Bionatura
Bionatura
Latin American Journal of Biotechnology and Life Sciences
Latin American Journal of Biotechnology and Life Sciences
Go to content
CS 2019.02.01.9
Files > Conference Series > 2019 > Humboldt Kolleg 2019
Bionatura Conference Series Vol 2. No 1. 2019
“Breaking Paradigms: Towards a Multi-, Inter- and Transdisciplinary Science” In commemoration of the 250th Anniversary of Alexander von Humboldt

INVESTIGATION / RESEARCH


Synthesis of new C75 derivatives, Fatty Acid Synthase inhibitors with cytotoxic properties

Kamil Makowskiabc*, Javier Arizab, Dolors Serrac, Jordi Garciab, Laura Herreroc, Marta Lópeza, Nicolás Montesdeocaa
Available in: http://dx.doi.org/10.21931/RB/CS/2019.02.01.9
 
 
 

 
ABSTRACT
 
The fatty-acid synthase (FAS) is the key enzyme that executes de novo fatty acid synthesis and is overexpressed in some human cancer tissues. C75 is a synthetic FAS inhibitor, which produces the death of cancer cells. In this work, we synthesized three derivatives of C75, two homologs with 1 and 2 carbons more in the beta position of the lactone and the other one with a change in the elongation of the aliphatic chain in gamma position from 8 to 15 carbons. The results from FAS inhibition and cytotoxic properties suggest that the performed changes in the derivatives decrease their activity.
 
Keywords: C75, Fatty acid synthase inhibitor, FAS, α-Methylene-γ-butyrolactones  
 
 
 
 
 
INTRODUCTION
 
Fatty acids (FAs) are essential for every living cell and have a wide diversity of biological functions. FAs are the main components of phospholipids, triglycerides, and other more complex lipids. Thus, cells need FAs for cellular membranes, energy storage or lipids for cellular signaling. Cancer cells have an increasing demand for fatty acids due to its high proliferation rate and the necessity of lipids as a building material for the cellular membranes. The FAs building blocks may come from exogenous sources, lipid storage or from de novo FA synthesis, nevertheless, in cancer cells, synthesis de novo is the main source of FAs 1.
 
Limiting FAs availability could result in a therapeutic strategy to kill cancer cells. One of the best ways to achieve this is by blocking the FA biosynthesis. The key enzyme in synthesis de novo of long-chain FA is a fatty acid synthase (FAS). Since FAS is overexpressed in many cancer cell lines pharmacological inhibition of that enzyme has a cytotoxic effect more powerful in cancer cell rather than normal cells and decrease the growth of tumors in animal models 2. Recently, imidazopyridine-based FAS inhibitors were discovered and TVB-2640 reach phase II of clinical trials demonstrating that FAS can be a viable target for cancer treatment3,4.
 
The C75 is a synthetic FAS inhibitor with a structure of α-methylene-γ-butyrolactone, which produce the death of cancer cells from many different tissues. In the majority of studies, the racemic C75 was used, however, it was demonstrated that (–)-C75 is responsible for FAS inhibition and do not produce a weight loss of treated animals as the racemic mixture5. In the more recent study, several alcoholic homologs of C75 were synthesized and (–)-UB006 was found as a better FAS inhibitor and cytotoxic agent with 40 times lower IC50 in some cancer line6.
 
In this work, we synthesized some derivatives of C75 with the aim of finding structural requirements for inhibition of FAS and new compounds with higher cytotoxic properties.
 
 
METHODOLOGY
 
The organic synthesis was carried out by standard procedures. A thin layer, column chromatography, melting point, NMR, I, and high-resolution mass spectrometry were used to characterize and identify the obtained products (data not shown).
 
The anti-carcinogenic activity of the different derivatives of C75 has been tested by two approaches: determining in vitro the inhibitory capacity on the FAS enzyme and checking the cytotoxic effect in different tumor cell lines. For the FAS activity assay, a spectrophotometric method was used using cytosolic hepatic extracts obtained from a rat. To evaluate the cytotoxic effect of the drugs, an MTT cytotoxicity assay was performed 6.
 

RESULTS
 
Three racemic derivatives of the C75 were synthesized, two homologs with 1 (UB207) and 2 (UB244) carbon more in the beta position of the lactone and the other with an elongated aliphatic chain in gamma position from 8 to 15 carbons (UB183).
 
 
 
Figure 1. Structure of the C75 and its synthesized derivatives.
 
 
UB183 was synthesized in a similar way as C757 but instead of nonanal hexadecanal was used which was obtained by oxidation of commercially hexadecanol using sodium hypochlorite and TEMPO (2,2,6,6-tetramethylpiperidine) as a catalyst. For purification purpose, the conversion of trans and a cis mixture of diastereomers intoselenoester derivatives, separation, and oxidation to recover double bond was necessary 8.
 


 
Figure 2. Synthetic route of UB183.
 
 
The trans lactone formation in the synthesis of UB207 was started from tricarballylic acid and was accomplished by Parker’s protocol 9. Subsequently, α-methylenantion was performed in two steps, carboxylation using methylmagnesium carbonate and decarboxylation - elimination using formaldehyde and N-methyl aniline to finally obtain the UB207.

 
Figure 3. Synthetic route of UB207.
 
 
The synthesis of UB244 was accomplished as previously 6 from dimethyl maleate.
 
 
 
Figure 4. Synthesis of UB244.
 
