Synthesis and structure-activity relationship of (1-halo-2-naphthyl) carbamate-based inhibitors of KIAA1363 (NCEH1/AADACL1)

Article abstract of DOI:10.1016/j.bmcl.2012.05.102

KIAA1363 is a serine hydrolase whose activity has been shown to be positively associated with tumor cell invasiveness. Thus, inhibitors of KIAA1363 represent a novel targeted therapy approach towards cancer. AX11890 ((1-bromo-2-naphthyl) N,N-dimethylcarbamate) was identified as a KIAA1363 inhibitor with an IC₅₀ value of 1.2 μM and was shown using ESI-MS to carbamylate the catalytic residue Ser¹⁹¹. SAR studies explored both substitution of the 1-bromo group and derivatization of the 6-position. Activity-based protein profiling demonstrated AX13057 inhibited tumor-localized KIAA1363 in SK-OV-3 xenograft-bearing mice.

Full text of DOI:10.1016/j.bmcl.2012.05.102

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5748–5751
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and structure–activity relationship of (1-halo-2-naphthyl)
carbamate-based inhibitors of KIAA1363 (NCEH1/AADACL1)
⇑
Kevin R. Shreder , Emme C. K. Lin, Jiangyue Wu, Julia Cajica, Christopher M. Amantea, Yi Hu,
Eric Okerberg, Heidi E. Brown, Lan M. Pham, De Michael Chung, Allister S. Fraser, Ethel McGee,
Jonathan S. Rosenblum, John W. Kozarich
ActivX Biosciences, Inc., 11025 N. Torrey Pines Road, La Jolla, CA 92037, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
KIAA1363 is a serine hydrolase whose activity has been shown to be positively associated with tumor cell
invasiveness. Thus, inhibitors of KIAA1363 represent a novel targeted therapy approach towards cancer.
AX11890 ((1-bromo-2-naphthyl) N,N-dimethylcarbamate) was identified as a KIAA1363 inhibitor with
Received 11 April 2012
Revised 24 May 2012
Accepted 29 May 2012
Available online 6 June 2012
an IC50 value of 1.2 l
M and was shown using ESI-MS to carbamylate the catalytic residue Ser191. SAR
studies explored both substitution of the 1-bromo group and derivatization of the 6-position. Activity-
based protein profiling demonstrated AX13057 inhibited tumor-localized KIAA1363 in SK-OV-3 xeno-
graft-bearing mice.
Keywords:
KIAA1363 inhibitor
Naphthyl carbamate
Carbamylation
Time-dependent
Xenograft
Ó 2012 Published by Elsevier Ltd.
KIAA1363 (gene name NCEH1; the protein is also known as
neutral cholesterol ester hydrolase 1 and arylacetamide deacetyl-
ase-like 1 [AADACL1]) is a serine hydrolase whose activity has been
shown to be positively associated with tumor cell invasiveness.1
KIAA1363 activity has also been found to be increased in clinical
breast tumor biopsies relative to normal breast tissue, and further-
more to be higher in prognostically worse ER/PR double negative
tumors than in double positive tumors.2 KIAA1363 mediates the
hydrolysis of 2-acetyl monoalkylglycerol, a metabolic intermediate
in an ether lipid pathway producing the biologically active lipid, al-
kyl-LPA.3 Inhibition of KIAA1363 by RNA knockdown or by use of
inhibitors has been shown to reduce alkyl-LPA and to disrupt cel-
lular processes such as migration3 or tumor growth in vivo.4 Thus,
inhibitors of KIAA1363 activity represent a novel targeted therapy
approach towards cancer. Various KIAA1363 inhibitors have been
reported in the literature,5–7 including the carbamate AS1153
(Fig. 1).
effort was the identification of AX13057 (Table 3), a tumor-pene-
trant KIAA1363 inhibitor.
