Development of potent and selective indomethacin analogues for the inhibition of AKR1C3 (type 5 17β-hydroxysteroid dehydrogenase/prostaglandin F synthase) in castrate-resistant prostate cancer

Article

pubs.acs.org/jmc

Development of Potent and Selective Indomethacin Analogues for

the Inhibition of AKR1C3 (Type 5 17β-Hydroxysteroid

Dehydrogenase/Prostaglandin F Synthase) in Castrate-Resistant

Prostate Cancer

Andy J. Liedtke,^†Adegoke O. Adeniji,^‡Mo Chen,^‡Michael C. Byrns,^‡Yi Jin,^‡David W. Christianson,^§

Lawrence J. Marnett,^†and Trevor M. Penning*^,‡

^†Departments of Biochemistry, Chemistry, and Pharmacology, Vanderbilt Institute of Chemical Biology, Center in Molecular

Toxicology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, United

States

^‡Department of Pharmacology and Center of Excellence in Environmental Toxicology, Perelman School of Medicine, University of

Pennsylvania, 1315 BRB II/III, 420 Curie Boulevard, Philadelphia, Pennsylvania 19104-6061, United States

^§Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, Pennsylvania 19104-6323, United States

S

* Supporting Information

ABSTRACT: Castrate-resistant prostate cancer (CRPC) is a

fatal, metastatic form of prostate cancer. CRPC is characterized

by reactivation of the androgen axis due to changes in

androgen receptor signaling and/or adaptive intratumoral

androgen biosynthesis. AKR1C3 is upregulated in CRPC

where it catalyzes the formation of potent androgens. This

makes AKR1C3 a target for the treatment of CRPC. AKR1C3

inhibitors should not inhibit AKR1C1/AKR1C2, which

inactivate 5α-dihydrotestosterone. Indomethacin, used to

inhibit cyclooxygenase, also inhibits AKR1C3 and displays

selectivity over AKR1C1/AKR1C2. Parallel synthetic strategies

were used to generate libraries of indomethacin analogues,

which exhibit reduced cyclooxygenase inhibitory activity but retain AKR1C3 inhibitory potency and selectivity. The lead

compounds inhibited AKR1C3 with nanomolar potency, displayed >100-fold selectivity over AKR1C1/AKR1C2, and blocked

testosterone formation in LNCaP-AKR1C3 cells. The AKR1C3·NADP⁺·2′-des-methyl-indomethacin crystal structure was

determined, and it revealed a unique inhibitor binding mode. The compounds reported are promising agents for the

development of therapeutics for CRPC.

subsequent approval by the FDA support the involvement of

adaptive androgen biosynthesis within the tumor in CRPC.⁷⁻¹⁰

However, abiraterone blocks glucocorticoid (cortisol) biosyn-

thesis. Abiraterone therefore needs to be coadministered with

the glucocorticoid, prednisone to limit adverse eﬀects. Since

AKR1C3 acts further downstream of CYP17, it does not play a

role in glucocorticoid biosynthesis, which in addition to its

intratumoral localization makes it an appealing therapeutic

target in CRPC.

AKR1C3 through its prostaglandin (PG) F_2αsynthase

activity also redirects the metabolism of PGD₂into 11β-

PGF_2αand can suppress the activation of PPARγ by preventing

the formation of putative PGJ₂ligands, which would favor

diﬀerentiation.¹¹⁻¹⁵Thus, AKR1C3 inhibition might be

INTRODUCTION

■

Aldo-keto reductase 1C3, also known as type 5 17β-

hydroxysteroid dehydrogenase, is a critical enzyme in androgen

biosynthesis within the prostate and has been implicated in the

development of castrate-resistant prostate cancer (CRPC).

CRPC is characterized by the intratumoral reactivation of

androgen receptor signaling caused by adaptive intratumoral

androgen biosynthesis and/or androgen receptor (AR)

ampliﬁcation leading to resistance to androgen deprivation

therapy.¹⁻³Elevated androgen levels within the tumor occur

due to the upregulation of key enzymes involved in androgen

biosynthesis.⁴⁻⁶AKR1C3 catalyzes the conversion of the weak

androgen precursors 4-androstene-3,17-dione (Δ⁴-AD) and 5α-

androstane-3,17-dione to the potent androgens testosterone

and 5α-dihydrotestosterone (DHT), respectively, and is one of

the most highly upregulated genes in CRPC, Figure 1.^4,5

The success in phase III clinical trials of abiraterone acetate

(Zytiga), a 17α-hydroxylase/17,20-lyase inhibitor, and its

Received: December 3, 2012

Published: February 22, 2013

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Figure 1. Representative reactions catalyzed by AKR1C3. AR = androgen receptor, FP = F prostanoid receptor, and PG = prostaglandin.

beneﬁcial in either hormone-dependent or hormone-independ-

ent prostate cancer.

dependent processes in human cell lines and murine

xenografts.^14,30However, the potential therapeutic usefulness

of indomethacin in the context of CRPC is limited because of

the undesired side eﬀects associated with chronic COX

inhibition. Despite this, indomethacin represents a vital lead

compound that can be used to develop selective AKR1C3

inhibitors that are stripped of COX inhibitory activity.

AKR1C1 and AKR1C2 are isoforms closely related to

AKR1C3 and are required to inactivate DHT in the

prostate.¹⁶⁻¹⁹It is therefore imperative that AKR1C3 be

inhibited selectively. Several selective AKR1C3 inhibitors have

recently been reported. These compounds include 3-

(phenylamino)benzoic acids,²⁰⁻²²baccharin,²³3-(3,4-dihydroi-

soquinolin-2(1H)-ylsulfonyl)benzoic acids,²⁴N-benzoylanthra-

nilic acids,²⁵and a thiazolidinedione²⁶that was discovered by

virtual screening (Figure 2).

Extensive SAR studies on indomethacin (Figure 3) have

shown that the p-chlorobenzoyl group, the 2′-methyl group,

Nonsteroidal anti-inﬂammatory drugs (NSAIDs) are potent

inhibitors of the AKR1C enzymes at therapeutically relevant

concentrations.^12,27−29In contrast to most NSAIDs, indome-

thacin inhibits AKR1C3 with little or no eﬀect on AKR1C1 and

AKR1C2. Indomethacin has been shown to inhibit AKR1C3-

Figure 3. Structure of indomethacin (1) is shown. Groups shown in

red are essential for COX inhibition and were modiﬁed to yield class I

analogues; class II compounds are 2′-des-methyl-indomethacin

analogues; and class III compounds are 3′-alkyl analogues.

and the 3′-acetic acid side chain are essential for COX

inhibition. Previous eﬀorts in our lab led to the discovery of N-

(4-chlorobenzoyl)-melatonin, which inhibited AKR1C3 with

potency comparable to that seen with indomethacin; moreover,

it was selective over AKR1C1 and AKR1C2, and it did not

inhibit COX.^31,32However, the therapeutic usefulness of this

Figure 2. Structures of other known selective AKR1C3 inhibitors.

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compound is limited due to its poor solubility and

bioavailability.

aliphatic acid group) as well as compounds 44 and 62 (Table 3;

class III reversed 2′-aliphatic acid/3′-alkyl or 2′-/3′-cyclized

acid substituents). For technical details, please refer to the cited

literature. Moreover, the procedure enabled simple scale-up

from milligram to multigram quantities (e.g., for lead

compounds 20 and 50), which is desirable for testing eﬃcacy

in murine xenograft models of CRPC.³⁰

In this study, we report the synthesis and structure−activity

relationship studies of three series of indomethacin analogues.

These compounds have been classiﬁed based on the presence

and position of the 2′-alkyl group and the 3′-aliphatic acid

group on the indole ring.³³Class I analogues have the 2′-alkyl,

3′-aliphatic acid arrangement on the indole ring of

indomethacin preserved, class II compounds are based on 2′-

des-methyl-indomethacin and retain the 3′-aliphatic acid group

on the indole ring but lack the 2′-methyl group, and class III

compounds have this arrangement reversed to give an aliphatic

acid group on the 2′-position and an alkyl group on the 3′-

positon of the indole ring (Figure 3).

This study led to the discovery of potent and selective

AKR1C3 inhibitors that are devoid of inhibitory activity against

AKR1C1, AKR1C2, and COX. Additionally, these compounds

block androgen synthesis in a prostate cancer cell line that

overexpresses AKR1C3 and have no agonist eﬀect on the AR.

The crystal structure of AKR1C3 in complex with 2′-des-

methyl-indomethacin is also reported, showing that this class of

compounds occupies a unique binding pose in the enzyme

active site.

Indomethacin analogues with conserved 5′-methoxy groups

and 4-chlorobenzoyl tails at their indole N¹were also

synthesized following the conventional reaction scheme,

Scheme 2, which was primarily adopted by Yamamoto³⁵for

assembly of 1. The key reagent for the underlying Fischer

indolization step, 4-chloro-N-(4-methoxyphenyl)-

benzohydrazide hydrochloride (D), was prepared according

to the reported literature procedures from the aryl hydrazine

precursor A via intermediates B and C.³⁶Target compounds 16

(Table 1; 3′-propionic acid group) and 20 (Table 2, 2′-des-

methyl-indomethacin) were readily obtained from D by

reﬂuxing with slight excess of 5-oxohexanoic acid E (here, R¹

= Me, n = 2) and 4-oxobutanoic acid E (R¹= H, n = 1) in

AcOH, respectively. Application of 4-oxohexanoic acid F (R¹

=Me, n = 1) as cyclization reagent quantitatively yielded the

reversed 2′-propionic acid/3′-alkyl indole derivative 44 as the

only isolable product.³⁷The purity of compounds 16, 20, and

44 after a simple ﬁltration and elutriation with water was 99%

based on LC−MS analysis. In contrast, reaction of D with

either 5-oxoheptanoic acid F (R¹= Me, n = 2) or 4-

oxoheptanoic acid F (R¹= Et, n = 1) in a trial to further extend

the aliphatic acid chain in position 2′ (for 45) or the 3′-alkyl

substituent (for 46) of the indole, led to isomer mixtures

containing only about 75% of the envisaged target structure (by

¹H NMR; compare Supporting Information). Mixtures of 45

and 46 of known composition were evaluated in the

biochemical screens as such. We later successfully isolated the

major indole regioisomer 47 (2′-propionic acid/3′-ethyl) from

mixture 46 with an acceptable sample/isomeric purity of 95%

by repeated trituration of the crude product precipitate with

diethyl ether.

RESULTS

■

Chemistry. Our search for novel AKR1C3 inhibitors was

aided by the availability of high-throughput chemistry and

analysis technologies. All three classes of compounds were

prepared using parallel synthesis approaches based on well-

established Fischer−indole chemistry. We reported previously

an eﬃcient three-step reaction sequence that is amenable to

rapid and high-yielding analogue production (outlined in

Scheme 1).³⁴The optimized route used indole alkanoic acid

Scheme 1. Optimized Fischer Indolization/N-Acylation/

Ester Cleavage Approach for Expedient Production of

Diﬀerent N-Acyl Indole Alkanoic Acid Analogues³⁴

Analogues 48 and 49 structurally derive from compound 44

and bear an additional α,α-dimethyl (48) or α-methyl group

(49) in their aliphatic acid chain. Their synthesis could be

accomplished by converting D with appropriately prebuilt keto

acids. However, these cyclization reagents, namely, 2,2-

dimethyl-4-oxohexanoic acid and 2-methyl-4-oxohexanoic

acid, were not commercially available and needed to be

prepared beforehand following existing protocols (for details,

see Supporting Information).^37,38Whereas analogue 49 could

be accumulated as solid and ﬁltered pure from the watery

reaction solution, compound 48 needed to be extracted into

dichloromethane and puriﬁed on silica gel. The purities of 48

(after ﬂash chromatography) and 49 (dried ﬁlter residue) were

judged to be 95% and >97%, respectively.

