Novel VilazodoneâTacrine Hybrids as Potential Multitarget-Directed

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Novel Vilazodone-Tacrine Hybrids as Potential Multitarget-Directed Ligands for the Treatment of Alzheimer’s Disease Accompanied with Depression: Design, Synthesis and Biological Evaluation Xiaokang Li, Huan Wang, Yixiang Xu, Wenwen Liu, Xiaoxia Qiu, Jin Zhu, Fei Mao, Haiyan Zhang, and Jian Li ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00259 • Publication Date (Web): 05 Sep 2017 Downloaded from http://pubs.acs.org on September 5, 2017

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ACS Chemical Neuroscience

Novel

Vilazodone-Tacrine

Hybrids

as

Potential

Multitarget-Directed Ligands for the Treatment of Alzheimer’s Disease Accompanied with Depression: Design, Synthesis and Biological Evaluation Xiaokang Li†,#, Huan Wang‡,§,#, Yixiang Xu†, Wenwen Liu†, Qi Gong‡, Wei Wang‡, Xiaoxia Qiu†, Jin Zhu†, Fei Mao†,*, Haiyan Zhang‡,*, Jian Li†,* †

Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China

University of Science and Technology, 130 Mei Long Road, Shanghai 200237, China ‡

CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica,

Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, China §

University of Chinese Academy of Science, No.19A Yuquan Road, Beijing 100049,

China #

These authors contributed equally to this work.

*To

whom

correspondence

should

be

addressed.

[email protected], [email protected].

ACS Paragon Plus Environment

[email protected],


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ABSTRACT: Depression is one of the most frequent psychiatric complications of Alzheimer’s disease (AD), affecting up to 50% of the patients. A novel series of hybrid molecules were designed and synthesized by combining the pharmacophoric features of vilazodone and tacrine as potential multitarget-directed ligands for the treatment of AD with depression. In vitro biological assays were conducted to evaluate the compounds, among the 30 hybrids, compound 1e showed relatively balanced profiles between acetylcholinesterase inhibition (IC50 = 3.319 ± 0.708 µM), 5-HT1A agonist (EC50 = 107 ± 37 nM) and 5-HT reuptake inhibition (IC50 = 76.3 ± 33 nM). Compound 1e displayed tolerable hepatotoxicity and moderate hERG inhibition activity, and could penetrate the blood–brain barrier in vivo. Furthermore, an oral intake of 30 mg/kg of 1e·HCl could significantly improve the cognitive function of scopolamine-induced amnesia mice and alleviate the depressive symptom in tail suspension test. The effectivity of 1e validates the rationality of our design strategy.

KEY WORDS: Alzheimer’s disease, Depression, Multitarget-Directed Ligand, ChE inhibition, 5-HT1A agonist, 5-HT reuptake inhibition.


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INTRODUCTION Alzheimer’s disease (AD) is a neurodegenerative disease characterized by a progressive decline in cognitive function and memory process. Its pathogenesis implicates multifactorial factors and has not been fully elucidated. It is estimated that currently over 44 million victims of AD exist in the world and commercial drugs are unable to meet the imperious market demands.1 Currently, the major clinical treatment choices are still acetylcholinesterase (AChE) inhibitors2 and an N-methyl-D-aspartic acid (NMDA) receptor antagonist.3 Many clinical trials based on candidates that aim at new targets have been actively boosted, but none prevail.4 The AD drug-development ecosystem requires strongly support. One of reasons for the difficult treatment of AD is that nearly all patients concurrently suffer from intricate psychological and neuropsychiatric complications,5 including delusion, hallucination, depression, mania, aggression, wandering, apathy and so on.6-8 Among them, depression is one of the most frequent comlication,9-12 with the prevalence of 20-50% depending on different studies and diagnostic criteria.12-16 Depression in AD results in greater damage to the quality of life and increases the burden of caregivers.17 Traditional single target drugs hardly respond to the complex complications, consequently, efficient symptomatic treatment should associate several drugs whose actions are oriented toward diverse symptoms involved in the disease. Therefore, it is a daring but significant attempt to develop a kind of drug candidates could simultaneously alleviate the amnesia and depression symptoms of AD. In fact, some groups have developed serotonin reuptake and AChE dual inhibitors for the potential use of AD with depression.18, 19 As reported, vilazodone is dual serotonin (5-hydeoxytryptamien, 5-HT) reuptake inhibitor and 5-HT1A receptor partial agonist.20 5-HT, a pivotal neurotransmitter widely existing throughout the brain, plays many important roles in normal brain functions including modulation of emotion, anxiety, mood states, vigor, memory and many crucial physiological functions.21,

