Methylene-Cycloalkylacetate (MCA) Scaffold-Based Compounds as

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Methylene-cycloalkylacetate (MCA) scaffoldbased compounds as novel neurotropic agents David Lankri, Dikla Haham, Adi Lahiani, Philip Lazarovici, and Dmitry Tsvelikhovsky ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00473 • Publication Date (Web): 21 Dec 2017 Downloaded from http://pubs.acs.org on December 22, 2017

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ACS Chemical Neuroscience

Methylene-cycloalkylacetate (MCA) scaffold-based compounds as novel neurotropic agents

David Lankri,† Dikla Haham,‡ Adi Lahiani,‡ Philip Lazarovici*‡ and Dmitry Tsvelikhovsky*†

School of Pharmacy, Institute for Drug Research, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 91120, Israel

Corresponding authors: ‡

Prof. Philip Lazarovici-School of Pharmacy Institute for Drug Research, Faculty of Medicine,

The Hebrew University of Jerusalem, POB 12065, Jerusalem 91120, Israel. E-mail: [email protected]; Tel: +972-2-6758729; Fax: +972-2-6757490. †

Dr. Dimitri Tsvelikhovsky- School of Pharmacy Institute for Drug Research, Faculty of

Medicine, The Hebrew University of Jerusalem, POB 12065, Jerusalem 91120, Israel. E-mail: [email protected]; Tel: +972-2-6757032; Fax: +972-2-6757076

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ABSTRACT One of the main symptoms in degenerative diseases is death of neuronal cell followed by the loss of neuronal pathways. In neuronal cultures, neurite outgrowths are cell sprouts capable of transforming into either axons or dendrites, to further form functional neuronal synaptic connections. Such connections have an important role in brain cognition, neuronal plasticity, neuronal survival and regeneration. Therefore, drugs that stimulate neurite outgrowth may be found beneficial in ameliorating neural degeneration. Here, we establish the existence of a unique family of methylene-cycloalkylacetate-based molecules (MCAs) that interface with neuronal cell properties, and operate as acceptable pharmacophores for a novel neurotropic (neurite outgrowth inducing) lead compounds. Using an established PC12 cell bioassay, we investigated the neurotropic effect of methylene-cycloalkylacetate compounds by comparison to NGF, a known neurotropic factor. Micrographs of the cells were collected by a light microscope camera, and digitized photographs were analyzed for compounds- induced neurotropic activity using NIH image protocol. The results indicate that the alkene element, integrated within the cycloalkylacetate core, is indispensable for neurotropic activity. The discovered lead compounds need further mechanistic investigation and may be improved towards development of a neurotropic drug.

Keywords: Methylene-cycloalkylacetate (MCA), pharmacophore, neurotropic activity, Df , PC12 cultures, NGF


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INTRODUCTION Development of natural or synthetic neurotrophic drugs, which prevent or delay cell death and preserve or induce neuronal pathways by stimulating formation of axons, dendrites and synaptic connections is an unmet clinical need.1 Therefore, development of neurotropic drugs, which can induce neuronal sprouts regeneration, may represent an important development in regenerative medicine.2 The continuing screening of novel neuroprotective, neurotrophic (promoting survival) and neurotropic (promoting neurite outgrowth) drugs by pharmaceutical companies, utilizes synthetic chemical platforms followed by screening to discover an effective lead compound.3 In addition to brain cortical and hippocampal neurons, a very popular neuronal model to investigate potential new drugs, consist of PC12 cell cultures.4 These cells are catecholaminergic, immortalized neurons that were cloned from a solid pheochromocytoma tumor of the adrenal gland of England Deaconess Hospital strain of rats injected with carcinogen.5 These neurosecretory cells contain mainly dopamine and norepinephrine, their doubling time is between 48-96 hours, have 40 chromosomes and can be easily propagated and cultured in vitro for screening chemicals with neurotropic activity.6 The PC12 cell line has been used in neuroscience research for studying neuronal signaling7 and has become the premiere model of choice for the study of neuronal differentiation. When treated with nanomolar concentrations of nerve growth factor (NGF), PC12 cells stop dividing, initiate short neurite processes (outgrowths) in the first 2 days of treatment, elongate significantly at seven days with sprouting (branching), and form synaptic connections between the neurites which, with time, become electrically excitable.6 NGF-neurotropic effects in the PC12 model can be conveniently measured using the fractal dimension (Df), a suitable parameter reflecting a dose-dependent elongation of neurite outgrowth (i.e. quantitative measurements of neuronal differentiation).8 For these reasons, PC12 cell cultures have been used intensively as a neuronal model for the discovery of neurotropic drugs.9 For example, several non-



