复旦大学：《药物设计学》课程教学资源（虚拟实验室课外实践）Exercises for Drug Design Courses_Practice-V SYNTHESIS, LIPOPHILICITY DETERMINATION, AND QSAR STUDY OF CHALCONES AND ANALOGS WITH ANTITUMORAL ACTIVITY

version date: 1 December 2006 EXERCISE V11 SYNTHESIS LIPOPHILICITY DETERMINATION AND QSAR STUDY OF CHALCONES AND ANALOGS WITH ANTITUMORAL ACTIVITY Mauricio Cabrera, Hugo Cerecetto, and Mercedes gonzalez Laboratorio de quimica Organica, Facultad de quimica/Facultad de Ciencias, UdelaR hcerecet@fg. edu. uy, megnzal@fg. edu.uy Objectives: 1)To apply a simple and safe synthetic procedure to prepare chalcones and analogs with activity against MCF-7 cells 2)To determine lipophilicity of these compounds by different methods (experimentally and theoreticall 3) To obtain correlations between lipophilicity and activity Synthetic procedures for derivatives 1-ll(see Table) W Y= H, Br, Cl, OMe, OCH2 Ph KOH/ MeOH Z=H. I OMe om temperature W=H. Br W X=H OH A mixture of the corresponding ketone(2-hydroxyacetophenone or acetophenone)(1 equiv ), the corresponding aldehyde (l equiv ) potassium hydroxide(10 equiv), and ethanol as solvent(50 mL gram of ketone) was stirred at room temperature during 24 h. After that, the mixture was acidified with aqueous solution of HCl (10 %) The precipitate was filtered off, washed with cold ethanol, and crystallized from methanol Lipophilicity determination Theoretical calculations The lipophilicity of the chalcone derivatives and analogs will be expressed as LogP. For the theoretical LogP(CLogP, Table) it could be employed using free programs from the Internet, or other kind of programs In the present proposal, the ClogP was calculated using Villar method, at AMI semiempirical method, implemented in Spartan 04, 1.0.1 version, suite of programs(Table Experimental determinations First, stock solutions of chalcones and analogs were prepared in pure acetone prior to use. The corresponding stock solutions were applied on precoated thin-layer chromatography (TLC) plates SIL RP-18W/UV254. The TLC was eluted with MeOH: physiological serum(80: 20, v/v). The plate were developed in a closed chromatographic tank, dried, and the spots were located under UV light The R values were averaged from two to three determinations, and converted into RM values via the relationship RM=log [(1/R-l] TLC was performed on precoated silica gel 60 F 254 TLC plates, and eluted with petroleum ether: ethyl acetate(80: 20, v/v). The plates were developed in a closed chromatography tank, dried and the spots were located under uv light

1 EXERCISE V.11 SYNTHESIS, LIPOPHILICITY DETERMINATION, AND QSAR STUDY OF CHALCONES AND ANALOGS WITH ANTITUMORAL ACTIVITY Mauricio Cabrera, Hugo Cerecetto, and Mercedes González Laboratorio de Química Orgánica, Facultad de Química/Facultad de Ciencias, UdelaR hcerecet@fq.edu.uy, megnzal@fq.edu.uy Objectives: 1) To apply a simple and safe synthetic procedure to prepare chalcones and analogs with activity against MCF-7 cells. 2).To determine lipophilicity of these compounds by different methods (experimentally and theoretically). 3) To obtain correlations between lipophilicity and activity. Synthetic procedures for derivatives 1–11 (see Table)1,i X O Y Z CHO KOH / MeOH room temperature X O Y Z X= H, OH W W Y= H, Br, Cl, OMe, OCH2Ph Z= H, I, OMe W= H, Br A mixture of the corresponding ketone (2-hydroxyacetophenone or acetophenone) (1 equiv), the corresponding aldehyde (1 equiv), potassium hydroxide (10 equiv), and ethanol as solvent (50 mL for each gram of ketone) was stirred at room temperature during 24 h. After that, the mixture was acidified with aqueous solution of HCl (10 %). The precipitate was filtered off, washed with cold ethanol, and crystallized from methanol. Lipophilicity determination Theoretical calculations2,3 The lipophilicity of the chalcone derivatives and analogs will be expressed as LogP. For the theoretical LogP (CLogP, Table) it could be employed using free programs from the Internet2 , or other kind of programs. In the present proposal, the CLogP was calculated using Villar method, at AM1 semiempirical method, implemented in Spartan’04, 1.0.1 version, suite of programs3 (Table). Experimental determinations4,i First, stock solutions of chalcones and analogs were prepared in pure acetone prior to use. The corresponding stock solutions were applied on precoated thin-layer chromatography (TLC) plates SIL RP-18W/UV254. The TLC was eluted with MeOH:physiological serum (80:20, v/v). The plates were developed in a closed chromatographic tank, dried, and the spots were located under UV light. The Rf values were averaged from two to three determinations, and converted into RM values via the relationship: RM = log [(1/Rf)–1] TLC was performed on precoated silica gel 60 F 254 TLC plates, and eluted with petroleum ether:ethyl acetate (80:20, v/v). The plates were developed in a closed chromatography tank, dried, and the spots were located under UV light. version date: 1 December 2006

