Synthesis of irreversibly binding bicalutamide analogs for imaging studies

Article abstract of DOI:10.1016/j.tetlet.2005.04.143

A new synthetic methodology for preparing radioactive androgen receptor binding compounds in order to determine receptor-ligands interactions has been developed.

Full text of DOI:10.1016/j.tetlet.2005.04.143

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 4821–4823
Synthesis of irreversibly binding bicalutamide analogs for
imaging studies
Vipin A. Nair,a Suni M. Mustafa,a Michael L. Mohler,a Jun Yang,b Leonid I. Kirkovsky,a
James T. Daltonb and Duane D. Millera,*
aDepartment of Pharmaceutical Sciences, University of Tennessee, The Health Science Center, 847 Monroe Avenue,
RM 227 C, Memphis, TN 38163, USA
bDivision of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, The Ohio State University, Columbus,
OH 43210, USA
Received 21 February2005; accepted 1 April 2005
Available online 31 May2005
Abstract—A new synthetic methodology for preparing radioactive androgen receptor binding compounds in order to determine
receptor–ligands interactions has been developed.
Ó 2005 Elsevier Ltd. All rights reserved.
Androgen receptor is an important cellular regulatory
protein, which plays a prominent role in numerous phys-
iological processes.1,2 Compounds that bind to the
androgen receptor have great therapeutic value in the
treatment of various conditions ranging from regulation
of male fertilityto prostate cancer. 3 In our continued ef-
forts to establish the mechanism of action of the tissue
selective androgen receptor modulator (SARM), which
we discovered in 1998 and to develop it as a potential
drug candidate for prostate cancer we synthesized a ser-
ies of iodo analogs of bicalutamide.4,5 These molecules,
which exhibited antagonistic activitywere found to
block the pharmacological effects of testosterone; and
were obtained byreplacing the trifluoromethyl and sul-
fonyl groups of bicalutamide by iodine and oxygen,
respectively, and introduction of chirality at the stereo-
genic center; the synthesis has been reported recently.5
This investigation was carried out primarilyto develop
a target molecule whose interaction with the androgen
receptor could be identified byhaving a tag or a label at-
tached to the molecule and to follow its course of action
byimaging techniques. Our attempts to label the mole-
cule byattaching a chromophore, which exhibits fluores-
cence did not turn out to be successful, because of the
low binding affinityto the receptor. Hence we decided
to incorporate a radioactive isotopic label (125I) to the
compound 2, which showed veryhigh binding affinity
to the receptor (Fig. 1).6
Earlier investigations have proved that the stereochemis-
tryat the chiral center, the nature of the functional
groups attached to the aromatic rings and the hetero-
atom linker playa prominent role in determining the
7–13
activityand affinity.
An isothiocyanate moiety had
been introduced at the 4th position of the aromatic B-
ring so as to provide an electrophilic center for an irre-
versiblybinding interaction between the receptor and
the ligand. Analogous agents have been shown to bind
irreversiblyproviding a theoretical basis to develop
these as prostate cancer therapeutics.14,15 Biological test-
ing of the isothiocyanate analogs of bicalutamide dem-
onstrated high binding affinityin exchange assays with
a radiolabeled high affinityandrogen receptor ligand
[3H]mibolerone, irreversible androgen receptor binding
in Scatchard analyses and potential growth inhibitory
activityagainst LNCaP, DU-145, PC-3, PPC-1, and
TSU prostate cancer cell lines.16,17 The advantages of
incorporation of the radioisotope 125I into an androgen
receptor ligand are threefold: (i) it allows radioimaging
of prostate tumor, (ii) provides an irreversiblybinding
ligand that should chemicallyablate androgen receptor
action in prostate, blocking cancer growth, and (iii) pro-
vides a synthetic strategy to incorporate other iodine
Keywords: Bicalutamide; Iodine; Androgen; Receptor; Radioactive.
*
Corresponding author. Tel.: +1 901 448 6026; fax: +1 901 448
3446; e-mail: dmiller@utmem.edu
0040-4039/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.04.143

4822
V. A. Nair et al. / Tetrahedron Letters 46 (2005) 4821–4823
NC
I
R
refluxing it with tert-butylcarbonate protected 4-amino-
phenol 9 and K2CO3 in 2-propanol medium to obtain
compound 10. The conversion of the intermediate 7 to
compound 10 through the epoxide formation was car-
ried out in a two-step, one-pot process wherein after
the epoxide was formed, the solvent was removed and
the resulting residue was immediatelycarried on to the
ring opening step.
