Discovery of selective and potent inhibitors of gram-positive bacterial thymidylate kinase (TMK)

Article abstract of DOI:10.1021/jm3011806

Thymidylate kinase (TMK) is an essential enzyme in bacterial DNA synthesis. The deoxythymidine monophosphate (dTMP) substrate binding pocket was targeted in a rational-design, structure-supported effort, yielding a unique series of antibacterial agents sh

Full text of DOI:10.1021/jm3011806

Article
pubs.acs.org/jmc
Discovery of Selective and Potent Inhibitors of Gram-Positive
Bacterial Thymidylate Kinase (TMK)
Gabriel Martínez-Botella,*,†,∥ John N. Breen,†,‡ James E. S. Duffy,§ Jacques Dumas,† Bolin Geng,†
Ian K. Gowers,§ Oluyinka M. Green,† Satenig Guler,† Martin F. Hentemann,† Felix A. Hernandez-Juan,§
Diane Joseph-McCarthy,† Sameer Kawatkar,† Nicholas A. Larsen,‡ Ovadia Lazari,§ James T. Loch,†
Jacqueline A. Macritchie,§ Andrew R. McKenzie,† Joseph V. Newman,† Nelson B. Olivier,‡
Linda G. Otterson,† Andrew P. Owens,§ Jon Read,‡ David W. Sheppard,§ and Thomas A. Keating†
†AstraZeneca Infection Innovative Medicines, 35 Gatehouse Drive, Waltham, Massachusetts 02451, United States
‡AstraZeneca Discovery Sciences, 35 Gatehouse Drive, Waltham, Massachusetts 02451, United States
§BioFocus, Chesterford Research Park, Saffron Walden CB10 1XL, U.K.
S
* Supporting Information
ABSTRACT: Thymidylate kinase (TMK) is an essential
enzyme in bacterial DNA synthesis. The deoxythymidine
monophosphate (dTMP) substrate binding pocket was
targeted in a rational-design, structure-supported effort,
yielding a unique series of antibacterial agents showing a
novel, induced-fit binding mode. Lead optimization, aided by
X-ray crystallography, led to picomolar inhibitors of both
Streptococcus pneumoniae and Staphylococcus aureus TMK.
MICs < 1 μg/mL were achieved against methicillin-resistant S. aureus (MRSA), S. pneumoniae, and vancomycin-resistant
Enterococcus (VRE). Log D adjustments yielded single diastereomers 14 (TK-666) and 46, showing a broad antibacterial
spectrum against Gram-positive bacteria and excellent selectivity against the human thymidylate kinase ortholog.
high-quality inhibitors of Gram-positive bacterial TMK employ-
ing a structure-guided approach.
INTRODUCTION
■
The discovery of novel antibacterial classes has proven
particularly challenging for the research community in both
industry and academia in the last few decades.1 The onset of
the genomic era and the promise of the discovery of novel
mechanisms have not translated into marketed drugs.2 The
need for new antibiotics to treat drug-resistant Gram-positive
infections is acute, especially against methicillin-resistant
Staphylococcus aureus and Staphylococcus epidermidis and
vancomycin-resistant Enterococcus faecium and Enterococcus
faecalis.3 In this context, structure-guided design approaches4
have yielded some initial success in addressing the resistance of
existing classes, for example the drug candidate Iclaprim,5 and
novel chemical classes of DNA gyrase inhibitors.6 We have
recently reported the first in vivo efficacious inhibitor of
thymidylate kinase (TMK) targeting Gram-positive bacteria.7
TMK is a nucleotide kinase that catalyzes the phosphorylation
of deoxythymidine monophosphate (dTMP) to deoxythymi-
dine diphosphate (dTDP) using ATP as a cosubstrate. This is a
necessary step in the biosynthesis of deoxythymidine
triphosphate (dTTP) for DNA synthesis. While dTMP can
be produced both de novo (from dUMP) and via salvage (from
thymidine), neither it nor dTDP can be imported into the cell
nor biosynthesized through an alternate path. This makes TMK
an essential enzyme and a very attractive target for therapeutic
intervention.8 This report describes the discovery of the first
CHEMISTRY
■
Piperidinylthymine analogs were synthesized as described in
Schemes 1, 2, and 3. The key intermediate, piperidinylthymine
1, was prepared using commercially available racemic- or (R)-
or (S)-tert-butyl 3-aminopiperidine-1-carboxylate (3) and 3-
methoxyacrylic acid (2) (Scheme 1). Standard reductive
amination conditions to couple 1 with benzaldehydes afforded
the analogs described in Tables 1 and 2. Scheme 2 shows the
synthesis for compounds in Table 3. 2-Fluoro-4-formylbenzo-
nitrile (5) and 1 afforded intermediate 6 under reductive
amination conditions using polymer-supported cyanoborohy-
dride. The addition of phenols, followed by the hydrolysis of
the cyano group in 7 gave the desired compounds in moderate
yields. In order to install an aliphatic side chain in the benzylic
carbon bridging the piperidine and the central phenyl ring,
addition of commercially available zincate 9 to acid chlorides
was performed routinely and in high yields (Scheme 3). The
resulting ketone 11a was transformed into mesylate 11b and
then coupled to 1 in moderate yields, though, in some
instances, this reaction led to elimination products. The ketone
Received: August 10, 2012
Published: October 8, 2012
© 2012 American Chemical Society
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Figure 1. The crystal structure of the Staphylococcus aureus thymidylate kinase (TMK) with bound dTMP (left)9 was the starting point for design of
lead inhibitor scaffolds (right). dTMP is depicted in orange, with the terminal phosphate located at the right-hand side of the panel. This phosphate
points in the direction of the substrate channel leading to the ATP binding site (not shown).
a
11a and protected 3-aminopiperidine could alternately be
coupled under standard reductive amination conditions and the
thymine ring built as described in Scheme 1. Installation of the
phenoxy ring was carried out under microwave conditions,
followed by subsequent hydrolysis of the benzonitrile under
aqueous basic conditions (Scheme 3). The diastereomeric
mixture 13 was then separated, affording pure diastereomers
(Scheme 3).
Scheme 1. Synthesis of TMK Inhibitors in Tables 1 and 2
RESULTS AND DISCUSSION
■
The X-ray structure of dTMP bound to S. aureus TMK9
provided the starting point for the rational design of thymine-
containing scaffolds (Figure 1). A set of deoxyribose-
replacement ring systems were designed from docking models,
and small libraries of each scaffold were synthesized by coupling
available R groups. These compounds were screened for
inhibition against the S. aureus and S. pneumoniae TMK
enzymes. While nearly all of the compounds synthesized were
inactive, as a result of this effort, thymine-based racemic
compound 15 was discovered (Figure 2). Compound 15 has
a
(a) (i) oxalyl chloride, AgOCN, 95 °C, 3 h; (ii) H2SO4, MeOH, 95
°C, 16 h, 40% overall; (b) 3-phenoxybenzaldehyde, trimethylammo-
nium cyanoborohydride, 22 °C, 16 h, 19%.
placing the phenoxy ring (ring D) into a newly revealed
hydrophobic pocket, created by movement of a helix and a loop
at the left-hand side of the binding pocket.7 This pocket is not
evident in the apo or dTMP structures.9 To verify the insight
gained from the crystal structure, several enantiomeric pairs
(27−30) were synthesized (Table 2), starting with enantio-
merically pure tert-butyl 3-aminopiperidine-1-carboxylate (2)
(Scheme 1). In all cases, the (S)-enantiomer was 12- to 100-
fold more potent against both S. pneumoniae and S. aureus
TMK enzymes than the (R)-enantiomer.
Figure 2. Structure of thymidylate kinase (TMK) racemic lead
inhibitor 15. The rings are lettered A−D for discussion in the text.
With the X-ray structure of (S)-16 in hand, binding was
improved by first modifying ring D. Two approaches were
employed: first, a virtual library of compounds with substituted
phenoxy rings was docked into the binding pocket of S. aureus
TMK.10 Results suggested that a great variety of substitutions
could be tolerated in this pocket. Based on this, a set of
compounds were synthesized and tested. However, little
improvement was observed in the activity compared to the
case of 16. Second, a more traditional Topliss method was also
employed in parallel to the above-mentioned structure-based
approach. The Topliss method11 is based on the assumption
that any substitution will modify the biological activity relative
to the parent compound as a result of changes in hydrophobic,
electronic, and steric effects. This approach resulted in
compounds with significant improvements in enzyme inhib-
ition. In particular, the 3-Cl and 3-Br analogs (22, 23) achieved
more than a 100-fold improvement against both S. pneumoniae
TMK and S. aureus TMK. Such an improvement most likely
resulted from a combination of desolvation and favorable
moderate affinity against S. pneumoniae TMK (IC50 = 3.4 μM)
and low affinity against S. aureus TMK (IC50 = 156 μM), but
given the dearth of other leads, it was pursued in a medicinal
chemistry program. An early survey of the phenoxy ring (Table
1) led to racemic compound 16, which was suitable for protein
crystallography, yielding the first structure of an inhibitor
bound to S. aureus and a distinct binding mode when compared
to dTMP7,9 (Figure 3). The X-ray structure showed the
thymine ring (ring A) retaining its position from dTMP, and
anchoring the inhibitor by creating multiple hydrogen bonds
with residues Arg70, Ser97, and Gln101, and π-stacking with
Phe66. Strikingly, a single enantiomer (S)-16 was found in the
crystal structure, which positions the compound to bend away
from the substrate binding channel rather than following it as
the dTMP substrate does. The piperidine (ring B) in this
enantiomer provided the correct geometry to enable the turn.
The middle phenyl ring (ring C) appeared to act as a spacer,
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Table 1. TMK Inhibition Activity of Analogues of 15
these two species may be attributed to the nature of the
hydrogen bond network formed between protein and
compound. The scaffold presented herein interacts with the
binding site such that rings A and D form a π-stacking
interaction with an aromatic residue: Tyr71 and Phe66 in S.
pneumoniae and S. aureus TMK, respectively.7 We hypothesize
that the hydroxyl group of S. pneumoniae Tyr71 forms a
hydrogen bond to the thymine carbonyl group. As the
hydrogen bonding energy between two uncharged groups is
estimated to be 0.5−1.8 kcal/mol,12 we ascribe the roughly 10-
fold difference in IC50 between the orthologs to the additional
interaction.