peptide natural compounds such as

phenylbutenoids10 and talaumidin,11 or polyphenolic

compounds such as flavonoids,12 were discovered to promote neurite outgrowth in micromolar concentrations in the PC12 neuronal model. However, limited structure-activity relationship and pharmacophore information has slowed the development of these lead compounds for clinical trials. The methylene-cycloalkylacetate structures (MCAs) can be frequently observed as scaffolds of various compounds and drugs of natural origin.13 Examples of these motifs have been identified among terpenoids,14 alkaloids,15 and antibiotics.16 Numerous studies have led to a wide variety of potential pharmaceutical candidates that share the methylene-cycloalkylacetate structure. Such a broad natural diversity makes them attractive targets for synthetic medicinal chemists. Some distinct examples, which all contain MCA frames, despite having different biological origins, are shown in Figure 1. Based on typical examples of natural products bearing methylenecycloalkylacetates, such as dysidolide, halmic acid, angolensate, etc. (Figure 1),17 a remarkable overlap in their structures becomes apparent. Recently, we reported general and collective syntheses of phylogenetically diverse tricyclic, spirolactones via controlled cyclizations of easily accessible common cycloalkylmethylene key precursor (1; Figure 2A).18 We have noticed that precursors, currently employed as platform for further syntheses of spirolactones, possess the capacity to act as cores of numerous natural products. The present study therefore, was inspired by assumption that synthetic methylene-cycloalkylacetate scaffolds, which are small, rigid, and highly reminiscent of natural scaffolds, could serve as operational ligands for development of a neurotropic lead compound. Many MCA-based natural products have been firmly established to demonstrate pharmacological activities.19 Thus, we were


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O

O

OH O

O

OH Me

HO2C Me

HO

O

O

O

RO

H

Me

O

Me Me

Me

Me

Me O

H

CO2Me Me

Me

R1

Me Me

Me

OH

Me

O

Methylenecycloalkylacetate common motif

O

ent-Dysidolide

ent-Halimic acid

Secocadinane extracted from A. annua

methyl-Angolensate

Figure 1. Natural products from diverse biological origins share methylene-cycloalkylacetate systems.

motivated to apply our designed architectures to the discovery of novel neurotropic compounds using the pheochromocytoma (PC12) cell neuronal model. We hypothesized that monocyclic dieneester 4 (Figure 2B), which can be readily synthesized,18 could serve as operationally acceptable pharmacophore for a novel neurotropic (neurite outgrowth inducing) lead compound.

A

Previous work: Methylene-cycloalkylacetate as a precursor for synthesis of spirolactones Methylenecycloalkylacetate precursor H (n)

X(n)

O OR

H

R = Me, Et or t-Bu I2

X(n)

(n)

O O

MeCN rt, 2 h

H

Pd2(dba)3 (7.5 mol%) SIMes (25 mol%)

O

(n)

O

X(n)

Cs2CO3 (1.1 equiv) MeCN, 100 °C, 16 h

I

Tricyclic spirolactone

1 Pd(OAc)2 (15 mol%), AgOAc (2.2 equiv.),

R=H

DMSO, 100 °C, 16h

B

Rapid accesss to methylene-cycloalkylacetate frame

O

R1

H

one-pot operation

(n)

R1

O

O

(n1)

KOt-Bu (2 equiv.) OR2

(n)

(ref. 21a)

2

MePPh3Br (2 equiv.)

O 50 3

(n2)

oC

H R1

(n1)

OR2

(n)

to r.t, THF, 3 h

R3

4

hydrolysis OR2 OH

(n2)

4a

R3

R1-3 = alk, Ar, alkene, N, O, S, heterocycle

Figure 2. Methodology developed for collective synthesis of methylene-cycloalkylacetates.