22

Research indicates the deficiency of



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serotonergic neurotransmissions is implicated with many psychiatric disorders, especially depression.23-26 Based on the serotonergic hypothesis of depression, the selective serotonin reuptake inhibitors (SSRIs), inhibiting the reuptake of 5-HT back into the varicosity of the nerve terminal, thus increasing the amount of synaptic 5-HT, has been developed as a kind of classical antidepressant drugs.27, 28 In addition, it has been widely reported the serotonergic system relates to cognitive and memory process,29-32 and extensive serotonergic denervation occurs in AD patients.33,

34

Therefore, developing molecules that enhance serotonin concentration of synaptic cleft has been treated as potential therapeutic strategy to slow the progression of AD.35 5-HT1A receptor, one of the 17 serotonin receptor subtypes, has long been reported implication in the pathogenesis and treatment of anxiety and depressive disorders.36-38 The 5-HT1A agonist activity of vilazodone contributes to speed up the onset time and extend the SSRI response, as well as less impairment of sexual function, this is the therapeutic advantages of vilazodone compared to other SSRIs.20, 39-41

Our group devoted to the research of old drug new use and the secondary development. Vilazodone,20 a marketed drug approved for major depressive disorder (MDD), was found possessing moderate AChE inhibition activity (IC50 = 21.3 ± 3.0 µM) in our recent high throughput screening (HTS) test with an in-house old drug library. This discovery gave us great inspiration that we could regard vilazodone as lead compound and enhance the AChE inhibitory potency by structural modification to develop a series of multifunctional ligands treating AD with depression. While, the most efficient method was of course to introduce an AChE pharmacophore of known AChE inhibitors (AChEIs) to the molecule of vilazodone. Tacrine was selected as the pharmacophore in view of its simple structure and structure-modifying accessibility. Although the hepatotoxicity of tacrine has limited its clinical use, numerous studies have shown that the safety can be improved by synthesizing tacrine derivatives or dimers.42-45 This is the origin of vilazodone-tacrine hybrids.


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Compounds 1-3 were conceived as structural compromise of vilazodone (antidepressant drug) and tacrine (anticholinergic drug), and this multitarget-directed ligands (MTDLs) profile seems to confer 1-3 the abilities to relieve the symptoms of cognitive impairment and depression simultaneously. Besides, this design strategy is a powerful combination of two CNS (Central nervous system) drugs, and may endow the hybrids with more potential druggability advantages. Based on this conjecture, a series of vilazodone-tacrine hybrids (1-3) were designed and synthesized in this article. Many biological tests have been conducted to evaluate these compounds as multifunctional anti-AD agents, and satisfying results were got. The optimal compound 1e displayed balanced antiamnesiac and antidepressant efficacy in two animal pharmacodynamics evaluation tests. Therefore, these vilazodone-tacrine hybrids could be treated as prospective prototypes in the research of innovative multifunctional drugs for AD, especially accompanied with psychological disorders like depression.

RESULTS AND DISCUSSION Designing Scheme The HTS test disclosed that antidepressant drug vilazodone possessed moderate AChE inhibitory activity. Considering AChEIs were the major choices for AD treatment and depression was the most common complication, vilazodone was expected to develop its new application as multifunctional anti-AD agent. The priority was to improve the deficiency of vilazodone in AChE inhibition, while the 5-HT1A agonist and 5-HT reuptake inhibition activities should be remained because they are beneficial for the therapy of accompanied depressive symptom. Tacrine was a famous AChEI with simple molecular structure, and many researchers have studied the effects of diverse tacrine derivatives in AD mode. Hence, we attempted to graft the tacrine derivative fragments to vilazodone molecule to promote AChE potency. Thanks to Merck KGaA’s intensive research on vilazodone discovery,46,

47

we obtained some valuable structure-activity relationships (SARs):



