Morpholylureas are a new class of potent and selective inhibitors of the type 5 17-β-hydroxysteroid dehydrogenase (AKR1C3)

Article abstract of DOI:10.1016/j.bmc.2013.12.050

Inhibitors of the aldo-keto reductase enzyme AKR1C3 are of interest as potential drugs for leukemia and hormone-related cancers A series of non-carboxylate morpholino(phenylpiperazin-1-yl)methanones were prepared by palladium-catalysed coupling of substit

Full text of DOI:10.1016/j.bmc.2013.12.050

Bioorganic & Medicinal Chemistry 22 (2014) 967–977
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Morpholylureas are a new class of potent and selective inhibitors
of the type 5 17-b-hydroxysteroid dehydrogenase (AKR1C3)
Jack U. Flanagan a,b, Graham J. Atwell a, Daniel M. Heinrich a, Darby G. Brooke a, Shevan Silva a,
Laurent J. M. Rigoreau c, Elisabeth Trivier c, Andrew P. Turnbull c, Tony Raynham c,
Stephen M. F. Jamieson a,b, William A. Denny a,b,
⇑
a Auckland Cancer Society Research Centre, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
b Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
c Cancer Research Technology Ltd, Wolfson Institute for Biomedical Research, The Cruciform Building, Gower St., London WC1E 6BT, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Inhibitors of the aldo–keto reductase enzyme AKR1C3 are of interest as potential drugs for leukemia and
hormone-related cancers. A series of non-carboxylate morpholino(phenylpiperazin-1-yl)methanones
were prepared by palladium-catalysed coupling of substituted phenyl or pyridyl bromides with the
known morpholino(piperazin-1-yl)methanone, and shown to be potent (IC50 ꢀ 100 nM) and very iso-
form-selective inhibitors of AKR1C3. Lipophilic electron-withdrawing substituents on the phenyl ring
were positive for activity, as was an H-bond acceptor on the other terminal ring, and the ketone moiety
(as a urea) was essential. These structure–activity relationships are consistent with an X-ray structure of
a representative compound bound in the AKR1C3 active site, which showed H-bonding between the car-
bonyl oxygen of the drug and Tyr55 and His117 in the ‘oxyanion hole’ of the enzyme, with the piperazine
bridging unit providing the correct twist to allow the terminal benzene ring to occupy the lipophilic
pocket and align with Phe311.
Received 18 October 2013
Revised 12 December 2013
Accepted 21 December 2013
Available online 2 January 2014
Keywords:
Aldo–keto reductase
Inhibitor
Crystal structure
Computer aided drug design
Morpholylureas
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
that the carboxylate group of the drugs form direct H-bonds to
Y55 and H117 residues in the ‘oxyanion hole’ of the enzyme.
The type 5 17-b-hydroxysteroid dehydrogenase (AKR1C3) is a
member of the aldo–keto reductase (AKR) superfamily of en-
zymes, responsible for reducing androst-4-ene-3,17-dione, es-
trone and progesterone to, respectively, the growth-stimulatory
More recently we have shown7 that 3-(3,4-dihydroisoquinolin-
2(1H)-ylsulfonyl)benzoic acids (e.g., 7) are a novel class of highly
potent and selective inhibitors of AKR1C3, with the carboxylate
adopting a similar orientation.
steroid hormones testosterone, 17b-estradiol and 20
a
-hydroxpro-
Since carboxylic acids are likely to be transported into cells by
carrier-mediated processes rather than passive diffusion,8 there
are potential advantages in finding non-carboxylate inhibitors,
and we have recently reported9 a new class of phenylpyrrolidin-
2-ones (e.g., 8) that achieve this without forming a direct interac-
tion with the enzymes oxyanion hole. A potential advantage of
these non-carboxylate inhibitors is that they show relatively high-
er activity than the carboxylate-based inhibitors in cell-based as-
says (relative to their enzyme inhibitory potencies), arguably
because of superior uptake/transport properties.
gesterone,1 making it a potential therapeutic target in both breast
and prostate cancer. Inhibitors of AKR1C3 have become of partic-
ular interest since it was suggested2 that the broad observation
that non-steroidal anti-inflammatory drugs (NSAIDs) appear to
protect against a variety of cancers3 may be due in part to their
demonstrated inhibition of AKR1C3. Several NSAIDs including flu-
fenamic acid (1), indomethacin (2), naproxen (3), meclofenamic
acid (4), S(+)-ibuprofen (5) and flurbiprofen (6),4 and their ana-
logues,5,6 are known to have AKR1 isoform inhibitory activity
(Fig. 1). Crystal structures of complexes of several NSAIDs,2,7 show
In a previously-described10 high-throughput screen we identi-
fied the morpholino(phenylpiperazin-1-yl)methanone 24 (Fig. 1)
as a novel, reasonably potent (IC50 ꢀ 100 nM) and very isoform-
selective non-carboxylate inhibitor of AKR1C3. In this paper we re-
port the synthesis and structure–activity relationship (SAR) studies
of a new class of morpholino(phenylpiperazin-1-yl)methanone
AKR1C3 inhibitors derived from this lead.
Abbreviations: AKR, aldo–keto reductase; COX, cyclo-oxygenase; DCM, dichlo-
romethane; DIPEA, diisopropylethylamine; DMSO, dimethyl sulfoxide; NADPH,
nicotinamide adenine dinucleotide phosphate; NSAID, non-steroidal anti-inflam-
matory drugs; PBD, Protein Data Bank; TFA, trifluoroacetic acid.
⇑
Corresponding author. Tel.: +64 9 923 6144; fax: +64 9 3737502.
E-mail address: b.denny@auckland.ac.nz (W.A. Denny).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.12.050

968
J. U. Flanagan et al. / Bioorg. Med. Chem. 22 (2014) 967–977
O
2. Chemistry
Me
CO2H
MeO
Me
CO2H
CO2H
H
N
The bulk of the compounds of Table 1 were prepared by palla-
dium-catalysed coupling of substituted phenyl or pyridyl bromides
with the known morpholino(piperazin-1-yl)methanone (64),
which was prepared as reported11 from tert-butyl piperazine-1-
carboxylate (62) and morpholine-4-carbonyl chloride (63)
(Scheme 1A). The 4-iodide 26 was prepared from Cu-catalysed hal-
ogen exchange of 4-bromide 25, and was in turn used to prepare
the 4-ethyne 21 via a Sonogashira reaction12 with TMS-acetylene
to give the TMS adduct 65 which was then deblocked with K2CO3
to give 21. The 4-ester 27 was hydrolysed to acid 28, which was
used to make amides 29–31. Reduction of the 4-nitro analogue
33 gave amine 66, which was in turn used to prepare acetamide
34 and sulfonamide 36. The methylene-linked analogue 41 was
prepared by reaction of mono-BOC piperazine (62) and 4-chloro-
benzaldehyde (67) to give 68, followed by coupling of this with
morpholine-4-carbonyl chloride (63) (Scheme 1B). The carbonyl-
linked analogue 42 was prepared by direct coupling of 64 with
4-chlorobenzoyl chloride (69) (Scheme 1C).
F3C
N
Cl
3
1: flufenamic acid
2: indomethacin
: naproxen
F
OMe
Me
Me
CO2H
Cl
CO2H
H
N
Me
CO2H
Cl
4
: meclofenamic acid
5
: S(+)-ibuprofen
6: flurbiprofen
Me
O
S
O
O2
O
HO2C
S
N
O
N
N
N
N
N
O
7
8
24
Cl
Figure 1. Representative inhibitors of AKR1C3.
The bulk of the compounds of Table 2 were prepared from
either 1-(4-chlorophenyl)piperazine (70) and appropriate carbonyl
chlorides, or from the corresponding 4-(4-chlorophenyl)pipera-
zine-1-carbonyl chloride (71) and appropriate amines. The sulfox-
ide 51 was prepared from the sulfide 50 by oxidation with H2O2.
The acid 55 was prepared by basic hydrolysis of ester 72. Hydrox-
yamide 56 was prepared from the intermediate t-butyl ester 73 by
hydrolysis with TFA to 74, coupling of this with 3-chloro-3-oxopro-
pyl acetate to give 75, followed by C2CO3-mediated hydrolysis
(Scheme 2).
Sulfonylurea 58 and thiourea 59 of Table 3 were prepared by
base-catalysed condensation of 1-(4-chlorophenyl)piperazine
(70) with morpholine-4-sulfonyl chloride (77) or morpholine-
4-carbothioyl chloride (78), respectively. Compounds 60 and
61 were similarly prepared from base-catalysed condensation of
morpholine-4-carbonyl chloride (63) with 1-(4-chlorophenyl)-
1,4-diazepane13 (79) or 2-(4-chlorophenyl)- 2,5-diazabicyclo
[2.2.1]heptane14 (80) respectively (Scheme 3).
Table 1
Structural and biological data for phenyl-substituted analogues
O
O
Cl
N
N
N
N
2
N
O
N
O
3
4
X
X
A
B
a
No.
Fm
X
AKR IC50
1C3
(lM)
1C1
>30
1C2
1C4
>30
1C3 (cells)b
9
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
B
B
H
>30
6.1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
2-Me
2-OMe
2-Cl
2-CF3
3-Cl
3-CONH2
4-Me
4-OMe
4-Ph
12.1
20.3
2.3
0.70
5.3
>30
0.36
0.35
>30
4.9
>30
>30
>30
>30
>30
>30
>30
>30
>30
3. Enzyme biochemistry and cell biology
>30
>30
>30
>30
>30
>30
The structures of the new compounds and their activities in the
enzyme and cellular assays are shown in Tables 1–3. The inhibition
assay of the compounds against the AKR1 isoforms C1–C4 was per-
formed with a competitive fluorescence assay, where a non-fluo-
rescent ketone probe (probe 5)15 selective for the AKR1C enzyme
isoforms is reduced to a fluorescent alcohol in the presence of
AKR1C enzyme and NADPH. Compounds with biochemical IC50 val-
4,5-Benz
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
3-Aza-4,5-benz
4-C„CH
4-CF3
4-F
4-Cl
4-Br
4-I
4-CO2Me
4-CO2H
4-CONH2
4-CONHMe
4-CONMe2
4-SO2Me
4-NO2
4-NHCOMe
4-NHSO2Me
2,4-diCl
2-Me, 4-Cl
2-CH2OH, 4-Cl
3-Aza, 4-Cl
2-Aza, 4-Br
CH2
2.9
0.66
0.068
0.50
0.11
0.14
0.067
>30
>30
>30
>30
>30
5.52
0.57
>30
>30
0.025
0.11
0.16
0.53
0.10
0.35
0.48
0.022
0.018
0.012
0.01
ues <0.3 lM, plus the two most potent morpholine-ring replace-
ments (56 and 57) were tested for activity at inhibiting the
AKR1C3-dependent aerobic reduction of the dinitrobenzamide
PR-104A to its hydroxylamine metabolite in an AKR1C3-over-
expressing HCT116 cell line. Compound IC50 values were calcu-
lated by fitting the inhibition data to a four-parameter logistic
sigmoidal dose–response curve using Prism 5.02 (GraphPad, La Jol-
la, CA, USA). The methanone compounds are competitive inhibitors
of AKR1C3; their effectiveness can be reduced by increasing sub-
strate concentration (data not shown).
