Tacrine–Trolox Hybrids: A Novel Class of Centrally Active

Oct 27, 2015 - National Institute of Mental Health, Topolova 748, 250 67 Klecany, Czech Republic. ⊥Center for Basic and Applied Research, Faculty of...
0 downloads 0 Views 4MB Size
Subscriber access provided by EPFL | Scientific Information and Libraries

Article

Tacrine – Trolox Hybrids: A Novel Class of Centrally Active, Non-Hepatotoxic Multi-Target-Directed Ligands Exerting Anticholinesterase and Antioxidant Activities with Low In Vivo Toxicity Eugenie Nepovimova, Jan Korabecny, Rafael Dolezal, Katerina Babkova, Ales Ondrejicek, Daniel Jun, Vendula Sepsova, Anna Horova, Martina Hrabinova, Ondrej Soukup, Neslihan Bukum, Petr Jost, Lubica Muckova, Jiri Kassa, David Malinak, Martin Andrs, and Kamil Kuca J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01325 • Publication Date (Web): 27 Oct 2015 Downloaded from http://pubs.acs.org on October 28, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 74

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

Tacrine – Trolox Hybrids: A Novel Class of Centrally Active, Non-Hepatotoxic Multi-Target-Directed Ligands Exerting Anticholinesterase and Antioxidant Activities with Low In Vivo Toxicity Eugenie Nepovimovaa,b,c,§, Jan Korabecnya,b,d,§, Rafael Dolezala,e, Katerina Babkovaa,b, Ales Ondrejicekg, Daniel Juna,b, Vendula Sepsovaa,b, Anna Horovaa,b, Martina Hrabinovaa,b, Ondrej Soukupa,b,d, Neslihan Bukuma,f, Petr Josta,b, Lubica Muckovab, Jiri Kassab, David Malinaka,c, Martin Andrsa,b, and Kamil Kucaa,e,* a

Biomedical Research Centre, University Hospital Hradec Kralove, Sokolska 581, 500 05

Hradec Kralove, Czech Republic. b

Department of Toxicology and Military Pharmacy, Faculty of Military Health Sciences,

University of Defence, Trebesska 1575, 500 01 Hradec Kralove, Czech Republic. c

Department of Intensive Medicine and Forensic Studies; Department of Physiology and

Pathophysiology, Faculty of Medicine, University of Ostrava, Syllabova 19, 703 00 Ostrava, Czech Republic. d

e

National Institute of Mental Health, Topolova 748, 250 67 Klecany, Czech Republic. Center for Basic and Applied Research, Faculty of Informatics and Management,

University of Hradec Kralove, Rokitanskeho 62, 500 03 Hradec Kralove, Czech Republic. f

Department of Chemistry, Faculty of Science, University of Hradec Kralove, Rokitanskeho

62, 500 03 Hradec Kralove, Czech Republic. g

Department of Pharmaceutical Chemistry and Drug Control, Faculty of Pharmacy in

Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, 500 05 Hradec Kralove, Czech Republic. §

E.N. and J.K. contributed equally.

ACS Paragon Plus Environment

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Coupling of two distinct pharmacophores – tacrine and trolox, endowed with different biological properties, afforded 21 hybrid compounds as novel multifunctional candidates against Alzheimer’s disease. Several of them showed improved inhibitory properties towards acetylcholinesterase (AChE) in relation to tacrine. These hybrids also scavenged free radicals. Molecular modeling studies in tandem with kinetic analysis exhibited that these hybrids target both catalytic active site as well as peripheral anionic site of AChE. In addition, incorporation of the moiety bearing antioxidant abilities displayed negligible toxicity on human hepatic cells. This striking effect was explained by formation of non-toxic metabolites after 1 h incubation in human liver microsomes system. Finally, tacrine – trolox hybrids exhibited low in vivo toxicity after i.m. administration in rats and potential to penetrate across blood-brain barrier. All of these outstanding in vitro results in combination with promising in vivo outcomes highlighted derivative 7u as the lead structure worthy of further investigation.

KEYWORDS: Alzheimer’s disease; Acetylcholinesterase inhibitors; Tacrine; Oxidative stress; Antioxidants; Trolox; MTDLs.

ACS Paragon Plus Environment

Page 2 of 74

Page 3 of 74

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

INTRODUCTION Alzheimer’s disease (AD) is a progressive, unremitting neurodegenerative disorder always resulting in death over a course that varies from 3 to 20 years.1 Clinically, the symptoms begin with brief lapses in ability to recall words or complete calculations. The end stage is a bed ridden, wheel chair bound, speechless, cachectic, incontinent, nursing home patient requiring total care.2 Among other neurodegenerative diseases AD stands out as the fourth leading cause of death in the Western countries and the most common cause of acquired dementia in elderly population.3 In line with an increase in average life expectancy, the number of affected persons is expected to grow up to 115.4 million cases worldwide by 2050,4 constituting thus from a macroeconomic and demographic point of view a huge problem. A great amount of experimental and clinical evidence indicates

that

AD

is

a

complex

disorder,

characterized

by

widespread

neurodegeneration of the CNS, with a major involvement of the cholinergic system, causing a progressive cognitive decline and dementia.5 Moreover, amyloid-β (Aβ) deposits in senile plaques and neurofibrillary tangles, mainly constituted of paired helical filaments of abnormally phosphorylated tau protein, have been identified as the main pathological hallmarks.6 Compelling lines of evidence support a concomitant role for oxidative stress, metal ion dysregulation, inflammation and cell cycle regulatory failure in AD pathogenesis.1,2 Trying to gather the pieces of the puzzle, scientists have reached a consensus that AD is a multifactorial disease with a polyetiology, where different factors set in motion a self-sustaining, amplifying cycle which culminates in neuronal cell death processes.7 Even though a robust effort is being made to elucidate the underlying mechanisms of the disease, the transitioning these findings into new therapeutic approaches is still challenging.

ACS Paragon Plus Environment

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

At this time, cholinesterase inhibitors and memantine (an NMDA receptor antagonist) still remain the drugs of choice.8 The first drug introduced on the market with the indication of AD was tacrine (1).9 Tacrine promotes an increase in the concentration and duration of action of synaptic acetylcholine, thus causing an enhancement of cholinergic neurotransmission through activation of synaptic nicotinic and muscarinic receptors.9 The prominence of 1 did not last for too long as it was withdrawn due to its hepatotoxicity and cholinergic side effects predominantly present upon the gastrointestinal tract.10 Therefore, over the past two decades a great deal of synthetic efforts has been expended to find tacrine derivatives with improved biochemical and/or pharmacological properties. One result of such endeavors was the development of 7methoxytacrine (2; 7-MEOTA) and 6-chlorotacrine (3). 7-MEOTA, originally introduced to the Armed Forces of the Czech Republic as an antidote against incapacitating compound 3-quinuclidinyl benzilate (QNB), is a centrally acting anticholinesterase agent which is biochemically slightly less efficient than tacrine.11 The main advantage of 2 is its low toxicity presumably due to a different metabolic degradation fate than that of 1.11 6Chlorotacrine represents a derivative with more favorable inhibitory effect on acetylcholinesterase comparing to its parent compound.12 Although tacrine does no longer belong to the mainstream of AD therapy, it is still, together with its derivatives, of interest to medicinal chemists in particular due to its lead-like character, synthetic accessibility, low molecular weight and relatively easy modification.8,12 Abundant human data consistently support the idea that oxidative stress occurs as a constant feature in AD brain pathology.13 Some recent evidence even indicates that this phenomenon is an early event contributing to initial progression of the neurodegenerative process.14 Additionally, increased levels of oxidative stress biomarkers (oxidized proteins, lipids, carbohydrates, and nucleic acids) detected in the

ACS Paragon Plus Environment

Page 4 of 74

Page 5 of 74

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

brains of AD patients strongly support the so called “oxidative stress hypothesis” of AD.13 Under physiological conditions, free radicals are normally produced as a part of well-characterized metabolic pathways in the course of cellular respiration. Generally, these unstable and highly reactive species are removed by specific detoxifying enzymes and kept at relatively low levels within the cells.15 However, in some situations their generation may exceed the endogenous ability of the body to destroy them. As a consequence, oxidative homeostasis alters and leads to oxidative stress.14 Moreover, in contrast with other organs, CNS is highly sensitive to oxidative stress as it is rich in peroxidizable fatty acids, has a high demand for oxygen and a relative paucity of classical antioxidant systems.13 From the therapeutic point of view, the most simplistic interpretation of the “oxidative stress hypothesis” of AD supposes that antioxidants administration to AD patients would generally be beneficial. Unfortunately, the real scenario is not so simple.13 A wide variety of antioxidants, for instance, vitamin C, vitamin E, β-carotene, selenium etc., have been reported as neuroprotectants in in vitro studies and animal models.16 However, the success of such experiments in clinical trials has been found elusive.13,16 Among the abovementioned antioxidants vitamin E or αtocopherol is the best known natural antioxidant. Despite its outstanding capacity to absorb reactive oxygen species (ROS), vitamin E exerts one relevant drawback – high lipophilicity which causes its relatively slow absorption by cells.17 To avoid the shortcomings conferred by inconvenient lipophilicity a water soluble analogue of vitamin E – trolox (±6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) – was synthesized by Scott et al..18 Additionally, more recently, Wu et al.19,20 found out that trolox protects different cell types (e.g. human myocytes, hepatocytes and erythrocytes) against oxyradicals generated in situ, giving trolox more preferences over vitamin E.

ACS Paragon Plus Environment

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Due to the complexity of AD and involvement of different proteins and/or events in its progression, the modulation of a single target might not be sufficient enough to produce the desired effect.21 Therefore, medicinal chemists have been prompted to invest their research efforts into design of molecules that would simultaneously interact with different diseased targets as a better strategy to block the course of multifactorial diseases rather than just reducing one symptom.22,23 Such molecules have been denoted “multi-target-directed ligands” (MTDLs).24 Moreover, unceasing identification of underlying targets critically involved in the neurotoxic cycle strongly indicates that the polypharmacy regimen is the only worthwhile future direction for AD drug research.7 In recent years, a huge number of articles describing specifically developed compounds exhibiting multiple pharmacological profile has been published25–32 and exhaustively reviewed7,21,22,24, suggesting MTDLs strategy as fruitful and worthy of further exploration.

Design With improved understanding of AD pathogenesis, there is no doubt that efficient AD therapy should be able to tackle on multiple fronts. Antioxidants are thought to offer a good possibility of combating neurodegeneration, in which free radicals play a significant role but are not the exclusive drivers.14 The drugs of choice in AD treatment still remain cholinesterase inhibitors conferring, however, only limited and transient benefits to patients. Thus, these two facts have prompted the medicinal chemists to design cholinesterase inhibitors endowed with antioxidant properties to hopefully increase the likelihood of success.16 In 2005, racemic lipocrine was at the forefront of this approach.16 This compound combined the structure of the natural antioxidant – lipoic acid – with a derivative of

ACS Paragon Plus Environment

Page 6 of 74

Page 7 of 74

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

tacrine in a single molecule.33 The results exceeded the expectations. Lipocrine revealed one of the most potent anticholinesterase effects ever (IC50 = 0.253 nM), furthermore, it reduced AChE-induced Aβ aggregation (IC50 = 45 μM) and protected human neuronal cells from ROS formation evoked by oxidative stress.33 Since 2005, the repertoire of dual-binding site AChE inhibitors possessing antioxidant activity has continued to grow. In particular, ferulic acid34, melatonin35 and many others have been exploited to afford hybrid molecules with a significantly improved pharmacological profile with respect to solely anticholinesterase and/or antioxidant templates. In the study presented herein, we turned our attention to compounds combining tacrine scaffolds (1-3) with trolox (4) in order to obtain more powerful multi-target-directed candidates. The rationale for their design was based on encouraging results showing cholinesterase inhibitors endowed with antioxidant properties to be able not only to act at different pathological steps of the neurodegenerative process, but also to produce additional cytoprotective effects. The latter attribute is in all probability ascribed in particular to the presence of antioxidant moieties. In parallel, the scientific group of Xie et al. has started to work on the same compounds.36 In contrast with us, they published fewer entities exploiting only tacrine moiety. The main contribution of our extended study has been tracking the effect of other tacrine scaffolds – safer or more potent (7MEOTA or 6-chlorotacrine, respectively), as well as observation of the improvement of the pharmacological profile in vivo, which runs in parallel to a pronounced decrease in toxicity.

Chart 1. Design Strategy toward Tacrine – Trolox Hybrids 7a-u

ACS Paragon Plus Environment

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

RESULTS AND DISCUSSION Chemistry Tacrine – trolox hybrids (7a-u) were synthesized following a sequence of reactions depicted in Scheme 1. Because the target molecules encompass three parts, the synthetic approach consisted firstly in preparation of tetrahydroacridine moieties (5a-c), subsequent introduction of the linker, and finally in connection with commercially available trolox moiety. 9-Chlorotacrines (5a-c) were prepared according to the previously reported protocols.37,38 In order to insert the side chain, different alkylenediamines reacted with 5a-c to form N1-(1,2,3,4-tetrahydroacridin-9-yl)alkane1,ω-diamine intermediates (6a-u).37,39–41 Finally, these intermediates (6a-u) were coupled with trolox in the presence of N-(3-dimethylaminopropyl)-N´-ethylcarbodiimide

ACS Paragon Plus Environment

Page 8 of 74

Page 9 of 74

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

hydrochloride (EDC.HCl) and 1-hydroxybenzotriazole hydrate (HOBt) to afford target compounds (7a-u) in yields (12 – 80%). All the tacrine – trolox hybrids (7a-u) showed analytical and spectroscopic data in good agreement with their structures. Structural characterization involved melting points, NMR spectroscopy and LC - mass spectrometry.

Scheme 1a. General Approach for Synthesis of Tacrine – Trolox Hybrids 7a-u

Cholinesterase Inhibition To determine the therapeutic potential of tacrine – trolox hybrids (7a-u) for the treatment of AD, their AChE and butyrylcholinesterase (BChE) inhibitory activities were assayed by the modified spectrophotometric method described by Ellman et al. using human recombinant AChE (hAChE; E.C. 3.1.1.7) and human serum BChE (hBChE; E.C. 3.1.1.8), respectively.42,43 The former mentioned cholinesterase represents a target of the approved anti-Alzheimer’s drugs, whereas the exact function of the latter is still not

ACS Paragon Plus Environment

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

fully understood.44 Several studies indicate that BChE may compensate for the lack of AChE observed in later stages of the disease, making its inhibition potentially therapeutically desirable.45 IC50 values, derived from triplicate experiments, and selectivity indexes (SI), expressed as the ratio of IC50 (hBChE)/IC50 (hAChE), are summarized in Table 1. To allow a comparison of the results tacrine, 7-methoxytacrine, 6-chlorotacrine and trolox were used as the reference compounds. Worth to note, trolox (4) did not inhibit either enzyme. It is evident, that all tacrine – trolox hybrids (7a-u) are effective inhibitors of hAChE with the IC50 values spanning from 13.29 to 0.08 μM. All the tested compounds could be divided into three families differing in the substitution on tacrine heterocycle. In the case of 7-methoxytacrine subset (7a-g), the IC50 values ranged in micromolar scale concomitantly being more potent inhibitors (except 7d) than their prototype – 7methoxytacrine. The class represented by non-substituted counterparts (7h-n) exerted better results compared to the first group displaying the inhibitory potencies in microto submicromolar concentrations, in similar fashion like tacrine. As one would expect, insertion of chlorine atom into the tetrahydroacridine system, affording derivatives 7ou, increased inhibitory activity towards hAChE showing IC50 values in submicro- to nanomolar scale. Nevertheless, none of novel hybrids overcame the hAChE inhibitory potency of 6-chlorotacrine. Such differences in potencies are a consequence of distinct thermodynamic interactions of tacrine fragments with the catalytic active site (CAS) and peripheral anionic site (PAS) of AChE. Modification of the spacer length between two nitrogen atoms did not affect the affinity for enzyme dramatically. According to the IC50 (hAChE) it appears, that either 3- or 8-carbon alkylene chain seems to be an optimal distance for a linker between the two pharmacophore groups. Although dual site binding to AChE is expected to increase the inhibitory potency relative to the monomeric parent

ACS Paragon Plus Environment

Page 10 of 74

Page 11 of 74

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

compounds (1-4) or intermediates (6a-u) from which they are constituted, this assumption is not always fulfilled. Indeed, the hAChE inhibitory activity of tacrine – trolox hybrids (7a-u) is comparable with that of the intermediates,46,47 suggesting that coupling with trolox scaffold does not obviously affect the extent of hAChE inhibitory potency. Moreover, all the novel compounds were assessed for their ability to inhibit the activity of hBChE. As the results indicate, the target compounds are potent inhibitors of hBChE with the IC50 values varying from micro- to nanomolar range. Structure – activity relationship towards hBChE revealed trends different from those observed for hAChE which became evident especially from low linear correlation between IC50 values of 7a7u for both enzymes (R2 = 0.03) Modifications of tacrine moiety demonstrated less pronounced differences in inhibitory potencies against hBChE. Furthermore, no significant dependency on the length of the tether was found for hBChE, highlighting compounds with 4-carbons in the linker as slightly more active. From the perspective of selectivity, a switch from hBChE to hAChE preference can be observed across the three families, as follows 7-methoxytacrine derivatives (7a-g) < non-substituted tacrine counterparts (7h-7n) < hybrids bearing 6-chloro moiety (7o7u). A comparison of selectivity towards hAChE inside single groups did not show any significant differences. Overall, hybrid 7u, derived from 6-chlorotacrine, 1,8diaminooctane and trolox, proved to be the best hAChE inhibitor of this series, with the IC50 of 80 nM that is 4-fold and even 125-fold better than that of 1 and 2, respectively. However, in comparison to 6-chlorotacrine it appeared to be 4-fold weaker. Additionally, 7u exerted moderate inhibitory activity against hBChE, indicating that this candidate could be effective in treating mild to moderate as well as severe forms of AD when the role of AChE takes over BChE.

