Imaging progesterone receptor in breast tumors: Synthesis and receptor binding affinity of fluoroalkyl-substituted analogues of Tanaproget

Article abstract of DOI:10.1021/jm100052k

The progesterone receptor (PR) is estrogen regulated, and PR levels in breast tumors can be used to predict the success of endocrine therapies targeting the estrogen receptor (ER). Tanaproget is a nonsteroidal progestin agonist with very high PR binding a

Full text of DOI:10.1021/jm100052k

J. Med. Chem. 2010, 53, 3349–3360 3349
DOI: 10.1021/jm100052k
Imaging Progesterone Receptor in Breast Tumors: Synthesis and Receptor Binding Affinity of
Fluoroalkyl-Substituted Analogues of Tanaproget
Hai-Bing Zhou,†,‡ Jae Hak Lee,† Christopher G. Mayne,† Kathryn E. Carlson,† and John A. Katzenellenbogen*,†
†Department of Chemistry, University of Illinois, 600 South Mathews Avenue, Urbana, Illinois 61801, and ‡State Key Laboratory of
Virology, College of Pharmacy, Wuhan University, Wuhan 430072, China
Received January 14, 2010
The progesterone receptor (PR) is estrogen regulated, and PR levels in breast tumors can be used to
predict the success of endocrine therapies targeting the estrogen receptor (ER). Tanaproget is a
nonsteroidal progestin agonist with very high PR binding affinity and excellent in vivo potency. When
appropriately radiolabeled, it might be used to image PR-positive breast tumors noninvasively by
positron emission tomography (PET). We describe the synthesis and PR binding affinities of a series of
fluoroalkyl-substituted 6-aryl-1,4-dihydrobenzo[d][1,3]oxazine-2-thiones, analogues of Tanaproget.
Some of these compounds have subnanomolar binding affinities, higher than that of either Tanaproget
itself or the high affinity PR ligand R5020. Structure-binding affinity relationships can be rationalized
by molecular modeling of ligand complexes with PR, and the enantioselectivity of binding has been
predicted. These compounds are being further evaluated as potential diagnostic PET imaging agents for
breast cancer, and enantiomerically pure materials of defined stereochemistry are being prepared.
Introduction
Diagnostic imaging of steroid receptors in breast tumors by
positron emission tomography (PET) is well established and
has been achieved using steroids labeled with fluorine-18. The
most extensive studies have been done using 16R-[18F]fluoro-
estradiol (FES) for imaging the estrogen receptor (ER).7-10 A
PR-basedradioligand, however, would have some advantages
over an ER-based one: there is a better correlation between
PR status and responsiveness to endocrine therapies than
there is with ER status;11-13 a PR-based ligand could be used
after the initiation of antiestrogen hormonal therapy, whereas
an ER-based one would not be useful when tumor ER is
saturated by the hormonal agent.14 Moreover, PR-based
ligands may benefit from the increased PR levels induced by
the transient agonistic effect of tamoxifen during the initial
course of tamoxifen treatment of breast tumor.15-17 In fact,
monitoring an increase in PR levels in a tumor by PET
imaging after a brief challenge with an estrogen could form
the basis of a new “estrogen challenge test” for assessing the
functionality oftumor ER. Such anestrogen challenge testhas
been done by monitoring changes in tumor uptake of
2-[18F]fluoro-2-deoxyglucose (FDG) either a week after the
initiation of tamoxifen therapy7,8 or a day after a dose of
estradiol.10 In both cases, an increase in FDG uptake after the
challenge was very highly predictive ofultimatepatient benefit
from endocrine therapies.7,8,10
The progesterone receptor (PRa) is an estrogen-regulated
protein found in female reproductive tissues and in many
breast tumors, and it can serve as a target for various endocrine
therapies, including the treatment of the breast cancer.1-6 PR
could also be used as a target for diagnostic imaging and
radiotherapy of breast cancer by the administration of a
suitably radiolabeled PR ligand that accumulates in receptor-
positive tumors, where it can be detected and quantified by
imaging. Such agents could be used to delineate the PR
positivity of tumors in vivo and in situ in a noninvasive,
comprehensive fashion, even in metastatic tumors and lymph
nodes that are inaccessible to surgical or needle biopsy. This
information can sometimes be used to evaluate tumor aggres-
siveness and predict the likelihood of responsiveness to endo-
crine therapies.7-10 In a related manner, a hormone receptor
ligand, labeled with a different radionuclide (e.g., an Auger
electron emitting isotope) that accumulates in a tumor through
a receptor-mediated uptake process (especially one like PR
that localizes activity in the nuclear chromatin fraction) could
deliver a cytotoxic dose of high linear energy transfer radiation
selectively to the tumor cells, ablating the tumor while limiting
widespread radiation toxicity. Therefore, the development of
PR ligands with potential for both diagnostic imaging and
radiotherapy is an area of great current interest.
Studies are currently underway with [18F]fluoro furanyl
norprogesterone (FFNP),18 a high affinity steroidal progestin
that appears to have promise as an agent for PET imaging of
PR. A number of related steroidal progestins labeled with
fluorine, bromine, and iodine have also been prepared.19-23
To broaden the types of progestins to be evaluated for PET
imaging of breast tumors, we embarked on a program to
identify structurally novel, nonsteroidal PR imaging agents
because such ligands can also have high receptor binding
*To whom correspondence should be addressed. Phone: 217-333-
6310. Fax: 217-333-7325. E-mail: jkatzene@uiuc.edu.
a Abbreviations: AR, androgen receptor; 9-BBN, 9-borabicyclo-
[3.3.1]nonane; CDI, carbonyl diimidazole; CSI, chlorosulfonyl iso-
cynate; DAST, diethylaminosulfur trifluoride; ER, estrogen receptor;
FES, 16R-[18F]fluoroestradiol; FFNP, fluoro furanyl norprogesterone;
GR, glucocorticoid receptor; PET, positron emission tomography;
RBA, relative binding affinity; PR, progesterone receptor; vdW,
van der Waals.
r
2010 American Chemical Society
Published on Web 03/31/2010
pubs.acs.org/jmc

3350 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Zhou et al.
affinity and good pharmacokinetics and they often have much
less cross reactivity with other steroid receptors.
Compounds 16 and 17 were prepared as shown in
Scheme 3. The unsymmetrical homoallylic alcohol 7 precur-
sor was prepared as a racemate by reaction of methyl ketone
6 with allylmagnesium chloride, followed by cyclization with
carbonyl diimidazole (CDI) in THF. A coupling reaction of
arylbromide 8 with N-BOC-pyrrole boronic acid in the
presence of Pd(0) gave pyrrole 9. The 5-cyano functional
group on pyrrole 10 was installed by treatment with chloro-
sulfonyl isocynate (CSI), followed by addition of DMF as a
quench. Oxidation of 10 with a catalytic amount of OsO4,
followed by treatment with NaIO4, afforded the aldehyde
11,28 which was reduced with sodium borohydride (NaBH4)
to give the alcohol 12. Deprotection of the BOC group on
the pyrrole nitrogen by thermolysis at 160 °C under neat
conditions then afforded the pyrrole 13. Fluoride substitu-
tion of 13 was accomplished by treatment with DAST
(diethylaminosulfur trifluoride). Methylation of 14 with
methyl iodide in the presence of potassium carbonate in
DMF, followed by thionation with Lawesson’s reagent in
toluene, gave target molecule 16 in good yield. Compound 17
was synthesized by direct thionation of 14 with Lawesson’s
reagent.
Fluoropropyl compounds 24 and 25, which have one more
carbon compared to fluoroethyl compounds 16 and 17, were
prepared as shown in Scheme 4. Treatment of the allyl
derivative 8 with 9-borabicyclo[3.3.1]nonane (9-BBN) af-
forded the corresponding alcohol 18,29 which was then
treated with the DAST to give the fluorine-substituted
compound 19. Fluoropropyl compound 19 was reacted with
N-BOC-pyrrole boronic acid in the presence of Pd(0) to
provide pyrrole 20, which was treated with CSI and DMF in
sequence to furnish nitrile compound 21. Removal of the
BOC group from pyrrole by heating neat at 160 °C gave
compound 22, and addition of methyl iodide to substitute on
the pyrrole nitrogen yielded methylated compound 23. The
desired compounds 24 and 25 were prepared by thionation
with Lawesson’s reagent in toluene, as shown in Scheme 4.
Progesterone Receptor Binding Affinities and Binding
Selectivity. The relative binding affinities of the tanaproget
derivatives as PR ligands were determined by a competitive
radiometric binding assay using [3H]R5020 as tracer and
R5020 as a standard, as previously described.19,30 The bind-
ing affinities are expressed as relative binding affinity (RBA)
values, with the RBA of the R5020 standard set to 100
(R5020 binds to PR with a Kd of 0.4 nM). The values given
are the average ( SD of two or more independent determi-
nations. The results are shown in Table 1. We also prepared a
sample of 1, and it was found to have an RBA value of 151,
which corresponds to the binding affinity reported by
Wyeth.24
Tanaproget (1), recently described by Wyeth Pharmaceu-
ticals,24,25 is a nonsteroidal progestin agonist, one of a number
of progestins of novel structure that have recently been
described.26 1 (Tanaproget) is constituted of two moieties, a
5-cyanopyrrole and a benzoxazin-2-thione, and it is a PR
agonist having an in vitro potency equivalent to that of the
beststeroidal progestins butsuperior in selectivity withrespect
to the other members of the steroid receptor family.24-27 We
anticipated that these characteristics might also make 1 a
highly effective and selective in vivo probe, which, when
appropriately radiolabeled with a positron-emitting radio-
nuclide, might be useful for PET imaging of PR-positive
breast tumors.