In our search for conserved residues across Gram-positive
bacteria, Arg48 was identified as an attractive side chain to
target. By the use of X-ray cocrystal structures and models, we
predicted that placing an acid group at the C-4 of ring C would
be optimal (Figure 4A). When synthesized and assayed, these
acid derivatives showed more than a 20-fold improvement in
IC50 (Table 3). The X-ray cocrystal structure of compound 34
(Figure 4B) showed the formation of two clear hydrogen bonds
between the carboxylate of 34 and the guanidinium of Arg48.
As shown in Table 3, these compounds showed excellent IC50
and MIC values against S. pneumoniae. The enzymatic IC50 and
MIC values for S. aureus were improved, but they remained
more than 10-fold weaker than those of S. pneumoniae.
In order to improve the binding affinity against both S.
pneumoniae and S. aureus TMK, the next area targeted was the
methylene linker between ring B and ring C. Based on the X-
ray structures and models, we hypothesized that adding a
substituent to this carbon should have a positive effect by
stabilizing the U-shaped binding conformation. The addition of
a small group, such as a methyl (35), led to an improvement in
the affinity for S. aureus TMK of more than 10-fold (Table 4).
Although the enzyme affinities for both S. pneumoniae and S.
aureus TMK were now comparable, the MICs for S. aureus were
relatively weaker than those for S. pneumoniae. At this point, we
established the link between enzyme inhibition and observed
MICs through resistant mutant generation, radioactive-
Figure 3. Crystal structure of (S)-16 bound to S. aureus TMK at 2.0 Å.
The green mesh represents the 2Fo − Fc electron density map
contoured to 1 σ. Racemic 16 was soaked with protein crystals, but
only the single enantiomer was found to be bound. Compared to the
binding mode of dTMP (Figure 1), the position and contacts of
thymine ring A are extremely well-conserved. However, piperidinyl
ring B, instead of executing a turn toward the substrate channel (off
right-hand side of panel) instead bends the molecule to the left, into a
newly formed, induced-fit binding pocket for ring D. This pocket is
not evident in the Figure 1 structure. Major changes in protein
structure to create this cryptic pocket include rotation of the Arg48
side chain, movement of the α2 helix (leftmost in figure), and
movement of the connecting loop at the top left of the figure. Phe66
forms a π-stack with rings A and D.
hydrophobic interactions. These inhibitors do, however, show a
higher affinity, ∼10-fold, against S. pneumoniae TMK versus S.
aureus TMK. The difference in compound potency between
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Table 2. TMK Inhibition Activity of Analogues of 4
Table 3. Activity of Analogues of 34
a
IC50 (nM)
MIC (μg/mL)
compd
R
Spn
Sau
log D
Spn SauMSQS/SauMRQR
8
Cl
1.9
1
73
27
−0.55
−0.51
−0.82
−1
1
1
32/32
31
32
33
34
Br
16/32
Me
F
8
114
339
120
4
64/128
128/128
64/64
28
10
32
4
CF3
−0.38
a
Bacterial strains from the AstraZeneca collection. MSQS: methicillin-
and quinolone-sensitive. MRQR: methicillin- and quinolone-resistant.
In an effort to improve whole-cell activity, we examined the
physical properties of the series. S. aureus membrane
permeability appears to be highly dependent on the physical
properties of the molecules. In our experience, there is a good
correlation between log D and the S. aureus MICs, with optimal
whole-cell activity observed for log D values between 1 and 2.10
Profiling of compounds 35−40 (Table 4) showed log D values
between −0.3 and 0.5. In order to increase the log D, a series of
compounds with substituents of increasing size and lipophilicity
at the carbon linker between rings B and C were designed using
calculated log D values and then synthesized (Scheme 3). As
the size of the substituent increased, the (R) configuration of
the new stereocenter became increasingly favored (Table 4,
39−43). The X-ray structure of compound 41 showed that the
binding mode was unchanged compared to our first X-ray
cocrystal structure with all major interactions maintained
(Figure 5). The source of affinity difference between the
(R,S) and (S,S) diastereomers may be explained by the ability
of the linker side chain in the (R) configuration to establish a
hydrophobic interaction with Val51 and Leu52. Under the
crystallization conditions, the enzyme also selectively bound the
(R,S)-diastereomer when the diastereomeric mixture of 41/42
was used.
As shown in Table 4, the MICs against S. aureus improved as
the log D increased from 0 to 2. For example, compound 47
exhibited excellent MICs against S. aureus (0.1−0.03 μg/mL)
and a log D value of 2. On the other hand, compound 51
exhibited only a modest improvement in S. aureus MIC (0.5
μg/mL) in spite of the highest log D (2.5), indicating a limit to
lipophilicity-induced gains in whole-cell activity for this series
(Figure 6).
a
Scheme 2. Synthesis of TMK Inhibitors in Table 3
Compounds 14 (TK-666)7 and 46 in Table 5 were profiled
against pathogens S. aureus, S. pneumoniae, Streptococcus
pyogenes, Staphylococcus epidermidis, and Enterococcus spp.,
showing excellent activity and demonstrating the value of a
novel antibacterial target and mechanism for addressing serious
exisiting clinical resistance mechanisms. Finally, for any novel
antibacterial target with an essential eukaryotic ortholog, it is
critical that selectivity be robust to avoid potential toxicity.
Several compounds were thus tested in the human TMK IC50
assay and showed exquisite (105−106) selectivity against the
human homologue as well as very low potential for cytotoxicity
in both human (A459) and yeast whole-cell assays (Table 6).
Compound 14 was further dosed in a murine S. aureus-infected
thigh model and demonstrated both efficacy (infection stasis at
a
(a) DMF, AcOH, polymer-BCN(H)3, 22 °C, 16 h, 28%; (b) 3-
chlorophenol, K2CO3, NMP, 150 °C, 15 min, 45%; (c) H2SO4, 150
°C, 30 min, 9%.
precursor incorporation assays, and tmk overexpression strains.7
The compounds were found to exert their growth-inhibitory
effect cleanly in both S. aureus and S. pneumoniae through TMK
inhibition.
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Figure 4. Structural characterization of the binding of 34 in S. aureus TMK. The addition of a carboxylate group to ring C was designed to
specifically interact with the side chain of conserved Arg48. This carboxylate drove significant improvements in potency and physical properties. (A)
Computational model of the carboxylate-Arg48 interaction predicted a single hydrogen bond. (B) Crystal structure, resolved to 1.7 Å. The carboxylic
acid interaction with Arg48 is revealed to be bidentate.
a
Scheme 3. Synthesis of TMK Inhibitors in Table 4
a
Pd(PPh3)4, THF; 22 °C, 16 h, 85%; (b) (i) NaBH4, MeOH, 22 °C, 2 h, 29%; (ii) MsCl, TEA, THF, 0 °C, 4 h; (c) (S)-1, 11b, DIEA, CH3CN, 70
°C, 20 h, 13%; (d) (i) 3-bromophenol, K2CO3, NMP, 150 °C, 30 min, 67%; (ii) NaOH, H2O, EtOH, 100 °C, 16 h, 77%; (e) purification/separation
by chiral chromatography, 15%.
150 mg/kg single dose, versus 40 mg/kg for comparator
levofloxacin) and tolerability (no observed adverse effects up to
and including the highest dose of 800 mg/kg/day).7
EXPERIMENTAL SECTION
■
General Experimental Details. All commercial reagents and
anhydrous solvents were obtained from commercial sources and were
used without further purification, unless otherwise specified.
LC-MS conditions: Method 1: Samples were analyzed by reversed
phase LC-MS using Waters Xterra C18 MS 100 mm × 4.6 mm, 5 μm
particle size columns; linear gradient from 5% to 95% acetonitrile in
water (10 mM ammonium hydrogen carbonate) over 5.5 min; flow
rate 2 mL/min; injection volume was 2−7 μL; UV detection via HP or
Waters DAD (210−400 nm range). Detection was based on ESCI in
positive and negative polarity using a Micromass ZQ single quadrapole
LC-MS or Quattro Micro LC-MS-MS, diode-array UV detector from
210 to 400 nm via HP or Waters DAD. Method 2: Samples were
analyzed by reversed phase LC-MS using Varian Polaris C18A, 2 mm
× 50 mm, 3 μm particle size columns. An Agilent HP1100
(Wimington, DE, USA) LC system was used with a gradient elusion
profile of 5−95% B over 4.5 min at 1 mL/min, then re-equilibration at
initial conditions to 6 min. Injection volume was 2 μL and column
temperature 30 °C. Mobile phase A was 0.1% formic acid in water and
mobile phase B 0.1% formic acid in acetonitrile. Detection was based
on electrospray ionization (ESI) in positive and negative polarity using
Waters ZQ mass spectrometer (Milford, MA, USA), diode-array UV
detector from 210 to 400 nm, and evaporative light-scattering detector
(Sedex 75, Sedere, Alfortville Cedex, France). Method 3: As Method 2,
except A was 10 mM ammonium acetate in (5/95 acetonitrile/water)
and B was acetonitrile.
CONCLUSIONS
■
In this report we have illustrated the use of X-ray
crystallography, computational methods, and traditional medic-
inal chemistry approaches to design the first potent, efficacious
inhibitors of Gram-positive bacterial thymidylate kinase. After a
small number of design cycles, the weak lead inhibitor 16 was
rapidly optimized into the highly selective picomolar inhibitor
14. While excellent cellular potency was easily obtained against
S. pneumoniae, improving cellular activity against S. aureus
proved to be more challenging due to the sensitivity of this
species to the physical properties of the inhibitor. By utilizing
the bridge carbon between rings B and C, log D adjustments
were made which allowed us to improve some enzymatic
potency but, more importantly, led to on-target, potent cellular
activity against S. aureus. Compounds 14 and 46 showed
excellent antibacterial spectrum against Gram-positive bacteria
and 105−106 selectivity versus the human thymidylate kinase
homologue. The in vivo target validation provided by
compound 147 against S. aureus provides great impetus for
the development of clinical candidates both in the series and for
the TMK target.
Accurate mass was done using a hybrid quadrupole time-of-flight
mass spectrometer (microTOFQ, Bruker Daltonics) in ESI+ mode.