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Suitable distance of a four saturated carbons chain between the indole ring and the basic nitrogen is critical, the indolebutylpiperazine fragment is an optimum for 5-HT1A binding and 5-HT reuptake inhibition, besides, the benzofuran-2-carboxamide fragment was appropriate site for structural modification and tolerated a diversity of substituents. As a consequence, benzofuran-2-carboxamide fragment was replaced by tacrine derivatives to produce the vilazodone-tacrine hybrids as depicted in Figure 1. Keeping the integrity of indolebutypiperazine fragment, compounds 1a-h were designed with diverse tacrine derivatives. For the sake of further improving the AChE inhibition, we tried to modify the 4-carbon chain and the diamine moiety (series 2 and 3). Based on above, a series of triple 5-HT1A agonist, 5-HT reuptake and AChE inhibitors were designed and synthesized as multifunctional anti-AD agents.

Figure 1. Designing scheme of vilazodone-tacrine hybrids as multitarget-directed potential anti-AD agents Chemistry The targeted compounds were synthesized as outlined in Schemes 1-2. In Scheme 1, the key intermediate 4a (commercial available) reacted with tacrine analogues 5a-h in the presence of Et3N to afford compounds 1a-h. As depicted in


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Scheme 2, the reaction of key indole intermediates 4b-k with tacrine analogues 5a, 5i-l provided target compounds 2a-n, the reaction with tacrine analogues 5e, 5m provided target compounds 3a-h. The synthesis of key intermediates 4b-k and 5a-m were described in supporting information (Schemes S1-7), the detailed structure of compounds 2a-3h were showed in Table 1. Compounds 1-3 were confirmed to have ≥ 95% pure (Table S1, Supporting Information), and were identified with non-PAINS on the web at http://fafdrugs3.mti.univparis-diderot.fr/ recommended by editors from the ACS (American Chemical Society).48 Scheme 1. Synthesis of Compounds 1a-ha

n

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a

Reagents and Conditions: (a) Et3N, KI, CH3CN, reflux.

Scheme 2. Synthesis of Compounds 2a-3ha



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a

Reagents and Conditions: (a) Et3N, KI, CH3CN, reflux; (b) NaH3B(CN), Na2SO4,

CH3OH:CH2Cl2 = 1:1, rt; (c) HOBt, EDCI, DIPEA, DMF, rt. Cholinesterase (ChE) Inhibitory Activity To determine the therapeutic potential of these vilazodone-tacrine hybrids for AD treatment, their anti-cholinesterase (anti-ChE) activities were evaluated by modified method of Ellman et al,49 using AChE from rat cortex and BuChE (butyrylcholinesterase) from rat serum, wherein tacrine and huperzine A (Hup A) were used as reference standards. The experimental data were presented as IC50 (µM) or, for poorly active compounds, as the percentage of inhibition at 40 µM. The results are summarized in Table 1. The IC50 values for rAChE inhibition ranged from greater than 40 µM to 0.098 µM. Compound 1a, having an indolebutylamine moiety of vilazodone and a tetrahydroacridine scaffold of tacrine, exhibited appropriate AChE inhibition, with an IC50 value of 1.134 µM. Introducing substituents on the benzene ring of tacrine unit decreased the potencies dramatically (1a vs 1b-c). Changing the quinoline moiety of tacrine unit to 1,8-naphthyridine moiety (X = H to X = N) was unadvisable (1a vs 1d,


