Design and Synthesis of Tubulin and Histone Deacetylase Inhibitor

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Design and Synthesis of Tubulin and Histone Deacetylase Inhibitor Based on isocombretastatin A-4 Diana Lamaa, Hsin-Ping Lin, Lena Zig, Cyril Bauvais, Guillaume Bollot, Jérôme Bignon, Helene Levaique, Olivier Pamlard, Joëlle Dubois, Mehdi Ouaissi, Martin Souce, Athena KASSELOURI, François Saller, Delphine Borgel, Chantal Jayat-Vignoles, Hazar AlMouhammad, Jean Feuillard, Karim Benihoud, Mouad Alami, and Abdallah Hamze J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00050 • Publication Date (Web): 13 Jul 2018 Downloaded from http://pubs.acs.org on July 13, 2018

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Journal of Medicinal Chemistry

Design and Synthesis of Tubulin and Histone Deacetylase Inhibitor Based on isocombretastatin A4 Diana Lamaa,a,‡ Hsin-Ping Lin,a,‡ Lena Zig,b Cyril Bauvais,c Guillaume Bollot,c Jérôme Bignon,d Helene Levaique,d Olivier Pamlard,d Joelle Dubois,d Mehdi Ouaissi,e Martin Souce,f Athena Kasselouri,f François Saller,g Delphine Borgel,g Chantal Jayat-Vignoles,h Hazar AlMouhammad,h Jean Feuillard,h,i Karim Benihoud,b Mouad Alami,*,a Abdallah Hamze*,a a

BioCIS, Univ. Paris-Sud, CNRS, équipe labellisée Ligue Contre le Cancer, Université Paris-Saclay, 92290,

Châtenay-Malabry, France b

Vectorologie et thérapeutiques anticancéreuses, UMR 8203 CNRS, Univ. Paris-Sud, Institut Gustave Roussy,

Université Paris-Saclay, Villejuif, France-94805 c

SYNSIGHT, 86 rue de Paris Orsay 91400, France

d

CIBI platform, Institut de Chimie des Substances Naturelles, UPR 2301, CNRS avenue de la terrasse, F-91198 Gif

sur Yvette, France e

CHRU Hôpital de Tours Trousseau, Service de chirurgie digestive, oncologique, endocrinienne et de

transplantation hépatique, avenue de la République, 37170 Chambray-lès-Tours f

Lip(Sys)², Chimie Analytique Pharmaceutique, Univ Paris-Sud, Université Paris-Saclay, F-92290 Châtenay-

Malabry, France. g

INSERM, UMR-S1176, University Paris-Saclay, F-94276 Le Kremlin-Bicêtre, France

h

Univ Limoges, Faculté de Médecine, CNRS UMR 7276, Laboratoire CRIBL, F-87025 Limoges, France

i

CHU Limoges, Hôpital Dupuytren, Service d'hématologie, F-87025 Limoges, France

1 ACS Paragon Plus Environment

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‡

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D.L. and L. H-S contributed equally to this work.

ABSTRACT

Designing multi-target drugs have raised considerable interest due to their advantages in the treatment of complex diseases such as cancer. Their design constitutes a challenge in antitumor drug discovery. The present study reports a dual inhibition of tubulin polymerization and HDAC activity. On the basis of 1,1-diarylethylenes (isoCA-4) and belinostat, a series of hybrid molecules was successfully designed and synthesized. In particular compounds, 5f and 5h were proven to be potent inhibitors of both tubulin polymerization and HDAC8 leading to excellent antiproliferative activity.

Keywords: histone deacetylase, tubulin, antiproliferative activity, multi-target drugs, dual inhibitor INTRODUCTION

In the last decade, multi-target drugs have gained considerable interest due to their advantages in the treatment of complex diseases. In cancer, because of the affected network complexity and the involvement of different cell types, a selective single target drug can rarely achieve its desired effect, and most curative cancer treatments are based on the combination of multiple effective drugs.1 However, the polypharmacology approach is often linked with side effects. Interestingly, when rationally designed drugs with multiple targets could have a superior therapeutic effect and display low side effect profile.2 Based on these challenges the paradigm of hybrid molecules with dual activity was developed. Hybridization of drugs is growing and undergoing development in both industry and academia,3 and their design is a niche in the large