In compounds 61 and 64, the 2′- and 3′-positions are

bridged by a fused six-membered aliphatic ring; the two

structures only vary in the position of the carboxylic acid

functionality attached to the cyclohexane ring. Although

compounds 61 and 64 formally represent 2,3,4,9-tetrahydro-

1H-carbazole-carboxylic acid derivatives, they were considered

possible AKR1C3 inhibitor candidates as well. The synthesis

followed the above-mentioned universal procedure in Scheme

2, except that diﬀerent cyclic keto acids G (for 61, p-keto; for

64, m-keto) were used for the ring-closure with the hydrazine

hydrochloride precursor D (Scheme 2). Both the symmetrical

methyl- or allyl esters as intermediates, featured maximum

synthetical ﬂexibility (e.g., variation of the indole 5′-substituents

or late-stage N-acylation), and was more sustainable than the

conventional pathway shown in Scheme 2 for quite a few of the

envisioned analogues, including 2′-des-methyl-indomethacin

(20). Compounds are characterized by either a 5′-methoxy or

5′-ﬂouro group on the indole ring, alternating 2′-/3′-(cyclo)-

aliphatic acid substituents and variable N¹-acyl moieties.

Utilization of Fischer indolization/N-acylation/(ester cleavage)

with microwave heating led to assembly of novel N-acyl indole

alkanoic acids or esters 2−13, 17, and 19 (Table 1; class I

analogues with 2′-alkyl and 3′-aliphatic acid groups), the 2′-des-

methyl derivatives 20−25, 42 and 43 (Table 2; class II 3′-

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Scheme 2. Classical Synthetic Strategy for the Preparation of N-(4-Chlorobenzoyl) Indole Alkanoic Acid Analogues and

a

Sulfonamide Derivatives as COOH Bioisosteres

a

Reagents and conditions: (i) 1. Et₃N, 2. CH₃CHO, toluene, 0 to 25 °C, 3 h; (ii) 4-chloro-benzoyl chloride, pyridine, 10 to 25 °C, 2 h; (iii) HCl (g),

CH₂Cl₂, 0°C to rt, 1 h; (iv) (cyclo)aliphatic keto acid, AcOH, 70-80 °C, 3 h; (v) 1,1′-carbonyldiimidazole (CDI), CH₂Cl₂, 0-5 °C, 2 h, then alkyl-/

arylsulfonamide, diazabicyclo[5.4.0]undec-7-ene (DBU), rt, 4-6 h; (vi) 1. oxalyl chloride, CH₂Cl₂, 0 °C to rt, 8 h, 2. alkyl-/arylsulfonamide, 1,2-DCE

or CH₂Cl₂, pyridine, rt, overnight.

4-oxocyclohexanecarboxylic acid and, more surprisingly, the

unsymmetrical 3-oxocyclohexanecarboxylic acid yielded a single

and clean reaction product (ﬁlterable precipitate) in high yield

(>82%; purity ∼99% by LC−MS).³⁹

as acid chlorides (to improve the eﬃciency of the reaction, e.g.,

for 15 and 27) prior to sulfonylamine addition.⁴⁰⁻⁴²After

acidic workup (to minimize amide hydrolysis) and evaporation

of the organic solvents, the residues were combined with

DMSO (1 mL each) and subjected to automated mass-directed

HPLC puriﬁcation with a set purity threshold of ≥95%.

The chemical structures of all intermediates and target

compounds were conﬁrmed and fully characterized by NMR

spectroscopy and mass spectrometry. The purity of the test

compounds was determined using liquid chromatography [UV

detection at 215 and 254 nm along with ELSD detection] and

was ≥95%, if not otherwise denoted (compare Experimental

Section and/or Supporting Information).

Strategy. The synthesized compounds were compared for

their ability to inhibit AKR1C3 over AKR1C2. AKR1C2 was

used in this comparison due to its similarity in amino acid

sequence and structure and because its inhibition is undesirable.

The ratio of the IC₅₀values for the two enzymes was computed

to generate a selectivity ratio. The higher the ratio, the more

selective the compound is in its ability to inhibit AKR1C3.

Class I Analogues. These compounds retain the 2′-alkyl,

3′-aliphatic acid substitution on the indole ring of indomethacin

and possess one or more of the following modiﬁcations:

substitution on the p-chlorobenzoyl group, replacement of the

5′-OCH₃group, esteriﬁcation/amidation of the free acid, and

modiﬁcation of the acetic acid side chain. Results from the

In order to enhance the desired biological (and physical)

properties of the compounds, (bio)isosteric replacements of the

free carboxylic acid functions were accomplished on

indomethacin (1), 2′-des-methyl-indomethacin (20), the 2′-

propionic acid/3′-methyl indole derivatives 44 and 51, and the

carbazole carboxylic acid analogue 61. Diﬀerent isostere

options were considered, and ﬁnally, the sulfonamide group

was chosen as it retained the essential acidic character of the

former COOH group. Moreover, this moiety facilitated the

introduction of further (sterically demanding) substituents at

the sulfone tail end, which might in turn aﬀect the size, shape,

conformation, inductive and mesomeric eﬀects, polarizability,

H-bond-formation capacity, lipophilicity, or pK_aof the new

analogues. A library of initially 30 sulfonamide derivatives was

generated as speciﬁed in Scheme 2 (right-hand side) applying

state-of-the-art methods of combinatorial chemistry: entries 14

and 15 (Table 1) represent class I analogues, entries 26−41

(Table 2) belong to class II, and ﬁnally compounds 50, 51, 52−

60, and 63 (Table 3) are members of structural class III (see

above). For the standard (one-pot) two-step amide coupling

reactions, the acid groups of starting compounds 1, 20, 44, 51,

and 61 were activated with carbonyldiimidazole or infrequently

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Table 1. Inhibitory Properties of Indomethacin Analogues on AKR1C3 and AKR1C2

AKR1C3/AKR1C2 screen of these compounds are shown in

Table 1. Indomethacin is a potent and selective inhibitor of

AKR1C3. It inhibited AKR1C3 with an IC₅₀value of 100 nM

and was over 300-fold selective over AKR1C2. To evaluate the

importance of substitution on the phenyl ring, the p-Cl

substituent of indomethacin was replaced with substituents of

varying electronic properties and lipophilicity. Introduction of

p-CH₃(2), p-CH₂Cl (3), or p-OCH₃(4) on the phenyl ring

did not signiﬁcantly alter either AKR1C3 potency or selectivity.

These compounds displayed IC₅₀values between 120 and 160

nM for AKR1C3 and were over 300-fold selective for AKR1C3

over AKR1C2. However, when the p-Cl group was substituted

with a p-CF₃group (5), the inhibitor lost both potency and

selectivity and was only 130-fold selective for AKR1C3. The

reduction in potency and selectivity was even more pronounced

with a smaller p-F (7) substitution and was reduced further

when the CF₃group was moved to the meta-position (6,

AKR1C3-IC₅₀> 2 μM). Removal of the p-chlorobenzoyl group

altogether to yield des-chlorobenzoyl-indomethacin 18 (a major

metabolite and decomposition product of indomethacin)

showed that inhibition potency and selectivity was completely

eliminated. These ﬁndings underscored the need for the N-

benzoyl ring as well as a suitable substitution at the para-

position of the phenyl ring for AKR1C3 inhibition.

Interchanging the 5′-OCH₃groups in compounds 1 and 6

with ﬂuorines at the same indole position in analogues 8 and 9

was detrimental to AKR1C3 selectivity as AKR1C3 inhibition

was mostly unchanged, while undesired AKR1C2 inhibition

was enhanced.

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Table 2. Inhibitory Properties of 2′-des-Methyl Indomethacin Analogues on AKR1C3 and AKR1C2

The free carboxylic acid, which exists predominantly as the

negatively charged carboxylate at physiological pH, is thought

to contribute to the general inhibition of AKR1C enzymes by

nonsteroidal anti-inﬂammatory drugs (NSAIDs). Consistent

with this, esteriﬁcation of indomethacin to give the methyl ester

(10) led to a 57-fold loss of inhibitory potency for AKR1C3

and a reduction in selectivity. This trend was also seen with all

other methyl esters 11−13 when compared to their

corresponding free acid derivatives (2, 4, and 5). Despite this

loss of potency, the rank order of inhibitory potency for the

methyl esters was similar to the free acids. The requirement for

an acid functionality for optimal AKR1C3 inhibition was

likewise apparent with compound 19. This indomethacin

fragment lacks the complete carboxyalkyl chain in position 3′ of

the indole and revealed a 36-fold lower inhibitory activity on

the target enzyme in comparison to 1. However, the

interactions of 19 and 1 with AKR1C2 were still comparable,

clearly indicating diﬀerences in the way the compounds bind to

AKR1C3 and AKR1C2. The AKR1C inhibition data for

compound 18 reconﬁrm the need for an intact N-acyl (here,

p-Cl-benzoyl) substituent. The decrease in inhibitor potency

(and selectivity) observed for AKR1C3 appeared to be less with

19 than with 18 (see above).

Further modiﬁcation of the acetic acid group of indometha-

cin yielded the N-(sulfonyl)acetamide analogues 14 and 15.

While the N-(methylsulfonyl)acetamide analogue, 14, led to a

22-fold reduction in inhibitory potency for AKR1C3 (IC₅₀

value for AKR1C3 = 2.24 μM), potency was partially restored

by the formation of the N-(triﬂuoromethylsulfonyl)acetamide

analogue 15, a compound with an IC₅₀value of 0.74 μM and

108-fold selectivity for AKR1C3.

Increasing the length of the aliphatic side chain of

indomethacin from -ethyl to -propyl (16) led to a 2-fold

reduction in AKR1C3 potency, but the compound retained

257-fold selectivity for AKR1C3 over AKR1C2 (Table 1).

Replacement of the 5′-OCH₃group of this compound with a

ﬂuorine atom (17) did not change the AKR1C3 inhibitory

potency, but this compound was a more potent AKR1C2

inhibitor than 16 leading to a loss of AKR1C3 selectivity.

Class II: 2′-des-Methyl-indomethacin Analogues.

Structurally, these compounds have a 3′aliphatic acid on the

indole ring, but the 2′-methyl group on the indole ring has been

eliminated. Compared to indomethacin, the parent compound

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Table 3. Inhibitory Properties of 3′-Alkyl Indomethacin Analogues on AKR1C3 and AKR1C2

in this class, 2′-des-methyl-indomethacin (20), was a less potent

and less selective AKR1C3 inhibitor (Table 2). It displayed an

IC₅₀value of 960 nM for AKR1C3 and an IC₅₀value of 100 μM

for AKR1C2, which translated to 100-fold selectivity for

AKR1C3. The loss in AKR1C3 potency with removal of the

2′-CH₃group was seen across most of the des-methyl-

indomethacin analogues when compared to their respective

class I analogues (e.g., 21 vs 10 and 23 vs 5). Within the triad

of class II compounds 22−24 with alternating substituents at

the benzoyl moiety, the potency and selectivity decreased in the

same rank order as discovered for the indomethacin counter-

parts 5−7 in Table 1 from p-CF₃> p-F > m-CF₃. One

exception with quite promising inhibitory qualities was the N-

(triﬂuoromethylsulfonyl)acetamide derivative, 27, which gave

an IC₅₀value of 210 nM for AKR1C3 and was 240-fold

selective for AKR1C3 over AKR1C2, while the corresponding

indomethacin analogue 15 was less potent and less selective.