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1e vs 1f). Replacement of cyclohexane ring to cyclopentane and cycloheptane ring only slightly affected the rAChE potencies (1a vs 1e, 1g), but the activity would completely lose if removing the cycloalkane ring (1a, 1e, 1g vs 1h). Compounds 2a-3h were designed by modification of the linker and indole substituents (R1). Introducing carbonyl groups on the four-carbon chain greatly improved the anti-ChE potencies (1a vs 2a-c, 1e vs 3a). Changing the length of four-carbon chain can effectively enhance the AChE inhibitory activities (2a vs 2d-e, 3a vs 3b-c, 3c was the most potent AChE inhibitor with IC50 value of 0.098 µM). Furthermore, replacement of the piperazine moiety to diverse diamine fragments didn’t brought inspiring results (2a vs 2f-i, 3a vs 3d). In the studied set of substituent effect on indole ring, concluding that replacement -CN was not tolerated, leading to an obvious decline in AChE potencies (2a vs 2j-n, 3a vs 3e-h). The IC50 values of these hybrids for rBuChE inhibition ranged from greater than 40 µM to 2.514 µM. In series 1, the important SAR is that the anti-rBuChE potency increased follow the order of 1e (cyclopentane ring), 1a (cyclohexane ring) and 1g (cycloheptane ring), and the other SARs of rBuChE inhibitions are similar with rAChE. In series 2, compounds 2a, 2d-j, and 2l showed moderate inhibitory activities. However, in series 3, most of the compounds displayed poor rBuChE potencies. Most of these vilazodone-tacrine hybrids exhibited relatively more potent potency toward rAChE than rBuChE, but there still no extremely significant selectivity (Table 1). It has been reported that the statuses of AChE and BuChE varied with the progression of the disease, where the role of AChE gradually decreased while BuChE progressively increased.50-52 Therefore, the balanced inhibition of both AChE and BuChE could be beneficial for the therapy of AD, and it is conceivable that nonselective ChE inhibitors may play an extraordinary therapeutic role in all the stages (early, medium and advanced) of this progressive diseases.



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Table 1: Structures, rAChE and rBuChE inhibitory activities, selectivity index for rAChE, and 5-HT1A agonist activities of compounds 1a-3h

R1

R2

R3

X

n

IC50 ± SDa (µM) for rAChE (inhibition % at 40 µM)

1a

CN

H

H

H

4

1.134 ± 0.083

9.822 ± 4.498

1b

CN

H

Cl

H

4

56%

27.27%

2937 ± 1659

1c

CN

OCH3

H

H

4

33%

11.65%

2840 ± 55

1d

CN

H

H

N

4

2.687 ± 0.393

21.44 ± 4.001

8.0

3414 ± 2322

1e

CN

H

H

H

3

3.319 ± 0.708

15.79 ± 0.263

4.8

107 ± 37

1f

CN

H

H

N

3

50.2%

48.78%

1g

CN

H

H

H

5

2.611 ± 0.351

2.514 ± 0.238

1h

CN

H

H

H

0

41.04%

30.85%

compd

Linker


IC50 ± SDa (µM) for rBuChE (inhibition % at 40 µM)

