Journal of Medicinal Chemistry
Article
130.3, 127.2, 117.6, 115.5, 106.9, 106.1, 98.9, 55.6, 51.9. The methyl
ester was then hydrolyzed to give 2d as a yellow solid (79% yield). 1H
NMR (DMSO, 500 MHz): δ = 12.91 (bs, 1H), 9.95 (s, 1H), 8.05
(s, 1H), 7.90 (d, J = 8.8 Hz, 1H), 7.82 (d, J = 8.1 Hz, 1H), 7.71 (d, J =
8.1 Hz, 1H), 7.59 (t, J = 8.1 Hz, 1H), 6.81 (d, J = 2.0 Hz, 1H), 6.52
(dd, J = 8.8, 2.0 Hz, 1H), 3.76 (s, 3H). 13C NMR (DMSO, 125 MHz):
δ = 169.3, 163.8, 148.7, 147.0, 142.3, 134.0, 130.7, 126.4, 116.7, 113.9,
107.4, 106.0, 99.2, 55.3. HRMS (ES) calcd for C14H14N2O5, 287.0668
(M − H−); found, 287.0656 (M − H−).
of CRPC, but it has to be coadministered with a glucocorticoid
to prevent the buildup of the potent mineralocorticoid,
desoxycorticosterone.6−8 Chronic use of glucocorticoids can
cause Cushing’s syndrome, immunosuppression, and diabetes.
Because AKR1C3 acts further downstream in the androgen
biosynthetic pathway, coadministration of AKR1C3 inhibitors
with a glucocorticoid is not necessary.
Taken together, we have systematically developed potent and
selective AKR1C3 inhibitors that are efficacious in a prostate
cancer model and are potential therapeutic agents for the
treatment of CRPC.
5-Acetyl-2-((4-(trifluoromethyl)phenyl)amino)benzoic Acid (3n).
As in 1c, methyl 5-acetyl-2-[[(trifluoromethyl)sulfonyl]oxy]benzoate
and 4-aminobenzotrifluoride were reacted to yield the methyl ester of
1
3n as a yellow solid (89% yield). H NMR (CDCl3, 500 MHz): δ =
10.13 (s, 1H), 8.64 (d, J = 2.2 Hz, 1H), 7.97 (dd, J = 8.9, 2.2 Hz, 1H),
7.63 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 8.6 Hz, 2H), 7.31 (d, J = 8.9 Hz,
1H), 3.97 (s, 3H), 2.57 (s, 3H). 13C NMR (CDCl3, 125 MHz): δ =
195.8, 168.7, 150.3, 142.9, 134.3, 133.7, 127.8, 127.0, 122.3, 113.8,
112.0, 52.5, 26.3. Hydrolysis of the ester gave 3n as a white solid (80%
EXPERIMENTAL SECTION
■
Chemistry. Solvents used for extraction and purification were
HPLC grade from Fisher. Unless otherwise indicated, all reactions
were run under an inert atmosphere of argon. Anhydrous toluene was
obtained via distillation from the appropriate drying agents.
Deuterated solvents were obtained from Cambridge Isotope
laboratories. Merck precoated silica gel plates (250 μm, 60 F254)
were used for analytical TLC. Spots were visualized using 254 nm
ultraviolet light, with either anisaldehyde or potassium permanganate
stains as visualizing agents. Chromatographic purifications were per-
formed on Sorbent Technologies silica gel (particle size 32−63 μm).
1H and 13C NMR spectra were recorded at 500 MHz, 360 and
125 MHz, and 90 MHz, respectively, in CDCl3 or (CD3)2SO on a Bruker
AM-500, DRX-500, or DRX-360 spectrometer. Chemical shifts are
reported relative to deuterated or 13C labeled chloroform (δ 7.27 for
1H, δ 77.23 for 13C) or deuterated or 13C labeled dimethyl sulfoxide
((δ 2.50 for 1H, δ 39.53 for 13C) as internal standards. High resolution
mass spectra were obtained by Dr. Rakesh Kohli at the University of
Pennsylvania Mass Spectrometry Service Center on an Autospec high
resolution double-focusing electrospray ionization/chemical ionization
spectrometer equipped with either DEC 11/73 or OPUS software data
system.