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field of drug discovery.4 Hybrid molecules are defined as new chemical entities which combine two distinct biologically active molecules, which could act as two distinct pharmacophores at different targets. The aim of this hybrid drug is to modulate, amplify, or exert dual-drug action.5 The main challenge in the development of dual molecules is the need to design a drug that modulates multiple disease targets simultaneously. Cancer treatment protocols are mostly designed to interfere with one or more mechanisms involved in cell proliferation. Thus, this heterogeneity open up a research opportunities for the multi-target drugs’ design and development. Histone deacetylases (HDACs), a class of enzyme which ensures the removal of acetyl groups from ε-N-acetyl lysine residues of histones, consequently play an important role in epigenetic regulations.6 They have essential role in many biological processes, largely through their repressive influence on transcription, which is responsible for regulation of proliferation, angiogenesis, migration, differentiation, and metastasis.7 Given the biological function of HDACs, it is not surprising that they play a highly important role in different diseases, and are the target of many drugs. Overexpression of HDACs is observed in various types of human tumors, including colorectal, gastric, liver breast, and lung cancers.8 Therefore, inhibition of HDACs has been approved as a successful strategy treating many cancer forms, and multiple products have reached the market as antitumor drugs, such as romidepsin (2004), vorinostat (2006), belinostat (2014), panobinostat (2015), and chidamide (2015). Over the past several years, a large number of clinical trials have been carried out, and some are on-going involving HDACi as a single therapy or in combination with other anticancer agents to improve their antitumor activities.9 Many studies have shown that HDACi could synergistically enhance the inhibitory effects of other antitumor agents, such as HSP90, topoisomerase, and tubulin inhibitors.10 3 ACS Paragon Plus Environment


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The tubulin/microtubule system plays an essential role in several primordial eukaryotic cellular processes such as cell division, motility, intracellular trafficking. It constitutes an important target for anticancer therapy,11 hence in recent years, new tubulin-binding agents have been under preclinical or clinical development. The tumor vascular disrupting agent combretastatin A4 (CA-4, Figure 1) is one of these classes of molecules and its association with carboplatin, paclitaxel, and bevacizumab, is currently under investigation in phase II of clinical trials in advanced non-squamous non-small-cell lung cancer.12 Its combination with pazopanib is in the phase 1b for the treatment of advanced ovarian cancer.13 The SARs study has shown that the optimal biological activity of CA-4 is linked to its cis-olefinic bond configuration.14 Unfortunately, the Z-configuration of CA-4 is associated with in vivo stability problems, due to its Z-E isomerization15 which currently remains the main obstacle to the broad clinical application of CA-4. From a chemical point of view, the pharmaceutical process of scaling-up of this molecule to produce the pure Z-enantiomer represents another weak point. Therefore, several attempts to modify this Z-olefinic bridge have been reported with the aim of preventing the isomerization problem. This mostly included the synthesis of restricted CA-4 analogs in which the Z double bond was incorporated into a heterocyclic system.16 In our ongoing effort to modify this link-bridge, we have recently reported a series of combretastatin A-4 analogs where isoCA-4 (Figure 1) emerged as the most potent agents with a comparable antiproliferative activity to CA4 without the isomerization problem.17 IsoCA-4 showed an excellent antitumor activity against a variety of cancer cell lines.17 Moreover, indolyl-based isocombretastatins also displayed potent cytotoxic activity (Figure 1).18 Recently, we have demonstrated that the nanocomposites formed between squalene-gemcitabine/isoCA-4 induced complete regression of tumors xenografted on nude mice.19


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A recent study has shown a synergic effect of the combination of microtubule-destabilizing agent vincristine and vorinostat (HDACi) in leukemia in vitro and in vivo,20 suggesting that vorinostat may alter microtubule dynamics through HDAC inhibition. Consequently, the discovery of a single molecule targeting both tubulin and HDAC proteins efficiently constitutes a significant challenge. Based on these results, isocombretastatins derivatives (3a or indoleisocombretastatin) and HDACi (vorinostat or belinostat) could be used as a good template to design dual tubulin/HDAC inhibitors. In this study, we have rationally designed and synthesized a series of novel isocombretastatinvorinostat or belinostat hybrids as dual antitumor agents. IsoCA-4 and vorinostat were first merged into new hybrid molecule 5a-b (Figure 1). Since structure-activity relationship studies on isoCA-4 derivatives21 have shown that the substitution at the hydroxyl group of isoCA-4 was tolerable, the linker bearing the hydroxamic acid group was attached to this position. Secondly, to probe the impact of the length and the nature of the linker on the activity, an olefinic linker derived from belinostat was used (5e-g), as well as an acetylenic linker 5c-d (Figure 1), and we finally tested the direct connection (no linker) between isoCA-4 and the zinc-binding group (ZBG). Third, a benzamide motif 5i was introduced replacing the hydroxamic acid ZBG. Lastly, in order to validate the importance of the ring B of isoCA-4 in the newly designed molecules, a series of indoleisocombretastatin were also designed and synthesized. These modifications are very interesting in terms of ligand-protein interactions, assessing new binding possibilities, as well as activity modulation.



