellow oil, yield 69%. 1H NMR (400 MHz, DMSO) δ 9.94 (s, 1H, -OH), 8.43 (d, J = 8.6 Hz, 1H), 8.15 - 8.13 (m, 1H, -CONH), 7.98 (d, J = 8.0 Hz, 1H), 7.84 - 7.80 (m, 2H), 7.57 7.53 (m, 1H), 7.35 (d, J = 8.6 Hz, 2H), 7.28 (d, J = 15.7 Hz, 1H), 6.80 (d, J = 8.6 Hz, 2H), 6.42 (d, J = 15.7 Hz, 1H), 3.90 - 3.85 (m, 2H), 3.20 - 3.15 (m, 2H), 3.00 (br, 2H), 2.67 (br, 2H), 1.81 (br, 4H), 1.78 - 1.73 (m, 2H), 1.56 - 1.49 (m, 2H). 13C NMR (101 MHz, DMSO) δ 165.33, 158.83, 155.55, 150.61, 138.37, 137.84, 132.39, 128.99, 128.99, 125.80, 124.96, 124.96, 119.10, 118.72, 115.70, 115.55, 115.55, 111.07, 46.82, 38.05, 27.88, 27.36, 26.24, 23.96, 21.42, 20.20. HRMS (ESI) m/z 416.2341 [M+H]+, calcd. for [C26H30N3O2]+, 416.2338. (E)-3-(4-hydroxyphenyl)-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)acrylamide (9d) Compound 9d was prepared according to the general procedure I, light yellow oil, yield 66%. 1H NMR (400 MHz, DMSO) δ 9.94 (s, 1H, -OH), 8.39 (d, J = 8.6 Hz, 1H), 8.10 - 8.08 (m, 1H, -CONH), 7.98 (d, J = 8.2 Hz, 1H), 7.83 - 7.78 (m, 2H), 7.56 7.52 (m, 1H), 7.34 (d, J = 8.6 Hz, 2H), 7.28 (d, J = 15.7 Hz, 1H), 6.79 (d, J = 8.6 Hz, 2H), 6.45 (d, J = 15.7 Hz, 1H), 3.84 - 3.79
ACCEPTED MANUSCRIPT (m, 2H), 3.14 - 3.12 (m, 2H), 3.00 (br, 2H), 2.65 (br, 2H), 1.80 (br, 4H), 1.73 - 1.70 (m, 2H), 1.45 - 1.42 (m, 2H), 1.33 - 1.32 (m, 4H). 13C NMR (101 MHz, DMSO) δ 165.28, 158.80, 155.41, 150.64, 138.28, 138.00, 132.29, 128.96, 128.96, 125.84, 124.92, 124.87, 119.24, 118.85, 115.69, 115.69, 115.56, 111.08, 47.04, 38.37, 29.77, 29.02, 27.95, 25.97, 25.70, 23.94, 21.43, 20.25. HRMS (ESI) m/z 444.2652 [M+H]+, calcd. for [C28H34N3O2]+, 444.2651. (E)-3-(4-hydroxyphenyl)-N-(8-((1,2,3,4-tetrahydroacridin-9-yl)amino)octyl)acrylamide (9e) Compound 9e was prepared according to the general procedure I, light yellow oil, yield 70%. 1H NMR (500 MHz, CD3OD) δ 8.29 (d, J = 8.7 Hz, 1H), 7.77 - 7.75 (m, 1H), 7.71 - 7.70 (m, 1H), 7.52 - 7.49 (m, 1H), 7.33 (d, J = 15.7 Hz, 1H), 7.27 (d, J = 8.7
RI PT
Hz, 2H), 6.68 (d, J = 8.6 Hz, 2H), 6.35 (d, J = 15.7 Hz, 1H), 3.87 - 3.84 (m, 2H), 3.23 - 3.20 (m, 2H), 2.94 - 2.92 (m, 2H), 2.61 2.59 (m, 2H), 1.90 - 1.86 (m, 4H), 1.80 - 1.74 (m, 2H), 1.51 - 1.49 (m, 2H), 1.39 - 1.31 (m, 8H). 13C NMR (101 MHz, DMSO) δ 165.22, 158.79, 155.52, 150.65, 138.28, 137.98, 132.39, 128.98, 128.98, 125.86, 124.95, 124.90, 119.22, 118.86, 115.69, 115.69, 115.57, 111.10, 47.16, 38.52, 30.72, 29.73, 29.11, 28.50, 27.93, 26.28, 25.91, 23.92, 21.42, 20.26. HRMS (ESI) m/z 472.2968 [M+H]+, calcd. for [C30H38N3O2]+, 472.2964. Due to rapid proton exchange with MeOH in CD3OD, the proton NH and phenol
SC
OH missed in the 1H NMR spectrum.
(E)-3-(4-hydroxy-3,5-dimethoxyphenyl)-N-(2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)acrylamide (9f) Compound 9f was prepared according to the general procedure I, white solid, yield 66%, mp > 210 °C. 1H NMR (500 MHz, DMSO) δ 8.86 (s, 1H, -OH), 8.57 (t, J = 5.9 Hz, 1H, -CONH), 8.50 (d, J = 8.7 Hz, 1H), 8.06 (br, 1H, -NH), 7.95 (dd, J = 8.5 Hz,
M AN U
0.9 Hz, 1H), 7.86 - 7.83 (m, 1H), 7.57 - 7.54 (m, 1H), 7.35 (d, J = 15.7 Hz, 1H), 6.84 (s, 2H), 6.47 (d, J = 15.7 Hz, 1H), 4.03 4.00 (m, 2H), 3.78 (s, 6H), 3.57 - 3.54 (m, 2H), 2.99 - 2.97 (m, 2H), 2.70 - 2.68 (m, 2H), 1.82 - 1.80 (m, 4H). 13C NMR (101 MHz, DMSO) δ 166.89, 155.96, 150.54, 148.04, 148.04, 139.93, 137.86, 137.56, 132.49, 125.18, 125.00, 124.95, 119.02, 118.54, 115.50, 111.23, 105.43, 105.43, 55.98, 55.98, 48.29, 27.83, 23.75, 21.42, 20.21. HRMS (ESI) m/z 448.2240 [M+H]+, calcd. for [C26H30N3O4]+, 448.2236.
(E)-3-(4-hydroxy-3,5-dimethoxyphenyl)-N-(3-((1,2,3,4-tetrahydroacridin-9-yl)amino)propyl)acrylamide (9g) Compound 9g was prepared according to the general procedure I, light yellow solid, yield 59%, mp > 210 °C. 1H NMR (400
TE D
MHz, DMSO) δ 8.82 (br, 1H, -OH), 8.15 (d, J = 8.3 Hz, 1H), 8.05 - 8.02 (m, 1H, -CONH), 7.72 - 7.70 (m, 1H), 7.53 - 7.50 (m, 1H), 7.36 - 7.34 (m, 1H), 7.35 (d, J = 15.7 Hz, 1H), 6.85 (s, 2H), 6.46 (d, J = 15.7 Hz, 1H), 5.57 (t, J = 6.3 Hz, 1H, -NH), 3.79 (s, 6H), 3.46 -3.41 (m, 2H), 3.26 - 3.21 (m, 2H), 2.92 - 2.89 (m, 2H), 2.75 - 2.73 (m, 2H), 1.82 - 1.80 (m, 4H), 1.76 - 1.69 (m, 2H). 13
C NMR (101 MHz, DMSO) δ 165.62, 157.93, 150.09, 148.05, 148.05, 146.85, 139.27, 137.33, 128.28, 127.81, 125.24, 123.27,
122.83, 120.28, 119.24, 115.99, 105.27, 105.27, 55.94, 55.94, 45.15, 36.16, 33.47, 30.77, 25.11, 22.73, 22.39. HRMS (ESI) m/z
EP
462.2397 [M+H]+, calcd. for [C27H32N3O4]+, 462.2393.
(E)-3-(4-hydroxy-3,5-dimethoxyphenyl)-N-(4-((1,2,3,4-tetrahydroacridin-9-yl)amino)butyl)acrylamide (9h) Compound 9h was prepared according to the general procedure I, light yellow oil, yield 66%. 1H NMR (400 MHz, DMSO) δ
AC C
8.79 (br, 1H, -OH), 8.13 (d, J = 8.4 Hz, 1H), 7.94 - 7.91 (m, 1H, -CONH), 7.70 (d, J = 8.4 Hz, 1H), 7.54 - 7.50 (m, 1H), 7.36 7.34 (m, 1H), 7.30 (d, J = 15.8 Hz, 1H), 6.83 (s, 2H), 6.44 (d, J = 15.7 Hz, 1H), 5.49 (br, 1H, -NH), 3.78 (s, 6H), 3.44 - 3.40 (m, 2H), 3.17 - 3.13 (m, 2H), 2.91 - 2.89 (m, 2H), 2.73 - 2.70 (m, 2H), 1.81- 1.76 (m, 4H), 1.61 - 1.56 (m, 2H), 1.51 - 1.46 (m, 2H). 13
C NMR (101 MHz, DMSO) δ 165.19, 157.63, 150.42, 148.05, 148.05, 146.52, 139.01, 137.26, 127.98, 127.89, 125.30, 123.26,
123.05, 120.08, 119.45, 115.70, 105.21, 105.21, 55.94, 55.94, 47.65, 38.36, 33.26, 28.09, 26.59, 25.00, 22.67, 22.31. HRMS (ESI) m/z 476.2549 [M+H]+, calcd. for [C28H34N3O4]+, 476.2549. (E)-3-(4-hydroxy-3,5-dimethoxyphenyl)-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)acrylamide (9i) Compound 9i was prepared according to the general procedure I, light yellow oil, yield 64%. 1H NMR (400 MHz, DMSO) δ 8.74 (br, 1H, -OH), 8.17 (d, J = 8.4 Hz, 1H), 7.92 - 7.89 (m, 1H, -CONH), 7.75 - 7.73 (m, 1H), 7.60 - 7.56 (m, 1H), 7.40- 7.36 (m, 1H), 7.30 (d, J = 15.6 Hz, 1H), 6.83 (s, 2H), 6.46 (d, J = 15.7 Hz, 1H), 5.90 (br, 1H, -NH), 3.78 (s, 6H), 3.52 - 3.47 (m, 2H), 3.15 - 3.10 (m, 2H), 2.91 - 2.90 (m, 2H), 2.71 - 2.68 (m, 2H), 1.81 - 1.80 (m, 4H), 1.61 - 1.57 (m, 2H), 1.43 - 1.40 (m, 2H), 1.30 - 1.29 (m, 4H). 13C NMR (101 MHz, DMSO) δ 165.13, 156.24, 151.46, 148.03, 148.03, 145.08, 138.91, 137.23, 128.83, 126.22, 125.31, 123.54, 123.43, 119.53, 119.19, 114.78, 105.19, 105.19, 55.93, 55.93, 47.72, 38.45, 32.23, 30.37, 29.08, 26.12, 25.95, 24.76,
ACCEPTED MANUSCRIPT 22.41, 21.93. HRMS (ESI) m/z 504.2867 [M+H]+, calcd. for [C30H38N3O4]+, 504.2862. (E)-3-(4-hydroxy-3,5-dimethoxyphenyl)-N-(8-((1,2,3,4-tetrahydroacridin-9-yl)amino)octyl)acrylamide (9j) Compound 9j was prepared according to the general procedure I, light yellow oil, yield 60%. 1H NMR (400 MHz, DMSO) δ 8.75 (br, 1H, -OH), 8.27 (d, J = 8.6 Hz, 1H), 7.97 - 7.94 (m, 1H, -CONH), 7.84 (d, J = 8.4 Hz, 1H), 7.71 - 7.67 (m, 1H), 7.47 - 7.43 (m, 1H), 7.30 (d, J = 15.6 Hz, 1H), 6.83 (s, 2H), 6.75 (br, 1H, -NH), 6.49 (d, J = 15.7 Hz, 1H), 3.78 (s, 6H), 3.66 - 3.61 (m, 2H), 3.15 - 3.10 (m, 2H), 2.95 (br, 2H), 2.67 (br, 2H), 1.81 (br, 4H), 1.66 - 1.61 (m, 2H), 1.40 - 1.39 (m, 2H), 1.29 - 1.24 (m, 8H). 13C NMR (101 MHz, DMSO) δ 165.15, 153.66, 153.35, 148.04, 148.04, 141.72, 138.88, 137.24, 130.47, 125.34, 124.15, 123.00,
21.96, 21.18. HRMS (ESI) m/z 532.3180 [M+H]+, calcd. for [C32H42N3O4]+, 532.3175. 4.4. General procedure II for the preparation of compounds 10a−10j
RI PT
123.00, 119.59, 117.52, 113.07, 105.20, 105.20, 55.95, 55.95, 47.50, 38.56, 30.27, 30.09, 29.11, 28.56, 28.56, 26.31, 26.06, 24.37,
To a solution of 9a−9j (0.516 mmol) in anhydrous DMF (10 mL) was added K2CO3 (0.774 mmol) and TMP-Br (0.619 mmol). The reaction mixture was stirred for 6 h at 60°C. It was filtered, concentrated and redissolved in CH2Cl2 (30 mL).Then it
SC
was washed with water (2 × 30 mL) and brine (30 mL), dried over sodium sulfate, concentrated under vacuum and purified by silica gel chromatography (CH2Cl2: MeOH=30:1) to afford corresponding target compound 10a−10j.