The synthesis of 1is relatively simple, and based on structure-
affinity relationships developed at Wyeth24 and a PR X-ray
crystal structure they obtained,25 it was known that the pyrrole
nitrogen can either be unsubstituted or substituted with small
groups, such as a methyl group (e.g., 1 itself). The geminal
dimethyl groups on the benzoxazin-2-thione ring can also be
replaced by larger groups; even substitution with a 2-thienyl
group gives a high affinity compound.24 By contrast, the ben-
zoxazin-2-thione N-H cannot be substituted because it forms an
important hydrogen bond interaction with an amino acid residue
in the binding pocket.25 The structure of 1and potential fluorine-
substituted derivatives are given in Scheme 1. The most promis-
ing sites for the attachment of groups bearing a positron-emitting
radionuclide are the pyrrole nitrogen (region 1) and the geminal
dimethyl groups (region 2) on the benzoxazinthione ring. In this
work, we describe the synthesis of various fluoroalkyl analogues
of 1 and assess their binding affinity for PR.
Results and Discussion
Synthesis. The N-fluoroalkyl substituted pyrrole nitrogen
derivatives 5a and 5b were synthesized as shown in Scheme 2.
The key intermediate 2 was prepared according to the
published procedure.24 Alkylation of 2 with the correspond-
ing fluoroalkyl reagents, followed by thionation with Law-
esson’s reagent in toluene, gave target molecules 5a and 5b in
high yield.
Scheme 1. Tanaproget (1) and Potential Fluorine-Substituted
Derivatives
The fluorine-substituted ligands show binding affinities to PR
that range from poor to excellent, and we found that the nature
of the substituent R1 on the pyrrole nitrogen and the substituent
Scheme 2. Synthesis of Fluorine-Substituted Derivatives 5a and 5ba
a Reaction conditions and reagents: (a) 2-FCH2CH2OTf (3a) or 3-FCH2CH2CH2OTf (3b), K2CO3, DMF, rt, 16 h, 74%, 84%; (b) Lawesson’s
reagent, toluene, reflux, 2 h, 78%, 82%.

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3351
Scheme 3. Synthesis of Fluorine-Substituted Derivatives 16 and 17a
a Reaction conditions and reagents: (a) allylmagnesium chloride, ether, -78 °C to rt, 12 h, 83%; (b) CDI, THF, rt, overnight, 89%; (c) N-BOC-pyrrole
boronic acid, Pd(PPh3)4, aq Na2CO3, toluene, reflux, 16 h, 73%; (d) CSI, DMF, THF, -78 °C, 1 h 30 min, 76%; (e) (i) OsO4, NMO, THF:water (10:1), rt,
5 h, (ii) NaIO4, THF:water (4:1), rt, 1 h, 80%; (f) NaBH4, EtOH, rt, 2 h, 82%; (g) neat, 160 °C, 20 min, 78%; (h) DAST, CH2Cl2, -78 °C, 1 h, 39%;
(i) Lawesson’s reagent, toluene, 120 °C, 2 h, 75-79%; (j) MeI, K2CO3, DMF, rt, 16 h, 75%.
Scheme 4. Synthesis of Fluorine-Substituted Derivatives 24 and 25a
a Reaction conditions and reagents: (a) 9-BBN, THF, rt, 20 h, 89%; (b) DAST, CH2Cl2, -78 °C, 1 h, 70%; (c) N-BOC-pyrrol boronic acid, Pd(PPh3)4,
aq K2CO3, toluene, 80 °C, 16 h, 72%; (d) CSI, DMF, THF, -78 °C, 1 h 30 min, 70%; (e) neat, 160 °C, 20 min, 68%; (f) MeI, K2CO3, DMF, 90 °C, 16 h,
78%; (g) Lawesson’s reagent, toluene, reflux, 2 h, 71-78%.
R2 on the benzoxazin-2-thione moiety both had significant
effects on PR binding affinity. While the methyl group on the
pyrrole nitrogen is well tolerated, larger fluoroalkyl substituents
at this position decreased the binding affinity considerably; e.g.,
the N-fluoroethyl compound 5a had an RBA of 18.5, and the
N-fluoropropyl compound 5b had a much lower binding affinity
(RBA = 0.99). The compounds with fluoroalkyl moieties on the
benzoxazin-2-thione template, R2, bound very well, with the
fluoroethyl moiety giving somewhat higher affinities for PR
(e.g., 16, RBA = 151; 17, RBA = 198) than the fluoropropyl
moiety (e.g., 24, RBA = 90.9; 25, RBA = 189).
nuclear receptors, most notably with the androgen receptor
(AR) and the glucocorticoid receptor (GR).31-37 Compound
1, however, has very low affinity for both AR and GR,24-27
as do some other nonsteroidal progestins.31,33,36,37 Notably,
our lead compound, the fluoropropyl Tanaproget analogue
(25) binds to AR with an affinity less than 0.04% that of the
potent androgen R1881 and to GR with less than 0.9% that
of the potent glucocorticoid dexamethasone. Thus, its PR
binding affinity as well as its binding selectivity are both
excellent.
Molecular Modeling of Structure-Binding Affinity Relation-
ships and Predicted Enantioselectivity. The binding affinities
for the Tanaproget analogues shown in Table 1 show a strong
It is well-known that steroidal progestins often have
substantial cross reactivity with other evolutionally related

3352 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Zhou et al.
dependence on the nature and position of the alkyl or
fluoroalkyl substituent on the pyrrole-benzoxazin-2-thione
ligand core. Thus, while the pyrrole N-methyl group of 1 is
well tolerated (1, RBA = 151), the larger fluoroalkyl substit-
uents are not (5a = 18.5, 5b = 0.99), indicating that the
ligand-binding pocket of PR has very limited tolerance for
more than a methyl group. By contrast, when fluoroethyl or
propyl substituents replace one of the C4 methyl groups of
the benzoxazin-2-thione unit, high affinity compounds are
produced, with three compounds (16, 17, 25) having equiva-
lent or higher binding affinity than that of compound 1 itself
and indicatingthatthisregion ofthe PRligand binding pocket
has good bulk tolerance, as suggested by prior work.24
Intriguingly, there appears to be some “reciprocity” between
the substituent on the pyrrole nitrogen and at the C4 position
such that larger groups (fluoroethyl and fluoropropyl) are
more readily accommodated at C4 when there is a smaller
group (H) on the pyrrole nitrogen. Also, it is of note that while
the pyrrole N-substituted compounds 5a and 5b are achiral, the
high affinity Tanaproget analogues modified at C4 position of
benzoxazin-2-thione are chiral but thus far have only been
studied as racemates. Thus, it is possible that one enantiomer
will have higher binding affinity than the other.
Table 1. Relative Binding Affinities (RBAs) of Tanaproget Derivativesa
To investigate further the structure-binding affinity corre-
lations we have uncovered, including the apparent reciprocity
between the pyrrole N-substituent and the C4-substituents,
and to explore possible enantioselectivity, we modeled our
new ligands into the ligand binding pocket of PR, using as a
guide the X-ray crystal structure of PR complexed with 1
(PDB accession code 1zuc). All structures from Table 1 were
dockedintothe ligandbindingpocket using AutodockVina,38
minimized, and further analyzed using commercial molecular
modeling software.3,30
First focusing on N-substitution at the pyrrole, we inves-
tigated the effects of substituent size on ligand conformation
by analyzing the torsional energetics of the ligand indepen-
dent of the protein. For the unsubstituted (N-H) analogue of
1, the torsional (tor), electronic (ele), and van der Waals
compdb
R1
R2
RBA (R5020=100)
Tanaproget (1) CH3
5a
CH3
CH3
151 ( 39
18.5 ( 5.2
0.99 ( 0.28
151 ( 13
198 ( 30
90.9 ( 27
189 ( 40
CH2CH2F
5b
CH2CH2CH2F CH3
16b
17b
24b
25b
CH3
H
CH2CH2F
CH2CH2F
CH3
H
CH2CH2CH2F
CH2CH2CH2F
a Relative binding affinity (RBA) values were determined by a compe-
titive radiometric binding assay, using [3H]R5020 as a tracer. For details,
see Methods and our prior publication.19,30 Values are expressed as
percentages relative to the affinity of the standard, R5020 = 100%. From
the binding affinity of R5020 (KD = 0.4 nM), one can calculate the KI of
these compounds (KI = 0.4 nM/[RBA] ꢀ 100). b Compounds 16, 17, 24,
and 25 were tested as racemates.
Figure 1. Ligand energy dependence on pyrrole-benzoxazin-2-thione core dihedral angle. At relatively small angles, several energy terms
contribute to the change in total ligand energy when rotating around the bond connecting the N-H pyrrole and benzoxazin-2-thione core (A).
N-Substitution at the pyrrole (B) changes these torsional dynamics by drastically increasing the magnitude of the vdW energy term. This effect
is magnified with increasing substituent size (C) and results in a shift to larger dihedral angles for the minimum energy conformation (C inset).
(Note differences in the energy scales on the different panels).

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3353
Figure 2. Docking poses for N-substituted analogues of Tanaproget. (A) The steric bulk of the pyrrole N-substituent for 1 (cyan), 5a (blue),
and 5b (red) induces an increasingly large dihedral angle, rotating the nitrile away from Gln725, and resulting in reduced hydrogen bonding.
(B) Calculating the interaction energy, however, suggests that small substituents (e.g., N-Me and N-EtF) have less rotational freedom but make
beneficial contacts with the receptor compared to unsubstituted analogues.
Shifting our focus to C4-substitution, we observed that
while docked poses for the N-Me and N-H series of
analogues show a conserved binding mode, the C4-sub-
stituent subtly affects ligand conformation and place-
ment. Overlaying the docked poses for compounds in the
N-Me (Figure 3) and N-H series (not shown) suggests that
the gem-dimethyl substituted compounds adopt an inter-
mediate position. As the size of the C4 substituent in-
creases, the compound rotates to a degree related to steric
bulk and in a direction that reflects the stereochemical
configuration.
Figure 3. Effects of methyl (cyan), fluoroethyl (blue), and fluoro-
propyl (red) substitution at C4 of Tanaproget. As substituent bulk
increases, the core of the R-enantiomer (A) is forced upward to
adopt a higher energy ligand conformation, while the S-enantiomer
(B) allows a downward rotation to a more energetically favorable
dihedral angle.