Method: 5−95% mobile phase B from 0.0 to 5.0 min, hold at 95%
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Table 4. Activity of Single Diastereomer Analogues of 41
a
Bacterial strains from the AstraZeneca collection. MSQS: methicillin- and quinolone-sensitive. MRQR: methicillin- and quinolone-resistant.
mobile phase B to 5.1 min. Mobile phase A: 0.1% formic acid in water,
B: 0.1% formic acid in acetonitrile. 1.0 mL/min flow, column Varian
Polaris C18-A 2.0 mm × 50 mm, 3.0 μm particle size. One μL of
sample was injected onto an HPLC using an externally calibrated
instrument for accurate mass measurement. 50 μL/min eluent from
LC and 50 μL/min make up solution (CH3CN/H2O 50/50) went
into MS.
1H NMR spectra (δ, ppm) were recorded using Bruker Advance
Ultrashield 300 MHz or Bruker DPX 400 MHz instruments.
Column chromatography was performed using Silcycle FLH-
R10030B Silisep cartridges (12−330 g). Preparative reversed phase
HPLC chromatography was carried out using a Waters Atlantis T3-
C18 column, 19 mm × 100 mm, 5 μm, a linear gradient from 10% to
90% CH3CN in H2O over 12 min (0.1% trifluoroacetic acid), and a
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Table 5. Gram-Positive Antibacterial Spectrum of Analogues
14 and 46
MIC (μg/mL)
a
bacterial strain
S. pneumoniae
(R,S)-14
(R,S)-46
0.02
0.01
Streptococcus pyogenes
0.08
0.04
S. aureus MSQS/S. aureus MRQR
Staphylococcus epidermidis
Enterococcus faecium (LRE)
Enterococcus faecalis (VRE)
0.25/0.25
0.25/0.1
0.5
2
0.25
1
0.25
0.06
a
Bacterial strains from AstraZeneca collection. MSQS: methicillin- and
quinolone-sensitive; MRQR: methicillin- and quinolone-resistant;
LRE: linezolid-resistant Enterococcus; VRE: vancomycin-resistant
Enterococcus.
Table 6. Eukaryotic Selectivity for Selected Analogues
IC50 (nM)
MIC (μg/mL)
a
Human TMK
Human A549
Candida albicans
Figure 5. Co-crystal structure of a single diastereomer (R,S)-41 and S.
aureus TMK at 2.0 Å, depicting optimized inhibitor binding to the
enzyme (IC50 = 1.2 nM). Key features are retention of thymine
binding interactions (ring A), hydrophobic packing of the isobutyl
group against conserved hydrophobic residues Val51/Leu52 (linker
between rings B and C), the bidentate salt bridge of the ring C
carboxylate to Arg48, and the cryptic hydrophobic pocket hosting the
chlorophenyl ring D. The π-stack of rings A and D with Phe66 is
maintained.
34
>200000
150000
150000
62000
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
(R,S)-35
(R,S)-39
(R,S)-14
(R,S)-44
(R,S)-46
43400
32000
a
Human A549: Human lung epithelial adenocarcinoma cell line.
of Sau-TMK protein in complex with related ligands were used as a
template for docking studies. The protein structures were prepared
using protein preparation wizard, and the ligands were prepared using
Ligprep utility in Maestro 8.5 (Schrodinger, LLC 2008, New York,
NY). Docked poses were minimized in OPLS 200114 force field as
implemented in Maestro 8.5.
Experimental Procedures and Characterization Data for
Analogues in Tables 1 and 2. 5-Methyl-1-(piperidin-3-yl)-
pyrimidine-2,4(1H,3H)-dione (1).15c Oxalyl chloride (24 mL) was
added to a mixture of (E)-3-methoxy-2-methylacrylic acid15 (2, 42 g,
0.25 mol, 1.0 equiv) in dichloromethane (250 mL) at 0 °C. The
mixture was stirred at room temperature for 1 h. The solvent was
removed under reduced pressure. The resultant residue was
redissolved in toluene (750 mL), and silver cyanate (48.75 g, 0.325
mol, 1.3 equiv) was added. The reaction mixture was stirred at 95 °C
for 3 h and allowed to cool to RT, and the inorganics were filtered.
Filtrate was added slowly to a solution of (S)-tert-butyl 3-amino-
piperidine-1-carboxylate (3, 50 g, 0.25 mol, 1.0 equiv) in dimethyl
formamide (100 mL). The mixture was stirred at room temperature
for 16 h. The solvent was removed under reduced pressure. The
residue was dissolved in diethyl ether (750 mL) and washed with
water (2 × 250 mL), 10% aqueous NaHCO3 (1 × 250 mL), and brine
(1 × 100 mL), dried over Na2SO4, and filtered, and the solvent was
removed under reduced pressure to afford (S)-tert-butyl (3-3-(((2E)-
3-methoxy-2-methylprop-2-enoyl)carbamoyl)amino)piperidine-1-car-
Figure 6. pMIC vs log D relationship for piperidinylthymine analogs
in Table 4. S. aureus appears to be more sensitive to log D variation
than S. pneumoniae, but both reach an MIC optimum with log D
between 1.5 and 1.8.
flow rate of 20 mL/min. Preparative chiral chromatography was
carried out as follows. HPLC: Chiralpak IC column, 30 mm × 250
mm, 5 μm; hexane (60%), methanol/ethanol (1:1) (40%), 0.5%
diethylamine; flow rate 40 mL/min. SFC: Chiralpak AD column, 30
mm × 250 mm, 5 μm; carbon dioxide (60%), isopropanol (40%); flow
rate 120 mL/min. Chiral analytical HPLC: Chiralpak IC column, 4.6
mm × 250 mm, 5 μm; hexane (60%), methanol/ethanol (1:1) (40%),
0.5% diethylamine; flow rate 1 mL/min. Chiral analytical SFC:
Chiralpak AD column, 4.6 mm × 250 mm, 5 μm; carbon dioxide
(60%), isopropanol (40%); flow rate 2.8 mL/min.
1
boxylate, as a pale yellow solid (53 g). H NMR (400 MHz, DMSO-
d6) δ 1.40 (s, 9H), 1.40−165 (m, 3H), 1.65 (s, 3H), 1.83 (m, 1H),
3.24 (m, 2H), 3.43 (m, 2H), 3.70 (m, 1H), 3.83 (s, 3H), 7.50 (s, 1H),
8.81 (d, 1H), 9.78 (s, 1H) ppm. A 1.69 N aqueous solution of sulfuric
acid (800 mL) was added to a mixture of (S)-(E)-tert-butyl 3-(3-(3-
methoxy-2-methylacryloyl)ureido)piperidine-1-carboxylate (53 g,
0.155 mol) and 1,4-dioxane (300 mL). The mixture was stirred at
95 °C for 16 h. The reaction mixture was allowed to cool to room
temperature and slowly neutralized with addition of solid sodium
hydrogen carbonate (200 g). The reaction mixture was concentrated
to dryness, and the residue was extracted with hot methanol. The
methanol was concentrated, and the residue was purified by column
chromatography to afford 1 as a pale yellow solid (20.7 g, 40% overall
The purity of the final compounds was assessed on the basis of
analytical LC-MS, and the results were greater than 95% unless
specified otherwise.
Molecular Modeling. Molecular docking was performed using
Glide version 5.013 in standard flexible docking mode. X-ray structures
yield from 2 steps). H NMR (400 MHz, CD3OD) δ 1.84−1.96 (m,
1
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Article
6H), 2.54 (dt, J = 2.60, 12.66 Hz, 1H), 2.74 (t, J = 11.60 Hz, 1H),
(s, 2H), 4.57 (m, 1H), 6.78 (m, 1H), 6.83 (m, 1H), 6.96 (m, 1H), 7.06
(m, 1H), 7.16 (d, 1H), 7.34 (m, 2H), 7.74 (s, 1H) ppm.
1
2.80−3.10 (m, 3H), 4.43−4.48 (m, 1H), 7.53 (s, 1H). H NMR (400
MHz, DMSO-d6): δ 1.60−1.68 (m, 1H), 1.76 (s, 3H), 1.78−1.85 (m,
3H), 2.54−2.60 (m, 1H), 2.85−2.94 (m, 3H), 2.98−3.06 (m,1H),
4.46−4.54 (m, 1H), 7.66 (s, 1H), 11.20 (br s, 1H) ppm.
(S)-1-(1-(3-(3-Chlorophenoxy)benzyl)piperidin-3-yl)-5-methylpyr-
imidine-2,4(1H,3H)-dione (22). LC-MS (Method 2) tR = 1.90 min, m/
1
z = 426.1 (M + 1). H NMR (300 MHz, CD3OD) δ 1.47−1.87 (m,
7H) 2.00−2.34 (m, 2H), 2.63 (br s, 1H), 2.68−2.84 (m, 1H), 3.47 (s,
2H), 4.45 (br s, 1H), 6.70−6.88 (m, 3H), 6.88−7.13 (m, 3H), 7.13−
7.32 (m, 2H), 7.64 (br s, 1H) ppm.
5-Methyl-1-(1-(3-phenoxybenzyl)piperidin-3-yl)pyrimidine-2,4-
(1H,3H)-dione (4). A mixture of 5-methyl-1-(piperidin-3-yl)-
pyrimidine-2,4(1H,3H)-dione (1, 100 mg, 0.48 mmol), 3-(phenoxy)-
benzaldehyde (285 mg, 1.44 mmol), and (polystyrylmethyl)-
trimethylammonium cyanoborohydride (200 mg, 0.8 mmol) in
dichloromethane/acetic acid (90:10 v/v, 4 mL) was stirred at room
temperature for 16 h. The reaction mixture was concentrated under
reduced pressure, and the crude product was purified by reverse phase
preparative HPLC to afford the title compound as a white solid (18
mg, 19%). LC-MS (Method 1) tR = 3.43 min, m/z = 392 (M + 1).
(S)-1-(1-(3-(3-Bromophenoxy)benzyl)piperidin-3-yl)-5-methylpyr-
imidine-2,4(1H,3H)-dione (23). LC-MS (Method 2) tR = 3.04 min, m/
z = 469.8 471.7 (M + 1, M + 3). HRMS calcd for C23H25BrN3O3
1
470.1074, found 470.1057. H NMR (400 MHz, DMSO-d6) δ 1.60
(m, 1H), 1.65−1.80 (s and m, 6H), 2.05 (t, 1H), 2.20 (t, 1H), 2.54
(m, 2H), 3.50 (s, 2H), 4.40 (m, 1H), 6.95 (m, 1H), 7.01 (m’s 2H),
7.15 (m’s, 2H), 7.33 (m, 2H), 7.37 (t, 1H), 7.71 (s, 1H), 11.2 (br s,
1H) ppm.
