Optimization of monocarboxylate transporter 1 blockers through analysis and modulation of atropisomer interconversion properties

Article abstract of DOI:10.1021/jm060995h

We have previously described a novel series of potent blockers of the monocarboxylate transporter, MCT1, which show potent immunomodulatory activity in an assay measuring inhibition of PMA/ionomycin-induced human PBMC proliferation. However, the preferred

Full text of DOI:10.1021/jm060995h

254
J. Med. Chem. 2007, 50, 254-263
Optimization of Monocarboxylate Transporter 1 Blockers through Analysis and Modulation of
Atropisomer Interconversion Properties
Simon D. Guile,†,* John R. Bantick,† Martin E. Cooper,†,§ David K. Donald,† Christine Eyssade,† Anthony H. Ingall,†
Richard J. Lewis,‡ Barrie P. Martin,† Rukhsana T. Mohammed,† Timothy J. Potter,‡ Rachel H. Reynolds,†
Stephen A. St-Gallay,‡ and Andrew D. Wright‡
Department of Medicinal Chemistry, AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, LE11 5RH, UK, and Department of
Physical & Metabolic Science, AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, LE11 5RH, UK
ReceiVed August 17, 2006
We have previously described a novel series of potent blockers of the monocarboxylate transporter, MCT1,
which show potent immunomodulatory activity in an assay measuring inhibition of PMA/ionomycin-induced
human PBMC proliferation. However, the preferred compounds had the undesirable property of existing as
a mixture of slowly interconverting rotational isomers. Here we show that variable temperature NMR is an
effective method of monitoring how alteration to the nature of the amide substituent can modulate the rate
of isomer exchange. This led to the design of compounds with increased rates of rotamer interconversion.
Moreover, some of these compounds also showed improved potency and provided a route to further
optimization.
Introduction
compounds require a heteroaryl group at the 6-position in
combination with an amide group at the 5-position.2 The
preferred 5-amide was found to be the (R)-hydroxypyrrolidine
amide. However, compounds containing this amide existed in
solution as a mixture of atropisomers6 (slowly equilibrating
conformational isomers), due to restricted rotations at the amide
(CO-N) and aryl-carbonyl (Ar-CO) bonds (Figure 1). For
quinoline 1 and the closely related fluoro analogue 2, we have
been able to isolate, assign conformations, and, in the case of
2, measure MCT1 potencies for each of the atropisomers.
Despite their good properties, progression of compounds such
as 1 as potential drugs would be complicated by the existence
of multiple conformational forms. These could present signifi-
cant challenges due to potential safety, analytical, and manu-
facturing concerns.7 There are numerous examples in the
literature of atropisomers encountered during drug discovery
programs.8-13 Reports describe how atropisomerism has been
simplified through symmetrization9 or eliminated by making
interconversion rapid.10 Alternatively, restricting rotation has
allowed isolation of a single conformer.11,12
An important component of the immune response is activation
of T cells following antigen challenge. However, undesirable
activation can lead to graft rejection following transplantation
and to autoimmune diseases such as rheumatoid arthritis.
We became interested in a report1 describing a series of
pyrrolopyrimidines as potential immunosuppressive agents.
From this lead, following an extensive structural investigation,
we identified a novel series of compounds exemplified by
quinoline amide 1.2 Furthermore, through compound-led target
identification3 using photoaffinity labeling and proteomic
characterization, we were able to show the previously unknown
molecular target of these compounds to be the monocarboxylate
transporter, MCT1. This was supported by a strong correlation
between binding at MCT1 and in Vitro immunomodulatory
activity in an assay measuring inhibition of PMA/ionomycin-
induced human PBMC proliferation.4
The monocarboxylate transporters are a family of proteins
which transport lactate and other small monocarboxylates.5 We
have shown that MCT1 expression is rapidly upregulated upon
T lymphocyte activation in order to meet the demand for lactate
efflux resulting from an increased glycolytic rate. Inhibition of
lactate efflux by potent blockade of lactate transport results in
the accumulation of lactate within the cell and feedback
inhibition of glycolysis. This suppression of cellular metabolism
results in the inability of T lymphocytes to sustain the rapid
rate of cell division occurring during the early immune response
to antigen recognition, without being cytotoxic. Blockade of
MCT1 is thus a novel mechanism of immunosuppression distinct
from current therapies.
To investigate the most suitable approach for our compounds,
we prepared a range of amides with different electronic and
steric properties. This led to an understanding of the factors
affecting rates of conformer interconversion allowing the design
of new amides which not only exhibited fast interconversion
but also had other advantages over the previously preferred
amides. We went on to make further modification resulting in
highly optimized drug-like compounds.
Chemistry
While the synthesis of quinoline 1 has been previously
described, the other target compounds 2-13 were prepared as
shown in Schemes 1-4 below.
The N-1 isopropyl compounds 3 and 4 were prepared as
shown in Scheme 1. The commercially available chloropyrim-
idinedione 14 was alkylated under forcing conditions with
isopropyl iodide to give a mixture of the O-alkylated and desired
N-alkylated products 15 and 16, respectively. The substituted
thiophene ring was incorporated in a three-step process to give
the ester 17 which was then saponified to the acid 18. Reaction
Quinoline amide 1 resulted from an optimization strategy
which focused on reducing logD of highly potent but lipophilic
leads. It was concluded that, in order to achieve a good balance
of properties, assisted by maintaining low lipophilicity, such
* Corresponding author: Tel: +44-1509-645308. Fax: +44-1509-
645512. e-mail: simon.guile@astrazeneca.com.
† Department of Medicinal Chemistry.
‡ Department of Physical & Metabolic Science.
§ Current address: 7TM Pharma A/S, Fremtidsvej 3, 2970 Horsholm,
Denmark.
10.1021/jm060995h CCC: $37.00 © 2007 American Chemical Society
Published on Web 12/21/2006

Atropisomer Modulation of MCT1 Blockers
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 255
interconversion which was confirmed by the ability to separate
the individual atropisomers by HPLC. These atropisomers could
be individually analyzed by LC-NMR because of their relatively
slow interconversion. The individual components were desig-
nated as 2a, 2b, 2c, and 2d in order of increasing HPLC
retention times (Figure 2).
LC-NMR14 analysis of 2 was performed and assignments of
the individual atropisomers were made using standard methods
(see Experimental Section). Assignments for 1 were made in a
similar way, largely on a mixture of the four atropisomers and
by reference to the assignments made for 2. Confirmation that
the elution order was the same for the atropisomers of 1 and 2
was made by NMR measurements on individual atropisomers
isolated by analytical scale HPLC. The resonances obtained in
this way for 1 and 2 are shown in Table 1. A comparison of
the 1D proton spectra of the isolated atropisomers of 2 is shown
in Figure 3.
Figure 1. Compounds 1 and 2 with arrows indicating bonds with
restricted rotation.