SIb

EC50 ± SDc (nM) for 5HT1A agonist

8.7

1002 ± 497

128 ± 25 0.96

1884 ± 556 16 ± 3

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2a

CN

H

H

H

4

0.300 ± 0.035

8.922 ± 1.156

29.7

6268 ± 1691

2b

CN

H

H

H

4

0.189 ± 0.028

16.64%

> 211.6

ndd

2c

CN

H

H

H

4

0.342 ± 0.056

20.26%

> 117.0

ndd

2d

CN

H

H

H

4

0.246 ± 0.039

22.91 ± 0.512

93.1

ndd

2e

CN

H

H

H

4

0.167 ± 0.033

15.15 ± 0.475

90.7

ndd

2f

CN

H

H

H

4

1.024 ± 0.198

6.529 ± 0.407

6.4

5018 ± 1028

2g

CN

H

H

H

4

0.469 ± 0.081

3.029 ± 0.205

6.5

ndd

2h

CN

H

H

H

4

1.436 ± 0.152

12.44 ± 0.468

8.7

ndd

2i

CN

H

H

H

4

1.918 ± 0.263

16.14 ± 1.007

8.4

ndd

2j

H

H

H

H

4

1.020 ± 0.226

14.9 ± 1.86

14.6

ndd

2k

Cl

H

H

H

4

3.380 ± 0.787

33.13%

> 11.8

ndd

2l

F

H

H

H

4

1.090 ± 0.275

10.6 ± 1.10

9.7

ndd

2m

COOCH3

H

H

H

4

0.687 ± 0.094

55.03%

> 58.2

ndd



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2n

OCH3

H

H

H

4

0.708 ± 0.068

52.53%

> 56.5

ndd

3a

CN

H

H

H

3

0.844 ± 0.096

33.37%

> 47.4

40000 ± 5400

3b

CN

H

H

H

3

0.477 ± 0.039

25.29%

> 83.9

ndd

3c

CN

H

H

H

3

0.098 ± 0.010

21.12%

> 408.2

ndd

3d

CN

H

H

H

3

2.790 ± 0.245

7.683 ± 0.310

2.8

ndd

3e

F

H

H

H

3

1.890 ± 0.252

51.83%

> 21.2

ndd

3f

H

H

H

H

3

3.280 ± 0.771

44.84%

> 12.2

ndd

3g

COOCH3

H

H

H

3

1.940 ± 0.447

34.40%

> 20.6

ndd

3h

OCH3

H

H

H

3

2.560 ± 0.448

54.65%

> 15.6

ndd

Tacrine

0.131 ± 0.016

0.081 ± 0.005

0.6

Hup A

0.119 ± 0.015

36.4 ± 1.838

306.9

Vilazod one 8-OHDPAT a

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21.3 ± 3.00

0.12 ± 0.005 5.78 ± 0.21

Results are expressed as the mean ± SD of at least three experiments. bSelectivity index for rAChE is defined as IC50(rBuChE)/IC50(rAChE).

c

Results are expressed as the mean ± SD of at least two experiments. dNot determined


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5-HT1A Agonist Activity In vitro 5-HT1A agonist activities of these hybrids were investigated to judge whether the newly designed compounds could retain the antidepressant function of vilazodone. Studies were conducted using Human Embryonic Kidney 293 (HEK293) cells transiently expressing relevant human 5-HT1A receptors. Vilazodone and 8-OH-DPAT were used as references. As exhibited in Table 1, the experimental data were presented as EC50 (nM) or, for poorly active compounds, as nd meaning not determined. The EC50 values showed that introduction of substituents on the benzene ring of tacrine unit was not tolerated (1a vs 1b-c), and replacement the quinoline moiety of tacrine unit to 1,8-naphthyridine moiety (X = H to X = N) was detrimental (1a vs 1d, 1e vs 1f). The size of naphthenic ring in tacrine unit seriously impacted the 5-HT1A affinity, a significant increase of agonist activity was observed follow the shrink of the cycle size: cycloheptane ring (1g) < cyclohexane ring (1a) < cyclopentane ring (1e) < no ring (1h). Retaining cyclohexane ring and cyclopentane ring fragment, compounds 2-3 were designed by modified the middle linker and indole substituent (R1). These derivatives (2a-3h) demonstrated remarkably decreased 5-HT1A agonist potencies (Table 1), despite some of them displaying excellent anti-ChE activities. 5-HT Reuptake Inhibitory Activity 5-HT transporter reuptake inhibition (RUI) assay was performed to evaluate whether these hybrids were able to inhibit the process of 5-HT transporter reuptake like vilazodone to exert antidepressant function. Studies were conducted using HEK293 cell line stably expressing the serotonin transporter protein. Vilazodone and citalopram were used as control. Considering their ChE inhibition and 5-HT1A agonist activities, 7 compounds were selected to conduct this assay, the results were shown in Table 2. Modification the middle linker significantly affected the 5-HT reuptake activities (1a vs 2a, 2i; 1e vs 3a, 3d). Keeping the integrity of vilazodone-like indolebutylamine fragment could maintain the activity at a considerable level (1a, 1e, 1g), and the potency enhanced following the augment of naphthenic ring: cycloheptane ring (1g) > cyclohexane ring (1a) > cyclopentane ring (1e). Table 2. The 5-HT reuptake inhibitory activities of selected compoundsa



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a

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compd

IC50 ± SD (nM) for RUI

compd

IC50 ± SD (nM) for RUI

1a

33.8 ± 5.3

3a

1238.4 ± 746.1

1e

76.3 ± 33

3d

1306.3 ± 801.5

1g

7.7 ± 6.4

Vilazodone

0.4 ± 0.1

2a

212.9 ± 14

Citalopram

8 ± 5.4

2i

530.9 ± 222.9

Results are expressed as the mean ± SD of at least two experiments.

