In the Laboratory edited by
The Microscale Laboratory
R. David Crouch Dickinson College Carlisle, PA 17013-2896
Microscale Synthesis and Spectroscopic Analysis of Flutamide, an Antiandrogen Prostate Cancer Drug
W
Ryan G. Stabile and Andrew P. Dicks* Department of Chemistry, University of Toronto, Toronto, Ontario, Canada, M5S 3H6; *
[email protected] Cancer of the prostate is the second leading cause of cancer deaths in American males, exceeded only by lung cancer. It is estimated that 221,000 new cases of the disease will be diagnosed during 2003, resulting in approximately 30,000 deaths (1). Steroid hormones such as testosterone and androsterone (androgens) enhance the proliferation of prostate tumors (2). Therapeutic agents that reduce androgen levels in target tissues (antiandrogens) are therefore of great importance to society. Two such prescription drugs (Figure 1) currently available for the prevention or treatment of prostate cancer are finasteride (marketed by Merck & Co. as Proscar; ref 3 ) and flutamide (marketed by Schering-Plough as Eulexin; ref 4). The high profile nature of cancer in the lives of students and relatively simple structure of nonsteroidal flutamide in-
H O
N C(CH3)3
CH3
NO2 CF3
CH3
H
H O
H N
N
H
H
C
H
CH(CH3)2
O Finasteride
Flutamide
Figure 1. Two drugs used for the treatment of prostate cancer.
spired the design of a microscale synthetic procedure. The experiment described involves a fundamental N-acylation reaction, converting a commercially available trisubstituted aromatic compound, 1, into its anilide derivative, 2 (flutamide, Scheme I). Such small scale acylations have been outlined previously encompassing the microscale preparation of aspirin (5) and acetanilide (6). The protocol allows for product isolation and purification within two hours while demonstrating basic organic laboratory methods of precipitation, vacuum filtration, and recrystallization. The reaction proceeds via a standard nucleophilic acyl substitution mechanism, initiating a discussion regarding the chemistry of carboxylic acid derivatives (7). An elective element that merits inclusion is the preparation of structural analogues of flutamide, by alteration of the acylating agent used. This leads to students generating different anilides, which exhibit varying degrees of antiandrogen activity. Melting point measurements and IR spectroscopy can distinguish the reaction products from one another. In addition other spectroscopic techniques (1H and 13C NMR, MS, UV) can be utilized to characterize flutamide (8). Spectral comparisons with authentic material are possible.1 The opportunity exists to demonstrate the concept of retrosynthetic analysis with respect to a modern pharmaceutical, which facilitates a discussion of activating–deactivating and directing effects of aromatic substituent groups. Biochemical students can research the in vivo activity of antiandrogens such as flutamide and interpret medicinal chemistry data. The experiment can therefore be adapted to illustrate many different and significant aspects of practical and theoretical organic chemistry, depending on the interests and academic level of students concerned. Optional Prelaboratory Assignment
NO2 NO2
O
CF3
(H3C)2HC
CF3
C Cl
pyridine, ∆
N H
NH2
C O
1
2
Scheme I. Synthesis of flutamide.
CH(CH3)2
Class interest is piqued on learning they will be synthesizing a pharmaceutical currently prescribed for the treatment of prostate cancer (or a structurally similar compound). Students are informed they are to undertake an N-acylation reaction between a 1,2,4-trisubstituted benzene derivative (substituent groups –NH2, –CF3, and –NO2) and an acid halide (Scheme II). The exact substitution pattern of aromatic starting material 1 is concealed from the class and deduced as a prelaboratory exercise. This is achieved by calculation of aromatic proton chemical shift values (δ) for four of the six possible isomeric 1,2,4-trisubstituted candidates, including 1. Computed chemical shifts for protons HA, HB, and HC are sig-
JChemEd.chem.wisc.edu • Vol. 80 No. 12 December 2003 • Journal of Chemical Education
1439
In the Laboratory NO2 NO2
O R
CF3
CF3
C Cl
pyridine, ∆
N H
NH2
δ(HA, HB, HC) = 7.26 + ∑i Zi
R O
R
2 (flutamide)
CH(CH3)2
3
(CH2)2CH3
4
CH2CH(CH3)2
5
(CH2)3CH3
Scheme II. Synthesis of structural analogues of flutamide.
CF3
NO2 BH
CF3
BH
NO2
CH
HA
CH
HA NH2
NH2
1 CF3
NH2
BH
NH2
BH
NO2
CH
HA
CH
HA
NO2
CF3
Figure 2. Some 1,2,4-substituted (trifluoromethyl)nitroanilines.