ACS Paragon Plus Environment

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 74

Table 1. In Vitro Results of Tacrine – Trolox Hybrids 7a-u and Reference Compounds 14 Compound

IC50 hAChE ± SEM (μM)a

IC50 hBChE ± SEM (μM)a

SI (hBChE/hAChE)b

Prediction of BBB penetration

DPPH assay EC50 ± SEM (μM)a

7a

4.36 ± 0.365

1.30 ± 0.173

0.30

CNS +/-

95.89 ± 4.56

CNS +/-

˃ 100.00

7b

1.54 ± 0.076

0.62 ± 0.032

0.40

7c

7.21 ± 0.550

0.29 ± 0.023

0.04

CNS +/-

˃ 100.00

CNS +/-

62.70 ± 1.22

7d

13.29 ± 1.269

0.40 ± 0.096

0.03

7e

6.61 ± 0.401

0.69 ± 0.039

0.10

CNS +/-

58.75 ± 1.23

7f

6.72 ± 0.374

0.70 ± 0.042

0.10

CNS -

59.01 ± 1.44

7g

3.48 ± 0.161

1.53 ± 0.105

0.44

CNS +/-

˃ 100.00

CNS +

˃ 100.00

7h

6.97 ± 0.315

0.06 ± 0.003

0.01

7i

0.26 ± 0.011

0.08 ± 0.004

0.31

CNS +/-

51.34 ± 4.27

7j

0.95 ± 0.028

0.08 ± 0.004

0.08

CNS -

˃ 100.00

CNS -

65.62 ± 2.31

7k

0.62 ± 0.021

0.56 ± 0.005

0.90

7l

0.36 ± 0.010

0.19 ± 0.010

0.53

CNS +/-

61.79 ± 3.24

7m

0.69 ± 0.022

0.13 ± 0.010

0.19

CNS +/-

˃ 100.00

CNS -

66.47 ± 3.36 67.83 ± 5.74

7n

0.69 ± 0.034

0.16 ± 0.011

0.23

7o

0.21 ± 0.009

0.02 ± 0.006

0.10

CNS +/-

7p

0.08 ± 0.002

0.77 ± 0.008

9.63

CNS +/-

48.83 ± 1.09

7q

0.15 ± 0.015

0.09 ± 0.003

0.60

CNS +/-

50.77 ± 6.91

7r

0.12 ± 0.010

0.13 ± 0.004

1.08

CNS +/-

49.76 ± 0.79

7s

0.10 ± 0.008

0.40 ± 0.015

4.00

CNS +/-

48.83 ± 1.09

7t

0.08 ± 0.003

0.70 ± 0.022

8.75

CNS +/-

44.24 ± 1.04

CNS +

44.09 ± 0.92

Tacrine

0.08 ± 0.003 0.32 ± 0.013

0.54 ± 0.037 0.08 ± 0.001

6.75 0.68

CNS +

˃ 100.00

7-MEOTA

10.00 ± 0.974

17.56 ± 0.795

1.76

CNS +

˃ 100.00

6-Chlorotacrine

0.02 ± 0.001

1.78 ± 0.097

100.68

CNS +

˃ 100.00

Trolox

186.40 ± 26.470

˃ 1000.00

n.d.c

CNS -

16.20 ± 0.42

7u

a

Results are expressed as the mean of at least three experiments. Selectivity for hAChE is determined as ratio IC50(hBChE)/IC50(hAChE). c Not determined. CNS + High BBB permeation predicted. CNS – Low BBB permeation predicted. CNS +/- Uncertain BBB permeation. b

Kinetic Assay As 7u displayed the highest activity towards hAChE, it was selected, as a representative compound of the series, for kinetic assay in order to obtain information about the mode of inhibition and binding site of target compounds. The mechanism of inhibition was analyzed by recording substrate concentration – enzyme velocity curves in the presence

ACS Paragon Plus Environment

Page 13 of 74

of different concentrations of 7u. Figure 1 demonstrates Lineweaver–Burk plots of tacrine – trolox hybrid 7u. Graphical analysis revealed both increasing slopes (decreased Vmax) and increasing intercepts (increased Kmax) at increasing concentration of compound 7u. This pattern indicates mixed-type inhibition (p˂0.05).48 Therefore, in agreement with the results of molecular modeling studies, 7u binds simultaneously to the catalytic site as well as to the peripheral anionic site of hAChE. From the perspective of AD therapy this is a highly desirable effect since aggregation of Aβ and subsequent neurotoxic cascade are catalyzed particularly by PAS of AChE.49 Replots of the slope versus concentration of 7u gave an estimate of the competitive inhibition constant, Ki, of 49.5 ± 19.1 nM, which is consistent with the IC50 (hAChE) value obtained above.

1.5×10 8

7u 100 nM 178 nM 316 nM 562 nM

-25000

-15000

v-1 (kat-1)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

1.0×10 8

5.0×10 7

-5000 -1

5000

15000

-1

[ATCh] (M )

Figure 1. Steady-state inhibition of AChE hydrolysis of acetylthiocholine (ATCh) by compound 7u. Lineweaver−Burk reciprocal plots of initial velocity and increasing substrate concentrations (0.078 − 0.625 mM) are presented. Lines were derived from a weighted least-squares analysis of the data points.

ACS Paragon Plus Environment

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Modeling Studies In order to find out the binding mode for the most active compound in each of three families, docking simulation studies within hAChE active site were conducted using AutoDock Vina 1.1.2.50 For the docking studies X-ray crystallographic structure of hAChE in complex with donepezil (PDB ID: 4EY7)51 was utilized. The structure of hAChE model was checked by Protein Preparation Wizard (Maestro Version 10.2.011, Schrödinger) to reveal missing atoms, bond angle and length deviations, improper torsion angles, steric clashes, isolated water clusters, etc. which could disturb the molecular docking calculations. The hAChE model passed all the important tests and hence it was submitted to a specific preparation in MGL Tools for docking in AutoDock Vina. Although the presence of structural water molecules plays an important role in formation of ligand–receptor complex, it is not straightforward to simulate such multi-particle system heuristically using AutoDock Vina.52 Therefore, our docking calculations approximate to the system ignoring the temperature and explicit solvent effects. However, the binding modes and energies obtained on such level of theory still provide principal information about the non-covalent interactions within the ligand-receptor complex and are well accepted in current academia as a fundamental elucidation of the structure-activity relationships (SAR). A considerably more appropriate approach to investigate the influence of the temperature and water in the molecular system is through molecular dynamics using suitable software and hardware. Notably, the flexibility and size of these ligands provided several solutions to energy minimization problem during the process of flexible molecular docking. A thorough inspection of the binding modes and energy values was necessary prior to selecting the putative binding modes that are discussed below. In general, the plausible binding

ACS Paragon Plus Environment

Page 14 of 74

Page 15 of 74

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

modes of 7b, 7i and 7u suggest that all these ligands are oriented from the bottom of the gorge, extending through mid-gorge region to the PAS of the enzyme (Figures 2 and 3). The studied ligands were used in a protonated form (pH = 7.4) as predicted by MavinSketch 6.2.0 software (http://www.chemaxon.com). Tacrine moieties of all the ligands are stacked between Trp286 and Tyr124 (7b, 7u) or Tyr341 (7i) of the PAS of the enzyme, while trolox units occupy the CAS in the proximity of the catalytic triad (i.e. Ser203, His447 and Glu334). Such ligand accommodation may explain why do the target compounds exert lower anticholinesterase potencies than other MTDLs recently published by our group.25,26 Interestingly, the inverse ligand lodging of tacrine - trolox subset with the tacrine moiety in the CAS and trolox unit in the PAS of the enzyme has been recently reported.36 The reason of this discrepancy might lie in: i) ligand and enzyme protonation (authors of the previous study probably omitted ligand as well as enzyme protonation); ii) length of the linker (the most active analogue contained six carbons in the alkylene linker, compared to three-carbon chained ligand 7i, iii) different setup of flexible residues and iv) restricted exhaustiveness of the calculations. More importantly, the results of performed docking studies are consistent with the outcomes of kinetic analysis for 7u that indicated mixed type inhibition. The estimated binding energies were comparable for all the ligands with minimal fluctuations around -15 kcal/mol (7b = -15.5 kcal/mol; 7i = -15.3 kcal/mol; 7u = -15.5 kcal/mol). These values suggest that all the ligands can be considered as very potent hAChE inhibitors. At the mouth of the gorge, the tricyclic tetrahydroacridine core of 7b is engaged in the formation of characteristic “sandwich-like” parallel π-π interactions between Tyr124 (3.6 Å) and Trp286 (3.6 Å). Moreover, Phe297 (3.8 Å) also contributes to ligand anchoring by formation of π-π interaction. Protonated nitrogen of tacrine moiety is

ACS Paragon Plus Environment

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

cation-π bound to Trp286 and Tyr72 (3.4 Å), the key residues of the PAS. Attached secondary amino group provides hydrogen contact to hydroxyl of Tyr124 (2.6 Å). Tyr341 (3.7 Å) and Phe297 (4.2 Å) contribute to stability of enzyme-7b complex by hydrophobic interactions. The trolox moiety is buried deep in catalytic region of hAChE adopting parallel π-π interactions with Trp86 (3.7 Å). Gly121 (4.1 Å) of the oxyanion hole is engaged in the weak polar contact with trolox motif. The presented pose depicts Figure 2A, 2B that also presumes hydrogen bonding between hydroxyl group of trolox and Tyr133 (2.9 Å). Finally, only some weak hydrophobic interactions might be seen between the catalytic triad residues and the trolox moiety. Steric clashes caused by the presence of 7-methoxy group of 7b might be responsible for a 180° rotation of tetrahydroacridine unit conferring slightly lower activity in comparison to 7i and 7u. Analogue 7i (Figure 2C, 2D) of tacrine - trolox subset exerts the highest affinity towards hAChE. It also contains three-membered alkylene chain between proximal trolox unit and distally lodged tetrahydroacridine scaffold. Tacrine moiety of 7i fits well within the PAS of hAChE in similar fashion as in the aforementioned enzyme-7b complex, however being 180° rotated. This includes parallel π-π as well as cation-π interactions with Trp286 (3.8 Å), Tyr72 (3.9 Å) and Tyr124 (3.8 Å). Tyr124 also revealed hydrogen bond accepting properties, in this case to hydrogen of amide group of 7i (2.2 Å). Similarly, Tyr341 (3.3 Å) is a hydrogen bond donor interacting with the amide group of 7i. The linker delineates the cavity mid-gorge and it is surrounded by several amino acid residues forming hydrophobic interactions (e.g. Phe297, Tyr341, Tyr124). Trolox unit occupies CAS of hAChE with close spatial orientation as 7b including hydrogen contact of hydroxyl with Tyr133 (2.6 Å) and parallel π-π interaction with Trp86 (3.6 Å). Even in this case, the catalytic triad seems to be unaffected.

ACS Paragon Plus Environment

Page 16 of 74

Page 17 of 74

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

The extended conformation of 7u (Figure 2E, 2F) has respective lengths of 20.6 Å from a tacrine unit wing till the end of trolox moiety, which is sufficient enough to cover both the PAS and CAS, leading this compound to act as a dual binding site inhibitor. The prediction of the binding mode of 7u prioritizes the position of tacrine moiety between Trp286 (3.6 Å) and Tyr124 (3.6 Å). In a similar way, protonated nitrogen of tacrine ring is stabilized by cation-π interactions with Tyr72 (3.6 Å) and Trp286. Chlorine atom attached to tetrahydroacridine scaffold might be responsible for the higher affinity compared to 7b and 7i through hydrophobic interactions in the PAS region. The alkylene linker fully spans the gorge of the enzyme and enables proper orientation of trolox to the CAS. Apart from prevailing π-π interactions between trolox moiety and Tyr337 (3.6 Å) and Tyr341 (3.8 Å), amide group of the tether shows hydrogen bond interactions with Ser203 (3.2 Å) as well as with His447 (4.2 Å) of the catalytic triad. These interactions with the catalytic triad, lacking in the case of 7b and 7i, may be a possible reason of prominent anticholinesterase activity of 7u.

ACS Paragon Plus Environment

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2. A, C, E: 2D representation of binding modes of the most potent compounds of 7-MEOTA - trolox (7b, A), tacrine - trolox (7i, C) and 6-chlorotacrine – trolox structural subsets (7u, E) in the hAChE active gorge obtained by flexible molecular docking. B, D, F: 3D overview of the best docked poses of 7b, 7i and 7u. Spatial orientation of residues involved in the ligand-enzyme interactions is illustrated with blue carbon atoms. The most active tacrine – trolox hybrids are displayed as sticks in green carbon atoms (7b, B), light-blue carbon atoms (7i, D) and purple carbon atoms (7u, F). The conformation of the rest of the enzyme is shown as light-grey ribbons. Black dashed lines display important interactions between ligands and enzyme (e.g. H-bond, π-π and cation-π interactions). Residues of the catalytic triad (Glu202, Ser203, His447) are depicted as yellow carbon sticks. Water molecules are omitted for the sake of clarity. Figures A, C and E were created with PoseView software (PoseView, http://poseview.zbh.uni-

ACS Paragon Plus Environment

Page 18 of 74

Page 19 of 74

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

hamburg.de/poseview/wizard, 2012); figures B, D and F were generated with PyMol 1.30.

Figure 3. Superimposition of the best docked poses of 7b (green carbon atoms), 7i (light-blue carbon atoms) and 7u (purple carbon atoms) in the active site of hAChE. The catalytic site of the enzyme is represented by Trp86 and Tyr337 (dark blue carbon atoms), whereas the peripheral anionic site of the enzyme is denoted by Trp286 (dark blue carbon atoms). Crystal structure of hAChE is represented in ribbon (light grey) diagram. Figure was created with PyMol 1.30.