1
HRMS calcd for C23H26N3O3 392.1969, found 392.1969. H NMR
(400 MHz, DMSO-d6) δ 1.55 (m, 1H), 1.68−1.80 (s, 6H), 2.05 (t,
1H), 2.20 (t, 1H), 2.70 (m, 2H), 3.55 (m, 2H), 4.45 (m, 1H), 6.95 (d,
1H), 7.00−7.20 (m, 5H), 7.35−7.42 (m, 3H), 7.74 (m, 1H), 11.23 (s,
1H) ppm.
(S)-1-(1-(3-(3-Chloro-4-methylphenoxy)benzyl)piperidin-3-yl)-5-
methylpyrimidine-2,4(1H,3H)-dione (24). LC-MS (Method 2) tR
=
3.19 min, m/z = 439.95, 441.88 (M + 1, M + 3). HRMS calcd for
C24H27ClN3O3 440.1735, found 440.1723. 1H NMR (400 MHz,
CD3OD) δ 1.66−1.90 (s and m, 7H), 2.23 (m, 1H), 2.33 (m, 4H),
2.73 (m, 1H), 2.85 (m, 1H), 3.55 (s, 2H), 4.55 (m, 1H), 6.83 (m, 1H),
6.83 (m, 1H), 6.91 (m, 1H), 6.94 (d, 1H), 7.00 (m, 1H), 7.11 (d, J =
7.83, 1H), 7.24 (d, J = 8.34, 1H), 7.33 (d, J = 7.83, 1H) 7.75 (s, 1H)
ppm.
5-Methyl-1-(1-(3-phenoxybenzyl)piperidin-3-yl)pyrimidine-2,4-
(1H,3H)-dione (15). LC-MS (Method 1) tR = 3.43 min, m/z = 392 (M
1
+ 1). HRMS calcd for C23H26N3O3 392.1969, found 392.1969. H
NMR (400 MHz, DMSO-d6) δ 1.55 (m, 1H), 1.68−1.80 (s, 6H), 2.05
(t, 1H), 2.20 (t, 1H), 2.70 (m, 2H), 3.55 (m, 2H), 4.45 (m, 1H), 6.95
(d, 1H), 7.00−7.20 (m, 5H), 7.35−7.42 (m, 3H), 7.74 (s, 1H), 11.23
(s, 1H) ppm.
(S)-1-(1-(3-(3-Chloro-5-fluorophenoxy)benzyl)piperidin-3-yl)-5-
methylpyrimidine-2,4(1H,3H)-dione (25). LC-MS (Method 1) tR
=
5-Methyl-1-(1-(3-(3-(trifluoromethyl)phenoxy)benzyl)piperidin-3-
yl)pyrimidine-2,4(1H,3H)-dione (16). LC-MS (Method 1) tR = 3.86
min, m/z = 460 (M + 1), 458 (M − 1). HRMS calcd for
C24H25F3N3O3 460.1843, found 460.1842. 1H NMR (400 MHz,
DMSO-d6) δ 1.57 (m, 1H), 1.66−1.83 (m, 3H), 1.83 (s, 3H), 2.08 (t,
1H), 2.24 (t, 1H), 2.69−2.83 (m, 2H), 3.57 (s, 2H), 4.38−4.46 (m,
1H), 7.02 (dd, 1H), 7.08 (s, 1H), 7.19 (d, 1H), 7.32 (d, 2H), 7.43 (t,
1H), 7.51 (d, 1H), 7.65 (t, 1H), 7.74 (s, 1H), 11.22 (s, 1H) ppm.
5-Methyl-1-{1-[3-(4-methylphenoxy)benzyl]piperidin-3-yl}-
pyrimidine-2,4(1H,3H)-dione (17). LC-MS (Method 2) tR = 2.64 min,
m/z = 406.4 (M + 1). HRMS calcd for C24H28N3O3 406.2125, found
406.2130. 1H NMR (400 MHz, CDCl3): δ 1.68−1.74 (m, 3H), 1.80−
1.82 (m, 1H), 1.90 (s, 3H), 2.34 (m, 5H), 2.58 (m, 1H), 2.77−2.79
(m, 1H), 3.49 (q, J = 13.20 Hz, 2H), 4.58−4.60 (m, 1H), 6.86−7.02
(m, 5H), 7.14 (d, J = 8.40 Hz, 2H), 7.23−7.28 (m, 1H), 7.69 (s, 1H),
8.12 (s, 1H) ppm.
1-(1-(3-(4-Chlorophenoxy)benzyl)piperidin-3-yl)-5-methylpyrimi-
dine-2,4(1H,3H)-dione (18). LC-MS (Method 2) tR = 2.71 min, m/z =
426, 428 (M + 1, M + 2). HRMS calcd for C23H25ClN3O3 426.1583,
found 426.1577. 1H NMR (400 MHz, CDCl3): δ 1.69−1.86 (m, 4H),
1.91 (s, 3H), 2.35 (m, 2H), 2.62 (m, 1H), 2.79−2.81 (m, 1H), 3.51 (q,
J =13.20 Hz, 2H), 4.58−4.60 (m, 1H), 6.89−6.90 (m, 3H), 7.01 (s,
1H), 7.06−7.08 (m, 1H), 7.28−7.32 (m, 3H), 7.62 (m, 1H), 8.13 (s,
1H) ppm.
1-(1-(3-(4-Methoxyphenoxy)benzyl)piperidin-3-yl)-5-methylpyri-
midine-2,4(1H,3H)-dione (19). LC-MS (Method 2) tR = 2.50 min, m/
z = 422.1 (M + 1). HRMS calcd for C24H28N3O4 422.2074, found
422.2072. 1H NMR (400 MHz, CDCl3): δ 1.66−1.75 (m, 3H), 1.81−
1.83 (m, 1H), 1.91 (s, 3H), 2.37−2.42 (m, 2H), 2.59 (m, 1H), 2.78
(dd, J =3.68, 11.34 Hz, 1H), 3.49 (q, J =13.20 Hz, 2H), 3.82 (s, 3H),
4.57−4.60 (m, 1H), 6.82 (dd, J = 1.84, 8.12 Hz, 1H), 6.88−6.93 (m,
3H), 6.96−7.00 (m, 3H), 7.22−7.25 (m, 1H), 7.67 (s, 1H), 8.05 (s,
1H) ppm.
(S)-5-Methyl-1-(1-(3-(m-tolyloxy)benzyl)piperidin-3-yl)-
pyrimidine-2,4(1H,3H)-dione (20). LC-MS (Method 2) tR = 2.70 min,
m/z = 406.2 (M). HRMS calcd for C24H28N3O3 484.1634, found
406.2105. 1H NMR (300 MHz, DMSO-d6) δ 1.60 (m, 1H), 1.65−1.80
(s and m, 6H), 2.05 (t, 1H), 2.20 (t, 1H), 2.54 (m, 2H), 3.50 (s, 2H),
4.40 (m, 1H), 6.95 (m, 1H), 7.01 (m’s, 2H), 7.15 (m’s, 2H), 7.33 (m,
2H), 7.37 (t, 1H), 7.71 (s, 1H), 11.2 (br s, 1H) ppm.
1.86 min, m/z = 444.1 (M + 1), 442.3 (M − 1). HRMS calcd for
C23H24ClFN3O3 444.1485, found 444.1487. 1H NMR (300 MHz,
CD3OD) δ 1.48−1.81 (m, 7H), 2.13 (br s, 1H), 2.24 (t, J = 10.17 Hz,
1H), 2.65 (d, J = 11.30 Hz, 1H), 2.76 (dd, J = 10.55, 3.77 Hz, 1H),
3.49 (s, 2H), 4.36−4.55 (m, 1H), 6.56 (d, J = 10.55 Hz, 1 H), 6.66 (s,
1H), 6.81 (d, J = 8.29 Hz, 1H), 6.90 (d, J = 8.29 Hz, 1H), 6.99 (s, 1H),
7.11 (d, J = 7.54 Hz, 1H), 7.30 (t, J = 7.91 Hz, 1H), 7.65 (s, 1H) ppm.
(S)-1-(1-(3-(3,5-Dichlorophenoxy)benzyl)piperidin-3-yl)-5-methyl-
pyrimidine-2,4(1H,3H)-dione (26). LC-MS (Method 2) tR = 1.92 min,
m/z = 460.1, 462.5 (M, M + 2). HRMS calcd for C23H24Cl2N3O3
1
460.1189, found 460.1184. H NMR (300 MHz, CD3OD) δ 1.83 (m,
7H), 2.28 (dt, 2H), 2.8 (dm, 2H), 3.58 (s, 2H), 4.55 (m, 1H), 6.89 (m,
2H), 6.93 (d, 1H), 7.08 (s, 1H), 7.15 (s, 1H), 7.28 (m, 1H), 7.39 (m,
1H), 7.74 (s, 1H) ppm.
(S)-1-(1-(3-(4-Chlorophenoxy)benzyl)piperidin-3-yl)-5-methylpyr-
imidine-2,4(1H,3H)-dione (27). LC-MS (Method 2) tR = 1.96 min, m/
z = 426.1 (M + 1). HRMS calcd for C23H25ClN3O3 426.1579, found
1
426.1570. H NMR (300 MHz, DMSO-d6) δ 1.79 (m, 7H), 2.82 (s,
1H), 3.17 (m, 1H), 3.42 (t, 2H), 4.32 (m, 2H), 4.72 (br t, 1H), 7.08
(s, 1H), 7.10 (s, 1H), 7.20 (d, 1H), 7.24 (s, 1H), 7.37 (d, 1H), 7.43−
7.55 (m, 4H), 9.9 (br s, 1H), 11.4 (s, 1H) ppm.