Examination of the shifts in Table 1 and Figure 3 reveals
interesting patterns in the data. For example, there are large
differences in chemical shift about the pyrrolidine ring between
equivalent protons in different atropisomers, and these differ-
ences can be as large as 1.4 ppm. There is also an interesting
symmetry between 7R, 7â and 10R, 10â. In rotamers a and b
the 10 protons are more deshielded than the 7 protons. This
can be interpreted as an indication of the orientation about the
amide bond caused by the expected anisotropic deshielding
effect on protons syn to the carbonyl.15 It is interesting to note
a small (2 Hz) difference in the geminal proton coupling
constants for the 7 protons dependent on the proposed orientation
relative to the amide carbonyl. Related conformational effects
of sp2 systems two bonds distant from a methylene group have
been described.16 A significant symmetry is also noted when
considering the faces of the pyrrolidine ring. Considering the
pairs of geminal protons 7, 9, and 10, without exception, the R
face is most deshielded in rotamers a and c. This suggests a
deshielding influence of the 4-carbonyl and implies that a and
c are related by rotation about both bonds. However, definitive
proof of the conformational identity of the atropisomers was
provided by a ROESY experiment on the equilibrium mixture.
This showed specific ROE cross-peaks from 11 to one of 7R,
7â, 10R, and 10â for each atropisomer, thus leading directly to
the conformation (Figure 4 and Table 2).
Figure 2. High-pressure liquid chromatogram (HPLC) of 2 showing
the resolution of the atropisomeric forms (2a-d).
of the dianion derived from 18 with 2-trifluoromethylbenzal-
dehyde, followed by deoxygenation, provided the acid 19.
Conversion to the acid chloride followed by reaction with
the appropriate amine gave amides 3 and 20. The latter
compound 20 was subsequently oxidized to the 1,3-thiazolidine
dioxide 4.
For compounds 5, 8, 11, 12, and 13 the intermediate esters
21c-g were prepared from the previously described 6-methyl-
5-ester 22,4 prepared analogously to 17. Radical bromination
provided key intermediate 23 which allowed introduction of the
appropriate heterocycle. To prepare azetidine 5, the ester 21c
was transformed directly to the amide followed by reaction with
methylamine to derive the 6-substituent. The other esters were
saponified to the acids 24d-g. For targets 6, 9, and 10 the
known acids 24a,b4 were prepared in a similar manner to 19.
Standard amide formation then gave the desired target com-
pounds 6 and 8-13 (Scheme 2 and Table 5 ).
For fluoroquinoline 2, the acid 254 was first converted to the
protected amide 26. Incorporation of the quinoline gave 27
which, following deoxygenation and deprotection, provided 2
(Scheme 3). The 6-O-linked compound 7 was prepared from
the 6-H compound 28.4 Chlorination provided the 6-chloro
compound 29 which was displaced with naphthol giving 30.
Ester hydrolysis to give acid 31 followed by amide formation
provided 7 (Scheme 4).
(b) Rate of Atropisomer Interconversion for 2a. 2a was
separated from the equilibrium mixture of 2 and re-equilibration
was followed at 37 °C by LC-NMR. The rate of conversion of
2a to 2d predominated, followed by a slower conversion of 2a/
2d to 2b/2c (Figure 5). Fitting first-order kinetics gave a rate
constant for conversion of 2a to 2d (amide bond rotation), and
an average value for conversion of 2a+2d to 2b+2c by either
rotation of the Ar-CO bond or a concerted mechanism. Half-
lives and activation energies at 37 °C were calculated as 60
min and 98 kJ/mol for rotation about the amide bond and 760
min and 105 kJ/mol for rotation about the Ar-CO bond (either
with or without a concerted rotation of the amide bond).
(c) Mechanism of Interconversion. In the literature,12b,17,18,19
there is much discussion as to whether atropisomer intercon-
version in related compounds proceeds via a concerted or
sequential mechanism. The concerted mechanism implies that
rotation about the carbonyl-aryl bond occurs most rapidly when
accompanied by a simultaneous rotation about the amide bond.
Such interdependence is referred to as geared or gated rotation
and is understood in terms of steric clashes on Ar-CO bond
rotation which are relieved by distortion (and concomitant
Results
(a) Structural Analysis of 1 and 2. For compounds contain-
ing the 5-(R)-hydroxypyrrolidine amide group (e.g., 1 and 2),
four atropisomers result from slow bond rotation since each bond
can assume two orientations (s-cis and s-trans for the amide;
axial-R and axial-S for the aryl-carbonyl). The four atropisomers
can be observed by high-pressure liquid chromatography
(HPLC) and NMR spectroscopy.
Rotation about the Ar-CO bond is restricted by a steric clash
between the core heterocycle and the amide moiety and
generates an axis of chirality in the molecule. Rotation about
the amide bond is restricted for electronic reasons and generates
configurational isomers.
Variable temperature NMR (VTNMR) on the fluoro quinoline
2 showed little line-broadening at 130 °C, suggesting very slow

256 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2
Guile et al.
Scheme 1a
a Reagents and conditions: (a) iPrI, K2CO3, DMPU, 81% (3:4 O:N-alkylated); (b) (i) NaSH, EtOH; (ii) ethyl 3-bromopyruvate, H2O; (iii) TiCl4, DCM,
16% (three steps); (c) NaOH, H2O, THF, MeOH, 95%; (d) (i) CF3C6H4CHO, LDA, THF, -78 °C; ii) TFA, triethylsilane, 55% (two steps); (e) (i) oxalyl
chloride, DMF (cat.), DCM; (ii) Me2NH, Na2CO3, H2O, 27% (two steps); (f) (i) oxalyl choride, DMF (cat.), DCM; (ii) thiazolidine, DCM; (g) mCPBA,
DCM, 23% (three steps).
Scheme 2a
a Reagents and conditions: (a) NBS, chloroform, 76%; (b) ‘imidazole’, NaH, DMF 51-60% or azaindole, NaH, ZnCl2, toluene, 49% or (i) zinc(II)
acetylacetonate, DCM; (ii) hydrazine, DCM, 92% (two steps); (c) NaOH, H2O, THF, MeOH, 76-92%; (d) (i) iPrMgCl, azetidine, THF, 28%; (ii) MeNH2,
H2O, MeOH, THF, 130 °C, 38%; (e) RR′NH, EDCI, HOBt, DCM or (i) oxalyl choride, DMF (cat.), DCM; (ii) RR′NH, DCM; 16-81%.
Scheme 3a
a Reagents and conditions: (a) (R)-3-hydroxypyrrolidine, EDCI, HOBt, DCM; (b) TBDMSCl, imidazole, DMF, 67% (two steps); (c) 6-fluoroquinoline-
4-carbaldehyde, LDA, THF, -78 °C, 83%; (d) Ac2O, TEA, DMAP, DCM; (e) H2, Pd/C, NaHCO3, H2O, MeOH, EtOAc; (f) CH3CN, HF(aq), 29% (three
steps).
rotation) of the amide bond. In an attempt to determine how
our compounds behaved, further kinetic measurements were
carried out.