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Figure 1. Chemical structures of CA-4, isoCA-4, and design of chimeric tubulin and HDAC inhibitors. RESULTS AND DISCUSSION

Chemistry N-tosylhydrazones were used as a source for the in situ generation of diazo substrates in the presence of a base.22 Recently, N-tosylhydrazones have emerged as a new type of versatile coupling partners for transition metal-catalyzed cross-coupling reactions, making this type of 6 ACS Paragon Plus Environment

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cross-coupling reactions practical in organic synthesis.23 N-Tosylhydrazones are easily derived from the corresponding ketones or aldehydes. Therefore, we found this methodology was perfectly suited for the preparation of the 1,1-diaryl ethylene scaffold of isoCA-4, as it results in the formation of the requisite double bond in a one-step procedure, in comparison to the classical Wittig reaction which requires many steps for synthesis. As depicted in Scheme 1, the key intermediate isoCA-4 was prepared in two steps. First, the coupling of the corresponding N-tosylhydrazone derivative 1a and aryl halide 2a under our previously reported conditions, using PdCl2(MeCN)2/dppf system.24 Secondly, the deprotection of OTBS group in the presence of K2CO3 in MeOH led to the formation of compound 3a, in good overall yield of 71%. The reaction of the phenol group of 3a with alkyl halides gave the corresponding alkyl aryl ethers 4a-c, in an 82, 97 and 85% yields respectively, which were converted quantitatively to the desired hydroxamic acids 5a, 5b, and 5c.

Scheme 1. Synthesis of esters derivatives 4a-c and compounds 5a-c. The synthesis of compounds 5d−j was carried out as shown in Scheme 2. Key intermediates 3b-d were prepared as above using N-tosylhydrazone partner 1a and di-halogenated aryl or heteroaryl derivatives 2b-d. In the case of electrophilic pyridine partner, the dppf ligand was found to be more efficient than Sphos. 7 ACS Paragon Plus Environment


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Compounds 4d-e containing alkyne function in meta-position of isoCA-4 were obtained by using standard Sonogashira coupling of intermediate 3b with the corresponding alkynes in excellent yield.25 Treatment of ester 4d-e with hydroxylamine gave the desired hydroxamic acid derivatives 5d-e. The acrylates derivatives 4f−h were obtained in good yield by reacting the corresponding aryl halides 3b-d with methyl or ethyl acrylate, using Mizoroki-Heck cross-coupling.26 Those compounds were converted into compounds 5f−h by treatment with NH2OH in MeOH. To attach the hydroxamic acid directly on the aromatic ring of the isoCA4, we initially prepared, ester 4i by reaction between aryl bromide 3b and ethyl chloroformate. This key intermediate 4i was used subsequently for the preparation of hydroxamic derivative 5i, but also for the formation of benzamide derivative 5j by reaction with o-phenylenediamine in the presence of EDCI/HOBt.