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
0.003
0.013
0.013
4. Crystallography and modelling
0.031
The structure of 24 bound in the AKR1C3 active site was deter-
mined by X-ray crystallography, as detailed previously10 (see
Table 4 for crystallographic parameters). Figure 2 shows that the
carbonyl interacts with the oxyanion hole residues Ty55 and
His117, while the terminal benzene ring aligns with Phe311 of
the SP1 pocket.16 The morpholine oxygen may contact Leu-54 via
CO
a
IC50 values were determined using a competitive fluorescence assay; see Sec-
tion 7. Values <1 M represent the mean for 2 or more determinations.
Only compounds with AKR1C3 IC50 values <0.3 M were evaluated in the cel-
lular assay.
l
b
l

J. U. Flanagan et al. / Bioorg. Med. Chem. 22 (2014) 967–977
969
O
O
O
i, ii
iii
A
Cl
+
N
N
N
NH
N
N
O
HN
O
O
N
N
O
64
R
63
62
O
9-
compounds
O
20, 22-25, 27,
32, 33, 36-40,
42 of Table 1
N
N
N
O
25
: R=Cl
iv
v
R
26: R=I
65
vi
21
compound
of Table 1
of Table 1
CSiMe3
: R=
vii
viii
N
27
28
compounds 29-31 of Table 1
O
N
N
x, xi
ix
34 35
compounds
,
33
O
66
H2N
B
C
Cl
Cl
xii
NH
41
compound
xiii
62
64
+
63
+
of Table 1
N
CHO
67
68
O
Cl
+
xiii
42
compound
of Table 1
Cl
69
Scheme 1. Reagents and conditions. (i) DIPEA, CH2Cl2; (ii) TFA; (iii) ArBr, Cs2CO3, BINAP, Pd(OAc)2, toluene, reflux; (iv) (1R,2R)-N1,N2-dimethylcyclohexane-1,2-diamine, CuI,
NaI, dioxane; (v) TMS-acetylene, CuI, (Ph3P)PdCl2, Et3N, DMF, then Cs2CO3, MeOH; (vi) K2CO3, MeOH; (vii) aqueous NaOH; (viii) (COCl)2, DMF, DCM, then RNH2/RRNH; (ix)
Pd-C, H2, THF; (x) Ac2O; (xi) MsCl; (xii) BBN, then Pd(dppf)Cl2, 4-bromo-1-chlorobenzene, K2CO3, DMF, then TFA; (xiii) DIPEA, DCM.
a structured water molecule. Analogues were docked with the core
of the molecule, represented by 9) restrained to the binding mode
of 24 and then used to find hotspots within the SP1 pocket that can
improve affinity.
a previous study.7 Of more interest were compounds 32 and 33,
bearing the polar but very electron-withdrawing substituents SO2-
Me and NO2, respectively, and which showed weak to moderate
activity (IC50s 5.5 and 0.57 lM, respectively).
The disubstituted compounds 36–40 showed patterns of activity
consistent with the above. The 2,4-dichloro analogue 36 was the
most potent compound in the series (IC50 0.025 lM), while the 2-
5. Results and discussion
CH2OH, 4-Cl compound 38 and the pyridine analogues 39 and 40
also had modest potency. Collectively these results suggested that
both the lipophilicity and electron-withdrawing properties of sub-
stituents might make positive contributions, and this was sup-
ported by a QSAR study (Eq. 1) of all the active compounds with
no H-bond donors in Table 1 for which descriptive parameter values
were available (compounds 9–14, 16–18, 21–26, 31, 33, 36, 37)
The inhibitory properties of the compounds for the four AKR1C
enzyme isoforms used a competitive fluorescence binding assay
where a non-fluorescent ketone probe is reduced to a fluorescent
alcohol in the presence of the AKR1C enzymes isoforms.10 Com-
pounds 9–39 explore SAR around the aromatic ring, which is
shown in the crystal structure of the 4-chloro compound 24 bound
to AKR1C3 (Fig. 2) to occupy the SP1 pocket bounded by the side
chains of Ser118, Met120, Asn167, Tyr216, Phe306, Phe311,
Tyr317, Pro318 and Tyr319 and the ordered water HOH28. The
Log IC50ðAKR1C3Þ ¼ ꢁ0:70ðꢂ0:24Þ
N = 19 R = 0.62 F = 4.64
r
ꢁ 0:70ðꢂ0:54Þ
p
þ 0:33
ð1Þ
unsubstituted analogue
10–13 showed good AKR1C3 selectivity and moderate to weak
potencies (IC50s 0.7–20 M). The 3-Cl analogue 14 also had modest
9 and the 2-substituted compounds
Compounds 43–58 in Table 3 explore the binding site for the
terminal substituent of the ketone moiety that binds the oxyanion
hole. In the crystal structure of 24 bound to AKR1C3 (Fig. 2) the
morpholine oxygen is within hydrogen bonding distance to a
structured water (HOH556) that is part of a network located in
the SP3 pocket, and the importance of this was examined. The
complete inactivity of compounds 43–47 suggest that there is a
requirement for a secondary aliphatic nitrogen (i.e., a urea moiety)
and an H-bond acceptor, although 48 and 49 show that mainte-
nance of only one of these can result in some level of AKR1C3 activ-
ity and (in the case of 49) isoform selectivity. The thiourea 50
l
activity, while the polar 3-CONH2 substituent in 15 abolished
activity. The 4-position was much more favoured (as illustrated
by the 2-, 3- and 4-Cl analogues 12, 14, 24 retrieving IC50s of 2.3,
5.3 and 0.011 lM, respectively), so 4-substituents were studied
in more detail (compounds 16–34). There was a clear distinction
between the utility of lipophilic (compounds 16–26; IC50s
0.07–4.9
l
M) and polar H-bond donor and acceptor containing
M) substituents. The
compounds (compounds 27–35; IC50s >30
l
relative effectiveness of these substituents suggests that interac-
tions with the lipophilic binding pocket are largely non-specific
van der Waals interactions, with the potential for little specific
H-bond contribution, although steric effects may also contribute
to the negative effect on activity.
shows a low level of inhibitory activity (9.1 lM), but the sulfoxide
51, the sulfone 52 and the N-methyl- and N-(2-hydroxyethyl)-
piperazines 53 and 54 were all inactive. The 4-carboxypiperidine
55 showed some minimal activity (IC50 22
2-hydroxyacetamide 56 was respectably active (IC50 0.46
along with the tetrahydroisoquinoline 57 (IC50 0.35 M).
l
M), while the
lM),
Exceptions included compound 18 (4-phenyl), which was
inactive, but this was likely due steric hindrance, a point noted in
l

970
J. U. Flanagan et al. / Bioorg. Med. Chem. 22 (2014) 967–977
Table 2
R
ii
N
Structural and biological data for morpholine ring replacements
51
compound
of Table 2
N
O
iv
43-50, 57
compounds
of Table 2
Cl
N
R
70: R=H
N
i
iii
71: R=COCl
compounds 52-54 of Table 2
Cl
O
a
v
No.
R
AKR IC50
1C3
(lM)
vi
CO2Me
N
N
55
compound
71
1C1
1C2
1C4
1C3b (cells)
of Table 2
N
N
72
Cl
43
44
45
46
47
48
49
50
51
O
>30
>30
>30
>30
>30
12.6
3.8
O
N
N
vii
Cl
x
56
compound
of Table 2
71
62
+
N
N
R
73
: R = CO2tBu
viii
ix
HN
HN
74: R = H
75: R = CO(CH2)2OCOMe
Scheme 2. Reagents and conditions: (i) Triphosgene, pyridine, DCM; (ii) HN-RR,
DIPEA, DCM; (iii) RRN-COCl, DIPEA, DCM. (iv) H2O2; (v) methyl piperidine-4-
carboxylate, DIPEA; (vi) MeOH, NaOH; (vii) DIPEA; (viii) TFA, DCM; (ix) 3-chloro-3-
oxopropyl acetate, DIPEA; (x) Cs2CO3.
N
N
>30
>30
>30
>30
>30
>30
S
S
N
N
9.1
Table 3
Structural and biological data for morpholides with varying linker groups
O
>30
a
No.
R
AKR IC50
1C2
(lM)
O
1C1
1C3
1C4
S
52
53
54
55
>30
>30
>30
21.9
N
N
O
O
O
S
NMe
N
N
N
58
>30
N
O
OH
N
N
Cl
S
CO2H
OH
N
N
N
59
60
61
>30
30
N
N
O
56
57
>30
>30
>30
>30
0.46
0.35
>30
>30
0.059
0.39
N
N
O
Cl
Cl
O
a
As for Table 1.
N
N
b
Only compounds with AKR1C3 IC50 values <0.5 lM were evaluated in the cel-
O
lular assay.
O
N
N
Analogues 58–61 in Table 3 demonstrate the importance of the
oxyanion binding moiety and the piperazine bridge that links this
to the aromatic ring. Displacing or replacing the ketone that makes
the two H-bond contacts with the Tyr55 and His117 residues in the
oxyanion hole with either the sulfone 58 or thioketone 59 abol-
ishes binding. The crystal structure (Fig. 2 and Table 4) also shows
the importance of the piperazine bridging unit in providing the
correct twist to allow the benzene ring to occupy the SP1
pocket.5,16 Altering this alignment with either a homo (60) or
bridged (61) piperazine respectively abolishes or greatly reduces
AKR1C3 inhibitory activity.
>30
>30
3.7
>30
O
N
Cl
a
As for Table 1.
