Potent and selective [2-imidazol-1-yl-2-(6-alkoxy-naphthalen-2-yl)-1- methyl-ethyl]-dimethyl-amines as retinoic acid metabolic blocking agents (RAMBAs)

Article abstract of DOI:10.1016/j.bmcl.2005.01.044

A series of [2-imidazol-1-yl-2-(6-alkoxy-naphthalen-2-yl)-1-methyl-ethyl]- dimethyl-amines were designed and synthesized as CYP26 inhibitors, serving as retinoic acid metabolic blocking agents (RAMBA's).

Full text of DOI:10.1016/j.bmcl.2005.01.044

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1669–1673
Potent and selective [2-imidazol-1-yl-2-(6-alkoxy-
naphthalen-2-yl)-1-methyl-ethyl]-dimethyl-amines as retinoic
acid metabolic blocking agents (RAMBAs)
Mark J. Mulvihill,a,* Julie L. C. Kan,a Patricia Beck,a Mark Bittner,b Cara Cesario,a
Andrew Cooke,b David M. Keane,a Anthony I. Nigro,a Christy Nillson,b Vanessa Smith,a
Mary Srebernak,b Feng-Lei Sun,b Michael Vrkljan,b Shannon L. Winski,b
Arlindo L. Castelhano,a David Emersonb and Neil Gibsona
aOSI Pharmaceuticals, Inc., 1 Bioscience Park Drive, Farmingdale, NY 11735, USA
bOSI Pharmaceuticals, Inc., 2860 Wilderness Place Boulder, CO 80301, USA
Received 22 November 2004; revised 14 January 2005; accepted 19 January 2005
Abstract—A series of [2-imidazol-1-yl-2-(6-alkoxy-naphthalen-2-yl)-1-methyl-ethyl]-dimethyl-amines were designed and synthesized
as CYP26 inhibitors, serving as retinoic acid metabolic blocking agents (RAMBAÕs).
Ó 2005 Elsevier Ltd. All rights reserved.
All-trans retinoic acid (atRA) (1), a naturally occurring
retinoid, is biosynthesized and present in a multitude of
human and mammalian tissues, and performs a crucial
role in the regulation of gene expression, cellular differ-
entiation, and proliferation of epithelial cells.1 Retinoids
have proven to be useful agents in tumor therapy and in
treating skin related diseases.2 Furthermore, all-trans
retinoic acid has been successfully used to treat acute
promyelocytic leukemia (APL) and acute myelogenous
leukemia (AML), changing the prognosis of APL from
a fatal leukemia to a highly curable disease.3
O
OH
X
1 (X = H, atRA)
2 (X = OH, 4-HO-atRA)
CYP26
Figure 1. Metabolism of atRA to 4-hydroxy-atRA by CYP26.
An alternative approach of potentiating endogenous
atRA can be through inhibition of CYP26 and may
avoid the frequency and severity of complications asso-
ciated with intensive high dose atRA therapy and may
provide an effective means of treatment following re-
lapse in cases where resistance emerges due to CYP26
upregulation. As a result, inhibitors of CYP26, also
known as retinoic acid metabolic blocking agents
(RAMBAs), have proven to be effective in blocking
the catabolic effects on atRA and have demonstrated
an increase in endogenous atRA levels.6 Liarozole (3,
IC50 = 6.0 lM) was the first generation CYP26 inhibitor
(Fig. 2) and in in vitro studies, suppressed the metabo-
lism of atRA in MCF7 cells.7 Additionally, liarozole
in conjunction with exogenously administered atRA,
displayed growth inhibition in MCF7, T47D, and
AT6.1 cells. In in vivo studies, liarozole (30 mg/kg
Despite the usefulness of atRA and retinoid compounds
in treating tumors and skin-related diseases, clinical uses
of atRA have been significantly hampered by the emerg-
ence of resistance, believed to be caused by the oxidative
catabolism to 4-hydroxy-atRA (2) by CYP26, an induci-
ble cytochrome P450 enzyme (Fig. 1).4 This tightly con-
trolled negative feedback mechanism limits the systemic
concentrations of atRA and thereby limits its biological
activity.5
Keywords: CYP26; All-trans retinoic acid (atRA); Retinoic acid meta-
bolic blocking agent (RAMBA); CYP26; Cancer.