In Vitro Antioxidant Properties In order to prove the radical scavenging activities of tacrine – trolox hybrids (7a-u) 1,1diphenyl-2-picryl-hydrazyl (DPPH) assay was used.53 Trolox itself was selected as a positive control, whereas tacrine and its modified counterparts served as negative standards as they show negligible radical-capturing abilities. Coupling of trolox with tacrine moieties caused decrease in the antioxidant activity compared with trolox. This

ACS Paragon Plus Environment

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

phenomenon may be explained by a conversion of free carboxylic group of 4 into amides making further rearrangement into appropriate quinones less feasible.54 Overall, all the target compounds displayed moderate to good antioxidant capacities, highlighting compound 7u, bearing 6-chlorotacrine, 8-carbon linker and trolox fragment, as the best antioxidant of this family.

In Vitro Blood–Brain Barrier Permeation Since one of the major requirements of anti-AD drugs is to reach their therapeutic targets, screening of the blood-brain barrier (BBB) penetration is of great importance. To investigate whether the target compounds 7a-u would be able to penetrate into the brain, a parallel artificial membrane permeation assay (PAMPA) was used. This simple and rapid model, described by Di et al.55, was slightly modified for hardly soluble compounds according to Xie et al..36 To determine the in vitro permeability (Pe) of tacrine – trolox hybrids (7a-u) as well as reference compounds (1-4) through a lipid extract of porcine brain PBS:EtOH (70:30) solution was used. It has been established that compounds with the Pe values above 4 x 10-6 cm.s-1 penetrate into CNS easily (CNS +), while derivatives with a Pe value below 2 x 10-6 cm.s-1 do not (CNS -). In hybrids having the permeability values between these boundary limits it is difficult to estimate, whether they cross BBB or not (CNS +/-).55 The assay was validated by donepezil and theophylline, which were tested as positive and negative control, respectively. The results of control compounds predicted correctly their real BBB permeability pointing out the reliability of the PAMPA experiment. The results of target hybrids are listed in Table 1 (for exact Pe values of single compounds see Table S1 of Supporting information).

ACS Paragon Plus Environment

Page 20 of 74

Page 21 of 74

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

The majority of the tested compounds displayed permeability values in the boundary limits, pointing out that it is unclear whether do they penetrate BBB by passive diffusion or not. Few exceptions – derivatives 7h and 7u – exerted Pe values over the above limits (4.4 and 5.0, respectively), indicating that they could easily cross BBB.

In Vitro Hepatotoxicity on HepG2 Cells As highlighted above, the biggest disadvantage of 1 and concurrently the main reason of its withdrawal from the market is its hepatotoxicity. To this end, to determine whether tacrine – trolox hybrids (7a, 7b, 7g, 7i, 7k, 7l, 7s, 7t and 7u) are more safe than their monomeric

parent

compounds

1-4

a

3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT) assay on human hepatoma cell line (HepG2) was carried out. Selection of the tested compounds (three representatives of each family, differing in the substitution on tacrine core) was performed pursuant to their anticholinesterase activities. The influence of selected and reference compounds on viability of HepG2 cells is summarized in Table 2. It is necessary to mention, that differences in non-toxic concentrations are due to limited solubility of the tested compounds. Based on the results observed after 24 h incubation several SAR propositions could be drawn. The most dramatic decrease in the cell viability was observed in 7-methoxy representatives, less significant in tacrine counterparts and as non-toxic proved to be hybrids carrying 6-chloro group (with the exception of 7s). Such results may sometimes be controversial, likewise in the case of 7-MEOTA. The IC50 value of 2 is lower than that of tacrine indicating higher in vitro cytotoxicity on hepatocytes. However, in vivo toxicity shows the inverse pattern, where tacrine is a more toxic drug. In vitro and in vivo toxicity may substantially differ due to the differences in pharmacokinetics and metabolism,

ACS Paragon Plus Environment

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

where biotransformation processes may play a crucial role in toxicity. The clincher of this statement is 7-MEOTA, that exerts slightly higher risk for the cells in vitro, but neither acute nor chronic hepatotoxicity has been observed in vivo.11 For this reason, 7u has been forwarded for further in vivo safety study as a non-toxic derivative with prominent in vitro results to confirm or to disprove the outcomes of aforementioned study.

Table 2. Results of In Vitro Hepatotoxicity on HepG2 cells IC50 HepG2 ± SEM (μM)d 7a 4.90 ± 0.53 7b non-toxice 7g 0.92 ± 0.08 7i 13.19 ± 0.60 7k 8.70 ± 0.01 7l 2.00 ± 0.03 7s 0.70 ± 0.02 7t non-toxicf 7u non-toxicf Tacrine 19.37 ± 0.75 7-Methoxytacrine 11.50 ± 0.30 6-Chlorotacrine 7.13 ± 0.11 Trolox non-toxicg d Results are expressed as the mean of at least three experiments. e Non-toxic at 2 μM concentration. f Non-toxic at 0.5 μM concentration. g Non-toxic at 512 μM concentration. Compound

Microsomal In Vitro Metabolism and Stability Analysis of Tacrine – Trolox Hybrid 7u All chemical compounds introduced to an organism are subjected to complex metabolic pathways which determine their life-time and fate under specific biochemical conditions. Metabolism as well as other pharmacokinetic processes (absorption, distribution and elimination) significantly participates in the active principle behind the

ACS Paragon Plus Environment

Page 22 of 74

Page 23 of 74

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

effect of every drug. Drug metabolism is of particular importance amongst the pharmacokinetic processes because it can considerably change the pharmacological profile of the drug, for instance, by rapid degradation of the parent chemical structure and/or by forming highly bioactive metabolites responsible for unexpected biological effects or toxicity.56 On the other hand, drug metabolism may be also considered as an effort of an organism to degrade and eliminate relatively high lipophilic substances through specific enzymatic systems. In order to facilitate the elimination of endogenous as well as exogenous substances, a series of biochemical reactions (i.e. modifications and conjugations) takes place to decrease the lipophilicity of xenobiotic compounds. Metabolism of xenobiotics is commonly divided into three phases: non-synthetic I. phase, subsequent II. phase of synthetic conjugations, and final III. phase of further modifications like splitting of the conjugates or acetylation. The first phase modifications lead generally to oxidation, reduction, hydrolysis or cyclization of the parent structure and are mainly catalyzed by liver cytochrome P-450 dependent oxidase system. In the present study, a metabolic experiment with human liver microsomes (HLM) was performed to identify I. phase metabolites and to evaluate the in vitro stability of compound 7u after 1 h incubation under conditions mimicking the human liver enzyme system. In order to get a quantitative insight into the metabolic processes, a calibration measurement on 8 triplicate concentration levels (0.5, 1, 5, 10, 25, 50, 75, 100 µg/mL) was carried out using LC-MS/MS system. Linearity of the calibration curve was confirmed with R2 = 0.9998, precision (given as relative standard deviation of the area under the peak) fluctuated between 0.15 – 0.97%, and accuracy of quantification (given as relative recovery) was 97 ± 6% over the whole concentration range (i.e. 0.5 - 100 µg/mL). The UV-chromatogram and basic chromatographic properties of the target compound 7u detected at 254 nm are illustrated in Figure S1 (Supporting information).

ACS Paragon Plus Environment

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Stability and formation of I. phase metabolites were investigated in microsomal incubation system containing 1 mg/mL concentration of microsomal enzymes. The metabolic in vitro experiment itself was performed at three different concentrations of compound 7u (1, 20, 50 µg/mL, and blank sample) for 1 h under standardized conditions. After quenching the metabolism with acetonitrile, the samples were analyzed by LC-MS/MS to identify the formed metabolites. A multiple reaction monitoring (MRM) approach combining full-scan and all-ions-fragmentation (AIF) in the range 100 – 1500 m/z was employed. Along with the tandem MS/MS spectra, UVchromatograms detected at 210, 254, 278 and 290 nm were recorded as well. The results of the metabolic assay at three different concentrations of 7u (blank sample, 1, 20 and 50 µg/mL) are depicted in Figure 4. The fragmentation of 7u in MS/MS and suggested chemical structures are outlined in Figure S2 (Supporting information).

Figure 4. UV-chromatograms (λ = 254nm) of microsomal incubation experiments. A – blank assay in the absence of 7u; B – metabolites formed at 1 µg/mL concentration of

ACS Paragon Plus Environment

Page 24 of 74

Page 25 of 74

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

7u; C - metabolites formed at 20 µg/mL concentration of 7u; D - metabolites formed at 50 µg/mL concentration of 7u. Identity of the peaks was determined from MS/MS spectra.

According to quantification against the calibration curve determined by UV (λ = 254nm), the samples after 1 h incubation in HLM system contained 58.18 ± 3.62% of the original 1 µg/mL concentration of the parent compound 7u, 85.55 ± 0.03% of 20 µg/mL and 85.42 ± 0.43% of 50 µg/mL. Generally, around 85% of the original amount of 7u retained unchanged after 1 h incubation in HLM system, with the exception of 1 µg/mL concentration where a substantial part of 7u was biotransformed. Analogical results would be observed in MS spectra of the samples, if extracted ion chromatograms (XIC) in the range 592.320-592.335 m/z (i.e. [M+H]+ of 7u) were analyzed (Figure 5). Corresponding to the principles of metabolism, the identified metabolites occurred at lower retention time in chromatograms than that of the parent structure 7u (Rt = 7.52 min).

Figure 5. Ion chromatograms in a full-scan mode resulting from LC-MS/MS analyses of metabolized samples. Compound 7u was exposed to HLM at three concentrations: 1, 20,

ACS Paragon Plus Environment

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

and 50 µg/mL. On the left side, there is a total-ion-current chromatogram (TIC), on the right side, there is extracted ion chromatogram related to the mass window of 592.320592.335 m/z which corresponds to the molecular weight of 7u ([M+H]+ = 592.33005). The highest peaks in the chromatograms eluting at 7.52 min are related to 7u.

Through the analysis of MS/MS spectra, 4 main metabolites (M1, M2, M3, M4) were revealed after 1 h incubation with HLM (Figure 6). M1 was revealed at Rt = 7.35 min with m/z=608.325 (z=1), M2 at Rt = 7.10 min with m/z=624.320 (z=1), M3 at Rt = 6.56 min with m/z=361.204 (z=1), and M4 at Rt = 6.52 min with m/z=375.183 (z=1). The relative abundance of metabolites determined from XIC chromatograms decreased in the following order: M1 (100%), M3 (8.6%), M2 (7.4%), M4 (3.1%) (Figure 7). All of four metabolites involved insertion of one or two oxygen atoms which were easily deciphered through mathematical prediction of molecular formula from the highresolution masses in Xcalibur 3.0.63. Nonetheless, a profound analysis of the MS/MS spectra using Mass Frontier 7.0 and Metworks 1.3 SP3 had to be performed in order to elucidate the chemical structures of the metabolites. The masses of proposed structures of found metabolites corresponded well with experimental MS/MS fragment masses, although further investigation, for instance, by NMR spectroscopy, should be accomplished to prove their real chemical identity. For the main metabolites and their fragments, the ion isotopic envelopes were examined as well (Figure S3, S4, S5, S6, S7, S8 of Supporting information). The MS/MS spectra of M1, M2, M3, and M4 are shown in Figure 8.

ACS Paragon Plus Environment

Page 26 of 74

Page 27 of 74

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

Figure 6. Proposed chemical structures of found metabolites in HLM enzymatic system after 1 h of incubation. The area under peak in XIC chromatograms decreased as follows: M1 (100%), M3 (8.6%), M2 (7.4%), M4 (3.1%).

ACS Paragon Plus Environment

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 7. Superimposed XIC chromatograms of found metabolites of 7u modified by HLM. The major metabolite M1 (yellow) was identified as an oxidation and hydroxylation product resulting from the reactivity of trolox moiety. M2 (blue) is a twice hydroxylated product of the parent structure that was further split and sequentially oxidized to an alcohol (M3, green) and carboxylic acid (M4, red).

ACS Paragon Plus Environment

Page 28 of 74

Page 29 of 74

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

Figure 8. MS/MS spectra of four main phase I metabolites of 7u. A – metabolite M1 identified as once hydroxylated parent structure (m/z=608.324, z=1); B – metabolite M2 identified as twice hydroxylated parent structure (m/z=624.319, z=1); C - metabolite M3 identified as split and hydroxylated parent structure (m/z=361.204, z=1); D metabolite M4 identified as split and carboxylated parent structure (m/z=375.183, z=1).

Increased occurrence of metabolite M1, identified as an oxidized and hydroxylated parent structure 7u, may be explained by chemical reactivity caused by trolox moiety. The antioxidant mechanism of trolox has been thoroughly investigated in the literature what helped to predict key chemical processes in metabolic experiments with HLM.54,57,58 In the first step of oxidation, 6-hydroxy function of trolox loses hydrogen atom to form phenoxy radical that rapidly rearranges to 2,4,5-trimethyl-3,6dioxocyclohexa-1,4-dien moiety with the radical electron localized at C2 carbon. According to Thomas and Bielski, this intermediate undergoes quick hydroxylation in aquatic

environment

resulting

in

2-hydroxy-2-methyl-4-(2,4,5-trimethyl-3,6-

ACS Paragon Plus Environment

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

dioxocyclohexa-1,4-dien-1-yl)butanoyl function.59 MS/MS analysis of M1 confirmed that similar oxidation process probably also occurs in case of 7u in the presence of HLM. However, no further quantitatively significant oxidation of trolox moiety like hydroperoxidation, epoxidation or secondary hydroxylation as observed by Bentayeb et al.54 has been detected. In M2, insertion of the second hydroxyl function was found in the aliphatic linker chain connecting 3 and 4 frameworks. Since in MS/MS spectra a complete series of fragments with CH2 to (CH2)8 bound to 6-chlorotacrine moiety and one fragment corresponding m/z to 6-chlorotacrine-(CH2)8O (m/z=359.188) was identified, we propose that the second hydroxyl group in M2 metabolite was located at C8 of the aliphatic linker chain. Analogical analysis of MS/MS spectra led us to characterize structures of M3 and M4 as a decomposed and oxidized products of M2, respectively. Interestingly, the second part of M2 containing trolox motif was not found in full-scan MS spectra what suggests that the fragment was metabolized by HLM system to smaller and/or to hardly protonable structures. On the other hand, M3 and M4 both contain basic nitrogen in 6-chlorotacrine moiety which allows the structures to be detected in positive mode of MS as protonized ions. M3 and M4 produced very similar MS/MS fragments what supports the hypothetical chemical structures that have been ascribed to them. In M3, the hydroxyl group was found located at the terminal C8 carbon of 6-chloro-N-octyl-1,2,3,4-tetrahydroacridin-9-amine. The same C8 carbon was further oxidized by HLM to carboxylic group providing M4. The split and oxidation of 7u brought about a drop in lipophilicity, however, the rate of formation of M3 and M4 seems to be rather slow to significantly support the elimination of the parent structure. Importantly, no significant metabolic changes in 6-chlorotacrine moiety were observed. Throughout MS/MS spectra only unaffected 6-chlorotacrine fragment (m/z=233.084) was revealed. In contrary to well-known metabolism of 1, where formation of tacrine-1-

ACS Paragon Plus Environment

Page 30 of 74

Page 31 of 74

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

ol, tacrine-2-ol, tacrine-4-ol, tacrine-7-ol, tacrine-1,3-diol or tacrine-1,7-diol was described as phase I metabolites, the 6-chlorotacrine substructure in 7u stayed stable after 1 h of incubation in HLM enzymatic system.60–64 However, only an extended research of a longer exposition to HLM as well as study of phase II metabolism could provide a deeper view of the metabolic fate of 7u. In present study, only major metabolites were detected and identified, although in the MS spectra several minor or traces of other products were found as well which unfortunately were very difficult to characterize. Thus, metabolites with the concentration lower than 1% comparing to the abundance of M1 were neglected. Nonetheless, we screened the full-scan MS spectra for 10 other non-specific metabolic changes of the parent structure 7u (i.e. introduction of 3 or 4 hydroxyl functions, demethylation, demethylation-oxidation, carboxylation, epoxidation, N-oxidation, hydroperoxidation, dechloration and dehydrogenation). Among the nonspecific metabolic changes, the ion intensities of 107 (about 1% of M1 intensity) were approached for structures corresponding to N-oxidation (m/z=607.317) and dehydrogenation (m/z=590.314) of the parent structure 7u. To sum up, the aforementioned data suggest that 7u may circulate in the organism for a long period as more than 85% of parent compound remain unchanged after 1 h incubation in HLM system providing thus enough time to cross BBB and reach the desired targets. Moreover, the results of the metabolic study are in consensus with the data obtained in hepatotoxicity assay as no toxic hydroxylated metabolite of tacrine emerges.

