Discovery and SAR of methylated tetrahydropyranyl derivatives as inhibitors of isoprenylcysteine carboxyl methyltransferase (ICMT)

Article abstract of DOI:10.1021/jm200249a

A series of tetrahydropyranyl (THP) derivatives has been developed as potent inhibitors of isoprenylcysteine carboxyl methyltransferase (ICMT) for use as anticancer agents. Structural modification of the submicromolar hit compound 3 led to the potent 3-methoxy substituted analogue 27. Further SAR development around the THP ring resulted in an additional 10-fold increase in potency, exemplified by analogue 75 with an IC₅₀ of 1.3 nM. Active and potent compounds demonstrated a dose-dependent increase in Ras cytosolic protein. Potent ICMT inhibitors also reduced cell viability in several cancer cell lines with growth inhibition (GI₅₀) values ranging from 0.3 to >100 μM. However, none of the cellular effects observed using ICMT inhibitors were as pronounced as those resulting from a farnesyltransferase inhibitor.

Full text of DOI:10.1021/jm200249a

ARTICLE
pubs.acs.org/jmc
Discovery and SAR of Methylated Tetrahydropyranyl Derivatives as
Inhibitors of Isoprenylcysteine Carboxyl Methyltransferase (ICMT)
Weston R. Judd,*,† Paul M. Slattum,† Khanh C. Hoang,† Leena Bhoite,† Liisa Valppu,‡ Glen Alberts,‡
Brita Brown,‡ Bruce Roth,‡ Kirill Ostanin,‡ Liwen Huang,‡ Daniel Wettstein,‡ Burt Richards,‡ and
J. Adam Willardsen†
†Department of Medicinal Chemistry and ‡Department of Discovery Biology, Myrexis, Inc., 305 Chipeta Way, Salt Lake City,
Utah 84108, United States
S Supporting Information
b
ABSTRACT: A series of tetrahydropyranyl (THP) derivatives
has been developed as potent inhibitors of isoprenylcysteine
carboxyl methyltransferase (ICMT) for use as anticancer
agents. Structural modification of the submicromolar hit com-
pound 3 led to the potent 3-methoxy substituted analogue 27.
Further SAR development around the THP ring resulted in an
additional 10-fold increase in potency, exemplified by analogue
75 with an IC50 of 1.3 nM. Active and potent compounds
demonstrated a dose-dependent increase in Ras cytosolic protein. Potent ICMT inhibitors also reduced cell viability in several
cancer cell lines with growth inhibition (GI50) values ranging from 0.3 to >100 μM. However, none of the cellular effects observed
using ICMT inhibitors were as pronounced as those resulting from a farnesyltransferase inhibitor.
’ INTRODUCTION
underway using FTIs in combination with standard cytotoxic
agents with the expectation that additive or synergistic activities
will be observed. Some beneficial activity has been seen in
patients with hematological malignancies, and several phase I
and II studies are being conducted to determine optimal dosing
regimen and patient selection criteria for treatment with FTIs.11
Over the past several years multiple reports have demon-
strated a key role for ICMT in Ras localization and transforma-
tion in rodent and human cell-based models.6,12ꢀ14 Small-
molecule inhibitors that specifically target ICMT may therefore
be beneficial in the treatment of cancers characterized by activated
Ras. An ICMT inhibitor, cysmethynil (Figure 1a), was recently
discovered by screening recombinant ICMT protein against a
chemical library of ∼10000 compounds.12 Cysmethynil is a rela-
tively potent in vitro inhibitor (IC50 = 2.4 μM) with modest
antiproliferative activity that selectively reduced growth of Icmt+/+
versus Icmtꢀ/ꢀ mouse embryonic fibroblasts.12 The membrane
localization of a GFP-Ras reporter protein was disrupted in a dose-
dependent manner in polarized MDCK cells treated with cysmethy-
nil. Moreover, the ability of Ras-overexpressing cells to form
colonies was sensitive to cysmethynil, and colony formation could
be rescued by overexpression of ICMT. Cysmethynil has also been
demonstrated to induce autophagic cell death in PC-3 cells via
reduction of mTOR signaling.15 Recently, analogues of cysmethynil
having improved in vitro potencies (IC50 ≈ 0.7 μM) and anti-
proliferative activities (GI50 ≈ 3 μM) have been reported.16
Proteins that contain a C-terminal CAAX (cysteine, aliphatic
amino acid, and X is one of several amino acids) motif regulate
numerous pathways that are critical for tumorigenesis. CAAX-
containing proteins are post-translationally modified via prenylation
of the cysteine residue by protein farnesyltransferase,1 removal of
the AAX motif by Ras-converting enzyme 1 (RCE1),2 and addition
of a methyl group to the modified cysteine residue by the enzyme
isoprenylcysteine carboxyl methyltransferase (ICMT).3 The faithful
modification of CAAX-containing proteins is essential for function
and, in certain cases, required for proper subcellular localization of
proteins. This is best exemplified with the Ras family of G proteins,
where decreased farnesyltransferase, RCE1, or ICMT activity results
in improper subcellular localization of Ras proteins, downstream
pathway inhibition, and loss of cellular transformation.4ꢀ6
Small-molecule inhibitors that disrupt Ras activity may be
beneficial for the treatment of cancers, as ∼30% of cancers have
activating Ras mutations. Several groups have successfully iden-
tified potent and selective farnesyltransferase inhibitors (FTIs)
that prevent Ras membrane localization and downstream path-
way activation.7,8 These compounds have shown potent activity
against Ras-activated tumor cell lines in cell culture and mouse
xenograft models.7,8 Unexpectedly, the FTIs also show potent
activity in cell lines that do not contain activated Ras, suggesting
that other targets such as CENP-E or CENP-F may be equally
important for phenotypes induced by FTI treatment.9
The results observed in preclinical work using FTIs as single
agents have not readily translated into benefit in several phase II
and III human studies.10 Numerous clinical trials are currently
Received: March 2, 2011
Published: June 10, 2011
r
2011 American Chemical Society
5031
dx.doi.org/10.1021/jm200249a J. Med. Chem. 2011, 54, 5031–5047
|

Journal of Medicinal Chemistry
ARTICLE
Figure 1. (a) Cysmethynil. (b) Farnesyltransferase inhibitor 4.
To identify novel small-molecule inhibitors of ICMT, we
screened a chemical library against human recombinant ICMT
enzyme. As a result, several distinct structural classes of compounds
were identified that ranged from low micromolar to submicromolar
potencies. The dimethyltetrahydropyranyl (THP) containing com-
pound 1 is one of two structurally similar HTS hits with low micro-
molar potency that were selected as a starting point for medicinal
chemistry efforts among 11 hit structural classes based on both struc-
tural novelty and AGGC (where AGGC is the prenylated sub-
strate N-acetyl-S-geranylgeranylcysteine) competitive mode of action
(IC50 = 3.5 μM, 1 ꢁ Km SAM; Figure 2). The AGGC-competitive
mode of inhibition is evidenced by an increase of Km but a lack of
significant effects on the maximal velocity of the reaction in the
presence of compound (Figure 2A). In this regard, 1 is similar to
cysmethynil, an indole-based ICMT inhibitor, which was shown to
be competitive with respect to a prenylated methyl acceptor substrate
but noncompetitive toward the second substrate, S-adenosylmethio-
nine (SAM).12 The currently available data for 1 do not allow us to
reliably distinguish between noncompetitive and uncompetitive
mechanisms with respect to SAM (Figure 2B). A relatively small
but significant increase in potency, which accompanied an increase in
SAM concentration from 1- to 10-fold excess over the respective Km,
may be indicative of uncompetitive behavior for this compound. If
confirmed by more thorough kinetic analyses, this observation would
support preferential binding of 1 to the SAM-occupied rather than
the substrate-free enzyme state. Such an assumption would be
consistent with the ordered kinetic mechanism with SAM being
the leading substrate, which has been demonstrated for ICMT.17 It is
also worth noting that, in contrast to cysmethynil, 1 does not
represent a time-dependent inhibitor as we did not observe any
significant increases in its inhibitory potency upon preincubation
with the target enzyme prior to activity assay (data not shown).
Subsequent substructure searches around 1 yielded dimethyl
THP derivatives 2 and 3, having an order of magnitude more
potent activity (IC50 = 0.12 and 0.31 μM), which were selected for
further optimization (Figure 3a). The potent ICMT inhibitors
derived from these efforts showed selective killing in K-Ras-trans-
formed normal rat kidney cells, modulation of Ras membrane
localization, and tumor cell line killing. However, all of the cellular
phenotypes resulting from ICMT inhibitor treatment were much less
pronounced than those observed with the farnesyltransferase inhi-
bitor 4 (FTI-2628, Figure 1b).18 Herein we describe the synthesis
and structureꢀactivity relationship (SAR) of one hit class that has, to
our knowledge, resulted in the most potent in vitro ICMT inhibitor
to date (IC50 = 1.3 nM).
Figure 2. Mechanistic analyses of ICMT inhibition by compound 1.
Enzymatic transmethylation of AGGC in the presence of SAM was
monitored using a fluorometric assay. (A) Effects of 3 μM test
compound on both Vmax and Km for AGGC. (B) Dependence of the
in vitro potency of 1 on SAM concentration.
potencies greater than that of the parent compound. We next focused
our efforts on analogues of compound 3. To simplify the synthesis of
these analogues, the N-furylethanone moiety was removed, leading
to compounds resembling aniline I (Figure 3b).
Synthesis of analogues I was accomplished according to the
general procedure outlined in Scheme 1.19 This general synthetic
approach enabled variation of both terminal rings (regions A and B)
as well as variations to the THP moiety (region C). With regard to
the latter, differential methyl substitution (i.e., des-, di- and tetra-
methyl) and variation of the ring heteroatom (affording THP,
tetrahydrothiopyranyl, and piperidine cores) were incorporated in
analogue preparation. The requisite ketones II were prepared as
described previously20 or were commercially available. Thus, con-
densation of ketones II with ethyl cyanoacetate provided the nitrile
ester intermediates III, which were subjected to copper-catalyzed
conjugate addition with various aryl Grignard reagents to afford the
’ CHEMISTRY
We began SAR investigations around the N-benzylfurancar-
boxamide compound 2(Figure 3a). Although some clear SAR trends
emerged from this endeavor (not shown), we were unable to achieve
5032
dx.doi.org/10.1021/jm200249a |J. Med. Chem. 2011, 54, 5031–5047

Journal of Medicinal Chemistry
ARTICLE
Figure 3. (a) Submicromolar dimethyltetrahydropyranyl ICMT inhibitors from HTS. (b) Simplified aniline analogues with regions of focus for SAR
indicated.
Scheme 1a
a Reagents and conditions: (i) ethyl cyanoacetate, NH4OAc, PhH, reflux; (ii) ArMgBr, CuBr S(Me)2, THF; (iii) KOH, HO(CH2)2OH, 190 °C; (iv)
3
LiAlH4, Et2O; (v) ArBr, Pd(dba)2, BINAP, NaO-t-Bu, PhMe, 80 °C; (vi) ArB(OH)2, DIPEA, Cu(OAc)2, CH2Cl2; (vii) ArF, K2CO3, DMF.
Scheme 2a
followed by coupling of the resulting acid 6 to various 3-sub-
stituted anilines via the mixed anhydride (Scheme 2).
Preparation of the urea and thiourea analogues was accom-
plished as depicted in Scheme 3. Thus, Grignard addition onto
ketone 8, followed by acid-mediated displacement of the resulting
alcohol 9 with sodium azide furnished tertiary azide 10.23 Urea
compounds 12a,b were readily obtained by reduction of azide 10,
followed by condensation of the resulting amine 11 with an aryl iso-
a Reagents and conditions: (i) conc AcOH/HCl, reflux; (ii) i-Bu-
cyanate. Thioureas 13aꢀd were prepared in an analogous manner
using the corresponding aryl isothiocyanate.
(CO2)Cl, DIEA, ArNH2.
The corresponding N-acyl and N-methyl analogues were prepared
from compounds 16 and 18 using standard acylation and alkylation
conditions, respectively (Scheme 4).
spirocyclic intermediates IV. Decarboxylation followed by reduction
of the resulting nitrile then provided the ethylamines VI that would
serve as the common building blocks for parallel synthesis of the
aniline analogues. The latter were formed through palladium-medi-
ated coupling with various aryl bromides21 or via copper-mediated
coupling with arylboronic acids.22 Alternatively, nucleophilic aro-
matic substitution of the corresponding aromatic fluoride was utilized
to arrive at the desired compounds I.
Modification of the intervening chain (region D) connecting
the THP moiety and region A was accomplished by preparation
of amide and urea/thiourea functionalized analogues as well as
N-substituted compounds (Schemes 2ꢀ4). Amide analogues
7aꢀc were prepared by hydrolysis of the nitrile intermediate 5,19
’ RESULTS AND DISCUSSION
Inhibition of recombinant human ICMT enzyme was deter-
mined by fluorometric and radiometric assays using N-acetyl-S-
geranylgeranylcysteine (AGGC) as a methyl-acceptor substrate.23,25
(Graphical data for the determination of ICMT Km and IC50 values
for a representative compound using these methods are given in the
Supporting Information.) Our initial efforts focused on SAR around
the aniline benzene ring of analogues I (region A, Figure 3b).
The unsubstituted analogue 16 was found to be nearly 10-fold
less potent than hit compound 3 (IC50 = 0.31 μM; Table 1).
5033
dx.doi.org/10.1021/jm200249a |J. Med. Chem. 2011, 54, 5031–5047

Journal of Medicinal Chemistry
ARTICLE
Scheme 3a
a Reagents and conditions: (i) PhMgBr, Et2O; (ii) NaN3, F3CCO2H, THF; (iii) LiAlH4, Et2O; (iv) ArNCO, THF; (v) ArNCS, THF.
Scheme 4a
a Reagents and conditions: (i) MeCOCl, DIEA, CH2Cl2; (ii) MeI, K2CO3, DMF.
Likewise, an o-methyl substituent resulted in a decrease in potency
against ICMT (cf. compound 17). In contrast, methyl substitution
in the 3- or 4-position (18 or 19) yielded compounds with
approximately 10-fold higher potencies relative to the parent
compound 3. The dimethyl-substituted analogues, 20 and 21, also
showed significant improvement in potency, albeit to a lesser degree
than the monosubstituted counterparts. A proportional decrease in
the inhibitory activity was observed with increased bulkiness of the
alkyl substituent. Thus, the ethyl substituted analogue 22 was
approximately 4-fold more potent than the 3- and 4-isopropyl
substituted compounds 23 and 24, which in turn were approxi-
mately 4-fold more potent than the 3- and 4-tert-butyl substituted
analogues 25 and 26, respectively. As observed for the methyl-
substituted analogues, the 3-substituted compounds were essentially
equipotent with the corresponding 4-substituted analogues bearing
the same alkyl substituent (i.e., 3-i-Pr vs 4-i-Pr, etc.).
A marked difference in potency was observed between the 3- and
4-methoxy-substituted analogues, with the former having signifi-
cantly higher activity against ICMT (cf. compounds 27 and 28).
Indeed, the 3-methoxy-substituted analogue 27 gave the most
significant improvement in potency among the initial series of
dimethyl THP compounds investigated (IC50 = 15 nM). On the
basis of the increase in potency obtained with 27 and the
4-methylaniline 19 (IC50 = 30 nM), we anticipated a commen-
surate improvement in activity with the 3-methoxy-4-methylani-
line 29. Although this compound displayed significant potency
against ICMT (IC50 = 90 nM), it proved to be less potent than
either of the corresponding monosubstituted analogues.
In general, the 3-position was more tolerant to substitution
relative to the 4-position of the aniline benzene (region A) ring.
A variety of meta-substituents were tolerated, with 3-OCF3, -CN,
-Cl,-F, and -NO2 analogues all displaying good potencies against
ICMT relative to the corresponding 4-substituted compounds.
In accord with previous observations, disubstituted compounds
exhibited diminished activities relative to the corresponding
(mono) 3-substitued analogues (cf. 40 vs 37). In the absence
of another substituent, fluorination of the region A benzene ring
generally resulted in improved potency, as the fluorinated
analogues 43ꢀ45 displayed greater inhibition than the unsub-
stituted compound 16. Region A fluorination in the presence of
another substituent gave mixed results with some analogues
showing improved potency (e.g., 47 and 48) while others
showed diminished potency (e.g., 46 and 49) relative to the
nonfluorinated analogues. Finally, more polar, potentially water-
solubilizing substituents generally led to diminished potencies
(e.g., compounds 55ꢀ59), possibly owing to the likely hydrophobic
environment of the enzyme, a feature necessary to accommodate
the lipophilic farnesyl moiety of post-translationally modified Ras
proteins. A representative analogue 18 (as well as others) was
further analyzed for its mode of ICMT inhibition and was found
to retain the AGGC-competitive mechanism as observed for the
original HTS hit 1 (see Supporting Information).
Having established 3-substitution (especially methoxy or
chloro) in region A as being optimal for potency against ICMT,
we proceeded to evaluate the effects of substitution in region B
(phenyl group adjacent to the THP moiety) with the objective to
further improve compound potency (Table 2). Region B analogues
5034
dx.doi.org/10.1021/jm200249a |J. Med. Chem. 2011, 54, 5031–5047

Journal of Medicinal Chemistry
ARTICLE
Table 1. In Vitro Potency of Dimethyl THP Region A
Analogues
Table 2. In Vitro Potency of Region B Analogues
compd
R1
R2
R3
R4
IC50 (μM)
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
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
F
2.70
H
H
3.6
Me
H
H
0.031
0.031
0.069
0.054
0.040
0.167
0.132
0.556
0.76
Me
Me
H
Me
Me
Et
H
i-Pr
H
H
i-Pr
H
t-Bu
H
t-Bu
H
OMe
H
0.015
0.27
OMe
Me
OMe
H
OMe
OMe
0.090
0.652
0.09
OCF3
H
OCF3
H
0.184
0.131
>5
NMe2
OH
H
CN
H
0.066
0.123
0.025
0.36
H
CN
H
Cl
H
Cl
Cl
H
0.17
Cl
Cl
0.181
0.308
0.049
0.55
Me
Cl
Cl
Me
H
H
H
H
F
F
H
0.052
0.168
0.3
of the 3-fluoro analogue 65 (IC50 = 8 nM), substitution in region B
was not tolerated, resulting in decreased potency relative to the corres-
ponding unsubstituted compound 27. This effect may be due to the
inability of the enzyme to accommodate additional steric bulk in this
region of the molecule. Replacement of the region B phenyl group
with thiophene resulted in compounds that were at least equipotent
with the corresponding phenyl analogues (66 vs 27), with significant
improvement observed in two cases (compounds 67 and 68).
Having established reasonable SAR around regions A and B of
the molecule, we focused our attention on the intervening chain
(region D) connecting the THP moiety and region A. In this regard,
we were particularly interested in modifications adjacent to the aniline
nitrogen, owing to ms/ms metabolic studies (data not shown)
which implicated the position as metabolically labile. Among the
analogues considered were the amides and ureas/thioureas
depicted in Table 3. However, none of the region D amide, urea,
or thiourea analogues were potent against ICMT. Moreover,
these compounds did not display any improved metabolic
stability relative to analogues having an unfunctionalized chain
(region D), indicating that the liability may be in the THP moiety
and/or the aromatic regions.
H
F
H
Me
OMe
OMe
Cl
H
H
H
H
F
F
0.16
F
0.031
0.47
F
H
H
Cl
H
H
H
H
H
H
H
H
Cl
F
0.025
0.032
0.38
H
H
H
H
H
H
H
H
H
H
CF3
H
NO2
H
0.026
0.682
13.1
H
NO2
SO2NH2
H
H
SO2N(CH3)2
NH(CO)Me
H
3.83
H
1.35
CO2Et
H
2.88
CO2H
>5
were synthesized according to Scheme 1, generally maintaining
3-methoxy or 3-chloro substitution in region A. With the exception
5035
dx.doi.org/10.1021/jm200249a |J. Med. Chem. 2011, 54, 5031–5047