We note that our results show that amide bond rotation has
the lowest energy, and the second process a somewhat higher
energy (by 7 kJ/mol). This contrasts to the results of Johnston17
and Albert12b who demonstrated that the concerted process was
the lowest energy, but is similar to the results of Clayden,18
though the difference in energy between the lowest energy
process (amide bond rotation) and concerted bond rotation was
found to be less.
(d) Potency Data for Separate Conformers. Following
separation of the four atropisomers of 2 (2a-d) by preparative
HPLC, the MCT1 potency of each conformer was measured
over a 24 h time course at room temperature (Table 3). At all
time points it can be seen that the most active conformer is 2c
followed by 2b with the other conformers, 2a and 2d, being
The individual atropisomers of 1 were separated by prepara-
tive HPLC and re-equilibration at 20 °C was followed by
analytical HPLC. Modelmaker v420 was used to analyze the data
(Figure 6). Although we were able to show similar rates of
conversion of 1a to 1d and 1b to 1c by rotation about the
amide bond, we were unable to determine whether rotation about
the Ar-CO bond followed a concerted or nonconcerted mech-
anism. Due to the large difference in rates between rotation
about the amide bond and the second process, adequate fits to
the data could be obtained by allowing either mechanism or
indeed both.

Atropisomer Modulation of MCT1 Blockers
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 257
Scheme 4a
a Reagents and conditions: (a) NCS, HCl, DCM, 55%; (b) 1-naphthol, Cs2CO3, NMP, 54%; (c) NaOH, H2O, THF, MeOH, 83%; (d) (R)-3-hydroxypyrrolidine,
EDCI, HOBt, DCM, 31%.
Table 1. 1H NMR Chemical Shifts of Pyrrolidine Resonances in Atropisomers of 1 and 2. The R Face Is Defined as That Bearing the OH Group
pr ot on
1a
1b
1c
1d
2a
2b
2c
2d
7 R
2.83 (dd,
2.96 (dt,
3.43 (dt,
3.48 (dt,
2.86 (dd,
2.92 (d,
3.43 (d,
3.41 (d,
J ) 11.2, 3.3 Hz) J ) 11.5, 1.6 Hz) J ) 13.1, 1.2 Hz) J ) 13.1, 1.6 Hz) J ) 11.5, 3.3 Hz) J ) 11.5 Hz) J ) 13.7 Hz) J ) 13.3 Hz)
7â
2.47 (dd,
3.37 (dd,
3.29 (dd,
3.72-3.76
4.53-4.55
1.75-1.80
2.54 (dd,
J ) 11.5,5.0 Hz)
3.91 (qn,
J ) 4.3 Hz)
1.80-1.85
ca. 3.30
ca. 3.30
3.69 (dd,
J ) 13.3, 4.5 Hz)
4.49-4.52
J ) 11.4, 5.4 Hz) J ) 11.7, 4.7 Hz) J ) 13.3, 4.9 Hz)
8â
3.86 (qn,
J ) 4.5 Hz)
1.78-1.85
4.40-4.42
4.24-4.26
4.38-4. 40
1.93-1. 98
4.27-4. 30
1.61-1. 65
9 R
1.95-2.01
1.61 (dddd,
J ) 12.9, 6.6,
3.4, 0.8 Hz)
1.25-1.32
1.72-1.77
9â
1.70-1.77
2.13-2.21
2.03 (dddd,
J ) 13.3, 9.2,
9.2, 4.7 Hz)
3.08-3.14
1.75-1.80
2.15-2. 20
1.30-1. 36
1.96-2.01
10 R
10 â
3.59 (dt,
J ) 12.1, 7.8 Hz)
3.25-3.32
3.63-3.67
3.78-3.81
3.10-3.18
3.58 (dt,
J ) 12.1, 8.2 Hz)
ca. 3.30
3.57-3. 62
3.74-3. 79
3.10-3. 18
2.40-2. 46
3.08-3.14
3.23-3.28
2.39 (ddd,
J ) 10.7, 8.2,
2.3 Hz)
3.24-3.27
Figure 4. Partial ROESY spectrum for mixture of atropsiomers of 1
showing key ROE cross-peaks (boxed) between protons 11 and protons
7 and 10.
Table 2. ROE Cross-peaks Observed from 11 to the Pyrrolidine Protons
and Deduced Conformation about the Amide and Ar-CO Bonds in
Atropisomers of 1
ROE
observed
conformation about
amide bond
conformation about
Ar-CO bond
Figure 3. Partial LC-NMR spectra for the four atropsiomers of 2
indicating the large changes in chemical shift observed.
rotamer
1a
1b
1c
1d
7â
7R
10â
10R
trans
trans
cis
axial-S
axial-R
axial-R
axial-S
less potent. Further interpretation is complicated by the fact that
interconversion about the amide bond at room temperature
occurs at a similar rate to the slow equilibration/slow on-rate
kinetics of binding which is apparent from the data shown.
(e) Affecting Speed of Rotation. In order to avoid potential
development issues arising from the atropisomeric nature of
compounds such as 1 and 2, alternative compounds were sought
with the goal of accelerating bond rotation. With a target
biological half-life of greater than 8 h we set as a criterion for
conformer interconversion a half-life of less than 15 min.
We required a screen to rapidly evaluate the compounds for
their atropisomeric properties, and variable temperature NMR
was used for this purpose. Compounds were dissolved in
cis
DMSO-d6 and NMR spectra obtained under automation at 25,
60, 90, and 130 °C. These spectra were examined, and
coalescence temperature and rotamer signal separation were
estimated. Using standard methods21 it was then possible to
estimate the half-life at 37 °C. Table 4 shows typical values
obtained for a range of temperatures and signal separations using
this methodology.
It is important to emphasize that the value of half-life obtained
is only an estimate, of sufficient accuracy for us to rank
compounds and assess structural changes. For example, no

258 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2
Guile et al.
Figure 5. Structural assignments for atropisomers 2a-d.
Figure 7. VTNMR of 5 showing different coalescence temperatures
for rotation about the amide and Ar-CO bonds.
6-position was also varied. It is assumed in this analysis that
all methylene-linked (hetero)aryl groups at the 6-position have
equal effect on rotation properties.
In order to validate the above approach, certain compounds
were examined by VTNMR in greater detail, and one example,
compound 5, is shown in Figure 7. For this molecule, the
methylene protons 10 are made diastereotopic by slow rotation
about the Ar-CO bond below 93 °C. However, the azetidine
protons (7 and 9) coalesce at 135 °C, indicating that for this
compound the barrier to rotation about the amide bond is higher
than that about the Ar-CO bond. Interestingly, for this
compound, the pair of azetidine protons on the same carbon
coalesce at virtually the same temperature as the methylene
protons (they have a similar separation). From this simple
observation we can deduce that the lower energy process is
rotation about the Ar-CO bond only, i.e. it is definitely not a
concerted rotation, which would coalesce protons on different
carbons.