An initial set of compounds (Table 1) was examined to explore
the SAR of the HTS hit AX11890. When the 1-bromo group was re-
moved (2)9 or moved to the 3-position (3) potency decreased.
Moving the bromine to the 6-position (4)9 resulted in a compound
with similar KIAA1363 inhibitory activity to AX11890. These re-
sults indicated that substitution at the 1 and 6 positions were
important drivers for potency. Truncated versions of the
AX11890 in which the unsubstituted phenyl ring was removed
(5)10 or replaced by two methyl groups (6) showed a dramatic
reduction in potency. This latter data was taken to demonstrate
that the bicyclic naphthalene core was an important pharmaco-
phore element.
Replacements for the 1-bromo group of AX11890 were exam-
ined to find analogs with improved potency (Table 2). The synthe-
As part of an effort to identify novel KIAA1363 inhibitors we
conducted a high throughput screen of an internal compound li-
brary which led to the identification of the carbamate AX11890
F
H
N
Br
O
(Fig. 1) as a 1.2 l
M inhibitor of KIAA1363.8 As part of a hit-to-lead
O
N
O
O
O
optimization effort, we set out to better understand the structure–
activity relationship (SAR) of the hit and to improve its potency by
modifying both the 1-position and 6-position. The result of this
O
AS115
(cis)
AX11890
⇑
Corresponding author.
E-mail address: kevins@activx.com (K.R. Shreder).
Figure 1. The structure of AS115 and the KIAA1363 HTS hit AX11890.
0960-894X/$ - see front matter Ó 2012 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmcl.2012.05.102

K. R. Shreder et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5748–5751
5749
Table 1
Table 3
KIAA1363 IC50 values (20 min pre-incubation) for the HTS hit AX11890 and
compounds 2–6
KIAA1363 IC50 values (20 min pre-incubation) for 6-position derivatives of the HTS
hit AX11890
R1
O
O
O
N
N
N
O
N
O
O
O
O
Br
Br
Br
R6
2
3
4
Compound
R6
R1
KIAA1363 IC50 (nM)
29,000
H
N
H3C
19
–Br
Br
O
H3C
H3C
O
N
H
O
N
H
N
O
O
20
21
22
–Br
–Br
–Br
1700
H
O
H
N
5
6
17,000
1500
O
Compound
KIAA1363 IC50
(
l
M)
H
N
AX11890
1.2
>100
55
0.92
>100
24
O
2
3
4
5
6
H
N
23
24
25
–Br
–Br
–Br
920
O
H
N
1300
11,000
O
Table 2
CH3
N
KIAA1363 IC50 values (20 min pre-incubation) for 1-position
variants of the HTS hit AX11890
H3C
O
R1
O
N
N
O
26
–Br
310
N
Compound
R1
KIAA1363 IC50 (lM)
O
27
AX13057
28
29
30
31
–CN
–CH2CH2CH3
–Br
-CN
–CH2CH2CH3
–Br
–Br
–Br
–Br
–Cl
–Cl
–Cl
150
130
50
150
150
80
AX11890
7
8
9
10
11
12
13
14
15
16
17
18
–Br
–CH3
1.2
23
6.8
3.4
7.5
21
–CH2CH3
–CH2CH2CH3
–CH2CH2CH2CH3
–CH(CH3)2
–NH2
–COCH3
–CO2Me
CN
69
11
3.9
>100
1.1
0.89
0.33
expanding the ring size of the N-cyclopropyl amide 21 resulted
in a concomitant increase in KIAA1363 inhibitory activity (com-
pare 21 through 24). Finally, annulation of the N,N-dimethylamide
25 gave the most potent inhibitor (26) among the 6-carboxamides
explored with a KIAA1363 IC50 of 310 nM.