Journal of Medicinal Chemistry
ARTICLE
Table 3. Amide and Urea Region D Analogues
Table 5. In Vitro Potencies of Enantiomers of 27
compd
X
Y
R
IC50 (μM)
7a
CH2
CH2
CH2
NH
NH
NH
NH
NH
NH
O
O
O
O
O
S
H
>5
>5
>5
>1
>1
>1
>1
>1
>1
7b
OMe
Cl
7c
12a
12b
13a
13b
13c
13d
OMe
Cl
a Absolute stereochemistry not determined, stereochemistry arbitrarily
assigned.
H
S
OMe
Cl
S
S
F
Table 6. In Vitro Potency of gem-Dimethyl vs des-Methyl
THP Analogues
Table 4. Effect of Aniline N-Substitution
compd
IC50 (μM)
R1
R2
compda
IC50 (μM)
compd
R1
R2
IC50 (μM)
69
70
71
72
73
3.25
0.777
3.83
3.25
0.46
Me
OMe
Cl
H
18
27
37
44
42
0.031
0.015
0.025
0.052
0.049
16
H
H
2.70
>10
H
14a
15
Me
Ac
H
H
H
H
2.34
0.031
9.85
F
H
18
Me
Me
Cl
Me
a Compounds 18ꢀ42 included for comparison.
14b
Me
To probe the effect of aniline nitrogen substitution, the corre-
sponding N-acylated and -methylated analogues were investigated.
As shown in Table 4, aniline N-methylation and -acylation resulted
in greatly diminished activities against ICMT, indicating a probable
H-bond donating role for the aniline.
The region A 3-methoxy-substituted compound 27 was
selected for further evaluation because of its impressive potency
against ICMT. Recognizing the chirality of the dimethyl THP
analogues, the enantiomers were separated by preparative chiral
HPLC (see Experimental Section) and tested in the in vitro assay.
Although the absolute stereochemistry was not determined, an
approximately 20-fold difference in activity was observed for the
respective enantiomers (Table 5).
We next investigated THP ring (region C) modifications,
specifically with regard to the effect of dimethyl substitution on in
vitro potency. Considering the chirality imparted by gem-di-
methylation, in addition to the relative ease of synthesis of analogues
unsubstituted around the THP moiety, we prepared the corre-
sponding des-methyl THP compounds starting from the com-
mercially available ketone according to Scheme 1. As shown in
Table 6, the in vitro potencies of the des-methyl analogues were
diminished 10- to >100-fold relative to the corresponding gem-
dimethyl THP analogues, illustrating the importance of THP
ring substitution.
Figure 4. Piperidine analogues.
piperidines. Analogues were prepared as in Scheme 1 from the
corresponding N-substituted piperidinones. A number of analo-
gues of the type shown in Figure 4 were investigated; however,
none were found to be potent against ICMT.
Having established some of the limitations associated with the
THP moiety (region C) and related analogues, particularly with
regard to THP ring substitution (see Table 6), we aimed to
determine the effect of additional substitution in this region of the
molecule in conjunction with further exploration of the ring
heteroatom. To this end, various tetramethyl-substituted analogues
were evaluated for potency against ICMT (Table 7). In general, the
tetramethyl THP analogues showed the highest potencies. Among
these compounds, the 3-methoxy analogue 75 displayed superior
potency (IC50 = 1.3 nM), being 10-fold more active than the
unsubstituted congener 74. Replacement of the THP oxygen with
either sulfur or nitrogen resulted in significantly decreased activity
Further exploration of the THP ring (region C) was pursued
through replacement of the THP moiety with various N-substituted
5036
dx.doi.org/10.1021/jm200249a |J. Med. Chem. 2011, 54, 5031–5047

Journal of Medicinal Chemistry
ARTICLE
(compounds 77ꢀ83), suggesting that retention of the ring oxygen
is necessary for reasonable potency against ICMT.
Treatment of HCT-116 cells with 74, 75, and farnesyltransferase
inhibitor (FTI) 4 resulted in a dose-dependent accumulation of
Ras in the cytosolic fraction (Figure 5A). The less potent 16 and
inactive analogue 76 did not appreciably increase cytosolic Ras
protein when compared to DMSO-treated cells. Appropriate
markers (consisting of the compartment-specific proteins, β-actin
for cytosolic and V-DAC for membrane) were included to ensure
integrity of the cytosolic and membrane fractions. The accumu-
lation of cytosolic Ras was most evident when cells were treated
with compound 75; less accumulation was seen with com-
pound 74 (Figure 5B). Additionally, treatment of HCT-116 cells
with ∼1 μM 75 or 4 resulted in comparable levels of Ras accumula-
tion in the cytosol. However, unlike FTI 4, which showed
additional Ras accumulation at higher concentrations, the maximal
cytosolic Ras levels using compounds 74 and 75 were lower than
that for FTI 4.
Accumulation of Cytosolic Ras after ICMT Inhibitor Treat-
ment. Disruption of ICMT activity reduces the amount of Ras
protein at the plasma membrane. This has been demonstrated by
both genetic knockout6 and pharmacological inhibition of ICMT.12
To investigate the effect of our series of ICMT inhibitors on
subcellular distribution of Ras, we evaluated multiple assay formats
including GFP-Ras reporter, immunostaining for endogenous Ras,
and subcellular fractionation. These studies identified fractionation of
membrane and cytosolic pools, followed by quantitative immuno-
blotting for Ras as the most quantitative measure of ICMT inhibition.
Table 7. In Vitro Potency of Tetramethyl Analogues
The membrane to cytosolic redistribution of Ras for FTI 4 in
our hands is consistent with published Ras redistribution IC50 values
(500ꢀ1000 nM) for 4 calculated from cell-based readouts.18 The
determination of Ras redistribution by the method illustrated in
Figure 5 is a measure of the cellular population as a whole. Thus,
while it may be possible to see single cells with no membrane bound
Ras, at the time point tested (18 h) in a heterogeneous population of
cells it is likely that not every cell will exhibit the same phenotype,
resulting in the population as a whole showing a mixture of
membrane and cytosolic Ras (e.g., appearance of a membrane Ras
protein band at 5 μM 4). A GFP-Ras reporter assay (not shown)
showed very similar results when using high content imaging analysis,
combining 1000 individual cells. These results suggest that inhibition
of the prenylation step (via farnesyltransferase inhibition) may have a
more pronounced effect on Ras localization than inhibition of the
methylation step.
compd
X
R1
R2
R3
IC50 (μM)
74
75
76
77
78
79
80
81
82
83
O
H
H
H
H
H
H
H
H
H
H
Me
H
H
0.015
0.0013
>5
O
OMe
Cl
O
3-Cl-Ph
S
H
H
H
H
H
H
H
H
>1
S
OMe
Cl
0.42
2.49
>1
S
NH
NH
NH
NH
OMe
Cl
>1
F
>1
Cl
>1
Figure 5. Accumulation and quantitation of Ras protein in cytosolic fraction after ICMT inhibitor treatment. (A) HCT-116 cells were seeded in a 10 cm plate
and treated for 18 h with indicated compounds. Cells were processed into cytosolic and membrane fractions using ProteoExtract subcellular proteome extraction kit
followed by separation on 12.5% PAGE gels. Proteins were detected using anti-p21 Ras antibody and compartment-specific antibodies, V-DAC and β-actin. (B)
Western blots from (A) were analyzed using the EpiChem3 UVP image analyzer. The band intensity for cytoplasmic Ras/β-actin was calculated for each treatment
and normalized to DMSO-treated cells. These results are representative of cytoplasmic Ras accumulation in HCT-116 cells as well as other cell lines tested.
5037
dx.doi.org/10.1021/jm200249a |J. Med. Chem. 2011, 54, 5031–5047

Journal of Medicinal Chemistry
ARTICLE
Table 8. Summary of ICMT Inhibitor Activity on Panel of Tumor Cell Linesa
GI50 (μM)
HCT-116,
K-Ras
MiaPaCa,
K-Ras
CCRF-CEM,
K-Ras
A549,
K-Ras
SKMel-2,
T24,
HL-60,
N-Ras
DU145,
WT
PC3,
WT
compd
in vitro IC50 (μM)
N-Ras
N-Ras
16
74
75
76
4
1
33
39
NT
3
36
36
85
NT
9
42
76
45
NT
2
35
0.015
0.001
>5
26
12
NT
NT
NT
NT
>100
>100
NT
43
17
20
19
0.3
12
3
13
>100
0.04
>100
0.0007
94
NT
0.0002
NT
0.002
>100
1
NT
0.0004
0.0009
a NT = not tested. WT = wild type.
Cell Viability after ICMT Inhibitor Treatment. To determine
if ICMT inhibitors reduced cell viability, we selected eight tumor
cell lines (with and without activating mutations in Ras) and
exposed them to various compounds. The potent ICMT inhibi-
tors 74 and 75 were modestly cytotoxic in all of the cell lines
tested independent of the Ras status, with GI50 values ranging from
0.3 to >100 μM (Table 8). The hematopoietic cell lines CCRF-
CEM and HL-60 were the most sensitive to ICMT inhibition. The
inactive analogue 76 did not reduce cell viability in any of the cell
lines tested. Unlike the ICMT inhibitors that showed modest
reduction in cell viability, treatment with FTI 4 resulted in potent
cytotoxicity in cell lines with Ras mutations (GI50 = 0.0002ꢀ
0.04 μM). The wild-type Ras DU145 and PC-3 cell lines were less
sensitive to 4 (GI50 = 1ꢀ2 μM). On the basis of these results,
inhibition of the prenylation step of CAAX protein processing with
4 was >1000-fold more effective in reducing cell viability than
inhibition of the methylation processing step with the class of small-
molecule ICMT inhibitors described herein.
It is conceivable that the modest cellular effects observed with
the potent THP analogues could be due to unfavorable compound
physical properties, particularly those affecting cellular permeability.
However, we do not believe that permeability factors for com-
pounds 74 (logD7.4 = 4.85) and 75 (logD7.4 = 4.83) account for a
lack of cell viability phenotype in the vast majority of cancer cell lines
tested, based on log D values, which are in the acceptable range of
Lipinski’s rules,26 and the fact that 75 is permeable in hematopoietic
cells and reduces viability. The activity of compound 75 in this cell
type is possibly due to off-target activity. The off-target may be
essential in suspension cells and not solid tumor lines. Additional
work would be required to confirm these statements.
inhibitors. This suggests that the final step (carboxyl methylation)
in post-translational processing of CAAX-containing proteins may
not be as essential as the first step (prenylation) in the biology of
proteins, such as Ras. Alternatively, it may suggest that FTIs
modulate additional non-CAAX motif-containing proteins that are
required for cell survival.
’ EXPERIMENTAL SECTION
Chemistry. General Methods and Materials. 1H NMR spectra
were recorded at 400 MHz. Chemical shifts are reported in parts per million
(ppm) downfield from TMS (0.00 ppm), and J coupling constants are
reported in hertz. HPLCꢀMS were run in ESI mode using an Xterra MS
C18 (Waters) 4.6 mm ꢁ 50 mm, 5 μm column. HPLC purity was
determined using a 4.6 mm ꢁ 150 mm Xterra C18 5 μm column, observ-
ing at both 203 and 280 nm. Both HPLCꢀMS and HPLC were reverse
phase with a MeCN/H2O (0.01% v/v TFA) gradient and a flow rate of
0.5 mL/min. All final compounds were g95% pure by HPLC at both 203
and 280 nm. MPLC purifications were performed using an Isco RF system
utilizing a hexane/EtOAc or DCM/MeOH gradient.
Ketones II were commercially available or were prepared according
to the literature.20
General Procedures of the Synthesis of Intermediates
IIIꢀVI.19 General Procedure for the Synthesis of Cyanoalk-
ylidene Ethyl Ester Intermediates III. The appropriate ketone
(10 mmol), ethyl cyanoacetate (15 mmol), and ammonium acetate
(1 mmol) were heated at reflux in 50 mL of toluene using a DeanꢀStark
trap. After 16ꢀ72 h of reflux, the solvent was removed and the residue
purified by MPLC.
General Procedure for the Synthesis of Intermediates IV.
The appropriate arylmagnesium bromide (3.34 mmol) was added to a
solution of a cyanoalkylidene ethyl ester intermediate III (2.84 mmol)
’ CONCLUSION
and a catalytic amount of CuBr S(Me)2 in 20 mL of THF at ꢀ78 °C.
3
The solution was allowed to warm to room temperature and stirred
overnight. The reaction was quenched with aqueous 1 N hydrochloric
acid, and the aqueous layer was extracted with ethyl acetate (3ꢁ). The
combined organic layers were dried (Na2SO4), filtered, and concen-
trated. The residue was purified by MPLC.
Several reports have demonstrated the importance of post-
translational modification of Ras proteins for tumor cell survival.12,15
We generated a series of potent ICMT inhibitors to disrupt the
terminal processing step of CAAX-containing proteins for potential
utility as chemotherapeutic agents. The potency of ICMT inhibition
in this series of compounds was increased 100-fold from hit
compound 2 (IC50 = 0.12 μM) to the tetramethyl-substituted
analogue 75 (IC50 = 1.3 nM). Kinetic analysis using HTS com-
pound 1 (as well as other representative compounds) suggests that
ICMT inhibition is competitive with respect to Ras protein
substrate. Mechanism-based cellular assays monitoring Ras subcel-
lular localization show a decrease in membrane localization upon
inhibitor treatment consistent with a role of ICMT in post-transla-
tional processing of CAAX-containing Ras. However, in contrast
to farnesyltransferase inhibition, we observe neither potent nor
Ras-dependent effects on viability with small-molecule ICMT
General Procedure for the Synthesis of Intermediates V
and VI. The appropriate cyano-1-arylacetic acid ethyl ester IV (2.0
mmol) and potassium hydroxide (4.0 mmol) were combined in 8 mL of
ethylene glycol and heated to 190 °C for 1.5 h. The reaction mixture was
poured into 90 mL of water and extracted with ethyl ether (3ꢁ). The
combined organic layers were washed with water (1ꢁ), brine (1ꢁ),
dried (Na2SO4), filtered, and concentrated. The residue was purified by
MPLC. The resulting 1-arylacetonitrile V was dissolved in ether (10 mL)
and cooled to 0 °C. Lithium aluminum hydride (2.0 equiv) was added
dropwise, and the reaction mixture was warmed to room temperature
and stirred for 2 h. The reaction was quenched by the sequential addition
of water (1 mL per 1.0 weight (g) equivalent LAH), 15% sodium
5038
dx.doi.org/10.1021/jm200249a |J. Med. Chem. 2011, 54, 5031–5047