(E)-N-(2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)-3-(4-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)acrylamide (10a)
M AN U
Compound 10a was prepared according to the general procedure II, light yellow solid, yield 79%, mp > 210 °C. 1H NMR (400 MHz, DMSO) δ 8.66 - 8.63 (m, 1H, -CONH), 8.51 (d, J = 8.7 Hz, 1H), 8.02 (br, 1H, -NH), 7.96 - 7.94 (m, 1H), 7.87 - 7.83 (m, 1H), 7.59 - 7.55 (m, 1H), 7.51 (d, J = 8.8 Hz, 2H), 7.41 (d, J = 15.8 Hz, 1H), 7.07 (d, J = 8.8 Hz, 2H), 6.50 (d, J = 15.8 Hz, 1H), 5.18 (s, 2H), 4.03 - 4.00 (m, 2H), 3.59 - 3.54 (m, 2H), 2.99 (br, 2H), 2.70 (br, 2H), 2.48 (s, 3H), 2.45 (s, 3H), 2.44 (s, 3H), 1.83 (br, 4H). 13C NMR (101 MHz, DMSO) δ 166.72, 159.48, 155.92, 150.98, 150.59, 149.24, 148.23, 145.08, 138.85, 137.91, 132.50, 129.13, 129.13, 127.65, 125.19, 124.99, 119.19, 119.10, 115.54, 115.15, 115.15, 111.31, 69.38, 48.17, 27.90, 23.79, 21.44, 21.20, 20.91, 20.25, 20.10. HRMS (ESI) m/z 522.2871 [M+H]+, calcd. for [C32H36N5O2]+, 522.2869.
TE D
(E)-N-(3-((1,2,3,4-tetrahydroacridin-9-yl)amino)propyl)-3-(4-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)acrylamide (10b)
Compound 10b was prepared according to the general procedure II, light yellow oil, yield 79%. 1H NMR (400 MHz, DMSO) δ 8.41 - 8.38 (m, 1H), 8.37 (br, 1H, -CONH), 7.95 - 7.92 (m, 1H), 7.92 - 7.90 (m, 1H), 7.78 - 7.74 (m, 1H), 7.54 - 7.52 (m, 1H, -NH), 7.50 - 7.48 (m, 2H), 7.36 (d, J = 15.8 Hz, 1H), 7.06 (d, J = 8.7 Hz, 2H), 6.51 (d, J = 15.8 Hz, 1H), 5.18 (s, 2H), 3.85 - 3.80
EP
(m, 2H), 3.29 - 3.24 (m, 2H), 2.97 (br, 2H), 2.70 (br, 2H), 2.48 (s, 3H), 2.44 (br, 6H), 1.90 - 1.87 (m, 2H), 1.80 (br, 4H). 13C NMR (101 MHz, DMSO) δ 165.59, 159.34, 154.60, 151.82, 150.97, 149.24, 148.22, 145.10, 139.40, 138.14, 131.58, 128.98, 128.98, 127.82, 124.65, 124.53, 120.72, 119.80, 116.34, 115.12, 115.12, 111.96, 69.38, 44.68, 35.86, 30.21, 28.85, 24.16, 21.65, 21.18,
AC C
20.90, 20.58, 20.09. HRMS (ESI) m/z 536.3030 [M+H]+, calcd. for [C33H38N5O2]+, 536.3026. (E)-N-(4-((1,2,3,4-tetrahydroacridin-9-yl)amino)butyl)-3-(4-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)acrylamide (10c)
Compound 10c was prepared according to the general procedure II, light yellow oil, yield 82%. 1H NMR (400 MHz, DMSO) δ 8.41 (d, J = 15.7 Hz, 1H), 8.14 - 8.11 (m, 1H, -CONH), 7.88 - 7.86 (m, 1H), 7.86 - 7.83 (m, 1H), 7.81 - 7.79 (m, 1H, -NH), 7.58 7.54 (m, 1H), 7.47 (d, J = 8.7 Hz, 2H), 7.33 (d, J = 15.7 Hz, 1H), 7.05 (d, J = 15.7 Hz, 2H), 6.51 (d, J = 15.8 Hz, 1H), 5.17 (s, 2H), 3.91 - 3.86 (m, 2H), 3.22 - 3.17 (m, 2H), 2.98 (br, 2H), 2.66 (br, 2H), 2.47 (s, 3H), 2.43 (br, 6H), 1.81 (br, 4H), 1.77 - 1.74 (m, 2H), 1.58 - 1.51 (m, 2H). 13C NMR (101 MHz, DMSO) δ 165.16, 159.28, 155.67, 150.96, 150.45, 149.24, 148.23, 145.12, 137.88, 137.69, 132.60, 128.92, 128.92, 127.89, 125.04, 125.04, 120.07, 118.92, 115.44, 115.11, 115.11, 111.12, 69.39, 45.62, 35.63, 27.86, 27.36, 26.24, 23.92, 21.37, 21.18, 20.90, 20.19, 20.09. HRMS (ESI) m/z 550.3190 [M+H]+, calcd. for [C34H40N5O2]+, 550.3182. (E)-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)-3-(4-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)acrylamide (10d)
ACCEPTED MANUSCRIPT Compound 10d was prepared according to the general procedure II, light yellow oil, yield 81%. 1H NMR (400 MHz, DMSO) δ 8.40 (d, J = 8.6 Hz, 1H), 8.08 - 8.06 (m, 1H, -CONH), 7.94 (d, J = 8.3 Hz, 1H), 7.85 - 7.81 (m, 1H), 7.75 (br, 1H, -NH), 7.58 7.54 (m, 1H), 7.48 (d, J = 8.7 Hz, 2H), 7.33 (d, J = 15.7 Hz, 1H), 7.06 (d, J = 8.7 Hz, 2H), 6.50 (d, J = 15.8 Hz, 1H), 5.18 (s, 2H), 3.87 - 3.82 (m, 2H), 3.16 - 3.12 (m, 2H), 3.00 (br, 2H), 2.66 (br, 2H), 2.48 (s, 3H), 2.45 (s, 3H), 2.44 (s, 3H), 1.82 (br, 4H), 1.74 1.71 (m, 2H), 1.46 - 1.43 (m, 2H), 1.34 (br, 4H). 13C NMR (101 MHz, DMSO) δ 165.04, 159.26, 155.50, 150.98, 150.67, 149.25, 148.23, 145.13, 138.02, 137.80, 132.41, 128.91, 128.91, 127.94, 124.94, 124.94, 120.20, 119.26, 115.58, 115.12, 115.12, 111.16, 69.38, 47.11, 38.40, 29.76, 28.99, 27.98, 25.97, 25.70, 23.90, 21.42, 21.19, 20.90, 20.27, 20.10. HRMS (ESI) m/z 578.3500
RI PT
[M+H]+, calcd. for [C36H44N5O2]+, 578.3495.
(E)-N-(8-((1,2,3,4-tetrahydroacridin-9-yl)amino)octyl)-3-(4-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)acrylamide (10e)
Compound 10e was prepared according to the general procedure II, light yellow oil, yield 85%. 1H NMR (400 MHz, DMSO) δ 8.33 (d, J = 8.5 Hz, 1H), 8.07 - 8.05 (m, 1H, -CONH), 7.90 (d, J = 8.1 Hz, 1H), 7.78 - 7.74 (m, 1H), 7.52 - 7.51 (m, 1H), 7.48 (d,
SC
J = 8.7 Hz, 2H), 7.33 (d, J = 15.7 Hz, 1H), 7.29 (br, 1H, -NH), 7.05 (d, J = 8.7 Hz, 2H), 6.51 (d, J = 15.8 Hz, 1H), 5.17 (s, 2H), 3.76 - 3.72 (m, 2H), 3.15 - 3.10 (m, 2H), 2.98 (br, 2H), 2.66 (br, 2H), 2.48 (s, 3H), 2.44 (s, 3H), 2.44 (s, 3H), 1.81 (br, 4H), 1.70 1.64 (m, 2H), 1.41 - 1.40 (m, 2H), 1.31 - 1.24 (m, 8H). 13C NMR (101 MHz, DMSO) δ 165.03, 159.25, 154.53, 152.09, 150.98, 149.26, 148.24, 145.13, 139.61, 137.79, 131.52, 128.91, 128.91, 127.97, 124.57, 120.98, 120.96, 120.23, 116.46, 115.11, 115.11,
M AN U
112.01, 69.39, 47.33, 38.57, 29.90, 29.10, 29.02, 28.53, 28.49, 26.30, 25.98, 24.12, 21.67, 21.19, 20.90, 20.69, 20.10. HRMS (ESI) m/z 606.3813 [M+H]+, calcd. for [C38H48N5O2]+, 606.3808.
(E)-3-(3,5-dimethoxy-4-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-N-(2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl) acrylamide (10f)
Compound 10f was prepared according to the general procedure II, light yellow solid, yield 74% mp 203.8-204.7 °C. 1H NMR (400 MHz, DMSO) δ 8.58 - 8.55 (m, 1H, -CONH), 8.51 (d, J = 8.7 Hz, 1H), 8.01 (br, 1H, -NH), 7.92 - 7.85 (m, 2H), 7.61 - 7.56 (m, 1H), 7.40 (d, J = 15.7 Hz, 1H), 6.89 (s, 2H), 6.59 (d, J = 15.7 Hz, 1H), 4.99 (s, 2H), 4.04 - 4.03 (m, 2H), 3.77 (br, 6H), 3.59-
TE D
3.58 (m, 2H), 2.99 (br, 2H), 2.74 - 2.71 (m, 2H), 2.59 (s, 3H), 2.44 (s, 3H), 2.40 (s, 3H), 1.85 (br, 4H). 13C NMR (101 MHz, DMSO) δ 166.55, 156.03, 153.29, 153.29, 150.60, 150.04, 150.04, 147.68, 145.83, 139.39, 137.90, 137.21, 132.67, 130.65, 125.26, 125.05, 120.92, 119.08, 115.51, 111.35, 104.96, 104.96, 73.53, 55.87, 55.87, 48.23, 27.91, 23.76, 21.43, 21.20, 20.83, 20.28, 20.02. HRMS (ESI) m/z 582.3078 [M+H]+, calcd. for [C34H40N5O4]+, 582.3080. (E)-3-(3,5-dimethoxy-4-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-N-(3-((1,2,3,4-tetrahydroacridin-9-yl)amino)propyl
EP
)acrylamide (10g)
Compound 10g was prepared according to the general procedure II, light yellow oil, yield 80%. 1H NMR (400 MHz, DMSO) δ 8.35 (d, J = 8.6 Hz, 1H), 8.29 (t, J = 5.0 Hz, 1H, -CONH), 7.86 (d, J =8.1 Hz, 1H), 7.77 - 7.73 (m, 1H), 7.52 - 7.48 (m, 1H), 7.36
AC C
(d, J = 15.7 Hz, 1H), 7.21 (br, 1H, -NH), 6.88 (s, 2H), 6.56 (d, J = 15.8 Hz, 1H), 4.99 (s, 2H), 3.79 - 3.77 (m, 6H), 3.33 - 3.30 (m, 2H), 3.29 - 3.26 (m, 2H), 2.97 (s, 2H), 2.74 - 2.72 (m, 2H), 2.59 (s, 3H), 2.44 (s, 3H), 2.40 (s, 3H), 1.91 - 1.88 (m, 2H), 1.86 1.83 (m, 4H). 13C NMR (101 MHz, DMSO) δ 165.39, 153.28, 153.28, 153.28, 150.58, 150.04, 147.67, 145.85, 138.71, 138.71, 137.06, 131.13, 130.84, 124.51, 124.36, 121.50, 121.50, 118.86, 116.98, 112.64, 104.84, 104.84, 73.52, 55.85, 55.85, 44.84, 35.98, 30.25, 29.59, 24.28, 21.82, 21.20, 20.89, 20.83, 20.02. HRMS (ESI) m/z 596.3235 [M+H]+, calcd. for [C35H42N5O4]+, 596.3237.