NO2 BH
CF3
HA
CH
Proton
δ Predicted
δ Observed
HA
7.09
7.04
HB
8.10
8.01
HC
6.97
6.79
NH2 Figure 3. Predicted and observed aromatic 1H chemical shifts for 3-trifluoromethyl-4-nitroaniline.
Table 1. Substituent Shift Constants Used To Calculate Aromatic 1H Chemical Shift Values Substituent
Shift Constant (Z, ppm) ortho
meta
para
᎑NH2
+0.95 ᎑ 0.75
+0.26 ᎑ 0.25
+0.38 ᎑ 0.65
᎑CF3
+0.32
+0.14
+0.20
᎑NO2
NOTE: Data from ref 9.
1440
(1)
C
1 Product
nificantly different from one compound to the next and allow for straightforward identification (Figure 2). Values of δ are determined according to the following equation (9),
where 7.26 ppm represents the chemical shift of benzene protons and Z values are substituent shift constants for different functional groups. Values of Z for a particular substituent are dependent on its orientation with respect to a ring proton (Table 1). An electron-donating amino functionality (shielding aromatic protons from an applied magnetic field) has negative Z constants, whereas electron-withdrawing groups such as nitro and trifluoromethyl have positive Z constants and exhibit a deshielding effect. Application of these values in eq 1 for 3-trifluoromethyl-4-nitroaniline, 1, predicts chemical shifts that are comparable with experimental measurements (Figure 3). Students are supplied with observed chemical shifts for 1 (or preferably the actual 1H NMR spectrum) at the beginning of the synthetic laboratory period. A positive identification of 1 is possible by comparing calculated δ values for the remaining three compounds in Figure 2. This exercise effectively illustrates the considerable electronic influence that each substituent group exhibits, impacting on the measured chemical shift of HA, HB, and HC. This can be discussed further after the synthesis when considering the 1H NMR spectrum of the reaction product, along with observable peak splitting due to ortho and meta proton coupling. Synthetic Overview
Synthesis of 2-Methyl-N-[3-trifluoromethyl-4nitrophenyl]propanamide (Flutamide) Flutamide, 2, is synthesized and purified on a microscale by modification of two literature procedures (10, 11). In a 10-mL Erlenmeyer flask, 3-trifluoromethyl-4-nitroaniline (1, 100 mg, 0.485 mmol) is dissolved in 2 mL of pyridine. The flask is capped with a rubber septum and cooled in an ice-bath for 10 min. 2-Methylpropanoyl chloride (70 µL, 0.668 mmol) is added dropwise through the septum whereupon a solution color change of red to orange is noted. The solution is then stirred and heated at 70 ⬚C in a water bath for 30 min. On cooling to room temperature, the reaction mixture is added to 100 mL of crushed ice兾water and vigorously stirred for a further 15 min. A yellow-brown solid precipitates during this period, which is collected by vacuum filtration. This crude product is recrystallized from toluene to yield flutamide as a yellow solid (typically 70–90 mg, 52– 67%): mp 109–111 ⬚C (lit. 110–111 ⬚C; ref 10) NMR (DMSO-d6, δ): 1.17 (d, 6H), 2.63 (septet, 1H), 8.05 (dd, 1H, J = 9.0 Hz, 2.2 Hz), 8.19 (d, 1H, J = 9.0 Hz), 8.30 (d, 1H, J = 2.1 Hz), 10.64 (s, 1H) 1H
NMR (DMSO-d6, δ): 19.8, 36.0, 117.8, 121.9, 122.8, 124.8, 128.4, 141.8, 144.8, 177.2 13C
IR (KBr disk, cm᎑1): 3360.2 (N⫺H), 1716.8 (C⫽O)
Journal of Chemical Education • Vol. 80 No. 12 December 2003 • JChemEd.chem.wisc.edu
In the Laboratory
Synthesis of N-[3-trifluoromethyl-4-nitrophenyl]butanamide, 3-Methyl-N-[3-trifluoromethyl-nitrophenyl]butanamide, and N-[3-trifluoromethyl-4-nitrophenyl]pentanamide Compounds 3, 4, and 5 can be synthesized by an identical method to that described for flutamide. The appropriate acid chloride (70 µL) is added to a solution of 3-trifluoromethyl-4-nitroaniline (1, 100 mg, 0.485 mmol) dissolved in pyridine. After isolation of the crude product in each instance, 3 is recrystallized from toluene and 4 and 5 from toluene兾hexanes to yield a yellow solid:
est is the change in aromatic proton chemical shifts observed on N-acylation (Scheme III). N-acylation has a dramatic deshielding effect that is most pronounced for adjacent ortho protons HA and HC. There exists an opportunity to rationalize this observation in terms of resonance electron withdrawal by the 2-methylpropanoyl group. Also noticeable is the marked downfield shift of aniline protons in 1, δ = 7.01, to δ = 10.64 in anilide 2. Aromatic proton coupling is apparent (Figure 4), which permits the calculation and interpretation of Jortho for HB and HC (9.0 Hz) and Jmeta for HA and HC (2.1 Hz; ref 12). Similar peak splitting is evident for 3-trifluoromethyl-4-nitroaniline.
