Research paper
Aegeline inspired synthesis of novel amino alcohol and thiazolidinedione hybrids with antiadipogenic activity in 3T3-L1 cells

https://doi.org/10.1016/j.ejmech.2017.11.041Get rights and content

Highlights

  • Compound 12c, a hybrid molecule of amino alcohol and thiazolidinedione is a potential anti-adipogenic compound.

  • Compound 12c inhibit adipogenesis in 3T3-L1 adipocyte.

  • Compound 12c inhibit mitotic clonal expansion by arresting cell cycle in S-phase.

  • Compound 12c provide a therapeutic target for the treatment of obesity.

Abstract

Excess adiposity is a hallmark of obesity, which is caused due to an imbalance between energy intake and energy consumed. Obesity is often associated with several metabolic disorders like dyslipidemia, cardiovascular diseases and type 2 diabetes. Earlier, our group had reported natural product Aegeline (amino-alcohol) isolated from the plant Aegle marmelos as an anti-diabetic and anti-dyslipidemic compound. With this background, we synthesized a series of novel amino alcohol and thiazolidinedione hybrid molecules and studied their antiadipogenic activity. As a result, we have identified a potent hybrid compound 12c as an inhibitor of adipocyte differentiation. The compound 12c inhibits lipid accumulation and adipogenesis in 3T3-L1 preadipocyte cell line. Exposure of compound 12c blocks mitotic clonal expansion and arrests cells in S-phase of cell cycle. Detailed analysis showed that compound 12c decreases expression of two major transcription factors that are involved in adipocyte differentiation, PPARγ, C/EBPα, and other adipogenesis associated genes like aP2 and FAS. Thus, we concluded that compound 12c shows potential ability to inhibit adipocyte differentiation which can be used therapeutically for the treatment of obesity and its associated metabolic disorders.

Introduction

Recently, obesity has become a serious health problem in the developed countries and it appears to be a menace all over the world. According to World Health Organization as updated in 2016, more than 1.9 billion adults are overweight and obese in 2014. Out of which, around 600 million people are obese. Obesity is a cluster of several medical complications such as dyslipidemia, visceral adiposity, high blood pressure and hypertension which contributes to increasing risk of type 2 diabetes, cardiovascular diseases, polycystic ovary syndrome and certain types of cancer [1], [2], [3], [4]. Obesity in addition with other metabolic alteration such as insulin resistance, hyperinsulinemia and dyslipidemia is found in several cases of polycystic ovary syndrome (PCOS) women. In particular, accumulating evidence suggests that InsR and compensatory hyperinsulinemia play a pivotal pathogenic role in the hyperandrogenism of many PCOS phenotypes, which in turn have a clear detrimental effect on chronic anovulation [5], [6]. It is associated with disproportion in energy intake and energy expenditure [7]. Obesity is characterized by increased adipose tissue mass that results from both hyperplasia (increase in number of adipocytes) and hypertrophy (increase in the size of adipocyte) [8].

The process of adipogenesis, differentiation of preadipocytes to mature adipocytes, is highly complex and involves various changes in gene expression and cellular morphology. Adipogenesis requires a well coordinated transcriptional and cellular program, including growth arrest and reentry of confluent preadipocytes to 2–3 rounds of division known as mitotic clonal division and initiation of transcriptional events for early and terminal phase of adipocyte differentiation [9], [10]. Among several transcription factors, CCAAT/enhancer-binding protein-α (C/EBP-α) and Peroxisome proliferator-activated receptor-γ (PPAR-γ) are two major factors that act as master regulators of adipocyte differentiation. PPAR-γ and C/EBP-α induce their own expression by positive feedback mechanism and also activate other genes required for adipocyte determination like lipoprotein lipase (LPL), fatty acid binding protein (aP2) and fatty acid synthase (FAS) [11], [12].

The 2,4-thiazolidinedione (TZD) is a versatile scaffold, it's embedded in a number of clinically used drugs. Over the years, an elaborative research on thiazolidinedione have been reported, as it can be used as antimicrobial [13], [14], antibacterial [15], anti-inflammatory [16], anticancer [17], [18], antihyperlipidemic [19] and anti-leishmanial agents [20]. Besides all these, TZDs are effective hypoglycemic drugs that are used to treat patients with type 2 diabetes. The TZD drugs show their hypoglycemic effect via increased insulin sensitivity and subsequently increased peripheral glucose utilization. PPARs (peroxisome proliferator-activated receptors) is a family of ligand activated transcription factor which regulate the expression of many genes involved in progression of several diseases such as dyslipidemia, obesity, type 2 diabetes, other metabolic syndromes and cancer. PPARs play a major role in glucose and lipid homeostasis [21], [22]. TZDs can activate nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) which stimulates genes involved in glucose and lipid metabolism [23], [24]. PPARγ is known to increase fatty acid uptake and lipogenesis and differentiate preadipocytes to mature lipid filled adipocytes [22], [25]. Activated PPARγ induces apoptosis of large fat cells in subcutaneous and visceral deposits in a mouse model. TZD activated PPARγ is associated with weight gain in humans due to increased fat distribution in subcutaneous adipose deposits [21]. PPARγ also shows negative and positive effects in different types of cancers [21], [22]. The effect of TZDs on improved insulin sensitivity and inflammation is associated with its protective effect against pancreatic ductal adenocarcinoma [22]. However, due to their side effects, TZDs are still in limited use. The major adverse effect of TZD is the gain in body weight due to increase in peripheral fat mass [26], [27], liver failure, heart failure and cancer [25].