(E)-3-(3,5-dimethoxy-4-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-N-(4-((1,2,3,4-tetrahydroacridin-9-yl)amino)butyl) acrylamide (10h) Compound 10h was prepared according to the general procedure II, light yellow oil, yield 81%. 1H NMR (400 MHz, DMSO) δ 8.32 (d, J = 8.6 Hz, 1H), 8.16 (br, 1H, -CONH), 7.87 (d, J = 8.5 Hz, 1H), 7.73 - 7.69 (m, 1H), 7.49 - 7.46 (m, 1H), 7.34 (d, J =15.7 Hz, 1H), 7.01 (br, 1H, -NH), 6.86 (s, 2H), 6.61 (d, J = 15.7 Hz, 1H), 4.97 (s, 2H), 3.75 (br, 6H), 3.73 - 3.71 (m, 2H), 3.21 3.17 (m, 2H), 2.96 (br, 2H), 2.68 (br, 2H), 2.58 (s, 3H), 2.43 (s, 3H), 2.39 (s, 3H), 1.81 (br, 4H), 1.75 - 1.68 (m, 2H), 1.56 - 1.49 (m, 2H). 13C NMR (101 MHz, DMSO) δ 164.97, 153.78, 153.29, 153.29, 153.11, 150.60, 150.07, 147.69, 145.88, 141.00, 138.41,
ACCEPTED MANUSCRIPT 136.98, 130.97, 130.89, 124.41, 124.34, 122.23, 121.86, 117.15, 112.72, 104.78, 104.78, 73.53, 55.85, 55.85, 47.14, 38.29, 29.82, 27.65, 26.34, 24.37, 21.90, 21.21, 20.99, 20.85, 20.04. HRMS (ESI) m/z 610.3389 [M+H]+, calcd. for [C36H44N5O4]+, 610.3393. (E)-3-(3,5-dimethoxy-4-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl) acrylamide (10i) Compound 10i was prepared according to the general procedure II, light yellow oil, yield 75%. 1H NMR (400 MHz, DMSO) δ 8.23 (d, J = 8.5 Hz, 1H), 8.03 (t, J = 5.5 Hz, 1H, -CONH), 7.78 (d, J = 8.2 Hz, 1H), 7.66 - 7.62 (m, 1H), 7.44 - 7.41 (m, 1H), 7.33 (d, J = 15.7 Hz, 1H), 6.87 (s, 2H), 6.58 (d, J = 15.7 Hz, 1H), 6.38 (br, 1H, -NH), 4.98 (s, 2H), 3.80 - 3.75 (m, 6H), 3.59 - 3.57 (m,
RI PT
2H), 3.17 - 3.12 (m, 2H), 2.94 - 2.92 (m, 2H) 2.70 - 2.68 (m, 2H), 2.59 (s, 3H), 2.43 (s, 3H), 2.39 (s, 3H), 1.81 (br, 4H), 1.64 1.61 (m, 2H), 1.44 - 1.41 (m, 2H), 1.31 (br, 4H). 13C NMR (101 MHz, DMSO) δ 164.83, 154.83, 153.27, 153.27, 152.51, 150.57, 150.05, 147.67, 145.86, 138.34, 138.34, 136.95, 130.97, 129.76, 124.50, 123.91, 123.84, 121.90, 118.28, 113.87, 104.76, 104.76, 73.52, 55.83, 55.83, 47.58, 38.50, 31.18, 30.24, 29.03, 26.10, 25.91, 24.58, 22.18, 21.53, 21.19, 20.83, 20.02. HRMS (ESI) m/z 638.3705 [M+H]+, calcd. for [C38H48N5O4]+, 638.3706.
SC
(E)-3-(3,5-dimethoxy-4-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-N-(8-((1,2,3,4-tetrahydroacridin-9-yl)amino)octyl) acrylamide (10j)
Compound 10j was prepared according to the general procedure II, light yellow oil, yield 83%. 1H NMR (400 MHz, DMSO) δ 8.27 (d, J = 8.6 Hz, 1H), 8.01 (t, J = 5.4 Hz, 1H, -CONH), 7.81 (d, J = 8.4 Hz, 1H), 7.73 - 7.69 (m, 1H), 7.49 - 7.45 (m, 1H), 7.34
M AN U
(d, J = 15.7 Hz, 1H), 6.87 (s, 2H), 6.79 (br, 1H, -NH), 6.58 (d, J = 15.7 Hz, 1H), 4.98 (s, 2H), 3.75 (br, 6H), 3.66 - 3.65 (m, 2H), 3.14 - 3.11 (m, 2H), 2.95 (br, 2H), 2.67 (br, 2H), 2.59 (s, 3H), 2.44 (s, 3H), 2.40 (s, 3H), 1.82 (br, 4H), 1.67 - 1.63 (m, 2H), 1.41 (br, 2H), 1.25 (br, 8H). 13C NMR (101 MHz, DMSO) δ 164.81, 153.48, 153.27, 153.27, 153.27, 150.58, 150.05, 147.68, 145.87, 138.34, 138.34, 136.95, 130.98, 124.22, 124.22, 122.83, 121.92, 117.44, 113.00, 109.64, 104.76, 104.76, 73.52, 55.83, 55.83, 47.51, 38.61, 30.77, 30.08, 29.07, 28.58, 28.58, 26.31, 26.07, 24.36, 21.94, 21.20, 21.16, 20.84, 20.02. HRMS (ESI) m/z 666.4018 [M+H]+, calcd. for [C40H52N5O4]+, 666.4019.
4.5. General procedure III for the preparation of compounds 10k−10o, 11a−11e and 12a−12e
TE D
To a stirred solution of 7a−7c (0.428 mmol), EDCI (0.514 mmol), HOBT (0.428 mmol) in anhydrous CH2Cl2 (10 mL) was added intermediate 3a−3e, the reaction mixture was stirred at room temperature for 12 h. It was diluted with CH2Cl2 (20 mL), washed successively with water (2 × 30 mL) and brine (30 mL), dried over sodium sulfate, concentrated under vacuum and purified by silica gel chromatography (CHCl3: MeOH = 20:1 ) to give compound 10k−10o, 11a−11e and 12a−12e. (E)-3-(3-methoxy-4-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-N-(2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)acry
EP
lamide (10k)
Compound 10k was prepared according to the general procedure III, white solid, yield 56%, mp > 210 °C. 1H NMR (500 MHz, DMSO+CD3OD) δ 8.43 (d, J = 8.7 Hz, 1H), 7.81 - 7.78 (m, 1H), 7.75 - 7.73 (m, 1H), 7.55 - 7.51 (m, 1H), 7.38 (d, J = 15.7 Hz,
AC C
1H), 7.11 (d, J = 1.5 Hz, 1H), 7.09 - 7.07 (m, 1H), 7.07 - 7.06 (m, 1H), 6.43 (d, J = 15.7 Hz, 1H), 5.12 (s, 2H), 4.05 - 4.03 (m, 2H), 3.75 (s, 3H), 3.60 - 3.57 (m, 2H), 2.93 - 2.90 (m, 2H), 2.68 - 2.66 (m, 2H), 2.47 (s, 3H), 2.42 (s, 3H), 2.42 (s, 3H), 1.85 1.82 (m, 4H). 13C NMR (101 MHz, DMSO+MeOD) δ 168.43, 157.15, 151.89, 151.25, 150.46, 150.41, 150.25, 149.15, 146.14, 140.75, 138.91, 133.27, 129.05, 125.91, 125.69, 122.15, 119.74, 119.46, 116.42, 114.58, 112.34, 111.28, 70.98, 56.01, 49.66, 46.78, 28.70, 24.40, 22.22, 21.32, 21.04, 21.04, 20.29. HRMS (ESI) m/z 552.2971 [M+H]+, calcd. for [C33H38N5O3]+, 552.2975. Due to rapid proton exchange with MeOH in CD3OD, the proton NH missed in the 1H NMR spectrum. (E)-3-(3-methoxy-4-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-N-(3-((1,2,3,4-tetrahydroacridin-9-yl)amino)propyl)ac rylamide (10l) Compound 10l was prepared according to the general procedure III, light yellow solid, yield 58%, mp 150.7-151.4 °C. 1H NMR (500 MHz, DMSO) δ 8.42 (d, J = 8.6 Hz, 1H), 8.38 - 8.36 (m, 1H, -CONH), 7.94 - 7.93 (m, 2H), 7.85 - 7.82 (m, 1H), 7.56 - 7.53 (m, 1H), 7.34 (d, J = 15.7 Hz, 1H), 7.16 (d, J = 1.7 Hz, 1H), 7.14 (d, J = 8.4 Hz, 1H), 7.10 (dd, J = 8.4 Hz, 1.7 Hz, 1H), 6.53 (d, J = 15.8 Hz, 1H), 5.15 (s, 2H), 3.91 - 3.89 (m, 2H), 3.77 (s, 3H), 3.29 - 3.26 (m, 2H), 2.99 (br, 2H), 2.69 (br, 2H), 2.48 (s, 3H), 2.45 (s, 3H), 2.44 (s, 3H), 1.94 - 1.89 (m, 2H), 1.81 (br, 4H). 13C NMR (101 MHz, DMSO) δ 165.61, 155.62, 150.95, 150.58,
ACCEPTED MANUSCRIPT 149.44, 149.29, 149.00, 148.15, 145.19, 138.51, 137.88, 132.41, 128.32, 124.95, 124.89, 121.02, 120.03, 119.15, 115.53, 113.79, 111.18, 110.52, 70.11, 55.53, 44.64, 35.81, 30.09, 27.89, 23.90, 21.41, 21.16, 20.86, 20.20, 20.05. HRMS (ESI) m/z 566.3128 [M+H]+, calcd. for [C34H40N5O3]+, 566.3131. (E)-3-(3-methoxy-4-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-N-(4-((1,2,3,4-tetrahydroacridin-9-yl)amino)butyl)acr ylamide (10m) Compound 10m was prepared according to the general procedure III, light yellow oil, yield 57%. 1H NMR (500 MHz, DMSO) δ 8.42 (d, J = 8.9 Hz, 1H), 8.16 - 8.13 (m, 1H, -CONH), 7.95 - 7.91 (m, 1H), 7.85 - 7.82 (m, 2H), 7.57 - 7.54 (m, 1H), 7.32 (d, J =
RI PT
15.7 Hz, 1H), 7.14 (d, J = 1.7 Hz, 1H), 7.13 (d, J = 8.6 Hz, 1H), 7.09 - 7.07 (m, 1H), 6.54 - 6.49 (m, 1H), 5.15 (s, 2H), 3.89 3.85 (m, 2H), 3.77 (s, 3H), 3.21 - 3.17 (m, 2H), 2.99 (br, 2H), 2.66 (br, 2H), 2.48 (s, 3H), 2.45 (s, 3H), 2.44 (s, 3H), 1.82 (br, 4H), 1.79 - 1.75 (m, 2H), 1.56 - 1.50 (m, 2H). 13C NMR (101 MHz, DMSO) δ 165.11, 155.54, 150.95, 150.67, 149.45, 149.29, 148.92, 148.15, 145.20, 138.19, 137.90, 132.39, 128.42, 124.95, 124.95, 120.98, 120.38, 119.17, 115.58, 113.78, 111.14, 110.40, 70.11, 55.51, 46.85, 38.11, 27.92, 27.36, 26.21, 23.94, 21.41, 21.17, 20.86, 20.21, 20.05. HRMS (ESI) m/z 580.3285 [M+H]+, calcd. for
SC
[C35H42N5O3]+, 580.3288.