version date: 1 December 2006 Correlations between lipophilicity and activity s it is well known, the lipophilicity of a drug plays a significant role in numerous biologi responses. So, the relationship between this property and the antitumoral activity(table)could be studied Plot the different forms of expression of the lipophilicity for this family of compounds( CLogP, RM, and R, Table)versus percentage of survival of MCF-7 cells treated with 100 HM of derivatives Study the relationships Percentage of survival of Derivative MCF-7 cells treated with CLogP CLogP R1C18 R 100μ M of derivatives2 partan Sio,e 4.59 4200.052095 4.59 4.20 0.017 4.46 3.93 0.070 n 3783.240.432nd 0.0520.85 1.2 3120.6300.45 6 4.31 4.60 0.2130.68 19 4.22 3.76 0.5250.91 8 4.98 4.59 0.194089 4854320.3080.89

2 Correlations between lipophilicity and activity As it is well known, the lipophilicity of a drug plays a significant role in numerous biological responses5 . So, the relationship between this property and the antitumoral activity (Table) could be studied. Plot the different forms of expression of the lipophilicity for this family of compounds (CLogP, RM, and Rf, Table) versus percentage of survival of MCF-7 cells treated with 100 µM of derivatives. Study the relationships. Derivative Percentage of survival of MCF-7 cells treated with 100 µM of derivativesa CLogP webb CLogP Spartanc RM C18d Rf SiO2 e OH O Br 1 43 4.59 4.20 –0.052 0.95 OH O Br 2 100 4.59 4.20 –0.017 ndf OH O Cl 3 41 4.46 3.93 –0.070 nd OH O OMe 4 10 3.78 3.24 –0.432 nd OH O OCH2Ph 5 56 5.39 4.98 –0.052 0.85 OH O OMe OMe 6 1.2 3.73 3.12 –0.630 0.45 OH O OMe I 7 96 4.31 4.60 –0.213 0.68 O 8 19 4.22 3.76 –0.525 0.91 O Br 9 44 4.98 4.59 –0.194 0.89 O Cl 10 56 4.85 4.32 –0.308 0.89 version date: 1 December 2006

version date: 1 December 2006 4.17 363-0.525074 Determined in Centro de Investigaciones en Farmacobiologia Aplicada(Pamplona, Spain). Dra. Adela Lopez de Cerain,Dr.AntonioMonge.http://www.molinspiration.com/cgi-bin/properties.viLlarmethodreference[3].silRp- 18W/UV254, MeOH: physiological serum (80: 20, v/v). SiO2, petroleum ether ethyl acetate (80: 20, V/v).nd: not determined Results In all of cases, like-quadratic correlations were obtained between lipophilicity and antitumoral activity. This is a typical behavior between this property and some kinds of activities 0.2616110.0024 8063028581100014 a86 survival MCF-7 cells MCF-7 R-Square SD N P 063300.1507110.0181 0.56910.129280.1219 MCF-7 The values that express lipophilicity of the compounds are linearly correlated

3 O OMe 11 30 4.17 3.63 –0.525 0.74 a Determined in Centro de Investigaciones en Farmacobiología Aplicada (Pamplona, Spain). Dra. Adela López de Ceráin, Dr. Antonio Monge. b http://www.molinspiration.com/cgi-bin/properties. c Villar method, reference [3]. d SIL RP- 18W/UV254, MeOH:physiological serum (80:20, v/v). e SiO2, petroleum ether:ethyl acetate (80:20, v/v). f nd: not determined. Results In all of cases, like-quadratic correlations were obtained between lipophilicity and antitumoral activity. This is a typical behavior between this property and some kinds of activities5 . 0 20 40 60 80 100 3,6 3,8 4,0 4,2 4,4 4,6 4,8 5,0 5,2 5,4 5,6 R-Square SD N P ------------------------------------------------ 0.7792 0.2616 11 0.0024 CLogP (web) % Survival MCF-7 cells 0 20 40 60 80 100 3,0 3,5 4,0 4,5 5,0 R-Square SD N P --------------------------------------------------- 0.8063 0.2858 11 0.0014 CLogP (Spartan) % Survival MCF-7 0 20 40 60 80 100 -0,7 -0,6 -0,5 -0,4 -0,3 -0,2 -0,1 0,0 R-Square SD N P ----------------------------------------------------- 0.6330 0.1507 11 0.0181 RM % Survival MCF-7 0 20 40 60 80 100 0,4 0,5 0,6 0,7 0,8 0,9 1,0 R-Square SD N P ---------------------------------------------------- 0.5691 0.1292 8 0.1219 Rf % Survival MCF-7 The values that express lipophilicity of the compounds are linearly correlated. version date: 1 December 2006