Using this VTNMR method to assess the rate of atropisomer
interconversion, we examined a number of other 5-amides which
had been prepared previously. In addition, in an effort to explain
the effect of structural changes on interconversion rate, experi-
mentally determined changes were compared with changes
predicted by torsional driving calculations. While these molec-
ular mechanics forcefields provide good estimates of steric
hindrance, there is no explicit electronic consideration with all
the amide bonds being treated equally. Therefore, any discrep-
ancy between measured and calculated rates suggests a signifi-
cant electronic influence.
Figure 6. Fit of model to experimental data obtained for the room
temperature interconversion of rotamers from 1a start point.
Table 3. Potency Data for Separate Conformersa
compd
0.5 h
1 h
6.6
507
22
4.9
67
3 h
2.8
172
8.5
1.7
31
24 h
2
15
489
46
13
118
0.81
18
3.0
0.66
4.1
2a
2b
2c
2d
a SPA assay (nM).2
Table 4. Approximate Half-Life (in minutes) at 37 °C According to
Rotamer Signal Separation and the Temperature (temp) at Which
Coalescence Is Observed
temp, ˚C
2 Hz
5 Hz
20 Hz
50 Hz
∼150
110
75
∼110
∼30
∼5
∼1
2.5
0.8
0.1
5 × 10-2
3 × 10-3
<7 × 10-4
0.1
3 × 10-2
7 × 10-3
<60
<2 × 10-2
<8 × 10-3
<2 × 10-3
The dimethyl amide 3 showed slow rotation as predicted,
being no less sterically demanding than the pyrrolidine amide
2. Both the sulfone 4 and the azetidine 5 amides showed
considerably faster interconversion, and even the diol analogue
6 showed some increase in interconversion rate relative to the
monohydroxy derivative 2. For the azetidine example we
rationalized this as a reduced steric clash from the azetidine
ring in agreement with the modeling. For the sulfone and diol
examples, reduced conjugation into the amide seemed the most
account has been made of the temperature dependence of ∆Gq;
coalescence temperatures and signal separations at coalescence
are only estimates. We assume we can treat the two bond
rotations independently and that rotamer populations are equal.
In some cases it was not possible to distinguish two distinct
coalescence processes, and a single half-life for both is given.
While exploring numerous 5-amides, the heterocycle at the

Atropisomer Modulation of MCT1 Blockers
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 259
Table 5. Structures, Rotamer Interconversion Rates, and Properties of Compounds 1-13a
a nd ) not determined. b Interconversion half-lives at 37 °C determined using the approximate VTNMR method (designated by > or ∼ symbols) or using
more accurate NMR measurements or other kinetic methods. c May be concerted in some cases. d SPA or filter binding assay.2,4 The standard deviations for
these assays were typically (10% of mean. eThe standard deviations for these assays were typically (50% of mean. f Assumed to be amide bond rotation.
g Assumed to be Ar-CO bond rotation.
likely cause for increased interconversion rate. While confor-
mational effects resulting in reduced steric interactions could
be an alternative explanation, this is not supported by the
modeling. Although the latter three compounds had appreciably
increased rotation rates, none had suitable potency for progres-
sion (Table 5).
in stabilizing the steric rotation transition state through resonance
(vinylogous carbamate). Rapid rotation was indeed observed
but further compounds of this type were not pursued, for while
7 had excellent activity at MCT1, its high logD resulted in poor
in Vitro metabolic stability and high human plasma protein
binding (>99% bound). Attempts to combine the oxygen link
with a suitable heterocycle were unsuccessful.
An alternative approach to fast interconversion was to replace
the 6-methylene linking group with a heteroatom. In the ether
7, it was expected that the oxygen link would provide less steric
encumbrance to rotation and would also benefit electronically
Returning to alternative amides, we speculated that an
alkoxyamide would have the desired properties for rapid
interconversion. The first example of this type to be prepared

260 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2
Guile et al.
was the ‘Weinreb amide’ 8.22 The structure of this alkoxyamide
8 was determined by single-crystal X-ray diffraction (see
Supporting Information), and this was used as a basis for the
modeling work described here. This structural modification had
a profound positive effect on rotation rate, reducing the barriers
to rotation about both the amide and Ar-CO bonds. Although
one explanantion for this effect could be reduced conjugation
in the amide bond resulting in more rapid rotation about both
restricted bonds, we rationalized this effect as being due to
reduced steric demand resulting from switching a methylene
unit to oxygen. The latter hypothesis was supported by modeling
data. Importantly, this rapidly interconverting compound 8 had
potency and other properties comparable with the atropisomeric
lead 1.
Combining features of the alkoxyamide motif with the
previously optimized (R)-hydroxypyrrolidine gave the (S)-
hydroxyisoxazolidine 9 which, as well as a suitable, fast rotation
rate, also showed excellent potency. Despite the higher logD
of (S)-hydroxyisoxazolidine 9 relative to (R)-hydroxypyrrolidine
1, both metabolic stability and CYP inhibition were at least as
good.
(f) Optimization of Hydroxyisoxazolidine-Containing Com-
pounds. Having found a nonatropisomeric amide with good
properties, further optimization was investigated. Initially, the
(S)-hydroxyisoxazolidine amide was combined with a range of
previously explored 6-arylmethyl groups and gave similar SAR.2
For example, 6-trifluoromethylbenzyl compound 10 was more
potent than the quinoline analogue 9 but also showed higher
CYP2C9 inhibition and lower metabolic stability. The azaindole
11 and methylaminobenzimidazole 12 containing derivatives
were similar in properties to quinoline 9, having slightly
improved metabolic stability.
Continuing with the strategy of lowering lipophilicity and
encouraged by the properties of monocyclic heterocycle 8,
further monoheterocyclic 6-substituents were explored. This
work resulted in the discovery of the dimethylpyrazole 13. This
compound is better than the starting compound 1 in each of the
properties indicated in Table 5. Besides having no atropisomer
issue, compound 13 exhibits excellent potency and metabolic
stability as well as low CYP inhibition. In addition, 13 is more
soluble than 1 (2.9 mg/mL vs 0.5 mg/mL, respectively).
The flow probe had a total volume of 110 µL and active volume
of 60 µL. Data were collected on the peaks of interest using the
stopped-flow method and the WET method23 to suppress the
resonances from residual methanol and protonated water. Suppres-
sion was achieved using “seduce” pulses of approximately 20 ms
duration. Separation of the atropisomers of 2 was carried out using
a Kromasil column (3.2 × 250 mm, 5 µm packing) and using an
isocratic method (60% CD3OD, 40% D2O, 0.025% ammonium
acetate), 0.5 mL/min, 20 °C. Peak detection was by UV at
280 nm.