(E)-3-(3-methoxy-4-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)acr ylamide (10n)
Compound 10n was prepared according to the general procedure III, light yellow oil, yield 55%. 1H NMR (500 MHz, DMSO) δ
M AN U
8.40 (d, J = 8.7 Hz, 1H), 8.08 - 8.06 (m, 1H, -CONH), 7.94 - 7.93 (m, 1H), 7.85 - 7.82 (m, 1H), 7.78 (br, 1H, -NH), 7.58 - 7.54 (m, 1H), 7.32 (d, J = 15.7 Hz, 1H), 7.15 (d, J = 1.7 Hz, 1H), 7.12 (d, J = 8.4 Hz, 1H), 7.07 (dd, J = 8.4 Hz, 1.7 Hz, 1H), 6.53 (d, J = 15.7 Hz, 1H), 5.14 (s, 2H), 3.86 - 3.82 (m, 2H), 3.76 (s, 3H), 3.16 - 3.13 (m, 2H), 2.99 (br, 2H), 2.65 (br, 2H), 2.48 (s, 3H), 2.45 (s, 3H), 2.44 (s, 3H), 1.82 (br, 4H), 1.75 - 1.69 (m, 2H), 1.46 - 1.41 (m, 2H), 1.36 - 1.30 (m, 4H). 13C NMR (101 MHz, DMSO) δ 165.01, 155.52, 150.94, 150.64, 149.44, 149.29, 148.89, 148.14, 145.20, 138.10, 137.95, 132.41, 128.47, 124.93, 124.93, 120.97, 120.49, 119.21, 115.55, 113.79, 111.13, 110.39, 70.11, 55.50, 47.10, 38.39, 29.74, 28.96, 27.94, 25.94, 25.68, 23.87, 21.39, 21.16, 20.86, 20.24, 20.05. HRMS (ESI) m/z 608.3598 [M+H]+, calcd. for [C37H46N5O3]+, 608.3601.
TE D
(E)-3-(3-methoxy-4-((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-N-(8-((1,2,3,4-tetrahydroacridin-9-yl)amino)octyl)acry lamide (10o)
Compound 10o was prepared according to the general procedure III, light yellow oil, yield 53%. 1H NMR (500 MHz, DMSO) δ 8.11 (d, J = 8.5 Hz, 1H), 7.98 - 7.96 (m, 1H, -CONH), 7.70 - 7.69 (m, 1H), 7.53 - 7.50 (m, 1H), 7.34 - 7.33 (m, 1H), 7.33 (d, J = 15.7 Hz, 1H), 7.16 (d, J = 1.7 Hz, 1H), 7.12 (d, J = 8.4 Hz, 1H), 7.08 (dd, J = 8.4 Hz, 1.7 Hz, 1H), 6.50 (d, J = 15.7 Hz, 1H), 5.53
EP
(br, 1H, -NH), 5.15 (s, 2H), 3.77 (s, 3H), 3.42 - 3.38 (m, 2H), 3.14 - 3.10 (m, 2H), 2.91 - 2.88 (m, 2H), 2.70 - 2.68 (m, 2H), 2.48 (s, 3H), 2.45 (s, 3H), 2.44 (s, 3H), 1.82 - 1.78 (m, 4H), 1.57 - 1.51 (m, 2H), 1.41 - 1.38 (m, 2H), 1.28 - 1.22 (m, 8H). 13C NMR (101 MHz, DMSO) δ 164.94, 157.45, 150.94, 150.54, 149.45, 149.30, 148.88, 148.14, 146.40, 145.21, 138.14, 128.48, 127.97,
AC C
127.75, 123.16, 123.06, 120.98, 120.46, 119.97, 115.52, 113.78, 110.37, 70.11, 55.48, 47.86, 38.55, 33.17, 30.47, 29.05, 28.61, 28.57, 26.27, 26.17, 24.91, 22.61, 22.28, 21.16, 20.85, 20.04. HRMS (ESI) m/z 636.3909 [M+H]+, calcd. for [C39H50N5O3]+, 636.3914.
(E)-3-(3,4-bis((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-N-(2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)acrylamide (11a)
Compound 11a was prepared according to the general procedure III, white solid, yield 72%, mp 112.1-113.0 °C. 1H NMR (500 MHz, DMSO) δ 8.64 - 8.62 (m, 1H, -CONH), 8.51 (d, J = 8.8 Hz, 1H), 8.01 (br, 1H, -NH), 7.94 - 7.93 (m, 1H), 7.87 - 7.84 (m, 1H), 7.58 - 7.55 (m, 1H), 7.41 (m, 2H), 7.16 -7.15 (m, 2H), 6.53 (d, J = 15.8 Hz, 1H), 5.16 (s, 4H), 4.13 - 4.02 (m, 2H), 3.59 3.56 (m, 2H), 2.99 (br, 2H), 2.70 (br, 2H), 2.43 (br, 6H), 2.42 (br, 6H), 2.41 (br, 3H), 2.41 (br, 3H), 1.83 - 1.82 (m, 4H). 13C NMR (101 MHz, DMSO) δ 166.72, 155.89, 150.93, 150.93, 150.68, 149.59, 149.46, 149.40, 148.17, 148.12, 148.09, 145.19, 145.13, 139.10, 138.04, 132.49, 128.14, 125.18, 124.97, 121.92, 119.60, 119.22, 115.59, 114.20, 113.13, 111.37, 70.29, 70.17, 48.25, 45.42, 27.97, 23.77, 21.44, 21.16, 21.16, 20.84, 20.83, 20.28, 19.99, 19.99. HRMS (ESI) m/z 672.3659 [M+H]+, calcd. for [C40H46N7O3]+, 672.3662.
ACCEPTED MANUSCRIPT (E)-3-(3,4-bis((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-N-(3-((1,2,3,4-tetrahydroacridin-9-yl)amino)propyl)acrylami de (11b) Compound 11b was prepared according to the general procedure III, light yellow solid, yield 71%, mp 68.7-69.5 °C. 1H NMR (500 MHz, DMSO) δ 8.43 - 8.41 (m, 1H), 8.37 (br, 1H, -CONH), 7.94 - 7.91 (m, 1H), 7.89 (br, 1H, -NH), 7.84 - 7.81 (m, 1H), 7.56 - 7.53 (m, 1H), 7.38 (d, J = 1.4 Hz, 1H), 7.34 (d, J = 15.7 Hz, 1H), 7.17 - 7.16 (m, 1H), 7.14 - 7.12 (m, 1H) 6.53 (dd, J = 15.8 Hz, 3.3 Hz, 1H), 5.16 (s, 4H), 3.92 - 3.88 (m, 2H), 3.30 - 3.27 (m, 2H), 2.99 (br, 2H), 2.69 (br, 2H), 2.43 (br, 12H), 2.41 (br, 6H), 1.95 - 1.89 (m, 2H), 1.82 (br, 4H). 13C NMR (101 MHz, DMSO) δ 165.60, 155.50, 150.89, 150.89, 150.72, 149.45, 149.45,
RI PT
149.38, 148.18, 148.09, 148.07, 145.20, 145.14, 138.39, 138.06, 132.31, 128.33, 124.92, 124.85, 121.72, 120.20, 119.33, 115.63, 114.23, 113.11, 111.27, 70.32, 70.19, 44.66, 35.90, 30.11, 28.00, 23.94, 21.45, 21.15, 20.83, 20.81, 20.25, 20.08, 19.98, 19.98. HRMS (ESI) m/z 686.3815 [M+H]+, calcd. for [C41H48N7O3]+, 686.3819.
(E)-3-(3,4-15bis((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-N-(4-((1,2,3,4-tetrahydroacridin-9-yl)amino)butyl)acrylam ide (11c)
SC
Compound 11c was prepared according to the general procedure III, light yellow solid, yield 85%, mp 67.5-68.4 °C. 1H NMR (500 MHz, DMSO) δ 8.41 (d, J = 8.6 Hz, 1H), 8.16 - 8.14 (m, 1H, -CONH), 7.92 (d, J = 8.6 Hz, 1H), 7.84 - 7.81 (m, 1H), 7.75 (br, 1H, -NH), 7.57 - 7.54 (m, 1H), 7.36 (d, J = 1.2 Hz, 1H), 7.31 (d, J = 15.7 Hz, 1H), 7.16 - 7.15 (m, 1H), 7.12 -7.11 (m, 1H), 6.52 (d, J = 15.8 Hz, 1H), 5.15 (s, 4H), 3.89 - 3.85 (m, 2H), 3.21 - 3.19 (m, 2H), 2.99 (br, 2H), 2.67 (br, 2H), 2.43 (br, 12H), 2.41
M AN U
(br, 6H), 1.82 (br, 4H), 1.77 - 1.73 (m, 2H), 1.56 - 1.50 (m, 2H). 13C NMR (101 MHz, DMSO) δ 165.09, 155.44, 150.90, 150.90, 150.86, 149.45, 149.38, 149.38, 148.17, 148.09, 148.07, 145.20, 145.15, 138.09, 138.09, 132.32, 128.42, 124.92, 124.92, 121.66, 120.52, 119.40, 115.70, 114.25, 113.03, 111.28, 70.31, 70.19, 46.90, 38.14, 28.07, 27.38, 26.24, 23.95, 21.44, 21.15, 21.15, 20.83, 20.81, 20.28, 19.98, 19.98. HRMS (ESI) m/z 700.3975 [M+H]+, calcd. for [C42H50N7O3]+, 700.3975. (E)-3-(3,4-bis((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)acrylamid e (11d)
Compound 11d was prepared according to the general procedure III, light yellow oil, yield 79%. 1H NMR (500 MHz, DMSO) δ
TE D
8.40 (d, J = 8.7 Hz, 1H), 8.11 - 8.08 (m, 1H, -CONH), 7.93 (d, J = 8.6 Hz, 1H), 7.84 - 7.81 (m, 1H), 7.77 (br, 1H, -NH), 7.57 7.54 (m, 1H), 7.37 (d, J = 1.5 Hz, 1H), 7.32 (d, J = 15.7 Hz, 1H), 7.16 - 7.14 (m, 1H), 7.12 - 7.10 (m, 1H), 6.54 (d, J = 15.8 Hz, 1H), 5.15 (s, 4H), 3.86 - 3.81 (m, 2H), 3.15 - 3.13 (m, 2H), 2.99 (br, 2H), 2.66 (br, 2H), 2.42 (br, 9H), 2.42 (br, 3H), 2.41 (br, 6H), 1.82 (br, 4H), 1.75 - 1.70 (m, 2H), 1.47 - 1.42 (m, 2H), 1.37 - 1.31 (m, 4H). 13C NMR (101 MHz, DMSO) δ 165.01, 155.49, 150.89, 150.89, 150.70, 149.45, 149.38, 149.34, 148.17, 148.09, 148.07, 145.20, 145.15, 138.02, 137.98, 132.38, 128.47, 124.91,
EP
124.91, 121.65, 120.66, 119.28, 115.58, 114.24, 113.00, 111.17, 70.31, 70.19, 47.12, 38.46, 29.76, 28.96, 27.98, 25.94, 25.69, 23.88, 21.41, 21.14, 21.14, 20.82, 20.81, 20.26, 19.97, 19.97. HRMS (ESI) m/z 728.4287 [M+H]+, calcd. for [C44H54N7O3]+, 728.4288.
AC C
(E)-3-(3,4-bis((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-N-(8-((1,2,3,4-tetrahydroacridin-9-yl)amino)octyl)acrylamide (11e)
Compound 11e was prepared according to the general procedure III, light yellow oil, yield 81%. 1H NMR (500 MHz, DMSO) δ 8.21 - 8.20 (m, 1H), 8.02 - 8.00 (m, 1H, -CONH), 7.76 (d, J = 8.5 Hz, 1H), 7.64 - 7.61 (m, 1H), 7.43 - 7.39 (m, 1H), 7.37 (d, J = 1.6 Hz, 1H), 7.32 (d, J = 15.7 Hz, 1H), 7.16 - 7.14 (m, 1H), 7.13 - 7.11 (m, 1H), 6.52 (d, J = 15.6 Hz, 1H), 6.29 (br, 1H, -NH), 5.16 (s, 2H), 5.15 (s, 2H), 3.57 - 3.54 (m, 2H), 3.14 - 3.11 (m, 2H), 2.93 - 2.91 (m, 2H), 2.69 - 2.67 (m, 2H), 2.43 (br, 9H), 2.42 (br, 9H) 1.82 - 1.79 (m, 4H), 1.64 - 1.58 (m, 2H), 1.43 - 1.38 (m, 2H), 1.30 - 1.24 (m, 8H). 13C NMR (101 MHz, DMSO) δ 164.96, 155.35, 152.14, 150.90, 150.90, 149.46, 149.40, 149.34, 148.18, 148.10, 148.08, 145.22, 145.17, 143.78, 138.02, 138.02, 129.40, 128.48, 125.10, 123.72, 123.65, 121.68, 120.63, 118.59, 114.23, 112.97, 70.29, 70.19, 47.68, 38.62, 31.53, 30.25, 29.06, 28.57, 28.57, 26.28, 26.10, 24.59, 22.24, 21.66, 21.15, 21.15, 20.83, 20.81, 19.98, 19.98. HRMS (ESI) m/z 756.4598 [M+H]+, calcd. for [C46H58N7O3]+, 756.4601. 3-(3,4-bis((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-2-hydroxy-N-(2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)pro panamide (12a)
ACCEPTED MANUSCRIPT Compound 12a was prepared according to the general procedure III, light yellow oil, yield 82%. 1H NMR (500 MHz, DMSO) δ 8.44 (d, J = 8.8 Hz, 1H), 8.35 (t, J = 5.9 Hz, 1H, -CONH), 7.93 - 7.91 (m, 1H), 7.84 - 7.81 (m, 2H), 7.55 - 7.52 (m, 1H), 7.01 (d, J = 1.7 Hz, 1H), 6.92 (d, J = 8.2 Hz, 1H), 6.71 (dd, J = 8.1 Hz, 1.5 Hz, 1H), 5.00 - 4.98 (m, 4H), 4.12 - 4.10 (m, 1H), 3.91 - 3.88 (m, 2H), 3.54 - 3.41 (m, 2H) 3.39 - 3.35 (m, 3H), 2.98 (br, 2H), 2.65 - 2.60 (m, 2H), 2.41 (br, 6H), 2.40 (s, 3H), 2.39 (s, 3H), 2.36 (s, 3H), 2.36 (s, 3H), 1.82 (br, 4H). 13C NMR (101 MHz, DMSO) δ 174.86, 155.70, 150.89, 150.72, 150.71, 150.66, 149.42, 149.37, 148.02, 148.02, 147.82, 146.73, 145.44, 145.22, 138.04, 132.39, 131.60, 125.23, 124.88, 122.47, 119.25, 115.52, 114.04,
(ESI) m/z 690.3768 [M+H]+, calcd. for [C40H48N7O4]+, 690.3768.