In Vivo Toxicity In addition to high activity, low in vitro hepatotoxicity, BBB permeation and the absence of potentially toxic hydroxylated metabolites of tacrine scaffold, overall low toxicity

ACS Paragon Plus Environment

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

would be of critical importance for drug-likeness of tacrine – trolox hybrid 7u. Therefore, to achieve the goal, its acute toxicity was evaluated. The study was performed on rats by the assessment of LD50 value and corresponding 95% confidence limits (95% CL) using probit-logarithmical analysis of death occurring within 48 h after i.m. administration of 7u, dissolved in corn oil, at five different doses with eight animals per dose.65 For the comparative purposes, the parent compound – 3 – administered under the same conditions was evaluated as well. The results are shown in Table 3. The LD50 value of newly developed tacrine – trolox hybrid 7u is at least 67-fold higher than that observed in 3, what is in striking line with the findings gained by in vitro assays. Unfortunately, the limitations in solubility of 7u did not allow determining a real value of LD50. However, despite this fact, it should be stressed that due to a low toxicity of 7u it can be used in relatively higher doses with no or minimum adverse effects compared to its parent compound – 6-chlorotacrine.

Table 3. Results of Acute Toxicity of 7u and Reference Compound 3 after i.m. Administration in Rats Compound

LD50 (mg/kg) ± 95% IS

7u

˃ 500

6-Chlorotacrine

7.5 (4.77 ± 10.7)

CONCLUSION Most of the antioxidants studied so far have had come to grief in AD clinical trials. Nevertheless, expanding research still continues to link oxidative stress to AD ethiology. Indisputably, redox dysregulation belongs to the key pathological pathways in a complex nature of AD pathogenesis although considerable evidence increasingly indicates that

ACS Paragon Plus Environment

Page 32 of 74

Page 33 of 74

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

merely hitting oxidative stress will not be enough to block the neurodegenerative process. Research has thus gradually moved from an oxidative-stress-centered approach to design of the antioxidants endowed with other relevant anti-AD abilities, which could in

tandem

contribute

to

combating

the

disease.16

Therefore,

endowing

anticholinesterase agents, currently being almost exclusive therapeutics for AD treatment, with antioxidant abilities represents an intriguing strategy for broadening the efficacy of not only AChE inhibitors but antioxidants as well. This assumption has been fully confirmed by the series of tacrine – trolox hybrids (7a-u) presented herein. Several of these compounds proved to have better cholinesterase inhibitory activity in relation to a reference compound – tacrine. However, achievement of potent inhibitors of the AChE CAS would not represent a significant improvement unless there is a concomitant blocking of the PAS of the enzyme which is associated with the neurotoxic cascade of AD through AChE-induced Aβ aggregation. The abovementioned requirement was confirmed by a kinetic analysis of the best inhibitor of the series 7u. Nevertheless, the suggestion that 7u could possibly inhibit Aβ-peptide fibril formation has to be unambiguously proven in additional investigations. Interestingly, molecular modeling studies, necessary to validate the results derived from the kinetic assay, revealed that 7u binds to the enzyme in inverse fashion comparing with other dual-binding-site inhibitors of AChE incorporating tacrine template, i.e. trolox moiety interacts with CAS, whereas tacrine framework binds to the PAS of the enzyme. As one of the building blocks in the design strategy of the target compounds was an antioxidant compound – trolox, it was expected that these hybrids would exert ROS scavenging ability as well. This was confirmed by the DPPH assay. The “key” assay in design of novel tacrine-based MTDLs is determination of their hepatotoxicity either in vitro or in vivo. In our case, the results exceeded the expectations, exerting non-toxic

ACS Paragon Plus Environment

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

effect on HepG2 cells in three of nine (7b, 7t and 7u) derivatives. In order to explain the non-hepatotoxic effect of 7u a metabolic assay was conducted. It disclosed that no potentially toxic hydroxylated derivative of tacrine scaffold emerges under experimental conditions. In addition, it was determined that more than 85% of the parent structure 7u remains unchanged after 1 h incubation in human liver microsomes system, providing thus enough time to cross BBB and reach the desired targets in CNS. In light of promising in vitro results, a final comment is deserved to in vivo evaluation of acute toxicity of 7u administered i.m. to rats. At concentration 500 mg/kg the animals did not exert any symptom of intoxication, comparing to parent compound – 6-chlorotacrine – which LD50 value is at least 67-fold lower than that of 7u. In conclusion, this work clearly validates the MTDLs strategy and demonstrates that structurally novel tacrine hybrids endowed with antioxidant properties may lead to an amplified anti-AD synergic effect. However, to obtain the proof of concept for the outstanding results of hybrid 7u further pharmacokinetic evaluation on animal models of AD is required.

EXPERIMENTAL SECTION Chemistry General Chemical Methods All the chemical reagents used were purchased from Sigma-Aldrich (Czech Republic). Solvents for synthesis were obtained from Penta chemicals Co. The course of the reactions was monitored by thin layer chromatography (TLC) on aluminium plates precoated with silica gel 60 F254 (Merck, Czech Republic) and then visualized by UV 254. Melting points were determined on a microheating stage PHMK 05 (VEB Kombinant Nagema, Radebeul, Germany) and are uncorrected. Uncalibrated purity was

ACS Paragon Plus Environment

Page 34 of 74

Page 35 of 74

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

ascertained by LC-UV using a reverse phase C18 chromatographic column. All the biologically tested compounds exhibited purity 96 – 99% at a wavelength 254 nm. NMR spectra of target compounds were recorded on Varian Mercury VX BB 300 (operating at 300 MHz for 1H and 75 MHz for 13C) or on Varian S500 spectrometer (operating at 500 MHz for 1H and 126 MHz for

13C;

Varian Comp. Palo Alto, USA). Chemical shifts are

reported in parts per million (ppm). Spin multiplicities are given as s (singlet), bs (broad singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quartet), or m (multiplet). The coupling constants (J) are reported in Hertz (Hz). High-resolution mass spectra (HRMS) were determined by Q Exactive Plus hybrid quadrupole-orbitrap spectrometer.

General Procedure for Synthesis of Tacrine – Trolox Hybrids (7a-u) Trolox (4, 0.50 g, 2.00 mmol), HOBt (0.27 g, 2.00 mmol) and appropriate N1-(1,2,3,4tetrahydroacridin-9-yl)alkane-1,ω-diamine (6a-u, 2.00 mmol) were treated with 50 mL of acetonitrile. Additional 15 mL of methanol were poured into mixture to complete dissolution. Finally EDC.HCl (0.38 g, 2.00 mmol) was added. The mixture was stirred at RT for 72 h. Crude product devoid of excessive solvent was purified by column chromatography, using ethyl acetate/methanol/triethylamine (50/1/0.2) as eluent. Evaporation of the solvent gave the desired product.

6-Hydroxy-N-{2-[(7methoxy-1,2,3,4-tetrahydroacridin-9-yl)amino]ethyl}-2,5,7,8-tetramethyl-3,4dihydro-2H-1-benzopyran-2-carboxamide (7a). Yield 74%; mp 69.0 – 71.3°C. Purity: 98%. 1H NMR (300 MHz, CDCl3-d) δ 7.76 (d, J = 9.1 Hz, 1H), 7.22 – 7.15 (m, 2H), 6.85 (bs, 1H), 4.23 (bs, 1H), 3.87 (s, 3H), 3.50 (m, 4H), 2.97 (t, J = 6.0 Hz, 2H), 2.65 (t, J = 5.9 Hz, 2H), 2.60 – 2.20 (m, 2H), 2.13 (s, 3H), 2.07 (s, 3H),

ACS Paragon Plus Environment

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

2.03 (s, 3H), 1.95 – 1.75 (m, 6H), 1.48 (s, 3H).

13C

NMR (75 MHz, CDCl3-d) δ 175.77,

156.20, 156.03, 149.29, 145.90, 143.81, 142.90, 129.91, 122.24, 121.59, 120.94, 120.27, 119.68, 117.63, 117.29, 101.18, 78.17, 55.45, 48.85, 40.39, 33.40, 29.57, 25.04, 24.04, 22.96, 22.65, 20.32, 12.39, 11.92, 11.46. HRMS [M+H]+: 504.2853 (calculated for [C30H38N3O4]+: 504.2818). 6-Hydroxy-N-{3-[(7-methoxy-1,2,3,4-tetrahydroacridin-9-yl)amino]propyl}2,5,7,8-tetramethyl-3,4-dihydro-2H-1-benzopyran-2-carboxamide (7b). Yield 69%; mp 96.6 – 100.2°C. Purity: 99%. 1H NMR (300 MHz, CDCl3-d) δ 7.79 (d, J = 9.1 Hz, 1H), 7.25 – 7.16 (m, 2H), 6.85 (t, J = 6.2 Hz, 1H), 4.38 (bs, 1H), 3.88 (s, 3H), 3.53 – 3.36 (m, 4H), 3.32 – 3.12 (m, 2H), 3.01 (t, J = 6.1 Hz, 2H), 2.73 (t, J = 6.0 Hz, 2H), 2.63 – 2.33 (m, 2H), 2.03 (s, 3 + 3H), 1.99 (s, 3H), 1.95 – 1.80 (m, 4H), 1.68 (q, J = 6.3 Hz, 2H), 1.54 (s, 3H).

13C

NMR (75 MHz, CDCl3-d) δ 175.21, 156.30, 156.17, 149.46, 145.65,

144.06, 143.06, 130.02, 121.87, 121.61, 120.48, 119.34, 118.30, 117.70, 100.88, 78.37, 55.39, 45.29, 36.89, 33.57, 31.06, 29.64, 25.20, 24.82, 23.05, 22.83, 20.54, 12.15, 11.85, 11.37. HRMS [M+H]+: 518.3002 (calculated for [C31H40N3O4]+: 518.2974). 6-Hydroxy-N-{4-[(7-methoxy-1,2,3,4-tetrahydroacridin-9-yl)amino]butyl}-2,5,7,8tetramethyl-3,4-dihydro-2H-1-benzopyran-2-carboxamide (7c). Yield 60%; mp 136.5 – 138.2°C. Purity: 99%. 1H NMR (300 MHz, CDCl3-d) δ 7.78 (d, J = 9.1 Hz, 1H), 7.22 - 7.17 (m, 1H), 7.15 (d, J = 2.7 Hz, 1H), 6.49 (t, J = 6.1 Hz, 1H), 3.88 (s, 3H), 3.36 – 3.24 (m, 4H), 2.99 (t, J = 6.1 Hz, 2H), 2.68 (t, J = 6.0 Hz, 2H), 2.61 – 2.26 (m, 2H), 2.16 (s, 3H), 2.14 (s, 3H), 2.06 (s, 3H), 1.92 – 1.80 (m, 6H), 1.67 – 1.33 (m, 3 + 4H). 13C

NMR (75 MHz, CDCl3-d) δ 174.46, 156.08, 149.61, 145.83, 144.05, 143.05, 129.95,

122.19, 121.50, 121.21, 120.15, 119.78, 117.85, 117.46, 101.54, 78.21, 55.41, 48.56, 38.57, 33.48, 29.53, 28.54, 27.13, 24.69, 24.36, 22.94, 22.69, 20.48, 12.42, 11.92, 11.49. HRMS [M+H]+: 532.3160 (calculated for [C32H42N3O4]+: 532.3131).

ACS Paragon Plus Environment

Page 36 of 74

Page 37 of 74

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

6-Hydroxy-N-{5-[(7-methoxy-1,2,3,4-tetrahydroacridin-9-yl)amino]pentyl}2,5,7,8-tetramethyl-3,4-dihydro-2H-1-benzopyran-2-carboxamide (7d). Yield 52%; mp 159.3 – 160.5°C. Purity: 99%. 1H NMR (300 MHz, CDCl3-d) δ 7.79 (d, J = 8.5 Hz, 1H), 7.24 – 7.15 (m, 2H), 6.44 (t, J = 6.0 Hz, 1H), 3.88 (s, 3H), 3.35 – 3.10 (m, 4H), 3.01 (t, J = 4.8 Hz, 2H), 2.69 (t, J = 4.9 Hz, 2H), 2.56 – 2.28 (m, 2H), 2.14 (s, 3 + 3H), 2.05 (s, 3H), 1.94 – 1.79 (m, 6H), 1.63 – 1.51 (m, 2H), 1.50 (s, 3H), 1.48 – 1.13 (m, 4H).

13C

NMR (75 MHz, CDCl3-d) δ 174.35, 156.11, 155.81, 149.76, 145.79, 144.09, 143.14, 130.01, 122.03, 121.49, 121.20, 120.15, 119.65, 117.90, 117.30, 101.68, 78.24, 55.41, 48.92, 38.64, 33.56, 31.30, 29.52, 29.35, 24.69, 24.49, 23.87, 22.97, 22.73, 20.50, 12.38, 11.92, 11.45. HRMS [M+H]+: 546.3322 (calculated for [C33H44N3O4]+: 546.3287). 6-Hydroxy-N-{6-[(7-methoxy-1,2,3,4-tetrahydroacridin-9-yl)amino]hexyl}-2,5,7,8tetramethyl-3,4-dihydro-2H-1-benzopyran-2-carboxamide (7e). Yield 36%; mp 64.1 – 66.9°C. Purity: 99%. 1H NMR (300 MHz, DMSO-d6) δ 7.61 (d, J = 9.1 Hz, 1H), 7.50 (bs, 1H), 7.38 (d, J = 2.7 Hz, 1H), 7.16 (dd, J = 9.1, 2.6 Hz, 1H), 5.19 (t, J = 6.6 Hz, 1H), 3.83 (s, 3H), 3.31 – 3.20 (m, 2H), 3.12 – 2.91 (m, 2H), 2.85 (t, J = 6.1 Hz, 2H), 2.69 (t, J = 6.0 Hz, 2H), 2.46 – 2.08 (m, 2H), 2.05 (s, 3H), 2.03 (s, 3H), 1.94 (s, 3H), 1.85 – 1.60 (m, 6H), 1.49 – 1.37 (m, 2H), 1.33 (s, 3H), 1.30 – 1.12 (m, 4H), 1.08 – 0.95 (m, 2H).

13C

NMR (75 MHz, DMSO-d6) δ 173.24, 155.76, 155.56, 149.64, 146.01, 144.04, 142.88, 130.03, 122.79, 121.46, 121.28, 120.40, 120.06, 117.28, 101.66, 77.41, 55.52, 47.71, 38.35, 33.44, 30.78, 29.65, 29.16, 26.27, 25.95, 25.50, 24.37, 23.03, 22.77, 20.32, 12.94, 12.20, 11.96. HRMS [M+H]+: 560.3475 (calculated for [C34H46N3O4]+: 560.3444). 6-Hydroxy-N-{7-[(7-methoxy-1,2,3,4-tetrahydroacridin-9-yl)amino]heptyl}2,5,7,8-tetramethyl-3,4-dihydro-2H-1-benzopyran-2-carboxamide (7f). Yield 34%; mp 58.3 – 60.8°C. Purity: 99%. 1H NMR (300 MHz, CDCl3-d) δ 7.82 – 7.77 (m, 1H), 7.23 – 7.17 (m, 2H), 6.43 (t, J = 5.9 Hz, 1H), 3.89 (s, 3H), 3.36 (t, J = 7.2 Hz, 2H), 3.29

ACS Paragon Plus Environment

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

– 3.06 (m, 2H), 2.99 (t, J = 5.0 Hz, 2H), 2.71 (t, J = 4.9 Hz, 2H), 2.60 – 2.28 (m, 2H), 2.15 (s, 3 + 3H), 2.06 (s, 3H), 1.93 – 1.77 (m, 6H), 1.65 – 1.52 (m, 2H), 1.50 (s, 3H), 1.44 – 1.04 (m, 8H).