NC
F
O
O
A
B
O
S
N
O
F C
3
N
H
H
OH
OH
O
2
1
R = F, Cl, Br, I
(R,S)-Bicalutamide
Figure 1.
To a mixture of compound 10 and tetrakis(triphenyl-
phosphine)palladium in anhydrous toluene under argon
atmosphere, hexamethyl ditin18 was introduced and re-
fluxed for 5 h. Workup of the reaction mixture followed
bypurification using flash column chromatography
afforded the trimethyl tin derivative 11. Equimolar
amounts of NaI and chloramine T were added to a solu-
tion of compound 11 in methanol and stirred at room
temperature for 30 min. The reaction mixture turned yel-
low during the course of the reaction; the completion of
the reaction was determined bythe absence of the start-
ing material in the reaction mixture. Subsequent washing
with sodium bicarbonate solution and extraction into
ethyl acetate afforded back the compound 10. The suc-
cess of this step ensured the repetition of the experiment
with radioactive Na125I so as to obtain the radioactive io-
dine incorporated analog of compound 10. The protec-
radioisotopes such as 131I, which could be investigated
in radioiodine therapyfor prostate cancer.
The synthetic procedure employed for the preparation
of target compound is depicted in Scheme 1. We recently
reported the syntheses of 4-amino-2-iodobenzonitrile 4
and (2R)-3-bromo-2-hydroxy-2-methylpropanoic acid 6
from 4-nitro-2-iodoaniline 3 and (D)-proline 5, respec-
tively.5 A solution of 6 in tetrahydrofuran was converted
to the corresponding acid chloride bythionyl chloride
under argon atmosphere at 0 °C. Addition of a solution
of 4 in tetrahydrofuran followed by overnight stirring at
room temperature yielded the (R)-propanamide deriva-
tive 7. A solution of compound 7 in acetone when
refluxed with K2CO3 for 2 h generated the correspond-
ing epoxide 8, which was subsequentlyopened up by
H N
2
NC
I
a
I
NO
NH
NC
2
2
NC
I
O
O
d
c
3
4
I
N
H
Br
N
O
H
OH
8
7
O
H
b
HO
Br
COOH
N
OH
H
5
6
NH-tBoc
HO
9
NH-tBoc
NH-tBoc
NC
f
NC
O
e
O
O
O
I
N
H
(Me) Sn
3
N
OH
10
H
OH
11
NH-tBoc
NH
2
NC
*I
NC
*I
g
h
O
O
O
O
N
H
N
H
OH
10
OH
12
NCS
NC
*I
O
i
O
N
H
OH
13
Scheme 1. Reagents and conditions: (a) (1) NaNO2, H2SO4, CuCN, NaCN, (2) HCl, SnCl2Æ2H2O, EtOH; (b) (1) methacryloyl chloride, NaOH,
acetone, (2) NBS, DMF, (3) HBr; (c) SOCl2, THF; (d) K2CO3, acetone; (e) K2CO3, 2-propanol; (f) Pd(PPh3)4, hexamethyl ditin, toluene; (g) NaI
(Na125I), chloramine T, MeOH; (h) acetyl chloride, absolute EtOH; (i) chloroform, thiophosgene, NaHCO3; *I = I or 125I.

V. A. Nair et al. / Tetrahedron Letters 46 (2005) 4821–4823
4823
9. Rosen, J.; Day, A.; Jones, T. K.; Nazdan, A. M.; Stein, R.
B. J. Med. Chem. 1995, 38, 4855.
10. Bohl, C. E.; Chang, C.; Mohler, M. L.; Chen, J.; Miller,
D. D.; Swaan, P. W.; Dalton, J. T. J. Med. Chem. 2004,
47, 3765.
tive group on the amino functionalitywas then removed
bydissolving it in absolute ethanol and treating it with
acetyl chloride under ice-cold conditions, followed by
stirring at room temperature for 2 h; which upon concen-
tration followed bywashing in aqueous medium and
extraction into ethyl acetate gave the free amine 12.