S
O
O
S
Cl
N
N
Cl
O
O
77
78
58
compound
of Table 3
59
To find SP1 pocket hot spots that improved the IC50, compounds
from Table 1 substituted around the phenyl ring that retrieved IC50
compound
of Table 3
70
i
i
values lower than that of compound 9 (6.1 lM) were docked into
the AKR1C3 active site using the X-ray determined binding mode
of compound 24 to position the core of the molecule. Compounds
with lipophilic substitutions at the 4-position retrieved IC50 values
NH
NH
N
N
80
79
less than 0.2 lM (22, 26, 36, 24, 25, 37, 38 and 40) and were pre-
Cl
i
compound 60
of Table 3
Cl
compound 61
63
dicted to probe a subsite within the SP1 pocket defined by the side
chains of amino acids Tyr319, Tyr317, Phe311, Met120 and Pro318,
with the larger halide based substitutions of (22 (Fig. 3) and 26)
of Table 3
i
i
Scheme 3. Reagents: (i) DIPEA, DCM.

J. U. Flanagan et al. / Bioorg. Med. Chem. 22 (2014) 967–977
971
Table 4
X-ray data collection and refinement statistics for compound 24
Data collection:
Refinement:
Resolution
Wavelength (Å)
Space group
1.54182
P212121
18.53–2.40 (2.46–2.40)
a
Rwork
0.219 (0.394)
0.297 (0.532)
13,488 (806)
702 (44)
30.0/31.1
26.0
30.3
0.0150
1.7795
97.0/0.3
6.3
b
Unit cell parameters (Å; °)
Resolution (Å)
Number of molecules/AU
Total number of reflections
Unique reflections
Multiplicity
58.0, 64.1, 96.4
18.53–2.40 (2.49–2.40)
1
47,000 (3213)
14,191 (1277)
3.3 (2.5)
97.6 (90.7)
12.3 (2.8)
0.068 (0.246)
49.9
Rfree
No. of working reflections
No. of free reflections
Average B factor protein main-chain/side-chain (Å2)
Average B factor ligands (Å2)
Average B factor water (Å2)
RMSD bond (Å)
Completeness (%)
Mean <I/
Rmerge
r
I>
RMSD angles (°)
Ramachandran most favoured/outliers (%)
Molprobity all-atom clash score
Wilson B (Å2)
Data were collected using a Rigaku Saturn 944+ CCD detector mounted on a Rigaku Micromax-007 HF rotating anode X-ray generator.
Numbers in parentheses represent data in the highest resolution shell.
a
Rwork
=
R
R
||F(obs)|ꢁ|F(calc)||/
R
|F(obs)| for the 95% of the reflection data used in refinement.
b
Rfree
=
||F(obs)|ꢁ|F(calc)||/
R|F(obs)| for the remaining 5%.
Figure 3. Predicted binding mode for 22 in the AKR1C3 active site. The oxy-anion
hole residues Tyr-55 and His117 are shown in white sticks, and NADP+ in purple
sticks. Compound 24 is shown in green sticks, and the interaction between its
central carbonyl group and the side chain hydroxyl group of Tyr55 is shown as a
black dashed line. The SP1 pocket region that the 4-phenyl-CF3 of 22 is predicted to
interact is shown in cyan sticks and 22 is shown in orange sticks.
Figure 2. Crystal structure of 24 complexed with AKR1C. PDB: Protein Data Bank
(PDB ID: 4HMN). Soaking experiment: AKR1C3+NADP+ crystal soaked with 5 mM
compound 24 for 3 days. Space group P212121; cell dimensions: a = 58.0, b = 64.1,
c = 96.4 Å. Resolution = 2.4 Å).
better suited to this site. This subsite is also used by the non-steroi-
dal anti-inflammatory drugs naproxen (PDB codes 3UFY, 3R58)
ibuprofen (PDB code 3R8G), zomepirac (PDB code 3R8H) and flur-
biprofen (PDB code 3R94).7 The 4-substituted 33 was predicted to
place an acceptor group close to the side chain hydroxyl of Tyr319,
part of the water binding site created by Glu192, Tyr216 and
Tyr319. The top ranked predicted mode binding for the 3-substi-
tuted, compound 13, consistent with the orientation of compound
24, placed the chloride toward the empty water binding site cre-
ated by Tyr216, Tyr319 and Glu192.
Comparison of the X-ray crystal structures of 24 and 7 (PDB
code 4FAM) bound in the AKR1C3 active site showed that
the N-linked phenyl group of 24 occupied a different region
of the SP1 binding pocket to the dihydroisoquinoline moiety of 7.
The side chains of Trp227 and Phe311 as well as the loop contain-
ing Phe311 adopted an alternate conformation to accommodate
the dihydroisoquinoline moiety. While the potency of 7 was re-
ported to be tolerant to many dihydroisoquinoline substitutions,10
modelling the likely binding modes of these analogues based on
that of 7 indicated that 5-position halide and amine substitutions
extended toward the same space as the 4-chloro substituted
phenyl ring of 24.
AKR1C isoform selectivity was determined for all compounds
with AKR1C3 IC50s <10
have minimal inhibitory activity for AKR1C1, 2 and 4 (IC50s
>30 M). A representative compound, 24, was also screened for
inhibition of COX-1 and COX-2, but was inactive in both assays
at 10 M.
Compounds of interest or with IC50s <0.3
lM, with all compounds tested found to
l
l
lM against isolated
AKR1C3 were also evaluated in an AKR1C3 overexpressing
HCT-116 human colon cancer cell line for their effectiveness in
inhibiting the 2-electron reduction of PR-104A, an exogenous
dinitrobenzamide substrate that is exclusively metabolised by
AKR1C3 under aerobic conditions to its cytotoxic 4-hydroxylamine
(PR-104H) and 4-amine (PR-104 M) metabolites17 as described
previously.10 For these compounds (22, 24–27, 36–38, 46,
56, 57), there was a broad rank order correlation between their
enzymatic and cellular activity, but an average 3-fold higher
potency in cells. This is in contrast with analogues of the carboxylic
acid 7 which, while showing a similar broad rank order correlation
between enzymatic and cellular activity, were on average 4-fold
less potent in cells than in isolated enzyme assays.10

972
J. U. Flanagan et al. / Bioorg. Med. Chem. 22 (2014) 967–977
Prism 5.02. Representative compounds were tested over repeat as-
6. Conclusions
says to ensure assay reproducibility.
While a considerable number of inhibitors of AKR1C3 have been
reported, the vast majority are carboxylates, where this moiety
binds to the ‘oxyanion hole’ of the enzyme. The present series pro-
vides a new class of non-carboxylate inhibitors that bind selec-
tively to AKR1C3 via the carbonyl oxygen of the central urea
linker. This activity is favored by lipophilic electron-withdrawing
substituents on the phenyl ring that probe specific regions of the
SP1 pocket and H-bond acceptors on the other terminal ring. This
is consistent with the X-ray structure of 24 bound in the AKR1C3
active site, which shows interactions between the carbonyl oxygen
of the drug and Tyr55 and His117 in the ‘oxyanion hole’ of the en-
zyme, with the piperazine bridging unit providing the correct twist
to allow the terminal benzene ring to occupy the lipophilic pocket
and align with Phe311. These neutral compounds had an average
3-fold higher potency for inhibition of AKR1C3 in cells compared
with isolated enzymes. Compound 24 has been used, in conjunc-
tion with the pan-AKR1 fluorogenic substrate coumberone, to eval-
uate AKR1C3 activity in human leukaemia cells.18
7.3. Chemistry
Combustion analyses were performed by the Campbell Micro-
analytical Laboratory, University of Otago, Dunedin, New Zealand.
Melting points were determined on an Electrothermal IA9100
melting point apparatus, and are as read. NMR spectra were mea-
sured on a Bruker Avance 400 spectrometer at 400 MHz for 1H and
are referenced to Me4Si. Chemical shifts and coupling constants are
recorded in units of ppm and Hertz, respectively. High-resolution
electron impact (HREIMS) and fast atom bombardment (HRFABMS)
mass spectra were determined on a VG-70SE mass spectrometer at
nominal 5000 resolution. High-resolution electrospray ionisation
(HRESIMS) and atmospheric pressure chemical ionisation (HRAPC-
IMS) mass spectra were determined on a Bruker micrOTOF-Q II
mass spectrometer. Low-resolution atmospheric pressure chemical
ionisation (APCI) mass spectra were measured for organic solutions
on a ThermoFinnigan Surveyor MSQ mass spectrometer, connected
to a Gilson autosampler. Thin-layer chromatography was carried
out on aluminium-backed silica gel plates (Merck 60 F254), with
exposure to I2, or by staining in permanganate and phosphomolyb-
dic acid dips and by UV light (254 nm & 365 nm). Column chroma-
tography was carried out on silica gel (Merck 230–400 mesh).
7. Experimental section
7.1. COX assays
These were were carried out by GVK Biosciences Ltd., Biology,
28A, IDA Nacharam, Hyderabad 500 076 India (www.gvkbio.com).
For details see Ref. 10.
7.3.1. Compounds of Table 1 (Scheme 1). (4-(4-
Chlorophenyl)piperazin-1-yl)(morpholino)methanone (24)
A solution of tert-butyl piperazine-1-carboxylate (62) (4.0 g,
21.5 mmol) in DCM (30 mL) was treated with DIPEA (5.61 mL, 1.5
equiv) and this solution was cooled to 0 °C and treated dropwise
with a solution of morpholine-4-carbonyl chloride (63) (3.85 g,
25.7 mmol) in DCM (10 mL). The mixture was stirred at 20 °C for
30 min, then diluted with DCM, washed with aqueous Na2CO3,
dried and evaporated. The residue was triturated with EtOH, then
dissolved in DCM (30 mL) and treated with TFA (30 mL). The solu-
tion was stirred at 20 °C for 2 h and evaporated. The residue was
shaken with DCM and 4 N NH4OH, the aqueous layer was back-ex-
tracted with DCM (4ꢃ) and the combined organic extracts were
dried and evaporated to give morpholino(piperazin-1-yl)metha-
none7 (64) (70%). 1H NMR (CDCl3) d 3.55 (t, J = 4.7 Hz, 4H), 3.14–
3.02 (m, 8H), 2.64 (t, J = 4.9 Hz, 4H), 2.25 (br s, 1H). HPLC purity
98.8%
A mixture of 64 (1.075 g, 5.5 mmol), 1-bromo-4-chlorobenzene
(1.03 g, 5.4 mmol), Cs2CO3 (2.28 g, 7 mmol) and BINAP (0.05 equiv)
in toluene (60 mL) was purged with N2, Pd(OAc)2 (110 mg,
0.22 mmol) was added and the mixture was stirred under reflux
for 16 h, then cooled, diluted with EtOAc, filtered and evaporated.
