Abstract
Background: Inhibition of cancer cell growth and low in vivo toxicity are two important criteria for the development of anti-cancer drugs. Curcumin is a promising candidate for developing novel anti-cancer drug analogs. The research group designed the 3,5-bis-(3,4,5- trimethoxybenzylidene)-1-methyl-piperidin-4-one analog of curcumin that significantly inhibited the growth of esophageal cancer cells in vivo. In this study, 81 curcumin analogs were synthesized, analyzed both in vitro and in vivo, and their structure activity relationships (SARs) were determined.
Methods: Based on the parent structure of curcumin, 81 analogs of 3,5-bis(substitutedbenzylidene)- piperidin-4-one compounds were designed and synthesized. Their anti-cancer activity in the human cancer cell lines was evaluated using the MTT assay, and in vivo toxicity was evaluated in mice. The SARs of selected compounds were analyzed.
Results and Discussion: Among the designed curcumin analogs, 61 compounds exerted anti-cancer effects higher than the parent compound in vitro; 23 compounds inhibited cell growth in the human cancer cell line at low concentrations (IC50 values below 1 μM). The acute toxicity of curcumin analogs was tested in mice; 13 compounds were selected, which did not show any obvious toxicity at doses as high as 25.0 mg/kg. The SARs of these shortlisted curcumin analogs were determined.
Conclusion: Twenty-three curcumin analogs exhibiting promising in vitro anti-cancer activity and low in vivo toxicity were designed. SAR analysis indicated the optimal functional groups in the molecule required for anti-cancer activity. This study not only suggested a useful strategy to design curcumin analogs for the development of anti-cancer drugs, but also revealed a group of curcumin analogs which could be further explored.
Keywords: Curcumin analogues, chemical synthesis, anti-cancer activity, low animal toxicity, satisfactory selectivity, structure activity relationships (SARs).
Graphical Abstract
[1]
Wei X, Du ZY, Cui XX, et al. Effects of cyclohexanone analogues of curcumin on growth, apoptosis and NF-κB activity in PC-3 human prostate cancer cells. Oncol Lett 2012; 4(2): 279-84.
[http://dx.doi.org/10.3892/ol.2012.710 PMID: 22844370]
[http://dx.doi.org/10.3892/ol.2012.710 PMID: 22844370]
[2]
Kunnumakkara AB, Guha S, Krishnan S, Diagaradjane P, Gelovani J, Aggarwal BB. Curcumin potentiates antitumor activity of gemcitabine in an orthotopic model of pancreatic cancer through suppression of proliferation, angiogenesis, and inhibition of nuclear factor-kappaB-regulated gene products. Cancer Res 2007; 67(8): 3853-61.
[http://dx.doi.org/10.1158/0008-5472.CAN-06-4257 PMID: 17440100]
[http://dx.doi.org/10.1158/0008-5472.CAN-06-4257 PMID: 17440100]
[3]
Seo JA, Kim B, Dhanasekaran DN, Tsang BK, Song YS. Curcumin induces apoptosis by inhibiting sarco/endoplasmic reticulum Ca2+ ATPase activity in ovarian cancer cells. Cancer Lett 2016; 371(1): 30-7.
[http://dx.doi.org/10.1016/j.canlet.2015.11.021 PMID: 26607901]
[http://dx.doi.org/10.1016/j.canlet.2015.11.021 PMID: 26607901]
[4]
Liu Z, Xie Z, Jones W, et al. Curcumin is a potent DNA hypomethylation agent. Bioorg Med Chem Lett 2009; 19(3): 706-9.
[http://dx.doi.org/10.1016/j.bmcl.2008.12.041 PMID: 19112019]
[http://dx.doi.org/10.1016/j.bmcl.2008.12.041 PMID: 19112019]
[5]
Hironori O, Xiao Z, Junko I, et al. Antitumor potential as anti-prostate cancer agents. J Med Chem 2002; 45: 5037-42.
[http://dx.doi.org/10.1021/jm020200g PMID: 12408714]
[http://dx.doi.org/10.1021/jm020200g PMID: 12408714]
[6]
Qiu X, Du Y, Lou B, et al. Synthesis and identification of new 4-arylidene curcumin analogues as potential anticancer agents targeting nuclear factor-κB signaling pathway. J Med Chem 2010; 53(23): 8260-73.