–Cl
–NO2
–I
When cyano (27), n-propyl (AX13057), and bromo (28) groups
were incorporated into the 6-position of AX11890, the most dra-
matic increases in potency were observed (7- to 21-fold). Indeed,
the most potent inhibitor identified in this study, the 1,6-dibromo
derivative 28 (KIAA1363 IC50 = 50 nM), was a fusion between the
sis of such analogs began with 1-substituted-2-naphthol deriva-
tives which were synthesized according to literature procedure11
or purchased commercially. Subsequent carbamylation with dim-
ethylcarbamyl chloride (Me2NCOCl) in Cs2CO3/acetone (see an
exemplary synthesis in Scheme 1) yielded the compounds in Table
2. Replacement of the 1-bromo group with linear or branched alkyl
groups (7–11), a primary amine (12), or various electron with-
drawing groups (13–15) offered only a decrease in potency relative
to the HTS hit. By contrast, the 1-chloro derivative 16 was equipo-
tent with AX11890. Only the nitro (17) and iodo (18) groups affor-
ded an increase in potency with the latter being the most potent
inhibitor in this series having a KIAA1363 IC50 value of 330 nM.
Derivatization of AX11890 at the 6-position was also explored
(Table 3). When several 6-carboxamide derivatives were synthe-
sized (Scheme 1) and examined in the KIAA1363 IC50 assay, SAR
data emerged indicating more hydrophobic or larger amides were
often preferred. For example, extending the N-methyl amide 19 to
N-heptyl (20) resulted in a 17-fold increase in potency. Similarly,
ꢀ1
lM KIAA1363 inhibitors AX11890 and 4. Three 1-chloro ana-
logs (29–31) of the most potent 1, 6-disubstituted inhibitors
described above were also investigated and found to be of similar
potency as their 1-bromo congeners. Thus, the 6-position was fur-
ther established as a driver for potency.
ESI-MS was used to confirm carbamylation as a mechanism of
action for AX11890. Incubation of a KIAA1363-expressing cell
membrane fraction with 100 lM AX11890 was followed by gel
filtration to remove any trace of small molecule inhibitor
detectable by MS. After tryptic digestion and MS2 sequencing, both
the un-modified active site peptide and the same peptide
N,N-dimethyl carbamylated at the active site serine 191 was
observed (see Supplementary data).12 Because covalent inhibitors
are often associated with time-dependent inhibition, we examined

5750
K. R. Shreder et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5748–5751
Br
OH
OH
a
MeO
1.0
MeO
O
O
0.5
0.0
Br
O
N
b
c
MeO
N
O
O
0
50
100
150
200
250
time (minutes)
Br
Br
O
O
N
O
Ra
N
d
O
O
HO
O
O
N
OH
Rb
decarbamylation
O
O
N
N
H
H
Scheme 1. Synthesis of 6-carboxamide derivatives of AX11890. Reagents and
conditions: (a) NBS (1.0 equiv), DMF, rt, 1 day, 89%; (b) Me2NCOCl (1.15 equiv),
Cs2CO3 (1 equiv), acetone, rt, 1 day, 89%; (c) LiOH (10 equiv), THF/H2O (1:1), rt,
1 day, 90%; (d) CDI (1 equiv), CH3CN then amine (2 equiv), rt, 3 days.