The residue was chromatographed on silica gel, with EtOAc/CH2Cl2
(2:1) eluting 24 (0.62 g. 37%), mp (EtOAc/pet ether) 121–123 °C. 1H
NMR (CDCl3) d 7.24 (br d, J = 4.5 Hz, 2H), 6.95 (br d, J = 4.5 Hz, 2H,
3.57 (t, J = 4,7 Hz, 4H), 3.32–3.25 (m, 4H), 3.20–3.08 (m, 8H). HPLC
purity 99.3%.
7.2. Measurement of AKR1C enzyme activity
Recombinant AKR1C protein was produced and purified as de-
scribed previously.10 A competitive fluorescence assay was used
to measure AKR1C enzyme activity, where a non-fluorescent ke-
tone probe (probe 5)15 selective for the AKR1C enzyme isoforms
is reduced to a fluorescent alcohol in the presence of AKR1C en-
zyme and NADPH. Briefly, purified protein (2
g/mL AKR1C2, 2 g/mL AKR1C3 and 5 g/mL AKR1C4) was
incubated with 40 M probe 5, test compounds and 50 M NADPH
lg/mL AKR1C1,
1
l
l
l
l
l
in an assay buffer of 10 mM MOPS (pH = 7.2), 130 mM NaCl, 1 mM
DTT and 0.01% Triton-X-100 for 1 h at 37 °C. The reaction was
stopped by addition of 35 mM NaOH and fluorescence was read
in a SpectraMax M2 microplate reader (Molecular Devices, Sunny-
vale, CA, USA) at excitation/emission wavelengths of 420/510 nM.
The compounds and known AKR1C3 inhibitors (flufenamic acid,
indomethacin, naproxen, meclofenamic acid, S(+)-ibuprofen and
flurbiprofen; Sigma–Aldrich, Auckland, New Zealand) were tested
at multiple concentrations between 0.1 nM and 100 lM in 2%
DMSO to generate AKR1C enzyme inhibition data. Compound
IC50 values were calculated by fitting the inhibition data to a
four-parameter logistic sigmoidal dose–response curve using
Prism 5.02 (GraphPad, La Jolla, CA, USA).
AKR1C3 activity was determined in HCT-116 cells engineered to
over-express AKR1C3 by measuring the major metabolite (PR-
104H) of PR-104A produced by 2-electron reduction under aerobic
conditions, a reaction catalysed selectively by AKR1C3.17 The syn-
thesis of PR-104A, PR-104H and tetradeuterated PR-104H,19,20 the
transfection and maintenance of the HCT-116/AKR1C3 cell line17
and the measurement of PR-104H by LC–MS/MS have been de-
scribed previously.21 The compounds were administered 2 h prior
7.3.2. Morpholino(4-phenylpiperazin-1-yl)methanone (9)
Similar reaction of 64 and 1-bromobenzene gave 9 (45%), mp
(hexane) 67–68 °C. 1H NMR (CDCl3) d 7.22 (br dd, J = 8.8, 7.3 Hz,
2H), 6.94 (br dd, J = 8.8, 1.0 Hz, 2H), 6.80 (t, J = 7.3 Hz, 1H), 3.58
(t, J = 4.7 Hz, 4H), 3.32–3.24 (m, 4H), 3.17 (t, J = 4.8 Hz, 4H), 3.15–
3.09 (m, 4H). HPLC purity 100%.
to 100
and 3
l
M PR-104A at multiple concentrations between 1 nM
7.3.3. Morpholino(4-(o-tolyl)piperazin-1-yl)methanone (10)
Similar reaction of 64 and 1-bromo-2-methylbenzene gave 10
(40%), mp (hexane) 134–135 °C. 7.20–7.11 (m, 2H), 7.04–6.93 (m,
2 H), 3.58 (t, J = 4.7 Hz, 4H), 3.32 (t, J = 4.8 Hz, 4H), 3.17 (t,
lM. PR-104H formation was quantitated against a PR-
104H standard curve ranging from 1 to 1000 nM. Compound IC50
values were calculated from four-parameter logistic sigmoidal
dose–response curves that were fitted to the inhibition data using

J. U. Flanagan et al. / Bioorg. Med. Chem. 22 (2014) 967–977
973
J = 4.7 Hz, 4H), 2,81 (t, J = 4,8 Hz, 4H), 2.26 (s, 3H). HPLC purity
99.0%.
7.3.12. Morpholino(4-(naphthalen-2-yl)piperazin-1-yl)methano
ne (19)
Similar reaction of 64 and 2-bromonaphthalene gave 19 (68%),
mp (EtOAc/pet ether) 149–152 °C. 1H NMR (CDCl3) d 7.79–7.69
(m, 3H), 7.42–7.34 (m, 2H), 7.30–7.24 (m, 1H), 7.18 (d, J = 2.3 Hz,
1H), 3.59 (t, J = 4.7 Hz, 4H), 3.39–3.32 (m, 4H), 3.28–3.16 (m, 4H).
HPLC purity 96.9%.
7.3.4. (4-(2-Methoxyphenyl)piperazin-1-yl)(morpholino)metha
none (11)
Similar reaction of 64 and 1-bromo-2-methoxybenzene gave 11
(65%), mp (EtOAc/pet ether) 113–114 °C. 1H NMR (CDCl3) d 6.98–
6.81 (m, 4H), 3.76 (s, 3H), 3.56 (t, J = 4.6 Hz, 4H), 3.33–3.20 (m, 4
H), 3.14 (t, J = 4.6 Hz, 4H), 2.91 (t, J = 4.7 Hz, 4H). HPLC purity 100%.
7.3.13. Morpholino(4-(quinolin-3-yl)piperazin-1-yl)methanone
(20)
Similar reaction of 64 and 3-bromoquinoline gave 20 (62%), mp
(EtOAc/pet ether) 156–157 °C. 1H NMR (CDCl3) d 8.87 (d, J = 2.9 Hz,
1H), 7.90–7.84 (m, 1H), 7.82–7.75 (m, 1H), 7.56 (d, J = 2.8 Hz, 1H),
7.54–7.46 (m, 2H), 3.59 (t, J = 4.7 Hz, 4H), 3.42–3.34 (m, 4H), 3.34–
3.26 (m, 4H), 3.20 (t, J = 4.7 Hz, 4H).m, 4H). HPLC purity 98.0%.
7.3.5. (4-(2-Chlorophenyl)piperazin-1-yl)(morpholino)methano
ne (12)
Similar reaction of 64 and 1-bromo-2-chlorobenzene gave 12
(48%), mp (EtOAc/pet ether) 137–139 °C. 1H NMR (CDCl3) d 7.42
(dd, J = 7.9, 1,5 Hz, 1H), 7.33–7.26 (m, 1H), 7.16 (dd, J = 8.1,
1.5 Hz, 1H), 7.06 (td, J = 7.6, 1.4 Hz, 1H), 3.58 (t, J = 4.7 Hz, 4H),
3.34 (t, J = 4.8 Hz, 4H), 3.17 (t, J = 4.7 Hz, 4H), 2.95 (t, J = 4.8 Hz,
4H). HPLC purity 100%.
7.3.14. (4-(4-Ethynylphenyl)piperazin-1-yl)(morpholino)metha
none (21)
A
mixture of (4-(4-iodophenyl)piperazin-1-yl)(morpho-
lino)methanone (26) (80 mg. 0.2 mmol), CuI (3 mg, 0.01 mmol)
and (Ph3P)2PdCl2 (104 mg, 0.01 mmol)) in dry THF (2 mL) was
flushed with N2. A solution of trimethylsilylacetylene (25 mg,
0.25 mmol) in Et3N (2 mL) was added and the reaction was stirred
at 20 °C for 15 h. The mixure was evaporated and the residue was
chromatographed on silica gel to give crude morpholino(4-(4-((tri-
methylsilyl)ethynyl)phenyl)piperazin-1-yl)methanone (65) (58%).
7.3.6. Morpholino(4-(2-(trifluoromethyl)phenyl)piperazin-1-yl)
methanone (13)
Similar reaction of 64 and 1-bromo-2-(trifluoromethyl)benzene
gave 13 (63%), mp (hexane) 110–112 °C. 1H NMR (CDCl3) d 7.71–
7.62 (m, 2H), 7.55 (d, J = 7.9 Hz, 1H), 7.35 (t, J = 7.6 Hz, 1H), 3.58
(t, J = 4.7 Hz, 4H), 3.32–3.20 (m, 4H), 3.17 (t, J = 4.7 Hz, 4H), 2.84
(t, J = 4.8 Hz, 4H). HPLC purity 88.1%.
A
mixture of 65 (41 mg, 0.11 mmol) and powdered K2CO3
(41 mg, 0.30 mmol) in MeOH (5 mL) was stirred at 20 °C for 3 h,
then evaporated to dryness. The residue was extracted with EtOAc
and the solution was filtered through silica gel to give 21 (30 mg,
91%), mp (EtOAc/pet ether) 159–161 °C. 1H NMR (CDCl3) d 7.31
(d, J = 8.8 Hz, 2H), 6.91 (d, J = 8.9 Hz, 2H), 3.93 (s, 1H), 3.60–3.54
(m, 4H), 3.32–3.24 (m, 4H), 3.24–3.14 (m, 8H). HPLC purity 99.0%.
7.3.7. (4-(3-Chlorophenyl)piperazin-1-yl)(morpholino)methano
ne (14)
Similar reaction of 64 and 1-bromo-3-chlorobenzene gave 14
(57%), mp (EtOAc/pet ether) 83–84 °C. 1H NMR (CDCl3) d 7.22 (t,
J = 8.1 Hz, 1H), 6.95 (t, J = 2.1 Hz, 1H), 6.92–6.87 (m, 1H), 6.83–
6.78 (m, 1H), 3.60–3.55 (m, 4H), 3.32–3.26 (m, 4H), 3.21–3.14
(m, 8H). HPLC purity 98.3%.