(R)-1-(1-(3-(4-Chlorophenoxy)benzyl)piperidin-3-yl)-5-methylpyr-
imidine-2,4(1H,3H)-dione (28). LC-MS (Method 2) tR = 1.82 min, m/
z = 428.3 (M + 1). HRMS calcd for C23H25ClN3O3 426.1579, found
426.1583. 1H NMR (300 MHz, CD3OD) δ 1.54−1.93 (m, 7H), 2.05−
2.42 (m, 2H), 2.63−2.96 (m, 2H), 3.55 (br s, 2H), 4.55 (d, J = 3.77
Hz, 1H), 6.78−7.05 (m, 4H), 7.10 (d, J = 7.54 Hz, 1H), 7.33 (d, J =
4.52 Hz, 3H), 7.72 (br s, 1H) ppm.
(S)-1-(1-(3-(3,4-Dichlorophenoxy)benzyl)piperidin-3-yl)-5-methyl-
pyrimidine-2,4(1H,3H)-dione (29). LC-MS (Method 2) tR = 2.29 min,
m/z = 460.0, 461.9 (M + 1 M + 3). HRMS calcd for C23H24Cl2N3O3
460.1189, found 460.1190. 1H NMR (300 MHz, DMSO-d6) δ 1.75 (s,
3H), 1.90 (m, 4H), 2.85 (m, 1H), 3.20 (m, 1H), 3.45 (t, 2H), 4.30 (m,
2H), 4.75 (t, 1H), 7.07 (m, 1H), 7.20 (d, 1H), 7.35 (m’s, 3H), 7.55
(m’s, 2H), 7.85 (d, 1H), 10.6 (br s, 1H), 11.35 (s, 1H) ppm.
(R)-1-(1-(3-(3,4-Dichlorophenoxy)benzyl)piperidin-3-yl)-5-meth-
ylpyrimidine-2,4(1H,3H)-dione (30). LC-MS (Method 2) tR = 2.04
min, m/z = 461.9 (M + 1), 458.3 (M − 1). HRMS calcd for
C23H24Cl2N3O3 460.1189, found 460.1187. 1H NMR (300 MHz,
CD3OD) δ 1.54−1.94 (m, 7H), 2.10−2.40 (m, 2H), 2.75 (d, J = 11.30
Hz, 1H), 2.80−2.92 (m, 1H), 3.20−3.45 (m, 2H), 3.45−3.67 (m, 2H),
4.54 (br s, 1H), 6.76−7.00 (m, 2H), 7.00−7.26 (m, 3H), 7.26−7.58
(m, 2H), 7.72 (br s, 1H) ppm.
(S)-1-(1-(3-(3-Fluorophenoxy)benzyl)piperidin-3-yl)-5-methylpyri-
midine-2,4(1H,3H)-dione (21). LC-MS (Method 2) tR = 2.77 min, m/
z = 410.1 (M + 1). 1H NMR (400 MHz, CD3OD) δ 1.60−1.88 (s and
m, 7H), 2.23 (t, 1H), 2.35 (t, 1H), 2.75 (m, 1H), 2.83 (m, 1H), 3.57
10017
dx.doi.org/10.1021/jm3011806 | J. Med. Chem. 2012, 55, 10010−10021

Journal of Medicinal Chemistry
Article
Experimental Procedures and Characterization Data for
Analogues in Table 3. (S)-2-Fluoro-4-((3-(5-methyl-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)piperidin-1-yl)methyl)benzonitrile (6).
(S)-5-Methyl-1-(piperidin-3-yl)pyrimidine-2,4(1H,3H)-dione (1,
1052 mg, 5.03 mmol), cyanoborohydride on resin (2515 mg, 5.03
mmol), and 2-fluoro-4-formylbenzonitrile (5, 500 mg, 3.35 mmol)
were suspended in DMF (10 mL). Acetic acid (0.500 mL) was added,
and the mixture was stirred at RT overnight. LCMS showed product.
The resin was removed by filtration, and the crude was then diluted in
ethyl acetate, washed with saturated aqueous sodium bicarbonate and
water, and then dried over anhydrous sodium sulfate. After the solvent
was removed under reduced pressure, the crude oil was purified by
reverse phase chromatography HPLC (acetonitrile, water, 0.1% TFA)
(5−50% acetonitrile). After the solvent was removed, a solid was
isolated and identified as 6 (323 mg, 28%). LC-MS (Method 1) tR =
1H), 7.17 (dd, J = 14.69, 7.16 Hz, 2H), 7.58 (s, 1H), 7.74 (d, J = 8.29
Hz, 1H) ppm.
4-((3-(5-Methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-
piperidin-1-yl)methyl)-2-(3-(trifluoromethyl)phenoxy)benzoic Acid
(34). LC-MS (Method 2) tR = 2.46 min, m/z = 504.2 (M + 1).
1
HRMS calcd for C25H25FN3O5 504.1741, found 504.1742. H NMR
(400 MHz, DMSO-d6): δ 1.48−1.53 (m, 1H), 1.65−1.71 (m, 3H),
1.73 (s, 3H), 2.03 (t, J = 10.04 Hz, 1H), 2.20 (t, J = 10.16 Hz, 1H),
2.66−2.76 (m, 2H), 3.57 (s, 2H), 4.35−4.39 (m, 1H), 7.09−7.14 (m,
3H), 7.29 (d, J = 8.08 Hz, 1H), 7.40 (d, J = 7.72 Hz, 1H), 7.56 (t, J =
7.88 Hz, 1H), 7.68 (s, 1H), 7.85 (d, J = 7.92 Hz, 1H), 11.19 (s, 1H),
12.88 (br s, 1H) ppm.
Experimental Procedures and Characterization Data for
Analogs in Table 4. 1-(4-Cyano-3-fluorophenyl)-(3,3-
dimethylbutyl)methanesulfonate (11b). (4-Cyano-3-fluorophenyl)-
zinc(II) bromide (9, 163 mL, 81.72 mmol) was added dropwise (over
20 min) to a suspension of 3,3-dimethylbutanoyl chloride (10, 10.37
mL, 74.29 mmol) and tetrakis(triphenylphosphine)palladium(0)
(2.146 g, 1.86 mmol) in tetrahydrofuran (200 mL) at room
temperature and under an atmosphere of nitrogen. The solution
turned from pale orange to dark red overnight. The crude was diluted
in ethyl acetate (200 mL) and washed with aqueous saturated sodium
bicarbonate, water, and aqueous 1 N HCl. The organic extract was
dried over anhydrous sodium sulfate, and the solvent was removed
under reduced pressure, yielding a thick reddish oil containing a shiny
solid. The crude oil was suspended in diethyl ether, and the solids were
removed by filtration. The crude was then purified by flash
chromatography on silica gel (330 g) eluting with mixtures of
hexanes/ethyl acetate (from 0 to 30% ethyl acetate in 53 min). The
main fraction was combined and the solvents were removed under
reduced pressure, yielding 4-(3,3-dimethylbutanoyl)-2-fluorobenzoni-
1
0.88 min, m/z = 342.0, 344.0 (M, M + 2). H NMR (300 MHz,
CD3OD) δ 1.82 (overlap m and s, 7H), 2.19 (m, 1H), 2.33 (t, J = 10.5
Hz, 1H), 2.78 (d, J = 10.5 Hz, 1H), 2.89 (dd, J = 10.5, 4.5 Hz, 1H),
3.67 (s, 2H), 4.59 (m, 1H), 7.39 (m, 2H), 7.71 (m, 2H) ppm.
(S)-2-(3-Chlorophenoxy)-4-((3-(5-methyl-2,4-dioxo-3,4-dihydro-
pyrimidin-1(2H)-yl)piperidin-1-yl)methyl)benzonitrile (7). 3-Chloro-
phenol (28.2 mg, 0.22 mmol), 6 (50 mg, 0.15 mmol), and anhydrous
potassium carbonate (20.18 mg, 0.15 mmol) were suspended in N-
methyl-2-pyrrolidinone (1 mL). The mixture was heated using a
microwave oven at 150 °C for 15 min. The reaction mixture was then
purified by preparative HPLC, eluting with mixtures of acetonitrile/
water/0.1% TFA. The fractions containing clean product were mixed,
and the solvent was removed under reduced pressure, yielding 7 (30.0
mg, 45%) as a solid. LC-MS (Method 1) tR = 1.79 min, m/z = 451.1
(M + 1), 449.2 (M − 1).
(S)-2-(3-Chlorophenoxy)-4-((3-(5-methyl-2,4-dioxo-3,4-dihydro-
pyrimidin-1(2H)-yl)piperidin-1-yl)methyl)benzoic Acid (8). 7 (30 mg,
0.07 mmol) was suspended in water (1 mL). Sulfuric acid (0.100 mL)
was added, and the sample was heated using microwave irradiation for
30 min at 150 °C. The crude was then purified using preparative
HPLC, eluting with mixtures of acetonitrile/water/TFA 0.1%. The
recovered clean fractions were combined, and the solvent was removed
using the lyophilizer, yielding 8 (3.40 mg, 9%) as an off white solid.
LC-MS (Method 1) tR = 1.42 min, m/z = 470.1, 468.2 (M + 1, M −
1
trile (11a, 13.80 g, 85%) as a semisolid (pale yellow). H NMR (300
MHz, CDCl3) δ 1.09 (s, 9H), 2.87 (s, 2H), 7.57−7.93 (m, 3H) ppm.
11a (1.5 g, 6.84 mmol) was dissolved in MeOH (10 mL). NaBH4
(0.227 g, 5.99 mmol) was added in small portions (gas evolution). The
reaction was stirred for 2 h. The crude was dissolved in ethyl acetate
and washed with 1 N HCl, saturated aqueous sodium bicarbonate, and
brine. The organic extract was dried over anhydrous sodium sulfate,
and the solvent was then removed under reduced pressure. The crude
oil was purified by flash chromatography on silica gel (40 g), eluting
with mixtures of hexanes/ethyl acetate (from 0 to 70% ethyl acetate in
25 min). The cleanest fractions were combined, and the solvent was
removed under reduced pressure, yielding 2-fluoro-4-(1-hydroxy-3,3-
dimethylbutyl)benzonitrile (0.433 g, 29%) as a colorless oil. 1H NMR
(300 MHz, CDCl3) δ 1.05 (s, 9H), 1.53 (dd, J = 14.8, 2.7 Hz, 1H), 1.7
(dd, J = 14.8, 8.9 Hz, 1H), 4.9 (dd, J = 8.8, 2.2 Hz, 1H), 7.2 (m, 2H,
obscured by CHCl3 signal), 7.6 (m, 1H) ppm. 2-Fluoro-4-(1-hydroxy-
3,3-dimethylbutyl)benzonitrile (433 mg, 1.9 mmol), and TEA (0.552
mL, 3.96 mmol) were dissolved in dichloromethane (5 mL) at 0 °C.