[http://dx.doi.org/10.1021/jm1004545 PMID: 21070043]
[http://dx.doi.org/10.1021/jm1004545 PMID: 21070043]
[7]
Lin L, Shi Q, Nyarko AK, et al. Antitumor agents. 250. Design and synthesis of new curcumin analogues as potential anti-prostate cancer agents. J Med Chem 2006; 49(13): 3963-72.
[http://dx.doi.org/10.1021/jm051043z PMID: 16789753]
[http://dx.doi.org/10.1021/jm051043z PMID: 16789753]
[8]
Zhou J, Geng G, Shi Q, Sauriol F, Wu JH. Design and synthesis of androgen receptor antagonists with bulky side chains for overcoming antiandrogen resistance. J Med Chem 2009; 52(17): 5546-50.
[http://dx.doi.org/10.1021/jm801218k PMID: 19725582]
[http://dx.doi.org/10.1021/jm801218k PMID: 19725582]
[9]
Wei X, Du ZY, Zheng X, Cui XX, Conney AH, Zhang K. Synthesis and evaluation of curcumin-related compounds for anticancer activity. Eur J Med Chem 2012; 53: 235-45.
[http://dx.doi.org/10.1016/j.ejmech.2012.04.005 PMID: 22551677]
[http://dx.doi.org/10.1016/j.ejmech.2012.04.005 PMID: 22551677]
[10]
Yadav B, Taurin S, Rosengren RJ, et al. Synthesis and cytotoxic potential of heterocyclic cyclohexanone analogues of curcumin. Bioorg Med Chem 2010; 18(18): 6701-7.
[http://dx.doi.org/10.1016/j.bmc.2010.07.063 PMID: 20728364]
[http://dx.doi.org/10.1016/j.bmc.2010.07.063 PMID: 20728364]
[11]
Bazzaro M, Anchoori RK, Mudiam MK, et al. α,β-Unsaturated carbonyl system of chalcone-based derivatives is responsible for broad inhibition of proteasomal activity and preferential killing of human papilloma virus (HPV) positive cervical cancer cells. J Med Chem 2011; 54(2): 449-56.
[http://dx.doi.org/10.1021/jm100589p PMID: 21186794]
[http://dx.doi.org/10.1021/jm100589p PMID: 21186794]
[12]
Qiu X, Liu Z, Shao WY, et al. Synthesis and evaluation of curcumin analogues as potential thioredoxin reductase inhibitors. Bioorg Med Chem 2008; 16(17): 8035-41.
[http://dx.doi.org/10.1016/j.bmc.2008.07.054 PMID: 18678491]
[http://dx.doi.org/10.1016/j.bmc.2008.07.054 PMID: 18678491]
[13]
Adams BK, Ferstl EM, Davis MC, et al. Synthesis and biological evaluation of novel curcumin analogs as anti-cancer and anti-angiogenesis agents. Bioorg Med Chem 2004; 12(14): 3871-83.
[http://dx.doi.org/10.1016/j.bmc.2004.05.006 PMID: 15210154]
[http://dx.doi.org/10.1016/j.bmc.2004.05.006 PMID: 15210154]
[14]
Tung LN, Song S, Chan KT, et al. Preclinical study of novel curcumin analogue SSC-5 using orthotopic tumor xenograft model for esophageal squamous cell carcinoma. Cancer Res Treat 2018; 50(4): 1362-77.
[http://dx.doi.org/10.4143/crt.2017.353 PMID: 29361818]
[http://dx.doi.org/10.4143/crt.2017.353 PMID: 29361818]
[15]
Chen L, Li Q, Weng B, et al. Design, synthesis, anti-lung cancer activity, and chemosensitization of tumor-selective MCACs based on ROS-mediated JNK pathway activation and NF-κB pathway inhibition. Eur J Med Chem 2018; 151: 508-19.
[http://dx.doi.org/10.1016/j.ejmech.2018.03.051 PMID: 29655083]
[http://dx.doi.org/10.1016/j.ejmech.2018.03.051 PMID: 29655083]
[16]
Wu J, Cai Z, Wei X, et al. Anti-lung cancer activity of the curcumin analog JZ534 in vitro. BioMed Res Int 2015.2015504529
[http://dx.doi.org/10.1155/2015/504529 PMID: 25977922]
[http://dx.doi.org/10.1155/2015/504529 PMID: 25977922]
[17]
Meiyanto E, Putri DDP, Susidarti RA, et al. Curcumin and its analogues (PGV-0 and PGV-1) enhance sensitivity of resistant MCF-7 cells to doxorubicin through inhibition of HER2 and NF-kB activation. Asian Pac J Cancer Prev 2014; 15(1): 179-84.