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RESULTS AND DISCUSSION The desired scaffolds (4 or 4a; Figure 2B) might be successfully constructed through the simple and straightforward sequence of synthetic transformations: α,α’-double enamine alkylation of cyclic ketone 2 (construction of intermediate 3); olefination (access to the collective precursor - 4); and hydrolysis. Following the established protocol, we prepared a series of compounds 5-21 featuring diverse electronic and steric characteristics (displaying varying ring architectures and functional group combinations; Figure 3).

Variation of R2 (H or Me)

A

O

O H

O

H

(n)

O

H OMe

OR2

O

H

H

OH

OH

OMe

R1 R3

Me

Me

Me

Me 5

Model MCA scaffold R1-3 = alk, Ar, alkene, N, O, S

B

Me

6

7

Variation of R1 (alk, Ar, alkene) O

O

O

H

H

H

OH

9

O

H

OH

C

8

10

OMe

Me Me Me

OH

11

12

Incorporation of heteroatoms into the cycloalkyl motif HO

O

O H

O

H

O

H

O

H

OR

OH

N

O

14 R = H 15 R = Me

16

OH S

EtO extension of chain length 13

D

17

Ketone variations O H

CN H

O H

O H

OMe

OMe

O

O

OMe

O

O

Me

Me Me 18

19

Figure 3. General architecture of methylene-cycloalkylacetates


20

21

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To examine whether the compact cycloalkyl scaffolds are endowed with neurotropic activity, we first screened a library of synthesized compounds 5-13, illustrated in Figure 3. The functional modifications of general methylene-cycloalkylacetate frame, in this case, were performed on the alkene-bearing chains (5-8), the cyclic domains (9-12) and the length of an acid chain (compound 13) according to the sequence demonstrated on Figure 2B. Prior to investigation of the activation profile of our library, using the PC12 cell neurotropic assay, we evaluated all compounds for cytotoxicity from 0.1 to 100 µM. Since the majority of compounds were not cytotoxic at 10 µM they were evaluated and compared at this concentration (Table 1). Compounds 5, 6, 7 and 13 were found the most active in inducing fast neurite outgrowth after two days of treatment, while after seven days of treatment a significant neurotropic activity was measured for compounds 5, 6, 7, 12 and 13 (Table 1 and Figures 4 and 5).

Neurotropic effect (mean ± SE) Compounda Df 5 6 7 8b 9 10 11 12 13 14 15b 16 17 18b 19 20 21

Two Days NGF (%)

0.42±0.01 0.14±0.15 0.12±0.14 n.a n.a n.a n.a n.a 0.39±0.01 n.a n.a n.a n.a n.a n.a n.a n.a

91.3 30.4 26.1

84.8

Df

Seven Days NGF (%)

0.20±0.17 0.20±0.17 0.20±0.17 n.a 0.01±0.01 n.a 0.01±0.01 0.17±0.16 0.45±0.01 n.a n.a n.a n.a n.a n.a n.a 0.01±0.01

43.5 43.5 43.5 2.2 2.2 37.0 97.8

2.2

Table 1. Neurotropic activity of the methylene-cycloalkylacetate novel derivatives. a

All compounds were evaluated at a concentration of 10 µM; n.a- not active. bPartial toxicity of about 30% of the

culture was observed at 10 µM.



1.2

1.0

Neurotropic activity Df 2 Days * 7 days *

*

*

0.8

0.6

0.4

0.2

16

17

18

19

20

21

15

20

14

19

13

18

12

17

11

16

10

15

9

14

8

13

9

7

12

8

6

11

7

5

10

6

Control NGF DMSO

5

NGF

0.0 Control/DMS O

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

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21

Compound

Figure 4. Neurotropic activity of MCAs at 10 µM after 2 and 7 days of treatment of PC12 cells.