O
Ser191
Figure 2. Plot of the diminishing signal intensity of the N,N-dimethyl carbamylated
KIAA1363 active site tryptic peptide (j) and the corresponding rise of the same un-
modified peptide (s) as a function of time. For the former, the best fit to a single
exponential decay is shown to yield a t1/2 value of 66 min. Below the plot is a
scheme showing the decarbamylation of the active site serine (i.e. conversion of j
to s).10
Table 4
KIAA1363 IC50 values measured within the same experiment (A: 20 min pre-
incubation time and B: 4 h pre-incubation time) for the HTS hit AX11890, carbamate
analogs of AX11890, and AS115. The last column is the ratio between the two pre-
incubation times for each compound and is shown as a comparative measure of time-
dependent inhibition
Table 5
Br
KIAA1363 inhibition measured using activity-based protein profiling with AX8891
after dosing compounds 27, 28, and AX13057 (po or ip, 100 mg/kg) to SK-OV-3
xenograft bearing female SCID mice
O
R
O
Compound
Route of administration
Tissue
% Inhibition KIAA1363
ID
R
KIAA1363
KIAA1363
A/B
27
28
AX13057
AX13057
AX13057
po
po
po
ip
Tumor
Tumor
Tumor
Tumor
Heart
14
18
22
64
66
IC50
(lM)—A
IC50
(lM)—B
N
N
1
1.8
1.7
1.1
6.1
17
ip
32
33
2.0
37
0.33
2.2
H
N
plotting the signal strength of the resultant carbamylated active
site peptide (see Fig. 2). When fit to a single-exponential, a t1/2 va-
lue of 66 min was measured for the N,N-dimethylcarbamylated
species, a relatively short value compared to the length of the
time-dependent assay. When this same set of experiments was
conducted with AS115, the expected active site carbamate was ob-
served and found not to decarbamylate after 4 h (data not shown).
On the continuum of covalent inhibitors, AX11890 can be
described as a covalent yet reversible inhibitor (i.e. pseudo-irre-
versible and substrate-like) whereas AS115 more closely approxi-
mates an irreversible inhibitor. In total the data demonstrates
the structure of carbamate can play a role in modulating the rate
of enzymatic decarbamylation, an observation noted before with
inhibitors of cholinesterase.14
Finally, the ability of selected KIAA1363 inhibitors (27, 28, and
AX13057) to inhibit KIAA1363 in SCID mice bearing SK-OV-3 hu-
man ovarian cancer xenografts was examined (see Table 5)15 Post
oral administration (100 mg/kg), all 3 compounds inhibited the
labeling of intratumoral human KIAA1363 by a fluorophosphonate
serine hydrolase activity-based probe. Because AX13057 gave the
highest level of inhibition (22%), it was re-examined via an intra-
peritoneal route of dosing to maximize exposure. Inhibition values
of 64% and 66% were measured for intratumoral human KIAA1363
and mouse heart KIAA1363, respectively. Inhibition of murine
heart KIAA1363 could also be examined as a pharmacodynamic
assay for in vivo inhibitor efficacy in non-tumor bearing mice.
H
N
34
35
28
27
1.5
30
37
H
N
0.71
36
42
1.1
38
54
N
AS115
5.4
0.10
the potency of AX11890 using both a 20 min and 4 h pre-incuba-
tion time. To our surprise, the KIAA1363 inhibitory activity of
AX11890 showed no significant increase in potency with a longer
pre-incubation time (Table 4). This behavior was in contrast to car-
bamate analogs of AX11890 (32–36)13, including the closely re-
lated azeditine carbamate 32, all of which exhibited time-
dependent inhibition. As a control, AS115 was examined and dem-
onstrated the largest degree of time-dependent inhibition with a
KIAA1363 IC50 value of 100 nM at the 4-h pre-incubation time. This
value was close to the reported AS115 KIAA1363 IC50 value of
150 nM determined using a serine hydrolase activity-based probe
assay.3
Decarbamylation of KIAA1363 could be monitored by denatur-
ing the covalently modified enzyme at different time points and

K. R. Shreder et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5748–5751
5751
reduced with DTT (1 M), and alkylated with iodoacetamide (1 M). Theses
samples were eluted through a Micro Bio-Gel P-30 column and the resulting
eluate digested with trypsin overnight. The digested samples were analyzed
with capillary HPLC (Agilent Technologies) and LTQ-linear Ion trap mass
spectrometer with nano-ESI source (Thermo Finnigan). Ten microlitres of each
In summary, the SAR studies described herein were used to
define the important pharmacophore elements of (1-halo-2-naph-
thyl) carbamates as a novel class of KIAA1363 inhibitors. Activity-
based protein profiling demonstrated that AX13057 inhibited
intratumoral KIAA1363 in SK-OV-3 xenograft-bearing mice. All
data taken together, (1-halo-2-naphthyl) carbamates offer promise
as a starting point in the development of KIAA1363 inhibitors. Lead
optimization based on this motif will be reported in due course.