The presence of a triﬂuoromethyl group on the acetamide

seems critical for AKR1C3 inhibition since the N-

(methylsulfonyl)acetamide analogue of des-methyl-indometha-

cin (26) displayed weak AKR1C3 inhibition and a total loss of

selectivity for AKR1C3. Further substitution on the N-

(sulfonyl)acetamide group with substituted phenyl groups or

heterocycles (28−41) did not signiﬁcantly improve AKR1C3

potency or selectivity, although analogues with bulky aromatic

rings and/or aromatic rings with substituents capable of

forming electrostatic interactions generally gave better

AKR1C3 inhibitors. Introduction of a 5′-F on the indole ring

of des-methyl-indomethacin to give 25 led to little or no

improvement in AKR1C3 potency and a 4-fold increase in

AKR1C2 inhibitory potency. This translated to a reduction in

AKR1C3 selectivity.

Finally, compounds 42 and 43 display structurally edited

versions of the parent molecules, 2′-des-methyl indomethacin

20 and its methyl ester 21, respectively, whose terminal aryl

rings of the original N-4-Cl-benzoyl substituents were truncated

leaving a linear propanoyl group in each case. Both derivatives

42 and 43 were about 2- to 2.5-fold less potent than their N-

benzoyl analogues 20 and 21 in inhibiting AKR1C3. The

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methyl ester 43 again was clearly inferior with respect to

AKR1C3 inhibition and selectivity.

potency and selectivity over the primary lead compound 44

within this series (IC₅₀= 340 nM).

Class III: 3′-Alkyl Indomethacin Analogues. The

compounds in this class have the acetic acid side chain of

indomethacin 1 replaced with a 3′-alkyl group and the 2′-CH₃

group substituted with an aliphatic acid or a substituted N-

(sulfonyl)acetamide, in eﬀect reversing the position of the

aliphatic acid and alkyl group on the indole ring of 1. Relative

to indomethacin and the class I inhibitor 16, compound 44,

with a 3′-CH₃group and a 2′-propionic acid substituent on the

indole ring, retained AKR1C3 potency with IC₅₀value of 130

nM (Table 3). This result indicated that AKR1C3 will tolerate

ﬂexibility in the position of the aliphatic acid side chain and

alkyl group on the indole ring. However, 44 was less selective

for AKR1C3 than both 1 and 16, being 111-fold selective for

AKR1C3 over AKR1C2. Introduction of an α-CH₃group on

the aliphatic acid side chain of 44 introduced a chiral center,

and the racemate of this compound, 49, displayed 2-fold lower

potency for AKR1C3 and an unchanged AKR1C2 potency

relative to 44. Removal of the chiral center by formation of the

geminal dimethyl analogue, 48, restored AKR1C3 potency.

Synthesis of the N-(methylsulfonyl)acetamide analogue, 50, led

to a 3-fold loss of AKR1C3 potency (340 nM) but an increase

in AKR1C3 selectivity (275-fold). However, its α-CH₃

analogue, 51, exhibited poor inhibition of AKR1C3. Replace-

ment of the methylsulfonyl group of 50 with a triﬂuorome-

thylsulfonyl group (52) also did not improve AKR1C3 potency

and selectivity in contrast to class I and II analogues, neither did

a series of further sulfonamides 53−60 with bulkier (hetero)-

aryl substituents attached to their sulfonyl tail end.

Inhibition of Other AKR1C Isoforms and COX

(Secondary Screens). Lead compounds were subsequently

counterscreened against other human AKR1C enzymes

(AKR1C1 and AKR1C4). A complete proﬁle of the inhibitory

properties of these compounds on all human AKR1C enzymes

is shown in Table 4. All the compounds tested had IC₅₀values

for AKR1C1 in the high micromolar range and showed

between 100- and 800-fold selectivity for AKR1C3 over

AKR1C1.

Table 4. Inhibitory Eﬀects of Compounds on Other AKR1C

Enzymes

IC₅₀values (μM)

a

compds

AKR1C3

AKR1C1

AKR1C2

AKR1C4

b

2

0.16

0.12

0.27

0.74

0.22

0.96

0.21

0.13

0.09

0.34

0.16

>100 (>625)

100 (833)

54.50 (336)

40.74 (329)

35.73 (134)

81.0 (108)

57.0 (257)

100 (100)

>100 (>625)

49.75 (415)

>100 (>370)

>100 (>135)

>100 (>455)

48.7 (357)

>100 (>478)

3.51 (27)

3

5

>100 (>370)

>100 (>135)

>100 (>455)

>100 (>100)

>100 (>478)

17.73 (136)

30.71 (341)

100 (296)

15

16

20

27

44

47

50

61

50.13 (240)

14.45 (111)

49.57 (538)

93.0 (275)

53.50 (331)

1.95 (22)

12.64 (37)

3.15 (20)

76.25 (477)

a

AKR1C3 and AKR1C2 data were taken from Tables 1−3 above.

Values in parentheses are IC₅₀ratios and represent inhibitor

b

The library also produced mixtures of 45 in which the 3′

substituent is methyl and the 2′ substituent is butyric acid or

the 3′ substituent is propionic acid and the 2′ substituent is a

propyl group. This mixture produced an IC₅₀value = 0.15 μM

for AKR1C3 and was 119-fold selective for the inhibition of

AKR1C3 over AKR1C2. Additionally, the library produced

mixtures of 46 in which either the 3′ substituent is ethyl and the

2′ substituent is propionic acid or the 3′ substituent is acetic

acid and the 2′ substituent is a propyl group. This mixture gave

an IC₅₀value of 70 nM for AKR1C3 that was 279-fold selective

for the inhibition of AKR1C3 over AKR1C2. The latter mixture

was then resolved and the active component identiﬁed as

compound 47.

Compound 47 was the most potent and selective compound

in the study, with an IC₅₀value of 90 nM for AKR1C3, and was

540-fold selective for AKR1C3 over AKR1C2. Cyclization of

the side chains at the 2′- and 3′-position on the indole ring of

47 to form the racemic tetrahydrocarbazole analogue with a 3′-

carboxylic acid group (61) did not produce a pronounced

change in either AKR1C3 potency or selectivity. This

compound gave an IC₅₀value of 160 nM for AKR1C3 and

showed a 330-fold selectivity for AKR1C3 over AKR1C2.

Changing the p-chloro substituent on the benzoyl ring of 61

with a m-CF₃group to give 62 fractionally improved the

selectivity to a factor of 360 by raising the IC₅₀value for

AKR1C2 inhibition more than the IC₅₀value for AKR1C3

inhibition. In contrast, replacement of the carboxylic acid

function of 61 by the isosteric N-(methylsulfonyl)acetamide

decreased the potency of 63 for AKR1C3 and drastically

reduced the selectivity to a factor of only 20. Compound 64, a

close analogue of the 2,3,4,9-tetrahydro-1H-carbazole-carbox-

ylic acid 61, but with the −COOH group in position 2 in the

six-membered cyclohexene ring, did not exhibit improved

selectivity for AKR1C3 over other AKR1C enzymes.

Also, all of the compounds tested had IC₅₀values between 50

to 100 μM for AKR1C4 and had over 130−600-fold selectivity

for AKR1C3 over AKR1C4 with the exception of the class III

compounds, 44, 47, 50, and 61, which showed less than 50-fold

selectivity.

Secondary screens also involved a screen against COX-1 and

COX-2 to reaﬃrm that compounds will not act as NSAIDs.

Initially we used a continuous colorimetric COX-1 assay that

monitored the oxidation of N,N,N,N-tetramethyl-1,4-phenyl-

enediamine (TMPD) when it is coupled to the formation of

PGH₂from PGG₂employing arachidonic acid (AA) as

substrate.

The activity of the lead compounds on oCOX-1 is shown in

Table 5. Indomethacin (1) potently inhibited COX-1 with an

IC₅₀of 20 nM. This value was consistent with reported

inhibitory potency of 1 for COX-1.⁴³All the other lead

compounds were at least 37-fold less potent than 1 on COX-1.

Compound 61 with IC₅₀value for COX-1 of 750 nM was the

Table 5. COX-1 Inhibitory Activity of Lead Compounds;

Data Derived from the Continuous Colorimetric Assay

AKR1C3 IC₅₀

COX 1 IC₅₀

COX 1 IC₅₀/

AKR1C3 IC₅₀

compd

(μM)

indomethacin

(1)

0.10

0.02

0.2

16

27

44

50

61

0.22

0.21

0.13

0.34

0.16

48.8

2.19

100

222

10

770

>294

5

>100

0.75

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Table 6. COX-1 and COX-2 Inhibitory Activities of Selected AKR1C3 Inhibitors; Data Derived from the Discontinuous

Radioactive TLC Assay

compd

oCOX 1 IC₅₀(μM)

0.05

wt mCOX 2 IC₅₀(μM)

0.20

COX 1 IC₅₀/COX 2 IC₅₀

indomethacin (1)

0.25

5.44

4.67

n/a

0.33

1.62

8

0.49

0.09

a

20

25

44

47

48

50

61

64

15.27 (P)

3.27 (P)

42.7% inhib. @ 25 μM

11% inhib. @ 25 μM

15.6% inhib. @ 25 μM (P)

24.2% inhib. @ 25 μM (P)

30.8% inhib. @ 25 μM (P)

1.0

35.6% inhib. @ 25 μM

17.6% inhib. @ 4 μM

7.0

33.7% inhib. @ 25 μM (P)

32.4% inhib. @ 25 μM (P)

3.02 (P @ 61.4%)

1.48 (P @ 62.6%)

2.4 (P @ 56.1%)

a

P = plateau; oCOX-1 = ovineCOX-1; mCOX-2 = murineCOX-2.

most potent COX-1 inhibitor among the lead compounds.

Surprisingly, 16, which contains a propyl side chain instead of

an ethyl side chain, was 2000-fold weaker as a COX-1 inhibitor

than 1. The 3′-alkyl analogues 44 and 50 did not display

signiﬁcant inhibitory activity on COX-1 at the highest inhibitor

concentration (100 μM) tested. Compared to their respective

AKR1C3 inhibitory potency, all the compounds with the

exception of 27 and 61, displayed over 200-fold selectivity for

AKR1C3 over COX-1.