13C

NMR (75 MHz, CDCl3-d) δ 174.28, 156.04, 155.80, 149.92, 145.78, 144.10,

143.08, 129.90, 122.08, 121.47, 121.18, 120.22, 119.71, 117.89, 117.22, 101.70, 78.23, 55.40, 49.12, 38.79, 33.49, 31.61, 29.51, 29.29, 28.98, 26.85, 26.32, 24.62, 24.50, 22.96, 22.71, 20.50, 12.42, 11.90, 11.49. HRMS [M+H]+: 574.3628 (calculated for [C35H48N3O4]+: 574.3600). 6-Hydroxy-N-{8-[(7-methoxy-1,2,3,4-tetrahydroacridin-9-yl)amino]octyl}-2,5,7,8tetramethyl-3,4-dihydro-2H-1-benzopyran-2-carboxamide (7g). Yield 14%; mp 43.2 – 45.8°C. Purity: 98%. 1H NMR (300 MHz, CDCl3-d) δ 7.86 – 7.74 (m, 1H), 7.24 - 7.17 (m, 2H), 6.44 (t, J = 5.9 Hz, 1H), 3.88 (s, 3H), 3.38 (t, J = 7.2 Hz, 2H), 3.29 – 3.07 (m, 2H), 3.05 – 2.96 (m, 2H), 2.75 – 2.67 (m, 2H), 2.62 – 2.26 (m, 2H), 2.15 (d, J = 1.6 Hz, 3 + 3H), 2.06 (s, 3H), 1.92 – 1.79 (m, 6H), 1.68 – 1.55 (m, 2H), 1.49 (s, 3H), 1.44 – 1.02 (m, 10H). 13C NMR (75 MHz, CDCl3-d) δ 174.26, 155.97, 155.77, 149.96, 145.78, 144.05, 143.00, 129.82, 122.06, 121.45, 121.10, 120.24, 119.67, 117.84, 117.06, 101.67, 78.18, 55.37, 49.07, 38.81, 33.44, 31.62, 29.49, 29.28, 29.17, 29.00, 26.86, 26.32, 24.62, 24.40, 22.94, 22.69, 20.47, 12.39, 11.89, 11.46. HRMS [M+H]+: 588.3795 (calculated for [C36H50N3O4]+: 588.3757). 6-Hydroxy-2,5,7,8-tetramethyl-N-{2-[(1,2,3,4-tetrahydroacridin-9yl)amino]ethyl}-3,4-dihydro-2H-1-benzopyran-2-carboxamide (7h). Yield 12%; mp 34.7 – 36.5°C. Purity: 99%. 1H NMR (300 MHz, CDCl3-d) δ 7.86 (d, J = 3.7 Hz, 1H), 7.84 (d, J = 3.8 Hz, 1H), 7.54 – 7.45 (m, 1H), 7.32 – 7.27 (m, 1H), 6.87 (t, J = 5.9 Hz, 1H), 4.44 (bs, 1H), 3.60 – 3.49 (m, 4H), 3.05 – 2.97 (m, 2H), 2.64 (t, J = 5.5 Hz, 2H), 2.61 – 2.24 (m, 2H), 2.12 (s, 3H), 2.06 (s, 3H), 2.03 (s, 3H), 1.95 – 1.80 (m, 6H), 1.50 (s, 3H).

13C

NMR (75 MHz, CDCl3-d) δ 175.83, 158.40, 150.37, 146.93, 145.88, 143.83,

ACS Paragon Plus Environment

Page 38 of 74

Page 39 of 74

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

128.34, 128.30, 123.84, 122.34, 122.14, 121.64, 119.97, 119.56, 117.67, 116.31, 78.23, 49.31, 40.29, 33.62, 29.58, 24.92, 24.17, 22.92, 22.59, 20.35, 12.38, 11.94, 11.45. HRMS [M+H]+: 474.2748 (calculated for [C29H36N3O3]+: 474.2712). 6-Hydroxy-2,5,7,8-tetramethylN-{3-[(1,2,3,4-tetrahydroacridin-9-yl)amino]propyl}-3,4-dihydro-2H-1-benzopyra n-2-carboxamide (7i). Yield 45%; slurry. Purity: 99%. 1H NMR (500 MHz, CDCl3-d) δ 7.91 (d, J = 8.3 Hz, 1H), 7.87 (d, J = 8.4 Hz, 1H), 7.55 – 7.50 (m, 1H), 7.35 – 7.30 (m, 1H), 6.73 (t, J = 6.3 Hz, 1H), 4.55 (bs, 1H), 3.53 – 3.23 (m, 4H), 3.03 (t, J = 6.0 Hz, 2H), 2.73 – 2.68 (m, 2H), 2.66 – 2.30 (m, 2H), 2.07 (s, 3 + 3H), 2.04 (s, 3H), 1.96 – 1.83 (m, 6H), 1.77 – 1.61 (m, 2H), 1.52 (s, 3H).

13C

NMR (126 MHz, CDCl3-d) δ 175.22, 158.37, 150.60, 146.86, 145.79, 143.94,

128.33, 128.14, 123.86, 122.40, 122.11, 121.53, 120.36, 119.59, 117.68, 116.80, 78.25, 45.38, 36.43, 33.58, 31.14, 29.60, 24.92, 24.53, 22.91, 22.60, 22.51, 12.24, 11.86, 11.39. HRMS [M+H]+: 488.2901 (calculated for [C30H38N3O3]+: 488.2869). 6-Hydroxy-2,5,7,8-tetramethyl-N-{4-[(1,2,3,4-tetrahydroacridin-9yl)amino]butyl}-3,4-dihydro-2H-1-benzopyran-2-carboxamide (7j). Yield 25%; mp 155.8 – 158.1°C. Purity: 99%. 1H NMR (300 MHz, CDCl3-d) δ 7.95 – 7.83 (m, 2H), 7.55 – 7.47 (m, 1H), 7.35 – 7.28 (m, 1H), 6.44 (t, J = 6.2 Hz, 1H), 3.37 (t, J = 7.6 Hz, 2H), 3.31 – 3.07 (m, 2H), 3.01 (t, J = 6.0 Hz, 2H), 2.65 (t, J = 5.9 Hz, 2H), 2.60 – 2.24 (m, 2H), 2.14 (s, 3 + 3H), 1.98 (s, 3H), 1.93 – 1.78 (m, 6H), 1.62 – 1.51 (m, 2H), 1.49 (s, 3H), 1.19 (q, J = 7.6 Hz, 2H).

13C

NMR (75 MHz, CDCl3-d) δ 174.39, 158.27, 150.69,

147.12, 145.82, 144.06, 128.34, 128.29, 123.58, 122.75, 122.10, 121.49, 120.06, 119.70, 117.87, 115.81, 78.22, 49.19, 38.62, 33.72, 31.26, 29.52, 29.28, 24.71, 24.46, 23.77, 22.92, 22.63, 12.40, 11.92, 11.46. HRMS [M+H]+: 502.3058 (calculated for [C31H40N3O3]+: 502.3025).

ACS Paragon Plus Environment

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

6-Hydroxy-2,5,7,8-tetramethyl-N-{5-[(1,2,3,4-tetrahydroacridin-9yl)amino]pentyl}-3,4-dihydro-2H-1-benzopyran-2-carboxamide (7k). Yield 26%; mp 54.8 – 58.9°C. Purity: 99%. 1H NMR (500 MHz, DMSO-d6) δ 8.07 (d, J = 8.5 Hz, 1H), 7.68 (d, J = 8.4 Hz, 1H), 7.50 (t, J = 7.6 Hz, 1H), 7.35 – 7.27 (m, 1H), 7.23 (t, J = 6.0 Hz, 1H), 5.34 (t, J = 6.5 Hz, 1H), 3.34 – 3.29 (m, 2H), 3.03 (t, J = 5.9 Hz, 2H), 2.88 (t, J = 6.3 Hz, 2H), 2.67 (t, J = 6.2 Hz, 2H), 2.47 – 2.07 (m, 2H), 2.04 (s, 3 + 3H), 1.96 (s, 3H), 1.87 – 1.62 (m, 8H), 1.41 – 1.33 (m, 4H), 1.31 (s, 3H). 13C NMR (126 MHz, DMSO-d6) δ 173.35, 158.03, 150.39, 147.02, 146.01, 144.03, 128.41, 128.04, 123.39, 123.17, 122.81, 121.35, 120.43, 120.38, 117.26, 116.02, 77.35, 47.76, 38.27, 33.68, 29.67, 27.87, 26.74, 25.29, 24.12, 22.96, 22.71, 22.63, 20.27, 12.95, 12.22, 11.97. HRMS [M+H]+: 516.3210 (calculated for [C32H42N3O3]+: 516.3182). 6-Hydroxy-2,5,7,8-tetramethyl-N-{6-[(1,2,3,4-tetrahydroacridin-9yl)amino]hexyl}-3,4-dihydro-2H-1-benzopyran-2-carboxamide (7l). Yield 22%; mp 147.7 – 149.8°C. Purity: 99%. 1H NMR (500 MHz, CDCl3-d) δ 7.94 (d, J = 8.6 Hz, 1H), 7.87 (d, J = 8.5 Hz, 1H), 7.56 – 7.47 (m, 1H), 7.38 – 7.29 (m, 1H), 6.43 (t, J = 6.0 Hz, 1H), 3.99 (bs, 1H), 3.41 (t, J = 7.3 Hz, 2H), 3.32 – 3.08 (m, 2H), 3.06 – 3.00 (m, 2H), 2.70 (t, J = 4.9 Hz, 2H), 2.64 – 2.31 (m, 2H), 2.17 (s, 3H), 2.16 (s, 3H), 2.07 (s, 3H), 1.94 – 1.80 (m, 6H), 1.60 – 1.52 (m, 2H), 1.51 (s, 3H), 1.45 – 1.33 (m, 2H), 1.33 – 1.24 (m, 2H), 1.16 – 1.07 (m, 2H).

13C

NMR (126 MHz, CDCl3-d) δ 174.27, 158.41, 150.65, 147.33,

145.77, 144.14, 128.53, 128.19, 123.54, 122.73, 122.07, 121.50, 120.20, 119.69, 117.91, 115.96, 78.25, 49.31, 38.68, 33.88, 31.53, 29.53, 29.31, 26.48, 26.16, 24.75, 24.53, 22.97, 22.70, 22.53, 12.38, 11.89, 11.44. HRMS [M+H]+: 530.3375 (calculated for [C33H44N3O3]+: 530.3338). 6-Hydroxy-2,5,7,8-tetramethyl-N-{7-[(1,2,3,4-tetrahydroacridin-9yl)amino]heptyl}-3,4-dihydro-2H-1-benzopyran-2-carboxamide (7m).

ACS Paragon Plus Environment

Page 40 of 74

Page 41 of 74

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

Yield 49%; mp 125.5 – 128.3°C. Purity: 99%. 1H NMR (500 MHz, CDCl3-d) δ 7.95 (d, J = 8.6 Hz, 1H), 7.89 (d, J = 8.5 Hz, 1H), 7.53 (t, J = 8.3 Hz, 1H), 7.34 (t, J = 8.2 Hz, 1H), 6.44 (t, J = 5.9 Hz, 1H), 3.95 (bs, 1H), 3.45 (t, J = 7.2 Hz, 2H), 3.31 – 3.11 (m, 2H), 3.05 (t, J = 3.7 Hz, 2H), 2.70 (t, J = 3.8 Hz, 2H), 2.65 – 2.32 (m, 2H), 2.18 (s, 3 + 3H), 2.09 (s, 3H), 1.95 – 1.83 (m, 6H), 1.65 – 1.57 (m, 2H), 1.52 (s, 3H), 1.46 – 1.35 (m, 2H), 1.35 – 1.28 (m, 2H), 1.28 – 1.19 (m, 2H), 1.17 – 1.08 (m, 2H). 13C NMR (126 MHz, CDCl3-d) δ 174.23, 158.42, 150.71, 147.38, 145.75, 144.16, 128.63, 128.19, 123.53, 122.76, 121.93, 121.53, 120.22, 119.56, 117.95, 115.93, 78.28, 49.45, 38.81, 33.91, 31.65, 29.54, 29.32, 28.93, 26.78, 26.34, 24.74, 24.50, 23.00, 22.73, 20.53, 12.39, 11.92, 11.46. HRMS [M+H]+: 544.3521 (calculated for [C34H46N3O3]+: 544.3495). 6-Hydroxy-2,5,7,8-tetramethyl-N-{8-[(1,2,3,4-tetrahydroacridin-9yl)amino]octyl}-3,4-dihydro-2H-1-benzopyran-2-carboxamide (7n). Yield 41%; mp 64.6 – 67.5°C. Purity: 99%. 1H NMR (500 MHz, CDCl3-d) δ 7.96 (d, J = 8.4 Hz, 1H), 7.89 (d, J = 8.4 Hz, 1H), 7.53 (t, J = 8.3 Hz, 1H), 7.34 (t, J = 8.3 Hz, 1H), 6.44 (t, J = 5.9 Hz, 1H), 3.96 (bs, 1H), 3.49 – 3.45 (m, 2H), 3.28 – 3.12 (m, 2H), 3.05 (t, J = 3.2 Hz, 2H), 2.71 (t, J = 3.3 Hz, 2H), 2.66 – 2.31 (m, 2H), 2.18 (s, 3 + 3H), 2.09 (s, 3H), 1.96 – 1.82 (m, 6H), 1.69 – 1.58 (m, 2H), 1.52 (s, 3H), 1.44 – 1.30 (m, 4H), 1.25 – 1.18 (m, 4H), 1.17 – 1.08 (m, 2H).

13C

NMR (126 MHz, CDCl3-d) δ 174.22, 158.40, 150.72, 147.39, 145.73,

144.15, 128.62, 128.18, 123.49, 122.77, 121.85, 121.54, 120.18, 119,47, 117.94, 115.81, 78.26, 49.43, 38.84, 33.92, 31.67, 29.54, 29.34, 29.15, 29.01, 26.80, 26.35, 24.75, 24.45, 23.01, 22.74, 20.51, 12.36, 11.91, 11.42. HRMS [M+H]+: 558.3685 (calculated for [C35H48N3O3]+: 558.3651). N-{2-[(6-Chloro-1,2,3,4-tetrahydroacridin-9-yl)amino]ethyl}-6-hydroxy-2,5,7,8tetramethyl-3,4-dihydro-2H-1-benzopyran-2-carboxamide (7o).

ACS Paragon Plus Environment

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 42 of 74

Yield 80%; mp 49.4 – 53.7°C. Purity: 99%. 1H NMR (500 MHz, CDCl3-d) δ 7.80 (d, J = 2.2 Hz, 1H), 7.77 (d, J = 9.0 Hz, 1H), 7.18 (dd, J = 9.0, 2.2 Hz, 1H), 6.86 (t, J = 6.0 Hz, 1H), 4.53 (bs, 1H), 3.59 – 3.51 (m, 4H), 3.00 (t, J = 5.7 Hz, 2H), 2.66 – 2.61 (m, 2H), 2.57 – 2.27 (m, 2H), 2.15 (s, 3H), 2.10 (s, 3H), 2.05 (s, 2 + 3H), 1.94 – 1.80 (m, 4H), 1.52 (s, 3H). 13C NMR (126 MHz, CDCl3-d) δ 176.00, 159.73, 150.37, 147.83, 145.84, 143.92, 133.89, 127.43, 124.35, 124.01, 122.16, 121.69, 119.58, 118.26, 117.75, 116.24, 78.30, 49.69, 40.33, 33.85, 29.63, 24.85, 24.22, 22.88, 22.55, 20.38, 12.37, 11.96, 11.43. HRMS [M+H]+: 508.2362 (calculated for [C29H35ClN3O3]+: 508.2322). N-{3-[(6-Chloro-1,2,3,4-tetrahydroacridin-9-yl)amino]propyl}-6-hydroxy-2,5,7,8tetramethyl-3,4-dihydro-2H-1-benzopyran-2-carboxamide (7p). Yield 78%; mp 50.2 – 53.3°C. Purity: 98%. 1H NMR (500 MHz, CDCl3-d) δ 7.87 (d, J = 9.1 Hz, 1H), 7.84 (d, J = 2.2 Hz, 1H), 7.25 (dd, J = 9.0, 2.2 Hz, 1H), 6.70 (t, J = 6.4 Hz, 1H), 3.51 – 3.20 (m, 4H), 3.06 – 3.00 (m, 2H), 2.74 – 2.67 (m, 2H), 2.67 – 2.33 (m, 2H), 2.11 (s, 3 + 3H), 2.06 (s, 3H), 1.94 – 1.82 (m, 6H), 1.76 – 1.59 (m, 2H), 1.55 (s, 3H).