Thereafter, to the solution of compound 12 in chloro-
form, thiophosgene, and sodium bicarbonate were added
at 0 °C and stirred overnight at room temperature to
yield the target compound 13.19 The synthetic strategy out-
lined herein provides a facile route to the development of
a new class of androgen receptor ligands with a tracer
that helps to identify, understand and establish the
mechanism of action along with a detailed insight to
the receptor–ligand binding interactions. Simultaneously
computational studies using molecular modeling tech-
niques are also being carried out bydocking the ligand
into the androgen receptor model that we developed,
so as to provide a theoretical rationale to substantiate
the experimental mechanistic observations of the binding
interactions and the resulting structure activityrelation-
ships. Efforts are ongoing to confirm the binding loci by
X-raycrystallographic techniques as well.
11. Morris, J. J.; Hughes, L. R.; Len, A. T.; Taylor, P. J.
J. Med. Chem. 1991, 34, 447.
12. James, K. D.; Ekwuribe, N. N. Synthesis 2002, 7, 850.
13. Yin, D.; Gao, W.; Kearby, J. D.; Xu, H.; Chung, K.; He,
Y.; Marhefka, C. A.; Veverka, K. A.; Miller, D. D.;
Dalton, J. T. J. Pharmacol. Exp. Ther. 2003, 304, 1334.
14. Kirkovsky, L.; Mukherjee, A.; Yin, D.; Dalton, J. T.;
Miller, D. D. J. Med. Chem. 2000, 43, 581.
15. Yin, D.; Xu, H.; Kirkovsky, L. I.; Miller, D. D.; Dalton, J.
T. J. Pharmacol. Exp. Ther. 2003, 304, 1323.
16. Mukherjee, A.; Kirkovsky, L. I.; Kimura, Y.; Marvel, M.
M.; Miller, D. D.; Dalton, J. T. Biochem. Pharmacol.
1999, 58, 1259.
17. Hwang, D. J.; Xu, H.; Mustafa, S. M.; Dalton, J. T.;
Miller, D. D. Synthesis of isothiocyanate derivatives of
irreversible selective androgen receptor modulators
(SARMs) and biological testing in prostate cancer cell
lines. Unpublished Results—Presented at the 229th Amer-
ican Chemical Society Meeting (Division of Medicinal
Chemistry), San Diego, 2005.
18. Hexamethyl ditin is highly toxic when inhaled or in
contact with skin and is also dangerous to the environ-
ment. Therefore it should be handled with adequate
precautions and care. For more information: ꢀToxicityof
tin and its compoundsꢁ: Winship, K. A. Adverse Drug
React. Acute Poison. Rev. 1988, 7, 19.
Acknowledgements
We thank the Department of Defense (DOD) for the
grant ꢀNovel Strategyfor Prostate Cancer Imaging: Syn-
thesis and Pharmacologyof Nonsteroidal Ligands ꢁ
DAMD17-01-1-0103 and the National Institute of
Health (NIH) for the grant ꢀNovel Irreversible Nonste-
roidal Selective Androgen Receptor Modulators
(SARMs) for Prostate Cancerꢁ DK 065227. The Ameri-
can Chemical Society, Division of Medicinal Chemistry
is gratefullyacknowledged for a pre-doctoral fellowship
for M.L.M.
19. Compound 10: Yield: 60%; NMR (1H, 300 MHz, CDCl3):
1.40 (m, 9H, 3CH3), 1.57 (s, 3H, CH3), 4.5 (d, 1H, CH),
4.7 (d, 1H, CH), 6.8 (m, 2H, ArH, J = 2.5 Hz, 8.8 Hz), 7.5
(m, 2H, ArH, J = 2.5 Hz, 8.8 Hz), 7.2 (m, 1H, ArH,
J = 0.6 Hz, 8.5 Hz), 7.8 (m, 1H, ArH, J = 2.4 Hz, 8.5 Hz),
8.2 (m, 1H, ArH, J = 0.6 Hz, 2.4 Hz), 8.0 (br, 1H, NH),
8.7 (br, 1H, NH); MS: 536.1 (MꢀH); C22H24IN3O5, calcd:
C, 49.17; H, 4.50; N, 7.82. Found: C, 49.10; H, 4.39; N,
25
7.76; ½aꢁD ꢀ25.4 (c 1.0, MeOH) 11: Yield: 53%; NMR (1H,
300 MHz, CDCl3): 0.9 (m, 9H, 3CH3), 1.40 (m, 9H,
3CH3), 1.52 (s, 3H, CH3), 4.6 (d, 1H, CH), 4.8 (d, 1H,
CH), 6.8 (m, 2H, ArH, J = 2.7 Hz, 8.6 Hz), 7.6 (m, 2H,
ArH, J = 2.7 Hz, 8.6 Hz), 7.4 (m, 1H, ArH, J = 0.7 Hz,
8.2 Hz), 7.8 (m, 1H, ArH, J = 2.3 Hz, 8.2 Hz), 7.9 (m, 1H,
ArH, J = 0.7 Hz, 2.3 Hz), 8.1 (br, 1H, NH), 8.7 (br, 1H,
NH); MS: 572.2 (MꢀH); C25H33N3O5Sn, calcd: C, 52.29;