The poses presented in Figure 3 imply that the stereo-
chemical configuration at C4 should have a marked effect on
ligand-protein interactions. The R-configuration directs the
substituent down in the binding pocket, where steric con-
straints force the core to rotate up and adopt a relatively
small dihedral angle (compare the dihedral angle of R/S-24
to 1 in Table 2). As clearly demonstrated in Figure 1B, the
shift to a smaller dihedral angle represents a higher-energy
ligand conformation. It is notable that N-H analogues follow
a similar trend, although with a smaller energy difference
that is obscured by the large energy range presented in
Figure 1A. The S-configuration, in contrast, directs the
substituent up in the pocket, causing the core to rotate
downward and adopt a more energetically favorable dihe-
dral angle. These qualitative observations are corroborated
by energy calculations whereby the total energy was calcu-
lated for each pose, and in all cases the S-stereoisomer for
each chiral analogue was lower in energy than the corre-
sponding R-stereoisomer (Table 2).
Finally, we set out to establish a model relating the
molecular consequences for both N- and C4-substitution
to the observed RBA values. Specifically, we wanted to
formalize the apparent reciprocity between the two sub-
stitution points, and to make predictions regarding the
enantioselectivity for our chiral analogues, doing so in a
simple yet functional manner. To this end, we initially
considered a number of models based on energy calcula-
tions. As illustrated by the calculated total energies in
Table 2, these models were capable of predicting the relative
order of the RBA values within each series (N-R, N-Me, and
N-H); however, all failed in cross series comparisons (e.g.,
5a vs 24 or 25). Ultimately, we developed a model focusing
on hydrogen bonding contacts.
(vdW) terms contribute significantly to the change in total
energy (Figure 1A); however, in all cases of N-substitution,
the change in ligand energy is dominated by vdW forces
(Figure 1B). Increasing the size of the substituent magnifies
this effect by increasing the ligand energy at small dihedral
angles (Figure 1C). By extension, the minimum energy
conformation is shifted to a larger dihedral angle as the
substituent size increases (Figure 1C inset).
The effect of dihedral angle on binding affinity becomes
obvious within the context of the protein, as shown for the
docked poses of 1, 5a, and 5b in Figure 2A. The rotation of
the pyrrole to accommodate larger substituents shifts the
position of the nitrile away from Gln725, an important
hydrogen bonding contact. Studying the energy associated
with the interaction between the ligand and the protein when
rotating the pyrrole in the binding pocket, however, sug-
gested that small substituents potentially make other non-
specific beneficial contacts, as shown in Figure 2B.
To ensure that any change in the interaction energy was
solely due to the substituent, we started with the docked
conformation of 5a (N-EtF) and modified the N-substituent
accordingly prior to each series of calculations. Comparing
the interaction energies between substituted (N-Me, N-EtF)
and unsubstituted (N-H) pyrroles shows that the substituted
compounds exist in a more narrow but deeper energy well
than the unsubstituted analogue, implying that the substi-
tuted pyrrole has less rotational freedom but makes more
positive contacts with the receptor.

3354 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Zhou et al.
Table 2. Calculated Binding Metrics for Docked Poses of Tanaproget Analogues
energy (kcal/mol)
meana H-bonding scoreb
individual average
series
NR
ligand
dihedral angle
individual
average
RBA
5a
5b
59.6
64.8
49.2
47.6
52.1
45.3
52.5
39
488.9
498.7
486.0
490.3
485.9
500.5
493.0
482.0
485.8
482.2
496.7
490.0
488.9
498.7
486.0
0.396
0.311
0.424
0.419
0.430
0.411
0.431
0.446
0.439
0.451
0.434
0.446
0.396
0.311
0.424
18.5
0.99
151
NMe
Tanaproget (1)
R-16
488.1
0.425
151
S-16
R-24
496.7
482.0
0.421
0.446
90.9
602
S-24
NH analogue of 1
NH
R-17
S-17
R-25
S-25
35.6
43
484.0
493.3
0.445
0.440
198
189
33.7
46.3
a Geometric mean. b Calculated using the “ligand interactions” module in MOE.
The large substituent at C4 restricts the rotational freedom of
the core, while N-Me substitution increases the internal vdW
energy of the ligand, resulting in a high-energy conformation
that precludes strong hydrogen bonding interactions. The
reduction of C4-substituent size allowing the core to rotate
to a more favorable position (ex. 15), or the removal of the
N-substituent reducing the energy associated with internal
torsion (ex. 25), results in increased hydrogen bonding scores
that correspond well to the observed higher RBA values.
This model also supports the prediction that the S-config-
uration of the C4-substitued systems will have higher affinity
than the R-stereoisomer.
While our model is rather simplistic, we obtain with it a
linear relationship between HB score and ln(RBA) having a
correlation coefficient over 0.95. This relationship accurately
predicts the relative order of binding affinity, and it gives
predicted RBA values within an average factor of 1.4 (and
always better than a factor of 2) relative to experimentally
determined ones, comparable to the error in experimentally
determined values (cf. Table 1). We suspect that the limita-
tions in our model could be reduced through the develop-
ment of a more complex model that includes a direct
treatment of energy calculations, the evaluation of nonspeci-
fic protein-ligand interactions, and multivariate analysis.
However, our very simple approach of analyzing ligand
binding affinity through the quantification of the hydrogen
bonding contacts is suitable for explaining the binding trends
of the compounds we have studied (Table 1), and it generates
a valuable predictive model for exploring enantioselectivity.
Figure 4. Linear Fit Relating the Calculated Hydrogen Bonding
Score to RBA.
From experimental SAR and PR-ligand crystal structures,
it has been well established that hydrogen bonding with
residues Gln725, Arg766, and Asn719 is a requirement for
tight binding. The remainder of the pocket is largely lipo-
philic and contributes to the binding affinity only through
vdW interactions, a notoriously difficult energy contribution
to calculate.39,40 On the basis of the position and interdepen-
dence of the hydrogen bonding partners, we modeled the
RBA values as a function of the geometric mean of calcu-
lated hydrogen bonding scores using the built-in “ligand
interactions” module of MOE.41 Linearization of this func-
tion yieldeda highlycorrelatedrelationship (Figure 4, slope =
43.8, R2 = 0.96), and the mean hydrogen bonding scores
correctly predict the relative order of binding affinity across
all three series, including extremely low (e.g., 5a, 5b) and high
(e.g., N-H analogue of 1) affinity compounds.
Conclusion
The application of this model to understand the observed
RBA values for compounds 5a and 5b is rather straight-
forward. The rotation of the pyrrole away from Gln725, as
highlighted in Figure 2, corresponds to the erosion of the
mean hydrogen bonding score (Table 2, series N-R) with
increased substituent size. The difference between RBA
values for the remainder of the compounds in Table 1 are
much smaller and the analysis correspondingly more subtle.
We propose that the ligand binds in a pose generally con-
served for all analogues; the ligand conformation strikes a
delicate balance between positioning the core to accommo-
date substitution at C4, maximizing hydrogen bonding con-
tacts and positive interactions between the pyrrole and
receptor and minimizing internal ligand energy.
We have synthesized a series of fluoroalkyl-substituted
6-aryl-1,4-dihydrobenzo-[d]-[1,3]oxazine-2-thiones, analogues of
the high affinity nonsteroidal progestin agonist 1(Tanaproget), as
potential diagnostic imaging agents using positron emission
tomography (PET). Some of the synthesized compounds showed
subnanomolar binding affinities, comparable to and even higher
than that of the known high affinity PR ligand R5020 and of 1
itself. We have been able to rationalize the distinct structure-
binding affinity relationships in this series by molecular modeling
of ligand internal energy and ligand-receptor hydrogen bonding
score, and our model yields an intriguing explanation for the
interaction between substituents at the two ends of the Tanapro-
get core as well as offering predictions of enantioselectivity.
Compounds with high binding affinities are currently being
evaluated as potential diagnostic imaging agents for PR in
breast tumors, and further work on the radiolabeling of some
The interplay between these interactions is best illustrated
by the marked decrease in RBA value for compound 24.

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Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3355
of these compounds and studies of their biodistribution and in
vivo PET imaging of mammary tumors in animals are cur-
rently underway and will be reported elsewhere. In addition,
work on the asymmetric synthesis and separation of the
racemates of the C4-substituted Tanaproget analogues are
progressing, and the result of the binding selectivities will be
reported in due course.
(s, 6H), 2.08-2.18 (m, 2H), 4.21 (t, 2H, J = 7.2 Hz), 4.32 (t, 1H,
J = 5.5, 47.0 Hz), 4.42 (t, 1H, J = 5.5, 47.0 Hz), 6.18 (d, 1H,
J = 4.0 Hz), 6.88 (d, 1H, J=4.0 Hz), 7.13 (d, 1H, J=2.0 Hz),
7.24-7.26 (m, 1H), 9.60 (brs, 1H). 13C NMR (125 MHz, CDCl3)
δ 28.3, 32.10 (d, 1C, J = 20.4 Hz), 43.13 (d, 1C, J = 3.9 Hz),
80.66 (d, 1C, J = 166.3 Hz), 83.21, 104.61, 110.46, 114.35,
115.37, 120.65, 124.25, 126.67, 127.11, 130.13, 134.61, 139.51,
153.25. MS m/e 327 (Mþ, 65%). HRMS (EI) calcd for
C18H18O2N3F 327.1383 (Mþ), found 327.1381.
General Procedure for the Synthesis of N-Fluoroalkyl Deriva-
tives. To a solution of 4 (1.0 mmol) in toluene (6 mL) was added
Lawesson’s reagent (275 mg, 0.7 mmol) and the reaction was
heated to 120 °C for 2 h. The reaction was cooled to RT, poured
into water (20 mL), and extracted with EtOAc (3 ꢀ 20 mL). The
organic layers were combined, washed with brine (20 mL), dried
over MgSO4, filtered, and concentrated in vacuo gave the crude
product (5).