Journal of Medicinal Chemistry
ARTICLE
hydroxide solution (1 mL per 1.0 weight (g) equivalent LAH), and water
(3 mL per 1.0 weight (g) equivalent LAH). The reaction mixture was
filtered through Celite and concentrated under vacuum to afford amine
intermediates VI.
N-[2-(2,2-Dimethyl-4-phenyltetrahydro-2H-pyran-4-yl)-
ethyl]-3,5-dimethylaniline (21). 21 was prepared according to
general procedure B using the appropriate aromatic bromide (19.8 mg,
28%). 1H NMR (CDCl3, 400 MHz) δ 7.33ꢀ7.40 (m, 3H), 7.22ꢀ7.28 (m,
2H), 6.29 (s, 1H), 5.94 (s, 2H), 3.76ꢀ3.88 (m, 2H), 3.15 (br s, 1H),
2.91ꢀ2.98 (m, 1H), 2.56ꢀ2.63 (m, 1H), 2.40ꢀ2.45 (m, 1H), 2.21 (dd, J =
13.5, 2.3 Hz, 1H), 2.16 (s, 6H), 1.86ꢀ1.94 (m, 1H), 1.64ꢀ1.74 (m, 3H),
1.20 (s, 3H), 0.67 (s, 3H). ESI-MS m/z 338 (M + H)+. HRMS: calcd,
338.247 84 (M + H)+; found, 338.248 77.
N-[2-(2,2-Dimethyl-4-phenyltetrahydro-2H-pyran-4-yl)-
ethyl]-3-ethylaniline (22). 22 was prepared according to general
procedure B using the appropriate aromatic bromide (13.1 mg, 18.5%). 1H
NMR (CDCl3, 400 MHz) δ 7.35ꢀ7.39 (m, 3H), 7.22ꢀ7.27 (m, 2H),
6.98ꢀ7.02 (m, 1H), 6.49 (d, J = 7.55 Hz, 1H), 6.14ꢀ6.17 (m, 2H),
3.76ꢀ3.89 (m, 2H), 3.20 (br s, 1H), 2.93ꢀ3.03 (m, 1H), 2.58ꢀ2.65 (m,
1H), 2.49 (q, J = 7.61 Hz, 2H), 2.40ꢀ2.46 (m, 1H), 2.22 (dd, J = 13.9, 2.3
Hz, 1H), 1.88ꢀ1.95 (m, 1H), 1.67ꢀ1.74 (m, 3H), 1.20 (s, 3H), 1.16 (t, J =
7.61 Hz, 3H), 0.67 (s, 3H). ESI-MS m/z 338 (M + H)+. HRMS: calcd,
338.247 84 (M + H)+; found, 338.248 57.
General Procedure A for the Synthesis of THP Ethylani-
lines: N-[2-(2,2-Dimethyl-4-phenyltetrahydro-2H-pyran-4-yl)-
ethyl]aniline (16). 2-(2,2-Dimethyl-4-phenyl-tetrahydropyran-4-
yl)ethylamine VI (50.0 mg, 0.21 mmol), phenylboronic acid (51.2 mg,
0.42 mmol), diisopropylethylamine (135 mg, 1.05 mmol), and copper-
(II) acetate (38.1 mg, 0.21 mmol) were combined in dichloromethane
(1.0 mL) and stirred overnight at room temperature. The solvent was
removed and the residue was purified by MPLC (silica, hexane/ethyl
acetate) to provide the title compound (19.4 mg, 30%). 1H NMR
(CDCl3, 400 MHz) δ 7.32ꢀ7.38 (m, 4H), 7.22ꢀ7.27 (m, 1H), 7.05ꢀ
7.10 (m, 2H), 6.63 (tt, J = 7.2, 1.1 Hz, 1H), 6.30ꢀ6.34 (m, 2H), 3.76ꢀ
3.90 (m, 2H), 3.22 (br s, 1H), 2.90ꢀ3.00 (m, 1H), 2.56ꢀ2.66 (m, 1H),
2.41ꢀ2.47 (m, 1H), 2.23 (dd, J = 13.8, 2.7 Hz, 1H), 1.88ꢀ1.96 (m, 1H),
1.66- 1.75 (m, 3H), 1.20 (s, 3H), 0.67 (s, 3H). ESI-MS m/z 310 (M + H)+.
HRMS: calcd, 310.216 54 (M + H)+; found, 310.217 04.
N-[2-(2,2-Dimethyl-4-phenyltetrahydro-2H-pyran-4-yl)-
ethyl]-3-isopropylaniline (23). 23 was prepared according to
general procedure B using the appropriate aromatic bromide (31.7 mg,
43%). 1H NMR (CDCl3, 400 MHz) δ 7.34ꢀ7.37 (m, 4H), 7.22ꢀ7.26 (m,
1H), 7.01 (t, J = 7.2 Hz, 1H), 6.52 (d, J = 7.2 Hz, 1H), 6.14ꢀ6.19 (m, 2H),
3.77ꢀ3.88 (m, 2H), 3.20 (br s, 1H), 2.93ꢀ3.00 (m, 1H), 2.73 (h, J = 6.9
Hz, 1H), 2.58ꢀ2.65 (m, 1H), 2.41ꢀ2.47 (m, 1H), 2.22 (dd, J = 13.9, 2.3
Hz, 1H), 1.89ꢀ1.96 (m, 1H), 1.66ꢀ1.75 (m, 3H), 1.20 (s, 3H), 1.18 (d, J=
6.9 Hz, 6H), 0.67 (s, 3H). ESI-MS m/z 352 (M + H)+. HRMS: calcd,
352.263 49 (M + H)+; found, 352.264 75.
N-[2-(2,2-Dimethyl-4-phenyltetrahydro-2H-pyran-4-yl)-
ethyl]-4-isopropylaniline (24). 24 was prepared according to
general procedure B using the appropriate aromatic bromide (28.3 mg,
38%). 1H NMR (CDCl3, 400 MHz) δ 7.33ꢀ7.36 (m, 4H), 7.21ꢀ7.25 (m,
1H), 6.93ꢀ6.96 (m, 2H), 6.27ꢀ6.30 (m, 2H), 3.77ꢀ3.88 (m, 2H), 3.13
(br s, 1H), 2.90ꢀ2.97 (m, 1H), 2.75 (h, J = 6.8 Hz, 1H), 2.54ꢀ2.63 (m,
1H), 2.40ꢀ2.47 (m, 1H), 2.22 (dd, J = 14.0, 2.4 Hz, 1H), 1.87ꢀ1.95 (m,
1H), 1.66ꢀ1.74 (m, 3H), 1.20 (s, 3H), 1.16 (d, J = 6.8 Hz, 6H), 0.67 (s,
3H). ESI-MS m/z 352 (M + H)+. HRMS: calcd, 352.263 49 (M + H)+;
found, 352.264 52.
3-tert-Butyl-N-[2-(2,2-dimethyl-4-phenyltetrahydro-2H-
pyran-4-yl)ethyl]aniline (25). 25 was prepared according to
general procedure B using the appropriate aromatic bromide (37.6 mg,
49%). 1H NMR (CDCl3, 400 MHz) δ 7.33ꢀ7.38 (m, 4H), 7.22ꢀ7.26 (m,
1H), 6.83 (d, J = 8.7 Hz, 1H), 6.14 (d, J = 2.7 Hz, 1H), 6.10 (dd, J = 8.7, 2.7
Hz, 1H), 3.76ꢀ3.87 (m, 2H), 3.09 (br s, 1H), 2.90ꢀ2.97 (m, 1H),
2.56ꢀ2.63 (m, 1H), 2.40ꢀ2.46 (m, 1H), 2.21 (dd, J = 14.0, 2.5 Hz, 1H),
2.12 (s, 3H), 2.10 (s, 3H), 1.85ꢀ1.94 (m, 1H), 1.64ꢀ1.74 (m, 3H), 1.20 (s,
3H), 0.67 (s, 3H). ESI-MS m/z 366 (M + H)+. HRMS: calcd, 366.279 14
(M + H)+; found, 366.280 44.
4-tert-Butyl-N-[2-(2,2-dimethyl-4-phenyltetrahydro-2H-
pyran-4-yl)ethyl]aniline (26). 26 was prepared according to
general procedure B using the appropriate aromatic bromide (21.2 mg,
27%). 1H NMR (400 MHz, CDCl3) δ 7.32ꢀ7.39 (m, 4H), 7.21ꢀ7.27 (m,
1H), 7.09ꢀ7.12 (m, 2H), 6.28ꢀ6.31 (m, 2H), 3.75ꢀ3.88 (m, 2H), 3.14
(br s, 1H), 2.90ꢀ2.98 (m, 1H), 2.55ꢀ2.62 (m, 1H), 2.40ꢀ2.46 (m, 1H),
2.22 (dd, J = 14.0, 2.6 Hz, 1H)), 1.87ꢀ1.95 (m, 1H), 1.67ꢀ1.74 (m, 3H),
1.24 (s, 9H), 1.20 (s, 3H), 0.67 (s, 3H). ESI-MS m/z 366 (M + H)+.
HRMS: calcd, 366.279 14 (M + 1); found, 366.279 11.
N-[2-(2,2-Dimethyl-4-phenyltetrahydro-2H-pyran-4-yl)-
ethyl]-2-methylaniline (17). 17 was prepared according to general
procedure A using the appropriate boronic acid (4.2 mg, 4.1%). H
1
NMR (CDCl3, 400 MHz) δ 7.32ꢀ7.48 (m, 4H), 7.20ꢀ7.27 (m, 1H),
6.95ꢀ7.04 (m, 2H), 6.95 (t, J = 8.38 Hz, 1H), 6.27 (d, J = 8.38 Hz, 1H),
3.76ꢀ3.91 (m, 2H), 2.91ꢀ3.00 (m, 1H), 2.62ꢀ2.73 (m, 1H), 2.46 (d,
J = 13.8 Hz, 1H), 2.24 (d, J = 13.8 Hz, 1H), 1.90ꢀ2.03 (m, 4H),
1.68ꢀ1.81 (m, 3H), 1.20 (s, 3H), 0.67 (s, 3H). ESI-MS m/z 324 (M +
H)+. HRMS: calcd, 324.232 19 (M + H)+; found, 324.231 04.
N-[2-(2,2-Dimethyl-4-phenyltetrahydro-2H-pyran-4-yl)-
ethyl]-3-methylaniline (18). 18 was prepared according to general
procedure A using the appropriate boronic acid (34.8 mg, 51%). 1H NMR
(CDCl3, 400 MHz) δ 7.33ꢀ7.40 (m, 3H), 7.22ꢀ7.28 (m, 2H), 6.97 (t, J =
7.75 Hz, 1H), 6.46 (d, J = 7.75 Hz, 1H), 6.11ꢀ6.16 (m, 2H), 3.76ꢀ3.91
(m, 2H), 3.20 (br s, 1H), 2.91ꢀ3.01 (m, 1H), 2.56ꢀ2.65 (m, 1H),
2.41ꢀ2.48 (m, 1H), 2.18ꢀ2.25 (m, 1H), 2.18 (s, 3H), 1.88ꢀ1.96 (m, 1H),
1.65- 1.77 (m, 3H), 1.20 (s, 3H), 0.67 (s, 3H). ESI-MS m/z 324 (M + H)+.
HRMS: calcd, 324.232 19 (M + H)+; found, 324.231 27.
N-[2-(2,2-Dimethyl-4-phenyltetrahydro-2H-pyran-4-yl)-
ethyl]-4-methylaniline (19). 19 was prepared according to general
procedure A using the appropriate boronic acid (12.0 mg, 12%). 1H NMR
(CDCl3, 400 MHz) δ 7.31ꢀ7.39 (m, 4H), 7.21ꢀ7.27 (m, 1H), 6.89 (d, J =
8.25 Hz, 2H), 6.26 (d, J= 8.25 Hz, 2H), 3.76ꢀ3.91 (m, 2H), 2.89ꢀ2.99 (m,
1H), 2.52ꢀ2.63 (m, 1H), 2.38ꢀ2.47 (m, 1H), 2.18ꢀ2.24 (m, 4H), 1.88ꢀ
1.96 (m, 1H), 1.65- 1.75 (m, 3H), 1.20 (s, 3H), 0.67 (s, 3H). ESI-MS m/z
324 (M + H)+. HRMS: calcd, 324.232 19 (M + H)+; found, 324.231 15.
General Procedure B for the synthesis of THP Ethylani-
lines: N-[2-(2,2-Dimethyl-4-phenyltetrahydro-2H-pyran-4-
yl)ethyl]-3,4-dimethylaniline (20). To a solution of bis(dibenzy-
lideneacetone)palladium(0) (Pd(dba)2, 1.0 mg, 0.0017 mmol), (()-BINAP
(1.0 mg, 0.017 mmol), and sodium tert-butoxide (28.2 mg, 0.294 mmol)
in N2-purged toluene (0.2 mL) was added 4-boromo-o-xylene (47.0 mg,
0.252 mmol) followed by a solution of 2-(2,2-dimethyl-4-phenyltetra-
hydropyran-4-yl)ethylamine VI (50.0 mg, 0.21 mmol) in N2-purged
toluene (0.2 mL). The vial was purged with N2, capped tightly, and the
mixture was stirred at 80 °C overnight. The reaction mixture was con-
centrated and purified by MPLC (silica, hexane/ethyl acetate) to afford the
title compound (20.4 mg, 29%). 1H NMR (CDCl3, 400 MHz) δ 7.35ꢀ
7.36 (m, 4H), 7.22ꢀ7.26 (m, 1H), 7.03 (t, J= 7.8 Hz, 1H), 6.68 (ddd, J=7.8,
1.9, 0.9 Hz, 1H), 6.37 (t, J = 1.9 Hz, 1H), 6.15 (ddd, J = 7.8, 2.5, 0.9 Hz,
1H), 3.77ꢀ3.88 (m, 2H), 3.20 (br s, 1H), 2.93ꢀ3.01 (m, 1H), 2.59ꢀ
2.66 (m, 1H), 2.42ꢀ2.48 (m, 1H), 2.23 (dd, J = 14.1, 2.5 Hz, 1H),
1.89ꢀ1.97 (m, 1H), 1.67ꢀ1.75 (m, 3H), 1.24 (s, 9H), 1.20 (s, 3H), 0.68
(s, 3H). ESI-MS m/z 338 (M + H)+. HRMS: calcd, 338.247 84 (M +
H)+; found, 338.247 45.
N-[2-(2,2-Dimethyl-4-phenyltetrahydro-2H-pyran-4-yl)-
ethyl]-3-methoxyaniline (27). 27 was prepared according to
general procedure B using the appropriate aromatic bromide (57.9 mg,
81%). 1H NMR (CDCl3, 400 MHz) δ 7.33ꢀ7.39 (m, 4H), 7.21ꢀ7.26 (m,
1H), 6.98 (t, J = 8.11 Hz, 1H), 6.20 (ddd, J = 8.1, 2.2, 0.7 Hz, 1H),
5.95 (ddd, J = 8.1, 2.2, 0.7 Hz, 1H), 5.89 (t, J = 2.25 Hz, 1H), 3.76ꢀ3.88
5039
dx.doi.org/10.1021/jm200249a |J. Med. Chem. 2011, 54, 5031–5047