[http://dx.doi.org/10.7314/APJCP.2014.15.1.179 PMID: 24528023]
[http://dx.doi.org/10.7314/APJCP.2014.15.1.179 PMID: 24528023]
[18]
Anand P, Thomas SG, Kunnumakkara AB, et al. Biological activities of curcumin and its analogues (Congeners) made by man and Mother Nature. Biochem Pharmacol 2008; 76(11): 1590-611.
[http://dx.doi.org/10.1016/j.bcp.2008.08.008 PMID: 18775680]
[http://dx.doi.org/10.1016/j.bcp.2008.08.008 PMID: 18775680]
[19]
Radhakrishna Pillai G, Srivastava AS, Hassanein TI, Chauhan DP, Carrier E. Induction of apoptosis in human lung cancer cells by curcumin. Cancer Lett 2004; 208(2): 163-70.
[http://dx.doi.org/10.1016/j.canlet.2004.01.008 PMID: 15142674]
[http://dx.doi.org/10.1016/j.canlet.2004.01.008 PMID: 15142674]
[20]
Kunnumakkara AB, Anand P, Aggarwal BB. Curcumin inhibits proliferation, invasion, angiogenesis and metastasis of different cancers through interaction with multiple cell signaling proteins. Cancer Lett 2008; 269(2): 199-225.
[http://dx.doi.org/10.1016/j.canlet.2008.03.009 PMID: 18479807]
[http://dx.doi.org/10.1016/j.canlet.2008.03.009 PMID: 18479807]
[21]
Srivastava RM, Singh S, Dubey SK, Misra K, Khar A. Immunomodulatory and therapeutic activity of curcumin. Int Immunopharmacol 2011; 11(3): 331-41.
[http://dx.doi.org/10.1016/j.intimp.2010.08.014 PMID: 20828642]
[http://dx.doi.org/10.1016/j.intimp.2010.08.014 PMID: 20828642]
[22]
Johnson JJ, Mukhtar H. Curcumin for chemoprevention of colon cancer. Cancer Lett 2007; 255(2): 170-81.
[http://dx.doi.org/10.1016/j.canlet.2007.03.005 PMID: 17448598]
[http://dx.doi.org/10.1016/j.canlet.2007.03.005 PMID: 17448598]
[23]
Gupta SC, Kim JH, Prasad S, Aggarwal BB. Regulation of survival, proliferation, invasion, angiogenesis, and metastasis of tumor cells through modulation of inflammatory pathways by nutraceuticals. Cancer Metastasis Rev 2010; 29(3): 405-34.
[http://dx.doi.org/10.1007/s10555-010-9235-2 PMID: 20737283]
[http://dx.doi.org/10.1007/s10555-010-9235-2 PMID: 20737283]
[24]
Ravindran J, Subbaraju GV, Ramani MV, Sung B, Aggarwal BB. Bisdemethylcurcumin and structurally related hispolon analogues of curcumin exhibit enhanced prooxidant, anti-proliferative and anti-inflammatory activities in vitro. Biochem Pharmacol 2010; 79(11): 1658-66.
[http://dx.doi.org/10.1016/j.bcp.2010.01.033 PMID: 20138025]
[http://dx.doi.org/10.1016/j.bcp.2010.01.033 PMID: 20138025]
[25]
Hua W-F, Fu Y-S, Liao Y-J, et al. Curcumin induces down-regulation of EZH2 expression through the MAPK pathway in MDA-MB-435 human breast cancer cells. Eur J Pharmacol 2010; 637(1-3): 16-21.
[http://dx.doi.org/10.1016/j.ejphar.2010.03.051 PMID: 20385124]
[http://dx.doi.org/10.1016/j.ejphar.2010.03.051 PMID: 20385124]
[26]
Costi R, Di Santo R, Artico M, et al. Cinnamoyl compounds as simple molecules that inhibit p300 histone acetyltransferase. J Med Chem 2007; 50(8): 1973-7.