5-(4,4-Dimethyl-2-thioxo-1,4-dihydro-2H-benzo[d][1,3]oxazin-
6-yl)-1-(2-fluoro-ethyl)-1H-pyrrole-2-carbonitrile (5a). Purified
by preparative thin-layer chromatography (5-30% EtOAc/hexane)
to give light-yellow needles (78% yield) that were recrystallized from
EtOAc/hexane, mp 229-231 °C. 1H NMR (500 MHz, acetone-d6)
δ 1.75 (s, 6H), 4.47 (t, 1H, J = 5.0, 25.5 Hz), 4.52 (t, 1H, J = 5.0,
25.5 Hz), 4.60 (t, 1H, J = 4.8, 47.5 Hz), 4.69 (t, 1H, J = 4.8, 47.5
Hz), 6.33 (dd, 1H, J = 4.0, 0.5 Hz), 7.00 (dd, 1H, J = 4.0, 0.5 Hz),
7.25 (d, 1H, J= 8.0 Hz), 7.47 (d, 1H, J=8.0Hz),7.49(s,1H),11.11
(brs, 1H). 13C NMR (125 MHz, acetone-d6) δ 26.8, 46.93 (d, 1C,
J = 20.4 Hz), 82.49 (d, 1C, J = 170.1 Hz), 83.48, 105.35, 110.73,
113.76, 114.68, 120.20, 124.91, 127.68, 127.78, 130.34, 132.77,
139.81, 184.19. 19F NMR (376 MHz, acetone-d6) δ -222.81. MS
m/e 329 (Mþ, 35%). HRMS (EI) calcd for C17H16ON3SF 329.0998
(Mþ), found 329.0997.
Experimental Procedures and Characterization Data
Materials and Methods. All reagents and solvents were ob-
tained from Aldrich. Tetrahydrofuran, diethyl ether, toluene,
and dichloromethane were obtained prior to use from a solvent-
dispensing system.42 Glassware was oven-dried, assembled
while hot, and cooled under an inert atmosphere. Unless other-
wise noted, all reactions were conducted in an inert atmosphere.
Reaction progress was monitored using analytical thin-layer
chromatography (TLC) on 0.25 mm Merck F-254 silica gel glass
plates. Visualization was achieved by either UV light (254 nm).
Flash chromatography was performed with neutral aluminum
(0.040-0.063 mm) packing and with silica gel (Merck, 230-400
mesh). Compounds Tanaproget (1) and 2 were synthesized
according to published procedures.24
The purity of all compounds for biological testing was
determined by HPLC method (see Supporting Information),
confirming >95% purity.
5-(4,4-Dimethyl-2-oxo-2,4-dihydro-1H-benzo[d][1,3]oxazin-6-
yl)-1H-pyrrole-2-carbonitrile (2). Compound 2 was prepared
according to the previous paper.17 NMR (500 MHz, CDCl3) δ
1.62 (s, 6H), 6.36 (dd, 1H, J = 3.5 Hz, 2.5 Hz), 6.74 (dd, 1H, J =
3.5 Hz, 2.5 Hz), 6.86 (d, 1H, J = 9.0 Hz), 7.43-7.41 (m, 2H),
8.66 (brs, 1H), 11.08 (brs, 1H). Registry number: 304853-99-4.
2-Fluoroethyl trifluoromethanesulfonate (3a). This compound
3a was prepared according to methods described in our prior
publication.43 NMR (500 MHz, CDCl3) δ 4.70-4.66 (m, 2H),
4.77-4.74 (m, 2H).
5-(4,4-Dimethyl-2-thioxo-1,4-dihydro-2H-benzo[d][1,3]oxazin-
6-yl)-1-(2-fluoro-propyl)-1H-pyrrole-2-carbonitrile (5b). Purified
by flash chromatography (10-30% EtOAc/hexane) to give a
yellow solid (82% yield) that was recrystallized from EtOAc, mp
219-221 °C. 1H NMR (500 MHz, CDCl3) δ 1.78 (s, 6H),
2.09-2.20 (m, 2H), 4.21 (t, 2H, J = 7.2 Hz), 4.33 (t, 1H, J =
5.5, 47.0 Hz), 4.42 (t, 1H, J = 5.5, 47.0 Hz), 6.20 (d, 1H, J =
4.0 Hz), 6.89 (d, 1H, J = 4.0 Hz), 7.02-7.04 (m, 1H), 7.16 (d, 1H,
Synthesis of N-Fluoroalkyl Derivatives 5. General Procedure
for the Synthesis of N-Fluoroalkyl Derivatives (4). To a solution
of 5-(4,4-dimethyl-2-oxo-1,4-dihydro-2H-3,1-benzoxazin-6-yl)-
1H-pyrrole-2-carbonitrile 2 (61 mg, 0.23 mmol) in anhydrous
DMF (5 mL) was added potassium carbonate (158 mg,
1.1 mmol) followed by fluoroalkyl triflate (0.21 mmol), and
the reaction was stirred for 16 h at room temperature. The
reaction mixture was poured into water (5 mL) and extracted
with EtOAc (2 ꢀ 20 mL). The organic layers were combined,
washed with brine (10 mL), dried over MgSO4, filtered, and
concentrated in vacuo. Purification of the concentrated filtrate
by flash column chromatography on silica gel (40% EtOAc/
hexane) or preparative thin-layer chromatography gave pro-
duct; further purification was effected by recrystallization.
5-(4,4-Dimethyl-2-oxo-1,4-dihydro-2H-benzo[d][1,3]oxazin-6-
yl)-1-(2-fluoro-ethyl)-1H-pyrrole-2-carbonitrile (4a). Purified by
preparative thin-layer chromatography (20% EtOAc/hexane)
to give colorless needles (74% yield) that were recrystallized
from EtOAc/hexane, mp 185-186 °C. 1H NMR (500 MHz,
acetone-d6) δ 1.71 (s, 6H), 4.45 (t, 1H, J = 5.0, 27.0 Hz), 4.51 (t,
1H, J = 5.0, 27.0 Hz), 4.59 (t, 1H, J = 5.0, 27.0 Hz), 4.69 (t, 1H,
J = 5.0, 27.0 Hz), 6.29 (d, 1H, J = 4.0 Hz), 6.98 (d, 1H, J = 4.0
Hz), 7.15 (d, 1H, J = 8.0 Hz), 7.40 (d, 1H, J = 8.0 Hz), 7.44 (s,
1H), 9.40 (brs, 1H). 13C NMR (125 MHz, acetone-d6) δ 27.7,
45.83 (d, 1C, J = 20.4 Hz), 81.89 (d, 1C, J = 172.3 Hz), 82.46,
103.45, 111.83, 113.71, 114.38, 120.16, 125.01, 127.33, 127.48,
130.24, 132.67, 139.36, 153.78. 19F NMR (376 MHz, CDCl3)
δ -222.83. MS m/e 313 (Mþ, 55%). HRMS (EI) calcd for
C17H16O2N3F 313.1227 (Mþ), found 313.1225.
J = 1.5 Hz), 7.33 (dd, 1H, J = 8.0, 1.5 Hz), 10.21 (brs, 1H). 13
C
NMR (125 MHz, CDCl3) δ 27.84, 32.09 (d, 1C, J = 19.4 Hz),
43.16 (d, 1C, J = 3.9 Hz), 80.61, (d, 1C, J = 166.1 Hz), 84.94,
104.98, 110.68, 114.21, 114.77, 120.72, 124.22, 127.55, 128.46,
130.23, 131.84, 139.05, 184.34. 19F NMR (376 MHz, CDCl3)
δ -221.63. MS m/e 343 (Mþ, 85%). HRMS (EI) calcd for
C18H18ON3SF 343.1155 (Mþ), found 343.1153.
Synthesis of Compounds 16 and 17. 4-Bromo-aminoacetophe-
none (6).44 To a solution of 2-aminoacetophenone (2.0 mg,
14.796 mmol) in CH2Cl2 (500 mL) was added slowly pyridine
hydrobromide perbromide (5.26 mg, 14.796 mmol) in 0 °C. The
reaction mixture was allowed warmed to RT for 24 h. The
reaction mixture was added water (200 mL) and extracted with
CH2Cl2 (50 mL ꢀ 3). Th combined organic layer was dried over
Na2SO4 and concentrated by rotary evaporator. The residue,
desired product 6 (2.97 g, 94%) as a yellow solid, was used in
next step without further purification. 1H NMR (500 MHz,
CDCl3) δ 2.65 (s, 3H), 6.29 (brs, 2H), 6.56 (d, 1H, J = 11.0 Hz),
7.34 (dd, 1H, J = 11.0 Hz, 3.0 Hz), 7.80 (d, 1H, J = 2.5 Hz). 13C
NMR (125 MHz, CDCl3) δ 27.7, 106.5, 118.9, 119.2, 134.0,
136.9, 149.0, 199.6. Registry number: 29124-56-9
2-(2-Amino-5-bromophenyl)pent-4-en-2-ol (7). To a solution
of 4-bromo-aminoacetophenone 6 (2.97 g, 13.875 mmol) in
THF (60 mL) was added allylmagnesium chloride (2.0 M,
20.81 mL, 41.624 mmol) at -78 °C under N2 atmosphere. The
reaction mixture was stirred for 4 h. The reaction mixture was
added water (300 mL) and extracted with EtOAc (50 mL ꢀ 3).
The combined organic layer was washed with aq NH4Cl
(30 mL ꢀ 3) and dried over Na2SO4. The resulting solution was
removed by reduced pressure. The residue was purified by flash
5-(4,4-Dimethyl-2-oxo-1,4-dihydro-2H-benzo[d][1,3]oxazin-6-
yl)-1-(2-fluoro-propyl)-1H-pyrrole-2-carbonitrile (4b). Purified
by flash chromatography (10-30% EtOAc/hexane) to give
colorless needles (81% yield) that were recrystallized from
1
EtOAc, mp 140-142 °C. H NMR (500 MHz, CDCl3) δ 1.75

3356 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Zhou et al.
column chromatography on silica gel (30% EtOAc/hexane) to give
allylated product 7 (3.479 g, 98%) as a pale-yellow oil. 1H NMR
(500 MHz, CDCl3) δ 1.70 (s, 3H), 2.66-2.74 (m, 1H), 2.89-2.94
(m, 1H), 4.84 (brs, 2H), 5.26-5.31 (m, 2H), 5.84-5.94 (m, 1H),
6.62 (d, 1H, J = 10.0 Hz), 7.23-7.27 (m, 2H). 13C NMR (125
MHz, CDCl3) δ 27.2, 44.4, 75.0, 109.2, 118.9, 119.3, 129.0, 130.5,
131.2, 133.3, 144.5. MS (EI): m/e 257 (M þ 2)þ, 255 (M)þ, 216,
214 (100), 198, 172, 158, 143, 136, 117. HRMS (EI) calcd for
C11H1479BrNO 255.0265, found 255.0259.