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MeO I

Br

NNHTs MeO

2b I

OMe

N

Method A PdCl2(MeCN)2 5%, dppf 10%, LiO-t-Bu 2,2 éq, dioxane 100°C, sealed tube

Br

+

MeO

Br

OMe

OMe 1a

Method B Pd(OAc)2 (5 mol %), SPhos (10 mol %), LiO-t-Bu 2,2 éq, dioxane 100°C

2c Br I 2d

MeO , 2b B m od fro eth m MeO from 2c, method A

N

Br

MeO

f m rom et 2 ho d d , B

OMe

OMe OMe 3b, 70%

OMe OMe 3c, 74% Br

MeO MeO

OMe OMe 3d, 78%

O n

O OEt

OEt

3b

PdCl2(PPh3)2/CuI Et3N/THF

MeO

NHOH

NH2OH/MeOH

OMe

MeO

OMe OMe n = 2; 5d, 85% n = 3; 5e, 82%

O OMe

MeO

MeO Pd(OAc)2/P(o-Tol)3 Et3N/THF, sealed tube

O OMe NH OH/MeOH 2

OMe

MeO

NHOH

MeO

OMe

OMe OMe 5f, 70%

4f, 82% O

O OMe

3c

n

MeO

OMe n = 2; 4d, 95% n = 3; 4e, 73%

O 3b

O

n

MeO

Pd(OAc)2/P(o-Tol)3 Et3N/THF, sealed tube

MeO

N

MeO

OMe OMe 4ga O

O OMe 1) NaOH/EtOH

MeO

2) EDCI, HOBt DMF/TEA NH2OH

MeO

N

NHOH OMe

OMe 5g, 58%

OEt

O

NHOH

O OEt

3d

Pd(OAc)2/P(o-Tol)3 Et3N/THF, sealed tube

Cl

NH2OH/MeOH

MeO

OMe

MeO MeO

OMe 4ha MeO MeO

tBuLi/THF

O OEt

OEt

NH2OH/MeOH

OMe OMe

OMe OMe 5h, 69%

O

O 3b

MeO

MeO

NHOH

MeO

4i, 36%

OMe OMe 5i, 77% H2N O

1) KOH 2) o-Phenylenediamine EDCI, HOBt, DMF/TEA

MeO

N H OMe

MeO OMe

5j, 60%



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Scheme 2. Synthesis of compounds 4d-i and 5d-j. aThe compound was used for the next step without any purification. Next, we used our standard conditions to perform the coupling of N-tosylhydrazone 1a and indole derivatives. The reaction is carried out under simple conditions and quickly leads to coupling products in less than an hour. The electrophilic partner is changed each time in order to vary the nature and the position of the substituent on the indole ring B, with the aim of studying the influence of substituents on biological activity.

Scheme 3. Synthesis of indoles-esters derivatives 4k-o. aThe compound was used for the next step without any purification. Hydroxamic derivatives of indoles 5k-o were synthesized as illustrated in Scheme 4. Ester 4k-n were converted into acids under basic conditions. Then, the coupling with hydroxylamine in the presence of EDC/HOBt gives the desired products in moderate yield after HPLC purification.


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Scheme 4. Synthesis of indoles derivatives 5k-o Biological results In vitro antiproliferative activity The synthesized compounds were assayed by MTS method for growth inhibition of human colon carcinoma cells (HCT116). With the aim of determining IC50 values, a treatment at eight different concentrations was used to determine the IC50 values for each compound in comparison to the control compound isoCA-4. The summarized results in Table 1 show that most of the tested compounds had a cytotoxic activity at micromolar concentrations or lower. Compounds evaluated in their ester form (cf, 4a-e), except for the compound 4f which displayed low antiproliferative activity on this cell line. An overall decrease in activity has been identified as the



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chain length of the linker reaches over five carbons seen with the compounds (cf, 4a vs 4b; 4b vs 4c; 4d vs 4e). The optimal activity was obtained with 3 carbons (compound 4f). However, potency was gained by switching from ester to hydroxamic acid group (cf, 5a vs 4a; 5b vs 4b; 5c vs 4c; 5d vs 4d; 5e vs 4e and 5f vs 4f).The compound bearing o-phenylenediaminebased zinc binding group 5j was turned out to be less active than the hydroxamic acid derivative (cf, 5j vs 5i). Above all 1,1-diarylethylene derivatives (4a-5j), compounds 5d, 5f, and 5h exhibited the best anti-proliferative activities. Compound 5f with an N-hydroxycinnamamide linker, in meta position, displayed more potent activity than its counterpart 5h. The introduction of a pyridine ring was probed systematically as a bioisostere of the phenyl ring. However, in our case, this modification led to a drop in the activity (cf, 5f vs 5g). Also, phenyl ring can be often replaced by a heteroaromatic ring. Accordingly, some new analogs of indole-isocombretastatin were also synthesized and evaluated. Again, the length and the position of the linker on the indole ring was studied, the results have shown that the best activity was obtained once more with an N-hydroxycinnamamide linker (cf, 5m, and 5n vs 5k-l, 5o). In addition, positioning this linker on C3 of the indole was more favorable for the activity than C2 position (cf, 5n vs 5m). Inhibition of tubulin polymerization. Compounds 4 and 5 were next evaluated to examine whether these derivatives were interacting with microtubules (Table 1). The results have clearly indicated that tubulin is the intracellular target of dual 1,1-diarylethylenes and indole-isocombretastatin analogs. Indeed, all efficient antiproliferative compounds were potent inhibitors of tubulin assembly with micromolar IC50 values. It is important to note that indole-isocombretastatin analogs (5k-o) exhibited less effective IC50 values against tubulin than their 1,1-diarylethylenes analogs (5d, 5f, and 5h).