 
The FAS inhibitory activity of the synthesized compounds was studied and compared with the racemic C75. All the synthesized derivatives show a lower inhibition profile of FAS when compared with C75 (Figure 5) which indicate small flexibility in distance between the carboxylic group and methylene. The length of the alkyl chain seems to be less important as decrease of inhibitory activity is not so drastic.  The cytotoxic activity was also studied and compared with C75 against some cell lines as ovarian - OVCAR3, prostate -PC3, colon - HCT116, and pancreas MiaPaca-2 as shown in Figure 6. The compounds UB183 and UB207 showed lower cytotoxic activity compared to C75 in all tested cell lines, which corresponds to their behavior against FAS. In contrast, the compound UB244 showed a slightly more cytotoxic activity than C75 especially in HCT116 (44.4±5.8 vs 22.3±2.5 µM) cell line suggesting that maybe another mechanism than FAS inhibition is involved in the cytotoxic activity of this compound, since UB244 present decrease in inhibition activity of FAS enzyme (460±44 µM vs 2952±717 µM).
 

 
Figure 5. Effects of (±)-UB183, (±)-UB207 and (±)-UB244 on in vitro FAS activity. Data are expressed as the mean ± SEM.
 
 
 
 

 
Figure 6. Cytotoxic effect of (±)-UB183, (±)-UB207 and (±)-UB244 against different tumor cells. The cells were incubated for 3 days with the molecules at increasing concentrations. The cytotoxicity was then determined by an MTT colorimetric assay. The results are expressed as the mean ± S.E.M of at least two experiments.
 
 
 
 
CONCLUSIONS
 
The elongation of the aliphatic chain or the introduction of larger groups in the β-position of the lactone causes a decrease in the inhibitory activity of FAS. UB244 present slightly higher cytotoxic activity in cell HCT116 cell line, however, another mechanism that FAS inhibition can be involved which need further investigation. All these findings contribute to the better understanding structure-activity relationship of FAS inhibition by C75 and his derivatives and may lead to discovery more specific and more potent FAS inhibitors, a possible drug for cancer treatment.
 
 
ACKNOWLEDGMENTS
 
This study was supported by the Ministry of Spain – MINECO (SAF2014-52223-C2-1-R to JG and DS, co-funded by the Fondos Europeos de Desarrollo Regional de la Unión Europea [FEDER] and SAF2013-45887-R to LH), the Centro de Investigación Biomédica en Red Fisiopatología de la Obesidad y la Nutrición (CIBEROBN) (Grant CB06/03/0001 to DS), the Generalitat de Catalunya (2014SGR465 to DS), Fundació Marató TV3 (201627-30-31 to DS) and the European Foundation for the Study of Diabetes (EFSD)/Janssen-Rising Star and L’Oréal-UNESCO “For Women in Science” research fellowships to LH. KM is grateful to IBUB (Universitat de Barcelona) for a fellowship.
 
 
REFERENCES
 
1         Yecies JL, Manning BD. Chewing the Fat on Tumor Cell Metabolism. Cell 2010. doi:10.1016/j.cell.2009.12.037.
 
2         Pizer ES, Chrest FJ, DiGiuseppe JA, Han WF. Pharmacological inhibitors of mammalian fatty acid synthase suppress DNA replication and induce apoptosis in tumor cell lines. Cancer Res 1998; 58: 4611–4615.
 
3         Jones SF, Infante JR. Molecular Pathways: Fatty Acid Synthase. Clin Cancer Res 2015; 21: 5434–5438. doi:10.1158/1078-0432.CCR-15-0126.
 
4         Menendez JA, Lupu R. Fatty acid synthase (FASN) as a therapeutic target in breast cancer. Expert Opin Ther Targets 2017; 21: 1001–1016. doi:10.1080/14728222.2017.1381087.
 
5         Makowski K, Mera P, Paredes D, Herrero L, Ariza X, Asins G et al. Differential pharmacologic properties of the two C75 enantiomers: (+)-C75 is a strong anorectic drug; (-)-C75 has antitumor activity. Chirality 2013; 25: 281–7. doi:10.1002/chir.22139.
 
6         Makowski K, Mir JF, Mera P, Ariza X, Asins G, Hegardt FG et al. (−)-UB006: A new fatty acid synthase inhibitor and cytotoxic agent without anorexic side effects. Eur J Med Chem 2017; 131: 207–221. doi:10.1016/j.ejmech.2017.03.012.
 
7         Kuhajda FP, Pizer ES, Li JN, Mani NS, Frehywot GL, Townsend CA. Synthesis and antitumor activity of an inhibitor of fatty acid synthase. Proc Natl Acad Sci U S A 2000; 97: 3450–4. doi:10.1073/pnas.050582897.
 
8         Sanchez C, Makowski K, Mera P, Farras J, Nicolas E, Herrero L et al. Convenient synthesis of C75, an inhibitor of FAS and CPT1. RSC Adv 2013; 3: 6564–6571. doi:10.1039/C3RA40913A.
 
9         Parker WL, Johnson F. Total synthesis of dl-avenaciolide. J Org Chem 1973; 38: 2489–2496. doi:10.1021/jo00954a015.
 

Received: 1 March 2019
 
Accepted: 5 jun 2019
 
 
 
 
Kamil Makowskiabc*, Javier Arizab, Dolors Serrac, Jordi Garciab, Laura Herreroc, Marta Lópeza, Nicolás Montesdeocaa
 
aSchool of Chemical Sciences and Engineering, Yachay Tech University. Ecuador. bDepartment of Inorganic and Organic Chemistry, Section of Organic Chemistry, Facultat de Química, Universitat of Barcelona, Spain. cDepartment of Biochemistry and Physiology, School of Pharmacy and Food Sciences, University of Barcelona, Barcelona, Spain. *Corresponding author e-mail: kmakowski@yachaytech.edu.ec
 
Back to content