4-Allyl-6-bromo-4-methyl-1Hbenzo[d][1,3]oxazine-2(4H)-one (8).
To a solution of 7 (3.91 g, 15.25 mmol) in THF (150 mL) was added
1,10-carbonyldiimidazole (2.72 g, 16.780 mmol) at RT and refluxed
for 48 h. The reaction mixture was cooled to RT and water was
added (200 mL). The reaction solution was extracted with EtOAc
(50 mL ꢀ 3). The combined organic layer was washed with aq
NH4Cl (30 mL ꢀ 3). The resulting solution was concentrated by
reduced pressure and purified by flash column chromatography
on silica gel (30% EtOAc/hexane) to provide benzo[d]oxaxines 8
(3.2 g, 74%) as a yellow oil. 1H NMR (500 MHz, CDCl3) δ 1.72 (s,
3H), 2.66-2.76 (m, 2H), 5.10 (s, 1H), 5.13 (d, 1H, J = 4.0 Hz),
5.69-5.78 (m, 1H), 6.93 (d, 1H, J = 8.0 Hz), 7.02-7.08 (m, 2H),
7.22 (td, 1H, J= 7.5 Hz, 1.5 Hz), 9.92 (s, 1H). 13CNMR(125MHz,
CDCl3) δ 26.2, 45.4, 84.7, 114.7, 119.8, 123.2, 123.8, 124.2, 128.8,
131.4, 134.1, 153.3. MS (EI): m/e 283 (M þ 2)þ, 281 (M)þ, 242,
240 (100), 224, 196, 161, 143, 115. HRMS (EI) calcd for
C12H1281BrNO2 283.0030, found 283.0031.
2-(4-Allyl-4-methyl-2-oxo-1,4-dihydro-2H-benzo[d][1,3]oxazin-
6-yl)-pyrrole-1-carboxylic acid tert-butyl ester (9). Purified by
flash chromatography (40-60% EtOAc/hexane) to give a light-
yellow solid (73%) that was recrystallized from EtOAc/hexane,
mp 95-97 °C. 1H NMR (500 MHz, CDCl3) δ 1.40 (s, 9H),1.72
(s, 3H), 2.66-2.73 (m, 2H), 5.12-5.15 (m, 2H), 5.73-5.80 (m,
1H), 6.15-6.16 (m, 1H), 6.21-6.22 (m, 1H), 6.89 (d, 1H, J = 8.0
Hz), 7.06 (d, 1H, J = 1.5 Hz), 7.22 (dd, 1H, J = 8.0, 1.5 Hz),
7.32-7.33 (m, 1H), 9.63 (brs, 1H). 13C NMR (125 MHz, CDCl3)
δ 26.51, 27.96, 45.82, 83.92, 85.01, 110.81, 114.16, 114.67, 120.19,
122.78, 123.87, 125.06, 129.79, 130.20, 131.74, 133.52, 134.42,
149.38, 153.30. MS m/e 368 (Mþ, 30%). HRMS (EI) calcd for
C21H24N2O4 368.1736 (Mþ), found 368.1737.
Na2SO3 was added. After the resulting mixture was stirred at RT for
1 h, it was worked up with EtOAc and water. The aqueous layer was
extracted with EtOAc (3 ꢀ 50 mL). The combined organic layer was
dried over Na2SO4. The solvent was evaporated under vacuum. The
residue was purified by flash chromatography using pure diethyl
ether to give 670 mg (80%) of diol.
To the above diol (341 mg, 0.805 mmol) was added THF (8 mL),
water (2 mL), and NaIO4 (343 mg, 1.61 mmol). After the resulting
mixture was stirred at RT for 1 h, it was worked up with diethyl
ether and water. The aqueous layer was extracted with diethyl ether
(3 ꢀ50 mL). The combined organic layer was washed with brine
and dried over Na2SO4. The solvent was evaporated under vac-
uum. The residue was purified by flash chromatography using
20% diethyl ether/petroleum ether to give aldehyde 11 as viscous
oil (65% yield). 1H NMR (500 MHz, CDCl3) δ1.52 (s, 9H), 1.82 (s,
3H), 3.04-3.13 (m, 2H), 6.22 (d, 1H, J = 4.0 Hz), 6.94-6.97 (m,
2H), 7.08 (d, 1H, J = 1.5 Hz), 7.25-7.27 (m, 1H), 9.44 (brs, 1H),
9.76 (s, 1H). 13C NMR (125 MHz, CDCl3) δ 18.92, 27.74, 52.71,
82.74, 87.64, 105.93, 113.50, 114.34, 114.89, 123.40, 124.71, 124.75,
128.17, 130.60, 134.36, 147.30, 150.97, 152.01, 198.53. MS m/e 395
(Mþ, 60%). HRMS (EI) calcd for C21H21O5N3 395.1481 (Mþ),
found 395.1480.
2-Cyano-5-[4-(2-hydroxy-ethyl)-4-methyl-2-oxo-1,4-dihydro-
2H-benzo[d][1,3]oxazin-6-yl]-pyrrole-1-carboxylic Acid tert-Butyl
Ester (12). To a solution of aldehyde 11 (138 mg, 0.35 mmol) in
MeOH (3 mL) at RT was added NaBH4 (39 mg, 1.05 mmol). The
reactionmixture was stirred for 2 h at RT. The mixture was added
5 mL of water and extracted with EtOAc several times. The
combined organic layer was washed with brine, dried MgSO4,
and concentrated in vacuo. Purified by flash chromatography
(40-60% EtOAc/hexane) to give 12 as a white solid (88% yield)
that was recrystallized from EtOAc/hexane, mp 145-147 °C. 1H
NMR (500 MHz, CDCl3) δ 1.52 (s, 9H), 1.75 (s, 3H), 2.22-2.33
(m, 2H), 3.80 (m, 2H), 6.23 (d, 1H, J = 4.0 Hz), 6.91 (d, 1H, J =
8.0 Hz), 6.97 (d, 1H, J = 4.0 Hz), 7.09 (d, 1H, J = 1.5 Hz), 7.22
(dd, 1H, J = 8.0, 1.5 Hz), 9.40 (brs, 1H). 13C NMR (125 MHz,
CDCl3) δ 27.59, 27.82, 43.05, 58.47, 85.08, 87.62, 105.80, 113.55,
114.21, 124.43, 124.72, 124.81, 127.76, 130.00, 134.55, 139.78,
147.43, 152.69. MS m/e 397 (Mþ, 70%). HRMS (EI) calcd for
C21H23O5N3 397.1638 (Mþ), found 397.1635.
2-(4-Allyl-4-methyl-2-oxo-1,4-dihydro-2H-benzo[d][1,3]oxazin-
6-yl)-5-cyano-pyrrole-1-carboxylic Acidtert-ButylEster(10). Toa
solution of benzo[d]oxazines 9 (700 mg, 1.900 mmol) in dried
THF (50 mL) was added chlorosulfonyl isocyanate (202 μL,
2.280 mmol) at -78 °C under N2 atmosphere and stirred for 2 h.
DMF (1 mL) was added to the reaction mixture, which allowed
warmed to RT for 1 h. The reaction mixture was added to water
(200 mL) and extracted with EtOAc (30 mL ꢀ 3). The combined
organic layer was washed with aq NH4Cl (50 mL) and dried over
Na2SO4. The resulting solution was concentrated by rotary
evaporator. The residue was purified by flash column chroma-
tography on silica gel (40% EtOAc/hexane) to obtain nitrilated
benzo[d ]oxazines 10 (478 mg, 64%) as a white solid, mp:
5-[4-(2-Hydroxy-ethyl)-4-methyl-2-oxo-1,4-dihydro-2H-benzo-
[d][1,3]oxazin-6-yl]-1H-pyrrole-2-carbonitrile (13). 2-Cyano-
5-[4-(2-hydroxy-ethyl)-4-methyl-2-oxo-1,4-dihydro-2H-benzo[d]-
[1,3]oxazin-6-yl]-pyrrole-1-carboxylic acid tert-butyl ester (12)
(397 mg, 1.0 mmol) was placed in a 25 mL round-bottomed flask
stopped with a rubber septum and equipped with nitrogen inlet
and a needle to allow gaseous outflow. A vigorous flow of
nitrogen was maintained as the flask was placed in an oil bath
and heated to 160 °C. After 20 min at this temperature, the flask
was removed from the oil bath and allowed to cool. The yellow
residue was purified by flash chromatography (40-60% EtOAc/
hexane) to give 13 as a yellow powder (169 mg, 57%) that was
1
1
recrystallized from EtOAc/hexane, mp 241-242 °C. H NMR
139.5-140.5 °C. H NMR (500 MHz, CDCl3) δ 1.51 (s, 9H),
(500 MHz, acetone-d6) δ 1.74 (s, 3H), 2.19-2.24 (m, 1H), 2.31-
2.33 (m, 1H), 3.63-3.70 (m, 2H), 6.66-6.67 (m, 1H), 6.94-6.95
(m, 1H), 7.05 (d, 1H, J = 8.5 Hz), 7.66 (dd, 1H, J = 8.5, 2.0 Hz),
7.71-7.72 (m, 1H), 9.26 (brs, 1H), 11.62 (brs, 1H). 13C NMR (125
MHz, acetone-d6) δ 26.74, 43.15, 57.33, 83.52, 101.24, 106.96,
114.45, 114.92, 114.99, 120.90, 120.99, 125.84, 126.11, 135.24,
137.31, 150.23. MS m/e 297 (Mþ, 90%), HRMS (EI) calcd for
C16H15O3N3 297.1113 (Mþ), found 297.1112.