Proton spectra were acquired on each separated atropisomer
(Figure 3), and COSY and ROESY spectra on selected isomers.
Assignment of the pyrrolidine protons to the R or â face was made
on the basis of ROE cross-peaks and coupling constants. ROESY
and eCOSY spectra were also acquired on an equilibrium mixture
of the atropisomers. These spectra enabled additional coupling
constants around the pyrrolidine ring to be measured, and the
assignment of the atropisomers to conformations about the amide
and Ar-CO bonds to be made.
Kinetics Measurements of 2. Atropisomer 2a was collected in
the NMR flow cell and equilibrated at 37 °C over 60 h. Integrating
NMR resonances in the aromatic region allowed the rate of
appearance of 2d to be followed (rotation about the amide bond).
The rate of appearance of 2b+2c (from 2a+2d) was taken as
representing rotation about the Ar-CO bond. As peaks for 2a and
2c were coincident, the amount of 2c present was estimated as being
the same as the amount of 2b (which could be integrated). The
rate of appearance of 2d and rates of appearance of 2b+2c were
fitted to an expression for reversible reaction kinetics, with the rate
of approach to equilibrium (k1+k-1) as the rate constant. This
simplified and approximate approach gave a reasonable fit (R2 )
74%, t1/2 ) 60 min ( 10 min) for rotation about the amide bond,
and a good fit (R2 ) 95%, t1/2 ) 12.7 h ( 0.6 h) for rotation about
the Ar-CO bond.
Kinetic Measurements and Atropisomer Identification of 1.
The atropisomers of 1 were isolated by HPLC on an Agilent 1100
series purification system using a Kromasil column (3.2 × 250 mm,
5 µm packing) and an isocratic method (60% CH3OH, 40% H2O,
0.025% ammonium acetate), 0.5 mL/min, 20 °C. Peak detection
was by UV at 280 nm. The kinetics of rotamer interconversion
were followed at 20 °C by HPLC using the same chromatographic
conditions; data was obtained from each of the four rotamer start-
points. ROESY and eCOSY spectra were acquired on an equilib-
rium mixture of 1 in 60% CD3OD, 40% D2O on a Bruker Avance
600 MHz spectrometer. This confirmed that the assignments for 1
were very similar to 2. Fractions of each atropisomer were collected,
and a portion of each was dried. Proton NMR spectra were acquired,
and this confirmed that the elution order for rotamers of 1 and 2
were identical.
Summary
Rapid VTNMR methods have been used to guide the
synthesis of nonatropisomeric MCT1 blockers. Combining
potency conferring, rapidly interconverting amides with further
logD lowering modifications has culminated in the identification
of potent MCT1 blockers, exemplified by pyrazole 13, pos-
sessing good drug-like properties.
Variable Temperature NMR. Variable temperature NMR was
performed on a Varian UnityInova spectrometer at 300 or 400 MHz,
usually under automation. Spectra at the different temperatures were
assessed as described in the text.
Chemistry. Reagents were obtained from Aldrich Chemical or
Lancaster Chemical companies and used without purification. High-
performance liquid chromatography (HPLC) grade solvents were
obtained from Fisher Scientific. Unless otherwise stated, reactions
were carried out at ambient temperature (18-25 °C) and under
positive nitrogen pressure with magnetic stirring. TLC was
performed on Merck silica gel 60 F254 plates and visualized under
UV light (254 nm) or by staining with potassium permanganate
(KMnO4). Flash chromatography was performed on E. Merck 230-
400 mesh silica gel 60. Preparative reverse phase (RP)HPLC
separations were performed using a Waters Symmetry, Novapak,
or Xterra column. Routine NMR spectra were recorded on a Varian
Unity spectrometer at a proton frequency of either 300 or 400 MHz.
Chemical shifts are expressed in ppm relative to TMS (1H, 0 ppm)
or CDCl3 (13C, 77.0 ppm); coupling constants are expressed in Hz.
Mass spectra were measured on either a VG 70-250S spectrometer
using electron impact ionization (EI) or on an Agilent 1100 MSD
G1946D spectrometer using electrospray ionization (ESI) or
A more detailed study of compounds 1, 2, and 5 has shown
that the fastest interconversion in each case, amide rotation for
the former two compounds and Ar-CO bond rotation for the
latter, proceeds without concerted rotation of the other bond.
This contrasts with some other literature reports. It is therefore
likely that the mechanism of atropisomer interconversion, i.e.,
whether concerted or not, is highly dependent on the exact nature
of substituents present on the compounds being studied.
Experimental Section
LC-NMR Separation and Analysis of 2. LC-NMR analysis of
2 was performed on a 600 MHz Varian Unity Inova Spectrometer
(Palo Alto) fitted with an HPLC-NMR system comprising a Varian
9012 pump, 9050 UV detector, and HPLC-NMR Analyte Collector
(Palo Alto, CA). A Jones Chromatography 7600 series solvent
degasser and 7971 Column Heater (Lakewood, CA) were also used.

Atropisomer Modulation of MCT1 Blockers
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 261
atmospheric pressure chemical ionization (APCI); generally only
ions which indicate the parent mass are reported.
acetate. The organic layer was dried (MgSO4), concentrated under
reduced pressure, and purified by flash chromatography. A mixture
of this compound and methylamine (2 M in THF, 0.6 mL, 1.2
mmol) in ethanol (6 mL) was heated in a pressurized vessel at 130
°C for 24 h. The reaction mixture was concentrated under reduced
pressure. The residue was purified by silica chromatography (elution
with 2% methanol in dichloromethane). The product obtained was
recrystallized from dichloromethane:iso-hexane to give compound
5 as a colorless solid (0.023 g, 38%). mp: 276-276.5 °C. 1H NMR
(300 MHz, CDCl3): δ 7.49 (1H, d, J ) 6.0 Hz), 7.17-7.07 (3H,
m), 6.75(1H, m), 5.3 (1H, s), 5.14 (1H, s), 4.3 (1H, m), 4.3-4.1
(2H, m), 3.8-3.7 (2H, m),3.6 (1H, m), 3.41 (3H, s), 3.07 (3H, d,
J ) 3.0 Hz), 2.3 (2H, m), 2.2 (1H, m), 0.92 (6H, d, J ) 9.0 Hz).
APCI-MS m/z: (pos) 481 [M + H]+. Anal. (C24H28N6O3S·0.8CH2-
Cl2) C, H, N, S.