[http://dx.doi.org/10.1021/jm060943s PMID: 17348637]
[http://dx.doi.org/10.1021/jm060943s PMID: 17348637]
[27]
Hossain M, Das S, Das U, Doroudi A, Zhu J, Dimmock JR. Novel hybrid molecules of 3,5-bis(benzylidene)-4-piperidones and dichloroacetic acid which demonstrate potent tumour-selective cytotoxicity. Bioorg Med Chem Lett 2020; 30(3): 126878-82.
[http://dx.doi.org/10.1016/j.bmcl.2019.126878 PMID: 31864797]
[http://dx.doi.org/10.1016/j.bmcl.2019.126878 PMID: 31864797]
[28]
Jung ME, Chen X-H, Wang C-Y, Li J, Zheng J. Preparation of bis-arylidene piperidones, cyclohexanones, piperazinones and analogs as inhibitors of the Wnt/beta-catenin pathway for the treatment of cancers U.S. Patent 6, 262, 3976, 2018.
[29]
ElNaggar AC, Saini U, Naidu S, et al. Anticancer potential of diarylidenyl piperidone derivatives, HO-4200 and H-4318, in cisplatin resistant primary ovarian cancer. Cancer Biol Ther 2016; 17(10): 1107-15.
[http://dx.doi.org/10.1080/15384047.2016.1210733 PMID: 27415751]
[http://dx.doi.org/10.1080/15384047.2016.1210733 PMID: 27415751]
[30]
Sumesh RV, Malathi A, Ranjith Kumar R. A facile tandem Michael addition/O-cyclization/elimination route to novel chromeno[3,2-c]pyridines. Mol Divers 2015; 19(2): 233-49.
[http://dx.doi.org/10.1007/s11030-015-9576-4 PMID: 25758540]
[http://dx.doi.org/10.1007/s11030-015-9576-4 PMID: 25758540]
[31]
Schmitt F, Gold M, Begemann G, Andronache I, Biersack B, Schobert R. Fluoro and pentafluorothio analogs of the antitumoral curcuminoid EF24 with superior antiangiogenic and vascular-disruptive effects. Bioorg Med Chem 2017; 25(17): 4894-903.
[http://dx.doi.org/10.1016/j.bmc.2017.07.039 PMID: 28774574]
[http://dx.doi.org/10.1016/j.bmc.2017.07.039 PMID: 28774574]
[32]
Rostamizadeh S, Hemmasi A, Zekri N. Magnetic amine-functionalized graphene oxide as a novel and recyclable bifunctional nanocatalyst for solvent-free synthesis of pyrano[3,2-c]pyridine derivatives. Nanochem Res 2017; 2: 29-41.
[33]
Eryanti Y, Hendra R, Herlina T, Zamri A, Supratman U. Synthesis of N-methyl-4-piperidone curcumin analogues and their cytotoxicity activity against T47D cell lines. Indones J Chem 2018; 18: 362-6.
[http://dx.doi.org/10.22146/ijc.24174]
[http://dx.doi.org/10.22146/ijc.24174]
[34]
Zhang Y, Liu Z, Wu J, et al. New MD2 inhibitors derived from curcumin with improved anti-inflammatory activity. Eur J Med Chem 2018; 148: 291-305.
[http://dx.doi.org/10.1016/j.ejmech.2018.02.008 PMID: 29466778]
[http://dx.doi.org/10.1016/j.ejmech.2018.02.008 PMID: 29466778]
[35]
Wu J, Zhang Y, Cai Y, et al. Discovery and evaluation of piperid-4-one-containing mono-carbonyl analogs of curcumin as anti-inflammatory agents. Bioorg Med Chem 2013; 21(11): 3058-65.
[http://dx.doi.org/10.1016/j.bmc.2013.03.057 PMID: 23611769]
[http://dx.doi.org/10.1016/j.bmc.2013.03.057 PMID: 23611769]
[36]
Zhao R-F, Hu J, Li F, Ye RF. Curcumin-like compounds as radical scavengers and their preparation CN Patent 1, 053, 6504, 2013.
[37]
Huang L, Wang J, Chen L, et al. Design, synthesis, and evaluation of NDGA analogues as potential anti-ischemic stroke agents. Eur J Med Chem 2018; 143: 1165-73.