7.3.15. Morpholino-(4-(4-(trifluoromethyl)phenyl)piperazin-1-
yl)methanone (22)
Similar reaction of 64 and 1-bromo-4-(trifluoromethyl)benzene
gave 22 (63%), mp (hexane) 117–118 °C. 1H NMR (CDCl3) d 7.52 (d,
J = 8.7 Hz, 2H), 7.06 (d, J = 8.7 Hz, 2H, 3.58 (t, J = 4.7 Hz, 4H), 3.32–
3.22 (m, 8H), 3.18 (t, J = 4.7 Hz, 4H. HPLC purity 99.8%.
7.3.8. 3-(4-(Morpholine-4-carbonyl)piperazin-1-yl)benzamide
(15)
Similar reaction of 64 and 3-bromobenzamide gave 15 (43%),
mp (CH2Cl2/pet ether) 239–242 °C. 1H NMR (CDCl3) d 7.88 (s,
2H), 7.43 (s, 2H), 7.33–7.20 (m, 3H), 7.09 (dt, J = 4.7 Hz, 1H), 3.58
(m, 4H), 3.22–3.13 (m. 8H). HPLC purity 97.9%.
7.3.16. (4-(4-Fluorophenyl)piperazin-1-yl)(morpholino)metha
none (23)
Similar reaction of 64 and 1-bromo-4-fluorobenzene gave 23
(86%), mp (hexane) 102–103 °C. 1H NMR (CDCl3) d 7.10–6.92 (m,
4H), 3.62–3.53 (m, 4H), 3.31–3.23 (m, 4H), 3.21, 3.14 (m, 4H),
3.10–3.02 (m, 4H). HPLC purity 98.7%.
7.3.9. Morpholino(4-(p-tolyl)piperazin-1-yl)methanone (16)
Similar reaction of 64 and 1-bromo-4-methylbenzene gave 16
(83%) mp (hexane) 97–98 °C. 1H NMR (CDCl3) d 7.03 (br d,
J = 8.3 Hz, 2H), 6.84 (be d, J = 8.6 Hz, 2H), 3.57 (t, J = 4.7 Hz, 4H),
3.31–3.23 (m, 4H), 3.17 (t, J = 4.7 Hz, 4H), 3.05 (t, J = 5.0 Hz, 4H),
2.20 (s, 3H). HPLC purity 96.2%.
7.3.17. (4-(4-Bromophenyl)piperazin-1-yl)(morpholino)
methanone (25)
Similar reaction of 64 and 1,4-dibromobenzene gave 25 (52%),
mp (EtOAc/pet ether) 145–147 °C. 1H NMR (CDCl3) d 7.35 (d,
J = 9.0 Hz, 2H), 6.90 (d, J = 9.1 Hz, 2H), 3–60–3.55 (m, 4H), 3.31–
3.22 (m, 4H) (after D2O), 3.20–3.10 (m, 8H). HPLC purity 97.3%.
7.3.10. (4-(4-Methoxyphenyl)piperazin-1-yl)(morpholino)meth
anone (17)
Similar reaction of 64 and 1-bromo-4-methoxybenzene gave 17
(52%), mp (hexane) 116–117 °C. 1H NMR (CDCl3) d 6.90 (br d,
J = 9.1 Hz, 2H), 6.82 (br d, J = 9.1 Hz, 2H), 3.68 (s, 3H), 3.60–3.55
(m, 4H), 3.31–3.24 (m, 4H), 3.20–3.13 (m, 4H), 3.02–2.95 (m,
4H). HPLC purity 99.4%.
7.3.18. (4-(4-Iodophenyl)piperazin-1-yl)(morpholino)methan
one (26)
A mixture of 25 (177 mg, 0.50 mmol), NaI (150 mg, 1.0 mmol),
CuI (5 mg, 0.025 mmol) and (1S,2S)-N1,N2-dimethylcyclohexane-
1,2-diamine (7 mg, 0.04 mmol) in dioxane (1 mL) was purged with
N2, then heated in a sealed tube at 110 °C for 48 h. The mixture was
evaporated, shaken with water and DCM, filtered and the organic
layer was dried and evaporated. The residue was dissolved in
EtOAc and filtered through silica gel to give 26 (75%), mp (EtOAc/
7.3.11. (4-(Biphenyl-4-yl)piperazin-1-yl)(morpholino)methan
one (18)
Similar reaction of 64 and 1-bromobiphenyl gave 18 (52%), mp
(EtOAc)185–186 °C. 1H NMR (CDCl3) d 7.63–7.57 (m, 2H), 7.55 (br
d, J = 8.8 Hz, 2H), 7.44–7.37 (m, 2H), 7.30–7.24 (m, 1H), 7.03 (br d,
J = 8.9 Hz, 2H), 3.58 (t, J = 4.7 Hz, 4H), 3.37–3.25 (m, 4H), 3.22–3.12
(m, 4H). HPLC purity 95.9%.

974
J. U. Flanagan et al. / Bioorg. Med. Chem. 22 (2014) 967–977
pet ether) 151–153 °C. 1H NMR (CDCl3) d 7.50 (d, J = 9.0 Hz, 2H),
6.79 (d, J = 9.0 Hz, 2H), 3.51–3.54 (m, 4H), 3.31–3.24 (m, 4H),
3.20–3.08 (m, 8H). HPLC purity 94.2%.
(br d, J = 9.4 Hz, 2H), 7.02 (br d, J = 9.5 MHz, 2H), 3.58 (t,
J = 4.7 Hz, 4H), 3.52–3.45 (m, 4H), 3.36–3.28 (m, 4H), 3.19 (t,
J = 4.7 Hz, 4H). HPLC purity 99.8%.
7.3.19. Methyl 4-(4-(morpholine-4-carbonyl)piperazin-1-yl)ben
zoate (27)
7.3.26. N-(4-(4-(Morpholine-4-carbonyl)piperazin-1-yl)phenyl)
acetamide (34)
Similar reaction of 64 and methyl 4-bromobenzoate gave 27
(81%), mp (EtOAc/pet ether) 149–150 °C. 1H NMR (CDCl3) d 7.80
(br t, J = 9.1 Hsz, 2H), 6.98 (br t, J = 9.1 Hz, 2H), 3.78 (s, 3H), 3.58
(t, J = 4.7 Hz, 4H), 3.36–3.28 (m, 8H), 3.18 (t, J = 4.7 Hz, 4H). HPLC
purity 98.8%.
A
solution of morpholino(4-(4-nitrophenyl)piperazin-1-
yl)methanone (33) (520 mg, 1.63 mmol) in THF (60 mL) was
hydrogenated over 5% Pd–C at 40 psi for 1 h. The catalyst was fil-
tered off and the solution of crude (4-(4-aminophenyl)piperazin-
1-yl)(morpholino)methanone (66) was treated with Ac2O
(333 mg, 3.26 mmol) and a trace of HClO4 at 20 °C for 5 min. The
mixture was then evaporated, and the residue was dissolved in
EtOAc, washed with aqueous Na2CO3 and evaporated. The crude
product was chromatographed on silica gel, eluting with EtOAc/
MeOH (9:1) to give 34 (92%), mp (EtOAc) 193–195 °C. 1H NMR
(CDCl3) d 9.67 (s, 1H), 7.42 (br d, J = 9.1 Hz, 2H), 6.88 (br d,
J = 9.1 Hz, 2H), 3.57 (t, J = 4.7 Hz, 4H), 3.31–3.23 (m, 4H) (after
D2O), 3.17 (t, J = 4.7 Hz, 4H), 3.08–3.01 (m, 4H), 1.99 (s, 3H). HPLC
purity 99.9%.
7.3.20. 4-(4-(Morpholine-4-carbonyl)piperazin-1-yl)benzoic
acid (28)
A solution of 27 (508 mg, 1.53 mmol) in MeOH/water (1:1,
3 mL) and 1 N aqueous NaOH (1.52 mL, 1 equiv) was heated under
reflux for 2 h, then diluted with water and extracted with DCM.
The aqueous layer was acidified with AcOH and the resulting solid
was collected and washed to give 28 (362 mg, 74%), mp (MeOH/
EtOAc) 240–243 °C. 1H NMR ((CD3)2SO) d 12.26 (s, 1H), 7.78 (br
d, J = 9.0 Hz, 2H), 6.96 (br d, J = 9.1 Hz, 2H), 3.58 (t, J = 4.7 Hz, 4H),
3.29 (s, 8H), 3.18 (t, J = 4.7 Hz, 4H), HPLC purity 98.6%.
7.3.27. N-(4-(4-(Morpholine-4-carbonyl)piperazin-1-yl)phenyl)
methanesulfonamide (35)
The crude amine (66) from evaporation of the hydrogenation of
33 (110 mg, 0.34 mmol) was dissolved in pyridine (2 mL) and
7.3.21. 4-(4-(Morpholine-4-carbonyl)piperazin-1-yl)benzamide
(29)
A suspension of 28 (200 mg, 0.62 mmol) in DCM (2 mL), (COCl)2
(20 mg, 2 equiv) and a trace of DMF was stirred at 20 °C for 30 min,
then evaporated to dryness. The residue of crude acid chloride was
dissolved in THF and the solution was cooled to 0 °C and treated
with excess NH3 gas, then evaporated to dryness. The residue
was partitioned between EtOAc and 2 N NH4OH, and the organic
layer was evaporated. The residue was chromatographed on silica
gel, eluting with EtOAc/MeOH (9:1) to give 29 (86%), mp (EtOAc/
pet ether) 255–257 °C. 1H NMR (CDCl3) d 7.76 (br d, J = 8.9 Hz,
2H), 7.68 (br s, 1H), 6.98 (br S, 1H), 6.98 (br d, J = 9,0 Hz, 2H),
3.56 (t, J = 4.7 Hz, 4H), 3.34–3.22 (m, 8H), 3.18 (t, J = 4.7 Hz, 2H).
HPLC purity 98.9%.
treated with MsCl (35 lL, 0.41 mmol) at 20 °C for 15 min. The
reaction was then diluted with aqueous KHCO3, extracted with
EtOAc, and the organic layer was washed, dried and evaporated.
The residue was chromatographed on silica gel, eluting with
EtOAc/MeOH (9:1), to give 35 (96 mg, 76%), mp (EtOAc/iPr2O)
189–192 °C. 1H NMR (CDCl3) d 9.24 (s, 1H), 7.09 (br d, J = 9.0 Hz,
2H), 6.93 (br d, J = 9.0 Hz, 2H), 3.60–3.55 (m, 4H), 3.32–3.26 (m,
4H), 3.20–3.15 (m. 4H), 3.11–3.06 (m, 4H), 2.57 (s, 3H). HPLC purity
99.6%.