Inhibition of Testosterone Formation in LNCaP-

AKR1C3 Cells. To evaluate the eﬃcacy of the lead compounds

in a prostate cancer cell model, we tested their ability to block

testosterone formation in LNCaP cells stably transfected with

AKR1C3.⁴⁶Cells were incubated with 0.1 μM Δ⁴-AD, and their

ability to block the formation of testosterone-17β-glucuronide

was measured (Figure 4). In the absence of the inhibitor, Δ⁴-

To further evaluate the residual COX-1 and COX-2

inhibitory activity of a subset of AKR1C3 inhibitors, we also

used a discontinuous ¹⁴C-AA TLC-based COX assay, where

samples were taken, the reaction stopped, and the concen-

tration of radioactive substrates/products determined on a plate

reader (for details see Supporting Information). Compounds

were tested at four distinct concentrations, 250 nM, 1000 nM, 4

μM, and 25 μM, on either ovine COX-1 or mouse COX-2, and

IC₅₀values (if applicable) were detected graphically from the

resulting dose−response curves. Results are compiled in Table

6. The AA concentration of 5 μM used in the latter assay

represents the K_mfor COX-1 and COX-2, which enables rapid,

reversible, and slow, tight-binding inhibitors to be detected.

Both assays are well established in our laboratories and have

been described earlier in the literature.^20,44

Unlike 1, its 5′-ﬂuoro equivalent 8 (Table 1) and to some

degree the class III inhibitors 61 and 64 (Table 3), the other

indole-based lead AKR1C3 inhibitors listed in Table 6, do not

strongly inhibit COX-1 or COX-2. As reported earlier, 2′-des-

methyl-indomethacin 20 is only a very weak competitive

inhibitor of both COX enzymes with a preference for COX-2.⁴⁵

The same proved true for its close analogue 25 (here, 5′-

OCH₃replaced by 5′-F) but without preference for either COX

enzyme. The best results from the standpoint of minimal COX

interaction were achieved with the 3′-alkyl indole derivatives

44, 47, 48 (free acids), and 50 (sulfonamide), whose percent

COX-1/-2 inhibition did not exceed 34% at 25 μM. In

particular, for compounds 44 (3′-methyl) and 47 (3′-ethyl),

with an unbranched 2′-aliphatic acid chain, the maximum

inhibition achieved was even lower. Compound 44 inhibited

oCOX-1 at 11% (25 μM) and mCOX-2 at <18% (4 μM). The

homo analogue 47 demonstrated 16% inhibition of oCOX-1 at

25 μM and a poor IC₅₀of 7 μM for mCOX-2. In many cases

the formation of a plateau at higher inhibitor concentrations

suggests that inhibition is readily reversible. Collectively,

compounds 16 (class I), compounds 20 and 27 (class II),

and compounds 44−47 and 50 (class III) are among the most

promising lead compounds not to inhibit COX-1 and COX-2.

Figure 4. Inhibitory eﬀect of indomethacin and indomethacin

analogues 44 and 50 on testosterone formation in LNCaP-AKR1C3

cells measured by radiochromatography.

AD is converted to testosterone and androsterone glucur-

onides. We found that there was robust inhibition of

testosterone formation when the cells were treated with 0.1

μM Δ⁴-AD in the presence of 30 μM 1, 44, and 50 (Figure 4).

Eﬀects of Lead Compounds on AR Reporter Gene

Assay. Since AR activation is involved in progression of CRPC,

we next evaluated the eﬀect of these compounds on AR

signaling using a luciferase reporter gene assay. Agonist activity

by these compounds would be deleterious in the context of

CRPC. When HeLa-AR3A-PSA-(ARE)₄-Luc13 cells (a kind gift

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from Dr. Elizabeth Wilson) were treated with the compounds

in the absence of DHT, none of the compounds displayed

agonist activity on the AR in the absence of DHT.

The compounds also did not enhance DHT-mediated AR

signaling (Figure 5). The EC₅₀value for DHT induction of the

Table 7. Data Collection and Reﬁnement Statistics for the

AKR1C3·NADP⁺·20 Complex (PDB ID: 4DBW)

data collection

resolution range (Å)

50.0−1.80

60585 (5607)

b

unique reﬂections measured

a

b

R_merge

0.050 (0.28)

b

I/σ (I)

19.8 (3.3)

b

completeness (%)

reﬁnement statistics

96.7 (89.8)

reﬂections used in reﬁnement/test set

56940/2893

c

R/R_free

0.178/0.233

d

protein atoms

5050

611

2

d

water molecules

d

NADP⁺molecules

d

inhibitor molecules

4

rmsd

bond lengths (Å)

bond angles (deg)

overall B-factor (Å²)

cofactor B-factor (Å²)

inhibitor B-factor (Å²)

0.005

1.0

28

21

34

e

Ramachandran statistics

allowed (%)

92.9

6.8

additionally allowed (%)

generously allowed (%)

disallowed (%)

Figure 5. Dose−response curve of 5α-dihydrotestosterone (5α-DHT)

in HeLa-AR3A-PSA-(ARE)₄-Luc13 cells in the presence and absence

of 10 μM lead compounds.

0.2

a

R_merge= Σ|I − ⟨I⟩|/ΣI, where I is the observed intensity and ⟨I⟩ is the

b

average intensity calculated for replicate data. The number in

parentheses refers to the outer 0.1 Å shell of data. Crystallographic R-

luciferase reporter in the presence and absence of the

indomethacin analogues was not signiﬁcantly diﬀerent. The

EC₅₀value for DHT induction of the luciferase reporter was

0.13 0.02 nM, while the values in the presence of 1, 27, 44,

and 50 were 0.14 0.02, 0.10 0.02, 0.15 0.03, and 0.14

0.05 nM, respectively (Figure 5). This data also indicates that

these compounds did not act as AR antagonists.

c

factor, R = Σ||F_o| − |F_c||/Σ|F_o| for reﬂections contained in the working

set. Free R-factor, R_free= Σ||F_o| − |F_c||/Σ|F_o| for reﬂections contained in

the test set excluded from reﬁnement. |F_o| and |F_c| are the observed

d

and calculated structure factor amplitudes, respectively. Per

e

asymmetric unit. Ramachandran statistics were calculated with

PROCHECK.⁵³

Crystal Structure of the AKR1C3·NADP⁺·20 (2′-des-

Methyl-indomethacin) Complex. The structure of AKR1C3

in complex with NADP⁺and 2′-des-methyl-indomethacin was

determined at a resolution of 1.8 Å by molecular replacement

(Table 7 and Figure 6).⁴⁷We found that there are two

monomers of AKR1C3 in each unit cell. Monomer A exhibited

a lower average B-factor compared to monomer B, but the two

monomers showed an almost identical conformation with an

rmsd of 0.23 Å. The cofactor binding cavity of AKR1C3 is not

perturbed upon binding of the inhibitor. NADP⁺maintains the

same binding position and conformation as in the other

AKR1C3 structures determined.⁴⁸⁻⁵¹The steroid/inhibitor

binding cavity of AKR1C3 is composed of ﬁve compartments:

oxyanion site (formed by Tyr55, His117, and NADP⁺), steroid

channel (Trp227 and Leu54), and three subpockets, SP1

(Ser118, Asn167, Phe306, Phe311, and Tyr319), SP2 (Trp86,

Leu122, Ser129, and Phe311), and SP3 (Tyr24, Glu192,

Ser221, and Tyr305).⁵²Two molecules of compound 20 are

found in each steroid/inhibitor binding cavity (Figure 6A). One

molecule interacts with the oxyanion site through hydrogen

bonding between the carboxylate group and the active site

residues Tyr55 and His117. The carboxylate group of this

molecule also lies on the face of the positively charged

nicotinamide ring of NADP⁺and makes a favorable charge−

charge interaction. The indole ring of this molecule is bound

between the two gate keepers of the steroid channel, Trp227

and Leu54, and the p-chorobenzoyl ring of the inhibitor

projects into the SP1 subpocket. This molecule is likely the ﬁrst

molecule of 20 to enter the active site considering its deeper

penetration in the cavity. The second molecule of 20 stacks on

the ﬁrst molecule through oﬀset stacked aromatic interactions

between the p-chorobenzoyl rings and edge-to-face aromatic

interactions between the indole rings. The indole ring of the

second molecule of 20 projects to the steroid channel. In

contrast to the crystal structure, the inhibition dose−response

curve of 20 for AKR1C3 exhibits a slope factor of 1, which

disagrees with multisite binding. Nonspeciﬁc binding or

occupancy of low aﬃnity binding sites by more than one

inhibitor molecule per enzyme active site can occur in the

presence of excess inhibitor used under the crystallization

conditions.

DISCUSSION

■

AKR1C3 is a critical enzyme in the intraprostatic biosynthesis

of androgens and has been implicated in the progression of

CRPC.⁴⁻⁶Therefore, AKR1C3 inhibition is expected to be an

eﬀective therapy in CRPC. We describe the synthesis and

structure−activity relationship studies of indomethacin-based

AKR1C3 inhibitors. On the basis of the known SAR for COX

inhibition, three classes of compounds were synthesized: (I)

analogues in which groups essential for COX-inhibition were

substituted; (II) 2′-des-methyl-indomethacin analogues in

which the 2′-CH₃group essential for tight binding to COX⁵⁴

was removed from the structure; and (III) 3′-alkyl analogues in

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Figure 6. Stereoview (A) and schematic (B) illustrations of the occupancy of the AKR1C3 active site by compound 2′-des-methyl-indomethacin

(20) (PDB ID: 4DBW). The ﬁrst molecule of 20 is anchored to the active site through hydrogen bonding to Tyr55 and His117. The second

molecule of 20 stacks on the ﬁrst molecule. Both molecules project their p-chorobenzoyl rings into the SP1 pocket. The distance between the 2′-

position on the indole ring of the ﬁrst molecule of 20 and the carboxamide group of the nicotinamide cofactor is only 3.5 Å (blue dash, panel A),

prohibiting indomethacin from assuming this binding pose. The simulated-annealing omit map of 20 is contoured at 2.3σ. Compound 20 is shown in

ball-and-stick representation with carbon atoms colored in black. The noncarbon atoms are colored as follows: oxygen = red, nitrogen = blue,

chlorine = green, and phosphorus = orange. Water molecules are shown as red spheres. Hydrogen bonds are indicated by red dashes.

which the acetic acid side chain essential for COX inhibition

was replaced with an alkyl group and the acid-side chain was

moved to the 2′-position.

with the pyrophosphate bridge of the cofactor (Figure 7A). As

the pH of the crystallization condition is increased to 7.5 (PDB

ID: 3UG8), deprotonation of the pyrophosphate bridge

weakened the interactions in SP3 so that the indole ring

rotates by ∼40° to permit the carboxylate group to interact

with the oxyanion site. In this binding pose, the 5′-OCH₃group

still remains in SP3, while the p-chlorobenzoyl ring enters SP1

(Figure 7B). In the AKR1C3·NADP⁺·2′-des-methyl-indome-

thacin complex (PDB ID: 4DBW), the carboxylate remains

anchored by the oxyanion hole, but the 5′-OCH₃group no

longer interacts with SP3. In addition, the orientation of the p-

chlorobenzoyl ring is altered so that it lies perpendicular to this

ring in the indomethacin structure determined at pH 7.5

(Figure 7B). Indomethacin cannot assume the binding pose of

2′-des-methyl-indomethacin since the 2′-methyl group of

indomethacin would clash with the nicotinamide headgroup

of the cofactor. In the AKR1C3·NADP⁺·Z-sulindac structure

(PDB ID: 3R7M), the indene ring is ﬂipped by 180° relative to

the indole ring in the 2′-des-methyl-indomethacin structure, and

the 4-methylsulﬁnyl group approaches SP2 (Figure 7C).