SAR Studies The SAR studies of a set of 30 hybrids provided important insights into the essential structural requirements for effective AChE inhibition, 5-HT1A agonist and 5-HT reuptake inhibition. An analysis of the data shown in Tables 1-2 reveals noteworthy observations of SARs for compounds 1a-h, 2a-n, 3a-h: (1) The indolebutylpiperazine fragment is crucial; modifying the 4-carbon chain and the piperazine moiety is disastrous for 5-HT1A agonist and 5-HT reuptake inhibition (1a vs 2a, 2i, 1e vs 3a, 3d), although it is largely enhanced the AChE inhibitory activities (2a-e, 3a-c). (2) The size of naphthenic ring in tacrine unit seriously affected the activities. The 5-HT1A agonist activity increases follow the shrink of the cycle size: cycloheptane ring (1g) < cyclohexane ring (1a) < cyclopentane ring (1e), but for 5-HT reuptake inhibition activity the order is inverse; AChE inhibitory activity is not sensitive for the cycle size change. (3) Introducing substituents on the benzene ring of tacrine unit is not tolerated for both AChE inhibition and 5-HT1A agonist activities(1a vs 1b-c). (4) Changing the quinoline moiety of tacrine unit to 1,8-naphthyridine moiety (X = H to X = N) is adverse for both AChE inhibition and 5-HT1A agonist activities (1a vs 1d, 1e vs 1f). In brief, the SARs are explicit. Compound 1e shows relatively balanced activities against the three targets. In Vitro Blood-Brain Barrier Permeation Assay and Drug-likeness Evaluation The first requirement of drugs playing curative effect is to reach their therapeutic targets, so crossing blood-brain barrier (BBB) is of great concern for developing anti-AD drugs. To explore whether the designed hybrids can penetrate into the brain, a parallel artificial membrane permeation assay (PAMPA) was performed as described by Di et al.53 The results presented in Table 3 indicated that most of the


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hybrids were capable of crossing BBB for their permeability values (Pe) were greater than the threshold for good BBB permeation (Pe > 3.08 × 10-6 cm s-1, CNS+). The experimental Pe values of the tested compounds could serve as significant references in the screening process of potential drug candidate. To determine the potential druggability of compounds 1a, 1e and 1g, their drug-likeness physicochemical properties were evaluated. As shown in Table 4, compounds 1a, 1e, 1g not only obey Lipinski’s rules but although fulfill other rules of CNS drugs.54-56 Compound 1e has small molecular weight, shows relatively low clogP, clogD7.4 values and high Pe value compared with 1a, 1g. Therefore, compound 1e was selected as the optimal one to conduct further animal experiments. Table 3. Permeability results from the PAMPA-BBB assay for selected compounds and their prediction of BBB penetration

compd

Pe (10-6 cm s-1)a

predictionb

compd

Pe (10-6 cm s-1)a

predictionb

1a

4.29 ± 0.600

CNS+

2l

8.87 ± 1.454

CNS+

1e

4.94 ± 0.175

CNS+

2m

1.02 ± 0.076

CNS−

1g

1.75 ± 0.059

CNS±

2n

4.40 ± 0.635

CNS+

2a

3.85 ± 0.091

CNS+

3a

2.03 ± 0.019

CNS±

2b

1.65 ± 0.125

CNS±

3b

1.77 ± 0.054

CNS±

2c

2.75 ± 0.118

CNS±

3c

1.68 ± 0.133

CNS±

2d

3.99 ± 0.199

CNS+

3d

4.57 ± 0.169

CNS+

2e

4.15 ± 1.348

CNS+

3e

4.36 ± 0.186

CNS+

2f

5.08 ± 0.410

CNS+

3f

5.45 ± 0.324

CNS+

2g

4.39 ± 0.319

CNS+

3g

0.74 ± 0.064

CNS−

2j

5.33 ± 0.417

CNS+

3h

3.46 ± 0.024

CNS+

a

Values are expressed as the mean ± SD of at least three independent experiments.

b

Compounds with permeabilities Pe > 3.08 × 10-6 cm s-1 could cross the BBB by

passive diffusion (CNS+), Pe < 1.13 × 10-6 cm s-1 could not cross the BBB (CNS−), 1.13 × 10-6 cm s-1 < Pe < 3.08 × 10-6 cm s-1 show uncertain BBB permeation (CNS±). Table 4. Drug-likeness properties of compounds 1a, 1e, 1ga compd

MW

HBAb

HBDb

H-bonds

RBb

tPSAb


clogPb

clogD7.4c


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 16 of 46

1a

463.63

5

1

6

6

58.95

6.03

4.32

1e

449.60

5

1

6

6

58.95

5.52

3.77

1g

477.66

5

6

6

58.95

6.54

4.88

rules a

< 500

1

d

< 10

e

e

< 450

d

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