5-[4-(2-Fluoro-ethyl)-4-methyl-2-oxo-1,4-dihydro-2H-benzo[d]-
[1,3]oxazin-6-yl]-1H- pyrrole-2-carbonitrile (14). A one-necked
round-bottom flask which contained 5-[4-(2-hydroxy-ethyl)-4-
methyl-2-oxo-1,4-dihydro-2H-benzo[d][1,3]oxazin-6-yl]-1H-pyr-
role-2-carbonitrile (13) (297 mg, 1 mmol) in CH2Cl2 (5 mL) was
cooled to -78 °C under nitrogen atmosphere. Diethylamino-
sulfur trifluoride (232 mg, 1.2 mmol) was dropped very slowly by
syringe at -78 °C. The cooling was removed, and the reaction
mixture was stirred for 1 h at RT. The reaction mixture was
1.72 (s, 3H), 2.66-2.75 (m, 2H), 5.13 (d, 1H, J = 2.0 Hz), 5.15 (s,
1H), 5.70-5.79 (m, 1H), 6.23 (d, 1H, J = 3.5 Hz), 6.91 (d, 1H,
J = 8.5 Hz), 6.97 (d, 1H, J = 3.5 Hz), 7.05 (d, 1H, J = 1.5 Hz),
7.22 (dd, 1H, J = 8.0 Hz, 2.0 Hz), 9.38 (s, 1H). 13C NMR
(125 MHz, CDCl3) δ 26.2, 27.5, 45.5, 84.7, 87.1, 105.5, 113.3,
113.8, 114.2, 120.2, 124.1, 124.4, 124.8, 127.4, 129.7, 131.1, 134.4,
139.6, 147.1, 152.6. MS (FAB): m/e 494.2 (M þ H)þ, 307.2, 298.1,
154.2 (100), 136.1, 106.8. EA calcd for C 67.16, H 5.89, N 10.68;
found C 66.43, H 5.82, N 10.47.
2-Cyano-5-[4-methyl-2-oxo-4-(2-oxo-ethyl)-1,4-dihydro-2H-
benzo[d][1,3]oxazin-6-yl]-pyrrole-1-carboxylic acid tert-butyl
ester (11). To a solution of 2-(4-allyl-4-methyl-2-oxo-1,4-dihydro-
2H-benzo[d][1,3]oxazin-6-yl)-5-cyano-pyrrole-1-carboxylic acid tert-
butyl ester 10 (770 mg, 1.96 mmol) in a mixed solvent of THF/water
(20 mL/2 mL) was added N-methylmorpholine-N-oxide (NMO,
276mg,2.35mmol),followedbyasolutionof4%aqOsO4 (0.25mL,
0039 mmol). After the solution was stirred at RT for 5 h, solid

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3357
cooled again to -78 °C, and MeOH (200 μL) was added. The
solution was additionally stirred for 30 min after removal of the
dry ice bath, and the reaction was quenched by saturated
NaHCO3 solution and extracted with excess EtOAc. The organic
layer was dried over Na2SO4 and concentrated. Purified by flash
chromatography (40-60% EtOAc/hexane) to give 14 as a yellow
solid (57% yield) that was recrystallized from EtOAc/hexane,
mp 96-98 °C. 1H NMR (500 MHz, acetone-d6) δ 1.76 (s, 3H),
2.41-2.56 (m, 2H), 4.50-4.72 (m, 2H), 6.68 (d, 1H, J = 4.0 Hz),
6.95 (d, 1H, J = 4.0 Hz), 7.07 (d, 1H, J = 8.5 Hz), 7.69 (dd, 1H,
J = 8.5, 2.0 Hz), 7.76 (d, 1H, J = 2.0 Hz), 9.32 (brs, 1H), 11.60
(brs, 1H). 13C NMR (125 MHz, acetone-d6) δ 26.58, 40.45 (d, 1C,
J = 20.4 Hz), 79.82 (d, 1C, J = 163.3 Hz), 82.71, 101.29, 107.04,
114.43, 115.11, 119.55, 120.89, 120.99, 125.63, 126.06, 126.25,
135.16, 155.82. MS m/e 299 (Mþ, 80%). HRMS (EI) calcd for
C16H14O2N3F 299.1070, found 299.1070.
To a solution of 4-allyl-6-bromo-4-methyl-1,4-dihydro-benzo[d]-
[1,3]oxazin-2-one (8) (983.5 mg, 3.50 mmol) in THF (30 mL) at
RT was added 9-borabicyclo[3.3.1]nonane (9-BBN; 21 mL, 0.5 M
solution in hexane, 10.50 mmol). The reaction mixture was stirred for
20 h. To the mixture were added, successively, EtOH (0.64 mL), 6 N
NaOH (0.215 mL), and 30% H2O2 (0.426 mL). The mixture was
heated at 50 °C for 1 h, cooled to RT, and extracted with EtOAc
several times. The combined organic layer was washed with brine,
dried over MgSO4, and concentrated in vacuo. Compound 18 was
purified by flash chromatography (40-60% EtOAc/hexane) to
1
give light-yellow oil (89% yield). H NMR (500 MHz, CDCl3) δ
1.63 (s, 3H), 1.75-2.01 (m, 4H), 3.51-3.52(m, 2H), 6.75(d, 1H, J=
8.0 Hz), 7.16 (d, 1H, J = 2.0 Hz), 7.28 (dd, 1H, J = 8.0, 2.0 Hz). 13
C
NMR (125 MHz, CDCl3) δ 26.99, 27.50, 35.78, 56.77, 84.93, 115.43,
116.67, 127.06, 132.45, 133.67, 152.77. MS m/e 301 (Mþþ2), 299
(Mþ), 240 (100), 233, 224, 145, 130. HRMS (EI) calcd for
C12H14O3N79Br 299.0157 (Mþ), found 299.0153.
5-[4-(2-Fluoro-ethyl)-4-methyl-2-oxo-1,4-dihydro-2H-benzo[d]-
[1,3]oxazin-6-yl]-1- methyl-1H-pyrrole-2-carbonitrile (15). To a
solution of 5-[4-(2-fluoro-ethyl)-4-methyl-2-oxo-1,4-dihydro-2H-
benzo[d][1,3]oxazin-6-yl]-1H-pyrrole-2-carbonitrile (14) (68 mg,
0.23 mmol) in anhydrous DMF (5 mL) was added potassium
carbonate (158 mg, 1.1 mmol) followed by methyl iodide (0.0129
mL, 0.21 mmol). The reaction mixture was stirred for 16 h at RT.
The reaction mixture was poured into water (10 mL) and
extracted with EtOAc (2 ꢀ 20 mL). The combined organic layer
was washed with brine (10 mL), dried over MgSO4, and concen-
trated in vacuo. Purification by flash chromatography (20-40%
EtOAc/hexane) gave 15 as a white solid (90% yield) that was
6-Bromo-4-(3-fluoro-propyl)-4-methyl-1,4-dihydro-benzo[d]-
[1,3]oxazin-2-one (19). Compound 19 was synthesized using the
procedure similar to that described for compound 14. Purifica-
tion by flash chromatography (10-30% EtOAc/hexane) gave a
light-yellow oil (70% yield). 1H NMR (500 MHz, CDCl3) δ 1.70
(s, 3H), 1.73-1.86 (m, 2H), 2.07-2.18 (m, 2H), 4.40 (t, 1H, J =
5.8, 47.5 Hz), 4.50 (t, 1H, J = 5.8, 47.5 Hz), 6.77 (d, 1H, J =
8.0 Hz), 7.20 (d, 1H, J = 2.0 Hz), 7.35 (dd, 1H, J = 8.0, 2.0 Hz),
9.46 (brs, 1H). 13C NMR (125 MHz, CDCl3) δ 25.08 (d, 1C, J =
20.4 Hz), 27.50, 36.78 (d, 1C, J = 3.9 Hz), 83.77 (d, 1C, J = 165.3
Hz), 84.93, 116.23, 116.66, 126.96, 132.25, 133.57, 152.71 (one
carbon missing as a result of overlap). MS m/e 303 (Mþþ2), 301
(Mþ), 240 (100), 224, 210, 196, 158, 143, 130. HRMS (EI) calcd
for C12H13O2N79BrF 301.0114, found 301.0115.
2-[4-(3-Fluoro-propyl)-4-methyl-2-oxo-1,4-dihydro-2H-benzo-
[d][1,3]oxazin-6-yl]- pyrrole-1-carboxylic Acid tert-Butyl Ester
(20). A solution of 6-bromo-4-(3-fluoro-propyl)-4-methyl-1,4-
dihydro-benzo[d][1,3]oxazin-2-one (19) (602 mg, 2 mmol) and
tetrakis(triphenylphosphine)palladium (0) (58 mg, 0.05 mmol)
in toluene (20 mL) was stirred under a flow of nitrogen for 25
min. To the solution was added sequentially 1-tert-butoxycar-
bonylpyrrole-2-boronic acid (824 mg, 3.9 mmol) in absolute
EtOH (5 mL) and potassium carbonate (539 mg, 3.9 mmol) in
water (5 mL). The mixture was heated to 80 °C for 16 h and
allowed to cool. The reaction mixture was poured into aqueous
saturated sodium bicarbonate solution (20 mL) and extracted
with EtOAc (3 ꢀ 20 mL). The organic layers were combined,
washed with water (20 mL) and brine (10 mL), and dried over
MgSO4. The solution was filtered, concentrated in vacuo, and
the residue was purified by flash chromatography (10-30%
EtOAc/hexane) to give 20 as a light-yellow solid (72% yield) that
was recrystallized from EtOAc/hexane, mp 87-89 °C. 1H NMR
(500 MHz, CDCl3) δ 1.41 (s, 9H), 1.72 (s, 3H), 1.81-1.90 (m, 2H),
2.08-2.21 (m, 2H), 4.39 (t, 1H, J = 7.5, 47.5 Hz), 4.48 (t, 1H, J =
7.5, 47.5 Hz), 6.15-6.15 (m, 1H), 6.19-6.22 (m, 1H), 6.90 (d, 1H,
J = 8.0 Hz), 7.08-7.10 (m, 1H), 7.22 (d, 1H, J = 8.0 Hz),
7.31-7.32 (m, 1H), 9.69 (brs, 1H). 13C NMR (125 MHz, CDCl3)
δ 25.75 (d, 1C, J = 20.4 Hz), 27.77, 27.96, 36.85 (d, 1C, J = 4.9
Hz), 83.97 (d, 1C, J = 164.3 Hz), 83.93, 85.51, 110.84, 114.32,
114.74, 122.83, 123.57, 124.88, 129.97, 130.18, 133.58, 134.37,
149.33, 153.28. MS m/e 388 (Mþ, 20%). HRMS (EI) calcd for
C21H25O4N2F 388.1798 (Mþ), found 388.1802.