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Table 1. Cytotoxic activity of compounds 4a-f and 5a-o against HCT116 cells.a compd

GI50 HCT116 (nM)b

IC50 ITP (µM) c

compd

GI50 HCT116 (nM)b

IC50 ITP (µM) c

4a

150 ± 20

5 ± 0.5

5f

1.5 ± 0.3

1.6 ± 0.3

5a

22 ± 0.58

2.4 ± 0.2

5g

37.2 ± 0.6

7.5 ± 1.2

4b

1000 ± 200

NAd

5h

8 ± 0.02

2.1 ± 0.4

5b

500 ± 52

NAd

5i

233 ± 22

20 ± 1.2

4c

3000 ± 150

NAd

5j

340 ±2

NAd

5c

2500 ± 100

NAd

5k

36 ± 4

81 ± 15

4d

1000 ± 100

NAd

5l

30 ± 1.5

7.3 ± 3.3

5d

8±2

1.7 ± 0.3

5m

20 ± 2.5

5.4 ± 1.5

4e

3000 ± 100

NAd

5n

14 ± 0.3

3.7 ± 0.5

5e

372 ± 5

18 ± 1.2

5o

393 ± 4.1

22 ± 2

4f

85 ± 4

4 ± 0.4

isoCA-4

2 ± 0.1

2.0 ± 0.3

a

HCT116 human colon carcinoma cells. bCompound concentration required to decrease cell growth by 50%; values represent the average ± sd of three experiments. c IC50 is the concentration of a compound that inhibits 50% of the rate of tubulin polymerization. d NA= not active at 200 µM.

Among all tested analogs, compounds 5f and 5h were as active as isoCA-4 against tubulin with an IC50 value of 1.5 and 2 µM respectively. This reinforces our idea that the phenyl ring is



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important and more tolerable than other heterocycles for potent anti-cancer activity in relation to a significant involvement of β-tubulin. Inhibition of HDAC. These preliminary cytotoxicity and inhibition of tubulin assembly results have shown that derivatives 5d, 5f, 5h, 5m and 5n (IC50 ≤ 20 nM) are promising drugs in this novel series. In this context, we next investigated their effects on the inhibition of HDAC. Profiling of these compounds was performed by Eurofins Cerep S.A. Compounds showing higher inhibition (or stimulation for assays run in basal conditions) than 50% are considered to represent significant effects of the test compounds. For example, in the enzyme assays, compound 5f have shown an inhibition above 50% on HDAC8 (95% at 1E-05 M), and HDAC11 (60% at 1E-05 M) (Figure 2). At the highest tested concentration, 5f caused light (< 50%) or no inhibition of other studied HDAC activities. On this basis, we have determined the IC50 for compound 5f on HDAC8 and HDAC11. At this point, testing the effect of our reference isoCA-4 on the inhibition of HDAC's seemed necessary to rule out the possibility of the effect being inherent in the tested structure. As expected, isoCA-4, not bearing a hydroxamic acid function (zinc chelator) does not inhibit the different HDACs, even at a high concentration of 10-5M (Figure 2). Accordingly, we continued our in vitro HDAC screening by examining the IC50 values of compounds 5d, 5f, 5h, 5m and 5n against recombinant human HDACs 6, 8, and 11. The results of these studies are summarized in Table 2. The most studied HDACi trichostatin A (TSA),27 Scriptaid and specific HDAC8i (PCI-34051) were used as a control. Overall, all tested compounds proved to be selective for HDAC8. Compounds 5f and 5n showed a close activity to TSA, and compound 5h possess a better HDACi activity compared to TSA. The SAR studies 14 ACS Paragon Plus Environment

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revealed that the conjugate bearing a long linker (C5, compound 5d) have a weak inhibition activity while the N-hydroxycinnamamide linker seems to have the optimal length. Also, we can notice the importance of the position of the linker on the aromatic ring (cf, compound 5f vs 5h and 5m vs 5n). These results are very encouraging, taking in account that the isozyme HDAC8 is involved in the pathogenesis of neuroblastoma and that its expression significantly correlates with advanced tumor stage.28



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Figure 2. Inhibition profile of selected compounds against HDAC isoforms 1-11. Selected compounds were used at 1.0-5 M.


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Table 2. In vitro inhibition of recombinant HDACs 6, 8 and 11 for selected compounds. IC50 (nM) Compds

a

HDAC6

HDAC8

HDAC11

5d

Nta

Nta

11000 ± 520

5f

15000 ± 1300

340 ± 26

10000 ± 850

5h

5000 ± 45

60 ± 10

8000 ± 100

5m

3800 ± 30

6200 ± 100

Nta

5n

6900 ± 120

240 ± 20

5000 ± 100

TSA

15 ± 1.4

280 ± 20

Nta

PCI-34051b

3100 ± 30

12 ± 2.2

Nta

Scriptaid

Nta

Nta

22000 ± 1000

isoCA-4

Nac

Nac

Nac

Nt = not tested. bPCI-34051 (CAS number = 950762-95-5). cNa = not active.