Most of the class I compounds would adopt the binding

conformations exempliﬁed by indomethacin. Since our

inhibition experiments were performed at pH 7.0, it is likely

that both pH 6 and pH 7.5 binding poses for indomethacin will

coexist in solution. The unsubstituted free acids inhibit

AKR1C3 with the highest potency and selectivity indicating

that the interaction between the binding sites and the

carboxylate is essential for inhibitor anchoring. Analogues

with an extended acid side chain at the 3′-position, i.e.,

replacement of the acetic acid chain by a propionic acid side

chain (compound 16), is also well tolerated suggesting there is

space available in the binding cavity to accommodate the

additional carbon atom, but larger subsitituents such as the N-

(sulfonyl)acetamide derivatives cannot be accommodated by

either of the two indomethacin binding poses observed in the

available crystal structures. Upon inspection of the AKR1C3

structures, we propose a ﬁfth binding pose for these bulky

analogues in which inhibitors extend their p-chorobenzoyl ring

This eﬀort led to the discovery of potent AKR1C3 inhibitors

that retain selectivity for AKR1C3 over other AKR1C enzymes

and have been stripped of COX inhibitory activity. These

compounds inhibit testosterone formation in a prostate cancer

cell line and display no agonist or antagonist activity at the

androgen receptor. The crystal structure of a lead compound,

2′-des-methyl-indomethacin (20), in complex with AKR1C3

was also obtained to elucidate enzyme−inhibitor interactions.

Of the compounds synthesized and tested, seven class I and

class II analogues had the required AKR1C3 selectivity and

potency. Many of the class III analogues had desirable AKR1C3

potency but were less selective for AKR1C3 over AKR1C4,

which plays a critical role in bile-acid biosynthesis.

Structural Consideration of AKR1C3 Inhibitor Potency

and Selectivity. Five crystal structures exist for AKR1C3

inhibitor complexes that are relevant to the present study:

AKR1C3·NADP⁺·indomethacin,^49,55AKR1C3·NADP⁺·2′-des-

methyl-indomethacin (20),⁴⁷and AKR1C3·NADP⁺·Z-sulin-

dac⁵⁵(Figure 7), which can be used to explain the binding

and selectivity of class I−III inhibitors. These structures show

that the active site of AKR1C3 contains three subpockets

(SP1−SP3), and depending on the inhibitor class, diﬀerent

subpockets may be occupied. At the base of the active site, an

oxyanion site exists and consists of Tyr55, His117, and Lys84,

which for most NSAID derivatives provides a counterion to

bind the carboxylic acid on these drugs.⁵⁵The steroid channel

extends from the oxyanion hole and is ﬂanked by Trp227 and

Leu54 on either side. Surprisingly, few NSAID derivatives fully

occupy the steroid channel.

Three AKR1C3·NADP⁺·indomethacin structures have been

reported in the Protein Data Bank.^49,55The structure obtained

at pH 6.0 (PDB ID: 1S2A) shows a unique binding pose, in

which the carboxylic acid, presumably ionized as the negatively

charged carboxylate, is not anchored in the oxyanion site but

instead projects into SP3, where it interacts with Gln222 and

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Figure 7. Binding poses of indomethacin analogues in AKR1C3. (A) Indomethacin (IMN, black) at pH 6 (PDB ID: 1S2A), where the carboxylate

group anchors the indole ring in the SP3 pocket by interacting with Q222 and a phosphate group of the pyrophosphate moiety of the cofactor. 2′-

des-Methyl-indomethacin (20, green, PDB ID: 4DBW) is superimposed as a reference showing how the oxyanion site is occupied by the carboxylate

group and the SP1 pocket is occupied by the p-chorobenzoyl ring. (B) Indomethacin (black) at pH 7.5 (PDB ID: 3UG8), where the carboxylate

group is tethered to the oxyanion site and the p-chorobenzoyl ring penetrates the SP1 pocket. Two alternative conformations exist for Trp227, each

with 50% occupancy. Note that the 5′-methoxy group on the indole ring points into SP3 so that the indole ring of indomethacin assumes a ∼120°

angle with the indole ring of 2′-des-methyl-indomethacin. (C) Z-Sulindac (black, PDB ID: 3R7M), where the carboxylate group is anchored to the

oxyanion site and the 4-methylsulﬁnyl group approaches the SP2 pocket. (D) The proposed ﬁfth binding pose illustrated by compound 27 (black,

generated by AutoDock Vina), where the N-(sulfonyl)acetamide group occupies the SP3 pocket, the p-chorobenzoyl ring occupies the SP1 pocket,

and the indole ring almost overlaps with the indole ring of 2′-des-methyl-indomethacin. Noncarbon atoms, water, and hydrogen bonds are presented

as described in Figure 6 where sulfur = yellow.

into the SP1, and the bulky 3-subsitituents would occupy the

SP3 (Figure 7D, generated by AutoDock Vina⁵⁶). The SP3

pocket is lined by polar/charged residues including Tyr24,

Gln222, Asp224, and Arg226, which supports the observation

that polar substituents are generally favored, and the very bulky

side chains may be exposed through an opening in the SP3

pocket into bulk solvent. This new binding pose is supported

by docking studies and can be applied to all the bulky analogues

in all three classes of inhibitors.

2′-des-methyl-indomethacin binding poses presented in any of

the crystal structures, but all these compounds could assume

the ﬁfth binding pose proposed. By contrast, class III

compounds containing a 3′-methyl/ethyl group may adopt

the Z-sulindac binding conformation since this conformation

imposes the least restriction on the size of the 2′ substitution.

Structural information of a AKR1C3·NADP⁺·class III inhibitor

would be invaluable to conﬁrm that this new binding pose

exists.

Only two class II compounds based on 2′-des-methyl-

indomethacin (20) are likely to adopt the binding pose of 20,

namely, compounds 21 and 25. Interaction with the oxyanion

site is again supported since the corresponding methyl esters

are weak inhibitors of AKR1C3. The N-(sulfonyl)-acetamide of

2′-des-methyl-indomethacin is too bulky to interact with the

oxyanion site and likely assumes the ﬁfth binding pose

proposed.

Promising Lead Compounds. Eleven compounds were

selected for further evaluation. The selection criteria was

AKR1C3 potency <1 μM and greater than 100-fold selectivity

for AKR1C3 over AKR1C2. On this list, ﬁve were class I

analogues, two were class II analogues, and four were class III

analogues. These compounds were all greater than 100-fold

selective for AKR1C3 over AKR1C1, ensuring that there would

be no interference with DHT inactivation within the prostate.

All the compounds were also selective for AKR1C3 over

AKR1C4. While the 3′-alkyl analogues were still over 20-fold

Class III compounds that have a bulky side chain at the 2′-

position cannot be easily superimposed on the indomethacin or

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spectrometer using chromatography. A purity of ≥95% (unless

otherwise indicated) for the ﬁnal compounds in this study was

conﬁrmed by analytical HPLC on either a Waters HPLC system with

PDA detector (set at λ = 254 nm) equipped with a Supelco Supelcosil

LC-18 reverse-phase column (15 cm × 3 mm, 5 μm) or the

aforementioned Agilent 1200 analytical LC−MS with UV detection at

λ = 215 and 254 nm along with ELCD detection (Stationary phase:

YMC J’sphere H-80 S-4 column (3.0 × 50 mm)), using two diﬀerent

gradient methods. The mobile phases were MeOH/0.01 M KH₂PO₄,

and ACN/H₂O (plus 0.1% TFA), respectively (for further details see

Supporting Information).

selective for AKR1C3 over AKR1C4, they displayed lower

AKR1C3 selectivity relative to AKR1C4 in comparison to the

class I and II analogues. AKR1C4 is primarily involved in bile

acid biosynthesis and steroid hormone biotransformation in the

liver, where it is predominantly expressed.^17,57,58The

consequence, if any, of the low selectivity of the 3-alkyl

analogues for AKR1C3 over AKR1C4 will need to be explored.

The absence of intrinsic agonist activity or positive

modulation of DHT-mediated AR transcription in the reporter

gene assay by all the lead compounds is important since either

eﬀect on the AR would counteract the eﬀect of AKR1C3

inhibition.

Preparation and Analytical Data of Exempliﬁed Test

Compounds. The general synthetic procedures A−D are speciﬁed

in the Supporting Information.

The compounds were also eﬃcacious in a prostate cancer cell

model where they produced robust inhibition of testosterone

formation when cells were treated with the AKR1C3 substrate

Δ⁴-AD. This indicates the compounds possess adequate

physicochemical properties to traverse the cell membrane

and, more importantly, are able to target AKR1C3 in a cellular

context. This is in agreement with the report of Cai et al., which

showed that indomethacin was eﬀective in vivo, in a castrate-

resistant prostate cancer setting at inhibiting tumor cell

proliferation.³⁰These ﬁndings support the potential therapeutic

usefulness of AKR1C3 inhibitors in CRPC. It equally indicates

that the indomethacin analogues and importantly those with

little or no COX activity such as the lead compounds reported

in this study should block the adaptive intratumoral biosyn-

thesis that drives CRPC, thereby inhibiting cancer progression

without any of the adverse eﬀects secondary to chronic COX

inhibition. Since the compounds identiﬁed in this study are

expected to have pharmacokinetic proﬁles comparable to that

of indomethacin, we anticipate that the lead compounds will be

well-tolerated for CRPC treatment.

In conclusion, we have discovered indomethacin-based

inhibitors of AKR1C3 that are devoid of activity against other

AKR1C isoforms, do not display COX inhibitory activity or AR

agonist activity, and are eﬀective in a prostate cancer cell model.

These compounds are useful therapeutic leads in the

management of CRPC. These compounds have the potential

to be superior therapeutic agents compared to abiraterone in

the management of CRPC because they do not interfere with

glucocorticoid biosynthesis and therefore would not require

coadministration with a glucocorticoid.

2-(1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-

(methylsulfonyl)acetamide (14). According to general procedure B,

the title compound was obtained from 2-(1-(4-chlorobenzoyl)-5-

methoxy-2-methyl-1H-indol-3-yl)acetic acid (IMN) (60 mg, 0.17

mmol) and methansulfonamide (16 mg, 0.17 mmol) after 20 h at

room temperature. The crude product was puriﬁed by ﬂash

chromatography (SiO₂, ethyl acetate/hexane gradient). Yield: 55 mg

1

(75%), a white solid. C₂₀H₁₉ClN₂O₅S, M_r= 434.89; H NMR (400

MHz, DMSO-d₆) δ 2.23 (s, 3H), 3.24 (s, 3H), 3.72 (s, 2H), 3.76 (s,

3H), 6.71 (dd, J = 2.4/8.8 Hz, 1H), 6.91 (d, J = 8.8 Hz, 1H), 7.08 (d, J

= 2.8 Hz, 1H), 7.63−7.69 (m, 4H); LC−MS (ESI) (method 2) tR

2.55 min (>99%, UV220, UV254), m/z 435.0 [M + H]⁺.