*
Corresponding author. Tel.: +1 631 962 0787; fax: +1 631 845
5671; e-mail addresses: mark.mulvihill@osip.com; mmulvihill@
osip.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.01.044

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M. J. Mulvihill et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1669–1673
core carrying a CYP26 selectivity handle at the 6-posi-
N
N
tion. The 2,6-disubstituted naphthalene core, from a 2-
D perspective, overlays in a complementary fashion with
the conjugated olefinic moiety of the tetraenoic acid side
chain of atRA. Additionally, a suitable tether at the 6-
position of the naphthyl core was proposed to mimic
that of the atRA side chain. With this design strategy
in mind, the synthesis of a series of [2-imidazol-1-yl-2-
(6-alkoxy-naphthalen-2-yl)-1-methyl-ethyl]-dimethyl-
amines (15–17) as shown in Schemes 1–3 was initiated.
Treatment of 2-bromo-6-methoxynaphthalene 6 with
magnesium generated a Grignard species which was
added to a solution of 2-chloropropionylchloride in
THF cooled to À78 °C to afford a-chloromethylketone
7 (Scheme 1).9 Conversion of 7 to the more reactive iodo-
ketone 8 via the Finkelstein reaction (NaI, acetone)
proceeded smoothly and subsequent alkylation with
dimethylamine cleanly afforded dimethylaminoketone
9 (direct reaction of chloromethylketone 7 with dimeth-
ylamine did not proceed as cleanly). Reduction of
aminoketone 9 with sodium borohydride afforded a
4:1 mixture of syn:anti aminoalcohol isomers whereas
hydrogenation afforded the reverse, a 4:1 ratio of anti:-
syn isomers (10). The isomers were easily separable by
column chromatography and were each independently
taken on to their respective imidazolyl final product by
reaction with CDI in acetonitrile. The syn-aminoalcohol
afforded the syn-imidazolylpropylamino product and
the anti-aminoalcohol afforded the anti-imidazolylpro-
N
N
N
N
N
N
H
S
N
H
Cl
3 (Liazozole)
Figure 2. CYP26 inhibitors (RAMBAs).
4 (R116010)
(b.i.d.)) showed 90% growth inhibition in both AT-6
androgen independent prostate adenocarcinoma
(R3227 Line) and in AT-6 androgen independent pros-
tate tumors in rats. Several other RAMBAs quickly fol-
lowed liarozole, one of which being R116010 (4, IC50 in
intact T47D cells = 8.7 nM) (Fig. 2) addressing the
potency toward CYP26, which liarozole lacked.8
To explore the therapeutic potential of increasing
endogenous levels of atRA through small molecule
CYP26 inhibitors, we prepared a novel class of highly
potent and CYP26 selective naphthyl-based inhibitors,
which show inhibition of proliferation of both T47D
and AT6.1 cells in vitro in combination with atRA.
Our inhibitor design fuses the heme-binding imidazolyl-
propylamino moiety of R116010 (4) with a naphthalene
O
O
Br
a
d
c
N
X
MeO
MeO
MeO
9
6
7 (X = Cl)
8 (X = I)
b
N
OH
N
e
N
N
MeO
MeO
10
11
Scheme 1. Reagents and conditions: (a) (i) Mg, THF; (ii) 2-chloropropionylchloride, À78 °C; (b) NaI, acetone; (c) dimethylamine, MeOH; (d)
NaBH4, MeOH; (e) CDI, CH3CN, 65 °C.
O
O
a
c
b
9
R1
N
N
O
HO
12
13
N
N
OH
d
R1
N
R1
N
O
O
14
15
Scheme 2. Reagents and conditions: (a) HBr/AcOH, 120 °C; (b) DIAD, Ph3P, R1OH; (c) NaBH4, MeOH; (d) CDI, CH3CN, 65 °C.

M. J. Mulvihill et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1669–1673
1671
N
N
N
N
a
R1
N
HO
Z
N
O
O
O
15
16
N
N
b
R2
N
Z
N
R3
O
O
17
Scheme 3. Reagents and conditions when R1 = Z–CO2R1a: (a) NaOH, THF/H2O; (b) EDC, HOBT, HNR2R3 or CDI, CH3CN, then HNR2R3.
pylamino derivative (11). The regio- and stereospecific
nature of the CDI reaction of aminoalcohols as well as
the mechanistic rational behind the reaction was deter-
mined through model studies conducted on (1S,2S)-
(+)-N-methylpseudoephedrine and (1S,2R)-(+)-N-meth-
ylephedrine and have been previously reported by our
research group.10
Cells were washed twice with PBS and scraped from
plates. Cells were pelleted and resuspended in homo-
genization buffer (0.1 M Tris–Cl, pH 7.4, 0.1 M DTT,
0.2 mM EDTA, 1.15% w/v KCl, 0.1 mM PMSF and
20% v/v glycerol). Microsomes were prepared by differ-
ential centrifugation of homogenized cells. Homogenate
was spun at 17,000g and the supernatant spun again at
100,000g. The pellet was resuspended in 25 mM potas-
sium phosphate, pH 7.4, 20% v/v glycerol and stored
at À80 °C.