Cytotoxic effect of compounds 5f and 5h on different tumor cell lines. To further determine the potential of these compounds, the most cytotoxic and HDAC8i derivatives 5f and 5h were evaluated on nine different cancer cell lines, which include human lung epithelial cells (A549), chronic myeloid leukemia cells (K562), doxorubicin-resistant K562 cells (K562R), human prostate cancer cells (PC3), human glioblastoma (U87-MG), breast cancer cells (MCF7), human primary pancreatic adenocarcinoma (BXPC3), human pancreatic 17 ACS Paragon Plus Environment


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carcinoma (MiaPaca2), and colon carcinoma cell lines (HT29). IsoCA-4, TSA and PCI-34051 were included as reference compounds for comparison. As depicted in Table 3, all of these compounds displayed good anti-proliferative potency with GI50 values ranging from 0.4 to 80 nM. Hybrid compounds 5f and 5h showed a higher anti-proliferative activity on all studied cells lines compared to the HDACi (TSA) and to the representative HDAC8 selective inhibitor PCI34051. The compound 5h was less active in term of cytotoxicity in comparison to the reference isoCA-4. On the other hand, compound 5f displayed more anti-proliferative efficiency compared to the controls compounds isoCA-4, TSA or PCI-34051. Interesting activity was obtained with 5f against doxorubicin-resistant K562R cells (60- and 2-fold higher than TSA and isoCA-4, respectively). The difference is even more important in the case of human glioblastoma U87MG29 (650- and 5-fold higher than TSA and isoCA-4, respectively). The screening results also revealed that compound 5f has the most potent inhibitory activity against human prostate cancer cells (PC3) cells (IC50 = 0.4 nM). This could be explained by the presence of an N-hydroxycinnamamide linker on meta position of the 1,1-diarylethylene which has an optimal inhibition of microtubule, and HDAC, resulting in high antiproliferative properties. In comparison to selective HDAC8i PCI3405, hybrid molecule 5f has a much higher antiproliferative activity on all tested tumor cell lines. This proves that the combination of the two effect: inhibition of tubulin polymerization and HDACi is beneficial for the antiproliferative effect.

To highlight the efficiency of these dual compounds, we investigated their potential in CA-4 refractory human colon adenocarcinoma cell line (HT-29, IC50 = 9137 nM).30 HT-29 cells are resistant to CA-4 due to the overexpression of the multidrug-resistance protein (MRP-1).31 As expected in Table 3, HT-29 cells were less sensitive to the combretastatin analogue isoCA-4 with


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an IC50 of 276 nM. TSA showed also a moderate activity. To our delight, compound 5f showed a marked activity and was l35-fold more active than isoCA-4 and 17-fold than TSA in the HT-29 cells suggesting a clear and definite advantage of this dual molecule (Figure 3). One can note that TSA (IC50 = 34 nM) seems to be less potent than 5f (IC50 = 2 nM), but more effective (% cell viability 15000

2970

> 20000

> 20000

> 10000

100 ± 36

200 ± 60

34.1 ± 2.96

0.04

±

0.02

5h

8 ± 0.02

22 ± 1.1

20 ± 0.5

80 ± 0.5

28 ± 0.2

isoCA-4

2 ± 0.2

10 ± 0.1

5.2 ± 0.2

2.2 ± 0.8

3.0 0.05

PCI-

2640 ±

34051b

1130

TSA a

35 ± 0.7

> 20000

80 ± 10

2010 ±

2200

850

1270

1100

88 ± 0.14

387 ± 88

262 ± 7.4

±

2660

±

2700 1038 ± 2

45 ± 5

GI50 = Compound concentration required to decrease cell growth by 50%; values represent the

average ± sd of three experiments. bPCI-34051 (CAS number = 950762-95-5).

100

C e ll v ia b ility (% )

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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Is o C A 4 + T S A 80

TSA 60

Is o C A 4

40

5f 5h

20 0 -1 1

-1 0

-9

-8

-7

-6

-5

lo g c o n c e n tr a tio n (M )

Figure 3. Effect of TSA, isoCA-4, TSA + isoCA-4, 5f and 5h on the HT-29 cells.