Methanesulfonyl chloride (0.169 mL, 2.18 mmol) was added, and the
reaction was stirred for 4 h. The reaction mixture was diluted with
dichloromethane, washed with 0.5 N HCl, saturated aqueous sodium
bicarbonate, and brine. The organic extract was dried over anhydrous
sodium sulfate, and the solvent was removed under reduced pressure,
yielding 11b as an oil which was used in the next step without further
1
1). HRMS calcd for C24H25ClN3O5 470.1477, found 470.1469. H
NMR (300 MHz, CD3OD) δ 1.89 (m, 4H), 2.08 (m, 3H), 3.0 (br t,
1H), 3.23 (apparent t, overlap with solvent signal, 1H), 3.5 (m, 2H),
4.38 (m, 2H), 4.67 (m, 1H), 6.9 (dd, J = 8.3, 1.1 Hz, 1H), 7.0 (t, J =
2.2 Hz, 1H), 7.13 (m, 1H), 7.24 (d, J = 1.5 Hz, 1H), 7.33 (m, 1H),
7.43 (m, 1H), 8.04 (d, J = 7.9 Hz, 1H) ppm.
(S)-2-(3-Bromophenoxy)-4-((3-(5-methyl-2,4-dioxo-3,4-dihydro-
pyrimidin-1(2H)-yl)piperidin-1-yl)methyl)benzoic Acid (31). LC-MS
(Method 2) tR = 1.45 min, m/z = 514.0, 516.0 (M + 1, M + 3), 512.3
(M − 1). HRMS calcd for C24H25BrN3O5 514.0972, found 514.0947.
1H NMR (300 MHz, CD3OD) δ 1.78−2.05 (m, 6H), 2.84 (t, 1H),
3.06 (t, 1H), 3.33 (m, 2H), 4.21 (m, 2H), 4.57 (br s, 1H), 6.85 (m,
1H), 7.03 (s, 1H), 7.15 (m, 1H), 7.34 (d, 1H), 7.39 (m, 1H), 7.92 (d,
1H) ppm.
(S)-4-((3-(5-Methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-
piperidin-1-yl)methyl)-2-(m-tolyloxy)benzoic Acid (32). LC-MS
(Method 2) tR = 1.38 min, m/z = 450.3 (M + 1), 448.34 (M − 1).
1
purification. H NMR (300 MHz, CDCl3) δ 1.01 (s, 9H), 1.61 (m,
1
HRMS calcd for C25H28N3O5 450.2023, found 450.2004. H NMR
overlap with water, 1H), 2.05 (dd, J = 15.1, 8.85 Hz, 1H), 2.84 (s, 3H),
5.71 (dd, J = 8.85, 3.58 Hz 1H), 7.29 (m, 2H, obscured by CHCl3
signal), 7.68 (dd, J = 8.10, 6.64 Hz, 1H) ppm.
(300 MHz, CD3OD) δ 1.78 (s, 3H), 1.82−2.14 (m, 3H), 2.21 (s, 2H),
2.87 (br s, 1H), 3.10 (t, J = 11.68 Hz, 1H), 3.29−3.46 (m, 2H), 4.11−
4.34 (m, 2H), 4.55 (dd, J = 10.55, 5.27 Hz, 1H), 6.68 (d, J = 7.54 Hz,
1H), 6.76 (s, 1H), 6.87 (d, J = 7.54 Hz, 1H), 7.02 (s, 1H), 7.13 (t, J =
7.54 Hz, 1H), 7.23 (d, J = 9.80 Hz, 1H), 7.32 (s, 1H), 7.88 (d, J = 8.29
Hz, 1H) ppm.
4-(3,3-Dimethyl-1-((S)-3-(5-methyl-2,4-dioxo-3,4-dihydropyrimi-
din-1-(2H)-yl)piperidin-1-yl)butyl)-2-fluorobenzonitrile (12). 11b
(4.1 g, 13.70 mmol), (S)-5-methyl-1-(piperidin-3-yl)pyrimidine-2,4-
(1H,3H)-dione (1, 7.16 g, 34.24 mmol), and DIEA (7.18 mL, 41.09
mmol) were dissolved in acetonitrile (40 mL). The mixture was heated
at 70 °C for 20 h. LCMS showed the desired product. The reaction
was diluted in ethyl acetate and washed with brine. The organic extract
was dried over anhydrous sodium sulfate. After removing the solvent
under reduced pressure, the crude was purified by flash chromatog-
raphy on silica gel (80 g), eluting with mixtures of hexanes/ethyl
acetate (from 0 to 100% ethyl acetate in 25 min). The cleanest
(S)-2-(3-Fluorophenoxy)-4-((3-(5-methyl-2,4-dioxo-3,4-dihydro-
pyrimidin-1(2H)-yl)piperidin-1-yl)methyl)benzoic Acid (33). LC-MS
(Method 2) tR = 1.3 min, m/z = 454.3 (M + 1), 452.2 (M − 1).
1
HRMS calcd for C24H25FN3O5 454.1773, found 454.1759. H NMR
(300 MHz, CD3OD) δ 1.47−1.81 (m, 7 H), 2.15 (br s, 1H), 2.28 (t, J
= 10.17 Hz, 1H), 2.68 (d, J = 10.55 Hz, 1H), 2.79 (dd, J = 10.55, 3.77
Hz, 1H), 3.54 (s, 2H), 4.44 (br s, 1H), 6.49−6.76 (m, 3H), 6.97 (s,
10018
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Journal of Medicinal Chemistry
Article
fractions were combined, and the solvent was removed under reduced
pressure, yielding 12 (0.710 g, 13%) as an off white solid. H NMR
6.82 (s, 1H), 6.88−7.05 (m, 2H), 7.19 (t, J = 8.29 Hz, 2 H), 7.49 (s,
1H), 7.78 (d, J = 8.29 Hz, 1H) ppm.
1
2-(3-Chlorophenoxy)-4-((S)-1-((S)-3-(5-methyl-2,4-dioxo-3,4-di-
hydropyrimidin-1(2H)-yl)piperidin-1-yl)ethyl)benzoic Acid (36). LC-
MS (Method 2) tR = 1.47 min, m/z = 484.0 (M + 1). HRMS calcd for
C25H27ClN3O5 484.1634, found 484.1620. 1H NMR (300 MHz,
CD3OD) δ 1.30 (d, J = 6.78 Hz, 3H), 1.44−1.83 (m, 7H), 2.08 (d, J =
12.06 Hz, 1H), 2.24 (t, J = 10.17 Hz, 1H), 2.68 (d, J = 12.06 Hz, 1H),
2.96 (d, J = 9.04 Hz, 1H), 3.57 (t, J = 6.40 Hz, 1H), 4.45 (br s, 1H),
6.65−6.86 (m, 2H), 6.87−7.01 (m, 2H), 7.06−7.26 (m, 2H), 7.55 (s,
1H), 7.78 (d, J = 8.29 Hz, 1H) ppm.
(300 MHz, CD3OD) δ 0.9 (bs, 9H), 1.56 (bt, 2H), 1.8 (m, 9H), 2.89
(dd, 1H), 2.95 (m, 1H), 3.88 (m, 1H), 4.53 (m, 1H), 7.3 (m, 2H),
7.52 (d, J = 18.6 Hz, 1H), 7.73 (m, 1H) ppm.
2-(3-Bromophenoxy)-4-(3,3-dimethyl-1-((S)-(5-methyl-2,4-dioxo-
3,4-dihydropyrimidin-1-(2H)-yl)piperidin-1-yl)butyl)benzoic Acid
(13). 12 (2.7 g, 6.55 mmol) was dissolved in NMP (20 mL). 3-
Bromophenol (1.699 g, 9.82 mmol) and K2CO3 (2.71 g, 19.64 mmol)
were added, and the mixture was split in two and heated in the
microwave at 150 °C for 30 min (two vials). The crudes were
combined and diluted with ethyl acetate and then washed with
aqueous saturated potassium bicarbonate and water. After drying over
anhydrous sodium sulfate, the solvent was removed under reduced
pressure, yielding a crude oil. The crude was then purified by flash
chromatography (220 g) eluting with a mixture of hexanes/ethyl
acetate (0−100% in 30 min). The fractions containing product were
combined and the solvent removed under reduced pressure, yielding
2-(3-bromophenoxy)-4-(3,3-dimethyl-1-((S)-3-(5-methyl-2,4-dioxo-
3,4-dihydropyrimidin-1(2H)-yl)piperidin-1-yl)butyl)benzonitrile (2.50
g, 67%) as a yellow foam/solid. tR = 2.58 min, m/z = 565.0, 567.0 (M
(3-Chlorophenoxy)-4-((R)-1-((S)-3-(5-methyl-2,4-dioxo-3,4-dihy-
dropyrimidin-1(2H)-yl)piperidin-1-yl)propyl)benzoic Acid (37). LC-
MS (Method 2) tR = 1.6 min, m/z = 500.0 (M + 1), 496.2 (M − 1).
1
HRMS calcd for C26H29ClN3O5 498.1790, found 498.1790. H NMR
(300 MHz, CD3OD) δ 0.83 (t, J = 7.16 Hz, 3H), 1.88 (s, 3H), 1.93−
2.11 (m, 3H), 2.11−2.43 (m, 3H), 2.89 (br s, 1H), 3.09 (br s, 1H),
3.46 (d, J = 10.55 Hz, 1H), 3.56 (br s, 1H), 4.38 (d, J = 7.54 Hz, 1H),
4.68 (br s, 1H), 6.88 (d, J = 8.29 Hz, 1H), 7.00 (s, 1H), 7.13 (d, J =
6.78 Hz, 1H), 7.26 (s, 1 H), 7.34 (t, J = 8.29 Hz, 1H), 7.39−7.56 (m,
2H), 8.09 (d, J = 8.29 Hz, 1H) ppm.
(3-Chlorophenoxy)-4-((S)-1-((S)-3-(5-methyl-2,4-dioxo-3,4-dihy-
dropyrimidin-1(2H)-yl)piperidin-1-yl)propyl)benzoic Acid (38). LC-
MS (Method 2) tR = 1.68 min, m/z = 498.1 (M + 1), 496.2 (M − 1).
1H NMR (300 MHz, CD3OD) δ 0.82 (t, 3H), 1.9 (s and m, 6H), 2.2
(br s, 1H), 3.08 (br s, 1H), 3.48 (br s, 1H), 3.70 (br s, 1H), 4.31 (m,
1H), 4.75 (m overlap with d-solvent signal, 1H), 6.9 (d, 1H), 7.01 (d,
1H), 7.15 (d, 1H), 7.23 (s, 1H), 7.34 (t, 1H), 7.45 (br s, 1H), 8.1 (d,
1H) ppm.