In order to explore various ether tethers, a naphthol
intermediate was required. Deprotection of methyl ether
9 with HBr/acetic acid afforded naphthol 12 (Scheme 2).
Alkylation of 12 with tert-butylbromoacetate using
potassium tert-butoxide as the base smoothly afforded
ether 13. However, these conditions with other alkyla-
ting agents afforded a mixture of both N- and O-alkyl-
ated products. We discovered that treatment of 12
under standard Mitsunobu conditions (PPh3, DIAD,
R1OH) cleanly afforded ethers 13. Reduction of ketone
group in 13 with NaBH4, afforded a 4:1 mixture of syn:
anti-aminoalcohols 14 as seen previously with amino-
ketone 9. Separation of the isomers and subsequent
reaction with CDI afforded imidazolylpropylamino
derivatives 15. In order to mimic the side chain of atRA,
terminal ester moieties (compound 15 where R1 = Z–
CO2R1a) were hydrolyzed to their respective carboxylic
acids 16 (Scheme 3). In some cases, the carboxylates
16 were converted to amides 17 via typical amide
coupling procedures (EDC/HOBT/HNR2R3 or CDI
then HNR2R3).
Enzymatic assays (HPLC biochemical CYP26 assay)
were performed in a total volume of 100 lL in a reaction
mixture composed of 100 mM Tris pH 7.4, 150 mM
KCl, 10 mM MgCl2, 2 mM NADPH, 40 nM 3H-atRA,
and varying concentrations of novel compound dis-
solved in DMSO such that the concentration in the reac-
tion is 0.1% final, and 20–30 lg of T47D microsomes.
The reactions were incubated at 37 °C for 30 min in
the dark. The reaction was stopped by the addition of
125 lL of acetonitrile, mixed and spun at 10,000g for
10 min. The supernatant was removed and atRA and
metabolites were separated on a C18 Waters Spherisorb
column with an in-line radiometric detector with a flow
rate of 1 mL/min and detected at 350 nM. The gradient
used was the mixture of 60 mM ammonium acetate,
pH 5.2/30% CH3OH, solvent
A
and solvent
B
(CH3OH). An increasing CH3OH gradient was run for
the separation of products by increasing solvent B.
The efficacy of compounds 15–17, as inhibitors of
CYP26 were confirmed in a number of in vitro assays.
A biochemical assay was performed using microsomal
preparations from T47D cells induced to express
CYP26. Enzymatic activity was measured as the conver-
sion of the radiolabeled substrate to its metabolites,
4-OH-atRA (4-hydroxy all-trans retinoic acid) and 4-
oxo-atRA (4-oxy retinoic acid) by reverse phase HPLC
separation. Inhibition of CYP26 activity in the presence
of variable inhibitor concentrations was used to deter-
mine IC50 values. Microsomal preparation from T47D
cells were conducted as follows: T47D cells were grown
in RPMI 1640 containing 10% FBS and 1% P/S, plated
and treated 16–25 h later with 5 lM atRA and allowed
to incubate for an additional 48 h before cell harvest.
Enzymatic assays to measure the inhibition of CYP3A4
activity was determined in 100 lL volume in a 96-well
black plate by the use of a fluorescence substrate (BD,
Gentest). Compounds were tested at various concentra-
tions in a reaction that contained 200 mM potassium
phosphate buffer, pH 7.4, 200 mM NADPH and
50 lM 7-benzyloxy-4-(trifluoromethyl)-coumarin. The
reaction was incubated at 37 °C for 45 min followed
by the addition of 37 lL of 80% acetonitrile in 0.5 M
Tris base to terminate the reaction. The plates were read
at excitation/emission of 405/535 nm, respectively.
The novel naphthalene analogs inhibited the prolifera-
tion of breast cancer and prostate cells in vitro. Experi-
ments were conducted on T47D breast cancer cell line

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M. J. Mulvihill et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1669–1673
and on the AT6.1 rat prostate adenocarcinoma cell line.
T47D cells were grown in RPMI 1640 containing 10%
FBS and 1% P/S. Cells were plated into 96-well culture
plates (2000 cells per well) in 100 lL of same medium.