3 mp 130–131 ⬚C (lit. 131–132 ⬚C; ref 10); IR (KBr disk, cm᎑1): 3361.2 (N⫺H), 1715.9 (C⫽O) 4 mp 102–103 ⬚C (lit. 103–104 ⬚C; ref 10); IR (KBr disk, cm᎑1): 3271.0 (N⫺H), 1676.9 (C⫽O) 5 mp 93–95 ⬚C (lit. 94–95 ⬚C; ref 10); IR (KBr disk, cm᎑1): 3367.0 (N⫺H), 1721.3 (C⫽O)
Hazards It is essential that all synthetic and purification procedures are carried out in an adequately ventilated fumehood. Pyridine has a foul odor and is related to long-term liver, kidney, and central nervous system damage. This compound is toxic by ingestion, inhalation, and skin absorption, as is toluene. Butanoyl, 2-methylpropanoyl, pentanoyl, and 3methylbutanoyl chlorides are all malodorous corrosive lachrymators that should be dispensed using an automatic delivery syringe. 3-Trifluoromethyl-4-nitroaniline is a skin irritant. The hazards associated with these reactants are reduced somewhat by the small quantities employed. The antiandrogenic products are harmful by ingestion, inhalation, and skin absorption. These compounds are possible teratogens and target the male reproductive system. All liquid reactants and solvents are either flammable (acid halides) or highly flammable (pyridine, toluene, hexanes). Students should therefore undertake all aspects of practical work wearing protective gloves, safety glasses, and a laboratory coat.
8.19
8.01
NO2
NO2 BH
CF3
BH
CF3
CH
HA
CH
HA
NH2 6.79
N H 8.05
7.04
C
8.30
CH(CH3)2
O
Scheme III. Aromatic 1H NMR chemical shifts of 3-trifluoromethyl4-nitroaniline and flutamide.
8.298
8.176
8.305 HB HA 8.205
8.073
8.065
Discussion A significant feature of this experiment is its adaptability towards differing organic chemistry curricula. At its simplest level it exposes introductory students to fundamental laboratory techniques such as solid product isolation and purification. If the option to synthesize structural analogues of flutamide (3, 4, and 5) has been adopted, compound identification can be accomplished by careful melting point measurements. The importance and role of flutamide as a treatment for prostate cancer provides an essential sense of real-world relevance to the material. The synthesis takes place via a nucleophilic acyl substitution mechanism and can be considered in terms of acid–halide reactivity. Students with a working knowledge of IR spectroscopy are able to comment on the characteristic spectral changes observed on transformation of an aniline into an anilide. The 1H NMR data of reactant and product(s) can facilitate an excellent discussion of shielding and deshielding effects and aromatic proton coupling, especially if the prelaboratory assignment has been undertaken. Of particular inter-
8.042 8.035
HC
8.4
8.2
30.22
29.82
8.0
30.63
Chemical Shift (ppm) Figure 4. flutamide.
1H
NMR spectrum (aromatic region, 300 MHz) of
JChemEd.chem.wisc.edu • Vol. 80 No. 12 December 2003 • Journal of Chemical Education
1441
In the Laboratory
A senior-level organic class will be equipped to devise retrosynthetic schemes for the synthesis of flutamide. Students are advised that the compound can be synthesized industrially using toluene as the aromatic starting material. They are also made aware that benzylic protons (such as those present in toluene) can be replaced successively by chlorine and fluorine atoms (Scheme IV). The transformation of (trichloromethyl)benzene into (trifluoromethyl)benzene (13) is not a reaction usually covered in general organic chemistry textbooks. This information enables the construction of a model retrosynthetic pathway (Scheme V). Several valuable synthetic concepts are illustrated by this approach. Nitration is directed to the desired position during the final synthetic step owing to the bulky N-2-methylpropanoyl moiety preventing ortho substitution. The –NH2 group has to be introduced indirectly by nitration and reduction of –NO 2 . The ability of a
trifluoromethyl group to promote meta substitution directs this nitration to the required position. Nitration of toluene would clearly produce an unwanted mixture of ortho and para isomers. The potential also exists to consider other reagents that could effect the N-acylation reaction that would be more appropriate than an acid halide in an industrial setting. More biologically oriented undergraduates can research the physiological mode of action of antiandrogens like flutamide. Such compounds are metabolized in vivo into an active hydroxylated form (14, 15), which binds effectively to prostate androgen receptors, preventing the uptake of testosterone. IR studies suggest the formation of a strong hydrogen bond between the hydroxyanilide and receptor site is of great significance, as is a highly electron deficient aromatic ring (16). The antiandrogenic activity of several substituted anilides has been quantified by animal testing (17), permitting student analysis of medicinal chemistry data. Summary
CH3
CCl3
Cl2
∆ HF, PCl5
CF3
Scheme IV. Synthesis of (trifluoromethyl)benzene from toluene.