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HDACi are well-known for their ability to trigger H2AX phosphorylation (γH2AX),32 a hallmark of DNA double-strand breaks. Therefore, we examine whether compounds 5f and 5h, which are able to inhibit HDAC activities, modulate γH2AX level. Compared to mock-treated cells, HT29 cells treated with compound 5f or 5h led to γH2AX induction (Figure 4). Interestingly, increase in drug concentration led to a dose-dependent increase in γH2AX.

Figure 4. γH2AX induction after treatment of colon carcinoma cells with compound 5f and 5h. HT29 cells were mock-treated or treated with compounds at the indicated doses. After 3 days, total H2AX, phosphorylated of H2AX (γH2AX) and actin were measured by western blot. The numbers indicate the level of γH2AX relative to mock-treated cells In addition to in vitro studies, HT29 colon carcinoma cell line was treated with compounds 5f and 5h as well as the reference compound PCI-34051, an HDAC8 specific inhibitor and the cell lysates were analyzed by western blot. Compared to mock-treated cells, cells treated with 5f and 5h displayed a strong increase in acetylation of SMC3, a well-known HDAC8 substrate, the increase being more prominent after 24h of treatment (Figure 5A). In contrast, no significant modification of α tubulin (a substrate of HDAC6) acetylation was found (Figure 5B). 21 ACS Paragon Plus Environment


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Altogether these data demonstrate that compounds 5f and 5h are able to inhibit selectively HDAC8 while avoiding inhibition of HDAC6.

Figure 5. Compounds 5f and 5h are both able to inhibit HDAC8 activity on SMC3 protein. HT29 colon carcinoma cells were mock-treated or treated with compounds 5f, 5h or PCI-34051 (25 µM) and cell lysates obtained after 6 or 24h were analyzed by western blot. (A) acetylated SMC3 and total SMC3; (B) acetylated α tubulin and total α tubulin. The numbers indicate the level of the acetylated form of a given protein relative to total level of the same protein. Molecular modelling Compounds 5f and 5h displaying the best antiproliferative-activity and effective inhibitions of both the tubulin polymerization and HDAC8 were selected for molecular modelling study. To elucidate the potential interactions of these compounds with the tubulin-microtubule system, docking experiments have been achieved in the colchicine binding site of tubulin (Figure 6).33 For compound 5f, classical hydrogen bond interaction between the Cysβ241 and the trimethoxyphenyl motif has been identified.34 Three additional hydrogen bonds were also observed between the hydroxamic chain of 5f, and the side chains of Tyrα224, Glnα11, and the carbonyl of the backbone of Glnβ247. The side chain of Leuβ248 also established two CH/π interactions with the two aromatic rings of 5f. 22 ACS Paragon Plus Environment

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Switching the linker from meta to ortho-position, in the molecule 5h led to a new orientation of the molecule in the colchicine binding site due to the steric hindrance near the double bond. A hydrogen bond between the methoxy group of the B-ring of 5h and the side chain of Thrα179 was observed.34 The carbonyl group of the hydroxamic acid interacts with NH function of the backbone of Serα178, while the hydroxyl group of the hydroxamic acid establishes two hydrogens bonds with the Thrβ353. Two CH/π interactions were also observed: one with the side chain of Leuβ248 and the aromatic B-ring of 5h, and the other between the OMe group of the Aring and the phenyl ring of Tyrα224.

Figure 6. Putative binding mode of compounds 5f, shown in orange sticks (left), and 5h, shown in blue sticks (right) in the colchicine binding site of tubulin (code PDB: 3HKC). Protein residues involved in the interaction are shown in yellow sticks (β chain) and pink sticks (⍺ chain), H-bond as blue dashed lines, apolar interactions as yellow dashed-lines.

In HDAC8, as expected, the hydroxamic acid group of compounds 5f and 5h coordinates the zinc atom in the catalytic site of HDAC8 (Figure 7). Compound 5f establishes four hydrogen bonds to the side chain of His142, His143, Asp178, and His180. Moreover, a CH/π interaction was detected between the side chain of Met274 and the aromatic A-ring of 5f. Similarly, 23 ACS Paragon Plus Environment


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compound 5h establishes three hydrogen bonds with His142, His143, and His180, and an additional hydrogen bond was also detected between the OMe group of B-ring and NH function of the backbone of Gly151.

Figure 7. Putative binding mode of compounds 5f (left) and 5h (right) in the binding site of HDAC8 (code PDB: 1T69). Protein residues involved in the interaction are shown in green sticks, zinc-ion as gray spheres, H-bond as blue dashed lines, CH/ߨ interactions as yellow dashed-lines. For clarity purposes, ߨ - ߨ stacking between compounds and Phe152 and Phe208 are not depicted here. Effect of compound 5f on the cell cycle Since compound 5f displayed the best antiproliferative activity, we have selected it for the cell cycle studies. As shown in Figure 8, compound 5f had a dramatic effect on the cell cycle, inducing cell cycle arrest at the G2/M phase in both HCT-116 and K562 cells. The arrest of the cell cycle was observed even at low concentration (5 nM).