sample was desalted and concentrated on
Bioresources). Peptides then were separated on a 0.18 Â 100 mm, C18, 5
reversed phase column with a gradient of 5% ACN, 0.1% formic acid to 50% ACN,
0.1% formic acid in 2 h with column flow of 1.8 L/min and injected to the LTQ
a peptide capTrap (Michrom
lm
l
mass spectrometer. Data was acquired in target MS/MS mode on the active site
peptide (ICISGDS191AGGNLAAALGQQFTQDASLK) and its carbamylated form
(ICISGD[S191-H+CONMe2]AGGNLAAALGQQFTQDASLK) with the following
settings: spray voltage: 1.8 kV, capillary temperature: 200 °C, capillary
voltage: 46 V, tube lens voltage: 120 v, relative collision energy: 35%. Peptide
fragments were identified with TurboSEQUEST (Thermo Finnigan). Post
incubation with AX11890 the signal intensity ratio of carbamylated active
site peptide to un-modified active site peptide was 13.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.05.
102.
13. These compounds were produced by the reaction of 1-bromo-2-naphthol with
4-nitrophenyl chloroformate (1 equiv) in DIPEA/DCM at 0 °C followed by
treatment with the appropriate amine (1 equiv) at room temperature
overnight. Secondary carbamates were found to exhibit chemical instability.
For example, the prolonged storage of such compounds in DMSO at À78 °C
resulted in decomposition. No such chemical instability was seen with tertiary
carbamates.
References and notes
1. Jessani, N.; Liu, Y.; Humphrey, M.; Cravatt, B. F. Proc. Natl. Acad. Sci. U.S.A. 2002,
99, 10335.
14. (a) Rampa, A.; Bisi, A.; Valenti, P.; Recanatini, M.; Cavalli, A.; Andrisano, V.;
Cavrini, V.; Fin, L.; Buriani, A.; Giusti, P. J. Med. Chem. 1998, 41, 3976; (b)
Rampa, A.; Piazzi, L.; Belluti, F.; Gobbi, S.; Bisi, A.; Bartolini, M.; Andrisano, V.;
Cavrini, V.; Cavalli, A.; Recanatini, M.; Valenti, P. J. Med. Chem. 2001, 44, 3810;
(c) Bartolini, M.; Cavrini, V.; Andrisano, V. J. Chromatogr., A 2007, 1144, 102.
15. In vivo experiments were carried out at Explora Biolabs (San Diego, CA) in
accordance with the guidelines of the Institutional Animal Care and Use
Committee. Female SCID mice bearing SK-OV-3 ovarian cancer xenografts of an
average volume [½ (length  width2)] of 115–700 mm3 were used for PD
2. Jessani, N.; Niessen, S.; Wei, B. Q.; Nicolau, M.; Humphrey, M. J. Y.; Han, W.;
Noh, D. Y.; Yates, J. R., III; Jeffrey, S. S.; Cravatt, B. F. Nat. Methods 2005, 2, 691.
3. Chiang, K. P.; Niessen, S.; Saghatelian, A.; Cravatt, B. F. Chem. Biol. 2006, 13,
1041.
4. Chang, J. W.; Nomura, D. K.; Cravatt, B. F. Chem. Biol. 2011, 18, 476.
5. Leung, D.; Hardouin, C.; Boger, D. L.; Cravatt, B. F. Nat. Biotechnol. 2003, 21, 687.
6. Li, W.; Blankman, J. L.; Cravatt, B. F. J. Am. Chem. Soc. 2007, 129, 9594.
7. Chang, J. W.; Moellering, R. E.; Cravatt, B. F. Angew. Chem., Int. Ed. 2012, 51, 966.
8. KIAA1363 IC50 assay: The human KIAA1363 cDNA clone was a kind gift from Dr.
Benjamin Cravatt (The Scripps Research Institute). KIAA1363 was expressed in
human 293T/17 cells (ATCC) and the membrane fraction was isolated by
ultracentrifugation at 100,000Âg for 1 h at 4 °C followed by resuspension using
sonication in 50 mM Tris pH 7.4. For the assay, membranes were diluted to a
final concentration of 0.1 mg/ml in 50 mM Tris pH 7.4 + 5 mM CHAPS.