6-[(6-Fluoroquinolin-4-yl)methyl]-5-{[(3R)-3-hydroxypyrro-
lidin-1-yl]carbonyl}-1-isobutyl-3-methylthieno[2,3-d]pyrimidine-
2,4(1H,3H)-dione (2). Acetic anhydride (0.13 mL, 1.3 mmol) was
added to a solution of compound 27 (0.57 g, 0.88 mmol),
triethylamine (0.25 mL, 1.8 mmol), and DMAP (0.01 g, 0.08 mmol)
in dichloromethane (5 mL) and the mixture stirred for 30 min. The
mixture was concentrated under reduced pressure. Methanol (5 mL),
ethyl acetate (2 mL), and saturated aqueous sodium bicarbonate
solution (1.2 mL) were added followed by 10% palladium on
charcoal (100 mg), and the mixture was stirred under hydrogen at
5 atm for 20 h. The reaction mixture was filtered through Celite
and concentrated under reduced pressure. The residue was dissolved
in acetonitrile (10 mL) and treated with 40% hydrofluoric acid (0.5
mL). After 18 h, the mixture was poured into saturated aqueous
sodium bicarbonate solution (50 mL) and extracted with ethyl
acetate (3 × 50 mL), dried (MgSO4), and concentrated under
reduced pressure. The residue was purified by flash chromatography
(elution with 14:1 dichloromethane:ethanol) and further by RPHPLC
5-{[(3R,4S)-3,4-Dihydroxypyrrolidin-1-yl]carbonyl}-1-isobu-
tyl-3-methyl-6-(quinolin-4-ylmethyl)thieno[2,3-d]pyrimidine-2,4-
(1H,3H)-dione (6). To a suspension of compound 24a4 (200 mg,
0.45 mmol) in dichloromethane (3 mL) was added 1-hydroxyben-
zotriazole hydrate (120 mg, 0.89 mmol), and the mixture was stirred
for 15 min. 1-Ethyl-3-(3′-dimethylaminopropyl)carbodiimide hy-
drochloride (171 mg, 0.89 mmol) was then added and stirring
continued for 30 min. (3R,4S)-Pyrrolidine-3,4-diol24 (125 mg, 0.90
mmol) was added, and the mixture was stirred for a further 3 h.
The mixture was poured into saturated sodium bicarbonate solution
(100 mL) and extracted with ethyl acetate, dried (MgSO4), and
concentrated under reduced pressure. The residue was purified by
flash chromatography (elution with 1:3 ethanol:dichloromethane).
The residue was further purified by RPHPLC to give compound 6
1
to give compound 2 as a colorless foam (0.13 g, 29%). H NMR
(300 MHz, DMSO-d6): δ 8.86-8.83 (m, 1H), 8.18-8.09 and 8.02
(m, 2H), 8.04 (d, J ) 8.0 Hz, 1H), 7.70 (td, J ) 8.7, 2.8 Hz, 1H),
7.55-7.45 (m, 1H), 5.02 and 4.99 and 4.93 and 4.85 (4 × d, 1H),
4.58-4.43 (m, 2H), 4.37-4.18 (m, 2H), 3.70-3.60 (m, 2H), 3.60-
3.08 (m, 1H), 3.20 (s, 3H), 2.95-2.68 (m, 1H), 2.19-2.06 (m,
1H), 2.00-1.64 (m, 2H), 0.93-0.81 (m, 6H), complex due to
atropisomerism (see text for assignment of individual rotamers).
APCI-MS m/z: (pos) 511 [M + H]+. Anal. (C26H27FN4O4S. H2O)
H, N; C: calcd, 58.98; found, 59.40; S calcd, 6.05; found, 5.58.
1-Isopropyl-N,N,3-trimethyl-2,4-dioxo-6-[2-(trifluoromethyl)-
benzyl]-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidine-5-carboxam-
ide (3). Oxalyl chloride (0.73 mL, 8.4 mmol) followed by DMF (2
drops) was added to compound 19 (1.2 g, 2.8 mmol) dissolved in
dichloromethane (30 mL). The reaction mixture was stirred for 90
min and then concentrated under reduced pressure. The residue was
dissolved in tetrahydrofuran (24 mL), and an aliquot (2 mL) of
this solution was added to dimethylamine (40% aq; 0.13 mL, 0.17
mmol) in a saturated solution of sodium carbonate (10 mL). The
reaction mixture was stirred for 2 h and then extracted with
dichloromethane providing compound 3 as a colorless solid (28
1
as a colorless solid (40 mg, 16%). mp: ∼120 °C. H NMR (400
MHz, DMSO-d6): δ 8.86 (d, J ) 4.0 Hz, 1H), 8.27 and 8.22 (d, J
) 8.0 Hz, 1H), 8.04 (d, J ) 8.0 Hz, 1H), 7.77 (t, J ) 8.0 Hz, 1H),
7.68-7.62 (m, 1H), 7.48 and 7.42 (d, J ) 4.0 Hz, 1H), 5.02 and
4.99 and 4.85 and 4.75 (br s, 2H), 4.49 (br s, 2H), 4.10-3.90 (m,
2H), 3.68-3.60 (m, 3H), 3.48-3.36 (m, 1H), 3.20 (s, 3H), 3.02-
2.98 (m, 1H), 2.95-2.92 and 2.82-2.78 (m, 1H), 2.12 (nonet, J )
7.0 Hz, 1H), 0.88-0.81 (m, 6H) - complex due to atropisomerism.
APCI-MS m/z: (pos) 509 [M + H]+. Anal. (C26H28N4O5S·2.2H2O)
C, H; N: calcd, 10.21; found, 11.70; S calcd, 5.83; found, 4.61.
5-{[(3R)-3-Hydroxypyrrolidin-1-yl]carbonyl}-1-isobutyl-3-
methyl-6-(1-naphthyloxy)thieno[2,3-d]pyrimidine-2,4(1H,3H)-di-
one (7). Prepared using the same method as for 6 but employing
acid 31 and (R)-3-hydroxypyrrolidine (colorless solid, 0.06 g, 31%
1
mg, 27%). H NMR (300 MHz, CDCl3): δ 7.67 (d, J) 6.0 Hz,
1H), 7.56-7.49 (m, 2H), 7.40-7.35 (t, J)7.4 Hz, 1H), 4.5 (br s,
1H), 4.25 (d, J) 16.0 Hz, 1H), 4.17 (d, J) 16.0 Hz, 1H), 3.36 (s,
3H), 3.15 (s, 3H), 2.87 (s, 3H), 1.56-1.52 (m, 6H); APCI-MS
m/z: (pos) 454 [M + H]+. Anal. (C21H22F3N3O3S) H, N, S; C calcd,
55.62; found, 56.28.
1
yield). mp: 165-172 °C. H-NMR (300 MHz, 130 °C, DMSO-
d6): δ 8.22-8.15 (m, 1H), 7.98-7.92 (m, 1H), 7.74 (d, J ) 8.3
Hz, 1H), 7.61-7.55 (m, 2H), 7.49-7.44 (m, 1H), 7.30 (d, J ) 7.7
Hz, 1H), 4.50-4.12 (m, 2H), 3.67 (d, J ) 7.5 Hz, 2H), 3.60-3.05
(m, 4H), 3.25 (s, 3H), 2.27-2.11 (m, 1H), 2.08-1.66 (m, 2H),
0.90 (d, J ) 6.7 Hz, 6H). APCI-MS m/z: (pos) 494 [M + H]+.