1
yield). H NMR (DMSO, 360 MHz): δ = 10.27 (s, 1H), 8.51 (d, J =
2.2 Hz, 1H), 7.99 (dd, J = 8.9, 2.2 Hz, 1H), 7.70 (d, J = 8.5 Hz, 2H),
7.49 (d, J = 8.3 Hz, 2H), 7.39 (d, J = 8.9 Hz, 1H). 13C NMR (DMSO,
90 MHz): δ = 195.3, 169.3, 148.9, 143.3, 134.0, 133.0, 127.4, 126.8,
126.7, 121.3, 114.1, 113.1, 26.2. HRMS (ES) calcd for C16H12F3NO3,
322.0691 (M − H−); found, 322.0677 (M − H−).
3-((3,5-Bis(trifluoromethyl)phenyl)amino)benzoic Acid (4zi). As in
1c, methyl 3-bromobenzoate and 3,5-bis(trifluoromethyl)aniline were
reacted to give the methyl ester of 4zi as a white solid (51% yield). 1H
NMR (CDCl3, 500 MHz): δ = 7.80 (s, 1H), 7.77 (d, J = 7.7 Hz, 1H),
7.45 (t, J = 7.8 Hz, 1H), 7.41 (s, 2H), 7.34−7.39 (m, 2H), 6.21 (bs,
1H), 3.94 (s, 3H). 13C NMR (CDCl3, 125 MHz): δ = 166.9, 144.9,
141.1, 133.2, 132.9, 132.1, 130.1, 124.8, 124.1, 121.2, 116.0, 114.0,
52.6. Hydrolysis of the ester gave 4ziii as a brown solid (75% yield).
1H NMR (DMSO, 500 MHz): δ = 9.13 (s, 1H), 7.72 (s, 1H), 7.59 (d,
J = 7.5 Hz, 1H), 7.52 (s, 2H), 7.47 (t, J = 7.7 Hz, 1H), 7.43 (m, 1H),
7.38 (s, 1H). 13C NMR (DMSO, 125 MHz): δ = 167.0, 145.6, 141.2,
132.4, 131.2, 129.9, 124.4, 123.2, 122.9, 122.3, 119.5, 114.7. HRMS
(ES) calcd for C15H9F6NO2, 348.0459 (M − H−); found, 348.0449
(M − H−).
Compound Characterization. All compounds were obtained in
milligram quantities. Details of the synthesis and characterization of a
compound from each class (1−5) is described below. Complete details
of the synthesis and physicochemical characterization of all
compounds are included in the Supporting Information. Structures
4-((3-Nitrophenyl)amino)benzoic Acid (5d). As in 1c, methyl 4-
bromobenzoate and 3-nitroaniline were reacted to give the methyl
1
ester of 5d as a yellow solid (57% yield). H NMR (DMSO, 500
of all reported compounds were confirmed by H and 13C NMR and
MHz): δ = 9.22 (s, 1H), 7.92 (t, J = 2.1 Hz, 1H), 7.88 (d, J = 8.8 Hz,
2H), 7.74 (m, 1H), 7.54−7.63 (m, 2H), 7.19 (d, J = 8.8 Hz, 2H), 3.81
(s, 3H). 13C NMR (DMSO, 125 MHz): δ = 165.9, 148.7, 146.8, 143.1,
131.1, 130.7, 123.9, 121.0, 115.6, 115.3, 111.7, 51.7. Hydrolysis of the
1
their mass confirmed by HRMS. Purity was determined by HPLC
analysis using a Waters 2690 Alliance system with an inline
photodiode array detector. All reported compounds had ≥95% purity
with the exception of 5l (94%).
1
ester gave 5d as a yellow solid (79% yield). H NMR (DMSO, 500
2-((2-Methoxyphenyl)amino)benzoic Acid (1c). Reaction of
methyl 2-(trifluoromethanesulfonyloxy)benzoate with o-anisidine
according to step A above provided the methyl ester of 1c as a
yellow oil (99% yield). 1H NMR (CDCl3, 500 MHz): δ = 9.44 (s, 1H),
7.97 (d, J = 8.0 Hz, 1H), 7.44 (d, J = 7.8 Hz, 1H), 7.30−7.36 (m, 2H),
7.05 (m, 1H), 6.92−7.00 (m, 2H), 6.75 (m, 1H), 3.92 (s, 3H), 3.90
(s, 3H). 13C NMR (CDCl3, 125 MHz): δ = 169.0, 151.8, 147.5, 134.1,
131.9, 130.4, 123.5, 120.9, 120.7, 117.4, 114.6, 113.0, 111.6, 56.0, 52.0.