Journal of Medicinal Chemistry
ARTICLE
(m, 2H), 3.71 (s, 3H), 3.23 (br s, 1H), 2.91ꢀ2.98 (m, 1H), 2.56ꢀ2.63 (m,
1H), 2.40ꢀ247 (m, 1H), 2.22 (dd, J = 13.9, 2.4 Hz, 1H), 1.88ꢀ1.95 (m,
1H), 1.66ꢀ1.74 (m, 3H), 1.20 (s, 3H), 0.67 (s, 3H). ESI-MS m/z340 (M +
H)+. HRMS: calcd, 340.227 11 (M + H)+; found, 340.227 10.
N-[2-(2,2-Dimethyl-4-phenyltetrahydro-2H-pyran-4-yl)-
ethyl]-4-methoxyaniline (28). 28 was prepared according to
general procedure A using the appropriate boronic acid (13.4 mg, 19%). 1H
NMR (CDCl3, 400 MHz) δ 7.31ꢀ7.40 (m, 4H), 7.21ꢀ7.27 (m, 1H),
6.66ꢀ6.72 (m, 2H), 6.28ꢀ6.33 (m, 2H), 3.72ꢀ3.91 (m, 2H), 3.70 (s, 3H),
2.87ꢀ2.96 (m, 1H), 2.53ꢀ2.60 (m, 1H), 2.41ꢀ2.48 (m, 1H), 2.23 (dd, J =
14.1, 2.2 Hz, 1H), 1.88ꢀ1.96 (m, 1H), 1.65ꢀ1.77 (m, 3H), 1.20 (s, 3H),
0.67 (s, 3H). ESI-MS m/z 340 (M + H)+. HRMS: calcd, 340.227 11 (M +
H)+; found, 340.226 42.
using the appropriate aromatic bromide. 1H NMR (CDCl3, 400 MHz) δ
7.26ꢀ7.18 (m, 2H), 7.15ꢀ7.09 (m, 2H), 6.97 (t, J = 8.0 Hz, 1H), 6.14
(ddd,J= 0.8, 2.4, 8.0 Hz, 1H), 6.08 (ddd, J= 0.8, 2.4, 8.0 Hz, 1H), 5.99 (t, J=
2.4 Hz, 1H), 3.73ꢀ3.58 (m, 2H), 3.14ꢀ3.07 (m, 1H), 3.00ꢀ2.93 (m, 1H),
2.01ꢀ1.90 (m, 6H), 1.32 (s, 3H), 1.31 (s, 3H). ESI-MS m/z326 (M + H)+.
HRMS: calcd, 326.211 46 (M + H)+; found, 326.212 16.
3-{[2-(2,2-Dimethyl-4-phenyltetrahydro-2H-pyran-4-yl)-
ethyl]amino}benzonitrile (35). 35 was prepared according to
general procedure B using the appropriate aromatic bromide (18.2 mg,
26%). 1H NMR (CDCl3, 400 MHz) δ 7.34ꢀ7.42 (m, 4H), 7.26ꢀ7.31 (m,
1H), 7.10 (t, J = 8.3 Hz, 1H), 6.86ꢀ6.88 (m 1H), 6.45 (ddd J = 8.3, 2.5, 0.9
Hz, 1H), 6.40 (dd, J = 2.0, 1.6 Hz, 1H), 3.78ꢀ3.89 (m, 2H), 3.43 (t, J = 5.8
Hz, 1H), 2.92ꢀ3.00 (m, 1H), 2.58ꢀ2.66 (m, 1H), 2.40ꢀ247 (m, 1H),
2.24 (dd, J = 14.0, 2.4 Hz, 1H), 1.88ꢀ1.95 (m, 1H), 1.65ꢀ1.73 (m, 3H),
1.20 (s, 3H), 0.67 (s, 3H). ESI-MS m/z 335 (M + H)+. HRMS: calcd,
335.211 79 (M + H)+; found, 335.212 11.
4-{[2-(2,2-Dimethyl-4-phenyltetrahydro-2H-pyran-4-yl)-
ethyl]amino}benzonitrile (36). 36 was prepared according to
general procedure B using the appropriate aromatic bromide (20.8 mg, 30%).
1H NMR (CDCl3, 400 MHz) δ 7.25ꢀ7.41 (m, 7H), 6.18ꢀ6.21 (m, 2H),
3.77ꢀ3.88 (m, 2H), 3.73 (t, J= 5.3 Hz, 1H), 2.96ꢀ3.04 (m, 1H), 2.63ꢀ2.71
(m, 1H), 2.40ꢀ2.46(m, 1H), 2.24(dd, J= 13.9, 2.6 Hz, 1H), 1.87ꢀ1.95(m,
1H), 1.65ꢀ1.74 (m, 3H), 1.20 (s, 3H), 0.67 (s, 3H). ESI-MS m/z 328 (M +
H)+. HRMS: calcd, 335.211 79 (M + H)+; found, 335.211 50.
N-[2-(2,2-Dimethyl-4-phenyltetrahydro-2H-pyran-4-yl)-
ethyl]-3-methoxy-4-methylaniline (29). 29 was prepared ac-
cording to general procedure A using the appropriate boronic acid. The
product was purified by HPLC and isolated as the trifluoroacetic acid salt
1
(17.5 mg, 18%). H NMR (CDCl3, 400 MHz) δ 7.32ꢀ7.40 (m, 4H),
7.22ꢀ7.27 (m, 1H), 7.09 (d, J = 8.0 Hz, 1H), 6.56 (d, J = 2.1 Hz, 1H), 6.51
(dd, J = 8.0, 2.1 Hz, 1H), 3.71ꢀ3.84 (m, 5H), 3.18 (dt, J = 12.3, 4.7 Hz,
1H), 2.60 (dt, J = 12.3, 4.7 Hz, 1H), 2.47 (dd, J = 14.2, 2.4 Hz, 1H), 2.32
(dd, J = 14.2, 2.4 Hz, 1H), 2.14 (s, 3H), 1.97 (dt, J = 12.7, 4.5 Hz, 1H),
1.50ꢀ1.80 (m, 3H), 1.16 (s, 3H), 0.62 (s, 3H). ESI-MS m/z 354 (M + H)+.
HRMS: calcd, 354.242 76 (M + H)+; found, 354.243 48.
N-[2-(2,2-Dimethyl-4-phenyltetrahydro-2H-pyran-4-yl)-
ethyl]-3,4-dimethoxyaniline (30). 30 was prepared according
to general procedure A using the appropriate aromatic bromide (18.1 mg,
23%). 1H NMR (CDCl3, 400 MHz) δ 7.33ꢀ7.38 (m, 4H), 7.21ꢀ7.27 (m,
1H), 6.65 (d, J = 8.8 Hz, 1H), 5.96 (d, J = 2.9 Hz, 1H), 5.86 (dd, J = 8.8, 2.9
Hz, 1H), 3.71ꢀ3.87 (m, 8H), 2.89ꢀ2.97 (m, 1H), 2.56ꢀ2.63 (m, 1H),
2.40ꢀ2.47 (m, 1H), 2.23 (dd, J = 14.0, 2.3 Hz, 1H), 1.88ꢀ1.96 (m, 1H),
1.66- 1.74 (m, 3H), 1.20 (s, 3H), 0.67 (s, 3H). ESI-MS m/z 370 (M + H)+.
HRMS: calcd, 370.237 67 (M + H)+; found, 370.238 00.
3-Chloro-N-[2-(2,2-dimethyl-4-phenyltetrahydro-2H-pyr-
an-4-yl)ethyl]aniline (37). 37 was prepared according to general
procedure A using the appropriate boronic acid (9.4 mg, 8.5%). H
NMR (CDCl3, 400 MHz) δ 7.33ꢀ7.42 (m, 4H), 7.24ꢀ7.30 (m, 1H),
6.96 (t, J = 8.16 Hz, 1H), 6.58 (dd, J = 8.2, 2.0 Hz, 1H), 6.25 (t, J = 12.0
Hz, 1H), 6.16 (dd, J = 8.2, 2.0 Hz, 1H), 3.72ꢀ3.91 (m, 2H), 3.30 (br s,
1H), 2.87ꢀ2.96 (m, 1H), 2.55ꢀ2.62 (m, 1H), 2.40ꢀ246 (m, 1H), 2.23
(dd, J = 13.8, 2.2 Hz, 1H), 1.86ꢀ1.95 (m, 1H), 1.64ꢀ1.74 (m, 3H), 1.20
(s, 3H), 0.67 (s, 3H). ESI-MS m/z 344 (M + H)+. HRMS: calcd,
344.177 57 (M + H)+; found, 344.176 57.
1
N-[2-(2,2-Dimethyl-4-phenyltetrahydro-2H-pyran-4-yl)-
ethyl]-3-(trifluoromethoxy)aniline (31). 31 was prepared ac-
cording to general procedure B using the appropriate aromatic bromide
4-Chloro-N-[2-(2,2-dimethyl-4-phenyltetrahydro-2H-pyr-
an-4-yl)ethyl]aniline (38). 38 was prepared according to general
1
1
(39.3 mg, 47%). H NMR (CDCl3, 400 MHz) δ 7.33ꢀ7.40 (m, 4H),
procedure A using the appropriate boronic acid (10.6 mg, 15%). H
7.24ꢀ7.28 (m, 1H), 7.04 (t, J = 9.0 Hz, 1H), 6.45 (d, J = 9.0 Hz, 1H), 6.19
(d, J = 9.0 Hz, 1H), 6.07 (s, 1H), 3.78ꢀ3.88 (m, 2H), 3.35 (br s, 1H),
2.91ꢀ2.99 (m, 1H), 2.56ꢀ2.66 (m, 1H), 2.44 (d, J = 14.0 Hz, 1H), 2.24 (d,
J = 14.0 Hz, 1H), 1.88ꢀ1.96 (m, 1H), 1.65ꢀ1.74 (m, 3H), 1.20 (s, 3H),
0.67 (s, 3H). ESI-MS m/z 394 (M + H)+. HRMS: calcd, 394.198 84 (M +
H)+; found, 394.199 15.
NMR (CDCl3, 400 MHz) δ 7.30ꢀ7.41 (m, 4H), 7.22ꢀ7.28 (m, 1H),
6.98ꢀ7.03 (m, 2H), 6.17ꢀ6.22 (m, 2H), 3.76ꢀ3.91 (m, 2H), 3.20 (br s,
1H), 2.91ꢀ3.01 (m, 1H), 2.56ꢀ2.65 (m, 1H), 2.41ꢀ2.48 (m, 1H), 2.23
(dd, J = 14.1, 2.2 Hz, 1H), 1.88ꢀ1.96 (m, 1H), 1.65ꢀ1.77 (m, 3H), 1.20
(s, 3H), 0.67 (s, 3H). ESI-MS m/z 344 (M + H)+. HRMS: calcd,
344.177 57 (M + H)+; found, 344.177 36.
N-[2-(2,2-Dimethyl-4-phenyltetrahydro-2H-pyran-4-yl)-
ethyl]-4-(trifluoromethoxy)aniline (32). 32 was prepared ac-
cording to general procedure B using the appropriate aromatic bromide
3,5-Dichloro-N-[2-(2,2-dimethyl-4-phenyltetrahydro-2H-
pyran-4-yl)ethyl]aniline (39). 39 was prepared according to general
procedure B using the appropriate aromatic bromide (66.7 mg, 84%). 1H
NMR (CDCl3, 400 MHz) δ 7.33ꢀ7.41 (m, 4H), 7.25ꢀ7.29 (m, 1H),
6.57 (t, J = 1.95 Hz, 1H), 6.10 (d, J = 1.95 Hz, 2H), 3.77ꢀ3.88 (m, 2H),
3.55 (t, J = 5.45 Hz, 1H), 2.87ꢀ2.96 (m, 1H), 2.54ꢀ2.62 (m, 1H),
2.39ꢀ2.45 (m, 1H), 2.23 (dd, J = 14.1, 2.7 Hz, 1H), 1.86ꢀ1.93 (m, 1H),
1.63ꢀ1.72 (m, 3H), 1.20 (s, 3H), 0.67 (s, 3H). ESI-MS m/z 379 (M +
H)+. HRMS: calcd, 378.138 60 (M + H)+; found, 378.136 82.
3,4-Dichloro-N-[2-(2,2-dimethyl-4-phenyltetrahydro-2H-
pyran-4-yl)ethyl]aniline (40). 40 was prepared according to gen-
eral procedure B using the appropriate aromatic bromide (44.5 mg, 56%).
1H NMR (CDCl3, 400 MHz) δ 7.33ꢀ7.39 (m, 4H), 7.23ꢀ7.28 (m, 1H),
6.89 (d, J = 8.2 Hz, 1H), 6.30 (d, J = 2.8 Hz, 1H), 6.12 (dd, J = 8.2, 2.8 Hz,
1H), 3.76ꢀ3.88 (m, 2H), 3.12 (br s, 1H), 2.88ꢀ2.95 (m, 1H), 2.53ꢀ2.61
(m, 1H), 2.40ꢀ2.46 (m, 1H), 2.18ꢀ2.24 (m, 4H), 1.86ꢀ1.93 (m, 1H),
1.64ꢀ1.73 (m, 3H), 1.20 (s, 3H), 0.67 (s, 3H). ESI-MS m/z378 (M + H)+.
HRMS: calcd, 378.138 60 (M + H)+; found, 378.138 16.
1
(45.9 mg, 57%). H NMR (CDCl3, 400 MHz) δ 7.33ꢀ7.391 (m, 4H),
7.24ꢀ7.28 (m, 1H), 6.92 (d, J = 8.11 Hz, 2H), 6.23 (d, J = 8.11 Hz, 2H),
3.77ꢀ3.91 (m, 2H), 3.27 (br s, 1H), 2.91ꢀ2.98 (m, 1H), 2.56ꢀ2.63 (m,
1H), 2.44 (d, J = 14.1 Hz, 1H), 2.24 (d, J = 14.1 Hz, 1H), 1.88ꢀ1.95 (m,
1H), 1.66ꢀ1.73 (m, 3H), 1.20 (s, 3H), 0.67 (s, 3H). ESI-MS m/z394 (M +
H)+. HRMS: calcd, 394.198 84 (M + H)+; found, 394.199 59.
N0-[2-(2,2-Dimethyl-4-phenyltetrahydro-2H-pyran-4-yl)-
ethyl]-N,N-dimethylbenzene-1,3-diamine (33). 33 was prepared
according to general procedure B using the appropriate aromatic bromide
1
(28.9 mg, 39%). H NMR (CDCl3, 400 MHz) δ 7.33ꢀ7.36 (m, 4H),
7.20ꢀ7.25 (m, 1H), 6.95 (t, J = 8.0 Hz, 1H), 6.09 (ddd, J = 8.0, 2.3, 0.7 Hz,
1H), 5.77 (ddd, J = 8.0, 2.3, 0.7 Hz, 1H), 5.71 (t, J = 2.3 Hz, 1H), 3.77ꢀ3.87
(m, 2H), 3.22 (br s, 1H), 2.93ꢀ3.02 (m, 1H), 2.85 (s, 6H), 2.58ꢀ2.68 (m,
1H), 2.41ꢀ2.47 (m, 1H), 2.22 (d, J = 14.0, 2.3 Hz, 1H), 1.89ꢀ1.96 (m,
1H), 1.67ꢀ1.74 (m, 3H), 1.20 (s, 3H), 0.67 (s, 3H). ESI-MS m/z353 (M +
H)+. HRMS: calcd, 353.258 74 (M + H)+; found, 353.259 59.
4-Chloro-N-[2-(2,2-dimethyl-4-phenyltetrahydro-2H-pyr-
an-4-yl)ethyl]-3-methylaniline (41). 41 was prepared according to
general procedure B using the appropriate aromatic bromide (31.0 mg,
3-[2-(2,2-Dimethyl-4-phenyltetrahydropyran-4-yl)ethyl-
amino]phenol (34). 34 was prepared according to general procedure B
5040
dx.doi.org/10.1021/jm200249a |J. Med. Chem. 2011, 54, 5031–5047

Journal of Medicinal Chemistry
ARTICLE
41%). 1H NMR (CDCl3, 400 MHz) δ 7.34ꢀ7.39 (m, 3H), 7.24ꢀ7.28
(m, 2H), 6.98ꢀ7.01 (m, 1H), 6.12ꢀ6.13 (m, 1H), 6.07 (dd, J = 8.7, 2.7
Hz, 1H), 3.77ꢀ3.88 (m, 2H), 3.16 (br s, 1H), 2.90ꢀ2.96 (m, 1H),
2.55ꢀ2.62 (m, 1H), 2.39ꢀ2.51(m, 1H), 2.16ꢀ2.24 (m, 4H), 1.86ꢀ1.94
(m, 1H), 1.64ꢀ1.73 (m, 3H), 1.20 (s, 3H), 0.67 (s, 3H). ESI-MS m/z
358 (M + H)+. HRMS: calcd, 358.193 22 (M + H)+; found, 358.194 56.
3-Chloro-N-[2-(2,2-dimethyl-4-phenyltetrahydro-2H-pyr-
an-4-yl)ethyl]-4-methylaniline (42). 42 was prepared according to
general procedure B using the appropriate aromatic bromide (36.5 mg,
49%). 1H NMR (CDCl3, 400 MHz) δ 7.33ꢀ7.39 (m, 4H), 7.23ꢀ7.28
(m, 1H), 6.89 (d, J = 8.2 Hz, 1H), 6.30 (d, J = 2.8 Hz, 1H), 6.12 (dd, J =
8.2, 2.8 Hz, 1H), 3.76ꢀ3.88 (m, 2H), 3.12 (br s, 1H), 2.88ꢀ2.95 (m,
1H), 2.53ꢀ2.61 (m, 1H), 2.40ꢀ2.46 (m, 1H), 2.18ꢀ2.24 (m, 4H),
1.86ꢀ1.93 (m, 1H), 1.64ꢀ1.73 (m, 3H), 1.20 (s, 3H), 0.67 (s, 3H).
ESI-MS m/z 358 (M + H)+. HRMS: calcd, 358.193 22 (M + H)+; found,
358.194 44.
[2-(2,2-Dimethyl-4-phenyltetrahydro-2H-pyran-4-yl)ethyl]-
(2-fluorophenyl)amine (43). 43 was prepared according to general
procedure B using the appropriate aromatic bromide. (CDCl3, 400 MHz)
δ 7.39ꢀ7.32 (m, 4H), 7.27ꢀ7.22 (m, 1H), 6.91ꢀ6.85 (m, 2H), 6.56ꢀ
6.51 (m, 1H), 6.31 (dt, J = 1.7, 8.6 Hz, 1H), 3.89ꢀ3.77 (m, 2H), 3.55 (br s,
1H), 2.97 (m, 1H), 2.61 (m, 1H), 2.44 (dq, J = 2.2, 14.0 Hz, 1H), 2.23 (dd,
J = 2.2, 14.0 Hz, 1H), 1.97ꢀ1.90 (m, 1H), 1.77ꢀ1.68 (m, 3H), 1.20 (s,
3H), 0.67 (s, 3H). ESI-MS m/z 328 (M + H)+. HRMS: calcd, 328.207 12
(M + H)+; found, 328.208 22.
N-[2-(2,2-Dimethyl-4-phenyltetrahydro-2H-pyran-4-yl)-
ethyl]-3-fluoroaniline (44). 44 was prepared according to general
procedure B using the appropriate aromatic bromide (33.3 mg, 48%). 1H
NMR (CDCl3, 400 MHz) δ 7.33ꢀ7.39 (m, 4H), 7.23ꢀ7.28 (m, 1H), 6.98
(dt, J = 8.3, 6.9 Hz, 1H), 6.30 (dt, J = 8.3, 2.4 Hz, 1H), 6.06 (m, 1H), 5.96
(dt, J = 11.6, 2.4 Hz, 1H), 3.77ꢀ3.88 (m, 2H), 3.22 (br s, 1H), 2.89ꢀ2.99
(m, 1H), 2.55ꢀ2.62 (m, 1H), 2.41ꢀ246 (m, 1H), 2.23 (dd, J = 13.8, 2.7
Hz, 1H), 1.87ꢀ1.95 (m, 1H), 1.66ꢀ1.74 (m, 3H), 1.20 (s, 3H), 0.67 (s,
3H). ESI-MS m/z 328 (M + H)+. HRMS: calcd, 328.207 12 (M + H)+;
found, 328.207 37.
N-[2-(2,2-Dimethyl-4-phenyltetrahydro-2H-pyran-4-yl)-
ethyl]-4-fluoroaniline (45). 45 was prepared according to general
procedure B using the appropriate aromatic bromide (40.6 mg, 59%). 1H
NMR (CDCl3, 400 MHz) δ 7.33ꢀ7.38 (m, 4H), 7.23ꢀ7.27 (m, 1H),
6.76ꢀ6.80 (m, 2H), 6.20ꢀ6.26 (m, 2H), 3.76ꢀ3.87 (m, 2H), 3.07 (br s,
1H), 2.88ꢀ2.95 (m, 1H), 2.53ꢀ2.60 (m, 1H), 2.41ꢀ2.46 (m, 1H), 2.23
(dd, J= 14.0, 2.4 Hz, 1H), 1.87ꢀ1.94 (m, 1H), 1.65ꢀ1.73 (m, 3H), 1.20 (s,
3H), 0.67 (s, 3H). ESI-MS m/z 328 (M + H)+. HRMS: calcd, 328.207 12
(M + H)+; found, 328.207 94.
[2-(2,2-Dimethyl-4-phenyltetrahydropyran-4-yl)ethyl]-
(2-fluoro-4-methylphenyl)amine (46). 46 was prepared ac-
cording to general procedure B using the appropriate aromatic bromide. 1H
NMR (CDCl3, 400 MHz) δ 7.39ꢀ7.32 (m, 4H), 7.26ꢀ7.21 (m, 1H),
6.74ꢀ6.66 (m, 2H), 6.21 (dd, J = 8.4, 9.0 Hz, 1H), 3.88ꢀ3.77 (m, 2H),
3.39 (br s, 1H), 2.99ꢀ2.90 (m, 1H), 2.63ꢀ2.54 (m, 1H), 2.43 (dq, J = 2.5,
14.0 Hz, 1H), 2.22 (dq, J = 2.5, 14.0 Hz, 1H), 2.19 (s, 3H), 1.96ꢀ1.88 (m,
1H), 1.76ꢀ1.68 (m, 3H), 1.20 (s, 3H), 0.67 (s, 3H). ESI-MS m/z342 (M +
H)+. HRMS: calcd, 342.222 77 (M + H)+; found, 342.223 35.
[2-(2,2-Dimethyl-4-phenyltetrahydropyran-4-yl)ethyl]-
(3-fluoro-4-methoxyphenyl)amine (47). 47 was prepared
according to general procedure B using the appropriate aromatic
bromide. 1H NMR (CDCl3, 400 MHz) δ 7.39ꢀ7.32 (m, 4H),
7.29ꢀ7.23 (m, 1H), 6.78ꢀ6.70 (m, 1H), 6.09 (dd, J = 2.8, 13.8
Hz, 1H), 6.01 (ddd, J = 1.3, 2.8, 8.6 Hz, 1H), 3.88ꢀ3.79 (m, 2H), 3.78
(s, 3H), 3.02 (br s, 1H), 2.93ꢀ2.86 (m, 1H), 2.58ꢀ2.51 (m, 1H),
2.43 (dq, J = 2.4, 14.0 Hz, 1H), 2.23 (dq, J = 2.4, 14.0 Hz, 1H),
1.93ꢀ1.86 (m, 1H), 1.73ꢀ1.64 (m, 3H), 1.20 (s, 3H), 0.67 (s, 3H).
ESI-MS m/z 358 (M + H)+. HRMS: calcd, 358.217 68 (M + H)+;
found, 358.218 08.
(3,5-Difluoro-4-methoxyphenyl)-[2-(2,2-dimethyl-4-phenyl-
tetrahydropyran-4-yl)ethyl]amine (48). 48 was prepared according
to general procedure B using the appropriate aromatic bromide. 1H NMR
(CDCl3, 400 MHz) δ 7.40ꢀ7.32 (m, 4H), 7.29ꢀ7.24 (m, 1H), 5.82ꢀ5.75
(m, 2H), 3.88ꢀ3.76 (m, 5H), 3.17 (t, J = 6.3 Hz, 1H), 2.91ꢀ2.82 (m, 1H),
2.58ꢀ2.50 (m, 1H), 1.92ꢀ1.85 (m, 1H), 1.72ꢀ1.63 (m, 3H), 2.42 (dq, J =
2.8, 14.0 Hz, 1H), 2.23 (dd, J = 2.8, 14.0, 1H), 1.20 (s, 3H), 0.67 (s, 3H).
ESI-MS m/z 376 (M + H)+. HRMS: calcd, 376.208 26 (M + H)+; found,
376.208 46.
(4-Chloro-3-fluorophenyl)-[2-(2,2-dimethyl-4-phenyltetra-
hydropyran-4-yl)ethyl]amine (49). 49 was prepared according to
general procedure B using the appropriate aromatic bromide. 1H NMR
(CDCl3, 400 MHz) δ 7.40ꢀ7.32 (m, 4H), 7.28ꢀ7.24 (m, 1H), 7.02ꢀ
6.98 (m, 2H), 6.02ꢀ5.97 (m, 2H), 3.88ꢀ3.77 (m, 2H), 3.29 (t, J = 5.4 Hz,
1H), 2.96ꢀ2.87 (m, 1H), 2.62ꢀ2.54 (m, 1H), 2.42 (dq, J = 2.4, 14.0 Hz,
1H), 2.23 (dd, J = 2.4, 14.0 Hz, 1H), 1.93ꢀ1.86 (m, 1H), 1.72ꢀ1.64 (m,
3H), 1.20 (s, 3H), 0.67 (s, 3H). ESI-MS m/z 362 (M + H)+. HRMS:
calcd, 362.168 15 (M + H)+; found 362.169 50.
(3-Chloro-4-fluorophenyl)-[2-(2,2-dimethyl-4-phenyltetra-
hydropyran-4-yl)ethyl]amine (50). 50 was prepared according to
general procedure B using the appropriate aromatic bromide. 1H NMR
(CDCl3, 400 MHz) δ 7.40ꢀ7.32 (m, 4H), 7.29ꢀ7.24 (m, 1H), 6.84 (t,
J = 8.9 Hz, 1H), 6.25 (dd, J = 3.1, 6.3 Hz, 1H), 6.10 (m, 1H), 3.89ꢀ3.77
(m, 2H), 3.12 (t, J = 5.0 Hz, 1H), 2.95ꢀ2.86 (m, 1H), 2.61ꢀ2.52 (dq, J =
2.4, 14.0 Hz, 1H), 2.23 (dd, J = 2.4, 14.0 Hz, 1H), 1.94ꢀ1.87 (m, 1H),
1.73ꢀ1.64 (m, 3H), 1.20 (s, 3H), 0.60 (s, 3H). ESI-MS m/z 362 (M +
H)+. HRMS: calcd, 362.168 15 (M + H)+; found, 362.169 18.
(5-Chloro-2-fluorophenyl)-[2-(2,2-dimethyl-4-phenyltetra-
hydropyran-4-yl)ethyl]amine (51). 51 was prepared according to
general procedure B using the appropriate aromatic bromide. 1H NMR
(CDCl3, 400 MHz) δ 7.40ꢀ7.32 (m, 4H), 7.29ꢀ7.24 (m, 1H), 6.78 (dd,
J = 8.6, 11.3 Hz, 1H), 6.49ꢀ6.46 (m, 1H), 6.21 (dd, J = 2.5, 7.6 Hz, 1H),
3.89ꢀ3.77 (m, 2H), 3.62 (q, J = 5.0 Hz, 1H), 2.99ꢀ2.90 (m, 1H),
2.63ꢀ2.55 (m, 1H), 2.44 (dq, J = 2.4, 14.0 Hz, 1H), 2.23 (dd, J = 2.4, 14.0
Hz, 1H), 1.98ꢀ1.90 (m, 1H), 1.75ꢀ1.66 (m, 3H), 1.20 (s, 3H), 0.67 (s,
3H). ESI-MS m/z 362 (M + H)+. HRMS: calcd, 362.168 15 (M + H)+;
found, 362.168 66.
N-[2-(2,2-Dimethyl-4-phenyltetrahydro-2H-pyran-4-yl)-
ethyl]-3-(trifluoromethyl)aniline (52). 52 was prepared ac-
cording to general procedure A using the appropriate boronic acid
1
(12.0 mg, 10%). H NMR (CDCl3, 400 MHz) δ 7.33ꢀ7.42 (m, 4H),
7.24ꢀ7.30 (m, 1H), 7.14 (t, J = 7.89 Hz, 1H), 6.85 (d, J = 7.89 Hz, 1H),
6.85 (s, 1H), 6.41 (d, J = 7.89 Hz, 1H), 3.72ꢀ3.91 (m, 2H), 3.40 (br s,
1H), 2.87ꢀ2.96 (m, 1H), 2.53ꢀ2.60 (m, 1H), 2.45 (dd, J = 14.1, 2.2 Hz,
1H), 2.23 (dd, J = 14.1, 2.2 Hz, 1H), 1.88ꢀ1.96 (m, 1H), 1.65ꢀ1.77 (m,
3H), 1.20 (s, 3H), 0.67 (s, 3H). ESI-MS m/z 378 (M + H)+. HRMS:
calcd, 378.203 93 (M + H)+; found, 378.204 93.
General Procedure C for the Synthesis of THP Ethylani-
lines: N-[2-(2,2-Dimethyl-4-phenyltetrahydro-2H-pyran-4-
yl)ethyl]-3-nitroaniline (53). 2-(2,2-Dimethyl-4-phenyltetrahydro-
pyran-4-yl)ethylamine VI (50.0 mg, 0.21 mmol), 1-fluoro-3-nitroben-
zene (35.5 mg, 0.252 mmol), and potassium carbonate (138 mg, 0.21
mmol) were combined in dimethylformamide (0.1 mL), and the mixture
was heated in a microwave at 150 °C for 5 min. The reaction mixture was
purified by MPLC (silica, hexane/ethyl acetate) to provide the title
compound (17.4 mg, 23%). 1H NMR (CDCl3, 400 MHz) δ 7.35ꢀ7.45
(m, 5H), 7.26ꢀ7.30 (m, 1H), 7.16 (t, J = 8.1 Hz, 1H), 7.08 (t, J = 2.3 Hz,
1H), 6.51 (ddd, J = 8.1, 2.3, 0.8 Hz, 1H), 3.79ꢀ3.90 (m, 2H), 3.52 (t, J =
5.7 Hz, 1H), 2.98ꢀ3.07 (m, 1H), 2.64ꢀ2.72 (m, 1H), 2.43ꢀ2.48 (m,
1H), 2.25 (d, J = 13.9, 2.4 Hz, 1H), 1.92ꢀ1.99 (m, 1H), 1.67ꢀ1.75 (m,
3H), 1.21 (s, 3H), 0.68 (s, 3H). ESI-MS m/z 355 (M + H)+. HRMS:
calcd, 355.201 62 (M + H)+; found, 355.201 57.
N-[2-(2,2-Dimethyl-4-phenyltetrahydro-2H-pyran-4-yl)-
ethyl]-4-nitroaniline (54). 54 was prepared according to general
5041
dx.doi.org/10.1021/jm200249a |J. Med. Chem. 2011, 54, 5031–5047