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Figure 8. Effects of compound 5f (blue) on cell cycle distribution in a) HCT116 and b) K562 cells at 5 and 10 nM, as determined by flow cytometry analysis (DMSO control in red). DNA content was assessed via propidium iodide staining. Cell death type by compound 5f Antiproliferative activity and cell cycle arrest at the G2/M phase was also observed for Burkittlymphoma (BL2) cells after treatment by compound 5f. As expected, again compound 5f showed an interesting antiproliferative activity against this cell line with an IC50 = 10 nM. To further characterize cell death type we analyzed phosphatidylserine externalization, an early event of apoptosis. Compound 5f behaves like a potent inducer of apoptosis, (Figure 9), a type of death that avoids inflammatory phenomena during anti-tumor treatments. Compound 5f seems to have a much more important cytotoxic effect on BL2 cells than the HDACi vorinostat and it was as toxic as the mitotic spindle poison isoCA-4.



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Figure 9. Burkitt-lymphoma BL2 cells were incubated with vorinostat, isoCA-4 or compound 5f at 10 nM for 48h. Percentage of apoptotic cells was evaluated by flow cytometry after labelling of external phophatidyl serine residues (Annexin-V-positive cells). Plasma membrane integrity is maintained during early apoptosis (propidium iodide negative cells). Evaluation of cytotoxicity in human non-cancer cells To demonstrate the safety profile of compound 5f, and to obtain a preliminary indication of the cytotoxic potential of this compound for normal human cells, we evaluated its activity on quiescent peripheral blood lymphocytes (PBL) isolated from two different healthy donors, and compared to isoCA-4 and vorinostat. In our hands, dose-response curves yielded an IC50 value of 1.7 µM for isoCA-4, 3.4 µM for vorinostat and 7.0 µM for compound 5f. These results indicate clearly the safety profile of 5f and confirm that this compound is not more cytotoxic than isoCA4 or vorinostat in quiescent PBLs.


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Immunofluorescence studies Considering the importance of the tubulin-microtubule system in the maintenance of cellular morphology and basic cellular functions, an assay involving the disruption of microtubule network was performed in order to reveal whether compound 5f could affect microtubule morphology in living cells. As shown in Figure 10, in the control experiment (DMSO), the microtubule network in the K562 cells exhibited normal organization, with microtubules extending from the central regions of the cell to the cell periphery. In contrast, after the exposure to 1 nM of compound 5f for 24h, the spindle formation showed distinct abnormalities and was heavily disrupted. The changes of the mitotic spindles were more clearly observed by increasing the concentration to 10 nM. In this case, the microtubule spindle has significantly shrunk, and a multinucleation phenomenon was also observed. These results have confirmed that compound 5f has dramatically disrupted the microtubule organization, and has interfered with the mitosis of K562 cells at low concentrations.



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Figure 10. Effect of 5f on microtubule network organization. K562 cells were treated with 5f for 24 h. After incubation, the cells were fixed and stained with monoclonal β-tubulin antibody (red), cell nuclei were stained with DAPI (blue). Physicochemical properties In order to ascertain the drug-likeness of our products, physicochemical properties of compounds 5f and 5h were assessed in parallel with the reference compound isoCA-4. Compounds 5f and 5h have shown to conform to Lipinski’s rule of five (Table 4). Additionally, 5f and 5h have shown an improved aqueous solubility compared to isoCA-4 indicating that the presence of the hydroxamic acid moiety fosters water solubility. As predicted, the experimental logP values were in the desirable druglike range (1 < logP < 3) assuring a good lipophilic-hydrophilic balance.


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Therefore, our findings showed that the presence of hydroxamic group could improve aqueous solubility with a comparative anti-proliferative activity of the isoCA-4. Table 4. Water solubility and physicochemical properties of compounds 5f, 5h and isoCA-4

a

Cmpd.

MWa

HBAb

HBDc

cLogPd

logPe

tPSAf

isoCA-4

316.35

5

1

2.86

2.16

57.16

11.62

5f

385.42

7

2

2.84

2.93

86.26

69.28

5h

385.42

7

2

2.84

2.13

86.26

88.33

RO5h

Design and Synthesis of Tubulin and Histone Deacetylase Inhibitor

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