By comparing to NGF effect, the most potent compounds were 5 > 13 > 6 > 7 and 13 > 5, 6, 7>12 after two and seven days of treatment, respectively. Therefore, we conclude that compounds 5 has a transient, acute effect on induction of neurite outgrowth, loosing 50% activity from day two to seven, while for compounds 6, 7, 12 and 13, the neurotropic activity was gradually increased from day two to seven, reminiscent of NGF neurotropic activity time course. Thus, we found that compact, methylene-cycloalkylacetate-based molecules could induce significant, NGF-like neurotropic activity, in PC12 neuronal cells. Interestingly, no activity was detected for compounds with substituted aromatic anchors (compounds 10 and 11), or 1,4-dimethylenated substrate (compound 9). After concluding that structures 5, 6, 7, 12 and 13 are endowed with neurotropic activity, we were compelled to examine the functionality of monocyclic variants integrated with heteroatoms. For this purpose, compounds 14−17 were designed according to the reported methodology,18 and their neurotropic activity was identically measured using the PC12 cell neurotropic assay. As shown in


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Figure 4 and Table 1, the incorporation of O, N or S atoms into the cyclic frame caused a complete loss of neurotropic activity.

Figure 5. Light microscopy images of PC12 cells after 7 days of treatment with 10 µM of different MCA compounds. Magnification X 100.

Based on these results, we were intrigued by the possibility of applying the established protocol to unsaturated variants of cycloalkylacetate substrates (Figure 2D). For these series of molecules (compounds 18-21), the cyclic alkene group was substituted by a ketone residue, and their neurotropic activity was again measured using the PC12 cell neurotropic assay. No significant neurotropic activity was detected for the above-mentioned compounds. This observation suggests that an alkene element is indispensable for the neurotropic activity. However, for compounds 14– 17, which resemble the topology of active compounds 5, 6, 7 and 12 and retain the double bond (see Figure 2), an introduction of heteroatoms within the central ring resulted with complete loss of



neurotropic activity. It should be noted that partial cytotoxic activity was detected for compounds 8, 15 and 18. Thus, due to interference with neurotropic activity, these MCA derivatives were excluded from the characterization.

CONCLUSION The methylene-cycloalkylacetate (MCA)-based molecules, demonstrated in this work, represent a novel scaffold for development of prospective neurotropic drugs. An alkene element, integrated within a cycloalkylacetate core, has been shown to act as a facilitating factor of intrinsic neurotropic activity of such structures. By employing this line of research, our ultimate aim is to single out a small molecule, bearing a potential for treatment of brain disorders, caused by insufficient trophic support. This is in line with the existing medicinal chemistry approaches, bolstering the search for small molecules capable of triggering regeneration and/or other neurotropic effects. If identified, these could further lead to a development of a full scale treatment of brain /neurological disorders. Examples of such neurotropic effects were reported in PC12 model using several natural products,3a derived from herbs/plants: diterpenenes from the plant Croton yanhuii;20 sesquiterpenes and iridoids from the plant Valeriana;21 sargaquinoic acid from the marine brown alga Sargassum macrocarpum,22 as well as prenylflavonoids, abundant in many plants.23 Along the same lines, neurotropic effects were found in PC12 cells using synthetic molecules such as catecholamine precursor dihydroxyphenylalanine (L-DOPA),24 N-propargyl caffeate amide,25 pyrimidine heterocyclic compounds,26 and synthetic peptides.27

EXPERIMENTAL SECTION General Information


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Unless otherwise stated, all reagents were purchased from commercial suppliers and used without further purification. DMEM medium, fetal calf (FCS) and horse (HS) serums, penicillin and streptomycin were purchased from Biological Industries (Beit Haemek, Afula Israel). Tissue culture grade mouse β-NGF, was purchased from Alomone Labs (Jerusalem, Israel). Solvents used in the reactions were distilled from appropriate drying agents prior to use. Reactions were monitored by thin-layer chromatography (TLC) on silica gel 60 F254 aluminium plates (Merck) and/or gas chromatography-mass spectrometry (GCMS). Visualization of compounds on TLC was accomplished by irradiation with UV light at 254 nm and/or vanillin stain. GCMS Analysis was performed with ‘Agilent 7820A’ gas chromatograph equipped with ‘Agilent 5975’ quadrupole mass selective detector, using Agilent HP-5MS capillary column (30 m, 0.25 mm, 0.25 µm film). Column chromatography was performed using silica gel 60 (particle size 0.040 - 0.063 mm) purchased from Sigma-Aldrich. Proton and carbon NMR spectra were recorded on Varian Mercury 300 MHz or Varian Mercury 500 MHz spectrometer in deuterated solvent. Proton chemical shifts are reported in ppm (å) relative to tetramethylsilane with the solvent resonance employed as the internal standard (CDCl3, δ 7.26 ppm).