RI PT
111.22, 72.02, 70.43, 70.37, 48.38, 45.35, 38.80, 28.05, 23.63, 21.45, 21.20, 21.19, 20.87, 20.83, 20.31, 19.99, 19.99. HRMS
3-(3,4-bis((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-2-hydroxy-N-(3-((1,2,3,4-tetrahydroacridin-9-yl)amino)propyl)pr opanamide (12b)
Compound 12b was prepared according to the general procedure III, light yellow oil, yield 81%. 1H NMR (500 MHz, CD3OD) δ 8.16 (d, J = 8.6 Hz, 1H), 7.70 - 7.67 (m, 2H), 7.42 - 7.40 (m, 1H), 7.05 (d, J = 1.8 Hz, 1H), 6.93 (d, J = 8.2 Hz, 1H), 6.84 (dd, J =
SC
8.2 Hz, 1.8 Hz, 1H), 5.04 (d, J = 1.2 Hz, 2H), 4.96 (br, 2H), 4.29 - 4.27 (m, 1H), 3.64 - 3.59 (m, 1H), 3.51 - 3.46 (m, 1H), 3.43 3.38 (m, 1H), 3.32 - 3.30 (m, 1H), 3.23- 3.17 (m, 1H), 3.01 - 2.98 (m, 1H), 2.92 - 2.90 (m, 2H), 2.86 - 2.82 (m, 1H), 2.66 - 2.65 (m, 2H), 2.43 (s, 3H), 2.42 (s, 3H), 2.40 (s, 3H), 2.40 (s, 6H), 2.35 (s, 3H), 1.85 - 1.84 (m, 4H), 1.82 - 1.78 (m, 2H). 13C NMR (101 MHz, MeOD) δ 176.74, 156.03, 154.19, 152.53, 152.46, 151.15, 151.13, 150.16, 150.07, 149.66, 148.76, 147.40, 147.33,
M AN U
142.42, 133.00, 132.45, 125.83, 125.53, 124.51, 122.80, 118.37, 118.06, 116.06, 114.21, 73.64, 71.97, 71.92, 45.90, 41.23, 36.82, 31.76, 30.99, 25.38, 23.29, 22.38, 21.36, 21.34, 21.17, 21.15, 20.43, 20.40. HRMS (ESI) m/z 704.3925 [M+H]+, calcd. for [C41H50N7O4]+, 704.3924.
3-(3,4-bis((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-2-hydroxy-N-(4-((1,2,3,4-tetrahydroacridin-9-yl)amino)butyl)pro panamide (12c)
Compound 12c was prepared according to the general procedure III, light yellow oil, yield 87%. 1H NMR (500 MHz, CD3OD) δ 7.92 (d, J = 8.6 Hz, 1H), 7.58 (d, J = 8.4 Hz, 1H), 7.49 - 7.46 (m, 1H), 7.05 - 7.02 (m, 1H), 7.00 (d, J = 1.7 Hz, 1H), 6.91 (d, J =
TE D
8.3 Hz, 1H), 6.77 (dd, J = 8.2 Hz, 1.7 Hz, 1H), 5.01 (s, 2H), 4.93 (s, 2H), 4.21 - 4.18 (m, 1H), 3.59 - 3.49 (m, 2H), 3.24 - 3.21 (m, 2H), 2.98 – 2.94 (m, 1H), 2.93 - 2.89 (m, 1H), 2.86 - 2.80 (m, 3H), 2.54 - 2.51 (m, 2H), 2.36 (s, 3H), 2.31 (s, 3H), 2.28 (s, 3H), 2.27 (s, 3H), 2.20 (s, 3H), 2.18 (s, 3H), 1.79 - 1.75 (m, 2H), 1.74 - 1.68 (m, 2H), 1.43 - 1.38 (m, 2H), 1.37 - 1.28 (m, 2H). 13C NMR (101 MHz, MeOD) δ 176.00, 155.58, 154.98, 152.55, 152.49, 151.20, 151.15, 150.20, 150.10, 149.55, 148.63, 147.29, 147.29, 143.09, 132.72, 131.96, 125.38, 125.25, 124.56, 123.32, 118.75, 117.55, 115.56, 114.40, 73.56, 71.85, 71.70, 49.09,
EP
41.18, 39.41, 31.44, 29.11, 27.84, 25.46, 23.41, 22.58, 21.30, 21.30, 21.07, 21.03, 20.42, 20.38. HRMS (ESI) m/z 718.4078 [M+H]+, calcd. for [C42H52N7O4]+, 718.4081.
3-(3,4-bis((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-2-hydroxy-N-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)pro
AC C
panamide (12d)
Compound 12d was prepared according to the general procedure III, light yellow oil, yield 85%. 1H NMR (500 MHz, DMSO) δ 8.31 (d, J = 8.7 Hz, 1H), 7.90 - 7.88 (m, 1H), 7.79 - 7.76 (m, 1H, -CONH), 7.71 - 7.68 (m, 1H), 7.55 (br, 1H, -NH), 7.50 - 7.47 (m, 1H), 7.02 (d, J = 1.6 Hz, 1H), 6.98 (d, J = 8.3 Hz, 1H), 6.74 (dd, J = 8.2 Hz, 1.5 Hz, 1H), 5.05 (s, 2H), 5.02 (s, 2H), 4.05 4.03 (m, 1H), 3.79 - 3.76 (m, 2H), 3.08 - 3.04 (m, 1H), 3.01 - 2.97 (m, 4H), 2.67 - 2.63 (m, 2H) 2.61 (br, 2H), 2.40 (s, 3H), 2.39 (s, 6H), 2.38 (s, 3H), 2.37 (s, 3H), 2.36 (s, 3H), 1.79 (br, 4H), 1.69 - 1.66 (m, 2H), 1.33 - 1.30 (m, 4H), 1.19 - 1.18 (m, 2H). 13C NMR (101 MHz, DMSO) δ 173.19, 155.02, 151.25, 150.79, 150.76, 150.68, 149.32, 149.32, 148.14, 148.03, 147.68, 146.55, 145.47, 145.43, 139.07, 131.93, 131.27, 124.81, 124.45, 122.19, 120.21, 115.74, 114.05, 111.45, 72.17, 70.46, 70.29, 47.31, 38.32, 37.93, 29.88, 29.00, 28.46, 25.85, 25.76, 23.96, 21.53, 21.45, 21.19, 20.85, 20.46, 20.12, 20.03, 20.01. HRMS (ESI) m/z 746.4388 [M+H]+, calcd. for [C44H56N7O4]+, 746.4394. 3-(3,4-bis((3,5,6-trimethylpyrazin-2-yl)methoxy)phenyl)-2-hydroxy-N-(8-((1,2,3,4-tetrahydroacridin-9-yl)amino)octyl)pro panamide (12e) Compound 12e was prepared according to the general procedure III, light yellow oil, yield 85%. 1H NMR (500 MHz, CD3OD) δ
ACCEPTED MANUSCRIPT 8.02 (d, J = 8.4 Hz, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.54 - 7.51 (m, 1H), 7.30 - 7.27 (m, 1H), 6.99 (d, J = 1.7 Hz, 1H), 6.93 (d, J = 8.3 Hz, 1H), 6.76 (dd, J = 8.2 Hz, 1.7 Hz, 1H), 5.04 (s, 2H), 5.00 (s, 2H), 4.17 - 4.15 (m, 1H), 3.58 - 3.54 (m, 2H), 3.16 - 3.10 (m, 1H), 3.03 - 2.98 (m, 1H), 2.97 - 2.93 (m, 1H), 2.91 -2.90 (m, 3H), 2.80 - 2.75 (m, 1H), 2.63 - 2.62 (m, 2H), 2.40 (s, 3H), 2.37 (br, 9H), 2.35 (s, 3H), 2.34 (s, 3H), 1.86 - 1.84 (m, 4H), 1.64 - 1.58 (m, 2H), 1.31 - 1.26 (m, 10H). 13C NMR (101 MHz, MeOD) δ 175.99, 156.63, 154.67, 152.53, 152.49, 151.23, 151.23, 150.21, 150.13, 149.76, 148.76, 147.54, 147.50, 145.23, 133.02, 131.10, 125.36, 125.17, 125.03, 124.34, 119.79, 117.94, 116.10, 115.31, 73.73, 72.01, 71.82, 49.53, 41.25, 39.86, 32.60, 32.02, 30.38, 30.17, 30.17, 27.74, 27.65, 25.69, 23.73, 23.09, 21.31, 21.29, 21.13, 21.10, 20.44, 20.41. HRMS (ESI) m/z 774.4708 [M+H]+,
RI PT
calcd. for [C46H60N7O4]+, 774.4707. 4.6. Biological activity 4.6.1. Cholinesterase inhibition assay in vitro
The inhibitory activity was measured by Ellman’s assay with slightly modifications [70]. Acetylcholinesterase (AChE from electric eel and human erythrocytes, EC 3.1.1.7.), Butyrylcholinesterase (BuChE from equine serum and human serum, EC
SC
3.1.1.8.), 5,5'-dithiobis(2-nitrobenzoic acid), acetylthiocholine (ATC), and butyrylthiocholine (BTC) iodides were purchased from Sigma-Aldrich. Enzyme solutions of eeAChE and eqBuChE, hAChE and hBuChE were prepared by dissolving the corresponding enzymes in Tris-HC1-pH 8.0 containing 0.1% w/v bovine serum albumin to give 0.22 and 0.3, 0.05 and 0.024 units/mL, respectively, each in 5 mL aliquots. DTNB (0.015 M) and ATC/BTC iodide (0.03 M) solutions were prepared in
concentrations (normally 10-5~10−11 M).