Journal of Medicinal Chemistry
ARTICLE
procedure C using the appropriate aromatic fluoride (18.3 mg, 25%).
1H NMR (CDCl3, 400 MHz) δ 7.95ꢀ7.99 (m, 2H), 7.34ꢀ7.42 (m,
4H), 7.26ꢀ7.31 (m, 1H), 6.13ꢀ6.17 (m, 2H), 4.02 (t, J = 5.2 Hz, 1H),
3.78ꢀ3.89 (m, 2H), 3.03ꢀ3.11 (m, 1H), 2.71ꢀ2.79 (m, 1H),
2.41ꢀ2.47 (m, 1H), 2.25 (dd, J = 14.0, 2.4 Hz, 1H), 1.90ꢀ1.97 (m,
1H), 1.66ꢀ1.77 (m, 3H), 1.21 (s, 3H), 0.68 (s, 3H). ESI-MS m/z 355
(M + H)+. HRMS: calcd, 355.201 62 (M + H)+; found, 355.201 34.
4-{[2-(2,2-Dimethyl-4-phenyltetrahydro-2H-pyran-4-yl)-
ethyl]amino}benzenesulfonamide (55). 55 was prepared ac-
cording to general procedure A using the appropriate boronic acid (12.8
mg, 16%). 1H NMR (CDCl3, 400 MHz) δ 7.38ꢀ7.50 (m, 6H), 7.26 (t,
J = 7.3 Hz, 1H), 6.28 (d, J = 7.3 Hz, 2H), 3.73ꢀ3.87 (m, 2H), 2.96 (dt, J =
11.9, 5.6 Hz, 1H), 2.50ꢀ2.57 (m, 2H), 2.33 (d, J = 14.5Hz, 1H), 2.33 (dd,
J = 11.8, 4.5 Hz, 1H), 1.64ꢀ1.70 (m, 3H), 1.17 (s, 3H), 0.65 (s, 3H). ESI-
MS m/z 389 (M + H)+. HRMS: calcd, 389.189 34 (M + H)+; found,
389.190 01.
3-{[2-(2,2-Dimethyl-4-phenyltetrahydro-2H-pyran-4-yl)-
ethyl]amino}-N,N-dimethylbenzenesulfonamide (56). 56
was prepared according to general procedure A using the appropriate
boronic acid. The product was purified by HPLC and isolated as the
trifluoroacetic acid salt (9.8 mg, 8.8%). 1H NMR (CDCl3, 400 MHz) δ
7.43ꢀ7.46 (m, 3H), 7.39 (t, J = 7.8 Hz, 1H), 7.24 (t, J = 7.4 Hz, 1H), 7.19
(t, J = 7.8 Hz, 1H), 6.86 (ddd, J = 7.4, 2.3, 0.8 Hz, 1H), 6.66 (t, J = 2.3 Hz,
1H), 6.53 (ddd, J = 7.4, 2.3, 0.8 Hz, 1H), 3.85 (dt, J = 12.1, 1.9 Hz, 1H),
3.73ꢀ3.78 (m, 1H), 2.90ꢀ2.97 (m, 1H), 2.47ꢀ2.60 (m, 9H), 1.89ꢀ1.98
(m, 1H), 1.64ꢀ1.72 (m, 3H), 1.18 (s, 3H), 0.66 (s, 3H). ESI-MS m/z 417
(M + H)+. HRMS: calcd, 417.220 64 (M + H)+; found, 417.219 79.
N-(3-{[2-(2,2-Dimethyl-4-phenyltetrahydro-2H-pyran-4-yl)-
ethyl]amino}phenyl)acetamide (57). 57 was prepared according to
general procedure A using appropriate boronic acid. The product was
purified by HPLC and isolated as the trifluoroacetic acid salt (19.5 mg,
19%). 1H NMR (CDCl3, 400 MHz) δ 7.60 (s, 1H), 7.35ꢀ7.40 (m, 4H),
7.22ꢀ7.30 (m, 2H), 7.15ꢀ7.18 (m, 1H), 6.71 (dd, J = 7.5, 1.4 Hz, 1H),
3.71ꢀ3.85 (m, 2H), 3.15 (dt, J = 11.9, 4.8 Hz, 1H), 2.59 (dt, J = 11.9, 4.8
Hz, 1H), 2.47 (dd, J = 14.0, 2.4 Hz, 1H), 2.33 (dd, J = 14.0, 2.4 Hz, 1H),
2.12 (s, 3H), 1.97 (dt, J = 12.3, 5.0 Hz, 1H), 1.77 (dt, J = 12.3, 5.0 Hz, 1H),
1.59ꢀ1.70 (m, 2H), 1.16(s, 3H), 0.63(s, 3H). ESI-MSm/z367 (M + H)+.
HRMS: calcd, 367.238 00 (M + H)+; found, 367.236 96.
Ethyl 4-{[2-(2,2-Dimethyl-4-phenyltetrahydro-2H-pyran-
4-yl)ethyl]amino}benzoate (58). 58 was prepared according to
general procedure C using the appropriate aromatic fluoride (40.7 mg,
51%). 1H NMR (CDCl3, 400 MHz) δ 7.74ꢀ7.78 (m, 2H), 7.33ꢀ7.41
(m, 4H), 7.25ꢀ7.29 (m, 1H), 6.20ꢀ6.24 (m, 2H), 4.31 (quint, J = 6.9
Hz, 2H), 3.78ꢀ3.88 (m, 2H), 3.67 (br s, 1H), 3.34ꢀ3.42 (m, 2H),
2.98ꢀ3.06 (m, 1H), 2.64ꢀ2.72 (m, 1H), 2.41ꢀ2.47 (m, 1H), 2.24 (dd,
J = 14.4, 2.7 Hz, 1H), 1.87ꢀ1.96 (m, 1H), 1.59ꢀ1.74 (m, 2H), 1.34 (t,
J = 6.9 Hz, 2H), 1.20 (s, 3H), 0.67 (s, 3H). ESI-MS m/z 382 (M + H)+.
HRMS: calcd, 382.237 67 (M + H)+; found, 382.238 22.
3-[2-(2,2-Dimethyl-4-phenyltetrahydropyran-4-yl)ethyl-
amino]benzoic Acid (59). 59 was prepared according to general
procedure B using the appropriate aromatic bromide. 1H NMR
(CDCl3, 400 MHz) δ 7.40ꢀ7.34 (m, 5H), 7.29ꢀ7.23 (m, 1H), 7.16
(t, J = 8.4 Hz, 1H), 7.06 (s, 1H), 6.50 (d, J = 8.4 Hz, 1H), 3.90ꢀ3.79 (m,
2H), 3.06ꢀ2.97 (m, 1H), 2.70ꢀ2.63 (m, 1H), 2.47 (d, J = 14.0 Hz,
1H), 2.42 (d, J = 14.0 Hz, 1H), 1.99ꢀ1.90 (m, 1H), 1.72ꢀ1.68 (m,
3H), 1.20 (s, 3H), 0.67 (s, 3H). ESI-MS m/z 354 (M + H)+. HRMS:
calcd, 354.206 37 (M + H)+; found, 354.207 28.
1H), 1.93ꢀ1.84 (m, 1H), 1.70ꢀ1.63 (m, 3H), 1.19 (s, 3H), 0.69 (s, 3H).
ESI-MS m/z 354 (M + H)+. HRMS: calcd, 354.242 76 (M + H)+; found,
354.241 64.
3-Methoxy-N-{2-[4-(4-methoxyphenyl)-2,2-dimethyltetra-
hydro-2H-pyran-4-yl]ethyl}aniline (61). 61 was prepared accord-
ing to general procedure B using the appropriate intermediate amine VI
and aromatic bromide (24.0 mg, 31%). 1H NMR (CDCl3, 400 MHz) δ
7.22ꢀ7.26 (m, 3H), 6.99 (t, J = 8.0 Hz, 1H), 6.89 (d, J = 8.0 Hz, 1H), 6.20
(ddd, J = 8.1, 2.2, 0.8 Hz, 1H), 5.95 (ddd, J = 8.1, 2.2, 0.8Hz, 1H), 5.90 (t, J
= 2.2 Hz, 1H), 3.74ꢀ3.85 (m, 5H), 3.72 (s, 3H), 3.25 (br s, 1H),
2.89ꢀ2.96 (m, 1H), 2.57ꢀ2.64 (m, 1H), 2.35ꢀ241(m, 1H), 2.17 (dd, J=
13.9, 2.3 Hz, 1H), 1.84ꢀ1.91 (m, 1H), 1.64ꢀ1.72 (m, 3H), 1.19 (s, 3H),
0.69 (s, 3H). ESI-MS m/z 370 (M + H)+. HRMS: calcd, 370.237 67 (M +
H)+; found, 370.236 24.
{2-[4-(4-Chlorophenyl)-2,2-dimethyltetrahydropyran-4-yl]-
ethyl}-(3-methoxyphenyl)amine (62). 62 was prepared according to
general procedure B using the appropriate intermediate amine VI and
aromatic bromide. 1H NMR(CDCl3, 400 MHz) δ7.33 (d, J = 8.7 Hz, 2H),
7.27 (d, J = 8.7 Hz, 2H), 7.00 (t, J = 8.2 Hz, 1H), 6.22 (ddd, J = 0.82, 2.4, 8.2
Hz, 1H), 5.89 (t, J = 2.4 Hz, 1H), 3.80ꢀ3.77 (m, 2H), 3.72 (s, 3H), 3.27 (s,
1H), 2.96ꢀ2.89 (m, 1H), 2.61ꢀ2.53 (m, 1H), 2.38 (dq, J = 2.1, 14.0 Hz,
1H), 2.17 (dd, J = 2.1, 14.0 Hz, 1H), 1.91ꢀ1.82 (m, 1H), 1.75ꢀ1.66 (m,
3H), 1.20 (s, 3H), 0.68 (s, 3H). ESI-MS m/z 374 (M + H)+. HRMS: calcd,
374.188 13 (M + H)+; found, 374.186 49.
{2-[4-(3-Chlorophenyl)-2,2-dimethyltetrahydropyran-4-yl]-
ethyl}-(3-methoxyphenyl)amine (63). 63 was prepared according to
general procedure B using the appropriate intermediate amine VI and
aromatic bromide. 1H NMR (CDCl3, 400 MHz) δ 7.35ꢀ7.22 (m, 5H),
7.00 (t, J = 6.8 Hz, 1H), 6.22 (d, J = 8.0 Hz, 1H), 5.98 (d, J = 8.2 Hz, 1H),
5.92 (m, 1H), 3.80 (m, 2H), 3.72 (s, 3H), 3.31 (br s, 1H), 2.97ꢀ2.91 (m,
1H), 2.62ꢀ2.55 (m, 1H), 2.44ꢀ2.35 (m, 1H), 2.15 (dd, J = 2.4, 14.0 Hz,
1H), 1.93ꢀ1.85 (m, 1H), 1.76ꢀ1.69 (m, 3H), 1.20 (s, 3H), 0.70 (s, 3H).
ESI-MS m/z 374 (M + H)+. HRMS: calcd, 374.188 13; found, 374.186 71.
{2-[4-(4-Fluorophenyl)-2,2-dimethyltetrahydropyran-4-yl]-
ethyl}-(3-methoxyphenyl)amine (64). 64 was prepared according to
general procedure B using the appropriate intermediate amine VI and
aromatic bromide. 1HNMR(CDCl3, 400 MHz) δ7.31ꢀ7.28 (m, 2H), 7.05
(t, J = 8.4 Hz, 1H), 7.00 (t, J = 8.4 Hz, 1H), 6.21 (ddd, J = 0.8, 2.4, 8.4 Hz,
1H), 5.97 (ddd, J = 0.8, 2.0, 8.0 Hz, 1H), 5.90 (t, J = 2.4 Hz, 1H), 3.81ꢀ3.77
(m, 2H), 3.72 (s, 3H), 3.28 (br s, 1H), 2.96ꢀ2.89 (m, 1H), 2.62ꢀ2.55 (m,
1H), 2.38 (dq, J = 2.4, 14.0 Hz, 1H), 2.15 (dd, J = 2.4, 14.0 Hz, 1H),
1.92ꢀ1.85 (m, 1H), 1.75ꢀ1.67 (m, 3H), 1.20 (s, 3H), 0.68 (s, 3H). ESI-MS
m/z358 (M + H)+. HRMS: calcd, 358.217 68 (M + H)+; found, 358.218 99.
{2-[4-(3-Fluoro-phenyl)-2,2-dimethyltetrahydropyran-4-yl]-
ethyl}-(3-methoxyphenyl)amine (65). 65 was prepared according to
general procedure B using the appropriate intermediate amine VI and
1
aromatic bromide. H NMR (CDCl3, 400 MHz) δ 7.36ꢀ7.30 (m, 1H),
7.13ꢀ7.11 (m, 1H), 7.06ꢀ6.92 (m, 3H), 6.21 (ddd, J= 0.8, 2.4, 8.4 Hz, 1H),
5.98 (ddd, J = 0.8, 2.4, 8.4 Hz, 1H), 5.91 (t, J = 2.4 Hz, 1H), 3.83ꢀ3.77 (m,
2H), 3.72 (s, 3H), 3.31 (br s, 1H), 2.98ꢀ2.91 (m, 1H), 2.62ꢀ2.54 (m, 1H),
2.37 (dq, J = 2.4, 14.0 Hz, 1H), 2.15 (dd, J = 2.4, 14.0 Hz, 1H), 1.93ꢀ1.86
(m, 1H), 1.76ꢀ1.68 (m, 3H), 1.20 (s, 3H), 0.69 (s, 3H). ESI-MS m/z 358
(M + H)+. HRMS: calcd, 358.217 68 (M + H)+; found, 358.217 57.
[2-(2,2-Dimethyl-4-thiophen-2-yltetrahydropyran-4-yl)-
ethyl]-(3-methoxyphenyl)amine (66). 66 was prepared according
to general procedure B using the appropriate intermediate amine VI and
aromatic bromide. 1H NMR (CDCl3, 400 MHz) δ 7.23 (dd, J = 0.4, 5.6
Hz, 1H), 7.01 (t, J = 8.0 Hz, 1H), 6.97 (m, 1H), 6.84 (dd, J = 1.2, 3.6 Hz,
1H), 6.22 (ddd, J = 0.8, 2.4, 8.4 Hz, 1H), 6.03 (ddd, J = 0.8, 2.0, 8.0 Hz,
1H), 5.97 (t, J = 2.4 Hz, 1H), 3.92 (dt, J = 2.0, 12.0 Hz, 1H), 3.79ꢀ3.74
(m, 1H), 3.73 (s, 3H), 3.32 (br s, 1H), 3.05ꢀ2.98 (m, 1H), 2.79ꢀ2.72
(m, 1H), 2.27 (dq, J = 2.4, 14.0 Hz, 1H), 2.11 (dd, J = 2.4, 14.0 Hz, 1H),
1.94ꢀ1.87 (m, 1H), 1.80ꢀ1.73 (m, 3H), 1.21 (s, 3H), 0.86 (s, 3H).
ESI-MSm/z 346 (M +H)+. HRMS: calcd, 346.183 53;found, 346.184 09.
[2-(2,2-Dimethyl-4-p-tolyltetrahydropyran-4-yl)ethyl]-(3-
methoxyphenyl)amine (60). 60 was prepared according to general
procedure A using the appropriate intermediate amine VI and boronic
acid. 1H NMR (CDCl3, 400 MHz) δ 7.23ꢀ7.10 (m, 4H), 6.99 (t, J = 8.2
Hz, 1H), 6.20 (dd, 2.4, 8.2 Hz, 1H), 5.96 (dd, 2.4, 8.2 Hz, 1H), 5.89 (t,
J = 2.4 Hz, 1H), 3.84ꢀ3.68 (m, 5H), 3.26 (br s, 1H), 2.97ꢀ2.90 (m,
1H), 2.64ꢀ2.57 (m, 1H), 2.44ꢀ2.31 (m, 4H), 2.20 (dd, J = 2.4, 14.0 Hz,
5042
dx.doi.org/10.1021/jm200249a |J. Med. Chem. 2011, 54, 5031–5047