13C

NMR (126

MHz, CDCl3-d) δ 175.36, 159.74, 150.58, 147.77, 145.74, 144.07, 133.90, 127.37, 124.45, 124.07, 122.02, 121.64, 119.50, 118.75, 117.80, 116.89, 78.35, 45.21, 36.25, 33.83, 31.22, 29.63, 24.92, 24.61, 22.89, 22.60, 20.52, 12.29, 11.94, 11.42. HRMS [M+H]+: 522.2510 (calculated for [C30H37ClN3O3]+: 522.2479). N-{4-[(6-Chloro1,2,3,4-tetrahydroacridin-9-yl)amino]butyl}-6-hydroxy-2,5,7,8-tetramethyl-3,4-di hydro-2H-1-benzopyran-2-carboxamide (7q). Yield 46%; mp 168.3 -171.4°C. Purity: 98%. 1H NMR (500 MHz, CDCl3-d) δ 7.85 (d, J = 2.2 Hz, 1H), 7.82 (d, J = 9.0 Hz, 1H), 7.26 (dd, J = 9.0, 2.2 Hz, 1H), 6.48 (t, J = 6.1 Hz, 1H), 3.86 (bs, 1H), 3.42 – 3.34 (m, 2H), 3.36 – 3.15 (m, 2H), 3.06 – 2.97 (m, 2H), 2.67 – 2.62 (m, 2H), 2.61 – 2.31 (m, 2H), 2.18 (s, 3H), 2.17 (s, 3H), 2.09 (s, 3H), 1.96 – 1.81 (m, 6H), 1.52

ACS Paragon Plus Environment

Page 43 of 74

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

(s, 3H), 1.52 – 1.42 (m, 4H).

13C

NMR (126 MHz, CDCl3-d) δ 174.47, 159.65, 150.50,

148.04, 145.66, 144.23, 133.94, 127.58, 124.34, 124.32, 121.75, 121.69, 119.34, 118.48, 118.03, 116.15, 78.35, 49.03, 38.53, 33.97, 29.58, 28.60, 27.08, 24.61, 24.48, 22.88, 22.61, 20.55, 12.33, 11.97, 11.38. HRMS [M+H]+: 536.2661 (calculated for [C31H39ClN3O3]+: 536.2635). N-{5-[(6-Chloro-1,2,3,4-tetrahydroacridin-9-yl)amino]pentyl}-6-hydroxy-2,5,7,8tetramethyl-3,4-dihydro-2H-1-benzopyran-2-carboxamide (7r). Yield 66%; mp 174.5- 178.0°C. Purity: 98%. 1H NMR (500 MHz, CDCl3-d) δ 7.90 – 7.85 (m, 2H), 7.29 – 7.24 (m, 1H), 6.44 (t, J = 6.0 Hz, 1H), 3.91 (bs, 1H), 3.38 (t, J = 7.3 Hz, 2H), 3.34 – 3.12 (m, 2H), 3.06 – 2.99 (m, 2H), 2.68 – 2.63 (m, 2H), 2.63 – 2.32 (m, 2H), 2.17 (s, 3 + 3H), 2.07 (s, 3H), 1.95 – 1.82 (m, 6H), 1.63 – 1.54 (m, 2H), 1.52 (s, 3H), 1.49 – 1.39 (m, 2H), 1.28 – 1.18 (m, 2H).

13C

NMR (126 MHz, CDCl3-d) δ 174.39, 159.56, 150.65,

148.06, 145.69, 144.23, 133.91, 127.52, 124.46, 124.20, 121.85, 121.63, 119.46, 118.40, 118.01, 115.84, 78.32, 49.35, 38.61, 33.95, 31.30, 29.56, 29.33, 24.58, 24.51, 23.79, 22.87, 22.60, 20.54, 12.34, 11.94, 11.40. HRMS [M+H]+: 550.2829 (calculated for [C32H41ClN3O3]+: 550.2792). N-{6-[(6-Chloro-1,2,3,4-tetrahydroacridin-9-yl)amino]hexyl}-6-hydroxy-2,5,7,8tetramethyl-3,4-dihydro-2H-1-benzopyran-2-carboxamide (7s). Yield 59%; mp 51.4 – 54.8°C. Purity: 98%. 1H NMR (500 MHz, CDCl3-d) δ 7.92 – 7.83 (m, 2H), 7.25 (dd, J = 9.1, 2.2 Hz, 1H), 6.42 (t, J = 6.0 Hz, 1H), 3.41 (t, J = 7.2 Hz, 2H), 3.35 – 3.09 (m, 2H), 3.07 – 2.97 (m, 2H), 2.70 – 2.63 (m, 2H), 2.63 – 2.33 (m, 2H), 2.18 (d, J = 2.5 Hz, 3 + 3H), 2.08 (s, 3H), 1.95 – 1.82 (m, 6H), 1.60 – 1.54 (m, 2H), 1.52 (s, 3H), 1.47 – 1.33 (m, 2H), 1.33 – 1.24 (m, 2H), 1.17 – 1.08 (m, 2H). 13C NMR (126 MHz, CDCl3-d) δ 174.28, 159.55, 150.72, 148.06, 145.65, 144.28, 133.90, 127.50, 124.47, 124.18, 121.86, 121.62, 119.50, 118.42, 118.04, 115.85, 78.34, 49.42, 38.68, 33.95, 31.57, 29.56, 29.36, 26.46,

ACS Paragon Plus Environment

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

26.15, 24.58, 22.89, 22.61, 20.56, 13.35, 12.35, 11.93, 11.41. HRMS [M+H]+: 564.2980 (calculated for [C33H43ClN3O3]+: 564.2948). N-{7-[(6-Chloro-1,2,3,4-tetrahydroacridin-9-yl)amino]heptyl}-6-hydroxy-2,5,7,8tetramethyl-3,4-dihydro-2H-1-benzopyran-2-carboxamide (7t). Yield 62%; mp 143.9 – 146.2°C. Purity: 98%. 1H NMR (500 MHz, CDCl3-d) δ 7.88 (d, J = 9.0 Hz, 1H), 7.84 (d, J = 2.2 Hz, 1H), 7.25 (dd, J = 9.0, 2.2 Hz, 1H), 6.44 (t, J = 6.0 Hz, 1H), 3.45 (t, J = 7.4 Hz, 2H), 3.31 – 3.11 (m, 2H), 3.02 (t, J = 5.8 Hz, 2H), 2.69 – 2.63 (m, 2H), 2.63 – 2.33 (m, 2H), 2.18 (d, J = 2.9 Hz, 3 + 3H), 2.09 (s, 3H), 1.95 – 1.82 (m, 6H), 1.65 – 1.57 (m, 2H), 1.52 (s, 3H), 1.45 – 1.34 (m, 2H), 1.33 – 1.20 (m, 4H), 1.12 (q, J = 7.5 Hz, 2H).

13C

NMR (126 MHz, CDCl3-d) δ 174.25, 159.47, 150.79, 148.01, 145.70, 144.21,

133.88, 127.43, 124.53, 124.13, 121.96, 121.56, 119.59, 118.36, 117.97, 115.72, 78.29, 49.52, 38.80, 33.89, 31.66, 29.55, 29.32, 28.90, 26.74, 26.32, 24.52, 22.86, 22.58, 20.53, 13.20, 12.40, 11.92, 11.46. HRMS [M+H]+: 578.3135 (calculated for [C34H45ClN3O3]+: 578.3105). N-{8-[(6-Chloro-1,2,3,4-tetrahydroacridin-9-yl)amino]octyl}-6-hydroxy-2,5,7,8tetramethyl-3,4-dihydro-2H-1-benzopyran-2-carboxamide (7u). Yield 73%; mp 57.3 – 60.5°C. Purity: 96%. 1H NMR (500 MHz, CDCl3-d) δ 7.89 (d, J = 9.1 Hz, 1H), 7.85 (d, J = 2.2 Hz, 1H), 7.25 (dd, J = 9.0, 2.2 Hz, 1H), 6.44 (t, J = 5.9 Hz, 1H), 3.47 (t, J = 7.2 Hz, 2H), 3.29 – 3.11 (m, 2H), 3.05 – 2.98 (m, 2H), 2.69 – 2.63 (m, 2H), 2.63 – 2.31 (m, 2H), 2.18 (d, J = 3.5 Hz, 3 + 3H), 2.09 (s, 3H), 1.95 – 1.82 (m, 6H), 1.67 – 1.58 (m, 2H), 1.51 (s, 3H), 1.44 – 1.29 (m, 4H), 1.29 – 1.15 (m, 4H), 1.15 – 1.07 (m, 2H). 13C NMR (126 MHz, CDCl3-d) δ 174.29, 159.41, 150.90, 147.96, 145.75, 144.25, 133.98, 127.37, 124.60, 124.16, 122.02, 121.61, 119.64, 118.31, 118.00, 115.61, 78.32, 49.52, 38.89, 33.86, 31.70, 29.60, 29.39, 29.15, 29.03, 26.79, 26.39, 24.55, 24.52, 22.90, 22.61, 20.57, 12.43, 11.96, 11.49. HRMS [M+H]+: 592.3294 (calculated for [C35H47ClN3O3]+: 592.3261).

ACS Paragon Plus Environment

Page 44 of 74

Page 45 of 74

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

Inhibition of Human AChE and BChE AChE and BChE inhibitory activities of tested compounds were determined using modified Ellman’s method and are expressed as IC50, i.e. concentration of compound required for 50% reduction in cholinesterase activity.42,43 Human recombinant AChE (hAChE; E.C. 3.1.1.7), human plasmatic BChE (hBChE; E.C. 3.1.1.8), 5,5´-dithiobis(2nitrobenzoic acid) (Ellman´s reagent; DTNB), phosphate buffer solution (PBS, pH 7.4), acetylthiocholine (ATCh) and butyrylthiocholine (BTCh) were purchased from SigmaAldrich (Czech Republic). Polystyrene Nunc 96-well microplates with flat bottom shape (ThermoFisher Scientific, USA) were used for the measuring purposes. All the assays were carried out in 0.1 M KH2PO4/K2HPO4 buffer, pH 7.4. Enzyme solutions were prepared at 2.0 U/mL in 2 mL aliquots. The assay medium (100 µL) consisted of 10 µL of enzyme, 40 µL of 0.1 M PBS (pH 7.4), 20 µL of 0.01 M DTNB, 10 µL of tested compound and 20 µL of 0.01 M substrate (ATCh or BTCh iodide solution). Assayed solutions of target compounds (10 µL, 10-3 – 10-9 M) were preincubated with corresponding cholinesterase for 5 min. The reaction was initiated by addition of 20 µL of substrate. The activity was determined by measuring of the increase in absorbance at 412 nm at 37°C in 2 min intervals using Multi-mode microplate reader Synergy 2 (Vermont, USA). Each concentration was assayed in triplicate. Percentage of inhibition (I) was calculated from the measured data as follows: I = ( 1−

ΔAi

indicates

∆Ai ∆A0

) × 100

absorbance

change

provided

by

cholinesterase

exposed

to

anticholinesterase compound. ΔA0 indicates absorbance change caused by intact cholinesterase, where phosphate buffer was applied in the same way as the

ACS Paragon Plus Environment

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

anticholinesterase compound. Software Microsoft Excel (Redmont, WA, USA) and GraphPad Prism version 5.02 for Windows (GraphPad Software, San Diego, CA, USA) were used for the statistical data evaluation.

Kinetic Study of AChE Inhibition Kinetic study of hAChE inhibition was performed by using Ellman’s method (described above). For the measurements following concentrations of substrate were used: 0.07813, 0.1563, 0.3125 and 0.625 mM. Vmax and Km values, respectively, of the Michaelis-Menten kinetics as well as Ki value were calculated by non-linear regression from the substrate velocity curves. Linear regression was used for calculation of Lineweaver-Burk plots. All calculations were performed using GraphPad Prism software.

Molecular Modeling Studies From the online PDB database (www.pdb.org) model of hAChE (PDB ID: 4EY7, resolution: 2.35 Å) was downloaded and prepared for flexible molecular docking by MGL Tools utilities.66 The preparation of this receptor involved removal of the surplus copies of the enzyme chains, non-bonded inhibitor (donepezil), addition of polar hydrogens and merging of non-polar ones. Default Gasteiger charges were assigned to all atoms. Flexible parts of the enzyme were determined by a spherical selection of residues (R = 11 Å) approximately around the center of the active site. In the same points the centers of the grid box of 33 × 33 × 33 Å were positioned. The rotatable bonds in the flexible residues were detected automatically by AutoDock Tools 1.5.4 program. Given the limitation of the program used for flexible molecular docking, water molecules had to be removed from the system. The flexible receptor part contained 40 residues for hAChE. Following xyz coordinates of the grid box center was applied: hAChE (10.698, -58.115, -

ACS Paragon Plus Environment

Page 46 of 74

Page 47 of 74

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

23.192). The studied ligands were firstly drawn in HyperChem 8.0, then manually protonated as suggested by MarvinSketch 6.2.0. software (http://www.chemaxon.com), geometrically optimized by semi-empirical quantum-chemistry PM3 method and stored as pdb files. The structures of the ligands were processed for docking in a similar way as abovementioned flexible parts of the receptor by AutoDock Tools 1.5.4 program. Molecular docking was carried out in AutoDock Vina 1.1.2 program utilizing computer resources of the Czech National Grid Infrastructure MetaCentrum. The search algorithm of AutoDock Vina efficiently combines a Markov chain Monte Carlo like method for the global search and a Broyden-Fletcher-Goldfarb-Shano gradient approach for the local search.50 It is a type of memetic algorithm based on interleaving stochastic and deterministic calculations.67 Each docking task was repeated 30 times with the exhaustiveness parameter set to 16, employing 16 CPU in parallel multithreading. From the obtained results, the solutions reaching the minimum predicted Gibbs binding energy were taken as the top-scoring modes. The graphic representations of the docked poses were rendered in PyMOL 1.3 (The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC.). 2D diagrams were generated using PoseView software (PoseView, http://poseview.zbh.uni-hamburg.de/poseview/wizard, 2012).

Evaluation of Antioxidant Activity Diphenyl-1-picrylhydrazyl stabile free radical assay (DPPH)53 is a simple method to determine antioxidant activity and is expressed as EC50, i.e. concentration of compound that causes 50% decrease in the DPPH activity. DPPH, methanol and trolox (as reference standard) were purchased from Sigma-Aldrich (Czech Republic). Polystyrene Nunc 96well microplates with flat bottom shape (ThermoFisher Scientific, USA) were used for the measuring purposes. All the assays were carried out in methanol. DPPH solution was

ACS Paragon Plus Environment

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

prepared at 0.2 mM concentration. The assay medium (200 µL) consisted of 100 µL of DPPH solution and 100 µL of tested compound (10-3 – 10-6 M). The reaction time constituted 30 minutes. The antioxidant activity was determined by measuring the increase in absorbance at 517 nm at laboratory temperature using Multi-mode microplate reader Synergy 2 (Vermont, USA).68 Each concentration was tested in triplicate. Software GraphPad Prism version 5 for Windows (GraphPad Software, San Diego, CA, USA) was used for statistical data evaluation.