M AN U
1×PBS. Stock solutions of the test compounds were prepared in DMSO and diluted with PBS to final concentration at least five
For measurement, 50 µL of the respective enzyme was added in a 96-well plate containing 20 µL of PBS and 20 µL of test compound solution, incubated for 5 min at 37 °C. Then, 100 µL of DTNB were added and incubated for 5 min at 37 °C in the dark. Finally, 10 µL of the substrate solution (ATC/BTC) was added and incubated for 3 min at 37 °C. The absorbance of each well was measured at 415 nm using a microplate reader. For the reference value, 20 µL of PBS replaced the test compound solution. For determining the blank value, additionally 100 µL of Tris-HC1 replaced the enzyme solution. Results are expressed
TE D
as the mean ± SD at least three different experiments. The inhibition curve was obtained by plotting percentage enzyme activity (100% for the reference) versus logarithm of test compound concentration. 4.6.2. Inhibition of Aβ42 self-aggregation
Inhibition of Aβ42 self-aggregation was measured using a Thioflavin T-binding assay. HFIP pretreated Aβ42 samples (Sigma-Aldrich) were resolubilized with PBS (pH = 7.4) to give a 40 µM solution. And ThT solution was prepared in PBS at a
EP
concentration of 10 µM. Then, 10 µL of 40 µM Aβ42 solution was added to each well containing 10 µL of different concentrations of test compounds (0.0064, 0.032, 0.16, 0.8, 4, 20, 100 µM). After incubated for 24h, the samples were diluted to a final volume of 200 µL with ThT solution. Fluorescence was measured on a Varioskan Flash spectral scanning multimode
AC C
reader (Thermo, Varioskan Flash 3001, USA) with excitation and emission wavelengths at 435 nm and 485 nm, respectively. 4.6.3. Atomic force microscope assay For the inhibition of Aβ42 aggregation experiment, 45 µL PBS was added in a 96-well plate containing 2.5 µL of 2 mM/L
(DMSO) Aβ42 stock solution and 2.5 µL (2 mM/L DMSO) of test compound solution, shaken for 12 hours. Then, the mixed solution was incubated for 4 days at 37 °C. The final concentration of Aβ42 and the compounds were 0.1 mM. For the control, 2.5 µL of DMSO replaced the test compound solution. Aliquots (5 µL) of the samples diluted 100-fold with PBS were dropped to the mica plate surface (about 0.5 cm2), naturally dried at ambient temperature. The aggregation state of Aβ42 were examined with atomic force microscope (Veeco Multi Mode 8/B0021, Veeco German) equipped with antimony doped silicon of conical shape (Veeco). The Aβ42 aggregation was scanned in intelligent mode and the force constant was monitored at 3 N/m. 4.6.4. Antioxidant activity in vitro The antioxidant activities of compounds 9i and 10i were evaluated by DPPH free-radical scavenging assay according to the method of Lee et al. with slightly modifications [34, 83]. Briefly, 95 mL of DPPH radical solution (300 mM) was added in a 96-well plate containing 5 mL of different concentrations of test compound dissolved in MeOH, and incubated for 30 min at
ACCEPTED MANUSCRIPT 37 °C in the dark. The absorbance of each well was measured at 517 nm using a microplate reader (Thermo, Varioskan Flash 3001, USA). 4.6.5. Cell viability assay on the PC12 cells CoCl2 was cytotoxic towards PC12 cells by production of reactive oxygen species (ROS) [79, 80]. Trolox, a strong antioxidant, which can effectively prevent oxidative neuronal death, was used as a reference compound at 10 µM. The PC12 cell line was purchased from Institute of Materia of Chinese Academy of Medical Science. Cells were cultured in RPMI 1640 medium supplemented with 5% (v/v) fetal bovine serum (FBS), 10% (v/v) heat inactivated horse serum and 100 U/mL
RI PT
penicillin-streptomycin (Thermo Technologies, USA) and incubated at 37 °C in a humidified atmosphere of 5% CO2. PC12 cells, maintained with serum-free media for 14 h, were placed on 96-well dishes pre-coated with poly-L-lysine at 7 × 104 cells/mL, differentiated by treated with NGF (50 ng/mL). Thereafter, the differentiated PC12 cells were pretreated with different concentrations of test compound (1.25, 2.5, 5 and 10 mM) for 36 h, respectively; then it was exposed to CoCl2 for 12 h. Cell viability was measured by using the MTT assay and the absorption was measured by a well plate reader at 490 nm (Thermo
SC
Multiskan GO, USA). For the neurotoxicity, test compounds was incubated for 48 h and measured by MTT assay. Cell viability was expressed as a percentage of the control. 4.6.6. Hepatotoxicity study in vitro
HepG2 cells (human hepatocellular liver carcinoma cell line purchased from Institute of Materia of Chinese Academy of
M AN U
Medical Science), were grown in DMEM supplemented with 10% FBS and 100 U/mL of penicillin-streptomycin (Thermo Technologies, USA) at 37 °C in a humidified atmosphere containing 5% CO2. For the experiments, cells (6 × 103 cells/well) were seeded in 96-well plate in complete medium. After 24 h, the medium was removed, and cells were exposed to the increasing concentrations of compound 9i or tacrine (1.25, 2.5, 5 and 10 µM) in DMEM for further 24 h. Cell survival was measured through MTT assay. 4.6.7. Hepatotoxicity study in vivo
Adult male ICR mice (weighing 22−25g) from Beijing HFK Bio-Technology.co., LTD (Beijing, China) were used to
TE D
perform experiments. Mice were housed in standard controlled conditions (22 ± 2 °C, 55 ± 10% humidity) with a reversed 12h light/dark cycle. Food and water were available ad libitum in the home cage. Tacrine hydrochloride was dissolved in CMC-Na solution (0.5 g CMC-Na in 100 mL of distilled water) and 3 mg/100 g b wt, corresponding to 12.82 µmol/100 g b wt, was given orally. 9i was dissolved in CMC-Na solution, and the equimolar dose corresponding to tacrine was administered. Heparinized serum was obtained 8, 24, and 36 h after dosing from the retro bulbar plexus to determine aspartate aminotransferase (AST) and
EP
alanine aminotransferase (ALT) activity, two indicators of a liver damage, using routine methods. All animal protocols were approved by the Animal Care and Use Committee of PUMC. 4.6.8. Kinetic characterization of AChE inhibition
AC C
The kinetic characterization of AChE was performed in the same manner with four different concentrations (0, 2.5, 5, 10 nm) of compound 9i. Lineweaver-Burk reciprocal plots were constructed by plotting 3/velocity against 1/ [substrate] at varying concentrations of the substrate acetylthiocholine iodide (0.05~0.5 mM). Data analysis was performed with Graph Pad Prism 5 software (San Diego, CA, USA). 4.6.9. Docking study
Molecular docking study was performed using Molecular Operating Environment (MOE) software version 2008.10
(Chemical Computing Group, Montreal, Canada). The X-ray crystal structure of TcAChE complexed with bis(7)-tacrine (PDB code: 2CKM), hAChE in complex with donepezil (PDB code: 4EY7) and hBuChE (PDB code: 1P0I) were obtained from the Protein Data Base (PDB). All water molecules in PDB files were removed and hydrogen atoms were subsequently added to the protein. Compound 9i was constructed using the builder interface of the MOE program and energy minimized using MMFF94x force field. Then 9i was docked into the active site of the protein by the ‘Triangle Matcher’ method. The Dock scoring was done using ASE scoring function and the Force field was selected as the refinement method. The retained best poses were analyzed using the MOE’s pose viewer utility.
ACCEPTED MANUSCRIPT Competing interests The authors declare that they have no competing interests. Acknowledgments We thank the Tianjin special support program for talent development, the key technologies R & D program of Tianjin (12ZCDZSY11900) and the Medical and Health Science and Technology Innovation Project of Chinese Academy of Medical Science (2017-I2M-3-021) for financial support of this study. We also thank Chinese Academy of Medical Sciences Peking
Authors’ contributions
RI PT
Union Medical College Information technology Center for their help in molecular modeling study.
Tianjun Liu and Guoliang Li designed the study; GuoLiang Li, Ge Hong, Xinxu Li, Zengping Xu, Lina Mao and Yan Zhang performed experiments; Guoliang Li analyzed data with the help of Tianjun Liu and Xizeng Feng; Tianjun Liu and Guoliang Li wrote the paper. All authors read and approved the final manuscript. References
SC
[1] J. Mendiola-Precoma, L.C. Berumen, K. Padilla, G. Garcia-Alcocer, Therapies for prevention and treatment of Alzheimer's disease, BioMed. Res. Int. 2016 (2016) 2589276.
[2] M. Prince, A. Wimo, M. Guerchet, G.C. Ali, Y.T. Wu, M. Prina, World Alzheimer Report 2015. The global impact of dementia. An analysis of prevalence, incidence, cost and trends, London: Alzheimer's Disease International, 2015.
M AN U
[3] Z. Wang, Y. Wang, W. Li, F. Mao, Y. Sun, L. Huang, X. Li, Design, synthesis, and evaluation of multitarget-directed selenium-containing clioquinol derivatives for the treatment of Alzheimer's disease, ACS Chem. Neurosci. 5 (2014) 952–962. [4] M.G. Savelieff, S. Lee, Y. Liu, M.H. Lim, Untangling amyloid-beta, tau, and metals in Alzheimer's disease, ACS Chem. Biol. 8 (2013) 856-865.
[5] S.S. Choi, S.R. Lee, S.U. Kim, H.J. Lee, Alzheimer's disease and stem cell therapy, Exp. Neurobiol. 23 (2014) 45–52. [6] C. Rochais, C. Lecoutey, F. Gaven, P. Giannoni, K. Hamidouche, D. Hedou, E. Dubost, D. Genest, S. Yahiaoui, T. Freret, V. Bouet, F. Dauphin, J. Sopkova de Oliveira Santos, C. Ballandonne, S. Corvaisier, A. Malzert-Freon, R. Legay, M. Boulouard, S.
TE D
Claeysen, P. Dallemagne, Novel multitarget-directed ligands (MTDLs) with acetylcholinesterase (AChE) inhibitory and serotonergic subtype 4 receptor (5-HT4R) agonist activities as potential agents against Alzheimer's disease: the design of donecopride, J. Med. Chem. 58 (2015) 3172–3187.
[7] M. Citron, Alzheimer's disease: strategies for disease modification, Nat. Rev. Drug. Discov. 9 (2010) 387–398. [8] C. Zhang, Q.Y. Du, L.D. Chen, W.H. Wu, S.Y. Liao, L.H. Yu, X.T. Liang, Design, synthesis and evaluation of novel
200–209.
EP
tacrine-multialkoxybenzene hybrids as multi-targeted compounds against Alzheimer's disease, Eur. J. Med. Chem. 116 (2016)
[9] A. Cavalli, M.L. Bolognesi, A. Minarini, M. Rosini, V. Tumiatti, M. Recanatini, C. Melchiorre, Multi-target-directed ligands
AC C
to combat neurodegenerative diseases, J. Med. Chem. 51 (2008) 347–372. [10] R. Leon, A.G. Garcia, J. Marco-Contelles, Recent advances in the multitarget-directed ligands approach for the treatment of Alzheimer's disease, Med. Res. Rev. 33 (2013) 139–189. [11] K. Xu, P. Wang, X. Xu, F. Chu, J. Lin, Y. Zhang, H. Lei, An overview on structural modifications of ligustrazine and biological evaluation of its synthetic derivatives, Res. Chem. Intermediat. (2013) doi:10.1007/s11164–013–1281–2. [12] M. Kim, S.O. Kim, M. Lee, J.H. Lee, W.S. Jung, S.K. Moon, Y.S. Kim, K.H. Cho, C.N. Ko, E.H. Lee, Tetramethylpyrazine, a natural alkaloid, attenuates pro-inflammatory mediators induced by amyloid beta and interferon-gamma in rat brain microglia, Eur. J. Pharmacol. 740 (2014) 504–511. [13] T.K. Kao, Y.C. Ou, J.S. Kuo, W.Y. Chen, S.L. Liao, C.W. Wu, C.J. Chen, N.N. Ling, Y.H. Zhang, W.H. Peng, Neuroprotection by tetramethylpyrazine against ischemic brain injury in rats, Neurochem. Int. 48 (2006) 166–176. [14] C. Zhang, S.Z. Wang, P.P. Zuo, X. Cui, J. Cai, Protective effect of tetramethylpyrazine on learning and memory function in D-galactose-lesioned mice, Chin. Med. Sci. J. 19 (2004) 180–184. [15] H. Chen, G. Li, P. Zhan, X. Liu, Ligustrazine derivatives. Part 5: design, synthesis and biological evaluation of novel
ACCEPTED MANUSCRIPT ligustrazinyloxy-cinnamic acid derivatives as potent cardiovascular agents, Eur. J. Med. Chem. 46 (2011) 5609–5615. [16] T. Zhang, J. Gu, L. Wu, N. Li, Y. Sun, P. Yu, Y. Wang, G. Zhang, Z. Zhang, Neuroprotective and axonal outgrowth-promoting effects of tetramethylpyrazine nitrone in chronic cerebral hypoperfusion rats and primary hippocampal neurons exposed to hypoxia, Neuropharmacology 118 (2017) 137–147. [17] C. Lu, J. Zhang, X. Shi, S. Miao, L. Bi, S. Zhang, Q. Yang, X. Zhou, M. Zhang, Y. Xie, Q. Miao, S. Wang, Neuroprotective effects of tetramethylpyrazine against dopaminergic neuron injury in a rat model of Parkinson's disease induced by MPTP, Int. J. Biol. Sci. 10 (2014) 350–357.