7.3.28. (4-(2,4-Dichlorophenyl)piperazin-1-yl)(morpholino)met
hanone (36)
Similar reaction of 64 and 1-bromo-2,4-dichlorobenzene gave
36 (65%), mp (EtOAc/pet ether) 135–136 °C. 1H NMR (CDCl3) d
7.55 (d, J = 2.5 Hz, 2H), 7.38 (dd, J = 8.7, 2.5 Hz, 1H), 7.17 (d,
J = 8.7 Hz, 1H), 3.58 (t, J = 4.7 Hz, 4H), 3.33 (t, J = 4.8 Hz, 4H), 3.16
(t, J = 4.7 Hz, 4H), 2.94 (t, J = 4.8 Hz, 4H). HPLC purity 98.9%.
7.3.22. N-Methyl-4-(4-(morpholine-4-carbonyl)piperazin-1-yl)
benzamide (30)
Similar treatment of the crude acid chloride of 28 with excess
aqueous methylamine gave 30 (82%), mp 217–220 °C. 1H NMR
(CDCl3) d 8.12 (d, J = 4.5 Hz, 1H), 7.72 (d, J = 9.0 Hz, 2H), 6.96 (d,
J = 9.0 Hz, 2H), 3.58 (t, J = 5.0 Hz, 4H), 3.38–3,22 (m, 4H), 3.20–
3.14 (m, 4H), 2.75 (d, J = 4.0 Hz, 3H). HPLC purity 98.3%.
7.3.29. (4-(4-Chloro-2-methylphenyl)piperazin-1-yl)(morpholi
no)methanone (37)
Similar reaction of 64 and 1-bromo-4-chloro-2-methylbenzene
gave 37 (61%), mp (EtOAc/pet ether) 129–131 °C. 1H NMR (CDCl3) d
7.24 (d, J = 2.3.Hz, 1H), 7.18 (dd, J = 8.5, 2.4 Hz, 1H), 7.02 (d,
J = 8.5 Hz, 1H), 3.58 (t, J = 4.6 Hz, 4H), 3.33–3.27 (m, 4H) after
D2O), 3.16 (t, J = 4.6 Hz, 4H), 2.79 (t, J = 4.7 Hz, 4H), 3.25 (s, 3H).
HPLC purity 99.6%.
7.3.23. N,N-Dimethyl-4-(4-(morpholine-4-carbonyl)piperazin-
1-yl)benzamide (31)
Similar treatment of the crude acid chloride of 28 with excess
dimethylamine in THF gave 31 (78%), mp (EtOC/pet ether) 130–
131 °C. 1H NMR (CDCl3)
d 7.31 (d, J = 9.5 Hz, 2H), 6.94 (d,
J = 9.5 Hz, 2H), 3.58 (t, J = 5.0 Hz, 2H), 3.73–3.68 (m, 4H), 3.63–
3.56 (m, 8H). HPLC purity 99.6%.
7.3.30. (4-(4-Chloro-2-(hydroxymethyl)phenyl)piperazin-1-yl)
(morpholino)methanone (38)
7.3.24. (4-(4-(Methylsulfonyl)phenyl)piperazin-1-yl)(morpholi
no)methanone (32)
Similar reaction of 64 and (2-bromo-5-chlorophenyl)methanol
gave 38 (8%), mp (EtOAc/pet ether) 160–162 °C. 1H NMR (CDCl3)
d 7.44 (d, J = 2.6 Hz, 1H), 7.24 (dd, J = 8.5, 2.7 Hz, 1H), 7.05 (d,
J = 8.6 Hz, 1H), 5.25 (t, J = 5.7 Hz, 1H), 4.53 (d, J = 5.7 Hz, 2H), 3.57
(t, J = 4.7 Hz, 4H), 3.33–3.27 (m, 4H), 3.16 (t, J = 4.7 Hz, 4H), 2.79
(t, J = 4.7 Hz, 4H). HPLC purity 98.5%.
Similar reaction of 64 and 1-bromo-4-(methylsulfonyl)benzene
gave 32 (45%), mp (EtOAc/pet ether) 180–181 °C. 1H NMR (CDCl3) d
7.69 (br d, J = 12 Hz, 2H), 7.07 (br d, J = 12 Hz, 2H), 3.58 (t,
J = 4.8 Hz, 2H), 3.38–3.27 (m, 8 H), 3.18 (t, J = 4.8 Hz, 2H), 3.09 (s,
3H). HPLC purity 99.6%.
7.3.31. (4-(6-Chloropyridin-3-yl)piperazin-1-yl)(morpholino)m
ethanone (39)
7.3.25. Morpholino(4-(4-nitrophenyl)piperazin-1-yl)methano
ne (33)
Similar reaction of 64 and 5-bromo-2-chloropyridine gave 39
Similar reaction of 64 and 1-bromo-4-nitrobenzene gave 33
(34%), mp (EtOAc/pet ether) 132–134 °C. 1H NMR (CDCl3) d 8.07
(48%), mp (EtOAc/iPr2O) 190–192 °C. 1H NMR (CDCl3) d 8.07

J. U. Flanagan et al. / Bioorg. Med. Chem. 22 (2014) 967–977
975
(d, J = 3.0 Hz, 1H), 7.43 (dd, J = 8.7, 3.2 Hz, 1H), 7.31 (d, J = 8.8 Hz,
1H), 3.60–3.54 m(m, 4 H), 3.32–3.26 (m., 4 H), 3.23–3.14 (m, 4H).
HPLC purity 98.0%.
J = 9.0 Hz, 2H), 6.96 (br d, J = 9.1 Hz, 2H), 3.68–3.51 (m, 4H),
3.19–3.01 (m, 4H), 2.68–2.55 (m, 1H), 1.76–1.58 (m, 5H), 1.40–
1.09 (m, 5H). HPLC purity 100%.
7.3.37. (4-(4-Chlorophenyl)piperazin-1-yl)(phenyl)methanone
(45)
Similar reaction of 70 and benzoyl chloride gave 45 (77%), mp
(EtOAc/pet ether) 81–82 °C. 1H NMR (CDCl3) d 7.50–7.40 (m, 5H),
7.25 (br d, J = 9.0 Hz, 2H), 6.96 (br d, J = 9.1 Hz, 2H), 3.86–3.36
(m, 4H), 3.18 (br s, 4H). HPLC purity 99.9%.
7.3.32. (4-(6-Bromopyridin-2-yl)piperazin-1-yl)(morpholino)
methanone (40)
Similar reaction of 64 and 2,5-dibromopyridine gave 40 (52%),
mp (EtOAc/pet ether) 141–142 °C. 1H NMR (CDCl3) d 8.18 (d,
J = 2.3 Hz, 1H), 7.70 (dd, J = 9.0, 2.3 Hz, 1H), 6.83 (d, J = 9.2 Hz,
1H), 3.61–3.54 (m, 4H), 3.52–3.45 (m, 4H), 3.18–3,22 (m, 4H),
3.20–3.14 (m, 4H). HPLC purity 99.3%.
7.3.38. 4-(4-Chlorophenyl)-N-phenylpiperazine-1-carboxamide
(46)
7.3.33. (4-(4-Chlorobenzyl)piperazin-1-yl)(morpholino)meth
anone (41)
Similar reaction of 70 (21 mg) and phenylisocyanate gave 46
(84%), mp (EtOAc/pet ether) 195–198 °C. 1H NMR (CDCl3) d 8.58
(s, 1H), 7.47 (br d, J = 8.6 Hz, 2H), 7.78–7.19 (m, 4H), 7.00 (br d,
J = 9.0 Hz, 2H), 6.94 (br d, J = 7.3 Hz, 1H), 3.59 (t, J = 5.1 Hz, 4H),
3.17 (t, J = 5.1 Hz, 4H). HPLC purity 99.4%.
A solution of tert-butyl piperazine-1-carboxylate (62) (2.0 g,
10.8 mmol) and 4-chlorobenzaldehyde (67) (1.28 g, 9.08 mmol)
in DCE (30 mL) were treated with NaBH(OAc)3 (3.12 g, 14.7 mmol)
and the mixure was stirred at 20 °C under N2 for 6 h, then diluted
with DCM and washed with 1 N aqueous NaOH. The organic layer
was dried and evaporated, and the residue was filtered through a
short column of silica gel. The crude product was dissolved in
DCM/TFA (1:1, 40 mL) and kept at 20 °C for 1 h, then evaporated.
The residue was dissolved in DCM and washed with 4 N NH4OH,
dried and evaporated. The residue was redissolved in 2 N aqueous
AcOH, and the aqueous layer was basified with concd NH4OH to
give crude 1-(4-chlorobenzyl)piperazine (68). A cold solution of
7.3.39. 4-(4-Chlorophenyl)-N-cyclohexylpiperazine-1-carbo
xamide (47)
Similar reaction of 70 and isocyanatocyclohexane gave 47
(90%), mp (EtOAc/pet ether) 166–169 °C. 1H NMR (CDCl3) d 7.23
(be d, J = 9.0 Hz, 2H), 6.96 (br d, J = 9.1 Hz, 2H), 6.22 (d, J = 7.7 Hz,
1H), 3.46–3.36 (m, 5H), 3.07 (t, J = 5.1 Hz, 4H), 1.79–1.51 (m, 5H),
1.30–1.00 (m, 5H), HPLC purity 99.5%.
68 (100 mg, 0.47 mmol) and DIPEA (20 lL, 2 equiv) in DCM
7.3.40. (4-(4-Chlorophenyl)piperazin-1-yl)(pyridin-4-yl)meth
anone (48)
(3 mL) was treated with morpholine-4-carbonyl chloride (63)
(110 mg, 0.74 mmol). The mixture was stirred at 20 °C for
30 min, then diluted with DCM, washed with Na2CO3, dried and
evaporated. Chromatography on silica gel, eluting with EtOAc/
MeOH (from 95:5) containing 0.5% concd NH4OH gave 41, mp
(hexane) 80–81 °C. 1H NMR (CDCl3) d 7.38 (br d, J = 8.5 Hz, 2H),
7.32 (br d, J = 8,5 Hz, 2H), 3.54 (t, J = 4.7 Hz, 4H), 3.47 (s, 2H),
3.15 (t, J = 4.9 Hz, 4H), 3.10 (t, J = 4.7 Hz, 4H), 2.33 (t, J = 4.9 Hz,
4H), HPLC purity 98.2%.