Membranes were pre-incubated with compound at room temperature prior
studies. Compounds were resuspended in PEG400 and 100 lL was dosed at
100 mg/kg according to average body weight. At 2 h after dosing, mice were
sacrificed and tumors and/or hearts were removed, weighed, and snap frozen
in liquid nitrogen. Tissues were individually homogenized in 50 mM Tris pH
7.4, and centrifuged at 1000Âg for 10 min at 4 °C. The supernatant was then
collected and briefly sonicated on ice. Protein concentrations were determined
using the BioRad Dc protein kit and adjusted typically to 5 mg/mL. An aliquot
to adding 200
experiments in which a 20 min pre-incubation was used, 200
present throughout the pre-incubation. For experiments in which a 4 h pre-
incubation was used, 200 DTNB was added at the time of substrate
addition. The final concentration of DMSO was 4% in a total reaction volume of
50 L. Reactions were allowed to proceed for 1 h at 37 °C, and absorbance at
l
M
2-thioacetyl MAGE substrate (Cayman Chemical). For
of each lysate was labeled with the activity-based probe AX8891 at 1 lM for
lM DTNB was
90 s. The reaction was terminated by the addition of an equal volume of
2ÂSDS–PAGE loading buffer and heating at 95 °C for 5 min. To determine total
and background (no KIAA1363 activity) labeling, equal volumes of protein-
adjusted lysates from control group animals were pooled and preincubated
lM
l
with DMSO or 10
lM AX13057 inhibitor, respectively, prior to activity-based
405 nm was measured using a Wallac Victor plate reader. All compounds were
tested in duplicate. IC50 values were determined using GraphPad Prism.
9. Previously synthesized, see: Gong, T. J.; Xiao, B.; Liu, Z. J.; Wan, J.; Xu, J.; Luo, D.
F.; Fu, Y.; Liu, L. Org. Lett. 2011, 13, 3235.
10. Previously synthesized, see: Yasuda, M.; Yoshioka, S.; Nakajima, H.; Chiba, K.;
Baba, A. Org. Lett. 2008, 10, 929.
11. 1-alkyl-2-naphthols: a) Carnduff, J.; Leppard, D. G. J. Chem. Soc., Perkin 1. 1976,
23, 2570; 1-iodo-2-naphthol: b) Iskra, J.; Stavber, S.; Zupan, M. Synthesis 1869,
2004, 11; 1-chloro-2-naphthol: c) Zhang, Y.; Shibatomi, K.; Yamamoto, H.
Synlett 2005, 2837.
probe labeling. Thirty-five
lg of each sample was loaded onto 12.5% SDS–PAGE
gels along with at least duplicate loadings of the total and background controls.
Resolved gels were directly scanned for fluorescence using a 605 nm bandpass
filter for detection on the Hitachi FMBIO IIe (MiraiBio). Fluorescence was
measured using ImageAnalysis software for KIAA1363 activity and expressed
as fluorescence volume units.
O
(5'-TAMRA)-NH-(CH2)10
P F
OEt
12. KIAA1363/293T/17 membrane in 50 mM Tris, pH 7.4 (0.15 mg in 50
incubated with 100 M of KIAA1363 inhibitor for 1 h at room temperature. The
resulting membranes (75 L) were eluted through Micro Bio-Gel P-30
column to remove excess inhibitor. The eluate was denatured with urea (6 M),
lL) was
l
AX8891
l
a