13

C chemical shifts are reported in ppm from

tetramethylsilane with the solvent resonance as the internal standard (CDCl3, δ 77.0 ppm). Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet), integration and coupling constants (Hz). Infrared (IR) spectra were recorded on a Thermo Fischer Scientific NICOLET iS10 spectrometer.

Methods

Cell Cultures - PC12 cells were grown in T-200 flasks in high glucose (4.5 gr/L) DMEM medium supplemented with 7% FCS, 7% horse serum and 1% penicillin and streptomycin. Cells were incubated at 370C in a humidified incubator containing 6 % CO2. All experiments were carried out in a clean room, according to ISO7 requirements (10,000 particles/m3).



Cell Seeding - Tissue culture Falcon plates were coated with 200 µg/ml collagen type 1 and placed under UV light for 30 minutes for sterilization. Thereafter, one ml of PC12 cell suspension (5000 cells/well) was applied in 12 or 24 well plate, respectively. The cultures were grown in the incubator two days before exposure to investigated compound.

Neurotropic activity (neurite outgrowth assay) - This experimental procedure contained two controls. The first consisted of untreated cells (negative control), representing the random effect: "noise signal"- the ability of cells to spontaneously grow neurite outgrowth which in seven days are of a length less than two-fold cell diameter. Also, negative controls consisted of cultures treated with 1% DMSO, solvent used to solubilize all compounds. The positive control consisted of cells treated with 50 ng/ml NGF, indicating maximal neurite outgrowth that can be achieved in this model. The experimental group consisted of PC12 cells treated with synthetic compounds. In each experiment, after two and seven days, six cultures were evaluated for neurotropic effect. These consecutive observations allow measurement of the progress of neurite outgrowth elongation and the percentage of cells with neurites.28

Analysis of the neurotropic activity - In order to assess neurotropic effect, the neurite outgrowth length in each culture was quantified. For this purpose, each culture was placed under an inverted microscope and photographed by the attached camera. Each well was photographed at three to five representative areas. After acquiring the photos, they were analyzed by ImageJ, NIH software. The neurite outgrowth was estimated by fractal dimension (Df), a statistical parameter that describes the fractional space (area and length) filled by neurite outgrowth. Df ranged from 0 to 1.20. This method estimates the amount of picsel covered by neurites/cells compared to empty space per square field, and therefore plotting log (number of square boxes) vs. log (size in pixels) relationship generates a linear curve with Df representing the slope of the curve. Every photograph that was taken, was opened in a Photoshop software and a new layer was generated on it. Using a 5-pixel wide digital pencil tool, all the outgrowths were marked and the layer with the markers (outgrowth


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network) was saved in a 0-255 gray scale as described in Fig. 2. Then, the saved layer was opened by ImageJ NIH software. The software "skeletonized" the layer, measured the length and complexity of every outgrowth in a fractal box count and calculated the fractional dimension parameter (Df).29

Figure 6. The method of neurotropic effect analysis after overlaying the neurite outgrowths. A. The original photo of the neurotropic effect taken by the light microscope camera at a magnification of X100; B. The overlaid neurite outgrowths skeletonized in a 0-225 gray scale by Image J in order to calculate Df

Cytotoxicity - Cell death was evaluated by morphological appearance of the cells and release of LDH into the medium, in the absence and presence of different concentrations of synthetic compounds as previously described.30 Statistics - Each experiment was performed in duplicates and repeated three to four times (n=6-8). By using SPSS software, one way ANOVA was performed for the fractional dimension of each compound, in order to evaluate the neurotropic effect. In case of significance, Bonferroni post-hoc analysis was performed. The results were considered significant when p

Methylene-Cycloalkylacetate (MCA) Scaffold-Based Compounds as

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