1
recrystallized from EtOAc/hexane, mp 160-162 °C. H NMR
(500 MHz, CDCl3) δ 1.79 (s, 3H), 2.38-2.51 (m, 2H), 3.71 (s, 3H),
4.51-4.74 (m, 2H), 6.19 (d, 1H, J = 4.0 Hz), 6.85 (d, 1H, J = 4.0
Hz), 6.90 (d, 1H, J = 8.0 Hz), 7.17 (m, 1H), 7.29 (dd, 1H, J = 8.0,
2.0 Hz), 8.06 (brs, 1H). 13C NMR (125 MHz, CDCl3) δ 26.58,
37.05, 41.33 (d, 1C, J = 20.4 Hz), 79.67 (d, 1C, J = 163.6 Hz),
83.01, 101.56, 106.98, 114.41, 115.17, 119.67, 121.03, 121.10,
125.37, 125.98, 126.12, 134.98, 155.62. MS m/e 313 (Mþ, 65%),
HRMS (EI) calcd for C17H16O2N3F 313.1227 (Mþ), found
313.1225.
5-[4-(2-Fluoro-ethyl)-4-methyl-2-thioxo-1,4-dihydro-2H-benzo-
[d][1,3]oxazin-6-yl]-1-methyl-1H-pyrrole-2-carbonitrile (16). Com-
pound 16 was synthesized using the procedure similar to that
described for compounds 5. Purification by preparative thin-layer
chromatography (30% EtOAc/hexane) gave a light-yellow solid
(65% yield), mp 172-174 °C. 1H NMR (500 MHz, acetone-d6) δ
2.36-2.56 (m, 2H), 3.73 (s, 3H), 4.49-4.78 (m, 2H), 6.21 (d, 1H,
J = 4.0 Hz), 6.86 (d, 1H, J = 4.0 Hz), 6.91 (d, 1H, J = 8.0 Hz),
7.20 (brs, 1H), 7.33-7.34 (m, 1H), 9.13 (brs, 1H). 13C NMR
(125 MHz, acetone-d6) δ 27.89, 37.45, 41.24 (d, 1C, J = 20.4 Hz),
80.66 (d, 1C, J = 163.6 Hz), 83.03, 101.55, 107.01, 114.43, 115.13,
119.78, 119.89, 121.56, 125.56, 126.12, 126.23, 135.00, 159.62. 19
F
NMR (376 MHz, acetone-d6) δ -218.56. MS m/e 329 (Mþ, 55%).
HRMS (EI) calcd for C17H16ON3SF 329.0998 (Mþ), found
329.0996.
5-[4-(2-Fluoro-ethyl)-4-methyl-2-thioxo-1,4-dihydro-2H-benzo-
[d][1,3]oxazin-6-yl]-1H-pyrrole-2-carbonitrile (17). Compound 17
was synthesized using the procedure similar to that described for
compounds 5. Purified by preparative thin-layer chromato-
graphy (10-35% EtOAc/hexane) to give a light-yellow solid (75%
yield), mp 104-106 °C. 1H NMR (500 MHz, acetone-d6) δ 1.82
(s, 3H), 2.40-2.58 (m, 2H), 4.51-4.76 (m, 2H), 6.73 (dd, 1H, J =
4.0, 2.0 Hz), 6.96 (dd, 1H, J = 4.0, 2.0 Hz), 7.75 (dd, 1H, J = 8.0,
2.0 Hz), 7.81 (d, 1H, J = 2.0 Hz), 10.96 (brs, 1H), 11.66 (brs, 1H).
13C NMR (125 MHz, acetone-d6) δ 25.98, 40.42 (d, 1C, J =
20.4 Hz), 80.32 (d, 1C, J = 163.3 Hz), 82.45, 101.12, 106.98,
114.22, 115.19, 119.95, 120.56, 121.09, 125.56, 126.09, 126.20,
134.16, 161.12. 19F NMR (376 MHz, acetone-d6) δ -218.73. MS
m/e 315 (Mþ, 20%). HRMS (EI) calcd for C16H14ON3SF
315.0842 (Mþ), found 315.0842.
2-Cyano-5-[4-(3-fluoro-propyl)-4-methyl-2-oxo-1,4-dihydro-2H-
benzo[d][1,3]oxazin-6-yl]-pyrrole-1-carboxylic Acid tert-Butyl Ester
(21). To a solution of 2-[4-(3-fluoro-propyl)-4-methyl-2-oxo-1,4-
dihydro-2H-benzo[d][1,3]oxazin-6-yl]-pyrrole-1-carboxylic acid
tert-butyl ester (20) (225 mg, 0.58 mmol) in THF (anhydrous,
5 mL) at -78 °C was added chlorosulfonyl isocyanate (0.066 mL,
0.67 mmol). After 90 min, DMF (0.9 mL, 11.6 mmol) was added,
and the reaction was allowed to warm to RT. The reaction
mixture waspouredintowater (10mL) andextracted withEtOAc
(2 ꢀ 20 mL). The organic layers were combined, washed with
brine (10 mL), dried over MgSO4, filtered, and concentrated in
Synthesis of Fluorine Compounds 24 and 25. 6-Bromo-4-(3-
hydroxy-propyl)-4-methyl-1,4-dihydro-benzo[d][1,3]oxazin-2-one (18).

3358 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8
Zhou et al.
vacuo. The residue was purified by flash chromatography
(10-30% EtOAc/hexane) to give 21 as a light-yellow solid
(76% yield) that was recrystallized from EtOAc/hexane, mp
87-89 °C. H NMR (500 MHz, CDCl3) δ 1.53 (s, 9H), 1.75
thin-layer chromatography (30% EtOAc/hexane) to give a
light-yellow solid (78% yield) that was recrystallized from
EtOAc/hexane, mp 103-105 °C. 1H NMR (500 MHz, acetone-
d6) δ 1.78 (s, 3H), 2.01-2.25 (m, 4H), 4.41-4.44 (m, 1H),
4.50-4.53 (m, 1H), 6.71 (dd, 1H, J = 4.0, 1.0 Hz), 6.96 (dd,
1H, J = 4.0, 1.0 Hz), 7.21 (d, 1H, J = 8.5 Hz), 7.73-7.76(m, 2H),
9.60 (brs, 1H), 11.62 (brs, 1H). 13C NMR (125 MHz, CDCl3) δ
25.08 (d, 1C, J = 20.3 Hz), 25.97, 36.33 (d, 1C, J = 4.0 Hz), 83.57
(d, 1C, J = 162.3 Hz), 85.85, 107.49, 114.67, 115.08, 120.76,
121.04, 125.99, 126.21, 127.88, 132.21, 135.21, 136.91, 160.56. MS
m/e 329 (Mþ, 15%). HRMS (EI) calcd for C17H16ON3SF
329.0998 (Mþ), found 329.0998.
Receptor Binding Affinity Assays. Relative binding affinities
for PR were determined by a competitive radiometric binding
assay as previously described,19,30 using 10 nM [3H]R5020 as
tracer ([17R-methyl-3H]-promegestone) (Perkin-Elmer, Boston,
MA), unlabeled R5020 as standard, and purified full length
progesterone receptor B from PanVera/Invitrogen (Carlsbad,
CA). Comparable experiments with AR30,45 used [3H]R1881
([17R-methyl-3H]-methyltrienolone) (Perkin-Elmer, Boston,
MA), unlabeled R1881, and purified recombinant rat androgen
receptor ligand binding domain (Pan Vera/Invitrogen, Carlsbad,
CA), and with GR,46 3H Dexamethasone ([1,2,4-3H]-dexametha-
sone (Amersham Biosciences, Piscataway, NJ), unlabeled dexa-
methasone, and adrenalectomized male rat liver cytosol. The
protein was incubated for buffer or several concentrations of
competitor for 18-24 h at 0 °C. Hydroxyapatite (BioRad,
Hercules, CA) was used to absorb the receptor-ligand com-
plexes, and free ligand was removed by washing with cold buffer.
The binding affinities are expressed as relative binding affinity
values with the RBA of the standard set to 100%. The values
given are the average ( range or SD of two or more independent
determinations. R5020 binds to PR with a KD of 0.4 nM, R1881
to AR with a KD of 0.6 nM, and Dexamethasone to GR with a KD
of 19 nM.
Incubations were for 18-24 h at 0 °C. Hydroxyapatite
(BioRad, Hercules, CA) was used to absorb the receptor-ligand
complexes, and free ligand was removed by washing with cold
buffer. The binding affinities are expressed as relative binding
affinity values with the RBA of R5020 set to 100%. The values
given are the average ( range or SD of two or more independent
determinations. R5020 binds to PR with a KD of 0.4 nM.
Molecular Modeling. Procedure for Docking Tanaproget (1)
and Analogues. The PR structure was obtained from the PDB
databank (1zuc) and prepared using the Molecular Operating
Environment (MOE). Explicit hydrogen atoms were added,
partial charges were computed using the MMFF94x force field,
and the receptor-ligand complex was minimized with a termi-
nation gradient of 0.5. All water molecules were then deleted
except the single water molecule hydrogen bound to Gln725 and
Arg766. Finally, the ligand was removed and the receptor
structure was processed using AutoDock Tools47 to define the
AD4 atom types and calculate Gasteiger charges.