There are many aspects of this experiment that generate stimulating and lively discussions. It can be tailored to meet the requirements of wide ranging organic chemistry courses with an emphasis placed on a number of different topics. These include spectroscopy (particularly 1H NMR and IR), reaction mechanisms, pharmaceutical synthesis (and retrosynthesis), and structure–activity relationships. Most importantly it provides students an opportunity to learn about a drug that is presently saving many lives throughout the world. Acknowledgments
NO2
We thank Ilya Gourevich for help with literature translation and express gratitude to the reviewers for instructive suggestions regarding the manuscript.
CF3 CF3
WSupplemental
N N H
C
H
CH(CH3)2
C
CH(CH3)2
O
Material
Instructions for the students, a prelaboratory assignment, notes for the instructor, and spectroscopic information are available in this issue of JCE Online.
O
Note
2 CF3
CF3
1. Authentic flutamide can be obtained from Sigma-Aldrich, product no. F-9397.
Literature Cited NO2
NH2
CF3
CCl3
Scheme V. Retrosynthetic analysis of flutamide.
1442
CH3
1. American Cancer Society Home Page. http://www.cancer.org/ docroot/lrn/lrn_0.asp (accessed Sep 2003). 2. Huggins, C.; Hodges, C. V. Cancer Res. 1941, 1, 293–297. 3. Wilde, M. I.; Goa, K. L. Drugs 1999, 57, 557–581. 4. Brogden, R. N.; Clissold, S. P. Drugs 1989, 38, 185–203. 5. Pandita, S.; Goyal, S. J. Chem. Educ. 1998, 75, 770. 6. Mayo, D. W.; Pike, R. M.; Trumper, P. K. In Microscale Organic Laboratory, 4th ed.; Wiley: New York, 1999; pp 304– 306. 7. For some examples, see (a) McMurry, J. In Organic Chemistry, 5th ed.; Brooks-Cole: Pacific Grove, CA, 2000; pp 857–
Journal of Chemical Education • Vol. 80 No. 12 December 2003 • JChemEd.chem.wisc.edu
In the Laboratory
8. 9.
10. 11.
860; (b) Brown, W.; Foote, C. In Organic Chemistry, 3rd ed.; Harcourt College: Philadelphia, PA, 2002; pp 652–653; (c) Solomons, G.; Fryhle, C. In Organic Chemistry, 7th ed.; Wiley: New York, 2001; pp 822–827. Sternal, R.; Nugara, N. Anal. Profiles Drug Subst. Excipients 2001, 27, 115–157. Pretsch, E.; Clerc, T.; Seibl, J.; Simon, W. In Spectral Data For Structure Determination Of Organic Compounds, 2nd ed.; Springer-Verlag: New York, 1989; pp H255–H260. Baker, J. W.; Bachman, G. L.; Schumacher, I.; Roman, D. P.; Tharp, A. L. J. Med. Chem. 1967, 10, 93–95. Neri, R. O.; Topliss, J. G. Substituted Anilides As Anti-Androgens. U.S. Patent 4144270, March 13, 1979.
12. Field, L. D.; Sternhell, S.; Kalman, J. R. In Organic Structures From Spectra, 2nd ed.; Wiley & Sons: New York, 2000; p 55. 13. Holt, L. C.; Mattison, E. L. Organic Fluorine Compounds. U. S. Patent 2005712, 1935. 14. Peets, E. A.; Henson, M. F.; Neri, R. O. Endocrinology 1974, 94, 532–540. 15. Tucker, H.; Crook, J. W.; Chesterson, G. J. J. Med. Chem. 1988, 31, 954–959. 16. Morris, J. J.; Hughes, L. R.; Glen, A. T.; Taylor, P. T. J. Med. Chem. 1991, 34, 447–455. 17. Varga, S. V.; Reznikov, A. G.; Bal’on, Y. G.; Lozinskii, M. O.; Smirnov, V. A. Probl. Endokrinol. 1983, 29, 67–71.
JChemEd.chem.wisc.edu • Vol. 80 No. 12 December 2003 • Journal of Chemical Education
1443