3-(1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)-

propanoic Acid (16). According to general procedure A, the title

compound was obtained from 4-chloro-N-(4-methoxyphenyl)-

benzohydrazide hydrochloride (50 mg, 0.16 mmol) and 5-oxohexanoic

acid (25 mg, 0.19 mmol) in AcOH (0.5 mL) after 3 h at 80 °C in 69%

1

yield (41 mg) as an oﬀ-white solid. C₂₀H₁₈ClNO₄, M_r= 371.81; H

NMR (400 MHz, DMSO-d₆) δ 2.19 (s, 3H, C2′-CH₃), 2.49 (t, J = 7.4

Hz, 2H, overlaid by DMSO-signal), 2.88 (t, J = 7.4 Hz, 2H), 3.77 (s,

3H, −OCH₃), 6.69 (dd, J = 2.6 Hz, 1H), 6.93 (d, J = 8.8 Hz, 1H), 7.06

(d, J = 2.4 Hz, 1H), 7.61−7.64 (m, 4H); LC−MS (ESI) (method 2)

tR 2.69 min (>99%, UV220, UV254), m/z 372.0 [M + H]⁺.

2-(1-(4-Chlorobenzoyl)-5-methoxy-1H-indol-3-yl)acetic Acid (20).

According to general procedure A, the title compound was obtained

from 4-chloro-N-(4-methoxyphenyl)benzohydrazide hydrochloride

(200 mg, 0.64 mmol) and 4-oxobutanoic acid (succinic semialdehyde,

15% solution in H₂O; 565 mg, 0.83 mmol) in AcOH (1.5 mL) after 3

h at 80 °C in 45% yield (99 mg) as an oﬀ-white solid. C₁₈H₁₄ClNO₄,

M_r= 343.76; ¹H NMR (400 MHz, DMSO-d₆) δ 3.67 (s, 2H), 3.81 (s,

3H), 6.99 (dd, J = 2.4/9.2 Hz, 1H), 7.12 (d, J = 2.4 Hz, 1H), 7.32 (s,

1H), 7.65−7.77 (m, 4H), 8.17 (d, J = 9.2 Hz, 1H); LC−MS (ESI)

(method 2) tR 2.60 min (>99%, ELSD), m/z 344.0 [M + H]⁺.

2-(1-(4-Chlorobenzoyl)-5-methoxy-1H-indol-3-yl)acetyl Chloride.

In accordance with general procedure C, oxalyl chloride (30 μL, 0.35

mmol) was added dropwise to a solution of 2-(1-(4-chlorobenzoyl)-5-

methoxy-1H-indol-3-yl)acetic acid 20 (100 mg, 0.29 mmol) in 2 mL of

dry CH₂Cl₂under argon. The reaction mixture was stirred overnight at

room temperature. The solvent was evaporated and the crude product

was washed with dry hexane (3 × 1 mL) and dried in vacuo to give the

title compound (pale white solid) in 95% yield (100 mg).

EXPERIMENTAL SECTION

■

Chemistry. General. All commercially available reagents and

anhydrous solvents were used as received. Microwave reactions were

carried out using a Biotage Initiator 8-EXP Microwave Synthesizer.

Analytical thin-layer chromatography was carried out using glass-

backed plates coated with ﬂuorescent silica gel 60 F₂₅₄from Whatman

(Partisil LK6D). Spots were visualized under natural light and UV

illumination at λ = 254 and 365 nm. Flash chromatography was

conducted on a Biotage SP1 automated ﬂash chromatography system

equipped with a ﬁxed wavelength UV detector (λ = 254 nm). Samples

were preabsorbed onto ready-made silica gel samplets and then

applied on normal-phase ﬂash chromatography cartridges (Biotage

KP-SIL, size according to requirements) eluting with a 0−100%

EtOAc/hexane (0.5% acetic acid) gradient. Where stated, puriﬁcation

was performed utilizing a custom high-throughput mass-directed

preparative (RP-) HPLC platform using an appropriate acetonitrile/

water gradient with TFA modiﬁer.⁵⁹¹H and ¹³C NMR spectra were

recorded at 400 and 100 MHz, respectively, using a Bruker AV-400

with sample changer (BACS 60). ¹⁹F NMR spectra were measured at

282 MHz using a Bruker AV-300. MS samples were analyzed on an

Agilent 1200 LC−MS system operating in single MS mode with

electrospray ionization. Samples were introduced into the mass

1

C₁₈H₁₃Cl₂NO₃, M_r= 362.21; H NMR (400 MHz, CDCl₃) δ 3.90

(s, 3H), 4.21 (s, 2H), 6.95 (d, J = 2.4 Hz, 1H), 7.04 (dd, J = 2.4/9.0

Hz, 1H), 7.29 (s, 1H), 7.52−7.55 (m, 2H), 7.67−7.71 (m, 2H), 8.29

(d, J = 9.2 Hz, 1H); LC−MS (ESI) (method 2) tR 2.72 min (>99%,

UV254), m/z 358.2 [M + H]⁺.

2-(1-(4-Chlorobenzoyl)-5-methoxy-1H-indol-3-yl)-N-

((triﬂuoromethyl)sulfonyl)acetamide (27). In accordance with

general procedure D, 80 mg (0.22 mmol) of 2-(1-(4-chlorobenzoyl)-

5-methoxy-1H-indol-3-yl)acetyl chloride and 49.4 mg (0.33 mmol) of

triﬂuoromethanesulfonamide were dissolved in 1.8 mL of 1,2-

dichloroethane (DCE) under stirring. Then 17.5 mg (0.22 mmol) of

pyridine was added, and the reaction was allowed to run at ambient

temperature until the starting material was consumed (∼4 h). After the

addition of 13 μL of AcOH, the organic solution was washed with

H₂O (3 × 2 mL), dried over Na₂SO₄, ﬁltered, and concentrated in

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vacuo. The crude product was puriﬁed by ﬂash chromatography (SiO₂,

ethyl acetate/hexane, 0.5% AcOH gradient) to aﬀord the title

compound in 70% yield (73 mg). C₁₉H₁₄ClF₃N₂O₅S, M_r= 474.84;

¹H NMR (400 MHz, DMSO-d₆) δ 3.44 (s, 2H), 3.79 (s, 3H), 6.95

(dd, J = 2.6/9.0 Hz, 1H), 7.14 9d, J = 2.4 Hz, 1H), 7.23 (s, 1H), 7.64−

7.66 (m, 2H), 7.72−7.74 (m, 2H), 8.15 (d, J = 8.8 Hz, 1H, C7′-H);

¹⁹F NMR (282 MHz, DMSO-d₆) δ −75.58 (s, −CF₃); LC−MS (ESI)

4-oxohexanoic acid (30 mg, 0.19 mmol) in AcOH (0.5 mL) after 3 h

at 80 °C, extraction into hot hexane, and subsequent puriﬁcation by

ﬂash chromatography in 36% yield (23 mg) as a yellow oil, which

crystallized constantly upon drying at high vacuum. C₂₂H₂₂ClNO₄, M_r

1

= 399.87; H NMR (400 MHz, DMSO-d₆) δ 1.03 (s, 6H), 2.21 (s,

3H), 3.25 (s, 2H), 3.76 (s, 3H), 6.52 (d, J = 9.2 Hz, 1H), 6.67 (dd, J =

2.6/9.0 Hz, 1H), 7.03 (d, J = 2.4 Hz, 1H), 7.64−7.68 (m, 4H); LC−

MS (ESI) (method 2) t_R2.89 min (95%, UV220, UV254), m/z 400.0

[M + H]⁺.

(method 2) tR 2.37 min (>99%, UV254, ELSD), m/z 475.0 [M + H]⁺.

2-(1-(4-Chlorobenzoyl)-5-methoxy-1H-indol-3-yl)-N-(naphtha-

len-2-ylsulfonyl)acetamide (29). According to general procedure B,

the title compound was obtained from 2-(1-(4-chlorobenzoyl)-5-

methoxy-1H-indol-3-yl)acetic acid 20 (30 mg, 0.087 mmol) and

naphthalene-2-sulfonamide (18.1 mg, 0.087 mmol) after 20 h at room

temperature. The crude product was puriﬁed by automated mass-

directed HPLC (RP-18, ACN/H₂O gradient). Yield: 21 mg (45%).

C₂₈H₂₁ClN₂O₅S, M_r= 532.99; ¹H NMR (400 MHz, DMSO-d₆) δ 3.64

(s, 3H), 3.65 (s, 2H), 6.87 (d, J = 2.4 Hz, 1H), 6.92 (dd, J = 2.6/9.0

Hz, 1H), 7.21 (s, 1H), 7.61−7.75 (m, 6H), 7.82 (dd, J = 2.0/8.8 Hz,

1H), 8.02−8.16 (m, 4H), 8.55 (d, J = 1.6 Hz, 1H); LC−MS (ESI)

(method 2) tR 3.01 min (98%, UV254, ELSD), m/z 533.1 [M + H]⁺.

2-(1-(4-chlorobenzoyl)-5-methoxy-1H-indol-3-yl)-N-((4-

(triﬂuoromethoxy)phenyl)sulfonyl)-acetamide (32). According to

general procedure B, the title compound was obtained from 2-(1-(4-

chlorobenzoyl)-5-methoxy-1H-indol-3-yl)acetic acid 20 (30 mg, 0.087

mmol) and 4-(triﬂuoromethoxy)benzene-sulfonamide (21 mg, 0.087

mmol) after 20 h at room temperature. The crude product was

puriﬁed by automated mass-directed HPLC (RP-18, ACN/H₂O

3-(1-(4-Chlorobenzoyl)-5-methoxy-3-methyl-1H-indol-2-yl)-2-

methylpropanoic Acid (49). According to general procedure A, the

title compound was obtained from 4-chloro-N-(4-methoxyphenyl)-

benzohydrazide hydrochloride (50 mg, 0.16 mmol) and 2-methyl-4-

oxohexanoic acid (28 mg, 0.19 mmol) in AcOH (0.5 mL) after 3 h at

80 °C in 49% yield (30 mg) as a beige solid. C₂₁H₂₀ClNO₄, M_r=

1

385.84; H NMR (400 MHz, DMSO-d₆) δ 1.01 (d, J = 6.8 Hz, 3H,

alpha-CH₃), 2.19 (s, 3H, C³−CH₃), 2.54 (s, J = 7.2 Hz, 1H, methin-

H), 2.91−3.19 (m, 2H, −CH₂−), 3.76 (s, 3H, −OCH₃), 6.46 (d, J =

9.2 Hz, 1H, C⁷−H), 6.64 (dd, J = 2.4/9.0 Hz, 1H, C⁶−H), 7.02 (d, J =

2.4 Hz, 1H, C⁴−H); LC−MS (ESI) (method 2) t_R2.75 min (>97%,

UV220, UV254, MSD), m/z 386.1 [M + H]⁺.

3-(1-(4-Chlorobenzoyl)-5-methoxy-3-methyl-1H-indol-2-yl)-N-

(methylsulfonyl)propanamide (50). According to general procedure

B, the title compound was obtained from 3-(1-(4-chlorobenzoyl)-5-

methoxy-3-methyl-1H-indol-2-yl)propanoic acid 44 (40 mg, 0.11

mmol) and methansulfonamide (10 mg, 0.11 mmol) after 20 h at

room temperature. The crude product was puriﬁed by ﬂash

chromatography (SiO₂, EtOAc/hexane gradient 1:1, 0.5% AcOH).