Anal. (C26H27N3O5S) C, H, N, S.
5-[(1,1-Dioxido-1,3-thiazolidin-3-yl)carbonyl]-1-isopropyl-3-
methyl-6-[2-(trifluoromethyl)benzyl]thieno[2,3-d]pyrimidine-
2,4(1H,3H)-dione (4). mCPBA (70-75%: 0.5 g, 2.2 mmol) was
added to crude compound 20 (0.15 g, 0.3 mmol) in dichloromethane
(5 mL) and stirred at room temperature for 24 h. The reaction
mixture was washed with 10% sodium metabisulfite solution, dried
(MgSO4), and concentrated under reduced pressure to give a brown
oil which was purified by flash chromatography (elution with 4:6
ethyl acetate:iso-hexane). Recrystallization from ethyl acetate:iso-
hexane gave 4 as a colorless solid (37 mg, 23%). mp. 194.4-195.6
°C. 1H NMR (300 MHz, CDCl3): δ 7.69 (d, J) 9.0 Hz, 1H), 7.55-
7.4 (m, 3H), 4.74 and 4.61(d, J) 12.0 Hz, 1H), 4.5-4.14 (m, 5H),
3.92-3.88 + 3.67-3.6 (m, 1H), 3.46-3.05 (m, 5H), 1.57-1.53
(m, 6H); ESI-MS m/z: (pos) 530 [M + H]+. Anal. (C22H22F3N3O5S2)
C, H, N; S calcd, 12.11; found, 11.16.
5-(Azetidin-1-ylcarbonyl)-1-isobutyl-3-methyl-6-{[2-(methyl-
amino)-1H-benzimidazol-1-yl]methyl}thieno[2,3-d]pyrimidine-
2,4(1H,3H)-dione (5). Isopropylmagnesium chloride (2 M in ether;
0.25 mL, 0.43 mmol) was added to azetidine (0.03 mL, 0.43 mmol)
in tetrahydrofuran. After 15 min, compound 21c (0.20 g, 0.43 mmol)
was added dropwise to the mixture. After 15 min further, azetidine
(0.03 mL, 1.29 mmol) and isopropylmagnesium chloride (2 M in
ether; 0.75 mL, 1.29 mmol) were added. After 15 min, the reaction
mixture was quenched with water and then extracted with ethyl
6-[(4,5-Dichloro-2-methyl-1H-imidazol-1-yl)methyl]-1-isobu-
tyl-N-methoxy-N,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydrothieno-
[2,3-d]pyrimidine-5-carboxa mide (8). Prepared using the same
method as for 3 but employing acid 24e and dimethylhydroxylamine
hydrochloride (colorless solid, 8.18 g, 79% yield). mp: 138-140
°C. 1H NMR (300 MHz, CDCl3): δ 5.23 (s, 2H), 3.72 (d, J ) 7.0
Hz, 2H), 3.55 (s, 3H), 3.23 (s, 3H), 3.16 (s, 3H), 2.32 (s, 3H), 2.21
(nonet, J ) 7.0 Hz, 1H), 0.92 d, J ) 7.0 Hz, 6H). APCI-MS m/z:
(pos) 488/490/492 [M + H]+. Anal. (C19H23Cl2N5O4S) C, H, N, S.
5-{[(4S)-4-Hydroxyisoxazolidin-2-yl]carbonyl}-1-isobutyl-3-
methyl-6-(quinolin-4-ylmethyl)thieno[2,3-d]pyrimidine-2,4(1H,3H)-
dione (9). Prepared using the same method as for 6 but employing
acid 24a and (S)-4-isoxazolidinol hydrochloride25 (colorless solid,
0.90 g, 81% yield). mp: 127-137 °C. 1H NMR (300 MHz, DMSO-
d6): δ 8.86 (d, J ) 5.0 Hz, 1H), 8.24 (d, J ) 7.0 Hz, 1H), 8.05 (d,
J ) 7.0 Hz, 1H), 7.78 (t, J ) 7.0 Hz, 1H), 7.63 (t, J ) 7.0 Hz,
1H), 7.46 (d, J ) 5.0 Hz, 1H), 5.54 (d, J ) 5.0 Hz, 1H), 4.79 (d,
J ) 5.0 Hz, 1H), 4.67-4.52 (m, 2H), 4.11-4.03 (m, 1H), 3.95-
3.89 (m, 1H), 3.82-3.80 (m, 1H), 3.67-3.54 (m, 3H),

262 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2
Guile et al.
(4) Murray, C. M.; Hutchinson, R.; Bantick, J. R.; Belfield, G. P.;
Benjamin, A. D.; Brazma, D.; Bundick, R. V.; Cook, I. D.; Craggs,
R. I.; Edwards, S.; Evans, L. R.; Harrison, R.; Holness, E.; Jackson,
A. P.; Jackson, C. G.; Kingston, L. P.; Perry, M. W. D.; Ross, A. R.
J.; Rugman, P. A.; Sidhu, S. S.; Sullivan, M.; Taylor-Fishwick, D.
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transporter (MCT) family: structure, function and regulation. Bio-
chem. J. 1999, 343, 281-299.
3.21 (s, 3H), 2.08 (nonet, J ) 6.8 Hz, 1H), 0.87 (d, J ) 6.5 Hz,
6H). APCI-MS m/z: (pos) 495 [M + H]+. Anal. (C25H26N4O5S)
C, H, N, S.
5-[(4S)-4-Hydroxy-2-isoxazolidinylcarbonyl]-3-methyl-1-(isobu-
tyl)-6-[2-(trifluoromethyl)phenylmethyl]-thieno[2,3-d]pyrimidine-
2,4(1H,3H)-dione (10). Prepared using the same method as for 6
but employing acid 24b and (S)-4-isoxazolidinol hydrochloride25
1
(colorless solid, 1.66 g, 70% yield). mp: 127-131 °C. H NMR
(300 MHz, DMSO-d6): δ 7.75 (d, J ) 7.5 Hz, 1H), 7.69-7.62
(m, 1H), 7.53-7.45 (m, 2H), 5.53-5.48 (m, 1H), 4.80-4.59 (m,
1H), 4.26-4.16 (m, 2H), 3.79-3.47 (m, 6H), 3.22 (s, 3H), 2.11
(quintet, J ) 6.8 Hz, 1H), 0.87 (d, J ) 6.5 Hz, 6H). APCI-MS
m/z: (pos) 512 [M + H]+. Anal. (C23H24F3N3O5S) C, H, N.