[http://dx.doi.org/10.1016/j.ejmech.2017.09.028 PMID: 29126723]
[http://dx.doi.org/10.1016/j.ejmech.2017.09.028 PMID: 29126723]
[38]
Al-Omar MA, Youssef KM, El-Sherbeny MA, Awadalla SAA, El-Subbagh HI. Synthesis and in vitro antioxidant activity of some new fused pyridine analogs. Arch Pharm (Weinheim) 2005; 338(4): 175-80.
[http://dx.doi.org/10.1002/ardp.200400953 PMID: 15864787]
[http://dx.doi.org/10.1002/ardp.200400953 PMID: 15864787]
[39]
Youssef KM, El-Sherbeny MA, El-Shafie FS, Farag HA, Al-Deeb OA, Awadalla SAA. Synthesis of curcumin analogues as potential antioxidant, cancer chemopreventive agents. Arch Pharm (Weinheim) 2004; 337(1): 42-54.
[http://dx.doi.org/10.1002/ardp.200300763 PMID: 14760627]
[http://dx.doi.org/10.1002/ardp.200300763 PMID: 14760627]
[40]
Deck LM, Hunsaker LA, Vander Jagt TA, Whalen LJ, Royer RE, Vander Jagt DL. Activation of anti-oxidant Nrf2 signaling by enone analogues of curcumin. Eur J Med Chem 2018; 143: 854-65.
[http://dx.doi.org/10.1016/j.ejmech.2017.11.048] [PMID: 29223100]
[http://dx.doi.org/10.1016/j.ejmech.2017.11.048] [PMID: 29223100]
[41]
Huber I, Rozmer Z, Gyöngyi Z, et al. Budán, F.; Horváth, P.; Kiss, E.; Perjési, P. Structure activity relationship analysis of antiproliferative cyclic C5-curcuminoids without DNA binding: Design, synthesis, lipophilicity and biological activity. J. Mol. Struct. 2020; 127661
[http://dx.doi.org/10.1016/j.molstruc.2019.127661]
[http://dx.doi.org/10.1016/j.molstruc.2019.127661]
[42]
Leonard NJ, Locke DM. γ-Pyridones by isomerization. Substituted 1-methyl-3,5-dibenzyl-4-pyridones. J Am Chem Soc 1955; 77: 1852-5.
[http://dx.doi.org/10.1021/ja01612a042]
[http://dx.doi.org/10.1021/ja01612a042]
[43]
Song S, Chen J, Pan W, Song H, Wan Y. 3,5-Bis(arylmethenyl)-1-methylpiperidin-4-one useful in treatment of cancer and its preparation CN Patent 1, 012, 8589, 2014.
[44]
George RF, Panda SS, Shalaby E-SM, Srour AM, Farag IA, Girgis AS. Synthesis and molecular modeling studies of indole-based antitumor agents. RSC Advances 2016; 6: 45434-51.
[http://dx.doi.org/10.1039/C6RA07061B]
[http://dx.doi.org/10.1039/C6RA07061B]
[45]
Jianzhang W, Bixia W, Peihong Q, et al. Synthesis, crystal structure, antitumor activity of spiro-heterocyclic mono-carbonyl analogues of curcumin. Youji Huaxue 2014; 34: 1573-81.
[http://dx.doi.org/10.6023/cjoc201402004]
[http://dx.doi.org/10.6023/cjoc201402004]
[46]
El-Hag FAA, Abdel-Hafez NA, Abbas EMH, El-Manawaty MA, El-Rashedy AA. Synthesis and antitumor activity of some new fused heterocyclic compounds. Russ J Gen Chem 2019; 89: 128-37.
[http://dx.doi.org/10.1134/S1070363219010237]
[http://dx.doi.org/10.1134/S1070363219010237]
[47]
Lai KK, Chan KT, Choi MY, et al. 14-3-3σ confers cisplatin resistance in esophageal squamous cell carcinoma cells via regulating DNA repair molecules. Tumour Biol 2016; 37(2): 2127-36.
[http://dx.doi.org/10.1007/s13277-015-4018-6 PMID: 26346170]
[http://dx.doi.org/10.1007/s13277-015-4018-6 PMID: 26346170]
[48]
Cao R, Chen Q, Hou X, et al. Synthesis, acute toxicities, and antitumor effects of novel 9-substituted β-carboline derivatives. Bioorg Med Chem 2004; 12(17): 4613-23.