Yield: 32 mg (66%), a bright yellow solid. C₂₁H₂₁ClN₂O₅S, M_r=

1

gradient). Yield: 25.4 mg (51%). C₂₅H₁₈ClF₃N₂O₆S, M_r= 566.93; H

NMR (400 MHz, DMSO-d₆) δ 3.66 (s, 2H), 3.74 (s, 3H), 6.89 (d, J =

2.4 Hz, 1H), 6.97 (dd, J = 2.6/9.0 Hz, 1H), 7.24 (s, 1H), 7.55 (dd, J =

0.8/8.8 Hz, 1H), 7.64−7.75 (m, 4H), 7.99−8.02 (m, 2H), 8.14 (d, J =

9.2 Hz, 1H); ¹⁹F NMR (282 MHz, DMSO-d₆) δ −54.88 (s, OCF₃);

LC−MS (ESI) (method 2) tR 3.08 min (98%, UV254, ELSD), m/z

567.0 [M + H]⁺.

1

448.92; H NMR (400 MHz, DMSO-d₆) δ 2.21 (s, 3H), 2.55 (t, J =

7.2 Hz, 2H), 3.11 (t, J = 7.2 Hz, 2H), 3.16 (s, 3H), 3.75 (s, 3H), 6.43

(d, J = 9.2 Hz, 1H), 6.64 (dd, J = 9.0 Hz, 1H), 7.02 (d, J = 2.4 Hz,

1H), 7.64−7.69 (m, 4H); LC−MS (ESI) (method 2) t_R2.74 min

(>96%, UV254, ELSD), m/z 249.1 [M + H]⁺.

3-(1-(4-Chlorobenzoyl)-5-methoxy-3-methyl-1H-indol-2-yl)-N-to-

sylpropanamide (53). According to general procedure B, the title

compound was obtained from 3-(1-(4-chlorobenzoyl)-5-methoxy-3-

methyl-1H-indol-2-yl)propanoic acid 44 (30 mg, 0.081 mmol) and 4-

methylbenzenesulfonamide (15.2 mg, 0.089 mmol) after 20 h at room

temperature. The crude product was puriﬁed by automated mass-

directed HPLC (RP-18, ACN/H₂O gradient). Yield: 23.1 mg (55%).

C₂₇H₂₅ClN₂O₅S, M_r= 525.02; ¹H NMR (400 MHz, DMSO-d₆) δ 2.07

(s, 3H), 2.35 (s, 3H), 2.49 (t, J = 7.2 Hz, 2H, partially overlaid by

solvent signal), 2.99 (t, J = 7.2 Hz, 2H), 3.76 (s, 3H), 6.37 (d, J = 9.2

Hz, 1H), 6.63 (dd, J = 2.6/9.0 Hz, 1H), 6.96 (d, J = 2.4 Hz, 1H), 7.31

(d, J = 8.0 Hz, 2H), 7.57−7.64 (m, 4H), 7.69 (d, J = 8.4 Hz, 2H);

LC−MS (ESI) (method 2) t_R3.09 min (95%, UV254, ELSD), m/z

525.1 [M + H]⁺.

3-(1-(4-Chlorobenzoyl)-5-methoxy-3-methyl-1H-indol-2-yl)-

propanoic Acid (44). According to general procedure A, the title

compound was obtained from 4-chloro-N-(4-methoxyphenyl)-

benzohydrazide hydrochloride (400 mg, 1.28 mmol) and 4-

oxohexanoic acid (200 mg, 1.54 mmol) in AcOH (4 mL) after 3 h

at 80 °C in 75% yield (368 mg) as an oﬀ-white solid. C₂₀H₁₈ClNO₄,

M_r= 371.81; ¹H NMR (400 MHz, DMSO-d₆) δ 2.20 (s, 3H), 2.46 (t,

J = 7.2 Hz, 2H), 3.08 (t, J = 7.6 Hz, 2H), 3.75 (s, 3H), 6.44 (d, J = 8.8

Hz), 6.63 (dd, J = 2.4/9.0 Hz, 1H), 7.01 (d, J = 2.4 Hz, 1H), 7.63−

7.68 (m, 4H); ¹³C NMR (100 MHz, DMSO-d₆) δ 8.78 (s, −CH₃),

21.61 (s, −CH₂−), 34.12 (s, −CH₂CO), 55.81 (s, −OCH₃), 101.93 (s,

C4′ indole), 111.97 (s, C6′ indole), 114.76 (s, C7′ indole), 116.11 (s,

C3′ indole), 129.53 (s, C3′/C5′ 4-Cl-benzoyl−), 130.63 (s), 131.58

(s), 131.62 (s, C2′/C6′ 4-Cl-benzoyl−), 134.42 (s), 137.09 (s), 138.14

(s), 155.83 (s, C5′ indole), 168.14 (s, >CO), 173.77 (s, −C

O(OH)); LC−MS (ESI) (method 2) t_R2.68 min (99%, UV254,

ELSD), m/z 372.1 [M + H]⁺.

3-(1-(4-Chlorobenzoyl)-5-methoxy-3-methyl-1H-indol-2-yl)-N-

((5-chlorothiophen-2-yl)sulfonyl)-propanamide (58). According to

general procedure B, the title compound was obtained from 3-(1-(4-

chlorobenzoyl)-5-methoxy-3-methyl-1H-indol-2-yl)propanoic acid 44

(30 mg, 0.081 mmol) and 5-chlorothiophene-2-sulfonamide (17.5 mg,

0.089 mmol) after 20 h at room temperature. The crude product was

puriﬁed by automated mass-directed HPLC (RP-18, ACN/H₂O

3-(1-(4-Chlorobenzoyl)-3-ethyl-5-methoxy-1H-indol-2-yl)-

propanoic Acid (47). The title compound was synthesized via general

procedure A as described for isomer mixture 46 (see Supporting

Information). H₂O was added. The watery reaction mixture was

extracted with methylene chloride (2×) and the combined organic

phases dried over Na₂SO₄. The solvent was removed under reduced

pressure and the residual beige solid repeatedly triturated with diethyl

ether. The resulting white precipitation was collected by ﬁltration

(ether wash phases abolished) and dried in vacuo to aﬀord 47 as a

1

gradient). Yield: 12.3 mg (28%). C₂₄H₂₀Cl₂N₂O₅S₂, M_r= 551.46; H

NMR (400 MHz, DMSO-d₆) δ 2.10 (s, 3H), 2.54 (t, J = 7.2 Hz, 2H),

3.06 (t, J = 7.2 Hz, 2H), 3.76 (s, 3H), 6.40 (d, J = 9.2 hz, 1H), 6.63

(dd, J = 2.6/9.0 Hz, 1H), 6.96 (d, J = 2.4 Hz, 1H), 7.21 (d, J = 4.0 Hz,

1H), 7.60 (d, J = 4.0 Hz, 1H), 7.64 (pseudo-s, 4H); LC−MS (ESI)

(method 2) t_R3.19 min (99%, UV254, ELSD), m/z 551.0 [M + H]⁺.

9-(4-Chlorobenzoyl)-6-methoxy-2,3,4,9-tetrahydro-1H-carba-

zole-3-carboxylic Acid (61). According to general procedure A, the

title compound was obtained from 4-chloro-N-(4-methoxyphenyl)-

benzohydrazide hydrochloride (50 mg, 0.16 mmol) and 4-

oxocyclohexanecarboxylic acid (27.2 mg, 0.19 mmol) in AcOH (0.5

mL) after 3 h at 80 °C in 83% yield (51 mg) as an oﬀ-white solid.

C₂₁H₁₈ClNO₄, M_r= 383.82; ¹H NMR (400 MHz, DMSO-d₆) δ 1.66−

1.76 (m, 1H), 2.02−2.08 (m, 1H), 2.52 (m, 2H, partially overlaid by

DMSO signal), 2.66−2.75 (m, 2H), 2.87−2.95 (m, 1H, methin-H),

1

single product. Yield: 36 mg, 29%. C₂₁H₂₀ClNO₄, M_r= 385.84; H

NMR (400 MHz, DMSO-d₆) δ 1.18 (t, J = 7.6 Hz, 3H), 2.46 (t, J = 7.6

Hz, 2H), 2.69 (q, J = 7.6 Hz, 2H), 3.09 (t, J = 7.6 Hz, 2H), 3.75 (s,

3H), 6.42 (d, J = 9.2 Hz, 1H), 6.63 (dd, J = 2.4/9.0 Hz, 1H), 7.03 (d, J

= 2.4 Hz, 1H), 7.63 (m, 4H); LC−MS (ESI) (method 2) t_R2.82 min

(95%, UV220, UV254), m/z 386.2 [M + H]⁺.

3-(1-(4-Chlorobenzoyl)-5-methoxy-3-methyl-1H-indol-2-yl)-2,2-

dimethylpropanoic Acid (48). According to general procedure A, the

title compound was obtained from 4-chloro-N-(4-methoxyphenyl)-

benzohydrazide hydrochloride (50 mg, 0.16 mmol) and 2,2-dimethyl-

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3.77 (s, 3H, −OCH₃), 6.74 (dd, J = 2.6/9.0 Hz, 1H), 7.02 (d, J = 2.8

Hz, 1H), 7.10 (d, J = 8.8 Hz, 1H), 7.61−7.67 (m, 4H, ar-H 4-Cl-

benzoyl); LC−MS (ESI) (method 2) t_R2.71 min (>99%, ELSD), m/z

384.1 [M + H]⁺.

which time the media is aspirated and fresh CSS media containing

0.1−100 nM DHT in the presence and absence of 10 μM lead

compound was added to the cells. The cells were then incubated for 20

h at 37 °C and 5% CO₂after which luciferase expression in the cells is

measured using the BrightGlo luciferase assay kit according to the

manufacturer’s instructions. Brieﬂy, after the incubation time, the cells

were allowed to equilibrate to room temperature. The luciferase

substrate was then added to the cells and the luminescence in each

well was measured on a BIOTEK Synergy 2 plate reader. The maximal

luciferase activity was seen with 100 nM DHT.

LNCaP-AKR1C3 Mediated Formation of Testosterone.

Androgen-dependent prostate cancer cells (LNCaP) were genetically

engineered to stably express AKR1C3 (LNCaP-AKR1C3).⁴⁶The cell

line was used to determine the ability of lead compounds to block the

conversion of Δ⁴-AD to testosterone. LNCaP-AKR1C3 cells were

plated in RPMI-1640 supplemented with 5% charcoal stripped serum,

1% Pen/strep, 2 mM ^L-glutamine, and 500 μg/mL G418 (CSS media).

After 24 h incubation, the media was aspirated and fresh CSS media

containing 0.1 μM [¹⁴C]-Δ⁴-AD with or without inhibitor was added.

The media was collected after 48 h treatment and analyzed for

testosterone levels. Brieﬂy, the collected media was extracted with

ethyl acetate. The aqueous fraction was then treated with β-

glucuronidase (200 units/mL at pH 6.6) for 24 h at 37 °C to liberate

conjugated testosterone and extracted with ethyl acetate. The organic

extracts were pooled and dried under vacuo. Steroid reference

standards and extracts dissolved in 100 μL of ethyl acetate were

applied to LK6D Silica TLC plates (Whatman Inc., Clifton, NJ). TLC

plates were developed using a dichloromethane/ethyl acetate (80:20

v/v) solution and were counted with a Bioscan System 200 plate

reader (Washington, DC).