Journal of Medicinal Chemistry
ARTICLE
[2-(2,2-Dimethyl-4-thiophen-2-yltetrahydropyran-4-yl)-
ethyl]phenylamine (67). 67 was prepared according to general
procedure B using the appropriate intermediate amine VI and aromatic
bromide. 1H NMR (CDCl3, 400 MHz) δ 7.27ꢀ7.24 (m, 1H), 7.11 (dt,
J = 1.2, 7.2 Hz, 2H), 6.98ꢀ6.96 (m, 1H), 6.85 (dd, J = 1.2, 3.2 Hz, 1H),
6.65 (tt, J = 1.2, 7.6 Hz, 1H), 6.41 (dd, J = 0.8, 8.8 Hz, 2H), 3.93 (dt, J = 2,
12.0 Hz, 1H), 3.77ꢀ3.75 (m, 1H), 3.30 (br s, 1H), 3.07ꢀ3.00 (m, 1H),
2.81ꢀ2.74 (m, 1H), 2.28 (dd, J = 2.0, 14.0 Hz, 1H), 2.11 (dd, J = 2.0, 14.0
Hz, 1H), 1.95ꢀ1.88 (m, 1H), 1.81ꢀ1.73 (m, 3H), 1.21 (s, 3H), 0.86 (s,
3H). ESI-MS m/z 316 (M + H)+. HRMS: calcd, 316.172 96 (M + H)+;
found, 316.171 58.
(3-Chlorophenyl)-[2-(2,2-dimethyl-4-thiophen-2-yltetra-
hydropyran-4-yl)ethyl]amine (68). 68 was prepared according to
general procedure B using the appropriate intermediate amine VI and
aromatic bromide. 1H NMR (CDCl3, 400 MHz) δ 7.25 (dd, J = 1.2, 5.2
Hz, 1H), 7.01ꢀ6.97 (m, 2H), 6.85 (dd, J = 1.2, 3.2 Hz, 1H), 6.60 (ddd, J =
0.8, 2.0, 7.6 Hz, 1H), 6.34 (t, 2.4 Hz, 1H), 6.25 (ddd, J= 0.8, 2.4, 8.4 Hz, 1H),
3.92 (dt, 1.6, 12.0 Hz, 1H), 3.79ꢀ3.75 (m, 1H), 3.37 (br s, 1H), 3.04ꢀ2.97
(m, 1H), 2.80ꢀ2.72 (m, 1H), 2.26 (dq, 2.4, 14.0 Hz, 1H), 2.11 (dd, 2.4, 14.0
Hz, 1H), 1.93ꢀ1.86 (m, 1H), 1.80ꢀ1.71 (m, 3H), 1.21 (s, 3H), 0.86 (s,
3H). ESI-MS m/z 350 (M + H)+. HRMS: calcd, 350.133 99 (M + H)+;
found, 350.133 78.
General Procedure D for the Synthesis of THP Aceta-
mides: 2-(2,2-Dimethyl-4-phenyltetrahydropyran-4-yl)-N-
phenylacetamide (7a). A mixture of nitrile 5 (458 mg, 2.0 mmol)
in concentrated HCl/acetic acid (2.0 mL/6.0 mL) was stirred at reflux for
48 h. The reaction mixture was cooled to room temperature, extracted with
ethyl acetate, and the combined organic extracts were concentrated under
vacuum. The carboxylic acid 6 thus obtained was dissolved in CH2Cl2
(10.0 mL). Diisopropylethylamine (0.70 mL, 4.0 mmol) was added, and the
resulting solution was cooled to 0 °C. To this solution was added isobutyl
chloroformate (0.40 mL, 3.0 mmol), and the reaction mixture was stirred at
0 °C for 20 min. Aniline (0.30 mL, 3.0 mmol) was then added, and the
reaction mixture was warmed to room temperature and stirred for 16 h. The
solvent was evaporated, and the residue was purified by silica gel MPLC. 1H
NMR (CDCl3, 400 MHz) δ 7.41ꢀ4.40 (m, 4H), 7.33ꢀ7.28 (m, 1H),
7.21ꢀ7.16 (m, 2H), 7.02ꢀ6.96 (m, 3H), 5.93 (s, 1H), 3.80 (dd, 2.8, 8.4 Hz,
2H), 2.61 (d, J = 12.8 Hz, 1H), 2.48 (dq, J = 2.4, 14.0 Hz, 1H), 2.43 (d, J =
12.8 Hz, 1H), 2.35 (dd, J = 2.4, 14.0 Hz, 1H), 1.94 (dt, J = 7.2, 14.4 Hz, 1H),
1.85 (d, J = 14.0 Hz, 1H), 1.26 (s, 3H), 0.71 (s, 3H). ESI-MS m/z 324 (M +
H)+. HRMS: calcd, 324.195 81 (M + H)+; found, 324.196 40.
2-(2,2-Dimethyl-4-phenyltetrahydropyran-4-yl)-N-(3-
methoxyphenyl)acetamide (7b). 7b was prepared according to
general procedure D using nitrile 5 and m-anisidine. 1H NMR (CDCl3,
400 MHz) δ 7.41 (d, J = 4.4 Hz, 4H), 7.35ꢀ7.30 (m, 1H), 7.06 (t, J = 8.0
Hz, 1H), 6.76 (t, J = 2.4 Hz, 1H), 6.56 (ddd, J = 0.8, 2.4, 8.0 Hz, 1H), 6.39
(ddd, J = 0.8, 2.4, 8.0 Hz, 1H), 5.88 (br s, 1H), 3.82ꢀ3.79 (m, 2H), 3.73
(s, 3H), 2.61 (d, J = 12.8 Hz, 1H), 2.50ꢀ2.45 (m, 1H), 2.42 (d, 12.4 Hz,
1H), 2.34 (dd, 2.4, 14.0Hz, 1H), 1.98ꢀ1.90 (m, 1H), 1.84 (d, J = 14.0 Hz,
1H), 1.24 (s, 3H), 0.71 (s, 3H). ESI-MS m/z 354 (M + H)+. HRMS:
calcd, 354.206 37 (M + H)+; found, 354.207 38.
Thurkauf et al.,23 phenylmagnesium bromide (2.5 mL, 3.0 M solution
in THF, 7.5 mmol) was added to a solution of ketone 8 (640 mg, 5.0 mmol)
in diethyl ether (20.0 mL) at 0 °C. The solution was allowed to warm to
room temperature and stirred for 12 h. The reaction mixture was quenched
by the addition of 1.0 N HCl and extracted with ethyl acetate (3ꢁ). The
combined organic extracts were washed with brine, dried (Na2SO4), filtered,
and concentrated. A portion of the resulting alcohol 9 (286 mg, 1.39 mmol)
was dissolved in THF (3.0 mL). NaN3 (270 mg, 4.17 mmol) was added, and
the resulting mixture was cooled to 0 °C. To this mixture was added
trifluoroacetic acid (788 mg, 6.90 mmol) dropwise over 5 min. The reaction
mixture was then allowed to warm to room temperature and stirred
overnight. The reaction mixture was partitioned between water and ether.
The layers were separated, and the organic layer was washed with water
(1ꢁ) and brine (1ꢁ). The organic layer was dried (Na2SO4), filtered, and
concentrated to give tertiary azide 10. The azide thus obtained (337 mg, 1.46
mmol) was dissolved in diethyl ether (20.0 mL), and the resulting solution
was cooled to 0 °C. Lithium aluminum hydride (2.19 mL, 2.0 M solution,
4.38 mmol) was then added, and the reaction mixture was stirred for 3 h at
0 °C. The reaction mixture was quenched by the sequential addition of water
(0.20 mL), 15% NaOH (0.20 mL), and water (0.60 mL); filtered through
Celite; and concentrated to give the tertiary amine 11. A portion of the
resulting amine (24.0 mg, 0.117 mmol) was dissolved in THF (0.5 mL).
3-Methoxyphenyl isocyanate (17.4 mg, 0.129 mmol) was added, and the
reaction mixture was stirred at room temperature overnight. The solvent was
removed, and the crude material was purified by silica gel MPLC. 1H NMR
(CD, 400 MHz) δ 7.45 (dd, J = 1.2, 8.5 Hz, 2H), 7.50ꢀ7.30 (m, 2H),
7.21ꢀ7.17 (m, 1H), 7.09 (t, J = 8.1 Hz, 1H), 7.02 (t, J = 2.3 Hz, 1H), 6.73
(ddd, 0.8, 2.0, 7.8 Hz, 1H), 6.50 (ddd, J = 0.8, 2.0, 8.2 Hz, 1H), 6.46 (s, 1H),
4.06 (dt, J = 2.2, 11.9 Hz, 1H), 3.75 (ddd, J = 2.8, 4.8, 12.5 Hz, 1H), 3.72 (s,
3H), 2.55 (dt, J = 2.2, 14.0 Hz, 1H), 2.16 (dt, J = 2.2, 14.0 Hz, 1H), 2.04
(ddd, J= 4.6, 11.7, 14.3 Hz, 1H), 1.96 (d, J= 14.3 Hz, 1H), 1.44 (s, 3H), 1.20
(s, 3H). ESI-MS m/z 355 (M + H)+. HRMS: calcd, 355.201 62 (M + H)+;
found, 355.200 77.
1-(3-Chlorophenyl)-3-(2,2-dimethyl-4-phenyltetrahydro-
pyran-4-yl)urea (12b). 12b was prepared according to general
1
procedure E using 3-chlorophenyl isocyanate. H NMR (CD3OD, 400
MHz) δ7.46ꢀ7.43 (m, 3H), 7.32 (t, J= 8.2 Hz, 2H), 7.20 (tt, J= 1.2, 7.4 Hz,
1H), 7.15 (d, J = 7.9 Hz, 1H), 7.10 (ddd, J = 1.1, 1.9, 8.2 Hz, 1H), 6.91 (ddd,
1.12, 1.92, 7.94 Hz, 1H), 6.49 (s, 1H), 4.10ꢀ4.01 (m, 1H), 3.77ꢀ3.72 (m,
1H), 2.55 (d, 13.9 Hz, 1H), 2.19ꢀ2.13 (m, 1H), 2.08ꢀ2.01 (m,1H), 1.96
(d, J = 14.2 Hz, 1H), 1.43 (s, 3H), 1.20 (s, 3H). ESI-MS m/z 359 (M + H)+.
HRMS: calcd, 359.152 08 (M + H)+; found, 359.151 68.
1-(2,2-Dimethyl-4-phenyltetrahydropyran-4-yl)-3-phenyl-
thiourea (13a). 13a was prepared according to general procedure E
using phenyl isothiocyanate. 1H NMR (CD3OD, 400 MHz) δ 7.50ꢀ7.43
(m, 2H), 7.41ꢀ7.33 (m, 2H), 7.31ꢀ7.20 (m, 4H), 7.18ꢀ7.13 (m, 1H),
4.06ꢀ3.96 (m, 1H), 3.76 (dq, J = 2.8, 12.4 Hz, 1H), 2.39ꢀ2.26 (m, 1H),
2.08ꢀ2.00 (m, 2H), 1.38 (s, 3H), 1.21 (s, 3H). ESI-MSm/z 359 (M + H)+.
HRMS: calcd, 341.167 16 (M + H)+; found, 341.167 16.
1-(2,2-Dimethyl-4-phenyltetrahydropyran-4-yl)-3-(3-
methoxyphenyl)thiourea (13b). 13b was prepared according
to general procedure E using 3-methoxyphenyl isothiocyanate. 1H NMR
(CD3OD, 400 MHz) δ 7.45 (d, J = 6.8 Hz, 2H), 7.41ꢀ7.33 (m, 2H),
7.29ꢀ7.16 (m, 2H), 6.72 (dd, J = 2.4, 8.0 Hz, 1H), 4.00 (t, J = 12.0 Hz,
1H), 3.78ꢀ3.72 (m, 4H), 2.41ꢀ2.29 (m, 1H), 2.08ꢀ2.01 (m, 2H), 1.37
(s, 3H), 1.20 (s, 3H). ESI-MS m/z 371 (M + H)+. HRMS: 371.178 78
(M + H)+; found, 371.178 23.
1-(3-Chlorophenyl)-3-(2,2-dimethyl-4-phenyltetrahydro-
pyran-4-yl)thiourea (13c). 13c was prepared according to general
procedure E using 3-chlorophenyl isothiocyanate. 1H NMR (CD3OD, 400
MHz) δ 7.52ꢀ7.42 (m, 2H), 7.39ꢀ7.32 (m, 2H), 7.23 (t, J = 8.0 Hz, 2H),
7.11ꢀ7.08 (m, 1H), 4.09ꢀ4.03 (m, 1H), 3.79ꢀ3.74 (m, 1H), 2.27 (d, J =
13.2 Hz, 1H), 2.08ꢀ2.03 (m, 2H), 1.43 (s, 3H), 1.22 (s, 3H). ESI-MS m/z
375 (M + H)+. HRMS: calcd, 375.129 24 (M + H)+; found, 375.128 74.
N-(3-Chlorophenyl)-2-(2,2-dimethyl-4-phenyltetrahydro-
pyran-4-yl)acetamide (7c). 7c was prepared according to general
procedure D using nitrile 5 and 3-chloroaniline. 1H NMR (CDCl3, 400
MHz) δ 7.40ꢀ7.42 (m, 4H), 7.30ꢀ7.35 (m, 1H), 7.13 (t, J = 2.0 Hz, 1H),
7.09 (t, J = 8.1 Hz, 1H), 6.98 (ddd, J = 1.1, 2.0, 8.1 Hz, 1H), 6.75 (ddd, 1.1,
2.0, 8.1 Hz, 1H), 5.89 (s, 1H), 3.81 (dd, J = 2.8, 8.1 Hz, 2H), 2.61 (d, J =
12.9 Hz, 1H), 2.49 (q, J = 2.4 Hz, 1H), 2.43 (d, J = 12.9 Hz, 1H), 2.35 (dd,
2.4, 14.2 Hz, 1H), 1.93 (dt, J = 8.0, 16.1Hz, 1H), 1.84 (d, J = 14.2 Hz, 1H),
1.24 (s, 3H), 0.71 (s, 3H). ESI-MS m/z 358 (M + H)+. HRMS: calcd,
358.156 83 (M + H)+; found, 358.156 98.
General Procedure E for the Synthesis of THP Urea
Analogues: 1-(2,2-Dimethyl-4-phenyltetrahydropyran-4-yl)-
3-(3-methoxphenyl)urea (12a). According to the procedure of
5043
dx.doi.org/10.1021/jm200249a |J. Med. Chem. 2011, 54, 5031–5047