1
+ 1, M + 3). H NMR (300 MHz, CDCl3) δ 0.86 (m, 9H), 1.76 (m,
9H), 2.58 (d, J = 10.9 Hz, 1H), 2.76 (d, J = 10.4 Hz, 1H), 2.8 (m, 1H),
3.64 (m, 1H), 4.56 (m, 1H), 6.76 (m, 1H), 7.03 (m, 2H), 7.17 (m,
1H), 7.31 (m, 2H), 7.66 (dd, J = 7.9, 2.3 Hz, 2H), 9.0 (d, J = 5.1 Hz,
1H) ppm. 2-(3-Bromophenoxy)-4-(3,3-dimethyl-1-((S)-3-(5-methyl-
2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)piperidin-1-yl)butyl)-
benzonitrile (2.5 g, 4.42 mmol) was then dissolved in ethanol (15 mL)
and water (15 mL). Sodium hydroxide (2.65 g, 66.31 mmol) was
added, and the mixture was heated at 100 °C for 16 h. The LCMS of
the crude showed only acid. The ethanol was removed under a stream
of nitrogen. The aqueous solution was treated with concentrated
aqueous HCl, and a solid was formed and isolated via filtration. The
solid was suspended in ethyl acetate and washed with water. The
organic fraction was isolated and dried over anhydrous sodium sulfate,
and the solvent was then removed under reduced pressure, yielding
the title compound 13 as an off white solid (2 g, 77%). LC-MS
2-(3-Chlorophenoxy)-4-((R)-1-((S)-3-(5-methyl-2,4-dioxo-3,4-di-
hydropyrimidin-1-(2H)-yl)piperidin-1-yl)butyl)benzoic Acid (39).
LC-MS (Method 3) tR = 1.8 min, m/z = 512.2, 510.1 (M + 1, M −
1
1). HRMS calcd for C29H31ClN3O5 512.1947, found 512.1936. H
NMR (300 MHz, CD3OD) δ. 0.81 (t, J = 7.16 Hz, 3 H), 0.98−1.30
(m, 2 H), 1.51−1.90 (m, 9 H), 2.11 (t, J = 10.17 Hz, 2 H), 2.70−2.90
(m, 2 H), 3.53 (dd, J = 9.04, 5.27 Hz, 1 H), 4.37 (d, J = 9.04 Hz, 1 H),
6.75 (d, J = 8.29, Hz, 1 H), 6.84 (d, J = 12.81 Hz, 2 H), 6.96 (d, J =
6.03 Hz, 1 H), 7.10 (d, J = 8.29 Hz, 1 H), 7.19 (t, J = 8.29 Hz, 1 H),
7.52 (s, 1 H), 7.77 (d, J = 7.54 Hz, 1 H) ppm. Diastereomeric ratio:
>99:1.
1
(Method 2) tR = 2.07 min, m/z = 584.1, 586.1 (M + 1, M + 3). H
NMR (300 MHz, CD3OD) δ 0.85 (s, 9H), 2.01 (m, 9H), 2.36 (br t,
1H), 2.8 (m, 1H), 3.06 (m, 1H), 3.55 (d, J = 11.3 Hz, 1H), 3.73 (d, J =
11.3 Hz, 1H), 4.61 (dd, J = 10.5, 7.9 Hz, 1H), 6.93 (m, 1H), 7.07 (m,
1H), 7.27 (m, 2H), 7.40 (m, 1H), 7.47 (d, J = 4.71 Hz, 1H), 7.60 (d, J
= 8.1 Hz, 1H), 8.09 (d, J = 8.1 Hz, 1H) ppm.
2-(3-Chlorophenoxy)-4-((S)-1-((S)-3-(5-methyl-2,4-dioxo-3,4-di-
hydropyrimidin-1(2H)-yl)piperidin-1-yl)butyl)benzoic Acid (40). LC-
MS (Method 2) tR = 1.86 min, m/z = 512.3 (M + 1), 510.2 (M − 1).
1
HRMS calcd for C27H31ClN3O5 512.1947, found 512.1936. H NMR
2-(3-Bromophenoxy)-4-((R)-3,3-dimethyl-1-((S)-(5-methyl-2,4-
dioxo-3,4-dihydropyrimidin-1-(2H)-yl)piperidin-1-yl)butyl)benzoic
Acid (14). Diastereomeric mixture 13 was subjected to HPLC
preparative chiral separation eluting with a mixture of hexanes
(60%), methanol/ethanol (1:1) (40%), and diethylamine (0.5%). The
title compound eluted second. After removing the solvent, the sample
(825 mg) contained residual diethyl amine that was removed by
preparative HPLC (XBridge C18 OBD, 19 mm × 150 mm, 5 μm; flow
rate 20 mL/min) eluting with a mixture of a linear gradient from 40 to
69% in 7.5 min methanol in H2O aqueous (0.2% NH4OH). After
lyophilizing the sample, the title compound 14 was obtained as an off
white solid (400 mg, 15%). LC-MS (Method 2) tR = 2.2 min, m/z =
584.0, 586.0 (M + 1, M + 3) HRMS calcd for C29H35BrN3O5
(300 MHz, CD3OD) δ 0.81−1.01 (m, 3H), 1.08−1.29 (m, 2H), 1.57−
1.97 (m, 9H), 2.12 (t, J = 10.17 Hz, 1H), 2.32 (t, J = 10.17 Hz, 1H),
2.86 (d, J = 11.30 Hz, 1H), 3.07 (d, J = 10.55 Hz, 1H), 3.61 (dd, J =
9.04, 5.27 Hz, 1H), 4.47−4.68 (m, 1H), 6.78−6.96 (m, 2 H), 6.99 (s,
1H), 7.07 (d, J = 7.54 Hz, 1H), 7.16−7.35 (m, 2H), 7.61 (s, 1H), 7.91
(d, J = 8.29 Hz, 1H) ppm. Diastereomeric ratio: >98:1.
2-(3-Chlorophenoxy)-4-((R)-3-methyl-1-((S)-3-(5-methyl-2,4-
dioxo-3,4-dihydropyrimidin-1(2H)-yl)piperidin-1-yl)butyl)benzoic
Acid (41). LC-MS (Method 2) tR = 1.9 min, m/z = 526.0 (M + 1).
1
HRMS calcd for C28H33ClN3O5 526.2103, found 526.2100. H NMR
(300 MHz, CD3OD) δ 0.79 (app t, 6H), 1.19 (app t, 1H), 1.31 (m,
1H), 1.66 (m, 6H), 1.75 (s, 3H), 2.11 (d, 2H), 2.86 (m, 3H), 3.64 (m,
1H), 4.38 (m, 1H), 6.75 (dd, 1H), 6.83 (m, 2H), 6.94 (m, 1H), 7.09
(d, 1H), 7.17 (app t, 1H), 7.47 (br s, 1H), 7.71 (d, 1H) ppm.
2-(3-Chlorophenoxy)-4-((S)-3-methyl-1-((S)-3-(5-methyl-2,4-
dioxo-3,4-dihydropyrimidin-1(2H)-yl)piperidin-1-yl)butyl)benzoic
Acid (42). LC-MS (Method 2) tR = 1.9 min, m/z = 526.0 (M + 1).
1
584.1755, found 584.1746. H NMR (300 MHz, CD3OD) δ 0.87 (s,
9H), 1.76 (m, 9H), 2.12 (m, 2H), 2.82 (d, J = 10.7 Hz, 1H), 3.0 (d, J =
7.2 Hz, 1H), 3.84 (t, J = 6.3 Hz, 1H), 4.49 (br s, 1H), 6.9 (m, 2H),
7.04 (d, J = 1.9 Hz, 1H), 7.18 (m, 3H), 7.53 (s, 1H), 7.79 (d, J = 7.91
Hz, 1H) ppm. Diastereomeric ratio: >98:1. Anal. Calcd for
C29H34BrN3O5·H2O·0.05C3H8O: C, 58.51; H, 6.09; N, 6.8. Found:
C, 58.41 ; H, 5.92; N, 6.90.
1
HRMS calcd for C28H33ClN3O5 526.2103, found 526.2091. H NMR
(300 MHz, CD3OD) δ 0.76 (app t, 6H), 1.19 (app t, 1H), 1.25 (m,
1H), 1.60 (m, 9H), 1.92 (m, 1H), 2.10 (m, 1H), 2.70 (m, 1H), 2.86
(m, 1H), 3.47 (m, 1H), 4.46 (m, 1H), 6.79 (m, 2H), 6.83 (m, 1H),
6.91 (m, 1H), 7.01 (d, 1H), 7.14 (app t, 1H), 7.49 (br s, 1H), 7.57 (d,
1H) ppm.
2-(3-Bromophenoxy)-4-((S)-3,3-dimethyl-1-((S)-3-(5-methyl-2,4-
dioxo-3,4-dihydropyrimidin-1(2H)-yl)piperidin-1-yl)butyl)benzoic
Acid (43). LC-MS (Method 2) tR = 2.0 min, m/z = 586.3 (M + 1),
584.3 (M − 1). HRMS calcd for C29H35BrN3O5 584.1755, found
2-(3-Chlorophenoxy)-4-((R)-1-((S)-3-(5-methyl-2,4-dioxo-3,4-di-
hydropyrimidin-1(2H)-yl)piperidin-1-yl)ethyl)benzoic Acid (35). LC-
MS (Method 2) tR = 1.46 min, m/z = 484.0 (M + 1), 482.2 (M − 1).