Prompted by these results, we planned to introduce β-amino alcohol pharmacophore to generate a hybrid compound with thiazolidinediones (Fig. 1). Earlier as a part of our drug discovery program, we reported Aegeline, an amino alcohol class of natural product, isolated from the leaves of Aegle marmelos, as a potent antidyslipidemic agent with predicted β3-AR activity in Quantitative Structre-Activity Relationship (QSAR) study [28]. Moreover, in addition to these results, we also reported synthetic derivatives of the amino alcohol (Aegeline) that show LDL oxidation and anti-oxidant property. β-amino alcohols are β3-adrenergic agonist that are known to activate β3-adrenergic signaling and promote lipolysis of stored triglycerides in both white and brown adipocytes [29]. β-amino alcohols are also reported in antihyperlidemic and anti-oxidant activity [30].

In continuation to our drug discovery program, we synthesized and evaluated a series of novel hybrids of amino alcohol and thiazolidinediones for the treatment of obesity. In the present study, we identified a promising compound 12c with anti adipogenic effect. The compound 12c inhibits preadipocyte to adipocyte differentiation via suppressing the expression of adipocyte specific genes such as PPARγ, C/EBPα, FAS and aP2. The compound 12c inhibits mitotic clonal expansion and arrests cells in S-phase of cell cycle. Thus, our study explored the possible molecular mechanism involved in the anti adipogenic activity of compound 12c.

Section snippets

Chemistry

Our strategy to synthesize the compounds 6a-6b, started with 4-hydroxybenzaldehyde (1), which was treated with epichlorohydrin to yield the compound 2 [31]. It was further reacted with benzonitrile (3a) and 3-methylbenzonitrile (3b) in the presence of BFOEt2 via Ritter reaction to afford compounds 4a-4b [30]. The compounds 4a-4b were converted into compounds 6a-6b on reaction with 2,4 thiazolidinedione (5) in methanol under refluxing conditions using piperidine as base [32], [33], [34], [35] (

Modeling studies of synthesized compounds against pharmacophore model

To establish the rationality, synthesized compounds were screened against the Common Feature Pharmacophore Model for Anti-adipogenic compounds (CFPMA) [44], out of which compound 12c was predicted as most active. Aegeline was used as internal standard compound as it was previously reported for anti-adipogenic activity [45]. The compound 12c was mapped on the developed pharmacophore model with the fit values of 2.96 and the reference compound Aegeline with fit values of 2.83. The observation

Cell culture

The mouse embryonic 3T3-L1 preadipocyte cell line was purchased from the American Type Culture Collection (ATCC). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM from Gibco) with 10% FBS (Cell Clone) and 1× Penicillin and Streptomycin (Gibco) at 37 °C and 5% CO2. After post confluency, cells were allowed to differentiate by adding differentiation media (MDI) composed of 0.5 mM 3-isobutyl-1-methylxantine, 250 nM dexamethasone, and 5 μg/ml insulin. The process of differentiation

Screening of series of thiazolidinedione (TZD) and β-amino alcohol hybrids and selection of compound 12c as anti-adipogenic compound in 3T3-L1 preadipocytes

Initially, we have prepared aminoalcohols (6a-6b and 12a-12d) and glycerol derivatives (8a-8d) and screened for their antiadipogenic activity in in vitro 3T3-L1 model of preadipocyte differentiation at a standard concentration of 20 μM. From these 10 compounds, none of the glycerol derivatives (8a-8d) showed activity. However, from aminoalcohols series (12a-12d), we identified compound 12c with 2-pyridyl ring and benzyl protected TZD showed the highest level of reduction in lipid accumulation

Conclusion

In conclusion, we carried out the synthesis of 18 hybrid compounds of thiazolidinedione and β-amino alcohol inspired by natural product Aegeline to identify potent inhibitor of adipocyte differentiation. Screening of all these compounds led to identify compound 12c as a potent inhibitor of adipocyte differentiation in 3T3-L1 cells as indicated by reduced lipid accumulation in adipocytes. Exposure of compound 12c blocks mitotic clonal expansion via arresting cells in S phase of cell cycle. The

General materials and instrumentations

All reagents were commercial and were used without further purification. Chromatography was carried on silica gel (60–120 and 100–200 mesh) using mixture of Chloroform and Methanol as eluents. Melting points were uncorrected. The 1H NMR, 2D-NMR (COSY, HMBC, HSQC) and 13C NMR spectra were determined on 200, 300, 400 MHz and 50, 75, 100 MHz, respectively, using CDCl3 CD3OD and DMSO-d6 as solvents and TMS as internal standard. All chemical shifts were given in ppm and multiplicity as s = singlet,

Conflicts of interest

The authors declare that they have no conflicts of interest.