Determination of In Vitro Blood-Brain Barrier Permeation Parallel artificial membrane permeation assay (PAMPA) was used according to modified protocol described by Di et al..55 Tested compounds were dissolved in the mixture of PBS pH 7.4 and ethanol (70:30) as described by Xie et al.36 to reach the final concentration in the donor well (50 or 100 µM). 300 µL of the solution were added to the donor wells (VA). The filter membrane was coated with porcine brain lipid (PBL) in dodecane (4 µL of 20 mg/mL of PBL in dodecane) and subsequently the acceptor well was filled with 300 µL of PBS pH 7.4 buffer (VD). The donor filter plate was carefully put on the acceptor plate so that a coated membrane was “in touch” with both donor solution and acceptor buffer. The set, where the tested compound diffused from the donor well through the lipid membrane (area = 0.28 cm2) into the acceptor well, was left intact for 18 h while the permeation occurred. The concentration of a drug in the wells was determined using the UV plate reader Biotek Synergy HT at the maximum absorption wavelength. Concentration of the compound was calculated from the standard curve and expressed as the permeability value (Pe) (Table S1 of Supporting information) according to following formula:69,70

ACS Paragon Plus Environment

Page 48 of 74

Page 49 of 74

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

Determination of Hepatotoxicity on HepG2 Cells The hepatotoxicity of tested compounds was evaluated using cell line HepG2 from human liver hepatocellular carcinoma (ATCC, Virginia, USA). These cells were seeded in 96-well plate at density 17x103 per well in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, USA) with 10% PBS (Gibco, USA) and were left attached overnight. The incubation was performed under conditions of 37°C, 5% CO2 and 80 – 95% air humidity. Stock solutions of tested compounds were prepared in DMSO (Sigma-Aldrich, USA) and then diluted in DMEM. The stock solutions in DMEM were serially diluted and added to cells in 96-well culture plates so that the final concentration of DMSO was less than 0.25%. The cell viability was detected using MTT assay described by Mosmann after 24 h incubation with tested compounds.71 After 24 h the medium was aspirated and 100 µL of MTT solution (0.5 mg/mL) in serum free DMEM medium was added to cells. The cells were then incubated for one hour. The medium was then aspirated and violet crystals of MTT formazan were dissolved in 100 μL of DMSO under shaking. The absorbance was measured with a microplate reader (Beckman Coulter Inc., California, USA) at a test wavelength of 570 nm. The IC50 values were calculated using four parametric non-linear regressions with a statistic software GraphPad Prism version 5.04 for Windows (GraphPad Software, San Diego, CA, USA). The data were obtained from three independent measurements. The IC50 values were expressed as mean ± SEM.

Microsomal In Vitro Metabolism and Stability Analysis

ACS Paragon Plus Environment

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

To determine the stability of the tested compound in microsomal system and to reveal possible metabolites of I. phase metabolism, a quick and easy protocol, using pooled human liver microsomes (HLM) and RapidStart NADPH Regenerating System, was proposed. Firstly, aliquotes of 25 µL of HLM and 50µL of RapidStart solution (diluted according to the supplier recommendation in ratio: 1.5 mL of the original RapidStart NADPH Regenerating System + 3.5 mL ultrapure water) were freely thawed at ambient laboratory temperature. The microsomal incubation system was formed as follows: 1) addition of 50 µL of RapidStart solution to 25 µL of HLM and incubation in rest for 5 min at laboratory temperature; 2) addition of 420 µL of ultrapure water and vortexing of the mixture (1000rpm) for 1min; 3) addition of 5 µL of a stock solution of the tested compound in DMSO; 4) incubation of the mixture in a thermo-stated shaker for 60 min at 37°C and 1000rpm; 5) quenching the metabolism by addition of 500 µL of cooled acetonitrile (-20°C); 6) relaxing for 5 min at -20°C; 7) centrifugation of the mixture at 21 000g for 20min at 10°C; 8) collection of 250 µL of the supernatant and LC-MS/MS analysis. The experiment with HLM was performed at three different concentrations of the tested compound in the final solution volume (i.e. 1 mL): 1 µg/mL, 20 µg/mL and 50 µg/mL. Due to stopping the metabolism by 500 µL of acetonitrile, the concentration of the assayed compound was changed by diluting factor of 2, but the theoretical concentrations of the analyte 1 µg/mL, 20 µg/mL and 50 µg/mL were related to 1mL of the final volume. One blank sample of the microsomal system was prepared in the same way with the exception of the third step (see above), when the sample was spiked with 5 µL of pure DMSO. For calibration, a series of 8 different concentrations of the target compound was analyzed by LC-MS/MS: 0.5, 1, 5, 10, 25, 50, 75, 100 µg/mL. The calibration series was prepared by diluting a stock solution of tested compound (in

ACS Paragon Plus Environment

Page 50 of 74

Page 51 of 74

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

methanol) in methanol. Working with µg/mL as a concentration unit was preferred since it is practically more straightforward. If needed, the concentrations in µg/mL can be easily transformed to µM/mL by dividing the value by molecular weight of the analyte. For LC-MS/MS analyses, the abovementioned Dionex UltiMate 3000 system coupled with a Q Exactive Plus hybrid quadrupole-orbitrap spectrometer was used. For elution, a ramp-gradient program, mixing ultrapure water (MFA) and acetonitrile (MFB), both acidified with 0.1% (v/v) of formic acid, was developed: 0 - 1.5 min 5% MFB, 1.5 - 9.5 min 5 - 100% MFB, 9.5 - 11.5 min 100% MFB, 11.5 - 11.5 min 5% MFB, 11.5 - 15.0 min 5% MFB. The injection volume was set to 5 µL, the column thermostat to 27°C, the flow rate to 0.2 mL/min. The detection was carried by a UV-detector operating at four wavelengths (210, 254, 278 and 290 nm) as well as by MS set to MRM acquisition mode. LC-MS/MS were performed in triplicate and simple statistics of the results was calculated.

Evaluation of Acute Toxicity Male albino Wistar rats weighing 180 – 210 g were obtained from VELAZ (Czech Republic). The animals were housed in a controlled environment (air-conditioning, T = 22 ± 2°C, 50 ± 10% relative humidity, 12 h dark/light cycle with light on at 07:00 a.m.). Food and water were given ad libitum. The rats were randomly divided into groups of eight animals. All the animal procedures followed the guidelines for the care and handling of laboratory animals and were approved by the Ethics Committee of the Faculty of Military Health Sciences in Hradec Kralove (Czech Republic). All the efforts were done to minimize animals’ suffering. Chemicals and solvents were purchased from Sigma-Aldrich (Czech Republic). Solutions of the tested compound were prepared in the

ACS Paragon Plus Environment

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

form of 10% emulsion of DMSO in corn oil and administered intramuscularly (i.m.) at a volume of 10 mL/kg body weight.

AUTHOR INFORMATION Corresponding author *

Kamil Kuca: phone: +420 495 832 923, +420 495 832 100; e-mail: [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENT This work was supported by MH CZ-DRO (University Hospital Hradec Kralove, No. 00179906), by the grant of Ministry of Defense “Long Term Organization Development Plan – 1011”, by specific research SV/FVZ201409, by the project Excellence 2015 of Faculty of Informatics and Management, University of Hradec Kralove, by Long Term Development Plan of Faculty of Medicine, University of Ostrava, by the project of National Institute of Mental Health (NIMH - CZ)“, grant number ED2.1.00/03.0078 and the European Regional Development Fund. The access to computing and storage facilities owned by parties and projects contributing to the National Grid Infrastructure MetaCentrum, provided under the program "Projects of Large Infrastructure for Research, Development, and Innovations" (LM2010005), is highly appreciated.

ABBREVIATIONS USED 7-MEOTA, 7-methoxytacrine; Aβ, amyloid-β; AChE, acetylcholinesterase; AD, Alzheimer’s disease; ATCh, acetylthiocholine; BBB, blood-brain barrier; BChE, butyrylcholinesterase; BTCh, butyrylthiocholine; CAS, catalytic anionic site; DMEM, Dulbecco’s Modified Eagle’s

ACS Paragon Plus Environment

Page 52 of 74

Page 53 of 74

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

Medium; DPPH, diphenyl-1-picrylhydrazyl; DTNB, 5,5´-dithiobis(2-nitrobenzoic acid); EDC.HCl, N-(3-dimethylaminopropyl)-N´-ethylcarbodiimide hydrochloride; HLM, human liver microsomes; HOBt, 1-hydroxybenzotriazole hydrate; MTDLs, multi-target-directed ligands; MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NMDA, Nmethyl-D-aspartate; PAS, peripheral anionic site; QNB, 3-quiniclidinyl benzilate; ROS, reactive oxygen species.

ASSOCIATED CONTENT Supporting Information Available: Permeability results from PAMPA-BBB assay for tacrine – trolox hybrids 7a-u and reference compounds 1-4, Materials and instrumentation of microsomal in vitro metabolism and stability analysis, Proposed structures of metabolites M1-M4 and their molecular fragments which were found in MS/MS spectra.

REFERENCES (1) Ballard, C.; Gauthier, S.; Corbett, A.; Brayne, C.; Aarsland, D.; Jones, E. Alzheimer’s Disease. Lancet 2011, 377, 1019–1031. (2) Blennow, K.; de Leon, M. J.; Zetterberg, H. Alzheimer’s Disease. Lancet 2006, 368, 387–403. (3) Ferri, C. P.; Prince, M.; Brayne, C.; Brodaty, H.; Fratiglioni, L.; Ganguli, M.; Hall, K.; Hasegawa, K.; Hendrie, H.; Huang, Y.; Jorm, A.; Mathers, C.; Menezes, P. R.; Rimmer, E.; Scazufca, M.; Alzheimer’s Disease International. Global Prevalence of Dementia: A Delphi Consensus Study. Lancet 2005, 366, 2112–2117.

ACS Paragon Plus Environment

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 54 of 74

(4) Prince, M.; Bryce, R.; Albanese, E.; Wimo, A.; Ribeiro, W.; Ferri, C. P. The Global Prevalence of Dementia: A Systematic Review and Metaanalysis. Alzheimer's Dement. 2013, 9, 63–75.e2. (5) Yankner, B. A. Mechanisms of Neuronal Degeneration in Alzheimer’s Disease. Neuron 1996, 16, 921–932. (6) Bossy-Wetzel, E.; Schwarzenbacher, R.; Lipton, S. A. Molecular Pathways to Neurodegeneration. Nat. Med. 2004, 10 Suppl, S2–S9. (7) Bolognesi, M. L.; Minarini, A.; Rosini, M.; Tumiatti, V.; Melchiorre, C. From Dual Binding Site Acetylcholinesterase Inhibitors to Multi-Target-Directed Ligands (MTDLs): A Step Forward in the Treatment of Alzheimer’s Disease. Mini Rev. Med. Chem. 2008, 8, 960–967. (8) Zemek, F.; Drtinova, L.; Nepovimova, E.; Sepsova, V.; Korabecny, J.; Klimes, J.; Kuca, K. Outcomes of Alzheimer’s Disease Therapy with Acetylcholinesterase Inhibitors and Memantine. Expert Opin. Drug Saf. 2014, 13, 759–774. (9) Summers, W. K. Tacrine (THA, Cognex(R)). J. Alzheimer's Dis. 2000, 2, 85–93. (10) Summers, W. K.; Koehler, A. L.; Marsh, G. M.; Tachiki, K.; Kling, A. Long-Term Hepatotoxicity of Tacrine. Lancet 1989, 1, 729. (11) Soukup, O.; Jun, D.; Zdarova-Karasova, J.; Patocka, J.; Musilek, K.; Korabecny, J.; Krusek, J.; Kaniakova, M.; Sepsova, V.; Mandikova, J.; Trejtnar, F.; Pohanka, M.; Drtinova, L.; Pavlik, M.; Tobin, G.; Kuca, K. A Resurrection of 7-MEOTA: A Comparison with Tacrine. Curr. Alzheimer Res. 2013, 10, 893–906. (12) Recanatini, M.; Cavalli, A.; Belluti, F.; Piazzi, L.; Rampa, A.; Bisi, A.; Gobbi, S.; Valenti, P.;

Andrisano,

V.;

Bartolini,

M.;

Cavrini,

V.

SAR

of

9-Amino-1,2,3,4-

Tetrahydroacridine-Based Acetylcholinesterase Inhibitors: Synthesis, Enzyme

ACS Paragon Plus Environment

Page 55 of 74

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

Inhibitory Activity, QSAR, and Structure-Based CoMFA of Tacrine Analogues. J. Med. Chem. 2000, 43, 2007–2018. (13) Praticò, D. Oxidative Stress Hypothesis in Alzheimer’s Disease: A Reappraisal. Trends Pharmacol. Sci. 2008, 29, 609–615. (14) Trippier, P. C.; Jansen Labby, K.; Hawker, D. D.; Mataka, J. J.; Silverman, R. B. Targetand Mechanism-Based Therapeutics for Neurodegenerative Diseases: Strength in Numbers. J. Med. Chem. 2013, 56, 3121–3147. (15) Lee, H. P.; Zhu, X.; Casadesus, G.; Castellani, R. J.; Nunomura, A.; Smith, M. A.; Lee, H.; Perry, G. Antioxidant Approaches for the Treatment of Alzheimer’s Disease. Expert Rev. Neurother. 2010, 10, 1201–1208. (16) Rosini, M.; Simoni, E.; Milelli, A.; Minarini, A.; Melchiorre, C. Oxidative Stress in Alzheimer’s Disease: Are We Connecting the Dots? J. Med. Chem. 2014, 57, 2821– 2831. (17) Zeng, L. H.; Wu, J.; Carey, D.; Wu, T. W. Trolox and Ascorbate: Are They Synergistic in Protecting Liver Cells in Vitro and in Vivo? Biochem. Cell Biol. 1991, 69, 198–201. (18) Scott, J. E.; Cort, W. M.; Harley, H.; Parrish, D. R.; Saucy, G. 6-Hydroxychroman-2Carboxylic Acids: Novel Antioxidants. J. Am. Oil Chem. Soc. 1974, 51, 200–203. (19) Wu, T. W.; Hashimoto, N.; Wu, J.; Carey, D.; Li, R. K.; Mickle, D. A.; Weisel, R. D. The Cytoprotective Effect of Trolox Demonstrated with Three Types of Human Cells. Biochem. Cell Biol. 1990, 68, 1189–1194. (20) Wu, T. W.; Hashimoto, N.; Au, J. X.; Wu, J.; Mickle, D. A.; Carey, D. Trolox Protects Rat Hepatocytes against Oxyradical Damage and the Ischemic Rat Liver from Reperfusion Injury. Hepatology 1991, 13, 575–580. (21) Rampa, A.; Belluti, F.; Gobbi, S.; Bisi, A. Hybrid-Based Multi-Target Ligands for the Treatment of Alzheimer’s Disease. Curr. Top. Med. Chem. 2011, 11, 2716–2730.

ACS Paragon Plus Environment

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(22) Bajda, M.; Guzior, N.; Ignasik, M.; Malawska, B. Multi-Target-Directed Ligands in Alzheimer’s Disease Treatment. Curr. Med. Chem. 2011, 18, 4949–4975. (23) Morphy, R.; Kay, C.; Rankovic, Z. From Magic Bullets to Designed Multiple Ligands. Drug Discov. Today 2004, 9, 641–651. (24) Cavalli, A.; Bolognesi, M. L.; Minarini, A.; Rosini, M.; Tumiatti, V.; Recanatini, M.; Melchiorre, C. Multi-Target-Directed Ligands to Combat Neurodegenerative Diseases. J. Med. Chem. 2008, 51, 347–372. (25) Nepovimova, E.; Uliassi, E.; Korabecny, J.; Peña-Altamira, L. E.; Samez, S.; Pesaresi, A.; Garcia, G. E.; Bartolini, M.; Andrisano, V.; Bergamini, C.; Fato, R.; Lamba, D.; Roberti, M.; Kuca, K.; Monti, B.; Bolognesi, M. L. Multitarget Drug Design Strategy: Quinone-Tacrine Hybrids Designed to Block Amyloid-β Aggregation and to Exert Anticholinesterase and Antioxidant Effects. J. Med. Chem. 2014, 57, 8576–8589. (26) Hamulakova, S.; Janovec, L.; Hrabinova, M.; Spilovska, K.; Korabecny, J.; Kristian, P.; Kuca, K.; Imrich, J. Synthesis and Biological Evaluation of Novel Tacrine Derivatives and Tacrine-Coumarin Hybrids as Cholinesterase Inhibitors. J. Med. Chem. 2014, 57, 7073–7084. (27) Spilovska, K.; Korabecny, J.; Horova, A.; Musilek, K.; Nepovimova, E.; Drtinova, L.; Gazova, Z.; Siposova, K.; Dolezal, R.; Jun, D.; Kuca, K. Design, Synthesis and in Vitro Testing of 7-Methoxytacrine-Amantadine Analogues: A Novel Cholinesterase Inhibitors for the Treatment of Alzheimer’s Disease. Med. Chem. Res. 2015, 24, 2645–2655. (28) Lee, S. K.; Park, M. K.; Jhang, H. E.; Yi, J.; Nahm, K.; Cho, D. W.; Ra, C. S.; Musilek, K.; Horova, A.; Korabecny, J.; Dolezal, R.; Jun, D.; Kuca, K. Preparation of 7-Methoxy Tacrine Dimer Analogs and Their In vitro/In Silico Evaluation as Potential Cholinesterase Inhibitors. Bull. Korean Chem. Soc. 2015, 36, 1654–1660.