[http://dx.doi.org/10.1016/j.bmc.2004.06.038 PMID: 15358288]
[http://dx.doi.org/10.1016/j.bmc.2004.06.038 PMID: 15358288]
[49]
Ahmed E-K, Muhamad M. Esam. R.A.; Samy, M.; Yaser, A.M. Aryl azide-sulfonamide hybrids induce cellular apoptosis: synthesis and preliminary screening of their cytotoxicity in human HCT116 and A549 cancer cell lines. Med Chem Res 2019; 28: 2088-98.
[http://dx.doi.org/10.1007/s00044-019-02438-x]
[http://dx.doi.org/10.1007/s00044-019-02438-x]
[50]
Zhou B, Hu Z-J, Zhang H-J, Li J-Q, Ding W-J, Ma Z-J. Bioactive staurosporine derivatives from the Streptomyces sp. NB-A13. Bioorg Chem 2019; 82: 33-40.
[http://dx.doi.org/10.1016/j.bioorg.2018.09.016 PMID: 30268972]
[http://dx.doi.org/10.1016/j.bioorg.2018.09.016 PMID: 30268972]
[51]
Wo X-D, Hong X-Q. k; Gao, C.-X.; Jin, M.-M.; Li, W.-L.; Ding, Z.-S.; Tang, L.-H. Long-period virulent test of curcumin. J Zhejiang College of TCM 2000; 24: 61-5.
[52]
Verma SK, Thareja S. Structure based comprehensive modelling, spatial fingerprints mapping and ADME screening of curcumin analogues as novel ALR2 inhibitors. PLoS One 2017; 12(4)e0175318
[http://dx.doi.org/10.1371/journal.pone.0175318 PMID: 28399135]
[http://dx.doi.org/10.1371/journal.pone.0175318 PMID: 28399135]
[53]
Karki SS, Das U, Umemura N, et al. 5-Bis-(3-alkylaminomethyl-4-hydroxybenzylidene)-4-piperidones: A novel class of potent tumor-selective cytotoxins. J Med Chem 2016; 59(2): 763-9.
[http://dx.doi.org/10.1021/acs.jmedchem.5b01706 PMID: 26727215]
[http://dx.doi.org/10.1021/acs.jmedchem.5b01706 PMID: 26727215]
[54]
Addala E, Rafiei H, Das S, et al. 3,5-Bis(3-dimethylaminomethyl-4-hydroxybenzylidene)-4-piperidone and related compounds induce glutathione oxidation and mitochondria-mediated cell death in HCT-116 colon cancer cells. Bioorg Med Chem Lett 2017; 27(16): 3669-73.
[http://dx.doi.org/10.1016/j.bmcl.2017.07.018 PMID: 28716495]
[http://dx.doi.org/10.1016/j.bmcl.2017.07.018 PMID: 28716495]
[55]
Jin R, Chen Q, Yao S, et al. Synthesis and anti-tumor activity of EF24 analogues as IKKβ inhibitors. Eur J Med Chem 2018; 144: 218-28.
[http://dx.doi.org/10.1016/j.ejmech.2017.11.077 PMID: 29351887]
[http://dx.doi.org/10.1016/j.ejmech.2017.11.077 PMID: 29351887]
[56]
Das S, Das U, Michel D, Gorecki DK, Dimmock JR. Novel 3,5-bis(arylidene)-4-piperidone dimers: potent cytotoxins against colon cancer cells. Eur J Med Chem 2013; 64: 321-8.
[http://dx.doi.org/10.1016/j.ejmech.2013.03.055 PMID: 23644215]
[http://dx.doi.org/10.1016/j.ejmech.2013.03.055 PMID: 23644215]
[57]
Lee EH-C, Lim SS-C, Yuen K-H, Lee C-Y. Curcumin and a hemi-analogue with improved blood-brain barrier permeability protect against amyloid-beta toxicity in Caenorhabditis elegans via SKN-1/Nrf activation. J Pharm Pharmacol 2019; 71(5): 860-8.
[http://dx.doi.org/10.1111/jphp.13052 PMID: 30515807]
[http://dx.doi.org/10.1111/jphp.13052 PMID: 30515807]
Article Metrics
12