9-(4-Chlorobenzoyl)-6-methoxy-N-(methylsulfonyl)-2,3,4,9-tetra-

hydro-1H-carbazole-3-carboxamide (63). According to general

procedure B, the title compound was obtained from 9-(4-

chlorobenzoyl)-6-methoxy-2,3,4,9-tetrahydro-1H-carbazole-3-carbox-

ylic acid 61 (20 mg, 0.052 mmol) and methansulfonamide (5 mg,

0.052 mmol) after 20 h at room temperature. The crude product was

puriﬁed by ﬂash chromatography (SiO₂, ethyl acetate/hexane

gradient). Yield: 19 mg (79%), a white solid. C₂₂H₂₁ClN₂O₅S, M_r=

460.93; ¹H NMR (400 MHz, DMSO-d₆) δ 1.63−1.73 (m, 1H), 2.03−

2.06 (m, 1H), 2.51−2.57 (m, 2H, partially overlaid by DMSO-signal),

2.65−2.76 (m, 2H), 2.88−2.91 (m, 1H), 3.25 (s, 3H, −SO₂−CH₃),

3.77 (s, 3H, −OCH₃), 6.74 (dd, J = 2.6/9.0 Hz, 1H), 7.03 (d, J = 2.8

Hz, 1H), 7.09 (d, J = 8.8 Hz, 1H), 7.61−7.68 (m, 4H); LC−MS (ESI)

(method 2) t_R2.65 min (>99%, UV220, UV254), m/z 461.0 [M +

H]⁺.

9-(4-Chlorobenzoyl)-6-methoxy-2,3,4,9-tetrahydro-1H-carba-

zole-2-carboxylic Acid (64). According to general procedure A, the

title compound was obtained from 4-chloro-N-(4-methoxyphenyl)-

benzohydrazide hydrochloride (50 mg, 0.16 mmol) and 3-

oxocyclohexanecarboxylic acid (27.2 mg, 0.19 mmol) in AcOH (0.5

mL) after 3 h at 80 °C in 82% yield (50 mg) as an oﬀ-white solid.

C₂₁H₁₈ClNO₄, M_r= 383.82; ¹H NMR (400 MHz, DMSO-d₆) δ 1.79−

1.87 (m, 1H), 2.11−2.14 (m, 1H), 2.57−2.75 (m, 5H), 6.73 (dd, J =

2.8/9.0 Hz, 1H), 6.97 (d, J = 2.8 Hz, 1H), 7.06 (d, J = 9.2 Hz, 1H),

7.62−7.69 (m, 4H); LC−MS (ESI) (method 2) t_R2.82 min (>99%,

ELSD), m/z 384.0 [M + H]⁺.

Enzyme Puriﬁcation. Homogenous recombinant enzymes

(AKR1C1-4, COX 1, and COX 2) used in this study were prepared

and puriﬁed as previously described.^{16,17,60−62}

AKR1C Isoforms. The potency of the compounds on all AKR

enzymes was determined by their ability to inhibit the NADP⁺

dependent oxidation of S-(+)-1,2,3,4-tetrahydro-1-naphthol (S-tetra-

lol) catalyzed by AKR1C isoforms, as previously described.^20,21The

concentration of S-tetralol used in the assays for each AKR1C enzyme

was equal to the K_mvalue for the respective enzyme so that the IC₅₀

values can be directly compared.

COX 1 and 2. The ability of compounds to inhibit COX activity

was determined by two diﬀerent test systems: a continuous

colorimetric assay that monitored the oxidation of N,N,N,N-

tetramethyl-1,4-phenylenediamine (TMPD) when it is coupled to

the formation of PGH₂from PGG₂using arachidonic acid (AA) as

substrate, and a discontinuous ¹⁴C-AA TLC-based COX assay, where

samples were taken, the reaction stopped, and the concentration of

radioactive substrates/products determined on a plate reader. Both

assays have been described earlier in literature.^20,44

IC₅₀Determination (Except for the Discontinuous COX

Assay, See Above). Initial velocities of an enzyme reaction measured

in the presence of varying concentrations of inhibitor were compared

to a solvent control to give percent inhibition values. IC₅₀values of

compounds was determined by ﬁtting the inhibition data using Graﬁt

5.0 software [y = (range)/[1 + (I/IC₅₀)S] + background] and obtained

from a single experiment with each inhibitor concentration run in

quadruplicate with the exception of COX assays that were conducted

in duplicate. Substrate, cofactor, and enzyme were titrated for each

experiment. Selectivity of a compound for AKR1C3 relative to another

tested enzyme is deﬁned as the ratio of the IC₅₀values for the tested

enzyme: IC₅₀value for AKR1C3; the higher the ratio, the greater the

selectivity for AKR1C3.

Crystallography. Crystals of the AKR1C3·NADP⁺·20 complex

were obtained using the hanging drop vapor diﬀusion method at 4 °C.

Drops typically contained 3.0 μL of protein solution (9.3 mg/mL

AKR1C3, 10 mM potassium phosphate (pH 7.0), 1.0 mM BME, 1.0

mM EDTA, 7% (v/v) DMSO, 1.33 mM 20, and 2.0 mM NADP⁺) and

3.0 μL of reservoir solution (0.1 M 2-(N-morpholino)ethanesulfonic

acid (pH 6.0) and 15% (w/v) polyethylene glycol 8000). Crystals

appeared and grew to a suitable size for diﬀraction in approximately 3

days. Prior to ﬂash-cooling, crystals were soaked for 5 min in a

cryoprotective solution containing 16% (w/v) Jeﬀamine ED-2001.

Crystals yielded diﬀraction data to 1.80 Å at beamline X29 (λ = 0.9795

Å) of the National Synchrotron Light Source at Brookhaven National

Laboratory (Upton, NY). Crystals belong to space group P1 with unit

cell parameters a = 47.35 Å, b = 49.09 Å, c = 83.44 Å, α = 74.30°, β =

87.04°, and γ = 69.95°. The unit cell contains two monomers of

AKR1C3. Data were integrated and scaled with HKL2000 and

Scalepack.⁶³Data collection and reduction statistics are reported in

Table 7.

The structure of the AKR1C3·NADP⁺·20 complex was solved by

molecular replacement performed with PHASER⁶⁴from the CCP4

suite of programs using the coordinates of the AKR1C3·-

NADP⁺·indomethacin complex (PDB: 1S2A)⁴⁹less ligand and solvent

molecules as a search probe. The programs CNS,⁶⁵PHENIX,⁶⁶and

COOT⁶⁷were used for reﬁnement and model ﬁtting. NADP⁺and 20

were built into the electron density map at the ﬁnal stage of

reﬁnement. The correct modeling of the ligands was conﬁrmed by

simulated annealing omit maps. The quality of the model was veriﬁed

with Molprobity and PROCHECK. The reﬁnement statistics are

reported in Table 7.

Androgen Receptor Reporter Gene Assay. HeLa cells that are

stably transfected with the AR and a luciferase reporter gene (HeLa-

AR3A-PSA-(ARE)4-Luc13 cells, a kind gift from Dr. Elizabeth

Wilson) were used for this assay. These cells are plated in a 96-well

plate at 1.5 × 10⁴cell/100 μL/well in phenol red free-MEM

supplemented with 5% charcoal stripped FBS, 1% Pen/Strep, 2 mM ^L-

glutamine, Geneticin (500 g/mL), and hygromycin (200 g/mL) (CSS

media) to maintain selection. The cells are incubated for 6−8 h after

ASSOCIATED CONTENT

■

S

* Supporting Information

Details of the synthesis and physicochemical characterization of

reported compounds. This material is available free of charge

via the Internet at http://pubs.acs.org.

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dx.doi.org/10.1021/jm3017656 | J. Med. Chem. 2013, 56, 2429−2446

Journal of Medicinal Chemistry

Article

(5) Montgomery, R. B.; Mostaghel, E. A.; Vessella, R.; Hess, D. L.;

Kalhorn, T. F.; Higano, C. S.; True, L. D.; Nelson, P. S. Maintenance

of intratumoral androgens in metastatic prostate cancer: a mechanism

for castration-resistant tumor growth. Cancer Res. 2008, 68, 4447−

4454.

AUTHOR INFORMATION

Corresponding Author

*Tel: 1-215-898-9445. Fax: 1-215-573-0200. E-mail: penning@

upenn.edu.

■

Author Contributions

A.J.L. and A.O.A. contributed equally to this work.

(6) Hofland, J.; van Weerden, W. M.; Dits, N. F.; Steenbergen, J.; van

Leenders, G. J.; Jenster, G.; Schroder, F. H.; de Jong, F. H. Evidence of

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Jarman, M. Hormonal impact of the 17α-hydroxylase/C(17,20)-lyase

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cancer. Br. J. Cancer 2004, 90, 2317−2325.

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Folkerd, E.; Messiou, C.; Molife, L. R.; Maier, G.; Thompson, E.;

Olmos, D.; Sinha, R.; Lee, G.; Dowsett, M.; Kaye, S. B.; Dearnaley, D.;

Kheoh, T.; Molina, A.; de Bono, J. S. Selective inhibition of CYP17

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Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

Supported by grant R01-CA89450 from the National Institutes

of Health (NIH) to L.J.M, by grants R01-CA90744 and P30-

ES013508 from the NIH and a Prostate Cancer Foundation

Challenge grant to T.M.P., by grant GM-056838 from the NIH

to D.W.C., and by grant F32-DK089827 awarded by the NIH

to M.C. A.J.L. was awarded a postdoctoral fellowship by the

German Research Foundation, DFG, for a research stay in the

United States (LI2019/1-1, Einzelprojekt). The X-ray crystallo-

graphic studies are based upon research conducted at beamline

X25 and X29 of the National Synchrotron Light Source.

Financial support for the National Synchrotron Light Source

comes principally from the Oﬃces of Biological and Environ-

mental Research and of Basic Energy Sciences of the US

Department of Energy, and from the NCRR of the NIH grant

number P41RR012408. We thank Ms. Ling Duan for help with

the metabolism studies and Mr. Kebreab Ghebreselasie for

assistance with the sensitive COX-1 and COX-2 activity

screens. We are also grateful to Mr. Nathan Kett and Mr.

Sichen Chang (Craig Lindsley Research Group) for the

puriﬁcation of compounds via the mass-directed HPLC system.

The compounds disclosed in this report are protected by a US

Provisional Patent Application No 61/548,004 ﬁled on October

21, 2011 and converted to an International Patent Application

No PCT/US1242-96 on October 17, 2012.

ABBREVIATIONS USED

■

AA, arachidonic acid; Δ⁴-AD, 4-androstene-3,17-dione; ADT,

androgen deprivation therapy; AKR, aldo-keto reductase; AR,

androgen receptor; BME, β-mercaptoethanol; COX, cyclo-

oxygenases (PGH₂-synthase I and II); CRPC, castrate resistant

prostate cancer; DHEA, dehydroepiandrosterone; DHT, 5α-

dihydrotestosterone; DMSO, dimethyl sulfoxide; FLU, ﬂufe-

namic acid; IMN, indomethacin; LC−MS, liquid chromatog-

raphy−mass spectrometry; N-PA, N-phenylanthranilates;

NSAIDs, nonsteroidal anti-inﬂammatory drugs; PSA, prostate

speciﬁc antigen; TLC, thin-layer chromatography

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Development of potent and selective indomethacin analogues for the inhibition of AKR1C3 (type 5 17β-hydroxysteroid dehydrogenase/prostaglandin F synthase) in castrate-resistant prostate cancer

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