ACS Paragon Plus Environment

Page 56 of 74

Page 57 of 74

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

(29) Shin, J. H.; Lee, Y. A.; Lee, J. K.; Lee, Y. B.; Cho, W.; Im, D. S.; Lee, J. H.; Yun, B. S.; Springer, J. E.; Gwag, B. J. Concurrent Blockade of Free Radical and Microsomal Prostaglandin E Synthase-1-Mediated PGE2 Production Improves Safety and Efficacy in a Mouse Model of Amyotrophic Lateral Sclerosis. J. Neurochem. 2012, 122, 952–961. (30) Li, S.; Guo, C.; Sun, X.; Li, Y.; Zhao, H.; Zhan, D.; Lan, M.; Tang, Y. Synthesis and Biological

Evaluation

of

Tetramethylpiperidine-N-Oxyl

Quinazoline as

and

Potential

Quinoline

Bearing

2,2,6,6-

Epidermal

Growth

Factor

receptor(EGFR) Tyrosine Kinase Inhibitors and EPR Bio-Probe Agents. Eur. J. Med. Chem. 2012, 49, 271–278. (31) Jin, H.; Randazzo, J.; Zhang, P.; Kador, P. F. Multifunctional Antioxidants for the Treatment of Age-Related Diseases. J. Med. Chem. 2010, 53, 1117–1127. (32) Hausler, N. E.; Devine, S. M.; McRobb, F. M.; Warfe, L.; Pouton, C. W.; Haynes, J. M.; Bottle, S. E.; White, P. J.; Scammells, P. J. Synthesis and Pharmacological Evaluation of Dual Acting Antioxidant A(2A) Adenosine Receptor Agonists. J. Med. Chem. 2012, 55, 3521–3534. (33) Rosini, M.; Andrisano, V.; Bartolini, M.; Bolognesi, M. L.; Hrelia, P.; Minarini, A.; Tarozzi, A.; Melchiorre, C. Rational Approach to Discover Multipotent AntiAlzheimer Drugs. J. Med. Chem. 2005, 48, 360–363. (34) Fang, L.; Kraus, B.; Lehmann, J.; Heilmann, J.; Zhang, Y.; Decker, M. Design and Synthesis of Tacrine-Ferulic Acid Hybrids as Multi-Potent Anti-Alzheimer Drug Candidates. Bioorg. Med. Chem. Lett. 2008, 18, 2905–2909. (35) Fernández-Bachiller, M. I.; Pérez, C.; Campillo, N. E.; Páez, J. A.; González-Muñoz, G. C.; Usán, P.; García-Palomero, E.; López, M. G.; Villarroya, M.; García, A. G.; Martínez, A.; Rodríguez-Franco, M. I. Tacrine-Melatonin Hybrids as Multifunctional Agents for

ACS Paragon Plus Environment

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Alzheimer’s Disease, with Cholinergic, Antioxidant, and Neuroprotective Properties. ChemMedChem 2009, 4, 828–841. (36) Xie, S.-S.; Lan, J.-S.; Wang, X.-B.; Jiang, N.; Dong, G.; Li, Z.-R.; Wang, K. D. G.; Guo, P.-P.; Kong, L.-Y. Multifunctional Tacrine-Trolox Hybrids for the Treatment of Alzheimer’s Disease with Cholinergic, Antioxidant, Neuroprotective and Hepatoprotective Properties. Eur. J. Med. Chem. 2015, 93, 42–50. (37) Hu, M.-K.; Lu, C.-F. A Facile Synthesis of Bis-Tacrine Isosteres. Tetrahedron Lett. 2000, 41, 1815–1818. (38) Korabecny, J.; Dolezal, R.; Cabelova, P.; Horova, A.; Hruba, E.; Ricny, J.; Sedlacek, L.; Nepovimova, E.; Spilovska, K.; Andrs, M.; Musilek, K.; Opletalova, V.; Sepsova, V.; Ripova, D.; Kuca, K. 7-MEOTA–donepezil like Compounds as Cholinesterase Inhibitors: Synthesis, Pharmacological Evaluation, Molecular Modeling and QSAR Studies. Eur. J. Med. Chem. 2014, 82, 426–438. (39) Spilovska, K.; Korabecny, J.; Kral, J.; Horova, A.; Musilek, K.; Soukup, O.; Drtinova, L.; Gazova, Z.; Siposova, K.; Kuca, K. 7-Methoxytacrine-Adamantylamine Heterodimers as Cholinesterase Inhibitors in Alzheimer’s Disease Treatment — Synthesis, Biological Evaluation and Molecular Modeling Studies. Molecules 2013, 18, 2397– 2418. (40) Szymanski, P.; Karpiński, A.; Mikiciuk-Olasik, E. Synthesis, Biological Activity and HPLC Validation of 1,2,3,4-Tetrahydroacridine Derivatives as Acetylcholinesterase Inhibitors. Eur. J. Med. Chem. 2011, 46, 3250–3257. (41) Szymański, P.; Markowicz, M.; Mikiciuk-Olasik, E. Synthesis and Biological Activity of Derivatives of Tetrahydroacridine as Acetylcholinesterase Inhibitors. Bioorg. Chem. 2011, 39, 138–142.

ACS Paragon Plus Environment

Page 58 of 74

Page 59 of 74

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

(42) Ellman, G. L.; Courtney, K. D.; Andres, V.; Feather-Stone, R. M. A New and Rapid Colorimetric Determination of Acetylcholinesterase Activity. Biochem. Pharmacol. 1961, 7, 88–95. (43) Talesa, V. N. Acetylcholinesterase in Alzheimer’s Disease. Mech. Ageing Dev. 2001, 122, 1961–1969. (44) Nordberg, A.; Ballard, C.; Bullock, R.; Darreh-Shori, T.; Somogyi, M. A Review of Butyrylcholinesterase as a Therapeutic Target in the Treatment of Alzheimer’s Disease. Prim. Care Companion CNS Disord. 2013, 15. DOI: 10.4088/PCC.12r01412. (45) Eckroat, T. J.; Green, K. D.; Reed, R. A.; Bornstein, J. J.; Garneau-Tsodikova, S. Investigation of the Role of Linker Moieties in Bifunctional Tacrine Hybrids. Bioorg. Med. Chem. 2013, 21, 3614–3623. (46) Chen, Y.; Sun, J.; Fang, L.; Liu, M.; Peng, S.; Liao, H.; Lehmann, J.; Zhang, Y. TacrineFerulic Acid-Nitric Oxide (NO) Donor Trihybrids as Potent, Multifunctional Acetyland Butyrylcholinesterase Inhibitors. J. Med. Chem. 2012, 55, 4309–4321. (47) Cornish-Bowden, A. A Simple Graphical Method for Determining the Inhibition Constants of Mixed, Uncompetitive and Non-Competitive Inhibitors. Biochem. J. 1974, 137, 143–144. (48) Inestrosa, N. C.; Alvarez, A.; Pérez, C. A.; Moreno, R. D.; Vicente, M.; Linker, C.; Casanueva, O. I.; Soto, C.; Garrido, J. Acetylcholinesterase Accelerates Assembly of Amyloid-Beta-Peptides into Alzheimer’s Fibrils: Possible Role of the Peripheral Site of the Enzyme. Neuron 1996, 16, 881–891. (49) Trott, O.; Olson, A. J. AutoDock Vina: Improving the Speed and Accuracy of Docking with a New Scoring Function, Efficient Optimization, and Multithreading. J. Comput. Chem. 2010, 31, 455–461.

ACS Paragon Plus Environment

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(50) Cheung, J.; Rudolph, M. J.; Burshteyn, F.; Cassidy, M. S.; Gary, E. N.; Love, J.; Franklin, M. C.; Height, J. J. Structures of Human Acetylcholinesterase in Complex with Pharmacologically Important Ligands. J. Med. Chem. 2012, 55, 10282–10286. (51) Dolezal, R.; Sobeslav, V.; Hornig, O.; Balik, L.; Korabecny, J.; Kuca, K. HPC Cloud Technologies for Virtual Screening in Drug Discovery. In Intelligent Information and Database Systems; Nguyen, N. T., Trawiński, B., Kosala, R., Eds.; Springer International Publishing Switzerland: Cham, 2015; Vol. 9012, pp 440–449. DOI: 10.1007/978-3-319-15705-4_43. (52) Molyneux, P. The Use of the Stable Free Radical Diphenylpicrylhydrazyl (DPPH) for Estimating Antioxidant Activity. Songklanakarin J. Sci. Technol. 2004, 26, 211–219. (53) Bentayeb, K.; Rubio, C.; Nerín, C. Study of the Antioxidant Mechanisms of Trolox and Eugenol with 2,2’-azobis(2-Amidinepropane)dihydrochloride Using Ultra-High Performance Liquid Chromatography Coupled with Tandem Mass Spectrometry. Analyst 2012, 137, 459–470. (54) Di, L.; Kerns, E. H.; Fan, K.; McConnell, O. J.; Carter, G. T. High Throughput Artificial Membrane Permeability Assay for Blood-Brain Barrier. Eur. J. Med. Chem. 2003, 38, 223–232. (55) Szultka, M.; Krzeminski, R.; Jackowski, M.; Buszewski, B. Identification of In Vitro Metabolites of Amoxicillin in Human Liver Microsomes by LC-ESI/MS. Chromatographia 2014, 77, 1027–1035. (56) Alberto, M. E.; Russo, N.; Grand, A.; Galano, A. A Physicochemical Examination of the Free Radical Scavenging Activity of Trolox: Mechanism, Kinetics and Influence of the Environment. Phys. Chem. Chem. Phys. 2013, 15, 4642–4650.

ACS Paragon Plus Environment

Page 60 of 74

Page 61 of 74

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

(57) Nakamura, M.; Hayashi, T. Oxidation Mechanism of Vitamin E Analogue (Trolox C, 6-Hydroxy-2,2,5,7,8-Pentamethylchroman)

and

Vitamin

E

by

Horseradish

Peroxidase and Myoglobin. Arch. Biochem. Biophys. 1992, 299, 313–319. (58) Thomas, M. J.; Bielski, B. H. J. Oxidation and Reaction of Trolox C, a Tocopherol Analogue, in Aqueous Solution. A Pulse-Radiolysis Study. J. Am. Chem. Soc. 1989, 111, 3315–3319. (59) Patocka, J.; Jun, D.; Kuca, K. Possible Role of Hydroxylated Metabolites of Tacrine in Drug Toxicity and Therapy of Alzheimer’s Disease. Curr. Drug Metab. 2008, 9, 332– 335. (60) Madden, S.; Woolf, T. F.; Pool, W. F.; Park, B. K. An Investigation into the Formation of Stable, Protein-Reactive and Cytotoxic Metabolites from Tacrine in Vitro. Studies with Human and Rat Liver Microsomes. Biochem. Pharmacol. 1993, 46, 13–20. (61) Spaldin, V.; Madden, S.; Pool, W. F.; Woolf, T. F.; Park, B. K. The Effect of Enzyme Inhibition on the Metabolism and Activation of Tacrine by Human Liver Microsomes. Br. J. Clin. Pharmacol. 1994, 38, 15–22. (62) Madden, S.; Spaldin, V.; Park, B. K. Clinical Pharmacokinetics of Tacrine. Clin. Pharmacokinet. 1995, 28, 449–457. (63) Larsen, J. T.; Hansen, L. L.; Spigset, O.; Brøsen, K. Fluvoxamine Is a Potent Inhibitor of Tacrine Metabolism in Vivo. Eur. J. Clin. Pharmacol. 1999, 55, 375–382. (64) Tallarida, R.; Murray, R. Manual of Pharmacological Calculation with Computer Programs; Springer-Verlag: New York, 1987. (65) Bajgar, J. Determination of acetylcholinesterase activity in human blood - possible modification for fields conditions. Vojen. Zdrav. Listy 1972, 41, 78–80.

ACS Paragon Plus Environment

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(66) Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell, D. S.; Olson, A. J. AutoDock4 and AutoDockTools4: Automated Docking with Selective Receptor Flexibility. J. Comput. Chem. 2009, 30, 2785–2791. (67) Liu, B.; Wang, L.; Jin, Y.-H. An Effective PSO-Based Memetic Algorithm for Flow Shop Scheduling. IEEE T. Syst. Man Cy. B 2007, 37, 18–27. (68) Pan, Y.; Chen, Y.; Li, Q.; Yu, X.; Wang, J.; Zheng, J. The Synthesis and Evaluation of Novel Hydroxyl Substituted Chalcone Analogs with in Vitro Anti-Free Radicals Pharmacological Activity and in Vivo Anti-Oxidation Activity in a Free RadicalInjury Alzheimer’s Model. Molecules 2013, 18, 1693–1703. (69) Sugano, K.; Hamada, H.; Machida, M.; Ushio, H. High Throughput Prediction of Oral Absorption: Improvement of the Composition of the Lipid Solution Used in Parallel Artificial Membrane Permeation Assay. J. Biomol. Screen. 2001, 6, 189–196. (70) Wohnsland, F.; Faller, B. High-Throughput Permeability pH Profile and HighThroughput Alkane/water Log P with Artificial Membranes. J. Med. Chem. 2001, 44, 923–930. (71) Mosmann, T. Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays. J. Immunol. Methods 1983, 65, 55–63.

ACS Paragon Plus Environment

Page 62 of 74

Page 63 of 74

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of Medicinal Chemistry

GRAFICAL ABSTRACT

ACS Paragon Plus Environment

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 64 of 74

Chart  1  

   

ACS Paragon Plus Environment

Page 65 of 74

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Journal of Medicinal Chemistry

Scheme  1  

   

ACS Paragon Plus Environment

Journal of Medicinal Chemistry

Figure  1   1.5×10 8

7u 100 nM 178 nM 316 nM 562 nM

-25000

-15000

v-1 (kat-1)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

1.0×10 8

5.0×10 7

-5000

[ATCh]-1 (M-1)

5000

15000

 

             

ACS Paragon Plus Environment

Page 66 of 74

Page 67 of 74

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Journal of Medicinal Chemistry

Figure  2  

 

ACS Paragon Plus Environment

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 68 of 74

Figure  3  

               

ACS Paragon Plus Environment

Page 69 of 74

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Journal of Medicinal Chemistry

Figure  4  

   

ACS Paragon Plus Environment

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 70 of 74

Figure  5  

             

ACS Paragon Plus Environment

Page 71 of 74

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Journal of Medicinal Chemistry

Figure  6  

   

ACS Paragon Plus Environment

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 72 of 74

Figure  7  

   

ACS Paragon Plus Environment

Page 73 of 74

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Journal of Medicinal Chemistry

Figure  8  

 

ACS Paragon Plus Environment

Journal of Medicinal Chemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Grafical  abstract  

 

ACS Paragon Plus Environment

Page 74 of 74