After attachment for 16–24 h, the vehicle (DMSO), or
atRA alone (at concentrations of 1 nM to 1 lM), or
atRA at these concentrations in combination with vary-
ing concentration of novel compound were added to
triplicate wells.11 Medium/treatments were repeated
3 days after the first treatment and measure of the
decrease in cell proliferation was measured 48 h later
using CellTiter-GloTM (Promega).
We discovered that a variety of tethers and substituents
were tolerated. While in some cases only a moderate de-
gree of selectivity for CYP3A4 was noted, we discovered
that a terminal carboxylate moiety in the case of com-
pounds 16f and 16h afforded a larger degree of selecti-
vity. The carboxylate moieties of the two lead
compounds, 16f and 16h, were converted to their respec-
tive amides (17a–f), however, a reduction in both
potency and selectivity (CYP3A4) was noted.
Compounds with a biochemical potency below 50 nM
were screened in a cellular assay (AT6.1 and T47D).
Representative lead compounds 16f and 16h exhibit
good cellular potency unlike the corresponding amides
(Table 2).
The method described above was also used for AT6.1
cells except that cells were plated at 1500 cells per well
and treatment was performed once with measure of
the decrease in cell proliferation 72 h post treatment.
AT6.1 cells were grown in RPMI 1640 containing 10%
FBS, 1% P/S and 250 nM dexamethasone.
The two lead compounds, 16f and 16h, were further pro-
filed in vivo for pharmacokinetic properties. Compound
16f had minimal bioavailability and was not pursued
further. Compound 16h displayed good PK properties
(female CD-1 mouse), including bioavailability of
99%,12 and favorable selectivity for CYP26 over
CYP3A4, 1A2, 2D6, and 2C9 (Table 3).
As shown in Table 1, a general trend emerged where the
syn-isomers were generally more potent than the anti-
isomers. The imidazoyl moiety proved to be a critical
element for CYP26 activity as seen by the lack of acti-
vity in the aminoalcohol precursors (14). Our chemistry
efforts focused around the ether tether (OR1/O–Z–
CO2H/O–Z–CONR2R3) while maintaining the dimeth-
ylaminopropylimidazole and naphthalene ring constant.
In conclusion, a series of [2-imidazol-1-yl-2-(6-alkoxy-
naphthalen-2-yl)-1-methyl-ethyl]-dimethyl-amines were
designed and synthesized as CYP26 inhibitors, serving
as retinoic acid metabolic blocking agents (RAMBAÕs).
Various substituted ether moieties at C6 of the naphthyl
Table 1. CYP26 potency and CYP3A4 selectivity of naphthyl derivatives
OR1/O–Z–CO2H/O–Z–CONR2R3
Stereochemistry
a
Biochemical IC50 (nM)
Compound
CYP26
CYP3A4
15a
15b
15c
15d
15e
15f
15g
15h
15i
(O)–CH2–Ph–4-(O-t-Bu)
(O)–CH2–Ph–4-(O-t-Bu)
(O)–CH2Ph
syn
anti
Mix
Mix
syn
anti
syn
anti
syn
anti
syn
anti
syn
anti
syn
Mix
Mix
syn
syn
anti
syn
anti
syn
anti
syn
syn
syn
syn
syn
syn
34.0
86.0
50.0
580.0
40.00
200.0
4.000
220.0
140.0
210.0
160.0
700.0
220.0
40.00
10.00
320.0
260.0
344.0
2200
(O)–CH2–Ph–4-(CH2CO2Me)
(O)–CH2–Ph–4-(OCH2CO2Me)
(O)–CH2–Ph–4-(OCH2CO2Me)
(O)–CH2–Ph–3-(CO2Me)
(O)–CH2–Ph–3-(CO2Me)
(O)–CH2C(CH3)3
100
19.0
153
7.00
18.0
21.4
73.0
16.0
85.0
15j
(O)–CH2C(CH3)3
15k
15l
(O)–CH2–Ph–4-(CO2Me)
(O)–CH2–Ph–4-(CO2Me)
(O)–CH2–Ph–4-(CN)
15m
15n
15o
16a
16b
16c
16d
16e
16f
16g
16h
16i
6.00
(O)–CH2–Ph–4-(CN)
(O)–CH2–Ph–4-(NO2)
(O)–CH2CO2H
71.0
77.0
335
25.0
8.00
(O)–CH2–Ph–4-(CH2CO2H)
(O)–CH2–Ph–4-(OCH2CO2H)
(O)–CH2–Ph–3-(CO2H)
(O)–CH2–Ph–3-(CO2H)
(O)–CH2–Ph–4-(CO2H)
(O)–CH2–Ph–4-(CO2H)
(O)–CH2C(CH3)2CO2H
(O)–CH2C(CH3)2CO2H
(O)–CH2–Ph–3-(CONH2)
(O)–CH2–Ph–3-(CONHCH3)
(O)–CH2–Ph–3-(CON(CH3)2)
(O)–CH2C(CH3)2CONH2
(O)–CH2C(CH3)2–(CONHCH3)
(O)–CH2C(CH3)2–(CON(CH3)2)
4.000
750.0
3790
3.50
6.30
3.30
1590
640.0
1180
6300
12.0
20.0
46.0
48.0
37.0
14.4
47.0
54.0
35.0
1488
17a
17b
17c
17d
17e
17f
481.0
397.0
357.0
1835
2010
2032
a Average of n > 2.