1
HRMS calcd for C25H27ClN3O5 484.1634, found 484.1618. H NMR
(300 MHz, CD3OD) δ 1.31 (d, J = 6.03 Hz, 3H), 1.47−1.68 (m, 2H),
1.68−1.86 (m, 5H), 1.99−2.35 (m, 2H), 2.82 (t, J = 10.55 Hz, 2H),
3.61 (d, J = 6.78 Hz, 1H), 4.40 (br s, 1H), 6.73 (d, J = 8.29 Hz, 1H),
10019
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Journal of Medicinal Chemistry
Article
1
584.1742. H NMR (300 MHz, CD3OD) δ 0.75 (s, 9H), 1.47−1.62
2-(3-Chlorophenoxy)-4-((R)-1-((S)-3-(5-methyl-2,4-dioxo-3,4-di-
hydropyrimidin-1(2H)-yl)piperidin-1-yl)-2-(tetrahydro-2H-pyran-4-
yl)ethyl)benzoic Acid (50). LC-MS (Method 2) tR = 1.60 min, m/z =
568.2 (M + 1), 566.3 (M − 1). HRMS calcd for C30H35ClN3O6
(m, 2H), 1.62−1.90 (m, 7H), 1.96 (br s, 1H), 2.16 (t, J = 10.55 Hz,
1H), 2.93 (dd, J = 16.77, 11.68 Hz, 2H), 3.80 (dd, J = 8.29, 3.96 Hz,
1H), 4.48 (br s, 1H), 6.73−6.84 (m, 1H), 6.84−7.00 (m, 2H), 7.02−
7.22 (m, 3H), 7.38 (s, 1H), 7.79 (d, J = 7.91 Hz, 1H) ppm.
Diastereomeric ratio: >99:1.
2-(3-Chlorophenoxy)-4-((R)-3,3-dimethyl-1-((S)-3-(5-methyl-2,4-
dioxo-3,4-dihydropyrimidin-1(2H)-yl)piperidin-1-yl)pentyl)benzoic
Acid (44). LC-MS (Method 2) tR = 2.1 min, m/z = 554.2, 552.2 (M +
1, M + 3). HRMS calcd for C30H37ClN3O5 554.2416, found 554.2410.
1H NMR (300 MHz, CD3OD) δ 0.74 (s, 3H), 0.77−0.83 (m, 3 H),
0.85 (s, 3H), 1.16−1.28 (m, 2H), 1.86−2.00 (m, 7H), 2.12 (br s, 1H),
2.24−2.37 (m, 1H), 2.67 (s, 1H), 2.84 (br s, 1H), 3.43−3.64 (m, 3 H),
4.50 (d, J = 11.11 Hz, 1H), 6.88 (dd, J = 8.10, 1.13 Hz, 1H), 6.90−6.94
(m, 1H), 7.12 (d, J = 7.91 Hz, 1H), 7.30−7.38 (m, 2H), 7.42 (s, 1H),
7.52 (d, J = 6.41 Hz, 1H), 8.07 (d, J = 8.10 Hz, 1H) ppm.
1
568.2209, found 568.2203. H NMR (300 MHz, CD3OD) δ 1.08−
1.33 (m, 3H), 1.41 (d, J = 12.62 Hz, 1H), 1.55 (d, J = 11.11 Hz, 3H),
1.61 (br s, 1H), 1.65−1.83 (m, 6H), 1.83−1.99 (m, 1H), 2.14 (t, J =
10.27 Hz, 1H), 2.76 (d, J = 10.93 Hz, 1H), 2.90 (d, J = 9.98 Hz, 1H),
3.55−3.68 (m, 1H), 3.76 (d, J = 11.87 Hz, 2H), 4.45 (br s, 1H), 6.69−
6.81 (m, 2H), 6.86 (s, 1H), 6.89−7.01 (m, 1H), 7.06−7.28 (m, 2 H),
7.44 (s, 1H), 7.79 (d, J = 7.91 Hz, 1H) ppm. Diastereomeric ratio:
>99:1.
(2-(3-Bromophenoxy)-4-(R)-(2-(1-adamantane)-1-((S)-3-(5-meth-
yl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)piperidin-1-yl)ethyl)-
benzoic Acid (51). LC-MS (Method 2) tR = 2.45 min, m/z = 662, 664
(M + 1, M + 3). HRMS calcd for C35H41BrN3O5 662.2224, found
1
662.2230. H NMR (300 MHz, DMSO-d6) δ 1.1−1.8 (m, 25H), 2.62
2-(3-Chlorophenoxy)-4-((R)-3-ethyl-1-((S)-3-(5-methyl-2,4-dioxo-
3,4-dihydropyrimidin-1(2H)-yl)piperidin-1-yl)pentyl)benzoic Acid
(45). LC-MS (Method 2) tR = 2.13 min, m/z = 554.5 (M + 1),
552.5 (M − 1). HRMS calcd for C30H37ClN3O5 554.2416, found
(d, 1H), 2.78 (m, 1H), 3.69 (m, 2H), 4.26 (m, 1H), 6.63 (s, 1H), 6.72
(m, 2H), 6.90 (d, 1H), 6.97 (d, 1H), 7.18 (t, 2H), 7.39 (d, 1H), 7.52
(s, 1H) ppm. Diastereomeric ratio: 99:1.
1
554.2415. H NMR (300 MHz, CD3OD) δ 0.59−0.82 (m, 6H), 0.93
ASSOCIATED CONTENT
(br s, 1H), 1.03−1.33 (m, 7H), 1.40−1.79 (m, 9H), 1.94−2.16 (m,
2H), 2.70−2.88 (m, 2H), 2.94 (q, J = 7.28 Hz, 2H), 3.48−3.66 (m,
1H), 4.38 (br s, 1H), 6.67−6.86 (m, 3H), 6.93 (d, J = 7.16 Hz, 1H),
7.06 (d, J = 7.91 Hz, 1H), 7.11−7.25 (m, 1H), 7.47 (s, 1H), 7.68 (d, J
= 7.72 Hz, 1H) ppm.
■
S
* Supporting Information
Enzymatic, MIC, and log D assay protocols as well as protein
crystallography methods and statistics tables. This material is
available free of charge via the Internet at http://pubs.acs.org.
PDB accession codes: 4HEJ (compound (S)-16); 4GSY (34);
4HDC (41).
2-(3-Bromophenoxy)-4-((R)-1-((S)-3-(5-methyl-2,4-dioxo-3,4-di-
hydropyrimidin-1(2H)-yl)piperidin-1-yl)hexyl)benzoic Acid (46). LC-
MS (Method 2) tR = 2.07 min, m/z = 584.2, 586.2 (M + 1, M + 3).
1
HRMS calcd for C29H35BrN3O5 584.1755, found 584.1754. H NMR
(300 MHz, CD3OD) δ 0.70−0.78 (m, 3H), 0.95−1.05 (m, 1H), 1.06−
1.21 (m, 5H), 1.56 (d, J = 10.55 Hz, 1H), 1.63−1.84 (m, 7H), 1.98−
2.07 (m, 1H), 2.17−2.27 (m, 2H), 2.76 (d, J = 12.06 Hz, 1H), 2.93−
2.99 (m, 1H), 3.49 (dd, J = 8.67, 5.65 Hz, 1H), 4.47 (br s, 1H), 6.78−
6.83 (m, 1H), 6.87 (d, J = 1.51 Hz, 1H), 6.92−6.95 (m, 1H), 7.09−
7.14 (m, 3H), 7.49 (s, 1H), 7.80 (d, J = 8.10 Hz, 1H) ppm.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: gabriel@sagerx.com. Phone: +1.617.299.8375.
Present Address
∥Sage Therapeutics, 215 First Street, Cambridge, MA 02141,
United States.
(2-(3-Bromophenoxy)-4-((R)-3-ethyl-3-methyl-1-((S)-3-(5-methyl-
2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)piperidin-1-yl)pentyl)-
benzoic Acid (47). LC-MS (Method 2) tR = 2.26 min, m/z = 612.1,
614.1 (M + 1, M + 3). HRMS calcd for C31H39BrN3O5 612.2068,
Notes
The authors declare no competing financial interest.
1
found 612.2075. H NMR (300 MHz, CD3OD) δ 0.69−0.81 (m, 9
H), 1.13−1.34 (m, 4H), 1.61 (d, J = 8.67 Hz, 2H), 1.74−1.89 (m,
7H), 1.90−2.00 (m, 1H), 2.17 (t, J = 10.46 Hz, 1H), 2.94 (br s, 2H),
3.76 (t, J = 6.03 Hz, 1H), 4.58 (br s, 1H), 6.83−6.99 (m, 2H), 7.03−
7.10 (m, 1H), 7.14−7.26 (m, 3H), 7.54 (s, 1H), 7.81 (d, J = 7.91 Hz,
1H) ppm.
ACKNOWLEDGMENTS
■
We thank Mahalingam Kannan and the team at Syngene
(Bangalore, India) for chemistry support. We thank AstraZe-
neca Infection colleagues Nancy DeGrace and Natascha
Bezdenejnih-Snyder for analytical support, Seth Ribe for
synthetic scale-up, Hongming Wang for modeling support,
Ann Boriack-Sjodin for structural biology support, Swati Prasad
for human TMK IC50s, and Selvi Pradeepan for coordinating
log D measurements.
2-(3-Chlorophenoxy)-4-((R)-3,3-dimethyl-1-((S)-3-(5-methyl-2,4-
dioxo-3,4-dihydropyrimidin-1(2H)-yl)piperidin-1-yl)hexyl)benzoic
Acid (48). LC-MS (Method 2) tR = 2.16 min, m/z = 568.1 (M + 1),
566.2 (M − 1). HRMS calcd for C31H39ClN3O5 568.2573, found
1
568.2562. H NMR (300 MHz, CD3OD) δ 0.64 (s, 3H), 0.69 (t, J =
6.78 Hz, 3H), 0.75 (s, 3H), 0.92−1.20 (m, 4H), 1.72−1.91 (m, 7H),
2.02 (d, J = 9.23 Hz, 1 H), 2.18−2.31 (m, 1H), 2.60 (br s, 1H), 2.85
(br s, 1H), 3.44 (d, J = 7.72 Hz, 1H), 3.52 (d, J = 10.74 Hz, 1H), 4.45
(d, J = 10.55 Hz, 1H), 4.55 (d, J = 7.54 Hz, 1H), 6.77 (ddd, J = 8.29,
2.45, 0.75 Hz, 1H), 6.80−6.84 (m, 1H), 7.01 (ddd, J = 8.01, 1.88, 0.85
Hz, 1H), 7.18−7.26 (m, 2 H), 7.29 (d, J = 1.13 Hz, 1H), 7.44 (dd, J =
8.01, 1.60 Hz, 1H), 7.97 (d, J = 8.10 Hz, 1H) ppm. Diastereomeric
ratio: >99:1
ABBREVIATIONS
■
TMK, thymidylate kinase; Spn, Streptococcus pneumoniae; Sau,
Staphylococcus aureus; MIC, minimum inhibitory concentration
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