Journal of Medicinal Chemistry
ARTICLE
1-(2,2-Dimethyl-4-phenyltetrahydropyran-4-yl)-3-(3-fluoro-
phenyl)thiourea (13d). 13d was prepared according to general proce-
dure E using 3-fluorophenyl isothiocyanate. 1H NMR (CD3OD, 400 MHz)
δ 7.49ꢀ7.42 (m, 2H), 7.40ꢀ7.31 (m, 2H), 7.28ꢀ7.20 (m, 2H), 6.98 (br s,
1H), 6.83 (t, J = 8.6 Hz, 1H), 4.05 (t, J = 11.7 Hz, 1H), 3.76 (d, J = 11.7 Hz,
1H), 2.31ꢀ2.23 (m, 1H), 2.10ꢀ1.99 (m, 3H), 1.42 (s, 3H), 1.22 (s, 3H).
ESI-MS m/z 359 (M + H)+. HRMS: calcd, 359.158 79 (M + H)+; found,
359.159 26.
N-[2-(2,2-Dimethyl-4-phenyltetrahydro-2H-pyran-4-yl)-
ethyl]-N-methylaniline (14a). Aniline 16 (26.7 mg, 0.086 mmol)
was combined with methyl iodide (24.5 mg, 0.172 mmol) and excess
potassium carbonate in DMF (1.0 mL), and the resulting mixture was stirred
at room temperature overnight. The reaction mixture was purified by MPLC
(silica, hexane/ethyl acetate) to afford the title compound (19.5 mg, 70%).
1H NMR (CDCl3, 400 MHz) δ 7.33ꢀ7.41 (m, 4H), 7.23ꢀ7.29 (m, 1H),
7.08ꢀ7.13 (m, 2H), 6.61 (tt, J = 7.3, 1.1 Hz, 1H), 6.31ꢀ6.35 (m, 2H),
3.76ꢀ3.90 (m, 2H), 3.08ꢀ3.18 (m, 1H), 2.73 (s,3H), 2.69ꢀ2.79 (m, 1H),
2.38ꢀ2.44 (m, 1H), 2.17 (dd, J = 13.8, 2.5 Hz, 1H), 1.61ꢀ1.85 (m, 4H),
1.20 (s, 3H), 0.76 (s, 3H). ESI-MS m/z 324 (M + H)+. HRMS: calcd,
324.232 19 (M + H)+; found, 324.231 35.
N-[2-(2,2-Dimethyl-4-phenyltetrahydro-2H-pyran-4-yl)-
ethyl]-N,3-dimethylaniline (14b). 14b was prepared according
to the procedure for 14a using 18. The crude material was purified by silica
gel MPLC (9.0 mg, 34%). 1H NMR (CDCl3, 400 MHz) δ 7.33ꢀ7.40 (m,
4H), 7.22ꢀ7.28 (m, 1H), 7.00 (t, J = 7.75 Hz, 1H), 6.50 (d, J = 7.75 Hz,
1H), 6.16 (dd, J= 8.2, 2.6 Hz, 1H), 6.08ꢀ6.10 (m, 1H), 3.76ꢀ3.91(m, 2H),
3.05ꢀ3.16 (m, 1H), 2.74ꢀ2.80 (m, 1H), 2.72 (s, 3H), 2.37ꢀ2.44 (m, 1H),
2.13ꢀ2.20 (m, 1H), 2.20 (s, 3H), 1.54ꢀ1.85 (m, 4H), 1.20 (s, 3H), 0.67 (s,
3H). ESI-MS m/z 338 (M + H)+. HRMS: calcd, 338.247 84 (M + H)+;
found, 338.247 35.
N-[2-(2,2-Dimethyl-4-phenyltetrahydro-2H-pyran-4-yl)-
ethyl]-N-phenylacetamide (15). To a solution of aniline 16
(13.3 mg, 0.043 mmol) and diisopropylethylamine (11.1 mg, 0.086 mmol)
in CH2Cl2 (0.5 mL) was added acetyl chloride (4.02 mg, 0.052 mmol)
dropwise. After being stirred at room temperature for 1 h, the reaction
mixture was diluted with CH2Cl2, washed with 1.0 N NaOH and water, and
then dried (Na2SO4), filtered, and concentrated. The residue was purified by
MPLC (silica, hexane/ethyl acetate) to give the title compound (7.7 mg,
51%). 1H NMR (CDCl3, 400 MHz) δ 7.14ꢀ7.36 (m, 8H), 6.94ꢀ6.96 (m,
2H), 3.71ꢀ3.83 (m, 2H), 3.62 (dt, J = 12.4, 4.7 Hz, 1H), 2.96ꢀ3.04 (m,
1H), 2.31ꢀ2.37 (m, 1H), 2.10 (dd, J = 13.8, 2.2 Hz, 1H), 1.84 (dt, J =
12.4,4.8 Hz, 1H), 1.74 (s, 3H), 1.59- 1.69 (m, 3H), 1.16 (s, 3H), 0.63 (s,
3H). ESI-MS m/z 352 (M + H)+. HRMS: calcd, 352.227 11 (M + H)+;
found, 352.226 33.
1H NMR (400 MHz, CDCl3) δ 7.41ꢀ7.37 (m, 2H), 7.31ꢀ7.26 (m, 3H),
7.02 (t, J = 8.0 Hz, 1H), 6.70 (d, J = 7.6 Hz, 1H), 6.4 (br s, 1H), 6.31 (d,
J = 8.0 Hz, 1H), 3.82ꢀ3.77 (m, 2H), 3.61ꢀ3.55 (m, 2H), 2.83ꢀ2.79 (m,
2H), 2.22ꢀ2.17 (m, 2H), 1.95ꢀ1.84 (m, 4H). ESI-MS m/z 316 (M + H)+.
HRMS: calcd, 316.146 27 (M + H)+; found, 316.146 13.
3-Fluoro-N-[2-(4-phenyltetrahydro-2H-pyran-4-yl)ethyl]-
aniline (72). 72 was prepared according to general procedure A using
the appropriate amine VI and 3-fluoro-1-bromobenzene (40 mg, 56%). 1H
NMR (400 MHz, CDCl3) δ 7.41ꢀ7.25 (m, 5H), 6.98 (dt, J = 6.8, 8.0 Hz,
1H), 6.30 (td, J = 8.4, 2.8 Hz, 1H), 6.07 (ddd, J = 8.0, 2.4, 0.8 Hz, 1H), 5.97
(dt, 11.6, 2.4 Hz, 1H), 3.79 (m, 2H), 3.59 (m, 2H), 3.36 (br s, 1H), 2.80 (m,
2H), 2.24ꢀ2.18 (m, 2H), 1.93ꢀ1.85 (m, 4H). ESI-MS m/z 300 (M + H)+.
HRMS: calcd, 300.175 82 (M + H)+; found, 300.176 16.
3-Chloro-4-methyl-N-[2-(4-phenyltetrahydro-2H-pyran-4-yl)-
ethyl]aniline (73). 73 was prepared according to general procedure A
using the appropriate amine VI and 4-bromo-2-chlorotoluene (32 mg, 41%).
1H NMR (400 MHz, CDCl3) δ 7.42ꢀ7.37 (m, 2H), 7.33ꢀ7.25 (m, 3H),
6.89 (d, J = 8.0 Hz, 1H), 6.31 (d, J = 2.4 Hz, 1H), 6.13 (dd, J = 8.4, 2.4 Hz,
1H), 3.82ꢀ3.77 (m, 2H), 3.61ꢀ3.55 (m, 2H), 3.16 (br s, 1H), 2.79 (t, J=7.2
Hz, 2H), 2.25ꢀ2.18 (m, 5H), 1.93ꢀ1.85 (m, 4H). ESI-MS m/z 330 (M +
H)+. HRMS: calcd, 330.161 92 (M + H)+; found, 330.160 94.
N-[2-(2,2,6,6-Tetramethyl-4-phenyltetrahydropyran-4-yl)-
ethyl]aniline (74). 74 was prepared according to general procedure B
using 2-(2,2,6,6-tetramethyl-4-phenyltetrahydropyran-4-yl)ethylamine VI
1
and the appropriate aromatic bromide. H NMR (CDCl3, 400 MHz) δ
7.43(d, J= 7.4 Hz, 2H), 7.34 (t, J= 7.4 Hz, 2H), 7.23 (tt, J= 1.1, 7.4 Hz, 1H),
7.08 (dd, 7.4, 8.6 Hz, 2H), 6.63 (tt, J= 1.1, 7.4 Hz, 1H), 6.31 (dd, 1.1, 8.6 Hz,
2H), 3.17(s, 1H), 2.82ꢀ2.78 (d, 2H), 2.45 (d, 14.1 Hz, 2H), 1.90ꢀ1.86 (m,
2H), 1.79 (d, J = 14.1 Hz, 2H), 1.30 (s, 3H), 1.07 (s, 3H). ESI-MS m/z 338
(M + H)+. HRMS: calcd, 338.247 84 (M + H)+; found, 338.248 06.
3-Methoxy-N-[2-(2,2,6,6-tetramethyl-4-phenyltetrahydro-
pyran-4-yl)ethyl]aniline (75). 75 was prepared according to general
procedure B using 2-(2,2,6,6-tetramethyl-4-phenyltetrahydropyran-4-yl)-
1
ethylamine VI and the appropriate aromatic bromide. H NMR (CDCl3,
400 MHz) δ 7.42 (dd, J = 1.2, 8.6 Hz, 2H), 7.33 (t, J = 8.2 Hz, 2H), 7.22 (tt,
J = 1.2, 7.2 Hz, 1H), 6.98 (t, J = 8.2, 1H), 6.20 (ddd, J = 0.8, 2.3, 8.3 Hz, 1H),
5.93 (ddd, 0.8, 2.3, 8.3 Hz, 1H), 5.88 (t, J=2.3Hz, 1H),3.71(s, 3H),3.20(br
s, 1H), 2.80ꢀ2.75 (m, 2H), 2.44 (d, J = 14.1 Hz, 2H), 1.89ꢀ1.85 (m, 2H),
1.78 (d, J = 14.1 Hz, 2H), 1.30 (s, 6H), 1.06 (s, 6H). ESI-MS m/z 368 (M +
H)+. HRMS: calcd, 368.258 41 (M + H)+; found, 368.259 83.
3-Chloro-N-(3-chlorophenyl)-N-[2-(2,2,6,6-tetramethyl-4-
phenyltetrahydropyran-4-yl)ethyl]aniline (76). 76 was pre-
pared according to general procedure B using 2-(2,2,6,6-tetramethyl-
4-phenyltetrahydropyran-4-yl)ethylamine VI and the appropriate aro-
1
3-Methyl-N-[2-(4-phenyltetrahydro-2H-pyran-4-yl)ethyl]-
aniline (69). 69 was prepared according to general procedure A using
the appropriate amine VI and 3-methylphenylboronic acid (22.0 mg,
34%). 1H NMR (400 MHz, CDCl3) δ 7.41ꢀ7.26 (m, 5H), 6.97 (t, J =
7.2 Hz, 1H), 6.46 (d, J = 7.2 Hz, 1H), 6.16 (s, 1H), 6.14 (s, 1H),
3.82ꢀ3.79 (m, 2H), 3.59 (t, J = 9.6 Hz, 2H), 2.82 (t, J = 6.8 Hz, 2H), 2.21
(br s, 5H), 1.91ꢀ1.87 (m, 4H). ESI-MS 296 (M + H)+. HRMS: calcd,
296.200 89 (M + H)+; found, 296.200 36.
3-Methoxy-N-[2-(4-phenyltetrahydro-2H-pyran-4-yl)ethyl]-
aniline (70). 70 was prepared according to general procedure B using the
appropriate amine VI and 3-bromoanisole (30 mg, 40%). 1H NMR (400
MHz, CDCl3) δ 7.40ꢀ7.22 (m, 5H), 6.99 (t, J = 8.0 Hz, 1H), 6.20 (dd, J =
8.4, 2.4 Hz, 1H), 5.96 (ddd, J = 8.4, 2.4, 0.8 Hz, 1H), 5.90 (t, J = 2.4 Hz,
1H), 3.82ꢀ3.73 (m, 2H), 3.71 (s, 3H), 3.61ꢀ3.55 (m, 2H), 3.27 (br s,
1H), 2.83ꢀ2.79 (m, 2H), 2.23ꢀ2.18 (m, 2H), 1.94ꢀ1.85 (m, 4H). ESI-
MS m/z 312 (M + H)+. HRMS: calcd, 312.195 81 (M + H)+; found,
312.195 91.
matic bromide. H NMR (CDCl3, 400 MHz) δ 7.45ꢀ7.37 (m, 4H),
7.31ꢀ7.27 (m, 1H), 7.06 (t, J = 8.4 Hz, 2H), 6.87 (ddd, J = 0.8, 2.0, 8.4
Hz, 2H), 6.64 (t, J = 2.0 Hz, 2H), 6.52 (ddd, J = 0.8, 2.0, 8.0 Hz, 2H),
7.32ꢀ7.28 (m, 2H), 2.40 (d, J = 14.4 Hz, 2H), 1.90ꢀ1.85 (m, 2H), 1.71
(d, 14.4 Hz, 2H), 1.26 (s, 6H), 1.05 (s, 6H). ESI-MS m/z 482 (M + H)+.
N-[2-(2,2,6,6-Tetramethyl-4-phenyltetrahydrothiopyran-
4-yl)ethyl]aniline (77). 77 was prepared according to general
procedure B using 2-(2,2,6,6-tetramethyl-4-phenyltetrahydrothiopyr-
an-4-yl)ethylamine VI and the appropriate aromatic bromide. 1H
NMR (CDCl3, 400 MHz) δ 7.46 (dd, J = 1.4, 8.8 Hz, 2H), 7.32 (dd,
J = 6.9, 7.1 Hz, 2H), 7.23 (tt, J = 1.2, 7.4 Hz, 1H), 7.07 (dd, J = 7.4, 8.6 Hz,
2H), 6.63 (tt, J = 0.8, 7.0 Hz, 1H), 6.28 (dt, J = 1.1, 7.8 Hz, 2H), 3.15 (br
s, 1H), 2.84ꢀ2.79 (m, 2H), 2.72 (d, J = 14.7 Hz, 2H), 1.83ꢀ1.78 (m,
4H), 1.37 (s, 6H), 1.22 (s, 6H). ESI-MS m/z 354 (M + H)+. HRMS:
calcd, 354.225 00 (M + H)+; found, 354.226 33.
3-Methoxy-N-[2-(2,2,6,6-tetramethyl-4-phenyltetrahydro-
thiopyran-4-yl)ethyl]aniline (78). 78 was prepared according to general
procedure B using 2-(2,2,6,6-tetramethyl-4-phenyltetrahydrothiopyran-4-yl)-
3-Chloro-N-[2-(4-phenyltetrahydro-2H-pyran-4-yl)ethyl]-
aniline (71). 71 was prepared according to general procedure A using the
appropriate amine VI and 3-chlorophenylboronic acid (34.0 mg, 15%).
1
ethylamine VI and the appropriate aromatic bromide. H NMR (CDCl3,
400 MHz) δ 7.45 (dd, J = 1.3, 8.6 Hz, 2H), 7.31 (t, J = 8.0 Hz, 2H), 7.22
5044
dx.doi.org/10.1021/jm200249a |J. Med. Chem. 2011, 54, 5031–5047