References and notes
1. Evans, R. M. Science 1988, 240, 889.
2. Van Dort, M. E.; Robins, D. M.; Wayburn, B. J. Med.
Chem. 2000, 43, 3344.
3. Tucker, H.; Chesterson, G. J. J. Med. Chem. 1988, 31, 885.
4. Dalton, J. T.; Mukherjee, A.; Zhu, Z.; Kirkovsky, L.;
Miller, D. D. Biochem. Biophys. Res. Commun. 1998, 244,
1.
5. Nair, V. A.; Mustafa, S. M.; Mohler, M. L.; Fisher, S. J.;
Dalton, J. T.; Miller, D. D. Tetrahedron Lett. 2004, 45,
9475.
6. Nair, V. A.; Mustafa, S. M.; Mohler, M. L.; Fisher, S. J.;
Dalton, J. T.; Miller, D. D., Studies on some selective
androgen receptor modulators. Unpublished Results—Pre-
sented at the 228th American Chemical SocietyMeeting
(Division of Medicinal Chemistry), Philadelphia, 2004.
7. Marhefka, C. A.; Gao, W.; Chung, K.; Kim, J.; He, Y.;
Yin, D.; Bohl, C.; Dalton, J. T.; Miller, D. D. J. Med.
Chem. 2004, 47, 993.
25
H, 5.79; N, 7.32. Found: C, 52.15; H, 5.70; N, 7.21; ½aꢁD
ꢀ37.5 (c 1.0, MeOH) 12: Yield: 42%; NMR (1H,
300 MHz, CDCl3): 1.48 (s, 3H, CH3), 4.4 (d, 1H, CH),
4.6 (d, 1H, CH), 6.4 (d, 2H, ArH, J = 2.8 Hz, 8.9 Hz), 6.6
(d, 2H, ArH, J = 2.8 Hz, 8.9 Hz), 7.2 (m, 1H, ArH,
J = 0.5 Hz, 8.5 Hz), 7.8 (m, 1H, ArH, J = 2.4 Hz, 8.5 Hz),
8.2 (m, 1H, ArH, J = 0.5 Hz, 2.4 Hz), 8.1 (br, 2H, NH2),
8.3 (br, 1H, NH); MS: 460.1 (M+Na+); C17H16IN3O3,
calcd: C, 46.70; H, 3.69; N, 29.02. Found: C, 46.65; H,
25
3.57; N, 29.1; ½aꢁD +54.3 (c 1.0, MeOH) 13: Yield: 35%;
NMR (1H, 300 MHz, CDCl3): 1.5 (s, 3H, CH3), 4.5 (d,
1H, CH), 4.7 (d, 1H, CH), 6.8 (m, 2H, ArH, J = 2.7 Hz,
9.0 Hz), 7.2 (m, 2H, ArH, J = 2.7 Hz, 9.0 Hz), 7.3 (m, 1H,
ArH, J = 0.5 Hz, 8.5 Hz), 7.8 (m, 1H, ArH, J = 2.4 Hz,
8.5 Hz), 8.2 (m, 1H, ArH, J = 0.5 Hz, 2.4 Hz), 8.5 (br, 1H,
NH); MS: 502.3 (M+Na+); C18H14IN3O3S, calcd: C,
45.11; H, 2.94; N, 8.77. Found: C, 45.15; H, 2.85; N,
8. Marhefka, C. A.; Moore, B. M., II; Bishop, T. C.;
Kirkovsky, L. I.; Mukherjee, A.; Dalton, J. T.; Miller, D.
D. J. Med. Chem. 2001, 44, 1729.
25
8.69; ½aꢁD +65.8 (c 1.0, MeOH).