version date: 1 December 2006 ◇ 02 a8 CLogP (web) CLogP (web) ◇ References 1)Song, LL, Kosmeder, J W;, Lee,SK, Gerhauser, C; Lantvit, D; Moon, R.C. Moriarty, R.B. Pezzuto, J M. Cancer Research. 59(1999)578-585 2)http://www.molinspiration.com/cgi-bin/properties 3)Spartan 04; Wavefunction, Inc. 18401 Von Karman Avenue, Suite 370. Irvine, California 92612 USA 4)a)Tsantili-Kakoulidou, A; Antoniadou-Vyza, A. Prog. Clin. Biol. Res. 291(1989)71-74. b) Denny, w.A.; Graham, J. A; Roberts, P. B, Anderson, R. F; Boyd, M., Lock, C.J. L; Wilson, W.R. J.Med.Chem,35(1992)4832-4841 5)C. Hansch, A. Leo, The hydrophobic parameter: measurement and calculation, in: Exploring QSAR Fundamentals and Applications in Chemistry and Biology, American Chemical Society, Washington, 1995, pp 97-124

4 3,6 3,8 4,0 4,2 4,4 4,6 4,8 5,0 5,2 5,4 5,6 3,0 3,5 4,0 4,5 5,0 CLogP (Spartan) CLogP (web) 3,6 3,8 4,0 4,2 4,4 4,6 4,8 5,0 5,2 5,4 5,6 -0,7 -0,6 -0,5 -0,4 -0,3 -0,2 -0,1 0,0 RM CLogP (web) 3,6 3,8 4,0 4,2 4,4 4,6 4,8 5,0 5,2 5,4 5,6 0,4 0,5 0,6 0,7 0,8 0,9 1,0 Rf CLogP (web) -0,7 -0,6 -0,5 -0,4 -0,3 -0,2 -0,1 0,0 0,4 0,5 0,6 0,7 0,8 0,9 1,0 Rf RM References 1) Song, L.L.; Kosmeder, J.W.; Lee, S.K.; Gerhäuser, C.; Lantvit, D.; Moon, R.C.; Moriarty, R.B.; Pezzuto, J.M. Cancer Research. 59 (1999) 578-585. 2) http://www.molinspiration.com/cgi-bin/properties. 3) Spartan'04; Wavefunction, Inc. 18401 Von Karman Avenue, Suite 370. Irvine, California 92612 USA. 4) a) Tsantili-Kakoulidou, A.; Antoniadou-Vyza, A. Prog. Clin. Biol. Res. 291 (1989) 71-74. b) Denny, W. A.; Graham, J. A.; Roberts, P. B.; Anderson, R. F.; Boyd, M.; Lock, C. J. L.; Wilson, W. R. J. Med. Chem., 35 (1992) 4832-4841. 5) C. Hansch, A. Leo, The hydrophobic parameter: measurement and calculation, in: Exploring QSAR. Fundamentals and Applications in Chemistry and Biology, American Chemical Society, Washington, 1995, pp 97-124. version date: 1 December 2006

version date: 1 December 2006 Hugo Cerecetto High standards in safety measures should be maintained in all work carried out in Medicinal Chemistry laboratories The handling of electrical instruments, heating elements, glass materials, dissolvents and other inflammable materials does not present a problem if the supervisor's instructions are carefully f This document has been supervised by Prof. Hugo Cerecetto (hcerecetafgedu uy) who has informed that no special risk (regarding toxicity, inflammability, explosions), outside ofthe standard risks pertaining to a Medicinal Chemistry laboratory exist when performing this exercise If your exercise involves any special risks, please inform the editor All the procedures must be done in a fume hood

5 Hugo Cerecetto hcerecet@fq.edu.uy High standards in safety measures should be maintained in all work carried out in Medicinal Chemistry Laboratories. The handling of electrical instruments, heating elements, glass materials, dissolvents and other inflammable materials does not present a problem if the supervisor’s instructions are carefully followed. This document has been supervised by Prof. Hugo Cerecetto (hcerecet@fq.edu.uy) who has informed that no special risk (regarding toxicity, inflammability, explosions), outside of the standard risks pertaining to a Medicinal Chemistry laboratory exist when performing this exercise. If your exercise involves any “special” risks, please inform the editor. Safety aspects i All the procedures must be done in a fume hood. version date: 1 December 2006