RI PT
[18] E.C. So, K.L. Wong, T.C. Huang, S.C. Tasi, C.F. Liu, Tetramethylpyrazine protects mice against thioacetamide-induced acute hepatotoxicity, J. Biomed. Sci. 9 (2002) 410–414.
[19] Y. Wang, X. Zhang, C. Xu, G. Zhang, Z. Zhang, P. Yu, L. Shan, Y. Sun, Y. Wang, Synthesis and biological evaluation of danshensu and tetramethylpyrazine conjugates as cardioprotective agents, Chem. Pharm. Bull. (Tokyo) 65 (2017) 381–388.
[20] P. Picone, D. Nuzzo, M. Di Carlo, Ferulic acid: a natural antioxidant against oxidative stress induced by oligomeric A-beta
SC
on sea urchin embryo, Biol. Bull. 224 (2013) 18–28.
[21] Y. Huang, M. Jin, R. Pi, J. Zhang, M. Chen, Y. Ouyang, A. Liu, X. Chao, P. Liu, J. Liu, C. Ramassamy, J. Qin, Protective effects of caffeic acid and caffeic acid phenethyl ester against acrolein-induced neurotoxicity in HT22 mouse hippocampal cells, Neurosci. Lett. 535 (2013) 146–151.
M AN U
[22] A. Abdel-Moneim, A.I. Yousef, S.M. Abd El-Twab, E.S. Abdel Reheim, M.B. Ashour, Gallic acid and p-coumaric acid attenuate type 2 diabetes-induced neurodegeneration in rats, Metab. Brain Dis. 32 (2017) 1279–1286. [23] F.N. Ekinci Akdemir, M. Albayrak, M. Calik, Y. Bayir, I. Gulcin, The protective effects of p-coumaric acid on acute liver and kidney damages induced by cisplatin, Biomedicines 5 (2017) pii: E18.
[24] S.Y. Park, G. Ahn, J.H. Um, E.J. Han, C.B. Ahn, N.Y. Yoon, J.Y. Je, Hepatoprotective effect of chitosan-caffeic acid conjugate against ethanol-treated mice, Exp. Toxicol. Pathol. 69 (2017) 618–624.
[25] W. Qu, H. Huang, K. Li, C. Qin, Danshensu-mediated protective effect against hepatic fibrosis induced by carbon
TE D
tetrachloride in rats, Pathol. Biol. (Paris) 62 (2014) 348–353.
[26] P. Wang, H. Zhang, F. Chu, X. Xu, J. Lin, C. Chen, G. Li, Y. Cheng, L. Wang, Q. Li, Y. Zhang, H. Lei, Synthesis and protective effect of new ligustrazine-benzoic acid derivatives against CoCl2-induced neurotoxicity in differentiated PC12 cells, Molecules 18 (2013) 13027–13042.
[27] G. Li, X. Xu, K. Xu, F. Chu, J. Song, S. Zhou, B. Xu, Y. Gong, H. Zhang, Y. Zhang, P. Wang, H. Lei, Ligustrazinyl amides: a
EP
novel class of ligustrazine-phenolic acid derivatives with neuroprotective effects, Chem. Cent. J. 9 (2015) 9. [28] G. Li, Y. Tian, Y. Zhang, Y. Hong, Y. Hao, C. Chen, P. Wang, H. Lei, A novel ligustrazine derivative T-VA prevents neurotoxicity in differentiated PC12 cells and protects the brain against ischemia injury in MCAO rats, Int. J. Mol. Sci. 16 (2015)
AC C
21759–21774.
[29] L. Ismaili, B. Refouvelet, M. Benchekroun, S. Brogi, M. Brindisi, S. Gemma, G. Campiani, S. Filipic, D. Agbaba, G. Esteban, M. Unzeta, K. Nikolic, S. Butini, J. Marco-Contelles, Multitarget compounds bearing tacrine- and donepezil-like structural and functional motifs for the potential treatment of Alzheimer's disease, Prog. Neurobiol. 151 (2017) 4–34. [30] N. Guzior, A. Wieckowska, D. Panek, B. Malawska, Recent development of multifunctional agents as potential drug candidates for the treatment of Alzheimer's disease, Curr. Med. Chem. 22 (2015) 373–404. [31] B. Sameem, M. Saeedi, M. Mahdavi, A. Shafiee, A review on tacrine-based scaffolds as multi-target drugs (MTDLs) for Alzheimer's disease, Eur. J. Med. Chem. 128 (2017) 332–345. [32] S. Hamulakova, L. Janovec, M. Hrabinova, P. Kristian, K. Kuca, M. Banasova, J. Imrich, Synthesis, design and biological evaluation of novel highly potent tacrine congeners for the treatment of Alzheimer's disease, Eur. J. Med. Chem. 55 (2012) 23– 31. [33] E.K. Reddy, C. Remya, K. Mantosh, A.M. Sajith, R.V. Omkumar, C. Sadasivan, S. Anwar, Novel tacrine derivatives exhibiting improved acetylcholinesterase inhibition: design, synthesis and biological evaluation, Eur. J. Med. Chem. 139 (2017)
ACCEPTED MANUSCRIPT 367–377. [34] S.S. Xie, J.S. Lan, X.B. Wang, N. Jiang, G. Dong, Z.R. Li, K.D. Wang, P.P. Guo, L.Y. Kong, Multifunctional tacrine-trolox hybrids for the treatment of Alzheimer's disease with cholinergic, antioxidant, neuroprotective and hepatoprotective properties, Eur. J. Med. Chem. 93 (2015) 42–50. [35] S.S. Xie, X.B. Wang, J.Y. Li, L. Yang, L.Y. Kong, Design, synthesis and evaluation of novel tacrine-coumarin hybrids as multifunctional cholinesterase inhibitors against Alzheimer's disease, Eur. J. Med. Chem. 64 (2013) 540–553. [36] J. Jerabek, E. Uliassi, L. Guidotti, J. Korabecny, O. Soukup, V. Sepsova, M. Hrabinova, K. Kuca, M. Bartolini, L.E.
RI PT
Pena-Altamira, S. Petralla, B. Monti, M. Roberti, M.L. Bolognesi, Tacrine-resveratrol fused hybrids as multi-target-directed ligands against Alzheimer's disease, Eur. J. Med. Chem. 127 (2017) 250–262.
[37] Y. Chen, J. Sun, L. Fang, M. Liu, S. Peng, H. Liao, J. Lehmann, Y. Zhang, Tacrine-ferulic acid-nitric oxide (NO) donor trihybrids as potent, multifunctional acetyl- and butyrylcholinesterase inhibitors, J. Med. Chem. 55 (2012) 4309–4321.
[38] P. Camps, X. Formosa, C. Galdeano, T. Gomez, D. Munoz-Torrero, M. Scarpellini, E. Viayna, A. Badia, M.V. Clos, A.
SC
Camins, M. Pallas, M. Bartolini, F. Mancini, V. Andrisano, J. Estelrich, M. Lizondo, A. Bidon-Chanal, F.J. Luque, Novel donepezil-based inhibitors of acetyl- and butyrylcholinesterase and acetylcholinesterase-induced beta-amyloid aggregation, J. Med. Chem. 51 (2008) 3588–3598.
[39] S. Butini, M. Brindisi, S. Brogi, S. Maramai, E. Guarino, A. Panico, A. Saxena, V. Chauhan, R. Colombo, L. Verga, E. De
M AN U
Lorenzi, M. Bartolini, V. Andrisano, E. Novellino, G. Campiani, S. Gemma, Multifunctional cholinesterase and amyloid Beta fibrillization modulators. Synthesis and biological investigation, ACS Med. Chem. Lett. 4 (2013) 1178–1182. [40] Q. Sun, D.Y. Peng, S.G. Yang, X.L. Zhu, W.C. Yang, G.F. Yang, Syntheses of coumarin-tacrine hybrids as dual-site acetylcholinesterase inhibitors and their activity against butylcholinesterase, abeta aggregation, and beta-secretase, Bioorg. Med. Chem. 22 (2014) 4784–4791.
[41] C. Lu, Q. Zhou, J. Yan, Z. Du, L. Huang, X. Li, A novel series of tacrine-selegiline hybrids with cholinesterase and monoamine oxidase inhibition activities for the treatment of Alzheimer's disease, Eur. J. Med. Chem. 62 (2013) 745–753.
TE D
[42] J.S. da Costa, J.P. Lopes, D. Russowsky, C.L. Petzhold, A.C. Borges, M.A. Ceschi, E. Konrath, C. Batassini, P.S. Lunardi, C.A. Goncalves, Synthesis of tacrine-lophine hybrids via one-pot four component reaction and biological evaluation as acetyland butyrylcholinesterase inhibitors, Eur. J. Med. Chem. 62 (2013) 556–563. [43] M. Khoobi, F. Ghanoni, H. Nadri, A. Moradi, M. Pirali Hamedani, F. Homayouni Moghadam, S. Emami, M. Vosooghi, R. Zadmard, A. Foroumadi, A. Shafiee, New tetracyclic tacrine analogs containing pyrano[2,3-c]pyrazole: efficient synthesis,
EP
biological assessment and docking simulation study, Eur. J. Med. Chem. 89 (2015) 296–303. [44] L. Pourabdi, M. Khoobi, H. Nadri, A. Moradi, F.H. Moghadam, S. Emami, M.M. Mojtahedi, I. Haririan, H. Forootanfar, A. Ameri, A. Foroumadi, A. Shafiee, Synthesis and structure-activity relationship study of tacrine-based pyrano[2,3-c]pyrazoles
AC C
targeting AChE/BuChE and 15-LOX, Eur. J. Med. Chem. 123 (2016) 298–308. [45] M. Eghtedari, Y. Sarrafi, H. Nadri, M. Mahdavi, A. Moradi, F. Homayouni Moghadam, S. Emami, L. Firoozpour, A. Asadipour, O. Sabzevari, A. Foroumadi, New tacrine-derived AChE/BuChE inhibitors: Synthesis and biological evaluation of 5-amino-2-phenyl-4H-pyrano[2,3-b]quinoline-3-carboxylates, Eur. J. Med. Chem. 128 (2017) 237–246. [46] L. Jalili-Baleh, H. Nadri, A. Moradi, S.N.A. Bukhari, M. Shakibaie, M. Jafari, M. Golshani, F. Homayouni Moghadam, L. Firoozpour, A. Asadipour, S. Emami, M. Khoobi, A. Foroumadi, New racemic annulated pyrazolo[1,2-b]phthalazines as tacrine-like AChE inhibitors with potential use in Alzheimer's disease, Eur. J. Med. Chem. 139 (2017) 280–289. [47] W. Luo, Y.P. Li, Y. He, S.L. Huang, D. Li, L.Q. Gu, Z.S. Huang, Synthesis and evaluation of heterobivalent tacrine derivatives as potential multi-functional anti-Alzheimer agents, Eur. J. Med. Chem. 46 (2011) 2609–2616. [48] R.J. Reiter, D.X. Tan, J.C. Mayo, R.M. Sainz, J. Leon, Z. Czarnocki, Melatonin as an antioxidant: biochemical mechanisms and pathophysiological implications in humans, Acta biochim. Pol. 50 (2003) 1129–1146. [49] M.I. Rodriguez-Franco, M.I. Fernandez-Bachiller, C. Perez, B. Hernandez-Ledesma, B. Bartolome, Novel tacrine-melatonin hybrids as dual-acting drugs for Alzheimer disease, with improved acetylcholinesterase inhibitory and antioxidant properties, J.
ACCEPTED MANUSCRIPT Med. Chem. 49 (2006) 459–462. [50] M.I. Fernandez-Bachiller, C. Perez, N.E. Campillo, J.A. Paez, G.C. Gonzalez-Munoz, P. Usan, E. Garcia-Palomero, M.G. Lopez, M. Villarroya, A.G. Garcia, A. Martinez, M.I. Rodriguez-Franco, Tacrine-melatonin hybrids as multifunctional agents for Alzheimer's disease, with cholinergic, antioxidant, and neuroprotective properties, ChemMedChem. 4 (2009) 828–841. [51] M.I. Fernandez-Bachiller, C. Perez, G.C. Gonzalez-Munoz, S. Conde, M.G. Lopez, M. Villarroya, A.G. Garcia, M.I. Rodriguez-Franco, Novel tacrine-8-hydroxyquinoline hybrids as multifunctional agents for the treatment of Alzheimer's disease, with neuroprotective, cholinergic, antioxidant, and copper-complexing properties, J. Med. Chem. 53 (2010) 4927–4937.