Acknowledgement

This work was supported by CSIR-CDRI Network project: “Towards holistic understanding of complex diseases: Unraveling the threads of complex disease (THUNDER) Project No: BSC0102. SS is supported by CSIR, AS is supported by CSIR-SRF, PY is supported by CSIR, SV is supported by ICMR-SRF. This is CDRI communication number 9596.

References (47)

  • T. Narender et al.

    Antihyperglycemic and antidyslipidemic agent from Aegle marmelos

    Bioorg. Med. Chem. Lett.

    (2007)
  • A.M. Cypess et al.

    Activation of human brown adipose tissue by a beta3-adrenergic receptor agonist

    Cell. Metab.

    (2015)
  • S. Sarkar et al.

    Synthesis of new N-acryl-1-amino-2-phenylethanol and N-acyl-1-amino-3-aryloxypropanols and evaluation of their antihyperlipidemic, LDL-oxidation and antioxidant activity

    Eur. J. Med. Chem.

    (2014)
  • E. Fullam et al.

    Analysis of beta-amino alcohols as inhibitors of the potential anti-tubercular target N-acetyltransferase

    Bioorg. Med. Chem. Lett.

    (2011)
  • R. Maccari et al.

    5-Arylidene-2,4-thiazolidinediones as inhibitors of protein tyrosine phosphatases

    Bioorg. Med. Chem.

    (2007)
  • L.A. Dakin et al.

    Discovery of novel benzylidene-1,3-thiazolidine-2,4-diones as potent and selective inhibitors of the PIM-1, PIM-2, and PIM-3 protein kinases

    Bioorg. Med. Chem. Lett.

    (2012)
  • S. Shah et al.

    Urea/thiourea catalyzed, solvent-free synthesis of 5-arylidenethiazolidine-2,4-diones and 5-arylidene-2-thioxothiazolidin-4-ones, Bioorg

    Med. Chem. Lett.

    (2012)
  • C.L. Viswanathan et al.

    Design, synthesis and evaluation of racemic 1-(4-hydroxyphenyl)-2-[3-(substituted phenoxy)-2-hydroxy-1-propyl]amino-1-propanol hydrochlorides as novel uterine relaxants

    Bioorg. Med. Chem. Lett.

    (2005)
  • J.E. Park et al.

    Synthesis and biological evaluation of 1-(2-hydroxy-3-phenyloxypropyl)piperazine derivatives as T-type calcium channel blockers

    Bioorg. Med. Chem. Lett.

    (2013)
  • S.E. Park et al.

    3-(3-Butylamino-2-hydroxy-propoxy)-1-hydroxy-xanthen-9-one acts as a topoisomerase IIalpha catalytic inhibitor with low DNA damage

    Eur. J. Med. Chem.

    (2013)
  • C.H. Tseng et al.

    Synthesis and anti-osteoporotic evaluation of certain 3-amino-2-hydroxypropoxyisoflavone derivatives

    Eur. J. Med. Chem.

    (2009)
  • L. Betti et al.

    Design, synthesis, and alpha(1)-adrenoceptor binding properties of new arylpiperazine derivatives bearing a flavone nucleus as the terminal heterocyclic molecular portion

    Bioorg. Med. Chem.

    (2004)
  • S. Varshney et al.

    Rohitukine inhibits in vitro adipogenesis arresting mitotic clonal expansion and improves dyslipidemia in vivo

    J. Lipid Res.

    (2014)
  • Cited by (20)

    • Recent advances in the development of active hybrid molecules in the treatment of cardiovascular diseases

      2022, Bioorganic and Medicinal Chemistry
      Citation Excerpt :

      The compound 39 also showed excellent binding affinity towards the calcium channel receptors and histamine H2 receptors and Kd values were found as 8.07 µM and 5.93 µM, respectively. Therefore, compound 39 was found to be a promising hybrid compound, which could provide clinically beneficial leads in the treatment of myocardial heart failure or other hypertensive disorders, owing to its good in vitro Ca2+-blocking action and an overall positive inotropic and chronotropic activity78 (Fig. 8). Besides the reported hybrid molecules, there are some small hybrid compounds that could be magic bullets in the treatment of CVDs and currently being used or have been evaluated in the clinical trials in the twentieth century.

    View all citing articles on Scopus
    1

    Both the authors contributed equally.

    View full text