M. J. Mulvihill et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1669–1673
Table 2. Cellular potency of lead naphthyl derivatives
1673
OR1/O–Z–CO2H/O–Z–CONR2R3
Stereochemistry
Cellular IC50 (nM)
AT6.1 T47D
a
Compound
17a
17b
17c
17d
17e
17f
16f
16h
(O)–CH2–Ph–3-(CONH2)
(O)–CH2–Ph–3-(CONHCH3)
(O)–CH2–Ph–3-(CON(CH3)2)
(O)–CH2C(CH3)2CONH2
(O)–CH2C(CH3)2–(CONHCH3)
(O)–CH2C(CH3)2–(CON(CH3)2)
(O)–CH2–Ph–4-(CO2H)
syn
syn
syn
syn
syn
syn
syn
syn
IAa
250
IAb
IAb
IAb
250
IAa
IAa
IAa
250
IAb
IAa
<100
300
<100
125
(O)–CH2C(CH3)2CO2H
a Inactive at 250 nM.
b Inactive at 1 lM.
Table 3. Compound 16h profile
181; (d) Kuijpers, A. L.; Van Pelt, P. T.; Bergers, M.;
Boegheim, P. J.; Den Bakker, J. E.; Siegenthaler, G.; Van
de Kerkhof, P. C.; Schalkwijk, J. Br. J. Dermatol. 1998,
139, 380.
Properties
Compound 16h
Biochemical potency (nM)
T47D cell activity (nM)
CYP3A4 (nM)
20
125
3. Clinical Oncology; Rubin, P., Ed., 8th ed.; W.B. Saunders
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6300
CYP1A2 (nM)
CYP2D6 (nM)
>5000
>5000
>5000
6.0
CYP2C9 (nM)
Cmax, 5 mg/kg p.o., (lM)
AUC, 5 mg/kg p.o., (ng h/mL)
Tmax, 5 mg/kg p.o. (h)
Terminal t1/2 (h)
4919
0.5
2.5
19.7
15
C0, 3 mg/kg i.v., (lM)
CL, 3 mg/kg i.v. (mL/min/kg)
Vss, 3 mg/kg i.v. (L/kg)
Oral bioavailability (F%)
3.1
99
ring were explored for potency against CYP26 and selec-
tivity versus other CYPÕs including CYP3A4. The carb-
oxylate alkyl moiety, conceived as a potential key
selectivity determinant, proved to limit general CYP
inhibition by these inhibitors. Utilization of structure–
activity relationships ultimately led to the discovery of
16h as a novel, potent, and selective CYP26 inhibitor
with excellent pharmacokinetic and physiochemical
properties. By suppressing CYP26-mediated resistance,
16h has the potential to modulate exposure to endoge-
nous or co-administered atRA, therefore improving
responses and providing
modality.
a therapeutic anticancer
Acknowledgements
We thank OSI Pharmaceuticals and Dr. Yongsheng Che
and Ms. Viorica M. Lazarescu for their analytical
support.
9. Sonawane, H. R.; Bellur, N. S.; Kulkarni, D. G.; Ayangar,
N. R. Tetrahedron 1994, 50, 1243.
10. Mulvihill, M. J.; Cesario, C.; Smith, V.; Beck, P.; Nigro,
A. J. Org. Chem. 2004, 69, 5124.
References and notes
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12. The oral PK was performed with compound 16h as the
bis-HCl salt, formulated in water and i.v. PK with the free
base compound formulated in 20% hydroxypropyl-b-
cyclodextrin.