Similar reaction of 70 and isonicotinoyl chloride gave 48 (91%),
mp (EtOAc/pet ether) 148–149 °C. 1H NMR (CDCl3) d 8.69 (dd,
J = 4.3, 1.6 Hz, 2H), 7.43 (dd, J = 4.4, 1.6 Hz, 2H), 7.25 (br d,
J = 9.0 Hz, 2H), 6.96 (br s, 2H), 3.76 (br s, 2H), 3.40 (br s, 2H),
3.25 (br s, 2H), 3.13 (br s, 2H), HPLC purity 99.7%.
7.3.41. (4-(4-Chlorophenyl)piperazin-1-yl)(piperidin-1-yl)met
hanone (49)
Similar reaction of 70 and piperidine-1-carbonyl chloride gave
49 (88%), mp (hexane) 100–101 °C. 1H NMR (CDCl3) d 7.23 (br d,
J = 9.0 Hz, 2H), 6.95 (br d, J = 9.1 Hz, 2H), 3–27–3–20 (m, 4H),
3.20–3.07 (m, 8H), 1.59–1.43 (m, 6H). HPLC purity 99.8%.
7.3.34. (4-(4-Chlorobenzoyl)piperazin-1-yl)(morpholino)meth
anone (42)
A solution of 64 (70 mg, 0.35 mmol) in DCM (3 mL) and DIPEA
(92 lL, 1.5 equiv) was treated at 0 °C with a solution of 4-chloro-
benzoyl chloride (69) (68 mg, 1.1 equiv) in DCM (1 mL). The mix-
ture was stirred at 20 °C for 15 min, then diluted with DCM,
washed with Na2CO3, evaporated and triturated with iPr2O to give
42 (89%), mp (EtOAc/pet ether) 147–149 °C. 1H NMR (CDCl3) d
7.54–7.49 (m, 4H), 7.47–7.43 (m, 4H) 3.55 (t, J = 5.0 Hz, 4H),
3.20–3.10 (m, 8H). HPLC purity 98.3%.
7.3.42. (4-(4-Chlorophenyl)piperazin-1-yl)(thiomorpholino)
methanone (50)
Similar reaction of 70 and thiomorpholine-4-carbonyl chloride
gave 50 (80%), mp (EtOAc/pet ether) 119–121 °C. 1H NMR (CDCl3)
d br d, J = 9.0 Hz, 2H), br d, J = 9.1 Hz, 2H), 3.47–3.40 (m, 4H),
3.29–3.22 (m, 4H), 3.16–3.08 (m, 4H), 2.63–2.57 (m, 4H). HPLC
purity 99.8%.
7.3.35. Compounds of Table 2 (Scheme 2). (4-(4-Chlorophenyl)
piperazin-1-yl)(tetrahydro-2H-pyran-4-yl)methanone (43)
7.3.43. (4-(4-Chlorophenyl)piperazin-1-yl)(1-oxido-4-thiomor
pholino)methanone (51)
A
solution of 1-(4-chlorophenyl)piperazine (70) (80 mg,
0.41 mmol) and DIPEA (0.13 mL, 2 equiv) in DCM (4 mL) was
treated at 0 °C with tetrahydro-2H-pyran-4-carbonyl chloride
(74 mg, 1.2 equiv) in DCM (1 mL) for 15 min. The mixture was
then diluted with DCM, washed with Na2CO3 and evaporated to
give 43 (115 mg, 91%), mp (EtOAc/pet ether) 153–154 °C. 1H
NMR (CDCl3) d 7.25 (br d, J = 9.0 Hz, 2H), 6.96 (br d, J = 9.1 Hz,
2H), 3.89–3.80 (m, 2H), 3.11–3.53 (m, 4H), 3.39 (td, J = 11.5,
2.7 Hz, 4H), 3.19–3.04 (m, 4H), 2.97–2.86 (m, 1H), 1.67–1.49 (m,
4H). HPLC purity 98.9%.
A solution of 50 (50 mg, 0.15 mmol) in EtOH (4 mL) was treated
with excess 27% aqueous H2O2 under reflux for 1 h, then evapo-
rated. The residue was chromatographed over silica gel, with
EtOAc/MeOH (85:15) eluting 51 (74%), mp (EtOAc/pet ether)
162–165 °C. 1H NMR (CDCl3) d 7.24 (br d, J = 9.0 Hz), 6.96 (brd d,
J = 9.1 Hz, 2H), 3.69–3.45 (m, 4H), 3.69–3.45 (m, 4H), 3.35–3.22
(m, 4H), 3.19–3.08 (m, 4H), 2.96–2.84 (m, 4H), 2.76–2.65 (m,
4H). HPLC purity 98.2%.
7.3.44. Thiomorpholine, 4-((4-chlorophenyl)-4-piperazinylcarb
onyl)-, 1,1-dioxide (52)
A solution of triphosgene (165 mg, 0.56 mmol) in DCM (4 mL)
and pyridine (0.26 mL, 3.3 mmol) at 20 °C was treated dropwise
over 20 min with a solution of 1-(4-chlorophenyl)piperazine (70)
7.3.36. (4-(4-Chlorophenyl)piperazin-1-yl)(cyclohexyl)met
hanone (44)
Similar reaction of 70 and cyclohexanecarbonyl chloride gave
44 (87%), mp (hexane) 79–81 °C. 1H NMR (CDCl3) d 7.24 (br d,

976
J. U. Flanagan et al. / Bioorg. Med. Chem. 22 (2014) 967–977
(295 mg, 1.5 mmol) and stirred at 20 °C for a further 3 h to give a
solution of crude 4-(4-chlorophenyl)piperazine-1-carbonyl
chloride22 (71) (258 mg, 1.0 mmol). This was added dropwise to
an ice-cold solution of thiomorpholine 1,1-dioxide23 (0.11 g,
0.81 mmol) and DIPEA (1.5 equiv) in DCM (4 mL), and the
mixture was allowed to warm to 20 °C over 1 h. The mixture was
diluted with water and the organic layer was separated,
washed with water, dried and evaporated. The crude product
was chromatographed on silica gel, eluting with EtOAc/DCM
(1:1) to give 52 (42%), mp (EtOAc/pet ether) 180–182 °C. 1H NMR
(CDCl3) d 7.24 (br d, J = 8.9 Hz, 2H), 6.96 (br d, J = 9.0 Hz, 2H),
3.67–3.53 (m, 4H), 3.40–3.28 (m, 4H), 3.23–3.07 (m, 8H). HPLC
purity 97.7%.
(62) (335 mg, 1.80 mmol) and DIPEA (0.52 mL, 2 equiv) in DCM
(2 mL). The mixture was stirred at 20 °C for 2 h, then diluted with
DCM, washed with aqueous Na2CO3, dried and evaporated to give
tert-butyl 4-(4-(4-chlorophenyl)piperazine-1-carbonyl)piperazine-
1-carboxylate (73) (292 mg, 48%), which was used directly. 1H NMR
(DMSO) d 7.24 (d, J = 9.0 Hz, 2H), 6.97 (d, J = 9.0 Hz, 2H), 3.37–2.26
(m, 8H), 3.18–3.10 (m, 8H), 1.41, s, 9H).
A solution of 73 (225 mg, 0.55 mmol) in DCM (10 mL) and TFA
(10 mL) was stirred at 20 °C for 3 h, then evaporated. The residue
was diluted with DCM, washed with 4 N NH4OH and evaporated,
and the residue from this was extracted with boiling hexane,
which cooling gave (4-(4-chlorophenyl)piperazin-1-yl)(piperazin-
1-yl)methanone (74) (88%), which was used directly. 1H NMR
(DMSO) d 7.24 (d, J = 9.0 Hz, 2H), 6.95 (s, J = 9.0 Hz, 2H), 3,21 (m,
4H), 3.15–3.06 (m, 8H), 2.69–2.62 (m, 4H).
7.3.45. (4-(4-Chlorophenyl)piperazin-1-yl)(4-methylpiperazin-
1-yl)methanone (53)
A solution of 74 (85 mg. 0.28 mmol) and DIPEA (72 lL, 1.5
equiv) in DCM (2 mL) was treated at 0 °C with a solution of
3-chloro-3-oxopropyl acetate (45 mg, 1.2 equiv) in DCM (1 mL).
The mixture was stirred at 20 °C for 10 min, then diluted with
DCM, washed with aqueous NaHCO3 and water, dried and
evaporated. The residue was triturated with iPr2O to give crude
3-(4-((4-(4-chlorophenyl)piperazin-1-yl)methyl)piperazin-1-yl)-
3-oxopropyl acetate (75) (82 mg, 73%), which was used directly. 1H
NMR (DMSO) d 7.23 (d, J = 9.0 Hz, 2H), 6.95 (d, J = 9.0 Hz, 2H), 3.41
(br s, 4H), 3.36 (br s, 4H), 3.30–3.24 (m, 4H), 3.21–3.11 (m, 4H),
3.03–3.09 (m, 4H), 2.06 (s, 3H).
A solution of crude 71 (50 mg, 0.19 mol) in DCM (3 mL) was
cooled to 0 °C and added to a cold solution of 1-methylpiperazine
(85 lL, 3 equiv) in DCM (2 mL). After stirring at 20 °C for 2 h the
mixture was diluted with DCM, washed with aqueous Na2CO3,
dried and evaporated. The residue was extracted with dilute aque-
ous AcOH, filtered and basified with aqueous Na2CO3 to precipitate
53 (32 mg, 42%), mp (EtOAc/pet ether) 130–132 °C. 1H NMR
(CDCl3) d 7.24 (br d, J = 9.0 Hz, 2H), 6.95 (br d, J = 9.1 Hz, 2H),
3.30–3.27 (m, 12H), 2.28 (t, J = 4.6 Hz, 4H), 2.17 (s, 3H). HPLC purity
94.6%.