Journal of Medicinal Chemistry
ARTICLE
(tt,J= 1.2, 7.3 Hz, 1H), 6.97 (t, J= 7.7 Hz, 1H), 6.20 (ddd, J= 0.8, 2.4, 8.3 Hz,
1H), 5.92 (ddd, J= 0.8, 2.0, 8.0 Hz, 1H), 5.87 (t, J= 2.3 Hz, 1H), 3.71 (s, 3H),
3.16 (br s, 1H), 2.82ꢀ2.78 (m, 2H), 2.71 (d, J = 14.5 Hz, 2H), 1.83ꢀ1.78
(m, 4H), 1.36 (s, 6H), 1.22 (s, 6H). ESI-MS m/z 384 (M + H)+. HRMS:
calcd, 384.235 56 (M + H)+; found, 384.234 82.
2.22 (dd, J = 13.9, 2.4 Hz, 1H), 1.88ꢀ1.95 (m, 1H), 1.66ꢀ1.74 (m, 3H),
1.20 (s, 3H), 0.67 (s, 3H). ESI-MS m/z 340 (M + H)+.
Peak 2 retention time 15.4 min (82% ee). H NMR (CDCl3, 400
MHz) δ 7.33ꢀ7.39 (m, 4H), 7.21ꢀ7.26 (m, 1H), 6.98 (t, J = 8.1 Hz,
1H), 6.20 (ddd, J = 8.1, 2.2, 0.7 Hz, 1H), 5.95 (ddd, J = 8.1, 2.2, 0.7 Hz,
1H), 5.89 (t, J = 2.2 Hz, 1H), 3.76ꢀ3.88 (m, 2H), 3.71 (s, 3H), 3.23 (br
s, 1H), 2.91ꢀ2.98 (m, 1H), 2.56ꢀ2.63 (m, 1H), 2.40ꢀ247 (m, 1H),
2.22 (dd, J = 13.9, 2.4 Hz, 1H), 1.88ꢀ1.95 (m, 1H), 1.66ꢀ1.74 (m, 3H),
1.20 (s, 3H), 0.67 (s, 3H). ESI-MS m/z 340 (M + H)+.
1
3-Chloro-N-[2-(2,2,6,6-tetramethyl-4-phenyltetrahydro-
thiopyran-4-yl)ethyl]aniline (79). 79 was prepared according to
general procedure B using 2-(2,2,6,6-tetramethyl-4-phenyltetrahydrothio-
pyran-4-yl)ethylamine VI and the appropriate aromatic bromide. 1H NMR
(CDCl3, 400 MHz) δ 7.48ꢀ7.45 (m, 2H), 7.36ꢀ7.31 (m, 2H), 7.27ꢀ7.22
(m, 1H), 6.95 (tt, J = 1.5, 6.5 Hz, 1H), 6.59ꢀ6.56 (m, 1H), 6.23ꢀ6.20 (m,
1H), 6.14ꢀ6.10 (m, 1H), 3.22 (br s, 1H), 2.83ꢀ2.76 (m, 2H), 2.71 (d, J =
14.7 Hz, 2H), 1.82ꢀ1.77 (m, 4H), 1.37 (s, 6H), 1.23 (s, 6H). ESI-MS m/z
388 (M + H)+. HRMS: calcd, 388.186 02 (M + H)+; found, 388.184 96.
3-Methoxy-N-[2-(2,2,6,6-tetramethyl-4-phenyl-4-piperidyl)-
ethyl]aniline (80). 80 was prepared according to general procedure B
using 2-(2,2,6,6-tetramethyl-4-phenylpiperidin-4-yl)ethylamine VI and the
appropriate aromatic bromide. 1H NMR (CD3OD, 400 MHz) δ 7.58 (d,
J= 8.0 Hz, 2H), 7.40 (t, J= 7.6 Hz, 2H), 7.31 (t, J= 7.6 Hz, 1H), 7.09 (td, J=
7.6, 2.4 Hz, 1H), 6.50 (m, 1H), 6.27 (m, 2H), 3.72 (s, 3H), 2.99 (d, J = 15.2
Hz, 2H), 2.86ꢀ2.82 (m, 2H), 1.81ꢀ1.77 (m, 2H), 1.72 (d, J = 15.2 Hz,
2H), 1.48 (s, 6H), 1.18 (s, 6H). ESI-MS m/z 367 (M + H)+. HRMS: calcd,
367.274 39 (M + H)+; found, 367.273 92.
3-Chloro-N-[2-(2,2,6,6-tetramethyl-4-phenyl-4-piperidyl)-
ethyl]aniline (81). 81 was prepared according to general procedure B
using 2-(2,2,6,6-tetramethyl-4-phenylpiperidin-4-yl)ethylamine VI and
the appropriate aromatic bromide. 1H NMR (CD3OD, 400 MHz) δ 7.61
(dd, J = 1.6, 8.8 Hz, 2H), 7.42 (t, J = 7.2 Hz, 2H), 7.32 (t, J = 7.6 Hz, 1H),
6.92 (t, J = 7.6 Hz, 1H), 6.47 (ddd, J = 8.0, 2.0, 0.8 Hz, 1H), 6.21 (t, J = 2.0
Hz, 1H), 6.17 (ddd, J = 8.4, 2.4, 0.8 Hz, 1H), 3.02 (d, J = 15.2, 2H),
2.75ꢀ2.71 (m, 2H), 1.74ꢀ1.69 (m, 4H), 1.48 (s, 6H), 1.20 (s, 6H). ESI-
MS m/z 371 (M + H)+. HRMS: calcd, 371.224 85 (M + H)+; found,
371.224 57.
3-Fluoro-N-[2-(2,2,6,6-tetramethyl-4-phenyl-4-piperidyl)-
ethyl]aniline (82). 82 was prepared according to general procedure B
using 2-(2,2,6,6-tetramethyl-4-phenylpiperidin-4-yl)ethylamine VI and
the appropriate aromatic bromide. 1H NMR (CD3OD, 400 MHz) δ 7.60
(dd, J = 1.6, 8.4 Hz, 2H), 7.42 (t, J = 7.2 Hz, 2H), 7.32 (t, J = 7.6 Hz, 1H),
6.96ꢀ6.91(m, 1H), 6.23ꢀ6.18 (m, 1H), 6.07 (ddd, J=0.8,2.0,8.0Hz, 1H),
5.93 (dt, J = 12.4, 2.4 Hz, 1H), 3.01 (d, J = 15.6, 2H), 2.75ꢀ2.71 (m, 2H),
1.75ꢀ1.70 (m, 4H), 1.48 (s, 6H), 1.20 (s, 6H). ESI-MS m/z 355 (M + H)+.
HRMS: calcd, 355.254 95 (M + H)+; found, 355.254 50.
3-Chloro-4-methyl-N-[2-(2,2,6,6-tetramethyl-4-phenyl-4-
piperidyl)ethyl]aniline (83). 83 was prepared according to general
procedure B using 2-(2,2,6,6-tetramethyl-4-phenylpiperidin-4-yl)ethylamine
VI and the appropriate aromatic bromide. 1H NMR (CD3OD, 400 MHz) δ
7.60 (dd, J = 1.2, 8.4 Hz, 2H), 7.42 (t, J = 7.2 Hz, 2H), 7.34ꢀ7.30 (m, 1H),
6.91 (d, J = 8.4 Hz, 1H), 6.35 (d, J = 2.4 Hz, 1H), 6.23 (dd, J = 2.8, 8.0 Hz,
1H), 3.01 (d, J= 15.6 Hz, 2H), 2.75ꢀ2.71 (m, 2H), 2.17 (s, 3H), 1.75ꢀ1.69
(m, 4H), 1.48 (s, 6H), 1.19 (s, 6H). ESI-MS m/z 385 (M + H)+. HRMS:
calcd, 385.240 50 (M + H)+; found, 385.240 01.
Biological Assays. ICMT in Vitro Analysis. The human ICMT
bearing His6-tag at the C-terminus was produced in a baculovirus-based
expression system using pVL1393 (Invitrogen) as a transfer vector. To
prepare the total membrane fraction of recombinant Sf9 cells, which was
used as the source of catalytically active ICMT, the cell lysates were
precleared by low speed centrifugation at 600g followed by ultracentrifuga-
tion at 125000g. The membrane pellets were stored in 50 mm Tris-HCl, pH
7.5, 150 mM NaCl, 1 mM EDTA, 10% glycerol supplemented with a
standard set of protease inhibitors (Roche Applied Science). An expression
plasmid for the N-terminally His-tagged S-adenosylhomocysteine hydrolase
(SAHH) from S. solfataricus, which served as a product detection enzyme for
monitoring ICMT activity, was constructed based on commercially available
vector pET28a (EMD Biosciences). The recombinant product was pro-
duced in an E. coli system and purified to apparent homogeneity by
immobilized metal affinity chromatography on Talon resin (Clontech).
A fluorometric, coupled-enzyme assay for SAM-dependent methyl-
transferases described by Collazo et al.24 was adapted for high-throughput
screening, hit confirmation, and kinetic analyses. Our standard assay protocol
involved incubation of 50 μL aliquots of 100 mM HEPES, pH 7.5, 5 mM
MgCl2, 0.001% Triton X-100, 1% DMSO, 10 μM SAM, 10 μM AGGC,
50 μg/mL SAHH, 20 μM ThioGlo1 supplemented by test compounds in
96-well microtiter plates for 1 h at room temperature. Under these condi-
tions, S-adenosylhomocysteine, a product of the ICMT-catalyzed reaction,
was quantitatively hydrolyzed to adenosine and homocysteine by SAHH
followed by nonenzymatic formation of the fluorescent adduct between
homocysteine and ThioGlo 1 reagent. The fluorescence intensities were
measured using Gemini XL plate reader with the following settings: λex
=
400 nm, λem = 515 nm. A counterassay intended for the elimination of false
positives, such as fluorescence quenchers and SAHH inhibitors, was con-
structed by substitution of SAH for both substrates and ICMT-containing
membrane fraction in the above assay mixture. A previously reported direct
radiometric assay25 was employed for confirmation of selected ICMT
inhibitors. In this case, the composition of assay buffer was essentially the
same as described above for the fluorometric method with the exceptions
that 10 μM [3H]SAM (∼80 mCi/mmol) was used as a substrate and both
SAHH and ThioGlo 1 were omitted from the assay solution.
Western Analysis of Ras Proteins in Cells Treated with
ICMT Inhibitors. HCT-116 cells were plated at 500 000 cells in 10 cm
plates and 2 days later treated with 4 (EMD Biosciences), ICMT
inhibitors, or DMSO. On day 3, cells were washed twice with Hank’s
buffered solution and the cells processed for subcellular fractionation
with the ProteoExtract subcellular proteome extraction kit (EMD
Biosciences). The cytosolic and membrane fractions (plasma and
organelle) were independently concentrated in Microcon 10 concen-
trators. An amount of 25 μg of each fraction was analyzed on a 12.5%
SDSꢀPAGE gel and immunoblotted with anti-p21 ras (catalog no.
Op40, EMD), anti-VDAC (catalog no. 529532, EMD), and anti-β-actin
(catalog no. A5441, Sigma) antibodies.
Chiral Separation of 27. N-{2-[(4S*)-2,2-Dimethyl-4-phenyl-
tetrahydro-2H-pyran-4-yl]ethyl}-3-methoxyaniline and N-{2-
[(4R*)-2,2-Dimethyl-4-phenyltetrahydro-2H-pyran-4-yl]ethyl}-
3-methoxyaniline (27). [2-(2,2-Dimethyl-4-phenyltetrahydropyran-4-
yl)ethyl]-(3-methoxyphenyl)amine (27) was separated into enantiomers
by chiral HPLC (Chiralpak IA; isopropanol/acetonitrile/ethanol, 11:39:31;
0.4% isopropylamine). Exact assignment of enantiomers has not been
completed.
Cell Viability Assays. Cells were cultured in DMEM or RPMI
medium (Invitrogen) supplemented with 10% fetal bovine serum
(Invitrogen), 1 mM HEPES, 1 mM pyruvate, 1 mM glutamate, and
nonessential amino acids. Cells were maintained at 37 °C in a humidified
5% CO2 atmosphere. For viability assays, cells were seeded at 500 cells/
well in 96-well plates (PerkinElmer) and treated with various concen-
trations of compound in DMSO. After 7 days of compound exposure,
1
Peak 1 retention time 14.7 min (82% ee). H NMR (CDCl3, 400
MHz) δ 7.33ꢀ7.39 (m, 4H), 7.21ꢀ7.26 (m, 1H), 6.98 (t, J = 8.1 Hz,
1H), 6.20 (ddd, J = 8.1, 2.2, 0.7 Hz, 1H), 5.95 (ddd, J = 8.1, 2.2, 0.7 Hz,
1H), 5.89 (t, J = 2.2 Hz, 1H), 3.76ꢀ3.88 (m, 2H), 3.71 (s, 3H), 3.23 (br
s, 1H), 2.91ꢀ2.98 (m, 1H), 2.56ꢀ2.63 (m, 1H), 2.40ꢀ247 (m, 1H),
5045
dx.doi.org/10.1021/jm200249a |J. Med. Chem. 2011, 54, 5031–5047

Journal of Medicinal Chemistry
ARTICLE
luminescent ATP-lite (PerkinElmer) was used to moniter cell viability.
Doseꢀresponse curves were generated with Prism software by plotting
percentage of relative light units reduction in compound-treated cells
relative to DMSO-treated cells.
the Selective Farnesyl Protein Transferase Inhibitor R115777 in Vivo
and in Vitro. Cancer Res. 2001, 61, 131–137.
(9) Schafer-Hales, K.; Iaconelli, J.; Snyder, J. P.; Prussia, A.; Nettles,
J. H.; El-Naggar, A.; Khuri, F. R.; Giannakakou, P.; Marcus, A. I. Farnesyl
Transferase Inhibitors Impair Chromosomal Maintenance in Cell Lines
and Human Tumors by Compromising CENP-E and CENP-F Func-
tion. Mol. Cancer Ther. 2007, 4, 1317–1328.
’ ASSOCIATED CONTENT
(10) Bishop, W. R.; Kirschmeier, P.; Baum, C. Farnesyl Transferase
Inhibitors: Mechanism of Action, Translational Studies and Clinical
Evaluation. Cancer Biol. Ther. 2003, 4 (Suppl. 1), S96–S104.
(11) Karp, J. E.; Lancet, J. E. Development of Farnesyltransferase
Inhibitors for Clinical Cancer Therapy: Focus on Hematologic Malignancies.
Cancer Invest. 2007, 6, 484–494.
S
Supporting Information. ICMT Km and IC50 determina-
b
tion of 75 using fluorometric and radiometric assays; graphical data
for mechanism of ICMT inhibition by 18. This material is available
free of charge via the Internet at http://pubs.acs.org.
(12) Winter-Vann, A. M.; Baron, R. A.; Wong, W.; dela Cruz, J.;
York, J. D.; Gooden, D. M.; Bergo, M. O.; Young, S. G.; Toone, E. J.;
Casey, P. J. A Small-Molecule Inhibitor of Isoprenylcysteine Carboxyl
Methyltransferase with Antitumor Activity in Cancer Cells. Proc. Natl.
Acad. Sci. U.S.A. 2005, 102, 4336–4341.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: 801-214-7877. Fax: 801-214-7993. E-mail: weston.judd@
myrexis.com.
(13) Svensson, A. W.; Casey, P. J.; Young, S. G.; Bergo, M. O. Genetic
and Pharmacologic Analyses of the Role of Icmt in Ras Membrane
Association and Function. Methods Enzymol. 2006, 407, 144–159.
(14) Bergo, M. O.; Gavino, B. J.; Hong, C.; Beigneux, A. P.; McMahon,
M.; Casey, P. J.; Young, S. G. Inactivation of Icmt Inhibits Transformation
by Oncogenic K-Ras and B-Raf. J. Clin. Invest. 2004, 113, 539–550.
(15) Wang, M.; Tan, W.; Zhou, J.; Leow, J.; Go, M.; Lee, H. S.;
Casey, P. J. A Small Molecule Inhibitor of Isoprenylcysteine Carbox-
ymethyltransferase Induces Autophagic Cell Ceath in PC3 Prostate
Cancer Cells. J. Biol. Chem. 2008, 283, 18678–18684.
’ ACKNOWLEDGMENT
We thank the Myrexis, Inc. analytical chemistry group for
assistance with purification and characterization of described
compounds, and Drs. Kraig M. Yager and Robert Carlson for
their support of this work.
’ ABBREVIATIONS USED
(16) Go, M.-L.; Leow, J. L.; Gorla, S. K.; Sch€uller, A. P.; Wang, M.;
Casey, P. J. Amino Derivatives of Indole as Potent Inhibitors of
Isoprenylcysteine Carboxyl Methyltransferase. J. Med. Chem. 2010,
53, 6838–6850.
(17) Baron, R. A.; Casey, P. J. Analysis of the Kinetic Mechanism of
Recombinant Human Isoprenylcysteine Carboxylmethyltransferase
(Icmt). BMC Biochem. 2004, 5, 19–30.
THP, tetrahydropyran; ICMT, isoprenylcysteine carboxyl
methyltransferase; SAR, structureꢀactivity relationship; FTI, farne-
syltransferase inhibitor; RCE-1, Ras-converting enzyme; CENP-E/F,
centromere-associated protein E/F; GFP, green fluorescent protein;
mTOR, mammalian target of rapamycin; HTS, high-throughput
screening; BINAP, 2,20-bis(diphenylphosphino)-1,10-binaphthalene;
DIPEA, N,N-diisopropylethylamine; SAM, S-adenosylmethionine;
SAHH, S-adenosylhomocysteine hydrolase
(18) (a) Carrico, D.; Ohkanda, J.; Kendrick, H.; Yokoyama, K.;
Blaskovich, M. A.; Bucher, C. J.; Buckner, F. S.; Van Voorhis, W. C.;
Chakrabarti, D.; Croft, S. L.; Gelb, M. H.; Sebti, S. M.; Hamilton, A. D. In
Vitro and in Vivo Antimalarial Activity of Peptidomimetic Protein Farne-
syltransferase Inhibitors with Improved Membrane Permeability. Bioorg.
Med. Chem. 2004, 12, 6517–6526. (b) Ohkanda, J.; Buckner, J. K.;
Lockman, J. W.; Yokoyama, K.; Carrico, D.; Eastman, R.; de Luca-Fradley,
K.; Davies, W.; Croft., S. L.; Van Voorhis, W. C.; Gelb, M. H.; Sebti, S. M.;
Hamilton, A. D. Design and Synthesis of Peptidomimetic Protein Farnesyl-
transferase Inhibitors as Anti-Trypanosoma brucei Agents. J. Med. Chem.
2004, 47, 432–445.
(19) (a) Haroutyunian, N. S.; Gharibain, K. M.; Hakopian, L. H.;
Tossunian, H. H.; Vartanian, S. H.; Chaoushian, K. A. Synthesis and Certain
Transformations of 2,2-Dialkyl-4-phenyl(benzyl)tetrahydropydranyl-4-
acetic Acids. Arm. Khim. Zh. 1986, 29, 438–444. (b) Arutyunyan, N. S.;
Akopyan, L. A.; Apoyan, N. A.; Tumadzhyan, A. E.; Vartanyan, S. A.
Synthesis and Study of the Antiinflammatory Properties of Tetrahydropyr-
an-Substituted γ-Aminopropanols. Pharm. Chem. J. 1989, 23, 144–146. (c)
Arutyunyan, N. S.; Akopyan, R. G.; Akopyan, N. E. Synthesis and Antic-
onvulsant Activity of Substituted Tetrahydropyran Amides and Amines.
Pharm. Chem. J. 1991, 25, 349–352.
(20) (a) Liljebris, C.; Martinsson, J.; Tedenborg, L.; Williams, M.;
Barker, E.; Duffy, J. E. S.; Nygren, A.; James, S. Synthesis and Biological
Activity of a Novel Class of Pyridazine Analogues as Non-Competitive
Reversible Inhibitors of Protein Tyrosine Phosphatase 1B (PTP1B). Bioorg.
Med. Chem. 2002, 10, 3197–3212. (b) Magnus, P; Mansley, T. Synthesis of
the ABCD-Rings of the Insecticidal Indole Alkaloid Nodulisporic Acid.
Tetrahedron Lett. 1999, 40, 6909–6912. (c) Johnson, P. Y.; Berchtold, G. A.
Photochemical Reactions of γ-Keto Sulfides. J. Org. Chem. 1970,
35, 584–592.
’ REFERENCES
(1) Casey, P. J.; Seabra, M. C. Protein Prenyltransferases. J. Biol.
Chem. 1996, 271, 5289–5292.
(2) Boyartchuk, V. L.; Ashby, M. N.; Rine, J. Modulation of Ras and
a-Factor Function by Carboxyl-Terminal Proteolysis. Science 1997,
275, 1796–1800.
(3) Clarke, S.; Vogel, J. P.; Deschenes, R. J.; Stock, J. Posttransla-
tional Modification of the Ha-Ras Oncogene Protein: Evidence for a
Third Class of Protein Carboxyl Methyltransferases. Proc. Natl. Acad. Sci.
U.S.A. 1988, 85, 4643–4637.
(4) Tamonoi, F.; Gau, C. L.; Jiang, C.; Edamatsu, H.; Kato-Stankiewicz,
K. Protein Farnesylation in Mammalian Cells: Effects of Farnesyl-
transferase Inhibitors on Cancer Cells. Cell. Mol. Life Sci. 2001, 58,
1636–1649.
(5) Kim, E.; Ambroziak, P.; Otto, J. C.; Taylor, B.; Ashby, M.;
Shannon, K.; Casey, P. J.; Young, S. G. Disruption of the Mouse Rce1
Gene Results in Defective Ras Processing and Mislocalization of Ras
within Cells. J. Biol. Chem. 1999, 274, 8383–8390.
(6) Bergo, M. O.; Leung, G. K.; Ambroziak, P.; Otto, J. C.; Casey,
P. J.; Young, S. G. Targeted Inactivation of the Isoprenylcysteine
Carboxyl Methyltransferase Gene Causes Mislocalization of K-Ras in
Mammalian Cells. J. Biol. Chem. 2000, 275, 17605–17610.
(7) Rose, W. C.; Lee, F. Y.; Fairchild, C. R.; Lynch, M.; Monticello,
T.; Kramer, R. A.; Manne, V. Preclinical Antitumor Activity of BMS-
214662, a Highly Apoptotic and Novel Farnesyltransferase Inhibitor.
Cancer Res. 2001, 61, 7507–7517.
(8) End, D. W.; Smets, G.; Todd, A. V.; Applegate, T. L.; Fuery, C. J.;
Angibaud, P.; Venet, M.; Sanz, G.; Poignet, H.; Skrzat, S.; Devine, A.;
Wouters, W.; Bowden, C. Characterization of the Antitumor Effects of
(21) Wolfe, J. P.; Buchwald, S. L. Scope and Limitations of the
Pd/BINAP-Catalyzed Amination of Aryl Bromides. J. Org. Chem. 2000,
65, 1144–1157.
5046
dx.doi.org/10.1021/jm200249a |J. Med. Chem. 2011, 54, 5031–5047

Journal of Medicinal Chemistry
ARTICLE
(22) Chan, D. M. T.; Monaco, K. L.; Wang, R.-P.; Winters, M. P.
New N- and O-Arylations with Phenylboronic Acids and Cupric Acetate.
Tetrahedron Lett. 1998, 39, 2933–2936.
(23) Thurkauf, A.; de Costa, B.; Yamaguchi, S.-I.; Mattson, M. V.;
Jacobson, A. E.; Rice, K. C.; Rogawski, M. A. Synthesis and Antic-
onvulsant Activity of 1-Phenylcyclohexylamine Analogues. J. Med. Chem.
1990, 33, 1452–1458.
(24) Collazo, E.; Couture, J.-F.; Bulfer, S.; Trievel, R. C. A Coupled
Fluorescent Assay for Histone Methyltransferases. Anal. Biochem. 2005,
342, 86–92.
(25) Kramer, K.; Harrington, E. O.; Lu, Q.; Bellas, R.; Newton, J.;
Sheahan, K. L.; Rounds, S. Isoprenylcysteine Carboxylmethyltransferase
Activity Modulates Endothelial Cell Apoptosis. Mol. Biol. Cell 2003,
14, 848–857.
(26) Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Freeney, P. J.
Experimental and Computational Approaches To Estimate Solubility
and Permeability in Drug Discovery and Development Settings. Adv.
Drug Delivery Rev. 1997, 23, 3–25.
5047
dx.doi.org/10.1021/jm200249a |J. Med. Chem. 2011, 54, 5031–5047