Hydrolysis of the ester according to step B provided 1c as a white solid
MHz): δ = 9.19 (s, 1H), 7.91 (s, 1H), 7.87 (d, J = 8.5 Hz, 2H), 7.72
(m, 1H), 7.50−7.63 (m, 2H), 7.18 (d, J = 8.5 Hz, 2H). 13C NMR
(DMSO, 125 MHz): δ = 167.0, 148.7, 146.3, 143.4, 131.2, 130.7,
123.6, 122.5, 115.7, 115.1, 111.4. HRMS (ES) calcd for C13H10N2O4,
257.0562 (M − H−); found, 257.0567 (M − H−).
Enzyme Purification. Homogenous recombinant enzymes
AKR1C1−4, AKR1B1, and AKR1B10 were prepared and purified as
previously described.14,16,48 COX-1 was purified to homogeneity from
ram seminal vesicles, and COX-2 was purified from baculovirus-
infected SF-21 cells as described previously.49,50 All AKR enzymes
were stored at −80 °C in 20 mM potassium phosphate buffer pH
7.0 containing 30% glycerol, 1 mM EDTA, and 1 mM β-mercaptoethanol.
Enzyme Activity Assays. AKR1C Isoforms. The potency of the
compounds was determined by measuring their ability to inhibit the
NADP+ dependent oxidation of S-(+)-1,2,3,4-tetrahydro-1-naphthol
(S-tetralol) catalyzed by AKR1C isoforms. The continuous assay was
conducted using a 96-well plate format and the reaction measured the
appearance of NADPH fluorimetrically (Exc/Emi, 340/460 nm) on
a BIOTEK Synergy 2 Multimode plate reader at 37 °C. The assay
mixture consisted of S-tetralol (in DMSO), inhibitor (in DMSO),
100 mM phosphate buffer, pH 7.0, 200 μM NADP+, and purified
recombinant enzyme to give a total volume of 200 μL and 4% DMSO.
The concentration of S-tetralol used in the assays for each AKR1C
isoform was equal to the KM value for the respective enzyme. This
enabled direct comparison of the IC50 values of the compounds on each
1
(53% yield). H NMR (DMSO, 500 MHz): δ = 12.93 (bs, 1H), 9.57
(s, 1H), 7.90 (m, 1H), 7.31−7.42 (m, 2H), 7.19 (d, J = 8.4 Hz, 1H),
7.00−7.11 (m, 2H), 6.94 (m, 1H), 6.76 (m, 1H), 3.82 (s, 3H). 13C
NMR (DMSO, 125 MHz): δ = 169.2, 150.5, 146.3, 133.6, 131.3,
128.9, 122.9, 120.1, 119.8, 116.7, 113.1, 112.4, 111.4, 55.2. HRMS
(ES) calcd for C14H13NO3, 242.0817 (M − H−); found, 242.0812
(M − H−).
4-Methoxy-2-((3-nitrophenyl)amino)benzoic Acid (2d). As in 1c,
methyl 4-methoxy-2-[[(trifluoromethyl)sulfonyl]oxy]benzoate and 3-
nitroaniline were reacted to give the methyl ester of 2d as a yellow oil
1
(93% yield). H NMR (CDCl3, 500 MHz): δ = 9.87 (s, 1H), 8.16
(s, 1H), 7.96 (d, J = 8.9 Hz, 1H), 7.88 (d, J = 8.0 Hz, 1H), 7.53 (d, J =
8.0 Hz, 1H), 7.48 (t, J = 8.0 Hz, 1H), 6.80 (d, J = 2.4 Hz, 1H), 6.43
(dd, J = 8.9, 2.4 Hz, 1H), 3.90 (s, 3H), 3.80 (s, 3H). 13C NMR
(CDCl3, 125 MHz): δ = 168.8, 164.7, 149.5, 148.1, 142.6, 134.0,
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dx.doi.org/10.1021/jm201547v | J. Med. Chem. 2012, 55, 2311−2323