RI PT
[52] M.I. Fernandez-Bachiller, C. Perez, L. Monjas, J. Rademann, M.I. Rodriguez-Franco, New tacrine-4-oxo-4H-chromene hybrids as multifunctional agents for the treatment of Alzheimer's disease, with cholinergic, antioxidant, and beta-amyloid-reducing properties, J. Med. Chem. 55 (2012) 1303–1317.
[53] S.S. Xie, X. Wang, N. Jiang, W. Yu, K.D. Wang, J.S. Lan, Z.R. Li, L.Y. Kong, Multi-target tacrine-coumarin hybrids: cholinesterase and monoamine oxidase B inhibition properties against Alzheimer's disease, Eur. J. Med. Chem. 95 (2015) 153–
SC
165.
[54] M.K. Hu, L.J. Wu, G. Hsiao, M.H. Yen, Homodimeric tacrine congeners as acetylcholinesterase inhibitors, J. Med. Chem. 45 (2002) 2277–2282.
[55] J.M. Roldan-Pena, D. Alejandre-Ramos, O. Lopez, I. Maya, I. Lagunes, J.M. Padron, L.E. Pena-Altamira, M. Bartolini, B.
M AN U
Monti, M.L. Bolognesi, J.G. Fernandez-Bolanos, New tacrine dimers with antioxidant linkers as dual drugs: Anti-Alzheimer's and antiproliferative agents, Eur. J. Med. Chem. 138 (2017) 761–773.
[56] L. Fang, D. Appenroth, M. Decker, M. Kiehntopf, C. Roegler, T. Deufel, C. Fleck, S. Peng, Y. Zhang, J. Lehmann, Synthesis and biological evaluation of NO-donor-tacrine hybrids as hepatoprotective anti-Alzheimer drug candidates, J. Med. Chem. 51 (2008) 713–716.
[57] M. Rosini, V. Andrisano, M. Bartolini, M.L. Bolognesi, P. Hrelia, A. Minarini, A. Tarozzi, C. Melchiorre, Rational approach to discover multipotent anti-Alzheimer drugs, J. Med. Chem. 48 (2005) 360–363.
TE D
[58] S. Thiratmatrakul, C. Yenjai, P. Waiwut, O. Vajragupta, P. Reubroycharoen, M. Tohda, C. Boonyarat, Synthesis, biological evaluation and molecular modeling study of novel tacrine-carbazole hybrids as potential multifunctional agents for the treatment of Alzheimer's disease, Eur. J. Med. Chem. 75 (2014) 21–30.
[59] J. Marco-Contelles, R. Leon, C. de Los Rios, A. Guglietta, J. Terencio, M.G. Lopez, A.G. Garcia, M. Villarroya, Novel multipotent tacrine-dihydropyridine hybrids with improved acetylcholinesterase inhibitory and neuroprotective activities as
EP
potential drugs for the treatment of Alzheimer's disease, J. Med. Chem. 49 (2006) 7607–7610. [60] L. Fang, B. Kraus, J. Lehmann, J. Heilmann, Y. Zhang, M. Decker, Design and synthesis of tacrine-ferulic acid hybrids as multi-potent anti-Alzheimer drug candidates, Bioorg. Med. Chem. Lett. 18 (2008) 2905–2909.
AC C
[61] Y. Fu, Y. Mu, H. Lei, P. Wang, X. Li, Q. Leng, L. Han, X. Qu, Z. Wang, X. Huang, Design, synthesis and evaluation of novel tacrine-ferulic acid hybrids as multifunctional drug candidates against Alzheimer's disease, Molecules 21 (2016) pii: E1338. [62] X. Chao, X. He, Y. Yang, X. Zhou, M. Jin, S. Liu, Z. Cheng, P. Liu, Y. Wang, J. Yu, Y. Tan, Y. Huang, J. Qin, S. Rapposelli, R. Pi, Design, synthesis and pharmacological evaluation of novel tacrine-caffeic acid hybrids as multi-targeted compounds against Alzheimer's disease, Bioorg. Med. Chem. Lett. 22 (2012) 6498–6502. [63] F. Mao, J. Li, H. Wei, L. Huang, X. Li, Tacrine-propargylamine derivatives with improved acetylcholinesterase inhibitory activity and lower hepatotoxicity as a potential lead compound for the treatment of Alzheimer's disease, J. Enzyme Inhib. Med. Chem. 30 (2015) 995–1001. [64] F. Mao, J. Chen, Q. Zhou, Z. Luo, L. Huang, X. Li, Novel tacrine-ebselen hybrids with improved cholinesterase inhibitory, hydrogen peroxide and peroxynitrite scavenging activity, Bioorg. Med. Chem. Lett. 23 (2013) 6737–6742. [65] X. Chen, K. Zenger, A. Lupp, B. Kling, J. Heilmann, C. Fleck, B. Kraus, M. Decker, Tacrine-silibinin codrug shows neuroand hepatoprotective effects in vitro and pro-cognitive and hepatoprotective effects in vivo, J. Med. Chem. 55 (2012) 5231–5242. [66] Z. Najafi, M. Mahdavi, M. Saeedi, E. Karimpour-Razkenari, R. Asatouri, F. Vafadarnejad, F.H. Moghadam, M. Khanavi, M.
ACCEPTED MANUSCRIPT Sharifzadeh, T. Akbarzadeh, Novel tacrine-1,2,3-triazole hybrids: In vitro, in vivo biological evaluation and docking study of cholinesterase inhibitors, Eur. J. Med. Chem. 125 (2017) 1200–1212. [67] A. Minarini, A. Milelli, V. Tumiatti, M. Rosini, E. Simoni, M.L. Bolognesi, V. Andrisano, M. Bartolini, E. Motori, C. Angeloni, S. Hrelia, Cystamine-tacrine dimer: a new multi-target-directed ligand as potential therapeutic agent for Alzheimer's disease treatment, Neuropharmacology 62 (2012) 997–1003. [68] M.A. Ceschi, J.S. da Costa, J.P.B. Lopes, V.S. Camara, L.F. Campo, A.C.A. Borges, C.A.S. Goncalves, D.F. de Souza, E.L. Konrath, A.L.M. Karl, I.A. Guedes, L.E. Dardenne, Novel series of tacrine-tianeptine hybrids: synthesis, cholinesterase
RI PT
inhibitory activity, S100B secretion and a molecular modeling approach, Eur. J. Med. Chem. 121 (2016) 758–772.
[69] N. Garcia-Font, H. Hayour, A. Belfaitah, J. Pedraz, I. Moraleda, I. Iriepa, A. Bouraiou, M. Chioua, J. Marco-Contelles, M.J. Oset-Gasque, Potent anticholinesterasic and neuroprotective pyranotacrines as inhibitors of beta-amyloid aggregation, oxidative stress and tau-phosphorylation for Alzheimer's disease, Eur. J. Med. Chem. 118 (2016) 178–192.
[70] W. Luo, Y.P. Li, Y. He, S.L. Huang, J.H. Tan, T.M. Ou, D. Li, L.Q. Gu, Z.S. Huang, Design, synthesis and evaluation of
SC
novel tacrine-multialkoxybenzene hybrids as dual inhibitors for cholinesterases and amyloid beta aggregation, Bioorg. Med. Chem. 19 (2011) 763–770.
[71] E. Nepovimova, E. Uliassi, J. Korabecny, L.E. Pena-Altamira, S. Samez, A. Pesaresi, G.E. Garcia, M. Bartolini, V. Andrisano, C. Bergamini, R. Fato, D. Lamba, M. Roberti, K. Kuca, B. Monti, M.L. Bolognesi, Multitarget drug design strategy:
Med. Chem. 57 (2014) 8576–8589.
M AN U
quinone-tacrine hybrids designed to block amyloid-beta aggregation and to exert anticholinesterase and antioxidant effects, J.
[72] J.S. Lan, S.S. Xie, S.Y. Li, L.F. Pan, X.B. Wang, L.Y. Kong, Design, synthesis and evaluation of novel tacrine-(beta-carboline) hybrids as multifunctional agents for the treatment of Alzheimer's disease, Bioorg. Med. Chem. 22 (2014) 6089–6104.
[73] J. Cen, H. Guo, C. Hong, J. Lv, Y. Yang, T. Wang, D. Fang, W. Luo, Development of tacrine-bifendate conjugates with improved cholinesterase inhibitory and pro-cognitive efficacy and reduced hepatotoxicity, Eur. J. Med. Chem. 144 (2017) 128–
TE D
136.
[74] E.H. Rydberg, B. Brumshtein, H.M. Greenblatt, D.M. Wong, D. Shaya, L.D. Williams, P.R. Carlier, Y.P. Pang, I. Silman, J.L. Sussman, Complexes of alkylene-linked tacrine dimers with Torpedo californica acetylcholinesterase: binding of bis5-tacrine produces a dramatic rearrangement in the active-site gorge, J. Med. Chem. 49 (2006) 5491–5500. [75] G.L. Ellman, K.D. Courtney, V. Andres, Jr., R.M. Feather-Stone, A new and rapid colorimetric determination of
EP
acetylcholinesterase activity, Biochem. Pharmacol. 7 (1961) 88–95. [76] X. Zha, D. Lamba, L. Zhang, Y. Lou, C. Xu, D. Kang, L. Chen, Y. Xu, L. Zhang, A. De Simone, S. Samez, A. Pesaresi, J. Stojan, M.G. Lopez, J. Egea, V. Andrisano, M. Bartolini, Novel tacrine-benzofuran hybrids as potent multitarget-directed ligands
AC C
for the treatment of Alzheimer's disease: design, synthesis, biological evaluation, and X-ray crystallography, J. Med. Chem. 59 (2016) 114–131.
[77] F. Cheng, W. Li, Y. Zhou, J. Shen, Z. Wu, G. Liu, P.W. Lee, Y. Tang, admetSAR: a comprehensive source and free tool for assessment of chemical ADMET properties, J. Chem Inf. Model. 52 (2012) 3099–3105. [78] N.A. Vyas, S.S. Bhat, A.S. Kumbhar, U.B. Sonawane, V. Jani, R.R. Joshi, S.N. Ramteke, P.P. Kulkarni, B. Joshi, Ruthenium(II) polypyridyl complex as inhibitor of acetylcholinesterase and Abeta aggregation, Eur. J. Med. Chem. 75 (2014) 375–381. [79] J.X. Chen, T. Zhao, D.X. Huang, Protective effects of edaravone against cobalt chloride-induced apoptosis in PC12 cells, Neurosci. Bull. 25 (2009) 67–74. [80] J. Hu, T.Z. Zhao, W.H. Chu, C.X. Luo, W.H. Tang, L. Yi, H. Feng, Protective effects of 20-hydroxyecdysone on CoCl(2)-induced cell injury in PC12 cells, J. Cell. Biochem. 111 (2010) 1512–1521. [81] S. Tranchimand, T. Tron, C. Gaudin, G. Iacazioa, First chemical synthesis of three natural depsides involved in flavonol catabolism and related to quercetinase catalysis, Synth. Commun. 36 (2006) 587–597.
ACCEPTED MANUSCRIPT [82] T. Kojima, N. Hirasa, D. Noguchi, T. Ishizuka, S. Miyazaki, Y. Shiota, K. Yoshizawa, S. Fukuzumi, Synthesis and characterization of ruthenium(II)-pyridylamine complexes with catechol pendants as metal binding sites, Inorg. Chem. 49 (2010) 3737–3745. [83] S.K. Lee, Z.H. Mbwambo, H. Chung, L. Luyengi, E.J. Gamez, R.G. Mehta, A.D. Kinghorn, J.M. Pezzuto, Evaluation of the
AC C
EP
TE D
M AN U
SC
RI PT
antioxidant potential of natural products, Comb. Chem. High Throughput Screen. 1 (1998) 35–46.
ACCEPTED MANUSCRIPT
Highlights 1. 35 novel tacrine-phenolic acid dihybrids and tacrine-phenolic acid-ligustrazine trihybrids were synthesized and screened for anti-AD purpose.
RI PT
2. 9i was the most potent AChE inhibitor (eeAChE, IC50 = 3.9 nM; hAChE, IC50 = 65.2 nM). 3. 9i could effectively block Aβ42 self-aggregation with a ratio of 47% at 20 µM. 4. 9i had potent neuroprotection towards PC12 cells while no neurotoxicity.
AC C
EP
TE D
M AN U
SC
5. 9i showed lower hepatotoxicity than tacrine.