1
(s, 3H), 1.78-1.88 (m, 2H), 2.07-2.13 (m, 1H), 2.16-2.22 (m,
1H), 4.40 (t, 1H, J = 5.75, 47.0 Hz), 4.49 (t, 1H, J = 5.75, 47.0
Hz), 6.24 (d, 1H, J = 3.5 Hz), 6.90 (d, 1H, J = 8.0 Hz), 6.97 (d,
1H, J = 3.5 Hz), 7.07 (d, 1H, J = 1.5 Hz), 7.23 (dd, 1H, J = 8.0,
1.5 Hz), 9.05 (brs, 1H). 13C NMR (125 MHz, CDCl3) δ 25.14 (d,
1C, J = 20.4 Hz), 27.66, 27.80, 36.81 (d, 1C, J = 4.0 Hz), 83.74 (d,
1C, J = 165.3 Hz), 85.38, 87.53, 105.85, 113.55, 114.19, 114.53,
124.21, 124.67, 124.88, 127.86, 129.97, 134.66, 139.74, 147.34,
152.49. 19F NMR (376 MHz, acetone-d6) δ -219.52. MS m/e 413
(Mþ, 75%). HRMS (EI) calcd for C22H24O4N3F 413.1551 (Mþ),
found 413.1750.
5-[4-(3-Fluoro-propyl)-4-methyl-2-oxo-1,4-dihydro-2H-benzo-
[d][1,3]oxazin-6-yl]-1H-pyrrole-2-carbonitrile (22). Compound
22 was synthesized using a procedure similar to that described
for compound 13. The desired compound was purified by flash
chromatography (40-60% EtOAc/hexane) to give 22 as a light-
yellow solid (68% yield) that was recrystallized from EtOAc/
hexane, mp 150-152 °C. 1H NMR (400 MHz, acetone-d6) δ 1.72
(s, 3H), 1.72-1.82 (m, 2H), 2.02-2.10 (m, 1H), 2.19-2.24 (m,
1H), 4.40 (t, 1H, J = 7.5, 48.0 Hz), 4.52 (t, 1H, J = 7.5, 48.0 Hz),
6.66 (dd, 1H, J = 4.0, 2.0 Hz), 6.94 (dd, 1H, J = 4.0, 2.0 Hz),
7.06 (d, 1H, J = 8.0 Hz), 7.66-7.71 (m, 2H), 9.31 (brs, 1H),
11.58 (brs, 1H). 13C NMR (100 MHz, acetone-d6) δ 25.12 (d, 1C,
J = 24.8 Hz), 26.58, 36.42 (d, 1C, J = 5.6 Hz), 83.50 (d, 1C, J =
163.1 Hz), 84.47, 101.25, 107.01, 114.47, 115.06, 120.90, 121.02,
125.68, 125.89, 126.22, 135.38, 137.25, 150.26. MS m/e 313 (Mþ,
70%). HRMS (EI) calcd for C17H16O2N3F 313.1227 (Mþ),
found 313.1225.
5-[4-(3-Fluoro-propyl)-4-methyl-2-oxo-1,4-dihydro-2H-benzo-
[d][1,3]oxazin-6-yl]-1-methyl-1H-pyrrole-2-carbonitrile (23). Com-
pound 23 was synthesized using a procedure similar to that
described for compound 15. The desired product was purified
by preparative thin-layer chromatography (10-35% EtOAc/
hexane) to give 23 as a light-yellow solid (95% yield) that was
1
recrystallized from EtOAc/hexane, mp 150-152 °C. H NMR
(500 MHz, CDCl3) δ 1.74 (s, 3H), 1.81-1.90 (m, 2H), 2.09-2.17
(m, 1H), 2.21-2.24 (m, 1H), 3.72 (s, 3H), 4.40-4.43 (m, 1H),
4.50-4.52 (m, 1H), 6.19 (d, 1H, J = 4.0 Hz), 6.85 (d, 1H, J =
4.0 Hz), 6.97 (d, 1H, J = 8.0 Hz), 7.10 (d, 1H, J = 1.5 Hz),
7.27-7.28 (m, 1H), 9.36 (brs, 1H). 13C NMR (125 MHz, CDCl3) δ
25.23 (d, 1C, J = 24.8 Hz), 27.77, 33.94, 36.72 (d, 1C, J = 5.6 Hz),
83.64 (d, 1C, J = 164.0 Hz), 85.42, 105.96, 110.01, 114.07, 114.34,
115.33, 124.79, 125.02, 126.95, 129.96, 134.75, 139.23, 161.46. MS
m/e 327 (Mþ, 65%). HRMS (EI) calcd for C18H18O2N3F
327.1383 (Mþ), found 327.1381.
5-[4-(3-Fluoro-propyl)-4-methyl-2-thioxo-1,4-dihydro-2H-benzo-
[d][1,3]oxazin-6-yl]-1-methyl-1H-pyrrole-2-carbonitrile (24). Com-
pound 24 was synthesized using the procedure similar to that
described for compounds 5. The desired product was purified by
preparative thin-layer chromatography (30% EtOAc/hexane) to
give a light-yellow solid (71% yield) that was recrystallized from
EtOAc/hexane, mp 72-74 °C. 1H NMR (500 MHz, CDCl3) δ 1.77
(s, 3H), 1.77-1.90 (m, 2H), 2.14-2.19 (m, 1H), 2.22-2.27 (m,
1H), 3.73 (s, 3H), 4.40-4.43 (m, 1H), 4.49-4.52 (m, 1H), 6.21 (d,
1H, J = 4.0 Hz), 6.86 (d, 1H, J = 4.0 Hz), 6.99-7.03 (m, 1H),
7.12-7.13 (m, 1H), 7.31-7.33 (m, 1H), 9.81 (brs, 1H). 13C NMR
(125 MHz, CDCl3) δ 25.28 (d, 1C, J = 20.4 Hz), 27.23, 34.03,
36.64 (d, 1C, J = 3.9 Hz), 83. 51 (d, 1C, J = 165.3 Hz), 87.08,
106.41, 110.30, 114.17, 114.38, 115.21, 119.85, 124.77, 128.72,
130.03, 134.39, 138.76, 161.28. 19F NMR (376 MHz, acetone-d6)
δ -219.76. MS m/e 343 (Mþ, 100%). HRMS (EI) calcd for
C18H18ON3SF 343.1155 (Mþ), found 343.1155.
All compounds in Table 1 were constructed in Sybyl 8.1.1,
cleaned up using the built-in Concord module,48 and minimized
using the Powell method with a termination gradient of
0.5 kcal/(mol A), 100K maximum iterations, and MMFF94
force fields and charges. The ligands were prepared for docking
using AutoDock Tools to assign AD4 atom types, calculate
Gasteiger charges, and set all rotatable bonds as active torsions.
Each ligand was docked into the receptor using AutoDock Vina.
The grid box was centered on the ligand in the original crystal
˚
˚
˚
structure and measured 18 A by 18 A by 22 A. To ensure that the
proper binding conformation was found, the exhaustiveness
parameter was set to 100 (default = 8, linear scale); all other
default settings were used.
5-[4-(3-Fluoro-propyl)-4-methyl-2-thioxo-1,4-dihydro-2H-benzo-
[d][1,3]oxazin-6-yl]-1H-pyrrole-2-carbonitrile (25). Compound 25
was synthesized using a procedure similar to that described for
compounds 5. The desired product was purified by preparative
The top five poses for each ligand were visually inspected in
MOE. Unreasonable poses were discarded, and the lowest
energy conformation of the remaining poses was selected for
further use. The selected pose and receptor structure were

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 8 3359
merged into a single file, all hydrogen atoms were explicitly
added, the partial charges were calculated using the MMFF94x
force field, and the protein-ligand complex was minimized. The
minimization was conducted in four stages: stage 1 minimized
the hydrogen atoms that were added to the structure in the
previous step, stage 2 minimized the ligand, stage 3 minimized
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˚
any residues within 4.5 A of the ligand, and stage 4 minimized
˚
both the ligand and any residues within 4.5 A. This worked up
structure was used for all other calculations and analyses.
Procedure for Calculating the Torsion Energetics of N-Sub-
stituted Analogues (Figure 1). The worked up structure was
opened in MOE and all atoms deleted except for the ligand.
The dihedral angle relating the pyrrole to the benzoxazin-2-
thione core was rotated from 0° to 90°, measuring each energy
term in 0.5° increments. The relative value of each term was
calculated by subtracting the minimum measured value.
Procedure for Calculating the Interaction Energy of the Protein-
Ligand Complex (Figure 2B). Similar to the above process, the
pyrrole was rotated with respect to the core by setting the
dihedral angle from 0° to 90°, measuring the interaction energy
in 0.5° increments. To ensure that any observed change in
the interaction energy was due solely to a difference in the
N-substitution, we started from the worked up pose of com-
pound 5a, deleting atoms as necessary to obtain N-Me (1) and
N-H (analogue of 1). The partial charges were recalculated using
MMFF94x force field prior to beginning the rotation.
Procedure for Calculating the Mean Hydrogen Bond Score
(Table 2). The hydrogen bonds formed between the ligand and
protein were scored using the “dock_HydrogenBonds” module
found in the standard scientific vector language (SVL) library
for MOE. This module forms the basis for the “ligand interac-
tions” feature accessible from MOE’s graphical user interface.
The mean score was subsequently calculated as the geometric
mean of the individual scores.
Acknowledgment. Supported by grants from the NIH
(PHS 5R37 DK015556). Funding for NMR and MS instru-
mentation is from the Keck Foundation, NIH, and NSF. We
are grateful to Dr. Sung Hoon Kim for helpful comments.
Supporting Information Available: Normal and reversed-
phase HPLC results and chromatograms of the fluoro-substi-
tuted compounds 5a-b, 16, 17, 24, and 25. This material is
available free of charge via the Internet at http://pubs.acs.org.
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