A solution of crude 75 (40 mg, 0.90 mmol) in MeOH (6 mL) was
treated with a solution of Cs2CO3 (50 mg, 1.5 equiv) in water
(1 mL). The mixture was stirred at 20 °C for 15 min, then diluted
with water, concentrated to remove the MeOH and extracted with
EtOAc. Evaporation of the organic layer gave 56 (92%), mp (EtOAc/
pet ether) 150–152 °C. 1H NMR (CDCl3) d 7.24 (br d, J = 9.0 Hz, 2H),
6.96 (br d, J = 9.1 Hz, 2H), 4.60 (t, J = 5.5 Hz, 1H), 4.09 (d, J = 5.5 Hz,
2H), 3.53–3.43 (m, 2H), 3.39–3.23 (m, 6H) after D2O. HPLC purity
98.1%.
7.3.46. (4-(4-Chlorophenyl)piperazin-1-yl)(4-(2-hydroxyethyl)
piperazin-1-yl)methanone (54)
A solution of crude 71 (70 mg, 0.27 mmol) in DCM (4 mL) was
cooled to 0 °C and treated dropwise with an ice-cold solution of
2-(piperazin-1-yl)ethanol (181 lL, 3 equiv) in DCM (2 mL). The
mixture was stirred at 20 °C for 2 h, diluted with DCM, washed
with aqueous Na2CO3, dried and evaporated. The residue was chro-
matographed on silica gel, eluting with EtOAc/MeOH (85:15) plus
1% NH3. The resulting product was triturated with iPr2O to give
54 (52%), mp (EtOAc/pet ether) 120–121 °C. 1H NMR (CDCl3) d
7.24 (br d, J = 9.0 Hz, 2H), 6.95 (br d, J = 9.1 Hz, 2H), 4.40 (t,
J = 5.3 Hz, 1H), 3.50 (q, J = 5.9 Hz, 2H), 3.29–3.22 (m, 4H), 3.21–
3.05 (m, 8H), 2.44–2.35 (m, 6H). HPLC purity 99.2%.
7.3.49. (4-(4-Chlorophenyl)piperazin-1-yl)(3,4-dihydroisoquino
lin-2(1H)-yl)methanone (57)
Solutions of 70 (75 mg, 0.38 mmol) and DIPEA (0.13 mL) in DCM
(3 mL) and 3,4-dihydroisoquinoline-2(1H)-carbonyl chloride (76)
(82 mg, 1.1 equiv) in DCM (1 mL) were mixed and stirred at
20 °C for 30 min, then diluted with DCM and water and the organic
layer was evaporated to give 57 (84%), mp (EtOAc/pet ether) 153–
154 °C. 1H NMR (CDCl3) d 7.24 (br d, J = 9.0 Hz, 2H), 7.18–7.12 (m,
4H), 6.97 (br d, J = 9.1 Hz, 2H), 4.41 (s, 2H), 3.46 (t, J = 5.9 Hz, 2H),
3.36–3.29 m (m, 4H), 3.20–3.13 (m, 4H), 2.84 (t, J = 5.8 Hz, 2H).
HPLC purity 98.8%.
7.3.47. 1-(4-(4-Chlorophenyl)piperazine-1-carbonyl)piperidine-
4-carboxylic acid (55)
A solution of crude 71 (300 mg, 1.16 mmol) in DCM (9 mL) was
cooled to 0 °C and treated dropwise with an ice-cold solution of
methyl piperidine-4-carboxylate (328 mg, 2.29 mmol) and DIPEA
(0.40 mL, 2,29 mmol) in DCM (3 mL). The mixture was stirred at
20 °C for 16 h, then diluted with DCM, washed with NaHCO3, dried
and evaporated. Chromatography on silica gel, melting with EtOAc/
DCM (1:1), gave crude methyl 1-(4-(4-chlorophenyl)piperazine-1-
carbonyl)piperidine-4-carboxylate (72) (217 mg, 70%) which was
used directly. A solution of ester 72 in MeOH (2 mL) and 1 N aque-
ous NaOH (0.5 mL) was heated under reflux for 1 h, then diluted
with water and concentrated to remove the MeOH. The concen-
trate was filtered and acidified with AcOH to give 55 (91%), mp
(EtOAc/pet ether) 192–194 °C. 1H NMR (CDCl3) d 12.23 (s, 1H),
7.24 (br d, J = 9.0 Hz, 2H), 6.95 (br d, J = 9.1 Hz, 2H), 3.61–3.51
(m, 2H), 3.30–3.21 (m, 4H), 3.16–3.07 (m, 4H), 2.87–2.76 (m,
2H), 2.46–2.36 (m, 1H), 1.86–1.74 (m, 2H), 1.56–1.42 (m, 2H). HPLC
purity 99.1%.
7.3.50. Compounds of Table 3 (Scheme 3). 4-((4-(4-Chlorophen
yl)piperazin-1-yl)sulfonyl)morpholine (58)
A solution of morpholine hydrochloride (6.0 g, 49 mmol) in
CHCl3 (20 mL) and SO2Cl2 (80 mL) was heated under reflux for
6 h, then evaporated under reduced pressure. The residue was dis-
solved in DCM, diluted with pet ether, filtered through Celite and
evaporated to give crude morpholine-4-sulfonyl chloride23 (77).
A
solution of 1-(4-chlorophenyl)piperazine (70) (48 mg,
0.24 mmol) in DCM (1 mL) was treated with crude 77 (50 mg,
0.27 mmol) in DCM 3 mL) and DIPEA (0.l mL). The mixture was
stirred for 2 h and diluted with DCM, washed with dilute aqueous
Na2CO3 and evaporated. The residue was dissolved in EtOAc, fil-
tered through Celite, evaporated and triturated with iPr2O to give
58 (77%), mp (EtOAc/pet ether) 152–153 °C. 1H NMR (CDCl3) d
7.26 (br d, J = 9.0 Hz, 2H), 6.97 (br d, J = 9.1 Hz, 2H), 3.66–3.58
(m, 4H), 3.33–3.22 (m, 4H) after D2O, 3.20–3.11 (m, 8H). HPLC pur-
ity 98.2%.
7.3.48. 1-(4-(4-(4-Chlorophenyl)piperazine-1-carbonyl)piper
azin-1-yl)-2-hydroxyethanone (56)
A solution of crude 71 (295 mg, 1.14 mmol) was cooled to 0 °C and
added dropwise to a solution of tert-butyl piperazine-1-carboxylate

J. U. Flanagan et al. / Bioorg. Med. Chem. 22 (2014) 967–977
977
7.3.51. (4-(4-Chlorophenyl)piperazin-1-yl)(morpholino)methan
ethione (59)
at default values and each ligand was subjected to 20 genetic algo-
rithm runs, and 20 predicted poses per compound were kept. The
top ranked poses were then used. Atom types were automatically
assigned.
A solution of 70 (90 mg, 0.46 mmol) and DIPEA (0.16 mL,
0.92 mmol) in DCM (3 mL) was cooled to 0 °C and treated with
morpholine-4-carbothioyl chloride24 (78) (83 mg, 0.5 mmol). The
mixture was stirred at 20 °C for 8 h, then diluted with DCM and
water and the organic layer was evaporated. The residue was dis-
solved in EtOAc and filtered through silica gel to give 59 (76%), mp
(EtOAc/pet ether) 157–160 °C. 1H NMR (CDCl3) d 7.25 (br d,
J = 9.0 Hz, 2H), 6.96 (br d, J = 9.1 Hz, 2H), 3.67–3.60 (m, 8H),
3.57–3.51 (m, 4H), 3.24–3.18 (m, 4H). HPLC purity 99.8%.
Acknowledgments
We thank Ms. Emma Hamilton for help with the enzyme stud-
ies and Dr. Chris Bunce for the AKR1C1, AKR1C2 and AKR1C4
expression vectors. We wish to acknowledge the contribution of
NeSI high-performance computing facilities. NZ’s national facilities
are provided by the NZ eScience Infrastructure and funded jointly
by NeSI’s collaborator institutions and through the Ministry of
Business, Innovation & Employment’s Research Infrastructure pro-
gramme. URL https://www.nesi.org.nz
7.3.52. (4-(4-Chlorophenyl)-1,4-diazepan-1-yl)
(morpholino)methanone (60)
A solution of 1-(4-chlorophenyl)-1,4-diazepane10 (79) (80 mg,
0.38 mmol) and DIPEA (0.13 mL, 2 equiv) in DCM (3 mL) was
cooled to 0 °C and treated with morpholine-4-carbonyl chloride
(63) (68 mg, 0.45 mmol) at 20 °C for 1 h. The mixture was washed
with aqueous Na2CO3 and the organic layer was evaporated. The
residue was dissolved in EtOAc and filtered through a column of
silica gel to give 60 (73%), mp (hexane) 70–73 °C. 1H NMR (CDCl3)
d 7.13 (br d, J = 9.1 Hz, 2H), 6.72 (br d, J = 9.1 Hz, 2H), 3.61 (t,
J = 5.5 Hz, 2H), 3.55–3.42 (m, 8H), 3.21 (t, J = 5.7 Hz, 2 H), 2.91 (t,
J = 4.7 Hz, 2H), 1.93–1.83 (m, 2H). HPLC purity 99.8%.
References and notes
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7.3.53. 5-(4-Chlorophenyl)-2,5-diazabicyclo[2.2.1]heptan-2-
yl)(morpholino)methanone (61)
Similar reaction of 2-(4-chlorophenyl)- 2,5-diazabicyclo[2.2.1]
heptane11 (80) (50 mg, 0.24 mmol) with morpholine-4-carbonyl
chloride (63) gave 61 (77%), mp (EtOAc/heptanes) 181–182 °C.
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7.4. Molecular modelling
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Protein structures were prepared for docking by the addition of
hydrogens using SYBYL-X2.1 (Tripos, St Louis), and the side chains
of His117, His194 and His314 were adjusted to improve hydrogen
bonding. All crystallographic waters were stripped prior to dock-
ing. Ligand preparation involved the generation of a single low en-
ergy conformation using the SYBYL protoplex and stereoplex
modules followed by energy minimisation using Gastieger Marsili
charge set with the Tripos forcefield for 1000 steps of Steep Des-
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gence set at 0.05) using a dielectric constant of 1 and the
distance dependent dielectric function. Docking was performed
using GOLDv5.2 with the ChemPLP scoring function and a 10 Å
cavity centred on 24 crystallised in the AKR1C3 active site. The grid
spacing for cavity detection and flood-fill steps was set at 0.25, and
ligands were constrained to the position of 24 using the heavy
atoms of the scaffold represented by 9 and a constraint weight
set at 20. All ligand flexibility terms were used along with the flip
option where available. Diverse solutions was turned on and used
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