5-{[(4S)-4-Hydroxyisoxazolidin-2-yl]carbonyl}-1-isobutyl-3-
methyl-6-(1H-pyrrolo[2,3-b]pyridin-3-ylmethyl)thieno[2,3-d]py-
rimidine-2,4(1H,3H)-dione (11). Prepared using the same method
as for 6 but employing acid 24f and (S)-4-isoxazolidinol hydro-
chloride25 (colorless solid, 0.06 g, 31% yield). mp: 159-161 °C.
1H NMR (300 MHz, DMSO-d6): δ 11.53 (br s, 1H), 8.20-8.18
(m, 1H), 7.97-7.90 (m, 1H), 7.44-7.41 (m, 1H), 7.02-6.99 (m,
1H), 5.55-5.50 (m, 1H), 4.80-4.60 (m, 1H), 4.18-4.00 (m, 3H),
3.90-3.75 (m, 2H), 3.68-3.53 (s, 3H), 3.21-3.20 (m, 3H), 2.13-
2.03 (m, 1H), 0.85-0.82 (m, 6H). APCI-MS m/z: (pos) 484 [M +
H]+. Anal. (C23H25N5O5S·0.38C4H8O2(ethyl acetate)) H, N, S; C:
calcd, 56.96; found, 56.13.
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(10) (a) Seto, S. Convenient synthesis of 7-aryl-3,4,5,6-tetrahydro-2H-
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rahydro-2H-pyrido[4,3-b]- and [2,3-b]-1,5-oxazocin-6-ones as NK1
antagonists. Bioorg. Med. Chem. Lett. 2005, 15, 1479-1484. (c) Seto,
S.; Tanioka, A.; Ikeda, M.; Izawa, S. 2-Substituted-4-aryl-6,7,8,9-
tetrahydro-5H-pyrimido[4,5-b][1,5]oxazocin-5-one as a structurally
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1488.
(11) (a) Ikeura, Y.; Ishichi, Y.; Tanaka, T.; Fujishima, A.; Murabayashi,
M.; Kawada, M.; Ishimaru, T.; Kamo, I.; Doi, T.; Natsugari, H.
Axially Chiral N-Benzyl-N,7-dimethyl-5-phenyl-1,7-naphthyridine-
6-carboxamide Derivatives as Tachykinin NK1 Receptor Antago-
nists: Determination of the Absolute Stereochemical Requirements.
J. Med. Chem. 1998, 41, 4232-4239. (b) Natsugari, H.; Ikeura, Y.;
Kamo, I.; Ishimaru, T.; Ishichi, Y.; Fujishima, A.; Tanaka, T.;
Kasahara, F.; Kawada, M.; Doi, T. Axially chiral 1,7-naphthyridine-
6-carboxamide derivatives as orally active tachykinin NK1 receptor
antagonists: synthesis, antagonistic activity, and effects on bladder
functions. J. Med. Chem. 1999, 42, 3982-3993. (c) Ishichi, Y.;
Ikeura, Y.; Natsugari, H. Amide-based atropisomers in tachykinin
NK1-receptor antagonists: synthesis and antagonistic activity of
axially chiral N-benzylcarboxamide derivatives of 2,3,4,5-tetrahydro-
6H-pyrido[2,3-b][1,5]oxazocin-6-one. Tetrahedron 2004, 60, 4481-
4490.
5-{[(4S)-4-Hydroxyisoxazolidin-2-yl]carbonyl}-1-isobutyl-3-
methyl-6-{[2-(methylamino)-1H-benzimidazol-1-yl]methyl}thieno-
[2,3-d]pyrimidine-2,4(1H,3 H)-dione (12). Prepared using the same
method as for 6 but employing acid 24d and (S)-4-isoxazolidinol
hydrochloride25 (colorless solid, 4.0 g, 58% yield). mp: 232 °C.
1H NMR (300 MHz, DMSO-d6): δ 7.29-7.17 (m, 2H), 7.00-
6.84 (m, 2H), 5.61 (d, J ) 3.8 Hz, 1H), 5.54-5.17 (m, 2H), 4.85-
4.63 (m, 1H), 4.15-3.98 (m, 1H), 3.95-3.52 (m, 5H), 3.21 (s,
3H), 2.96-2.91 (m, 3H), 2.14-1.99 (m, 1H), 0.88-0.81 (m, 6H).
APCI-MS m/z: (pos) 513 [M + H]+. Anal. (C24H28N6O5S·1.5H2O)
C, H, N, S.
(S)-6-[(3,5-Dimethyl-1H-pyrazol-4-yl)methyl]-5-{[4-hydroxy-
isoxazolidin-2-yl]carbonyl}-1-isobutyl-3-methylthieno[2,3-d]py-
rimidine-2,4(1H,3H)-dione (13). Prepared using the same method
as for 6 but employing acid 24 g and (S)-4-isoxazolidinol
hydrochloride25 (colorless solid, 2.1 g, 62% yield). mp: 198-202
°C. 1H NMR (300 MHz, DMSO-d6): δ 12.12 (s, 1H), 5.52 (d, J )
3.8 Hz, 1H), 4.79-4.52 (m, 1H), 4.12-3.46 (m, 8H), 3.21 (s, 1H),
3.19 (s, 2H), 2.17-2.00 (m, 7H), 0.87 (d, J ) 6.7 Hz, 6H). APCI-
MS m/z: (pos) 462 [M + H]+. Anal. (C25H27N5O5S) C, H, N, S.
Acknowledgment. The authors thank Y. Whitehead for
binding data, B. Stensland for X-ray structure determination,
and J. Hackett for assistance with HPLC separations.
(12) (a) Albert, J. S.; Aharony, D.; Andisik, D.; Barthlow, H.; Bernstein,
P. R.; Bialecki, R. A.; Dedinas, R.; Dembofsky, B. T.; Hill, D.;
Kirkland, K.; Koether, G. M.; Kosmider, B. J.; Ohnmacht, C.; Palmer,
W.; Potts, W.; Rumsey, W.; Shen, L.; Shenvi, A.; Sherwood, S.;
Warwick, P. J.; Russell, K. Design, Synthesis, and SAR of Tachykinin
Antagonists: Modulation of Balance in NK1/NK2 Receptor Antago-
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Ohnmacht, C.; Bernstein, P. R.; Rumsey, W. L.; Aharony, D.;
Alelyunas, Y.; Russell, D. J.; Potts, W.; Sherwood, S. A.; Shen, L.;
Dedinas, R. F.; Palmer, W. E.; Russell, K. Structural Analysis and
Optimization of NK1 Receptor Antagonists through Modulation of
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as selective NK1 antagonists. Bioorg. Med. Chem. 2004, 12, 2653-
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Potts, W.; Evenden, J. L. Design and optimization of cyclized NK1
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Supporting Information Available: Combustion analysis data
for compounds 2-13, experimental methods for compounds 16-
20, 21c-f, 23, 24e-g, 26, 27, and 29-31, and crystallographic
data for compound 8. This material is available free of charge via
the Internet at http:// pubs.acs.org.
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